Engineered BCG boosts Vγ9Vδ2 T cells via HMBPP production

Tuberculosis remains a major global health threat, and the century-old live-attenuated vaccine strain BCG fails to prevent pulmonary infection in adults. Faced with this urgent need for a more effective vaccine, a team led by Jeffery S. Cox set out to rethink how BCG might be improved. Instead of only adding new antigens, the researchers used a synthetic biology approach to change what BCG makes inside the bacterium. They focused on making more of a small molecule called (E)-4-hydroxy-3-methyl-but-2-enyl pyrophosphate (HMBPP), an intermediate in the bacterial methylerythritol phosphate (MEP) pathway. HMBPP is not made by human cells but is a natural product of many bacteria, and it is a potent activator of a special class of immune cells called Vγ9Vδ2 T cells. These Vγ9Vδ2 T cells are found in higher-order primates and can protect against Mycobacterium tuberculosis infection. By engineering BCG to make more of this self-nonself signal, the team hoped to recruit an innate recognition mechanism in people that has not been intentionally engaged by vaccine design before.

To design their modified BCG strains, the researchers performed synteny analyses of more than 63 mycobacterial species and surveyed 356 genomes to understand how isoprenoid biosynthetic genes are organized. They found that isoprenoid biosynthetic genes are not operonic across all 356 surveyed genomes, although some genes are frequently found in pairs. Using those insights, they built synthetic loci aimed at driving specific overproduction of HMBPP through the MEP pathway. The team then tested these recombinant BCG strains in an in vitro stimulation assay to measure human Vγ9Vδ2 T cell expansion. BCG strains expressing a synthetic MEP locus significantly enhanced Vγ9Vδ2 T cell expansion compared with the wild-type vaccine strain. Separately, overexpression of the HMBPP synthase GcpE alone potently induced Vγ9Vδ2 T cell expansion and did so without causing downregulation of other pathway genes. Together these approaches enabled accumulation of HMBPP and helped overcome feedback inhibition in the MEP pathway.

The work shows a novel route to improving how BCG stimulates the immune system by exploiting a natural recognition pathway rather than only adding conventional antigens. By boosting production of (E)-4-hydroxy-3-methyl-but-2-enyl pyrophosphate (HMBPP), the engineered strains more effectively engage Vγ9Vδ2 T cells, a population known to have protective activity against Mycobacterium tuberculosis. The study presents two practical engineering strategies: one that reconstitutes a synthetic MEP locus to increase flux through the pathway and another that overexpresses the HMBPP synthase GcpE to raise the specific ligand level. Both approaches address feedback inhibition that normally limits HMBPP accumulation. While these results come from laboratory assays, they provide a clear proof of principle that synthetic biology can rewire BCG metabolism to harness innate-like T cell responses. If these findings translate into animal models and clinical testing, they could open a new avenue for making BCG or BCG-derived vaccines more effective at preventing pulmonary tuberculosis in adults.