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Introduction: Post-acute sequelae of COVID-19 affects the quality of life of many COVID-19 survivors, yet the etiology of post-acute sequelae of COVID-19 remains unknown. We aimed to determine if persistent inflammation and ongoing T-cell activation during convalescence were a contributing factor to the pathogenesis of post-acute sequelae of COVID-19. Methods: We evaluated 67 individuals diagnosed with COVID-19 by nasopharyngeal polymerase chain reaction for persistent symptoms during convalescence at separate time points occurring up to 180 days post-diagnosis. Fifty-two of these individuals were evaluated longitudinally. We obtained whole blood samples at each study visit, isolated peripheral blood mononuclear cells, and stained for multiple T cell activation markers for flow cytometry analysis. The activation states of participants' CD4+ and CD8+ T-cells were next analyzed for each of the persistent symptoms. Results: Overall, we found that participants with persistent symptoms had significantly higher levels of inflammation at multiple time points during convalescence when compared to those who fully recovered from COVID-19. Participants with persistent dyspnea, forgetfulness, confusion, and chest pain had significantly higher levels of proliferating effector T-cells (CD8+Ki67+), and those with chest pain, joint pain, difficulty concentrating, and forgetfulness had higher levels of regulatory T-cells (CD4+CD25+). Additionally, those with dyspnea had significantly higher levels of CD8+CD38+, CD8+ Granzyme B+, and CD8+IL10+ cells. A retrospective comparison of acute phase inflammatory markers in adults with and without post-acute sequelae of COVID-19 showed that CD8+Ki67+ cells were significantly higher at the time of acute illness (up to 14 days post-diagnosis) in those who developed persistent dyspnea. Discussion: These findings suggest continued CD8+ T-cell activation following SARS-CoV-2 infection in adults experiencing post-acute sequelae of COVID-19 and that the increase in T regulatory cells for a subset of these patients represents the ongoing attempt by the host to reduce inflammation.
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COVID-19 , Humanos , Adulto , COVID-19/complicaciones , Linfocitos T CD8-positivos , Estudios Retrospectivos , Convalecencia , Leucocitos Mononucleares , Antígeno Ki-67 , Síndrome Post Agudo de COVID-19 , Calidad de Vida , SARS-CoV-2 , Linfocitos T CD4-Positivos , Estudios de Cohortes , Complejo CD3 , Progresión de la Enfermedad , Inflamación , Proliferación Celular , Sobrevivientes , Disnea , Dolor en el PechoRESUMEN
INTRODUCTION: Lassa virus is a priority pathogen for vaccine research and development, however the duration of cellular immunity and protection in Lassa fever (LF) survivors remains unclear. METHODS: We investigated Lassa virus specific CD8+ T cell responses in 93 LF survivors. Peripheral blood mononuclear cells from these individuals were infected with recombinant vesicular stomatitis virus encoding Lassa virus antigens and virus specific T cell responses were measured after 18-hour incubation. Participants who had undetectable CD8+ T cell response underwent further analysis using a 10-day T cell proliferation assays to evaluate for low T cell precursor frequency. RESULTS: Forty-five of the 93 LF survivors did not have a Lassa virus specific CD8+ T cell response. Of those with responses and a known date of onset of LF (N = 11), 9 had LF within the last ten years. Most participants without a measurable CD8+ T cell response were more than 10 years removed from a clinical history of LF (N = 14/16). Fourteen of 21 patients (67%) with undetectable CD8+ T cell response had a measurable Lassa virus specific CD8+ T cell response with the 10-day assay. DISCUSSION: Despite reports of strong CD8+ T cell responses during acute Lassa virus infection, circulating Lassa virus-specific CD8+ T cells declined to undetectable levels in most Lassa fever survivors after ten years when evaluated with an 18-hour T cell stimulation. However, when Lassa virus-specific T cells were expanded prior to restimulation, a Lassa virus-specific CD8+ T cell response could be detected in many if the samples that were negative in the 18-hour stimulation assay, suggesting that prolonged cellular immunity does exist in Lassa fever survivors at low frequencies.
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Fiebre de Lassa , Células Precursoras de Linfocitos T , Humanos , Virus Lassa , Leucocitos Mononucleares , Inmunidad , Linfocitos T CD8-positivosRESUMEN
Remarkable potency has been demonstrated for mRNA vaccines in reducing the global burden of the ongoing COVID-19 pandemic. An alternative form of the mRNA vaccine is the self-amplifying mRNA (sa-mRNA) vaccine, which encodes an alphavirus replicase that self-amplifies the full-length mRNA and SARS-CoV-2 spike (S) transgene. However, early-phase clinical trials of sa-mRNA COVID-19 vaccine candidates have questioned the potential of this platform to develop potent vaccines. We examined the immune gene response to a candidate sa-mRNA vaccine against COVID-19, ARCT-021, and compared our findings to the host response to other forms of vaccines. In blood samples from healthy volunteers that participated in a phase I/II clinical trial, greater induction of transcripts involved in Toll-like receptor (TLR) signalling, antigen presentation and complement activation at 1 day post-vaccination was associated with higher anti-S antibody titers. Conversely, transcripts involved in T-cell maturation at day 7 post-vaccination informed the magnitude of eventual S-specific T-cell responses. The transcriptomic signature for ARCT-021 vaccination strongly correlated with live viral vector vaccines, adjuvanted vaccines and BNT162b2 1 day post-vaccination. Moreover, the ARCT-021 signature correlated with day 7 YF17D live-attenuated vaccine transcriptomic responses. Altogether, our findings show that sa-mRNA vaccination induces innate immune responses that are associated with the development of adaptive immunity from other forms of vaccines, supporting further development of this vaccine platform for clinical application.
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Platelets play a role not only in hemostasis and thrombosis, but also in inflammation and innate immunity. We previously reported that an activated form of tyrosyl-tRNA synthetase (YRSACT) has an extratranslational activity that enhances megakaryopoiesis and platelet production in mice. Here, we report that YRSACT mimics inflammatory stress inducing a unique megakaryocyte (MK) population with stem cell (Sca1) and myeloid (F4/80) markers through a mechanism dependent on Toll-like receptor (TLR) activation and type I interferon (IFN-I) signaling. This mimicry of inflammatory stress by YRSACT was studied in mice infected by lymphocytic choriomeningitis virus (LCMV). Using Sca1/EGFP transgenic mice, we demonstrated that IFN-I induced by YRSACT or LCMV infection suppressed normal hematopoiesis while activating an alternative pathway of thrombopoiesis. Platelets of inflammatory origin (Sca1/EGFP+) were a relevant proportion of those circulating during recovery from thrombocytopenia. Analysis of these "inflammatory" MKs and platelets suggested their origin in myeloid/MK-biased hematopoietic stem cells (HSCs) that bypassed the classical MK-erythroid progenitor (MEP) pathway to replenish platelets and promote recovery from thrombocytopenia. Notably, inflammatory platelets displayed enhanced agonist-induced activation and procoagulant activities. Moreover, myeloid/MK-biased progenitors and MKs were mobilized from the bone marrow, as evidenced by their presence in the lung microvasculature within fibrin-containing microthrombi. Our results define the function of YRSACT in platelet generation and contribute to elucidate platelet alterations in number and function during viral infection.
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Ataxias Espinocerebelosas , Trombocitopenia , Trombosis , Tirosina-ARNt Ligasa , Virosis , Ratones , Animales , Trombopoyesis , Ratones TransgénicosRESUMEN
A self-transcribing and replicating RNA (STARR)-based vaccine (LUNAR-COV19) has been developed to prevent SARS-CoV-2 infection. The vaccine encodes an alphavirus-based replicon and the SARS-CoV-2 full-length spike glycoprotein. Translation of the replicon produces a replicase complex that amplifies and prolongs SARS-CoV-2 spike glycoprotein expression. A single prime vaccination in mice led to robust antibody responses, with neutralizing antibody titers increasing up to day 60. Activation of cell-mediated immunity produced a strong viral antigen-specific CD8+ T lymphocyte response. Assaying for intracellular cytokine staining for interferon (IFN)γ and interleukin-4 (IL-4)-positive CD4+ T helper (Th) lymphocytes as well as anti-spike glycoprotein immunoglobulin G (IgG)2a/IgG1 ratios supported a strong Th1-dominant immune response. Finally, single LUNAR-COV19 vaccination at both 2 µg and 10 µg doses completely protected human ACE2 transgenic mice from both mortality and even measurable infection following wild-type SARS-CoV-2 challenge. Our findings collectively suggest the potential of LUNAR-COV19 as a single-dose vaccine.
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Anticuerpos Neutralizantes/biosíntesis , Anticuerpos Antivirales/biosíntesis , Vacunas contra la COVID-19/administración & dosificación , COVID-19/prevención & control , SARS-CoV-2/efectos de los fármacos , Glicoproteína de la Espiga del Coronavirus/inmunología , Vacunas Sintéticas/administración & dosificación , Alphavirus/genética , Alphavirus/inmunología , Enzima Convertidora de Angiotensina 2/genética , Enzima Convertidora de Angiotensina 2/inmunología , Animales , Linfocitos T CD8-positivos/efectos de los fármacos , Linfocitos T CD8-positivos/inmunología , Linfocitos T CD8-positivos/virología , COVID-19/inmunología , COVID-19/patología , COVID-19/virología , Vacunas contra la COVID-19/biosíntesis , Vacunas contra la COVID-19/genética , Vacunas contra la COVID-19/inmunología , Femenino , Expresión Génica , Humanos , Inmunidad Celular/efectos de los fármacos , Inmunidad Humoral/efectos de los fármacos , Interferón gamma/genética , Interferón gamma/inmunología , Interleucina-4/genética , Interleucina-4/inmunología , Ratones , Ratones Transgénicos , Replicón/inmunología , SARS-CoV-2/inmunología , SARS-CoV-2/patogenicidad , Glicoproteína de la Espiga del Coronavirus/química , Glicoproteína de la Espiga del Coronavirus/genética , Células TH1/efectos de los fármacos , Células TH1/inmunología , Células TH1/virología , Transgenes , Resultado del Tratamiento , Vacunación/métodos , Vacunas Sintéticas/biosíntesis , Vacunas Sintéticas/genética , Vacunas Sintéticas/inmunología , Vacunas de ARNmRESUMEN
Lassa fever (LF) causes multisystem disease and has a fatality rate <70%. Severe cases exhibit abnormal coagulation, endothelial barrier disruption, and dysfunctional platelet aggregation but the underlying mechanisms remain poorly understood. In Sierra Leone during 2015-2018, we assessed LF patients' day-of-admission plasma samples for levels of proteins necessary for coagulation, fibrinolysis, and platelet function. P-selectin, soluble endothelial protein C receptor, soluble thrombomodulin, plasminogen activator inhibitor 1, ADAMTS-13, von Willebrand factor, tissue factor, soluble intercellular adhesion molecule 1, and vascular cell adhesion molecule 1 were more elevated in LF patients than in controls. Endothelial protein C receptor, thrombomodulin, intercellular adhesion molecule 1, plasminogen activator inhibitor 1, D-dimer, and hepatocyte growth factor were higher in fatal than nonfatal LF cases. Platelet disaggregation occurred only in samples from fatal LF cases. The impaired homeostasis and platelet dysfunction implicate alterations in the protein C pathway, which might contribute to the loss of endothelial barrier function in fatal infections.
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Plaquetas/patología , Endotelio/fisiopatología , Fiebre de Lassa , Adolescente , Adulto , Anciano , Coagulación Sanguínea , Niño , Preescolar , Femenino , Fibrinólisis , Humanos , Lactante , Fiebre de Lassa/diagnóstico , Fiebre de Lassa/epidemiología , Masculino , Persona de Mediana Edad , Sierra Leona , Adulto JovenRESUMEN
BACKGROUND: Ebola virus (EBOV) disease has killed thousands of West and Central Africans over the past several decades. Many who survive the acute disease later experience post-Ebola syndrome, a constellation of symptoms whose causative pathogenesis is unclear. METHODS: We investigated EBOV-specific CD8+ and CD4+ T-cell responses in 37 Sierra Leonean EBOV disease survivors with (n = 19) or without (n = 18) sequelae of arthralgia and ocular symptoms. Peripheral blood mononuclear cells were infected with recombinant vesicular stomatitis virus encoding EBOV antigens. We also studied the presence of EBOV-specific immunoglobulin G, antinuclear antibodies, anti-cyclic citrullinated peptide antibodies, rheumatoid factor, complement levels, and cytokine levels in these 2 groups. RESULTS: Survivors with sequelae had a significantly higher EBOV-specific CD8+ and CD4+ T-cell response. No differences in EBOV-specific immunoglobulin G, antinuclear antibody, or anti-cyclic citrullinated peptide antibody levels were found. Survivors with sequelae showed significantly higher rheumatoid factor levels. CONCLUSION: EBOV-specific CD8+ and CD4+ T-cell responses were significantly higher in Ebola survivors with post-Ebola syndrome. These findings suggest that pathogenesis may occur as an immune-mediated disease via virus-specific T-cell immune response or that persistent antigen exposure leads to increased and sustained T-cell responses.
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Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD8-positivos/inmunología , Ebolavirus/inmunología , Fiebre Hemorrágica Ebola/inmunología , Adulto , Anticuerpos Antivirales/inmunología , Antígenos Virales/inmunología , Femenino , Técnica del Anticuerpo Fluorescente , Fiebre Hemorrágica Ebola/patología , Humanos , Inmunidad Celular , Masculino , Sierra Leona/epidemiología , SobrevivientesRESUMEN
Early and robust T cell responses have been associated with survival from Lassa fever (LF), but the Lassa virus-specific memory responses have not been well characterized. Regions within the virus surface glycoprotein (GPC) and nucleoprotein (NP) are the main targets of the Lassa virus-specific T cell responses, but, to date, only a few T cell epitopes within these proteins have been identified. We identified GPC and NP regions containing T cell epitopes and HLA haplotypes from LF survivors and used predictive HLA-binding algorithms to identify putative epitopes, which were then experimentally tested using autologous survivor samples. We identified 12 CD8-positive (CD8+) T cell epitopes, including epitopes common to both Nigerian and Sierra Leonean survivors. These data should be useful for the identification of dominant Lassa virus-specific T cell responses in Lassa fever survivors and vaccinated individuals as well as for designing vaccines that elicit cell-mediated immunity.IMPORTANCE The high morbidity and mortality associated with clinical cases of Lassa fever, together with the lack of licensed vaccines and limited and partially effective interventions, make Lassa virus (LASV) an important health concern in its regions of endemicity in West Africa. Previous infection with LASV protects from disease after subsequent exposure, providing a framework for designing vaccines to elicit similar protective immunity. Multiple major lineages of LASV circulate in West Africa, and therefore, ideal vaccine candidates should elicit immunity to all lineages. We therefore sought to identify common T cell epitopes between Lassa fever survivors from Sierra Leone and Nigeria, where distinct lineages circulate. We identified three such epitopes derived from highly conserved regions within LASV proteins. In this process, we also identified nine other T cell epitopes. These data should help in the design of an effective pan-LASV vaccine.
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Linfocitos T CD8-positivos/inmunología , Epítopos de Linfocito T/química , Fiebre de Lassa/inmunología , Virus Lassa/inmunología , Nucleoproteínas/inmunología , Proteínas del Envoltorio Viral/inmunología , Adolescente , Secuencia de Aminoácidos , Animales , Anticuerpos Antivirales/biosíntesis , Antígenos Virales/química , Antígenos Virales/genética , Antígenos Virales/inmunología , Linfocitos T CD8-positivos/virología , Niño , Epítopos de Linfocito T/genética , Epítopos de Linfocito T/inmunología , Femenino , Genes Reporteros , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/inmunología , Antígenos HLA-DQ/genética , Antígenos HLA-DQ/inmunología , Haplotipos , Interacciones Huésped-Patógeno/genética , Interacciones Huésped-Patógeno/inmunología , Humanos , Sueros Inmunes/análisis , Memoria Inmunológica , Fiebre de Lassa/genética , Fiebre de Lassa/patología , Virus Lassa/patogenicidad , Masculino , Nigeria , Nucleoproteínas/genética , Sierra Leona , Sobrevivientes , Proteínas del Envoltorio Viral/genética , Adulto JovenRESUMEN
Lassa virus infects hundreds of thousands of people each year across rural West Africa, resulting in a high number of cases of Lassa fever (LF), a febrile disease associated with high morbidity and significant mortality. The lack of approved treatments or interventions underscores the need for an effective vaccine. At least four viral lineages circulate in defined regions throughout West Africa with substantial interlineage nucleotide and amino acid diversity. An effective vaccine should be designed to elicit Lassa virus specific humoral and cell mediated immunity across all lineages. Most current vaccine candidates use only lineage IV antigens encoded by Lassa viruses circulating around Sierra Leone, Liberia, and Guinea but not Nigeria where lineages I-III are found. As previous infection is known to protect against disease from subsequent exposure, we sought to determine whether LF survivors from Nigeria and Sierra Leone harbor memory T cells that respond to lineage IV antigens. Our results indicate a high degree of cross-reactivity of CD8+ T cells from Nigerian LF survivors to lineage IV antigens. In addition, we identified regions within the Lassa virus glycoprotein complex and nucleoprotein that contributed to these responses while T cell epitopes were not widely conserved across our study group. These data are important for current efforts to design effective and efficient vaccine candidates that can elicit protective immunity across all Lassa virus lineages.
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Antígenos Virales/inmunología , Linfocitos T CD8-positivos/inmunología , Epítopos de Linfocito T/inmunología , Virus Lassa/inmunología , África Occidental , Reacciones Cruzadas , Femenino , Humanos , Masculino , Especificidad de la EspecieRESUMEN
Lymphocytic choriomeningitis virus (LCMV) WE variant 2.2 (v2.2) generated a high level of the major mouse urinary protein: MUP. Mice infected with LCMV WE v54, which differed from v2.2 by a single amino acid in the viral glycoprotein, failed to generate MUP above baseline levels found in uninfected controls. Variant 54 bound at 2.5 logs higher affinity to the LCMV receptor α-dystroglycan (α-DG) than v2.2 and entered α-DG-expressing but not α-DG-null cells. Variant 2.2 infected both α-DG-null or -expressing cells. Variant 54 infected more dendritic cells, generated a negligible CD8 T cell response, and caused a persistent infection, while v2.2 generated cytotoxic T lymphocytes (CTLs) and cleared virus within 10 days. By 20 days postinfection and through the 80-day observation period, significantly higher amounts of MUP were found in v2.2-infected mice. Production of MUP was dependent on virus-specific CTL as deletion of such cells aborted MUP production. Furthermore, MUP production was not elevated in v2.2 persistently infected mice unless virus was cleared following transfer of virus-specific CTL.
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Linfocitos T CD8-positivos/inmunología , Regulación de la Expresión Génica/inmunología , Coriomeningitis Linfocítica/inmunología , Virus de la Coriomeningitis Linfocítica/inmunología , Proteínas/inmunología , Animales , Distroglicanos/inmunología , Coriomeningitis Linfocítica/patología , RatonesRESUMEN
Understanding of T cell exhaustion and successful therapy to restore T cell function was first described using Clone (Cl) 13 variant selected from the lymphocytic choriomeningitis virus (LCMV) Armstrong (ARM) 53b parental strain. T cell exhaustion plays a pivotal role in both persistent infections and cancers of mice and humans. C57BL/6, BALB, SWR/J, A/J, 129, C3H, and all but one collaborative cross (CC) mouse strain following Cl 13 infection have immunosuppressed T cell responses, high PD-1, and viral titers leading to persistent infection and normal life spans. In contrast, the profile of FVB/N, NZB, PL/J, SL/J, and CC NZO mice challenged with Cl 13 is a robust T cell response, high titers of virus, PD-1, and Lag3 markers on T cells. These mice all die 7 to 9 d after Cl 13 infection. Death is due to enhanced pulmonary endothelial vascular permeability, pulmonary edema, collapse of alveolar air spaces, and respiratory failure. Pathogenesis involves abundant levels of Cl 13 receptor alpha-dystroglycan on endothelial cells, with high viral replication in such cells leading to immunopathologic injury. Death is aborted by blockade of interferon-1 (IFN-1) signaling or deletion of CD8 T cells.
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Linfocitos T CD8-positivos , Interferón Tipo I , Coriomeningitis Linfocítica , Virus de la Coriomeningitis Linfocítica/fisiología , Replicación Viral/genética , Animales , Antígenos CD/genética , Antígenos CD/metabolismo , Linfocitos T CD8-positivos/metabolismo , Linfocitos T CD8-positivos/patología , Humanos , Interferón Tipo I/genética , Interferón Tipo I/metabolismo , Coriomeningitis Linfocítica/genética , Coriomeningitis Linfocítica/metabolismo , Coriomeningitis Linfocítica/patología , Ratones , Receptor de Muerte Celular Programada 1/genética , Receptor de Muerte Celular Programada 1/metabolismo , Proteína del Gen 3 de Activación de LinfocitosRESUMEN
The recent Ebola epidemic exemplified the importance of understanding and controlling emerging infections. Despite the importance of T cells in clearing virus during acute infection, little is known about Ebola-specific CD8+ T cell responses. We investigated immune responses of individuals infected with Ebola virus (EBOV) during the 2013-2016 West Africa epidemic in Sierra Leone, where the majority of the >28,000 EBOV disease (EVD) cases occurred. We examined T cell memory responses to seven of the eight Ebola proteins (GP, sGP, NP, VP24, VP30, VP35, and VP40) and associated HLA expression in survivors. Of the 30 subjects included in our analysis, CD8+ T cells from 26 survivors responded to at least one EBOV antigen. A minority, 10 of 26 responders (38%), made CD8+ T cell responses to the viral GP or sGP. In contrast, 25 of the 26 responders (96%) made response to viral NP, 77% to VP24 (20 of 26), 69% to VP40 (18 of 26), 42% (11 of 26) to VP35, with no response to VP30. Individuals making CD8+ T cells to EBOV VP24, VP35, and VP40 also made CD8+ T cells to NP, but rarely to GP. We identified 34 CD8+ T cell epitopes for Ebola. Our data indicate the immunodominance of the EBOV NP-specific T cell response and suggest that its inclusion in a vaccine along with the EBOV GP would best mimic survivor responses and help boost cell-mediated immunity during vaccination.
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Anticuerpos Antivirales/inmunología , Linfocitos T CD8-positivos/inmunología , Ebolavirus/inmunología , Epidemias , Antígenos HLA/inmunología , Fiebre Hemorrágica Ebola/inmunología , Adolescente , Adulto , Anticuerpos Antivirales/sangre , Antígenos Virales/inmunología , Epítopos de Linfocito T/inmunología , Femenino , Antígenos HLA/sangre , Fiebre Hemorrágica Ebola/sangre , Fiebre Hemorrágica Ebola/epidemiología , Fiebre Hemorrágica Ebola/prevención & control , Humanos , Masculino , Nucleoproteínas/inmunología , Sierra Leona , Sobrevivientes , Vacunación/métodos , Proteínas Virales/inmunología , Adulto JovenRESUMEN
Arenaviruses exist worldwide and can cause hemorrhagic fever and neurologic disease. A single glycoprotein expressed on the viral surface mediates entry into target cells. This glycoprotein, termed GPC, contains a membrane-associated signal peptide, a receptor-binding subunit termed GP1 and a fusion-mediating subunit termed GP2. Although GPC is a critical target of antibodies and vaccines, the structure of the metastable GP1-GP2 prefusion complex has remained elusive for all arenaviruses. Here we describe the crystal structure of the fully glycosylated prefusion GP1-GP2 complex of the prototypic arenavirus LCMV at 3.5 Å. This structure reveals the conformational changes that the arenavirus glycoprotein must undergo to cause fusion and illustrates the fusion regions and potential oligomeric states.
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Virus de la Coriomeningitis Linfocítica/química , Glicoproteínas de Membrana/química , Proteínas del Envoltorio Viral/química , Animales , Línea Celular , Cristalografía por Rayos X , Drosophila , Glicosilación , Humanos , Coriomeningitis Linfocítica/metabolismo , Coriomeningitis Linfocítica/virología , Virus de la Coriomeningitis Linfocítica/fisiología , Glicoproteínas de Membrana/metabolismo , Modelos Moleculares , Conformación Proteica , Multimerización de Proteína , Señales de Clasificación de Proteína , Subunidades de Proteína/química , Subunidades de Proteína/metabolismo , Proteínas del Envoltorio Viral/metabolismo , Internalización del VirusRESUMEN
UNLABELLED: Hemorrhagic fever arenaviruses (HFAs) pose important public health problems in regions where they are endemic. Concerns about human-pathogenic arenaviruses are exacerbated because of the lack of FDA-licensed arenavirus vaccines and because current antiarenaviral therapy is limited to an off-label use of ribavirin that is only partially effective. We have recently shown that the noncoding intergenic region (IGR) present in each arenavirus genome segment, the S and L segments (S-IGR and L-IGR, respectively), plays important roles in the control of virus protein expression and that this knowledge could be harnessed for the development of live-attenuated vaccine strains to combat HFAs. In this study, we further investigated the sequence plasticity of the arenavirus IGR. We demonstrate that recombinants of the prototypic arenavirus lymphocytic choriomeningitis virus (rLCMVs), whose S-IGRs were replaced by the S-IGR of Lassa virus (LASV) or an entirely nonviral S-IGR-like sequence (Ssyn), are viable, indicating that the function of S-IGR tolerates a high degree of sequence plasticity. In addition, rLCMVs whose L-IGRs were replaced by Ssyn or S-IGRs of the very distantly related reptarenavirus Golden Gate virus (GGV) were viable and severely attenuated in vivo but able to elicit protective immunity against a lethal challenge with wild-type LCMV. Our findings indicate that replacement of L-IGR by a nonviral Ssyn could serve as a universal molecular determinant of arenavirus attenuation. IMPORTANCE: Hemorrhagic fever arenaviruses (HFAs) cause high rates of morbidity and mortality and pose important public health problems in regions where they are endemic. Implementation of live-attenuated vaccines (LAVs) will represent a major step to combat HFAs. Here we document that the arenavirus noncoding intergenic region (IGR) has a high degree of plasticity compatible with virus viability. This observation led us to generate recombinant LCMVs containing nonviral synthetic IGRs. These rLCMVs were severely attenuated in vivo but able to elicit protective immunity against a lethal challenge with wild-type LCMV. These nonviral synthetic IGRs can be used as universal molecular determinants of arenavirus attenuation for the rapid development of safe and effective, as well as stable, LAVs to combat HFA.
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ADN Intergénico , Virus de la Coriomeningitis Linfocítica/genética , Virus de la Coriomeningitis Linfocítica/patogenicidad , Mutagénesis Insercional , Recombinación Genética , Vacunas Virales/inmunología , Animales , Infecciones por Arenaviridae/patología , Infecciones por Arenaviridae/prevención & control , Modelos Animales de Enfermedad , Virus Lassa/genética , Virus de la Coriomeningitis Linfocítica/fisiología , Ratones Endogámicos C57BL , Viabilidad Microbiana , Análisis de Supervivencia , Vacunas Atenuadas/administración & dosificación , Vacunas Atenuadas/genética , Vacunas Atenuadas/inmunología , Vacunas Virales/administración & dosificación , Vacunas Virales/genéticaRESUMEN
Although type I interferon (IFN-I) is thought to be beneficial against microbial infections, persistent viral infections are characterized by high interferon signatures suggesting that IFN-I signaling may promote disease pathogenesis. During persistent lymphocytic choriomeningitis virus (LCMV) infection, IFNα and IFNß are highly induced early after infection, and blocking IFN-I receptor (IFNAR) signaling promotes virus clearance. We assessed the specific roles of IFNß versus IFNα in controlling LCMV infection. While blockade of IFNß alone does not alter early viral dissemination, it is important in determining lymphoid structure, lymphocyte migration, and anti-viral T cell responses that lead to accelerated virus clearance, approximating what occurs during attenuation of IFNAR signaling. Comparatively, blockade of IFNα was not associated with improved viral control, but with early dissemination of virus. Thus, despite their use of the same receptor, IFNß and IFNα have unique and distinguishable biologic functions, with IFNß being mainly responsible for promoting viral persistence.
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Interferón-alfa/metabolismo , Interferón beta/metabolismo , Coriomeningitis Linfocítica/inmunología , Coriomeningitis Linfocítica/virología , Virus de la Coriomeningitis Linfocítica/inmunología , Transducción de Señal , Animales , Modelos Animales de Enfermedad , Ratones Endogámicos C57BL , Ratones NoqueadosRESUMEN
Many persistent viral infections are characterized by a hypofunctional T cell response and the upregulation of negative immune regulators. These events occur days after the initiation of infection. However, the very early host-virus interactions that determine the establishment of viral persistence remain poorly uncharacterized. Here we show that to establish persistence, LCMV must counteract an innate anti-viral immune response within eight hours after infection. While the virus triggers cytoplasmic RNA sensing pathways soon after infection, LCMV counteracts this pathway through a rapid increase in viral titers leading to a dysfunctional immune response characterized by a high cytokine and chemokine expression profile. This altered immune environment allows for viral replication in the splenic white pulp as well as infection of immune cells essential to an effective anti-viral immune response. Our findings illustrate how early events during infection critically dictate the characteristics of the immune response to infection and facilitate either virus control and clearance or persistence.
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Interacciones Huésped-Patógeno/fisiología , Coriomeningitis Linfocítica/inmunología , Virosis , Animales , Células Cultivadas , Chlorocebus aethiops , Enfermedad Crónica , Cricetinae , Tolerancia Inmunológica , Inmunidad Innata , Coriomeningitis Linfocítica/virología , Virus de la Coriomeningitis Linfocítica/inmunología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Bazo/inmunología , Factores de Tiempo , Células Vero , Virosis/inmunología , Virosis/virología , Latencia del Virus/inmunología , Replicación Viral/inmunologíaRESUMEN
The outcome of a viral infection reflects the balance between virus virulence and host susceptibility. The clone 13 (Cl13) variant of lymphocytic choriomeningitis virus--a prototype of Old World arenaviruses closely related to Lassa fever virus--elicits in C57BL/6 and BALB/c mice abundant negative immunoregulatory molecules, associated with T-cell exhaustion, negligible T-cell-mediated injury, and high virus titers that persist. Conversely, here we report that in NZB mice, despite the efficient induction of immunoregulatory molecules and high viremia, Cl13 generated a robust cytotoxic T-cell response, resulting in thrombocytopenia, pulmonary endothelial cell loss, vascular leakage, and death within 6-8 d. These pathogenic events required type I IFN (IFN-I) signaling on nonhematopoietic cells and were completely abrogated by IFN-I receptor blockade. Thus, IFN-I may play a prominent role in hemorrhagic fevers and other acute virus infections associated with severe vascular pathology, and targeting IFN-I or downstream effector molecules may be an effective therapeutic approach.
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Interferón Tipo I/metabolismo , Fiebre de Lassa/virología , Enfermedades Vasculares/virología , Animales , Lavado Broncoalveolar , Línea Celular , Cricetinae , Citocinas/metabolismo , Femenino , Virus Lassa , Coriomeningitis Linfocítica/virología , Virus de la Coriomeningitis Linfocítica/metabolismo , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Endogámicos NZB , Ratones Transgénicos , Transducción de Señal , Células Madre/química , Linfocitos T Citotóxicos/virología , Activación ViralRESUMEN
During persistent viral infections, chronic immune activation, negative immune regulator expression, an elevated interferon signature, and lymphoid tissue destruction correlate with disease progression. We demonstrated that blockade of type I interferon (IFN-I) signaling using an IFN-I receptor neutralizing antibody reduced immune system activation, decreased expression of negative immune regulatory molecules, and restored lymphoid architecture in mice persistently infected with lymphocytic choriomeningitis virus. IFN-I blockade before and after establishment of persistent virus infection resulted in enhanced virus clearance and was CD4 T cell-dependent. Hence, we demonstrate a direct causal link between IFN-I signaling, immune activation, negative immune regulator expression, lymphoid tissue disorganization, and virus persistence. Our results suggest that therapies targeting IFN-I may help control persistent virus infections.
Asunto(s)
Infecciones por Arenaviridae/inmunología , Infecciones por Arenaviridae/virología , Interferón Tipo I/metabolismo , Virus de la Coriomeningitis Linfocítica/inmunología , Virus de la Coriomeningitis Linfocítica/fisiología , Transducción de Señal , Animales , Anticuerpos Antivirales/sangre , Infecciones por Arenaviridae/patología , Antígeno B7-H1/metabolismo , Linfocitos T CD4-Positivos/inmunología , Citocinas/metabolismo , Células Dendríticas/inmunología , Células Dendríticas/virología , Femenino , Tolerancia Inmunológica , Interferón Tipo I/inmunología , Interleucina-10/metabolismo , Linfocitos/inmunología , Linfocitos/virología , Masculino , Ratones , Ratones Endogámicos C57BL , Receptor de Interferón alfa y beta/inmunología , Receptor de Interferón alfa y beta/metabolismo , Bazo/inmunología , Bazo/patología , ViremiaRESUMEN
A recent publication indicated that overexpression of Axl, a cellular receptor that negatively regulates Toll-like receptor signaling, enhanced the entry of viruses pseudotyped with the glycoprotein of lymphocytic choriomeningitis virus (LCMV) in vitro. In testing the biological relevance of these observations, we found differences in neither viral kinetics between LCMV infections of Axl(-/-) and wild-type mice nor T-cell responses prior to spontaneous viral clearance. Thus, Axl is not required for productive LCMV infection of mice.
Asunto(s)
Infecciones por Arenaviridae/fisiopatología , Arenavirus/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Tirosina Quinasas Receptoras/metabolismo , Internalización del Virus , Animales , Arenavirus/fisiología , Fluorescencia , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Proteínas Proto-Oncogénicas/genética , Proteínas Tirosina Quinasas Receptoras/genética , Tirosina Quinasa del Receptor AxlRESUMEN
Plasmacytoid dendritic cells (pDCs) are the major producers of type I IFN in response to viral infection and have been shown to direct both innate and adaptive immune responses in vitro. However, in vivo evidence for their role in viral infection is lacking. We evaluated the contribution of pDCs to acute and chronic virus infection using the feeble mouse model of pDC functional deficiency. We have previously demonstrated that feeble mice have a defect in TLR ligand sensing. Although pDCs were found to influence early cytokine secretion, they were not required for control of viremia in the acute phase of the infection. However, T cell priming was deficient in the absence of functional pDCs and the virus-specific immune response was hampered. Ultimately, infection persisted in feeble mice. We conclude that pDCs are likely required for efficient T cell priming and subsequent viral clearance. Our data suggest that reduced pDC functionality may lead to chronic infection.