Clinical Focus


  • Infectious Disease

Administrative Appointments


  • Associate Dean for Basic and Translational Research, Stanford University School of Medicine (2021 - Present)
  • Co-Director, Stanford MSTP (2022 - Present)
  • Associate Director, Stanford Medical Scientist Training Program (MSTP) (2014 - 2021)
  • Fellow, Stanford Center for Innovation in Global Health (CIGH) (2015 - Present)

Honors & Awards


  • NIDA Avant Garde Award for HIV Research, NIH/NIAID (2018)
  • Outstanding Investigator Award, Western Society for Clinical Investigation (2018)
  • Chan Zuckerberg Investigator, Chan Zuckerberg Biohub (2017)
  • Fellow, Infectious Diseases Society of America (2017)
  • Investigator in the Pathogenesis of Infectious Diseases, Burroughs Wellcome Foundation (2017)
  • Outstanding Investigator Award, Western Society for Clinical Investigation (2017)
  • Elected Member, American Society for Clinical Investigation (2016)
  • Tashia and John Morgridge Faculty Scholar in Pediatric Translational Medicine, Stanford Child Health Research Institute (2015)
  • Clinical Scientist Development Award, Doris Duke Charitable Foundation (2013)
  • Faculty Scholar, Donald E. and Delia B. Baxter Foundation (2013)
  • NIH Director's New Innovator Award, NIH (2013)
  • McCormick Faculty Award, Stanford University School of Medicine, Office of Diversity and Leadership (2012)
  • Outstanding Faculty Mentor Award, Stanford Immunology (2012)
  • Young Investigator Award, Arnold and Mabel Beckman Foundation (2012)
  • ICAAC Young Investigator Award, American Society for Microbiology (2010)
  • Young and Early Career Investigator, Enterprise-OCTAVE Workshop on Correlates of Vaccine Protection to HIV (2010)
  • New Investigator Award, University of Washington Center for AIDS Research (2009)
  • Young Investigator Award, AIDS Vaccine, Seattle, WA (2007)
  • Outstanding Consultant, Infectious Diseases, MedCon (2003)

Boards, Advisory Committees, Professional Organizations


  • Member, Infectious Diseases Society of America (2013 - Present)
  • Member, American Association of Immunologists (2012 - Present)
  • Member, American Society for Microbiology (2009 - Present)

Professional Education


  • Medical Education: University of Washington Registration and Transcripts Office (2001) WA
  • PhD Training: University of Washington School of Medicine (1999) WA
  • Residency: University of Washington Medical Center Dept of Medicine (2003) WA
  • Fellowship: University of Washington Infectious Disease Program (2007) WA
  • Board Certification: American Board of Internal Medicine, Infectious Disease (2006)
  • PhD, University of Washington, Immunology (1999)
  • BS, University of California, Davis, Biochemistry (1993)

Current Research and Scholarly Interests


Our goal is to develop new methods to prevent and control infectious diseases through better understanding of human immunology. We have several major areas of ongoing investigation.

Understanding the diversity and biology of human natural killer (NK) cells.
Our interest in NK cells stems from their ability to directly lyse infected and tumor cells and to mediate antibody-dependent cellular cytotoxicity, acting as a bridge between innate and adaptive immune responses. Our recent study demonstrated that human NK cells are much more diverse than previously appreciated, with both genetic and environmental determinants. We are currently examining how this diversity is regulated and its implications for viral immunity in both healthy and diseased states.

Defining the role of NK cells in viral immunity.
Vaccination is one of the most effective methods to prevent morbidity and mortality related to infectious diseases, yet there are many viral infections for which durable, broadly cross-protective vaccines remain desperately needed. Recent data indicating that NK cells may be capable of immunologic memory raises the possibility that we could harness NK cells to fight viruses. Current projects in the laboratory are focused on better understanding how human NK cells recognize and control infection with HIV-1, influenza, West Nile Virus, and Epstein Barr Virus.

Immune signatures of human pregnancy.
Pregnant women are at increased risk of contracting viruses including HIV and influenza, and are more susceptible to severe complications once infected. A major focus of the laboratory is to define the immune mechanisms that contribute to viral susceptibility in pregnant women. These investigations focus broadly on T cell, antibody, and NK cell responses to viruses during pregnancy, and use infection and vaccination as models. In addition, we are also studying the role of immunity in preterm birth.

Clinical Trials


  • Genetic and Environmental Factors in the Response to Influenza Vaccination Not Recruiting

    The purpose of the study is to investigate and compare the immune responses to influenza vaccination in monozygotic (identical) and dizygotic (fraternal) twins to determine the roles of genetics and environment in the response to flu vaccination.

    Stanford is currently not accepting patients for this trial. For more information, please contact Cornelia L Dekker, MD, 650-724-4437.

    View full details

2022-23 Courses


Stanford Advisees


All Publications


  • Multi-omic profiling reveals widespread dysregulation of innate immunity and hematopoiesis in COVID-19. The Journal of experimental medicine Wilk, A. J., Lee, M. J., Wei, B., Parks, B., Pi, R., Martinez-Colon, G. J., Ranganath, T., Zhao, N. Q., Taylor, S., Becker, W., Stanford COVID-19 Biobank, Jimenez-Morales, D., Blomkalns, A. L., O'Hara, R., Ashley, E. A., Nadeau, K. C., Yang, S., Holmes, S., Rabinovitch, M., Rogers, A. J., Greenleaf, W. J., Blish, C. A. 2021; 218 (8)

    Abstract

    Our understanding of protective versus pathological immune responses to SARS-CoV-2, the virus that causes coronavirus disease 2019 (COVID-19), is limited by inadequate profiling of patients at the extremes of the disease severity spectrum. Here, we performed multi-omic single-cell immune profiling of 64 COVID-19 patients across the full range of disease severity, from outpatients with mild disease to fatal cases. Our transcriptomic, epigenomic, and proteomic analyses revealed widespread dysfunction of peripheral innate immunity in severe and fatal COVID-19, including prominent hyperactivation signatures in neutrophils and NK cells. We also identified chromatin accessibility changes at NF-kappaB binding sites within cytokine gene loci as a potential mechanism for the striking lack of pro-inflammatory cytokine production observed in monocytes in severe and fatal COVID-19. We further demonstrated that emergency myelopoiesis is a prominent feature of fatal COVID-19. Collectively, our results reveal disease severity-associated immune phenotypes in COVID-19 and identify pathogenesis-associated pathways that are potential targets for therapeutic intervention.

    View details for DOI 10.1084/jem.20210582

    View details for PubMedID 34128959

  • A single-cell atlas of the peripheral immune response in patients with severe COVID-19. Nature medicine Wilk, A. J., Rustagi, A., Zhao, N. Q., Roque, J., Martinez-Colon, G. J., McKechnie, J. L., Ivison, G. T., Ranganath, T., Vergara, R., Hollis, T., Simpson, L. J., Grant, P., Subramanian, A., Rogers, A. J., Blish, C. A. 2020

    Abstract

    There is an urgent need to better understand the pathophysiology of Coronavirus disease 2019 (COVID-19), the global pandemic caused by SARS-CoV-2, which has infected more than three million people worldwide1. Approximately 20% of patients with COVID-19 develop severe disease and 5% of patients require intensive care2. Severe disease has been associated with changes in peripheral immune activity, including increased levels of pro-inflammatory cytokines3,4 that may be produced by a subset of inflammatory monocytes5,6, lymphopenia7,8 and T cell exhaustion9,10. To elucidate pathways in peripheral immune cells that might lead to immunopathology or protective immunity in severe COVID-19, we applied single-cell RNA sequencing (scRNA-seq) to profile peripheral blood mononuclear cells (PBMCs) from seven patients hospitalized for COVID-19, four of whom had acute respiratory distress syndrome, and six healthy controls. We identify reconfiguration of peripheral immune cell phenotype in COVID-19, including a heterogeneous interferon-stimulated gene signature, HLA class II downregulation and a developing neutrophil population that appears closely related to plasmablasts appearing in patients with acute respiratory failure requiring mechanical ventilation. Importantly, we found that peripheral monocytes and lymphocytes do not express substantial amounts of pro-inflammatory cytokines. Collectively, we provide a cell atlas of the peripheral immune response to severe COVID-19.

    View details for DOI 10.1038/s41591-020-0944-y

    View details for PubMedID 32514174

  • Charge-altering releasable transporters enable phenotypic manipulation of natural killer cells for cancer immunotherapy. Blood advances Wilk, A. J., Weidenbacher, N. L., Vergara, R. n., Haabeth, O. A., Levy, R. n., Waymouth, R. M., Wender, P. A., Blish, C. A. 2020; 4 (17): 4244–55

    Abstract

    Chimeric antigen receptor (CAR) natural killer (NK) cells are an emerging cell therapy with promising results in oncology trials. However, primary human NK cells are difficult to transfect, hampering both mechanistic studies and clinical applications of NK cells. Currently, NK cell CAR modification relies on viral vectors or cell activation. The former raises cost and tolerability issues, while the latter alters NK cell biology. Here, we report that readily synthesized and inexpensive nonviral charge-altering releasable transporters (CARTs) efficiently transfect primary human NK cells with messenger RNA without relying on NK cell activation. Compared with electroporation, CARTs transfect NK cells more efficiently, better preserve cell viability, and cause minimal reconfiguration of NK cell phenotype and function. We use CARTs to generate cytotoxic primary anti-CD19 CAR NK cells, demonstrating this technology can drive clinical applications of NK cells. To our knowledge, CARTs represent the first efficacious transfection technique for resting primary human NK cells that preserves NK cell phenotype and can enable new biological discoveries and therapeutic applications of this understudied lymphocyte subset.

    View details for DOI 10.1182/bloodadvances.2020002355

    View details for PubMedID 32898247

  • Zika Virus Infection Induces Cranial Neural Crest Cells to Produce Cytokines at Levels Detrimental for Neurogenesis. Cell host & microbe Bayless, N. L., Greenberg, R. S., Swigut, T., Wysocka, J., Blish, C. A. 2016; 20 (4): 423-428

    Abstract

    Zika virus (ZIKV) infection during pregnancy is linked to microcephaly, which is attributed to infection of developing brain structures. ZIKV infects neural progenitor cells in vitro, though its effects on other developmentally relevant stem cell populations, including cranial neural crest cells (CNCCs), have not been assessed. CNCCs give rise to most cranial bones and exert paracrine effects on the developing brain. Here, we report that CNCCs are productively infected by ZIKV, but not by the related dengue virus. ZIKV-infected CNCCs undergo limited apoptosis but secrete cytokines that promote death and drive aberrant differentiation of neural progenitor cultures. Addition of two such cytokines, LIF or VEGF, at levels comparable to those secreted by ZIKV-infected CNCCs is sufficient to recapitulate premature neuronal differentiation and apoptotic death of neural progenitors. Thus, our results suggest that CNCC infection by ZIKV may contribute to associated embryopathies through signaling crosstalk between developing face and brain structures.

    View details for DOI 10.1016/j.chom.2016.09.006

    View details for PubMedID 27693308

    View details for PubMedCentralID PMC5113290

  • Human NK cell repertoire diversity reflects immune experience and correlates with viral susceptibility. Science translational medicine Strauss-Albee, D. M., Fukuyama, J., Liang, E. C., Yao, Y., Jarrell, J. A., Drake, A. L., Kinuthia, J., Montgomery, R. R., John-Stewart, G., Holmes, S., Blish, C. A. 2015; 7 (297): 297ra115-?

    Abstract

    Innate natural killer (NK) cells are diverse at the single-cell level because of variegated expressions of activating and inhibitory receptors, yet the developmental roots and functional consequences of this diversity remain unknown. Because NK cells are critical for antiviral and antitumor responses, a better understanding of their diversity could lead to an improved ability to harness them therapeutically. We found that NK diversity is lower at birth than in adults. During an antiviral response to either HIV-1 or West Nile virus, NK diversity increases, resulting in terminal differentiation and cytokine production at the cost of cell division and degranulation. In African women matched for HIV-1 exposure risk, high NK diversity is associated with increased risk of HIV-1 acquisition. Existing diversity may therefore decrease the flexibility of the antiviral response. Collectively, the data reveal that human NK diversity is a previously undefined metric of immune history and function that may be clinically useful in forecasting the outcomes of infection and malignancy.

    View details for DOI 10.1126/scitranslmed.aac5722

    View details for PubMedID 26203083

  • Enhanced natural killer-cell and T-cell responses to influenza A virus during pregnancy. Proceedings of the National Academy of Sciences of the United States of America Kay, A. W., Fukuyama, J., Aziz, N., Dekker, C. L., Mackey, S., Swan, G. E., Davis, M. M., Holmes, S., Blish, C. A. 2014; 111 (40): 14506-14511

    Abstract

    Pregnant women experience increased morbidity and mortality after influenza infection, for reasons that are not understood. Although some data suggest that natural killer (NK)- and T-cell responses are suppressed during pregnancy, influenza-specific responses have not been previously evaluated. Thus, we analyzed the responses of women that were pregnant (n = 21) versus those that were not (n = 29) immediately before inactivated influenza vaccination (IIV), 7 d after vaccination, and 6 wk postpartum. Expression of CD107a (a marker of cytolysis) and production of IFN-γ and macrophage inflammatory protein (MIP) 1β were assessed by flow cytometry. Pregnant women had a significantly increased percentage of NK cells producing a MIP-1β response to pH1N1 virus compared with nonpregnant women pre-IIV [median, 6.66 vs. 0.90% (P = 0.0149)] and 7 d post-IIV [median, 11.23 vs. 2.81% (P = 0.004)], indicating a heightened chemokine response in pregnant women that was further enhanced by the vaccination. Pregnant women also exhibited significantly increased T-cell production of MIP-1β and polyfunctionality in NK and T cells to pH1N1 virus pre- and post-IIV. NK- and T-cell polyfunctionality was also enhanced in pregnant women in response to the H3N2 viral strain. In contrast, pregnant women had significantly reduced NK- and T-cell responses to phorbol 12-myristate 13-acetate and ionomycin. This type of stimulation led to the conclusion that NK- and T-cell responses during pregnancy are suppressed, but clearly this conclusion is not correct relative to the more biologically relevant assays described here. Robust cellular immune responses to influenza during pregnancy could drive pulmonary inflammation, explaining increased morbidity and mortality.

    View details for DOI 10.1073/pnas.1416569111

    View details for PubMedID 25246558

  • Genetic and environmental determinants of human NK cell diversity revealed by mass cytometry. Science translational medicine Horowitz, A., Strauss-Albee, D. M., Leipold, M., Kubo, J., Nemat-Gorgani, N., Dogan, O. C., Dekker, C. L., Mackey, S., Maecker, H., Swan, G. E., Davis, M. M., Norman, P. J., Guethlein, L. A., Desai, M., Parham, P., Blish, C. A. 2013; 5 (208): 208ra145-?

    Abstract

    Natural killer (NK) cells play critical roles in immune defense and reproduction, yet remain the most poorly understood major lymphocyte population. Because their activation is controlled by a variety of combinatorially expressed activating and inhibitory receptors, NK cell diversity and function are closely linked. To provide an unprecedented understanding of NK cell repertoire diversity, we used mass cytometry to simultaneously analyze 37 parameters, including 28 NK cell receptors, on peripheral blood NK cells from 5 sets of monozygotic twins and 12 unrelated donors of defined human leukocyte antigen (HLA) and killer cell immunoglobulin-like receptor (KIR) genotype. This analysis revealed a remarkable degree of NK cell diversity, with an estimated 6000 to 30,000 phenotypic populations within an individual and >100,000 phenotypes in the donor panel. Genetics largely determined inhibitory receptor expression, whereas activation receptor expression was heavily environmentally influenced. Therefore, NK cells may maintain self-tolerance through strictly regulated expression of inhibitory receptors while using adaptable expression patterns of activating and costimulatory receptors to respond to pathogens and tumors. These findings further suggest the possibility that discrete NK cell subpopulations could be harnessed for immunotherapeutic strategies in the settings of infection, reproduction, and transplantation.

    View details for DOI 10.1126/scitranslmed.3006702

    View details for PubMedID 24154599

  • The human disease gene LYSET is essential for lysosomal enzyme transport and viral infection. Science (New York, N.Y.) Richards, C. M., Jabs, S., Qiao, W., Varanese, L. D., Schweizer, M., Mosen, P. R., Riley, N. M., Klüssendorf, M., Zengel, J. R., Flynn, R. A., Rustagi, A., Widen, J. C., Peters, C. E., Ooi, Y. S., Xie, X., Shi, P. Y., Bartenschlager, R., Puschnik, A. S., Bogyo, M., Bertozzi, C. R., Blish, C. A., Winter, D., Nagamine, C. M., Braulke, T., Carette, J. E. 2022: eabn5648

    Abstract

    Lysosomes are key degradative compartments of the cell. Transport to lysosomes relies on GlcNAc-1-phosphotransferase-mediated tagging of soluble enzymes with mannose 6-phosphate (M6P). GlcNAc-1-phosphotransferase deficiency leads to the severe lysosomal storage disorder mucolipidosis II (MLII). Several viruses require lysosomal cathepsins to cleave structural proteins and thus depend on functional GlcNAc-1-phosphotransferase. Here, we used genome-scale CRISPR screens to identify Lysosomal Enzyme Trafficking factor (LYSET) as essential for infection by cathepsin-dependent viruses including SARS-CoV-2. LYSET deficiency resulted in global loss of M6P tagging and mislocalization of GlcNAc-1-phosphotransferase from the Golgi complex to lysosomes. Lyset knockout mice exhibited MLII-like phenotypes and human pathogenic LYSET alleles failed to restore lysosomal sorting defects. Thus, LYSET is required for correct functioning of the M6P trafficking machinery, and mutations in LYSET can explain the phenotype of the associated disorder.

    View details for DOI 10.1126/science.abn5648

    View details for PubMedID 36074821

  • Deconvoluting complex correlates of COVID-19 severity with a multi-omic pandemic tracking strategy. Nature communications Parikh, V. N., Ioannidis, A. G., Jimenez-Morales, D., Gorzynski, J. E., De Jong, H. N., Liu, X., Roque, J., Cepeda-Espinoza, V. P., Osoegawa, K., Hughes, C., Sutton, S. C., Youlton, N., Joshi, R., Amar, D., Tanigawa, Y., Russo, D., Wong, J., Lauzon, J. T., Edelson, J., Mas Montserrat, D., Kwon, Y., Rubinacci, S., Delaneau, O., Cappello, L., Kim, J., Shoura, M. J., Raja, A. N., Watson, N., Hammond, N., Spiteri, E., Mallempati, K. C., Montero-Martín, G., Christle, J., Kim, J., Kirillova, A., Seo, K., Huang, Y., Zhao, C., Moreno-Grau, S., Hershman, S. G., Dalton, K. P., Zhen, J., Kamm, J., Bhatt, K. D., Isakova, A., Morri, M., Ranganath, T., Blish, C. A., Rogers, A. J., Nadeau, K., Yang, S., Blomkalns, A., O'Hara, R., Neff, N. F., DeBoever, C., Szalma, S., Wheeler, M. T., Gates, C. M., Farh, K., Schroth, G. P., Febbo, P., deSouza, F., Cornejo, O. E., Fernandez-Vina, M., Kistler, A., Palacios, J. A., Pinsky, B. A., Bustamante, C. D., Rivas, M. A., Ashley, E. A. 2022; 13 (1): 5107

    Abstract

    The SARS-CoV-2 pandemic has differentially impacted populations across race and ethnicity. A multi-omic approach represents a powerful tool to examine risk across multi-ancestry genomes. We leverage a pandemic tracking strategy in which we sequence viral and host genomes and transcriptomes from nasopharyngeal swabs of 1049 individuals (736 SARS-CoV-2 positive and 313 SARS-CoV-2 negative) and integrate them with digital phenotypes from electronic health records from a diverse catchment area in Northern California. Genome-wide association disaggregated by admixture mapping reveals novel COVID-19-severity-associated regions containing previously reported markers of neurologic, pulmonary and viral disease susceptibility. Phylodynamic tracking of consensus viral genomes reveals no association with disease severity or inferred ancestry. Summary data from multiomic investigation reveals metagenomic and HLA associations with severe COVID-19. The wealth of data available from residual nasopharyngeal swabs in combination with clinical data abstracted automatically at scale highlights a powerful strategy for pandemic tracking, and reveals distinct epidemiologic, genetic, and biological associations for those at the highest risk.

    View details for DOI 10.1038/s41467-022-32397-8

    View details for PubMedID 36042219

  • Programmable antivirals targeting critical conserved viral RNA secondary structures from influenza A virus and SARS-CoV-2. Nature medicine Hagey, R. J., Elazar, M., Pham, E. A., Tian, S., Ben-Avi, L., Bernardin-Souibgui, C., Yee, M. F., Moreira, F. R., Rabinovitch, M. V., Meganck, R. M., Fram, B., Beck, A., Gibson, S. A., Lam, G., Devera, J., Kladwang, W., Nguyen, K., Xiong, A., Schaffert, S., Avisar, T., Liu, P., Rustagi, A., Fichtenbaum, C. J., Pang, P. S., Khatri, P., Tseng, C., Taubenberger, J. K., Blish, C. A., Hurst, B. L., Sheahan, T. P., Das, R., Glenn, J. S. 2022

    Abstract

    Influenza A virus's (IAV's) frequent genetic changes challenge vaccine strategies and engender resistance to current drugs. We sought to identify conserved and essential RNA secondary structures within IAV's genome that are predicted to have greater constraints on mutation in response to therapeutic targeting. We identified and genetically validated an RNA structure (packaging stem-loop 2 (PSL2)) that mediates in vitro packaging and in vivo disease and is conserved across all known IAV isolates. A PSL2-targeting locked nucleic acid (LNA), administered 3d after, or 14d before, a lethal IAV inoculum provided 100% survival in mice, led to the development of strong immunity to rechallenge with a tenfold lethal inoculum, evaded attempts to select for resistance and retained full potency against neuraminidase inhibitor-resistant virus. Use of an analogous approach to target SARS-CoV-2, prophylactic administration of LNAs specific for highly conserved RNA structures in the viral genome, protected hamsters from efficient transmission of the SARS-CoV-2 USA_WA1/2020 variant. These findings highlight the potential applicability of this approach to any virus of interest via a process we term 'programmable antivirals', with implications for antiviral prophylaxis and post-exposure therapy.

    View details for DOI 10.1038/s41591-022-01908-x

    View details for PubMedID 35982307

  • Phenotypes of disease severity in a cohort of hospitalized COVID-19 patients: Results from the IMPACC study. EBioMedicine Ozonoff, A., Schaenman, J., Jayavelu, N. D., Milliren, C. E., Calfee, C. S., Cairns, C. B., Kraft, M., Baden, L. R., Shaw, A. C., Krammer, F., van Bakel, H., Esserman, D. A., Liu, S., Sesma, A. F., Simon, V., Hafler, D. A., Montgomery, R. R., Kleinstein, S. H., Levy, O., Bime, C., Haddad, E. K., Erle, D. J., Pulendran, B., Nadeau, K. C., Davis, M. M., Hough, C. L., Messer, W. B., Higuita, N. I., Metcalf, J. P., Atkinson, M. A., Brakenridge, S. C., Corry, D., Kheradmand, F., Ehrlich, L. I., Melamed, E., McComsey, G. A., Sekaly, R., Diray-Arce, J., Peters, B., Augustine, A. D., Reed, E. F., Altman, M. C., Becker, P. M., Rouphael, N. 2022; 83: 104208

    Abstract

    Better understanding of the association between characteristics of patients hospitalized with coronavirus disease 2019 (COVID-19) and outcome is needed to further improve upon patient management.Immunophenotyping Assessment in a COVID-19 Cohort (IMPACC) is a prospective, observational study of 1164 patients from 20 hospitals across the United States. Disease severity was assessed using a 7-point ordinal scale based on degree of respiratory illness. Patients were prospectively surveyed for 1 year after discharge for post-acute sequalae of COVID-19 (PASC) through quarterly surveys. Demographics, comorbidities, radiographic findings, clinical laboratory values, SARS-CoV-2 PCR and serology were captured over a 28-day period. Multivariable logistic regression was performed.The median age was 59 years (interquartile range [IQR] 20); 711 (61%) were men; overall mortality was 14%, and 228 (20%) required invasive mechanical ventilation. Unsupervised clustering of ordinal score over time revealed distinct disease course trajectories. Risk factors associated with prolonged hospitalization or death by day 28 included age ≥ 65 years (odds ratio [OR], 2.01; 95% CI 1.28-3.17), Hispanic ethnicity (OR, 1.71; 95% CI 1.13-2.57), elevated baseline creatinine (OR 2.80; 95% CI 1.63- 4.80) or troponin (OR 1.89; 95% 1.03-3.47), baseline lymphopenia (OR 2.19; 95% CI 1.61-2.97), presence of infiltrate by chest imaging (OR 3.16; 95% CI 1.96-5.10), and high SARS-CoV2 viral load (OR 1.53; 95% CI 1.17-2.00). Fatal cases had the lowest ratio of SARS-CoV-2 antibody to viral load levels compared to other trajectories over time (p=0.001). 589 survivors (51%) completed at least one survey at follow-up with 305 (52%) having at least one symptom consistent with PASC, most commonly dyspnea (56% among symptomatic patients). Female sex was the only associated risk factor for PASC.Integration of PCR cycle threshold, and antibody values with demographics, comorbidities, and laboratory/radiographic findings identified risk factors for 28-day outcome severity, though only female sex was associated with PASC. Longitudinal clinical phenotyping offers important insights, and provides a framework for immunophenotyping for acute and long COVID-19.NIH.

    View details for DOI 10.1016/j.ebiom.2022.104208

    View details for PubMedID 35952496

  • An intranasal ASO therapeutic targeting SARS-CoV-2. Nature communications Zhu, C., Lee, J. Y., Woo, J. Z., Xu, L., Nguyenla, X., Yamashiro, L. H., Ji, F., Biering, S. B., Van Dis, E., Gonzalez, F., Fox, D., Wehri, E., Rustagi, A., Pinsky, B. A., Schaletzky, J., Blish, C. A., Chiu, C., Harris, E., Sadreyev, R. I., Stanley, S., Kauppinen, S., Rouskin, S., Näär, A. M. 2022; 13 (1): 4503

    Abstract

    The COVID-19 pandemic is exacting an increasing toll worldwide, with new SARS-CoV-2 variants emerging that exhibit higher infectivity rates and that may partially evade vaccine and antibody immunity. Rapid deployment of non-invasive therapeutic avenues capable of preventing infection by all SARS-CoV-2 variants could complement current vaccination efforts and help turn the tide on the COVID-19 pandemic. Here, we describe a novel therapeutic strategy targeting the SARS-CoV-2 RNA using locked nucleic acid antisense oligonucleotides (LNA ASOs). We identify an LNA ASO binding to the 5' leader sequence of SARS-CoV-2 that disrupts a highly conserved stem-loop structure with nanomolar efficacy in preventing viral replication in human cells. Daily intranasal administration of this LNA ASO in the COVID-19 mouse model potently suppresses viral replication (>80-fold) in the lungs of infected mice. We find that the LNA ASO is efficacious in countering all SARS-CoV-2 "variants of concern" tested both in vitro and in vivo. Hence, inhaled LNA ASOs targeting SARS-CoV-2 represents a promising therapeutic approach to reduce or prevent transmission and decrease severity of COVID-19 in infected individuals. LNA ASOs are chemically stable and can be flexibly modified to target different viral RNA sequences and could be stockpiled for future coronavirus pandemics.

    View details for DOI 10.1038/s41467-022-32216-0

    View details for PubMedID 35922434

  • Genome-wide bidirectional CRISPR screens identify mucins as host factors modulating SARS-CoV-2 infection. Nature genetics Biering, S. B., Sarnik, S. A., Wang, E., Zengel, J. R., Leist, S. R., Schafer, A., Sathyan, V., Hawkins, P., Okuda, K., Tau, C., Jangid, A. R., Duffy, C. V., Wei, J., Gilmore, R. C., Alfajaro, M. M., Strine, M. S., Nguyenla, X., Van Dis, E., Catamura, C., Yamashiro, L. H., Belk, J. A., Begeman, A., Stark, J. C., Shon, D. J., Fox, D. M., Ezzatpour, S., Huang, E., Olegario, N., Rustagi, A., Volmer, A. S., Livraghi-Butrico, A., Wehri, E., Behringer, R. R., Cheon, D., Schaletzky, J., Aguilar, H. C., Puschnik, A. S., Button, B., Pinsky, B. A., Blish, C. A., Baric, R. S., O'Neal, W. K., Bertozzi, C. R., Wilen, C. B., Boucher, R. C., Carette, J. E., Stanley, S. A., Harris, E., Konermann, S., Hsu, P. D. 2022

    Abstract

    Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) causes a range of symptoms in infected individuals, from mild respiratory illness to acute respiratory distress syndrome. A systematic understanding of host factors influencing viral infection is critical to elucidate SARS-CoV-2-host interactions and the progression of Coronavirus disease 2019 (COVID-19). Here, we conducted genome-wide CRISPR knockout and activation screens in human lung epithelial cells with endogenous expression of the SARS-CoV-2 entry factors ACE2 and TMPRSS2. We uncovered proviral and antiviral factors across highly interconnected host pathways, including clathrin transport, inflammatory signaling, cell-cycle regulation, and transcriptional and epigenetic regulation. We further identified mucins, a family of high molecular weight glycoproteins, as a prominent viral restriction network that inhibits SARS-CoV-2 infection in vitro and in murine models. These mucins also inhibit infection of diverse respiratory viruses. This functional landscape of SARS-CoV-2 host factors provides a physiologically relevant starting point for new host-directed therapeutics and highlights airway mucins as a host defense mechanism.

    View details for DOI 10.1038/s41588-022-01131-x

    View details for PubMedID 35879412

  • 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

  • Broad-spectrum CRISPR-mediated inhibition of SARS-CoV-2 variants and endemic coronaviruses in vitro. Nature communications Zeng, L., Liu, Y., Nguyenla, X. H., Abbott, T. R., Han, M., Zhu, Y., Chemparathy, A., Lin, X., Chen, X., Wang, H., Rane, D. A., Spatz, J. M., Jain, S., Rustagi, A., Pinsky, B., Zepeda, A. E., Kadina, A. P., Walker, J. A., Holden, K., Temperton, N., Cochran, J. R., Barron, A. E., Connolly, M. D., Blish, C. A., Lewis, D. B., Stanley, S. A., La Russa, M. F., Qi, L. S. 2022; 13 (1): 2766

    Abstract

    A major challenge in coronavirus vaccination and treatment is to counteract rapid viral evolution and mutations. Here we demonstrate that CRISPR-Cas13d offers a broad-spectrum antiviral (BSA) to inhibit many SARS-CoV-2 variants and diverse human coronavirus strains with >99% reduction of the viral titer. We show that Cas13d-mediated coronavirus inhibition is dependent on the crRNA cellular spatial colocalization with Cas13d and target viral RNA. Cas13d can significantly enhance the therapeutic effects of diverse small molecule drugs against coronaviruses for prophylaxis or treatment purposes, and the best combination reduced viral titer by over four orders of magnitude. Using lipid nanoparticle-mediated RNA delivery, we demonstrate that the Cas13d system can effectively treat infection from multiple variants of coronavirus, including Omicron SARS-CoV-2, in human primary airway epithelium air-liquid interface (ALI) cultures. Our study establishes CRISPR-Cas13 as a BSA which is highly complementary to existing vaccination and antiviral treatment strategies.

    View details for DOI 10.1038/s41467-022-30546-7

    View details for PubMedID 35589813

  • TNF-alpha+ CD4+ Tcells dominate the SARS-CoV-2 specific T cell response in COVID-19 outpatients and are associated with durable antibodies. Cell reports. Medicine van der Ploeg, K., Kirosingh, A. S., Mori, D. A., Chakraborty, S., Hu, Z., Sievers, B. L., Jacobson, K. B., Bonilla, H., Parsonnet, J., Andrews, J. R., Press, K. D., Ty, M. C., Ruiz-Betancourt, D. R., de la Parte, L., Tan, G. S., Blish, C. A., Takahashi, S., Rodriguez-Barraquer, I., Greenhouse, B., Singh, U., Wang, T. T., Jagannathan, P. 2022: 100640

    Abstract

    Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-specific CD4+ Tcells are likely important in immunity against coronavirus 2019 (COVID-19), but our understanding of CD4+ longitudinal dynamics following infection and of specific features that correlate with the maintenance of neutralizing antibodies remains limited. Here, we characterize SARS-CoV-2-specific CD4+ Tcells in a longitudinal cohort of 109 COVID-19 outpatients enrolled during acute infection. The quality of the SARS-CoV-2-specific CD4+ response shifts from cells producing interferon gamma (IFNgamma) to tumor necrosis factor alpha (TNF-alpha) from 5days to 4months post-enrollment, with IFNgamma-IL-21-TNF-alpha+ CD4+ Tcells the predominant population detected at later time points. Greater percentages of IFNgamma-IL-21-TNF-alpha+ CD4+ Tcells on day 28 correlate with SARS-CoV-2-neutralizing antibodies measured 7months post-infection (⍴= 0.4, p= 0.01). mRNA vaccination following SARS-CoV-2 infection boosts both IFNgamma- and TNF-alpha-producing, spike-protein-specific CD4+ Tcells. These data suggest that SARS-CoV-2-specific, TNF-alpha-producing CD4+ Tcells may play an important role in antibody maintenance following COVID-19.

    View details for DOI 10.1016/j.xcrm.2022.100640

    View details for PubMedID 35588734

  • Facile discovery of surrogate cytokine agonists. Cell Yen, M., Ren, J., Liu, Q., Glassman, C. R., Sheahan, T. P., Picton, L. K., Moreira, F. R., Rustagi, A., Jude, K. M., Zhao, X., Blish, C. A., Baric, R. S., Su, L. L., Garcia, K. C. 2022

    Abstract

    Cytokines are powerful immune modulators that initiate signaling through receptor dimerization, but natural cytokines have structural limitations as therapeutics. We present a strategy to discover cytokine surrogate agonists by using modular ligands that exploit induced proximity and receptor dimer geometry as pharmacological metrics amenable to high-throughput screening. Using VHH and scFv to human interleukin-2/15, type-I interferon, and interleukin-10 receptors, we generated combinatorial matrices of single-chain bispecific ligands that exhibited diverse spectrums of functional activities, including potent inhibition of SARS-CoV-2 by surrogate interferons. Crystal structures of IL-2R:VHH complexes revealed that variation in receptor dimer geometries resulted in functionally diverse signaling outputs. This modular platform enabled engineering of surrogate ligands that compelled assembly of an IL-2R/IL-10R heterodimer, which does not naturally exist, that signaled through pSTAT5 on T and natural killer (NK) cells. This "cytokine med-chem" approach, rooted in principles of induced proximity, is generalizable for discovery of diversified agonists for many ligand-receptor systems.

    View details for DOI 10.1016/j.cell.2022.02.025

    View details for PubMedID 35325595

  • Gastrointestinal Perforation in a patient with Anti-nuclear Matrix Protein 2 Antibody Positive Dermatomyositis. Arthritis care & research Valenzuela, A., Rieger, K. E., Blish, C. A., Chung, L., Fiorentino, D. 2022

    View details for DOI 10.1002/acr.24879

    View details for PubMedID 35287251

  • The immunology and immunopathology of COVID-19. Science (New York, N.Y.) Merad, M., Blish, C. A., Sallusto, F., Iwasaki, A. 2022; 375 (6585): 1122-1127

    Abstract

    Considerable research effort has been made worldwide to decipher the immune response triggered upon severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infections, identify the drivers of severe and fatal COVID-19, and understand what leads to the prolongation of symptoms after disease resolution. We review the results of almost 2 years of COVID-19 immunology research and discuss definitive findings and remaining questions regarding our understanding of COVID-19 pathophysiology. We discuss emerging understanding of differences in immune responses seen in those with and without Long Covid syndrome, also known as post-acute sequelae of SARS-CoV-2. We hope that the knowledge gained from this COVID-19 research will be applied in studies of inflammatory processes involved in critical and chronic illnesses, which remain a major unmet need.

    View details for DOI 10.1126/science.abm8108

    View details for PubMedID 35271343

  • Comparative analysis of cell-cell communication at single-cell resolution. bioRxiv : the preprint server for biology Wilk, A. J., Shalek, A. K., Holmes, S., Blish, C. A. 2022

    Abstract

    Inference of cell-cell communication (CCC) from single-cell RNA-sequencing data is a powerful technique to uncover putative axes of multicellular coordination, yet existing methods perform this analysis at the level of the cell type or cluster, discarding single-cell level information. Here we present Scriabin a" a flexible and scalable framework for comparative analysis of CCC at single-cell resolution. We leverage multiple published datasets to show that Scriabin recovers expected CCC edges and use spatial transcriptomic data to validate that the recovered edges are biologically meaningful. We then apply Scriabin to uncover co-expressed programs of CCC from atlas-scale datasets, validating known communication pathways required for maintaining the intestinal stem cell niche as well as previously unappreciated modes of intercellular communication. Finally, we utilize single-cell communication networks calculated using Scriabin to follow communication pathways that operate between timepoints in longitudinal datasets, highlighting bystander cells as important initiators of inflammatory reactions in acute SARS-CoV-2 infection. Our approach represents a broadly applicable strategy to leverage single-cell resolution data maximally toward uncovering CCC circuitry and rich niche-phenotype relationships in health and disease.

    View details for DOI 10.1101/2022.02.04.479209

    View details for PubMedID 35169794

  • Innovative vaccine approaches-a Keystone Symposia report. Annals of the New York Academy of Sciences Cable, J., Rappuoli, R., Klemm, E. J., Kang, G., Mutreja, A., Wright, G. J., Pizza, M., Castro, S. A., Hoffmann, J. P., Alter, G., Carfi, A., Pollard, A. J., Krammer, F., Gupta, R. K., Wagner, C. E., Machado, V., Modjarrad, K., Corey, L., B Gilbert, P., Dougan, G., Lurie, N., Bjorkman, P. J., Chiu, C., Nemes, E., Gordon, S. B., Steer, A. C., Rudel, T., Blish, C. A., Sandberg, J. T., Brennan, K., Klugman, K. P., Stuart, L. M., Madhi, S. A., Karp, C. L. 1800

    Abstract

    The rapid development of COVID-19 vaccines was the result of decades of research to establish flexible vaccine platforms and understand pathogens with pandemic potential, as well as several novel changes to the vaccine discovery and development processes that partnered industry and governments. And while vaccines offer the potential to drastically improve global health, low-and-middle-income countries around the world often experience reduced access to vaccines and reduced vaccine efficacy. Addressing these issues will require novel vaccine approaches and platforms, deeper insight how vaccines mediate protection, and innovative trial designs and models. On June 28-30, 2021, experts in vaccine research, development, manufacturing, and deployment met virtually for the Keystone eSymposium "Innovative Vaccine Approaches" to discuss advances in vaccine research and development.

    View details for DOI 10.1111/nyas.14739

    View details for PubMedID 35029310

  • 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

  • Association Between SARS-CoV-2 RNAemia and Postacute Sequelae of COVID-19. Open forum infectious diseases Ram-Mohan, N., Kim, D., Rogers, A. J., Blish, C. A., Nadeau, K. C., Blomkalns, A. L., Yang, S. 2022; 9 (2): ofab646

    Abstract

    Determinants of Post-Acute Sequelae of COVID-19 are not known. Here we show that 83.3% of patients with viral RNA in blood (RNAemia) at presentation were symptomatic in the post-acute phase. RNAemia at presentation successfully predicted PASC, independent of patient demographics, worst disease severity, and length of symptoms.

    View details for DOI 10.1093/ofid/ofab646

    View details for PubMedID 35111870

    View details for PubMedCentralID PMC8802799

  • Deep Phenotypic Analysis of Blood and Lymphoid T and NK Cells From HIV+ Controllers and ART-Suppressed Individuals. Frontiers in immunology George, A. F., Luo, X., Neidleman, J., Hoh, R., Vohra, P., Thomas, R., Shin, M., Lee, M. J., Blish, C. A., Deeks, S. G., Greene, W. C., Lee, S. A., Roan, N. R. 2022; 13: 803417

    Abstract

    T and natural killer (NK) cells are effector cells with key roles in anti-HIV immunity, including in lymphoid tissues, the major site of HIV persistence. However, little is known about the features of these effector cells from people living with HIV (PLWH), particularly from those who initiated antiretroviral therapy (ART) during acute infection. Our study design was to use 42-parameter CyTOF to conduct deep phenotyping of paired blood- and lymph node (LN)-derived T and NK cells from three groups of HIV+ aviremic individuals: elite controllers (N = 5), and ART-suppressed individuals who had started therapy during chronic (N = 6) vs. acute infection (N = 8), the latter of which is associated with better outcomes. We found that acute-treated individuals are enriched for specific subsets of T and NK cells, including blood-derived CD56-CD16+ NK cells previously associated with HIV control, and LN-derived CD4+ T follicular helper cells with heightened expansion potential. An in-depth comparison of the features of the cells from blood vs. LNs of individuals from our cohort revealed that T cells from blood were more activated than those from LNs. By contrast, LNs were enriched for follicle-homing CXCR5+ CD8+ T cells, which expressed increased levels of inhibitory receptors and markers of survival and proliferation as compared to their CXCR5- counterparts. In addition, a subset of memory-like CD56brightTCF1+ NK cells was enriched in LNs relative to blood. These results together suggest unique T and NK cell features in acute-treated individuals, and highlight the importance of examining effector cells not only in blood but also the lymphoid tissue compartment, where the reservoir mostly persists, and where these cells take on distinct phenotypic features.

    View details for DOI 10.3389/fimmu.2022.803417

    View details for PubMedID 35154118

  • The B.1.427/1.429 (epsilon) SARS-CoV-2 variants are more virulent than ancestral B.1 (614G) in Syrian hamsters. PLoS pathogens Carroll, T., Fox, D., van Doremalen, N., Ball, E., Morris, M. K., Sotomayor-Gonzalez, A., Servellita, V., Rustagi, A., Yinda, C. K., Fritts, L., Port, J. R., Ma, Z. M., Holbrook, M. G., Schulz, J., Blish, C. A., Hanson, C., Chiu, C. Y., Munster, V., Stanley, S., Miller, C. J. 2022; 18 (2): e1009914

    Abstract

    As novel SARS-CoV-2 variants continue to emerge, it is critical that their potential to cause severe disease and evade vaccine-induced immunity is rapidly assessed in humans and studied in animal models. In early January 2021, a novel SARS-CoV-2 variant designated B.1.429 comprising 2 lineages, B.1.427 and B.1.429, was originally detected in California (CA) and it was shown to have enhanced infectivity in vitro and decreased antibody neutralization by plasma from convalescent patients and vaccine recipients. Here we examine the virulence, transmissibility, and susceptibility to pre-existing immunity for B 1.427 and B 1.429 in the Syrian hamster model. We find that both variants exhibit enhanced virulence as measured by increased body weight loss compared to hamsters infected with ancestral B.1 (614G), with B.1.429 causing the most marked body weight loss among the 3 variants. Faster dissemination from airways to parenchyma and more severe lung pathology at both early and late stages were also observed with B.1.429 infections relative to B.1. (614G) and B.1.427 infections. In addition, subgenomic viral RNA (sgRNA) levels were highest in oral swabs of hamsters infected with B.1.429, however sgRNA levels in lungs were similar in all three variants. This demonstrates that B.1.429 replicates to higher levels than ancestral B.1 (614G) or B.1.427 in the oropharynx but not in the lungs. In multi-virus in-vivo competition experiments, we found that B.1. (614G), epsilon (B.1.427/B.1.429) and gamma (P.1) dramatically outcompete alpha (B.1.1.7), beta (B.1.351) and zeta (P.2) in the lungs. In the nasal cavity, B.1. (614G), gamma, and epsilon dominate, but the highly infectious alpha variant also maintains a moderate size niche. We did not observe significant differences in airborne transmission efficiency among the B.1.427, B.1.429 and ancestral B.1 (614G) and WA-1 variants in hamsters. These results demonstrate enhanced virulence and high relative oropharyngeal replication of the epsilon (B.1.427/B.1.429) variant in Syrian hamsters compared to an ancestral B.1 (614G) variant.

    View details for DOI 10.1371/journal.ppat.1009914

    View details for PubMedID 35143587

  • Natural Killer Cell Receptors and Ligands Are Associated With Markers of HIV-1 Persistence in Chronically Infected ART Suppressed Patients. Frontiers in cellular and infection microbiology Ivison, G. T., Vendrame, E., Martinez-Colon, G. J., Ranganath, T., Vergara, R., Zhao, N. Q., Martin, M. P., Bendall, S. C., Carrington, M., Cyktor, J. C., McMahon, D. K., Eron, J., Jones, R. B., Mellors, J. W., Bosch, R. J., Gandhi, R. T., Holmes, S., Blish, C. A., ACTG 5321 Team 2022; 12: 757846

    Abstract

    The latent HIV-1 reservoir represents a major barrier to achieving a long-term antiretroviral therapy (ART)-free remission or cure for HIV-1. Natural Killer (NK) cells are innate immune cells that play a critical role in controlling viral infections and have been shown to be involved in preventing HIV-1 infection and, in those who are infected, delaying time to progression to AIDS. However, their role in limiting HIV-1 persistence on long term ART is still uncharacterized. To identify associations between markers of HIV-1 persistence and the NK cell receptor-ligand repertoire, we used twin mass cytometry panels to characterize the peripheral blood NK receptor-ligand repertoire in individuals with long-term antiretroviral suppression enrolled in the AIDS Clinical Trial Group A5321 study. At the time of testing, participants had been on ART for a median of 7 years, with virological suppression <50 copies/mL since at most 48 weeks on ART. We found that the NK cell receptor and ligand repertoires did not change across three longitudinal samples over one year-a median of 25 weeks and 50 weeks after the initial sampling. To determine the features of the receptor-ligand repertoire that associate with markers of HIV-1 persistence, we performed a LASSO normalized regression. This analysis revealed that the NK cell ligands CD58, HLA-B, and CRACC, as well as the killer cell immunoglobulin-like receptors (KIRs) KIR2DL1, KIR2DL3, and KIR2DS4 were robustly predictive of markers of HIV-1 persistence, as measured by total HIV-1 cell-associated DNA, HIV-1 cell-associated RNA, and single copy HIV-RNA assays. To characterize the roles of cell populations defined by multiple markers, we augmented the LASSO analysis with FlowSOM clustering. This analysis found that a less mature NK cell phenotype (CD16+CD56dimCD57-LILRB1-NKG2C-) was associated with lower HIV-1 cell associated DNA. Finally, we found that surface expression of HLA-Bw6 measured by CyTOF was associated with lower HIV-1 persistence. Genetic analysis revealed that this was driven by lower HIV-1 persistence in HLA-Bw4/6 heterozygotes. These findings suggest that there may be a role for NK cells in controlling HIV-1 persistence in individuals on long-term ART, which must be corroborated by future studies.

    View details for DOI 10.3389/fcimb.2022.757846

    View details for PubMedID 35223535

  • Stereotypic Expansion of T Regulatory and Th17 Cells during Infancy Is Disrupted by HIV Exposure and Gut Epithelial Damage. Journal of immunology (Baltimore, Md. : 1950) Dzanibe, S., Lennard, K., Kiravu, A., Seabrook, M. S., Alinde, B., Holmes, S. P., Blish, C. A., Jaspan, H. B., Gray, C. M. 2021

    Abstract

    Few studies have investigated immune cell ontogeny throughout the neonatal and early pediatric period, when there is often increased vulnerability to infections. In this study, we evaluated the dynamics of two critical T cell populations, T regulatory (Treg) cells and Th17 cells, over the first 36 wk of human life. First, we observed distinct CD4+ T cells phenotypes between cord blood and peripheral blood, collected within 12 h of birth, showing that cord blood is not a surrogate for newborn blood. Second, both Treg and Th17 cells expanded in a synchronous fashion over 36 wk of life. However, comparing infants exposed to HIV in utero, but remaining uninfected, with HIV-unexposed uninfected control infants, there was a lower frequency of peripheral blood Treg cells at birth, resulting in a delayed expansion, and then declining again at 36 wk. Focusing on birth events, we found that Treg cells coexpressing CCR4 and alpha4beta7 inversely correlated with plasma concentrations of CCL17 (the ligand for CCR4) and intestinal fatty acid binding protein, IL-7, and CCL20. This was in contrast with Th17 cells, which showed a positive association with these plasma analytes. Thus, despite the stereotypic expansion of both cell subsets over the first few months of life, there was a disruption in the balance of Th17 to Treg cells at birth likely being a result of gut damage and homing of newborn Treg cells from the blood circulation to the gut.

    View details for DOI 10.4049/jimmunol.2100503

    View details for PubMedID 34819390

  • The proximal proteome of 17 SARS-CoV-2 proteins links to disrupted antiviral signaling and host translation. PLoS pathogens Meyers, J. M., Ramanathan, M., Shanderson, R. L., Beck, A., Donohue, L., Ferguson, I., Guo, M. G., Rao, D. S., Miao, W., Reynolds, D., Yang, X., Zhao, Y., Yang, Y., Blish, C., Wang, Y., Khavari, P. A. 2021; 17 (10): e1009412

    Abstract

    Viral proteins localize within subcellular compartments to subvert host machinery and promote pathogenesis. To study SARS-CoV-2 biology, we generated an atlas of 2422 human proteins vicinal to 17 SARS-CoV-2 viral proteins using proximity proteomics. This identified viral proteins at specific intracellular locations, such as association of accessary proteins with intracellular membranes, and projected SARS-CoV-2 impacts on innate immune signaling, ER-Golgi transport, and protein translation. It identified viral protein adjacency to specific host proteins whose regulatory variants are linked to COVID-19 severity, including the TRIM4 interferon signaling regulator which was found proximal to the SARS-CoV-2 M protein. Viral NSP1 protein adjacency to the EIF3 complex was associated with inhibited host protein translation whereas ORF6 localization with MAVS was associated with inhibited RIG-I 2CARD-mediated IFNB1 promoter activation. Quantitative proteomics identified candidate host targets for the NSP5 protease, with specific functional cleavage sequences in host proteins CWC22 and FANCD2. This data resource identifies host factors proximal to viral proteins in living human cells and nominates pathogenic mechanisms employed by SARS-CoV-2.

    View details for DOI 10.1371/journal.ppat.1009412

    View details for PubMedID 34597346

  • The B.1.427/1.429 (epsilon) SARS-CoV-2 variants are more virulent than ancestral B.1 (614G) in Syrian hamsters. bioRxiv : the preprint server for biology Carroll, T., Fox, D., van Doremalen, N., Ball, E., Morris, M. K., Sotomayor-Gonzalez, A., Servellita, V., Rustagi, A., Yinda, C. K., Fritts, L., Port, J. R., Ma, Z., Holbrook, M., Schulz, J., Blish, C. A., Hanson, C., Chiu, C. Y., Munster, V., Stanley, S., Miller, C. J. 2021

    Abstract

    As novel SARS-CoV-2 variants continue to emerge, it is critical that their potential to cause severe disease and evade vaccine-induced immunity is rapidly assessed in humans and studied in animal models. In early January 2021, a novel variant of concern (VOC) designated B.1.429 comprising 2 lineages, B.1.427 and B.1.429, was originally detected in California (CA) and shown to enhance infectivity in vitro and decrease antibody neutralization by plasma from convalescent patients and vaccine recipients. Here we examine the virulence, transmissibility, and susceptibility to pre-existing immunity for B 1.427 and B 1.429 in the Syrian hamster model. We find that both strains exhibit enhanced virulence as measured by increased body weight loss compared to hamsters infected with ancestral B.1 (614G), with B.1.429 causing the most body weight loss among all 3 lineages. Faster dissemination from airways to parenchyma and more severe lung pathology at both early and late stages were also observed with B.1.429 infections relative to B.1. (614G) and B.1.427 infections. In addition, subgenomic viral RNA (sgRNA) levels were highest in oral swabs of hamsters infected with B.1.429, however sgRNA levels in lungs were similar in all three strains. This demonstrates that B.1.429 replicates to higher levels than ancestral B.1 (614G) or B.1.427 in the upper respiratory tract (URT) but not in the lungs. In multi-virus in-vivo competition experiments, we found that epsilon (B.1.427/B.1.429) and gamma (P.1) dramatically outcompete alpha (B.1.1.7), beta (B.1.351) and zeta (P.2) in the lungs. In the URT gamma, and epsilon dominate, but the highly infectious alpha variant also maintains a moderate size niche. We did not observe significant differences in airborne transmission efficiency among the B.1.427, B.1.429 and ancestral B.1 (614G) variants in hamsters. These results demonstrate enhanced virulence and high relative fitness of the epsilon (B.1.427/B.1.429) variant in Syrian hamsters compared to an ancestral B.1 (614G) strain.Author Summary: In the last 12 months new variants of SARS-CoV-2 have arisen in the UK, South Africa, Brazil, India, and California. New SARS-CoV-2 variants will continue to emerge for the foreseeable future in the human population and the potential for these new variants to produce severe disease and evade vaccines needs to be understood. In this study, we used the hamster model to determine the epsilon (B.1.427/429) SARS-CoV-2 strains that emerged in California in late 2020 cause more severe disease and infected hamsters have higher viral loads in the upper respiratory tract compared to the prior B.1 (614G) strain. These findings are consistent with human clinical data and help explain the emergence and rapid spread of this strain in early 2021.

    View details for DOI 10.1101/2021.08.25.457626

    View details for PubMedID 34462750

  • Integrated analysis of multimodal single-cell data. Cell Hao, Y., Hao, S., Andersen-Nissen, E., Mauck, W. M., Zheng, S., Butler, A., Lee, M. J., Wilk, A. J., Darby, C., Zager, M., Hoffman, P., Stoeckius, M., Papalexi, E., Mimitou, E. P., Jain, J., Srivastava, A., Stuart, T., Fleming, L. M., Yeung, B., Rogers, A. J., McElrath, J. M., Blish, C. A., Gottardo, R., Smibert, P., Satija, R. 2021

    Abstract

    The simultaneous measurement of multiple modalities represents an exciting frontier for single-cell genomics and necessitates computational methods that can define cellular states based on multimodal data. Here, we introduce "weighted-nearest neighbor" analysis, an unsupervised framework to learn the relative utility of each data type in each cell, enabling an integrative analysis of multiple modalities. We apply our procedure to a CITE-seq dataset of 211,000 human peripheral blood mononuclear cells (PBMCs) with panels extending to 228 antibodies to construct a multimodal reference atlas of the circulating immune system. Multimodal analysis substantially improves our ability to resolve cell states, allowing us to identify and validate previously unreported lymphoid subpopulations. Moreover, we demonstrate how to leverage this reference to rapidly map new datasets and to interpret immune responses to vaccination and coronavirus disease 2019 (COVID-19). Our approach represents a broadly applicable strategy to analyze single-cell multimodal datasets and to look beyond the transcriptome toward a unified and multimodal definition of cellular identity.

    View details for DOI 10.1016/j.cell.2021.04.048

    View details for PubMedID 34062119

  • Physical Properties of COVID-19 Acute Respiratory Distress Syndrome (ARDS) Sputum Pacheco-Navarro, A., Kratochvil, M. J., Kaber, G., Roque, J., Blish, C., Yang, S., Nadeau, K. C., Heilshorn, S. C., Milla, C. E., Rogers, A., Bollyky, P. AMER THORACIC SOC. 2021
  • Clinician Prognostic Scoring System Reveals COVID-19 Patients' Prognosis More Likely to Be Limited by Acute Illness Relative to Traditional ICU Patients Moore, A. R., Pienkos, S., Nadeau, K. C., Blish, C., Yang, S., Wilson, J. G., Levitt, J. E., Rogers, A., Stanford COVID-19 Biobank AMER THORACIC SOC. 2021
  • Synthetic Siglec-9 Agonists Inhibit Neutrophil Activation Associated with COVID-19 ACS CENTRAL SCIENCE Delaveris, C. S., Wilk, A. J., Riley, N. M., Stark, J. C., Yang, S. S., Rogers, A. J., Ranganath, T., Nadeau, K. C., Blish, C. A., Bertozzi, C. R., Stanford COVID-19 Biobank 2021; 7 (4): 650-657
  • CytoGLMM: conditional differential analysis for flow and mass cytometry experiments. BMC bioinformatics Seiler, C., Ferreira, A., Kronstad, L. M., Simpson, L. J., Le Gars, M., Vendrame, E., Blish, C. A., Holmes, S. 2021; 22 (1): 137

    Abstract

    BACKGROUND: Flow and mass cytometry are important modern immunology tools for measuring expression levels of multiple proteins on single cells. The goal is to better understand the mechanisms of responses on a single cell basis by studying differential expression of proteins. Most current data analysis tools compare expressions across many computationally discovered cell types. Our goal is to focus on just one cell type. Our narrower field of application allows us to define a more specific statistical model with easier to control statistical guarantees.RESULTS: Differential analysis of marker expressions can be difficult due to marker correlations and inter-subject heterogeneity, particularly for studies of human immunology. We address these challenges with two multiple regression strategies: a bootstrapped generalized linear model and a generalized linear mixed model. On simulated datasets, we compare the robustness towards marker correlations and heterogeneity of both strategies. For paired experiments, we find that both strategies maintain the target false discovery rate under medium correlations and that mixed models are statistically more powerful under the correct model specification. For unpaired experiments, our results indicate that much larger patient sample sizes are required to detect differences. We illustrate the CytoGLMM R package and workflow for both strategies on a pregnancy dataset.CONCLUSION: Our approach to finding differential proteins in flow and mass cytometry data reduces biases arising from marker correlations and safeguards against false discoveries induced by patient heterogeneity.

    View details for DOI 10.1186/s12859-021-04067-x

    View details for PubMedID 33752595

  • Peginterferon Lambda-1a for treatment of outpatients with uncomplicated COVID-19: a randomized placebo-controlled trial. Nature communications Jagannathan, P. n., Andrews, J. R., Bonilla, H. n., Hedlin, H. n., Jacobson, K. B., Balasubramanian, V. n., Purington, N. n., Kamble, S. n., de Vries, C. R., Quintero, O. n., Feng, K. n., Ley, C. n., Winslow, D. n., Newberry, J. n., Edwards, K. n., Hislop, C. n., Choong, I. n., Maldonado, Y. n., Glenn, J. n., Bhatt, A. n., Blish, C. n., Wang, T. n., Khosla, C. n., Pinsky, B. A., Desai, M. n., Parsonnet, J. n., Singh, U. n. 2021; 12 (1): 1967

    Abstract

    Type III interferons have been touted as promising therapeutics in outpatients with coronavirus disease 2019 (COVID-19). We conducted a randomized, single-blind, placebo-controlled trial (NCT04331899) in 120 outpatients with mild to moderate COVID-19 to determine whether a single, 180 mcg subcutaneous dose of Peginterferon Lambda-1a (Lambda) within 72 hours of diagnosis could shorten the duration of viral shedding (primary endpoint) or symptoms (secondary endpoint). In both the 60 patients receiving Lambda and 60 receiving placebo, the median time to cessation of viral shedding was 7 days (hazard ratio [HR] = 0.81; 95% confidence interval [CI] 0.56 to 1.19). Symptoms resolved in 8 and 9 days in Lambda and placebo, respectively, and symptom duration did not differ significantly between groups (HR 0.94; 95% CI 0.64 to 1.39). Both Lambda and placebo were well-tolerated, though liver transaminase elevations were more common in the Lambda vs. placebo arm (15/60 vs 5/60; p = 0.027). In this study, a single dose of subcutaneous Peginterferon Lambda-1a neither shortened the duration of SARS-CoV-2 viral shedding nor improved symptoms in outpatients with uncomplicated COVID-19.

    View details for DOI 10.1038/s41467-021-22177-1

    View details for PubMedID 33785743

  • 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

  • SARS-CoV-2 subgenomic RNA kinetics in longitudinal clinical samples Open Forum Infectious Diseases Verma, R., Kim, E., Martinez, G., Jagannathan, ., Rustagi, A., Parsonnet, J., Bonilla, H., Khosla, C., Holubar, M., Subramanian, A., Singh, ., Maldonado, Y., Blish, C., Andrews, J. 2021

    View details for DOI 10.1093/ofid/ofab310

  • SARS-CoV-2 RNAemia predicts clinical deterioration and extrapulmonary complications from COVID-19. Clinical infectious diseases : an official publication of the Infectious Diseases Society of America Ram-Mohan, N. n., Kim, D. n., Zudock, E. J., Hashemi, M. M., Tjandra, K. C., Rogers, A. J., Blish, C. A., Nadeau, K. C., Newberry, J. A., Quinn, J. V., O'Hara, R. n., Ashley, E. n., Nguyen, H. n., Jiang, L. n., Hung, P. n., Blomkalns, A. L., Yang, S. n. 2021

    Abstract

    The determinants of COVID-19 disease severity and extrapulmonary complications (EPCs) are poorly understood. We characterized relationships between SARS-CoV-2 RNAemia and disease severity, clinical deterioration, and specific EPCs.We used quantitative (qPCR) and digital (dPCR) PCR to quantify SARS-CoV-2 RNA from plasma in 191 patients presenting to the Emergency Department (ED) with COVID-19. We recorded patient symptoms, laboratory markers, and clinical outcomes, with a focus on oxygen requirements over time. We collected longitudinal plasma samples from a subset of patients. We characterized the role of RNAemia in predicting clinical severity and EPCs using elastic net regression.23.0% (44/191) of SARS-CoV-2 positive patients had viral RNA detected in plasma by dPCR, compared to 1.4% (2/147) by qPCR. Most patients with serial measurements had undetectable RNAemia within 10 days of symptom onset, reached maximum clinical severity within 16 days, and symptom resolution within 33 days. Initially RNAaemic patients were more likely to manifest severe disease (OR 6.72 [95% CI, 2.45 - 19.79]), worsening of disease severity (OR 2.43 [95% CI, 1.07 - 5.38]), and EPCs (OR 2.81 [95% CI, 1.26 - 6.36]). RNA load correlated with maximum severity (r = 0.47 [95% CI, 0.20 - 0.67]).dPCR is more sensitive than qPCR for the detection of SARS-CoV-2 RNAemia, which is a robust predictor of eventual COVID-19 severity and oxygen requirements, as well as EPCs. Since many COVID-19 therapies are initiated on the basis of oxygen requirements, RNAemia on presentation might serve to direct early initiation of appropriate therapies for the patients most likely to deteriorate.

    View details for DOI 10.1093/cid/ciab394

    View details for PubMedID 33949665

  • A historical perspective on ACE2 in the COVID-19 era. Journal of human hypertension Bhalla, V., Blish, C. A., South, A. M. 2020

    View details for DOI 10.1038/s41371-020-00459-3

    View details for PubMedID 33318644

  • Progenitor identification and SARS-CoV-2 infection in human distal lung organoids. Nature Salahudeen, A. A., Choi, S. S., Rustagi, A., Zhu, J., van Unen, V., de la O, S. M., Flynn, R. A., Margalef-Catala, M., Santos, A. J., Ju, J., Batish, A., Usui, T., Zheng, G. X., Edwards, C. E., Wagar, L. E., Luca, V., Anchang, B., Nagendran, M., Nguyen, K., Hart, D. J., Terry, J. M., Belgrader, P., Ziraldo, S. B., Mikkelsen, T. S., Harbury, P. B., Glenn, J. S., Garcia, K. C., Davis, M. M., Baric, R. S., Sabatti, C., Amieva, M. R., Blish, C. A., Desai, T. J., Kuo, C. J. 2020

    Abstract

    The distal lung contains terminal bronchioles and alveoli that facilitate gas exchange. Three-dimensional in vitro human distal lung culture systems would strongly facilitate investigation of pathologies including interstitial lung disease, cancer, and SARS-CoV-2-associated COVID-19 pneumonia. We generated long-term feeder-free, chemically defined culture of distal lung progenitors as organoids derived from single adult human alveolar epithelial type II (AT2) or KRT5+ basal cells. AT2 organoids exhibited AT1 transdifferentiation potential while basal cell organoids developed lumens lined by differentiated club and ciliated cells. Single cell analysis of basal organoid KRT5+ cells revealed a distinct ITGA6+ITGB4+ mitotic population whose proliferation further segregated to a TNFRSF12Ahi subfraction comprising ~10% of KRT5+ basal cells, residing in clusters within terminal bronchioles and exhibiting enriched clonogenic organoid growth activity. Distal lung organoids were created with apical-out polarity to display ACE2 on the exposed external surface, facilitating SARS-CoV-2 infection of AT2 and basal cultures and identifying club cells as a novel target population. This long-term, feeder-free organoid culture of human distal lung, coupled with single cell analysis, identifies unsuspected basal cell functional heterogeneity and establishes a facile in vitro organoid model for human distal lung infections including COVID-19-associated pneumonia.

    View details for DOI 10.1038/s41586-020-3014-1

    View details for PubMedID 33238290

  • Profiling of the Human Natural Killer Cell Receptor-Ligand Repertoire. Journal of visualized experiments : JoVE Vendrame, E., McKechnie, J. L., Ranganath, T., Zhao, N. Q., Rustagi, A., Vergara, R., Ivison, G. T., Kronstad, L. M., Simpson, L. J., Blish, C. A. 2020

    Abstract

    Natural killer (NK) cells are among the first responders to viral infections. The ability of NK cells to rapidly recognize and kill virally infected cells is regulated by their expression of germline-encoded inhibitory and activating receptors. The engagement of these receptors by their cognate ligands on target cells determines whether the intercellular interaction will result in NK cell killing. This protocol details the design and optimization of two complementary mass cytometry (CyTOF) panels. One panel was designed to phenotype NK cells based on receptor expression. The other panel was designed to interrogate expression of known ligands for NK cell receptors on several immune cell subsets. Together, these two panels allow for the profiling of the human NK cell receptor-ligand repertoire. Furthermore, this protocol also details the process by which we stain samples for CyTOF. This process has been optimized for improved reproducibility and standardization. An advantage of CyTOF is its ability to measure over 40 markers in each panel, with minimal signal overlap, allowing researchers to capture the breadth of the NK cell receptor-ligand repertoire. Palladium barcoding also reduces inter-sample variation, as well as consumption of reagents, making it easier to stain samples with each panel in parallel. Limitations of this protocol include the relatively low throughput of CyTOF and the inability to recover cells after analysis. These panels were designed for the analysis of clinical samples from patients suffering from acute and chronic viral infections, including dengue virus, human immunodeficiency virus (HIV), and influenza. However, they can be utilized in any setting to investigate the human NK cell receptor-ligand repertoire. Importantly, these methods can be applied broadly to the design and execution of future CyTOF panels.

    View details for DOI 10.3791/61912

    View details for PubMedID 33283785

  • Mass Cytometry Analysis of the NK Cell Receptor-Ligand Repertoire Reveals Unique Differences between Dengue-Infected Children and Adults. ImmunoHorizons McKechnie, J. L., Beltran, D., Ferreira, A. M., Vergara, R., Saenz, L., Vergara, O., Estripeaut, D., Arauz, A. B., Simpson, L. J., Holmes, S., Lopez-Verges, S., Blish, C. A. 2020; 4 (10): 634–47

    Abstract

    Dengue virus (DENV) is a significant cause of morbidity in many regions of the world, with children at the greatest risk of developing severe dengue. NK cells, characterized by their ability to rapidly recognize and kill virally infected cells, are activated during acute DENV infection. However, their role in viral clearance versus pathogenesis has not been fully elucidated. Our goal was to profile the NK cell receptor-ligand repertoire to provide further insight into the function of NK cells during pediatric and adult DENV infection. We used mass cytometry to phenotype isolate NK cells and PBMCs from a cohort of DENV-infected children and adults. Using unsupervised clustering, we found that pediatric DENV infection leads to a decrease in total NK cell frequency with a reduction in the percentage of CD56dimCD38bright NK cells and an increase in the percentage of CD56dimperforinbright NK cells. No such changes were observed in adults. Next, we identified markers predictive of DENV infection using a differential state test. In adults, NK cell expression of activation markers, including CD69, perforin, and Fas-L, and myeloid cell expression of activating NK cell ligands, namely Fas, were predictive of infection. In contrast, increased NK cell expression of the maturation marker CD57 and myeloid cell expression of inhibitory ligands, such as HLA class I molecules, were predictive of pediatric DENV infection. These findings suggest that acute pediatric DENV infection may result in diminished NK cell activation, which could contribute to enhanced pathogenesis and disease severity.

    View details for DOI 10.4049/immunohorizons.2000074

    View details for PubMedID 33067399

  • Cytokine profile in plasma of severe COVID-19 does not differ from ARDS and sepsis. JCI insight Wilson, J. G., Simpson, L. J., Ferreira, A., Rustagi, A., Roque, J. A., Asuni, A., Ranganath, T., Grant, P. M., Subramanian, A. K., Rosenberg-Hasson, Y., Maecker, H., Holmes, S., Levitt, J. E., Blish, C., Rogers, A. J. 2020

    Abstract

    BACKGROUND: Elevated levels of inflammatory cytokines have been associated with poor outcomes among COVID-19 patients. It is unknown, however, how these levels compare to those observed in critically ill patients with ARDS or sepsis due to other causes.METHODS: We used a luminex assay to determine expression of 76 cytokines from plasma of hospitalized COVID-19 patients and banked plasma samples from ARDS and sepsis patients. Our analysis focused on detecting statistical differences in levels of 6 cytokines associated with cytokine storm (IL-1b, IL-1RA, IL-6, IL-8, IL-18, and TNFalpha) between patients with moderate COVID-19, severe COVID-19, and ARDS or sepsis.RESULTS: 15 hospitalized COVID-19 patients, 9 of whom were critically ill, were compared to critically ill patients with ARDS (n = 12) or sepsis (n = 16). There were no statistically significant differences in baseline levels of IL-1b, IL-1RA, IL-6, IL-8, IL-18, and TNFalpha between patients with COVID-19 and critically ill controls with ARDS or sepsis.CONCLUSIONS: Levels of inflammatory cytokines were not higher in severe COVID-19 patients than in moderate COVID-19 or critically ill patients with ARDS or sepsis in this small cohort. Broad use of immunosuppressive therapies in ARDS has failed in numerous Phase 3 studies; use of these therapies in unselected patients with COVID-19 may be unwarranted.FUNDING: A.J.R.: Stanford ICU Biobank NHLBI K23 HL125663. C.A.B.: Burroughs Wellcome Fund Investigators in the Pathogenesis of Infectious Diseases #1016687; NIH/NIAID U19AI057229-16 (PI MM Davis); Stanford Maternal Child Health Research Institute; Chan Zuckerberg Biohub.

    View details for DOI 10.1172/jci.insight.140289

    View details for PubMedID 32706339

  • Clinical characteristics associated with COVID-19 severity in California. Journal of clinical and translational science Rubin, S. J., Falkson, S. R., Degner, N. R., Blish, C. 2020; 5 (1): e3

    Abstract

    Given the rapidly progressing coronavirus disease 2019 (COVID-19) pandemic, this report on a US cohort of 54 COVID-19 patients from Stanford Hospital and data regarding risk factors for severe disease obtained at initial clinical presentation is highly important and immediately clinically relevant. We identified low presenting oxygen saturation as predictive of severe disease outcomes, such as diagnosis of pneumonia, acute respiratory distress syndrome, and admission to the intensive care unit, and also replicated data from China suggesting an association between hypertension and disease severity. Clinicians will benefit by tools to rapidly risk stratify patients at presentation by likelihood of progression to severe disease.

    View details for DOI 10.1017/cts.2020.40

    View details for PubMedID 34192044

    View details for PubMedCentralID PMC7274026

  • Enhancing natural killer cell function with gp41-targeting bispecific antibodies to combat HIV infection. AIDS (London, England) Ramadoss, N. S., Zhao, N. Q., Richardson, B. A., Grant, P. M., Kim, P. S., Blish, C. A. 2020

    Abstract

    OBJECTIVE(S): To develop and evaluate the activity of bispecific antibodies (bsAbs) to enhance NK cell antibody-dependent cellular cytotoxicity (ADCC) against HIV-infected cells.DESIGN: These bsAbs are based on patient-derived antibodies targeting the conserved gp41 stump of HIV Env, and also incorporate a high affinity single chain variable fragment (scFv) targeting the activating receptor CD16 on NK cells. Overall, we expect the bsAbs to provide increased affinity and avidity over their corresponding monoclonal antibodies, allowing for improved ADCC activity against Env-expressing target cells.METHODS: bsAbs and their corresponding mAbs were expressed in 293T cells and purified. The binding of bsAbs and mAbs to their intended targets was determined using Bio-Layer Interferometry, as well as flow cytometry-based binding assays on in vitro infected cells. The ability of these bsAbs to improve NK cell activity against HIV-infected cells was tested using in vitro co-culture assays, using flow cytometry and calcein release to analyze NK cell degranulation and target cell killing, respectively.RESULTS: The bsAbs bound gp41 with similar affinity to their corresponding mAbs, and had increased affinity for CD16. The bsAbs also bound to primary CD4 T cells infected in vitro with two different strains of HIV. In addition, the bsAbs induce increased NK cell degranulation and killing of autologous HIV-infected CD4 T cells.CONCLUSIONS: Based on their in vitro killing efficacy, bsAbs may provide a promising strategy to improve NK-mediated immune targeting of infected cells during HIV infection.

    View details for DOI 10.1097/QAD.0000000000002543

    View details for PubMedID 32287071

  • Reinvigorating NIH Grant Peer Review. Immunity Crotty, S. n., Blish, C. n., Cadwell, K. n., Chi, H. n., Goldrath, A. n., Green, D. n., Kaech, S. M., Krummel, M. n., Pepper, M. n., Rothlin, C. V., Wherry, E. J. 2020; 52 (1): 1–3

    View details for DOI 10.1016/j.immuni.2019.12.016

    View details for PubMedID 31940266

  • Safety of ACE-I and ARB medications in COVID-19: A retrospective cohort study of inpatients and outpatients in California Journal of Clinical and Translational Science Rubin, S. J., Falkson, S. R., Degner, N. R., Blish, C. A. 2020: e8

    Abstract

    There is significant interest in the use of angiotensin converting enzyme inhibitors (ACE-I) and angiotensin II receptor blockers (ARB) in coronavirus disease 2019 (COVID-19) and concern over potential adverse effects since these medications upregulate the severe acute respiratory syndrome coronavirus 2 host cell entry receptor ACE2. Recent studies on ACE-I and ARB in COVID-19 were limited by excluding outpatients, excluding patients by age, analyzing ACE-I and ARB together, imputing missing data, and/or diagnosing COVID-19 by chest computed tomography without definitive reverse transcription polymerase chain reaction (RT-PCR), all of which are addressed here.We performed a retrospective cohort study of 1023 COVID-19 patients diagnosed by RT-PCR at Stanford Hospital through April 8, 2020 with a minimum follow-up time of 14 days to investigate the association between ACE-I or ARB use with outcomes.Use of ACE-I or ARB medications was not associated with increased risk of hospitalization, intensive care unit admission, or death. Compared to patients with charted past medical history, there was a lower risk of hospitalization for patients on ACE-I (odds ratio (OR) 0.43; 95% confidence interval (CI) 0.19-0.97; P = 0.0426) and ARB (OR 0.39; 95% CI 0.17-0.90; P = 0.0270). Compared to patients with hypertension not on ACE-I or ARB, patients on ARB medications had a lower risk of hospitalization (OR 0.09; 95% CI 0.01-0.88; P = 0.0381).These findings suggest that the use of ACE-I and ARB is not associated with adverse outcomes and may be associated with improved outcomes in COVID-19, which is immediately relevant to care of the many patients on these medications.

    View details for DOI 10.1017/cts.2020.489

    View details for PubMedCentralID PMC7605244

  • Influenza-Induced Interferon Lambda Response Is Associated With Longer Time to Delivery Among Pregnant Kenyan Women. Frontiers in immunology Seiler, C. n., Bayless, N. L., Vergara, R. n., Pintye, J. n., Kinuthia, J. n., Osborn, L. n., Matemo, D. n., Richardson, B. A., John-Stewart, G. n., Holmes, S. n., Blish, C. A. 2020; 11: 452

    Abstract

    Specific causes of preterm birth remain unclear. Several recent studies have suggested that immune changes during pregnancy are associated with the timing of delivery, yet few studies have been performed in low-income country settings where the rates of preterm birth are the highest. We conducted a retrospective nested case-control evaluation within a longitudinal study among HIV-uninfected pregnant Kenyan women. To characterize immune function in these women, we evaluated unstimulated and stimulated peripheral blood mononuclear cells in vitro with the A/California/2009 strain of influenza to understand the influenza-induced immune response. We then evaluated transcript expression profiles using the Affymetrix Human GeneChip Transcriptome Array 2.0. Transcriptional profiles of sufficient quality for analysis were obtained from 54 women; 19 of these women delivered <34 weeks and were defined as preterm cases and 35 controls delivered >37 weeks. The median time to birth from sample collection was 13 weeks. No transcripts were significantly associated with preterm birth in a case-control study of matched term and preterm birth (n = 42 women). In the influenza-stimulated samples, expression of IFNL1 was associated with longer time to delivery-the amount of time between sample collection and delivery (n = 54 women). A qPCR analysis confirmed that influenza-induced IFNL expression was associated with longer time to delivery. These data indicate that during pregnancy, ex vivo influenza stimulation results in altered transcriptional response and is associated with time to delivery in cohort of women residing in an area with high preterm birth prevalence.

    View details for DOI 10.3389/fimmu.2020.00452

    View details for PubMedID 32256497

    View details for PubMedCentralID PMC7089959

  • Characterization of the Impact of Daclizumab Beta on Circulating Natural Killer Cells by Mass Cytometry. Frontiers in immunology Ranganath, T. n., Simpson, L. J., Ferreira, A. M., Seiler, C. n., Vendrame, E. n., Zhao, N. n., Fontenot, J. D., Holmes, S. n., Blish, C. A. 2020; 11: 714

    Abstract

    Daclizumab beta is a humanized monoclonal antibody that binds to CD25 and selectively inhibits high-affinity IL-2 receptor signaling. As a former treatment for relapsing forms of multiple sclerosis (RMS), daclizumab beta induces robust expansion of the CD56bright subpopulation of NK cells that is correlated with the drug's therapeutic effects. As NK cells represent a heterogeneous population of lymphocytes with a range of phenotypes and functions, the goal of this study was to better understand how daclizumab beta altered the NK cell repertoire to provide further insight into the possible mechanism(s) of action in RMS. We used mass cytometry to evaluate expression patterns of NK cell markers and provide a comprehensive assessment of the NK cell repertoire in individuals with RMS treated with daclizumab beta or placebo over the course of 1 year. Treatment with daclizumab beta significantly altered the NK cell repertoire compared to placebo treatment. As previously reported, daclizumab beta significantly increased expression of CD56 on total NK cells. Within the CD56bright NK cells, treatment was associated with multiple phenotypic changes, including increased expression of NKG2A and NKp44, and diminished expression of CD244, CD57, and NKp46. These alterations occurred broadly across the CD56bright population, and were not associated with a specific subset of CD56bright NK cells. While the changes were less dramatic, CD56dim NK cells responded distinctly to daclizumab beta treatment, with higher expression of CD2 and NKG2A, and lower expression of FAS-L, HLA-DR, NTB-A, NKp30, and Perforin. Together, these data indicate that the expanded CD56bright NK cells share features of both immature and mature NK cells. These findings show that daclizumab beta treatment is associated with unique changes in NK cells that may enhance their ability to kill autoreactive T cells or to exert immunomodulatory functions.

    View details for DOI 10.3389/fimmu.2020.00714

    View details for PubMedID 32391016

    View details for PubMedCentralID PMC7194113

  • Identification of the first cases of complete CD16A deficiency: Association with persistent EBV infection. The Journal of allergy and clinical immunology Pérez-Portilla, A. n., Moraru, M. n., Blázquez-Moreno, A. n., Kolb, P. n., Bravo García-Morato, M. n., Ranganath, T. n., Esteso, G. n., Gianelli, C. n., Rodríguez-Pena, R. n., Lozano-Rodríguez, R. n., Torres-Canizales, J. M., Blish, C. A., Vales-Gomez, M. n., Hengel, H. n., Vilches, C. n., López-Granados, E. n., Reyburn, H. T. 2020

    View details for DOI 10.1016/j.jaci.2019.11.049

    View details for PubMedID 31953104

  • Symptomatic SARS-CoV-2 infections display specific IgG Fc structures. medRxiv : the preprint server for health sciences Chakraborty, S. n., Edwards, K. n., Buzzanco, A. S., Memoli, M. J., Sherwood, R. n., Mallajosyula, V. n., Xie, M. M., Gonzalez, J. n., Buffone, C. n., Kathale, N. n., Providenza, S. n., Jagannathan, P. n., Andrews, J. R., Blish, C. A., Krammer, F. n., Dugan, H. n., Wilson, P. C., Pham, T. D., Boyd, S. D., Zhang, S. n., Taubenberger, J. K., Morales, T. n., Schapiro, J. M., Parsonnet, J. n., Wang, T. T. 2020

    Abstract

    The ongoing severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic has caused a public health crisis that is exacerbated by our poor understanding of correlates of immunity. SARS-CoV-2 infection can cause Coronavirus Disease 2019 (COVID-19), with a spectrum of symptoms ranging from asymptomatic carriage to life threatening pneumonia and cytokine dysregulation [1-3]. Although antibodies have been shown in a variety of in vitro assays to promote coronavirus infections through mechanisms requiring interactions between IgG antibodies and Fc gamma receptors (FcγRs), the relevance of these observations to coronavirus infections in humans is not known [4-7]. In light of ongoing clinical trials examining convalescent serum therapy for COVID-19 patients and expedited SARS-CoV-2 vaccine testing in humans, it is essential to clarify the role of antibodies in the pathogenesis of COVID-19. Here we show that adults with PCR-diagnosed COVID-19 produce IgG antibodies with a specific Fc domain repertoire that is characterized by reduced fucosylation, a modification that enhances interactions with the activating FcγR, FcγRIIIa. Fc fucosylation was reduced when compared with SARS-CoV-2-seropositive children and relative to adults with symptomatic influenza virus infections. These results demonstrate an antibody correlate of symptomatic SARS-CoV-2 infections in adults and have implications for novel therapeutic strategies targeting FcγRIIIa pathways.

    View details for DOI 10.1101/2020.05.15.20103341

    View details for PubMedID 32511463

    View details for PubMedCentralID PMC7252581

  • Treated HIV Infection Alters Phenotype but Not HIV-Specific Function of Peripheral Blood Natural Killer Cells. Frontiers in immunology Zhao, N. Q., Ferreira, A., Grant, P. M., Holmes, S., Blish, C. A. 2020; 11: 829

    Abstract

    Natural killer (NK) cells are the predominant antiviral cells of the innate immune system, and may play an important role in acquisition and disease progression of HIV. While untreated HIV infection is associated with distinct alterations in the peripheral blood NK cell repertoire, less is known about how NK phenotype is altered in the setting of long-term viral suppression with antiretroviral therapy (ART), as well as how NK memory can impact functional responses. As such, we sought to identify changes in NK cell phenotype and function using high-dimensional mass cytometry to simultaneously analyze both surface and functional marker expression of peripheral blood NK cells in a cohort of ART-suppressed, HIV+ patients and HIV- healthy controls. We found that the NK cell repertoire following IL-2 treatment was altered in individuals with treated HIV infection compared to healthy controls, with increased expression of markers including NKG2C and CD2, and decreased expression of CD244 and NKp30. Using co-culture assays with autologous, in vitro HIV-infected CD4 T cells, we identified a subset of NK cells with enhanced responsiveness to HIV-1-infected cells, but no differences in the magnitude of anti-HIV NK cell responses between the HIV+ and HIV- groups. In addition, by profiling of NK cell receptors on responding cells, we found similar phenotypes of HIV-responsive NK cell subsets in both groups. Lastly, we identified clusters of NK cells that are altered in individuals with treated HIV infection compared to healthy controls, but found that these clusters are distinct from those that respond to HIV in vitro. As such, we conclude that while chronic, treated HIV infection induces a reshaping of the IL-2-stimulated peripheral blood NK cell repertoire, it does so in a way that does not make the repertoire more HIV-specific.

    View details for DOI 10.3389/fimmu.2020.00829

    View details for PubMedID 32477342

  • Natural killer cell phenotype is altered in HIV-exposed seronegative women. PloS one Zhao, N. Q., Vendrame, E., Ferreira, A., Seiler, C., Ranganath, T., Alary, M., Labbe, A., Guedou, F., Poudrier, J., Holmes, S., Roger, M., Blish, C. A. 2020; 15 (9): e0238347

    Abstract

    Highly exposed seronegative (HESN) individuals present a unique setting to study mechanisms of protection against HIV acquisition. As natural killer (NK) cell activation and function have been implicated as a correlate of protection in HESN individuals, we sought to better understand the features of NK cells that may confer protection. We used mass cytometry to phenotypically profile NK cells from a cohort of Beninese sex workers and healthy controls. We found that NK cells from HESN women had increased expression of NKG2A, NKp30 and LILRB1, as well as the Fc receptor CD16, and decreased expression of DNAM-1, CD94, Siglec-7, and NKp44. Using functional assessments of NK cells from healthy donors against autologous HIV-infected CD4+ T cells, we observed that NKp30+ and Siglec-7+ cells had improved functional activity. Further, we found that NK cells from HESN women trended towards increased antibody-dependent cellular cytotoxicity (ADCC) activity; this activity correlated with increased CD16 expression. Overall, we identify features of NK cells in HESN women that may contribute to protection from HIV infection. Follow up studies with larger cohorts are warranted to confirm these findings.

    View details for DOI 10.1371/journal.pone.0238347

    View details for PubMedID 32870938

  • Human B cell clonal expansion and convergent antibody responses to SARS-CoV-2. bioRxiv : the preprint server for biology Nielsen, S. C., Yang, F. n., Jackson, K. J., Hoh, R. A., Röltgen, K. n., Stevens, B. n., Lee, J. Y., Rustagi, A. n., Rogers, A. J., Powell, A. E., Najeeb, J. n., Otrelo-Cardoso, A. R., Yost, K. E., Daniel, B. n., 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

    During virus infection B cells are critical for the production of antibodies and protective immunity. Here we show that the human B cell compartment in patients with diagnostically confirmed SARS-CoV-2 and clinical COVID-19 is rapidly altered with the 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 extensive convergence of antibody sequences across SARS-CoV-2 patients, highlighting stereotyped naïve 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 other zoonotic spillover coronaviruses.

    View details for DOI 10.1101/2020.07.08.194456

    View details for PubMedID 32676593

    View details for PubMedCentralID PMC7359515

  • B cell clonal expansion and convergent antibody responses to SARS-CoV-2. Research square Nielsen, S. C., Yang, F. n., Hoh, R. A., Jackson, K. J., Roeltgen, K. n., Lee, J. Y., Rustagi, A. n., Rogers, A. J., Powell, A. E., Kim, P. S., Wang, T. T., Pinsky, B. n., Blish, C. A., Boyd, S. D. 2020

    Abstract

    During virus infection B cells are critical for the production of antibodies and protective immunity. Establishment of a diverse antibody repertoire occurs by rearrangement of germline DNA at the immunoglobulin heavy and light chain loci to encode the membrane-bound form of antibodies, the B cell antigen receptor. Little is known about the B cells and antigen receptors stimulated by the novel human coronavirus SARS-CoV-2. Here we show that the human B cell compartment in patients with diagnostically confirmed SARS-CoV-2 and clinical COVID-19 is rapidly altered with the early recruitment of B cells expressing a limited subset of V genes, and extensive activation of IgG and IgA subclasses without significant somatic mutation. We detect expansion of B cell clones as well as convergent antibodies with highly similar sequences across SARS-CoV-2 patients, highlighting stereotyped naïve responses to this virus. A shared convergent B cell clonotype in SARS-CoV-2 infected patients was previously seen in patients with SARS. These findings offer molecular insights into shared features of human B cell responses to SARS-CoV-2 and other zoonotic spillover coronaviruses.

    View details for DOI 10.21203/rs.3.rs-27220/v1

    View details for PubMedID 32702737

    View details for PubMedCentralID PMC7336706

  • TIGIT is upregulated by HIV-1 infection and marks a highly functional adaptive and mature subset of natural killer cells. AIDS (London, England) Vendrame, E. n., Seiler, C. n., Ranganath, T. n., Zhao, N. Q., Vergara, R. n., Alary, M. n., Labbé, A. C., Guédou, F. n., Poudrier, J. n., Holmes, S. n., Roger, M. n., Blish, C. A. 2020

    Abstract

    Our objective was to investigate the mechanisms that govern natural killer (NK) cell responses to HIV, with a focus on specific receptor-ligand interactions involved in HIV recognition by NK cells.We first performed a mass cytometry-based screen of NK cell receptor expression patterns in healthy controls and HIV individuals. We then focused mechanistic studies on the expression and function of T cell immunoreceptor with Ig and ITIM domains (TIGIT).The mass cytometry screen revealed that TIGIT is upregulated on NK cells of untreated HIV women, but not in antiretroviral-treated women. TIGIT is an inhibitory receptor that is thought to mark exhausted NK cells; however, blocking TIGIT did not improve anti-HIV NK cell responses. In fact, the TIGIT ligands CD112 and CD155 were not upregulated on CD4 T cells in vitro or in vivo, providing an explanation for the lack of benefit from TIGIT blockade. TIGIT expression marked a unique subset of NK cells that express significantly higher levels of NK cell activating receptors (DNAM-1, NTB-A, 2B4, CD2) and exhibit a mature/adaptive phenotype (CD57, NKG2C, LILRB1, FcRγ, Syk). Furthermore, TIGIT NK cells had increased responses to mock-infected and HIV-infected autologous CD4 T cells, and to PMA/ionomycin, cytokine stimulation and the K562 cancer cell line.TIGIT expression is increased on NK cells from untreated HIV individuals. Although TIGIT does not participate directly to the response to HIV-infected cells, it marks a population of mature/adaptive NK cells with increased functional responses.

    View details for DOI 10.1097/QAD.0000000000002488

    View details for PubMedID 32028328

  • The Innate Immune System: Fighting on the Front Lines or Fanning the Flames of COVID-19? Cell host & microbe McKechnie, J. L., Blish, C. A. 2020

    Abstract

    The coronavirus disease 2019 (COVID-19) pandemic caused by SARS-CoV-2 has had devastating global impacts and will continue to have dramatic effects on public health for years to come. A better understanding of the immune response to SARS-CoV-2 will be critical for the application and development of therapeutics. The degree to which the innate immune response confers protection or induces pathogenesis through a dysregulated immune response remains unclear. In this review, we discuss what is known about the role of the innate immune system during SARS-CoV-2 infection, suggest directions for future studies, and evaluate proposed COVID-19 immunomodulating therapeutics.

    View details for DOI 10.1016/j.chom.2020.05.009

    View details for PubMedID 32464098

  • Synthetic Siglec-9 Agonists Inhibit Neutrophil Activation Associated with COVID-19. ChemRxiv : the preprint server for chemistry Delaveris, C. n., Wilk, A. n., Riley, N. n., Stark, J. n., Yang, S. n., Rogers, A. n., Ranganath, T. n., Nadeau, K. n., Blish, C. n., Bertozzi, C. n. 2020

    Abstract

    Severe cases of coronavirus disease 2019 (COVID-19), caused by infection with SARS-Cov-2, are characterized by a hyperinflammatory immune response that leads to numerous complications. Production of proinflammatory neutrophil extracellular traps (NETs) has been suggested to be a key factor in inducing a hyperinflammatory signaling cascade, allegedly causing both pulmonary tissue damage and peripheral inflammation. Accordingly, therapeutic blockage of neutrophil activation and NETosis, the cell death pathway accompanying NET formation, could limit respiratory damage and death from severe COVID-19. Here, we demonstrate that synthetic glycopolymers that activate the neutrophil checkpoint receptor Siglec-9 suppress NETosis induced by agonists of viral toll-like receptors (TLRs) and plasma from patients with severe COVID-19. Thus, Siglec-9 agonism is a promising therapeutic strategy to curb neutrophilic hyperinflammation in COVID-19.
    .

    View details for DOI 10.26434/chemrxiv.13378148

    View details for PubMedID 33469569

    View details for PubMedCentralID PMC7814829

  • SARS-CoV-2 RNAaemia predicts clinical deterioration and extrapulmonary complications from COVID-19. medRxiv : the preprint server for health sciences Ram-Mohan, N. n., Kim, D. n., Zudock, E. J., Hashemi, M. M., Tjandra, K. C., Rogers, A. J., Blish, C. A., Nadeau, K. C., Newberry, J. A., Quinn, J. V., O'Hara, R. n., Ashley, E. n., Nguyen, H. n., Jiang, L. n., Hung, P. n., Blomkalns, A. L., Yang, S. n. 2020

    Abstract

    The determinants of COVID-19 disease severity and extrapulmonary complications (EPCs) are poorly understood. We characterise the relationships between SARS-CoV-2 RNAaemia and disease severity, clinical deterioration, and specific EPCs.We used quantitative (qPCR) and digital (dPCR) PCR to quantify SARS-CoV-2 RNA from nasopharyngeal swabs and plasma in 191 patients presenting to the Emergency Department (ED) with COVID-19. We recorded patient symptoms, laboratory markers, and clinical outcomes, with a focus on oxygen requirements over time. We collected longitudinal plasma samples from a subset of patients. We characterised the role of RNAaemia in predicting clinical severity and EPCs using elastic net regression.23·0% (44/191) of SARS-CoV-2 positive patients had viral RNA detected in plasma by dPCR, compared to 1·4% (2/147) by qPCR. Most patients with serial measurements had undetectable RNAaemia 10 days after onset of symptoms, but took 16 days to reach maximum severity, and 33 days for symptoms to resolve. Initially RNAaemic patients were more likely to manifest severe disease (OR 6·72 [95% CI, 2·45 - 19·79]), worsening of disease severity (OR 2·43 [95% CI, 1·07 - 5·38]), and EPCs (OR 2·81 [95% CI, 1·26 - 6·36]). RNA load correlated with maximum severity ( r = 0·47 [95% CI, 0·20 - 0·67]).dPCR is more sensitive than qPCR for the detection of SARS-CoV-2 RNAaemia, which is a robust predictor of eventual COVID-19 severity and oxygen requirements, as well as EPCs. Since many COVID-19 therapies are initiated on the basis of oxygen requirements, RNAaemia on presentation might serve to direct early initiation of appropriate therapies for the patients most likely to deteriorate.NIH/NIAID (Grants R01A153133, R01AI137272, and 3U19AI057229 - 17W1 COVID SUPP #2) and a donation from Eva Grove.Evidence before this study: The varied clinical manifestations of COVID-19 have directed attention to the distribution of SARS-CoV-2 in the body. Although most concentrated and tested for in the nasopharynx, SARS-CoV-2 RNA has been found in blood, stool, and numerous tissues, raising questions about dissemination of viral RNA throughout the body, and the role of this process in disease severity and extrapulmonary complications. Recent studies have detected low levels of SARS-CoV-2 RNA in blood using either quantitative reverse transcriptase real-time PCR (qPCR) or droplet digital PCR (dPCR), and have associated RNAaemia with disease severity and biomarkers of dysregulated immune response.Added value of this study: We quantified SARS-CoV-2 RNA in the nasopharynx and plasma of patients presenting to the Emergency Department with COVID-19, and found an array-based dPCR platform to be markedly more sensitive than qPCR for detection of SARS-CoV-2 RNA, with a simplified workflow well-suited to clinical adoption. We collected serial plasma samples during patients' course of illness, and showed that SARS-CoV-2 RNAaemia peaks early, while clinical condition often continues to worsen. Our findings confirm the association between RNAaemia and disease severity, and additionally demonstrate a role for RNAaemia in predicting future deterioration and specific extrapulmonary complications.Implications of all the available evidence: Variation in SARS-CoV-2 RNAaemia may help explain disparities in disease severity and extrapulmonary complications from COVID-19. Testing for RNAaemia with dPCR early in the course of illness may help guide patient triage and management.

    View details for DOI 10.1101/2020.12.19.20248561

    View details for PubMedID 33398290

    View details for PubMedCentralID PMC7781329

  • 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
  • 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

  • Universal Principled Review: A Community-Driven Method to Improve Peer Review. Cell Krummel, M., Blish, C., Kuhns, M., Cadwell, K., Oberst, A., Goldrath, A., Ansel, K. M., Chi, H., O'Connell, R., Wherry, E. J., Pepper, M., Future Immunology Consortium, Brodsky, I., Chang, J., Arron, J. R., Haining, N., Bhattacharya, D., Anderson, M., Rothlin, C. V., Schwab, S., Belkaid, Y., Molofsky, A., Savage, P., Mucida, D., Iwasaki, A., Victora, G., Ansel, K. M., Hamerman, J., Masopust, D., Barton, G., Kaech, S., Woodruff, P., Stetson, D. B., Scharschmidt, T. C., Kedl, R., Zuniga, E. I., Hoffmann, A., Williams, M., Mayer-Barber, K. D., Shin, S., Bensinger, S., Lu, L., Looney, M., Round, J. L., Amigorena, S., Yewdell, J., Sun, J., Harty, J. T. 2019; 179 (7): 1441–45

    Abstract

    Despite being a staple of our science, the process of pre-publication peer review has few agreed-upon standards defining its goals or ideal execution. As a community of reviewers and authors, we assembled an evaluation format and associated specific standards for the process as we think it should be practiced. We propose that we apply, debate, and ultimately extend these to improve the transparency of our criticism and the speed with which quality data and ideas become public.

    View details for DOI 10.1016/j.cell.2019.11.029

    View details for PubMedID 31835023

  • HLA Upregulation During Dengue Virus Infection Suppresses the Natural Killer Cell Response FRONTIERS IN CELLULAR AND INFECTION MICROBIOLOGY McKechnie, J. L., Beltran, D., Pitti, A., Saenz, L., Arauz, A. B., Vergara, R., Harris, E., Lanier, L. L., Blish, C. A., Lopez-Verges, S. 2019; 9
  • HLA Upregulation During Dengue Virus Infection Suppresses the Natural Killer Cell Response. Frontiers in cellular and infection microbiology McKechnie, J. L., Beltrán, D., Pitti, A., Saenz, L., Araúz, A. B., Vergara, R., Harris, E., Lanier, L. L., Blish, C. A., López-Vergès, S. 2019; 9: 268

    Abstract

    Dengue virus (DENV) is the most prevalent mosquito-borne virus in the world and a major cause of morbidity in the tropics and subtropics. Upregulation of HLA class I molecules has long been considered a feature of DENV infection, yet this has not been evaluated in the setting of natural infection. Natural killer (NK) cells, an innate immune cell subset critical for mounting an early response to viral infection, are inhibited by self HLA class I, suggesting that upregulation of HLA class I during DENV infection could dampen the NK cell response. Here we addressed whether upregulation of HLA class I molecules occurs during in vivo DENV infection and, if so, whether this suppresses the NK cell response. We found that HLA class I expression was indeed upregulated during acute DENV infection across multiple cell lineages in vivo. To better understand the role of HLA class I upregulation, we infected primary human monocytes, a major target of DENV infection, in vitro. Upregulation of total HLA class I is dependent on active viral replication and is mediated in part by cytokines and other soluble factors induced by infection, while upregulation of HLA-E occurs in the presence of replication-incompetent virus. Importantly, blocking DENV-infected monocytes with a pan-HLA class I Fab nearly doubles the frequency of degranulating NK cells, while blocking HLA-E does not significantly improve the NK cell response. These findings demonstrate that upregulation of HLA class I during DENV infection suppresses the NK cell response, potentially contributing to disease pathogenesis.

    View details for DOI 10.3389/fcimb.2019.00268

    View details for PubMedID 31396492

    View details for PubMedCentralID PMC6663972

  • A novel human IL2RB mutation results in T and NK cell-driven immune dysregulation JOURNAL OF EXPERIMENTAL MEDICINE Fernandez, I. Z., Baxter, R. M., Garcia-Perez, J. E., Vendrame, E., Ranganath, T., Kong, D. S., Lundquist, K., Nguyen, T., Ogolla, S., Black, J., Galambos, C., Gumbart, J. C., Dawany, N., Kelsen, J. R., de Zoeten, E. F., Quinones, R., Eissa, H., Verneris, M. R., Sullivan, K. E., Rochford, R., Blish, C. A., Kedl, R. M., Dutmer, C. M., Hsieh, E. Y. 2019; 216 (6): 1255–67
  • Human natural killer cells mediate adaptive immunity to viral antigens SCIENCE IMMUNOLOGY Nikzad, R., Angelo, L. S., Aviles-Padilla, K., Le, D. T., Singh, V. K., Bimler, L., Vukmanovic-Stejic, M., Vendrame, E., Ranganath, T., Simpson, L., Haigwood, N. L., Blish, C. A., Akbar, A. N., Paust, S. 2019; 4 (35)
  • Pregnancy-Induced Alterations in NK Cell Phenotype and Function. Frontiers in immunology Le Gars, M., Seiler, C., Kay, A. W., Bayless, N. L., Starosvetsky, E., Moore, L., Shen-Orr, S. S., Aziz, N., Khatri, P., Dekker, C. L., Swan, G. E., Davis, M. M., Holmes, S., Blish, C. A. 2019; 10: 2469

    Abstract

    Pregnant women are particularly susceptible to complications of influenza A virus infection, which may result from pregnancy-induced changes in the function of immune cells, including natural killer (NK) cells. To better understand NK cell function during pregnancy, we assessed the ability of the two main subsets of NK cells, CD56dim, and CD56bright NK cells, to respond to influenza-virus infected cells and tumor cells. During pregnancy, CD56dim and CD56bright NK cells displayed enhanced functional responses to both infected and tumor cells, with increased expression of degranulation markers and elevated frequency of NK cells producing IFN-gamma. To better understand the mechanisms driving this enhanced function, we profiled CD56dim and CD56bright NK cells from pregnant and non-pregnant women using mass cytometry. NK cells from pregnant women displayed significantly increased expression of several functional and activation markers such as CD38 on both subsets and NKp46 on CD56dim NK cells. NK cells also displayed diminished expression of the chemokine receptor CXCR3 during pregnancy. Overall, these data demonstrate that functional and phenotypic shifts occur in NK cells during pregnancy that can influence the magnitude of the immune response to both infections and tumors.

    View details for DOI 10.3389/fimmu.2019.02469

    View details for PubMedID 31708922

  • Maintaining a Robust Pipeline of Future Physician-Scientists JOURNAL OF INFECTIOUS DISEASES Blish, C. A. 2018; 218: S40–S43

    Abstract

    Perhaps the most dramatic finding in the 2014 National Institutes of Health Physician-Scientist Workforce Working Group Report is the aging of the physician-scientist workforce. There are currently 1.6-fold more physician-scientists over the age of 61 than under the age of 50, indicating that our pipeline of physician-scientists is insufficient to maintain current numbers. Several factors likely contribute to this leaky pipeline, including the long training periods, poor compensation during training, diminished funding odds for young investigators, and lack of role models, particularly for women and underrepresented minorities. This perspective will present several ideas for how training programs can play a role in assuring a robust pipeline of future physician scientists.

    View details for PubMedID 30124975

    View details for PubMedCentralID PMC6093428

  • Differential Induction of IFN-alpha and Modulation of CD112 and CD54 Expression Govern the Magnitude of NK Cell IFN-gamma Response to Influenza A Viruses. Journal of immunology (Baltimore, Md. : 1950) Kronstad, L. M., Seiler, C., Vergara, R., Holmes, S. P., Blish, C. A. 2018

    Abstract

    In human and murine studies, IFN-gamma is a critical mediator immunity to influenza. IFN-gamma production is critical for viral clearance and the development of adaptive immune responses, yet excessive production of IFN-gamma and other cytokines as part of a cytokine storm is associated with poor outcomes of influenza infection in humans. As NK cells are the main population of lung innate immune cells capable of producing IFN-gamma early in infection, we set out to identify the drivers of the human NK cell IFN-gamma response to influenza A viruses. We found that influenza triggers NK cells to secrete IFN-gamma in the absence of T cells and in a manner dependent upon signaling from both cytokines and receptor-ligand interactions. Further, we discovered that the pandemic A/California/07/2009 (H1N1) strain elicits a seven-fold greater IFN-gamma response than other strains tested, including a seasonal A/Victoria/361/2011 (H3N2) strain. These differential responses were independent of memory NK cells. Instead, we discovered that the A/Victoria/361/2011 influenza strain suppresses the NK cell IFN-gamma response by downregulating NK-activating ligands CD112 and CD54 and by repressing the type I IFN response in a viral replication-dependent manner. In contrast, the A/California/07/2009 strain fails to repress the type I IFN response or to downregulate CD54 and CD112 to the same extent, which leads to the enhanced NK cell IFN-gamma response. Our results indicate that influenza implements a strain-specific mechanism governing NK cell production of IFN-gamma and identifies a previously unrecognized influenza innate immune evasion strategy.

    View details for PubMedID 30143589

  • Diversification of human NK cells: Lessons from deep profiling. Journal of leukocyte biology Wilk, A. J., Blish, C. A. 2018

    Abstract

    NK cells are innate lymphocytes with important roles in immunoregulation, immunosurveillance, and cytokine production. Originally defined on the functional basis of their "natural" ability to lyse tumor targets and thought to be a relatively homogeneous group of lymphocytes, NK cells possess a remarkable degree of phenotypic and functional diversity due to the combinatorial expression of an array of activating and inhibitory receptors. Diversification of NK cells is multifaceted: mechanisms of NK cell education that promote self-tolerance result in a heterogeneous repertoire that further diversifies upon encounters with viral pathogens. Here, we review the genetic, developmental, and environmental sources of NK cell diversity with a particular focus on deep profiling and single-cell technologies that will enable a more thorough and accurate dissection of this intricate and poorly understood lymphocyte lineage.

    View details for PubMedID 29350874

  • Humanized mouse model supports development, function, and tissue residency of human natural killer cells PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Herndler-Brandstetter, D., Shan, L., Yao, Y., Stecher, C., Plajer, V., Lietzenmayer, M., Strowig, T., de Zoete, M. R., Palm, N. W., Chen, J., Blish, C. A., Frleta, D., Gurer, C., Macdonald, L. E., Murphy, A. J., Yancopoulos, G. D., Montgomery, R. R., Flavell, R. A. 2017; 114 (45): E9626–E9634

    Abstract

    Immunodeficient mice reconstituted with a human immune system represent a promising tool for translational research as they may allow modeling and therapy of human diseases in vivo. However, insufficient development and function of human natural killer (NK) cells and T cell subsets limit the applicability of humanized mice for studying cancer biology and therapy. Here, we describe a human interleukin 15 (IL15) and human signal regulatory protein alpha (SIRPA) knock-in mouse on a Rag2-/- Il2rg-/- background (SRG-15). Transplantation of human hematopoietic stem and progenitor cells into SRG-15 mice dramatically improved the development and functional maturation of circulating and tissue-resident human NK and CD8+ T cells and promoted the development of tissue-resident innate lymphoid cell (ILC) subsets. Profiling of human NK cell subsets by mass cytometry revealed a highly similar expression pattern of killer inhibitory receptors and other candidate molecules in NK cell subpopulations between SRG-15 mice and humans. In contrast to nonobese diabetic severe combined immunodeficient Il2rg-/- (NSG) mice, human NK cells in SRG-15 mice did not require preactivation but infiltrated a Burkitt's lymphoma xenograft and efficiently inhibited tumor growth following treatment with the therapeutic antibody rituximab. Our humanized mouse model may thus be useful for preclinical testing of novel human NK cell-targeted and combinatory cancer immunotherapies and for studying how they elicit human antitumor immune responses in vivo.

    View details for PubMedID 29078283

  • Redefining Memory: Building the Case for Adaptive NK Cells JOURNAL OF VIROLOGY Paust, S., Blish, C. A., Reeves, R. 2017; 91 (20)

    Abstract

    Classically, natural killer (NK) cells have been defined by nonspecific innate killing of virus-infected and tumor cells. However, burgeoning evidence suggests that the functional repertoire of NK cells is far more diverse than has been previously appreciated, thus raising the possibility that there may be unexpected functional specialization and even adaptive capabilities among NK cell subpopulations. Some of the first evidence that NK cells respond in an antigen-specific fashion came from experiments revealing that subpopulations of murine NK cells were able to respond to a specific murine cytomegalovirus (MCMV) protein and that in the absence of T and B cells, murine NK cells also mediated adaptive immune responses to a secondary challenge with specific haptens. These data have been followed by demonstrations of NK cell memory of viruses and viral antigens in mice and primates. Herein, we discuss different forms of NK cell antigen specificity and how these responses may be tuned to specific viral pathogens, and we provide assessment of the current literature that may explain molecular mechanisms of the novel phenomenon of NK cell memory.

    View details for PubMedID 28794018

    View details for PubMedCentralID PMC5625515

  • Zika virus infection induces cranial neural crest cells to produce cytokines at levels detrimental for neurogenesis Blish, C. SPRINGER. 2017: S9–S10
  • Mass Cytometry Analytical Approaches Reveal Cytokine-Induced Changes in Natural Killer Cells. Cytometry. Part B, Clinical cytometry Vendrame, E., Fukuyama, J., Strauss-Albee, D. M., Holmes, S., Blish, C. A. 2017; 92 (1): 57-67

    Abstract

    Natural killer (NK) cells have antiviral and antitumor activity that could be harnessed for the treatment of infections and malignancies. To maintain cell viability and enhance antiviral and antitumor effects, NK cells are frequently treated with cytokines. Here we performed an extensive assessment of the effects of cytokines on the phenotype and function of human NK cells.We used cytometry by time-of-flight (CyTOF) to evaluate NK cell repertoire changes after stimulation with interleukin (IL)-2, IL-15 or a combination of IL- 12/IL-15/IL-18. To analyze the high dimensional CyTOF data, we used several statistical and visualization tools, including viSNE (Visualization of t-Distributed Stochastic Neighbor Embedding), Citrus (Cluster identification, characterization, and regression), correspondence analysis, and the Friedman-Rafsky test.All three treatments (IL-2, IL-15, and IL-12/IL-15/IL-18) increase expression of CD56 and CD69. The effects of treatment with IL-2 and IL-15 are nearly indistinguishable and characterized principally by increased expression of surface markers including CD56, NKp30, NKp44, and increased expression of functional markers, such as perforin, granzyme B, and MIP-1β. The combination of IL-12/IL-15/IL- 18 induces a profound shift in the repertoire structure, decreasing expression of CD16, CD57, CD8, NKp30, NKp46, and NKG2D, and dramatically increasing expression of IFN-γ.CyTOF provides insights into the effects of cytokines on the phenotype and function of NK cells, which could inform future research efforts and approaches to NK cell immunotherapy. There are several analytical approaches to CyTOF data, and the appropriate method should be carefully selected based on which aspect of the dataset is being explored. This article is protected by copyright. All rights reserved.

    View details for DOI 10.1002/cyto.b.21500

    View details for PubMedID 27933717

  • The newborn human NK cell repertoire is phenotypically formed but functionally reduced CYTOMETRY PART B-CLINICAL CYTOMETRY Strauss-Albee, D. M., Liang, E. C., Ranganath, T., Aziz, N., Blish, C. A. 2017; 92 (1): 33-41

    Abstract

    Infection is a leading cause of death worldwide in babies under 1 month of age. Better vaccines and therapeutics are desperately needed for this vulnerable population.Because newborns rely heavily on the innate immune system, we evaluated cell phenotype and function of some of the earliest cellular responders during infection, natural killer (NK) cells. We used mass cytometry to provide a comprehensive comparison of NK cells from umbilical cord blood and adult peripheral blood.In unsupervised analyses, including viSNE and principal component analysis, the structure of the cord blood and adult NK cell repertoires are highly similar, distinguishable mainly by maturity-related markers expressed on rare subpopulations of cells. However, in functional analyses, cord blood NK cells show reduced degranulation and cytokine production following target recognition, as well as antibody-dependent cell-mediated cytotoxicity and apoptosis induction in targets.These findings show that the structure of the NK cell repertoire is intact at birth, suggesting great potential for vaccine and therapeutic strategies targeting this cell population. © 2016 International Clinical Cytometry Society.

    View details for DOI 10.1002/cyto.b.21485

    View details for Web of Science ID 000394980300005

  • The natural killer cell response to West Nile virus in young and old individuals with or without a prior history of infection. PloS one Yao, Y., Strauss-Albee, D. M., Zhou, J. Q., Malawista, A., Garcia, M. N., Murray, K. O., Blish, C. A., Montgomery, R. R. 2017; 12 (2)

    Abstract

    West Nile virus (WNV) typically leads to asymptomatic infection but can cause severe neuroinvasive disease or death, particularly in the elderly. Innate NK cells play a critical role in antiviral defenses, yet their role in human WNV infection is poorly defined. Here we demonstrate that NK cells mount a robust, polyfunctional response to WNV characterized by cytolytic activity, cytokine and chemokine secretion. This is associated with downregulation of activating NK cell receptors and upregulation of NK cell activating ligands for NKG2D. The NK cell response did not differ between young and old WNV-naïve subjects, but a history of symptomatic infection is associated with more IFN-γ producing NK cell subsets and a significant decline in a specific NK cell subset. This NK repertoire skewing could either contribute to or follow heightened immune pathogenesis from WNV infection, and suggests that NK cells could play an important role in WNV infection in humans.

    View details for DOI 10.1371/journal.pone.0172625

    View details for PubMedID 28235099

    View details for PubMedCentralID PMC5325267

  • NKG2A-Expressing Natural Killer Cells Dominate the Response to Autologous Lymphoblastoid Cells Infected with Epstein-Barr Virus FRONTIERS IN IMMUNOLOGY Hatton, O., Strauss-Albee, D. M., Zhao, N. Q., Haggadone, M. D., Pelpola, J. S., Krams, S. M., Martinez, O. M., Blish, C. A. 2016; 7

    Abstract

    Epstein-Barr virus (EBV) is a human γ-herpesvirus that establishes latency and lifelong infection in host B cells while achieving a balance with the host immune response. When the immune system is perturbed through immunosuppression or immunodeficiency, however, these latently infected B cells can give rise to aggressive B cell lymphomas. Natural killer (NK) cells are regarded as critical in the early immune response to viral infection, but their role in controlling expansion of infected B cells is not understood. Here, we report that NK cells from healthy human donors display increased killing of autologous B lymphoblastoid cell lines (LCLs) harboring latent EBV compared to primary B cells. Coculture of NK cells with autologous EBV(+) LCL identifies an NK cell population that produces IFNγ and mobilizes the cytotoxic granule protein CD107a. Multi-parameter flow cytometry and Boolean analysis reveal that these functional cells are enriched for expression of the NK cell receptor NKG2A. Further, NKG2A(+) NK cells more efficiently lyse autologous LCL than do NKG2A(-) NK cells. More specifically, NKG2A(+)2B4(+)CD16(-)CD57(-)NKG2C(-)NKG2D(+) cells constitute the predominant NK cell population that responds to latently infected autologous EBV(+) B cells. Thus, a subset of NK cells is enhanced for the ability to recognize and eliminate autologous, EBV-infected transformed cells, laying the groundwork for harnessing this subset for therapeutic use in EBV(+) malignancies.

    View details for DOI 10.3389/fimmu.2016.00607

    View details for Web of Science ID 000389857900001

    View details for PubMedID 28018364

    View details for PubMedCentralID PMC5156658

  • Increased Proinflammatory Responses of Monocytes and Plasmacytoid Dendritic Cells to Influenza A Virus Infection During Pregnancy JOURNAL OF INFECTIOUS DISEASES Le Gars, M., Kay, A. W., Bayless, N. L., Aziz, N., Dekker, C. L., Swan, G. E., Davis, M. M., Blish, C. A. 2016; 214 (11): 1666-1671

    Abstract

    Pregnancy-induced alterations in immunity may contribute to the increased morbidity associated with influenza A virus infection during pregnancy. We characterized the immune response of monocytes and plasmacytoid dendritic cells (pDCs) to influenza A virus infection in 21 pregnant and 21 nonpregnant women. In pregnant women, monocytes and pDCs exhibit an exaggerated proinflammatory immune response to 2 strains of influenza A virus, compared with nonpregnant women, characterized by increased expression of major histocompatibility complex class II (approximately 2.0-fold), CD69 (approximately 2.2-fold), interferon γ-induced protein 10 (approximately 2.0-fold), and macrophage inflammatory protein 1β (approximately 1.5-fold). This enhanced innate inflammatory response during pregnancy could contribute to pulmonary inflammation following influenza A virus infection.

    View details for DOI 10.1093/infdis/jiw448

    View details for Web of Science ID 000393128800008

    View details for PubMedID 27655870

    View details for PubMedCentralID PMC5144734

  • The newborn human NK cell repertoire is phenotypically formed but functionally reduced. Cytometry. Part B, Clinical cytometry Strauss-Albee, D. M., Liang, E. C., Ranganath, T., Aziz, N., Blish, C. A. 2016

    Abstract

    Infection is a leading cause of death worldwide in babies under 1 month of age. Better vaccines and therapeutics are desperately needed for this vulnerable population.Because newborns rely heavily on the innate immune system, we evaluated cell phenotype and function of some of the earliest cellular responders during infection, natural killer (NK) cells. We used mass cytometry to provide a comprehensive comparison of NK cells from umbilical cord blood and adult peripheral blood.In unsupervised analyses, including viSNE and principal component analysis, the structure of the cord blood and adult NK cell repertoires are highly similar, distinguishable mainly by maturity-related markers expressed on rare subpopulations of cells. However, in functional analyses, cord blood NK cells show reduced degranulation and cytokine production following target recognition, as well as antibody-dependent cell-mediated cytotoxicity and apoptosis induction in targets.These findings show that the structure of the NK cell repertoire is intact at birth, suggesting great potential for vaccine and therapeutic strategies targeting this cell population. © 2016 International Clinical Cytometry Society.

    View details for DOI 10.1002/cyto.b.21485

    View details for PubMedID 27718327

  • Natural Killer Cells Display Functional and Receptor Diversity in Response to Alloantigen. Hatton, O., Shawler, T., Blish, C., Martinez, O., Krams, S. WILEY-BLACKWELL. 2016: 721
  • Natural Killer Cell Diversity in Viral Infection: Why and How Much? Pathogens & immunity Blish, C. A. 2016; 1 (1): 165-192

    Abstract

    Natural killer cells are a diverse group of innate lymphocytes that are specialized to rapidly respond to cancerous or virus-infected cells. NK cell function is controlled by the integration of signals from activating and inhibitory receptors expressed at the cell surface. Variegated expression patterns of these activating and inhibitory receptors at the single cell level leads to a highly diverse NK cell repertoire. Here I review the factors that influence NK cell repertoire diversity and its functional consequences for our ability to fight viruses.

    View details for PubMedID 27635417

  • Human NK Cell Diversity in viral infection: Ramifications of Ramification FRONTIERS IN IMMUNOLOGY Strauss-Albee, D. M., Blish, C. A. 2016; 7

    Abstract

    Natural killer (NK) cells are a unique lymphocyte lineage with remarkable agility in the rapid destruction of virus-infected cells. They are also the most poorly understood class of lymphocyte. A spectrum of activating and inhibitory receptors at the NK cell surface leads to an unusual and difficult-to-study mechanism of cellular recognition, as well as a very high capacity for diversity at the single-cell level. Here, we review the evidence for the role of NK cells in the earliest stage of human viral infection, and in its prevention. We argue that single-cell diversity is a logical evolutionary adaptation for their position in the immune response and contributes to their ability to kill virus-infected cells. Finally, we look to the future, where emerging single-cell technologies will enable a new generation of rigorous and clinically relevant studies on NK cells accounting for all of their unique and diverse characteristics.

    View details for DOI 10.3389/fimmu.2016.00066

    View details for Web of Science ID 000371276400001

    View details for PubMedID 26973646

    View details for PubMedCentralID PMC4776076

  • Application of Mass Cytometry (CyTOF) for Functional and Phenotypic Analysis of Natural Killer Cells. Methods in molecular biology (Clifton, N.J.) Kay, A. W., Strauss-Albee, D. M., Blish, C. A. 2016; 1441: 13-26

    Abstract

    Mass cytometry is a novel platform for high-dimensional phenotypic and functional analysis of single cells. This system uses elemental metal isotopes conjugated to monoclonal antibodies to evaluate up to 42 parameters simultaneously on individual cells with minimal overlap between channels. The platform can be customized for analysis of both phenotypic and functional markers. Here, we will describe methods to stain, collect, and analyze intracellular functional markers and surface phenotypic markers on natural killer cells.

    View details for DOI 10.1007/978-1-4939-3684-7_2

    View details for PubMedID 27177653

  • Pregnancy Does Not Attenuate the Antibody or Plasmablast Response to Inactivated Influenza Vaccine. journal of infectious diseases Kay, A. W., Bayless, N. L., Fukuyama, J., Aziz, N., Dekker, C. L., Mackey, S., Swan, G. E., Davis, M. M., Blish, C. A. 2015; 212 (6): 861-870

    Abstract

    Inactivated influenza vaccine (IIV) is recommended during pregnancy to prevent influenza infection and its complications in pregnant women and their infants. However, the extent to which pregnancy modifies the antibody response to vaccination remains unclear, and prior studies have focused primarily on hemagglutinin inhibition (HI) titers. A more comprehensive understanding of how pregnancy modifies the humoral immune response to influenza vaccination will aid in maximizing vaccine efficacy.Healthy pregnant women and control women were studied prior to, 7 days after, and 28 days after vaccination with IIV. HI titers, microneutralization (MN) titers, and the frequency of circulating plasmablasts were evaluated in pregnant versus control women.Pregnant women and control women mount similarly robust serologic immune responses to IIV, with no significant differences for any influenza strain in postvaccination geometric mean HI or MN titers. HI and MN titers correlate, though MN titers demonstrate more robust changes pre- versus postvaccination. The induction of circulating plasmablasts is increased in pregnant women versus controls (median fold-change 2.60 vs 1.49 [interquartile range, 0.94-7.53 vs 0.63-2.67]; P = .03).Pregnant women do not have impaired humoral immune responses to IIV and may have increased circulating plasmablast production compared to control women.

    View details for DOI 10.1093/infdis/jiv138

    View details for PubMedID 25740957

    View details for PubMedCentralID PMC4548461

  • Intrinsic retroviral reactivation in human preimplantation embryos and pluripotent cells. Nature Grow, E. J., Flynn, R. A., Chavez, S. L., Bayless, N. L., Wossidlo, M., Wesche, D. J., Martin, L., Ware, C. B., Blish, C. A., Chang, H. Y., Pera, R. A., Wysocka, J. 2015; 522 (7555): 221-225

    Abstract

    Endogenous retroviruses (ERVs) are remnants of ancient retroviral infections, and comprise nearly 8% of the human genome. The most recently acquired human ERV is HERVK(HML-2), which repeatedly infected the primate lineage both before and after the divergence of the human and chimpanzee common ancestor. Unlike most other human ERVs, HERVK retained multiple copies of intact open reading frames encoding retroviral proteins. However, HERVK is transcriptionally silenced by the host, with the exception of in certain pathological contexts such as germ-cell tumours, melanoma or human immunodeficiency virus (HIV) infection. Here we demonstrate that DNA hypomethylation at long terminal repeat elements representing the most recent genomic integrations, together with transactivation by OCT4 (also known as POU5F1), synergistically facilitate HERVK expression. Consequently, HERVK is transcribed during normal human embryogenesis, beginning with embryonic genome activation at the eight-cell stage, continuing through the emergence of epiblast cells in preimplantation blastocysts, and ceasing during human embryonic stem cell derivation from blastocyst outgrowths. Remarkably, we detected HERVK viral-like particles and Gag proteins in human blastocysts, indicating that early human development proceeds in the presence of retroviral products. We further show that overexpression of one such product, the HERVK accessory protein Rec, in a pluripotent cell line is sufficient to increase IFITM1 levels on the cell surface and inhibit viral infection, suggesting at least one mechanism through which HERVK can induce viral restriction pathways in early embryonic cells. Moreover, Rec directly binds a subset of cellular RNAs and modulates their ribosome occupancy, indicating that complex interactions between retroviral proteins and host factors can fine-tune pathways of early human development.

    View details for DOI 10.1038/nature14308

    View details for PubMedID 25896322

  • Immunogenicity and clinical efficacy of influenza vaccination in pregnancy FRONTIERS IN IMMUNOLOGY Kay, A. W., Blish, C. A. 2015; 6: 1-9

    Abstract

    Pregnant women are at high risk from influenza due to disproportionate morbidity, mortality, and adverse pregnancy outcomes following infection. As such, they are classified as a high-priority group for vaccination. However, changes in the maternal immune system required to accommodate the allogeneic fetus may alter the immunogenicity of influenza vaccines. A large number of studies have evaluated the safety of the influenza vaccine. Here, we will review available studies on the immunogenicity and efficacy of the influenza vaccine during pregnancy, focusing on both humoral and cellular immunity.

    View details for DOI 10.3389/fimmu.2015.00289

    View details for Web of Science ID 000357155700001

    View details for PubMedCentralID PMC4455389

  • IL-2 DEPENDENT NATURAL KILLER CELL RESPONSES TO ALLOGENEIC AND AUTOLOGOUS EPSTEIN-BARR VIRUS (EBV) plus B CELL LYMPHOMAS Hatton, O., Hadad, U., Blish, C., Esquivel, C., Krams, S., Martinez, O. WILEY-BLACKWELL. 2015: 93
  • Pregnancy does not attenuate the antibody or plasmablast response to inactivated influenza vaccine Bayless, N., Kay, A., Fukuayama, J., Aziz, N., Dekker, C., Mackey, S., Swan, G., Davis, M., Holmes, S., Blish, C. AMER ASSOC IMMUNOLOGISTS. 2015
  • IL-2-Dependent Natural Killer Cell Responses to Epstein-Barr Virus B Cell Lymphomas Involve the NKG2A/NKG2C Axis Hatton, O., Strauss-Albee, D., Blish, C., Esquivel, C., Krams, S., Martines, O. WILEY-BLACKWELL. 2015
  • Delayed BCG vaccination--time to take a shot. journal of infectious diseases Kay, A. W., Blish, C. A. 2015; 211 (3): 335-337

    View details for DOI 10.1093/infdis/jiu435

    View details for PubMedID 25108029

  • Immunogenicity and Clinical Efficacy of Influenza Vaccination in Pregnancy. Frontiers in immunology Kay, A. W., Blish, C. A. 2015; 6: 289-?

    Abstract

    Pregnant women are at high risk from influenza due to disproportionate morbidity, mortality, and adverse pregnancy outcomes following infection. As such, they are classified as a high-priority group for vaccination. However, changes in the maternal immune system required to accommodate the allogeneic fetus may alter the immunogenicity of influenza vaccines. A large number of studies have evaluated the safety of the influenza vaccine. Here, we will review available studies on the immunogenicity and efficacy of the influenza vaccine during pregnancy, focusing on both humoral and cellular immunity.

    View details for DOI 10.3389/fimmu.2015.00289

    View details for PubMedID 26089824

  • Coordinated Regulation of NK Receptor Expression in the Maturing Human Immune System JOURNAL OF IMMUNOLOGY Strauss-Albee, D. M., Horowitz, A., Parham, P., Blish, C. A. 2014; 193 (10): 4871-4879

    Abstract

    NK cells are responsible for recognizing and killing transformed, stressed, and infected cells. They recognize a set of non-Ag-specific features termed "altered self" through combinatorial signals from activating and inhibitory receptors. These NKRs are also expressed on CD4(+) and CD8(+) T cells, B cells, and monocytes, although a comprehensive inventory of NKR expression patterns across leukocyte lineages has never been performed. Using mass cytometry, we found that NKR expression patterns distinguish cell lineages in human peripheral blood. In individuals with high levels of CD57, indicative of a mature immune repertoire, NKRs are more likely to be expressed on non-NK cells, especially CD8(+) T cells. Mature NK and CD8(+) T cell populations show increased diversity of NKR surface expression patterns, but with distinct determinants: mature NK cells acquire primarily inhibitory receptors, whereas CD8(+) T cells attain a specific subset of both activating and inhibitory receptors, potentially imbuing them with a distinct functional role. Concurrently, monocytes show decreased expression of the generalized inhibitory receptor leukocyte Ig-like receptor subfamily b member 1, consistent with an increased activation threshold. Therefore, NKR expression is coordinately regulated as the immune system matures, resulting in the transfer of "altered self" recognition potential among leukocyte lineages. This likely reduces Ag specificity in the mature human immune system, and implies that vaccines and therapeutics that engage both its innate and adaptive branches may be more effective in the settings of aging and chronic infection.

    View details for DOI 10.4049/jimmunol.1401821

    View details for Web of Science ID 000345023400015

    View details for PubMedCentralID PMC4225175

  • Association between Latent Proviral Characteristics and Immune Activation in Antiretrovirus-Treated Human Immunodeficiency Virus Type 1-Infected Adults. Journal of virology Liang, E. C., Sceats, L., Bayless, N. L., Strauss-Albee, D. M., Kubo, J., Grant, P. M., Furman, D., Desai, M., Katzenstein, D. A., Davis, M. M., Zolopa, A. R., Blish, C. A. 2014; 88 (15): 8629-8639

    Abstract

    Generalized immune activation during HIV infection is associated with an increased risk of cardiovascular disease, neurocognitive disease, osteoporosis, metabolic disorders, and physical frailty. The mechanisms driving this immune activation are poorly understood, particularly for individuals effectively treated with antiretroviral medications. We hypothesized that viral characteristics such as sequence diversity may play a role in driving HIV-associated immune activation. We therefore sequenced proviral DNA isolated from peripheral blood mononuclear cells from HIV-infected individuals on fully suppressive antiretroviral therapy. We performed phylogenetic analyses, calculated viral diversity and divergence in the env and pol genes, and determined coreceptor tropism and the frequency of drug resistance mutations. Comprehensive immune profiling included quantification of immune cell subsets, plasma cytokine levels, and intracellular signaling responses in T cells, B cells, and monocytes. These antiretroviral therapy-treated HIV-infected individuals exhibited a wide range of diversity and divergence in both env and pol genes. However, proviral diversity and divergence in env and pol, coreceptor tropism, and the level of drug resistance did not significantly correlate with markers of immune activation. A clinical history of virologic failure was also not significantly associated with levels of immune activation, indicating that a history of virologic failure does not inexorably lead to increased immune activation as long as suppressive antiretroviral medications are provided. Overall, this study demonstrates that latent viral diversity is unlikely to be a major driver of persistent HIV-associated immune activation.Chronic immune activation, which is associated with cardiovascular disease, neurologic disease, and early aging, is likely to be a major driver of morbidity and mortality in HIV-infected individuals. Although treatment of HIV with antiretroviral medications decreases the level of immune activation, levels do not return to normal. The factors driving this persistent immune activation, particularly during effective treatment, are poorly understood. In this study, we investigated whether characteristics of the latent, integrated HIV provirus that persists during treatment are associated with immune activation. We found no relationship between latent viral characteristics and immune activation in treated individuals, indicating that qualities of the provirus are unlikely to be a major driver of persistent inflammation. We also found that individuals who had previously failed treatment but were currently effectively treated did not have significantly increased levels of immune activation, providing hope that past treatment failures do not have a lifelong "legacy" impact.

    View details for DOI 10.1128/JVI.01257-14

    View details for PubMedID 24850730

    View details for PubMedCentralID PMC4135944

  • Natural Killer Cell Responses to Autologous and Allogeneic Epstein-Barr Virus (EBV)(+) B Cell Lymphomas Hatton, O., Hadad, U., Strauss-Albee, D., Blish, C., Esquivel, C., Krams, S., Martinez, O. LIPPINCOTT WILLIAMS & WILKINS. 2014: 245–46
  • Systemic Cytokine Levels Show Limited Correlation With Risk of HIV-1 Acquisition. Journal of acquired immune deficiency syndromes Lehman, D. A., Ronen, K., Blish, C. A., Baeten, J. M., Jalalian-Lechak, Z., Jaoko, W., Mandaliya, K., Richardson, B. A., McClelland, R. S., Overbaugh, J. 2014; 66 (2): 135-139

    Abstract

    It has been hypothesized that immune activation and inflammation may increase HIV-1 susceptibility and that cytokines may be useful biomarkers for risk. Within a prospective cohort, we conducted a nested case-control analysis of plasma cytokine levels among women who acquired HIV-1 <3 months after sampling, compared to three different control groups. We observed associations between lower IL-6 and IL-10 and higher IL-7 levels with HIV-1 acquisition, however these associations were inconsistent when comparing to different control groups. Inconsistent results within our study and among prior studies suggest that reproducible findings are needed before cytokines are useful biomarkers for HIV-1 susceptibility.

    View details for DOI 10.1097/QAI.0000000000000104

    View details for PubMedID 24413043

  • Natural Killer Cell Responses to Autologous and Allogeneic Epstein-Barr Virus (EBV)(+) B Cell Lymphomas Hatton, O., Hadad, U., Strauss-Albee, D., Blish, C., Esquivel, C., Krams, S., Martinez, O. WILEY-BLACKWELL. 2014: 245–46
  • Association between Cellular Immune Activation, Target Cell Frequency, and Risk of Human Immunodeficiency Virus Type 1 Superinfection JOURNAL OF VIROLOGY Blish, C. A., Dogan, O. C., Jaoko, W., McClelland, R. S., Mandaliya, K., Odem-Davis, K., Richardson, B. A., Overbaugh, J. 2014; 88 (10): 5894-5899

    Abstract

    We performed a case-control study of women at risk of HIV-1 superinfection to understand the relationship between immune activation and HIV-1 acquisition. An increase in the frequency of HIV-1 target cells, but not in other markers of T cell activation, was associated with a 1.7-fold increase in the odds of superinfection. This suggests that HIV-1 acquisition risk is influenced more by the frequency of target cells than by the generalized level of immune activation.

    View details for DOI 10.1128/JVI.00187-14

    View details for Web of Science ID 000335446400062

    View details for PubMedID 24623424

  • Antibody-dependent cell-mediated virus inhibition antibody activity does not correlate with risk of HIV-1 superinfection. Journal of acquired immune deficiency syndromes Forthal, D. N., Landucci, G., Chohan, B., Richardson, B. A., McClelland, R. S., Jaoko, W., Blish, C., Overbaugh, J. 2013; 63 (1): 31-33

    Abstract

    Previous studies of HIV-infected women with high-risk behavior have indicated that neither neutralizing antibody nor cellular immunity elicited by an initial HIV-1 infection is associated with protection against superinfection with a different HIV-1 strain. Here, we measured antibody-dependent cell-mediated virus inhibition (ADCVI) antibody activity in the plasma of 12 superinfected cases and 36 singly infected matched controls against 2 heterologous viruses. We found no association between plasma ADCVI activity and superinfection status. ADCVI antibody activity against heterologous virus elicited by the original infection may not contribute to preventing a superinfecting HIV-1.

    View details for DOI 10.1097/QAI.0b013e3182874d41

    View details for PubMedID 23344546

  • Quantifying the unanticipated diversity of the human NK cell repertoire Strauss-Albee, D., Horowitz, A., Dogan, O., Mackey, S., Swan, G., Dekker, C., Davis, M., Parham, P., Blish, C. AMER ASSOC IMMUNOLOGISTS. 2013
  • Genital Inflammation Predicts HIV-1 Shedding Independent of Plasma Viral Load and Systemic Inflammation JAIDS-JOURNAL OF ACQUIRED IMMUNE DEFICIENCY SYNDROMES Blish, C. A., McClelland, R. S., Richardson, B. A., Jaoko, W., Mandaliya, K., Baeten, J. M., Overbaugh, J. 2012; 61 (4): 436-440

    Abstract

    In women, genital HIV-1 RNA levels predict the risk of HIV-1 transmission independent of plasma viral load. To better understand the factors that contribute to genital HIV-1 shedding, we evaluated the relationships between genital and plasma cytokine concentrations and HIV-1 RNA levels. Vaginal, but not plasma, levels of interferon gamma-induced protein 10 (IP-10) were significantly associated with vaginal viral load, independent of plasma viral load. Thus, efforts to decrease HIV-1 transmission must take into account the role of local inflammation, which is not necessarily reflected in plasma measurements.

    View details for DOI 10.1097/QAI.0b013e31826c2edd

    View details for Web of Science ID 000311083200008

    View details for PubMedID 22878424

    View details for PubMedCentralID PMC3494808

  • HIV-1 Transmission Goes Retro (Steps Back) JOURNAL OF INFECTIOUS DISEASES Blish, C. A. 2012; 206 (9): 1336-1338

    View details for DOI 10.1093/infdis/jis506

    View details for Web of Science ID 000309930700003

    View details for PubMedID 22997234

  • Measurements of Immune Responses for Establishing Correlates of Vaccine Protection Against HIV AIDS RESEARCH AND HUMAN RETROVIRUSES Burgers, W. A., Manrique, A., Masopust, D., McKinnon, L. R., Reynolds, M. R., Rolland, M., Blish, C., Chege, G. K., Curran, R., Fischer, W., Herrera, C., Sather, D. N. 2012; 28 (7): 641-648

    Abstract

    Well-defined correlates of protective immunity are an essential component of rational vaccine development. Despite years of basic science and three HIV vaccine efficacy trials, correlates of immunological protection from HIV infection remain undefined. In December 2010, a meeting of scientists engaged in basic and translational work toward developing HIV-1 vaccines was convened. The goal of this meeting was to discuss current opportunities and optimal approaches for defining correlates of protection, both for ongoing and future HIV-1 vaccine candidates; specific efforts were made to engage young scientists. We discuss here the highlights from the meeting regarding the progress made and the way forward for a protective HIV-1 vaccine.

    View details for DOI 10.1089/aid.2011.0239

    View details for Web of Science ID 000305810900002

    View details for PubMedID 21861777

    View details for PubMedCentralID PMC3380381

  • Cellular immune responses and susceptibility to HIV-1 superinfection: a case-control study AIDS Blish, C. A., Dogan, O. C., Jaoko, W., McClelland, R. S., Mandaliya, K., Odem-Davis, K. S., Richardsonb, B. A., Overbaugh, J. 2012; 26 (5): 643-646

    Abstract

    A case-control study was performed to determine the effects of HIV-1-specific cellular immune responses on the odds of acquiring a second HIV-1 infection (superinfection). Changes in the frequency of cytokine-producing or cytolytic CD8+ or CD4+ T cells were not associated with significant alterations in the odds of superinfection, suggesting that HIV-1 specific cellular immune responses at the level induced by chronic infection do not appear to significantly contribute to protection from HIV-1 superinfection.

    View details for DOI 10.1097/QAD.0b013e3283509a0b

    View details for Web of Science ID 000301333000015

    View details for PubMedID 22210637

    View details for PubMedCentralID PMC3511787

  • The impact of HIV-1 infection and exposure on natural killer (NK) cell phenotype in Kenyan infants during the first year of life. Frontiers in immunology Slyker, J. A., Lohman-Payne, B., John-Stewart, G. C., Dong, T., Mbori-Ngacha, D., Tapia, K., Atzberger, A., Taylor, S., Rowland-Jones, S. L., Blish, C. A. 2012; 3: 399-?

    Abstract

    Natural killer (NK) cells play an important role in the containment of HIV replication during primary infection, though their functions are impaired during chronic HIV infection. Infants experience more rapid HIV disease progression than adults, but contributions of infant NK cells to containing HIV infection are unknown. The aim of this study was to determine the impact of HIV infection on infant NK cell phenotype by evaluating samples and data from a cohort study of women and their infants, conducted in Nairobi, Kenya between 1999 and 2003. The percentage and phenotype of NK cells was evaluated longitudinally by multi-parameter flow cytometry over the first year of life in HIV-infected (HIV+, = 16), HIV-exposed uninfected (HIV-EU, n = 6), and healthy unexposed controls (HIV-, n = 4). At birth, NK subset distributions based on expression of CD56 and CD16 did not differ between HIV+, HIV-EU, or HIV- infants. However, HIV infection was associated with a subsequent decline in NK cells as a percentage of total lymphocytes (p < 0.001), and an expanding proportion of CD56-CD16+ NK cells (p < 0.001). Activated CD38(bright)CD69+ NK cells were more frequent in the HIV+ infants, followed by HIV-EU and HIV- infants, in both CD56(dim) (p = 0.005) and CD56(bright) compartments (p = 0.03). HIV infection and exposure was also associated with a significant decline in the percentage of perforin-expressing NK cells in the CD56(dim) compartment over the first year of life, with HIV+ infants losing approximately 2.5% (p < 0.001) and HIV-EU infants losing 3.0% (p = 0.01) of perforin+ cells per month. Thus, infant HIV infection is associated with alterations in NK cell subsets, activation, and cytolytic potential that could contribute to their poor control over HIV infection. Furthermore, exposure to HIV infection in infants who escaped infection is also associated with alterations in NK cells that may contribute to the reduced ability to fight infections that is observed in HIV-EU infants.

    View details for DOI 10.3389/fimmu.2012.00399

    View details for PubMedID 23293640

  • The impact of HIV-1 infection and exposure on natural killer (NK) cell phenotype in Kenyan infants during the first year of life FRONTIERS IN IMMUNOLOGY Slyker, J. A., Lohman-Payne, B., John-Stewart, G. C., Dong, T., Mbori-Ngacha, D., Tapia, K., Atzberger, A., Taylor, S., Rowland-Jones, S. L., Blish, C. A. 2012; 3

    Abstract

    Natural killer (NK) cells play an important role in the containment of HIV replication during primary infection, though their functions are impaired during chronic HIV infection. Infants experience more rapid HIV disease progression than adults, but contributions of infant NK cells to containing HIV infection are unknown. The aim of this study was to determine the impact of HIV infection on infant NK cell phenotype by evaluating samples and data from a cohort study of women and their infants, conducted in Nairobi, Kenya between 1999 and 2003. The percentage and phenotype of NK cells was evaluated longitudinally by multi-parameter flow cytometry over the first year of life in HIV-infected (HIV+, = 16), HIV-exposed uninfected (HIV-EU, n = 6), and healthy unexposed controls (HIV-, n = 4). At birth, NK subset distributions based on expression of CD56 and CD16 did not differ between HIV+, HIV-EU, or HIV- infants. However, HIV infection was associated with a subsequent decline in NK cells as a percentage of total lymphocytes (p < 0.001), and an expanding proportion of CD56-CD16+ NK cells (p < 0.001). Activated CD38(bright)CD69+ NK cells were more frequent in the HIV+ infants, followed by HIV-EU and HIV- infants, in both CD56(dim) (p = 0.005) and CD56(bright) compartments (p = 0.03). HIV infection and exposure was also associated with a significant decline in the percentage of perforin-expressing NK cells in the CD56(dim) compartment over the first year of life, with HIV+ infants losing approximately 2.5% (p < 0.001) and HIV-EU infants losing 3.0% (p = 0.01) of perforin+ cells per month. Thus, infant HIV infection is associated with alterations in NK cell subsets, activation, and cytolytic potential that could contribute to their poor control over HIV infection. Furthermore, exposure to HIV infection in infants who escaped infection is also associated with alterations in NK cells that may contribute to the reduced ability to fight infections that is observed in HIV-EU infants.

    View details for DOI 10.3389/fimmu.2012.00399

    View details for Web of Science ID 000209501300391

    View details for PubMedCentralID PMC3533178

  • The role of amino acid changes in the human immunodeficiency virus type 1 transmembrane domain in antibody binding and neutralization VIROLOGY Lovelace, E., Xu, H., Blish, C. A., Strong, R., Overbaugh, J. 2011; 421 (2): 235-244

    Abstract

    The detailed interactions between antibodies and the HIV-1 envelope protein that lead to neutralization are not well defined. Here, we show that several conservative substitutions in the envelope gp41 led to a ~100 fold increase in neutralization sensitivity to monoclonal antibodies (MAbs) that target gp41: 4E10 and 2F5. Substitution at position 675 alone did not impact neutralization susceptibility to MAbs that recognize more distal sites in gp120 (b12, VRC01, PG9). However, changes at position 675 in conjunction with Thr to Ala at position 569 increased the neutralization sensitivity to all gp41 and gp120 MAbs and plasma, in some cases by more than 1000-fold. Interestingly, the T569A change had a dramatic effect on b12 binding, but no effect on neutralization sensitivity. This finding suggests that antibody neutralization may occur through a multi-step pathway that includes distinct changes in envelope conformation that may affect binding but not neutralization susceptibility.

    View details for DOI 10.1016/j.virol.2011.09.032

    View details for Web of Science ID 000297183900017

    View details for PubMedID 22029936

  • The Breadth and Potency of Passively Acquired Human Immunodeficiency Virus Type 1-Specific Neutralizing Antibodies Do Not Correlate with the Risk of Infant Infection JOURNAL OF VIROLOGY Lynch, J. B., Nduati, R., Blish, C. A., Richardson, B. A., Mabuka, J. M., Jalalian-Lechak, Z., John-Stewart, G., Overbaugh, J. 2011; 85 (11): 5252-5261

    Abstract

    Although a major goal of human immunodeficiency virus type 1 (HIV-1) vaccine efforts is to elicit broad and potent neutralizing antibodies (NAbs), there are no data that directly demonstrate a role for such NAbs in protection from HIV-1 infection in exposed humans. The setting of mother-to-child transmission provides an opportunity to examine whether NAbs provide protection from HIV-1 infection because infants acquire passive antibodies from their mothers prior to exposure to HIV-1 through breastfeeding. We evaluated the characteristics of HIV-1-specific NAbs in 100 breast-fed infants of HIV-1-positive mothers who were HIV-1 negative at birth and monitored them until age 2. A panel of eight viruses that included variants representative of those in the study region as well as more diverse strains was used to determine the breadth of the infant NAbs. From their mothers, infants acquired broad and potent NAbs that were capable of recognizing heterologous circulating HIV-1 variants of diverse subtypes, but the presence of NAbs of broad HIV-1 specificity was not associated with transmission risk. There was also no correlation between responses to any particular virus tested, which included a range of diverse variants that demonstrated different neutralization profiles, including recognition by specific antibodies with known epitope targets. The eight viruses tested exhibited neutralization profiles to a variety of monoclonal antibodies (2F5, PG9, and VRC01) similar to those of viruses present in pregnant women in the cohort. These results suggest that the breadth and potency of the heterologous antibody response in exposed infants, measured against a virus panel comprised of variants typical of those circulating in the population, does not predict protection.

    View details for DOI 10.1128/JVI.02216-10

    View details for Web of Science ID 000290298700003

    View details for PubMedID 21411521

  • Hormonal Contraception and HIV-1 Transmission AMERICAN JOURNAL OF REPRODUCTIVE IMMUNOLOGY Blish, C. A., Baeten, J. M. 2011; 65 (3): 302-307

    Abstract

    Safe and effective contraceptive choices are essential for women with HIV-1 infection and at risk for HIV-1 infection. Epidemiological and laboratory-based studies suggest that hormonal contraception may influence HIV-1 transmission. Several large studies in high-risk populations indicate that hormonal contraceptive use may modestly increase the risk of HIV-1 acquisition. In addition, HIV-1-infected users of hormonal contraceptives may be more infectious to their uninfected partners, although no studies have directly measured HIV-1 transmission risk from women to men. However, several studies failed to demonstrate a link between contraceptive use and HIV-1 acquisition or transmission, and interpretation of many studies limited by methodological considerations, such as infrequent measurements of contraceptive exposure and HIV-1 status. As a result, many questions remain, and high-quality studies remain needed. It is clear that hormonal contraceptives are not protective against HIV-1 infection and that dual protection with condoms should be the goal for women using hormonal contraception.

    View details for DOI 10.1111/j.1600-0897.2010.00930.x

    View details for Web of Science ID 000287037200017

    View details for PubMedID 21087338

  • Changes in Plasma Cytokines after Treatment of Ascaris lumbricoides Infection in Individuals with HIV-1 Infection JOURNAL OF INFECTIOUS DISEASES Blish, C. A., Sangare, L., Herrin, B. R., Richardson, B. A., John-Stewart, G., Walson, J. L. 2010; 201 (12): 1816-1821

    Abstract

    Albendazole treatment of individuals with human immunodeficiency virus type 1 (HIV-1) and Ascaris lumbricoides co-infection has led to significantly improved CD4(+) cell counts and a trend for lower plasma HIV-1 RNA levels in a previous randomized placebo-controlled trial. To define mechanisms by which deworming contributed to changes in markers of HIV-1 disease progression, plasma cytokine levels were evaluated. Albendazole treatment, compared with placebo, was associated with significantly decreased plasma interleukin (IL) 10 levels (P = .01)ot associated with significant changes in levels of IL-1beta, IL-2, IL-4, IL-5, IL-6, IL-7, IL-8, IL-12p70, IL-13, interferon gamma, tumor necrosis factor alpha, or thymic stromal lymphopoietin. Treatment of A. lumbricoides co-infection may delay HIV-1 disease progression by reducing helminth-induced, IL-10-mediated immunosuppression.

    View details for DOI 10.1086/652784

    View details for Web of Science ID 000277687900007

    View details for PubMedID 20441516

  • Comparative Immunogenicity of Subtype A Human Immunodeficiency Virus Type 1 Envelope Exhibiting Differential Exposure of Conserved Neutralization Epitopes JOURNAL OF VIROLOGY Blish, C. A., Sather, D. N., Sellhorn, G., Stamatatos, L., Sun, Y., Srivastava, I., Barnett, S. W., Cleveland, B., Overbaugh, J., Hu, S. 2010; 84 (5): 2573-2584

    Abstract

    Development of broadly cross-reactive neutralizing antibodies (NAbs) remains a major goal of HIV-1 vaccine development, but most candidate envelope immunogens have had limited ability to cross-neutralize heterologous strains. To evaluate the immunogenicity of subtype A variants of HIV-1, rabbits were immunized with pairs of closely related subtype A envelopes from the same individual. In each immunogen pair, one variant was readily neutralized by a variety of monoclonal antibodies and plasma antibodies, while the other was neutralization resistant, suggesting differences in the exposures of key epitopes. The breadth of the antibody response was evaluated against subtype A, B, C, and D variants of HIV-1. The specificity of the immunogen-derived neutralizing antibody response was also compared to that of the infected individuals from whom these variants were cloned. None of the immunogens produced broad neutralizing antibodies in immunized animals, and most of the neutralizing antibodies were directed to the variable loops, particularly the V3 loop. No detectable antibodies to either of the potentially exposed conserved epitopes, the membrane proximal external region, or the CD4 binding site were found with immunized rabbits. In contrast, relatively little of the neutralizing activity within the plasma samples of the infected individuals was directed to linear epitopes within the variable loops. These data indicate that immunogens designed to expose conserved regions did not enhance generation of broadly neutralizing antibodies in comparison with the immunogens that failed to expose those regions using this immunization approach.

    View details for DOI 10.1128/JVI.01687-09

    View details for Web of Science ID 000274330300035

    View details for PubMedID 20015987

  • Breadth of Neutralizing Antibody Response to Human Immunodeficiency Virus Type 1 Is Affected by Factors Early in Infection but Does Not Influence Disease Progression JOURNAL OF VIROLOGY Piantadosi, A., Panteleeff, D., Blish, C. A., Baeten, J. M., Jaoko, W., McClelland, R. S., Overbaugh, J. 2009; 83 (19): 10269-10274

    Abstract

    The determinants of a broad neutralizing antibody (NAb) response and its effect on human immunodeficiency virus type 1 (HIV-1) disease progression are not well defined, partly because most prior studies of a broad NAb response were cross-sectional. We examined correlates of NAb response breadth among 70 HIV-infected, antiretroviral-naïve Kenyan women from a longitudinal seroincident cohort. NAb response breadth was measured 5 years after infection against five subtype A viruses and one subtype B virus. Greater NAb response breadth was associated with a higher viral load set point and greater HIV-1 env diversity early in infection. However, greater NAb response breadth was not associated with a delayed time to a CD4(+) T-cell count of <200, antiretroviral therapy, or death. Thus, a broad NAb response results from a high level of antigenic stimulation early in infection, which likely accounts for prior observations that greater NAb response breadth is associated with a higher viral load later in infection.

    View details for DOI 10.1128/JVI.01149-09

    View details for Web of Science ID 000269614300059

    View details for PubMedID 19640996

  • Cross-Subtype Neutralization Sensitivity despite Monoclonal Antibody Resistance among Early Subtype A, C, and D Envelope Variants of Human Immunodeficiency Virus Type 1 JOURNAL OF VIROLOGY Blish, C. A., Jalalian-Lechak, Z., Rainwater, S., Nguyen, M., Dogan, O. C., Overbaugh, J. 2009; 83 (15): 7783-7788

    Abstract

    The human immunodeficiency virus type 1 (HIV-1) variants that are transmitted to newly infected individuals are the primary targets of interventions, such as vaccines and microbicides, aimed at preventing new infections. Newly acquired subtype A, B, and C variants have been the focus of neutralization studies, although many of these viruses, particularly of subtypes A and B, represent viruses circulating more than a decade ago. In order to better represent the global diversity of transmitted HIV-1 variants, an additional 31 sexually transmitted Kenyan HIV-1 env genes, representing several recent infections with subtype A, as well as subtypes A/D, C, and D, were cloned, and their neutralization profiles were characterized. Most env variants were resistant to neutralization by the monoclonal antibodies (MAbs) b12, 4E10, 2F5, and 2G12, suggesting that targeting the epitopes of these MAbs may not be effective against variants that are spreading in areas of endemicity. However, significant cross-subtype neutralization by plasma was observed, indicating that there may be other epitopes, not yet defined by the limited available MAbs, which could be recognized more broadly.

    View details for DOI 10.1128/JVI.00673-09

    View details for Web of Science ID 000267747400043

    View details for PubMedID 19474105

  • Human Immunodeficiency Virus Type 1 Superinfection Occurs despite Relatively Robust Neutralizing Antibody Responses JOURNAL OF VIROLOGY Blish, C. A., Dogan, O. C., Derby, N. R., Nguyen, M., Chohan, B., Richardson, B. A., Overbaugh, J. 2008; 82 (24): 12094-12103

    Abstract

    Superinfection by a second human immunodeficiency virus type 1 (HIV-1) strain indicates that gaps in protective immunity occur during natural infection. To define the role of HIV-1-specific neutralizing antibodies (NAbs) in this setting, we examined NAb responses in 6 women who became superinfected between approximately 1 to 5 years following initial infection compared to 18 women with similar risk factors who did not. Although superinfected individuals had less NAb breadth than matched controls at approximately 1 year postinfection, no significant differences in the breadth or potency of NAb responses were observed just prior to the second infection. In fact, four of the six subjects had relatively broad and potent NAb responses prior to infection by the second strain. To more specifically examine the specificity of the NAbs against the superinfecting virus, these variants were cloned from five of the six individuals. The superinfecting variants did not appear to be inherently neutralization resistant, as measured against a pool of plasma from unrelated HIV-infected individuals. Moreover, the superinfected individuals were able to mount autologous NAb responses to these variants following reinfection. In addition, most superinfected individuals had NAbs that could neutralize their second viral strains prior to their reinfection, suggesting that the level of NAbs elicited during natural infection was not sufficient to block infection. These data indicate that preventing infection by vaccination will likely require broader and more potent NAb responses than those found in HIV-1-infected individuals.

    View details for DOI 10.1128/JVI.01730-08

    View details for Web of Science ID 000261164000011

    View details for PubMedID 18842728

  • Enhancing exposure of HIV-1 neutralization epitopes through mutations in gp41 PLOS MEDICINE Blish, C. A., Nguyen, M., Overbaugh, J. 2008; 5 (1): 90-103

    Abstract

    The generation of broadly neutralizing antibodies is a priority in the design of vaccines against HIV-1. Unfortunately, most antibodies to HIV-1 are narrow in their specificity, and a basic understanding of how to develop antibodies with broad neutralizing activity is needed. Designing methods to target antibodies to conserved HIV-1 epitopes may allow for the generation of broadly neutralizing antibodies and aid the global fight against AIDS by providing new approaches to block HIV-1 infection. Using a naturally occurring HIV-1 Envelope (Env) variant as a template, we sought to identify features of Env that would enhance exposure of conserved HIV-1 epitopes.Within a cohort study of high-risk women in Mombasa, Kenya, we previously identified a subtype A HIV-1 Env variant in one participant that was unusually sensitive to neutralization. Using site-directed mutagenesis, the unusual neutralization sensitivity of this variant was mapped to two amino acid mutations within conserved sites in the transmembrane subunit (gp41) of the HIV-1 Env protein. These two mutations, when introduced into a neutralization-resistant variant from the same participant, resulted in 3- to >360-fold enhanced neutralization by monoclonal antibodies specific for conserved regions of both gp41 and the Env surface subunit, gp120, >780-fold enhanced neutralization by soluble CD4, and >35-fold enhanced neutralization by the antibodies found within a pool of plasmas from unrelated individuals. Enhanced neutralization sensitivity was not explained by differences in Env infectivity, Env concentration, Env shedding, or apparent differences in fusion kinetics. Furthermore, introduction of these mutations into unrelated viral Env sequences, including those from both another subtype A variant and a subtype B variant, resulted in enhanced neutralization susceptibility to gp41- and gp120-specific antibodies, and to plasma antibodies. This enhanced neutralization sensitivity exceeded 1,000-fold in several cases.Two amino acid mutations within gp41 were identified that expose multiple discontinuous neutralization epitopes on diverse HIV-1 Env proteins. These exposed epitopes were shielded on the unmodified viral Env proteins, and several of the exposed epitopes encompass desired target regions for protective antibodies. Env proteins containing these modifications could act as a scaffold for presentation of such conserved domains, and may aid in developing methods to target antibodies to such regions.

    View details for DOI 10.1371/journal.pmed.0050009

    View details for Web of Science ID 000254928700017

    View details for PubMedID 18177204

  • Transmission of HIV-1 in the face of neutralizing antibodies CURRENT HIV RESEARCH Blish, C. A., Blay, W. A., Haigwood, N. L., Overbaughl, J. 2007; 5 (6): 578-587

    Abstract

    In most cases of HIV-1 transmission, only a subset of variants is transmitted from the index case to the newly infected individual. Understanding the characteristics of these transmitted variants may aid in developing new methods to halt the spread of HIV-1. Studies evaluating the genotypic and antigenic properties of transmitted variants have provided insights into how the selective pressures applied during different modes of transmission uniquely imprint the infecting viruses. In the setting of sexual transmission, variants with increased neutralization sensitivity appeared to be selected during transmission in discordant subtype C-infected couples, although transmitted variants from different risk groups and HIV-1 subtypes did not demonstrate increased neutralization sensitivity, suggesting this may not be a consistent feature of transmitted variants. Studies of both mother to child transmission (MTCT) and superinfection, where preexisting NAbs are present at the time of exposure, provide opportunities to analyze whether the breadth and potency of the NAb response influence the incidence of new infections. MTCT resulted in selection for variants that were resistant to maternal antibodies, suggesting that maternal antibodies can protect the baby from those variants that are susceptible to the antibodies present. There are some data to suggest that poor neutralizing antibody (NAb) responses are present in cases of superinfection, although these data are preliminary. Defining the characteristics of the viruses transmitted in the presence and absence of NAbs as well as defining the NAb responses that fail to protect from infection during MTCT and superinfection may provide critical insights into the antibody responses that are needed for effective vaccines and other prophylactic therapeutics.

    View details for Web of Science ID 000253633600008

    View details for PubMedID 18045114

  • HIV-1 subtype A envelope variants from early in infection have variable sensitivity to neutralization and to inhibitors of viral entry AIDS Blish, C. A., Nedellec, R., Mandaliya, K., Mosier, D. E., Overbaugh, J. 2007; 21 (6): 693-702

    Abstract

    An effective HIV-1 vaccine or microbicide must block the transmitted virus variants that initially establish a new infection; consequently, it is critical that such viruses be isolated and characterized.To evaluate HIV-1 envelope variants from early in infection from individuals infected heterosexually with subtype A HIV-1 for their sensitivity to antibody-mediated neutralization and to inhibitors of viral entry.Full-length subtype A HIV-1 envelope clones from 28-75 days postinfection were used to generate pseudoviruses for infection studies. The susceptibility of these pseudoviruses to neutralization by autologous and heterologous plasma and by monoclonal antibodies was examined. The sensitivity of these pseudoviruses to PSC-RANTES and TAK-779, inhibitors of CCR5, and to soluble CD4 (sCD4) was also evaluated.Pseudoviruses with subtype A HIV-1 envelopes from early in infection demonstrated a broad range of neutralization sensitivities to both autologous and heterologous plasma. However, neutralization by the monoclonal antibodies b12, 2G12, 4E10 and 2F5 was generally poor; notably, none of the 14 early virus variants were neutralized by 2G12 and only one was neutralized by b12. Viruses bearing these early CCR5-using envelopes were generally sensitive to the CCR5 inhibitors PSC-RANTES and TAK-779, but they demonstrated more variable sensitivity to sCD4.These subtype A HIV-1 variants, representing the viruses that must be blocked by antibody-based prevention strategies, vary in their susceptibility to neutralization. A subset of these HIV-1 variants from early in infection will be useful for screening candidate vaccines and microbicides.

    View details for Web of Science ID 000245637500007

    View details for PubMedID 17413690

  • Anergic CD8(+) T cells can persist and function in vivo JOURNAL OF IMMUNOLOGY Blish, C. A., Dillon, S. R., Farr, A. G., Fink, P. J. 1999; 163 (1): 155-164

    Abstract

    Using a mouse model system, we demonstrate that anergic CD8+ T cells can persist and retain some functional capabilities in vivo, even after the induction of tolerance. In TCR Vbeta5 transgenic mice, mature CD8+Vbeta5+ T cells transit through a CD8lowVbeta5low deletional intermediate during tolerance induction. CD8low cells are characterized by an activated phenotype, are functionally compromised in vitro, and are slated for deletion in vivo. We now demonstrate that CD8low cells derive from a proliferative compartment, but do not divide in vivo. CD8low cells persist in vivo with a t1/2 of 3-5 days, in contrast to their in vitro t1/2 of 0.5-1 day. During this unexpectedly long in vivo life span, CD8low cells are capable of producing IFN-gamma in vivo despite their inability to proliferate or to kill target cells in vitro. CD8low cells also accumulate at sites of inflammation, where they produce IFN-gamma. Therefore, rather than withdrawing from the pool of functional CD8+ T cells, anergic CD8low cells retain a potential regulatory role despite losing their capacity to proliferate. The ability of anergic cells to persist and function in vivo adds another level of complexity to the process of tolerance induction in the lymphoid periphery.

    View details for Web of Science ID 000080973700023

    View details for PubMedID 10384112

  • Chronic modulation of the TCR repertoire in the lymphoid periphery JOURNAL OF IMMUNOLOGY Blish, C. A., Gallay, B. J., Turk, G. L., Kline, K. V., Wheat, W., Fink, P. J. 1999; 162 (6): 3131-3140

    Abstract

    Using TCR V beta 5 transgenic mice as a model system, we demonstrate that the induction of peripheral tolerance can mold the TCR repertoire throughout adult life. In these mice, three distinct populations of peripheral T cells are affected by chronic selective events in the lymphoid periphery. First, CD4+V beta 5+ T cells are deleted in the lymphoid periphery by superantigens encoded by mouse mammary tumor viruses-8 and -9 in an MHC class II-dependent manner. Second, mature CD8+V beta 5+ T cells transit through a CD8lowV beta 5low deletional intermediate during tolerance induction by a process that depends upon neither mouse mammary tumor virus-encoded superantigens nor MHC class II expression. Third, a population of CD4-CD8-V beta 5+ T cells arises in the lymphoid periphery in an age-dependent manner. We analyzed the TCR V alpha repertoire of each of these cellular compartments in both V beta 5 transgenic and nontransgenic C57BL/6 mice as a function of age. This analysis revealed age-related changes in the expression of V alpha families among different cellular compartments, highlighting the dynamic state of the peripheral immune repertoire. Our work indicates that the chronic processes maintaining peripheral T cell tolerance can dramatically shape the available TCR repertoire.

    View details for Web of Science ID 000079105000004

    View details for PubMedID 10092762

  • LOSS OF CELL-CYCLE CONTROLS IN APOPTOTIC LYMPHOBLASTS OF THE BURSA OF FABRICIUS MOLECULAR BIOLOGY OF THE CELL Neiman, P. E., BLISH, C., HEYDT, C., Loring, G., THOMAS, S. J. 1994; 5 (7): 763-772

    Abstract

    Lymphoblasts of the normal embryonic follicles of the chicken bursa of Fabricius undergo rapid apoptosis when exposed to gamma-radiation or when cell-cell contacts are disrupted by mechanical dispersion in short term culture. We have observed previously that overexpression of v-myc sensitizes preneoplastic bursal lymphoblasts to induction of cell death, whereas resistance to induced cell death is acquired during progression to neoplasia. In this study we observed extensive DNA degradation in the large majority of the lymphoblast population within the first hour after dispersion-induced apoptosis. Paradoxically these cells continued to progress into S-phase with the bulk of DNA cleavage and death occurring in S-phase cells (i.e., in cells with more than 2C and less than 4C DNA content). We confirmed the S phase status of apoptotic cells by determining that detection of nuclear cyclin A in individual cells also corresponded with detection of DNA breakage. Levels of cyclin E, cyclin E-dependent H1 histone kinase, and p53 proteins were maintained during dispersion-induced DNA cleavage. gamma-radiation failed either to inhibit cell cycle progression or to raise p53 levels in dispersed bursal lymphoblasts. In intact bursal follicles low doses of gamma-radiation induced p53 whereas higher, apoptosis-inducing doses failed to induce p53 or prevent G1 to S-phase progression. These results suggest that normal DNA damage-induced cell cycle checkpoint controls are lost or overridden when apoptosis is induced in bursal lymphoblasts.

    View details for Web of Science ID A1994PC03800005

    View details for PubMedID 7812045