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Immune escape caused by genetic variations of SARS-CoV-2 S protein immunogenic epitopes affects the efficiency of monoclonal antibody-based therapy of COVID-19. Therefore, predicting the effects of these variations on immune escape is important to adapt rapidly anti SARS-CoV-2 Mab therapy. We herein describe a computational method to evaluate the neutralizing power a monoclonal antibody specific of a given SARS-CoV-2 variant and to compare it to its potential neutralizing power of others and emergent variants. The method’s calls for building in silico complex between the spike protein of a SARS-CoV-2 variant and a neutralizing antibody, analyzing the molecular interactions pattern and calculating the binding energy. This data is assigned a neutralizing value of 100% to which can be compared the neutralization value of any SARS-CoV-2 variant determined after molecular replacement in the complex of the RBD sequence with the RBD of this variant. Application of this method to the class 3 neutralizing antibody Sotrovimab and 24 variants and subvariants showed that the affinity binding and neutralizing power, decreased gradually with new variants. This method is of interest to adapt the use of therapeutic antibodies to the treatment of emerging variants. It could be applied to antibody-based treatment of other viral infections.

Background: The epidermal growth factor receptor (EGFR) was the first molecular marker to be targeted successfully in monoclonal antibodies-based cancer immunotherapy. The human EGFR displays two common natural genetic variants (R521K) located in the molecule extracellular CR2 domain. This polymorphism affects the outcome of cancer immunotherapy using anti-EGFR mAbs.Results: In this paper, we report the production, purification and characterization of recombinant forms of the EGFR/CR2-R and CR2-K variants as Glutathione S-transferase (GST) fusion protein in E. Coli BL21. We used these two proteins to generate three different murine monoclonal antibodies to the EGFR CR2 domain (anti-CR2R, anti-CR2K and anti-CR2-RK). We carried out the molecular characterization of the anti-CR2-RK mAb. Analysis using Western blot, ELISA and Immunohistochemistry of various tumor tissues samples, showed that anti-CR2RK mAb was specific of the human EGFR CR2 extracellular domain and recognizes equally both the CR2-K and CR2-R natural genetic variants. In addition, the affinity binding of anti-CR2-RK mAb, as determined by Surface Plasmon Resonance, was equal to 27.7KD μM. Conclusions: We produced recombinant forms of the human EGFR CR2 domain R and K variants and generated mAbs that discriminate between them. These mAbs can be engineered into novel cancer therapeutic and or diagnostic tools tailored to the patients EGFR genetic makeup.

We have previously reported Israa, immune-system-released activating agent, as a novel gene nested in intron 8 of the mouse Zmiz1 gene. We have also shown that Israa encodes for a novel FYN-binding protein and might be involved in the regulation of T-cell activation. In this report, we demonstrate that Israa gene product regulates the expression of a pool of genes involved in T-cell activation and signaling. Real time PCR and GFP knock-in expression analysis showed that Israa is transcribed and expressed in the spleen mainly by CD3+CD8+ cells as well as in the thymus by CD3+ (DP and DN), CD4+SP and CD8+SP cells at different developmental stages. We also showed that Israa is downregulated in T-cells following activation of T-cell receptor. Using yeast two-hybrid analysis, we identified ELF1, a transcription factor involved in T-cell regulation, as an ISRAA-binding partner. Transcriptomic analysis of an EL4 cell line overexpressing ISRAA revealed differential expression of several genes involved in T-cell signaling, activation and development. Among these genes, Prkcb, Mib2, Fos, Ndfip2, Cxxc5, B2m, Gata3 and Cd247 were upregulated whereas Itk, Socs3, Tigit, Ifng, Il2ra and FoxJ1 were downregulated. Our findings support the existence in mouse of a novel FYN-related T-cell regulation pathway involving the product of an intron-nested gene.

We previously reported Israa (immune-system-released activating agent), a novel gene nested in intron 6 of the mouse Zmiz1 gene. Zmiz1 is involved in several functions such as fertility and T cell development and its knockout leads to non-viable embryos. We also reported ISRAA's expression in lymphoid organs, particularly in the thymus CD3+ T cells during all developmental stages. In addition, we showed that ISRAA is a binding partner of Fyn and Elf-1 and regulates the expression of T cell activation-related genes in vitro. In this paper, we report the generation and characterization of an Israa−/− constitutive knockout mouse. The histological study shows that Israa−/− mice exhibit thymus and spleen hyperplasia. Israa−/− derived T cells showed increased proliferation compared to the wild-type mice T cells. Moreover, gene expression analysis revealed a set of differentially expressed genes in the knockout and wild-type animals during thymus development (mostly genes of T cell activation pathways). Immunological phenotyping of the thymocytes and splenocytes of Israa−/- showed no difference with those of the wild-type. Moreover, we observed that knocking out the Zmiz1 intron embedded Israa gene does not affect mice fertility, thus does not disturb this Zmiz1 function. The characterization of the Israa−/- mouse confirms the role ISRAA plays in the expression regulation of genes involved in T cell activation established in vitro. Taken together, our findings point toward a potential functional interrelation between the intron nested Israa gene and the Zmiz1 host gene in regulating T cell activation. This constitutively Israa−/− mice can be a good model to study T cell activation and to investigate the relationship between host and intron-nested genes., Highlights • Israa gene is the first reported intron-nested gene involved in T cell activation in mouse. • Israa−/− derived T cells showed increased proliferation compared to the wild-type mice T cells. • Israa knockdown perturbates expression of genes involved in T cell activation.

SARS-CoV-2 infectivity is largely determined by the virus Spike protein binding to the ACE2 receptor. Meanwhile, marked infection rate differences were reported between populations and individuals. To understand the disease dynamic, we developed a computational approach to study the implications of both SARS-CoV-2 RBD mutations and ACE2 polymorphism on the stability of the virus-receptor complex. We used the 6LZG PDB RBD/ACE2 3D model, the mCSM platform, the LigPlot+ and PyMol software to analyze the data on SARS-CoV-2 mutations and ACE variants retrieved from GISAID and Ensembl/GnomAD repository. We observed that out of 351 RBD point mutations, 83% destabilizes the complex according to free energy (ΔΔG) differences. We also spotted variations in the patterns of polar and hydrophobic interactions between the mutations occurring in 15 out of 18 contact residues. Similarly, comparison of the effect on the complex stability of different ACE2 variants showed that the pattern of molecular interactions and the complex stability varies also according to ACE2 polymorphism. We infer that it is important to consider both ACE2 variants and circulating SARS-CoV-2 RBD mutations to assess the stability of the virus-receptor association and evaluate infectivity. This approach might offers a good molecular ground to mitigate the virus spreading.

Additional file 2: A list of EGFR gene variants. The previously reported alterations found in the CR2 domain of EGFR gene in patients and healthy control samples from the Arabian peninsula region.

Additional file 4: Polar interactions between wild type and mutated EGFR with EGF (untethered monomer, 3NJP). A) Wild CR1/CR2 shows 5 polar interactions. B) CR1/CR2-R521K showing 4 polar interactions. C) CR1/CR2-V550M showing 4 polar interactions. (Blue residues represent CR1 domain, green residues represent CR2 domain).

Additional file 3: Polar interactions between wild type and mutated EGFR with EGF (untethered monomer, 3NJP). A) Wild EGF/EGFR complex shows 15 polar interactions. B) EGF/EGFR-V550M showing 1 missing and 1 extra polar interaction. (Blue residues represent EGFR wild type; green residues represent EGF wild type).

A harmonized learning outcome is eagerly needed when it comes to teaching complex knowledge, particularly concepts that can be contingent on different perceptions and understanding. No teaching method is currently available, however, about achieving a harmonized learning outcome of puzzling knowledge. To fill this educational gap, we developed an incrementally innovative learning-centered method; the consensual cooperative-learning method (CCL) and tested it on a group of executives enrolled in an innovation management PhD program. This paper describes the CCL method and highlights the potential of combining and building upon self-learning methods in achieving harmonized acquisition of sophisticated knowledge.

We previously reported that immunoreactivity of recombinant CFP32 (Rv0577), a virulence factor of Mycobacterium tuberculosis, was higher when produced in transformed Pichia pastoris as compared to transformed E. coli. In this study, we show that this difference is partly due to the N-glycosylation of the recombinant CFP32 (rCFP32) by the yeast Pichia pastoris. In addition, SDS-PAGE and western blotting analysis of Mycobacterium bovis BCG and yeast-produced rCFP32 showed the presence of a band corresponding to a homodimeric state of the protein, unlike that of rCFP32 produced in E. coli. Computational modeling indicates that a single cysteine residue at position 193 of each monomer might bond to stabilize the homodimeric state of CFP32. Computational study showed that this residue is buried inside the protein core of E. coli-produced rCFP32 suggesting that rCFP32 may adopt a different folding in P. pastoris and BCG, in which C193 is solvent exposed. Surprisingly, an enzyme-linked immunosorbent assay using a generated monoclonal antibody (14D4) reveals the presence of a differential epitope that appears to be the consequence of the protein dimerization of the yeast- and BCG-, but not E.coli– produced, CFP32 recombinant form. We conclude that, in addition to N-glycosylation, homodimeric folding significantly enhances the immunoreactivity of rCFP32 and may these post-translational modifications may factor into the structure and function of native M. tuberculosis CFP32.

Protein Disulfide Isomerase (PDI) was originally discovered fifty years ago as the first protein folding catalyst and isolated from rat liver [1]. It was demonstrated early on that PDI acts as a dithiol–disulfide oxidoreductase capable of reducing, oxidizing and isomerizing disulfide bonds. Independently of its redox activity, PDI can also act as a vital cellular defense against the intracellular accumulation of misfolded proteins via its chaperone activity [2].

The interaction between antibodies and Immune cells surface FcγRIIIa (CD16a) receptor triggers a variety of immune responses including antibody-dependent cell-mediated cytotoxicity, antibody neutralization, phagocytosis, inflammation and tissue injury. Recent studies showed that IgG1 upper hinge region and FcγRs polymorphism play a major role in the interaction with Fcγ receptors and in the stability of the immune complex hence, in mounting strong inflammatory response. To further investigate this issue, we developed a tool box of IgG1 Fc isoforms to depict the affinity between mutated IgG1 Fc regions and extracellular domain variants (V158F) of CD16a. Our strategy consisted of designing different random upper-hinge mutated variants of IgG1 Fc domain, reproducing the naturally occurring two variants of CD16a and producing all of them as recombinant fusion proteins in Pichia Pastoris. The interactions were assayed using the Surface Plasmon Resonance (Biacore) method along with an in silico analysis to identify the major interaction and key residues that underline the affinity between the Fc region and CD16a variants. Our data showed that the affinity of the Fc region to the CD16a is strongly correlated to polar interactions. This molecular engineering approach yielded an IgG1Fc mutant with enhanced binding affinity to CD16a F158 variant.

Single nucleotide polymorphisms (SNPs) are useful genetic markers to investigate the onset of multiple sclerosis (MS). A genome wide association study identified 7 SNPs associated with interferon‑β therapy response, however, not with MS risk in a Spanish population. To investigate these findings in a different cohort, the 7 SNPs were investigated in an Arabian Gulf population. The SNPs were analyzed in 268 subjects (156 patients and 112 healthy volunteers) from the Arabian Gulf region using restriction fragment length polymorphism-polymerase chain reaction (PCR) and KBioscience Competitive Allele Specific PCR genotyping methods. Associations between the SNPs and MS were investigated using logistic regression. The present study observed, for the first time, that in an Arabian Gulf population, the ZFAT rs733254 polymorphism (T>G) is a gender‑specific risk marker for MS. ZFAT was associated with MS in women but not in men. The G variant was highly associated with the risk of MS [odds ratio (OR)=2.38 and 95% confidence interval (CI), 1.45‑3.91); P=0.0014]. Whereas variant T was a significantly protective factor [OR=0.420 (95% CI, 0.25‑0.69); P=0.0014, recessive model]. The findings of the present study provide a genetic basis for the gender‑associated susceptibility to MS. In addition, this MS-associated rs733254 SNP may predict MS onset in females from the Arabian Gulf population.

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.