Increased level and longevity of protective immune responses induced by DNA vaccine expressing the HIV-1 Env glycoprotein when combined with IL-21 and IL-15 gene delivery

Abstract

We investigated the ability of a plasmid-derived IL-21 delivered alone or in combination with the IL-15 gene to regulate immune responses to the HIV-1 envelope (Env) glycoprotein induced by DNA vaccination. Mice were injected with the gp140DeltaCFI(HXB2/89.6) vector expressing a modified Env glycoprotein with C-terminal mutations intended to mimic a fusion intermediate, in which the most divergent region encoding the variable V1, V2, and V3 domains of CXCR4-tropic HxB2 virus was replaced with the dual-tropic 89.6 viral strain. Using a recombinant vaccinia virus expressing 89.6 Env glycoprotein (vBD3) in a mouse challenge model, we observed that IL-21 plasmid produced sustained resistance to viral transmission when injected 5 days after DNA vaccination. Moreover, IL-21 in a synergistic manner with IL-15 expression vector augmented the vaccine-induced recall responses to the vBD3 challenge compared with those elicited by immunization in the presence of either cytokine alone. The synergistic combination of IL-21 and IL-15 plasmids promoted expansion of CD8+CD127+ memory T cell pools specific for a subdominant HLA-A2-restricted Env(121-129) epitope (KLTPLCVTL). Our results also show that coimmunization with IL-21 and IL-15 plasmid combination resulted in enhanced CD8+ T cell function that was partially independent of CD4+ T cell help in mediating protection against vBD3 challenge. Furthermore, the use of IL-21 and IL-15 genes was able to increase Ab-dependent cellular cytotoxicity and complement-dependent lysis of Env-expressing target cells through augmentation of Env-specific IgG Ab levels. These data indicate that the plasmid-delivered IL-21 and IL-15 can increase the magnitude of the response to DNA vaccines.

FIGURE 1

Expression of the gp140ΔCFI_HxB2/89.6 Env glycoprotein and induction of neutralizing Ab responses. A, Schematic representation of the gp140ΔCFI_HxB2/89.6 expression construct. In the HIV-1 gp140ΔCFI_HxB2/89.6 chimeric vector, the region from residues 105 to 363 in gp140ΔCFI_HXB2 was replaced with the corresponding sequence from the HIV-1_89.6 Env glycoprotein (in gray). Abbreviations for the restriction enzyme sites NsiI and Bsu36I are denoted as N and B, respectively. B, Expression of gp140ΔCFI_HxB2/89.6 and gp140ΔCFI_HXB2 glycoproteins in culture supernatants from 293T transfectants. Control cells were transfected with the backbone plasmid (lane 1). The oligomeric states of secreted gp140ΔCFI_HXB2 and gp140ΔCFI_HxB2/89.6 glycoproteins were confirmed by BN-PAGE on 4–12% Bis-Tris NuPAGE gel (lanes 2 and 3, respectively). C, Generation of neutralizing Ab response by the gp140ΔCFI_HxB2/89.6 vaccine in rabbits. Sera were collected from three rabbits before immunization and after the prime/boost immunization with the gp140ΔCFI_HXB2 DNA construct and recombinant protein as described in Materials and Methods. Both preimmune and postimmune sera were tested at a 1/10 dilution for neutralizing activity against a panel of HIV-1 isolates. Neutralizing Ab titers were analyzed as previously described (54). SDs are indicated.

FIGURE 2

Protection against challenge with vBD3 vaccinia virus following immunization with the gp140ΔCFI_HxB2/89.6 construct alone or in combination with a plasmid-encoded IL-21. A, BALB/c mice were immunized i.m. with DNA without an insert (sham vector) or a vector expressing gp140ΔCFI_HxB2/89.6 Env (10, 30, 100, or 300 µg/mouse). Five weeks after the third immunization, the mice were challenged i.p. with 2 × 10⁷ PFU of vBD3 or vSC8. Five days later, the mice were sacrificed, ovaries removed, homogenized, and assayed for vaccinia virus titers by plating serial 10-fold dilutions on HuTK⁻143 cells, staining with crystal violet, and counting plaques at each dilution. B, BALB/c mice were immunized with the gp140ΔCFI_HxB2/89.6 construct alone or in combination with a plasmid-encoded IL-21 (10, 20, or 40 µg/mouse) before a challenge with vBD3 or vSC8 virus. The vaccinia titers are presented as the mean log₁₀ ± SD (error bars) of PFU per ovaries of five mice per group. *, p < 0.05 and **, p < 0.01.

FIGURE 3

Effect of IL-21 delivery relative to the gp140ΔCFI_HxB2/89.6 DNA immunization on the level and duration of the Env vaccine-induced protection against challenge with vBD3 virus. A, BALB/c mice were immunized three times with the gp140ΔCFI_HxB2/89.6 construct alone or in combination with IL-21. The IL-21 plasmid was injected simultaneously with the Env vaccine (day 0), or 5 and 15 days later. The immunized mice were challenged with 2 × 10⁷ PFU of vBD3 (■) and vSC8 (□) 1 mo after the third immunization. The viral titers in ovaries were analyzed 5 days after the challenge and tested for vaccinia titers on a monolayer of HuTK⁻143 cell line. B, The titers of vBD3 and vSC8 in ovaries of gp140ΔCFI_HxB2/89.6-immunized and IL-21-treated mice were analyzed 3 mo after vaccination. C, The titers of vBD3 and vSC8 in ovaries of gp140ΔCFI_HxB2/89.6-immunized and IL-21-treated mice were analyzed 6 mo after vaccination. The values are presented as the mean log₁₀ ± SD (error bars) of PFU per ovaries of five mice per group. *, p < 0.05; **, p < 0.01; and ***, p < 0.001.

FIGURE 4

Effect of IL-15 and IL-21 genes on the Env vaccine-induced CD8⁺ T cell responses. A, Protection against vBD3 challenge. Five weeks after the third immunization with the gp140ΔCFI_HxB2/89.6 construct in the presence, absence or combination of IL-15 and IL-21 genes, BALB/c mice were challenged i.p. with 2 × 10⁷ PFU of vBD3 (■) or vSC8 (□). Control mice were immunized with the sham vector. Five days later, mice were sacrificed, ovaries removed, homogenized, and assayed for vaccinia virus titers. The titers are presented as the mean log₁₀ ± SD (error bars) of PFU per ovaries of five mice per group. B, Analysis of Env-specific CTL responses in mice immunized with the gp140ΔCFI_89.6 construct in the presence, absence or combination of IL-15 and IL-21 expression vectors. The Env-specific CTL response was analyzed in a standard ⁵¹Cr release assay against P815 cells infected with vBD3 (solid symbols) or vSC8 (open symbols). All determinations were made in triplicate samples, and the SD was <10%. Results are presented as the means ± SD (error bars) of three independent experiments. C, The numbers of IFN-γ-secreting CD8⁺ T cells in splenocyte cultures established from mice immunized with the gp140ΔCFI_HxB2/89.6 construct were determined by ELISPOT assay with vBD3-infected P815 cells. Control cells derived from mice immunized with the sham plasmid. Results are presented as the means ± SD (error bars) of at least three independent experiments. The frequencies of IFN-γ-secreting cells were determined by regression analysis from a curve generated by plotting the number of spots vs the number of effector cells. *, p < 0.05; **, p < 0.01; and ***, p < 0.001.

FIGURE 5

Expression of CD44, CD62L, and CD127 Ags on the cell surface of Env_121–129/HLA-A*0201 tetramer⁺CD8⁺ splenocytes in the HLA-A2/K^b transgenic mice immunized with the gp140ΔCFI_HxB2/89.6 DNA vaccine alone or in combination with IL-15 and IL-21 genes. A, The HLA-A2/K^b-transgenic mice were immunized three times with the gp140ΔCFI_HxB2/89.6 DNA vaccine. The phenotypic analysis of splenocytes was performed on 5 wk after the last booster immunization. B, The gp140ΔCFI_HxB2/89.6-immunized HLA-A2/K^b-transgenic mice were injected with IL-15 and IL-21 genes as described in Materials and Methods. The phenotypic analysis of splenocytes was performed on 5 wk after the last booster immunization. Histograms depict vaccine-induced CD8⁺T cells gated first for lymphocytes by forward scatter (FSC) and side scatter (SSC) and then for Env_121–129 peptide-specific CD8⁺ T cells by tetramer and anti-CD8 mAb staining. Background staining was assessed using an isotype control Ab. The percent and mean fluorescent intensity (MFI) of CD44, CD62L, and CD127 molecules on Env_121–129 peptide-specific CD8⁺ T cells are given. Arrows indicate the source of cells in the histograms. Data are from one representative experiment of three performed.

FIGURE 6

IL-21 acts synergistically with IL-51 in expanding the vaccine-induced CD8⁺ T cell responses to the subdominant Env_121–129 epitope during vBD3 challenge. A, The phenotypic analysis of Env_121–129 peptide-specific CD8⁺ T cells in splenocytes of the HLA-A2/K^b transgenic mice immunized with gp140ΔCFI_HxB2/89.6 DNA vaccine alone was performed on day 5 of vBD3 challenge. B, The effect of coimmunization with IL-15 and IL-21 genes on the expansion of the vaccine-induced CD8⁺ T cell responses to the subdominant Env_121–129 epitope was analyzed 5 days after vBD3 challenge. Histograms depict vaccine-induced CD8⁺ T cells gated first for lymphocytes by forward scatter (FSC) and side scatter (SSC) and then for Env_121–129 peptide-specific CD8⁺ T cells by tetramer and anti-CD8 mAb staining. Background staining was assessed using an isotype control Ab. The percent and MFI of CD44, CD62L, and CD127 on Env_121–129 peptide-specific CD8⁺ T cells are given. Arrows indicate the source of cells in the histograms. The results are representative of four independent experiments.

FIGURE 7

The generation of functional CD8⁺ T cells in gp140ΔCFI_HxB2/89.6-immunized mice requires CD4⁺ T cells during priming and is partially restored by coimmunization with IL-15 and IL-21 genes in a model of CD4⁺ Th cell depletion. A, BALB/c mice were depleted of CD4⁺ T cells before vaccination with the gp140ΔCFI_HxB2/89.6 construct by i.p. injection of anti-CD4 mAb. Additional groups of mice were depleted of CD8⁺ T cells by treatment with anti-CD8 mAb. The Ab treatment continued every 5 days during the entire period of three immunizations and until the challenge studies with vBD3 (■) and vSC8 (□) vaccinia viruses performed 1 mo later. B, BALB/c mice were depleted of CD4⁺ T cells before vaccination with the gp140ΔCFI_HxB2/89.6 construct and treatment with the combination of IL-15 and IL-21 genes by i.p. injection of anti-CD4 mAb. In all experiments, IL-15 was delivered together with the vaccine, whereas IL-21 expression vector was injected 5 days later. Additional groups of mice were depleted of CD8⁺ T cells by treatment with anti-CD8 mAb. The vaccinia titers are presented as the mean log₁₀± SD (error bars) of PFU per ovaries of four mice per group. *, p < 0.05; ***, p < 0.0001.

FIGURE 8

Env-specific Ab responses induced by vaccination of BALB/c mice with the gp140ΔCFI_89.6 construct in the presence, absence, or combination of IL-15 and IL-21 genes. A, BALB/c mice were immunized three times with the gp140ΔCFI_HxB2/89.6 construct in the presence, absence, or combination of IL-15 and IL-21 genes. In all experiments, IL-15 was delivered together with the vaccine, whereas IL-21 expression vector was injected 5 days later. The titers of Env-specific IgM Abs in sera of gp140ΔCFI_89.6-immunized mice were determined after each immunization by ELISA using alkaline phosphatase-conjugated secondary Abs. B, The titers of Env-specific IgG Abs were analyzed in the same sera of the gp140ΔCFI_89.6 construct-immunized mice. C, The titers of Env-specific IgG subclasses were determined in sera after the third immunization with the gp140ΔCFI_89.6 construct in the presence, absence, or combination of IL-15 and IL-21 genes. Results are presented as the means ± SD (error bars) of three independent experiments. *, p < 0.05.

FIGURE 9

ADCC and CDC against Env-expressing cells mediated by sera of mice immunized with the gp140ΔCFI_89.6 construct in the presence, absence or combination of IL-15 and IL-21 genes. A, ADCC was measured using vBD3-infected (solid symbols) and vSC8-infected (open symbols) target cells and serial dilutions of sera collected 3 wk after the third immunization with the Env vaccine in the presence, absence, or combination of IL-15 and IL-21 genes. B, The complement-mediated lysis of vBD3-infected (black symbols) and vSC8-infected (white symbols) target cells was determined using the same sera as for the ADCC assay. All determinations were made in triplicate samples, and the SD was <10%. Results are presented as the means ± SD (error bars) of three independent experiments.