Immunoprotectivity of HLA-A2 CTL peptides derived from respiratory syncytial virus fusion protein in HLA-A2 transgenic mouse

Abstract

Identification of HLA-restricted CD8+ T cell epitopes is important to study RSV-induced immunity and illness. We algorithmically analyzed the sequence of the fusion protein (F) of respiratory syncytial virus (RSV) and generated synthetic peptides that can potentially bind to HLA-A*0201. Four out of the twenty-five 9-mer peptides tested: peptides 3 (F33-41), 13 (F214-222), 14 (F273-281), and 23 (F559-567), were found to bind to HLA-A*0201 with moderate to high affinity and were capable of inducing IFN-γ and IL-2 secretion in lymphocytes from HLA-A*0201 transgenic (HLA-Tg) mice pre-immunized with RSV or recombinant adenovirus expressing RSV F. HLA-Tg mice were immunized with these four peptides and were found to induce both Th1 and CD8+ T cell responses in in vitro secondary recall. Effector responses induced by these peptides were observed to confer differential protection against live RSV challenge. These peptides also caused better recovery of body weight loss induced by RSV. A significant reduction of lung viral load was observed in mice immunized with peptide 23, which appeared to enhance the levels of inflammatory chemokines (CCL17, CCL22, and IL-18) but did not increase eosinophil infiltration in the lungs. Whereas, significant reduction of infiltrated eosinophils induced by RSV infection was found in mice pre-immunized with peptide 13. Our results suggest that HLA-A2-restricted epitopes of RSV F protein could be useful for the development of epitope-based RSV vaccine.

Figure 1. Induction of IFN-γ and IL-2 in splenocytes from RSV F peptide immunized HLA-A*0201 transgenic mice upon secondary recall.

Splenocytes harvested on day 30 from HLA-A-Tg B6 mice primed and boosted at 20 days interval intranasally with 10⁷ pfu of rAd-F0 (□) or 10⁴ pfu of RSV-B1 (▪)were restimulated with 2 µg of the individual RSV F peptide or 10 µg/ml Con A for 5 days in the presence of murine IL-2 . After stimulation, 5×10⁵ splenocytes were seeded to anti-IFN-γ (A) or anti-IL-2 (B) capture antibody coated ELISPOT plates for 2 days for ELISPOT assay as described in Materials and Methods. Cytokine-positive immunospots were developed and the results are expressed as the number of immunospots +/−2 standard deviations for each experimental group. Data is representative of results derived from two independent experiments, each with five mice per group.

Figure 2. Epitope-specific CD4⁺ and CD8⁺ T-cell activation in peptide-immunized HLA-A*0201 transgenic mice.

Splenocytes were isolated on day 17 from mice immunized twice subcutaneously with the peptides 3, 13, 14, 17, 23, or vehicle at day 0 and day 10. The splenocytes were labeled with CFSE and cultured in the presence or absence of 10 µg/mL of the respective peptides for 8 days. Proliferation of CD4⁺ (A) or CD8⁺ (B) lymphocytes in response to the different CD8 epitopes was analyzed by flow cytometry using anti-CD4 or CD8 antibodies conjugated with PE-Cy5. Results are presented as cell division index (CDI) as described in the Materials and Methods. (C) The splenocytes were stimulated in vitro with or without the peptides and were stained with anti-CD8 antibody conjugated with FITC, and then fixed and stained for intracellular IFN-γ using PE-conjugated anti-IFN-γ antibody. The percentage of CD8⁺ IFN-γ⁺ T cells was calculated. *(p<0.05) and **(p<0.01) indicate they are significantly different from the unstimulated splenocytes. Data is representative of results derived from three independent experiments.

Figure 3. Determination of lung viral load and gain of lost body weight.

Mice were immunized twice intranasally with vehicle (▪), peptide 3 (◊), peptide 13 (▴),peptide 14 (▽), or peptide 23 (□) before being intranasally challenged with 10⁷ pfu of live RSV B1. (A) The viral load in the lungs of individual mice was determined 4 days after challenge by real-time RT-PCR to quantitate RSV N gene expression as described in the Materials and Methods. 10 mice per group were used and the results are expressed as the relative expression of N gene normalized to GAPDH gene expression for each mouse. *(p<0.05) indicates they are significantly different from the vehicle-immunized group. (B) The body weight of each mouse was recorded daily for 9 days after virus challenge. Results are expressed as % (mean) for 5 mice in each experimental group. Two independent experiments were performed and data from one is shown. P value <0.05 calculated for peptide 3, peptide 13, peptide 14, and peptide 23 indicates they are significantly different compared to vehicle-immunized control.

Figure 4. Expression of proinflammatory chemokines in the lungs of HLA-A*0201 transgenic mice immunized with CD8 epitopes and challenged with RSV.

At day 4 post RSV challenge, lung RNA was extracted from the individual mice subcutaneously immunized twice with IFA-emulsified peptides or vehicle (IFA only). RNA was subjected to quantitative expression analysis of CCL11 (A), CCL17 (B), CCL22 (C), IL-13 (D), IL-17 (E), and IL-18 (F) by real-time RT-PCR using specific primers. GAPDH was used as internal control. The results are representative of the relative expression of the target gene normalized to GAPDH expression for the individual mouse. *(p<0.05) indicates the treatment is significantly different from the vehicle-immunized control. Similar results were obtained from two independent experiments, each with six mice per group, and one of them is shown.

Figure 5. Induction of pulmonary IFN-γ and CTL activity in peptide immunized transgenic mice challenged with RSV.

At day 4 post RSV infection, lung homogenates were prepared and the following were measured. (A) IFN-γ expression by ELISA, and (B) the number of CD8⁺ T cells in the lungs by flow cytometry using PE-cy5-labeled anti-CD8 antibody. (C). Enumeration of CTL activity in the lungs of mice immunized with CD8 peptide epitopes and challenged with RSV. Effector lymphocytes isolated from the lungs of mice immunized with peptide 3 (▪), peptide 13 (▴), peptide 14 (▾), or peptide 23 (•) were cultured and supplemented with murine IL-2 in the presence of 2 µg of the same peptide for 5 days. In parallel, cultured pulmonary lymphocytes from vehicle-immunized mice were stimulated with 2 µg of peptide 3 (□), peptide 13 (Δ), peptide 14 (▽), or peptide 23 (ο), respectively, for 4 days. DCs isolated from the tibia of HLA-B6 mice were pulsed with 20 µg per mL of the individual peptides for 2 hours at 37°C and labeled with CFSE and used as targets in the in vitro CTL assay. Un-pulsed DCs served as negative control. The viable effector cells were co-cultured with 10⁴ peptide-loaded target DCs cells at effector∶target ratios of 50∶1, 10∶1, and 0∶1 for five hours. The cell mixtures were labeled with 7-AAD and analyzed by flow cytometry. Results are expressed as mean percentage of 7-AAD/CFSE positive cells normalized with un-pulsed DCs. Six mice were taken in each group. The result is a representative of two independent experiments.

Figure 6. Eosinophil infiltration into the lungs of peptide-immunized transgenic mice upon RSV challenge.

Immunohistochemical analysis of lung sections with anti-major basic protein antibody specific for eosinophils followed by the HRP-conjugated anti-rat antibody was performed. (A) Pictures from the section of normal mouse lung (a), or vehicle- (b), peptide 3- (c), 13- (d), 14- (e) and 23- (f) immunized mouse lung section. (B) Quantitative representation of eosinophil count per section of peptide- or vehicle-immunized HLA-transgenic mice at day 4 post RSV challenge. Twenty bright field pictures from each lung mesenchymal region were examined and the number of eosinophils was counted under 200× magnification. The mean number of eosinophils in each group, six mice per group, is represented. Similar results from two independent experiments were obtained and one of the results is shown.