Enhanced induction of SARS-CoV nucleocapsid protein-specific immune response using DNA vaccination followed by adenovirus boosting in BALB/c mice

Abstract

Objective: To investigate immunogenicity in the induction of humoral and cellular immune responses to genetic vaccines of the recombinant severe acute respiratory syndrome-associated coronavirus (SARS-CoV)-N gene expressing the same protein plasmid, pcDNA3.1-N, and replication-defective adenoviral vector, rAd-N, in a pcDNA3.1-N prime-rAd-N boost regimen and the reverse sequence in a rAd-N prime-pcDNA3.1-N boost regimen.

Method: After the mice had been immunized intramuscularly and/or intraperitoneally with pcDNA3.1-N and rAd-N in prime-triple boost immunization, humoral and cellular immune responses were detected.

Results: After detection, different levels of anti-N humoral and cellular responses are shown compared to controls. The humoral immune response was induced more effectively by the DNA priming and recombinant adenovirus boosting regimen and the reverse sequence of heterogeneous combinations. There is a significant difference between heterogeneous and homologous vaccinations. However, the cytotoxic T lymphocyte (CTL) response was not significantly altered by the different prime-boost immunizations or the recombinant adenovirus of pcDNA3.1-N prime-rAd-N boost regimen alone, but lymphoproliferation and interferon-gamma (IFN-gamma) secretion were all enhanced by heterologous combination immunizations compared to homologous combinations. For the reverse sequence immunization regimen, lymphoproliferation, IFN-gamma and CTL responses were all significantly weaker compared with pcDNA3.1-N prime-rAd-N boost regimen.

Conclusion: Taken together, of all the combinations, the prime-triple boost immunization of pcDNA3.1-N/pcDNA3.1-N/pcDNA3.1-N/rAd-N can effectively induce SARS-CoV-N-specific and strong humoral and cellular immune responses in mice. The present results suggest that DNA immunization followed by recombinant adenovirus boosting could be used as a potential SARS-CoV vaccine in the induction of an enhanced humoral and cellular immune response.

Fig. 1a

I, II, III and IV show that mice were immunized in the week indicated; I' shows that blood and spleens were sampled, and sera and splenocytes were prepared in the week indicated in order to detect the antibody, lymphoproliferation, IFN-γ and CTL responses specific to SARS-CoV-N protein. b I, II, III and IV show that mice were immunized in the week indicated; I', II', III', IV' and V' show that blood and spleens were sampled, and sera and splenocytes were prepared in the week indicated in order to detect the antibody, lymphoproliferation, IFN-γ and CTL responses specific to SARS-CoV-N protein.

Fig. 2

Western blot analysis for N protein expression and SARS-CoV-N-specific IgG antibodies. a Western blot analysis for N protein expression. Lane 1: Lysates of SP2/0 cells transfected with pcDNA3.1-N. Lane 2: Lysates of SP2/0 cells transfected with pcDNA3.1 as negative control. Lane 3: Lysates of Vero cells infected with rAd-N. Lane 4: Lysates of Vero cells infected with rAd-LacZ as negative control. b Seven sera pools from mice of groups 1-7 were used as primary antibodies and purified N protein as antigens to perform the Western blot analysis. To lanes 1-5 were added sera from experimental groups, and to lanes 6 and 7 were added sera from the 2 groups of negative controls. c Five sera pools from mice of groups 8-12 were used as primary antibodies and purified N protein as antigens to perform the Western blot analysis. To lanes 1-3 were added sera from experimental groups, and to lanes 4 and 5 were added sera from the 2 groups of negative controls.

Fig. 3

ELISA assay of SARS-CoV N-protein-specific IgG antibodies. Mice (groups 1-7 (a) and groups 8-12 b)) were immunized with twelve combinations (shown in tables 1 and 2) in prime-triple boost immunization at 100 μg/mouse of pcDNA3.1-N or pcDNA3.1 and at 1.55 × 10⁹ or 2.4 × 10⁸ pfu/mouse of rAd-N or rAd-LacZ at 2-week intervals. ELISA was used to measure IgG antibody titers in sera at a dilution of 1:100 of individual mice and data are expressed as mean ± SD based on the OD_{450 nm} values of 5 mice in each group.

Fig. 4

Lymphoproliferative response assay. a Splenocytes from a single mouse each of groups 1-7 were stimulated in vitro: with 5 μg/ml of purified recombinant N protein in a well of a 96-well plate as experimental groups, labeled 1-7; with 10 μg/ml of PHA as positive control, labeled P₁–P₇, and without stimulation as negative controls, labeled N₁–N₇. b Splenocytes from a single mouse each of groups 8-12 were stimulated in vitro: with 5 μg/ml of purified recombinant N protein in a well of a 96-well plate as experimental groups, labeled 1-5; with 10 μg/ml of PHA as positive controls, labeled 7, and without stimulation as negative controls, labeled 6. Proliferation responses were detected by the MTT method and calculated as means of triplicate wells. Data are reported as the mean value ± SD of OD_{550 nm} of 5 or 2 mice in each group.

Fig. 5

ELISA assay of IFN-γ secretion. IFN-γ levels were measured in splenocyte culture supernatants from a single mouse of each immunized group (groups 1-7 (a), groups 8- 12 (b)) using a standard ELISA assay. A commercially available mouse IFN-γ ELISA kit was used according to the manufacturer's instructions. IFN-γ amounts are reported as the mean value ± SD of 5 or 2 mice in each group.

Fig. 6

LDH-release assay for CTLs. Splenocytes from mice of groups 1-7 (a) were prepared in week 8, and groups 8-12 (b) were prepared in weeks 0, 2, 4, 6 and 8, respectively. Splenocytes from a single mouse of each immunized group were re-stimulated in vitro with N protein-pressible SP2/0 cells for 6 days and tested for cytolytic activity by the LDH-release assay. Cytolytic activity is defined as the specific lysis percentage and is detected at 25:1, 50:1 and 100:1 of E:T cell ratios. Data on the specific lysis percentage of each group were calculated as the mean of 5 or 2 mice in each group and each experimental point represented the mean of triplicate wells.