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 Qβ-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.

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.

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.

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.

Human papillomavirus (HPV) is a globally prevalent sexually-transmitted pathogen, responsible for most cases of cervical cancer. HPV vaccination rates remain suboptimal, partly due to the need for multiple doses, leading to a lack of compliance and incomplete protection. To address the drawbacks of current HPV vaccines, we used a scalable manufacturing process to prepare implantable polymer-protein blends for single-administration with sustained delivery. Peptide epitopes from HPV16 capsid protein L2 were conjugated to the virus-like particles derived from bacteriophage Qβ, to enhance their immunogenicity. The HPV-Qβ particles were then encapsulated into poly(lactic-co-glycolic acid) (PLGA) implants, using a benchtop melt-processing system. The implants facilitated the slow and sustained release of HPV-Qβ particles without the loss of nanoparticle integrity, during high temperature melt processing. Mice vaccinated with the implants generated IgG titers comparable to the traditional soluble injections and achieved protection in a pseudovirus neutralization assay. HPV-Qβ implants offer a new vaccination platform; because the melt-processing is so versatile, the technology offers the opportunity for massive upscale into any geometric form factor. Notably, microneedle patches would allow for self-administration in the absence of a healthcare professional, within the developing world. The Qβ technology is highly adaptable, allowing the production of vaccine candidates and their delivery devices for multiple strains or types of viruses.

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 Qβ 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-1β, 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.

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 Qβ virus-like particles (VLPs) displaying a highly conserved epitope from the L2 protein of HPV (Qβ-HPV). Polymeric microneedle delivery of Qβ-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 Qβ VLP and its delivery technology is a plug-and-play system that could serve as a universal platform with a broad range of applications. Qβ 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.

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 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 Qβ. 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 Qβ VLPs could be stockpiled, and epitopes exchanged to target new mutants or emergent diseases as the need arises.