Effects of MIP-1 alpha, MIP-3 alpha, and MIP-3 beta on the induction of HIV Gag-specific immune response with DNA vaccines

Abstract

Transfection of DNA vaccines with chemokines may recruit dendritic cells (DCs) locally to capture the antigenic genes and their gene products to generate enhanced CD8(+) cytotoxic T lymphocytes (CTLs). In this study, we investigated the effects of macrophage inflammatory protein (MIP)-1 alpha, MIP-3 alpha, and MIP-3beta on human immunodeficiency virus (HIV) Gag DNA vaccination. The chemokine plasmids markedly enhanced the local infiltration of inflammatory cells and increased the presence of CD11c(+) B7.2(+)-activated DCs. MIP-1 alpha and MIP-3 alpha were potent adjuvants in augmenting CTLs and afforded strong protection to immunized animals against challenge with vaccinia virus expressing Gag (vv-Gag). However, decreased humoral response was observed. MIP-3beta plasmid did not dramatically alter immunity. The chemokine inoculation time with respect to DNA vaccine priming was also investigated. The injection of pMIP-3 alpha three days before Gag plasmid (pGag) vaccination markedly increased specific CTLs compared with simultaneous injection and led to higher protection against vv-Gag. Immunity was also shifted toward a T-helper type-1 (Th1) response. In contrast, inoculation with pMIP-3 alpha three days after pGag vaccination shifted immunity toward a Th2 response. Our data suggest that administration of a chemokine with DNA vaccines offers a valuable strategy to modulate the efficacy and polarization of specific immunity and that chemokine-antigen timing is critical in determining overall biological effects.

Figure 1. Macrophage inflammatory protein (MIP)-3α and MIP-3β recruit immature and mature dendritic cells (DCs), respectively

The ability of the expression products from plasmid MIP (pMIP)-1α, pMIP-3α, or pMIP-3β to attract (a) murine immature DCs and (b) murine mature DCs was evaluated in a chemotaxis chamber assay. Results are expressed as the total number of migrated cells in response to the chemo-attractants. Supernatants of untransfected human embryonic kidney 293 cells were used as negative controls.

Figure 2. The injection of macrophage inflammatory protein (MIP) plasmids leads to local recruitment of inflammatory cells

(a) Hematoxylin and eosin staining was carried out 3 days after injection of the MIP cassettes into mouse tibialis muscle. (b) The type of cells was measured at the site of injection 5 days after inoculation: (A) naïve muscle; (B) muscle injected with phosphate-buffered saline; (C) muscle injected with Gag plasmid (pGag); muscle injected with 10 μg of chemokine encoding the constructs (D) plasmid MIP (pMIP)-1α, (E) pMIP-3α, and (F) pMIP-3β. The infiltrated inflammatory cells were isolated from the muscle and stained with fluorescein isothiocyanate–labeled anti-CD11c antibody and phycoerythrin-labeled anti-CD86 (B7.2) antibody, and analyzed by FACScan.

Figure 3. The immuno-adjuvant effects of the macrophage inflammatory protein (MIP) plasmids on Gag plasmid (pGag) vaccination

Determination of (a) Gag-specific humoral responses, (b) interferon-γ (IFN-γ) expression by enzyme-linked immunosorbent spot (ELISPOT), (c) cytotoxic T lymphocyte (CTL) response, and (d) IFN-γ production by T cells in immunized BALB/c mice. Mice were intramuscularly immunized twice, at weeks 0 and 2, with 50 μg of pGag alone or in conjunction with 50 μg of one of the MIP plasmids pMIP-1α, pMIP-3α, or pMIP-3β. Animals were killed 2 weeks after the second immunization, and sera and splenocytes were harvested. Gag-specific enzyme-linked immunosorbent assay was used to measure specific immunoglobulin G (IgG), IgG1, and IgG2a levels, and the splenocytes were studied for IFN-γ production using standard ELISPOT assay. Enriched T cells of the splenocytes were used for CTL assay. T cells were cultured at 5 × 10⁶ cells/ml in the presence of interleukin-2 (IL-2) and p7g peptide for 5 days. Culture supernatants were collected and assayed for cytokine secretion using enzyme-linked immunosorbent assay kits for mouse IFN-γ and IL-4. E:T ratio, effector-to-target-cell ratio.

Figure 4. Administration of macrophage inflammatory protein-3α plasmid (pMIP-3α) 3 days before Gag plasmid (pGag) vaccination leads to enhanced cytotoxic T lymphocyte (CTL) response and interferon-γ (IFN-γ) production

Determination of (a) Gag-specific CTL response, (b) Gag-specific humoral response, and (c) IFN-γ production by T cells in immunized BALB/c mice. Mice were intramuscularly immunized twice, at weeks 0 and 2, with 50 μg of pGag alone or in conjunction with 50 μg of pMIP-3α either 3 days before, simultaneous with, or 3 days after pGag vaccination. Sera were collected 2 weeks after the second immunization. An enzyme-linked immunosorbent assay was employed to measure specific immunoglobulin G (IgG), IgG1, and IgG2a concentrations. Animals were killed 2 weeks after the second immunization, and enriched T cells of splenocytes were studied for CTLs. T cells were cultured at 5 × 10⁶ cells/ml in the presence of interleukin-2 (IL-2) and p7g peptide for 5 days. Culture supernatants were collected and assayed for cytokine secretion using enzyme-linked immunosorbent assay kits for mouse IFN-γ and IL-4.

Figure 5. Inoculation with macrophage inflammatory protein-3α plasmid (pMIP-3α) 3 days ahead of Gag plasmid (pGag) immunization results in improved interferon-γ (IFN-γ) expression in specific T cells

IFN-γ expression in Gag-specific T cells determined by (a) enzyme-linked immunosorbent spot (ELISPOT) and (b) intracellular staining. Mice were intramuscularly immunized twice, at weeks 0 and 2, with 50 μg of pGag alone or in conjunction with 50 μg of pMIP-3α, either 3 days before, simultaneous with, or 3 days after pGag vaccination. Animals were killed 2 weeks after the second immunization, and the splenocytes were harvested. Splenocytes were cultured for 30 hours in the absence or presence of 2 μg/ml of p7g peptide, and cells producing IFN-γ were detected using standard ELISPOT assay. For intracellular IFN-γ staining, two-color analysis on CD8 and IFN-γ was performed on splenocytes that had been previously stimulated at 5 × 10⁶ cells/ml for 30 hours with 2 μg/ml of p7g peptide. vv-Gag, vaccinia virus expressing Gag; SFC, spot-forming cell.

Figure 6. Vaccination with Gag plasmid (pGag) and macrophage inflammatory proteins (MIPs) could protect animals from recombinant vaccinia virus expressing Gag (vv-Gag)

BALB/c mice were immunized twice with 50 μg of pGag alone or in conjunction with 50 μg of plasmid MIP (pMIP)-1α, pMIP-3α, or pMIP-3β (upper panels). Two weeks after the second immunization, mice were challenged with (a) vv-Gag or (b) vaccinia virus expressing LacZ (vv-LacZ). In a separate experiment, groups of mice were immunized twice with 50 μg of pGag alone or in conjunction with 50 μg of pMIP-3α either 3 days before, simultaneous with, or 3 days after pGag vaccination (lower panels). Two weeks after the second immunization, animals were challenged with (c) vv-Gag or (d) vv-LacZ. The virus titer was determined 5 days after challenge on BSC-1 cells (epithelial cells of African green monkey kidney).