Sublingual administration of an adenovirus serotype 5 (Ad5)-based vaccine confirms Toll-like receptor agonist activity in the oral cavity and elicits improved mucosal and systemic cell-mediated responses against HIV antigens despite preexisting Ad5 immunity

Abstract

HIV/AIDS continue to devastate populations worldwide. Recent studies suggest that vaccines that induce beneficial immune responses in the mucosal compartment may improve the efficacy of HIV vaccines. Adenovirus serotype 5 (Ad5)-based vectors remain a promising platform for the development of effective vaccines. In an effort to improve the efficacy of Ad5-based vaccines, even in the presence of preexisting Ad5 immunity, we evaluated the potential for an Ad5-based HIV vaccine to induce antigen-specific immune responses following sublingual (s.l.) administration, a route not previously tested in regard to Ad-based vaccines. s.l. vaccination with an Ad5-based HIV-Gag vaccine resulted in a significant induction of Gag-specific cytotoxic T-lymphocyte (CTL) responses in both the systemic and the mucosal compartment. We also show that s.l. immunization not only avoided preexisting Ad5 immunity but also elicited a broad repertoire of antigen-specific CTL clones. Additionally, we confirm for the first time that oral delivery of a vaccine expressing a potent Toll-like receptor (TLR) agonist can stimulate innate immune responses through induction of cytokines and chemokines and activation of NK cells, NKT cells, and macrophages in vivo. These results positively correlated with improved antigen-specific CTL responses. These results could be achieved both in Ad5-naïve mice and in mice with preexisting immunity to Ad5. The simplicity of the s.l. vaccination regimen coupled with augmentation of TLR-dependent pathways active in the oral cavity makes s.l. delivery a promising method for HIV vaccine development specifically, as well as for many other vaccine applications in general.

FIG. 1.

HIV-Gag-specific T-cell responses in naïve and Ad5-preimmune C57BL/6 mice following o.g. or s.l. administration of Ad5-HIV-Gag. Naïve or Ad5-preimmune C57BL/6 mice (n = 6 for all treatment groups) were immunized with 1 × 10¹⁰ vps of Ad5-HIV-Gag by either the s.l. or the o.g. route of delivery. (A) Fourteen days postvaccination, IFN-γ ELISpot analyses were used to quantify HIV-Gag-specific T-cell responses in splenocyte preparations. Cells were stimulated with 4 different peptides as indicated. Spot-forming cells (SFCs) are reported per 1 × 10⁶ total cells plated per well. The induction of T-cell responses was considered significant if the average number of SFCs recalled with the indicated peptide was statistically higher than the number of SFCs from cells incubated in medium alone (no peptide), as determined using a homoscedastic two-tailed t test (*, P < 0.05; **, P < 0.01). In naïve mice, the average numbers of background SFCs derived from splenocytes incubated in medium alone were 2.8 and 4.3 for o.g. and s.l. preparations, respectively. In Ad5-preimmune mice, the average numbers of background SFCs derived from splenocytes incubated in medium alone were 2.8 and 4.8 for o.g. and s.l. preparations, respectively. Significant differences between o.g. and s.l. groups for one peptide (e.g., 304740 in naïve mice) are indicated (#, P < 0.05; ##, P < 0.01) and were calculated using one-way analysis of variance (ANOVA) followed by a Newman-Keuls post hoc test using data from all groups incubated with that peptide. Bars represent means ± standard errors (SE). (B) IL-2 ELISpot analyses were used to quantify HIV-Gag-specific T-cell responses in splenocyte preparations. The average numbers of background splenocyte SFCs from naïve mice were 0.5 and 1 for o.g.- and s.l.-treated mice, respectively. The average numbers of background splenocyte SFCs from Ad5-preimmune mice were 1.1 and 5 for o.g.- and s.l.-treated mice, respectively. (C) IFN-γ ELISpot analyses were used to quantify HIV-Gag-specific T-cell responses in SMLN preparations. Statistics were completed as described for panel A. In naïve mice, the average numbers of background SFCs derived from SMLN cells incubated in medium alone were 2.3 and 1.5 for o.g.- and s.l.-treated mice, respectively. In Ad5-preimmune mice, the average numbers of background SFCs derived from SMLN cells incubated in medium alone were 2.1 and 3 for o.g.- and s.l.-treated mice, respectively.

FIG. 2.

Ad5-specific T-cell responses in SMLNs and spleens of C57BL/6 mice with or without preexisting Ad5 immunity. Naïve or Ad5-preimmune C57BL/6 mice (n = 6 for both s.l. and o.g. treatment groups) were immunized with 1 × 10¹⁰ vps of Ad5-HIV-Gag by either the s.l. or the o.g. route of delivery. Fourteen days postvaccination, IFN-γ (A) and IL-2 (B) ELISpot analyses were used to quantify Ad5-specific T-cell responses in both SMLN and splenocyte preparations. Cells were stimulated with heat-inactivated Ad5 particles. Spot-forming cells (SFCs) are reported per 1 × 10⁶ total cells plated per well. Each symbol represents one sample, and the horizontal line indicates the mean of the presented data. The induction of T-cell responses was considered significant if the average number of SFCs recalled with the indicated peptide was statistically higher than the number of SFCs derived from cells incubated in medium alone, as determined using a homoscedastic two-tailed t test (*, P < 0.05; **, P < 0.01). The average numbers of background SFCs are reported in the legend for Fig. 1.

FIG. 3.

Ad5-GFP and Ad5-GFP/rEA induced NK, NKT, B-cell, and CD4⁺ T-cell activation in the spleen in vivo. C57BL/6 mice (n = 3 for all groups) were either mock treated or treated s.l. with 1 × 10¹⁰ vps of either Ad5-GFP or Ad5-GFP/rEA premixed with 1 × 10¹⁰ vps of Ad5-HIV-Gag. Lymphocytes from spleen tissue were harvested 14 h after s.l. treatment and stained for expression of surface markers specific to NK cells (A), NKT cells (B), B cells (C), or CD4⁺ T cells (D) and FACS sorted. Bars represent means ± SE. Data were analyzed using one-way ANOVA followed by a Newman-Keuls post hoc test. Significant differences from mock-injected mice are indicated by number symbols: #, P < 0.05; ##, P < 0.01; ###, P < 0.001. Significant differences between the indicated groups are shown by asterisks: *, P < 0.05; **, P < 0.01; ***, P < 0.001.

FIG. 4.

Ad5-GFP and Ad5-GFP/rEA induced macrophage activation in spleens in vivo. C57BL/6 mice (n = 3 for all groups) were either mock treated or treated s.l. with 1 × 10¹⁰ vps of either Ad5-GFP or Ad5-GFP/rEA premixed with 1 × 10¹⁰ vps of Ad5-HIV-Gag. Lymphocytes from spleen tissue were harvested 14 h after s.l. treatment and stained for expression of surface markers specific to Cd11b⁺ macrophages and expression of CD80, CD86, and MHC-II. Bars represent means ± SE. Data were analyzed using one-way ANOVA followed by a Newman-Keuls post hoc test. Significant differences from mock-injected mice are indicated by number symbols: #, P < 0.05; ###, P < 0.001. Significant differences between the indicated groups are shown by asterisks: *, P < 0.05; ***, P < 0.001. Representative histograms are found at right. MFI, mean fluorescence intensity.

FIG. 5.

Heightened Ad5-GFP/rEA induced cytokine responses. Naïve or Ad5-preimmune C57BL/6 mice (n = 6 for all groups) were either mock treated or treated s.l. with 1 × 10¹⁰ vps of either Ad5-GFP or Ad5-GFP/rEA premixed with 1 × 10¹⁰ vps of Ad5-HIV-Gag. Plasma was harvested, and cytokines and chemokines were measured at 6 h postinfection. Bars represent means ± SE. Data were analyzed using one-way ANOVA followed by a Newman-Keuls post hoc test. Significant differences from identically treated naïve animals are indicated by number symbols: #, P < 0.05; ##, P < 0.01; ###, P < 0.001. Significant differences between the indicated groups are shown by asterisks: *, P < 0.05; **, P < 0.01; ***, P < 0.001. ND ×3, not detectable in 3 out of 6 samples.

FIG. 6.

Increased HIV-Gag-specific CMI responses in naïve and Ad5-immune mice coadministered Ad5-HIV-Gag and Ad5-GFP/rEA. Naïve or Ad5-preimmune C57BL/6 mice (n = 6 for all groups) were either mock treated or treated s.l. with 1 × 10¹⁰ vps of either Ad5-GFP or Ad5-GFP/rEA premixed with 1 × 10¹⁰ vps of Ad5-HIV-Gag. Lymphocytes from spleen (A and C) and SMLN (B and D) tissue were harvested 14 days after s.l. treatment. IFN-γ and IL-2 ELISpot analyses were used to quantify HIV-Gag-specific T-cell responses in splenocyte and SMLN preparations. Cells were stimulated with 4 different peptides as indicated. SFCs in each well were background subtracted from the number in the appropriate unstimulated wells. SFCs are reported per 1 × 10⁶ total cells plated per well. Bars represent means ± SE. Asterisks indicate a significant difference between Ad5-GFP- and Ad5-GFP/rEA-treated mice (*, P < 0.05; **, P < 0.01; ***, P < 0.001), as determined using a homoscedastic two-tailed t test. Number symbols indicate a significant reduction in levels for Ad5-preimmune mice compared to those for identically treated naïve mice, as determined by one-way ANOVA followed by a Newman-Keuls post hoc test, inclusive of all data obtained with stimulation by the indicated peptide (#, P < 0.05; ##, P < 0.01).