Virology

Group Leader: Luisa L. Villa Dr. Luisa L. Villa

Infection with Human papillomavirus (HPV) high-risk types is the primary cause of cervical cancer, one of the leading causes of women death worldwide. Dr. Luisa Villa's Virology Group is conducting a study with one of the longest follow-up of about 2,000 women from a high-risk area for cervical cancer in Brazil. The information stemming from this large longitudinal study of the natural history of HPV infections and risk of cervical cancer is providing seminal information for the development of new prevention measures to control cervical cancer, including prophylactic HPV vaccines that are presently in phase III trials to determine their efficacy. In addition, a large international prospective study of men, the HPV Infection in Men Study (HIM), has been initiated. The information collected will be useful in developing vaccination strategies targeting men to prevent HPV related cancers both in men and women, as well as answering fundamental questions relative to the natural history of male HPV infection.

Some of the evidences accumulated by the group show that measurement of viral load can identify women at risk of developing cervical cancer precursor lesions. Furthermore, it has been shown that both episomes and integrated genomes of HPV-16 are found in cervical lesion of different grades and that intratypic variation of HPV-16 is an important predictor of progression to clinically relevant cervical lesions probably due to enhanced promoter activity of certain HPV-16 variants. We observed that non-European variants of HPVs 16 and 18 are epidemiologically associated with an increased risk of persistent infection and development of cervical lesion. We have also shown that concerning HPV-18 LCR intratypic nucleotide variability, the Asian-Amerindian B18-3 isolate was transcriptionally more active than some European isolates tested. Furthermore, we observed that the HPV-18 prototype LCR sequence was more active than the HPV-16 prototype. HPV life cycle is synchronized to epithelial differentiation probably due to the differential expression of cellular transcriptional factors in the different cell layers. Thus, we are currently analyzing the transcriptional activity of HPVs 16 and 18 molecular variants in organotypic cultures. Finally, it has been observed that some alleles and haplotypes of MHC Class II genes are related to HPV infection prevalence and persistence.

A high proportion of women of reproductive age are infected HPV. However, the majority of these infections are transient and only a low proportion of infected women develop cervical cancer. Epidemiological and molecular studies have shown that the outcome of HPV infections is influenced by local cell-mediated immune response. The proinflammatory cytokine tumor necrosis factor- (TNF) is one of the main mediators of skin and mucosa inflammation. We have shown that this cytokine inhibits both normal and HPV-16 immortalized human keratinocytes proliferation. On the other hand, HPV-18 immortalized and HPV-16 or -18 transformed keratinocytes are resistant to TNF. The different tumorigenic capacity between HPV-containing cell lines may be linked to alterations in gene expression profiles. Using microarray platform we have identified groups of genes that clearly differentiate TNF-sensitive (normal and HPV-16 immortalized keratinocytes) from TNF-resistant (HPV-18 immortalized keratinocytes) samples. The identification of potential tumor-progression markers associated to HPV-induced TNF resistance, could contribute to the characterization of lesions with higher risk of evolving to malignancy and in developing of therapeutical strategies. Besides, we have observed that acute expression of high-risk HPV E7 is enough to confer resistance to TNF cytostatic effect both in monolayer and organotypic cultures. Experiments are under way in order to determine the role of high-risk HPV oncogenes in HPV-mediated TNF resistance. Finally, the role of HPV in activating the AKT pathway has been investigated in the past few years. This pathway is activated upon cell attachment and growth factor signaling transmitting signals to the cell nucleus to inhibit apoptosis and thereby increase cell survival during proliferation. We are studying the influence of HPV-16 oncoproteins in targeting this pathway to increase cell proliferation and cell surviving.

High-risk human papillomavirus genomes are present in almost all cases of cervical cancer. Presence of antibodies or cellular response against epitopes of viral proteins has already been described. However, the role of humoral response in HPV infection is still unclear. Phage display constitutes a robust method for characterization of peptides specifically recognized by antibodies, which originally would recognize "in vivo" tumor antigens. We applied this technology to identify peptide sequences recognized by serum antibodies from patients exposed to HPV. We are currently analyzing the diagnostic potential of these sequences in series of sera of women from the Ludwig/McGill cohort. Besides, the peptides identified are being tested in tissue microarrays containing series of cervical cancer samples. In addition, these peptides have been used to immunize mice challenged with tumor cells in order to investigate their potential therapeutic effects.

Our laboratory is also studying cellular immune responses against HPV associated tumors. Our proposed model consists of mouse chimeras where RAG deficient mice bearing HPV16 associated tumors are recipients for C57 black6 wild type donor cells. Donor cells consist of mixed or pure lymph node and spleen lymphocytes or bone marrow cells from C57 black6 mice. With this approach we intend to investigate tumor infiltrating populations, cytokine expression modulation, effect of adaptative response and specific cells populations on tumor progression, cross-talk between innate and adaptative response in mice bearing HPV16 associated tumors.

Publications

Luisa Lina Villa Laura Sichero Maria Cecília Costa Maria Antonieta Ávilla Andreoli Enrique M. Boccardo Lara Termini Andrea Trevisan Silvaneide Aparecida Ferreira Raquel Hessel Lenice Galan de Paula José Carlos Mann João Simão Pereira Sobrinho M. Stella Leme Hering M. Luiza Baggio Garcia Pinto Aline Bolpetti Ana Paula Lepique Carina Victoria Manzini Baldi Tatiana Rabachini

Staff Members

Virology Group staff members