Adenosine deaminase co-immunization reverses age-associated immunosenescence by restoring germinal center T follicular helper cell function

Older adults experience disproportionate morbidity and mortality from Clostridioides difficile infection (CDI); however, existing toxoid- and receptor binding domain (RBD)-based vaccines elicit suboptimal protection in aged hosts due to the age-associated defects in CD4 ⁺ T cell function, T follicular helper (T _FH ) cell activation, and antibody quality. We evaluated whether adenosine deaminase (ADA), an enzymatic immune modulator that degrades immunosuppressive adenosine, and improves GC T _FH differentiation and survival, could reverse these age-related impairments when co-delivered with DNA vaccine plasmids targeting toxin A and B RBDs (pRBD). In aged mice, pRBD vaccination alone produced markedly reduced toxin-specific effector/memory CD4 ⁺ T cells, diminished T _FH activation, and poor toxin A neutralization compared to vaccinated young mice. Co-immunization with plasmid-encoded adenosine deaminase-1 (pADA) restored toxin-specific CD4 ⁺ T cell generation and cytokine production, activation-induced marker (AIM) T _FH responses, and antibody-mediate toxin neutralization to levels comparable to young adults. Mechanistically, pADA co-immunization was associated with the reduction of CXCR4 on germinal center (GC) T _FH cells—an age-related defect linked to impaired GC positioning and diminished B cell help—suggesting that ADA improves humoral quality by correcting GC T _FH mislocalization. These immune enhancements corresponded with improved clinical outcomes in morbidity, mortality, and weight-loss following C. difficile spore challenge of aged mice. Finally, pADA significantly reduced adenosine levels in aged lymph nodes, implicating a potential enzymatic-based regulation of GC immunosenescence. Together, these findings identify ADA as a metabolic adjuvant capable of reversing key features of vaccine immunosenescence and highlight adenosine dependent CXCR4 regulation as a tractable axis for improving vaccine efficacy in older populations.