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Human papilloma virus type 16 (HPV16) is the most prevalent etiologic agent associated with cervical cancer, and its early proteins E5, E6 and E7 play important roles in cervical epithelium transformation to cervical intraepithelial neoplasia and even cervical cancer. Hence, these oncoproteins are ideal target antigens for developing immunotherapeutic vaccines against HPV-associated infection and cervical cancer. Currently, multi-epitope vaccines have been a promising strategy for immunotherapy for viral infection or cancers. In this study, the E5aa28-46, E6aa37-57 and E7aa26-57 peptides were selected and linked to form a novel multi-epitopes vaccine (E765m), which was inserted into the major immune dominant region (MIR) of hepatitis B virus core antigen (HBc) to construct a HBc-E765m chimeric virus-like particles (cVLPs). The immunogenicity and immunotherapeutic effect of the cVLPs vaccine was evaluated in immunized mice and a tumor-bearing mouse model. The results showed that HBc-E765m cVLPs elicited high E5-, E6- and E7- specific CTL and serum IgG antibody responses, and also relatively high levels of the cytokines IFN-γ, IL-4 and IL-5. More importantly, the cVLPs vaccine significant suppressed tumor growth in mice bearing E5-TC-1 tumors. Our findings provide strong evidence that this novel HBc-E765m cVLPs vaccine could be a candidate vaccine for specific immunotherapy in HPV16-associated cervical intraepithelial neoplasia or cervical cancer.

Parvovirus B19 (B19) belongs to the Erythroparvovirus genus and is known to cause the fifth disease in children. Primary infection of pregnant women is associated with a high risk of hydrops fetalis and stillbirth due to severe fetal anemia. Virus-like particle (VLP) vaccine candidates for B19 have been developed, although none have been approved so far. The B19 phospholipase A2 domain (B19 PLA2), located in the VP1 unique region, is believed to be associated with adverse inflammatory reactions, and previous effective attempts to improve this vaccine modality inserted a mutation to impair the PLA2 activity of VLPs. In this study, we designed VLPs with a deletion mutant of PLA2 (⊿PLA2 B19 VLP), devoid of PLA2 activity, and confirmed their immunogenicity and safe use in vivo. These results were supported by the lack of histological inflammatory reactions at the site of immunization or the production of IL-6 in ⊿PLA2 B19 VLP-immunized mice, that were observed in mice immunized with B19 VLPs. CD4

Persistent infection of high-risk human papillomavirus (HPV) is a leading cause of some cancers, including cervical cancer. However, with over 20 carcinogenic HPV types, it is difficult to design a multivalent vaccine that can offer complete protection. Here, we describe the design and optimization of a HPV51/69/26 triple-type chimeric virus-like particle (VLP) for vaccine development. Using E. coli and a serial N-terminal truncation strategy, we created double- and triple-type chimeric VLPs through loop-swapping at equivalent surface loops. The lead candidate, H69-51BC-26FG, conferred similar particulate properties as that of its parental VLPs and comparable immunogenicity against HPV51, -69 and -26. When produced in a GMP-like facility, these H69-51BC-26FG VLPs were verified to have excellent qualities for the development of a multivalent HPV vaccine. This study showcases an amenable way to create a single VLP using type-specific epitope clustering for the design of a triple-type vaccine.

Inactivated influenza vaccines have struggled to provide consistent protection in older individuals. Circumventing immune senescence, an aging of the immune response characterized by weak humoral responses to vaccines, and unchecked inflammation during infection require novel immunization strategies. Plant-based virus-like particles (VLPs) bearing recombinant hemagglutinin proteins have been shown to provide protection in older animals in preclinical challenge studies, despite eliciting relatively low or absent humoral responses. The nature of the cellular response induced by these vaccines and its evolution during infection have not yet been fully characterized, however. Using a murine model that recapitulates features of human immune senescence, we assessed T cell responses to vaccination with a VLP bearing the hemagglutinin of H1N1/California 07/2009 (H1-VLP) before and after challenge in young and aged BALB/c mice (2 and 18 mo old, respectively). We report that two i.m. doses of H1-VLP (3 μg) vaccine 21 d apart generated H1-specific Th1 and Th2 cells associated with the prevention of prolonged pulmonary inflammation and mortality in both adult and aged mice. While investigating the regulation of cellular immunity, we identified a unique IL-1R1+ tissue-adapted regulatory T cell population in the lungs of both H1-VLP–vaccinated adult and aged mice, suggesting a novel regulatory T cell population associated with vaccine-mediated protection. Collectively, this study provides preclinical evidence that the plant-based H1-VLP vaccine may act, in part, by preventing exacerbated immune responses against influenza A.

In this study, we present the first integrated and continuous downstream process for the production of microbial virus-like particle vaccines. Modular murine polyomavirus major capsid VP1 with integrated J8 antigen was used as a model virus-like particle vaccine. The integrated continuous downstream process starts with crude cell lysate and consists of a flow-through chromatography step followed by periodic counter-current chromatography (PCC) (bind-elute) using salt-tolerant mixed-mode resin and subsequent in-line assembly. The automated process showed a robust behavior over different inlet feed concentrations ranging from 1.0 to 3.2 mg ml

The highly contagious SARS-CoV-2 is mainly transmitted by respiratory droplets and aerosols. Consequently, people are required to wear masks and maintain a social distance to avoid spreading of the virus. Despite the success of the commercially available vaccines, the virus is still uncontained globally. Given the tropism of SARS-CoV-2, a mucosal immune reaction would help to reduce viral shedding and transmission locally. Only seven out of hundreds of ongoing clinical trials are testing the intranasal delivery of a vaccine against COVID-19.In the current study, we evaluated the immunogenicity of a traditional vaccine platform based on virus-like particles (VLPs) displaying RBD of SARS-CoV-2 for intranasal administration in a murine model. The candidate vaccine platform, CuMVCuMVOur data demonstrate that intranasal administration of CuMV

The sequence diversity of HIV-1 is the biggest hurdle for the design of a prophylactic vaccine. Mosaic (Mos) antigens consisting of synthetically shuffled epitopes from various HIV-1 strains are currently tested in the clinical vaccine trial Mosaico (NCT03964415). Besides adenovirus vectors encoding variants of Mos.Gag-Pol and soluble Mos.Env proteins, the Mosaico vaccine entails vectors mediating gene transfer and expression of the membrane-anchored Env-variant Mos2S.Env. We thus examined whether the expression of mosaic Gag mediates the formation of virus-like particles (VLPs). Mos1.Gag- and Mos2.Gag-VLP-formation was readily detected using Western blot- and electron microscopic-analysis. Upon co-expression of both mosaic Gag variants with Mos2S.Env, incorporation of Env into Gag-formed VLPs was observed. The display of the respective neutralization-sensitive target epitopes on Mos2S.Env-decorated VLPs was demonstrated employing a panel of broadly neutralizing antibodies (bNAbs) in a VLP-capture assay. This opens new perspectives for future HIV vaccine designs.

INTRODUCTION: Vaccination can be effective defense against many infectious agents and the corresponding diseases. Discoveries elucidating the mechanisms of the immune system have given hopes to developing vaccines against diseases recalcitrant to current treatment/prevention strategies. One such finding is the ability of immunogenic biological nanoparticles to powerfully boost the immunogenicity of poorer antigens conjugated to them with virus-like particle (VLP)-based vaccines as a key example. VLPs take advantage of the well-defined molecular structures associated with sub-unit vaccines and the immunostimulatory nature of conjugate vaccines. AREAS COVERED: In this review, we will discuss how advances in understanding the immune system can inform VLP-based vaccine design and how VLP-based vaccines have uncovered underlying mechanisms in the immune system. EXPERT OPINION: As our understanding of mechanisms underlying the immune system increases, that knowledge should inform our vaccine design. Testing of proof-of-concept vaccines in the lab should seek to elucidate the underlying mechanisms of immune responses. The integration of these approaches will allow for VLP-based vaccines to live up to their promise as a powerful plug-and-play platform for next generation vaccine development.

Virus-like particles (VLPs) induce strong humoral and cellular responses and have formed the basis of some currently licensed vaccines. Here, we present the method used for the production of R21, a VLP-based anti-sporozoite malaria vaccine, under current Clinical Good Manufacturing Practice regulations (cGMP). Previous preclinical studies in BALB/c mice showed that R21 produced almost complete protection against sporozoite challenge with transgenic Plasmodium berghei parasites. Here, we have modified the preclinical production process to enable the production of sufficient quantities of highly pure, clinical-grade material for use in human clinical trials. The R21 construct was re-engineered to include a C-tag to allow affinity-based separation from the major contaminant alcohol oxidase 1 (AOX 1, ~74 kDa). To our knowledge, this is the first use of C-tag technology to purify a VLP vaccine candidate for use in human clinical trials. The R21 vaccine has shown high-level efficacy in an African Phase IIb trial, and multiple clinical trials are underway to assess the safety and efficacy of the vaccine. Our findings support the future use of C-tag platform technologies to enable cGMP-compliant biomanufacturing of high purity yeast-expressed VLP-based vaccines for early phase clinical trials when clinical grade material is required in smaller quantities in a quick time frame.

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.

Although antivirals are important tools to control severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection, effective vaccines are essential to control the current coronavirus disease 2019 (COVID-19) pandemic. Plant-derived virus-like particle (VLP) vaccine candidates have previously demonstrated immunogenicity and efficacy against influenza. Here, we report the immunogenicity and protection induced in rhesus macaques by intramuscular injections of a VLP bearing a SARS-CoV-2 spike protein (CoVLP) vaccine candidate formulated with or without Adjuvant System 03 (AS03) or cytidine-phospho-guanosine (CpG) 1018. Although a single dose of the unadjuvanted CoVLP vaccine candidate stimulated humoral and cell-mediated immune responses, booster immunization (at 28 days after priming) and adjuvant administration significantly improved both responses, with higher immunogenicity and protection provided by the AS03-adjuvanted CoVLP. Fifteen micrograms of CoVLP adjuvanted with AS03 induced a polyfunctional interleukin-2 (IL-2)-driven response and IL-4 expression in CD4 T cells. Animals were challenged by multiple routes (i.e., intratracheal, intranasal, and ocular) with a total viral dose of 106 plaque-forming units of SARS-CoV-2. Lower viral replication in nasal swabs and bronchoalveolar lavage fluid (BALF) as well as fewer SARS-CoV-2-infected cells and immune cell infiltrates in the lungs concomitant with reduced levels of proinflammatory cytokines and chemotactic factors in the BALF were observed in animals immunized with the CoVLP adjuvanted with AS03. No clinical, pathologic, or virologic evidence of vaccine-associated enhanced disease was observed in vaccinated animals. The CoVLP adjuvanted with AS03 was therefore selected for vaccine development and clinical trials.

Background SARS‐CoV‐2 caused one of the most devastating pandemics in the recent history of mankind. Due to various countermeasures, including lock‐downs, wearing masks, and increased hygiene, the virus has been controlled in some parts of the world. More recently, the availability of vaccines, based on RNA or adenoviruses, has greatly added to our ability to keep the virus at bay; again, however, in some parts of the world only. While available vaccines are effective, it would be desirable to also have more classical vaccines at hand for the future. Key feature of vaccines for long‐term control of SARS‐CoV‐2 would be inexpensive production at large scale, ability to make multiple booster injections, and long‐term stability at 4℃. Methods Here, we describe such a vaccine candidate, consisting of the SARS‐CoV‐2 receptor‐binding motif (RBM) grafted genetically onto the surface of the immunologically optimized cucumber mosaic virus, called CuMVTT‐RBM. Results Using bacterial fermentation and continuous flow centrifugation for purification, the yield of the production process is estimated to be >2.5 million doses per 1000‐litre fermenter run. We demonstrate that the candidate vaccine is highly immunogenic in mice and rabbits and induces more high avidity antibodies compared to convalescent human sera. The induced antibodies are more cross‐reactive to mutant RBDs of variants of concern (VoC). Furthermore, antibody responses are neutralizing and long‐lived. In addition, the vaccine candidate was stable for at least 14 months at 4℃. Conclusion Thus, the here presented VLP‐based vaccine may be a good candidate for use as conventional vaccine in the long term., In this study, we describe a novel conventional COVID‐19 vaccine that consists of the RBM of SARS‐CoV‐2 genetically grafted onto the surface of our optimized cucumber‐mosaic virus‐like particles. We demonstrate that the vaccine candidate (mCuMVTT‐RBM) is highly immunogenic in mice and rabbits, can efficiently neutralize SARS‐CoV‐2, and is stable and highly scalable. The induced antibodies show cross‐reactivity with VoC.

Virus-like particle (VLPs) vaccines have been extensively studied due to their good immunogenicity and safety; however, they highly rely on cold-chain storage and transportation. Nanotechnology of bio-mineralization as a useful strategy has been employed to improve the thermal stability and immunogenicity of VLPs. A zeolitic imidazole framework (ZIF-8), a core-shell structured nanocomposite, was applied to encapsulate foot-and-mouth disease virus (FMDV) VLPs. It was found that the ZIF-8 shell enhanced the heat resistance of VLPs and promoted their ability to be taken up by cells and escape from lysosomes. The VLPs-ZIF-8 easily activated antigen-presenting cells (APCs), triggered higher secretion levels of cytokines, and elicited stronger immune responses than VLPs alone even after being treated at 37 °C for 7 days. This platform has good potential in the development of VLP-based vaccine products without transportation.

Virus-like particles (VLPs) are nano-scale particles that are morphologically similar to a live virus but which lack a genetic component. Since the pandemic spread of COVID-19, much focus has been placed on coronavirus (CoV)-related VLPs. CoVs contain four structural proteins, though the minimum requirement for VLP formation differs among virus species. CoV VLPs are commonly produced in mammalian and insect cell systems, sometimes in the form of chimeric VLPs that enable surface display of CoV epitopes. VLPs are an ideal model for virological research and have been applied as vaccines and diagnostic reagents to aid in clinical disease control. This review summarizes and updates the research progress on the characteristics of VLPs from different known CoVs, mainly focusing on assembly, in vitro expression systems for VLP generation, VLP chimerism, protein-based nanoparticles and their applications in basic research and clinical settings, which may aid in development of novel VLP vaccines against emerging coronavirus diseases such as SARS-CoV-2.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection resulting in the coronavirus disease 2019 (COVID-19) has afflicted tens of millions of people in a worldwide pandemic. A recently developed recombinant Plant-Derived Virus-Like Particle Vaccine candidate for COVID-19 (CoVLP) formulated with AS03 has been shown to be well-tolerated and highly immunogenic in healthy adults. Since the target population for the vaccine includes women of childbearing potential, the objective of the study was to evaluate any untoward prenatal and postnatal effects of AS03-adjuvanted CoVLP administered intramuscularly to Sprague-Dawley female rats before cohabitation for mating (22 and 8 days prior) and during gestation (Gestation Days [GD] 6 and 19). The embryo-fetal development (EFD) cohort was subjected to cesarean on GD 21 and the pre/post-natal (PPN) cohort was allowed to naturally deliver. Effects of AS03-adjuvanted CoVLP was evaluated on pregnant rats, embryo-fetal development (EFD), during parturition, lactation and the development of the F1 offspring up to weaning Vaccination with AS03-adjuvanted CoVLP induced an antibody response in F0 females and anti-SARS-CoV-2 spike-specific maternal antibodies were detected in the offspring at the end of the gestation and lactation periods. Overall, there was no evidence of untoward effects of AS03-adjuvanted CoVLP on the fertility or reproductive performance of the vaccinated F0 females. There was no evidence of untoward effects on embryo-fetal development (including teratogenicity), or early (pre-weaning) development of the F1 offspring. These results support the acceptable safety profile of the AS03-adjuvanted CoVLP vaccine for administration to women of childbearing potential.

After its emergence in late 2019 SARS-CoV-2 was declared a pandemic by the World Health Organization on 11 March 2020 and has claimed more than 2.8 million lives. There has been a massive global effort to develop vaccines against SARS-CoV-2 and the rapid and low cost production of large quantities of vaccine is urgently needed to ensure adequate supply to both developed and developing countries. Virus-like particles (VLPs) are composed of viral antigens that self-assemble into structures that mimic the structure of native viruses but lack the viral genome. Thus they are not only a safer alternative to attenuated or inactivated vaccines but are also able to induce potent cellular and humoral immune responses and can be manufactured recombinantly in expression systems that do not require viral replication. VLPs have successfully been produced in bacteria, yeast, insect and mammalian cell cultures, each production platform with its own advantages and limitations. Plants offer a number of advantages in one production platform, including proper eukaryotic protein modification and assembly, increased safety, low cost, high scalability as well as rapid production speed, a critical factor needed to control outbreaks of potential pandemics. Plant-based VLP-based viral vaccines currently in clinical trials include, amongst others, Hepatitis B virus, Influenza virus and SARS-CoV-2 vaccines. Here we discuss the importance of plants as a next generation expression system for the fast, scalable and low cost production of VLP-based vaccines.

Introduction: Virus-like particles (VLPs), self-assembled multiprotein structures, can stimulate robust immune responses due to their structural similarity to native virions that allow the presentation of multiple copies of the target epitopes. Utilizing VLPs as vaccine platforms to present exogenous antigens is a promising and challenging approach in the vaccine development field. This study investigates the potential of the truncated hepatitis E virus (HEV) capsid as a VLP platform to present foreign antigens. Methods: The S and M domains of the HEV capsid protein were selected as the optimal carrier (CaSM). The exogenous antigen Seq8 containing 3 neutralizing epitopes from 3 different foot-and-mouth disease virus (FMDV) strains was linked to the C-terminal of CaSM to construct a chimeric VLP (CaSM-Seq8). The chimeric particles were produced in Escherichia coli, and their morphology, physicochemical properties, antigenicity, and immunogenicity were analyzed. Results: Morphological analysis showed that CaSM-Seq8 self-assembled into VLPs similar to CaSM VLPs (∼26 nm in diameter) but smaller than native HEV virions. Further, the thermal stability and the resistance to enzymatic proteolysis of Seq8 were enhanced when it was attached to the CaSM carrier. The antigenicity analysis revealed a more robust reactivity against anti-FMDV antibodies when Seq8 was presented on CaSM particles. Upon injection into mice, FMDV-specific IgGs induced by CaSM-Seq8 appeared earlier, increased faster, and maintained higher levels for a longer time than those induced by Seq8 alone or the inactivated FMDV vaccine. Conclusion: This study demonstrated the potential of utilizing the truncated HEV capsid as an antigen-presenting platform for the development of chimeric VLP immunogens.

Site-specific protein modifications are vital for biopharmaceutical drug development. Gluconoylation is a non-enzymatic, post-translational modification of N-terminal HisTags. We report high-yield, site-selective in vitro α-aminoacylation of peptides, glycoproteins, antibodies, and virus-like particles (VLPs) with azidogluconolactone at pH 7.5 in 1 h. Conjugates slowly hydrolyse, but diol-masking with borate esters inhibits reversibility. In an example, we multimerise azidogluconoylated SARS-CoV-2 receptor-binding domain (RBD) onto VLPs via click-chemistry, to give a COVID-19 vaccine. Compared to yeast antigen, HEK-derived RBD was immunologically superior, likely due to observed differences in glycosylation. We show the benefits of ordered over randomly oriented multimeric antigen display, by demonstrating single-shot seroconversion and best virus-neutralizing antibodies. Azidogluconoylation is simple, fast and robust chemistry, and should accelerate research and development.

Graphical abstract, Virus-like particles (VLPs) can play important roles in prevention and therapy for infectious diseases and cancer. Here we describe recent advances in rational construction of VLP assemblies, as well as new approaches to enhance long-lasting antibody and CD8+ T cell responses. DNA origami and computational protein design identified optimal spacing of antigens. Chemical biology advances enabled simple and irreversible VLP decoration with protein or polysaccharide antigens. Mosaic VLPs co-displayed antigens to generate cross-reactive antibodies against different influenza strains and coronaviruses. The mode of action of adjuvants inside VLPs was established through knock-outs and repackaging of innate immune stimuli. VLPs themselves showed their power as adjuvants in cancer models. Finally, landmark clinical results were obtained against malaria and the SARS-CoV-2 pandemic.

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.

Background This study is the first clinical trial for a parenteral non-replicating rotavirus vaccine developed using virus-like particle (VLP) technology. Methods This open-labeled, randomized, placebo-controlled trial was conducted in two parts: Part A (a first-in-human study in Australian adults) and Part B (ascending dose and descending age in South African adults, toddlers and infants). In Part A, two cohorts of 10 adults were assigned to receive a single intramuscular injection of 1 of 2 escalating dose levels of the rotavirus VLP (Ro-VLP) vaccine (7 μg or 21 μg) or placebo. In Part B, one cohort of 10 adults was assigned to receive a single injection of the Ro-VLP vaccine (21 μg) or placebo, two cohorts of 10 toddlers were assigned to receive 2 injections of 1 of 2 escalating dose levels of the Ro-VLP vaccine (7 μg or 21 μg) or placebo 28 days apart, and three cohorts of 20 infants were assigned to receive 3 injections of 1 of 3 escalating dose levels of the Ro-VLP vaccine (2.5 μg, 7 μg or 21 μg) or placebo or 2 doses of oral Rotarix 28 days apart. Safety, reactogenicity and immunogenicity were assessed. Results There were no safety or tolerability concerns after administration of the Ro-VLP vaccine. The Ro-VLP vaccine induced an anti-G1P[8] IgG response in infants 4 weeks after the second and third doses. Neutralizing antibody responses against homologous G1P[8] rotavirus were higher in all Ro-VLP infant groups than in the placebo group 4 weeks after the third dose. No heterotypic immunity was elicited by the Ro-VLP vaccine. Conclusions The Ro-VLP vaccine was well tolerated and induced a homotypic immune response in infants, suggesting that this technology platform is a favorable approach for a parenteral non-replicating rotavirus vaccine. Clinical Trial Registration: NCT03507738.

Strategies to prevent the recurrence of poliovirus (PV) after eradication may utilise non-infectious, recombinant virus-like particle (VLP) vaccines. Despite clear advantages over inactivated or attenuated virus vaccines, instability of VLPs can compromise their immunogenicity. Glutathione (GSH), an important cellular reducing agent, is a crucial co-factor for the morphogenesis of enteroviruses, including PV. We report cryo-EM structures of GSH bound to PV serotype 3 VLPs showing that it can enhance particle stability. GSH binds the positively charged pocket at the interprotomer interface shown recently to bind GSH in enterovirus F3 and putative antiviral benzene sulphonamide compounds in other enteroviruses. We show, using high-resolution cryo-EM, the binding of a benzene sulphonamide compound with a PV serotype 2 VLP, consistent with antiviral activity through over-stabilizing the interprotomer pocket, preventing the capsid rearrangements necessary for viral infection. Collectively, these results suggest GSH or an analogous tight-binding antiviral offers the potential for stabilizing VLP vaccines.

As the SARS-CoV-2 pandemic evolves, vaccine evaluation needs to include consideration of both durability and cross-reactivity. This report expands on previously reported results from a Phase 1 trial of an AS03-adjuvanted, plant-based coronavirus-like particle (CoVLP) displaying the spike (S) glycoprotein of the ancestral SARS-CoV-2 virus in healthy adults (NCT04450004). Humoral and cellular responses against the ancestral strain were evaluated 6 months post-second dose (D201) as secondary outcomes. Independent of dose, all vaccinated individuals retain binding antibodies, and ~95% retain neutralizing antibodies (NAb). Interferon gamma and interleukin-4 responses remain detectable in ~94% and ~92% of vaccinees respectively. In post-hoc analyses, variant-specific (Alpha, Beta, Delta, Gamma and Omicron) NAb were assessed at D42 and D201. Using a live virus neutralization assay, broad cross-reactivity is detectable against all variants at D42. At D201, cross-reactive antibodies are detectable in almost all participants against Alpha, Gamma and Delta variants (94%) and the Beta variant (83%) and in a smaller proportion against Omicron (44%). Results are similar with the pseudovirion assay. These data suggest that two doses of 3.75 µg CoVLP+AS03 elicit a durable and cross-reactive response that persists for at least 6 months post-vaccination.

Licensed L1-VLP-based immunizations against high-risk mucosal human papillomavirus (HPV) types have been a great success in reducing anogenital cancers, although they are limited in their cross-protection against HPV types not covered by the vaccine. Further, their utility in protection against cutaneous HPV types, of which some contribute to non-melanoma skin cancer (NMSC) development, is rather low. Next generation vaccines achieve broadly cross-protective immunity against highly conserved sequences of L2. In this exploratory study, we tested two novel HPV vaccine candidates, HPV16 RG1-VLP and CUT-PANHPVAX, in the preclinical natural infection model Mastomys coucha. After immunization with either vaccines, a mock control or MnPV L1-VLPs, the animals were experimentally infected and monitored. Besides vaccine-specific seroconversion against HPV L2 peptides, the animals also developed cross-reactive antibodies against the cutaneous Mastomys natalensis papillomavirus (MnPV) L2, which were cross-neutralizing MnPV pseudovirions in vitro. Further, both L2-based vaccines also conferred in vivo protection as the viral loads in plucked hair after experimental infection were lower compared to mock-vaccinated control animals. Importantly, the formation of neutralizing antibodies, whether directed against L1-VLPs or L2, was able to prevent skin tumor formation and even microscopical signs of MnPV infection in the skin. For the first time, our study shows the proof-of-principle of next generation L2-based vaccines even across different PV genera in an infection animal model with its genuine PV. It provides fundamental insights into the humoral immunity elicited by L2-based vaccines against PV-induced skin tumors, with important implications to the design of next generation HPV vaccines.

Influenza virus hemagglutinin (HA) stem is currently regarded as an extremely promising immunogen for designing universal influenza vaccines. The appropriate antigen-presenting vaccine vector would be conducive to increasing the immunogenicity of the HA stem antigen. In this study, we generated chimeric virus-like particles (cVLPs) co-displaying the truncated C-terminal of DnaK from Escherichia coli and H1 stem or full-length H1 antigen using the baculovirus expression system. Transmission electronic micrography revealed the expression and presentation of H1 stem antigens on the surface of VLPs. Vaccinations of mice with the H1 stem cVLPs induced H1-specific immune responses and provided heterologous immune protection in vivo, which was more effective than vaccinations with VLPs displaying H1 stem alone in protecting mice against weight loss as well as increasing survival rates after lethal influenza viral challenge. The results indicate that the incorporation of the truncated C-terminal of DnaK as an adjuvant protein into the cVLPs significantly enhances the H1-specific immunity and immune protection. We have explicitly identified the VLP platform as an effective way of expressing HA stem antigen and revealed that chimeric VLP is an vaccine vector for developing HA stem-based universal influenza vaccines.

Introduction Zika virus disease received little attention until its recent explosive emergence around the globe. The devastating consequences of this pandemic include congenital Zika syndrome (CZS) and the neurological autoimmune disorder Guillain-Barre syndrome. These potential outcomes prompted massive efforts to understand the course of Zika infection and to develop therapeutic and prophylactic strategies for treatment and prevention of disease. Area covered Preclinical and clinical data demonstrate that a safe and efficacious vaccine for protection against Zika virus infection is possible in the near future. Nevertheless, significant knowledge gaps regarding the outcome of a mass vaccination strategy exist and must be addressed. Zika virus circulates in flavivirus-endemic regions, an ideal Zika vaccine should avoid the potential of antibody-dependent enhancement from exposure to dengue virus. Prevention of CZS is the primary goal for immunization, and the vaccine must provide protection against intrauterine transmission for use during pregnancy and in women of childbearing age. Ideally, a vaccine should also prevent sexual transmission of the virus through mucosal protection. Expert opinion This review describes current vaccine approaches against Zika virus with particular attention to the application of virus-like particle (VLP) technology as a strategy for solving the challenges of Zika virus immunization.

Congenital tremor (CT) type A-II in piglets is a worldwide disease caused by an emerging atypical porcine pestivirus (APPV). Preparation and evaluation of vaccines in laboratory animals is an important preliminary step toward prevention and control of the disease. Here, virus-like particles (VLPs) of APPV were prepared and VLPs vaccine was evaluated in BALB/c mice. Purified Erns and E2 proteins expressed in E. coli were allowed to self-assemble into VLPs, which had the appearance of hollow spherical particles with a diameter of about 100 nm by transmission electron microscopy (TEM). The VLPs induced strong antibody responses and reduced the viral load in tissues of BALB/c mice. The data from animal challenge experiments, RT-PCR, and immunohistochemical analysis demonstrated that BALB/c mice are an appropriate laboratory model for APPV. These results suggest the feasibility of using VLPs as a vaccine for the prevention and control of APPV and provide useful information for further study of APPV in laboratory animals.

Virus-like particles (VLPs) are particulate structures, which are applied as vaccines or delivery vehicles. VLPs assemble from subunits, named capsomeres, composed of recombinantly expressed viral structural proteins. During downstream processing, in vivo-assembled VLPs are typically dis- and reassembled to remove encapsulated impurities and to improve particle morphology. Disassembly is achieved in a high-pH solution and by the addition of a denaturant or reducing agent. The optimal disassembly conditions depend on the VLP amino acid sequence and structure, thus requiring material-consuming disassembly experiments. To this end, we developed a low-volume and high-resolution disassembly screening that provides time-resolved insight into the VLP disassembly progress. In this study, two variants of C-terminally truncated hepatitis B core antigen were investigated showing different disassembly behaviors. For both VLPs, the best capsomere yield was achieved at moderately high urea concentration and pH. Nonetheless, their disassembly behaviors differed particularly with respect to disassembly rate and aggregation. Based on the high-throughput screening results, a diafiltration-based disassembly process step was developed. Compared with mixing-based disassembly, it resulted in higher yields of up to 0.84 and allowed for integrated purification. This process step was embedded in a filtration-based process sequence of disassembly, capsomere separation, and reassembly, considerably reducing high-molecular-weight species.

Rabies is an ancient zoonotic disease that still causes the death of over 59,000 people worldwide each year. The rabies lyssavirus encodes five proteins, including the envelope glycoprotein and the matrix protein. RVGP is the only protein exposed on the surface of viral particle, and it can induce immune response with neutralizing antibody formation. RVM has the ability to assist with production process of virus-like particles. VLPs were produced in recombinant baculovirus system. In this work, two recombinant baculoviruses carrying the RVGP and RVM genes were constructed. From the infection and coinfection assays, we standardized the best multiplicity of infection and the best harvest time. Cell supernatants were collected, concentrated, and purified by sucrose gradient. Each step was used for protein detection through immunoassays. Sucrose gradient analysis enabled to verify the separation of VLPs from rBV. Through the negative contrast technique, we visualized structures resembling rabies VLPs produced in insect cells and rBV in the different fractions of the sucrose gradient. Using ELISA to measure total RVGP, the recovery efficiency of VLPs at each stage of the purification process was verified. Thus, these results encourage further studies to confirm whether rabies VLPs are a promising candidate for a veterinary rabies vaccine.

Porcine circovirus type 2 (PCV2) is a non-enveloped, icosahedral virus of the Circoviridae family, with a small, circular, single-stranded DNA genome. PCV2 infections cause substantial economic losses in the pig industry worldwide. Currently, commercially produced PCV2 vaccines are expensive, whereas plant-based expression systems can produce recombinant proteins at low cost for use as vaccines. In this study, recombinant PCV2 capsid protein (rCap) was transiently expressed in Nicotiana benthamiana and purified by metal affinity chromatography, with a yield of 102 mg from 1 kg plant leaves. Electron microscopy confirmed that purified rCap self-assembled into virus-like particles (VLPs) at neutral pH. It was shown to provoke a strong immune response in guinea pigs. The results indicate that plant systems can enable production of large amounts of proteins to serve as candidates for subunit vaccines.

Yellow fever (YF) is a life-threatening viral disease endemic in parts of Africa and Latin America. Although there is a very efficacious vaccine since the 1930s, YF still causes 29,000-60,000 annual deaths. During recent YF outbreaks there were issues of vaccine shortage of the current egg-derived vaccine; rare but fatal vaccine adverse effects occurred; and cases were imported to Asia, where the circulating mosquito vector could potentially start local transmission. Here we investigated the production of YF virus-like particles (VLPs) using stably transfected HEK293 cells. Process intensification was achieved by combining sequential FACS (fluorescence-activated cell sorting) rounds to enrich the stable cell pool in terms of high producers and the use of perfusion processes. At shaken-tube scale, FACS enrichment of cells allowed doubling VLP production, and pseudoperfusion cultivation (with daily medium exchange) further increased VLP production by 9.3-fold as compared to batch operation mode. At perfusion bioreactor scale, the use of an inclined settler as cell retention device showed operational advantages over an ATF system. A one-step steric exclusion chromatography purification allowed significant removal of impurities and is a promising technique for future integration of upstream and downstream operations. Characterization by different techniques confirmed the identity and 3D-structure of the purified VLPs.

SARS coronavirus 1 (SARS-CoV-1) causes a respiratory infection that can lead to acute respiratory distress characterized by inflammation and high levels of cytokines in the lung tissue. In this study we constructed a herpes simplex virus 1 replication-defective mutant vector expressing SARS-CoV-1 spike protein as a potential vaccine vector and to probe the effects of spike protein on host cells. The spike protein expressed from this vector is functional in that it localizes to the surface of infected cells and induces fusion of ACE2-expressing cells. In immunized mice, the recombinant vector induced antibodies that bind to spike protein in an ELISA assay and that show neutralizing activity. The spike protein expressed from this vector can induce the expression of cytokines in an ACE2-independent, MyD88-dependent process. These results argue that the SARS-CoV-1 spike protein intrinsically activates signaling pathways that induce cytokines and contribute directly to the inflammatory process of SARS.

Vaccines are typically stored under refrigerated conditions (2–8 °C) to limit potency and efficacy loss. Maintaining the cold chain is costly and prone to vaccine wastage due to unexpected changes in the storage conditions. The development of formulations conferring enhanced stability can extend vaccine shelf-life and facilitate storage under non-refrigerated conditions, thus simplifying the distribution process and reducing vaccine wastage. In this study, the suitability of a natural deep eutectic system (NADES) consisting of trehalose and glycerol (TGly) for storage of influenza hemagglutinin (HA)-displaying virus-like particles (VLPs) was successfully demonstrated. TGly was efficient in maintaining the activity and physical integrity of HA-VLPs for up to 4 h at 50 °C (accelerated stability study), with half-life values ≈ 20–34 h for the best TGly formulations. Importantly, improved storage is achieved at increasingly higher TGly concentrations, hence confirming the importance of TGly content in its protective capacity. In addition, HA-VLPs were stable in TGly for over one month at room temperature (short-term stability study), with no impact on HA titer or particle size distribution. This work highlights the potential of NADES to improve stability of VLP-based vaccines, showing promising protective capacity under non-refrigerated conditions and short-term thermal stress, and thus having a notable impact on vaccine’s cold chain.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) emerged into the human population in late 2019 and caused the global COVID-19 pandemic. SARS-CoV-2 has spread to more than 215 countries and infected many millions of people. Despite the introduction of numerous governmental and public health measures to control disease spread, infections continue at an unabated pace, suggesting that effective vaccines and antiviral drugs will be required to curtail disease, end the pandemic, and restore societal norms. Here, we review the current developments in antibody and vaccine countermeasures to limit or prevent disease.

Several severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) vaccines are being deployed, but the global need greatly exceeds the supply, and different formulations might be required for specific populations. Here we report Day 42 interim safety and immunogenicity data from an observer-blinded, dose escalation, randomized controlled study of a virus-like particle vaccine candidate produced in plants that displays the SARS-CoV-2 spike glycoprotein (CoVLP: NCT04450004). The co-primary outcomes were the short-term tolerability/safety and immunogenicity of CoVLP formulations assessed by neutralizing antibody (NAb) and cellular responses. Secondary outcomes in this ongoing study include safety and immunogenicity assessments up to 12 months after vaccination. Adults (18–55 years, n = 180) were randomized at two sites in Quebec, Canada, to receive two intramuscular doses of CoVLP (3.75 μg, 7.5 μg, and 15 μg) 21 d apart, alone or adjuvanted with AS03 or CpG1018. All formulations were well tolerated, and adverse events after vaccination were generally mild to moderate, transient and highest in the adjuvanted groups. There was no CoVLP dose effect on serum NAbs, but titers increased significantly with both adjuvants. After the second dose, NAbs in the CoVLP + AS03 groups were more than tenfold higher than titers in Coronavirus 2019 convalescent sera. Both spike protein-specific interferon-γ and interleukin-4 cellular responses were also induced. This pre-specified interim analysis supports further evaluation of the CoVLP vaccine candidate., Safety and immunogenicity results in humans of a two-dose SARS-CoV-2 vaccine made from plants support further assessment of potential efficacy.

The importance of developing new vaccine technologies towards versatile platforms that can cope with global virus outbreaks has been evidenced with the most recent severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic. Virus-like particles (VLPs) are a highly immunogenic, safe, and robust approach that can be used to base several vaccine candidates on. Particularly, HIV-1 Gag VLPs is a flexible system comprising a Gag core surrounded by a lipid bilayer that can be modified to present diverse types of membrane proteins or antigens against several diseases, like influenza, dengue, West Nile virus, or human papillomavirus, where it has been proven successful. The size distribution and structural characteristics of produced VLPs vary depending on the cell line used to produce them. In this study, we established an analytical method of characterization for the Gag protein core and clarified the current variability of Gag stoichiometry in HIV-1 VLPs depending on the cell-based production platform, directly determining the number of Gag molecules per VLP in each case. Three Gag peptides have been validated to quantify the number of monomers using parallel reaction monitoring, an accurate and fast, mass-spectrometry-based method that can be used to assess the quality of the produced Gag VLPs regardless of the cell line used. An average of 3617 ± 17 monomers per VLP was obtained for HEK293, substantially varying between platforms, including mammalian and insect cells. This offers a key advantage in quantification and quality control methods to characterize VLP production at a large scale to accelerate new recombinant vaccine production technologies.

Cervical cancer is the fourth most common cancer among women. Human papilloma virus (HPV) is the most common cause of cervical cancer which accounts for 5% of all human cancers and results in about 528000 cases and 266000 deaths every year. HPV vaccines are considered the most effective strategy for the prevention of HPV infection and cervical carcinoma. Since 2006, three prophylactic vaccines against HPV have been available on the market, including bivalent vaccines, quadrivalent vaccines, and nine-valent vaccines. Among them, nine-valent vaccines have been reported to be the most effective. They can prevent 97% of the high-grade pre-cancer lesions. Virus-like particles (VLPs), which are arranged as 360 copies of capsid proteins L1, are the only antigens of the HPV vaccine. Nine-valent HPV vaccines are prepared by mixing nine types of VLPs with adjuvants. Thus, the quality of the VLPs, including their stability and content in the HPV bulk, is very important for developing HPV vaccines. In this study, a method was developed for the determination of the nine types of VLPs (HPV6/11/16/18/31/33/45/52/58) in HPV bulk by size exclusion chromatography (SEC). The parameters of this method were optimized in terms of column brand, pore size of stationary phase particles, buffer concentration, and pH value. SHIMSEN Ankylo SEC-300 column (300 mm×7.8 mm, 3 μm) combined with a buffer aqueous solution containing 300 mmol/L NaCl and 50 mmol/L phosphate (pH 7.0) was utilized to separate the VLPs from the matrix since a narrow peak shape and good repeatability for VLPs could be obtained with this column and mobile phase. The optimized method had a wide linear range, good repeatability (RSDs of peak area were not more than 5.0%), and a satisfactory sensitivity (LOQs in the range of 4.58-15.24 μg/mL). The optimized method was used to determine the VLPs in the HPV bulk. The LOQs of the current method were much lower than the content of the nine types of VLPs in the HPV bulk, indicating that this method was sensitive enough for the determination of the nine types of VLPs in the HPV bulk. The method was also used to determine the VLPs in an HPV bulk that had been stored at 4 ℃ for one week. A decrease in the nine types of VLPs in the range of 10.0%-62.6% was observed after they were stored at 4 ℃ for one week. An HPV vaccine was prepared by mixing the VLPs with an adjuvant. Thereafter, the VLPs were adsorbed on the surface of the adjuvant. The developed method was applied to determine the free VLPs in twelve batches of HPV vaccines from three different manufacturers. No obvious free protein was detected in the twelve batches of the HPV vaccines from the three manufacturers, indicating that VLPs from these manufactures react well with their aluminum adjuvant. Folin-phenol (Lowry assay) is commonly used for the determination of proteins in vaccines. It is based on the reduction of phosphomolybdotungstic mixed acid chromogen in the phosphomolybdotungstic reagent, which results in an absorbance maximum at 650 nm. The Lowry method was sensitive to interfering substances. Most interfering substances caused a lower color yield, while some detergents caused a slight increase in color. To reduce the effect of the interfering substances, a procedure for precipitating the proteins was usually required before the sample was tested. Thus, the Lowry assay is complex, time-consuming, and of low selectivity. Compared to the Lowry method, the method we developed is simpler and more automatic. It is a high-throughput method of determining VLPs. It can be used to determine VLPs in HPV bulk and free VLPs in HPV vaccines.

Background Our previous study developed ATRQβ-001 vaccine, which targets peptide ATR001 from angiotensin Ⅱ (Ang Ⅱ) receptor type 1 (AT1R). The ATRQβ-001 vaccine could induce the production of anti-ATR001 monoclonal antibody (McAb-ATR) and inhibit atherosclerosis without feedback activation of the renin-angiotensin system (RAS). This study aims at investigating the underexploited mechanisms of McAb-ATR in ameliorating atherosclerosis. Methods AT1R–KO HEK293T cell lines were constructed to identify the specificity of McAb-ATR and key sites of ATRQβ-001 vaccine. Beta-arrestin1 knock-out (Arrb1−/−) mice, Beta-arrestin2 knock-out (Arrb2−/−) mice, and low-density lipoprotein receptor knock-out (LDLr−/−) mice were used to detect potential signaling pathways affected by McAb-ATR. The role of McAb-ATR in beta-arrestin and G proteins (Gq or Gi2/i3) signal transduction events was also investigated. Results McAb-ATR could specifically bind to the Phe182-His183-Tyr184 site of AT1R second extracellular loop (ECL2). The anti-atherosclerotic effect of McAb-ATR disappeared in LDLr−/− mice transplanted with Arrb2−/− mouse bone marrow (BM) and BM-derived macrophages (BMDMs) from Arrb2−/− mice. Furthermore, McAb-ATR inhibited beta-arrestin2-dependent extracellular signal regulated kinase1/2 (ERK1/2) phosphorylation, and promoted beta-arrestin2-mediated nuclear factor kappa B p65 (NFκB p65) inactivity. Compared with conventional AT1R blockers (ARBs), McAb-ATR did not inhibit Ang Ⅱ-induced uncoupling of heterotrimeric G proteins (Gq or Gi2/i3) and Gq-dependent intracellular Ca2+ release, nor cause RAS feedback activation. Conclusions Through regulating beta-arrestin2, McAb-ATR ameliorates atherosclerosis without affecting Gq or Gi2/i3 pathways. Due to high selectivity for AT1R and biased interaction with beta-arrestin2, McAb-ATR could serve as a novel strategy for treating atherosclerosis.

Zika virus (ZIKV), a flavivirus transmitted primarily by infected mosquitos, can cause neurological symptoms such as Guillian–Barré syndrome and microcephaly. We developed several vaccinia virus (VACV) vaccine candidates for ZIKV based on replication-inducible VACVs (vINDs) expressing ZIKV pre-membrane (prM) and envelope (E) proteins (vIND-ZIKVs). These vIND-ZIKVs contain elements of the tetracycline operon and replicate only in the presence of tetracyclines. The pool of vaccine candidates was narrowed to one vIND-ZIKV containing a novel mutation in the signal peptide of prM that led to higher expression and secretion of E and production of virus-like particles, which was then tested for safety, immunogenicity, and efficacy in mice. vIND-ZIKV grows to high titers in vitro in the presence of doxycycline (DOX) but is replication-defective in vivo in the absence of DOX, causing no weight loss in mice. C57BL/6 mice vaccinated once with vIND-ZIKV in the absence of DOX (as a replication-defective virus) developed robust levels of E-peptide-specific IFN-γ-secreting splenocytes and anti-E IgG titers, with modest levels of serum-neutralizing antibodies. Vaccinated mice treated with anti-IFNAR1 antibody were completely protected from ZIKV viremia post-challenge after a single dose of vIND-ZIKV. Furthermore, mice with prior immunity to VACV developed moderate anti-E IgG titers that increased after booster vaccination, and were protected from viremia only after two vaccinations with vIND-ZIKV.

This paper aims to detect the antigens in porcine circovirus type 2 (PCV2) vaccines by high-performance size-exclusion chromatography (HPSEC) coupled with multi-angle laser light scattering (MALLS). With purified inactivated PCV2 and PCV2 virus-like particles (VLP) as references, two inactivated vaccines (a and b) and two VLP vaccines (c and d) for PCV2 from four manufacturers were analyzed by HPSEC-MALLS after demulsification. The antigen peaks in HPSEC-MALLS were identified by PCV2 antigen test strips, Western blotting and transmission electron microscope (TEM). The repeatability and linearity of the method were investigated. The results showed the virus antigens in the two inactivated vaccines were eluted at about 13.3 min in HPSEC. The molecular weight of these antigens was 2.61×10

The ongoing COVID-19 pandemic has contributed largely to the global vaccine disparity. Development of protein subunit vaccines can help alleviate shortages of COVID-19 vaccines delivered to low-income countries. Here, we evaluated the efficacy of a three-dose virus-like particle (VLP) vaccine composed of hepatitis B surface antigen (HBsAg) decorated with the receptor binding domain (RBD) from the Wuhan or Beta SARS-CoV-2 strain adjuvanted with either aluminum hydroxide (alum) or squalene in water emulsion (SWE). RBD HBsAg vaccines were compared to the standard two doses of Pfizer mRNA vaccine. Alum-adjuvanted vaccines were composed of either HBsAg conjugated with Beta RBD alone (β RBD HBsAg+Al) or a combination of both Beta RBD HBsAg and Wuhan RBD HBsAg (β/Wu RBD HBsAg+Al). RBD vaccines adjuvanted with SWE were formulated with Beta RBD HBsAg (β RBD HBsAg+SWE) or without HBsAg (β RBD+SWE). Both alum-adjuvanted RBD HBsAg vaccines generated functional RBD IgG against multiple SARS-CoV-2 variants of concern (VOC), decreased viral RNA burden, and lowered inflammation in the lung against Alpha or Beta challenge in K18-hACE2 mice. However, only β/Wu RBD HBsAg+Al was able to afford 100% survival to mice challenged with Alpha or Beta VOC. Furthermore, mice immunized with β RBD HBsAg+SWE induced cross-reactive neutralizing antibodies against major VOC of SARS-CoV-2, lowered viral RNA burden in the lung and brain, and protected mice from Alpha or Beta challenge similarly to mice immunized with Pfizer mRNA. However, RBD+SWE immunization failed to protect mice from VOC challenge. Our findings demonstrate that RBD HBsAg VLP vaccines provided similar protection profiles to the approved Pfizer mRNA vaccines used worldwide and may offer protection against SARS-CoV-2 VOC.

Following the development of various types of vaccines, the use of adjuvants to boost vaccine efficacy has become a focus of research. Aluminum hydroxide (alum), the most commonly used adjuvant, induces a certain immune response and ensures safety in human trials. However, alum mainly induces only a Th2 response; its Th1 response is weak. Thus, we previously developed a single-stranded ribose nucleic acid (ssRNA) adjuvant that induces a Th1 response through toll-like receptors. Here, we explored whether 10-valent human papilloma virus (HPV)-like particle (VLP) vaccine formulated with ssRNA adjuvant and alum helped to enhance immune response and maintained memory response. The mice were immunized intramuscularly twice at 2 week intervals and were inoculated 4 days after the second boost (after about 1 year). The antibody response and T cell activation were measured by Elispot, ELISA using harvested serum and splenocytes. The 10-valent HPV VLP vaccine formulated with ssRNA adjuvant and alum increased the antigen-specific immune response more than alum used alone. It increased each type-specific IgG1/IgG2a titer, and antigen-specific IFN-γ cells. Furthermore, the ssRNA adjuvant with alum induced memory response. In memory response, each type-specific IgG1/IgG2c, IFN-γ, and IL-6 cytokine, and neutralizing antibodies were increased by the ssRNA adjuvant with alum. Overall, the ssRNA adjuvant with alum induced memory responses and balanced Th1/Th2 responses. The ssRNA adjuvant and alum may help to enhance prophylactic vaccine efficacy.

Virus-like particles (VLPs) are nanostructures assemble from viral proteins. Besides widely used for vaccine development, VLPs have also been explored as nanocarriers for cargo delivery as they combine the key advantages of viral and non-viral vectors. While it protects cargo molecules from degradation, the VLP has good cell penetrating property to mediate cargo passing the cell membrane and released into cells, making the VLP an ideal tool for intracellular delivery of biomolecules and drugs. Great progresses have been achieved and multiple challenges are still on the way for broad applications of VLP as delivery vectors. Here we summarize current advances and applications in VLP as a delivery vector. Progresses on delivery of different types of biomolecules as well as drugs by VLPs are introduced, and the strategies for cargo packaging are highlighted which is one of the key steps for VLP mediated intracellular delivery. Production and applications of VLPs are also briefly reviewed, with a discussion on future challenges in this rapidly developing field.

In this study, we demonstrate how encapsulating a conserved influenza ectodomain matrix-2 protein virus-like particle (M2e5x VLP) into a pre-crosslinked bovine serum albumin (BSA) polymeric matrix enhances in vitro antigen immunogenicity and in vivo efficacy. The spray-dried M2e5x VLP-loaded BSA microparticles (MPs) showed enhanced stimulation of antigen presenting cells (APCs), as confirmed through nitrite production and increased antigen–cell interactions seen in real time using live-cell imaging. Next, to further boost the immunogenicity of M2e5x VLP microparticles, M2e5x MPs were combined with Alhydrogel® and monophosphoryl lipid-A (MPL-A®) adjuvant microparticles. M2e5x VLP MPs and the combination VLP M2e5x VLP + Alhydrogel® + MPL-A® MPs elicited a significant increase in the expression of antigen-presenting molecules in dendritic cells compared to M2e5x VLP alone. Lastly, for preliminary evaluation of in vivo efficacy, the vaccine was administered in mice through the skin using an ablative laser. The M2e5x VLP + Alhydrogel® + MPL-A® MPs were shown to induce high levels of M2e-specific IgG antibodies. Further, a challenge with live influenza revealed heightened T-cell stimulation in immune organs of mice immunized with M2e5x VLP + Alhydrogel® + MPL-A® MPs. Hence, we utilized the advantages of both VLP and polymeric delivery platforms to enhance antigen immunogenicity and adaptive immunity in vivo.

H9N2 and H3N2 are the two most important subtypes of low pathogenic avian influenza viruses (LPAIV) because of their ongoing threat to the global poultry industry and public health. Although commercially available inactivated H9N2 vaccines are widely used in the affected countries, endemic H9N2 avian influenza remains uncontrolled. In addition, there is no available avian H3N2 vaccine. Influenza virus-like particles (VLPs) are one of the most promising vaccine alternatives to traditional egg-based vaccines. In this study, to increase the immunogenic content of VLPs to reduce production costs, we developed chimeric bivalent VLPs (cbVLPs) co-displaying hemagglutinin (HA) and neuraminidase (NA) of H9N2 and H3N2 viruses with the Gag protein of bovine immunodeficiency virus (BIV) as the inner core using the Bac-to-Bac baculovirus expression system. The results showed that a single immunization of chickens with 40μg/0.3mL cbVLPs elicited an effective immune response and provided complete protection against H9N2 and H3N2 viruses. More importantly, cbVLPs with accompanying serological assays can successfully accomplish the strategy of differentiating infected animals from vaccinated animals (DIVA), making virus surveillance easier. Therefore, this cbVLP vaccine candidate would be a promising alternative to conventional vaccines, showing great potential for commercial development.

Here, rabbits were immunized with a virus-like particle (VLP) vaccine prepared by expressing 239 amino acids of the swine hepatitis E virus (HEV)-3 capsid protein using a baculovirus system. Thirty specific-pathogen-free rabbits were divided into five groups (negative and positive control and 10, 50, and 100 μg VLP-vaccinated). Positive control group rabbits showed viremia and fecal viral shedding, whereas rabbits vaccinated with 10 μg VLP showed transient fecal viral shedding, and rabbits vaccinated with 50 and 100 μg VLP did not show viremia or fecal viral shedding. Serum anti-HEV antibody titers increased in a dose-dependent manner. Anti-HEV antibody titers were significantly higher (p < 0.05) in 100 μg VLP-vaccinated rabbits than in the negative control rabbits at week 4. Anti-HEV antibody titers were significantly higher in 50 and 10 μg VLP-vaccinated rabbits than in the negative control rabbits at weeks 8 and 11, respectively. Serum IFN-γ and IL-12 levels were significantly higher (p < 0.01) in rabbits vaccinated with 50 and 100 μg VLP than in the negative control rabbits at weeks 4 and 6. Liver tissues of 50 and 100 μg VLP-vaccinated rabbits displayed significantly less (p < 0.05) fibrosis than those of the positive control rabbits. The prepared VLP vaccine demonstrated dose-dependent immunogenicity sufficient for inducing anti-HEV antibody production, thus protecting rabbits against swine HEV-3.

Virus-like particles (VLPs) are self-assembled viral proteins that represent a superior form of antigens in vaccine formulations. To enhance immunogenicity, adjuvants, especially the aluminum salts (Alum), are essentially formulated in VLP vaccines. However, Alum only induce biased humoral immune responses that limits further applications of VLP-based vaccines. To stimulate more balanced immunity, we, herein, develop a one-step strategy of using VLPs as the biotemplates to synthesize raspberry-like silica-adjuvanted VLP@Silica nanovaccines. Hepatitis B surface antigen (HBsAg) VLPs and human papillomavirus type 18 (HPV 18) VLPs are selected as model templates. Circular dichroism (CD) and affinity analyses demonstrate that HBsAg VLPs in the nanovaccines maintain their secondary structure and immunogenicity, respectively. VLP@Silica promote silica dissolution-induced lysosomal escape and cytosolic delivery of antigens, and enhance the secretion of both Th1 and Th2 type cytokines in murine bone marrow-derived dendritic cells (BMDCs). Additionally, they could improve antigen trafficking and mediate DC activation in draining lymph nodes (DLNs). Vaccination study demonstrate that both HBsAg VLP@Silica and HPV 18 VLP@Silica nanovaccines induce enhanced antigen-specific antibody productions and T-cell mediated adaptive immune responses. This design strategy can utilize VLPs derived from a diversity of viruses or their variants as templates to construct both prophylactic and therapeutic vaccines with improved immunogenicity.

Human alphapapillomaviruses (αHPV) infect genital mucosa, and a high-risk subset is a necessary cause of cervical cancer. Licensed L1 virus-like particle (VLP) vaccines offer immunity against the nine most common αHPV associated with cervical cancer and genital warts. However, vaccination with an αHPV L2-based multimer vaccine, α11-88x5, protected mice and rabbits from vaginal and skin challenge with diverse αHPV types. While generally clinically inapparent, human betapapillomaviruses (βHPV) are possibly associated with cutaneous squamous cell carcinoma (CSCC) in epidermodysplasia verruciformis (EV) and immunocompromised patients. Here we show that α11-88x5 vaccination protected wild type and EV model mice against HPV5 challenge. Passive transfer of antiserum conferred protection independently of Fc receptors (FcR) or Gr-1+ phagocytes. Antisera demonstrated robust antibody titers against ten βHPV by L1/L2 VLP ELISA and neutralized and protected against challenge by 3 additional βHPV (HPV49/76/96). Thus, unlike the licensed vaccines, α11-88x5 vaccination elicits broad immunity against αHPV and βHPV.

Background Middle East respiratory syndrome coronavirus (MERS-CoV) causes severe respiratory disease in humans, with a case fatality rate of approximately 35%, thus posing a considerable threat to public health. The lack of approved vaccines or antivirals currently constitutes a barrier in controlling disease outbreaks and spread. Methods In this study, using a mammalian expression system, which is advantageous for maintaining correct protein glycosylation patterns, we constructed chimeric MERS-CoV virus-like particles (VLPs) and determined their immunogenicity and protective efficacy in mice. Results Western blot and cryo-electron microscopy analyses demonstrated that MERS-CoV VLPs were efficiently produced in cells co-transfected with MERS-CoV spike (S), envelope, membrane and murine hepatitis virus nucleocapsid genes. We examined their ability as a vaccine in a human dipeptidyl peptidase 4 knock-in C57BL/6 congenic mouse model. Mice immunized with MERS VLPs produced S-specific antibodies with virus neutralization activity. Furthermore, MERS-CoV VLP immunization provided complete protection against a lethal challenge with mouse-adapted MERS-CoV and improved virus clearance in the lung. Conclusions Overall, these data demonstrate that MERS-CoV VLPs have excellent immunogenicity and represent a promising vaccine candidate.

The family of human papillomaviruses (HPV) includes over 400 genotypes. Genus α genotypes generally infect the anogenital mucosa, and a subset of these HPV are a necessary, but not sufficient, cause of cervical cancer. Of the 13 high-risk (HR) and 11 intermediate-risk (IR) HPV associated with cervical cancer, genotypes 16 and 18 cause 50% and 20% of cases, respectively, whereas HPV16 dominates in other anogenital and oropharyngeal cancers. A plethora of βHPVs are associated with cutaneous squamous cell carcinoma (CSCC), especially in sun-exposed skin sites of epidermodysplasia verruciformis (EV), AIDS, and immunosuppressed patients. Licensed L1 virus-like particle (VLP) vaccines, such as Gardasil 9, target a subset of αHPV but no βHPV. To comprehensively target both α- and βHPVs, we developed a two-component VLP vaccine, RG2-VLP, in which L2 protective epitopes derived from a conserved αHPV epitope (amino acids 17 to 36 of HPV16 L2) and a consensus βHPV sequence in the same region are displayed within the DE loop of HPV16 and HPV18 L1 VLP, respectively. Unlike vaccination with Gardasil 9, vaccination of wild-type and EV model mice (Tmc6(Δ/Δ) or Tmc8(Δ/Δ)) with RG2-VLP induced robust L2-specific antibody titers and protected against β-type HPV5. RG2-VLP protected rabbits against 17 αHPV, including those not covered by Gardasil 9. HPV16- and HPV18-specific neutralizing antibody responses were similar between RG2-VLP- and Gardasil 9-vaccinated animals. However, only transfer of RG2-VLP antiserum effectively protected naive mice from challenge with all βHPVs tested. Taken together, these observations suggest RG2-VLP’s potential as a broad-spectrum vaccine to prevent αHPV-driven anogenital, oropharyngeal, and βHPV-associated cutaneous cancers. IMPORTANCE Licensed preventive HPV vaccines are composed of VLPs derived by expression of major capsid protein L1. They confer protection generally restricted to infection by the αHPVs targeted by the up-to-9-valent vaccine, and their associated anogenital cancers and genital warts, but do not target βHPV that are associated with CSCC in EV and immunocompromised patients. We describe the development of a two-antigen vaccine protective in animal models against known oncogenic αHPVs as well as diverse βHPVs by incorporation into HPV16 and HPV18 L1 VLP of 20-amino-acid conserved protective epitopes derived from minor capsid protein L2.