Distinct motifs in the E protein are required for SARS-CoV-2 virus particle formation and lysosomal deacidification in host cells.

IMPORTANCE: Severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2), the virus responsible for coronavirus disease 2019 (COVID-19), has caused a global public health crisis. The E protein, a structural protein found in this virus particle, is also known to be a viroporin. As such, it forms oligomeric ion channels or pores in the host cell membrane. However, the relationship between these two functions is poorly understood. In this study, we showed that the roles of E protein in virus particle and viroporin formation are distinct. This study contributes to the development of drugs that inhibit SARS-CoV-2 virus particle formation. Additionally, we designed a highly sensitive and high-throughput virus-like particle detection system using the HiBiT tag, which is a useful tool for studying the release of SARS-CoV-2.

A hepatitis B virus core antigen-based virus-like particle vaccine expressing SARS-CoV-2 B and T cell epitopes induces epitope-specific humoral and cell-mediated immune responses but confers limited protection against SARS-CoV-2 infection.

The hepatitis B virus core antigen (HBcAg) tolerates insertion of foreign epitopes and maintains its ability to self-assemble into virus-like particles (VLPs). We constructed a ∆HBcAg-based VLP vaccine expressing three predicted severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) B and T cell epitopes and determined its immunogenicity and protective efficacy. The recombinant ∆HBcAg-SARS-CoV-2 protein was expressed in Escherichia coli, purified, and shown to form VLPs. K18-hACE2 transgenic C57BL/6 mice were immunized intramuscularly with ∆HBcAg VLP control (n = 15) or ∆HBcAg-SARS-CoV-2 VLP vaccine (n = 15). One week after the 2nd booster and before virus challenge, five ∆HBcAg-SARS-CoV-2 vaccinated mice were euthanized to evaluate epitope-specific immune responses. There is a statistically significant increase in epitope-specific Immunoglobulin G (IgG) response, and statistically higher interleukin 6 (IL-6) and monocyte chemoattractant protein-1 (MCP-1) expression levels in ∆HBcAg-SARS-CoV-2 VLP-vaccinated mice compared to ∆HBcAg VLP controls. While not statistically significant, the ∆HBcAg-SARS-CoV-2 VLP mice had numerically more memory CD8+ T-cells, and 3/5 mice also had numerically higher levels of interferon gamma (IFN-γ) and tumor necrosis factor (TNF). After challenge with SARS-CoV-2, ∆HBcAg-SARS-CoV-2 immunized mice had numerically lower viral RNA loads in the lung, and slightly higher survival, but the differences are not statistically significant. These results indicate that the ∆HBcAg-SARS-CoV-2 VLP vaccine elicits epitope-specific humoral and cell-mediated immune responses but they were insufficient against SARS-CoV-2 infection.

Bioengineered Bovine Papillomavirus L1 Protein Virus-like Particle (VLP) Vaccines for Enhanced Induction of CD8 T Cell Responses through Cross-Priming.

Safe and effective T cell vaccines are needed for the treatment or prevention of cancers as well as infectious agents where vaccines for neutralizing antibodies have performed poorly. Recent research highlights an important role for tissue-resident memory T cells (TRM cells) in protective immunity and the role of a subset of dendritic cells that are capable of cross-priming for the induction of TRM cells. However, efficient vaccine technologies that operate through cross-priming and induce robust CD8+ T cell responses are lacking. We developed a platform technology by genetically engineering the bovine papillomavirus L1 major capsid protein to insert a polyglutamic acid/cysteine motif in place of wild-type amino acids in the HI loop. Virus-like particles (VLPs) are formed by self-assembly in insect cells infected with a recombinant baculovirus. Polyarginine/cysteine-tagged antigens are linked to the VLP by a reversible disulfide bond. The VLP possesses self-adjuvanting properties due to the immunostimulatory activity of papillomavirus VLPs. Polyionic VLP vaccines induce robust CD8+ T cell responses in peripheral blood and tumor tissues. A prostate cancer polyionic VLP vaccine was more efficacious than other vaccines and immunotherapies for the treatment of prostate cancer in a physiologically relevant murine model and successfully treated more advanced diseases than the less efficacious technologies. The immunogenicity of polyionic VLP vaccines is dependent on particle size, reversible linkage of the antigen to the VLP, and an interferon type 1 and Toll-like receptor (TLR)3/7-dependent mechanism.

Pseudotyping Improves the Yield of Functional SARS-CoV-2 Virus-like Particles (VLPs) as Tools for Vaccine and Therapeutic Development.

Virus-like particles (VLPs) have been proposed as an attractive tool in SARS-CoV-2 vaccine development, both as (1) a vaccine candidate with high immunogenicity and low reactogenicity and (2) a substitute for live virus in functional and neutralization assays. Though multiple SARS-CoV-2 VLP designs have already been explored in Sf9 insect cells, a key parameter ensuring VLPs are a viable platform is the VLP spike yield (i.e., spike protein content in VLP), which has largely been unreported. In this study, we show that the common strategy of producing SARS-CoV-2 VLPs by expressing spike protein in combination with the native coronavirus membrane and/or envelope protein forms VLPs, but at a critically low spike yield (~0.04-0.08 mg/L). In contrast, fusing the spike ectodomain to the influenza HA transmembrane domain and cytoplasmic tail and co-expressing M1 increased VLP spike yield to ~0.4 mg/L. More importantly, this increased yield translated to a greater VLP spike antigen density (~96 spike monomers/VLP) that more closely resembles that of native SARS-CoV-2 virus (~72-144 Spike monomers/virion). Pseudotyping further allowed for production of functional alpha (B.1.1.7), beta (B.1.351), delta (B.1.617.2), and omicron (B.1.1.529) SARS-CoV-2 VLPs that bound to the target ACE2 receptor. Finally, we demonstrated the utility of pseudotyped VLPs to test neutralizing antibody activity using a simple, acellular ELISA-based assay performed at biosafety level 1 (BSL-1). Taken together, this study highlights the advantage of pseudotyping over native SARS-CoV-2 VLP designs in achieving higher VLP spike yield and demonstrates the usefulness of pseudotyped VLPs as a surrogate for live virus in vaccine and therapeutic development against SARS-CoV-2 variants.

Investigating the Human Intestinal DNA Virome and Predicting Disease-Associated Virus-Host Interactions in Severe Myalgic Encephalomyelitis/Chronic Fatigue Syndrome (ME/CFS).

Understanding how the human virome, and which of its constituents, contributes to health or disease states is reliant on obtaining comprehensive virome profiles. By combining DNA viromes from isolated virus-like particles (VLPs) and whole metagenomes from the same faecal sample of a small cohort of healthy individuals and patients with severe myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS), we have obtained a more inclusive profile of the human intestinal DNA virome. Key features are the identification of a core virome comprising tailed phages of the class Caudoviricetes, and a greater diversity of DNA viruses including extracellular phages and integrated prophages. Using an in silico approach, we predicted interactions between members of the Anaerotruncus genus and unique viruses present in ME/CFS microbiomes. This study therefore provides a framework and rationale for studies of larger cohorts of patients to further investigate disease-associated interactions between the intestinal virome and the bacteriome.

SARS-CoV-2 viral budding and entry can be modeled using BSL-2 level virus-like particles.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) was first discovered in December 2019 in Wuhan, China, and expeditiously spread across the globe causing a global pandemic. Research on SARS-CoV-2, as well as the closely related SARS-CoV-1 and MERS coronaviruses, is restricted to BSL-3 facilities. Such BSL-3 classification makes SARS-CoV-2 research inaccessible to the majority of functioning research laboratories in the United States; this becomes problematic when the collective scientific effort needs to be focused on such in the face of a pandemic. However, a minimal system capable of recapitulating different steps of the viral life cycle without using the virus' genetic material could increase accessibility. In this work, we assessed the four structural proteins from SARS-CoV-2 for their ability to form virus-like particles (VLPs) from human cells to form a competent system for BSL-2 studies of SARS-CoV-2. Herein, we provide methods and resources of producing, purifying, fluorescently and APEX2-labeling of SARS-CoV-2 VLPs for the evaluation of mechanisms of viral budding and entry as well as assessment of drug inhibitors under BSL-2 conditions. These systems should be useful to those looking to circumvent BSL-3 work with SARS-CoV-2 yet study the mechanisms by which SARS-CoV-2 enters and exits human cells.

New Advances in Using Virus-like Particles and Related Technologies for Eukaryotic Genome Editing Delivery.

The designer nucleases, including Zinc Finger Nuclease (ZFN), Transcription Activator-Like Effector Nuclease (TALEN), and Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR-associated (CRISPR/Cas), have been widely used for mechanistic studies, animal model generation, and gene therapy development. Clinical trials using designer nucleases to treat genetic diseases or cancers are showing promising results. Despite rapid progress, potential off-targets and host immune responses are challenges to be addressed for in vivo uses, especially in clinical applications. Short-term expression of the designer nucleases is necessary to reduce both risks. Currently, delivery methods enabling transient expression of designer nucleases are being pursued. Among these, virus-like particles as delivery vehicles for short-term designer nuclease expression have received much attention. This review will summarize recent developments in using virus-like particles (VLPs) for safe delivery of gene editing effectors to complement our last review on the same topic. First, we introduce some background information on how VLPs can be used for safe and efficient CRISPR/Cas9 delivery. Then, we summarize recently developed virus-like particles as genome editing vehicles. Finally, we discuss applications and future directions.

Angiomotin regulates budding and spread of Ebola virus.

The Ebola virus (EBOV) VP40 matrix protein (eVP40) orchestrates assembly and budding of virions in part by hijacking select WW-domain-bearing host proteins via its PPxY late (L)-domain motif. Angiomotin (Amot) is a multifunctional PPxY-containing adaptor protein that regulates angiogenesis, actin dynamics, and cell migration/motility. Amot also regulates the Hippo signaling pathway via interactions with the WW-domain-containing Hippo effector protein Yes-associated protein (YAP). In this report, we demonstrate that endogenous Amot is crucial for positively regulating egress of eVP40 virus-like particles (VLPs) and for egress and spread of authentic EBOV. Mechanistically, we show that ectopic YAP expression inhibits eVP40 VLP egress and that Amot co-expression rescues budding of eVP40 VLPs in a dose-dependent and PPxY-dependent manner. Moreover, results obtained with confocal and total internal reflection fluorescence microscopy suggested that Amot's role in actin organization and dynamics also contributes to promoting eVP40-mediated egress. In summary, these findings reveal a functional and competitive interplay between virus and host proteins involving the multifunctional PPxY-containing adaptor Amot, which regulates both the Hippo pathway and actin dynamics. We propose that our results have wide-ranging implications for understanding the biology and pathology of EBOV infections.

Viruses go modular.

The WW domain is a modular protein structure that recognizes the proline-rich Pro-Pro-x-Tyr (PPxY) motif contained in specific target proteins. The compact modular nature of the WW domain makes it ideal for mediating interactions between proteins in complex networks and signaling pathways of the cell (e.g. the Hippo pathway). As a result, WW domains play key roles in a plethora of both normal and disease processes. Intriguingly, RNA and DNA viruses have evolved strategies to hijack cellular WW domain-containing proteins and thereby exploit the modular functions of these host proteins for various steps of the virus life cycle, including entry, replication, and egress. In this review, we summarize key findings in this rapidly expanding field, in which new virus-host interactions continue to be identified. Further unraveling of the molecular aspects of these crucial virus-host interactions will continue to enhance our fundamental understanding of the biology and pathogenesis of these viruses. We anticipate that additional insights into these interactions will help support strategies to develop a new class of small-molecule inhibitors of viral PPxY-host WW-domain interactions that could be used as antiviral therapeutics.

Bluetongue Virus Particles as Nanoreactors for Enzyme Delivery and Cancer Therapy.

The side effects of chemotherapy can be reduced by targeting tumor cells with an enzyme (or the corresponding gene) that converts a nontoxic prodrug into a toxic drug inside the tumor cells, also killing the surrounding tumor cells via the bystander effect. Viruses are the most efficient gene delivery vehicles because they have evolved to transfer their own nucleic acids into cells, but their efficiency must be balanced against the risks of infection, the immunogenicity of nucleic acids, and the potential for genomic integration. We therefore tested the effectiveness of genome-free virus-like particles (VLPs) for the delivery of Herpes simplex virus 1 thymidine kinase (HSV1-TK), the most common enzyme used in prodrug conversion therapy. HSV1-TK is typically delivered as a gene, but in the context of VLPs, it must be delivered as a protein. We constructed VLPs and smaller core-like particles (CLPs) based on Bluetongue virus, with HSV1-TK fused to the inner capsid protein VP3. TK-CLPs and TK-VLPs could be produced in large quantities in plants. The TK-VLPs killed human glioblastoma cells efficiently in the presence of ganciclovir, with an IC50 value of 14.8 µM. Conversely, CLPs were ineffective because they remained trapped in the endosomal compartment, in common with many synthetic nanoparticles. VLPs are advantageous because they can escape from endosomes and therefore allow HSV1-TK to access the cytosolic adenosine triphosphate (ATP) required for the phosphorylation of ganciclovir. The VLP delivery strategy of TK protein therefore offers a promising new modality for the treatment of cancer with systemic prodrugs such as ganciclovir.

Nucleic acid-induced dimerization of HIV-1 Gag protein.

HIV-1 Gag is a highly flexible multidomain protein that forms the protein lattice of the immature HIV-1 virion. In vitro, it reversibly dimerizes, but in the presence of nucleic acids (NAs), it spontaneously assembles into virus-like particles (VLPs). High-resolution structures have revealed intricate details of the interactions of the capsid (CA) domain of Gag and the flanking spacer peptide SP1 that stabilize VLPs, but much less is known about the assembly pathway and the interactions of the highly flexible NA-binding nucleocapsid (NC) domain. Here, using a novel hybrid fluorescence proximity/sedimentation velocity method in combination with calorimetric analyses, we studied initial binding events by monitoring the sizes and conformations of complexes of Gag with very short oligonucleotides. We observed that high-affinity binding of oligonucleotides induces conformational changes in Gag accompanied by the formation of complexes with a 2:1 Gag/NA stoichiometry. This NA-liganded dimerization mode is distinct from the widely studied dimer interface in the CA domain and from protein interactions arising in the SP1 region and may be mediated by protein-protein interactions localized in the NC domain. The formation of the liganded dimer is strongly enthalpically driven, resulting in higher dimerization affinity than the CA-domain dimer. Both detailed energetic and conformational analyses of different Gag constructs revealed modulatory contributions to NA-induced dimerization from both matrix and CA domains. We hypothesize that allosterically controlled self-association represents the first step of VLP assembly and, in concert with scaffolding along the NA, can seed the formation of two-dimensional arrays near the NA.

Development and fit-for-purpose verification of an LC-MS method for quantitation of hemagglutinin and neuraminidase proteins in influenza virus-like particle vaccine candidates.

Recombinant influenza Virus-Like Particle (VLP) vaccines are promising vaccine candidates to prevent influenza, contain two major viral antigenic glycoproteins, Hemagglutinin (HA) and Neuraminidase (NA), on the surface of recombinant VLPs. Accurate quantitation of the mass of these antigenic proteins is important to ensure the product quality and proper dosing. Currently, Single Radial Immunodiffusion (SRID) is a recognized assay for determination of the HA immuno-reactive concentration (potency) in vaccine products, based on immuno-reactivity of HA with strain-specific antisera. The SRID assay, however, requires availability of strain-specific and properly calibrated reagents, which can be time-consuming to generate and calibrate. In addition, the assay is not suitable for quantitation of low abundant proteins, such as NA. In order to accelerate the overall production cycle, we have developed and optimized a high-resolution (HR) LC-MS method for absolute quantitation of both HA and NA protein concentrations in influenza VLP vaccine candidates. In this work, we present the method development, optimization and verification of its suitability for the intended purpose, as a prerequisite for its potential application in Quality Control, by assessing specificity, precision and accuracy, detection characteristics, and dynamic linear range. The method can be also used for other HA/NA containing preparations including in-process samples, purified proteins, whole virus preparations, nano-particle and egg-based vaccine preparations, or for calibration of SRID reference antigens.

A novel rapid modularized hepatitis B core virus-like particle-based platform for personalized cancer vaccine preparation via fixed-point coupling.

Personalized cancer vaccine which targets neoepitopes shows great promise for cancer treatment. However, rapid preparation is a critical challenge for clinical application of personalized cancer vaccine. Genetic recombination and chemical modification are a time-consuming "trial and error" pattern for making vaccines. Here we first constructed a platform for peptide vaccine preparation by inserting SpyCatcher into the major immunodominant region (MIR) of hepatitis B core protein (HBc) (1-183). The resulted recombinant protein HBc(1-183)-SpyCatcher (HBc(1-183)-S) assembled to virus-like particles (VLPs) and readily bound to SpyTag conjugated with OVA epitope peptides by just mixing, forming HBc(1-183)-S-OVA. HBc(1-183)-S-OVA VLPs effectively induced dendritic cell maturation. Our further results indicated that HBc(1-183)-S-OVA VLPs vaccination inhibited tumor growth in both prophylactic and treatment ways in E.G7-OVA tumor bearing mice by generating significant OVA-specific cytotoxic T lymphocyte responses. Our study provides a simple, rapid, efficient and universal HBc-based platform for the preparation of personalized cancer vaccine.

A therapeutic HPV16 E7 vaccine in combination with active anti-FGF-2 immunization synergistically elicits robust antitumor immunity in mice.

FGF-2 accumulates in many tumor tissues and is closely related to the development of tumor angiogenesis and the immunosuppressive microenvironment. This study aimed to investigate whether active immunization against FGF-2 could modify antitumor immunity and enhance the efficacy of an HPV16 E7-specific therapeutic vaccine. Combined immunization targeting both FGF-2 and E7 significantly suppressed tumor growth, which was accompanied by significantly increased levels of IFN-γ-expressing splenocytes and effector CD8 T cells and decreased levels of immunosuppressive cells such as regulatory T cells (Tregs) and myeloid-derived suppressor cells(MDSCs) in both the spleen and tumor; in addition, the levels of FGF-2 and neovascularization in tumors were decreased in the mice receiving the combined immunization, and tumor cell apoptosis was promoted. The combination of an HPV16 E7-specific vaccine and active immunization against FGF-2 significantly enhances antitumor immune responses in mice with TC-1 tumors, indicating a promising strategy for tumor immunotherapy.

Induction of protective immune response to intranasal administration of influenza virus-like particles in a mouse model.

Human influenza A viruses (IAVs) cause global pandemics and epidemics, which remains a nonignorable serious concern for public health worldwide. To combat the surge of viral outbreaks, new treatments are urgently needed. Here, we design a new vaccine based on virus-like particles (VLPs) and show how intranasal administration of this vaccine triggers protective immunity, which can be exploited for the development of new therapies. H1N1 VLPs were produced in baculovirus vectors and were injected into BALB/c mice by the intramuscular (IM) or intranasal (IN) route. We found that there were significantly higher inflammatory cell and lymphocyte concentrations in bronchoalveolar lavage samples and the lungs of IN immunized mice; however, the IM group had little signs of inflammatory responses. On the basis of our results, immunization with H1N1 influenza VLP elicited a strong T cell immunity in BALB/c mice. Despite T cell immunity amplification after both IN and IM vaccination methods in mice, IN-induced T cell responses were significantly more intense than IM-induced responses, and this was likely related to an increased number of both CD11bhigh and CD103+ dendritic cells in mice lungs after IN administration of VLP. Furthermore, evaluation of interleukin-4 and interferon gamma cytokines along with several chemokine receptors showed that VLP vaccination via IN and IM routes leads to a greater CD4+ Th1 and Th2 response, respectively. Our findings indicated that VLPs represent a potential strategy for the development of an effective influenza vaccine; however, employing relevant routes for vaccination can be another important part of the universal influenza vaccine puzzle.

Virus-like particles in a new vaccination approach against infectious laryngotracheitis.

Gallid alphaherpesvirus 1 (syn. infectious laryngotracheitis virus; ILTV) is the causative agent of infectious laryngotracheitis, a respiratory disease of chickens causing substantial economic losses in the poultry industry every year. Currently, the most efficient way to achieve protection against infection is immunization with live-attenuated vaccines. However, this vaccination strategy entails the risk of generating new pathogenic viruses resulting from spontaneous mutations or from recombination with field strains. This work presents a new approach based on virus-like particles (VLPs) displaying ILTV glycoproteins B (gB) or G (gG) on their surface. The main focus of this pilot study was to determine the tolerability of VLPs delivered in ovo and intramuscularly (i.m.) into chickens and to investigate the nature of the immune response elicited. The study revealed that the new vaccines were well tolerated in hybrid layer chicks independent of the administration method (in ovo or i.m.). Upon in ovo injection, vaccination with VLP-gG led to an antibody response, while a cellular immune response in VLP-gB-immunized chickens was hardly detectable. Since the administration of VLPs had no visible side effects in vivo and was shown to elicit an antibody-based immune response, we anticipate that VLPs will become a valuable platform for the development of new safe vaccines for poultry.

Prevalence of antibodies against BKPyV subtype I and IV in kidney transplant recipients and in the general Czech population.

Active infection with BK polyomavirus (BKPyV) may cause serious complications in transplantation settings. Recently, the level of BKPyV IgG seroreactivity in graft donors has been shown to predict viremia and BKPyV-associated nephropathy in kidney transplant (KTx) recipients. Pretransplantation testing of the donor and recipient BKPyV serostatus could, therefore, identify patients at high risk. For the development of serological immunoassays, antibody response to the predominant BKPyV subtypes (BKPyV-I and BKPyV-IV) was studied using virus-like particle (VLP)-based enzyme-linked immunosorbent assay (ELISA). VLPs made from the capsid protein, VP1, derived from BKPyV-I and BKPyV-IV subtypes were produced using a baculovirus expression system and used as antigens. The tests were used for IgG antibody determination in 50 KTx recipients and 111 healthy blood donors. While 87% of samples reacted with mixed BKPyV-I and BKPyV-IV antigens, only 49% of samples were reactive in both ELISA tests when using BKPyV-I or BKPyV-IV antigens separately. Twenty-seven percent of healthy blood donors and 26% of KTx recipients were reactive only with BKPyV-I, while 9% and 20% were reactive only with BKPyV-IV, respectively. To determine the specificities of the antigens, selected seropositive samples were retested after preadsorption with soluble BKPyV-I, BKPyV-IV, or JC polyomavirus antigens. The experiments confirmed that recombinant VP1 VLP-based ELISAs predominantly detected BKPyV type-specific antibodies. The results imply that anti-BKPyV antibody ELISA tests should contain a mixture of subtype-specific VLP-based antigens instead of antigen derived from the most prevalent BKPyV-I subtype. The tests can be used for serological surveys of BKPyV infection and improved KTx patient management.

Preparation of virus-like particles for porcine circovirus type 2 by YeastFab Assembly.

Porcine circovirus type 2 (PCV2) poses a genuine threat to pig industry. An effective vaccine production against the pandemic is desirable. The aim of this study was to construct recombination yeast strains with PCV2 Cap protein. We adopt to YeastFab Assembly method to synthesize transcriptional units in a single tube by piecing up promoter, open reading frame, and terminator in S. cerevisiae. Two yeast recombinants were successfully constructed using GPD and TEF2 promoters, respectively, to express PCV2 by secreting Cap protein in vitro. Electronic microscope observation demonstrated that the yeast-derived PCV2 Cap protein could self-assembles into 18-nm-diameter virus-like particles (VLPs). The yield of two different recombination yeasts containing GPD and TEF2 promoters were 12, 25 µg/ml, respectively. Our results showed that it is feasible to use S. cerevisiae as a safe and simple system to produce PCV2 virus-like particles. This indicated that there is possibility of obtaining PCV2 VLP vaccine by homologous recombination in yeast genome, and Cap protein was secreted into the cultural supernatant which can be used as a potential oral vaccine to protect pigs from PCV2-infection.

Production and Immunogenicity of FeLV Gag-Based VLPs Exposing a Stabilized FeLV Envelope Glycoprotein.

The envelope glycoprotein (Env) of retroviruses, such as the Feline leukemia virus (FeLV), is the main target of neutralizing humoral response, and therefore, a promising vaccine candidate, despite its reported poor immunogenicity. The incorporation of mutations that stabilize analogous proteins from other viruses in their prefusion conformation (e.g., HIV Env, SARS-CoV-2 S, or RSV F glycoproteins) has improved their capability to induce neutralizing protective immune responses. Therefore, we have stabilized the FeLV Env protein following a strategy based on the incorporation of a disulfide bond and an Ile/Pro mutation (SOSIP) previously used to generate soluble HIV Env trimers. We have characterized this SOSIP-FeLV Env in its soluble form and as a transmembrane protein present at high density on the surface of FeLV Gag-based VLPs. Furthermore, we have tested its immunogenicity in DNA-immunization assays in C57BL/6 mice. Low anti-FeLV Env responses were detected in SOSIP-FeLV soluble protein-immunized animals; however, unexpectedly no responses were detected in the animals immunized with SOSIP-FeLV Gag-based VLPs. In contrast, high humoral response against FeLV Gag was observed in the animals immunized with control Gag VLPs lacking SOSIP-FeLV Env, while this response was significantly impaired when the VLPs incorporated SOSIP-FeLV Env. Our data suggest that FeLV Env can be stabilized as a soluble protein and can be expressed in high-density VLPs. However, when formulated as a DNA vaccine, SOSIP-FeLV Env remains poorly immunogenic, a limitation that must be overcome to develop an effective FeLV vaccine.

Insect Cell-Based Quadrivalent Seasonal Influenza Virus-like Particles Vaccine Elicits Potent Immune Responses in Mice.

Influenza viruses can cause highly infectious respiratory diseases, posing noteworthy epidemic and pandemic threats. Vaccination is the most cost-effective intervention to prevent influenza and its complications. However, reliance on embryonic chicken eggs for commercial influenza vaccine production presents potential risks, including reductions in efficacy due to HA gene mutations and supply delays due to scalability challenges. Thus, alternative platforms are needed urgently to replace egg-based methods and efficiently meet the increasing demand for vaccines. In this study, we employed a baculovirus expression vector system to engineer HA, NA, and M1 genes from seasonal influenza strains A/H1N1, A/H3N2, B/Yamagata, and B/Victoria, generating virus-like particle (VLP) vaccine antigens, H1N1-VLP, H3N2-VLP, Yamagata-VLP, and Victoria-VLP. We then assessed their functional and antigenic characteristics, including hemagglutination assay, protein composition, morphology, stability, and immunogenicity. We found that recombinant VLPs displayed functional activity, resembling influenza virions in morphology and size while maintaining structural integrity. Comparative immunogenicity assessments in mice showed that our quadrivalent VLPs were consistent in inducing hemagglutination inhibition and neutralizing antibody titers against homologous viruses compared to both commercial recombinant HA and egg-based vaccines (Vaxigrip). The findings highlight insect cell-based VLP vaccines as promising candidates for quadrivalent seasonal influenza vaccines. Further studies are worth conducting.