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
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
Human papillomaviruses (HPVs) are responsible for the most common human sexually transmitted viral infections. Infection with high-risk HPVs, particularly HPV16, is associated with the development of cervical cancer. The papillomavirus L1 major capsid protein, the basis of the currently marketed vaccines, self-assembles into virus-like particles (VLPs). Here, we describe the expression, purification and characterization of recombinant HPV16 L1 produced by a methylotrophic yeast. A codon-optimized HPV16 L1 gene was cloned into a non-integrative expression vector under the regulation of a methanol-inducible promoter and used to transform competent Pichia pastoris cells. Purification of L1 protein from yeast extracts was performed using heparin-sepharose chromatography, followed by a disassembly/reassembly step. VLPs could be assembled from the purified L1 protein, as demonstrated by electron microscopy. The display of conformational epitopes on the VLPs surface was confirmed by hemagglutination and hemagglutination inhibition assays and by immuno-electron microscopy. This study has implications for the development of an alternative platform for the production of a papillomavirus vaccine that could be provided by public health programs, especially in resource-poor areas, where there is a great demand for low-cost vaccines.
Assuntos
Proteínas do Capsídeo/metabolismo , Papillomavirus Humano 16/metabolismo , Proteínas Oncogênicas Virais/metabolismo , Pichia/virologia , Western Blotting , Proteínas do Capsídeo/isolamento & purificação , Transformação Celular Viral/fisiologia , Eletroforese em Gel de Poliacrilamida , Regulação Viral da Expressão Gênica , Testes de Inibição da Hemaglutinação , Papillomavirus Humano 16/ultraestrutura , Microscopia Eletrônica de Transmissão , Proteínas Oncogênicas Virais/isolamento & purificação , Infecções por Papillomavirus/metabolismo , Pichia/metabolismoRESUMO
Rabbit hemorrhagic disease (RHD) is a contagious and highly lethal viral disease of rabbits that spreads rapidly and infects animals by nasal, conjunctival and oral routes. Therefore, this experiment was undertaken to study the immune response generated after intranasal (i.n.) vaccination with the recombinant VP60 capsid protein from rabbit hemorrhagic disease virus (RHDV) expressed at high levels in Pichia pastoris. Groups of BALB/c mice were immunized with three doses of purified VP60 protein (Group 1), VP60 formulated within the cell debris fraction of the transformed yeast (Group 2) and placebo (Group 3) by intranasal route. Mice were also intramuscularly injected with purified VP60 protein (Group 4). A rapid antibody response specific against rabbit hemorrhagic disease virus was observed in all the experimental groups, except in Group 3, as detected by ELISA. The highest titers were found 60 days after the first immunization. Mice from Group 1 showed the highest IgG response (p<0.05) and the most balanced profile of IgG1, IgG2a and IgG2b subclasses. IgA titers specific to the virus were found only in animals from this group, which also developed the highest specific lymphocyte proliferative response. Interferon-gamma (IFN-gamma) and interleukin-12 (IL-12) gene expression was also detected after an ex vivo-specific stimulation of mice from Groups 1 and 4. These data demonstrated the capacity of VP60 protein expressed in P. pastoris to elicit a potent humoral and cell-mediated immune response following an intranasal immunization scheme.
Assuntos
Anticorpos Antivirais/sangue , Citocinas/biossíntese , Vírus da Doença Hemorrágica de Coelhos/imunologia , Proteínas Estruturais Virais/imunologia , Vacinas Virais/imunologia , Administração Intranasal , Animais , Anticorpos Antivirais/biossíntese , Infecções por Caliciviridae/prevenção & controle , Infecções por Caliciviridae/veterinária , Citocinas/genética , Relação Dose-Resposta Imunológica , Ensaio de Imunoadsorção Enzimática/métodos , Ensaio de Imunoadsorção Enzimática/veterinária , Feminino , Regulação Viral da Expressão Gênica , Imunidade Celular , Imunoglobulina G/biossíntese , Imunoglobulina G/sangue , Camundongos , Camundongos Endogâmicos BALB C , Pichia/genética , Pichia/imunologia , Pichia/virologia , Coelhos , Distribuição Aleatória , Proteínas Recombinantes/administração & dosagem , Proteínas Recombinantes/imunologia , Fatores de Tempo , Vacinas Sintéticas/administração & dosagem , Vacinas Sintéticas/imunologia , Proteínas Estruturais Virais/administração & dosagem , Vacinas Virais/administração & dosagemRESUMO
The plasmidic stability of methyltrophic yeast Pichia pastoris expressing the recombinant protein E of dengue virus 4 was analyzed. To this end, the number of generations from the growth process in petri plaque to the propagation in zaranda was estimated, as well as the fermentation process. Besides, in the selected colonies the integration pattern was determined by Dot-Blot and neuclotide sequence of the gene E of dengue virus 4. This study allowed to prove the conservation and integrity of the aminoacid sequence of protein E, despite the genetic changes produced by molecular yeast mechanisms. On the other hand, it was also part of the control and checking of the primary bank of yeast cells that contains the gene of interest used at present in the process of expression of protein E of dengue virus 4.
Assuntos
DNA Recombinante , Vírus da Dengue/genética , Pichia/genética , Pichia/virologia , Proteínas do Envelope Viral/genética , Sequência de Aminoácidos , Células Cultivadas , DNA Fúngico/análise , Fermentação , Expressão Gênica , Immunoblotting , Modelos Teóricos , Pichia/crescimento & desenvolvimento , Pichia/metabolismo , Reação em Cadeia da Polimerase , Proteínas Recombinantes/genéticaRESUMO
Little is known about the mechanism of hepatitis C virion assembly. So the capacity of the entire Hepatitis C virus core protein (HCcAg) produced in Pichia pastoris to form particles either in its native soluble state or after detergent treatment of HCcAg associated to cell debris were studied. Size exclusion chromatography suggested that HCcAg assembled into high molecular weight structures. HCcAg was also specifically recognized by a serum from a chronic HCV carrier patient. This antigen migrated with buoyant density values similar to those obtained for native nucleocapsid particles from infected patients when analyzed using sucrose density gradient centrifugation. The analysis by electron microscopy of purified HCcAg showed aggregates resembling virus-like particles (VLPs) with an average diameter of 30 nm. These results indicated that the HCcAg obtained from P. pastoris assembled into VLPs resembling HCV nucleocapsid particles in a mature stage. Such HCcAg aggregates characterized here could be a valuable tool to elucidate the mechanisms of HCV nucleocapsid assembly.