*3.1. Design of DNA Vaccines Targeting EBNA1, LMP1, and LMP2A*

We designed consensus optimized DNA vaccines targeting the oncogenic EBV latent proteins commonly seen in malignancies, which are EBNA1, LMP1, and LMP2A. Consensus immunogens can focus the immune response towards conserved regions of important antigens, allowing for increased T cell cross reactivity as well as partially compensating for minor variability in the vaccine targeted antigens [48–50]. Consensus sequences using *GD1* (type 1), *B95-8* (type 1), and *AG876* (type 2) EBV genes were generated for all 3 antigens (Figure 1A) to optimize the ability of the vaccines to elicit immune responses against all common viral strains, which are phylogenetically similar [51]. Modifications were made to remove repetitive sequences and to ablate oncogenic properties inherent to the proteins while preserving the structures of the antigens (Figure 1B). EBNA1vax had repetitive sequence removed, and all three antigens had amino acids modified to abrogate functional regions and cell signaling pathways (Appendix A Figure A1). Phylogenetic trees show close relationships between the vaccine antigens and known sequences from viral isolates (Figure 1C). Large deletions

were made to repetitive sequences when engineering EBNA1vax, leading to divergence from known EBNA1 sequences and the long branch away in the diagram, although the retained sequences are well-conserved. The LMP vaccines lie well within their phylogenetic trees, with LMP1 demonstrating roughly 10-fold more diversity than LMP2A. This conservation supports the likelihood that the targeted changes will elicit immune responses against native EBV antigens, as we have described in the clinic for HPV [26,27], Ebola [52], and Zika [53]. However, formal testing in animal models and evaluation in humans is important.

**Figure 1.** Design and expression of EBNA1vax, LMP1vax, and LMP2Avax vaccine antigens. (**A**) Diagram showing the similarity of the consensus sequence of the EBNA1, LMP1, and LMP2A vaccines, generated from the sequences of EBV strains B95-8, AG876, and GD1. The vaccine antigen designs use a SynCon sequence embedded in a pVAX plasmid. (**B**) Modifications were made to the consensus vaccine antigens to avoid potentially oncogenic properties and repetitive sequences. (**C**) Phylogenic trees showing relationship of vaccines to known EBV latent protein sequences. (**D**) Western blots showing the expression of vaccine antigens in untransfected cells (left columns) and cells transfected with the DNA vaccine (right columns). Beta-actin was used as a loading control. (**E**) Immunofluorescence images showing expression of the vaccine antigens in 293T cells, with cytoplasmic EBNA1vax, LMP1vax on the outer membrane, and LMP2Avax showing a vesicular localization. Antigens are labeled in green, and DAPI (4- ,6-diamidino-2-phenylindole) shows the nucleus in blue. Scale bars are 10 μm.

#### *3.2. In Vitro Expression of DNA Vaccines*

293T cells were transfected with the vaccine DNA plasmids to test for expression of the designed synthetic DNA constructs. Western blots of lysates from the transfected cells showed bands for EBNA1vax, LMP1vax and LMP2Avax vaccines close to their predicted molecular weights (Figure 1D). We performed immunofluorescence on the transfected 293T cells to further evaluate the expression and localization of the constructs. These studies confirmed expression of all 3 proteins, with LMP2Avax

showing its characteristic granular distribution and LMP1vax displaying membrane expression (Figure 1E). Interestingly, EBNA1vax was found in the cytoplasm instead of with the typical nuclear localization of EBNA1. This difference may be due to the changes to the consensus sequence aimed at avoiding sequence repeats and specific changes in the functional domains that affect the ability of EBNA1vax to bind to DNA, suggesting that the encoded changes result in attenuation.
