Human Papillomavirus Type 16 L1 Research Articles

Human Papillomavirus Type 16 L2 Gene Sequence Variation Analysis in Indonesian Cervical Cancer Specimens.

Background: Human papillomavirus type 16 (HPV16) is the most prevalent etiology of cervical cancer in Indonesian women. The L2 minor capsid protein has considerable potential as a broad-protective antigen target of the cervical cancer vaccine strategies, yet the data on L2 gene variation is still minimal. In this research, we determined the variations of the HPV16 L2 gene sequences in Indonesian cervical cancer specimens. Methods:We cross-sectionally observed 23 DNA isolates of HPV16 positive cervical cancer specimens stored in the laboratory of the Center for Diagnostic and Research on Infectious Diseases (PDRPI Lab), Faculty of Medicine, Universitas Andalas, Padang, Indonesia. We detected and amplified the HPV16 L2 gene sequences in the samples, followed by sequencing, DNA alignment, single nucleotide polymorphisms (SNPs) analysis, and phylogenetic tree reconstruction. Results:As many as 35 SNPs were found, consist of 18 synonymous SNPs (sSNPs) and 17 non-synonymous SNPs (nsSNPs). Amino acid variations were mostly detected at S269P (100%) and L330F (43.48%) with no variation in the immuno-protective region near L2 N-terminus. A total of 5 HPV16 phylogenetic sub-lineages were found closely related to A1 (n=5), A2 (n=12), A3 (n=2), A4 (n=3), and C (n=1). Conclusion:The variations of HPV16 L2 gene sequences are mainly located in the central region of the L2 sequences, and the cross-protective region near the L2 N-terminus is remarkably conserved. This study should enhance the information about HPV16 L2 gene variation in Indonesia.

Overexpression of m6A-factors METTL3, ALKBH5, and YTHDC1 alters HPV16 mRNA splicing

We report that overexpression of the m6A-demethylase alkB homolog 5 RNA demethylase (ALKBH5) promoted production of intron retention on the human papillomavirus type 16 (HPV16) E6 mRNAs thereby promoting E6 mRNA production. ALKBH5 also altered alternative splicing of the late L1 mRNA by an exon skipping mechanism. Knock-down of ALKBH5 had the opposite effect on splicing of these HPV16 mRNAs. Overexpression of the m6A-methylase methyltransferase-like protein 3 (METLL3) induced production of intron-containing HPV16 E1 mRNAs over spliced E2 mRNAs and altered HPV16 L1 mRNA splicing in a manner opposite to ALKBH5. Overexpression of the nuclear m6A-“reader” YTH domain-containing protein 1 (YTHDC1), enhanced retention of the E6-encoding intron and promoted E6 mRNA production. We also show that HPV16 mRNAs are bound to YTHDC1 in human cells and that YTHDC1 affected splicing of HPV16 E6/E7 mRNAs produced from the episomal form of the HPV16 genome. Finally, we show that HPV16 mRNAs are m6A-methylated in tonsillar cancer cells. In summary, HPV16 mRNAs are methylated in HPV16-infected tonsillar cancer cells and overexpression of m6A-“writer” METTL3, m6A-“eraser” ALKBH5 and the m6A-“reader” YTHDC1 affected HPV16 mRNA splicing, suggesting that m6A plays an important role in the HPV16 gene expression program, at least in cancer cells.

Inducible expression of human papillomavirus-16 L1 capsomeres in the plastomes of Nicotiana tabacum: Transplastomic plants develop normal flowers and pollen.

Human papillomavirus type-16 (HPV-16) is the major HPV type involved in causing cervical cancer among women. The disease burden is high in developing and underdeveloped countries. Previously, the constitutive expression of HPV-16 L1 protein led to male sterility in transplastomic tobacco plants. Here, the HPV-16 L1 gene was expressed in chloroplasts of Nicotiana tabacum under the control of an ethanol-inducible promoter, trans-activated by nucleus-derived signal peptide. Plants containing nuclear component were transformed with transformation vector pEXP-T7-L1 by biolistic gun. The transformation and homoplasmic status of transformed plants was verified by polymerase chain reaction and Southern blotting, respectively. Protein was induced by spraying 5% ethanol for 7 consecutive days. The correct folding of L1 protein was confirmed by antigen-capture ELISA using a conformation-specific antibody. The L1 protein accumulated up to 3μg/g of fresh plant material. The L1 protein was further purified using affinity chromatography. All transplastomic plants developed normal flowers and produced viable seeds upon self-pollination. Pollens also showed completely normal structure under light microscope and scanning electron microscopy. These data confirm the use of the inducible expression as plant-safe approach for expressing transgenes in plants, especially those genes that cause detrimental effects on plant growth and morphology.

In silico prediction of T‐cell and B‐cell epitopes of human papillomavirus type 16 L1 protein

Human papillomavirus type 16 (HPV-16) is one of the most important cause of developing cervical cancer. Therefore, effective epitope-based vaccine design for HPV-16 would be of major medical benefit. The aim of our study was to identify B- and T-cell epitopes of HPV-16 L1 protein. In this study, the HPV-16 L1 gene was isolated from HPV recovered from five vaginal swab samples using specific primers and finally sequenced. The ExPASy translate tool (http://web.expasy.org/translate/) was used to convert nucleotide sequence into amino acid sequence. Bioinformatic analysis was employed to predict suitable B- and T-cell epitopes and immunogenicity, allergenicity, and toxicity of predicted epitopes were then evaluated. Afterward, the selected T-cell epitopes were docked using Molegro Virtual Docker software. The two epitopes 207 AMDFTTLQA215 and 200 MVDTGFGAM208 have showed a very strong binding affinity to HLA-A0201 and HLA-B3501 molecules, respectively. Outcome of B-cell epitope prediction showed that epitope 475 KAKPKFTLGKRK ATPTTSSTSTTAKRKK502 contained overlapped epitope, which might be the epitope associated with the production of neutralizing antibody response. Based on this finding, the predicted B- and T-cell epitopes are promising targets for epitope-based vaccine development against HPV-16. Further in vivo and in vitro experiments are needed to confirm our findings.

Characterization of major capsid protein (L1) variants of Human papillomavirus type 16 by cervical neoplastic status in Indian women: Phylogenetic and functional analysis.

The etiological role of infection with Human papillomavirus type 16 (HPV16) in cervical cancer is well established. HPV16 variants, classified based on less than 10% nucleotide variations in the major capsid (L1 ORF) are known to contribute to persistent infection leading to cancer development. L1 protein forms the cornerstone of HPV structure and antigenicity. In the present study, HPV16 L1 variants were characterized by cervical lesion grade and variations in sequences were correlated to structure and function. The L1 gene was analyzed in 152 HPV16 positive cervical samples obtained from Indian women using polymerase chain reaction-directed sequencing. Phylogenetic analysis was carried out for lineage typing. Sixty-one SNPs were detected in L1 genes resulting in 20 nonsynonymous amino acid substitutions of which N56T, N92T, L158F, V178G, N181I, K236T, K443Q, K454T, and K475R are reported in Indian isolates for the first time. The substitutions N181T, T353P, and T389S were significantly associated with high-grade cervical disease. The predominance of lineage A (A1-A4, 84.96%) was observed among the isolates, while the D3 sublineage showed significant association with high-grade cervical lesions. No evidence for recombination and the positive selection was obtained. These substitutions, when mapped on three-dimensional structure, revealed that 11 and 4 substitutions are part of experimentally validated B- and T-cell epitopes, of which T266A and N285T were common to both types of epitopes and may impact HPV vaccine efficacy. The variants identified through this study have the potential to serve as translational leads for designing diagnostic probes and vaccines.

Neutralization sites of human papillomavirus-6 relate to virus attachment and entry phase in viral infection

ABSTRACT Human papillomavirus type 6 (HPV6) is the major etiologic agent of genital warts and recurrent respiratory papillomatosis. Although the commercial HPV vaccines cover HPV6, the neutralization sites and mode for HPV6 are poorly understood. Here, we identify the HPV6 neutralization sites and discriminate the inhibition of virus attachment and entry by three potent neutralizing antibodies (nAbs), 5D3, 17D5, and 15F7. Mutagenesis assays showed that these nAbs predominantly target surface loops BC, DE, and FG of HPV6 L1. Cryo-EM structures of the HPV6 pseudovirus (PsV) and its immune complexes revealed three distinct binding modalities – full-occupation-bound to capsid, top-center-bound-, and top-rim-bound to pentamers – and illustrated a structural atlas for three classes of antibody-bound footprints that are located at center-distal ring, center, and center-proximal ring of pentamer surface for 5D3, 17D5, and 15F7, respectively. Two modes of neutralization were identified: mAb 5D3 and 17D5 block HPV PsV from attaching to the extracellular matrix (ECM) and the cell surface, whereas 15F7 allows PsV attachment but prohibits PsV from entering the cell. These findings highlight three neutralization sites of HPV6 L1 and outline two antibody-mediated neutralization mechanisms against HPV6, which will be relevant for HPV virology and antiviral inhibitor design. Highlights Major neutralization sites of HPV6 were mapped on the pseudovirus cryo-EM structure mAb 15F7 binds HPV6 capsid with a novel top-rim binding modality and confers a post-attachment neutralization mAb 17D5 binds capsid in top-centre manner but unexpectedly prevents virus from attachment to cell surface

Mapping the B cell epitopes within the major capsid protein L1 of human papillomavirus type 16

Persistent infection with human papillomavirus type16 (HPV16) has much association with the development of cervical cancer. L1 is the major capsid protein of HPV, it has been well investigated as a potential vaccine candidate. However, B cell epitopes present on L1 have not been well characterized. To identify the potential B-cell antigenic epitopes within HPV16 L1 protein, sixteen serial overlapping truncations (H1–H16) covering the whole region were expressed in E. coli and used in mice immunization. The mice antisera were tested in ELISA binding, IFA and HI assays. Finally, four fragments (H2, H4, H11, H12) were found to contain B cell epitopes of HPV16 L1 protein in ELISA and IFA assays, three fragments (H2, H3, H9) might contain neutralizing epitopes of HPV16 L1 protein in HI assay. Among them, H11 and H12 fragments contain B cell epitopes have never been reported before, and H3 was found as hemagglutination inhibition epitope for the first time. This work provides new insights to B cell epitopes on HPV16 L1 protein. Several new epitopes were identified and may provide some guidance for HPV16 subunit vaccine design. The results of this study might open new perspectives on the antibody–antigen reaction and have important implications for the development of epitopes-based protective HPV16 vaccines.

Inhibition of Human Papillomavirus Type 16 Infection Using an RNA Aptamer.

Human papillomavirus type 16 (HPV16) DNA has been found in ∼50% of cervical tumors worldwide. HPV infection starts with the binding of the virus capsid to heparan sulfate (HS) receptors exposed on the surface of epithelial basal layer keratinocytes. Previously, our group isolated a high-affinity RNA aptamer (Sc5c3) specific for HPV16 L1 virus-like particles (VLPs). In this study, we report the inhibition of HPV16 infection by Sc5c3 in a pseudovirus (PsVs) model. 293TT cells were infected by HPV16 PsVs containing the yellow fluorescent protein (YFP) as reporter gene. Incubation of HPV16 PsVs with Sc5c3 before infection resulted in a dose-dependent decrease in YFP fluorescence, suggesting infection inhibition. Aptamer degradation by RNase A restored PsVs infectivity, supporting the previous observation that Sc5c3 aptamer can inhibit infection. VLP mutants with removed HS binding sites were used in binding assays to elucidate the Sc5c3 blocking mechanism; however, no binding difference was observed between wild-type and mutant VLPs, suggesting that pseudoinfection inhibition relies on mechanisms additional to electrostatic HS binding site interaction. A DNA/RNA Sc5c3 version also inhibited HPV PsVs infection, suggesting that a modified, nuclease-resistant Sc5c3 may be used to inhibit HPV16 infection in vivo.

Naturally Occurring Single Amino Acid Substitution in the L1 Major Capsid Protein of Human Papillomavirus Type 16: Alteration of Susceptibility to Antibody-Mediated Neutralization.

Each vaccine for human papillomavirus type 16 (HPV16) has been developed on the basis of a single variant, and whether these vaccines can prevent infection due to naturally occurring variants was not clear. To examine this question, constructs of 39 naturally occurring single amino acid substitutions in L1 were generated for pseudovirion production, based on the analysis of 1204 HPV16 L1 protein sequences from the National Center for Biotechnology Information and Papilloma Virus Episteme. Thirty-one of 39 HPV16 L1 mutants produced infectious pseudovirions that exhibited similar particle-to-infectivity ratios, compared with reference pseudovirions. Twenty-one of 31 pseudovirion-producing mutants showed different susceptibilities to monoclonal antibodies, with 6 resulting in complete loss of reactivity to some of the tested monoclonal antibodies. The vaccinated sera neutralized all 31 variants. Mean neutralization titers of most variants changed by approximately 4-fold, compared with the reference pseudovirions, with the C428W and K430Q mutations displaying 9-fold and 11-fold lower susceptibilities, respectively, to neutralization by the sera than the reference pseudovirions. These results suggest that the current HPV vaccines may not offer equal protection against all of the naturally occurring HPV16 variants discovered so far.

Heterogeneous Nuclear Ribonucleoprotein C Proteins Interact with the Human Papillomavirus Type 16 (HPV16) Early 3′-Untranslated Region and Alleviate Suppression of HPV16 Late L1 mRNA Splicing

In order to identify cellular factors that regulate human papillomavirus type 16 (HPV16) gene expression, cervical cancer cells permissive for HPV16 late gene expression were identified and characterized. These cells either contained a novel spliced variant of the L1 mRNAs that bypassed the suppressed HPV16 late, 5'-splice site SD3632; produced elevated levels of RNA-binding proteins SRSF1 (ASF/SF2), SRSF9 (SRp30c), and HuR that are known to regulate HPV16 late gene expression; or were shown by a gene expression array analysis to overexpress the RALYL RNA-binding protein of the heterogeneous nuclear ribonucleoprotein C (hnRNP C) family. Overexpression of RALYL or hnRNP C1 induced HPV16 late gene expression from HPV16 subgenomic plasmids and from episomal forms of the full-length HPV16 genome. This induction was dependent on the HPV16 early untranslated region. Binding of hnRNP C1 to the HPV16 early, untranslated region activated HPV16 late 5'-splice site SD3632 and resulted in production of HPV16 L1 mRNAs. Our results suggested that hnRNP C1 controls HPV16 late gene expression.

Human Papillomavirus 16 E6 Antibodies in Individuals without Diagnosed Cancer: A Pooled Analysis.

The increasing incidence of oropharyngeal cancer in many developed countries has been attributed to human papillomavirus type 16 (HPV16) infections. Recently, HPV16 E6 serology has been identified as a promising early marker for oropharyngeal cancer. Therefore, characterization of HPV16 E6 seropositivity among individuals without cancer is warranted. A total of 4,666 controls were pooled from several studies of cancer and HPV seropositivity, all tested within the same laboratory. HPV16 E6 seropositive controls were classified as having (i) moderate [mean fluorescent intensity (MFI) ≥ 484 and <1,000] or (ii) high seroreactivity (MFI ≥ 1,000). Associations of moderate and high HPV16 E6 seroreactivity with (i) demographic risk factors; and seropositivity for (ii) other HPV16 proteins (E1, E2, E4, E7, and L1), and (iii) E6 proteins from non-HPV16 types (HPV6, 11, 18, 31, 33, 45, and 52) were evaluated. Thirty-two (0.7%) HPV16 E6 seropositive controls were identified; 17 (0.4%) with moderate and 15 (0.3%) with high seroreactivity. High HPV16 E6 seroreactivity was associated with former smoking [odds ratio (OR), 5.5; 95% confidence interval (CI), 1.2-51.8], and seropositivity against HPV16 L1 (OR, 4.8; 95% CI, 1.3-15.4); E2 (OR, 7.7; 95% CI, 1.4-29.1); multiple HPV16 proteins (OR, 25.3; 95% CI, 2.6-119.6 for three HPV16 proteins beside E6) and HPV33 E6 (OR, 17.7; 95% CI, 1.9-81.8). No associations were observed with moderate HPV16 E6 seroreactivity. High HPV16 E6 seroreactivity is rare among individuals without diagnosed cancer and was not explained by demographic factors. Some HPV16 E6 seropositive individuals without diagnosed HPV-driven cancer, especially those with seropositivity against other HPV16 proteins, may harbor a biologically relevant HPV16 infection.

Characterization of two new monoclonal antibodies against human papillomavirus type 16 L1 protein.

BackgroundHuman papillomavirus type 16 (HPV16) infection is implicated in cervical carcinogenesis. This study aimed to characterize two new monoclonal antibodies (mAbs) against HPV L1 protein.MethodsThe immunocompetence of AE3 and AG7 mAbs for HPV L1 protein was evaluated by Western blot analysis, immunostaining, hemagglutination inhibition assay, and ELISA. The heavy chain variable region (VH) and light chain variable region (VL) of AE3 and AG7 mAbs were sequenced and analyzed.ResultsBoth mAbs specifically recognized HPV16 L1 and virus-like particles (VLPs). Both the affinity and the titer of AE3 mAb were higher than that of AG7. There were differences in sequences in the complementary determining regions (CDR) 2 and 3 of VL, as well as in the CDR1 and CDR3 of VH. The two mAbs have distinct predicted three-dimensional structures.ConclusionsWe characterized two mAbs neutralizing antibodies for HPV L1 protein, which would help develop genetic-engineered neutralizing antibodies against HPV16 for diagnostic and therapeutic purposes.

Suppression of HPV-16 late L1 5′-splice site SD3632 by binding of hnRNP D proteins and hnRNP A2/B1 to upstream AUAGUA RNA motifs

Human papillomavirus type 16 (HPV-16) 5′-splice site SD3632 is used exclusively to produce late L1 mRNAs. We identified a 34-nt splicing inhibitory element located immediately upstream of HPV-16 late 5′-splice site SD3632. Two AUAGUA motifs located in these 34 nt inhibited SD3632. Two nucleotide substitutions in each of the HPV-16 specific AUAGUA motifs alleviated splicing inhibition and induced late L1 mRNA production from episomal forms of the HPV-16 genome in primary human keratinocytes. The AUAGUA motifs bind specifically not only to the heterogeneous nuclear RNP (hnRNP) D family of RNA-binding proteins including hnRNP D/AUF, hnRNP DL and hnRNP AB but also to hnRNP A2/B1. Knock-down of these proteins induced HPV-16 late L1 mRNA expression, and overexpression of hnRNP A2/B1, hnRNP AB, hnRNP DL and the two hnRNP D isoforms hnRNP D37 and hnRNP D40 further suppressed L1 mRNA expression. This inhibition may allow HPV-16 to hide from the immune system and establish long-term persistent infections with enhanced risk at progressing to cancer. There is an inverse correlation between expression of hnRNP D proteins and hnRNP A2/B1 and HPV-16 L1 production in the cervical epithelium, as well as in cervical cancer, supporting the conclusion that hnRNP D proteins and A2/B1 inhibit HPV-16 L1 mRNA production.

HPV16 L1 and L2 DNA methylation predicts high‐grade cervical intraepithelial neoplasia in women with mildly abnormal cervical cytology

DNA methylation changes in human papillomavirus type 16 (HPV16) DNA are common and might be important for identifying women at increased risk of cervical cancer. Using recently published data from Costa Rica we developed a classification score to differentiate women with cervical intraepithelial neoplasia grade 2 or 3 (CIN2/3) from those with no evident high-grade lesions. Here, we aim to investigate the performance of the score using data from the UK. Exfoliated cervical cells at baseline and 6-months follow-up were analyzed in 84 women selected from a randomized clinical trial of women undergoing surveillance for low-grade cytology. Selection of women for the methylation study was based on detectable HPV16 in the baseline sample. Purified DNA was bisulfite converted, amplified and pyrosequenced at selected CpG sites in the viral genome (URR, E6, L1 and L2), with blinding of laboratory personnel to the clinical data. The primary measure was a predefined score combining the mean methylation in L1 and any methylation in L2. At the second follow-up visit, 73/84 (87%) women were HPV16 positive and of these 25 had a histopathological diagnosis of CIN2/3. The score was significantly associated with CIN2/3 (area under curve = 0.74, p = 0.002). For a cutoff with 92% sensitivity, colposcopy could have been avoided in 40% (95% CI 27–54%) of HPV16 positive women without CIN2/3; positive predictive value was 44% (32–58%) and negative predictive value was 90% (71–97%). We conclude that quantitative DNA methylation assays could help to improve triage among HPV16 positive women.

Identification of B cell epitopes reactive to human papillomavirus type-16L1- derived peptides

BackgroundPersistent infection of human papillomavirus (HPV) types 16 and 18 causes cervical cancer. To better understand immune responses to the prophylactic vaccine, HPV 16/18 L1 virus-like particles (HPV-VLPs), we investigated B cell epitopes of HPV16 L1-derived peptides.MethodsSera from mice immunized with HPV-16/18 L1 VLPs were analyzed for their IgG titers against 10 different HPV16 L1-derived peptides (20-mer) that contain human leukocyte antigen (HLA)-class I A-2, A-24 and class II DR.ResultsOne 20-mer peptide at positions 300 to 319 was identified as a common B cell epitope in both Balb/c (H-2d) and C57BL/6 (H-2b) mice. Mapping analysis showed that the 10-amino-acid sequence at positions 304to 313 was an immunogenic portion. It is of note that the binding capability of this 10-mer peptide to the HLA-A2 and HLA-A24 molecules was confirmed by the HLA class I stabilization assay. In addition, one unique 20-mer was determined as a B cell epitope in each strain.ConclusionsThese results might provide new information for better understanding of immune responses to HPV 16 L1.

Influence of Physiologic Folate Deficiency on Human Papillomavirus Type 16 (HPV16)-harboring Human Keratinocytes in Vitro and in Vivo

Although HPV16 transforms infected epithelial tissues to cancer in the presence of several co-factors, there is insufficient molecular evidence that poor nutrition has any such role. Because physiological folate deficiency led to the intracellular homocysteinylation of heterogeneous nuclear ribonucleoprotein E1 (hnRNP-E1) and activated a nutrition-sensitive (homocysteine-responsive) posttranscriptional RNA operon that included interaction with HPV16 L2 mRNA, we investigated the functional consequences of folate deficiency on HPV16 in immortalized HPV16-harboring human (BC-1-Ep/SL) keratinocytes and HPV16-organotypic rafts. Although homocysteinylated hnRNP-E1 interacted with HPV16 L2 mRNA cis-element, it also specifically bound another HPV16 57-nucleotide poly(U)-rich cis-element in the early polyadenylation element (upstream of L2L1 genes) with greater affinity. Together, these interactions led to a profound reduction of both L1 and L2 mRNA and proteins without effects on HPV16 E6 and E7 in vitro, and in cultured keratinocyte monolayers and HPV16-low folate-organotypic rafts developed in physiological low folate medium. In addition, HPV16-low folate-organotypic rafts contained fewer HPV16 viral particles, a similar HPV16 DNA viral load, and a much greater extent of integration of HPV16 DNA into genomic DNA when compared with HPV16-high folate-organotypic rafts. Subcutaneous implantation of 18-day old HPV16-low folate-organotypic rafts into folate-replete immunodeficient mice transformed this benign keratinocyte-derived raft tissue into an aggressive HPV16-induced cancer within 12 weeks. Collectively, these studies establish a likely molecular linkage between poor folate nutrition and HPV16 and predict that nutritional folate and/or vitamin-B(12) deficiency, which are both common worldwide, will alter the natural history of HPV16 infections and also warrant serious consideration as reversible co-factors in oncogenic transformation of HPV16-infected tissues to cancer.

Influence of Codon Bias on Heterologous Production of Human Papillomavirus Type 16 Major Structural Protein L1 in Yeast

Heterologous gene expression is dependent on multistep processes involving regulation at the level of transcription, mRNA turnover, protein translation, and posttranslational modifications. Codon bias has a significant influence on protein yields. However, sometimes it is not clear which parameter causes observed differences in heterologous gene expression as codon adaptation typically optimizes many sequence properties at once. In the current study, we evaluated the influence of codon bias on heterologous production of human papillomavirus type 16 (HPV-16) major structural protein L1 in yeast by expressing five variants of codon-modified open reading frames (OFRs) encoding HPV-16 L1 protein. Our results showed that despite the high toleration of various codons used throughout the length of the sequence of heterologously expressed genes in transformed yeast, there was a significant positive correlation between the gene's expression level and the degree of its codon bias towards the favorable codon usage. The HPV-16 L1 protein expression in yeast can be optimized by adjusting codon composition towards the most preferred codon adaptation, and this effect most probably is dependent on the improved translational elongation.

Genetic variability in the major capsid L1 protein of human papillomavirus type 16 (HPV-16) and 18 (HPV-18)

HPV-16 and HPV-18 infections result in nearly 73% of cervical cancers worldwide. The L1 protein comprising HPV vaccine formulations elicit high-titre neutralizing antibodies. The aim of this study was to detect L1 HPV-16 and HPV-18 gene polymorphisms and analyze intratypic variations. HPV-16 (n=29) and HPV-18 (n=5) L1 gene sequences were obtained from cervical samples harvested from Italian women. Phylogenetic trees were constructed using the Neighbor-Joining and the Kimura 2-parameters methods (MEGA software). To estimate selection pressures acting on the L1 gene, codon-specific non-synonymous (dN) and synonymous (dS) substitutions were inferred using the Nei–Gojobori method and Jukes–Cantor model (MEGA software) and integrated analyses carried out using SLAC, FEL and REL methodologies. All the HPV-16 L1 sequences analyzed fell into the European branch (99.4–99.7% similarity). Thirty-four single nucleotide changes were observed and 18 (52.9%) were non-synonymous mutations (7/18 were identified in sequences encoding an immunodominant loop and one occurred in the sequence encoding the α-4 domain associated with VLP conformation). There was no evidence of positive selection in the sequence alignment of L1 HPV-16 genes (P-value<0.1). One mutation was identified in a negatively selected codon. HPV-18 L1 analyzed sequences fell into two phylogenetic branches: the HPV-18 European branch (99.5–100% similarity) and the HPV-18 African branch (99.8% similarity). Nine single nucleotide changes were observed and 4/9 (44.5%) of these nucleotide mutations were non-synonymous and one was present in a sequence encoding the immunodominant FG loop. There was no evidence of positive selection in the sequence alignment of L1 HPV-18 genes (P-value<0.1). This study identified polymorphisms of undefined biological activity in HPV-16 and HPV-18 L1 sequences. Information regarding the genetic diversity of HPV-16 and HPV-18 L1 gene sequences may help define the oncogenic potential of respective strains and to better understand immune escape mechanisms.

Serine/arginine-rich protein 30c activates human papillomavirus type 16 L1 mRNA expression via a bimodal mechanism

Two splice sites on the human papillomavirus type 16 (HPV-16) genome are used exclusively by the late capsid protein L1 mRNAs: SD3632 and SA5639. These splice sites are suppressed in mitotic cells. This study showed that serine/arginine-rich protein 30c (SRp30c), also named SFRS9, activated both SD3632 and SA5639 and induced production of L1 mRNA. Activation of HPV-16 L1 mRNA splicing by SRp30c required an intact arginine/serine-repeat (RS) domain of SRp30c. In addition to this effect, SRp30c could enhance L1 mRNA production indirectly by inhibiting the early 3'-splice site SA3358, which competed with the late 3'-splice site SA5639. SRp30c bound directly to sequences downstream of SA3358, suggesting that SRp30c inhibited the enhancer at SA3358 and caused a redirection of splicing to the late 3'-splice site SA5639. This inhibitory effect of SRp30c was independent of its RS domain. These results suggest that SRp30c can activate HPV-16 L1 mRNA expression via a bimodal mechanism: directly by stimulating splicing to late splice sites and indirectly by inhibiting competing early splice sites.

Maltosylated polyethylenimine-based triple nanocomplexes of human papillomavirus 16L1 protein and DNA as a vaccine co-delivery system

To improve vaccine delivery, we herein designed a co-delivery system using a protein antigen and its encoding plasmid linked in nanocomplexes via maltosylated PEI (mPEI). Cationic mPEI was electrostatically complexed to a plasmid encoding the human papillomavirus (HPV) type 16L1 protein (pHPV16L1), and further complexed to a maltose binding protein (MBP)-fused human papillomavirus type 16L1 fusion protein (HPV16L1-MBP). The HPV16L1-MBP/mPEI/pHPV16L1 complexes were characterized by gel-retardation properties, zeta potentials and sizes. The intracellular co-delivery of protein and plasmid DNA vaccines was significantly higher for mPEI-based triple nanocomplexes than for a simple physical mixture of the proteins and DNA. Moreover, the cellular delivery of plasmid DNA using mPEI-based triple nanocomplexes resulted in higher expression levels comparable to those obtained using dual complexes of mPEI and the plasmid DNA. In vivo, co-immunization of mice with HPV16L1-MBP/mPEI/pHPV16L1 nanocomplexes triggered the highest levels of humoral immune responses among various vaccination groups. Moreover, the mPEI-based nanocomplexes significantly enhanced the number of interferon-γ producing CD8 + T cells compared with the use of mixed proteins and plasmid DNA. These results suggest that the effective cellular co-delivery of MBP-fused antigen proteins and plasmid DNA using maltosylated PEI-based triple nanocomplexes could enhance the immunogenicity of HPV16L1 vaccines.