A human T-cell leukemia virus type 1 regulatory element enhances the immunogenicity of human immunodeficiency virus type 1 DNA vaccines in mice and nonhuman primates

Abstract

Plasmid DNA vaccines elicit potent and protective immune responses in numerous small-animal models of infectious diseases. However, their immunogenicity in primates appears less potent. Here we investigate a novel approach that optimizes regulatory elements in the plasmid backbone to improve the immunogenicity of DNA vaccines. Among various regions analyzed, we found that the addition of a regulatory sequence from the R region of the long terminal repeat from human T-cell leukemia virus type 1 (HTLV-1) to the cytomegalovirus (CMV) enhancer/promoter increased transgene expression 5- to 10-fold and improved cellular immune responses to human immunodeficiency virus type 1 (HIV-1) antigens. In cynomolgus monkeys, DNA vaccines containing the CMV enhancer/promoter with the HTLV-1 R region (CMV/R) induced markedly higher cellular immune responses to HIV-1 Env from clades A, B, and C and to HIV-1 Gag-Pol-Nef compared with the parental DNA vaccines. These data demonstrate that optimization of specific regulatory elements can substantially improve the immunogenicity of DNA vaccines encoding multiple antigens in small animals and in nonhuman primates. This strategy could therefore be explored as a potential method to enhance DNA vaccine immunogenicity in humans.

FIG. 1.

In vitro expression of HIV-1 Env gp145 ΔCFI from various DNA expression vectors. (A). Schematic diagram of the alternative cellular and viral regulatory elements in eukaryotic expression plasmids. (B). Western blot analysis of 3T3 cells transfected with empty parental 1012 (CMV) (lane 1) or those expressing HIV Env gp145 ΔCFI with the CMV (lane 2), CMV/R (lane 3), mUB (lane 4), mUB/R (lane 5), RSV (lane 6), or RSV/R (lane 7).

FIG. 2.

Immunogenicity of plasmid DNA vaccines expressing HIV-1 Env gp145ΔCFI in mice. Mice (n = 5/group) were immunized with 50 μg of the parental 1012 DNA vaccine or the CMV/R, RSV/R, mUB, or mUB/R DNA vaccines expressing HIV-1 Env gp145 ΔCFI at weeks 0, 2, and 6. After the third immunization, splenocytes were assessed for Env-specific cellular immune responses by IFN-γ and TNF-α ICS assays. (A) % CD3⁺CD4⁺ IFN-γ/TNF-α⁺ and (B) % CD3⁺CD8⁺ IFN-γ/TNF-α⁺ splenocytes are shown.

FIG. 3.

Immunogenicity of CMV/R plasmid DNA vaccines expressing HIV-1 Gag-Pol-Nef in mice. Mice (n = 8/group) were immunized with 50 μg of the parental 1012 DNA vaccine or the CMV/R DNA vaccine expressing HIV-1 Gag-Pol-Nef at weeks 0 and 6. Splenocytes were assessed for Gag-, Pol-, and Nef-specific cellular immune responses by IFN-γ ELISPOT assays 3 weeks after the (A) initial and (B) week 6 boost immunizations. (C) Splenocytes depleted of CD8⁺ T lymphocytes were also assessed in similar ELISPOT assays following the boost immunization.

FIG. 4.

Immunogenicity of multivalent CMV/R DNA vaccines expressing HIV-1 antigens in cynomolgus monkeys. Cynomolgus monkeys (n = 6/group) were immunized with the four-plasmid multivalent (A) 1012 DNA vaccines or (B) CMV/R DNA vaccines expressing HIV-1 Env gp145 ΔCFI from clades A, B, and C and a Gag-Pol-Nef fusion protein. (C) A third group of monkeys was immunized with a six-plasmid multivalent CMV/R DNA vaccine expressing HIV-1 Env gp145 ΔCFI from clades A, B, and C and separate gag, pol, and nef genes. All monkeys received 8 mg total DNA at weeks 0, 4, and 8. IFN-γ ELISPOT responses were assessed 2 weeks after the second immunization at week 6.

FIG. 5.

Mean responses to multivalent CMV/R DNA vaccines expressing HIV-1 antigens in cynomolgus monkeys. (A to C) Cynomolgus monkeys were vaccinated as described in the legend to Fig. 4. Mean and standard errors of IFN-γ ELISPOT responses in these groups of animals were assessed at weeks 0, 2, 6, 10, and 12 following immunization.