RESUMO
We currently have an incomplete understanding of why only a fraction of human antibodies that bind to flaviviruses block infection of cells. Here we define the footprint of a strongly neutralizing human monoclonal antibody (mAb G9E) with Zika virus (ZIKV) by both X-ray crystallography and cryo-electron microscopy. Flavivirus envelope (E) glycoproteins are present as homodimers on the virion surface, and G9E bound to a quaternary structure epitope spanning both E protomers forming a homodimer. As G9E mainly neutralized ZIKV by blocking a step after viral attachment to cells, we tested if the neutralization mechanism of G9E was dependent on the mAb cross-linking E molecules and blocking low-pH triggered conformational changes required for viral membrane fusion. We introduced targeted mutations to the G9E paratope to create recombinant antibodies that bound to the ZIKV envelope without cross-linking E protomers. The G9E paratope mutants that bound to a restricted epitope on one protomer poorly neutralized ZIKV compared to the wild-type mAb, demonstrating that the neutralization mechanism depended on the ability of G9E to cross-link E proteins. In cell-free low pH triggered viral fusion assay, both wild-type G9E, and epitope restricted paratope mutant G9E bound to ZIKV but only the wild-type G9E blocked fusion. We propose that, beyond antibody binding strength, the ability of human antibodies to cross-link E-proteins is a critical determinant of flavivirus neutralization potency.
Assuntos
Infecção por Zika virus , Zika virus , Humanos , Zika virus/genética , Epitopos , Anticorpos Neutralizantes , Anticorpos Antivirais , Subunidades Proteicas , Microscopia Crioeletrônica , Proteínas do Envelope Viral/genética , Anticorpos MonoclonaisRESUMO
Host kinases play essential roles in the host cell cycle, innate immune signaling, the stress response to viral infection, and inflammation. Previous work has demonstrated that coronaviruses specifically target kinase cascades to subvert host cell responses to infection and rely upon host kinase activity to phosphorylate viral proteins to enhance replication. Given the number of kinase inhibitors that are already FDA approved to treat cancers, fibrosis, and other human disease, they represent an attractive class of compounds to repurpose for host-targeted therapies against emerging coronavirus infections. To further understand the host kinome response to betacoronavirus infection, we employed multiplex inhibitory bead mass spectrometry (MIB-MS) following MERS-CoV and SARS-CoV-2 infection of human lung epithelial cell lines. Our MIB-MS analyses revealed activation of mTOR and MAPK signaling following MERS-CoV and SARS-CoV-2 infection, respectively. SARS-CoV-2 host kinome responses were further characterized using paired phosphoproteomics, which identified activation of MAPK, PI3K, and mTOR signaling. Through chemogenomic screening, we found that clinically relevant PI3K/mTOR inhibitors were able to inhibit coronavirus replication at nanomolar concentrations similar to direct-acting antivirals. This study lays the groundwork for identifying broad-acting, host-targeted therapies to reduce betacoronavirus replication that can be rapidly repurposed during future outbreaks and epidemics. The proteomics, phosphoproteomics, and MIB-MS datasets generated in this study are available in the Proteomics Identification Database (PRIDE) repository under project identifiers PXD040897 and PXD040901.
Assuntos
COVID-19 , Hepatite C Crônica , Coronavírus da Síndrome Respiratória do Oriente Médio , Humanos , Antivirais/farmacologia , Inibidores de MTOR , Fosfatidilinositol 3-Quinases , SARS-CoV-2 , Replicação Viral , Coronavírus da Síndrome Respiratória do Oriente Médio/fisiologia , Serina-Treonina Quinases TORRESUMO
We describe the molecular-level composition of polyclonal immunoglobulin G (IgG) anti-spike antibodies from ancestral severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection, vaccination, or their combination ("hybrid immunity") at monoclonal resolution. Infection primarily triggers S2/N-terminal domain (NTD)-reactive antibodies, whereas vaccination mainly induces anti-receptor-binding domain (RBD) antibodies. This imprint persists after secondary exposures wherein >60% of ensuing hybrid immunity derives from the original IgG pool. Monoclonal constituents of the original IgG pool can increase breadth, affinity, and prevalence upon secondary exposures, as exemplified by the plasma antibody SC27. Following a breakthrough infection, vaccine-induced SC27 gained neutralization breadth and potency against SARS-CoV-2 variants and zoonotic viruses (half-maximal inhibitory concentration [IC50] â¼0.1-1.75 nM) and increased its binding affinity to the protective RBD class 1/4 epitope (dissociation constant [KD] < 5 pM). According to polyclonal escape analysis, SC27-like binding patterns are common in SARS-CoV-2 hybrid immunity. Our findings provide a detailed molecular definition of immunological imprinting and show that vaccination can produce class 1/4 (SC27-like) IgG antibodies circulating in the blood.
RESUMO
We used plasma IgG proteomics to study the molecular composition and temporal durability of polyclonal IgG antibodies triggered by ancestral SARS-CoV-2 infection, vaccination, or their combination ("hybrid immunity"). Infection, whether primary or post-vaccination, mainly triggered an anti-spike antibody response to the S2 domain, while vaccination predominantly induced anti-RBD antibodies. Immunological imprinting persisted after a secondary (hybrid) exposure, with >60% of the ensuing serological response originating from the initial antibodies generated during the first exposure. We highlight one instance where hybrid immunity arising from breakthrough infection resulted in a marked increase in the breadth and affinity of a highly abundant vaccination-elicited plasma IgG antibody, SC27. With an intrinsic binding affinity surpassing a theoretical maximum (K D < 5 pM), SC27 demonstrated potent neutralization of various SARS-CoV-2 variants and SARS-like zoonotic viruses (IC 50 â¼0.1-1.75 nM) and provided robust protection in vivo . Cryo-EM structural analysis unveiled that SC27 binds to the RBD class 1/4 epitope, with both VH and VL significantly contributing to the binding interface. These findings suggest that exceptionally broad and potent antibodies can be prevalent in plasma and can largely dictate the nature of serological neutralization. HIGHLIGHTS: ⪠Infection and vaccination elicit unique IgG antibody profiles at the molecular level⪠Immunological imprinting varies between infection (S2/NTD) and vaccination (RBD)⪠Hybrid immunity maintains the imprint of first infection or first vaccination⪠Hybrid immune IgG plasma mAbs have superior neutralization potency and breadth.
RESUMO
Whole virus-based inactivated SARS-CoV-2 vaccines adjuvanted with aluminum hydroxide have been critical to the COVID-19 pandemic response. Although these vaccines are protective against homologous coronavirus infection, the emergence of novel variants and the presence of large zoonotic reservoirs harboring novel heterologous coronaviruses provide significant opportunities for vaccine breakthrough, which raises the risk of adverse outcomes like vaccine-associated enhanced respiratory disease. Here, we use a female mouse model of coronavirus disease to evaluate inactivated vaccine performance against either homologous challenge with SARS-CoV-2 or heterologous challenge with a bat-derived coronavirus that represents a potential emerging disease threat. We show that inactivated SARS-CoV-2 vaccines adjuvanted with aluminum hydroxide can cause enhanced respiratory disease during heterologous infection, while use of an alternative adjuvant does not drive disease and promotes heterologous viral clearance. In this work, we highlight the impact of adjuvant selection on inactivated vaccine safety and efficacy against heterologous coronavirus infection.
Assuntos
Hidróxido de Alumínio , Vacinas contra COVID-19 , COVID-19 , SARS-CoV-2 , Vacinas de Produtos Inativados , Animais , Vacinas contra COVID-19/imunologia , Vacinas contra COVID-19/administração & dosagem , Feminino , COVID-19/prevenção & controle , COVID-19/imunologia , COVID-19/virologia , Camundongos , Vacinas de Produtos Inativados/imunologia , SARS-CoV-2/imunologia , Hidróxido de Alumínio/administração & dosagem , Modelos Animais de Doenças , Adjuvantes Imunológicos/administração & dosagem , Adjuvantes de Vacinas , Anticorpos Antivirais/imunologia , Camundongos Endogâmicos BALB C , Humanos , Coronavírus Relacionado à Síndrome Respiratória Aguda Grave/imunologiaRESUMO
Group 2B ß-coronaviruses (sarbecoviruses) have caused regional and global epidemics in modern history. Here, we evaluate the mechanisms of cross-sarbecovirus protective immunity, currently less clear yet important for pan-sarbecovirus vaccine development, using a panel of alphavirus-vectored vaccines covering bat to human strains. While vaccination does not prevent virus replication, it protects against lethal heterologous disease outcomes in both severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and clade 2 bat sarbecovirus challenge models. The spike vaccines tested primarily elicit a highly S1-specific homologous neutralizing antibody response with no detectable cross-virus neutralization. Rather, non-neutralizing antibody functions, mechanistically linked to FcgR4 and spike S2, mediate cross-protection in wild-type mice. Protection is lost in FcR knockout mice, further supporting a model for non-neutralizing, protective antibodies. These data highlight the importance of FcR-mediated cross-protective immune responses in universal pan-sarbecovirus vaccine designs.
Assuntos
Alphavirus , COVID-19 , Quirópteros , Coronavírus Relacionado à Síndrome Respiratória Aguda Grave , Vacinas Virais , Humanos , Animais , Camundongos , Anticorpos Antivirais , SARS-CoV-2 , COVID-19/prevenção & controle , Anticorpos Neutralizantes , VacinaçãoRESUMO
Two group 2B ß-coronaviruses (sarbecoviruses) have caused regional and global epidemics in modern history. The mechanisms of cross protection driven by the sarbecovirus spike, a dominant immunogen, are less clear yet critically important for pan-sarbecovirus vaccine development. We evaluated the mechanisms of cross-sarbecovirus protective immunity using a panel of alphavirus-vectored vaccines covering bat to human strains. While vaccination did not prevent virus replication, it protected against lethal heterologous disease outcomes in both SARS-CoV-2 and clade 2 bat sarbecovirus HKU3-SRBD challenge models. The spike vaccines tested primarily elicited a highly S1-specific homologous neutralizing antibody response with no detectable cross-virus neutralization. We found non-neutralizing antibody functions that mediated cross protection in wild-type mice were mechanistically linked to FcgR4 and spike S2-binding antibodies. Protection was lost in FcR knockout mice, further supporting a model for non-neutralizing, protective antibodies. These data highlight the importance of FcR-mediated cross-protective immune responses in universal pan-sarbecovirus vaccine designs.