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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.

The three human pathogenic ebolaviruses: Zaire (EBOV), Bundibugyo (BDBV), and Sudan (SUDV) virus, cause severe disease with high fatality rates. Epitopes of ebolavirus glycoprotein (GP) recognized by antibodies with binding breadth for all three ebolaviruses are of major interest for rational vaccine design. In particular, the heptad repeat 2 –membrane-proximal external region (HR2-MPER) epitope is relatively conserved between EBOV, BDBV, and SUDV GP and targeted by human broadly-neutralizing antibodies. To study whether this epitope can serve as an immunogen for the elicitation of broadly-reactive antibody responses, protein design in Rosetta was employed to transplant the HR2-MPER epitope identified from a co-crystal structure with the known broadly-reactive monoclonal antibody (mAb) BDBV223 onto smaller scaffold proteins. From computational analysis, selected immunogen designs were produced as recombinant proteins and functionally validated, leading to the identification of a sterile alpha motif (SAM) domain displaying the BDBV-HR2-MPER epitope near its C terminus as a promising candidate. The immunogen was fused to one component of a self-assembling, two-component nanoparticle and tested for immunogenicity in rabbits. Robust titers of cross-reactive serum antibodies to BDBV and EBOV GPs and moderate titers to SUDV GP were induced following immunization. To confirm the structural composition of the immunogens, solution NMR studies were conducted and revealed structural flexibility in the C-terminal residues of the epitope. Overall, our study represents the first report on an epitope-focused immunogen design based on the structurally challenging BDBV-HR2-MPER epitope.

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 to Ebola virus glycoprotein (EBOV GP) represent an important correlate of the vaccine efficiency and infection survival. Both neutralization and some of the Fc-mediated effects are known to contribute the protection conferred by antibodies of various epitope specificities. At the same time, the role of the complement system remains unclear. Here, we compare complement activation by two groups of representative monoclonal antibodies (mAbs) interacting with the glycan cap (GC) or the membrane-proximal external region (MPER) of GP. Binding of GC-specific mAbs to GP induces complement-dependent cytotoxicity (CDC) in the GP-expressing cell line via C3 deposition on GP in contrast to MPER-specific mAbs. In the mouse model of EBOV infection, depletion of the complement system leads to an impairment of protection exerted by one of the GC-specific, but not MPER-specific mAbs. Our data suggest that activation of the complement system represents an important mechanism of antiviral protection by GC antibodies. An analysis of the complement activation by human monoclonal antibodies to Ebola virus glycoprotein suggests the distinct mechanisms for antibodies targeting the glycan cap versus antibodies against the membrane-proximal external region.

Selection and development of monoclonal antibody (mAb) therapeutics against pathogenic viruses depends on certain functional characteristics. Neutralization potency, or the half-maximal inhibitory concentration (IC50) values, is an important characteristic of candidate therapeutic antibodies. Structural insights into the bases of neutralization potency differences between antiviral neutralizing mAbs are lacking. In this report, we present cryo-electron microscopy (EM) reconstructions of three anti-Eastern equine encephalitis virus (EEEV) neutralizing human mAbs targeting overlapping epitopes on the E2 protein, with greater than 20-fold differences in their respective IC50 values. From our structural and biophysical analyses, we identify several constraints that contribute to the observed differences in the neutralization potencies. Cryo-EM reconstructions of EEEV in complex with these Fab fragments reveal structural constraints that dictate intravirion or intervirion cross-linking of glycoprotein spikes by their IgG counterparts as a mechanism of neutralization. Additionally, we describe critical features for the recognition of EEEV by these mAbs including the epitope–paratope interaction surface, occupancy, and kinetic differences in on-rate for binding to the E2 protein. Each constraint contributes to the extent of EEEV inhibition for blockade of virus entry, fusion, and/or egress. These findings provide structural and biophysical insights into the differences in mechanism and neutralization potencies of these antibodies, which help inform rational design principles for candidate vaccines and therapeutic antibodies for all icosahedral viruses.

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.

Eastern equine encephalitis virus (EEEV) is a mosquito-transmitted alphavirus with a high case mortality rate in humans. EEEV is a biodefence concern because of its potential for aerosol spread and the lack of existing countermeasures. Here, we identify a panel of 18 neutralizing murine monoclonal antibodies (mAbs) against the EEEV E2 glycoprotein, several of which have ‘elite’ activity with 50 and 99% effective inhibitory concentrations (EC 50 and EC 99 ) of less than 10 and 100 ng ml −1 , respectively. Alanine-scanning mutagenesis and neutralization escape mapping analysis revealed epitopes for these mAbs in domains A or B of the E2 glycoprotein. A majority of the neutralizing mAbs blocked infection at a post-attachment stage, with several inhibiting viral membrane fusion. Administration of one dose of anti-EEEV mAb protected mice from lethal subcutaneous or aerosol challenge. These experiments define the mechanistic basis for neutralization by protective anti-EEEV mAbs and suggest a path forward for treatment and vaccine design. Neutralizing murine monoclonal antibodies against the Eeastern equine encephalitis virus target the E2 glycoprotein, block infection at a post-attachment stage by inhibiting viral membrane fusion and protect mice from lethal challenge.

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.

Alphaviruses cause a debilitating arthritogenic or encephalitic disease in humans and can result in millions of cases worldwide. Additionally, the encephalitic alphaviruses have potential for use as bioterrorism agents. There are no licensed vaccines or antiviral drugs to combat these viruses. To design therapeutic candidates against alphaviruses, an understanding of the human humoral response is key. Through characterization of human anti-alphavirus monoclonal antibodies (mAbs) from two survivors of natural Eastern equine encephalitis virus (EEEV), two main mechanisms of neutralization were identified. Neutralizing E2-specific mAbs potently neutralize EEEV through stabilization of virus particles to inhibit virus entry into host cells. In contrast, neutralizing E1-specific mAbs inhibit virus egress through recognition of cryptic epitopes on intact virus particles that become exposed during virus maturation on the surface of infected cells. Both mechanisms of neutralization can protect against subcutaneous or stringent aerosol EEEV challenge. Given the cryptic nature of these epitopes, regions of the E1 glycoprotein are highly conserved (i.e., fusion loop), which can be targeted by mAbs for cross-reactivity, cross-neutralization, and cross-protection. In addition to neutralization, other mAb-mediated mechanisms appear to be involved, such as interaction with macrophages through Fc and complement receptors, to aid in in vivo efficacy against alphavirus infection. Altogether, a landscape of the human humoral response against alphaviruses, with a special focus on EEEV, was characterized during these studies to help inform rationale vaccine design and identify therapeutic candidates against alphaviruses.