In situ vaccination with cowpea mosaic virus nanoparticles suppresses metastatic cancer.

Cowpea mosaic virus (CPMV) is currently in the development pipeline for multiple biomedical applications, including cancer immunotherapy. In particular the application of CPMV as in situ vaccine has shown promise; here the plant viral nanoparticle is used as an adjuvant and is injected directly into a tumor to reverse immunosuppression and prime systemic anti-tumor immunity. Efficacy of this CPMV-based cancer immunotherapy has been demonstrated in multiple tumor mouse models and canine cancer patients. However, while CPMV is non-infectious to mammals, it is infectious to legumes and therefore, from a safety perspective, it is desired to develop non-infectious CPMV formulations. Non-infectious virus-like particles of CPMV devoid of nucleic acids have been produced; nevertheless, efficacy of such empty CPMV nanoparticles does not match efficacy of nucleic acid-laden CPMV. The multivalent capsid activates the innate immune system through pathogen pattern recognition receptors (PRRs) such as toll-like receptors (TLRs); the RNA cargo provides additional signaling through TLR-7/8, which boosts the efficacy of this adjuvant. Therefore, in this study, we set out to develop RNA-laden, but non-infectious CPMV. We report inactivation of CPMV using UV light and chemical inactivation using β-propiolactone (βPL) or formalin. 7.5 J cm−2 UV, 50 mM βPL or 1 mM formalin was determined to be sufficient to inactivate CPMV and prevented plant infection. We compared the immunogenicity of native CPMV and inactivated CPMV formulations in vitro and in vivo using RAW-Blue™ reporter cells and a murine syngeneic, orthotropic melanoma model (using B16F10 cells and C57BL6 mice). While the in vitro assay indicated activation of the RAW-Blue™ reporter cells by formaldehyde and UV-inactivated CPMV at levels comparable to native CPMV; βPL-inactivated CPMV appeared to have diminished activity. Tumor mouse model experiments indicate potent efficacy of the chemically-inactivated CPMV (UV-treated CPMV was not tested) leading to tumor regression and increased survival; efficacy was somewhat reduced when compared to CPMV, however these samples outperformed the empty CPMV nanoparticles. These results will facilitate the translational development of safe and potent CPMV-based cancer immunotherapies.

Conventional chemo- and radiotherapy are largely ineffective for metastatic melanoma and emerging immune checkpoint inhibitors, while promising, remain ineffective in the majority of patients and carry associated autoimmune side effects. We hypothesized that treatment of lung tumor-bearing mice with a viral-like nanoparticle could modulate the lung immune environment and prevent the development of B16F10 metastatic-like lesions. We report here that inhalation of a self-assembling viral-like particle derived from the cowpea mosaic virus (CPMV) suppresses the development of tumors in the lungs of intravenous challenged mice. The disparity in tumor burden between CPMV- and PBS-treated mice was pronounced, and the effect was immune-mediated as it was not seen in Ifn-γ-/-, Il-12-/-, and NOD/scid/Il2Rγ-/- mice. CPMV nanoparticles were rapidly taken up by antigen-presenting cells that had trafficked to the lung following treatment and resulted in their activation and cytokine/chemokine production. CPMV particles are manufactured in plants and purified of nucleic acids and therefore are nonreplicative and free of endotoxin and other pattern recognition receptor agonists. CPMV nanoparticles are highly scalable, stable, nontoxic, and modifiable with drugs and antigens. These properties, combined with their inherent immunogenicity and significant efficacy against a poorly immunogenic tumor, present CPMV as an attractive novel immunotherapy against cancer metastatic to the lung.

Cancer immunotherapy approaches have emerged as novel treatment regimens against cancer. A particularly interesting avenue is the concept of in situ vaccination, where immunostimulatory agents are introduced into an identified tumor to overcome local immunosuppression and, if successful, mount systemic antitumor immunity. We had previously shown that nanoparticles from cowpea mosaic virus (CPMV) are highly potent in inducing long-lasting antitumor immunity when used as an in situ vaccine in various tumor mouse models. Here we asked whether the nanoparticles from tobacco mosaic virus (TMV) could also be applied as an in situ vaccine and, if so, whether efficacy or mechanism of immune-activation would be affected by the nanoparticle size (300 × 18 nm native TMV vs 50 × 18 nm short TMV nanorods), shape (nanorods vs spherical TMV, termed SNP), or state of assembly (assembled TMV rod vs free coat protein, CP). Our studies indicate that CPMV, but less so TMV, elicits potent antitumor immunity after intratumoral treatment of dermal melanoma (B16F10 using C57BL/6 mice). TMV and TMVshort slowed tumor growth and increased survival time, however, at significantly lower potency compared to that of CPMV. There were no apparent differences between TMV, TMVshort, or the SNP indicating that the aspect ratio does not necessarily play a role in plant viral in situ vaccines. The free CPs did not elicit an antitumor response or immunostimulation, which may indicate that a multivalent assembly is required to trigger an innate immune recognition and activation. Differential potency of CPMV vs TMV can be explained with differences in immune-activation: data indicate that CPMV stimulates an antitumor response through recruitment of monocytes into the tumor microenvironment (TME), establishing signaling through the IFN-γ pathway, which also leads to recruitment of tumor-infiltrated neutrophils (TINs) and natural killer (NK) cells. Furthermore, the priming of the innate immune system also mounts an adaptive response with CD4+ and CD8+ T cell recruitment and establishment of effector memory cells. While the TMV treatment also lead to the recruitment of innate immune cells as well as T cells (although to a lesser degree), key differences were noted in cyto/chemokine profiling with TMV inducing a potent immune response early on characterized by strong pro-inflammatory cytokines, primarily IL-6. Together, data indicate that some plant viral nanotechnology platforms are more suitable for application as in situ vaccines than others; understanding the intricate differences and underlying mechanism of immune-activation may set the stage for clinical development of these technologies.

Intratumoral immunotherapy (ITIT) delivers immune-stimulating agents or treatments directly into solid tumors to reverse the immunosuppressive nature of the tumor microenvironment (TME) and enables local and systemic antitumor immunity. However, optimal clinical strategies for ITIT are not yet fully understood. Cowpea mosaic virus (CPMV) is a plant virus that is not infectious in mammals but is a powerful multi-toll-like receptor (TLR) agonist which stimulates potent antitumor immunity when used in ITIT. ITIT by transfection of interleukin-12 (IL-12) expressing plasmids into melanoma tumors via in vivo electroporation is being assessed in Phase II clinical trials to treat metastatic disease. We investigated the combination of CPMV with IL-12 plasmid electroporation to generate systemic (abscopal) immunity superior to that from either monotherapy alone. Mice were inoculated with two B16-F10 melanoma or MC38 colorectal cancer tumors and one tumor on each flank. One tumor was treated once per week for 2 weeks with IL-12 electroporation and CPMV intratumoral injection. IL-12 electroporation following CPMV injection greatly reduced the efficacy of CPMV by disrupting its viral structure. Conversely, electroporation of IL-12 prior to CPMV injection led to total clearance of 100% of treated tumors and significantly greater suppression of distant, untreated tumors compared to either monotherapy or the reverse treatment order (CPMV IT injection, followed by IL-12 electroporation). This superior abscopal effect led to a significantly improved survival. Our novel treatment strategy offers significant translational value in treating solid tumors by improving the local and systemic efficacy of IL-12 electroporation or CPMV alone.

The current study investigates the use of an immunostimulatory plant virus nanoparticle called the cowpea mosaic virus (CPMV) in both the prophylaxis and treatment of metastatic breast cancer and melanoma. Metastatic cancer remains to this day one of the most difficult areas in oncology to treat and diagnose. Current standardized treatments consist of systemically administered chemotherapeutics, which are not targeted to the diseased areas and oftentimes cause significant side effects. Therefore, novel treatments such as targeted immunotherapies are under development. The S100A9 protein is upregulated in the tumor microenvironment and it plays an important role in tumor metastasis, aggressiveness, and tumor immunosuppression. It is present not only in the pre-metastatic tumor environment, but is also heavily secreted and expressed after metastatic tumor establishment has occurred. This makes S100A9 a prime target in both prophylactic and therapeutic settings; yet, targeted immunotherapies against S100A9 have never previously been explored. CPMV has been shown in the past to be immunostimulatory when injected directly into the tumor with the ability to recruit immune cells for tumor cell killing. However, metastatic tumors cannot be treated through intratumoral injections; therefore, the current work utilizes systemically-administered S100A9-targeted CPMV nanoparticles. Biodistribution studies indicate that S100A9-targeted CPMV homes to the lung while confocal imaging of S100A9 and S100A9-targeted CPMV within the lungs of tumor-inoculated mice demonstrates co-localization. We further demonstrate using flow cytometry that after CPMV homes to the lungs, immune cells such as neutrophils and dendritic cells are recruited to the lungs, and macrophages are polarized towards their antitumor M1 phenotype. Prophylaxis efficacy studies where CPMV is injected before the tumors show that the S100A9-targeted CPMV causes tumor rejection in both metastatic breast cancer and melanoma but that native CPMV does not produce a significant effect. When used as a treatment, the targeted CPMV, similarly shows efficacy against these two cancer models with native CPMV again showing ineffectiveness. The S100A9-targeted CPMV holds great potential as a targeted immunotherapy both prophylactically (e.g. after primary tumor debulking to reduce the onset of metastases) and therapeutically after tumor establishment has occurred. Citation Format: Young Hun Chung, Jooneon Park, Hui Cai, Nicole F. Steinmetz. Prophylaxis and treatment of metastatic breast cancer and melanoma using S100A9-targeted cowpea mosaic virus nanoparticles [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 297.

Cowpea mosaic virus (CPMV) is a potent immunogenic adjuvant and epitope display platform for the development of vaccines against cancers and infectious diseases, including coronavirus disease 2019. However, the proteinaceous CPMV nanoparticles are rapidly degraded in vivo. Multiple doses are therefore required to ensure long-lasting immunity, which is not ideal for global mass vaccination campaigns. Therefore, we formulated CPMV nanoparticles in injectable hydrogels to achieve slow particle release and prolonged immunostimulation. Liquid formulations were prepared from chitosan and glycerophosphate (GP) before homogenization with CPMV particles at room temperature. The formulations containing high-molecular-weight chitosan and 0–4.5 mg mL–1 CPMV gelled rapidly at 37 °C (5–8 min) and slowly released cyanine 5-CPMV particles in vitro and in vivo. Importantly, when a hydrogel containing CPMV displaying severe acute respiratory syndrome coronavirus 2 spike protein epitope 826 (amino acid 809–826) was administered to mice as a single subcutaneous injection, it elicited an antibody response that was sustained over 20 weeks, with an associated shift from Th1 to Th2 bias. Antibody titers were improved at later time points (weeks 16 and 20) comparing the hydrogel versus soluble vaccine candidates; furthermore, the soluble vaccine candidates retained Th1 bias. We conclude that CPMV nanoparticles can be formulated effectively in chitosan/GP hydrogels and are released as intact particles for several months with conserved immunotherapeutic efficacy. The injectable hydrogel containing epitope-labeled CPMV offers a promising single-dose vaccine platform for the prevention of future pandemics as well as a strategy to develop long-lasting plant virus-based nanomedicines.

Plant viruses are naturally occurring nanoparticles and adjuvants that interact with the mammalian immune system. This property can be harnessed in vaccines and immunotherapy. We have previously demonstrated that intratumoral immunotherapy with cowpea mosaic virus (CPMV) stimulates systemic and durable antitumor immunity in mouse tumor models and canine cancer patients. Here we compared the antitumor efficacy of CPMV with potato virus X (PVX) using a mouse model B-cell lymphoma (A20 and BALB/c mice). Despite their diverse morphologies and physiochemical properties, both plant viruses elicited systemic and long-lasting antitumor immune memory, preventing the recurrence of A20 lymphoma in rechallenge experiments. Data indicate differences in the underlying mechanism: CPMV persists longer in the tumor microenvironment (TME) compared to PVX; CPMV is a potent and multivalent toll-like receptor (TLR) agonist (activating TLRs 2, 4 and 7) while PVX may only weakly engage with TLR7. While CPMV and PVX recruit myeloid cells (neutrophils)─CPMV also recruits macrophages. Data further indicate that antiviral T cells may play a role in antitumor efficacy in the case of CPMV immunotherapy, however this may not be the case for PVX. Regardless of the mechanism of action, both CPMV and PVX elicited a durable antitumor response against a B-cell lymphoma tumor model and thus are intratumoral immunotherapy candidates for clinical development.

Despite advances in laparoscopic surgery combined with neoadjuvant and adjuvant therapy, colon cancer management remains challenging in oncology. Recurrence of cancerous tissue locally or in distant organs (metastasis) is the major problem in colon cancer management. Vaccines and immunotherapies hold promise in preventing cancer recurrence through stimulation of the immune system. We and others have shown that nanoparticles from plant viruses, such as cowpea mosaic virus (CPMV) nanoparticles, are potent immune adjuvants for cancer vaccines and serve as immunostimulatory agents in the treatment or prevention of tumors. While being noninfectious toward mammals, CPMV activates the innate immune system through recognition by pattern recognition receptors (PRRs). While the particulate structure of CPMV is essential for prominent immune activation, the proteinaceous architecture makes CPMV subject to degradation in vivo; thus, CPMV immunotherapy requires repeated injections for optimal outcome. Frequent intraperitoneal (IP) injections however are not optimal from a clinical point of view and can worsen the patient's quality of life due to the hospitalization required for IP administration. To overcome the need for repeated IP injections, we loaded CPMV nanoparticles in injectable chitosan/glycerophosphate (GP) hydrogel formulations, characterized their slow-release potential, and assessed the antitumor preventative efficacy of CPMV-in-hydrogel single dose versus soluble CPMV (single and prime-boost administration). Using fluorescently labeled CPMV-in-hydrogel formulations, in vivo release data indicated that single IP injection of the hydrogel formulation yielded a gel depot that supplied intact CPMV over the study period of 3 weeks, while soluble CPMV lasted only for one week. IP administration of the CPMV-in-hydrogel formulation boosted with soluble CPMV for combined immediate and sustained immune activation significantly inhibited colon cancer growth after CT26 IP challenge in BALB/c mice. The observed antitumor efficacy suggests that CPMV can be formulated in a chitosan/GP hydrogel to achieve prolonged immunostimulatory effects as single-dose immunotherapy against colon cancer recurrence. The present findings illustrate the potential of injectable hydrogel technology to accommodate plant virus nanoparticles to boost the translational development of effective antitumor immunotherapies.

Virus nanoparticles are supramolecular structures containing a nucleic acid (either DNA or RNA) core surrounded by a protein layer. In this article, we study the stability of cowpea mosaic virus (CPMV) nanoparticles on mica, Au and graphite surfaces under applied load by atomic force microscope in the tapping mode. The CPMV nanoparticles adsorbed on the hydrophilic mica and Au surfaces are intact, while on the hydrophobic graphite surface, the CPMV nanoparticles undergo the morphological changes from spherical to ellipsoidal and doughnut shapes with the increase in applied load. An understanding of the stability of the CPMV nanoparticles on substrates is critical in developing some of their applications in nanotechnology.

Viral immunotherapies are being recognized in cancer treatment, with several currently approved or undergoing clinical testing. While contemporary approaches have focused on oncolytic viral therapies, our efforts center on the development of plant virus-based cancer immunotherapies. In a previous work, we demonstrated the potent efficacy of the cowpea mosaic virus (CPMV), a plant virus that does not replicate in animals, applied as an in situ vaccine. CPMV is an immunostimulatory drug candidate, and intratumoral administration remodels the tumor microenvironment leading to activation of local and systemic antitumor immunity. Efficacy has been demonstrated in multiple tumor mouse models and canine cancer patients. As wild-type CPMV is infectious toward various legumes and because shedding of infectious virus from patients may be an agricultural concern, we developed UV-inactivated CPMV (termed inCPMV) which is not infectious toward plants. We report that as a monotherapy, wild-type CPMV outperforms inCPMV in mouse models of dermal melanoma or disseminated colon cancer. Efficacy of inCPMV is less than that of CPMV and similar to that of RNA-free CPMV. Immunological investigation using knockout mice shows that inCPMV does not signal through TLR7 (toll-like receptor); structure-function studies indicate that the RNA is highly cross-linked and therefore unable to activate TLR7. Wild-type CPMV signals through TLR2, -4, and -7, whereas inCPMV more closely resembles RNA-free CPMV which signals through TLR2 and -4 only. The structural features of inCPMV explain the increased potency of wild-type CPMV through the triple pronged TLR activation. Strikingly, when inCPMV is used in combination with an anti-OX40 agonist antibody (administered systemically), exceptional efficacy was demonstrated in a bilateral B16F10 dermal melanoma model. Combination therapy, with in situ vaccination applied only into the primary tumor, controlled the progression of the secondary, untreated tumors, with 10 out of 14 animals surviving for at least 100 days post tumor challenge without development of recurrence or metastatic disease. This study highlights the potential of inCPMV as an in situ vaccine candidate and demonstrates the power of combined immunotherapy approaches. Strategic immunocombination therapies are the formula for success, and the combination of in situ vaccination strategies along with therapeutic antibodies targeting the cancer immunity cycle is a particularly powerful approach.

Cowpea mosaic virus (CPMV) is a plant virus that has been developed for multiple biomedical and nanotechnology applications, including immunotherapy. Two key platforms are available: virus nanoparticles (VNPs) based on the complete CMPV virion, including the genomic RNA, and virus-like nanoparticles (VLPs) based on the empty CPMV (eCPMV) virion. It is unclear whether these platforms differ in terms of immunotherapeutic potential. We therefore compared their physicochemical properties and immunomodulatory activities following in situ vaccination of an aggressive ovarian tumor mouse model (ID8-Defb29/Vegf-A). In physicochemical terms, CPMV and eCPMV were very similar, and both significantly increased the survival of tumor-bearing mice and showed promising antitumor efficacy. However, they demonstrated distinct yet overlapping immunostimulatory effects due to the presence of virus RNA in wild-type particles, indicating their suitability for different immunotherapeutic strategies. Specifically, we found that the formulations had similar effects on most secreted cytokines and immune cells, but the RNA-containing CPMV particles were uniquely able to boost populations of potent antigen-presenting cells, such as tumor-infiltrating neutrophils and activated dendritic cells. Our results will facilitate the development of CPMV and eCPMV as immunotherapeutic vaccine platforms with tailored responses.IMPORTANCE The engagement of antiviral effector responses caused by viral infection is essential when using viruses or virus-like particles (VLPs) as an immunotherapeutic agent. Here, we compare the chemophysical and immunostimulatory properties of wild-type cowpea mosaic virus (CPMV) (RNA containing) and eCPMV (RNA-free VLPs) produced from two expression systems (agrobacterium-based plant expression system and baculovirus-insect cell expression). CPMV and eCPMV could each be developed as novel adjuvants to overcome immunosuppression and thus promote tumor regression in ovarian cancer (and other tumor types). To our knowledge, this is the first study to define the immunotherapeutic differences between CPMV and eCPMV, which is essential for the further development of biomedical applications for plant viruses and the selection of rational combinations of immunomodulatory reagents.

Infusion of imaging and therapeutic molecules into the plant virus-based carrier cowpea mosaic virus: Cargo-loading and delivery

Cowpea mosaic virus (CPMV) has been used as a nanoparticle platform for biomedical applications including vaccine development, in vivo vascular imaging, and tissue-targeted delivery. A better understanding of the mechanisms of CPMV targeting and cell internalization would enable enhanced targeting and more effective delivery. Previous studies showed that, following binding and internalization by mammalian cells, CPMV localizes in a perinuclear late-endosome compartment where it remains for as long as several days. To further investigate endocytic trafficking of CPMV within the cell, we used multiple approaches including pharmacologic inhibition of pathways and colocalization with endocytic vesicle compartments. CPMV internalization was clathrin-independent and utilized a combination of caveolar endocytosis and macropinocytosis pathways for entry. CPMV particles colocalized with Rab5(+) early endosomes to traffic ultimately to a lysosomal compartment. These studies facilitate the further development of effective intracellular drug-delivery strategies using CPMV.

Peritoneal metastases (PMs) occur due to the metastasis of gynecological and gastrointestinal cancers such as ovarian, colon, pancreatic, or gastric tumors. PM outgrowth is often fatal, and patients with PMs have a median survival of 6 months. Cowpea mosaic virus (CPMV) has been shown, when injected intratumorally, to act as an immunomodulator reversing the immunosuppressive tumor microenvironment, therefore turning cold tumors hot and priming systemic antitumor immunity. However, not all tumors are injectable, and PMs especially will require targeted treatments to direct CPMV toward the disseminated tumor nodules. Toward this goal, we designed and tested a CPMV nanoparticle targeted to S100A9, a key immune mediator for many cancer types indicated in cancer growth, invasiveness, and metastasis. Here, we chose to use an intraperitoneal (IP) colon cancer model, and analysis of IP gavage fluid demonstrates that S100A9 is upregulated following IP challenge. S100A9-targeted CPMV particles displaying peptide ligands specific for S100A9 homed to IP-disseminated tumors, and treatment led to improved survival and decreased tumor burden. Targeting CPMV to S100A9 improves preclinical outcomes and harbors the potential of utilizing CPMV for the treatment of IP-disseminated diseases.

BackgroundCowpea Mosaic Virus (CPMV) is increasingly being used as a nanoparticle platform for multivalent display of molecules via chemical bioconjugation to the capsid surface. A growing variety of applications have employed the CPMV multivalent display technology including nanoblock chemistry, in vivo imaging, and materials science. CPMV nanoparticles can be inexpensively produced from experimentally infected cowpea plants at high yields and are extremely stable. Although CPMV has not been shown to replicate in mammalian cells, uptake in mammalian cells does occur in vitro and in vivo. Thus, inactivation of the virus RNA genome is important for biosafety considerations, however the surface characteristics and chemical reactivity of the particles must be maintained in order to preserve chemical and structural functionality.Methodology/Principal FindingsShort wave (254 nm) UV irradiation was used to crosslink the RNA genome within intact particles. Lower doses of UV previously reported to inactivate CPMV infectivity inhibited symptoms on inoculated leaves but did not prohibit systemic virus spread in plants, whereas higher doses caused aggregation of the particles and an increase in chemical reactivity further indicating broken particles. Intermediate doses of 2.0–2.5 J/cm2 were shown to maintain particle structure and chemical reactivity, and cellular binding properties were similar to CPMV-WT.ConclusionsThese studies demonstrate that it is possible to inactivate CPMV infectivity while maintaining particle structure and function, thus paving the way for further development of CPMV nanoparticles for in vivo applications.