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 role of 6-Aminonicotinamide in the resistance of peritoneal macrophages against Leishmania major infection in BALB/c mouse

Background: The objective of this study was to investigate the relationship between glucose-6-phosphate dehydrogenase inhibition in macrophages treated with 6-Aminonicotinamide, the amount of nitric oxide (NO) production and the resistance of infected macrophages against Leishmania major infection. Methods: Peritoneal macrophages of BALB/c mice were isolated and treated with different concentrations (1.25, 2.5, 5, 10 mM) of 6-aminonicotinamide. After 24 hours, the viability of treated macrophages was measured by MTT assay at 540 nm. G6PD activity was measured in the cell extracts 24 hours later. Macrophages were then infected with leishmanial amastigotes and after 18 hours NO production was determined using Griess-reagent. In order to study the inhibition of macrophage activity, 5 mM concentration of 6-AN was used and number of leishmanial amastigotes was recorded in these cells from day 1 to7. Results: Different concentrations of 6-AN were shown to cause a significant increase in cell death and decrease in G6PD activity and NO production in macrophages. Also, the number of amastigotes in macrophages was increased significantly (p < 0.05). Conclusion: The concentration of 6-aminonicotinamide and G6PD activity affect the viability of BALB/c mice peritoneal macrophages through production of NO. Inhibition of G6PD activity leads to decreased leishmani-cidal activity of mouse peritoneal macrophages

Induction of protective immunity against toxoplasmosis in mice by immunization with a plasmid encoding Toxoplama gondii ROP1 gene

Toxoplasma gondii is an obligate intracellular protozoan that is a causative agent of toxoplasmosis, a disease which may result in a spectrum of consequences. It has been shown that DNA vaccine can be effective in partial protection against this parasite. In the present work, a single DNA vaccine containing ROP1 was evaluated against T. gondii infection in Balb/c mice. To enhance the immune response, alum was used as an adjuvant. After intramuscular immunization, cytokine and antibody assays and mortality rate were evaluated. The results showed that mice immunized by pcROP1 with or without alum produced high Th1 immune response compared with control groups. This type of DNA vaccine prolonged slightly the survival time. The current study showed that ROP1 DNA vaccine can induced partial protective response against toxoplasmosis.Keywords: Toxoplasma gondii, DNA vaccine, ROP1gene, immunity, mic