RESUMEN
Members of the Sarbecovirus subgenus of Coronaviridae have twice caused deadly threats to humans. There is increasing concern about the rapid mutation of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which has evolved into multiple generations of epidemic variants in 3 years. Broad neutralizing antibodies are of great importance for pandemic preparedness against SARS-CoV-2 variants and divergent zoonotic sarbecoviruses. Here, we analyzed the structural conservation of the receptor-binding domain (RBD) from representative sarbecoviruses and chose S2H97, a previously reported RBD antibody with ideal breadth and resistance to escape, as a template for computational design to enhance the neutralization activity and spectrum. A total of 35 designs were purified for evaluation. The neutralizing activity of a large proportion of these designs against multiple variants was increased from several to hundreds of times. Molecular dynamics simulation suggested that extra interface contacts and enhanced intermolecular interactions between the RBD and the designed antibodies are established. After light and heavy chain reconstitution, AI-1028, with five complementarity determining regions optimized, showed the best neutralizing activity across all tested sarbecoviruses, including SARS-CoV, multiple SARS-CoV-2 variants, and bat-derived viruses. AI-1028 recognized the same cryptic RBD epitope as the parental prototype antibody. In addition to computational design, chemically synthesized nanobody libraries are also a precious resource for rapid antibody development. By applying distinct RBDs as baits for reciprocal screening, we identified two novel nanobodies with broad activities. These findings provide potential pan-sarbecovirus neutralizing drugs and highlight new pathways to rapidly optimize therapeutic candidates when novel SARS-CoV-2 escape variants or new zoonotic coronaviruses emerge. IMPORTANCE The subgenus Sarbecovirus includes human SARS-CoV, SARS-CoV-2, and hundreds of genetically related bat viruses. The continuous evolution of SARS-CoV-2 has led to the striking evasion of neutralizing antibody (NAb) drugs and convalescent plasma. Antibodies with broad activity across sarbecoviruses would be helpful to combat current SARS-CoV-2 mutations and longer term animal virus spillovers. The study of pan-sarbecovirus NAbs described here is significant for the following reasons. First, we established a structure-based computational pipeline to design and optimize NAbs to obtain more potent and broader neutralizing activity across multiple sarbecoviruses. Second, we screened and identified nanobodies from a highly diversified synthetic library with a broad neutralizing spectrum using an elaborate screening strategy. These methodologies provide guidance for the rapid development of antibody therapeutics against emerging pathogens with highly variable characteristics.
Asunto(s)
Anticuerpos Antivirales , Anticuerpos ampliamente neutralizantes , Coronavirus Relacionado al Síndrome Respiratorio Agudo Severo , Anticuerpos de Dominio Único , Animales , Humanos , Anticuerpos Antivirales/biosíntesis , Anticuerpos Antivirales/química , Anticuerpos Antivirales/metabolismo , Anticuerpos ampliamente neutralizantes/biosíntesis , Anticuerpos ampliamente neutralizantes/química , Anticuerpos ampliamente neutralizantes/metabolismo , Quirópteros , COVID-19/virología , SARS-CoV-2/genética , SARS-CoV-2/inmunología , Coronavirus Relacionado al Síndrome Respiratorio Agudo Severo/inmunología , Coronavirus Relacionado al Síndrome Respiratorio Agudo Severo/metabolismo , Estructura Terciaria de Proteína , Modelos Moleculares , Unión ProteicaRESUMEN
The conserved coronavirus fusion peptide is a target of broadly neutralizing antibodies.
Asunto(s)
Alphacoronavirus , Anticuerpos Antivirales , Betacoronavirus , Anticuerpos ampliamente neutralizantes , Péptidos , Glicoproteína de la Espiga del Coronavirus , Proteínas Virales de Fusión , Anticuerpos Antivirales/química , Anticuerpos Antivirales/inmunología , Anticuerpos ampliamente neutralizantes/química , Anticuerpos ampliamente neutralizantes/inmunología , Péptidos/química , Péptidos/inmunología , Glicoproteína de la Espiga del Coronavirus/química , Glicoproteína de la Espiga del Coronavirus/inmunología , Proteínas Virales de Fusión/química , Proteínas Virales de Fusión/inmunología , Pruebas de Neutralización , Humanos , Betacoronavirus/inmunología , Alphacoronavirus/inmunología , Complejo Antígeno-Anticuerpo/química , Infecciones por Coronavirus/prevención & controlRESUMEN
The conformationally dynamic HIV-1 envelope trimer (Env) is the target of broadly neutralizing antibodies (bnAbs) that block viral entry. Single-molecule Förster resonance energy transfer (smFRET) has revealed that HIV-1 Env exists in at least three conformational states on the virion. Prior to complete host-receptor engagement (State 3), Env resides most prevalently in the smFRET-defined State 1, which is preferentially recognized by most bnAbs that are elicited by natural infection. smFRET has also revealed that soluble trimers containing prefusion-stabilizing disulfide and isoleucine-to-proline substitutions reside primarily in State 2, which is a required intermediate between States 1 and 3. While high-resolution Env structures have been determined for States 2 and 3, the structure of these trimers in State 1 is unknown. To provide insight into the State 1 structure, here we characterized antigenic differences between smFRET-defined states and then correlated these differences with known structural differences between States 2 and 3. We found that cell surface-expressed Env was enriched in each state using state-enriching antibody fragments or small-molecule virus entry inhibitors and then assessed binding to HIV-1 bnAbs preferentially binding different states. We observed small but consistent differences in binding between Env enriched in States 1 and 2, and a more than 10-fold difference in binding to Env enriched in these states versus Env enriched in State 3. We conclude that structural differences between HIV-1 Env States 1 and 3 are likely more than 10-fold greater than those between States 1 and 2, providing important insight into State 1.
Asunto(s)
Infecciones por VIH , VIH-1 , Productos del Gen env del Virus de la Inmunodeficiencia Humana , Anticuerpos ampliamente neutralizantes/química , Anticuerpos ampliamente neutralizantes/metabolismo , Anticuerpos Anti-VIH , VIH-1/metabolismo , Humanos , Conformación Proteica , Productos del Gen env del Virus de la Inmunodeficiencia Humana/química , Productos del Gen env del Virus de la Inmunodeficiencia Humana/metabolismoRESUMEN
Understanding broadly neutralizing sarbecovirus antibody responses is key to developing countermeasures against SARS-CoV-2 variants and future zoonotic sarbecoviruses. We describe the isolation and characterization of a human monoclonal antibody, designated S2K146, that broadly neutralizes viruses belonging to SARS-CoV- and SARS-CoV-2-related sarbecovirus clades which use ACE2 as an entry receptor. Structural and functional studies show that most of the virus residues that directly bind S2K146 are also involved in binding to ACE2. This allows the antibody to potently inhibit receptor attachment. S2K146 protects against SARS-CoV-2 Beta challenge in hamsters and viral passaging experiments reveal a high barrier for emergence of escape mutants, making it a good candidate for clinical development. The conserved ACE2-binding residues present a site of vulnerability that might be leveraged for developing vaccines eliciting broad sarbecovirus immunity.
Asunto(s)
Enzima Convertidora de Angiotensina 2/metabolismo , Anticuerpos Antivirales/inmunología , Betacoronavirus/inmunología , Anticuerpos ampliamente neutralizantes/inmunología , COVID-19/terapia , SARS-CoV-2/inmunología , Glicoproteína de la Espiga del Coronavirus/inmunología , Enzima Convertidora de Angiotensina 2/química , Animales , Anticuerpos Monoclonales/química , Anticuerpos Monoclonales/inmunología , Anticuerpos Monoclonales/metabolismo , Anticuerpos Monoclonales/uso terapéutico , Anticuerpos Antivirales/química , Anticuerpos Antivirales/metabolismo , Afinidad de Anticuerpos , Anticuerpos ampliamente neutralizantes/química , Anticuerpos ampliamente neutralizantes/metabolismo , Anticuerpos ampliamente neutralizantes/uso terapéutico , COVID-19/inmunología , Reacciones Cruzadas , Microscopía por Crioelectrón , Epítopos , Humanos , Evasión Inmune , Mesocricetus , Modelos Moleculares , Imitación Molecular , Mutación , Conformación Proteica , Dominios Proteicos , Receptores de Coronavirus/química , Receptores de Coronavirus/metabolismo , Glicoproteína de la Espiga del Coronavirus/química , Glicoproteína de la Espiga del Coronavirus/genética , Glicoproteína de la Espiga del Coronavirus/metabolismoRESUMEN
The high genetic diversity of hepatitis C virus (HCV) complicates effective vaccine development. We screened a cohort of 435 HCV-infected individuals and found that 2%-5% demonstrated outstanding HCV-neutralizing activity. From four of these patients, we isolated 310 HCV antibodies, including neutralizing antibodies with exceptional breadth and potency. High neutralizing activity was enabled by the use of the VH1-69 heavy-chain gene segment, somatic mutations within CDRH1, and CDRH2 hydrophobicity. Structural and mutational analyses revealed an important role for mutations replacing the serines at positions 30 and 31, as well as the presence of neutral and hydrophobic residues at the tip of the CDRH3. Based on these characteristics, we computationally created a de novo antibody with a fully synthetic VH1-69 heavy chain that efficiently neutralized multiple HCV genotypes. Our findings provide a deep understanding of the generation of broadly HCV-neutralizing antibodies that can guide the design of effective vaccine candidates.
Asunto(s)
Anticuerpos ampliamente neutralizantes/genética , Hepacivirus/inmunología , Anticuerpos contra la Hepatitis C/genética , Linfocitos B/inmunología , Anticuerpos ampliamente neutralizantes/química , Anticuerpos ampliamente neutralizantes/inmunología , Regiones Determinantes de Complementariedad/química , Regiones Determinantes de Complementariedad/genética , Regiones Determinantes de Complementariedad/inmunología , Epítopos , Femenino , Genotipo , Hepacivirus/genética , Hepatitis C/inmunología , Anticuerpos contra la Hepatitis C/química , Anticuerpos contra la Hepatitis C/inmunología , Humanos , Cadenas Pesadas de Inmunoglobulina/química , Cadenas Pesadas de Inmunoglobulina/genética , Cadenas Pesadas de Inmunoglobulina/inmunología , Masculino , Persona de Mediana Edad , Mutación , Proteínas del Envoltorio Viral/química , Proteínas del Envoltorio Viral/inmunologíaRESUMEN
The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) Omicron variant of concern evades antibody-mediated immunity that comes from vaccination or infection with earlier variants due to accumulation of numerous spike mutations. To understand the Omicron antigenic shift, we determined cryo-electron microscopy and x-ray crystal structures of the spike protein and the receptor-binding domain bound to the broadly neutralizing sarbecovirus monoclonal antibody (mAb) S309 (the parent mAb of sotrovimab) and to the human ACE2 receptor. We provide a blueprint for understanding the marked reduction of binding of other therapeutic mAbs that leads to dampened neutralizing activity. Remodeling of interactions between the Omicron receptor-binding domain and human ACE2 likely explains the enhanced affinity for the host receptor relative to the ancestral virus.
Asunto(s)
Enzima Convertidora de Angiotensina 2/química , Anticuerpos Antivirales/química , Evasión Inmune , Receptores de Coronavirus/química , SARS-CoV-2/química , SARS-CoV-2/inmunología , Glicoproteína de la Espiga del Coronavirus/química , Sustitución de Aminoácidos , Enzima Convertidora de Angiotensina 2/metabolismo , Anticuerpos Monoclonales/química , Anticuerpos Monoclonales/inmunología , Anticuerpos Monoclonales/metabolismo , Anticuerpos Antivirales/inmunología , Anticuerpos Antivirales/metabolismo , Deriva y Cambio Antigénico , Anticuerpos ampliamente neutralizantes/química , Anticuerpos ampliamente neutralizantes/inmunología , Anticuerpos ampliamente neutralizantes/metabolismo , Microscopía por Crioelectrón , Cristalografía por Rayos X , Humanos , Modelos Moleculares , Mutación , Unión Proteica , Conformación Proteica , Dominios Proteicos/genética , Dominios y Motivos de Interacción de Proteínas/genética , Receptores de Coronavirus/metabolismo , SARS-CoV-2/genética , SARS-CoV-2/fisiología , Glicoproteína de la Espiga del Coronavirus/genética , Glicoproteína de la Espiga del Coronavirus/inmunología , Glicoproteína de la Espiga del Coronavirus/metabolismoRESUMEN
Deep mining of B cell repertoires of HIV-1-infected individuals has resulted in the isolation of dozens of HIV-1 broadly neutralizing antibodies (bNAbs). Yet, it remains uncertain whether any such bNAbs alone are sufficiently broad and potent to deploy therapeutically. Here, we engineered HIV-1 bNAbs for their combination on a single multispecific and avid molecule via direct genetic fusion of their Fab fragments to the human apoferritin light chain. The resulting molecule demonstrated a remarkable median IC50 value of 0.0009 µg/mL and 100% neutralization coverage of a broad HIV-1 pseudovirus panel (118 isolates) at a 4 µg/mL cutoff-a 32-fold enhancement in viral neutralization potency compared to a mixture of the corresponding HIV-1 bNAbs. Importantly, Fc incorporation on the molecule and engineering to modulate Fc receptor binding resulted in IgG-like bioavailability in vivo. This robust plug-and-play antibody design is relevant against indications where multispecificity and avidity are leveraged simultaneously to mediate optimal biological activity.
Asunto(s)
Anticuerpos Neutralizantes/inmunología , Afinidad de Anticuerpos/inmunología , Anticuerpos Anti-VIH/inmunología , Pruebas de Neutralización , Ingeniería de Proteínas , Anticuerpos Neutralizantes/química , Anticuerpos ampliamente neutralizantes/química , Anticuerpos ampliamente neutralizantes/inmunología , Epítopos/química , Epítopos/inmunología , Anticuerpos Anti-VIH/química , Anticuerpos Anti-VIH/genética , VIH-1/inmunología , Humanos , Modelos Moleculares , Pruebas de Neutralización/métodos , Conformación Proteica , Ingeniería de Proteínas/métodos , Relación Estructura-ActividadRESUMEN
The low abundance of envelope spikes and the inability of IgG to aggregate virions render HIV-1 an inadequate target for antibody-mediated clearance by phagocytes. In an attempt to improve the ability of antibody to mediate the internalization of HIV-1 virions, we generated multimers of the broadly neutralizing HIV-1-specific monoclonal antibody (MAb) VRC01 using site-directed mutagenesis of the Fc segment. We then measured virion internalization using primary human monocytes and neutrophils. We found that, in the absence of complement, immune complexes consisting of HIV-1 virions and VRC01 multimers were slightly more efficiently internalized than were complexes formed with monomeric VRC01. The presence of complement, however, greatly augmented internalization of immune complexes formed with the multimeric MAb but had little impact on monomeric MAb-mediated internalization. Multimerization and the presence of complement overcome the limited ability of monomeric antibody to mediate internalization of HIV-1 virions and may thus provide a therapeutic approach to clearing virus. IMPORTANCE Antibody-mediated internalization of HIV-1 by phagocytes, a potential mechanism for clearing virus, is very inefficient. In an effort to improve viral clearance, we produced a multimeric form of the broadly neutralizing monoclonal antibody VRC01. We found that VRC01 antibody multimers (primarily hexamers) were only slightly more efficient in mediating HIV-1 internalization than was monomeric VRC01. However, the addition of complement resulted in substantially greater internalization of multimer-opsonized virus. In contrast, complement had little if any impact on internalization of monomer-opsonized virus. Therefore, antibody multimerization in combination with complement may overcome the limited ability of monomeric antibody to mediate internalization of HIV-1 virions. Our findings may provide a therapeutic approach to clearing virus.
Asunto(s)
Proteínas del Sistema Complemento/inmunología , Anticuerpos Anti-VIH/inmunología , VIH-1/inmunología , Fagocitosis/inmunología , Virión/inmunología , Complejo Antígeno-Anticuerpo/química , Complejo Antígeno-Anticuerpo/genética , Complejo Antígeno-Anticuerpo/inmunología , Anticuerpos ampliamente neutralizantes/química , Anticuerpos ampliamente neutralizantes/genética , Anticuerpos ampliamente neutralizantes/inmunología , Anticuerpos Anti-VIH/química , Anticuerpos Anti-VIH/genética , Proteína gp41 de Envoltorio del VIH/inmunología , Humanos , Monocitos/inmunología , Mutación , Neutrófilos/inmunología , Multimerización de Proteína , Receptores Fc/genética , Receptores Fc/inmunologíaRESUMEN
Antibodies with exceptional breadth and potency have been elicited in some individuals during natural HIV-1 infection. Elicitation and affinity maturation of broadly neutralizing antibodies (bnAbs) is therefore the central goal of HIV-1 vaccine development. The functional properties of bnAbs also make them attractive as immunotherapeutic agents, which has led to their production and optimization for passive immunotherapy. This process requires in vitro manufacturing and monitoring of any heterogeneous expression, especially when subpopulations of antibodies are produced with varying levels of biological activity. Post-translational modification (PTM) of antibodies can contribute to heterogeneity and is the focus of this study. Specifically, we have investigated cysteinylation in a bnAb lineage (PCDN family) targeting the N332-glycan supersite on the surface envelope glycoprotein (Env) of HIV-1. This PTM is defined by capping of unpaired cysteine residues with molecular cysteine. Through chromatography and mass spectrometry, we were able to characterize subpopulations of cysteinylated and non-cysteinylated antibodies when expressed in mammalian cells. The crystal structures of two PCDN antibodies represent the first structures of a cysteinylated antibody and reveal that the cysteinylation in this case is located in CDRH3. Biophysical studies indicate that cysteinylation of these HIV-1 antibodies does not interfere with antigen binding, which has been reported to occur in other cysteinylated antibodies. As such, these studies highlight the need for further investigation of cysteinylation in anti-HIV and other bnAbs.
Asunto(s)
Anticuerpos Antivirales/química , Anticuerpos ampliamente neutralizantes/química , Cisteína/química , VIH-1/inmunología , Procesamiento Proteico-Postraduccional , Anticuerpos Monoclonales/química , Cristalografía , Células HEK293 , HumanosRESUMEN
The third variable loop region (V3 loop) on gp120 plays an important role in cellular entry of HIV-1. Its interaction with the cellular CD4 and coreceptors is an important hallmark in facilitating the bridging by gp41 and subsequent fusion of membranes for transfer of viral genetic material. Further, the virus phenotype determines the cell tropism via respective co- receptor binding. Thus, coreceptor binding motif of envelope is considered to be a potent anti-viral drug target for viral entry inhibition. However, its high variability in sequence is the major hurdle for developing inhibitors targeting the region. In this study, we have used an in silico Virtual Screening and "Fragment-based" method to design small molecules based on the gp120 V3 loop interactions with a potent broadly neutralizing human monoclonal antibody, 447-52D. From the in silico analysis a potent scaffold, 1,3,5-triazine was identified for further development. Derivatives of 1,3,5-triazine with specific functional groups were designed and synthesized keeping the interaction with co-receptor intact. Finally, preliminary evaluation of molecules for HIV-1 inhibition on two different virus strains (clade C, clade B) yielded IC50 < 5.0 µM. The approach used to design molecules based on broadly neutralizing antibody, was useful for development of target specific potent antiviral agents to prevent HIV entry. The study reported promising inhibitors that could be further developed and studied.
Asunto(s)
Fármacos Anti-VIH/farmacología , Anticuerpos ampliamente neutralizantes/farmacología , Diseño de Fármacos , Proteína gp120 de Envoltorio del VIH/antagonistas & inhibidores , VIH-1/efectos de los fármacos , Fármacos Anti-VIH/síntesis química , Fármacos Anti-VIH/química , Anticuerpos ampliamente neutralizantes/química , Relación Dosis-Respuesta a Droga , Proteína gp120 de Envoltorio del VIH/metabolismo , VIH-1/metabolismo , Humanos , Pruebas de Sensibilidad Microbiana , Estructura Molecular , Relación Estructura-ActividadRESUMEN
Potent neutralizing SARS-CoV-2 antibodies often target the spike protein receptor-binding site (RBS), but the variability of RBS epitopes hampers broad neutralization of multiple sarbecoviruses and drifted viruses. Here, using humanized mice, we identified an RBS antibody with a germline VH gene that potently neutralized SARS-related coronaviruses, including SARS-CoV and SARS-CoV-2 variants. X-ray crystallography revealed coordinated recognition by the heavy chain of non-RBS conserved sites and the light chain of RBS with a binding angle mimicking the angiotensin-converting enzyme 2 (ACE2) receptor. The minimum footprints in the hypervariable region of RBS contributed to the breadth of neutralization, which was enhanced by immunoglobulin G3 (IgG3) class switching. The coordinated binding resulted in broad neutralization of SARS-CoV and emerging SARS-CoV-2 variants of concern. Low-dose therapeutic antibody treatment in hamsters reduced the virus titers and morbidity during SARS-CoV-2 challenge. The structural basis for broad neutralizing activity may inform the design of a broad spectrum of therapeutics and vaccines.
Asunto(s)
Anticuerpos ampliamente neutralizantes/inmunología , Reacciones Cruzadas/inmunología , SARS-CoV-2/inmunología , Animales , Betacoronavirus/inmunología , Sitios de Unión de Anticuerpos , Anticuerpos ampliamente neutralizantes/química , Anticuerpos ampliamente neutralizantes/uso terapéutico , COVID-19/prevención & control , COVID-19/terapia , COVID-19/virología , Cricetinae , Humanos , Cambio de Clase de Inmunoglobulina , Fragmentos Fab de Inmunoglobulinas/química , Fragmentos Fab de Inmunoglobulinas/metabolismo , Inmunoglobulina G/química , Inmunoglobulina G/inmunología , Ratones , Dominios Proteicos , Glicoproteína de la Espiga del Coronavirus/química , Glicoproteína de la Espiga del Coronavirus/inmunología , Glicoproteína de la Espiga del Coronavirus/metabolismoRESUMEN
Interventions against variants of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are urgently needed. Stable and potent nanobodies (Nbs) that target the receptor binding domain (RBD) of SARS-CoV-2 spike are promising therapeutics. However, it is unknown if Nbs broadly neutralize circulating variants. We found that RBD Nbs are highly resistant to variants of concern (VOCs). High-resolution cryoelectron microscopy determination of eight Nb-bound structures reveals multiple potent neutralizing epitopes clustered into three classes: Class I targets ACE2-binding sites and disrupts host receptor binding. Class II binds highly conserved epitopes and retains activity against VOCs and RBDSARS-CoV. Cass III recognizes unique epitopes that are likely inaccessible to antibodies. Systematic comparisons of neutralizing antibodies and Nbs provided insights into how Nbs target the spike to achieve high-affinity and broadly neutralizing activity. Structure-function analysis of Nbs indicates a variety of antiviral mechanisms. Our study may guide the rational design of pan-coronavirus vaccines and therapeutics.
Asunto(s)
Anticuerpos ampliamente neutralizantes/inmunología , Epítopos/inmunología , SARS-CoV-2/inmunología , Anticuerpos de Dominio Único/inmunología , Enzima Convertidora de Angiotensina 2/química , Enzima Convertidora de Angiotensina 2/metabolismo , Anticuerpos Monoclonales/química , Anticuerpos Monoclonales/inmunología , Anticuerpos Monoclonales/metabolismo , Sitios de Unión , Anticuerpos ampliamente neutralizantes/química , Anticuerpos ampliamente neutralizantes/clasificación , Anticuerpos ampliamente neutralizantes/metabolismo , COVID-19/prevención & control , Epítopos/química , Epítopos/metabolismo , Humanos , Modelos Moleculares , Mutación , Unión Proteica , SARS-CoV-2/genética , Anticuerpos de Dominio Único/química , Anticuerpos de Dominio Único/clasificación , Anticuerpos de Dominio Único/metabolismo , Glicoproteína de la Espiga del Coronavirus/química , Glicoproteína de la Espiga del Coronavirus/genética , Glicoproteína de la Espiga del Coronavirus/inmunología , Glicoproteína de la Espiga del Coronavirus/metabolismo , Relación Estructura-Actividad , Tratamiento Farmacológico de COVID-19RESUMEN
The rational development of norovirus vaccine candidates requires a deep understanding of the antigenic diversity and mechanisms of neutralization of the virus. Here, we isolate and characterize a panel of broadly cross-reactive naturally occurring human monoclonal IgMs, IgAs and IgGs reactive with human norovirus (HuNoV) genogroup I or II (GI or GII). We note three binding patterns and identify monoclonal antibodies (mAbs) that neutralize at least one GI or GII HuNoV strain when using a histo-blood group antigen (HBGA) blocking assay. The HBGA blocking assay and a virus neutralization assay using human intestinal enteroids reveal that the GII-specific mAb NORO-320, mediates HBGA blocking and neutralization of multiple GII genotypes. The Fab form of NORO-320 neutralizes GII.4 infection more potently than the mAb, however, does not block HBGA binding. The crystal structure of NORO-320 Fab in complex with GII.4 P-domain shows that the antibody recognizes a highly conserved region in the P-domain distant from the HBGA binding site. Dynamic light scattering analysis of GII.4 virus-like particles with mAb NORO-320 shows severe aggregation, suggesting neutralization is by steric hindrance caused by multivalent cross-linking. Aggregation was not observed with the Fab form of NORO-320, suggesting that this clone also has additional inhibitory features.
Asunto(s)
Anticuerpos Antivirales/inmunología , Anticuerpos ampliamente neutralizantes/inmunología , Reacciones Cruzadas , Norovirus/inmunología , Secuencia de Aminoácidos , Anticuerpos Monoclonales/química , Anticuerpos Monoclonales/inmunología , Anticuerpos Monoclonales/metabolismo , Anticuerpos Antivirales/química , Anticuerpos Antivirales/metabolismo , Variación Antigénica , Sitios de Unión , Antígenos de Grupos Sanguíneos/metabolismo , Anticuerpos ampliamente neutralizantes/química , Anticuerpos ampliamente neutralizantes/metabolismo , Infecciones por Caliciviridae/inmunología , Infecciones por Caliciviridae/virología , Proteínas de la Cápside/química , Proteínas de la Cápside/genética , Proteínas de la Cápside/inmunología , Proteínas de la Cápside/metabolismo , Cristalografía por Rayos X , Mapeo Epitopo , Genotipo , Humanos , Fragmentos Fab de Inmunoglobulinas/química , Fragmentos Fab de Inmunoglobulinas/inmunología , Fragmentos Fab de Inmunoglobulinas/metabolismo , Norovirus/genética , Unión Proteica , Dominios ProteicosRESUMEN
The recent emergence of SARS-CoV-2 variants of concern1-10 and the recurrent spillovers of coronaviruses11,12 into the human population highlight the need for broadly neutralizing antibodies that are not affected by the ongoing antigenic drift and that can prevent or treat future zoonotic infections. Here we describe a human monoclonal antibody designated S2X259, which recognizes a highly conserved cryptic epitope of the receptor-binding domain and cross-reacts with spikes from all clades of sarbecovirus. S2X259 broadly neutralizes spike-mediated cell entry of SARS-CoV-2, including variants of concern (B.1.1.7, B.1.351, P.1, and B.1.427/B.1.429), as well as a wide spectrum of human and potentially zoonotic sarbecoviruses through inhibition of angiotensin-converting enzyme 2 (ACE2) binding to the receptor-binding domain. Furthermore, deep-mutational scanning and in vitro escape selection experiments demonstrate that S2X259 possesses an escape profile that is limited to a single substitution, G504D. We show that prophylactic and therapeutic administration of S2X259 protects Syrian hamsters (Mesocricetus auratus) against challenge with the prototypic SARS-CoV-2 and the B.1.351 variant of concern, which suggests that this monoclonal antibody is a promising candidate for the prevention and treatment of emergent variants and zoonotic infections. Our data reveal a key antigenic site that is targeted by broadly neutralizing antibodies and will guide the design of vaccines that are effective against all sarbecoviruses.
Asunto(s)
Anticuerpos Monoclonales/inmunología , Anticuerpos Monoclonales/uso terapéutico , Anticuerpos Antivirales/inmunología , Anticuerpos ampliamente neutralizantes/inmunología , Anticuerpos ampliamente neutralizantes/uso terapéutico , COVID-19/prevención & control , SARS-CoV-2/clasificación , SARS-CoV-2/inmunología , Animales , Anticuerpos Monoclonales/química , Anticuerpos Antivirales/química , Anticuerpos Antivirales/uso terapéutico , Anticuerpos ampliamente neutralizantes/química , COVID-19/inmunología , COVID-19/virología , Reacciones Cruzadas/inmunología , Modelos Animales de Enfermedad , Femenino , Humanos , Evasión Inmune/genética , Evasión Inmune/inmunología , Mesocricetus/inmunología , Mesocricetus/virología , Mutación , Pruebas de Neutralización , SARS-CoV-2/química , SARS-CoV-2/genética , Zoonosis Virales/inmunología , Zoonosis Virales/prevención & control , Zoonosis Virales/virologíaRESUMEN
Stimulating broadly neutralizing antibodies (bnAbs) directly from germline remains a barrier for HIV vaccines. HIV superinfection elicits bnAbs more frequently than single infection, providing clues of how to elicit such responses. We used longitudinal antibody sequencing and structural studies to characterize bnAb development from a superinfection case. BnAb QA013.2 bound initial and superinfecting viral Env, despite its probable naive progenitor only recognizing the superinfecting strain, suggesting both viruses influenced this lineage. A 4.15 Å cryo-EM structure of QA013.2 bound to native-like trimer showed recognition of V3 signatures (N301/N332 and GDIR). QA013.2 relies less on CDRH3 and more on framework and CDRH1 for affinity and breadth compared to other V3/glycan-specific bnAbs. Antigenic profiling revealed that viral escape was achieved by changes in the structurally-defined epitope and by mutations in V1. These results highlight shared and novel properties of QA013.2 relative to other V3/glycan-specific bnAbs in the setting of sequential, diverse antigens.
Asunto(s)
Anticuerpos ampliamente neutralizantes/inmunología , Anticuerpos ampliamente neutralizantes/aislamiento & purificación , Anticuerpos Anti-VIH/inmunología , Infecciones por VIH/inmunología , Polisacáridos/inmunología , Sobreinfección/inmunología , Anticuerpos ampliamente neutralizantes/química , Anticuerpos ampliamente neutralizantes/genética , Microscopía por Crioelectrón , Epítopos/genética , Epítopos/inmunología , Femenino , Células HEK293 , VIH-1 , Humanos , Modelos Moleculares , Mutación , Polisacáridos/químicaRESUMEN
An ideal therapeutic anti-SARS-CoV-2 antibody would resist viral escape1-3, have activity against diverse sarbecoviruses4-7, and be highly protective through viral neutralization8-11 and effector functions12,13. Understanding how these properties relate to each other and vary across epitopes would aid the development of therapeutic antibodies and guide vaccine design. Here we comprehensively characterize escape, breadth and potency across a panel of SARS-CoV-2 antibodies targeting the receptor-binding domain (RBD). Despite a trade-off between in vitro neutralization potency and breadth of sarbecovirus binding, we identify neutralizing antibodies with exceptional sarbecovirus breadth and a corresponding resistance to SARS-CoV-2 escape. One of these antibodies, S2H97, binds with high affinity across all sarbecovirus clades to a cryptic epitope and prophylactically protects hamsters from viral challenge. Antibodies that target the angiotensin-converting enzyme 2 (ACE2) receptor-binding motif (RBM) typically have poor breadth and are readily escaped by mutations despite high neutralization potency. Nevertheless, we also characterize a potent RBM antibody (S2E128) with breadth across sarbecoviruses related to SARS-CoV-2 and a high barrier to viral escape. These data highlight principles underlying variation in escape, breadth and potency among antibodies that target the RBD, and identify epitopes and features to prioritize for therapeutic development against the current and potential future pandemics.
Asunto(s)
Anticuerpos ampliamente neutralizantes/inmunología , COVID-19/virología , Reacciones Cruzadas/inmunología , Evasión Inmune , SARS-CoV-2/clasificación , SARS-CoV-2/inmunología , Glicoproteína de la Espiga del Coronavirus/química , Glicoproteína de la Espiga del Coronavirus/inmunología , Adulto , Anciano , Animales , Anticuerpos Monoclonales/química , Anticuerpos Monoclonales/inmunología , Anticuerpos Antivirales/química , Anticuerpos Antivirales/inmunología , Afinidad de Anticuerpos , Anticuerpos ampliamente neutralizantes/química , COVID-19/inmunología , Vacunas contra la COVID-19/química , Vacunas contra la COVID-19/inmunología , Línea Celular , Cricetinae , Epítopos de Linfocito B/química , Epítopos de Linfocito B/genética , Epítopos de Linfocito B/inmunología , Femenino , Humanos , Evasión Inmune/genética , Evasión Inmune/inmunología , Masculino , Mesocricetus , Persona de Mediana Edad , Modelos Moleculares , SARS-CoV-2/química , SARS-CoV-2/genética , Glicoproteína de la Espiga del Coronavirus/genética , Vacunología , Tratamiento Farmacológico de COVID-19RESUMEN
Three variable 2 (V2) loops of HIV-1 envelope glycoprotein (Env) trimer converge at the Env apex to form the epitope of an important classes of HIV-1 broadly neutralizing antibodies (bNAbs). These V2-glycan/apex antibodies are exceptionally potent but less broad (â¼60 to 75%) than many other bNAbs. Their CDRH3 regions are typically long, acidic, and tyrosine sulfated. Tyrosine sulfation complicates efforts to improve these antibodies through techniques such as phage or yeast display. To improve the breadth of CAP256-VRC26.25 (VRC26.25), a very potent apex antibody, we adapted and extended a B cell display approach. Specifically, we used CRISPR/Cas12a to introduce VRC26.25 heavy- and light-chain genes into their respective loci in a B cell line, ensuring that each cell expresses a single VRC26.25 variant. We then diversified these loci through activation-induced cytidine deaminase-mediated hypermutation and homology-directed repair using randomized CDRH3 sequences as templates. Iterative sorting with soluble Env trimers and further randomization selected VRC26.25 variants with successively improving affinities. Three mutations in the CDRH3 region largely accounted for this improved affinity, and VRC26.25 modified with these mutations exhibited greater breadth and potency than the original antibody. Our data describe a broader and more-potent form of VRC26.25 as well as an approach useful for improving the breadth and potency of antibodies with functionally important posttranslational modifications.
Asunto(s)
Anticuerpos ampliamente neutralizantes/inmunología , Anticuerpos Anti-VIH/inmunología , VIH-1/inmunología , Linfocitos B/inmunología , Anticuerpos ampliamente neutralizantes/química , Anticuerpos ampliamente neutralizantes/genética , Anticuerpos Anti-VIH/química , Anticuerpos Anti-VIH/genética , Infecciones por VIH/inmunología , Infecciones por VIH/virología , VIH-1/genética , Humanos , Ingeniería de Proteínas , Productos del Gen env del Virus de la Inmunodeficiencia Humana/genética , Productos del Gen env del Virus de la Inmunodeficiencia Humana/inmunologíaRESUMEN
Broadly neutralizing antibodies (bNAbs) are the focus of increasing interest for human immunodeficiency virus type 1 (HIV-1) prevention and treatment. Although several bNAbs are already under clinical evaluation, the development of antibodies with even greater potency and breadth remains a priority. Recently, we reported a novel strategy for improving bNAbs against the CD4-binding site (CD4bs) of gp120 by engraftment of the elongated framework region 3 (FR3) from VRC03, which confers the ability to establish quaternary interactions with a second gp120 protomer. Here, we applied this strategy to a new series of anti-CD4bs bNAbs (N49 lineage) that already possess high potency and breadth. The resultant chimeric antibodies bound the HIV-1 envelope (Env) trimer with a higher affinity than their parental forms. Likewise, their neutralizing capacity against a global panel of HIV-1 Envs was also increased. The introduction of additional modifications further enhanced the neutralization potency. We also tried engrafting the elongated CDR1 of the heavy chain from bNAb 1-18, another highly potent quaternary-binding antibody, onto several VRC01-class bNAbs, but none of them was improved. These findings point to the highly selective requirements for the establishment of quaternary contact with the HIV-1 Env trimer. The improved anti-CD4bs antibodies reported here may provide a helpful complement to current antibody-based protocols for the therapy and prevention of HIV-1 infection.IMPORTANCE Monoclonal antibodies represent one of the most important recent innovations in the fight against infectious diseases. Although potent antibodies can be cloned from infected individuals, various strategies can be employed to improve their activity or pharmacological features. Here, we improved a lineage of very potent antibodies that target the receptor-binding site of HIV-1 by engineering chimeric molecules containing a fragment from a different monoclonal antibody. These engineered antibodies are promising candidates for development of therapeutic or preventive approaches against HIV/AIDS.
Asunto(s)
Sitios de Unión de Anticuerpos , Anticuerpos ampliamente neutralizantes/inmunología , Antígenos CD4/metabolismo , Anticuerpos Anti-VIH/inmunología , Proteína gp120 de Envoltorio del VIH/química , Proteína gp120 de Envoltorio del VIH/inmunología , VIH-1/inmunología , Ingeniería de Proteínas , Anticuerpos Monoclonales/química , Anticuerpos Monoclonales/genética , Anticuerpos Monoclonales/inmunología , Anticuerpos Monoclonales/uso terapéutico , Sitios de Unión , Sitios de Unión de Anticuerpos/inmunología , Anticuerpos ampliamente neutralizantes/química , Anticuerpos ampliamente neutralizantes/genética , Anticuerpos ampliamente neutralizantes/uso terapéutico , Antígenos CD4/química , Epítopos/química , Epítopos/inmunología , Anticuerpos Anti-VIH/química , Anticuerpos Anti-VIH/genética , Anticuerpos Anti-VIH/uso terapéutico , Proteína gp120 de Envoltorio del VIH/metabolismo , Infecciones por VIH/prevención & control , Infecciones por VIH/terapia , Humanos , Modelos Moleculares , Mutación , Unión Proteica , Multimerización de Proteína , Subunidades de Proteína/químicaRESUMEN
Seasonal influenza vaccines confer protection against specific viral strains but have restricted breadth that limits their protective efficacy. The H1 and H3 subtypes of influenza A virus cause most of the seasonal epidemics observed in humans and are the major drivers of influenza A virus-associated mortality. The consequences of pandemic spread of COVID-19 underscore the public health importance of prospective vaccine development. Here, we show that headless hemagglutinin (HA) stabilized-stem immunogens presented on ferritin nanoparticles elicit broadly neutralizing antibody (bnAb) responses to diverse H1 and H3 viruses in nonhuman primates (NHPs) when delivered with a squalene-based oil-in-water emulsion adjuvant, AF03. The neutralization potency and breadth of antibodies isolated from NHPs were comparable to human bnAbs and extended to mismatched heterosubtypic influenza viruses. Although NHPs lack the immunoglobulin germline VH1-69 residues associated with the most prevalent human stem-directed bnAbs, other gene families compensated to generate bnAbs. Isolation and structural analyses of vaccine-induced bnAbs revealed extensive interaction with the fusion peptide on the HA stem, which is essential for viral entry. Antibodies elicited by these headless HA stabilized-stem vaccines neutralized diverse H1 and H3 influenza viruses and shared a mode of recognition analogous to human bnAbs, suggesting that these vaccines have the potential to confer broadly protective immunity against diverse viruses responsible for seasonal and pandemic influenza infections in humans.
Asunto(s)
Glicoproteínas Hemaglutininas del Virus de la Influenza/inmunología , Vacunas contra la Influenza/inmunología , Primates/inmunología , Animales , Anticuerpos Antivirales/biosíntesis , Anticuerpos Antivirales/química , Complejo Antígeno-Anticuerpo/química , Anticuerpos ampliamente neutralizantes/biosíntesis , Anticuerpos ampliamente neutralizantes/química , COVID-19 , Ferritinas/química , Ferritinas/inmunología , Glicoproteínas Hemaglutininas del Virus de la Influenza/química , Glicoproteínas Hemaglutininas del Virus de la Influenza/genética , Humanos , Vacunas contra la Influenza/administración & dosificación , Vacunas contra la Influenza/química , Gripe Humana/inmunología , Gripe Humana/virología , Macaca fascicularis , Modelos Moleculares , Nanopartículas/química , Pandemias , Primates/virología , Estructura Cuaternaria de Proteína , SARS-CoV-2 , Investigación Biomédica TraslacionalRESUMEN
Accurate predictive modeling of antibody-antigen complex structures and structure-based antibody design remain major challenges in computational biology, with implications for biotherapeutics, immunity, and vaccines. Through a systematic search for high-resolution structures of antibody-antigen complexes and unbound antibody and antigen structures, in conjunction with identification of experimentally determined binding affinities, we have assembled a non-redundant set of test cases for antibody-antigen docking and affinity prediction. This benchmark more than doubles the number of antibody-antigen complexes and corresponding affinities available in our previous benchmarks, providing an unprecedented view of the determinants of antibody recognition and insights into molecular flexibility. Initial assessments of docking and affinity prediction tools highlight the challenges posed by this diverse set of cases, which includes camelid nanobodies, therapeutic monoclonal antibodies, and broadly neutralizing antibodies targeting viral glycoproteins. This dataset will enable development of advanced predictive modeling and design methods for this therapeutically relevant class of protein-protein interactions.