Development of toxin-antitoxin self-destructive bacteria, aimed for salmonella vaccination.

The most common source of foodborne Salmonella infection in humans is poultry eggs and meat, such that prevention of human infection is mostly achieved by vaccination of farm animals. While inactivated and attenuated vaccines are available, both present drawbacks. This study aimed to develop a novel vaccination strategy, which combines the effectiveness of live-attenuated and safety of inactivated vaccines by construction of inducible self-destructing bacteria utilizing toxin-antitoxin (TA) systems. Hok-Sok and CeaB-CeiB toxin-antitoxin systems were coupled with three induction systems aimed for activating cell killing upon lack of arabinose, anaerobic conditions or low concentration of metallic di-cations. The constructs were transformed into a pathogenic Salmonella enterica serovar Enteritidis strain and bacteria elimination was evaluated in vitro under specific activating conditions and in vivo following administration to chickens. Four constructs induced bacterial killing under the specified conditions, both in growth media and within macrophages. Cloacal swabs of all chicks orally administered transformed bacteria had no detectable levels of bacteria within 9 days of inoculation. By day ten, no bacteria were identified in the spleen and liver of most birds. Antibody immune response was raised toward TA carrying Salmonella which resembled response toward the wildtype bacteria. The constructs described in this study led to self-destruction of virulent Salmonella enteritidis both in vitro and in inoculated animals within a period which is sufficient for the induction of a protective immune response. This system may serve as a safe and effective live vaccine platform against Salmonella as well as other pathogenic bacteria.

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.

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-γ 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.

Microbial and viral chitinases: Attractive biopesticides for integrated pest management.

The negative impact of the massive use of synthetic pesticides on the environment and on human health has stimulated the search for environment-friendly practices for controlling plant diseases and pests. Among them, biocontrol, which relies on using beneficial organisms or their products (bioactive molecules and/or hydrolytic enzymes), holds the greatest promise and is considered a pillar of integrated pest management. Chitinases are particularly attractive to this purpose since they have fungicidal, insecticidal, and nematicidal activities. Here, current knowledge on the biopesticidal action of microbial and viral chitinases is reviewed, together with a critical analysis of their future development as biopesticides.