RESUMO
The presence of hepatitis C virus (HCV) negative strand RNA in extrahepatic compartments based on PCR detection assays has been suggested in many reports with a very heterologous detection rate (from 0 to 100%). In this study, we have analyzed the presence of HCV negative strand in hepatic (liver biopsies, n = 20) and extrahepatic (sera, n = 32; PBMC, n = 26 and fresh bone marrow cells, n = 8) compartments from infected patients with three different reverse transcriptase (RT)-PCR-based assays using primers located in the 5' noncoding region, with or without a tag selected to display different viral loads (10(5)-3 x 10(7) genomic equivalent/ml or gram) and viral genotypes (n = 5). Using synthetic as well as biological templates, we could document extensive artifactual detection of negative strand RNA, due to self priming and mispriming events, even either 5' noncoding region primer pair was used, whereas both artifacts were dramatically reduced (mispriming) or eliminated (selfpriming) using CAP-based RT-PCR assay. Mispriming artifacts were directly correlated to the titer of positive strand RNA present in the sample. Using the CAP-PCR assay, the presence of HCV negative strand RNA was found in 75% of livers (16:20) and only 8% of PBMC, independent of the genotype involved, but could not be documented in sera (0:32) and fresh bone marrow cells (0:6). These findings suggest that caution regarding the type of RT-PCR assay used and the level of HCV positive strand RNA present in the biological sample analyzed has to be taken to avoid false identification of viral reservoirs. The findings suggest that hematopoietic peripheral cells can support HCV replication, although in a very limited number of carriers.
Assuntos
Medula Óssea/virologia , Hepatite C/virologia , Reação em Cadeia da Polimerase/métodos , RNA Viral/isolamento & purificação , Artefatos , Sequência de Bases , Primers do DNA , Hepacivirus/crescimento & desenvolvimento , Hepatite C/sangue , Hepatite C/genética , Humanos , Leucócitos Mononucleares/virologia , Fígado/virologia , Dados de Sequência Molecular , Sensibilidade e EspecificidadeRESUMO
We analyzed different vaccine approaches aimed at enhancing CD4(+)- and CD8(+)-dependent responses against hepatitis C virus (HCV) core antigen. Specific DNA vectors expressing various forms of the core in fusion with the ubiquitin or the lysosome-associated membrane protein (LAMP) were generated. These expressed the full-length wildtype core; the full-length core expressed as a covalent fusion with the ubiquitin; the full-length core expressed as a noncovalent fusion with the ubiquitin and containing a N-stabilizing or N-destabilizing residue; and the full-length core expressed as a fusion with the LAMP sequence. In vitro expression levels of the different plasmids differed by as much as tenfold. After injection into mice, none of the plasmids yielded a detectable antibody response, whereas core-specific cytotoxic T-lymphocyte (CTL) activity could be observed with all plasmids as long as 21 weeks postimmunization. No increase in CTL activity (ranging from 7% to 34% specific lysis) was observed with the ubiquitin-fusion-expressed core antigens compared with the wildtype core. The lowest CTL activity (< 5% specific lysis) was observed with the LAMP fusion. This vector was nonetheless unable to induce a detectable proliferative response. Screening of 10 different putative CTL peptide epitopes failed to reveal newly targeted epitopes when the core-fusion plasmids were used compared with the wildtype core-expressing plasmid. These data underline the difficulty in optimizing anti-core cellular immune response using molecular targeting strategies in DNA-based vaccination.
Assuntos
Apresentação de Antígeno , Hepacivirus/imunologia , Antígenos de Histocompatibilidade Classe II/imunologia , Antígenos de Histocompatibilidade Classe I/imunologia , Vacinas de DNA/imunologia , Proteínas do Core Viral/imunologia , Animais , Antígenos CD/biossíntese , Antígenos CD/genética , Antígenos CD/metabolismo , Transporte Biológico , Células Cultivadas , Citocinas/biossíntese , Citocinas/imunologia , Mapeamento de Epitopos , Feminino , Hepacivirus/genética , Hepatite C/imunologia , Hepatite C/prevenção & controle , Hepatite C/virologia , Antígenos da Hepatite C/biossíntese , Antígenos da Hepatite C/genética , Antígenos da Hepatite C/imunologia , Ativação Linfocitária/imunologia , Proteínas de Membrana Lisossomal , Glicoproteínas de Membrana/biossíntese , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Sinais Direcionadores de Proteínas , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/imunologia , Proteínas Recombinantes de Fusão/metabolismo , Linfócitos T Citotóxicos/imunologia , Ubiquitinas/biossíntese , Ubiquitinas/genética , Ubiquitinas/metabolismo , Vacinas de DNA/genética , Proteínas do Core Viral/biossíntese , Proteínas do Core Viral/genética , Vacinas contra Hepatite Viral/genética , Vacinas contra Hepatite Viral/imunologiaRESUMO
Interactive glycoproteins present on the surface of viral particles represent the main target of neutralizing antibodies. The ability of DNA vaccination to induce antibodies directed at such structures was investigated by using eight different expression plasmids engineered either to favor or to prevent interaction between the hepatitis C virus (HCV) envelope glycoproteins E1 and E2. Independently of the injection route (intramuscular or intraepidermal), plasmids expressing antigens capable of forming heterodimers presumed to be the prebudding form of the HCV envelope protein complex failed to induce any significant, stable antibodies following injection in mice. In sharp contrast, high titers of antibodies directed at both conformational and linear determinants were induced by using plasmids expressing severely truncated antigens that have lost the ability to form native complexes. In addition, only a truncated form of E2 induced antibodies reacting against the hypervariable region 1 of E2 (specifically with the C-terminal part of it) known to contain a neutralization site. When injected intraepidermally into small primates, the truncated E2-encoding plasmid induced antibodies able to neutralize in vitro the binding of a purified E2 protein onto susceptible cells. Because such antibodies have been associated with viral clearance in both humans and chimpanzees, these findings may have important implications for the development of protective immunity against HCV.
Assuntos
DNA Viral/imunologia , Hepacivirus/imunologia , Anticorpos Anti-Hepatite C/biossíntese , Vacinas de DNA/imunologia , Proteínas do Envelope Viral/imunologia , Vacinas contra Hepatite Viral/imunologia , Animais , Células CHO , Cricetinae , Citocinas/biossíntese , Mapeamento de Epitopos , Feminino , Anticorpos Anti-Hepatite C/imunologia , Antígenos da Hepatite C/imunologia , Humanos , Imunoglobulina G/imunologia , Isotipos de Imunoglobulinas/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Testes de Neutralização , Plasmídeos , Saguinus , Baço/imunologia , Células Tumorais Cultivadas , Vacinação , Proteínas do Envelope Viral/genéticaRESUMO
We have explored different domains within the hepatitis C virus (HCV) 5' noncoding region as potential targets for inhibition of HCV translation by antisense oligodeoxynucleotides (ODNs). Inhibition assays were performed with two different cell-free systems, rabbit reticulocyte lysate and wheat germ extract, and three types of chemical structures for the ODNs were evaluated: natural phosphodiesters (beta-PO), alpha-anomer phosphodiesters (alpha-PO), and phosphorothioates (PS). A total of six original ODNs, displaying sequence-specific inhibition ranging from 62 to 96%, that mapped in the pyrimidine-rich tract (nucleotides [nt] 104 to 127) and in the initiator AUG codon (nt 338 to 357) were identified. Two ODNs, which were targeted at the initiatory AUG (nt 341 to 367 and 351 to 377) and which had been previously described as active against genotype 1b and 2a sequences, were shown to exhibit inhibition of expression (> 95%) of a type 1a sequence. Control experiments with the irrelevant chloramphenicol acetyltransferase sequence as a marker and randomized ODNs demonstrated that levels of inhibition associated with the use of PS compounds (of as much as 94%) were mainly due to nonspecific effects. Both alpha- and beta-PO ODNs were found equally active, and no difference could be seen in the activity of beta-PO when it was tested in either rabbit reticulocyte lysate or wheat germ extract, suggesting that RNase H-independent mechanisms may be involved in the inhibitions observed. However, specific RNA cleavage products generated from beta-PO inhibition experiments could be identified, indicating that, with these compounds, control of translation also involves RNase H-dependent mechanisms. This study further delimits the existence of favorable target sequences for the action of ODNs within the HCV 5' noncoding region and indicates the possibility of using nuclease-resistant alpha-PO compounds in cellular studies.
Assuntos
Regulação Viral da Expressão Gênica/efeitos dos fármacos , Genes Virais/efeitos dos fármacos , Hepacivirus/genética , Oligonucleotídeos Antissenso/farmacologia , Animais , Sequência de Bases , Hepacivirus/efeitos dos fármacos , Dados de Sequência Molecular , Oligonucleotídeos Antissenso/síntese química , Plasmídeos/genética , Biossíntese de Proteínas/efeitos dos fármacos , Coelhos , Ribonuclease H/metabolismo , Transcrição Gênica/efeitos dos fármacosRESUMO
Different delivery routes of plasmid DNA may result in the induction of differential humoral and cellular immunity. We have studied the influence of two main routes of plasmid injection, performed intramuscularly and intraepidermally using a gene gun, for the induction of immune responses specific to hepatitis C virus (HCV) envelope protein E2. Three plasmids expressing different immunogenic domains of E2 (amino acids [aa] 384443, aa 504-555, and aa 384-746) were injected into BALB/c mice according to five different protocols using various combinations of intramuscular (i.m.) or intraepidermal (i.e.) primary and booster injections. Seroconversion rates, antibody titers and isotypes, epitope recognition, and T-helper (Th) release cytokine profiles were analyzed. Antibody titers and epitope recognition were linked to either or both the nature of the immunogen expressed and the delivery route chosen. In all cases, the lowest antibody titers were obtained using single i.m.-based protocols. Independently of the antibody titers generated, only some specific i.e.-combined delivery routes induced antibodies able to recognize determinants located in the N-terminal of E2 (aa 384411 and aa 411437) and mimicked by synthetic peptides. By contrast, the antibody isotypes and the splenic cytokine production identified were independent of the plasmids used and the delivery route implemented. All conditions resulted in Th-1 like responses suggested by the exclusive detection of IgG2a and 2b antibodies and the production of interferon gamma (INF-gamma) but no interleukin-4 (IL-4). Overall, our results suggest that the combination of i.m. and i.e. delivery routes provides the most efficient way to induce a broad immune response against HCV-E2.
Assuntos
DNA Viral/administração & dosagem , Hepacivirus/metabolismo , Plasmídeos/genética , Proteínas do Envelope Viral/genética , Proteínas do Envelope Viral/imunologia , Animais , Formação de Anticorpos/fisiologia , Biolística , Citocinas/biossíntese , DNA Viral/farmacologia , Epitopos/imunologia , Feminino , Anticorpos Anti-Hepatite C/análise , Imunização/métodos , Injeções , Injeções Intradérmicas , Injeções Intramusculares , Camundongos , Camundongos Endogâmicos BALB C , Baço/citologia , Baço/imunologia , Baço/metabolismo , Proteínas do Envelope Viral/metabolismoRESUMO
BACKGROUND/METHODS: To characterize the repertoire of T-cell epitopes on the hepatitis C virus (HCV) core protein, we studied major histocompatibility complex (MHC) class I binding of 75 decapeptides on 20 human B-cell lines and murine spleen cells using a flow cytometric assay. The results were compared with MHC class I stabilization on T2 cells, the SYFPEITHI algorithm, and known T-cell epitopes from the literature. RESULTS: Binding of peptides proved to be specific for MHC class I molecules. We observed peak fluorescence signals at positions amino acids (aa) 35-44, aa 87-96, aa 131-140, and aa 167-176 in virtually all HLA-A2-positive cell lines. These sites corresponded to T-cell epitopes predicted by SYFPEITHI and the positions of known T-cell epitopes, whereas T2 stabilization was at variance for two peptides. The assay was applied to HLA-A2-negative cells and murine spleen cells without further modification, and identified additional peptides, corresponding to known T-cell epitopes. CONCLUSIONS: Peptide binding to different MHC class I alleles can be mapped rapidly by a flow cytometric assay and enables a first orientation on the sites of possible T-cell epitopes. Application of this assay to HCV core suggests a rather limited repertoire of epitopes in the Caucasoid population.
Assuntos
Mapeamento de Epitopos/métodos , Epitopos de Linfócito T/metabolismo , Citometria de Fluxo/métodos , Antígenos de Histocompatibilidade Classe I/metabolismo , Proteínas do Core Viral/imunologia , Proteínas do Core Viral/metabolismo , Animais , Linfócitos B/imunologia , Ligação Competitiva , Biotinilação , Células Cultivadas , Epitopos de Linfócito T/imunologia , Corantes Fluorescentes , Antígenos de Histocompatibilidade Classe I/imunologia , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Oligopeptídeos/imunologia , Oligopeptídeos/metabolismo , Ligação Proteica , Baço/imunologia , Linfócitos T Citotóxicos/imunologia , Linfócitos T Citotóxicos/metabolismoRESUMO
Replicating and nonreplicating nucleic acid-based vaccines as well as Semliki Forest-recombinant Viruses (rSFVs) were evaluated for the development of a vaccine against hepatitis C virus (HCV). Replicating SFV-DNA vaccines (pSFV) and rSFVs expressing HCV core or E2 antigens were compared with classical CMV-driven plasmids (pCMV) in single or bimodal vaccine protocols. In vitro experiments indicated that all vaccine vectors produced the HCV antigens but to different levels depending on the antigen expressed. Both replicating and nonreplicating core-expressing plasmids induced, upon injection in mice, specific comparable CTL responses ranging from 10 to 50% lysis (E:T ratio 100:1). Comparison of different injection modes (intramuscular versus intraepidermal) and the use of descalating doses of DNA (1-100 microgram) did not show an increased efficacy of the core-SFV plasmid compared with the CMV-driven one. Surprisingly, rSFVs yielded either no detectable anticore CTL or very low anti-E2 antibody titers following either single or bimodal administration together with CMV-expressing counterparts. Prime-boost experiments revealed, in all cases, the superiority of DNA-based only vaccines. The anti-E2 antibody response was evaluated using three different assays which indicated that all generated anti-E2 antibodies were targeted at similar determinants. This study emphasizes the potential of DNA-based vaccines for induction of anti-HCV immune responses and reveals an unexpected and limited benefit of SFV-based vaccinal approaches in the case of HCV core and E2.