Effects of antigen and genetic adjuvants on immune responses to human immunodeficiency virus DNA vaccines in mice

Abstract

The effects of genetic adjuvants on humoral and cell-mediated immunity to two human immunodeficiency virus antigens, Env and Nef, have been examined in mice. Despite similar levels of gene expression and the same gene delivery vector, the immune responses to these two gene products differed following DNA immunization. Intramuscular immunization with a Nef expression vector plasmid generated a humoral response and antigen-specific gamma interferon (IFN-gamma) production but little cytotoxic-T-lymphocyte (CTL) immunity. In contrast, immunization with an Env vector stimulated CTL activity but did not induce a high-titer antibody response. The ability to modify these antigen-specific immune responses was investigated by coinjection of DNA plasmids encoding cytokine and/or hematopoietic growth factors, interleukin-2 (IL-2), IL-12, IL-15, Flt3 ligand (FL), and granulocyte-macrophage colony-stimulating factor (GM-CSF). Coadministration of these genes largely altered the immune responses quantitatively but not qualitatively. IL-12 induced the greatest increase in IFN-gamma and immunoglobulin G responses to Nef, and GM-CSF induced the strongest IFN-gamma and CTL responses to Env. A dual approach of expanding innate immunity by administering the FL gene, together with a cytokine that enhances adaptive immune responses, IL-2, IL-12, or IL-15, generated the most potent immune response at the lowest doses of Nef antigen. These findings suggest that intrinsic properties of the antigen determine the character of immune reactivity for this method of immunization and that specific combination of innate and adaptive immune cytokine genes can increase the magnitude of the response to DNA vaccines.

FIG. 1.

Antigen plays a role in determining the induction of predominant CTL or antibody responses. Mice were intramuscularly immunized with plasmid DNA expressing Nef or Env. (A) Ten days after the third immunization, the cytolytic activity of spleen cells from mice was tested against the relevant stably transfected target cells (left), BC14H cells which express gp160, and P815-Nef cells which express Nef, or against peptide-pulsed P815 target cells (right) as detailed in Materials and Methods. The standard deviation for each triplicate point was ≤10%. Mice were injected with Env expression vectors encoding gp160 (left) or gp150 (right), respectively. (B) The antibody response was determined by immunoprecipitation followed by Western blot analysis with horseradish peroxidase-conjugated goat anti-mouse IgG (Santa Cruz Biotechnology Inc.). Each lane corresponds to the serum from an animal immunized with either the control vector (lanes 2 and 5) or a plasmid that expresses either Env or Nef (lanes 3 and 6). Mouse polyclonal serum to gp160 (HIV-1 V3 monoclonal [IIIB-V3-13], National Institutes of Health AIDS Reagent Program) and monoclonal antibody to Nef (Intracel) were used as positive controls (lanes 1 and 4, respectively).

FIG. 2.

IFN-γ responses following coimmunization of DNA expressing Nef with DNA expressing cytokine or hematopoietic factors. Mice were intramuscularly immunized with DNA without an insert (control) or plasmid expressing Nef alone (80 μg and 20 μg of pNGVL per mouse) or with Nef plus IL-2, IL-15, IL-12, GM-CSF, or FL (20 μg/mouse); FL and IL-2, IL-15, or IL-12 (10 μg of each cytokine/mouse), Nef, and FL on the first immunization and Nef and IL-2 or IL-12 on the second and third immunizations (2+3); or empty vector (pNGVL). Two weeks after the third immunization, spleens from immunized mice were restimulated in vitro with Nef protein in the form of a GST fusion-tagged protein at 5 (A) or 1 (B) μg/ml. IFN-γ secretion was assessed by ELISA in supernatants removed at 72 h. Responses are mean (plus standard deviation) concentrations, with the statistically insignificant background subtracted, for 5 to 16 mice per group. *, P < 0.05; **, P < 0.01; ***, P < 0.005 versus pNGVL-Nef-immunized mice at the same in vitro dose of Nef protein.

FIG. 3.

Coinjection with plasmids expressing immunomodulator molecules enhances the induction of serum anti-Nef specific IgG antibodies. Mice were immunized as described in the legend to Fig. 2. The levels of anti-Nef antibodies, total IgG, and IgG subclasses were determined by ELISA on serum samples recovered 2 weeks after the second (A) and third (B) immunizations. The results are mean (plus standard deviation) endpoint titers for 5 to 16 mice per group. *, P < 0.05 versus IgG1 titer observed in the same group of mice (A) or P < 0.05 versus pNGVL-Nef-immunized mice (B).

FIG. 4.

IFN-γ responses following coimmunization of DNA expressing HIV gp160 with DNA expressing cytokine or hematopoietic factors. Mice were intramuscularly immunized with DNA expressing gp160 alone (80 μg and 20 μg of pNGVL per mouse) or with gp160 plus plasmid expressing IL-2, IL-15, IL-12, GM-CSF, or FL (20 μg/mouse); FL and IL-2, IL-15, or IL-12 (10 μg of each cytokine/mouse); or empty vector (pNGVL). Two weeks after the third immunization, spleens from immunized mice were restimulated in vitro with medium alone or with the Env V3 loop peptide at a concentration of 40, 20, 10, or 5 μM or with concanavalin A (ConA). IFN-γ secretion was assessed by ELISA in supernatants removed at 72 h. The responses are mean (plus standard deviation) concentrations for 5 to 16 mice per group. *, P < 0.05; ‡, P < 0.01; ¶, P < 0.005 versus pNGVL-gp160-immunized mice at the same in vitro dose of V3 peptide.

FIG. 5.

IFN-γ responses due to restimulation with recombinant gp120 following coimmunization of DNA expressing HIV gp160 with DNA expressing cytokine or hematopoietic factors. Mice were immunized as described in the legend to Fig. 4. In addition to restimulation of spleen cells with V3 peptide, cells were also restimulated with recombinant gp120 at a concentration of 5 or 1 μg/ml. The responses are mean (plus standard deviation) concentrations, with the background subtracted, for five mice per group. *, P < 0.05; **, P < 0.01 versus pNGVL-gp160-immunized mice at the same in vitro dose of gp120 protein.

FIG. 6.

CTL responses following coimmunization of DNA expressing HIV gp160 with DNA expressing cytokine or hematopoietic factors. Mice were intramuscularly immunized with DNA expressing gp160 alone or with gp160 plus IL-2, IL-15, IL-12, GM-CSF, FL, or empty vector (pNGVL). Two weeks after the third immunization, cytolytic activities of restimulated pools of spleen cells from four mice were tested against P815 target cells incubated with the V3 loop peptide or with medium alone. The results are expressed as net percent specific lysis, obtained by subtracting the percent specific lysis of unpulsed P815 target cells (range, 0 to 10%) from the lysis of peptide-bearing cells.