CD4+ T cell cytokine responses to the DAR-901 booster vaccine in BCG-primed adults: A randomized, placebo-controlled trial

Abstract

Background: DAR-901 is an inactivated whole cell tuberculosis booster vaccine, prepared using a new scalable, broth-grown method from the master cell bank of SRL172, a vaccine previously shown to prevent tuberculosis. This study examined whether DAR-901 (a) induces CD4+ T cell cytokine profiles previously proposed as correlates of protection and (b) has a specific vaccine-induced immunological signature compared to BCG or placebo.

Methods: We analysed CD4+ T cell cytokine immune responses from 10 DAR-901 recipients, 9 BCG recipients and 9 placebo recipients from the Phase I DAR-901 MDES trial. In that study, HIV-negative, IGRA-negative participants with prior BCG immunization were randomized (double-blind) to receive three intradermal injections of DAR-901 or saline placebo or two injections of saline placebo followed by an intradermal injection of BCG. Antigen-specific functional and phenotypic CD4+ T cell responses along with effector phenotype of responder cells were measured by intracellular cytokine staining.

Results: DAR-901 recipients exhibited increased DAR-901 antigen-specific polyfunctional or bifunctional T cell responses compared to baseline. Vaccine specific CD4+ IFNγ, IL2, TNFα and any cytokine responses peaked at 7 days post-dose 3. Th1 responses predominated, with most responder cells exhibiting a polyfunctional effector memory phenotype. BCG induced greater CD4+ T cell responses than placebo while the more modest DAR-901 responses did not differ from placebo. Neither DAR-901 nor BCG induced substantial or sustained Th17 /Th22 cytokine responses.

Conclusion: DAR-901, a TB booster vaccine grown from the master cell bank of SRL 172 which was shown to prevent TB, induced low magnitude polyfunctional effector memory CD4+ T cell responses. DAR-901 responses were lower than those induced by BCG, a vaccine that has been shown ineffective as a booster to prevent tuberculosis disease. These results suggest that induction of higher levels of CD4+ cytokine stimulation may not be a critical or pre-requisite characteristic for candidate TB vaccine boosters.

Trial registration: ClinicalTrials.gov NCT02063555.

Conflict of interest statement

The authors have declared that no competing interests exist.

Figures

Fig 1. Schematic vaccination timeline of the total vaccinated subjects (n = 28) included in this study.

Subjects within each group were randomized 1:1:3 to receive three doses of saline placebo-group 1 or two of saline followed by BCG (1–8 x 106 CFU)-group 2, or three injections of DAR-901 7 x 106 CFU / for 1 mg)-group 3. Blood samples for CD4+ T cell cytokine expression analyses were collected at baseline/pre-dose 1, pre-dose 2 and pre-dose 3 (responses measured prior to administration of respective treatment) and at 7,28,56 and 180-days after dose 3.

Fig 2. Total CD4+ T cell cytokine positive production by DAR-901, BCG and placebo vaccinated individuals in response to Mtb lysate stimulation at various study timepoints.

CD4+ T cell expression of IFNγ (a), IL2 (b), TNFα (c), IL17(d), IL22 (e) and any IFNγ+, IL2+,TNFα+ cytokine (f) and any IFNγ+, IL2+,IL17+,IL22+ cytokine (g) in response to Mtb lysate stimulant in the DAR-901 (n = 10), BCG (n = 9) and Placebo (n = 9) vaccinated subject was assessed. The median CD4+ T cell cytokine responses (+IQR) are shown at each visit, after subtraction of the unstimulated levels. Treatment-specific immune responses were calculated by AUC analyses for the longitudinal immune responses for individual participants within each treatment group. The Kruskal-Wallis test (with Dunn’s post hoc test) was used to assess for differences between groups from 7-days until 180-days post dose 3 in total cytokine-producing CD4+ T cells (results shown above). Total median cytokine producing CD4+ T cell responses were compared for differences at baseline, at 7-days post dose 3 and at 180-days post dose 3) using Wilcoxon signed-rank test (results shown in S1 Table).

Fig 3. Longitudinal kinetics of DAR-901 lysate-specific CD4+ T cell responses in the DAR-901 compared to placebo vaccinated subjects.

Plotted graphs represent the median for each group after subtraction of background responses, and error bars represent the IQR. Frequencies of DAR-901 lysate-specific CD4+ T cells co-expressing IFNγ, TNFα and IL2 (a) or combinations between IFNγ, TNFα, IL2 (b,c,d) at each timepoint is shown for each treatment group (DAR-901 n = 10; Placebo n = 9). For each CD4+ T cell subset the AUC was compared using Mann-Whitney U test for differences between placebo and DAR-901 treatment groups (results shown above), whilst Wilcoxon signed-rank test was used for comparison of median responses at baseline versus all post-dose 3 timepoints within the DAR-901 vaccinated group (results shown in S2 Table).

Fig 4. CD4+ T cell functional profiles of vaccine specific responses in DAR-901 and BCG vaccinated individuals.

Side to side comparison of DAR-901 responses to DAR-901 lysate (n = 10) and BCG responses to BCG stimulant (n = 9). Pie charts represent relative proportions of respectivevaccine-specific CD4+ T cells co-producing combinations of A) IFNγ, TNFα and IL2, and B) combinations of IFNγ, IL2, IL17 and IL22 among the DAR-901 and BCG vaccinated subjects at the peak (7-days post-dose 3) and final time point (180-days post-dose 3). Frequencies of each gate were determined using Boolean gating. DAR-901 vaccine responses predominantly comprise of polyfunctional combinations of IFNγ+TNFα+IL2+ producing CD4+ T cells which were sustained until the final study point (Key: G = IFNγ, 2 = IL2, T = TNFα, 17 = IL17, 22 = IL22).

Fig 5. Memory phenotype of MTB and vaccine-specific CD4+ IFNγ+TNFα+IL2+ T- cell responses induced by either DAR-901 vaccine or BCG at 7 days and 180-dayss post-dose 3.

Memory differentiation state was based on CD45RO versus CCR7 expression to distinguish between naïve T cells, central memory (TCM), effector memory (TEM) and T cell effector memory re-expressing CD45RA/terminally differentiated effector memory (TEMRA) populations. Box plots represent the T cell memory proportions comparing the frequency of vaccine-elicited CD4 memory T cells in peripheral blood in response to DAR-901 and BCG antigen re-stimulation for the respective vaccinated group. Statistical comparison using Mann-Whitney U test was used to determine the significance of differences in the proportions of memory CD4+ T cells between the two treatment groups (DAR-901 n = 10, BCG n = 9).