RESUMO
BACKGROUND: It has been shown that women positive for HPV 16 and HPV 18 have an increased risk of high-grade cervical intraepithelial neoplasia (CIN) compared with women positive for other high-risk (HR) HPV types. In addition, HPV 18 and HPV 45 have been closely linked to aggressive and difficult to detect adenocarcinomas. OBJECTIVES: To develop a test based on the Hybrid Capture technology capable of specifically detecting the most important carcinogenic HPV types; 16, 18, and 45. STUDY DESIGN: The assay is based on Hybrid Capture technology utilizing a mixture of short type-specific oligoribonucleotides to detect HPV types 16, 18, or 45. The assay utilizes no target amplification and shares workflow and critical reagents with the Digene HC2 HPV screening assay. Studies to evaluate specificity, performance of the test in comparison to HC2, and capability to detect a single genotype in the presence of multiple infections are described. Specificity was evaluated analytically using a panel of HR- and LR-HPV types to illustrate cross-reactivity. Performance in comparison to the HC2 test was evaluated by testing aliquots of the same prepared samples by the genotyping test and HC2. Ability to detect a single genotype during multiple infections was modeled by detecting HPV 16 plasmid in the presence of HPV 6 or HPV 31 at high copy numbers. RESULTS: The proposed genotyping assay specifically detects HPV 16, 18, and 45 with an analytical sensitivity of 5,000 copies per assay. The assay is highly specific and does not detect other tested high-risk or low-risk types at 10(8) copies per reaction. Utility of the genotyping test was demonstrated using clinical samples collected in Digene Specimen Transport Medium (STM) and results were confirmed by PCR. CONCLUSIONS: The target-amplification free assay provides a genotyping method for highly specific detection of HPV 16, 18, and 45 without the complexity of PCR technology.
Assuntos
Colo do Útero/virologia , DNA Viral/genética , Técnicas de Diagnóstico Molecular/métodos , Hibridização de Ácido Nucleico/métodos , Papillomaviridae/classificação , Papillomaviridae/isolamento & purificação , Infecções por Papillomavirus/diagnóstico , Virologia/métodos , Reações Cruzadas , Feminino , Genótipo , Papillomavirus Humano 16/classificação , Papillomavirus Humano 16/genética , Papillomavirus Humano 16/isolamento & purificação , Papillomavirus Humano 18/classificação , Papillomavirus Humano 18/genética , Papillomavirus Humano 18/isolamento & purificação , Humanos , Papillomaviridae/genética , Infecções por Papillomavirus/virologia , Sensibilidade e EspecificidadeRESUMO
A mixture of DNA plasmids expressing five Plasmodium falciparum pre-erythrocyte-stage antigens was administered with or without a DNA plasmid encoding human granulocyte-macrophage colony-stimulating factor (hGM-CSF) as an immune enhancer. After DNA immunization, antigen-specific gamma interferon (IFN-gamma) responses were detected by ELISPOT in 15/31 volunteers to multiple class I- and/or class II-restricted T-cell epitopes derived from all five antigens. Responses to multiple epitopes (=7) were detected simultaneously in some volunteers. By 4 weeks after challenge with P. falciparum parasites, 23/31 volunteers had positive IFN-gamma responses and the magnitude of responses was increased from 2- to 143-fold. Nonetheless, none was protected against malaria. Volunteers who received hGM-CSF had a reduced frequency of IFN-gamma responses to class I peptides compared to those who only received plasmids expressing P. falciparum proteins before challenge (3/23 versus 3/8; P = 0.15) or after parasite challenge (4/23 versus 5/8; P = 0.015) but not to class II peptides before or after challenge. The responses to one antigen (P. falciparum circumsporozoite protein [PfCSP]) were similar among volunteers who received the five-gene mixture compared to volunteers who only received the PfCSP DNA plasmid in a previous study. In summary, DNA-primed IFN-gamma responses were boosted in humans by exposure to native antigen on parasites, coadministration of a plasmid expressing hGM-CSF had a negative effect on boosting of class I-restricted IFN-gamma responses, and there was no evidence that immunization with PfCSP DNA in the mixture reduced T-cell responses to PfCSP compared to when it was administered alone.
Assuntos
Antígenos de Protozoários/imunologia , Interferon gama/sangue , Vacinas Antimaláricas/imunologia , Malária Falciparum/imunologia , Plasmodium falciparum/imunologia , Vacinas de DNA/imunologia , Animais , Antígenos de Protozoários/administração & dosagem , Antígenos de Protozoários/genética , Mordeduras e Picadas , Culicidae , Fator Estimulador de Colônias de Granulócitos e Macrófagos/administração & dosagem , Fator Estimulador de Colônias de Granulócitos e Macrófagos/genética , Fator Estimulador de Colônias de Granulócitos e Macrófagos/imunologia , Humanos , Imunização , Vacinas Antimaláricas/administração & dosagem , Plasmídeos , Linfócitos T/imunologia , Resultado do Tratamento , Vacinas de DNA/administração & dosagemRESUMO
Vaccine-induced protection against diseases like malaria, AIDS, and cancer may require induction of Ag-specific CD8(+) and CD4(+) T cell and Ab responses in the same individual. In humans, a recombinant Plasmodium falciparum circumsporozoite protein (PfCSP) candidate vaccine, RTS,S/adjuvant system number 2A (AS02A), induces T cells and Abs, but no measurable CD8(+) T cells by CTL or short-term (ex vivo) IFN-gamma ELISPOT assays, and partial short-term protection. P. falciparum DNA vaccines elicit CD8(+) T cells by these assays, but no protection. We report that sequential immunization with a PfCSP DNA vaccine and RTS,S/AS02A induced PfCSP-specific Abs and Th1 CD4(+) T cells, and CD8(+) cytotoxic and Tc1 T cells. Depending upon the immunization regime, CD4(+) T cells were involved in both the induction and production phases of PfCSP-specific IFN-gamma responses, whereas, CD8(+) T cells were involved only in the production phase. IFN-gamma mRNA up-regulation was detected in both CD45RA(-) (CD45RO(+)) and CD45RA(+)CD4(+) and CD8(+) T cell populations after stimulation with PfCSP peptides. This finding suggests CD45RA(+) cells function as effector T cells. The induction in humans of the three primary Ag-specific adaptive immune responses establishes a strategy for developing immunization regimens against diseases in desperate need of vaccines.