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

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

Human papillomaviruses (HPV) are causally associated with cervical cancer and genital warts. Lack of permissive and productive cell cultures for HPV has hindered the study of HPV and evaluation of virus-neutralizing antibodies. So generation of infectious virions in vitro is highly desirable. In this report, we got high titer infectious HPV16 pseudovirions by calcium phosphate co-transfection of codon optimized HPV16 capsid genes L1 and L2 and reporter plasmids into 293FT cell line. Electron micrograph indicated that the pseudovirions were morphologically similar with the intact HPV16 virions. To evaluate the feasibility of using the pseudovirions to identify neutralizing monoclonal antibodies (mAbs), pseudovirions were incubated with 2-fold gradient dilution of the well identified mAbs V5, E70, U4 and D9 and then used to infect 293FT cells preplated in 96-well tissue culture plate. The infection of pseudovirions could be inhibited by neutralizing mAbs V5, E70 and U4 that recognize surface conformational epitopes on L1 VLP, but not by mAb D9 that is reactive to a linear epitope buried in VLP, which indicated that the pseudovirions could be used to evaluate the neutralization efficiency of mono- and polyclonal antibodies. The pseudovirions were then employed to identify neutralizing mAbs from 18 mAbs generated previously in our lab, 8 of which were conformational and 10 were linear. PD1 and 3D10, both of which recognized conformational epitopes on L1 VLP, had obviously strong neutralizing efficiency, with the neutralizing titer reached 81,920 and 20,480 respectively, while none of the linear mAbs were neutralizing, which reflected that rare linear mAbs have neutralization activity. The mechanism of PD1 and 3D10 block the infection of HPV16 pseudovirions need to be further studied. The technologies about generation of HPV16 pseudovirions and screening neutralizing mAb in our report are economical and efficient, can be easily used in large scale. They pave the way for rapid and precise evaluation of the protection efficiency of our prophylactic HPV vaccine being developed now.
Animals ; Antibodies, Monoclonal ; immunology ; Biomimetics ; Cell Line ; Epitopes ; immunology ; Human papillomavirus 16 ; immunology ; physiology ; Lipids ; genetics ; Neutralization Tests ; Transfection ; Viral Vaccines ; immunology ; Virion ; immunology

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

OBJECTIVE: To determine the relationship between serum antibody against HPV16 E4 and cervical cancer, and to construct HPV 16 E4 protein expression vector as an antigen to detect its corresponding serum antibody among different populations. METHODS: HPV16 E4 early gene was ligated into pRSET-A expression vector. The constructed plasmids were transformed into BL21 (DE3)cells, and induced to express HPV 16 E4 protein by isopropylthio-beta-D-galactoside (IPTG). The expressed E4 inclusions were denatured, purified through Ni-column, and renatured. After the activity was revealed, antibodies against HPV 16 E4 in the sera from healthy women and patients with chronic cervicitis and cervical cancer were respectively determined by enzyme-linked immunosorbent assay (ELISA) using the fusion protein as the antigen. RESULTS: HPV 16 E4 fusion protein of Mr 15*10(3) was expressed by pRSET-16E4 after IPTG induction. The fusion protein accounted for 30% of the total bacterial proteins and expressed as inclusive body. After purification with Ni-NTA agarose resin, the recombinant protein revealed purity of 95%, and activity of the renatured protein was identified by ELISA. The serum antibody-positive rate of HPV 16 E4 was 10.00%, 39.13% and 28.13%, respectively in 80 healthy women, 46 chronic cervicitis patients, and 32 cervical cancer patients. The antibody-positive rate in cervical cancer patients and chronic cervicitis patients were significantly higher than that in healthy women (P<0.01), while the difference between the antibody-positive rate in cervical cancer patients and chronic cervicitis patients was not significant. CONCLUSION HPV 16 E4 protein expressed from pRSET-A/BL21 can be used in serological studies on cervical cancer-related HPV infection. Serum antibody against HPV16 E4 is present in a significantly higher percentage in cervical cancer and chronic cervicitis patients than in healthy women.
Adult ; Aged ; Antibodies, Viral ; blood ; Female ; Genetic Vectors ; Human papillomavirus 16 ; genetics ; immunology ; Humans ; Middle Aged ; Oncogene Proteins, Viral ; biosynthesis ; genetics ; immunology ; Papillomavirus Infections ; immunology ; virology ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; Uterine Cervical Neoplasms ; immunology ; virology ; Uterine Cervicitis ; virology

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

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

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 develop human papillomavirus (HPV) 16 DNA vaccine for the treatment of HPV16 infection and its related tumors.

METHODSHPV16 oncogene E7 was modified by combined approaches including insertion and replication of specific region of E7 gene, murine codon optimization, and point-mutation at transforming regions of the E7 protein. The resulting artificial gene, named as mE7, was obtained by gene synthesis. The mE7 gene was then genetically fused to murine CD40 ligand (CD40L) by overlapping PCR to form the mE7/CD40L fusion gene. The mE7/CD40L gene was inserted into pVR1012 plasmid and then immunized C57/BL6 mice intramuscularly. The E7-specific IFN-gamma-secreting CD8+ T cells were analyzed with EIISPOT, and E7-specific antibody was measured by indirect ELISA. FACS assays were performed to analyze the activation of E7-specific Th cells. Mice were vaccinated, followed by tumor challenged or challenged before immunization. Tumor growth was observed.

RESULTSThe mE7 DNA vaccine elicited an increased E7-specific antibody level (P < 0.01), E7-specific IFN-gamma-secreting CD8+ T (P < 0.01), and CD4+ T cells number (P < 0.05), compared with those of mice immunized with wE7 gene. Furthermore, the mE7/CD40L DNA vaccine elicited an increased number of E7-specific IFN-gamma secreting CD8+ T cell compared with that of mice immunized with mE7 gene (P < 0.01); however, no significant differences were found between mice immunized with the mE7 gene and mE7/CD40L fusion gene in the E7-specific antibody production and Th cell activation. In the preventive experiment, all mice received the mE7 or mE7/CD40L remained tumor-free 7 weeks after challenges with TC-1 tumor cells, while the wE7 group exhibited tumor growth within 2 weeks. In the therapeutic experiment, all the mice in the wE7 group exhibited tumor growth within 8 days, while among mice receiving the mE7 and mE7/CD40L, 30% and 45% of mice remained tumor-free after TC-1 challenge, respectively. HE staining of tumor tissues showed copious lymphocytes infiltration around tumor cells in mE7 and mE7/CD40L mice with regression of tumor growth.

CONCLUSIONSThe mE7 DNA vaccine increases the E7-specific humoral and cellular immune responses, and the fusion of CD40L to mE7 gene enhances the specific immune responses and anti-tumor effects against HPV16 E7-expressing murine tumors. mE7/CD40L may therefore be a suitable and promising target for HPV16 therapeutic vaccine.

Animals ; CD40 Antigens ; genetics ; immunology ; Cancer Vaccines ; genetics ; immunology ; therapeutic use ; Cell Line, Tumor ; Gene Fusion ; Human papillomavirus 16 ; immunology ; Immunity, Cellular ; Immunity, Humoral ; Mice ; Mice, Inbred C57BL ; Neoplasm Transplantation ; Papillomavirus E7 Proteins ; genetics ; immunology ; Papillomavirus Vaccines ; genetics ; immunology ; therapeutic use ; Vaccines, DNA ; genetics ; immunology ; therapeutic use

OBJECTIVETo construct recombinant adenovirus containing codon-modified HPV16L1 gene, and evaluate systemic and mucosal immunological responses induced after immunization with the recombinant virus.

METHODSThe recombinant adenovirus rAd-mod.HPV16L1 was constructed by Admax kit. The C57 BL/6 mice were immunized by purified rAd-mod.HPV16L1 through different inoculation routes. The immunological effect was evaluated by testing the specific neutralizing antibodies in sera and vaginal secretions of immunized mice through indirect ELISA and neutralization assay based HPV pseudovirus.

RESULTSThe result showed that intramuscular immunization could induce good systemic immunity, but the mucosal immunity was too weak, and immunization via intranasal route could induce satisfactory immunity both in sera and vaginal secretions, while intravaginal immunization failed to induce any specific immunological responses either in sera or vaginal secretions.

CONCLUSIONThe recombinant adenovirus containing codon- modified HPV16L1 gene was successfully constructed. Immunization through intranasal route could induce satisfactory immunity both in sera and vaginal secretions, while intramuscular immunization could only induce high titer of neutralizing antibodies in sera.

Adenoviridae ; genetics ; Animals ; Antibodies, Viral ; analysis ; immunology ; Antibody Specificity ; Capsid Proteins ; genetics ; immunology ; Codon ; genetics ; DNA, Recombinant ; genetics ; Female ; Genetic Engineering ; Human papillomavirus 16 ; genetics ; immunology ; Mice ; Mice, Inbred C57BL ; Oncogene Proteins, Viral ; genetics ; immunology ; Vaccination