Expression by Streptomyces lividans of the Rat α Integrin CD11b A-Domain as a Secreted and Soluble Recombinant Protein

We already reported the use of a long synthetic signal peptide (LSSP) to secrete the Streptomyces sp. TO1 amylase by Streptomyces lividans strain. We herein report the expression and secretion of the rat CD11b A-domain using the same LSSP and S. lividans as host strain. We have used the Escherichia coli/Streptomyces shuttle vector pIJ699 for the cloning of the A-domain DNA sequence downstream of LSSP and under the control of the constitutive ermE-up promoter of Streptomyces erythraeus. Using this construct and S. lividans as a host strain, we achieved the expression of 8 mg/L of soluble secreted recombinant form of the A-domain of the rat leukocyte β2 integrin CD11/CD18 alpha M subunit (CD11b). This secreted recombinant CD11b A-domain reacted with a function blocking antibody showing that this protein is properly folded and probably functional. These data support the capability of Streptomyces to produce heterologous recombinant proteins as soluble secreted form using the “LSSP” synthetic signal peptide

Humoral and cellular immune responses to modified hepatitis B plasmid DNA vaccine in mice

Purpose: To evaluate the immunogenicity and types of immune response of a quality-controlled modified recombinant hepatitis B surface antigen (HBsAg) plasmid encoding HBsAg in mice.Methods: The characterized plasmid DNA was used in the immunization of Balb/c mice. Three groups of mice were intramuscularly injected with three different concentrations (50, 25 and 10 μg/100 μL) of the modified plasmid. Humoral immune response was monitored by enzyme-linked immunosorbent assay (ELISA), while cellular immune response was investigated by analysis of spleen cytokine profile (TNFα, IFN γ and IL2) as well as CD69 expression level in CD4 and CD8 positive cells.Results: In general, the activated CD4 cells showing intracellular cytokines were higher than CD8 positive population of cells (p < 0.05). These findings indicate that the vaccine induced both a humoral and cellular immunity. Cytokine profile also showed high levels of TNFα, IFN γ and IL2 and CD69 expression in the group of animals immunized at a dose of 10 μg when compared to control group (p < 0.05).Conclusion: A 10 μg dose intramuscular injection of the modified DNA-based vaccine encoding HBsAg in mice induces both high humoral and cellular immune responses.Keywords: Hepatitis B virus, Plasmid DNA, Vaccine, Spleen cytokines, Humoral and cellular immune response

AI is a viable alternative to high throughput screening: a 318-target study

: High throughput screening (HTS) is routinely used to identify bioactive small molecules. This requires physical compounds, which limits coverage of accessible chemical space. Computational approaches combined with vast on-demand chemical libraries can access far greater chemical space, provided that the predictive accuracy is sufficient to identify useful molecules. Through the largest and most diverse virtual HTS campaign reported to date, comprising 318 individual projects, we demonstrate that our AtomNet® convolutional neural network successfully finds novel hits across every major therapeutic area and protein class. We address historical limitations of computational screening by demonstrating success for target proteins without known binders, high-quality X-ray crystal structures, or manual cherry-picking of compounds. We show that the molecules selected by the AtomNet® model are novel drug-like scaffolds rather than minor modifications to known bioactive compounds. Our empirical results suggest that computational methods can substantially replace HTS as the first step of small-molecule drug discovery

Precision and personalized vaccines needed to face COVID-19 pandemic

Among the abounding lessons we learned from the SARS-C0V-2 pandemic is the uttermost determinant that people are not equal before the severity of COVID-19. Indeed, the disease course differs with age, gender, ethnicity, underlying clinical conditions and virus variants. Other diseases modifying factors are associated with genetic traits such as those driving the immune response, the blood groups, the coagulation system and the ACE2 receptor variants [1-4]

A model to study the effects of a viral inactivator (beta-propiolactone) on DNA ligation and gene expression in E. coli and Cos cells.

International audienceAn experimental model to study the effects of viral inactivators on the biological properties of DNA was developed. Beta-propiolactone (betaPL) was used in this model and its effects on ligation, transfer and gene expression of naked DNA were assessed. Evidence that betaPL impairs these two major DNA functions are presented. The amounts of betaPL that alter or abolish gene expression and prevent DNA cohesive ends ligation were determined. Based on these observations, it was concluded that this experimental approach could be used to study the effects on the biological properties of DNA of other inactivators used in vaccine preparations

A Central Nervous System-Dependent Intron-Embedded Gene Encodes a Novel Murine Fyn Binding Protein.

The interplay between the nervous and immune systems is gradually being unraveled. We previously reported in the mouse the novel soluble immune system factor ISRAA, whose activation in the spleen is central nervous system-dependent. We also showed that ISRAA plays a role in modulating anti-infection immunity. Herein, we report the genomic description of the israa locus, along with some insights into the structure-function relationship of the protein. Our findings revealed that israa is nested within intron 6 of the mouse zmiz1 gene. Protein sequence analysis revealed a typical SH2 binding motif (Y102TEV), with Fyn being the most likely binding partner. Docking simulation showed a favorable conformation for the ISRAA-Fyn complex, with a specific binding mode for the binding of the YTEV motif to the SH2 domain. Experimental studies showed that in vitro, recombinant ISRAA is phosphorylated by Fyn at tyrosine 102. Cell transfection and pull-down experiments revealed Fyn as a binding partner of ISRAA in the EL4 mouse T-cell line. Indeed, we demonstrated that ISRAA downregulates T-cell activation and the phosphorylation of an activation tyrosine (Y416) of Src-family kinases in mouse splenocytes. Our observations highlight ISRAA as a novel Fyn binding protein that is likely to be involved in a signaling pathway driven by the nervous system

Expression in Pichia pastoris of a recombinant scFv form of MAb 107, an anti human CD11b integrin antibody

International audienceWe herein report the production in the methylotrophic yeast Pichia pastoris of a recombinant scFv form of monoclonal antibody 107 [MAb 107]. This antibody is directed to the leukocyte adhesion molecule CR3 (CD11b/CD18) and is specific of the CD11bA domain. MAb 107 is a ligand mimic of CR3 that blocks the adhesion of leukocyte and their transendothelial migration (diapedesis), a feature that confers this MAb a potential anti inflammatory activity. Expression of scFv 107 as a myc-(His)6 secreted fusion protein was achieved in the yeast P. pastoris using plasmid pICZαB. A spontaneous specific cleavage of the myc-His tag was consistently observed regardless of the culture conditions and the addition of different concentrations of casaminoacids in the medium. Loss of the myc-His tag did not affect the binding activity of scFv 107. This loss is due either to a specific proteolysis or to a mechanical cleavage. Optimization of the production of scFv 107 in shake flasks was carried out using an L16 array Taguchi design. A level of purified scFv-myc-His fusion protein estimated at 40 mg/L was reached at pH 6 and a temperature of 27 °C. The production of large amounts of recombinant scFv form of MAb 107 is essential for further molecular studies of the interactions between CR3 and its ligands, mainly those mediated by the CD11b A domain. It will also facilitate the investigation of its anti inflammatory activity in vivo

Use of Taguchi's methods as a basis to optimize hybridoma cell line growth and antibody production in a spinner flask

International audienceTaguchi's methods were used for the design of an experimental strategy aimed at optimizing cell density and monoclonal antibody (mAb) production from a spinner flask hybridoma culture. 23G11 is an antibody to the human leukocyte adhesion molecule, CR3 or beta 2 integrin (CD11b/CD18). It recognizes specifically the A-domain of the alpha subunit CD11b. Anti beta 2 integrin monoclonal antibodies hold a great potential for preventing inflammation mediated tissue injuries. An L8 orthogonal experimental design was used to investigate four different culture components: stirring speed, nature of serum, concentration of serum and nature of media (RPMI 1640 or RPMI 1640 supplemented with glucose and glutamine). The experiments were conducted using two levels for each factor studied and a direct ELISA test was used to estimate the level of antibody production. Statistical analysis of the collected data pointed to the stirring speed and serum concentration, and the interaction between these parameters, as the components that affected cell growth. Antibody production was affected by these factors and by the nature of medium but also by the following two interactions: stirring speed/nature of serum and stirring speed/concentration of serum. This study emphasizes the value of using Taguchi's methods as a basis for optimization of mAb production from a hybridoma culture, in cost effective and significantly less labor intensive ways