RESUMEN
Spatial organization and conformational changes of antibodies may significantly affect their biological functions. We assessed the effect of mutual organization of the two VH H domains within bispecific antibodies recognizing human TNF and the surface molecules of murine myeloid cells (F4/80 or CD11b) on TNF retention and inhibition. TNF-neutralizing properties in vitro and in vivo of MYSTI-2 and MYSTI-3 antibodies were compared with new variants with interchanged VH H domains and different linker sequences. The most effective structure of MYSTI-2 and MYSTI-3 proteins required the Ser/Gly-containing 'superflexible' linker. The orientation of the modules was crucial for the activity of the proteins, but not for MYSTI-3 with the Pro/Gln-containing 'semi-rigid' linker. Our results may contribute toward the development of more effective drug prototypes.
Asunto(s)
Células Mieloides/efectos de los fármacos , Inhibidores del Factor de Necrosis Tumoral/farmacología , Animales , Anticuerpos Biespecíficos/farmacología , Enfermedad Hepática Inducida por Sustancias y Drogas , Galactosamina , Humanos , Lipopolisacáridos , Macrófagos/efectos de los fármacos , Ratones , Tasa de Supervivencia , Factor de Necrosis Tumoral alfa/farmacologíaRESUMEN
Systemic TNF neutralization can be used as a therapy for several autoimmune diseases. To evaluate the effects of cell type-restricted TNF blockade, we previously generated bispecific antibodies that can limit TNF secretion by myeloid cells (myeloid cell-specific TNF inhibitors or MYSTIs). In this study several such variable domain (VH) of a camelid heavy-chain only antibody-based TNF inhibitors were compared in relevant experimental models, both in vitro and in vivo. Pretreatment with MYSTI-2, containing the anti-F4/80 module, can restrict the release of human TNF (hTNF) from LPS-activated bone marrow-derived macrophage (BMDM) cultures of humanized TNF knock-in (mice; hTNFKI) more effectively than MYSTI-3, containing the anti-CD11b module. MYSTI-2 was also superior to MYSTI-3 in providing in vivo protection in acute toxicity model. Finally, MYSTI-2 was at least as effective as Infliximab in preventing collagen antibody-induced arthritis. This study demonstrates that a 33 kDa bispecific mini-antibody that specifically restricts TNF secretion by macrophages is efficient for amelioration of experimental arthritis.
Asunto(s)
Anticuerpos Monoclonales/farmacología , Artritis Experimental/terapia , Antígeno CD11b/antagonistas & inhibidores , Proteínas de Unión al Calcio/antagonistas & inhibidores , Cadenas Pesadas de Inmunoglobulina/farmacología , Células Progenitoras Mieloides/efectos de los fármacos , Receptores Acoplados a Proteínas G/antagonistas & inhibidores , Inhibidores del Factor de Necrosis Tumoral/farmacología , Animales , Antirreumáticos/farmacología , Artritis Experimental/genética , Artritis Experimental/inmunología , Artritis Experimental/patología , Antígeno CD11b/genética , Antígeno CD11b/inmunología , Proteínas de Unión al Calcio/genética , Proteínas de Unión al Calcio/inmunología , Expresión Génica , Humanos , Infliximab/farmacología , Lipopolisacáridos/farmacología , Macrófagos/efectos de los fármacos , Macrófagos/inmunología , Ratones , Ratones Transgénicos , Células Progenitoras Mieloides/inmunología , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/inmunología , Factor de Necrosis Tumoral alfa/genética , Factor de Necrosis Tumoral alfa/inmunologíaRESUMEN
In spite of successful therapeutic neutralization of proinflammatory cytokines in several autoimmune diseases, such therapy is not entirely free of side effects. The main reason relates to the fact that cytokine signaling may have protective components that need to be spared. Several approaches to achieve a less damaging cytokine inhibition are being explored. In our experimental studies we are using bispecific reagents based on VHH-modules from the heavy-chain-only antibodies to limit bioavailability of TNF and IL-6 produced by myeloid cells. After evaluation of their properties in vitro and in vivo we argue that these types of reagents may have an advantage over systemic blockers.
Asunto(s)
Enfermedades Autoinmunes/metabolismo , Autoinmunidad , Citocinas/metabolismo , Células Mieloides/metabolismo , Animales , Enfermedades Autoinmunes/inmunología , Disponibilidad Biológica , HumanosRESUMEN
Binding of native bacterial protein SlyD to metal affinity matrices remains a major problem in affinity purification of His-tagged recombinant proteins from Escherichia coli cells. In this study, four novel E. coli strains that lack the expression of SlyD/SlyX, were engineered using λ-red mediated chromosomal deletion. The resultant mutant E. coli strains allow us to obtain SlyD-free proteins immediately after metal affinity chromatography, and eliminate additional purification processes. As a model protein, bispecific antibodies composed of anti-F4/80 VHH module and anti-TNF VHH module (MYSTI-2) were used. Using this protein we have shown that the SlyD/SlyX-deficient E. coli strains allow us to obtain a fully functional protein.
Asunto(s)
Escherichia coli/metabolismo , Isomerasa de Peptidilprolil/deficiencia , Proteínas Recombinantes/aislamiento & purificación , Proteínas de Escherichia coli/metabolismo , Citometría de Flujo , Humanos , Isomerasa de Peptidilprolil/metabolismo , Factor de Necrosis Tumoral alfa/antagonistas & inhibidores , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
Upregulation of the expression of tumor necrosis factor (TNF-α, TNF) has a significant role in the development of autoimmune diseases. The fluorescent antibodies binding TNF may be used for personalized therapy of TNF-dependent diseases as a tool to predict the response to anti-TNF treatment. We generated recombinant fluorescent proteins consisting of the anti-TNF module based on the variable heavy chain (VHH) of camelid antibodies fused with the far-red fluorescent protein Katushka (Kat). Two types of anti-TNF VHH were developed: one (BTN-Kat) that was bound both human or mouse TNF, but did not neutralize their activity, and a second (ITN-Kat) that was binding and neutralizing human TNF. BTN-Kat does not interfere with TNF biological functions and can be used for whole-body imaging. ITN-Kat can be evaluated in humanized mice or in cells isolated from humanized mice. It is able to block human TNF (hTNF) activities both in vitro and in vivo and may be considered as a prototype of a theranostic agent for autoimmune diseases.
RESUMEN
Proinflammatory cytokines, such as TNF, IL-6, and IL-1, play pathogenic roles in multiple diseases and are attractive targets for biologic drugs. Because proinflammatory cytokines possess non-redundant protective and immunoregulatory functions, their systemic neutralization carries the potential for unwanted side effects. Therefore, next-generation anti-cytokine therapies would seek to selectively neutralize pathogenic cytokine signaling, leaving normal function intact. Fortunately, the biology of proinflammatory cytokines provides several such opportunities. Here, we discuss various applications of bispecific antibodies targeting cytokines with specific focus on selective TNF neutralization targeted directly to the surface of specific populations of monocytes and macrophages. These bispecific antibodies combine an anti-TNF VHH with VHHs or scFvs directed against abundant surface molecules on myeloid cells and serve to limit the bioavailability of TNF produced by these cells. Such reagents may become prototypes of a novel class of anti-cytokine biologics.
RESUMEN
Overexpression of TNF contributes to pathogenesis of multiple autoimmune diseases, accounting for a remarkable success of anti-TNF therapy. TNF is produced by a variety of cell types, and it can play either a beneficial or a deleterious role. In particular, in autoimmunity pathogenic TNF may be derived from restricted cellular sources. In this study we evaluated the feasibility of cell-type-restricted TNF inhibition in vivo. To this end, we engineered MYSTI (Myeloid-Specific TNF Inhibitor)--a recombinant bispecific antibody that binds to the F4/80 surface molecule on myeloid cells and to human TNF (hTNF). In macrophage cultures derived from TNF humanized mice MYSTI could capture the secreted hTNF, limiting its bioavailability. Additionally, as evaluated in TNF humanized mice, MYSTI was superior to an otherwise analogous systemic TNF inhibitor in protecting mice from lethal LPS/D-Galactosamine-induced hepatotoxicity. Our results suggest a novel and more specific approach to inhibiting TNF in pathologies primarily driven by macrophage-derived TNF.
Asunto(s)
Anticuerpos Biespecíficos/inmunología , Antígenos de Diferenciación/inmunología , Enfermedad Hepática Inducida por Sustancias y Drogas/terapia , Macrófagos Peritoneales/metabolismo , Factor de Necrosis Tumoral alfa/antagonistas & inhibidores , Sustitución de Aminoácidos , Animales , Anticuerpos Biespecíficos/genética , Anticuerpos Neutralizantes/genética , Anticuerpos Neutralizantes/inmunología , Especificidad de Anticuerpos , Reacciones Antígeno-Anticuerpo , Antígenos de Diferenciación/genética , Antígenos de Superficie/inmunología , Camelus/inmunología , Enfermedad Hepática Inducida por Sustancias y Drogas/etiología , Enfermedad Hepática Inducida por Sustancias y Drogas/inmunología , Enfermedad Hepática Inducida por Sustancias y Drogas/prevención & control , Regiones Determinantes de Complementariedad/química , Regiones Determinantes de Complementariedad/genética , Regiones Determinantes de Complementariedad/inmunología , Galactosamina/toxicidad , Genes Sintéticos , Humanos , Células L , Macrófagos Peritoneales/inmunología , Ratones , Mutación , Distribución Aleatoria , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/inmunología , Anticuerpos de Cadena Única/genética , Anticuerpos de Cadena Única/inmunología , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
Zbed4, a member of the BED subclass of Zinc-finger proteins, is expressed in cone photoreceptors and glial Müller cells of human retina whereas it is only present in Müller cells of mouse retina. To characterize structural and functional properties of Zbed4, enough amounts of purified protein were needed. Thus, recombinant Zbed4 was expressed in E. coli and its refolding conditions optimized for the production of homogenous and functionally active protein. Zbed4's secondary structure, determined by circular dichroism spectroscopy, showed that this protein contains 32% α-helices, 18% ß-sheets, 20% turns and 30% unordered structures. CASTing was used to identify the target sites of Zbed4 in DNA. The majority of the DNA fragments obtained contained poly-Gs and some of them had, in addition, the core signature of GC boxes; a few clones had only GC-boxes. With electrophoretic mobility shift assays we demonstrated that Zbed4 binds both not only to DNA and but also to RNA oligonucleotides with very high affinity, interacting with poly-G tracts that have a minimum of 5 Gs; its binding to and GC-box consensus sequences. However, the latter binding depends on the GC-box flanking nucleotides. We also found that Zbed4 interacts in Y79 retinoblastoma cells with nuclear and cytoplasmic proteins Scaffold Attachment Factor B1 (SAFB1), estrogen receptor alpha (ERα), and cellular myosin 9 (MYH9), as shown with immunoprecipitation and mass spectrometry studies as well as gel overlay assays. In addition, immunostaining corroborated the co-localization of Zbed4 with these proteins. Most importantly, in vitro experiments using constructs containing promoters of genes directing expression of the luciferase gene, showed that Zbed4 transactivates the transcription of those promoters with poly-G tracts.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , ADN/genética , ADN/metabolismo , Proteínas Recombinantes/metabolismo , Transactivadores/metabolismo , Transcripción Genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Sitios de Unión , Línea Celular Tumoral , Citoplasma/metabolismo , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/genética , Escherichia coli/genética , Regulación de la Expresión Génica , Células HEK293 , Humanos , Ratones , Modelos Moleculares , Datos de Secuencia Molecular , Proteínas Nucleares/metabolismo , Regiones Promotoras Genéticas/genética , Replegamiento Proteico , Estructura Secundaria de Proteína , Transporte de Proteínas , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Retina/metabolismo , Especificidad por Sustrato , Transactivadores/química , Transactivadores/genética , Factores de TranscripciónRESUMEN
An RNA-based, non-cytopathic replicon vector system, based on the flavivirus Kunjin, has shown considerable promise as a new vaccine delivery system. Here we describe the testing in mice of four different SIVmac239 gag vaccines delivered by Kunjin replicon virus-like-particles. The four vaccines encoded the wild type gag gene, an RNA-optimised gag gene, a codon-optimised gag gene and a modified gag-pol gene construct. The vaccines behaved quite differently for induction of effector memory and central memory responses, for mediation of protection, and with respect to insert stability, with the SIV gag-pol vaccine providing the optimal performance. These results illustrate that for an RNA-based vector the RNA sequence of the antigen can have profound and unforeseen consequences on vaccine behaviour.
Asunto(s)
Productos del Gen gag/genética , Productos del Gen gag/inmunología , Replicón , Vacunas contra el SIDAS/genética , Vacunas contra el SIDAS/inmunología , Virus del Nilo Occidental/genética , Animales , Femenino , Vectores Genéticos , Interferón gamma/biosíntesis , Ratones , Ratones Endogámicos BALB C , Bazo/inmunología , Análisis de Supervivencia , Linfocitos T/inmunologíaRESUMEN
Envelope protein E2 of human hepatitis C virus (HCV) is an attractive component of a prototype HCV vaccine. Delivered by DNA immunogens, E2 evokes specific immune response of Th1-type, failing to induce either considerable antibody production, or T-helper cell proliferation. We aimed at modulating the immunogenic performance of E2 gene by changing the mode of protein expression in eukaryotic cells. Plasmids were constructed encoding full-length E2 and nonstructural protein 1 (p7) fused to either 13 or 38 C-terminal amino acids (aa) of HCV E1 that contain second hydrophobic segment of E1 stop-transfer signal, or a complete E1 stop-transfer signal with duplicated second hydrophobic segment. Injected into BALB/c mice, E2/p7 genes induced potent antibody and T-helper cell response targeted against hypervariable region 1, aa 472-586 of E2, and a novel epitope at aa 774-796 of p7. Profile of cytokines secreted by proliferating mouse splenocytes stimulated in vitro with E2- and p7-derived peptides, indicated mixed Th1/Th2 type of immune response. Thus, the full-length E2 and p7 genes supplied in one cassette were both immunogenic. E2/p7 containing a complete E1 stop-transfer signal with prolonged membrane spanning domain was superior to the shorter E2/p7 version in terms of both antibody and cellular immunogenicity. Optimal performance of HCV E2 could thus be achieved without the aid of external/heterologous signals by easing, through modification of the E2 signal sequence, the release of E2 from the rough ER while retaining full-length E2 and p7 sequences. This finding may help to improve the Th2 performance of HCV envelope genes as prototype vaccines.
Asunto(s)
Genes Virales , Hepacivirus/inmunología , Señales de Clasificación de Proteína/fisiología , Proteínas del Envoltorio Viral/inmunología , Vacunas contra Hepatitis Viral/inmunología , Secuencia de Aminoácidos , Animales , Células COS , Línea Celular Transformada , Transformación Celular Viral , Chlorocebus aethiops , Escherichia coli/genética , Variación Genética , Células HeLa , Hepacivirus/genética , Humanos , Inyecciones Intramusculares , Ratones , Ratones Endogámicos BALB C , Datos de Secuencia Molecular , Plásmidos , Proteínas del Envoltorio Viral/química , Proteínas del Envoltorio Viral/genética , Proteínas Virales/inmunologíaRESUMEN
Structural and functional characterization of the multidrug transporter, MexB, of Pseudomonas aeruginosa is significantly restricted due to a low yield of approximately 0.1 mg/L of culture from natural sources. To facilitate structural studies of this medically important transporter protein, we developed a large-scale system for expression of the genetically engineered recombinant, MexB, in the Escherichia coli cell. Using the system, the eventual yield of MexB attained was about 10mg/L of culture. The optimized purification protocol in the presence of dodecyl beta-D-maltoside allowed isolation of highly homogeneous MexB. The oligomeric state of the protein in detergent solution has been characterized to verify that the native state of the purified protein has been preserved. The molecular mass of the protein-detergent complex was found to be 380-450kDa. The MexB-dodecyl beta-d-maltoside mass ratio was determined to be 1.8 +/- 0.05. Taking into account the monomeric MexB molecular mass deduced from its amino acid sequence (112.8 kDa), we concluded that the purified MexB exists as the homotrimer in the surfactant solution. Circular dichroism analysis of MexB showed dominance of the alpha-helix structures. High yield, homogeneity, and stability of MexB position it as a good candidate for structural and functional characterization.
Asunto(s)
Proteínas de la Membrana Bacteriana Externa/química , Proteínas de la Membrana Bacteriana Externa/aislamiento & purificación , Proteínas de Transporte de Membrana/química , Proteínas de Transporte de Membrana/aislamiento & purificación , Proteínas de la Membrana Bacteriana Externa/genética , Secuencia de Bases , Escherichia coli/genética , Expresión Génica , Vectores Genéticos , Proteínas de Transporte de Membrana/genética , Datos de Secuencia Molecular , Plásmidos , Pseudomonas aeruginosa/enzimología , Proteínas Recombinantes/biosíntesis , Regulación hacia ArribaRESUMEN
We developed an Escherichia coli expression system for overproduction of a highly toxic membrane protein that is impossible to overexpress by traditionally used approaches. The method is based on combination of the genetic modifications of a bicistronic expression plasmid, stabilization of a synthesized protein, and selection of a compatible expression host. This enabled us to enhance the expression level of a toxic membrane protein 30-50 times compared with expression in the native state and to obtain 3-5mg of a highly purified functionally active protein per liter of culture. We describe the method for the amplified expression of membrane proteins, using the Pseudomonas aeruginosa multidrug resistance protein, MexY, as an example. The amplified MexY was correctly folded in the cytoplasmic membrane of the E. coli without forming inclusion bodies. This method can be applicable to the large-scale expression of the other problematic membrane proteins that are otherwise extremely difficult to overproduce.
Asunto(s)
Regulación Bacteriana de la Expresión Génica , Proteínas de la Membrana/metabolismo , Proteínas de la Membrana Bacteriana Externa/química , Proteínas de la Membrana Bacteriana Externa/genética , Proteínas de la Membrana Bacteriana Externa/metabolismo , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Secuencia de Bases , Resistencia a Múltiples Medicamentos , Escherichia coli/genética , Escherichia coli/metabolismo , Técnicas de Transferencia de Gen , Vectores Genéticos , Proteínas de la Membrana/genética , Proteínas de Transporte de Membrana/química , Proteínas de Transporte de Membrana/genética , Proteínas de Transporte de Membrana/metabolismo , Plásmidos/genética , Pseudomonas aeruginosa/genética , Pseudomonas aeruginosa/metabolismoRESUMEN
The interferon (IFN) response is the first line of defense against viral infections, and the majority of viruses have developed different strategies to counteract IFN responses in order to ensure their survival in an infected host. In this study, the abilities to inhibit IFN signaling of two closely related West Nile viruses, the New York 99 strain (NY99) and Kunjin virus (KUN), strain MRM61C, were analyzed using reporter plasmid assays, as well as immunofluorescence and Western blot analyses. We have demonstrated that infections with both NY99 and KUN, as well as transient or stable transfections with their replicon RNAs, inhibited the signaling of both alpha/beta IFN (IFN-alpha/beta) and gamma IFN (IFN-gamma) by blocking the phosphorylation of STAT1 and its translocation to the nucleus. In addition, the phosphorylation of STAT2 and its translocation to the nucleus were also blocked by KUN, NY99, and their replicons in response to treatment with IFN-alpha. IFN-alpha signaling and STAT2 translocation to the nucleus was inhibited when the KUN nonstructural proteins NS2A, NS2B, NS3, NS4A, and NS4B, but not NS1 and NS5, were expressed individually from the pcDNA3 vector. The results clearly demonstrate that both NY99 and KUN inhibit IFN signaling by preventing STAT1 and STAT2 phosphorylation and identify nonstructural proteins responsible for this inhibition.
Asunto(s)
Proteínas de Unión al ADN/antagonistas & inhibidores , Interferones/farmacología , Transducción de Señal , Transactivadores/antagonistas & inhibidores , Proteínas no Estructurales Virales/metabolismo , Virus del Nilo Occidental/patogenicidad , Animales , Línea Celular , Chlorocebus aethiops , Proteínas de Unión al ADN/metabolismo , Regulación de la Expresión Génica , Humanos , Factor de Transcripción STAT1 , Factor de Transcripción STAT2 , Transactivadores/metabolismo , Células Vero , Virus del Nilo Occidental/clasificaciónRESUMEN
The tripartite xenobiotic-antibiotic transporter of Pseudomonas aeruginosa consists of the inner membrane transporter (e.g., MexB, MexY), the periplasmic membrane-fusion-protein (e.g., MexA, MexX), and the outer membrane channel protein (e.g., OprM). These subunits were assumed to assemble into a transporter unit during export of the substrates. However, subunit interaction and their specificity in native form remained to be elucidated. To address these important questions, we analyzed the role of the individual subunits for the assembly of MexAB-OprM by pull-down assay tagging only one of the subunits. We found stable MexA-MexB-OprM complex without chemical cross-linking that withstand all purification procedures. Results of bi-partite interactions analysis showed tight association between MexA and OprM in the absence of MexB, whereas the expression systems lacking MexA failed to co-purify MexB or OprM. None of the heterologous subunit combinations such as MexA+MexY(his)+OprM and MexX+MexB(his)+OprM showed interaction. These results implied that the membrane fusion protein is central to the tripartite xenobiotic transporter assembly.
Asunto(s)
Proteínas de la Membrana Bacteriana Externa/metabolismo , Farmacorresistencia Bacteriana/fisiología , Proteínas de Transporte de Membrana/metabolismo , Pseudomonas aeruginosa/metabolismo , Escherichia coli/metabolismo , Mapeo de Interacción de ProteínasRESUMEN
Numerous attempts to induce immunity against HCV core (HCV-C) by DNA immunization met serious difficulties in optimizing T-helper cell and antibody responses. Immunomodulatory properties of HCV-C could be blamed that seem to be dependent on the genotype of HCV source. Here, we characterized HCV-C gene from HCV 1b isolate 274933RU. Eukaryotic expression of HCV-C was effectively driven by CMVIE, while human elongation factor 1 alpha promoter directed low levels of HCV-C expression. C57BL/6 mice were immunized with CMVIE-driven HCV-C gene, and assessed for specific antibody production, T-cell proliferation and cytokine secretion. The number and proportion of CD19+, CD3+, CD3+/CD4+, and CD3+/CD8+ splenocytes in HCV-C gene recipients was evaluated by flow cytometry. A significant mounting drop in CD3+/CD4+ T-cell counts occurred in HCV-C gene-recipients as compared to the controls. Despite that, 75% of mice exhibited core-specific cellular reactivity revealed as high proliferative responses to HCV-C and HCV-C peptides. Stimulated T-cells secreted predominantly IFN-gamma and IL-2. A shift of epitope specificity was observed with the early response being broad, and the late limited to the HCV-C C-terminus. Thus, we demonstrate both T-cell immunogenicity and T-cell modulation by core of HCV 1b. Immune modulation by HCV core may affect host ability to mount long-lasting cellular and antibody response and should be dealt with in designing core-based HCV vaccines.
Asunto(s)
Linfocitos T CD4-Positivos/inmunología , Hepatitis C/inmunología , Linfocitos T/inmunología , Proteínas del Núcleo Viral/inmunología , Vacunas Virales/inmunología , Células 3T3 , Animales , Citometría de Flujo , Inmunización , Inmunoglobulina G/biosíntesis , Ratones , Ratones Endogámicos C57BL , Oocitos , Plásmidos/inmunología , Bazo/citología , Bazo/inmunología , Proteínas Virales/inmunología , Xenopus laevisRESUMEN
Nonstructural protein 3 (NS3) of human hepatitis C virus (HCV) is a conserved multi-functional protein essential for replication and translation of viral RNA and polyprotein processing. Early T-cell response against NS3 is capable of restricting viremia. We aimed at characterizing the immunogenicity in gene immunization of the conserved regions of NS3 critical for protein folding and activity. C57BL/6 mice were injected with NS3 gene of Russian HCV 1b isolate 274933RU. Immunization did not exert any overt histological changes and had no long-term effects on the immune status of NS3 gene-recipients. The immune response in NS3 gene-recipients was screened by antibody ELISA, T-cell proliferation test and immune assays for specific cytokine production. T-lymphocytes of NS3 gene-recipients proliferated in response to peptides representing conserved regions of protease and ATPase/helicase. Stimulated T-lymphocytes produced IL-2, and in response to protease-derived peptides, also IFN-gamma. Potent and long-lasting antibody response was raised against conserved NS3 regions including "Greek-key" motif of protease, motifs II, V and polynucleotide-binding domains of ATPase/helicase. Thus, gene immunization effectively targeted conserved regions critical for NS3 protease and helicase function. In type and specificity, immune response of NS3 gene-immunized mice mimicked immunity achieved in the acute self-limiting HCV infection of human and primates and in virus-exposed healthy individuals, indicating promiscuity of NS3 as immunogen.