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 (TFH) cell activation, and antibody quality. We evaluated whether adenosine deaminase (ADA), an enzymatic immune modulator that degrades immunosuppressive adenosine, and improves GC TFH 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 TFH 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) TFH 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) TFH cells--an age-related defect linked to impaired GC positioning and diminished B cell help--suggesting that ADA improves humoral quality by correcting GC TFH 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.