RESUMO
Nipah virus (NiV) represents a significant pandemic threat with zoonotic transmission from bats-to-humans with almost annual regional outbreaks characterized by documented human-to-human transmission and high fatality rates. Currently, no vaccine against NiV has been approved. Structure-based design and protein engineering principles were applied to stabilize the fusion (F) protein in its prefusion trimeric conformation (pre-F) to improve expression and increase immunogenicity. We covalently linked the stabilized pre-F through trimerization domains at the C-terminus to three attachment protein (G) monomers, forming a chimeric design. These studies detailed here focus on mRNA delivery of NiV immunogens in mice, assessment of mRNA immunogen-specific design elements and their effects on humoral and cellular immunogenicity. The pre-F/G chimera elicited a strong neutralizing antibody response and a superior NiV-specific Tfh and other effector T cell response compared to G alone across both the mRNA and protein platforms. These findings enabled final candidate selection of pre-F/G Fd for clinical development.
Assuntos
Antígenos Virais/genética , Lipossomos/administração & dosagem , Nanopartículas/administração & dosagem , Vírus Nipah/imunologia , Proteínas do Envelope Viral/genética , Proteínas Virais de Fusão/genética , Vacinas Virais/administração & dosagem , Vacinas de mRNA/administração & dosagem , Animais , Antígenos Virais/imunologia , Feminino , Imunoglobulina G/sangue , Camundongos , Parcerias Público-Privadas , RNA Mensageiro/administração & dosagem , Linfócitos T/imunologia , Proteínas do Envelope Viral/imunologia , Proteínas Virais de Fusão/imunologiaRESUMO
Vaccine-associated enhanced respiratory disease (VAERD) was previously observed in some preclinical models of severe acute respiratory syndrome (SARS) and MERS coronavirus vaccines. We used the SARS coronavirus 2 (SARS-CoV-2) mouse-adapted, passage 10, lethal challenge virus (MA10) mouse model of acute lung injury to evaluate the immune response and potential for immunopathology in animals vaccinated with research-grade mRNA-1273. Whole-inactivated virus or heat-denatured spike protein subunit vaccines with alum designed to elicit low-potency antibodies and Th2-skewed CD4+ T cells resulted in reduced viral titers and weight loss post challenge but more severe pathological changes in the lung compared to saline-immunized animals. In contrast, a protective dose of mRNA-1273 induced favorable humoral and cellular immune responses that protected from viral replication in the upper and lower respiratory tract upon challenge. A subprotective dose of mRNA-1273 reduced viral replication and limited histopathological manifestations compared to animals given saline. Overall, our findings demonstrate an immunological signature associated with antiviral protection without disease enhancement following vaccination with mRNA-1273.
Assuntos
Vacinas contra COVID-19/imunologia , COVID-19/imunologia , COVID-19/prevenção & controle , Interações Hospedeiro-Patógeno/imunologia , SARS-CoV-2/imunologia , Vacinas Sintéticas/imunologia , Animais , Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/imunologia , Biópsia , Vacinas contra COVID-19/administração & dosagem , Modelos Animais de Doenças , Humanos , Imunoglobulina G , Imuno-Histoquímica , Camundongos , Avaliação de Resultados em Cuidados de Saúde , RNA Mensageiro , Glicoproteína da Espícula de Coronavírus/imunologia , Subpopulações de Linfócitos T/imunologia , Subpopulações de Linfócitos T/metabolismo , Vacinas Sintéticas/administração & dosagem , Vacinas de mRNARESUMO
The emergence of highly transmissible SARS-CoV-2 variants of concern (VOCs) that are resistant to therapeutic antibodies highlights the need for continuing discovery of broadly reactive antibodies. We identified four receptor binding domain-targeting antibodies from three early-outbreak convalescent donors with potent neutralizing activity against 23 variants, including the B.1.1.7, B.1.351, P.1, B.1.429, B.1.526, and B.1.617 VOCs. Two antibodies are ultrapotent, with subnanomolar neutralization titers [half-maximal inhibitory concentration (IC50) 0.3 to 11.1 nanograms per milliliter; IC80 1.5 to 34.5 nanograms per milliliter). We define the structural and functional determinants of binding for all four VOC-targeting antibodies and show that combinations of two antibodies decrease the in vitro generation of escape mutants, suggesting their potential in mitigating resistance development.
Assuntos
Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/imunologia , COVID-19/imunologia , SARS-CoV-2/imunologia , SARS-CoV-2/patogenicidade , Glicoproteína da Espícula de Coronavírus/imunologia , Enzima de Conversão de Angiotensina 2/antagonistas & inibidores , Enzima de Conversão de Angiotensina 2/metabolismo , Anticorpos Neutralizantes/química , Anticorpos Neutralizantes/metabolismo , Anticorpos Antivirais/química , Anticorpos Antivirais/metabolismo , Afinidade de Anticorpos , Reações Antígeno-Anticorpo , COVID-19/virologia , Humanos , Evasão da Resposta Imune , Fragmentos Fab das Imunoglobulinas/imunologia , Fragmentos Fab das Imunoglobulinas/metabolismo , Mutação , Testes de Neutralização , Domínios Proteicos , Receptores de Coronavírus/antagonistas & inibidores , Receptores de Coronavírus/metabolismo , SARS-CoV-2/genética , Glicoproteína da Espícula de Coronavírus/química , Glicoproteína da Espícula de Coronavírus/genética , Glicoproteína da Espícula de Coronavírus/metabolismoRESUMO
The emergence of highly transmissible SARS-CoV-2 variants of concern (VOC) that are resistant to therapeutic antibodies highlights the need for continuing discovery of broadly reactive antibodies. We identify four receptor-binding domain targeting antibodies from three early-outbreak convalescent donors with potent neutralizing activity against 12 variants including the B.1.1.7 and B.1.351 VOCs. Two of them are ultrapotent, with sub-nanomolar neutralization titers (IC50 <0.0006 to 0.0102 µ g/mL; IC80 < 0.0006 to 0.0251 µ g/mL). We define the structural and functional determinants of binding for all four VOC-targeting antibodies, and show that combinations of two antibodies decrease the in vitro generation of escape mutants, suggesting potential means to mitigate resistance development. These results define the basis of therapeutic cocktails against VOCs and suggest that targeted boosting of existing immunity may increase vaccine breadth against VOCs.
RESUMO
SARS-CoV-2 first emerged in the human population in late 2019 in Wuhan, China, and in a matter of months, spread across the globe resulting in the Coronavirus Disease 19 (COVID-19) pandemic and substantial economic fallout. SARS-CoV-2 is transmitted between humans via respiratory particles, with infection presenting a spectrum of clinical manifestations ranging from asymptomatic to respiratory failure with multiorgan dysfunction and death in severe cases. Prior experiences with human pathogenic coronaviruses and respiratory virus diseases in general have revealed an important role for cellular immunity in limiting disease severity. Here, we review some of the key mechanisms underlying cell-mediated immunity to respiratory viruses and summarize our current understanding of the functional capacity and role of SARS-CoV-2-specific T cells following natural infection and vaccination.
Assuntos
Vacinas contra COVID-19/uso terapêutico , COVID-19/imunologia , COVID-19/prevenção & controle , SARS-CoV-2/imunologia , Linfócitos T/imunologia , Vacinas contra COVID-19/imunologia , Humanos , VacinaçãoRESUMO
A vaccine for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is needed to control the coronavirus disease 2019 (COVID-19) global pandemic. Structural studies have led to the development of mutations that stabilize Betacoronavirus spike proteins in the prefusion state, improving their expression and increasing immunogenicity1. This principle has been applied to design mRNA-1273, an mRNA vaccine that encodes a SARS-CoV-2 spike protein that is stabilized in the prefusion conformation. Here we show that mRNA-1273 induces potent neutralizing antibody responses to both wild-type (D614) and D614G mutant2 SARS-CoV-2 as well as CD8+ T cell responses, and protects against SARS-CoV-2 infection in the lungs and noses of mice without evidence of immunopathology. mRNA-1273 is currently in a phase III trial to evaluate its efficacy.
Assuntos
Betacoronavirus/imunologia , Infecções por Coronavirus/imunologia , Infecções por Coronavirus/prevenção & controle , Pandemias/prevenção & controle , Pneumonia Viral/imunologia , Pneumonia Viral/prevenção & controle , Vacinas Virais/imunologia , Vacina de mRNA-1273 contra 2019-nCoV , Animais , Anticorpos Neutralizantes/imunologia , Betacoronavirus/genética , Linfócitos T CD8-Positivos/imunologia , COVID-19 , Vacinas contra COVID-19 , Ensaios Clínicos Fase III como Assunto , Infecções por Coronavirus/genética , Infecções por Coronavirus/virologia , Feminino , Pulmão/imunologia , Pulmão/virologia , Camundongos , Mutação , Nariz/imunologia , Nariz/virologia , Pneumonia Viral/virologia , RNA Mensageiro/genética , RNA Viral/genética , SARS-CoV-2 , Células Th1/imunologia , Receptor 4 Toll-Like/agonistas , Receptor 4 Toll-Like/imunologia , Vacinas Virais/química , Vacinas Virais/genéticaRESUMO
A SARS-CoV-2 vaccine is needed to control the global COVID-19 public health crisis. Atomic-level structures directed the application of prefusion-stabilizing mutations that improved expression and immunogenicity of betacoronavirus spike proteins. Using this established immunogen design, the release of SARS-CoV-2 sequences triggered immediate rapid manufacturing of an mRNA vaccine expressing the prefusion-stabilized SARS-CoV-2 spike trimer (mRNA-1273). Here, we show that mRNA-1273 induces both potent neutralizing antibody and CD8 T cell responses and protects against SARS-CoV-2 infection in lungs and noses of mice without evidence of immunopathology. mRNA-1273 is currently in a Phase 2 clinical trial with a trajectory towards Phase 3 efficacy evaluation.
RESUMO
Avian influenza vaccines exhibit poor immunogenicity in humans. We hypothesized that one factor underlying weak B cell responses was sequence divergence between avian and seasonal influenza hemagglutinin proteins, thus limiting the availability of adequate CD4 T cell help. To test this, a novel chimeric hemagglutinin protein (cH7/3) was derived, comprised of the stem domain from seasonal H3 hemagglutinin and the head domain from avian H7. Immunological memory to seasonal influenza was established in mice, through strategies that included seasonal inactivated vaccines, Flumist, and synthetic peptides derived from the H3 stalk domain. After establishment of memory, mice were vaccinated with H7 or cH7/3 protein. The cH7/3 Ag was able to recall H3-specific CD4 T cells, and this potentiated CD4 T cell response was associated with enhanced early germinal center response and rapid elicitation of Abs to H7, including Abs specific for the H7 head domain. These results suggest that in pandemic situations, inclusion of CD4 T cell epitopes from seasonal viruses have the potential to overcome the poor immunogenicity of avian vaccines by helping B cells and conferring greater subtype-specific Ab response to viral HA.
Assuntos
Formação de Anticorpos/imunologia , Linfócitos T CD4-Positivos/imunologia , Memória Imunológica/imunologia , Vacinas contra Influenza/imunologia , Influenza Aviária/imunologia , Influenza Humana/imunologia , Animais , Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/imunologia , Aves/imunologia , Aves/virologia , Epitopos de Linfócito T/imunologia , Feminino , Glicoproteínas de Hemaglutininação de Vírus da Influenza/imunologia , Humanos , Vírus da Influenza A/imunologia , Camundongos , Infecções por Orthomyxoviridae/imunologia , Estações do Ano , Vacinas de Produtos Inativados/imunologiaRESUMO
Technologies that define the atomic-level structure of neutralization-sensitive epitopes on viral surface proteins are transforming vaccinology and guiding new vaccine development approaches. Previously, iterative rounds of protein engineering were performed to preserve the prefusion conformation of the respiratory syncytial virus (RSV) fusion (F) glycoprotein, resulting in a stabilized subunit vaccine candidate (DS-Cav1), which showed promising results in mice and macaques. Here, phase I human immunogenicity data reveal a more than 10-fold boost in neutralizing activity in serum from antibodies targeting prefusion-specific surfaces of RSV F. These findings represent a clinical proof of concept for structure-based vaccine design, suggest that development of a successful RSV vaccine will be feasible, and portend an era of precision vaccinology.
Assuntos
Imunogenicidade da Vacina , Infecções por Vírus Respiratório Sincicial/prevenção & controle , Vacinas contra Vírus Sincicial Respiratório/química , Vacinas contra Vírus Sincicial Respiratório/imunologia , Vírus Sincicial Respiratório Humano/imunologia , Proteínas Virais de Fusão/química , Proteínas Virais de Fusão/imunologia , Adolescente , Adulto , Anticorpos Neutralizantes/sangue , Anticorpos Antivirais/sangue , Mapeamento de Epitopos , Humanos , Pessoa de Meia-Idade , Adulto JovemRESUMO
This 20-color flow cytometry panel was designed to resolve the cellular heterogeneity of antigen-presenting cells and was optimized for lymph node tissue. Reagents were carefully selected and optimized for identification of B cells (B220), neutrophils (Ly6G), monocytes and macrophages (Ly6C, CD169, F4/80), and dendritic cells (XCR1, CD172a, CD11c, I-A/I-E, CD24, CD64, pDCA-1, CD103, CD11b). Inclusion of additional functional markers involved in cell migration (CCR7), co-stimulation (CD80), and adhesion (ICAM-1) enabled further phenotypic characterization. Finally, this panel has been tested and is compatible with fluorescently labeled antigens such as Alexa Fluor 488 (Ax488) for the study of antigen-bearing cells in vivo. Published 2019. This article is a U.S. Government work and is in the public domain in the USA.
Assuntos
Células Apresentadoras de Antígenos/citologia , Citometria de Fluxo/métodos , Animais , Antígenos CD/metabolismo , Galinhas , Cor , Células Dendríticas/citologia , Imunofluorescência , CamundongosRESUMO
Ferrets are an ideal animal model in which to study the transmission of respiratory viruses as well as disease progression and vaccine efficacy because of their close anatomical and physiological resemblances to humans. However, a paucity of reagents and standardized procedures has hampered research progress, especially for studying cell-mediated immunity. The approaches described here-leukocyte isolation from whole blood and secondary lymphoid tissues-are generalizable, highly reproducible, and deliver single cell suspensions with excellent cell viability. Importantly, we have now developed assays to quantify key cellular components and antigen-specific T cell responses at the single cell level from multiple tissue compartments following influenza infection in ferrets. Collectively, these methods were instrumental in flow cytometry studies that revealed alterations in immune cell composition and distribution across lymphoid tissues following viral infection. Furthermore, sorting of T cell populations and peptide restimulation ex vivo in cytokine ELISpot assays has provided novel insight into the influenza-specific CD4 and CD8 T cell repertoire. The detailed procedures for these techniques are described in this chapter and can likely be adapted for the analyses of responses to many respiratory pathogens.
Assuntos
Interações Hospedeiro-Patógeno , Imunidade Celular , Infecções por Orthomyxoviridae/imunologia , Infecções por Orthomyxoviridae/virologia , Orthomyxoviridae/imunologia , Animais , Citocinas/metabolismo , ELISPOT , Furões , Citometria de Fluxo , Humanos , Influenza Humana/imunologia , Influenza Humana/virologia , Leucócitos Mononucleares/imunologia , Leucócitos Mononucleares/metabolismo , Depleção Linfocítica , Infecções por Orthomyxoviridae/metabolismoRESUMO
CD4 T cells convey a number of discrete functions to protective immunity to influenza, a complexity that distinguishes this arm of adaptive immunity from B cells and CD8 T cells. Although the most well recognized function of CD4 T cells is provision of help for antibody production, CD4 T cells are important in many aspects of protective immunity. Our studies have revealed that viral antigen specificity is a key determinant of CD4 T cell function, as illustrated both by mouse models of infection and human vaccine responses, a factor whose importance is due at least in part to events in viral antigen handling. We discuss research that has provided insight into the diverse viral epitope specificity of CD4 T cells elicited after infection, how this primary response is modified as CD4 T cells home to the lung, establish memory, and after challenge with a secondary and distinct influenza virus strain. Our studies in human subjects point out the challenges facing vaccine efforts to facilitate responses to novel and avian strains of influenza, as well as strategies that enhance the ability of CD4 T cells to promote protective antibody responses to both seasonal and potentially pandemic strains of influenza.
Assuntos
Antígenos Virais/imunologia , Linfócitos T CD4-Positivos/imunologia , Glicoproteínas de Hemaglutininação de Vírus da Influenza/imunologia , Memória Imunológica/imunologia , Vírus da Influenza A Subtipo H1N1/imunologia , Influenza Humana/imunologia , Animais , Anticorpos Antivirais/imunologia , Linfócitos B , Modelos Animais de Doenças , Humanos , CamundongosRESUMO
One of the major contributions to protective immunity to influenza viruses that is provided by virus-specific CD4 T cells is delivery of effector function to the infected lung. However, there is little known about the selection and breadth of viral epitope-specific CD4 T cells that home to the lung after their initial priming. In this study, using a mouse model of influenza A infection and an unbiased method of epitope identification, the viral epitope-specific CD4 T cells elicited after infection were identified and quantified. We found that a very diverse specificity of CD4 T cells is primed by infection, including epitopes from hemagglutinin, neuraminidase, matrix protein, nucleoprotein, and non-structural protein-1. Using peptide-specific cytokine EliSpots, the diversity and immunodominance hierarchies established in the lung-draining lymph node were compared with specificities of CD4 T cells that home to the lung. Our studies revealed that CD4 T cells of all epitope specificities identified in peripheral lymphoid tissue home back to the lung and that most of these lung-homing cells are localized within the tissue rather than the pulmonary vasculature. There is a striking shift of CD4 T cell functionality that enriches for IFN-γ production as cells are primed in the lymph node, enter the lung vasculature, and finally establish residency in the tissue, but with no apparent shifts in their functional avidity. We conclude that CD4 T cells of broad viral epitope specificity are recruited into the lung after influenza infection, where they then have the opportunity to encounter infected or antigen-bearing antigen-presenting cells.