*3.3. Inbred Mice Produced Significant Responses to Latent Protein DNA Vaccines*

In vivo immune responses to EBNA1vax, LMP1vax, and LMP2Avax were examined in BALB/c and C57BL/6 mice. The animals were vaccinated with either the empty vector, individual EBNA1vax, LMP1vax, or LMP2Avax vaccine antigens, or a combination vaccine incorporating all three plasmids. Groups of five mice received biweekly vaccinations, and a week after the second dose they were sacrificed to have their splenocytes collected for analysis (Figure 2A).

**Figure 2.** DNA vaccination produces strong cellular responses in inbred mice. (**A**) Vaccination schedule to test the immunogenicity of latent proteins in inbred mice. 2 doses of individual or combined latent protein vaccines (vax) were given to groups of 5 BALB/c or C57BL/6 mice two weeks apart, with mouse splenocytes being harvested one week after the final dose (sac). (**B**) Cellular responses of BALB/c and

C57BL/6 mice measured using IFNγ ELISPOT after overnight stimulation with peptide pools. Responses were minimal for LMP1vax, but much larger for EBNA1vax and LMP2Avax. (**C**) Cellular response measured by flow cytometry. IFNγ staining of cells was measured following their stimulation with latent protein peptides. Pooled EBNA1, LMP1, and LMP2A peptides were used for stimulation. (**D**) The gating of representative examples of the BALB/c CD8 data is shown. Peptide stimulated splenocytes from a mouse vaccinated with the combination vaccine are shown on the left, and control cells left in media are shown on the right. \**p* < 0.05, \*\**p* < 0.01, ns: not significant.

IFNγ responses to latent protein peptide pools were evaluated using an ELISPOT assay (Figure 2B). Splenocytes from mice vaccinated with EBNA1vax generated an average of 81 spot forming units (sfu) per million cells for the individual vaccine and 104 sfu for the combined triple vaccine in BALB/c mice, an insignificant difference. A more robust 340 sfu were observed for the same vaccine in C57BL/6 mice, whereas the combination vaccine was much less immunogenic, suggesting that other antigens in the mixture were more a focus of the immune response. LMP2Avax generated responses in both mouse strains as an individual vaccination and in combination with the other antigens. BALB/c mice showed 102 sfu for the individual vaccine and 80 sfu for the combined, and C57BL/6 mice exhibited 83 sfu for LMP2A vax alone and 178 sfu in combination. LMP1vax produced a more modest response of 15 sfu in BALB/c mice that was only notable in the combination vaccine and not observed in the C57BL/6 animals. The modifications that were made to LMP1vax may have limited its immunogenicity. Additional engineering was undertaken to enhance the immunity of the LMP1 antigen. Modified constructs involved the inclusion of an IgE leader sequence coincident with truncation of the N-terminal native sequence, as well as inclusion by gene fusion of tetanus toxoid fragments as part of the ORF. Two constructs were made, one with a short peptide fragment inserted at the C-terminus (LMP1tt30) and the other with a 256 amino acid fragment inserted after the leader sequence (LMP1ttDOM). This design improved the immunity generated by the fusion antigen vaccine (Appendix A Figure A2).

Evaluation of IFNγ by flow cytometry was showed that CD8 cells were driving the immune response (Figure 2C). The triple vaccine generated more robust CD4 and CD8 responses in BALB/c mice, with greater CD8 responses than in the C57BL/6 mice. Overall, the responses induced appeared to be more potent for the induction of CD8 T cell immunity, with a smaller percentage of CD4 T cell induction, suggesting the vaccine is CD8 T cell biased. Gating for the flow cytometry data is shown in Figure 2D.
