The expression of membrane protein augments the specific responses induced by SARS-CoV nucleocapsid DNA immunization.
Nucleocapsid protein plays a critical role in SARS-CoV pathogenesis, and high-level anti-nucleocapsid antibodies are detected in the patients infected by severe acute respiratory syndrome-associated coronavirus (SARS-CoV). Several studies have shown that there exists an interaction between nucleocapsid (N) and membrane (M) protein. In this paper, we investigate whether the expression of membrane protein can affect the immune responses induced by nucleocapsid DNA immunization. Two recombinant plasmids containing M and N coding sequence were constructed. Moreover, in order to get the antigen for ELISA and in vitro stimulation assay, N protein were expressed and purified from E. coli bacteria. Injection of 20mug of the mixture of pVAX1-M and pVAX1-N into the Balb/c mice could elicit the humoral and cellular responses. The ELISA analysis using the N antigen or inactivated SARS-CoV particles as capture antigen showed that co-injection of SARS-M could enhance N-induced antibody production, especially IgG2a subclass. After lymphocytes were stimulated with 10mug/ml purified N antigen, The CD4+ and CD8+ T cells of N and M plus N group were increased compared with those of control groups, and the M protein could augment the activation of lymphocytes induced by N DNA vaccine. Cytokine ELISA analysis revealed that co-injection of M could enhance the levels of IFN-gamma, IL-2 release induced by N antigen. Further experiments in field mouse also support the claim that membrane protein can augment the N-specific immune responses. Virus challenge test was conducted in BSL3 bio safety laboratory with Brandt's vole SARS-CoV model, and the results indicated that co-immunization of M and N antigens could reduce the mortality and pathological changes in lung from the virus infection.
Shi
Shu-Qun
SQ
State Key Laboratory of Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing 100080, PR China.
Peng
Jing-Pian
JP
Li
Yin-Chuan
YC
Qin
Chuan
C
Liang
Guo-Dong
GD
Xu
Li
L
Yang
Ying
Y
Wang
Jin-Ling
JL
Sun
Quan-Hong
QH
D000818:Animals; D000914:Antibodies, Viral / Q000276:immunology; D000936:Antigen-Antibody Complex / Q000276:immunology; D003411:Arvicolinae / Q000276:immunology; D015496:CD4-Positive T-Lymphocytes / Q000166:cytology / Q000378:metabolism; D018414:CD8-Positive T-Lymphocytes / Q000166:cytology / Q000378:metabolism; D049109:Cell Proliferation; D016207:Cytokines / Q000378:metabolism; D004279:DNA, Viral / Q000235:genetics; D005434:Flow Cytometry; D015870:Gene Expression; D008168:Lung / Q000166:cytology; D008565:Membrane Proteins / Q000235:genetics / Q000276:immunology* / Q000378:metabolism*; D051379:Mice; D008807:Mice, Inbred BALB C; D019590:Nucleocapsid Proteins / Q000235:genetics* / Q000276:immunology* / Q000302:isolation & purification; D045473:Severe acute respiratory syndrome-related coronavirus / Q000235:genetics* / Q000276:immunology*; D019444:Vaccines, DNA / Q000276:immunology*
D000914:Antibodies, Viral; D000936:Antigen-Antibody Complex; D016207:Cytokines; D004279:DNA, Viral; D008565:Membrane Proteins; D019590:Nucleocapsid Proteins; D019444:Vaccines, DNA