Production of L1 protein from different types of HPV in Pichia pastoris using an integrative vector

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.

Immunohistochemical staining of Langerhans cells in HPV-positive and HPV-negative cases of oral squamous cells carcinoma

The Human Papillomavirus (HPV) has been strongly implicated in development of some cases of oral squamous cell carcinoma (OSCC). However, the immunological system somehow reacts against the presence of this virus. Among the cells involved in such mechanism of defense Langerhans cells (LC) stand out, which are responsible for processing and presenting antigens. OBJECTIVES: The purposes of this study were to investigate the presence of HPV DNA and to evaluate the immunohistochemical reactivity for Langerhans cells between HPV-positive and HPV-negative OSCC. Twenty-seven cases of OSSC were evaluated. MATERIAL AND METHODS: DNA was extracted from paraffin-embedded tissue samples and amplified by Polymerase Chain Reaction (PCR) for the detection of HPV DNA. Viral typing was performed by dot blot hybridization. Immunohistochemistry was performed by the Streptavidin-biotin technique. RESULTS: From the 27 cases, 9 (33.3 percent) were HPV-positive and 18 (66.0 percent) HPV-negative. HPV 18 was the most prevalent viral type (100 percent cases) and infection with HPV-16 (co-infection) was detected in only 1 case. In the OSCC specimens examined, immunoreactivity to S-100 antibody was detected in all cases, with a mean number of 49.48±30.89 Langerhans cells positive for immunostaining. The mean number of immunostained Langerhans cells was smaller in the HPV-positive cases (38 cells/case) than in the HPV-negative cases (42.5 cells/case), but this difference was not significant (p=0.38). CONCLUSIONS: The low frequency of detection of HPV DNA in OSCC indicates a possible participation of the virus in the development and progression of only a subgroup of these tumors. There was no association between the immunohistochemical labeling for Langerhans cells (S-100+) and HPV infection of in OSSC. These findings suggest that the presence of HPV in such OSCC cases could not alter the immunological system, particularly the Langerhans cells.

Genotipos de virus papiloma humano (VPH) en pacientes con cáncer cervico-uterino en un hospital público y una clínica privada de Santiago, Chile / Human papillomavirus (HPV) genotypes in cervix uterine cancer patients in a public hospital and private clinic from Santiago, Chile

We compared HPV genotypes among squamous cervical cancer samples from a public hospital (n = 55) and a private clinic (n = 35 cases) of Santiago. Paraffin-embedded specimens were analyzed by PCR followed by an immunoenzimatic assay. Reverse line blotting was used for the identification of 36 HPV genotypes. We found HPVDNAm 94.4 percent of all cancers. Single mfections: HPV16: 40.0 percent, (clinic 37.1 percent, hospital 41.8 percent) VPH18:7.8 percent (clinic 2.9 percent, hospital 10.9 percent); single+multiple mfections: VPH16: 61.1 percent (clinic 53.1 percent, hospital 71.7 percent), VPH18: 34.4 percent (clinic 21.9 percent, hospital 45.2 percent). HPV16 orHPV18 occurredin 75.6 percent of cases, higher inthe hospital than the clinic (87.3 percent-95 percent CI: 84.9-96.3 - and 57. l percent-95 percent CI: 46.6-66 - respectively, p = 0.002). Other genotypes in single mfections: HPV 26, 31, 33, 45, 58, 67; in co-mfections: HPV 35,52,56,59 and 66. HPV16 but specially HPV 18 were significantly more frequent in the public hospital; 75.6 percent of squamous cervical cancer were associated to the vaccine preventable HPV16/18.

Se comparan los genotipos de VPH en casos de cáncer cérvico-uterino escamocelular de una clínica privada (n: 35) y de un hospital público (n: 55) atendidos entre 1996 y 2006 en Santiago, Chile. Se analizaron por RPC y ensayo inmunoenzimático muestras tumorales en bloques de parafina, genotipificándose con reverse Une blotting para 36 genotipos de VPH. Se detectó VPH en 94,4 por ciento de los casos: infecciones únicas por: VPH 16: 40,0 por ciento>, (clínica 37,1 por ciento, hospital 41,8 por ciento) VPH 18: 7,8 por ciento (clínica 2,9 por ciento, hospital 10,9 por ciento); total de infecciones por VPH 16 61,1 por ciento (clínica 53,1 por ciento, hospital 71,7 por ciento), por VPH 18 34,4 por ciento (clínica 21,9 por ciento, hospital 45,2 por ciento). Co-infección: VPH 16/18 75,6 por ciento (clínica 57,1 por ciento; IC95 por ciento = 46,6-66,0 hospital 87,3 por ciento; IC95 por ciento = 84,9-96,3, p = 0,002). Se identificó otros 11 genotipos oncogénicos en infecciones únicas (VPH: 26, 31, 33, 45, 58, 67) o en co-infección con VPH 16/18 (VPH: 35, 52, 56, 59, 66). VPH 16 y VPH 18 fueron significativamente más frecuentes en el hospital público, particularmente VPH18; 75,6 por ciento> de los cánceres se asociaron a los genotipos VPH 16/18, tipos prevenibles por vacuna.

Declaración del Comité Consultivo de Inmunizaciones de la Sociedad Chilena de Infectología respecto a la vacuna anti-virus papiloma humano: septiembre 2008 / Human papillomavirus vaccine: statement of the Consultive Committee of Immunizations on behalf of The Chilean Infectious Diseases Society: September 2008

The article briefly reviews the epidemiology of human papillomavirus (HPV) infection and associated diseases in the world and in Chile and the scientific information of the licensed HPV vaccines: Gardasil® and Cervarix®. Considering the available information, the Consultive Committee of Immunizations of the Chilean Society of Infectious Diseases, summarizes its conclusions and makes recommendations for vaccination against HPV in Chile.

El artículo revisa brevemente la epidemiología de las infecciones por virus papiloma humano (VPH) y las enfermedades asociadas, tanto en el mundo como en Chile y la información científica de las vacunas contra VPH licenciadas: Gardasil® y Cervarix®. Considerando la información disponible, el Comité Consultivo de Inmunizaciones de la Sociedad Chilena de Infectología, resume sus conclusiones y hace sus recomendaciones para la vacunación contra VPH en Chile.

Virus papiloma humano y cáncer cérvico-uterino: [editorial] / Papillomavirus and cervical cancer in Chile: [editorial]

Molecular, clinical and epidemiolagical studies have established beyond doubt that human papiloma viruses (HPV) cause cervical cancer. The virus is also associated with genital warts and other less common cancers in oropharynx, vulva, vagina and penis. Worldwide, VPH genotypes 16 and 18 are the most common high risk genotypes, detected in near 70 percent of women with cervical cáncer. The discovery of a cause-effect relationship between several carcinogenic microorganisms and cancer open avenues for new diagnostic, treatment and prevention strategies. In this issue of Revista Médica de Chile, two papers on HPV are presented. Guzman and colleagues demonstrate that HPV can be detected in 66 percent to 77 percent of healthy male adolescents by polymerase chain reaction and that positivity depends on the site of the penis that is sampled. These results support the role of male to female transmission of high risk HPVs in Chile and should lead to even more active educational campaigns. The second paper provides recommendations for HPV vaccine use in Chile, generated by the Immunization Advisory Committee of the Chilean Infectious Disease Society. To issue these recommendations, the Committee analyzes the epidemiological information available on HPV infection and cervical cancer in Chile, vaccine safety and effectiveness data, and describes cost-effectiveness studies. Taking into account that universal vaccination is controversial the Committee favors vaccine use in Chile and it's incorporation into a national program. However, there is an indication that the country requires the implementation of an integrated surveillance approach including cross matching of data obtained from HPV genotype surveillance, monitoring of vaccination coverage, and surveillance of cervical cáncer The final decision of universal vaccine use in Chile should be based on a through analysis of information.

Immunohistochemical localization of human papilloma virus in conjunctival neoplasias: a retrospective study.

BACKGROUND: The extent of association of human papilloma virus (HPV) in human conjunctival neoplasias has been debated in studies originating from different parts of the world, but no substantial evidence has been generated on Indian subjects. This prompted us to carry out a retrospective study on conjunctival neoplasias diagnosed over the past 12 years. MATERIALS AND METHODS: Histopathological and immunohistochemical analysis of 65 specimens of ocular neoplasias and 30 normal controls diagnosed between 1991 and 2002 at a tertiary eye care hospital, was undertaken. Formalin-fixed, paraffin-embedded tissues were reviewed for confirming histopathological diagnosis, presence of koilocytosis and changes related to actinic keratosis. Immunohistochemical analysis was done using HPV-specific monoclonal antibodies. Clinicopathological correlation and the association of HPV antigen with the histopathological features were performed. RESULTS: Out of the 65 cases analyzed, 35 were papillomas and 30 were ocular surface squamous neoplasias (OSSN). The mean age was 48 years with a male preponderance. Histologically, koilocytosis was observed in 17.1% of papillomas and 36.6% of OSSN. Actinic keratosis was present in 33% of OSSN. Immunohistochemically 17.1% conjunctival papillomas stained positive for HPV antigen, all cases of OSSN were negative for HPV. There was no correlation between koilocytosis or actinic keratosis and the detection of HPV antigen. CONCLUSIONS: The association between HPV and conjunctival neoplasias is variable in different geographical areas and also depends on the methods of detection used. This study warrants the need for applying more advanced techniques at a molecular level to determine the possible etiology of HPV in conjunctival neoplasias among Asian-Indians.