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

Due to the increasing incidence of cancer, there is a need to develop new platforms that can combat this disease. Cancer immunotherapy is a platform that takes advantage of the immune system to recognize and eradicate tumors and metastases. Our lab has identified a plant virus nanoparticle, cowpea mosaic virus (CPMV) as a promising approach for cancer immunotherapy. When administered intratumorally, CPMV relieves the immune system of tumor-induced immunosuppression and reprograms the tumor microenvironment into an activated state to launch systemic antitumor immunity. The efficacy of CPMV has been tested in many tumor models and in canine cancer patients with promising results: tumor shrinkage, systemic efficacy (abscopal effect), and immune memory to prevent recurrence. To translate this drug candidate from the bench to the clinic, studies that investigate the safety, pharmacology, and toxicity are needed. In this work, we describe the efficacy of CPMV against a metastatic ovarian tumor model and investigate the biodistribution of CPMV after single or repeated intraperitoneal administration in tumor-bearing and healthy mice. CPMV shows good retention in the tumor nodules and broad bioavailability with no apparent organ toxicity based on histopathology. Data indicate persistence of the viral RNA, which remains detectable 2 weeks post final administration, a phenomenon also observed with some mammalian viral infections. Lastly, while protein was not detected in stool or urine, RNA was shed through excretion from mice; however, there was no evidence that RNA was infectious to plants. Taken together, the data indicate that systemic administration results in broad bioavailability with no apparent toxicity. While RNA is shed from the subjects, data suggest agronomical safety. This data is consistent with prior reports and provides support for translational efforts.

Neoadjuvant Intratumoral Immunotherapy with Cowpea Mosaic Virus Induces Local and Systemic Antitumor Efficacy in Canine Mammary Cancer Patients.

The lack of optimal models to evaluate novel agents is delaying the development of effective immunotherapies against human breast cancer (BC). In this prospective open label study, we applied neoadjuvant intratumoral immunotherapy with empty cowpea mosaic virus-like particles (eCPMV) to 11 companion dogs diagnosed with canine mammary cancer (CMC), a spontaneous tumor resembling human BC. We found that two neoadjuvant intratumoral eCPMV injections resulted in tumor reduction in injected tumors in all patients and in noninjected tumors located in the ipsilateral and contralateral mammary chains of injected dogs. Tumor reduction was independent of clinical stage, tumor size, histopathologic grade, and tumor molecular subtype. RNA-seq-based analysis of injected tumors indicated a decrease in DNA replication activity and an increase in activated dendritic cell infiltration in the tumor microenvironment. Immunohistochemistry analysis demonstrated significant intratumoral increases in neutrophils, T and B lymphocytes, and plasma cells. eCPMV intratumoral immunotherapy demonstrated antitumor efficacy without any adverse effects. This novel immunotherapy has the potential for improving outcomes for human BC patients.

COVID-19 vaccines based on viral nanoparticles displaying a conserved B-cell epitope show potent immunogenicity and a long-lasting antibody response.

The COVID-19 pandemic caused by SARS-CoV-2 sparked intensive research into the development of effective vaccines, 50 of which have been approved thus far, including the novel mRNA-based vaccines developed by Pfizer and Moderna. Although limiting the severity of the disease, the mRNA-based vaccines presented drawbacks, such as the cold chain requirement. Moreover, antibody levels generated by these vaccines decline significantly after 6 months. These vaccines deliver mRNA encoding the full-length spike (S) glycoprotein of SARS-CoV-2, but must be updated as new strains and variants of concern emerge, creating a demand for adjusted formulations and booster campaigns. To overcome these challenges, we have developed COVID-19 vaccine candidates based on the highly conserved SARS CoV-2, 809-826 B-cell peptide epitope (denoted 826) conjugated to cowpea mosaic virus (CPMV) nanoparticles and bacteriophage Qß virus-like particles, both platforms have exceptional thermal stability and facilitate epitope delivery with inbuilt adjuvant activity. We evaluated two administration methods: subcutaneous injection and an implantable polymeric scaffold. Mice received a prime-boost regimen of 100 µg per dose (2 weeks apart) or a single dose of 200 µg administered as a liquid formulation, or a polymer implant. Antibody titers were evaluated longitudinally over 50 weeks. The vaccine candidates generally elicited an early Th2-biased immune response, which stimulates the production of SARS-CoV-2 neutralizing antibodies, followed by a switch to a Th1-biased response for most formulations. Exceptionally, vaccine candidate 826-CPMV (administered as prime-boost, soluble injection) elicited a balanced Th1/Th2 immune response, which is necessary to prevent pulmonary immunopathology associated with Th2 bias extremes. While the Qß-based vaccine elicited overall higher antibody titers, the CPMV-induced antibodies had higher avidity. Regardless of the administration route and formulation, our vaccine candidates maintained high antibody titers for more than 50 weeks, confirming a potent and durable immune response against SARS-CoV-2 even after a single dose.

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

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 Qß, a platform technology currently undergoing clinical trials for in situ vaccination; we found that 60% of patients present with anti-Qß 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.

Dual Functionalization of Nanoparticles for Generating Corona-Free and Noncytotoxic Silica Nanoparticles.

Protein coronas form on the surfaces of nanomaterials in biological fluids. This layer of proteins affects the properties of nanomaterials, altering their behavior and masking engineered functionality. The use of nonfouling moieties reduces or prevents corona formation; however, these ligands typically complicate functionalization. We present here a surface modification strategy for silica nanoparticles using specific molar ratios of zwitterionic and amine moieties. Through proper balance of ligands, we were able to generate particles that featured reactive "handles", while retaining nonfouling properties, high hemocompatibility, and low cytotoxicity.