Juan Morales

A truncated variant of the hepatitis C virus core induces a slow but potent immune response in mice following DNA immunization

Vaccination of BALB/c mice with pIDKCo, a plasmid containing the coding sequence for the first 176 amino acids of the hepatitis C virus (HCV) core protein, induced both humoral and cellular specific immune responses. Particularly, the level of anti-core antibodies increased slowly with time up to a mean value above 1:8000 that was generally superior than that found in anti-HCV positive individuals. Six out of nine anti-HCV positive human sera were able to inhibit at different extent the binding of mouse anti-core sera to a recombinant capsid protein. Our results show that it is possible to elicit a potent humoral and cellular immune response against the HCV core antigen in mice following DNA immunization.

Improvement of human interferon HUIFNα2 and HCV core protein expression levels in Escherichia coli but not of HUIFNα8 by using the tRNAAGA/AGG

Abstract High-level expression from one particular heterologous gene in Escherichia coli generally requires the optimization of codon usage. Genes encoding for Hepatitis C virus core protein (HCcAg), human interferon α2 and 8 subtypes (HUIFNα2 and HUIFNα8) show a high content of AGA/AGG codons. These are encoded by the product of the dnaY gene in E. coli. The proteins used in this work have a high therapeutic value and were used as models for studying the effects of these rare codons on the efficiency of heterologous gene expression in E. coli. Expression plasmids were constructed to express any of these proteins and the dnaY gene product simultaneously in E. coli. After dnaY gene expression, HCcAg, and HUIFNα2 expression levels increased 5 and 3 times, respectively. However, HUIFNα8 expression was barely detected either supplying or not the additional dnaY gene product. These results suggest that the high frequency of AGA/AGG codons present in the HCcAg and HUIFNα2 genes could be one of the factors limiting its expression in E. coli. Nevertheless, for HUIFNα8 it seems that other factors prevail upon the lack of dnaY product. Data presented here for HCcAg and HUIFNα2 expressions proved the value of this approach to obtain therapeutic proteins in E. coli.

Enhancement of the immune response generated against hepatitis C virus envelope proteins after DNA vaccination with polyprotein-encoding plasmids

Plasmids expressing variants of the hepatitis C virus (HCV) core, E1 and E2 proteins individually or as polyproteins were administered to BALB/c mice. All plasmids induced a detectable and specific antibody response. Antibody titres against core, E1 and E2 proteins, 19 weeks after primary immunization, ranged from 1:50 to 1:4500 depending on the inoculated plasmid and the HCV antigen evaluated. Constructs expressing HCV envelope proteins as polyprotein variants including the core amino acid region induced statistically stronger antibody responses than plasmids encoding individual E1 and E2 proteins. Particularly, the pIDKE2 plasmid, expressing the first 650 amino acids in the viral polyprotein, induced a potent and multispecific antibody and lymphoproliferative response against HCV core, E1 and E2 proteins. Anti‐E2 antibodies generated by pIDKE2 immunization were cross‐reactive to hypervariable region‐1 peptides from different genotypes. Immunization with the pIDKE2 also generated a positive cellular immune response against the core antigen, determined by interferon‐γ enzyme‐linked immunospot (ELISPOT) assay, and induced detectable levels of interferon‐γ but not interleukin‐4 in vaccinated mice. The detection of both antibody and cytotoxic T‐lymphocyte responses, potentially targeted to circulating or cell‐infecting virions respectively, in mice vaccinated with the pIDKE2 plasmid is very attractive for the effective eradication of HCV infection.

Processing of the Hepatitis C virus precursor protein expressed in the methylotrophic yeast Pichia pastoris

The expression and processing of the Hepatitis C virus core protein (HCcAg) were analyzed in the methylotrophic yeast Pichia pastoris. Two proteins with 21 (p21) and 23 kDa (p23) were detected in immunoblot with a serum from a chronic carrier patient, as the major products for HCcAg. Both proteins, p21 and p23, produced by proteolytic processing in P. partoris, share the same N-terminal region and reacted with a monoclonal antibody towards the first 35 amino acids of HCcAg. The proteolytic processing of the recombinant polypeptide, having the HCcAg and the first 148 aa of E1 protein, was also confirmed by immunoblot analysis using mAbs with HCcAg and E1 specificities, respectively. The 32 kDa glycosilated E1 protein was then immuno-identified, as well as the processed HCcAg. These data demonstrated the usefulness of P. pastoris, as expression system, to study the processing of HCV structural proteins.

Multicenter evaluation of tendency-oriented perimetry (TOP) using the G1 grid.

Purpose The G1-TOP program is a short automated perimetric strategy which sub-divides the G1 grid of 59 points into four sub-grids. Each point is tested only once, but each patients response is used to modify that particular point and the surrounding ones from the remaining sub-grids. This study compared the results of the G1-TOP program with the Standard Bracketing strategy. Methods Eleven participating institutions provided data from 213 patients (406 eyes). The main group consisted of 284 glaucomas and 55 glaucoma suspects. Other groups included 31 eyes with neurological disorders, 20 with chorioretinal lesions and 16 normal eyes. Mean age was 62.7 ± 15.4 (range 14–88) years. All subjects had previous perimetric experience and visual acuity better than 0.5. Examination included G1-Standard Bracketing and G1-TOP testing, in interchangeable order, with the Octopus 1-2-3 perimeter. Results The correlation coefficient for mean defect (MD) was 0.95. Standard error (YX) for MD, square root of loss variance (LV) and individual thresholds were 1.86 dB, 1.29 dB, and 4.72 dB, respectively. Mean sensitivity values were similar (difference 0.04 ± 1.87 dB) (p>0.05). Mean duration for G1-TOP was 2.19 ± 0.26 min, while G1-Standard Bracketing took 11.51 ± 1.52 min (ratio 1/5.1, or a net reduction of 80.4%). The sensitivity of G1-TOP versus G1-Standard Bracketing was: glaucoma 77.1/78.5, glaucoma suspects 38.2/47.3, neurological disorders 87.1/87.1 and chorioretinal lesions 80.0/85.0. Conclusions The G1-TOP program gave very similar results to G1-Standard Bracketing in only 20% of the time required by the standard strategy.

Hepatitis C virus (HCV) core protein enhances the immunogenicity of a co-delivered DNA vaccine encoding HCV structural antigens in mice

In the present study, recombinant HCV (hepatitis C virus) core proteins enhanced the immune response elicited by a co‐delivered DNA vaccine encoding HCV core and envelope proteins. A mixture of the plasmid pIDKE2 and Co.173, a protein comprising the first 173 amino acids of HCV core, in particular induces a strong humoral response, including antibodies that recognized peptides representing hypervariable region I from different viral isolates. Moreover, positive lymphoproliferative responses against the HCV structural proteins, encoded by the plasmid, were detected after two doses with this mixture. When the HCV core protein used in the mixture with pIDKE2 was Co.120, a protein comprising the first 120 amino acids of the viral antigen, a strong humoral response and a positive lymphoproliferative response were also detected. The effectiveness of this formulation was tested in vivo by measuring the protection against infection with a recombinant vaccinia virus expressing HCV core protein. A 2 log reduction in vaccinia‐virus titre was observed in mice immunized with the mixture of pIDKE2 and Co.120. Humoral and cellular immune responses elicited for the mixture of pIDKE2 with either Co.173 or Co.120 was stronger and more diverse than those generated by individual components. In conclusion, our results indicate that formulations comprising both DNA constructs and protein subunit vaccine candidates are able to elicit strong humoral and cellular immunity against several antigens. Particularly, HCV core protein might be used as a feasible vehicle/adjuvant for DNA vaccines.

Expression and immunological evaluation of the Escherichia coli -derived hepatitis C virus envelope E1 protein

Immunological response against envelope protein E1 is very important in natural hepatitis C virus (HCV) infection, although it is insufficient to clear the viraemia. The HCV genomic region encoding the first 149 amino acids of the envelope E1 protein (E1340, amino acids 192–340) was expressed in Escherichia coli (to a level of 30% of the whole cellular proteins) and purified to 85%. We measured the immune response in rabbits and mice as well as the reactivity against 37 human sera raised against the whole recombinant protein and E1‐encoding peptides. From this, 51.1% of human sera were found to react with E1340. High‐level antibodies against E1340 were obtained in rabbits and mice when immunized. These antibodies had a similar peptide‐recognition pattern to that described previously for human sera. The most reactive region was located at the N‐terminus of the E1 protein. Cellular immunity in mice was evaluated by delayed‐type hypersensitivity assay. It revealed the induction of a CD4+ T‐cell‐mediated response by this protein. This E1340 protein and the animal‐derived anti‐E1 sera are immunological tools that could aid in the monitoring and development of anti‐HCV therapies.

Immunization with a DNA vaccine encoding the hepatitis-C-virus structural antigens elicits a specific immune response against the capsid and envelope proteins in rabbits and Macaca irus (crab-eating macaque monkeys)

In the present study, we evaluated the capability of the plasmid pIDKE2, encoding the HCV (hepatitis C virus) structural proteins Core, E1 and E2, to induce immune response against HCV antigens after injection into rabbits and Macaca irus (crab‐eating macaque). Animals were immunized intramuscularly with different amounts of plasmid on weeks 0, 3 and 8. Monkeys received a booster dose on week 46. All rabbits immunized with pIDKE2 generated a positive antibody response and, particularly in rabbits immunized with 2 mg, antibody titres reached values above 1:1500 and 1:400 against the core and the envelope proteins, respectively, 28 weeks after primary immunization. The antibody response in monkeys developed slowly, but antibody titres greater than 1:3500 against HCV structural antigens were detected at week 52. Moreover, anti‐E2 antibodies recognized synthetic peptides covering the HVR‐1 (hypervariable region‐1) from different isolates corresponding to different genotypes. Additionally, a specific lymphoproliferative response against Core and E2 was detected in two out of three monkeys immunized with pIDKE2. The other monkey had a specific proliferative response to E1. Taking all these data together, immunization with pIDKE2 is able to elicit both humoral and cellular immunity against HCV structural antigens in animal models other than mice.

Repeated administration of hepatitis C virus core-encoding plasmid to mice does not necessarily increase the immune response generated against this antigen

DNA immunization is a promising approach in generating immune responses to infectious pathogens in many different animal models. In an effort to augment the anti‐[hepatitis C virus (HCV) core] immune response, generated after DNA immunization, the importance of vaccination regimen regarding dose and boosting was investigated in the present study. Balb/c mice were intramuscularly injected with an expression plasmid encoding a truncated variant comprising amino acids 1–176 of the HCV core protein. The highest anti‐core antibody titres (1:3700) were detected in mice inoculated with 50–100 μg of core‐encoding plasmid. Additionally, we demonstrated that antibody levels induced by a single injection of DNA could be further increased by boosting with a second injection of DNA three weeks after primary immunization. However, administration of additional doses or lengthening of the resting period between inoculations resulted in similar or even weaker anti‐core antibody responses. A similar anti‐(HCV core) lymphoproliferative response was also detected in animals that had the highest level of anti‐core antibodies. These results indicate that, in clinical trials, vaccination regimen might be a critical factor in generating optimal anti‐(HCV core) immune responses after genetic immunization.

Additives and Protein-DNA Combinations Modulate the Humoral Immune Response Elicited by a Hepatitis C Virus Core-encoding Plasmid in Mice

Humoral and cellular immune responses are currently induced against hepatitis C virus (HCV) core following vaccination with core-encoding plasmids. However, the anti-core antibody response is frequently weak or transient. In this paper, we evaluated the effect of different additives and DNA-protein combinations on the anti-core antibody response. BALB/c mice were intramuscularly injected with an expression plasmid (pIDKCo), encoding a C-terminal truncated variant of the HCV core protein, alone or combined with CaCl2, PEG 6000, Freunds adjuvant, sonicated calf thymus DNA and a recombinant core protein (Co. 120). Mixture of pIDKCo with PEG 6000 and Freunds adjuvant accelerated the development of the anti-core Ab response. Combination with PEG 6000 also induced a bias to IgG2a subclass predominance among anti-core antibodies. The kinetics, IgG2a/IgG1 ratio and epitope specificity of the anti-core antibody response elicited by Co. 120 alone or combined with pIDKCo was different regarding that induced by the pIDKCo alone. Our data indicate that the antibody response induced following DNA immunization can be modified by formulation strategies.