HPV Research at the São Paulo Branch
Luisa Lina Villa
In the early 80’s, we exploited the availability of cervical and penile cancer samples from the Cancer Hospital, where the São Paulo Branch of the LICR is located, to address a burning question at that time: how frequent are Human Papillomavirus (HPV) DNA sequences in genital tumors? Despite the low sensitivity molecular assays available, it became clear that indeed these viral genomes were very often found in such tumors. Together with many studies performed around the world, these findings helped pave the way for the establishment of HPV as the causative agent of cervical cancer (by LICR Scientific Committee Member, Dr. Harald zur Hausen) about a decade later. In the mean-time, the discovery of a high proportion of HPV infections in asymptomatic women led to the launch of natural history (epidemiological) studies aimed at understanding the risk correlates for HPV-associated cervical disease.
In 1993 we began a large cohort study to verify the hypothesis that persistent HPV infection increases risk of low grade and high-grade cervical lesions in a population of low-income women in São Paulo, Brazil. The ‘Ludwig/McGill cohort’ is one of the largest longitudinal studies of the natural history of HPV infections and risk of cervical cancer. (The ‘McGill’ part of the name pays tribute to the contributions of Dr. Eduardo Franco, an epidemiologist who worked at the São Paulo Branch and then went to McGill University in Canada, where he is the Director of the Department of Oncology and Epidemiology). Using PCR-based methods, we were able to show that most HPV infections are transient and of little clinical significance. However, the small proportion of women who harbor persistent HPV infections stand at a much greater risk of subsequent cervical neoplasia, indicating that persistent, not transient, HPV infections are the actual biologic precursor in cervical carcinogenesis. Needless to say, this finding was important for understanding the clinical significance of HPV DNA testing results and set policies for inclusion of some form of HPV testing in cervical cancer prevention internationally. Another relevant surrogate marker for HPV persistence was found in our studies to be viral load. Measurements of HPV copy numbers over time can predict women at higher risk of developing cervical cancer precursors. Furthermore, higher viral loads correlate with integration of the viral genome in disease risk determination.
We have also shown that intratypic variation of HPV-16 and HPV-18 is an important predictor of progression to clinically relevant cervical lesions. Variation in the regulatory region of most molecular variants correlates with an enhanced promoter activity of the viral early region when compared to the prototype. This could account for an enhanced transcription of E6 and E7 oncogenes that could explain in part the differential oncogenic potential associated epidemiologically with certain variants of these two very common high-risk HPVs.
Another important aspect addressed by our prospective study deals with the immune responses associated with HPV infections and risk of cervical neoplasia. We have described positive and negative associations between some alleles and haplotypes of MHC Class II genes (HLA-DRB1, -DQA1, and –DQB1) and cervical cancer, precursor lesions, and HPV infections. Concerning humoral responses, both serum IgG levels and more recently neutralizing antibodies (Nabs) were tested in the specimens from our cohort study. Both are markers of exposure to HPV, with NAbs against HPV-16 being a more specific indicator of HPV-16 infection than total IgG titers. Additional studies are underway, including different approaches to better characterize the immune responses to HPV infections in animal models and to unravel the pathways involved in the anti-proliferative effect exerted by TNF in HPV-16 and -18 immortalized cell lines. Similar instruments to those applied to the Ludwig/McGill cohort will be used to assess exposure of HPV in the male genital area, aiming to further our understanding of HPV infections in men so that effective programs can be developed to reduce HPV anogenital warts and the cancer burden in both men (penile and anal) and women (cervical).
We are also investigating the level of micronutrients and antioxidants in the sera from women of the Ludwig/McGill cohort. We observed that consumption of certain carotenoids, as well as higher endogenous retinoic acid concentrations leading to increases in serum levels, may decrease the duration of an oncogenic HPV infection thereby decreasing risk of cervical disease in high-risk women. This is particularly important in the developing world, where changes in diet are more feasible and less costly than treating the infection.
Understanding the epidemiology of cervical HPV infection is an important first step towards the development of strategies for preventing genital HPV disease and, ultimately, cervical cancer. Results from our HPV natural history studies were seminal in facilitating the design and implementation of clinical trials of prophylactic vaccines against HPV. Our involvement with Merck’s HPV vaccine clinical program resulted in a recent publication in The Lancet Oncology that demonstrated for the first time the safety, immunogenicity and efficacy of a quadrivalent HPV L1 vaccine containing Virus-like particles (VLPs) of the two main causative agents of genital warts – types 6 and 11 – and of two high-risk types responsible for about 70% of all cervical cancers, namely HPV types 16 and 18. This phase II trial was conducted in Brazil, from where most young volunteers were accrued, in the USA and Northern Europe. Over the two and a half years of follow-up after vaccination, the combined incidence of persistent infection from HPV 6, 11, 16, or 18 and related genital disease including new cervical pre-cancers and genital warts was reduced by 90 percent in women that received the vaccine compared with placebo among women who were naïve to the relevant HPV types at baseline. Although the study was not originally designed or powered to assess vaccine efficacy on the disease endpoints separately, efficacy of this vaccine against cervical pre-cancers and genital warts caused by the HPV types 16, 18, 6 and 11 was 100 percent. Data from phase III clinical trials, currently underway with over 25,000 participants enrolled worldwide, are expected to be available later this year.
It is within this very positive and vivid scenario that I foresee a continuation of our contributions to the fields of molecular epidemiology and immunology of HPV. Along 20 years of research I have counted on several brilliant students and collaborators, both in Brazil and abroad, whose cooperation and dedication to this program is gratefully acknowledged. I am grateful to Dr. Ricardo Brentani, our Director, for his continuous inspiration and support.
Luisa Lina Villa
Leader, Virology Group
LICR São Paulo Branch