Cinar, M. S.; Adams, T. M.; Ozdilek, A.; Avci, F.

Nucleic acid vaccines, including mRNA platforms, represent a transformative approach by inducing adaptive immunity against in situ-expressed antigens. Tuberculosis (TB), caused by Mycobacterium tuberculosis (Mtb), remains a leading global health threat. The only licensed TB vaccine, Bacillus Calmette-Guerin (BCG), offers limited protection against pulmonary TB in adults and adolescents. Underscoring the urgent need for more effective vaccination strategies. However, clinical trials targeting the Mtb Ag85 complex proteins as nucleic acid vaccines have failed to confer protective immunity. We hypothesized that the glycosylation of Mtb immunogens expressed in mammalian hosts as a post-translational modification impairs their immunogenicity and antigenicity. Here, we show that Ag85B expressed in human cells undergoes aberrant N-glycosylation, leading to significantly dampened antigenicity compared to its native, non-glycosylated bacterial counterpart. Site-directed mutagenesis to eliminate N-glycosylation motifs restored immune recognition and response. We then elucidated the molecular and cellular mechanisms by which host-derived glycosylation can impair the efficacy of nucleic acid vaccines, particularly targeting non-viral pathogens. This insight provides a critical foundation for the rational design of next-generation nucleic acid vaccines, where controlling or circumventing undesirable post-translational modifications may be essential to elicit robust protective immunity.