The 19-kD antigen and protective immunity in a murine model of tuberculosis.

V. V. YEREMEEV, I. V. LYADOVA, B. V. NIKONENKO, A. S. APT, C. ABOU-ZEID*, J. INWALD* &D. B. YOUNG*Laboratory for Immunogenetics, Central Institute for Tuberculosis, Moscow, Russia, and *Department of InfectiousDiseases and Microbiology, Imperial College School of Medicine, London, UK(Accepted for publication 18 January 2000)SUMMARYThe 19-kD antigen is a cell wall-associated lipoprotein present in Mycobacterium tuberculosis and inbacille Calmette–Gue´rin (BCG) vaccine strains. Expression of the 19-kD antigen as a recombinantprotein in two saprophytic mycobacteria—M. vaccae and M. smegmatis—resulted in abrogation oftheir ability to confer protection against M. tuberculosis in a murine challenge model, and in their abilityto prime a DTH response to cross-reactive mycobacterial antigens. Induction of an immune response tothe 19-kD antigen by an alternative approach of DNA vaccination had no effect on subsequentM. tuberculosis challenge. These results are consistent with a model in which the presence of the 19-kDprotein has a detrimental effect on the efficacy of vaccination with live mycobacteria. Targetedinactivation of genes encoding selected antigens represents a potential route towards development ofimproved vaccine candidates.Keywords tuberculosis mouse model recombinant vaccines immune responseINTRODUCTIONThe 19-kD protein was originally identified as a major antigen ofMycobacterium tuberculosis on the basis of its recognition bymurine MoAbs raised against crude bacterial extracts [1].Subsequently, this antigen was shown to be the target of bothantibody and T cell responses in the course of tuberculosisinfection and M. bovis bacille Calmette–Gue´rin (BCG) vaccina-tion in humans [2–4]. Related proteins are present in some slow-growing mycobacterial species [5], but database screening hasfailed to identify homologues in other organisms, and thebiological function of the 19-kD protein remains to be determined.Rapid-growing non-pathogenic mycobacteria, such as M. smeg-matis and M. vaccae, have no endogenous homologue of the 19-kD antigen, but readily express the protein following transforma-tion with the appropriate gene from M. tuberculosis [6,7]. Therecombinant protein expressed in mycobacterial systems resem-bles the native antigen in its modification by post-translationalacylation and glycosylation [6,7].In previous experiments to explore novel tuberculosisvaccines, we compared the effect of immunization of mice withM. vaccae alone with that of M. vaccae expressing the 19-kDantigen [7]. Despite induction of a strong type 1 immune responsespecific to the 19-kD protein, we observed an unexpectedreduction in efficacy of vaccination with the 19-kD recombinantin comparison with control M. vaccae in low-dose aerosol and inhigh-dose i.v. challenge models [7]. This paradoxical result has, toour knowledge, no analogues amongst a variety of mycobacterialantigen vaccine candidates studied so far. There are two ways inwhich the anti-vaccine effect may have been generated. First, theexistence of a type 1 response to the 19-kD antigen at the time ofchallenge might have the effect of exacerbating the M. tubercu-losis infection. Alternatively, the presence of the 19-kD antigen inthe vaccine preparation might interfere with the induction ofevents associated with protection.To explore this phenomenon further, in the present study wecompared the effect of the 19-kD antigen in two differentmycobacterial delivery systems (live M. vaccae and M. smegma-tis), and examined the consequences of induction of a type 1response to the 19-kD antigen by an alternative procedure of DNAvaccination. The results are consistent with the hypothesis that thepresence of the 19-kD antigen alters the response to vaccinationwith mycobacterial vectors.MATERIALS AND METHODSRecombinant mycobacterial strainsMycobacterium vaccae NCTC11659 (supplied by ProfessorJ. Stanford, University College London Medical School, London,UK) was grown at 308C on Middlebrook 7H11 (Difco, Detroit, MI)agar plates or with shaking in 7H9 medium (Difco) supplementedwith 2% glucose. Mycobacterium smegmatis mc