Polyfunctional CD4+ T cell responses to a set of pathogenic arenaviruses provide broad population coverage

Background. Several arenaviruses cause severe hemorrhagic fever and aseptic meningitis in humans for which no licensed vaccines are available. A major obstacle for vaccine development is pathogen heterogeneity within the Arenaviridae family. Evidence in animal models and humans indicate that T cell and antibody-mediated immunity play important roles in controlling arenavirus infection and replication. Because CD4+T cells are needed for optimal CD8+T cell responses and to provide cognate help for B cells, knowledge of epitopes recognized by CD4+T cells is critical to the development of an effective vaccine strategy against arenaviruses. Thus, the goal of the present study was to define and characterize CD4+T cell responses from a broad repertoire of pathogenic arenaviruses (including lymphocytic choriomeningitis, Lassa, Guanarito, Junin, Machupo, Sabia, and Whitewater Arroyo viruses) and to provide determinants with the potential to be incorporated into a multivalent vaccine strategy. Results. By inoculating HLA-DRB1*0101 transgenic mice with a panel of recombinant vaccinia viruses, each expressing a single arenavirus antigen, we identified 37 human HLA-DRB1*0101-restricted CD4+T cell epitopes from the 7 antigenically distinct arenaviruses. We showed that the arenavirus-specific CD4+T cell epitopes are capable of eliciting T cells with a propensity to provide help and protection through CD40L and polyfunctional cytokine expression. Importantly, we demonstrated that the set of identified CD4+T cell epitopes provides broad, non-ethnically biased population coverage of all 7 arenavirus species targeted by our studies. Conclusions. The identification of CD4+T cell epitopes, with promiscuous binding properties, derived from 7 different arenavirus species will aid in the development of a T cell-based vaccine strategy with the potential to target a broad range of ethnicities within the general population and to protect against both Old and New World arenavirus infection. © 2010 Kotturi et al; licensee BioMed Central Ltd

RSV-specific airway resident memory CD8+ T cells and differential disease severity after experimental human infection

In animal models, resident memory CD8+ T (Trm) cells assist in respiratory virus elimination but their importance in man has not been determined. Here, using experimental human respiratory syncytial virus (RSV) infection, we investigate systemic and local virus-specific CD8+ T cell responses in adult volunteers. Having defined the immunodominance hierarchy, we analyze phenotype and function longitudinally in blood and by serial bronchoscopy. Despite rapid clinical recovery, we note surprisingly extensive lower airway inflammation with persistent viral antigen and cellular infiltrates. Pulmonary virus-specific CD8+ T cells display a CD69+CD103+ Trm phenotype and accumulate to strikingly high frequencies into convalescence without continued proliferation. These are more highly differentiated but express fewer cytotoxicity markers than in blood, but their abundance prior to infection correlates with protection from more severe disease

A Multivalent and Cross-Protective Vaccine Strategy against Arenaviruses Associated with Human Disease

Arenaviruses are the causative pathogens of severe hemorrhagic fever and aseptic meningitis in humans, for which no licensed vaccines are currently available. Pathogen heterogeneity within the Arenaviridae family poses a significant challenge for vaccine development. The main hypothesis we tested in the present study was whether it is possible to design a universal vaccine strategy capable of inducing simultaneous HLA-restricted CD8+ T cell responses against 7 pathogenic arenaviruses (including the lymphocytic choriomeningitis, Lassa, Guanarito, Junin, Machupo, Sabia, and Whitewater Arroyo viruses), either through the identification of widely conserved epitopes, or by the identification of a collection of epitopes derived from multiple arenavirus species. By inoculating HLA transgenic mice with a panel of recombinant vaccinia viruses (rVACVs) expressing the different arenavirus proteins, we identified 10 HLA-A02 and 10 HLA-A03-restricted epitopes that are naturally processed in human antigen-presenting cells. For some of these epitopes we were able to demonstrate cross-reactive CD8+ T cell responses, further increasing the coverage afforded by the epitope set against each different arenavirus species. Importantly, we showed that immunization of HLA transgenic mice with an epitope cocktail generated simultaneous CD8+ T cell responses against all 7 arenaviruses, and protected mice against challenge with rVACVs expressing either Old or New World arenavirus glycoproteins. In conclusion, the set of identified epitopes allows broad, non-ethnically biased coverage of all 7 viral species targeted by our studies

Tracking of CD4⁺ T cell responses during virulent <i>Salmonella</i> infection.

<p>C57BL/6 mice were infected orally with virulent <i>Salmonella</i> Typhimurium and at day 7, 14, 21 and 28 post-infection CD4⁺ T cells were isolated from the spleen and restimulated with dendritic cells that were either loaded with different <i>Salmonella</i> peptide epitopes (AhpC_154–168, EutC_243–257, STM1540_262–276 and FliC_429–443) or not loaded with peptide (medium). (A) Graphs show the number of IFN-γ⁺ spots per 2×10⁵ CD4⁺ T cells, as measured by ELISPOT assays, for each epitope at the indicated timepoints post-infection. (B) Graph shows similar data as in (A) depicted as the IFN-γ⁺ spot frequency in time. (C) Graph shows the number of IFN-γ⁺ spots per 2×10⁵ CD4⁺ T cells that were purified from mesenteric lymph nodes (MLN) at day 28 post-infection. All graphs show mean and SEM of 5–6 mice per group. Experiments were performed twice with similar results.</p

Polyfunctional CD4⁺ T Cell Responses to Immunodominant Epitopes Correlate with Disease Activity of Virulent <em>Salmonella</em>

<div><p><em>Salmonella enterica</em> serovars are intracellular bacteria capable of causing typhoid fever and gastroenteritis of significant morbidity and mortality worldwide. Current prophylactic and therapeutic treatment is hampered by the emergence of multidrug-resistant (MDR) strains of <em>Salmonella</em>, and vaccines provide only temporal and partial protection in vaccinees. To develop more effective <em>Salmonella</em> vaccines, it is important to understand the development of protective adaptive immunity to virulent <em>Salmonella</em>. Here we report the identification of novel CD4⁺ T cell peptide epitopes, which are conserved among <em>Salmonella</em> serovars. Immunization of <em>Salmonella</em>-infected mice with these peptide epitopes reduces the burden of <em>Salmonella</em> disease. Furthermore, we show that distinct polyfunctional (interferon-γ⁺, tumor necrosis factor⁺, and interleukin-2⁺) <em>Salmonella</em>-specific CD4⁺ T cell responses develop with respect to magnitude and kinetics. Moreover, we found that CD4⁺ T cell responses against immunodominant epitopes are predictive for active <em>Salmonella</em> disease. Collectively, these data could contribute to improved diagnosis of <em>Salmonella</em>-related diseases and rational design of <em>Salmonella</em> vaccines.</p> </div

Development of polyfunctional CD4⁺ T cell responses during <i>Salmonella</i> infection.

<p>C57BL/6 mice were infected orally with 1×10⁷ virulent <i>Salmonella</i> Typhimurium and at day 14 post-infection the cytokine profiles of <i>Salmonella</i>-specific CD4⁺ T cells were determined by polychromatic flow cytometry. (A) Shown are representative analyses of the IFN-γ and TNF cytokine profiles gated on CD3⁺CD4⁺ T cells, which were stimulated with dendritic cells that were either loaded with peptides (i.e. FliC_429–443, STM1540_262–276, AhpC_154–168 or EutC_243–257) or not loaded with peptide (medium). The percentage of IFN-γ⁺TNF⁺ within the CD3⁺CD4⁺ T cell population is indicated. (B) Histogram plots show the percentage of IL-2 positive cells within the double IFN-γ⁺TNF⁺ (blue line) and single IFN-γ⁺ (filled grey histograms) population. (C) Shown is the IFN-γ mean fluorescence intensity (MFI) ± SEM of epitope-specific triple IFN-γ⁺TNF⁺IL-2⁺, double IFN-γ⁺TNF⁺ and single IFN-γ⁺ CD3⁺CD4⁺ T cells. (D) Shown are representative analyses of the intracellular IL-10, IL-17A and IFN-γ expression of CD3⁺CD4⁺ T cells, which were stimulated with dendritic cells that were loaded with STM1540_262–276 peptide or not loaded with peptide (medium). The numbers indicate the percentage of cells that are positive for the indicated cytokines in each quadrant. Four independent experiments were performed with four mice per experiment.</p

<i>Salmonella</i> infection induces splenomegaly and activation of T cells.

<p>C57BL/6 mice were infected orally with 1×10⁷ virulent <i>Salmonella</i> Typhimurium (strain LT2). At day 7, 14, 21 and 28 post-infection, (A) the total number of splenocytes and the splenic weight were determined. (B) Graphs show the total number of splenic CD4⁺ (Foxp3⁻) and CD8⁺ T cells and the percentage of KLRG1 within these populations over time. Data are shown as mean ± SEM of 5–7 mice per timepoint. (C) Representative histogram plots show the KLRG1 expression on splenic CD4⁺ (Foxp3⁻) and CD8⁺ T cells at the indicated timepoints post-infection. Numbers indicate the percentage of KLRG1⁺ cells within the CD4⁺ (Foxp3⁻) or CD8⁺ T cell population. (D) Graphs show the percentage of CD69⁺ and CD62L⁻ within the CD4⁺ (Foxp3⁻) and CD8⁺ T cell populations over time. Each data point shows mean ± SEM for 5 mice per group. (E) Representative dot plots show the KLRG1 <i>versus</i> CD69 or the KLRG1 <i>versus</i> CD62L expression on splenic CD4⁺ (Foxp3⁻) and CD8⁺ T cells at day 14 post-infection. The percentage of positive cells within each quadrant is indicated. Experiments were performed twice with similar results.</p

CD4⁺ T cell epitopes to virulent <i>Salmonella</i>.

a<p>Indicated is the amino-acid position in <i>Salmonella</i> Typhimurium strain LT2 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0043481#pone.0043481-McClelland1" target="_blank">[28]</a>.</p>b<p>Mice were infected orally with 1×10⁷<i>Salmonella</i> Typhimurium (strain LT2). After 18 days, CD4⁺ T cells were tested in ELISPOT assays for IFN-γ production. Data is presented as Stimulation Index (SI), calculated as the number of spot forming cells after stimulation with peptide-loaded dendritic cells divided by spot forming cells in background.</p