Competitive adsorption on gold nanoparticles for human papillomavirus 16 L1 protein detection by LDI-MS.

The detection of the HPV L1 protein provides information about the infection status of the virus, reflects the replication status of the HPV virus in cervical cells, and helps understand the regression and progress of cervical lesions. Herein, we report a novel laser desorption ionization mass spectrometry (LDI MS) method for the sensitive detection of the HPV 16 L1 protein, based on non-covalent competitive adsorption between the HPV 16 L1 aptamer and melamine on gold nanoparticles (AuNPs). The intensity of the MS signal corresponding to the mass tag shows a linear relationship with the HPV 16 L1 concentration in the range 2-80 ng mL-1, with a limit of detection (LOD) of 58.8 pg mL-1. Using this method, the HPV 16 L1 protein is quantitatively analyzed in both clinical and vaccine samples. The described method is simple and has high sensitivity and good reliability.

Genital HPV infection among heterosexual and homosexual male attendees of sexually transmitted diseases clinic in Beijing, China.

Human papillomavirus (HPV) has been identified as etiologic agent of various cancers for both men and women. However, HPV vaccine has not been recommended for men in China by far. To provide more evidences to promote HPV vaccination among males at high-risk of infection, this study investigated genital HPV genotypes among male attendees of sexually transmitted disease (STD) clinic. Male attendees (⩾18 years old) were recruited from STD clinic of Beijing Ditan Hospital. Data on sociodemographic characteristics and self-reported sexual behaviors were collected based on questionnaire. Genital swab specimens were collected for HPV genotypes. Finally, a total of 198 eligible participants were included in the study. Nearly half of them were infected with at least one type of HPV. The prevalence of genital infection among participants with only heterosexual behaviors (50·91%, 56/110) was significantly higher than those with only homosexual behaviors (36·36%, 32/88) (P < 0·001). However, the distribution pattern of the most frequently observed HPV subtypes were found to be similar between these two subgroups. HPV31, HPV18, HPV16 and HPV58 were the most frequently identified high-risk types and HPV11, HPV6, HPV81 and HPV61 were the most frequently observed low-risk types. Our results, although need further verification by larger sample size, suggested that currently available HPV vaccines covered most prevalent HPV types observed in Chinese men. As HPV vaccine has been approved for application in females in China, molecular epidemiological studies and intervention studies among high-risk males should be promoted as well.

[Quantitative analysis of nine types of virus-like particles in human papilloma virus bulk by size-exclusion chromatography].

Cervical cancer is the fourth most common cancer among women. Human papilloma virus (HPV) is the most common cause of cervical cancer which accounts for 5% of all human cancers and results in about 528000 cases and 266000 deaths every year. HPV vaccines are considered the most effective strategy for the prevention of HPV infection and cervical carcinoma. Since 2006, three prophylactic vaccines against HPV have been available on the market, including bivalent vaccines, quadrivalent vaccines, and nine-valent vaccines. Among them, nine-valent vaccines have been reported to be the most effective. They can prevent 97% of the high-grade pre-cancer lesions. Virus-like particles (VLPs), which are arranged as 360 copies of capsid proteins L1, are the only antigens of the HPV vaccine. Nine-valent HPV vaccines are prepared by mixing nine types of VLPs with adjuvants. Thus, the quality of the VLPs, including their stability and content in the HPV bulk, is very important for developing HPV vaccines. In this study, a method was developed for the determination of the nine types of VLPs (HPV6/11/16/18/31/33/45/52/58) in HPV bulk by size exclusion chromatography (SEC). The parameters of this method were optimized in terms of column brand, pore size of stationary phase particles, buffer concentration, and pH value. SHIMSEN Ankylo SEC-300 column (300 mm×7.8 mm, 3 µm) combined with a buffer aqueous solution containing 300 mmol/L NaCl and 50 mmol/L phosphate (pH 7.0) was utilized to separate the VLPs from the matrix since a narrow peak shape and good repeatability for VLPs could be obtained with this column and mobile phase. The optimized method had a wide linear range, good repeatability (RSDs of peak area were not more than 5.0%), and a satisfactory sensitivity (LOQs in the range of 4.58-15.24 µg/mL). The optimized method was used to determine the VLPs in the HPV bulk. The LOQs of the current method were much lower than the content of the nine types of VLPs in the HPV bulk, indicating that this method was sensitive enough for the determination of the nine types of VLPs in the HPV bulk. The method was also used to determine the VLPs in an HPV bulk that had been stored at 4 ℃ for one week. A decrease in the nine types of VLPs in the range of 10.0%-62.6% was observed after they were stored at 4 ℃ for one week. An HPV vaccine was prepared by mixing the VLPs with an adjuvant. Thereafter, the VLPs were adsorbed on the surface of the adjuvant. The developed method was applied to determine the free VLPs in twelve batches of HPV vaccines from three different manufacturers. No obvious free protein was detected in the twelve batches of the HPV vaccines from the three manufacturers, indicating that VLPs from these manufactures react well with their aluminum adjuvant. Folin-phenol (Lowry assay) is commonly used for the determination of proteins in vaccines. It is based on the reduction of phosphomolybdotungstic mixed acid chromogen in the phosphomolybdotungstic reagent, which results in an absorbance maximum at 650 nm. The Lowry method was sensitive to interfering substances. Most interfering substances caused a lower color yield, while some detergents caused a slight increase in color. To reduce the effect of the interfering substances, a procedure for precipitating the proteins was usually required before the sample was tested. Thus, the Lowry assay is complex, time-consuming, and of low selectivity. Compared to the Lowry method, the method we developed is simpler and more automatic. It is a high-throughput method of determining VLPs. It can be used to determine VLPs in HPV bulk and free VLPs in HPV vaccines.

Simultaneous quantification of major capsid protein of human papillomavirus 16 and human papillomavirus 18 in multivalent human papillomavirus vaccines by liquid chromatography-tandem mass spectrometry.

Human papillomavirus (HPV) vaccination is the most effective mean to prevent HPV infection and cervical carcinoma. Licensed HPV prophylactic vaccines are formulated to contain a defined amount of different major capsid protein (L1), the critical antigen to elicit protection. No method is currently available to simultaneously quantify individual L1s in multivalent vaccines, presenting a daunting challenge for the quality control of HPV vaccines. Here, HPV16 and HPV18 L1 can be analyzed by liquid chromatography-tandem mass spectrometry (LC-MS/MS) using tryptic digestion without pre-digestion reduction and alkylation in multiple reaction monitoring (MRM) mode. Two signature peptides were selected to be the markers of the two L1s and can be well separated within 5.1 min. Their linear calibration curves were both obtained in the range of 20-500 nmol/L (R2 > 0.990). To HPV16 L1, intra/inter assay precisions and accuracies of the assay were below 11% and between 83.96-113.57%. While for HPV18 L1, they were below 12% and between 81.40-103.49%. In addition, the limits of quantitation (LOQ) were as low as 2.8 nmol/L for HPV16 L1 and 1.7 nmol/L for HPV18 L1, respectively, representing about 68 and 112 times more sensitive than those obtained with Smith Bicinchoninic Acid (BCA) assay. This LC-MS/MS method can be applied to the quantification of both bulk products and the final multivalent vaccines. This method is superior to the current assays in terms of sensitivity, specificity, precision, accuracy and throughput; it could become the method of choice for absolute quantification of proteins in multivalent vaccines.

Microbicidas para la prevención de infecciones de transmisión sexual: estado actual y estrategias para la evaluación preclínica de nuevos candidatos / Microbicides for preventing sexually transmitted infections: current status and strategies for preclinical evaluation of new candidates

Los microbicidas constituyen una nueva herramienta, todavía en proceso de investigación, que podrían ayudar en la prevención de la infección por los virus de la inmunodeficiencia humana (Human immunodeficiency virus: HIV) y de otras infecciones de transmisión sexual (ITS). Una serie de evidencias ha demostrado que la complejidad de la transmisión sexual de patógenos virales requiere de la identificación de compuestos capaces de bloquear los eventos tempranos del ciclo de infección viral. En este manuscrito hacemos una revisión exhaustiva de las diferentes estrategias que se han estudiado o se están considerando para prevenir ITS mediante el uso de microbicidas, haciendo particular énfasis en aquellos con el potencial de bloquear la infección por el HIV. También se revisa el proceso complejo de evaluación preclínica que se requiere para llegar a estudios en humanos y se concluye con un breve análisis de las estrategias que podrían formar parte del futuro inmediato en la investigación de microbicidas

Microbicides are a new tool, still under investigation, which could help prevent infection by the human immunodeficiency virus (HIV) and other sexually transmitted infections (STIs). Increasing evidence shows that the complexity of sexual transmission of viral pathogens requires the identification of compounds able to block the early events during the cycle of viral infection. In this manuscript we provide a comprehensive review of the different microbicide strategies that have been studied or are currently being considered for STI prevention, particularly emphasizing those having the potential to block HIV infection. The manuscript also reviews the complex process that is required to conduct future clinical studies in humans and concludes with a brief discussion of the strategies that could be part of the immediate future in microbicide research

[Clone of HPV58 E6 gene and analysis of HLA-DQB1 03 restricted T-cell epitopes].

AIM: To clone HPV58 E6 gene and analyze the HLA-DQB1 03-restricted T cell epitopes on E6 protein. METHODS: Total genome DNA was isolated from a cervical cancer sample. The HPV58 E6 gene was amplified by PCR and inserted into pGEM-T Easy vector. The recombinant plasmid was identified by restriction endonuclease analysis and sequencing. HLA-DQB1 03-restricted T cell epitopes on E6 protein were predicted and analyzed by the position-specific scoring matrix, support vector machine theory and prediction algorithm for proteasomal cleavages. RESULTS: A HPV58 E6 gene was successfully cloned and submitted to GenBank (EF060239). Epitope 47(FADLRIAYRDGNPFA) and Epitope 102(RCIICQRPLCPQEKK) were theoretic HLA-DQB1 03-restricted T cell epitopes on E6 protein. CONCLUSION: These two epitopes could serve as candidates for screening and identification of the vaccine against HPV58 infection in the further study.

Vacinas contra o HPV:uma visão crítica

Devemos ter prudência e precaução para a utilização desta vaciana, pois não estamos diante de uma doença que se supõe um grave e urgente problema de saúde pública e para a qual não se dispõe de nenhum método de tratamento ou preventivo efetivo.Desconhecemos seus efeitos a longo e curto prazo e,até o momento,não há provas que demonstrem que a vacinação dessas pacientes reduzirá a sua probabilidade de ter câncer cervical.Por fim, seria de bom alvitre solicitarmos o "consentimento informado" aos pais de pré-adolescentes,e de adoslecentes,e de adultos,que optarem por utilizar a vacina,explicitando que a vacina provavelmente lhes protegerá contra o HPV,que não está claro por quanto tempo durará a proteção,ou se erá efeitos colaterais nocivos para a sua saúde.

Tipificación del VPH en cáncer de cuello uterino: en la población venezolana / Typification of HPV in the uterine cervical cancer: in the venezuelan population

Realizar la tipificación del virus del papiloma humano en pacientes con cáncer de cuello uterino en la población venezolana. Se incluyeron 53 pacientes con diagnóstico de cáncer de cuello uterino entre abril de 2004 y noviembre de 2005. Se realizó tipificación del virus del papiloma humano mediante la reacción en cadena de la polimerasa para los tipos 6,11,16,18,31 y 35. se analizaron otras variables como edad, estadio y tipo histológico. La edad promedio fue 44,79 años. La mayoría de las pacientes tenían diagnóstico de cáncer localmente avanzado (43,18 por ciento St IIb, 40,90 por ciento St IIIb y 2,27 por ciento St IVa). El tipo histológico predominante fue carcinoma epidermoide en 86,36 por ciento de adenocarcinoma en 11,36 por ciento y carcinoma adenoescamoso en 2,27 por ciento. Se evidenció la presencia de secuencias de ADN asociados a virus del papiloma humano en 28 pacientes (52,83 por ciento). se identificaron secuecias de ADN asociadas a virus del papiloma humano 16 en 13 pacientes (24,52 por ciento), a virus del papiloma humano 18 en 4 pacientes (7,54 por ciento), virus del papiloma humano 31 en 3 (5,66 por ciento), virus del papiloma humano 6 en 2 (3,77 por ciento y virus del papiloma humano 11, virus del papiloma humano 33 y virus del papiloma humano 35 en un caso respectivamente (1,88 por ciento). Los resultados obtenidos en la tipificación del virus del papiloma humano en nuestras pacientes no reflejan aquellos reportados en otros paìses y plantean la necesidad de realizar estudios epidemiológicos a gran escala y que incluyen otros tipos de alto riesgo.

Identify and typify human papilloma virus in patients with cancer of the uterine cervix in the venezuelan population. We included 53 patients with diagnosis of cancer of the uterine cervix between April 2004 and November 2005. Polymerase chain reaction was used to typify human papilloma virus types 6, 11, 16, 18, 31, 33 and 35. Other variables such as age, tumor stage and histology were analized. RESULTS: Mean age was 44.79 years. The majority of patients had diagnosis of locally advanced uterine cervix cancer (43.18 % St IIb, 40.90 % St IIIb and 2.27 % St IVa). The predominant histology was epidermoid carcinoma in 83.36 % of cases, followed by adenocarcinoma in 11.36 % and adenosquamous carcinoma in 2.27 %. When human papilloma virus identification and typification was performed using PCR, the presence of DNA sequences associated with human papilloma virus were not found in 28 patients (52.83 %). DNA sequences associaed with human papilloma virus type 16 were identified in 13 patients (24.52 %), with human papilloma virus type 18 in 4 patients (7.54 %), human papilloma virus 31 in 3 (5.66 %), human papilloma virus 6 in 2 (3.77 %) and human papilloma virus 11, human papilloma virus 33 and human papilloma virus 35 in one case respectively (1.88 %). The results obtained with human papilloma virus identification and typification in our patient population is different from those reported in other countries. Epidemiological studies that include other high risk virus types must be performed.

Vacuna frente al VPH / No disponible

No disponible

Vacuna contra el virus del papiloma humano (VPH) / Vaccine against human papillomavirus (HPV)

El virus papiloma humano (VPH) como principal responsable del cáncer cérvico uterino tiene gran impacto en salud humana. Actualmente existen dos vacunas disponibles comercialmente, de reconocida eficacia, que se están usando en varios países, e incluso informalmente en cierta población en Chile. El MINSAL ha planteado la necesidad de hacer un estudio sobre la pertinencia de incorporar dicha vacuna al Programa Nacional de Inmunizaciones (PNI), solicitando formalmente la opinión del Comité Asesor en Vacunas y Estrategias de Inmunización (CAVEI). Con dicho objeto el CAVEI organizó un Subcomité para analizar el tema, encabezado por la Dra. Catterina Ferreccio, una reconocida especialista en la materia. A partir de Marzo 2012 se empezó a discutir en el CAVEI la conveniencia de incorporar la vacuna VPH al PNI Se invitó a representantes de Merck Sharp & Dohme y GlaxoSmithKline empresas que comercializan las dos vacunas existentes en el mercado, Gardasil® y Cervarix®, respectivamente a exponer las características de ellas ante los miembros del CAVEI el 9 de Mayo. Posteriormente, se ha discutido el tema en varias reuniones, llegando a la conclusión que en Chile están las condiciones técnicas y económicas para plantear la incorporación de la vacuna VPH al PNI. En estas circunstancias, se encargó a la Dra. Ferreccio que presentara un informe fundamentado de la recomendación que el CAVEI debe hacer al MINSAL, para lo cual ella contó con la colaboración de la Dra. Patricia Roessler y de Vanessa Van De Wyngard. Las recomendaciones que a continuación se presentan llevan implícitas la necesidad de vigilancia. Como la vacunas de contra VPH tienen poco tiempo de aplicación y la investigación respectiva sigue en curso, algunas indicaciones pueden variar a la luz de nuevos resultados, por lo que debe estarse permanentemente atentos a actualización de las mismas.