Computationally restoring the potency of a clinical antibody against Omicron.

The COVID-19 pandemic underscored the promise of monoclonal antibody-based prophylactic and therapeutic drugs1-3 and revealed how quickly viral escape can curtail effective options4,5. When the SARS-CoV-2 Omicron variant emerged in 2021, many antibody drug products lost potency, including Evusheld and its constituent, cilgavimab4-6. Cilgavimab, like its progenitor COV2-2130, is a class 3 antibody that is compatible with other antibodies in combination4 and is challenging to replace with existing approaches. Rapidly modifying such high-value antibodies to restore efficacy against emerging variants is a compelling mitigation strategy. We sought to redesign and renew the efficacy of COV2-2130 against Omicron BA.1 and BA.1.1 strains while maintaining efficacy against the dominant Delta variant. Here we show that our computationally redesigned antibody, 2130-1-0114-112, achieves this objective, simultaneously increases neutralization potency against Delta and subsequent variants of concern, and provides protection in vivo against the strains tested: WA1/2020, BA.1.1 and BA.5. Deep mutational scanning of tens of thousands of pseudovirus variants reveals that 2130-1-0114-112 improves broad potency without increasing escape liabilities. Our results suggest that computational approaches can optimize an antibody to target multiple escape variants, while simultaneously enriching potency. Our computational approach does not require experimental iterations or pre-existing binding data, thus enabling rapid response strategies to address escape variants or lessen escape vulnerabilities.

Therapeutic administration of a cross-reactive mAb targeting the fusion glycoprotein of Nipah virus protects nonhuman primates.

No licensed vaccines or therapies exist for patients infected with Nipah virus (NiV), although an experimental human monoclonal antibody (mAb) cross-reactive to the NiV and Hendra virus (HeV) G glycoprotein, m102.4, has been tested in a phase 1 trial and has been provided under compassionate use for both HeV and NiV exposures. NiV is a highly pathogenic zoonotic paramyxovirus causing regular outbreaks in humans and animals in South and Southeast Asia. The mortality rate of NiV infection in humans ranges from 40% to more than 90%, making it a substantial public health concern. The NiV G glycoprotein mediates host cell attachment, and the F glycoprotein facilitates membrane fusion and infection. We hypothesized that a mAb against the prefusion conformation of the F glycoprotein may confer better protection than m102.4. To test this, two potent neutralizing mAbs against NiV F protein, hu1F5 and hu12B2, were compared in a hamster model. Hu1F5 provided superior protection to hu12B2 and was selected for comparison with m102.4 for the ability to protect African green monkeys (AGMs) from a stringent NiV challenge. AGMs were exposed intranasally to the Bangladesh strain of NiV and treated 5 days after exposure with either mAb (25 milligrams per kilogram). Whereas only one of six AGMs treated with m102.4 survived until the study end point, all six AGMs treated with hu1F5 were protected. Furthermore, a reduced 10 milligrams per kilogram dose of hu1F5 also provided complete protection against NiV challenge, supporting the upcoming clinical advancement of this mAb for postexposure prophylaxis and therapy.

Functional and antigenic landscape of the Nipah virus receptor binding protein.

Nipah virus recurrently spills over to humans, causing fatal infections. The viral receptor-binding protein (RBP or G) attaches to host receptors and is a major target of neutralizing antibodies. Here we use deep mutational scanning to measure how all amino-acid mutations to the RBP affect cell entry, receptor binding, and escape from neutralizing antibodies. We identify functionally constrained regions of the RBP, including sites involved in oligomerization, along with mutations that differentially modulate RBP binding to its two ephrin receptors. We map escape mutations for six anti-RBP antibodies, and find that few antigenic mutations are present in natural Nipah strains. Our findings offer insights into the potential for functional and antigenic evolution of the RBP that can inform the development of antibody therapies and vaccines.

Potent HPIV3-neutralizing IGHV5-51 Antibodies Identified from Multiple Individuals Show L Chain and CDRH3 Promiscuity.

Human parainfluenza virus 3 (HPIV3) is a widespread pathogen causing severe and lethal respiratory illness in at-risk populations. Effective countermeasures are in various stages of development; however, licensed therapeutic and prophylactic options are not available. The fusion glycoprotein (HPIV3 F), responsible for facilitating viral entry into host cells, is a major target of neutralizing Abs that inhibit infection. Although several neutralizing Abs against a small number of HPIV3 F epitopes have been identified to date, relatively little is known about the Ab response to HPIV3 compared with other pathogens, such as influenza virus and SARS-CoV-2. In this study, we aimed to characterize a set of HPIV3-specific Abs identified in multiple individuals for genetic signatures, epitope specificity, neutralization potential, and publicness. We identified 12 potently neutralizing Abs targeting three nonoverlapping epitopes on HPIV3 F. Among these, six Abs identified from two different individuals used Ig heavy variable gene IGHV 5-51, with five of the six Abs targeting the same epitope. However, despite the use of the same H chain variable (VH) gene, these Abs used multiple different L chain variable genes (VL) and diverse H chain CDR 3 (CDRH3) sequences. Together, these results provide further information about the genetic and functional characteristics of HPIV3-neutralizing Abs and suggest the existence of a reproducible VH-dependent Ab response associated with VL and CDRH3 promiscuity. Understanding sites of HPIV3 F vulnerability and the genetic and molecular characteristics of Abs targeting these sites will help guide efforts for effective vaccine and therapeutic development.

Discovery and Characterization of a Pan-betacoronavirus S2-binding antibody.

Three coronaviruses have spilled over from animal reservoirs into the human population and caused deadly epidemics or pandemics. The continued emergence of coronaviruses highlights the need for pan-coronavirus interventions for effective pandemic preparedness. Here, using LIBRA-seq, we report a panel of 50 coronavirus antibodies isolated from human B cells. Of these antibodies, 54043-5 was shown to bind the S2 subunit of spike proteins from alpha-, beta-, and deltacoronaviruses. A cryo-EM structure of 54043-5 bound to the pre-fusion S2 subunit of the SARS-CoV-2 spike defined an epitope at the apex of S2 that is highly conserved among betacoronaviruses. Although non-neutralizing, 54043-5 induced Fc-dependent antiviral responses, including ADCC and ADCP. In murine SARS-CoV-2 challenge studies, protection against disease was observed after introduction of Leu234Ala, Leu235Ala, and Pro329Gly (LALA-PG) substitutions in the Fc region of 54043-5. Together, these data provide new insights into the protective mechanisms of non-neutralizing antibodies and define a broadly conserved epitope within the S2 subunit.

A broadly reactive antibody targeting the N-terminal domain of SARS-CoV-2 spike confers Fc-mediated protection.

Most neutralizing anti-SARS-CoV-2 monoclonal antibodies (mAbs) target the receptor binding domain (RBD) of the spike (S) protein. Here, we characterize a panel of mAbs targeting the N-terminal domain (NTD) or other non-RBD epitopes of S. A subset of NTD mAbs inhibits SARS-CoV-2 entry at a post-attachment step and avidly binds the surface of infected cells. One neutralizing NTD mAb, SARS2-57, protects K18-hACE2 mice against SARS-CoV-2 infection in an Fc-dependent manner. Structural analysis demonstrates that SARS2-57 engages an antigenic supersite that is remodeled by deletions common to emerging variants. In neutralization escape studies with SARS2-57, this NTD site accumulates mutations, including a similar deletion, but the addition of an anti-RBD mAb prevents such escape. Thus, our study highlights a common strategy of immune evasion by SARS-CoV-2 variants and how targeting spatially distinct epitopes, including those in the NTD, may limit such escape.

Homotypic antibodies target novel E glycoprotein domains after natural DENV 3 infection/vaccination.

The envelope (E) glycoprotein is the primary target of type-specific (TS) neutralizing antibodies (nAbs) after infection with any of the four distinct dengue virus serotypes (DENV1-4). nAbs can be elicited to distinct structural E domains (EDs) I, II, or III. However, the relative contribution of these domain-specific antibodies is unclear. To identify the primary DENV3 nAb targets in sera after natural infection or vaccination, chimeric DENV1 recombinant encoding DENV3 EDI, EDII, or EDIII were generated. DENV3 EDII is the principal target of TS polyclonal nAb responses and encodes two or more neutralizing epitopes. In contrast, some were individuals vaccinated with a DENV3 monovalent vaccine-elicited serum TS nAbs targeting each ED in a subject-dependent fashion, with an emphasis on EDI and EDIII. Vaccine responses were also sensitive to DENV3 genotypic variation. This DENV1/3 panel allows the measurement of serum ED TS nAbs, revealing differences in TS nAb immunity after natural infection or vaccination.

SARS-CoV-2 antibodies from children exhibit broad neutralization and belong to adult public clonotypes.

From the beginning of the COVID-19 pandemic, children have exhibited different susceptibility to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection, reinfection, and disease compared with adults. Motivated by the established significance of SARS-CoV-2-neutralizing antibodies in adults, here we characterize SARS-CoV-2-specific antibody repertoires in a young cohort of individuals aged from 5 months to 18 years old. Our results show that neutralizing antibodies in children possess similar genetic features compared to antibodies identified in adults, with multiple antibodies from children belonging to previously established public antibody clonotypes in adults. Notably, antibodies from children show potent neutralization of circulating SARS-CoV-2 variants that have cumulatively resulted in resistance to virtually all approved monoclonal antibody therapeutics. Our results show that children can rely on similar SARS-CoV-2 antibody neutralization mechanisms compared to adults and are an underutilized source for the discovery of effective antibody therapeutics to counteract the ever-evolving pandemic.

Aerosol delivery of SARS-CoV-2 human monoclonal antibodies in macaques limits viral replication and lung pathology.

Passively administered monoclonal antibodies (mAbs) given before or after viral infection can prevent or blunt disease. Here, we examine the efficacy of aerosol mAb delivery to prevent infection and disease in rhesus macaques inoculated with the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) Delta variant via intranasal and intratracheal routes. SARS-CoV-2 human mAbs or a human mAb directed to respiratory syncytial virus (RSV) are nebulized and delivered using positive airflow via facemask to sedated macaques pre- and post-infection. Nebulized human mAbs are detectable in nasal, oropharyngeal, and bronchoalveolar lavage (BAL) samples. SARS-CoV-2 mAb treatment significantly reduces levels of SARS-CoV-2 viral RNA and infectious virus in the upper and lower respiratory tracts relative to controls. Reductions in lung and BAL virus levels correspond to reduced BAL inflammatory cytokines and lung pathology. Aerosolized antibody therapy for SARS-CoV-2 could be effective for reducing viral burden and limiting disease severity.

Developing a Prototype Pathogen Plan and Research Priorities for the Alphaviruses.

The Togaviridae family, genus, Alphavirus, includes several mosquito-borne human pathogens with the potential to spread to near pandemic proportions. Most of these are zoonotic, with spillover infections of humans and domestic animals, but a few such as chikungunya virus (CHIKV) have the ability to use humans as amplification hosts for transmission in urban settings and explosive outbreaks. Most alphaviruses cause nonspecific acute febrile illness, with pathogenesis sometimes leading to either encephalitis or arthralgic manifestations with severe and chronic morbidity and occasional mortality. The development of countermeasures, especially against CHIKV and Venezuelan equine encephalitis virus that are major threats, has included vaccines and antibody-based therapeutics that are likely to also be successful for rapid responses with other members of the family. However, further work with these prototypes and other alphavirus pathogens should target better understanding of human tropism and pathogenesis, more comprehensive identification of cellular receptors and entry, and better understanding of structural mechanisms of neutralization.

Blocking of ebolavirus spread through intercellular connections by an MPER-specific antibody depends on BST2/tetherin.

Ebola virus (EBOV) and Bundibugyo virus (BDBV) belong to the family Filoviridae and cause a severe disease in humans. We previously isolated a large panel of monoclonal antibodies from B cells of human survivors from the 2007 Uganda BDBV outbreak, 16 survivors from the 2014 EBOV outbreak in the Democratic Republic of the Congo, and one survivor from the West African 2013-2016 EBOV epidemic. Here, we demonstrate that EBOV and BDBV are capable of spreading to neighboring cells through intercellular connections in a process that depends upon actin and T cell immunoglobulin and mucin 1 protein. We quantify spread through intercellular connections by immunofluorescence microscopy and flow cytometry. One of the antibodies, BDBV223, specific to the membrane-proximal external region, induces virus accumulation at the plasma membrane. The inhibiting activity of BDBV223 depends on BST2/tetherin.

Host range, transmissibility and antigenicity of a pangolin coronavirus.

The pathogenic and cross-species transmission potential of SARS-CoV-2-related coronaviruses (CoVs) remain poorly characterized. Here we recovered a wild-type pangolin (Pg) CoV GD strain including derivatives encoding reporter genes using reverse genetics. In primary human cells, PgCoV replicated efficiently but with reduced fitness and showed less efficient transmission via airborne route compared with SARS-CoV-2 in hamsters. PgCoV was potently inhibited by US Food and Drug Administration approved drugs, and neutralized by COVID-19 patient sera and SARS-CoV-2 therapeutic antibodies in vitro. A pan-Sarbecovirus antibody and SARS-CoV-2 S2P recombinant protein vaccine protected BALB/c mice from PgCoV infection. In K18-hACE2 mice, PgCoV infection caused severe clinical disease, but mice were protected by a SARS-CoV-2 human antibody. Efficient PgCoV replication in primary human cells and hACE2 mice, coupled with a capacity for airborne spread, highlights an emergence potential. However, low competitive fitness, pre-immune humans and the benefit of COVID-19 countermeasures should impede its ability to spread globally in human populations.

Multifunctional human monoclonal antibody combination mediates protection against Rift Valley fever virus at low doses.

The zoonotic Rift Valley fever virus (RVFV) can cause severe disease in humans and has pandemic potential, yet no approved vaccine or therapy exists. Here we describe a dual-mechanism human monoclonal antibody (mAb) combination against RVFV that is effective at minimal doses in a lethal mouse model of infection. We structurally analyze and characterize the binding mode of a prototypical potent Gn domain-A-binding antibody that blocks attachment and of an antibody that inhibits infection by abrogating the fusion process as previously determined. Surprisingly, the Gn domain-A antibody does not directly block RVFV Gn interaction with the host receptor low density lipoprotein receptor-related protein 1 (LRP1) as determined by a competitive assay. This study identifies a rationally designed combination of human mAbs deserving of future investigation for use in humans against RVFV infection. Using a two-pronged mechanistic approach, we demonstrate the potent efficacy of a rationally designed combination mAb therapeutic.

A highly potent human neutralizing antibody prevents vertical transmission of Rift Valley fever virus in a rat model.

Rift Valley fever virus (RVFV) is an emerging mosquito-transmitted virus that circulates in livestock and humans in Africa and the Middle East. Outbreaks lead to high rates of miscarriages in domesticated livestock. Women are also at risk of vertical virus transmission and late-term miscarriages. MAb RVFV-268 is a highly potent recombinant neutralizing human monoclonal antibody that targets RVFV. Here we show that mAb RVFV-268 reduces viral replication in rat placenta explant cultures and prevents vertical transmission in a rat model of congenital RVF. Passive transfer of mAb RVFV-268 from mother to fetus occurs as early as 6 h after administration and persists through 24 h. Administering mAb RVFV-268 2 h prior to RVFV challenge or 24 h post-challenge protects the dams and offspring from RVFV infection. These findings support mAb RVFV-268 as a pre- and post-infection treatment to subvert RVFV infection and vertical transmission, thus protecting the mother and offspring.

Potent cross-neutralization of respiratory syncytial virus and human metapneumovirus through a structurally conserved antibody recognition mode.

Respiratory syncytial virus (RSV) and human metapneumovirus (hMPV) infections pose a significant health burden. Using pre-fusion conformation fusion (F) proteins, we isolated a panel of anti-F antibodies from a human donor. One antibody (RSV-199) potently cross-neutralized 8 RSV and hMPV strains by recognizing antigenic site III, which is partially conserved in RSV and hMPV F. Next, we determined the cryoelectron microscopy (cryo-EM) structures of RSV-199 bound to RSV F trimers, hMPV F monomers, and an unexpected dimeric form of hMPV F. These structures revealed how RSV-199 engages both RSV and hMPV F proteins through conserved interactions of the antibody heavy-chain variable region and how variability within heavy-chain complementarity-determining region 3 (HCDR3) can be accommodated at the F protein interface in site-III-directed antibodies. Furthermore, RSV-199 offered enhanced protection against RSV A and B strains and hMPV in cotton rats. These findings highlight the mechanisms of broad neutralization and therapeutic potential of RSV-199.

Mechanistic basis for potent neutralization of Sin Nombre hantavirus by a human monoclonal antibody.

Rodent-borne hantaviruses are prevalent worldwide and upon spillover to human populations, cause severe disease for which no specific treatment is available. A potent antibody response is key for recovery from hantavirus infection. Here we study a highly neutralizing human monoclonal antibody, termed SNV-42, which was derived from a memory B cell isolated from an individual with previous Sin Nombre virus (SNV) infection. Crystallographic analysis demonstrates that SNV-42 targets the Gn subcomponent of the tetrameric (Gn-Gc)4 glycoprotein assembly that is relevant for viral entry. Integration of our 1.8 Å structure with the (Gn-Gc)4 ultrastructure arrangement indicates that SNV-42 targets the membrane-distal region of the virus envelope. Comparison of the SNV-42 paratope encoding variable genes with inferred germline gene segments reveals high sequence conservation, suggesting that germline-encoded antibodies inhibit SNV. Furthermore, mechanistic assays reveal that SNV-42 interferes with both receptor recognition and fusion during host-cell entry. This work provides a molecular-level blueprint for understanding the human neutralizing antibody response to hantavirus infection.

A chikungunya virus-like particle vaccine induces broadly neutralizing and protective antibodies against alphaviruses in humans.

Chikungunya virus (CHIKV) is a mosquito-transmitted alphavirus that causes epidemics of acute and chronic musculoskeletal disease. Here, we analyzed the human B cell response to a CHIKV-like particle-adjuvanted vaccine (PXVX0317) from samples obtained from a phase 2 clinical trial in humans (NCT03483961). Immunization with PXVX0317 induced high levels of neutralizing antibody in serum against CHIKV and circulating antigen-specific B cells up to 6 months after immunization. Monoclonal antibodies (mAbs) generated from peripheral blood B cells of three PXVX0317-vaccinated individuals on day 57 after immunization potently neutralized CHIKV infection, and a subset of these inhibited multiple related arthritogenic alphaviruses. Epitope mapping and cryo-electron microscopy defined two broadly neutralizing mAbs that uniquely bind to the apex of the B domain of the E2 glycoprotein. These results demonstrate the inhibitory breadth and activity of the human B cell response induced by the PXVX0317 vaccine against CHIKV and potentially other related alphaviruses.

Corrigendum: Convergent antibody responses are associated with broad neutralization of hepatitis C virus.

[This corrects the article DOI: 10.3389/fimmu.2023.1135841.].

Glycan masking in vaccine design: Targets, immunogens and applications.

Glycan masking is a novel technique in reverse vaccinology in which sugar chains (glycans) are added on the surface of immunogen candidates to hide regions of low interest and thus focus the immune system on highly therapeutic epitopes. This shielding strategy is inspired by viruses such as influenza and HIV, which are able to escape the immune system by incorporating additional glycosylation and preventing the binding of therapeutic antibodies. Interestingly, the glycan masking technique is mainly used in vaccine design to fight the same viruses that naturally use glycans to evade the immune system. In this review we report the major successes obtained with the glycan masking technique in epitope-focused vaccine design. We focus on the choice of the target antigen, the strategy for immunogen design and the relevance of the carrier vector to induce a strong immune response. Moreover, we will elucidate the different applications that can be accomplished with glycan masking, such as shifting the immune response from hyper-variable epitopes to more conserved ones, focusing the response on known therapeutic epitopes, broadening the response to different viral strains/sub-types and altering the antigen immunogenicity to elicit higher or lower immune response, as desired.

Convergent antibody responses are associated with broad neutralization of hepatitis C virus.

Introduction: Early development of broadly neutralizing antibodies (bNAbs) targeting the hepatitis C virus (HCV) envelope glycoprotein E2 is associated with spontaneous clearance of infection, so induction of bNAbs is a major goal of HCV vaccine development. However, the molecular antibody features important for broad neutralization are not known. Methods: To identify B cell repertoire features associated with broad neutralization, we performed RNA sequencing of the B cell receptors (BCRs) of HCV E2-reactive B cells of HCV-infected individuals with either high or low plasma neutralizing breadth. We then produced a monoclonal antibody (mAb) expressed by pairing the most abundant heavy and light chains from public clonotypes identified among clearance, high neutralization subjects. Results: We found distinctive BCR features associated with broad neutralization of HCV, including long heavy chain complementarity determining region 3 (CDRH3) regions, specific VH gene usage, increased frequencies of somatic hypermutation, and particular VH gene mutations. Most intriguing, we identified many E2-reactive public BCR clonotypes (heavy and light chain clones with the same V and J-genes and identical CDR3 sequences) present only in subjects who produced highly neutralizing plasma. The majority of these public clonotypes were shared by two subjects who cleared infection. A mAb expressing the most abundant public heavy and light chains from these clearance, high neutralization subjects had features enriched in high neutralization clonotypes, such as increased somatic hypermutation frequency and usage of IGHV1-69, and was cross-neutralizing. Discussion: Together, these results demonstrate distinct BCR repertoires associated with high plasma neutralizing capacity. Further characterization of the molecular features and function of these antibodies can inform HCV vaccine development.