Vaccine-induced immune responses against both Gag and Env improve control of simian immunodeficiency virus replication in rectally challenged rhesus macaques

<div><p>The ability to control lentivirus replication may be determined, in part, by the extent to which individual viral proteins are targeted by the immune system. Consequently, defining the antigens that elicit the most protective immune responses may facilitate the design of effective HIV-1 vaccines. Here we vaccinated four groups of rhesus macaques with a heterologous vector prime/boost/boost/boost (PBBB) regimen expressing the following simian immunodeficiency virus (SIV) genes: <i>env</i>, <i>gag</i>, <i>vif</i>, <i>rev</i>, <i>tat</i>, and <i>nef</i> (Group 1); <i>env</i>, <i>vif</i>, <i>rev</i>, <i>tat</i>, and <i>nef</i> (Group 2); <i>gag</i>, <i>vif</i>, <i>rev</i>, <i>tat</i>, and <i>nef</i> (Group 3); or <i>vif</i>, <i>rev</i>, <i>tat</i>, and <i>nef</i> (Group 4). Following repeated intrarectal challenges with a marginal dose of the neutralization-resistant SIVmac239 clone, vaccinees in Groups 1–3 became infected at similar rates compared to control animals. Unexpectedly, vaccinees in Group 4 became infected at a slower pace than the other animals, although this difference was not statistically significant. Group 1 exhibited the best post-acquisition virologic control of SIV infection, with significant reductions in both peak and chronic phase viremia. Indeed, 5/8 Group 1 vaccinees had viral loads of less than 2,000 vRNA copies/mL of plasma in the chronic phase. Vaccine regimens that did not contain <i>gag</i> (Group 2), <i>env</i> (Group 3), or both of these inserts (Group 4) were largely ineffective at decreasing viremia. Thus, vaccine-induced immune responses against both Gag and Env appeared to maximize control of immunodeficiency virus replication. Collectively, these findings are relevant for HIV-1 vaccine design as they provide additional insights into which of the lentiviral proteins might serve as the best vaccine immunogens.</p></div

Immune correlates analysis of virologic control of SIVmac239 replication.

<p>Three vaccine-induced immune parameters were used for this analysis: the log-transformed titers of gp140-binding antibodies in Groups 1 and 2 at the time of SIV challenge (A and B); the total frequency of SIV-specific CD8+ T-cell responses in Groups 1–4 at the time of SIV challenge (C and D); and the total of frequency of SIV-specific CD4+ T-cell responses in Groups 1–4 at the time of SIV challenge (E and F). These variables were compared with each animal’s peak (A, C, and E) or setpoint (B, D, and F) VLs using the Spearman rank correlation test. Groups 1, 2, 3, and 4 are color coded in pink, green, beige, and black, respectively, and each symbol denotes one monkey.</p

Outcome of repeated marginal dose SIVmac239 IR challenges.

<p>A) Challenge scheme. Macaques were exposed to SIV on day 0 and subsequently bled on days 7 and 10. Plasma collected at these two occasions was assayed for the presence of SIV RNA and a decision was made as to whether or not challenge the animals on day 14. Macaques that remained aviremic on both days 7 and 10 were re-challenged, whereas monkeys with a positive VL on either of these days were not re-challenged. For logistical reasons, vaccinees in Groups 1 and 2, as well as four monkeys in Group 5 (r04156, r02116, r05027, and r09027) were challenged for the first time at wk 14 post rRRV. The Group 3 and Group 4 vaccinees, as well as the remaining monkeys in Group 5 (r08037, r08040, r05092, and rh2313) were challenged for the first time at wk 20 post rRRV. B) Kinetics of SIVmac239 acquisition in macaques in Groups 1–5. Individual animals in each of Groups 1–4 are depicted along with the challenge that infected them (filled circles). Animal r09046 was challenged 11 times because it was aviremic on days 7 and 10 following the 10^th SIV exposure. However, a subsequent analysis revealed that this animal was viremic on day 14 post challenge #10 (i.e., also the day of the 11^th SIV challenge), indicating that it likely acquired SIV infection after the 10^th exposure. This is depicted in panel B as an additional empty circle (non-infecting exposure) placed in front of r09046 at challenge #11. Animal r08030 in Group 3 resisted 22 IR challenges with SIVmac239 (the last three at 2,000 TCID₅₀).</p

Animal characteristics.

<p>Animal characteristics.</p

ADCC titers in vaccinated macaques in Groups 1 and 2.

<p>Plasma collected from vaccinated macaques in Groups 1 (A) and 2 (B) at the time of the first SIV challenge was screened for ADCC activity against SIVmac239-infected target cells (pink, green, and blue lines). SHIV_AD8-E0-infected cells were used as internal controls for non-specific killing (black lines). The decrease in relative light units indicates the loss of virus-infected cells in the presence of an NK cell line during the duration of the assay. Dashed lines denote 50% activity. C) Plasma from an SIV-infected rhesus macaque (382–03) with a defined ADCC titer against SIVmac239-infected cells was used as a positive control for these measurements.</p

Plasma virus concentrations after SIVmac239 infection.

<p>Viral load (VL) traces for individual animals in Group 1 (A), Group 2 (B), Group 3 (C), Group 4 (D), and Group 5 (E). F) Median VLs for Groups 1–5. The median peak (G) and setpoint (H) VLs of Groups 1–4 were compared to those of Group 5 using the Mann-Whitney test. VLs were log-transformed and correspond to the number of vRNA copies/mL of plasma. The dotted lines in all the graphs are for reference only and indicate a VL of 10³ vRNA copies/mL. The dashed lines are also for reference only and denote a VL of 10⁶ vRNA copies/mL. Groups 1, 2, 3, 4, and 5 are color coded in pink, green, beige, black, and red, respectively. Lines represent medians and each symbol corresponds to one vaccinee.</p

Vaccine-induced CD8+ T-cell responses directed against Mamu-A*01- or Mamu-A*02-restricted epitopes in macaques in Groups 1–4.

<p>Four macaques in each of Groups 1–4 expressed either <i>Mamu-A*01</i> or <i>Mamu-A*02</i> (<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006529#ppat.1006529.t001" target="_blank">Table 1</a>). Using the appropriate fluorochrome-labeled MHC-I tetramers for each group, we monitored the ontogeny of vaccine-induced CD8+ T-cell responses specific for SIV epitopes restricted by Mamu-A*01 (A-D) and Mamu-A*02 (E-H). Each panel shows the magnitude of vaccine-induced CD8+ T-cells detected by individual MHC-I tetramers in animals in Groups 1–4. The following Mamu-A*01-restricted epitopes were evaluated: A) Gag CM9 (aa 181–189), B) Env CL9 (aa 233–241), C) Vif VL10 (aa 100–109), and D) Tat SL8 (aa 28–35). The following Mamu-A*02-restricted epitopes were evaluated: E) Gag GY9 (aa 71–79), F) Env RY9 (aa 296–304), G) Vif WY8 (aa 97–104), and H) Nef YY9 (aa 159–167). The times of each vaccination (vertical dashed black lines) and when the two SIVmac239 IR challenge rounds were started (vertical solid red lines) are shown in each graph. Macaques in Groups 1, 2, 3, and 4 are color coded in pink, green, beige, and black, respectively.</p

ICS analysis of vaccine-induced T-cell responses in Groups 1–4 at the time of SIV challenge.

<p>CD8+ and CD4+ T-cell responses were measured in PBMC by ICS using pools of peptides (15mers overlapping by 11 aa) spanning the appropriate SIVmac239 proteins. Peptides spanning the Rev and Tat proteins were tested together in the same in tubes. The percentages of responding CD4+ or CD8+ T-cells displayed in all panels were calculated by adding the frequencies of positive responses producing any combination of three immunological functions (IFN-γ, TNF-α, and CD107a). The magnitude and specificity of vaccine-induced CD8+ and CD4+ T-cell responses are shown for Group 1 (A), Group 2 (B), Group 3 (C), and Group 4 (D). The Kruskal-Wallis test was used for these comparisons and no difference was detected for CD8+ T-cell responses (<i>P</i> = 0.63). A significant difference in CD4+ T-cell responses was detected by this approach (<i>P</i> = 0.03), which was subsequently investigated by pairwise comparisons using the Mann-Whitney test. This statistically significant difference stemmed from higher levels of SIV-specific CD4+ T-cells in Group 1 compared to Group 2 (<i>P</i> = 0.004). Groups 1, 2, 3, and 4 are color coded in pink, green, beige, and black, respectively. Lines represent medians, and each symbol corresponds to one vaccinee. N.T., not tested.</p

Kaplan-Meier rate of infection after repeated IR challenges with SIVmac239.

<p>Macaques in Groups 1–5 were inoculated intrarectally with 200 TCID₅₀ of SIVmac239 every other week (light shade of gray). Since the Group 6 vaccinee r08030 remained uninfected after 19 challenges with 200 TCID₅₀, the dose of the inoculum was increased 10-fold (2,000 TCID₅₀) in the three subsequent exposures (dark shade of gray). Macaque r08030 still remained uninfected after these three high-dose SIV challenges and was not re-challenged. The rate of SIV infection in Groups 1–4 was not significantly different than that of Group 5 (<i>P</i> > 0.34).</p