Papaya Mosaic Virus Coat Protein Research Articles

Modulation of Antigen Display on PapMV Nanoparticles Influences Its Immunogenicity.

Background: The papaya mosaic virus (PapMV) vaccine platform is a rod-shaped nanoparticle made of the recombinant PapMV coat protein (CP) self-assembled around a noncoding single-stranded RNA (ssRNA) template. The PapMV nanoparticle induces innate immunity through stimulation of the Toll-like receptors (TLR) 7 and 8. The display of the vaccine antigen at the surface of the nanoparticle, associated with the co-stimulation signal via TLR7/8, ensures a strong stimulation of the immune response, which is ideal for the development of candidate vaccines. In this study, we assess the impact of where the peptide antigen is fused, whether at the surface or at the extremities of the nanoparticles, on the immune response directed to that antigen. Methods: Two different peptides from influenza A virus were used as model antigens. The conserved M2e peptide, derived from the matrix protein 2 was chosen as the B-cell epitope, and a peptide derived from the nucleocapsid was chosen as the cytotoxic T lymphocytes (CTL) epitope. These peptides were coupled at two different positions on the PapMV CP, the N- (PapMV-N) or the C-terminus (PapMV-C), using the transpeptidase activity of Sortase A (SrtA). The immune responses, both humoral and CD8+ T-cell-mediated, directed to the peptide antigens in the two different fusion contexts were analyzed and compared. The impact of coupling density at the surface of the nanoparticle was also investigated. Conclusions: The results demonstrate that coupling of the peptide antigens at the N-terminus (PapMV-N) of the PapMV CP led to an enhanced immune response to the coupled peptide antigens as compared to coupling to the C-terminus. The difference between the two vaccine platforms is linked to the enhanced capacity of the PapMV-N vaccine platform to stimulate TLR7/8. We also demonstrated that the strength of the immune response increases with the density of coupling at the surface of the nanoparticles.

A versatile papaya mosaic virus (PapMV) vaccine platform based on sortase-mediated antigen coupling

BackgroundFlexuous rod-shaped nanoparticles made of the coat protein (CP) of papaya mosaic virus (PapMV) have been shown to trigger innate immunity through engagement of toll-like receptor 7 (TLR7). PapMV nanoparticles can also serve as a vaccine platform as they can increase the immune response to fused peptide antigens. Although this approach shows great potential, fusion of antigens directly to the CP open reading frame (ORF) is challenging because the fused peptides can alter the structure of the CP and its capacity to self assemble into nanoparticles—a property essential for triggering an efficient immune response to the peptide. This represents a serious limitation to the utility of this approach as fusion of small peptides only is tolerated.ResultsWe have developed a novel approach in which peptides are fused directly to pre-formed PapMV nanoparticles. This approach is based on the use of a bacterial transpeptidase (sortase A; SrtA) that can attach the peptide directly to the nanoparticle. An engineered PapMV CP harbouring the SrtA recognition motif allows efficient coupling. To refine our engineering, and to predict the efficacy of coupling with SrtA, we modeled the PapMV structure based on the known structure of PapMV CP and on recent reports revealing the structure of two closely related potexviruses: pepino mosaic virus (PepMV) and bamboo mosaic virus (BaMV). We show that SrtA can allow the attachment of long peptides [Influenza M2e peptide (26 amino acids) and the HIV-1 T20 peptide (39 amino acids)] to PapMV nanoparticles. Consistent with our PapMV structural model, we show that around 30% of PapMV CP subunits in each nanoparticle can be fused to the peptide antigen. As predicted, engineered nanoparticles were capable of inducing a strong antibody response to the fused antigen. Finally, in a challenge study with influenza virus, we show that mice vaccinated with PapMV-M2e are protected from infection.ConclusionsThis technology will allow the development of vaccines harbouring long peptides containing several B and/or T cell epitopes that can contribute to a broad and robust protection from infection. The design can be fast, versatile and can be adapted to the development of vaccines for many infectious diseases as well as cancer vaccines.

Engineering of the PapMV vaccine platform with a shortened M2e peptide leads to an effective one dose influenza vaccine

The emergence of highly virulent influenza strains and the risks of pandemics as well as the limited efficiency of the current seasonal vaccines are important public health concerns. There is a major need for new influenza vaccines that would be broadly cross-protective. The ectodomain of matrix protein 2 (M2e) is highly conserved amongst different influenza strains and could be used as a broad spectrum antigen. To overcome its low immunogenicity we have fused a short peptide epitope derived from the human consensus sequence of M2e (amino acids 6–14, EVETPIRNE) to the N-terminus of papaya mosaic virus coat protein. The fusion harboring coat proteins were assembled around a single stranded RNA into virus-like particles (PapMV-sM2e). The resulting PapMV-sM2e rod-shaped particle was stable and indistinguishable from regular PapMV particles. A single intramuscular immunization with PapMV-sM2e was sufficient to mount appreciable levels of CD4 dependent M2e specific total IgG and IgG2a antibody in mice sera. PapMV-sM2e proved to be self-adjuvanting since the addition of PapMV as an exogenous adjuvant did not result in significantly improved antibody titers. In addition, we confirmed the adjuvant property of PapMV-sM2e using the trivalent inactivated flu vaccine as antigen and demonstrated that the newly engineered nanoparticles areas efficacious as an adjuvant than the original PapMV nanoparticles. Upon infection with a sub-lethal dose of influenza, PapMV-sM2e vaccinated animals were completely protected from virus induced morbidity and mortality. Mice immunized with decreasing amounts of PapMV-sM2e and challenged with a more stringent dose of influenza virus displayed dose-dependent levels of protection. Seventy percent of the mice immunized once with the highest dose of PapMV-sM2e survived the challenged. The survival of the mice correlated mainly with the levels of anti-M2e IgG2a antibodies obtained before the infection. These results demonstrate that PapMV-sM2e can be an important component of a broadly cross-reactive influenza vaccine.

Engineering of papaya mosaic virus (PapMV) nanoparticles with a CTL epitope derived from influenza NP

BackgroundThe ever-present threat of infectious disease, e.g. influenza pandemics, and the increasing need for new and effective treatments in immunotherapy are the driving forces that motivate research into new and innovative vaccine platforms. Ideally, such platforms should trigger an efficient CTL response, be safe, and easy to manufacture. We recently developed a novel nanoparticle adjuvant comprised of papaya mosaic virus (PapMV) coat protein (CP) assembled around an RNA. The PapMV nanoparticle is an efficient vaccine platform in which the peptide antigen is fused to the C-terminus of the PapMV CP, leading to nanoparticles presenting surface-exposed epitope. The fusion stabilizes the epitope and improves its immunogenicity. We found recently that C-terminal fusions are not always efficient, depending on the nature of the peptide fused to the platform.ResultsWe chose a CTL epitope derived from the nucleocapsid (NP) of influenza virus (NP147-155) for this proof-of-concept demonstration. Recombinant nanoparticles harbouring a fusion at the N-terminus were more efficient in triggering a CTL response. Efficacy appeared to be linked to the stability of the nanoparticles at 37°C. We also showed that discs—smaller than nanoparticles—made of 20 subunits of PapMV CP are less efficient for induction of a CTL response in mice, revealing that assembly of the recombinant PapMV CP into nanoparticles is crucial to triggering an efficient CTL response.ConclusionThe point of fusion on the PapMV vaccine platform is critical to triggering an efficient CTL response. Efficacy is linked to nanoparticle stability; nanoparticles must be stable at 37°C but remain susceptible to cellular proteases to ensure efficient processing of the CTL epitope by cells of the immune system. The results of this study improve our understanding of the PapMV vaccine platform, which will facilitate the design of efficient vaccines to various infectious threats.

Crystal Structure of the Coat Protein of the Flexible Filamentous Papaya Mosaic Virus

Papaya mosaic virus (PapMV) is a filamentous plant virus that belongs to the Alphaflexiviridae family. Flexible filamentous viruses have defied more than two decades of effort in fiber diffraction, and no high-resolution structure is available for any member of the Alphaflexiviridae family. Here, we report our structural characterization of PapMV by X-ray crystallography and cryo-electron microscopy three-dimensional reconstruction. We found that PapMV is 135Å in diameter with a helical symmetry of ~10 subunits per turn. Crystal structure of the C-terminal truncated PapMV coat protein (CP) reveals a novel all-helix fold with seven α-helices. Thus, the PapMVCP structure is different from the four-helix-bundle fold of tobacco mosaic virus in which helix bundling dominates the subunit interface in tobacco mosaic virus and conveys rigidity to the rod virus. PapMV CP was crystallized as an asymmetrical dimer in which one protein lassoes the other by the N-terminal peptide. Mutation of residues critical to the inter-subunit lasso interaction abolishes CP polymerization. The crystal structure suggests that PapMV may polymerize via the consecutive N-terminal loop lassoing mechanism. The structure of PapMV will be useful for rational design and engineering of the PapMV nanoparticles into innovative vaccines.

Engineering of Papaya Mosaic Virus (PapMV) Nanoparticles through Fusion of the HA11 Peptide to Several Putative Surface-Exposed Sites

Papaya mosaic virus has been shown to be an efficient adjuvant and vaccine platform in the design and improvement of innovative flu vaccines. So far, all fusions based on the PapMV platform have been located at the C-terminus of the PapMV coat protein. Considering that some epitopes might interfere with the self-assembly of PapMV CP when fused at the C-terminus, we evaluated other possible sites of fusion using the influenza HA11 peptide antigen. Two out of the six new fusion sites tested led to the production of recombinant proteins capable of self assembly into PapMV nanoparticles; the two functional sites are located after amino acids 12 and 187. Immunoprecipitation of each of the successful fusions demonstrated that the HA11 epitope was located at the surface of the nanoparticles. The stability and immunogenicity of the PapMV-HA11 nanoparticles were evaluated, and we could show that there is a direct correlation between the stability of the nanoparticles at 37°C (mammalian body temperature) and the ability of the nanoparticles to trigger an efficient immune response directed towards the HA11 epitope. This strong correlation between nanoparticle stability and immunogenicity in animals suggests that the stability of any nanoparticle harbouring the fusion of a new peptide should be an important criterion in the design of a new vaccine.

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.

Purification and biochemical characterization of a monomeric form of papaya mosaic potexvirus coat protein

Papaya mosaic virus (PapMV) is a flexuous rod shape virus made of 1400 subunits that assemble around a plus sense genomic RNA. The structure determination of PapMV and of flexuous viruses in general is a major challenge for both NMR and X-ray crystallography. In this report, we present the characterization of a truncated version of the PapMV coat protein (CP) that is suitable for NMR study. The deletion of the N-terminal 26 amino acids of the PapMV CP (CP 27–215) generates a monomer that can be expressed to high level and easily purified for production of an adequate NMR sample. The RNA gel shift assay showed that CP 27–215 lost its ability to bind RNA in vitro, suggesting that the multimerization of the subunit is important for this function. The fusion of a 6× His tag at the C-terminus improved the solubility of the monomer and allowed its concentration to 0.2 mM. The CD spectra of the truncated and the wild-type proteins were similar, suggesting that both proteins are well ordered and have a similar secondary structure. CP 27–215 was 15N labeled for NMR studies and a 2D 1H– 15N-HSQC spectrum confirmed the presence of a well-ordered structure and the monomeric form of the protein. These results show that CP 27–215 is amenable to a complete and exhaustive NMR study that should lead to the first three-dimensional structure determination of a flexuous rod shape virus.

Crystallization and Preliminary X-ray Analysis of Papaya Mosaic Virus Coat Protein

Papaya mosaic virus coat protein has been treated with trypsin and a large fragment of the intact protein has been crystallized in space group P 3121 or P3221 (unit cell dimensions: a = b = 110 Å, c = 237 Å.). The crystals diffract to 3·5 Å resolution. Crystals of the untreated protein have also been grown. The untreated protein crystals diffract to 4 Å resolution, but have a large mosaic spread. They have the same space group as the trypsin-treated protein crystals, but a much smaller unit cell (a = b = 72 Å, c = 240 Å).

The primary structure of papaya mosaic virus coat protein: a revision.

The presence of an acetyl blocking group at the N-terminus of the coat protein of papaya mosaic virus has been identified by FAB mass spectrometry. Furthermore, we have found that the N-terminal sequence of the protein is four amino-acid residues (AC-Ser-Lys-Ser-Ser-) longer than that previously reported, while Glu instead of Gln is the C-terminal residue. The present paper shows that PMV-protein is made up of 215 amino acid residues, with a molecular mass of 22,960 Da.

The primary structure of papaya mosaic virus coat protein

The amino acid sequence of the coat protein of the potexvirus papaya mosaic virus (PMV) has been determined, using a combination of the Edman degradation procedure (manual and automated) and mass spectrometry. The amino acid sequence is compared with that of the coat protein of potato virus X (PVX) as reported (S. Yu. Morozov, V. M. Zakhariev, B. K. Chernov, V. S. Prasolov, Yu. V. Kozlov, J. G. Atabekov, and K. G. Skyabin, Dokl. Acad. Nauk, SSSR 271, 211–215,1983). PMV has 73 of 211 amino acid residues in common with PVX and has 25 fewer residues in the polypeptide chain. Also 10 of a possible 16 proline residues are in similar positions in both proteins, including a sequence of 3 prolines at the carboxyl end. Furthermore, the 2 cysteine residues in PMV correspond with 2 of the 3 cysteines in PVX.

Circular dichroism studies of papaya mosaic virus coat protein and its polymers

The near-u.v. circular dichroism of papaya mosaic virus coat protein is dominated by intrasubunit interactions involving tryptophan and, to a much lesser extent, phenylalanine. The conformations of the 14 S protein discs at pH 4.0, pH 6.0 and pH 8.0 are judged to be similar by circular dichroism. At pH 8.0, however, the conformation of the protein is influenced by the extent of aggregation, which is reflected in the intensity, but not the shape, of the circular dichroism spectrum. The increase in intensity level that accompanies aggregation at pH 8.0 mainly appears to reflect modifications of tryptophanyl interactions. On the other hand, no spectral changes result when discs polymerize to form helical tubes at pH 4.0, indicating that a major conformational change is not requisite for helix formation. The above results are interpreted in view of the various protein and virus assembly reactions in which papaya mosaic virus coat protein participates. Denaturation of the coat protein results in quenching of optical activity in the near-u.v. region, and also in marked changes in the u.v. spectrum of the protein. These results indicate that the majority of the tryptophan and tyrosine residues are buried within a hydrophobic environment in the undenatured protein.