Two-component spike nanoparticle vaccine protects macaques from SARS-CoV-2 infection.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic is continuing to disrupt personal lives, global healthcare systems, and economies. Hence, there is an urgent need for a vaccine that prevents viral infection, transmission, and disease. Here, we present a two-component protein-based nanoparticle vaccine that displays multiple copies of the SARS-CoV-2 spike protein. Immunization studies show that this vaccine induces potent neutralizing antibody responses in mice, rabbits, and cynomolgus macaques. The vaccine-induced immunity protects macaques against a high-dose challenge, resulting in strongly reduced viral infection and replication in the upper and lower airways. These nanoparticles are a promising vaccine candidate to curtail the SARS-CoV-2 pandemic.

A Site of Vulnerability on the Influenza Virus Hemagglutinin Head Domain Trimer Interface.

Here, we describe the discovery of a naturally occurring human antibody (Ab), FluA-20, that recognizes a new site of vulnerability on the hemagglutinin (HA) head domain and reacts with most influenza A viruses. Structural characterization of FluA-20 with H1 and H3 head domains revealed a novel epitope in the HA trimer interface, suggesting previously unrecognized dynamic features of the trimeric HA protein. The critical HA residues recognized by FluA-20 remain conserved across most subtypes of influenza A viruses, which explains the Ab's extraordinary breadth. The Ab rapidly disrupted the integrity of HA protein trimers, inhibited cell-to-cell spread of virus in culture, and protected mice against challenge with viruses of H1N1, H3N2, H5N1, or H7N9 subtypes when used as prophylaxis or therapy. The FluA-20 Ab has uncovered an exceedingly conserved protective determinant in the influenza HA head domain trimer interface that is an unexpected new target for anti-influenza therapeutics and vaccines.

Systematic Analysis of Monoclonal Antibodies against Ebola Virus GP Defines Features that Contribute to Protection.

Antibodies are promising post-exposure therapies against emerging viruses, but which antibody features and in vitro assays best forecast protection are unclear. Our international consortium systematically evaluated antibodies against Ebola virus (EBOV) using multidisciplinary assays. For each antibody, we evaluated epitopes recognized on the viral surface glycoprotein (GP) and secreted glycoprotein (sGP), readouts of multiple neutralization assays, fraction of virions left un-neutralized, glycan structures, phagocytic and natural killer cell functions elicited, and in vivo protection in a mouse challenge model. Neutralization and induction of multiple immune effector functions (IEFs) correlated most strongly with protection. Neutralization predominantly occurred via epitopes maintained on endosomally cleaved GP, whereas maximal IEF mapped to epitopes farthest from the viral membrane. Unexpectedly, sGP cross-reactivity did not significantly influence in vivo protection. This comprehensive dataset provides a rubric to evaluate novel antibodies and vaccine responses and a roadmap for therapeutic development for EBOV and related viruses.

Human anti-N1 monoclonal antibodies elicited by pandemic H1N1 virus infection broadly inhibit HxN1 viruses in vitro and in vivo.

Neuraminidase (NA) is one of the two influenza virus surface glycoproteins, and antibodies that target it are an independent correlate of protection. However, our current understanding of NA antigenicity is incomplete. Here, we describe human monoclonal antibodies (mAbs) from a patient with a pandemic H1N1 virus infection in 2009. Two mAbs exhibited broad reactivity and inhibited NA enzyme activity of seasonal H1N1 viruses circulating before and after 2009, as well as viruses with avian or swine N1s. The mAbs provided robust protection from lethal challenge with human H1N1 and avian H5N1 viruses in mice, and both target an epitope on the lateral face of NA. In summary, we identified two broadly protective NA antibodies that share a novel epitope, inhibited NA activity, and provide protection against virus challenge in mice. Our work reaffirms that NA should be included as a target in future broadly protective or universal influenza virus vaccines.

Immunization-Elicited Broadly Protective Antibody Reveals Ebolavirus Fusion Loop as a Site of Vulnerability.

While neutralizing antibodies are highly effective against ebolavirus infections, current experimental ebolavirus vaccines primarily elicit species-specific antibody responses. Here, we describe an immunization-elicited macaque antibody (CA45) that clamps the internal fusion loop with the N terminus of the ebolavirus glycoproteins (GPs) and potently neutralizes Ebola, Sudan, Bundibugyo, and Reston viruses. CA45, alone or in combination with an antibody that blocks receptor binding, provided full protection against all pathogenic ebolaviruses in mice, guinea pigs, and ferrets. Analysis of memory B cells from the immunized macaque suggests that elicitation of broadly neutralizing antibodies (bNAbs) for ebolaviruses is possible but difficult, potentially due to the rarity of bNAb clones and their precursors. Unexpectedly, germline-reverted CA45, while exhibiting negligible binding to full-length GP, bound a proteolytically remodeled GP with picomolar affinity, suggesting that engineered ebolavirus vaccines could trigger rare bNAb precursors more robustly. These findings have important implications for developing pan-ebolavirus vaccine and immunotherapeutic cocktails.

Antibodies from a Human Survivor Define Sites of Vulnerability for Broad Protection against Ebolaviruses.

Experimental monoclonal antibody (mAb) therapies have shown promise for treatment of lethal Ebola virus (EBOV) infections, but their species-specific recognition of the viral glycoprotein (GP) has limited their use against other divergent ebolaviruses associated with human disease. Here, we mined the human immune response to natural EBOV infection and identified mAbs with exceptionally potent pan-ebolavirus neutralizing activity and protective efficacy against three virulent ebolaviruses. These mAbs recognize an inter-protomer epitope in the GP fusion loop, a critical and conserved element of the viral membrane fusion machinery, and neutralize viral entry by targeting a proteolytically primed, fusion-competent GP intermediate (GPCL) generated in host cell endosomes. Only a few somatic hypermutations are required for broad antiviral activity, and germline-approximating variants display enhanced GPCL recognition, suggesting that such antibodies could be elicited more efficiently with suitably optimized GP immunogens. Our findings inform the development of both broadly effective immunotherapeutics and vaccines against filoviruses.

Cross-Reactive and Potent Neutralizing Antibody Responses in Human Survivors of Natural Ebolavirus Infection.

Recent studies have suggested that antibody-mediated protection against the Ebolaviruses may be achievable, but little is known about whether or not antibodies can confer cross-reactive protection against viruses belonging to diverse Ebolavirus species, such as Ebola virus (EBOV), Sudan virus (SUDV), and Bundibugyo virus (BDBV). We isolated a large panel of human monoclonal antibodies (mAbs) against BDBV glycoprotein (GP) using peripheral blood B cells from survivors of the 2007 BDBV outbreak in Uganda. We determined that a large proportion of mAbs with potent neutralizing activity against BDBV bind to the glycan cap and recognize diverse epitopes within this major antigenic site. We identified several glycan cap-specific mAbs that neutralized multiple ebolaviruses, including SUDV, and a cross-reactive mAb that completely protected guinea pigs from the lethal challenge with heterologous EBOV. Our results provide a roadmap to develop a single antibody-based treatment effective against multiple Ebolavirus infections.

Analysis of a Therapeutic Antibody Cocktail Reveals Determinants for Cooperative and Broad Ebolavirus Neutralization.

Structural principles underlying the composition of protective antiviral monoclonal antibody (mAb) cocktails are poorly defined. Here, we exploited antibody cooperativity to develop a therapeutic mAb cocktail against Ebola virus. We systematically analyzed the antibody repertoire in human survivors and identified a pair of potently neutralizing mAbs that cooperatively bound to the ebolavirus glycoprotein (GP). High-resolution structures revealed that in a two-antibody cocktail, molecular mimicry was a major feature of mAb-GP interactions. Broadly neutralizing mAb rEBOV-520 targeted a conserved epitope on the GP base region. mAb rEBOV-548 bound to a glycan cap epitope, possessed neutralizing and Fc-mediated effector function activities, and potentiated neutralization by rEBOV-520. Remodeling of the glycan cap structures by the cocktail enabled enhanced GP binding and virus neutralization. The cocktail demonstrated resistance to virus escape and protected non-human primates (NHPs) against Ebola virus disease. These data illuminate structural principles of antibody cooperativity with implications for development of antiviral immunotherapeutics.

Vaccination with Glycan-Modified HIV NFL Envelope Trimer-Liposomes Elicits Broadly Neutralizing Antibodies to Multiple Sites of Vulnerability.

The elicitation of broadly neutralizing antibodies (bNAbs) against the HIV-1 envelope glycoprotein (Env) trimer remains a major vaccine challenge. Most cross-conserved protein determinants are occluded by self-N-glycan shielding, limiting B cell recognition of the underlying polypeptide surface. The exceptions to the contiguous glycan shield include the conserved receptor CD4 binding site (CD4bs) and glycoprotein (gp)41 elements proximal to the furin cleavage site. Accordingly, we performed heterologous trimer-liposome prime:boosting in rabbits to drive B cells specific for cross-conserved sites. To preferentially expose the CD4bs to B cells, we eliminated proximal N-glycans while maintaining the native-like state of the cleavage-independent NFL trimers, followed by gradual N-glycan restoration coupled with heterologous boosting. This approach successfully elicited CD4bs-directed, cross-neutralizing Abs, including one targeting a unique glycan-protein epitope and a bNAb (87% breadth) directed to the gp120:gp41 interface, both resolved by high-resolution cryoelectron microscopy. This study provides proof-of-principle immunogenicity toward eliciting bNAbs by vaccination.

Electron-Microscopy-Based Epitope Mapping Defines Specificities of Polyclonal Antibodies Elicited during HIV-1 BG505 Envelope Trimer Immunization.

Characterizing polyclonal antibody responses via currently available methods is inherently complex and difficult. Mapping epitopes in an immune response is typically incomplete, which creates a barrier to fully understanding the humoral response to antigens and hinders rational vaccine design efforts. Here, we describe a method of characterizing polyclonal responses by using electron microscopy, and we applied this method to the immunization of rabbits with an HIV-1 envelope glycoprotein vaccine candidate, BG505 SOSIP.664. We detected known epitopes within the polyclonal sera and revealed how antibody responses evolved during the prime-boosting strategy to ultimately result in a neutralizing antibody response. We uncovered previously unidentified epitopes, including an epitope proximal to one recognized by human broadly neutralizing antibodies as well as potentially distracting non-neutralizing epitopes. Our method provides an efficient and semiquantitative map of epitopes that are targeted in a polyclonal antibody response and should be of widespread utility in vaccine and infection studies.

Multifunctional Pan-ebolavirus Antibody Recognizes a Site of Broad Vulnerability on the Ebolavirus Glycoprotein.

Ebolaviruses cause severe disease in humans, and identification of monoclonal antibodies (mAbs) that are effective against multiple ebolaviruses are important for therapeutics development. Here we describe a distinct class of broadly neutralizing human mAbs with protective capacity against three ebolaviruses infectious for humans: Ebola (EBOV), Sudan (SUDV), and Bundibugyo (BDBV) viruses. We isolated mAbs from human survivors of ebolavirus disease and identified a potent mAb, EBOV-520, which bound to an epitope in the glycoprotein (GP) base region. EBOV-520 efficiently neutralized EBOV, BDBV, and SUDV and also showed protective capacity in relevant animal models of these infections. EBOV-520 mediated protection principally by direct virus neutralization and exhibited multifunctional properties. This study identified a potent naturally occurring mAb and defined key features of the human antibody response that may contribute to broad protection. This multifunctional mAb and related clones are promising candidates for development as broadly protective pan-ebolavirus therapeutic molecules.

Fully synthetic platform to rapidly generate tetravalent bispecific nanobody-based immunoglobulins.

Nanobodies bind a target antigen with a kinetic profile similar to a conventional antibody, but exist as a single heavy chain domain that can be readily multimerized to engage antigen via multiple interactions. Presently, most nanobodies are produced by immunizing camelids; however, platforms for animal-free production are growing in popularity. Here, we describe the development of a fully synthetic nanobody library based on an engineered human VH3-23 variable gene and a multispecific antibody-like format designed for biparatopic target engagement. To validate our library, we selected nanobodies against the SARS-CoV-2 receptor-binding domain and employed an on-yeast epitope binning strategy to rapidly map the specificities of the selected nanobodies. We then generated antibody-like molecules by replacing the VH and VL domains of a conventional antibody with two different nanobodies, designed as a molecular clamp to engage the receptor-binding domain biparatopically. The resulting bispecific tetra-nanobody immunoglobulins neutralized diverse SARS-CoV-2 variants with potencies similar to antibodies isolated from convalescent donors. Subsequent biochemical analyses confirmed the accuracy of the on-yeast epitope binning and structures of both individual nanobodies, and a tetra-nanobody immunoglobulin revealed that the intended mode of interaction had been achieved. This overall workflow is applicable to nearly any protein target and provides a blueprint for a modular workflow for the development of multispecific molecules.

Complementary antibody lineages achieve neutralization breadth in an HIV-1 infected elite neutralizer.

Broadly neutralizing antibodies (bNAbs) have remarkable breadth and potency against most HIV-1 subtypes and are able to prevent HIV-1 infection in animal models. However, bNAbs are extremely difficult to induce by vaccination. Defining the developmental pathways towards neutralization breadth can assist in the design of strategies to elicit protective bNAb responses by vaccination. Here, HIV-1 envelope glycoproteins (Env)-specific IgG+ B cells were isolated at various time points post infection from an HIV-1 infected elite neutralizer to obtain monoclonal antibodies (mAbs). Multiple antibody lineages were isolated targeting distinct epitopes on Env, including the gp120-gp41 interface, CD4-binding site, silent face and V3 region. The mAbs each neutralized a diverse set of HIV-1 strains from different clades indicating that the patient's remarkable serum breadth and potency might have been the result of a polyclonal mixture rather than a single bNAb lineage. High-resolution cryo-electron microscopy structures of the neutralizing mAbs (NAbs) in complex with an Env trimer generated from the same individual revealed that the NAbs used multiple strategies to neutralize the virus; blocking the receptor binding site, binding to HIV-1 Env N-linked glycans, and disassembly of the trimer. These results show that diverse NAbs can complement each other to achieve a broad and potent neutralizing serum response in HIV-1 infected individuals. Hence, the induction of combinations of moderately broad NAbs might be a viable vaccine strategy to protect against a wide range of circulating HIV-1 viruses.

Influenza hemagglutinin-specific IgA Fc-effector functionality is restricted to stalk epitopes.

In this study, we utilized a panel of human immunoglobulin (Ig) IgA monoclonal antibodies isolated from the plasmablasts of eight donors after 2014/2015 influenza virus vaccination (Fluarix) to study the binding and functional specificities of this isotype. In this cohort, isolated IgA monoclonal antibodies were primarily elicited against the hemagglutinin protein of the H1N1 component of the vaccine. To compare effector functionalities, an H1-specific subset of antibodies targeting distinct epitopes were expressed as monomeric, dimeric, or secretory IgA, as well as in an IgG1 backbone. When expressed with an IgG Fc domain, all antibodies elicited Fc-effector activity in a primary polymorphonuclear cell-based assay which differs from previous observations that found only stalk-specific antibodies activate the low-affinity FcγRIIIa. However, when expressed with IgA Fc domains, only antibodies targeting the stalk domain showed Fc-effector activity in line with these previous findings. To identify the cause of this discrepancy, we then confirmed that IgG signaling through the high-affinity FcγI receptor was not restricted to stalk epitopes. Since no corresponding high-affinity Fcα receptor exists, the IgA repertoire may therefore be limited to stalk-specific epitopes in the context of Fc receptor signaling.

Multimerization- and glycosylation-dependent receptor binding of SARS-CoV-2 spike proteins.

Receptor binding studies on sarbecoviruses would benefit from an available toolkit of recombinant spike proteins, or domains thereof, that recapitulate receptor binding properties of native viruses. We hypothesized that trimeric Receptor Binding Domain (RBD) proteins would be suitable candidates to study receptor binding properties of SARS-CoV-1 and -2. Here we created monomeric and trimeric fluorescent RBD proteins, derived from adherent HEK293T, as well as in GnTI-/- mutant cells, to analyze the effect of complex vs high mannose glycosylation on receptor binding. The results demonstrate that trimeric, complex glycosylated proteins are superior in receptor binding compared to monomeric and immaturely glycosylated variants. Although differences in binding to commonly used cell lines were minimal between the different RBD preparations, substantial differences were observed when respiratory tissues of experimental animals were stained. The RBD trimers demonstrated distinct ACE2 expression profiles in bronchiolar ducts and confirmed the higher binding affinity of SARS-CoV-2 over SARS-CoV-1. Our results show that complex glycosylated trimeric RBD proteins are attractive to analyze sarbecovirus receptor binding and explore ACE2 expression profiles in tissues.

Prominent Neutralizing Antibody Response Targeting the Ebolavirus Glycoprotein Subunit Interface Elicited by Immunization.

The severe death toll caused by the recent outbreak of Ebola virus disease reinforces the importance of developing ebolavirus prevention and treatment strategies. Here, we have explored the immunogenicity of a novel immunization regimen priming with vesicular stomatitis virus particles bearing Sudan Ebola virus (SUDV) glycoprotein (GP) that consists of GP1 & GP2 subunits and boosting with soluble SUDV GP in macaques, which developed robust neutralizing antibody (nAb) responses following immunizations. Moreover, EB46, a protective nAb isolated from one of the immune macaques, is found to target the GP1/GP2 interface, with GP-binding mode and neutralization mechanism similar to a number of ebolavirus nAbs from human and mouse, indicating that the ebolavirus GP1/GP2 interface is a common immunological target in different species. Importantly, selected immune macaque polyclonal sera showed nAb specificity similar to EB46 at substantial titers, suggesting that the GP1/GP2 interface region is a viable target for ebolavirus vaccine.Importance: The elicitation of sustained neutralizing antibody (nAb) responses against diverse ebolavirus strains remains as a high priority for the vaccine field. The most clinically advanced rVSV-ZEBOV vaccine could elicit moderate nAb responses against only one ebolavirus strain, EBOV, among the five ebolavirus strains, which last less than 6 months. Boost immunization strategies are desirable to effectively recall the rVSV vector-primed nAb responses to prevent infections in prospective epidemics, while an in-depth understanding of the specificity of immunization-elicited nAb responses is essential for improving vaccine performance. Here, using non-human primate animal model, we demonstrated that booster immunization with a stabilized trimeric soluble form of recombinant glycoprotein derived from the ebolavirus Sudan strain following the priming rVSV vector immunization led to robust nAb responses that substantially map to the subunit interface of ebolavirus glycoprotein, a common B cell repertoire target of multiple species including primates and rodents.

A natural mutation between SARS-CoV-2 and SARS-CoV determines neutralization by a cross-reactive antibody.

Epitopes that are conserved among SARS-like coronaviruses are attractive targets for design of cross-reactive vaccines and therapeutics. CR3022 is a SARS-CoV neutralizing antibody to a highly conserved epitope on the receptor binding domain (RBD) on the spike protein that is able to cross-react with SARS-CoV-2, but with lower affinity. Using x-ray crystallography, mutagenesis, and binding experiments, we illustrate that of four amino acid differences in the CR3022 epitope between SARS-CoV-2 and SARS-CoV, a single mutation P384A fully determines the affinity difference. CR3022 does not neutralize SARS-CoV-2, but the increased affinity to SARS-CoV-2 P384A mutant now enables neutralization with a similar potency to SARS-CoV. We further investigated CR3022 interaction with the SARS-CoV spike protein by negative-stain EM and cryo-EM. Three CR3022 Fabs bind per trimer with the RBD observed in different up-conformations due to considerable flexibility of the RBD. In one of these conformations, quaternary interactions are made by CR3022 to the N-terminal domain (NTD) of an adjacent subunit. Overall, this study provides insights into antigenic variation and potential cross-neutralizing epitopes on SARS-like viruses.

Structural and functional evaluation of de novo-designed, two-component nanoparticle carriers for HIV Env trimer immunogens.

Two-component, self-assembling nanoparticles represent a versatile platform for multivalent presentation of viral antigens. Computational design of protein nanoparticles with differing sizes and geometries enables combination with antigens of choice to test novel multimerization concepts in immunization strategies where the goal is to improve the induction and maturation of neutralizing antibody lineages. Here, we describe detailed antigenic, structural, and functional characterization of computationally designed tetrahedral, octahedral, and icosahedral nanoparticle immunogens displaying trimeric HIV envelope glycoprotein (Env) ectodomains. Env trimers, based on subtype A (BG505) or consensus group M (ConM) sequences and engineered with SOSIP stabilizing mutations, were fused to an underlying trimeric building block of each nanoparticle. Initial screening yielded one icosahedral and two tetrahedral nanoparticle candidates, capable of presenting twenty or four copies of the Env trimer. A number of analyses, including detailed structural characterization by cryo-EM, demonstrated that the nanoparticle immunogens possessed the intended structural and antigenic properties. When the immunogenicity of ConM-SOSIP trimers presented on a two-component tetrahedral nanoparticle or as soluble proteins were compared in rabbits, the two immunogens elicited similar serum antibody binding titers against the trimer component. Neutralizing antibody titers were slightly elevated in the animals given the nanoparticle immunogen and were initially more focused to the trimer apex. Altogether, our findings indicate that tetrahedral nanoparticles can be successfully applied for presentation of HIV Env trimer immunogens; however, the optimal implementation to different immunization strategies remains to be determined.

Potent anti-influenza H7 human monoclonal antibody induces separation of hemagglutinin receptor-binding head domains.

Seasonal influenza virus infections can cause significant morbidity and mortality, but the threat from the emergence of a new pandemic influenza strain might have potentially even more devastating consequences. As such, there is intense interest in isolating and characterizing potent neutralizing antibodies that target the hemagglutinin (HA) viral surface glycoprotein. Here, we use cryo-electron microscopy (cryoEM) to decipher the mechanism of action of a potent HA head-directed monoclonal antibody (mAb) bound to an influenza H7 HA. The epitope of the antibody is not solvent accessible in the compact, prefusion conformation that typifies all HA structures to date. Instead, the antibody binds between HA head protomers to an epitope that must be partly or transiently exposed in the prefusion conformation. The "breathing" of the HA protomers is implied by the exposure of this epitope, which is consistent with metastability of class I fusion proteins. This structure likely therefore represents an early structural intermediate in the viral fusion process. Understanding the extent of transient exposure of conserved neutralizing epitopes also may lead to new opportunities to combat influenza that have not been appreciated previously.

Pre-fusion structure of a human coronavirus spike protein.

HKU1 is a human betacoronavirus that causes mild yet prevalent respiratory disease, and is related to the zoonotic SARS and MERS betacoronaviruses, which have high fatality rates and pandemic potential. Cell tropism and host range is determined in part by the coronavirus spike (S) protein, which binds cellular receptors and mediates membrane fusion. As the largest known class I fusion protein, its size and extensive glycosylation have hindered structural studies of the full ectodomain, thus preventing a molecular understanding of its function and limiting development of effective interventions. Here we present the 4.0 Å resolution structure of the trimeric HKU1 S protein determined using single-particle cryo-electron microscopy. In the pre-fusion conformation, the receptor-binding subunits, S1, rest above the fusion-mediating subunits, S2, preventing their conformational rearrangement. Surprisingly, the S1 C-terminal domains are interdigitated and form extensive quaternary interactions that occlude surfaces known in other coronaviruses to bind protein receptors. These features, along with the location of the two protease sites known to be important for coronavirus entry, provide a structural basis to support a model of membrane fusion mediated by progressive S protein destabilization through receptor binding and proteolytic cleavage. These studies should also serve as a foundation for the structure-based design of betacoronavirus vaccine immunogens.