Review: A systematic review of virus-like particles of coronavirus: Assembly, generation, chimerism and their application in basic research and in the clinic.

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.

Efficacy of a low-dose candidate malaria vaccine, R21 in adjuvant Matrix-M, with seasonal administration to children in Burkina Faso: a randomised controlled trial.

BACKGROUND: Stalled progress in controlling Plasmodium falciparum malaria highlights the need for an effective and deployable vaccine. RTS,S/AS01, the most effective malaria vaccine candidate to date, demonstrated 56% efficacy over 12 months in African children. We therefore assessed a new candidate vaccine for safety and efficacy. METHODS: In this double-blind, randomised, controlled, phase 2b trial, the low-dose circumsporozoite protein-based vaccine R21, with two different doses of adjuvant Matrix-M (MM), was given to children aged 5-17 months in Nanoro, Burkina Faso-a highly seasonal malaria transmission setting. Three vaccinations were administered at 4-week intervals before the malaria season, with a fourth dose 1 year later. All vaccines were administered intramuscularly into the thigh. Group 1 received 5 µg R21 plus 25 µg MM, group 2 received 5 µg R21 plus 50 µg MM, and group 3, the control group, received rabies vaccinations. Children were randomly assigned (1:1:1) to groups 1-3. An independent statistician generated a random allocation list, using block randomisation with variable block sizes, which was used to assign participants. Participants, their families, and the local study team were all masked to group allocation. Only the pharmacists preparing the vaccine were unmasked to group allocation. Vaccine safety, immunogenicity, and efficacy were evaluated over 1 year. The primary objective assessed protective efficacy of R21 plus MM (R21/MM) from 14 days after the third vaccination to 6 months. Primary analyses of vaccine efficacy were based on a modified intention-to-treat population, which included all participants who received three vaccinations, allowing for inclusion of participants who received the wrong vaccine at any timepoint. This trial is registered with ClinicalTrials.gov, NCT03896724. FINDINGS: From May 7 to June 13, 2019, 498 children aged 5-17 months were screened, and 48 were excluded. 450 children were enrolled and received at least one vaccination. 150 children were allocated to group 1, 150 children were allocated to group 2, and 150 children were allocated to group 3. The final vaccination of the primary series was administered on Aug 7, 2019. R21/MM had a favourable safety profile and was well tolerated. The majority of adverse events were mild, with the most common event being fever. None of the seven serious adverse events were attributed to the vaccine. At the 6-month primary efficacy analysis, 43 (29%) of 146 participants in group 1, 38 (26%) of 146 participants in group 2, and 105 (71%) of 147 participants in group 3 developed clinical malaria. Vaccine efficacy was 74% (95% CI 63-82) in group 1 and 77% (67-84) in group 2 at 6 months. At 1 year, vaccine efficacy remained high, at 77% (67-84) in group 1. Participants vaccinated with R21/MM showed high titres of malaria-specific anti-Asn-Ala-Asn-Pro (NANP) antibodies 28 days after the third vaccination, which were almost doubled with the higher adjuvant dose. Titres waned but were boosted to levels similar to peak titres after the primary series of vaccinations after a fourth dose administered 1 year later. INTERPRETATION: R21/MM appears safe and very immunogenic in African children, and shows promising high-level efficacy. FUNDING: The European & Developing Countries Clinical Trials Partnership, Wellcome Trust, and National Institute for Health Research Oxford Biomedical Research Centre.

Next generation methodology for updating HA vaccines against emerging human seasonal influenza A(H3N2) viruses.

While vaccines remain the best tool for preventing influenza virus infections, they have demonstrated low to moderate effectiveness in recent years. Seasonal influenza vaccines typically consist of wild-type influenza A and B viruses that are limited in their ability to elicit protective immune responses against co-circulating influenza virus variant strains. Improved influenza virus vaccines need to elicit protective immune responses against multiple influenza virus drift variants within each season. Broadly reactive vaccine candidates potentially provide a solution to this problem, but their efficacy may begin to wane as influenza viruses naturally mutate through processes that mediates drift. Thus, it is necessary to develop a method that commercial vaccine manufacturers can use to update broadly reactive vaccine antigens to better protect against future and currently circulating viral variants. Building upon the COBRA technology, nine next-generation H3N2 influenza hemagglutinin (HA) vaccines were designed using a next generation algorithm and design methodology. These next-generation broadly reactive COBRA H3 HA vaccines were superior to wild-type HA vaccines at eliciting antibodies with high HAI activity against a panel of historical and co-circulating H3N2 influenza viruses isolated over the last 15 years, as well as the ability to neutralize future emerging H3N2 isolates.

Virus-like particles: preparation, immunogenicity and their roles as nanovaccines and drug nanocarriers.

Virus-like particles (VLPs) are virus-derived structures made up of one or more different molecules with the ability to self-assemble, mimicking the form and size of a virus particle but lacking the genetic material so they are not capable of infecting the host cell. Expression and self-assembly of the viral structural proteins can take place in various living or cell-free expression systems after which the viral structures can be assembled and reconstructed. VLPs are gaining in popularity in the field of preventive medicine and to date, a wide range of VLP-based candidate vaccines have been developed for immunization against various infectious agents, the latest of which is the vaccine against SARS-CoV-2, the efficacy of which is being evaluated. VLPs are highly immunogenic and are able to elicit both the antibody- and cell-mediated immune responses by pathways different from those elicited by conventional inactivated viral vaccines. However, there are still many challenges to this surface display system that need to be addressed in the future. VLPs that are classified as subunit vaccines are subdivided into enveloped and non- enveloped subtypes both of which are discussed in this review article. VLPs have also recently received attention for their successful applications in targeted drug delivery and for use in gene therapy. The development of more effective and targeted forms of VLP by modification of the surface of the particles in such a way that they can be introduced into specific cells or tissues or increase their half-life in the host is likely to expand their use in the future. Recent advances in the production and fabrication of VLPs including the exploration of different types of expression systems for their development, as well as their applications as vaccines in the prevention of infectious diseases and cancers resulting from their interaction with, and mechanism of activation of, the humoral and cellular immune systems are discussed in this review.

Special Issue "Virus-Like Particle Vaccines".

Virus-like particles (VLPs) have become a key tool for vaccine developers and manufacturers [...].

Zika Virus-Like Particle (VLP) vaccine displaying Envelope (E) protein CD loop antigen elicits protective and specific immune response in a murine model.

Zika virus (ZIKV) is a mosquito-borne flavivirus associated with Congenital Zika Syndrome (CZS), reflecting a wide range of congenital abnormalities in fetuses and infants infected with ZIKV before birth. ZIKV infections have also been associated with the neurological autoimmune disorder known as Guillian-Barré syndrome (GBS). To date, no vaccines or antiviral strategies are licensed for ZIKV. We used rational design to develop a novel ZIKV vaccine candidate using a Woodchuck Hepatitis core Antigen (WHcAg) Virus-Like Particle (VLP) scaffold for displaying selected antigens from the ZIKV Envelope (E) protein. A Zika-VLP vaccine candidate containing the CD Loop sub-structural domain from ZIKV E protein Domain III (WHcAg CD Loop) elicited a strong immune response in a murine model. Analysis of serum immunoglobulins demonstrated induction of both Th1- and Th2- mediated immune response. No cross-reacting antibodies were detected between Zika, dengue and yellow fever virus, demonstrating a high level of specificity for the ZIKV CD Loop antigen. Immunization with the WHcAg CD Loop vaccine candidate demonstrated immunoprotection in a murine model of ZIKV infection, stimulating protective antibodies associated with antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) activities. The WHcAg CD Loop candidate may represent a safer vaccine for preventing antibody dependent enhancement (ADE).

Vaccination against the Epstein-Barr virus.

Epstein-Barr virus (EBV) was the first human tumor virus being discovered and remains to date the only human pathogen that can transform cells in vitro. 55 years of EBV research have now brought us to the brink of an EBV vaccine. For this purpose, recombinant viral vectors and their heterologous prime-boost vaccinations, EBV-derived virus-like particles and viral envelope glycoprotein formulations are explored and are discussed in this review. Even so, cell-mediated immune control by cytotoxic lymphocytes protects healthy virus carriers from EBV-associated malignancies, antibodies might be able to prevent symptomatic primary infection, the most likely EBV-associated pathology against which EBV vaccines will be initially tested. Thus, the variety of EBV vaccines reflects the sophisticated life cycle of this human tumor virus and only vaccination in humans will finally be able to reveal the efficacy of these candidates. Nevertheless, the recently renewed efforts to develop an EBV vaccine and the long history of safe adoptive T cell transfer to treat EBV-associated malignancies suggest that this oncogenic γ-herpesvirus can be targeted by immunotherapies. Such vaccination should ideally implement the very same immune control that protects healthy EBV carriers.

Virus-Like Particle-Mediated Vaccination against Interleukin-13 May Harbour General Anti-Allergic Potential beyond Atopic Dermatitis.

Virus-like particle (VLP)-based anti-infective prophylactic vaccination has been established in clinical use. Although validated in proof-of-concept clinical trials in humans, no VLP-based therapeutic vaccination against self-proteins to modulate chronic disease has yet been licensed. The present review summarises recent scientific advances, identifying interleukin-13 as an excellent candidate to validate the concept of anti-cytokine vaccination. Based on numerous clinical studies, long-term elimination of IL-13 is not expected to trigger target-related serious adverse effects and is likely to be safer than combined targeting of IL-4/IL-13. Furthermore, recently published results from large-scale trials confirm that elimination of IL-13 is highly effective in atopic dermatitis, an exceedingly common condition, as well as eosinophilic esophagitis. The distinctly different mode of action of a polyclonal vaccine response is discussed in detail, suggesting that anti-IL-13 vaccination has the potential of outperforming monoclonal antibody-based approaches. Finally, recent data have identified a subset of follicular T helper cells dependent on IL-13 which selectively trigger massive IgE accumulation in response to anaphylactoid allergens. Thus, prophylactic IL-13 vaccination may have broad application in a number of allergic conditions.

Enhanced protective immune response of foot-and-mouth disease vaccine through DNA-loaded virus-like particles.

Foot-and-mouth disease virus (FMDV) is the etiological agent of a highly contagious disease that affects cloven-hoofed animals. Virus-like particles (VLPs) can induce a robust immune response and deliver DNA and small molecules. In this study, a VLP-harboring pcDNA3.1/P12A3C plasmid was generated, and the protective immune response was characterized. Guinea pigs were injected with VLPs, naked DNA vaccine, DNA-loaded VLPs, or phosphate-buffered saline twice subcutaneously at four-week intervals. Results demonstrated that the VLPs protected the naked DNA from DNase degeneration and delivered the DNA into the cells in vitro. The DNA-loaded VLPs and the VLPs alone induced a similar level of specific antibodies (P > 0.05) except at 49 dpv (P < 0.05). The difference in interferon-γ was consistent with that in specific antibodies. The levels of neutralizing antibodies induced by the DNA-loaded VLPs were significantly higher than those of other samples (P < 0.01). Similarly, the lymphocyte proliferation by using DNA-loaded VLPs was significantly higher than those using other formulas after booster immunization. Vaccination with DNA-loaded VLPs provided higher protection (100%) against viral challenge compared with vaccination with VLPs (75%) and DNA vaccine (25%). This study suggested that VLPs can be used as a delivery carrier for DNA vaccine. In turn, the DNA vaccine can enhance the immune response and prolong the serological duration of the VLP vaccine. This phenomenon contributes in providing complete protection against the FMDV challenge in guinea pigs and can be valuable in exploring novel nonreplicating vaccines and controlling FMD in endemic countries worldwide.

Recent Advances in the Use of Plant Virus-Like Particles as Vaccines.

Vaccination is one of the most effective public health interventions of the 20th century. All vaccines can be classified into different types, such as vaccines against infectious diseases, anticancer vaccines and vaccines against autoimmune diseases. In recent decades, recombinant technologies have enabled the design of experimental vaccines against a wide range of diseases using plant viruses and virus-like particles as central elements to stimulate protective and long-lasting immune responses. The analysis of recent publications shows that at least 97 experimental vaccines have been constructed based on plant viruses, including 71 vaccines against infectious agents, 16 anticancer vaccines and 10 therapeutic vaccines against autoimmune disorders. Several plant viruses have already been used for the development of vaccine platforms and have been tested in human and veterinary studies, suggesting that plant virus-based vaccines will be introduced into clinical and veterinary practice in the near future.

All the small things: How virus-like particles and liposomes modulate allergic immune responses.

Recent years have seen a dramatic increase in the range of applications of virus-like nanoparticle (VNP)- and liposome-based antigen delivery systems for the treatment of allergies. These platforms rely on a growing number of inert virus-backbones or distinct lipid formulations and intend to engage the host's innate and/or adaptive immune system by virtue of their co-delivered immunogens. Due to their particulate nature, VNP and liposomal preparations are also capable of breaking tolerance against endogenous cytokines, Igs, and their receptors, allowing for the facile induction of anti-cytokine, anti-IgE, or anti-FcεR antibodies in the host. We here discuss the "pros and cons" of inducing such neutralizing autoantibodies. Moreover, we cover another major theme of the last years, i.e., the engineering of non-anaphylactogenic particles and the elucidation of the parameters relevant for the specific trafficking and processing of such particles in vivo. Finally, we put the various technical advances in VNP- and liposome-research into (pre-)clinical context by referring and critically discussing the relevant studies performed to treat allergic diseases.

In silico/In vivo analysis of high-risk papillomavirus L1 and L2 conserved sequences for development of cross-subtype prophylactic vaccine.

Human papillomavirus (HPV) is the most common sexually transmitted infection in the world and the main cause of cervical cancer. Nowadays, the virus-like particles (VLPs) based on L1 proteins have been considered as the best candidate for vaccine development against HPV infections. Two commercial HPV (Gardasil and Cervarix) are available. These HPV VLP vaccines induce genotype-limited protection. The major impediments such as economic barriers especially gaps in financing obstructed the optimal delivery of vaccines in developing countries. Thus, many efforts are underway to develop the next generation of vaccines against other types of high-risk HPV. In this study, we developed DNA constructs (based on L1 and L2 genes) that were potentially immunogenic and highly conserved among the high-risk HPV types. The framework of analysis include (1) B-cell epitope mapping, (2) T-cell epitope mapping (i.e., CD4+ and CD8+ T cells), (3) allergenicity assessment, (4) tap transport and proteasomal cleavage, (5) population coverage, (6) global and template-based docking, and (7) data collection, analysis, and design of the L1 and L2 DNA constructs. Our data indicated the 8-epitope candidates for helper T-cell and CTL in L1 and L2 sequences. For the L1 and L2 constructs, combination of these peptides in a single universal vaccine could involve all world population by the rate of 95.55% and 96.33%, respectively. In vitro studies showed high expression rates of multiepitope L1 (~57.86%) and L2 (~68.42%) DNA constructs in HEK-293T cells. Moreover, in vivo studies indicated that the combination of L1 and L2 DNA constructs without any adjuvant or delivery system induced effective immune responses, and protected mice against C3 tumor cells (the percentage of tumor-free mice: ~66.67%). Thus, the designed L1 and L2 DNA constructs would represent promising applications for HPV vaccine development.

Delivery of plasmid DNA to shrimp hemocytes by Infectious hypodermal and hematopoietic necrosis virus (IHHNV) nanoparticles expressed from a baculovirus insect cell system.

Virus-like particles (VLPs) are potential containers for delivery of therapeutic agents at the nanoscale. In this study, the capsid protein of Infectious hypodermal and hematopoietic necrosis virus (IHHNV) was expressed in a baculovirus insect cell system. The 37-kDa recombinant protein containing the hexahistidine residues (His Tag) at N-terminal was purified using immobilized metal affinity chromatography (IMAC) and assembled into VLPs with a diameter of 23 ±â€¯3 nm analyzed by transmission electron microscopy. We also verified that disassembly/reassembly of IHHNV-VLPs was controlled in the presence and absence of DTT. The efficiency of IHHNV-VLPs to encapsulate plasmid DNA was about 48.2%, and the VLPs encapsulating the pcDNA3.1(+)-EGFP plasmid DNA could recognize the primary shrimp hemocytes and deliver the loaded plasmid into cells by detection of expressed enhanced green fluorescent protein (EGFP). These results implied that the IHHNV-VLPs might be a good candidate for packaging and delivery of expressible plasmid DNA, and may produce an antiviral product in shrimp cells for gene therapy.

Virus-like particles containing multiple antigenic proteins of Toxoplasma gondii induce memory T cell and B cell responses.

Although the efforts to develop vaccine against Toxoplasma gondii infection were made for decades, there is currently no licensed vaccine available for humans. Upon discovering a number of T or B cell epitope regions from T. gondii IMC, ROP18 and MIC8, multi-antigen VLPs or combination VLPs were generated. Mice immunized with multi-antigen VLPs or combination VLPs were challenge infected with T. gondii (ME49). T. gondii-specific IgG, IgG isotypes and IgA antibody responses, memory T and B cell responses and protection were evaluated. All the mice survived upon T. gondii challenge infection by multi-antigen VLPs vaccination. Vaccinated mice elicited higher levels of parasite-specific IgG and IgG2a antibody responses in sera, IgA antibody responses in feces, CD4+ and CD8+ T cell responses, and cytokines (IFN-γ, IL-10) responses compared to combination VLPs. In particular, the multi-antigen VLPs vaccination showed significantly higher levels of antibody secreting cell (ASC) responses, CD4+ and CD8+ effector memory T cells, and memory B cells than combination VLPs. Multi-antigen VLPs vaccination showed significant reduction of brain cyst counts and size, and all mice survived. Prediction and analysis of epitopes have indicated that IMC, ROP18 and MIC8 showed partially overlapping epitopes for T and B cells. Our results indicated that antibody responses, memory T and B cells induced by multi-antigen VLPs vaccination might contribute to the complete protection upon T. gondii (ME49) challenge infection.

Foot-and-mouth disease vaccines: recent updates and future perspectives.

Foot-and-mouth disease (FMD) is a major worldwide viral disease in animals, affecting the national and international trade of livestock and animal products and leading to high economic losses and social consequences. Effective control measures of FMD involve prevention through vaccination with inactivated vaccines. These inactivated vaccines, unfortunately, require short-term protection and cold-chain and high-containment facilities. Major advances and pursuit of hot topics in vaccinology and vectorology are ongoing, involving peptide vaccines, DNA vaccines, live vector vaccines, and novel attenuated vaccines. DIVA capability and marker vaccines are very important in differentiating infected animals from vaccinated animals. This review focuses on updating the research progress of these novel vaccines, summarizing their merits and including ideas for improvement.

The ATRQß-001 vaccine improves cardiac function and prevents postinfarction cardiac remodeling in mice.

We invented the ATRQß-001 hypertension vaccine, which targeted angiotensin II type 1 receptor (AT1R) and showed a desirable blocking effect for AT1R. The purpose of this study was to investigate whether the ATRQß-001 vaccine could improve cardiac function and prevent cardiac remodeling after acute myocardial infarction (AMI). C57BL/6 male mice were randomly assigned into four groups: sham + VLP, MI + VLP, MI + ATRQß-001, and MI + valsartan. Mice were administered Qß virus-like particle (Qß-VLP, 100 µg/time), ATRQß-001 vaccine (100 µg/time), and valsartan (6 mg/kg/day) before AMI, which was induced by permanently ligating the left anterior descending coronary artery. The effect of the ATRQß-001 vaccine on cardiac function and cardiac remodeling was observed by following up for 1 week, 4 weeks, and 12 weeks post MI. The ATRQß-001 vaccine significantly reduced sudden cardiac death and increased survival rates (compared with MI + VLP, 80% versus 55% and mean estimate (days) 68.4 ± 7.0 versus 47.8 ± 8.9, respectively; p = 0.046) post MI. Echocardiography showed that the ATRQß-001 vaccine remarkably improved cardiac function (left ventricular ejection fraction, 24.8 ± 7.0% versus 13.2 ± 3.8%, p = 0.005) post MI. Histological analysis revealed that the ATRQß-001 vaccine obviously mitigated myocardial inflammation, apoptosis, and fibrosis after AMI. Further, the ATRQß-001 vaccine significantly inhibited the TGF-ß1/Smad2/3 signaling pathway. Assessment of the renin-angiotensin system (RAS) demonstrated that the ATRQß-001 vaccine did not cause obvious feedback of circulating RAS, but prominently attenuated the expression of AT1R, compared with the other groups at 4 and 12 weeks after AMI. In conclusion, the ATRQß-001 vaccine decreased mortality and improved cardiac function and remodeling after AMI.

Use of plant viruses and virus-like particles for the creation of novel vaccines.

In recent decades, the development of plant virology and genetic engineering techniques has resulted in the construction of plant virus-based vaccines for protection against different infectious agents, cancers and autoimmune diseases in both humans and animals. Interaction studies between plant viruses and mammalian organisms have suggested that plant viruses and virus-like particles (VLPs) are safe and noninfectious to humans and animals. Plant viruses with introduced antigens are powerful vaccine components due to their strongly organized, repetitive spatial structure; they can elicit strong immune responses similar to those observed with infectious mammalian viruses. The analysis of published data demonstrated that at least 73 experimental vaccines, including 61 prophylactic and 12 therapeutic vaccines, have been constructed using plant viruses as a carrier structure for presentation of different antigens. This information clearly demonstrates that noninfectious viruses are also applicable as vaccine carriers. Moreover, several plant viruses have been used for platform development, and corresponding vaccines are currently being tested in human and veterinary clinical trials. This review therefore discusses the main principles of plant VLP vaccine construction, emphasizing the physical, chemical, genetic and immunological aspects. Results of the latest studies suggest that several plant virus-based vaccines will join the list of approved human and animal vaccines in the near future.

Non-clinical safety assessment of repeated intramuscular administration of an EV-A71 VLP vaccine in rabbits.

A candidate hand, foot, and mouth disease vaccine comprising of human enterovirus A71 (EV-A71) virus-like particles (VLPs) was tested in rabbits to evaluate the potential local and systemic effects of this vaccine. The rabbits received more than double the full human dose and one additional dose according to the n + 1 recommended scheme. The three doses were given mixed with Alhydrogel adjuvant as intramuscular (IM) injections. Vaccinations were well-tolerated, with no indication of overt toxicity in any parameter observed. An EV-A71 specific immune response in the form of antibodies that specifically reacted with the virus capsid proteins VP1 and VP0, the complete VLP, and EV-A71 viruses of different subgenotypes to that of the vaccine could be demonstrated. A boosting effect in the form of higher EV-A71 specific antibody titers was observed after the subsequent doses, and these enhanced titers were shown to be statistically significant in one-way ANOVA analyses. Fortnightly intramuscular administration of EV-A71 VLP vaccine did not result in any test article-related changes in immunotoxicity as defined by increased serum IL-6, and in general IL-6 concentrations remained below the lower limit of quantitation for the majority of animals throughout the study. Although increased indicators of inflammation at the injection site were observed in animals sacrificed immediately after the last vaccination, these largely reversed at the end of the recovery phase. No findings suggestive of systemic or delayed toxicity were recorded in this independently conducted study. In conclusion, repeated IM administration of the EV-A71 VLP vaccine were locally and systemically well-tolerated in rabbits and immunogenic, supporting the clinical development of the vaccine.