3.1. Vaccine trial design
Twenty four Indian-origin rhesus monkeys were distributed into three experimental and one control group, each consisting of six animals. On week 0, six monkeys in the first experimental group received priming immunizations with 1011 viral particles (vp) of rAdHu5 expressing HIV-1 clade B Env and 1011 vp of rAdHu5 expressing SIVmac239 Gag-Pol, administered intramuscularly. Six monkeys in the second experimental group were primed by the same route with 1011 vp of rAdC7 expressing HIV-1 clade B Env and 1011 vp of rAdC7 expressing SIVmac239 Gag-Pol. The third experimental group was primed with 1011 vp each of rAdC68 expressing HIV-1 clade B Env and SIVmac239 Gag-Pol. Monkeys in the control group received inoculations with 2×1011 vp of rAd5 expressing rabies virus glycoprotein. At week 29, monkeys in all three experimental groups were boosted with 1011 vp of rAdC1/C5-HIV-1 clade B Env and 1011 vp of rAdC1/C5-SIVmac239 Gag, administered by the intramuscular route. Monkeys in the control group were inoculated with 2×1011 vp of rAdC1/C5 expressing rabies virus glycoprotein.
3.2. Vaccine-elicited cellular and humoral immune responses
The cellular immune responses elicited by these vaccine constructs were assessed using pooled peptide IFN-γ ELISpot assays and intracellular cytokine assays (ICS). As shown in , vaccine-elicited ELISpot responses to HIV-1 Env and SIV Pol peptides were detected in the PBMC of all experimentally vaccinated monkeys 2 weeks after the first inoculation. Monkeys that were primed with rAdHu5 vaccine constructs showed a response to SIV Gag and higher magnitude IFN-γ responses to HIV-1 clade B Env and SIV Pol antigens than the two groups of monkeys that received immunizations with the chimpanzee adenovirus vectors, rAdC7 and rAdC68. Monkeys receiving immunizations with rAdC7 and rAdC68 had comparable magnitude IFN-γ ELISpot responses to all three antigens tested. But the higher magnitude IFN-γ ELISpot responses seen in rAdHu5-primed monkeys than the other two groups of monkeys were not statistically significant (Mann-Whitney test).
Figure 1 Vaccine-elicited cellular immune responses to SIVmac239 Gag, SIVmac239 Pol and HIV-1 HXBc2 Env (clade B). PBMC were isolated at weeks 2 (Post-prime), 31 (Post-boost) and 47 (Day of challenge) post-immunization and assessed in IFN-γ ELISpot assays (more ...)
At week 29, monkeys in all three experimental groups were boosted with 1011 vp of rAdC1/C5-clade B Env and 1011 vp of rAdC1/C5-SIV Gag. rAdC1/C5 was chosen as a boosting immunogen because AdC1/C5 uses CD46 as receptor and not CAR, which is used by Ad5, AdC7 and AdC68. We reasoned that the use of a genetically disparate viral vector might prove a better boosting immunogen. PBMC IFN-γ ELISpot responses to SIV Gag, SIV Pol and HIV-1 clade B Env peptides were assessed in all 3 groups of animals at week 31, 2 weeks following the boost. Monkeys primed with rAdHu5 had a 4-fold increase in their IFN-γ ELISpot responses to SIV Gag protein, whereas responses to HIV-1 Env were of same magnitudes as observed following the priming immunization (). Similarly, monkeys primed with either rAdC7 or rAdC68 showed almost 10-fold increases in their IFN-γ ELISpot responses to SIV Gag and a marginal increase in their responses to the HIV-1 Env protein. As SIV Pol was not included in the construct used in the boost, none of these groups of monkeys showed any increase in their responses to SIV Pol (). Total IFN-γ ELISpot responses in the rAdHu5-primed group were significantly higher than those in the rAdC68-primed group (p=0.03). The difference in magnitude of responses between rAdHu5-primed monkeys and the rAdC7-primed monkeys did not achieve statistical significance (Mann-Whitney test).
These IFN-γ ELISpot responses declined slowly in all groups of experimentally immunized monkeys, and as shown in , by week 47, eighteen weeks following the last boost, there was a 1.5-2-fold decrease from the peak responses in the total spot forming cell responses. Monkeys in the rAdHu5-primed group had less of a decline in their total ELISpot responses than the groups primed with chimpanzee adenovirus vectors () and had significantly higher responses than the rAdC68-primed group (p=0.026) but not significantly different than rAdC7-primed group.
As a pooled peptide ELISpot assay only assesses the secretion of IFN-γ by peptide-stimulated T lymphocytes, we sought to assess secretion of other cytokines by these lymphocytes using a flow cytometry-based intracellular cytokine assay. Two weeks following the boost, peripheral blood lymphocytes from the monkeys were exposed to overlapping peptides spanning the SIV Gag protein, and the fractions of CD4+ or CD8+ T cells producing IFN-γ, IL-2 and TNF-α were determined by intracellular cytokine staining. No statistically significant differences were noted in the frequencies of cytokine-producing CD4+ T lymphocytes between the three groups of vaccinated monkeys (, upper panel). As shown in (lower panel), the two groups of monkeys that were primed with recombinant chimpanzee adenovirus vectors showed very comparable frequencies of cytokine-producing CD8+ T lymphocytes. However, a trend toward higher frequencies of IFN-γ- and TNF-α-producing CD8+ lymphocytes was seen in monkeys that were primed with rAdHu5 (, lower panel) but these differences did not achieve statistical significance. Due to limitations in lymphocytes available for performing the cytokine analysis, we chose to assess only the SIV Gag-specific responses.
Figure 2 IFN-γ, IL-2 and TNF-α secretion by CD4+ (A) and CD8+ (B) T lymphocytes were measured by intracellular cytokine staining following stimulation of PBMC with peptide pools spanning the SIV Gag protein two weeks following the boost. The bars (more ...)
To assess the vaccine-elicited humoral immune responses, serum samples from the vaccinated and control monkeys were analyzed for HIV-1 Env neutralizing antibodies. No HIV-1 Env-specific neutralization above background was detected in sera of the vaccinated monkeys at the time of peak vaccine-elicited immunity (data not shown).
3.3. Cellular and humoral immune responses following viral challenge
Eighteen weeks after the final immunization all monkeys were challenged with 50 MID50 of cell-free SHIV-89.6P by the intravenous route. Since virus-specific cellular immune responses post-challenge contribute to the early control of viremia, we monitored the relative magnitudes of the virus-specific cellular immune responses in these infected monkeys. Two weeks post-challenge, the magnitudes of the total IFN-γ ELISpot responses were slightly higher in the group of monkeys primed with rAdC68. The rAdHu5- and rAdC7-primed groups had comparable total ELISpot responses (). The control animals had much lower ELISpot responses. Therefore, although the vaccine-elicited pre-challenge peak and plateau immune responses were greater in the group of monkeys primed with rAdHu5 than in the groups receiving immunizations with the recombinant chimpanzee adenovirus vectors, the post-challenge peak secondary responses were comparable in magnitude in all 3 groups of experimentally vaccinated animals.
Figure 3 PBMC IFN-γ ELISpot responses in the vaccinated and control monkeys following viral challenge. Freshly isolated PBMC were assessed for IFN-γ ELISpot responses after in vitro exposure to peptide pools spanning the SIVmac239 Gag, SIVmac239 (more ...)
IFN-γ ELISpot responses were also assessed at the end of the acute phase of infection, at week 4 following viral challenge. As shown in , rAdHu5- and rAdC68-primed groups of monkeys had a marginal contraction in their total ELISpot responses compared with those measured at the peak of viremia. In contrast, the rAdC7-primed monkeys had no contraction in their ELISpot responses from those measured at 2 weeks post-challenge. Twenty four weeks later, at week 28 following challenge, there was little further contraction in the ELISpot responses in these cohorts of monkeys (). The monkeys in all three vaccinated groups had comparable magnitudes spot forming cell responses at this time.
Emergence of neutralizing antibody response against the challenge virus was monitored in all groups of monkeys following infection (). At four weeks post-challenge, 13 of 18 experimentally vaccinated monkeys had detectable ID50 NAb titers against SHIV-89.6P, and all 24 animals developed a detectable response within six weeks of infection. No significant differences were seen between the vaccinated and control groups of monkeys in either the kinetics or the magnitude of ID50 neutralization titers. On week 10 following challenge, all vaccinated and control monkeys showed moderate titer of neutralizing antibody responses specific for 89.6P envelope. To assess the breadth of the neutralizing antibody responses, sera from all animals were tested against a heterologous panel of clade B viruses. As shown in no significant differences were observed following challenge between vaccine groups in the evolution of neutralizing antibodies against the challenge virus. It is interesting to note that although no significant differences were observed between vaccine groups against the majority of viruses in the panel of heterologous clade B viruses, the rAdHu5-rAdC1/C7 group showed a significantly higher neutralization titer against SF162.LS than the other two groups (p= 0.026, Mann-Whitney test).
Figure 4 Post-challenge NAb responses against SHIV-89.6P. Serum samples were obtained from vaccinated and control monkeys on the day of SHIV-89.6P challenge and subsequent time points following infection. Serial dilutions of samples were tested for NAb activity (more ...)
Figure 5 Tier 1 and Tier 2 virus neutralization. Plasma samples were obtained from vaccinated monkeys six weeks following SHIV-89.6P challenge. Serial dilutions of samples were tested for NAb activity against a panel of Tier 1 and Tier 2 HIV-1 pseudoviruses. Data (more ...)
3.4. Plasma viral RNA levels and CD4 T cell counts
Viral replication in the SHIV-89.6P challenged monkeys was assessed by quantitating plasma viral RNA levels. Log copies of plasma viral RNA per milliliter of plasma for each animal in each of the four groups are shown and the median values for each group of animals for each sampling time are shown in the solid line in each (). The median values of the peak plasma viral RNA levels in each of the 3 groups of experimentally vaccinated monkeys were 6.5 (rAdHu5-rAdC1/C5), 6.0 (rAdC7-rAdC1/C5), and 7.0 (rAdC68-rAdC1/C5) log copies of viral RNA per milliliter of plasma. Median viral load of the monkeys in all three experimentally vaccinated groups was significantly lower than that of the control group (median 7.65 log copies) [p= 0.01 (rAdHu5-rAdC1/C5), 0.0002 (rAdC7-rAdC1/C5), 0.015 (rAdC68-rAdC1/C5), Mann-Whitney test]. Comparisons between the three experimental groups did not reveal significant differences in their peak viral loads.
Figure 6 Plasma viral RNA levels following intravenous challenge with SHIV-89.6P. These values were determined by an ultrasensitive branched DNA amplification assay with a detection limit of 125 copies/ml. Peak viremia values represent plasma viral RNA levels (more ...)
The plasma viral RNA levels in each of the experimentally vaccinated and control monkeys were also assessed following challenge during the post-acute period, defined for each monkey as the median value of six determinations performed on specimens obtained between days 35 and 70 following challenge. The median plasma viral RNA levels during this post-acute period in each of the 3 groups of experimentally vaccinated monkeys were 3.7 (rAdHu5-rAdC1/C5), 3.16 (rAdC7-rAdC1/C5), and 4.13 (rAdC68-rAdC1/C5) and that of the control group was 4.8 log copies of viral RNA per milliliter of plasma. Monkeys in two of the experimentally vaccinated groups had significantly lower plasma viremia than the control vaccinees [p= 0.01 (rAdHu5-rAdC1/C5), p = 0.004 (rAdC7-rAdC1/C5), Mann-Whitney test] (, Post-acute period panel). The plasma viral RNA levels in the rAdC68-primed monkeys and the control monkeys were not significantly different during this period. No significant differences in plasma viral RNA levels were observed between the groups of vaccinated monkeys during the post-acute infection period.
The rAdHu5-rAdC1/C5- and rAdC7-rAdC1/C5- vaccinated monkeys had lower long-term set point plasma viral RNA levels than the control monkeys. The set point plasma viral RNA values used for evaluation of each monkey were the median of eight data points obtained between days 84 and 224 post-challenge. Using these set point values, the six control monkeys had a median value of 4.5 log copies/ml. The median plasma viral RNA levels at set point in the experimentally vaccinated monkeys were 2.85 (rAdHu5-rAdC1/C5), 2.5 (rAdC7-rAdC1/C5), and 3.86 (rAdC68-rAdC1/C5) log copies/ml (, Long-term set point panel). Therefore, at set point, the two groups of vaccinated animals (rAdHu5-rAdC1/C5 and rAdC7-rAdC1/C5) had 1.5–1.8 logs lower plasma viral RNA levels than the control animals [p= 0.015 (rAdHu5-rAdC1/C5), p = 0.009 (rAdC7-rAdC1/C5), Mann-Whitney test]. The AdC68-primed monkeys did not have statistically significant differences in their long-term set point plasma viral RNA levels when compared with the control animals.
Peripheral blood CD4+ T lymphocyte counts were also measured in all the monkeys to assess the vaccine-mediated clinical protection against the pathogenic SHIV-89.6P challenge. All six control monkeys had a greater loss of peripheral blood CD4+ T lymphocytes than the experimentally vaccinated animals by day 14 after challenge (, Peak panel). During the post-acute period (days 35 through 70 following challenge), all six control monkeys showed a profound loss of peripheral blood CD4+ T lymphocytes. In contrast, all experimentally vaccinated animals had a transient decline in peripheral blood CD4+ T lymphocytes detected by day 70 following challenge, but this cell loss substantially reversed in 17 of the 18 monkeys.
Figure 7 Peripheral blood CD4+ T lymphocytes following SHIV-89.6P challenge. The values were determined by multiplying the percentage of CD3+ T lymphocytes that were CD4+ by the total lymphocyte counts. Peak (top panel) represents the CD4+ T lymphocyte counts (more ...)
To quantitate the CD4+ T lymphocyte counts of the monkeys at the time of long-term set-point plasma viremia, the median peripheral blood CD4+ T lymphocyte count was determined between days 84 and 224 post-challenge for each animal. Using these values, the control monkeys had a median CD4+ T lymphocyte count of 171 and the experimentally vaccinated animals had median CD4+ T lymphocyte counts of 596 (rAdHu5-rAdC1/C5), 672 (rAdC7-rAdC1/C5), and 407 (rAdC68-rAdC1/C5) [p= 0.04 (rAdHu5-rAdC1/C5), p = 0.015 (rAdC7-rAdC1/C5), Mann-Whitney test]. The median CD4+ T lymphocyte count for the rAdC68-primed monkeys was not significantly different than that of the control group of monkeys. Therefore, the CD4+ T lymphocyte counts in these monkeys reflected their plasma viral RNA levels post-challenge.