Dendritic cells infected by recombinant rabies virus vaccine vector expressing HIV-1 Gag are immunogenic even in the presence of vector-specific immunity

Abstract

Dendritic cells (DC) are the most potent antigen presenting cells whose ability to interact with T cells, B cells and NK cells has led to their extensive use in vaccine design. Here, we designed a DC-based HIV-1 vaccine using an attenuated rabies virus vector expressing HIV-1 Gag (RIDC-Gag). To test this, BALB/c mice were immunized with RIDC-Gag, and the primary, secondary as well as humoral immune responses were monitored. Our results indicate that RIDC-Gag stimulated HIV-1 Gag-specific immune responses in mice. When challenged with vaccinia virus (VV) expressing HIV-1 Gag, they elicited a potent Gag-specific recall response characterized by CD8+ T cells expressing multiple cytokines that were capable of specifically lysing Gag-pulsed target cells. Moreover, RIDC-Gag also enhanced CD8+ T cell responses via a homologous prime-boost regimen. These results show that a DC-based vaccine using live RV is immunogenic and a potential candidate for a therapeutic HIV-1 vaccine.

Figure 1. Characterization of RIDCs

Bone marrow from BALB/c mice was differentiated in the presence of 10ng/ml GM-CSF for a period of 7 days (See materials and methods). Primary BMDCs were confirmed by expression of CD11c (a). BMDCs were infected with SPBN-GFP at an MOI of 10. After 48h, GFP positive cells were identified by immunofluorescence (b). To identify viral protein expression, cells were stained with a FITC-tagged antibody against RV-N and analyzed by flow cytometry (c). The viability of the RV, RV-Gag infected and uninfected BMDCs from 5 indepdent mice was determined by 7-AAD staining versus FSC – early apoptotic cell populations were identified as R2, whereas R3 denotes live cells(d). The average apoptic BMDCs from n=5 independent mice is denoted by the bar graphs at 24 (e) and 36hpi (f). Apoptotic NA cells were included as a positive control.

Figure 2. RIDCs are functional APCs

BMDCs were infected with RV at an MOI of 10. The maturation phenotype of RIDCs was determined 48hpi by FACS analysis of CD80 and CD86 expression, with numerical inserts of the Geometric mean fluorescence (GMF) (a). Presentation of antigen by RIDCs was analyzed after infection of BMDCs from C57BL/6 mice with either RV-OVA or RV at MOI-10. 48hpi the cells were harvested and stained with an antibody against the MHCI Kb SIINFEKL complex. Cells pulsed with OVA (SIINFEKL) peptide were used as a positive control.(b). BALB/c derived RIDC-Gag were incubated with CFSE pulsed splenocytes from C57BL/6 mice in an allogeneic MLR for 5 days as described in materials and methods. CFSE dilution in CD4 and CD8 populations were determined by GMF intensities and compared to splenocytes incubated with uninfected DCs (c).

Figure 3. Mice immunized with RIDC-based HIV-1 vaccine exhibit a Gag-specific primary immune response

BALB/c mice were immunized with 4 –5 × 10⁵ DCs (uninfected, RIDCs or RIDC-Gag) or 1 × 10⁶ ffu of live RV-Gag. Primary immune responses were monitored seven and eleven days post immunization. Spleens were harvested, stained and analyzed as shown in the representative charts (a). CD8 T cells were analyzed for the expression CD44^hi, CD62L^lo and AMQMLKETI HIV-1 Gag tetramer (a–c). IFN-γ expression after AMQMLKETI HIV-1 Gag peptide stimulation was detected in an ELISpot assay 7 days post immunization and represented as the mean with the standard error of the mean (SEM) (n=4: Uninfected, RV-Gag) and (n=3 RIDC and RIDC-Gag). (d). The data presented here is representative of two independent experiments. Statistical analyses were performed using one-way ANOVA followed by Bonferroni post-test. P<0.05 * and P<0.001 ***.

Figure 4. Mice immunized with RIDC-Gag mount a recall response

Previously immunized BALB/c mice were rested for 60 days followed by a challenge with a dose of 1 × 10⁶ pfu of VV-Gag. The resulting cellular immune responses were analyzed five days post challenge. Splenocytes were obtained from the three immunization groups (Uninfected DC, RIDC and RIDC-Gag) and stained for the expression of CD8, AMQMLKETI HIV-1 Gag tetramer, CD44^hi, and CD62L^lo (a–b). Functional expression of IFN-γ determined via ELISpot (c) and ICS (d). Functional expression of TNF-α, IL2, IL6, and IL10 by CD8 T cells was determined ICS (d). The data presented here is representative of at least three independent experiments. Statistical analyses were performed using one-way ANOVA followed by Bonferroni post test. P<0.05 *, P<0.01 ** and P<0.001 ***.

Figure 5. Gag specific immune response is not due to secondary infection by RV-Gag

RIDC-Gag and -ΔG-Gag infected at MOI-10 were stained with antibodies against RV-N and CD86 and analyzed by flow (a). Mice were primed with either RIDC-Gag or RIDC-ΔG-Gag. Thirty days post prime, the mice were challenged with a dose of 1 × 10⁶ pfu of VV-Gag. Activated and AMQMLKETI specific CD8 T cell induction was determined by FACS analysis (b–c). Functional expression of inflammatory cytokines by antigen pulsed splenocytes was determined by IFN-γ ELISpot (d), presented as the mean with the SEM (n=2: Uninfected, n=3: RIDC-Gag and n=5: RIDC. ΔG-Gag), as well as ICS (IFN-γ, TNF-α, IL6) (e). The data presented is representative of two repeat experiments. Statistical analyses were performed using one-way ANOVA followed by Bonferroni post-test.

Figure 6. RIDC induced CD8 T cells are cytotoxic

P815 target cells were pulsed overnight with either AMQMKLETI peptide or Env (89.6 R10) peptide. The target cells were then harvested and stained with Calcein as described in materials and methods. P815 target cells were incubated in different E:T ratios (100:1, 33:1, 11:1, 3.7:1) with splenocytes obtained from mice first immunized with uninfected DCs, RIDC-Gag or RIDC.ΔG.Gag and then challenged with 1 × 10⁶ pfu of VV-Gag. After the 3hr incubation, supernatants were collected and the amount of fluorescence read to determine the percentage of lysed cells (see materials and methods).

Figure 7. RIDC-Gag enhance cellular responses in a homologous prime-boost regimen

BALB/c mice were immunized with 1 × 10⁶ ffu RV-Gag or 5 × 10⁵ RIDC-Gag and rested for at least 21 days. The mice received booster shots of either RV-Gag or RIDC-Gag and the cellular immune responses analyzed. The cells were gated as shown (a). On day 11 post boost, AMQMLKETI tetramer specific CD8 T cells (b) and IFN-γ producing cells in the presence of AMQMLKETI peptide were quantified in an ELISpot assay (c) and presented as the mean (n=2: Naive, n=3: RV-Gag/RV-Gag, n=5: RV-Gag/RIDC-Gag and n=5:RIDC-Gag/RIDC.Gag). Statistical analyses were performed using two-tailed unpaired t-tests with welch’s correction where necessary.

Figure 8. Mice immunized with RIDCs mount a RV vector specific humoral immune response

BALB/c mice were immunized with either 1 × 10⁵ ffu live RV expressing HIV-1 Gag (i.m.) or 5 × 10⁵ RIDC-Gag (i.v.). Eye bleeds of eight mice per group were done 7, 14 and 21 days post prime. The OD (490nm) readings of total anti RV-G IgG levels in the immunized mice were measured. The results from 1:450, 1:1350, 1:4050 dilutions were charted to monitor the development of the humoral immune response (a). The ratio of anti RV-G IgG2a to IgG1 antibodies induced 14 days post immunization was measured (b). Statistical analyses were performed using two-tailed unpaired t-tests, P<0.05 *, P<0.01 ** and P<0.001 ***. The mean and SEM are presented (n=8 per group).