Poxvirus modulation of type I interferon (IFN) production
Over the past decades, major progress has been made toward understanding how viruses are recognized by the immune system to trigger antiviral immune responses and how viruses evade host defense mechanisms. Several viral-sensing molecules have been identified, including Toll-like receptors (TLRs), RIG-I like receptors (RLRs), and cytosolic DNA sensors. Both TLR-dependent and TLR-independent sensing mechanisms leading to interferon (IFN) induction in the host cells have been identified for host detection of poxviruses. We have reported that infection with attenuated vaccinia virus with deletion of virulence factor E3 (a Z-DNA/dsRNA binding protein) induces type I IFN production by epithelial cells via a cytosolic dsRNA sensing mechanism dependent on mitochondrial antiviral signaling protein (MAVS; an adaptor for RIG-I and MDA5) and the transcription factor IRF3. The C-terminal dsRNA binding domain of E3 plays an inhibitory role. We have recently reported that myxoma virus, a Leporipoxvirus that causes lethal myxomatosis in European rabbits, induces type I IFN production in plasmacytoid dendritic cells, via a TLR9/MyD88-, IRF5/IRF7-, and IFNAR-dependent pathway. This pathway is inhibited by the N-terminal Z-DNA binding domain of E3, which is missing in the ortholog M029 protein expressed by myxoma virus. In conventional dendritic cells, infection of the highly attenuated modified vaccinia virus Ankara (MVA), the current vaccine for smallpox, induces type I IFN production via an IRF3-dependent mechanism involving DNA sensing. This pathway is also inhibited by the Z-DNA binding domain of E3. Therefore, poxvirus infection is detected by various host innate antiviral sensing mechanisms in a cell-type-specific manner. Current studies focus on the identification of novel components of poxviral sensing pathways, mechanisms of viral immune evasion by vaccinia E3 and other viral immunomodulatory proteins, and the role of an innate immune sensing pathway in host defense against poxvirus infection.
Poxvirus modulation of autophagy
We have developed a new line of research on poxviral modulation of autophagy. Autophagy, or self-eating, is a cellular response to nutrient starvation or other environmental cues and stress involving degradation of its cellular components through the lysosomal apparatus. The hallmark of autophagy is the formation of autophagosomes and autolysosomes. Autophagy proceeds in several stages: initiation, vesicle nucleation, vesicle elongation and maturation, and vesicle fusion with late endosomes and lysosomes followed by degradation of the vesicle contents. The initiation of autophagy involves inhibition of TOR (target of rapamycin), leading to the activation of Atg1/ULK1. Vesicle nucleation occurs through the activation of Class III phosphatidylinositol 3-kinase (PI3K or Vps34) to generate phosphatidylinositol-3-phosphate (PIP3) via the formation of a multiprotein complex including Beclin-1 (mammalian orthologue of Atg6). Vesicle elongation requires two ubiquitin-like conjugation systems. One system involves the covalent conjugation of Atg12 to Atg5. The other system involves the conjugation of phosphatidylethanolamine (PE) to Atg8 (LC3 in mammals). Lipid conjugation leads to the conversion of the soluble form of LC3 (LC3-I) to the autophagic-vesicle-associated form (LC3-II). LC3-II is used as a marker for autophagy because its lipidation and specific recruitment to autophagosomes provide a shift from diffuse to punctate staining pattern under light microscopy and increase its electrophorectic mobility compared with LC3-I using western blot analysis.
Autophagy plays important roles in host antiviral innate and adaptive immune responses. Many viruses have evolved strategies to evade autophagy. How poxvirus modulates autophagy is unclear. We find that infection with attenuated vaccinia virus MVA or myxoma virus induces autophagy whereas infection with wild-type vaccinia does not. Autophagy induction is critically linked to host sensing of poxvirus infection and type I IFN induction. We are currently investigating the cross-talk between autophagy and DNA sensing pathways, the mechanisms mediating poxviral induction or inhibition of autophagy, and the role of autophagy machinery in host defense against poxvirus infection.
Poxvirus as oncolytic and immunotherapy for melanoma
Advanced melanoma is largely refractory to conventional therapies, including chemotherapy and radiation. The discovery of somatic mutations in the serine-threonine kinase BRAF in about 50 percent of melanomas opened the door for targeted therapy in this disease. Early clinical trials with BRAF inhibitors showed remarkable but not sustainable responses in patients with melanomas with BRAF mutation. Therefore, alternative treatment strategies for patients with wild-type BRAF and with BRAF inhibitor-resistant tumors are urgently needed.
Poxviruses hold promise as oncolytic and immunotherapeutic agents for cancers. Our goal is to develop attenuated vaccinia viruses for melanoma therapy. Our preliminary studies indicate the use of attenuated vaccinia viruses might provide a novel strategy to achieve melanoma cell killing and to initiate innate and adaptive immune responses against melanoma. We currently are investigating the mechanisms of induction of innate immune responses and apoptosis in melanoma cells by attenuated vaccinia virus ∆E3L in human and murine melanoma cells and the immunological mechanisms mediating tumor eradication through intratumoral injection of viruses in murine melanoma models. We also investigate the therapeutic efficacy of the combination of virotherapy and blockade of cytotoxic T lymphocyte-associated antigen 4 (CTLA-4), a key negative regulator of T cell activation, in animal models. Clinical trials of fully humanized anti-CTLA-4 monoclonal antibodies have shown impressive results with durable responses and improved survival in advanced melanoma patients, which led to the recent FDA approval for the treatment of unresectable or metastatic melanoma. We propose that infection of tumor cells with attenuated vaccinia results in tumor cell death, release of tumor antigens, and the recruitment of inflammatory cell infiltrates leading to the induction of tumor specific adaptive immunity, which is enhanced in the presence of CTLA-4 blockade.