Increased immune response elicited by DNA vaccination with a synthetic gp120 sequence with optimized codon usage

Abstract

DNA vaccination elicits humoral and cellular immune responses and has been shown to confer protection against several viral, bacterial, and parasitic pathogens. Here we report that optimized codon usage of an injected DNA sequence considerably increases both humoral and cellular immune responses. We recently generated a synthetic human immunodeficiency virus type 1 gp120 sequence in which most wild-type codons were replaced with codons from highly expressed human genes (syngp120). In vitro expression of syngp120 is considerably increased in comparison to that of the respective wild-type sequence. In BALB/c mice, DNA immunization with syngp120 resulted in significantly increased antibody titers and cytotoxic T-lymphocyte reactivity, suggesting a direct correlation between expression levels and the immune response. Moreover, syngp120 is characterized by rev-independent expression and a low risk of recombination with viral sequences. Thus, synthetic genes with optimized codon usage represent a novel strategy to increase the efficacy and safety of DNA vaccination.

FIG. 1

Codon bias in the HIV-1 envelope gene. Codon frequencies in the HIV-1 envelope gene (black boxes) and in highly expressed human genes (shaded boxes) were calculated by use of standard software from the University of Wisconsin Genetics Computer Group and sequences derived from the Los Alamos National Library database. Codon frequencies were tabulated with 24 different HIV-1 envelope sequences from the following isolates: Ada, Ant70, Br0141, Br0259, JFL, JRCSF, JY1, LAI, M12199, MaI, MN, MVP5180, RF, Rw0914, SF2, SF162, SF33, T8659, Th1412, Ug0205, Ug0317, Ug0378, ZAM20, and Z6649. Codon frequencies from highly expressed cellular genes are listed according to the work of Cherry (10). The most frequently used codon for every amino acid is underlined.

FIG. 2

Increased expression of syngp120 in various mammalian cell lines in vitro. 293T (adenovirus-transformed human kidney cells), HeLa (human cervix carcinoma cells), NIH 3T3 (mouse fibroblasts), and COS-7 (African green monkey kidney cells) were transiently transfected by calcium phosphate coprecipitation with plasmids carrying genes coding for either wild-type gp120MN (wt) or syngp120 (syn). Culture supernatants of radioactively labelled cells were immunoprecipitated with a human antiserum derived from an HIV-1-infected individual and analyzed on a reducing SDS–7% PAGE.

FIG. 3

Increased humoral immune responses of BALB/c mice immunized with syngp120. ELISA analysis of sera from mice immunized with plasmid DNA encoding either syngp120 (filled symbols) or wild-type gp120 MN (open symbols). IgG antibody reactivity against gp120 in sera from DNA-inoculated mice was measured by an ELISA. Mice were either immunized once (wt 1, wt 2, syn 1, and syn 2) or immunized and boosted three times after 2, 4, and 6 weeks (wt 3, wt 4, syn 3, and syn 4). Sera were drawn from the tail vein 3 (wt 1, wt 2, syn 1, and syn 2) or 10 (wt 3, wt 4, syn 3, and syn 4) weeks after the initial immunization. OD₄₀₅, optical density at 405 nm; wt, wild type; syn, syngp120.

FIG. 4

Increased humoral immune responses of BALB/c mice immunized with syngp120 independently of the regulatory protein Rev. Western blot analysis of sera from mice immunized with pCdm7 (lanes 1 to 3), gp120MNrre and pCMV-rev (lanes 4 to 9), or syngp120v3LAIrre and pCMV-rev (lanes 10 to 15) is shown. Western blot strips prepared with the HIV-1 MvP899 isolate were reacted with sera derived from either DNA-injected mice (lanes 1 to 15) or an HIV-1-infected individual (lane 16). Mice were immunized twice, and serum was collected 12 weeks after the initial immunization. wt, wild type.

FIG. 5

Increased cellular immune responses of BALB/c mice immunized with syngp120. CTL assay of spleen cells from DNA-injected mice against target cells loaded with titrated amounts of the nonamer peptide GPGRAFVTI. BALB/c mice were immunized with 40 μg of either pCDM7 (CDM71 to CDM73; negative control), syngp120v3LAIrre (syn1 to syn4), or gp120LAIrre (wt1 to wt4) and 10 μg of pCMV-rev, boosted twice after 6 and 30 weeks, and sacrified after an additional 3 weeks. Spleen cells were restimulated with interleukin 2 and peptide-loaded B7 cells in vitro. Data are mean percentages of three replicate cultures. Killing was antigen specific and not caused by NK cells, as wells without peptide did not show any chromium release (<5% specific release in all mice).

FIG. 6

Time course analysis of sera from mice immunized with either wild-type gp120 or syngp120 sequences. Time course of antibody reactivity against gp120 in sera from BALB/c mice immunized with either syngp120 (syn IIIb 1 and syn IIIb 2) or wild-type gp120 (wt IIIb 1 and wt IIIb 2). Mice were immunized and boosted three times after 2, 4, and 11 weeks with 50 μg of plasmid DNA in both anterior tibial muscles (arrows). Serum was drawn 6 and 15 (wt IIIb 1, wt IIIb 2, syn IIIb 1, and syn IIIb 2) and 23 (wt IIIb 1 and wt IIIb 2) weeks after the initial immunization from the tail vein of immunized mice. IgG antibody reactivity against gp120 in sera from DNA-inoculated mice was measured by an ELISA and is shown for a serum dilution of 1:100. OD₄₀₅, optical density at 405 nm.