Safety and Immunogenicity of an In Vivo Muscle Electroporation Delivery System for DNA-hsp65 Tuberculosis Vaccine in Cynomolgus Monkeys.

A Bacille Calmette-Guérin (BCG) is still the only licensed vaccine for the prevention of tuberculosis, providing limited protection against Mycobacterium tuberculosis infection in adulthood. New advances in the delivery of DNA vaccines by electroporation have been made in the past decade. We evaluated the safety and immunogenicity of the DNA-hsp65 vaccine administered by intramuscular electroporation (EP) in cynomolgus macaques. Animals received three doses of DNA-hsp65 at 30-day intervals. We demonstrated that intramuscular electroporated DNA-hsp65 vaccine immunization of cynomolgus macaques was safe, and there were no vaccine-related effects on hematological, renal, or hepatic profiles, compared to the pre-vaccination parameters. No tuberculin skin test conversion nor lung X-ray alteration was identified. Further, low and transient peripheral cellular immune response and cytokine expression were observed, primarily after the third dose of the DNA-hsp65 vaccine. Electroporated DNA-hsp65 vaccination is safe but provides limited enhancement of peripheral cellular immune responses. Preclinical vaccine trials with DNA-hsp65 delivered via EP may include a combination of plasmid cytokine adjuvant and/or protein prime-boost regimen, to help the induction of a stronger cellular immune response.

TNFR1 single nucleotide polymorphisms are not associated with cervical HPV-induced pre-malignant lesion but regulate in situ cervical TNFR1 expression.

TNF-α is involved in HPV infection control by triggering cell signaling through binding in specific receptors TNFR1 and TNFR2. Genetic polymorphisms in these receptors may influence TNF-α signaling. Herein, we investigated TNFR1 rs767455 and rs2234649 single nucleotide polymorphisms, and TNFR1 protein expression in cervical squamous intraepithelial lesions (SIL) to identify their role in cervical pre-malignant development. SIL patients (n = 179) and healthy volunteers (n = 227) were enrolled for TNFR1 genotyping analysis by PCR-RFLP in blood samples and TNFR1 protein expression in cervical tissue by immunohistochemistry. No statistical differences regard genotypes and allelic frequencies for both polymorphisms were observed. Cervical TNFR1-expressing cells were rare in epithelium and basal layer regardless the groups. However, a progressive increase in infiltrating cells was observed in the stromal area, mainly in high SIL (HSIL) group compared to low SIL (LSIL, p < 0.001) and control (p < 0.001) groups. TNFR1-expressing cells frequency was higher in TNFR1 rs767455AG/GG (p < 0.001), and in rs2234649AA (p < 0.001) genotypes carries in HSIL subgroup. These data indicated that TNFR1-expression is abrogated in cervical epithelium, where HPV-induced pre-malignant lesion occurs, increasing its frequency in inflammatory cells in stroma, and is genetically controlled by TNFR1 rs767455AG/GG and rs234649AA genotypes. These biomarkers may be useful to identify cervical precancerous lesions progression.

Associação de polimorfismos e expressão gênica de marcadores de apoptose ligados carcinogênese cervical induzida pelo hpv em mulheres infectadas ou não pelo HIV / Association of polymorphisms and gene expression of apoptosis markers linked cervical carcinogenesis induced hpv in women infected with HIV or not

A frequência observada desses SNPs estavam em equilíbrio de Hardy–Weinberg nos casos e controles. Nenhuma diferença estatística na frequência genotípica e alélica do polimorfismo de BCL-2 (-938C>A) foi observada entre casos e controles. Contudo, uma forte associação foi identificada na freqüência do genótipo GG de BAX (-248G>A) entre os casos e controles. Quando os grupos de LSIL e HSIL foram comparados, nenhuma diferença estatística foi observada, indicando que o genótipo GG pode influenciar no aparecimento de lesões cervicais, mas não na gravidade da doença. Analisamos a expressão de RNAm de genes associados a apoptose (BAX, BCL2, p53 e pRb) e citotoxicidade (perforina e Fas). Quando a expressão de RNAm de Perforina e Fas foram avaliadas, diferença estatística foi observadas em pacientes portadoras de NIC III e câncerObservamos diferença estatística entre os controles e todos os casos, quando analisamos a expressão de RNAm de p53, mas não de pRb, sendo observada diferença apenas nos controles e NIC II e III. Na analise de RNAm de BCL2, notamos uma baixa expressão no grupo de câncer quando comparado com NIC II, III e controle. Em conclusão, a infecção pelo HIV pode induzir redução na degranulação de células inflamatórias, corroborando para a progressão de infecção pelo HPV, e que carreadoras do alelo G na região promotora de BAX (-248G>A) pode estar associado ao desenvolvimento de NIC quando comparado com as carreadoras do alelo A, possuindo papel protetor; contudo o alelo G não está correlacionado com a gravidade da doença...

Cell-mediated cytotoxicity plays an important role in the regulation of HPVinfection, and in tumor formation. Apoptosis is regulated by different pathways involving a number of genes that either promote (BAX gene)or inhibit (BCL2 gene) the cell death. We evaluated the presence of cervical inflammatory cells, apoptotic (Bax, Bcl-2, FasL, NOS2, perforin) markers and the degranulating expressing cell marker (CD107a) from HPV and HPV/HIV women. Higher per centage of cervical CD4 + and CD8 + T cells were observed in both groups, with lower CD4+ T cells count observed in HPV/HIV women. There were few FasL, Bax and Bcl - 2 inflammatory cervical expressing cells in both groups. NOS2 expression was present especially in the epithelium basal layer on keratinocytes in both groups. Perforin was identified in few cervical cells. However, CD107a was detected in the epithelium basal layer and stroma, meanly on HPV women. We determined whether the SNPs of BCL2(-938C>A) and BAX ( - 248G>A) promoters are associated with risk of cervical intraepithelial neoplasia (CIN) outcome. The observed genotype frequencies of these SNPs were in agreement with Hardy – Weinberg equilibrium in cases and controlgroups. No statistical difference in genotype and allelic frequency ofBCL - 2 ( - 938C>A) polymorphism was observed between all cases and control groups. However, a strong association was identified in GG genotype frequency of BAX ( - 248G>A) polymorphism between cases and controls. When LSIL an d HSILgroups were compared, no statistical difference was observed, indicating thatGG genotype may influence the risk of CIN but not the lesion severity. Weanalyzed the mRNA expression of apoptosis - associated genes (BAX, BCL2, p53 and pRb ) and cytotoxicity -related genes (perforin and Fas). When Fas and Perforin mRNA expressions were evaluated, the statistical difference betweenCIN III and cancer was seen...

Immune response in cervical dysplasia induced by human papillomavirus: the influence of human immunodeficiency virus-1 co-infection -- review.

Human immunodeficiency virus (HIV-1) has become an important risk factor for human papillomavirus (HPV) infection and the development of HPV associated lesions in the female genital tract. HIV-1 may also increase the oncogenicity of high risk HPV types and the activation of low risk types. The Center for Disease Control and Prevention declared invasive cervical cancer an acquired immunodeficiency virus (AIDS) defining illness in HIV positive women. Furthermore, cervical cancer happens to be the second most common female cancer worldwide. The host's local immune response plays a critical factor in controlling these conditions, as well as in changes in the number of professional antigen-presenting cells, cytokine, and MHC molecules expression. Also, the production of cytokines may determine which arm of the immune response will be stimulated and may influence the magnitude of immune protection. Although there are many studies describing the inflammatory response in HPV infection, few data are available to demonstrate the influence of the HIV infection and several questions regarding the cervical immune response are still unknown. In this review we present a brief account of the current understanding of HIV/HPV co-infection, emphasizing cervical immune response.

Distribution of immune cell subsets and cytokine-producing cells in the uterine cervix of human papillomavirus (HPV)-infected women: influence of HIV-1 coinfection.

The aim of this study was to characterize the immune system profile in the uterine cervix of 17 human papillomavirus (HPV)-infected women, compared with 17 whom were coinfected with HIV-1. Five histologically normal cervices in immunocompetent women were used as controls. HPV infection was associated with a marked increase in cells expressing interleukin (IL)-6, interferon gamma (IFN-gamma), and tumor necrosis factor alpha (TNF-alpha). Coinfection by HPV and HIV-1 led to decreased expression of IL-6, TNF-alpha, and IFN-gamma. However, coinfection led to increased numbers of cells expressing IL-4, IL-10, and IL-8. Compared with the histologically normal cervices, increased numbers of macrophages (CD68, RFD7) and T lymphocytes (CD4, CD8) were seen in HPV-infected cervices; coinfection with HIV-1 was associated with a higher number of CD8 cells and lower number of CD68 cells. HPV DNA localized exclusively to the dysplastic squamous cells, whereas HIV-1 RNA was detected mainly in CD68-positive stromal cells. In conclusion, this study shows differential expression of various cytokines and classes of inflammatory cells, relative to HIV-1 infection and HPV coinfection, which may relate to the risk of transmission of HIV-1 and increased risk of cervical cancer in these women.

Immune response in cervical dysplasia induced by human papillomavirus: the influence of human immunodeficiency virus-1 co-infection - review

Human immunodeficiency virus (HIV-1) has become an important risk factor for human papillomavirus (HPV) infection and the development of HPV associated lesions in the female genital tract. HIV-1 may also increase the oncogenicity of high risk HPV types and the activation of low risk types. The Center for Disease Control and Prevention declared invasive cervical cancer an acquired immunodeficience virus (AIDS) defining illness in HIV positive women. Furthermore, cervical cancer happens to be the second most common female cancer worldwide. The host's local immune response plays a critical factor in controlling these conditions, as well as in changes in the number of professional antigen-presenting cells, cytokine, and MHC molecules expression. Also, the production of cytokines may determine which arm of the immune response will be stimulated and may influence the magnitude of immune protection. Although there are many studies describing the inflammatory response in HPV infection, few data are available to demonstrate the influence of the HIV infection and several questions regarding the cervical immune response are still unknown. In this review we present a brief account of the current understanding of HIV/HPV co-infection, emphasizing cervical immune response.