Induction of Th1 type response by DNA vaccinations with N, M, and E genes against SARS-CoV in mice - PubMed (original) (raw)

Induction of Th1 type response by DNA vaccinations with N, M, and E genes against SARS-CoV in mice

Huali Jin et al. Biochem Biophys Res Commun. 2005.

Abstract

Vaccination against the SARS-CoV infection is an attractive means to control the spread of viruses in public. In this study, we employed a DNA vaccine technology with the levamisole, our newly discovered chemical adjuvant, to generate Th1 type of response. To avoid the enhancement antibody issue, genes encoding the nucleocapsid, membrane, and envelope protein of SARS-CoV were cloned and their expressions in mammalian cells were determined. After the intramuscular introduction into animals, we observed that the constructs of the E, M, and N genes could induce high levels of specific antibodies, T cell proliferations, IFN-gamma, DTH responses, and in vivo cytotoxic T cells activities specifically against SARS-CoV antigens. The highest immune responses were generated by the construct encoding the nucleocapsid protein. The results suggest that the N, M, and E genes could be used as the targets to prevent SARS-CoV infection in the DNA vaccine development.

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Figures

Fig. 1

Fig. 1

Eukaryotic expression of DNA vaccines in HeLa cells. Total RNA was extracted from HeLa cells 12 h after transfected with pcD3d/E (lane 1), pcD3d/M (lane 2), pcD3d/N (lane 3), and pcD3d (lane 4). The RNAs were used to perform RT-PCR with specific primers, respectively. Positive band of each construct was observed in the transfected sample.

Fig. 2

Fig. 2

Antibody responses to SARS-CoV mice (five per group) that were bled on day 14, 28, 42, and 56 after initial immunization. The mean titers with standard deviation (SD) of antibodies in five animals against CoV were evaluated by ELISA with 2 μg/ml of chemically killed SARS-CoV as antigen coated on each well in a 96-well plate and by using an absolute ratio of Post/Naïve serum at a cutoff of 2.1. *Indicates values that are statistically significant at a p value <0.05 by Student’s t test compared with the group of control vector.

Fig. 3

Fig. 3

T cell proliferation with MTS colorimetric detection. Single suspension of splenocytes and lymphocytes of the immunized mice was isolated 2 weeks after the second immunization and stimulated in vitro either with the killed SARS-CoV for test groups or an unrelevant protein, BSA for negative control, or with Con A as the positive control. Splenocytes from a group of normal mice were stimulated with chemically killed SARS-CoV in vitro which served as the sham control. The stimulation index was derived from the value of test group divided by medium control group. (A) T cell proliferation responses from the spleen of the animals. (B) T cell proliferation responses from the spleen of the animals. *Indicates values that are statistically significant at a p value <0.05 by Student’s t test compared with all other groups.

Fig. 4

Fig. 4

In vivo CTL assay. To analyze SARS-CoV-specific cytotoxicity in vivo, a 1:1 mixture of 107 cells of each target cell population was injected i.v. into naïve BALB/c mice 7 days after the second injection with the DNA vaccine. After 4 h, the mice were killed and the splenocytes were analyzed for the presence of CFSEhigh and CFSElow target cell populations. The percentage of specific lysis was then determined as described in Materials and methods. (A) Representative histogram plot of lymph node cells obtained from the mice injected with SARS-CoV DNA vaccines 4 h after transfer of CFSE-labeled target cells. (B–E) The SARS-CoV-specific cytotoxic responses in the lymph nodes are shown from mice previously injected by the pcD3d vector control, pcD3d/E, pcD3d/M, and pcD3d/N constructs, respectively. Each datum has been repeated twice.

Fig. 5

Fig. 5

Semi-quantitative RT-PCR assay for cytokine gene expression. Total RNA was isolated from spleen of the immunized groups or normal mice (3 for each group). The levels of the cytokines IL-2, IFN-γ, IL-4, and IL-10 were semi-quantitatively measured by a competitive RT-PCR with the addition of the 150 pg/ml of the pQRS competitor after the normalization of tested cDNA concentration to a constant amount of HPRT as described in Materials and methods.

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