Biology subjects

Shahar, E.

Publications and source records attributed to Shahar, E..

3 recordsLinked to original sources

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↗