A 2-Dose AERAS-402 Regimen Boosts CD8+ Polyfunctionality in HIV-Negative, BCG-Vaccinated Recipients

Abstract

Despite the widespread use of BCG, tuberculosis (TB) remains a global threat. Existing vaccine candidates in clinical trials are designed to replace or boost BCG which does not provide satisfying long-term protection. AERAS-402 is a replication-deficient Ad35 vaccine encoding a fusion protein of the M. tuberculosis (Mtb) antigens 85A, 85B, and TB10.4. The present phase I trial assessed the safety and immunogenicity of AERAS-402 in participants living in India - a highly TB-endemic area. Healthy male participants aged 18-45 years with a negative QuantiFERON-TB Gold in-tube test (QFT) were recruited. Enrolled participants (n=12) were randomized 2:1 to receive two intramuscular injections of either AERAS-402 (3 x 10¹⁰ viral particles [vp]); (n=8) or placebo (n=4) on study days 0 and 28. Safety and immunogenicity parameters were evaluated for up to 182 days post the second injection. Immunogenicity was assessed by a flow cytometry-based intracellular cytokine staining (ICS) assay and transcriptional profiling. The latter was examined using dual-color-Reverse-Transcriptase-Multiplex-Ligation-dependent-Probe-Amplification (dc-RT MLPA) assay. AERAS-402 was well tolerated, and no vaccine-related serious adverse events were recorded. The vaccine-induced CD8⁺ T-cell responses were dominated by cells co-expressing IFN-γ, TNF-α, and IL-2 ("polyfunctional" cells) and were more robust than CD4⁺ T-cell responses. Five genes (CXCL10, GNLY, IFI35, IL1B and PTPRCv2) were differentially expressed between the AERAS-402-group and the placebo group, suggesting vaccine-induced responses. Further, compared to pre-vaccination, three genes (CLEC7A, PTPRCv1 and TAGAP) were consistently up-regulated following two doses of vaccination in the AERAS-402-group. No safety concerns were observed for AERAS-402 in healthy Indian adult males. The vaccine-induced predominantly polyfunctional CD8⁺ T cells in response to Ag85B, humoral immunity, and altered gene expression profiles in peripheral blood mononuclear cells (PBMCs) indicative of activation of various immunologically relevant biological pathways.

Keywords: tuberculosis; AERAS-402; Phase 1 trial; T-cell responses; TB vaccine; transcriptional profiling.

Figure 1

Study flow chart.

Figure 2

T cell responses to vaccine-encoded antigens. PBMCs were thawed, rested overnight, and stimulated for 5-6 hours with DMSO (negative control), SEB (positive control), or peptide pools corresponding to the vaccine antigens Ag85A (A, B), Ag85B (C, D), or TB10.4 (E, F). Specimens were then stained for viability, phenotypic markers, and intracellular cytokine expression and evaluated by flow cytometry. Data were analyzed using FlowJo software to generate cytokine Boolean gates. Each gate was subjected to DMSO subtraction to remove background. Negative results following DMSO-subtraction were set to zero. The sum of these gates was then used to determine the total cytokine response for CD4+ (A, C, E) and CD8+ (B, D, F) T cells for the AERAS-402-vaccinated (grey bars) or placebo control (black bars) groups. Bars are plotted for each group and time point for the total response (any cytokine alone or in combination for IFN-γ or IL-2 or TNF-α). Bars represent the 25^th to 75^th percentile, with the cross bar representing the median response. The mean is indicated by an “x” and the error bars represent the minimum and maximum responses.

Figure 3

CD8⁺ cytokine responses to Ag85B. PBMCs were thawed, rested overnight, and stimulated for 5-6 hours with DMSO (negative control), SEB (positive control), or peptide pools corresponding to the vaccine antigens Ag85A, Ag85B, or TB10.4. Specimens were then stained for viability, phenotypic markers, and intracellular cytokine expression and evaluated by flow cytometry. Data was analyzed using Flow Jo software to generate cytokine Boolean gates. Each gate was subjected to DMSO subtraction to remove background. Negative results following background subtraction are set to zero. The sum of these gates was then used to determine the total cytokine response for vaccinated (A) and placebo control (B) groups or plotted by functionality to assess the polyfunctional response (C). Lines (A, B) are plotted for each individual subject. CD8⁺ Single-Gate (Boolean; C) ICS responses for Ag85B at Study Day are shown for vaccinated (gray bars) and placebo (black bars) groups. Bars represent the 25^th to 75^th percentile, with the cross bar representing the median response. The Mean is indicated by an “x” and the error bars represent the minimum and maximum responses.

Figure 4

Mann-Whitney test was applied. Dot-plot graph depicting genes that were differentially expressed between the AERAS-402 and placebo recipients. Day 7 (A); day 35 (B); day 42 (C, D); day 182 (E).

Figure 5

Improved visualizations on the result page of WebGestalt. (A) Bar chart shows enrichment ratio or normalized enrichment score of results with direction. (B) Directed acyclic graph representation with colored nodes depicting corresponding biological process of the enriched genes in the input gene set. Functional enrichment analysis of genes using FunRich. (C) Bar graph of biological pathways in percentage of genes are shown; the blue bar represents the percentage of genes, the yellow bar represents the reference p-value=0.05 and the red bar that depicts the exact p-value.