A dendritic cell targeted vaccine induces long-term HIV-specific immunity within the gastrointestinal tract

Abstract

Despite significant therapeutic advances for HIV-1 infected individuals, a preventative HIV-1 vaccine remains elusive. Studies focusing on early transmission events, including the observation that there is a profound loss of gastrointestinal (GI) CD4(+) T cells during acute HIV-1 infection, highlight the importance of inducing HIV-specific immunity within the gut. Here we report on the generation of cellular and humoral immune responses in the intestines by a mucosally administered, dendritic cell (DC) targeted vaccine. Our results show that nasally delivered α-CD205-p24 vaccine in combination with polyICLC, induced polyfunctional immune responses within naso-pulmonary lymphoid sites that disseminated widely to systemic and mucosal (GI tract and the vaginal epithelium) sites. Qualitatively, while α-CD205-p24 prime-boost immunization generated CD4(+) T-cell responses, heterologous prime-boost immunization with α-CD205-p24 and NYVAC gag-p24 generated high levels of HIV-specific CD4(+) and CD8(+) T cells within the GI tract. Finally, DC-targeting enhanced the amplitude and longevity of vaccine-induced immune responses in the GI tract. This is the first report of a nasally delivered, DC-targeted vaccine to generate HIV-specific immune responses in the GI tract and will potentially inform the design of preventative approaches against HIV-1 and other mucosal infections.

Figure 1. Intranasal immunization with α–CD205-p24 and poly ICLC delivered i.n and i.p elicit antigen specific IFN-γ⁺CD4⁺ T cell responses in the gastrointestinal (GI) lamina propria

C57Bl/6 mice were immunized with α-CD205-p24 fusion mAb (5µg) and poly ICLC (50µg). The vaccine was delivered i.p., i.n., i.v., s.c. or i.m in a prime boost manner. α-CD205-empty mAb (5µg) delivered i.p. served as control. Mononuclear cells were isolated from the small intestinal lamina propria (SILP) and spleen one-week post vaccine boost. IFN-γ secretion in response to HIV p24 (immunizing) or p17 (control) peptide pools was evaluated by intracellular cytokine staining. (A) Shows the schema of immunization. (B) FACS plots from a representative experiment. (C, D) Data from three independent experiments (5 mice per group) illustrating the mean levels of IFN-γ-producing CD4⁺ T cells in the (c) SILP and (d) Spleen. Background (p17) subtracted p24 data are shown. Statistical comparisons to α-CD205-empty (Ctrl) mice are shown. (E) ELISPOT quantification of p24-specific IgA⁺ cells in the GI tract following i.n. and i.p. immunization. α-CD205-empty mAb (5µg) delivered i.p. served as control. Mean data from two experiments, 5 mice per group. Statistical comparisons to α-CD205-empty (Ctrl) mice are shown. (F, G) ELISA quantification of p24-specific (f) IgA and (g) IgG in the serum from the above experiments. Error bars show mean ± SD. ***=p<0.001

Figure 2. Following intranasal immunization, p24 immune responses are generated within nasal and lung tissues and disseminate to mucosal and systemic sites

(A – F) C57Bl/6 mice were vaccinated i.n. with α-CD205-p24 fusion mAb (5µg) and poly ICLC (50µg) and p24-specific immune responses were evaluated one week post boost. (A) FACS plots from a representative experiment, illustrating the induction of IFN-γ⁺CD4⁺ cells in the lung, mediastinal LN (Med LN) and nose. (B) Mean data from three independent experiments (5 mice per group). Statistical comparisons to p17 (control) are shown. (C) FACS plots from a representative experiment, illustrating the induction of IFN-γ⁺CD4⁺ cells in the GI effector (CLP and SILP) and inductive (Peyer’s patch- PP and mesenteric LN- MLN) sites. (D) Mean data from three independent experiments (5 mice per group). Statistical comparisons to p17 (control) are shown. (E) FACS plots from a representative experiment, illustrating the induction of IFN-γ⁺CD4⁺ cells in the spleen and vaginal mucosa. (F) Mean data from three independent experiments (5 mice per group). Statistical comparisons to p17 (control) are shown. Error bars show mean ± SD. ***=p<0.001

Figure 3. Classical DCs are essential for inducing systemic and mucosal immune responses to intranasal immunization

Wild type (WT) and zbtb46^DTR mice were immunized i.n. with α–CD205-p24 fusion mAb (5µg) and poly ICLC (50µg) in a prime-boost manner. Diphtheria toxin (DT), (0.5µg) was administered i.p., 48 hours prior to immunization and every five days post vaccination. Additional doses of DT were administered on day 28 and 4 days post-boost. IFN-γ secretion in response to HIV gag p24 (immunizing) or p17 (control) peptide pools was evaluated one week post-boost by intracellular cytokine staining. (A) Shows the schema of immunization (B) FACS plots from a representative experiment, illustrating the induction of IFN-γ⁺CD4⁺ cells in the lung, spleen, SILP and CLP. (C) Mean data from three independent experiments (5 mice per group) is shown. Statistical comparisons between p24 levels in WT and zbtb46^DTR mice are shown. Error bars show mean ± SD. *=p<0.05, **=p<0.01, ***=p<0.001

Figure 4. DC targeting enhances the amplitude of anti-p24 immune responses in the GI tract compared to untargeted protein vaccination

C57Bl/6 mice were immunized i.n. with polyICLC, plus either α–CD205-p24 fusion mAb or untargeted p24 protein, both delivered i.n. in a prime-boost regimen, in escalating doses of 0.5µg, 5µg and 15µg. Mean data from three independent experiments (5 mice per group) is shown in (A-C) where in response to HIV gag-p24 or p17 peptide pools, IFN-γ⁺CD4⁺ T cells were quantified in the SILP (a), lung (b) and CLP (c). Background (p17) subtracted p24 data are shown. Additionally, statistical comparisons between α-CD205-p24 fusion mAb and p24 are shown. Error bars show mean ± SD. *=p<0.05, **=p<0.01, ***=p<0.001

Figure 5. Intranasal immunization with α–CD205-p24 induced predominantly antigen specific Th1 and Th17 responses in the GI tract

C57Bl/6 mice were immunized i.n. with α–CD205-p24 fusion mAb (5µg) and poly ICLC (50µg) in a prime-boost regimen. One-week post boost, mononuclear cells were isolated from the SILP, spleen, Med LN and CLP and re-stimulated with either p24 or p17 peptide pools and α-CD28 (1µg/ml) for 20 hours. Multiplexed ELISA was used to quantify the levels of secreted IFN-γ, IL-17, IL-6, IL-1β, IL-2, MIP-1α and MIP-1β. Mean data from two individual experiments is shown (5 mice per experiment). Statistical comparisons to p17 control are shown. Error bars show mean ± SD. *=p<0.05, **=p<0.01, ***=p<0.001

Figure 6. α–CD205-p24 vaccine delivered i.n. induces long term memory in effector sites of the GI tract

C57Bl/6 mice were immunized in a prime-boost regimen with polyICLC plus either α–CD205-p24 fusion mAb (5µg) or p24 (15µg, the highest dose of untargeted p24 vaccine was chosen). Mice were sacrificed 25 weeks post boost and lymphocytes were isolated from the spleen, lung and the effector sites (SILP and CLP) of the GI tract. IFN-γ⁺CD4⁺ T cells were examined by flow cytometry. (A, B) FACS plots from a representative experiment showing antigen specific IFN-γ⁺CD4⁺ T cells in the spleen, lung, SILP and CLP. (C) Mean data from three independent experiments is shown (5 mice per group). Background (p17) subtracted p24 data are shown. Statistical comparisons between α-CD205-p24 fusion mAb and p24 are shown. Error bars show mean ± SD. *p<0.05, **p<0.01, ***p<0.001

Figure 7. Heterologous α–CD205-p24 prime, NYVAC gag/pol/nef boost immunization, generates p24-specific CD8⁺ T cells in the systemic and mucosal compartments

F1 hybrid (cross between BALB/c females and C57Bl/6 males; heterozygous at all loci) mice were immunized with α–CD205-p24 fusion mAb (5µg), poly ICLC (50µg) and α-CD40 (25µg). After 4 weeks, the mice were administered 10⁷ PFU NYVAC gag/pol/nef delivered i.n. After 2 weeks of boost, IFN-γ secretion was evaluated in CD8⁺ T cells. (A) Shows the schema of immunization. (B) FACS plots from a representative experiment, illustrating the induction of IFN-γ⁺CD8⁺ cells in the lung, vaginal tract, blood and spleen. (C) Mean data from three independent experiments (5 mice per group) summarizing IFN-γ⁺CD8⁺ T cells in the lung, Blood, Spleen and vagina. Statistical comparisons to p17 (control) are shown. Error bars show mean ± SD. *=p<0.05, ***=p<0.001