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

Analysis of the immunomodulatory properties of two heat-killed mycobacterial preparations in a human whole blood model

The significant role played by mycobacteria in modulating immune responses through enhancing the crosstalk between innate and adaptive immunity has been highlighted in several studies. Owing to their unique antigenic profile, heat killed (HK) preparations of rapid-growing mycobacteria, currently undergoing clinical development, have been assessed as adjuvant therapy in various diseases. The purpose of this study is to investigate the regulation of leukocyte surface receptors, in whole blood from healthy donors, following in vitro stimulation with HK Mycobacterium vaccae (M. vaccae) or M. obuense. We have demonstrated the ability of both mycobacterial preparations to target monocytes and neutrophils and to regulate the surface expression of selected adhesion receptors, antigen-presenting and costimulatory receptors, pattern recognition receptors, complement and Fc receptors, as well as cytokine/chemokine receptors. Toll-like receptors (TLRs) 1 and 2 were also shown to be involved in mediating the M. obuense-induced upregulation of selected surface receptors on monocytes. Whole blood stimulation with M. vaccae or M. obuense resulted in a significant increase in the secretion of a specific set of cytokines and chemokines. Both mycobacterial preparations induced strong antigen-specific proliferative responses in peripheral blood mononuclear cells. Collectively, our data shows that M. vaccae and M. obuense have the potential to act as potent immunomodulators. Future research based on these findings may reveal novel immune pathways induced by these preparations with potential implication for their use in diverse immunotherapeutic approaches

HIV-Specific CD4 T Cell Responses to Different Viral Proteins Have Discordant Associations with Viral Load and Clinical Outcome

A successful prophylactic vaccine is characterized by long-lived immunity, which is critically dependent on CD4 T cell-mediated helper signals. Indeed, most licensed vaccines induce antigen-specific CD4 T cell responses, in addition to high-affinity antibodies. However, despite the important role of CD4 T cells in vaccine design and natural infection, few studies have characterized HIV-specific CD4 T cells due to their preferential susceptibility to HIV infection. To establish at the population level the impact of HIV-specific CD4 T cells on viral control and define the specificity of HIV-specific CD4 T cell peptide targeting, we conducted a comprehensive analysis of these responses to the entire HIV proteome in 93 subjects at different stages of HIV infection. We show that HIV-specific CD4 T cell responses were detectable in 92% of individuals and that the breadth of these responses showed a significant inverse correlation with the viral load (P = 0.009, R = −0.31). In particular, CD4 T cell responses targeting Gag were robustly associated with lower levels of viremia (P = 0.0002, R = −0.45). Importantly, differences in the immunodominance profile of HIV-specific CD4 T cell responses distinguished HIV controllers from progressors. Furthermore, Gag/Env ratios were a potent marker of viral control, with a high frequency and magnitude of Gag responses and low proportion of Env responses associated with effective immune control. At the epitope level, targeting of three distinct Gag peptides was linked to spontaneous HIV control (P = 0.60 to 0.85). Inclusion of these immunogenic proteins and peptides in future HIV vaccines may act as a critical cornerstone for enhancing protective T cell responses

Polyfunctional responses by human T cells result from sequential release of cytokines

The release of cytokines by T cells defines a significant part of their functional activity in vivo, and their ability to produce multiple cytokines has been associated with beneficial immune responses. To date, time-integrated end-point measurements have obscured whether these polyfunctional states arise from the simultaneous or successive release of cytokines. Here, we used serial, time-dependent, single-cell analysis of primary human T cells to resolve the temporal dynamics of cytokine secretion from individual cells after activation ex vivo. We show that multifunctional, Th1-skewed cytokine responses (IFN-γ, IL-2, TNFα) are initiated asynchronously, but the ensuing dynamic trajectories of these responses evolve programmatically in a sequential manner. That is, cells predominantly release one of these cytokines at a time rather than maintain active secretion of multiple cytokines simultaneously. Furthermore, these dynamic trajectories are strongly associated with the various states of cell differentiation suggesting that transient programmatic activities of many individual T cells contribute to sustained, population-level responses. The trajectories of responses by single cells may also provide unique, time-dependent signatures for immune monitoring that are less compromised by the timing and duration of integrated measures.National Institute of Allergy and Infectious Diseases (U.S.) (grant no. 1U19AI089992)National Institute of Allergy and Infectious Diseases (U.S.) (grant no. 5P01AI045757)National Institute of Allergy and Infectious Diseases (U.S.) (grant no. 1R21AI088590)National Institute of Allergy and Infectious Diseases (U.S.) (grant no. 1RC1AI086152