The Breadth of Synthetic Long Peptide Vaccine-Induced CD8⁺ T Cell Responses Determines the Efficacy against Mouse Cytomegalovirus Infection

<div><p>There is an ultimate need for efficacious vaccines against human cytomegalovirus (HCMV), which causes severe morbidity and mortality among neonates and immunocompromised individuals. In this study we explored synthetic long peptide (SLP) vaccination as a platform modality to protect against mouse CMV (MCMV) infection in preclinical mouse models. In both C57BL/6 and BALB/c mouse strains, prime-booster vaccination with SLPs containing MHC class I restricted epitopes of MCMV resulted in the induction of strong and polyfunctional (i.e., IFN-γ⁺, TNF⁺, IL-2⁺) CD8⁺ T cell responses, equivalent in magnitude to those induced by the virus itself. SLP vaccination initially led to the formation of effector CD8⁺ T cells (KLRG1^hi, CD44^hi, CD127^lo, CD62L^lo), which eventually converted to a mixed central and effector-memory T cell phenotype. Markedly, the magnitude of the SLP vaccine-induced CD8⁺ T cell response was unrelated to the T cell functional avidity but correlated to the naive CD8⁺ T cell precursor frequency of each epitope. Vaccination with single SLPs displayed various levels of long-term protection against acute MCMV infection, but superior protection occurred after vaccination with a combination of SLPs. This finding underlines the importance of the breadth of the vaccine-induced CD8⁺ T cell response. Thus, SLP-based vaccines could be a potential strategy to prevent CMV-associated disease.</p></div

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

Secondary expansion potential of SLP-induced CD8⁺ T cells upon MCMV challenge.

<p>M45_985-993 and m139_419-426 epitope specific CD8⁺ T cells were isolated from the spleen at day 60 after booster vaccination and infection of SLP vaccinated and MCMV infected CD45.1 mice. 1 × 10⁴ antigen-specific CD8⁺ T cells were adoptively transferred into naive C57BL/6 (CD45.2) recipient mice. Recipient mice were subsequently infected i.p. with 5 × 10⁴ PFU MCMV-Smith. The total numbers of the donor derived M45_985-993 and m139_419-426 CD8⁺ T cells were determined in the spleen at day 5 post challenge. Data represent mean values + SEM (n = 5). Experiments were performed twice with similar outcome. *, P<0.05; **, P<0.01.</p

Prime-boost SLP vaccination provokes the induction of robust CD8⁺ T cell responses analogous to MCMV infection.

<p><b>(A)</b> The magnitude of the CD8⁺ T cell responses specific to the indicated epitopes was determined in blood by MHC class I tetramer staining at day 7 post booster vaccination with SLPs and at day 7 post MCMV infection in C57BL/6 mice and in BALB/c mice. Representative flow cytometry plots show MHC class I tetramer (Tm) staining within the CD8⁺ T cell population. Numbers represent the percentage of Tm+ cells within the total CD8⁺ T cell population. <b>(B)</b> Longitudinal analysis of the epitope-specific CD8⁺ T cell responses induced by either SLP vaccination or MCMV infection in blood. Data represents mean values ± SEM (n = 6 per group). <b>(C)</b> Percentages and total numbers of splenic SLP and MCMV-specific CD8⁺ T cells during the acute phase (at day 7 post booster vaccination and day 8 and after MCMV infection) and memory phase (at day 60 post booster vaccination and day 60 post MCMV infection) are shown. Data represents mean values + SEM (n = 6 per group), and are representative of three independent experiments. *, P< 0.05; **, P<0.01; ***, P<0.001.</p

Efficacy of single SLP vaccines against acute MCMV infection.

<p>Unvaccinated (naive), SLP vaccinated and MCMV infected C57BL/6 and BALB/c mice were challenged at day 60 post vaccination/infection with 5 × 10⁴ PFU and 5 × 10³ PFU salivary gland-derived MCMV Smith, respectively. At day 5 post challenge, spleen, liver, and lungs were isolated and the viral genome copies were determined by qPCR. The viral titres of individual <b>(A)</b> C57BL/6 and <b>(B)</b> BALB/c mice are depicted (n = 5–8 per group). Mean ± SEM is indicated. Dashed line represents the detection limit as measured in naive mice. Experiments were performed twice with similar outcome. *, P< 0.05; **, P<0.01; ***, P<0.001; ns, not significant.</p

Enhanced efficacy against CMV infection by combinatorial use of distinct SLP vaccines.

<p>C57BL/6 mice were vaccinated with single m139 and M38 SLPs, a combination of these SLPs, and a mixture of five different (M45, M57, m139, M38, IE3) SLPs. <b>(A)</b> Kinetics of the antigen-specific CD8⁺ T cells in the blood. Data shown are mean values ± SEM (n = 8) <b>(B)</b> Phenotypic profile of the (combinatorial) SLP vaccine-induced CD8⁺ T cells in blood at day 7 (acute) and day 60 (memory) post booster vaccination. Data represent mean values (n = 8). Experiment was performed twice with similar outcome. <b>(C)</b> Unvaccinated (naive), (combined) SLP vaccinated and MCMV infected C57BL/6 mice were challenged 65 day post booster-vaccination/infection with 5 × 10⁴ PFU salivary gland-derived MCMV Smith. At day 5 post challenge, liver, lungs and spleen were isolated and the viral genome copies were determined by qPCR. The viral titres of individual mice are depicted (n = 6–8 per group). <b>(D)</b> Splenic antigen-specific CD8⁺ T cells were sorted at day 60 post booster-vaccination of single m139_419-426 or M38_316-323 SLP vaccinated CD45.1 mice. 1 × 10⁴ m139_419-426, 1 × 10⁴ M38_316-323 or 0.5 × 10⁴ m139_419-426 plus 0.5 × 10⁴ M38_316-323 CD8⁺ T cells were adoptively transferred into naive CD45.2 recipient mice. Subsequently, recipient mice were infected i.p. with 5 × 10⁴ PFU MCMV-Smith. At day 5 post infection spleen and liver were isolated and the viral genome copies were determined by qPCR. The viral titres of individual mice are depicted. Mean ± SEM is also shown. The detection limit was below 1000 genome copies as measured in naive mice. Experiments were performed twice with similar outcome. Statistical difference is indicated (*, P<0.05; **, P<0.01; ***, P<0.001; ****, P<0.0001; ns, not significant) as compared to the unvaccinated group unless otherwise indicated.</p

SLP vaccination elicits polyfunctional CD8⁺ T cells.

<p>Following SLP vaccination or MCMV infection the cytokine polyfunctionality of splenic CD8⁺ T cells was determined after peptide restimulation. Representative plots show IFN-γ versus TNF production at <b>(A)</b> day 8 (acute phase) and <b>(B)</b> day 60 (memory phase) post booster vaccination and post MCMV infection. Pie charts depict the percentages of the single (IFN-γ), double (IFN-γ/TNF) and triple (IFN-γ/TNF/IL-2) cytokine producers of each antigen-specific T cell population upon peptide stimulation. Data represents mean values, and are representative of three independent experiments (n = 4–5 per group). Statistics of the results depicted in these pie charts are reported in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005895#ppat.1005895.s005" target="_blank">S4 Fig</a>.</p

The T cell precursor frequency but not the functional avidity of the SLP vaccine-induced CD8⁺ T cells predicts the magnitude of the response.

<p><b>(A)</b> Splenocytes from SLP vaccinated (n = 4–5 mice per epitope) and MCMV infected C57BL/6 mice (n = 4–5 mice) were isolated during the acute (day 8 post booster SLP vaccination and MCMV infection) and memory (day 60 post booster SLP vaccination and MCMV infection) phase and were restimulated with various peptide concentrations in the presence of brefeldin A. The percentage of IFN-γ producing CD8⁺ T cells was measured and normalized to the response at the highest peptide concentration. <b>(B)</b> Similar description as in (A) for experiments performed with BALB/c mice. Note that the M45_507-515 CD8⁺ T cell response during MCMV infection was too low to obtain accurate results. Shown are the functional avidity curves and are representative of at least 2 independent experiments. Data represents mean values. <b>(C)</b> Absolute numbers of epitope specific T cell precursors present in spleen and lymph nodes of naive C57BL/6 and BALB/c mice were determined by tetramer staining. Each symbol represents an individual mouse. Data represents mean values ± SEM (n = 4–6 per group). Experiments were performed twice with similar outcome.</p

Phenotypic heterogeneity of SLP vaccine-induced CD8⁺ T cells.

<p><b>(A)</b> Peripheral blood from either SLP immunized (day 7 after booster peptide vaccination) or MCMV infected C57BL/6 and BALB/c mice (day 7 post infection) were stained with MHC class I tetramers and for cell surface molecules. Representative plots show CD127 versus KLRG1 expression on tetramer-positive CD8⁺ T cells. <b>(B)</b> Cell-surface characteristics of tetramer-positive CD8⁺ T cells in blood and spleen at day 8 post booster SLP vaccination and day 8 after MCMV infection. <b>(C)</b> Peripheral blood from either SLP immunized (day 60 after booster peptide vaccination) or MCMV infected C57BL/6 and BALB/c mice (day 60 post infection) were stained with MHC class I tetramers and for cell surface molecules. Representative plots show CD127 versus KLRG1 expression on tetramer-positive CD8⁺ T cells. <b>(D)</b> Cell-surface characteristics of antigen-specific CD8⁺ T cells in blood and spleen at day 60 post booster SLP vaccination and day 60 after MCMV infection. Data represents mean values, and are representative of three independent experiments (n = 6 per group).</p

CD4⁺ T cells reduce the burden of virulent <i>Salmonella</i> infection.

<p>C57BL/6 mice were infected orally with 1×10⁷ virulent <i>Salmonella</i> Typhimurium. (A) At day 7, 14, 21 and 28 post-infection, the <i>Salmonella</i> CFU in the spleen were determined. Data show the mean ± SEM of 5–7 mice per timepoint. (B) At the onset of <i>Salmonella</i> infection, CD4⁺ or CD8⁺ T cell populations were depleted with anti-CD4 (clone GK1.5) or anti-CD8 (clone 2.43) monoclonal antibodies and at day 14 post-infection recoverable <i>Salmonella</i> CFU were determined. Graph shows mean + SEM for 5–6 mice per group. Statistical significance between groups is indicated (Mann-Whitney test, *<i>p</i><0.05). Experiments were performed twice with similar results.</p