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1.
Vaccine ; 25(42): 7339-53, 2007 Oct 16.
Artigo em Inglês | MEDLINE | ID: mdl-17875349

RESUMO

We designed and evaluated in HLA-class I transgenic mouse models a hepatitis C virus (HCV) T cell-based MVA vectored vaccine expressing three viral antigens known to be targets of potent CD8+- and CD4+-mediated responses. An accelerated (3 week-based) vaccination induced specific CD8+ T cells harboring two effector functions (cytolytic activity - both in vitro and in vivo- and production of IFNgamma) as well as specific CD4+ T cells recognizing all three vaccine antigens. Responses were long lasting (6 months), boostable by a fourth MVA vaccination and in vivo cross-reactive as demonstrated in a surrogate Listeria-based challenge assay. This candidate vaccine has now moved into clinical trials.


Assuntos
Hepacivirus/imunologia , Esquemas de Imunização , Linfócitos T/imunologia , Vacinas contra Hepatite Viral/administração & dosagem , Sequência de Aminoácidos , Animais , Linfócitos T CD8-Positivos/imunologia , Reações Cruzadas , Vetores Genéticos , Antígeno HLA-A2/genética , Antígeno HLA-B7/genética , Hepacivirus/genética , Antígenos da Hepatite C/genética , Humanos , Imunização Secundária , Listeria monocytogenes/genética , Listeria monocytogenes/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Transgênicos , Linfócitos T Citotóxicos/imunologia , Vacinas Sintéticas/administração & dosagem , Vacinas Sintéticas/genética , Vacinas Sintéticas/imunologia , Vaccinia virus/genética , Vacinas contra Hepatite Viral/genética , Vacinas contra Hepatite Viral/imunologia
2.
Vaccine ; 24(16): 3153-64, 2006 Apr 12.
Artigo em Inglês | MEDLINE | ID: mdl-16481078

RESUMO

To develop a vaccine against hepatitis C virus, we synthesized four long peptides from nonstructural proteins NS3, NS4 and NS5B containing HLA-class I and class II epitopes mainly inducing responses in natural infection. In HLA-A2.1 transgenic mice, the four peptides primed higher CTL responses to 6:7 minimal HLA-A2 epitopes than those induced by the minimal epitopes. HLA-A2.1/HLA-DR1 transgenic mice immunized with one peptide, containing a class II epitope implicated in viral resolution, developed IFNgamma-producing CD4+-T and CD8+-T cells. These peptides recalled HCV-specific IFNgamma-producing cells from HCV-infected patients' PBMC. This support the selection of these domains for inclusion in a vaccine formulation.


Assuntos
Linfócitos T CD4-Positivos/imunologia , Epitopos/imunologia , Hepacivirus/imunologia , Memória Imunológica , Linfócitos T Citotóxicos/imunologia , Vacinas contra Hepatite Viral/imunologia , Proteínas não Estruturais Virais/imunologia , Adulto , Animais , Citotoxicidade Imunológica , Epitopos de Linfócito T/imunologia , Antígeno HLA-A2/genética , Antígeno HLA-DR1/genética , Hepatite C/imunologia , Humanos , Interferon gama/biossíntese , Camundongos , Camundongos Transgênicos , Pessoa de Meia-Idade , Proteínas Recombinantes/administração & dosagem , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Proteínas não Estruturais Virais/administração & dosagem , Proteínas não Estruturais Virais/genética
3.
J Virol ; 78(1): 187-96, 2004 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-14671100

RESUMO

Prophylactic hepatitis C virus (HCV) vaccine trials with human volunteers are pending. There is an important need for immunological end points which correlate with vaccine efficacy and which do not involve invasive procedures, such as liver biopsies. By using a multicomponent DNA priming-protein boosting vaccine strategy, naïve chimpanzees were immunized against HCV structural proteins (core, E1, and E2) as well as a nonstructural (NS3) protein. Following immunization, exposure to the heterologous HCV 1b J4 subtype resulted in a peak of plasma viremia which was lower in both immunized animals. Compared to the naïve infection control and nine additional historical controls which became chronic, vaccinee 2 (Vac2) rapidly resolved the infection, while the other (Vac1) clearly controlled HCV infection. Immunization induced antibodies, peptide-specific gamma interferon (IFN-gamma), protein-specific lymphoproliferative responses, IFN-gamma, interleukin-2 (IL-2), and IL-4 T-helper responses in both vaccinees. However, the specificities were markedly different: Vac2 developed responses which were lower in magnitude than those of Vac1 but which were biased towards Th1-type cytokine responses for E1 and NS3. This proof-of-principle study in chimpanzees revealed that immunization with a combination of nonstructural and structural antigens elicited T-cell responses associated with an alteration of the course of infection. Our findings provide data to support the concept that the quality of the response to conserved epitopes and the specific nature of the peripheral T-helper immune response are likely pivotal factors influencing the control and clearance of HCV infection.


Assuntos
Hepacivirus/imunologia , Hepatite C Crônica/prevenção & controle , Células Th1/imunologia , Células Th2/imunologia , Vacinas contra Hepatite Viral/imunologia , Animais , Citocinas/biossíntese , Hepacivirus/patogenicidade , Anticorpos Anti-Hepatite C/sangue , Antígenos da Hepatite C/imunologia , Hepatite C Crônica/imunologia , Humanos , Imunização , Ativação Linfocitária/imunologia , Pan troglodytes , Proteínas do Core Viral/imunologia , Vacinas contra Hepatite Viral/administração & dosagem , Proteínas não Estruturais Virais/imunologia
4.
Hepatology ; 34(6): 1206-17, 2001 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-11732011

RESUMO

The immunogenicity of the Hepatitis C virus (HCV) nonstructural protein 3 (NS3) was investigated using different DNA-based strategies and a preclinical mouse model transgenic for the HLA-A2.1 molecule. Plasmids expressing NS3 either as a wild-type protein, as a fusion with murine lysosome-associated-membrane protein-1 specific sequences, or under the control of the Semliki Forest virus replicase were evaluated in vitro and in vivo. All plasmids were shown to express the expected size protein. These 3 NS3-expressing vaccines induced overall comparable levels of CTLs when measured at different times postvaccination although mice injected with the NS3-LAMP expressing plasmid showed a particularly homogeneous and overall vigorous response (specific lysis ranged from 60% to 90 % for an E:T ratio of 33.3:1 with a mean CTL precursor frequency of 1:2.10(5) cells). Out of the four HLA-A2.1-restricted NS3 epitopes previously described in HCV infected patients (aa 1073-1081, aa 1406-1415; aa 1169-1177 and aa 1287-1296), the NS3-DNA generated CTLs were predominantly targeted at the aa 1073-1081 epitope. Peptide-based immunization showed that the mouse repertoire was intact for all epitopes tested except one (aa 1287-1296). In conclusion, the 3 NS3-DNA vaccines although based on different mode of action, shared a comparable efficacy at inducing CTL. Surprisingly, the breadth of such response was restricted to a single, major epitope.


Assuntos
Epitopos/imunologia , Antígeno HLA-A2/análise , Linfócitos T Citotóxicos/imunologia , Vacinas de DNA/imunologia , Proteínas não Estruturais Virais/imunologia , Células 3T3 , Animais , Formação de Anticorpos , Feminino , Antígeno HLA-A2/genética , Imunização , Interferon gama/biossíntese , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C3H , Camundongos Transgênicos/genética , Linfócitos T/metabolismo
5.
J Virol ; 75(24): 12088-97, 2001 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-11711599

RESUMO

Deglycosylation of viral glycoproteins has been shown to influence the number of available epitopes and to modulate immune recognition of antigens. We investigated the role played by N-glycans in the immunogenicity of hepatitis C virus (HCV) E1 envelope glycoprotein, a naturally poor immunogen. Eight plasmids were engineered, encoding E1 protein mutants in which the four N-linked glycosylation sites of the protein were mutated separately or in combination. In vitro expression studies showed an influence of N-linked glycosylation on expression efficiency, instability, and/or secretion of the mutated proteins. Immunogenicity of the E1 mutants was studied in BALB/c mice following intramuscular and intraepidermal injection of the plasmids. Whereas some mutations had no or only minor effects on the antibody titers induced, mutation of the fourth glycosylation site (N4) significantly enhanced the anti-E1 humoral response in terms of both seroconversion rates and antibody titers. Moreover, antibody induced by the N4 mutant was able to recognize HCV-like particles with higher titers than those induced by the wild-type construct. Epitope mapping indicated that the E1 mutant antigens induced antibody directed at two major domains: one, located at amino acids (aa) 313 to 332, which is known to be reactive with sera from HCV patients, and a second one, located in the N-terminal domain of E1 (aa 192 to 226). Analysis of the induced immune cellular response confirmed the induction of gamma interferon-producing cells by all mutants, albeit to different levels. These results show that N-linked glycosylation can limit the antibody response to the HCV E1 protein and reveal a potential vaccine candidate with enhanced immunogenicity.


Assuntos
Anticorpos Antivirais/biossíntese , Proteínas do Envelope Viral/imunologia , Sequência de Aminoácidos , Animais , Feminino , Glicosilação , Imunização , Imunoglobulina G/biossíntese , Imunoglobulina G/classificação , Interferon gama/biossíntese , Camundongos , Camundongos Endogâmicos BALB C , Dados de Sequência Molecular , Mutação , Proteínas do Envelope Viral/química , Vacinas contra Hepatite Viral/imunologia
6.
Virology ; 276(2): 259-70, 2000 Oct 25.
Artigo em Inglês | MEDLINE | ID: mdl-11040118

RESUMO

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.


Assuntos
Hepacivirus/imunologia , Vírus da Floresta de Semliki/genética , Vacinas de DNA/imunologia , Proteínas do Core Viral/imunologia , Proteínas do Envelope Viral/imunologia , Vacinas contra Hepatite Viral/imunologia , Animais , DNA Viral/biossíntese , Desenho de Fármacos , Feminino , Anticorpos Anti-Hepatite B/biossíntese , Anticorpos Anti-Hepatite B/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Vacinas de DNA/genética , Vacinas Sintéticas/genética , Vacinas Sintéticas/imunologia
7.
J Virol ; 73(9): 7497-504, 1999 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-10438839

RESUMO

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/genética
8.
J Gen Virol ; 80 ( Pt 4): 887-896, 1999 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-10211957

RESUMO

The hepatitis C virus (HCV) genome encodes two membrane-associated envelope glycoproteins (E1 and E2), which are released from the viral polyprotein precursor by host signal peptidase cleavages. These glycoproteins interact to form a noncovalent heterodimeric complex, which is retained in the endoplasmic reticulum. HCV glycoproteins, E1 and E2, are heavily modified by N-linked glycosylation. A recent study has revealed that upon partial deglycosylation with endoglycosidase H only four of the five potential glycosylation sites of HCV glycoprotein E1 are utilized. In this work, the unused glycosylation site on the E1 glycoprotein was identified and the influence of N-linked glycosylation on the formation of the HCV glycoprotein complex was studied by expressing a panel of E1 glycosylation mutants in HepG2 cells. Each of the five potential N-linked glycosylation sites, located at amino acid positions 196, 209, 234, 305 and 325, respectively, on the HCV polyprotein, was mutated separately as well as in combination with the other sites. Expression of the mutated E1 proteins in HepG2 cells indicated that the fifth glycosylation site is not used for the addition of N-linked oligosaccharides and the Pro immediately following the sequon (Asn-Trp-Ser) precludes core glycosylation. The effect of each mutation on the formation of noncovalent E1E2 complexes was also analysed. As determined with the use of a conformation-sensitive monoclonal antibody, mutations at positions N2 and N3 had no, or only minor, effects on the assembly of the E1E2 complex, whereas a mutation at position N1 and predominantly at position N4 dramatically reduced the efficiency of the formation of noncovalent E1E2 complexes.


Assuntos
Glicoproteínas/metabolismo , Hepacivirus/química , Proteínas do Envelope Viral/metabolismo , Animais , Proteínas de Ligação ao Cálcio/metabolismo , Calnexina , Linhagem Celular , Glicosilação , Camundongos , Mutagênese Sítio-Dirigida , Conformação Proteica , Coelhos , Relação Estrutura-Atividade
9.
Virology ; 249(1): 32-41, 1998 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-9740774

RESUMO

Both linear and conformational determinants of hepatitis C virus (HCV) are believed to be involved in viral neutralization. After immortalization of B cells from HCV chronically infected patients with Epstein-Barr virus, we obtained two polyclonal lymphoblastoid cell lines (LCL) secreting human monoclonal antibodies (HMabs). One clone was derived from a patient infected with a genotype 4 isolate while the second was isolated from a genotype 1b-infected patient. Immunoprecipitation studies, Western blot, and immunofluorescence analysis, peptide scanning, and ELISA studies indicated that the HMabs (1) recognized conformation-dependent determinant(s), (2) were capable of recognizing genotype 1a and 1b derived antigens, and (3) were able to precipitate noncovalently associated E1E2 complexes believed to exist on the surface of virion particles. The HMab derived from the genotype 4-infected patient was in addition shown to neutralize the in vitro binding of recombinant E2 protein onto susceptible cells suggesting a potential for in vivo neutralization. These data indicate that anti-E2 antibodies directed at conserved conformational-dependent determinant(s) exist in chronic HCV infection.


Assuntos
Anticorpos Antivirais/imunologia , Hepacivirus/imunologia , Proteínas do Envelope Viral/imunologia , Anticorpos Monoclonais/imunologia , Sítios de Ligação/imunologia , Mapeamento de Epitopos , Humanos , Epitopos Imunodominantes
10.
Hepatology ; 28(1): 237-44, 1998 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-9657118

RESUMO

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/metabolismo
11.
J Virol ; 71(9): 7101-9, 1997 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-9261444

RESUMO

Plasmids expressing different domains of the hepatis C virus (HCV) envelope E2 glycoprotein from a genotype 1a isolate were constructed to compare the immunogenic potential of E2 in nucleic acid-based immunizations. One plasmid, pCIE2t, expressed a C-terminally truncated form of E2, while others, pS2.SE2A to pS2.SE2E, encoded the adjacent 60-amino-acid (aa) sequences of E2 (inserts A to E) expressed as a fusion with the hepatitis B virus surface antigen. BALB/c mice were given injections of the plasmids intramuscularly (i.m.) or intraepidermally (i.e.) via a gene gun (biolistic introduction), and induced humoral immune responses were evaluated. The i.e. injections resulted in higher seroconversion rates and antibody titers, up to 100-fold, than did the i.m. injections (P = 0.01 to 0.04). Three restricted immunogenic domains, E2A (aa 384 to 443), E2C (aa 504 to 555), and E2E (aa 609 to 674), that yielded antibody titers ranging from 1:59 to > 1:43,700 could be identified. Subtype 1a- and 1b-derived E2 antigens and synthetic peptides were used in Western blot and enzyme-linked immunosorbent assay analyses, which revealed that the cross-reactivity of the plasmid-induced antibodies was linked both to the type of antigen expressed and to the injection mode. Induced anti-E2 antibodies could immunoprecipitate noncovalent E1E2 complexes believed to exist on the surface of HCV virions. This study allowed us to identify restricted immunogenic domains within E2 and demonstrated that different routes of injection of HCV E2 plasmids can result in quantitatively and qualitatively different humoral immune responses.


Assuntos
Hepacivirus/imunologia , Anticorpos Anti-Hepatite C/imunologia , Plasmídeos , Vacinas de DNA/imunologia , Proteínas do Envelope Viral/genética , Vacinas contra Hepatite Viral/imunologia , Animais , Biolística , Mapeamento de Epitopos , Hepacivirus/genética , Antígenos da Hepatite C/genética , Imunização , Injeções Intramusculares , Camundongos , Camundongos Endogâmicos BALB C , Vacinas de DNA/genética
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