RESUMO
Chronic cytomegalovirus (CMV) infection leads to long-term maintenance of extraordinarily large CMV-specific T cell populations. The magnitude of this so-called 'memory inflation' is thought to mainly depend on antigenic stimulation during the chronic phase of infection. However, by mapping the long-term development of CD8+ T cell families derived from single naive precursors, we find that fate decisions made during the acute phase of murine CMV infection can alter the level of memory inflation by more than 1,000-fold. Counterintuitively, a T cell family's capacity for memory inflation is not determined by its initial expansion. Instead, those rare T cell families that dominate the chronic phase of infection show an early transcriptomic signature akin to that of established T central memory cells. Accordingly, a T cell family's long-term dominance is best predicted by its early content of T central memory precursors, which later serve as a stem-cell-like source for memory inflation.
Assuntos
Evolução Clonal/imunologia , Interações Hospedeiro-Patógeno/imunologia , Memória Imunológica , Subpopulações de Linfócitos T/imunologia , Subpopulações de Linfócitos T/metabolismo , Viroses/etiologia , Viroses/metabolismo , Doença Aguda , Animais , Biomarcadores , Doença Crônica , Citomegalovirus/imunologia , Infecções por Citomegalovirus/imunologia , Infecções por Citomegalovirus/virologia , Perfilação da Expressão Gênica , Humanos , Imunofenotipagem , Camundongos , Muromegalovirus/imunologiaRESUMO
Upon viral infection, natural killer (NK) cells expressing certain germline-encoded receptors are selected, expanded, and maintained in an adaptive-like manner. Currently, these are thought to differentiate along a common pathway. However, by fate mapping of single NK cells upon murine cytomegalovirus (MCMV) infection, we identified two distinct NK cell lineages that contributed to adaptive-like responses. One was equivalent to conventional NK (cNK) cells while the other was transcriptionally similar to type 1 innate lymphoid cells (ILC1s). ILC1-like NK cells showed splenic residency and strong cytokine production but also recognized and killed MCMV-infected cells, guided by activating receptor Ly49H. Moreover, they induced clustering of conventional type 1 dendritic cells and facilitated antigen-specific T cell priming early during MCMV infection, which depended on Ly49H and the NK cell-intrinsic expression of transcription factor Batf3. Thereby, ILC1-like NK cells bridge innate and adaptive viral recognition and unite critical features of cNK cells and ILC1s.
Assuntos
Imunidade Adaptativa/imunologia , Linhagem da Célula/imunologia , Infecções por Herpesviridae/imunologia , Imunidade Inata/imunologia , Células Matadoras Naturais/imunologia , Animais , Feminino , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , MuromegalovirusRESUMO
Cytomegalovirus (CMV) induces a unique T cell response, where antigen-specific populations do not contract, but rather inflate during viral latency. It has been proposed that subclinical episodes of virus reactivation feed the inflation of CMV-specific memory cells by intermittently engaging T cell receptors (TCRs), but evidence of TCR engagement has remained lacking. Nuclear factor of activated T cells (NFAT) is a family of transcription factors, where NFATc1 and NFATc2 signal downstream of TCR in mature T lymphocytes. We show selective impacts of NFATc1 and/or NFATc2 genetic ablations on the long-term inflation of MCMV-specific CD8+ T cell responses despite largely maintained responses to acute infection. NFATc1 ablation elicited robust phenotypes in isolation, but the strongest effects were observed when both NFAT genes were missing. CMV control was impaired only when both NFATs were deleted in CD8+ T cells used in adoptive immunotherapy of immunodeficient mice. Transcriptome analyses revealed that T cell intrinsic NFAT is not necessary for CD8+ T cell priming, but rather for their maturation towards effector-memory and in particular the effector cells, which dominate the pool of inflationary cells.
Assuntos
Infecções por Citomegalovirus , Muromegalovirus , Animais , Camundongos , Muromegalovirus/fisiologia , Linfócitos T CD8-Positivos , Citomegalovirus , Receptores de Antígenos de Linfócitos T , Memória ImunológicaRESUMO
Recent emergence of SARS-CoV-1 variants demonstrates the potential of this virus for targeted evolution, despite its overall genomic stability. Here we show the dynamics and the mechanisms behind the rapid adaptation of SARS-CoV-2 to growth in Vero E6 cells. The selective advantage for growth in Vero E6 cells is due to increased cleavage efficiency by cathepsins at the mutated S1/S2 site. S1/S2 site also constitutes a heparan sulfate (HS) binding motif that influenced virus growth in Vero E6 cells, but HS antagonist did not inhibit virus adaptation in these cells. The entry of Vero E6-adapted virus into human cells is defective because the mutated spike variants are poorly processed by furin or TMPRSS2. Minor subpopulation that lack the furin cleavage motif in the spike protein rapidly become dominant upon passaging through Vero E6 cells, but wild type sequences are maintained at low percentage in the virus swarm and mediate a rapid reverse adaptation if the virus is passaged again on TMPRSS2+ human cells. Our data show that the spike protein of SARS-CoV-2 can rapidly adapt itself to available proteases and argue for deep sequence surveillance to identify the emergence of novel variants. IMPORTANCE Recently emerging SARS-CoV-2 variants B.1.1.7 (alpha variant), B.1.617.2 (delta variant), and B.1.1.529 (omicron variant) harbor spike mutations and have been linked to increased virus pathogenesis. The emergence of these novel variants highlights coronavirus adaptation and evolution potential, despite the stable consensus genotype of clinical isolates. We show that subdominant variants maintained in the virus population enable the virus to rapidly adapt to selection pressure. Although these adaptations lead to genotype change, the change is not absolute and genomes with original genotype are maintained in the virus swarm. Thus, our results imply that the relative stability of SARS-CoV-2 in numerous independent clinical isolates belies its potential for rapid adaptation to new conditions.
Assuntos
COVID-19/metabolismo , Furina/metabolismo , SARS-CoV-2/fisiologia , Serina Endopeptidases/metabolismo , Glicoproteína da Espícula de Coronavírus/metabolismo , Adaptação Fisiológica , Animais , COVID-19/genética , COVID-19/virologia , Chlorocebus aethiops , Efeito Citopatogênico Viral , Furina/genética , Células HEK293 , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Mutação , SARS-CoV-2/genética , Serina Endopeptidases/genética , Glicoproteína da Espícula de Coronavírus/genética , Células Vero , Replicação ViralRESUMO
Viral immune evasion is currently understood to focus on deflecting CD8 T cell recognition of infected cells by disrupting antigen presentation pathways. We evaluated viral interference with the ultimate step in cytotoxic T cell function, the death of infected cells. The viral inhibitor of caspase-8 activation (vICA) conserved in human cytomegalovirus (HCMV) and murine CMV (MCMV) prevents the activation of caspase-8 and proapoptotic signaling. We demonstrate the key role of vICA from either virus, in deflecting antigen-specific CD8 T cell-killing of infected cells. vICA-deficient mutants, lacking either UL36 or M36, exhibit greater susceptibility to CD8 T cell control than mutants lacking the set of immunoevasins known to disrupt antigen presentation via MHC class I. This difference is evident during infection in the natural mouse host infected with MCMV, in settings where virus-specific CD8 T cells are adoptively transferred. Finally, we identify the molecular mechanism through which vICA acts, demonstrating the central contribution of caspase-8 signaling at a point of convergence of death receptor-induced apoptosis and perforin/granzyme-dependent cytotoxicity.
Assuntos
Infecções por Citomegalovirus/imunologia , Citomegalovirus/imunologia , Interações entre Hospedeiro e Microrganismos/imunologia , Evasão da Resposta Imune , Linfócitos T Citotóxicos/imunologia , Animais , Apoptose/imunologia , Caspase 8/genética , Caspase 8/metabolismo , Linhagem Celular , Técnicas de Cocultura , Citomegalovirus/patogenicidade , Infecções por Citomegalovirus/virologia , Modelos Animais de Doenças , Fibroblastos , Granzimas/metabolismo , Antígenos de Histocompatibilidade Classe I/imunologia , Antígenos de Histocompatibilidade Classe I/metabolismo , Humanos , Camundongos , Camundongos Knockout , Muromegalovirus/genética , Muromegalovirus/imunologia , Muromegalovirus/metabolismo , Mutagênese , Perforina/genética , Perforina/metabolismo , Receptores de Morte Celular/metabolismo , Transdução de Sinais/imunologia , Linfócitos T Citotóxicos/metabolismo , Imagem com Lapso de Tempo , Proteínas Virais/genética , Proteínas Virais/imunologia , Proteínas Virais/metabolismoRESUMO
Congenital cytomegalovirus (cCMV) is the most common intrauterine infection, leading to infant neurodevelopmental disabilities. An improved knowledge of correlates of protection against cCMV is needed to guide prevention strategies. Here, we employ an ex vivo model of human CMV (HCMV) infection in decidual tissues of women with and without preconception immunity against CMV, recapitulating nonprimary vs. primary infection at the authentic maternofetal transmission site. We show that decidual tissues of women with preconception immunity against CMV exhibit intrinsic resistance to HCMV, mounting a rapid activation of tissue-resident memory CD8+ and CD4+ T cells upon HCMV reinfection. We further reveal the role of HCMV-specific decidual-tissue-resident CD8+ T cells in local protection against nonprimary HCMV infection. The findings could inform the development of a vaccine against cCMV and provide insights for further studies of the integrity of immune defense against HCMV and other pathogens at the human maternal-fetal interface.
Assuntos
Infecções por Citomegalovirus , Citomegalovirus , Lactente , Humanos , Feminino , Linfócitos T CD8-Positivos , Células T de Memória , FetoRESUMO
Current vaccines against COVID-19 elicit immune responses that are overall strong but wane rapidly. As a consequence, the necessary booster shots have contributed to vaccine fatigue. Hence, vaccines that would provide lasting protection against COVID-19 are needed, but are still unavailable. Cytomegaloviruses (CMVs) elicit lasting and uniquely strong immune responses. Used as vaccine vectors, they may be attractive tools that obviate the need for boosters. Therefore, we tested the murine CMV (MCMV) as a vaccine vector against COVID-19 in relevant preclinical models of immunization and challenge. We have previously developed a recombinant MCMV vaccine vector expressing the spike protein of the ancestral SARS-CoV-2 (MCMVS). In this study, we show that the MCMVS elicits a robust and lasting protection in young and aged mice. Notably, spike-specific humoral and cellular immunity was not only maintained but also even increased over a period of at least 6 months. During that time, antibody avidity continuously increased and expanded in breadth, resulting in neutralization of genetically distant variants, like Omicron BA.1. A single dose of MCMVS conferred rapid virus clearance upon challenge. Moreover, MCMVS vaccination controlled two variants of concern (VOCs), the Beta (B.1.135) and the Omicron (BA.1) variants. Thus, CMV vectors provide unique advantages over other vaccine technologies, eliciting broadly reactive and long-lasting immune responses against COVID-19.
Assuntos
Anticorpos Antivirais , Vacinas contra COVID-19 , COVID-19 , SARS-CoV-2 , Glicoproteína da Espícula de Coronavírus , Animais , SARS-CoV-2/imunologia , SARS-CoV-2/genética , Camundongos , Vacinas contra COVID-19/imunologia , COVID-19/prevenção & controle , COVID-19/imunologia , Glicoproteína da Espícula de Coronavírus/imunologia , Glicoproteína da Espícula de Coronavírus/genética , Anticorpos Antivirais/imunologia , Anticorpos Antivirais/sangue , Muromegalovirus/imunologia , Muromegalovirus/genética , Feminino , Anticorpos Neutralizantes/imunologia , Anticorpos Neutralizantes/sangue , Camundongos Endogâmicos BALB C , Humanos , Vetores Genéticos , Imunidade Celular , Imunidade Humoral , Modelos Animais de DoençasRESUMO
Vaccines are central to controlling the coronavirus disease 2019 (COVID-19) pandemic but the durability of protection is limited for currently approved COVID-19 vaccines. Further, the emergence of variants of concern (VoCs) that evade immune recognition has reduced vaccine effectiveness, compounding the problem. Here, we show that a single dose of a murine cytomegalovirus (MCMV)-based vaccine, which expresses the spike (S) protein of the virus circulating early in the pandemic (MCMVS), protects highly susceptible K18-hACE2 mice from clinical symptoms and death upon challenge with a lethal dose of D614G SARS-CoV-2. Moreover, MCMVS vaccination controlled two immune-evading VoCs, the Beta (B.1.135) and the Omicron (BA.1) variants in BALB/c mice, and S-specific immunity was maintained for at least 5 months after immunization, where neutralizing titers against all tested VoCs were higher at 5-months than at 1-month post-vaccination. Thus, cytomegalovirus (CMV)-based vector vaccines might allow for long-term protection against COVID-19.
RESUMO
The development of antibody therapies against SARS-CoV-2 remains a challenging task during the ongoing COVID-19 pandemic. All approved therapeutic antibodies are directed against the receptor binding domain (RBD) of the spike, and therefore lose neutralization efficacy against emerging SARS-CoV-2 variants, which frequently mutate in the RBD region. Previously, phage display has been used to identify epitopes of antibody responses against several diseases. Such epitopes have been applied to design vaccines or neutralize antibodies. Here, we constructed an ORFeome phage display library for the SARS-CoV-2 genome. Open reading frames (ORFs) representing the SARS-CoV-2 genome were displayed on the surface of phage particles in order to identify enriched immunogenic epitopes from COVID-19 patients. Library quality was assessed by both NGS and epitope mapping of a monoclonal antibody with a known binding site. The most prominent epitope captured represented parts of the fusion peptide (FP) of the spike. It is associated with the cell entry mechanism of SARS-CoV-2 into the host cell; the serine protease TMPRSS2 cleaves the spike within this sequence. Blocking this mechanism could be a potential target for non-RBD binding therapeutic anti-SARS-CoV-2 antibodies. As mutations within the FP amino acid sequence have been rather rare among SARS-CoV-2 variants so far, this may provide an advantage in the fight against future virus variants.
Assuntos
Bacteriófagos , COVID-19 , Anticorpos Neutralizantes , Anticorpos Antivirais , Formação de Anticorpos , Bacteriófagos/metabolismo , Epitopos , Humanos , Pandemias , SARS-CoV-2/genética , Glicoproteína da Espícula de CoronavírusRESUMO
Global pandemics caused by influenza or coronaviruses cause severe disruptions to public health and lead to high morbidity and mortality. There remains a medical need for vaccines against these pathogens. CMV (cytomegalovirus) is a ß-herpesvirus that induces uniquely robust immune responses in which remarkably large populations of antigen-specific CD8+ T cells are maintained for a lifetime. Hence, CMV has been proposed and investigated as a novel vaccine vector for expressing antigenic peptides or proteins to elicit protective cellular immune responses against numerous pathogens. We generated two recombinant murine CMV (MCMV) vaccine vectors expressing hemagglutinin (HA) of influenza A virus (MCMVHA) or the spike protein of severe acute respiratory syndrome coronavirus 2 (MCMVS). A single injection of MCMVs expressing either viral protein induced potent neutralizing antibody responses, which strengthened over time. Importantly, MCMVHA-vaccinated mice were protected from illness following challenge with the influenza virus, and we excluded that this protection was due to the effects of memory T cells. Conclusively, we show here that MCMV vectors induce not only long-term cellular immunity but also humoral responses that provide long-term immune protection against clinically relevant respiratory pathogens.
Assuntos
Vacinas contra COVID-19/imunologia , COVID-19/prevenção & controle , Glicoproteínas de Hemaglutininação de Vírus da Influenza/imunologia , Imunidade Humoral , Vírus da Influenza A/imunologia , Vacinas contra Influenza/imunologia , Infecções por Orthomyxoviridae/prevenção & controle , SARS-CoV-2/imunologia , Glicoproteína da Espícula de Coronavírus/imunologia , Vacinação/métodos , Animais , Anticorpos Neutralizantes/sangue , Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/sangue , Anticorpos Antivirais/imunologia , COVID-19/virologia , Chlorocebus aethiops , Citomegalovirus/imunologia , Cães , Feminino , Células HEK293 , Humanos , Imunidade Celular , Células Madin Darby de Rim Canino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Infecções por Orthomyxoviridae/virologia , Células VeroRESUMO
To fully understand the function of cytomegalovirus (CMV) genes, it is imperative that they are studied in the context of infection. Therefore, the targeted deletion of individual viral genes and the comparison of these loss-of-function viral mutants to the wild-type virus allow for the identification of the relevance and role for a particular gene in the viral replication cycle. Targeted CMV mutagenesis has made huge advances over the past 20 years. The cloning of CMV genomes into Escherichia coli as bacterial artificial chromosomes (BAC) allows for not only quick and efficient deletion of viral genomic regions, individual genes, or single-nucleotide exchanges in the viral genome but also the insertion of heterologous genetic sequences for gain-of-function approaches. The conceptual advantage of this strategy is that it overcomes the restrictions of recombinant technologies in cell culture systems. Namely, recombination in infected cells occurs only in a few clones, and their selection is not possible if the targeted genes are relevant for virus replication and are not able to compete for growth against the unrecombined parental viruses. On the other hand, BAC mutagenesis enables the selection for antibiotic resistance in E. coli, providing selective growth advantage to the recombined genomes and thus clonal selection of viruses with even extremely poor fitness. Here we describe the methods used for the generation of a CMV BAC, targeted mutagenesis of BAC clones, and transfection of human cells with CMV BAC DNA in order to reconstitute the viral infection process.
Assuntos
Cromossomos Artificiais Bacterianos/genética , Clonagem Molecular/métodos , Citomegalovirus/genética , Células Cultivadas , Escherichia coli/genética , Genes Virais/genética , Genoma Viral/genética , Humanos , Mutagênese/genética , Transfecção/métodos , Replicação Viral/genéticaRESUMO
Adoptive transfer of T cells expressing a transgenic T cell receptor (TCR) has the potential to revolutionize immunotherapy of infectious diseases and cancer. However, the generation of defined TCR-transgenic T cell medicinal products with predictable in vivo function still poses a major challenge and limits broader and more successful application of this "living drug." Here, by studying 51 different TCRs, we show that conventional genetic engineering by viral transduction leads to variable TCR expression and functionality as a result of variable transgene copy numbers and untargeted transgene integration. In contrast, CRISPR/Cas9-mediated TCR replacement enables defined, targeted TCR transgene insertion into the TCR gene locus. Thereby, T cell products display more homogeneous TCR expression similar to physiological T cells. Importantly, increased T cell product homogeneity after targeted TCR gene editing correlates with predictable in vivo T cell responses, which represents a crucial aspect for clinical application in adoptive T cell immunotherapy.
Assuntos
Edição de Genes , Genes Codificadores dos Receptores de Linfócitos T , Imunoterapia , Linfócitos T/imunologia , Animais , Linhagem Celular , Membrana Celular/metabolismo , Feminino , Humanos , Masculino , Camundongos Endogâmicos NOD , Transcrição GênicaRESUMO
The novel betacoronavirus severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2) causes a form of severe pneumonia disease called coronavirus disease 2019 (COVID-19). To develop human neutralizing anti-SARS-CoV-2 antibodies, antibody gene libraries from convalescent COVID-19 patients were constructed and recombinant antibody fragments (scFv) against the receptor-binding domain (RBD) of the spike protein were selected by phage display. The antibody STE90-C11 shows a subnanometer IC50 in a plaque-based live SARS-CoV-2 neutralization assay. The in vivo efficacy of the antibody is demonstrated in the Syrian hamster and in the human angiotensin-converting enzyme 2 (hACE2) mice model. The crystal structure of STE90-C11 Fab in complex with SARS-CoV-2-RBD is solved at 2.0 Šresolution showing that the antibody binds at the same region as ACE2 to RBD. The binding and inhibition of STE90-C11 is not blocked by many known emerging RBD mutations. STE90-C11-derived human IgG1 with FcγR-silenced Fc (COR-101) is undergoing Phase Ib/II clinical trials for the treatment of moderate to severe COVID-19.
Assuntos
Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/imunologia , SARS-CoV-2/patogenicidade , Glicoproteína da Espícula de Coronavírus/genética , COVID-19/virologia , Humanos , Mutação/genética , Peptidil Dipeptidase A/metabolismo , Ligação Proteica , Domínios Proteicos/genética , Glicoproteína da Espícula de Coronavírus/imunologiaRESUMO
Apoptosis is an important defense mechanism mounted by the immune system to control virus replication. Hence, cytomegaloviruses (CMV) evolved and acquired numerous anti-apoptotic genes. The product of the human CMV (HCMV) UL36 gene, pUL36 (also known as vICA), binds to pro-caspase-8, thus inhibiting death-receptor apoptosis and enabling viral replication in differentiated THP-1 cells. In vivo studies of the function of HCMV genes are severely limited due to the strict host specificity of cytomegaloviruses, but CMV orthologues that co-evolved with other species allow the experimental study of CMV biology in vivo. The mouse CMV (MCMV) homolog of the UL36 gene is called M36, and its protein product (pM36) is a functional homolog of vICA that binds to murine caspase-8 and inhibits its activation. M36-deficient MCMV is severely growth impaired in macrophages and in vivo. Here we show that pUL36 binds to the murine pro-caspase-8, and that UL36 expression inhibits death-receptor apoptosis in murine cells and can replace M36 to allow MCMV growth in vitro and in vivo. We generated a chimeric MCMV expressing the UL36 ORF sequence instead of the M36 one. The newly generated MCMVUL36 inhibited apoptosis in macrophage lines RAW 264.7, J774A.1, and IC-21 and its growth was rescued to wild type levels. Similarly, growth was rescued in vivo in the liver and spleen, but only partially in the salivary glands of BALB/c and C57BL/6 mice. In conclusion, we determined that an immune-evasive HCMV gene is conserved enough to functionally replace its MCMV counterpart and thus allow its study in an in vivo setting. As UL36 and M36 proteins engage the same molecular host target, our newly developed model can facilitate studies of anti-viral compounds targeting pUL36 in vivo.