Structural features of HtpGMtb and HtpG-ESAT6Mtb vaccine antigens against tuberculosis: Molecular determinants of antigenic synergy and cytotoxicity modulation

Vaccine development against tuberculosis is an urgent need as the only available vaccine, M. bovis Bacillus Calmette Guerin (BCG), is unable to provide significant protection in adults. Among newly identified antigens, Rv2299c is an excellent candidate for the rational design of an effective multi-antigenic TB vaccine. Also, when fused to the T cell antigen ESAT6, it becomes highly effective in boosting BCG immunization and it adopts low cytotoxicity compared to ESAT6. We here characterize these proteins by coupling various biophysical techniques to cytofluorimetry and computational studies. Altogether, our data provide an experimental evidence of the role of Rv2299c as a dimeric and highly thermostable molecular chaperone, here denoted as HtpG. Molecular dynamics simulations show that ATP rigidly anchors the ATP-binding loop in a conformation incompatible with the structure of the free enzyme. We also show that HtpG dimeric state is an important molecular feature for the improved antigenic and cytotoxic properties of HtpG-ESAT6. Indeed, structural features of HtpG-ESAT6 show that not only does this molecule combine the antigenic properties of HtpG and ESAT6, but HtpG locks ESAT6 in a dimeric state, thus improving its cytotoxicity properties. The data presented here provide solid basis for the rational design of upgraded antigens.