3D bioprinting cowpea mosaic virus as an immunotherapy depot for ovarian cancer prevention in a preclinical mouse model.

Implantable polymeric hydrogels loaded with immunostimulatory cowpea mosaic virus (CPMV) were fabricated using digital light processing (DLP) printing technology. The CPMV-laden hydrogels were surgically implanted into the peritoneal cavity to serve as depots for cancer slow-release immunotherapy. Sustained release of CPMV within the intraperitoneal space alleviates the need for repeated dosing and we demonstrated efficacy against ovarian cancer in a metastatic mouse model.

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.

Treatment of Hepatocellular Carcinoma by Multimodal In Situ Vaccination Using Cryoablation and a Plant Virus Immunostimulant.

PURPOSE: To test the hypothesis that cryoablation combined with intratumoral immunomodulating nanoparticles from cowpea mosaic virus (CPMV) as an in situ vaccination approach induces systemic antitumoral immunity in a murine model of hepatocellular carcinoma (HCC). MATERIALS AND METHODS: Mice with bilateral, subcutaneous RIL-175 cell-derived HCCs were randomized to 4 groups: (a) phosphate-buffered saline (control), (b) cryoablation only (Cryo), (c) CPMV-treated only (CPMV), and (d) cryoablation plus CPMV-treated (Cryo + CPMV) (N = 11-14 per group). Intratumoral CPMV was administered every 3 days for 4 doses, with cryoablation performed on the third day. Contralateral tumors were monitored. Tumor growth and systemic chemokine/cytokine levels were measured. A subset of tumors and spleens were harvested for immunohistochemistry (IHC) and flow cytometry. One- or 2-way analysis of variance was performed for statistical comparisons. A P value of <.05 was used as the threshold for statistical significance. RESULTS: At 2 weeks after treatment, the Cryo and CPMV groups, alone or combined, outperformed the control group in the treated tumor; however, the Cryo + CPMV group showed the strongest reduction and lowest variance (1.6-fold ± 0.9 vs 6.3-fold ± 0.5, P < .0001). For the untreated tumor, only Cryo + CPMV significantly reduced tumor growth compared with control (9.2-fold ± 0.9 vs 17.8-fold ± 2.1, P = .01). The Cryo + CPMV group exhibited a transient increase in interleukin-10 and persistently decreased CXCL1. Flow cytometry revealed natural killer cell enrichment in the untreated tumor and increased PD-1 expression in the spleen. Tumor-infiltrating lymphocytes increased in Cryo + CPMV-treated tumors by IHC. CONCLUSIONS: Cryoablation and intratumoral CPMV, alone or combined, demonstrated potent efficacy against treated HCC tumors; however, only cryoablation combined with CPMV slowed the growth of untreated tumors, consistent with an abscopal effect.

Integrating plant molecular farming and materials research for next-generation vaccines.

Biologics - medications derived from a biological source - are increasingly used as pharmaceuticals, for example, as vaccines. Biologics are usually produced in bacterial, mammalian or insect cells. Alternatively, plant molecular farming, that is, the manufacture of biologics in plant cells, transgenic plants and algae, offers a cheaper and easily adaptable strategy for the production of biologics, in particular, in low-resource settings. In this Review, we discuss current vaccination challenges, such as cold chain requirements, and highlight how plant molecular farming in combination with advanced materials can be applied to address these challenges. The production of plant viruses and virus-based nanotechnologies in plants enables low-cost and regional fabrication of thermostable vaccines. We also highlight key new vaccine delivery technologies, including microneedle patches and material platforms for intranasal and oral delivery. Finally, we provide an outlook of future possibilities for plant molecular farming of next-generation vaccines and biologics.

Cowpea Mosaic Virus Nanoparticle Vaccine Candidates Displaying Peptide Epitopes Can Neutralize the Severe Acute Respiratory Syndrome Coronavirus.

The development of vaccines against coronaviruses has focused on the spike (S) protein, which is required for the recognition of host-cell receptors and thus elicits neutralizing antibodies. Targeting conserved epitopes on the S protein offers the potential for pan-beta-coronavirus vaccines that could prevent future pandemics. We displayed five B-cell epitopes, originally identified in the convalescent sera from recovered severe acute respiratory syndrome (SARS) patients, on the surface of the cowpea mosaic virus (CPMV) and evaluated these formulations as vaccines. Prime-boost immunization of mice with three of these candidate vaccines, CPMV-988, CPMV-1173, and CPMV-1209, elicited high antibody titers that neutralized the severe acute respiratory syndrome coronavirus (SARS-CoV) in vitro and showed an early Th1-biased profile (2-4 weeks) transitioning to a slightly Th2-biased profile just after the second boost (6 weeks). A pentavalent slow-release implant comprising all five peptides displayed on the CPMV elicited anti-S protein and epitope-specific antibody titers, albeit at a lower magnitude compared to the soluble formulations. While the CPMV remained intact when released from the PLGA implants, processing results in loss of RNA, which acts as an adjuvant. Loss of RNA may be a reason for the lower efficacy of the implants. Finally, although the three epitopes (988, 1173, and 1209) that were found to be neutralizing the SARS-CoV were 100% identical to the SARS-CoV-2, none of the vaccine candidates neutralized the SARS-CoV-2 in vitro suggesting differences in the natural epitope perhaps caused by conformational changes or the presence of N-linked glycans. While a cross-protective vaccine candidate was not developed, a multivalent SARS vaccine was developed. The technology discussed here is a versatile vaccination platform that can be pivoted toward other diseases and applications that are not limited to infectious diseases.

The in vivo fate of tobacco mosaic virus nanoparticle theranostic agents modified by the addition of a polydopamine coat.

Plant virus nanoparticles (VNPs) have multiple advantages over their synthetic counterparts including the cost-effective large-scale manufacturing of uniform particles that are easy to functionalize. Tobacco mosaic virus (TMV) is one of the most promising VNP scaffolds, reflecting its high aspect ratio and ability to carry and/or display multivalent therapeutic ligands and contrast agents. Here we investigated the circulation, protein corona, immunogenicity, and organ distribution/clearance of TMV particles internally co-labeled with cyanine 5 (Cy5) and chelated gadolinium (Gd) for dual tracking by fluorescence imaging and optical emission spectrometry, with or without an external coating of polydopamine (PDA) to confer photothermal and photoacoustic capabilities. The PDA-coated particles (Gd-Cy5-TMV-PDA) showed a shorter plasma circulation time and broader distribution to organs of the reticuloendothelial system (liver, lungs, and spleen) than uncoated Gd-Cy5-TMV particles (liver and spleen only). The Gd-Cy5-TMV-PDA particles were surrounded by 2-10-fold greater protein corona (containing mainly immunoglobulins) compared to Gd-Cy5-TMV particles. However, the enzyme-linked immunosorbent assay (ELISA) revealed that PDA-coated particles bind 2-fold lesser to anti-TMV antibodies elicited by particle injection than uncoated particles, suggesting that the PDA coat enables evasion from systemic antibody surveillance. Gd-Cy5-TMV-PDA particles were cleared from organs after 8 days compared to 5 days for the uncoated particles. The slower tissue clearance of the coated particles makes them ideal for theranostic applications by facilitating sustained local delivery in addition to multimodal imaging and photothermal capabilities. We have demonstrated the potential of PDA-coated proteinaceous nanoparticles for multiple biomedical applications.

Tobacco mosaic virus for the targeted delivery of drugs to cells expressing prostate-specific membrane antigen.

Prostate-specific membrane antigen (PSMA) is a membrane-bound protein that is preferentially expressed in the prostate gland and induced in many prostate cancers, making it an important target for new diagnostics and therapeutics. To improve the efficacy of nanoparticle formulations for the imaging and/or eradication of prostate cancer, we synthesized the PSMA-binding glutamic acid derivative DUPA and conjugated it to the external surface of tobacco mosaic virus (TMV) particles. DUPA-targeted TMV was subsequently loaded with the antineoplastic agent mitoxantrone (MTO) or conjugated internally with the fluorescent dye cyanine 5 (Cy5). We found that TMV particles could be efficiently decorated with DUPA and loaded with MTO or Cy5 while maintaining structural integrity. DUPA-targeted TMV particles were able to bind more efficiently to the surface of PSMA+ LNCaP cells compared to non-targeted TMV; but there was little difference in binding efficiency between targeted and untargeted TMV when we tested PSMA- PC3 cells (both cell lines are prostate cancer cell lines). DUPA-targeted TMV particles were internalized by LNCaP cells enabling drug delivery. Finally, we loaded the DUPA-targeted TMV particles and untargeted control particles with MTO to test their cytotoxicity against LNCaP cells in vitro. The cytotoxicity of the TMV-MTO particles (IC50 = 10.2 nM) did not differ significantly from that of soluble MTO at an equivalent dose (IC50 = 12.5 nM) but the targeted particles (TMV-DUPA-MTO) were much more potent (IC50 = 2.80 nM). The threefold increase in cytotoxicity conferred by the DUPA ligand suggests that MTO-loaded, DUPA-coated TMV particles are promising as a therapeutic strategy for PSMA+ prostate cancer and should be advanced to preclinical testing in mouse models of prostate cancer.

Plant Viruses and Bacteriophage-Based Reagents for Diagnosis and Therapy.

Viral nanotechnology exploits the prefabricated nanostructures of viruses, which are already abundant in nature. With well-defined molecular architectures, viral nanocarriers offer unprecedented opportunities for precise structural and functional manipulation using genetic engineering and/or bio-orthogonal chemistries. In this manner, they can be loaded with diverse molecular payloads for targeted delivery. Mammalian viruses are already established in the clinic for gene therapy and immunotherapy, and inactivated viruses or virus-like particles have long been used as vaccines. More recently, plant viruses and bacteriophages have been developed as nanocarriers for diagnostic imaging, vaccine and drug delivery, and combined diagnosis/therapy (theranostics). The first wave of these novel virus-based tools has completed clinical development and is poised to make an impact on clinical practice.

The unique potency of Cowpea mosaic virus (CPMV) in situ cancer vaccine.

The immunosuppressive tumor microenvironment enables cancer to resist immunotherapies. We have established that intratumoral administration of plant-derived Cowpea mosaic virus (CPMV) nanoparticles as an in situ vaccine overcomes the local immunosuppression and stimulates a potent anti-tumor response in several mouse cancer models and canine patients. CPMV does not infect mammalian cells but acts as a danger signal that leads to the recruitment and activation of innate and subsequently, adaptive immune cells. In the present study we addressed whether other icosahedral viruses or virus-like particles (VLPs) of plant, bacteriophage and mammalian origin can be similarly employed as intratumoral immunotherapy. Our results indicate that CPMV in situ vaccine outperforms Cowpea chlorotic mottle virus (CCMV), Physalis mosaic virus (PhMV), Sesbania mosaic virus (SeMV), bacteriophage Qß VLPs, or Hepatitis B virus capsids (HBVc). Furthermore, ex vivo and in vitro assays reveal unique features of CPMV that makes it an inherently stronger immune stimulant.

Affinity of plant viral nanoparticle potato virus X (PVX) towards malignant B cells enables cancer drug delivery.

Non-Hodgkin's B cell lymphomas (NHL) include a diverse set of neoplasms that constitute ∼90% of all lymphomas and the largest subset of blood cancers. While chemotherapy is the first line of treatment, the efficacy of contemporary chemotherapies is hampered by dose-limiting toxicities. Partly due to suboptimal dosing, ∼40% of patients exhibit relapsed or refractory disease. Therefore more efficacious drug delivery systems are urgently needed to improve survival of NHL patients. In this study we demonstrate a new drug delivery platform for NHL based on the plant virus Potato virus X (PVX). We observed a binding affinity of PVX towards malignant B cells. In a metastatic mouse model of NHL, we show that systemically administered PVX home to tissues harboring malignant B cells. When loaded with the chemotherapy monomethyl auristatin (MMAE), the PVX nanocarrier enables effective delivery of MMAE to human B lymphoma cells in a NHL mouse model leading to inhibition of lymphoma growth in vivo and improved survival. Thus, PVX nanoparticle is a promising drug delivery platform for B cell malignancies.

COVID-19 vaccine development and a potential nanomaterial path forward.

The COVID-19 pandemic has infected millions of people with no clear signs of abatement owing to the high prevalence, long incubation period and lack of established treatments or vaccines. Vaccines are the most promising solution to mitigate new viral strains. The genome sequence and protein structure of the 2019-novel coronavirus (nCoV or SARS-CoV-2) were made available in record time, allowing the development of inactivated or attenuated viral vaccines along with subunit vaccines for prophylaxis and treatment. Nanotechnology benefits modern vaccine design since nanomaterials are ideal for antigen delivery, as adjuvants, and as mimics of viral structures. In fact, the first vaccine candidate launched into clinical trials is an mRNA vaccine delivered via lipid nanoparticles. To eradicate pandemics, present and future, a successful vaccine platform must enable rapid discovery, scalable manufacturing and global distribution. Here, we review current approaches to COVID-19 vaccine development and highlight the role of nanotechnology and advanced manufacturing.

Active Delivery of VLPs Promotes Anti-Tumor Activity in a Mouse Ovarian Tumor Model.

Virus-like nanoparticles (VLPs) have been used as an attractive means in cancer immunotherapy because of their unique intrinsic immunostimulatory properties. However, for treating metastatic tumors in the peritoneal cavity, such as ovarian cancer, multiple injections of therapy are needed due to the large peritoneal space and fast excretion of therapy. Here, it is reported on the development of active VLP delivery vehicles for the treatment of peritoneal ovarian tumors using biocompatible Qß VLPs-loaded Mg-based micromotors. The autonomous propulsion of such Qß VLPs-loaded Mg-micromotors in the peritoneal fluid enables active delivery of intact immunostimulatory Qß VLPs to the peritoneal space of ovarian tumor bearing mice, greatly enhancing the local distribution and retention of Qß VLPs. Such improved distribution and longer retention time of Qß in the peritoneal cavity leads to enhanced immunostimulation and therefore increased survival rate of tumor-bearing mice compared to a passive Qß treatment. For clinical translation, the active delivery of VLPs holds great promise for tumor immunotherapy toward the treatment of different types of primary and metastatic tumors in the peritoneal cavity.

Antibody Response against Cowpea Mosaic Viral Nanoparticles Improves In Situ Vaccine Efficacy in Ovarian Cancer.

Cancer immunotherapies are designed to facilitate recognition and elimination of transformed cells by the immune system. We have established the immunotherapeutic efficacy of the plant virus cowpea mosaic virus (CPMV) as an in situ vaccine in several syngeneic tumor mouse models as well as in companion dogs with metastatic melanoma. Intratumoral injection of CPMV modulates the local tumor microenvironment to relieve immunosuppression and potentiate antitumor immunity. The viral nucleocapsid that drives this antitumor immunity, however, also is a potent immunogen itself, and thus immune response in the form of anti-CPMV antibodies is expected during the treatment based on repeat administrations. Moreover, being part of the food chain, pre-existing antibodies to plant viruses may be prevalent. The presence of such pre-existing anti-CPMV immunity could potentially impact immunotherapeutic efficacy of the in situ vaccine and could have translational implications. To address such concerns, this study evaluated the efficacy of CPMV in situ vaccine in the presence of pre-existing antibodies in a syngeneic mouse model of ovarian cancer. Our results indicate that prior exposure to CPMV had no negative impact on the efficacy of CPMV in situ vaccine. Strikingly, an improved efficacy of CPMV in situ vaccine was observed. This study therefore presents an important milestone in the translational development of plant viral-based in situ vaccines and alleviates concerns about the presence of anti-CPMV antibodies, which are developed during the course of treatment but have no impact on immunotherapeutic efficacy.

Active Microneedle Administration of Plant Virus Nanoparticles for Cancer in situ Vaccination Improves Immunotherapeutic Efficacy.

The solid tumor microenvironment (TME) poses a significant structural and biochemical barrier to immunotherapeutic agents. To address the limitations of tumor penetration and distribution, and to enhance antitumor efficacy of immunotherapeutics, we present here an autonomous active microneedle (MN) system for the direct intratumoral (IT) delivery of a potent immunoadjuvant, cowpea mosaic virus nanoparticles (CPMV) in vivo. In this active delivery system, magnesium (Mg) microparticles embedded into active MNs react with the interstitial fluid in the TME, generating a propulsive force to drive the nanoparticle payload into the tumor. Active delivery of CPMV payload into B16F10 melanomas in vivo demonstrated substantially more pronounced tumor regression and prolonged survival of tumor-bearing mice compared to that of passive MNs and conventional needle injection. Active MN administration of CPMV also enhanced local innate and systemic adaptive antitumor immunity. Our approach represents an elaboration of conventional CPMV in situ vaccination, highlighting substantial immune-mediated antitumor effects and improved therapeutic efficacy that can be achieved through an active and autonomous delivery system-mediated CPMV in situ vaccination.

The Antitumor Efficacy of CpG Oligonucleotides is Improved by Encapsulation in Plant Virus-Like Particles.

Oligodeoxynucleotides (ODNs) with CpG motifs have potent immunostimulatory effects on many subsets of immune cells. For example, Class B CpG-ODNs, such as ODN1826 induce the phagocytic activity of macrophages by activating the Toll-like receptor 9 signaling pathway. Systemic ODN delivery results in unfavorable pharmacokinetic profiles and can trigger adverse effects. To address this issue, plant virus-like particles (VLPs) are developed for the targeted delivery of ODN1826 to tumor-associated macrophages (TAMs). ODN1826 is encapsulated by the in vitro disassembly and reassembly of Cowpea chlorotic mottle virus (CCMV), producing VLPs that are structurally analogous to the native virus. The encapsulation of ODN1826 in CCMV-derived VLPs promotes ODN uptake by TAMs ex vivo and significantly enhance their phagocytic activity. The antitumor activity of the VLPs in vivo is also evaluated, revealing that the direct injection of ODN1826 VLPs into established tumors induces a robust antitumor response by increasing the phagocytic activity of TAMs in the tumor microenvironment. CCMV encapsulation significantly enhances the efficacy of ODN1826 compared to the free drug, slowing tumor growth and prolonging survival in mouse models of colon cancer and melanoma.

Cowpea Mosaic Virus (CPMV)-Based Cancer Testis Antigen NY-ESO-1 Vaccine Elicits an Antigen-Specific Cytotoxic T Cell Response.

Cancer vaccines are promising adjuvant immunotherapies that can stimulate the immune system to recognize tumor-associated antigens and eliminate the residual or recurring disease. The aberrant and restricted expression of highly immunogenic cancer testis antigen NY-ESO-1 in several malignancies, including triple-negative breast cancer, melanoma, myelomas, and ovarian cancer, makes NY-ESO-1 an attractive antigenic target for cancer vaccines. This study describes a NY-ESO-1 vaccine based on a bio-inspired nanomaterial platform technology, specifically a plant virus nanoparticle. The 30 nm icosahedral plant virus cowpea mosaic virus (CPMV) displaying multiple copies of human HLA-A2 restricted peptide antigen NY-ESO-1157-165 exhibited enhanced uptake and activation of antigen-presenting cells and stimulated a potent CD8+ T cell response in transgenic human HLA-A2 expressing mice. CD8+ T cells from immunized mice exhibited antigen-specific proliferation and cancer cell cytotoxicity, highlighting the potential application of a CPMV-NY-ESO-1 vaccine against NY-ESO-1+ malignancies.

Built-In Active Microneedle Patch with Enhanced Autonomous Drug Delivery.

The use of microneedles has facilitated the painless localized delivery of drugs across the skin. However, their efficacy has been limited by slow diffusion of molecules and often requires external triggers. Herein, an autonomous and degradable, active microneedle delivery platform is introduced, employing magnesium microparticles loaded within the microneedle patch, as the built-in engine for deeper and faster intradermal payload delivery. The magnesium particles react with the interstitial fluid, leading to an explosive-like rapid production of H2 bubbles, providing the necessary force to breach dermal barriers and enhance payload delivery. The release kinetics of active microneedles is evaluated in vitro by measuring the amount of IgG antibody (as a model drug) that passed through phantom tissue and a pigskin barrier. In vivo experiments using a B16F10 mouse melanoma model demonstrate that the active delivery of anti-CTLA-4 (a checkpoint inhibitor drug) results in greatly enhanced immune response and significantly longer survival. Moreover, spatially resolved zones of active and passive microneedles allow a combinatorial rapid burst response along with slow, sustained release, respectively. Such versatile and effective autonomous dynamic microneedle delivery technology offers considerable promise for a wide range of therapeutic applications, toward a greatly enhanced outcome, convenience, and cost.

Cowpea Mosaic Virus Immunotherapy Combined with Cyclophosphamide Reduces Breast Cancer Tumor Burden and Inhibits Lung Metastasis.

Patients with metastatic triple-negative breast cancer (TNBC) have a poor prognosis, so new therapies or drug combinations that achieve more effective and durable responses are urgently needed. Here, a combination therapy using cowpea mosaic virus (CPMV) and low doses of cyclophosphamide (CPA) is developed with remarkable synergistic efficacy against 4T1 mouse tumors in vivo. The combination therapy not only attenuates the growth of primary tumor and increases survival, but also suppresses distant tumor growth and reduces lung metastasis. Mechanistic analysis indicates that the combination of CPMV and CPA increases the secretion of several cytokines, activates antigen-presenting cells, increases the abundance of tumor infiltrating T cells, and systematically reverses the immunosuppression. These results show that the combination of CPMV in situ vaccination with chemotherapy may become a potent new strategy for the treatment of TNBC.

Plant Virus-Like Particle In Situ Vaccine for Intracranial Glioma Immunotherapy.

Despite aggressive multi-modality treatment with surgery, radiation and chemotherapies, malignant glioma inevitably recurs and has dismal survival rates. Recent progress in immunotherapy has led to a resurgence of interest, and immunotherapies are being investigated for treatment of glioma. However, the unique brain anatomy and a highly immunosuppressive glioma microenvironment pose significant challenges to achieving efficacy. Thus, there is a critical need for assessment of next-generation immunotherapies for glioma. In this study, we have investigated the efficacy of the nanoparticle platform technology based on plant-derived Cowpea mosaic virus like particles (empty CPMV or eCPMV) to instigate a potent immune response against intracranial glioma. CPMV immunotherapy has been shown to efficiently reverse the immunosuppressive tumor microenvironments in pre-clinical murine models of dermal melanoma and metastatic melanoma, metastatic breast cancer, intraperitoneal ovarian cancer and in canine patients with oral melanoma. In the present study, we demonstrate that in situ administration of CPMV immunotherapy in the setting of glioma can effectively recruit unique subset of effector innate and adaptive immune cells to the brain parenchyma while reducing immune suppressive cellular population, leading to regression of intracranial glioma. The in situ CPMV nanoparticle vaccine offers a potent yet safe and localized immunotherapy for intracranial glioma.

Heterologous Prime-Boost Enhances the Antitumor Immune Response Elicited by Plant-Virus-Based Cancer Vaccine.

New cancer vaccine strategies are required to vanquish the self-tolerance and elicit robust immune responses against tumor-associated antigens and/or neoantigens. Contemporary approaches in nanomedicine center on the use of a single nanocarrier modified with multiple copies of multiple different functional domains, e.g., epitopes for vaccines. Therefore, we set out to develop a combinatorial approach toward the next-generation concept of epitope delivery: a prime-boost strategy in which the same epitope is delivered using different nanocarriers. We tested this concept in the setting of HER2+ breast cancer. We synthesized HER2-based cancer vaccines using three icosahedral plant viruses as carriers and evaluated the immune response as a result of repetitive, homologous immunization using BALB/c mice. Two of the vaccines induced a Th2-predominant response and the other a Th1-predominant response. To enhance the immunogenicity of the vaccines, we developed a heterologous prime-boost strategy with each of the vaccines administered only once, yielding higher titers of HER2-specific immunoglobulins and increasing the toxicity of the antisera toward cancer cells. The prime-boost also induced a Th1-predominant response. An in vivo tumor challenge showed that the prime-boost regimen reduced tumor growth and improved survival in mice. This novel strategy to elicit robust immune responses against weakly immunogenic antigens in principle could be broadly applicable to cancers and other diseases.