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221 Comparative Analysis of Expression and Immunogenicity of HIV-1 Group-Specific Antigen Pr55gag by Conventional Plasmid Vectors Versus Semliki-Forest-Virus-Derived Vectors
J. Zhang*1,2 , J. Wild2, K. Bieler2, M. Graf2, L. Deml2, H. Wolf2 , P. Liljeström3, R. Wagner2, and Y. Shao1
1Natl. Ctr. for AIDS Prevention and Control, Chinese Academy for Preventive Med., China; 2Inst. of Med. Microbiology, Univ. of Regensburg, Germany; and 3Microbiology and Tumorbiology Ctr., Karolinska Inst., Stockholm, Sweden
Background: To investigate if DNA replicon derived from Semliki Forest Virus (SFV) can be used as a potential candidate vector in HIV vaccine development, the expression and immunogenicity of Pr55gag directed by SFV-derived DNA replicon versus conventional DNA vaccine vector were comparatively studied.
Methods: HIV-1 subtype B wild-type gag as well as a codon usage optimized gag gene were cloned into 2 vector systems, i.e., SFV replicon vector and conventional pCDNA3.1(+) vector. 293T, H1299, C2C12, and BHK-21 cells were transfected by calcium coprecipitation technique. In cell Pr55gag expression and virus-like particle release were detected by Western blot and p24 ELISA assays. BALB/c mice were intramuscularly immunized and immunogenicity was comparatively studied.
Results: Rev independent Pr55gag expression directed by SFV-wtgag construct was observed in 293T, H1299, C2C12, and BHK-21 cell lines, whereas no Gag expression and Gag-specific immune responses were induced by pC-wtgag. Although with a lower transfection efficiency, both of SFV-wtgag and SFV-syngag constructs initiated sufficient Gag expression in all tested cells. However, Pr55gag virus-like particle releases from SFV-Gag expression construct transfected cells were not as efficient as that of pC-syngag plasmid. In intramuscular vaccinated BALB/c mice, neither SFV-layered plasmid nor conventional DNA construct elicited Gag-specific immune response at low injection doses (0.1 and 1.0 mug). In high DNA dose (10, 30, and 100mug) immunized mice, pC-syngag plasmid induced higher serum Gag-specific Ig titer with predominant IgG2a antibody and higher level interferon-gamma secretion from splenocytes, compared with SFV-wtgag and SFV-syngag constructs. More seraconversion mice and higher anti-Gag Ig were observed in SFV-syngag immunized mice than that of SFV-wtgag construct.
Conclusions: The SFV-layered vector has no advantage over pCDNA3.1(+) vector in inducing GAG-specific immunogenicity in BALB/c model. The results suggested that the consequence of DNA vaccine by using SFV replicon might be influenced by the nature of expressed antigen. The potential immunogenicity of codon-usage optimized synthetic gag gene was further proved in SFV layered vector system.
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