The orthopoxvirus (OPXV) genus is home to many severe mammalian pathogens, including monkeypox (MPXV), variola (VARV), cowpox (CPXV), and ectromelia (ECTV). Though vaccines have proven effective in preventing OPXV disease, limited distribution and underutilization leave much of the world’s population vulnerable to circulating OPXV infection (e.g., MPXV). To date, several primary mechanisms contributing to OPXV pathogenicity remain unknown or ill-defined, and many gene families within OPXV genomes have poorly defined functions.

The B22 family consists of large surface glycoproteins conserved across most virulent OPXVs. The Eisenlohr lab has previously demonstrated that C15, a protein in the B22 family, is necessary for the lethality of ECTV in a BALB/c mouse infection model as well as efficient replication in C57BL/6 mice. This suggests the protein is an essential virulence factor. Additionally, previous work has demonstrated the capacity for C15 to inhibit both the activation of CD4+ T cells and the degranulation of natural killer (NK) cells.

Despite these discoveries, relatively little is known about the molecular mechanisms that mediate immunomodulation by C15 at both the innate and adaptive immune response levels. Specifically, our lab is interested in uncovering the main molecular target of C15 in the host cell as well as mapping the protein’s topography to determine the regions necessary for immunological inhibition.

We previously generated a stable cell line expressing mouse MHC II molecules (IAb and IEd), their transactivator (CIITA), and C15-HA using the Sleeping Beauty transposon system in B6 mice-derived fibroblasts. The Tet-on expression system allows us to induce C15 expression via overnight incubation with doxycycline. The cell line’s C15 surface expression was verified using flow cytometry, and a MUG assay confirmed the ability of the expressed C15 protein to inhibit T cell activation in NP45 and NA110 hybridomas.

My project focused on optimizing an immunoprecipitation (IP) protocol in order to isolate the HA-tagged C15 protein and its binding partners using the generated B6-CIITA-IEd-C15-HA cell line. Initial attempts using an HA-specific IP kit were successful in pulling down C15, and future work will focus on performing mass spectrometric analysis on the eluates in order to quantitatively determine the binding partners of C15. Additionally, we hope to perform an MHC II immunoprecipitation, followed by immunoblotting with antibodies against HA.

We also generated a recombinant ECTV including the N-terminus of C15 (amino acids 1-456) using a CRISPR/Cas9 selection system with sgRNA GFP. A plasmid containing the N-terminus and a V5 tag was transfected into BSC-1 cells infected with ECTVC15KO, a viral construct in which the C15 coding region was replaced with GFP. This recombination event occurs with very low frequency. As poxviruses do not contain DNA repair machinery, exposure to the GFP sgRNA causes abortive replication, which allows us to select against nonrecombinant, GFP-containing viruses. Three rounds of selection with GFP sgRNA were successfully performed to isolate plaques containing the recombinant ECTV-C15aa1-456. Future work involves screening and purifying rECTV-C15aa1-456 and testing its ability to inhibit CD4+ T cell activation in vitro and NK cell function in vivo.