Nathalie Majeau

Effect of mutations K97A and E128A on RNA binding and self assembly of papaya mosaic potexvirus coat protein

Papaya mosaic potexvirus (PapMV) coat protein (CP) was expressed (CPΔN5) in Escherichia coli and showed to self assemble into nucleocapsid like particles (NLPs). Twenty per cent of the purified protein was found as NLPs of 50 nm in length and 80% was found as a multimer of 450 kDa (20 subunits) arranged in a disk. Two mutants in the RNA binding domain of the PapMV CP, K97A and E128A showed interesting properties. The proteins of both mutants could be easily purified and CD spectra of these proteins showed secondary and tertiary structures similar to the WT protein. The mutant K97A was unable to self assemble and bind RNA. On the contrary, the mutant E128A showed an improved affinity for RNA and self assembled more efficiently in NLPs. E128A NLPs were longer (150 nm) than the recombinant CPΔN5 and 100% percent of the protein was found as NLPs in bacteria. E128A NLPs were more resistant to digestion by trypsin than the CPΔN5 but were more sensitive to denaturation by heat. We discuss the possible role of K97 and E128 in the assembly of PapMV.

Improvement of the Trivalent Inactivated Flu Vaccine Using PapMV Nanoparticles

Commercial seasonal flu vaccines induce production of antibodies directed mostly towards hemaglutinin (HA). Because HA changes rapidly in the circulating virus, the protection remains partial. Several conserved viral proteins, e.g., nucleocapsid (NP) and matrix proteins (M1), are present in the vaccine, but are not immunogenic. To improve the protection provided by these vaccines, we used nanoparticles made of the coat protein of a plant virus (papaya mosaic virus; PapMV) as an adjuvant. Immunization of mice and ferrets with the adjuvanted formulation increased the magnitude and breadth of the humoral response to NP and to highly conserved regions of HA. They also triggered a cellular mediated immune response to NP and M1, and long-lasting protection in animals challenged with a heterosubtypic influenza strain (WSN/33). Thus, seasonal flu vaccine adjuvanted with PapMV nanoparticles can induce universal protection to influenza, which is a major advancement when facing a pandemic.

Palmitoylation of hepatitis C virus core protein is important for virion production

Hepatitis C virus core protein is the viral nucleocapsid of hepatitis C virus. Interaction of core with cellular membranes like endoplasmic reticulum (ER) and lipid droplets (LD) appears to be involved in viral assembly. However, how these interactions with different cellular membranes are regulated is not well understood. In this study, we investigated how palmitoylation, a post-translational protein modification, can modulate the targeting of core to cellular membranes. We show that core is palmitoylated at cysteine 172, which is adjacent to the transmembrane domain at the C-terminal end of core. Site-specific mutagenesis of residue Cys172 showed that palmitoylation is not involved in the maturation process carried out by the signal peptide peptidase or in the targeting of core to LD. However, palmitoylation was shown to be important for core association with smooth ER membranes and ER closely surrounding LDs. Finally, we demonstrate that mutation of residue Cys172 in the J6/JFH1 virus genome clearly impairs virion production.

Translating innate response into long-lasting antibody response by the intrinsic antigen-adjuvant properties of papaya mosaic virus

Identifying the properties of a molecule involved in the efficient activation of the innate and adaptive immune responses that lead to long‐lasting immunity is crucial for vaccine and adjuvant development. Here we show that the papaya mosaic virus (PapMV) is recognized by the immune system as a pathogen‐associated molecular pattern (PAMP) and as an antigen in mice (Pamptigen). A single immunization of PapMV without added adjuvant efficiently induced both cellular and specific long‐lasting antibody responses. PapMV also efficiently activated innate immune responses, as shown by the induction of lipid raft aggregation, secretion of pro‐inflammatory cytokines, up‐regulation of co‐stimulatory molecules on dendritic cells and macrophages, and long‐lasting adjuvant effects upon the specific antibody responses to model antigens. PapMV mixed with Salmonella enterica serovar Typhi (S. typhi) outer membrane protein C increased its protective capacity against challenge with S. typhi, revealing the intrinsic adjuvant properties of PapMV in the induction of immunity. Antigen‐presenting cells loaded with PapMV efficiently induced antibody responses in vivo, which may link the innate and adaptive responses observed. PapMV recognition as a Pamptigen might be translated into long‐lasting antibody responses and protection observed. These properties could be used in the development of new vaccine platforms.

Structure and dynamics of the N-terminal half of hepatitis C virus core protein: an intrinsically unstructured protein.

Hepatitis C virus core protein plays an important role in the assembly and packaging of the viral genome. We have studied the structure of the N-terminal half of the core protein (C82) which was shown to be sufficient for the formation of nucleocapsid-like particle (NLP) in vitro and in yeast. Structural bioinformatics analysis of C82 suggests that it is mostly unstructured. Circular dichroism and structural NMR data indicate that C82 lacks secondary structure. Moreover, NMR relaxation data shows that C82 is highly disordered. These results indicate that the N-terminal half of the HCV core protein belongs to the growing family of intrinsically unstructured proteins (IUP). This explains the tendency of the hepatitis C virus core protein to interact with several host proteins, a well-documented characteristic of IUPs.

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.

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.

Improvement of the PapMV nanoparticle adjuvant property through an increased of its avidity for the antigen (influenza NP)

The principal caveat of existing influenza vaccine is their failure to provide long-term protection. This lack of efficiency is caused by persistent (drift) and dramatic (shift) antigenic changes on the major surface proteins, the main target of protective immunity generated by traditional vaccines. Alternatively, vaccination with most conserved protein, like the nucleoprotein (NP) can stimulate immunity against multiple serotypes and could potentially provides an extended protection. The NP antigen contains more than 90% protein sequence homology among influenza A isolates and it also contains dominant CTL targets epitopes that made this antigen an attractive target for developing universal vaccine. However, NP protein is a weak antigen and need the use of adjuvant to increase its immunogenicity. We have developed an innovative high avidity VLP (HAV) nanoparticle to improve its adjuvant property to the NP antigen. The nanoparticles are derived from papaya mosaic virus capsid protein (PapMV CP) produced in a bacteria expression system. We generated the HAV by adding an affinity peptide directed to the NP protein at the surface of the VLPs. The fusions of the affinity peptide to PapMV VLPs increased the avidity of PapMV VLPs to NP protein. This modification enhanced the humoral and the IFN-γ response directed to NP. Moreover, the immunity generated by the HAV adjuvanted NP vaccine improved the protection of vaccinated mice to a challenge with influenza virus. The protection was characterized by accelerated virus elimination after the onset of infection and rapid recovery of the vaccinated animals.

Mapping the surface-exposed regions of papaya mosaic virus nanoparticles

In general, the structure of the papaya mosaic virus (PapMV) and other members of the potexviruses is poorly understood. Production of PapMV coat proteins in a bacterial expression system and their self‐assembly in vitro into nanoparticles is a very useful tool to study the structure of this virus. Using recombinant PapMV nanoparticles that are similar in shape and appearance to the plant virus, we evaluated surface‐exposed regions by two different methods, immunoblot assay and chemical modification with 1‐ethyl‐3‐(3‐dimethylaminopropyl)carbodiimide or diethyl‐pyrocarbonate followed by mass spectrometry. Three regions were targeted by the two techniques. The N‐ and C‐termini were shown to be surfaced exposed as expected. However, the region 125–136 was revealed for the first time as the major surface‐exposed region of the nanoparticles. The presence of linear peptides at the surface was finally confirmed using antibodies directed to those peptides. It is likely that region 125–136 plays a key role in the lifecycle of PapMV and other members of the potexvirus group.

Interfering with hepatitis C virus assembly in vitro using affinity peptides directed towards core protein

Viral assembly is a crucial key step in the life cycle of every virus. In the case of Hepatitis C virus (HCV), the core protein is the only structural protein to interact directly with the viral genomic RNA. Purified recombinant core protein is able to self-assemble in vitro into nucleocapsid-like particles upon addition of a structured RNA, providing a robust assay with which to study HCV assembly. Inhibition of self-assembly of the C170 core protein (first 170 amino acids) was tested using short peptides derived from the HCV core, from HCV NS5A protein, and from diverse proteins (p21 and p73) known to interact with HCV core protein. Interestingly, peptides derived from the core were the best inhibitors. These peptides are derived from regions of the core predicted to be involved in the interaction between core subunits during viral assembly. We also demonstrated that a peptide derived from the C-terminal end of NS5A protein moderately inhibits the assembly process.