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Vaccines containing minigenes - isolated antigenic epitopes encoded by short open reading frames - can, under certain circumstances, confer protective immunity upon the vaccinee. Here we evaluate the efficacy of the minigene vaccine approach using DNA immunization and find that, to be immunogenic, a minigene-encoded epitope requires a perfect "Kozak" translational initiation region. In addition, using intracellular cytokine staining, we show that immunization with a plasmid encoding a full-length protein induces epitope-specific CD8(+) T cells which are detectable directly ex vivo, and constitute approximately 2% of the vaccinee's splenic CD8(+) T cells. In contrast, such cells are undetectable directly ex vivo in recipients of a minigene vaccine. Nevertheless, the minigene plasmid does induce a low number of epitope-specific CD8(+) T cells, which can be amplified to detectable levels by in vivo stimulation. Indeed, 4 days after in vivo stimulation (by virus infection), all vaccinated mice - regardless of whether they had been vaccinated with the minigene or with the full-length gene - had similar numbers of epitope-specific CD8(+) T cells. However, despite these strong responses at 4 days post-infection, recipients of the minigene vaccine showed no enhanced ability to limit virus replication and dissemination. We therefore observe a dichotomy; minigene vaccinees are not protected, despite the presence of strong virus-specific immune responses at 4 days post-challenge. We suggest that the protective benefits of vaccination exert themselves very soon - perhaps within minutes or hours - after virus challenge. If the vaccine-induced immune response is too low to achieve this early protective effect, virus-specific T cells will expand rapidly, but ineffectually, leading to the strong but non-protective response measured at 4 days post-infection. Thus, vaccine-induced immunity should be monitored very early in infection, since the extent to which these responses may later be amplified is largely irrelevant to the protection observed.

A panel of five neutralizing monoclonal antibodies was generated from mice immunized with an attenuated strain of Mengo virus. Four of the antibodies were used to select mutants of Mengo virus which were able to escape neutralization by the selecting antibody, but it was not possible to select mutants which could escape neutralization by the fifth antibody. The capsid coding region of the RNA genome of each mutant was directly sequenced to identify the mutation(s) responsible for the neutralization escape phenotype. These results are compared to those of a previous study in which immunogenic determinants recognized by neutralizing antibodies generated against pentameric capsid subunits were located on the external surface of the Mengo virion. We have confirmed the existence of the previously identified immunogenic determinant in VP3 (site 2) as well as an immunodominant determinant in VP2 (site 1). Two previously uncharacterized determinants, located in surface loops of VP1 (sites 3 and 4A), were also identified. None of the mutations conferring the neutralization escape phenotype was found near the surface depressions on the virion which are believed to be the receptor binding sites.

A set of four monoclonal antibodies which neutralized the infectivity of Mengo virus was used to select 20 non-neutralizable (escape) mutants. Altered amino acids were identified by sequence analyses of the capsid-coding regions of the mutant virus genomes. Mutations were found predominantly in proteins VP2 and VP3, while mutations in VP1 were detected only as second mutations. The Mengo virus VP2 mutations at amino acid residues 2144, 2145, 2147, and 2148 align with site Nlm II in human rhinovirus-14 and site 2 in polioviruses 1 and 3. The mutation at 2075 as well as those at 3057, 3061, and 3068 in VP3 correspond to site 3 in poliovirus. These alignments notwithstanding, the results of cross-neutralization experiments indicate the existence of a single composite neutralization site on the Mengo virion. Considering the three-dimensional structure of the Mengo capsid, the amino acids which are altered in the escape mutants are all exposed on the outer surface and none are found in the "pit," the probable site for binding of a cellular receptor. The VP3 mutations are located in the VP3 "knob" and the VP2 mutations on a nearby ridge. Together these mutations define a set of epitopes within a single composite antigenic determinant which forms a crescent-shaped area around the three-fold icosahedral axes of the Mengo virion.