Human Papillomavirus L1 Capsid Protein Research Articles

A CRR2-Dependent sRNA Sequence Supports Papillomavirus Vaccine Expression in Tobacco Chloroplasts

Human papillomavirus (HPV) infection is the leading cause of cervical cancer, and vaccination with HPV L1 capsid proteins has been successful in controlling it. However, vaccination coverage is not universal, particularly in developing countries, where 80% of all cervical cancer cases occur. Cost-effective vaccination could be achieved by expressing the L1 protein in plants. Various efforts have been made to produce the L1 protein in plants, including attempts to express it in chloroplasts for high-yield performance. However, manipulating chloroplast gene expression requires complex and difficult-to-control expression elements. In recent years, a family of nuclear-encoded, chloroplast-targeted RNA-binding proteins, the pentatricopeptide repeat (PPR) proteins, were described as key regulators of chloroplast gene expression. For example, PPR proteins are used by plants to stabilize and translate chloroplast mRNAs. The objective is to demonstrate that a PPR target site can be used to drive HPV L1 expression in chloroplasts. To test our hypothesis, we used biolistic chloroplast transformation to establish tobacco lines that express two variants of the HPV L1 protein under the control of the target site of the PPR protein CHLORORESPIRATORY REDUCTION2 (CRR2). The transgenes were inserted into a dicistronic operon driven by the plastid rRNA promoter. To determine the effectiveness of the PPR target site for the expression of the HPV L1 protein in the chloroplasts, we analyzed the accumulation of the transgenic mRNA and its processing, as well as the accumulation of the L1 protein in the transgenic lines. We established homoplastomic lines carrying either the HPV18 L1 protein or an HPV16B Enterotoxin::L1 fusion protein. The latter line showed severe growth retardation and pigment loss, suggesting that the fusion protein is toxic to the chloroplasts. Despite the presence of dicistronic mRNAs, we observed very little accumulation of monocistronic transgenic mRNA and no significant increase in CRR2-associated small RNAs. Although both lines expressed the L1 protein, quantification using an external standard suggested that the amounts were low. Our results suggest that PPR binding sites can be used to drive vaccine expression in plant chloroplasts; however, the factors that modulate the effectiveness of target gene expression remain unclear. The identification of dozens of PPR binding sites through small RNA sequencing expands the set of expression elements available for high-value protein production in chloroplasts.

Useful cytological confirmation of HPV 13 in lesional mucosa enhances diagnosis of focal epithelial hyperplasia

An 11-year-old female presented with multiple oral lesions for several months. Histopathological findings suggested focal epithelial hyperplasia (FEH), also known as Heck disease. FEH is strongly associated with Human papillomavirus (HPV), especially genotypes 13 and 32. An oral swab of a mucosal lesion was subsequently obtained for cytology, immunohistochemistry and in situ hybridization. In addition, in situ hybridization and immunohistochemistry were also performed retrospectively on the biopsy specimen for correlation. The cytology specimen showed squamous cells with enlarged, slightly atypical nuclei and rare perinuclear halos. The histology findings included papillomatosis with acanthosis, mild nuclear atypia and focal perinuclear halos. The immunohistochemistry for the consensus HPV L1 capsid protein was found in both the cytology and biopsy specimens indicating that the lesion was HPV-related. High viral copy numbers of HPV 13 were detected by in situ hybridization in both the cytology and histology specimens. Although histologic features of FEH have been well characterized in the literature, to our knowledge, this is the first case to describe in FEH with adjunct immunohistochemistry and in situ hybridization results. Furthermore, these findings assisted in our diagnosis since the patient's clinical presentation was a diagnostic challenge with smooth dome-shaped papules instead of the typically described flat-topped verrucous lesions seen in FEH. In summary, our case reveals that there is a high concordance between the HPV 13 detection in the cytology and histology of FEH, and that performing cytology in addition to histology can be used to optimize diagnostic evaluation towards appropriate patient care.

Expression of Chimeric HPV-HIV Protein L1P18 in Pichia pastoris; Purification and Characterization of the Virus-like Particles.

Currently, three human papillomavirus (HPV) vaccines are already licensed and all of them are based on virus-like particles (VLPs) of HPV L1 capsid protein but not worldwide accessible. While about 38.0 million people were living with HIV in 2019, only 68% of HIV-infected individuals were accessing antiretroviral therapy as of the end of June 2020 and there is no HIV vaccine yet. Therefore, safe, effective, and affordable vaccines against those two viruses are immediately needed. Both HPV and HIV are sexually transmitted infections and one of the main access routes is the mucosal genital tract. Thus, the development of a combined vaccine that would protect against HPV and HIV infections is a logical effort in the fight against these two major global pathogens. In this study, a recombinant Pichia pastoris producing chimeric HPV-HIV L1P18 protein intracellularly was constructed. After cell disruption, the supernatant was collected, and the VLPs were purified by a combination of ammonium sulfate precipitation, size exclusion chromatography, ultracentrifugation, and ultrafiltration. At the end of purification process, the chimeric VLPs were recovered with 96% purity and 9.23% overall yield, and the morphology of VLPs were confirmed by transmission electron microscopy. This work contributes towards the development of an alternative platform for production of a bivalent vaccine against HPV and HIV in P. pastoris.

Research Progress of HPV L1 Capsid Protein in Prediction of Cervical Lesions

Cervical cancer is one of the most common malignant gynecological tumors and has the second highest incidence of all malignancies in females. Chronic and persistent infection with High Risk Human Papillomavirus (HR-HPV) is the main cause of cervical cancer. There is a distinct lack of methodology by which to determine whether cervical epithelial dysplasia is cancerous following HPV infection. HPV L1 capsid protein is a major structural protein of human papillomavirus (HPV)?and it is the main target of the local cellular immune response aiming to combat human papillomavirus after HPV infection within cervical cells. Greater understanding of HPV L1 capsid protein and its association with cervical cytology, histopathology, patient age and human papillomavirus viral load has the potential to contribute toward improved the improved diagnosis and management of cervical cancer, providing useful information for gynecological clinicians in the hope of improving patient treatment and quality of life. This article reviews the predictive utility of HPV L1 capsid protein for cervical lesions.

P120 catenin recruits HPV to γ-secretase to promote virus infection.

During internalization and trafficking, human papillomavirus (HPV) moves from the cell surface to the endosome where the transmembrane protease γ-secretase promotes insertion of the viral L2 capsid protein into the endosome membrane. Protrusion of L2 through the endosome membrane into the cytosol allows the recruitment of cytosolic host factors that target the virus to the Golgi en route for productive infection. How endosome-localized HPV is delivered to γ-secretase, a decisive infection step, is unclear. Here we demonstrate that cytosolic p120 catenin, likely via an unidentified transmembrane protein, interacts with HPV at early time-points during viral internalization and trafficking. In the endosome, p120 is not required for low pH-dependent disassembly of the HPV L1 capsid protein from the incoming virion. Rather, p120 is required for HPV to interact with γ-secretase–an interaction that ensures the virus is transported along a productive route. Our findings clarify an enigmatic HPV infection step and provide critical insights into HPV infection that may lead to new therapeutic strategies against HPV-induced diseases.

Utility of human papillomavirus L1 capsid protein and HPV test as prognostic markers for cervical intraepithelial neoplasia 2+ in women with persistent ASCUS /LSIL cervical cytology.

Objective: Efficient and highly predictive biomarkers reflecting the prognosis of persistent atypical squamous cells of unknown significance(ASCUS) and low grade squamous intraepithelial lesion(LSIL)s are unavailable and need to be developed urgently. We aimed to develop a predictive model for diagnosis of cervical intraepithelial neoplasia(CIN)2+ by analyzing the immunocytochemical expression of the HPV L1 capsid protein in patients with persistent ASCUS and LSIL with a high risk of HPV infection.Methods: Cervical cytology samples comprising (70 ASCUS and 215 LSIL Pap smears) were analyzed. Immunocytochemical identification of the HPV L1 capsid protein in cervical cytology samples was performed. Expression levels of HPV L1 capsid protein in cervical cytology samples were measured, and the correlation between HPV L1 expression and cervical pathologic diagnosis was evaluated. The risk for CIN2+ was calculated using the results of immunocytochemistry and the HPV DNA test.Results: Negative results for HPV L1 immunochemistry test were more frequently observed in CIN2+, and expression of the HPV L1 capsid protein was higher in CIN1 or cervicitis (Fisher's exact test, p<0.05). Diagnosis rates for CIN2+ were highest for the combination of HPV L1 capsid protein immunocytochemistry, cytology and HPV test when compared with other combinations (Akaike information criterion (AIC): 191.7, Schwarz criterion(SC): 206.3, p<0.001).Conclusion: Absence of HPV L1 capsid expression and presence of HPV type 16 or 18 infection are reliable predictors of progression to CIN2+ in patients showing persistent ASCUS and LSIL.

Human papillomavirus DNA, HPV L1 capsid protein and p16INK4a protein as markers to predict cervical lesion progression.

Cervical cancer is the most common malignant tumors in women leading to serious morbidity and mortality worldwide, especially among developing countries. A main cause of the disease is the high-risk human papillomavirus (HR-HPV) infection. HSIL usually progress to cervical cancer, and low-grade lesions, including LSIL and ASCUS, mostly turn to normal or benign lesions, but there are still a small number of patients who will progress to HSIL. Up to now there is no efficient biomarker clinically available to predict people with high risk to progress into HSIL. This study was conducted to evaluate the value of human papillomavirus (HPV) DNA, p16INK4a protein, and HPV L1 capsid protein in predicting HSIL and minimizing unnecessary colposcopy treatments. 1222 patients with HR-HPV infection or with abnormal Thinprep cytologic test (TCT) were chosen to conduct colposcopy in the cervical out-patient clinic of Shanghai First Maternity and Infant Hospital affiliated to Shanghai Tongji University from June 2014 to January 2017. TCT, cervical biopsy, HPV DNA and HPVL1 were performed on all patients. 110 patients were selected to detect p16INK4a protein. Hybrid capture 2 (HC-2) was used to detect HPV DNA, and their subgroups using gene typing system. Immunohistochemical technology was used to detect HPV L1 and p16. HPV DNA was positive in 1097 cases, with the positive rate of 89.7% (1097/1222). In particular, the positive expression rates of HPV DNA were 82.3, 95.7, 96.6 and 100% in Normal/CC, LSIL, HSIL and cervical cancer groups, respectively (p < 0.001). HPV L1 was negative in 781 cases with HR-HPV infection, and the overall negative rate is 71.1%. In patients with Normal/CC, LSIL and HSIL, the negative expression rates of HPV L1 were 91.3, 40 and 81.2%, respectively (p value < 0.001). In the 110 patients, HPV L1 was negative in 98.1% (53/54) of Normal/CC, 42.9% (12/28) of LSIL and 85.1% (23/27) of HSIL (p value = 0.0043). P16-positive rates in patients with Normal/CC, LSIL and HSIL were 33.3% (18/54), 75% (21/28) and 96.2% (26/27), respectively (p value < 0.001). 18 out of 28 cases express low positive (+) in LSIL, 25 out of 27 cases express strong positive (3+) in HSIL. Patients with L1(-)p16(+) including 18.5% (10/54) of normal/cervicitis, 60.7% (17/28) of LSIL and 85.1% (23/27) of HSIL (p value < 0.005). Furthermore, patients with L1(-)p16(1+) included 37% (10/27) of normal/cervicitis 59.3% (16/27) of LSIL and 3.7% (1/27) of HSIL; patients with L1(-)p16(2+) consisted of 0% of normal/cervicitis/LSIL and 100% (1/1) of HSIL; patients with L1(-)p16(3+) were composed of 0% of normal/cervicitis, 4.5% (1/22) of LSIL and 95.5% (21/22) of HSIL (p value < 0.005) (Table6). With the increase in the degree of the cervical lesions, the expression of HPV DNA and p16 is up-regulated while HPV L1 protein is down-regulated. HPV DNA, HPV L1 and p16 are useful markers for the prediction of HSIL. Combined detection of these three markers has important potential to predicting HSIL and minimizing unnecessary colposcope examination.

Abstract LB-135: Biomarker utility of the E2/E6 ratio and L1 immunoreactivity for determining HPV16-positive ≥CIN2 lesions and cervical squamous cell carcinoma

Abstract Objectives: Human papillomavirus (HPV)16 is the most carcinogenic HPV genotype. The objective of this study was to identify whether expression of the HPV16 L1 capsid protein and the E2/E6 ratio—both determined by cervical cytology—could be used as biomarkers of cervical pre-malignancy and cancer. Methods: Cervical specimens were obtained from 226 patients with HPV16 single infection at Seoul St. Mary's Hospital. A Cytoactiv®HPV L1 screening set was prepared to detect the L1 capsid protein, and real-time polymerase chain reaction (PCR) was performed to determine the E2/E6 ratio of HPV16 DNA. Cytology results then were compared to results of conventional histologic analysis of cervical tissues (cervical intraepithelial neoplasia [CIN]1-3 and squamous cell carcinoma [SCC]) to evaluate the association. Results: We found that the mean E2/E6 ratios of specimens graded as SCC (0.355) and of those graded as CIN2/3 (0.481) were significantly lower than the mean E2/E6 ratios for specimens graded as CIN1 (0.736) and those regarded as normal (0.792) (P &amp;lt; 0.05). Cytologic detection of HPV L1 enabled prediction of ≥CIN2 cervical lesions and differentiation of CIN2/3 from SCC (P &amp;lt; 0.05). Based on findings expressed as area under the receiver operating characteristic curve (AUC), the combination of HPV L1 immunohistochemistry and the E2/E6 ratio predicted ≥CIN2 cervical lesions (AUC, 0.871; 95% confidence interval, 0.826-0.917) better than either individual test. Conclusions: To our knowledge, this study is the first to validate the expression of the HPV L1 capsid protein and a decreased HPV E2/E6 ratio as valuable predictive markers of ≥CIN2 cervical lesions, thereby discriminating cervical pre-malignancy and cancer. Cervical cytology can be analyzed on an outpatient basis with noninvasive procedures, in contrast to conventional histologic analysis, which is invasive. Moreover, cervical cytology can be conducted longitudinally to evaluate disease progression/regression without repeated invasive procedures. Citation Format: Youn Jin Choi, Sung-Jong Lee. Biomarker utility of the E2/E6 ratio and L1 immunoreactivity for determining HPV16-positive ≥CIN2 lesions and cervical squamous cell carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr LB-135.

E2/E6 ratio and L1 immunoreactivity as biomarkers to determine HPV16-positive high-grade squamous intraepithelial lesions (CIN2 and 3) and cervical squamous cell carcinoma.

ObjectiveHuman papillomavirus (HPV) 16 is the most carcinogenic HPV genotype. We investigated if HPV16 L1 capsid protein and E2/E6 ratio, evaluated by cervical cytology, may be used as biomarkers of ≥cervical intraepithelial neoplasia (CIN) 2 lesions.MethodsCervical specimens were obtained from 226 patients with HPV16 single infection. Using cytology specimen, L1 capsid protein and E2/E6 ratio were detected and the results were compared with those of the conventional histologic analysis of cervical tissues (CIN1–3 and squamous cell carcinoma [SCC]) to evaluate the association.ResultsThe L1 positivity of CIN2/3 was significantly lower than that of normal cervical tissue (p<0.001) and SCC demonstrated significantly lower L1 positivity than CIN1 (p<0.001). The mean E2/E6 ratios of specimens graded as SCC (0.356) and CIN2/3 (0.483) were significantly lower than those of specimens graded as CIN1 (0.786) and normal (0.793) (p<0.05). We observed that area under the receiver operating characteristic curve (AUC) for E2/E6 ratio (0.844; 95% confidence interval [CI]=0.793–0.895) was higher than that for L1 immunochemistry (0.636; 95% CI=0.562–0.711). A combination of E2/E6 ratio and L1 immunocytochemistry analyses showed the highest AUC (0.871; 95% CI=0.826–0.917) for the prediction of ≥CIN2 lesions.ConclusionTo our knowledge, this is the first study to validate HPV L1 capsid protein expression and decreased HPV E2/E6 ratio as valuable predictive markers of ≥CIN2 cervical lesions. Cervical cytology may be analyzed longitudinally on an outpatient basis with noninvasive procedures as against invasive conventional histologic analysis.

Human papillomavirus and Epstein-Barr virus in the pathogenesis of inverted papilloma and associated sinonasal carcinoma

To determine whether Epstein-Barr virus and human papillomavirus (HPV) types 6, 11, 16, 18, 35, and 43 DNA can be present in inverted papilloma (IP) and associated sinonasal carcinoma. The investigation was carried out using tissue samples obtained during surgery from 76 cases of IP and 4 cases of sinonasal carcinoma that had developed in the presence of IP. PCR was performed in 41 cases of IP and in 4 cases of sinonasal carcinoma; immunohistochemistry was done in 15 cases of IP to identify HPV L1 capsid protein. HPV6 DNA was detected in 20 cases; three of them were simultaneously found to have also HPV11; 1 case had only HPV11. HPV types 16, 18, 35, and 45 and Epstein-Barr virus were not detected. PCR revealed HPV types 16 and 35 in one of the 4 cases of sinonasal carcinoma associated with IP. Immunohistochemical study identified HPV L1 capsid protein in the epitheliocytes and stromal infiltrating lymphocytes and macrophages in two cases of IP with the highest concentration of HPV DNA as evidenced by PCR, as well as in one case of sinonasal carcinoma. Taking into account the low HPV DNA concentration detected only for types 6 and 11, as well as the immunohistochemical detection of HPV L1 in the tumor stromal lymphocytes and macrophages and some basal epitheliocytes in individual cases, it can be assumed that HPV does not play a crucial role as an etiological factor for IP. PCR revealed oncogenic HPV types 16 and 35 in sinonasal carcinoma associated with IP only in one case, which makes it untimely to claim that this virus plays a very special role in the etiology of this carcinoma. Epstein-Barr virus was not found in any case.

A proper triage for detecting women with high-risk human papillomavirus genotypes other than HPV16/18

ObjectiveTo evaluate the effect of human papillomavirus (HPV) L1 capsid protein detection in cervical exfoliated cells as a proper triage for women with high-risk human papillomavirus (hrHPV) genotypes other than HPV 16/18. Study designFrom January 2013 to June 2015, a total of 513 women aged 30–65 years infected with non-16/18 hrHPV were enrolled into the study. Primary HPV testing, HPV 16/18 genotyping and Papanicolaou (Pap) test were performed in all eligible women. HPV L1 capsid protein was detected by immunocytochemistry in cervical exfoliated cells. All hrHPV positive women underwent colposcopy and further biopsy if indicated. Relationships between HPV L1 capsid protein expression and histologic diagnosis, as well as cytology were analyzed. ResultsThe positive expression rate of HPV L1 capsid protein in CIN2+ group was significantly lower than that in CIN1 or better group (16.3% vs. 66.7%, P=0.000). Compared with the Pap test, HPV L1 detection achieved higher sensitivity (83.7% vs. 51.2%, P=0.000), higher negative predictive value (NPV) (95.3% vs. 88.6%, P=0.002), and significant lower specificity (66.7% vs.76.1%, P=0.002) while identifying CIN2+. Compared with the Pap test, HPV L1 detection achieved higher sensitivity (89.7% vs. 51.7%, P=0.008), higher NPV (99.0% vs. 96.2%, P=0.043), and significant lower specificity (61.2% vs.73.8%, P=0.000) while identifying CIN3+. ConclusionBecause of its higher sensitivity and NPV than that of Pap test, HPV L1 capsid protein detection in cervical exfoliated cells reduces the missed identification of high-grade cervical intraepithelial neoplasia (CIN) in women with hrHPV genotypes other than HPV 16/18. HPV L1 capsid protein detection could be a potential triage for women with non-16/18 hrHPV infection.

Clinical significance of CK7, HPV-L1, and koilocytosis for patients with cervical low-grade squamous intraepithelial lesions: a retrospective analysis

Most cervical low-grade squamous intraepithelial lesions (LSILs) do not progress to high-grade squamous intraepithelial lesions (HSILs); however, reliable biomarkers that predict LSIL progression are lacking. We investigated the association of cytokeratin 7 (CK7), human papillomavirus-L1 capsid protein (HPV-L1), and koilocytosis with clinical outcomes of patients with LSIL. CK7, HPV-L1, Ki67, and p16-INK4A expression was determined in 72 cervical LSIL and 28 HSIL biopsy samples; koilocytosis was evaluated by reviewing biopsy slides. Fifty patients with LSIL received follow-up. CK7, HPV-L1, and koilocytosis were detected in 48.6%, 44.4%, and 52.0% of LSIL tissues and in 78.6%, 10.7%, and 64.3% of HSIL tissues, respectively. Lesion grade was correlated directly with CK7 expression (P=.007) and inversely with HPV-L1 expression (P=.004). CK7 expression in LSILs was correlated inversely with HPV-L1 expression and directly with p16-INK4A and Ki67 status. Furthermore, koilocytosis was significantly associated with HPV-L1 and p16-INK4A expression. Progression to cervical intraepithelial lesions of grades ≥2 occurred in 34% of cases. CK7 negativity and HPV-L1 positivity were significantly associated with lower HSIL progression rates. HPV-L1-positive and CK7-negative LSILs showed significantly lower progression rates compared with HPV-L1-negative and CK7-positive LSILs (6.3% v-positive cases showed a significantly lower progression rate (17.6%) compared with nonkoilocytic and HPV-L1-negative cases (50%). CK7-negative, HPV-L1-positive, and koilocytic LSILs showed a progression rate of 7.7%. Koilocytosis and p16-INK4A were not significantly associated with clinical outcomes. Hence, evaluating HPV-L1, CK7, and koilocytosis profiles combined may be more reliable for LSIL prognostication.

HPV L1 and P16 Expression in CIN1 to Predict Future CIN2.

The goal of this study was to use the biomarkers human papillomavirus (HPV) L1 and p16 to develop an algorithm that could triage the individual patient with CIN1 at risk for progression. A total of 82 patients initially diagnosed with CIN1 at Peking University Shenzhen Hospital in China had their initial and follow-up paraffin-embedded tissue blocks immune-stained for HPV L1 capsid protein and p16. For CIN1, any staining of abnormal epithelium was considered positive. All patients were followed until they developed CIN2+ or for ≥3 years. About 38 patients regressed (HPV-, Cytology-), 17 persisted (CIN1), and 27 progressed (≥CIN2+). At initial diagnosis, HPV L1 capsid protein was expressed in 42.7% of the CIN1 cases. There was no difference in L1 expression among the 3 groups. However, p16-positive staining in the progression group was significantly higher than in the regression group (P<0.05). In the regression group, the proportion of HPV L1-/p16- category was significantly higher than that in the progression group. In the progression group, when CIN1 lesions progressed to CIN2+, the L1-positive rate was significantly decreased from 51.9% to 18.5%, the p16+/HPV L1+ rate decreased from the initial (44.4%) to the final diagnosis (14.8%), and the p16+/HPV L1- rate increased from the initial (25.9%) to the final diagnosis (66.7%). P16 expression is a clear risk factor for the progression of CIN1. The p16-/HPV L1- pattern was significantly associated with the regression of CIN1. Moving from CIN1 to CIN2+ over time, p16+/HPV L1+ decreased, and p16+/HPV L1- increased. Unfortunately, our objective of finding a sensitive and specific triage algorithm for the individual patient with CIN1 was not achieved.

Human Papillomavirus E2 Regulates SRSF3 (SRp20) To Promote Capsid Protein Expression in Infected Differentiated Keratinocytes.

The human papillomavirus (HPV) life cycle is tightly linked to differentiation of the infected epithelial cell, suggesting a sophisticated interplay between host cell metabolism and virus replication. Previously, we demonstrated in differentiated keratinocytes in vitro and in vivo that HPV type 16 (HPV16) infection caused increased levels of the cellular SR splicing factors (SRSFs) SRSF1 (ASF/SF2), SRSF2 (SC35), and SRSF3 (SRp20). Moreover, the viral E2 transcription and replication factor that is expressed at high levels in differentiating keratinocytes could bind and control activity of the SRSF1 gene promoter. Here, we show that the E2 proteins of HPV16 and HPV31 control the expression of SRSFs 1, 2, and 3 in a differentiation-dependent manner. E2 has the greatest transactivation effect on expression of SRSF3. Small interfering RNA depletion experiments in two different models of the HPV16 life cycle (W12E and NIKS16) and one model of the HPV31 life cycle (CIN612-9E) revealed that only SRSF3 contributed significantly to regulation of late events in the virus life cycle. Increased levels of SRSF3 are required for L1 mRNA and capsid protein expression. Capsid protein expression was regulated specifically by SRSF3 and appeared independent of other SRSFs. Taken together, these data suggest a significant role of the HPV E2 protein in regulating late events in the HPV life cycle through transcriptional regulation of SRSF3 expression. Human papillomavirus replication is accomplished in concert with differentiation of the infected epithelium. Virus capsid protein expression is confined to the upper epithelial layers so as to avoid immune detection. In this study, we demonstrate that the viral E2 transcription factor activates the promoter of the cellular SRSF3 RNA processing factor. SRSF3 is required for expression of the E4(^)L1 mRNA and so controls expression of the HPV L1 capsid protein. Thus, we reveal a new dimension of virus-host interaction crucial for production of infectious virus. SRSF proteins are known drug targets. Therefore, this study provides an excellent basis for developing strategies to regulate capsid protein production in the infected epithelium and the production of new virions.

Immunohistochemistry and Polymerase Chain Reaction for Detection Human Papilloma Virus in Warts: A Comparative Study.

BackgroundImmunohistochemistry and polymerase chain reaction (PCR) are the most widely used methods for the detection of viruses. PCR is known to be a more sensitive and specific method than the immunohistochemical method at this time, but PCR has the disadvantages of high cost and skilled work to use widely. With the progress of technology, the immunohistochemical methods used in these days has come to be highly sensitive and actively used in the diagnostic fields.ObjectiveTo evaluate and compare the usefulness of immunohistochemistry and PCR for detection human papilloma virus (HPV) in wart lesions.MethodsNine biopsy samples of verruca vulgaris and 10 of condyloma accuminatum were examined. Immunohistochemical staining using monoclonal antibody to HPV L1 capsid protein and PCR were done for the samples. DNA sequencing of the PCR products and HPV genotyping were also done.ResultsHPV detection rate was 78.9% (88.9% in verruca vulgaris, 70.0% in condyloma accuminatum) on immunohistochemistry and 100.0% for PCR. HPV-6 genotype showed a lower positivity rate on immunohistochemistry (50.0%) as compared to that of the other HPV genotypes.ConclusionImmunohistochemistry for HPV L1 capsid protein showed comparable sensitivity for detection HPV. Considering the high cost and great effort needed for the PCR methods, we can use immunohistochemistry for HPV L1 capsid protein with the advantage of lower cost and simple methods for HPV detection.

Safety and Efficacy Data on Vaccines and Immunization to Human Papillomavirus.

Since the discovery of the causal association between human papillomavirus (HPV) and cervical cancer, efforts to develop an effective prophylactic vaccine to prevent high-risk HPV infections have been at the forefront of modern medical research. HPV causes 530,000 cervical cancer cases worldwide, which is the second most common cause of cancer deaths in women; a worldwide collaboration among epidemiologists, molecular biologists, vaccinologists, virologists, and clinicians helped lead to the development of two highly effective prophylactive HPV vaccines. The first, Gardasil, is a quadrivalent vaccine made up of recombinant HPV L1 capsid proteins from the two high-risk HPV types (16/18) responsible for 70% of cervical cancer cases as well as two low-risk HPV types (6/11) which are the causative agent for genital warts. The second, Cervarix, is a bivalent vaccine that was FDA approved three years after Gardasil and is also composed of L1 capsid proteins from HPV types 16/18. This review article focuses on the safety and efficacy data of both FDA-approved vaccines, as well as highlighting a few advances in future HPV vaccines that show promise in becoming additional treatment options for this worldwide disease.

Clinical significance of HPV L1 capsid protein detection in cervical exfoliated cells in high-risk HPV positive women

To explore the clinical significance of human papillomavirus L1 capsid protein detection in cervical exfoliated cells in high-risk HPV positive women. From November 2012 to June 2013, 386 high-risk HPV positive (detected by hybrid capture II) cases were enrolled as eligible women from Huzhou Maternity & Child Care Hospital and Women's Hospital, School of Medicine, Zhejiang University. All eligible women underwent liquid-based cytology (ThinPrep) followed by colposcopy. Biopsies were taken if indicated. Cervical exfoliated cells were collected for HPV L1 capsid protein detection by immunocytochemistry. Expression of HPV L1 capsid protein in groups with different histological diagnosis were compared, and the role of HPV L1 capsid protein detection in cervical exfoliated cells in cervical lesions screening was accessed. Total 386 enrolled eligible women were finally diagnosed histologically as follwed: 162 normal cervix, 94 low-grade squamous intraepithelial lesion (LSIL), 128 high-grade squamous intraepithelial lesion (HSIL) and 2 squamous cervical cancer (SCC). The positive expression rate of HPV L1 in HSIL+ (HSIL or worse) group was significantly lower than that in LSIL- (LSIL or better) group (19.2% vs 66.4%, P=0.000). While identifying HSIL+ in HPV positive cases and compared with cytology, HPV L1 detection resulted in significant higher sensitivity (80.77% vs 50.77%, P=0.000) and negative predictive value (NPV; 87.18% vs 76.47%, P=0.004), significant lower specificity (66.41% vs 81.25%, P=0.000), and comparable positive predictive value (PPV; 54.97% vs 57.89%, P=0.619). To identify HSIL+ in HPV-positive/cytology-negative women, the sensitivity, specificity, PPV, and NPV of HPV L1 detection were 87.50%, 61.54%, 41.18%, and 94.12% respectively, while 80.00%, 86.36%, 80.00% and 86.36% respectively in HPV-positive/atypical squamous cell of undetermined significance (ASCUS) women. HPV L1 capsid detection in cervical exfoliated cells have a role in cervical lesions screening in high-risk HPV positive women, and may be a promising triage for high-risk HPV-positive/cytology-negative or ASCUS women.

Study of clinical application of HPV L1 capsid protein combined with HPV typing and TCT detection in diagnosis and treatment of cervical lesion

Objective To investigate the expression of human papilloma virus L1 (HPV L1)capsid protein in cervical lesions and different human papillomavirus (HPV) subtypes, and to guide clinical triage management and best individual treatment. Methods Retrospective analysis of 2012 January to 2014 January in Jiaxing Hospital of Traditional Chinese Medicine gynecology clinic for HPV L1 protein combined with HPV type, and liquid-based cytology test (TCT) of 176 patients data. Results The positive expression rate of HPV L1 protein with TCT examination in the negative for intraepithelial lesion or malignancy (NILM), atypical squamous cells of undetermined significance(ASCUS), low-grade squamous intraepithe lial lesion (LSIL), atypical squamous cells not except high lesion (ASC-H), high-grade squamous intraepithelial lesion (HSIL), and squamous-cell carcinoma (SCC) was 28.99%, 44.19%, 64.44%, 22.22%, 12.50%, and 0, respectively. No significant differences were found between the NILM and ASCUS groups (P>0.05). The positive expression rate of HPV L1 protein in LSIL group was the highest, and it was statistically significantly different from ASC-H and HSIL groups (χ2=3.88, 5.50, P 0.05); and statistically significant difference was found between CIN Ⅰ group and CIN Ⅱ, CIN Ⅲ group (χ2=4.53, 5.56, P 0.05). Conclusions Detection of HPV L1 protein is of clinical value to evaluate the risk of cervical lesions. HPV L1 protein combined with HPV type and TCT detection is helpful for traffic management and personalized treatment, and benefit patients with cervical lesions. Key words: Papillomaviridae/ME; Viral envelope proteins/ME; Cytological techniques; Uterine cervical diseases/ME/DI/TH

New strategies in advanced cervical cancer: from angiogenesis blockade to immunotherapy.

Cervical cancer remains unique among solid tumor malignancies. Persistent infection with oncogenic subtypes of the human papillomavirus (HPV) results in carcinogenesis, predominantly occurring at the cervical transformation zone where endocervical columnar cells undergo metaplasia to a stratified squamous epithelium. The molecular cascade involving viral oncoproteins, E6 and E7 and their degradative interactions with cellular tumor suppressor gene products, p53 and pRb, respectively, has been precisely delineated. The precursor state of cervical neoplasia may last for years allowing for ready detection through successful screening programs in developed countries using cervical cytology and/or high-risk HPV DNA testing. Prophylactic HPV L1 capsid protein vaccines using virus-like-particle technology have been developed to prevent primary infection by the most common high-risk HPVs (16 and 18). Women who lack access to health care and those who undergo sporadic screening remain at risk. Although radical surgery (including fertility-sparing surgery) is available for patients with early-stage cancers, and chemoradiation plus high-dose-rate brachytherapy can cure the majority of those with locally advanced disease, patients with metastatic and nonoperable recurrent cervical cancer constitute a high-risk population with an unmet clinical need. On August 14, 2014, the FDA approved the antiangiogenesis drug bevacizumab for women with advanced cervical cancer. This review will highlight advances in translational science, antiangiogenesis therapy and immunotherapy for advanced disease.

HPV L1 capsid protein detection in high-risk human papillomavirus-positive cervical smears

Background: The L1 capsid protein is a viral nuclear protein that encapsidates the human papillomavirus (HPV) DNA to build new infectious particles. Previous studies in immune-competent patients have shown that detectable L1 protein in lowgrade squamous intraepithelial lesion (LSIL) smears is associated with remission in 60-75% of cases. Thus, the test can reduce interventions by approximately 75%. Objective: This study was performed to evaluate the use of HPV L1 capsid protein detection on cytology samples in a population with relatively high human immunodeficiency virus (HIV) prevalence and with known high-risk HPV (hrHPV) DNA results. Setting: Samples were obtained during a cervical cancer screening study at primary healthcare clinics in the Tshwane district, Gauteng. The HIV prevalence of the target group was estimated to be between 20% and 30%. Method: Conventional cervical cytology smears of 575 women were microscopically assessed and diagnosed. In addition, women were tested for the presence of HPV DNA on a cervical or vaginal sample. Immunocytochemical analysis was performed on morphologically abnormal smears and on a 52 control group smears reported to be negative for HPV DNA and morphological abnormalities. The detection of L1 capsid protein was carried out with the Cytoactiv® HPV L1 screening set, an antibody-based immunocytochemical stain. Results: A cytological diagnosis of LSIL was made in 19 women (3.3%), high-grade squamous intraepithelial lesions (HSILs) in 42 (7.5%) and malignancy in 1 (0.2%). Of the LSIL cases, 10 of 19 (52.6%) stained positive for the presence of the L1 protein, while only one of 42 HSIL (2.4%) cases stained positive. Three hundred and four cases (52.9%) tested positive for hrHPV DNA, including women with LSIL, HSIL and malignancy. All of the control cases stained negative for the L1 capsid protein. Conclusion: hrHPV was not a useful triage test for women with abnormal cellular morphology in this population. Promising results were reported for Cytoactiv® immunostaining as a triage test for patients with LSIL, but it added no value to samples known to be HPV-negative or reported to be morphologically negative. The immunostaining of smears reported to be HSIL or worse were almost universally negative, supporting the diagnostic accuracy of cytology and the expectation of persistence or progression of these lesions.