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1.
J Nanobiotechnology ; 14(1): 43, 2016 Jun 10.
Artigo em Inglês | MEDLINE | ID: mdl-27282291

RESUMO

BACKGROUND: The addition of an adjuvant to a vaccine is a promising approach to increasing strength and immunogenicity towards antigens. Despite the fact that adjuvants have been used in vaccines for decades, their mechanisms of action and their influence on the kinetics of the immune response are still not very well understood. The use of papaya mosaic virus (PapMV) nanoparticles-a novel TLR7 agonist-was recently shown to improve and broaden the immune response directed to trivalent inactivated flu vaccine (TIV) in mice and ferrets. RESULTS: We investigated the capacity of PapMV nanoparticles to increase the speed of the immune response toward TIV. PapMV nanoparticles induced a faster and stronger humoral response to TIV that was measured as early as 5 days post-immunization. The addition of PapMV nanoparticles was shown to speed up the differentiation of B-cells into early plasma cells, and increased the growth of germinal centers in a CD4+ dependent manner. TIV vaccination with PapMV nanoparticles as an adjuvant protected mice against a lethal infection as early as 10 days post-immunization. CONCLUSION: In conclusion, PapMV nanoparticles are able to accelerate a broad humoral response to TIV. This property is of the utmost importance in the field of vaccination, especially in the case of pandemics, where populations need to be protected as soon as possible after vaccination.


Assuntos
Adjuvantes Imunológicos/uso terapêutico , Formação de Anticorpos , Vacinas contra Influenza/uso terapêutico , Vírus do Mosaico/imunologia , Nanopartículas/uso terapêutico , Infecções por Orthomyxoviridae/prevenção & controle , Vacinas de Produtos Inativados/uso terapêutico , Adjuvantes Imunológicos/química , Animais , Anticorpos Antivirais/imunologia , Linfócitos B/imunologia , Linfócitos B/virologia , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/virologia , Carica/virologia , Feminino , Imunização , Vacinas contra Influenza/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Vírus do Mosaico/química , Nanopartículas/química , Nanopartículas/virologia , Orthomyxoviridae/imunologia , Infecções por Orthomyxoviridae/imunologia , Vacinas de Produtos Inativados/imunologia
2.
Vaccine ; 33(51): 7245-7253, 2015 Dec 16.
Artigo em Inglês | MEDLINE | ID: mdl-26549362

RESUMO

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.


Assuntos
Portadores de Fármacos , Vacinas contra Influenza/administração & dosagem , Vacinas contra Influenza/imunologia , Potexvirus/genética , Vacinas de Partículas Semelhantes a Vírus/administração & dosagem , Vacinas de Partículas Semelhantes a Vírus/imunologia , Proteínas da Matriz Viral/imunologia , Animais , Anticorpos Antivirais/sangue , Proteínas do Capsídeo/genética , Modelos Animais de Doenças , Relação Dose-Resposta Imunológica , Imunoglobulina G/sangue , Vacinas contra Influenza/genética , Injeções Intramusculares , Camundongos Endogâmicos BALB C , Infecções por Orthomyxoviridae/patologia , Infecções por Orthomyxoviridae/prevenção & controle , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/imunologia , Análise de Sobrevida , Vacinas Sintéticas/administração & dosagem , Vacinas Sintéticas/genética , Vacinas Sintéticas/imunologia , Vacinas de Partículas Semelhantes a Vírus/genética , Proteínas da Matriz Viral/genética
3.
J Mol Biol ; 422(2): 263-73, 2012 Sep 14.
Artigo em Inglês | MEDLINE | ID: mdl-22659319

RESUMO

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.


Assuntos
Proteínas do Capsídeo/química , Carica/virologia , Sequência de Aminoácidos , Proteínas do Capsídeo/metabolismo , Carica/metabolismo , Microscopia Crioeletrônica , Cristalografia por Raios X , Dados de Sequência Molecular , Vírus de Plantas/genética , Vírus de Plantas/metabolismo
4.
Vaccine ; 28(34): 5617-26, 2010 Aug 02.
Artigo em Inglês | MEDLINE | ID: mdl-20600515

RESUMO

Chimeric VLPs made of papaya mosaic virus (PapMV) trigger a CTL response through antigenic presentation of epitopes on MHC class I. Here, a chimeric VLP composed of malva mosaic virus (MaMV) was shown to share similar properties. We demonstrated the capacity of both VLPs to enter human APCs. The chimeric constructions were cross-presented in CD40-activated B lymphocytes leading to in vitro expansion of antigen-specific T lymphocytes. We showed that high concentrations of chimeric MaMV induced cell death, suggesting that some modifications can trigger collateral effects in vitro. Results suggest that potexvirus VLPs are an attractive vaccine platform for inducing a CTL response.


Assuntos
Apresentação de Antígeno , Apresentação Cruzada , Epitopos/imunologia , Potexvirus/imunologia , Sequência de Aminoácidos , Linfócitos B/imunologia , Antígenos CD40/imunologia , Proteínas do Capsídeo/imunologia , Proliferação de Células , Clonagem Molecular , Humanos , Dados de Sequência Molecular , Engenharia de Proteínas , Linfócitos T Citotóxicos/imunologia
5.
FEBS J ; 273(1): 14-25, 2006 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-16367744

RESUMO

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/fisiologia
6.
Protein Expr Purif ; 47(1): 273-80, 2006 May.
Artigo em Inglês | MEDLINE | ID: mdl-16310377

RESUMO

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.


Assuntos
Proteínas do Capsídeo/química , Proteínas do Capsídeo/isolamento & purificação , Carica/virologia , Potexvirus/química , Sequência de Aminoácidos , Proteínas do Capsídeo/genética , Clonagem Molecular , Escherichia coli , Dados de Sequência Molecular , Potexvirus/genética
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