Papilomavírus humano: novas abordagens epidemiológicas, diagnósticas e perspectivas vacinais

Abstract

Introduction: HPV infection is the most common sexually transmitted disease in humans, which can cause benign diseases (warty lesion) or malignant as the anogenital cancer, oral cancer and, with the highest incidence, cervical cancer. Overall, the main HPV detection method is based on cytological evaluation of Papanicolaou; however, false-negative results and unsatisfactory sampling complicate the disease diagnosis and prevention. In this context, the investigation of new diagnosis platforms and prophylaxys has great interest. Objectives: The aim of this study was to conduct an epidemiological study of HPV infection in the women population of southeastern Brazil and then propose new diagnostic and vaccine approaches. Methodology: Genital samples of 5,223 women were evaluated by a new molecular tool based on PCR (Nested Multiplex PCR - NMPCR), using a cocktail of primers for simultaneous detection of 38 different HPV types, in single tube. Another molecular approach was proposed by Phage Display technique to select the IgA binding peptides from cervical samples of patients with HPV infection. In silico (Linear and 3D bioinformatics) and in vitro (ELISA, Slot blot and Bioelectrode) analysis were performed to validate the selected phages and synthetic peptides in cervical (n=91) and salivary specimens (n=66). The sensitivity and specificity parameters of HPV diagnosis were determined by ROC curve. To test the vaccine potential of synthetic peptides, in silico (3D bioinformatic), in vivo (female BALB/c mice immunization) or in vitro analyzes (MTT, ELISA, splenocytes culture, CBA and Neutralization assay) were carried out. Results: Among the 5,223 women evaluated, there was a prevalence of 58.9% of HPV infection, especially the types 53, 52 and 06, showing the relevance to propose new alternatives for HPV detection and prevention. In molecular approach of Phage Display, thirty-two distinct phage clones were selected against IgA from positive HPV cervical samples. Phage C.B1 showed higher reactivity against HPV samples compared to the negative control group by ELISA. This peptide is a putative epitope of HPV major capsid protein (L1) and has distinguished efficiently infection from HPV controls in cervical and saliva samples (p <0.0001), with high sensitivity (above 95%) and specificity (above 71%) in both fluids. The C.B1 peptide was also successfully incorporated (p<0.05) onto a graphite bioelectrode for HPV direct diagnosis in saliva. In the vaccine approach, three peptides (PEP1, PEP3 and PEP4) showed no toxicity in murine macrophages. Immunizations showed potent induction of serum IgG antibodies production by PEP3, and increase in IgA titer in the vaginal mucosa under PEP1 and PEP3 stimuli. The evaluation of cellular immune response indicated that the PEP1 and, particularly, the PEP3 chimeric peptide were able to polarize the immune response towards to Th1 profile and sensitize the cells to a pro-inflammatory \"status\", suitable for antiviral activity. The HPV-16 PsVs neutralization assay showed that PEP3 was able to reduce the infection by 49%. Conclusions: Overall, the studies presented here showed a high prevalence of HPV among Brazilian women by viral types still little explored in the literature. We selected mimotopes by Phage Display tool capable of detecting HPV in both saliva samples as in the cervical secretion, providing a low cost, simple and non-invasive diagnostics for population screening. In addition, the mimotopes selected have attractive applicability in vaccine formulations, but further studies are still needed.