Detecção de DNA de HPV no plasma para potencial identificação precoce de recidiva de câncer do colo de útero

O câncer do colo do útero constitui a terceira neoplasia maligna mais comum na população feminina, com aproximadamente 520 mil novos casos e 260 mil óbitos por ano e origina-se a partir da infecção genital persistente pelo Papiloma Vírus Humano (HPV) oncogênico. Os principais HPVs considerados de alto risco oncogênico são os tipos HPV-16 e 18, responsáveis por cerca de 70% de todos os casos de cânceres cervicais (CC) no mundo. Pacientes com CC apresentam taxa de recidiva variando de 8% a 49%. Dentro de dois anos de seguimento, 62% a 89% das recidivas são detectadas. Atualmente, os testes usados para detecção de recidiva são a citopatologia da cúpula vaginal e exames de imagem, porém ainda não estão disponíveis testes específicos. O DNA livre-circulante (cf-DNA) representa um biomarcador não-invasivo facilmente obtido no plasma e soro. Vários estudos mostram ser possível detectar e quantificar ácidos nucléicos no plasma de pacientes com câncer e que as alterações no cfDNA potencialmente refletem mudanças que ocorrem durante a tumorigênese. Essa ferramenta diagnóstica não-invasiva pode ser útil no rastreio, prognóstico e monitoramento da resposta ao tratamento do câncer. Portanto, o desenvolvimento e a padronização de testes laboratoriais não invasivos capazes de identificar marcadores tumorais e diagnosticar precocemente a recidiva da doença aumentam a chance de cura através da utilização dos tratamentos preconizados. Sendo assim, este estudo tem o objetivo de detectar o DNA de HPV no plasma de pacientes com CC para avaliar sua potencial utilidade como marcador precoce de recidiva. Um fragmento de tumor e sangue de pacientes com CC, atendidas no ICESP e HC de Barretos, foram coletados antes do tratamento. Entraram no estudo 137 pacientes nas quais o tumor foi positivo para HPV-16 ou 18, sendo 120 amostras positivas para HPV-16 (87,6%), 12 positivas para HPV-18 (8,8%) e cinco positivas para HPV-16 e 18 (3,6%). A média de idade das pacientes deste estudo foi de 52,5 anos. Plasma de 131 pacientes com CC da data do diagnóstico e de 110 pacientes do seguimento foram submetidas ao PCR em Tempo Real HPV tipo específico. A presença do DNA de HPV no plasma pré-tratamento foi observada em 58,8% (77/131) com carga viral variando de 204 cópias/mL a 2.500.000 cópias/mL. A positividade de DNA no plasma pré-tratamento aumentou com o estadio clínico do tumor: I - 45,2%, II - 52,5%, III - 80,0% e IV - 76,9%, (p=0,0189). A presença do DNA de HPV no plasma pós-tratamento foi observada em 27,3% (30/110). A média de tempo das recidivas foi de 3,1 anos (2,7 - 3,5 anos). O DNA de HPV foi positivo até 460 dias antes do diagnóstico clínico da recidiva. As pacientes com DNA de HPV no plasma apresentaram pior prognóstico, tanto sobrevida como o tempo livre de doença, em relação às que foram negativas. Nas pacientes com CC a presença de HPV no plasma de seguimento pode ser um marcador precoce útil para o monitoramento da resposta terapêutica e detecção de pacientes com risco aumentado de recidiva e progressão da doença.

Variabilidade intratípica de HPV 16 em relação à origem étnica e HLA de uma população de alto risco para o câncer do colo do útero

A infecção por papilomavirus é a principal causa de desenvolvimento de neoplasias intraepiteliais cervicais (NIC) e câncer do colo do útero (CCU). Estudos epidemiológicos têm demonstrado que a persistência do genoma viral encontra-se associado a variantes moleculares específicas de papilomavirus humano (HPV) de alto risco. As moléculas HLA de classe II têm um importante papel na resposta imune. Associações entre HLA e CCU ou infecção por HPV tem sido demonstrado em diferentes populações. O nosso objetivo foi verificar se a variabilidade de HLA-DRB1 e DQB1 estavam associada ao CCU e NIC III em mulheres de Belém, uma população formada pelos 3 principais grupos étnicos humanos e uma área de alto risco para o CCU no Norte do Brasil. Foi investigada a existência de diferenças na distribuição de alelos HLA entre mulheres com CCU e NIC III portadoras de diferentes variantes de HPV-16 e mulheres citologicamente normais. Os genes HLA DQB1 e DRB1 foram tipados pelo método de PCR-SSO em 95 casos e 287 controles de mulheres com citologia normal atendidas em um centro de prevenção do colo do útero na mesma cidade. As variantes de HPV-16 foram tipadas por sequenciamento de um fragmento da região controladora do genoma viral (LCR). O polimorfismo na posição 350 do gene E6 foi tipado baseado em um protocolo de hibridização em pontos, para identificar a alteração na posição 350T→G. A magnitude das associações foi estimada por odds ratio (OR) e os respectivos intervalos de confiança (IC), ajustados para potenciais fatores de confusão. Uma associação positiva foi observada entre CCU e os haplótipos DRB1* 150 l-DQB1*0602, DRB1*04-DQB1*0301 e DRB1*1602-DQB1*0301. Ao contrário, DRB1*01-DQB1*0501 mostrou um efeito protetor. Os alelos DRB1*0804, DQB1*0402 apresentaram efeito protetor contra positividade por HPV. O alelo DQB1*0502 e o grupo DRB1*15 foram positivamente associados. Os nossos resultados mostram que as associações positivas de DRB1*1501 e DRB1*1602 podem ser atribuídas a variantes asiático-americanas quando comparado a variantes européias. O risco conferido a DRB1*1501 foi encontrado associado tanto a variantes E6350G quanto a variantes E6350T, entretanto, o maior efeito foi devido às variantes E6250T. A associação positiva de DRB1*1602 foi significativa somente no grupo de mulheres positivas para E6350G. Estes resultados estão de acordo com a composição étnica da população estudada bem como um maior potencial oncogênico de certas variantes. Nossos dados sugerem que a contribuição dos alelos HLA na susceptibilidade genética ao CCU difere de acordo com a distribuição das variantes de HPV em uma dada região geográfica ou grupo étnico.

A polytope DNA vaccine elicits multiple effector and memory CTL responses and protects against human papillomavirus 16 E7-expressing tumour

Vaccine-induced CD8 T cells directed to tumourspecific antigens are recognised as important components of protective and therapeutic immunity against tumours. Where tumour antigens have pathogenic potential or where immunogenic epitopes are lost from tumours, development of subunit vaccines consisting of multiple individual epitopes is an attractive alternative to immunising with whole tumour antigen. In the present study we investigate the efficacy of two DNA-based multiepitope('polytope') vaccines containing murine (H-2(b)) and human (HLA-A* 0201)-restricted epitopes of the E7 oncoprotein of human papillomavirus type 16, in eliciting tumour-protective cytotoxic T-lymphocyte (CTL) responses. We show that the first of these polytopes elicited powerful effector CTL responses ( measured by IFN-gamma ELISpot) and long-lived memory CTL responses ( measured by functional CTL assay and tetramers) in immunised mice. The responses could be boosted by immunisation with a recombinant vaccinia virus expressing the polytope. Responses induced by immunisation with polytope DNA alone partially protected against infection with recombinant vaccinia virus expressing the polytope. Complete protection was afforded against challenge with an E7-expressing tumour, and reduced growth of nascent tumours was observed. A second polytope differing in the exact composition and order of CTL epitopes, and lacking an inserted endoplasmic reticulum targeting sequence and T-helper epitope, induced much poorer CTL responses and failed to protect against tumour challenge. These observations indicate the validity of a DNA polytope vaccine approach to human papillomavirus E7 - associated carcinoma, and underscore the importance of design in polytope vaccine construction.

God's gift to women: The human papillomavirus vaccine

An Australian newspaper recently bestowed Ian Frazer the title of God's gift to women for his research team's part in developing a vaccine to help control cervical cancer. Here Frazer discusses this work and the science behind the vaccine.

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.

Reformulation of a thermostable broadly protective recombinant vaccine against human papilloma virus

The causal relationship between Human Papilloma Virus (HPV) infection and cervical cancer has motivated the development, and further improvement, of prophylactic vaccines against this virus. 70% of cervical cancers, 80% of which in low-resources countries, are associated to HPV16 and HPV18 infection, with 13 additional HPV types, classified as high-risk, responsible for the remaining 30% of tumors. Current vaccines, Cervarix® (GlaxoSmithKline) and Gardasil®(Merk), are based on virus-like particles (VLP) obtained by self-assembly of the major capsid protein L1. Despite their undisputable immunogenicity and safety, the fact that protection afforded by these vaccines is largely limited to the cognate serotypes included in the vaccine (HPV 16 and 18, plus five additional viral types incorporated into a newly licensed nonavalent vaccine) along with high production costs and reduced thermal stability, are pushing the development of 2nd generation HPV vaccines based on minor capsid protein L2. The increase in protection broadness afforded by the use of L2 cross-neutralizing epitopes, plus a marked reduction of production costs due to bacterial expression of the antigens and a considerable increase in thermal stability could strongly enhance vaccine distribution and usage in low-resource countries. Previous studies from our group identified three tandem repeats of the L2 aa. 20-38 peptide as a strongly immunogenic epitope if exposed on the scaffold protein thioredoxin (Trx). The aim of this thesis work is the improvement of the Trx-L2 vaccine formulation with regard to cross-protection and thermostability, in order to identify an antigen suitable for a phase I clinical trial. By testing Trx from different microorganisms, we selected P. furiosus thioredoxin (PfTrx) as the optimal scaffold because of its sustained peptide epitope constraining capacity and striking thermal stability (24 hours at 100°C). Alternative production systems, such as secretory Trx-L2 expression in the yeast P. pastoris, have also been set-up and evaluated as possible means to further increase production yields, with a concomitant reduction of production costs. Limitations in immune-responsiveness caused by MHC class II polymorphisms –as observed, for example, in different mouse strains- have been overcome by introducing promiscuous T-helper (Th) epitopes, e.g., PADRE (Pan DR Epitope), at both ends of PfTrx. This allowed us to obtain fairly strong immune responses even in mice (C57BL/6) normally unresponsive to the basic Trx-L2 vaccine. Cross-protection was not increased, however. I thus designed, produced and tested a novel multi-epitope formulation consisting of 8 and 11 L2(20-38) epitopes derived from different HPV types, tandemly joined into a single thioredoxin molecule (“concatemers”). To try to further increase immunogenicity, I also fused our 8X and 11X PfTrx-L2 concatemers to the N-terminus of an engineered complement-binding protein (C4bp), capable to spontaneously assemble into ordered hepatmeric structures, previously validated as a molecular adjuvant. Fusion to C4bp indeed improved antigen presentation, with a fairly significant increase in both immunogenicity and cross-protection. Another important issue I addressed, is the reduction of vaccine doses/treatment, which can be achieved by increasing immunogenicity, while also allowing for a delayed release of the antigen. I obtained preliminary, yet quite encouraging results in this direction with the use of a novel, solid-phase vaccine formulation, consisting of the basic PfTrx-L2 vaccine and its C4bp fusion derivative adsorbed to mesoporus silica-rods (MSR).

Genital human papillomavirus: Women's knowledge and attitudes of this sexually transmitted disease

The purpose of this study was to investigate women's knowledge and attitudes regarding genital human papillomavirus (n=100). Using a descriptive design, the Health Education Questionnaire was administered to 100 female patients (Mean Age = 33, SD = 7.17) at a physicians office in South Florida. The results indicated a lack of knowledge regarding genital human papillomavirus with 21 patients (21%) reported having knowledge and 79 (79%) having never heard of this disease. In addition, the group familiar with genital human papillomavirus also possessed a low level of knowledge with only 57% acknowledging an association of genital human papillomavirus and cervical cancer, 52% aware that a pap smear can detect the virus, 42% knowing that antibiotics can not treat the disease and 57% aware that it is not associated with a family history. An association was found between attitudes and health seeking behaviors. Subjects stating that they would take all measures to prevent genital human papillomavirus, were more likely to have a pap smear within the last year (Chi-square (1) = 4.33, p < .05). Higher levels of education and income were associated with increased knowledge regarding genital human papillomavirus when subjects were categorized according to sociodemographic characteristic (Chi-square (1) =9.45, p < .05; Chi-square (1) = 6.75, p < .05). There was no significant correlation between knowledge and ethnicity, marital status or age. Findings indicated the need for improved education and promotion of positive attitudes regarding human papillomaviurs in order to improve health seeking behaviors among women.

Efficacy, safety, and immunogenicity of the human papillomavirus 16/18 AS04-adjuvanted vaccine in women older than 25 years : 7-year follow-up of the phase 3, double-blind, randomised controlled VIVIANE study

Acknowledgments The VIVIANE study was funded and coordinated by GlaxoSmithKline Biologicals SA, which also covered all costs associated with development and publication of this report. We thank all study participants and their families. We gratefully acknowledge the work of the central and local study coordinators, and staff members of the sites who participated in this study. Writing support services were provided by Mary Greenacre (An Sgriobhadair, Isle of Barra, UK), on behalf of GSK Vaccines; editing and publication coordination services were provided by Jérôme Leemans (Keyrus Biopharma, Lasne, Belgium), Stéphanie Delval (XPE Pharma and Science, Wavre, Belgium), and Matthieu Depuydt (Business Decision Life Sciences, Brussels, Belgium), on behalf of GSK Vaccines

Longitudinal Incidence Study of Clinically-Relevant HPV and Vaccine-Type HPV in Young Online Female Daters

Thesis (Master's)--University of Washington, 2016-08