Scott D. Boyd, MD PhD

All Publications

• Antibody and B Cell Responses to SARS-CoV-2 Infection and Vaccination: The End of the Beginning. Annual review of pathology Roltgen, K., Boyd, S. D. 2023

  Abstract

  As the COVID-19 pandemic has evolved during the past years, interactions between human immune systems, rapidly mutating and selected SARS-CoV-2 viral variants, and effective vaccines have complicated the landscape of individual immunological histories. Here, we review some key findings for antibody and B cell-mediated immunity, including responses to the highly mutated omicron variants; immunological imprinting and other impacts of successive viral antigenic variant exposures on antibody and B cell memory; responses in secondary lymphoid and mucosal tissues and non-neutralizing antibody-mediated immunity; responses in populations vulnerable to severe disease such as those with cancer, immunodeficiencies, and other comorbidities, as well as populations showing apparent resistance to severe disease such as many African populations; and evidence of antibody involvement in postacute sequelae of infection or long COVID. Despite the initial phase of the pandemic ending, human populations will continue to face challenges presented by this unpredictable virus. Expected final online publication date for the Annual Review of Pathology: Mechanisms of Disease, Volume 19 is January 2024. Please see http://www.annualreviews.org/page/journal/pubdates for revised estimates.

  View details for DOI 10.1146/annurev-pathmechdis-031521-042754

  View details for PubMedID 37738512

• Influenza virus infection history shapes antibody responses to influenza vaccination. Nature medicine Auladell, M., Phuong, H. V., Mai, L. T., Tseng, Y., Carolan, L., Wilks, S., Thai, P. Q., Price, D., Duong, N. T., Hang, N. L., Thanh, L. T., Thuong, N. T., Huong, T. T., Diep, N. T., Bich, V. T., Khvorov, A., Hensen, L., Duong, T. N., Kedzierska, K., Anh, D. D., Wertheim, H., Boyd, S. D., Good-Jacobson, K. L., Smith, D., Barr, I., Sullivan, S., van Doorn, H. R., Fox, A. 2022

  Abstract

  Studies of successive vaccination suggest that immunological memory against past influenza viruses may limit responses to vaccines containing current strains. The impact of memory induced by prior infection is rarely considered and is difficult to ascertain, because infections are often subclinical. This study investigated influenza vaccination among adults from the Ha Nam cohort (Vietnam), who were purposefully selected to include 72 with and 28 without documented influenza A(H3N2) infection during the preceding 9 years (Australian New Zealand Clinical Trials Registry 12621000110886). The primary outcome was the effect of prior influenza A(H3N2) infection on hemagglutinin-inhibiting antibody responses induced by a locally available influenza vaccine administered in November 2016. Baseline and postvaccination sera were titrated against 40 influenza A(H3N2) strains spanning 1968-2018. At each time point (baseline, day 14 and day 280), geometric mean antibody titers against 2008-2018 strains were higher among participants with recent infection (34 (29-40), 187 (154-227) and 86 (72-103)) than among participants without recent infection (19 (17-22), 91 (64-130) and 38 (30-49)). On days 14 and 280, mean titer rises against 2014-2018 strains were 6.1-fold (5.0- to 7.4-fold) and 2.6-fold (2.2- to 3.1-fold) for participants with recent infection versus 4.8-fold (3.5- to 6.7-fold) and 1.9-fold (1.5- to 2.3-fold) for those without. One of 72 vaccinees with recent infection versus 4 of 28 without developed symptomatic A(H3N2) infection in the season after vaccination (P=0.021). The range of A(H3N2) viruses recognized by vaccine-induced antibodies was associated with the prior infection strain. These results suggest that recall of immunological memory induced by prior infection enhances antibody responses to inactivated influenza vaccine and is important to attain protective antibody titers.

  View details for DOI 10.1038/s41591-022-01690-w

  View details for PubMedID 35177857

• Multiple early factors anticipate post-acute COVID-19 sequelae. Cell Su, Y., Yuan, D., Chen, D. G., Ng, R. H., Wang, K., Choi, J., Li, S., Hong, S., Zhang, R., Xie, J., Kornilov, S. A., Scherler, K., Pavlovitch-Bedzyk, A. J., Dong, S., Lausted, C., Lee, I., Fallen, S., Dai, C. L., Baloni, P., Smith, B., Duvvuri, V. R., Anderson, K. G., Li, J., Yang, F., Duncombe, C. J., McCulloch, D. J., Rostomily, C., Troisch, P., Zhou, J., Mackay, S., DeGottardi, Q., May, D. H., Taniguchi, R., Gittelman, R. M., Klinger, M., Snyder, T. M., Roper, R., Wojciechowska, G., Murray, K., Edmark, R., Evans, S., Jones, L., Zhou, Y., Rowen, L., Liu, R., Chour, W., Algren, H. A., Berrington, W. R., Wallick, J. A., Cochran, R. A., Micikas, M. E., ISB-Swedish COVID-19 Biobanking Unit, Wrin, T., Petropoulos, C. J., Cole, H. R., Fischer, T. D., Wei, W., Hoon, D. S., Price, N. D., Subramanian, N., Hill, J. A., Hadlock, J., Magis, A. T., Ribas, A., Lanier, L. L., Boyd, S. D., Bluestone, J. A., Chu, H., Hood, L., Gottardo, R., Greenberg, P. D., Davis, M. M., Goldman, J. D., Heath, J. R. 2022

  Abstract

  Post-acute sequelae of COVID-19 (PASC) represent an emerging global crisis. However, quantifiable risk factors for PASC and their biological associations are poorly resolved. We executed a deep multi-omic, longitudinal investigation of 309 COVID-19 patients from initial diagnosis to convalescence (2-3months later), integrated with clinical data and patient-reported symptoms. We resolved four PASC-anticipating risk factors at the time of initial COVID-19 diagnosis: type 2 diabetes, SARS-CoV-2 RNAemia, Epstein-Barr virus viremia, and specific auto-antibodies. In patients with gastrointestinal PASC, SARS-CoV-2-specific and CMV-specific CD8+ Tcells exhibited unique dynamics during recovery from COVID-19. Analysis of symptom-associated immunological signatures revealed coordinated immunity polarization into four endotypes, exhibiting divergent acute severity and PASC. We find that immunological associations between PASC factors diminish over time, leading to distinct convalescent immune states. Detectability of most PASC factors at COVID-19 diagnosis emphasizes the importance of early disease measurements for understanding emergent chronic conditions and suggests PASC treatment strategies.

  View details for DOI 10.1016/j.cell.2022.01.014

  View details for PubMedID 35216672

• Early non-neutralizing, afucosylated antibody responses are associated with COVID-19 severity. Science translational medicine Chakraborty, S., Gonzalez, J. C., Sievers, B. L., Mallajosyula, V., Chakraborty, S., Dubey, M., Ashraf, U., Cheng, B. Y., Kathale, N., Tran, K. Q., Scallan, C., Sinnott, A., Cassidy, A., Chen, S. T., Gelbart, T., Gao, F., Golan, Y., Ji, X., Kim-Schulze, S., Prahl, M., Gaw, S. L., Gnjatic, S., Marron, T. U., Merad, M., Arunachalam, P. S., Boyd, S. D., Davis, M. M., Holubar, M., Khosla, C., Maecker, H. T., Maldonado, Y., Mellins, E. D., Nadeau, K. C., Pulendran, B., Singh, U., Subramanian, A., Utz, P. J., Sherwood, R., Zhang, S., Jagannathan, P., Tan, G. S., Wang, T. T. 1800: eabm7853

  Abstract

  A damaging inflammatory response is implicated in the pathogenesis of severe coronavirus disease 2019 (COVID-19), but mechanisms contributing to this response are unclear. In two prospective cohorts, early non-neutralizing, afucosylated IgG antibodies specific to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) were associated with progression from mild to more severe COVID-19. In contrast to the antibody structures that were associated with disease progression, antibodies that were elicited by mRNA SARS-CoV-2 vaccines were instead highly fucosylated and enriched in sialylation, both modifications that reduce the inflammatory potential of IgG. To study the biology afucosylated IgG immune complexes, we developed an in vivo model that revealed that human IgG-Fc gamma receptor (FcgammaR) interactions could regulate inflammation in the lung. Afucosylated IgG immune complexes isolated from COVID-19 patients induced inflammatory cytokine production and robust infiltration of the lung by immune cells. By contrast, vaccine-elicited IgG did not promote an inflammatory lung response. Together, these results show that IgG-FcgammaR interactions are able to regulate inflammation in the lung and may define distinct lung activities associated with the IgG that are associated with severe COVID-19 and protection against infection with SARS-CoV-2.

  View details for DOI 10.1126/scitranslmed.abm7853

  View details for PubMedID 35040666

• Durability of immune responses to the BNT162b2 mRNA vaccine MED Suthar, M. S., Arunachalam, P. S., Hu, M., Reis, N., Trisal, M., Raeber, O., Chinthrajah, S., Davis-Gardner, M. E., Manning, K., Mudvari, P., Boritz, E., Godbole, S., Henry, A. R., Douek, D. C., Halfmann, P., Kawaoka, Y., Boyd, S. D., Davis, M. M., Zarnitsyna, V. I., Nadeau, K., Pulendran, B. 2022; 3 (1): 25-27

  View details for DOI 10.1016/j.medj.2021.12.005

  View details for Web of Science ID 000758831100004

• Antibodies elicited by SARS-CoV-2 infection or mRNA vaccines have reduced neutralizing activity against Beta and Omicron pseudoviruses. Science translational medicine Sievers, B. L., Chakraborty, S., Xue, Y., Gelbart, T., Gonzalez, J. C., Cassidy, A. G., Golan, Y., Prahl, M., Gaw, S. L., Arunachalam, P. S., Blish, C. A., Boyd, S. D., Davis, M. M., Jagannathan, P., Nadeau, K. C., Pulendran, B., Singh, U., Scheuermann, R. H., Frieman, M. B., Vashee, S., Wang, T. T., Tan, G. S. 1800: eabn7842

  Abstract

  Multiple severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants that possess mutations associated with increased transmission and antibody escape have arisen over the course of the current pandemic. Although the current vaccines have largely been effective against past variants, the number of mutations found on the Omicron (B.1.1.529) spike protein appear to diminish the protection conferred by pre-existing immunity. Using vesicular stomatitis virus (VSV) pseudoparticles expressing the spike protein of several SARS-CoV-2 variants, we evaluated the magnitude and breadth of the neutralizing antibody response over time in individuals after infection and in mRNA-vaccinated individuals. We observed that boosting increases the magnitude of the antibody response to wildtype (D614), Beta, Delta, and Omicron variants; however, the Omicron variant was the most resistant to neutralization. We further observed that vaccinated healthy adults had robust and broad antibody responses whereas responses may have been reduced in vaccinated pregnant women, underscoring the importance of learning how to maximize mRNA vaccine responses in pregnant populations. Findings from this study show substantial heterogeneity in the magnitude and breadth of responses after infection and mRNA vaccination and may support the addition of more conserved viral antigens to existing SARS-CoV-2 vaccines.

  View details for DOI 10.1126/scitranslmed.abn7842

  View details for PubMedID 35025672

• Immune imprinting, breadth of variant recognition, and germinal center response in human SARS-CoV-2 infection and vaccination. Cell Röltgen, K., Nielsen, S. C., Silva, O., Younes, S. F., Zaslavsky, M., Costales, C., Yang, F., Wirz, O. F., Solis, D., Hoh, R. A., Wang, A., Arunachalam, P. S., Colburg, D., Zhao, S., Haraguchi, E., Lee, A. S., Shah, M. M., Manohar, M., Chang, I., Gao, F., Mallajosyula, V., Li, C., Liu, J., Shoura, M. J., Sindher, S. B., Parsons, E., Dashdorj, N. J., Dashdorj, N. D., Monroe, R., Serrano, G. E., Beach, T. G., Chinthrajah, R. S., Charville, G. W., Wilbur, J. L., Wohlstadter, J. N., Davis, M. M., Pulendran, B., Troxell, M. L., Sigal, G. B., Natkunam, Y., Pinsky, B. A., Nadeau, K. C., Boyd, S. D. 2022

  Abstract

  During the SARS-CoV-2 pandemic, novel and traditional vaccine strategies have been deployed globally. We investigated whether antibodies stimulated by mRNA vaccination (BNT162b2), including third-dose boosting, differ from those generated by infection or adenoviral (ChAdOx1-S and Gam-COVID-Vac) or inactivated viral (BBIBP-CorV) vaccines. We analyzed human lymph nodes after infection or mRNA vaccination for correlates of serological differences. Antibody breadth against viral variants is lower after infection compared with all vaccines evaluated but improves over several months. Viral variant infection elicits variant-specific antibodies, but prior mRNA vaccination imprints serological responses toward Wuhan-Hu-1 rather than variant antigens. In contrast to disrupted germinal centers (GCs) in lymph nodes during infection, mRNA vaccination stimulates robust GCs containing vaccine mRNA and spike antigen up to 8 weeks postvaccination in some cases. SARS-CoV-2 antibody specificity, breadth, and maturation are affected by imprinting from exposure history and distinct histological and antigenic contexts in infection compared with vaccination.

  View details for DOI 10.1016/j.cell.2022.01.018

  View details for PubMedID 35148837

• Direct comparison of antibody responses to four SARS-CoV-2 vaccines in Mongolia. Cell host & microbe Dashdorj, N. J., Wirz, O. F., Roltgen, K., Haraguchi, E., Buzzanco, A. S., Sibai, M., Wang, H., Miller, J. A., Solis, D., Sahoo, M. K., Arunachalam, P. S., Lee, A. S., Shah, M. M., Liu, J., Byambabaatar, S., Bat-Ulzii, P., Enkhbat, A., Batbold, E., Zulkhuu, D., Ochirsum, B., Khurelsukh, T., Dalantai, G., Burged, N., Baatarsuren, U., Ariungerel, N., Oidovsambuu, O., Bungert, A. S., Genden, Z., Yagaanbuyant, D., Mordorj, A., Pulendran, B., Chinthrajah, S., Nadeau, K. C., Jardetzky, T., Wilbur, J. L., Wohlstadter, J. N., Sigal, G. B., Pinsky, B. A., Boyd, S. D., Dashdorj, N. D. 2021

  Abstract

  Different SARS-CoV-2 vaccines are approved in various countries, but few direct comparisons of the antibody responses they stimulate have been reported. We collected plasma specimens in July 2021 from 196 Mongolian participants fully vaccinated with one of four COVID-19 vaccines: Pfizer/BioNTech, AstraZeneca, Sputnik V, and Sinopharm. Functional antibody testing with a panel of nine SARS-CoV-2 viral variant receptor binding domain (RBD) proteins revealed marked differences in vaccine responses, with low antibody levels and RBD-ACE2 blocking activity stimulated by the Sinopharm and Sputnik V vaccines in comparison to the AstraZeneca or Pfizer/BioNTech vaccines. The Alpha variant caused 97% of infections in Mongolia in June and early July 2021. Individuals who recover from SARS-CoV-2 infection after vaccination achieve high antibody titers in most cases. These data suggest that public health interventions such as vaccine boosting, potentially with more potent vaccine types, may be needed to control COVID-19 in Mongolia and worldwide.

  View details for DOI 10.1016/j.chom.2021.11.004

  View details for PubMedID 34861167

• Antibody and B cell responses to SARS-CoV-2 infection and vaccination. Cell host & microbe Roltgen, K., Boyd, S. D. 2021

  Abstract

  Antibodies, and the B cell and plasma cell populations responsible for their production, are key components of the human immune system's response to SARS-CoV-2, which has caused the coronavirus disease 2019 (COVID-19) pandemic. Here, we review findings addressing the nature of antibody responses against SARS-CoV-2 and their role in protecting from infection or modulating COVID-19 disease severity. In just over a year, much has been learned, and replicated in independent studies, about human immune responses to this pathogen, contributing to the development of effective vaccines. Nevertheless, important questions remain about the duration and effectiveness of antibody responses, differences between immunity derived from infection compared to vaccination, the cellular basis for serological findings, and the extent to which viral variants will escape from current immunity.

  View details for DOI 10.1016/j.chom.2021.06.009

  View details for PubMedID 34174992

• Shared B cell memory to coronaviruses and other pathogens varies in human age groups and tissues. Science (New York, N.Y.) Yang, F., Nielsen, S. C., Hoh, R. A., Roltgen, K., Wirz, O. F., Haraguchi, E., Jean, G. H., Lee, J., Pham, T. D., Jackson, K. J., Roskin, K. M., Liu, Y., Nguyen, K., Ohgami, R. S., Osborne, E. M., Nadeau, K. C., Niemann, C. U., Parsonnet, J., Boyd, S. D. 2021

  Abstract

  Vaccination and infection promote the formation, tissue distribution, and clonal evolution of B cells, which encode humoral immune memory. We evaluated convergent antigen-specific antibody genes of similar sequences shared between individuals in pediatric and adult blood, and deceased organ donor tissues. B cell memory varied for different pathogens. Polysaccharide antigen-specific clones were not exclusive to the spleen. Adults had higher clone frequencies and greater class-switching in lymphoid tissues than blood, while pediatric blood had abundant class-switched convergent clones. Consistent with reported serology, pre-pandemic children had class-switched convergent clones to SARS-CoV-2 with weak cross-reactivity to other coronaviruses, while adult blood or tissues showed few such clones. The results highlight the prominence of early childhood B cell clonal expansions and cross-reactivity for future responses to novel pathogens.

  View details for DOI 10.1126/science.abf6648

  View details for PubMedID 33846272

• Local immune response to food antigens drives meal-induced abdominal pain. Nature Aguilera-Lizarraga, J., Florens, M. V., Viola, M. F., Jain, P., Decraecker, L., Appeltans, I., Cuende-Estevez, M., Fabre, N., Van Beek, K., Perna, E., Balemans, D., Stakenborg, N., Theofanous, S., Bosmans, G., Mondelaers, S. U., Matteoli, G., Ibiza Martinez, S., Lopez-Lopez, C., Jaramillo-Polanco, J., Talavera, K., Alpizar, Y. A., Feyerabend, T. B., Rodewald, H., Farre, R., Redegeld, F. A., Si, J., Raes, J., Breynaert, C., Schrijvers, R., Bosteels, C., Lambrecht, B. N., Boyd, S. D., Hoh, R. A., Cabooter, D., Nelis, M., Augustijns, P., Hendrix, S., Strid, J., Bisschops, R., Reed, D. E., Vanner, S. J., Denadai-Souza, A., Wouters, M. M., Boeckxstaens, G. E. 2021

  Abstract

  Up to 20% of people worldwide develop gastrointestinal symptoms following a meal1, leading to decreased quality of life, substantial morbidity and high medical costs. Although the interest of both the scientific and lay communities in this issue has increased markedly in recent years, with the worldwide introduction of gluten-free and other diets, the underlying mechanisms of food-induced abdominal complaints remain largely unknown. Here we show that a bacterial infection and bacterial toxins can trigger an immune response that leads to the production of dietary-antigen-specific IgE antibodies in mice, which are limited to the intestine. Following subsequent oral ingestion of the respective dietary antigen, an IgE- and mast-cell-dependent mechanism induced increased visceral pain. This aberrant pain signalling resulted from histamine receptor H1-mediated sensitization of visceral afferents. Moreover, injection of food antigens (gluten, wheat, soy and milk) into the rectosigmoid mucosa of patients with irritable bowel syndrome induced local oedema and mast cell activation. Our results identify and characterize a peripheral mechanism that underlies food-induced abdominal pain, thereby creating new possibilities for the treatment of irritable bowel syndrome and related abdominal pain disorders.

  View details for DOI 10.1038/s41586-020-03118-2

  View details for PubMedID 33442055

• Modeling human adaptive immune responses with tonsil organoids. Nature medicine Wagar, L. E., Salahudeen, A. n., Constantz, C. M., Wendel, B. S., Lyons, M. M., Mallajosyula, V. n., Jatt, L. P., Adamska, J. Z., Blum, L. K., Gupta, N. n., Jackson, K. J., Yang, F. n., Röltgen, K. n., Roskin, K. M., Blaine, K. M., Meister, K. D., Ahmad, I. N., Cortese, M. n., Dora, E. G., Tucker, S. N., Sperling, A. I., Jain, A. n., Davies, D. H., Felgner, P. L., Hammer, G. B., Kim, P. S., Robinson, W. H., Boyd, S. D., Kuo, C. J., Davis, M. M. 2021

  Abstract

  Most of what we know about adaptive immunity has come from inbred mouse studies, using methods that are often difficult or impossible to confirm in humans. In addition, vaccine responses in mice are often poorly predictive of responses to those same vaccines in humans. Here we use human tonsils, readily available lymphoid organs, to develop a functional organotypic system that recapitulates key germinal center features in vitro, including the production of antigen-specific antibodies, somatic hypermutation and affinity maturation, plasmablast differentiation and class-switch recombination. We use this system to define the essential cellular components necessary to produce an influenza vaccine response. We also show that it can be used to evaluate humoral immune responses to two priming antigens, rabies vaccine and an adenovirus-based severe acute respiratory syndrome coronavirus 2 vaccine, and to assess the effects of different adjuvants. This system should prove useful for studying critical mechanisms underlying adaptive immunity in much greater depth than previously possible and to rapidly test vaccine candidates and adjuvants in an entirely human system.

  View details for DOI 10.1038/s41591-020-01145-0

  View details for PubMedID 33432170

• Systems vaccinology of the BNT162b2 mRNA vaccine in humans. Nature Arunachalam, P. S., Scott, M. K., Hagan, T., Li, C., Feng, Y., Wimmers, F., Grigoryan, L., Trisal, M., Edara, V. V., Lai, L., Chang, S. E., Feng, A., Dhingra, S., Shah, M., Lee, A. S., Chinthrajah, S., Sindher, S. B., Mallajosyula, V., Gao, F., Sigal, N., Kowli, S., Gupta, S., Pellegrini, K., Tharp, G., Maysel-Auslender, S., Hamilton, S., Aoued, H., Hrusovsky, K., Roskey, M., Bosinger, S. E., Maecker, H. T., Boyd, S. D., Davis, M. M., Utz, P. J., Suthar, M. S., Khatri, P., Nadeau, K. C., Pulendran, B. 2021

  Abstract

  The emergency use authorization of two mRNA vaccines in less than a year since the emergence of SARS-CoV-2 represents a landmark in vaccinology1,2. Yet, how mRNA vaccines stimulate the immune system to elicit protective immune responses is unknown. Here we used a systems vaccinology approach to comprehensively profile the innate and adaptive immune responses of 56 healthy volunteers vaccinated with the Pfizer-BioNTech mRNA vaccine. Vaccination resulted in robust production of neutralizing antibodies (nAbs) against the parent Wuhan strain and, to a lesser extent, the B.1.351 strain, and significant increases in antigen-specific polyfunctional CD4 and CD8 T cells after the second dose. Booster vaccination stimulated a strikingly enhanced innate immune response compared to primary vaccination, evidenced by a greater: (i) frequency of CD14+CD16+ inflammatory monocytes; (ii) concentration of plasma IFN-g; (iii) transcriptional signature of innate antiviral immunity. Consistent with these observations, single-cell transcriptomics analysis demonstrated a ~100-fold increase in the frequency of a myeloid cell cluster, enriched in interferon-response transcription factors (TFs) and reduced in AP-1 TFs, following secondary immunization. Finally, we identified distinct innate pathways associated with CD8 T cell and nAb responses, and show that a monocyte-related signature correlates with the nAb response against the B.1.351 variant strain. Collectively, these data provide insights into immune responses induced by mRNA vaccination and demonstrate its capacity to prime the innate immune system to mount a more potent response following booster immunization.

  View details for DOI 10.1038/s41586-021-03791-x

  View details for PubMedID 34252919

• Aberrant B cell repertoire selection associated with HIV neutralizing antibody breadth. Nature immunology Roskin, K. M., Jackson, K. J., Lee, J., Hoh, R. A., Joshi, S. A., Hwang, K., Bonsignori, M., Pedroza-Pacheco, I., Liao, H., Moody, M. A., Fire, A. Z., Borrow, P., Haynes, B. F., Boyd, S. D. 2020

  Abstract

  A goal of HIV vaccine development is to elicit antibodies with neutralizing breadth. Broadly neutralizing antibodies (bNAbs) to HIV often have unusual sequences with long heavy-chain complementarity-determining region loops, high somatic mutation rates and polyreactivity. A subset of HIV-infected individuals develops such antibodies, but it is unclear whether this reflects systematic differences in their antibody repertoires or is a consequence of rare stochastic events involving individual clones. We sequenced antibody heavy-chain repertoires in a large cohort of HIV-infected individuals with bNAb responses or no neutralization breadth and uninfected controls, identifying consistent features of bNAb repertoires, encompassing thousands of B cell clones per individual, with correlated T cell phenotypes. These repertoire features were not observed during chronic cytomegalovirus infection in an independent cohort. Our data indicate that the development of numerous B cell lineages with antibody features associated with autoreactivity may be a key aspect in the development of HIV neutralizing antibody breadth.

  View details for DOI 10.1038/s41590-019-0581-0

  View details for PubMedID 31959979

• Defining the features and duration of antibody responses to SARS-CoV-2 infection associated with disease severity and outcome. Science immunology Röltgen, K. n., Powell, A. E., Wirz, O. F., Stevens, B. A., Hogan, C. A., Najeeb, J. n., Hunter, M. n., Wang, H. n., Sahoo, M. K., Huang, C. n., Yamamoto, F. n., Manohar, M. n., Manalac, J. n., Otrelo-Cardoso, A. R., Pham, T. D., Rustagi, A. n., Rogers, A. J., Shah, N. H., Blish, C. A., Cochran, J. R., Jardetzky, T. S., Zehnder, J. L., Wang, T. T., Narasimhan, B. n., Gombar, S. n., Tibshirani, R. n., Nadeau, K. C., Kim, P. S., Pinsky, B. A., Boyd, S. D. 2020; 5 (54)

  Abstract

  SARS-CoV-2-specific antibodies, particularly those preventing viral spike receptor binding domain (RBD) interaction with host angiotensin-converting enzyme 2 (ACE2) receptor, can neutralize the virus. It is, however, unknown which features of the serological response may affect clinical outcomes of COVID-19 patients. We analyzed 983 longitudinal plasma samples from 79 hospitalized COVID-19 patients and 175 SARS-CoV-2-infected outpatients and asymptomatic individuals. Within this cohort, 25 patients died of their illness. Higher ratios of IgG antibodies targeting S1 or RBD domains of spike compared to nucleocapsid antigen were seen in outpatients who had mild illness versus severely ill patients. Plasma antibody increases correlated with decreases in viral RNAemia, but antibody responses in acute illness were insufficient to predict inpatient outcomes. Pseudovirus neutralization assays and a scalable ELISA measuring antibodies blocking RBD-ACE2 interaction were well correlated with patient IgG titers to RBD. Outpatient and asymptomatic individuals' SARS-CoV-2 antibodies, including IgG, progressively decreased during observation up to five months post-infection.

  View details for DOI 10.1126/sciimmunol.abe0240

  View details for PubMedID 33288645

• Origins and clonal convergence of gastrointestinal IgE+ B cells in human peanut allergy. Science immunology Hoh, R. A., Joshi, S. A., Lee, J. Y., Martin, B. A., Varma, S. n., Kwok, S. n., Nielsen, S. C., Nejad, P. n., Haraguchi, E. n., Dixit, P. S., Shutthanandan, S. V., Roskin, K. M., Zhang, W. n., Tupa, D. n., Bunning, B. J., Manohar, M. n., Tibshirani, R. n., Fernandez-Becker, N. Q., Kambham, N. n., West, R. B., Hamilton, R. G., Tsai, M. n., Galli, S. J., Chinthrajah, R. S., Nadeau, K. C., Boyd, S. D. 2020; 5 (45)

  Abstract

  B cells in human food allergy have been studied predominantly in the blood. Little is known about IgE+ B cells or plasma cells in tissues exposed to dietary antigens. We characterized IgE+ clones in blood, stomach, duodenum, and esophagus of 19 peanut-allergic patients, using high-throughput DNA sequencing. IgE+ cells in allergic patients are enriched in stomach and duodenum, and have a plasma cell phenotype. Clonally related IgE+ and non-IgE-expressing cell frequencies in tissues suggest local isotype switching, including transitions between IgA and IgE isotypes. Highly similar antibody sequences specific for peanut allergen Ara h 2 are shared between patients, indicating that common immunoglobulin genetic rearrangements may contribute to pathogenesis. These data define the gastrointestinal tract as a reservoir of IgE+ B lineage cells in food allergy.

  View details for DOI 10.1126/sciimmunol.aay4209

  View details for PubMedID 32139586

• Influenza vaccine-induced human bone marrow plasma cells decline within a year after vaccination. Science (New York, N.Y.) Davis, C. W., Jackson, K. J., McCausland, M. M., Darce, J. n., Chang, C. n., Linderman, S. L., Chennareddy, C. n., Gerkin, R. n., Brown, S. J., Wrammert, J. n., Mehta, A. K., Cheung, W. C., Boyd, S. D., Waller, E. K., Ahmed, R. n. 2020

  Abstract

  A universal vaccine against influenza would ideally generate protective immune responses that are not only broadly reactive against multiple influenza strains, but also long-lasting. Because long-term serum antibody levels are maintained by bone marrow plasma cells (BMPC), we investigated the production and maintenance of these cells after influenza vaccination. We found increased numbers of influenza-specific BMPC four weeks after immunization with the seasonal inactivated influenza vaccine, but numbers returned to near their pre-vaccination levels after one year. This decline was driven by the loss of BMPC induced by the vaccine, while pre-existing BMPC were maintained. Our results suggest that most BMPC generated by influenza vaccination in adults are short-lived. Designing strategies to enhance their persistence will be a key challenge for the next generation of influenza vaccines.

  View details for DOI 10.1126/science.aaz8432

  View details for PubMedID 32792465

• Human B Cell Clonal Expansion and Convergent Antibody Responses to SARS-CoV-2. Cell host & microbe Nielsen, S. C., Yang, F. n., Jackson, K. J., Hoh, R. A., Röltgen, K. n., Jean, G. H., Stevens, B. A., Lee, J. Y., Rustagi, A. n., Rogers, A. J., Powell, A. E., Hunter, M. n., Najeeb, J. n., Otrelo-Cardoso, A. R., Yost, K. E., Daniel, B. n., Nadeau, K. C., Chang, H. Y., Satpathy, A. T., Jardetzky, T. S., Kim, P. S., Wang, T. T., Pinsky, B. A., Blish, C. A., Boyd, S. D. 2020

  Abstract

  B cells are critical for the production of antibodies and protective immunity to viruses. Here we show that patients infected with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) who develop coronavirus disease 2019 (COVID-19) display early recruitment of B cells expressing a limited subset of IGHV genes, progressing to a highly polyclonal response of B cells with broader IGHV gene usage and extensive class switching to IgG and IgA subclasses with limited somatic hypermutation in the initial weeks of infection. We identify convergence of antibody sequences across SARS-CoV-2-infected patients, highlighting stereotyped naive responses to this virus. Notably, sequence-based detection in COVID-19 patients of convergent B cell clonotypes previously reported in SARS-CoV infection predicts the presence of SARS-CoV/SARS-CoV-2 cross-reactive antibody titers specific for the receptor-binding domain. These findings offer molecular insights into shared features of human B cell responses to SARS-CoV-2 and SARS-CoV.

  View details for DOI 10.1016/j.chom.2020.09.002

  View details for PubMedID 32941787

• Proinflammatory IgG Fc structures in patients with severe COVID-19 Nature Immunology Chakraborty, S., Gonzales, J., Edwards, K., Mallajosyulla, V., Buzzanco, A. S., Sherwood, R., Buffone, C., Kathale, N., Providenza, S., Xie, M. M., Andrews, J. R., Blish, C. A., Singh, U., Dugan, H., Wilson, P. C., Pham, T. D., Boyd, S. D., Nadeau, K. C., Pinsky, B. A., Zhang, S., Memoli, M. J., Taubenberger, J. K., Morales, T., Schapiro, J. M., Tan, G. S., et al 2020

  View details for DOI 10.1038/s41590-020-00828-7

• Shaping of infant B cell receptor repertoires by environmental factors and infectious disease. Science translational medicine Nielsen, S. C., Roskin, K. M., Jackson, K. J., Joshi, S. A., Nejad, P., Lee, J., Wagar, L. E., Pham, T. D., Hoh, R. A., Nguyen, K. D., Tsunemoto, H. Y., Patel, S. B., Tibshirani, R., Ley, C., Davis, M. M., Parsonnet, J., Boyd, S. D. 2019; 11 (481)

  Abstract

  Antigenic exposures at epithelial sites in infancy and early childhood are thought to influence the maturation of humoral immunity and modulate the risk of developing immunoglobulin E (IgE)-mediated allergic disease. How different kinds of environmental exposures influence B cell isotype switching to IgE, IgG, or IgA, and the somatic mutation maturation of these antibody pools, is not fully understood. We sequenced antibody repertoires in longitudinal blood samples in a birth cohort from infancy through the first 3 years of life and found that, whereas IgG and IgA show linear increases in mutational maturation with age, IgM and IgD mutations are more closely tied to pathogen exposure. IgE mutation frequencies are primarily increased in children with impaired skin barrier conditions such as eczema, suggesting that IgE affinity maturation could provide a mechanistic link between epithelial barrier failure and allergy development.

  View details for PubMedID 30814336

• Shaping of infant B cell receptor repertoires by environmental factors and infectious disease SCIENCE TRANSLATIONAL MEDICINE Nielsen, S. A., Roskin, K. M., Jackson, K. L., Joshi, S. A., Nejad, P., Lee, J., Wagar, L. E., Pham, T. D., Hoh, R. A., Nguyen, K. D., Tsunemoto, H. Y., Patel, S. B., Tibshirani, R., Ley, C., Davis, M. M., Parsonnet, J., Boyd, S. D. 2019; 11 (481)

  View details for DOI 10.1126/scitranslmed.aat2004

  View details for Web of Science ID 000460307000001

• Longitudinal Analysis of the Human B Cell Response to Ebola Virus Infection. Cell Davis, C. W., Jackson, K. J., McElroy, A. K., Halfmann, P. n., Huang, J. n., Chennareddy, C. n., Piper, A. E., Leung, Y. n., Albariño, C. G., Crozier, I. n., Ellebedy, A. H., Sidney, J. n., Sette, A. n., Yu, T. n., Nielsen, S. C., Goff, A. J., Spiropoulou, C. F., Saphire, E. O., Cavet, G. n., Kawaoka, Y. n., Mehta, A. K., Glass, P. J., Boyd, S. D., Ahmed, R. n. 2019

  Abstract

  Ebola virus (EBOV) remains a public health threat. We performed a longitudinal study of B cell responses to EBOV in four survivors of the 2014 West African outbreak. Infection induced lasting EBOV-specific immunoglobulin G (IgG) antibodies, but their subclass composition changed over time, with IgG1 persisting, IgG3 rapidly declining, and IgG4 appearing late. Striking changes occurred in the immunoglobulin repertoire, with massive recruitment of naive B cells that subsequently underwent hypermutation. We characterized a large panel of EBOV glycoprotein-specific monoclonal antibodies (mAbs). Only a small subset of mAbs that bound glycoprotein by ELISA recognized cell-surface glycoprotein. However, this subset contained all neutralizing mAbs. Several mAbs protected against EBOV disease in animals, including one mAb that targeted an epitope under evolutionary selection during the 2014 outbreak. Convergent antibody evolution was seen across multiple donors, particularly among VH3-13 neutralizing antibodies specific for the GP1 core. Our study provides a benchmark for assessing EBOV vaccine-induced immunity.

  View details for DOI 10.1016/j.cell.2019.04.036

  View details for PubMedID 31104840

• High-fat diet induces systemic B-cell repertoire changes associated with insulin resistance. Mucosal immunology Pham, T. D., Chng, M. H., Roskin, K. M., Jackson, K. J., Nguyen, K. D., Glanville, J., Lee, J., Engleman, E. G., Boyd, S. D. 2017

  Abstract

  The development of obesity-associated insulin resistance is associated with B-lymphocyte accumulation in visceral adipose tissue (VAT) and is prevented by B-cell ablation. To characterize potentially pathogenic B-cell repertoires in this disorder, we performed high-throughput immunoglobulin (Ig) sequencing from multiple tissues of mice fed high-fat diet (HFD) and regular diet (RD). HFD significantly changed the biochemical properties of Ig heavy-chain complementarity-determining region-3 (CDRH3) sequences, selecting for IgA antibodies with shorter and more hydrophobic CDRH3 in multiple tissues. A set of convergent antibodies of highly similar sequences found in the VAT of HFD mice but not RD mice showed significant somatic mutation, suggesting a response shared between mice to a common antigen or antigens. These findings indicate that a simple high-fat dietary intervention has a major impact on mouse B-cell repertoires, particularly in adipose tissues.Mucosal Immunology advance online publication, 19 April 2017; doi:10.1038/mi.2017.25.

  View details for DOI 10.1038/mi.2017.25

  View details for PubMedID 28422186

• Identifying specificity groups in the T cell receptor repertoire. Nature Glanville, J. n., Huang, H. n., Nau, A. n., Hatton, O. n., Wagar, L. E., Rubelt, F. n., Ji, X. n., Han, A. n., Krams, S. M., Pettus, C. n., Haas, N. n., Arlehamn, C. S., Sette, A. n., Boyd, S. D., Scriba, T. J., Martinez, O. M., Davis, M. M. 2017

  Abstract

  T cell receptor (TCR) sequences are very diverse, with many more possible sequence combinations than T cells in any one individual. Here we define the minimal requirements for TCR antigen specificity, through an analysis of TCR sequences using a panel of peptide and major histocompatibility complex (pMHC)-tetramer-sorted cells and structural data. From this analysis we developed an algorithm that we term GLIPH (grouping of lymphocyte interactions by paratope hotspots) to cluster TCRs with a high probability of sharing specificity owing to both conserved motifs and global similarity of complementarity-determining region 3 (CDR3) sequences. We show that GLIPH can reliably group TCRs of common specificity from different donors, and that conserved CDR3 motifs help to define the TCR clusters that are often contact points with the antigenic peptides. As an independent validation, we analysed 5,711 TCRβ chain sequences from reactive CD4 T cells from 22 individuals with latent Mycobacterium tuberculosis infection. We found 141 TCR specificity groups, including 16 distinct groups containing TCRs from multiple individuals. These TCR groups typically shared HLA alleles, allowing prediction of the likely HLA restriction, and a large number of M. tuberculosis T cell epitopes enabled us to identify pMHC ligands for all five of the groups tested. Mutagenesis and de novo TCR design confirmed that the GLIPH-identified motifs were critical and sufficient for shared-antigen recognition. Thus the GLIPH algorithm can analyse large numbers of TCR sequences and define TCR specificity groups shared by TCRs and individuals, which should greatly accelerate the analysis of T cell responses and expedite the identification of specific ligands.

  View details for PubMedID 28636589

• Defining antigen-specific plasmablast and memory B cell subsets in human blood after viral infection or vaccination NATURE IMMUNOLOGY Ellebedy, A. H., Jackson, K. J., Kissick, H. T., Nakaya, H. I., Davis, C. W., Roskin, K. M., McElroy, A. K., Oshansky, C. M., Elbein, R., Thomas, S., Lyon, G. M., Spiropoulou, C. F., Mehta, A. K., Thomas, P. G., Boyd, S. D., Ahmed, R. 2016; 17 (10): 1226-?

  Abstract

  Antigen-specific B cells bifurcate into antibody-secreting cells (ASCs) and memory B cells (MBCs) after infection or vaccination. ASCs (plasmablasts) have been extensively studied in humans, but less is known about B cells that become activated but do not differentiate into plasmablasts. Here we have defined the phenotype and transcriptional program of a subset of antigen-specific B cells, which we have called 'activated B cells' (ABCs), that were distinct from ASCs and were committed to the MBC lineage. We detected ABCs in humans after infection with Ebola virus or influenza virus and also after vaccination. By simultaneously analyzing antigen-specific ASCs and ABCs in human blood after vaccination against influenza virus, we investigated the clonal overlap and extent of somatic hypermutation (SHM) in the ASC (effector) and ABC (memory) lineages. Longitudinal tracking of vaccination-induced hemagglutinin (HA)-specific clones revealed no overall increase in SHM over time, which suggested that repeated annual immunization might have limitations in enhancing the quality of influenza-virus-specific antibody.

  View details for DOI 10.1038/ni.3533

  View details for PubMedID 27525369

• Broadening Horizons: New Antibodies Against Influenza. Cell Jackson, K. J., Boyd, S. D. 2016; 166 (3): 532-533

  Abstract

  Seasonal influenza vaccine formulation efforts struggle to keep up with viral antigenic variation. Two studies now report engineered or naturally occurring human antibodies targeting the influenza hemagglutinin (HA) stem, with exceptional neutralizing breadth (Joyce et al., 2016; Kallewaard et al., 2016). Antibodies with similar structural features are elicited in multiple subjects, suggesting that modified vaccine regimens could provide broad protection.

  View details for DOI 10.1016/j.cell.2016.07.023

  View details for PubMedID 27471961

• Maturation Pathway from Germline to Broad HIV-1 Neutralizer of a CD4-Mimic Antibody. Cell Bonsignori, M., Zhou, T., Sheng, Z., Chen, L., Gao, F., Joyce, M. G., Ozorowski, G., Chuang, G., Schramm, C. A., Wiehe, K., Alam, S. M., Bradley, T., Gladden, M. A., Hwang, K., Iyengar, S., Kumar, A., Lu, X., Luo, K., Mangiapani, M. C., Parks, R. J., Song, H., Acharya, P., Bailer, R. T., Cao, A., Druz, A., Georgiev, I. S., Kwon, Y. D., Louder, M. K., Zhang, B., Zheng, A., Hill, B. J., Kong, R., Soto, C., Mullikin, J. C., Douek, D. C., Montefiori, D. C., Moody, M. A., Shaw, G. M., Hahn, B. H., Kelsoe, G., Hraber, P. T., Korber, B. T., Boyd, S. D., Fire, A. Z., Kepler, T. B., Shapiro, L., Ward, A. B., Mascola, J. R., Liao, H., Kwong, P. D., Haynes, B. F. 2016; 165 (2): 449-463

  Abstract

  Antibodies with ontogenies from VH1-2 or VH1-46-germline genes dominate the broadly neutralizing response against the CD4-binding site (CD4bs) on HIV-1. Here, we define with longitudinal sampling from time-of-infection the development of a VH1-46-derived antibody lineage that matured to neutralize 90% of HIV-1 isolates. Structures of lineage antibodies CH235 (week 41 from time-of-infection, 18% breadth), CH235.9 (week 152, 77%), and CH235.12 (week 323, 90%) demonstrated the maturing epitope to focus on the conformationally invariant portion of the CD4bs. Similarities between CH235 lineage and five unrelated CD4bs lineages in epitope focusing, length-of-time to develop breadth, and extraordinary level of somatic hypermutation suggested commonalities in maturation among all CD4bs antibodies. Fortunately, the required CH235-lineage hypermutation appeared substantially guided by the intrinsic mutability of the VH1-46 gene, which closely resembled VH1-2. We integrated our CH235-lineage findings with a second broadly neutralizing lineage and HIV-1 co-evolution to suggest a vaccination strategy for inducing both lineages.

  View details for DOI 10.1016/j.cell.2016.02.022

  View details for PubMedID 26949186

• Diversification of the antigen-specific T cell receptor repertoire after varicella zoster vaccination. Science translational medicine Qi, Q., Cavanagh, M. M., Le Saux, S., Namkoong, H., Kim, C., Turgano, E., Liu, Y., Wang, C., Mackey, S., Swan, G. E., Dekker, C. L., Olshen, R. A., Boyd, S. D., Weyand, C. M., Tian, L., Goronzy, J. J. 2016; 8 (332): 332ra46-?

  Abstract

  Diversity and size of the antigen-specific T cell receptor (TCR) repertoire are two critical determinants for successful control of chronic infection. Varicella zoster virus (VZV) that establishes latency during childhood can escape control mechanisms, in particular with increasing age. We examined the TCR diversity of VZV-reactive CD4 T cells in individuals older than 50 years by studying three identical twin pairs and three unrelated individuals before and after vaccination with live attenuated VZV. Although all individuals had a small number of dominant T cell clones, the breadth of the VZV-specific repertoire differed markedly. A genetic influence was seen for the sharing of individual TCR sequences from antigen-reactive cells but not for repertoire richness or the selection of dominant clones. VZV vaccination favored the expansion of infrequent VZV antigen-reactive TCRs, including those from naïve T cells with lesser boosting of dominant T cell clones. Thus, vaccination does not reinforce the in vivo selection that occurred during chronic infection but leads to a diversification of the VZV-reactive T cell repertoire. However, a single-booster immunization seems insufficient to establish new clonal dominance. Our results suggest that repertoire analysis of antigen-specific TCRs can be an important readout to assess whether a vaccination was able to generate memory cells in clonal sizes that are necessary for immune protection.

  View details for DOI 10.1126/scitranslmed.aaf1725

  View details for PubMedID 27030598

• IgH sequences in common variable immune deficiency reveal altered B cell development and selection. Science translational medicine Roskin, K. M., Simchoni, N., Liu, Y., Lee, J., Seo, K., Hoh, R. A., Pham, T., Park, J. H., Furman, D., Dekker, C. L., Davis, M. M., James, J. A., Nadeau, K. C., Cunningham-Rundles, C., Boyd, S. D. 2015; 7 (302): 302ra135-?

  Abstract

  Common variable immune deficiency (CVID) is the most common symptomatic primary immune deficiency, affecting ~1 in 25,000 persons. These patients suffer from impaired antibody responses, autoimmunity, and susceptibility to lymphoid cancers. To explore the cellular basis for these clinical phenotypes, we conducted high-throughput DNA sequencing of immunoglobulin heavy chain gene rearrangements from 93 CVID patients and 105 control subjects and sorted naïve and memory B cells from 13 of the CVID patients and 10 of the control subjects. The CVID patients showed abnormal VDJ rearrangement and abnormal formation of complementarity-determining region 3 (CDR3). We observed a decreased selection against antibodies with long CDR3s in memory repertoires and decreased variable gene replacement, offering possible mechanisms for increased patient autoreactivity. Our data indicate that patient immunodeficiency might derive from both decreased diversity of the naïve B cell pool and decreased somatic hypermutation in memory repertoires. The CVID patients also exhibited an abnormal clonal expansion of unmutated B cells relative to the controls. Although impaired B cell germinal center activation is commonly viewed as causative in CVID, these data indicate that CVID B cells diverge from controls as early as the pro-B stage, cell and suggest possible explanations for the increased incidence of autoimmunity, immunodeficiency, and lymphoma CVID patients.

  View details for DOI 10.1126/scitranslmed.aab1216

  View details for PubMedID 26311730

  View details for PubMedCentralID PMC4584259

• Single B-cell deconvolution of peanut-specific antibody responses in allergic patients. The Journal of allergy and clinical immunology Hoh, R. A., Joshi, S. A., Liu, Y., Wang, C., Roskin, K. M., Lee, J. Y., Pham, T., Looney, T. J., Jackson, K. J., Dixit, V. P., King, J., Lyu, S. C., Jenks, J., Hamilton, R. G., Nadeau, K. C., Boyd, S. D. 2015

  Abstract

  The frequencies, cellular phenotypes, epitope specificity, and clonal diversity of allergen-specific B cells in patients with food allergy are not fully understood but are of major pathogenic and therapeutic significance.We sought to characterize peanut allergen-specific B-cell populations and the sequences and binding activities of their antibodies before and during immunotherapy.B cells binding fluorescently labeled Ara h 1 or Ara h 2 were phenotyped and isolated by means of flow cytometric sorting from 18 patients at baseline and 13 patients during therapy. Fifty-seven mAbs derived from allergen-binding single B cells were evaluated by using ELISA, Western blotting, and peptide epitope mapping. Deep sequencing of the B-cell repertoires identified additional members of the allergen-specific B-cell clones.Median allergen-binding B-cell frequencies were 0.0097% (Ara h 1) or 0.029% (Ara h 2) of B cells in baseline blood from allergic patients and approximately 3-fold higher during immunotherapy. Five of 57 allergen-specific cells belonged to clones containing IgE-expressing members. Almost all allergen-specific antibodies were mutated, and binding to both conformational and linear allergen epitopes was detected. Increasing somatic mutation of IgG4 members of a clone was seen in immunotherapy, whereas IgE mutation levels in the clone did not increase.Most peanut allergen-binding B cells isolated by means of antigen-specific flow sorting express mutated and isotype-switched antibodies. Immunotherapy increases their frequency in the blood, and even narrowly defined allergen epitopes are recognized by numerous distinct B-cell clones in a patient. The results also suggest that oral immunotherapy can stimulate somatic mutation of allergen-specific IgG4.

  View details for DOI 10.1016/j.jaci.2015.05.029

  View details for PubMedID 26152318

• Predicting vaccine responsiveness. Cell host & microbe Boyd, S. D., Jackson, K. J. 2015; 17 (3): 301-307

  Abstract

  Vaccination has saved many lives and prevented needless suffering from disease, but it is not always effective. Immune responses are a highly "personalized" aspect of an individual's biology, as they are subject to germline genetic influences but are embodied in cell populations that continuously sample the environment. Additionally, immunity is shaped by memory of prior infectious diseases and other antigenic exposures. Here, we review examples of recent technical advances and insights into human vaccine responses that are helping to define the features associated with successful vaccination and that may enable a more predictive vaccinology in the future.

  View details for DOI 10.1016/j.chom.2015.02.015

  View details for PubMedID 25766292

• B-cell repertoire responses to varicella-zoster vaccination in human identical twins. Proceedings of the National Academy of Sciences of the United States of America Wang, C., Liu, Y., Cavanagh, M. M., Le Saux, S., Qi, Q., Roskin, K. M., Looney, T. J., Lee, J., Dixit, V., Dekker, C. L., Swan, G. E., Goronzy, J. J., Boyd, S. D. 2015; 112 (2): 500-505

  Abstract

  Adaptive immune responses in humans rely on somatic genetic rearrangements of Ig and T-cell receptor loci to generate diverse antigen receptors. It is unclear to what extent an individual's genetic background affects the characteristics of the antibody repertoire used in responding to vaccination or infection. We studied the B-cell repertoires and clonal expansions in response to attenuated varicella-zoster vaccination in four pairs of adult identical twins and found that the global antibody repertoires of twin pair members showed high similarity in antibody heavy chain V, D, and J gene segment use, and in the length and features of the complementarity-determining region 3, a major determinant of antigen binding. These twin similarities were most pronounced in the IgM-expressing B-cell pools, but were seen to a lesser extent in IgG-expressing B cells. In addition, the degree of antibody somatic mutation accumulated in the B-cell repertoire was highly correlated within twin pair members. Twin pair members had greater numbers of shared convergent antibody sequences, including mutated sequences, suggesting similarity among memory B-cell clonal lineages. Despite these similarities in the memory repertoire, the B-cell clones used in acute responses to ZOSTAVAX vaccination were largely unique to each individual. Taken together, these results suggest that the overall B-cell repertoire is significantly shaped by the underlying germ-line genome, but that stochastic or individual-specific effects dominate the selection of clones in response to an acute antigenic stimulus.

  View details for DOI 10.1073/pnas.1415875112

  View details for PubMedID 25535378

  View details for PubMedCentralID PMC4299233

• Immunoglobulin gene insertions and deletions in the affinity maturation of HIV-1 broadly reactive neutralizing antibodies. Cell host & microbe Kepler, T. B., Liao, H., Alam, S. M., Bhaskarabhatla, R., Zhang, R., Yandava, C., Stewart, S., Anasti, K., Kelsoe, G., Parks, R., Lloyd, K. E., Stolarchuk, C., Pritchett, J., Solomon, E., Friberg, E., Morris, L., Karim, S. S., Cohen, M. S., Walter, E., Moody, M. A., Wu, X., Altae-Tran, H. R., Georgiev, I. S., Kwong, P. D., Boyd, S. D., Fire, A. Z., Mascola, J. R., Haynes, B. F. 2014; 16 (3): 304-313

  Abstract

  Induction of HIV-1 broad neutralizing antibodies (bnAbs) is a goal of HIV-1 vaccine development but has remained challenging partially due to unusual traits of bnAbs, including high somatic hypermutation (SHM) frequencies and in-frame insertions and deletions (indels). Here we examined the propensity and functional requirement for indels within HIV-1 bnAbs. High-throughput sequencing of the immunoglobulin (Ig) VHDJH genes in HIV-1 infected and uninfected individuals revealed that the indel frequency was elevated among HIV-1-infected subjects, with no unique properties attributable to bnAb-producing individuals. This increased indel occurrence depended only on the frequency of SHM point mutations. Indel-encoded regions were generally proximal to antigen binding sites. Additionally, reconstruction of a HIV-1 CD4-binding site bnAb clonal lineage revealed that a large compound VHDJH indel was required for bnAb activity. Thus, vaccine development should focus on designing regimens targeted at sustained activation of bnAb lineages to achieve the required SHM and indel events.

  View details for DOI 10.1016/j.chom.2014.08.006

  View details for PubMedID 25211073

  View details for PubMedCentralID PMC4163498

• Diversity and clonal selection in the human T-cell repertoire. Proceedings of the National Academy of Sciences of the United States of America Qi, Q., Liu, Y., Cheng, Y., Glanville, J., Zhang, D., Lee, J., Olshen, R. A., Weyand, C. M., Boyd, S. D., Goronzy, J. J. 2014; 111 (36): 13139-13144

  Abstract

  T-cell receptor (TCR) diversity, a prerequisite for immune system recognition of the universe of foreign antigens, is generated in the first two decades of life in the thymus and then persists to an unknown extent through life via homeostatic proliferation of naïve T cells. We have used next-generation sequencing and nonparametric statistical analysis to estimate a lower bound for the total number of different TCR beta (TCRB) sequences in human repertoires. We arrived at surprisingly high minimal estimates of 100 million unique TCRB sequences in naïve CD4 and CD8 T-cell repertoires of young adults. Naïve repertoire richness modestly declined two- to fivefold in healthy elderly. Repertoire richness contraction with age was even less pronounced for memory CD4 and CD8 T cells. In contrast, age had a major impact on the inequality of clonal sizes, as estimated by a modified Gini-Simpson index clonality score. In particular, large naïve T-cell clones that were distinct from memory clones were found in the repertoires of elderly individuals, indicating uneven homeostatic proliferation without development of a memory cell phenotype. Our results suggest that a highly diverse repertoire is maintained despite thymic involution; however, peripheral fitness selection of T cells leads to repertoire perturbations that can influence the immune response in the elderly.

  View details for DOI 10.1073/pnas.1409155111

  View details for PubMedID 25157137

• HIV-1 Envelope gp41 Antibodies Can Originate from Terminal Ileum B Cells that Share Cross-Reactivity with Commensal Bacteria. Cell host & microbe Trama, A. M., Moody, M. A., Alam, S. M., Jaeger, F. H., Lockwood, B., Parks, R., Lloyd, K. E., Stolarchuk, C., Scearce, R., Foulger, A., Marshall, D. J., Whitesides, J. F., Jeffries, T. L., Wiehe, K., Morris, L., Lambson, B., Soderberg, K., Hwang, K., Tomaras, G. D., Vandergrift, N., Jackson, K. J., Roskin, K. M., Boyd, S. D., Kepler, T. B., Liao, H., Haynes, B. F. 2014; 16 (2): 215-226

  Abstract

  Monoclonal antibodies derived from blood plasma cells of acute HIV-1-infected individuals are predominantly targeted to the HIV Env gp41 and cross-reactive with commensal bacteria. To understand this phenomenon, we examined anti-HIV responses in ileum B cells using recombinant antibody technology and probed their relationship to commensal bacteria. The dominant ileum B cell response was to Env gp41. Remarkably, a majority (82%) of the ileum anti-gp41 antibodies cross-reacted with commensal bacteria, and of those, 43% showed non-HIV-1 antigen polyreactivity. Pyrosequencing revealed shared HIV-1 antibody clonal lineages between ileum and blood. Mutated immunoglobulin G antibodies cross-reactive with both Env gp41 and microbiota could also be isolated from the ileum of HIV-1 uninfected individuals. Thus, the gp41 commensal bacterial antigen cross-reactive antibodies originate in the intestine, and the gp41 Env response in HIV-1 infection can be derived from a preinfection memory B cell pool triggered by commensal bacteria that cross-react with Env.

  View details for DOI 10.1016/j.chom.2014.07.003

  View details for PubMedID 25121750

  View details for PubMedCentralID PMC4294419

• Human responses to influenza vaccination show seroconversion signatures and convergent antibody rearrangements. Cell host & microbe Jackson, K. J., Liu, Y., Roskin, K. M., Glanville, J., Hoh, R. A., Seo, K., Marshall, E. L., Gurley, T. C., Moody, M. A., Haynes, B. F., Walter, E. B., Liao, H., Albrecht, R. A., García-Sastre, A., Chaparro-Riggers, J., Rajpal, A., Pons, J., Simen, B. B., Hanczaruk, B., Dekker, C. L., Laserson, J., Koller, D., Davis, M. M., Fire, A. Z., Boyd, S. D. 2014; 16 (1): 105-114

  Abstract

  B cells produce a diverse antibody repertoire by undergoing gene rearrangements. Pathogen exposure induces the clonal expansion of B cells expressing antibodies that can bind the infectious agent. To assess human B cell responses to trivalent seasonal influenza and monovalent pandemic H1N1 vaccination, we sequenced gene rearrangements encoding the immunoglobulin heavy chain, a major determinant of epitope recognition. The magnitude of B cell clonal expansions correlates with an individual's secreted antibody response to the vaccine, and the expanded clones are enriched with those expressing influenza-specific monoclonal antibodies. Additionally, B cell responses to pandemic influenza H1N1 vaccination and infection in different people show a prominent family of convergent antibody heavy chain gene rearrangements specific to influenza antigens. These results indicate that microbes can induce specific signatures of immunoglobulin gene rearrangements and that pathogen exposure can potentially be assessed from B cell repertoires.

  View details for DOI 10.1016/j.chom.2014.05.013

  View details for PubMedID 24981332

  View details for PubMedCentralID PMC4158033

• Effects of Aging, Cytomegalovirus Infection, and EBV Infection on Human B Cell Repertoires JOURNAL OF IMMUNOLOGY Wang, C., Liu, Y., Xu, L. T., Jackson, K. J., Roskin, K. M., Pham, T. D., Laserson, J., Marshall, E. L., Seo, K., Lee, J., Furman, D., Koller, D., Dekker, C. L., Davis, M. M., Fire, A. Z., Boyd, S. D. 2014; 192 (2): 603-611

  Abstract

  Elderly humans show decreased humoral immunity to pathogens and vaccines, yet the effects of aging on B cells are not fully known. Chronic viral infection by CMV is implicated as a driver of clonal T cell proliferations in some aging humans, but whether CMV or EBV infection contributes to alterations in the B cell repertoire with age is unclear. We have used high-throughput DNA sequencing of IGH gene rearrangements to study the BCR repertoires over two successive years in 27 individuals ranging in age from 20 to 89 y. Some features of the B cell repertoire remain stable with age, but elderly subjects show increased numbers of B cells with long CDR3 regions, a trend toward accumulation of more highly mutated IgM and IgG Ig genes, and persistent clonal B cell populations in the blood. Seropositivity for CMV or EBV infection alters B cell repertoires, regardless of the individual's age: EBV infection correlates with the presence of persistent clonal B cell expansions, whereas CMV infection correlates with the proportion of highly mutated Ab genes. These findings isolate effects of aging from those of chronic viral infection on B cell repertoires and provide a baseline for understanding human B cell responses to vaccination or infectious stimuli.

  View details for DOI 10.4049/jimmunol.1301384

  View details for Web of Science ID 000329224000006

  View details for PubMedID 24337376

• An autoreactive antibody from an SLE/HIV-1 individual broadly neutralizes HIV-1. The Journal of clinical investigation Bonsignori, M. n., Wiehe, K. n., Grimm, S. K., Lynch, R. n., Yang, G. n., Kozink, D. M., Perrin, F. n., Cooper, A. J., Hwang, K. K., Chen, X. n., Liu, M. n., McKee, K. n., Parks, R. J., Eudailey, J. n., Wang, M. n., Clowse, M. n., Criscione-Schreiber, L. G., Moody, M. A., Ackerman, M. E., Boyd, S. D., Gao, F. n., Kelsoe, G. n., Verkoczy, L. n., Tomaras, G. D., Liao, H. X., Kepler, T. B., Montefiori, D. C., Mascola, J. R., Haynes, B. F. 2014

  Abstract

  Broadly HIV-1-neutralizing antibodies (BnAbs) display one or more unusual traits, including a long heavy chain complementarity-determining region 3 (HCDR3), polyreactivity, and high levels of somatic mutations. These shared characteristics suggest that BnAb development might be limited by immune tolerance controls. It has been postulated that HIV-1-infected individuals with autoimmune disease and defective immune tolerance mechanisms may produce BnAbs more readily than those without autoimmune diseases. In this study, we identified an HIV-1-infected individual with SLE who exhibited controlled viral load (<5,000 copies/ml) in the absence of controlling HLA phenotypes and developed plasma HIV-1 neutralization breadth. We collected memory B cells from this individual and isolated a BnAb, CH98, that targets the CD4 binding site (CD4bs) of HIV-1 envelope glycoprotein 120 (gp120). CH98 bound to human antigens including dsDNA, which is specifically associated with SLE. Anti-dsDNA reactivity was also present in the patient's plasma. CH98 had a mutation frequency of 25% and 15% nt somatic mutations in the heavy and light chain variable domains, respectively, a long HCDR3, and a deletion in the light chain CDR1. The occurrence of anti-dsDNA reactivity by a HIV-1 CD4bs BnAb in an individual with SLE raises the possibility that some BnAbs and SLE-associated autoantibodies arise from similar pools of B cells.

  View details for DOI 10.1172/JCI73441

  View details for PubMedID 24614107

• Convergent antibody signatures in human dengue. Cell host & microbe Parameswaran, P., Liu, Y., Roskin, K. M., Jackson, K. K., Dixit, V. P., Lee, J., Artiles, K. L., Zompi, S., Vargas, M. J., Simen, B. B., Hanczaruk, B., McGowan, K. R., Tariq, M. A., Pourmand, N., Koller, D., Balmaseda, A., Boyd, S. D., Harris, E., Fire, A. Z. 2013; 13 (6): 691-700

  Abstract

  Dengue is the most prevalent mosquito-borne viral disease in humans, and the lack of early prognostics, vaccines, and therapeutics contributes to immense disease burden. To identify patterns that could be used for sequence-based monitoring of the antibody response to dengue, we examined antibody heavy-chain gene rearrangements in longitudinal peripheral blood samples from 60 dengue patients. Comparing signatures between acute dengue, postrecovery, and healthy samples, we found increased expansion of B cell clones in acute dengue patients, with higher overall clonality in secondary infection. Additionally, we observed consistent antibody sequence features in acute dengue in the highly variable major antigen-binding determinant, complementarity-determining region 3 (CDR3), with specific CDR3 sequences highly enriched in acute samples compared to postrecovery, healthy, or non-dengue samples. Dengue thus provides a striking example of a human viral infection where convergent immune signatures can be identified in multiple individuals. Such signatures could facilitate surveillance of immunological memory in communities.

  View details for DOI 10.1016/j.chom.2013.05.008

  View details for PubMedID 23768493

  View details for PubMedCentralID PMC4136508

• Diagnostic applications of high-throughput DNA sequencing. Annual review of pathology Boyd, S. D. 2013; 8: 381-410

  Abstract

  Advances in DNA sequencing technology have allowed comprehensive investigation of the genetics of human beings and human diseases. Insights from sequencing the genomes, exomes, or transcriptomes of healthy and diseased cells in patients are already enabling improved diagnostic classification, prognostication, and therapy selection for many diseases. Understanding the data obtained using new high-throughput DNA sequencing methods, choices made in sequencing strategies, and common challenges in data analysis and genotype-phenotype correlation is essential if pathologists, geneticists, and clinicians are to interpret the growing scientific literature in this area. This review highlights some of the major results and discoveries stemming from high-throughput DNA sequencing research in our understanding of Mendelian genetic disorders, hematologic cancer biology, infectious diseases, the immune system, transplant biology, and prenatal diagnostics. Transition of new DNA sequencing methodologies to the clinical laboratory is under way and is likely to have a major impact on all areas of medicine.

  View details for DOI 10.1146/annurev-pathol-020712-164026

  View details for PubMedID 23121054

• Measurement and Clinical Monitoring of Human Lymphocyte Clonality by Massively Parallel V-D-J Pyrosequencing Science Translational Medicine Boyd SD, Marshall EL, Merker JD, Maniar JM, Zhang LN, Sahaf B, Jones CD, Simen BB, Hanczaruk B, Nguyen KD, Nadeau KC, Egholm M, Miklos DB, Zehnder JL, Fire AZ 2009; 1 (12)
• Ultrasensitive allele inference from immune repertoire sequencing data with MiXCR. Genome research Mikelov, A., Nefedev, G., Tashkeev, A., Rodriguez, O. L., Ortmans, D. A., Skatova, V., Izraelson, M., Davydov, A. N., Poslavsky, S., Rahmouni, S., Watson, C. T., Chudakov, D. M., Boyd, S. D., Bolotin, D. A. 2024

  Abstract

  Allelic variability in the adaptive immune receptor loci, which harbor the gene segments that encode B cell and T cell receptors (BCR/TCR), is of critical importance for immune responses to pathogens and vaccines. Adaptive immune receptor repertoire sequencing (AIRR-seq) has become widespread in immunology research making it the most readily available source of information about allelic diversity in immunoglobulin (IG) and T cell receptor (TR) loci. Here we present a novel algorithm for extra-sensitive and specific variable (V) and joining (J) gene allele inference, allowing reconstruction of individual high-quality gene segment libraries. The approach can be applied for inferring allelic variants from peripheral blood lymphocyte BCR and TCR repertoire sequencing data, including hypermutated isotype-switched BCR sequences, thus allowing high-throughput novel allele discovery from a wide variety of existing datasets. The developed algorithm is a part of the MiXCR software. We demonstrate the accuracy of this approach using AIRR-seq paired with long-read genomic sequencing data, comparing it to a widely used algorithm, TIgGER. We applied the algorithm to a large set of IG heavy chain (IGH) AIRR-seq data from 450 donors of ancestrally diverse population groups, and to the largest reported full-length TCR alpha and beta chain (TRA; TRB) AIRR-seq dataset, representing 134 individuals. This allowed us to assess the genetic diversity within the IGH, TRA and TRB loci in different populations and to establish a database of alleles of V and J genes inferred from AIRR-seq data and their population frequencies with free public access through an online database.

  View details for DOI 10.1101/gr.278775.123

  View details for PubMedID 39433438

• HLA-DR+regulatory T cells and IL-10 are associated with success or failure of desensitization outcomes. Allergy Zhou, X., Dunham, D., Sindher, S. B., Long, A., Fernandes, A., Chang, I., Assa'ad, A., Pongracic, J., Spergel, J. M., Tam, J., Tilles, S., Wang, J., Boyd, S. D., Chinthrajah, R. S., Nadeau, K. C. 2024

  Abstract

  BACKGROUND: Omalizumab (XOLAIR)-assisted multi-food oral immunotherapy (mOIT) has been shown to safely, effectively, and rapidly desensitize patients with multiple food allergies. In our clinical trial (NCT02626611) on omalizumab-assisted mOIT, different desensitization outcomes (success or failure of desensitization) were observed following a period of either continued or discontinued mOIT. However, the association between the immunological changes induced by omalizumab-assisted mOIT and desensitization outcomes has not yet been fully elucidated. In this study, due to the key roles of regulatory T (Treg) cells and the type 2 helper T cell (Th2) pathway in immune tolerance to food allergens, we aimed to characterize their association with the desensitization outcomes of omalizumab-assisted mOIT.METHODS: Mass cytometry and multiplex cytokine assays were performed on blood samples obtained from participants with allergies to peanut, cashew, or milk in our phase 2 clinical study (NCT02626611). Comprehensive statistical and bioinformatic analyses were conducted on high-dimensional cytometry-based single-cell data and high-throughput multiplex cytokine data.RESULTS: Our results demonstrated that the frequency of HLA-DR+ Treg cells, and the production of Th2 cytokines (IL-4, IL-5, IL-13, and IL-9) as well as the immunoregulatory cytokine IL-10 by peripheral blood mononuclear cells (PBMCs) was significantly increased in cultures with allergen compared to cultures with media alone at baseline (Week 0). We also observed increased frequency of allergen responsive HLA-DR+ Treg cells and enhanced production of IL-10 by PBMCs in participants who achieved successful desensitization compared to those with failure of desensitization. However, the production of Th2 cytokines by PBMCs did not show significant differences between participants with different desensitization outcomes (success vs. failure of desensitization), despite omalizumab-assisted mOIT inducing a significant reduction in the production of Th2 cytokines.CONCLUSIONS: We demonstrated that the frequency of HLA-DR+ Treg cells and IL-10 cytokine production by PBMCs are associated with desensitization outcomes of omalizumab-assisted mOIT. These findings suggest potential immunological parameters that could be targeted to enhance desensitization success rates.

  View details for DOI 10.1111/all.16311

  View details for PubMedID 39291303

• Gestational SARS-CoV-2 Infection in a Ugandan Birth Cohort: High Incidence, Mild Maternal Disease, and Evidence of Association with Transient Infant Stunting. The American journal of tropical medicine and hygiene Jacobson, K. B., Roltgen, K., Lam, B., Nayebare, P., Kakuru, A., Kizza, J., Aguti, M., Nankya, F., Briggs, J., Takahashi, S., Greenhouse, B., Rodriguez-Barraquer, I., van der Ploeg, K., Wohlstadter, J. N., Sigal, G. B., Roh, M. E., Nankabirwa, J. I., Cuu, G., Gaw, S. L., Rosenthal, P. J., Kamya, M. R., Ssewanyana, I., Dorsey, G., Boyd, S. D., Jagannathan, P. 2024

  Abstract

  Many questions remain about the prevalence and effects of SARS-CoV-2 infection in malaria-endemic African countries like Uganda, particularly in vulnerable groups such as pregnant women. We describe SARS-CoV-2 immunoglobulin (Ig)G and IgM antibody responses and clinical outcomes in mother-infant dyads enrolled in malaria chemoprevention trials in Uganda. From December 2020-February 2022, among 400 unvaccinated pregnant women enrolled at 12-20 weeks gestation and followed through delivery, 128 (32%) were seronegative for anti-SARS-CoV-2 IgG and IgM at enrollment and delivery, 80 (20%) were infected prior to or early in pregnancy, and 192 (48%) were infected or re-infected with SARS-CoV-2 during pregnancy. We observed preferential binding of plasma IgG to Wuhan-Hu-1-like antigens in individuals seroconverting up to early 2021, and to Delta variant antigens in a subset of individuals in mid-2021. Breadth of IgG binding to all variants improved over time, consistent with affinity maturation of the antibody response in the cohort. No women experienced severe respiratory illness during the study. SARS-CoV-2 infection in early pregnancy was associated with lower median length-for-age Z-score at age 3 months compared with no infection or late pregnancy infect (-1.54 versus -0.37 and -0.51, P = 0.009). These findings suggest that pregnant Ugandan women experienced high levels of SARS-CoV-2 infection without severe respiratory illness. Variant-specific serology testing demonstrated evidence of antibody affinity maturation at the population level. Early gestational SARS-CoV-2 infection was associated with transient shorter stature in early infancy. Further research should explore the significance of this finding and define targeted measures to prevent infection in pregnancy.

  View details for DOI 10.4269/ajtmh.23-0801

  View details for PubMedID 39288758

• Association of cytotoxic effector memory CD8+ T cells with sustained unresponsiveness after peanut oral immunotherapy. Allergy Seastedt, H., Han, X., Fernandes, A., Galli, S. J., Boyd, S. D., Nadeau, K. C., Manohar, M., Chinthrajah, S. 2024

  View details for DOI 10.1111/all.16307

  View details for PubMedID 39244697

• Elucidating allergic reaction mechanisms in response to SARS-CoV-2 mRNA vaccination in adults. Allergy Shah, M. M., Layhadi, J. A., Hourcade, D. E., Fulton, W. T., Tan, T. J., Dunham, D., Chang, I., Vel, M. S., Fernandes, A., Lee, A. S., Liu, J., Arunachalam, P. S., Galli, S. J., Boyd, S. D., Pulendran, B., Davis, M. M., O'Hara, R., Park, H., Mitchell, L. M., Akk, A., Patterson, A., Jerath, M. R., Monroy, J. M., Ren, Z., Kendall, P. L., Durham, S. R., Fedina, A., Gibbs, B. F., Agache, I., Chinthrajah, S., Sindher, S. B., Heider, A., Akdis, C. A., Shamji, M. H., Pham, C. T., Nadeau, K. C. 2024

  Abstract

  During the COVID-19 pandemic, novel nanoparticle-based mRNA vaccines were developed. A small number of individuals developed allergic reactions to these vaccines although the mechanisms remain undefined.To understand COVID-19 vaccine-mediated allergic reactions, we enrolled 19 participants who developed allergic events within 2 h of vaccination and 13 controls, nonreactors. Using standard hemolysis assays, we demonstrated that sera from allergic participants induced stronger complement activation compared to nonallergic subjects following ex vivo vaccine exposure.Vaccine-mediated complement activation correlated with anti-polyethelyne glycol (PEG) IgG (but not IgM) levels while anti-PEG IgE was undetectable in all subjects. Depletion of total IgG suppressed complement activation in select individuals. To investigate the effects of vaccine excipients on basophil function, we employed a validated indirect basophil activation test that stratified the allergic populations into high and low responders. Complement C3a and C5a receptor blockade in this system suppressed basophil response, providing strong evidence for complement involvement in vaccine-mediated basophil activation. Single-cell multiome analysis revealed differential expression of genes encoding the cytokine response and Toll-like receptor (TLR) pathways within the monocyte compartment. Differential chromatin accessibility for IL-13 and IL-1B genes was found in allergic and nonallergic participants, suggesting that in vivo, epigenetic modulation of mononuclear phagocyte immunophenotypes determines their subsequent functional responsiveness, contributing to the overall physiologic manifestation of vaccine reactions.These findings provide insights into the mechanisms underlying allergic reactions to COVID-19 mRNA vaccines, which may be used for future vaccine strategies in individuals with prior history of allergies or reactions and reduce vaccine hesitancy.

  View details for DOI 10.1111/all.16231

  View details for PubMedID 39033312

• Allergen-specific B cell responses in oral immunotherapy-induced desensitization, remission, and natural outgrowth in cow's milk allergy. Allergy Satitsuksanoa, P., van de Veen, W., Tan, G., Lopez, J. F., Wirz, O., Jansen, K., Sokolowska, M., Mirer, D., Globinska, A., Boonpiyathad, T., Schneider, S. R., Barletta, E., Spits, H., Chang, I., Babayev, H., Tahralı, İ., Deniz, G., Yücel, E. Ö., Kıykım, A., Boyd, S. D., Akdis, C. A., Nadeau, K., Akdis, M. 2024

  Abstract

  Antigen-specific memory B cells play a key role in the induction of desensitization and remission to food allergens in oral immunotherapy and in the development of natural tolerance (NT). Here, we characterized milk allergen Bos d 9-specific B cells in oral allergen-specific immunotherapy (OIT) and in children spontaneously outgrowing cow's milk allergy (CMA) due to NT.Samples from children with CMA who received oral OIT (before, during, and after), children who naturally outgrew CMA (NT), and healthy individuals were received from Stanford biobank. Bos d 9-specific B cells were isolated by flow cytometry and RNA-sequencing was performed. Protein profile of Bos d 9-specific B cells was analyzed by proximity extension assay.Increased frequencies of circulating milk allergen Bos d 9-specific B cells were observed after OIT and NT. Milk-desensitized subjects showed the partial acquisition of phenotypic features of remission, suggesting that desensitization is an earlier stage of remission. Within these most significantly expressed genes, IL10RA and TGFB3 were highly expressed in desensitized OIT patients. In both the remission and desensitized groups, B cell activation-, Breg cells-, BCR-signaling-, and differentiation-related genes were upregulated. In NT, pathways associated with innate immunity characteristics, development of marginal zone B cells, and a more established suppressor function of B cells prevail that may play a role in long-term tolerance. The analyses of immunoglobulin heavy chain genes in specific B cells demonstrated that IgG2 in desensitization, IgG1, IgA1, IgA2, IgG4, and IgD in remission, and IgD in NT were predominating. Secreted proteins from allergen-specific B cells revealed higher levels of regulatory cytokines, IL-10, and TGF-β after OIT and NT.Allergen-specific B cells are essential elements in regulating food allergy towards remission in OIT-received and naturally resolved individuals.

  View details for DOI 10.1111/all.16220

  View details for PubMedID 38989779

• Epstein-Barr virus-associated post-transplant lymphoproliferative disorders in pediatric transplantation: A prospective multicenter study in the United States. Pediatric transplantation Tajima, T., Martinez, O. M., Bernstein, D., Boyd, S. D., Gratzinger, D., Lum, G., Sasaki, K., Tan, B., Twist, C. J., Weinberg, K., Armstrong, B., Desai, D. M., Mazariegos, G. V., Chin, C., Fishbein, T. M., Tekin, A., Venick, R. S., Krams, S. M., Esquivel, C. O. 2024; 28 (4): e14763

  Abstract

  Epstein-Barr virus (EBV)-associated post-transplant lymphoproliferative disorders (PTLD) is the most common malignancy in children after transplant; however, difficulties for early detection may worsen the prognosis.The prospective, multicenter, study enrolled 944 children (≤21 years of age). Of these, 872 received liver, heart, kidney, intestinal, or multivisceral transplants in seven US centers between 2014 and 2019 (NCT02182986). In total, 34 pediatric EBV+ PTLD (3.9%) were identified by biopsy. Variables included sex, age, race, ethnicity, transplanted organ, EBV viral load, pre-transplant EBV serology, immunosuppression, response to chemotherapy and rituximab, and histopathological diagnosis.The uni-/multivariable competing risk analyses revealed the combination of EBV-seropositive donor and EBV-naïve recipient (D+R-) was a significant risk factor for PTLD development (sub-hazard ratio: 2.79 [1.34-5.78], p = .006) and EBV DNAemia (2.65 [1.72-4.09], p < .001). Patients with D+R- were significantly more associated with monomorphic/polymorphic PTLD than those with the other combinations (p = .02). Patients with monomorphic/polymorphic PTLD (n = 21) had significantly more EBV DNAemia than non-PTLD patients (p < .001) and an earlier clinical presentation of PTLD than patients with hyperplasias (p < .001), within 6-month post-transplant. Among non-liver transplant recipients, monomorphic/polymorphic PTLD were significantly more frequent than hyperplasias in patients ≥5 years of age at transplant (p = .01).D+R- is a risk factor for PTLD and EBV DNAemia and associated with the incidence of monomorphic/polymorphic PTLD. Intensive follow-up of EBV viral load within 6-month post-transplant, especially for patients with D+R- and/or non-liver transplant recipients ≥5 years of age at transplant, may help detect monomorphic/polymorphic PTLD early in pediatric transplant.

  View details for DOI 10.1111/petr.14763

  View details for PubMedID 38682750

• An organism-wide atlas of hormonal signaling based on the mouse lemur single-cell transcriptome. Nature communications Liu, S., Ezran, C., Wang, M. F., Li, Z., Awayan, K., Long, J. Z., De Vlaminck, I., Wang, S., Epelbaum, J., Kuo, C. S., Terrien, J., Krasnow, M. A., Ferrell, J. E. 2024; 15 (1): 2188

  Abstract

  Hormones mediate long-range cell communication and play vital roles in physiology, metabolism, and health. Traditionally, endocrinologists have focused on one hormone or organ system at a time. Yet, hormone signaling by its very nature connects cells of different organs and involves crosstalk of different hormones. Here, we leverage the organism-wide single cell transcriptional atlas of a non-human primate, the mouse lemur (Microcebus murinus), to systematically map source and target cells for 84 classes of hormones. This work uncovers previously-uncharacterized sites of hormone regulation, and shows that the hormonal signaling network is densely connected, decentralized, and rich in feedback loops. Evolutionary comparisons of hormonal genes and their expression patterns show that mouse lemur better models human hormonal signaling than mouse, at both the genomic and transcriptomic levels, and reveal primate-specific rewiring of hormone-producing/target cells. This work complements the scale and resolution of classical endocrine studies and sheds light on primate hormone regulation.

  View details for DOI 10.1038/s41467-024-46070-9

  View details for PubMedID 38467625

  View details for PubMedCentralID 1540572

• Impaired innate and adaptive immune responses to BNT162b2 SARS-CoV-2 vaccination in systemic lupus erythematosus. JCI insight Sarin, K. Y., Zheng, H., Chaichian, Y., Arunachalam, P. S., Swaminathan, G., Eschholz, A., Gao, F., Wirz, O. F., Lam, B., Yang, E., Lee, L. W., Feng, A., Lewis, M. A., Lin, J., Maecker, H. T., Boyd, S. D., Davis, M. M., Nadeau, K. C., Pulendran, B., Khatri, P., Utz, P. J., Zaba, L. C. 2024; 9 (5)

  Abstract

  Understanding the immune responses to SARS-CoV-2 vaccination is critical to optimizing vaccination strategies for individuals with autoimmune diseases, such as systemic lupus erythematosus (SLE). Here, we comprehensively analyzed innate and adaptive immune responses in 19 patients with SLE receiving a complete 2-dose Pfizer-BioNTech mRNA vaccine (BNT162b2) regimen compared with a control cohort of 56 healthy control (HC) volunteers. Patients with SLE exhibited impaired neutralizing antibody production and antigen-specific CD4+ and CD8+ T cell responses relative to HC. Interestingly, antibody responses were only altered in patients with SLE treated with immunosuppressive therapies, whereas impairment of antigen-specific CD4+ and CD8+ T cell numbers was independent of medication. Patients with SLE also displayed reduced levels of circulating CXC motif chemokine ligands, CXCL9, CXCL10, CXCL11, and IFN-γ after secondary vaccination as well as downregulation of gene expression pathways indicative of compromised innate immune responses. Single-cell RNA-Seq analysis reveals that patients with SLE showed reduced levels of a vaccine-inducible monocyte population characterized by overexpression of IFN-response transcription factors. Thus, although 2 doses of BNT162b2 induced relatively robust immune responses in patients with SLE, our data demonstrate impairment of both innate and adaptive immune responses relative to HC, highlighting a need for population-specific vaccination studies.

  View details for DOI 10.1172/jci.insight.176556

  View details for PubMedID 38456511

• Corrigendum: Advances and potential of omics studies for understanding the development of food allergy. Frontiers in allergy Sindher, S. B., Chin, A. R., Aghaeepour, N., Prince, L., Maecker, H., Shaw, G. M., Stevenson, D., Nadeau, K. C., Snyder, M., Khatri, P., Boyd, S. D., Winn, V. D., Angst, M. S., Chinthrajah, R. S. 2024; 5: 1373485

  Abstract

  [This corrects the article DOI: 10.3389/falgy.2023.1149008.].

  View details for DOI 10.3389/falgy.2024.1373485

  View details for PubMedID 38464397

  View details for PubMedCentralID PMC10921899

• Single cell multi-omic analysis identifies key genes differentially expressed in innate lymphoid cells from COVID-19 patients. Frontiers in immunology Kaushik, A., Chang, I., Han, X., He, Z., Komlosi, Z. I., Ji, X., Cao, S., Akdis, C. A., Boyd, S., Pulendran, B., Maecker, H. T., Davis, M. M., Chinthrajah, R. S., DeKruyff, R. H., Nadeau, K. C. 2024; 15: 1374828

  Abstract

  Introduction: Innate lymphoid cells (ILCs) are enriched at mucosal surfaces where they respond rapidly to environmental stimuli and contribute to both tissue inflammation and healing.Methods: To gain insight into the role of ILCs in the pathology and recovery from COVID-19 infection, we employed a multi-omics approach consisting of Abseq and targeted mRNA sequencing to respectively probe the surface marker expression, transcriptional profile and heterogeneity of ILCs in peripheral blood of patients with COVID-19 compared with healthy controls.Results: We found that the frequency of ILC1 and ILC2 cells was significantly increased in COVID-19 patients. Moreover, all ILC subsets displayed a significantly higher frequency of CD69-expressing cells, indicating a heightened state of activation. ILC2s from COVID-19 patients had the highest number of significantly differentially expressed (DE) genes. The most notable genes DE in COVID-19 vs healthy participants included a) genes associated with responses to virus infections and b) genes that support ILC self-proliferation, activation and homeostasis. In addition, differential gene regulatory network analysis revealed ILC-specific regulons and their interactions driving the differential gene expression in each ILC.Discussion: Overall, this study provides mechanistic insights into the characteristics of ILC subsets activated during COVID-19 infection.

  View details for DOI 10.3389/fimmu.2024.1374828

  View details for PubMedID 39026668

• Acute Respiratory Illness Is Associated with Memory T Cell Differentiation and Other Immune Cell Changes in an Age-Associated Manner. ImmunoHorizons Ugale, S. S., Holmes, T. H., Maysel-Auslender, S., Boyd, S. D., Dekker, C. L., Davis, M. M., Maecker, H. T. 2023; 7 (9): 611-618

  Abstract

  Respiratory viruses such as influenza are encountered multiple times through infection and/or vaccination and thus have the potential to shape immune cell phenotypes over time. In particular, memory T cell compartments may be affected, as both CD4+ and CD8+ T cell responses likely contribute to viral control. In this study, we assessed immune phenotypes using cytometry by time of flight in the peripheral blood of 22 humans with acute respiratory illness and 22 age-matched noninfected controls. In younger infected individuals (1-19 y of age), we found decreased B and NK cell frequencies and a shift toward more effector-like CD4+ and CD8+ T cell phenotypes, compared with young healthy controls. Significant differences between noninfected and infected older individuals (30-74 y of age) were not seen. We also observed a decrease in naive CD4+ T cells and CD27+CD8+ T cells as well as an increase in effector memory CD8+ T cells and NKT cells in noninfected individuals with age. When cell frequencies were regressed against age for infected versus noninfected subjects, significant differences in trends with age were observed for multiple cell types. These included B cells and various subsets of CD4+ and CD8+ T cells. We conclude that acute respiratory illness drives T cell differentiation and decreases circulating B cell frequencies preferentially in young compared with older individuals.

  View details for DOI 10.4049/immunohorizons.2300050

  View details for PubMedID 37707792

• Germline-encoded specificities and the predictability of the B cell response. PLoS pathogens C Vieira, M., Palm, A. E., Stamper, C. T., Tepora, M. E., Nguyen, K. D., Pham, T. D., Boyd, S. D., Wilson, P. C., Cobey, S. 2023; 19 (8): e1011603

  Abstract

  Antibodies result from the competition of B cell lineages evolving under selection for improved antigen recognition, a process known as affinity maturation. High-affinity antibodies to pathogens such as HIV, influenza, and SARS-CoV-2 are frequently reported to arise from B cells whose receptors, the precursors to antibodies, are encoded by particular immunoglobulin alleles. This raises the possibility that the presence of particular germline alleles in the B cell repertoire is a major determinant of the quality of the antibody response. Alternatively, initial differences in germline alleles' propensities to form high-affinity receptors might be overcome by chance events during affinity maturation. We first investigate these scenarios in simulations: when germline-encoded fitness differences are large relative to the rate and effect size variation of somatic mutations, the same germline alleles persistently dominate the response of different individuals. In contrast, if germline-encoded advantages can be easily overcome by subsequent mutations, allele usage becomes increasingly divergent over time, a pattern we then observe in mice experimentally infected with influenza virus. We investigated whether affinity maturation might nonetheless strongly select for particular amino acid motifs across diverse genetic backgrounds, but we found no evidence of convergence to similar CDR3 sequences or amino acid substitutions. These results suggest that although germline-encoded specificities can lead to similar immune responses between individuals, diverse evolutionary routes to high affinity limit the genetic predictability of responses to infection and vaccination.

  View details for DOI 10.1371/journal.ppat.1011603

  View details for PubMedID 37624867

• Genetic variation in the immunoglobulin heavy chain locus shapes the human antibody repertoire. Nature communications Rodriguez, O. L., Safonova, Y., Silver, C. A., Shields, K., Gibson, W. S., Kos, J. T., Tieri, D., Ke, H., Jackson, K. J., Boyd, S. D., Smith, M. L., Marasco, W. A., Watson, C. T. 2023; 14 (1): 4419

  Abstract

  Variation in the antibody response has been linked to differential outcomes in disease, and suboptimal vaccine and therapeutic responsiveness, the determinants of which have not been fully elucidated. Countering models that presume antibodies are generated largely by stochastic processes, we demonstrate that polymorphisms within the immunoglobulin heavy chain locus (IGH) impact the naive and antigen-experienced antibody repertoire, indicating that genetics predisposes individuals to mount qualitatively and quantitatively different antibody responses. We pair recently developed long-read genomic sequencing methods with antibody repertoire profiling to comprehensively resolve IGH genetic variation, including novel structural variants, single nucleotide variants, and genes and alleles. We show that IGH germline variants determine the presence and frequency of antibody genes in the expressed repertoire, including those enriched in functional elements linked to V(D)J recombination, and overlapping disease-associated variants. These results illuminate the power of leveraging IGH genetics to better understand the regulation, function, and dynamics of the antibody response in disease.

  View details for DOI 10.1038/s41467-023-40070-x

  View details for PubMedID 37479682

• Detection of SARS-CoV-2 Antibodies in Immunoglobulin Products. The journal of allergy and clinical immunology. In practice Cousins, K., Sano, K., Lam, B., Roltgen, K., Bhavsar, D., Singh, G., Jeong, S., Aboelregal, N., Ho, H., Boyd, S., Krammer, F., Cunningham-Rundles, C. 2023

  Abstract

  BACKGROUND: For patients with primary antibody deficiency, the first line of therapy is replacement with immunoglobulin (Ig) products. Prior to the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic, Ig products did not contain antibodies with specificity for this virus, and there have been limited data on the antibodies present in the Ig products in current use.OBJECTIVE: To quantitatively examine SARS-CoV-2 antibodies in current immunoglobulin products.METHODS: 142 unique lots of 11 different Ig products intended for intravenous (IV) and/or subcutaneous (SC) delivery were examined for IgG binding activities against recombinant SARS-CoV-2 receptor binding domain (RBD), spike, and nucleocapsid proteins (NP) by enzyme-linked immunosorbent assays (ELISA). Additionally, to assess functionality, 48 of these unique lots were assessed for their ability to inhibit the variants SARS-CoV-2 Ancestral, Alpha, Beta, Delta, and Omicron spike binding to angiotensin converting enzyme 2 (ACE2).RESULTS: Significantly increased antibody values were observed for products manufactured after the year 2020 (expiration dates 2023-2024), as compared to Ig products before 2020 (pre-pandemic). Sixty and eighty-five percent of the Ig products with expiration dates of 2023 and 2024 were positive for antibody to SARS-CoV-2 proteins, respectively. AUC values were significantly higher in products with later expiration dates. Later dates of expiration were also strongly correlated with inhibition of ACE2 binding activity, however a decline in inhibition activity was observed with later variants.CONCLUSION: Overall, more recent Ig products (expiration dates: 2023 - 2025) contained significantly higher binding and inhibition activities against SARS-CoV-2 proteins, as compared to earlier, or pre-pandemic products. Normal donor SARS-CoV-2 antibodies are capable of inhibiting ACE2-binding activities and may provide a therapeutic benefit for patients who do not make a robust vaccine response.

  View details for DOI 10.1016/j.jaip.2023.05.005

  View details for PubMedID 37182564

• Advances and potential of omics studies for understanding the development of food allergy. Frontiers in allergy Sindher, S. B., Chin, A. R., Aghaeepour, N., Prince, L., Maecker, H., Shaw, G. M., Stevenson, D. K., Nadeau, K. C., Snyder, M., Khatri, P., Boyd, S. D., Winn, V. D., Angst, M. S., Chinthrajah, R. S. 2023; 4: 1149008

  Abstract

  The prevalence of food allergy continues to rise globally, carrying with it substantial safety, economic, and emotional burdens. Although preventative strategies do exist, the heterogeneity of allergy trajectories and clinical phenotypes has made it difficult to identify patients who would benefit from these strategies. Therefore, further studies investigating the molecular mechanisms that differentiate these trajectories are needed. Large-scale omics studies have identified key insights into the molecular mechanisms for many different diseases, however the application of these technologies to uncover the drivers of food allergy development is in its infancy. Here we review the use of omics approaches in food allergy and highlight key gaps in knowledge for applying these technologies for the characterization of food allergy development.

  View details for DOI 10.3389/falgy.2023.1149008

  View details for PubMedID 37034151

  View details for PubMedCentralID PMC10080041

• Spheromers reveal robust T cell responses to the Pfizer/BioNTech vaccine and attenuated peripheral CD8+ T cell responses post SARS-CoV-2 infection. Immunity Gao, F., Mallajoysula, V., Arunachalam, P. S., van der Ploeg, K., Manohar, M., Röltgen, K., Yang, F., Wirz, O., Hoh, R., Haraguchi, E., Lee, J. Y., Willis, R., Ramachandiran, V., Li, J., Kathuria, K. R., Li, C., Lee, A. S., Shah, M. M., Sindher, S. B., Gonzalez, J., Altman, J. D., Wang, T. T., Boyd, S. D., Pulendran, B., Jagannathan, P., Nadeau, K. C., Davis, M. M. 2023

  Abstract

  T cells are a critical component of the response to SARS-CoV-2, but their kinetics after infection and vaccination are insufficiently understood. Using "spheromer" peptide-MHC multimer reagents, we analyzed healthy subjects receiving two doses of the Pfizer/BioNTech BNT162b2 vaccine. Vaccination resulted in robust spike-specific T cell responses for the dominant CD4+ (HLA-DRB1∗15:01/S191) and CD8+ (HLA-A∗02/S691) T cell epitopes. Antigen-specific CD4+ and CD8+ T cell responses were asynchronous, with the peak CD4+ T cell responses occurring 1 week post the second vaccination (boost), whereas CD8+ T cells peaked 2 weeks later. These peripheral T cell responses were elevated compared with COVID-19 patients. We also found that previous SARS-CoV-2 infection resulted in decreased CD8+ T cell activation and expansion, suggesting that previous infection can influence the T cell response to vaccination.

  View details for DOI 10.1016/j.immuni.2023.03.005

  View details for PubMedID 36996809

  View details for PubMedCentralID PMC10017386

• Clonal evolution and stereotyped sequences of human IgE lineages in aeroallergen-specific immunotherapy. The Journal of allergy and clinical immunology Hoh, R. A., Thörnqvist, L., Yang, F., Godzwon, M., King, J. J., Lee, J. Y., Greiff, L., Boyd, S. D., Ohlin, M. 2023

  Abstract

  Allergic disease reflects specific inflammatory processes initiated by interaction between allergen and allergen-specific IgE. Specific immunotherapy (SIT) is an effective long-term treatment option, but the mechanisms by which SIT provides desensitization are not well understood.To characterize IgE sequences expressed by allergen-specific B cells over a 3-year longitudinal study of patients with aeroallergies undergoing SIT.Allergen-specific IgE-expressing clones were identified using combinatorial scFv libraries and tracked in PBMC and nasal biopsies over a 3-year period with antibody gene repertoire sequencing. Characteristics of private IgE-expressing clones were compared with those of stereotyped or "public" IgE responses to the grass pollen allergen Phl p 2.Members of the same allergen-specific IgE lineages were observed in nasal biopsies and blood, and lineages detected at baseline persisted in blood and nasal biopsies after 3 years of SIT, including B cells that express IgE. Evidence of progressive class-switch recombination (CSR) to IgG subclasses was observed after 3 years of SIT. A common stereotyped Phl p 2-specific antibody heavy chain sequence was detected in multiple donors. The amino acid residues enriched in IgE stereotyped sequences from seropositive donors were analyzed with machine learning and k-mer motif discovery. Stereotyped IgE sequences had lower overall rates of somatic hypermutation and antigen selection than scFv-derived allergen-specific sequences or IgE sequences of unknown specificity.Longitudinal tracking of rare circulating and tissue-resident allergen-specific IgE+ clones demonstrates persistence of allergen-specific IgE+ clones, progressive CSR to IgG subtypes, and distinct maturation of a stereotyped Phl p 2-clonotype.

  View details for DOI 10.1016/j.jaci.2023.02.009

  View details for PubMedID 36828082

• Mutations In Latent Membrane Protein 1 of Epstein-Barr Virus are Associated with Increased Risk for Post-Transplant Lymphoproliferative Disorder in Children. American journal of transplantation : official journal of the American Society of Transplantation and the American Society of Transplant Surgeons Martinez, O. M., Krams, S. M., Robien, M. A., Lapasaran, M. G., Arvedson, M. P., Reitsma, A., Balachandran, Y., Harris-Arnold, A., Weinberg, K., Boyd, S. D., Armstrong, B., Trickey, A., Twist, C. J., Gratzinger, D., Tan, B., Brown, M., Chin, C., Desai, D. M., Fishbein, T. M., Mazariegos, G. V., Tekin, A., Venick, R. S., Bernstein, D., Esquivel, C. O. 2023

  Abstract

  Epstein-Barr virus (EBV)+ post-transplant lymphoproliferative disorder (PTLD) results in significant morbidity and mortality in pediatric transplant recipients. Identifying individuals at increased risk of EBV+ PTLD could influence clinical management of immunosuppression and other therapies, improving post-transplant outcomes. A seven-center prospective, observational clinical trial of 872 pediatric transplant recipients evaluated the presence of mutations at position 212 and 366 of EBV latent membrane protein 1 (LMP1) as an indicator of risk for EBV+ PTLD (Clinical Trials: NCT02182986). DNA was isolated from peripheral blood of EBV+ PTLD cases and matched controls (1:2 nested case-control), and the cytoplasmic tail of LMP1 sequenced. Thirty-four participants reached the primary endpoint of biopsy-proven EBV+ PTLD. DNA was sequenced from 32 PTLD cases and 62 matched controls. Both LMP1 mutations were present in 31/32 PTLD cases (96.9%) and in 45/62 matched controls (72.6%) (p=0.005, OR=11.7, 95% CI 1.5, 92.6). The presence of both G212S and S366T carries a nearly 12-fold increased risk for development of EBV+ PTLD. Conversely, transplant recipients without both LMP1 mutations carry a very low risk of PTLD. Analysis of mutations at positions 212 and 366 of LMP1 can be informative in stratifying patients for risk of EBV+ PTLD.

  View details for DOI 10.1016/j.ajt.2023.02.014

  View details for PubMedID 36796762

• Allergy: Mechanistic insights into new methods of prevention and therapy SCIENCE TRANSLATIONAL MEDICINE Akdis, C. A., Akdis, M., Boyd, S. D., Sampath, V., Galli, S. J., Nadeau, K. C. 2023; 15 (679)

  View details for Web of Science ID 000932397900005

• Reinfection with SARS-CoV-2 and Waning Humoral Immunity: A Case Report. Vaccines Goldman, J. D., Wang, K., Röltgen, K., Nielsen, S. C., Roach, J. C., Naccache, S. N., Yang, F., Wirz, O. F., Yost, K. E., Lee, J. Y., Chun, K., Wrin, T., Petropoulos, C. J., Lee, I., Fallen, S., Manner, P. M., Wallick, J. A., Algren, H. A., Murray, K. M., Hadlock, J., Chen, D., Dai, C. L., Yuan, D., Su, Y., Jeharajah, J., Berrington, W. R., Pappas, G. P., Nyatsatsang, S. T., Greninger, A. L., Satpathy, A. T., Pauk, J. S., Boyd, S. D., Heath, J. R. 2022; 11 (1)

  Abstract

  Recovery from COVID-19 is associated with production of anti-SARS-CoV-2 antibodies, but it is uncertain whether these confer immunity. We describe viral RNA shedding duration in hospitalized patients and identify patients with recurrent shedding. We sequenced viruses from two distinct episodes of symptomatic COVID-19 separated by 144 days in a single patient, to conclusively describe reinfection with a different strain harboring the spike variant D614G. This case of reinfection was one of the first cases of reinfection reported in 2020. With antibody, B cell and T cell analytics, we show correlates of adaptive immunity at reinfection, including a differential response in neutralizing antibodies to a D614G pseudovirus. Finally, we discuss implications for vaccine programs and begin to define benchmarks for protection against reinfection from SARS-CoV-2.

  View details for DOI 10.3390/vaccines11010005

  View details for PubMedID 36679852

• CD8+ T cell differentiation status correlates with the feasibility of sustained unresponsiveness following oral immunotherapy. Nature communications Kaushik, A., Dunham, D., Han, X., Do, E., Andorf, S., Gupta, S., Fernandes, A., Kost, L. E., Sindher, S. B., Yu, W., Tsai, M., Tibshirani, R., Boyd, S. D., Desai, M., Maecker, H. T., Galli, S. J., Chinthrajah, R. S., DeKruyff, R. H., Manohar, M., Nadeau, K. C. 2022; 13 (1): 6646

  Abstract

  While food allergy oral immunotherapy (OIT) can provide safe and effective desensitization (DS), the immune mechanisms underlying development of sustained unresponsiveness (SU) following a period of avoidance are largely unknown. Here, we compare high dimensional phenotypes of innate and adaptive immune cell subsets of participants in a previously reported, phase 2 randomized, controlled, peanut OIT trial who achieved SU vs. DS (no vs. with allergic reactions upon food challenge after a withdrawal period; n=21 vs. 30 respectively among total 120 intent-to-treat participants). Lower frequencies of naive CD8+ T cells and terminally differentiated CD57+CD8+ T cell subsets at baseline (pre-OIT) are associated with SU. Frequency of naive CD8+ T cells shows a significant positive correlation with peanut-specific and Ara h 2-specific IgE levels at baseline. Higher frequencies of IL-4+ and IFNgamma+ CD4+ T cells post-OIT are negatively correlated with SU. Our findings provide evidence that an immune signature consisting of certain CD8+ T cell subset frequencies is potentially predictive of SU following OIT.

  View details for DOI 10.1038/s41467-022-34222-8

  View details for PubMedID 36333296

• Exceptional diversity and selection pressure on coronavirus host receptors in bats compared to other mammals. Proceedings. Biological sciences Frank, H. K., Enard, D., Boyd, S. D. 2022; 289 (1979): 20220193

  Abstract

  Pandemics originating from non-human animals highlight the need to understand how natural hosts have evolved in response to emerging human pathogens and which groups may be susceptible to infection and/or potential reservoirs to mitigate public health and conservation concerns. Multiple zoonotic coronaviruses, such as severe acute respiratory syndrome-associated coronavirus (SARS-CoV), SARS-CoV-2 and Middle Eastern respiratory syndrome-associated coronavirus (MERS-CoV), are hypothesized to have evolved in bats. We investigate angiotensin-converting enzyme 2 (ACE2), the host protein bound by SARS-CoV and SARS-CoV-2, and dipeptidyl-peptidase 4 (DPP4 or CD26), the host protein bound by MERS-CoV, in the largest bat datasets to date. Both the ACE2 and DPP4 genes are under strong selection pressure in bats, more so than in other mammals, and in residues that contact viruses. Additionally, mammalian groups vary in their similarity to humans in residues that contact SARS-CoV, SARS-CoV-2 and MERS-CoV, and increased similarity to humans in binding residues is broadly predictive of susceptibility to SARS-CoV-2. This work augments our understanding of the relationship between coronaviruses and mammals, particularly bats, provides taxonomically diverse data for studies of how host proteins are bound by coronaviruses and can inform surveillance, conservation and public health efforts.

  View details for DOI 10.1098/rspb.2022.0193

  View details for PubMedID 35892217

• SARS-CoV-2 Brain Regional Detection, Histopathology, Gene Expression, and Immunomodulatory Changes in Decedents with COVID-19. Journal of neuropathology and experimental neurology Serrano, G. E., Walker, J. E., Tremblay, C., Piras, I. S., Huentelman, M. J., Belden, C. M., Goldfarb, D., Shprecher, D., Atri, A., Adler, C. H., Shill, H. A., Driver-Dunckley, E., Mehta, S. H., Caselli, R., Woodruff, B. K., Haarer, C. F., Ruhlen, T., Torres, M., Nguyen, S., Schmitt, D., Rapscak, S. Z., Bime, C., Peters, J. L., Alevritis, E., Arce, R. A., Glass, M. J., Vargas, D., Sue, L. I., Intorcia, A. J., Nelson, C. M., Oliver, J., Russell, A., Suszczewicz, K. E., Borja, C. I., Cline, M. P., Hemmingsen, S. J., Qiji, S., Hobgood, H. M., Mizgerd, J. P., Sahoo, M. K., Zhang, H., Solis, D., Montine, T. J., Berry, G. J., Reiman, E. M., Roltgen, K., Boyd, S. D., Pinsky, B. A., Zehnder, J. L., Talbot, P., Desforges, M., DeTure, M., Dickson, D. W., Beach, T. G. 2022

  Abstract

  Brains of 42 COVID-19 decedents and 107 non-COVID-19 controls were studied. RT-PCR screening of 16 regions from 20 COVID-19 autopsies found SARS-CoV-2 E gene viral sequences in 7 regions (2.5% of 320 samples), concentrated in 4/20 subjects (20%). Additional screening of olfactory bulb (OB), amygdala (AMY) and entorhinal area for E, N1, N2, RNA-dependent RNA polymerase, and S gene sequences detected one or more of these in OB in 8/21 subjects (38%). It is uncertain whether these RNA sequences represent viable virus. Significant histopathology was limited to 2/42 cases (4.8%), one with a large acute cerebral infarct and one with hemorrhagic encephalitis. Case-control RNAseq in OB and AMY found more than 5000 and 700 differentially expressed genes, respectively, unrelated to RT-PCR results; these involved immune response, neuronal constituents, and olfactory/taste receptor genes. Olfactory marker protein-1 reduction indicated COVID-19-related loss of OB olfactory mucosa afferents. Iba-1-immunoreactive microglia had reduced area fractions in cerebellar cortex and AMY, and cytokine arrays showed generalized downregulation in AMY and upregulation in blood serum in COVID-19 cases. Although OB is a major brain portal for SARS-CoV-2, COVID-19 brain changes are more likely due to blood-borne immune mediators and trans-synaptic gene expression changes arising from OB deafferentation.

  View details for DOI 10.1093/jnen/nlac056

  View details for PubMedID 35818336

• Anti-nucleocapsid antibody levels and pulmonary comorbid conditions are linked to post-COVID-19 syndrome. JCI insight Jia, X., Cao, S., Lee, A. S., Manohar, M., Sindher, S. B., Ahuja, N., Artandi, M., Blish, C. A., Blomkalns, A. L., Chang, I., Collins, W. J., Desai, M., Din, H. N., Do, E., Fernandes, A., Geng, L. N., Rosenberg-Hasson, Y., Mahoney, M. R., Glascock, A. L., Chan, L. Y., Fong, S. Y., Phelps, M., Raeber, O., Purington, N., Röltgen, K., Rogers, A. J., Snow, T., Wang, T. T., Solis, D., Vaughan, L., Verghese, M., Maecker, H., Wittman, R., Puri, R., Kistler, A., Yang, S., Boyd, S. D., Pinsky, B. A., Chinthrajah, S., Nadeau, K. C. 2022; 7 (13)

  Abstract

  BACKGROUNDProlonged symptoms after SARS-CoV-2 infection are well documented. However, which factors influence development of long-term symptoms, how symptoms vary across ethnic groups, and whether long-term symptoms correlate with biomarkers are points that remain elusive.METHODSAdult SARS-CoV-2 reverse transcription PCR-positive (RT-PCR-positive) patients were recruited at Stanford from March 2020 to February 2021. Study participants were seen for in-person visits at diagnosis and every 1-3 months for up to 1 year after diagnosis; they completed symptom surveys and underwent blood draws and nasal swab collections at each visit.RESULTSOur cohort (n = 617) ranged from asymptomatic to critical COVID-19 infections. In total, 40% of participants reported at least 1 symptom associated with COVID-19 six months after diagnosis. Median time from diagnosis to first resolution of all symptoms was 44 days; median time from diagnosis to sustained symptom resolution with no recurring symptoms for 1 month or longer was 214 days. Anti-nucleocapsid IgG level in the first week after positive RT-PCR test and history of lung disease were associated with time to sustained symptom resolution. COVID-19 disease severity, ethnicity, age, sex, and remdesivir use did not affect time to sustained symptom resolution.CONCLUSIONWe found that all disease severities had a similar risk of developing post-COVID-19 syndrome in an ethnically diverse population. Comorbid lung disease and lower levels of initial IgG response to SARS-CoV-2 nucleocapsid antigen were associated with longer symptom duration.TRIAL REGISTRATIONClinicalTrials.gov, NCT04373148.FUNDINGNIH UL1TR003142 CTSA grant, NIH U54CA260517 grant, NIEHS R21 ES03304901, Sean N Parker Center for Allergy and Asthma Research at Stanford University, Chan Zuckerberg Biohub, Chan Zuckerberg Initiative, Sunshine Foundation, Crown Foundation, and Parker Foundation.

  View details for DOI 10.1172/jci.insight.156713

  View details for PubMedID 35801588

• Integrated plasma proteomic and single-cell immune signaling network signatures demarcate mild, moderate, and severe COVID-19. Cell reports. Medicine Feyaerts, D., Hédou, J., Gillard, J., Chen, H., Tsai, E. S., Peterson, L. S., Ando, K., Manohar, M., Do, E., Dhondalay, G. K., Fitzpatrick, J., Artandi, M., Chang, I., Snow, T. T., Chinthrajah, R. S., Warren, C. M., Wittman, R., Meyerowitz, J. G., Ganio, E. A., Stelzer, I. A., Han, X., Verdonk, F., Gaudillière, D. K., Mukherjee, N., Tsai, A. S., Rumer, K. K., Jacobsen, D. R., Bjornson-Hooper, Z. B., Jiang, S., Saavedra, S. F., Valdés Ferrer, S. I., Kelly, J. D., Furman, D., Aghaeepour, N., Angst, M. S., Boyd, S. D., Pinsky, B. A., Nolan, G. P., Nadeau, K. C., Gaudillière, B., McIlwain, D. R. 2022: 100680

  Abstract

  The biological determinants underlying the range of coronavirus 2019 (COVID-19) clinical manifestations are not fully understood. Here, over 1,400 plasma proteins and 2,600 single-cell immune features comprising cell phenotype, endogenous signaling activity, and signaling responses to inflammatory ligands are cross-sectionally assessed in peripheral blood from 97 patients with mild, moderate, and severe COVID-19 and 40 uninfected patients. Using an integrated computational approach to analyze the combined plasma and single-cell proteomic data, we identify and independently validate a multi-variate model classifying COVID-19 severity (multi-class area under the curve [AUC]training = 0.799, p = 4.2e-6; multi-class AUCvalidation = 0.773, p = 7.7e-6). Examination of informative model features reveals biological signatures of COVID-19 severity, including the dysregulation of JAK/STAT, MAPK/mTOR, and nuclear factor κB (NF-κB) immune signaling networks in addition to recapitulating known hallmarks of COVID-19. These results provide a set of early determinants of COVID-19 severity that may point to therapeutic targets for prevention and/or treatment of COVID-19 progression.

  View details for DOI 10.1016/j.xcrm.2022.100680

  View details for PubMedID 35839768

• Cellular and humoral immune response to SARS-CoV-2 vaccination and booster dose in immunosuppressed patients: An observational cohort study. Journal of clinical virology : the official publication of the Pan American Society for Clinical Virology Yang, L. M., Costales, C., Ramanathan, M., Bulterys, P. L., Murugesan, K., Schroers-Martin, J., Alizadeh, A. A., Boyd, S. D., Brown, J. M., Nadeau, K. C., Nadimpalli, S. S., Wang, A. X., Busque, S., Pinsky, B. A., Banaei, N. 2022; 153: 105217

  Abstract

  BACKGROUND: Humoral and cellular immune responses to SARS-CoV-2 vaccination among immunosuppressed patients remain poorly defined, as well as variables associated with poor response.METHODS: We performed a retrospective observational cohort study at a large Northern California healthcare system of infection-naive individuals fully vaccinated against SARS-CoV-2 (mRNA-1273, BNT162b2, or Ad26.COV2.S) with clinical SARS-CoV-2 interferon gamma release assay (IGRA) ordered between January through November 2021. Humoral and cellular immune responses were measured by anti-SARS-CoV-2 S1 IgG ELISA (anti-S1 IgG) and IGRA, respectively, following primary and/or booster vaccination.RESULTS: 496 immunosuppressed patients (54% female; median age 50 years) were included. 62% (261/419) of patients had positive anti-S1 IgG and 71% (277/389) had positive IGRA after primary vaccination, with 20% of patients having a positive IGRA only. Following booster, 69% (81/118) had positive anti-S1 IgG and 73% (91/124) had positive IGRA. Factors associated with low humoral response rates after primary vaccination included anti-CD20 monoclonal antibodies (P<0.001), sphingosine 1-phsophate (S1P) receptor modulators (P<0.001), mycophenolate (P=0.002), and B cell lymphoma (P=0.004); those associated with low cellular response rates included S1P receptor modulators (P<0.001) and mycophenolate (P<0.001). Of patients who had poor humoral response to primary vaccination, 35% (18/52) developed a significantly higher response after the booster. Only 5% (2/42) of patients developed a significantly higher cellular response to the booster dose compared to primary vaccination.CONCLUSIONS: Humoral and cellular response rates to primary and booster SARS-CoV-2 vaccination differ among immunosuppressed patient groups. Clinical testing of cellular immunity is important in monitoring vaccine response in vulnerable populations.

  View details for DOI 10.1016/j.jcv.2022.105217

  View details for PubMedID 35714462

• Regulation of the BCR signalosome by the class II peptide editor, H2-M, affects the development and repertoire of innate-like B cells. Cell reports Ghosh, D., Pham, T. D., Nanaware, P. P., Sengupta, D., Adler, L. N., Li, C. G., He, X., O'Mara, M. E., Kantor, A. B., Nguyen, K. D., Yang, Y., Eisenlohr, L. C., Jensen, P. E., Herzenberg, L. A., Stern, L. J., Boyd, S. D., Ghosn, E. E., Mellins, E. D. 1800; 38 (4): 110200

  Abstract

  The non-classical Major Histocompatibility Complex class II (MHCII) protein, H2-M, edits peptides bound to conventional MHCII in favor of stable peptide/MHCII (p/MHCII) complexes. Here, we show that H2-M deficiency affects B-1 cell survival, reduces cell renewal capacity, and alters immunoglobulin repertoire, allowing for the selection of cells specific for highly abundant epitopes, but not low-frequency epitopes. H2-M-deficient B-1 cells have shorter CDR3 length, higher content of positively charged amino acids, shorter junctional regions, less mutation frequency, and a skewed clonal distribution. Mechanistically, H2-M loss reduces plasma membrane p/MHCII association with B cell receptors (BCR) on B-1 cells and diminishes integrated BCR signal strength, a key determinant of B-1 cell selection, maturation, and maintenance. Thus, H2-M:MHCII interaction serves as a cell-intrinsic regulator of BCR signaling and influences the selection of the B-1 cell clonal repertoire.

  View details for DOI 10.1016/j.celrep.2021.110200

  View details for PubMedID 35081339

• Safety, immunogenicity, and protection provided by unadjuvanted and adjuvanted formulations of a recombinant plant-derived virus-like particle vaccine candidate for COVID-19 in nonhuman primates. Cellular & molecular immunology Pillet, S., Arunachalam, P. S., Andreani, G., Golden, N., Fontenot, J., Aye, P. P., Roltgen, K., Lehmicke, G., Gobeil, P., Dube, C., Trepanier, S., Charland, N., D'Aoust, M., Russell-Lodrigue, K., Monjure, C., Blair, R. V., Boyd, S. D., Bohm, R. P., Rappaport, J., Villinger, F., Landry, N., Pulendran, B., Ward, B. J. 1800

  Abstract

  Although antivirals are important tools to control severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection, effective vaccines are essential to control the current coronavirus disease 2019 (COVID-19) pandemic. Plant-derived virus-like particle (VLP) vaccine candidates have previously demonstrated immunogenicity and efficacy against influenza. Here, we report the immunogenicity and protection induced in rhesus macaques by intramuscular injections of a VLP bearing a SARS-CoV-2 spike protein (CoVLP) vaccine candidate formulated with or without Adjuvant System 03 (AS03) or cytidine-phospho-guanosine (CpG) 1018. Although a single dose of the unadjuvanted CoVLP vaccine candidate stimulated humoral and cell-mediated immune responses, booster immunization (at 28 days after priming) and adjuvant administration significantly improved both responses, with higher immunogenicity and protection provided by the AS03-adjuvanted CoVLP. Fifteen micrograms of CoVLP adjuvanted with AS03 induced a polyfunctional interleukin-2 (IL-2)-driven response and IL-4 expression in CD4 T cells. Animals were challenged by multiple routes (i.e., intratracheal, intranasal, and ocular) with a total viral dose of 106 plaque-forming units of SARS-CoV-2. Lower viral replication in nasal swabs and bronchoalveolar lavage fluid (BALF) as well as fewer SARS-CoV-2-infected cells and immune cell infiltrates in the lungs concomitant with reduced levels of proinflammatory cytokines and chemotactic factors in the BALF were observed in animals immunized with the CoVLP adjuvanted with AS03. No clinical, pathologic, or virologic evidence of vaccine-associated enhanced disease was observed in vaccinated animals. The CoVLP adjuvanted with AS03 was therefore selected for vaccine development and clinical trials.

  View details for DOI 10.1038/s41423-021-00809-2

  View details for PubMedID 34983950

• Gastrointestinal γδ T cells reveal differentially expressed transcripts and enriched pathways during peanut oral immunotherapy. Allergy Zhang, W., Krishna Dhondalay, G., Liu, T. A., Kaushik, A., Hoh, R., Kwok, S., Kambham, N., Fernandez-Becker, N. Q., Andorf, S., Desai, M., Galli, S. J., Boyd, S. D., Nadeau, K. C., Manohar, M., DeKruyff, R. H., Chinthrajah, R. S. 2022

  View details for DOI 10.1111/all.15250

  View details for PubMedID 35143054

• Long Term Accuracy of SARS-CoV-2 Interferon-γ Release Assay and its Application in Household Investigation. Clinical infectious diseases : an official publication of the Infectious Diseases Society of America Murugesan, K., Jagannathan, P., Altamirano, J., Maldonado, Y. A., Bonilla, H. F., Jacobson, K. B., Parsonnet, J., Andrews, J. R., Shi, R. Z., Boyd, S., Pinsky, B. A., Singh, U., Banaei, N. 2022

  Abstract

  An immunodiagnostic assay that sensitively detects a cell-mediated immune response to SARS-CoV-2 is needed for epidemiological investigation and for clinical assessment of T cell-mediated immune response to vaccines, particularly in the context of emerging variants that might escape antibody responses.The performance of a whole blood interferon-gamma (IFN-γ) release assay (IGRA) for the detection of SARS-CoV-2 antigen-specific T cells was evaluated in COVID-19 convalescents tested serially up to 10 months post-infection and in healthy blood donors. SARS-CoV-2 IGRA was applied in contacts of households with index cases. Freshly collected blood in the lithium heparin tube was left unstimulated, stimulated with a SARS-CoV-2 peptide pool, and stimulated with mitogen.The overall sensitivity and specificity of IGRA were 84.5% (153/181; 95% confidence interval [CI] 79.0-89.0) and 86.6% (123/142; 95% CI;80.0-91.2), respectively. The sensitivity declined from 100% (16/16; 95% CI 80.6-100) at 0.5-month post-infection to 79.5% (31/39; 95% CI 64.4-89.2) at 10 months post-infection (P<0.01). The IFN-γ response remained relatively robust at 10 months post-infection (3.8 vs. 1.3 IU/mL, respectively). In 14 households, IGRA showed a positivity rate of 100% (12/12) and 65.2% (15/23), and IgG of 50.0% (6/12) and 43.5% (10/23) in index cases and contacts, respectively, exhibiting a difference of +50% (95% CI +25.4-+74.6) and +21.7% (95% CI, +9.23-+42.3), respectively. Either IGRA or IgG was positive in 100% (12/12) of index cases and 73.9% (17/23) of contacts.The SARS-CoV-2 IGRA is a useful clinical diagnostic tool for assessing cell-mediated immune response to SARS-CoV-2.

  View details for DOI 10.1093/cid/ciac045

  View details for PubMedID 35079772

• Host microRNAs are decreased in pediatric solid-organ transplant recipients during EBV+ Post-transplant Lymphoproliferative Disorder. Frontiers in immunology Sen, A., Enriquez, J., Rao, M., Glass, M., Balachandran, Y., Syed, S., Twist, C. J., Weinberg, K., Boyd, S. D., Bernstein, D., Trickey, A. W., Gratzinger, D., Tan, B., Lapasaran, M. G., Robien, M. A., Brown, M., Armstrong, B., Desai, D., Mazariegos, G., Chin, C., Fishbein, T. M., Venick, R. S., Tekin, A., Zimmermann, H., Trappe, R. U., Anagnostopoulos, I., Esquivel, C. O., Martinez, O. M., Krams, S. M. 2022; 13: 994552

  Abstract

  Post-transplant lymphoproliferative disorder (PTLD) is a serious complication of solid organ transplantation. Predisposing factors include primary Epstein-Barr virus (EBV) infection, reactivation of EBV in recipient B cells, and decreased T cell immunity due to immunosuppression. In our previous studies EBV infection was demonstrated to markedly alter the expression of host B cell microRNA (miR). Specifically, miR-194 expression was uniquely suppressed in EBV+ B cell lines from PTLD patients and the 3'untranslated region of IL-10 was determined to be targeted by miR-194. Although EBV has been shown to regulate host miR expression in B cell lymphoma cell lines, the expression of miRs in the circulation of patients with EBV-associated PTLD has not been studied. The objective of this study was to determine if changes in miR expression are associated with EBV+ PTLD. In this study, we have shown that miR-194 is significantly decreased in EBV+PTLD tumors and that additional miRs, including miRs-17, 19 and 106a are also reduced in EBV+PTLD as compared to EBV-PTLD. We quantitated the levels of miRs-17, 19, 106a, 155, and 194 in the plasma and extracellular vesicles (EV; 50-70 nm as determined by nanoparticle tracking analysis) from pediatric recipients of solid organ transplants with EBV+ PTLD+ that were matched 1:2 with EBV+ PTLD- pediatric transplant recipients as part of the NIH-sponsored Clinical Trials in Organ Transplantation in Children, (CTOTC-06) study. Levels of miRs-17, 19, 106a, and 194 were reduced in the plasma and extracellular vesicles (EV) of EBV+ PTLD+ group compared to matched controls, with miRs-17 (p = 0.034; plasma), miRs-19 (p = 0.029; EV) and miR-106a (p = 0.007; plasma and EV) being significantly reduced. Similar levels of miR-155 were detected in the plasma and EV of all pediatric SOT recipients. Importantly, ~90% of the cell-free miR were contained within the EV supporting that EBV+ PTLD tumor miR are detected in the circulation and suggesting that EVs, containing miRs, may have the potential to target and regulate cells of the immune system. Further development of diagnostic, mechanistic and potential therapeutic uses of the miRs in PTLD is warranted.

  View details for DOI 10.3389/fimmu.2022.994552

  View details for PubMedID 36304469

• Maternal and Infant Immune Repertoire Sequencing Analysis Identifies Distinct Ig and TCR Development in Term and Preterm Infants. Journal of immunology (Baltimore, Md. : 1950) Le, B. L., Sper, R., Nielsen, S. C., Pineda, S., Nguyen, Q., Lee, J., Boyd, S. D., MacKenzie, T. C., Sirota, M. 2021

  Abstract

  Preterm labor (PTL) is the leading cause of neonatal morbidity and mortality worldwide. Whereas many studies have investigated the maternal immune responses that cause PTL, fetal immune cell activation has recently been raised as an important contributor to the pathogenesis of PTL. In this study, we analyzed lymphocyte receptor repertoires in maternal and cord blood from 14 term and 10 preterm deliveries, hypothesizing that the high prevalence of infection in patients with PTL may result in specific changes in the T cell and B cell repertoires. We analyzed TCR beta-chain (TCR-beta) and IgH diversity, CDR3 lengths, clonal sharing, and preferential usage of variable and joining gene segments. Both TCR-beta and IgH repertoires had shorter CDR3s compared with those in maternal blood. In cord blood samples, we found that CDR3 lengths correlated with gestational age, with shorter CDR3s in preterm neonates suggesting a less developed repertoire. Preterm cord blood displayed preferential usage of a number of genes. In preterm pregnancies, we observed significantly higher prevalence of convergent clones between mother/baby pairs than in term pregnancies. Together, our results suggest the repertoire of preterm infants displays a combination of immature features and convergence with maternal TCR-beta clones compared with that of term infants. The higher clonal convergence in PTL could represent mother and fetus both responding to a shared stimulus like an infection. These data provide a detailed analysis of the maternal-fetal immune repertoire in term and preterm patients and contribute to a better understanding of neonate immune repertoire development and potential changes associated with PTL.

  View details for DOI 10.4049/jimmunol.2100566

  View details for PubMedID 34654689

• SARS-CoV-2 Nucleocapsid Plasma Antigen for Diagnosis and Monitoring of COVID-19. Clinical chemistry Wang, H., Hogan, C. A., Verghese, M., Solis, D., Sibai, M., Huang, C., Roltgen, K., Stevens, B. A., Yamamoto, F., Sahoo, M. K., Zehnder, J., Boyd, S. D., Pinsky, B. A. 2021

  Abstract

  BACKGROUND: Detection of SARS-CoV-2 nucleocapsid antigen in blood has been described, but the diagnostic and prognostic role of antigenemia is not well understood. This study aimed to determine the frequency, duration, and concentration of nucleocapsid antigen in plasma and its association with COVID-19 severity.METHODS: We utilized an ultrasensitive electrochemiluminescence immunoassay targeting SARS-CoV-2 nucleocapsid antigen to evaluate 777 plasma samples from 104 individuals with COVID-19. We compared plasma antigen to respiratory nucleic acid amplification testing (NAAT) in 74 individuals with COVID-19 from samples collected ± 1day of diagnostic respiratory NAAT, and in 52 SARS-CoV-2-negative individuals. We used Kruskal-Wallis tests, multivariable logistic regression, and mixed-effects modeling to evaluate whether plasma antigen concentration was associated with disease severity.RESULTS: Plasma antigen had 91.9% (95% CI 83.2-97.0%) clinical sensitivity and 94.2% (84.1-98.8%) clinical specificity. Antigen-negative plasma samples belonged to patients with later respiratory cycle thresholds (Ct) when compared with antigen-positive plasma samples. Median plasma antigen concentration (log10 fg/mL) was 5.4 (IQR 3.9-6.0) in outpatients, 6.0 (5.4-6.5) in inpatients, and 6.6 (6.1-7.2) in intensive care unit (ICU) patients. In models adjusted for age, sex, diabetes, and hypertension, plasma antigen concentration at diagnosis was associated with ICU admission (OR 2.8 [95% CI 1.2-6.2], P=.01), but not with non-ICU hospitalization. Rate of antigen decrease was not associated with disease severity.CONCLUSIONS: SARS-CoV-2 plasma nucleocapsid antigen exhibited comparable diagnostic performance to upper respiratory NAAT, especially among those with late respiratory Ct. In addition to currently available tools, antigenemia may facilitate patient triage to optimize intensive care utilization.

  View details for DOI 10.1093/clinchem/hvab216

  View details for PubMedID 34605900

• Effective Viral Vector SARS-CoV-2 Booster Vaccination in a Patient with Rheumatoid Arthritis after Initial Ineffective mRNA Vaccine Response. Arthritis & rheumatology (Hoboken, N.J.) Baker, M. C., Mallajosyula, V., Davis, M. M., Boyd, S. D., Nadeau, K. C., Robinson, W. H. 2021

  Abstract

  Managing patients with rheumatic disease during the COVID-19 pandemic has posed a unique challenge. Immunosuppressed patients are at an increased risk for developing severe COVID-19 and may not derive full protection from the vaccine (1-5). Thus, it is paramount we develop strategies whereby rheumatic disease patients can be protected from the pandemic virus and its variants.

  View details for DOI 10.1002/art.41978

  View details for PubMedID 34514750

• Estimated SARS-CoV-2 Seroprevalence in US Patients Receiving Dialysis 1 Year After the Beginning of the COVID-19 Pandemic. JAMA network open Anand, S., Montez-Rath, M., Han, J., Cadden, L., Hunsader, P., Kerschmann, R., Beyer, P., Boyd, S. D., Garcia, P., Dittrich, M., Block, G. A., Parsonnet, J., Chertow, G. M. 2021; 4 (7): e2116572

  Abstract

  Importance: Seroprevalence studies complement data on detected cases and attributed deaths in assessing the cumulative spread of the SARS-CoV-2 virus.Objective: To estimate seroprevalence of SARS-CoV-2 antibodies in patients receiving dialysis and adults in the US in January 2021 before the widespread introduction of COVID-19 vaccines.Design, Setting, and Participants: This cross-sectional study used data from the third largest US dialysis organization (US Renal Care), which has facilities located nationwide, to estimate SARS-CoV-2 seroprevalence among US patients receiving dialysis. Remainder plasma (ie, plasma that would have otherwise been discarded) of all patients receiving dialysis at US Renal Care facilities from January 1 to 31, 2021, was tested for SARS-CoV-2 antibodies. Patients were excluded if they had a documented dose of SARS-CoV-2 vaccination or if a residence zip code was missing from electronic medical records. Crude seroprevalence estimates from this sample (January 2021) were standardized to the US adult population using the 2018 American Community Survey 1-year estimates and stratified by age group, sex, self-reported race/ethnicity, neighborhood race/ethnicity composition, neighborhood income level, and urban or rural status. These data and case detection rates were then compared with data from a July 2020 subsample of patients who received dialysis at the same facilities.Exposures: Age, sex, race/ethnicity, and region of residence as well as neighborhood race/ethnicity composition, poverty, population density, and urban or rural status.Main Outcomes and Measures: The spike protein receptor-binding domain total antibody assay (Siemens Healthineers; manufacturer-reported sensitivity of 100% and specificity of 99.8%) was used to estimate crude SARS-CoV-2 seroprevalence in the unweighted sample, and then the estimated seroprevalence rates for the US dialysis and adult populations were calculated, adjusting for age, sex, and region.Results: A total of 21 464 patients (mean [SD] age, 63.1 [14.2] years; 12 265 men [57%]) were included in the unweighted sample from January 2021. The patients were disproportionately older (aged 65-79 years, 7847 [37%]; aged ≥80 years, 2668 [12%]) and members of racial/ethnic minority groups (Hispanic patients, 2945 [18%]; non-Hispanic Black patients, 4875 [29%]). Seroprevalence of SARS-CoV-2 antibodies was 18.9% (95% CI, 18.3%-19.5%) in the sample, with a seroprevalence of 18.7% (95% CI, 18.1%-19.2%) standardized to the US dialysis population, and 21.3% (95% CI, 20.3%-22.3%) standardized to the US adult population. In the unweighted sample, younger persons (aged 18-44 years, 25.9%; 95% CI, 24.1%-27.8%), those who self-identified as Hispanic or living in Hispanic neighborhoods (25.1%; 95% CI, 23.6%-26.4%), and those living in the lowest-income neighborhoods (24.8%; 95% CI, 23.2%-26.5%) were among the subgroups with the highest seroprevalence. Little variability was observed in seroprevalence by geographic region, population density, and urban or rural status in the January 2021 sample (largest regional difference, 1.2 [95% CI, 1.1-1.3] higher odds of seroprevalence in residents of the Northeast vs West).Conclusions and Relevance: In this cross-sectional study of patients receiving dialysis in the US, fewer than 1 in 4 patients had evidence of SARS-CoV-2 antibodies 1 year after the first case of SARS-CoV-2 infection was detected in the US. Results standardized to the US population indicate similar prevalence of antibodies among US adults. Vaccine introduction to younger individuals, those living in neighborhoods with a large population of racial/ethnic minority residents, and those living in low-income neighborhoods may be critical to disrupting the spread of infection.

  View details for DOI 10.1001/jamanetworkopen.2021.16572

  View details for PubMedID 34251441

• Antibody Response to COVID-19 Vaccination in Patients Receiving Dialysis. Journal of the American Society of Nephrology : JASN Anand, S., Montez-Rath, M., Han, J., Garcia, P., Cadden, L., Hunsader, P., Kerschmann, R., Beyer, P., Dittrich, M., Block, G., Boyd, S., Parsonnet, J., Chertow, G. 2021

  View details for DOI 10.1681/ASN.2021050611

  View details for PubMedID 34117129

• Serial SARS-CoV-2 Receptor-Binding Domain Antibody Responses in Patients Receiving Dialysis. Annals of internal medicine Anand, S., Montez-Rath, M. E., Han, J., Garcia, P., Cadden, L., Hunsader, P., Kerschmann, R., Beyer, P., Boyd, S. D., Chertow, G. M., Parsonnet, J. 2021

  Abstract

  BACKGROUND: Assessing the evolution of SARS-CoV-2 immune response among patients receiving dialysis can define its durability in a highly clinically relevant context because patients receiving dialysis share the characteristics of persons most susceptible to SARS-CoV-2 infection.OBJECTIVE: To evaluate the persistence of SARS-CoV-2 receptor-binding domain (RBD) IgG in seroprevalent patients receiving dialysis.DESIGN: Prospective.SETTING: Nationwide sample from dialysis facilities.PATIENTS: 2215 patients receiving dialysis who had evidence of SARS-CoV-2 infection as of July 2020.MEASUREMENTS: Remainder plasma from routine monthly laboratories was used to measure semiquantitative RBD IgG index value over 6 months.RESULTS: A total of 2063 (93%) seroprevalent patients reached an assay detectable response (IgG index value ≥1). Most (n = 1323, 60%) had responses in July with index values classified as high (IgG ≥10); 1003 (76%) remained within this stratum. Adjusted median index values declined slowly but continuously (July vs. December values were 21 vs. 13; P < 0.001). The trajectory of the response did not vary by age group, sex, race/ethnicity, or diabetes status. Patients without an assay detectable response (n = 137) were more likely to be White and in the younger (18 to 44 years) or older (≥80 years) age groups and less likely to have diabetes and hypoalbuminemia.LIMITATION: Lack of data on symptoms or reverse transcriptase polymerase chain reaction diagnosis, cohort of persons who survived infection, and use of a semiquantitative assay.CONCLUSION: Despite impaired immunity, most seropositive patients receiving dialysis maintained RBD antibody levels over 6 months. A slow and continual decline in median antibody levels over time was seen, but no indication that subgroups with impaired immunity had a shorter-lived humoral response was found.PRIMARY FUNDING SOURCE: Ascend Clinical Laboratories.

  View details for DOI 10.7326/M21-0256

  View details for PubMedID 34000201

• Increased viral variants in children and young adults with impaired humoral immunity and persistent SARS-CoV-2 infection: A consecutive case series. EBioMedicine Truong, T. T., Ryutov, A., Pandey, U., Yee, R., Goldberg, L., Bhojwani, D., Aguayo-Hiraldo, P., Pinsky, B. A., Pekosz, A., Shen, L., Boyd, S. D., Wirz, O. F., Roltgen, K., Bootwalla, M., Maglinte, D. T., Ostrow, D., Ruble, D., Han, J. H., Biegel, J. A., Li, M., Huang, C., Sahoo, M. K., Pannaraj, P. S., O'Gorman, M., Judkins, A. R., Gai, X., Dien Bard, J. 2021; 67: 103355

  Abstract

  BACKGROUND: There is increasing concern that persistent infection of SARS-CoV-2 within immunocompromised hosts could serve as a reservoir for mutation accumulation and subsequent emergence of novel strains with the potential to evade immune responses.METHODS: We describe three patients with acute lymphoblastic leukemia who were persistently positive for SARS-CoV-2 by real-time polymerase chain reaction. Viral viability from longitudinally-collected specimens was assessed. Whole-genome sequencing and serological studies were performed to measure viral evolution and evidence of immune escape.FINDINGS: We found compelling evidence of ongoing replication and infectivity for up to 162 days from initial positive by subgenomic RNA, single-stranded RNA, and viral culture analysis. Our results reveal a broad spectrum of infectivity, host immune responses, and accumulation of mutations, some with the potential for immune escape.INTERPRETATION: Our results highlight the potential need to reassess infection control precautions in the management and care of immunocompromised patients. Routine surveillance of mutations and evaluation of their potential impact on viral transmission and immune escape should be considered.

  View details for DOI 10.1016/j.ebiom.2021.103355

  View details for PubMedID 33915337

• Adjuvanting a subunit COVID-19 vaccine to induce protective immunity. Nature Arunachalam, P. S., Walls, A. C., Golden, N., Atyeo, C., Fischinger, S., Li, C., Aye, P., Navarro, M. J., Lai, L., Edara, V. V., Roltgen, K., Rogers, K., Shirreff, L., Ferrell, D. E., Wrenn, S., Pettie, D., Kraft, J. C., Miranda, M. C., Kepl, E., Sydeman, C., Brunette, N., Murphy, M., Fiala, B., Carter, L., White, A. G., Trisal, M., Hsieh, C., Russell-Lodrigue, K., Monjure, C., Dufour, J., Spencer, S., Doyle-Meyer, L., Bohm, R. P., Maness, N. J., Roy, C., Plante, J. A., Plante, K. S., Zhu, A., Gorman, M. J., Shin, S., Shen, X., Fontenot, J., Gupta, S., O'Hagan, D. T., Van Der Most, R., Rappuoli, R., Coffman, R. L., Novack, D., McLellan, J. S., Subramaniam, S., Montefiori, D., Boyd, S. D., Flynn, J. L., Alter, G., Villinger, F., Kleanthous, H., Rappaport, J., Suthar, M. S., King, N. P., Veesler, D., Pulendran, B. 2021

  Abstract

  The development of a portfolio of COVID-19 vaccines to vaccinate the global population remains an urgent public health imperative1. Here we demonstrate the capacity of a subunit vaccine, comprising the SARS-CoV-2 spike receptor binding domain displayed on a protein nanoparticle (RBD-NP), to stimulate robust and durable neutralizing antibody (nAb) responses and protection against SARS-CoV-2 in non-human primates. We evaluated five adjuvants including Essai O/W 1849101, a squalene-in-water emulsion; AS03, an alpha-tocopherol-containing oil-in-water emulsion; AS37, a TLR-7 agonist adsorbed to Alum; CpG1018-Alum, a TLR-9 agonist formulated in Alum; and Alum. RBD-NP immunization with AS03, CpG1018-Alum, AS37 or Alum induced substantial nAb and CD4 T cell responses, and conferred protection against SARS-CoV-2 infection in the pharynges, nares and bronchoalveolar lavage. Live-virus nAb response was maintained up to 180 days post-vaccination with RBD/AS03, and correlated with protection. RBD-NP immunization cross-neutralized the B.1.1.7 variant efficiently but showed a reduced response against the B.1.351 variant. While RBD-NP/AS03 demonstrated a 4.5-fold reduction in neutralization of B.1.351, the RBD-NP/AS37 group showed a 16-fold reduction, suggesting differences in the breadth of the nAb response induced by these adjuvants. Furthermore, RBD-NP/AS03 was as immunogenic as a prefusion stabilized Spike immunogen (Hexapro) adjuvanted with AS03. These data highlight the efficacy of the adjuvanted RBD-NP vaccine in promoting protective immunity against SARS-CoV-2, and have paved the way for the clinical evaluation of this vaccine in Phase I/II clinical trials (NCT04742738 and NCT04750343).

  View details for DOI 10.1038/s41586-021-03530-2

  View details for PubMedID 33873199

• SARS-CoV-2 IgG Seropositivity and Acute Asymptomatic Infection Rate Among Firefighter First Responders in an Early Outbreak County in California. Prehospital emergency care : official journal of the National Association of EMS Physicians and the National Association of State EMS Directors Newberry, J. A., Gautreau, M., Staats, K., Carrillo, E., Mulkerin, W., Yang, S., Kohn, M. A., Matheson, L., Boyd, S. D., Pinsky, B. A., Blomkalns, A. L., Strehlow, M. C., D'Souza, P. A. 2021: 1–10

  Abstract

  Objective: Firefighter first responders and other emergency medical services (EMS) personnel have been among the highest risk healthcare workers for illness during the SARS-CoV-2 pandemic. We sought to determine the rate of seropositivity for SARS-CoV-2 IgG antibodies and of acute asymptomatic infection among firefighter first responders in a single county with early exposure in the pandemic.Methods: We conducted a cross-sectional study of clinically active firefighters cross-trained as paramedics or EMTs in the fire departments of Santa Clara County, California. Firefighters without current symptoms were tested between June and August 2020. Our primary outcomes were rates of SARS-CoV-2 IgG antibody seropositivity and SARS-CoV-2 RT-PCR swab positivity for acute infection. We report cumulative incidence, participant characteristics with frequencies and proportions, and proportion positive and associated relative risk (with 95% confidence intervals).Results: We enrolled 983 out of 1339 eligible participants (response rate: 73.4%). Twenty-five participants (2.54%, 95% CI 1.65-3.73) tested positive for IgG antibodies and 9 (0.92%, 95% CI 0.42-1.73) tested positive for SARS-CoV-2 by RT-PCR. Our cumulative incidence, inclusive of self-reported prior positive PCR tests, was 34 (3.46%, 95% CI 2.41-4.80).Conclusion: In a county with one of the earliest outbreaks in the United States, the seroprevalence among firefighter first responders was lower than that reported by other studies of frontline health care workers, while the cumulative incidence remained higher than that seen in the surrounding community.

  View details for DOI 10.1080/10903127.2021.1912227

  View details for PubMedID 33819128

• Immune Changes Beyond Th2 Pathways During Rapid Multifood Immunotherapy enabled with Omalizumab. Allergy Manohar, M., Dunham, D., Gupta, S., Yan, Z., Zhang, W., Minnicozzi, S., Kirkey, M., Bunning, B., Chowdhury, R. R., Galli, S. J., Boyd, S. D., Kost, L. E., Chinthrajah, R. S., Desai, M., Oettgen, H. C., Maecker, H. T., Yu, W., DeKruyff, R. H., Andorf, S., Nadeau, K. C. 2021

  Abstract

  BACKGROUND: Multifood Oral Immunotherapy (mOIT) with adjunctive anti-IgE (omalizumab, Xolair ) treatment affords safe, effective, and rapid desensitization to multiple foods, although the specific immune mechanisms mediating this desensitization remain to be fully elucidated.METHODS: Participants in our phase 2 mOIT trial (NCT02643862) received omalizumab from baseline to week 16 and mOIT from week 8 to week 36. We compared the immune profile of PBMCs and plasma taken at baseline, week 8 and week 36 using high-dimensional mass cytometry, component-resolved diagnostics, the indirect basophil activation test, and Luminex.RESULTS: We found (i) decreased frequency of IL4+ peanut-reactive CD4+ T cells and a marked downregulation of GPR15 expression and CXCR3 frequency among gammadelta and CD8+ T cell subsets at week 8 during the initial, omalizumab-alone induction phase; (ii) significant upregulation of the skin-homing receptor CCR4 in peanut-reactive CD4+ T and Th2 effector memory (EM) cells and of cutaneous lymphocyte-associated antigen (CLA) in peanut-reactive CD8+ T and CD8+ EM cells (iii) downregulation of CD86 expression among antigen-presenting cell subsets; and (iv) reduction in pro-inflammatory cytokines, notably IL-17, at week 36 post-OIT. We also observed significant attenuation of the Th2 phenotype post-OIT, defined by downregulation of IL-4 peanut-reactive T cells and OX40 in Th2EM cells, increased allergen component-specific IgG4/IgE ratio, and decreased allergen-driven activation of indirectly sensitized basophils.CONCLUSIONS: This exploratory study provides novel comprehensive insight into the immune underpinnings of desensitization through omalizumab-facilitated mOIT. Moreover, this study provides encouraging results to support the complex immune changes that can be induced by OIT.

  View details for DOI 10.1111/all.14833

  View details for PubMedID 33782956

• Antibody Response to COVID-19 vaccination in Patients Receiving Dialysis. medRxiv : the preprint server for health sciences Anand, S., Montez-Rath, M. E., Han, J., Garcia, P., Cadden, L., Hunsader, P., Kerschmann, R., Beyer, P., Dittrich, M., Block, G. A., Boyd, S. D., Parsonnet, J., Chertow, G. M. 2021

  Abstract

  Patients receiving dialysis may mount impaired responses to COVID19 vaccination.We report antibody response to vaccination from 1140 patients without, and 493 patients with pre-vaccination SARS-CoV-2 RBD antibody. We used commercially available assays (Siemens) to test remainder plasma monthly in association with vaccination date and type, and assess prevalence of absent total receptor binding antibody, and absent or attenuated (index value < 10) semiquantitative receptor binding domain IgG index values. We used Poisson regression to evaluate risk factors for absent or attenuated response to vaccination.Among patients who were seronegative versus seropositive before vaccination, 62% and 56% were ≥65 years old, 20% and 24% were Hispanic, and 22% and 23% were Black. Median IgG index values rose steadily over time, and were higher among the seropositive than in the seronegative patients after completing vaccination (150 [25 th , 75 th percentile 23.2, 150.0] versus 41.6 [11.3, 150.0]). Among 610 patients who completed vaccination (assessed ≥14 days later, median 29 days later), the prevalence of absent total RBD response, and absent and attenuated semiquantitative IgG response was 4.4% (95% CI 3.1, 6.4%), 3.4% (2.4, 5.2%), and 14.3% (11.7, 17.3%) respectively. Risk factors for absent or attenuated response included longer vintage of end-stage kidney disease, and lower pre-vaccination serum albumin.More than one in five patients receiving dialysis had evidence of an attenuated immune response to COVID19 vaccination.Patients receiving dialysis face high likelihood of severe COVID19; at the same time, vaccination may be less efficacious, as prior data indicate impaired immune responses to influenza and Hepatitis B vaccination. We found that 22% of patients receiving dialysis had suboptimal responses to vaccination, irrespective of whether or not they had evidence of prior SARS-CoV-2 infection. Poorer health status and longer duration of end-stage kidney disease increased likelihood of suboptimal response. Ongoing vigilance for COVID19 in dialysis facilities and studies of modified vaccination dosing schedules will be critical to protecting patients receiving dialysis.

  View details for DOI 10.1101/2021.05.06.21256768

  View details for PubMedID 34013281

  View details for PubMedCentralID PMC8132255

• Use of Outpatient-Derived COVID-19 Convalescent Plasma in COVID-19 Patients Before Seroconversion. Frontiers in immunology Wirz, O. F., Roltgen, K., Stevens, B. A., Pandey, S., Sahoo, M. K., Tolentino, L., Verghese, M., Nguyen, K., Hunter, M., Snow, T. T., Singh, A. R., Blish, C. A., Cochran, J. R., Zehnder, J. L., Nadeau, K. C., Pinsky, B. A., Pham, T. D., Boyd, S. D. 2021; 12: 739037

  Abstract

  Background: Transfusion of COVID-19 convalescent plasma (CCP) containing high titers of anti-SARS-CoV-2 antibodies serves as therapy for COVID-19 patients. Transfusions early during disease course was found to be beneficial. Lessons from the SARS-CoV-2 pandemic could inform early responses to future pandemics and may continue to be relevant in lower resource settings. We sought to identify factors correlating to high antibody titers in convalescent plasma donors and understand the magnitude and pharmacokinetic time course of both transfused antibody titers and the endogenous antibody titers in transfused recipients.Methods: Plasma samples were collected up to 174 days after convalescence from 93 CCP donors with mild disease, and from 16 COVID-19 patients before and after transfusion. Using ELISA, anti-SARS-CoV-2 Spike RBD, S1, and N-protein antibodies, as well as capacity of antibodies to block ACE2 from binding to RBD was measured in an in vitro assay. As an estimate for viral load, viral RNA and N-protein plasma levels were assessed in COVID-19 patients.Results: Anti-SARS-CoV-2 antibody levels and RBD-ACE2 blocking capacity were highest within the first 60 days after symptom resolution and markedly decreased after 120 days. Highest antibody titers were found in CCP donors that experienced fever. Effect of transfused CCP was detectable in COVID-19 patients who received high-titer CCP and had not seroconverted at the time of transfusion. Decrease in viral RNA was seen in two of these patients.Conclusion: Our results suggest that high titer CCP should be collected within 60 days after recovery from donors with past fever. The much lower titers conferred by transfused antibodies compared to endogenous production in the patient underscore the importance of providing CCP prior to endogenous seroconversion.

  View details for DOI 10.3389/fimmu.2021.739037

  View details for PubMedID 34594341

• Efficient Identification of High-Titer Anti-Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) Antibody Plasma Samples by Pooling Method. Archives of pathology & laboratory medicine Nguyen, K. D., Wirz, O. F., Röltgen, K., Pandey, S., Tolentino, L., Boyd, S. D., Pham, T. D. 2021

  Abstract

  The ongoing coronavirus disease 2019 (COVID-19) pandemic caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has elicited a surge in demand for serological testing to identify previously infected individuals. In particular, antibody testing is crucial in identifying COVID-19 convalescent plasma (CCP), which has been approved by the Food and Drug Administration (FDA) under the Emergency Use Authorization (EUA) for use as passive immune therapy for hospitalized patients infected with COVID-19. Currently, high-titer CCP can be qualified by Ortho's Vitros COVID-19 IgG antibody test (VG).To explore the use of an efficient testing method to identify high-titer CCP for use in treating COVID-19 infected patients and track COVID-19 positivity over time.We evaluated an ELISA-based method that detects antibodies specific to the SARSCoV-2 receptor binding domain (RBD) with individual and pooled plasma samples and compared its performance against VG. Using the pooled RBD-ELISA (P-RE) method, we also screened over 10,000 longitudinal healthy blood donor samples to assess seroprevalence.P-RE demonstrates 100% sensitivity in detecting FDA-defined high-titer samples when compared to VG. Overall sensitivity of P-RE when compared to VG and our individual sample RBD-ELISA (I-RE) were 83% and 56%, respectively. When screening 10,218 healthy blood donor samples by P-RE, we found the seroprevalence correlated with the local infection rates with a correlation coefficient of 0.21 (P< .001).Pooling plasma samples can be used to efficiently screen large populations for individuals with high-titer anti-RBD antibodies, important for CCP identification.

  View details for DOI 10.5858/arpa.2021-0215-SA

  View details for PubMedID 34101801

• Plasma as an alternative COVID-19 diagnostic specimen in a hospitalized patient negative for SARS-CoV-2 by nasopharyngeal swab. Diagnostic microbiology and infectious disease Lawrence, L. n., Stevens, B. A., Sahoo, M. K., Huang, C. n., Yamamoto, F. n., Röltgen, K. n., Wirz, O. n., Zehnder, J. n., Shi, R. Z., Boyd, S. D., Schoolnik, G. n., Pinsky, B. A., Hogan, C. A. 2021; 100 (3): 115365

  Abstract

  We present the case of an inpatient with pneumonia and repeatedly negative nasopharyngeal SARS-CoV-2 testing. In such challenging cases, alternative diagnostic options include lower respiratory tract and plasma SARS-CoV-2 RNA testing, of which the latter may be particularly useful where bronchoscopy is deferred due to clinical factors or transmission risk.

  View details for DOI 10.1016/j.diagmicrobio.2021.115365

  View details for PubMedID 33865070

• Evaluation of SARS-CoV-2 total antibody detection via a lateral flow nanoparticle fluorescence immunoassay. Journal of clinical virology : the official publication of the Pan American Society for Clinical Virology Sibai, M. n., Solis, D. n., Röltgen, K. n., Stevens, B. A., Mfuh, K. O., Sahoo, M. K., Shi, R. Z., Zehnder, J. n., Boyd, S. D., Pinsky, B. A. 2021; 139: 104818

  Abstract

  The coronavirus disease 2019 (COVID-19) endgame may benefit from simple, accurate antibody testing to characterize seroprevalence and immunization coverage.To evaluate the performance of the lateral flow QIAreach anti-SARS-CoV-2 Total rapid nanoparticle fluorescence immunoassay compared to reference isotype-specific IgG, IgM, and IgA SARS-CoV-2 ELISA using S1 or receptor binding domain (RBD) as antigens.A diagnostic comparison study was carried out using 154 well-characterized heparin plasma samples. Agreement between assays was assessed by overall, positive, and negative percent agreement and Cohen's kappa coefficient.Overall agreement between the QIAreach anti-SARS-CoV-2 Total and any anti-spike domain (S1 or RBD) antibody isotype was 96.0 % (95 % CI 89.8-98.8), the positive percent agreement was 97.6 % (95 % CI 91.0-99.9), the negative percent agreement was 88.2 % (95 % CI 64.4-98.0). The kappa coefficient was 0.86 (95 % CI 0.72 to 0.99).The QIAreach anti-SARS-CoV-2 Total rapid antibody test provides comparable performance to high-complexity, laboratory-based ELISA.

  View details for DOI 10.1016/j.jcv.2021.104818

  View details for PubMedID 33932848

• Case-Control Study of Individuals with Discrepant Nucleocapsid and Spike Protein SARS-CoV-2 IgG Results. Clinical chemistry Wang, H. n., Wiredja, D. n., Yang, L. n., Bulterys, P. L., Costales, C. n., Röltgen, K. n., Manalac, J. n., Yee, J. n., Zehnder, J. n., Shi, R. Z., Boyd, S. D., Pinsky, B. A. 2021

  Abstract

  Laboratory-based methods for SARS-CoV-2 antibody detection vary widely in performance. However, there are limited prospectively-collected data on assay performance, and minimal clinical information to guide interpretation of discrepant results.Over a two-week period, 1080 consecutive plasma samples submitted for clinical SARS-CoV-2 IgG testing were tested in parallel for anti-nucleocapsid IgG (anti-N, Abbott) and anti-spike IgG (anti-S1, EUROIMMUN). Chart review was conducted for samples testing positive or borderline on either assay, and for an age/sex-matched cohort of samples negative by both assays. CDC surveillance case definitions were used to determine clinical sensitivity/specificity and conduct receiver operating characteristics curve analysis.There were 52 samples positive by both methods, 2 positive for anti-N only, 34 positive for anti-S1 only, and 27 borderline for anti-S1. Of the 34 individuals positive for anti-S1 alone, 8 (24%) had confirmed COVID-19. No anti-S1 borderline cases were positive for anti-N or had confirmed/probable COVID-19. The anti-N assay was less sensitive (84.2% [95% CI 72.1-92.5%] versus 94.7% [95% CI 85.4-98.9%]) but more specific (99.2% [95% CI 95.5-100%] versus 86.9% [95% CI 79.6-92.3%]) than anti-S1. Abbott anti-N sensitivity could be improved to 96.5% with minimal effect on specificity if the index threshold was lowered from 1.4 to 0.6.Real-world concordance between different serologic assays may be lower than previously described in retrospective studies. These findings have implications for the interpretation of SARS-CoV-2 IgG results, especially with the advent of spike antigen-targeted vaccination, as a subset of patients with true infection are anti-N negative and anti-S1 positive.

  View details for DOI 10.1093/clinchem/hvab045

  View details for PubMedID 33720347

• SARS-CoV-2 Seroprevalence in Healthcare Personnel in Northern California Early in the COVID-19 Pandemic. Infection control and hospital epidemiology Rosser, J. I., Roltgen, K., Dymock, M., Shepard, J., Martin, A., Hogan, C. A., Blomkalns, A., Mathew, R., Parsonnet, J., Pinsky, B. A., Maldonado, Y. A., Boyd, S. D., Chang, S., Holubar, M., Stanford Healthcare COVID-19 Workforce Response Group 2020: 1–27

  Abstract

  OBJECTIVE: We aimed to assess the magnitude of unidentified SARS-CoV-2 infections in our healthcare personnel (HCP) early in the COVID-19 pandemic and evaluate risk factors for infection in order to identify areas for infection control practice improvement in a northern California academic medical center.METHODS: We reviewed the anti-SARS-CoV-2 receptor binding domain (RBD) IgG serologic test results and self-reported risk factors for seropositivity among 10,449 asymptomatic HCP who underwent voluntary serology testing between April 20 and May 20, 2020.RESULTS: In total, 136 employees (1.3%) tested positive for SARS-CoV-2 IgG. This included 41 (30.1%) individuals who had previously tested positive for SARS-CoV-2 by nasopharyngeal reverse transcription polymerase chain reaction (RT-PCR) between March 13 and April 16, 2020. In multivariable analysis, employees of Hispanic ethnicity (OR = 2.01; 95% CI = 1.22-3.46) and those working in environmental services/food services/patient transport (OR = 4.81; 95% CI = 2.08-10.30) were at increased risk for seropositivity compared to other groups. Employees reporting a household contact with COVID-19 were also at higher risk for seropositivity (OR = 3.25; 95% CI = 1.47-6.44), but those with a work exposure were not (OR = 1.27; 95% CI = 0.58-2.47). Importantly, one-third of seropositive individuals reported no prior symptoms, no suspected exposures, and no prior positive RT-PCR test.CONCLUSION: In this study, SARS-CoV-2 seropositivity among HCP early in the northern California epidemic appeared to be quite low and was more likely attributable to community rather than occupational exposure.

  View details for DOI 10.1017/ice.2020.1358

  View details for PubMedID 33292895

• Transcriptional changes in peanut-specific CD4+ T cells over the course of oral immunotherapy. Clinical immunology (Orlando, Fla.) Wang, W., Lyu, S., Ji, X., Gupta, S., Manohar, M., Dhondalay, G. K., Chinthrajah, S., Andorf, S., Boyd, S. D., Tibshirani, R., Galli, S. J., Nadeau, K. C., Maecker, H. T. 2020: 108568

  Abstract

  Oral immunotherapy (OIT) can successfully desensitize allergic individuals to offending foods such as peanut. Our recent clinical trial (NCT02103270) of peanut OIT allowed us to monitor peanut-specific CD4+ T cells, using MHC-peptide Dextramers, over the course of OIT. We used a single-cell targeted RNAseq assay to analyze these cells at 0, 12, 24, 52, and 104 weeks of OIT. We found a transient increase in TGFbeta-producing cells at 52 weeks in those with successful desensitization, which lasted until 117 weeks. We also performed clustering and identified 5 major clusters of Dextramer+ cells, which we tracked over time. One of these clusters appeared to be anergic, while another was consistent with recently described TFH13 cells. The other 3 clusters appeared to be Th2 cells by their coordinated production of IL-4 and IL-13, but they varied in their expression of STAT signaling proteins and other markers. A cluster with high expression of STAT family members also showed a possible transient increase at week 24 in those with successful desensitization. Single cell TCRalphabeta repertoire sequences were too diverse to track clones over time. Together with increased TGFbeta production, these changes may be mechanistic predictors of successful OIT that should be further investigated.

  View details for DOI 10.1016/j.clim.2020.108568

  View details for PubMedID 32783912

• Adjuvanted H5N1 influenza vaccine enhances both cross-reactive memory B cell and strain-specific naive B cell responses in humans. Proceedings of the National Academy of Sciences of the United States of America Ellebedy, A. H., Nachbagauer, R., Jackson, K. J., Dai, Y., Han, J., Alsoussi, W. B., Davis, C. W., Stadlbauer, D., Rouphael, N., Chromikova, V., McCausland, M., Chang, C. Y., Cortese, M., Bower, M., Chennareddy, C., Schmitz, A. J., Zarnitsyna, V. I., Lai, L., Rajabhathor, A., Kazemian, C., Antia, R., Mulligan, M. J., Ward, A. B., Fremont, D. H., Boyd, S. D., Pulendran, B., Krammer, F., Ahmed, R. 2020

  Abstract

  There is a need for improved influenza vaccines. In this study we compared the antibody responses in humans after vaccination with an AS03-adjuvanted versus nonadjuvanted H5N1 avian influenza virus inactivated vaccine. Healthy young adults received two doses of either formulation 3 wk apart. We found that AS03 significantly enhanced H5 hemagglutinin (HA)-specific plasmablast and antibody responses compared to the nonadjuvanted vaccine. Plasmablast response after the first immunization was exclusively directed to the conserved HA stem region and came from memory B cells. Monoclonal antibodies (mAbs) derived from these plasmablasts had high levels of somatic hypermutation (SHM) and recognized the HA stem region of multiple influenza virus subtypes. Second immunization induced a plasmablast response to the highly variable HA head region. mAbs derived from these plasmablasts exhibited minimal SHM (naive B cell origin) and largely recognized the HA head region of the immunizing H5N1 strain. Interestingly, the antibody response to H5 HA stem region was much lower after the second immunization, and this suppression was most likely due to blocking of these epitopes by stem-specific antibodies induced by the first immunization. Taken together, these findings show that an adjuvanted influenza vaccine can substantially increase antibody responses in humans by effectively recruiting preexisting memory B cells as well as naive B cells into the response. In addition, we show that high levels of preexisting antibody can have a negative effect on boosting. These findings have implications toward the development of a universal influenza vaccine.

  View details for DOI 10.1073/pnas.1906613117

  View details for PubMedID 32661157

• Persistent detection of SARS-CoV-2 RNA in patients and healthcare workers with COVID-19. Journal of clinical virology : the official publication of the Pan American Society for Clinical Virology Gombar, S., Chang, M., Hogan, C. A., Zehnder, J., Boyd, S., Pinsky, B. A., Shah, N. H. 2020; 129: 104477

  Abstract

  BACKGROUND: Current guidelines for returning health care workers (HCW) to service after a positive SARS-CoV-2 RT-PCR test and ceasing of transmission precautions for patients is based on two general strategies. A test-based strategy that requires negative respiratory RT-PCR tests obtained after the resolution of symptoms. Alternatively, due to the limited availability of testing, many sites employ a symptom-based strategy that recommends excluding HCW from the workforce and keeping patients on contact precautions until a fixed period of time has elapsed from symptom recovery. The underlying assumption of the symptom-based strategy is that waiting for a fixed period of time is a surrogate for negative RT-PCR testing, which itself is a surrogate for the absence of shedding infectious virus.OBJECTIVES: To better understand the appropriate length of symptom based return to work and contact precaution strategies.STUDY DESIGN: We performed an observational analysis of 150 patients and HCW that transitioned from RT-PCR SARS-CoV-2 positive to negative over the course of 2 months at a US academic medical center.RESULTS: We found that the average time to transition from RT-PCR positive to negative was 24 days after symptom onset and 10 % remained positive even 33 days after symptom onset. No difference was seen in HCW and patients.CONCLUSIONS: These findings suggest until definitive evidence of the length of infective viral shedding is obtained that the fixed length of time before returning to work or ceasing contract precautions be revised to over one-month.

  View details for DOI 10.1016/j.jcv.2020.104477

  View details for PubMedID 32505778

• SARS-CoV-2 RNAemia in a Healthy Blood Donor 40 Days After Respiratory Illness Resolution. Annals of internal medicine Pham, T. D., Huang, C. n., Wirz, O. F., Röltgen, K. n., Sahoo, M. K., Layon, A. n., Pandey, S. n., Foung, S. K., Boyd, S. D., Pinsky, B. A. 2020

  View details for DOI 10.7326/L20-0725

  View details for PubMedID 32678685

• Gastrointestinal Eosinophil Responses in a Longitudinal, Randomized Trial of Peanut Oral Immunotherapy. Clinical gastroenterology and hepatology : the official clinical practice journal of the American Gastroenterological Association Wright, B. L., Fernandez-Becker, N. Q., Kambham, N. n., Purington, N. n., Cao, S. n., Tupa, D. n., Zhang, W. n., Sindher, S. B., Rank, M. A., Kita, H. n., Katzka, D. A., Shim, K. P., Bunning, B. J., Doyle, A. D., Jacobsen, E. A., Tsai, M. n., Boyd, S. D., Manohar, M. n., Chinthrajah, R. S. 2020

  Abstract

  Gastrointestinal side effects are common during oral immunotherapy (OIT) and eosinophilic esophagitis (EoE) is a potential complication. We aimed to characterize eosinophilic gastrointestinal responses to peanut OIT, in which peanut protein is given orally, with incremental increases in dose over time.Twenty adults with IgE-mediated peanut allergy were randomly assigned to groups given peanut OIT (n=15) or placebo (n=5); 1 additional subject withdrew before randomization. Serial gastrointestinal biopsies were collected at baseline (n=21, 0 weeks), following dose escalation (n=10, 52 weeks), and during the maintenance phase (n=11, 104 weeks). Endoscopic findings were characterized using the EoE endoscopic reference score. Biopsies were assessed for eosinophils per high-power field (eos/hpf) and other pathology features using EoE histologic scoring system scores. We performed immunohistochemical analyses of eosinophil peroxidase deposition, quantified using automated image analysis.At baseline, no subjects reported current gastrointestinal symptoms. However, 3 of the 21 subjects (14%) had esophageal peak eosinophil counts ≥15 eos/hpf and all subjects had dilated intercellular spaces (DIS). OIT induced or exacerbated esophageal eosinophilia (EE) at 52 weeks in most subjects (peak eosinophil counts >5 eos/hpf in 6 of 7 patients [86%]; peak eosinophil counts ≥15 eos/hpf in 4 of 7 patients [57%]). One subject met clinicopathologic criteria for EoE and withdrew; no significant changes in esophageal peak eosinophil counts were observed in the placebo group. EE in the OIT group corresponded with significant increases in EoE histologic scoring system scores and deposition of eosinophil peroxidase. In 4 of 6 participants (67%), OIT-induced EE and gastrointestinal eosinophilia resolved by the end of the maintenance phase. Gastrointestinal symptoms were not clearly associated with EE or gastrointestinal eosinophilia.In this pilot study, we found that peanut OIT-induced EE and gastrointestinal eosinophilia are usually transient and are not always associated with gastrointestinal symptoms. Clinicaltrials.gov no: NCT02103270.

  View details for DOI 10.1016/j.cgh.2020.05.019

  View details for PubMedID 32434067

• Large-Scale Testing of Asymptomatic Healthcare Personnel for Severe Acute Respiratory Syndrome Coronavirus 2. Emerging infectious diseases Hogan, C. A., Gombar, S. n., Wang, H. n., Röltgen, K. n., Shi, R. Z., Holubar, M. n., Chang, S. I., Lee, G. M., Boyd, S. D., Zehnder, J. n., Pinsky, B. A. 2020; 27 (1)

  Abstract

  Large-scale, 1-time testing of >12,000 asymptomatic healthcare personnel in California, USA, during April-June 2020 showed that prevalence of severe acute respiratory syndrome coronavirus 2 was low (<1%). Testing might identify asymptomatic and presymptomatic persons, including some with high viral burden, enabling prompt implementation of measures to limit nosocomial spread.

  View details for DOI 10.3201/eid2701.203892

  View details for PubMedID 33256889

• Histology-Independent Signature Distinguishes Kikuchi-Fujimoto Disease/Systemic Lupus Erythematosus-Associated Lymphadenitis From Benign and Malignant Lymphadenopathies. American journal of clinical pathology Scott, G. D., Kumar, J. n., Oak, J. S., Boyd, S. D., Raess, P. W., Gratzinger, D. A. 2020

  Abstract

  Kikuchi-Fujimoto disease (KFD) and systemic lupus erythematosus (SLE) are benign entities with histologic features that raise concern about malignancy and infection. We searched for a histology-independent KFD/SLE signature relying on only immunophenotype and basic clinical characteristics.A histology-independent KFD/SLE signature was generated using 975 excised lymph nodes with flow immunophenotyping, including 16 cases of KFD/SLE. This signature was then evaluated in 1,198 fine-needle aspiration (FNA) specimens.The top flow cytometry discriminant for KFD/SLE was uniform CD38+ expression on CD19+ events. Immunohistochemistry demonstrated nodules of IgD+, IgM- B cells surrounding necrotizing and activated T-cell areas. A signature combining 6 flow cytometry criteria with age and sample site had a positive predictive value of 88% for KFD/SLE, which had a prevalence of 1.6%. All 4 signature-positive FNA cases with follow-up excision were KFD/SLE. At a second institution, 4 of 5 KFD/SLE cases passed the top discriminant.A flow cytometry signature combined with age and biopsy site identifies KFD/SLE independent of histology, suggesting a shared immune composition and independently confirming that KFD/SLE represents a distinct entity. Unexpectedly, an IgD+CD38+ small B-cell population is a distinctive feature of KFD/SLE, suggesting a possible pathologic role for anergic/autoreactive B cells.

  View details for DOI 10.1093/ajcp/aqaa036

  View details for PubMedID 32367142

• Oral Immunotherapy and Basophil and Mast Cell Reactivity in Food Allergy. Frontiers in immunology Paranjape, A. n., Tsai, M. n., Mukai, K. n., Hoh, R. A., Joshi, S. A., Chinthrajah, R. S., Nadeau, K. C., Boyd, S. D., Galli, S. J. 2020; 11: 602660

  Abstract

  Basophil activation tests (BATs) can closely monitor, in vitro, a patient's propensity to develop type I hypersensitivity reactions. Because of their high specificity and sensitivity, BATs have become promising diagnostic tools, especially in cases with equivocal clinical histories, skin prick test results, and/or levels of specific IgE to allergen extracts. BATs also are useful as tools for monitoring the effects of treatment, since oral immunotherapy (OIT) studies report a diminution in patients' basophil responsiveness over the course of OIT. This review will discuss the BAT findings obtained before, during, and after OIT for food allergy. We will mainly focus on the association of basophil responsiveness, and alterations in basophil surface markers, with clinical outcomes and other clinical features, such as blood levels of specific IgG and IgE antibodies. The detailed analysis of these correlations will ultimately facilitate the use of BATs, along with other blood biomarkers, to differentiate short-term desensitization versus sustained unresponsiveness and to improve treatment protocols. Given the critical anatomic location of mast cells adjacent to the many IgE+ plasma cells found in the gastrointestinal tissues of allergic individuals, we will also discuss the role of gastrointestinal mast cells in manifestations of food allergies.

  View details for DOI 10.3389/fimmu.2020.602660

  View details for PubMedID 33381123

  View details for PubMedCentralID PMC7768812

• Biology and dynamics of B cells in the context of IgE-mediated food allergy. Allergy Satitsuksanoa, P. n., Daanje, M. n., Akdis, M. n., Boyd, S. D., van de Veen, W. n. 2020

  Abstract

  An increasing number of people suffer from IgE-mediated food allergies. The immunological mechanisms that cause IgE-mediated food allergy have been extensively studied. B cells play a key role in the development of IgE-mediated food allergies through the production of allergen-specific antibodies. While this particular function of B cells has been known for many years, we still do not fully understand the mechanisms that regulate the induction and maintenance of allergen-specific IgE production. It is still not fully understood where in the body IgE class switch recombination of food allergen-specific B cells occurs, and what processes are involved in the immunological memory of allergen-specific IgE responses. B cells can also contribute to the regulation of allergen-specific immune responses through other mechanisms such as antigen presentation and cytokine production. Recent technological advances have enabled highly detailed analysis of small subsets of B cells down to the single cell level. In this review, we provide an overview of the current knowledge on the biology of B cells in relation to IgE-mediated food allergies.

  View details for DOI 10.1111/all.14684

  View details for PubMedID 33274454

• Recent progress in the analysis of alphabetaT cell and B cell receptor repertoires. Current opinion in immunology Davis, M. M., Boyd, S. D. 2019; 59: 109–14

  Abstract

  T cell receptors (TCRs) and B cell receptors (BCRs) are vertebrate evolution's best answer to the threat of microbial pathogens that can evolve much faster than ourselves. These antigen receptors are generated during T cell or B cell development by combinatorial rearrangement of germline genome V, D and J gene segments, and with junctional residues capable of enormous diversity. For decades the complexity of these receptor repertoires has limited their analysis, but advances in DNA sequencing technology and an array of complementary tools have now made their study much more tractable, filling a major gap in our ability to understand immunology as a system. Here, we summarize the recent approaches and discoveries that are enabling these advances, with some suggestions as to what may lie ahead.

  View details for DOI 10.1016/j.coi.2019.05.012

  View details for PubMedID 31326777

• New technologies and applications in infant B cell immunology CURRENT OPINION IN IMMUNOLOGY Nielsen, S., Boyd, S. 2019; 57: 53–57

  View details for DOI 10.1016/j.coi.2018.12.005

  View details for Web of Science ID 000469153400009

• CLONALITY: POINT ESTIMATION ANNALS OF APPLIED STATISTICS Tian, L., Liu, Y., Fire, A. Z., Boyd, S. D., Olshen, R. A. 2019; 13 (1): 113–31

  View details for DOI 10.1214/18-AOAS1197

  View details for Web of Science ID 000464000700005

• New technologies and applications in infant B cell immunology. Current opinion in immunology Nielsen, S. C., Boyd, S. D. 2019; 57: 53–57

  Abstract

  The human immune system changes dramatically with age, and early life exposures to pathogens and environmental antigens begin the formation of immune memory which influences subsequent responses later in life. To study infant immunity, sample-sparing experimental methods that extract maximal data from small samples of blood or other tissues are needed; fortunately, recent developments in high-throughput sequencing and multiplexed labeling and measurement of markers on cells are well-suited to these tasks. Here, we review some recent studies of infant immune responses to infectious disease, highlighting similarities and differences between infants and adults, and identifying important questions for future research. Recent clinical trials in food allergy have revealed the critical role of immunological events in the first year of life that determine an individual's risk of developing peanut allergy; these also warrant thorough evaluation using the new immune monitoring tools.

  View details for PubMedID 30825678

• VDJbase: an adaptive immune receptor genotype and haplotype database. Nucleic acids research Omer, A. n., Shemesh, O. n., Peres, A. n., Polak, P. n., Shepherd, A. J., Watson, C. T., Boyd, S. D., Collins, A. M., Lees, W. n., Yaari, G. n. 2019

  Abstract

  VDJbase is a publicly available database that offers easy searching of data describing the complete sets of gene sequences (genotypes and haplotypes) inferred from adaptive immune receptor repertoire sequencing datasets. VDJbase is designed to act as a resource that will allow the scientific community to explore the genetic variability of the immunoglobulin (Ig) and T cell receptor (TR) gene loci. It can also assist in the investigation of Ig- and TR-related genetic predispositions to diseases. Our database includes web-based query and online tools to assist in visualization and analysis of the genotype and haplotype data. It enables users to detect those alleles and genes that are significantly over-represented in a particular population, in terms of genotype, haplotype and gene expression. The database website can be freely accessed at https://www.vdjbase.org/, and no login is required. The data and code use creative common licenses and are freely downloadable from https://bitbucket.org/account/user/yaarilab/projects/GPHP.

  View details for DOI 10.1093/nar/gkz872

  View details for PubMedID 31602484

• Sustained outcomes in oral immunotherapy for peanut allergy (POISED study): a large, randomised, double-blind, placebo-controlled, phase 2 study. Lancet (London, England) Chinthrajah, R. S., Purington, N. n., Andorf, S. n., Long, A. n., O'Laughlin, K. L., Lyu, S. C., Manohar, M. n., Boyd, S. D., Tibshirani, R. n., Maecker, H. n., Plaut, M. n., Mukai, K. n., Tsai, M. n., Desai, M. n., Galli, S. J., Nadeau, K. C. 2019

  Abstract

  Dietary avoidance is recommended for peanut allergies. We evaluated the sustained effects of peanut allergy oral immunotherapy (OIT) in a randomised long-term study in adults and children.In this randomised, double-blind, placebo-controlled, phase 2 study, we enrolled participants at the Sean N Parker Center for Allergy and Asthma Research at Stanford University (Stanford, CA, USA) with peanut allergy aged 7-55 years with a positive result from a double-blind, placebo-controlled, food challenge (DBPCFC; ≤500 mg of peanut protein), a positive skin-prick test (SPT) result (≥5 mm wheal diameter above the negative control), and peanut-specific immunoglobulin (Ig)E concentration of more than 4 kU/L. Participants were randomly assigned (2·4:1·4:1) in a two-by-two block design via a computerised system to be built up and maintained on 4000 mg peanut protein through to week 104 then discontinued on peanut (peanut-0 group), to be built up and maintained on 4000 mg peanut protein through to week 104 then to ingest 300 mg peanut protein daily (peanut-300 group) for 52 weeks, or to receive oat flour (placebo group). DBPCFCs to 4000 mg peanut protein were done at baseline and weeks 104, 117, 130, 143, and 156. The pharmacist assigned treatment on the basis of a randomised computer list. Peanut or placebo (oat) flour was administered orally and participants and the study team were masked throughout by use of oat flour that was similar in look and feel to the peanut flour and nose clips, as tolerated, to mask taste. The statistician was also masked. The primary endpoint was the proportion of participants who passed DBPCFCs to a cumulative dose of 4000 mg at both 104 and 117 weeks. The primary efficacy analysis was done in the intention-to-treat population. Safety was assessed in the intention-to-treat population. This trial is registered at ClinicalTrials.gov, NCT02103270.Between April 15, 2014, and March 2, 2016, of 152 individuals assessed, we enrolled 120 participants, who were randomly assigned to the peanut-0 (n=60), peanut-300 (n=35), and placebo groups (n=25). 21 (35%) of peanut-0 group participants and one (4%) placebo group participant passed the 4000 mg challenge at both 104 and 117 weeks (odds ratio [OR] 12·7, 95% CI 1·8-554·8; p=0·0024). Over the entire study, the most common adverse events were mild gastrointestinal symptoms, which were seen in 90 of 120 patients (50/60 in the peanut-0 group, 29/35 in the peanut-300 group, and 11/25 in the placebo group) and skin disorders, which were seen in 50/120 patients (26/60 in the peanut-0 group, 15/35 in the peanut-300 group, and 9/25 in the placebo group). Adverse events decreased over time in all groups. Two participants in the peanut groups had serious adverse events during the 3-year study. In the peanut-0 group, in which eight (13%) of 60 participants passed DBPCFCs at week 156, higher baseline peanut-specific IgG4 to IgE ratio and lower Ara h 2 IgE and basophil activation responses were associated with sustained unresponsiveness. No treatment-related deaths occurred.Our study suggests that peanut OIT could desensitise individuals with peanut allergy to 4000 mg peanut protein but discontinuation, or even reduction to 300 mg daily, could increase the likelihood of regaining clinical reactivity to peanut. Since baseline blood tests correlated with week 117 treatment outcomes, this study might aid in optimal patient selection for this therapy.National Institute of Allergy and Infectious Diseases.

  View details for DOI 10.1016/S0140-6736(19)31793-3

  View details for PubMedID 31522849

• Baseline Gastrointestinal Eosinophilia Is Common in Oral Immunotherapy Subjects With IgE-Mediated Peanut Allergy FRONTIERS IN IMMUNOLOGY Wright, B. L., Fernandez-Becker, N. Q., Kambham, N., Purington, N., Tupa, D., Zhang, W., Rank, M. A., Kita, H., Shim, K. P., Bunning, B. J., Doyle, A. D., Jacobsen, E. A., Boyd, S. D., Tsai, M., Maecker, H., Manohar, M., Galli, S. J., Nadeau, K. C., Chinthrajah, R. 2018; 9

  View details for DOI 10.3389/fimmu.2018.02624

  View details for Web of Science ID 000450901300001

• Baseline Gastrointestinal Eosinophilia Is Common in Oral Immunotherapy Subjects With IgE-Mediated Peanut Allergy. Frontiers in immunology Wright, B. L., Fernandez-Becker, N. Q., Kambham, N., Purington, N., Tupa, D., Zhang, W., Rank, M. A., Kita, H., Shim, K. P., Bunning, B. J., Doyle, A. D., Jacobsen, E. A., Boyd, S. D., Tsai, M., Maecker, H., Manohar, M., Galli, S. J., Nadeau, K. C., Chinthrajah, R. S. 2018; 9: 2624

  Abstract

  Rationale: Oral immunotherapy (OIT) is an emerging treatment for food allergy. While desensitization is achieved in most subjects, many experience gastrointestinal symptoms and few develop eosinophilic gastrointestinal disease. It is unclear whether these subjects have subclinical gastrointestinal eosinophilia (GE) at baseline. We aimed to evaluate the presence of GE in subjects with food allergy before peanut OIT. Methods: We performed baseline esophagogastroduodenoscopies on 21 adults before undergoing peanut OIT. Subjects completed a detailed gastrointestinal symptom questionnaire. Endoscopic findings were assessed using the Eosinophilic Esophagitis (EoE) Endoscopic Reference Score (EREFS) and biopsies were obtained from the esophagus, gastric antrum, and duodenum. Esophageal biopsies were evaluated using the EoE Histologic Scoring System. Immunohistochemical staining for eosinophil peroxidase (EPX) was also performed. Hematoxylin and eosin and EPX stains of each biopsy were assessed for eosinophil density and EPX/mm2 was quantified using automated image analysis. Results: All subjects were asymptomatic. Pre-existing esophageal eosinophilia (>5 eosinophils per high-power field [eos/hpf]) was present in five participants (24%), three (14%) of whom had >15 eos/hpf associated with mild endoscopic findings (edema, linear furrowing, or rings; median EREFS = 0, IQR 0-0.25). Some subjects also demonstrated basal cell hyperplasia, dilated intercellular spaces, and lamina propria fibrosis. Increased eosinophils were noted in the gastric antrum (>12 eos/hpf) or duodenum (>26 eos/hpf) in 9 subjects (43%). EPX/mm2 correlated strongly with eosinophil counts (r = 0.71, p < 0.0001). Conclusions: Pre-existing GE is common in adults with IgE-mediated peanut allergy. Eosinophilic inflammation (EI) in these subjects may be accompanied by mild endoscopic and histologic findings. Longitudinal data collection during OIT is ongoing.

  View details for DOI 10.3389/fimmu.2018.02624

  View details for PubMedID 30524424

  View details for PubMedCentralID PMC6261984

• Global fingerprint of humans on the distribution of Bartonella bacteria in mammals. PLoS neglected tropical diseases Frank, H. K., Boyd, S. D., Hadly, E. A. 2018; 12 (11): e0006865

  Abstract

  As humans move and alter habitats, they change the disease risk for themselves, their commensal animals and wildlife. Bartonella bacteria are prevalent in mammals and cause numerous human infections. Understanding how this genus has evolved and switched hosts in the past can reveal how current patterns were established and identify potential mechanisms for future cross-species transmission. We analyzed patterns of Bartonella transmission and likely sources of spillover using the largest collection of Bartonella gltA genotypes assembled, including 67 new genotypes. This pathogenic genus likely originated as an environmental bacterium and insect commensal before infecting mammals. Rodents and domestic animals serve as the reservoirs or at least key proximate host for most Bartonella genotypes in humans. We also find evidence of exchange of Bartonella between phylogenetically distant domestic animals and wildlife, likely due to increased contact. Care should be taken to avoid contact between humans, domestic animals and wildlife to protect the health of all.

  View details for PubMedID 30439961

• Global fingerprint of humans on the distribution of Bartonella bacteria in mammals PLOS NEGLECTED TROPICAL DISEASES Frank, H. K., Boyd, S. D., Hadly, E. A. 2018; 12 (11)

  View details for DOI 10.1371/journal.pntd.0006865

  View details for Web of Science ID 000452162500019

• Gut Mucosal Antibody Responses and Implications for Food Allergy. Frontiers in immunology Hoh, R. A., Boyd, S. D. 2018; 9: 2221

  Abstract

  The gastrointestinal mucosa is a critical environmental interface where plasma cells and B cells are exposed to orally-ingested antigens such as food allergen proteins. It is unclear how the development of B cells and plasma cells in the gastrointestinal mucosa differs between healthy humans and those with food allergy, and how B cells contribute to, or are affected by, the breakdown of oral tolerance. In particular, the antibody gene repertoires associated with symptomatic allergy have only begun to be characterized in full molecular detail. Here, we review literature concerning B cells and plasma cells in the gastrointestinal system in the context of food allergy, with a focus on human studies.

  View details for DOI 10.3389/fimmu.2018.02221

  View details for PubMedID 30319658

  View details for PubMedCentralID PMC6170638

• Gut Mucosal Antibody Responses and Implications for Food Allergy FRONTIERS IN IMMUNOLOGY Hoh, R. A., Boyd, S. D. 2018; 9

  View details for DOI 10.3389/fimmu.2018.02221

  View details for Web of Science ID 000445781800001

• Human adaptive immune receptor repertoire analysis-Past, present, and future. Immunological reviews Nielsen, S. C., Boyd, S. D. 2018; 284 (1): 9–23

  Abstract

  The genes encoding adaptive immune antigen receptors, namely the immunoglobulins expressed in membrane-bound or secreted forms by B cells, and the cell surface T cell receptors, are unique in human biology because they are generated by combinatorial rearrangement of the genomic DNA. The diversity of receptors so generated in populations of lymphocytes enables the human immune system to recognize antigens expressed by pathogens, but also underlies the pathological specificity of autoimmune diseases and the mistargeted immunity in allergies. Several recent technological developments, foremost among them the invention of high-throughput DNA sequencing instruments, have enabled much deeper and thorough evaluation of clones of human B cells and T cells and the antigen receptors they express during physiological and pathogenic immune responses. The evolutionary struggles between host adaptive immune responses and populations of pathogens are now open to greater scrutiny, elucidation of the underlying reasons for successful or failed immunity, and potential predictive modeling, than ever before. Here we give an overview of the foundations, recent progress, and future prospects in this dynamic area of research.

  View details for PubMedID 29944765

• Human adaptive immune receptor repertoire analysis-Past, present, and future IMMUNOLOGICAL REVIEWS Nielsen, S. A., Boyd, S. D. 2018; 284 (1): 9-23

  View details for DOI 10.1111/imr.12667

  View details for Web of Science ID 000436250700002

• Dynamics of Viral and Host Immune Cell MicroRNA Expression during Acute Infectious Mononucleosis FRONTIERS IN MICROBIOLOGY Kaul, V., Weinberg, K. I., Boyd, S. D., Bernstein, D., Esquivel, C. O., Martinez, O. M., Krams, S. M. 2018; 8

  View details for DOI 10.3389/fmicb.2017.02666

  View details for Web of Science ID 000419879800001

• Food allergy and omics. The Journal of allergy and clinical immunology Dhondalay, G. K., Rael, E. n., Acharya, S. n., Zhang, W. n., Sampath, V. n., Galli, S. J., Tibshirani, R. n., Boyd, S. D., Maecker, H. n., Nadeau, K. C., Andorf, S. n. 2018; 141 (1): 20–29

  Abstract

  Food allergy (FA) prevalence has been increasing over the last few decades and is now a global health concern. Current diagnostic methods for FA result in a high number of false-positive results, and the standard of care is either allergen avoidance or use of epinephrine on accidental exposure, although currently with no other approved treatments. The increasing prevalence of FA, lack of robust biomarkers, and inadequate treatments warrants further research into the mechanism underlying food allergies. Recent technological advances have made it possible to move beyond traditional biological techniques to more sophisticated high-throughput approaches. These technologies have created the burgeoning field of omics sciences, which permit a more systematic investigation of biological problems. Omics sciences, such as genomics, epigenomics, transcriptomics, proteomics, metabolomics, microbiomics, and exposomics, have enabled the construction of regulatory networks and biological pathway models. Parallel advances in bioinformatics and computational techniques have enabled the integration, analysis, and interpretation of these exponentially growing data sets and opens the possibility of personalized or precision medicine for FA.

  View details for PubMedID 29307411

• Dynamics of Viral and Host Immune Cell MicroRNA Expression during Acute Infectious Mononucleosis. Frontiers in microbiology Kaul, V. n., Weinberg, K. I., Boyd, S. D., Bernstein, D. n., Esquivel, C. O., Martinez, O. M., Krams, S. M. 2017; 8: 2666

  Abstract

  Epstein-Barr virus (EBV) is the etiological agent of acute infectious mononucleosis (IM). Since acute IM is a self-resolving disease with most patients regaining health in 1-3 weeks there have been few studies examining molecular signatures in early acute stages of the disease. MicroRNAs (miRNAs) have been shown, however, to influence immune cell function and consequently the generation of antibody responses in IM. In this study, we performed a comprehensive analysis of differentially expressed miRNAs in early stage uncomplicated acute IM. miRNAs were profiled from patient peripheral blood obtained at the time of IM diagnosis and at subsequent time points, and pathway analysis performed to identify important immune and cell signaling pathways. We identified 215 differentially regulated miRNAs at the most acute stage of infection when the patients initially sought medical help. The number of differentially expressed miRNAs decreased to 148 and 68 at 1 and 2 months post-primary infection, with no significantly changed miRNAs identified at 7 months post-infection. Interferon signaling, T and B cell signaling and antigen presentation were the top pathways influenced by the miRNAs associated with IM. Thus, a dynamic and regulated expression profile of miRNA accompanies the early acute immune response, and resolution of infection, in IM.

  View details for PubMedID 29379474

  View details for PubMedCentralID PMC5775229

• Immune monitoring for precision medicine in allergy and asthma. Current opinion in immunology Boyd, S. D., Hoh, R. A., Nadeau, K. C., Galli, S. J. 2017; 48: 82–91

  Abstract

  'Precision Medicine' embodies the analyses of extensive data collected from patients and their environments to identify and apply patient-specific prophylactic strategies and medical treatments to improve clinical outcomes and healthcare cost-effectiveness. Many new methods have been developed for evaluating the activity of the human immune system. Such 'immune monitoring' approaches are now being used in studies of allergy and asthma in the hope of identifying better correlates of disease status, predictors of therapeutic outcomes, and potential side-effects of treatment. Together with analyses of family histories, genetic and other biometric data, and measurements of exposures to environmental and other risk factors for developing or exacerbating disease, immune monitoring approaches promise to enable 'Precision Medicine' for allergic diseases and asthma.

  View details for DOI 10.1016/j.coi.2017.08.007

  View details for PubMedID 28889067

  View details for PubMedCentralID PMC5743231

• Amino Acid Changes in the HIV-1 gp41 Membrane Proximal Region Control Virus Neutralization Sensitivity. EBioMedicine Bradley, T., Trama, A., Tumba, N., Gray, E., Lu, X., Madani, N., Jahanbakhsh, F., Eaton, A., Xia, S. M., Parks, R., Lloyd, K. E., Sutherland, L. L., Scearce, R. M., Bowman, C. M., Barnett, S., Abdool-Karim, S. S., Boyd, S. D., Melillo, B., Smith, A. B., Sodroski, J., Kepler, T. B., Alam, S. M., Gao, F., Bonsignori, M., Liao, H. X., Moody, M. A., Montefiori, D., Santra, S., Morris, L., Haynes, B. F. 2016

  Abstract

  Most HIV-1 vaccines elicit neutralizing antibodies that are active against highly sensitive (tier-1) viruses or rare cases of vaccine-matched neutralization-resistant (tier-2) viruses, but no vaccine has induced antibodies that can broadly neutralize heterologous tier-2 viruses. In this study, we isolated antibodies from an HIV-1-infected individual that targeted the gp41 membrane-proximal external region (MPER) that may have selected single-residue changes in viral variants in the MPER that resulted in neutralization sensitivity to antibodies targeting distal epitopes on the HIV-1 Env. Similarly, a single change in the MPER in a second virus from another infected-individual also conferred enhanced neutralization sensitivity. These gp41 single-residue changes thus transformed tier-2 viruses into tier-1 viruses that were sensitive to vaccine-elicited tier-1 neutralizing antibodies. These data demonstrate that Env amino acid changes within the MPER bnAb epitope of naturally-selected escape viruses can increase neutralization sensitivity to multiple types of neutralizing antibodies, and underscore the critical importance of the MPER for maintaining the integrity of the tier-2 HIV-1 trimer.

  View details for DOI 10.1016/j.ebiom.2016.08.045

  View details for PubMedID 27612593

• Deep sequencing and human antibody repertoire analysis CURRENT OPINION IN IMMUNOLOGY Boyd, S. D., Crowe, J. E. 2016; 40: 103-109

  Abstract

  In the past decade, high-throughput DNA sequencing (HTS) methods and improved approaches for isolating antigen-specific B cells and their antibody genes have been applied in many areas of human immunology. This work has greatly increased our understanding of human antibody repertoires and the specific clones responsible for protective immunity or immune-mediated pathogenesis. Although the principles underlying selection of individual B cell clones in the intact immune system are still under investigation, the combination of more powerful genetic tracking of antibody lineage development and functional testing of the encoded proteins promises to transform therapeutic antibody discovery and optimization. Here, we highlight recent advances in this fast-moving field.

  View details for DOI 10.1016/j.coi.2016.03.008

  View details for Web of Science ID 000377836700015

  View details for PubMedID 27065089

• Persistence and evolution of allergen-specific IgE repertoires during subcutaneous specific immunotherapy JOURNAL OF ALLERGY AND CLINICAL IMMUNOLOGY Levin, M., King, J. J., Glanville, J., Jackson, K. J., Looney, T. J., Hoh, R. A., Mari, A., Andersson, M., Greiff, L., Fire, A. Z., Boyd, S. D., Ohlin, M. 2016; 137 (5): 1535-1544

  Abstract

  Specific immunotherapy (SIT) is the only treatment with proved long-term curative potential in patients with allergic disease. Allergen-specific IgE is the causative agent of allergic disease, and antibodies contribute to SIT, but the effects of SIT on aeroallergen-specific B-cell repertoires are not well understood.We sought to characterize the IgE sequences expressed by allergen-specific B cells and track the fate of these B-cell clones during SIT.We used high-throughput antibody gene sequencing and identification of allergen-specific IgE with combinatorial antibody fragment library technology to analyze immunoglobulin repertoires of blood and the nasal mucosa from aeroallergen-sensitized subjects before and during the first year of subcutaneous SIT.Of 52 distinct allergen-specific IgE heavy chains from 8 allergic donors, 37 were also detected by using high-throughput antibody gene sequencing of blood samples, nasal mucosal samples, or both. The allergen-specific clones had increased persistence, higher likelihood of belonging to clones expressing other switched isotypes, and possibly larger clone size than the rest of the IgE repertoire. Clone members in nasal tissue showed close mutational relationships.In the future, combining functional binding studies, deep antibody repertoire sequencing, and information on clinical outcomes in larger studies might aid assessment of SIT mechanisms and efficacy.

  View details for DOI 10.1016/j.jaci.2015.09.027

  View details for Web of Science ID 000376180200030

  View details for PubMedID 26559321

  View details for PubMedCentralID PMC5010428

• Molecular and cellular mechanisms of food allergy and food tolerance. journal of allergy and clinical immunology Chinthrajah, R. S., Hernandez, J. D., Boyd, S. D., Galli, S. J., Nadeau, K. C. 2016; 137 (4): 984-997

  Abstract

  Ingestion of innocuous antigens, including food proteins, normally results in local and systemic immune nonresponsiveness in a process termed oral tolerance. Oral tolerance to food proteins is likely to be intimately linked to mechanisms that are responsible for gastrointestinal tolerance to large numbers of commensal microbes. Here we review our current understanding of the immune mechanisms responsible for oral tolerance and how perturbations in these mechanisms might promote the loss of oral tolerance and development of food allergies. Roles for the commensal microbiome in promoting oral tolerance and the association of intestinal dysbiosis with food allergy are discussed. Growing evidence supports cutaneous sensitization to food antigens as one possible mechanism leading to the failure to develop or loss of oral tolerance. A goal of immunotherapy for food allergies is to induce sustained desensitization or even true long-term oral tolerance to food allergens through mechanisms that might in part overlap with those associated with the development of natural oral tolerance.

  View details for DOI 10.1016/j.jaci.2016.02.004

  View details for PubMedID 27059726

• Successful immunotherapy induces previously unidentified allergen-specific CD4+ T-cell subsets. Proceedings of the National Academy of Sciences of the United States of America Ryan, J. F., Hovde, R., Glanville, J., Lyu, S., Ji, X., Gupta, S., Tibshirani, R. J., Jay, D. C., Boyd, S. D., Chinthrajah, R. S., Davis, M. M., Galli, S. J., Maecker, H. T., Nadeau, K. C. 2016; 113 (9): E1286-95

  Abstract

  Allergen immunotherapy can desensitize even subjects with potentially lethal allergies, but the changes induced in T cells that underpin successful immunotherapy remain poorly understood. In a cohort of peanut-allergic participants, we used allergen-specific T-cell sorting and single-cell gene expression to trace the transcriptional "roadmap" of individual CD4+ T cells throughout immunotherapy. We found that successful immunotherapy induces allergen-specific CD4+ T cells to expand and shift toward an "anergic" Th2 T-cell phenotype largely absent in both pretreatment participants and healthy controls. These findings show that sustained success, even after immunotherapy is withdrawn, is associated with the induction, expansion, and maintenance of immunotherapy-specific memory and naive T-cell phenotypes as early as 3 mo into immunotherapy. These results suggest an approach for immune monitoring participants undergoing immunotherapy to predict the success of future treatment and could have implications for immunotherapy targets in other diseases like cancer, autoimmune disease, and transplantation.

  View details for DOI 10.1073/pnas.1520180113

  View details for PubMedID 26811452

• DJ Pairing during VDJ Recombination Shows Positional Biases That Vary among Individuals with Differing IGHD Locus Immunogenotypes. Journal of immunology Kidd, M. J., Jackson, K. J., Boyd, S. D., Collins, A. M. 2016; 196 (3): 1158-1164

  Abstract

  Human IgH diversity is influenced by biases in the pairing of IGHD and IGHJ genes, but these biases have not been described in detail. We used high-throughput sequencing of VDJ rearrangements to explore DJ pairing biases in 29 individuals. It was possible to infer three contrasting IGHD-IGHJ haplotypes in nine of these individuals, and two of these haplotypes include deletion polymorphisms involving multiple contiguous IGHD genes. Therefore, we were able to explore how the underlying genetic makeup of the H chain locus influences the formation of particular DJ pairs. Analysis of nonproductive rearrangements demonstrates that 3' IGHD genes tend to pair preferentially with 5' IGHJ genes, whereas 5' IGHD genes pair preferentially with 3' IGHJ genes; the relationship between IGHD gene pairing frequencies and IGHD gene position is a near linear one for each IGHJ gene. However, striking differences are seen in individuals who carry deletion polymorphisms in the D locus. The absence of different blocks of IGHD genes leads to increases in the utilization frequencies of just a handful of genes, and these genes have no clear positional relationships to the deleted genes. This suggests that pairing frequencies may be influenced by additional complex positional relationships that perhaps arise from chromatin structure. In contrast to IGHD gene usage, IGHJ gene usage is unaffected by the IGHD gene-deletion polymorphisms. Such an outcome would be expected if the recombinase complex associates with an IGHJ gene before associating with an IGHD gene partner.

  View details for DOI 10.4049/jimmunol.1501401

  View details for PubMedID 26700767

• Human B-cell isotype switching origins of IgE JOURNAL OF ALLERGY AND CLINICAL IMMUNOLOGY Looney, T. J., Lee, J., Roskin, K. M., Hoh, R. A., King, J., Glanville, J., Liu, Y., Pham, T. D., Dekker, C. L., Davis, M. M., Boyd, S. D. 2016; 137 (2): 579-?

  Abstract

  B cells expressing IgE contribute to immunity against parasites and venoms and are the source of antigen specificity in allergic patients, yet the developmental pathways producing these B cells in human subjects remain a subject of debate. Much of our knowledge of IgE lineage development derives from model studies in mice rather than from human subjects.We evaluate models for isotype switching to IgE in human subjects using immunoglobulin heavy chain (IGH) mutational lineage data.We analyzed IGH repertoires in 9 allergic and 24 healthy adults using high-throughput DNA sequencing of 15,843,270 IGH rearrangements to identify clonal lineages of B cells containing members expressing IgE. Somatic mutations in IGH inherited from common ancestors within the clonal lineage are used to infer the relationships between B cells.Data from 613,641 multi-isotype B-cell clonal lineages, of which 592 include an IgE member, are consistent with indirect switching to IgE from IgG- or IgA-expressing lineage members in human subjects. We also find that these inferred isotype switching frequencies are similar in healthy and allergic subjects.We found evidence that secondary isotype switching of mutated IgG1-expressing B cells is the primary source of IgE in human subjects, with lesser contributions from precursors expressing other switched isotypes and rarely IgM or IgD, suggesting that IgE is derived from previously antigen-experienced B cells rather than naive B cells that typically express low-affinity unmutated antibodies. These data provide a basis from which to evaluate allergen-specific human antibody repertoires in healthy and diseased subjects.

  View details for DOI 10.1016/j.jaci.2015.07.014

  View details for Web of Science ID 000369235500028

• Design of a Genomics Curriculum: Competencies for Practicing Pathologists. Archives of pathology & laboratory medicine Laudadio, J., McNeal, J. L., Boyd, S. D., Le, L. P., Lockwood, C., McCloskey, C. B., Sharma, G., Voelkerding, K. V., Haspel, R. L. 2015; 139 (7): 894-900

  Abstract

  Context .- The field of genomics is rapidly impacting medical care across specialties. To help guide test utilization and interpretation, pathologists must be knowledgeable about genomic techniques and their clinical utility. The technology allowing timely generation of genomic data is relatively new to patient care and the clinical laboratory, and therefore, many currently practicing pathologists have been trained without any molecular or genomics exposure. Furthermore, the exposure that current and recent trainees receive in this field remains inconsistent. Objective .- To assess pathologists' learning needs in genomics and to develop a curriculum to address these educational needs. Design .- A working group formed by the College of American Pathologists developed an initial list of genomics competencies (knowledge and skills statements) that a practicing pathologist needs to be successful. Experts in genomics were then surveyed to rate the importance of each competency. These data were used to create a final list of prioritized competencies. A subset of the working group defined subtopics and tasks for each competency. Appropriate delivery methods for the educational material were also proposed. Results .- A final list of 32 genomics competency statements was developed. A prioritized curriculum was created with designated subtopics and tasks associated with each competency. Conclusions .- We present a genomics curriculum designed as a first step toward providing practicing pathologists with the competencies needed to practice successfully.

  View details for DOI 10.5858/arpa.2014-0253-CP

  View details for PubMedID 26125429

• Laboratory and Data Analysis Methods for Characterization of Human B Cell Repertoires by High-Throughput DNA Sequencing. Methods in molecular biology (Clifton, N.J.) Wang, C., Liu, Y., Roskin, K. M., Jackson, K. J., Boyd, S. D. 2015; 1343: 219-233

  Abstract

  High-throughput DNA sequencing techniques have greatly accelerated the pace of research into the repertoires of antibody and T cell receptor gene rearrangements that confer antigen specificity to adaptive immune responses. Studies of aging-related changes in human B cell repertoires have benefited from the ability to detect and quantify thousands to millions of B cell clones in human samples, and study the mutational lineages and isotype switching relationships within each clonal lineage. Correlation of repertoire analysis with antibody gene data from antigen-specific B cells is poised to give much greater insight into clinically relevant B cell responses and memory storage. Here, we describe strategies for preparing and analyzing human antibody gene libraries for studying B cell repertoires.

  View details for DOI 10.1007/978-1-4939-2963-4_17

  View details for PubMedID 26420720

• High-Throughput DNA Sequencing Analysis of Antibody Repertoires ANTIBODIES FOR INFECTIOUS DISEASES Boyd, S. D., Joshi, S. A., Crowe, J. E., Boraschi, D., Rappuoli, R. 2015: 345-362

  View details for DOI 10.1128/microbiolspec.AID-0017-2014

  View details for Web of Science ID 000697764300021

• A Balanced Look at the Implications of Genomic (and Other "Omics") Testing for Disease Diagnosis and Clinical Care. Genes Boyd, S. D., Galli, S. J., Schrijver, I., Zehnder, J. L., Ashley, E. A., Merker, J. D. 2014; 5 (3): 748-766

  Abstract

  The tremendous increase in DNA sequencing capacity arising from the commercialization of "next generation" instruments has opened the door to innumerable routes of investigation in basic and translational medical science. It enables very large data sets to be gathered, whose interpretation and conversion into useful knowledge is only beginning. A challenge for modern healthcare systems and academic medical centers is to apply these new methods for the diagnosis of disease and the management of patient care without unnecessary delay, but also with appropriate evaluation of the quality of data and interpretation, as well as the clinical value of the insights gained. Most critically, the standards applied for evaluating these new laboratory data and ensuring that the results and their significance are clearly communicated to patients and their caregivers should be at least as rigorous as those applied to other kinds of medical tests. Here, we present an overview of conceptual and practical issues to be considered in planning for the integration of genomic methods or, in principle, any other type of "omics" testing into clinical care.

  View details for DOI 10.3390/genes5030748

  View details for PubMedID 25257203

• High-Throughput DNA Sequencing Analysis of Antibody Repertoires. Microbiology spectrum Boyd, S. D., Joshi, S. A. 2014; 2 (5)

  Abstract

  New high-throughput DNA sequencing (HTS) technologies developed in the past decade have begun to be applied to the study of the complex gene rearrangements that encode human antibodies. This article first reviews the genetic features of Ig loci and the HTS technologies that have been applied to human repertoire studies, then discusses key choices for experimental design and data analysis in these experiments and the insights gained in immunological and infectious disease studies with the use of these approaches.

  View details for PubMedID 26104353

• Comprehensive whole-genome sequencing of an early-stage primary myelofibrosis patient defines low mutational burden and non-recurrent candidate genes. Haematologica Merker, J. D., Roskin, K. M., Ng, D., Pan, C., Fisk, D. G., King, J. J., Hoh, R., Stadler, M., Okumoto, L. M., Abidi, P., Hewitt, R., Jones, C. D., Gojenola, L., Clark, M. J., Zhang, B., Cherry, A. M., George, T. I., Snyder, M., Boyd, S. D., Zehnder, J. L., Fire, A. Z., Gotlib, J. 2013; 98 (11): 1689-1696

  Abstract

  In order to identify novel somatic mutations associated with classic BCR/ABL1-negative myeloproliferative neoplasms, we performed high-coverage genome sequencing of DNA from peripheral blood granulocytes and cultured skin fibroblasts from a patient with MPL W515K-positive primary myelofibrosis. The primary myelofibrosis genome had a low somatic mutation rate, consistent with that observed in similar hematopoietic tumor genomes. Interfacing of whole-genome DNA sequence data with RNA expression data identified three somatic mutations of potential functional significance: a nonsense mutation in CARD6, implicated in modulation of NF-kappaB activation; a 19-base pair deletion involving a potential regulatory region in the 5'-untranslated region of BRD2, implicated in transcriptional regulation and cell cycle control; and a non-synonymous point mutation in KIAA0355, an uncharacterized protein. Additional mutations in three genes (CAP2, SOX30, and MFRP) were also evident, albeit with no support for expression at the RNA level. Re-sequencing of these six genes in 178 patients with polycythemia vera, essential thrombocythemia, and myelofibrosis did not identify recurrent somatic mutations in these genes. Finally, we describe methods for reducing false-positive variant calls in the analysis of hematologic malignancies with a low somatic mutation rate. This trial is registered with ClinicalTrials.gov (NCT01108159).

  View details for DOI 10.3324/haematol.2013.092379

  View details for PubMedID 23872309

• Human lymphocyte repertoires in ageing. Current opinion in immunology Boyd, S. D., Liu, Y., Wang, C., Martin, V., Dunn-Walters, D. K. 2013; 25 (4): 511-515

  Abstract

  Deterioration of adaptive immunity with ageing may reflect changes in the repertoire of T cells and B cells available to respond to antigenic challenges, due to altered proportions and absolute numbers of lymphocyte subpopulations as well as changes in the repertoire of antigen receptor genes expressed by these cells. High-throughput DNA sequencing (HTS) now facilitates examination of immunoglobulin and T cell receptor gene rearrangements, and initial studies using these methods to study immune system ageing in humans have demonstrated age-related alterations in the receptor populations within lymphocyte subsets, as well as in repertoires responding to vaccination. Accurate measurement of repertoire diversity remains an experimental challenge. Studies of larger numbers of human subjects, analysis of defined lymphocyte subpopulations including antigen-specific populations, and controlling for factors such as chronic viral infections, will be important for gaining additional understanding of the impact of ageing on human lymphocyte populations.

  View details for DOI 10.1016/j.coi.2013.07.007

  View details for PubMedID 23992996

• Co-evolution of a broadly neutralizing HIV-1 antibody and founder virus. Nature Liao, H., Lynch, R., Zhou, T., Gao, F., Alam, S. M., Boyd, S. D., Fire, A. Z., Roskin, K. M., Schramm, C. A., Zhang, Z., Zhu, J., Shapiro, L., Mullikin, J. C., Gnanakaran, S., Hraber, P., Wiehe, K., Kelsoe, G., Yang, G., Xia, S., Montefiori, D. C., Parks, R., Lloyd, K. E., Scearce, R. M., Soderberg, K. A., Cohen, M., Kamanga, G., Louder, M. K., Tran, L. M., Chen, Y., Cai, F., Chen, S., Moquin, S., Du, X., Joyce, M. G., Srivatsan, S., Zhang, B., Zheng, A., Shaw, G. M., Hahn, B. H., Kepler, T. B., Korber, B. T., Kwong, P. D., Mascola, J. R., Haynes, B. F. 2013; 496 (7446): 469-476

  Abstract

  Current human immunodeficiency virus-1 (HIV-1) vaccines elicit strain-specific neutralizing antibodies. However, cross-reactive neutralizing antibodies arise in approximately 20% of HIV-1-infected individuals, and details of their generation could provide a blueprint for effective vaccination. Here we report the isolation, evolution and structure of a broadly neutralizing antibody from an African donor followed from the time of infection. The mature antibody, CH103, neutralized approximately 55% of HIV-1 isolates, and its co-crystal structure with the HIV-1 envelope protein gp120 revealed a new loop-based mechanism of CD4-binding-site recognition. Virus and antibody gene sequencing revealed concomitant virus evolution and antibody maturation. Notably, the unmutated common ancestor of the CH103 lineage avidly bound the transmitted/founder HIV-1 envelope glycoprotein, and evolution of antibody neutralization breadth was preceded by extensive viral diversification in and near the CH103 epitope. These data determine the viral and antibody evolution leading to induction of a lineage of HIV-1 broadly neutralizing antibodies, and provide insights into strategies to elicit similar antibodies by vaccination.

  View details for DOI 10.1038/nature12053

  View details for PubMedID 23552890

• Selective Immunophenotyping for Diagnosis of B-cell Neoplasms: Immunohistochemistry and Flow Cytometry Strategies and Results APPLIED IMMUNOHISTOCHEMISTRY & MOLECULAR MORPHOLOGY Boyd, S. D., Natkunam, Y., Allen, J. R., Warnke, R. A. 2013; 21 (2): 116-131

  Abstract

  Determining the immunophenotype of hematologic malignancies is now an indispensable part of diagnostic classification, and can help to guide therapy, or to predict clinical outcome. Diagnostic workup should be guided by morphologic findings and evaluate clinically important markers, but ideally should avoid the use of overly broad panels of immunostains that can reveal incidental findings of uncertain significance and give rise to increased costs. Here, we outline our approach to diagnosis of B-cell neoplasms, combining histologic and clinical data with tailored panels of immunophenotyping reagents, in the context of the 2008 World Health Organization classification. We present data from cases seen at our institution from 2004 through 2008 using this approach, to provide a practical reference for findings seen in daily diagnostic practice.

  View details for DOI 10.1097/PAI.0b013e31825d550a

  View details for Web of Science ID 000315464500004

  View details for PubMedID 22820658

• Integration of Genomic Medicine into Pathology Residency Training The Stanford Open Curriculum JOURNAL OF MOLECULAR DIAGNOSTICS Schrijver, I., Natkunam, Y., Galli, S., Boyd, S. D. 2013; 15 (2): 141-148

  Abstract

  Next-generation sequencing methods provide an opportunity for molecular pathology laboratories to perform genomic testing that is far more comprehensive than single-gene analyses. Genome-based test results are expected to develop into an integral component of diagnostic clinical medicine and to provide the basis for individually tailored health care. To achieve these goals, rigorous interpretation of high-quality data must be informed by the medical history and the phenotype of the patient. The discipline of pathology is well positioned to implement genome-based testing and to interpret its results, but new knowledge and skills must be included in the training of pathologists to develop expertise in this area. Pathology residents should be trained in emerging technologies to integrate genomic test results appropriately with more traditional testing, to accelerate clinical studies using genomic data, and to help develop appropriate standards of data quality and evidence-based interpretation of these test results. We have created a genomic pathology curriculum as a first step in helping pathology residents build a foundation for the understanding of genomic medicine and its implications for clinical practice. This curriculum is freely accessible online.

  View details for DOI 10.1016/j.jmoldx.2012.11.003

  View details for Web of Science ID 000315841600001

  View details for PubMedID 23313248

• New tools for classification and monitoring of autoimmune diseases NATURE REVIEWS RHEUMATOLOGY Maecker, H. T., Lindstrom, T. M., Robinson, W. H., Utz, P. J., Hale, M., Boyd, S. D., Shen-Orr, S. S., Fathman, C. G. 2012; 8 (6): 317-328

  Abstract

  Rheumatologists see patients with a range of autoimmune diseases. Phenotyping these diseases for diagnosis, prognosis and selection of therapies is an ever increasing problem. Advances in multiplexed assay technology at the gene, protein, and cellular level have enabled the identification of 'actionable biomarkers'; that is, biological metrics that can inform clinical practice. Not only will such biomarkers yield insight into the development, remission, and exacerbation of a disease, they will undoubtedly improve diagnostic sensitivity and accuracy of classification, and ultimately guide treatment. This Review provides an introduction to these powerful technologies that could promote the identification of actionable biomarkers, including mass cytometry, protein arrays, and immunoglobulin and T-cell receptor high-throughput sequencing. In our opinion, these technologies should become part of routine clinical practice for the management of autoimmune diseases. The use of analytical tools to deconvolve the data obtained from use of these technologies is also presented here. These analyses are revealing a more comprehensive and interconnected view of the immune system than ever before and should have an important role in directing future treatment approaches for autoimmune diseases.

  View details for DOI 10.1038/nrrheum.2012.66

  View details for PubMedID 22647780

• The Inference of Phased Haplotypes for the Immunoglobulin H Chain V Region Gene Loci by Analysis of VDJ Gene Rearrangements JOURNAL OF IMMUNOLOGY Kidd, M. J., Chen, Z., Wang, Y., Jackson, K. J., Zhang, L., Boyd, S. D., Fire, A. Z., Tanaka, M. M., Gaeta, B. A., Collins, A. M. 2012; 188 (3): 1333-1340

  Abstract

  The existence of many highly similar genes in the lymphocyte receptor gene loci makes them difficult to investigate, and the determination of phased "haplotypes" has been particularly problematic. However, V(D)J gene rearrangements provide an opportunity to infer the association of Ig genes along the chromosomes. The chromosomal distribution of H chain genes in an Ig genotype can be inferred through analysis of VDJ rearrangements in individuals who are heterozygous at points within the IGH locus. We analyzed VDJ rearrangements from 44 individuals for whom sufficient unique rearrangements were available to allow comprehensive genotyping. Nine individuals were identified who were heterozygous at the IGHJ6 locus and for whom sufficient suitable VDJ rearrangements were available to allow comprehensive haplotyping. Each of the 18 resulting IGHV│IGHD│IGHJ haplotypes was unique. Apparent deletion polymorphisms were seen that involved as many as four contiguous, functional IGHV genes. Two deletion polymorphisms involving multiple contiguous IGHD genes were also inferred. Three previously unidentified gene duplications were detected, where two sequences recognized as allelic variants of a single gene were both inferred to be on a single chromosome. Phased genomic data brings clarity to the study of the contribution of each gene to the available repertoire of rearranged VDJ genes. Analysis of rearrangement frequencies suggests that particular genes may have substantially different yet predictable propensities for rearrangement within different haplotypes. Together with data highlighting the extent of haplotypic variation within the population, this suggests that there may be substantial variability in the available Ab repertoires of different individuals.

  View details for DOI 10.4049/jimmunol.1102097

  View details for Web of Science ID 000299690200048

  View details for PubMedID 22205028

• High-throughput VDJ sequencing for quantification of minimal residual disease in chronic lymphocytic leukemia and immune reconstitution assessment PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Logan, A. C., Gao, H., Wang, C., Sahaf, B., Jones, C. D., Marshall, E. L., Buno, I., Armstrong, R., Fire, A. Z., Weinberg, K. I., Mindrinos, M., Zehnder, J. L., Boyd, S. D., Xiao, W., Davis, R. W., Miklos, D. B. 2011; 108 (52): 21194-21199

  Abstract

  The primary cause of poor outcome following allogeneic hematopoietic cell transplantation (HCT) for chronic lymphocytic leukemia (CLL) is disease recurrence. Detection of increasing minimal residual disease (MRD) following HCT may permit early intervention to prevent clinical relapse; however, MRD quantification remains an uncommon diagnostic test because of logistical and financial barriers to widespread use. Here we describe a method for quantifying CLL MRD using widely available consensus primers for amplification of all Ig heavy chain (IGH) genes in a mixture of peripheral blood mononuclear cells, followed by high-throughput sequencing (HTS) for disease-specific IGH sequence quantification. To achieve accurate MRD quantification, we developed a systematic bioinformatic methodology to aggregate cancer clone sequence variants arising from systematic and random artifacts occurring during IGH-HTS. We then compared the sensitivity of IGH-HTS, flow cytometry, and allele-specific oligonucleotide PCR for MRD quantification in 28 samples collected from 6 CLL patients following allogeneic HCT. Using amplimer libraries generated with consensus primers from patient blood samples, we demonstrate the sensitivity of IGH-HTS with 454 pyrosequencing to be 10(-5), with a high correlation between quantification by allele-specific oligonucleotide PCR and IGH-HTS (r = 0.85). From the same dataset used to quantify MRD, IGH-HTS also allowed us to profile IGH repertoire reconstitution after HCT-information not provided by the other MRD methods. IGH-HTS using consensus primers will broaden the availability of MRD quantification in CLL and other B cell malignancies, and this approach has potential for quantitative evaluation of immune diversification following transplant and nontransplant therapies.

  View details for DOI 10.1073/pnas.1118357109

  View details for Web of Science ID 000298479900065

  View details for PubMedID 22160699

  View details for PubMedCentralID PMC3248502

• Initial antibodies binding to HIV-1 gp41 in acutely infected subjects are polyreactive and highly mutated JOURNAL OF EXPERIMENTAL MEDICINE Liao, H., Chen, X., Munshaw, S., Zhang, R., Marshall, D. J., Vandergrift, N., Whitesides, J. F., Lu, X., Yu, J., Hwang, K., Gao, F., Markowitz, M., Heath, S. L., Bar, K. J., Goepfert, P. A., Montefiori, D. C., Shaw, G. C., Alam, S. M., Margolis, D. M., Denny, T. N., Boyd, S. D., Marshal, E., Egholm, M., Simen, B. B., Hanczaruk, B., Fire, A. Z., Voss, G., Kelsoe, G., Tomaras, G. D., Moody, M. A., Kepler, T. B., Haynes, B. F. 2011; 208 (11): 2237-2249

  Abstract

  The initial antibody response to HIV-1 is targeted to envelope (Env) gp41, and is nonneutralizing and ineffective in controlling viremia. To understand the origins and characteristics of gp41-binding antibodies produced shortly after HIV-1 transmission, we isolated and studied gp41-reactive plasma cells from subjects acutely infected with HIV-1. The frequencies of somatic mutations were relatively high in these gp41-reactive antibodies. Reverted unmutated ancestors of gp41-reactive antibodies derived from subjects acutely infected with HIV-1 frequently did not react with autologous HIV-1 Env; however, these antibodies were polyreactive and frequently bound to host or bacterial antigens. In one large clonal lineage of gp41-reactive antibodies, reactivity to HIV-1 Env was acquired only after somatic mutations. Polyreactive gp41-binding antibodies were also isolated from uninfected individuals. These data suggest that the majority of gp41-binding antibodies produced after acute HIV-1 infection are cross-reactive responses generated by stimulating memory B cells that have previously been activated by non-HIV-1 antigens.

  View details for DOI 10.1084/jem.20110363

  View details for Web of Science ID 000296537800011

  View details for PubMedID 21987658

  View details for PubMedCentralID PMC3201211

• Determinants of nucleosome organization in primary human cells NATURE Valouev, A., Johnson, S. M., Boyd, S. D., Smith, C. L., Fire, A. Z., Sidow, A. 2011; 474 (7352): 516-U148

  Abstract

  Nucleosomes are the basic packaging units of chromatin, modulating accessibility of regulatory proteins to DNA and thus influencing eukaryotic gene regulation. Elaborate chromatin remodelling mechanisms have evolved that govern nucleosome organization at promoters, regulatory elements, and other functional regions in the genome. Analyses of chromatin landscape have uncovered a variety of mechanisms, including DNA sequence preferences, that can influence nucleosome positions. To identify major determinants of nucleosome organization in the human genome, we used deep sequencing to map nucleosome positions in three primary human cell types and in vitro. A majority of the genome showed substantial flexibility of nucleosome positions, whereas a small fraction showed reproducibly positioned nucleosomes. Certain sites that position in vitro can anchor the formation of nucleosomal arrays that have cell type-specific spacing in vivo. Our results unveil an interplay of sequence-based nucleosome preferences and non-nucleosomal factors in determining nucleosome organization within mammalian cells.

  View details for DOI 10.1038/nature10002

  View details for Web of Science ID 000291939700050

  View details for PubMedID 21602827

  View details for PubMedCentralID PMC3212987

• Benchmarking the performance of human antibody gene alignment utilities using a 454 sequence dataset BIOINFORMATICS Jackson, K. J., Boyd, S., Gaeta, B. A., Collins, A. M. 2010; 26 (24): 3129-3130

  Abstract

  Immunoglobulin heavy chain genes are formed by recombination of genes randomly selected from sets of IGHV, IGHD and IGHJ genes. Utilities have been developed to identify genes that contribute to observed VDJ rearrangements, but in the absence of datasets of known rearrangements, the evaluation of these utilities is problematic. We have analyzed thousands of VDJ rearrangements from an individual (S22) whose IGHV, IGHD and IGHJ genotype can be inferred from the dataset. Knowledge of this genotype means that the Stanford_S22 dataset can serve to benchmark the performance of IGH alignment utilities.We evaluated the performance of seven utilities. Failure to partition a sequence into genes present in the S22 genome was considered an error, and error rates for different utilities ranged from 7.1% to 13.7%.Supplementary data includes the S22 genotypes and alignments. The Stanford_S22 dataset and an evaluation tool is available at http://www.emi.unsw.edu.au/~ihmmune/IGHUtilityEval/.

  View details for DOI 10.1093/bioinformatics/btq604

  View details for Web of Science ID 000284947700019

  View details for PubMedID 21036814

• Individual Variation in the Germline Ig Gene Repertoire Inferred from Variable Region Gene Rearrangements JOURNAL OF IMMUNOLOGY Boyd, S. D., Gaeta, B. A., Jackson, K. J., Fire, A. Z., Marshall, E. L., Merker, J. D., Maniar, J. M., Zhang, L. N., Sahaf, B., Jones, C. D., Simen, B. B., Hanczaruk, B., Nguyen, K. D., Nadeau, K. C., Egholm, M., Miklos, D. B., Zehnder, J. L., Collins, A. M. 2010; 184 (12): 6986-6992

  Abstract

  Individual variation in the Ig germline gene repertoire leads to individual differences in the combinatorial diversity of the Ab repertoire, but the study of such variation has been problematic. The application of high-throughput DNA sequencing to the study of rearranged Ig genes now makes this possible. The sequencing of thousands of VDJ rearrangements from an individual, either from genomic DNA or expressed mRNA, should allow their germline IGHV, IGHD, and IGHJ repertoires to be inferred. In addition, where previously mere glimpses of diversity could be gained from sequencing studies, new large data sets should allow the rearrangement frequency of different genes and alleles to be seen with clarity. We analyzed the DNA of 108,210 human IgH chain rearrangements from 12 individuals and determined their individual IGH genotypes. The number of reportedly functional IGHV genes and allelic variants ranged from 45 to 60, principally because of variable levels of gene heterozygosity, and included 14 previously unreported IGHV polymorphisms. New polymorphisms of the IGHD3-16 and IGHJ6 genes were also seen. At heterozygous loci, remarkably different rearrangement frequencies were seen for the various IGHV alleles, and these frequencies were consistent between individuals. The specific alleles that make up an individual's Ig genotype may therefore be critical in shaping the combinatorial repertoire. The extent of genotypic variation between individuals is highlighted by an individual with aplastic anemia who appears to lack six contiguous IGHD genes on both chromosomes. These deletions significantly alter the potential expressed IGH repertoire, and possibly immune function, in this individual.

  View details for DOI 10.4049/jimmunol.1000445

  View details for Web of Science ID 000278516700047

  View details for PubMedID 20495067

• A Comparison of Two Methods for Screening CEBPA Mutations in Patients with Acute Myeloid Leukemia JOURNAL OF MOLECULAR DIAGNOSTICS Ahn, J., Seo, K., Weinberg, O., Boyd, S. D., Arber, D. A. 2009; 11 (4): 319-323

  Abstract

  The goal of the study was to compare the performance of a fluorescence-based multiplex PCR fragment analysis to a direct sequencing method for detecting CEBPA mutations in patients with acute myeloid leukemia. Thirty-three samples were selected from a larger study of 107 cases of acute myeloid leukemia by screening for CEBPA mutations by sequence analysis. Of ten identified mutations, six (insertions and deletions) were detected by both sequencing and fragment methods. The fragment analysis method did not detect the remaining four base substitutions because the method cannot detect changes that result in identically sized products. The multiplex PCR fragment length analysis method therefore failed to detect substitution mutations accounting for 40% of total CEBPA mutations in our patient set. Our results indicate that fragment length analysis should not be used in isolation, and that direct sequencing is required to evaluate CEBPA gene mutational status in acute myeloid leukemia. A combination of the two assays may offer some advantages, chiefly in permitting more sensitive detection by fragment length analysis of insertions and deletions.

  View details for DOI 10.2353/jmoldx.2009.080121

  View details for Web of Science ID 000267562000008

  View details for PubMedID 19525338

  View details for PubMedCentralID PMC2710708

• Features of hemolysis due to Clostridium perfringens infection INTERNATIONAL JOURNAL OF LABORATORY HEMATOLOGY Boyd, S. D., Mobley, B. C., Regula, D. P., Arber, D. A. 2009; 31 (3): 364-367

  Abstract

  Infection by Clostridium perfringens can be an unsuspected cause of hemolysis in emergency room patients. Historically, this condition has been associated with wound contamination and other tissue infections. We report the case of an autistic patient who presented to our emergency department with a distended abdomen and hemolysis of unknown etiology. The patient had no history of recent surgery. Exploration of the abdomen revealed a hepatic abscess. Blood cultures tested culture positive for C. perfringens. We present images demonstrating the salient features of the peripheral blood smear in cases of this uncommon but deadly cause of hemolysis.

  View details for DOI 10.1111/j.1751-553X.2007.01018.x

  View details for Web of Science ID 000265407400013

  View details for PubMedID 18177433

• Everything you wanted to know about small RNA but were afraid to ask LABORATORY INVESTIGATION Boyd, S. D. 2008; 88 (6): 569-578

  Abstract

  MicroRNAs are a class of recently discovered small RNA molecules that regulate other genes in the human genome. Studies in human cells and model organisms have begun to reveal the mechanisms of microRNA activity, and the wide range of normal physiological functions they influence. Their alteration in pathologic states from cancer to cardiovascular disease is also increasingly clear. A review of current evidence for the role of these molecules in human health and disease will be helpful to pathologists and medical researchers as the fascinating story of these small regulators continues to unfold.

  View details for DOI 10.1038/labinvest.2008.32

  View details for Web of Science ID 000256114600001

  View details for PubMedID 18427554

• Alloimmunization to red blood cell antigens affects clinical outcomes in liver transplant patients LIVER TRANSPLANTATION Boyd, S. D., Stenard, F., Lee, D. K., Goodnough, L. T., Esquivel, C. O., Fontaine, M. J. 2007; 13 (12): 1654-1661

  Abstract

  Transfusion therapy of liver transplant patients remains a challenge. High volumes of intraoperative blood transfusion have been shown to increase the risk of poor graft or patient survival. We conducted a retrospective study of 209 consecutive liver transplant cases at our institution. Only patients receiving their first liver transplant, with no other simultaneous organ transplants, were included. Cox proportional hazard modeling was used to identify clinical variables correlated with postoperative patient mortality. Statistically significant variables for poor patient survival were the number of red blood cell and plasma units transfused, a history of red blood cell alloantibodies, and the immunosuppressive regimen used. History of pregnancy also approached statistical significance but was less robust than the other 3 variables. Our findings suggest that blood transfusion and immune modulation greatly affect the survival of patients after liver transplantation.

  View details for DOI 10.1002/It.21241

  View details for PubMedID 18044783

• An intact HDM2 RING-finger domain is required for nuclear exclusion of p53 NATURE CELL BIOLOGY Boyd, S. D., Tsai, K. Y., Jacks, T. 2000; 2 (9): 563-568

  Abstract

  The p53 tumour-suppressor protein is negatively regulated by HDM2. Recent reports indicate that the leucine-rich nuclear-export sequence (NES) of HDM2 enables it to shuttle to the cytoplasm, and that this activity is required for degradation of p53. However, it is unclear whether HDM2 is involved in nuclear export of p53, partly because p53 has itself been shown to contain a functional NES within its tetramerization domain. Here we show that co-expression of HDM2 with green fluorescent protein (GFP)-tagged p53 causes redistribution of p53 from the nucleus to the cytoplasm of the cell. This activity is dependent on binding of p53 to HDM2, and requires an intact p53 NES, but is independent of the HDM2 NES. A mutant of the HDM2 RING-finger domain that is unable to ubiquitinate p53 does not cause relocalization of p53, indicating that ubiquitin ligation or other activities of this region of HDM2 may be necessary for its regulation of p53 localization.

  View details for Web of Science ID 000089250600006

  View details for PubMedID 10980695

• The role of CTLA-4 in regulating Th2 differentiation JOURNAL OF IMMUNOLOGY OOSTERWEGEL, M. A., Mandelbrot, D. A., Boyd, S. D., Lorsbach, R. B., Jarrett, D. Y., Abbas, A. K., Sharpe, A. H. 1999; 163 (5): 2634-2639

  Abstract

  To examine the role of CTLA-4 in Th cell differentiation, we used two newly generated CTLA-4-deficient (CTLA-4-/-) mouse strains: DO11. 10 CTLA-4-/- mice carrying a class II restricted transgenic TCR specific for OVA, and mice lacking CTLA-4, B7.1 and B7.2 (CTLA-4-/- B7.1/B7.2-/- ). When purified naive CD4+ DO11.10 T cells from CTLA-4-/- and wild-type mice were primed and restimulated in vitro with peptide Ag, CTLA-4-/- DO11.10 T cells developed into Th2 cells, whereas wild-type DO11.10 T cells developed into Th1 cells. Similarly, when CTLA-4-/- CD4+ T cells from mice lacking CTLA-4, B7. 1, and B7.2 were stimulated in vitro with anti-CD3 Ab and wild-type APC, these CTLA-4-/- CD4+ T cells produced IL-4 even during the primary stimulation, whereas CD4+ cells from B7.1/B7.2-/- mice did not produce IL-4. Upon secondary stimulation, CD4+ T cells from CTLA-4-/- B7.1/B7.2-/- mice secreted high levels of IL-4, whereas CD4+ T cells from B7.1/B7.2-/- mice produced IFN-gamma. In contrast to the effects on CD4+ Th differentiation, the absence of CTLA-4 resulted in only a modest effect on T cell proliferation, and increased proliferation of CTLA-4-/- CD4+ T cells was seen only during secondary stimulation in vitro. Administration of a stimulatory anti-CD28 Ab in vivo induced IL-4 production in CTLA-4-/- B7.1/B7.2-/- but not wild-type mice. These studies demonstrate that CTLA-4 is a critical and potent inhibitor of Th2 differentiation. Thus, the B7-CD28/CTLA-4 pathway plays a critical role in regulating Th2 differentiation in two ways: CD28 promotes Th2 differentiation while CTLA-4 limits Th2 differentiation.

  View details for Web of Science ID 000082125400039

  View details for PubMedID 10453003

• Inhibition of cyclin-dependent kinase 2 by p21 is necessary for retinoblastoma protein-mediated G(1) arrest after gamma-irradiation PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Brugarolas, J., Moberg, K., Boyd, S. D., Taya, Y., Jacks, T., Lees, J. A. 1999; 96 (3): 1002-1007

  Abstract

  In mammalian cells, activation of certain checkpoint pathways as a result of exposure to genotoxic agents results in cell cycle arrest. The integrity of these arrest pathways is critical to the ability of the cell to repair mutations that otherwise might compromise viability or contribute to deregulation of cellular growth and proliferation. Here we examine the mechanism through which DNA damaging agents result in a G1 arrest that depends on the tumor suppressor p53 and its transcriptional target p21. By using primary cell lines lacking specific cell cycle regulators, we demonstrate that this pathway functions through the growth suppressive properties of the retinoblastoma protein (pRB) tumor suppressor. Specifically, gamma-irradiation inhibits the phosphorylation of pRB at cyclin-dependent kinase 2-specific, but not cyclin-dependent kinase 4-specific, sites in a p21-dependent manner. Most importantly, we show that pRB is a critical component of this DNA damage checkpoint. These data indicate that the p53 --> p21 checkpoint pathway uses the normal cell cycle regulatory machinery to induce the accumulation of the growth suppressive form of pRB and suggest that loss of pRB during the course of tumorigenesis disrupts the function of an important DNA damage checkpoint.

  View details for Web of Science ID 000078484100038

  View details for PubMedID 9927683

• CTLA4Ig prevents lymphoproliferation and fatal multiorgan tissue destruction in CTLA-4-deficient mice JOURNAL OF IMMUNOLOGY Tivol, E. A., Boyd, S. D., Mckeon, S., Borriello, F., Nickerson, P., Strom, T. B., Sharpe, A. H. 1997; 158 (11): 5091-5094

  Abstract

  Mice lacking CTLA-4 develop a fatal spontaneous lymphoproliferative disease with massive lymphocytic infiltrates and tissue destruction in many organs. CTLA-4-deficient (-/-) splenocytes and lymph node cells proliferate without added stimuli in vitro. We report here that CTLA4Ig treatment of CTLA-4 -/- mice prevents lymphoproliferation and fatal multiorgan tissue damage in vivo and proliferation of CTLA-4 -/- splenocytes and lymph node cells in vitro. Therefore, stimulation via CD28-B7 interactions appears necessary for CTLA-4 -/- T cell proliferation and the production of lymphoproliferative disease in vivo. When CTLA4Ig treatment is terminated, CTLA-4 -/- T cells become activated and lymphoproliferative disease develops. The lack of long term protective effects of CTLA4Ig treatment suggests that CTLA-4 is needed for the induction and or maintenance of tolerance.

  View details for Web of Science ID A1997XA06300007

  View details for PubMedID 9164923

• B7-1 and B7-2 have overlapping, critical roles in immunoglobulin class switching and germinal center formation IMMUNITY Borriello, F., Sethna, M. P., Boyd, S. D., Schweitzer, A. N., Tivol, E. A., Jacoby, D., Strom, T. B., Simpson, E. M., Freeman, G. J., Sharpe, A. H. 1997; 6 (3): 303-313

  Abstract

  Humoral immune responses were characterized in mouse strains lacking either or both B7 molecules. Mice deficient in both B7-1 and B7-2 failed to generate antigen-specific IgG1 and IgG2a responses and lacked germinal centers when immunized by a number of routes and even in the presence of complete Freund's adjuvant. These results demonstrate that B7-mediated signaling plays a critical role in germinal center formation and immunoglobulin class switching in vivo. Mice lacking only B7-1 or B7-2 mounted high-titer antigen-specific IgG responses when immunized in complete Freund's adjuvant, indicating that B7-1 and B7-2 can have overlapping, compensatory functions for IgG responses. When immunized intravenously without adjuvant, B7-2-deficient mice failed to switch antibody isotypes or form germinal centers, whereas B7-1-deficient mice gave antibody responses comparable with wild-type mice. Thus, B7-2 has an important role in initiating antibody responses in the absence of adjuvant, but the induction of B7-1 by adjuvant in B7-2-deficient mice can compensate for the absence of B7-2.

  View details for Web of Science ID A1997XC61800010

  View details for PubMedID 9075931