The genome organization of the novel human papillomavirus type 108 (HPV108), isolated from a low-grade cervical lesion, deviates from those of other HPVs in lacking an E6 gene. The three related HPV types HPV103, HPV108, and HPV101 were isolated from cervicovaginal cells taken from normal genital mucosa (HPV103) and low-grade (HPV108) and high-grade cervical (HPV101) intraepithelial neoplasia (Z. Chen, M. Schiffman, R. Herrero, R. DeSalle, and R. D. Burk, Virology 360:447-453, 2007, and this report). Their unusual genome organization, against the background of considerable phylogenetic distance from the other HPV types usually associated with lesions of the genital tract, prompted us to investigate whether HPV108 E7 per se is sufficient to induce the above-mentioned clinical lesions. Expression of HPV108 E7 in organotypic keratinocyte cultures increases proliferation and apoptosis, focal nuclear polymorphism, and polychromasia. This is associated with irregular intra-and extracellular lipid accumulation and loss of the epithelial barrier. These alterations are linked to HPV108 E7 binding to pRb and inducing its decrease, an increase in PCNA expression, and BrdU incorporation, as well as increased p53 and p21 CIP1 protein levels. A delay in keratin K10 expression, increased expression of keratins K14 and K16, and loss of the corneal proteins involucrin and loricrin have also been noted. These modifications are suggestive of infection by a high-risk papillomavirus.A large number of human papillomavirus (HPV) types have been associated with benign and malignant lesions of the genital tract. High-risk papillomaviruses encode two oncoproteins, E6 and E7, which independently immortalize human keratinocytes, although their combined actions have complementary and synergistic effects (5,22,23,35,43). High-risk E7 protein binds to a multitude of cellular proteins in vitro. Probably its most important action is targeting the pocket protein pRb for degradation and thereby allowing uncontrolled cell cycle progression (30,34). More recent studies, however, have indicated that the pRb-independent functions of E7 are sufficient to induce disruption of terminal differentiation and mild hyperplasia (3). The key role of high-risk E6, on the other hand, is to inactivate the oncosuppressive protein p53 functionally by inducing its degradation through the ubiquitin-proteosome pathway (31). Expression of high-risk E7 alone stabilizes the p53 protein, leading to increased levels (12), although its transcriptional activity is disturbed (32).The genome organizations of the majority of human-pathogenic papillomaviruses are characterized by an early region containing five genes (E1, E2, E4, E6, and E7) and two genes (L1 and L2) in the late region. The HPV types of the genus Alphapapillomavirus usually harbor an E5 gene in the region between the early and late genes, whereas this gene is absent in the HPV types commonly associated with cutaneous lesions and grouped in the genera Betapapillomavirus, Gammapapillomavirus, Deltapapillomavirus, ...

Induction of effective immune responses may help prevent cancer progression. Tumor-specific antigens, such as those of human papillomaviruses involved in cervical cancer, are targets with limited intrinsic immunogenicity. Here we show that immunization with low doses (10 6 infectious units/dose) of a recombinant human adenovirus type 5 encoding a fusion of the E7 oncoprotein of human papillomavirus type 16 to the carboxyl terminus of the surface antigen of hepatitis B virus (HBsAg) induces remarkable E7-specific humoral and cellular immune responses. The HBsAg/E7 fusion protein assembled efficiently into virus-like particles, which stimulated antibody responses against both carrier and foreign antigens, and evoked antigen-specific kill of an indicator cell population in vivo. Antibody and T-cell responses were significantly higher than those induced by a control adenovirus vector expressing wild-type E7. Such responses were not affected by preexisting immunity against either HBsAg or adenovirus. These data demonstrate that the presence of E7 on HBsAg particles does not interfere with particle secretion, as it occurs with bigger proteins fused to the C terminus of HBsAg, and results in enhancement of CD8 ؉ -mediated T-cell responses to E7. Thus, fusion to HBsAg is a convenient strategy for developing cervical cancer therapeutic vaccines, since it enhances the immunogenicity of E7 while turning it into an innocuous secreted fusion protein.Tumor cells of certain types of cancer express proteins, designated as tumor-specific antigens (TSAs), which are not present in nontumor cells. In neoplasias caused by oncoviruses, such as cervical cancers associated with human papillomavirus type 16 (HPV-16) and liver cancers caused by the hepatitis B and C viruses, the viral proteins represent TSAs. A natural mechanism for elimination of chronically infected or transformed cells is activation of cytotoxic T lymphocytes (CTLs) specific for the viral proteins. However, such proteins, are in general weak immunogens and do not induce adequate activation of antigen-specific T cells.The E6 and E7 products of HPV-16 induce transformation by blocking p53 and retinoblastoma (Rb)-mediated cell cycle control pathways, respectively, and by activating cyclins E and A (44). These proteins are constitutively expressed, albeit at low levels, in preneoplastic as well as cancer tissues and, therefore, represent persistent TSAs. Several lines of evidence suggest that E7 may be an effective immunological target for vaccines against oncogenic HPVs. Cell-mediated immunity to E7 has been demonstrated in HPV-mediated intraepithelial lesions of the uterine cervix (2, 31). Cytolytic T cells to HPV-16 E7 have been found in the blood of women with HPV-16-positive cervical neoplasia (20), and lymphoproliferative responses to E7 were found to inversely correlate with viral load (21). In addition, most cervical intraepithelial lesions caused by HPV regress spontaneously, and the phenomenon is accompanied by macrophage and CD4 ϩ

HPV Direct Flow CHIP is a newly developed test for identifying 18 high-risk and 18 low-risk human papillomavirus (HPV) genotypes. It is based on direct PCR from crude-cell extracts, automatic flow-through hybridization, and colorimetric detection. The aim of this study was to evaluate the performance of HPV Direct Flow CHIP in the analysis of 947 samples from routine cervical screening or the follow-up of abnormal Pap smears. The specimens were dry swab samples, liquid-based cytology samples, or formalin-fixed paraffin-embedded tissues. The genotype distribution was in agreement with known epidemiological data for the Spanish population. Three different subgroups of the samples were also tested by Linear Array (LA) HPV Genotyping Test (n=108), CLART HPV2 (n=82), or Digene Hybrid Capture 2 (HC2) HPV DNA Test (n=101). HPV positivity was 73.6% by HPV Direct Flow CHIP versus 67% by LA, 65.9% by HPV Direct Flow CHIP versus 59.8% by CLART, and 62.4% by HPV Direct Flow CHIP versus 42.6% by HC2. HPV Direct Flow CHIP showed a positive agreement of 88.6% with LA (k=0.798), 87.3% with CLART (k=0.818), and 68.2% with HC2 (k=0.618). In conclusion, HPV Direct Flow CHIP results were comparable with those of the other methods tested. Although further investigation is needed to compare the performance of this new test with a gold-standard reference method, these preliminary findings evidence the potential value of HPV Direct Flow CHIP in HPV vaccinology and epidemiology studies.

The novel HPV Direct Flow CHIP commercial system for Human Papillomavirus (HPV) genotyping is based on rapid PCR and automatic reverse dot blot hybridization to genotype-specific probes, allowing the detection of 36 HPV genotypes. This study examined the performance of HPV Direct Flow CHIP in formalin-fixed paraffin-embedded (FFPE) samples (n= 99). Each sample was analyzed both by Direct PCR, using crude cell extracts without DNA purification, and by conventional PCR, using purified DNA. Pair-wise analysis of the results demonstrated strong concordance between the results obtained with the two protocols, although a slightly higher rate of multiple infections was detected by conventional PCR. In summary, HPV Direct Flow CHIP achieves effective HPV detection from FFPE samples with both Direct PCR and Conventional PCR protocols.