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Layer-by-layer microparticle (LbL-MP) fabrication was used to produce synthetic vaccines presenting a fusion peptide containing RSV G protein CX3C chemokine motif and a CD8 epitope of the RSV matrix protein 2 (GM2) with or without a covalently linked TLR2 agonist (Pam3.GM2). Immunization of BALB/c mice with either GM2 or Pam3.GM2 LbL-MP in the absence of adjuvant elicited G-specific antibody responses and M2-specific CD8+ T-cell responses. Following challenge with RSV, mice immunized with the GM2 LbL-MP vaccine developed a Th2-biased immune response in the lungs with elevated levels of IL-4, IL-5, IL-13, and eotaxin in the bronchoalveolar lavage (BAL) fluid and a pulmonary influx of eosinophils. By comparison, mice immunized with the Pam3.GM2 LbL-MP vaccine had considerably lower to non-detectable levels of the Th2 cytokines and chemokines and very low numbers of eosinophils in the BAL fluid post-RSV challenge. In addition, mice immunized with the Pam3.GM2 LbL-MP also had higher levels of RSV G-specific IgG2a and IgG2b in the post-challenge BAL fluid compared to those immunized with the GM2 LbL-MP vaccine. While both candidates protected mice from infection following challenge, as evidenced by the reduction or elimination of RSV plaques, the inclusion of the TLR2 agonist yielded a more potent antibody response, greater protection, and a clear shift away from Th2/eosinophil responses. Since the failure of formalin-inactivated RSV (FI-RSV) vaccines tested in the 1960s has been hypothesized to be partly due to the ablation of host TLR engagement by the vaccine and inappropriate Th2 responses upon subsequent viral infection, these findings stress the importance of appropriate engagement of the innate immune response during initial exposure to RSV G CX3C.
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