rHRV-DNA prime-boost vaccination elicits robust CMI

A robust CMI to HIV Gag appears to correlate with control of HIV infection in humans12 and non-human primates22,23. We previously showed that vaccination of mice with 3 doses of pVAX-Gag-PRF elicited broad, poly-functional Gag-specific CMI able to control EcoHIV after challenge24, and that vaccination with pVAX-sTat-IMX313 elicited high titre anti-Tat responses that also controlled EcoHIV infection post challenge25. Moreover, Tat shows adjuvant activities and the ability to broaden CTL responses without affecting Th2 responses when included together Gag- or Env-based HIV vaccines26,27. Therefore, we wished to compare the efficacy of rHRV-DNA vaccination with that of wt-HRV-A1/pVAX or 3X pVAX-Gag-Tat vaccination. Initially, we used the IFN-γ ELISpot assay (and Gag or Tat peptide pools) to compare CMI responses in splenocytes harvested 14 days after the final vaccine inoculation. This experiment showed that the Gag-specific responses to the different peptide pools ranged from 169 to 427 and 72 to 278 (mean SFU/106 cells), in animals vaccinated with rHRV-DNA and 3X pVAX-Gag-Tat, respectively (Fig. 1A–D). These responses were detected in splenocytes stimulated with all 5 Gag peptide pools suggesting that each of the 5 Gag fragments encoded in the rHRV-Gag/Tat cocktail vaccine was expressed in vivo and thus contributed to the resultant Gag-specific responses. Splenocytes from the wt-HRV/pVAX vaccination control group showed responses which were considerably lower (<22 mean IFN-γ SFU) than those from unstimulated cells from vaccinated mice (mean IFN-γ SFU = 25), thus the net IFN-γ responses in this vaccination group is considered zero. The combined Gag response was ~2 fold higher after vaccination with rHRV-DNA compared to 3X pVAX-Gag-Tatvaccination (1147 and 554, respectively, p = 0.0175) as shown in Fig. 1E. Similarly, wt-HRV/pVAX vaccination failed to induce Tat-specific CMI responses above background (mean IFN-γ SFU = 0), but high magnitude Tat-specific CMI responses were readily detected after rHRV-DNA vaccination (mean IFN-γ SFU = 190) as shown in Fig. 1F. Furthermore, the Tat-specific CMI responses elicited by rHRV-DNA were ~3.9 fold higher than those elicited by 3X pVAX-Gag-Tat (190 compared to 49 SFU, respectively, p = 0.0041) as shown in Fig. 1F.

Figure 1 rHRV-DNA vaccination elicits robust CMI in the spleen. Mice (n = 7) received either 2 doses (containing 5 × 106 TCID 50 per dose per animal) of wild-type (wt)-HRV-A1 followed by 50 μg pVAX (vaccination control group) or 2 doses of rHRV-Gag/Tat (containing 5 × 106 TCID 50 per dose per animal) followed by a single ID dose of 50 μg of a DNA cocktail (pVAX-Gag-Tat) containing equimolar concentrations of pVAX-sTat-IMX313 and pVAX-Gag-PRF. This vaccination regimen is referred to as rHRV-DNA prime-boost vaccination. Other mice were vaccinated with 3 ID doses (50 μg/dose per animal) of pVAX-Gag-PRF/pVAX-sTat-IMX313 and referred to as 3X pVAX-Gag-Tat vaccination. Splenocytes were collected 14 days after the final dose and restimulated in duplicate with overlapping peptides representing the entire Gag protein. (A) Gag peptide pool 1, (B) Gag peptide pool 2, (C) Gag peptide pool 3 and (D) Gag peptide pool 4 in an IFN-γ ELIspot. (E) Total Gag responses depicted in pools 1–4. (F) Tat-specific responses from cells stimulated with the complete Tat peptide pool. Splenocytes were also stained with the H-2Kd-Gag 197–205 tetramer for 1 h at room temperature and the number of tetramer-positive CD8+ T cells was analyzed by flow cytometry (G). The data are representative of 2 independent experiments (n = 7) and are plotted as mean SFU per 106 splenocytes (±SEM). Each symbol represents an individual mouse. *p < 0.05, **p < 0.01 and ***p < 0.001 (Mann–Whitney U test). Full size image

As IFN-γ ELISpot assays involve extensive in vitro stimulation of effector cells, which can cause activation-induced cell death28, splenocytes from immunized mice were stained with the H-2Kd-restricted Gag 197–205 tetramer to directly enumerate the Gag-specific CD8+ T cells present in vivo. The mean number of tetramer-positive CD8+ T cells following wt-HRV-pVAX vaccination was less than background from unstained cells, thus the net number of tetramer positive cells in this vaccination group is considered zero. Moreover, tetramer binding CD8+ T cells (mean of tetramer positive CD8+ T cells = 45921) could be readily detected after rHRV-DNA vaccination and these were also significantly higher than those generated following 3X pVAX-Gag-Tat vaccination (45921 vs 20814, P = 0.0023) as shown in Fig. 1G. Collectively, these results illustrate that rHRV-DNA is more effective at inducing Gag and Tat-specific CMI than wt-HRV-pVAX or 3X pVAX-Gag-Tat vaccination.

rHRV-DNA elicits superior systemic poly-functional CMI

T cells capable of producing multiple anti-viral cytokines (eg. IFN-γ and TNF-α) and/or survival/proliferative cytokines (eg. IL-2) i.e. poly-functional T cells, are desirable outcomes for HIV-1 vaccine development as the presence of these cells appears to correlate with improved control of HIV infection in humans12,29 and SIV infection in non-human primates22,23. The data showed that KdGag 197–205 -specific CD8+ T cells from all rHRV-DNA vaccinated animals responded to vaccination by producing IL-2, IFN-γ or TNF-α, whereas responses from wt-HRV-A1/pVAX vaccinated animals did not surpass background from unstimulated cells (Fig. 2A–C). Furthermore, the number of CD8+ T cells producing IFN-γ, IL-2 or TNF-α was ~2 fold (mean 46318 vs 22679, P = 0.0006), ~5 fold (38928 vs 7712, P = 0.0006) and ~3 fold (39007 vs 12738, P = 0.0012) higher following rHRV-DNA than after 3X pVAX-Gag-Tat vaccination, respectively (Fig. 2A–C).

Figure 2 rHRV-DNA elicits superior systemic poly-functional CMI. Mice were vaccinated as described in the legend to Fig. 1 and splenocytes harvested 14 days after the final dose. The cells were stimulated for 1 h with 5 μg/ml of the H-2Kd-restricted Gag 197–205 immuno-dominant peptide, in the presence of protein transport inhibitor (Brefeldin A, eBiosciences) for a further 6 h and cytokine production then analyzed by flow cytometry. We gated on CD8+ T cells to assess the number of CD8+ T cells producing (A) IFN-γ, (B) TNF-α, (C) IL-2, (D) IFN-γ and TNF-α, (E) TNF-α and IL-2, (F) IFN-γ and IL-2 and (G) IFN-γ, TNF-α and IL-2 after stimulation.The data are representative of 2 independent experiments, plotted as mean (n = 7) ± SEM and each symbol represents an individual mouse. *p < 0.05, **p < 0.01, ***p < 0.001 and ns, p > 0.05 (Mann–Whitney U test). Full size image

The number of Gag-specificCD8+ T cells that concurrently produced at least 2 cytokines (poly-functional CD8+ T cells) following KdGag 197–205 peptide stimulation was also determined. Vaccination with rHRV-DNA resulted in ~9-fold more cells producing IFN-γ and TNF-α than 3X pVAX-Gag-Tat vaccination (1259 vs 140, P = 0.0006) as shown in Fig. 2D. Similarly, the number of cells that produced IL-2 and TNF-α was ~6 fold higher in rHRV-DNA vaccinated mice (4240 vs 707, P = 0.0012) as shown in Fig. 2E, while the number of cells producing IFN-γ and IL-2 was ~2.5 fold (1836 vs 523, P = 0.0041) higher (Fig. 2F). The number of triple cytokine- (IFN-γ, IL-2 and TNF-α) producing CD8+ T cells was ~3.3 fold higher after rHRV-DNA vaccination (1398 vs 427, P = 0.0070) as shown in Fig. 2G.

We then examined the number of CD8+ cells with an effector memory T cell (T EM ) phenotype. Effector memory is vital to establish antigen-specific recall responses22,30 and is required for an effective HIV vaccine. As T EM are marked by CD44 expression31, we determined the number of CD44hiCD8+ T cells which synthesised cytokines after splenocytes were stimulated with the KdGag 197–205 peptide.

The number of CD44hiCD8+ T cells that produced IFN-γ, TNF-α or IL-2 was 3–4 fold higher in the rHRV-DNA-than in the 3X pVAX-Gag-Tat vaccination group (Supplementary Fig. S1A–C). Although the number of double and triple positive CD44hiCD8+ T cells was modest after rHRV-DNA or 3X pVAX-Gag-Tat vaccination, it was significantly higher in the rHRV-DNA group (Supplementary Fig. S1D–G, respectively). In summary, the data also illustrate that rHRV-DNA is more effective at inducing poly-functional Gag-specific CD8+ T cell responses in the spleen than wt-HRV-pVAX or 3X pVAX-Gag-Tat vaccination.

rHRV-DNA vaccination elicits superior poly-functional CMI in the gut mucosa

The vast majority of CD4+ T cell death during acute HIV-1 infection occurs in the gut and thus poly-functional CMI responses at the gut mucosa are likely to significantly reduce CD4+ T cell death32. Vaccination with wt-HRV-A1/pVAX failed to induce KdGag 197–205 -specific CD8+ T cell responses above background from unstimulated lymphocytes prepared from mesenteric lymph nodes, whereas high numbers of Gag-specific cells were readily detected following rHRV-DNA vaccination (Fig. 3A–G). Furthermore, the number of mono-functional (Fig. 3A–C) and poly-functional cells (Fig. 3D–G) generated after rHRV-DNA was significantly higher than that after 3X pVAX-Gag-Tat vaccination. The number of poly-functional T EM cells derived from the mesenteric lymph nodes produced following rHRV-DNA or 3X pVAX-Gag-Tat vaccination was generally modest, although rHRV-DNA appeared to be more effective than 3X pVAX-Gag-Tat (Supplementary Fig. S2). Overall, consistent with the splenocyte data, rHRV-DNA induced poly-functional Gag-specific CD8+ T cell responses in the gut more effectively than wt-HRV-pVAX or 3X pVAX-Gag-Tat vaccination.

Figure 3 rHRV-DNA vaccination elicits superior poly-functional CMI in the gut mucosa. Mice were vaccinated as described in the legend to Fig. 1 and lymphocytes prepared from mesenteric lymph nodes harvested 14 days after the final dose. The lymphocytes were stimulated for 1 h with 5 μg/ml of the H-2Kd-restricted Gag 197–205 immuno-dominant peptide and then cultured in the presence of Brefeldin A for a further 6 h. Cytokine production was analyzed by flow cytometry. We gated on CD8+ T cells to assess the number of CD8+ T cells producing (A) IFN-γ, (B) TNF-α, (C) IL-2, (D) IFN-γ and TNF-α, (E) TNF-α and IL-2, (F) IFN-γ and IL-2 and (G) IFN-γ, TNF-α and IL-2 after stimulation. The data are representative of 2 independent experiments, plotted as mean (n = 7) ± SEM and each symbol represents an individual mouse. *p ≤ 0.05, **p ≤ 0.01 and ***p ≤ 0.001 (Mann–Whitney U test). Full size image

rHRV-DNA vaccination elicits superior Tat-specific humoral responses

High titre Tat-specific serum IgG appears to correlate with control of HIV infection in humans11,33 and SIV in macaques34,35. Therefore, we compared the titres of Tat-specific IgG generated after vaccination with wt-HRV-A1/pVAX, rHRV-DNA or 3X pVAX-Gag-Tat. Tat-specific IgG was detected in serum samples from all vaccinated mice (except in the wt-HRV-A1/pVAX group) with mean reciprocal endpoint titres ranging from 270 to 65610 in the rHRV-DNA vaccinated group and from 30 to 810 in the 3X pVAX-Gag-Tat vaccinated group (Fig. 4A). In the rHRV-DNA vaccinated group, anti-Tat IgG was detectable in samples after the first rHRV dose with a mean titre of 236, but was undetectable in the 3X pVAX-Gag-Tat vaccinated group at this time point. After the 2nd dose, the mean anti-Tat antibody titre in serum from the rHRV-DNA vaccinated group increased by ~65 fold (from 236 to 15312) and was considerably higher than that in serum from the 3X pVAX-Gag-Tat group (mean titer = 9). Administration of a single DNA booster dose increased the IgG titres by ~1.2 fold (from 15312 to 17704) in the rHRV-DNA group and by ~38 fold (from 9 to 338) in the 3X pVAX-Gag-Tat vaccinated group. The differences in serum IgG titres were statistically significant at each time point between the two vaccinated groups (Fig. 4A). The OD generated by serum from wt-HRV/pVAX vaccinated animals was lower than the cut-off, thus the antibody titre in this vaccination group was considered to be zero.

Figure 4 rHRV-DNA vaccination elicits superior Tat-specific humoral responses. Mice were vaccinated as described in the legend to Fig. 1. Blood and CVL samples were collected 1 day before each vaccination and 14 days after the final dose. (A) ELISA results showing serum log 10 anti-Tat IgG titres. (B) ELISA results showing anti-Tat sIgA titres. Data are plotted as mean (n = 7) ± SEM and are representative of 2 independent experiments. *p ≤ 0.05 and ***p ≤ 0.001 (Mann–Whitney U test). Full size image

We also examined CVL samples from vaccinated animals for Tat-specific mucosal sIgA, as the presence of antigen-specific sIgA in blood or at the mucosa appears to correlate with protection in humans36,37. We detected modest levels of sIgA (mean titre 20) in CVL samples collected after the DNA boost from all mice in the rHRV-DNA group, but not from mice in the wt-HRV/pVAX or 3X pVAX-Gag-Tat vaccinated group (Fig. 4B). Most surprisingly, sIgA was not detected in samples prior to the DNA boost, indicating that this was required for the rHRV-Gag/Tat vaccine to induce detectable mucosal sIgA. On the whole, the results described above indicate that rHRV-DNA is more effective at inducing robust Gag and Tat-specific CMI as well as Tat-specific humoral immune responses at the mucosa than wt-HRV-pVAX or 3X pVAX-Gag-Tat vaccination.

rHRV-DNA controls the EcoHIV viral load post-challenge

EcoHIV is a murine HIV challenge model and has been used previously to evaluate the efficacy of candidate HIV vaccines24,38. However, there is no documented evidence that EcoHIV can infect mice via the intravaginal route which accounts for a majority of HIV transmissions6, and thus the IP route is a convenient route to successfully deliver the virus24,38. EcoHIV encodes the complete range of HIV proteins, including Tat, from the HIV-1 Clade B NL4-3 strain, except for the HIV gp120 which was replaced with gp80 from the murine leukaemia virus39. The virus spreads from the primary site of infection to the spleen and the brain in a manner reminiscent of HIV infection39.

Consequently, we examined the protective efficacy of wt-HRV/pVAX, rHRV-DNA or 3X pVAX-Gag-Tat vaccination against EcoHIV challenge. All animals were challenged 10 days after the final vaccination, then culled 7 days later and splenocytes, PECs and peripheral blood samples collected to quantify the viral load (VL) by qRT PCR. The results were normalised to the RPL13a house-keeping gene as described previously24,38. Vaccination with rHRV-DNA or 3X pVAX-Gag-Tat significantly reduced the EcoHIV VL by ~204 fold (p = 0.0006) and ~8 fold (p = 0.0006) in PECs (Fig. 5A), ~62 fold (p = 0.0006) and ~11 fold (p = 0.0006) in splenocytes (Fig. 5B), and by ~40 fold (p = 0.0006) and ~7.5 (p = 0.0006) in blood (Fig. 5C), respectively, compared to vaccination with wt-HRV/pVAX. Furthermore, rHRV-DNA vaccination showed superior control of EcoHIV infection compared to 3X pVAX-Gag-Tat vaccination and reduced the VL by ~25 fold (p = 0.0006) in PECs, ~5.5 fold (p = 0.0286) in splenocytes and by ~5.4 fold (p = 0.0035) in peripheral blood as shown in Fig. 5A–C. Taken together, the data suggest that the rHRV-DNA regimen can also be used to achieve effective virologic control of EcoHIV-1 infection.