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Specificity profiling is a requirement for monoclonal antibodies (mAbs) and antibody-directed biotherapeutics such as CAR-T cells prior to initiating human trials. However, traditional approaches to assess the specificity of mAbs, primarily tissue cross-reactivity studies, have been unreliable, leading to off-target binding going undetected. Here, we review the emergence of cell-based protein arrays as an alternative and improved assessment of mAb specificity. Cell-based protein arrays assess binding across the full human membrane proteome, ~6,000 membrane proteins each individually expressed in their native structural configuration within live or unfixed cells. Our own profiling indicates a surprisingly high off-target rate across the industry, with 33% of lead candidates displaying off-target binding. Moreover, about 20% of therapeutic mAbs in clinical development and currently on the market display off-target binding. Case studies and off-target rates at different phases of biotherapeutic drug approval suggest that off-target binding is likely a major cause of adverse events and drug attrition.
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Anticuerpos Monoclonales , Análisis por Matrices de Proteínas , Humanos , Anticuerpos Monoclonales/uso terapéutico , Anticuerpos Monoclonales/inmunología , Análisis por Matrices de Proteínas/métodos , Especificidad de Anticuerpos , Animales , Unión ProteicaRESUMEN
Influenza B viruses (IBVs) comprise a substantial portion of the circulating seasonal human influenza viruses. Here, we describe the isolation of human monoclonal antibodies (mAbs) that recognized the IBV neuraminidase (NA) glycoprotein from an individual following seasonal vaccination. Competition-binding experiments suggested the antibodies recognized two major antigenic sites. One group, which included mAb FluB-393, broadly inhibited IBV NA sialidase activity, protected prophylactically in vivo, and bound to the lateral corner of NA. The second group contained an active site mAb, FluB-400, that broadly inhibited IBV NA sialidase activity and virus replication in vitro in primary human respiratory epithelial cell cultures and protected against IBV in vivo when administered systemically or intranasally. Overall, the findings described here shape our mechanistic understanding of the human immune response to the IBV NA glycoprotein through the demonstration of two mAb delivery routes for protection against IBV and the identification of potential IBV therapeutic candidates.
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Anticuerpos Monoclonales , Anticuerpos Antivirales , Virus de la Influenza B , Gripe Humana , Neuraminidasa , Neuraminidasa/inmunología , Humanos , Virus de la Influenza B/inmunología , Animales , Anticuerpos Monoclonales/inmunología , Anticuerpos Antivirales/inmunología , Gripe Humana/inmunología , Gripe Humana/prevención & control , Vacunas contra la Influenza/inmunología , Ratones , Infecciones por Orthomyxoviridae/inmunología , Infecciones por Orthomyxoviridae/prevención & control , Proteínas Virales/inmunología , Replicación Viral/efectos de los fármacosRESUMEN
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) continues to evolve, escape coronavirus disease 2019 therapeutics and vaccines, and jeopardize public health. To combat SARS-CoV-2 antigenic escape, we developed a rapid, high-throughput pipeline to discover monospecific VHH antibodies and iteratively develop VHH-Fc-VHH bispecifics capable of neutralizing emerging SARS-CoV-2 variants. By panning VHH single-domain phage libraries against ancestral or beta spike proteins, we discovered high-affinity VHH antibodies with unique target epitopes. Combining two VHHs into a tetravalent bispecific construct conferred broad neutralization activity against multiple variants and was more resistant to antigenic escape than the monospecific antibody alone. Following the rise of the Omicron variant, a VHH in the original bispecific construct was replaced with another VHH discovered against the Omicron BA.1 receptor binding domain; the resulting bispecific exhibited neutralization against both BA.1 and BA.5 sublineage variants. A heavy chain-only tetravalent VHH-Fc-VHH bispecific platform derived from humanized synthetic libraries held a myriad of unique advantages: (i) synthetic preconstructed libraries minimized risk of liabilities and maximized discovery speed, (ii) VHH scaffolds allowed for a modular "plug-and-play" format that could be rapidly iterated upon as variants of concern arose, (iii) natural dimerization of single VHH-Fc-VHH polypeptides allowed for straightforward bispecific production and purification methods, and (iv) multivalent approaches enhanced avidity boosting effects and neutralization potency, and conferred more robust resistance to antigenic escape than monovalent approaches against specific variants. This iterative platform of rapid VHH discovery combined with modular bispecific design holds promise for long-term viral control efforts.
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The repeat emergence of SARS-CoV-2 variants of concern (VoC) with decreased susceptibility to vaccine-elicited antibodies highlights the need to develop next-generation vaccine candidates that confer broad protection. Here we describe the antibody response induced by the SARS-CoV-2 Spike Ferritin Nanoparticle (SpFN) vaccine candidate adjuvanted with the Army Liposomal Formulation including QS21 (ALFQ) in non-human primates. By isolating and characterizing several monoclonal antibodies directed against the Spike Receptor Binding Domain (RBD), N-Terminal Domain (NTD), or the S2 Domain, we define the molecular recognition of vaccine-elicited cross-reactive monoclonal antibodies (mAbs) elicited by SpFN. We identify six neutralizing antibodies with broad sarbecovirus cross-reactivity that recapitulate serum polyclonal antibody responses. In particular, RBD mAb WRAIR-5001 binds to the conserved cryptic region with high affinity to sarbecovirus clades 1 and 2, including Omicron variants, while mAb WRAIR-5021 offers complete protection from B.1.617.2 (Delta) in a murine challenge study. Our data further highlight the ability of SpFN vaccination to stimulate cross-reactive B cells targeting conserved regions of the Spike with activity against SARS CoV-1 and SARS-CoV-2 variants.
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Nanopartículas , Coronavirus Relacionado al Síndrome Respiratorio Agudo Severo , Animales , Ratones , Anticuerpos Neutralizantes , Macaca mulatta , Vacunación , Anticuerpos Antivirales , Anticuerpos Monoclonales , Vacunas contra la COVID-19 , Ferritinas , Glicoproteína de la Espiga del Coronavirus/genéticaRESUMEN
To exploit highly conserved and difficult drug targets, including multipass membrane proteins, monoclonal antibody discovery efforts increasingly rely on the advantages offered by divergent species such as rabbits, camelids, and chickens. Here, we provide an overview of antibody discovery technologies, analyze gaps in therapeutic antibodies that stem from the historic use of mice, and examine opportunities to exploit previously inaccessible targets through discovery now possible in alternate species. We summarize the clinical development of antibodies raised from divergent species, discussing how these animals enable robust immune responses against highly conserved binding sites and yield antibodies capable of penetrating functional pockets via long HCDR3 regions. We also discuss the value of pan-reactive molecules often produced by these hosts, and how these antibodies can be tested in accessible animal models, offering a faster path to clinical development.
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Anticuerpos Monoclonales , Pollos , Animales , Ratones , Conejos , Epítopos , Anticuerpos Monoclonales/uso terapéuticoRESUMEN
Sequential dengue virus (DENV) infections often generate neutralizing antibodies against all four DENV serotypes and sometimes, Zika virus. Characterizing cross-flavivirus broadly neutralizing antibody (bnAb) responses can inform countermeasures that avoid enhancement of infection associated with non-neutralizing antibodies. Here, we used single cell transcriptomics to mine the bnAb repertoire following repeated DENV infections. We identified several new bnAbs with comparable or superior breadth and potency to known bnAbs, and with distinct recognition determinants. Unlike all known flavivirus bnAbs, which are IgG1, one newly identified cross-flavivirus bnAb (F25.S02) was derived from IgA1. Both IgG1 and IgA1 versions of F25.S02 and known bnAbs displayed neutralizing activity, but only IgG1 enhanced infection in monocytes expressing IgG and IgA Fc receptors. Moreover, IgG-mediated enhancement of infection was inhibited by IgA1 versions of bnAbs. We demonstrate a role for IgA in flavivirus infection and immunity with implications for vaccine and therapeutic strategies.
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Flavivirus , Infección por el Virus Zika , Virus Zika , Humanos , Anticuerpos ampliamente neutralizantes , Transcriptoma , Anticuerpos Neutralizantes , Inmunoglobulina G , Inmunoglobulina A , Anticuerpos AntiviralesRESUMEN
Zika virus (ZIKV) is an emerging pathogen that causes devastating congenital defects. The overlapping epidemiology and immunologic cross-reactivity between ZIKV and dengue virus (DENV) pose complex challenges to vaccine design, given the potential for antibody-dependent enhancement of disease. Therefore, classification of ZIKV-specific antibody targets is of notable value. From a ZIKV-infected rhesus macaque, we identify ZIKV-reactive B cells and isolate potent neutralizing monoclonal antibodies (mAbs) with no cross-reactivity to DENV. We group these mAbs into four distinct antigenic groups targeting ZIKV-specific cross-protomer epitopes on the envelope glycoprotein. Co-crystal structures of representative mAbs in complex with ZIKV envelope glycoprotein reveal envelope-dimer epitope and unique dimer-dimer epitope targeting. All four specificities are serologically identified in convalescent humans following ZIKV infection, and representative mAbs from all four groups protect against ZIKV replication in mice. These results provide key insights into ZIKV-specific antigenicity and have implications for ZIKV vaccine, diagnostic, and therapeutic development.
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Virus del Dengue , Dengue , Vacunas Virales , Infección por el Virus Zika , Virus Zika , Humanos , Animales , Ratones , Anticuerpos Neutralizantes , Epítopos , Macaca mulatta , Anticuerpos Antivirales , Anticuerpos Monoclonales , Vacunas Virales/uso terapéutico , Proteínas del Envoltorio Viral/químicaRESUMEN
Novel therapeutic monoclonal antibodies (MAbs) must accommodate comprehensive breadth of activity against diverse sarbecoviruses and high neutralization potency to overcome emerging variants. Here, we report the crystal structure of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) receptor binding domain (RBD) in complex with MAb WRAIR-2063, a moderate-potency neutralizing antibody with exceptional sarbecovirus breadth, that targets the highly conserved cryptic class V epitope. This epitope overlaps substantially with the spike protein N-terminal domain (NTD) -interacting region and is exposed only when the spike is in the open conformation, with one or more RBDs accessible. WRAIR-2063 binds the RBD of SARS-CoV-2 WA-1, all variants of concern (VoCs), and clade 1 to 4 sarbecoviruses with high affinity, demonstrating the conservation of this epitope and potential resiliency against variation. We compare structural features of additional class V antibodies with their reported neutralization capacity to further explore the utility of the class V epitope as a pan-sarbecovirus vaccine and therapeutic target. IMPORTANCE Characterization of MAbs against SARS-CoV-2, elicited through vaccination or natural infection, has provided vital immunotherapeutic options for curbing the COVID-19 pandemic and has supplied critical insights into SARS-CoV-2 escape, transmissibility, and mechanisms of viral inactivation. Neutralizing MAbs that target the RBD but do not block ACE2 binding are of particular interest because the epitopes are well conserved within sarbecoviruses and MAbs targeting this area demonstrate cross-reactivity. The class V RBD-targeted MAbs localize to an invariant site of vulnerability, provide a range of neutralization potency, and exhibit considerable breadth against divergent sarbecoviruses, with implications for vaccine and therapeutic development.
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Anticuerpos Antivirales , COVID-19 , Epítopos , Coronavirus Relacionado al Síndrome Respiratorio Agudo Severo , Humanos , Anticuerpos Monoclonales/química , Anticuerpos Monoclonales/metabolismo , Anticuerpos Neutralizantes/química , Anticuerpos Neutralizantes/metabolismo , Anticuerpos Antivirales/química , Anticuerpos Antivirales/metabolismo , Epítopos/química , SARS-CoV-2/química , SARS-CoV-2/metabolismo , Coronavirus Relacionado al Síndrome Respiratorio Agudo Severo/química , Dominios Proteicos , Cristalografía por Rayos X , Estructura Cuaternaria de Proteína , Modelos Moleculares , Línea CelularRESUMEN
Understanding the human antibody response to emerging viral pathogens is key to epidemic preparedness. As the size of the B cell response to a pathogenic-virus-protective antigen is poorly defined, we perform deep paired heavy- and light-chain sequencing in Ebola virus glycoprotein (EBOV-GP)-specific memory B cells, allowing analysis of the ebolavirus-specific antibody repertoire both genetically and functionally. This approach facilitates investigation of the molecular and genetic basis for the evolution of cross-reactive antibodies by elucidating germline-encoded properties of antibodies to EBOV and identification of the overlap between antibodies in the memory B cell and serum repertoire. We identify 73 public clonotypes of EBOV, 20% of which encode antibodies with neutralization activity and capacity to protect mice in vivo. This comprehensive analysis of the public and private antibody repertoire provides insight into the molecular basis of the humoral immune response to EBOV GP, which informs the design of vaccines and improved therapeutics.
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Ebolavirus , Fiebre Hemorrágica Ebola , Humanos , Animales , Ratones , Anticuerpos Neutralizantes , Anticuerpos Antivirales , Formación de Anticuerpos , Prevalencia , Glicoproteínas/genéticaRESUMEN
Sequential dengue virus (DENV) infections often generate neutralizing antibodies against all four DENV serotypes and sometimes, Zika virus. Characterizing cross-flavivirus broadly neutralizing antibody (bnAb) responses can inform countermeasure strategies that avoid infection enhancement associated with non-neutralizing antibodies. Here, we used single cell transcriptomics to mine the bnAb repertoire following secondary DENV infection. We identified several new bnAbs with comparable or superior breadth and potency to known bnAbs, and with distinct recognition determinants. Unlike all known flavivirus bnAbs, which are IgG1, one newly identified cross-flavivirus bnAb (F25.S02) was derived from IgA1. Both IgG1 and IgA1 versions of F25.S02 and known bnAbs displayed neutralizing activity, but only IgG1 enhanced infection in monocytes expressing IgG and IgA Fc receptors. Moreover, IgG-mediated enhancement of infection was inhibited by IgA1 versions of bnAbs. We demonstrate a role for IgA in flavivirus infection and immunity with implications for vaccine and therapeutic strategies.
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Despite rapid and ongoing vaccine and therapeutic development, SARS-CoV-2 continues to evolve and evade, presenting a need for next-generation diverse therapeutic modalities. Here we show that nurse sharks immunized with SARS-CoV-2 recombinant receptor binding domain (RBD), RBD-ferritin (RFN), or spike protein ferritin nanoparticle (SpFN) immunogens elicit a set of new antigen receptor antibody (IgNAR) molecules that target two non-overlapping conserved epitopes on the spike RBD. Representative shark antibody variable NAR-Fc chimeras (ShAbs) targeting either of the two epitopes mediate cell-effector functions, with high affinity to all SARS-CoV-2 viral variants of concern, including the divergent Omicron strains. The ShAbs potently cross-neutralize SARS-CoV-2 WA-1, Alpha, Beta, Delta, Omicron BA.1 and BA.5, and SARS-CoV-1 pseudoviruses, and confer protection against SARS-CoV-2 challenge in the K18-hACE2 transgenic mouse model. Structural definition of the RBD-ShAb01-ShAb02 complex enabled design and production of multi-specific nanobodies with enhanced neutralization capacity, and picomolar affinity to divergent sarbecovirus clade 1a, 1b and 2 RBD molecules. These shark nanobodies represent potent immunotherapeutics both for current use, and future sarbecovirus pandemic preparation.
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COVID-19 , Coronavirus Relacionado al Síndrome Respiratorio Agudo Severo , Anticuerpos de Dominio Único , Animales , Ratones , Anticuerpos Neutralizantes , Anticuerpos Antivirales , COVID-19/prevención & control , Epítopos , Ferritinas/genética , Fragmentos Fc de Inmunoglobulinas , Ratones Transgénicos , SARS-CoV-2/genética , Glicoproteína de la Espiga del Coronavirus/genética , TiburonesRESUMEN
Humanity has faced three recent outbreaks of novel betacoronaviruses, emphasizing the need to develop approaches that broadly target coronaviruses. Here, we identify 55 monoclonal antibodies from COVID-19 convalescent donors that bind diverse betacoronavirus spike proteins. Most antibodies targeted an S2 epitope that included the K814 residue and were non-neutralizing. However, 11 antibodies targeting the stem helix neutralized betacoronaviruses from different lineages. Eight antibodies in this group, including the six broadest and most potent neutralizers, were encoded by IGHV1-46 and IGKV3-20. Crystal structures of three antibodies of this class at 1.5-1.75-Å resolution revealed a conserved mode of binding. COV89-22 neutralized SARS-CoV-2 variants of concern including Omicron BA.4/5 and limited disease in Syrian hamsters. Collectively, these findings identify a class of IGHV1-46/IGKV3-20 antibodies that broadly neutralize betacoronaviruses by targeting the stem helix but indicate these antibodies constitute a small fraction of the broadly reactive antibody response to betacoronaviruses after SARS-CoV-2 infection.
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COVID-19 , SARS-CoV-2 , Animales , Cricetinae , Anticuerpos Monoclonales , Brotes de Enfermedades , Mesocricetus , Anticuerpos Antivirales , Anticuerpos Neutralizantes , Glicoproteína de la Espiga del Coronavirus/genéticaRESUMEN
The tight junction protein claudin 6 (CLDN6) is differentially expressed on cancer cells with almost no expression in healthy tissue. However, achieving therapeutic MAb specificity for this 4 transmembrane protein is challenging because it is nearly identical to the widely expressed CLDN9, with only 3 extracellular amino acids different. Most other CLDN6 MAbs, including those in clinical development are cross-reactive with CLDN9, and several trials have now been stopped. Here we isolated rare MAbs that bind CLDN6 with up to picomolar affinity and display minimal cross-reactivity with CLDN9, 22 other CLDN family members, or across the human membrane proteome. Amino acid-level epitope mapping distinguished the binding sites of our MAbs from existing clinical-stage MAbs. Atomic-level epitope mapping identified the structural mechanism by which our MAbs differentiate CLDN6 and CLDN9 through steric hindrance at a single molecular contact point, the γ carbon on CLDN6 residue Q156.
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The E1 and E2 envelope proteins of hepatitis C virus (HCV) form a heterodimer that drives virus-host membrane fusion. Here, we analyze the role of each amino acid in E1E2 function, expressing 545 individual alanine mutants of E1E2 in human cells, incorporating them into infectious viral pseudoparticles, and testing them against 37 different monoclonal antibodies (MAbs) to ascertain full-length translation, folding, heterodimer assembly, CD81 binding, viral pseudoparticle incorporation, and infectivity. We propose a model describing the role of each critical residue in E1E2 functionality and use it to examine how MAbs neutralize infection by exploiting functionally critical sites of vulnerability on E1E2. Our results suggest that E1E2 is a surprisingly fragile protein complex where even a single alanine mutation at 92% of positions disrupts its function. The amino-acid-level targets identified are highly conserved and functionally critical and can be exploited for improved therapies and vaccines.
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Hepacivirus , Hepatitis C , Alanina , Anticuerpos Monoclonales , Humanos , Proteínas del Envoltorio Viral , Internalización del VirusRESUMEN
Zika virus (ZIKV) is a global public health concern due to its ability to cause congenital Zika syndrome and lack of approved vaccine, therapeutic, or other control measures. We discovered eight novel rabbit monoclonal antibodies (MAbs) that bind to distinct ZIKV envelope protein epitopes. The majority of the MAbs were ZIKV specific and targeted the lateral ridge of the envelope (E) protein domain III, while the MAb with the highest neutralizing activity recognized a putative quaternary epitope spanning E protein domains I and III. One of the non-neutralizing MAbs specifically recognized ZIKV precursor membrane protein (prM). Somatic hypermutation of immunoglobulin variable regions increases antibody affinity maturation and triggers antibody class switching. Negative correlations were observed between the somatic hypermutation rate of the immunoglobulin heavy-chain variable region and antibody binding parameters such as equilibrium dissociation constant, dissociation constant, and half-maximal effective concentration value of MAb binding to ZIKV virus-like particles. Complementarity-determining regions recognize the antigen epitopes and are scaffolded by canonical framework regions. Reversion of framework region amino acids to the rabbit germ line sequence decreased anti-ZIKV MAb binding activity of some MAbs. Thus, antibody affinity maturation, including somatic hypermutation and framework region mutations, contributed to the binding and function of these anti-ZIKV MAbs. IMPORTANCE ZIKV is a global health concern against which no vaccine or therapeutics are available. We characterized eight novel rabbit monoclonal antibodies recognizing ZIKV envelope and prM proteins and studied the relationship between somatic hypermutation of complementarity-determining regions, framework regions, mutations, antibody specificity, binding, and neutralizing activity. The results contribute to understanding structural features and somatic mutation pathways by which potent Zika virus-neutralizing antibodies can evolve, including the role of antibody framework regions.
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Anticuerpos Monoclonales , Anticuerpos Antivirales , Hipermutación Somática de Inmunoglobulina , Virus Zika , Animales , Anticuerpos Monoclonales/genética , Anticuerpos Neutralizantes/genética , Anticuerpos Antivirales/genética , Regiones Determinantes de Complementariedad/genética , Epítopos/genética , Mutación , Conejos , Proteínas del Envoltorio Viral/genética , Proteínas del Envoltorio Viral/inmunología , Virus Zika/inmunologíaRESUMEN
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.
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COVID-19 , SARS-CoV-2 , Animales , Anticuerpos Neutralizantes , Anticuerpos Antivirales , COVID-19/genética , Humanos , Ratones , SARS-CoV-2/genética , Glicoproteína de la Espiga del Coronavirus/genéticaRESUMEN
Coronavirus disease 2019 (COVID-19) is an evolving global public health crisis in need of therapeutic options. Passive immunization of monoclonal antibodies (mAbs) represents a promising therapeutic strategy capable of conferring immediate protection from SARS-CoV-2 infection. Herein, we describe the discovery and characterization of neutralizing SARS-CoV-2 IgG and VHH antibodies from four large-scale phage libraries. Each library was constructed synthetically with shuffled complementarity-determining region loops from natural llama and human antibody repertoires. While most candidates targeted the receptor-binding domain of the S1 subunit of SARS-CoV-2 spike protein, we also identified a neutralizing IgG candidate that binds a unique epitope on the N-terminal domain. A select number of antibodies retained binding to SARS-CoV-2 variants Alpha, Beta, Gamma, Kappa and Delta. Overall, our data show that synthetic phage libraries can rapidly yield SARS-CoV-2 S1 antibodies with therapeutically desirable features, including high affinity, unique binding sites, and potent neutralizing activity in vitro, and a capacity to limit disease in vivo.
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Anticuerpos Neutralizantes/inmunología , Anticuerpos Antivirales/inmunología , COVID-19/inmunología , Técnicas de Visualización de Superficie Celular , Inmunoglobulina G/inmunología , Biblioteca de Péptidos , SARS-CoV-2/inmunología , Anticuerpos de Dominio Único/inmunología , Glicoproteína de la Espiga del Coronavirus/inmunología , Animales , Anticuerpos Neutralizantes/genética , Anticuerpos Neutralizantes/metabolismo , Anticuerpos Neutralizantes/farmacología , Anticuerpos Antivirales/genética , Anticuerpos Antivirales/metabolismo , Especificidad de Anticuerpos , Sitios de Unión de Anticuerpos , COVID-19/metabolismo , COVID-19/prevención & control , COVID-19/virología , Chlorocebus aethiops , Modelos Animales de Enfermedad , Epítopos , Femenino , Interacciones Huésped-Patógeno , Inmunoglobulina G/genética , Inmunoglobulina G/metabolismo , Inmunoglobulina G/farmacología , Mesocricetus , SARS-CoV-2/patogenicidad , Anticuerpos de Dominio Único/genética , Anticuerpos de Dominio Único/metabolismo , Anticuerpos de Dominio Único/farmacología , Células VeroRESUMEN
Eastern equine encephalitis virus (EEEV), western equine encephalitis virus (WEEV) and Venezuelan equine encephalitis virus (VEEV) can cause fatal encephalitis in humans and equids. Some MAbs to the E1 glycoprotein are known to be cross-reactive, weakly neutralizing in vitro but can protect from disease in animal models. We investigated the mechanism of neutralization of VEEV infection by the broadly cross-reactive E1-specific MAb 1A4B-6. 1A4B-6 protected 3-week-old Swiss Webster mice prophylactically from lethal VEEV challenge. Likewise, 1A4B-6 inhibited virus growth in vitro at a pre-attachment step after virions were incubated at 37 °C and inhibited virus-mediated cell fusion. Amino acid residue N100 in the fusion loop of E1 protein was identified as critical for binding. The potential to elicit broadly cross-reactive MAbs with limited virus neutralizing activity in vitro but that can inhibit virus entry and protect animals from infection merits further exploration for vaccine and therapeutic developmental research.
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Anticuerpos Antivirales/inmunología , Virus de la Encefalitis Equina Venezolana/inmunología , Virus de la Encefalitis Equina Venezolana/metabolismo , Encefalomielitis Equina Venezolana/inmunología , Encefalomielitis Equina Venezolana/virología , Proteínas del Envoltorio Viral/inmunología , Replicación Viral/efectos de los fármacos , Alphavirus/inmunología , Infecciones por Alphavirus/inmunología , Secuencia de Aminoácidos , Animales , Anticuerpos Monoclonales/inmunología , Anticuerpos Neutralizantes/inmunología , Línea Celular , Chlorocebus aethiops , Reacciones Cruzadas , Encefalomielitis Equina Venezolana/terapia , Glicoproteínas/inmunología , Inmunoterapia , Ratones , Unión Proteica , Células Vero , Proteínas del Envoltorio Viral/metabolismo , Virión/inmunología , Virión/metabolismoRESUMEN
Prevention of viral escape and increased coverage against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants of concern require therapeutic monoclonal antibodies (mAbs) targeting multiple sites of vulnerability on the coronavirus spike glycoprotein. Here we identify several potent neutralizing antibodies directed against either the N-terminal domain (NTD) or the receptor-binding domain (RBD) of the spike protein. Administered in combinations, these mAbs provided low-dose protection against SARS-CoV-2 infection in the K18-human angiotensin-converting enzyme 2 mouse model, using both neutralization and Fc effector antibody functions. The RBD mAb WRAIR-2125, which targets residue F486 through a unique heavy-chain and light-chain pairing, demonstrated potent neutralizing activity against all major SARS-CoV-2 variants of concern. In combination with NTD and other RBD mAbs, WRAIR-2125 also prevented viral escape. These data demonstrate that NTD/RBD mAb combinations confer potent protection, likely leveraging complementary mechanisms of viral inactivation and clearance.