Targeting the vaginal mucosa with human papillomavirus pseudovirion vaccines delivering simian immunodeficiency virus DNA

Abstract

The majority of HIV infections occur via mucosal transmission. Vaccines that induce memory T and B cells in the female genital tract may prevent the establishment and systemic dissemination of HIV. We tested the immunogenicity of a vaccine that uses human papillomavirus (HPV)-based gene transfer vectors, also called pseudovirions (PsVs), to deliver SIV genes to the vaginal epithelium. Our findings demonstrate that this vaccine platform induces gene expression in the genital tract in both cynomolgus and rhesus macaques. Intravaginal vaccination with HPV16, HPV45, and HPV58 PsVs delivering SIV Gag DNA induced Gag-specific Abs in serum and the vaginal tract, and T cell responses in blood, vaginal mucosa, and draining lymph nodes that rapidly expanded following intravaginal exposure to SIV(mac251.) HPV PsV-based vehicles are immunogenic, which warrant further testing as vaccine candidates for HIV and may provide a useful model to evaluate the benefits and risks of inducing high levels of SIV-specific immune responses at mucosal sites prior to SIV infection.

FIGURE 1

HPV transduces macaque epithelium and induces antibodies to HPV. Cynomolgus macaques were vaccinated intra-vaginally with HPV pseudovirions expressing either RFP or SIV Gag. (A) Schematic showing the design of the study, eight cynomolgus macaques were vaccinated with HPV16 and HPV45, thirty days apart. Blood (black bars) and tissues (diamonds) were sampled pre and post vaccination. In order to obtain sufficient samples to analyze the female genital tract, animals were serially sacrificed at 37, 44, or fifty six days post vaccination (stars). (B) Forty-eight hours post vaccination a camera fitted to an endoscope was used to visualize the cervix and vaginal tract. Successful HPV vaccination was confirmed by the detection of RFP in each animal. Shown is the level of in vivo fluorescence from HPV-RFP PsVs measured in a representative animal. (C) Serum IgG to the HPV16 and 45 capsid L1 measured fourteen-fifty six days post vaccination. The vaginal epithelia were disrupted by 4 methods using nonoxynol 9 (N9). N9 was prepared as a 10% gel and delivered once (Gel 1X white bars), twice, (Gel 2X hatched bars), or twice in combination with abrasion using a cytobrush (Gel 2X+cytobrush, black bars). In addition, we used a 12.5% foam application of N9 also given twice (Foam striped bars).

FIGURE 2

Intravaginal vaccination with HPV PsVs-Gag induces both cell mediated and humoral immune responses to SIV Gag. (A) Western blot showing the expression of the SIV Gag poly-protein (p55) in 293TT-cells following transduction with HPV16–SIV Gag constructs (lanes 2 & 3). Recombinant Gag p27 was loaded as a positive control in lane 1, degradation products of the recombinant protein are seen below the p27 band. Lysates from non-transduced cells were run as a negative control (lane 4). (B) IFN-γ responses measured by ELISpot after stimulation with SIV Gag peptides. (C) SIV Gag serum titers of IgG measured post HPV vaccination. (D) SIV Gag-specific IgA as a function of total IgA in vaginal secretions.

FIGURE 3

SIV Gag specific cytokine production in systemic and mucosal tissues. (A) Representative flow cytometric plots showing the frequency of IL-2 production in unstimulated or Gag stimulated CD4⁺ T-cells. (B) PBMCs from HPV-SIVGag vaccinated macaques were stimulated with Gag peptides. The frequency of CD4⁺ T-cells (left) and CD8⁺ T-cells (right) producing IFN-γ and IL2 (cytokine+) is shown. Arrows above indicate the approximate time each HPV vaccine was administered. (C) Mononuclear cells were isolated from the cervix and vaginal tract and stimulated with Gag peptides. However, in some animals sufficient cervical mononuclear cells were not obtained to perform stimulations. The frequency of CD4⁺ T-cells (left) and CD8⁺ T-cells (right) producing cytokines is shown. (D) At sacrifice, the genital draining obturator lymph nodes were collected from vaccinated macaques. Mononuclear cells were isolated and stimulated with SIV Gag peptides. The frequency of CD4⁺ T-cells (left) or CD8⁺ T-cells (right) producing cytokines is shown.

FIGURE 4

HPV PsVs are immunogenic in rhesus macaques, recruit CD4⁺ and CD8⁺ T-cells to the site of vaccination and induce vaginal humoral responses. (A) Schematic showing the vaccination and sampling schedule in rhesus macaques. Macaques were vaccinated on days 0, 30, and 121 with HPV16, 45, and 58, respectively. Blood (black squares) and tissues (white diamonds) were collected pre and post HPV vaccination. (B) Western blot showing the expression of SIV Gag polyprotein p55 and processed p27 protein in 293TT-cells transduced with HPV16 and HPV45 Gag-Pro constructs (lanes 2 & 3). Non-transduced 293TT lysates was used as a negative control (lane 1). (C) Vaginal biopsies were obtained prior to and one-week post HPV vaccination, and paraffin embedded. A representative example of immunohistochemical staining performed on embedded tissue, stained for CD4 (red) Ki67 (green) and nuclear material stained by dapi (blue) prior to (left) and post (right) HPV vaccination. (D) The absolute number of CD4⁺ and CD8⁺ T-cells enumerated from vaginal biopsies pre (white bars) and post vaccination (black bars). A repeated measure analysis of variance demonstrated that the difference is statistically significant (p=0.0049 and p=0.012). (E) The absolute number of Ki67⁺ CD4⁺ and Ki67⁺ CD8⁺ cells in vaginal biopsies pre and post HPV vaccination. A repeated measure analysis of variance demonstrated that the difference is statistically significant (p=0.040 and p=0.034) (F) SIV Gag-specific IgA as a function of total IgA in vaginal secretions. (G) SIV Gag-specific IgG as a function of total IgG in vaginal secretions.

FIGURE 5

SIV specific Memory T-cell responses to HPV PsVs (A) Antigen experienced CD95⁺CD28 ^(+/-) CD8⁺ T- cells from PBMC’s were gated and the frequency of SIVGagCM9⁺ cells assessed post HPV vaccination. Animals treated with N9 foam are depicted in hatched bars, while N9 gel treated animals are in black bars. Arrows above indicate the times when each HPV vaccine was administered. (B) Intracellular cytokine staining indicating the frequency of CD107⁺(hatched), IL-2 (white), IFN-γ and/or TNF-α (black) producing CD4⁺ T-cells from Gag stimulated PBMC’s in unvaccinated (left) foam treated (middle) and gel treated (right) animals. (C) Intracellular cytokine staining indicating the frequency of CD107⁺, IL-2⁺, IFN-γ and/or TNF-α producing CD8⁺ T-cells from Gag stimulated PBMC’s.

FIGURE 6

Intravaginal exposure to SIV induces an expansion of vaccine-primed immune responses and does not exacerbate virus replication. (A) Study design showing SIV exposure six weeks post the final HPV vaccine. Animals were sacrificed at one or two weeks post infection (indicated by crosses), and blood and tissues collected at sacrifice. (B) Plasma viral load in unvaccinated and vaccinated macaques one and two weeks post infection. (C) Cell associated viral load in the vagina quantified as the number of SIV DNA copies/10⁶ cells. (D) Representative flow cytometric plots (top panel) showing the frequency of SIVGagCM9 positive memory CD8⁺ T-cells in the blood, genital draining obturator lymph node, cervix, vagina, and rectum two weeks post challenge. Both intraepithelial and lamina propria CD8⁺T-cells were obtained from the female genital tract. The bottom panel shows the average frequency of SIV specific Gag CM9 CD95⁺CD8⁺ T-cells from vaccinated animals. (E) Average IFN-γ producing spot forming mononuclear cells from axillary, obturator lymph nodes, and blood after stimulation with overlapping peptides spanning SIV Gag (left) or SIV Env (right) from vaccinated and unvaccinated controls, two weeks post challenge. A repeated measure analysis of variance demonstrated that the difference in IFN- γ production from the obturator lymph node between vaccinated and non-vaccinated animals is statistically significant (p=0.034).