Immunization with a Mixture of HIV Env DNA and VLP Vaccines Augments Induction of CD8 T Cell Responses

Abstract

The immune response induced by immunization with HIV Env DNA and virus-like particle (VLP) vaccines was investigated. Immunization with the HIV Env DNA vaccine induced a strong CD8 T cell response but relatively weak antibody response against the HIV Env whereas immunization with VLPs induced higher levels of antibody responses but little CD8 T cell response. Interestingly, immunization with a mixture the HIV Env DNA and VLP vaccines induced enhanced CD8 T cell and antibody responses. Further, it was observed that the mixing of DNA and VLP vaccines during immunization is necessary for augmenting induction of CD8 T cell responses and such augmentation of CD8 T cell responses was also observed by mixing the HIV Env DNA vaccine with control VLPs. These results show that immunization with a mixture of DNA and VLP vaccines combines advantages of both vaccine platforms for eliciting high levels of both antibody and CD8 T cell responses.

Figure 1

Characterization of SHIV 89.6 VLPs. SHIV 89.6 VLPs were produced by coinfection of Sf9 cells with rBVs expressing HIV 89.6 Env and SIVmac239 Gag proteins at the MOI (multiplicity of infection) of 5 and 2, respectively, and purified as described in Section 2. SIV Gag VLPs were produced by infection of Sf9 cells with rBV expressing SIVmac239 Gag proteins and purified similarly. (a) Characterization of SHIV 89.6 VLPs by Western blot. 5 μg total proteins were taken from each VLP preparation and analyzed by SDS-PAGE followed by Western blot using rabbit-anti-gp120 antibody for detection of HIV Env proteins and monkey-anti-SIV fordetection of the SIV Gag proteins. Lanes 1, SHIV 89.6 VLP; 2, SIV Gag-only VLP. (b) Comparison of HIV 89.6 Env amount in VLP preparations by a quantitative ELISA. The amount of HIV Env proteins in SHIV 89.6 VLP preparations was determined by a sandwich ELISA. ELISA plates were coated with a sheep-anti-gp120 antibody as the capture antibody, followed by addition of serial 2-fold dilutions of SHIV 89.6 VLPs lysed by 1% Triton X-100. The amount of HIV Env bound to the plate was then detected by sera from HIV-infected patients as the detecting antibody, followed by addition of HRP-conjugated Goat-antihuman antibody and development of color. Lysed SIVmac239 Gag VLPs were used as controls. A standard curve for the amount of HIV Env was obtained by adding purified HIV 89.6 Gp120 mixed with SIVmac239 Gag VLP lysed by 1% Triton X-100 to the sheep-anti-gp120 antibody-coated ELISA plate. The amount of HIV Env proteins was then calculated based on the obtained standard curve and then expressed as nanograms (ng) of HIV Env in 1 μg VLP preparation. VLP1, VLP2, and VLP3 represent SHIV 89.6 VLPs produced from three different batches and Gag-VLP represents the control SIVmac239 Gag only VLP preparation. (c) Negative staining and EM examination of SHIV 89.6 VLPs. Purified SHIV 89.6 VLPs were stained with 1% uranyl acetate followed by examination under a transmission electron microscope.

Figure 2

Schematic diagram of immunization study design. Six groups of mice (groups of 6) were immunized with different vaccine preparations as indicated. Group 1: a mixture of 50 μg DNA vector pCAGGS and 50 μg SIV Gag-VLPs; Group 2: 50 μg HIV Env-DNA; Group 3: 50 μg SHIV 89.6 VLPs; Group 4: a mixture of 50 μg HIV Env-DNA and 50 μg SHIV 89.6 VLPs; Group 5: simultaneous injection with 50 μg HIV Env-DNA and 50 μg SHIV 89.6 VLPs at separate sites; Group 6: a mixture of 50 μg pCAGGS DNA vector and 50 μg SHIV 89.6 VLPs; Group 7: a mixture of 50 μg HIV Env-DNA and 50 μg SIV Gag-VLPs. Mice were immunized twice by intramuscular injections at weeks 0 and 4, and sacrificed at week 6 (two weeks after the second immunization) to collect blood samples and prepare splenocytes for analysis of immune responses.

Figure 3

Enhanced CD8 T cell responses by immunization with a mixture of HIV Env-DNA and SHIV 89.6 VLPs but not by simultaneous injection at separate sites. Groups of mice (6 per group) were immunized by intramuscular injection at weeks 0 and 4 with different vaccine preparations as shown in Figure 2. Two weeks after the second immunization, mouse splenocytes were prepared and stimulated by the peptide IGPGRAFYAR corresponding to the dominant CD8 epitope in the HIV Env for Balb/c mice. The percentages of IFNγ-producing CD8 T cells were analyzed by intracellular-cytokine staining and flow cytometry. (a) Representative results of FACS analysis for IFNγ-producing CD8 T cells from each immunization group stimulated with the peptide IGPGRAFYAR. Numbers in lower-right boxes represent percentages of IFNγ staining positive CD8 T cells. Background levels of IFNγ-producing CD8 T cells similar to Group 1 were obtained for all samples stimulated with an irrelevant peptide AMQMLKETI (data not shown). (b) Percentages of IFNγ staining positive CD8 T cells for each immunization group after stimulation with the peptide IGPGRAFYAR. Error bars represent standard deviations for each group. ∗ indicates the groups with significantly higher levels of CD8 T cell responses than Group 2 that received the HIV Env-DNA vaccine only (P < .05).

Figure 4

Comparison of antibody responses induced by immunization with the HIV Env-DNA and SHIV 89.6 VLP vaccines in different combinations. Mice were immunized by different vaccine preparations as described in Figure 2. Sera were collected at 2 weeks after the second immunization and analyzed for antibodies specific for HIV 89.6 gp120 by ELISA. The levels of antibody responses are expressed as the quantity (ng) of antibodies binding to HIV 89.6 gp120 in 1 mL sera from each mouse. (a) Total IgG antibodies against gp120. (b) IgG1 subtype antibodies against gp120. (c) IgG2a subtype antibodies against gp120. Error bars indicate the standard deviations for each immunization group. ∗ indicates the groups with significantly higher levels of antibody responses than Group 2 that received the HIV Env-DNA vaccine only (P < .05).

Figure 5

(a) DNA molecules bind to VLPs in the DNA/VLP mixture. HIV 89.6 Env-DNA, SHIV 89.6 VLPs, or their mixtures were loaded onto a two-layer sucrose cushion, with 500 μl 30% sucrose at bottom and 2 mL 20% sucrose above, followed by ultracentrifugation. After centrifugation, 10 μl from the 30% cusion of each sample was loaded onto a 1% agarose gel, and after electrophoresis, the gel was stained with Ethidium Bromide followed by destaining and then visualization of DNA by UV light. Marker: DNA molecular weight marker (lambda DNA Hind III digest); VLP: 50 μg SHIV 89.6 VLPs; DNA: 50 μg HIV Env-DNA; DNA/VLP: 50 μg HIV Env-DNA mixed with 50 μg SHIV: 89.6 VLPs; DNA/VLP (lysed): 50 μg HIV Env-DNA mixed with 50 μg SHIV 89.6 VLPs that were lysed with 1% Triton X-100. (b) SHIV 89.6 VLPs stimulate cytokine secretion by BMDCs. BMDCs were prepared as described in Section 2 and incubated with different stimulants in triplicates. DCs incubated with culture medium only were used as negative controls and DCs incubated with LPS (10 ng/mL) were used as positive controls. To ensure that stimulation of DC by DNA or VLPs is not due to contamination of endotoxin, the DCs were also incubated with the same stimulants that have been heat-treated at 100°C for 30 minutes. Cell-free supernatants were harvested 24 hours after incubation at 37°C in 5%  CO₂ and assayed for the levels (pg/mL) of IL-6, IL-12, and TNF-alpha by ELISA. Error bars represent standard deviations and the results shown represent typical results obtained from two different stimulation experiments. Medium, cell culture medium (negative control): LPS, 10 ng/mL (positive control); DNA: HIV Env-DNA (50 μg/mL); VLP: SHIV 89.6 VLPs (10 μg/mL); DNA-VLP: a mixture of HIV Env-DNA (50 μg/mL) and SHIV 89.6 VLPs (10 μg/mL). (c) Maturation of BMDCs after stimulation by SHIV 89.6 VLP vaccines. BMDCs were incubated with different stimulants as described above. After stimulation, the BMDCs were stained for surface expression of CD11c, CD80, and CD86 and then analyzed by flow cytometry. The results are presented as histograms for CD80 (upper panel) and CD86 (lower panel) for CD11c positive cells. Medium: cell culture medium (negative control); LPS: 10 ng/mL (positive control); DNA: HIV Env-DNA (50 μg/mL); VLP: SHIV 89.6 VLPs (10 μg/mL); DNA-VLP: a mixture of HIV Env-DNA (50 μg/mL) and SHIV 89.6 VLPs (10 μg/mL).