Eliane Campos Coimbra

Susceptibility to cervical cancer: an overview.

Cervical cancer is the second most common cancer in females worldwide. It is well-established that Human Papillomavirus (HPV) infections play a critical role in the development of cervical cancer. However, a large number of women infected with oncogenic HPV types will never develop cervical cancer. Thus, there are several external environment and genetic factors involved in the progression of a precancerous lesion to invasive cancer. In this review article, we addressed possible susceptible phenotypes to cervical cancer, focusing on host genome and HPV DNA variability, multiple HPV infections, co-infection with other agents, circulating HPV DNA and lifestyle.

Molecular targets of HPV oncoproteins: potential biomarkers for cervical carcinogenesis.

Cervical cancer is the second most common cancer among women worldwide and is responsible for 275,000 deaths each year. Persistent infection with high-risk human papillomavirus (HR-HPV) is an essential factor for the development of cervical cancer. Although the process is not fully understood, molecular mechanisms caused by HPV infection are necessary for its development and reveal a large number of potential biomarkers for diagnosis and prognosis. These molecules are host genes and/or proteins, and cellular microRNAs involved in cell cycle regulation that result from disturbed expression of HR-HPV E5, E6 and E7 oncoproteins. One of the current challenges in medicine is to discover potent biomarkers that can correctly diagnose cervical premalignant lesions and standardize clinical management. Currently, studies are showing that some of these molecules are potential biomarkers of cervical carcinogenesis, and it is possible to carry out a more accurate diagnosis and provide more appropriate follow-up treatment for women with cervical dysplasia. In this paper, we review recent research studies on cell cycle molecules deregulated by HPV infections, as well as their potential use for cervical cancer screening.

Prospects of molecularly-targeted therapies for cervical cancer treatment.

Cervical cancer is the third most common cancer among women worldwide and is responsible for 275.000 deaths each year. The development of cervical cancer has been linked to cell cycle disturbances caused by persistent expression of high- risk HPV oncoproteins (E5, E6 and E7), which modulate the expression of host genes and cellular microRNAs. An estimated 5 million women throughout the world are currently infected by HPV and several of them will develop invasive cervical cancer. Despite failures in conventional screening tests, approved therapies have no direct effect on HPV infection. In view of this, effective therapy for cervical cancer is still urgently needed, particularly in developing countries where more than 85% of fatal cases occur. In this paper we review the current molecular targeted therapies which are being explored and may have a significant impact on the treatment of HPV- related cervical dysplasia and carcinoma.

Quantifying mRNA and microRNA with qPCR in cervical carcinogenesis: a validation of reference genes to ensure accurate data.

A number of recent studies have catalogued global gene expression patterns in a panel of normal, tumoral cervical tissues so that potential biomarkers can be identified. The qPCR has been one of the most widely used technologies for detecting these potential biomarkers. However, few studies have investigated a correct strategy for the normalization of data in qPCR assays for cervical tissues. The aim of this study was to validate reference genes in cervical tissues to ensure accurate quantification of mRNA and miRNA levels in cervical carcinogenesis. For this purpose, some issues for obtaining reliable qPCR data were evaluated such as the following: geNorm analysis with a set of samples which meet all of the cervical tissue conditions (Normal + CIN1 + CIN2 + CIN3 + Cancer); the use of individual Ct values versus pooled Ct values; and the use of a single (or multiple) reference genes to quantify mRNA and miRNA expression levels. Two different data sets were put on the geNorm to assess the expression stability of the candidate reference genes: the first dataset comprised the quantities of the individual Ct values; and the second dataset comprised the quantities of the pooled Ct values. Moreover, in this study, all the candidate reference genes were analyzed as a single “normalizer”. The normalization strategies were assessed by measuring p16INK4a and miR-203 transcripts in qPCR assays. We found that the use of pooled Ct values, can lead to a misinterpretation of the results, which suggests that the maintenance of inter-individual variability is a key factor in ensuring the reliability of the qPCR data. In addition, it should be stressed that a proper validation of the suitability of the reference genes is required for each experimental setting, since the indiscriminate use of a reference gene can also lead to discrepant results.

Secretory expression of Porcine Circovirus Type 2 capsid protein in Pichia pastoris

Porcine circovirus type 2 (PCV2) is associated with postweaning multisystemic wasting syndrome (PMWS). The PCV2 capsid (Cap) protein is a leading antigen candidate for vaccine and serological diagnostic testing, due to its immunogenic properties. In this study, the codon-optimized PCV2 Cap gene was cloned into a pPICZαA vector for secretory expression in the methylotrophic yeast Pichia pastoris after methanol induction. The screening of recombinant yeasts was followed by detection of the recombinant Cap (rCap) protein by Western blot, using sera from pigs naturally infected with PCV2. The rCap secreted protein was used without prior purification as a coating antigen in the ELISA test, with high discrimination between PCV2-positive and negative sera. These results reveal a high confidence in the specific immunoreactivity of the secreted antigen and show the antigenicity of the recombinant protein. The feasibility of the P. pastoris expression system for the production of PCV2 Cap as secreted protein and its apparent bioactivity, suggests there are good prospects for the use of this antigen in the investigation of PCV2 infections and testing for vaccine purposes.

Development of an IP-Free Biotechnology Platform for Constitutive Production of HPV16 L1 Capsid Protein Using the Pichia pastoris PGK1 Promoter

The human papillomavirus (HPV) L1 major capsid protein, which forms the basis of the currently available vaccines against cervical cancer, self-assembles into virus-like particles (VLPs) when expressed heterologously. We report the development of a biotechnology platform for HPV16 L1 protein expression based on the constitutive PGK1 promoter (PPGK1) from the methylotrophic yeast Pichia pastoris. The L1 gene was cloned under regulation of PPGK1 into pPGKΔ3 expression vector to achieve intracellular expression. In parallel, secretion of the L1 protein was obtained through the use of an alternative vector called pPGKΔ3α, in which a codon optimized α-factor signal sequence was inserted. We devised a work-flow based on the detection of the L1 protein by dot blot, colony blot, and western blot to classify the positive clones. Finally, intracellular HPV VLPs assembly was demonstrated for the first time in yeast cells. This study opens up perspectives for the establishment of an innovative platform for the production of HPV VLPs or other viral antigens for vaccination purposes, based on constitutive expression in P. pastoris.

Production of Equine Infectious Anemia Virus (EIAV) antigen in Pichia pastoris

Equine Infectious Anemia (EIA) is a persistent lentivirus infection of horses which causes a chronic clinical condition with worldwide importance in veterinary medicine. The p26 protein is usually prepared for use as an antigen in serological tests for EIA diagnosis since it is a well-conserved gene sequence and very immunogenic. In view of the ability of yeast to make post-translational modifications of proteins, this study was carried out to allow Pichia pastoris to be used for the expression of a synthetic codon-optimized EIAV p26 gene. The gene was cloned into pPICZαA vector after appropriate enzymatic digestion. P. pastoris clones transformed with the pPICZαAp26 construction were induced to produce the recombinant p26 protein (rp26) under the regulation of alcohol oxidase 1 promoter by adding methanol to the culture medium. The p26 gene expression was detected by RT-PCR and the production of rp26 was confirmed by dot blotting, Western blotting, ELISA and AGID. The P. pastoris expression system was capable of producing a functional EIAV p26 protein that can be used directly in the functionality tests without requiring laborious purification or recovery steps. This is the first reported study of EIAV p26 protein production in yeast cells.

Vaccine Strategies against Human Papillomavirus: A Discussion Focused on Developing Countries

Cervical cancer is the second most common form of cancer among women, and responsible for 274,000 deaths each year, most of which occur in developing countries. Persistent infection with high-risk human papillomavirus (HR-HPV) is an essential factor in the development of cervical cancer and also a contributory factor in other types of cancer. The current prophylactic HPV vaccines provide protection against the -16 and -18 genotypes which are most commonly associated with cervical cancer worldwide. However, the increased costs of these vaccines inhibit their implementation in developing countries, affecting their viability. Moreover, a therapeutic vaccine is needed for women who are already infected by HPV and/or affected by HPV-related cancer. A number of innovative approaches to combat and treat HPV infection are currently being studied and some of these will be consider in this work, together with the development of new vaccines, especially in seriously affected areas located in developing countries. At the same time, there will be a discussion of the issues involved in carrying out effective HPV vaccination programs; these will take account of financial constraints, the lack of adequate infrastructure and the competing priorities, that are found in the surrounding social context of the developing countries.

Human Papillomavirus-Related Cancers

Cancer is a public health problem occupying the first and second place in number of deaths in developed and developing countries, respectively. Since the last century, the relationship between infection and cancer has been established in animals and more recently in several human cancers. Currently known that 15–20 % of cancers in the world of infectious origin, many of them related to viral infections. The human papillomavirus (HPV) stands out for its association with confirmed cervical cancer and the large volume of evidence that relate to the head and neck cancer. In addition, there is evidence of their relationship with breast cancers, lung and prostate. However, they are still required more detailed research that aim to clarify the possible mechanisms involved in these processes related to carcinogenic HPV.This chapter discusses the main molecular characteristics of HPV and its relationship with cancers using for this the infective models described by recent studies, the mechanisms of tumor progression, forms of diagnosis and therapy.