Self-assembled Virus-like Particles Research Articles

Recombinantly expressed virus-like particles (VLPs) of canine circovirus for development of an indirect ELISA.

Canine circovirus (CanineCV) is an emerging pathogen in domestic dogs, detected in multiple countries in association with varying clinical and pathological presentations including diarrhoea, vasculitis, granulomatous inflammation, and respiratory signs. Understanding the pathology of CanineCV is confounded by the fact that it has been detected in asymptomatic dogs as well as in diseased dogs concurrently infected with known pathogens. Recombinantly expressed self-assembling Virus-like particles (VLPs) lack viral genomic material but imitate the capsid surface conformations of wild type virion, allowing arrays of biological applications including subunit vaccine development and immunodiagnostics. In this study, full length CanineCV capsid gene was expressed in Escherichia coli followed by two-step purification process to yield soluble capsid protein in high concentration. Transmission electron microscopy (TEM) confirmed the capsid antigen self-assembled into 17-20nm VLPs in glutathione S-transferase (GST) buffer, later utilised to develop an indirect enzyme-linked immunosorbent assay (iELISA). The respective sensitivity and specificity of the proposed iELISA were 94.10% and 88.40% compared with those obtained from Western blot. The mean OD450 value for western blot positive samples was 1.22 (range 0.12-3.39) and negative samples was 0.21 (range 0.07-0.41). An optimal OD450 cut-off of 0.35 was determined by ROC curve analysis. Median inter-assay and intra-assay validation revealed that the iELISA test results were reproducible with coefficients of variation 7.70 (range 5.6-11.9) and 4.21 (range 1.2-7.4). Our results demonstrated that VLP-based iELISA is a highly sensitive method for serological diagnosis of CanineCV infections in dogs, suitable for large-scale epidemiological studies.

Nanoparticles and Antiviral Vaccines.

Viruses have threatened human lives for decades, causing both chronic and acute infections accompanied by mild to severe symptoms. During the long journey of confrontation, humans have developed intricate immune systems to combat viral infections. In parallel, vaccines are invented and administrated to induce strong protective immunity while generating few adverse effects. With advancements in biochemistry and biophysics, different kinds of vaccines in versatile forms have been utilized to prevent virus infections, although the safety and effectiveness of these vaccines are diverse from each other. In this review, we first listed and described major pathogenic viruses and their pandemics that emerged in the past two centuries. Furthermore, we summarized the distinctive characteristics of different antiviral vaccines and adjuvants. Subsequently, in the main body, we reviewed recent advances of nanoparticles in the development of next-generation vaccines against influenza viruses, coronaviruses, HIV, hepatitis viruses, and many others. Specifically, we described applications of self-assembling protein polymers, virus-like particles, nano-carriers, and nano-adjuvants in antiviral vaccines. We also discussed the therapeutic potential of nanoparticles in developing safe and effective mucosal vaccines. Nanoparticle techniques could be promising platforms for developing broad-spectrum, preventive, or therapeutic antiviral vaccines.

Proof of concept for a single-dose Group B Streptococcus vaccine based on capsular polysaccharide conjugated to Qβ virus-like particles

A maternal vaccine to protect neonates against Group B Streptococcus invasive infection is an unmet medical need. Such a vaccine should ideally be offered during the third trimester of pregnancy and induce strong immune responses after a single dose to maximize the time for placental transfer of protective antibodies. A key target antigen is the capsular polysaccharide, an anti-phagocytic virulence factor that elicits protective antibodies when conjugated to carrier proteins. The most prevalent polysaccharide serotypes conjugated to tetanus or diphtheria toxoids have been tested in humans as monovalent and multivalent formulations, showing excellent safety profiles and immunogenicity. However, responses were suboptimal in unprimed individuals after a single shot, the ideal schedule for vaccination during the third trimester of pregnancy. In the present study, we obtained and optimized self-assembling virus-like particles conjugated to Group B Streptococcus capsular polysaccharides. The resulting glyco-nanoparticles elicited strong immune responses in mice already after one immunization, providing pre-clinical proof of concept for a single-dose vaccine.

Exploring the Landscape of the PP7 Virus-like Particle for Peptide Display.

Self-assembling virus-like particles (VLPs) can tolerate a wide degree of genetic and chemical manipulation to their capsid protein to display a foreign molecule polyvalently. We previously reported the successful incorporation of foreign peptide sequences in the junction loop and onto the C-terminus of PP7 dimer VLPs, as these regions are accessible for surface display on assembled capsids. Here, we report the implementation of a library-based approach to test the assembly tolerance of PP7 dimer capsid proteins to insertions or terminal extensions of randomized 15-mer peptide sequences. By performing two iterative rounds of assembly-based selection, we evaluated the degree of favorability of all 20 amino acids at each of the 15 randomized positions. Deep sequencing analysis revealed a distinct preference for the inclusion of hydrophilic peptides and negatively charged amino acids (Asp and Glu) and the exclusion of positively charged peptides and bulky and hydrophobic amino acid residues (Trp, Phe, Tyr, and Cys). Within the libraries tested here, we identified 4000 to 22,000 unique 15-mer peptide sequences that can successfully be displayed on the surface of the PP7 dimer capsid. Overall, the use of small initial libraries consisting of no more than a few million members yielded a significantly larger number of unique and assembly-competent VLP sequences than have been previously characterized for this class of nucleoprotein particle.

ESCRT recruitment to SARS-CoV-2 spike induces virus-like particles that improve mRNA vaccines

Prime-boost regimens for COVID-19 vaccines elicit poor antibody responses against Omicron-based variants and employ frequent boosters to maintain antibody levels. We present a natural infection-mimicking technology that combines features of mRNA- and proteinnanoparticle-based vaccines through encoding self-assembling enveloped virus-like particles (eVLPs). eVLP assembly is achieved by inserting an ESCRT- and ALIX-binding region (EABR) into the SARS-CoV-2 spike cytoplasmic tail, which recruits ESCRT proteins to induce eVLP budding from cells. Purified spike-EABR eVLPs presented densely arrayed spikes and elicited potent antibody responses in mice. Two immunizations with mRNA-LNP encoding spike-EABR elicited potent CD8+ Tcell responses and superior neutralizing antibody responses against original and variant SARS-CoV-2 compared with conventional spike-encoding mRNA-LNP and purified spike-EABR eVLPs, improving neutralizing titers >10-fold against Omicron-based variants for 3months post-boost. Thus, EABR technology enhances potency and breadth of vaccine-induced responses through antigen presentation on cell surfaces and eVLPs, enabling longer-lasting protection against SARS-CoV-2 and other viruses.

Two-component vaccine consisting of virus-like particles displaying hepatitis C virus envelope protein 2 oligomers

Development of B-cell-based hepatitis C virus (HCV) vaccines that induce broadly neutralizing antibodies (bNAbs) is hindered by extensive sequence diversity and low immunogenicity of envelope glycoprotein vaccine candidates, most notably soluble E2 (sE2). To overcome this, we employed two-component approaches using self-assembling virus-like particles (cVLPs; component 1), displaying monomeric or oligomeric forms of HCV sE2 (sE2mono or sE2oligo; component 2). Immunization studies were performed in BALB/c mice and the neutralizing capacity of vaccine-induced antibodies was tested in cultured-virus-neutralizations, using HCV of genotypes 1–6. sE2-cVLP vaccines induced significantly higher levels of NAbs (p = 0.0065) compared to corresponding sE2 vaccines. Additionally, sE2oligo-cVLP was superior to sE2mono-cVLP in inducing bNAbs. Interestingly, human monoclonal antibody AR2A had reduced binding in ELISA to sE2oligo-cVLP compared with sE2mono-cVLP and competition ELISA using mouse sera from vaccinated animals indicated that sE2oligo-cVLP induced significantly less non-bNAbs AR2A (p = 0.0043) and AR1B (p = 0.017). Thus, cVLP-displayed oligomeric sE2 shows promise as an HCV vaccine candidate.

Intracellular Delivery of Micron-Sized Magnetic Particles through a Virus Infection Pathway.

Micron-sized magnetic particles (M-MPs) have low toxicity, strong magnetic signals, and long-term retention capability, which are significant advantages for their application in biomedical imaging. Unfortunately, M-MPs are only internalized by few cell types, such as macrophages and phagocytes, and because of this lack of active intracellular delivery, their applications are restricted. The emergence of self-assembled virus-like particles (VLPs) offers a viable approach to drive M-MPs into cells, although the specific mechanism has not been revealed. In this study, we investigated in detail the intracellular pathway of M-MPs mediated by VLPs using a fluorescence co-localization method. The results indicated that the intracellular movement of M-MPs was consistent with the virus infection pathway, specifically caveolae-dependent endocytosis, transportation through microtubules, and accumulation in the endoplasmic reticulum. This study provides experimental support for the active transport of M-MPs into other cell types, thereby further extending their applications.

Development of a synthetic nanoparticle vaccine presenting the HIV-1 envelope glycoprotein

Two-component self-assembling virus-like particles (VLPs) are promising scaffolds for achieving high-density display of HIV-1 envelope (gp140) trimers, which can improve the induction of neutralising antibodies (NAbs). In this study gp140 was displayed on the surface of VLPs formed by the AP205 phage coat protein. The CAP256 SU gp140 antigen was selected as the patient who this virus was isolated from developed broadly neutralising antibodies (bNAbs) shortly after superinfection with this virus. The CAP256 SU envelope is also sensitive to several bNAbs and has shown enhanced reactivity for certain bNAb precursors. A fusion protein comprising the HIV-1 CAP256 SU gp140 and the SpyTag (ST) (gp140-ST) was produced in HEK293 cells, and trimers were purified to homogeneity using gel filtration. SpyCatcher (SC)-AP205 VLPs were produced in Escherichia coli and purified by ultracentrifugation. The gp140-ST trimers and the SC-AP205 VLPs were mixed in varying molar ratios to generate VLPs displaying the glycoprotein (AP205-gp140-ST particles). Dynamic light scattering, negative stain electron microscopy and 2D classification indicated that gp140-ST was successfully bound to the VLPs, although not all potential binding sites were occupied. The immunogenicity of the coupled VLPs was evaluated in a pilot study in rabbits. One group was injected four times with coupled VLPs, and the second group was primed with DNA vaccines expressing Env and a mosaic Gag, followed by modified vaccinia Ankara expressing the same antigens. The animals were then boosted twice with coupled VLPs. Encouragingly, gp140-ST displayed on SC-AP205 VLPs was an effective boost to heterologously primed rabbits, leading to induction of autologous Tier 2 neutralising antibodies in 2/5 rabbits. However, four inoculations of coupled VLPs alone failed to elicit any Tier 2 antibodies. These results demonstrate that the native-like structure of HIV-1 envelope trimers and selection of a geometrically-suitable nanoparticle scaffold to achieve a high-density display of the trimers are important considerations that could improve the effect of nanoparticle-displayed gp140.

Norovirus-VLPs expressing pre-erythrocytic malaria antigens induce functional immunity against sporozoite infection

Despite the development of prophylactic anti-malarial drugs and practices to prevent infection, malaria remains a health concern. Preclinical testing of novel malaria vaccine strategies achieved through rational antigen selection and novel particle-based delivery platforms is yielding encouraging results. One such platform, self-assembling virus-like particles (VLP) is safer than attenuated live viruses, and has been approved as a vaccination tool by the FDA. We explore the use of Norovirus sub-viral particles lacking the natural shell (S) domain forming the interior shell but that retain the protruding (P) structures of the native virus as a vaccine vector. Epitope selection and their surface display has the potential to focus antigen specific immune responses to crucial epitopes. Recombinant P-particles displaying epitopes from two malaria antigens, Plasmodium falciparum (Pf) CelTOS and Plasmodium falciparum (Pf) CSP, were evaluated for immunogenicity and their ability to confer protection in a murine challenge model. Immune responses induced in mice resulted either in sterile protection (displaying PfCelTOS epitopes) or in antibodies with functional activity against sporozoites (displaying PfCSP epitopes) in an in vitro liver-stage development assay (ILSDA). These results are encouraging and support further evaluation of this platform as a vaccine delivery system.

Targeted Delivery of Nanovaccine to Dendritic Cells via DC-Binding Peptides Induces Potent Antiviral Immunity in vivo.

BackgroundDendritic cell (DC) targeted antigen delivery is a promising strategy to enhance vaccine efficacy and delivery of therapeutics. Self-assembling peptide-based nanoparticles and virus-like particles (VLPs) have attracted extensive interest as non-replicating vectors for nanovaccine design, based on their unique properties, including molecular specificity, biodegradability and biocompatibility. DCs are specialized antigen-presenting cells involved in antigen capture, processing, and presentation to initiate adaptive immune responses. Using DC-specific ligands for targeted delivery of antigens to DCs may be utilized as a promising strategy to drive efficient and strong immune responses.MethodsIn this study, several candidates for DC-binding peptides (DCbps) were individually integrated into C-terminal of porcine circovirus type 2 (PCV2) Cap, a viral protein that could self-assemble into icosahedral VLPs with 60 subunits. The immunostimulatory adjuvant activity of DC-targeted VLPs was further evaluated in a vaccine model of PCV2 Cap.ResultsWith transmission electron microscopy (TEM), E. coli expressed Cap-DCbp fusion proteins were observed self-assembled into highly ordered VLPs. Further, in dynamic light scattering (DLS) analysis, chimeric VLPs exhibited similar particle size uniformity and narrow size distribution as compared to wild type Cap VLPs. With a distinctly higher targeting efficiency, DCbp3 integrated Cap VLPs (Cap-DCbp3) displayed enhanced antigen uptake and increased elicitation of antigen presentation-related factors in BM-DCs. Mice subcutaneously immunized with Cap-DCbp3 VLPs exhibited significantly higher levels of Cap-specific antibodies, neutralizing antibodies and intracellular cytokines than those with other DCbp integrated or wild type Cap VLPs without any DCbp. Interestingly, Cap-DCbp3 VLPs vaccine induces robust cellular immune response profile, including the efficient production of IFN-γ, IL-2 and IL-10. Meanwhile, the improved proliferation index in lymphocytes with Cap-DCbp3 was also detected as compared to other VLPs.ConclusionThis study described the potential of DC-binding peptides for further improved antigen delivery and vaccine efficacy, explainning nanovaccine optimization in relation to a range of emerging and circulating infectious pathogens.

Finger printing human norovirus-like particles by capillary isoelectric focusing with whole column imaging detection

Owing to the limitation of in vitro culture of human noroviruses (HuNoVs), the development of HuNoV vaccines has to depend on the self-assembling virus-like particles (VLPs) with capsid protein expression. The heterogeneity of artificial VLPs exert an impact on the immunogenicity, and should be considered as one of the factors in vaccine evaluation. In this study, we biochemically finger print the HuNoV VLPs with different genogroups, genotypes and sub-genotypes which constitute for a candidate vaccine, by using capillary isoelectric focusing with whole column imaging detection (cIEF-WCID). The electropherograms of GI.1, GII.3, GII.4 and GII.17 VLPs in fluorescence signal were described in the monomer VP1 forms after degenerated by 8 M urea. The four HuNoV VLPs showed different properties in electropherogram finger prints. The finger prints were also reproducible within a certain concentration range (approx. 150 ∼ 20 ug/ml). This method can also tell the changes of pI finger-print patterns when the expired HoNoV VLPs were tested. In conclusion, cIEF-WCID shows great promise for evaluating the production consistency of HuNoV VLP vaccine.

Nanovaccines against Animal Pathogens: The Latest Findings.

Nowadays, safe and efficacious vaccines represent powerful and cost-effective tools for global health and economic growth. In the veterinary field, these are undoubtedly key tools for improving productivity and fighting zoonoses. However, cases of persistent infections, rapidly evolving pathogens having high variability or emerging/re-emerging pathogens for which no effective vaccines have been developed point out the continuing need for new vaccine alternatives to control outbreaks. Most licensed vaccines have been successfully used for many years now; however, they have intrinsic limitations, such as variable efficacy, adverse effects, and some shortcomings. More effective adjuvants and novel delivery systems may foster real vaccine effectiveness and timely implementation. Emerging vaccine technologies involving nanoparticles such as self-assembling proteins, virus-like particles, liposomes, virosomes, and polymeric nanoparticles offer novel, safe, and high-potential approaches to address many vaccine development-related challenges. Nanotechnology is accelerating the evolution of vaccines because nanomaterials having encapsulation ability and very advantageous properties due to their size and surface area serve as effective vehicles for antigen delivery and immunostimulatory agents. This review discusses the requirements for an effective, broad-coverage-elicited immune response, the main nanoplatforms for producing it, and the latest nanovaccine applications for fighting animal pathogens.

Production of capsid proteins of rat hepatitis E virus in Escherichia coli and characterization of self-assembled virus-like particles

Rat hepatitis E virus (HEV) has been isolated from wild rats worldwide and the potential of zoonotic transmission has been documented. Escherichia coli (E. coli) is utilized as an effective system for producing HEV-like particles. However, the production of rat HEV ORF2 proteins in E. coli forming virus-like particles (VLPs) has not yet been reported. In this study, nine rat HEV ORF2 proteins of the ratELOMB-131L strain with truncated N- and C-termini (amino acids 339–594, 349–594, 351–594, 354–594, 357–594, 357–599, 357–604, 357–609, and 357–614 of ORF2 protein) were expressed in E. coli and the 357–614 protein self-assembled most efficiently. A bioanalyzer showed that the purified 357–614 protein has a molecular weight of 33.5 kDa and a purity of 93.2%. Electron microscopy revealed that the purified 33.5 kDa protein formed VLPs with a diameter of 21–52 (average 32) nm, and immunoelectron microscopy using an anti-rat HEV ORF2 monoclonal antibody (TA7014) indicated that the observed VLPs were derived from rat HEV ORF2. The VLPs attached to and entered the PLC/PRF/5 cells and blocked the neutralization of rat HEV by TA7014, suggesting that the VLPs possess the antigenic structure of infectious rat HEV particles. In addition, rat HEV VLPs showed high immunogenicity in mice. The present results would be useful for future studies on the development of VLP-based vaccines for HEV prevention in a rat model and for the prevention of rat HEV infection in humans.

Immunogenicity and Protective Activity of Pigeon Circovirus Recombinant Capsid Protein Virus-Like Particles (PiCV rCap-VLPs) in Pigeons (Columba livia) Experimentally Infected with PiCV.

Pigeon circovirus (PiCV) is the most recurrent virus diagnosed in pigeons and is among the major causative agents of young pigeon disease syndrome (YPDS). Due to the lack of an established laboratory protocol for PiCV cultivation, development of prophylaxis is hampered. Alternatively, virus-like particles (VLPs), which closely resemble native viruses but lack the viral genetic material, can be generated using a wide range of expression systems and are shown to have strong immunogenicity. Therefore, the use of VLPs provides a promising prospect for vaccine development. In this study, transfected human embryonic kidney (HEK-293) cells, a mammalian expression system, were used to express the PiCV capsid protein (Cap), which is a major component of PiCV and believed to contain antibody epitopes, to obtain self-assembled VLPs. The VLPs were observed to have a spherical morphology with diameters ranging from 12 to 26 nm. Subcutaneous immunization of pigeons with 100 µg PiCV rCap-VLPs supplemented with water-in-oil-in-water (W/O/W) adjuvant induced specific antibodies against PiCV. Observations of the cytokine expression and T-cell proliferation levels in spleen samples showed significantly higher T-cell proliferation and IFN- γ expression in pigeons immunized with VLPs compared to the controls (p < 0.05). Experimentally infected pigeons that were vaccinated with VLPs also showed no detectable viral titer. The results of the current study demonstrated the potential use of PiCV rCap-VLPs as an effective vaccine candidate against PiCV.

Switch from Polymorphic to Homogenous Self-Assembly of Virus-Like Particles of Simian Virus 40 through Double-Cysteine Substitution.

Self-assembled virus-like particles (VLPs) hold great potential as natural nanomaterials for applications in many fields. For such purposes, monodisperse size distribution is a desirable property. However, the VLPs of simian virus 40 (SV40), a representative VLP platform, are characterized by polymorphism. In an attempt to eliminate the polymorphism, 15 mutants of the VLP subunit (VP1) are constructed through the substitution of double cysteines at the VP1 pentamer interfaces, generating a group of VLPs with altered size distributions. One of the mutants, SS2 (L102C/P300C), specifically forms homogenous T= 1-like tiny VLPs of 24 ± 3nm in diameter. Moreover, the stability of the SS2 VLPs is markedly enhanced compared with that of wild-type VLPs. The homogeneous self-assembly and stability enhancement of SS2 VLPs can be attributed to the new disulfide bonds contributed by Cys102 and Cys300, which are identified by mass spectrometry and explored by molecular dynamics simulations. Endocytosis inhibition assays indicate that SS2 VLPs, like the polymorphic wild-type VLPs, preserve the multipathway feature of cellular uptake. SS2 VLPs may serve as an evolved version of SV40 VLPs in future studies and applications. The findings of this work would be useful for the design and fabrication of VLP-based materials and devices.

Nanomedicine strategies to target coronavirus.

With the severe acute respiratory syndrome coronavirus (SARS-CoV) in 2002, the middle east respiratory syndrome CoV (MERS-CoV) in 2012 and the recently discovered SARS-CoV-2 in December 2019, the 21st first century has so far faced the outbreak of three major coronaviruses (CoVs). In particular, SARS-CoV-2 spread rapidly over the globe affecting nearly 25.000.000 people up to date. Recent evidences pointing towards mutations within the viral spike proteins of SARS-CoV-2 that are considered the cause for this rapid spread and currently around 300 clinical trials are running to find a treatment for SARS-CoV-2 infections. Nanomedicine, the application of nanocarriers to deliver drugs specifically to a target sites, has been applied for different diseases, such as cancer but also in viral infections. Nanocarriers can be designed to encapsulate vaccines and deliver them towards antigen presenting cells or function as antigen-presenting carriers themselves. Furthermore, drugs can be encapsulated into such carriers to directly target them to infected cells. In particular, virus-mimicking nanoparticles (NPs) such as self-assembled viral proteins, virus-like particles or liposomes, are able to replicate the infection mechanism and can not only be used as delivery system but also to study viral infections and related mechanisms. This review will provide a detailed description of the composition and replication strategy of CoVs, an overview of the therapeutics currently evaluated in clinical trials against SARS-CoV-2 and will discuss the potential of NP-based vaccines, targeted delivery of therapeutics using nanocarriers as well as using NPs to further investigate underlying biological processes in greater detail.

Design and Generation of Self-Assembling Peptide Virus-like Particles with Intrinsic GPCR Inhibitory Activity.

Synthetic analogs of the second transmembrane domain (TM) containing a portion of the extracellular loop 1 of G-protein-coupled receptors (GPCR) can serve as biased antagonists of the corresponding receptor. Analogs with negative charges added to the extracellular end self-assemble into round structures. Addition of polyethylene glycol chains of defined length to the C-terminus of the peptides prevents super aggregation and results in highly uniform particles that can fuse with cell membranes spontaneously. Added PEG chains slow down cell fusion, while attachment of receptor ligands to the surface of particles results in receptor-mediated membrane fusion and cell-selective delivery. Critical assembly concentration of TM peptide particles is in the nanomolar range and thus requires nontraditional methods of determination. In this chapter, we outline sequence selection and design of self-assembling GPCR antagonists, methods of the preparation of the nanoparticles, and biophysical methods of particle characterization. The protocols allow for straightforward rational design, generation, and characterization of self-assembling GPCR antagonists for a variety of applications.

Geometric architecture of viruses.

In the current SARS-CoV-2 disease (COVID-19) pandemic, the structural understanding of new emerging viruses in relation to developing effective treatment and interventions are very necessary. Viruses present remarkable differences in geometric shapes, sizes, molecular compositions and organizations. A detailed structural knowledge of a virion is essential for understanding the mechanisms of capsid assembly/disassembly, antigenicity, cell-receptor interaction, and designing therapeutic strategies. X-ray crystallography, cryo-electron microscopy and molecular simulations have elucidated atomic-level structure of several viruses. In view of this, a recently determined crystal structure of SARS-CoV-2 nucleocapsid has revealed its architecture and self-assembly very similar to that of the SARS-CoV-1 and the Middle-East respiratory syndrome virus (MERS-CoV). In structure determination, capsid symmetry is an important factor greatly contributing to its stability and balance between the packaged genome and envelope. Since the capsid protein subunits are asymmetrical, the maximum number of inter-subunit interactions can be established only when they are arranged symmetrically. Therefore, a stable capsid must be in a perfect symmetry and lowest possible free-energy. Isometric virions are spherical but geometrically icosahedrons as compared to complex virions that are both isometric and helical. Enveloped icosahedral or helical viruses are very common in animals but rare in plants and bacteria. Icosahedral capsids are defined by triangulation number (T = 1, 3, 4, 13, etc.), i.e., the identical equilateral-triangles formed of subunits. Biologically significant defective capsids with or without nucleic acids are common in enveloped alpha-, flavi- and hepadnaviruses. The self-assembling, stable and non-infectious virus-like particles have been widely exploited as vaccine candidates and therapeutic molecules delivery vehicles.

Preparation of Chicken Anemia Virus (CAV) Virus-Like Particles and Chicken Interleukin-12 for Vaccine Development Using a Baculovirus Expression System.

Chicken infectious anemia (CIA) is a poultry disease that causes huge economic losses in the poultry industry worldwide. Commercially available CIA vaccines are derived from wild-type chicken anemia viruses (CAVs) by serial passage in cells or chicken embryos. However, these vaccinal viruses are not completely attenuated; therefore, they can be transmitted vertically and horizontally, and may induce clinical symptoms in young birds. In this study, we sought to eliminate these issues by developing a subunit vaccine exploiting the CAV structural proteins, engineering recombinant baculovirus-infected Spodoptera frugiperda (Sf9) cells that contained both the viral protein 1 (VP1) and VP2 of CAV. Moreover, we produced single-chain chicken interleukin-12 (chIL-12) in the same system, to serve as an adjuvant. The recombinant VP1 was recognized by chicken anti-CAV polyclonal antibodies in Western blotting and immunofluorescence assays, and the bioactivity of the recombinant chIL-12 was confirmed by stimulating interferon-γ (IFN-γ) secretion in chicken splenocytes. Furthermore, the ability of the recombinant VP1 to generate self-assembling virus-like particles (VLPs) was confirmed by transmission electron microscopy. Specific pathogen-free (SPF) chickens inoculated with VLPs and co-administered the recombinant chIL-12 induced high CAV-specific antibodies and cell-mediated immunity. Taken together, the VLPs produced by the baculovirus expression system have the potential to be a safe and effective CIA vaccine. Finally, we demonstrated the utility of recombinant chIL-12 as an adjuvant for poultry vaccine development.

Synthetic self-assembling ADDomer platform for highly efficient vaccination by genetically encoded multiepitope display.

Self-assembling virus-like particles represent highly attractive tools for developing next-generation vaccines and protein therapeutics. We created ADDomer, an adenovirus-derived multimeric protein-based self-assembling nanoparticle scaffold engineered to facilitate plug-and-play display of multiple immunogenic epitopes from pathogens. We used cryo-electron microscopy at near-atomic resolution and implemented novel, cost-effective, high-performance cloud computing to reveal architectural features in unprecedented detail. We analyzed ADDomer interaction with components of the immune system and developed a promising first-in-kind ADDomer-based vaccine candidate to combat emerging Chikungunya infectious disease, exemplifying the potential of our approach.