Whole-Inactivated Influenza Virus Is a Potent Adjuvant for Influenza Peptides Containing CD8+ T Cell Epitopes

Influenza peptide antigens coding for conserved T cell epitopes have the capacity to induce cross-protective influenza-specific immunity. Short peptide antigens used as a vaccine, however, often show poor immunogenicity. In this study, we demonstrate that whole-inactivated influenza virus (WIV) acts as an adjuvant for influenza peptide antigens, as shown by the induction of peptide-specific CD8+ T cells in HLA-A2.1 transgenic mice upon vaccination with the influenza-M1-derived GILGFVFTL peptide (GIL), formulated with WIV. By screening various concentrations of GIL and WIV, we found that both components contributed to the GIL-specific T cell response. Whereas co-localization of the peptide antigen and WIV adjuvant was found to be important, neither physical association between peptide and WIV nor fusogenic activity of WIV were relevant for the adjuvant effect of WIV. We furthermore show that WIV may adjuvate T cell responses to a variety of peptides, using pools of either conserved wild-type influenza peptides or chemically altered peptide ligands. This study shows the potential of WIV as an adjuvant for influenza peptides. The simple formulation process and the solid safety record of WIV make this an attractive adjuvant for T cell peptides, and may also be used for non-influenza antigens

Existencia y unicidad de solución y comportamiento asintótico para la ecuación de onda con condición de frontera del tipo Neumann y disipación localmente distribuido

En este trabajo se estudia la existencia y unicidad de solución de la ecuación de la onda con condiciones de frontera del tipo Neumann, con disipación localmente distribuida usando el método de Faedo Galerkin. Además analiza el decaimiento no exponencial de la energía asociado al sistema planteado. Se hacen las estimativas correspondientes basándose en propiedades del espacio donde se encuentra la solución de la ecuación, así como los teoremas correspondientes al sistema estudiado.Tesi

Corrigendum: Bacillus Calmette–Guérin-Induced Trained Immunity Is Not Protective for Experimental Influenza A/Anhui/1/2013 (H7N9) Infection in Mice

Avian influenza A of the subtype H7N9 has been responsible for almost 1,600 confirmed human infections and more than 600 deaths since its first outbreak in 2013. Although sustained human-to-human transmission has not been reported yet, further adaptations to humans in the viral genome could potentially lead to an influenza pandemic, which may have severe consequences due to the absence of pre-existent immunity to this strain at population level. Currently there is no influenza A (H7N9) vaccine available. Therefore, in case of a pandemic outbreak, alternative preventive approaches are needed, ideally even independent of the type of influenza virus outbreak. Bacillus Calmette–Guérin (BCG) is known to induce strong heterologous immunological effects, and it has been shown that BCG protects against non-related infection challenges in several mouse models. BCG immunization of mice as well as human induces trained innate immune responses, resulting in increased cytokine responses upon subsequent ex vivo peripheral blood mononuclear cell restimulation. We investigated whether BCG (Statens Serum Institut-Denmark)-induced trained immunity may protect against a lethal avian influenza A/Anhui/1/2013 (H7N9) challenge. Here, we show that isolated splenocytes as well as peritoneal macrophages of BCG-immunized BALB/c mice displayed a trained immunity phenotype resulting in increased innate cytokine responses upon ex vivo restimulation. However, after H7N9 infection, no significant differences were found between the BCG immunized and the vehicle control group at the level of survival, weight loss, pulmonary influenza A nucleoprotein staining, or histopathology. In conclusion, BCG-induced trained immunity did not result in protection in an oseltamivir-sensitive influenza A/Anhui/1/2013 (H7N9) challenge mouse model

Chemical Modification of Influenza CD8+ T-Cell Epitopes Enhances Their Immunogenicity Regardless of Immunodominance.

T cells are essential players in the defense against infection. By targeting the MHC class I antigen-presenting pathway with peptide-based vaccines, antigen-specific T cells can be induced. However, low immunogenicity of peptides poses a challenge. Here, we set out to increase immunogenicity of influenza-specific CD8+ T cell epitopes. By substituting amino acids in wild type sequences with non-proteogenic amino acids, affinity for MHC can be increased, which may ultimately enhance cytotoxic CD8+ T cell responses. Since preventive vaccines against viruses should induce a broad immune response, we used this method to optimize influenza-specific epitopes of varying dominance. For this purpose, HLA-A*0201 epitopes GILGFVFTL, FMYSDFHFI and NMLSTVLGV were selected in order of decreasing MHC-affinity and dominance. For all epitopes, we designed chemically enhanced altered peptide ligands (CPLs) that exhibited greater binding affinity than their WT counterparts; even binding scores of the high affinity GILGFVFTL epitope could be improved. When HLA-A*0201 transgenic mice were vaccinated with selected CPLs, at least 2 out of 4 CPLs of each epitope showed an increase in IFN-γ responses of splenocytes. Moreover, modification of the low affinity epitope NMLSTVLGV led to an increase in the number of mice that responded. By optimizing three additional influenza epitopes specific for HLA-A*0301, we show that this strategy can be extended to other alleles. Thus, enhancing binding affinity of peptides provides a valuable tool to improve the immunogenicity and range of preventive T cell-targeted peptide vaccines

Binding affinity dose-response curves of CPLs and WT peptides.

<p>The IC₅₀ curves of the selected CPLs show increased HLA binding affinity compared to IC₅₀ curves of the corresponding WT-peptides. To generate IC₅₀ curves the FP-based competition assay was performed using threefold peptide dilutions in the presence of a standard amount of tracer peptide. Shown are averages and their standard deviation of three independent experiments. Curves of CPLs are shifted to the left compared to WT peptides, indicating that a lower dose of CPLs is needed to inhibit tracer binding.</p

Flow cytometry analysis on CD8⁺ T cell responses of CPL- and WT-vaccinated mice.

<p>Dot plots showing IFN-γ production by CD8⁺ T cells of mice vaccinated with 75 nmol of either WT peptide or CPL (G1, F5, N53 and N172). In the upper panel, the respective WT-peptide control of that particular experiment is shown. In the lower panel, the CPL-induced IFN-γ responses are shown. Spleen cells (2*10⁶/well) were stimulated O/N with 1 nmol/well WT peptide. Highest responders of each group are shown. Vaccination with F5 and N53 induced the largest improvement in IFN-γ production compared to WT peptide-vaccinated mice. Negative control (mock stimulated splenocytes) had an average of 0.07% with a SD of 0.1%.</p

Summary of pre-selection experiments.

<p>Summary of pre-selection experiments.</p

Vaccination with CPLs shows enhanced IFN-γ responses in vivo compared to vaccination with WT peptide.

<p>Mice were vaccinated with different doses of WT peptide or CPLs on day 0 and day 21 and two weeks later spleen cells were isolated and restimulated with homologous peptides or WT peptide. Responses were measured by IFN-γ ELISpot after 16 hours stimulation with 0.1 nmol peptide/well. Mice were vaccinated with mock (not shown), 10, 25, 50 or 100 nmol of WT GILGFVFTL or with the indicated CPLs. Spleen cells were restimulated with homologous <b>(A)</b> or WT <b>(B)</b> peptide. Overall, responses were highest after stimulation with CPL G1. For FMYSDFHFI mice were vaccinated with mock (not shown), 25, 50 or 75 nmol of WT peptide or the indicated CPLs. Cells were restimulated for 16 hours with homologous <b>(C)</b> or WT <b>(D)</b> peptide. Three out of four CPLs (F5, F100 and F111) induced higher responses compared to WT-peptide vaccination. For NMLSTVLGV mice were mock vaccinated or vaccinated with a dose of 75 nmol of WT peptide or respective CPLs. Spleen cells were restimulated with homologous <b>(E)</b> or WT <b>(F)</b> peptide. CPL N172 induced most T cells that responded to homologous stimulation, whereas N53 induced most T cells responding to WT peptide. Mock-vaccinated mice in experiments shown in Fig 2A-2D showed comparable responses to mock-vaccinated mice in Fig 2E-2F. Fig 2A-2D depict three mice per dose. Data in Fig 2E and 2F are derived from 7–8 mice per group, with the exception of the mock, for which three mice were included. Bars are min to max, with line at mean. Data were statistically analyzed using a Mann-Whitney test. * p<0.05; *** p<0.001 compared to the WT equivalent.</p