A recombinant virus-like particle vaccine against adenovirus-7 induces a potent humoral response.

Adenoviruses (AdVs) cause infections in humans that range from mild to severe, and can cause outbreaks particularly in close contact settings. Several human AdV types have been identified, which can cause a wide array of clinical manifestations. AdV types 4 and 7 (AdV-4 and AdV-7), which are among the most commonly circulating types in the United States, are known to cause acute respiratory disease that can result in hospitalization and rarely, death. Currently, the only vaccines approved for use in humans are live virus vaccines against AdV-4 and AdV-7, though these vaccines are only authorized for use in U.S. military personnel. While they are efficacious, use of these live virus vaccines carries considerable risks of vaccine-associated viral shedding and recombination. Here, we present an alternative vaccination strategy against AdV-7 using the virus-like particle platform (AdVLP-7). We describe the production of stable recombinant AdVLP-7, and demonstrate that AdVLP-7 is structurally analogous to wild-type AdV-7 virions (WT AdV-7). Preclinical immunogenicity studies in mice show that AdVLP-7 elicits a potent humoral immune response, comparable to that observed in mice immunized with WT AdV-7. Specifically, AdVLP-7 induces high titers of antibodies against AdV-7-specific antigens that can effectively neutralize AdV-7.

Novel and efficient method for the reconstruction of adenoviruses through isothermal assembly and its potential applications.

Adenovirus based vectors are useful tools for vaccine development, gene therapy, and oncolytic virotherapy. Here we describe a novel approach for the genetic engineering of any portion of the adenovirus genome and the reconstruction of either fully replication competent or defective virions. This innovative strategy is rapid, effective and suitable for the manipulation of the entire genome broadening the spectrum of potential applications for the adenovirus system. Our strategy involved insertion of restriction enzyme recognition sequences absent in the native virus into the termini of the adenovirus genome in order to facilitate recovery. These restriction enzyme sites, together with the two inverted terminal repeats and packaging sequences, were synthesized and then subcloned into the pBR322 vector. The remaining internal portion of the adenovirus genome was separated and amplified via PCR into six segments, of which groups of two were joined together by PCR and then subcloned into pBR322 plasmids. During the PCR reaction, an overlap of 30-40 bp was added to the termini of the adjacent fragments, allowing for the subsequent isothermal assembly and correct orientation of all fragments. This approach allows for the genetic modification of each genomic fragment according to the specific research goals, (e.g., deletion, substitution, addition, etc.) To recreate the entire viral genome, the four engineered fragments (each comprised of two adenovirus genomic sections) as well as the pBR322 backbone, were reassembled into a single construct utilizing an isothermal assembly reaction. Finally, the reassembled plasmid containing the entire genome was linearized and transfected into HEK293 cells to recover the complete reconstructed adenoviral vector. Using this approach, we have successfully generated two recombinant reporter adenoviruses, one of human adenovirus serotype 14 and another of serotype 55. The E3 region was replaced by the reporter genes (GFP and Luciferase) to visualize and track the recovery process. Subsequent infection of A549 cells with these reconstructed adenovirus vectors demonstrated that they were replication competent. This method shortens the viral reconstruction process because the one-step isothermal assembly requires less than 4 days, and recombinant adenovirus recovery occurs within 10 days. This new method allows for single or multiple genetic modifications within any portion of the viral genome and can be used to construct or manipulate any adenovirus whose complete genome sequence is known.

Immunogenicity and Efficacy of Monovalent and Bivalent Formulations of a Virus-Like Particle Vaccine against SARS-CoV-2.

Virus-like particles (VLPs) offer great potential as a safe and effective vaccine platform against SARS-CoV-2, the causative agent of COVID-19. Here, we show that SARS-CoV-2 VLPs can be generated by expression of the four viral structural proteins in a mammalian expression system. Immunization of mice with a monovalent VLP vaccine elicited a potent humoral response, showing neutralizing activity against multiple variants of SARS-CoV-2. Subsequent immunogenicity and efficacy studies were performed in the Golden Syrian hamster model, which closely resembles the pathology and progression of COVID-19 in humans. Hamsters immunized with a bivalent VLP vaccine were significantly protected from infection with the Beta or Delta variant of SARS-CoV-2. Vaccinated hamsters showed reduced viral load, shedding, replication, and pathology in the respiratory tract. Immunized hamsters also showed variable levels of cross-neutralizing activity against the Omicron variant. Overall, the VLP vaccine elicited robust protective efficacy against SARS-CoV-2. These promising results warrant further study of multivalent VLP vaccines in Phase I clinical trials in humans.

Current development of Zika virus vaccines with special emphasis on virus-like particle technology.

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-Barré 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.

Structural characterization of the PCV2d virus-like particle at 3.3 Šresolution reveals differences to PCV2a and PCV2b capsids, a tetranucleotide, and an N-terminus near the icosahedral 3-fold axes.

Porcine circovirus 2 (PCV2) has a major impact on the swine industry. Eight PCV2 genotypes (a-h) have been identified using capsid sequence analysis. PCV2d has been designated as the emerging genotype. The cryo-electron microscopy molecular envelope of PCV2d virus-like particles identifies differences between PCV2a, b and d genotypes that accompany the emergence of PCV2b from PCV2a, and PCV2d from PCV2b. These differences indicate that sequence analysis of genotypes is insufficient, and that it is important to determine the PCV2 capsid structure as the virus evolves. Structure-based sequence comparison demonstrate that each genotype possesses a unique combination of amino acids located on the surface of the capsid that undergo substitution. We also demonstrate that the capsid N-terminus moves in response to increasing amount of nucleic acid packaged into the capsid. Furthermore, we model a tetranucleotide between the 5- and 2-fold axes of symmetry that appears to be responsible for capsid stability.

Dengue-2 virus-like particle (VLP) based vaccine elicits the highest titers of neutralizing antibodies when produced at reduced temperature.

A dengue vaccine capable of rapidly eliciting a robust and balanced immunity against the four virus serotypes after only a few immunizations is greatly needed. We describe a new strategy to develop dengue vaccines based on the assembly of virus-like particles (VLPs) utilizing the structural proteins CprME together with a modified complex of the NS2B/NS3 protease, which enhances particle formation and yield. These VLPs are produced in mammalian cells and resemble native dengue virus as demonstrated by negative staining and immunogold labelling electron microscopy (EM). We found that VLPs produced at lower temperature (31 °C) were recognized by conformational monoclonal antibodies (MAbs) 4G2, 3H5 and C10 whereas VLPs produced at higher temperature (37 °C) were not recognized by these MAbs. To investigate the significance of these conformational discrepancies in vaccine performance, we tested the immunogenicity of VLP vaccines produced at 31 °C or 37 °C. Mice immunized with the VLP vaccine produced at 31 °C (VLP-31 °C) elicited the highest titer of neutralizing antibodies when compared to those elicited by equivalent doses of the vaccine produced at 37 °C (VLP-37 °C), inactivated dengue virus vaccine or to the titer of a human anti-dengue-2 convalescence serum reference. Our results demonstrate that the conformation of the E protein displayed on the VLP vaccine plays a critical role in the induction of highly neutralizing antibodies. These findings will guide development of a tetravalent vaccine capable of eliciting a robust and balanced neutralizing response against the four-dengue serotypes regardless of background immunity.

Adjuvant formulations for virus-like particle (VLP) based vaccines.

The development of virus-like particle (VLP) technology has had an enormous impact on modern vaccinology. In order to optimize the efficacy and safety of VLP-based vaccines, adjuvants are included in most vaccine formulations. To date, most licensed VLP-based vaccines utilize the classic aluminum adjuvant compositions. Certain challenging pathogens and weak immune responder subjects may require further optimization of the adjuvant formulation to maximize the magnitude and duration of the protective immunity. Indeed, novel classes of adjuvants such as liposomes, agonists of pathogen recognition receptors, polymeric particles, emulsions, cytokines and bacterial toxins, can be used to further improve the immunostimulatory activity of a VLP-based vaccine. This review describes the current advances in adjuvant technology for VLP-based vaccines directed at viral diseases, and discusses the basic principles for designing adjuvant formulations for enhancing the vaccine immunogenicity.

Zika virus-like particle (VLP) based vaccine.

The newly emerged mosquito-borne Zika virus poses a major public challenge due to its ability to cause significant birth defects and neurological disorders. The impact of sexual transmission is unclear but raises further concerns about virus dissemination. No specific treatment or vaccine is currently available, thus the development of a safe and effective vaccine is paramount. Here we describe a novel strategy to assemble Zika virus-like particles (VLPs) by co-expressing the structural (CprME) and non-structural (NS2B/NS3) proteins, and demonstrate their effectiveness as vaccines. VLPs are produced in a suspension culture of mammalian cells and self-assembled into particles closely resembling Zika viruses as shown by electron microscopy studies. We tested various VLP vaccines and compared them to analogous compositions of an inactivated Zika virus (In-ZIKV) used as a reference. VLP immunizations elicited high titers of antibodies, as did the In-ZIKV controls. However, in mice the VLP vaccine stimulated significantly higher virus neutralizing antibody titers than comparable formulations of the In-ZIKV vaccine. The serum neutralizing activity elicited by the VLP vaccine was enhanced using a higher VLP dose and with the addition of an adjuvant, reaching neutralizing titers greater than those detected in the serum of a patient who recovered from a Zika infection in Brazil in 2015. Discrepancies in neutralization levels between the VLP vaccine and the In-ZIKV suggest that chemical inactivation has deleterious effects on neutralizing epitopes within the E protein. This along with the inability of a VLP vaccine to cause infection makes it a preferable candidate for vaccine development.

Novel Respiratory Syncytial Virus-Like Particle Vaccine Composed of the Postfusion and Prefusion Conformations of the F Glycoprotein.

Respiratory syncytial virus (RSV) is the leading cause of severe respiratory disease in infants and children and represents an important global health burden for the elderly and the immunocompromised. Despite decades of research efforts, no licensed vaccine for RSV is available. We have developed virus-like particle (VLP)-based RSV vaccines assembled with the human metapneumovirus (hMPV) matrix protein (M) as the structural scaffold and the RSV fusion glycoprotein (F) in either the postfusion or prefusion conformation as its prime surface immunogen. Vaccines were composed of postfusion F, prefusion F, or a combination of the two conformations and formulated with a squalene-based oil emulsion as adjuvant. Immunization with these VLP vaccines afforded full protection against RSV infection and prevented detectable viral replication in the mouse lung after challenge. Analyses of lung cytokines and chemokines showed that VLP vaccination mostly induced the production of gamma interferon (IFN-γ), a marker of the Th1-mediated immune response, which is predominantly required for viral protection. Conversely, immunization with a formalin-inactivated RSV (FI-RSV) vaccine induced high levels of inflammatory chemokines and cytokines of the Th2- and Th17-mediated types of immune responses, as well as severe lung inflammation and histopathology. The VLP vaccines showed restricted production of these immune mediators and did not induce severe bronchiolitis or perivascular infiltration as seen with the FI-RSV vaccine. Remarkably, analysis of the serum from immunized mice showed that the VLP vaccine formulated using a combination of postfusion and prefusion F elicited the highest level of neutralizing antibody and enhanced the Th1-mediated immune response.

A novel intranasal virus-like particle (VLP) vaccine designed to protect against the pandemic 1918 influenza A virus (H1N1).

We have prepared a virus-like particle (VLP) vaccine bearing the surface glycoproteins HA and NA of the 1918 influenza A virus by infecting Sf9 cells with a quadruple recombinant baculovirus that expresses the four influenza proteins (HA, NA, M1, and M2) required for the assembly and budding of the VLPs. The presence of HA and M1 in the purified VLPs was confirmed by Western blot, and that of NA by a neuraminidase enzymatic assay. For in vivo studies, the 1918 VLP vaccine was formulated with or without an oligonucleotide containing two CpG motifs and administered in two doses 2 wk apart via the intranasal route. The antibody titers in mice immunized with VLP vaccines were higher than in mice vaccinated with an inactivated swine virus (H1N1) control, when CHO cells expressing 1918 HA were used as antigen. The opposite result was obtained when disrupted swine virus was the antigen for the ELISA test. Vaccine efficacy was evaluated by challenging immunized mice with the 1918 antigenically related influenza virus A/swine/Iowa/15/30 (H1N1) and measuring viral titers in the upper and lower respiratory tract. Mice immunized with VLP vaccine plus CpG demonstrated significantly lower viral titers in the nose and lungs than did the control on days 2 and 4 postchallenge and completely cleared the virus by day 6. Furthermore, they did not show symptoms of disease although there was a minor decrease in body weight. Mice vaccinated with VLP alone also demonstrated significantly lower viral titers in the nose and lungs than did the placebo group as well as the inactivated virus group on days 4 and 6 postchallenge. These results suggest that it is feasible to make a safe and immunogenic vaccine to protect against the extremely virulent 1918 virus, using a novel and safe cell-based technology.

Virus-like particle vaccine conferred complete protection against a lethal influenza virus challenge.

We have previously demonstrated the formation and release of influenza virus-like particles (VLPs) from the surface of Sf9 cells infected with either a quadruple baculovirus recombinant that simultaneously expresses the influenza structural proteins hemagglutinin (HA), neuraminidase (NA), matrix 1 (M1), and matrix 2 (M2), or a combination of single recombinants that include the M1 protein. In this work, we present data on the immunogenicity and protective efficacy afforded by VLPs (formed by M1 and HA) after immunization of mice. VLP vaccine ( approximately 1 microg HA) were formulated with or without IL-12 as adjuvant and administered twice, at 2-week intervals, by either intranasal instillation or intramuscular injection. All VLP-vaccinated and influenza-immunized control mice demonstrated high antibody titers to the HA protein; however, intranasal instillation of VLPs elicited antibody titers that were higher than those induced by either intramuscular inoculation of VLPs or intranasal inoculation with two sub-lethal doses of the challenge influenza virus (control group). Antibody responses were enhanced when VLP vaccine was formulated with IL12 as adjuvant. All mice were challenged with 5 LD50 of a mouse-adapted influenza A/Hong Kong/68 (H3N2) virus. Intramuscular administration of VLP vaccine formulated with or without IL-12 afforded 100% protection against a lethal influenza virus challenge. Similarly, intranasal instillation of VLP vaccine alone protected 100% of the mice, whereas VLP formulated with IL-12 protected 90% of the vaccinated mice. Not only do these results suggest a novel approach to the development of VLP vaccines for diverse influenza virus strains, but also the creation of multivalent vaccines by decoration of the surface of the VLPs with antigens from other pathogens.

Virus-like particle (VLP) vaccine conferred complete protection against a lethal influenza virus challenge.

We have previously demonstrated the formation and release of influenza virus-like particles (VLPs) from the surface of Sf9 cells infected with either a quadruple baculovirus recombinant that simultaneously expresses the influenza structural proteins hemagglutinin (HA), neuraminidase (NA), matrix 1 (M1) and M2, or a combination of single recombinants that include the M1 protein. In this work, we present data on the immunogenicity and protective efficacy afforded by VLPs (formed by M1 and HA) following immunization of mice. VLP vaccine (approximately 1 microg HA) were formulated with or without IL-12 as adjuvant and administered twice, at two weeks intervals, by either intranasal instillation or intramuscular injection. All VLP-vaccinated and influenza-immunized control mice demonstrated high antibody titers to the HA protein; however, intranasal instillation of VLPs elicited antibody titers that were higher than those induced by either intramuscular inoculation of VLPs or intranasal inoculation with two sub-lethal doses of the challenge influenza virus (control group). Antibody responses were enhanced when VLP vaccine was formulated with IL12 as adjuvant. All mice were challenged with 5 LD50 of a mouse-adapted influenza A/Hong Kong/68 (H3N2) virus. Intramuscular administration of VLP vaccine formulated with or without IL-12 afforded 100% protection against a lethal influenza virus challenge. Similarly, intranasal instillation of VLP vaccine alone protected 100% of the mice, whereas VLP formulated with IL-12 protected 90% of the vaccinated mice. Not only do these results suggest a novel approach to the development of VLP vaccines for diverse influenza virus strains, but also the creation of multivalent vaccines by decoration of the surface of the VLPs with antigens from other pathogens.