rBCG Induces Strong Antigen-Specific T Cell Responses in Rhesus Macaques in a Prime-Boost Setting with an Adenovirus 35 Tuberculosis Vaccine Vector

BACKGROUND: BCG vaccination, combined with adenoviral-delivered boosts, represents a reasonable strategy to augment, broaden and prolong immune protection against tuberculosis (TB). We tested BCG (SSI1331) (in 6 animals, delivered intradermally) and a recombinant (rBCG) AFRO-1 expressing perfringolysin (in 6 animals) followed by two boosts (delivered intramuscullary) with non-replicating adenovirus 35 (rAd35) expressing a fusion protein composed of Ag85A, Ag85B and TB10.4, for the capacity to induce antigen-specific cellular immune responses in rhesus macaques (Macaca mulatta). Control animals received diluent (3 animals). METHODS AND FINDINGS: Cellular immune responses were analyzed longitudinally (12 blood draws for each animal) using intracellular cytokine staining (TNF-alpha, IL-2 and IFN-gamma), T cell proliferation was measured in CD4(+), CD8alpha/beta(+), and CD8alpha/alpha(+) T cell subsets and IFN-gamma production was tested in 7 day PBMC cultures (whole blood cell assay, WBA) using Ag85A, Ag85B, TB10.4 recombinant proteins, PPD or BCG as stimuli. Animals primed with AFRO-1 showed i) increased Ag85B-specific IFN-gamma production in the WBA assay (median >400 pg/ml for 6 animals) one week after the first boost with adenoviral-delivered TB-antigens as compared to animals primed with BCG (<200 pg/ml), ii) stronger T cell proliferation in the CD8alpha/alpha(+) T cell subset (proliferative index 17%) as compared to BCG-primed animals (proliferative index 5% in CD8alpha/alpha(+) T cells). Polyfunctional T cells, defined by IFN-gamma, TNF-alpha and IL-2 production were detected in 2/6 animals primed with AFRO-1 directed against Ag85A/b and TB10.4; 4/6 animals primed with BCG showed a Ag85A/b responses, yet only a single animal exhibited Ag85A/b and TB10.4 reactivity. CONCLUSION: AFRO-1 induces qualitatively and quantitatively different cellular immune responses as compared with BCG in rhesus macaques. Increased IFN-gamma-responses and antigen-specific T cell proliferation in the CD8alpha/alpha+ T cell subset represents a valuable marker for vaccine-take in BCG-based TB vaccine trials

The Novel Tuberculosis Vaccine, AERAS-402, Induces Robust and Polyfunctional CD4+ and CD8+ T Cells in Adults

Rationale: AERAS-402 is a novel tuberculosis vaccine designed to boost immunity primed by bacillus Calmette-Guérin (BCG), the only licensed vaccine

Study Timeline and Sampling Schedule.

<p>NHPs were boosted with AERAS-402 fifteen and twenty-seven weeks after the prime with BCG or AFRO-1, Animals in group 1 were primed with BCG, animals in group 2 with the recombinant BCG (AFRO-1) which combines endosomal escape, TB10.4 expression and over-expression of Ag85A and Ag85B. Animals in both groups were boosted with the non-replicating adenovirus 35 AERAS-402 which expresses the Ag85A, Ag85B and TB10.4 fusion protein. Animals in group 3 received the diluent (control group).</p

Prime with BCG or AFRO-1 induces a different IFN-γ production profile in response to <i>Mtb</i> antigen stimulation.

<p>The median of IFN-γ production (measured by ELISA) in whole blood cultures for each group in response to different <i>Mtb</i> antigen stimulation was assessed. Stronger IFN-γ production was seen in animals primed with AFRO-1 in response to Ag85A and Ag85B, as compared to animals primed with BCG one week after the first boost with AERAS-402.</p

Prime with AFRO-1 induces proliferation of Ag85B-specific T cells in CD4⁺ and CD8alpha/alpha⁺ T cells.

<p>The median of the proliferation index (% of blasts in response to antigen stimulation - % of blasts in negative control) in response to <i>Mtb</i> antigens was determined by flow cytometric analysis. Differential expansion of T cell subsets was gauged by gating on T cell subsets, i.e. CD4⁺, CD8alpha/beta⁺ and CD8alpha/alpha⁺. Animals primed with AFRO-1 showed stronger proliferation in response to Ag85B stimulation within CD4⁺ T cells (A) and CD8alpha/alpha⁺ T cells (B) as compared to animals primed with BCG one week after the first boost with AERAS-402. No difference was detectable between animals primed with AFRO-1 or BCG in the CD8alpha/beta⁺ T cell compartment (C).</p