Differences in transcriptional activity of human papillomavirus type 6 molecular variants in recurrent respiratory papillomatosis

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Nucleotide and phylogenetic analysis of human papillomavirus types 6 and 11 isolated from recurrent respiratory papillomatosis in Brazil

There are few studies about the distribution of natural molecular variants of low-risk HPVs. Our aim was to evaluate the E6 early gene variability among HPV-6 and HPV-11 isolates detected in recurrent respiratory papillomatosis (RRP) samples obtained in a cohort of Brazilian patients. We also performed a phylogenetic analysis in order to compare nucleotide sequences identified in our study with previously reported isolates from different anatomic sites (laryngeal papillomas, genital warts, cervical cancer and anal swabs) obtained from other parts of the world to determine the phylogenetic relationships of variants detected in Brazil. The complete coding region of the E6 gene of 25 samples was cloned and sequenced: 18 isolates of HPV-6 (72%) and 7 isolates of HPV-11 (28%). A total of four different HPV-6 genomic variants and two HPV-11 genomic variants was identified. It was not possible to correlate specific variants with disease severity. Phylogenetic trees for both HPV types were constructed enclosing both E6 sequences detected in our study and formerly published sequences. In both phylogenetic trees, the sequences from Brazil did not group together. We could not establish a geographical association between HPV-6 or HPV-11 variants, unlike HPV-16 and HPV-18. © 2013 Elsevier B.V.

Post Hoc Analysis Of The Patricia Randomized Trial Of The Efficacy Of Human Papillomavirus Type 16 (hpv-16)/hpv-18 As04-adjuvanted Vaccine Against Incident And Persistent Infection With Nonvaccine Oncogenic Hpv Types Using An Alternative Multiplex Type-specific Pcr Assay For Hpv Dna.

The efficacy of the human papillomavirus type 16 (HPV-16)/HPV-18 AS04-adjuvanted vaccine against cervical infections with HPV in the Papilloma Trial against Cancer in Young Adults (PATRICIA) was evaluated using a combination of the broad-spectrum L1-based SPF10 PCR-DNA enzyme immunoassay (DEIA)/line probe assay (LiPA25) system with type-specific PCRs for HPV-16 and -18. Broad-spectrum PCR assays may underestimate the presence of HPV genotypes present at relatively low concentrations in multiple infections, due to competition between genotypes. Therefore, samples were retrospectively reanalyzed using a testing algorithm incorporating the SPF10 PCR-DEIA/LiPA25 plus a novel E6-based multiplex type-specific PCR and reverse hybridization assay (MPTS12 RHA), which permits detection of a panel of nine oncogenic HPV genotypes (types 16, 18, 31, 33, 35, 45, 52, 58, and 59). For the vaccine against HPV types 16 and 18, there was no major impact on estimates of vaccine efficacy (VE) for incident or 6-month or 12-month persistent infections when the MPTS12 RHA was included in the testing algorithm versus estimates with the protocol-specified algorithm. However, the alternative testing algorithm showed greater sensitivity than the protocol-specified algorithm for detection of some nonvaccine oncogenic HPV types. More cases were gained in the control group than in the vaccine group, leading to higher point estimates of VE for 6-month and 12-month persistent infections for the nonvaccine oncogenic types included in the MPTS12 RHA assay (types 31, 33, 35, 45, 52, 58, and 59). This post hoc analysis indicates that the per-protocol testing algorithm used in PATRICIA underestimated the VE against some nonvaccine oncogenic HPV types and that the choice of the HPV DNA testing methodology is important for the evaluation of VE in clinical trials. (This study has been registered at ClinicalTrials.gov under registration no. NCT00122681.).

Human papillomavirus type 6b virus-like particles are able to activate the Ras-MAP kinase pathway and induce cell proliferation

The initial step in viral infection is the attachment of the virus to the host cell via an interaction with its receptor. We have previously shown that a receptor for human papillomavirus is the alpha6 integrin. The alpha6 integrin is involved in the attachment of epithelial cells with the basement membrane, but recent evidence suggests that ligation of many integrins results in intracellular signaling events that influence cell proliferation. sere we present evidence that exposure of A431 human epithelial cells to human papillomavirus type 6b L1 virus-like particles (VLPs) results in a dose-dependent increase in cell proliferation, as measured by bromodeoxyuridine incorporation. This proliferation is Lost if VLPs are first denatured or incubated with a monoclonal antibody against L1 protein. The MEK1 inhibitor PB98059 inhibits the VLP-mediated increase in fell proliferation, suggesting involvement of the Ras-MAP kinase pathway, Indeed, VLP binding results in rapid phosphorylation of the beta4 integrin upon tyrosine residues and subsequent recruitment of the adapter protein She to beta4, Within 30 min, the activation of Ras, Raf, and Erk2 was observed. Finally, the upregulation of c-myc mRNA was observed at 60 min, These data indicate that human papillomavirus type 6b is able to signal cells via the Ras-MAP kinase pathway to induce cell proliferation. We hypothesize that such a mechanism would allow papillomaviruses to infect hosts more successfully by increasing the potential pool of cells they are able to infect via the initiation of proliferation in resting keratinocyte stem and suprabasal cells.

Codon modified human papillomavirus type 16 E7 DNA vaccine enhances cytotoxic T-lymphocyte induction and anti-tumour activity

Polynucleotide immunisation with the E7 gene of human papillomavirus (HPV) type 16 induces only moderate levels of immune response, which may in part be due to limitation in E7 gene expression influenced by biased HPV codon usage. Here we compare for expression and immunogenicity polynucleotide expression plasmids encoding wild-type (pWE7) or synthetic codon optimised (pHE7) HPV16 E7 DNA. Cos-1 cells transfected with pHE7 expressed higher levels of E7 protein than similar cells transfected with pW7. C57BL/6 mice and F1 (C57X FVB) E7 transgenic mice immunised intradermally with E7 plasmids produced high levels of anti-E7 antibody. pHE7 induced a significantly stronger E7-specific cytotoxic T-lymphocyte response than pWE7 and 100% tumour protection in C57BL/6 mice, but neither vaccine induced CTL in partially E7 tolerant K14E7 transgenic mice. The data indicate that immunogenicity of an E7 polynucleotide vaccine can be enhanced by codon modification. However, this may be insufficient for priming E7 responses in animals with split tolerance to E7 as a consequence of expression of E7 in somatic cells. (C) 2002 Elsevier Science (USA).

Global Genomic Diversity of Human Papillomavirus 6 Based on 724 Isolates and 190 Complete Genome Sequences.

Human papillomavirus type 6 (HPV6) is the major etiological agent of anogenital warts and laryngeal papillomas and has been included in both the quadrivalent and nonavalent prophylactic HPV vaccines. This study investigated the global genomic diversity of HPV6, using 724 isolates and 190 complete genomes from six continents, and the association of HPV6 genomic variants with geographical location, anatomical site of infection/disease, and gender. Initially, a 2,800-bp E5a-E5b-L1-LCR fragment was sequenced from 492/530 (92.8%) HPV6-positive samples collected for this study. Among them, 130 exhibited at least one single nucleotide polymorphism (SNP), indel, or amino acid change in the E5a-E5b-L1-LCR fragment and were sequenced in full. A global alignment and maximum likelihood tree of 190 complete HPV6 genomes (130 fully sequenced in this study and 60 obtained from sequence repositories) revealed two variant lineages, A and B, and five B sublineages: B1, B2, B3, B4, and B5. HPV6 (sub)lineage-specific SNPs and a 960-bp representative region for whole-genome-based phylogenetic clustering within the L2 open reading frame were identified. Multivariate logistic regression analysis revealed that lineage B predominated globally. Sublineage B3 was more common in Africa and North and South America, and lineage A was more common in Asia. Sublineages B1 and B3 were associated with anogenital infections, indicating a potential lesion-specific predilection of some HPV6 sublineages. Females had higher odds for infection with sublineage B3 than males. In conclusion, a global HPV6 phylogenetic analysis revealed the existence of two variant lineages and five sublineages, showing some degree of ethnogeographic, gender, and/or disease predilection in their distribution. IMPORTANCE: This study established the largest database of globally circulating HPV6 genomic variants and contributed a total of 130 new, complete HPV6 genome sequences to available sequence repositories. Two HPV6 variant lineages and five sublineages were identified and showed some degree of association with geographical location, anatomical site of infection/disease, and/or gender. We additionally identified several HPV6 lineage- and sublineage-specific SNPs to facilitate the identification of HPV6 variants and determined a representative region within the L2 gene that is suitable for HPV6 whole-genome-based phylogenetic analysis. This study complements and significantly expands the current knowledge of HPV6 genetic diversity and forms a comprehensive basis for future epidemiological, evolutionary, functional, pathogenicity, vaccination, and molecular assay development studies.

International collaborative proficiency study of Human Papillomavirus type 16 serology.

We performed an international proficiency study of Human Papillomavirus (HPV) type 16 serology. A common methodology for serology based on virus-like particle (VLP) ELISA was used by 10 laboratories in 6 continents. The laboratories used the same VLP reference reagent, which was selected as the most stable, sensitive and specific VLP preparation out of VLPs donated from 5 different sources. A blinded proficiency panel consisting of 52 serum samples from women with PCR-verified HPV 16-infection, 11 control serum samples from virginal women and the WHO HPV 16 International Standard (IS) serum were distributed. The mean plus 3 standard deviations of the negative control serum samples was the most generally useful "cut-off" criterion for distinguishing positive and negative samples. Using sensitivity of at least 50% and a specificity of 100% as proficiency criteria, 6/10 laboratories were proficient. In conclusion, an international Standard Operating Procedure for HPV serology, an international reporting system in International Units (IU) and a common "cut-off" criterion have been evaluated in an international HPV serology proficiency study.

Immune Responses and Therapeutic Antitumor Effects of an Experimental DNA Vaccine Encoding Human Papillomavirus Type 16 Oncoproteins Genetically Fused to Herpesvirus Glycoprotein D

Recombinant adenovirus or DNA vaccines encoding herpes simplex virus type 1 (HSV-1) glycoprotein D (gD) genetically fused to human papillomavirus type 16 (HPV-16) oncoproteins (E5, E6, and E7) induce antigen-specific CD8(+) T-cell responses and confer preventive resistance to transplantable murine tumor cells (TC-1 cells). In the present report, we characterized some previously uncovered aspects concerning the induction of CD8(+) T-cell responses and the therapeutic anticancer effects achieved in C57BL/6 mice immunized with pgD-E7E6E5 previously challenged with TC-1 cells. Concerning the characterization of the immune responses elicited in mice vaccinated with pgD-E7E6E5, we determined the effect of the CD4(+) T-cell requirement, longevity, and dose-dependent activation on the E7-specific CD8(+) T-cell responses. In addition, we determined the priming/boosting properties of pgD-E7E6E5 when used in combination with a recombinant serotype 68 adenovirus (AdC68) vector encoding the same chimeric antigen. Mice challenged with TC-1 cells and then immunized with three doses of pgD-E7E6E5 elicited CD8(+) T-cell responses, measured by intracellular gamma interferon (IFN-gamma) and CD107a accumulation, to the three HPV-16 oncoproteins and displayed in vivo antigen-specific cytolytic activity, as demonstrated with carboxyfluorescein diacetate succinimidyl ester (CFSE)-labeled target cells pulsed with oligopeptides corresponding to the H-2D(b)-restricted immunodominant epitopes of the E7, E6, or E5 oncoprotein. Up to 70% of the mice challenged with 5 x 10(5) TC-1 cells and immunized with pgD-E7E6E5 controlled tumor development even after 3 days of tumor cell challenge. In addition, coadministration of pgD-E7E6E5 with DNA vectors encoding pGM-CSF or interleukin-12 (IL-12) enhanced the therapeutic antitumor effects for all mice challenged with TC-1 cells. In conclusion, the present results expand our previous knowledge on the immune modulation properties of the pgD-E7E6E5 vector and demonstrate, for the first time, the strong antitumor effects of the DNA vaccine, raising promising perspectives regarding the development of immunotherapeutic reagents for the control of HPV-16-associated tumors.

The human papillomavirus type 16 E6 and E7 oncoproteins cooperate to induce mitotic defects and genomic instability by uncoupling centrosome duplication from the cell division cycle

Loss of genomic integrity is a defining feature of many human malignancies, including human papillomavirus (HPV)-associated preinvasive and invasive genital squamous lesions. Here we show that aberrant mitotic spindle pole formation caused by abnormal centrosome numbers represents an important mechanism in accounting for numeric chromosomal alterations in HPV-associated carcinogenesis. Similar to what we found in histopathological specimens, HPV-16 E6 and E7 oncoproteins cooperate to induce abnormal centrosome numbers, aberrant mitotic spindle pole formation, and genomic instability. The low-risk HPV-6 E6 and E7 proteins did not induce such abnormalities. Whereas the HPV-16 E6 oncoprotein has no immediate effects on centrosome numbers, HPV-16 E7 rapidly induces abnormal centrosome duplication. Thus our results suggest a model whereby HPV-16 E7 induces centrosome-related mitotic disturbances that are potentiated by HPV-16 E6.

Assembly of Human Papillomavirus Type-16 Virus-Like Particles: Multifactorial Study of Assembly and Competing Aggregation

Pentameric capsomeres of human papillomavirus capsid protein L1 expressed in Escherichia coli self-assemble into virus-like particles (VLPs) in vitro. A multifactorial experimental design was used to explore a wide range of solution conditions to optimize the assembly process. The degree of assembly was measured using an enzyme-linked immunosorbent assay, and a high-throughput turbidity assay was developed to monitor competing aggregation. The presence of zinc ions in the assembly buffer greatly increased the incidence of aggregation and had to be excluded from the experiment for meaningful analysis. Assembly of VLPs was optimal at a pH of about 6.5, calcium and sodium ions had no measurable effect, and dithiothreitol and glutathione inhibited assembly. Tryptophan fluorescence spectroscopy demonstrated that an increase in urea concentration reduced the rate of VLP formation but had no effect on the final concentration of assembled VLPs. This study demonstrates the use of the hanging-drop vapor-diffusion crystallization method to screen for conditions that promote aggregation and the use of tryptophan fluorescence spectroscopy for real-time monitoring of the assembly process.

Post translational modifications of recombinant human Papillomavirus type 6b major capsid protein

We have determined the post-translational modifications of the major capsid protein, L1 of human papillomavirus (HPV) type 6b. Since this virus cannot be cultured in the laboratory to obtain sufficient material for a study, a recombinant L1 protein produced in a vaccinia virus expression system was used in this investigation. Our results show that this protein is phosphorylated at serine residues and is also glycosylated. No myristoylation or palmitoylation was detected. The fraction of L1 protein incorporated into virus-like particles was not glycosylated. Since recombinant L1 protein is a potential human vaccine candidate, knowledge of the post-translation modifications of this protein may prove useful for the design of anti-HPV vaccines. (C) 1999 Elsevier Science B.V. All rights reserved.

Differences in the post-translational modifications of human papillomavirus type 6b major capsid protein expressed from a baculovirus system compared with a vaccinia virus system

Virus-like particles (VLPs) are being currently investigated in vaccines against viral infections in humans. There are different recombinant-protein-expression systems available for obtaining the necessary VLP preparation for vaccination. However, the differences in post-translational modifications of the recombinant proteins obtained and their differences in efficacy in eliciting an anti-viral response in vaccines are not well established. In this study we have compared the posttranslational modifications of human papillomavirus type-6b major capsid protein L1 (HPV 6bL1) expressed using recombinant baculovirus (rBV) in Sf9 (Spodoptera frugiperda) insect cells, with the protein expressed using recombinant vaccinia virus (rVV) in CV-1 kidney epithelial cells, Two-dimensional gel electrophoresis of biosynthetically labelled rBV-expressed HPV 6bL1 showed several post-translationally modified variants of the protein, whereas rVV-expressed HPV 6bL1 showed only a few variants. Phosphorylations were detected at threonine and serine residues for the L1 expressed from rBV compared with phosphorylation at serine residues only for the L1 expressed from rVV. HPV 6bL1 expressed using rBV incorporated [H-3]mannose and [H-3]galactose, whereas HPV 6bL1 expressed using rVV incorporated only [H-3]galactose. We conclude that post-translational modification of recombinant HPV 6bL1 can differ according to the system used for its expression. Since recombinant L1 protein is a potential human-vaccine candidate, the implication of the observed differences in post-translational modifications on immunogenicity of L1 VLPs warrants investigation.

E7 oncoprotein of human papillomavirus type 16 expressed constitutively in the epidermis has no effect on E7-specific B- or Th-repertoires or on the immune response induced or sustained after immunization with E7 protein

A line of FVB (H-2(q)) mice transgenic for the E6/E7 open reading frames of Human Papillomavirus type 16 driven from the alpha-A crystallin promoter expresses E7 mRNA in lens and skin epithelium. E7 protein is detectable in adult skin, coinciding with the development or inflammatory skin disease, which progresses to papillomata and squamous carcinomata in some mice. By examining the outcome of parenteral immunization with E7 protein, we sought to determine whether endogenous expression of E7 in skin had induced a preexisting immune outcome, i.e., specific immunity or tolerance, or whether the mice remain naive (''ignorant'') to E7. Our data show that the antibody response to defined E7 B-epitopes, the proliferative response to Th epitopes, and the delayed-type hypersensitivity (DTH) response to whole E7 did not differ between groups or young and old E6/E7 transgenic mice (likely having different degrees of lifetime exposure to E7 protein) or between E6/E7-transgenic and nontransgenic parental strain control mice. Although an E7-specific CTL response could not be induced in the H-2(q) background of these mice, incorporation of a D-b allele into the genome allowed comparison of D-b-restricted CTL responses in E6/E7 transgenic and nontransgenic mice. Experiments indicated that the E7-immunization-induced CTL response did not differ significantly between E6/E7 transgenic and nontransgenic mice. We interpret these results to indicate that in spite of expression of E7 protein in adult skin, E6/E7 transgenic mice remain immunologically naive (ignorant) of E7 epitopes presented by immunization. (C) 1997 Academic Press.

Low level expression of human papillomavirus type 16 (HPV16) E6 in squamous epithelium does not elicit E6 specific B- or T-helper immunological responses, or influence the outcome of immunisation with E6 protein

Mice transgenic for E6/E7 oncogenes of Human Papillomavirus type 16 display life-long expression of E6 in lens and skin epithelium, and develop inflammatory skin disease late in life, which progresses to papillomata and squamous carcinoma in some mice. We asked whether endogenous expression of E6 induced a specific immunological outcome, i.e. immunity or tolerance, or whether the mice remained immunologically naive to E6. We show that prior to the onset of skin disease, E6 transgenic mice did not develop a spontaneous E6-directed antibody response, nor did they display T-cell proliferative responses to dominant T-helper epitope peptides within E6. In contrast, old mice in which skin disease had arisen, developed antibodies to E6. We also show that following immunisation with E6, specific antibody responses did not differ significantly among groups of EB-transgenic mice of different ages (and therefore of different durations and amounts of exposure to endogenous E6), and non-transgenic controls. Additionally, E6 immunisation-induced T-cell proliferative responses were similar in E6-transgenic and non-transgenic mice. These data are consistent with the interpretation that unimmunised Eb-transgenic mice that have not developed inflammatory skin disease remain immunologically naive to E6 at the B- and Th levels. There are implications for E6-mediated tumorigenesis in humans, and for the development of putative E6 therapeutic vaccines. (C) 2001 Elsevier Science B.V. All rights reserved.

Nonspecific down-regulation of CD8+ T-Cell responses in mice expressing human Papillomavirus Type 16 E7 oncoprotein from the keratin-14 promoter

The E7 oncoprotein of human papillomavirus 16 (HPV16) transforms basal and suprabasal cervical epithelial cells and is a tumor-specific antigen in cervical carcinoma, to which immunotherapeutic strategies aimed at cytotoxic T-lymphocyte (CTL) induction are currently directed. By quantifying major histocompatibility complex class I tetramer-binding T cells and CTL in mice expressing an HPV16 E7 transgene from the keratin-l l (K14) promoter in basal and suprabasal keratinocytes and in thymic cortical epithelium, we show that antigen responsiveness of both E7- and non-E7-specific CD8(+) cells is down-regulation compared to non-E7 transgenic control mice. We show that the effect is specific for E7, and not another transgene, expressed from the K14 promoter, Down-regulation did not involve deletion of CD8(+) T cells of high affinity or high avidity, and T-cell receptor (TCR) VP-chain usage and TCR receptor density were similar in antigen-responsive cells from E7 transgenic and non-E7 transgenic mice. These data indicate that E7 expressed chronically from the K14 promoter nonspecifically down-regulates CD8+ T-cell responses. The in vitro data correlated with the failure of immunized E7 transgenic mice to control the growth of an E7-expressing tumor challenge, We have previously shown that E7-directed CTL down-regulation correlates with E7 expression in peripheral but not thymic epithelium (T, Dean et al., J, Virol. 73:6166-6170, 1999), The findings have implications for the immunological consequences of E7-expressing tumor development and E7-directed immunization strategies. Generically, the findings illustrate a T-cell immunomodulatory function for a virally encoded human oncoprotein.