In Vivo Fate of Cowpea Mosaic Virus In Situ Vaccine: Biodistribution and Clearance

Abstract

Cowpea mosaic virus (CPMV) is a nucleoprotein nanoparticle that functions as a highly potent immunomodulator when administered intratumorally and is used as an in situ vaccine. CPMV in situ vaccination remodels the tumor microenvironment and primes a highly potent, systemic, and durable antitumor immune response against the treated and untreated, distant metastatic sites (abscopal effect). Potent efficacy was demonstrated in multiple tumor mouse models and, most importantly, in canine cancer patients with spontaneous tumors. Data indicate that presence of anti-CPMV antibodies are not neutralizing and that in fact opsonization leads to enhanced efficacy. Plant viruses are part of the food chain, but to date, there is no information on human exposure to CPMV. Therefore, patient sera were tested for the presence of immunoglobulins against CPMV, and indeed, >50% of deidentified patient samples tested positive for CPMV antibodies. To get a broader sense of plant virus exposure and immunogenicity in humans, we also tested sera for antibodies against tobacco mosaic virus (>90% patients tested positive), potato virus X (<20% patients tested positive), and cowpea chlorotic mottle virus (no antibodies were detected). Further, patient sera were analyzed for the presence of antibodies against the coliphage Qβ, a platform technology currently undergoing clinical trials for in situ vaccination; we found that 60% of patients present with anti-Qβ antibodies. Thus, data indicate human exposure to CPMV and other plant viruses and phages. Next, we thought to address agronomical safety; i.e., we examined the fate of CPMV after intratumoral treatment and oral gavage (to mimic consumption by food). Because live CPMV is used, an important question is whether there is any evidence of shedding of infectious particles from mice or patients. CPMV is noninfectious toward mammals; however, it is infectious toward plants including black-eyed peas and other legumes. Biodistribution data in tumor-bearing and healthy mice indicate little leaching from tumors and clearance via the reticuloendothelial system followed by biliary excretion. While there was evidence of shedding of RNA in stool, there was no evidence of infectious particles when plants were challenged with stool extracts, thus indicating agronomical safety. Together these data aid the translational development of CPMV as a drug candidate for cancer immunotherapy.

Pharmacology of a Plant Virus Immunotherapy Candidate for Peritoneal Metastatic Ovarian Cancer

“…ELISA assay was performed as described previously. 11 For CPMVspecific serum, IgM antibody titers were determined, following multiple administration of sCy5-CPMV in healthy and tumorbearing animals (plasma from first, third, and sixth time points was analyzed). Plasma collected prior to the first sCy5-CPMV ip injection (prebleed) was used as nai ̈ve control, and the successive bleeds were compared to prebleed for changing antibody levels.…”

“…13,14 Intratumorally, CPMV resides within the tumor for days with minimal leaching into other organs. 11 In this study, we set out to investigate the pharmacology of CPMV when administered intraperitoneally in tumor-free and tumor-bearing mice; an intraperitoneal disseminated (metastatic) ovarian tumor model (ID8-Defb29-Vegf) was used. 12 In particular, we set up an assessment of biodistribution after single and repeat administration of CPMV (Figure 1).…”

“…10 CPMV primarily infects legumes and black-eyed peas through a common insect vector. 11 CPMV is immunogenic and generates B-and T-cell responses when administered in mice models. 12 Indeed, CPMV antibodies are also common in the human population, which indicates its prevalence and human exposure through the food chain or agricultural practices.…”

TLR Agonists Delivered by Plant Virus and Bacteriophage Nanoparticles for Cancer Immunotherapy

“…When administered intratumorally (i.t. ), CPMV is a potent in situ vaccine that activates TLR2, TLR4, and TRL7, and the resulting anti-tumor immune responses have been investigated in the context of colon cancer, breast cancer, glioma, ovarian cancer, and melanoma. − CPMV has also been used as a delivery platform for proteins, peptides, small-molecule drugs, and imaging agents through the direct modification of the capsid. Cowpea chlorotic mottle virus (CCMV) is structurally similar to CPMV but has no anti-tumor efficacy .…”

Inactivated Cowpea Mosaic Virus for In Situ Vaccination: Differential Efficacy of Formalin vs UV-Inactivated Formulations

“…If infectious, CPMV is shed from (canine) patients that could potentially lead to plant infection. While there is currently no evidence that infectious CPMV particles are shed from animal models or humans after ingestion, 16 there are reports that plant viral shedding is detectable in human feces. 17 CPMV infects black-eyed peas and other crops that are grown in the United States.…”