Cowpea Mosaic Virus Outperforms Other Members of the Secoviridae as In Situ Vaccine for Cancer Immunotherapy

Abstract

In situ vaccination for cancer immunotherapy uses intratumoral administration of small molecules, proteins, nanoparticles, or viruses that activate pathogen recognition receptors (PRRs) to reprogram the tumor microenvironment and prime systemic antitumor immunity. Cowpea mosaic virus (CPMV) is a plant virus that─while noninfectious toward mammals─activates mammalian PRRs. Application of CPMV as in situ vaccine (ISV) results in a potent and durable efficacy in tumor mouse models and canine patients; data indicate that CPMV outperforms small molecule PRR agonists and other nonrelated plant viruses and virus-like particles (VLPs). In this work, we set out to compare the potency of CPMV versus other plant viruses from the Secoviridae. We developed protocols to produce and isolate cowpea severe mosaic virus (CPSMV) and tobacco ring spot virus (TRSV) from plants. CPSMV, like CPMV, is a comovirus with genome and protein homology, while TRSV lacks homology and is from the genus nepovirus. When applied as ISV in a mouse model of dermal melanoma (using B16F10 cells and C57Bl6J mice), CPMV outperformed CPSMV and TRSV─again highlighting the unique potency of CPMV. Mechanistically, the increased potency is related to increased signaling through toll-like receptors (TLRs)─in particular, CPMV signals through TLR2, 4, and 7. Using knockout (KO) mouse models, we demonstrate here that all three plant viruses signal through the adaptor molecule MyD88─with CPSMV and TRSV predominantly activating TLR2 and 4. CPMV induced significantly more interferon β (IFNβ) compared to TRSV and CPSMV; therefore, IFNβ released upon signaling through TLR7 may be a differentiator for the observed potency of CPMV-ISV. Additionally, CPMV induced a different temporal pattern of intratumoral cytokine generation characterized by significantly increased inflammatory cytokines 4 days after the second of 2 weekly treatments, as if CPMV induced a "memory response". This higher, longer-lasting induction of cytokines may be another key differentiator that explains the unique potency of CPMV-ISV.