EF1α Promoter Research Articles

Stable transformation of Babesia bigemina and Babesia bovis using a single transfection plasmid

Babesia bigemina and Babesia bovis, are the two major causes of bovine babesiosis, a global neglected disease in need of improved methods of control. Here, we describe a shared method for the stable transfection of these two parasites using electroporation and blasticidin/blasticidin deaminase as a selectable marker. Stably transfected B. bigemina and B. bovis were obtained using a common transfection plasmid targeting the enhanced green fluorescent protein-BSD (egfp-bsd) fusion gene into the elongation factor-1α (ef-1α) locus of B. bigemina and B. bovis under the control of the B. bigemina ef-1α promoter. Sequencing, Southern blotting, immunoblotting and immunofluorescence analysis of parasite-infected red blood cells, demonstrated that the egfp-bsd gene was expressed and stably integrated solely into the ef-1α locus of both, B. bigemina and B. bovis. Interestingly, heterologous B. bigemina ef-1α sequences were able to drive integration into the B. bovis genome by homologous recombination, and the stably integrated B. bigemina ef-1α-A promoter is fully functional in B. bovis. Collectively, the data provides a new tool for genetic analysis of these parasites, and suggests that the development of vaccine platform delivery systems based on transfected B. bovis and B. bigemina parasites using homologous and heterologous promoters is feasible.

RNAi-Mediated Loss of Function of Xenopus Immune Genes by Transgenesis.

Generation of transgenic frogs through the stable integration of foreign DNA into the genome is well established in Xenopus This protocol describes the combination of transgenesis with stable RNA interference as an efficient reverse genetic approach to study gene function in Xenopus Initially developed in the fish medaka and later adapted to Xenopus, this transgenic method uses the I-SceI meganuclease, a "rare-cutter" endonuclease with an 18 bp recognition sequence. In this protocol, transgenic X. laevis with knocked down expression of a specific gene are generated using a double promoter expression cassette. This cassette, which is flanked by I-SceI recognition sites, contains the shRNA of choice under the control of the human U6 promoter and a green fluorescent protein (GFP) reporter gene under the control of the human EF-1α promoter. Prior to microinjection the plasmid is linearized by digestion with I-SceI and the entire reaction is then microinjected into one-cell stage eggs. The highly stringent recognition sequence of I-SceI is thought to maintain the linearized plasmid in a nonconcatamerized state, which promotes random integration of the plasmid transgene in the genome. The injected embryos are reared until larval stage 56 and then screened for GFP expression by fluorescence microscopy and assessed for effective knockdown by quantitative RT-PCR using a tail biopsy. Typically, the I-SceI meganuclease transgenesis technique results in 35%-50% transgenesis efficiency, a high survival rate (>35%) and bright nonmosaic GFP expression. A key advantage of this technique is that the high efficiency and nonmosaic transgene expression permit the direct use of F0 animals.

A Promising Recombinant Herpesvirus of Turkeys Vaccine Expressing PmpD-N of Chlamydia psittaci Based on Elongation Factor-1 Alpha Promoter.

The obligate intracellular Gram-negative bacterium Chlamydia psittaci often causes avian chlamydiosis and influenza-like symptoms in humans. However, the commercial subunit C. psittaci vaccine could only provide a partial protection against avian chlamydiosis due to poor cellular immune response. In our previous study, a recombinant herpesvirus of turkeys (HVT)-delivered vaccine against C. psittaci and Marek’s disease based on human cytomegalovirus (CMV) promoter (rHVT-CMV-pmpD) was developed and provided an effective protection against C. psittaci disease with less lesions and reduced chlamydial loads. In this study, we developed another recombinant HVT vaccine expressing the N-terminal fragment of PmpD (PmpD-N) based on human elongation factor-1 alpha (EF-1α) promoter (rHVT-EF-pmpD) by modifying the HVT genome within a bacterial artificial chromosome. The related characterization of rHVT-EF-pmpD was evaluated in vitro in comparison with that of rHVT-CMV-pmpD. The expression of PmpD-N was determined by western blot. Under immunofluorescence microscopy, PmpD-N protein of both two recombinant viruses was located in the cytoplasm and on the cell surface. Growth kinetics of rHVT-EF-pmpD was comparable to that of rHVT-CMV-pmpD, and the growth rate of rHVT-EF-pmpD was apparently higher than that of rHVT-CMV-pmpD on 48, 72, and 120 h postinfection. Macrophages activated by rHVT-EF-pmpD could produce more nitric oxide and IL-6 than that activated by rHVT-CMV-pmpD. In this study, a recombinant HVT vaccine expressing PmpD-N based on EF-1α promoter was constructed successfully, and a further research in vivo was needed to analyze the vaccine efficacy.

Identification and characterization of interchangeable cross-species functional promoters between Babesia gibsoni and Babesia bovis

The development of transgenic techniques has been reported in many protozoan parasites over the past few years. We recently established a successful transient transfection system for Babesia gibsoni based on Bg 5′-ef-1α promoter. This study investigated 6 homologous and 6 heterologous promoters for B. gibsoni and B. bovis and identified novel interchangeable cross-species functional promoters between B. gibsoni and B. bovis. Ten out of twelve promoters had heterologous promoter function. In particular, Bg 5′-ef-1α and Bg 5′-actin heterologous promoters resulted in a significantly higher luciferase activity than Bb 5′-ef-1α homologous promoter in B. bovis. The present study showed that Bg 5′-actin promoted the highest luciferase activity in both B. gibsoni and B. bovis. The study further indicates that heterologous promoter function widely exists between B. gibsoni and B. bovis. This finding is an important step for future stable transfection construct design and for the production of vaccines based on transfected B. gibsoni and B. bovis parasites.

Adaptation of vectors and drug-inducible systems for controlled expression of transgenes in the tumor microenvironment

Biological therapies based on recombinant proteins such as antibodies or cytokines are continuously improving the repertoire of treatments against cancer. However, safety and efficacy of this approach is often limited by inappropriate biodistribution and pharmacokinetics of the proteins when they are administered systemically. Local administration of gene therapy vectors encoding these proteins would be a feasible alternative if they could mediate long-term and controlled expression of the transgene after a single intratumoral administration. We describe a new vector platform specially designed for this purpose. Different combinations of transactivators and promoters were evaluated to obtain a fully humanized inducible system responsive to the well-characterized drug mifepristone. The optimal transactivator conformation was based on DNA binding domains from the chimeric protein ZFHD1 fused to the progesterone receptor ligand binding domain and the NFkb p65 activation domain. The expression of this hybrid transactivator under the control of the elongation factor 1α (EF1α) or the chimeric CAG promoters ensured functionality of the system in a variety of cancer types. Expression cassettes with luciferase as a reporter gene were incorporated into High-Capacity adenoviral vectors (HC-Ad) for in vivo evaluation. Systemic administration of the vectors into C57BL/6 mice revealed that the vector based on the EF1α promoter (HCA-EF-ZP) allows tight control of transgene expression and remains stable for at least two months, whereas the CAG promoter suffers a progressive inactivation. Using an orthotopic pancreatic cancer model in syngeneic C57BL/6 mice we show that the local administration of HCA-EF-ZP achieves better tumor/liver ratio of luciferase production than the intravenous route. However, regional spread of the vector led to substantial transgene expression in peritoneal organs. We reduced this leakage through genetic modification of the vector capsid to display RGD and poly-lysine motifs in the fiber knob. Safety and antitumor effect of this gene therapy platform was demonstrated using interleukin-12 as a therapeutic gene. In conclusion, we have developed a new tool that allows local, sustained and controlled production of therapeutic proteins in tumors.

Safety and Efficacy of AAV Retrograde Pancreatic Ductal Gene Delivery in Normal and Pancreatic Cancer Mice

Recombinant adeno-associated virus (rAAV)-mediated gene delivery shows promise to transduce the pancreas, but safety/efficacy in a neoplastic context is not well established. To identify an ideal AAV serotype, route, and vector dose and assess safety, we have investigated the use of three AAV serotypes (6, 8, and 9) expressing GFP in a self-complementary (sc) AAV vector under an EF1α promoter (scAAV.GFP) following systemic or retrograde pancreatic intraductal delivery. Systemic delivery of scAAV9.GFP transduced the pancreas with high efficiency, but gene expression did not exceed >45% with the highest dose, 5 × 1012 viral genomes (vg). Intraductal delivery of 1 × 1011 vg scAAV6.GFP transduced acini, ductal cells, and islet cells with >50%, ∼48%, and >80% efficiency, respectively, and >80% pancreatic transduction was achieved with 5 × 1011 vg. In a KrasG12D-driven pancreatic cancer mouse model, intraductal delivery of scAAV6.GFP targeted acini, epithelial, and stromal cells and exhibited persistent gene expression 5 months post-delivery. In normal mice, intraductal delivery induced a transient increase in serum amylase/lipase that resolved within a day of infusion with no sustained pancreatic inflammation or fibrosis. Similarly, in PDAC mice, intraductal delivery did not increase pancreatic intraepithelial neoplasia progression/fibrosis. Our study demonstrates that scAAV6 targets the pancreas/neoplasm efficiently and safely via retrograde pancreatic intraductal delivery.

Generation of DKK1 transgenic Tibet minipigs by somatic cell nuclear transfer (SCNT).

Hairless mice have been widely applied in skin-related researches, while hairless pigs will be a useful model for skin-related study and other biomedical researches. Dickkopf-related protein 1 (DKK1) is inhibitor of Wnt signaling pathway. Transgenic mice expressing DKK1 transgene under control of a human keratin 14 (K14) promoter display hairless phenotype, which encouraged us to generate transgenic minipigs expressing pig DKK1 transgene under control of K14 promoter and finally achieve hairless minipigs. To generate transgenic cloned pigs, we constructed the lentiviral expression vector pERKDZG which contains two independent expression cassettes, the transcription of Tibet minipig DKK1 and EGFP genes are driven by K14 promoter, while mRFP is regulated under the control of Ef-1α promoter. Prior to generating the transgenic pig, the functionality of pERKDZG construct was verified by fluorescence assay and via checking pDKK1 expression. Subsequently, lentiviruses harboring ERKDZG transgene infected porcine embryonic fibroblasts (PEFs), followed by sorting RFP-positive PEFs by flow cytometry to obtain the purified PEFs carrying ERKDZG, designated DKK1-PEFs as donor cells used for somatic cell nuclear transfer (SCNT). Finally, we obtained 3 DKK1 transgenic cloned pigs with skin-specific expression of pDKK1 and EGFP transgenes, but unfortunately, DKK1 transgenic cloned pigs don't display hairless phenotype as expected. Taken together, we achieve DKK1 transgenic cloned pigs with skin-specific expression of pDKK1 transgene which provide a pig model for exploring DKK1 gene functions in pigs.

Baculovirus-induced recombinant protein expression in human mesenchymal stromal stem cells: A promoter study

Human mesenchymal stem cells (hMSCs) are the current workhorses of regenerative medicine and gene therapy. The corresponding vectors are usually based on lentiviruses, adenoviruses, retroviruses or adeno-associated viruses, but recently they have been joined by baculoviruses, which are more widely known for their role in the development of pesticides and vaccines. Here we show that gene transfer to an immortalized human mesenchymal stroma cell line can be achieved by baculovirus transduction. We also compared the performance of five different constitutive promoters controlling GFP expression. The human elongation factor 1α promoter (EF1α) achieved the strongest GFP expression, whereas the mouse phosphoglycerate kinase 1 promoter (PGK) was the weakest. The human EF1α promoter is therefore recommended for the regulation of genes introduced into hMSC-TERTs by baculovirus vectors.

Foreign gene expression and induction of antibody response by recombinant fowl adenovirus-9-based vectors with exogenous promoters.

Fowl adenoviruses (FAdVs) are promising vectors for poultry vaccines and gene therapy. The commonly used human cytomegalovirus (CMV) promoter in recombinant FAdV-9 viruses (recFAdV-9s) leads to foreign gene expression that elicits an antibody response. Despite its strength, studies have shown that the CMV promoter is prone to silencing by methylation hampering the in vivo application of vectors containing this promoter. Therefore, to improve our virus vector system and circumvent potential limitations of silencing, we engineered recFAdV-9s with foreign gene expression cassettes carrying the CMV enhancer/chicken β-actin (CAG) or the human elongation factor 1 alpha (EF1α) promoters with or without the posttranscriptional regulatory element of woodchuck hepatitis virus (WPRE). Chicken hepatoma cells (CH-SAH) infected with recFAdV-9s carrying either CAG or EF1α promoters expressed higher levels of foreign protein than those infected with recFAdV-9 carrying the CMV promoter. Incorporation of the WPRE element rendered lower gene expression regardless of promoter type. Surprisingly, most chickens inoculated with recFAdV-9 containing the CMV promoter had the highest antibody response to foreign protein compared to other promoters. Our findings suggest the importance of promoter selection for candidate virus vector vaccines based on humoral immune response rather than foreign protein expression levels in vitro.

CRISPR/Cas9-Mediated Knockin Application in Cell Therapy: A Non-viral Procedure for Bystander Treatment of Glioma in Mice.

The use of non-viral procedures, together with CRISPR/Cas9 genome-editing technology, allows the insertion of single-copy therapeutic genes at pre-determined genomic sites, overcoming safety limitations resulting from random gene insertions of viral vectors with potential for genome damage. In this study, we demonstrate that combination of non-viral gene delivery and CRISPR/Cas9-mediated knockin via homology-directed repair can replace the use of viral vectors for the generation of genetically modified therapeutic cells. We custom-modified human adipose mesenchymal stem cells (hAMSCs), using electroporation as a transfection method and CRISPR/Cas9-mediated knockin for the introduction and stable expression of a 3 kb DNA fragment including the eGFP (selectable marker) and a variant of the herpes simplex virus 1 thymidine kinase genes (therapeutic gene), under the control of the human elongation factor 1 alpha promoter in exon 5 of the endogenous thymidine kinase 2 gene. Using a U87 glioma model in SCID mice, we show that the therapeutic capacity of the new CRISPR/Cas9-engineered hAMSCs is equivalent to that of therapeutic hAMSCs generated by introduction of the same therapeutic gene by transduction with a lentiviral vector previously published by our group. This strategy should be of general use to other applications requiring genetic modification of therapeutic cells.

One-Step piggyBac Transposon-Based CRISPR/Cas9 Activation of Multiple Genes.

Neural cell fate is determined by a tightly controlled transcription regulatory network during development. The ability to manipulate the expression of multiple transcription factors simultaneously is required to delineate the complex picture of neural cell development. Because of the limited carrying capacity of the commonly used viral vectors, such as lentiviral or retroviral vectors, it is often challenging to perform perturbation experiments on multiple transcription factors. Here we have developed a piggyBac (PB) transposon-based CRISPR activation (CRISPRa) all-in-one system, which allows for simultaneous and stable endogenous transactivation of multiple transcription factors and long non-coding RNAs. As a proof of principle, we showed that the PB-CRISPRa system could accelerate the differentiation of human induced pluripotent stem cells into neurons and astrocytes by triggering endogenous expression of different sets of transcription factors. The PB-CRISPRa system has the potential to become a convenient and robust tool in neuroscience, which can meet the needs of a variety of in vitro and in vivo gain-of-function applications.

PhiC31 integrase can improve the efficiency of different construct designs for monoclonal antibody expression in CHO cells

ObjectivesSeveral 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 resultsIn 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. ConclusionsOur 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.

Selection of an optimal promoter for gene transfer in normal B cells.

Gene transfer into normal quiescent human B cells is a challenging procedure. The present study aimed to investigate whether it is possible to increase the levels of transgene expression by using various types of promoters to drive the expression of selected genes-of-interest. To produce lentiviral particles, the present study used the 2nd generation psPAX2 packaging vector and the vesicular stomatitis virus -expressing envelope vector pMD2.G. Subsequently, lentiviral vectors were generated containing various promoters, including cytomegalovirus (CMV), elongation factor-1 alpha (EF1α) and spleen focus-forming virus (SFFV). The present study was unable to induce satisfactory transduction efficiency in quiescent normal B cells; however, infection of normal B cells with Epstein-Barr virus resulted in increased susceptibility to lentiviral transduction. In addition, the SFFV promoter resulted in a higher level of transgene expression compared with CMV or EF1α promoters. As a proof-of concept that this approach allows for stable gene expression in normal B cells, the present study used bicistronic lentiviral vectors with genes encoding fluorescent reporter proteins, as well as X-box binding protein-1 and binding immunoglobulin protein.

Enhancement of glycoprotein-based DNA vaccine for viral hemorrhagic septicemia virus (VHSV) via addition of the molecular adjuvant, DDX41

The use of molecular adjuvants to improve the immunogenicity of DNA vaccines has been thoroughly studied in recent years. Glycoprotein (G)-based DNA vaccines had been proven to be effective in combating infection against Rhabdovirus (especially infectious hematopoietic necrosis virus, IHNV) in salmonids. DDX41 is a helicase known to induce antiviral and inflammatory responses by inducing a type I IFN innate immune response. To gain more information regarding G-based DNA vaccines in olive flounder (Paralicthys olivaceus), we tried to develop a more efficient G-based DNA vaccine by adding a molecular adjuvant, DDX41. We designed a DNA vaccine in which the VHSV glycoprotein (G-protein) and DDX41 were driven by the EF-1α and CMV promoters, respectively. Olive flounders were intramuscularly immunized with 1 μg of plasmids encoding the G-based DNA vaccine alone (pEF-G), the molecular adjuvant alone (pEF-D), or the vaccine-adjuvant construct (pEF-GD). At two different time points, 15 and 30 days later, the fish were intraperitoneally infected with VHSV (100 μL; 1 × 106 TCID50/mL). Our assays revealed that the plasmid constructs showed up-regulated expression of IFN-1 and its associated genes at day 3 post-vaccination in both kidney and spleen samples. Specifically, pEF-GD showed statistically higher expression of immune response genes than pEF-G and pEF-D treated group (p < 0.05/p < 0.001). After VHSV challenge, the fish group treated with pEF-GD showed higher survival rate than the pEF-G treated group, though difference was not statistically significant in the 15 dpv challenged group however in the 30 dpv challenged group, the difference was statistically significant (p < 0.05). Together, these results clearly demonstrate that DDX41 is an effective adjuvant for the G-based DNA vaccine in olive flounder. Our novel findings could facilitate the development of more effective DNA vaccines for the aquaculture industry.

Developmental tracing of oocyte development in gonadal soma-derived factor deficiency medaka (Oryzias latipes) using a transgenic approach.

Gonadal soma-derived factor (gsdf) is reported to be a male initiator in medaka based on loss- and gain- of function via targeted disruption, or transgenic over-expression. However, little is known about how gsdf promotes undifferentiated gonad entry into male pathways or prevents entry into the female pathway. We utilized a visible folliculogenesis system with a reporter cassette of dual-color fluorescence expression to identify difference between oocyte development from wildtype and gsdf deficiency medaka. A red fluorescent protein (RFP) is driven by a major component of the synaptonemal complex (SYCP3) promoter which enables RFP expression solely in oocytes after the onset of meiosis, and a histone 2b-EGFP fused protein (H2BEGFP) under the control of an elongation factor (EF1α) promoter, wildly used as a mitotic reporter of cell cycle. This mitosis-meiosis visible switch revealed that early meiotic oocytes present in gsdf deficiency were more than those in wildtype ovaries, corresponding to the decrease of inhibin expression detected by real-time qPCR analysis, suggesting gsdf is tightly involved in the process of medaka oocyte development at early stage.

In Vivo Tracking of Chemokine Receptor CXCR4-Engineered Mesenchymal Stem Cell Migration by Optical Molecular Imaging.

CXCR4, the stromal cell-derived factor-1 receptor, plays an important role in the migration of hematopoietic progenitor/stem cells to injured and inflamed areas. Noninvasive cell tracking methods could be useful for monitoring cell fate. Therefore, in this study, we evaluated the efficacy of an intravenous infusion of genetically engineered mesenchymal stem cells (MSCs) overexpressing CXC chemokine receptor 4 (CXCR4) to home to the tumor, by optical imaging. We constructed a retroviral vector containing CXCR with dual reporter genes, eGFP and Fluc2, under the control of an EF1α promoter (pBABE-EF1α-CXCR4-eGFP-IRES-Fluc2). We also developed an eGFP-Fluc2 construct in the Retro-X retroviral vector (Retro-X-eGFP-Fluc2). MSCs were transduced with retroviruses to generate CXCR4-overexpressing MSCs (MSC-CXCR4/Fluc2) and MSCs (MSC/Fluc2). CXCR4 mRNA and protein expression was confirmed by RT-PCR and Western blotting, respectively, and it was higher in MSC-CXCR4/Fluc2 than in naive MSCs. eGFP expression was confirmed by confocal microscopy. The transfected MSC-CXCR4/Fluc2 cells showed higher migratory capacity than naive MSCs observed in Transwell migration assay. The in vivo migration of CXCR4-overexpressing MSCs to MDAMB231/Rluc tumor model by BLI imaging was also confirmed. Intravenous delivery of genetically modified MSCs overexpressing CXCR4 with a Fluc2 reporter gene may be a useful, noninvasive BLI imaging tool for tracking cell fate.

Construction and Testing of Two Distinct Humanized CD19-Specific Chimeric Antigen Receptors (CARs) for the Treatment of B-Cell Malignancies

Introduction: Modificiation of T cells using CD19-specific chimeric antigen receptor (CAR) therapy has produced dramatic responses against a number of hematologic malignancies in multiple clinical trials. To date, most of the CARs studied in clinical trials are derived from mouse single chain fragment variable (scFv), which can elicit an immune response when infused into human patients and thereby can limit the persistency of CAR-T cells. Indeed, a subset of patients with limited persistency of infused CAR-Ts has been observed in clinical trials. However, this can be overcome by utilizing the humanized scFv in CAR design. Here, we constructed two new CD19-specific CARs, which are derived from the scFv of two distinct humanized CD19 antibody clones and compared them with the widely used CD19-CAR derived from a mouse scFv (FMC63).Methods: Lentiviral constructs encoding FMC63-BBZ CAR was generated based on the published sequence of FMC63 scFV and cloned in to a lentiviral vector driven by an EF1α promoter. The FMC63-BBZ CAR sequence was fused in frame with T2A ribosomal skip sequence followed by a puromycin resistance gene (PAC). Humanized CD19-Specific CARs were generated by replacing the FMC63 sequence with two humanized CD19-specific scFv sequences that were derived from two distinct humanized monoclonal antibodies. 293FT cells were used to make lentiviruses and the concentrated viruses were used to infect T-cells from healthy donors, NK92MI and Jurkat-NFAT-GFP (JNG) cells. Surface expressions of CARs were detected by protein-L staining using flow cytometry. Cytolytic activity of CAR-engineered T and NK cells was measured using a 4 hour co-culture assay. Cytokines (IL-2, IFN-γ and TNF-α) were measured from the co-cultured supernatants by ELISA.Results: We were able to infect primary-T cells, NK92MI and JNG cells using the FMC63 and humanized CAR constructs, and the surface expression of CARs was comparable between them. Co-culture of JNG cells stably expressing the FMC63 or humanized CARs with CD19+ve Raji (Burkitt Lymphoma), Nalm6 (Pre- B-cell acute lymphoblastic leukemia) and BV173 (Chronic Myeloid Leukemia) cancer cells lead to robust expression of GFP (indicative of activated NFAT signaling), CD69 (an activation marker for cytotoxic cells), and a rapid increase in the secretion of IL-2. None of these effects were observed in unmodified JNG cells or with the co-culture of JNG cells stably expressing FMC63 or humanized CARs with CD19-ve HL60 (Acute Promyelocytic Leukemia) and U937 (Histiocytic Lymphoma) cancer cells. Next, we made stable NK92MI cells expressing FMC63, humanized CD19-specific CARs, and a control CAR, followed by cellular cytotoxicity assay. Both FMC63-CAR and humanized CD19-CARs expressing NK92MI cells readily eliminated CD19+ve cancer cells, whereas they had no discernible effect on CD19-ve cancer cells. Furthermore, parental or a control CAR expressing NK92MI cells are without any effect, either with CD19+ve or CD19-ve cancer cells, thereby strictly highlighting the comparable specificity of our humanized CD19-CARs with FMC63-CAR against CD19+ve cells. More importantly, co-culture of NK92MI cells stably expressing FMC63-CAR and humanized CD19-CARs with direct patient samples [CD19+ve B-ALLs and CD19-ve Acute Myelogenous Leukemia’s (AMLs)] eliminated only the patient B-ALLs, but without any effect on patient AMLs. Also, increased surface expression of CD69 and robust secretion of cytokines (IFN-γ and TNF-α) were observed only when FMC63 or humanized CD19-CARs expressing NK92MI cells were incubated with CD19+ve patient B-ALLs. Finally, expression of FMC63and humanized CD19-CARs in primary donor T-cells also resulted in the elimination CD19+ve cancer cells along with a significant increase in the secretion of cytokines. Most importantly, both humanized CD19-specific CARs significantly increased the survival of CD19+ve cancer cells bearing NSG mice, whereas the unmodified T-cells or the T-cells expressing a non-specific CAR were without any effect.Conclusion: Our extensive pre-clinical study clearly shows that humanized CD19-specific CAR-T or NK cells exhibit comparable potency with the widely used FMC63-CAR, which provides the basis for the clinical testing of these humanized CARs in CD19+ve malignancies, thereby allaying the possible concern on immune mediated rejection of mouse scFv based CD19-CAR therapy. DisclosuresNo relevant conflicts of interest to declare.

Transfected Babesia bovis Expressing a Tick GST as a Live Vector Vaccine.

The Rhipicephalus microplus tick is a notorious blood-feeding ectoparasite of livestock, especially cattle, responsible for massive losses in animal production. It is the main vector for transmission of pathogenic bacteria and parasites, including Babesia bovis, an intraerythrocytic apicomplexan protozoan parasite responsible for bovine Babesiosis. This study describes the development and testing of a live B. bovis vaccine expressing the protective tick antigen glutathione-S-transferase from Haemaphysalis longicornis (HlGST). The B. bovis S74-T3B parasites were electroporated with a plasmid containing the bidirectional Ef-1α (elongation factor 1 alpha) promoter of B. bovis controlling expression of two independent genes, the selectable marker GFP-BSD (green fluorescent protein–blasticidin deaminase), and HlGST fused to the MSA-1 (merozoite surface antigen 1) signal peptide from B. bovis. Electroporation followed by blasticidin selection resulted in the emergence of a mixed B. bovis transfected line (termed HlGST) in in vitro cultures, containing parasites with distinct patterns of insertion of both exogenous genes, either in or outside the Ef-1α locus. A B. bovis clonal line termed HlGST-Cln expressing intracellular GFP and HlGST in the surface of merozoites was then derived from the mixed parasite line HlGST using a fluorescent activated cell sorter. Two independent calf immunization trials were performed via intravenous inoculation of the HlGST-Cln and a previously described control consisting of an irrelevant transfected clonal line of B. bovis designated GFP-Cln. The control GFP-Cln line contains a copy of the GFP-BSD gene inserted into the Ef-1α locus of B. bovis in an identical fashion as the HIGST-Cln parasites. All animals inoculated with the HlGST-Cln and GFP-Cln transfected parasites developed mild babesiosis. Tick egg fertility and fully engorged female tick weight was reduced significantly in R. microplus feeding on HlGST-Cln-immunized calves. Collectively, these data show the efficacy of a transfected HlGST-Cln B. bovis parasite to induce detectable anti-glutathione-S-transferase antibodies and a reduction in tick size and fecundity of R. microplus feeding in experimentally inoculated animals.

Identification of interchangeable cross-species function of elongation factor-1 alpha promoters in Babesia bigemina and Babesia bovis.

BackgroundTick-borne Babesia bigemina is responsible for acute and potentially lethal hemolytic disease in cattle. The development of genetic manipulation tools necessary to the better understanding of parasite biology is currently limited by the lack of a complete parasite genome and experimental tools such as transfection. Effective promoters, required to regulate expression of transgenes, such as the elongation factor-1 alpha (ef-1α), have been identified in other apicomplexans such as Babesia bovis and Plasmodium falciparum. MethodsThe B. bigemina ef-1a locus was defined by searching a partial genome library of B. bigemina (Sanger Institute). Presence of an intron in the 5’ untranslated region was determined by 5’ Rapid Amplification of cDNA Ends (RACE) analysis. Promoter activity was determined by measurement of luciferase expression at several time points after electroporation, efficiency of transfections and normalization of data was determined by quantitative PCR and by the percentage of parasitized erythrocytes.ResultsThe ef-1α locus contains two identical head to head ef-1α genes separated by a 1.425 kb intergenic (IG) region. Significant sequence divergence in the regions upstream of the inverted repeats on each side of the B. bigemina IG region suggest independent regulation mechanisms for controlling expression of each of the two ef-1α genes. Plasmid constructs containing the 5’ and 3’ halves of the IG regions controlling the expression of the luciferase gene containing a 3’ region of a B. bigemina rap-1a gene, were generated for the testing of luciferase activity in transiently transfected parasites. Both halves of the ef-1α IG region tested showed the ability to promote high level production of luciferase. Moreover, both B. bigemina ef-1α promoters are also active in transiently transfected B. bovis and conversely, a B. bovis ef-1α promoter is active in transiently transfected B. bigemina. ConclusionsCollectively these data demonstrate the existence of two distinct promoters with homologous and heterologous promoter function in B. bigemina and B. bovis which is described for the first time in Babesia species. This study is of significance for development of interspecies stable transfection systems for B. bigemina and for B. bovis.Electronic supplementary materialThe online version of this article (doi:10.1186/s13071-016-1859-9) contains supplementary material, which is available to authorized users.

Constitutive overexpressed type I interferon induced downregulation of antiviral activity in medaka fish (Oryzias latipes)

In fish, as well as vertebrates, type I interferons (IFNs) are important cytokines that help to provide innate, antiviral immunity. Although low amounts of IFN are constitutively secreted under normal physiological conditions, long-term and excessive IFN stimulation leads to reduced sensitivity to the IFN signal. This provides a negative feedback mechanism that prevents inappropriate responses and autoimmunity. At present, however, neither IFN desensitization nor the normal physiological role of constitutive IFN are well characterized in fish. The objective here was therefore to produce and characterize a transgenic medaka fish (Oryzias latipes), designated IFNd-Tg, that constitutively overexpressed the IFNd gene. A dual promoter expression vector was constructed for overexpression of IFNd under an EF1α promoter and a DsRed reporter gene under control of a γF-crystaline promoter. The phenotype of the IFNd-Tg fish had a lower response to poly(I:C) and increased susceptibility to nervous necrosis virus (NNV) infection compared to wild-type (WT). Furthermore, transduction of IFN signals for STAT1b, STAT2 and IRF9 were down-regulated in the IFNd-Tg fish, and expression levels of RLR signal molecules (MDA5, MITA, IRF1 and IRF3) were lower than in WT. The constitutive overexpression of IFNd resulted in desensitization of IFN-stimulation, apparently due to downregulation of IFN signal transduction, and this caused increased susceptibility to NNV.