RESUMO
Cobalt-porphyrin phospholipid displays recombinant protein antigens on liposome surfaces via antigen polyhistidine-tag (His-tag), and when combined with monophosphorylated lipid A and QS-21 yields the "CPQ" vaccine adjuvant system. In this proof of principle study, CPQ was used to generate vaccine prototypes that elicited antibodies for two different alphaviruses (AV). Mice were immunized with computationally designed, His-tagged, physicochemical property consensus (PCPcon) protein antigens representing the variable B-domain of the envelope protein 2 (E2) from the serotype specific Venezuelan Equine Encephalitis Virus (VEEVcon) or a broad-spectrum AV-antigen termed EVCcon. The CPQ adjuvant enhanced the antigenicity of both proteins without eliciting detectable anti-His-tag antibodies. Antibodies elicited from mice immunized with antigens admixed with CPQ showed orders-of-magnitude higher levels of antigen-specific IgG compared to alternative control adjuvants. The ELISA results correlated with antiviral activity against VEEV strain TC83 and more weakly to Chikungunya virus 118/25. Thus, display of E.coli-produced His-tagged E2 protein segments on the surface of immunogenic liposomes elicits high levels of antigen-specific and AV neutralizing antibodies in mice with vaccination, while facilitating vaccine preparation and providing dose-sparing potential.
Assuntos
Adjuvantes Imunológicos , Alphavirus , Anticorpos Antivirais , Antígenos Virais , Lipossomos , Proteínas do Envelope Viral , Vacinas Virais , Animais , Anticorpos Antivirais/imunologia , Camundongos , Lipossomos/imunologia , Alphavirus/imunologia , Antígenos Virais/imunologia , Proteínas do Envelope Viral/imunologia , Vacinas Virais/imunologia , Vacinas Virais/administração & dosagem , Adjuvantes Imunológicos/administração & dosagem , Vírus da Encefalite Equina Venezuelana/imunologia , Feminino , Anticorpos Neutralizantes/imunologia , Vírus Chikungunya/imunologia , Camundongos Endogâmicos BALB C , Imunoglobulina G/imunologia , Imunoglobulina G/sangueRESUMO
The mitochondrial anti-viral signaling (MAVS) protein is an intermediary adaptor protein of retinoic acid-inducible gene-1 (RIG-I) like receptor (RLR) signaling, which activates the transcription factor interferon (IFN) regulatory factor 3 (IRF3) and NF-kB to produce type I IFNs. MAVS expression has been reported in different fish species, but few studies have shown its functional role in anti-viral responses to fish viruses. In this study, we used the transcription activator-like effector nuclease (TALEN) as a gene editing tool to disrupt the function of MAVS in Chinook salmon (Oncorhynchus tshawytscha) embryonic cells (CHSE) to understand its role in induction of interferon I responses to infections with the (+) RNA virus salmonid alphavirus subtype 3 (SAV-3), and the dsRNA virus infectious pancreatic necrosis virus (IPNV) infection. A MAVS-disrupted CHSE clone with a 7-aa polypeptide (GVFVSRV) deletion mutation at the N-terminal of the CARD domain infected with SAV-3 resulted in significantly lower expression of IRF3, IFNa, and ISGs and increased viral titer (1.5 log10) compared to wild-type. In contrast, the IPNV titer in MAVS-disrupted cells was not different from the wild-type. Furthermore, overexpression of salmon MAVS in MAVS-disrupted CHSE cells rescued the impaired type I IFN-mediated anti-viral effect against SAV-3.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal , Infecções por Alphavirus , Alphavirus , Doenças dos Peixes , Vírus da Necrose Pancreática Infecciosa , Transdução de Sinais , Replicação Viral , Animais , Vírus da Necrose Pancreática Infecciosa/fisiologia , Vírus da Necrose Pancreática Infecciosa/imunologia , Alphavirus/imunologia , Alphavirus/fisiologia , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/imunologia , Doenças dos Peixes/imunologia , Doenças dos Peixes/virologia , Infecções por Alphavirus/imunologia , Infecções por Alphavirus/virologia , Salmão/virologia , Salmão/imunologia , Proteínas de Peixes/genética , Proteínas de Peixes/imunologia , Proteínas de Peixes/metabolismo , Fator Regulador 3 de Interferon/metabolismo , Fator Regulador 3 de Interferon/genética , Infecções por Birnaviridae/imunologia , Infecções por Birnaviridae/veterinária , Infecções por Birnaviridae/virologiaRESUMO
Self-amplifying mRNA (SAM) vaccines can be rapidly deployed in the event of disease outbreaks. A legitimate safety concern is the potential for recombination between alphavirus-based SAM vaccines and circulating viruses. This theoretical risk needs to be assessed in the regulatory process for SAM vaccine approval. Herein, we undertake extensive in vitro and in vivo assessments to explore recombination between SAM vaccine and a wide selection of alphaviruses and a coronavirus. SAM vaccines were found to effectively limit alphavirus co-infection through superinfection exclusion, although some co-replication was still possible. Using sensitive cell-based assays, replication-competent alphavirus chimeras were generated in vitro as a result of rare, but reproducible, RNA recombination events. The chimeras displayed no increased fitness in cell culture. Viable alphavirus chimeras were not detected in vivo in C57BL/6J, Rag1-/- and Ifnar-/- mice, in which high levels of SAM vaccine and alphavirus co-replicated in the same tissue. Furthermore, recombination between a SAM-spike vaccine and a swine coronavirus was not observed. In conclusion we state that although the ability of SAM vaccines to recombine with alphaviruses might be viewed as an environmental safety concern, several key factors substantially mitigate against in vivo emergence of chimeric viruses from SAM vaccine recipients.
Assuntos
Alphavirus , Recombinação Genética , Vacinas de mRNA , Animais , Camundongos , Alphavirus/genética , Alphavirus/imunologia , Camundongos Endogâmicos C57BL , Humanos , Receptor de Interferon alfa e beta/genética , Replicação Viral , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/imunologia , Vacinas Sintéticas/imunologia , Vacinas Sintéticas/efeitos adversos , Camundongos Knockout , SARS-CoV-2/genética , SARS-CoV-2/imunologia , Vacinas Virais/imunologia , Vacinas Virais/genética , Vacinas Virais/efeitos adversosRESUMO
Getah virus (GETV) is a re-emerging mosquito-borne alphavirus that is highly pathogenic, mainly to pigs and horses. There are no vaccines or treatments available for GETV in swine in China. Therefore, the development of a simple, rapid, specific, and sensitive serological assay for GETV antibodies is essential for the prevention and control of GETV. Current antibody monitoring methods are time-consuming, expensive, and dependent on specialized instrumentation, and these features are not conducive to rapid detection in clinical samples. To address these problem, we developed immunochromatographic test strips (ICTS) using eukaryotically expressed soluble recombinant p62-E1 protein of GETV as a labelled antigen, which has good detection sensitivity and no cross-reactivity with other common porcine virus-positive sera. The ICTS is highly compatible with IFA and ELISA and can be stored for 1 month at 37 °C and for at least 3 months at room temperature. Hence, p62-E1-based ICTS is a rapid, accurate, and convenient method for rapid on-site detection of GETV antibodies. KEY POINTS: ⢠We established a rapid antibody detection method that can monitor GETV infection ⢠We developed colloidal gold test strips with high sensitivity and specificity ⢠The development of colloidal gold test strips will aid in the field serologic detection of GETV.
Assuntos
Alphavirus , Anticorpos Antivirais , Coloide de Ouro , Sensibilidade e Especificidade , Animais , Coloide de Ouro/química , Anticorpos Antivirais/sangue , Anticorpos Antivirais/imunologia , Alphavirus/imunologia , Suínos , Cromatografia de Afinidade/métodos , Infecções por Alphavirus/diagnóstico , Infecções por Alphavirus/imunologia , Doenças dos Suínos/diagnóstico , Doenças dos Suínos/virologia , Fitas Reagentes , China , Ensaio de Imunoadsorção Enzimática/métodosRESUMO
The development and persistence of antibody secreting cells (ASC) after antigenic challenge remain inadequately understood in teleosts. In this study, intraperitoneal (ip) injection of Atlantic salmon (Salmo salar) with salmonid alphavirus (WtSAV3) increased the total ASC response, peaking 3-6 weeks post injection (wpi) locally in the peritoneal cavity (PerC) and in systemic lymphoid tissues, while at 13 wpi the response was only elevated in PerC. At the same time point a specific ASC response was induced by WtSAV3 in PerC and systemic tissues, with the highest frequency in PerC, suggesting a local role. Inactivated SAV (InSAV1) induced comparatively lower ASC responses in all sites, and specific serum antibodies were only induced by WtSAV3 and not by InSAV1. An InSAV1 boost did not increase these responses. Expression of immune marker genes implies a role for PerC adipose tissue in the PerC immune response. Overall, the study suggests the Atlantic salmon PerC as a secondary immune site and an ASC survival niche.
Assuntos
Infecções por Alphavirus , Alphavirus , Anticorpos Antivirais , Células Produtoras de Anticorpos , Doenças dos Peixes , Cavidade Peritoneal , Salmo salar , Animais , Salmo salar/imunologia , Salmo salar/virologia , Alphavirus/imunologia , Infecções por Alphavirus/imunologia , Infecções por Alphavirus/veterinária , Infecções por Alphavirus/virologia , Cavidade Peritoneal/citologia , Doenças dos Peixes/imunologia , Doenças dos Peixes/virologia , Células Produtoras de Anticorpos/imunologia , Anticorpos Antivirais/sangue , Anticorpos Antivirais/imunologia , Injeções Intraperitoneais/veterináriaRESUMO
Replicating RNA, including self-amplifying RNA (saRNA) and trans-amplifying RNA (taRNA), holds great potential for advancing the next generation of RNA-based vaccines. Unlike in vitro transcribed mRNA found in most current RNA vaccines, saRNA or taRNA can be massively replicated within cells in the presence of RNA-amplifying enzymes known as replicases. We recently demonstrated that this property could enhance immune responses with minimal injected RNA amounts. In saRNA-based vaccines, replicase and antigens are encoded on the same mRNA molecule, resulting in very long RNA sequences, which poses significant challenges in production, delivery, and stability. In taRNA-based vaccines, these challenges can be overcome by splitting the replication system into two parts: one that encodes replicase and the other that encodes a short antigen-encoding RNA called transreplicon. Here, we review the identification and use of transreplicon RNA in alphavirus research, with a focus on the development of novel taRNA technology as a state-of-the art vaccine platform. Additionally, we discuss remaining challenges essential to the clinical application and highlight the potential benefits related to the unique properties of this future vaccine platform.
Assuntos
Alphavirus , RNA Viral , Desenvolvimento de Vacinas , Animais , Humanos , Alphavirus/genética , Alphavirus/imunologia , Infecções por Alphavirus/imunologia , Infecções por Alphavirus/virologia , RNA Viral/genética , Vacinas Virais/imunologia , Vacinas Virais/genética , Replicação ViralRESUMO
IMPORTANCE: Alphaviruses threaten public health continuously, and Getah virus (GETV) is a re-emerging alphavirus that can potentially infect humans. Approved antiviral drugs and vaccines against alphaviruses are few available, but several host antiviral factors have been reported. Here, we used GETV as a model of alphaviruses to screen for additional host factors. Tetrachlorodibenzo-p-dioxin-inducible poly(ADP ribose) polymerase was identified to inhibit GETV replication by inducing ubiquitination of the glycoprotein E2, causing its degradation by recruiting the E3 ubiquitin ligase membrane-associated RING-CH8 (MARCH8). Using GETV as a model virus, focusing on the relationship between viral structural proteins and host factors to screen antiviral host factors provides new insights for antiviral studies on alphaviruses.
Assuntos
Alphavirus , Interações entre Hospedeiro e Microrganismos , Proteínas de Transporte de Nucleosídeos , Poli(ADP-Ribose) Polimerases , Transcriptoma , Humanos , Alphavirus/crescimento & desenvolvimento , Alphavirus/imunologia , Glicoproteínas/metabolismo , Proteínas de Transporte de Nucleosídeos/genética , Proteínas de Transporte de Nucleosídeos/metabolismo , Poli(ADP-Ribose) Polimerases/genética , Poli(ADP-Ribose) Polimerases/metabolismo , Ubiquitinação , Proteínas Estruturais Virais/metabolismo , Replicação ViralRESUMO
Arboviruses have two ecological transmission cycles: sylvatic and urban. For some, the sylvatic cycle has not been thoroughly described in America. To study the role of wildlife in a putative sylvatic cycle, we sampled free-ranging bats and birds in two arbovirus endemic locations and analyzed them using molecular, serological, and histological methods. No current infection was detected, and no significant arbovirus-associated histological changes were observed. Neutralizing antibodies were detected against selected arboviruses. In bats, positivity in 34.95% for DENV-1, 16.26% for DENV-2, 5.69% for DENV-3, 4.87% for DENV-4, 2.43% for WNV, 4.87% for SLEV, 0.81% for YFV, 7.31% for EEEV, and 0.81% for VEEV was found. Antibodies against ZIKV were not detected. In birds, PRNT results were positive against WNV in 0.80%, SLEV in 5.64%, EEEV in 8.4%, and VEEV in 5.63%. An additional retrospective PRNT analysis was performed using bat samples from three additional DENV endemic sites resulting in a 3.27% prevalence for WNV and 1.63% for SLEV. Interestingly, one sample resulted unequivocally WNV positive confirmed by serum titration. These results suggest that free-ranging bats and birds are exposed to not currently reported hyperendemic-human infecting Flavivirus and Alphavirus; however, their role as reservoirs or hosts is still undetermined.
Assuntos
Alphavirus/imunologia , Animais Selvagens/imunologia , Anticorpos Antivirais/sangue , Aves/imunologia , Quirópteros/imunologia , Flavivirus/imunologia , Estudos Soroepidemiológicos , Infecções por Alphavirus/epidemiologia , Infecções por Alphavirus/veterinária , Animais , Anticorpos Neutralizantes/sangue , Doenças das Aves/epidemiologia , Costa Rica/epidemiologia , Vírus da Dengue/imunologia , Reservatórios de Doenças , Feminino , Infecções por Flavivirus/epidemiologia , Infecções por Flavivirus/veterinária , Humanos , Masculino , Testes de Neutralização , PrevalênciaRESUMO
Despite vaccination, pancreas disease (PD) caused by salmonid alphavirus (SAV) has been the economically most important virus disease in salmon farming in Ireland, Scotland, and Norway. A vaccine based on DNA plasmid has been authorized to be used in Norwegian aquaculture since 2018. DNA vaccination of plasmids expressed subcellular viral proteins have been shown its particular protective effect against SAV3 that surface expression of the E2 protein with the whole viral protein construct, yielding a more effective vaccine. The chapter describes methods to design and test the sublocalization of expressed viral protein and the performance evaluation of vaccines against SAV3 infection in Atlantic salmon.
Assuntos
Infecções por Alphavirus , Alphavirus , Doenças dos Peixes , Salmonidae , Vacinas de DNA , Alphavirus/genética , Alphavirus/imunologia , Infecções por Alphavirus/prevenção & controle , Infecções por Alphavirus/veterinária , Animais , Doenças dos Peixes/prevenção & controle , Proteínas ViraisRESUMO
Eastern equine encephalitis virus (EEEV), western equine encephalitis virus (WEEV) and Venezuelan equine encephalitis virus (VEEV) can cause fatal encephalitis in humans and equids. Some MAbs to the E1 glycoprotein are known to be cross-reactive, weakly neutralizing in vitro but can protect from disease in animal models. We investigated the mechanism of neutralization of VEEV infection by the broadly cross-reactive E1-specific MAb 1A4B-6. 1A4B-6 protected 3-week-old Swiss Webster mice prophylactically from lethal VEEV challenge. Likewise, 1A4B-6 inhibited virus growth in vitro at a pre-attachment step after virions were incubated at 37 °C and inhibited virus-mediated cell fusion. Amino acid residue N100 in the fusion loop of E1 protein was identified as critical for binding. The potential to elicit broadly cross-reactive MAbs with limited virus neutralizing activity in vitro but that can inhibit virus entry and protect animals from infection merits further exploration for vaccine and therapeutic developmental research.
Assuntos
Anticorpos Antivirais/imunologia , Vírus da Encefalite Equina Venezuelana/imunologia , Vírus da Encefalite Equina Venezuelana/metabolismo , Encefalomielite Equina Venezuelana/imunologia , Encefalomielite Equina Venezuelana/virologia , Proteínas do Envelope Viral/imunologia , Replicação Viral/efeitos dos fármacos , Alphavirus/imunologia , Infecções por Alphavirus/imunologia , Sequência de Aminoácidos , Animais , Anticorpos Monoclonais/imunologia , Anticorpos Neutralizantes/imunologia , Linhagem Celular , Chlorocebus aethiops , Reações Cruzadas , Encefalomielite Equina Venezuelana/terapia , Glicoproteínas/imunologia , Imunoterapia , Camundongos , Ligação Proteica , Células Vero , Proteínas do Envelope Viral/metabolismo , Vírion/imunologia , Vírion/metabolismoRESUMO
Mayaro virus (MAYV) is an alphavirus endemic to both Latin America and the Caribbean. Recent reports have questioned the ability of MAYV and its close relative, Chikungunya virus (CHIKV), to generate cross-reactive, neutralizing antibodies to one another. Since CHIKV was introduced to South America in 2013, discerning whether individuals have cross-reactive antibodies or whether they have had exposures to both viruses previously has been difficult. Using samples obtained from people infected with MAYV prior to the introduction of CHIKV in the Americas, we performed neutralizing assays and observed no discernable neutralization of CHIKV by sera from patients previously infected with MAYV. These data suggest that a positive CHIKV neutralization test cannot be attributed to prior exposure to MAYV and that previous exposure to MAYV may not be protective against a subsequent CHIKV infection.
Assuntos
Infecções por Alphavirus/diagnóstico , Infecções por Alphavirus/epidemiologia , Anticorpos Neutralizantes/sangue , Anticorpos Antivirais/sangue , Febre de Chikungunya/diagnóstico , Febre de Chikungunya/epidemiologia , Alphavirus/imunologia , Infecções por Alphavirus/imunologia , Infecções por Alphavirus/virologia , Febre de Chikungunya/imunologia , Febre de Chikungunya/virologia , Vírus Chikungunya/imunologia , Reações Cruzadas , Humanos , Soros Imunes/química , Testes de Neutralização , Peru/epidemiologiaRESUMO
Chikungunya virus (CHIKV) and Mayaro virus (MAYV) are closely related members of the Semliki Forest virus antigenic complex classified as belonging to the genus Alphavirus of the family Togaviridae. These viruses cause human disease, with sudden fever and joint inflammation that can persist for long periods. CHIKV is the causative agent of large outbreaks worldwide, and MAYV infection represents a growing public health concern in Latin America, causing sporadic cases and geographically limited outbreaks. Considering the relationship between CHIKV and MAYV, the present study aimed to evaluate if preexisting CHIKV immunity protects against MAYV infection. Immunocompetent C57BL/6 mice were intraperitoneally infected with CHIKV and, 4 weeks later, they were infected with MAYV in their hind paw. We observed that the preexistence of CHIKV immunity conferred partial cross-protection against secondary MAYV infection, reducing disease severity, tissue viral load, and histopathological scores. Interestingly, CHIKV antibodies from humans and mice showed low cross-neutralization to MAYV, but neutralizing activity significantly increased after secondary infection. Furthermore, depletion of adaptive immune cells (CD4+ T, CD8+ T, and CD19+ B cells) did not alter the cross-protection phenotype, suggesting that distinct cell subsets or a combination of adaptive immune cells stimulated by CHIKV are responsible for the partial cross-protection against MAYV. The reduction of proinflammatory cytokines, such as interferon gamma (IFN-γ), in animals secondarily infected by MAYV, suggests a role for innate immunity in cross-protection. Our findings shed light on how preexisting immunity to arthritogenic alphaviruses may affect secondary infection, which may further develop relevant influence in disease outcome and viral transmission. IMPORTANCE Mosquito-borne viruses have a worldwide impact, especially in tropical climates. Chikungunya virus has been present mostly in developing countries, causing millions of infections, while Mayaro virus, a close relative, has been limited to the Caribbean and tropical regions of Latin America. The potential emergence and spread of Mayaro virus to other high-risk areas have increased the scientific community's attention to an imminent worldwide epidemic. Here, we designed an experimental protocol of chikungunya and Mayaro virus mouse infection, which develops a measurable and quantifiable disease that allows us to make inferences about potential immunological effects during secondary virus infection. Our results demonstrate that previous chikungunya virus infection is able to reduce the severity of clinical outcomes during secondary Mayaro infection. We provide scientific understanding of immunological features during secondary infection with the closely related virus, thus assisting in better comprehending viral transmission and the pathological outcome of these diseases.
Assuntos
Infecções por Alphavirus/imunologia , Infecções por Alphavirus/prevenção & controle , Vírus Chikungunya/imunologia , Proteção Cruzada/imunologia , Alphavirus/imunologia , Infecções por Alphavirus/patologia , Animais , Anticorpos Antivirais/imunologia , Febre de Chikungunya/virologia , Modelos Animais de Doenças , Epidemias , Feminino , Inflamação , Camundongos , Camundongos Endogâmicos C57BL , Carga ViralRESUMO
Arthritogenic alphaviruses are globally distributed, mosquito-transmitted viruses that cause rheumatological disease in humans and include Chikungunya virus (CHIKV), Mayaro virus (MAYV), and others. Although serological evidence suggests that some antibody-mediated heterologous immunity may be afforded by alphavirus infection, the extent to which broadly neutralizing antibodies that protect against multiple arthritogenic alphaviruses are elicited during natural infection remains unknown. Here, we describe the isolation and characterization of MAYV-reactive alphavirus monoclonal antibodies (mAbs) from a CHIKV-convalescent donor. We characterized 33 human mAbs that cross-reacted with CHIKV and MAYV and engaged multiple epitopes on the E1 and E2 glycoproteins. We identified five mAbs that target distinct regions of the B domain of E2 and potently neutralize multiple alphaviruses with differential breadth of inhibition. These broadly neutralizing mAbs (bNAbs) contain few somatic mutations and inferred germline-revertants retained neutralizing capacity. Two bNAbs, DC2.M16 and DC2.M357, protected against both CHIKV- and MAYV-induced musculoskeletal disease in mice. These findings enhance our understanding of the cross-reactive and cross-protective antibody response to human alphavirus infections.
Assuntos
Infecções por Alphavirus/imunologia , Anticorpos Monoclonais/isolamento & purificação , Anticorpos Amplamente Neutralizantes/imunologia , Alphavirus/imunologia , Alphavirus/patogenicidade , Animais , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/farmacologia , Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/imunologia , Artrite/etiologia , Artrite/imunologia , Artrite/virologia , Anticorpos Amplamente Neutralizantes/isolamento & purificação , Anticorpos Amplamente Neutralizantes/farmacologia , Febre de Chikungunya/virologia , Vírus Chikungunya/imunologia , Vírus Chikungunya/patogenicidade , Reações Cruzadas , Epitopos/imunologia , Células Germinativas/imunologia , Glicoproteínas/imunologia , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BLRESUMO
Human pathogens belonging to the Alphavirus genus, in the Togaviridae family, are transmitted primarily by mosquitoes. The signs and symptoms associated with these viruses include fever and polyarthralgia, defined as joint pain and inflammation, as well as encephalitis. In the last decade, our understanding of the interactions between members of the alphavirus genus and the human host has increased due to the re-appearance of the chikungunya virus (CHIKV) in Asia and Europe, as well as its emergence in the Americas. Alphaviruses affect host immunity through cytokines and the interferon response. Understanding alphavirus interactions with both the innate immune system as well as the various cells in the adaptive immune systems is critical to developing effective therapeutics. In this review, we summarize the latest research on alphavirus-host cell interactions, underlying infection mechanisms, and possible treatments.
Assuntos
Infecções por Alphavirus , Alphavirus , Alphavirus/imunologia , Alphavirus/patogenicidade , Infecções por Alphavirus/epidemiologia , Infecções por Alphavirus/prevenção & controle , Infecções por Alphavirus/virologia , Animais , Humanos , Vacinas Virais/imunologiaRESUMO
Alphaviruses are emerging, mosquito-transmitted pathogens that cause musculoskeletal and neurological disease in humans. Although neutralizing antibodies that inhibit individual alphaviruses have been described, broadly reactive antibodies that protect against both arthritogenic and encephalitic alphaviruses have not been reported. Here, we identify DC2.112 and DC2.315, two pan-protective yet poorly neutralizing human monoclonal antibodies (mAbs) that avidly bind to viral antigen on the surface of cells infected with arthritogenic and encephalitic alphaviruses. These mAbs engage a conserved epitope in domain II of the E1 protein proximal to and within the fusion peptide. Treatment with DC2.112 or DC2.315 protects mice against infection by both arthritogenic (chikungunya and Mayaro) and encephalitic (Venezuelan, Eastern, and Western equine encephalitis) alphaviruses through multiple mechanisms, including inhibition of viral egress and monocyte-dependent Fc effector functions. These findings define a conserved epitope recognized by weakly neutralizing yet protective antibodies that could be targeted for pan-alphavirus immunotherapy and vaccine design.
Assuntos
Alphavirus/imunologia , Anticorpos Antivirais/imunologia , Sequência Conservada/imunologia , Epitopos/imunologia , Proteínas Virais/imunologia , Infecções por Alphavirus/imunologia , Infecções por Alphavirus/virologia , Sequência de Aminoácidos , Animais , Anticorpos Monoclonais/imunologia , Febre de Chikungunya/imunologia , Febre de Chikungunya/virologia , Vírus Chikungunya/imunologia , Chlorocebus aethiops , Mapeamento de Epitopos , Epitopos/química , Humanos , Masculino , Camundongos Endogâmicos C57BL , Modelos Biológicos , Monócitos/metabolismo , Células Vero , Proteínas Virais/química , Liberação de VírusRESUMO
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
Mayaro virus (MAYV), which causes mayaro fever, is endemic to limited regions of South America that may expand due to the possible involvement of Aedes spp. mosquitoes in its transmission. Its effective control will require the accurate identification of infected individuals, which has been restricted to nucleic acid-based tests due to similarities with other emerging members of the Alphavirus genus of the Togaviridae family; both in structure and clinical symptoms. Serological tests have a more significant potential to expand testing at a reasonable cost, and their performance primarily reflects that of the antigen utilized to capture pathogen-specific antibodies. Here, we describe the assembly of a synthetic gene encoding multiple copies of antigenic determinants mapped from the nsP1, nsP2, E1, and E2 proteins of MAYV that readily expressed as a stable chimeric protein in bacteria. Its serological performance as the target in ELISAs revealed a high accuracy for detecting anti-MAYV IgM antibodies. No cross-reactivity was observed with serum from seropositive individuals for dengue, chikungunya, yellow fever, Zika, and other infectious diseases as well as healthy individuals. Our data suggest that this bioengineered antigen could be used to develop high-performance serological tests for MAYV infections.
Assuntos
Infecções por Alphavirus/diagnóstico , Alphavirus/imunologia , Epitopos/imunologia , Infecções por Togaviridae/diagnóstico , Aedes/virologia , Alphavirus/patogenicidade , Infecções por Alphavirus/imunologia , Infecções por Alphavirus/transmissão , Infecções por Alphavirus/virologia , Animais , Ensaio de Imunoadsorção Enzimática , Epitopos/genética , Epitopos/ultraestrutura , Feminino , Genes Sintéticos/genética , Genes Sintéticos/imunologia , Humanos , Imunoglobulina M/imunologia , Masculino , Testes Sorológicos , América do Sul/epidemiologia , Togaviridae/isolamento & purificação , Togaviridae/patogenicidade , Infecções por Togaviridae/imunologia , Infecções por Togaviridae/transmissão , Infecções por Togaviridae/virologiaRESUMO
Alphaviruses have a single-stranded, positive-sense RNA genome that contains two open reading frames encoding either the non-structural or the structural genes. Upon infection, the genomic RNA is translated into the non-structural proteins (nsPs). NsPs are required for viral RNA replication and transcription driven from the subgenomic promoter (sgP). Transfection of an RNA encoding the luciferase gene under the control of the sgP into cells enabled the detection of replication-competent chikungunya virus (CHIKV) or Mayaro virus (MAYV) with high sensitivity as a function of the induced luciferase activity. This assay principle was additionally used to analyze virus-neutralizing antibodies in sera and might be an alternative to standard virus neutralization assays based on virus titration or the use of genetically modified tagged viruses.
Assuntos
Alphavirus/imunologia , Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/sangue , RNA Viral/genética , Testes Sorológicos/métodos , Alphavirus/classificação , Infecções por Alphavirus/sangue , Infecções por Alphavirus/diagnóstico , Infecções por Alphavirus/imunologia , Animais , Linhagem Celular , Vírus Chikungunya/genética , Reações Cruzadas , Humanos , Imunoglobulina G/sangue , Imunoglobulina M/sangue , Luciferases/genética , Camundongos , Camundongos Endogâmicos BALB C , Sensibilidade e Especificidade , Testes Sorológicos/normasRESUMO
Mayaro virus (MAYV) is an emerging arbovirus of the Americas that may cause a debilitating arthritogenic disease. The biology of MAYV is not fully understood and largely inferred from related arthritogenic alphaviruses. Here, we present the structure of MAYV at 4.4 Å resolution, obtained from a preparation of mature, infective virions. MAYV presents typical alphavirus features and organization. Interactions between viral proteins that lead to particle formation are described together with a hydrophobic pocket formed between E1 and E2 spike proteins and conformational epitopes specific of MAYV. We also describe MAYV glycosylation residues in E1 and E2 that may affect MXRA8 host receptor binding, and a molecular "handshake" between MAYV spikes formed by N262 glycosylation in adjacent E2 proteins. The structure of MAYV is suggestive of structural and functional complexity among alphaviruses, which may be targeted for specificity or antiviral activity.