Chimeric Porcine Parvovirus VP2 Virus-like Particles with Epitopes of South African Serotype 2 Foot-and-Mouth Disease Virus Elicits Specific Humoral and Cellular Responses in Mice.

Southern Africa Territories 2 (SAT2) foot-and-mouth disease (FMD) has crossed long-standing regional boundaries in recent years and entered the Middle East. However, the existing vaccines offer poor cross-protection against the circulating strains in the field. Therefore, there is an urgent need for an alternative design approach for vaccines in anticipation of a pandemic of SAT2 Foot-and-mouth disease virus (FMDV). The porcine parvovirus (PPV) VP2 protein can embed exogenous epitopes into the four loops on its surface, assemble into virus-like particles (VLPs), and induce antibodies and cytokines to PPV and the exogenous epitope. In this study, chimeric porcine parvovirus VP2 VLPs (chimeric PPV-SAT2-VLPs) expressing the T-and/or B-cell epitopes of the structural protein VP1 of FMDV SAT2 were produced using the recombinant pFastBac™ Dual vector of baculoviruses in Sf9 and HF cells We used the Bac-to-Bac system to construct the recombinant baculoviruses. The VP2-VLP--SAT2 chimeras displayed chimeric T-cell epitope (amino acids 21-40 of VP1) and/or the B-cell epitope (amino acids 135-174) of SAT FMDV VP1 by substitution of the corresponding regions at the N terminus (amino acids 2-23) and/or loop 2 and/or loop 4 of the PPV VP2 protein, respectively. In mice, the chimeric PPV-SAT2-VLPs induced specific antibodies against PPV and the VP1 protein of SAT2 FMDV. The VP2-VLP-SAT2 chimeras induced specific antibodies to PPV and the VP1 protein specific epitopes of FMDV SAT2. In this study, as a proof-of-concept, successfully generated chimeric PPV-VP2 VLPs expressing epitopes of the structural protein VP1 of FMDV SAT2 that has a potential to prevent FMDV SAT2 and PPV infection in pigs.

Optimized production of full-length PCV2d virus-like particles in Escherichia coli: A cost-effective and high-yield approach for potential vaccine antigen development.

Porcine circovirus type 2 (PCV2) is a globally prevalent infectious pathogen affecting swine, with its capsid protein (Cap) being the sole structural protein critical for vaccine development. Prior research has demonstrated that PCV2 Cap proteins produced in Escherichia coli (E. coli) can form virus-like particles (VLPs) in vitro, and nuclear localization signal peptides (NLS) play a pivotal role in stabilizing PCV2 VLPs. Recently, PCV2d has emerged as an important strain within the PCV2 epidemic. In this study, we systematically optimized the PCV2d Cap protein and successfully produced intact PCV2d VLPs containing NLS using E. coli. The recombinant PCV2d Cap protein was purified through affinity chromatography, yielding 7.5 mg of recombinant protein per 100 ml of bacterial culture. We augmented the conventional buffer system with various substances such as arginine, ß-mercaptoethanol, glycerol, polyethylene glycol, and glutathione to promote VLP assembly. The recombinant PCV2d Cap self-assembled into VLPs approximately 20 nm in diameter, featuring uniform distribution and exceptional stability in the optimized buffer. We developed the vaccine and immunized pigs and mice, evaluating the immunogenicity of the PCV2d VLPs vaccine by measuring PCV2-IgG, IL-4, TNF-α, and IFN-γ levels, comparing them to commercial vaccines utilizing truncated PCV2 Cap antigens. The HE staining and immunohistochemical tests confirmed that the PCV2 VLPs vaccine offered robust protection. The results revealed that animals vaccinated with the PCV2d VLPs vaccine exhibited high levels of PCV2 antibodies, with TNF-α and IFN-γ levels rapidly increasing at 14 days post-immunization, which were higher than those observed in commercially available vaccines, particularly in the mouse trial. This could be due to the fact that full-length Cap proteins can assemble into more stable PCV2d VLPs in the assembling buffer. In conclusion, our produced PCV2d VLPs vaccine elicited stronger immune responses in pigs and mice compared to commercial vaccines. The PCV2d VLPs from this study serve as an excellent candidate vaccine antigen, providing insights for PCV2d vaccine research.

In situ bio-mineralized Mn nanoadjuvant enhances anti-influenza immunity of recombinant virus-like particle vaccines.

Virus like particles (VLPs) have been well recognized as one of the most important vaccine platforms due to their structural similarity to natural viruses to induce effective humoral and cellular immune responses. Nevertheless, lack of viral nucleic acids in VLPs usually leads the vaccine candidates less efficient in provoking innate immune against viral infection. Here, we constructed a biomimetic dual antigen hybrid influenza nanovaccines THM-HA@Mn with robust immunogenicity via in situ synthesizing a stimulator of interferon genes (STING) agonist Mn3O4 inside the cavity of a recombinant Hepatitis B core antigen VLP (HBc VLP) having fused SpyTag and influenza M2e antigen peptides (Tag-HBc-M2e, THM for short), followed by conjugating a recombinant hemagglutinin (rHA) antigen on the surface of the nanoparticles through SpyTag/SpyCatcher ligating. Such inside Mn3O4 immunostimulator-outside rHA antigen design, together with the chimeric M2e antigen on the HBc skeleton, enabled the synthesized hybrid nanovaccines THM-HA@Mn to well imitate the spatial distribution of M2e/HA antigens and immunostimulant in natural influenza virus. In vitro cellular experiments indicated that compared with the THM-HA antigen without Mn3O4 and a mixture vaccine consisting of THM-HA + MnOx, the THM-HA@Mn hybrid nanovaccines showed the highest efficacies in dendritic cells uptake and in promoting BMDC maturation, as well as inducing expression of TNF-α and type I interferon IFN-ß. The THM-HA@Mn also displayed the most sustained antigen release at the injection site, the highest efficacies in promoting the DC maturation in lymph nodes and germinal center B cells activation in the spleen of the immunized mice. The co-delivery of immunostimulant and antigens enabled the THM-HA@Mn nanovaccines to induce the highest systemic antigen-specific antibody responses and cellular immunogenicity in mice. Together with the excellent colloid dispersion stability, low cytotoxicity, as well as good biosafety, the synthetic hybrid nanovaccines presented in this study offers a promising strategy to design VLP-based vaccine with robust natural and adaptive immunogenicity against emerging viral pathogens.

Enhancing antibody responses by multivalent antigen display on thymus-independent DNA origami scaffolds.

Protein-based virus-like particles (P-VLPs) are commonly used to spatially organize antigens and enhance humoral immunity through multivalent antigen display. However, P-VLPs are thymus-dependent antigens that are themselves immunogenic and can induce B cell responses that may neutralize the platform. Here, we investigate thymus-independent DNA origami as an alternative material for multivalent antigen display using the receptor binding domain (RBD) of the SARS-CoV-2 spike protein, the primary target of neutralizing antibody responses. Sequential immunization of mice with DNA-based VLPs (DNA-VLPs) elicits protective neutralizing antibodies to SARS-CoV-2 in a manner that depends on the valency of the antigen displayed and on T cell help. Importantly, the immune sera do not contain boosted, class-switched antibodies against the DNA scaffold, in contrast to P-VLPs that elicit strong B cell memory against both the target antigen and the scaffold. Thus, DNA-VLPs enhance target antigen immunogenicity without generating scaffold-directed immunity and thereby offer an important alternative material for particulate vaccine design.

Surface Cross-Linking by Macromolecular Tethers Enhances Virus-like Particles' Resilience to Mucosal Stress Factors.

Virus-like particles (VLPs) are emerging as nanoscaffolds in a variety of biomedical applications including delivery of vaccine antigens and cargo such as mRNA to mucosal surfaces. These soft, colloidal, and proteinaceous structures (capsids) are nevertheless susceptible to mucosal environmental stress factors. We cross-linked multiple capsid surface amino acid residues using homobifunctional polyethylene glycol tethers to improve the persistence and survival of the capsid to model mucosal stressors. Surface cross-linking enhanced the stability of VLPs assembled from Acinetobacter phage AP205 coat proteins in low pH (down to pH 4.0) and high protease concentration conditions (namely, in pig and mouse gastric fluids). Additionally, it increased the stiffness of VLPs under local mechanical indentation applied using an atomic force microscopy cantilever tip. Small angle X-ray scattering revealed an increase in capsid diameter after cross-linking and an increase in capsid shell thickness with the length of the PEG cross-linkers. Moreover, surface cross-linking had no effect on the VLPs' mucus translocation and accumulation on the epithelium of in vitro 3D human nasal epithelial tissues with mucociliary clearance. Finally, it did not compromise VLPs' function as vaccines in mouse subcutaneous vaccination models. Compared to PEGylation without cross-linking, the stiffness of surface cross-linked VLPs were higher for the same length of the PEG molecule, and also the lifetimes of surface cross-linked VLPs were longer in the gastric fluids. Surface cross-linking using macromolecular tethers, but not simple conjugation of these molecules, thus offers a viable means to enhance the resilience and survival of VLPs for mucosal applications.

Bac to Bac System Efficiency for Preparing HPV Type 16 Virus-Like Particle Vaccine.

Today, the human papillomavirus (HPV) L1 protein is the main target in the construction of prophylactic HPV vaccines. The production of virus-like particles (VLPs) that closely resemble the natural structure of the HPV16 virus and induce high levels of virus-neutralizing antibodies in animals and humans is facilitated by the expression of HPV16-L1 protein in eukaryotic cells. The Bac-to-Bac system has been previously used to produce high levels of recombinant proteins. In this study, we utilized this expression system to generate HPV16-L1 VLPs in Spodoptra frugipedra (Sf9) insect cells. The wild-type L1 gene of papillomavirus type 16 was selected from Gene Bank and placed in bacmid structure after codon optimization using pFast Bac vector. The recombinant baculovirus containing HPV-16/L1 gene was then provided using the Bac-to-Bac system. It should be mentioned that the vector was transfected into the Sf9 cell. The cells were then lysed and the expression of L1 protein was revealed by SDS-PAGE and confirmed by Western Blot. The L1 purification was performed through Ni-NTA chromatography. The VLP formation of papillomavirus L1 protein was visualized by transmission electron microscopy. The expressed recombinant L1 was ~60 KD on SDS-PAGE which was identified in western blot by a specific anti-L1 monoclonal antibody. The electron microscopy confirmed the assembly of VLPs. Results of this study showed that the production of this protein at the industrial level can be optimized using a baculovirus/Sf9 system. The characteristics and advantages of this system are promising and it is a suitable candidate for protein synthesis.

Development of a Tag/Catcher-mediated capsid virus-like particle vaccine presenting the conserved Neisseria gonorrhoeae SliC antigen that blocks human lysozyme.

Virus-like particles (VLPs) are promising nanotools for the development of subunit vaccines due to high immunogenicity and safety. Herein, we explored the versatile and effective Tag/Catcher-AP205 capsid VLP (cVLP) vaccine platform to address the urgent need for the development of an effective and safe vaccine against gonorrhea. The benefits of this clinically validated cVLP platform include its ability to facilitate unidirectional, high-density display of complex/full-length antigens through an effective split-protein Tag/Catcher conjugation system. To assess this modular approach for making cVLP vaccines, we used a conserved surface lipoprotein, SliC, that contributes to the Neisseria gonorrhoeae defense against human lysozyme, as a model antigen. This protein was genetically fused at the N- or C-terminus to the small peptide Tag enabling their conjugation to AP205 cVLP, displaying the complementary Catcher. We determined that SliC with the N-terminal SpyTag, N-SliC, retained lysozyme-blocking activity and could be displayed at high density on cVLPs without causing aggregation. In mice, the N-SliC-VLP vaccines, adjuvanted with AddaVax or CpG, induced significantly higher antibody titers compared to controls. In contrast, similar vaccine formulations containing monomeric SliC were non-immunogenic. Accordingly, sera from N-SliC-VLP-immunized mice also had significantly higher human complement-dependent serum bactericidal activity. Furthermore, the N-SliC-VLP vaccines administered subcutaneously with an intranasal boost elicited systemic and vaginal IgG and IgA, whereas subcutaneous delivery alone failed to induce vaginal IgA. The N-SliC-VLP with CpG (10 µg/dose) induced the most significant increase in total serum IgG and IgG3 titers, vaginal IgG and IgA, and bactericidal antibodies.

Self-Assembled Virus-Like Particle Vaccines via Fluorophilic Interactions Enable Infection Mimicry and Immune Protection.

Influenza epidemics persistently threaten global health. Vaccines based on virus-like particles (VLPs), which resemble the native conformation of viruses, have emerged as vaccine candidates. However, the production of VLPs via genetic engineering remains constrained by challenges such as low yields, high costs, and being time consuming. In this study, a novel VLP platform is developed that could mimic infection and confer influenza protection through fluorination-driven self-assembly. The VLPs closely mimick the key steps in viral infection including dendritic cell (DC) attachment and pH-responsive endo-lysosomal escape, which enhances DC maturation and antigen cross-presentation. It is also observed that the VLPs migrate from the injection site to the draining lymph nodes efficiently. Immunization with VLPs triggers both Th1 and Th2 cellular responses, thereby inducing an improved CD8+ T cell response along with strong antigen-specific antibody responses. In several infected mouse models, VLP vaccines ameliorate weight loss, lung virus titers, pulmonary pathologies, and confer full protection against H1N1, H6N2, H9N2, and mixed influenza viruses. Therefore, the results support the potential of VLPs as an effective influenza vaccine with improved immune potency against infection. A methodology to generate VLPs based on fluorophilic interactions, which can be a general approach for development of pathogenic VLPs, is reported.

Immunogenicity and protective efficacy of human metapneumovirus virus-like particles produced by a recombinant baculovirus in mice.

BACKGROUND: Human metapneumovirus (HMPV) causes respiratory tract infections among infant, elderly, and immunocompromised patients, with significant mortality. Currently no licensed vaccines or therapeutic agents of HMPV exist. METHODS: HMPV virus-like particle (VLP) was constructed by co-expressing fusion protein of HMPV and matrix 1 protein of influenza virus using the baculovirus expression. Mice were immunized with VLP with or without aluminum hydroxide (alum) adjuvant by intramuscular route respectively. Sera were determined for titers of IgG and neutralizing antibody. Splenic lymphocytes were determined by IFN-γ and IL-4 ELISPOT. Mice were challenged with HMPV, and protective efficacy was evaluated. RESULTS: We generated HMPV VLP in baculovirus expression system. After three times immunization, IgG antibody titers induced by VLP formulated with or without alum adjuvant group were 273,066 ± 100,331 and 136,533 ± 47,269 respectively, there was no difference (p Ëƒ 0.05); the neutralizing antibody titers vaccinated with VLP plus with alum adjuvant (266 ± 92) were higher than those of the VLP alone group (106 ± 37). For IFN-γ, mice vaccinated with VLP with or without alum adjuvant are 151 ± 36.4 and 77.0 ± 17.1SFC/106 respectively, there was difference (p = 0.03); For IL-4, they are 261.3 ± 38.7 versus 125.67 ± 29.78SFC/106 respectively, the difference was significant (p = 0.009). After challenge, in pathological analysis, the overall lesion scores in the VLP plus with and without alum adjuvant were 3.25 and 5.6 respectively, those of control group is 8. For immunohistochemical analyses, the average optical density of the lungs in the VLP immunized group containing adjuvant (9.07 ± 1.74) was lower than that in the VLP group without adjuvant (12.83 ± 2.31, p = 0.14). CONCLUSIONS: This is the first study to demonstrate that HMPV VLP was successfully prepared in the baculovirus expression system. HMPV VLP could induce specific humoral and cellular immune responses as well as protective efficacy, and aluminum hydroxide may be an effective adjuvant in mice.

SpyTag/SpyCatcher display of influenza M2e peptide on norovirus-like particle provides stronger immunization than direct genetic fusion.

Introduction: Virus-like particles (VLPs) are similar in size and shape to their respective viruses, but free of viral genetic material. This makes VLP-based vaccines incapable of causing infection, but still effective in mounting immune responses. Noro-VLPs consist of 180 copies of the VP1 capsid protein. The particle tolerates C-terminal fusion partners, and VP1 fused with a C-terminal SpyTag self-assembles into a VLP with SpyTag protruding from its surface, enabling conjugation of antigens via SpyCatcher. Methods: To compare SpyCatcher-mediated coupling and direct peptide fusion in experimental vaccination, we genetically fused the ectodomain of influenza matrix-2 protein (M2e) directly on the C-terminus of norovirus VP1 capsid protein. VLPs decorated with SpyCatcher-M2e and VLPs with direct M2 efusion were used to immunize mice. Results and discussion: We found that direct genetic fusion of M2e on noro-VLP raised few M2e antibodies in the mouse model, presumably because the short linker positions the peptide between the protruding domains of noro-VLP, limiting its accessibility. On the other hand, adding aluminum hydroxide adjuvant to the previously described SpyCatcher-M2e-decorated noro-VLP vaccine gave a strong response against M2e. Surprisingly, simple SpyCatcher-fused M2e without VLP display also functioned as a potent immunogen, which suggests that the commonly used protein linker SpyCatcher-SpyTag may serve a second role as an activator of the immune system in vaccine preparations. Based on the measured anti-M2e antibodies and cellular responses, both SpyCatcher-M2e as well as M2e presented on the noro-VLP via SpyTag/Catcher show potential for the development of universal influenza vaccines.

Chimaeric plant-produced bluetongue virus particles as potential vaccine candidates.

Bluetongue virus (BTV) causes bluetongue disease in ruminants and sheep. The current live attenuated and inactivated vaccines available for prevention pose several risks, and there is thus a need for vaccines that are safer, economically viable, and effective against multiple circulating serotypes. This work describes the development of recombinant virus-like particle (VLP) vaccine candidates in plants, which are assembled by co-expression of the four BTV serotype 8 major structural proteins. We show that substitution of a neutralising tip domain of BTV8 VP2 with that of BTV1 VP2 resulted in the assembly of VLPs that stimulated serotype-specific antibodies as well as virus-specific neutralising antibodies.

Development of a novel virus-like particle-based vaccine for preventing tick-borne encephalitis virus infection.

Tick-borne encephalitis virus (TBEV) is an important tick-borne pathogen that poses as a serious public health concern. The coverage and immunogenicity of the currently available vaccines against TBEV are relatively low; therefore, it is crucial to develop novel and effective vaccines against TBEV. The present study describes a novel strategy for the assembly of virus-like particles (VLPs) by co-expressing the structural (core/prM/E) and non-structural (NS2B/NS3Pro) proteins of TBEV. The efficacy of the VLPs was subsequently evaluated in C57BL/6 mice, and the resultant IgG serum could neutralize both Far-Eastern and European subtypes of TBEV. These findings indicated that the VLP-based vaccine elicited the production of cross-subtype reactive antibodies. The VLPs provided protection to mice lacking the type I interferon receptor (IFNAR-/-) against lethal TBEV challenge, with undetectable viral load in brain and intestinal tissues. Furthermore, the group that received the VLP vaccine did not exhibit significant pathological changes and the inflammatory factors were significantly suppressed compared to the control group. Immunization with the VLP vaccine induced the production of multiple-cytokine-producing antiviral CD4+ T cells in vivo, including TNF-α+, IL-2+, and IFN-γ+ T cells. Altogether, the findings suggest that noninfectious VLPs can serve as a potentially safe and effective vaccine candidate against diverse subtypes of TBEV.

The position of Spy Tag/Catcher system in hepatitis B core protein particles affects the immunogenicity and stability of the synthetic vaccine.

Presenting exogenous antigens on virus-like particles (VLPs) through "plug-and-display" decoration strategies based on SpyTag/SpyCatcher isopeptide bonding have emerged as attractive technology for vaccine synthesis. However, whether the position of ligation site in VLPs will impose effects on immunogenicity and physiochemical properties of the synthetic vaccine remains rarely investigated. Here in the present work, the well-established hepatitis B core (HBc) protein was used as chassis to construct dual-antigen influenza nanovaccines, with the conserved epitope peptides derived from extracellular domain of matrix protein M2 (M2e) and hemagglutinin (HA) as target antigens. The M2e antigen was genetically fused to the HBc in the MIR region, together with the SpyTag peptide, which was fused either in the MIR region or at the N-terminal of the protein, so that a recombinant HA antigen (rHA) linked to SpyCatcher can be displayed on it, at two different localizations. Both synthetic nanovaccines showed ability in inducing strong M2e and rHA-specific antibodies and cellular immunogenicity; nevertheless, the one in which rHA was conjugated by N-terminal Tag ligation, was superior to another one synthesized by linking the rHA to MIR region SpyTagged-HBc in all aspects, including higher antigen-specific immunogenicity responses, lower anti-HBc carrier antibody, as well as better dispersion stability. Surface charge and hydrophobicity properties of the two synthetic nanovaccines were analyzed, results revealed that linking the rHA to MIR region SpyTagged-HBc lead to more significant and disadvantageous alteration in physiochemical properties of the HBc chassis. This study will expand our knowledge on "plug-and-display" decoration strategies and provide helpful guidance for the rational design of HBc-VLPs based modular vaccines by using SpyTag/Catcher synthesis.

Downstream process design for Gag HIV-1 based virus-like particles.

Virus-like particles-based vaccines have been gaining interest in recent years. The manufacturing of these particles includes their production by cell culture followed by their purification to meet the requirements of its final use. The presence of host cell extracellular vesicles represents a challenge for better virus-like particles purification, because both share similar characteristics which hinders their separation. The present study aims to compare some of the most used downstream processing technologies for capture and purification of virus-like particles. Four steps of the purification process were studied, including a clarification step by depth filtration and filtration, an intermediate step by tangential flow filtration or multimodal chromatography, a capture step by ion exchange, heparin affinity and hydrophobic interaction chromatography and finally, a polishing step by size exclusion chromatography. In each step, the yields were evaluated by percentage of recovery of the particles of interest, purity, and elimination of main contaminants. Finally, a complete purification train was implemented using the best results obtained in each step. A final concentration of 1.40 × 1010 virus-like particles (VLPs)/mL with a purity of 64% after the polishing step was achieved, with host cell DNA and protein levels complaining with regulatory standards, and an overall recovery of 38%. This work has resulted in the development of a purification process for HIV-1 Gag-eGFP virus-like particles suitable for scale-up.

Construction, expression and assemble of EMCV VLPs and their potency evaluation.

Encephalomycarditis virus (EMCV) is an essential pathogen with a broad host range and causes enormous economic losses to the pig industry worldwide. Here, we constructed and assembled the EMCV virus-like particles (VLPs) in vitro and verified high efficiency of virus protection. Results showed that the proteins auto-assembled into VLPs successfully in vitro. The animal experiments revealed that high-titer antibody production is triggered by VLPs. Meanwhile, the mice challenged with EMCV were obviously protected. The protection rate of group VLPs with the adjuvant was 75%, while that of the VLPs group was 62.5% compared to the control. These findings indicate that recombinant EMCV VLPs have a remarkable anti-EMCV effect and could be a new vaccine candidate for the control of EMCV infection.

Advancing usability of an influenza hemagglutinin virus-like particle vaccine expressing a chimeric cytokine.

Vaccine efficacy of conventional influenza vaccines depend on the antigenic similarity between the selected vaccine strain and annual epidemic strain. Since the influenza virus evolves yearly, a vaccine which is independent from viral antigenic mutation is desired. We have developed chimeric cytokine (CC) and hemagglutinin (HA) incorporated virus-like particle (CCHA-VLP) as a universal influenza vaccine candidate. Using mouse models, it was shown that the vaccine provided broad-based protective activity against several types of human and avian influenza A viruses. In this report, nasal immunization and mixture form (CC- and HA-VLP) were tested to improve usability of this vaccine. Immunogenicity was evaluated by induction of IgG, IgA, and IFN-γ secreting cells. Protective activity was measured as mouse survival rate against lethal challenge with H1N1 and H5N1 viruses and against H3N2 virus by lung viral titer. Nasal immunization showed low immunogenicity and low protective efficacy, but the addition of a sesame oil adjuvant improved vaccine efficacy. Mixture form of CC- and HA-VLP showed comparable or higher vaccine efficacy when compared to the incorporated form, CCHA-VLP. These results contribute to improved usability, such as needle-less administration and easy HA subtypes alteration.

Site-Specific Modification of Virus-Like Particles for Exogenous Tumor Antigen Display and Minimizing Preexisting Immunity.

The widespread preexisting immunity against virus-like particles (VLPs) seriously limits the applications of VLPs as vaccine vectors. Enabling technology for exogenous antigen display should not only ensure the assembly ability of VLPs and site-specific modification, but also consider the effect of preexisting immunity on the behavior of VLPs in vivo. Here, combining genetic code expansion technique and synthetic biology strategy, a site-specific modification method for hepatitis B core (HBc) VLPs via incorporating azido-phenylalanine into the desired positions is described. Through modification position screening, it is found that HBc VLPs incorporated with azido-phenylalanine at the main immune region can effectively assemble and rapidly conjugate with the dibenzocycolctyne-modified tumor-associated antigens, mucin-1 (MUC1). The site-specific modification of HBc VLPs not only improves the immunogenicity of MUC1 antigens but also shields the immunogenicity of HBc VLPs themselves, thereby activating a strong and persistent anti-MUC1 immune response even in the presence of preexisting anti-HBc immunity, which results in the efficient tumor elimination in a lung metastatic mouse model. Together, these results demonstrate the site-specific modification strategy enabled HBc VLPs behave as a potent antitumor vaccine and this strategy to manipulate immunogenicity of VLPs may be suitable for other VLP-based vaccine vectors.

Comparative Efficacy of Mayaro Virus-Like Particle Vaccines Produced in Insect or Mammalian Cells.

Mayaro virus (MAYV) is a mosquito-transmitted alphavirus that causes often debilitating rheumatic disease in tropical Central and South America. There are currently no licensed vaccines or antiviral drugs available for MAYV disease. Here, we generated Mayaro virus-like particles (VLPs) using the scalable baculovirus-insect cell expression system. High-level secretion of MAYV VLPs in the culture fluid of Sf9 insect cells was achieved, and particles with a diameter of 64 to 70 nm were obtained after purification. We characterize a C57BL/6J adult wild-type mouse model of MAYV infection and disease and used this model to compare the immunogenicity of VLPs from insect cells with that of VLPs produced in mammalian cells. Mice received two intramuscular immunizations with 1 µg of nonadjuvanted MAYV VLPs. Potent neutralizing antibody responses were generated against the vaccine strain, BeH407, with comparable activity seen against a contemporary 2018 isolate from Brazil (BR-18), whereas neutralizing activity against chikungunya virus was marginal. Sequencing of BR-18 illustrated that this virus segregates with genotype D isolates, whereas MAYV BeH407 belongs to genotype L. The mammalian cell-derived VLPs induced higher mean neutralizing antibody titers than those produced in insect cells. Both VLP vaccines completely protected adult wild-type mice against viremia, myositis, tendonitis, and joint inflammation after MAYV challenge. IMPORTANCE Mayaro virus (MAYV) is associated with acute rheumatic disease that can be debilitating and can evolve into months of chronic arthralgia. MAYV is believed to have the potential to emerge as a tropical public health threat, especially if it develops the ability to be efficiently transmitted by urban mosquito vectors, such as Aedes aegypti and/or Aedes albopictus. Here, we describe a scalable virus-like particle vaccine against MAYV that induced neutralizing antibodies against a historical and a contemporary isolate of MAYV and protected mice against infection and disease, providing a potential new intervention for MAYV epidemic preparedness.

Immunogenicity of Wild Type and Mutant Hepatitis B Surface Antigen Virus-like Particles (VLPs) in Mice with Pre-Existing Immunity against the Wild Type Vector.

Virus-like particles (VLPs), composed of the small hepatitis B virus surface antigen (HBsAgS), are the antigenic components of the hepatitis B virus (HBV) vaccine and represent the backbones for a chimeric anti-malaria vaccine and various vaccine candidates. Biological vectors have to face pre-existing anti-vector immune responses due to previous immune exposure. Vector recognition after natural infections or vaccinations can result in unwarranted outcomes, with compromising effects on clinical outcomes. In order to evaluate the impact of a pre-existing anti-HBsAgS immune response, we developed mutant VLPs composed of subunits with reduced HBsAgS-specific antigenicity. The insertion of a Plasmodium falciparum circumsporozoite protein (CSP)-derived epitope as a read-out allowed the assessment of wild type (wt) and mutant VLPs in the context of a pre-existing immune response. Mutant and wt VLP platforms with a CSP-epitope insert are immunogenic and have the ability to generate anti-CSP antibody responses in both naïve BALB/c mice and mice with a pre-existing anti-HBsAgS immune response, but with superior anti-CSP responses in mice with a pre-existing immunity. The data indicate that previous HBsAgS exposure facilitates enhanced antibody responses against foreign epitopes delivered by the HBsAgS platform, and, in this context, the state of immune sensitization alters the outcome of subsequent vaccinations.

Systematic engineering of virus-like particles to identify self-assembly rules for shifting particle size.

Virus-like particles (VLPs) are promising scaffolds for biomaterials as well as diagnostic and therapeutic applications. However, there are some key challenges to be solved, such as the ability to engineer alternate sizes for varied use cases. To this end, we created a library of MS2 VLP variants at two key residues in the coat protein which have been implicated as important to controlling VLP size and geometry. By adapting a method for systematic mutagenesis coupled with size-based selections and high-throughput sequencing as a readout, we developed a quantitative assessment of two residues in MS2 coat protein that govern the size shift in MS2 VLPs. We then applied the strategy to the equivalent residues in Qß VLPs, an MS2 homolog, and demonstrate that the analogous pair of residues are also able to impact Qß VLP size and shape. These results underscore the power of fitness landscapes in identifying critical features for assembly.