HPV induced R-loop formation represses innate immune gene expression while activating DNA damage repair pathways.

R-loops are trimeric nucleic acid structures that form when an RNA molecule hybridizes with its complementary DNA strand, displacing the opposite strand. These structures regulate transcription as well as replication, but aberrant R-loops can form, leading to DNA breaks and genomic instability if unresolved. R-loop levels are elevated in many cancers as well as cells that maintain high-risk human papillomaviruses. We investigated how the distribution as well as function of R-loops changed between normal keratinocytes and HPV positive cells derived from a precancerous lesion of the cervix (CIN I). The levels of R-loops associated with cellular genes were found to be up to 10-fold higher in HPV positive cells than in normal keratinocytes while increases at ALU1 elements increased by up to 500-fold. The presence of enhanced R-loops resulted in altered levels of gene transcription, with equal numbers increased as decreased. While no uniform global effects on transcription due to the enhanced levels of R-loops were detected, genes in several pathways were coordinately increased or decreased in expression only in the HPV positive cells. This included the downregulation of genes in the innate immune pathway, such as DDX58, IL-6, STAT1, IFN-ß, and NLRP3. All differentially expressed innate immune genes dependent on R-loops were also associated with H3K36me3 modified histones. Genes that were upregulated by the presence of R-loops in HPV positive cells included those in the DNA damage repair such as ATM, ATRX, and members of the Fanconi Anemia pathway. These genes exhibited a linkage between R-loops and H3K36me3 as well as γH2AX histone marks only in HPV positive cells. These studies identify a potential link in HPV positive cells between DNA damage repair as well as innate immune regulatory pathways with R-loops and γH2AX/H3K36me3 histone marks that may contribute to regulating important functions for HPV pathogenesis.

Detection of cytokines in cervicovaginal lavage in HIV-infected women and its association with high-risk human papillomavirus.

Background: Women living with HIV/AIDS (WLHA) have an increased prevalence of high-risk HPV infection (HR-HPV) and cervical intraepithelial neoplasia (CIN) and a greater risk of cervical cancer despite access to a new generation of antiretroviral therapy. The aim of this study is to evaluate the concentrations of different cytokines involved in the local immune response in WLHA, which is fundamental for understanding the pathogenesis of HPV-related cancer in this population. Methods: IL-1ß, IL-2, IL-4, IL-6, IL-10, IFN-γ, TNF-α, IP-10, GM-CSF, and MIP-1α were investigated in the cervicovaginal lavage (CVL) of 106 WLHA attending at Hospital Universitario Professor Edgard Santos in Salvador, Bahia, Brazil, during the period December 2019 to April 2023 by Luminex®. All participants were also tested for Chlamydia trachomatis and Neisseria gonorrhoeae and underwent colposcopy, Pap smear, and Nugent score. HIV plasma viral load (VL) and CD4 cell count were performed for all WLHA. Results: In this study, 22.6% (24/106) of WLHA were infected with HR-HPV. A higher proportion of patients with HR-HPV (66.7%) had detectable levels of IL-10 than those negative ones (40.2%, p = 0.02). More premenopausal women had either IL-6 (51.4%) or IP-10 (58.3%) than those in menopausal status (26.5% for IL-6 and 32.4% for IP-10, p = 0.013 and p = 0.011, respectively). Vaginosis was negatively associated with detection of IP-10 (24.2% vs. 61.4%, p < 0.001) and INF-γ (39.4% vs. 68.6%, p = 0.005). A positive association was detected for IL-1ß (66.7 vs. 37.1%, p = 0.005) and IL-10 (63.6% vs. 37.1%, p = 0.01). VL and CD4 were not associated with the studied cytokines. Conclusion: We demonstrated a positive association between IL-10 and HPV infection in CVL, suggesting the predominance of the Th2 response in HIV/HPV co-infected patients. However, further studies with longer follow-up will be needed to evaluate the association of IL-10 with HPV infection, CIN, and cervical cancer in WLHA.

Allelic loss of HLA class I facilitates evasion from immune surveillance in cervical intraepithelial neoplasia.

Loss of heterozygosity (LOH) has been reported to occur in HLA regions in cervical intraepithelial neoplasia (CIN) and cervical cancer. However, the details of how this is related to the progression of CIN have been unclear. In this study, we examined the human papillomavirus (HPV) antigen-presenting capacity of people with CIN and the significance of LOH of HLA class I in the progression of CIN. It was shown that differences in antigen-presenting capacity among each case depended on HLA types, not HPV genotypes. Focusing on the HLA type, there was a positive correlation between antigen-presenting capacity against HPV and the frequency of allelic loss. Furthermore, the lost HLA-B alleles had a higher HPV antigen-presenting capacity than intact alleles. In addition, frequency of LOH of HLA class I was significantly higher in advanced CIN (CIN2-3) than in cervicitis or early-stage CIN (CIN1): around half of CIN2-3 had LOH of any HLA class I. Moreover, the antigen-presenting capacity against E5, which is the HPV proteins that facilitate viral escape from this immune surveillance by suppressing HLA class I expression, had the most significant impact on the LOH in HLA-B. This study suggests that HPV evades immune surveillance mechanisms when host cells lose the capacity for antigen presentation by HLA class I molecules, resulting in long-term infection and progression to advanced lesions.

Randomized Phase II Trial of Imiquimod with or without 9-Valent HPV Vaccine versus Observation in Patients with High-grade Pre-neoplastic Cervical Lesions (NCT02864147).

PURPOSE: We report the results of a randomized phase II trial of imiquimod, a topical immune-response modulator versus imiquimod plus a 9-valent human papillomavirus (HPV) vaccine (9vHPV) versus clinical surveillance in cervical intraepithelial neoplasia (CIN2/3) patients. PATIENTS AND METHODS: We randomly allocated 133 patients with untreated CIN2/3 in equal proportions to a 4-month treatment with self-applied vaginal suppositories containing imiquimod (Arm B) or imiquimod plus a 9vHPV (Arm C) versus clinical surveillance (Arm A). The main outcome was efficacy, defined as histologic regression to CIN1 or less. Secondary outcomes were HPV clearance and tolerability. Exploratory objectives included the comparison of cervical CD4/CD8 T-cell infiltration at baseline, mid-study, and posttreatment by flow cytometry among study arms. RESULTS: Of the 114 evaluable patients 77% and 23% harbored CIN2 and CIN3, respectively. Regression to CIN1 or less was observed in 95% of patients in the imiquimod group (Arm B) compared with 79% in the control/surveillance (Arm A); P = 0.043 and 84% in the imiquimod+9vHPV group (Arm C; P = 0.384 vs. Arm A). Neither of the treatment-arm differences from Arm A reached the prespecified α = 0.025 significance level. No significant differences were noted in the secondary outcome of rate of HPV clearance. The number of tissue-resident memory CD4/CD8 T cells in cytobrush samples demonstrated a >5-fold increase in Arm B/imiquimod when compared with Arm A/surveillance (P < 0.01). In contrast, there was no significant difference in T-cell responses among participants in Arm C when compared with Arm A. Imiquimod treatment was well tolerated. CONCLUSIONS: Although imiquimod induced a higher regression to CIN1 or less and significant increases in CD4/CD8 T cells infiltrating the cervix, it did not meet its prespecified statistical outcome for efficacy. A higher regression rate than expected was observed in the surveillance arm of this prospective trial. Future clinical trials with imiquimod targeting CIN3 patients are warranted.

The Role of Neutrophil-Lymphocytes Ratio in the Prognosis of CIN2+ Recurrence after Excisional Treatment.

OBJECTIVES: The main risk factor involved in CIN2+ recurrence after treatment is the HPV persistent infection. The dysregulation of the immune system permits only HR-HPVs to become persistent infections, to promote cancer development, and to increase the risk of recurrence after treatment. Therefore, there is a shift to a Th2-type cytokine pattern during the carcinogenesis pathway; for this reason, the neutrophil-lymphocytes ratio (NLR) could be a marker of this immunological change. The study aimed to analyse the predictive role of NLR in the recurrence of high-grade CIN (CIN2+) after excisional treatment in a real-world life setting of patients treated for CIN2+. DESIGN: This study wascross-sectional study. PARTICIPANTS/MATERIALS, SETTING, METHODS: We examined a retrospective database of 444 patients, who attended the colposcopy service of our department from 2011 to 2020 due to an abnormal screening Pap smear, and we compared the clinical characteristics to NLR performed at the time of diagnosis. All analysed patients were treated according to an established protocol (colposcopy every 6 months for the first 2 years and every year for over 3 years) and HPV-DNA test and cervical biopsy were performed at entry and the end of follow-up. All patients underwent a blood sample examination, including complete white blood cell counts and collecting neutrophil and lymphocyte values expressed as 103/mL. RESULTS: The sensitivity (SE) and specificity (SP) of the NLR cut-off point of 1.34 for the diagnosis of CIN2+ recurrence were 0.76 and 0.67, respectively. We found that CIN2+ recurrences were significantly higher in patients with NLR <1.34 (3.7% vs. 0.6%, p = 0.033) and the 5-year recurrence-free survival was higher in patients with NLR ≥1.34 (97% vs. 93%, p = 0.030). LIMITATIONS: Firstly, the retrospective analysis and low incidence of recurrence may limit the conclusions. Second, for the retrospective design of the study, we did not take into consideration the patient's comorbidities and habits (smoking) that may influence the NLR. On the other hand, the median duration of follow-up in our study was 26 months (IQR: 22-31), which fully reflects the incidence of recurrences. CONCLUSIONS: It is well known that CIN2+ lesions are sustained by deregulation of the immune system caused by persistent HPV infection, which may lead to cervical cancer. Among the actors underlying dysregulation of immunity, lymphocytes are involved in the permission of persistent infection and for this reason, NRL could be a reliable and cost-effective biomarker in predicting the risk of recurrence, especially for high-grade cervical lesions.

Effectiveness and immune responses of focused ultrasound ablation for cervical intraepithelial neoplasia.

PURPOSE: To investigate the safety, efficacy, and the immune responses of focused ultrasound in cervical intraepithelial neoplasia (CIN). METHODS: Patients with biopsy-confirmed CIN were recruited for focused ultrasound treatment and asked to return during 3-6 and 6-12 months post-treatment to receive cervical cytology, high-risk human papilloma virus (HPV) detection, and colposcopy. The effective rate was evaluated within 3-6 months, whereas the recurrence rate was evaluated within 6-12 months. Cervicovaginal lavage and cervical tissue were sampled before and 3-6 months after treatment. The expression of interferon gamma (IFN-γ), endoplasmic reticulum aminopeptidase 1 (ERAP1), human leucocyte antigen I (HLA-I), cluster of differentiation 4 (CD4), and cluster of differentiation 8 (CD8) in the cervical tissue were observed by immunohistochemistry. Immunoglobulin A (IgA) and interleukin 10 (IL-10) levels in the cervicovaginal lavage were detected by enzyme-linked immunosorbent assay. Comparisons were made in immune analyte levels before and after treatment. RESULTS: We analyzed the results of 154 patients. The effective rate at 3-6 months was 96.8%. The recurrence rate at 6-12 months was 2.0%. The eradication rate of HPV was 72.4% at 3-6 months and 81.0% at 6-12 months. No serious adverse reactions and complications were observed. After treatment, a higher expression of ERAP1 was observed (p < 0.05). Significant down-regulation of IgA and IL-10 were detected (each p < 0.05). However, the expression of CD4, CD8, HLA-I, as well as the release of IFN-γ, did not reach statistical significance (each p > 0.05). CONCLUSIONS: Focused ultrasound is an effective and safe therapy for treating CIN, which could improve the local immune milieu of the cervix to some extent.

Therapeutic DNA Vaccines against HPV-Related Malignancies: Promising Leads from Clinical Trials.

In 2014 and 2021, two nucleic-acid vaccine candidates named MAV E2 and VGX-3100 completed phase III clinical trials in Mexico and U.S., respectively, for patients with human papillomavirus (HPV)-related, high-grade squamous intraepithelial lesions (HSIL). These well-tolerated but still unlicensed vaccines encode distinct HPV antigens (E2 versus E6+E7) to elicit cell-mediated immune responses; their clinical efficacy, as measured by HSIL regression or cure, was modest when compared with placebo or surgery (conization), but both proved highly effective in clearing HPV infection, which should help further optimize strategies for enhancing vaccine immunogenicity, toward an ultimate goal of preventing malignancies in millions of patients who are living with persistent, oncogenic HPV infection but are not expected to benefit from current, prophylactic vaccines. The major roadblocks to a highly efficacious and practical product remain challenging and can be classified into five categories: (i) getting the vaccines into the right cells for efficient expression and presentation of HPV antigens (fusion proteins or epitopes); (ii) having adequate coverage of oncogenic HPV types, beyond the current focus on HPV-16 and -18; (iii) directing immune protection to various epithelial niches, especially anogenital mucosa and upper aerodigestive tract where HPV-transformed cells wreak havoc; (iv) establishing the time window and vaccination regimen, including dosage, interval and even combination therapy, for achieving maximum efficacy; and (v) validating therapeutic efficacy in patients with poor prognosis because of advanced, recurrent or non-resectable malignancies. Overall, the room for improvements is still large enough that continuing efforts for research and development will very likely extend into the next decade.

Role of FoxP3-positive regulatory T-cells in regressive and progressive cervical dysplasia.

PURPOSE: Forkhead Box Protein 3 (FoxP3) is known as a key mediator in the immunosuppressive function of regulatory T-cells (Tregs). The aim of our study was to investigate whether FoxP3-positive Tregs have the potential to act as an independent predictor in progression as well as in regression of cervical intraepithelial neoplasia, especially in patients with intermediate cervical intraepithelial neoplasia (CIN II). METHODS: Nuclear FoxP3 expression was immunohistochemically analysed in 169 patient samples (CIN I, CIN II with regressive course, CIN II with progressive course, CIN III). The median numbers were calculated for each slide and correlated with the histological CIN grade. Statistical analysis was performed by SPSS 26 (Mann-Whitney U test, Spearman's rank correlation). RESULTS: An increased FoxP3 expression in CIN II with progression could be detected in comparison to CIN II with regression (p = 0.003). Total FoxP3 expression (epithelium and dysplasia-connected stroma) was higher in more advanced CIN grades (p < 0.001 for CIN I vs. CIN II; p = 0.227 for CIN II vs. CIN III). A positive correlation could be detected between FoxP3-positive cells in epithelium and total FoxP3 expression (Spearman's Rho: 0,565; p < 0.01). CONCLUSION: Expression of FoxP3 could be a helpful predictive factor to assess the risks of CIN II progression. As a prognosticator for regression and progression in cervical intraepithelial lesions it might thereby help in the decision process regarding surgical treatment vs. watchful waiting strategy to prevent conisation-associated risks for patients in child-bearing age. In addition, the findings support the potential of Tregs as a target for immune therapy in cervical cancer patients.

Human papilloma virus infection of uterine cervix and spectrum of cervical pathology in human immunodeficiency virus/AIDS.

BACKGROUND: Human papilloma virus (HPV) is one of the most common causes of sexually transmitted viral diseases worldwide. High-risk HPV types such as HPV16 and 18 are known to cause cervical dysplasia and carcinoma. In human immunodeficiency virus (HIV)-positive individual, chance of HPV coinfection and risk of cervical dysplasia/carcinoma have been found to be significantly more than in HIV-negative individuals. AIM: In this institution-based, cross-sectional, observational study, we aim to find out the relationship of HPV infection of the uterine cervix with cervical dysplasia and neoplasia in HIV-infected/AIDS patients. MATERIALS AND METHODS: Conventional Pap smears were taken from HIV-infected individuals admitted in the department of gynecology and obstetrics and reported by the Bethesda system. A second sample was sent to the virology unit of ICMR for detection and typing of HPV. Control samples were taken from HIV-negative individuals. RESULTS: Fifty HIV-positive patients were included in this study. On cervical Pap smear examination, 32 cases were cytologically benign and 18 cases showed atypical cytomorphology. Twenty-four cases were HPV positive, among which 16 were cytologically atypical and 8 were benign. HPV 16 was the most common subtype (50%) followed by HPV 18 (37.5%) and others (12.5%) in HIV-positive patients. Chance of cervical dysplasia increased with age independent of HIV infection and with progressive lower CD4 count. Koilocytosis was a significant predictor of HPV infection. Majority of patients were asymptomatic. Peak incidence of HPV infection occurred in reproductive age group (20-40 years). The association between HIV and HPV coinfection (P = 0.002) and between HPV infection and cytology atypia (P < 0.0001) was statistically significant. CONCLUSION: Present study highlights the necessity of routine cervical Pap smear screening in HIV infected reproductive age-group women. Early detection enables dysplasia to revert or be effectively managed.

The vaginal microbiota and innate immunity after local excisional treatment for cervical intraepithelial neoplasia.

BACKGROUND: Vaginal microbiota (VMB) composition is altered in women with cervical intra-epithelial neoplasia (CIN) compared to healthy controls and is associated with disease progression. However, the impact of CIN excision on the VMB and innate immunity is not known. This observational study aims to explore the impact of CIN excision on the VMB, antimicrobial peptides (AMP) and proinflammatory cytokines. METHODS: We sampled 103 non-pregnant, premenopausal women at the time of excisional treatment for CIN and at their 6-month follow-up visit. A further 39 untreated controls with normal cytology were also sampled. We used metataxonomics to group vaginal swab samples into community state types (CSTs) and ELISA to quantify cytokine and AMP levels in matched vaginal secretions. Analyses were performed to compare the bacterial composition and immune analyte levels before and after CIN excision and in healthy controls. RESULTS: Women with CIN had significantly higher rates of Lactobacillus species depletion pre-treatment compared to healthy controls (CST IV 21/103, 20% vs 1/39, 3%, p = 0.0081). Excision did not change the VMB composition, with CST IV remaining significantly more prevalent after excision compared to untreated, healthy controls (CST IV 19/103, 20% vs 1/39, 3%, p = 0.0142). Prevotella bivia and Sneathia amnii were significantly higher in samples before treatment compared to untreated controls, and Prevotella bivia remained significantly higher amongst the treated, with less Lactobacillus crispatus compared to untreated controls. IL-1ß and IL-8 remained significantly elevated pre- (p < 0.0001 and p = 0.0014, respectively) and post-treatment (p < 0.0001 and p = 0.0035, respectively) compared to untreated controls. Levels of human beta-defensin-1 and secretory leukocyte protease inhibitor were both significantly reduced following CIN excision (p < 0.0001); however, their levels remained lower than controls post-treatment. CONCLUSIONS: Women with CIN have an increased prevalence of Lactobacillus sp. depletion, high-diversity VMB composition, and higher levels of proinflammatory cytokines and AMPs compared to normal controls. Surgical excision of the disease reduces levels of vaginal AMPs but does not alter VMB composition or cytokine levels. These findings suggest that women with CIN have an inherent predisposition to a high-diversity proinflammatory environment that is not corrected by disease excision. The failure to re-establish a Lactobacillus-enriched CST may explain why women remain at high risk of pre-invasive and invasive disease recurrence.

Cytokines profiles in cervical mucosa in patients with cervical high-risk human papillomavirus infection.

INTRODUCTION: The purpose of this study was to detect the expression of local cytokines in cervical mucosa between patients with transient and persistent HR-HPV infection with or without CIN. METHODOLOGY: A total of 150 patients who were diagnosed as HR-HPV infection in Tianjin Central Hospital of Obstetrics and Gynecology from January 2016 to December 2016 were included in this study. The expression levels of 9 cytokines in 150 patients with HR-HPV infection, including interleukin (IL)-1ß, IL-4, IL-6, IL-8, IL-10, IL-12, IL-12p70, IL-21, interferon (IFN)-γ and tumor necrosis factor (TNF)-α, were simultaneously measured by using a multiplex immunoassay. Moreover, HR-HPV genotype was performed by using pyrosequencing. The association between cytokines and HPV genotype was also investigated. RESULTS: There was a statistically significant difference in IL-1ß level between patients with HPV transient infection and HPV persistent infection (p = 0.041). There were statistically significant differences in the levels of IL-1ß, IL-10, IL-21 and TNF-α between patients with low grade squamous intraepithelial lesion (LSIL) and high grade squamous intraepithelial lesion (HSIL) (p = 0.011, p = 0.008, p = 0.046 and p = 0.019, respectively). CONCLUSIONS: Pro-inflammatory cytokines, IL-1ß and TNF-α, and Th2 type cytokines, IL-10 and IL-21, became stronger in cervical mucosa with the progression of CIN. IL-1ß may be advantageous for HR-HPV persistent infection.

Prognostic role of immunohistochemical overexpression of the p16 protein in women under the age of 35 and diagnosed with HSIL (CIN2) subjected to "cervix sparing" excision.

OBJECTIVE: To evaluate the role of immunohistochemical staining overexpression of p16 protein (p16 IHC) as a prognostic factor of persistence or recurrence of intraepithelial disease after excision procedure in young women diagnosed with HSIL (CIN2). PATIENTS AND METHODS: 62 women with a histological diagnosis of HSIL (CIN2) subjected to "cervix sparing" excisional procedure were included in this retrospective study. All had age less than or equal to 35 years, negative history of immunosuppression, available follow-up, and assessment of the resection margins state. Immunohistochemical staining for the p16 protein was evaluated on reviewed and confirmed HSIL (CIN2) histological specimens with negative resection margins. The post-treatment follow-up, including cytology, colposcopy, and histology, ranged from a minimum of 6 months to a maximum of 60 months. The persistence or recurrence of SIL during the follow-up period was based on histologic referral and defined as "the presence of SIL", "the presence of HSIL" and "progression to HSIL (CIN3)". RESULTS: 31/62 patients were positive for immunostaining (p16 IHC+), and 31/62 were negative (p16 IHC-). Persistence or recurrence after excision occurred more frequently within the p16 IHC+ than in p16 IHC- group, both as SIL (29% p16 IHC- vs. 32.3% p16 IHC+, p = 0.783) and HSIL (6.5% p16 IHC- vs. 12.9% p16 IHC+, p = 0.671). None of the patients in the p16 IHC- group showed progression to CIN3 for the entire observation period, whereas 9.7% of p16 IHC+ women progressed to CIN3 lesion (p = 0.042). The p16 IHC positivity showed a significant association with progression to CIN3 in 5 years of follow-up (p = 0.029) and with the presence of SIL after two years of follow-up (p = 0.031). The differences between the two groups increased after two years post-treatment: the p16 IHC- patients still had SIL only in 3.2% of cases and no longer had HSIL, while the p16 IHC+ women still showed SIL in 19.4% and HSIL in 6.5% of cases. The negative predictive value (NPV) of p16 IHC in predicting SIL's presence after treatment increased with the severity of the lesion (NPV for SIL 70.97%, for HSIL 93.55%, for CIN3 100%). CONCLUSIONS: The study suggests that young patients with p16 IHC- HSIL (CIN2) have a better post-excisional course of the cervical intraepithelial disease compared to p16 IHC+ women and that p16 IHC could have prognostic utility during the long-term follow-up, especially in forecasting progression to CIN3 in consideration of the high NPV (up to 100%). The efficacy of the adjuvant HPV vaccination in the management of HSIL (CIN2) p16+ young women is to be evaluated as part of the fertility-sparing treatment.

High-Grade Cervical Intraepithelial Neoplasia (CIN) Associates with Increased Proliferation and Attenuated Immune Signaling.

Implementation of high-risk human papilloma virus (HPV) screening and the increasing proportion of HPV vaccinated women in the screening program will reduce the percentage of HPV positive women with oncogenic potential. In search of more specific markers to identify women with high risk of cancer development, we used RNA sequencing to compare the transcriptomic immune-profile of 13 lesions with cervical intraepithelial neoplasia grade 3 (CIN3) or adenocarcinoma in situ (AIS) and 14 normal biopsies from women with detected HPV infections. In CIN3/AIS lesions as compared to normal tissue, 27 differential expressed genes were identified. Transcriptomic analysis revealed significantly higher expression of a number of genes related to proliferation, (CDKN2A, MELK, CDK1, MKI67, CCNB2, BUB1, FOXM1, CDKN3), but significantly lower expression of genes related to a favorable immune response (NCAM1, ARG1, CD160, IL18, CX3CL1). Compared to the RNA sequencing results, good correlation was achieved with relative quantitative PCR analysis for NCAM1 and CDKN2A. Quantification of NCAM1 positive cells with immunohistochemistry showed epithelial reduction of NCAM1 in CIN3/AIS lesions. In conclusion, NCAM1 and CDKN2A are two promising candidates to distinguish whether women are at high risk of developing cervical cancer and in need of frequent follow-up.

HPV-16 E7-Specific Cellular Immune Response in Women With Cervical Intraepithelial Lesion Contributes to Viral Clearance: A Cross-Sectional and Longitudinal Clinical Study.

High-risk human papillomavirus (HPV) infection is the cause of almost all cervical cancers. HPV16 is one of the main risk subtypes. Although screening programs have greatly reduced the prevalence of cervical cancer in developed countries, current diagnostic tests cannot predict if mild lesions may progress into invasive lesions or not. In the current cross-sectional and longitudinal clinical study, we found that the HPV16 E7-specific T cell response in peripheral blood mononuclear cells of HPV16-infected patients is related to HPV16 clearance. It contributes to protecting the squamous interaepithelial lesion (SIL) from further malignant development. Of the HPV16 infected women enrolled (n = 131), 42 had neither intraepithelial lesion nor malignancy (NILM), 33 had low-grade SIL, 39 had high-grade SIL, and 17 had cervical cancer. Only one of 17 (5.9%) cancer patients had a positive HPV16 E7-specific T cell response, dramatically lower than the groups of precancer patients. After one year of follow-up, most women (28/33, 84.8%) with persistent HPV infection did not exhibit a HPV16 E7-specific T cell response. Furthermore, 3 malignantly progressed women, one progressed to high-grade SIL and two progressed to low-grade SIL, were negative to the HPV16 E7-specific T cell response. None of the patients with a positive HPV16 E7-specific T cell response progressed to further deterioration. Our observation suggests that HPV16 E7-specific T cell immunity is significant in viral clearance and contributes in protection against progression to malignancy.

CD4+ and CD8+ cell populations in HIV-positive women with cervical squamous intra-epithelial lesions and squamous cell carcinoma.

INTRODUCTION: This study aimed to analyse cervical lymphocytic populations in HIV+ and HIV- patients and correlate different cervical lesions with HIV viral load and presence of high-risk HPV types. MATERIAL AND METHODS: A total of 132 histological specimens from 40 HIV+ and 72 HIV- patients were evaluated for CD4+ and CD8+ T cell distribution, presence of high-risk HPV types, peripheral blood HIV viral load and CD4+/CD8+ ratio. RESULTS: High-grade squamous intraepithelial lesions (HSIL) and squamous cell carcinoma (SCC) from HIV+ patients had lower CD4+ T cell scores compared with HIV- patients. In all lesion groups, HIV+ patients presented higher epithelial and stromal CD8+ T cell scores. HIV viral load was more often detectable in patients with SCC than in those with low-grade squamous intraepithelial lesion (LSIL) (p = 0.0409). HSIL HIV+ patients had lower circulating CD4+ T cell counts (p = 0.0434) and CD4+/CD8+ ratio (p = 0.0378) compared with LSIL HIV+ patients. High-risk HPV types other than 16 and 18/45 were more prevalent in the HIV+ group. DISCUSSION: These results support an imbalance between cervical CD4+ and CD8+ T lymphocytes of HIV+ patients with SIL and SCC, with increased CD8+ infiltrate density with lesion severity, even in patients with immune system recovery under cART.

Correlation between HPV-negative cervical lesions and cervical microenvironment.

OBJECTIVE: To investigate the correlation between high-risk human papillomavirus (HR HPV)-negative cervical lesions and cervical microenvironment in Inner Mongolia, China, and to find the pathogenic factors of HR HPV-negative cervical lesions. MATERIALS AND METHODS: 74 cases of HR HPV-negative healthy women and 80 cases of patients with cervical lesions (28 cases of LSIL, 49 cases of HSIL and 3 cases of CSCC) were selected as the study group; 26 cases of HPV-positive women and 352 cases of patients with cervical lesions (108 cases of LSIL, 214 cases of HSIL and 30 cases of CSCC) were control group. Questionnaires were collected from the study group and the control group and specimens were collected. Gram staining, hematoxylin and eosin staining microscopy, and substrate colorimetry method were used to detect vaginal micro-ecological indicators; ELISA was used to detect the concentration of SIgA, IgG, IL-2 and IL-10 in vaginal lavage fluid. Genetic testing was used to detect HPV, mycoplasma, and chlamydia infection. The changes of vaginal micro-ecology evaluation index and local immune factor concentration in healthy women and cervical lesions of all grades in the study group and the control group were compared. RESULTS: Patients with cervical lesions, compared with healthy women, had a decrease in dominant lactobacilli and dysbacteriosis (P < 0.05), and this trend became more apparent as the disease progressed. The diversity and concentration of the flora in the HPV-negative group increased, the abnormal composition ratio decreased, and the HPV-positive group showed the opposite trend. As the lesion progressed, H2O2 decreased first and then increased, and the overall trend of SNa, LE, GUS, and GADP increased. The infection rate of trichomoniasis, BV and chlamydia increased and infection rate of Candida decreased. Also, compared with healthy women, patients with cervical lesions showed changes in immune factor concentration (P < 0.05). As the lesion progressed, IL-2 decreased, IL-10 increased, and IL-2/IL-10 decreased. However, IL-2 expression in HPV-negative group was higher than HSIL. SIgA was significantly lower in patients with cervical lesions than in healthy women. IgG had an upward trend in the HPV positive group. CONCLUSION: This study showed that vaginal micro-ecological imbalance and weakening of local cervical immune function are important reasons for the development of cervical lesions. It is expected to inhibit the development of cervical lesions by regulating the balance of vaginal micro-ecology and enhancing local immune function. By detecting Lactobacillus vaginalis, pre-enzyme, IL-2, IL-10, SIgA, it can guide the further diversion of HPV-positive women and predict the development direction of cervical lesions after HPV infection.

Understanding HLA-G driven journey from HPV infection to cancer cervix: Adding missing pieces to the jigsaw puzzle.

Human Papillomavirus (HPV) is a vital risk-factor for cancer cervix. However, persistent HPV infection results in cervical cancer in only a minority. Probably, HPV subdues the host immune response for persistence, which includes augmentation of HLA-G and plausibly aids in progression to cervical cancer. HLA-G, which comprises of membrane and soluble form, downregulates the host's immune response and generate tolerance. The current study aimed to analyze both forms of HLA-G in fresh tissue and plasma of women with HPV-infected and uninfected cervix and cancer cervix using Western blot and ELISA. The study cohort included 30 women with cervical carcinoma and equal number with normal cervix and 6 with HPV infected cervix. We observed a significant upregulation of membranous HLA-G expression in HPV infected cervix and cervical carcinoma (P < 0.001). Interestingly, the pairwise comparison of HLA-G tissue protein expression of the normal cervix and cervical carcinoma, as well as the normal cervix with HPV infected cervix, was significant (P < 0.001). Levels of soluble HLA-G were significantly raised in carcinoma cervix. We observed a progressive increase in HLA-G protein expression in HPV infected cervix and cervical carcinoma. These findings compel us to hypothesize that the upregulation of HLA-G expression favors the persistence of HPV in a microenvironment of a submissive host response. This progressive upregulation further leads to cervical cancer. Thus elimination of HPV infection seems to be a desirable proposition to prevent cervical cancer. In the absence of antiviral therapy for HPV, exploration of HLA-G antibody-based therapeutic strategies appear promising.

Cross-Protective IgG and IgA Antibodies against Oncogenic and Non-Oncogenic HPV Genotypes.

OBJECTIVE: The aim of the study was to describe the course of IgG/IgA immune response in women immunized with bivalent vaccine and in women non-vaccinated with HPV infection, as well as evaluating the cross-protection against non-vaccine HPV types. METHODS: Serum and cervical mucus samples were collected from infected and vaccinated women for HPV detection/genotyping and for detection of IgG/IgA anti-HPV/VLP (Virus-like Particles) by ELISA. RESULTS: The median absorbance detected in serum samples for anti-HPV-IgG antibodies was higher in vaccinated women when compared to HPV infected women (p <0.01), however, the median absorbance in cervical mucus samples for anti-HPV-IgA was higher in infected women when compared to vaccinated women (p <0.01). Additionally, our analyses also provided additional evidence for cross-protective efficacy of the HPV-16/18 vaccine against HPV-82, -6, -11, -13, -61, -72 and -74. CONCLUSION: The IgG antibodies were significantly more detected in the serum of vaccinated women, while the IgA was found in greater quantities in cervical samples from those infected by the virus. In addition, there is evidence that the bivalent vaccine provides cross-protection against other non-oncogenic viral subtypes.
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Extending Human Papillomavirus (HPV) vaccination beyond female adolescents and after treatment for high grade CIN: the Italian HPV Study Group (IHSG) review and position paper.

OBJECTIVE: Human PapillomaVirus (HPV) vaccination has been introduced in recent years in clinical practice as the most effective primary prevention strategy for cervical cancer and HPV-induced lesions, either pre-malignant or benign. Since its introduction, HPV vaccination has been progressively demonstrated as extremely effective in preventing extra-genital and male diseases also; furthermore, non only adolescents but adult subjects have been investigated and reported as positively responding to vaccine immunostimulation. More recently, effectiveness of post-treatment vaccine administration has been preliminarily investigated with very promising results in terms of decreased recurrences. On this basis, we report an Italian-focused picture of the state of the art and take a position in favour of the extension of HPV vaccination to male adolescents, to older age groups and to already treated subjects.

Association of genomic variants at the human leukocyte antigen locus with cervical cancer risk, HPV status and gene expression levels.

The human leukocyte antigen (HLA) locus on chromosome 6 has been reported to be associated with cervical cancer. We investigated two independent single-nucleotide polymorphisms in a large case-control series of cervical dysplasia and carcinoma that has been newly established by the German Cervigen Consortium, comprising a total of 2481 cases and 1556 healthy females. We find significant associations for both variants, rs9272117 at HLA-DQA1 and rs2844511 at MICA and HCP5, with cervical disease. Both variants showed evidence of association with invasive cervical cancer (rs9272117: OR 0.89, 95% CI 0.79-0.99, P = .036; rs2844511: OR 1.17, 95% CI 1.04-1.31, P = .008) and with high-grade dysplasia (rs9272117: OR 0.78, 95% CI 0.70-0.87, P = 7.1 × 10-6 ; rs2844511: OR 1.13, 95% CI 1.01-1.26, P = .035), as well as in a combined analysis of both groups (rs9272117: OR 0.83, 95% CI 0.75-0.91, P = 6.9 × 10-5 ; rs2844511: OR 1.14, 95% CI 1.04-1.26, P = .005). Variant rs2844511, but not rs9272117, also showed modest evidence of association with low-grade dysplasia (OR 1.26, 95% CI 1.04-1.54, P = .019). In case-only analyses, rs2844511 tended to predict HPV status (P = .044) and rs9272117 tended to associate with HPV16 (P = .022). RNA studies in cervical samples showed a significant correlation in the transcript levels of MICA, HCP5 and HLA-DQA1, suggesting extensive co-regulation. All three genes were upregulated in HPV16-positive samples. In stratified analyses, rs9272117 was associated with HLA-DQA1 levels, specifically in HPV-positive samples, while rs2844511 was associated with MICA and HCP5 levels. The risk allele of rs2844511 was required for correlations between MICA or HCP5 with HLA-DQA1. Altogether, our results support 6p21.32-33 as the first consistent cervical cancer susceptibility locus and provide evidence for a link between genetic risk variants, HPV16 status and transcript levels of HLA-DQA1, HCP5 and MICA, which may contribute to tumor immune evasion.