Prime-boost immunization schedules based on influenza virus and vaccinia virus vectors potentiate cellular immune responses against human immunodeficiency virus Env protein systemically and in the genitorectal draining lymph nodes

Abstract

Vaccines that elicit systemic and mucosal immune responses should be the choice to control human immunodeficiency virus (HIV) infections. We have previously shown that prime-boost immunizations with influenza virus Env and vaccinia virus (VV) WR Env recombinants induced an enhanced systemic CD8(+) T-cell response against HIV-1 Env antigen. In this report, we analyzed in BALB/c mice after priming with influenza virus Env the ability of two VV recombinants expressing HIV-1 Env B (VV WR Env and the highly attenuated modified VV Ankara [MVA] Env) to boost cellular immune responses in the spleen and in the lymph nodes draining the genital and rectal tracts. Groups of mice were primed by the intranasal route with 10(4) PFU of influenza virus Env and boosted 14 days later by the intraperitoneal or intranasal route with 10(7) PFU of MVA Env or VV WR Env, while the control group received two immunizations with influenza virus Env. We found that the combined immunization (Flu/VV) increased more than 60 times the number of gamma interferon-specific CD8(+) T cells compared to the Flu/Flu scheme. Significantly, boosting with MVA Env by the intraperitoneal route induced a response 1.25 or 2.5 times (spleen or genital lymph nodes) higher with respect to that found after the boost with VV WR Env. Mice with an enhanced CD8(+) T-cell response also had an increased Th1/Th2 ratio, evaluated by the cytokine pattern secreted following in vitro restimulation with gp160 protein and by the specific immunoglobulin G2a (IgG2a)/IgG1 ratio in serum. By the intranasal route recombinant WR Env booster gave a more efficient immune response (10 and 1.3 times in spleen and genital lymph nodes, respectively) than recombinant MVA Env. However, the scheme influenza virus Env/MVA Env increased four times the response in the spleen, giving a low but significant response in the genital lymph nodes compared with a single intranasal immunization with MVA Env. These results demonstrate that the combination Flu/MVA in prime-booster immunization regimens is an effective vaccination approach to generate cellular immune responses to HIV antigens at sites critical for protective responses.

FIG. 1.

Evaluation of specific cellular immune responses against HIV-1 Env antigen. Quantification of Env peptide specific IFN-γ-secreting CD8⁺ T cells. Four BALB/c mice per group were first intranasally (i.n.) immunized with 10⁴ PFU of influenza virus Env, and 14 days later the animals were intranasally boosted with 10⁴ PFU of influenza virus Env or intraperitoneally (i.p.) boosted with 10⁷ PFU of VV WR Env or MVA Env. Cell suspensions of the spleens (A) or genitorectal lymph nodes (B) obtained 14 days after the immunization were evaluated for Env-specific IFN-γ-secreting cells by ELISPOT assay. The number of antigen-specific IFN-γ-secreting cells with standard deviation from triplicate cultures is shown. The pattern of cytokine secretion after gp160 restimulation of cell suspensions of spleens (C) or genitorectal lymph nodes (D) was determined. After 72 h of culture, cell culture supernatants were harvested and evaluated for IFN-γ and IL-4 by ELISA. Bars represent the median ± standard deviation of triplicate samples.

FIG. 2.

Induction of anti-V3 Env antibodies and IgG2a/IgG1 ratios after immunization with influenza virus and VV vectors. Sera from mice of the experiment described in Fig. 1 were evaluated for specific anti-gp160 antibodies 14 days after booster. Reactivity of individual serum samples against V3 peptide was assayed by ELISA. (A) Absorbances for IgG1 and IgG2a subclasses in 1:50 serum dilutions from mice of the different groups (absorbance from sera of nonimmunized mice was subtracted). (B) IgG2a/IgG1 ratios in the different groups.

FIG. 3.

Comparison of levels of expression of gp160 between cells infected with VV WR Env and MVA Env. (A) Western blot. 3T3 and BHK-21 cells were infected (5 PFU/cell) with VV WR Env or MVA Env, and at various times postinfection cells were collected and Env expression was analyzed by Western blot with a specific anti-gp160 IIIB antibody. (B) Immunofluorescence analysis under permeable conditions. 3T3 cells were infected (5 PFU/cell) with recombinant VV WR Env, MVA Env, or MVAluc, and at 24 h postinfection cells were fixed, permeabilized, and incubated with polyclonal gp120 IIIB antibody to show Env, with antibody against wheat germ to show the Golgi, or with To-Pro to show the DNA. To the right is the color merging to show colocalization of gp160 and Golgi compartments. (C) Immunofluorescence analysis under nonpermeable conditions. 3T3 cells were infected (1 PFU/cell) with recombinant VV WR Env, MVA Env, or MVAluc, and at 18 h postinfection cells were fixed, nonpermeabilized, and incubated with rabbit polyclonal gp120 IIIB antibody.

FIG. 4.

Evaluation of the CD8⁺ T-cell responses against Env after a mucosal immunization scheme. Four BALB/c mice per group were first intranasally immunized with 10⁴ PFU of influenza virus Env, and 14 days later they were intranasally boosted with 10⁷ PFU of VV WR Env or MVA Env. Fourteen days after boosting, Env peptide-specific IFN-γ-secreting cells in spleen cells (A) and genitorectal lymph nodes (B) were quantified. Shown are the mean numbers of antigen-specific IFN-γ-secreting cells with standard deviation from triplicate cultures. (C) Potentiation of the CD8⁺ T-cell anti-Env response induced after intranasal MVA Env immunization by priming mice with influenza virus Env. Splenocytes of both groups were obtained 14 days after immunization and evaluated by ELISPOT. Shown are the mean numbers of antigen-specific IFN-γ-secreting cells with standard deviation from triplicate cultures.

FIG. 5.

Pattern of specific cytokine secretion after mucosal immunization. Cell suspensions of spleens (A) or genitorectal lymph nodes (B) of mice in Fig. 4, obtained 14 days after the booster, were in vitro restimulated with gp160 or RPMI (negative control), and 72 h later cell culture supernatants were harvested and evaluated for IFN-γ and IL-4 by ELISA. Bars represent the mean with standard deviation of triplicates from cell cultures gp160-stimulated; nonspecific cytokine levels found in negative controls were subtracted.

FIG. 6.

Booster effect of poxvirus vectors in heterologous prime-boost schemes is dependent on the VV strain and dose of virus vector inoculated. Groups of four BALB/c mice were immunized as indicated. Fourteen days after the booster immunization, the number of IFN-γ-secreting CD8⁺ T cells in the spleen from the different groups of mice was evaluated by ELISPOT. The VV vector (MVA Env and VV WR Env) dose applied was 5 × 10⁷ PFU/mouse (A and B) or 10⁷ PFU/mouse (C and D). Shown are the mean numbers of antigen-specific IFN-γ-secreting cells with standard deviation from triplicate cultures. Panel E shows the increase obtained in the specific CD8⁺ T-cell response after the heterologous prime-boost scheme, represented as the ratio of heterologous prime-boost response versus the homologous prime-boost response.

FIG. 7.

Increase in the level of antibodies against β-galactosidase after a second dose with recombinant VV. Fourteen days after intraperitoneal immunization with one or two doses of the indicated recombinant VV vectors, VV WR Env (white bars) or MVA Env (black bars), pooled sera from four mice were analyzed to evaluate the specific antibodies against β-galactosidase (a recombinant gene expressed from both recombinant VV) by standard ELISA. (A) Levels of anti-β-galactosidase (β-gal) IgG (optical density of 1:200 dilution of sera). (B) Bars represent, in optical densities, the relative increase (optical density of 1:200 serum dilution after second immunization/optical density of 1:200 serum dilution after one immunization) in the level of antibody (Ab) against β-galactosidase after the second immunization with recombinant VV. Data are representative of three different ELISA determinations.