Increased immunogenicity of HIV-1 p24 and gp120 following immunization with gp120/p24 fusion protein vaccine expressing alpha-gal epitopes

Abstract

Developing an effective HIV-1 vaccine will require strategies to enhance antigen presentation to the immune system. In a previous study we demonstrated a marked increase in immunogenicity of the highly glycosylated HIV-1 gp120 protein following enzymatic addition of alpha-gal epitopes to the carbohydrate chains. In the present study we determined whether gp120(alphagal) can also serve as an effective platform for targeting other HIV-1 proteins to APC and thus increase immunogenicity of both proteins. For this purpose we produced a recombinant fusion protein between gp120 and the HIV-1 matrix p24 protein (gp120/p24). Multiple alpha-gal epitopes were synthesized enzymatically on the gp120 portion of the fusion protein to generate a gp120(alphagal)/p24 vaccine. Immune responses to gp120(alphagal)/p24 compared to gp120/p24 vaccine lacking alpha-gal epitopes were evaluated in alpha1,3galactosyltransferase knockout (KO) mice. These mice lack alpha-gal epitopes and, therefore, are capable of producing the anti-Gal antibody. T cell responses to p24, as assessed by ELISPOT and by CD8+ T cells intracellular staining assays for IFNgamma, was on average 12- and 10-fold higher, respectively, in gp120(alphagal)/p24 immunized mice than in mice immunized with gp120/p24. In addition, cellular and humoral immune responses against gp120 were higher by 10-30-fold in mice immunized with gp120(alphagal)/p24 than in gp120/p24 immunized mice. Our data suggest that the alpha-gal epitopes on the gp120 portion of the fusion protein can significantly augment the immunogenicity of gp120, as well as that of the fused viral protein which lacks alpha-gal epitopes. This strategy of anti-Gal mediated targeting to APC may be used for production of effective HIV-1 vaccines comprised of various viral proteins fused to gp120.

Figure 1

(A) Schematic representation of the fusion gene used for production of gp120/p24. Note: the p24 gene was fused to the C terminus of gp120 in order to keep the codon-optimized t-PA leader signal proximally upstream of the gp120 gene. (B) Detection of the gp120/p24 fusion protein by Western blots. Western blot stained with rabbit anti-gp120 (left lane) or rabbit anti-p24 antibodies (right lane). Note that each antibody detected a band of the expected size of the gp120/p24 protein.

Figure 2. Binding of monoclonal anti-Gal to gp120/p24 and to gp120_gal/p24 in ELISA

Binding of the monoclonal anti-Gal M86 to gp120/p24 (○); gp120_αgal/p24 (●) and to α-gal BSA that expresses 10 synthetic α-gal epitopes (■), as measured by ELISA with different amounts of glycoproteins coating the ELISA wells.

Figure 3. ELISPOT analysis of IFNγ secretion by T cells in KO mice immunized with gp120/p24 or gp120_αgal/p24, and in response to p24 peptide

(A) or gp120 peptide (B). Presentation of ELISPOT data of 6 mice immunized twice with gp120/p24 (mice #1-#6) and 6 mice immunized twice with gp120_αgal/p24 (mice #7-#12) as the number of spots per 10⁶ splenocytes. Splenocytes were stimulated with 5 μg of p24 peptide. Data are presented as mean ± standard deviation of triplicate wells.

Figure 3. ELISPOT analysis of IFNγ secretion by T cells in KO mice immunized with gp120/p24 or gp120_αgal/p24, and in response to p24 peptide

(A) or gp120 peptide (B). Presentation of ELISPOT data of 6 mice immunized twice with gp120/p24 (mice #1-#6) and 6 mice immunized twice with gp120_αgal/p24 (mice #7-#12) as the number of spots per 10⁶ splenocytes. Splenocytes were stimulated with 5 μg of p24 peptide. Data are presented as mean ± standard deviation of triplicate wells.

Figure 4. Analysis of intracellular staining of IFNγ in CD8+ T cells from KO mice immunized with gp120/p24 or gp120_αgal/p24 in response to p24 peptide

ICS analysis of IFNγ production in CD8+ T cells in response to p24 peptide from mice immunized with gp120/p24 (left panels mice #1 - #5) and mice immunized with gp120_αgal/p24 (right panels mice #6 - #10). Lymphocytes were stained for CD3+, CD8+ membrane markers and intracellular IFNγ. Gated CD3+/CD8+ positive events were analyzed for IFNγ production. The % of CD8+ T cells with intracellular IFNγ is indicated in the upper right quadrant for each mouse. Note that 4 of the 5 mice immunized with gp120_αgal/p24 (#6-#9) displayed higher proportions of IFNγ positive CD8+ T cells in comparison to mice immunized with gp120/p24.

Figure 5. Anti-gp120 response in KO mice immunized with gp120/p24 or gp120_αgal/p24

A. Production of anti-gp120 antibodies in KO mice immunized twice two weeks apart in KO mice either with gp120/p24 (○) or gp120_αgal/p24 (●). Note that KO mice immunized with gp120/p24 produced low titers of anti-gp120 antibodies or completely lacked such antibodies, whereas extensive anti-gp120 antibody production was observed in mice immunized with gp120_αgal/p24. B. The mean values ± standard deviation calculated from Figure 5A.

Figure 6. HIV neutralization activity in mice immunized with gp120/p24 or gp120_αgal/p24

HIV neutralization activity in mice immunized with gp120/p24 (mice 1 to 5) or gp120_αgal/p24 (mice 6 to 10). Titer is defined as the reciprocal of the serum dilution displaying 50% neutralization