Characterization of T cell responses to co-administered hookworm vaccine candidates Na-GST-1 and Na-APR-1 in healthy adults in Gabon

Abstract

Two hookworm vaccine candidates, Na-GST-1 and Na-APR-1, formulated with Glucopyranosyl Lipid A (GLA-AF) adjuvant, have been shown to be safe, well tolerated, and to induce antibody responses in a Phase 1 clinical trial (Clinicaltrials.gov NCT02126462) conducted in Gabon. Here, we characterized T cell responses in 24 Gabonese volunteers randomized to get vaccinated three times with Na-GST-1 and Na-APR-1 at doses of 30μg (n = 8) or 100μg (n = 10) and as control Hepatitis B (n = 6). Blood was collected pre- and post-vaccination on days 0, 28, and 180 as well as 2-weeks after each vaccine dose on days 14, 42, and 194 for PBMCs isolation. PBMCs were stimulated with recombinant Na-GST-1 or Na-APR-1, before (days 0, 28 and 180) and two weeks after (days 14, 42 and 194) each vaccination and used to characterize T cell responses by flow and mass cytometry. A significant increase in Na-GST-1 -specific CD4+ T cells producing IL-2 and TNF, correlated with specific IgG antibody levels, after the third vaccination (day 194) was observed. In contrast, no increase in Na-APR-1 specific T cell responses were induced by the vaccine. Mass cytometry revealed that, Na-GST-1 cytokine producing CD4+ T cells were CD161+ memory cells expressing CTLA-4 and CD40-L. Blocking CTLA-4 enhanced the cytokine response to Na-GST-1. In Gabonese volunteers, hookworm vaccine candidate, Na-GST-1, induces detectable CD4+ T cell responses that correlate with specific antibody levels. As these CD4+ T cells express CTLA-4, and blocking this inhibitory molecules resulted in enhanced cytokine production, the question arises whether this pathway can be targeted to enhance vaccine immunogenicity.

Fig 1. Vaccination and sampling time points.

Fig 2. Cytokine responses to Na-GST-1 vaccination.

(A) The frequency of IL-2 and TNF producing CD4⁺ T cells in response to Na-GST-1 stimulation, over time compared to baseline (day 0), in control (green line), low dose (orange line) and high dose group (blue line), using linear mixed model for statistical analysis. Cells producing cytokines are expressed as percentage of CD4⁺ T cells. The mean and standard deviation of cytokine producing cells is given for each time point and each vaccine group. (B) Frequency of CD4⁺ T cells producing IL-2 or TNF alone or together in response to Na-GST-1 on day 0 and day 194, in the high dose (100 μg) group. Data are presented as boxplots representing the median, 1^st and 3^rd quantile. Whiskers are extending to the maximum/minimum, no further than 1.5x the IQR (interquartile range). Wilcoxon one-tailed paired test was performed for comparison between day 0 and day 194. (C) Correlation between the change induced by vaccination in Na-GST-1 specific IgG antibody levels and the frequency of TNF producing CD4⁺ T cells, in the high dose group (Spearman correlation test rho = 0.83, p = 0.003). The change in IgG and cytokine producing cells was determine by subtracting the baseline value (day 0) from day 194 value. The antibody levels are given as arbitrary unit (AU). The frequency of cytokine producing cells in (A), (B) and (C) was determined as percentage of total number of CD4⁺ T cell. (*) indicates the significance p≤0.05 in (A) and (B).

Fig 3. Na-GST-1 specific cell phenotype.

(A) HSNE embedding of 1.1 million CD4⁺ T cells at day 194 in Na-GST-1 high dose (100 μg) recipients (n = 3), depicting 5 subsets. The colour represents arcsin5- transformed expression values of indicated markers. (B) Heatmap summarizing the median expression of markers present on identified clusters of CD4⁺ CD161⁺ T cell subset (subset 5). The colours are the same as for the HSNE plots in A. (C) Bar plots depicting the frequency of clusters producing cytokines relative to CD4⁺ CD161⁺ T cells. (D) Cluster partitioning of CD4⁺ CD161⁺ T cells and HSNE plots highlighting cluster 15, which has an increased expression of both CD40-L and CTLA-4.

Fig 4. Antigen specific cytokine production following blocking of CTLA-4.

Analysis of TNF production in response to Na-GST-1 in cells treated with anti-CTLA-4. Data are presented as boxplots showing the median and IQR. Whiskers are extending to the maximum/minimum, no further than 1.5x the IQR. Individual values are shown as points. The change in TNF production in response to Na-GST-1 was determine by subtracting baseline (day 0) value from day 194 value when either medium, control IgG or anti CTLA-4 was used. A significant increase in TNF production was observed after CTLA-4 blocking compared to IgG isotype or medium control. Statistical significance was determined using Kruskal Wallis test. (*) indicates the significance p≤0.05.