Mechanism of Action through an IFN Type I - Independent Responses to Influenza Subunit Vaccine MF 59 and Pam 3 CSK 4 Boost Adaptive

The innate immune pathways induced by adjuvants required to increase adaptive responses to influenza subunit vaccines are not well characterized. We profiled different TLR-independent (MF59 and alum) and TLR-dependent (CpG, resiquimod, and Pam3CSK4) adjuvants for the ability to increase the immunogenicity to a trivalent influenza seasonal subunit vaccine and to tetanus toxoid (TT) in mouse. Although all adjuvants boosted the Ab responses to TT, only MF59 and Pam3CSK4 were able to enhance hemagglutinin Ab responses. To identify innate immune correlates of adjuvanticity to influenza subunit vaccine, we investigated the gene signatures induced by each adjuvant in vitro in splenocytes and in vivo in muscle and lymph nodes using DNA microarrays. We found that flu adjuvanticity correlates with the upregulation of proinflammatory genes and other genes involved in leukocyte transendothelial migration at the vaccine injection site. Confocal and FACS analysis confirmed that MF59 and Pam3CSK4 were the strongest inducers of blood cell recruitment in the muscle compared with the other adjuvants tested. Even though it has been proposed that IFN type I is required for adjuvanticity to influenza vaccines, we found that MF59 and Pam3CSK4 were not good inducers of IFN-related innate immunity pathways. By contrast, resiquimod failed to enhance the adaptive response to flu despite a strong activation of the IFN pathway in muscle and lymph nodes. By blocking IFN type I receptor through a mAb, we confirmed that the adjuvanticity of MF59 and Pam3CSK4 to a trivalent influenza vaccine and to TT is IFN independent. T here are different types of influenza virus vaccines with distinct immunogenicity profiles (1–3). Live-attenuated virus vaccines induce a subclinical infection and therefore are very efficacious (4). Formalin-inactivated whole-virus vaccines (WV) contain viral RNA, which activates IFN type I responses that are required to boost the adaptive immune responses (5). Subunit virus vaccines contain hemagglutinin (HA) and neuraminidase Ags, next to some residual structural proteins (M1 and NP), but lack most of the viral RNA, except from some residual RNA that may be associated with NP (6). These vaccines are safer compared with WV, but they are also less immunogenic and therefore require an adjuvant when administered to previously unexposed or immunocompromised subjects. It has been proposed that the best strategy to boost subunit flu vaccine formulation is to restore IFN type I-associated activities that are lost during the purification process. Accordingly, coadministration of type I rIFN with a subunit influenza vaccine in mice …