A SARS-CoV-2 oral vaccine development strategy based on the attenuated Salmonella type III secretion system.

AIMS: This study aimed to provide a safe, stable and efficient SARS-CoV-2 oral vaccine development strategy based on the type III secretion system of attenuated Salmonella and a reference for the development of a SARS-CoV-2 vaccine. METHODS AND RESULTS: The attenuated Salmonella mutant ΔhtrA-VNP was used as a vector to secrete the antigen SARS-CoV-2 based on the type III secretion system (T3SS). The Salmonella pathogenicity island 2 (SPI-2)-encoded T3SS promoter (sifB) was screened to express heterologous antigens (RBD, NTD, S2), and the SPI-2-encoded secretion system (sseJ) was employed to secrete this molecule (psifB-sseJ-antigen, abbreviated BJ-antigen). Both immunoblotting and fluorescence microscopy revealed effective expression and secretion of the antigen into the cytosol of macrophages in vitro. The mixture of the three strains (BJ-RBD/NTD/S2, named AisVax) elicited a marked increase in the induction of IgA or IgG S-protein Abs after oral gavage, intraperitoneal and subcutaneous administration. Flow cytometric analysis proved that AisVax caused T-cell activation, as shown by a significant increase in CD44 and CD69 expression. Significant production of IgA or IgG N-protein Abs was also detected by using psifB-sseJ-N(FL), indicating the universality of this strategy. CONCLUSIONS: Delivery of multiple SARS-CoV-2 antigens using the type III secretion system of attenuated Salmonella ΔhtrA-VNP is a potential COVID-19 vaccine strategy. SIGNIFICANCE AND IMPACT OF THE STUDY: The attenuated Salmonella strain ΔhtrA-VNP showed excellent performance as a vaccine vector. The Salmonella SPI-2-encoded T3SS showed highly efficient delivery of SARS-COV-2 antigens. Anti-loss elements integrated into the plasmid stabilized the phenotype of the vaccine strain. Mixed administration of antigen-expressing strains improved antibody induction.

Hygienic assessment of digestate from a high solids anaerobic co-digestion of sewage sludge with biowaste by testing Salmonella Typhimurium, Escherichia coli and SARS-CoV-2.

Anaerobic digestion is a consolidated technology to convert sewage sludge and other organic wastes into biogas and a nutrient-rich fertilizer (i.e. digestate). The origin of sewage sludge does not exclude the potential presence of pathogens (e.g. Salmonella spp. and SARS-CoV-2) in mature digestate that hence could represent a source of sanitary concerns when it is spread on soil for agriculture purpose. Therefore, an experimental study aimed at proving the sanitizing effect of a full scale thermophilic high solids anaerobic digestion process was conducted by monitoring the hygienic characteristics of mature digestate. Although Salmonella spp. was detected in the sewage sludge fed to the full scale plant, the anaerobic digestion treatment demonstrated sanitization capacity since the monitored pathogens were never found in the mature digestate over the entire duration of the monitoring survey. Furthermore, tests on the regrowth of Salmonella Typhimurium and Escherichia coli, artificially inoculated on mature digestate, were also conducted under both anaerobic and aerobic conditions with the aim to assess the effectiveness of mature digestate as microbial growth medium. Concentrations of Salmonella Typhimurium and Escherichia coli were drastically reduced after a short time of incubation under anaerobic process and the two microorganisms already resulted undetectable after 24-48 h, whereas, under aerobic conditions, two microorganisms' concentrations were stably high for longer than 10 days. The combination of no free oxygen, high temperature, anaerobic metabolites (e.g. total ammonium nitrogen, and volatile fatty acids) production, bacteria competition and lack of nutritional elements in mature digestate considerably reduced in 24-48 h the sanitary risks associated to accidently contaminated digestate. Furthermore, a SARS-CoV-2 monitoring survey on mature digestate during 13 months, resulted in the absence of the virus RNA in the analyzed digestate.

Carrying out pseudo dual nucleic acid detection from sample to visual result in a polypropylene bag with CRISPR/Cas12a.

Amplification-based nucleic acid detection is widely employed in food safety, medical diagnosis and environment monitoring. However, conventional nucleic acid analysis has to be carried out in laboratories because of requiring expensive instruments and trained personnel. If people could do nucleic acid detection at home by themselves, the application of nucleic acid detection would be greatly accelerated. We herein reported a polypropylene (PP) bag-based method for convenient detection of nucleic acids in the oil-sealed space. The PP bag has three chambers which are responsible for lysis, washing and amplification/detection, respectively. After adding sample, nucleic acids are adsorbed on magnetic particles (MPs) and moved into these three chambers successively through immiscible oil channel by an external magnet. Combined with isothermal amplification, the PP bag can be incubated in a water bath or milk warmer and acted as a reaction tube. With highly specific CRISPR technology, Salmonella typhimurium (St) and SARS-CoV-2 can be visually detected in these PP bags within 1 h, indicating its potential household application. To further improve the reliability of nucleic acid testing at home, a logic decision method is introduced by detecting both target and endogenous reference gene. Positive/negative/invalid detection result can be obtained by chronologically adding the CRISPR reagents of target and endogenous reference gene. We anticipate that this PP bag can provide a novel toolkit for nucleic acid detection in people's daily life.

Protective Role of Glutathione against Peroxynitrite-Mediated DNA Damage During Acute Inflammation.

Inflammation is an immune response to protect against various types of infections. When unchecked, acute inflammation can be life-threatening, as seen with the current coronavirus pandemic. Strong oxidants, such as peroxynitrite produced by immune cells, are major mediators of the inflammation-associated pathogenesis. Cellular thiols play important roles in mitigating inflammation-associated macromolecular damage including DNA. Herein, we have demonstrated a role of glutathione (GSH) and other thiols in neutralizing the effect of peroxynitrite-mediated DNA damage through stable GSH-DNA adduct formation. Our observation supports the use of thiol supplements as a potential therapeutic strategy against severe COVID-19 cases and a Phase II (NCT04374461) open-label clinical trial launched in early May 2020 by the Memorial Sloan Kettering Cancer Center.