This study aims to establish a time-resolved fluorescence immunochromatography method for simultaneous determination of human papillomavirus (HPV) type 16 E6 and L1 protein concentrations. The amount of lanthanide microsphere-labeled antibodies, the concentration of coated antibodies, and the reaction time were optimized, and then a test strip for the simultaneous determination of the protein concentrations was prepared. The performance of the detection method was evaluated based on the concordance of the results from clinical practice. The optimal conditions were 8 μg and 10 μg of HPV16 L1 and E6-labeled antibodies, respectively, 1.5 mg/mL coated antibodies, and reaction for 10 min. The detection with the established method for L1 and E6 proteins showed the linear ranges of 5-320 ng/mL and 2-64 ng/mL and the lowest limits of detection of 1.78 ng/mL and 1.09 ng/mL, respectively. There was no cross reaction with human immunodeficiency virus (HIV), treponema pallidum (TP), or HPV18 E6 and L1 proteins. The average recovery rate of the established method was between 97% and 107%. The test strip prepared in this study showed the sensitivity, specificity, and diagnostic accuracy of 97.46%, 90.57%, and 95.32%, respectively, in distinguishing patients with cervical cancer and precancerous lesions from healthy subjects, with the area under the curve (AUC) of 0.980 1 and 95% Confidence Interval (CI) of 0.956 5 to 1.000 0. The time-resolved fluorescence immunochromatography combined with the test strips prepared in this study showed high sensitivity, high accuracy, simple operation, and rapid reaction in the quantitation of HPV16 E6 and L1 proteins. It thus can be used as an auxiliary method for the diagnosis and early screening of cervical cancer and precancerous lesions and the assessment of disease course.
Oncogene Proteins, Viral/immunology* ; Humans ; Chromatography, Affinity/methods* ; Female ; Human papillomavirus 16 ; Repressor Proteins/immunology* ; Capsid Proteins/immunology* ; Papillomavirus Infections/diagnosis* ; Fluorescence ; Uterine Cervical Neoplasms/virology*

PURPOSE: To identify new immunogenic HLA-A*33;03-restricted epitopes from the human papillomavirus (HPV) 16 E7 protein for immunotherapy against cervical cancer. MATERIALS AND METHODS: We synthesized fourteen overlapping 15-amino acid peptides and measured intracellular interferon-γ (IFN-γ) production in PBMC and CD8+ cytotoxic T lymphocytes (CTLs) after sensitization with these peptides using flow cytometry and ELISpot assay. The immunogenicity of epitopes was verified using a ⁵¹Cr release assay with SNU1299 cells. RESULTS: Among the fourteen 15-amino acid peptides, E7₄₉₋₆₃ (RAHYNIVTFCCKCDS) demonstrated the highest IFN-γ production from peripheral blood mononuclear cells (PBMCs), and CD8+ CTLs sensitized with E7₄₉₋₆₃ showed higher cytotoxic effect against SNU1299 cells than did CD8+ CTLs sensitized with other peptides or a negative control group. Thirteen 9- or 10-amino acid overlapping peptides spanning E7₄₉₋₆₃, E7₅₀₋₅₉ (AHYNIVTFCC), and E7₅₂₋₆₁ (YNIVTFCCKC) induced significantly higher IFN-γ production and cytotoxic effects against SNU1299 cells than the other peptides and negative controls, and the cytotoxicity of E7₅₀₋₅₉- and E7₅₂₋₆₁-sensitized PBMCs was induced via the cytolytic effect of CD8+ CTLs. CONCLUSION: We identified E7₅₀₋₅₉ and E7₅₂₋₆₁ as novel HPV 16 E7 epitopes for HLA-A*33;03. CD8+ CTL sensitized with these peptides result in an antitumor effect against cervical cancer cells. These epitopes could be useful for immune monitoring and immunotherapy for cervical cancer and HPV 16-related diseases including anal cancer and oropharyngeal cancer.
Amino Acid Sequence ; CD8-Positive T-Lymphocytes/immunology/metabolism ; Epitopes/*immunology/therapeutic use ; Female ; *HLA-A Antigens ; Human papillomavirus 16/*immunology ; Humans ; *Immunotherapy ; Interferon-gamma/analysis/*biosynthesis ; Leukocytes, Mononuclear/immunology/metabolism ; T-Lymphocytes, Cytotoxic/immunology/metabolism ; Uterine Cervical Neoplasms/*therapy

OBJECTIVETo understand the distributions of DNA and neutralizing antibodies of human papillomavirus (HPV)16, 18 in 18-45 year-old women.

METHODSTotally, 1494 women were enrolled through multistage random sampling in Funing, Jiangsu province. Cervical exfoliated cells were collected from them for HPV DNA testing, and serum samples were taken from them for the detection of HPV16, 18 neutralizing antibodies by using pseudovirion-based neutralization assay(PBNA).

RESULTSAmong the 1494 women, 28(1.9%) and 188(12.6%) were positive for DNA and neutralizing antibody of HPV16 respectively, and 15(1.0%) and 60(4.0%) were positive for DNA and neutralizing antibody of HPV18, respectively. There were no significant differences in the detection rates of DNA and neutralizing antibody of HPV16, 18 among different age groups. About 16.7% of the women were infected with HPV16, 18, or both.

CONCLUSIONIn Funing county of Jiangsu province, most women aged 18-45 years has no immunity to HPV16 and 18, indicating that they are appropriate targets for HPV 16/18 vaccination.

Adolescent ; Adult ; Antibodies, Neutralizing ; isolation & purification ; Antibodies, Viral ; isolation & purification ; China ; DNA, Viral ; isolation & purification ; Female ; Human papillomavirus 16 ; immunology ; Human papillomavirus 18 ; immunology ; Humans ; Middle Aged ; Papillomavirus Infections ; prevention & control ; Papillomavirus Vaccines ; Young Adult

OBJECTIVETo explore the distribution of serum antibodies against human papillomavirus (HPV) 16/18 among women at high-risk for cervical cancer.

METHODSAll women when tested positive for anyone of the cervical cancer screening programs, from Xinmi county of Henan province in 2011, were recruited as the subjects of this study. Cervical exfoliated cells were collected, using cervical brush for HPV DNA testing, and 10 ml venous blood was drawn for HPV-16, 18 serum antibodies testing, by enzyme-linked immunosorbent assay.

RESULTSAmong the 952 women under study, 230 cases (24.2%)showed HPV DNA positive, with positivity rates of HPV16 and 18 L1 virus-like particle (VLP)antibodies as 23.2% and 6.5%, respectively. The overall positivity rate of any type of HPV16, 18 VLP antibodies was 26.8% . Geometric means of HPV16, 18 VLP antibody titers were 79.1 (Yangshengtang Unit,YU/ml) and 125.0(YU/ml). Positivity rate of HPV16 antibody was significantly associated with age, viral load of HPV DNA, and cervical lesion severity (P < 0.05). Seropositivity of HPV18 was also increasing with the increase of viral load (P < 0.01) with different cervical lesion significantly showing different titer of HPV18 antibody (P < 0.01). Based on the results of HPV DNA detection among the two years of study, women with HPV persistent infection showed significant higher positive rate of HPV16/18 antibodies than women who did not have HPV infection or emerging infection (P < 0.001). When comparing to those women without HPV infection, the ones with transient infection showed higher seropositivity rates on both HPV16 antibodies and titer of HPV16 antibody (P < 0.001).

CONCLUSIONSeroprevalence rates on HPV16 and 18 among the unvaccinated high-risk women in Henan were high. Prevalence of both HPV16 and 18 antibodies were correlated with age, viral load, cervical lesion and history of infection.Women with high viral load, high grade cervical lesion or history of infection would more likely to be seropositive.

Adult ; Aged ; Antibodies, Viral ; blood ; China ; epidemiology ; Female ; Human papillomavirus 16 ; immunology ; Human papillomavirus 18 ; immunology ; Humans ; Middle Aged ; Papillomavirus Infections ; epidemiology ; Seroepidemiologic Studies ; Uterine Cervical Neoplasms ; virology

Human papillomavirus (HPV)-induced cervical cancer is the second most common cancer among women worldwide. Despite the encouraging development of the preventive vaccine for HPV, a vaccine for both prevention and therapy or pre-cancerous lesions remains in high priority. Thus far, most of the HPV therapeutic vaccines are focused on HPV E6 and E7 oncogene. However these vaccines could not completely eradicate the lesions. Recently, HPV E5, which is considered as an oncogene, is getting more and more attention. In this study, we predicted the epitopes of HPV16 E5 by bioinformatics as candidate peptide, then, evaluated the efficacy and chose an effective one to do the further test. To evaluate the effect of vaccine, rTC-1 (TC-1 cells infected by rAAV-HPV16E5) served as cell tumor model and rTC-1 loading mice as an ectopic tumor model. We prepared vaccine by muscle injection. The vaccine effects were determined by evaluating the function of tumor-specific T cells by cell proliferation assay and ELISPOT, calculating the tumor volume in mice and estimating the survival time of mice. Our in vitro and in vivo studies revealed that injection of E5 peptide+CpG resulted in strong cell-mediated immunity (CMI) and protected mice from tumor growth, meanwhile, prolonged the survival time after tumor cell loading. This study provides new insights into HPV16 E5 as a possible target on the therapeutic strategies about cervical cancer.
Adult ; Aged ; Amino Acid Sequence ; Animals ; Cancer Vaccines ; administration & dosage ; immunology ; Cell Line ; Cell Line, Tumor ; Dependovirus ; genetics ; Female ; Gene Expression Regulation, Viral ; immunology ; Genetic Vectors ; genetics ; Human papillomavirus 16 ; genetics ; immunology ; Humans ; Mice ; Mice, Inbred C57BL ; Middle Aged ; Neoplasms, Experimental ; immunology ; prevention & control ; virology ; Oncogene Proteins, Viral ; genetics ; immunology ; Papillomavirus Infections ; immunology ; prevention & control ; virology ; Papillomavirus Vaccines ; administration & dosage ; immunology ; Survival Analysis ; T-Lymphocytes ; immunology ; metabolism ; Tumor Burden ; immunology ; Uterine Cervical Neoplasms ; immunology ; prevention & control ; virology ; Vaccines, Subunit ; administration & dosage ; immunology

Globally, about 70% of cervical cancers are associated with human papillomavirus (HPV)-16 or HPV-18 infection. A meta-analysis of epidemiologic studies in China showed that HPV was present in 98% of cervical cancer samples. The HPV-16/18 AS04-adjuvanted vaccine Cervarix has shown a high level of protection against HPV-16/18 infections and associated cervical lesions. This phase I trial (NCT00549900) assessed the safety, tolerability, and immunogenicity of the vaccine in Chinese. Thirty healthy Chinese females, aged 15 to 45 years with a median age of 29.5 years, received three doses of Cervarix in Months 0, 1, and 6. Safety was assessed via recording solicited local and systemic symptoms within 7 days and unsolicited symptoms within 30 days after each vaccination. Serious adverse events, new onset of chronic diseases, and other medically significant conditions were recorded throughout this trial. As an exploratory objective, HPV-16/18 antibody titers were determined by enzyme-linked immunosorbent assay in serum samples collected in Months 0 and 7. Pain at the injection site was the most frequently reported local symptom. Two subjects reported medically significant adverse events. Both cases were assessed as unrelated to vaccination by the investigator. In Month 7, 100% seroconversion was observed for both anti-HPV-16 and anti-HPV-18 with high geometric mean antibody titers. HPV-16/18 AS04-adjuvanted vaccine, evaluated for the first time in Chinese females, was generally well tolerated and immunogenic, as previously shown in global studies.
Adjuvants, Immunologic ; Adolescent ; Adult ; Antibodies, Viral ; blood ; Asian Continental Ancestry Group ; China ; Female ; Human papillomavirus 16 ; immunology ; Human papillomavirus 18 ; immunology ; Humans ; Middle Aged ; Papillomavirus Infections ; immunology ; prevention & control ; virology ; Papillomavirus Vaccines ; administration & dosage ; adverse effects ; therapeutic use ; Uterine Cervical Neoplasms ; immunology ; prevention & control ; virology ; Young Adult

OBJECTIVETo explore the relationship between human papillomavirus(HPV) infection and expression of human leukocyte antigen class I (HLA-I) family genes (HLA-A, B and C) in cervical cancers of Uighur women, and to investigate their effect on cervical cancer progression.

METHODSFresh tissue samples of 78 Uighur women with cervical squamous carcinoma, cervical intraepithelial neoplasia (CIN) or benign cervicitis were selected. HLA-A, B and C expression and HPV infection were analyzed using RT-PCR and HPV gene chips, respectively.

RESULTSThere was a tendency of increasing the total loss of HLA-A, B and C mRNA as the cervical lesions became more aggressive. Loss of HLA-I mRNA in CIN (I, II and III) and cervical squamous carcinoma was 70.0% (14/20) and 84.8% (39/46) respectively. Poorly differentiated cervical carcinomas had the highest HLA-I expression loss (90.6%). In contrast, HLA-I mRNA loss was seen in only 8% of cases of cervicitis. Moreover, it was found that high risk HPV 16 infection was strongly correlated with the loss HLA-I mRNA expression (r = 0.803, P < 0.01).

CONCLUSIONSThe loss of HLA-I gene expression is strongly correlated with HPV-16 infection, and may serve as a biomarker of cervical cancer progression in Uighur women.

Adult ; Aged ; Carcinoma, Squamous Cell ; ethnology ; genetics ; immunology ; virology ; Cervical Intraepithelial Neoplasia ; ethnology ; genetics ; immunology ; virology ; China ; ethnology ; Female ; HLA Antigens ; genetics ; metabolism ; HLA-A Antigens ; genetics ; metabolism ; HLA-B Antigens ; genetics ; metabolism ; HLA-C Antigens ; genetics ; metabolism ; Human papillomavirus 16 ; isolation & purification ; Humans ; Middle Aged ; Papillomavirus Infections ; ethnology ; genetics ; immunology ; virology ; RNA, Messenger ; metabolism ; Uterine Cervical Neoplasms ; ethnology ; genetics ; immunology ; virology ; Uterine Cervicitis ; ethnology ; genetics ; immunology ; virology

OBJECTIVETo investigate the association between HPV infection and HLA-DQB1 alleles polymorphism in the cervical carcinogenesis in Uyghur women in southern Xinjiang.

METHODSTwenty-one subtypes of HPV and the 5 alleles of HLA-DQB1 were detected from cervical tissues of 190 cervical cancer cases and 190 normal subjects by flow-through hybridization and gene chip (HybriMax) assay and polymerase chain reaction sequence-specific oligonucleotide technique (PCR-SSO).

RESULTS(1) The positive rate of HPV in cervical cancer and control groups were 70.0% and 8.9%, respectively. HPV16 was the most common type in HPV positive cervical cancer patients with a rate of 64.7%, followed by HPV18 (2.6%), HPV68 (2.1%), HPV45 (1.6%), HPV58 (1.6%), HPV39 (1.6%), HPV31 (1.1%), HPV56 (1.1%), HPV59 (0.5%), HPV53 (0.5%) and HPV6 (0.5%), respectively. The positive rates of HPV and HPV16 in the cervical cancer group were significantly higher than that in the control group. (2) The frequency of HLA-DQB1*03 was significantly lower than that in the control group (OR = 0.683, 95%CI = 0.505 - 0.923). The frequency of other HLA alleles had no significant differences between the cervical cancer group and the control group. (3) In the cervical cancer group, the frequency of HLA-DQB1*06 in both HPV positive and HPV16 positive cases were significantly higher than that in both the HPV and HPV16 negative cases.

CONCLUSIONHPV16 is the most common type in both the cervical cancer and control groups. It appears that HLA-DQB1*03 may be a protective gene in the cervical carcinogenesis in southern Xinjiang Uyghur women, and the HLA-DQB1*06 is a susceptibility gene to HPV/HPV16 infection in Uyghur women. The study of HLA alleles in the cervical carcinogenesis in Uyghur women may play an important role in the intervention research of cervical cancer.

Adult ; Aged ; Alleles ; China ; ethnology ; Ethnic Groups ; genetics ; Female ; Gene Frequency ; Genetic Predisposition to Disease ; HLA-DQ Antigens ; genetics ; HLA-DQ beta-Chains ; Human papillomavirus 16 ; isolation & purification ; Humans ; Middle Aged ; Papillomaviridae ; isolation & purification ; Papillomavirus Infections ; genetics ; virology ; Polymorphism, Genetic ; Uterine Cervical Neoplasms ; genetics ; immunology ; virology ; Young Adult

HPV16 L1 gene was amplified from HPV16 positive vaginal secretion sample by PCR, and inserted into pTO-T7 to obtain the recombinant expression vector pTO-T7-HPV16-L1. Then, the pTO-T7-HPV16-L1 was transformed into E. coil strain ER2566 and the recombinant protein HPV16 L1 was expressed in soluble form. After purification by ammonium sulfate precipitation, ion-exchange chromatography, and hydrophobic interaction chromatography, the recombinant protein HPV16 L1 had a purity of more than 98%. By removing DTT, purified HPV16 L1 proteins self-assembled in vitro into VLPs with the diameter of 50 nm. The vaccination experiments on experimental animals showed the VLPs could elicit high titer of neutralizing antibodies against HPV 16. HPV16 VLPs with high immunogenicity and high purity can be produced easily and effectively from an E. coli expression system in the study, and thus can be used in structure investigation and HPV16 vaccine development.
Animals ; Antibodies, Viral ; immunology ; ultrastructure ; Capsid Proteins ; genetics ; immunology ; isolation & purification ; Goats ; Human papillomavirus 16 ; genetics ; immunology ; ultrastructure ; Humans ; Male ; Oncogene Proteins, Viral ; genetics ; immunology ; isolation & purification ; Papillomavirus Infections ; immunology ; virology ; Rabbits ; Recombinant Proteins ; genetics ; immunology ; Vaccination ; Virion ; genetics ; immunology

OBJECTIVETo study the effects of DNA vaccine transdermal delivery with microneedle array.

METHODSThe pcDNA3.1-HPV16E7 recombinant vector acting as gene vaccine was established. The infiltration quantity of pcDNA3.1-HPV16E7 getting across the microchannels generated by microneedle arrays in vitro was observed. 30 BALB/c mice were divided into 3 groups (experimental group, in vain plasmid group, negative control). Each group had 10 mice. Then immunized BALB/c mice with a dose of 200 microg with microneedle array every two weeks. The control groups did the same as that as the study groups. Two weeks after the third immunization, the serum and lymphocytes were separated to detect the functions of humoral immunity with indirect immunofluorescence test, while, the functions of cellular immunity with lymphocyte transformation test was also detected.

RESULTSThe DNA vaccine could easily get across the microchannels generated by microneedle arrays in vitro. Moreover, the course was permanent and the whole infiltration quantity was comparatively high, reaching 0.73819 mg/cm2 at the 30th hour. And among immunized BALB/c mouse, DNA vaccine transdermal delivery with microneedle array could induce specific antibodies. Lymphocyte transformation test showed that there was significant difference for the lymphocyte transformation rate between experiment (the average of lymphocyte transformation rate was 47.25%) and control group (the average of lymphocyte transformation rate was 30.00%) (chi2 = 12.903, P < 0.001). Also, the difference was found between in vain plasmid group (the average of lymphocyte transformation rate was 43.00%) and negative control(chi2 = 7.292, P = 0.007). While, no difference was observed in the experimental group and in vain plasmid group (chi2 = 0.817, P = 0.366).

CONCLUSIONThe DNA vaccine combined administering with microneedle array might get across the microchannels generated by microneedle arrays in vitro and induce humoral and cellular immune response in vivo.

Administration, Cutaneous ; Animals ; Human papillomavirus 16 ; genetics ; immunology ; Injections ; Mice ; Mice, Inbred BALB C ; Skin Absorption ; Vaccines, DNA ; administration & dosage ; immunology