• Journal of Pharmaceutical Investigation
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• 제31권3호
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• pp.185-190
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• 2001

Human papillomavirus (HPV) infection is known to cause cervical cancers. Human papillomavirus-like particles (VLP) have been studied as preventive vaccines of cervical cancers. To develop VLP as a therapeutic gene carrier, we studied the method to encapsulate cytokine genes in virus-like particles. HPV type 16 capsid L1 genes were amplified by polymerase chain reaction and cloned into T vector. L1 gene was then inserted into baculovirus transfer vector. The clone of baculovirus encoding L1 gene was isolated and used to express L1 protein in Sf 21 insect cells. VLP were purified by CsCl density gradient and ultracentrifugation. VLP were disassembled to capsomer units by treatment of a reducing agent. Given that interleukin-2 (IL-2) genes have been used in anticancer gene therapy and as a molecular adjuvant, IL-2 cytokine plasmids were chosen as a model gene. IL-2 plasmids were incubated with the disassembled capsomer suspension. To reassemble the particles, the mixture of capsomers and cytokine plasmids was dialyzed. The disassembly and reassembly of VLP were confirmed by transmission electron microscopy. The entrapment of cytokine plasmids in reassembled VLP was tested by the stability of plasmids against DNase I. After treatment of reassembled virus-like particles with DNase I, discrete IL-2 DNA band was observed. Our results indicate that IL-2 cytokine plasmid (3.5 kb size) can be encapsulated in the virus-like particles, suggesting the potential of VLP as a gene delivery system. Moreover, VLP containing the adjuvant cytokine plasmids might function as more effective subunit vaccines.

• Asian Pacific Journal of Cancer Prevention
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• 제13권6호
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• pp.2643-2646
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• 2012

Objectives: Human papillomavirus (HPV) is the major etiological agent of cervical cancer, a leading cause of morbidity and mortality in women worldwide. Screening strategies for reducing the burden of HPV-mediated carcinogenesis are emerging as an effective means for cervical cancer control and prevention in developing countries. Our study, therefore, aimed to identify HPV infection status in North Indian women during random population screening. Methodology: Cervical/vaginal exfoliated cells and/or Pap smear specimens were collected from 890 women of North Indian ethnicity residing in Lucknow and adjoining areas, during random population screening from June 2009-March 2012. HPV viral loads in clinical specimens were determined by the Hybrid Capture (hc)-2 HPV DNA assay, and subsequently, positive/negative/borderline HPV status was calculated. Results: The HPV incidence in the present study was 11.7%. 751 out of a total of 890 women (84.4%) participating in our HPV screening program were HPV negative (HPV -), 104 (11.7%) tested positive (HPV +) while 35 (3.9%) showed borderline (HPV $^*$) infection status. Furthermore, in the HPV + subjects (N=104), 18 (17.3%) showed strong positivity. We observed that HPV positivity tends to increase with age in North Indian women; the higher the viral load with increasing age, higher is the susceptibility to HPV-mediated cervical cancer. Conclusions: HPV viral load/genotyping may help in identifying women at risk of developing cervical cancer. However, cost-effective HPV screening protocols with a wider population coverage are warranted so as to reduce the burden of cervical cancer in women worldwide in the vaccine-era.

• Asian Pacific Journal of Cancer Prevention
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• 제15권16호
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• pp.6887-6892
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• 2014

The oncogenic role of human papillomavirus (HPV) in triggering cervical cancer, the second most common cancer in women worldwide, is well established. Romania ranks in first place in Europe in terms of the incidence of cervical cancer. Geographical widespread data on HPV type-distribution are essential for estimating the impact of HPV vaccines and cervical cancer screening programmes. In this study we aimed to identify the prevalence of HPV genotypes and to establish correlations with abnormal cervical cytology among the female population of Brasov County, Romania. A total of 1,000 women aged 17.3-57 years, attending routine cervical examination in the Obstetrics and Gynecology Hospital of Brasov, Romania, and undergoing both cytological examination and HPV genotyping were screened. Infection with 35 different HPV genotypes was detected in 39.6% of cytological specimens. Overall HPV infections were highest in young women under 25 years (p<0.0001), in which cervical cytological abnormalities also reached the highest prevalence. Patients infected by HPV-16 or HPV-18 showed the highest prevalence of cervical cytological abnormalities. Some 48.2% of women with abnormal cytology were infected with high-risk HPV types whereas less than 3% of them were infected only with low-risk HPV types. Our study showed that the prevalence of high-risk HPV infection among Romanian women is higher compared to other studies in other geographic areas. Thus, we consider that in areas where there is an increased prevalence of high-risk HPV infections, HPV genotyping should be performed in all women aged between 18 and 45 years, and Pap test should be performed every 6 months in women with high-risk HPV infection, even those with previous normal cervical cytology.

• Asian Pacific Journal of Cancer Prevention
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• 제15권6호
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• pp.2719-2725
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• 2014

Background: Secreted frizzled-related protein (SFRP) genes, new tumor suppressor genes, are negative regulators of the Wnt pathway whose alteration is associated with various tumors. In ovarian cancer, SFRPs genes promoter methylation can lead to gene inactivation. This study investigated mechanisms of SFRP and adenomatous polyposis coli (APC) genes silencing in ovarian cancer infected with high risk human papillomavirus. Materials and Methods: DNA was extracted from 200 formalin-fixed paraffin-embedded ovarian cancer and their normal adjacent tissues (NAT) and DNA methylation was detected by methylation specific PCR (MSP). High risk human papillomavirus (HPV) was detected by nested PCR with consensus primers to amplify a broad spectrum of HPV genotypes. Results: The percentages of SFRP and APC genes with methylation were significantly higher in ovarian cancer tissues infected with high risk HPV compared to NAT. The methylated studied genes were associated with suppression in their gene expression. Conclusion: This finding highlights the possible role of the high risk HPV virus in ovarian carcinogenesis or in facilitating cancer progression by suppression of SFRP and APC genes via DNA methylation.

• Asian Pacific Journal of Cancer Prevention
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• 제15권6호
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• pp.2447-2451
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• 2014

Human papillomavirus (HPV) is the primary etiologic agent of cervical cancer. Consideration of safety and non human leukocyte antigen restriction, protein vaccine has become the most likely form of HPV therapeutic vaccine, although none have so far been reported as effective. Since tumor cells consistently express the two proteins E6 and E7, most therapeutic vaccines target one or both of them. In this study, we fabricated DC vaccines by transducing replication-defective recombinant adenoviruses expressing E6/E7 fusion gene of HPV-16, to investigate the lethal effects of specific cytotoxic T lymphocytes (CTL) against CaSki cells in vitro. Mouse immature dendritic cells (DC) were generated from bone marrow, and transfected with pAd-E6/E7 to prepare a DC vaccine and to induce specific CTL. The surface expression of CD40, CD68, MHC II and CD11c was assessed by flow cytometry (FCM), and the lethal effects of CTL against CaSki cells were determined by DAPI, FCM and CCK-8 methods. Immature mouse DC was successfully transfected by pAd-E6/E7 in vitro, and the transfecting efficiency was 40%-50%. A DC vaccine was successfully prepared and was used to induce specific CTL. Experimental results showed that the percentage of apoptosis and killing rate of CaSki cells were significantly increased by coculturing with the specific CTL (p <0.05). These results illustrated that a DC vaccine modified by HPV-16 E6/E7 gene can induce apoptosis of CaSki cells by inducing CTL, which may be used as a new strategy for biological treatment of cervical cancer.

• Journal of Microbiology and Biotechnology
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• 제25권6호
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• pp.936-940
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• 2015

Human papillomavirus (HPV) type 52 is a high-risk HPV responsible for cervical cancer. HPV type 52 is common around the world and is the most common in some Asian regions. The available prophylactic HPV vaccines protect only from HPV types 16 and 18. Supplementing economical vaccines that target HPV type 52 may satisfactorily complement available prophylactic vaccines. A codon-adapted HPV 52 L1 gene was expressed in the methylotrophic yeast Hansenula polymorpha, which is used as an industrial platform for economical hepatitis B surface antigen particle production in China. We found that the recombinant proteins produced in this expression system could form virus-like particles (VLPs) with diameters of approximately 50 nm. This study suggests that the HPV 52 VLPs produced in this platform may satisfactorily complement available prophylactic vaccines in fighting against HPVs prevalent in Asia.

• Journal of Microbiology and Biotechnology
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• 제18권2호
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• pp.350-354
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• 2008

In order to investigate the prevalence of sexually transmitted viruses such as human papillomavirus (HPV) and herpes simplex virus (HSV) in Korean commercial sex workers (CSWs), we selected 188 CSWs (age range 20-44 years, median age 24 years) who regularly visited one public health center in Seoul, Korea. HPV genotypes were analyzed by using a HPV DNA Chip, and an enzyme-linked immunosorbent assay (ELISA) was used to detect type-specific IgG against HSV2 antibody identifying seropositivity for HSV2 infection. Polymerase chain reaction (PCR) was performed with specific primers to detect HPV and HSV1/2 in cervical swabs from the CSWs. The prevalence of HPV infection was 83.5% in 188 cervical swab specimens and the main high-risk HPV genotypes were HPV16, 18, 56, and 58. The principal low-risk HPV genotypes were HPV6 and 11. The prevalence of HSV1/2 DNA was 13.8% and HSV2 seroprevalence was 86.2%. These results suggest that high frequencies of HPV and HSV2 infection might contribute to the rapid spread of STD viruses in CSWs in Korea. Additionally, an understanding of why high-risk HPV genotypes are so prevalent could provide guidelines for prophylactic vaccine development in Korea.

• 한국미생물·생명공학회지
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• 제46권2호
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• pp.171-179
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• 2018

Human papillomavirus (HPV) plays a critical role in the development of cervical carcinoma. This study analyzed the efficiency of multiplex real-time PCR in detecting and identifying HPV genotypes in samples from women who visited a Korean hospital for checkups. Cervical swab specimens were obtained from women who attended a checkup at the Health Improvement Center of Hospital in Dankook University Cheonan, South Korea and were referred for an HPV genotyping test between January and September 2014. A total of 1703 cervical swab specimens were collected consecutively during this period. PCR results were compared with those of the traditional cytological assay for the same population. Among the 1,703 specimens, 19.91% were HPV positive, of which 14.50% indicated a single infection and 5.40% indicated multiple infections. However, cytology identified only 2.52% of positive cases, including 1.23% cases of atypical squamous cells of undetermined significance, 1% of low grade squamous intra-epithelial lesion, and 0.29% of high grade squamous intra-epithelial lesion. The rate of high-risk and low-risk HPV in the abnormal cytology group was 48 and 23, respectively, and 274 and 136 in the normal group, respectively. HPV types 56, 52, 43 were the most prevalent in that order. Our results confirm the efficiency of the HPV DNA assay for the detection of 28 different HPV genotypes with reasonable sensitivity. A screening strategy that comprises the HPV DNA assay and cytology would help overcome the low sensitivity of a cytological diagnosis.

• Asian Pacific Journal of Cancer Prevention
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• 제15권21호
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• pp.9361-9365
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• 2014

Background: To estimate the pre-vaccination distribution of human papillomavirus (HPV) types among women from urban Tunis. Materials and Methods: A total of 611 women aged 18-69 years were enrolled in three local gynaecological outpatient departments. All underwent a gynaecological examination with Pap test and dry swab for HPV detection and typing performed by linear array genotyping test (Roche). Cytological examination was conducted on conventional Pap smears. Results: HPV DNA was found in 6.5% of the women; the most frequent HPV types were HPV 16 and HPV 11 at 3.27% and 1.96%, respectively. The second most frequent high risk (HR) HPV type was HPV 58 (0.82%) followed by HPV 18, HPV 31 and HPV 33 found in only 0.33% of women. Single infections with HPV types, targeted by the quadrivalent vaccine (6, 11, 16, and 18), were detected in 3.6 % of the study patients (55% of positive women). HPV infection was found in 3.83% of women with normal cytology and in 47.4% of women with cytological abnormalities. No statistically significant trend in prevalence by age group emerged for any HPV type or for high or low risk types. Conclusions: These data show a relatively low prevalence of HPV infection in women from urban Tunis with a high proportion of HPV16 and HPV58. This should be considered in the upcoming screening programs and vaccination strategy.

• 대한의생명과학회지
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• 제12권4호
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• pp.399-403
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• 2006

Cervical cancer is one of the most prevalent cancers developed in women worldwide, and human papillomavirus (HPV) type 16 is the most common agent linked to human cerivical carcinoma. Viral oncogenes E6 and E7 are selectively ratined and expressed in carcinoma cells infected with human papillomavirus type 16 and cooperated with each other in immortalization and transformation of primary keratinocytes. Because of HPV oncogenesis mechanism was not completely solved, the more studies be required thoroughly. In the present study, to investigate the telomere independent role of telomerase in HPV oncogenesis, we constructed the E6 mutant, E7, E6/E7 and hTERT over-expressed stable cells with a telomerase negative cell line, SW13. Expressions of Inserted genes were measured by RT-PCR. E6, E7 and hTERT genes were well expressed in each cell lines comparing with the control groups. By analyzing the cell morphology under the microscope, hTERT clone size was a more smaller than the mock control but oncogene expressed clones were slightly lengthened the marginal region. In addition, hTERT cells has also, a tendency of brief dividing time compared to the mock control. To determine whether telomerase activity associated with a HPV oncogenesis by oncoprotein expression, we performed the PCR based TRAP assay and Northern blot analysis. In TRAP assay data, telomerase activities in hTERT and oncogene clones were more increased than the mock control. In addition, SW13/ E6/E7 cells appeared a extremely increased activity than any other clones. Induced TERT mRNA by E6/E7 wasn't, however, detected in Northern blotting. In conclusion, these findings suggest that telomerase activity closely associated the HPV oncogenesis and E6/E7 co-expression is a most important factor of telomerase activity.