Peter S. Kim

All Publications

• Design of Epitope-FocusedVaccines via Epitope Cleavage ACS CENTRAL SCIENCE Pham, D. M., Costello, R. M., Bruun, T. U. J., Kim, P. S. 2026

  View details for DOI 10.1021/acscentsci.6c00476

  View details for Web of Science ID 001824478900001

• Oriented Multivalent Display Drives Consistent Serum Immunodominance to the Ebola Virus Glycoprotein. ACS central science Zheng, C., Rubio, A. A., Vasquez, S., Pham, D., Pan, Z., Barnes, C. O., Kim, P. S. 2026; 12 (1): 100-110

  Abstract

  Despite the vast diversity of B cell repertoires, serum antibody responses during viral infection often focus on a limited set of epitopesa phenomenon known as immunodominance. This inherent bias establishes a hierarchy of epitope responses, which often facilitates viral immune evasion and presents a major challenge for universal vaccine design. It remains unclear whether serum immunodominance is primarily driven by antigen-intrinsic properties or by the spatial constraints imposed by virion-bound antigen presentation. Here, using Ebola virus glycoprotein (GP) as a model system, we found that trimeric GP elicited varied epitope hierarchies between individual animals during primary immunization. In contrast, multivalent GP presentation on either a vesicular stomatitis virus or ferritin nanoparticlesin the native orientation found on the Ebola viruselicited highly consistent and more refined epitope hierarchies across multiple mice and guinea pigs. These findings reveal a key role of oriented multivalent presentation in shaping serum immunodominance. The striking consistency of epitope hierarchy among individuals suggests that oriented multivalent presentation may promote more uniform immune protection at the population level, beyond increasing the magnitude of antibody binding and neutralizing responses.

  View details for DOI 10.1021/acscentsci.5c01886

  View details for PubMedID 41625225

  View details for PubMedCentralID PMC12856675

• Development of ACE2-tropic-betacoronavirus therapeutics for future pandemic preparedness. Nature communications Utz, A., Armbrust, M., Nguyen, T. T., Morris, M. K., Matthews, C. O., Kompella, P., Cao, Z., Ha, J. W., Violette, A., Brewer, R. C., Lanz, T. V., Robinson, W. H., Xu, D., Hanson, C., Hugenmatter, A., Kim, P. S. 2025

  Abstract

  A major challenge during viral pandemics is the ability to develop therapeutics whose efficacy can withstand viral genetic evolution. During the COVID-19 pandemic, five SARS-CoV-2 monoclonal antibody (mAb) therapeutics were rendered ineffective within a period of 2 years, leading to the U.S. FDA revoking their emergency use authorization. Here, we describe ReconnAb-multimers, a new therapeutic design that broadly and potently neutralize all tested betacoronaviruses that use host ACE2 as their receptor to enter cells. These ReconnAb-multimers have potent neutralization efficacy via avidity, enhanced breadth via a new pan-betacoronavirus-binding antibody that targets a highly conserved epitope on SARS-CoV-2 spike protein, and the potential for clinical development by using a catalytically inactive ACE2 component. We demonstrate that ReconnAb-multimers neutralize all SARS-CoV-2 pseudoviruses and authentic viral variants of concern (VOC) tested, with similar or higher potency than mAbs previously approved by the FDA; neutralize related pandemic-potential betacoronaviruses, including SARS-CoV, WIV1-CoV, PRD-0038, and merbecovirus HKU5-CoV-2; and despite a short half-life, protect female mice against authentic viral challenge with Omicron variant XBB.1.5. Our results highlight ReconnAb-multimers as a broad and highly potent therapeutic that could potentially withstand viral escape against current and future betacoronaviruses that require host ACE2 as a receptor.

  View details for DOI 10.1038/s41467-025-66805-6

  View details for PubMedID 41318533

• Exceptionally broad HIV-1 neutralization via bispecific antibody-mediated prepositioning. Proceedings of the National Academy of Sciences of the United States of America Kim, S., Travisano, K. A., Wilder, B., Palomares, M., Cao, Z., Seaman, M. S., Kim, P. S. 2025; 122 (40): e2517311122

  Abstract

  Antibodies that recognize the conserved prehairpin intermediate (PHI) of class I viral membrane-fusion proteins typically show limited neutralization and have not been considered promising therapeutic agents. We previously developed a bispecific antibody (bsAb), iMab/D5_AR, directed toward both the gp41 N-heptad repeat (NHR) that is exposed within the HIV-1 PHI and toward CD4, the HIV-1 receptor on T cells. CD4-binding led to prepositioning of the bsAb at the site of viral fusion, enhancing its neutralization potency and achieving 95% breadth (IC80 < 5 μg/mL) against a panel of 119 pseudotyped, multiclade HIV-1 viruses. In the current study, we engineered a bsAb against NHR that also targets CCR5, one of two HIV-1 coreceptors on T cells. This optimized bsAb design further improves neutralization potency and achieves 100% neutralization breadth against the 119-member pseudotyped virus panel, including those resistant to CD4-binding iMab/D5_AR. Considering that nearly all initial HIV-1 infections occur via CCR5-tropic viruses, we expect our redesigned bsAb targeting CCR5 to be an effective prophylactic agent. These findings further support the rationale for pursuing the NHR as a therapeutic target for HIV-1 and lay the groundwork for a new class of engineered broadly neutralizing antibodies.

  View details for DOI 10.1073/pnas.2517311122

  View details for PubMedID 41026814

• Utilizing Machine Learning to Improve Neutralization Potency of an HIV-1 Antibody Targeting the gp41 N-Heptad Repeat. ACS chemical biology Filsinger Interrante, M. V., Tang, S., Kim, S., Shanker, V. R., Hie, B. L., Bruun, T. U., Wu, W., Pak, J. E., Fernandez, D., Kim, P. S. 2025

  Abstract

  The N-heptad repeat (NHR) of the HIV-1 gp41 prehairpin intermediate (PHI) is an attractive potential vaccine target with high sequence conservation across diverse strains. However, despite the potency of NHR-targeting peptides and clinical efficacy of the NHR-targeting entry inhibitor enfuvirtide, no potently neutralizing NHR-directed monoclonal antibodies (mAbs) nor antisera have been identified or elicited to date. The lack of potent NHR-binding mAbs both dampens enthusiasm for vaccine development efforts at this target and presents a barrier to performing passive immunization experiments with NHR-targeting antibodies. To address this challenge, we previously developed an improved variant of the NHR-directed mAb D5, called D5_AR, which is capable of neutralizing diverse tier-2 viruses. Building on that work, here we present the 2.7Å-crystal structure of D5_AR bound to NHR mimetic peptide IQN17. We then utilize protein language models and supervised machine learning to generate small (n < 100) libraries of D5_AR variants that are subsequently screened for improved neutralization potency. We identify a variant with 5-fold improved neutralization potency, D5_FI, which is the most potent NHR-directed monoclonal antibody characterized to date and exhibits broad neutralization of tier-2 and -3 pseudoviruses as well as replicating R5 and X4 challenge strains. Additionally, our work highlights the ability of protein language models to efficiently identify improved mAb variants from relatively small libraries.

  View details for DOI 10.1021/acschembio.5c00035

  View details for PubMedID 40540236

• A broad antibody with enhanced HIV-1 neutralization via bispecific antibody-mediated prepositioning. Nature communications Kim, S., Radford, C. E., Xu, D., Zhong, J., Do, J., Pham, D. M., Travisano, K. A., Filsinger Interrante, M. V., Bruun, T. U., Rezek, V., Wilder, B., Palomares, M., Seaman, M. S., Kitchen, S. G., Bloom, J. D., Kim, P. S. 2025; 16 (1): 4617

  Abstract

  Antibodies targeting the highly conserved prehairpin intermediate (PHI) of class I viral membrane-fusion proteins are generally weakly neutralizing and are not considered viable therapeutic agents. We previously demonstrated that antibodies targeting the gp41 N-heptad repeat (NHR), which is transiently exposed in the HIV-1 PHI, exhibit enhanced broad neutralization in cells expressing the Fc receptor, FcγRI. To enhance neutralization in cells lacking FcγRI, we here develop a bispecific antibody (bsAb) by fusing an NHR-targeting antibody to an antibody against CD4, the HIV-1 receptor on T cells. The bsAb provides a 5000-fold neutralization enhancement and shows unprecedented neutralization breadth compared to existing broadly neutralizing antibodies. Importantly, the bsAb reduces viral load in HIV-1-infected humanized male mice, and viral envelope sequencing under bsAb pressure revealed an NHR mutation that potentially impairs viral fitness. These findings validate the NHR as a potential HIV-1 therapeutic target, setting the stage for a new class of broadly neutralizing antibodies.

  View details for DOI 10.1038/s41467-025-60035-6

  View details for PubMedID 40383778

  View details for PubMedCentralID 2517140

• Engineering a SARS-CoV-2 Vaccine Targeting the Receptor-Binding Domain Cryptic-Face via Immunofocusing. ACS central science Bruun, T. U., Do, J., Weidenbacher, P. A., Utz, A., Kim, P. S. 2024; 10 (10): 1871-1884

  Abstract

  The receptor-binding domain (RBD) of the SARS-CoV-2 spike protein is the main target of neutralizing antibodies. Although they are infrequently elicited during infection or vaccination, antibodies that bind to the conformation-specific cryptic face of the RBD display remarkable breadth of binding and neutralization across Sarbecoviruses. Here, we employed the immunofocusing technique PMD (protect, modify, deprotect) to create RBD immunogens (PMD-RBD) specifically designed to focus the antibody response toward the cryptic-face epitope recognized by the broadly neutralizing antibody S2X259. Immunization with PMD-RBD antigens induced robust binding titers and broad neutralizing activity against homologous and heterologous Sarbecovirus strains. A serum-depletion assay provided direct evidence that PMD successfully skewed the polyclonal antibody response toward the cryptic face of the RBD. Our work demonstrates the ability of PMD to overcome immunodominance and refocus humoral immunity, with implications for the development of broader and more resilient vaccines against current and emerging viruses with pandemic potential.

  View details for DOI 10.1021/acscentsci.4c00722

  View details for PubMedID 39463836

  View details for PubMedCentralID PMC11503491

• Engineering a SARS-CoV-2 Vaccine Targeting the Receptor-Binding Domain Cryptic-Face via Immunofocusing ACS CENTRAL SCIENCE Bruun, T. U. J., Do, J., Weidenbacher, P., Utz, A., Kim, P. S. 2024

  View details for DOI 10.1021/acscentsci.4c00722

  View details for Web of Science ID 001315141300001

• Unsupervised evolution of protein and antibody complexes with a structure-informed language model. Science (New York, N.Y.) Shanker, V. R., Bruun, T. U., Hie, B. L., Kim, P. S. 2024; 385 (6704): 46-53

  Abstract

  Large language models trained on sequence information alone can learn high-level principles of protein design. However, beyond sequence, the three-dimensional structures of proteins determine their specific function, activity, and evolvability. Here, we show that a general protein language model augmented with protein structure backbone coordinates can guide evolution for diverse proteins without the need to model individual functional tasks. We also demonstrate that ESM-IF1, which was only trained on single-chain structures, can be extended to engineer protein complexes. Using this approach, we screened about 30 variants of two therapeutic clinical antibodies used to treat severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. We achieved up to 25-fold improvement in neutralization and 37-fold improvement in affinity against antibody-escaped viral variants of concern BQ.1.1 and XBB.1.5, respectively. These findings highlight the advantage of integrating structural information to identify efficient protein evolution trajectories without requiring any task-specific training data.

  View details for DOI 10.1126/science.adk8946

  View details for PubMedID 38963838

• Interstitial macrophages are a focus of viral takeover and inflammation in COVID-19 initiation in human lung. The Journal of experimental medicine Wu, T. T., Travaglini, K. J., Rustagi, A., Xu, D., Zhang, Y., Andronov, L., Jang, S., Gillich, A., Dehghannasiri, R., Martinez-Colon, G. J., Beck, A., Liu, D. D., Wilk, A. J., Morri, M., Trope, W. L., Bierman, R., Weissman, I. L., Shrager, J. B., Quake, S. R., Kuo, C. S., Salzman, J., Moerner, W. E., Kim, P. S., Blish, C. A., Krasnow, M. A. 2024; 221 (6)

  Abstract

  Early stages of deadly respiratory diseases including COVID-19 are challenging to elucidate in humans. Here, we define cellular tropism and transcriptomic effects of SARS-CoV-2 virus by productively infecting healthy human lung tissue and using scRNA-seq to reconstruct the transcriptional program in "infection pseudotime" for individual lung cell types. SARS-CoV-2 predominantly infected activated interstitial macrophages (IMs), which can accumulate thousands of viral RNA molecules, taking over 60% of the cell transcriptome and forming dense viral RNA bodies while inducing host profibrotic (TGFB1, SPP1) and inflammatory (early interferon response, CCL2/7/8/13, CXCL10, and IL6/10) programs and destroying host cell architecture. Infected alveolar macrophages (AMs) showed none of these extreme responses. Spike-dependent viral entry into AMs used ACE2 and Sialoadhesin/CD169, whereas IM entry used DC-SIGN/CD209. These results identify activated IMs as a prominent site of viral takeover, the focus of inflammation and fibrosis, and suggest targeting CD209 to prevent early pathology in COVID-19 pneumonia. This approach can be generalized to any human lung infection and to evaluate therapeutics.

  View details for DOI 10.1084/jem.20232192

  View details for PubMedID 38597954

• Stabilized trimeric peptide immunogens of the complete HIV-1 gp41 N- heptad repeat and their use as HIV-1 vaccine candidates PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Wu, C., Raheem, I. T., Nahas, D. D., Citron, M., Kim, P. S., Montefiori, D. C., Ottinger, E. A., Hepler, R. W., Hrin, R., Patel, S. B., Soisson, S. M., Joyce, J. G. 2024; 121 (22): e2317230121

  Abstract

  Efforts to develop an HIV-1 vaccine include those focusing on conserved structural elements as the target of broadly neutralizing monoclonal antibodies. MAb D5 binds to a highly conserved hydrophobic pocket on the gp41 N-heptad repeat (NHR) coiled coil and neutralizes through prevention of viral fusion and entry. Assessment of 17-mer and 36-mer NHR peptides presenting the D5 epitope in rodent immunogenicity studies showed that the longer peptide elicited higher titers of neutralizing antibodies, suggesting that neutralizing epitopes outside of the D5 pocket may exist. Although the magnitude and breadth of neutralization elicited by NHR-targeting antigens are lower than that observed for antibodies directed to other epitopes on the envelope glycoprotein complex, it has been shown that NHR-directed antibodies are potentiated in TZM-bl cells containing the FcγRI receptor. Herein, we report the design and evaluation of covalently stabilized trimeric 51-mer peptides encompassing the complete gp41 NHR. We demonstrate that these peptide trimers function as effective antiviral entry inhibitors and retain the ability to present the D5 epitope. We further demonstrate in rodent and nonhuman primate immunization studies that our 51-mer constructs elicit a broader repertoire of neutralizing antibody and improved cross-clade neutralization of primary HIV-1 isolates relative to 17-mer and 36-mer NHR peptides in A3R5 and FcγR1-enhanced TZM-bl assays. These results demonstrate that sensitive neutralization assays can be used for structural enhancement of moderately potent neutralizing epitopes. Finally, we present expanded trimeric peptide designs which include unique low-molecular-weight scaffolds that provide versatility in our immunogen presentation strategy.

  View details for DOI 10.1073/pnas.2317230121

  View details for Web of Science ID 001237231900006

  View details for PubMedID 38768344

  View details for PubMedCentralID PMC11145295

• Design of universal Ebola virus vaccine candidates via immunofocusing. Proceedings of the National Academy of Sciences of the United States of America Xu, D., Powell, A. E., Utz, A., Sanyal, M., Do, J., Patten, J. J., Moliva, J. I., Sullivan, N. J., Davey, R. A., Kim, P. S. 2024; 121 (7): e2316960121

  Abstract

  The Ebola virus causes hemorrhagic fever in humans and poses a significant threat to global public health. Although two viral vector vaccines have been approved to prevent Ebola virus disease, they are distributed in the limited ring vaccination setting and only indicated for prevention of infection from orthoebolavirus zairense (EBOV)-one of three orthoebolavirus species that have caused previous outbreaks. Ebola virus glycoprotein GP mediates viral infection and serves as the primary target of neutralizing antibodies. Here, we describe a universal Ebola virus vaccine approach using a structure-guided design of candidates with hyperglycosylation that aims to direct antibody responses away from variable regions and toward conserved epitopes of GP. We first determined the hyperglycosylation landscape on Ebola virus GP and used that to generate hyperglycosylated GP variants with two to four additional glycosylation sites to mask the highly variable glycan cap region. We then created vaccine candidates by displaying wild-type or hyperglycosylated GP variants on ferritin nanoparticles (Fer). Immunization with these antigens elicited potent neutralizing antisera against EBOV in mice. Importantly, we observed consistent cross-neutralizing activity against Bundibugyo virus and Sudan virus from hyperglycosylated GP-Fer with two or three additional glycans. In comparison, elicitation of cross-neutralizing antisera was rare in mice immunized with wild-type GP-Fer. These results demonstrate a potential strategy to develop universal Ebola virus vaccines that confer cross-protective immunity against existing and emerging filovirus species.

  View details for DOI 10.1073/pnas.2316960121

  View details for PubMedID 38319964

• Vaccine design via antigen reorientation. Nature chemical biology Xu, D., Carter, J. J., Li, C., Utz, A., Weidenbacher, P. A., Tang, S., Sanyal, M., Pulendran, B., Barnes, C. O., Kim, P. S. 2024

  Abstract

  A major challenge in creating universal influenza vaccines is to focus immune responses away from the immunodominant, variable head region of hemagglutinin (HA-head) and toward the evolutionarily conserved stem region (HA-stem). Here we introduce an approach to control antigen orientation via site-specific insertion of aspartate residues that facilitates antigen binding to alum. We demonstrate the generalizability of this approach with antigens from Ebola, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and influenza viruses and observe enhanced neutralizing antibody responses in all cases. We then reorient an H2 HA in an 'upside-down' configuration to increase the exposure and immunogenicity of HA-stem. The reoriented H2 HA (reoH2HA) on alum induced stem-directed antibodies that cross-react with both group 1 and group 2 influenza A subtypes. Electron microscopy polyclonal epitope mapping (EMPEM) revealed that reoH2HA (group 1) elicits cross-reactive antibodies targeting group 2 HA-stems. Our results highlight antigen reorientation as a generalizable approach for designing epitope-focused vaccines.

  View details for DOI 10.1038/s41589-023-01529-6

  View details for PubMedID 38225471

  View details for PubMedCentralID 9345323

• Bringing immunofocusing into focus. NPJ vaccines Musunuri, S., Weidenbacher, P. A., Kim, P. S. 2024; 9 (1): 11

  Abstract

  Immunofocusing is a strategy to create immunogens that redirect humoral immune responses towards a targeted epitope and away from non-desirable epitopes. Immunofocusing methods often aim to develop "universal" vaccines that provide broad protection against highly variant viruses such as influenza virus, human immunodeficiency virus (HIV-1), and most recently, severe acute respiratory syndrome coronavirus (SARS-CoV-2). We use existing examples to illustrate five main immunofocusing strategies-cross-strain boosting, mosaic display, protein dissection, epitope scaffolding, and epitope masking. We also discuss obstacles for immunofocusing like immune imprinting. A thorough understanding, advancement, and application of the methods we outline here will enable the design of high-resolution vaccines that protect against future viral outbreaks.

  View details for DOI 10.1038/s41541-023-00792-x

  View details for PubMedID 38195562

• Author Correction: A ferritin-based COVID-19 nanoparticle vaccine that elicits robust, durable, broad-spectrum neutralizing antisera in non-human primates. Nature communications Weidenbacher, P. A., Sanyal, M., Friedland, N., Tang, S., Arunachalam, P. S., Hu, M., Kumru, O. S., Morris, M. K., Fontenot, J., Shirreff, L., Do, J., Cheng, Y. C., Vasudevan, G., Feinberg, M. B., Villinger, F. J., Hanson, C., Joshi, S. B., Volkin, D. B., Pulendran, B., Kim, P. S. 2023; 14 (1): 6211

  View details for DOI 10.1038/s41467-023-42061-4

  View details for PubMedID 37798288

• Formulation development and comparability studies with an aluminum-salt adjuvanted SARS-CoV-2 spike ferritin nanoparticle vaccine antigen produced from two different cell lines. Vaccine Kumru, O. S., Sanyal, M., Friedland, N., Hickey, J. M., Joshi, R., Weidenbacher, P., Do, J., Cheng, Y. C., Kim, P. S., Joshi, S. B., Volkin, D. B. 2023

  Abstract

  The development of safe and effective second-generation COVID-19 vaccines to improve affordability and storage stability requirements remains a high priority to expand global coverage. In this report, we describe formulation development and comparability studies with a self-assembled SARS-CoV-2 spike ferritin nanoparticle vaccine antigen (called DCFHP), when produced in two different cell lines and formulated with an aluminum-salt adjuvant (Alhydrogel, AH). Varying levels of phosphate buffer altered the extent and strength of antigen-adjuvant interactions, and these formulations were evaluated for their (1) in vivo performance in mice and (2) in vitro stability profiles. Unadjuvanted DCFHP produced minimal immune responses while AH-adjuvanted formulations elicited greatly enhanced pseudovirus neutralization titers independent of ∼100%, ∼40% or ∼10% of the DCFHP antigen adsorbed to AH. These formulations differed, however, in their in vitro stability properties as determined by biophysical studies and a competitive ELISA for measuring ACE2 receptor binding of AH-bound antigen. Interestingly, after one month of 4°C storage, small increases in antigenicity with concomitant decreases in the ability to desorb the antigen from the AH were observed. Finally, we performed a comparability assessment of DCFHP antigen produced in Expi293 and CHO cells, which displayed expected differences in their N-linked oligosaccharide profiles. Despite consisting of different DCFHP glycoforms, these two preparations were highly similar in their key quality attributes including molecular size, structural integrity, conformational stability, binding to ACE2 receptor and mouse immunogenicity profiles. Taken together, these studies support future preclinical and clinical development of an AH-adjuvanted DCFHP vaccine candidate produced in CHO cells.

  View details for DOI 10.1016/j.vaccine.2023.08.037

  View details for PubMedID 37620203

• Efficient evolution of human antibodies from general protein language models. Nature biotechnology Hie, B. L., Shanker, V. R., Xu, D., Bruun, T. U., Weidenbacher, P. A., Tang, S., Wu, W., Pak, J. E., Kim, P. S. 2023

  Abstract

  Natural evolution must explore a vast landscape of possible sequences for desirable yet rare mutations, suggesting that learning from natural evolutionary strategies could guide artificial evolution. Here we report that general protein language models can efficiently evolve human antibodies by suggesting mutations that are evolutionarily plausible, despite providing the model with no information about the target antigen, binding specificity or protein structure. We performed language-model-guided affinity maturation of seven antibodies, screening 20 or fewer variants of each antibody across only two rounds of laboratory evolution, and improved the binding affinities of four clinically relevant, highly mature antibodies up to sevenfold and three unmatured antibodies up to 160-fold, with many designs also demonstrating favorable thermostability and viral neutralization activity against Ebola and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pseudoviruses. The same models that improve antibody binding also guide efficient evolution across diverse protein families and selection pressures, including antibiotic resistance and enzyme activity, suggesting that these results generalize to many settings.

  View details for DOI 10.1038/s41587-023-01763-2

  View details for PubMedID 37095349

  View details for PubMedCentralID 4410700

• A ferritin-based COVID-19 nanoparticle vaccine that elicits robust, durable, broad-spectrum neutralizing antisera in non-human primates. Nature communications Weidenbacher, P. A., Sanyal, M., Friedland, N., Tang, S., Arunachalam, P. S., Hu, M., Kumru, O. S., Morris, M. K., Fontenot, J., Shirreff, L., Do, J., Cheng, Y. C., Vasudevan, G., Feinberg, M. B., Villinger, F. J., Hanson, C., Joshi, S. B., Volkin, D. B., Pulendran, B., Kim, P. S. 2023; 14 (1): 2149

  Abstract

  While the rapid development of COVID-19 vaccines has been a scientific triumph, the need remains for a globally available vaccine that provides longer-lasting immunity against present and future SARS-CoV-2 variants of concern (VOCs). Here, we describe DCFHP, a ferritin-based, protein-nanoparticle vaccine candidate that, when formulated with aluminum hydroxide as the sole adjuvant (DCFHP-alum), elicits potent and durable neutralizing antisera in non-human primates against known VOCs, including Omicron BQ.1, as well as against SARS-CoV-1. Following a booster ~one year after the initial immunization, DCFHP-alum elicits a robust anamnestic response. To enable global accessibility, we generated a cell line that can enable production of thousands of vaccine doses per liter of cell culture and show that DCFHP-alum maintains potency for at least 14 days at temperatures exceeding standard room temperature. DCFHP-alum has potential as a once-yearly (or less frequent) booster vaccine, and as a primary vaccine for pediatric use including in infants.

  View details for DOI 10.1038/s41467-023-37417-9

  View details for PubMedID 37069151

  View details for PubMedCentralID 9225255

• Formulation development and comparability studies with an aluminum-salt adjuvanted SARS-CoV-2 Spike ferritin nanoparticle vaccine antigen produced from two different cell lines. bioRxiv : the preprint server for biology Kumru, O. S., Sanyal, M., Friedland, N., Hickey, J., Joshi, R., Weidenbacher, P., Do, J., Cheng, Y. C., Kim, P. S., Joshi, S. B., Volkin, D. B. 2023

  Abstract

  The development of safe and effective second-generation COVID-19 vaccines to improve affordability and storage stability requirements remains a high priority to expand global coverage. In this report, we describe formulation development and comparability studies with a self-assembled SARS-CoV-2 spike ferritin nanoparticle vaccine antigen (called DCFHP), when produced in two different cell lines and formulated with an aluminum-salt adjuvant (Alhydrogel, AH). Varying levels of phosphate buffer altered the extent and strength of antigen-adjuvant interactions, and these formulations were evaluated for their (1) in vivo performance in mice and (2) in vitro stability profiles. Unadjuvanted DCFHP produced minimal immune responses while AH-adjuvanted formulations elicited greatly enhanced pseudovirus neutralization titers independent of ∼100%, ∼40% or ∼10% of the DCFHP antigen adsorbed to AH. These formulations differed, however, in their in vitro stability properties as determined by biophysical studies and a competitive ELISA for measuring ACE2 receptor binding of AH-bound antigen. Interestingly, after one month of 4°C storage, small increases in antigenicity with concomitant decreases in the ability to desorb the antigen from the AH were observed. Finally, we performed a comparability assessment of DCFHP antigen produced in Expi293 and CHO cells, which displayed expected differences in their N-linked oligosaccharide profiles. Despite consisting of different DCFHP glycoforms, these two preparations were highly similar in their key quality attributes including molecular size, structural integrity, conformational stability, binding to ACE2 receptor and mouse immunogenicity profiles. Taken together, these studies support future preclinical and clinical development of an AH-adjuvanted DCFHP vaccine candidate produced in CHO cells.

  View details for DOI 10.1101/2023.04.03.535447

  View details for PubMedID 37066156

  View details for PubMedCentralID PMC10103975

• Structure-guided stabilization improves the ability of the HIV-1 gp41 hydrophobic pocket to elicit neutralizing antibodies. The Journal of biological chemistry Bruun, T. J., Tang, S., Erwin, G., Deis, L., Fernandez, D., Kim, P. 2023: 103062

  Abstract

  The hydrophobic pocket found in the N-heptad repeat (NHR) region of HIV-1 gp41 is a highly conserved epitope that is the target of various HIV-1 neutralizing monoclonal antibodies. Although the high conservation of the pocket makes it an attractive vaccine candidate, it has been challenging to elicit potent anti-NHR antibodies via immunization. Here, we solved a high-resolution structure of the NHR mimetic IQN17, and, consistent with previous ligand-bound gp41 pocket structures, we observed remarkable conformational plasticity of the pocket. The high malleability of this pocket led us to test whether we could improve the immunogenicity of the gp41 pocket by stabilizing its conformation. We show that the addition of five amino acids at the C-terminus of IQN17, to generate IQN22, introduces a stabilizing salt bridge at the base of the peptide that rigidifies the pocket. Mice immunized with IQN22 elicited higher avidity antibodies against the gp41 pocket and a more potent, albeit still weak, neutralizing response against HIV-1 compared to IQN17. Stabilized epitope-focused immunogens could serve as the basis for future HIV-1 fusion-inhibiting vaccines.

  View details for DOI 10.1016/j.jbc.2023.103062

  View details for PubMedID 36841484

• HIV-1 prehairpin intermediate inhibitors show efficacy independent of neutralization tier. Proceedings of the National Academy of Sciences of the United States of America Bell, B. N., Bruun, T. U., Friedland, N., Kim, P. S. 2023; 120 (8): e2215792120

  Abstract

  HIV-1 strains are categorized into one of three neutralization tiers based on the relative ease by which they are neutralized by plasma from HIV-1-infected donors not on antiretroviral therapy; tier-1 strains are particularly sensitive to neutralization while tier-2 and tier-3 strains are increasingly difficult to neutralize. Most broadly neutralizing antibodies (bnAbs) previously described target the native prefusion conformation of HIV-1 Envelope (Env), but the relevance of the tiered categories for inhibitors targeting another Env conformation, the prehairpin intermediate, is not well understood. Here, we show that two inhibitors targeting distinct highly conserved regions of the prehairpin intermediate have strikingly consistent neutralization potencies (within ~100-fold for a given inhibitor) against strains in all three neutralization tiers of HIV-1; in contrast, best-in-class bnAbs targeting diverse Env epitopes vary by more than 10,000-fold in potency against these strains. Our results indicate that antisera-based HIV-1 neutralization tiers are not relevant for inhibitors targeting the prehairpin intermediate and highlight the potential for therapies and vaccine efforts targeting this conformation.

  View details for DOI 10.1073/pnas.2215792120

  View details for PubMedID 36795752

• Enhancing HIV-1 Neutralization by Increasing the Local Concentration of Membrane-Proximal External Region-Directed Broadly Neutralizing Antibodies. Journal of virology Kim, S., Filsinger Interrante, M. V., Kim, P. S. 2022: e0164722

  Abstract

  Broadly neutralizing antibodies (bNAbs) against the membrane-proximal external region (MPER) of the gp41 component of the human immunodeficiency virus type 1 (HIV-1) envelope (Env) are characterized by long, hydrophobic, heavy chain complementarity-determining region 3s (HCDR3s) that interact with the MPER and some viral membrane lipids to achieve increased local concentrations. Here, we show that increasing the local concentration of MPER-directed bNAbs at the cell surface via binding to the high-affinity Fc receptor FcγRI potentiates their ability to prevent viral entry in a manner analogous to the previously reported observation wherein the lipid-binding activity of MPER bNAbs increases their concentration at the viral surface membrane. However, binding of MPER-directed bNAb 10E8 to FcγRI abolishes the neutralization synergy that is seen with the N-heptad repeat (NHR)-targeting antibody D5_AR and NHR-targeting small molecule enfuvirtide (T20), possibly due to decreased accessibility of the NHR in the FcγRI-10E8-MPER complex. Taken together, our results suggest that lipid-binding activity and FcγRI-mediated potentiation function in concert to improve the potency of MPER-directed bNAbs by increasing their local concentration near the site of viral fusion. Therefore, lipid binding may not be a strict requirement for potent neutralization by MPER-targeting bNAbs, as alternative methods can achieve similar increases in local concentrations while avoiding potential liabilities associated with immunologic host tolerance. IMPORTANCE The trimeric glycoprotein Env, the only viral protein expressed on the surface of HIV-1, is the target of broadly neutralizing antibodies and the focus of most vaccine development efforts. Broadly neutralizing antibodies targeting the membrane proximal external region (MPER) of Env show lipid-binding characteristics, and modulating this interaction affects neutralization. In this study, we tested the neutralization potencies of variants of the MPER-targeting antibody 10E8 with different viral-membrane-binding and host FcγRI-binding capabilities. Our results suggest that binding to both lipid and FcγRI improves the neutralization potency of MPER-directed antibodies by concentrating the antibodies at sites of viral fusion. As such, lipid binding may not be uniquely required for MPER-targeting broadly neutralizing antibodies, as alternative methods to increase local concentration can achieve similar improvements in potency.

  View details for DOI 10.1128/jvi.01647-22

  View details for PubMedID 36541800

• Human sperm TMEM95 binds eggs and facilitates membrane fusion. Proceedings of the National Academy of Sciences of the United States of America Tang, S., Lu, Y., Skinner, W. M., Sanyal, M., Lishko, P. V., Ikawa, M., Kim, P. S. 2022; 119 (40): e2207805119

  Abstract

  Tmem95 encodes a sperm acrosomal membrane protein, whose knockout has a male-specific sterility phenotype in mice. Tmem95 knockout murine sperm can bind to, but do not fuse with, eggs. How TMEM95 plays a role in membrane fusion of sperm and eggs has remained elusive. Here, we utilize a sperm penetration assay as a model system to investigate the function of human TMEM95. We show that human TMEM95 binds to hamster egg membranes, providing evidence for a TMEM95 receptor on eggs. Using X-ray crystallography, we reveal an evolutionarily conserved, positively charged region of TMEM95 as a putative receptor-binding surface. Amino acid substitutions within this region of TMEM95 ablate egg-binding activity. We identify monoclonal antibodies against TMEM95 that reduce the number of human sperm fused with hamster eggs in sperm penetration assays. Strikingly, these antibodies do not block binding of sperm to eggs. Taken together, these results provide strong evidence for a specific, receptor-mediated interaction of sperm TMEM95 with eggs and suggest that this interaction may have a role in facilitating membrane fusion during fertilization.

  View details for DOI 10.1073/pnas.2207805119

  View details for PubMedID 36161911

• Converting non-neutralizing SARS-CoV-2 antibodies into broad-spectrum inhibitors. Nature chemical biology Weidenbacher, P. A., Waltari, E., de Los Rios Kobara, I., Bell, B. N., Morris, M. K., Cheng, Y., Hanson, C., Pak, J. E., Kim, P. S. 2022

  Abstract

  Omicron and its subvariants have rendered most authorized monoclonal antibody-based treatments for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) ineffective, highlighting the need for biologics capable of overcoming SARS-CoV-2 evolution. These mostly ineffective antibodies target variable epitopes. Here we describe broad-spectrum SARS-CoV-2 inhibitors developed by tethering the SARS-CoV-2 receptor, angiotensin-converting enzyme 2 (ACE2), to known non-neutralizing antibodies that target highly conserved epitopes in the viral spike protein. These inhibitors, called receptor-blocking conserved non-neutralizing antibodies (ReconnAbs), potently neutralize all SARS-CoV-2 variants of concern (VOCs), including Omicron. Neutralization potency is lost when the linker joining the binding and inhibitory ReconnAb components is severed. In addition, a bi-functional ReconnAb, made by linking ACE2 to a bi-specific antibody targeting two non-overlapping conserved epitopes, defined here, shows sub-nanomolar neutralizing activity against all VOCs, including Omicron and BA.2. Given their conserved targets and modular nature, ReconnAbs have the potential to act as broad-spectrum therapeutics against SARS-CoV-2 and other emerging pandemic diseases.

  View details for DOI 10.1038/s41589-022-01140-1

  View details for PubMedID 36076082

• Simplified Purification of Glycoprotein-Modified Ferritin Nanoparticles for Vaccine Development. Biochemistry Weidenbacher, P., Musunuri, S., Powell, A. E., Tang, S., Do, J., Sanyal, M., Kim, P. S. 2022

  Abstract

  Ferritin-based, self-assembling protein nanoparticle vaccines are being developed against a range of viral pathogens, including SARS-CoV-2, influenza, HIV-1, and Epstein-Barr virus. However, purification of these nanoparticles is often laborious and requires customization for each potential nanoparticle vaccine. We propose that the simple insertion of a polyhistidine tag into exposed flexible loops on the ferritin surface (His-Fer) can mitigate the need for complex purifications and enable facile metal-chelate-based purification, thereby allowing for optimization of early stage vaccine candidates. Using sequence homology and computational modeling, we identify four sites that can accommodate insertion of a polyhistidine tag and demonstrate purification of both hemagglutinin-modified and SARS-CoV-2 spike-modified ferritins, highlighting the generality of the approach. A site at the 4-fold axis of symmetry enables optimal purification of both protein nanoparticles. We demonstrate improved purification through modulating the polyhistidine length and optimizing both the metal cation and the resin type. Finally, we show that purified His-Fer proteins remain multimeric and elicit robust immune responses similar to those of their wild-type counterparts. Collectively, this work provides a simplified purification scheme for ferritin-based vaccines.

  View details for DOI 10.1021/acs.biochem.2c00241

  View details for PubMedID 35960597

• Chemically Modified Bacterial Sacculi as a Vaccine Microparticle Scaffold. ACS chemical biology Weidenbacher, P. A., Rodriguez-Rivera, F. P., Sanyal, M., Visser, J. A., Do, J., Bertozzi, C. R., Kim, P. S. 2022

  Abstract

  Vaccine scaffolds and carrier proteins increase the immunogenicity of subunit vaccines. Here, we developed, characterized, and demonstrated the efficacy of a novel microparticle vaccine scaffold comprised of bacterial peptidoglycan (PGN), isolated as an entire sacculi. The PGN microparticles contain bio-orthogonal chemical handles allowing for site-specific attachment of immunogens. We first evaluated the purification, integrity, and immunogenicity of PGN microparticles derived from a variety of bacterial species. We then optimized PGN microparticle modification conditions; Staphylococcus aureus PGN microparticles containing azido-d-alanine yielded robust conjugation to immunogens. We then demonstrated that this vaccine scaffold elicits comparable immunostimulation to the conventional carrier protein, keyhole limpet hemocyanin (KLH). We further modified the S. aureus PGN microparticle to contain the SARS-CoV-2 receptor-binding domain (RBD)─this conjugate vaccine elicited neutralizing antibody titers comparable to those elicited by the KLH-conjugated RBD. Collectively, these findings suggest that chemically modified bacterial PGN microparticles are a conjugatable and biodegradable microparticle scaffold capable of eliciting a robust immune response toward an antigen of interest.

  View details for DOI 10.1021/acschembio.2c00140

  View details for PubMedID 35412807

• Mechanisms of innate and adaptive immunity to the Pfizer-BioNTech BNT162b2 vaccine. Nature immunology Li, C., Lee, A., Grigoryan, L., Arunachalam, P. S., Scott, M. K., Trisal, M., Wimmers, F., Sanyal, M., Weidenbacher, P. A., Feng, Y., Adamska, J. Z., Valore, E., Wang, Y., Verma, R., Reis, N., Dunham, D., O'Hara, R., Park, H., Luo, W., Gitlin, A. D., Kim, P., Khatri, P., Nadeau, K. C., Pulendran, B. 2022

  Abstract

  Despite the success of the BNT162b2 mRNA vaccine, the immunological mechanisms that underlie its efficacy are poorly understood. Here we analyzed the innate and adaptive responses to BNT162b2 in mice, and show that immunization stimulated potent antibody and antigen-specific T cell responses, as well as strikingly enhanced innate responses after secondary immunization, which was concurrent with enhanced serum interferon (IFN)-gamma levels 1d following secondary immunization. Notably, we found that natural killer cells and CD8+ T cells in the draining lymph nodes are the major producers of this circulating IFN-gamma. Analysis of knockout mice revealed that induction of antibody and T cell responses to BNT162b2 was not dependent on signaling via Toll-like receptors 2, 3, 4, 5 and 7 nor inflammasome activation, nor the necroptosis or pyroptosis cell death pathways. Rather, the CD8+ T cell response induced by BNT162b2 was dependent on type I interferon-dependent MDA5 signaling. These results provide insights into the molecular mechanisms by which the BNT162b2 vaccine stimulates immune responses.

  View details for DOI 10.1038/s41590-022-01163-9

  View details for PubMedID 35288714

• Evolutionary velocity with protein language models predicts evolutionary dynamics of diverse proteins. Cell systems Hie, B. L., Yang, K. K., Kim, P. S. 2022

  Abstract

  The degree to which evolution is predictable is a fundamental question in biology. Previous attempts to predict the evolution of protein sequences have been limited to specific proteins and to small changes, such as single-residue mutations. Here, we demonstrate that by using a protein language model to predict the local evolution within protein families, we recover a dynamic "vector field" of protein evolution that we call evolutionary velocity (evo-velocity). Evo-velocity generalizes to evolution over vastly different timescales, from viral proteins evolving over years to eukaryotic proteins evolving over geologic eons, and can predict the evolutionary dynamics of proteins that were not used to develop the original model. Evo-velocity also yields new evolutionary insights by predicting strategies of viral-host immune escape, resolving conflicting theories on the evolution of serpins, and revealing a key role of horizontal gene transfer in the evolution of eukaryotic glycolysis.

  View details for DOI 10.1016/j.cels.2022.01.003

  View details for PubMedID 35120643

• Multimerization of Ebola GPDeltamucin on protein nanoparticle vaccines has minimal effect on elicitation of neutralizing antibodies. Frontiers in immunology Powell, A. E., Xu, D., Roth, G. A., Zhang, K., Chiu, W., Appel, E. A., Kim, P. S. 2022; 13: 942897

  Abstract

  Ebola virus (EBOV), a member of the Filoviridae family of viruses and a causative agent of Ebola Virus Disease (EVD), is a highly pathogenic virus that has caused over twenty outbreaks in Central and West Africa since its formal discovery in 1976. The only FDA-licensed vaccine against Ebola virus, rVSV-ZEBOV-GP (Ervebo), is efficacious against infection following just one dose. However, since this vaccine contains a replicating virus, it requires ultra-low temperature storage which imparts considerable logistical challenges for distribution and access. Additional vaccine candidates could provide expanded protection to mitigate current and future outbreaks. Here, we designed and characterized two multimeric protein nanoparticle subunit vaccines displaying 8 or 20 copies of GPDeltamucin, a truncated form of the EBOV surface protein GP. Single-dose immunization of mice with GPDeltamucin nanoparticles revealed that neutralizing antibody levels were roughly equivalent to those observed in mice immunized with non-multimerized GPDeltamucin trimers. These results suggest that some protein subunit antigens do not elicit enhanced antibody responses when displayed on multivalent scaffolds and can inform next-generation design of stable Ebola virus vaccine candidates.

  View details for DOI 10.3389/fimmu.2022.942897

  View details for PubMedID 36091016

• Hydrogel-Based Slow Release of a Receptor-Binding Domain Subunit Vaccine Elicits Neutralizing Antibody Responses Against SARS-CoV-2. Advanced materials (Deerfield Beach, Fla.) Gale, E. C., Powell, A. E., Roth, G. A., Meany, E. L., Yan, J., Ou, B. S., Grosskopf, A. K., Adamska, J., Picece, V. C., d'Aquino, A. I., Pulendran, B., Kim, P. S., Appel, E. A. 2021: e2104362

  Abstract

  The development of effective vaccines that can be rapidly manufactured and distributed worldwide is necessary to mitigate the devastating health and economic impacts of pandemics like COVID-19. The receptor-binding domain (RBD) of the SARS-CoV-2 spike protein, which mediates host cell entry of the virus, is an appealing antigen for subunit vaccines because it is efficient to manufacture, highly stable, and a target for neutralizing antibodies. Unfortunately, RBD is poorly immunogenic. While most subunit vaccines are commonly formulated with adjuvants to enhance their immunogenicity, clinically-relevant adjuvants Alum, AddaVax, and CpG/Alum are found unable to elicit neutralizing responses following a prime-boost immunization. Here, it has been shown that sustained delivery of an RBD subunit vaccine comprisingCpG/Alumadjuvant in an injectable polymer-nanoparticle (PNP) hydrogelelicited potentanti-RBD andanti-spikeantibody titers, providing broader protection against SARS-CoV-2 variants of concern compared to bolus administration of the same vaccine and vaccines comprising other clinically-relevant adjuvant systems. Notably, a SARS-CoV-2 spike-pseudotyped lentivirus neutralization assay revealed that hydrogel-based vaccines elicited potent neutralizing responses when bolus vaccines didnot. Together, these results suggest that slow delivery of RBD subunit vaccines with PNP hydrogels can significantly enhance the immunogenicity of RBD and induce neutralizing humoral immunity.

  View details for DOI 10.1002/adma.202104362

  View details for PubMedID 34651342

• New-onset IgG autoantibodies in hospitalized patients with COVID-19. Nature communications Chang, S. E., Feng, A., Meng, W., Apostolidis, S. A., Mack, E., Artandi, M., Barman, L., Bennett, K., Chakraborty, S., Chang, I., Cheung, P., Chinthrajah, S., Dhingra, S., Do, E., Finck, A., Gaano, A., GeSSner, R., Giannini, H. M., Gonzalez, J., Greib, S., Gundisch, M., Hsu, A. R., Kuo, A., Manohar, M., Mao, R., Neeli, I., Neubauer, A., Oniyide, O., Powell, A. E., Puri, R., Renz, H., Schapiro, J., Weidenbacher, P. A., Wittman, R., Ahuja, N., Chung, H., Jagannathan, P., James, J. A., Kim, P. S., Meyer, N. J., Nadeau, K. C., Radic, M., Robinson, W. H., Singh, U., Wang, T. T., Wherry, E. J., Skevaki, C., Luning Prak, E. T., Utz, P. J. 2021; 12 (1): 5417

  Abstract

  COVID-19 is associated with a wide range of clinical manifestations, including autoimmune features and autoantibody production. Here we develop three protein arrays to measure IgG autoantibodies associated with connective tissue diseases, anti-cytokine antibodies, and anti-viral antibody responses in serum from 147 hospitalized COVID-19 patients. Autoantibodies are identified in approximately 50% of patients but in less than 15% of healthy controls. When present, autoantibodies largely target autoantigens associated with rare disorders such as myositis, systemic sclerosis and overlap syndromes. A subset of autoantibodies targeting traditional autoantigens or cytokines develop de novo following SARS-CoV-2 infection. Autoantibodies track with longitudinal development of IgG antibodies recognizing SARS-CoV-2 structural proteins and a subset of non-structural proteins, but not proteins from influenza, seasonal coronaviruses or other pathogenic viruses. We conclude that SARS-CoV-2 causes development of new-onset IgG autoantibodies in a significant proportion of hospitalized COVID-19 patients and are positively correlated with immune responses to SARS-CoV-2 proteins.

  View details for DOI 10.1038/s41467-021-25509-3

  View details for PubMedID 34521836

• A derivative of the D5 monoclonal antibody that targets the gp41 N-heptad repeat of HIV-1 with broad tier-2 neutralizing activity. Journal of virology Rubio, A. A., Filsinger Interrante, M. V., Bell, B. N., Brown, C. L., Bruun, T. U., LaBranche, C. C., Montefiori, D. C., Kim, P. S. 2021

  Abstract

  HIV-1 infection is initiated by the viral glycoprotein Env, which, after interaction with cellular coreceptors, adopts a transient conformation known as the pre-hairpin intermediate (PHI). The N-heptad repeat (NHR) is a highly conserved region of gp41 exposed in the PHI; it is the target of the FDA-approved drug enfuvirtide and of neutralizing monoclonal antibodies (mAbs). However, to date these mAbs have only been weakly effective against tier-1 HIV-1 strains, which are most sensitive to neutralizing antibodies. Here, we engineered and tested 11 IgG variants of D5, an anti-NHR mAb, by recombining previously described mutations in four of D5's six antibody complementarity-determining regions. One variant, D5_AR, demonstrated 6-fold enhancement in ID50 against lentivirus pseudotyped with HXB2 Env. D5_AR exhibited weak cross-clade neutralizing activity against a diverse set of tier-2 HIV-1 viruses, which are less sensitive to neutralizing antibodies than tier-1 viruses and are the target of current antibody-based vaccine efforts. In addition, the neutralization potency of D5_AR IgG was greatly enhanced in target cells expressing FcgammaRI, with ID50 values below 0.1 mug/mL; this immunoglobulin receptor is expressed on macrophages and dendritic cells, which are implicated in the early stages of HIV-1 infection of mucosal surfaces. D5 and D5_AR have equivalent neutralization potency in IgG, Fab, and scFv formats, indicating that neutralization is not impacted by steric hindrance. Taken together, these results provide support for vaccine strategies that target the PHI by eliciting antibodies against the gp41 NHR and support investigation of anti-NHR mAbs in non-human primate passive immunization studies.ImportanceDespite advances in anti-retroviral therapy, HIV remains a global epidemic and has claimed more than 32 million lives. Accordingly, developing an effective HIV vaccine remains an urgent public health need. The gp41 N-heptad repeat (NHR) of the HIV-1 pre-hairpin intermediate (PHI) is highly conserved (>90%) and is inhibited by the FDA-approved drug enfuvirtide, making it an attractive vaccine target. However, to date anti-NHR antibodies have not been potent. Here, we engineered D5_AR, a more potent variant of the anti-NHR antibody D5, and established its ability to inhibit HIV-1 strains that are more difficult to neutralize and are more representative of circulating strains (tier-2 strains). The neutralizing activity of D5_AR was greatly potentiated in cells expressing FcgammaRI; FcgammaRI is expressed on cells that are implicated at the earliest stages of sexual HIV-1 transmission. Taken together, these results bolster efforts to target the gp41 NHR and the PHI for vaccine development.

  View details for DOI 10.1128/JVI.02350-20

  View details for PubMedID 33980592

• Neutralizing antibodies targeting the SARS-CoV-2 receptor binding domain isolated from a naive human antibody library. Protein science : a publication of the Protein Society Bell, B. N., Powell, A. E., Rodriguez, C., Cochran, J. R., Kim, P. S. 2021

  Abstract

  Infection with SARS-CoV-2 elicits robust antibody responses in some patients, with a majority of the response directed at the receptor binding domain (RBD) of the spike surface glycoprotein. Remarkably, many patient-derived antibodies that potently inhibit viral infection harbor few to no mutations from the germline, suggesting that naive antibody libraries are a viable means for discovery of novel SARS-CoV-2 neutralizing antibodies. Here, we used a yeast surface-display library of human naive antibodies to isolate and characterize three novel neutralizing antibodies that target the RBD: one that blocks interaction with angiotensin-converting enzyme 2 (ACE2), the human receptor for SARS-CoV-2, and two that target other epitopes on the RBD. These three antibodies neutralized SARS-CoV-2 spike-pseudotyped lentivirus with IC50 values as low as 60ng/mL in vitro. Using a biolayer interferometry-based binding competition assay, we determined that these antibodies have distinct but overlapping epitopes with antibodies elicited during natural COVID-19 infection. Taken together, these analyses highlight how in vitro selection of naive antibodies can mimic the humoral response in vivo, yielding neutralizing antibodies and various epitopes that can be effectively targeted on the SARS-CoV-2 RBD. This article is protected by copyright. All rights reserved.

  View details for DOI 10.1002/pro.4044

  View details for PubMedID 33586288

• A Single Immunization with Spike-Functionalized Ferritin Vaccines Elicits Neutralizing Antibody Responses against SARS-CoV-2 in Mice. ACS central science Powell, A. E., Zhang, K., Sanyal, M., Tang, S., Weidenbacher, P. A., Li, S., Pham, T. D., Pak, J. E., Chiu, W., Kim, P. S. 2021; 7 (1): 183–99

  Abstract

  The development of a safe and effective SARS-CoV-2 vaccine is a public health priority. We designed subunit vaccine candidates using self-assembling ferritin nanoparticles displaying one of two multimerized SARS-CoV-2 spikes: full-length ectodomain (S-Fer) or a C-terminal 70 amino-acid deletion (SDeltaC-Fer). Ferritin is an attractive nanoparticle platform for production of vaccines, and ferritin-based vaccines have been investigated in humans in two separate clinical trials. We confirmed proper folding and antigenicity of spike on the surface of ferritin by cryo-EM and binding to conformation-specific monoclonal antibodies. After a single immunization of mice with either of the two spike ferritin particles, a lentiviral SARS-CoV-2 pseudovirus assay revealed mean neutralizing antibody titers at least 2-fold greater than those in convalescent plasma from COVID-19 patients. Additionally, a single dose of SDeltaC-Fer elicited significantly higher neutralizing responses as compared to immunization with the spike receptor binding domain (RBD) monomer or spike ectodomain trimer alone. After a second dose, mice immunized with SDeltaC-Fer exhibited higher neutralizing titers than all other groups. Taken together, these results demonstrate that multivalent presentation of SARS-CoV-2 spike on ferritin can notably enhance elicitation of neutralizing antibodies, thus constituting a viable strategy for single-dose vaccination against COVID-19.

  View details for DOI 10.1021/acscentsci.0c01405

  View details for PubMedID 33527087

• The high-affinity immunoglobulin receptor FcgammaRI potentiates HIV-1 neutralization via antibodies against the gp41 N-heptad repeat. Proceedings of the National Academy of Sciences of the United States of America Montefiori, D. C., Filsinger Interrante, M. V., Bell, B. N., Rubio, A. A., Joyce, J. G., Shiver, J. W., LaBranche, C. C., Kim, P. S. 2021; 118 (3)

  Abstract

  The HIV-1 gp41 N-heptad repeat (NHR) region of the prehairpin intermediate, which is transiently exposed during HIV-1 viral membrane fusion, is a validated clinical target in humans and is inhibited by the Food and Drug Administration (FDA)-approved drug enfuvirtide. However, vaccine candidates targeting the NHR have yielded only modest neutralization activities in animals; this inhibition has been largely restricted to tier-1 viruses, which are most sensitive to neutralization by sera from HIV-1-infected individuals. Here, we show that the neutralization activity of the well-characterized NHR-targeting antibody D5 is potentiated >5,000-fold in TZM-bl cells expressing FcgammaRI compared with those without, resulting in neutralization of many tier-2 viruses (which are less susceptible to neutralization by sera from HIV-1-infected individuals and are the target of current antibody-based vaccine efforts). Further, antisera from guinea pigs immunized with the NHR-based vaccine candidate (ccIZN36)3 neutralized tier-2 viruses from multiple clades in an FcgammaRI-dependent manner. As FcgammaRI is expressed on macrophages and dendritic cells, which are present at mucosal surfaces and are implicated in the early establishment of HIV-1 infection following sexual transmission, these results may be important in the development of a prophylactic HIV-1 vaccine.

  View details for DOI 10.1073/pnas.2018027118

  View details for PubMedID 33431684

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

  Abstract

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

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

  View details for PubMedID 33432170

• Accuracy of serological testing for SARS-CoV-2 antibodies: first results of a large mixed-method evaluation study. Allergy Brigger, D., Horn, M. P., Pennington, L. F., Powell, A. E., Siegrist, D., Weber, B., Engler, O., Piezzi, V., Damonti, L., Iseli, P., Hauser, C., Froehlich, T. K., Villiger, P. M., Bachmann, M. F., Leib, S. L., Bittel, P., Fiedler, M., Largiader, C., Marschall, J., Stalder, H., Kim, P. S., Jardetzky, T. S., Eggel, A., Nagler, M. 2020

  Abstract

  BACKGROUND: Serological immunoassays that can identify protective immunity against SARS-CoV-2 are needed to adapt quarantine measures, assess vaccination responses, and evaluate donor plasma. To date, however, the utility of such immunoassays remains unclear. In a mixed-design evaluation study, we compared the diagnostic accuracy of serological immunoassays that are based on various SARS-CoV-2 proteins and assessed the neutralizing activity of antibodies in patient sera.METHODS: Consecutive patients admitted with confirmed SARS-CoV-2 infection were prospectively followed alongside medical staff and biobank samples from winter 2018/2019. An in-house enzyme-linked immunosorbent assay utilizing recombinant receptor-binding domain (RBD) of the SARS-CoV-2 spike protein was developed and compared to three commercially available enzyme-linked immunosorbent assays (ELISAs) targeting the nucleoprotein (N), the S1 domain of the spike protein (S1) and a lateral flow immunoassay (LFI) based on full-length spike protein. Neutralization assays with live SARS-CoV-2 were performed.RESULTS: One-thousand four-hundred and seventy-seven individuals were included comprising 112 SARS-CoV-2 positives (defined as a positive real-time PCR result; prevalence 7.6%). IgG seroconversion occurred between day 0 and day 21. While the ELISAs showed sensitivities of 88.4% for RBD, 89.3% for S1, and 72.9% for N protein, the specificity was above 94% for all tests. Out of 54 SARS-CoV-2 positive individuals, 96.3% showed full neutralization of live SARS-CoV-2 at serum dilutions ≥1:16, while none of the 6 SARS-CoV-2 negative sera revealed neutralizing activity.CONCLUSIONS: ELISAs targeting RBD and S1 protein of SARS-CoV-2 are promising immunoassays which shall be further evaluated in studies verifying diagnostic accuracy and protective immunity against SARS-CoV-2.

  View details for DOI 10.1111/all.14608

  View details for PubMedID 32997812

• 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

• 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

• 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

• Functional Enrichment and Analysis of Antigen-Specific Memory B Cell Antibody Repertoires in PBMCs. Frontiers in immunology Waltari, E., McGeever, A., Friedland, N., Kim, P. S., McCutcheon, K. M. 2019; 10: 1452

  Abstract

  Phenotypic screening of antigen-specific antibodies in human blood is a common diagnostic test for infectious agents and a correlate of protection after vaccination. In addition to long-lived antibody secreting plasma cells residing in the bone marrow, memory B cells are a latent source of antigen-experienced, long-term immunity that can be found at low frequencies in circulating peripheral blood mononuclear cells (PBMCs). Assessing the genotype, clonal frequency, quality, and function of antibodies resulting from an individual's persistent memory B cell repertoire can help inform the success or failure of immune protection. Using in vitro polyclonal stimulation, we functionally expand the memory repertoire from PBMCs and clonally map monoclonal antibodies from this population. We show that combining deep sequencing of stimulated memory B cell repertoires with retrieving single antigen-specific cells is a promising approach in evaluating the latent, functional B cell memory in PBMCs.

  View details for DOI 10.3389/fimmu.2019.01452

  View details for PubMedID 31293598

  View details for PubMedCentralID PMC6603168

• Identification of HIV gp41-specific antibodies that mediate killing of infected cells PLOS PATHOGENS Williams, K. L., Stumpf, M., Naiman, N., Ding, S., Garrett, M., Gobillot, T., Vezina, D., Dusenbury, K., Ramadoss, N. S., Basom, R., Kim, P. S., Finzi, A., Overbaugh, J. 2019; 15 (2)

  View details for DOI 10.1371/journal.ppat.1007572

  View details for Web of Science ID 000459972100024

• A high-affinity human PD-1/PD-L2 complex informs avenues for small-molecule immune checkpoint drug discovery. Proceedings of the National Academy of Sciences of the United States of America Tang, S. n., Kim, P. S. 2019

  Abstract

  Immune checkpoint blockade of programmed death-1 (PD-1) by monoclonal antibody drugs has delivered breakthroughs in the treatment of cancer. Nonetheless, small-molecule PD-1 inhibitors could lead to increases in treatment efficacy, safety, and global access. While the ligand-binding surface of apo-PD-1 is relatively flat, it harbors a striking pocket in the murine PD-1/PD-L2 structure. An analogous pocket in human PD-1 may serve as a small-molecule drug target, but the structure of the human complex is unknown. Because the CC' and FG loops in murine PD-1 adopt new conformations upon binding PD-L2, we hypothesized that mutations in these two loops could be coupled to pocket formation and alter PD-1's affinity for PD-L2. Here, we conducted deep mutational scanning in these loops and used yeast surface display to select for enhanced PD-L2 binding. A PD-1 variant with three substitutions binds PD-L2 with an affinity two orders of magnitude higher than that of the wild-type protein, permitting crystallization of the complex. We determined the X-ray crystal structures of the human triple-mutant PD-1/PD-L2 complex and the apo triple-mutant PD-1 variant at 2.0 Å and 1.2 Å resolution, respectively. Binding of PD-L2 is accompanied by formation of a prominent pocket in human PD-1, as well as substantial conformational changes in the CC' and FG loops. The structure of the apo triple-mutant PD-1 shows that the CC' loop adopts the ligand-bound conformation, providing support for allostery between the loop and pocket. This human PD-1/PD-L2 structure provide critical insights for the design and discovery of small-molecule PD-1 inhibitors.

  View details for DOI 10.1073/pnas.1916916116

  View details for PubMedID 31727844

• Protect, modify, deprotect (PMD): A strategy for creating vaccines to elicit antibodies targeting a specific epitope. Proceedings of the National Academy of Sciences of the United States of America Weidenbacher, P. A., Kim, P. S. 2019

  Abstract

  In creating vaccines against infectious agents, there is often a desire to direct an immune response toward a particular conformational epitope on an antigen. We present a method, called protect, modify, deprotect (PMD), to generate immunogenic proteins aimed to direct a vaccine-induced antibody (Ab) response toward an epitope defined by a specific monoclonal Ab (mAb). The mAb is used to protect the target epitope on the protein. Then the remaining exposed surfaces of the protein are modified to render them nonimmunogenic. Finally, the epitope is deprotected by removal of the mAb. The resultant protein is modified at surfaces other than the target epitope. We validate PMD using a well-characterized antigen, hen egg white lysozyme, then demonstrate the utility of PMD using influenza virus hemagglutinin (HA). We use an mAb to protect a highly conserved epitope on the stem domain of HA. Exposed surface amines are then modified with short polyethylene glycol chains. The resultant antigen shows markedly reduced binding to mAbs that target the head region of HA, while maintaining binding to mAbs at the epitope of interest. This antigenic preference is also observed with yeast cells displaying Ab fragments. Antisera from guinea pigs immunized with the PMD-modified HA show increased cross-reactivity with HAs from other influenza strains, compared with antisera obtained with unmodified HA trimers. PMD has the potential to direct an Ab response at high resolution and could be used in combination with other such strategies. There are many attractive targets for the application of PMD.

  View details for PubMedID 31028143

• FLASH: a next-generation CRISPR diagnostic for multiplexed detection of antimicrobial resistance sequences. Nucleic acids research Quan, J. n., Langelier, C. n., Kuchta, A. n., Batson, J. n., Teyssier, N. n., Lyden, A. n., Caldera, S. n., McGeever, A. n., Dimitrov, B. n., King, R. n., Wilheim, J. n., Murphy, M. n., Ares, L. P., Travisano, K. A., Sit, R. n., Amato, R. n., Mumbengegwi, D. R., Smith, J. L., Bennett, A. n., Gosling, R. n., Mourani, P. M., Calfee, C. S., Neff, N. F., Chow, E. D., Kim, P. S., Greenhouse, B. n., DeRisi, J. L., Crawford, E. D. 2019

  Abstract

  The growing prevalence of deadly microbes with resistance to previously life-saving drug therapies is a dire threat to human health. Detection of low abundance pathogen sequences remains a challenge for metagenomic Next Generation Sequencing (NGS). We introduce FLASH (Finding Low Abundance Sequences by Hybridization), a next-generation CRISPR/Cas9 diagnostic method that takes advantage of the efficiency, specificity and flexibility of Cas9 to enrich for a programmed set of sequences. FLASH-NGS achieves up to 5 orders of magnitude of enrichment and sub-attomolar gene detection with minimal background. We provide an open-source software tool (FLASHit) for guide RNA design. Here we applied it to detection of antimicrobial resistance genes in respiratory fluid and dried blood spots, but FLASH-NGS is applicable to all areas that rely on multiplex PCR.

  View details for DOI 10.1093/nar/gkz418

  View details for PubMedID 31114866