Isolation of OmpA gene from Salmonella typhimurium and transformation into alfalfa in order to develop an edible plant based vaccine

The outer membrane protein A (OmpA) of Salmonella typhimurium may contribute to immunity and virulence in livestock animals. Introduction of this gene in forage crops like alfalfa may be an alternative and effective way to produce animal edible vaccine. In the present study, the OmpA gene was obtained after polymerase chain reaction (PCR) amplification and sequencing. We successfully identified the complete ORF encoding this protein. In order to express OmpA protein in alfalfa, the gene was insertedinto a plant expression vector PBI121.The recombinant OmpA was  expressed in Escherichia coli TG1. The new construct was used to transform the Agrobacterium tumefaciens Strain LBA4404 before plant transformation. Transgenic alfalfa plants were then developed by introducing OmpA gene in the plant genome under the control of Camv35s promoter and for the first time we expressed this protein in alfalfa. Releasing this one new transgenic variety may be a considerable progress towards release varieties which enables the production of edible vaccine.Key words: Outer membrane protein A (OmpA), sub cloning, plantibodies, transgenic alfalfa, bioreactor, edible vaccine

Development of specific nanobodies (VHH) for CD19 immunotargeting of human B-Lymphocytes

Objective(s): CD19 is a transmembrane glycoprotein of immunoglobulin superfamily. In order to treat lymphoma, monoclonal antibodies (mAb) can target different antigens, including CD19, CD20 and CD22 on the surface of B-cells. Along with biotechnology progress, a new generation of antibodies is introduced, with the purpose of eliminating the defects of the previous generation. Among the most developed one are nanobodies (Nb). Nbs are a unique kind of camelid single domain antibody fragments with a broad range of medical applications. Unique physicochemical properties of Nbs have made them ideal candidates for therapeutic and diagnostic applications. Materials and Methods: An immune gene library was created, and several CD19 specific Nbs were selected through antigen panning process, and their molecular properties as well as specificity, sensitivity, affinity and immunoreactivity against CD19 positive and negative cells were evaluated. Results: The Nb library was prepared with 7.2 �107 members. We managed to isolate a panel of CD19- specific Nbs after the last round of selection with the affinity of isolated Nbs being estimated at the standard range of 15-35 nM. Sequence analysis of positive clones was indicative of the fact that 12 variable sequences were confirmed. Of all these 12 clones, 2 clones with the greatest level signal in ELISA underwent subsequent analysis. Our sequencing results indicated high sequence homology (approximately 90) between the Nb and Homa variable immunoglobulin domains. Conclusion: Specific Nbs possess the potential to be used as novel therapeutic approaches in order to treat autoimmune diseases and B-cell lymphoma. © 2018, Mashhad University of Medical Sciences. All rights reserved

Development of specific nanobodies (VHH) for CD19 immunotargeting of human B-Lymphocytes

Objective(s): CD19 is a transmembrane glycoprotein of immunoglobulin superfamily. In order to treat lymphoma, monoclonal antibodies (mAb) can target different antigens, including CD19, CD20 and CD22 on the surface of B-cells. Along with biotechnology progress, a new generation of antibodies is introduced, with the purpose of eliminating the defects of the previous generation. Among the most developed one are nanobodies (Nb). Nbs are a unique kind of camelid single domain antibody fragments with a broad range of medical applications. Unique physicochemical properties of Nbs have made them ideal candidates for therapeutic and diagnostic applications. Materials and Methods: An immune gene library was created, and several CD19 specific Nbs were selected through antigen panning process, and their molecular properties as well as specificity, sensitivity, affinity and immunoreactivity against CD19 positive and negative cells were evaluated. Results: The Nb library was prepared with 7.2 �107 members. We managed to isolate a panel of CD19- specific Nbs after the last round of selection with the affinity of isolated Nbs being estimated at the standard range of 15-35 nM. Sequence analysis of positive clones was indicative of the fact that 12 variable sequences were confirmed. Of all these 12 clones, 2 clones with the greatest level signal in ELISA underwent subsequent analysis. Our sequencing results indicated high sequence homology (approximately 90) between the Nb and Homa variable immunoglobulin domains. Conclusion: Specific Nbs possess the potential to be used as novel therapeutic approaches in order to treat autoimmune diseases and B-cell lymphoma. © 2018, Mashhad University of Medical Sciences. All rights reserved

The effect of superparamagnetic iron oxide nanoparticles surface engineering on relaxivity of magnetoliposome

The purpose of this work is evaluating the effect of ultra small superparamagnetic iron oxide nanoparticles (USPIONs) coatings on encapsulation efficiency in liposomes and cellular cytotoxicity assay. Moreover, we assessed the effects of surface engineering on the relaxivity of magnetoliposome nanoparticles in order to create a targeted reagent for the intelligent diagnosis of cancers by MRI. For estimating the effect of nanoparticle coatings on encapsulation, several kinds of USPIONs coated by dextran, PEG5000 and citrate were used. All kinds of samples are monodispersed and below 100 ± 10 nm and the coatings of USPIONs have no significant effect on magnetoliposome diameter. The coating of USPIONs could have effect on percentage of encapsulation. The dextran coated USPIONs have more stability and quality accordingly the encapsulation increased up to 92, then the magnetoliposome nano particles have been targeted by Herceptin and anti-HER2 VHH, separately. Over storage period of four weeks the resulting particles were stable and physico-chemical properties such as size and zetapotential did not show any significant changes. The relaxivity of contrast agents was measured using a 1.5 T MRI. The r2/r1 ratio was more than two for all samples which demonstrate the negative contrast enhancing of all SPION embedded specimens. The high ratio of r2/r1 as well as high r2 is the best combination of a negative contrast agent as it is obtained for pure magnetite. The value of r2/r1 for all other samples including Herceptin targeted magnetoliposome, anti-HER2 VHH targeted magnetoliposome and non-targeted magnetoliposome were between ~21 to ~28, which show the magnetite embedded samples have enough negative contrast to be detectable by MRI. Therefore the HER2 targeted magnetoliposomes are a good and stable candidate as contrast agents in clinical radiology and biomedical research with minimal cytotoxicity and biocompatibility effects. Copyright © 2016 John Wiley & Sons, Ltd. Copyright © 2016 John Wiley & Sons, Ltd

Properties, production, and applications of camelid single-domain antibody fragments

Camelids produce functional antibodies devoid of light chains of which the single N-terminal domain is fully capable of antigen binding. These single-domain antibody fragments (VHHs or Nanobodies®) have several advantages for biotechnological applications. They are well expressed in microorganisms and have a high stability and solubility. Furthermore, they are well suited for construction of larger molecules and selection systems such as phage, yeast, or ribosome display. This minireview offers an overview of (1) their properties as compared to conventional antibodies, (2) their production in microorganisms, with a focus on yeasts, and (3) their therapeutic applications

Single domain antibodies: promising experimental and therapeutic tools in infection and immunity

Antibodies are important tools for experimental research and medical applications. Most antibodies are composed of two heavy and two light chains. Both chains contribute to the antigen-binding site which is usually flat or concave. In addition to these conventional antibodies, llamas, other camelids, and sharks also produce antibodies composed only of heavy chains. The antigen-binding site of these unusual heavy chain antibodies (hcAbs) is formed only by a single domain, designated VHH in camelid hcAbs and VNAR in shark hcAbs. VHH and VNAR are easily produced as recombinant proteins, designated single domain antibodies (sdAbs) or nanobodies. The CDR3 region of these sdAbs possesses the extraordinary capacity to form long fingerlike extensions that can extend into cavities on antigens, e.g., the active site crevice of enzymes. Other advantageous features of nanobodies include their small size, high solubility, thermal stability, refolding capacity, and good tissue penetration in vivo. Here we review the results of several recent proof-of-principle studies that open the exciting perspective of using sdAbs for modulating immune functions and for targeting toxins and microbes