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

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

Properties of MHC Class I Presented Peptides That Enhance Immunogenicity

<div><p>T-cells have to recognize peptides presented on MHC molecules to be activated and elicit their effector functions. Several studies demonstrate that some peptides are more immunogenic than others and therefore more likely to be T-cell epitopes. We set out to determine which properties cause such differences in immunogenicity. To this end, we collected and analyzed a large set of data describing the immunogenicity of peptides presented on various MHC-I molecules. Two main conclusions could be drawn from this analysis: First, in line with previous observations, we showed that positions P4–6 of a presented peptide are more important for immunogenicity. Second, some amino acids, especially those with large and aromatic side chains, are associated with immunogenicity. This information was combined into a simple model that was used to demonstrate that immunogenicity is, to a certain extent, predictable. This model (made available at <a href="http://tools.iedb.org/immunogenicity/" target="_blank">http://tools.iedb.org/immunogenicity/</a>) was validated with data from two independent epitope discovery studies. Interestingly, with this model we could show that T-cells are equipped to better recognize viral than human (self) peptides. After the past successful elucidation of different steps in the MHC-I presentation pathway, the identification of variables that influence immunogenicity will be an important next step in the investigation of T-cell epitopes and our understanding of cellular immune responses.</p></div

Data acquisition and handling oversight.

<p>Data was collected from four different sources (see <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1003266#s4" target="_blank">Methods</a>). The first panel shows how many pMHCs were derived from each data set and their respective MHC restrictions and immunogenicity status. Data from all sets was combined, the number of non-redundant 9mers with respect to the host in which the data was obtained is shown in the second panel.</p

Amino acid characteristics of immunogenic peptides presented on HLA class I molecules.

<p>Sets of amino acids were counted in immunogenic and non-immunogenic peptides based on size, aromaticity, acidity and charge, and enrichments were determined (see <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1003266#s4" target="_blank">Methods</a>). The association of these characteristics (e.g. size) with immunogenicity was tested by comparing the distributions in one extreme of a characteristic with the distribution in the other extreme of that characteristic (e.g. large versus small) using Fisher's exact test. This way, one test is performed per characteristic.</p

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

Cross-validation of the immunogenicity model.

<p>Two-thirds of the data were used for making the immunogenicity model (see <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1003266#s4" target="_blank">methods</a>) and one-third for cross-validation. The average ROC (thick grey line) of 25 of such cross-validations (thin lines) are plotted. The average AUC was 0.65.</p