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Current advances in antibody engineering driving the strongest growth area in biotherapeutic agents development. Affinity improvement that is mainly important for biological activity and clinical efficacy of therapeutic antibodies, has still remained a challenging task. In the human body, during a course of immune response affinity maturation increase antibody activity by several rounds of somatic hypermutation and clonal selection in the germinal center. The final outputs are antibodies representing higher affinity and specificity against a particular antigen. In the realm of biotechnology, exploring of mutations which improve antibody affinity while preserving its specificity and stability is an extremely time-consuming and laborious process. Recent advances in computational algorithms and DNA sequencing technologies help researchers to redesign antibody structure to achieve desired properties such as improved binding affinity. In this review, we briefly described the principle of affinity maturation and different corresponding in vitro techniques. Also, we recapitulated the most recent advancements in the field of antibody affinity maturation including computational approaches and next-generation sequencing (NGS).

In recent decades, immunotoxins have attracted significant attention in treatment of a wide range of diseases including cancers due to their natural origins and their role in blocking crucial pathways within the cells. Ribosome inactivating proteins (RIPs) are efficient molecules in blocking protein synthesis through interactions with ribosomal rRNA molecules. cDNA molecule encoding HER2 scFv antibody fragment originated from trastuzumab attached to the mature alpha luffin gene fragment was subcloned into pET28a expression vector and expressed in different

A number of mutations in the epidermal growth factor receptor (EGFR) have been identified that imparts resistance to anti-EGFR monoclonal antibodies (mAbs) in clinical and preclinical samples. Primary or acquired resistance to targeted therapy will eventually limit the clinical benefit of anticancer mAbs. The aim of the current study was to perform computational analysis to investigate the structural implications of the EGFR somatic mutations on its complexes with the four anti-EGFR mAbs (Cetuximab, Panitumumab, Necitumumab, and Matuzumab). Docking analysis and molecular dynamics (MD) simulations were performed to understand the plausible structural and dynamical implications caused by somatic mutations available in the Catalogue of Somatic Mutations in Cancer database on the EGFR and anti-EGFR mAbs. We found that EGFRS492R and EGFRV441I in complex with Cetuximab, EGFRR377S and EGFRS447Y in complex with Panitumumab, and EGFRV441I in complex with Necitumumab have a weakest binding affinity in comparison to EGFRWT in complex with the relevant mAb. Taken together with the results obtained from docking analysis and MD simulations, the present findings may suggest that, the S492R and V441I mutations confer resistance to Cetuximab, R377S and S447Y mutations mediate resistance to Panitumumab and finally, V441I mutation also confers resistance to Necitumumab.

Background: Leishmania major (L. major) superoxide dismutase B1 (SODB1) is an important enzyme for parasite survival. Previous studies have shown that SODB1 is highly immunogenic and sera from patients infected with Leishmania react with recombinant SODB1 (rSODB1). Objectives: In the present study, the protective effect of immunization with recombinant SODB1 (rSODB1) against the infection with Leishmania major was evaluated in a mice model. Methods: Three groups of BALB/c mice received rSODB1 mixed with complete Freund’s adjuvant (immunized group), adjuvant (control I) or PBS alone (control II). This protocol was repeated 15 days later. Three weeks after the second booster immunization, four mice from each group were sacrificed to measure in vitro cytokines production by splenocytes; remaining mice were challenged using meta-cyclic promastigotes. Eight weeks later, the infected mice were sacrificed, and their splenocytes were re-stimulated with rSODB1; then, cytokine levels were assayed by the enzyme linked immunosorbent assay (ELISA). Results: Immunization of mice with rSODB1 plus adjuvant elicited high level of IFN-γ, low level of IL-5 production, and a significant increase in IL-10 level, as compared to controls. Post-challenge re-stimulation of splenocytes also showed a polarized TH1 response accompanied by higher levels of IL-10 in immunized mice with rSODB1 plus adjuvant compared to controls. Conclusions: Although immunization with recombinant SODB1 plus adjuvant induced a strong TH1 response, which was identified by high-level IFN-y production, regarding the increase in footpad swelling and lesions size, it could be concluded that immunization with rSODB1 plus adjuvant was not able to provide protection against Leishmania infection in the presence of high level of IL-10 production.

Production of insulin-like growth factor 1 (IGF1) in Escherichia coli mostly results in the formation of inclusion bodies. In the present study, IGF1 was fused to disulfide bond oxidoreductase A (DsbA) and expressed in SHuffle™ T7 strain, in order to obtain correctly folded protein. Soluble expression and IMAC purification of DsbA-IGF1 were optimized by applying the Box-Behnken design of response surface methodology. The optimization greatly increased concentration of soluble protein from 317 to 2600 mg/L, and IMAC yield from 400 to 1900 mg/L. Results of ANOVA showed induction OD600 and temperature had significant effects on the soluble protein expression while isopropyl-β-d thiogalactoside, in the concentrations tested, displayed no significant effect. Moreover, the three parameters of the binding buffer including, pH, concentration of NaCl, and imidazole displayed significant effects on the IMAC yield. Then, purified DsbA-IGF1 was cleaved by human rhinovirus 3C protease, and authentic IGF1 was obtained in flow through of a subtractive IMAC. Final polishing of the protein by reversed-phase HPLC yielded IGF1 with purity of 96%. The quality attributes of purified IGF1 such as purity, identity, molecular size, molecular weight, secondary structure, and biological activity were assessed and showed to be comparable to the standard IGF1. The final yield of purified IGF1 was estimated to be 120 ± 18 mg from 1 L of the culture. Our results demonstrated a simple and easily scalable strategy for production of large amounts of bioactive IGF1 by rational designing soluble protein expression, and further optimization of expression and purification methods.

Rapid growth in the therapeutic antibody market leads to a drastic development of antibody engineering to optimize biophysical properties. One of the main focuses of biopharmaceutical researches was the expansion of different approaches for affinity maturation of antibodies to enhance biological activity. Adalimumab (D2E7) is an anti-tumor necrosis factor alpha (TNFα) antibody used in the treatment of some autoimmune disorders like rheumatoid arthritis, psoriasis, etc. In this study, by engineering complementary determining regions (CDRs) of D2E7 antibody, we produced a new variant of the antibody with improved affinity and potency to TNFα. We designed four D2E7 mutants that harbored a single point mutation in their CDRs. The native antibody, lc-A94K, lc-A50Y, lc-A33R, and lc-A92R mutant models were transiently produced and characterized. Data showed mutation of ALA to LYS in CDR3 of D2E7 antibody generated new hydrogen bonds with TNFα, thus the lc-A94K model revealed significantly higher binding activity (EC50) and kinetic affinity (KD) to its antigen, in comparison to the wild antibody. Moreover, this model was found to have significantly stronger biological activity (IC50) to activate antibody-dependent cell-mediated cytotoxicity in the mouse fibroblast L929 cells. Secondary structure analysis demonstrated the mutation has no inappropriate conformational impact on the beta and alpha structures of lc-A94K antibody. In conclusion, our study revealed that it is possible to manipulate antibody’s CDRs to increase affinity and potency by single point mutation and also preserve basic structure of the original antibody during CDRs engineering to avoid adverse effects of CDRs mutations on specificity and stability.

Antibody-drug conjugates are now of considerable interest and are recommended for the treatment of cancers. Linkers are having a crucial role in potency and efficacy of these drugs. Herein, for the first time, we have used a water-soluble poly-ethylene glycol based linker (succinimidyl-[(N-maleimido propionamido)-diethyleneglycol] [SM(PEG)2]) for lysine amide coupling of DM1 drug to trastuzumab considering evaluation of the effect of using a hydrophilic linker on physicochemical and biological properties of the resulting conjugate in comparison to the conjugate containing succinimidyl 4-(N-maleimidomethyl) cyclohexane-1-carboxylate (SMCC) linker, which has a relative hydrophobic nature. The physicochemical properties of synthesized conjugates were investigated in terms of drug to antibody ratio, size variants and free drug quantities. In vitro biological activity of trastuzumab-DM1 conjugates was assessed on breast cancer cell lines expressing different levels of HER2 using binding affinity, antiproliferative, apoptosis, and antibody-dependent cell-mediated cytotoxicity (ADCC) assays. Synthesized conjugate containing hydrophilic linker, showed higher drug to antibody ratio, no aggregated form and higher cellular toxicity in comparison to SMCC bearing conjugate. Binding affinity and ADCC potential of conjugates was not affected upon the usage of hydrophilic linker. In conclusion, application of SM(PEG)2 for coupling of DM1 to trastuzumab enhance desirable characteristics of the resulting conjugate.

Traditional methods to generate CHO cell lines rely on random integration(s) of the gene of interest and result in unpredictable and unstable protein expression. In comparison, site-specific recombination methods increase the recombinant protein expression by inserting transgene at a locus with specific expression features. PhiC31 serine integrase, catalyze unidirectional integration that occurs at higher frequency in comparison with the reversible integration carried out by recombinases such as Cre. In this study, using different ratios of phiC31 serine integrase, we evaluated the phiC31 mediated gene integration for expression of a humanized IgG1 antibody (mAb0014) in CHO-S cells. Light chain (LC) and heavy chain (HC) genes were expressed in one operon under EF1α promoter and linked by internal ribosome entry site (IRES) element. The clonal selection was carried out by limiting dilution. Targeted integration approach increased recombinant protein yield and stability in cell pools. The productivity of targeted cell pools was about 4 mg/L and about 40 µg/L in the control cell pool. The number of integrated transgenes was about 19 fold higher than the control cells pools. Our results confirmed that the phiC31 integrase leads to mAb expression in more than 90% of colonies. The productivity of the PhiC31 integrated cell pools was stable for three months in the absence of selection as compared with conventional transfection methods. Hence, utilizing PhiC31 integrase can increase protein titer and decrease the required time for protein expression. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:1570-1576, 2016.

Human cutaneous leishmaniasis is a disease caused by eukaryotic single-celled Leishmania species, the developmental program of which relies upon blood-feeding adult female sand flies and their dominant mammal blood sources, namely wild rodents in area where human beings exert more or less transient activities. The recourse to model rodents - namely laboratory mice such as C57BL/6 mice - has allowed extracted the immune signatures that account for the healing of the transient cutaneous lesion that develops at the site where Leishmania major promastigotes were delivered. Indeed, if the latter mice are exposed to a second inoculum of L. major promastigotes, no lesion will develop in the secondary skin site remodeled as a niche for a low size intracellular L. major amastigote population. Moreover, IFN-γ dominates over IL-10 in the supernatant of cultures of PBMCs -prepared from blood sampled from human beings who healed from a cutaneous lesion- and incubated with L. major class I Nuclease LmaCIN, a protein highly expressed in the cell-cycling amastigote population which is dominant by macrophages. Altogether, these datasets were strong incentive to promote research aimed to design and monitor efficacy of L. major amastigote protein-based vaccines in pre-clinical settings. Using L. major enzyme class I nuclease (LmaCIN) expressed in the L. major cell-cycling amastigote population hosted by macrophages, BALB/c mice were immunized three times with either rLmaCIN plus LPD nanoparticles (LPD-rLmaCIN), or rLmaCIN-CpG DNA or free rLmaCIN and dextrose. The following parameters: footpad swelling, splenic L. major load, L. major binding IgGs and cytokine profiles of rLmaCIN- reactive T lymphocytes were then compared. Once coadminstered with LPD, rLmaCIN allow BALB/c mice to display delayed onset of skin lesion at the challenge inoculation site and delayed L. major dissemination from the challenged site to the spleen. Thus, the LPD-rLmaCIN is shown to display some promising features out of three formulations inoculated to the BALB/c mouse immunization.

Recombinant antibodies have emerged over the last few decades as the fastest growing class of therapeutic proteins for autoimmune diseases. Post-translation modifications of antibodies produced by human cell lines are highly consistent with those existing in natural human proteins and this is a major advantage of utilizing these cell lines. Cinorra is a biosimilar form of the antibody Adalimumab, which is an antagonist of TNF-α used for the treatment of autoimmune diseases. Adalimumab and Cinorra were produced by stable expression from CHO cells. The aim of this study was to select HEK cells as a host for producing Adalimumab to reveal whether the antibody produced by this human-derived cell line has similar characterization to Cinorra. Adalimumab was transiently produced in HEK-293T cells, characterized and analyzed for its properties. Circular dichroism spectroscopy confirmed a strong structural similarity of the expressed antibody with Cinorra. Likewise its binding activity and kinetic affinity to TNF-α (EC50 = 416.5 ng/ml, KD = 3.89 E-10 M,) were highly similar to that of Cinorra (EC50 = 421.2 ng/ml and KD = 3.34 E-10 M,). Additionally there was near identical neutralization of TNF-α-mediated cellular cytotoxicity (IC50 of the expressed = 4.93 nM; IC50 of Cinorra = 4.5 nM). Results indicate that Adalimumab produced by HEK-293T cells possesses a similarly efficient function and biological activity to Cinorra. Consequently, human-derived host cells with human post-translational modifications might potentially provide a basis for the development of Adalimumab with pharmaceutical properties for research and therapeutic use.

Background: The increase of the protein expression via ribosomal manipulation is one of the suggested cellular mechanisms involved in EnBase fed-batch mode of cultivation. However, this system has not been implemented for cytotoxic proteins.Objectives: Here, the expression pattern of α-Luffin, a ribosome inactivation protein (RIP) with an innate toxicity, was investigated in EnBase system and the effect of low temperature cultivation on the increase of α-Luffin solubility was determined.Materials and Methods: The encoding cDNA for mature α-Luffin was synthesized and subcloned into pET28a plasmid under the control of T7 promoter. The E. coli expression yield in EnBase® Flo fed-batch system was compared with traditional batch mode at two temperatures: 25 °C and 30 °C. Sampling was performed at several time intervals and solubility of recombinant-protein was checked on SDS-PAGE in pellet and supernatant samples. The purification of recombinant protein was performed by Ni-NTA column.Results: In fed-batch cultivation mode, the early incubation time was desirable at 30 °C whereas the maximum amount of soluble α-Luffin was achieved from the extended protein synthesis period (12 and 24h post induction) at 25 °C.Conclusions: Our founding showed that EnBase had a greater efficacy in producing higher soluble protein ratios compared to batch cultivation growth rate, however for cytotoxic proteins, incubation temperature and time need to be optimized. Owing to the advantages of natural toxins from RIP family for producing anticancer immune-conjugates, well optimization of this protein expression is of importance regarding industrial aspects. The optimized condition proposed here is promising in terms of large scale soluble production of α-Luffin without the need for refolding.

Eptacog alfa (NovoSeven®) is a vitamin K-dependent recombinant Factor VIIa produced by genetic engineering from baby hamster kidney (BHK) cells as a single peptide chain of 406 residues. After activation, it consists of a light chain (LC) of 152 amino and a heavy chain (HC) of 254 amino acids. Recombinant FVIIa undergoes many post-translational modifications (PTMs). The first ten glutamic acids of the N-terminal moiety are γ-carboxylated, Asn145 and Asn322 are N-glycosylated, and Ser52 and Ser60 are O-glycosylated. A head-to-head biosimilarity study was conducted for the originator and the first biosimilar AryoSeven™ to evaluate comparable bioengineering. Physicochemical properties were analyzed based on mass spectrometry, including intact mass, PTMs and higher-order structure. Both biotherapeutics exhibit a batch-to-batch variability in their N-glycan profiles. N-Glycopeptide analysis with UHPLC- QTOF-MSE confirmed N-glycosylation sites as well as two different O-glycopeptide sites. Ser60 was found to be O-fucosylated and Ser52 had O-glucose or O-glucose-(xylose)1,2 motifs as glycan variants. Ion mobility spectrometry (TWIMS) and NMR spectroscopy data affirm close similarity of the higher-order structure of both biologicals. Potency of the biodrugs was analyzed by a coagulation assay demonstrating comparable bioactivity. Consequently, careful process optimization led to a stable production process of the biopharmaceuticals.

Leishmaniasis is a major public health problem. Despite numerous attempts, yet there is no effective vaccine against human leishmaniasis, mainly due to a lack of an effective vaccine delivery system as well as adjuvant.The aim of this study was to evaluate the ability of recombinant glycoprotein 63 (rgp63) as a model of Leishmania antigen, entrapped in liposome-polycation-DNA (LPD) complexes nanoparticles in inducing cell mediated immune (CMI) response and protecting against L. major in BALB/c mice.To this end, the abundant leishmania promastigote cell surface glycoprotein, gp63, was entrapped in nano-sized LPD (CpG) particles, (LPD (CpG)-rgp63), and BALB/c mice were immunized three times with either (LPD (CpG)-rgp63) or rgp63-CpG DNA or LPD (CpG) or free rgp63 and dextrose 5%. Various parameters including footpad thickness, splenic load of L. major parasites, rgp63-binding IgGs and also cytokine levels of rgp63-reactive T lymphocytes were then compared among different vaccinated animals.The lowest number of parasites in spleen, the higher levels of IgG2a after challenge infection, the minimal footpad swelling and high level of IFN-γ secretion, all indicated that adjuvants and antigen-delivery systems are essential in modifying immune responses; as mice received LPD (CpG)-rgp63 induced immune response stronger than the other groups.This study demonstrates that LPD nanoparticle is a promising and adaptable delivery system which could be modified towards specific vaccine targets to induce a more potent immune response in combination with rgp63.

Recombinant human factor VII (rh-FVII) is produced by engineered BHK cells at low dose. Accordingly, establishment of a precise method is crucial to reliably measuring expression level of this protein during manufacturing pro-cesses. We developed and established a reproducible sandwich enzyme-linked immunosorbent assay (ELISA) method for measuring amount of FVII during biopharmaceutical in upstream and downstream processes of Aryo-Seven. A sandwich ELISA was designed using two different high affinity mon-oclonal antibodies (mAb1 and mAb2) against h-FVII. The bounded FVII to the first antibody was revealed by the use of a second mouse anti-FVII monoclo-nal antibody (1F1-B11), labeled with HRP that binds to another antigenic determinant of the FVII. Then, validation was done by determination of spec-ificity, linearity, accuracy, precision, reproducibility, detection and quantifica-tion limit and robustness according to ICH Q2 (R1) guideline. In developed ELISA, no interference was found between FVII and proteins derived from BHK which commonly exist in the supernatant. Linear range of detection was from 25-1.56 ng/mL with P-Value

Fibrinolytic agents are widely used in treatment of the thromboembolic disorders. The new generations like recombinant tissue plasminogen activator (t-PA, alteplase) are not showing promising results in clinical practice in spite of displaying specific binding to fibrin in vitro. Vampire bat plasminogen activator (b-PA) is a plasminogen activator with higher fibrin affinity and specificity in comparison to t-PA resulting in reduced probability of hemorrhage. b-PA is also resistant to plasminogen activator inhibitor-1 (PAI-1) showing higher half-life compared to other variants of t-PA. However, its non-human origin was a driving force to design a human t-PA with favorable properties of b-PA. In the present study, we designed a chimeric t-PA with desirable b-PA properties and this new molecule was called as CT-b. The construct was prepared through kringle 2 domain removal and replacement of t-PA finger domain with b-PA one. In addition, the KHRR sequence at the initial part of protease domain was replaced by four alanine residues. The novel construct was integrated in Pichia pastoris genome by electroporation. Catalytic activity was investigated in the presence and absence of fibrin. The purified protein was analyzed by western blot. Fibrin binding and PAI resistance assays were also conducted. The activity of the recombinant protein in the presence of fibrin was 1560 times more than its activity in the absence of fibrin, showing its higher specificity to fibrin. The fibrin binding of CT-b was 1.2 fold more than t-PA. In addition, it was inhibited by PAI enzyme 44% less than t-PA. Although the presented data demonstrate a promising in vitro activity, more in vivo studies are needed to confirm the therapeutic advantage of this novel plasminogen activator.

International audience; Human tissue plasminogen activator (t-PA) plays a pivotal role in the treatment of acute myocardial infarction, ischemic stroke, and deep vein thrombosis. It has the benefit of generating no adverse effects such as fibrinogen depletion, systemic hemorrhage, and immunologic reactions. Human t-PA is a serine-protease enzyme containing 527 amino acid residues in five structural domains. The correct folding of t-PA requires the correct pairing of 17 disulfide bridges in the molecule. A gene encoding full-length human t-PA was cloned into pPICZαA expression vector downstream of alcohol oxidase promoter and α-mating signal sequence from Saccharomyces cerevisiae and flush with the kex2 cleavage site to express the protein with a native N terminus. The methylotrophic yeast, Pichia pastoris GS115 strain, was transformed with this cassette, and methanol utilizing (mut+) transformants were selected for production and secretion of human t-PA into culture media. SDS-PAGE and Western blot analysis showed the expressed bands of t-PA protein. Zymography test indicated suitable folding and proper function of the expressed recombinant human t-PA in conversion of plasminogen to plasmin and gelatin lysis. Amidolytic activity test showed the amidolytic activity of 1,650 IU/ml. The results of this study concluded that P. pastoris methylotrophic yeast can be a suitable alternative for mammalian and prokaryotic expression systems to produce t-PA.

Summary Identification of autoimmune processes and introduction of new autoantigens involved in the pathogenesis of multiple sclerosis (MS) can be helpful in the design of new drugs to prevent unresponsiveness and side effects in patients. To find significant changes, we evaluated the autoantibody repertoires in newly diagnosed relapsing–remitting MS patients (NDP) and those receiving disease-modifying therapy (RP). Through a random peptide phage library, a panel of NDP- and RP-specific peptides was identified, producing two protein data sets visualized using Gephi, based on protein-–protein interactions in the STRING database. The top modules of NDP and RP networks were assessed using Enrichr. Based on the findings, a set of proteins, including ATP binding cassette subfamily C member 1 (ABCC1), neurogenic locus notch homologue protein 1 (NOTCH1), hepatocyte growth factor receptor (MET), RAF proto-oncogene serine/threonine-protein kinase (RAF1) and proto-oncogene vav (VAV1) was found in NDP and was involved in over-represented terms correlated with cell-mediated immunity and cancer. In contrast, transcription factor RelB (RELB), histone acetyltransferase p300 (EP300), acetyl-CoA carboxylase 2 (ACACB), adiponectin (ADIPOQ) and phosphoenolpyruvate carboxykinase 2 mitochondrial (PCK2) had major contributions to viral infections and lipid metabolism as significant events in RP. According to these findings, further research is required to demonstrate the pathogenic roles of such proteins and autoantibodies targeting them in MS and to develop therapeutic agents which can ameliorate disease severity.

Since physiological and pathological processes occur at nano-environments, nanotechnology has considered as an efficient tool for designing of next generation specific biomolecules with enhanced pharmacodynamic and pharmacodynamic properties. In the current investigation, by control of the size and hydrodynamic volume at the nanoscale, for the first time, physicochemical and pharmacokinetic properties of an anti-VEGFA nanobody was remarkably improved by attachment of a Proline-Alanine-Serine (PAS) rich sequence. The results elucidated unexpected impressive effects of PAS sequence on physicochemical properties especially on size, hydrodynamics radius, and even solubility of nanobody. CD analysis revealed an increment in random coil structure of the PASylated protein in comparison to native one without any change in charge state or binding kinetic parameters of nanobody assessed by isoelectric focusing and surface plasmon resonance measurements, respectively. In vitro biological activities of nanobody were not affected by coupling of the PAS sequence. In contrast, the terminal half-life was significantly increased by a factor of 14 for the nanobody-PAS after single dose IV injection to the mice. Our study demonstrated that the control of size in the design of small therapeutic proteins has a promising effect on the stability and solubility, in addition to their physiochemical and pharmacokinetic properties. The designed new anti-VEGFA nanobody could promise a better therapeutic agent with a long administration intervals and lower dose, which in turn leads to a better patient compliance. Size adjustment of an anti-VEGF nanobody at the nanoscale by the attachment of a natural PAS polymer remarkably improves physicochemical properties, as well as a pharmacokinetic profile without any change in biological activity of the miniaturized antibody.

Establishing stable Chinese Hamster Ovary (CHO) cells producing monoclonal antibodies (mAbs) usually pass through the random integration of vectors to the cell genome, which is sensitive to gene silencing. One approach to overcome this issue is to target a highly transcribed region in the genome. Transposons are useful devices to target active parts of genomes, and PiggyBac (PB) transposon can be considered as a good option. In the present study, three PB transposon donor vectors containing both heavy and light chains were constructed, one contained independent expression cassettes while the others utilized either an Internal Ribosome Entry Site (IRES) or 2A element to express mAb. Conventional cell pools were created by transferring donor vectors into the CHO cells, whereas transposon-based cells were generated by transfecting the cells with donor vectors with a companion of a transposase-encoding helper vector, with 1:2.5 helper/donor vectors ratio. To evaluate the influence of helper/donor vectors ratio on expression, the second transposon-based cell pools were generated with 1:5 helper/donor ratio. Expression levels in the transposon-based cells were two to five -folds more than those created by conventional method except for the IRES-mediated ones, in which the observed difference increased more than 100-fold. The results were dependent on both donor vector design and vectors ratios.

Because of high mortality caused by cardiovascular diseases, various fibrinolytic agents with diverse pharmacokinetic and pharmacodynamic properties have been developed. A novel mutated chimeric tissue plasminogen activator (mt-PA) was developed by the removal of first three domains of t-PA, insertion of GHRP sequence and mutation towards resistance to plasminogen activator inhibitor-1 (PAI-1). Mt-PA protein was expressed in Expi293F cells. The expression level of mt-PA was found to be 5000 IU/mL. Following purification, the pharmacokinetic properties of mt-PA were evaluated in three doses in rats. Data related to mt-PA were best fitted to two compartment model. With the increase in dose, the Area Under the plasma concentration-time Curve (AUC0→∞) increased. The elimination half-life (t1/2) of mt-PA was in the range of 19.1–26.1 min in three doses while that of Alteplase was 8.3 min. The plasma clearance (CLp) of mt-PA ranged from 3.8 to 5.9 mL/min in three doses, which was several times lower than that of Alteplase (142.6 mL/min). The mean residence time (MRT) of mt-PA ranged from 23.3–31.8 min in three doses, which was 4–5 times greater than that of Alteplase (6 min). Mt-PA showed extended half-life and mean residence time and is a good candidate for further clinical studies.

Background As the demand for monoclonal antibodies (mAb) increases, more efficient expression methods are required for their manufacturing process. Transcriptional gene silencing is a common phenomenon in recombinant cell lines which leads to expression reduction and instability. There are reports on improved antibody expression in ubiquitous chromatin opening element (UCOE) containing both heavy and light chain gene constructs. Here we investigate the impact of having these elements as part of the light chain, heavy chain or both genes during cell line development. In this regard, non-UCOE and UCOE vectors were constructed and stable Chinese hamster ovary (CHO) cell pools were generated by different vector combinations. Results Expression analysis revealed that all UCOE cell pools had higher antibody yields compared to non-UCOE cells, Moreover the most optimal expression was obtained by cells containing just the UCOE on heavy chain. In terms of stability, it was shown that the high level of expression was kept consistence for more than four months in these cells whereas the expression titers were reduced in the other UCOE pools. Conclusions In conclusion, UCOE significantly enhanced the level and stability of antibody expression and the use of this element with heavy chain provided more stable cell lines with higher production level.

Thrombolytic therapy by plasminogen activators (PAs) has been a main goal in the treatment of acute myocardial infarction. Despite improved outcomes of currently available thrombolytic therapies, all these agents have different drawbacks that may result in less than optimal outcomes. In order to make tissue plasminogen activator (tPA) more potent, while being more resistant to plasminogen activator inhibitor-1 (PAI-1) and having a higher affinity to fibrin, a new chimeric-truncated form of tPA (CT tPA) was designed and expressed in Pichia pastoris. This novel variant consists of a finger domain of Desmoteplase, an epidermal growth factor (EGF) domain, a kringle 1 (K1) domain, a kringle 2 (K2) domain, in which the lysine binding site (LBS) was deleted, and a protease domain, where the four amino acids lysine 296, arginine 298, arginine 299, and arginine 304 were substituted by aspartic acid. The chimera CT tPA showed 14-fold increase in its activity in the presence of fibrin compared to the absence of fibrin. Furthermore, CT tPA showed about 10-fold more potency than commercially available full-length tPA (Actylase(®)) and provided 1.2-fold greater affinity to fibrin. A residual activity of only 68 % was observed after incubation of Actylase(®) with PAI-1, however, 91 % activity remained for CT tPA. These promising findings suggest that the novel CT tPA variant might be an acceptable PA with superior characteristics and properties.

Objectives Several types of expression vectors have been used for recombinant protein expression in Chinese hamster ovary cells (CHO) which usually result in variable and unstable levels of expression. Methods and results In this study, we have compared the mAb0014 expression level of single ORF/IRES vector and dual ORF vector in the presence and absence of phiC31 integrase targeting system. Both expression vectors contain an elongation factor 1α (EF1α) promoter upstream of LC and harboring an attB site. CHO-S cells were co-transfected with single ORF/IRES or dual ORF vectors along with a phiC31 integrase expression vector which can catalyze recombination between attB site and pseudo- attP sites presented in the mammalian genome. Our results demonstrated that dual ORF vector in the presence of phiC31 integrase expression vectors (+FC31 2P) generated more recombinant antibody in comparison to its negative control (-FC31 2P). Moreover, both of +FC31 2P and -FC31 2P cell pools yield higher recombinant protein in comparison to single ORF/IRES vector (FC31 IRES) cell pools. Stability of expression in phiC31 co-transfected cell pools (+FC31 2P and +FC31 IRES) had no considerable changes. Conclusions Our results indicated that the dual ORF vector using integrase can support the generation of cell lines with stable transgene expression at an elevated mAb relative to single ORF/IRES vector.

BMPs are osteoinductive proteins which are used in treatment of acute fractures. Large quantities of recombinant proteins are usually needed to achieve efficacy in the clinic. This translates to severe complications and high costs. Different strategies have been developed to improve the efficacy and safety of BMPs. Modification of the heparin-binding site in order to increase the local retention time of the morphogen is one of these approaches. Aiming at further improvement in properties of BMP-7, a novel form of this protein was designed and expressed successfully in Chinese Hamster Ovarian (CHO) cells. Substitution of the Bone morphogenetic protein-7 N-terminus by the heparin-binding site of Bone morphogenetic protein-2 was carried out to increase the heparin binding capacity of the novel protein. It was found that the novel variant, retained its in vitro biological activity and the heparin binding capacity of this protein was approximately 20% higher than that of the wild-type at a protein concentration of 100 ng/mL. The novel protein as the first variant of hBMP-7 with the enriched heparin-binding site may offer more advantages in clinical use as compared to the existing commercial form.

In recent years, monoclonal antibodies (mAbs) have been developed as powerful therapeutic and diagnostic agents and Chinese hamster ovary (CHO) cells have emerged as the dominant host for the recombinant expression of these proteins. A critical step in recombinant expression is the utilization of strong promoters, such as the Chinese Hamster Elongation Factor-1α (CHEF-1) promoter. To compare the strengths of CHEF with cytomegalovirus (CMV) promoter for mAb expression in CHO cells, four bicistronic vectors bearing either internal ribosome entry site (IRES) or Furin/2A (F2A) sequences were designed. The efficiency of these promoters was evaluated by measuring level of expressed antibody in stable cell pools. Our results indicated that CHEF promoter-based expression of mAbs was 2.5 fold higher than CMV-based expression in F2A-mediated vectors. However, this difference was less significant in IRES-mediated mAb expressing cells. Studying the stability of the F2A expression system in the course of 18 weeks, we observed that the cells having CHEF promoter maintained their antibody expression at higher level than those transfected with CMV promoter. Further analyses showed that both IRES-mediated vectors, expressed mAbs with correct size, whereas in antibodies expressed via F2A system heterogeneity of light chains were detected due to incomplete furin cleavage. Our findings indicated that the CHEF promoter is a viable alternative to CMV promoter-based expression in F2A-mediated vectors by providing both higher expression and level of stability.

Chinese hamster ovary (CHO) cells are the most commonly used host system for the expression of high quality recombinant proteins. However, the development of stable, high-yielding CHO cell lines is a major bottleneck in the industrial manufacturing of therapeutic proteins. Therefore, different strategies such as the generation of more efficient expression vectors and establishment of genetically engineered host cells have been employed to increase the efficiency of cell line development. In order to examine the possibility of generating improved CHO host cells, cell line engineering approaches were developed based on ceramide transfer protein (CERT), and X-box binding protein 1s (XBP1s).CHO cells were transfected with CERT S132A, a mutant variant of CERT which is resistant to phosphorylation, or XBP1s expression plasmids, and then stable cell pools were generated. Transient expression of t-PA was examined in engineered cell pools in comparison to un-modified CHO host cells.Overexpression of CERT S132A led to the enhancement of recombinant tissue plasminogen activator (t-PA) expression in transient expression by 50%. On the other hand, it was observed that the ectopic expression of the XBP1s, did not improve the t-PA expression level.The results obtained in this study indicate successful development of the improved CHO host cells through CERT S132A overexpression.

International audience; The tumor-associated glycoprotein 72 (TAG-72) is a membrane mucin whose over-expression is correlated with advanced tumor stage and increased invasion and metastasis. In this study, we identified a panel of four nanobodies, single variable domains of dromedary heavy-chain antibodies that specifically recognize the TAG-72 antigen. All selected nanobodies were shown to selectively bind to this cancer-related molecule with low-nanomolar affinities and do not cross-react with other antigens, such as MUC1 or HER2. Furthermore, they can detect TAG-72 in concentrations as low as 5 U/ml which is valuable in sensitive detection of this molecule in cancerous patients. Cell ELISA experiments proved their ability for binding to the native target antigen on TAG-72 expressing cells while not showing any reactivity to HT-29 cells, a TAG-72-negative cell line. Using competition studies, we found that each nanobody recognizes a distinct epitope on the TAG-72 antigen that is different from the one recognized by the mouse anti-TAG-72 antibody, CC49. Considering their high specificity, reduced immunogenicity and multi-targeting behavior, these oligoclonal nanobodies represent a promising tool to target TAG-72 over-expressing tumor cells.

International audience; An important modification of thrombolytic agents is resistance to plasminogen activator inhibitor-1 (PAI-1). In previous studies, a new truncated PAI-1-resistant variant was developed based on deletion of the first three domains in t-PA and the substitution of KHRR 128-131 amino acids with AAAA in the truncated t-PA. The novel variant expressed in a static culture system of Chinese Hamster Ovary (CHO) DG44 cells exhibited a higher resistance to PAI-1 when compared with the full-length commercial drug; Actylase. In the present study, the truncatedmutant protein was expressed in CHO DG44 cells in 50 ml orbital shaking bioreactors. The final yield of the truncatedmutant in the culture was 752 IU/ml, representing a 63% increase compared with the static culture system. Therefore, these results suggest that using the combined features of a transient and stable expression system is feasible for the production of novel recombinant proteins in the quantities needed for preclinical studies.

Background: The hemagglutinin molecule of influenza virus is considered as an ideal model to study biological processes as well as the effect of glycosylation on the function of glycoproteins. Objectives: The large subunit of the influenza virus A/New Caledonia/20/99 (H1N1) hemagglutinin (HA1) was expressed in recombinant Escherichia coli containing the glycosylation system of Campylobacter jejuni. This viral glycoprotein contains glycosylation motifs recognized by prokaryotic and eukaryotic oligosaccharyltransferases. Methods: In order to express the hemagglutinin large subunit gene, the gene was amplified using reverse transcription polymerase chain reaction (RT-PCR), and it was cloned in pET22b for periplasmic expression. Results: Western blotting and lectin blotting bands confirmed glycosylation of the HA1 in recombinant E. coli. Conclusion: Such a successful accomplishment of hemagglutinin expression in recombinant E. coli can be used to construct carbohydrates in hemagglutinin molecules of different strains in order to produce effective antigens for vaccine and rapid diagnostic kits against new emerging viruses.

Background and Aims: Glycoprotein B (gB) is among the most important immunogenic proteins of HSV-1 that is now being used as recombinant vaccine to induce protectively against the virus. It is believed that gB is responsible for induction of neutralizing antibody and up to 90% of CTL (Cytotoxic T Cell) activity against HSV-1 in some haplotypes. Methods: In the present study the antibody titer induced by a gB based DNA vaccine and the protective effect of the candidate vaccine in BALB/c mice in comparison with those induced by the whole virus were evaluated. DNA plasmid carrying full-length of gB-1 was constructed and its ability to express the protein was confirmed by immunofluorescence test. Results: Humoral immune response and protective effect of the constructed vector were evaluated in BALB/c mice. Conclusion: The neutralizing antibody titer induced by the DNA vaccine was significantly lower than the one induced by the whole virus, although, both of vaccines could confer 100% protectively against HSV-1 challenge.

Background and Aims: Since 1998, Iranian poultry industry has been affected by avian influenza (AI) virus, subtype H9N2. The association of high mortality and case report of H5N1 and H9N2 influenza virus in wild birds in recent years raised the suspicion of a possible new genetic modified AI virus. Methods: Partial nucleotide sequences and deduced amino acid of hemagglutinin (HA) genes of 4 H9N2 influenza viruses isolated from broilers in Iran in 2007 were genetically analyzed. Results: The isolates possessed two types of amino acid motif -R-S-S-RlG-L- and -R-S-NRlG-L- at the cleavage site of HA. "-R-S-N-RlG-L in Iranian isolates is the same as the motif previously reported in Israel. Glycosylation site of the virus has been missed in some isolates. Receptor binding site of the isolates were found similar to the human H9N2 isolates. Conclusion: Based on published reports for high similarity of H9N2 with human H5N1 isolates, it is important to consider the potential of Iranian avian influenza viruses to infect human.

Two gonadotropin-releasing hormone (GnRH) isoforms were identified in the beluga (Huso huso) brain by cDNA sequencing: prepro-mammalian GnRH (mGnRH) and prepro-chicken GnRH-II (cGnRH-II). The nucleotide sequences of the beluga mGnRH and cGnRH-II precursors are 273 and 258 base pairs (bp) long, encoding peptides of 91 and 86 amino acids, respectively. To investigate the effect of methylmercury (MeHg) on GnRH gene expression, animals were fed with four diets containing increasing levels of MeHg (0 mg kg(-1) [control]; 0.76 mg kg(-1) [low]; 7.8 mg kg(-1) [medium]; 16.22 mg kg(-1) [high]) for 32 days. The effects of MeHg on brain GnRH mRNA levels were evaluated by real-time PCR. A significant decrease in brain mGnRH and cGnRH-II mRNA levels were detected in fish receiving high dietary MeHg dose compared to controls on day 11 (P0.05). On day 18 and 32, all treatment groups had significantly lower brain mGnRH and cGnRH-II mRNA levels compared to the control group (P0.05). These findings demonstrate a disruptive role of MeHg on the level of brain mGnRH and cGnRH-II mRNAs in immature beluga.

BACKGROUND: Leishmaniasis recidivans (LR) is a rare phenomenon in the world with high morbidity in children. METHODS: Overall 22 838 school children were examined during 1994-2006. Diagnosis was performed by combination of methods as clinical appearance, direct smears, cultures, polymerase chain reaction (PCR) and histology. RESULTS: Ninety-eight cases were diagnosed as LR with duration of lesions varying from 2 to 8 years and diameter of lesions 1-5 cm, yellowish-brown appearance with papules around or in the scar. Most of the lesions (95%) were on the face. No amastigote was found in direct smears. Identification of nine random isolates by PCR confirmed all species to be L. tropica. Tissue sections showed typical granulomatous reactions with various inflammatory cells but no visible amastigote was seen. CONCLUSIONS: The presence of LR as an important cause of morbidity has future implications for treatment regimens and immunoprophylaxis.

Hepatitis B virus (HBV) viral load and its genotype play important roles in clinical outcome, management of disease and response to antiviral therapy. In many parts of the world such as Europe or the Middle East, distinguishing HBV genotype D from non-D is most relevant for treatment decisions, because genotype D-infected patients respond poorly to interferon-based therapeutic regimens. Here, we developed an in-house real-time PCR to concordantly assess HBV genotype (D vs non-D) based on melt curve analysis and quantify the viral load. Genotype distinction was established with control plasmids of all HBV genotypes and validated with 57 clinical samples from patients infected with six different HBV genotypes. Our in-house real-time PCR assay could discriminate HBV genotype D from non-D using single-step melt curve analysis with a 2 °C difference in the melt curve temperature in all samples tested. Viral load quantification was calibrated with the WHO HBV international standard, demonstrating an excellent correlation with a commercial kit (r = 0.852; P < 0.0001) in a linear range from 3.2 × 10(2) to 3.2 × 10(10) IU/mL. In conclusion, we developed a rapid, simple and cost-effective method to simultaneously quantify and distinguish HBV genotypes D from non-D with a single-step PCR run and melt curve analysis. This assay should be a useful diagnostic alternative to aid clinical decisions about initiation and choice of antiviral therapy, especially in geographical regions with a high prevalence of HBV genotype D.

Liposomes as a lipid-based system have been shown to be an effective adjuvant formulation. In this study, the role of liposome charge in induction of a Th1 type of immune response and protection against leishmaniasis in BALB/c mice was studied. Liposomes containing rgp63 were prepared by Dehydration-Rehydration Vesicle (DRV) method. Neutral liposomes consisted of dipalmitoylphosphatidylcholine and cholesterol. Positively and negatively charged liposomes were prepared by adding dimethyldioctadecylammonium bromide (DDAB) or dicetyl phosphate (DCP) to the neutral liposome formulation, respectively. Female BALB/c mice were immunized subcutaneously with negatively, positively charged or neutral liposomes encapsulated with rgp63, rgp63 in soluble form or PBS, three times in 3week intervals. The extent of protection and type of immune response generated were studied in different groups of mice. The group of mice immunized with rgp63 encapsulated in neutral liposomes showed a significantly (P

GB virus C (GBV-C) infection is frequent in patients infected with the human immunodeficiency virus (HIV) due to similar transmission routes of these viruses. The aim of this study was to determine the rate of infection and genotypic characteristics of GBV-C in this population. The presence of GBV-C RNA was determined in serum samples of 106 patients infected with HIV by reverse transcriptase-nested polymerase chain reaction. GBV-C genotypes were determined by direct sequencing. Hepatitis B surface antigen (HBsAg), hepatitis B surface antibody (anti-HBs), hepatitis C antibody (anti-HCV), alanine aminotransferase (ALT) levels, HIV viral load and CD4+ count were also tested in all patients. The overall prevalence of GBV-C infection was 11.3% in HIV patients. There was no significant difference between patients with and without GBV-C infection regarding age, sex, route of transmission, viral load, ALT levels, HBV and HCV co-infection and treatment with antiretroviral drugs. 66.7% of patients with GBV-C had a CD4+ count ≥200 and 33.3% had a CD4+ count

Antinociceptive potency of opioids is greater against various noxious stimuli in animals with peripheral inflammation. Opioid agonists stimulate activation of G-protein-coupled receptor. Changes in the resting levels of G-protein subtypes could have an effect on intracellular signalling pathways. The present study was designed to investigate the effects of analgesic morphine treatment on the level G-protein subunits mRNA in the presence and absence of inflammation. Our results showed that the carrageenan administration increased G-protein subunits. Administration of analgesic dose of morphine alone and in the presence of inflammation induced different alterations in the levels of G-protein mRNA. Taken together, the results obtained using real time RT-PCR suggested that G-protein genes expression levels following the acute administration of morphine between animals with and without inflammation could influence, at least in part, analgesic responsiveness.

CpG oligodeoxynucleotides (CpG ODN) are known to be a potent immunoadjuvant for a wide range of antigens. The aim of this study was to evaluate the role of CpG ODN co-encapsulated with rgp63 antigen in cationic liposomes (Lip-rgp63-CpG ODN) in immune response enhancement and protection in BALB/c mice against leishmaniasis. Lip-rgp63-CpG ODN prepared by using dehydration-rehydration vesicle (DRV) method significantly inhibited (P

Background: The optimization of bioprocess conditions towards improved growth profile and productivity yield is considered of great importance in biopharmaceutical manufacturing. Peptones as efficient sources of nutrients have been studied for their effect on media development; however, their role on metabolic pathway is not well understood. Methods: In the present study, the effect of different concentration of peptones on a recombinant Chinese hamster ovary (CHO) cell line grown in three serum-free suspension cultures was determined. Six peptones of different origins and available amino acid profiles were investigated regarding their impact on cell growth, productivity, and metabolic pathways changes. Results: In optimized feeding strategies, increases of 136% and 159% in volumetric productivity (for a low-nutrient culture media) and 55% (for a high-nutrient culture media) were achieved. Furthermore, particular sources of peptones with specific amino acid profile developed preferential results for each different culture medium. Two peptones, SoyA2SC and SoyE-110, were the only hydrolysates that showed production improvement in all three media. Casein Peptone plus Tryptone N1 and SoyA3SC showed different improved results based on their implemented concentration for each individual basal medium. Conclusion: The amino acid profile of peptones may provide clues to identify the most effective feeding strategies for recombinant CHO cells.

Recently, Pichia pastoris has been the focal point of interest as an expression system for production of many recombinant proteins. The study and optimization of feeding strategy are of major importance to achieve maximum volumetric productivity in fed-batch cultivations. Among different feeding strategies used in P. pastoris fed-batch cultures, those trying to maintain a constant specific growth rate have usually resulted in superior productivities. The objective of the present study was to investigate and optimize the co-feeding of glycerol and methanol to attain maximum expression of t-PA in P. pastoris fed-batch cultures with constant specific growth rate. The experiments were designed by response surface methodology, considering the specific feeding rates of methanol and glycerol as independent variables. In each experiment, glycerol and methanol were fed according to a predetermined equation to maintain a constant specific growth rate. It was found that with glycerol feeding for higher specific growth rates, the inhibitory properties of glycerol are more pronounced, while the best expression level was achieved when the ratio of µ set glycerol to that of methanol was around 1.67. In all specific growth rates tested, almost a similar ratio of the specific glycerol feeding rate to that of methanol led to the maximum protein production and activity. The statistical model predicted the optimal operating conditions for µ set glycerol and that of methanol to be 0.05 and 0.03 h(-1), respectively. Applying the optimum strategy, maximum of 52 g/L biomass, 300 mg/L t-PA and 340,000 IU/mL enzyme activity were obtained.

The objective of this study was expression of a recombinant fusion protein p24–gp41 to gain a proper folding pattern of the proteins which could be recognized by specific antibodies against human immunodeficiency virus type 1 (HIV-1) for development of a reliable serodiagnostic kit. Serodiagnostic method using enzyme-linked immunosorbent assay (ELISA) with the expressed recombinant fusion protein p24–gp41 was carried out to test the sensitivity and specificity of the protein using human sera and various reference panels from Boston Biomedica Inc. (BBI). The level of the expression was determined to be 30% and the final recovery from fermentation and purification process was calculated as 80 mg/L with more than 98% purity. The developed ELISA assay was demonstrated to have 100 and 99.5% sensitivity and specificity, respectively, detecting anti-HIV-1 antibody using 900 positive and 10,000 negative human sera. The developed assay showed reliable results in comparison with other reference HIV ELISA kits using various BBI panels as well. In conclusion, the recombinant fusion protein p24–gp41 was expressed and used to develop a serodiagnostic kit for screening of the HIV-1 with high sensitivity (100%) and specificity (99.5%) which could be useful for screening large groups of blood donors.

In spite of hepatitis B virus (HBV) vaccination, HBV infection remains an important public health problem worldwide. Although the HBV genotype distribution has been determined in some parts of South Central Asia, no survey has been conducted to determine the HBV genotype in Afghanistan. Twelve Afghan patients infected with HBV living in Afghanistan were enrolled in this study. Partial HBsAg and basic core promoter, precore, and core (BCP/preC/C) regions were amplified and subjected for direct sequencing. In parallel, precore G1896A mutation was also determined by an amplification-created restriction site method. Results revealed HBV genotype D (95% bootstrap value), sub-genotype D1 (98% bootstrap value), and subtype ayw2 in all Afghan isolates. Afghan isolates clustered in a separate branch in the D1 sub-genotype called D1', while supported by 82% bootstrap value. The percentage of intra-genotypic distance among Afghan isolates was 1.05% and inter-genotypic distance with the other genotype D was 2.87% and with other genotypes was 7.50%-11.1%. The wild-type, mixed infection, and precore mutant were found in six, two, and four HBV isolates, respectively. The A1762T/G1764A BCP dual mutation was found in one isolate. Three isolates presented single mutation in the BCP dual mutation region, whereas two showed a novel G1764T mutation. In conclusion, this preliminary study revealed HBV genotype D, sub-genotype D1, and subtype ayw2 of HBV among hepatitis B infected patients from Afghanistan. Further investigation should be carried out.

The most commonly occurring hepatitis B virus (HBV) mutation is the G to A mutation at nucleotide 1896 in the precore region. The aim of this study was to develop a novel accurate amplification created restriction site (ACRS) method for determination of the TGG wild type and the TAG precore mutant HBV variants. Two conserved and consensus specific and diagnostic primers introducing BstXI and XagI cleavage sites were designed in order to determine the G1896 wild type and the A1896 precore mutant HBV variants in all HBV genotypes. The results of the ACRS method were compared with sequencing data. With the ACRS method, three different patterns could be distinguished for the wild type, the precore mutant and mixed infection HBV variants. The results of the ACRS method on 30 HBV isolates revealed the TAG precore mutant in 50% (15/30), the TGG wild type variant in 30% (9/30) and the mixed infection in 20% (6/30). The sequencing data of these samples were in agreement with the ACRS results. The ACRS method is a rapid and cost-effective technique for detecting both the TGG wild type and the TAG HBV precore mutant variants. It can be carried out for follow-up of G1896A precore mutant variant in hepatitis B virus infected subjects at routine molecular diagnostic laboratories.

International audience; Recombinant tissue plasminogen activator (rt-PA) is one of the most important thrombolytic agents for treating cardiovascular obstructions such as stroke. Glycoprotein rt-PA is a serine protease, consisting of 527 amino acids of which 35 are cysteine residues. A variety of recombinant protein expression systems have been developed for heterologous gene expression in prokaryotic and eukaryotic hosts. In recent years, Leishmania tarentolae has been considered because of its safety aspects and special attributes in expression of complex proteins. In this study, two expression cassettes, each one including two copies of t-PA cDNA, were used for integration into the L. tarentolae genome by electroporation. Transformed clones were selected in the presence of appropriate antibiotics. Expression of active rt-PA was confirmed by Western blot and Zymography tests. Real-time PCR analysis was applied to investigate the presence of multiple t-PA gene copies in the parasite genome. Correlation of t-PA gene dosage and production rate was confirmed with real-time PCR. It was shown that the expression level of rt-PA in L. tarentolae is at least 480 IU/mL of culture media. This concentration of rt-PA is seven times higher than what was reported in previous studies in L. tarentolae and some other eukaryotic systems.

Introduction: This study compared the efficacy of Aryoseven with Novoseven to control bleeding episodes in patients with hemophilia A with inhibitors. Methods: Sixty-six patients were randomized into 2 groups, with 4 consecutive block randomization. These groups received Aryoseven and Novoseven dosages of 90 to 120 μg/kg intravenously every 2 hours. Results: Median (interquartile range) level of factor VIII (FVIII) inhibitor in groups A and B was 15.0 and 19.0 Bethesda Unit (BU) preadministration. Bleeding onset in group A was 1246 ± 1104 minutes and in group B was 2301 ± 1693 minutes ( P = .311). The Kavakli global response scores and treatment success rate was comparable in both the groups. The side effects in groups A (9.7%) and B (2.9%) were comparable. Conclusion: Biosimilar recombinant activated FVII is found to be as effective as Novoseven in the treatment of acute joint bleeding in patients with hemophilia with inhibitors. Its usage will decrease the gaps in hemophilia.

Background Transient Gene Expression (TGE) gained popularity over the last decade as a rapid method for the production of milligram to gram quantities of recombinant proteins for preclinical studies in biophama industry. Thereby, the optimization of the TGE technique for Chinese hamster ovary (CHO) as the dominant host for the production of biotherapeutics is of great interest to reach the values for Human Embryo Kidney-293 (HEK-293) cells in terms of transfection efficiencies and production titers. TGE efficiencies are cell line and vector dependant. Methods In transfection efficiency optimization experiments, different starting cell densities, different amounts of plasmid DNA and PEI transfection reagent were investigated to achieve the best conditions leading to maximum transfection efficiencies. Furthermore, in order to investigate the effect of peptone feeding on transfection efficiency, three different sources of peptones with the greatest effect in the CD DG44 basal media were selected; Casein Tryptone N1, Soy petone A2SC and Soy peptone E110. Results The transfection strategy performed here was able to make an outstanding increase in transfection efficiency of CHO DG44 cell line transfected with pTracer-SV40-mutated t-PA plasmid from 3.6% in our starting non-optimized condition to 66.93% in finally optimized situation. Moreover, peptone feeding strategy used here was successful to increase volumetric productivities up to 37%. In addition, the amounts of both PEI and plasmid DNA were reduced up to 66% and 25%, respectively compared to our previous protocol. Conclusion Here we described an optimization process for TGE in suspension-adapted CHO cells based on Polyethylenimine (PEI)/DNA concentration, DNA: PEI ratio, starting cell densities and peptone feeding strategy.

Objectives of this study were to test the cytokine gene expression in peripheral blood mononuclear cells (PBMCs) from cases with nonhealing and healing cutaneous leishmaniasis (CL) in response to in vitro stimulation of recombinant gp63 (rgp63) and soluble Leishmania antigen (SLA). Healing and nonhealing cases are, respectively, defined as recovered from disease and refractory to various treatments. To evaluate the type of immunological response, mRNA transcription level for interleukin (IL)-4, IL-10, IL-12 and interferon (IFN)-γ were determined using semiquantitative reverse transcription–polymerase chain reaction (RT–PCR) technique in PBMCs of these volunteers. The results clearly demonstrated a high level of IL-4 expression in nonhealing cases of CL and a low expression level of transcripts for IFN-γ and IL-12. In contrast, a high level of IFN-γ and IL-12 expression and a low level of IL-4 and IL-10 expression were detected in the healing cases. These findings not only support the balance of Th1/Th2 cytokines in the inducing predominant profile in healing and nonhealing cases, but it may also show the potential of rgp63 as a proper immunogen which might induce protective responses.

International audience; Methylmercury (MeHg) is the most toxic form of mercury which is bioaccumulated in the aquatic food chain. It has been shown that one of the main targets of MeHg toxicity is the brain, but there is little knowledge of the molecular mechanisms of its toxic effects. In this work we used a proteomics analysis to determine the changes in the brain proteome of juvenile beluga (Huso huso) exposed to dietary MeHg. The juvenile beluga were fed the diet containing 0.8 ppm MeHg for 70 days. Proteins of the brain tissue were analyzed using two-dimensional electrophoresis and MALDI-TOF/TOF mass spectrometry. We found eight proteins with significant altered expression level in the fish brain exposed to MeHg. These proteins are involved in different cell functions including cell metabolism, protein folding, cell division, and signal transduction. Our results support the idea that MeHg exerts its toxicity through oxidative stress induction and apoptotic effects. They also suggest that chronic MeHg exposure would induce an important metabolic deficiency in the brain. These findings provide basic information to understand possible mechanisms of MeHg toxicity in aquatic ecosystems.