Santiago Dueñas-Carrera

Center for Genetic Engineering and Biotechnology, La Habana, Ciudad de La Habana, Cuba

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Publications (55)88.1 Total impact

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    ABSTRACT: Hepatitis C virus (HCV) infection is a worldwide health problem. Vaccines against this pathogen are not available and advances in this field are limited due to the high genetic variability of the virus, inaccessibility of animal models and incomplete definition of immunological correlates of protection. In the present work, a chimeric protein, Eq1, encompassing HCV amino acids regions from structural antigens, was generated. Eq1 was expressed in GC-366 bacterial cells. After cell disruption Eq1 was purified from the insoluble fraction by sequential steps of differential solubilization and metal chelating affinity chromatography. Eq1 was specifically recognized by anti-HCV positive human sera. Moreover, immunization of BALB/c mice with different doses of Eq1 formulated either in Alum or Freund's incomplete adjuvant elicited both, humoral and cellular specific immune responses. Doses of 20 μg of Eq1 induced the strongest cell mediated immune responses and only the formulation of this dose in Alum elicited neutralizing antibody response against heterologous cell culture HCV (HCVcc). All these data together indicate that Eq1 is immunogenic in mice and might be an interesting component of vaccine candidates against HCV infection. This article is protected by copyright. All rights reserved.
    Biotechnology and Applied Biochemistry 02/2014; · 1.35 Impact Factor
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    ABSTRACT: HCV is a worldwide health problem despite the recent advances in the development of more effective therapies. No preventive vaccine is available against this pathogen. However, non-sterilizing immunity has been demonstrated and supports the potential success of HCV vaccines. Induction of cross-neutralizing antibodies and T cell responses targeting several conserved epitopes, have been related to hepatitis C virus (HCV) clearance. Therefore, in this work, the immunogenicity of a preparation (MixprotHC) based on protein variants of HCV Core, E1, E2 and NS3 was evaluated in mice and monkeys. IgG from MixprotHC immunized mice and monkeys neutralized the infectivity of heterologous HCVcc. Moreover, strong CD4+ and CD8+ T cells proliferative and IFN-γ secretion responses were elicited against HCV proteins. Remarkably, immunization with MixprotHC induced control of viremia in a surrogate challenge model in mice. These results suggest that MixprotHC might constitute an effective immunogen against HCV in humans with potential for reducing the likelihood of immune escape and viral persistence.
    Vaccine 01/2014; · 3.77 Impact Factor
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    ABSTRACT: To analyze hepatitis C virus (HCV)-specific immune responses in chronically infected patients under triple therapy with interferon-α (IFN-α) plus ribavirin and CIGB-230. CIGB-230 was administered in different schedules with respect to IFN-α plus ribavirin therapy. Paired serum and peripheral blood mononuclear cells (PBMC) samples from baseline and end of treatment were analyzed. The HCV-specific humoral response was tested by enzyme-linked immunosorbent assay, neutralizing antibodies were evaluated by cell culture HCV neutralization assays, PBMC proliferation was assayed by carboxyfluorescein succinimidyl ester staining and IFN-γ secretion was assessed by enzyme-linked immunospot. Data on virological and histological response and their association with immune variables are also provided. From week 12 to week 48, all groups of patients showed a significant reduction in mean leukocyte counts. Statistically significant reductions in antibody titers were frequent, but only individuals immunized with CIGB-230 as early add-on treatment sustained the core-IgG response, and the neutralizing antibody response was enhanced only in patients receiving CIGB-230. Cell-mediated immune responses also tended to decline, but significant reductions in IFN-γ secretion and total absence of core-specific lymphoproliferation were exclusive of the control group. Only CIGB-230-immunized individuals showed de novo induced lymphoproliferative responses against the structural antigens. Importantly, it was demonstrated that the quality of the CIGB-230-induced immune response depended on the number of doses and timing of administration in relation to the antiviral therapy. Specifically, the administration of 6 doses of CIGB-230 as late add-on to therapy increased the neutralizing antibody activity and the de novo core-specific IFN-γ secretion, both of which were associated with the sustained virological response. CIGB-230, combined with IFN-α-based therapy, modifies the immune response in chronic patients. The study provides evidence for the design of more effective therapeutic vaccine interventions against HCV.
    World Journal of Gastroenterology 01/2014; 20(1):148-62. · 2.55 Impact Factor
  • Biotecnologia Aplicada 12/2012; 29(4):271-274.
  • Liz Alvarez-Lajonchere, Santiago Dueñas-Carrera
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    ABSTRACT: Hepatitis C virus (HCV) infects approximately 3% of global population. This pathogen is one of the main causes of chronic viral hepatitis, cirrhosis, and liver cancer, as well as the principal reason for liver transplant in Western countries. Therapy against HCV infection is effective in only half of treated patients. There is no vaccine available against HCV. Some vaccine candidates have reached the clinical trials but several factors, including the incomplete definition of immunological correlates of protection and treatment-related clearance have slowed down vaccine development. Precisely, the present review discusses the state of the art in the establishment of parameters related with immunity against HCV. Validity and limitations of the information accumulated from chimpanzees and other animal models, analysis of studies in humans infected with HCV, and relevance of aspects like type, strength, duration, and specificity of immune response related to successful outcome are evaluated in detail. Moreover, the immune responses induced in some clinical trials with vaccine candidates resemble the theoretical immunological correlates, raising questions about the validity of those correlates. When all facts are taken together, complete definition of immunological correlates for protection or treatment-related clearance is an urgent priority. A limited or wrong criterion with respect to this relevant matter might cause incorrect vaccine design and selection of immunization strategies or erroneous clinical evaluation.
    International Reviews Of Immunology 06/2012; 31(3):223-42. · 5.73 Impact Factor
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    ABSTRACT: Hepatitis C virus (HCV) genotypes are relevant to epidemiological questions, vaccine development, and clinical management of chronic HCV infection. In the present work, we aimed at investigating HCV genotype, variability and genetic history of HCV isolates in Cuba from a sample of chronically infected patients. A prospective study, involving 73 Cuban anti-HCV positive patients, was carried out. RT-PCR and phylogenetic analysis was employed to determine HCV genotypes. Divergence dates and demographic parameters in a Bayesian coalescent framework were estimated, as implemented in BEAST v1.4.8. HCV RNA was undetectable in 15 patients that received antiviral therapy. All HCV RNA positive patients, 58, were infected with genotype 1, three of them with subtype 1a and 55 with subtype 1b. The analysis of the DNA sequence coding for a core fragment, spanning nt positions 435-816 (relative to strain H77), revealed high percent (96.7% +/- 0.8%) nucleotide identity within Cuban HCV subtype 1b sequences. However, 56.7% and 20% of 30 analyzed individuals had changes in the core region in a six-month interval, at the nucleotide and amino acid level, respectively. Mutations involving aa changes were mainly found in the region encompassed between aa 70 and 106 of the core protein, with only one isolate showing a point mutation at position 43. Interestingly, some of the observed changes seem to be reversions and might in fact contribute to reducing the variability of this region. The estimated date for the most recent common ancestor of HCV genotype 1b Cuban isolates is 1969 (CI, 1953 to 1977). Analysis of HCV core encoding sequences from chronic patients reveals mutability of genotype 1b isolates in Cuba, which seem to be predominant and rapidly multiplied during the eighty decade of last century, and might limit the benefits obtained from current antiviral therapy.
    European review for medical and pharmacological sciences 11/2011; 15(11):1320-7. · 1.09 Impact Factor
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    ABSTRACT: Several studies advocate for the induction of strong cellular immune response against viral antigens, as well as neutralizing antibodies against HCV E2 envelope protein, as a success criteria. However, the influence of several antigens against each others in a protein preparation and the impact on the immunogenicity, have not been explored yet. In the present study a factorial design was used to generate several HCV vaccine preparations with the structural core, E1 and E2 proteins as antigens. An optimal antigen composition was chosen based on the lymphoproliferative response against HCV as well as protection against challenge with a surrogate virus in BALBC/c mice. The protein ratio (1:160:160), with 0.1 μg of Co.120-16.7 μg of E1.340-16.7 μg E2.680 (Co-E1-E2) was selected as the optimal composition to induce a functional immune response in mice. Additionally, strong humoral immune response against the 412-438 amino acid region from HCV E2 protein was detected when African green monkeys were immunized with Co-E1-E2. This region includes a conserved epitope, involved in T cell lymphoproliferative response against Co.120 and E2.680 proteins as well as in HCV neutralization. The results evidenced the relevance of proportion between HCV structural antigens in vaccine preparations for eliciting successful immune response.
    Biotecnologia Aplicada 01/2011; 28(3):180-182.
  • Yalena Amador-Cañizares, Santiago Dueñas-Carrera
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    ABSTRACT: Approximately 170 million people are infected with the hepatitis C virus (HCV) worldwide. Infection with this pathogen is persistent in more than 80% of cases, frequently developing severe forms of liver damage such as cirrhosis and hepatocellular carcinoma. No preventive vaccine is available against HCV, and current treatment based on the combination of pegylated interferon and ribavirin is effective in ∼55% of patients infected with genotype 1, the most prevalent genotype. This review analyzes several factors influencing the achievement of a sustained virological response, namely undetectable HCV RNA at 6 months after conclusion of therapy. Particularly, the relevant issue of age and duration of infection is discussed in detail. Indeed, the final decision for starting treatment should be a case-by-case point. However, the cost-benefit analysis seems to indicate that in patients who are motivated and without contraindications, starting the treatment as early as possible is probably the best choice for success.
    Journal of interferon & cytokine research: the official journal of the International Society for Interferon and Cytokine Research 11/2010; 30(11):817-24. · 1.63 Impact Factor
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    ABSTRACT: HCV (hepatitis C virus) infection is among the leading causes of chronic liver disease, but currently there is no vaccine available. Data have accumulated about the importance of targeting different HCV antigens in vaccine candidate preparations. Here, a surface response study to select the optimal ratio of recombinant HCV structural antigens in a vaccine preparation, capable of generating in vivo functional cellular immune response in mice, was performed. The immunogenicity of the selected HCV structural protein mixture (Co-E1-E2) in mice and African green monkeys, after five doses of immunization, was also demonstrated. Specific T-cell proliferative response against HCV structural antigens was induced in vaccinated mice. Moreover, on challenge with recombinant HCV VV (vaccinia virus), all mice controlled the viraemia and 80% were protected. On the other hand, monkeys immunized with Co-E1-E2 developed antibodies, specifically directed to region 412-438 of E2 protein, that include an epitope implicated in HCV neutralization, in addition to a specific proliferative response against HCV Core and E2 proteins. These results indicated that the optimal amount and ratio of HCV recombinant proteins should be taken into account to elicit a successful immune response against HCV and therefore have important implications for vaccine design.
    Biotechnology and Applied Biochemistry 07/2010; 56(3):111-8. · 1.35 Impact Factor
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    ABSTRACT: CIGB-230, a mixture of a DNA plasmid expressing hepatitis C virus (HCV) structural antigens and a HCV recombinant capsid protein, has demonstrated to elicit strong immune responses in animals. The present study evaluated the plasmid biodistribution after the administration of CIGB-230 in mice, as well as toxicity of this vaccine candidate in rats. In the biodistribution study, mice received single or repeated intramuscular injections of CIGB-230, 50 microg of plasmid DNA mixed with 5 microg of Co.120 protein. Plasmid presence was assessed in ovaries, kidney, liver, pancreas, mesenteric ganglion, blood, and muscle of the injection site by a qualitative polymerase chain reaction. The toxicology evaluation included treatment groups receiving doses 5, 15, or 50 times higher, according to the body weight, than the expected therapeutic clinical dose. During the first hour after repeated inoculation, a promiscuous distribution was observed. However, 3 months later, plasmid could not be detected in any tissue. There was an absence of detectable adverse effects on key toxicology parameters and no damage evidenced in inspected organs and tissues. These results indicate that CIGB-230 is nontoxic at local and systemic levels and no concerns about persistence are observed, which support clinical testing of this vaccine candidate against HCV.
    Human & Experimental Toxicology 10/2009; 28(8):479-91. · 1.31 Impact Factor
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    ABSTRACT: In the present study, we evaluated the safety of CIGB-230, a novel vaccine candidate based on the mixture of a plasmid for DNA immunization, expressing hepatitis C virus (HCV) structural antigens, with a recombinant HCV Core protein. Fifteen HCV chronically-infected volunteers with detectable levels of HCV RNA genotype 1b, who were nonresponders to previous treatment with interferon plus ribavirin, were intramuscularly injected with CIGB-230 on weeks 0, 4, 8, 12, 16 and 20. Individuals were also immunized at weeks 28, 32 and 36 with a recombinant vaccine against hepatitis B. Adverse events were recorded and analyzed. Blood samples were taken every 4 weeks up to month 12 for hematological, biochemical, virological and immunological analysis. All patients completed the treatment with CIGB-230. Adverse events were only slight (83.6%) or moderate (16.4%). No significant differences in hematological and biochemical parameters, including serum aminotransferases, were detected between the baseline and post-treatment state. Induction of a CD4+ T lymphocyte response against a particular region in HCV E1, spanning amino acids 230-312 in HCV polyprotein, was detected in 42.8% of patients during treatment with CIGB-230. The ability of T cells to proliferate in response to mitogenic stimulation was not weakened. Most individuals (78.6%) were seroprotected after anti-hepatitis B vaccination and 42.8% were hyper-responders (antibody titers > 100 UI/ml). No anti-mitochondrial, anti-nuclear and anti-extractable nuclear antigen antibodies were generated during immunization with CIGB-230. Vaccination with CIGB-230 in HCV chronically-infected individuals was safe, well tolerated and did not impair the ability to respond to non-HCV antigens.
    The Journal of Gene Medicine 10/2009; 12(1):107-16. · 2.16 Impact Factor
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    Liz Alvarez-Lajonchere, Santiago Dueñas-Carrera
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    ABSTRACT: Hepatitis C virus (HCV) causes chronic infection in approximately two thirds of cases, leading to chronic hepatitis, liver cirrhosis and hepatocellular carcinoma in a substantial proportion of the 170 million HCV-infected individuals worldwide. As there is neither prophylactic nor therapeutic vaccines for this virus, the research in this area is of special importance. Several vaccine candidates have been evaluated in pre-clinic, but only a few have reached the clinical evaluation. DNA immunization is one of the most evaluated approaches to obtain an effective vaccine against HCV infection. In the last few years a group of technical refinements in DNA vaccines has allowed to increase their immunogenicity. Two DNA vaccine candidates against HCV have already reached clinical evaluation, and are well tolerated and immunogenic in HCV-chronically infected individuals. The main results, opportunities and challenges of DNA immunization against HCV are discussed further in the present commentary. DNA vaccination is already a valuable tool for research HCV. Further improvements in formulation and delivery devices might be sufficient for becoming a real alternative in HCV vaccine.
    Human vaccines 09/2009; 5(8):568-71. · 3.14 Impact Factor
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    ABSTRACT: In the present work, immunogenicity of recombinant in vitro assembled hepatitis C virus core particles, HCcAg.120-VLPs, either alone or in combination with different adjuvants was evaluated in BALB/c mice. HCcAg.120-VLPs induced high titers of anti-HCcAg.120 antibodies and virus-specific cellular immune responses. Particularly, HCcAg.120-VLPs induced specific delayed type hypersensitivity, and generated a predominant T helper 1 cytokine pro file in immunized mice. In addition, HCcAg.120-VLPs prime splenocytes proliferate in vitro against different HCcAg.120-specific peptides, depending on either the immunization route or the adjuvant used. Remarkably, immunization with HCcAg.120-VLPs/Montanide ISA888 formulation resulted in a significant control of vaccinia virus titer in mice after challenge with a recombinant vaccinia virus expressing HCV core protein, vvCore. Animals immunized with this formulation had a marked increase in the number of IFN-gamma producing spleen cells, after stimulation with P815 cells infected with vvCore. These results suggest the use of recombinant HCV core particles as components of therapeutic or preventive vaccine candidates against HCV.
    Biological research 02/2009; 42(1):41-56. · 1.13 Impact Factor
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    Liz Alvarez-Lajonchere, Santiago Dueñas-Carrera
    Human Vaccines - HUM VACCINES. 01/2009; 5(8):568-571.
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    ABSTRACT: In the present study, antibody and peripheral blood mononuclear cells (PBMC) proliferative responses against hepatitis C virus (HCV) antigens were evaluated in HCV chronically infected patients. Paired serum and PBMC samples were taken six months apart from 34 individuals, either treated or not, and tested by enzyme-linked immunosorbent assay (ELISA) and carboxyfluorescein succinimidyl ester staining. Over 70% of the patients showed specific IgG and IgM against capsid, E1 and NS3, while HVR-1 was recognized by half of the patients. An increase in the levels of the anti-capsid IgM (P = 0.027) and IgG (P = 0.0006) was observed in six-month samples, compared to baseline. Similarly, a significantly higher percent of patients had detectable IgA reactivity to capsid (P = 0.017) and NS3 (P = 0.005) after six months, compared to baseline. Particularly, IgA against structural antigens positively correlated with hepatic damage (P = 0.036). IgG subclasses evaluation against capsid and NS3 revealed a positive recognition mediated by IgG1 in more than 80% of the individuals. On the contrary, less than 30% of the patients showed a positive proliferative response either of CD4+ or CD8+ T cells, being the capsid poorly recognized. These results confirm that while the cellular immune response is narrow and weak, a broad and vigorous humoral response occurs in HCV chronic infection. The observed correlation between IgA and hepatic damage may have diagnostic significance, although it warrants further confirmation.
    World Journal of Gastroenterology 12/2008; 14(44):6844-52. · 2.55 Impact Factor
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    ABSTRACT: Because the acquisition of in vitro transcription kits, for production of RNA standards, might not be affordable for some small laboratories, the current work describes some alternatives to the commercial Promega protocols. Yields of tested reactions were all higher than the ones reported for the standard Riboprobe kit (1mug RNA/mug template, 1x) and the optimized variant for high yields (5-10x). They were also as good as the ones described for the high RNA production kit RiboMAX (10-20x), exceeding them in approximately 70% of reactions. Our best results, achieved in a 500-mul format, were three to five times higher than the ones reported for the Promega kit.
    Analytical Biochemistry 11/2008; 385(1):179-81. · 2.58 Impact Factor
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    ABSTRACT: Hepatitis C virus (HCV) is a worldwide health problem. No vaccine is available against this pathogen and therapeutic treatments currently in use are of limited efficacy. In the present study, the immunogenicity of the therapeutic vaccine candidate CIGB-230, based on the mixture of pIDKE2, a plasmid expressing HCV structural antigens, with a recombinant HCV core protein, Co.120, was evaluated. CIGB-230 was administered by intramuscular injection on weeks 0, 4, 8, 12, 16 and 20 to 15 HCV-chronically infected individuals, non-responders to previous treatment with interferon (IFN) plus ribavirin. Interestingly, following the final immunization, neutralizing antibody responses against heterologous viral pseudoparticles were modified in eight individuals, including six de novo responders. In addition, 73% of vaccinees exhibited specific T cell proliferative response and T cell IFN-gamma secretory response 24 weeks after primary immunization with CIGB-230. Furthermore, 33.3% of individuals developed de novo cellular immune response against HCV core and the number of patients (46.7% at the end of treatment) with cellular immune response against more than one HCV structural antigen increased during vaccination (P = 0.046). In addition, despite persistent detection of HCV RNA, more than 40% percent of vaccinated individuals improved or stabilized liver histology, particularly reducing fibrosis, which correlated with cellular immune response against more than one HCV antigen (P = 0.0053). In conclusion, CIGB-230 is a promising candidate for effective therapeutic interventions based on its ability for enhancing the immune response in HCV chronically infected individuals.
    Journal of Viral Hepatitis 11/2008; 16(3):156-67. · 3.08 Impact Factor
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    ABSTRACT: HCV (hepatitis C virus) is a worldwide health problem nowadays. No preventive vaccine is available against this pathogen, and therapeutic treatments currently in use have important drawbacks, including limited efficacy. In the present work a recombinant fowlpox virus, FPCoE1, expressing a truncated HCV core–E1 polyprotein, was generated. FPCoE1 virus generally failed to elicit a humoral immune response against HCV antigens in BALB/c mice. By contrast, mice inoculated with FPCoE1 elicited a positive interferon-γ secretion response against HCV core in ex-vivo ELISPOT (enzyme-linked immunospot) assays. Remarkably, mice inoculated with FPCoE1 significantly controlled viraemia in a surrogate challenge model with vvRE, a recombinant vaccinia virus expressing HCV structural antigens. In fact, 40 % of the mice had no detectable levels of vvRE in their ovaries. Administration of FPCoE1 in vervet monkeys [Chlorocebus (formerly Cercophitecus) aethiops sabaeus] induced lymphoproliferative response against HCV core and E1 proteins in 50 % of immunized animals. Monkeys immunized with FPCoE1 had no detectable levels of vvRE in their blood, whereas monkeys inoculated with FP9, the negative control virus, had detectable levels of vvRE in blood up to 7 days after challenge. In conclusion, recombinant fowlpox virus FPCoE1 is able to induce an anti-HCV immune response in mice and monkeys. This ability could be rationally employed to develop effective strategies against HCV infection by using FPCoE1 in combination with other vaccine candidates or antiviral treatments.
    Biotechnology and Applied Biochemistry 01/2008; 51:97-105. · 1.35 Impact Factor
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    ABSTRACT: Currently, there are no effective tools to fight multiple pathogens due to the insufficient knowledge on their components and the limited arsenal of vaccine alternatives. In the present paper, combinations of recombinant viral antigens and nucleic acids are studied using electron microscopy, electrophoresis, nuclease digestion and sucrose or cesium chloride gradients. We describe new data on morphology, size, and the increased stability of protein particles composed of viral antigens in close association with nucleic acids. We demonstrate, for the first time, that particles of recombinant HCV viral core proteins are able to interact with plasmid molecules for DNA immunization, forming complexes of particles with increased density and size. Additionally, the increased immunogenicity of these recombinant viral protein-plasmid DNA mixtures for DNA immunization was evidenced when administered in animal models, compared to the individual components. In addition, we discuss the possible mechanisms behind the enhancement of the immune response, based on the experimental evidence of the interaction between mixture components and their properties. The use of recombinant viral proteins interacting with nucleic acids as the active principle, and simultaneously as an adjuvant or molecular vehicle for DNA vaccines, has important implications for the development of new effective strategies to prevent and treat diseases caused by different pathogens.
    Biotecnologia Aplicada 07/2007; 24(3-4-ISSN 1027-2852):311-314.
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    ABSTRACT: Production of immunogenic hepatitis C virus (HCV) envelope proteins will assist in the future development of preventive or therapeutics applications. Only properly folded monomeric E2 protein is able to bind a putative cellular co-receptor CD81, but this interaction may modulate cell immune function. Recombinant E2 proteins, similar to the native form, but lacking undesirable immunoregulatory features, might be promising components of vaccine candidates against HCV. To obtain E2 suitable for structural as well as functional studies, a recombinant E2 variant (E2680) was produced in Pichia pastoris cells. E2680, comprising amino acids 384 to 680 of the HCV polyprotein, was secreted into the culture supernatant in the N-glycosilated form and was mainly composed of disulfide-linked multimers. Both monomeric and oligomeric forms of E2680 were recognized by conformational-sensitive MAb H53. In addition, antibodies in sera from 70% of HCVpositive patients were reactive against E2680. By immunizing E2680 in BALB/c mice, both a specific cellular immune response and anti-E2680 IgG antibody titers of 1:200,000 were induced. Our data suggest that recombinant E2680 could be useful to successfully induce strong anti-HCV immunity.
    Molecular Biotechnology 04/2007; 35(3):225-35. · 2.26 Impact Factor