OBJECTIVEHuman papillomavirus 6 (HPV 6) causes genital warts, a common sexually transmitted disease. L1-capsids protein is a highly promising vaccine candidate that has entered phase II clinical trial. But the existing methodologies for producing L1-capsids in insect cells is expensive for use in developing countries. As methylotrophic yeast,the Pichia pastoris expression system offers economy,and high expression levels. Over-expression of HPV6-L1 protein in P. pastoris was the purpose of this study.

METHODSThe whole L1 gene with preferred codons for P. pastoris was rebuilt and A-T rich regions were abolished, Cloning into pPIC3.5K,electroporation of KM71, in vivo screen of multiple inserts by G418 resistance, PCR analysis of pichia integrants, BMGY/BMMY are used for induction and expression of L1 proteins.

RESULTSThree clones were found to produce L1 protein which can be identified with Western blot. Compared with L1 protein from E.coli, pichia-produced L1 has some glycosylation. Reacting strongly with MabH6B10.5 in indirect immunofluorescence assay indicated that L1 protein expressed in pichia cell holds its native conformational epitopes which is important for vaccine use. A total 125 microg pure L1 protein could be obtained from 1L cultures through ion-exchange and Ni-NTA chromatography.

CONCLUSIONHPV type 6 L1 protein expressed in Pichia pastoris will facilitate the HPV vaccine development and structure-function study.

Capsid Proteins ; biosynthesis ; genetics ; isolation & purification ; Cloning, Molecular ; Genes ; Papillomaviridae ; genetics ; Pichia ; genetics ; metabolism ; Viral Proteins

AIMTo pretreat sperm at various temperatures before exposure to human papillomavirus (HPV) 16 DNA fragments and to assess the efficiency of HPV carrier sperm to transfect cumulus cells.

METHODSCumulus cells from follicular aspirates were obtained, pooled and divided into culture dishes containing Sybr Gold-stained HPV DNA carrying sperm that were either pretreated at 4 degree C, 37 degree C or 40 degree C (n = 5). The cells were incubated in 5% CO(2) in air mixture at 37 degree C for 24 hours. The efficiency of sperm to take up fluorescent HPV DNA was determined at hour 0. After incubation, cumulus cell viability was assessed using the eosin method and the percentages of fluorescent cumulus cells determined.

RESULTSOver half of all the cumulus cells became fluorescent with the highest percentage in the 37 degree C group. Sperm pretreated at 4 degree C had the greatest amount of HPV DNA fragments. Total sperm motility was similar for the 3 pretreatment groups. There were no differences in cumulus viability among the groups.

CONCLUSIONSperm pretreated at 37 degree C transferred the greatest amount of fluorescent HPV DNA fragments to the cumulus cells. The HPV DNA was observed in the nuclear and cytoplasmic compartments. The data suggested the possibility of sperm as a vector for the transmission of HPV DNA to the cumulus cells surrounding ovulated oocytes, which might lead to early implantation failures.

DNA, Viral ; pharmacokinetics ; Humans ; Male ; Papillomaviridae ; genetics ; Spermatozoa ; physiology ; Temperature ; Transfection ; methods

OBJECTIVE: To analyze the characteristics of cervical microecology in late reproductive-age women with cervical lesions and explore new methods for preventing cervical lesions. METHODS: Cervical smears were obtained from a total of 147 women of late reproductive age, including 24 with high-risk HPV infection (HR-HPV), 27 with low-grade squamous intra-epithelial lesions (LSIL), 36 with high-grade squamous intra-epithelial lesions (HSIL), 35 with cervical cancer (CC) and 25 healthy women. llumina MiSeq sequencing of V3-V4 region of the 16S rRNA gene amplicons was used to characterize the vaginal microbiota of the women. OTUs analysis of the valid data was performed, and the α-diversity (Chao1, Simpson's Index and Shannon Index) and β-diversity (T-test, weighted UniFrac β diversity, and MetaStat analysis) were evaluated. RESULTS: Dilution curve and species accumulation boxplot validated the quality of the samples. OTUs analysis of the 5 groups demonstrated that cervical bacterial genus consisted primarily of CONCLUSIONS The abundance of
Female ; Humans ; Microbiota ; Papillomaviridae ; Papillomavirus Infections ; RNA, Ribosomal, 16S/genetics* ; Uterine Cervical Neoplasms ; Vaginal Smears

OBJECTIVE: To investigate the correlation of cyclin D1 (CCND1) G870A single nucleotide polymorphism (SNP) with radiotherapy response in patients with high risk human papillomavirus (HR-HPV) related cervical cancer.
 METHODS: A total of 273 patients with cervical cancer, who were confirmed by histopathology and hybrid capture 2 (HC-2) assay and treated by radiotherapy, were enrolled for this study. The correlation of CCND1 G870A polymorphism with tumor response in patients was assessed.
 RESULTS: Compared with patients with AA genotype, the patients with GG genotype and AA genotype showed lower sensitivity to radio-therapy treatment (adjusted ORGA=2.69, 95% CI 1.28-5.67 and adjusted ORGG=3.28, 95% CI 1.47-7.29, respectively), an increase in risks of recurrence/metastasis (adjusted ORGA=2.52, 95% CI 1.12-5.63 and adjusted ORGG=3.95, 95% CI 1.68-9.26, respectively), and shorter recurrence/metastasis-free survival (PGA=0.010 and PGG=0.045).
 CONCLUSION G870A polymorphism is a frequent variation that could be used for evaluate the radio-sensitivity and prognosis for patients with HR-HPV related cervical cancer.
Cyclin D1 ; genetics ; Female ; Genotype ; Humans ; Papillomaviridae ; Polymorphism, Single Nucleotide ; Prognosis ; Uterine Cervical Neoplasms ; genetics ; radiotherapy ; virology

Objective: To investigate the distribution of the gene subtypes of human papillomavirus (HPV) in male patients with condyloma acuminatum (CA) and analyze the characteristics of the gene subtypes. METHODS: We extracted genomic DNA of the HPV virus from the genital tissue of 70 male CA patients, detected the DNA subtypes of HPV using the PCR-reverse dot hybridization technique, and analyzed the rates of different subtypes identified and their characteristics of distribution in different age groups. RESULTS: The male HPV-positive patients were mainly infected at the age of 20－39 years, primarily with high- and low-risk mixed infection of various subtypes, which accounted for 61.54% in the 20- to 29-year-olds and 42.86% in the 30- to 39-year-olds. Among the 70 CA patients, 22 HPV subtypes were identified, the top five subtypes including HPV 11 (21.08%), HPV 6 (19.46%), HPV 42 (6.49%), HPV 59 (6.49%) and HPV 53 (5.95%); 20 infected with a single subtype (28.57%), 19 with two subtypes (27.14%) and 31 with three or more (44.29%); and 30 infected with a low-risk single subtype (42.86%) and 40 with both high- and low-risk multiple subtypes (57.14%). CONCLUSIONS Male patients with CA are mainly infected with HPV 11 and HPV 6, with a significantly higher rate of multi-subtype than single-subtype infection, and the multi-subtype patients chiefly with high- and low-risk mixed infection. Men aged 20－39 years old are most commonly affected by CA.
Adult ; Condylomata Acuminata/virology* ; DNA, Viral/genetics* ; Genotype ; Humans ; Male ; Papillomaviridae/genetics* ; Papillomavirus Infections/virology* ; Young Adult

Objectives: To analyze the type and distribution characteristics of human papillomavirus (HPV) infection along with cervical cytology in middle-aged and elderly women in Guangxi and to provide a basis for the prevention and treatment of cervical cancer in elderly women. Methods: 21 subtypes of HPV and cervical cytology of women over 45-year-old visiting the First Affiliated Hospital of Guangxi Medical University from January 2019 to December 2020 were collected. They were divided into two groups by age, 45-64 years group and over 65 years group. The HPV, HR-HPV, and multiple HPV infection prevalence were analyzed, as well as HPV genotypes, the age distribution of HPV infection rate, and cervical cytology. Results: A total of 6 657 eligible women were included. 6 238 women were in the 45-64 years group, with a HPV prevalence about 20.86% (1 301), while 419 women were in the over 65 years group, with a HPV prevalence about 32.94% (138). The age-associated HPV and HR-HPV prevalence increased with the age, peaking at the age group of 70-74 years (P<0.001). The most prevalent genotype was HPV52, and the infection rate was 5.3% (353), followed by HPV16 and HPV 58, about 4.63% (308) and 3.08% (205) respectively. The majority cytology of HPV-positive middle-aged and elderly women was normal. 8.70% (88) of them were ASC-US, 6.52% (66) for HSIL, 4.55% (46) for LSIL, and 2.96% (30) for ASC-H, and 0.10% (1) for SCC. Compared to middle-aged women, elderly women had a lower negative cytology rate, 69.79% (67) vs. 77.95% (714), but a higher HSIL rate, 13.54% (13) vs. 5.79% (53) (P<0.05). Conclusions: HPV and HR-HPV prevalence of elderly women in a medical center of Guangxi are higher than those of middle-aged women. The most prevalent genotype is HPV16 in elderly women, followed by HPV52 and HPV58.
Aged ; China/epidemiology* ; Female ; Hospitals ; Human papillomavirus 16 ; Humans ; Male ; Middle Aged ; Papillomaviridae/genetics* ; Papillomavirus Infections/genetics* ; Uterine Cervical Neoplasms

OBJECTIVETo explore the genotypes of human papilloma virus (HPV) in male patients with condyloma acuminate in Lishui area of Zhejiang Province, China.

METHODSUsing HybriMax, we identified the genotypes of HPV of the verruca samples from 110 male condyloma acuminate patients aged 16 - 65 years in Lishui area.

RESULTSHPV infection was detected in 107 (97.27%) of the condyloma acuminate patients, including 76 cases (71.02%) of simple infection and 31 cases (28.97%) of multiple infection. The peak age of infection was 21 - 40 years old. Totally, 14 genotypes of HPV were identified, including the high-risk genotypes as HPV16, 53, 52, 33, CP8304, 58, 51, 45, 66, 68, and 59, which accounted for 44.85% of the cases, and the low-risk ones as HPV6, 11, and 19 (43), with an infection rate of 93.45%.

CONCLUSIONThe low-risk genotype is a dominant type of HPV infection and the rates of high-risk genotype and multiple infection are high among the male condyloma acuminate patients in Lishui area.

Adolescent ; Adult ; Aged ; China ; Condylomata Acuminata ; virology ; Genotype ; Humans ; Male ; Middle Aged ; Papillomaviridae ; genetics ; Papillomavirus Infections ; virology ; Risk ; Young Adult

OBJECTIVETo establish a method for rapid detection and sub-typing of human papilloma virus (HPV).

METHODSWe utilized the piezoelectric genosensor (PG) technique, which is a combination of the piezoelectric biosensor and gene chips for HPV identification in 22 recurrent biopsy specimens and 22 corresponding original biopsy specimens. The control samples came from normal tissue of healthy persons. A combined reaction took place on the sensor surface between the target genes and probes. The frequency of the piezoelectric sensor will decrease when such reactions occur, and the frequency decrease depends on the concentration of the target gene. Specimens were also analyzed with conventional PCR and dot blot.

RESULTSOf the 22 recurrent specimens, 15 contained HPV6 DNA, 2 HPV11 DNA, and 4 HPV16 DNA. Only one specimen was negative. All the 22 original specimens were positive: 17 harbored HPV6 DNA, 3 sequence homologous HPV11 DNA, and 2 HPV16 DNA. No HPV18 DNA was detected in any specimen. When compared with PCR and dot blot analysis, the results were essentially the same except for one specimen, which was shown to contain other sub-types of HPV.

CONCLUSIONOur results show that the piezoelectric genosensor technique is a rapid and specific method to analyze HPV.

Biosensing Techniques ; DNA, Viral ; analysis ; Humans ; Papillomaviridae ; classification ; genetics ; Papillomavirus Infections ; virology ; Polymerase Chain Reaction ; Tumor Virus Infections ; virology

OBJECTIVETo investigate the prevalence and subtype distribution of human papillomavirus (HPV) infection and its correlation with age among women in Beijing urban area, and provide some epidemiological evidence for the clinical application of HPV vaccines.

METHODSWe collected cervical specimens from 1999 women in the Outpatient Department of our hospital, performed genetyping of HPV-DNA, and analyzed the incidence of HPV infection in different age groups.

RESULTSHPV infection was detected in 502 (25.2%) of the 1999 women patients, with 391 (19.6%) cases of high-risk HPV, which included 326 (83.4%, 326/391) cases of single infection. HPV-16 was the most common type (21.2%, 69/326), followed by HPV-52 (19.3%, 63/326) and HPV-58 (16.0%, 52/326). The prevalence of HPV infection was the highest among the women aged 41 -50 years and the lowest among those over 60 years.

CONCLUSIONThe subtype- and age-specific distribution of HPV infection among women in Beijing urban area shows an obvious heterogeneity, which deserves due consideration in the clinical application of HPV vaccines.

Adolescent ; Adult ; Age Distribution ; Aged ; China ; epidemiology ; Female ; Genotype ; Humans ; Middle Aged ; Papillomaviridae ; classification ; genetics ; Papillomavirus Infections ; epidemiology ; Young Adult

OBJECTIVETo develop a recombinant vaccinia virus vaccine expressing HPV58 E7 and to determine its immuno-protective activity in mice bearing HPV58 E7+ tumor.

METHODSE7 DNA was amplified and cloned from a plasmid containing HPV58 E7 genome by PCR. To abolish its transforming activity, the nucleotides coding for amino acid residues at positions 24 and 92 were modified by site-directed mutagenesis so that cysteine was substituted by glycine. Balb/c 3T3 cells were transfected with mE7. The expression of E7 protein by the mE7-transfected Balb/c cells was confirmed by immunofluorescence staining. The transfected cells were observed in vitro for anchorage-independent growth and tumorigenesis in nude mice. Recombinant E7 vaccinia virus vaccine was constructed by homologous recombination of HPV58 E7 vaccinia expression plasmid and vaccinia virus (Tiantan stain). The immuno-protective activity of the vaccines was determined by tumor growth inhibition and cytotoxic T lymphocytes (CTL) induction in vaccine-immunized syngeneic mice.

RESULTSSubstitution of cysteine by glycine at both positions 24 and 92 of HPV58 E7 abolished its transforming activity. Growth of HPV E7+ tumor in mice immunized with the recombinant vaccinia virus expressing HPV58 E7 was inhibited, and the surviving time of the immunized mice was prolonged. CTL activity was induced as revealed by in vitro cytotoxicity assay using E7+ tumor cells as target cells.

CONCLUSIONSHPV58 E7, with its transforming potential abolished, may be used as vaccine for immunotherapy of patients with HPV 58 related cancers.

Animals ; Mice ; Mice, Inbred BALB C ; Mutagenesis, Site-Directed ; Oncogene Proteins, Viral ; genetics ; Papillomaviridae ; genetics ; immunology ; T-Lymphocytes, Cytotoxic ; immunology ; Transfection ; Vaccines, Synthetic ; immunology ; Vaccinia virus ; genetics ; immunology