RESUMO
There is pressing urgency to understand the pathogenesis of the severe acute respiratory syndrome coronavirus clade 2 (SARS-CoV-2), which causes the disease COVID-19. SARS-CoV-2 spike (S) protein binds angiotensin-converting enzyme 2 (ACE2), and in concert with host proteases, principally transmembrane serine protease 2 (TMPRSS2), promotes cellular entry. The cell subsets targeted by SARS-CoV-2 in host tissues and the factors that regulate ACE2 expression remain unknown. Here, we leverage human, non-human primate, and mouse single-cell RNA-sequencing (scRNA-seq) datasets across health and disease to uncover putative targets of SARS-CoV-2 among tissue-resident cell subsets. We identify ACE2 and TMPRSS2 co-expressing cells within lung type II pneumocytes, ileal absorptive enterocytes, and nasal goblet secretory cells. Strikingly, we discovered that ACE2 is a human interferon-stimulated gene (ISG) in vitro using airway epithelial cells and extend our findings to in vivo viral infections. Our data suggest that SARS-CoV-2 could exploit species-specific interferon-driven upregulation of ACE2, a tissue-protective mediator during lung injury, to enhance infection.
Assuntos
Células Epiteliais Alveolares/metabolismo , Enterócitos/metabolismo , Células Caliciformes/metabolismo , Interferon Tipo I/metabolismo , Mucosa Nasal/citologia , Peptidil Dipeptidase A/genética , Adolescente , Células Epiteliais Alveolares/imunologia , Enzima de Conversão de Angiotensina 2 , Animais , Betacoronavirus/fisiologia , COVID-19 , Linhagem Celular , Células Cultivadas , Criança , Infecções por Coronavirus/virologia , Enterócitos/imunologia , Células Caliciformes/imunologia , Infecções por HIV/imunologia , Humanos , Influenza Humana/imunologia , Interferon Tipo I/imunologia , Pulmão/citologia , Pulmão/patologia , Macaca mulatta , Camundongos , Mycobacterium tuberculosis , Mucosa Nasal/imunologia , Pandemias , Peptidil Dipeptidase A/metabolismo , Pneumonia Viral/virologia , Receptores Virais/genética , SARS-CoV-2 , Serina Endopeptidases/metabolismo , Análise de Célula Única , Tuberculose/imunologia , Regulação para CimaRESUMO
Humoral responses in coronavirus disease 2019 (COVID-19) are often of limited durability, as seen with other human coronavirus epidemics. To address the underlying etiology, we examined post mortem thoracic lymph nodes and spleens in acute SARS-CoV-2 infection and observed the absence of germinal centers and a striking reduction in Bcl-6+ germinal center B cells but preservation of AID+ B cells. Absence of germinal centers correlated with an early specific block in Bcl-6+ TFH cell differentiation together with an increase in T-bet+ TH1 cells and aberrant extra-follicular TNF-α accumulation. Parallel peripheral blood studies revealed loss of transitional and follicular B cells in severe disease and accumulation of SARS-CoV-2-specific "disease-related" B cell populations. These data identify defective Bcl-6+ TFH cell generation and dysregulated humoral immune induction early in COVID-19 disease, providing a mechanistic explanation for the limited durability of antibody responses in coronavirus infections, and suggest that achieving herd immunity through natural infection may be difficult.
Assuntos
Infecções por Coronavirus/imunologia , Centro Germinativo/imunologia , Pneumonia Viral/imunologia , Linfócitos T Auxiliares-Indutores/imunologia , Idoso , Idoso de 80 Anos ou mais , Linfócitos B/imunologia , COVID-19 , Feminino , Centro Germinativo/patologia , Humanos , Masculino , Pessoa de Meia-Idade , Pandemias , Proteínas Proto-Oncogênicas c-bcl-6/genética , Proteínas Proto-Oncogênicas c-bcl-6/metabolismo , Baço/imunologia , Baço/patologia , Fator de Necrose Tumoral alfa/metabolismoRESUMO
Broadly neutralizing antibodies (bnAbs) targeting the hemagglutinin (HA) stem of influenza A viruses (IAVs) tend to be effective against either group 1 or group 2 viral diversity. In rarer cases, intergroup protective bnAbs can be generated by human antibody paratopes that accommodate the conserved glycan differences between the group 1 and group 2 stems. We applied germline-engaging nanoparticle immunogens to elicit a class of cross-group bnAbs from physiological precursor frequency within a humanized mouse model. Cross-group protection depended on the presence of the human bnAb precursors within the B cell repertoire, and the vaccine-expanded antibodies enriched for an N55T substitution in the CDRH2 loop, a hallmark of the bnAb class. Structurally, this single mutation introduced a flexible fulcrum to accommodate glycosylation differences and could alone enable cross-group protection. Thus, broad IAV immunity can be expanded from the germline repertoire via minimal antigenic input and an exceptionally simple antibody development pathway.
Assuntos
Anticorpos Neutralizantes , Anticorpos Antivirais , Vírus da Influenza A , Vacinas contra Influenza , Infecções por Orthomyxoviridae , Vacinação , Animais , Camundongos , Humanos , Anticorpos Antivirais/imunologia , Vacinas contra Influenza/imunologia , Vírus da Influenza A/imunologia , Anticorpos Neutralizantes/imunologia , Infecções por Orthomyxoviridae/imunologia , Infecções por Orthomyxoviridae/prevenção & controle , Glicoproteínas de Hemaglutininação de Vírus da Influenza/imunologia , Substituição de Aminoácidos , Linfócitos B/imunologia , Influenza Humana/imunologia , Influenza Humana/prevenção & controle , Anticorpos Amplamente Neutralizantes/imunologiaRESUMO
Antibody paratopes are formed by hypervariable complementarity-determining regions (CDRH3s) and variable gene-encoded CDRs. The latter show biased usage in human broadly neutralizing antibodies (bnAbs) against both HIV and influenza virus, suggesting the existence of gene-endowed targeting solutions that may be amenable to pathway amplification. To test this, we generated transgenic mice with human CDRH3 diversity but simultaneously constrained to individual user-defined human immunoglobulin variable heavy-chain (VH) genes, including IGHV1-69, which shows biased usage in human bnAbs targeting the hemagglutinin stalk of group 1 influenza A viruses. Sequential immunization with a stalk-only hemagglutinin nanoparticle elicited group 1 bnAbs, but only in IGHV1-69 mice. This VH-endowed response required minimal affinity maturation, was elicited alongside pre-existing influenza immunity, and when IGHV1-69 B cells were diluted to match the frequency measured in humans. These results indicate that the human repertoire could, in principle, support germline-encoded bnAb elicitation using a single recombinant hemagglutinin immunogen.
Assuntos
Anticorpos Antivirais/metabolismo , Linfócitos B/imunologia , Anticorpos Amplamente Neutralizantes/metabolismo , Vírus da Influenza A/fisiologia , Vacinas contra Influenza/imunologia , Influenza Humana/imunologia , Receptores de Antígenos de Linfócitos B/genética , Animais , Anticorpos Antivirais/genética , Afinidade de Anticorpos , Anticorpos Amplamente Neutralizantes/genética , Regiões Determinantes de Complementaridade/genética , Mutação em Linhagem Germinativa/genética , Glicoproteínas de Hemaglutininação de Vírus da Influenza/imunologia , Humanos , Imunidade Humoral , Imunização Secundária , Cadeias Pesadas de Imunoglobulinas/genética , Camundongos , Camundongos Transgênicos , Nanopartículas , Engenharia de ProteínasRESUMO
In cells, asparagine/N-linked glycans are added to glycoproteins cotranslationally, in an attachment process that supports proper folding of the nascent polypeptide. We found that following pruning of N-glycan by the amidase PNGase F, the principal influenza vaccine antigen and major viral spike protein hemagglutinin (HA) spontaneously reattached N-glycan to its de-N-glycosylated positions when the amidase was removed from solution. This reaction, which we term N-glycanation, was confirmed by site-specific analysis of HA glycoforms by mass spectrometry prior to PNGase F exposure, during exposure to PNGase F, and after amidase removal. Iterative rounds of de-N-glycosylation followed by N-glycanation could be repeated at least three times and were observed for other viral glycoproteins/vaccine antigens, including the envelope glycoprotein (Env) from HIV. Covalent N-glycan reattachment was nonenzymatic as it occurred in the presence of metal ions that inhibit PNGase F activity. Rather, N-glycanation relied on a noncovalent assembly between protein and glycan, formed in the presence of the amidase, where linearization of the glycoprotein prevented this retention and subsequent N-glycanation. This reaction suggests that under certain experimental conditions, some glycoproteins can organize self-glycan addition, highlighting a remarkable self-assembly principle that may prove useful for re-engineering therapeutic glycoproteins such as influenza HA or HIV Env, where glycan sequence and structure can markedly affect bioactivity and vaccine efficacy.
Assuntos
Vacinas contra a AIDS , Vacinas contra Influenza , Influenza Humana , Antígenos HIV , Humanos , Peptídeo-N4-(N-acetil-beta-glucosaminil) Asparagina Amidase , PolissacarídeosRESUMO
Distinguishing safety from danger is necessary for survival, but is aberrant in individuals with post-traumatic stress disorder (PTSD). While PTSD is more prevalent in women than men, research on sex differences in safety learning is limited. Here, female rats demonstrated greater fear discrimination than males in a CS+/CS- paradigm. To determine if this sex difference transferred to fear inhibition, rats were tested for conditioned inhibition in a summation test with the CS+ and CS- presented in compound; no sex difference emerged. The results suggest sex differences in the neural mechanisms of discrimination learning but not recall of a fear inhibitor.
Assuntos
Condicionamento Psicológico/fisiologia , Discriminação Psicológica/fisiologia , Medo/fisiologia , Inibição Psicológica , Rememoração Mental/fisiologia , Caracteres Sexuais , Análise de Variância , Animais , Sinais (Psicologia) , Eletrochoque , Feminino , Reação de Congelamento Cataléptica/fisiologia , Generalização Psicológica/fisiologia , Masculino , Testes Psicológicos , Ratos Sprague-DawleyRESUMO
Nanoparticle (NP) vaccine formulations promote immune responses through multiple mechanisms. We recently reported that mannose-binding lectin (MBL) triggers trafficking of glycosylated HIV Env-immunogen NPs to lymph node follicles. Here, we investigate effects of MBL and complement on NP forms of HIV and other viral antigens. MBL recognition of oligomannose on gp120 nanoparticles significantly increases antigen accumulation in lymph nodes and antigen-specific germinal center (GC) responses. MBL and complement also mediate follicular trafficking and enhance GC responses to influenza, HBV, and HPV particulate antigens. Using model protein nanoparticles bearing titrated levels of glycosylation, we determine that mannose patches at a minimal density of 2.1 × 10-3 mannose patches/nm2 are required to trigger follicular targeting, which increases with increasing glycan density up to at least â¼8.2 × 10-3 patches/nm2. Thus, innate immune recognition of glycans has a significant impact on humoral immunity, and these findings provide a framework for engineering glycan recognition to optimize vaccine efficacy.
Assuntos
Sistemas de Liberação de Medicamentos/métodos , HIV-1/imunologia , Lectina de Ligação a Manose/imunologia , Animais , Antígenos/metabolismo , Antígenos Virais/imunologia , Proteínas do Sistema Complemento/metabolismo , Feminino , Centro Germinativo/metabolismo , Glicosilação , HIV-1/efeitos dos fármacos , Humanos , Imunidade Humoral/imunologia , Masculino , Manose , Camundongos , Camundongos Endogâmicos C57BL , Sistemas de Liberação de Fármacos por Nanopartículas/farmacologia , Nanopartículas , Polissacarídeos/metabolismoRESUMO
Initial exposure to a pathogen elicits an adaptive immune response to control and eradicate the threat. Interrogating the abundance and specificity of the naive B cell repertoire drives understanding of how to mount protective responses. Here, we isolated naive B cells from eight seronegative human donors targeting the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) receptor binding domain (RBD). Single-cell B cell receptor (BCR) sequencing identified diverse gene usage and no restriction on complementarity determining region length. A subset of recombinant antibodies produced by naive B cell precursors bound to SARS-CoV-2 RBD and engaged circulating variants including B.1.1.7, B.1.351, and B.1.617.2, as well as preemergent bat-derived coronaviruses RaTG13, SHC104, and WIV1. By structural characterization of a naive antibody in complex with SARS-CoV-2 spike, we identified a conserved mode of recognition shared with infection-induced antibodies. We found that representative naive antibodies could signal in a B cell activation assay, and by using directed evolution, we could select for a higher-affinity RBD interaction, conferred by a single amino acid change. The minimally mutated, affinity-matured antibodies also potently neutralized SARS-CoV-2. Understanding the SARS-CoV-2 RBDspecific naive repertoire may inform potential responses capable of recognizing future SARS-CoV-2 variants or emerging coronaviruses, enabling the development of pan-coronavirus vaccines aimed at engaging protective germline responses.
Assuntos
Linfócitos B/citologia , Linfócitos B/imunologia , Coronavirus/imunologia , SARS-CoV-2/imunologia , Anticorpos Neutralizantes/imunologia , Antígenos Virais/imunologia , Linfócitos B/metabolismo , COVID-19/imunologia , Vacinas contra COVID-19/imunologia , Epitopos , Humanos , Ativação Linfocitária , SARS-CoV-2/classificação , SARS-CoV-2/metabolismo , Glicoproteína da Espícula de Coronavírus/química , Glicoproteína da Espícula de Coronavírus/metabolismoRESUMO
Exposure to a pathogen elicits an adaptive immune response aimed to control and eradicate. Interrogating the abundance and specificity of the naive B cell repertoire contributes to understanding how to potentially elicit protective responses. Here, we isolated naive B cells from 8 seronegative human donors targeting the SARS-CoV-2 receptor-binding domain (RBD). Single B cell analysis showed diverse gene usage with no restricted complementarity determining region lengths. We show that recombinant antibodies engage SARS-CoV-2 RBD, circulating variants, and pre-emergent coronaviruses. Representative antibodies signal in a B cell activation assay and can be affinity matured through directed evolution. Structural analysis of a naive antibody in complex with spike shows a conserved mode of recognition shared with infection-induced antibodies. Lastly, both naive and affinity-matured antibodies can neutralize SARS-CoV-2. Understanding the naive repertoire may inform potential responses recognizing variants or emerging coronaviruses enabling the development of pan-coronavirus vaccines aimed at engaging germline responses.
RESUMO
Saponins are potent and safe vaccine adjuvants, but their mechanisms of action remain incompletely understood. Here, we explored the properties of several saponin formulations, including immune-stimulatory complexes (ISCOMs) formed by the self-assembly of saponin and phospholipids in the absence or presence of the Toll-like receptor 4 agonist monophosphoryl lipid A (MPLA). We found that MPLA self-assembles with saponins to form particles physically resembling ISCOMs, which we termed saponin/MPLA nanoparticles (SMNP). Saponin-containing adjuvants exhibited distinctive mechanisms of action, altering lymph flow in a mast celldependent manner and promoting antigen entry into draining lymph nodes. SMNP was particularly effective, exhibiting even greater potency than the compositionally related adjuvant AS01B in mice, and primed robust germinal center B cell, TFH, and HIV tier 2 neutralizing antibodies in nonhuman primates. Together, these findings shed new light on mechanisms by which saponin adjuvants act to promote the immune response and suggest that SMNP may be a promising adjuvant in the setting of HIV, SARS-CoV-2, and other pathogens.
Assuntos
Imunidade Adaptativa/efeitos dos fármacos , Adjuvantes Imunológicos/farmacologia , Linfa/efeitos dos fármacos , Saponinas/farmacologia , Receptores Toll-Like/agonistas , Animais , Linfócitos B/imunologia , Linfócitos T CD4-Positivos/imunologia , Feminino , Linfa/fisiologia , Macaca mulatta , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Nanopartículas , Ratos , Ratos WistarRESUMO
Humoral responses in COVID-19 disease are often of limited durability, as seen with other human coronavirus epidemics. To address the underlying etiology, we examined postmortem thoracic lymph nodes and spleens in acute SARS-CoV-2 infection and observed the absence of germinal centers, a striking reduction in Bcl-6+ germinal center B cells but preservation of AID+ B cells. Absence of germinal centers correlated with an early specific block in Bcl-6+TFH cell differentiation together with an increase in T-bet+TH1 cells and aberrant extra-follicular TNF-a accumulation. Parallel peripheral blood studies revealed loss of transitional and follicular B cells in severe disease and accumulation of SARS-CoV-2-specific "disease-related" B cell populations. These data identify defective Bcl-6+TFH cell generation and dysregulated humoral immune induction early in COVID-19 disease, providing a mechanistic explanation for the limited durability of antibody responses in coronavirus infections and suggest that achieving herd immunity through natural infection may be difficult. Funding: This work was supported by NIH U19 AI110495 to SP, NIH R01 AI146779 to AGS, NIH R01AI137057 and DP2DA042422 to DL, BMH was supported by NIGMS T32 GM007753, TMC was supported by T32 AI007245. Funding for these studies from the Massachusetts Consortium of Pathogen Readiness, the Mark and Lisa Schwartz Foundation and Enid Schwartz is also acknowledged. Conflict of Interest: None. Ethical Approval: This study was performed with the approval of the Institutional Review Boards at the Massachusetts General Hospital and the Brigham and Women's Hospital.