Recent developments in miRNA based recombinant protein expression in CHO.

It is widely accepted that the growing demand for recombinant therapeutic proteins has led to the expansion of the biopharmaceutical industry and the development of strategies to increase recombinant protein production in mammalian cell lines such as SP2/0 HEK and particularly Chinese hamster ovary cells. For a long time now, most investigations have been focused on increasing host cell productivity using genetic manipulating of cellular processes like cell cycle, apoptosis, cell growth, protein secretory and other pathways. In recent decades MicroRNAs beside different genetic engineering tools (e.g., TALEN, ZFN, and Crisper/Cas) have attracted further attention as a tool in the genetic engineering of host cells to increase protein expression levels. Their ability to simultaneously target multiple mRNAs involved in one or more cellular processes made them a favorable tool in this field. Accordingly, this study aimed to review the methods of selecting target miRNA for cell line engineering, miRNA gain- or loss-of-function strategies, examples of laboratory and pilot studies in this field and discussed advantages and disadvantages of this technology.

Optimization of culture conditions for high-level expression of soluble and active tumor necrosis factor-α in E. coli.

Anti-TNF inhibitors exert their therapeutic effect by inhibition of the excessive amounts of TNF-α within the body. Recombinant TNF-α should be produced in a soluble refolded form to investigate the effectiveness and efficiency of anti-TNF-α compounds. In this research, the designed cassette was subcloned in the pET28a expression vector and expressed in E. coli BL21 (DE3). The identity of the protein was confirmed through SDS-PAGE and Western blotting. After optimizing expression conditions, protein purification was performed using native Ni-NTA affinity chromatography. The biological activity of the soluble recombinant TNF-α was investigated using MTT assay. Also, the affinity of an anti-TNF-α agent, Altebrel, was investigated against the expressed protein through ELISA. Optimization of TNF-α expression conditions represented that the highest expression could be achieved at 37 °C using 0.5 mM IPTG 6 h post-induction. The recombinant protein represented an inhibitory effect on the L929 murine fibroblast cell line and was successfully detected by Altebrel in ELISA. Binding kinetics were also studied using Cimzia as an anti-TNF-α molecule and 7.2 E-13M was calculated as the equilibrium dissociation constant value (KD). The significant expression level of the recombinant protein in the soluble form, its high purity, and assessment of its biological activity showed that the expressed protein could be used in tests of ELISA and MTT to assess the activity of anti-TNF-α agents.

Enhanced truncated-t-PA (CT-b) expression in high-cell-density fed-batch cultures of Pichia pastoris through optimization of a mixed feeding strategy by response surface methodology.

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.

Development of Genetically Modified Chinese Hamster Ovary Host Cells for the Enhancement of Recombinant Tissue Plasminogen Activator Expression.

BACKGROUND: 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). METHODS: 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. RESULTS: 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. CONCLUSION: The results obtained in this study indicate successful development of the improved CHO host cells through CERT S132A overexpression.

Multiple myeloma cell-derived exosomes promote favorable tumor functional performance by polarizing macrophages toward M2-like cells.

It has long been hypothesized that leukemic cells are able to modulate the fate of resident cells in the tumor microenvironment (TME) toward either supporting or immunosuppressive cells for the development of tumors. Exosomes can be a potential culprit in imposing tumor desire. There is evidence about the impact of tumor-derived exosomes on different immune cells in different malignancies. However, findings about macrophages are contradictory. Here, we evaluated the potential influence of multiple myeloma (MM)-cell-derived exosomes on the polarization of macrophages by examining hallmarks of M1 and M2 macrophages. After treatment of M0 macrophages with isolated exosomes (from U266B1), gene expression (Arg-1, IL-10, TNF-α and IL-6), immunophenotyping markers (CD206), cytokine secretion (IL-10 and IL-6), nitric oxide (NO) production, and redox potentiality of target cells were assessed. Our results revealed significantly increased expression of the genes involved in the development of M2-like cells but not M1 cells. The CD 206 marker and IL-10 protein levels were significantly increased at different time points. The expression of IL-6 mRNA and IL-6 protein secretion did not change significantly. MM-cell-derived exosomes induced significant changes in NO production and intracellular ROS levels in M0 cells.

Overexpression of miR-32 in Chinese hamster ovary cells increases production of Fc-fusion protein.

The demand for industrial genetically modified host cells were increased with the growth of the biopharmaceutical market. Numerous studies on improving host cell productivity have shown that altering host cell growth and viability through genetic engineering can increase recombinant protein production. During the last decades, it was demonstrated that overexpression or downregulation of some microRNAs in Chinese Hamster Ovary (CHO) cells as the host cell in biopharmaceutical manufacturing, can improve their productivity. The selection of microRNA targets has been based on their previously identified role in human cancers. MicroRNA-32 (miR-32), which is conserved between humans and hamsters (Crisetulus griseus), was shown to play a role in the regulation of cell proliferation and apoptosis in some human cancers. In this study, we investigated the effect of miR-32 overexpression on the productivity of CHO-VEGF-trap cells. Our results indicated that stable overexpression of miR-32 could dramatically increase the productivity of CHO cells by 1.8-fold. It also significantly increases cell viability, batch culture longevity, and cell growth. To achieve these results, following the construction of a single clone producing an Fc-fusion protein, we transfected cells with a pLexJRed-miR-32 plasmid to stably produce the microRNA and evaluate the impact of mir-32 overexpression on cell productivity, growth and viability in compare with scrambled control. Our findings highlight the application of miRNAs as engineering tools and indicated that miR-32 could be a target for engineering CHO cells to increase cell productivity.

Computational and Experimental Evaluation of Linker Peptides and Thioredoxin Fusion Tag in CD20-rituximab Specific Interactions.

Background: Overexpression of CD20 protein on the surface of B cells in lymphoma can be targeted by several anti-CD20 molecules. The development of accessible interactive epitopes is more favorable than the full-length transmembrane CD20 in the affinity assessment of anti-CD20 monoclonal antibodies (mAbs). Methods: The sequence of these epitopes was extracted, and the effects of different linker peptides and the location of histidine (His)-tag were computationally analyzed. The impact of thioredoxin (Trx)-tag on the folding of the selected construct and its interaction with rituximab was further investigated. The two final expression cassettes were expressed in Escherichia coli after optimization of culture conditions for incubation temperature, post-induction time, optical density at the induction time, and concentration of the inducer. ELISA evaluated the binding affinity of rituximab towards the recombinant proteins. Results: By homology modeling studies, C-terminal His-tagged structures represented more desirable folded structures. Validation of the models revealed that CD20 extracellular domain linked by the G4S polypeptide had better stereochemical quality and structural compatibility. It was selected due to its more effective interaction with rituximab showing the highest dissociation constant of 5.8E-09M, which improved after the fusion of Trx-tag (7.1E-10M). The most influential parameters in the expression of the two selected proteins were post-induction temperature and optical density at the induction time. Homemade ELISA assays revealed a slightly higher affinity of rituximab towards the Trx-CD20 protein than the CD20/G4S molecule. Conclusions: Experimental in vitro studies confirmed the computationally calculated affinity of rituximab towards the two designed CD20 constructs. Also, the cell-based binding assessment of anti-CD20 mAbs could be substituted by the engineered extracellular domain of human CD20 protein.

Development and characterization of single domain monoclonal antibody against programmed cell death ligand-1; as a cancer inhibitor candidate.

Objectives: One of the important interactions in controlling the human immune system is the reaction between checkpoint proteins such as programmed cell death-1 (PD-1) and its ligand, PD-L1. These are negative immunoregulatory molecules that promote immune evasion of tumor cells. PD-L1 expression is an immune-mediated mechanism used by various malignant cells in order to down-regulate the immune system. Checkpoint inhibitors (CPIs) are a new class of anti-cancer agents that stimulate immune cells to elicit an antitumor response by blocking the ligand and receptor interactions. Nanobody (Nb) as a new type of antibody fragment, has some potential as CPI. Materials and Methods: A female camel was immunized with recombinant PD-L1 protein, nanobody library was constructed and PD-L1 specific Nb was selected. The selected Nb was characterized in terms of affinity, specificity, and binding potency in ELISA, Western blotting, and flow cytometry. Results: Developed nanobody, A22 binds to its cognate target with high specificity and affinity. Western blot and flow cytometry techniques showed that nanobody A22 was able to specifically detect and attach to human PD-L1 protein on the cell surface and in the cell lysate. MTT assay showed the inhibitory effect of PD-L1 by specific Nb on A431 and HEK293 cells, with no cytotoxic effect on cell growth. Conclusion: The results highlighted the potential of anti-PD-L1 Nb as a novel therapeutic in cancer therapy without undesirable cytotoxicity.

Expression of a novel chimeric truncated t-PA in CHO cells based on in silico experiments.

Tissue plasminogen activator (t-PA) is one of the fibrin-specific serine proteases that play a crucial role in the fibrinolytic system. The rapid clearance of the drug from the circulation, caused by its active uptake in the liver, has lead to complicated clinical applications. Different forms of plasminogen activators have been developed to treat thrombotic disease. Deletion of the first three domains of t-PA by gene manipulation techniques has shown a significant increase in its plasma half life. In order to compensate the disadvantage of higher bleeding risk, a novel chimeric truncated form of t-PA with 394 amino acids and more fibrin affinity compared to the truncated form was designed to be expressed in Chinese Hamster Ovarian (CHO) cells. The recombinant chimeric plasminogen activator consists of kringle 2 and serine protease (K2S) domains of t-PA, namely GHRP-SYQ-K2S. The level of expression was found to be 752 IU/ml with 566,917 IU/mg specific activity, based on amidolytic activity. The fibrin binding of this novel chimeric truncated t-PA was 86% of the full length t-PA at a fibrinogen concentration of 0.2 mg/ml. This could be a promising approach with more desirable pharmacodynamic properties compared to existing commercial forms.

Molecular cloning of cDNA of mammalian and chicken II gonadotropin-releasing hormones (mGnRHs and cGnRH-II) in the beluga (Huso huso) and the disruptive effect of methylmercury on gene expression.

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 (P < 0.05). On day 18 and 32, all treatment groups had significantly lower brain mGnRH and cGnRH-II mRNA levels compared to the control group (P < 0.05). These findings demonstrate a disruptive role of MeHg on the level of brain mGnRH and cGnRH-II mRNAs in immature beluga.

Prevalence of viral hepatitis and molecular analysis of HBV among voluntary blood donors in west Iran.

To determine the prevalence of viral hepatitis infection and hepatitis B virus (HBV) molecular characterization, 11,200 blood donors from citizens of Shahrekord (a city located in west of Iran) were investigated. Results showed HBsAg-positive in 1.78% of persons (n=200), anti-HDV-positive in 3% of HBsAg-positive cases (n=6) and anti-HCV-positive in 0.67% of donors (n=76). HBV phylogenetic analysis disclosed HBV genotype D, sub-genotype D1, and subtype ayw2. Amino acid mapping of the HBV pol region revealed various HBV drug-resistance mutations though donors had no antiviral therapy. In conclusion, this study demonstrated notable viral hepatitis seroprevalence rate in blood donors in west of Iran.

Enhancement of Expression Level of Modified t-PA (TNKase) in Leishmania tarentolae by Induction System

Background: The expression of bio-therapeutic proteins in mammalian cells, such as CHO, attains high homogeneity related to post-translational modifications. Although CHO remains the most popular cell line for bestselling biotherapeutic proteins on the market, there are still drawbacks such as expensive culture media, long time line, and high drug cost. Recently, researches on a novel Leishmania protozoan system have confirmed that this low-level eukaryote could represent a competitive alternative to the mammalian cell lines. Methods: The full length of coding sequence of modified tPA TNKase (tenecteplase) was synthesized and cloned into an inducible expression vector of L. tarentolae T7-TR cells. Results: The expression of the construct was driven by a Tet-inducible promoter. A Leishmania secretory signal sequence was also added to the expression cassette to facilitate the release of the recombinant protein into the medium. The secretory recombinant protein was analyzed and confirmed by SDS-PAGE and Western blot analyses. The expression level of TNKase in this novel system of L. tarentolae was 810 IU/mL after induction, which means that the percentage of expression increases two times compared to previous models in L. tarentolae. The TNKase activity was comparable with Activase. Conclusion: Our results suggested that expressed TNK (modified tPA) is functionally compatible with Activase regarding their effect on fibrinolysis. Given the post-translational modification similarities between mammalian and L. tarentolae, it is speculated that this system is capable of producing complex proteins such as tPA similar to mammalian system, with easier manipulation and non-expensive method.

Expression of human tissue plasminogen activator in the trypanosomatid protozoan Leishmania tarentolae.

A variety of recombinant protein expression systems have been developed for heterologous genes in both prokaryotic and eukaryotic systems such as bacteria, yeast, mammals, insects, transgenic animals and transgenic plants. Also, it has been reported that Leishmania tarentolae, a trypanosomatid protozoan parasite of the white-spotted wall gecko (Tarentola annularis), has the capability of expressing heterologous genes. Trypanosomatidae are rich in glycoproteins, which can account for more than 10% of total protein. The oligosaccharide structures of their glycoproteins are similar to those of mammals with N-linked galactose, and sialic acid residues. For a variety of reasons, including the glycosylation patterns and the secondary structures of some of these proteins, synthesis in eukaryotic system is highly preferable. In addition, formation of native disulfide bonds in complex eukaryotic proteins is tremendously important. In the present study, we tried to express the tPA (tissue plasminogen activator) gene in L. tarentolae. This protein is a thrombolytic agent with 527 amino acid residues. tPA possesses serine-protease activity, with 35 cysteine residues that participate in the formation of 17 disulfide bonds. We have used an expression cassette, including the alpha intergenic regions of Leishmania major and two sites at the 3'- and 5'-ends, for homologous recombination in L. tarentolae, in addition to antibiotic-resistant genes. Southern-blot analysis showed that the human tPA gene had been inserted into the genome of the parasite. The expression of the tPA at the mRNA and protein levels was confirmed. It was shown that the expressed tPA in this system was 70 i.u. (international units)/ml of culture media, which is much higher than levels reported previously in other systems.

A serological screening assay of human immunodeficiency virus type 1 antibodies based on recombinant protein p24-gp41 as a fusion protein expressed in Escherichia coli.

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.

A novel accurate amplification created restriction site method for determination of the wild type and the precore mutant hepatitis B virus variants.

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.

Expression of a novel chimeric-truncated tPA in Pichia pastoris with improved biochemical properties.

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.

Design of a novel chimeric tissue plasminogen activator with favorable Vampire bat plasminogen activator properties.

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.

Engineering the cellular protein secretory pathway for enhancement of recombinant tissue plasminogen activator expression in Chinese hamster ovary cells: effects of CERT and XBP1s genes.

Cell line development is the most critical and also the most time-consuming step in the production of recombinant therapeutic proteins. In this regard, a variety of vector and cell engineering strategies have been developed for generating high-producing mammalian cells; however, the cell line engineering approach seems to show various results on different recombinant protein producer cells. In order to improve the secretory capacity of a recombinant tissue plasminogen activator (t-PA)-producing Chinese hamster ovary (CHO) cell line, we developed cell line engineering approaches based on the ceramide transfer protein (CERT) and X-box binding protein 1 (XBP1) genes. For this purpose, CERT S132A, a mutant form of CERT that is resistant to phosphorylation, and XBP1s were overexpressed in a recombinant t-PA-producing CHO cell line. Overexpression of CERT S132A increased the specific productivity of t-PA-producing CHO cells up to 35%. In contrast, the heterologous expression of XBP1s did not affect the t-PA expression rate. Our results suggest that CERTS132A- based secretion engineering could be an effective strategy for enhancing recombinant t- PA production in CHO cells.

Immune responses against a new HIV-1 p24-gp41/pCAGGS-IL-12 DNA vaccine in Balb/c mice.

BACKGROUND: Development of an effective vaccine is highly needed in order to restrict the AIDS pandemic. DNA vaccines initiate both arms of immunity without the potential of causing disease. HIV-1 p24 and gp41 (gag and env) proteins play important roles in viral pathogenesis and are effective candidates for immune induction and vaccine design. OBJECTIVE: In this study, new DNA vaccine candidates constructed from HIV-1 fused p24-gp41 or gp41 alone were evaluated in Balb/c mice for induction of cellular and humoral immune responses. METHODS: Recombinant plasmids, pcDNA3.1/Hygro expression vector containing immunogenic sequences of fused p24-gp41 or gp41alone were produced. Dendrosome used as a system for carrying vectors in laboratory animals, and an IL-12 containing vector (pCAGGS-IL-12) was co-immunized with the p24-gp41 vector as a genetic adjuvant. Induction of effective immune responses against the designed vectors as DNA vaccine candidates in Balb/c mice was evaluated. Levels of total antibodies, IgG isotypes (IgG2a and IgG1); IFN-γ and IL-4 were measured by ELISA. MTT assay was used to evaluate lymphoproliferation. RESULTS: The results confirmed that the immunogenic epitopes of both p24 and gp41 genes are highly effective inducers of immune responses, and administration of fused p24-gp41 alone or along with IL-12 resulted in further enhancement of immune responses. Group 4 that received fused fragments (p24-gp41) along with an IL-12 expressing vector demonstrated a significantly higher Stimulation Index (SI) and IFN-γ production (p<0.0001) with a significant increase in IgG2a/IgG1 ratio, indicating the stimulation of CMI towards Th1. Although gp41 containing vector (group 6) also showed significant increases in both proliferation and IFN-γ production, the responses were persistently lower than that of p24-gp41 containing vectors. Total antibody production was highest in group 6 as expected. CONCLUSION: Dendrosome proved to be an efficient carrier of recombinant plasmids constructed in this study. Further studies are necessary to evaluate these constructs as HIV vaccine candidates.

Combined TGE-SGE expression of novel PAI-1-resistant t-PA in CHO DG44 cells using orbitally shaking disposable bioreactors.

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.