Mycobacterium Candidates against Immune Subdominant Antigens as Vaccine

Unlike most pathogens, many of the immunodominant epitopes from Mycobacterium tuberculosis are under purifying selection. This startling ﬁnding suggests that M. tuberculosis may gain an evolutionary advantage by focusing the human immune response against selected proteins. Although the implications of this to vaccine development are incompletely understood, it has been suggested that inducing strong Th1 responses against Ags that are only weakly recognized during natural infection may circumvent this evasion strategy and increase vaccine efﬁcacy. To test the hypothesis that subdominant and/or weak M. tuberculosis Ags are viable vaccine candidates and to avoid complications because of differential immunodominance hierarchies in humans and experimental animals, we deﬁned the immunodominance hierarchy of 84 recombinant M. tuberculosis proteins in experimentally infected mice. We then combined a subset of these dominant or subdominant Ags with a Th1 augmenting adjuvant, glucopyranosyl lipid adjuvant in stable emulsion, to assess their immunogenicity in M. tuberculosis– naive animals and protective efﬁcacy as measured by a reduction in lung M. tuberculosis burden of infected animals after prophylactic vaccination. We observed little correlation between immunodominance during primary M. tuberculosis infection and vaccine efﬁcacy, conﬁrming the hypothesis that subdominant and weakly antigenic M. tuberculosis proteins are viable vaccine candidates. Finally, we developed two fusion proteins based on strongly protective subdominant fusion proteins. When paired with the glucopyranosyl lipid adjuvant in stable emulsion, these fusion proteins elicited robust Th1 responses and limited pulmonary M. tuberculosis for at least 6 wk postinfection with a single immunization. These ﬁndings expand the potential pool of M. tuberculosis proteins that can be Recombinant His-tagged protein products were isolated under native (soluble recombinant proteins) or denaturing (8 M urea) conditions using Ni-nitrilotriacetic acid metal ion afﬁnity chromatography according to the manufacturer’s instructions (Qiagen). Further puriﬁcation by ion exchange and/or size exclusion chromatography was used as necessary to obtain . 95% purity of M. tuberculosis proteins, as determined by visualization using SDS-PAGE. Puriﬁed protein fractions were combined and dialyzed against 20 mM Tris (pH 8.0), concentrated using Amicon Ultra 10-kDa molecular mass cutoff centrifugal ﬁlters (Millipore), and quantiﬁed using a bicinchoninic acid protein assay (Pierce). LPS contamination was evaluated by the Limulus amebocyte lysate assay (Cambrex). All the recombinant proteins used in this study showed residual endotoxin levels , 100 endotoxin units/mg protein.Thefusion proteins ID91 and ID97 were constructed in the same manner as we previously reported for ID93 (9). In brief, the genes for Rv1886, Rv3478, and Rv3619 were attached to the genes for either Rv2389 (ID91) or Rv2875 (ID97) using restriction site linkers and cloned into the pET28a vector. Fusion proteins were expressed in E. coli , puriﬁed under denaturing conditions by chromatography on DEAE and Q Sepharose columns, and analyzed by SDS-PAGE on a 4–20% Tris glycine gel (Invitrogen). The absence of E. coli contamination was conﬁrmed by immunoblotting with HRP-conjugated rabbit polyclonal anti– E. coli Ab (1:1000; ViroStat). Residual LPS contamination was determined to be , 15 endotoxin units/mg protein by the Limulus amebocyte lysate assay (Cambrex).