RESUMO
Human papillomavirus (HPV) infection is the most common sexually transmitted disease in the world and is related to the etiology of cervical cancer. The most common high-risk HPV types are 16 and 18; however, the second most prevalent type in the Midwestern region of Brazil is HPV-33. New vaccine strategies against HPV have shown that virus-like particles (VLP) of the major capsid protein (L1) induce efficient production of antibodies, which confer protection against the same viral type. The methylotrophic yeast Pichia pastoris is an efficient and inexpensive expression system for the production of high levels of heterologous proteins stably using a wild-type gene in combination with an integrative vector. It was recently demonstrated that P. pastoris can produce the HPV-16 L1 protein by using an episomal vector associated with the optimized L1 gene. However, the use of an episomal vector is not appropriate for protein production on an industrial scale. In the present study, the vectors were integrated into the Pichia genome and the results were positive for L1 gene transcription and protein production, both intracellularly and in the extracellular environment. Despite the great potential for expression by the P. pastoris system, our results suggest a low yield of L1 recombinant protein, which, however, does not make this system unworkable. The achievement of stable clones containing the expression cassettes integrated in the genome may permit optimizations that could enable the establishment of a platform for the production of VLP-based vaccines.
Assuntos
Alphapapillomavirus/imunologia , Proteínas do Capsídeo/biossíntese , Proteínas Oncogênicas Virais/biossíntese , Pichia/metabolismo , Alphapapillomavirus/genética , Anticorpos Antivirais/imunologia , Proteínas do Capsídeo/genética , Transformação Celular Viral/fisiologia , Eletroforese em Gel de Poliacrilamida , Regulação Viral da Expressão Gênica , Proteínas Oncogênicas Virais/genética , Vacinas contra Papillomavirus/imunologia , Pichia/genética , Pichia/virologia , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Background & objective: Recombinant DNA technology allows expression of the human papillomavirus (HPV) major capsid protein (L1) in heterologous expression systems and the recombinant protein self assembles to virus-like particles (VLP). We took up this study to produce recombinant HPV-16 L1 in yeast, establish the process of recombinant L1 derived VLP preparation and develop an ELISA using VLP as the antigen for serological evaluation of anti HPV-16 L1 antibody status. Methods: Complete HPV-16 L1 was amplified from genomic DNA of an esophageal cancer biopsy, cloned and the protein was expressed in a galactose-inducible Saccharomyces cerevisiae expression system. Self assembled VLP was purified by a two-step density gradient centrifugation process and the VLP preparation used to test its suitability in developing an ELISA. Results: The recombinant protein was predominantly a ~55 KD species with distinct immunoreactivity and formed VLP as confirmed by electron microscopy. An ELISA using the VLP showed its efficacy in appropriate immunoreactivity to serum/plasma IgG. Interpretation & conclusions: Recombinant HPV-16 capsid protein derived VLP was produced and the VLP antigen based ELISA can be used to probe serological association of HPV with different clinical conditions. The VLP technology can be improved further and harnessed for future vaccine development efforts in the country.
Assuntos
Anticorpos Antivirais/análise , Antígenos Virais/biossíntese , Antígenos Virais/genética , Proteínas do Capsídeo/biossíntese , Proteínas do Capsídeo/genética , Proteínas do Capsídeo/imunologia , Ensaio de Imunoadsorção Enzimática , Papillomavirus Humano 16/genética , Papillomavirus Humano 16/imunologia , Papillomavirus Humano 16/ultraestrutura , Humanos , Proteínas Oncogênicas Virais/biossíntese , Proteínas Oncogênicas Virais/genética , Proteínas Oncogênicas Virais/imunologia , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Saccharomyces cerevisiae/genéticaRESUMO
The capsid of the foot and mouth disease (FMD) virus carries the epitopes that are critical for inducing the immune response. In an attempt to enhance the specific immune response, plasmid DNA was constructed to express VP1/interleukin-1alpha (IL-1alpha) and precursor capsid (P1) in combination with 2A (P1-2A)/IL-1alpha under the control of the human cytomegalovirus (HCMV) immediateearly promoter and intron. After DNA transfection into MA104 (monkey kidney) cells, Western blotting and an immunofluorescence assay were used to confirm the expression of VP1 or P1-2A and IL-1alpha. Mice were inoculated with the encoding plasmids via the intradermal route, and the IgG1 and IgG2a levels were used to determine the immune responses. These results show that although the immunized groups did not carry a high level of neutralizing antibodies, the plasmids encoding the VP1/ IL-1alpha, and P1-2A /IL-1alpha fused genes were effective in inducing an enhanced immune response.