Novel mucosal DNA-MVA HIV vaccination in which DNA-IL-12 plus cholera toxin B subunit (CTB) cooperates to enhance cellular systemic and mucosal genital tract immunity

Abstract

Induction of local antiviral immune responses at the mucosal portal surfaces where HIV-1 and other viral pathogens are usually first encountered remains a primary goal for most vaccines against mucosally acquired viral infections. Exploring mucosal immunization regimes in order to find optimal vector combinations and also appropriate mucosal adjuvants in the HIV vaccine development is decisive. In this study we analyzed the interaction of DNA-IL-12 and cholera toxin B subunit (CTB) after their mucosal administration in DNA prime/MVA boost intranasal regimes, defining the cooperation of both adjuvants to enhance immune responses against the HIV-1 Env antigen. Our results demonstrated that nasal mucosal DNA/MVA immunization schemes can be effectively improved by the co-delivery of DNA-IL-12 plus CTB inducing elevated HIV-specific CD8 responses in spleen and more importantly in genital tract and genito-rectal draining lymph nodes. Remarkably, these CTL responses were of superior quality showing higher avidity, polyfunctionality and a broader cytokine profile. After IL-12+CTB co-delivery, the cellular responses induced showed an enhanced breadth recognizing with higher efficiency Env peptides from different subtypes. Even more, an in vivo CTL cytolytic assay demonstrated the higher specific CD8 T-cell performance after the IL-12+CTB immunization showing in an indirect manner its potential protective capacity. Improvements observed were maintained during the memory phase where we found higher proportions of specific central memory and T memory stem-like cells T-cell subpopulations. Together, our data show that DNA-IL-12 plus CTB can be effectively employed acting as mucosal adjuvants during DNA prime/MVA boost intranasal vaccinations, enhancing magnitude and quality of HIV-specific systemic and mucosal immune responses.

Figure 1. Nasal co-administration of DNA-IL-12 plus CTB cooperate to induce an enhancement effect in the cellular immune responses against HIV-1 Env antigen during DNA/MVA immunization schemes.

Groups of four to six BALB/c mice were inoculated as described in the immunization schemes (Table 1). (A) Ten days after the booster dose, specific cellular immune responses (IFN-γ secreting CD8 T-cells) against Env were quantified by ELISPOT using pooled cells from spleen, cervical lymph nodes (CLNs) and iliac lymph nodes (ILNs). Bars represent the average number of specific IFN-γ spots forming units (SFU)/10⁶ cells +SD of a representative experiment from 2 to 6 performed. Each determination was evaluated in duplicate or triplicate and background values from negative control wells were subtracted. Statistical differences between indicated groups: *p<0.05; **p<0.01; ***p<0.001; by one-way ANOVA test and Bonferroni's correction post-test. (B) Summarize of the results obtained in all the experiments performed. Left bars (white) represent the mean fold increments in the magnitude of the specific cellular immune response compared to the control group. Right bars (grey) represent the percentage of experiments with adjuvant efficacy (%AE); %AE: represents a significant increase in the specific cellular immune response induced compared to the control group.

Figure 2. Nasal co-administration of IL-12 plus CTB improved the quality of the mucosal specific cellular immune responses in the genital tract.

Groups of six BALB/c mice were immunized following the control: (DNA-EnvB/MVA-EnvB: grey bars) and IL-12₅₀+CTB (DNA-EnvB+DNA-IL-12+CTB/MVA-EnvB+CTB: black bars) immunization schemes described in Table 1. Ten days after the MVA booster dose specific cellular immune responses against Env peptide were quantified by ELISPOT assay (IFN-γ and IL-2 secreting CD8+ T-cells) in (A) genital tract and (B) iliac lymph nodes (ILNs, left panel). Data are representative of three independent experiments with comparable results. (B, right panel) Iliac lymph-node (ILNs) cells were stimulated with specific peptide during 72 h and cytokines were measured in culture supernatants using the mouse Th1/Th2 cytokine cytometric bead array (CBA). Bars represent the average values +SD of duplicate determinations from pooled samples. Background values of negative control (RPMIc) were subtracted. Responses were considered as positives if they were above the cut-off values (RPMI +3SD). Statistical differences between groups: **p<0.01; ***p<0.001 by Student's T test.

Figure 3. T-cell response improvements afforded by DNA-IL-12 plus CTB administration during DNA/MVA immunizations were maintained during the memory phase of the adaptive response.

Thirty or 53 days post- immunization, Env specific IFNγ and IL-2 secreting T-cells were evaluated by ELISPOT using pooled cells from six mice in (A) spleen, (B) iliac lymph nodes (ILNs) and (C) genital tract for DNA-EnvB/MVA-EnvB control group (grey bars), and DNA-EnvB+DNA-IL-12₅₀+CTB/MVA-EnvB+CTB group (black bars). Bars represent the average of spot forming units (SFU)/10⁶ cells +SD for duplicate or triplicate wells. Background values of negative control wells were subtracted. Data are representative of three (30 days) and two (53 days) independent experiments. Statistical differences between groups: *p<0.05; **p<0.01; ***p<0.001 by Student's T test.

Figure 4. Mucosal IL-12 plus CTB modulates the T-cell capacities of cytotoxicity and specific cytokine pattern production in spleen and genital tract.

Quality of the response was evaluated in pooled samples from six mice immunized as DNA-EnvB/MVA-EnvB (grey bars) and DNA-EnvB+DNA-IL-12₅₀+CTB/MVA-EnvB+CTB (black bars) groups. (A) Ten days after the MVA boost, the proportion of cytokine secreting (IFN-γ, TNF-α and IL-2) and cytotoxic (CD107a/b) specific CD8+ T-cells against V3 loop were determinated by ICS in splenocytes. (B) Considering the total of specific CD 8⁺ T-cells, percentages of monofunctional (1fx, white), bifunctional (2fx, grey) and trifunctional (3fx, black) cells are represented. (C) Percentages of specific CD8 T-cells detailing the function performed. (D) The same analysis performed in (A) and (C) was done for genital tract cells. The percentage of specific CD8 T-cells able to secrete IFN-γ, TNF-α, or with cytotoxicity capacity (CD107a/b+ cells) and polyfunctionality are depicted in the left and right panel respectively. Bars represent the average +SD for duplicate of pooled cells from six mice. Statistical differences between groups: *p<0.05; **p<0.01; ***p<0.001 by Student's T test (E) 53 days after the boost dose, splenocytes from immunized mice were stimulated with V3 loop peptide during 72 h and cytokines were measured in culture supernatants using the mouse Th1/Th2 cytokine cytometric bead array (CBA). (F) 30 and 53 days after the MVA boost, splenocytes were stimulated with recombinant Gp-120 protein and cytokines were measured as in (E). Bars represent the average values +SD of duplicate determinations from pooled samples. Background values of negative control (RPMIc) were subtracted. Responses were considered as positive if they were above the cut-off values (RPMI +3SD).

Figure 5. CD8⁺ T-cell quality measured by its proliferative potential and its ability to recognized cross-reactive peptides.

Specific cellular immune responses were analyzed at 10, 30 or 53 days after the immunization in groups of six BALB/c mice. Pooled splenocytes of DNA-EnvB/MVA-EnvB (grey bars) and DNA-EnvB+DNA-IL-12₅₀+CTB/MVA-EnvB+CTB (black bars) groups were evaluated. (A) CFSE-labeled cells were stimulated in vitro during 4 days with the specific V3 loop peptide and then stained with surface antibodies (CD3 and CD8). Proliferating CFSE low CD8 T-cells was determined by flow cytometry. A representative dot plot is depicted (left panel) and percentage of proliferating cells were analyzed (right panel). (B) Ten and 53 days after immunization, splenocytes were stimulated with different pools of Env PTE peptides and evaluated by ELISPOT. Bars represented the accumulated number of IFN-γ spot forming units (SFU)/10⁶ cells for the total PTE peptide-pools. (C) Magnitude of the response against the individual PTE peptide-pools is depicted. (B) and (C): Data are representative of two independent experiments. Bars represent the average +SD for duplicate samples. Background values were subtracted in each case. Statistical differences between groups: *p<0.05; **p<0.01; ***p<0.001 by Student's T test. (D) Scheme indicating the gp160 region included in the different PTE peptide pools.

Figure 6. Study of T-cell functional avidity and in vivo T-cell specific killing activity.

Pooled splenocytes from control and IL-12₅₀+CTB groups (three to six mice/group), were analyzed for functional avidity of the V3- loop specific T-cells at (A) 10 and (B) 30 days after immunization, by ELISPOT using serial dilutions of the peptide from 20 to 0.00002 µg/ml for triplicate wells. Data represents the average percentage of the maximal response. SD50: sensitizing dose of peptide required to yield 50% of maximal T-cell triggering IFN-γ production. (C) In vivo cytotoxicity was analyzed adoptive transfer of control CFSElow and Env-peptide pulsed CFSEhigh dye cells. At 10 and 30 days after immunization, individual spleens of mice from naïve (I), DNA-EnvB/MVA-EnvB (II) and DNA-EnvB+DNA-IL-12₅₀+CTB/MVA-EnvB+CTB (III) groups were analyzed at the indicated times after adoptive transfer. A representative histogram is depicted in the left panel. Dots represent each analyzed mice. Lines represent median Statistical differences between groups: *p<0.05; **p<0.01 by Mann-Whitney test.

Figure 7. Characterization of specific memory T cell subpopulations induced.

Thirty days after immunization, pooled-splenocytes of six BALB/c mice were CFSE- labeled and stimulated with V₃-peptide during 4 days. Then, cells were stained with surface marker (CD8, CD44 and CD62L) and analyzed by flow cytometer. CD8 T-cells with specific proliferative capacity (evaluated by the CFSE dilution assay) were classified in four memory subpopulations. (A) Bars represent the percentage of specific CD8 T-cell with a phenotype compatible with: T stem cells memory (T_SCM; CD44L-CD62L+), T central memory (T_CM; CD44+ CD62L+), T effector memory (T_EM; CD44+ CD62L-) and T terminal effector (T_TEM; CD44- CD62L-). (B) Pie charts show the percentage of specific CD8 T-cells expressing the indicated phenotype. Statistical differences between groups are indicated: *p<0.05; ***p<0.001 by Student's T test.