Equine papillomavirus type 1: complete nucleotide sequence and characterization of recombinant virus-like particles composed of the EcPV-1 L1 major capsid protein

Equus caballus papillomavirus type 1 (EcPV-1) was isolated from a cutaneous papilloma, the most common neoplasm in horses. The complete EcPV-1 nucleotide sequence and genomic organization were determined. Phylogenetic analysis showed that EcPV-1 is a close-to-root papillomavirus, with only distant relationships to the fibropapillomaviruses and the benign cutaneous papillomaviruses. To produce EcPV-1 virus-like particles (VLPs), the EcPV-1 L1 major capsid protein was expressed in insect cells using a recombinant baculovirus vector. The self-assembled EcPV-1 VLPs were morphologically indistinguishable from wild type papillomavirus virions. Monoclonal antibodies were developed against intact and denatured EcPV-1 VLPs. When tested by ELISA, all monoclonal antibodies produced against intact (#18) and some against denatured EcPV-1 VLPs (#16) reacted with intact EcPV-1 VLPs only, demonstrating that the VLPs carry type-specific conformational as well as linear epitopes on their surface. Recombinant EcPV-1 VLPs offer the potential of a noninfectious vaccine to prevent and eradicate equine cutaneous papillomatosis.     

High avidity binding of engineered papaya mosaic virus virus-like particles to resting spores of Plasmodiophora brassicae

Papaya mosaic virus (PapMV) like particles (VLPs) were used as a platform for fusion of affinity peptides binding to resting spores of Plasmodiophora brassicae-a major pathogen of crucifers. Three peptides with specific affinity to the target were isolated and cloned at the C-terminus of the PapMV coat protein (CP), generating three different high avidity VLPs. The peptides were exposed at the surface of the VLPs and their avidity to resting spores of P. brassicae was measured by flow cytometry. NLP-A, with the peptide DPAPRPR, showed the highest avidity. The binding avidity of NLP-A to P. brassicae spores was comparable to that of a polyclonal antibody. NLP-A was also shown to be more specific than the antibody. Fusion of the affinity peptide to a monomeric form (mCP) of the CP [Lecours, K., Tremblay, M.-H., Laliberté Gagné, M.-E., Gagné, S.M., Leclerc, D., 2006. Purification and biochemical characterization of a monomeric form of papaya mosaic potexvirus coat protein. Protein Express. Purific. 47, 273-280] generated a fusion protein that was unable to assemble into VLPs, and mCP-A fusions failed to bind resting spores. The avidity of VLP-A was increased by adding a glycine spacer between the C-terminus of the PapMV CP and the peptide, and improved even further by using a duplicated A peptide in the fusion protein. The use of high avidity VLPs has advantages over polyclonal antibodies because of target specificity. VLPs offers the specificity of monoclonal antibodies but can be more easily generated using the powerful selection of phage display.     

Induction of HIV neutralizing antibodies against the MPER of the HIV envelope protein by HA/gp41 chimeric protein-based DNA and VLP vaccines

Several conserved neutralizing epitopes have been identified in the HIV Env protein and among these, the MPER of gp41 has received great attention and is widely recognized as a promising target. However, little success has been achieved in eliciting MPER-specific HIV neutralizing antibodies by a number of different vaccine strategies. We investigated the ability of HA/gp41 chimeric protein-based vaccines, which were designed to enhance the exposure of the MPER in its native conformation, to induce MPER-specific HIV neutralizing antibodies. In characterization of the HA/gp41 chimeric protein, we found that by mutating an unpaired Cys residue (Cys-14) in its HA1 subunit to a Ser residue, the modified chimeric protein HA-C14S/gp41 showed increased reactivity to a conformation-sensitive monoclonal antibody against HA and formed more stable trimers in VLPs. On the other hand, HA-C14S/gp41 and HA/gp41 chimeric proteins expressed on the cell surfaces exhibited similar reactivity to monoclonal antibodies 2F5 and 4E10. Immunization of guinea pigs using the HA-C14S/gp41 DNA or VLP vaccines induced antibodies against the HIV gp41 as well as to a peptide corresponding to a segment of MPER at higher levels than immunization by standard HIV VLPs. Further, sera from vaccinated guinea pigs were found to exhibit HIV neutralizing activities. Moreover, sera from guinea pigs vaccinated by HA-C14S/gp41 DNA and VLP vaccines but not the standard HIV VLPs, were found to neutralize HIV pseudovirions containing a SIV-4E10 chimeric Env protein. The virus neutralization could be blocked by a MPER-specific peptide, thus demonstrating induction of MPER-specific HIV neutralizing antibodies by this novel vaccine strategy. These results show that induction of MPER-specific HIV neutralizing antibodies can be achieved through a rationally designed vaccine strategy.     

Biochemical characterization of a smaller form of recombinant Norwalk virus capsids assembled in insect cells.

The expression of the single capsid protein of Norwalk virus (NV) in Spodoptera frugiperda (Sf9) insect cells infected with recombinant baculovirus results in the assembly of virus-like particles (VLPs) of two sizes, the predominant 38-nm, or virion-size VLPs, and smaller, 23-nm VLPs. Here we describe the purification and biochemical characterization of the 23-nm VLPs. The 23-nm VLPs were purified to 95% homogeneity from the medium of Sf9 cultures by isopycnic CsCl gradient centrifugation followed by rate-zonal centrifugation in sucrose gradients. The compositions of the purified 23- and 38-nm VLPs were compared by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and protein immunoblots. VLPs of both sizes showed a doublet at 58 kDa, the size of the full-length capsid protein. Upon alkaline treatment, the 23-nm VLPs underwent dissociation into soluble intermediates that were able to reassemble into 23- and 38-nm VLPs upon dialysis, suggesting that the assembly of both types of structures has a common pathway. Antigenic and biochemical properties of the 38- and 23-nm VLPs were examined and found to be conserved. Immunoprecipitation assays using polyclonal and monoclonal antibodies indicated that immunodominant epitopes on the capsid protein as well as conformational epitopes are conserved in the two types of particles. The trypsin cleavage site at residue 227 was protected in the assembled particles of both sizes but exposed after alkaline dissociation. These results, and the conservation of the binding activity of both forms of recombinant NV VLPs to cultured cells (L. J. White, J. M. Ball, M. E. Hardy, T. N. Tanaka, N. Kitamoto, and M. K. Estes, J. Virol. 70:6589-6597, 1996), suggest that the tertiary folding of the capsid protein responsible for these properties is conserved in the two structures. We hypothesize that the 23-nm VLPs are formed when 60 units of the NV capsid protein assembles into a structure with T=1 symmetry.     

Quantitative Disassembly and Reassembly of Human Papillomavirus Type 11 Viruslike Particles In Vitro

The human papillomavirus (HPV) capsid is primarily composed of a structural protein denoted L1, which forms both pentameric capsomeres and capsids composed of 72 capsomeres. The L1 protein alone is capable of self-assembly in vivo into capsidlike structures referred to as viruslike particles (VLPs). We have determined conditions for the quantitative disassembly of purified HPV-11 L1 VLPs to the level of capsomeres, demonstrating that disulfide bonds alone are essential to maintaining long-term HPV-11 L1 VLP structure at physiological ionic strength. The ionic strength of the disassembly reaction was also important, as increased NaCl concentrations inhibited disassembly. Conversely, chelation of cations had no effect on disassembly. Quantitative reassembly to a homogeneous population of 55-nm, 150S VLPs was reliably achieved by the re-formation of disulfide linkages following removal of reducing agent at near-neutral pH and moderate NaCl concentration. HPV-11 L1 VLPs could also be dissociated by treatment with carbonate buffer at pH 9.6, but VLPs could not be regenerated following carbonate treatment. When probed with conformationally sensitive and/or neutralizing monoclonal antibodies, both capsomeres generated by disulfide reduction of purified VLPs and reassembled VLPs formed from capsomeres upon removal of reducing agents exhibited epitopes found on the surface of authentic HPV-11 virions. Antisera raised against either purified VLP starting material or reassembled VLPs similarly neutralized infectious HPV-11 virions. The ability to disassemble and reassemble VLPs in vitro and in bulk allows basic features of capsid assembly to be studied and also opens the possibility of packaging selected exogenous compounds within the reassembled VLPs.     

Enhancement of vaccine potency by fusing modified LTK63 into human papillomavirus type 16 chimeric virus-like particles

To enhance the immunogenicity of human papillomavirus 16 (HPV 16) virus-like particles (VLPs), the modified adjuvant, mLTK63, was fused to the C-terminus of HPV 16 L2 protein. Coexpression of HPV 16 L1 and L2-mLTK63 proteins in insect cells led to the efficient assembly of HPV 16 L1/L2-mLTK63 chimeric VLPs (cVLPs), which combined the antigen and adjuvant as a unit. Compared with HPV 16 L1/L2 VLPs, the HPV 16 L1/L2-mLTK63 cVLPs had similar structural biology characteristics and binding activities with the cell surface receptors and HPV 16-specific neutralizing monoclonal antibodies. Intramuscular immunization of BALB/c mice with the HPV 16 L1/L2-mLTK63 cVLPs could induce higher titers of HPV 16-specific long-lasting neutralizing serum antibodies and stronger splenocyte proliferation, Th1- and Th2-type cytokines and CD4(+) Th responses than HPV 16 L1/L2 VLPs. The results suggested that it is possible to enhance the immunogenicity of HPV VLP vaccines via a strategy of fusing effective adjuvant protein into cVLPs.     

呈现新型冠状病毒RBD抗原病毒样颗粒的表达与鉴定*

目的:以乙型肝炎病毒核心抗原HBcAg为载体,构建呈现新冠病毒刺突蛋白受体结合域的病毒样颗粒,并鉴定其免疫原性,为新冠病毒疫苗的开发提供新思路。方法:在乙型肝炎病毒核心蛋白氨基酸编码序列第78和81位插入新冠病毒刺突蛋白受体结合域(RBD),并通过柔性linker(G4S)3进行连接,序列优化后将融合基因克隆到原核表达载体pET-28a(+),转化表达菌Rosetta,在自诱导培养基中诱导表达,菌体破碎后经蔗糖密度梯度离心,透析浓缩的方法纯化病毒样颗粒。SDS-PAGE、Western blot、透射电子显微镜检测和鉴定VLPs。将制备的VLPs与佐剂等比例混合经皮下免疫BALB/c小鼠,ELISA检测小鼠血清中特异性抗体,分析该HBc-RBD VLPs的免疫原性。结果:在自诱导培养基中,大肠埃希菌可表达部分可溶的VLPs,经蔗糖密度梯度离心纯化后在透射电子显微镜下可以观察到病毒样颗粒的存在。动物实验表明HBc-RBD VLPs刺激小鼠产生了特异性抗体。结论:在原核表达系统中成功表达了展示RBD抗原的VLPs,并通过小鼠实验初步验证了免疫原性,为新冠病毒疫苗的研发提供了新方向。     

Inhibition of Marburg Virus Budding by Nonneutralizing Antibodies to the Envelope Glycoprotein

The envelope glycoprotein (GP) of Marburg virus (MARV) and Ebola virus (EBOV) is responsible for virus entry into host cells and is known as the only target of neutralizing antibodies. While knowledge about EBOV-neutralizing antibodies and the mechanism for the neutralization of infectivity is being accumulated gradually, little is known about antibodies that can efficiently regulate MARV infectivity. Here we show that MARV GP-specific monoclonal antibodies AGP127-8 (IgG1) and MGP72-17 (IgM), which do not inhibit the GP-mediated entry of MARV into host cells, drastically reduced the budding and release of progeny viruses from infected cells. These antibodies similarly inhibited the formation of virus-like particles (VLPs) consisting of GP, the viral matrix protein, and nucleoprotein, whereas the Fab fragment of AGP127-8 showed no inhibitory effect. Morphological analyses revealed that filamentous VLPs were bunched on the surface of VLP-producing cells cultured in the presence of the antibodies. These results demonstrate a novel mechanism of the antibody-mediated inhibition of MARV budding, in which antibodies arrest unformed virus particles on the cell surface. Our data lead to the idea that such antibodies, like classical neutralizing antibodies, contribute to protective immunity against MARV and that the “classical” neutralizing activity is not the only indicator of a protective antibody that may be available for prophylactic and therapeutic use.     

Expression and characterization of virus-like particles containing rubella virus structural proteins.

Rubella virus (RV) virions contain two envelope glycoproteins (E1 and E2) and a capsid protein (C). Noninfectious RV-like particles (VLPs) containing three structural proteins were expressed in a BHK cell line (BHK-24S) by using an inducible promoter. These VLPs were found to resemble RV virons in terms of their size, their morphology, and some biological activities. In immunoblotting studies, VLPs were found to bind similarly to native RV virions with 10 of a panel of 12 RV-specific murine monoclonal antibodies. Immunization of mice with VLPs induced specific antibody responses against RV structural proteins as well as virus-neutralizing and hemagglutination-inhibiting antibodies. After immunization of mice with VLPs, in vitro challenge of isolated lymphocytes with inactivated RV and individual RV structural proteins stimulated proliferation. Our data suggest the possibility of using VLPs as immunogens for serodiagnostic assays and RV vaccines.     

VLPs Displaying a Single L2 Epitope Induce Broadly Cross-Neutralizing Antibodies against Human Papillomavirus

Background

Virus-like Particles (VLPs) display can be used to increase the immunogenicity of heterologous antigens. Here, we report the use of a bacteriophage MS2-based VLP display platform to develop a monovalent vaccine targeting a broadly neutralizing epitope in the minor capsid protein human papillomavirus (HPV) that provides broad protection from diverse HPV types in a mouse pseudovirus infection model.

Methodology/Principal Findings

Peptides spanning a previously described cross-neutralizing epitope from HPV type 16 were genetically inserted at the N-terminus of MS2 bacteriophage coat protein. Three of the four recombinant L2-coat proteins assembled into VLPs. L2-VLPs elicited high-titer anti-L2 antibodies in mice, similar to recombinant VLPs that we had previously made in which the L2 peptide was displayed on a surface-exposed loop on VLPs of a related bacteriophage, PP7. Somewhat surprisingly, L2-MS2 VLPs elicited antibodies that were much more broadly cross-reactive with L2 peptides from diverse HPV isolates than L2-PP7 VLPs. Similarly, mice immunized with L2-MS2 VLPs were protected from genital and cutaneous infection by highly diverse HPV pseudovirus types.

Conclusion/Significance

We show that peptides can be displayed in a highly immunogenic fashion at the N-terminus of MS2 coat protein VLPs. A VLP-based vaccine targeting HPV L2 elicits broadly cross-reactive and cross-protective antibodies to heterologous HPV types. L2-VLPs could serve as the basis of a broadly protective second generation HPV vaccine.     

HIV-1 virus-like particles bearing pure env trimers expose neutralizing epitopes but occlude nonneutralizing epitopes

Hypothetically, since native HIV-1 Env trimers are exclusively recognized by neutralizing antibodies, they might induce the neutralizing antibodies in a vaccine setting. This idea has not been evaluated due to the difficulty of separating trimers from nonfunctional Env (uncleaved gp160 and gp41 stumps). The latter are immunodominant and induce nonneutralizing antibodies. We previously showed that nonfunctional Env can be selectively cleared from virus-like particle (VLP) surfaces by enzyme digests (E. T. Crooks, T. Tong(,) K. Osawa, and J. M. Binley, J.Virol. 85:5825, 2011). Here, we investigated the effects of these digests on the antigenicity of VLPs and their sensitivity to neutralization. Before digestion, WT VLPs (bearing wild-type Env) and UNC VLPs (bearing uncleaved gp160) were recognized by various Env-specific monoclonal antibodies (MAbs), irrespective of their neutralizing activity, a result which is consistent with the presence of nonfunctional Env. After digestion, only neutralizing MAbs recognized WT VLPs, consistent with selective removal of nonfunctional Env (i.e., "trimer VLPs"). Digests eliminated the binding of all MAbs to UNC VLPs, again consistent with removal of nonfunctional Env. An exception was MAb 2F5, which weakly bound to digested UNC VLPs and bald VLPs (bearing no Env), perhaps due to lipid cross-reactivity. Trimer VLPs were infectious, and their neutralization sensitivity was largely comparable to that of undigested WT VLPs. However, they were ～100-fold more sensitive to the MAbs 4E10 and Z13e1, suggesting increased exposure of the gp41 base. Importantly, a scatterplot analysis revealed a strong correlation between MAb binding and neutralization of trimer VLPs. This suggests that trimer VLPs bear essentially pure native trimer that should allow its unfettered evaluation in a vaccine setting.     

Attachment and entry of recombinant Norwalk virus capsids to cultured human and animal cell lines.

Norwalk virus (NV) is the prototype strain of a group of noncultivable human caliciviruses responsible for epidemic outbreaks of acute gastroenteritis. While these viruses do not grow in tissue culture cells or animal models, expression of the capsid protein in insect cells results in the self-assembly of recombinant Norwalk virus-like particles (rNV VLPs) that are morphologically and antigenically similar to native NV. We have used these rNV VLPs to examine virus-cell interactions. Binding and internalization of VLPs to cultured human and animal cell lines were studied in an attempt to identify potentially susceptible cell lines for virus propagation in vitro and to determine if early events in the replication cycle were responsible for the narrow host range and restriction of virus growth in cell culture. Radiolabeled VLPs specifically bound to a saturable number of binding molecules on the cell surface of 13 cell lines from different origins, including human intestine (differentiated and undifferentiated Caco-2) and insect (Spodoptera frugiperda 9) ovary. Differentiated Caco-2 cells bound significantly more rNV VLPs than the other cell lines. Variations in the amount of bound VLPs among the different cell lines did not correlate with the tissue or species of origin. VLP binding was specific, as determined by competition experiments with unlabeled rNV VLPs; however, only 1.4 to 6.8% of the specifically prebound radiolabeled VLPs became internalized into cells. Blocking experiments using polygonal and monoclonal anti-rNV sera and specific antipeptide sera were performed to map the domains on rNV VLPs involved in binding to cells. One monoclonal antibody (NV8812) blocked binding of rNV VLPs to human and animal cell lines. The binding site of monoclonal antibody NV8812 was localized to the C-terminal 300 to 384 residues of the capsid protein by immunoprecipitation with truncated and cleaved forms of the capsid protein. These data suggest that the C-terminal region of the capsid protein is involved in specific binding of rNV VLPs to cells.     

Effects of downstream processing on structural integrity and immunogenicity in the manufacture of papillomavirus type 16 L1 virus-like particles

There is increasing demand for virus-like particles (VLPs) as a platform for prophylactic vaccine production. However, little attention has been paid to how downstream processing affects the structure and immunogenicity of the VLPs. In this study, we compared three methods of purifying human papillomavirus type 16 (HPV16) VLPs, each including the same cation-exchange chromatography (CEC) step. Method T-1 uses both ammonium sulfate precipitation (ASP) and a step to remove precipitated contaminating proteins (SRPC) prior to CEC, while T-2 uses only the SRPC step prior to CEC and T-3 includes neither step. We compared the structural integrity and immunogenicity of the HPV16 VLPs resulting from these three methods. All three preparations were highly pure. However, the final yields of the VLPs obtained with T-2 were 1.5 and 2 fold higher than with T-1 and T-3, respectively. With respect to structural integrity, T-1 and T-2 HPV16 VLPs had smaller hydrodynamic diameters and higher reactivity towards monoclonal anti-HPV16 neutralizing antibodies than T-3 VLPs, indicating higher potentials of T-1 and T-2 VLPs for eliciting anti-HPV16 neutralizing antibodies. Moreover, it was confirmed that the T-1 and T-2 HPV16 VLPs elicit anti-HPV16 neutralizing antibodies more efficiently than T-3 HPV16 VLPs do in mice immunizations: the abilities for eliciting neutralizing antibodies were in the order T-2 VLP > T-1 VLP > T-3 VLP. We conclude that the process design for purifying HPV VLPs is a critical determinant of the quality of the final product.     

Probing the structural states of human immunodeficiency virus type 1 pr55gag by using monoclonal antibodies

Gag-FP (fluorescent protein) fusion constructs are commonly used to study human immunodeficiency virus type 1 assembly, yielding diffuse signals throughout the cytoplasm along with punctate signals routinely described as virus-like particles (VLPs) representing assembled but unprocessed Gag. However, these particles cannot be accurately described as VLPs, since fluorescence microscopy cannot provide structural resolution. We demonstrate here that the inability of a monoclonal p24 antibody to bind its cognate epitope when unprocessed Gag is assembled distinguishes VLPs from unassembled, monomeric Gag. Furthermore, we show that assembled and unassembled Gag punctate signals travel along microtubules. These monoclonal antibody studies provide a new tool for examining retroviral assembly.     

Identification of human papillomavirus type 16 L1 surface loops required for neutralization by human sera

The variable surface loops on human papillomavirus (HPV) virions required for type-specific neutralization by human sera remain poorly defined. To determine which loops are required for neutralization, a series of hybrid virus-like particles (VLPs) were used to adsorb neutralizing activity from HPV type 16 (HPV16)-reactive human sera before being tested in an HPV16 pseudovirion neutralization assay. The hybrid VLPs used were composed of L1 sequences of either HPV16 or HPV31, on which one or two regions were replaced with homologous sequences from the other type. The regions chosen for substitution were the five known loops that form surface epitopes recognized by monoclonal antibodies and two additional variable regions between residues 400 and 450. Pretreatment of human sera, previously found to react to HPV16 VLPs in enzyme-linked immunosorbent assays, with wild-type HPV16 VLPs and hybrid VLPs that retained the neutralizing epitopes reduced or eliminated the ability of sera to inhibit pseudovirus infection in vitro. Surprisingly, substitution of a single loop often ablated the ability of VLPs to adsorb neutralizing antibodies from human sera. However, for all sera tested, multiple surface loops were found to be important for neutralizing activity. Three regions, defined by loops DE, FG, and HI, were most frequently identified as being essential for binding by neutralizing antibodies. These observations are consistent with the existence of multiple neutralizing epitopes on the HPV virion surface.     

A Japanese encephalitis virus peptide present on Johnson grass mosaic virus-like particles induces virus-neutralizing antibodies and protects mice against lethal challenge

Protection against Japanese encephalitis virus (JEV) is antibody dependent, and neutralizing antibodies alone are sufficient to impart protection. Thus, we are aiming to develop a peptide-based vaccine against JEV by identifying JEV peptide sequences that could induce virus-neutralizing antibodies. Previously, we have synthesized large amounts of Johnson grass mosaic virus (JGMV) coat protein (CP) in Escherichia coli and have shown that it autoassembled to form virus-like particles (VLPs). The envelope (E) protein of JEV contains the virus-neutralization epitopes. Four peptides from different locations within JEV E protein were chosen, and these were fused to JGMV CP by recombinant DNA methods. The fusion protein autoassembled to form VLPs that could be purified by sucrose gradient centrifugation. Immunization of mice with the recombinant VLPs containing JEV peptide sequences induced anti-peptide and anti-JEV antibodies. A 27-amino-acid peptide containing amino acids 373 to 399 from JEV E protein, present on JGMV VLPs, induced virus-neutralizing antibodies. Importantly, these antibodies were obtained without the use of an adjuvant. The immunized mice showed significant protection against a lethal JEV challenge.     

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.     

Self-Assembly and Release of Peste des Petits Ruminants Virus-Like Particles in an Insect Cell-Baculovirus System and Their Immunogenicity in Mice and Goats

Peste des petits ruminants (PPR) is an acute, febrile, viral disease of small ruminants that has a significant economic impact. For many viral diseases, vaccination with virus-like particles (VLPs) has shown considerable promise as a prophylactic approach; however, the processes of assembly and release of peste des petits ruminants virus (PPRV) VLPs are not well characterized, and their immunogenicity in the host is unknown. In this study, VLPs of PPRV were generated in a baculovirus system through simultaneous expression of PPRV matrix (M) protein and hemaglutin in (H) or fusion (F) protein. The released VLPs showed morphology similar to that of the native virus particles. Subcutaneous injection of these VLPs (PPRV-H, PPRV-F) into mice and goats elicited PPRV-specific IgG production, increased the levels of virus neutralizing antibodies, and promoted lymphocyte proliferation. Without adjuvants, the immune response induced by the PPRV-H VLPs was comparable to that obtained using equivalent amounts of PPRV vaccine. Thus, our results demonstrated that VLPs containing PPRV M protein and H or F protein are potential “differentiating infected from vaccinated animals” (DIVA) vaccine candidates for the surveillance and eradication of PPR.     

Mosaic RNA Phage VLPs Carrying Domain III of the West Nile Virus E Protein

The virus-neutralising domain III (DIII) of the West Nile virus glycoprotein E was exposed on the surface of RNA phage AP205 virus-like particles (VLPs) in mosaic form. For this purpose, a 111 amino acid sequence of DIII was added via amber or opal termination codons to the C-terminus of the AP205 coat protein, and mosaic AP205-DIII VLPs were generated by cultivation in amber- or opal-suppressing Escherichia coli strains. After extensive purification to 95 % homogeneity, mosaic AP205-DIII VLPs retained up to 11–16 % monomers carrying DIII domains. The DIII domains appeared on the VLP surface because they were fully accessible to anti-DIII antibodies. Immunisation of BALB/c mice with AP205-DIII VLPs resulted in the induction of specific anti-DIII antibodies, of which the level was comparable to that of the anti-AP205 antibodies generated against the VLP carrier. The AP205-DIII-induced anti-DIII response was represented by a significant fraction of IgG2 isotype antibodies, in contrast to parallel immunisation with the DIII oligopeptide, which failed to induce IgG2 isotype antibodies. Formulation of AP-205-DIII VLPs in alum adjuvant stimulated the level of the anti-DIII response, but did not alter the fraction of IgG2 isotype antibodies. Mosaic AP205-DIII VLPs could be regarded as a promising prototype of a putative West Nile vaccine.