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. 2015 Mar 12:5:8925.
doi: 10.1038/srep08925.

Primate immune responses to HIV-1 Env formulated in the saponin-based adjuvant AbISCO-100 in the presence or absence of TLR9 co-stimulation

Paola Martinez  1 , Christopher Sundling  1 , Sijy O'Dell  2 , John R Mascola  2 , Richard T Wyatt  3 , Gunilla B Karlsson Hedestam  1
Affiliations

Primate immune responses to HIV-1 Env formulated in the saponin-based adjuvant AbISCO-100 in the presence or absence of TLR9 co-stimulation

Paola Martinez et al. Sci Rep. .

Abstract

Protein-based vaccines require adjuvants to achieve optimal responses. Toll-like receptor (TLR) 9 agonists were previously shown to improve responses to protein-based vaccines, such as the Hepatitis B virus vaccine formulated in alum. Here, we used CpG-C together with the clinically relevant saponin-based adjuvant AbISCO-100/Matrix-M (AbISCO), to assess if TLR9 co-stimulation would quantitatively or qualitatively modulate HIV-1 envelope glycoprotein (Env)-specific B and T cell responses in rhesus macaques. The macaques were inoculated with soluble Env trimers in AbISCO, with or without the addition of CpG-C, using an interval similar to the Hepatitis B virus vaccine. Following a comprehensive evaluation of antigen-specific responses in multiple immune compartments, we show that the Env-specific circulating IgG, memory B cells and plasma cells displayed similar kinetics and magnitude in the presence or absence of CpG-C and that there was no apparent difference between the two groups in the elicited HIV-1 neutralizing antibody titers or antigen-specific CD4+ T cell responses. Importantly, the control of SHIV viremia was significantly improved in animals from both Env-immunized groups relative to adjuvant alone controls, demonstrating the potential of AbISCO to act as a stand-alone adjuvant for Env-based vaccines.

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Figures

Figure 1
Figure 1. Kinetics of the Env-specific IgG response in periphery and mucosa after immunization.
Animals were divided into three experimental groups as follows: Env formulated in AbISCO-100 (AbISCO) and ODN2395 (AbISCO+CpG) (n = 6), Env formulated in AbISCO (n = 6) and AbISCO and ODN2395 alone (Control) (n = 6). (A) Inoculations were given three times, at weeks 0, 8 and 32 (black arrows). Blood (red arrows), bone marrow (blue arrows), and vaginal and rectal lavage (green arrows) were sampled at the indicated time point. (B) Binding of Env-specific IgG represented as log10 of OD50 titers (left panel), and half-life during the long-term interval (right panel); each dot represent an animal and the lines represent a group, AbISCO+CpG (blue) and AbISCO (red). There was no difference in the Env-specific plasma antibody titers at any time point, as assessed by two-way ANOVA followed by Bonferroni multiple comparison post-test. (C, D) Mucosal responses presented as % Env-specific IgG of total IgG in the sample, were evaluated for vaginal (C) and rectal (D) lavages at four different time points (left panels) with error bars representing the standard error of the mean; AbISCO+CpG (blue) and AbISCO (red). Positive correlations between the mucosa Ab frequencies and the circulating Ab titers was found through non-parametric Spearman (r); linear regression indicates a good fit (right panels).
Figure 2
Figure 2. Env-specific ASC frequency in the periphery and in the bone marrow.
A and B left diagrams show the experimental setup for the analysis of peripheral memory B cells (A) and bone marrow-resident plasma cells (B). Memory B cell analysis requires in vitro mitogenic activation for differentiation into ASC, contrary to plasma cells that spontaneously secrete Ab and already defined as ASC. Kinetics of the response plotted as ASC per 1 × 106 cells (middle panels) and frequency of cells producing Env-specific IgG in relation to total IgG (right panels). Circles indicate group mean (n = 6). Quantification of the cells against: total IgG in the AbISCO group (orange circle, dotted line) and AbISCO+CpG group (orange circle, solid line); Env-specific in the AbISCO group (red) and AbISCO+CpG group (blue); and the negative control, Ovalbumin (black). The same color scheme was used in A and B. Because there was no difference between the AbISCO and AbISCO+CpG groups, these were pooled and responses at the different time points evaluated with Two-way ANOVA followed by Bonferroni's post-test for multiple comparisons. NS = non-significant. ****p < 0.0001.
Figure 3
Figure 3. Env-specific CD4+ T cells frequency and cytokine secretion.
Frequencies of cytokine-producing CD4+ T cells (IFN-γ, IL-2, TNF-α) after in vitro stimulation of PBMCs with 15-mer peptides spanning YU2 gp140-F. (A) Gating strategy from one representative animal (K17: in the AbISCO+CpG group) sampled two weeks after the second immunization is shown. Living, single lymphocytes (upper panels), were gated on CD3 and CD4 (middle panels) and then independently for IFN-γ, IL-2 or TNF-α positive populations (lower panels). (B) Kinetics of the frequency of cytokine producing Env-specific CD4+ T cells for each animal two weeks after the first, second and third immunization. Data from six individual animals were plotted after medium subtraction. (C) Total cytokine-producing Env-specific CD4+ T cells were subdivided in seven populations based on the production of one, two or three cytokines (IFN-γ, IL-2, TNF-α). Data from two weeks after the first and the second immunizations (n = 12) after medium subtraction are shown and analyzed with Mann-Whitney test. NS = non-significant.
Figure 4
Figure 4. Plasma neutralizing activity against a panel of tier 1 and tier 2 Env-pseudoviruses.
HIV-1 neutralizing activity in plasma collected two weeks after the third immunization was measured against a panel of tier 1 and tier 2 Env- pseudovirus isolates. Neutralization capacity against CD4-induced epitopes was measured in the presence and absence of soluble CD4. Data from individual animals are shown as the reciprocal dilution giving 50% neutralization (ID50 titer). Color scale is given according to the ID50 neutralization titer of plasma with white representing the absence of neutralization, yellow moderate, orange medium and red high.
Figure 5
Figure 5. Plasma neutralization of the SHIV-SF162P4 challenge stock.
Neutralizing activity against the SHIV-SF162P4 challenge stock was determined in plasma sampled two weeks following the third immunization. (A) Data from individual animals are shown as the reciprocal dilution giving 50% neutralization (ID50 titer). (B) Sera from animals in both the AbISCO and AbISCO+CpG groups displayed significantly higher neutralizing Ab titer against SHIV-SF162P4 compared to Control (*p < 0.05, **<0.01) with no difference between the groups (p > 0.05). Statistics were evaluated with the Kruskal-Wallis test followed by Dunn's test for multiple comparisons. NS = non-significant.
Figure 6
Figure 6. Evaluation of SHIV-SF61P4 viral load in infected macaques.
Viral load (copies per ml) was determined by one-step Q-RT-PCR in plasma sampled weekly, for seven weeks, following vaginal challenge with 100 TCID50 SHIV-SF162P4 viruses. The limit of detection was 100 viral RNA copies per ml plasma. (A) Viral load curves are shown for individual Env-immunized macaques in the AbISCO (red, n = 5) or AbISCO+CpG (blue, n = 6) groups. (B) Group viral load curves are shown (mean ± SD) for AbISCO (red n = 5), AbISCO+CpG (blue, n = 6) and Control (black, n = 9) over a seven week period. Statistical differences were evaluated with Two-way ANOVA followed by Bonferroni's post-test. Red stars indicate AbISCO vs Control (2 w post challenge, p < 0.00001; 3 w post challenge, p = 0.0053) and Blue stars indicate AbISCO+CpG vs Control (2 w post challenge, p = 0.0.0001; 3 w post challenge, p = 0.0032). (C, D) The cumulative viral load over the seven weeks of monitoring was determined by quantifying the area under curve. In (C) the AbISCO (red) and AbISCO+CpG (blue) groups were compared with the Control group and in (D) the pooled AbISCO and AbISCO+CpG groups, referred to as “All Env-immunized” (red/blue circles, n = 11) were compared with the Control group (black, n = 9) using the Kruskal-Wallis test followed by Dunn's post-test (C; p = 0.0321 and p = 0.0155 respectively) and the Mann-Whitney test (D; p = 0.0005). Significant differences were determined as *p < 0.05, **p < 0.01, ***p < 0.001 and ****p < 0.0001. Box plots show median (line), mean (plus), 25–75% (box) and min-max (whiskers). The same color scheme was used throughout the figure.
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