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Studies on Ebola virus disease (EVD) survivors and clinical studies on Ebola virus (EBOV) vaccine candidates have pinpointed the importance of a strong antibody response in protection and survival from EBOV infection. However, little is known about the T cell responses to EBOV or EBOV vaccines. We used HLA-A*02:01 (HLA-A2) transgenic mice to study HLA-A2-specific T cell responses elicited following vaccination with EBOV glycoprotein (EBOV-GP) presented with three different systems: (i) recombinant protein (rEBOV-GP), (ii) vesicular stomatitis replication-competent recombinant virus (VSV-EBOV-GP), and (iii) modified vaccinia Ankara virus recombinant (MVA-EBOV-GP). T cells from immunized animals were analyzed using peptide pools representing the entire GP region and individual peptides. Regardless of the vaccine formulation, we identified a minimal 9mer epitope containing an HLA-A2 motif (FLDPATTS), which was confirmed through HLA-A2 binding affinity and immunization studies. Using binding prediction software, we identified substitutions surrounding position 9 (S9V, P10V, and Q11V) that predicted enhanced binding to the HLA-A2 molecule. This enhanced binding was confirmed through in vitro binding studies and enhanced potency was shown with in vivo immunization studies using the enhanced sequences and the wild-type sequence. Of note, in silico studies predicted the enhanced 9mer epitope carrying the S9V substitution as the best overall HLA-A2 epitope for the full-length EBOV-GP. These results suggest that EBOV-GP-S9V and EBOV-GP-P10V represent more potent in vivo immunogens. Identification and enhancement of EBOV-specific human HLA epitopes could lead to the development of tools and reagents to induce more robust T cell responses in human subjects. IMPORTANCE Vaccine efficacy and immunity to viral infection are often measured by neutralizing antibody titers. T cells are specialized subsets of immune cells with antiviral activity, but this response is variable and difficult to track. We showed that the HLA-A2-specific T cell response to the Ebola virus glycoprotein can be enhanced significantly by a single residue substitution designed to improve an epitope binding affinity to one of the most frequent MHC alleles in the human population. This strategy could be applied to improve T cell responses to Ebola vaccines designed to elicit antibodies and adapted to target MHC alleles of populations in regions where endemic infections, like Ebola virus disease, are still causing outbreaks with concerning pandemic potential.
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Aminoácidos , Ebolavirus , Epítopos de Linfocito T , Glicoproteínas , Fiebre Hemorrágica Ebola , Aminoácidos/metabolismo , Animales , Anticuerpos Neutralizantes , Anticuerpos Antivirales , Vacunas contra el Virus del Ébola/genética , Ebolavirus/genética , Epítopos de Linfocito T/metabolismo , Antígeno HLA-A2/genética , Antígeno HLA-A2/metabolismo , Humanos , Ratones , Proteínas Recombinantes , Virus Vaccinia , VesiculovirusRESUMEN
Supplementing influenza vaccines with additional protective antigens such as neuraminidase (NA) is a promising strategy for increasing the breadth of the immune response. Here, we improved the immunogenicity and stability of secreted recombinant NA (rNA) tetramers by covalently conjugating them onto the surface of AP205 capsid virus-like particles (cVLPs) using a Tag/Catcher ligation system. cVLP display increased the induction of IgG2a subclass anti-NA antibodies, which exhibited cross-reactivity with an antigenically distant homologous NA. It also reduced the single dose rNA amounts needed for protection against viral challenge in mice, demonstrating a dose-sparing effect. Moreover, effective cVLP-display was achieved across different NA subtypes, even when the conjugation was performed shortly before administration. Notably, the rNA-cVLP immunogenicity was retained upon mixing or co-administering with commercial vaccines. These results highlight the potential of this approach for bolstering the protective immune responses elicited by influenza vaccines.
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In this study, we evaluated the immunogenicity and protective immunity of in vitro transcribed Venezuelan equine encephalitis virus (VEEV TC-83 strain) self-amplifying RNA (saRNA) encoding the SARS-CoV-2 spike (S) protein in wild type (S-WT) and stabilized pre-fusion conformations (S-PP). Immunization with S-WT and S-PP saRNA induced specific neutralizing antibody responses in both K18-Tg hACE2 (K18) and BALB/c mice, as assessed using SARS-CoV-2 pseudotyped viruses. Protective immunity was assessed in challenge experiments. Two immunizations with S-WT and S-PP induced protective immunity, evidenced by lower mortality, lower weight loss and more than one log10 lower subgenomic virus RNA titers in the upper and lower respiratory tracts in both K18 and BALB/c mice. Histopathologic examination of lungs post-challenge showed that immunization with S-WT and S-PP resulted in a higher degree of immune cell infiltration and inflammatory changes, compared with control mice, characterized by high levels of T- and B-cell infiltration. No substantial differences were found in the presence and localization of eosinophils, macrophages, neutrophils, and natural killer cells. CD4 and CD8 T-cell depletion post immunization resulted in reduced lung inflammation post challenge but also prolonged virus clearance. These data indicate that immunization with saRNA encoding the SARS-CoV-2 S protein induces immune responses that are protective following challenge, that virus clearance is associated with pulmonary changes caused by T-cell and B-cell infiltration in the lungs, but that this T and B-cell infiltration plays an important role in viral clearance.
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COVID-19 , SARS-CoV-2 , Glicoproteína de la Espiga del Coronavirus , Vacunas Virales , gammaglobulinas , Animales , Humanos , Ratones , Anticuerpos Neutralizantes , Anticuerpos Antivirales , COVID-19/prevención & control , Inmunización , Glicoproteína de la Espiga del Coronavirus/genéticaRESUMEN
CD8(+) T-cell immune response to liver antigens is often functionally diminished or absent. This may occur via deletion of these autoaggressive T-cells, through the acquisition of an anergic phenotype, or via active suppression mediated by other cell populations. We generated a double transgenic model in which mice express CD8(+) T-cells specific for the lymphocytic choriomeningitis virus nucleoprotein (LCMV-NP) and LCMV-NP as a hepatic neo-autoantigen, to study the immunological response of potentially liver antigen autoaggressive CD8(+) T-cells. Autoreactive transgenic CD8(+) T-cells were analyzed for functionality and cytotoxic effector status. Despite severe peripheral deletion of liver-specific CD8(+) T-cells, a fraction of autoreactive NP-specific CD8(+) T-cells accumulate in liver, resulting in hepatocyte injury and production of auto-antibodies in both male and female mice. NP-specific intrahepatic T-cells showed capacity to proliferate, produce cytokines and up-regulate activation markers. These data provide in vivo evidence that autoreactive CD8(+) T-cells are activated in the liver and developed an inflammatory process, but require additional factors to cause severe autoimmune destruction of hepatocytes. Our new model will provide a valuable tool for further exploration of the immunological response involved in inflammatory liver diseases, including autoimmune hepatitis.
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Linfocitos T CD8-positivos/inmunología , Hepatocitos/inmunología , Hígado/inmunología , Nucleoproteínas/metabolismo , Animales , Antígenos Virales/genética , Antígenos Virales/inmunología , Apoptosis/genética , Apoptosis/inmunología , Células Cultivadas , Citotoxicidad Inmunológica/genética , Modelos Animales de Enfermedad , Femenino , Hepatitis Autoinmune/inmunología , Hepatocitos/patología , Virus de la Coriomeningitis Linfocítica , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Nucleoproteínas/genética , Nucleoproteínas/inmunología , Especificidad de Órganos/genética , Especificidad de Órganos/inmunología , Transgenes/genéticaRESUMEN
INTRODUCTION: A hepatitis B vaccination (HepB) series with an initial dose of hepatitis B immune globulin (HBIG) is the recommended prophylaxis for infants born to mothers with chronic hepatitis B virus (HBV) infection and for HBV-exposed persons without known protection. The HepB and HBIG are administered at different sites (limbs). Instances of HepB and HBIG administered at the same site are documented but the impact on immune responses to HepB remains unanswered. METHODS: Newborn and adult BALB/c mice received one dose of HepB at time zero alone or with HBIG in the same or different sites, followed by 2 additional doses of HepB at 3 and 10 weeks (newborn mice) or 4 and 16 weeks (adult mice). To study memory responses mice were given a 4th, booster, dose of HepB at 26 weeks and B cells analyzed. RESULTS: Administration of HepB with HBIG resulted in reduced responses to HepB following the first 2 doses, regardless of site, compared to mice that received HepB only. Lower levels of antibody to HBV surface antigen (anti-HBs) were observed at the end of the 3-dose series (p < 0.0001) in all groups of newborn mice that received HepB and HBIG. In adult mice, this difference was only seen when HepB and HBIG were delivered at the same site. However, following a HepB booster at 26 weeks, HBsAg-specific B-cell expansion and memory phenotype were not impacted by initial HBIG administration CONCLUSION: Administration of HBIG with HepB can delay and reduce responses to HepB in mice. Our findings suggest that the initial circulating levels of HBIG could prevent infection despite an impaired response to vaccine and support the current recommendation of assessing seroprotection after series completion for infants born to HBV carrier mothers, including in cases where vaccine and HBIG are administered incorrectly at the same site.
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Vacunas contra Hepatitis B , Hepatitis B , Inmunoglobulinas , Animales , Ratones , Hepatitis B/prevención & control , Anticuerpos contra la Hepatitis B/sangre , Antígenos de Superficie de la Hepatitis B , Vacunas contra Hepatitis B/administración & dosificación , Vacunas contra Hepatitis B/uso terapéutico , Hepatitis B Crónica/prevención & control , Inmunoglobulinas/administración & dosificaciónRESUMEN
Identifying the type of diabetogenic CD8+ T cells that initiate autoimmune diabetes (AID) is a critical step in designing appropriate strategies for the early detection of beta cell-directed autoimmunity and its progression to diabetes. We generated a novel double transgenic (Tg) mouse model on the naturally diabetes resistant C57Bl/6 background, co-expressing two transgenes including a specific TCR anti-lymphocytic choriomeningitis virus nucleoprotein (LCMV-NP) carried by CD8+ T cells and LCMV-NP (as neo-self antigen) expressed by pancreatic beta cells. The resulting double Tg mouse showed partial thymic deletion of the NP-specific CD8+ T cells. The escaping autoreactive NP-specific CD8+ T cells joining the periphery were activated and gained effector functions. Both male and female mice mounted anti-NP antibodies, but only one-fourth adult males spontaneously developed AID. Significant upregulation of the CD44 and CD122 markers as compared to healthy male and female mice characterized the phenotype of diabetogenic CD8+ T cells in diabetic male mice. We also show that only 10% of these CD8+ T cells expressed programmed death 1 receptor (PD-1). Together, these results suggest that in our double Tg mouse model, Ag-specific effector CD44+CD122+PD-1-CD8+ T cell subpopulation is associated with the pathogenesis of AID.