Immunization with SARS-CoV S DNA vaccine generates memory CD4+ and CD8+ T cell immune responses

Abstract

An effective vaccine for severe acute respiratory syndrome (SARS) will probably require the generation and maintenance of both humoral and cellular immune responses. It has been reported that after natural infection in humans and immunization in animals with SARS-CoV vaccine, antibody is produced and persistent for a long period of time. In the present study, mice were immunized i.m. with SARS-CoV S DNA vaccine, and three different methods (ELISA, ELISPOT and FACS) were used to evaluate the immune responses when the cells were stimulated in vitro with a pool of peptides overlapping entire SARS spike protein. The results show that prime-immunization with SARS-CoV S DNA vaccine can induce both CD4(+) and CD8(+) T cell responses. Boosting with the same vaccine enhances CD4(+) and CD8(+) T cell responses in both lymphoid and nonlymphoid organs and were persistent over two months. The SARS-CoV S-specific CD4(+) and CD8(+) T cells were CD62L(-), a marker for memory cells, and -30 to 50% of the cells expressed IL-7Ralpha (CD127), a marker for the capacity of effector cells to develop into memory cells. In addition, immunization with the DNA vaccine elicited high levels of antibody production. Taken together, these data demonstrate that immunization with SARS-CoV S DNA vaccine can generate antigen-specific humoral and cellular immune responses that may contribute to long-term protection.

Fig. 1

Frequency of SARS-CoV S-specific IFN-γ producing cells are increased following prime and boost DNA immunization. Mice were vaccinated in a similar manner as described. One week after boost, cells from spleen and lymph node were cultured at a density of 2 × 10⁵ cells/well with 1 ug/ml of pooled peptide and anti-CD28 in the 96 well pre-coated ELISPOT plate. After incubation for 16 h, ELISPOT assay was performed as described in Section 2. The empty circles represent the results in the absence of peptides and solid circles represent the results in the presence of peptides. ^*P < 0.05; ^**P < 0.01; n.s. P > 0.05.

Fig. 2

SARS-CoV S-specific CD4⁺ and CD8⁺ T cells are generated following prime and boost vaccination. Mice were vaccinated as described in Fig. 1. Cells were prepared from lymph nodes (LN), spleen and lungs, and incubated with a pool of SARS-CoV S peptides and anti-CD28 for 5 h. CD4⁺ and CD8⁺ T cells were first gated (A), IFN-γ and IL-2-producing CD4⁺ and CD8⁺ T cells were determined by intracellular cytokines staining as described in Section 2 following prime (B) and boost (C) vaccination. The frequency of IFN-γ⁺ and IL-2⁺ cells was indicated as percentage of CD4⁺ and CD8⁺ T cells.

Fig. 3

Immunization via i.m. results in higher frequency of SARS-CoV S-specific IFN-γ producing cells than that of s.c. Mice were immunized via either i.m. or s.c. with SARS-CoV S DNA vaccine. One week after boost, cells from lymph nodes, spleen and lungs were cultured and stained as described in Fig. 2. The results were expressed as a mean percentage (three to five mice) of IFN-γ-producing cells in CD4⁺ T cells (A) and CD8⁺ T cells (B).

Fig. 4

Distinct populations of SARS-CoV S specific IFN-γ and IL-2-producing CD4⁺ and CD8⁺ T cells are persistent in lymphoid and non-lymphoid organs following prime and boost DNA vaccination. Mice were vaccinated as indicated in Fig. 2 following prime (A) and boost (B) DNA vaccination. Cells were prepared from lymph nodes (LN), spleens and lungs, and incubated with a pool of SARS-CoV S peptides and anti-CD28 for 5 h. CD4⁺ and CD8⁺ T cells were first gated, the frequency of IFN-γ and/or IL-2-producing cells were analyzed within the population of CD4⁺ and CD8⁺ T cells. Results are representative of three separate experiments with similar results. The numbers at the corner in each sample represent the percentage of cytokine-producing cells.

Fig. 5

Long-term persistence of effector/memory CD4⁺ and CD8⁺ T cells following DNA vaccination. Mice were vaccinated as indicated in Fig. 2. Cells were prepared from lymph nodes (LN), spleens and lungs 8 weeks after final boost vaccination, and incubated with a pool of SARS-CoV S peptides and anti-CD28 for 5 h. CD4⁺ and CD8⁺ T cells were first gated. The frequency of IFN-γ⁺-CD4⁺ and CD8⁺-T cells (A) or IL-2⁺-CD4⁺ and CD8⁺-T cells (B) were analyzed within the population of IL-7Rα⁺ and CD62L⁺ cells. The numbers at the corner in each sample represent the percentage of positive cells.

Fig. 6

SARS-CoV S protein-specific antibody responses following DNA vaccination. BALB/c mice were immunized i.m. as described in Section 2. Sera from normal and immunized mice were collected at day 7 after boost vaccination. Antibody titers in sera were determined at different dilutions by ELISA specific for SARS-S protein. Data are expressed as the mean value ± S.D. for each group. A non-parametric two-tailed t-test was used for statistical analysis with similar result; ^*P < 0.01.