HPV L1 Protein Research Articles

Myosin 9 and N-glycans jointly regulate human papillomavirus entry

Persistent high-risk HPV infection is closely associated with cervical cancer development, and there is no drug targeting HPV on the market at present, so it is particularly important to understand the interaction mechanism between HPV and the host which may provide the novel strategies for treating HPV diseases. HPV can hijack cell surface heparan sulfate proteoglycans (HSPGs) as primary receptors. However, the secondary entry receptors for HPV remain elusive. We identify myosin-9 (NMHC-IIA) as a host factor that interacts with HPV L1 protein and mediates HPV internalization. Efficient HPV entry required myosin-9 redistribution to the cell surface regulated by HPV-hijacked MEK-MLCK signaling. Myosin-9 maldistribution by ML-7 or ML-9 significantly inhibited HPV pseudoviruses infection invitro and invivo. Meanwhile, N-glycans, especially the galactose chains, may act as the decoy receptors for HPV, which can block the interaction of HPV to myosin-9 and influence the way of HPV infection. Taken together, we identify myosin-9 as a novel functional entry receptor for high-risk HPV both invitro and invivo, and unravel the new roles of myosin-9 and N-glycans in HPV entry, which provides the possibilities for host targets of antiviral drugs.

Simultaneous Detection of the HPV L1 Gene and the Human β-Globin Gene in the Blood Components of Cervical Cancer Patients Living in Yakutia

Background: To create a test for the early detection of cervical cancer (CC), we conducted exploratory studies on detecting HPV genes and genes of the human β-globin locus in plasma (Pl) and RBC suspension (RBCsus) samples from patients with newly detected CC (NDCC). Methods and Results: Smears of venous blood containing K3-EDTA from five anonymous patients aged from 45 to 55 years (residents of Yakutia), with NDCC were obtained. Three types of blood component samples were prepared – plasma (Pl), red blood cell (RBC) suspension (RBCsus), and the erythrocyte fraction treated with trypsin (RBCsus-Try). To detect circulating cell-free DNA (cfDNA) in NDCC patients, we studied the presence of genes corresponding to the HPV L1 protein region and genes of the human β-globin locus by real-time PCR (qPCR) using appropriate primers. The genes of β-globin locus and HPV L1 were detected in Pl of NDCC patients in 20% of cases, and in RBCsus in 60% of cases. The amplified gene products using primers were present in RBCsus-Try in only one patient (20% of cases). In patients with uncertain amplification, electrophoresis showed the absence of amplified products in Pl and their presence in RBCsus. Conclusion: In NDCC patients, HPV L1 and β-globin genes can be detected in both Pl and RBCsus. In addition, in RBC samples, these genes were detected more often than in plasma samples, and no absolute absence of amplification products was observed in RBC samples. The research needs to be continued.

Engineering for an HPV 9-valent vaccine candidate using genomic constitutive over-expression and low lipopolysaccharide levels in Escherichia coli cells

BackgroundThe various advantages associated with the growth properties of Escherichia coli have justified their use in the production of genetically engineered vaccines. However, endotoxin contamination, plasmid vector instability, and the requirement for antibiotic supplementation are frequent bottlenecks in the successful production of recombinant proteins that are safe for industrial-scaled applications. To overcome these drawbacks, we focused on interrupting the expression of several key genes involved in the synthesis of lipopolysaccharide (LPS), an endotoxin frequently responsible for toxicity in recombinant proteins, to eliminate endotoxin contamination and produce better recombinant proteins with E. coli.ResultsOf 8 potential target genes associated with LPS synthesis, we successfully constructed 7 LPS biosynthesis-defective recombinant strains to reduce the production of LPS. The endotoxin residue in the protein products from these modified E. coli strains were about two orders of magnitude lower than that produced by the wild-type strain. Further, we found that 6 loci—lpxM, lpxP, lpxL, eptA, gutQ and kdsD—were suitable for chromosomal integrated expression of HPV L1 protein. We found that a single copy of the expression cassette conferred stable expression during long-term antibiotic-free cultivation as compared with the more variable protein production from plasmid-based expression. In large-scale fermentation, we found that recombinant strains bearing 3 to 5 copies of the expression cassette had 1.5- to 2-fold higher overall expression along with lower endotoxin levels as compared with the parental ER2566 strain. Finally, we engineered and constructed 9 recombinant E. coli strains for the later production of an HPV 9-valent capsid protein with desirable purity, VLP morphology, and antigenicity.ConclusionsReengineering the LPS synthesis loci in the E. coli ER2566 strain through chromosomal integration of expression cassettes has potential uses for the production of a 9-valent HPV vaccine candidate, with markedly reduced residual endotoxin levels. Our results offer a new strategy for recombinant E. coli strain construction, engineering, and the development of suitable recombinant protein drugs.

Worldwide genetic variations in high-risk human papillomaviruses capsid L1 gene and their impact on vaccine efficiency.

Human papillomavirus is the most common sexually transmitted infection. It is associated with different cancers, mainly cervical cancer, which remains the fourth most frequent cancer among women worldwide; it is also related to anogenital (anus, vulvar, vagina, and penis) and oropharyngeal cancers. Vaccination against HPV infection is the major way of prevention, and it has demonstrated impressive efficacy in reducing cervical cancer incidence. Nowadays, all the licensed HPV recombinant vaccines were designed based on HPV major capsid L1 protein. However, some variations in the HPV L1 gene sequence may induce structural changes within the L1 protein, which may alter the affinity and interaction of monoclonal antibodies (MAbs) with L1 protein epitopes, and influence host immune response and recognition. Hence, the importance of accuracy in delineating epitopes relevant to vaccine design and defining genetic variations within antigenic regions in the L1 gene to predict its impact on prophylactic vaccine efficiency. The present review reports the sequence variations in HR-HPV L1 gene isolates from different countries around the world, which may help to understand the effect of HPV L1 gene variations on vaccine efficiency. Research studies were retrieved from PubMed, Google Scholar, Science direct, and the National Center for Biotechnology Information (NCBI) database. A total of 31 articles describing genetic variations within the major capsid L1 gene and conducted in Africa, Europe, America and Asia were found. Only 26 studies conducted on HPV16, 18, 31, 33, 58, 45 and 52 which are the targets of HPV prophylactic vaccines, and which reported genetic variations within the L1 gene, were selected and evaluated in this review. We found a total of 87, 49, 11, 7, 22, 3, and 17 non-synonymous single nucleotide polymorphisms (SNPs) within HPV16, HPV18, HPV31, HPV58, HPV45, and HPV52 L1 gene, respectively. Four mutations were frequently observed in HPV16 L1 sequences: T353P in the HI loop, H228D in the EF loop, T266A in the FG loop, and T292A in the FG loop. Two mutations in HPV58 L1 sequences: T375N in the HI loop and L150F in the DE loop. Three mutations in HPV33 L1 sequences: T56N in the BC loop, G133S in the DE loop, T266K in the FG loop. Other mutations were found in HPV18, HPV45, and HPV52 L1 sequences. Some were found in different countries, and others were specific to a given population. Furthermore, some variations were located on peptide binding epitopes and lead to a modification of epitopes, which may influence MAbs interactions. Others need further investigations due to the lack of studies. This study investigated the major capsid L1 genetic diversity of HPV16, 18, 31, 33, 58, 45, and 52 circulating in different populations around the world. Further investigations should be conducted to confirm their effect on immunogenicity and prophylactic vaccine efficiency.

Competitive adsorption on gold nanoparticles for human papillomavirus 16 L1 protein detection by LDI-MS.

The detection of the HPV L1 protein provides information about the infection status of the virus, reflects the replication status of the HPV virus in cervical cells, and helps understand the regression and progress of cervical lesions. Herein, we report a novel laser desorption ionization mass spectrometry (LDI MS) method for the sensitive detection of the HPV 16 L1 protein, based on non-covalent competitive adsorption between the HPV 16 L1 aptamer and melamine on gold nanoparticles (AuNPs). The intensity of the MS signal corresponding to the mass tag shows a linear relationship with the HPV 16 L1 concentration in the range 2-80 ng mL-1, with a limit of detection (LOD) of 58.8 pg mL-1. Using this method, the HPV 16 L1 protein is quantitatively analyzed in both clinical and vaccine samples. The described method is simple and has high sensitivity and good reliability.

Development and characterization of an HPV18 detection kit using two novel HPV18 type-specific monoclonal antibodies

BackgroundHPV 18 is one of the most prevalent oncogenic types, only second to HPV 16, and included in the licensed vaccines on the market. In this study, we describe the production and characterization of a panel of monoclonal antibodies (mAb) to HPV18.MethodsThe immunocompetence of 1B1 and 4C2 mAbs for HPV L1 protein was evaluated by SDS-PAGE analysis, neutralization assays, affinity identification, and ELISA. The 1B1 and 4C2 genes were sequenced and analyzed. Finally, the detection kit with the two mAbs was assessed for linearity, repeatability and specificity.ResultsBoth mAbs specifically recognized HPV18 L1 and virus-like particles (VLPs). These mAbs are conformation-neutralizing antibodies that have high affinity and type specificity. Based on these characteristics of these mAbs, we developed an ELISA kit for specifically detecting HPV 18 antigen. We showed that this kit displayed good linearity, repeatability and sensitivity for detecting HPV18 L1 pentamer and HPV18 VLP.ConclusionsWe characterized two monoclonal neutralizing antibodies for HPV L1 protein, and developed an ELISA kit for specifically detecting HPV 18 antigen. This newly developed kit can be used to monitor the potency of HPV vaccines throughout the entire production process as well as preliminary analysis of HPV18 infections.

Naturally occurring capsid protein variants L1 of human papillomavirus genotype 16 in Morocco.

HPV L1 protein is a corner stone in HPV structure, it's involved in the formation of the viral capsid; widely used as a systematic material and considered as the main component in vaccines development and production. The present study aims to characterize genetic variation of L1 gene of HPV 16 specimens and to evaluate in silico the impact of major variants on the epitope change affecting its conformational structure. A fragment of L1 gene from 35 HPV 16 confirmed specimens were amplified by PCR and sequenced. Overall, five amino acids residues changes were reported: T390P in 16 specimens, M425I and M431I in 2 cases, insertion of Serine at 460 and aspartic acid deletion at position 477 in all analyzed cases. The 3D generated model showed that T389P amino acid substitution is located in the H-I loop; the two substitutions M424I and M430I are both located in the H2 helice. The Serine insertion and aspartic acid deletion are located in the H4 helice and B-C loop, respectively. Superimposition of sequences' structures showed that they share a very similar conformation highlighting that the reported amino acids variations don't affect the structure of the L1 protein. However T389P, located in the H-I loop identified as an immunogenetic region of L1 capsid, was reported in 51.4% of cases could interact with vaccines induced monoclonal antibodies suggesting a potential impact on the efficacy of available anti-HPV vaccines.

Development of a bio-electrochemical immunosensor based on the immobilization of SPINNTKPHEAR peptide derived from HPV-L1 protein on a gold electrode surface

A novel bio-electrochemical immunosensor for sensitive and selective detection of anti-peptide antibodies of HPV was fabricated by immobilization of a peptide (SPINNTKPHEAR) (1) linked to a 6-aminohexanoic (Ahx) residue and ferrocene (Fc) on a gold electrode surface, used as sensing interface. The Fc–Ahx–peptide (Fc–Ahx–SPINNTKPHEAR) (1AFc) was obtained by solid phase peptide synthesis by using the Fmoc/tBu strategy. Peptides were purified by preparative HPLC, characterized by MS, MALDI-TOF, and Circular Dichroism and their purity was evaluated by using analytical HPLC. The electrochemical behavior of the modified electrode was examined by cyclic voltammetry in phosphate buffer solution (PBS) in order to evaluate the redox behavior of the ferrocene moiety. The influence of anti-peptide antibodies on the voltammetric response of the modified electrode was investigated by comparing results obtained with pre-immune (control) and post immune serum samples at similar dilution factor. Changes in such behavior upon addition of the serum samples to PBS suggested that the fabricated bioelectrochemical sensor was able to recognize the interaction between anti-peptide antibodies and the immobilized peptide (1AFc) with high selectivity and sensitivity. Such influence increased as the dilution decreased, but the effect was less pronounced at relatively high concentrated solutions owing to the effect of the sample matrix. Notwithstanding, the proposed biosensor can clearly detect the target anti-peptide antibodies at a significant large concentration range.

A fluorescence-enhanced inorganic probe to detect the peptide and capsid protein of human papillomavirus in vitro

A europium-substituted polyoxometalate (EuW10) could be used as a fluorescence-enhanced probe to detect the recombinant HPV L1 protein in vitro.

Carcinogenic HPV infection in the cervical squamo-columnar junction.

Recent studies have suggested the involvement of a unique population of cells at the cervical squamo-columnar junction (SCJ) in the pathogenesis of early (squamous intraepithelial lesion or SIL) and advanced (squamous cell and adeno-carcinomas) cervical neoplasia. However, there is little evidence to date showing that SCJ cells harbour carcinogenic HPV or are instrumental in the initial phases of neoplasia. This study was designed to (1) determine if normal-appearing SCJ cells contained evidence of carcinogenic HPV infection and (2) trace their transition to early SIL. Sections of cervix from high-risk reproductive age women were selected and SCJ cells were analysed by using several techniques which increasingly implicated HPV infection: HPV DNA (genotyping and in situ hybridization)/RNA (PCR), immunostaining for HPV16 E2 (an early marker of HPV infection), p16(ink4), Ki67, and HPV L1 protein. In 22 cases with a history of SIL and no evidence of preneoplastic lesion in the excision specimen, HPV DNA was isolated from eight of ten with visible SCJ cells, six of which were HPV16/18 DNA-positive. In five of these latter cases, the SCJ cells were positive for p16(ink4) and/or HPV E2. Transcriptionally active HPV infection (E6/E7 mRNAs) was also detected in microdissected SCJ cells. Early squamous atypia associated with the SCJ cells demonstrated in addition diffuse p16(ink4) immunoreactivity, elevated proliferative index, and rare L1 antigen positivity. We present for the first time direct evidence that normal-appearing SCJ cells can be infected by carcinogenic HPV. They initially express HPV E2 and their progression to SIL is heralded by an expanding metaplastic progeny with increased proliferation and p16(ink4) expression. Whether certain SCJs are more vulnerable than others to carcinogenic HPV genotypes and what variables determine transition to high-grade SIL remain unresolved, but the common event appears to be a vulnerable cell at the SCJ.

A coding variant in TMC8 (EVER2) is associated with high risk HPV infection and head and neck cancer risk.

HPV infection is a causal agent in many epithelial cancers, yet our understanding of genetic susceptibility to HPV infection and resultant cancer risk is limited. Epidermodysplasia Verruciformis is a rare condition of extreme susceptibility to cutaneous HPV infection primarily attributable to mutations in TMC6 and TMC8. Genetic variation in the TMC6/TMC8 region has been linked to beta-type HPV infection and squamous cell carcinoma of the skin, cervical cancer, HPV persistence and progression to cervical cancer. Here, we have tested the hypothesis that the common TMC8 SNP rs7208422 is associated with high-risk HPV infection and risk of head and neck squamous cell carcinoma (HNSCC). Seropositivity to the HPV L1 protein (HPV16, 18, 11, 31, 33, 35, 45, 52, 58) was measured in 514 cases and 452 population-based controls. Genotype was significantly associated with seropositivity to HPV18 L1 (OR TT vs AA = 0.48, 95% CI = 0.22–0.99) and borderline significantly associated with HPV16 L1 (OR TT vs AA = 0.58, 95% CI = 0.22–1.17). There was a consistent inverse association between TMC8 genotype and infection with other HPV types, including statistically significant associations for HPV31 and HPV52. Consistent with these results, the variant T genotype was associated with a reduced risk of HNSCC (ORAT: 0.63, 95% CI 0.45–0.89, ORTT: 0.54, 95% CI 0.36–0.81), even among subjects seronegative for all HPV types (ORAT: 0.71, 95% CI 0.45–1.11, ORTT: 0.54, 95% CI 0.31–0.93). Our data indicate that common genetic variation in TMC8 is associated with high-risk HPV infection and HNSCC etiology.

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.

Expression of human papillomavirus is correlated with Ki-67 and COX-2 expressions in keratocystic odontogenic tumor.

The aim of the current study was to investigate the presence of human papillomavirus (HPV) and evaluate its association with Ki-67 and cyclooxygenase-2 (COX-2) expressions in keratocystic odontogenic tumor (KCOT). Nineteen cases were included in the present study. Conventional PCR method and immunohistochemical analysis were performed for the detection of HPV-DNA and HPV-L1 capsid protein. Moreover, the expressions of Ki-67 and COX-2 proteins were analyzed immunohistochemically. HPV-DNA was detected in 36.8% (7/19) of tumor samples, whilst HPV-L1 protein was identified in 68.4% (13/19) of them. The Kappa coefficient statistical test showed a moderate agreement (κ 0.424) between PCR and IHC assays for HPV detection. Expression of HPV-DNA was positively correlated with Ki-67 and COX-2 expressions (p < 0.05), whereas HPV-L1 positive staining was positively correlated with COX-2 (p < 0.05) and highly associated with those of Ki-67 (p < 0.01). There was no significant correlation between the presence of HPV and the recurrence of the studied lesions. The results of the current study showed that active HPV infection was present in the odontogenic epithelium of KCOT, and it was associated with increased proliferation rate and COX-2 expression. These findings suggest that HPV may have a role in the pathogenesis and aggressiveness of KCOT. Based on these conclusions, we recommend further investigations of HPV vaccine or antiviral therapy and COX-2 inhibitors as nonsurgical options in the prevention and management of KCOT.

Expression pattern of HPV L1 in different cervical lesion tissues

Objective To explore the factors influencing the HPV L1 protein expression in tumor tissues and to understand the expression pattern of HPV L1 protein in different cervical lesion tissues.Methods Different cervical lesion tissues were collected to detect genotypes and genome physical states of HPV 16 and 18.ELISA,Western blotting analysis,and immunohistochemical staining were used to examine the expression of HPV L1 protein in the tissues.Results A total of 48 specimens were collected,with 42 being HPV16positive and 3being HPV18 positive.In HPV16 positive specimens,25 harbored exclusively the episomal form,7carried mixed forms,and 10 had integrated type.All the HPV18 positive specimens belonged to the integrated type.The results of ELISA and Western blotting analysis displayed that HPV L1 expression decreased with the aggravation of the lesion severities.The immunohistochemistry findings showed that L1 positive reactions were of different forms and varied with different physical states of viral genome,differentiation degrees,and progression of lesion tissues.Conclusion HPV L1 expression in cervical lesion tissues has its structural foundation,and the expression levels are closely related to many factors,including the physical state of virus genome in lesion tissues,differentiation degrees of tumor tissue,and the state of virus gene sequence being inserted into host ones.

Effectiveness of Toki s criteria and determination of variables for identification of HPV L1 protein in oral lesions

Objectives: To evaluate the effectiveness of Toki’s criteria in identifying the HPV L1 protein in oral lesions with the use of immunohistochemistry (IHC) and to determine which criteria optimize such identification. Study Design: Retrospective study of 277 cases diagnosed as HPV lesions at 22 years. Tests of sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV), kappa coefficients, and chi2 values, as well as two logistic regression analyses (p≤0.05), were conducted. Results: Of the lesions studied, 96.4% (267 of 277) were positive for HPV using Toki’s criteria and 28.5% (79 of 277) were positive for L1 by IHC. Toki’s criteria showed sensitivity=93.67%, specificity=2.53%, PPV=6.99%, and NPV=46.55%. Neither concordance nor statistically significant associations were observed between both tests. The logistic regression of Toki’s criteria was useful in the diagnosis of L1, correctly classified 71.8% of the lesions positive for L1, and showed a Hosmer-Lemeshow adjustment of p=0.614 and a Nagelkerke’s coefficient of determination of 6.8%. The explanatory variables statistically significant at p≤0.05 were dyskeratosis (p=0.01) and papillomatosis (p=0.04). Forty-nine independent variables (clinical and histopathologic) were involved in the second regression analysis. The model correctly classified 85.2% of the lesions and showed a Hosmer-Lemeshow adjustment of p=0.696 and a Nagelkerke’s coefficient of determination of 60.2%. The explanatory variables statistically significant atp≤0.05 were: age younger than 35 years (p=0.001), multiple lesions (p=0.031), hyperorthokeratosis (p=0.019), focal intracellular edema (p=0.002), and the presence of 1 to more than 5 cells with degenerative changes in their nucleus (p=0.048). Conclusions: Toki’s criteria are not adequate to make a diagnosis of lesions by HPV in the mouth, but the logistic regression analysis showed clinical and histopathologic variables which optimize the identification of lesions through the L1 protein. However, a PCR study is advisable when the presence of high-risk HPV is suspected. Key words:HPV, Toki’s criteria.

English

p16 is a tumor suppressor protein and also a cyclin-dependent kinase inhibitor and HPV-L1 (L1) is a capsidic protein that is expressed in the early, productive phase of cervical carcinogenesis. To provide evidence for the evaluation of endometrial intraepithelial neoplasia (CIN), the expressions of p16 and HPVL1 in different cervical diseases were determined and their relationship was explored. Immunohistochemistry was performed to detect the expressions of p16 and HPVL1 in women positive for HPV among whom 38, 32, 28, 33 and 10 had CIN I, CIN II, CIN III, cervical squamous cell carcinoma (SCC) and cervical adenocarcinoma, respectively. Fourteen healthy subjects were recruited as controls. Results showed the positive rate of p16 in cervicitis, CIN I, CIN II, CIN III,cervical SCC and cervical adenocarcinoma patients was 0, 26.3, 81.2, 96.4, 100 and 90%, respectively, and that of HPVL1 was 100, 65.8, 13.8, 0, 0 and 0%, respectively. p16(-)/HPVL1(+) was frequently found in patients with cervicitis or cervical lesions of low grade and p16(+)/HPVL1(-) was often noted in women with CIN III or cervical cancer. The cervical lesions of patients with p16(-)/HPVL1(-) or p16(+)/HPVL1(+) might have no progression or undergo degeneration. Detection of p16 and HPVL1 expressions plays an important role in the prediction of early cervical cancer and its progression. Key words: Cervical tumor, cervical intraepithelial neoplasia, human papilloma virus, HPVL1 protein, p16.

HPV L1 capsid protein expression in squamous intraepithelial lesions of cervix uteri and its relevance to disease outcome

The purpose of this study was to investigate the usefulness of immunocytochemical detection of HPV L1 capsid protein expression in predicting the course of cervical intraepithelial neoplasia. It is known that most of the low grade dysplastic lesions of cervix uteri regress spontaneously and only some will progress to high grade dysplastic lesions. HPV L1 capsid protein represents about 90% of the total protein on the surface of the virus and can be detected in mild to moderate dysplasia and rarely in severe dysplasia. Pap smears from 65 women, in whom diagnoses of LSIL (n = 43) and HSIL (n = 22) were made on cytology and histology specimens, were immunocytochemically stained using antibody against HPV L1capsid protein. The results of immunocytochemical analysis were correlated with the outcome during the 24-month follow-up. p value <0.05 was considered significant. The immunostaining reaction for L1 capsid protein was positive in 28 cases (65.1%) of LSIL while 15 (34.9%) cases of LSIL and all of the 22 cases of HSIL were negative (p < 0.001). After 24 months of follow-up, among the 28 L1-positive LSIL cases, we found a 60.7% (17/28) spontaneous regression rate, whereas in the 15 L1-negative LSIL patients, the regression rate was 33.3% (5/15). Out of the 22 HSIL cases, 13.6% (3/22) had regression. Our data support that immunocytochemical detection of HPV-L1 protein could present prognostic information about the evolution of early dysplastic cervical lesions and can be useful in predicting their biologic potential.

Expression of human papillomavirus type 16 (HPV16) L1 protein in Pichia pastoris

To investigate the production of human papillomavirus type 16 (HPV16) L1 structural protein in Pichia pastoris , the effect of different gene constructs and varying cultivation conditions on production levels were assessed. The highest volumetric production levels in P. pastoris were obtained with a human codon-optimised gene construct. This construct displayed both the lowest codon adaptation index (CAI) and the highest guanine-cytosine (GC) content of the three constructs investigated. Dissolved oxygen (DO) controlled cultivations produced superior results compared to a constant methanol feeding (CMF) strategy, delivering the highest volumetric HPV L1 protein concentration (104.8 µg.l -1 ) at a 20% DO set-point. This was concomitant with an increased biomass concentration in the DO controlled cultivations.

Etiologic role of human papillomavirus infection in bladder carcinoma

The authors elucidated an etiologic role of human papillomavirus (HPV) infection in carcinoma of the bladder. One hundred seventeen of 224 patients with bladder carcinoma who were treated between 1997 and 2009 were enrolled in this study. The presence of HPV DNA was tested on frozen carcinoma tissues that were obtained by transurethral resection using a polymerases chain reaction-based method. Localization of HPV was observed on archival tissue specimens by in situ hybridization (ISH) for high-risk HPV DNA. Cyclin-dependent kinase (CDK) inhibitor 2A (inhibits CDK4) (p16-INK4a) and minichromosome maintenance protein-7 (mcm-7)-surrogate markers for high-risk HPV-E7 oncoprotein-and HPV-L1 (capsid) protein expression were evaluated by immunohistochemistry. HPV types 16, 18, 31, 33, 52, and 58, and an unknown HPV type were detected in 18 of 117 samples (15%) from patients with bladder carcinoma. HPV16 was identified in 6 samples, HPV18 was identified in 4 samples, and HPV33 was identified in 3 samples. All were single HPV type infections. HPV was detected in 38% (12 of 28) of histologic grade 1 bladder carcinomas, 8.5% (6 of 71) of grade 2 bladder carcinomas, and in 0% (0 of 18) of grade 3 bladder carcinomas. Multivariate analysis indicated that younger age (<60 years; odds ratio [OR], 10.9; 95% confidence interval [CI], 2.6-45.3) and grade 1 tumors (OR, 4.5; 95% CI, 1.2-17.0) were associated with HPV infection. ISH analysis indicated that high-risk HPV DNA was localized in the nuclei of tumor cells of all HPV-positive samples. p16-INK4a and mcm-7 were expressed in 94% and 89% of HPV-positive carcinoma cells, respectively. HPV-L1 protein expression, which suggested reproductive HPV infection, was not observed in any carcinoma. The current results indicated that high-risk HPV is likely to be a causative agent of some low-grade bladder carcinomas that develop in younger patients.

An optimized yeast cell-free system: Sufficient for translation of human papillomavirus 58 L1 mRNA and assembly of virus-like particles

We developed a yeast cell-free system suitable for in vitro translation of human papillomavirus 58 (HPV58) L1 mRNA. This system was systematically optimized resulting in enhanced translation efficiency. The optimal concentrations of potassium and magnesium ions observed were specific to the HPV58 L1 protein production. Supplementation with sucrose in the preparation of the yeast lysate greatly enhanced its stability. After optimization, protein production in this system was significantly superior to that produced by the rabbit reticulocyte (RRL) system. Finally, we demonstrated for the first time that virus-like particles (VLPs) were assembled from HPV58 L1 capsid protein in the yeast cell-free system. Thus, the system described here is a powerful tool for the HPV L1 protein production and will be useful for the study of VLP assembly and DNA encapsulation.