It is unlikely that overexpression

It is unlikely that overexpression of Ag85A and/or Ag85B was responsible for the VZV reactivations, as extensive pre-clinical and clinical use of these CB-5083 in other experimental TB vaccines has been completed without reports of zoster. Overexpression of the Rv3407 antigen has not been previously studied in any other experimental TB vaccine clinical trial. However, we were unable to detect evidence of Rv3407-specific immunity induced by AERAS-422 (data not shown), suggesting only low levels of the antigen were produced in vivo. Although type I IFN (IFN-α/IFN-β) induces important anti-viral effects and type II IFN exerts immunogenic activity against intracellular pathogens, both type I and type II IFNs appear to have counter-regulatory activities. Type I IFN can exacerbate Mtb infections in mice associated with reduced CD4+ Th1 cell responses (Manca et al., 2001) and further impair the ability of human macrophages to control the growth of intracellular mycobacteria (Bouchonnet et al., 2002; Teles et al., 2013). The molecular signature of active TB disease has been characterized as potent type I IFN signaling in human neutrophils (Berry et al., 2010) and demonstrated to reduce type II IFNR expression at the molecular level (Rayamajhi et al., 2010). Pre-vaccination IFN-γ responses stimulated by BCG in vitro culture with PBMC, as demonstrated in all three vaccine groups in our study (Fig. 3B), have been seen previously and interpreted as either innate immune responses induced by BCG in NK cells or other nonspecific cells, or background memory T cell immunity directed against cross-reactive antigens expressed by environmental organisms other than Mtb (Hoft et al., 2002; Kemp et al., 1996; Hoft et al., 1998; Hoft et al., 1999a; Hoft et al., 1999b; Hoft et al., 2008). Although increases in BCG-induced responses in all three groups post-vaccination were not statistically significant (likely due to the high variability of responses and small sample size), these innate or cross-reactive background responses may be as important for predisposing subjects to an immunopathologic event such as VZV reactivation as any vaccine-induced immune responses. A new anti-IFN-γ-specific autoimmune phenomenon has been identified that is associated with expression of certain HLA-DR/DQ alleles during dissemination of both mycobacterial infection and VZV reactivation (Kampitak et al., 2011; Browne et al., 2012; Chi et al., 2013), suggesting a role for type II IFN responses in suppression of Mtb replication and VZV reactivation. Combined with the high level BCG-induced IFN-γ responses detected in the two volunteers who developed zoster, these data engendered the possibility that these volunteers may possess anti-IFN-γ antibodies that neutralized cytokine function. Such an autoimmune response could lead to exaggerated IFN-γ responses trying to compensate for neutralizing antibody activity. The two zoster-presenting volunteers expressed HLA class II alleles previously associated with anti-IFN-γ autoimmunity, but so did other volunteers that did not develop zoster. Also, IFN-γ neutralizing activity was not detected in plasma harvested either pre- or post-vaccination from the volunteers who developed zoster, indicating that anti-IFN-γ autoimmunity was unlikely to be responsible for a defect in IFN-γ effector function. Despite the lack of detected autoimmunity, the similarity of the Class II HLA alleles between the two zoster-presenting volunteers, combined with complete absence of similar Class I alleles, suggests an association of VZV reactivation with Class II. The mechanisms of this potential association are not understood. Whether too much type II IFN may inhibit type I IFN responses involved in maintenance of VZV latency is unknown. However, both volunteers who developed zoster displayed hypersensitive IFN-γ responses after whole blood stimulation with BCG in vitro pre- and post-vaccination, responses that were five-ten fold higher than those observed in other high dose AERAS-422 recipients pre-vaccination and that increased post-vaccination. Future research should investigate negative effects of type II IFN on type I IFN signaling and functional effects.