Priming with SARS CoV S DNA and boosting with SARS CoV S epitopes specific for CD4+ and CD8+ T cells promote cellular immune responses

Abstract

Cellular immune response plays an important role in antiviral immunity. In our previous study, immunization of mice with severe acute respiratory syndrome coronavirus (SARS CoV) spike (S) DNA vaccine could induce both humoral and cellular immunity in response to a pool of entire overlapping S peptides. Identification of functional dominant epitopes in SARS CoV S protein for T cells is crucial for further understanding of cellular immune responses elicited by SARS CoV S DNA vaccine. In present study, mice were immunized with SARS CoV S DNA vaccine. Subsequently, a pool of 17-19 mers overlapped SARS CoV S peptides, which served as immunogens, were scanned to identify the specific epitopes for T cells. Two H-2(d) restricted CD4(+) T epitopes, N60 (S435-444) and P152 (S1111-1127), and two H-2(d) restricted CD8(+) T cell epitopes, N50 (S365-374) and P141 (S1031-1047) were identified by three different methods, enzyme-linked immunosorbent assay (ELISA), enzyme linked immunospot assay (ELISPOT) and fluorescence activated cell sorter (FACS). The dominant CD4(+) T cell epitope (N60) and CD8(+) T cell epitope (N50) located in the receptor-binding domain (RBD) of SARS CoV S protein, which mediated virus combining and fusing to susceptible cells. Importantly, our novel finding is that mice primed with SARS S DNA vaccine and boosted with T cell epitopes (N50 and N60) could promote antigen specific CD4(+) and CD8(+) T cell immune responses. Our study provides valuable information for the design of vaccine for SARS study.

Fig. 1

Determination of epitopes by IFN-γ production. (A) BALB/c mice were immunized i.m. by SARS CoV S DNA. One to two weeks after the final boost immunization, splenocytes were prepared and stimulated with or without peptide pools. Seventy-two hours later, supernatants were collected and levels of IFN-γ were detected by ELISA. Results represented the ratio of IFN-γ levels induced by a single pool (1–9) compared to the mixed S peptide pool in the same experiment. Each open circle represents mean value of an independent experiment. Cross bar represents the average results of three to five times experiments. Each individual peptide in pool 3 (B) and pool 8 (C) was screened as described previously. Experiments were done in triplicate. The solid bars represent the mean values + standard deviation. “0” represents non-stimulated control.

Fig. 2

Verification of potential SARS CoV S epitopes. Potential SARS CoV S epitopes P50, P51, P59 and P60 in pool 3, and P141, P144, P151 and P152 in pool 8 were used to stimulate splenocytes from SARS CoV S DNA immunized BALB/c mice. IFN-γ production was detected by ELISA (A) and ELISPOT (B), respectively. Each symbol represents the results of an individual experiment (n = 3–7). In addition, intracellular cytokine staining (C) was performed to determine CD4⁺ or CD8⁺ T cell population. “0” represents the non-peptide stimulated control. Numbers at the corner in each sample represent the percentage of positive cells. Representative results of three independent experiments were shown.

Fig. 3

Identification of new synthetic 10aa CD4 and CD8 epitopes. The overlapped amino acids between P50 and P51 (N50), and between P59 and P60 (N60) were synthesized. Peptides N50 and N60 were used to stimulate splenocytes from SARS CoV S DNA vaccine-immunized mice. P50 and P60 were used as positive controls, respectively. ELISA (A) and ELISPOT (B) were performed as described above. Furthermore, N50 and 60 were serial diluted to stimulate splenocytes from the DNA immunized mice, ELISPOT (C) was performed to detect numbers of antigen specific IFN-γ producing cells. Experiments were carried out in duplicate and representative results were shown. “0” represents non-peptide-cultured negative control.

Fig. 4

Synergistic roles of peptide N50 and N60 in H-2^b and H-2^d restricted mice. BALB/c and C57BL/6 mice were immunized by SARS CoV S DNA vaccine as described previously. One to two weeks after final boost vaccination, N50 and N60 were administrated alone or combined to stimulate splenocytes from both kinds of heterogeneous mice at the same times. ELISA (A), ELISPOT (B) and FACS (C) were performed to detect IFN-γ. “0” represents non-peptide contained negative control. Experiments were done in duplicate and representative results were shown.

Fig. 5

Characterization of N50 and N60 specific effector/memory CD4⁺ and CD8⁺ T cells. Mice were vaccinated as described previously . Two months after the final boost vaccination, cells were prepared from lymph node (LN), spleen and lung, and incubated with N50 and N60 plus anti-CD28 for 5 h. FACS was performed. CD4⁺ and CD8⁺ T cells were first gated and frequencies of IFN-γ⁺ CD4⁺ and CD8⁺ T cells were analyzed in the population of IL-7R⁺ and CD62L⁺ cells. Numbers at the corner in each sample represent the percentage of positive cells. Experiments were done in triplicate and representative results were shown.

Fig. 6

N50 and N60 can elicit antigen specific immune responses in vivo. BALB/c mice were primed twice with the DNA vaccines. Three weeks later, mice were divided into four groups (n = 4), injected by peptides (N50 and N60, 50 μg of each) plus CpG ODN (25 μg), peptide, CpG ODN and PBS, respectively. One to two weeks after boost vaccination, single cell suspensions were prepared from lymph node (LN), spleen and lung. Cells were stimulated by peptide N50 and N60 plus anti-CD28. ELISA (A) and ELISPOT (B) were performed to detect the antigen specific IFN-γ levels and cells in each group. FACS was performed to detect the frequencies of antigen specific IFN-γ and/or IL-2 producing cells in CD4⁺ (C) and CD8⁺ (D) T cell populations, respectively. Experiments were done in duplicate and representative results were shown.

Fig. 7

Production of N50 and N60 specific IgG. Pre-coating the 96 well plate with peptide N50 (open circle), N60 (open triangle), K9I (open diamond, negative control) and truncate S protein (open squad, positive control), sera from SARS CoV S DNA immunized BALB/c mice were 10-folds dilutions and added to wells. Optical density (OD) values under 450 nm were detected. The result represents one of three independent experiments.