*3.4. CD8 Cellular Responses Were Robust in Outbred CD-1 Mice*

To further study these immunogens in a more relevant outbred animal model, we next vaccinated CD-1 mice and compared their responses to control-vaccinated animals. These mice were vaccinated three times at two-week intervals, and immune studies were performed a week after the final vaccination (Figure 3A). Cellular responses were once again more robust for EBNA1 and LMP2A than for LMP1, as was again observed in the inbred mouse models. However, stimulation with each of the latent protein peptide pools produced some responses, as measured by IFNγ ELISPOT (Figure 3B). CD8 responses were dominant when the splenocytes were analyzed by flow cytometry, and CD4 responses were lower (Figure 3C). The CD-1 response supports the CD8 potency of this vaccine approach.

**Figure 3.** Cellular responses produced by combination vaccine in outbred CD-1 mice. (**A**) Vaccination schedule in outbred CD-1 mice. Mice were vaccinated with a combination of EBNA1vax, LMP1vax, and LMP2Avax three times at biweekly intervals, followed by harvesting of their splenocytes a week after the final vaccination. (**B**) Cellular responses to respective peptide pools, shown by IFNγ ELISPOT. (**C**) Plots showing CD4 or CD8 responses of CD-1 mice immunized with the triple vaccine or empty vector (pVax), stimulated with pooled peptides derived from EBNA1, LMP2A and LMP1. Cellular responses are driven by CD8+ cells, as shown by flow cytometry following stimulation of splenocytes.
