Biology subjects

Yadid, I.

Publications and source records attributed to Yadid, I..

7 recordsLinked to original sources

bioRxiv2026

Dual Immune Checkpoint and Cytokine Receptor Modulation by an Engineered Human CTLA-4/IL-10 Bispecific Fusion Protein

Bispecific fusion proteins represent a unique strategy for developing precision therapeutics. By linking functional domains from distinct proteins, these biomolecules can engage multiple targets, enhancing both therapeutic efficacy and safety. Unlike bispecific antibodies, low-molecular-weight fusion proteins offer distinct advantages, including reduced immunogenicity and superior tissue penetration due to their relatively compact size and structure. Such a profile is particularly valuable in managing complex inflammatory diseases, where modulating multiple pathways is required to impart an effective anti-inflammatory effect. Among the various regulators of immune signaling, the cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) and interleukin-10 (IL-10) play imperative roles in immune suppression through their interactions with CD80/86 and IL-10R, respectively. While Fc-fused CTLA-4 is a clinically approved drug (e.g., Abatacept), the clinical development of IL-10 has been hampered by unpredictable immunostimulatory side effects. Here, we engineered a bispecific fusion protein linking the extracellular domain of CTLA-4 to IL-10. We successfully expressed the protein in E. coli as an N-terminal GST-tagged variant and refolded it from the inclusion bodies. Additionally, we achieved soluble expression of an Fc-tagged variant in mammalian CHO cells. Both origins demonstrated binding to their cognate receptors, CD80 and IL-10R. Finally, the fusion protein demonstrated a T cell-inhibitory effect by reducing Interferon-{gamma} (IFN{gamma}) secretion levels in an in vitro human Virus-Specific T cells (VSTs) model. This innovative protein engineering offers a promising strategy for addressing unmet clinical needs in autoimmune and inflammatory diseases.

bioengineering↗

bioRxiv2024

Curing of a field strain of Salmonella enterica serovar Infantis isolated from poultry from its highly stable pESI like plasmid

Salmonella enterica serovar Infantis (S. Infantis) is an important emerging pathogen, associated with poultry and poultry products and related to an increasing number of human infections in many countries. A concerning trend among S. Infantis isolates is the presence of plasmid-mediated multi-drug resistance. In many instances, the genes responsible for this resistance are carried on a megaplasmid known as the plasmid of emerging S. Infantis (pESI) or pESI like plasmids. Plasmids can be remarkably stable due to the presence of multiple replicons and post-segregational killing systems (PSKs), which contribute to their maintenance within bacterial populations. To enhance our understanding of S. Infantis and its multidrug resistance determinants toward the development of new vaccination strategies, we have devised a method for targeted plasmid curing. This approach effectively overcomes plasmid addiction by leveraging the temporal overproduction of specific antitoxins coupled with the deletion of the partition region. By employing this strategy, we successfully generated a plasmid-free strain from a field isolate derived from S. Infantis 119944. This method provides valuable tools for studying S. Infantis and its plasmid-borne multidrug resistance mechanisms.

microbiology↗

bioRxiv2024

Engineering of methionine-auxotroph Escherichia coli via parallel evolution of two enzymes from Corynebacterium glutamicum's direct-sulfurylation pathway enables its recovery in minimal medium

Methionine biosynthesis relies on the sequential catalysis of multiple enzymes. Escherichia coli, the main bacteria used in research and industry for protein production and engineering, utilizes the three-step trans-sulfurylation pathway catalyzed by L-homoserine O-succinyl transferase, cystathionine gamma synthase and cystathionine beta lyase to convert L-homoserine to L-homocysteine. However, most bacteria employ the two-step direct-sulfurylation pathway involving L-homoserine O-acetyltransferases and O-acetyl homoserine sulfhydrylase. We previously showed that a methionine-auxotroph E. coli strain (MG1655) with deletion of metA, encoding for L-homoserine O-succinyl transferase, and metB, encoding for cystathionine gamma synthase, could be complemented by introducing the genes metX, encoding for L-homoserine O-acetyltransferases and metY, encoding for O-acetyl homoserine sulfhydrylase, from various sources, thus altering the Escherichia coli methionine biosynthesis metabolic pathway to direct-sulfurylation. However, introducing metX and metY from Corynebacterium glutamicum failed to complement methionine auxotrophy. Herein, we generated a randomized genetic library based on the metX and metY of Corynebacterium glutamicum and transformed it into a methionine-auxotrophic E. coli strain lacking the metA and metB genes. Through multiple enrichment cycles, we successfully isolated active clones capable of growing in M9 minimal media without external methionine supplementation. The dominant metX mutations in the evolved methionine-autotrophs Escherichia coli were L315P and H46R. Interestingly, we found that a metY gene encoding only the N-terminus 106 out of 438 amino acids of the wild-type MetY enzyme is functional and supports the growth of the methionine auxotroph. Recloning the new genes into the original plasmid and transforming them to methionine auxotroph Escherichia coli validated their functionality. These results show that directed enzyme-evolution enables the fast engineering of new active variants within the Escherichia coli methionine direct-sulfurylation pathway, leading to efficient complementation.

biochemistry↗

bioRxiv2023

Conversion of methionine biosynthesis in E. coli from trans- to direct-sulfurylation enhances extracellular methionine levels

Methionine is an essential amino acid in mammals and a critical metabolite in all organisms. As such, various applications, including food, feed, and pharmaceuticals, necessitate the addition of L-methionine. Although amino acids and other metabolites are commonly produced through bacterial fermentation, high-yield biosynthesis of L-methionine remains a significant challenge due to the strict cellular regulation of the biosynthesis pathway. As a result, methionine is produced primarily synthetically, resulting in a racemic mixture of D,L-methionine. This study aimed to enhance methionine bio-production yields in E. coli by replacing its highly regulated trans-sulfurylation pathway with the more common direct-sulfurylation pathway used by other bacteria. To this end, we generated an auxotroph E. coli strain (MG1655) by simultaneously deleting metA and metB genes and complementing them with metX and metY from different bacteria. Complementation of the genetically modified E. coli with metX/metY from Cyclobacterium marinum or Deinococcus geothermalis, together with the deletion of the global repressor metJ and overexpression of the transporter YjeH, resulted in a substantial increase of up to 126 and 160-fold methionine relative to the wild-type strain, respectively, and accumulation of up to 700 mg/L using minimal MOPS medium and 2 ml culture. Our findings provide a method to study methionine biosynthesis and a chassis for enhancing L-methionine production by fermentation. HighlightsO_LIReplacement of E. coli metA and metB with metX and metY recovered its growth C_LIO_LIThe engineered E. coli has a 160-fold increase in extracellular methionine levels C_LIO_LISelection of different metX and metY leads to varying growth rates and enhanced methionine levels C_LI

biochemistry↗

bioRxiv2022

Production and secretion of recombinant proteins using the endotoxin-free, Gram-negative bacterium Sphingobium japonicum

Gram-negative bacteria are common and efficient protein expression systems, yet their outer membrane endotoxins can elicit undesirable toxic effects, limiting their applicability for parenteral therapeutic applications, e.g., production of vaccine components. In the bacterial genus Sphingomonas from the Alphaproteobacteria class, lipopolysaccharide (LPS) endotoxins are replaced with non-toxic glycosphingolipids (GSL), rendering it an attractive alternative for therapeutic protein production. To explore the use of Sphingomonas as a safe expression system for production of proteins for therapeutic applications, in this study, Sphingobium japonicum (SJ) injected live into embryonated hen eggs proved safe and nontoxic. Multimeric viral polypeptides derived from Newcastle disease virus (NDV) designed for expression in SJ, yielded soluble proteins which were specifically recognized by antibodies raised against the whole virus. In addition, native signal peptides (SP) motifs identified using whole-genome computerized analysis and coupled to secreted proteins in SJ induced secretion of Amy and mCherry gene products. Relative to the same genes expressed without an SP, SP 104 increased secretion of Amy (3.7-fold) and mCherry (16.3-fold) proteins and yielded accumulation of up to 80{micro}g/L of the later in the culture medium. Taken together, the presented findings demonstrate the potential of this unique LPS-free Gram-negative bacterial family to serve as an important tool for protein expression for both research and biotechnological purposes, including for the development of novel vaccines. ImportanceThe Gram-negative, LPS-free Sphingomonas genus can serve as an effective and safe platform for therapeutic protein expression and secretion and bears potential as a live bacteria delivery system for protein vaccines.

molecular biology↗

bioRxiv2021

Protection against avian coronavirus conferred by oral vaccination with live bacteria secreting LTB-fused viral proteins

The devastating impact of infectious bronchitis (IB) triggered by the IB virus (IBV), on poultry farms is generally curbed by livestock vaccination with live attenuated or inactivated vaccines. Yet, this approach is challenged by continuously emerging variants and by time limitations of vaccine preparation techniques. This work describes the design and evaluation of an anti-IBV vaccine comprised of E. coli expressing and secreting viral spike 1 subunit (S1) and nucleocapsid N-terminus and C-terminus polypeptides fused to heat-labile enterotoxin B (LTB) (LS1, LNN, LNC, respectively). Following chicken oral vaccination, anti-IBV IgY levels and cellular-mediated immunity as well as protection against virulent IBV challenge, were evaluated 14 days following the booster dose. Oral vaccination induced IgY levels that exceeded those measured following vaccination with each component separately. Following exposure to inactivated IBV, splenocytes isolated from chicks orally vaccinated with LNN or LNC -expressing bacteria, showed a higher percentage of CD8+ cells as compared to splenocytes isolated from chicks vaccinated with wild type or LTB-secreting E. coli and to chicks subcutaneously vaccinated. Significant reduction in viral load and percent of shedders in the vaccinated chicks was evident starting 3 days following challenge with 107.5 EID50/ml virulent IBV. Taken together, orally delivered LTB-fused IBV polypeptide-expressing bacteria induced virus-specific IgY antibody production and was associated with significantly shorter viral shedding on challenge with a live IBV. The proposed vaccine design and delivery route promise an effective and rapidly adaptable means of protecting poultry farms from devastating IB outbreaks. HighlightsO_LIMucosal vaccination was shown particularly beneficial against respiratory viruses. C_LIO_LIAn anti-IBV vaccine composed of three IBV polypeptides fused to LTB was designed. C_LIO_LIVaccine composed of bacteria secreting polypeptides was orally delivered. C_LIO_LIVaccine induced specific immune responses and shortened viral shedding duration. C_LI

immunology↗

bioRxiv2021

Oral subunit SARS-CoV-2 vaccine induces systemic neutralizing IgG, IgA and cellular immune responses and can boost neutralizing antibody responses primed by an injected vaccine

The rapid spread of the COVID-19 pandemic, with its devastating medical and economic impacts, triggered an unprecedented race toward development of effective vaccines. The commercialized vaccines are parenterally administered, which poses logistic challenges, while adequate protection at the mucosal sites of virus entry is questionable. Furthermore, essentially all vaccine candidates target the viral spike (S) protein, a surface protein that undergoes significant antigenic drift. This work aimed to develop an oral multi-antigen SARS-CoV-2 vaccine comprised of the receptor binding domain (RBD) of the viral S protein, two domains of the viral nucleocapsid protein (N), and heat-labile enterotoxin B (LTB), a potent mucosal adjuvant. The humoral, mucosal and cell-mediated immune responses of both a three-dose vaccination schedule and a heterologous subcutaneous prime and oral booster regimen were assessed in mice and rats, respectively. Mice receiving the oral vaccine compared to control mice showed significantly enhanced post-dose-3 virus-neutralizing antibody, anti-S IgG and IgA production and N-protein-stimulated IFN-{gamma} and IL-2 secretion by T cells. When administered as a booster to rats following parenteral priming with the viral S1 protein, the oral vaccine elicited markedly higher neutralizing antibody titres than did oral placebo booster. A single oral booster following two subcutaneous priming doses elicited serum IgG and mucosal IgA levels similar to those raised by three subcutaneous doses. In conclusion, the oral LTB-adjuvanted multi-epitope SARS-CoV-2 vaccine triggered versatile humoral, cellular and mucosal immune responses, which are likely to provide protection, while also minimizing technical hurdles presently limiting global vaccination, whether by priming or booster programs. HighlightsO_LIMigVax-101 is a multi-epitope oral vaccine for SARS-CoV-2. C_LIO_LIMigVax-101 elicits neutralizing IgG and IgA production and cellular responses in mice C_LIO_LIMigVax-101 serves as an effective booster in rats to a parenteral anti-S1 vaccine. C_LI

immunology↗