Alphavirus replicon-based adjuvants enhance the immunogenicity and effectiveness of Fluzone® in rhesus macaques

Venezuelan equine encephalitis virus replicon particles (VRP) without a transgene (null VRP) have been used to adjuvant effective humoral [1], cellular [2], and mucosal [3] immune responses in mice. To assess the adjuvant activity of null VRP in the context of a licensed inactivated influenza virus vaccine, rhesus monkeys were immunized with Fluzone® alone or Fluzone® mixed with null VRP and then challenged with a human seasonal influenza isolate, A/Memphis/7/2001 (H1N1). Compared to Fluzone® alone, Fluzone®Â + null VRP immunized animals had stronger influenza-specific CD4+ T cell responses (4.4 fold) with significantly higher levels of virus-specific IFN-γ (7.6 fold) and IL-2 (5.3 fold) producing CD4+ T cells. Fluzone®Â + null VRP immunized animals also had significantly higher plasma anti-influenza IgG (p < 0.0001, 1.3 log) and IgA (p < 0.05, 1.2 log) levels. In fact, the mean plasma anti-influenza IgG titers after one Fluzone®Â + null VRP immunization was 1.2 log greater (p < 0.04) than after two immunizations with Fluzone® alone. After virus challenge, only Fluzone®Â + null VRP immunized monkeys had a significantly lower level of viral replication (p < 0.001) relative to the unimmunized control animals. Although little anti-influenza antibody was detected in the respiratory secretions after immunization, strong anamnestic anti-influenza IgG and IgA responses were present in secretions of the Fluzone®Â + null VRP immunized monkeys immediately after challenge. There were significant inverse correlations between influenza RNA levels in tracheal lavages and plasma anti-influenza HI and IgG anti-influenza antibody titers prior to challenge. These results demonstrate that null VRP dramatically improve both the immunogenicity and protection elicited by a licensed inactivated influenza vaccine.