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1.
Virol J ; 3: 83, 2006 Oct 02.
Artigo em Inglês | MEDLINE | ID: mdl-17014700

RESUMO

BACKGROUND: Infections with papillomaviruses induce type-specific immune responses, mainly directed against the major capsid protein, L1. Based on the propensity of the L1 protein to self-assemble into virus-like particles (VLPs), type-specific vaccines have already been developed. In order to generate vaccines that target a broader spectrum of HPV types, extended knowledge of neutralizing epitopes is required. Despite the association of human papillomavirus type 33 (HPV33) with cervical carcinomas, fine mapping of neutralizing conformational epitopes on HPV33 has not been reported yet. By loop swapping between HPV33 and HPV16 capsid proteins, we have identified amino acid sequences critical for the binding of conformation-dependent type-specific neutralizing antibodies to surface-exposed hyper variable loops of HPV33 capsid protein L1. RESULTS: Reactivities of monoclonal antibodies (mAbs) H33.B6, H33.E12, H33.J3 and H16.56E with HPV16:33 and HPV33:16 hybrid L1 VLPs revealed the complex structures of their conformational epitopes as well as the major residues contributing to their binding sites. Whereas the epitope of mAb H33.J3 was determined by amino acids (aa) 51-58 in the BC loop of HPV33 L1, sequences of at least two hyper variable loops, DE (aa 132-140) and FGb (aa 282-291), were found to be essential for binding of H33.B6. The epitope of H33.E12 was even more complex, requiring sequences of the FGa loop (aa 260-270), in addition to loops DE and FGb. CONCLUSION: These data demonstrate that neutralizing epitopes in HPV33 L1 are mainly located on the tip of the capsomere and that several hyper variable loops contribute to form these conformational epitopes. Knowledge of the antigenic structure of HPV is crucial for designing hybrid particles as a basis for intertypic HPV vaccines.


Assuntos
Proteínas do Capsídeo/química , Proteínas do Capsídeo/imunologia , Epitopos/imunologia , Papillomaviridae/química , Papillomaviridae/imunologia , Anticorpos Antivirais/imunologia , Linhagem Celular , Regiões Determinantes de Complementaridade/imunologia , Epitopos/química , Humanos , Modelos Moleculares , Testes de Neutralização , Papillomaviridae/classificação , Conformação Proteica
2.
PLoS One ; 7(9): e44505, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-22973456

RESUMO

Inefficient intracellular protein trafficking is a critical issue in the pathogenesis of a variety of diseases and in recombinant protein production. Here we investigated the trafficking of factor VIII (FVIII), which is affected in the coagulation disorder hemophilia A. We hypothesized that chemical chaperones may be useful to enhance folding and processing of FVIII in recombinant protein production, and as a therapeutic approach in patients with impaired FVIII secretion. A tagged B-domain-deleted version of human FVIII was expressed in cultured Chinese Hamster Ovary cells to mimic the industrial production of this important protein. Of several chemical chaperones tested, the addition of betaine resulted in increased secretion of FVIII, by increasing solubility of intracellular FVIII aggregates and improving transport from endoplasmic reticulum to Golgi. Similar results were obtained in experiments monitoring recombinant full-length FVIII. Oral betaine administration also increased FVIII and factor IX (FIX) plasma levels in FVIII or FIX knockout mice following gene transfer. Moreover, in vitro and in vivo applications of betaine were also able to rescue a trafficking-defective FVIII mutant (FVIIIQ305P). We conclude that chemical chaperones such as betaine might represent a useful treatment concept for hemophilia and other diseases caused by deficient intracellular protein trafficking.


Assuntos
Fator VIII/metabolismo , Hemofilia A/metabolismo , Chaperonas Moleculares/metabolismo , Análise de Variância , Animais , Betaína/metabolismo , Betaína/farmacologia , Western Blotting , Células CHO , Cricetinae , Cricetulus , Fator VIII/genética , Citometria de Fluxo , Vetores Genéticos , Hemofilia A/tratamento farmacológico , Humanos , Lentivirus , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Microscopia de Fluorescência , Dobramento de Proteína , Transporte Proteico/efeitos dos fármacos , Transporte Proteico/fisiologia , Proteínas Recombinantes/biossíntese
3.
Hum Gene Ther ; 20(4): 325-36, 2009 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-19267665

RESUMO

Cell membrane-anchored (ma) antiviral peptides derived from the C-terminal heptad repeat of the HIV-1 transmembrane glycoprotein gp41 (C-peptides) and expressed from retroviral vectors were shown to efficiently inhibit HIV-1 entry into target cells. Here, we analyzed the influence of the vector backbone, the scaffold modules that anchor the peptide to the membrane and the length of the C-peptide on expression level and antiviral activity. In general, antiviral activity was determined primarily by the density of the C-peptide on the cell surface. By influencing expression levels, the scaffold elements indirectly also determined antiviral activity. Additional direct effects of the scaffold on antiviral activity were minor. At comparable expression levels, the elongated C-peptide (maC46) was found to be more potent than the shorter maC36. On the basis of these findings, a dose-response assay was established that quantifies antiviral activity relative to the expression level of the antiviral gene product. Taken together, these data demonstrate the importance of analyzing the efficacy of therapeutic genes relative to the dose of the gene product.


Assuntos
Antivirais/farmacologia , Peptídeo C/farmacologia , Membrana Celular/metabolismo , Proteínas/metabolismo , Sequência de Aminoácidos , Peptídeo C/química , Linhagem Celular , Membrana Celular/efeitos dos fármacos , Relação Dose-Resposta a Droga , Vetores Genéticos/genética , Infecções por HIV/virologia , Humanos , Dados de Sequência Molecular , Retroviridae/genética , Transgenes , Internalização do Vírus/efeitos dos fármacos
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