An Alphavirus-Based Adjuvant Enhances Serum and Mucosal Antibodies, T Cells, and Protective Immunity to Influenza Virus in Neonatal Mice

Neonatal immune responses to infection and vaccination are biased toward TH2 at the cost of proinflammatory TH1 responses needed to combat intracellular pathogens. However, upon appropriate stimulation, the neonatal immune system can induce adult-like TH1 responses. Here we report that a new class of vaccine adjuvant is especially well suited to enhance early life immu-nity. The GVI3000 adjuvant is a safe, nonpropagating, truncated derivative of Venezuelan equine encephalitis virus that targets dendritic cells (DCs) in the draining lymph node (DLN) and produces intracellular viral RNA without propagating to other cells. RNA synthesis strongly activates the innate immune response so that in adult animals, codelivery of soluble protein antigens induces robust humoral, cellular, andmucosal responses. The adjuvant properties of GVI3000 were tested in a neonatal BALB/c mouse model using inactivated influenza virus (iFlu). After a single immunization, mice immunized with iFlu with the GVI3000 adjuvant (GVI3000-adjuvanted iFlu) had significantly higher and sustained influenza virus-specific IgG antibodies, mainly IgG2a (TH1), compared to the mice immunized with antigen only. GVI3000 significantly increased antigen-specific CD4 and CD8 T cells, primedmucosal immune responses, and enhanced protection from lethal challenge. As seen in adult mice, the GVI3000 adjuvant increased the DC population in the DLNs, caused activation andmaturation of DCs, and induced proinflam-matory cytokines and chemokines in the DLNs soon after immunization, including gamma interferon (IFN-), tumor necrosi