Quantitative human papillomavirus 16 and 18 levels in incident infections and cervical lesion development

Human papillomavirus (HPV) RNA levels may be a more sensitive early indicator of predisposition to carcinogenesis than DNA levels. We evaluated whether levels of HPV‐16 and HPV‐18 DNA and messenger RNA (mRNA) in newly detected infections are associated with cervical lesion development. Female university students were recruited from 1990 to 2004. Cervical samples for HPV DNA, HPV mRNA, and Papanicolaou testing were collected tri‐annually, and women were referred for colposcopically directed biopsy when indicated. Quantitative real‐time polymerase chain reaction of L1 and E7 DNA and E7 mRNA was performed on samples from women with HPV‐16 and HPV‐18 infections that were incidently detected by consensus PCR. Adjusting for other HPV types, increasing E7 cervical HPV‐16 mRNA levels at the time of incident HPV‐16 DNA detection were associated with an increased risk of cervical intraepithelial neoplasia grade 2–3 (HR per 1 log₁₀ increase in mRNA = 6.36, 95% CI = 2.00–20.23). Increasing HPV‐16 mRNA levels were also associated with an increased risk of cervical squamous intraepithelial lesions; the risk was highest at the incident positive visit and decreased over time. Neither HPV‐16 E7 DNA levels nor HPV‐18 E7 DNA nor mRNA levels were significantly associated with cervical lesion development. Report of >1 new partner in the past 8 months (relative to no new partners) was associated with increased HPV mRNA (viral level ratio [VLR] = 10.05, 95% CI = 1.09–92.56) and increased HPV DNA (VLR = 16.80, 95% CI = 1.46–193.01). In newly detected HPV‐16 infections, increasing levels of E7 mRNA appear to be associated with an increased risk of developing cervical pre‐cancer. J. Med. Virol. 81:713–721, 2009 © 2009 Wiley‐Liss, Inc.     

Effectiveness of HPV 16 viral load and the E2/E6 ratio for the prediction of cervical cancer risk among Chinese women

The effectiveness of the E2/E6 ratio, the state of viral genome integration and the viral load of human papillomavirus 16 (HPV 16) in predicting the risk of cervical cancer among Chinese women was investigated. Quantitative PCRs for the E2/E6 ratio and the viral load were performed on 85 cervical cancer samples and 55 HPV 16 positive healthy controls. The integrated form of the viral genome was found in 10.9% control samples and in 26.4% cervical cancer samples (P = 0.02). The majority of the cervical cancer (63.2%) and control samples (60%) were mixed forms. The E2/E6 ratio was associated with a high risk of cervical cancer (OR = 7.29, P = 9.55E?6). The integrated form (OR = 6.54, P = 0.005) and mixed form (OR = 2.93, P = 0.042) increased the risk of cervical cancer. The mean viral load in cervical cancer samples (37,371 ± 227,135) was higher than that in the controls (4,619 ± 27,079; P = 0.011). Additionally, the viral load increased along with the cervical cancer progression from the International Federation of Gynecology and Obstetrics (FIGO) stage I (12,337 ± 25,604) to stage II (67,453 ± 319,821). Compared with the state of viral genome integration (area under the receiver operating characteristic curve (AUC) = 0.743) or the viral load (AUC = 0.694), the E2/E6 ratio improved the effectiveness of the risk prediction of cervical cancer (AUC = 0.777), with the sensitivity (specificity) 81.2% (71.7%). The state of viral genome integration and the viral load of HPV 16 were important factors for the risk prediction of cervical cancer among Chinese women, and the E2/E6 ratio had a better cervical cancer risk prediction with age adjustment. J. Med. Virol. 85:646–654, 2013. © 2013 Wiley Periodicals, Inc.     

Human papillomavirus (HPV) E6/E7 mRNA as a triage test after detection of HPV 16 and HPV 18 DNA

High‐risk human papillomavirus (HPV) DNA detection provides high sensitivity but low specificity for moderate‐grade cervical intraepithelial neoplasia or worse histological identification. A prospective study evaluated mRNA testing efficacy for predicting this histological diagnosis in case of HPV 16 and/or 18 DNA detection. A total of 165 endocervical samples harboring HPV 16 and/or 18 DNA were tested with NucliSENS‐EasyQ® HPV E6/E7‐mRNA‐assay (Biomerieux, Marcy l´Etoile, France). Women with cytological alterations were referred to colposcopy (n = 111). Moderate‐grade cervical intraepithelial neoplasia or worse was diagnosed in 25.8% of women presenting atypical squamous cells of undetermined significance or low‐grade squamous intraepithelial lesions and in 89.8% of women with high‐grade squamous intraepithelial lesions. mRNA sensitivity was 81.3% and 84.1%, respectively. Specificity was 52.2%, and 80.0%, respectively. Negative predictive value (NPV) was 88.9% in undetermined or low‐grade squamous lesions. Positive predictive value (PPV) was 97.4% in high‐grade squamous lesions. mRNA reduced colposcopies by 44.3% in undetermined or low‐grade squamous lesions. Direct treatment of mRNA‐positive cases reduced 77.5% of colposcopies in high‐grade squamous lesions. Women without cytological alterations were followed for 18 months (n = 35), and moderate‐grade cervical intraepithelial neoplasia or worse was diagnosed in 34.3%; mRNA sensitivity and specificity were 83.3% and 86.9%, respectively. PPV and NPV were 76.9% and 90.9%, respectively for predicting moderate‐grade cervical intraepithelial neoplasia or worse in 18 months. mRNA reduced the number of visits for follow‐up in 62.2%. In conclusion, NucliSENS‐EasyQ® HPV E6/E7‐mRNA‐assay (Biomerieux) can serve as a triage test in case of HPV 16 and/or 18 DNA detection. J. Med. Virol. 85: 1063–1068, 2013. © 2013 Wiley Periodicals, Inc.     

Identification of a novel human papillomavirus type 16 E1 gene variant with potentially reduced oncogenicity

The human papillomavirus (HPV) 16 genome has been studied extensively, although no study has focused on the E1 gene that is implicated in viral DNA replication. After analyzing the E1 region of HPV 16 genomes in 429 cervical samples, 11.2% were found to contain a 63 nucleotides duplication in this region. Sequence analysis of the E6 and the E7 regions has shown that all samples containing this duplication were related to E6‐G350 variant of the HPV 16 (Chi square test, P = 0.0012). A comparison of cervical lesion severity of the examinees having regular or variant E1 genes has shown that the variant group had a significantly (Fischer's exact test, P = 0.0401) lower percentage of high grade disease cases, suggesting that this particular duplication might reduce the oncogenic potential of HPV 16, and also might clarify the differences of E6‐G350 variant oncogenicity observed in European populations. Albeit, a decreased incidence of high grade cervical lesions can be linked to the prevalence of multiple HPV infection, the additional decrease of those cases with the variant E1 gene versus those without (10.5% and 18.6%, respectively) can only be ascribed to the effect of this particular HPV variant. Further research is needed to clarify the biology of these HPV 16 E1 variants. J. Med. Virol. 80:2134–2140, 2008. © 2008 Wiley‐Liss, Inc.     

Viral load and physical status of human papillomavirus (HPV) 18 in cervical samples from female sex workers infected with HPV 18 in Burkina Faso

Viral DNA load and physical status might be predictive of either high‐grade cervical lesions or disease progression among women infected by human papillomavirus (HPV) 16, but these virological markers have rarely been studied in HPV 18 infections. The relationships between HPV 18 DNA load, viral genome physical status and cervical squamous intraepithelial lesions were analyzed among female sex workers infected with HPV18 in Burkina Faso. HPV 18 E2 and E6 genes were quantitated by real‐time PCR. Among 21 women infected with HPV 18, 67% of whom were HIV‐1‐seropositive, 11 (52.4%) had a normal cytology, 8 (38.1%) had low‐grade squamous intraepithelial lesions, and 2 (9.5%) had high‐grade squamous intraepithelial lesions. Total viral load and integrated viral load were higher in women with squamous intraepithelial lesions than in women with normal cytology (P = 0.01 for both parameters). Total viral load and integrated viral load were higher in HIV‐1‐seropositive women than in those who were not infected with HIV (P = 0.01, and P, 0.01, respectively). Total viral load or integrated viral load >1,000 copies/ng of DNA were more frequent in women with squamous intraepithelial lesions than in women with normal cytology (7/10 vs. 1/11; P = 0.007) and in HIV‐1‐seropositive women (8/14 vs. 0/7 in HIV‐uninfected women; P = 0.02). Both HPV 18 DNA and integrated DNA loads might represent markers of cervical lesions. Prospective evaluations are needed to establish the value of these parameters to predict high‐grade lesion or lesion progression. J. Med. Virol. 81:1786–1791, 2009. © 2009 Wiley‐Liss, Inc.     

Prevalence and viral load of oncogenic human papillomavirus types associated with cervical carcinoma in a population of North Italy

A cross‐sectional study was carried out in a population of North Italy to determine the prevalence of eight oncogenic human papillomavirus (HPV) types most commonly found in cervical carcinoma and to study the relationship between HPV DNA loads and severity of disease. A total of 597 cervical samples obtained from patients with pathological findings (n = 472) and from women with normal cytology (n = 125) were analyzed by means of normalized Real‐time PCR assays to quantify HPV‐16, ‐18, ‐31, ‐45, and ‐33 group (including ‐33, ‐52, ‐58, ‐67); the normalization of oncogenic HPV viral load was carried out by quantitation of a single copy gene. The two most common oncogenic HPV types found were 16 and 31 (24.3% and 22.9% of pathological samples, respectively); multiple infections were demonstrated in 22% of pathological samples. Overall, the HPV total viral load was found to increase with increasing severity of associated lesions, although a stronger association was observed only for HPV‐31 and HPV‐16 (γ = 0.49 and 0.41, respectively) as compared to HPV‐18 and ‐33 group (γ = 0.19 and 0.02, respectively). However, we found that high levels of HPV‐31 or 33 group DNA could be prognostic of minor oncogenic risk for high‐grade squamous intraepithelial lesions (H‐SIL) (age adjusted odds ratio [AORs] = 1.57 and 1.26, respectively) than HPV‐16 and HPV‐18 (AORs = 30 and 8, respectively). The AORs also increased with HPV total viral load and reached a maximum of AORs = 15.7. Thus, HPV load is a type‐dependent risk marker for the development of H‐SIL. J. Med. Virol. 81:278–287, 2009. © 2008 Wiley‐Liss, Inc.     

Analyses of human papillomavirus genotypes and viral loads in anogenital warts

Condylomata acuminata (genital warts) are the most common sexually transmitted viral diseases. These lesions are caused by infection with mucosal human papillomaviruses (HPVs). However, there is limited information on HPV strain distribution involved in the molecular pathogenesis of these lesions. To address this, the strain prevalence and the frequency of multiple HPV infections were determined in wart tissue obtained from 31 patients attending a wart clinic. These lesions were bisected and subjected to parallel DNA and mRNA extractions. HPV-type prevalence and incidence of multiple infections were determined by the Roche Linear Array assay. qPCR compared HPV 6, 11, 16, and 18 viral loads and RT-qPCR measured HPV 6 and 11 E6 genomic expression levels. Seventy-one percent of these samples were infected with multiple HPVs. Only one sample was negative for HPV 6 or 11 DNA. Forty-eight percent of samples were positive for a high risk (oncogenic) HPV. The results show that multiple infections in tissue are frequent and the subsequent analysis of HPV 6 and 11 E6 DNA viral loads suggested that other HPVs could be causing lesions. Further analysis of HPV 6/11 E6 mRNA levels showed that there was no discernable relationship between HPV 6 E6 DNA viral load and relative HPV 6 or 11 E6 mRNA levels thereby questioning the relevance of viral load to lesion causality.     

Viral load and genomic integration of HPV 16 in cervical samples from HIV-1-infected and uninfected women in Burkina Faso

The relationships between human papillomavirus type 16 (HPV 16) viral load, HPV 16 integration status, human immunodeficiency virus type 1 (HIV-1) status, and cervical cytology were studied among women enrolled in a cohort of female sex workers in Burkina Faso. The study focused on 24 HPV 16-infected women. The HPV 16 viral load in cervical samples was determined by real-time PCR. Integration ratio was estimated as the ratio between E2 and E6 genes DNA copy numbers. Integrated HPV16 viral load was defined as the product of HPV 16 viral load by the integration ratio. High HPV 16 viral load and high integration ratio were more frequent among women with squamous intraepithelial lesions compared with women with normal cytology (33% vs. 11%, and 33% vs. 0%, respectively), and among women with high-grade squamous intraepithelial lesions compared with women without high-grade squamous intraepithelial lesions (50% vs. 17%, and 50% vs. 11%, respectively). High HPV 16 DNA load, but not high integration ratio, was also more frequent among HIV-1-positive women (39% vs. 9%; and 23% vs. 18%, respectively). The absence of statistical significance of these differences might be explained by the small study sample size. High-integrated HPV 16 DNA load was significantly associated with the presence of high-grade squamous intraepithelial lesions (50% vs. 5%, P = 0.03) in univariate and multivariate analysis (adjusted odds-ratio: 19.05; 95% confidence interval (CI), 1.11-328.3, P = 0.03), but not with HIV-1 or other high-risk HPV types (HR-HPV). Integrated HPV 16 DNA load may be considered as a useful marker of high-grade cervical lesions in HPV 16-infected women.     

Evaluation of the performance of the novel PapilloCheck® HPV genotyping test by comparison with two other genotyping systems and the HC2 test

The novel PapilloCheck® genotyping test was compared with SPF10 PCR LiPav1 and PGMY09/11 on hybrid capture 2 (HC2)‐pretested samples. From results of 826 cervical samples detection rates and kappa values for the tests were calculated using a HPV type consensus definition. With PapilloCheck® HPV types 53, 56, and 33 were found with a sensitivity of 100%. The lowest detection rate was observed for HPV 35 (72.2%). The SPF10 PCR LiPav1 was found to be 100% positive for HPV 18, 31, 53, 56, and 35 and lowest for HPV 59 (81%). The PGMY09/11 system detected only HPV 59 at 100% detection rate and showed lowest sensitivity for HPV 56 (40.5%). Multiple infection rates ranged from 25.8% (PGMY09/11 PCR‐LBA), over 39.5% (PapilloCheck®) to 55.9% (SPF10 PCR LiPav1). In samples with higher viral DNA load detection rates and concordance between the genotyping tests increases. The kappa values in comparison to the HPV consensus type ranged from k = 0.21 to k = 0.82 for comparing SPF10 PCR with the HPV consensus type, while values for PGMY09/11 PCR ranged from k = 0 to k = 0.96 and were best for the PapilloCheck® (k = 0.49–0.98). Detection rates for the identification of high‐grade cervical intraepithelial neoplasia (CIN2+) ranged from 93.7% (PGMY09/11 PCR) to 98.4% (PapilloCheck®, SPF10 PCR, HC2). In conclusion, this study shows that the PapilloCheck® give comparable results to established PCR methods. However, these results also show a necessity for the standardization of genotype‐specific HPV detection assays. J. Med. Virol. 82:605–615, 2010. © 2010 Wiley‐Liss, Inc.     

Comparing triage algorithms using HPV DNA genotyping,HPV E7 mRNA detection and cytology in high-risk HPV DNA-positive women

BackgroundHigh-risk human papillomavirus (hrHPV) DNA positive women require triage testing to identify those with high-grade cervical intraepithelial neoplasia or cancer (≥CIN2).ObjectiveComparing three triage algorithms (1) E7 mRNA testing following HPV16/18/31/33/45/52/58 genotyping (E7 mRNA test), (2) HPV16/18 DNA genotyping and (3) cytology, for ≥CIN2 detection in hrHPV DNA-positive women.Study designhrHPV DNA-positive women aged 18–63 years visiting gynecology outpatient clinics were included in a prospective observational cohort study. From these women a cervical scrape and colposcopy-directed biopsies were obtained. Cervical scrapes were evaluated by cytology, HPV DNA genotyping by bead-based multiplex genotyping of GP5+6+-PCR-products, and presence of HPV16/18/31/33/45/52/58 E7 mRNA using nucleic acid sequence-based amplification (NASBA) in DNA positive women for respective HPV types. Sensitivities and specificities for ≥CIN2 were compared between E7 mRNA test and HPV16/18 DNA genotyping in the total group (n = 348), and E7 mRNA test and cytology in a subgroup of women referred for non-cervix-related gynecological complaints (n = 133).ResultsSensitivity for ≥CIN2 of the E7 mRNA test was slightly higher than that of HPV16/18 DNA genotyping (66.9% versus 60.9%; ratio 1.10, 95% CI: 1.0002–1.21), at similar specificity (54.8% versus 52.3%; ratio 1.05, 95% CI: 0.93–1.18). Neither sensitivity nor specificity of the E7 mRNA test differed significantly from that of cytology (sensitivity: 68.8% versus 75.0%; ratio 0.92, 95% CI: 0.72–1.17; specificity: 59.4% versus 65.3%; ratio 0.91, 95% CI: 0.75–1.10).ConclusionFor detection of ≥CIN2 in hrHPV DNA-positive women, an algorithm including E7 mRNA testing following HPV16/18/31/33/45/52/58 DNA genotyping performs similar to HPV16/18 DNA genotyping or cytology.     

Analysis of human papillomavirus type 16 (HPV16) DNA load and physical state for identification of HPV16-infected women with high-grade lesions or cervical carcinoma

Integration of human papillomavirus (HPV) DNA into the host cell genome is a frequent event in cervical carcinogenesis, even though this phenomenon does not seem to be mandatory for cervical cancer development. Our objective was to describe the load and physical state of HPV type 16 (HPV16) DNA in a series of cervical samples representative of the natural history of cervical cancer. We used a combination of three real-time PCR assays targeting E6, E2, and albumin genes to calculate HPV16 load (E6 and albumin) and the E2/E6 ratio as a surrogate of integration. This method was applied to 173 HPV16-positive cervical samples. Results show that viral load increases with the lesion grade (from 102 HPV16 DNA copies per 10³ cells in normal samples up to 56,354 copies per 10³ cells in cancers), while E2/E6 ratio decreases (from 1 in normal samples down to 0.36 in cancers). We propose that, according to this technique, an HPV16 viral load of higher than 22,000 copies/10³ cells or an E2/E6 ratio of lower than 0.50 allows the identification of women with prevalent high-grade lesions or worse with a high specificity. In conclusion, both viral load and E2/E6 ratio, used in combination with an appropriate cutoff value, are suitable to screen women with prevalent cervical intraepithelial neoplasia grade 2 or 3 or cancer. Therefore, these assays would be useful in addition to routine HPV testing to more accurately identify women with (pre)cancerous lesions.     

E6 and E7 variants of human papillomavirus‐16 and ‐52 in Japan,the Philippines,and Vietnam

Human papillomavirus (HPV) has several intragenotypic variants with different geographical and ethnic distributions. This study aimed to elucidate the distribution patterns of E6 and E7 (E6/E7) intragenotypic variants of HPV type 16 (HPV‐16), which is most common worldwide, and HPV‐52, which is common in Asian countries such as Japan, the Philippines, and Vietnam. In previous studies, genomic DNA samples extracted from cervical swabs were collected from female sex workers in these three countries and found to be positive for HPV‐16 or HPV‐52. Samples were amplified further for their E6/E7 genes using type‐specific primers and analyzed genetically. Seventy‐nine HPV‐16 E6/E7 genes were analyzed successfully and grouped into three lineages: European (Prototype), European (Asian), and African‐2. The prevalences of HPV‐16 European (Prototype)/European (Asian) lineages were 19.4%/80.6% (n = 31) in Japan, 75.0%/20.8% (n = 24) in the Philippines, and 0%/95.8% (n = 24) in Vietnam. The 109 HPV‐52 E6/E7 genes analyzed successfully were grouped into four lineages, A–D; the prevalences of lineages A/B/C/D were, respectively, 5.1%/92.3%/0%/2.6% in Japan (n = 39), 34.4%/62.5%/0%/3.1% in the Philippines (n = 32), and 15.8%/73.7%/7.9%/2.6% in Vietnam (n = 38). The distribution patterns of HPV‐16 and HPV‐52 lineages in these countries differed significantly (P < 0.000001 and P = 0.0048, respectively). There was no significant relationship between abnormal cervical cytology and either HPV‐16 E6/E7 lineages or specific amino acid mutations, such as E6 D25E, E6 L83V, and E7 N29S. Analysis of HPV‐16 and HPV‐52 E6/E7 genes can be a useful molecular‐epidemiological tool to distinguish geographical diffusion routes of these HPV types in Asia. J. Med. Virol. 85: 1069–1076, 2013. © 2013 Wiley Periodicals, Inc.     

Comparison of the performance in detection of HPV infections between the high‐risk HPV genotyping real time PCR and the PCR‐reverse dot blot assays

A new multiplex real‐time PCR assay, the high‐risk HPV genotyping real time PCR assay (HR HPV RT‐PCR), has been developed to detect 15 high‐risk HPV types with respective viral loads. In this report, a total of 684 cervical specimens from women diagnosed with vaginitis were assessed by the HR HPV RT‐PCR and the PCR reaction and reverse dot blot (PCR‐RDB) assays, using a PCR‐sequencing method as a reference standard. A total coincidence of 97.7% between the HR HPV RT PCR and the PCR‐RDB assays was determined with a Kappa value of 0.953. The HR HPV RT PCR assay had sensitivity, specificity, and concordance rates (accuracy) of 99.7%, 99.7%, and 99.7%, respectively, as confirmed by PCR‐sequencing, while the PCR‐RDB assay had respective rates of 98.8%, 97.1%, and 98.0%. The overall rate of HPV infection, determined by PCR‐sequencing, in women diagnosed with vaginitis was 49.85%, including 36.26% of single infection and 13.6% of multiple infections. The most common infections among the 15 high‐risk HPV types in women diagnosed with vaginitis were HPV‐52, HPV‐16, and HPV‐58, with a total detection rate of 10.23%, 7.75%, and 5.85%, respectively. We conclude that the HR HPV RT PCR assay exhibits better clinical performance than the PCR‐RDB assay, and is an ideal alternative method for HPV genotyping. In addition, the HR HPV RT PCR assay provides HPV DNA viral loads, and could serve as a quantitative marker in the diagnosis and treatment of single and multiple HPV infections.     

Frequency and clinical significance of human beta-herpesviruses in cervical samples from Italian women

Human papillomaviruses (HPVs) are necessary, but not sufficient, for the development of cervical cancer (CC). Human beta-herpesviruses (beta-HHVs) have been suggested as possible cofactors in the oncogenesis of CC. In this cross-sectional study, the prevalence and possible association of cytomegalovirus (CMV), HHV-6 and -7 with HPV presence was investigated by quantitative real-time PCR assays in cervical samples obtained from 208 italian women. The two most common high-risk HPV types found were 31 and 16. Overall, the positive rates for CMV, HHV-6 and HHV-7 were 66%, 25%, and 6%, respectively. In particular, the prevalence of CMV was found to be extremely high irrespective of either the cytological category or HPV positivity. The prevalence of HHV-6 DNA was significantly higher in high-grade squamous intraepithelial lesions (HSIL) respect to normal women (P < 0.017); by contrast, the prevalence HHV-7 DNA was generally low and not associated with SIL. Copresence of CMV and HHV-6 DNA was found to be significantly higher in patients with SIL respect to normal women (P < 0.05). No correlation was demonstrated between the viral load of all three beta-HHVs and the different cytological stages or with the HPV presence. A few patients with severe disease however showed very high viral loads which for HHV-6 may be indicative of viral integration. In conclusion, this study suggests that CMV and HHV-7 alone are probably not implicated in the oncogenesis of CC whilst HHV-6 alone or together with CMV may contribute to the development of CC.     

CD4+ T cells against human papillomavirus-18 E7 in patients with high-grade cervical lesions associate with the absence of the virus in the cervix

Cervical neoplastic lesions are associated with infection by high‐risk human papilloma‐viruses (HPV). The two genotypes most frequently found in the lesions are HPV‐16 and HPV‐18 with a prevalence of 50–60% and 15–18%, respectively. The E6 and E7 viral oncoproteins are involved in the transformation process and represent foreign antigens for the host. We previously reported that anti‐HPV‐18 E6 CD4⁺ T cells are present in patients with high‐grade HPV‐18‐expressing cervical lesions but also in 50% of the total consecutive patients tested, independently of the HPV type carried. These results indicated that HPV‐18 E6 is immunogenic and suggested that all responsive patients, irrespective of the HPV expressed, had encountered HPV‐18 and cleared the infection. Here, we investigated anti‐HPV‐18 E7 CD4⁺ T‐cell immunity in a cohort of 23 HPV‐18 E6‐responsive patients. We found that, although E7‐specific CD4⁺ T cells were present in all women, a robust T helper type (Th1)/Th2 type response against E7 was associated with HPV‐18‐negative status, suggesting that indeed these patients might have cleared the virus. In agreement with this hypothesis, we found strong anti‐E7 CD4⁺ T‐cell immunity in 20% of 24 healthy donors without evidence of disease. In contrast, a robust Th1/Th2 type response against E6 but not E7 correlated with a lack of disease relapse and/or infection recurrence but did not discriminate between HPV‐18‐positive and HPV‐18‐negative patients. Collectively, our data suggest different roles for anti‐HPV‐18 E6 and E7 CD4⁺ T cells in anti‐viral and anti‐tumour immunity.     

Distribution and viral load of eight oncogenic types of human papillomavirus (HPV) and HPV 16 integration status in cervical intraepithelial neoplasia and carcinoma.

Current human papillomavirus (HPV) DNA testing using pooled probes, although sensitive, lacks specificity in predicting the risk of high-grade cervical intraepithelial neoplasia (CIN 2/3) progression. To evaluate selected HPV genotyping, viral load, and viral integration status as potential predictive markers for CIN progression, we performed HPV genotyping in formalin-fixed, paraffin-embedded cervical tissue with cervical carcinoma (29 cases) and CINs (CIN 1, 27 cases; CIN 2, 28 cases; CIN 3, 33 cases). General HPVs were screened using consensus primers GP5+/GP6+ and PGMY09/11. HPV genotyping and viral load measurement were performed using quantitative real-time PCR for eight oncogenic HPV types (16, 18, 31, 33, 35, 45, 52, and 58). HPV 16 viral integration status was evaluated by measuring HPV 16 E2/E6 ratio. We observed that HPV DNA positivity increased in parallel with the severity of CINs and carcinoma, with 59% positivity in CIN 1, 68% in CIN 2, 76% in CIN 3, and 97% in carcinoma (P trend=0.004). The eight oncogenic HPV types were significantly associated with CIN 2/3 (81%) and carcinoma (93%) (odds ratio (OR), 15.0; 95% confidence interval (CI), 5.67-39.76; P<0.0001) compared with the unknown HPV types (OR, 2.87; 95% CI, 0.89-9.22; P=0.08). HPV 16 was the predominant oncogenic HPV type in CIN 2/3 (51%) and carcinoma (71%) and integrated significantly more frequently in carcinoma than in CIN 2/3 (P=0.004). No significant differences in viral load were observed across the disease categories. Our findings suggest that selected genotyping for the eight oncogenic HPV types might be useful in separating women with a higher risk of CIN progression from those with a minimal risk. We also conclude that the HPV 16 integration status has potential to be a marker for risk assessment of CIN progression.