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The protective human antibody response to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) focuses on the spike (S) protein, which decorates the virion surface and mediates cell binding and entry. Most SARS-CoV-2 protective antibodies target the receptor-binding domain or a single dominant epitope (\"supersite\") on the N-terminal domain (NTD). Using the single B cell technology called linking B cell receptor to antigen specificity through sequencing (LIBRA-Seq), we isolated a large panel of NTD-reactive and SARS-CoV-2-neutralizing antibodies from an individual who had recovered from COVID-19. We found that neutralizing antibodies against the NTD supersite were commonly encoded by the IGHV1-24 gene, forming a genetic cluster representing a public B cell clonotype. However, we also discovered a rare human antibody, COV2-3434, that recognizes a site of vulnerability on the SARS-CoV-2 S protein in the trimer interface (TI) and possesses a distinct class of functional activity. COV2-3434 disrupted the integrity of S protein trimers, inhibited the cell-to-cell spread of the virus in culture, and conferred protection in human angiotensin-converting enzyme 2-transgenic (ACE2-transgenic) mice against the SARS-CoV-2 challenge. This study provides insight into antibody targeting of the S protein TI region, suggesting this region may be a site of virus vulnerability.

Eastern equine encephalitis virus (EEEV) is a mosquito-transmitted alphavirus that can cause severe encephalitis in humans and horses with a high case fatality rate. There are no licensed EEEV vaccines or therapeutics for human use, warranting the need to better understand the human immune response against EEEV. Here we present a cryo-EM reconstruction of the chimeric virus, Sindbis (SINV)/EEEV, in complex with a potently neutralizing and efficacious intact human IgG1 antibody in a mouse model of infection and disease. This antibody requires bivalency to recognize a quaternary epitope on the E2 glycoprotein and cross-links two virus spikes across the icosahedral two-fold axis through a unique binding mode. Kinetic analysis of the binding interaction provides insights into this distinguishing feature. Mechanistically, the antibody inhibits viral entry into cells through blockade of receptor binding and early fusion events but does not block egress, thereby, exclusively targeting an epitope found on intact virions. The discovery of the quaternary epitope and unique binding mode recognized by this antibody together advance our understanding of the complexity of antibody-antigen interactions and can aid in vaccine design to elicit recognition of distinct epitopes of clinically relevant alphaviruses. Structural analyses of antibody-virus complexes offer critical insights into immune recognition mechanisms. In this report, we present how a patient-derived IgG recognizes a quaternary epitope on EEEV particles through strictly bivalent interactions.

Marburg virus (MARV) disease is lethal, with fatality rates up to 90%. Neutralizing antibodies (Abs) are promising drug candidates to prevent or treat the disease. Current efforts are focused in part on vaccine development to induce such MARV-neutralizing Abs. We analyzed the antibody repertoire from healthy unexposed and previously MARV-infected individuals to assess if naïve repertoires contain suitable precursor antibodies that could become neutralizing with a limited set of somatic mutations. We computationally searched the human Ab variable gene repertoire for predicted structural homologs of the neutralizing Ab MR78 that is specific to the receptor binding site (RBS) of MARV glycoprotein (GP). Eight Ab heavy-chain complementarity determining region 3 (HCDR3) loops from MARV-naïve individuals and one from a previously MARV-infected individual were selected for testing as HCDR3 loop chimeras on the MR78 Ab framework. Three of these chimerized antibodies bound to MARV GP. We then tested a fulllength native Ab heavy chain encoding the same 17-residue-long HCDR3 loop that bound to the MARV GP the best among the chimeric Abs tested. Despite only 57% amino acid sequence identity, the Ab from a MARV-naïve donor recognized MARV GP and possessed neutralizing activity against the virus. Crystallization of both chimeric and full-length native heavy chain-containing Abs provided structural insights into the mechanism of binding for these types of Abs. Our work suggests that the MARV GP RBS is a promising candidate for epitope-focused vaccine design to induce neutralizing Abs against MARV.

Hantaviruses are high-priority emerging pathogens carried by rodents and transmitted to humans by aerosolized excreta or, in rare cases, person-to-person contact. While infections in humans are relatively rare, mortality rates range from 1 to 40% depending on the hantavirus species. There are currently no FDA-approved vaccines or therapeutics for hantaviruses, and the only treatment for infection is supportive care for respiratory or kidney failure. Additionally, the human humoral immune response to hantavirus infection is incompletely understood, especially the location of major antigenic sites on the viral glycoproteins and conserved neutralizing epitopes. Here, we report antigenic mapping and functional characterization for four neutralizing hantavirus antibodies. The broadly neutralizing antibody SNV-53 targets an interface between Gn/Gc, neutralizes through fusion inhibition and cross-protects against the Old World hantavirus species Hantaan virus when administered pre- or post-exposure. Another broad antibody, SNV-24, also neutralizes through fusion inhibition but targets domain I of Gc and demonstrates weak neutralizing activity to authentic hantaviruses. ANDV-specific, neutralizing antibodies (ANDV-5 and ANDV-34) neutralize through attachment blocking and protect against hantavirus cardiopulmonary syndrome (HCPS) in animals but target two different antigenic faces on the head domain of Gn. Determining the antigenic sites for neutralizing antibodies will contribute to further therapeutic development for hantavirus-related diseases and inform the design of new broadly protective hantavirus vaccines.

Avian H7N9 influenza viruses cause sporadic outbreaks of human infections and threaten to cause a major pandemic. The breadth of B cell responses to natural infection and the dominant antigenic sites recognized during first exposure to H7 HA following infection are incompletely understood. Here, we studied the B cell response to H7 HA of 2 individuals who had recovered from natural H7N9 virus infection. We used competition binding, hydrogen-deuterium mass spectrometry, and single-particle negative stain electron microscopy to identify the patterns of molecular recognition of the antibody responses to H7 HA. We found that circulating H7-reactive B cells recognized a diverse antigenic landscape on the HA molecule, including HA head domain epitopes in antigenic sites A and B and in the trimer interface-II region and epitopes in the stem region. Most H7 antibodies exhibited little heterosubtypic breadth, but many recognized a wide diversity of unrelated H7 strains. We tested the antibodies for functional activity and identified clones with diverse patterns of inhibition, including neutralizing, hemagglutination- or egress-inhibiting, or HA trimer-disrupting activities. Thus, the human B cell response to primary H7 natural infection is diverse, highly functional, and broad for recognition of diverse H7 strains.

The ongoing pandemic of coronavirus disease 2019 (COVID-19), which is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is a major threat to global health 1 and the medical countermeasures available so far are limited 2 , 3 . Moreover, we currently lack a thorough understanding of the mechanisms of humoral immunity to SARS-CoV-2 4 . Here we analyse a large panel of human monoclonal antibodies that target the spike (S) glycoprotein 5 , and identify several that exhibit potent neutralizing activity and fully block the receptor-binding domain of the S protein (S RBD ) from interacting with human angiotensin-converting enzyme 2 (ACE2). Using competition-binding, structural and functional studies, we show that the monoclonal antibodies can be clustered into classes that recognize distinct epitopes on the S RBD , as well as distinct conformational states of the S trimer. Two potently neutralizing monoclonal antibodies, COV2-2196 and COV2-2130, which recognize non-overlapping sites, bound simultaneously to the S protein and neutralized wild-type SARS-CoV-2 virus in a synergistic manner. In two mouse models of SARS-CoV-2 infection, passive transfer of COV2-2196, COV2-2130 or a combination of both of these antibodies protected mice from weight loss and reduced the viral burden and levels of inflammation in the lungs. In addition, passive transfer of either of two of the most potent ACE2-blocking monoclonal antibodies (COV2-2196 or COV2-2381) as monotherapy protected rhesus macaques from SARS-CoV-2 infection. These results identify protective epitopes on the S RBD and provide a structure-based framework for rational vaccine design and the selection of robust immunotherapeutic agents. An analysis identifies human monoclonal antibodies that potently neutralize wild-type SARS-CoV-2 and protect animals from disease, including two that synergize in a cocktail, suggesting that these could be candidates for use as therapeutic agents for the treatment of COVID-19 in humans.

Antibodies are a principal determinant of immunity for most RNA viruses and have promise to reduce infection or disease during major epidemics. The novel coronavirus SARS-CoV-2 has caused a global pandemic with millions of infections and hundreds of thousands of deaths to date 1 , 2 . In response, we used a rapid antibody discovery platform to isolate hundreds of human monoclonal antibodies (mAbs) against the SARS-CoV-2 spike (S) protein. We stratify these mAbs into five major classes on the basis of their reactivity to subdomains of S protein as well as their cross-reactivity to SARS-CoV. Many of these mAbs inhibit infection of authentic SARS-CoV-2 virus, with most neutralizing mAbs recognizing the receptor-binding domain (RBD) of S. This work defines sites of vulnerability on SARS-CoV-2 S and demonstrates the speed and robustness of advanced antibody discovery platforms. A platform for rapid antibody discovery enabled the isolation of hundreds of human monoclonal antibodies against SARS-CoV-2 and the prioritization of potent antibody candidates for clinical trials in patients with COVID-19.

Influenza type A viruses (IAVs) remain an extraordinary burden to global public health and regularly circulate through human populations. This investigation describes the isolation of human mAbs from an individual with a substantial history of influenza exposure via vaccination and natural infection. From these mAbs, a clonally expanded B cell lineage was identified that recognizes the IAV neuraminidase (NA) glycoprotein and binds near the NA active site of H3N2 viruses to inhibit sialidase activity. Further characterization found that some somatically mutated members of this lineage exhibited cross-reactive binding to recombinant N1and N9 antigens, suggesting that heterosubtypic reactivity was acquired through somatic mutation. Two candidate mAbs from this family - FluA-168 and FluA-173 - potently inhibited IAV replication in vitro and protected against lethality in vivo. The results of this study contribute to our understanding of cross-reactivity between IAV subtypes in response to diverse exposure patterns and identified 2 mAbs as potential therapeutic candidates for IAV infection.

Influenza type A viruses (IAVs) remain an extraordinary burden to global public health and regularly circulate through human populations. This investigation describes the isolation of human mAbs from an individual with a substantial history of influenza exposure via vaccination and natural infection. From these mAbs, a clonally expanded B cell lineage was identified that recognizes the IAV neuraminidase (NA) glycoprotein and binds near the NA active site of H3N2 viruses to inhibit sialidase activity. Further characterization found that some somatically mutated members of this lineage exhibited cross-reactive binding to recombinant N1 and N9 antigens, suggesting that heterosubtypic reactivity was acquired through somatic mutation. Two candidate mAbs from this family - FluA-168 and FluA-173 - potently inhibited IAV replication in vitro and protected against lethality in vivo. The results of this study contribute to our understanding of cross-reactivity between IAV subtypes in response to diverse exposure patterns and identified 2 mAbs as potential therapeutic candidates for IAV infection.

Protein folding in the endoplasmic reticulum (ER) happens with the help of ER-resident proteins called chaperones. However, when UBX2 is deleted in Saccharomyces cerevisiae, some of these chaperones, namely Kar2p and Pdi1p, are secreted outside the cell at uncommonly high levels. This is noteworthy since none of the other proteins involved in Ubx2p’s primary known function demonstrate the same secretion phenotype when deleted. Ubx2p is also known to regulate desaturated lipids in the cell’s membranes, so in order to investigate this unusual secretion phenotype, ubx2Δ mutant cells, WT cells, and ubx2Δ transformed with either full-length UBX2 or a construct lacking one of its two primary domains were assayed for secretion of chaperone proteins after growth on YPD and YPD with oleate. Oleate reduced differences in chaperone secretion between ubx2Δ and WT cells, suggesting that it is Ubx2p’s role in lipid regulation that results in the secretion defect when it is removed.