The choice of resin-bound ligand affects the structure and immunogenicity of column-purified human papillomavirus type 16 virus-like particles

Cell growth conditions and purification methods are important in determining biopharmaceutical activity. However, in studies aimed at manufacturing virus-like particles (VLPs) for the purpose of creating a prophylactic vaccine and antigen for human papillomavirus (HPV), the effects of the presence of a resin-bound ligand during purification have never been investigated. In this study, we compared the structural integrity and immunogenicity of two kinds of VLPs derived from HPV type 16 (HPV16 VLPs): one VLP was purified by heparin chromatography (hHPV16 VLP) and the other by cation-exchange chromatography (cHPV16 VLP). The reactivity of anti-HPV16 neutralizing monoclonal antibodies (H16.V5 and H16.E70) towards hHPV16 VLP were significantly higher than the observed cHPV16 VLP reactivities, implying that hHPV16 VLP possesses a greater number of neutralizing epitopes and has a greater potential to elicit anti-HPV16 neutralizing antibodies. After the application of heparin chromatography, HPV16 VLP has a higher affinity for H16.V5 and H16.E70. This result indicates that heparin chromatography is valuable in selecting functional HPV16 VLPs. In regard to VLP immunogenicity, the anti-HPV16 L1 IgG and neutralizing antibody levels elicited by immunizations of mice with hHPV16 VLPs were higher than those elicited by cHPV16 VLP with and without adjuvant. Therefore, the ability of hHPV16 VLP to elicit humoral immune responses was superior to that of cHPV16 VLP. We conclude that the specific chromatographic technique employed for the purification of HPV16 VLPs is an important factor in determining the structural characteristics and immunogenicity of column-purified VLPs.     

Virus-Like Particle Vaccine Protects against 2009 H1N1 Pandemic Influenza Virus in Mice

Background

The 2009 influenza pandemic and shortages in vaccine supplies worldwide underscore the need for new approaches to develop more effective vaccines.

Methodology/Principal Findings

We generated influenza virus-like particles (VLPs) containing proteins derived from the A/California/04/2009 virus, and tested their efficacy as a vaccine in mice. A single intramuscular vaccination with VLPs provided complete protection against lethal challenge with the A/California/04/2009 virus and partial protection against A/PR/8/1934 virus, an antigenically distant human isolate. VLP vaccination induced predominant IgG2a antibody responses, high hemagglutination inhibition (HAI) titers, and recall IgG and IgA antibody responses. HAI titers after VLP vaccination were equivalent to those observed after live virus infection. VLP immune sera also showed HAI responses against diverse geographic pandemic isolates. Notably, a low dose of VLPs could provide protection against lethal infection.

Conclusion/Significance

This study demonstrates that VLP vaccination provides highly effective protection against the 2009 pandemic influenza virus. The results indicate that VLPs can be developed into an effective vaccine, which can be rapidly produced and avoid the need to isolate high growth reassortants for egg-based production.     

Proteomic characterization of influenza H5N1 virus-like particles and their protective immunogenicity

Recombinant virus-like particles (VLPs) have been shown to induce protective immunity. Despite their potential significance as promising vaccine candidates, the protein composition of VLPs produced in insect cells has not been well characterized. Here we report a proteomic analysis of influenza VLPs containing hemagglutinin (HA) and matrix M1 proteins from a human isolate of avian influenza H5N1 virus (H5 VLPs) produced in insect cells using the recombinant baculovirus expression system. Comprehensive proteomic analysis of purified H5 VLPs identified viral proteins and 37 additional host-derived proteins, many of which are known to be present in other enveloped viruses. Proteins involved in different cellular structures and functions were found to be present in H5 VLPs including those from the cytoskeleton, translation, chaperone, and metabolism. Immunization with purified H5 VLPs induced protective immunity, which was comparable to the inactivated whole virus containing all viral components. Unpurified H5 VLPs containing excess amounts of noninfluenza soluble proteins also conferred 100% protection against lethal challenge although lower immune responses were induced. These results provide important implications consistent with the idea that VLP production in insect cells may involve similar cellular machinery as other RNA enveloped viruses during synthesis, assembly, trafficking, and budding processes.     

Systemic, Mucosal, and Heterotypic Immune Induction in Mice Inoculated with Venezuelan Equine Encephalitis Replicons Expressing Norwalk Virus-Like Particles

Norwalk-like viruses (NLVs) are a diverse group of single-stranded, nonenveloped, positive-polarity RNA viruses and are the leading cause of epidemic acute gastroenteritis in the United States. In this study, the major capsid gene of Norwalk virus, the prototype NLV, has been cloned and expressed in mammalian cells using a Venezuelan equine encephalitis (VEE) replicon expression system. Upon infection of baby hamster kidney (BHK) cells with VEE replicon particles (VRPs), the Norwalk virus capsid proteins self-assemble to generate high titers of Norwalk virus-like particles (VLPs) that are morphologically and antigenically analogous to wild-type Norwalk virus. Mice inoculated subcutaneously with VRPs expressing the Norwalk virus capsid protein (VRP-NV1) developed systemic and mucosal immune responses to Norwalk VLPs, as well as heterotypic antibody responses to the major capsid protein from another genogroup I NLV strain (NCFL) isolated from a recent outbreak. A second Norwalk virus capsid clone (NV2) containing three amino acid codon mutations from the NV1 clone was also expressed using VEE replicons (VRP-NV2), but upon infection of BHK cells failed to confer VLP self-assembly. Mice inoculated with VRP-NV2 elicited reduced systemic and mucosal immune responses to Norwalk VLPs, demonstrating the importance and potential utility of endogenous VLP presentation for maximum immune induction. Inoculation with either VRP-NV1 or VRP-NV2 resulted in serum antibody responses far superior to the induction in mice dosed orally with VLPs that were prepared using the VEE-NV1 replicon construct, a regimen similar to current models for NLV vaccination. Expression of NLV VLPs in mammalian cells offers a powerful approach for the design of novel NLV vaccines, either alone or in combination with current vaccination models.     

Virus-like particles: passport to immune recognition

Virus-like particles (VLPs) are formed by the self-assembly of envelope and/or capsid proteins from many viruses. In many cases such VLPs have structural characteristics and antigenicity similar to the parental virus, and some have already proven successful as vaccines against the cognate virus infection. The structural components of some VLPs have also proven amenable to the insertion or fusion of foreign antigenic sequences, allowing the production of chimeric VLPs exposing the foreign antigen on their surface. Other VLPs have been used as carriers for foreign antigens, including non-protein antigens, via chemical conjugation. This review outlines some of the advantages, disadvantages, and technical considerations for the use of a wide range of VLP systems in vaccine development.     

Incorporation of glycosylphosphatidylinositol-anchored granulocyte- macrophage colony-stimulating factor or CD40 ligand enhances immunogenicity of chimeric simian immunodeficiency virus-like particles

The rapid worldwide spread of human immunodeficiency virus (HIV) mandates the development of successful vaccination strategies. Since live attenuated HIV is not accepted as a vaccine due to safety concerns, virus-like particles (VLPs) offer an attractive safe alternative because they lack the viral genome yet they are perceived by the immune system as a virus particle. We hypothesized that adding immunostimulatory signals to VLPs would enhance their efficacy. To accomplish this we generated chimeric simian immunodeficiency virus (SIV) VLPs containing either glycosylphosphatidylinositol (GPI)-anchored granulocyte-macrophage colony-stimulating factor (GM-CSF) or CD40 ligand (CD40L) and investigated their biological activity and ability to enhance immune responses in vivo. Immunization of mice with chimeric SIV VLPs containing GM-CSF induced SIV Env-specific antibodies as well as neutralizing activity at significantly higher levels than those induced by standard SIV VLPs, SIV VLPs containing CD40L, or standard VLPs mixed with soluble GM-CSF. In addition, mice immunized with chimeric SIV VLPs containing either GM-CSF or CD40L showed significantly increased CD4(+)- and CD8(+)-T-cell responses to SIV Env, compared to standard SIV VLPs. Taken together, these results demonstrate that the incorporation of immunostimulatory molecules enhances humoral and cellular immune responses. We propose that anchoring immunostimulatory molecules into SIV VLPs can be a promising approach to augmenting the efficacy of VLP antigens.     

Binding and internalization of human papillomavirus type 33 virus-like particles by eukaryotic cells.

Infection of cells by human papillomaviruses (HPVs) associated with malignant genital lesions has not been studied because of the lack of an in vitro system and the unavailability of virions. We have now used virus-like particles (VLPs) of HPV type 33 to analyze the initial events in the interaction of the HPV capsid with cell lines. Binding of VLPs to HeLa cells was observed in biochemical assays and by immunofluorescence. VLP binding was inhibited by antisera raised against VLPs but not by monoclonal antibodies recognizing either L1 or L2 epitopes accessible on VLPs. Under saturating conditions, approximately 2 x 10(4) VLPs were bound per cell, with a dissociation constant of about 100 pM. VLPs composed of L1 alone bound as well as VLPs composed of both capsid proteins, indicating that L2 is not required for initial binding. VLPs dissociated into capsomers did not bind, demonstrating that intercapsomer contacts are required. Neither capsomers nor simian virus 40 virions competed with VLP binding. Uptake of VLPs by small and smooth endocytic vesicles was demonstrated by immunoelectron microscopy. Cellular binding of VLPs was sensitive to trypsin but not to sialidase, N-glycosidase, or octyl-beta-D-glycopyranoside treatment, suggesting that a cell surface protein is involved in the VLP binding. Cell lines originating from a variety of tissues and organisms as distantly related as insects and humans bound VLPs with similar efficiency and specificity. Therefore, the putative receptor mediating VLP attachment should be highly conserved and cannot be responsible for the species and tissue specificity of HPVs.     

Influenza virus hemagglutinin and neuraminidase, but not the matrix protein, are required for assembly and budding of plasmid-derived virus-like particles

For influenza virus, we developed an efficient, noncytotoxic, plasmid-based virus-like particle (VLP) system to reflect authentic virus particles. This system was characterized biochemically by analysis of VLP protein composition, morphologically by electron microscopy, and functionally with a VLP infectivity assay. The VLP system was used to address the identity of the minimal set of viral proteins required for budding. Combinations of viral proteins were expressed in cells, and the polypeptide composition of the particles released into the culture media was analyzed. Contrary to previous findings in which matrix (M1) protein was considered to be the driving force of budding because M1 was found to be released copiously into the culture medium when M1 was expressed by using the vaccinia virus T7 RNA polymerase-driven overexpression system, in our noncytotoxic VLP system M1 was not released efficiently into the culture medium. Additionally, hemagglutinin (HA), when treated with exogenous neuraminidase (NA) or coexpressed with viral NA, could be released from cells independently of M1. Incorporation of M1 into VLPs required HA expression, although when M1 was omitted from VLPs, particles with morphologies similar to those of wild-type VLPs or viruses were observed. Furthermore, when HA and NA cytoplasmic tail mutants were included in the VLPs, M1 failed to be efficiently incorporated into VLPs, consistent with a model in which the glycoproteins control virus budding by sorting to lipid raft microdomains and recruiting the internal viral core components. VLP formation also occurred independently of the function of Vps4 in the multivesicular body pathway, as dominant-negative Vps4 proteins failed to inhibit influenza VLP budding.     

Different applications of virus‐like particles in biology and medicine: Vaccination and delivery systems

Virus‐like particles (VLPs) mimic the whole construct of virus particles devoid of viral genome as used in subunit vaccine design. VLPs can elicit efficient protective immunity as direct immunogens compared to soluble antigens co‐administered with adjuvants in several booster injections. Up to now, several prokaryotic and eukaryotic systems such as insect, yeast, plant, and E. coli were used to express recombinant proteins, especially for VLP production. Recent studies are also generating VLPs in plants using different transient expression vectors for edible vaccines. VLPs and viral particles have been applied for different functions such as gene therapy, vaccination, nanotechnology, and diagnostics. Herein, we describe VLP production in different systems as well as its applications in biology and medicine. © 2015 Wiley Periodicals, Inc. Biopolymers 105: 113–132, 2016.     

Cell selective targeting of a simian virus 40 virus-like particle conjugated to epidermal growth factor

Simian virus 40 (SV40) virus-like particles (VLPs) are efficient nanocarriers for gene delivery. VLPs conjugated to human epidermal growth factor (hEGF) were prepared and the cell selectivity of the VLP was examined using human epithelial carcinoma A431 cells, which overexpress the EGF receptor. The endocytic efficiency was determined by the level of Gaussia luciferase activity from the encapsulated protein in hEGF-conjugated VLPs. EGF receptor-mediated endocytosis of hEGF-conjugated VLPs was significantly increased and was confirmed by fluorescence imaging using mCherry encapsulated in hEGF-conjugated VLPs. These results suggest that VLPs of SV40 conjugated to a specific ligand could be used for cell selective gene delivery.     

Fluorescence Fluctuation Spectroscopy on Viral-Like Particles Reveals Variable Gag Stoichiometry

Fluorescence fluctuation spectroscopy determines the brightness, size, and concentration of fluorescent particles from the intensity bursts generated by individual particles passing through a small observation volume. Brightness provides a measure of the number of fluorescently labeled proteins within a complex and has been used previously to determine the stoichiometry of small oligomers in cells. We extend brightness analysis to large macromolecular protein complexes containing thousands of proteins and determine their stoichiometry. This study investigates viral-like particles (VLP) formed from human immunodeficiency virus type 1 (HIV-1) Gag protein expressed in COS-1 cells using fluorescence fluctuation spectroscopy to determine the stoichiometry of HIV-1 Gag within the particles. Control experiments establish that the stoichiometry and size of VLPs are not influenced by labeling of HIV-1 Gag with a fluorescent protein. The experiments further show that the brightness scales linearly with the amount of labeled Gag within the particle. Brightness analysis shows that the Gag stoichiometry of VLPs formed in COS-1 cells is not constant, but varies with the amount of transfected DNA plasmid. We observed HIV-1 Gag stoichiometries ranging from ∼750 to ∼2500, whereas the size of the VLPs remains unchanged. This result indicates that large areas of the VLP membrane are void of Gag protein. Therefore, a closed layer of HIV-1 Gag at the membrane is not required for VLP production. This study shows that brightness analysis has the potential to become an important tool for investigating large molecular complexes by providing quantitative information about their size and composition.     

Dengue virus-like particles: construction and application

Virus-like particles (VLPs) are shell-like viruses that lack virus-specific genetic materials. Many viral-structured proteins can assemble into VLPs, which mimic the overall structure of virus particles and can elicit strong immune responses in a host. Dengue viruses (DENVs), from the genus Flavivirus, are transmitted to humans through the bites of an infected Aedes mosquito. DENVs cause several diseases that prevailed mainly in tropical and subtropical areas. However, effective treatment measures and preventive strategies for dengue diseases are still lacking. The present minireview summarized the assembly and maturation of DENVs, the strategies and effective factors for dengue VLP construction, and the application of DENV VLPs.     

An Epstein-Barr Virus Mutant Produces Immunogenic Defective Particles Devoid of Viral DNA

Virus-like particles (VLPs) from hepatitis B and human papillomaviruses have been successfully used as preventative vaccines against these infectious agents. These VLPs consist of a self-associating capsid polymer formed from a single structure protein and are devoid of viral DNA. Since virions from herpesviruses consist of a large number of molecules of viral and cellular origin, generating VLPs from a subset of these would be a particularly arduous task. Therefore, we have adopted an alternative strategy that consists of producing DNA-free defective virus particles in a cell line infected by a herpesvirus mutant incapable of packaging DNA. We previously reported that an Epstein-Barr virus (EBV) mutant devoid of the terminal repeats (ΔTR) that act as packaging signals in herpesviruses produces substantial amounts of VLPs and of light particles (LPs). However, ΔTR virions retained some infectious genomes, and although these mutants had lost their transforming abilities, this poses potential concerns for clinical applications. Therefore, we have constructed a series of mutants that lack proteins involved in maturation and assessed their ability to produce viral DNA-free VLP/LPs. Some of the introduced mutations were deleterious for capsid maturation and virus production. However, deletion of BFLF1/BFRF1A or of BBRF1 resulted in the production of DNA-free VLPs/LPs. The ΔBFLF1/BFRF1A viruses elicited a potent CD4⁺ T-cell response that was indistinguishable from the one obtained with wild-type controls. In summary, the defective particles produced by the ΔBFLF1/BFRF1A mutant fulfill the criteria of efficacy and safety expected from a preventative vaccine.     

Norwalk virus-like particle hemagglutination by binding to h histo-blood group antigens

Noroviruses are a major cause of epidemic acute nonbacterial gastroenteritis worldwide. Here we report our discovery that recombinant Norwalk virus virus-like particles (rNV VLPs) agglutinate red blood cells (RBCs). Since histo-blood group antigens are expressed on gut mucosa as well as RBCs, we used rNV VLP hemagglutination (HA) as a model system for studying NV attachment to cells in order to help identify a potential NV receptor(s). rNV VLP HA is dependent on low temperature (4 degrees C) and acidic pH. Of the 13 species of RBCs tested, rNV VLPs hemagglutinated only chimpanzee and human RBCs. The rNV VLPs hemagglutinated all human type O (11 of 11), A (9 of 9), and AB (4 of 4) RBCs; however, few human type B RBC samples (4 of 14) were hemagglutinated. HA with periodate- and neuraminidase-treated RBCs indicated that rNV VLP binding was carbohydrate dependent and did not require sialic acid. The rNV VLPs did not hemagglutinate Bombay RBCs (zero of seven) that lack H type 2 antigen, and an anti-H type 2 antibody inhibited rNV VLP HA of human type O RBCs. These data indicated that the H type 2 antigen functions as the rNV VLP HA receptor on human type O RBCs. The rNV VLP HA was also inhibited by rNV VLP-specific monoclonal antibody 8812, an antibody that inhibits VLP binding to Caco-2 cells. Convalescent-phase sera from NV-infected individuals showed increased rNV VLP HA inhibition titers compared to prechallenge sera. In carbohydrate binding assays, the rNV VLPs bound to synthetic Lewis d (Le(d)), Le(b), H type 2, and Le(y) antigens, and these antigens also inhibited rNV VLP HA of human type O RBCs. Overall, our results indicate that carbohydrate antigens in the gut are a previously unrecognized factor in NV pathogenesis.     

Identification of the alpha6 integrin as a candidate receptor for papillomaviruses.

Papillomaviruses (PVs) bind in a specific and saturable fashion to a range of epithelial and other cell lines. Treatment of cells with trypsin markedly reduces their ability to bind virus particles, suggesting that binding is mediated via a cell membrane protein. We have investigated the interaction of human PV type 6b L1 virus-like particles (VLPs) with two epithelial cell lines, CV-1 and HaCaT, which bind VLPs, and a B-cell line (DG75) previously shown not to bind VLPs. Immunoprecipitation of a mixture of PV VLPs with [35S]methionine-labeled cell extracts and with biotin-labeled cell surface proteins identified four proteins from CV-1 and HaCaT cells of 220, 120, 87, and 35 kDa that reacted with VLPs and were not present in DG75 cells. The alpha6beta4 integrin complex has subunits corresponding to the VLP precipitated proteins, and the tissue distribution of this complex suggested that it was a candidate human PV receptor. Monoclonal antibodies (MAbs) to the alpha6 or beta4 integrin subunits precipitated VLPs from a mixture of CV-1 cell proteins and VLPs, whereas MAbs to other integrin subunits did not. An alpha6 integrin-specific MAb (GoH3) inhibited VLP binding to CV-1 and HaCaT cells, whereas an anti-beta4 integrin MAb and a range of integrin-specific and other MAbs did not. Furthermore, human laminin, the natural ligand for the alpha6beta4 integrin, was able to block VLP binding. By use of sections of monkey esophagus, the distribution of alpha6 integrin expression in the basal epithelium was shown to coincide with the distribution of bound VLPs. Taken together, these data suggest that VLPs bind specifically to the alpha6 integrin subunit and that integrin complexes containing alpha6 integrin complexed with either beta1 or beta4 integrins may act as a receptor for PV binding and entry into epithelial cells.     

A virus-type specific serological diagnosis of flavivirus infection using virus-like particles

Many flaviviruses are emerging and reemerging pathogens, such as West Nile virus (WNV), dengue virus (DENV), yellow fever virus (YFV), and Japanese encephalitis virus. Serological assay is the dominant method for diagnosis of flavivirus infections in human. Because antibodies generated during flavivirus infections cross-react with other flavivirus members, plaque reduction neutralization test (PRNT) is the only available assay to determine the infecting flavivirus type.Since PRNT requires culturing raw viruses, it must be performed in biosafety level-3 or level-4 containment for many flaviviruses, and takes more than ten days to complete. To overcome these problems, we have developed flavivirus viral-like particles (VLPs) that could be used to replace raw viruses in the neutralization assay. The VLPs were prepared by trans packaging a luciferase-reporting replicon with viral structural proteins. This novel assay involves three simple steps: (ⅰ) VLPs from a panel of flaviviruses are incubated with flavivirus-infected sera at 37℃ for 1 h; (ⅱ)the neutralized VLPs are used to infect Vero cells; and (ⅲ) the infected cells are measured for luciferase activities at 22 h post-infection. The virus type whose VLP is most efficiently neutralized by the serum specimen (as quantified by the luciferase activities) is the etiologic agent. As a proof-of-concept, we show that a WNV-infected mouse serum neutralized the WNV VLP more efficiently and selectively than the DENV and YFV VLPs. Our results demonstrate that the VLP neutralization assay maintains the "gold standard" of the classic PRNT; importantly, it shortens the assay time from ＞10 days to ＜1 day, and can be performed in biosafety level-2 facility.     

Exploiting the yeast L-A viral capsid for the in vivo assembly of chimeric VLPs as platform in vaccine development and foreign protein expression

A novel expression system based on engineered variants of the yeast (Saccharomyces cerevisiae) dsRNA virus L-A was developed allowing the in vivo assembly of chimeric virus-like particles (VLPs) as a unique platform for a wide range of applications. We show that polypeptides fused to the viral capsid protein Gag self-assemble into isometric VLP chimeras carrying their cargo inside the capsid, thereby not only effectively preventing proteolytic degradation in the host cell cytosol, but also allowing the expression of a per se cytotoxic protein. Carboxyterminal extension of Gag by T cell epitopes from human cytomegalovirus pp65 resulted in the formation of hybrid VLPs that strongly activated antigen-specific CD8(+) memory T cells ex vivo. Besides being a carrier for polypeptides inducing antigen-specific immune responses in vivo, VLP chimeras were also shown to be effective in the expression and purification of (i) a heterologous model protein (GFP), (ii) a per se toxic protein (K28 alpha-subunit), and (iii) a particle-associated and fully recyclable biotechnologically relevant enzyme (esterase A). Thus, yeast viral Gag represents a unique platform for the in vivo assembly of chimeric VLPs, equally attractive and useful in vaccine development and recombinant protein production.     

Antibody Persistence in Adults Two Years after Vaccination with an H1N1 2009 Pandemic Influenza Virus-Like Particle Vaccine

The influenza virus is a human pathogen that causes epidemics every year, as well as potential pandemic outbreaks, as occurred in 2009. Vaccination has proven to be sufficient in the prevention and containment of viral spreading. In addition to the current egg-based vaccines, new and promising vaccine platforms, such as cell culture-derived vaccines that include virus-like particles (VLPs), have been developed. VLPs have been shown to be both safe and immunogenic against influenza infections. Although antibody persistence has been studied in traditional egg-based influenza vaccines, studies on antibody response durations induced by VLP influenza vaccines in humans are scarce. Here, we show that subjects vaccinated with an insect cell-derived VLP vaccine, in the midst of the 2009 H1N1 influenza pandemic outbreak in Mexico City, showed antibody persistence up to 24 months post-vaccination. Additionally, we found that subjects that reported being revaccinated with a subsequent inactivated influenza virus vaccine showed higher antibody titres to the pandemic influenza virus than those who were not revaccinated. These findings provide insights into the duration of the antibody responses elicited by an insect cell-derived pandemic influenza VLP vaccine and the possible effects of subsequent influenza vaccination on antibody persistence induced by this VLP vaccine in humans.     

Newcastle disease virus-like particles as a platform for the development of vaccines for human and agricultural pathogens

Vaccination is the single most effective way to control viral diseases. However, many currently used vaccines have safety concerns, efficacy issues or production problems. For other viral pathogens, classic approaches to vaccine development have, thus far, been unsuccessful. Virus-like particles (VLPs) are increasingly being considered as vaccine candidates because they offer significant advantages over many currently used vaccines or developing vaccine technologies. VLPs formed with structural proteins of Newcastle disease virus, an avian paramyxovirus, are a potential vaccine candidate for Newcastle disease in poultry. More importantly, these VLPs are a novel, uniquely versatile VLP platform for the rapid construction of effective vaccine candidates for many human pathogens, including genetically complex viruses and viruses for which no vaccines currently exist.