We describe a DNA vaccine strategy that allows antigens to be produced in vivo in the context of an alphaviral replicon. Mice immunized with such vectors developed humoral and cellular immune responses at higher levels than mice that received a conventional DNA vaccine vector. Immunized animals acquired protective immunity to lethal influenza challenge. Compared with traditional DNA vaccine strategies in which vectors are persistent and the expression constitutive, the expression mediated by the alphaviral vector was transient and lytic. As a result, biosafety risks such as chromosomal integration, and the induction of immunological tolerance, could be circumvented.

Our results provide a surprising new explanation for the 6K doublet, demand a fundamental reinterpretation of existing data on the alphavirus 6K protein, and open the way for future progress in the further characterization of the 6K and TF proteins. The results have implications for alphavirus biology, virion structure, viroporins, ribosomal frameshifting, and bioinformatic identification of novel frameshift-expressed genes, both in viruses and in cellular organisms.

We explored the role of Peyer's patch (PP) dendritic cell (DC) populations in the induction of immune responses to reovirus strain type 1 Lang (T1L). Immunofluorescence staining revealed the presence of T1L structural (σ1) and nonstructural (σNS) proteins in PPs of T1L-infected mice. Cells in the follicle-associated epithelium contained both σ1 and σNS, indicating productive viral replication. In contrast, σ1, but not σNS, was detected in the subepithelial dome (SED) in association with CD11c+/CD8α−/CD11blo DCs, suggesting antigen uptake by these DCs in the absence of infection. Consistent with this possibility, PP DCs purified from infected mice contained σ1, but not σNS, and PP DCs from uninfected mice could not be productively infected in vitro. Furthermore, σ1 protein in the SED was associated with fragmented DNA by terminal deoxy-UTP nick-end labeling staining, activated caspase-3, and the epithelial cell protein cytokeratin, suggesting that DCs capture T1L antigen from infected apoptotic epithelial cells. Finally, PP DCs from infected mice activated T1L-primed CD4+ T cells in vitro. These studies show that CD8α−/CD11blo DCs in the PP SED process T1L antigen from infected apoptotic epithelial cells for presentation to CD4+ T cells, and therefore demonstrate the cross-presentation of virally infected cells by DCs in vivo during a natural viral infection.

In genetic vaccination, recipients are immunized with antigen-encoding nucleic acid, usually DNA. This study addressed the possibility of using the recombinant alpha virus RNA molecule, which replicates in the cytoplasm of transfected cells, as a novel approach for genetic vaccination. Mice were immunized with recombinant Semliki Forest virus RNA-encoding envelope proteins from one of 3 viruses: influenza A virus, a tickborne flavivirus (louping ill virus), or respiratory syncytial virus (RSV). Serologic analyses showed that antigen-specific antibody responses were elicited. IgG isotyping indicated that predominantly Th1 type immune responses were induced after immunization with RSV F protein-encoding RNA, which is relevant for protection against RSV infection. Challenge infection showed that RNA immunization had elicited significant levels of protection against the 3 model virus diseases.