Peripheral Blood T-lymphocyte Phenotypes in Mother-Child Pairs Stratified by the Maternal HPV Status: Persistent HPV16 vs. HPV-Negative: A Case-Control Study.

Only few studies exist on the phenotype distribution of peripheral blood lymphocytes concerning persistent oral HPV infection. T-lymphocyte subsets were phenotyped in women who had persistent genital or oral HPV16 infection, using HPV-negative women as a reference group. A subset of 42 mothers and their children (n = 28), were stratified into two groups according to the mothers' HPV status. PBMCs from previously cryopreserved venous samples were immunophenotyped by flow cytometry. Proportions of the CD4+ or CD8+ lymphocytes by their immunophenotype subsets were compared between HPV-positive and -negative mothers and their children. The mean rank distribution of CD8+ memory cells was significantly higher among mothers with persistent genital HPV16 infection. The median levels of both the antigen-presenting CD4+ cells and activated CD8+ cells were significantly lower in mothers with persistent oral HPV16 infection. When oral and genital HPV16-persistors were analyzed as a group, a marker of terminal effector cells was significantly increased as compared to HPV-negative women. Significantly higher levels of activated CD4+, CD8+ and circulating CD8+ memory cells were found among children whose mothers had persistent oral HPV16 infection. Persistent HPV16 infections are associated with changes in peripheral blood T-lymphocyte subsets. The mother's persistent oral HPV16 infection possibly results in immune alterations in her offspring.

Interferon-γ and IL-5 associated cell-mediated immune responses to HPV16 E2 and E6 distinguish between persistent oral HPV16 infections and noninfected mucosa.

OBJECTIVES: Natural history of human papillomavirus (HPV) infection in the head and neck region is poorly understood, and their impact on collective HPV-specific immunity is not known. MATERIALS AND METHODS: In this study, we have performed a systematic analysis of HPV16-specific cell-mediated immunity (CMI) in 21 women with known oral and genital HPV DNA status and HPV serology (Ab) based on 6-year follow-up data. These women being a subgroup from the Finnish Family HPV Study were recalled for blood sampling to be tested for their CMI-responses to HPV16 E2, E6, and E7 peptides. RESULTS: The results showed that HPV16 E2-specific lymphocyte proliferation was more prevalent in women who tested HPV16 DNA negative in oral mucosa and were either HPV16 seropositive or negative than in HPV16 DNA+/Ab+ women (p = 0.046 and p = 0.035). In addition, the HPV16 DNA-/Ab- women most often displayed E6-specific proliferation (p = 0.020). Proportional cytokine profiles indicated that oral HPV16-negative women were characterized by prominent IFN-γ and IL-5 secretion not found in women with persisting oral HPV16 (p = 0.014 and p = 0.040, respectively). CONCLUSIONS: Our results indicate that the naturally arising immune response induced by oral HPV infections displays a mixed Th1/Th2/Th17 cytokine profile while women with persisting oral HPV16 might have an impaired HPV16-specific CMI, shifted partly toward a Th2 profile, similarly as seen earlier among patients with high-grade genital HPV lesions. Thus, the lack of HPV 16 E2 and E6 specific T memory cells and Th2 cytokines might also predispose women for persistent oral HPV16 infection which might be related to the risk of cancer.

Human papillomavirus 16-specific cell-mediated immunity in children born to mothers with incident cervical intraepithelial neoplasia (CIN) and to those constantly HPV negative.

OBJECTIVES: HPV infections are detected in sexually naive children. This has raised the question about the role of early HPV infections in either protecting or predisposing to further HPV infections. HPV16-specific cell-mediated immunity (CMI) was studied in 10 case-children born to mothers with an incident cervical intraepithelial neoplasia (CIN) diagnosed during their 14-year follow-up (FU), and in 21 children born to mothers, who remained constantly HPV-negative (controls). The mean age of children was 12.3 years. METHODS: Peripheral blood mononuclear cells were isolated from blood and stimulated with peptide pools covering HPV16 E2, E6 and E7. Proliferation of lymphocytes, their secretion of cytokines, and the frequency of regulatory T-cells were determined. The results were correlated with the HPV status and analyzed in a nested case-control setting. RESULTS: All children, except two controls, displayed CMI against HPV16 E2, E6 and/or E7 peptides associated with type 1 and 2 cytokine secretion. Only two statistically significant differences were found in the nested case-control setting; (1) case-children had a higher TNF-α response to HPV16 E2 (p = 0.004) than controls and (2) controls had no response to HPV16 E7.2 peptide pool while 3/10 case-children had (p = 0.013). Totally, 50 and 57 % of the cases and controls, respectively, had HPV positive oral samples at some FU-visit. In addition, the children without any HPV antibodies before the age of 6 months showed proliferative responses of PBMC after HPV16 exposure more frequently than other children (p = 0.045). CONCLUSIONS: HPV16-specific CMI is common in young, sexually inexperienced children. This suggests that oral HPV infections occur frequently in children. Our results might also explain the previous findings that half of healthy adults demonstrate HPV-specific CMI irrespective of their partner/sexual status.

Cell mediated immunity against HPV16 E2, E6 and E7 peptides in women with incident CIN and in constantly HPV-negative women followed-up for 10-years.

BACKGROUND: Virus-specific cell-mediated immunity (CMI) plays a role in the outcome of genital HPV infections. To cast further light on the question why most women clear their HPV infection while others develop high-grade cervical intraepithelial neoplasia (CIN), we analyzed HPV16 E2-, E6- and E7 -specific CMI in women who developed CIN during a 10-year follow-up of the Finnish Family HPV cohort. METHODS: Overlapping 30-35 mer peptides covering the entire HPV16 E2-, E6- and E7 protein sequences were used for defining the lymphocyte proliferation capacity, cytokine production (IL-2, IL-5, IL-10, IL-17A, IFN-γ and TNF-α) and numbers of HPV16 -specific CD4+ CD25+ Foxp3+ regulatory T-cells in 10 women who developed CIN, and in 22 control women who tested constantly HPV-negative during the follow-up. HPV-specific CMI was related to the demographic data including sexual behavior, smoking and alcohol consumption. RESULTS: Women with CIN and their controls had similar T-cell mediated immunity against HPV16 E2, E6 and E7 peptide pools. However, nearly fourfold higher T-cell reactivity against common antigens was found in the CIN women than in the healthy donors (p = 0.001). HPV16 E6 stimulation resulted in higher IL-17A secretion in the controls than in the CIN women (p = 0.035). Smoking and use of alcohol affected the T-cell response to common antigens but not to HPV peptides (p = 0.032 and 0.045, respectively). CONCLUSION: While both the CIN women and controls exhibited an HPV16-specific CMI, IL-17A might be of importance in HPV induced pathology. The hyper-responsiveness of the CIN patients to common antigens needs further studies. Smoking and alcohol had no effect on HPV-specific CMI.

Human papillomavirus 16 E2-, E6- and E7-specific T-cell responses in children and their mothers who developed incident cervical intraepithelial neoplasia during a 14-year follow-up of the Finnish Family HPV cohort.

BACKGROUND: Human papillomavirus (HPV) infection has traditionally been regarded as a sexually transmitted disease (STD), but recent evidence implicates that an infected mother can transmit HPV to her newborn during pregnancy, at delivery, perinatal period or later. Given the lack of any studies on HPV-specific immune responses in children, we conducted HPV16-specific cell-mediated immune (CMI) monitoring of the mother-child pairs with known oral and genital HPV follow-up (FU) data since the delivery. In the Finnish Family HPV Study, 10 out of 331 mothers developed incident cervical intraepithelial neoplasia (CIN) during their 14-year FU. Our hypothesis according to the common dogma is that there is no HPV16 specific immune response in offspring of the CIN mother as she/he has not started the sexual life yet. METHODS: We used overlapping 30-35 mer peptides covering the entire HPV16 E2, E6 and E7 protein sequences. Assays for lymphocyte proliferation capacity, cytokine production and HPV16-specific Foxp3 + CD25 + CD4+ regulatory T-cells were performed. RESULTS: HPV16-specific proliferative T-cell responses were broader in children than in their mothers. Nine of 10 children had responses against both E2 peptide pools compared to only 4 of the 10 mothers. Six of the 10 children and only 2 mothers displayed reactivity to E6 and/or E7. The cytokine levels of IL-2 (p = 0.023) and IL-5 (p = 0.028) induced by all peptide pools, were also higher among children than their mothers. The children of the mothers with incident CIN3 had significantly higher IFN-γ (p = 0.032) and TNF-α (p = 0.008) levels than other children. CONCLUSIONS: Our study is the first to show that also children could have HPV-specific immunity. These data indicate that the children have circulating HPV16-specific memory T-cells which might have been induced by previous HPV16 exposure or ongoing HPV 16 infection.

Human papillomavirus genotypes present in the oral mucosa of newborns and their concordance with maternal cervical human papillomavirus genotypes.

OBJECTIVES: To elucidate the concordance of human papillomavirus (HPV) genotypes between the mother and her newborn and to identify risk factors for the vertical transmission of HPV. STUDY DESIGN: HPV genotypes present in 329 pregnant women, their newborns, cord blood, and placenta samples were determined by molecular techniques, including using pure DNA for nested polymerase chain reaction. HPV antibodies were tested using multiplex HPV serology. Kappa statistics and the Wilcoxon test were used to assess concordance, and regression analysis was used to calculate ORs and 95% CIs. RESULTS: HPV DNA was detected in 17.9% of oral samples from newborns and in 16.4% of the cervical samples of the mothers. At delivery, mother-newborn pairs had similar HPV-genotype profiles, but this concordance disappeared in 2 months. Oral HPV carriage in newborns was most significantly associated with the detection of HPV in the placenta (OR=14.0; 95% CI, 3.7-52.2; P=.0001). The association between status of the cord blood and oral HPV was also significant at delivery (OR=4.7; 95% CI, 1.4-15.9; P=.015) but disappeared within 1 month. HPV antibodies in infants were of maternal origin (OR=68; 95% CI, 20.1-230.9; P=.0001). CONCLUSIONS: HPV is prevalent in oral samples from newborns. The genotype profile of newborns was more restricted than that of the maternal cervical samples. The close maternal-newborn concordance could indicate that an infected mother transmits HPV to her newborn via the placenta or cord blood.

Molecular markers implicating early malignant events in cervical carcinogenesis.

BACKGROUND: Human papillomavirus can induce a stepwise progression of precursor lesions to carcinoma. Sensitive and specific molecular markers are needed to identify the cervical lesions (CIN) at risk for this progression. hTERT activation could be one indicator of a point of no return in malignant progression. METHODS: The UT-DEC-1 cell line is an in vitro model for the study of human papillomavirus-induced progression. Using molecular mining, nine potential genes interlinking hTERT and viral oncogene expression with the phenotypical features of CIN2 were identified. After preliminary testing with real-time PCR, five genes were selected for further analysis: hTERT, DKC1, Bcl-2, S100A8, and S100A9. These proteins were also tested in a series of 120 CIN lesions using immunohistochemistry. RESULTS: Analysis of the mRNA expression of these genes at different cell passages revealed three time points with significant changes. hTERT, Bcl-2, and S100A9 were also overexpressed in CIN lesions, and the expression pattern changed during the progression toward CIN3 lesions. CONCLUSIONS: These identified time points that were combined with the mRNA overexpression of target genes matched events previously shown to be important in the progression toward malignancy: (a) the viral integration into the cell genome and episome loss; (b) the selection of cells with an acquired growth advantage and ability to maintain telomerase activity; and (c) the final stage of malignancy with permanently upregulated telomerase. IMPACT: hTERT, Bcl-2, and S100A9 together might compose a potential prognostic marker panel for the assessment of CIN lesions. These results, however, need further validation in prospective clinical settings. (c)2010 AACR.