Abstract
Adenovirus vector-based vaccine is a promising approach to protect HIV infection. However, a recent phase IIb clinical trial using the vector did not show its protective efficacy against HIV infection. To improve the vaccine, we explored the transgene protein expression and its immunogenicity using optimized codon usage, promoters and adaptors. We compared protein expression and immunogenicity of adenovirus vector vaccines carrying native or codon usage-optimized HIV-1 clade C gag and env genes expression cassettes driven by different promoters (CMV, CMVi, and CA promoters) and adapters (IRES and F2A). The adenovirus vector vaccine containing optimized gag gene produced higher Gag protein expression and induced higher immune responses than the vector containing native gag gene in mice. Furthermore, CA promoter generated higher transgene expression and elicited higher immune responses than other two popularly used promoters (CMV and CMVi). The second gene expression using F2A adaptor resulted in higher protein expression and immunity than that of using IRES and direct fusion protein. Taken together, the adenovirus vector containing the expression cassette with CA promoter, optimized HIV-1 clade C gene and an F2A adaptor produced the best protein expression and elicited the highest transgene-specific immune responses. This finding would be promising for vaccine design and gene therapy.
Highlights
The human immunodeficiency virus (HIV), responsible for acquired immune deficiency syndrome (AIDS), is classified into type 1 and type 2
To compare the expression efficiency of the HIV clade C Gag protein among several gag expression cassettes (Figure 1) in vitro, HeLa cells were transduced with plasmids or infected with Ad vectors (Ad-cytomegalovirus immediate-early gene (CMV)-gagopt, Ad-CMV (intron A) sequence (CMVi)-gag, Ad-CMVi-gagopt, or Ad-chicken b-actin intron sequence (CA)-gagopt) (Figure 1)
After two days of incubation, Gag protein expression was analyzed by western blotting with an antiHIV clade C gag p24 monoclonal antibody (mAb), and the intensity of Gag was quantified with Image J software. p55Gag and two or three extra proteins containing p24 were detected (Figures 2A and 2B)
Summary
The human immunodeficiency virus (HIV), responsible for acquired immune deficiency syndrome (AIDS), is classified into type 1 and type 2. HIV type 1 (HIV-1) is further classified into 11 phylogenetically related genetic subtypes (clades) from A to K. HIV-1 clade B is dominant in developed countries, such as North America and Western Europe, and has been the primary focus of vaccine development. HIV-1 clade C is the most dominant subtype (approximately half of HIV infected people) [3] in the world, especially in developing countries such as India [4], China [5], and the Sub-Saharan African countries [6]. The development of a vaccine against HIV-1 clade C is urgently required
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