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Protein Sci ; 24(5): 633-42, 2015 May.
Artigo em Inglês | MEDLINE | ID: mdl-25559821


Equine lentivirus receptor 1 (ELR1) has been identified as a functional cellular receptor for equine infectious anemia virus (EIAV). Herein, recombinant ELR1 and EIAV surface glycoprotein gp90 were respectively expressed in Drosophila melanogaster S2 cells, and purified to homogeneity by Ni-NTA affinity chromatography and gel filtration chromatography. Gel filtration chromatography and analytical ultracentrifugation (AUC) analyses indicated that both ELR1 and gp90 existed as individual monomers in solution and formed a complex with a stoichiometry of 1:1 when mixed. The structure of ELR1 was first determined with the molecular replacement method, which belongs to the space group P42 21 2 with one molecule in an asymmetric unit. It contains eight antiparallel ß-sheets, of which four are in cysteine rich domain 1 (CRD1) and two are in CRD2 and CRD3, respectively. Alignment of ELR1 with HVEM and CD134 indicated that Tyr61, Leu70, and Gly72 in CRD1 of ELR1 are important residues for binding to gp90. Isothermal titration calorimetry (ITC) experiments further confirmed that Leu70 and Gly72 are the critical residues.

Lentivirus Equinos/química , Glicoproteínas de Membrana/química , Estrutura Secundária de Proteína , Receptores Virais/química , Proteínas Recombinantes/química , Animais , Drosophila melanogaster , Anemia Infecciosa Equina/genética , Anemia Infecciosa Equina/virologia , Cavalos/virologia , Vírus da Anemia Infecciosa Equina/patogenicidade , Glicoproteínas de Membrana/biossíntese , Glicoproteínas de Membrana/genética , Complexos Multiproteicos/química , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/genética
BMC Cell Biol ; 11: 73, 2010 Sep 23.
Artigo em Inglês | MEDLINE | ID: mdl-20863390


BACKGROUND: Adipose-Derived Stromal Cells have been shown to have multiple lineage differentiation properties and to be suitable for tissues regeneration in many degenerative processes. Their use has been proposed for the therapy of joint diseases and tendon injuries in the horse. In the present report the genetic manipulation of Equine Adipose-Derived Stromal Cells has been investigated. RESULTS: Equine Adipose-Derived Stromal Cells were successfully virally transduced as well as transiently and stably transfected with appropriate parameters, without detrimental effect on their differentiation properties. Moreover, green fluorescent protein alone, fused to neo gene, or co-expressed as bi-cistronic reporter constructs, driven by viral and house-keeping gene promoters, were tested. The better expressed cassette was employed to stably transfect Adipose-Derived Stromal Cells for cell therapy purposes. Stably transfected Equine Adipose-Derived Stromal Cells with a heterologous secreted viral antigen were able to immunize horses upon injection into the lateral wall of the neck. CONCLUSION: This study provides the methods to successfully transgenize Adipose-Derived Stromal Cells both by lentiviral vector and by transfection using optimized constructs with suitable promoters and reporter genes. In conclusion these findings provide a working platform for the delivery of potentially therapeutic proteins to the site of cells injection via transgenized Equine Adipose-Derived Stromal Cells.

Artropatias/terapia , Infecções por Lentivirus/terapia , Células Estromais/transplante , Transdução Genética/métodos , Transfecção/métodos , Tecido Adiposo/citologia , Animais , Antígenos Virais/genética , Antígenos Virais/imunologia , Antígenos Virais/metabolismo , Diferenciação Celular/genética , Clonagem Molecular , Estudos de Viabilidade , Terapia Genética , Regeneração Tecidual Guiada , Cavalos , Imunidade/genética , Imunização , Artropatias/patologia , Infecções por Lentivirus/genética , Infecções por Lentivirus/imunologia , Lentivirus Equinos , Transplante de Células-Tronco , Células Estromais/imunologia , Células Estromais/metabolismo , Células Estromais/patologia
Proc Natl Acad Sci U S A ; 104(38): 15105-10, 2007 Sep 18.
Artigo em Inglês | MEDLINE | ID: mdl-17846425


Lentiviral envelope antigenic variation and associated immune evasion are believed to present major obstacles to effective vaccine development. Although this perception is widely assumed by the scientific community, there is, to date, no rigorous experimental data assessing the effect of increasing levels of lentiviral Env variation on vaccine efficacy. It is our working hypothesis that Env is, in fact, a primary determinant of vaccine effectiveness. We previously reported that a successful experimental attenuated equine infectious anemia virus vaccine, derived by mutation of the viral S2 accessory gene, provided 100% protection from disease after virulent virus challenge. Here, we sought to comprehensively test our hypothesis by challenging vaccinated animals with proviral strains of defined, increasing Env variation, using variant envelope SU genes that arose naturally during experimental infection of ponies with equine infectious anemia virus. The reference attenuated vaccine combined with these variant Env challenge strains facilitated evaluation of the protection conferred by ancestral immunogens, because the Env of the attenuated vaccine is a direct ancestor to the variant proviral strain Envs. The results demonstrated that ancestral Env proteins did not impart broad levels of protection against challenge. Furthermore, the results displayed a significant inverse linear correlation of Env divergence and protection from disease. This study demonstrates potential obstacles to the use of single isolate ancestral Env immunogens. Finally, these findings reveal that relatively minor Env variation can pose a substantial challenge to lentiviral vaccine immunity, even when attenuated vaccines are used that, to date, achieve the highest levels of vaccine protection.

Variação Antigênica , Produtos do Gene env/imunologia , Vacinas Virais/imunologia , Animais , Anticorpos Antivirais/imunologia , Anemia Infecciosa Equina/imunologia , Anemia Infecciosa Equina/prevenção & controle , Feminino , Cavalos , Vírus da Anemia Infecciosa Equina/imunologia , Lentivirus Equinos/patogenicidade , Masculino , Fatores de Tempo , Proteínas Virais/imunologia , Vacinas Virais/administração & dosagem , Virulência
Immunogenetics ; 55(7): 508-14, 2003 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-12942208


Three moderately to broadly recognized equine infectious anemia virus (EIAV) peptides that contained helper T-lymphocyte (Th) 1 epitopes were previously identified. Although lipopeptide immunization was only weakly immunostimulatory in a preliminary study, as measured by T-lymphocyte proliferation responses, it was of interest to define additional broadly recognized Th1 epitopes to include in future immunization trials. Using broadly cross-reactive and conserved Th epitopes known in the related human immunodeficiency virus-1 (HIV-1) and binding motifs defined in human leukocyte antigen DR molecules as guides, this work identified three new peptides containing Th1 epitopes recognized by 60-75% of EIAV infected horses. The observed similarity across species of major histocompatibility complex (MHC) class II binding motifs and the conservation of Th peptides between related viruses should allow easier targeting of Th epitope regions in less well characterized pathogens and/or in species whose MHC class II molecules are poorly defined.

Proteínas de Ligação a DNA/imunologia , HIV-1/imunologia , Antígenos HLA-DR/imunologia , Lentivirus Equinos/imunologia , Peptídeos/imunologia , Fatores de Transcrição/imunologia , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos , Cavalos/imunologia , Humanos