Enhanced immunogenicity in mouse by recombinant BCG prime and protein boost based on latency antigen Rv1733c and/or...

Mycobacterium bovis bacillus Calmette-Guérin (BCG) is the only licensed vaccine against tuberculosis (TB), but it fails to prevent the establishment and reactivation of latent TB. Overexpression of latency and reactivation antigens by BCG may enhance its efficacy against latent infection and subsequent reactivation. This study aimed to construct recombinant BCG strains (rBCGs) overexpressing the latency antigen Rv1733c and/or resuscitation promoting factor E (RpfE).

Episomal (pJH222) or integrative (pJH223) plasmids-either empty or encoding rv1733c or rpfE-were transformed into a lysine auxotroph strain of BCG for lysine complementation. The resulting rBCGs were genetically and phenotypically characterized. Female BALB/c mice underwent a six-month vaccination regimen consisting of a homologous prime-boost immunisation with the rBCGs, followed by a heterologous boost with Rv1733c and/or RpfE recombinant proteins. Splenocytes from immunised mice were stimulated ex vivo with purified protein derivative (PPD) or the corresponding recombinant protein(s) to measure secreted cytokines.

The rBCGs carrying episomal recombinant plasmids encoding rv1733c or rpfE elicited higher levels of antigen-specific interferon-gamma (IFN-γ) compared to empty-vector controls. rBCG strains with integrative constructs encoding either antigen induced higher levels of tumour necrosis factor-alpha relative to the sham, wild-type BCG, and empty-vector groups. All rBCGs expressing either or both antigens generated more favourable IFN-γ to interleukin (IL)-10 ratios upon stimulation with their respective antigen(s) than with PPD. While two rBCGs elicited detectable IL-4 responses, the rBCGs expressing both antigens did not induce IL-4.

This study demonstrates promising immunogenicity of the latency antigen Rv1733c and the resuscitation antigen RpfE, highlighting their potential to enhance BCG efficacy through a tailored prime-boost vaccination strategy.