Lineage analysis of human papillomavirus types 31 and 45 in cervical samples of Iranian women.

Knowing the regional lineages/sublineages of human papillomavirus 31 (HPV 31) and 45 would be of great importance for further evolutionary, epidemiological, and biological analysis. In this regard, to characterize more common lineages and sublineages of HPV 31 and 45, the sequence variations of E6 gene were investigated in normal, premalignant, and malignant samples collected from the cervix in Iran. In total, 54 HPV 31- and 24 HPV 45-positive samples were analyzed by hemi-nested polymerase chain reaction (PCR) and nested-PCR, respectively. All PCR products were subjected to direct sequencing analysis. The results indicated that all three lineages A, B, and C were detected in HPV 31-positive samples; among which HPV 31 lineage A was dominant as it was found in 66.7% of all samples. HPV 31 lineages B and C were identified in 5.5% and 27.8% of samples, respectively. In HPV 45-infected samples, lineage B comprised of 62.5% of all samples and the remaining 37.5%  belonged to lineage A. In conclusion, our findings showed that lineage A of HPV 31 was predominant in Iran. Lineage B of HPV 45 was also dominant among Iranian women. However, further studies with larger sample size should be addressed to estimate the pathogenicity risk of HPV 31 or HPV 45 lineages/sublineages in the development of cervical cancer among Iranian women.

Functional evaluation of human papillomavirus type 31 long control region variants.

Persistent infections by high-risk human papillomavirus (HR-HPV) are a necessary condition, but not sufficient for cervical cancer development. Genetic variants of HR-HPV appear to be related to the risk of persistent infections. The study performed a functional evaluation of variants of the HPV-31 promoter region (LCR). For this, cloning and subcloning of variants HPV-31/UFPE-21 HPV-31/UFPE-89, HPV-31/UFPE-66, E2 gene and prototype HPV-31 were performed. Transfection with different concentrations of E2 was done and the concentration of 25 ng was determined to be ideal for LCR activation. HPV-31/UFPE-21 and HPV-31/UFPE-89 have a greater ability to alter Nluc reporter gene expression levels and HPV-31/UFPE-66 showed decreased levels of gene expression of Nluc reporter gene compared to control. Statistical analysis showed a significant difference between the polymorphic LCR regions and the control (p < 0.0001). A more refined profile of variants of HPV-31 and its importance for the prognosis of cervical lesions begins to be drawn.

The Synthesis of Main Capsid Protein of Anogenital Type HPV6 L1 in Plant Expression System on the Basis of Tomato Fruits.

The anogenital type HPV6 L1 major capsid protein was synthesized in a plant expression system on the basis of tomato fruits. The content of HPV6 L1 reached 380 µg per 1 mg of total soluble protein of raw fruit mass, which was represented as a single band with a molecular mass of 56 kDa on the SDS electrophoregram. When orally administrated to mice, the vaccine material from the tomato fruit transgenic for HPV6 L1 induced highly effective antibody immune response with a high titer. The cross-reactivity during the interaction of the antibody to the HPV6 L1 protein from peripheral blood serum of mice vaccinated with HPV6 L1 with the antigenic proteins HPV16 L1, HPV18 L1, HPV31 L1, and HPV45 L1 was found. This is promising for creating a vaccine with a broad reactivity against dangerous anogenital papillomatoses and cervical cancer.

How to triage HPV positive cases: Results of four million females.

OBJECTIVE: To evaluate the Turkey's nationwide HPV DNA screening program on the basis of first 4 million screened women. METHODS: Women over age 30 were invited for screening via HPV DNA and conventional cytology. Single visit screen strategy was used to collect for both screening and triage (extended genotyping and conventional pap-smear). RESULTS: A total of 4,099,230 patients had attended to HPV DNA cancer screening. 4.39% were found to be HPV DNA positive. The most common HPV type was 16, followed by 51, 31, 52, 56 and 18 at all age intervals and geographic regions. Cytology results were reported as "normal" in (69.2%), "inadequate sampling" in (16.6%) and as "abnormal (≥ASC-US)" in the remaining. Current Turkish screening with HPV DNA (referral to colposcopy with HPV 16 or 18 or any smear abnormality ≥ASC-US) gives overall PPV of 24.3% for ≥CIN2. Only Pap-Smear triage revealed PPV of 26.4% for ≥ASC-US thresholds. Comparison of different triage methods for ≥CIN2+ according to different HPV genotype revealed a PPV of 32,6% for HPV 16; 15,3% for HPV 18. This figure was 34.4%, 19.3%, 15.3% and 14.0% for HPV 33, 31, 45 and 35; respectively. CONCLUSION: This study involves the largest series in the world summarizing a real-world experience with primary HPV DNA screening and triage with a single visit. The results show the feasibility and applicability of such screening method in developing countries with acceptable colposcopy referral rates. Among triage tests, only pap-smear seems to be effective without a need for extended genotyping.

Long-term Cross-reactivity Against Nonvaccine Human Papillomavirus Types 31 and 45 After 2- or 3-Dose Schedules of the AS04-Adjuvanted Human HPV-16/18 Vaccine.

This analysis focused on long-term cross-reactive immunogenicity against nonvaccine human papillomavirus (HPV) types 31 and 45 following 2 doses of AS04-adjuvanted HPV-16/18 vaccine in girls aged 9-14 years or following 3 doses in women aged 15-25 years, for up to 3 years (HPV-070 study) and up to 5 years (HPV-048 study) after the first vaccination. Both schedules elicited antibodies against HPV-31 and HPV-45 up to 5 years after first dose. The antibody concentration was similar in young girls as compared to women. Specific CD4+ T-cell and B-cell responses to HPV-31 and HPV-45 at month 36 were similar across groups. Clinical trials registration: NCT01381575 and NCT00541970.

CpG methylation in human papillomavirus (HPV) type 31 long control region (LCR) in cervical infections associated with cytological abnormalities.

The mechanisms that regulate papillomavirus gene expression include DNA methylation. The transcription of papillomavirus oncogenes E6 and E7 is controlled by certain regulatory elements in the LCR, which include binding sites for the E2 protein, a viral regulator of oncogene expression. In HPV-31-infected exfoliated cervical cells, the CpG methylation of the entire LCR was determined by next-generation sequencing after bisulfite modification. Six of the 22 cases had methylated CpG sites in the HPV-31 LCR, including position 7479 and/or 7485, at the promoter distal E2 binding site, thus suggesting a potential regulatory mechanism for papillomavirus transcription.

Cervical intraepithelial neoplasia grade 2 or worse in Galicia, Spain: HPV 16 prevalence and vaccination impact.

INTRODUCTION: The etiology of cervical intraepithelial neoplasia grade 2 or worse (CIN2+) can influence the efficacy of Public Health preventive strategies. This study aimed to determine the high-risk papillomavirus (HR-HPV) prevalence in CIN2+ cases in unvaccinated women in Galicia (Spain), the expected impact of bivalent vaccination, and the distribution of HPV 16 in squamous lesions. MATERIAL AND METHODS: Ninety-four histologically confirmed cases of CIN2+ (2009-2010) were retrospectively studied: 23 CIN2, 58 CIN3- squamous carcinoma in situ (CIN3-CIS), 5 adenocarcinoma in situ (AIS), and 8 invasive squamous cervical cancer (SCC). Linear Array HPV Genotyping Test (Roche Diagnostics, Mannheim, Germany) was performed on the cervical specimens. Bivalent vaccination impact was calculated, based on regional vaccination coverage data, local HR-HPV prevalence, and reported efficacy (direct and cross-protection) of the vaccine. RESULTS: HR-HPV prevalence was 96.8%. The most frequent genotypes were HPV 16 (48.8-58.2%) and HPV 31 (9.3%-12.1%), considering single infections or single-multiple infections, respectively (hierarchical attribution). In squamous lesions, HPV 16 prevalence in women younger than 45 years of age increased in severe lesions (CIN3-CIS/SCC, OR 4.2), and was higher than in older women (OR 5.5). The vaccine could reduce the cumulative incidence of CIN2+ by 50.6% (direct protection), or by 62.7% (direct and cross-protection). CONCLUSION: HPV vaccination could have a great impact in women younger than 45 years of age due to the high prevalence of HPV 16 in their lesions.

Differences in integration frequencies and APOBEC3 profiles of five high-risk HPV types adheres to phylogeny.

Persistent infection with Human Papillomavirus (HPV) is responsible for almost all cases of cervical cancers, and HPV16 and HPV18 associated with the majority of these. These types differ in the proportion of viral minor nucleotide variants (MNVs) caused by APOBEC3 mutagenesis as well as integration frequencies. Whether these traits extend to other types remains uncertain. This study aimed to investigate and compare genomic variability and chromosomal integration in the two phylogenetically distinct Alpha-7 and Alpha-9 clades of carcinogenic HPV types. The TaME-seq protocol was employed to sequence cervical cell samples positive for HPV31, HPV33 or HPV45 and combine these with data from a previous study on HPV16 and HPV18. APOBEC3 mutation signatures were found in Alpha-9 (HPV16/31/33) but not in Alpha-7 (HPV18/45). HPV45 had significantly more MNVs compared to the other types. Alpha-7 had higher integration frequency compared to Alpha-9. An increase in integration frequency with increased diagnostic severity was found for Alpha-7. The results highlight important differences and broaden our understanding of the molecular mechanisms behind cervical cancer induced by high-risk HPV types from the Alpha-7 and Alpha-9 clades.

Detection of high-risk human papillomavirus infection and treatment of high-grade vaginal intraepithelial neoplasia: A single-institution study.

OBJECTIVE: To identify high-risk HPV (hrHPV) genotypes associated with high-grade vaginal intraepithelial neoplasia (VaIN), and evaluate the efficacy of various treatments for high-grade VaIN. METHODS: A retrospective review of outcomes among women diagnosed with VaIN after vaginal punch biopsy conducted due to an abnormal Papanicolaou smear or positive test for hrHPV at a hospital in Seoul, Korea, from 2013 to 2018. Logistic regression was used to identify variables associated with abnormal pathologic outcomes. RESULTS: Among 389 women included in the study, 58 were diagnosed with high-grade VaIN, including VaIN stage 2 (n = 37), VaIN stage 3 (n = 16), carcinoma in situ of the vagina (n = 3), and squamous carcinoma of the vagina (n = 2). In multivariate logistic regression analysis, risk of high-grade VaIN and cancer was higher among women with abnormal cytology (odds ratio [OR], 1.88; 95% confidence interval [CI], 1.47-2.47), any hrHPV infection (OR, 8.75; 95% CI, 1.14-67.31), HPV16 infection (OR, 5.71; 95% CI, 2.57-12.68), or HPV31 infection (OR, 4.37; 95% CI, 1.45-13.11). CONCLUSION: The findings suggest that infection with hrHPV, especially HPV16 and HPV31, is significantly associated with high-grade VaIN. Regarding treatment modalities, ablative or excisional treatments showed good efficacy against pathologic regression of high-grade VaIN.

Phylogenomic Analysis of Human Papillomavirus Type 31 and Cervical Carcinogenesis: A Study of 2093 Viral Genomes.

Human papillomavirus (HPV) type 31 (HPV31) is closely related to the most carcinogenic type, HPV16, but only accounts for 4% of cervical cancer cases worldwide. Viral genetic and epigenetic variations have been associated with carcinogenesis for other high-risk HPV types, but little is known about HPV31. We sequenced 2093 HPV31 viral whole genomes from two large studies, one from the U.S. and one international. In addition, we investigated CpG methylation in a subset of 175 samples. We evaluated the association of HPV31 lineages/sublineages, single nucleotide polymorphisms (SNPs) and viral methylation with cervical carcinogenesis. HPV31 A/B clade was >1.8-fold more associated with cervical intraepithelial neoplasia grade 3 and cancer (CIN3+) compared to the most common C lineage. Lineage/sublineage distribution varied by race/ethnicity and geographic region. A viral genome-wide association analysis identified SNPs within the A/B clade associated with CIN3+, including H23Y (C626T) (odds ratio = 1.60, confidence intervals = 1.17-2.19) located in the pRb CR2 binding-site within the E7 oncogene. Viral CpG methylation was higher in lineage B, compared to the other lineages, and was most elevated in CIN3+. In conclusion, these data support the increased oncogenicity of the A/B lineages and suggest variation of E7 as a contributing risk factor.

A ten-year study of immunogenicity and safety of the AS04-HPV-16/18 vaccine in adolescent girls aged 10-14 years.

This study assessed long-term immunogenicity and safety following 3 doses of AS04-adjuvanted human papillomavirus (HPV)-16/18 L1 virus-like particle (VLP) vaccine in females 10-14 years old. Girls included in the immunogenicity subset in the primary controlled, observer-blinded, randomized study (NCT00196924) who received 3 doses were invited for a 10-year follow-up (NCT00316706 and NCT00877877). Serum antibody responses against HPV-16/18 (vaccine types) and HPV-31/45 (non-vaccine types) were measured by enzyme-linked immunosorbent assay (ELISA) using type-specific VLP as coating antigens. Serious adverse events (SAEs) and pregnancy information were recorded. At Month (M) 120, all subjects (N = 418, according-to-protocol immunogenicity cohort) were seropositive for anti-HPV-16/18 antibodies. Geometric mean titers (GMTs) were 1589.9 ELISA Units [EU]/mL (95% confidence interval [CI]: 1459.8-1731.6) for anti-HPV-16 and 597.2 EU/mL (95% CI: 541.7-658.5) for anti-HPV-18 in subjects seronegative at baseline for the type analyzed. Post hoc mathematical modeling predicted a durability ≥50 years for anti-HPV-16 and anti-HPV-18. For the non-vaccine humoral type response, all initially seronegative subjects had seroconverted at M7, with anti-HPV-31 GMT of 2030.5 EU/mL (95% CI: 1766.2-2334.4) and anti-HPV-45 GMT of 2300.8 EU/mL (95% CI: 2036.8-2599.0). At M120, 87.7% and 85.1% remained seropositive for anti-HPV-31 with GMT of 242.9 EU/mL (95% CI: 201.4-293.0) and anti-HPV-45 with GMT of 204.7 EU/mL (95% CI: 170.0-246.6). During the 10-year follow-up, no SAEs or abnormal pregnancy outcomes were causally related to vaccination. Three doses of the AS04-HPV-16/18 vaccine induced high and sustained antibody response against HPV-16,18,31 and 45 in girls aged 10-14 years during the 10-year follow-up, with an acceptable long-term safety profile.

Genetic variability in E5, E6, E7 and L1 genes of human papillomavirus type 31.

Human papillomavirus (HPV) type 31 is an important pathogenic subtype associated with cervical cancer. The aims of the present study were to analyze E5, E6, E7 and L1 gene mutations of HPV­31 among females, and to elucidate the evolutionary associations between them. In total, 87 positive samples were collected. The E5, E6, E7 and L1 genes were amplified by polymerase chain reaction and sequenced. Subsequently, two phylogenetic trees were constructed from the nucleotide sequences of the E5, E6 and E7 and the L1 variants of HPV­31. In total, 31 mutation sites of E5, E6 and E7 genes were identified, of which 16 were non­synonymous. T4053A (F80I), C285T (H60Y), C520T (A138V) and A743G (K62E) were the most common non­synonymous mutations. A total of 30 mutation sites of L1 genes were identified, of which four were non­synonymous. The most common non­synonymous mutations of L1 genes were A6350G (T29A) and C6372A (T36N). By phylogenetic analysis, A and C variants were most frequently detected, while B variants were less frequently detected in this population. The sequence variation data obtained in the present study provides a foundation for future research regarding HPV­induced oncogenesis, and may prove valuable for developing diagnostic probes and in the design of HPV vaccines for targeted populations.

Displaying 31RG-1 peptide on the surface of HPV16 L1 by use of a human papillomavirus chimeric virus-like particle induces cross-neutralizing antibody responses in mice.

Current available human papillomavirus (HPV) vaccines are based on the major capsid protein L1 virus-like particles (VLPs), which mainly induce type-specific neutralizing antibodies against vaccine types. Continuing to add more types of VLPs in a vaccine raises the complexity and cost of production which remains the principal impediment to achieve broad implementation of HPV vaccines, particularly in developing regions. In this study, we constructed 16L1-31L2 chimeric VLP (cVLP) by displaying HPV31 L2 aa.17-38 on the h4 coil surface region of HPV16 L1, and assessed its immunogenicity in mouse model. We found that the cVLP adjuvanted with alum plus monophosphoryl lipid A could induce cross-neutralizing antibody responses against 16 out of 17 tested HPV pseudoviruses, and the titer against HPV16 was as high as that was induced by HPV16 L1VLP (titer > 105), more importantly, titers over 103 were observed against two HR-HPVs including HPV31 (titer, 2,200) and -59 (titer, 1,013), among which HPV59 was not covered by Gardasil-9, and medium or low titers of cross-neutralizing antibodies against other 13 tested HPV pseudoviruses were also observed. Our data demonstrate that 16L1-31L2 cVLP is a promising candidate for the formulation of broader spectrum HPV vaccines.

Frequency of Human Papilloma Virus (HPV) subtypes 31,33,35,39 and 45 among Yemeni women with cervical cancer.

BACKGROUND: Identification of different HPV subtypes in unidentified communities provides sufficient information for screening and monitoring potential impact of a vaccination program. Therefore, the aim of this study was to screen for the presence of HPVs subtypes 31,33,35,39 and 45 among Yemeni women with Cervical Cancer. METHODOLOGY: A total of 200 (150 malignant and 50 benign) tissue samples were obtained from Yemeni women with cervical cancer, were investigated for the presence of HPV subtypes 31,33,35,39 and 45 by Polymerase Chain Reaction (PCR). RESULTS: Of the 150 cervical cancer tissue specimens, HPV 31, HPV 33, HPV35, HPV 39 and HPV45 were identified in 10/150 (6.7 %), 6/150 (4 %), 6/150 (4 %), 5/150 (3.3 %) and 10/150 (6.7 %), respectively. The frequency of these HPV subtypes among Yemeni women with cervical cancer was 24 %. CONCLUSION: HPV 31, HPV 33, HPV35, HPV 39 and HPV45 were prevalent among Yemeni women with cervical cancer.

Durable immunity to oncogenic human papillomaviruses elicited by adjuvanted recombinant Adeno-associated virus-like particle immunogen displaying L2 17-36 epitopes.

Vaccination with the minor capsid protein L2, notably the 17-36 neutralizing epitope, induces broadly protective antibodies, although the neutralizing titers attained in serum are substantially lower than for the licensed L1 VLP vaccines. Here we examine the impact of other less reactogenic adjuvants upon the induction of durable neutralizing serum antibody responses and protective immunity after vaccination with HPV16 and HPV31 L2 amino acids 17-36 inserted at positions 587 and 453 of VP3, respectively, for surface display on Adeno-Associated Virus 2-like particles [AAVLP (HPV16/31L2)]. Mice were vaccinated three times subcutaneously with AAVLP (HPV16/31L2) at two week intervals at several doses either alone or formulated with alum, alum and MPL, RIBI adjuvant or Cervarix. The use of adjuvant with AAVLP (HPV16/31L2) was necessary in mice for the induction of L2-specific neutralizing antibody and protection against vaginal challenge with HPV16. While use of alum was sufficient to elicit durable protection (>3 months after the final immunization), antibody titers were increased by addition of MPL and RIBI adjuvants. To determine the breadth of immunity, rabbits were immunized three times with AAVLP (HPV16/31L2) either alone, formulated with alum±MPL, or RIBI adjuvants, and after serum collection, the animals were concurrently challenged with HPV16/31/35/39/45/58/59 quasivirions or cottontail rabbit papillomavirus (CRPV) at 6 or 12 months post-immunization. Strong protection against all HPV types was observed at both 6 and 12 months post-immunization, including robust protection in rabbits receiving the vaccine without adjuvant. In summary, vaccination with AAVLP presenting HPV L2 17-36 epitopes at two sites on their surface induced cross-neutralizing serum antibody, immunity against HPV16 in the genital tract, and long-term protection against skin challenge with the 7 most common oncogenic HPV types when using a clinically relevant adjuvant.

Neutralizing and cross-neutralizing antibody titres induced by bivalent and quadrivalent human papillomavirus vaccines in the target population of organized vaccination programmes.

Aim of this investigator-initiated study was to evaluate and compare the titres of neutralizing and cross-neutralizing antibodies (NAbs) induced by the bivalent (Cervarix(®)) and quadrivalent (Gardasil(®)) HPV vaccines in a cohort of girls aged 11-13 years from organized vaccination programmes. To this aim, HPV16 and HPV18 NAbs were measured by pseudovirion-based neutralization assays in serum collected at 1-6 months after the third vaccine dose in 107 girls vaccinated with Cervarix(®) and 126 vaccinated with Gardasil(®), while HPV31 and HPV45 cross-NAbs were tested in the first 50 consecutive girls of both vaccine groups. The results of this study demonstrated that all vaccinated girls developed HPV16 and HPV18 NAbs, with the exception of two Gardasil(®) vaccinees with undetectable HPV18 NAbs. Geometric mean titres (GMTs) of both HPV16 and HPV18 NAbs were significantly higher in Cervarix(®) than in Gardasil(®) vaccinees [HPV16 NAb GMT 22,136 (95% CI, 18,811-26,073) vs 5092 (4230-6151), respectively; P<0.0001; HPV18 NAb GMT 11,962 (9536-14,363) vs 1804 (1574-2110), respectively; P<0.0001]. Cross-NAbs to HPV31 and HPV45 were detected more frequently Cervarix(®) (HPV31 NAb positivity rates 92.7% and 36%, respectively; P<0.05) than in Gardasil(®) vaccinees (HPV45 NAb positivity rates 56% and 6%, respectively; P<0.0001). The titres of cross-NAbs against HPV31 and HPV45 were also significantly higher in Cervarix(®) than in Gardasil(®) vaccinees [HPV31 NAb GMT 157.2 (95% CI, 92-269) vs 13.0 (6.5-25.8), respectively; P<0.0001; HPV45 NAb GMT 4.7 (2.1-10.2) vs 1.3 (0.3-3.1), respectively; P<0.01]. In conclusion, in adolescent girls vaccinated within organized vaccination programmes, HPV vaccines drive the generation not only of NAbs to HPV vaccine types, but also of cross-NAbs. The bivalent vaccine induced significantly higher HPV16 and HPV18 NAb titres and more frequently and at higher titre HPV31 and HPV45 cross-NAbs than the quadrivalent vaccine.