Pluronic F127 "nanoarmor" for stabilization of Cowpea mosaic virus immunotherapy.

Our lab demonstrated that intratumoral Cowpea mosaic virus (CPMV) is a potent antitumor immunotherapy when used as in situ vaccine. As we pave the way for human clinical translation, formulation chemistry needs to be optimized for long-term storage of the drug candidate. In this work, CPMV was nanoengineered with Pluronic F127 to realize liquid and gel formulations which mitigate structural changes and RNA release during long-term storage. We evaluated the CPMV-F127 formulations for their stability and biological activity through a combination of in vitro assays and efficacy in vivo using a B16F10 murine melanoma model. Results demonstrate that both F127 liquid and gel formulations preserve CPMV structure and function following extended periods of thermal incubation at 4°C, 25°C, and 37°C. Heat-incubated CPMV without formulation resulted in structural changes and inferior in vivo efficacy. In stark contrast, in vivo efficacy was preserved when CPMV was formulated and protected with the F127 "nanoarmor."

Viral nanoparticle vaccines against S100A9 reduce lung tumor seeding and metastasis.

Metastatic cancer accounts for 90% of all cancer-related deaths and continues to be one of the toughest challenges in cancer treatment. A growing body of data indicates that S100A9, a major regulator of inflammation, plays a central role in cancer progression and metastasis, particularly in the lungs, where S100A9 forms a premetastatic niche. Thus, we developed a vaccine against S100A9 derived from plant viruses and virus-like particles. Using multiple tumor mouse models, we demonstrate the effectiveness of the S100A9 vaccine candidates in preventing tumor seeding within the lungs and outgrowth of metastatic disease. The elicited antibodies showed high specificity toward S100A9 without cross-reactivity toward S100A8, another member of the S100A family. When tested in metastatic mouse models of breast cancer and melanoma, the vaccines significantly reduced lung tumor nodules after intravenous challenge or postsurgical removal of the primary tumor. Mechanistically, the vaccines reduce the levels of S100A9 within the lungs and sera, thereby increasing the expression of immunostimulatory cytokines with antitumor function [(interleukin) IL-12 and interferonγ] while reducing levels of immunosuppressive cytokines (IL-10 and transforming growth factorß). This also correlated with decreased myeloid-derived suppressor cell populations within the lungs. This work has wide-ranging impact, as S100A9 is overexpressed in multiple cancers and linked with poor prognosis in cancer patients. The data presented lay the foundation for the development of therapies and vaccines targeting S100A9 to prevent metastasis.

Two decades of vaccine development against atherosclerosis.

Atherosclerosis is an immune-mediated chronic inflammatory disease that leads to the development of fatty plaques in the arterial walls, ultimately increasing the risk of thrombosis, stroke, and myocardial infarction. The immune response in this complex disease is both atheroprotective and pro-atherogenic, involving both innate and adaptive immunity. Current treatments include the adjustment of lifestyle factors, cholesterol-lowering drugs such as statins, and immunotherapy, whereas vaccine development has received comparatively little attention. In this review, we discuss the potential of antigen-specific vaccination as a preventative approach based on more than 20 years of research and innovation. Vaccination targets include proteins that are more abundant in atherosclerotic patients, such as oxidized low-density lipoprotein (LDL), apolipoprotein B-100, proprotein convertase subtilisin/kexin type-9 serine protease (PCSK9), cholesteryl ester transfer protein (CETP), and heat shock proteins HSP60 and HSP65. Immunization with such proteins or their peptide epitopes has been shown to induce T-cell activation, produce antigen-specific antibodies, reduce the size of atherosclerotic lesions, and/or reduce serum cholesterol levels. Vaccination against atherosclerosis therefore offers a new strategy to address the burden on healthcare systems caused by cardiovascular disease, the leading cause of death worldwide.

A co-formulated vaccine of irradiated cancer cells and cowpea mosaic virus improves ovarian cancer rejection.

Ovarian cancer ranks fifth in cancer deaths amongst women, and most patients are diagnosed with late-stage and disseminated diseases. Surgical debulking and chemotherapy remove most of the tumor burden and provide a short period of remission; however, most patients experience cancer relapse and eventually succumb to the disease. Therefore, there is an urgent need for the development of vaccines to prime anti-tumor immunity and prevent its recurrence. Here we developed vaccine formulations composed of a mixture of irradiated cancer cells (ICCs, providing the antigen) and cowpea mosaic virus (CPMV) adjuvants. More specifically we compared the efficacy of co-formulated vs. mixtures of ICCs and CPMV. Specifically, we compared co-formulations where the ICCs and CPMV are bonded through natural CPMV-cell interactions or chemical coupling vs. mixtures of PEGylated CPMV and ICCs, where PEGylation of CPMV prevents ICC interactions. Flow cytometry and confocal imaging provided insights into the composition of the vaccines and their efficacy was tested using a mouse model of disseminated ovarian cancer. 67% of the mice receiving the co-formulated CPMV-ICCs survived the initial tumor challenge, and 60% of the surviving mice rejected tumors in a re-challenge experiment. In stark contrast, simple mixtures of the ICCs and (PEGylated) CPMV adjuvants were ineffective. Overall, this study highlights the importance of the co-delivery of cancer antigens and adjuvants in ovarian cancer vaccine development.

Production of cytoplasmic type citrus leprosis virus-like particles by plant molecular farming.

Many plant virus-like particles (VLPs) utilized in nanotechnology are 30-nm icosahedrons. To expand the VLP platforms, we produced VLPs of Cytoplasmic type citrus leprosis virus (CiLV-C) in Nicotiana benthamiana. We were interested in CiLV-C because of its unique bacilliform shape (60-70 nm × 110-120 nm). The CiLV-C capsid protein (p29) gene was transferred to the pTRBO expression vector transiently expressed in leaves. Stable VLPs were formed, as confirmed by agarose gel electrophoresis, transmission electron microscopy and size exclusion chromatography. Interestingly, the morphology of the VLPs (15.8 ± 1.3 nm icosahedral particles) differed from that of the native bacilliform particles indicating that the assembly of native virions is influenced by other viral proteins and/or the packaged viral genome. The smaller CiLV-C VLPs will also be useful for structure-function studies to compare with the 30-nm icosahedrons of other VLPs.

A single-dose Qß VLP vaccine against S100A9 protein reduces atherosclerosis in a preclinical model.

The standard therapy for cardiovascular disease (CVD) is the administration of statins to reduce plasma cholesterol levels, but this requires lifelong treatment. We developed a CVD vaccine candidate that targets the pro-inflammatory mediator calprotectin by eliciting antibodies against the S100A9 protein. The vaccine, based on bacteriophage Qß virus-like particles (VLPs) displaying S100A9 peptide epitopes, was formulated as a slow-release PLGA:VLP implant by hot-melt extrusion. The single-dose implant elicited S100A9-specific antibody titers comparable to a three-dose injection schedule with soluble VLPs. In an animal model of CVD (ApoE-/- mice fed on a high-fat diet), the implant reduced serum levels of calprotectin, IL-1ß, IL-6 and MCP-1, resulting in less severe aortic lesions. This novel implant was therefore able to attenuate atherosclerosis over a sustained period and offers a novel and promising strategy to replace the repetitive administration of statins for the treatment of CVD.

Multivalent Display of ApoAI Peptides on the Surface of Tobacco Mosaic Virus Nanotubes Improves Cholesterol Efflux.

Atherosclerosis is a progressive cardiovascular disease in which cholesterol-rich plaques build up within arteries, increasing the risk of thrombosis, myocardial infarction, and stroke. One promising therapeutic approach is the use of high-density lipoprotein (HDL) biomimetic formulations based on ApoAI peptides that promote cholesterol efflux from plaques, ultimately leading to cholesterol excretion. Here, we describe the multivalent display of ApoAI peptides on the surface of protein nanotubes derived from the plant virus tobacco mosaic virus (TMV) and protein nanoparticles using virus-like particles from bacteriophage Qß. Bioconjugation yielded ApoAI conjugates varying in size and morphology. We tested ABCA1-mediated cholesterol efflux using macrophage foam cells, the mitigation of reactive oxygen species in endothelial cells, and wound healing in endothelial cells. We found that the multivalent ApoAI platform, in particular the TMV-based nanotube, significantly improved the efficacy of cholesterol efflux compared to free peptides, Qß nanoparticle formulations, and traditional HDL therapy. Finally, to better understand the mechanistic basis of enhanced cholesterol efflux, we used confocal microscopy to show that while native TMV was taken up by cells, TMV-ApoAI remained at the exterior of foam cell membranes and efflux was documented using fluorescent cholesterol. Together, these data highlight that high aspect ratio materials with multivalent display of ApoAI peptides offer unique capabilities promoting efficient cholesterol efflux and may find applications in cardiovascular therapy.

Photothermal immunotherapy of melanoma using TLR-7 agonist laden tobacco mosaic virus with polydopamine coat.

Photothermal therapy (PTT) is a promising cancer treatment that debulks tumors locally while priming immune responses. However, PTT as a standalone treatment approach often has limited systemic efficacy, motivating the development of synergistic combination approaches. Toward this goal, herein, the tobacco mosaic virus (TMV) was loaded with a small molecule immunomodulator, toll-like receptor 7 agonist (1V209), and its surface was coated with photothermal biopolymer polydopamine (PDA). The resulting 1V209-laden and PDA-coated TMV was used to treat B16F10 dermal melanoma in C57BL/6 mice. 1V209-TMV-PDA was intratumorally injected and irradiated using an 808-nm near infrared laser. 60 % of the mice receiving PTT with intratumoral 1V209-TMV-PDA + laser remained alive at the end point - in contrast to only 20 % survivors were observed in the control group. Immunological analysis indicates systemic anti-tumor immunity being induced by the combination therapy with a greater number of tumor-specific T cells (as determined by a splenocyte assay). This study highlights the potential of TMV versatility as a multifunctional nano-platform for combined PTT-immunotherapy.

Injectable Slow-Release Hydrogel Formulation of a Plant Virus-Based COVID-19 Vaccine Candidate.

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.

Dissolving Microneedle Delivery of a Prophylactic HPV Vaccine.

Prophylactic vaccines capable of preventing human papillomavirus (HPV) infections are still inaccessible to a vast majority of the global population due to their high cost and challenges related to multiple administrations performed in a medical setting. In an effort to improve distribution and administration, we have developed dissolvable microneedles loaded with a thermally stable HPV vaccine candidate consisting of Qß virus-like particles (VLPs) displaying a highly conserved epitope from the L2 protein of HPV (Qß-HPV). Polymeric microneedle delivery of Qß-HPV produces similar amounts of anti-HPV16 L2 IgG antibodies compared to traditional subcutaneous injection while delivering a much smaller amount of intradermal dose. However, a dose sparing effect was found. Furthermore, immunization yielded neutralizing antibody responses in a HPV pseudovirus assay. The vaccine candidate was confirmed to be stable at room temperature after storage for several months, potentially mitigating many of the challenges associated with cold-chain distribution. The ease of self-administration and minimal invasiveness of such microneedle patch vaccines may enable wide-scale distribution of the HPV vaccine and lead to higher patient compliance. The Qß VLP and its delivery technology is a plug-and-play system that could serve as a universal platform with a broad range of applications. Qß VLPs may be stockpiled for conjugation to a wide range of epitopes, which are then packaged and delivered directly to the patient via noninvasive microneedle patches. Such a system paves the way for rapid distribution and self-administration of vaccines.

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.

A single-dose, implant-based, trivalent virus-like particle vaccine against "cholesterol checkpoint" proteins.

Cardiovascular disease is the number one cause of death globally. Lowering cholesterol levels in plasma is the mainstay therapy; however lifelong treatment and adverse effects call for improved therapeutic interventions. We developed a trivalent vaccine candidate targeting proprotein convertase subtilisin/kexin-9 (PCSK9), apolipoprotein B (ApoB), and cholesteryl ester transfer protein (CETP). Vaccine candidates were developed using bacteriophage Qß-based virus-like particles (VLPs) displaying antigens of PCKS9, ApoB, and CETP, respectively. Vaccine candidate mixtures were formulated as slow-release PLGA:VLP implants using hot-melt extrusion. The delivery of the trivalent vaccine candidate via the implant produced antibodies against the cholesterol checkpoint proteins at levels comparable to a three-dose injection schedule with soluble mixtures. The reduction in PCSK9 and ApoB levels in plasma, inhibition of CETP (in vitro), and total plasma cholesterol decrease was achieved. All-together, we present a platform technology for a single-dose multi-target vaccination platform targeting cholesterol checkpoint proteins.

Trivalent Subunit Vaccine Candidates for COVID-19 and Their Delivery Devices.

The COVID-19 pandemic highlights the need for platform technologies enabling rapid development of vaccines for emerging viral diseases. The current vaccines target the SARS-CoV-2 spike (S) protein and thus far have shown tremendous efficacy. However, the need for cold-chain distribution, a prime-boost administration schedule, and the emergence of variants of concern (VOCs) call for diligence in novel SARS-CoV-2 vaccine approaches. We studied 13 peptide epitopes from SARS-CoV-2 and identified three neutralizing epitopes that are highly conserved among the VOCs. Monovalent and trivalent COVID-19 vaccine candidates were formulated by chemical conjugation of the peptide epitopes to cowpea mosaic virus (CPMV) nanoparticles and virus-like particles (VLPs) derived from bacteriophage Qß. Efficacy of this approach was validated first using soluble vaccine candidates as solo or trivalent mixtures and subcutaneous prime-boost injection. The high thermal stability of our vaccine candidates allowed for formulation into single-dose injectable slow-release polymer implants, manufactured by melt extrusion, as well as microneedle (MN) patches, obtained through casting into micromolds, for prime-boost self-administration. Immunization of mice yielded high titers of antibodies against the target epitope and S protein, and data confirms that antibodies block receptor binding and neutralize SARS-CoV and SARS-CoV-2 against infection of human cells. We present a nanotechnology vaccine platform that is stable outside the cold-chain and can be formulated into delivery devices enabling single administration or self-administration. CPMV or Qß VLPs could be stockpiled, and epitopes exchanged to target new mutants or emergent diseases as the need arises.

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.

Nanocarriers for the Delivery of Medical, Veterinary, and Agricultural Active Ingredients.

Nanocarrier-based delivery systems can be used to increase the safety and efficacy of active ingredients in medical, veterinary, or agricultural applications, particularly when such ingredients are unstable, sparingly soluble, or cause off-target effects. In this review, we highlight the diversity of nanocarrier materials and their key advantages compared to free active ingredients. We discuss current trends based on peer-reviewed research articles, patent applications, clinical trials, and the nanocarrier formulations already approved by regulatory bodies. Although most nanocarriers have been engineered to combat cancer, the number of formulations developed for other purposes is growing rapidly, especially those for the treatment of infectious diseases and parasites affecting humans, livestock, and companion animals. The regulation and prohibition of many pesticides have also fueled research to develop targeted pesticide delivery systems based on nanocarriers, which maximize efficacy while minimizing the environmental impact of agrochemicals.