RESUMO
Plant-virus-based vaccines have emerged as a promising avenue in vaccine development. This report describes the engineering of an innovative vaccine platform using the papaya mosaic virus (PapMV) capsid protein (CP) as a carrier protein and a C-terminal fused hepatitis C virus (HCV) E2 epitope as the immunogenic target. Two antigen organizations of the PapMV-based vaccines were tested: a virus-like-particle (VLP; PapMVCP-E2) and a monomeric form (PapMVCP(27-215)-E2). While the two forms of the vaccine were both shown to be actively internalized in vitro in bone-marrow-derived antigen presenting cells (APCs), immunogenicity was demonstrated to be strongly dependent on antigen organization. Indeed, C3H/HeJ mice injected twice with the multimeric VLP vaccine showed a long-lasting humoral response (more than 120 days) against both the CP and the fused HCV E2 epitope. The antibody profile (production of IgG1, IgG2a, IgG2b, IgG3) suggests a Th1/Th2 response. Immunogenicity of the PapMV vaccine platform was not observed when the monomer PapMVCP-E2 was injected. These results demonstrate for the first time the potential of the PapMV vaccine platform and the critical function of multimerization in its immunogenicity.
Assuntos
Carica/virologia , Epitopos/imunologia , Engenharia Genética , Hepacivirus/imunologia , Vírus do Mosaico/genética , Proteínas do Envelope Viral/imunologia , Vacinas Virais/imunologia , Animais , Células da Medula Óssea/citologia , Proteínas do Capsídeo/genética , Proteínas do Capsídeo/metabolismo , Células Dendríticas/citologia , Células Dendríticas/imunologia , Hepacivirus/genética , Anticorpos Anti-Hepatite C/imunologia , Antígenos da Hepatite C/genética , Antígenos da Hepatite C/imunologia , Humanos , Camundongos , Vírus do Mosaico/fisiologia , RNA/genética , Proteínas do Envelope Viral/genética , Vacinas Virais/química , Vacinas Virais/genéticaRESUMO
Papaya mosaic potexvirus (PapMV) coat protein (CP) was expressed (CPdeltaN5) 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 CPdeltaN5 and 100% percent of the protein was found as NLPs in bacteria. E128A NLPs were more resistant to digestion by trypsin than the CPdeltaN5 but were more sensitive to denaturation by heat. We discuss the possible role of K97 and E128 in the assembly of PapMV.
Assuntos
Proteínas do Capsídeo/genética , Vírus do Mosaico/genética , Mutação , Potexvirus/genética , RNA Viral/metabolismo , Montagem de Vírus/genética , Sequência de Aminoácidos , Sítios de Ligação , Cromatografia em Gel , Dicroísmo Circular , Escherichia coli/genética , Escherichia coli/metabolismo , Imuno-Histoquímica , Dados de Sequência Molecular , Vírus do Mosaico/metabolismo , Vírus do Mosaico/fisiologia , Nucleocapsídeo/química , Nucleocapsídeo/genética , Nucleocapsídeo/metabolismo , Potexvirus/fisiologia , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Alinhamento de Sequência , Temperatura , Tripsina/metabolismo , Montagem de Vírus/fisiologiaRESUMO
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 (CP27-215) 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 CP27-215 lost its ability to bind RNA in vitro, suggesting that the multimerization of the subunit is important for this function. The fusion of a 6x 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. CP27-215 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 CP27-215 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.
