Muscle macrophage regenerative response after squalene-adjuvanted influenza vaccination drives Th2-skewed response and is reduced with age

Squalene-based adjuvants like MF59, and its research alternative AddaVax, induce transient muscle injury, but their mechanisms downstream of muscle injury remain unclear. We show that an AddaVax-adjuvanted quadrivalent inactivated influenza virus vaccine (QIV) intramuscular injection triggers muscle regeneration-like immune processes and increases CX3CR1+Ly6C+ macrophages in the muscle and inguinal lymph nodes by day 4 post-injection. This leads to a Th2 skewed vaccine response with higher levels of vaccine specific IgG1 titers, and Th2-associated cytokines in the lungs 5 days after subsequent influenza viral challenge. In aged mice, the macrophage recruitment and polarization is diminished, which is consistent with age-associated muscle mass loss, reflecting the age-related decline in muscle regeneration. Unlike young mice, aged mice exhibit a reduction in magnitude and skewing of AddaVax-mediated Th2 responses to QIV. We found that adoptive transfer of bone marrow-derived macrophages derived from young mice into aged mice at the moment of vaccination leads to their infiltration into the injected muscle, where they collect vaccine antigens, drain to the lymph node, and enhance the Th2 response, recapitulating the young host response in an older host. However, rescuing the Th2-skewing effects of AddaVax alone was not sufficient to enhance protection against mismatched subsequent influenza viral infection in aged mice, suggesting additional factors at play in the diminished vaccine response in aged hosts. This underscores the importance of the macrophage-driven muscle regenerative response in the mechanism of action for squalene-based adjuvants like AddaVax and emphasizes the need to study how muscle damage and regenerative pathways in intramuscular vaccine responses contribute to vaccine effectiveness.