Immunogenicity of a lentiviral-based DNA vaccine driven by the 5'LTR of the naturally attenuated caprine arthritis encephalitis virus (CAEV) in mice and macaques

Abstract

Increasing the safety and the efficacy of existing HIV vaccines is one of the strategies that could help to promote the development of a vaccine for human use. We developed a HIV DNA vaccine (Δ4-SHIVKU2) that has been shown to induce potent polyfunctional HIV-specific T cell responses following a single dose immunization of mice and macaques. Δ4-SHIVKU2 also induced protection when immunized macaques were challenged with homologous pathogenic viruses. In the present study, our aim was to examine whether a chimeric HIV DNA vaccine (CAL-Δ4-SHIVKU2) whose genome is driven by the LTR of the goat lentivirus, caprine arthritis encephalitis (CAEV) expresses efficiently the vaccine antigens and induces potent immune responses in animal models for HIV vaccine. Data of radioimmunoprecipitation assays clearly show that this chimeric genome drives efficient expression of all HIV antigens in the construct. In addition, evaluation of the p24 Gag protein in the supernatant of HEK-293-T cells transfected in parallel with Δ4-SHIVKU2 and CAL-Δ4-SHIVKU2 showed no difference suggesting that these two LTRs are inducing equally the expression of the viral genes. Immunization of mice and macaques using our single dose immunization regimen resulted in induction of similar IFN-γ ELISPOT responses in Δ4-SHIVKU2- and CAL-Δ4-SHIVKU2-treated mice. Similar profiles of T cell responses were also detected both in mice and macaques when multiparametric flow cytometry analyses were performed. Since CAEV LTR is not dependent of Tat to drive viral gene expression and is not functional for integration with HIV integrase, this new vector increases the safety and efficacy of our vaccine vectors and vaccination strategy.

Figure 1

Design of Δ4SHIVku2 and CAL-Δ4SHIVku2 DNA vaccine genomes. CAL-Δ4SHIVku2 DNA was derived from Δ4SHIVku2 by removing the SIV 5' LTR sequences and ligating the CAEV 5' LTR. Oligonucleotide primers specific to the extremities of CAEV LTR were used to amplify the 0.45 kb PCR product from the molecular clone of CAEV. The resulting fragment was double digested with EcoR1 and Nar1 restriction enzymes and then used for ligation with the gel-purified EcoR1-Nar1-digested 9 kb fragment from Δ4SHIVku2. The resultant molecular construct was named CAL-Δ4SHIVku2.

Figure 2

Viral protein expression in transfected HEK 293T cells (A) Culture medium samples of cells transfected with HEK 293T with Δ4SHIVku2 and CAL-Δ4SHIVku2 constructs were harvested at 14 h and 24 h post-transfection and examined by ELISA for detection of HIV Gag p24. Triplicate measurements were performed and the results are representative of two independent experiments. (B) After forty-eight hours, proteins of transfected HEK 293T cells were labeled with ³⁵S-methionine and then viral proteins were immunoprecipitated from the cell lysate (C) and the supernatant fluid (S) with hyperimmune monkey anti-SHIV plasma. Sizes of the major proteins are indicated in kDa.

Figure 3. IFN-γ ELISPOT responses in mice immunized with Δ4SHIVku2 and CAL-Δ4SHIVku2 DNA vaccines

At two weeks post-immunization of BALB/c mice with a single dose of 200 μg of CAL-Δ4-SHIVku2 or Δ4-SHIVku2 DNA vaccine, splenocytes were isolated and examined for IFN-γ ELISPOT responses using pools of Gag, Env, TRN (Tat+Rev and Nef combined) HIV peptides. The data show the mean of the measurements and the standard deviation (represented by the errors bars) obtained by 5 immunized animals in each group. SFC: spot-forming cells. No significant difference was observed between the two different DNA vaccines.

Figure 4. HIV-specific IFN-γ producing and proliferating CD8⁺ T cells in mice immunized with CAL-Δ4SHIVku2 DNA vaccine

Pools of splenocytes from 3 mice immunized with a single dose of 200 μg of CAL-Δ4-SHIVku2 at two to four weeks post-immunization were used to detect and evaluate specific CD8⁺ T cell responses to HIV antigens. CFSE-labeled splenocytes were cultured for 5 days with specific HIV peptides (Gag, Env, TRN) or in absence of antigens (medium only). After 5 days, cells were harvested and restimulated for 6h with indicated antigens (Medium or Ag (5d) Ag (6h)) in the presence of co-stimulatory antibodies and Brefeldin A. Restimulated cells were surface labeled with anti-CD3, CD8, -CD4 antibodies in presence of EMA (for exclusion of dead cells) and subsequently fixed, permeabilized and stained with anti-IFN-γ and anti-IL-2 mAbs. Cells were gated on low FSC/SSC, EMA-, CD3⁺ and high CD8⁺ population and data were displayed as two color dot plots (IFN-γ vs IL-2 (upper row) and IFN-γ vs CFSE (lower row)). Frequencies of IFN-γ producing cells (upper number in each plot) and those of cells proliferating only (lower number in low row plots) are indicated. Results are representative of 2–3 independent experiments.

Figure 5. HIV-specific IFN-γ producing and proliferating CD8⁺ T cells in macaque immunized with CAL-Δ4SHIVku2 DNA vaccine

Macaque 1 and 2 were immunized with a single dose of 30 mg of CAL-Δ4SHIVku2 DNA vaccine. At the indicated pre-immunization and post-immunization times, PBMCs were collected, labeled with CFSE, cultured, restimulated and stained using the same procedure as that described for mouse in Fig. 4. The proportion of cells producing IFN-γ contour plot, upper number) and proliferating (CFSE dilution; contour plot, lower number) in response to specific HIV antigens are presented for the two macaques at the pre-immunization time point and at the peak of the primary vaccine induced-immune response (between 2 and 4 weeks post-immunization, PI). A red arrow is pointing the discrete proportion of cells producing IFN-γ and proliferating in response to Gag antigen in the macaque 1.