RESUMO
Alphaviruses cause severe arthritogenic or encephalitic disease. The E1 structural glycoprotein is highly conserved in these viruses and mediates viral fusion with host cells. However, the role of antibody responses to the E1 protein in immunity is poorly understood. We isolated E1-specific human monoclonal antibodies (mAbs) with diverse patterns of recognition for alphaviruses (ranging from Eastern equine encephalitis virus [EEEV]-specific to alphavirus cross-reactive) from survivors of natural EEEV infection. Antibody binding patterns and epitope mapping experiments identified differences in E1 reactivity based on exposure of epitopes on the glycoprotein through pH-dependent mechanisms or presentation on the cell surface prior to virus egress. Therapeutic efficacy in vivo of these mAbs corresponded with potency of virus egress inhibition in vitro and did not require Fc-mediated effector functions for treatment against subcutaneous EEEV challenge. These studies reveal the molecular basis for broad and protective antibody responses to alphavirus E1 proteins.
Assuntos
Alphavirus/imunologia , Anticorpos Antivirais/imunologia , Reações Cruzadas/imunologia , Proteínas Virais/imunologia , Liberação de Vírus/fisiologia , Animais , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/isolamento & purificação , Anticorpos Neutralizantes/imunologia , Antígenos Virais/imunologia , Linhagem Celular , Vírus Chikungunya/imunologia , Vírus da Encefalite Equina do Leste/imunologia , Encefalomielite Equina/imunologia , Encefalomielite Equina/virologia , Mapeamento de Epitopos , Feminino , Cavalos , Humanos , Concentração de Íons de Hidrogênio , Articulações/patologia , Masculino , Camundongos Endogâmicos C57BL , Modelos Biológicos , Ligação Proteica , RNA Viral/metabolismo , Receptores Fc/metabolismo , Temperatura , Vírion/metabolismo , Internalização do VírusRESUMO
Eastern equine encephalitis virus (EEEV) is one of the most virulent viruses endemic to North America. No licensed vaccines or antiviral therapeutics are available to combat this infection, which has recently shown an increase in human cases. Here, we characterize human monoclonal antibodies (mAbs) isolated from a survivor of natural EEEV infection with potent (<20 pM) inhibitory activity of EEEV. Cryo-electron microscopy reconstructions of two highly neutralizing mAbs, EEEV-33 and EEEV-143, were solved in complex with chimeric Sindbis/EEEV virions to 7.2 Å and 8.3 Å, respectively. The mAbs recognize two distinct antigenic sites that are critical for inhibiting viral entry into cells. EEEV-33 and EEEV-143 protect against disease following stringent lethal aerosol challenge of mice with highly pathogenic EEEV. These studies provide insight into the molecular basis for the neutralizing human antibody response against EEEV and can facilitate development of vaccines and candidate antibody therapeutics.
Assuntos
Aerossóis/administração & dosagem , Anticorpos Monoclonais/imunologia , Anticorpos Antivirais/imunologia , Vírus da Encefalite Equina do Leste/imunologia , Encefalomielite Equina/imunologia , Encefalomielite Equina/prevenção & controle , Adulto , Animais , Anticorpos Monoclonais/isolamento & purificação , Anticorpos Neutralizantes/imunologia , Antígenos Virais/imunologia , Microscopia Crioeletrônica , Modelos Animais de Doenças , Vírus da Encefalite Equina do Leste/ultraestrutura , Encefalomielite Equina/virologia , Epitopos/química , Feminino , Glicoproteínas/imunologia , Humanos , Camundongos , Modelos Moleculares , Mutagênese/genética , Testes de Neutralização , Ligação Proteica , Domínios Proteicos , Proteínas Recombinantes/imunologia , Sindbis virus/imunologia , Vírion/imunologia , Vírion/ultraestrutura , Internalização do VírusRESUMO
Hendra (HeV) and Nipah (NiV) viruses are emerging zoonotic pathogens in the Henipavirus genus causing outbreaks of disease with very high case fatality rates. Here, we report the first naturally occurring human monoclonal antibodies (mAbs) against HeV receptor binding protein (RBP). All isolated mAbs neutralized HeV, and some also neutralized NiV. Epitope binning experiments identified five major antigenic sites on HeV-RBP. Animal studies demonstrated that the most potent cross-reactive neutralizing mAbs, HENV-26 and HENV-32, protected ferrets in lethal models of infection with NiV Bangladesh 3 days after exposure. We solved the crystal structures of mAb HENV-26 in complex with both HeV-RBP and NiV-RBP and of mAb HENV-32 in complex with HeV-RBP. The studies reveal diverse sites of vulnerability on RBP recognized by potent human mAbs that inhibit virus by multiple mechanisms. These studies identify promising prophylactic antibodies and define protective epitopes that can be used in rational vaccine design.
Assuntos
Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/imunologia , Vírus Hendra/imunologia , Henipavirus/imunologia , Testes de Neutralização , Vírus Nipah/imunologia , Receptores Virais/imunologia , Sequência de Aminoácidos , Animais , Anticorpos Monoclonais/química , Anticorpos Monoclonais/isolamento & purificação , Antígenos Virais/imunologia , Sítios de Ligação , Ligação Competitiva , Encéfalo/patologia , Quirópteros/virologia , Reações Cruzadas/imunologia , Cristalografia por Raios X , Efrina-B2/metabolismo , Feminino , Furões/virologia , Humanos , Interferometria , Fígado/patologia , Modelos Moleculares , Ligação Proteica , Conformação Proteica , Domínios Proteicos , Receptores Virais/química , Receptores Virais/metabolismoRESUMO
Here, we describe the discovery of a naturally occurring human antibody (Ab), FluA-20, that recognizes a new site of vulnerability on the hemagglutinin (HA) head domain and reacts with most influenza A viruses. Structural characterization of FluA-20 with H1 and H3 head domains revealed a novel epitope in the HA trimer interface, suggesting previously unrecognized dynamic features of the trimeric HA protein. The critical HA residues recognized by FluA-20 remain conserved across most subtypes of influenza A viruses, which explains the Ab's extraordinary breadth. The Ab rapidly disrupted the integrity of HA protein trimers, inhibited cell-to-cell spread of virus in culture, and protected mice against challenge with viruses of H1N1, H3N2, H5N1, or H7N9 subtypes when used as prophylaxis or therapy. The FluA-20 Ab has uncovered an exceedingly conserved protective determinant in the influenza HA head domain trimer interface that is an unexpected new target for anti-influenza therapeutics and vaccines.
Assuntos
Anticorpos Monoclonais Murinos/imunologia , Anticorpos Antivirais/imunologia , Epitopos/imunologia , Glicoproteínas de Hemaglutininação de Vírus da Influenza/imunologia , Vírus da Influenza A/imunologia , Vacinas contra Influenza/imunologia , Infecções por Orthomyxoviridae , Animais , Cães , Células Madin Darby de Rim Canino , Camundongos , Infecções por Orthomyxoviridae/imunologia , Infecções por Orthomyxoviridae/patologia , Infecções por Orthomyxoviridae/prevenção & controleRESUMO
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.
Assuntos
Anticorpos Monoclonais , Anticorpos Antivirais , Vírus da Influenza B , Influenza Humana , Neuraminidase , Neuraminidase/imunologia , Humanos , Vírus da Influenza B/imunologia , Animais , Anticorpos Monoclonais/imunologia , Anticorpos Antivirais/imunologia , Influenza Humana/imunologia , Influenza Humana/prevenção & controle , Vacinas contra Influenza/imunologia , Camundongos , Infecções por Orthomyxoviridae/imunologia , Infecções por Orthomyxoviridae/prevenção & controle , Proteínas Virais/imunologia , Replicação Viral/efeitos dos fármacosRESUMO
Monkeypox (MPXV) and cowpox (CPXV) are emerging agents that cause severe human infections on an intermittent basis, and variola virus (VARV) has potential for use as an agent of bioterror. Vaccinia immune globulin (VIG) has been used therapeutically to treat severe orthopoxvirus infections but is in short supply. We generated a large panel of orthopoxvirus-specific human monoclonal antibodies (Abs) from immune subjects to investigate the molecular basis of broadly neutralizing antibody responses for diverse orthopoxviruses. Detailed analysis revealed the principal neutralizing antibody specificities that are cross-reactive for VACV, CPXV, MPXV, and VARV and that are determinants of protection in murine challenge models. Optimal protection following respiratory or systemic infection required a mixture of Abs that targeted several membrane proteins, including proteins on enveloped and mature virion forms of virus. This work reveals orthopoxvirus targets for human Abs that mediate cross-protective immunity and identifies new candidate Ab therapeutic mixtures to replace VIG.
Assuntos
Anticorpos Monoclonais/imunologia , Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/imunologia , Especificidade de Anticorpos , Infecções por Poxviridae/imunologia , Varíola Bovina/imunologia , Vírus da Varíola Bovina/imunologia , Reações Cruzadas , Humanos , Leucócitos Mononucleares/imunologia , Mpox/imunologia , Monkeypox virus/imunologia , Varíola/imunologia , Vacínia/imunologia , Vaccinia virus/imunologia , Vírus da Varíola/imunologiaRESUMO
Recent studies have suggested that antibody-mediated protection against the Ebolaviruses may be achievable, but little is known about whether or not antibodies can confer cross-reactive protection against viruses belonging to diverse Ebolavirus species, such as Ebola virus (EBOV), Sudan virus (SUDV), and Bundibugyo virus (BDBV). We isolated a large panel of human monoclonal antibodies (mAbs) against BDBV glycoprotein (GP) using peripheral blood B cells from survivors of the 2007 BDBV outbreak in Uganda. We determined that a large proportion of mAbs with potent neutralizing activity against BDBV bind to the glycan cap and recognize diverse epitopes within this major antigenic site. We identified several glycan cap-specific mAbs that neutralized multiple ebolaviruses, including SUDV, and a cross-reactive mAb that completely protected guinea pigs from the lethal challenge with heterologous EBOV. Our results provide a roadmap to develop a single antibody-based treatment effective against multiple Ebolavirus infections.
Assuntos
Anticorpos Monoclonais/imunologia , Anticorpos Neutralizantes/imunologia , Ebolavirus/imunologia , Doença pelo Vírus Ebola/imunologia , Sobreviventes , Animais , Reações Cruzadas , Modelos Animais de Doenças , Mapeamento de Epitopos , Cobaias , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Microscopia Eletrônica , Modelos Moleculares , Mutagênese , UgandaRESUMO
Ebolaviruses cause severe disease in humans, and identification of monoclonal antibodies (mAbs) that are effective against multiple ebolaviruses are important for therapeutics development. Here we describe a distinct class of broadly neutralizing human mAbs with protective capacity against three ebolaviruses infectious for humans: Ebola (EBOV), Sudan (SUDV), and Bundibugyo (BDBV) viruses. We isolated mAbs from human survivors of ebolavirus disease and identified a potent mAb, EBOV-520, which bound to an epitope in the glycoprotein (GP) base region. EBOV-520 efficiently neutralized EBOV, BDBV, and SUDV and also showed protective capacity in relevant animal models of these infections. EBOV-520 mediated protection principally by direct virus neutralization and exhibited multifunctional properties. This study identified a potent naturally occurring mAb and defined key features of the human antibody response that may contribute to broad protection. This multifunctional mAb and related clones are promising candidates for development as broadly protective pan-ebolavirus therapeutic molecules.
Assuntos
Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/farmacologia , Anticorpos Neutralizantes/imunologia , Anticorpos Neutralizantes/farmacologia , Anticorpos Antivirais/imunologia , Anticorpos Antivirais/farmacologia , Ebolavirus/imunologia , Glicoproteínas/imunologia , Doença pelo Vírus Ebola/imunologia , Células 3T3 , Adulto , Animais , Células CHO , Linhagem Celular , Chlorocebus aethiops , Cricetulus , Modelos Animais de Doenças , Drosophila , Feminino , Furões , Cobaias , Doença pelo Vírus Ebola/prevenção & controle , Doença pelo Vírus Ebola/virologia , Humanos , Imunoglobulina G/imunologia , Células Jurkat , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Knockout , Células THP-1 , Células VeroRESUMO
The human genome contains approximately 20 thousand protein-coding genes1, but the size of the collection of antigen receptors of the adaptive immune system that is generated by the recombination of gene segments with non-templated junctional additions (on B cells) is unknown-although it is certainly orders of magnitude larger. It has not been established whether individuals possess unique (or private) repertoires or substantial components of shared (or public) repertoires. Here we sequence recombined and expressed B cell receptor genes in several individuals to determine the size of their B cell receptor repertoires, and the extent to which these are shared between individuals. Our experiments revealed that the circulating repertoire of each individual contained between 9 and 17 million B cell clonotypes. The three individuals that we studied shared many clonotypes, including between 1 and 6% of B cell heavy-chain clonotypes shared between two subjects (0.3% of clonotypes shared by all three) and 20 to 34% of λ or κ light chains shared between two subjects (16 or 22% of λ or κ light chains, respectively, were shared by all three). Some of the B cell clonotypes had thousands of clones, or somatic variants, within the clonotype lineage. Although some of these shared lineages might be driven by exposure to common antigens, previous exposure to foreign antigens was not the only force that shaped the shared repertoires, as we also identified shared clonotypes in umbilical cord blood samples and all adult repertoires. The unexpectedly high prevalence of shared clonotypes in B cell repertoires, and identification of the sequences of these shared clonotypes, should enable better understanding of the role of B cell immune repertoires in health and disease.
Assuntos
Anticorpos/genética , Anticorpos/imunologia , Linfócitos B/imunologia , Células Clonais/imunologia , Receptores de Antígenos de Linfócitos B/imunologia , Adulto , Sequência de Aminoácidos , Anticorpos/química , Antígenos/imunologia , Linfócitos B/citologia , Linfócitos B/metabolismo , Sequência de Bases , Células Clonais/citologia , Células Clonais/metabolismo , Feminino , Sangue Fetal/citologia , Sangue Fetal/imunologia , Voluntários Saudáveis , Humanos , Recém-Nascido , Masculino , Receptores de Antígenos de Linfócitos B/química , Receptores de Antígenos de Linfócitos B/genética , Análise de Sequência de DNARESUMO
Rift Valley fever virus (RVFV), an emerging arboviral and zoonotic bunyavirus, causes severe disease in livestock and humans. Here, we report the isolation of a panel of monoclonal antibodies (mAbs) from the B cells of immune individuals following natural infection in Kenya or immunization with MP-12 vaccine. The B cell responses of individuals who were vaccinated or naturally infected recognized similar epitopes on both Gc and Gn proteins. The Gn-specific mAbs and two mAbs that do not recognize either monomeric Gc or Gn alone but recognized the hetero-oligomer glycoprotein complex (Gc+Gn) when Gc and Gn were coexpressed exhibited potent neutralizing activities in vitro, while Gc-specific mAbs exhibited relatively lower neutralizing capacity. The two Gc+Gn-specific mAbs and the Gn domain A-specific mAbs inhibited RVFV fusion to cells, suggesting that mAbs can inhibit the exposure of the fusion loop in Gc, a class II fusion protein, and thus prevent fusion by an indirect mechanism without direct fusion loop contact. Competition-binding analysis with coexpressed Gc/Gn and mutagenesis library screening indicated that these mAbs recognize four major antigenic sites, with two sites of vulnerability for neutralization on Gn. In experimental models of infection in mice, representative mAbs recognizing three of the antigenic sites reduced morbidity and mortality when used at a low dose in both prophylactic and therapeutic settings. This study identifies multiple candidate mAbs that may be suitable for use in humans against RVFV infection and highlights fusion inhibition against bunyaviruses as a potential contributor to potent antibody-mediated neutralization.
Assuntos
Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/imunologia , Vírus da Febre do Vale do Rift/imunologia , Proteínas Virais de Fusão/imunologia , Animais , Anticorpos Monoclonais/imunologia , Linhagem Celular Tumoral , Células Cultivadas , Chlorocebus aethiops , Epitopos/química , Epitopos/imunologia , Células HEK293 , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Células Vero , Proteínas Virais de Fusão/químicaRESUMO
Escherichia coli is one of the most frequent human pathogens, increasingly exhibits antimicrobial resistance, and has complex interactions with the host immune system. E. coli exposure or infection can result in the generation of antibodies specific for outer membrane protein A (OmpA), a multifunctional porin. We identified four OmpA-specific naturally occurring antibodies from healthy human donor B cells and assessed their interactions with E. coli and OmpA. These antibodies are highly specific for OmpA, exhibiting no cross-reactivity to a strain lacking ompA and retaining binding to both laboratory and clinical isolates of E. coli in enzyme-linked immunosorbent assay (ELISA) and immunofluorescence assays. One monoclonal antibody (Mab), designated ECOL-11, is specific for the extracellular N-terminal porin domain of OmpA and induces growth phase-specific bacterial aggregation. This aggregation is not induced by the fragment antigen binding (Fab) form of the MAb, suggesting the importance of bivalency for this aggregating activity. ECOL-11 decreases adhesion and phagocytosis of E. coli by RAW 264.7 macrophage-like cells, possibly by inhibiting the adhesion functions of OmpA. Despite this in vitro phenotype, organ E. coli burdens were not altered by antibody prophylaxis in a murine model of lethal E. coli septic shock. Our findings support the importance of OmpA at the host-pathogen interface and begin to explore the implications and utility of E. coli-specific antibodies in human hosts.
Assuntos
Infecções por Escherichia coli , Sepse , Animais , Anticorpos Antibacterianos/metabolismo , Anticorpos Monoclonais , Proteínas da Membrana Bacteriana Externa/genética , Escherichia coli/genética , Humanos , Camundongos , Porinas/metabolismoRESUMO
Ross River fever is a mosquito-transmitted viral disease that is endemic to Australia and the surrounding Pacific Islands. Ross River virus (RRV) belongs to the arthritogenic group of alphaviruses, which largely cause disease characterized by debilitating polyarthritis, rash, and fever. There is no specific treatment or licensed vaccine available, and the mechanisms of protective humoral immunity in humans are poorly understood. Here, we describe naturally occurring human mAbs specific to RRV, isolated from subjects with a prior natural infection. These mAbs potently neutralize RRV infectivity in cell culture and block infection through multiple mechanisms, including prevention of viral attachment, entry, and fusion. Some of the most potently neutralizing mAbs inhibited binding of RRV to Mxra8, a recently discovered alpahvirus receptor. Epitope mapping studies identified the A and B domains of the RRV E2 protein as the major antigenic sites for the human neutralizing antibody response. In experiments in mice, these mAbs were protective against cinical disease and reduced viral burden in multiple tissues, suggesting a potential therapeutic use for humans.
Assuntos
Infecções por Alphavirus/prevenção & controle , Anticorpos Monoclonais , Anticorpos Neutralizantes , Anticorpos Antivirais , Proteínas do Capsídeo/imunologia , Epitopos/imunologia , Ross River virus/imunologia , Proteínas do Envelope Viral/imunologia , Infecções por Alphavirus/imunologia , Infecções por Alphavirus/patologia , Animais , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/farmacologia , Anticorpos Neutralizantes/imunologia , Anticorpos Neutralizantes/farmacologia , Anticorpos Antivirais/imunologia , Anticorpos Antivirais/farmacologia , Chlorocebus aethiops , Feminino , Humanos , Camundongos , Pessoa de Meia-Idade , Células VeroRESUMO
Zika virus (ZIKV) is an emerging mosquito-transmitted flavivirus that can cause severe disease, including congenital birth defects during pregnancy. To develop candidate therapeutic agents against ZIKV, we isolated a panel of human monoclonal antibodies from subjects that were previously infected with ZIKV. We show that a subset of antibodies recognize diverse epitopes on the envelope (E) protein and exhibit potent neutralizing activity. One of the most inhibitory antibodies, ZIKV-117, broadly neutralized infection of ZIKV strains corresponding to African and Asian-American lineages. Epitope mapping studies revealed that ZIKV-117 recognized a unique quaternary epitope on the E protein dimer-dimer interface. We evaluated the therapeutic efficacy of ZIKV-117 in pregnant and non-pregnant mice. Monoclonal antibody treatment markedly reduced tissue pathology, placental and fetal infection, and mortality in mice. Thus, neutralizing human antibodies can protect against maternal-fetal transmission, infection and disease, and reveal important determinants for structure-based rational vaccine design efforts.
Assuntos
Anticorpos Neutralizantes/imunologia , Doenças Fetais/prevenção & controle , Transmissão Vertical de Doenças Infecciosas/prevenção & controle , Replicação Viral/imunologia , Infecção por Zika virus/imunologia , Infecção por Zika virus/virologia , Zika virus/crescimento & desenvolvimento , Zika virus/imunologia , África , América , Animais , Anticorpos Monoclonais/química , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/uso terapêutico , Anticorpos Neutralizantes/química , Anticorpos Neutralizantes/uso terapêutico , Anticorpos Antivirais/química , Anticorpos Antivirais/imunologia , Anticorpos Antivirais/uso terapêutico , Especificidade de Anticorpos , Ásia , Linfócitos B/imunologia , Modelos Animais de Doenças , Mapeamento de Epitopos , Feminino , Doenças Fetais/imunologia , Doenças Fetais/virologia , Feto/imunologia , Feto/virologia , Humanos , Masculino , Camundongos , Modelos Moleculares , Placenta/imunologia , Placenta/virologia , Gravidez , Multimerização Proteica , Taxa de Sobrevida , Proteínas Virais/química , Proteínas Virais/imunologia , Vacinas Virais/química , Vacinas Virais/imunologia , Infecção por Zika virus/patologiaRESUMO
The human B cell response to natural filovirus infections early after recovery is poorly understood. Previous serologic studies suggest that some Ebola virus survivors exhibit delayed antibody responses with low magnitude and quality. Here, we sought to study the population of individual memory B cells induced early in convalescence. We isolated monoclonal antibodies (MAbs) from memory B cells from four survivors treated for Ebola virus disease (EVD) 1 or 3 months after discharge from the hospital. At the early time points postrecovery, the frequency of Ebola-specific B cells was low and dominated by clones that were cross-reactive with both Ebola glycoprotein (GP) and with the secreted GP (sGP) form. Of 25 MAbs isolated from four donors, only one exhibited neutralization activity. This neutralizing MAb, designated MAb EBOV237, recognizes an epitope in the glycan cap of the surface glycoprotein. In vivo murine lethal challenge studies showed that EBOV237 conferred protection when given prophylactically at a level similar to that of the ZMapp component MAb 13C6. The results suggest that the human B cell response to EVD 1 to 3 months postdischarge is characterized by a paucity of broad or potent neutralizing clones. However, the neutralizing epitope in the glycan cap recognized by EBOV237 may play a role in the early human antibody response to EVD and should be considered in rational design strategies for new Ebola virus vaccine candidates.IMPORTANCE The pathogenesis of Ebola virus disease (EVD) in humans is complex, and the mechanisms contributing to immunity are poorly understood. In particular, it appears that the quality and magnitude of the human B cell response early after recovery from EVD may be reduced compared to most viral infections. Here, we isolated human monoclonal antibodies from B cells of four survivors of EVD at 1 or 3 months after hospital discharge. Ebola-specific memory B cells early in convalescence were low in frequency, and the antibodies they encoded demonstrated poor neutralizing potencies. One neutralizing antibody that protected mice from lethal infection, EBOV237, was identified in the panel of 25 human antibodies isolated. Recognition of the glycan cap epitope recognized by EBOV237 suggests that this antigenic site should be considered in vaccine design and treatment strategies for EVD.
Assuntos
Anticorpos Monoclonais/imunologia , Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/imunologia , Linfócitos B/imunologia , Ebolavirus/imunologia , Doença pelo Vírus Ebola/imunologia , Memória Imunológica , Sobreviventes , Proteínas do Envelope Viral/imunologia , Feminino , Humanos , Masculino , Estados UnidosRESUMO
The nutrient metal iron plays a key role in the survival of microorganisms. The iron-regulated surface determinant (Isd) system scavenges heme-iron from the human host, enabling acquisition of iron in iron-deplete conditions in Staphylococcus aureus during infection. The cell surface receptors IsdB and IsdH bind hemoproteins and transfer heme to IsdA, the final surface protein before heme-iron is transported through the peptidoglycan. To define the human B-cell response to IsdA, we isolated human monoclonal antibodies (mAbs) specific to the surface Isd proteins and determined their mechanism of action. We describe the first isolation of fully human IsdA and IsdH mAbs, as well as cross-reactive Isd mAbs. Two of the identified IsdA mAbs worked in a murine septic model of infection to reduce bacterial burden during staphylococcal infection. Their protection was a result of both heme-blocking and Fc-mediated effector functions, underscoring the importance of targeting S. aureus using diverse mechanisms.
Assuntos
Anticorpos Monoclonais/imunologia , Antígenos de Bactérias/imunologia , Hemeproteínas/imunologia , Infecções Estafilocócicas/imunologia , Staphylococcus aureus/imunologia , Animais , Proteínas de Bactérias , Modelos Animais de Doenças , Feminino , Humanos , Espectrometria de Massa com Troca Hidrogênio-Deutério , Camundongos , Camundongos Endogâmicos BALB C , Receptores de Superfície Celular/imunologiaRESUMO
Antibodies are essential for immunity against Ehrlichia chaffeensis, and protective mechanisms involve blocking of ehrlichial attachment or complement and Fcγ-receptor-dependent destruction. In this study, we determined that major outer membrane protein 1 (OMP-19) hypervariable region 1 (HVR1)-specific human monoclonal antibodies (huMAbs) are protective through conventional extracellular neutralization and, more significantly, through a novel intracellular TRIM21-mediated mechanism. Addition of OMP-1-specific huMAb EHRL-15 (IgG1) prevented infection by blocking attachment/entry, a mechanism previously reported; conversely, OMP-1-specific huMAb EHRL-4 (IgG3) engaged intracellular TRIM21 and initiated an immediate innate immune response and rapid intracellular degradation of ehrlichiae. EHRL-4-TRIM21-mediated inhibition was significantly impaired in TRIM21 knockout THP-1 cells. EHRL-4 interacted with cytosolic Fc receptor TRIM21, observed by confocal microscopy and confirmed by co-immunoprecipitation. E. chaffeensis-EHRL-4-TRIM21 complexes caused significant upregulation of proinflammatory cytokine/chemokine transcripts and resulted in rapid (<30 min) nuclear accumulation of NF-κB and TRIM21 and ehrlichial destruction. We investigated the role of TRIM21 in the autophagic clearance of ehrlichiae in the presence of EHRL-4. Colocalization between EHRL-4-opsonized ehrlichiae, polyubiquitinated TRIM21, autophagy regulators (ULK1 and beclin 1) and effectors (LC3 and p62), and lysosome-associated membrane protein 2 (LAMP2) was observed. Moreover, autophagic flux defined by conversion of LC3I to LC3II and accumulation and degradation of p62 was detected, and EHRL-4-mediated degradation of E. chaffeensis was abrogated by the autophagy inhibitor 3-methyladenine. Our results demonstrate that huMAbs are capable of inhibiting E. chaffeensis infection by distinct effector mechanisms: extracellularly by neutralization and intracellularly by engaging TRIM21, which mediates a rapid innate immune response that mobilizes the core autophagy components, triggering localized selective autophagic degradation of ehrlichiae.
Assuntos
Antígenos de Bactérias/imunologia , Proteínas da Membrana Bacteriana Externa/genética , Proteínas da Membrana Bacteriana Externa/imunologia , Ehrlichia chaffeensis/imunologia , Ribonucleoproteínas/genética , Adenina/análogos & derivados , Adenina/farmacologia , Anticorpos Monoclonais/imunologia , Anticorpos Neutralizantes/imunologia , Antígenos de Bactérias/genética , Autofagia/imunologia , Aderência Bacteriana/genética , Sistemas CRISPR-Cas , Linhagem Celular Tumoral , Ehrlichia chaffeensis/genética , Técnicas de Inativação de Genes , Humanos , Imunidade Humoral/imunologia , NF-kappa B/genética , Células THP-1RESUMO
BACKGROUND & AIMS: Human noroviruses are responsible for approximately 200,000 deaths worldwide each year. In 2012, the GII.4 Sydney strain emerged and became the major circulating norovirus strain associated with human disease. Our understanding of the human norovirus-specific antibody response is limited because few human monoclonal antibodies (mAbs) to noroviruses have been described, and there are no functional assays to measure virus neutralization. We studied the antibody-mediated response to the genogroup (G) II.4 strain by isolating mAbs to GII.4 from infected patients and developing virus neutralization assays. METHODS: We used a robust human hybridoma technique to isolate mAbs from patients previously infected with norovirus and identified mAbs that blocked virus binding to cell receptors, using virus-like particles to test blockade ability. We tested the ability of select mAbs to neutralize live human noroviruses using stem cell-derived human enteroids. RESULTS: We isolated a panel of 25 IgG or IgA human mAbs that recognized norovirus GII.4 Sydney 2012 and determined their potential to block virus binding to cell receptors. In competition binding studies, most antibodies recognized 3 major antigenic sites on the GII.4 Sydney 2012 protruding (P) domain. CONCLUSIONS: We isolated and characterized human mAbs that neutralize live human norovirus GII.4 Sydney 2012-the predominant strain responsible for recent outbreaks. Analyses of these antibodies identified neutralizing epitopes; further studies will provide insight into the human immune response to this deadly virus.
Assuntos
Anticorpos Monoclonais/imunologia , Anticorpos Antivirais/imunologia , Infecções por Caliciviridae/virologia , Norovirus/imunologia , Anticorpos Monoclonais/isolamento & purificação , Anticorpos Antivirais/isolamento & purificação , Humanos , Ligação ViralRESUMO
The four dengue virus (DENV) serotypes are mosquito-borne flaviviruses responsible for dengue fever and dengue hemorrhagic fever. People exposed to DENV develop antibodies (Abs) that strongly neutralize the serotype responsible for infection. Historically, infection with DENV serotype 4 (DENV4) has been less common and less studied than infections with the other three serotypes. However, DENV4 has been responsible for recent large and sustained epidemics in Asia and Latin America. The neutralizing antibody responses and the epitopes targeted against DENV4 have not been characterized in human infection. In this study, we mapped and characterized epitopes on DENV4 recognized by neutralizing antibodies in people previously exposed to DENV4 infections or to a live attenuated DENV4 vaccine. To study the fine specificity of DENV4 neutralizing human antibodies, B cells from two people exposed to DENV4 were immortalized and screened to identify DENV-specific clones. Two human monoclonal antibodies (MAbs) that neutralized DENV4 were isolated, and their epitopes were finely mapped using recombinant viruses and alanine scan mutation array techniques. Both antibodies bound to quaternary structure epitopes near the hinge region between envelope protein domain I (EDI) and EDII. In parallel, to characterize the serum neutralizing antibody responses, convalescence-phase serum samples from people previously exposed to primary DENV4 natural infections or a monovalent DENV4 vaccine were analyzed. Natural infection and vaccination also induced serum-neutralizing antibodies that targeted similar epitope domains at the EDI/II hinge region. These studies defined a target of neutralizing antigenic site on DENV4 targeted by human antibodies following natural infection or vaccination.IMPORTANCE The four serotypes of dengue virus are the causative agents of dengue fever and dengue hemorrhagic fever. People exposed to primary DENV infections develop long-term neutralizing antibody responses, but these principally recognize only the infecting serotype. An effective vaccine against dengue should elicit long-lasting protective antibody responses to all four serotypes simultaneously. We and others have defined antigenic sites on the envelope (E) protein of viruses of dengue virus serotypes 1, 2, and 3 targeted by human neutralizing antibodies. The epitopes on DENV4 E protein targeted by the human neutralizing antibodies and the mechanisms of serotype 4 neutralization are poorly understood. Here, we report the properties of human antibodies that neutralize dengue virus serotype 4. People exposed to serotype 4 infections or a live attenuated serotype 4 vaccine developed neutralizing antibodies that bound to similar sites on the viral E protein. These studies have provided a foundation for developing and evaluating DENV4 vaccines.
Assuntos
Anticorpos Neutralizantes/sangue , Anticorpos Antivirais/sangue , Linfócitos B/imunologia , Vírus da Dengue/imunologia , Dengue/prevenção & controle , Imunidade Adaptativa , Aedes , Animais , Anticorpos Antivirais/efeitos dos fármacos , Linhagem Celular , Dengue/imunologia , Dengue/virologia , Mapeamento de Epitopos , Humanos , Memória Imunológica , Ligação Proteica , Domínios Proteicos , Vacinação , Vacinas Atenuadas/imunologia , Vacinas Virais/imunologiaRESUMO
The 2-component leukotoxin LukAB is critical for Staphylococcus aureus targeting and killing of human neutrophils ex vivo and is produced in the setting of human infection. We report 3 LukAB-specific human monoclonal antibodies (mAbs) with distinct mechanisms of toxin neutralization and in vivo efficacy. Three hybridomas secreting mAbs with anti-LukAB activity (designated SA-13, -15, and -17) were generated from B cells obtained from a 12-year-old boy with S. aureus osteomyelitis. Each of the 3 mAbs neutralized LukAB-mediated neutrophil toxicity, exhibited differing levels of potency, recognized different antigenic sites on the toxin, and displayed at least 2 distinct mechanisms for cytotoxic inhibition. SA-15 bound exclusively to the dimeric form of the toxin, suggesting that human B cells recognize epitopes on the dimerized form of LukAB during natural infection. Both SA-13 and SA-17 bound the LukA monomer and the LukAB dimer. Although all 3 mAbs potently neutralized cytotoxicity, only SA-15 and SA-17 significantly inhibited toxin association with the cell surface. Treatment with a 1:1 mixture of mAbs SA-15 and SA-17 resulted in significantly lower bacterial colony counts in heart, liver, and kidneys in a murine model of S. aureus sepsis. These data describe the isolation of diverse and efficacious antitoxin mAbs.
Assuntos
Anticorpos Antibacterianos/sangue , Anticorpos Monoclonais/sangue , Proteínas de Bactérias/imunologia , Leucocidinas/imunologia , Neutrófilos/imunologia , Infecções Estafilocócicas/microbiologia , Animais , Linfócitos B/imunologia , Criança , Feminino , Humanos , Hibridomas , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Análise de Regressão , Staphylococcus aureusRESUMO
UNLABELLED: The proposed antibody-dependent enhancement (ADE) mechanism for severe dengue virus (DENV) disease suggests that non-neutralizing serotype cross-reactive antibodies generated during a primary infection facilitate entry into Fc receptor bearing cells during secondary infection, resulting in enhanced viral replication and severe disease. One group of cross-reactive antibodies that contributes considerably to this serum profile target the premembrane (prM) protein. We report here the isolation of a large panel of naturally occurring human monoclonal antibodies (MAbs) obtained from subjects following primary DENV serotype 1, 2, or 3 or secondary natural DENV infections or following primary DENV serotype 1 live attenuated virus vaccination to determine the antigenic landscape on the prM protein that is recognized by human antibodies. We isolated 25 prM-reactive human MAbs, encoded by diverse antibody-variable genes. Competition-binding studies revealed that all of the antibodies bound to a single major antigenic site on prM. Alanine scanning-based shotgun mutagenesis epitope mapping studies revealed diverse patterns of fine specificity of various clones, suggesting that different antibodies use varied binding poses to recognize several overlapping epitopes within the immunodominant site. Several of the antibodies interacted with epitopes on both prM and E protein residues. Despite the diverse genetic origins of the antibodies and differences in the fine specificity of their epitopes, each of these prM-reactive antibodies was capable of enhancing the DENV infection of Fc receptor-bearing cells. IMPORTANCE: Antibodies may play a critical role in the pathogenesis of enhanced DENV infection and disease during secondary infections. A substantial proportion of enhancing antibodies generated in response to natural dengue infection are directed toward the prM protein. The fine specificity of human prM antibodies is not understood. Here, we isolated a panel of dengue prM-specific human monoclonal antibodies from individuals after infection in order to define the mode of molecular recognition by enhancing antibodies. We found that only a single antibody molecule can be bound to each prM protein at any given time. Distinct overlapping epitopes were mapped, but all of the epitopes lie within a single major antigenic site, suggesting that this antigenic domain forms an immunodominant region of the protein. Neutralization and antibody-dependent enhanced replication experiments showed that recognition of any of the epitopes within the major antigenic site on prM was sufficient to cause enhanced infection of target cells.