Safety Studies of Pneumococcal Endolysins Cpl-1 and Pal

Abstract

Bacteriophage-derived endolysins have gained increasing attention as potent antimicrobial agents and numerous publications document the in vivo efficacy of these enzymes in various rodent models. However, little has been documented about their safety and toxicity profiles. Here, we present preclinical safety and toxicity data for two pneumococcal endolysins, Pal and Cpl-1. Microarray, and gene profiling was performed on human macrophages and pharyngeal cells exposed to 0.5 µM of each endolysin for six hours and no change in gene expression was noted. Likewise, in mice injected with 15 mg/kg of each endolysin, no physical or behavioral changes were noted, pro-inflammatory cytokine levels remained constant, and there were no significant changes in the fecal microbiome. Neither endolysin caused complement activation via the classic pathway, the alternative pathway, or the mannose-binding lectin pathway. In cellular response assays, IgG levels in mice exposed to Pal or Cpl-1 gradually increased for the first 30 days post exposure, but IgE levels never rose above baseline, suggesting that hypersensitivity or allergic reaction is unlikely. Collectively, the safety and toxicity profiles of Pal and Cpl-1 support further preclinical studies.

Phage Lytic Protein LysRODI Prevents Staphylococcal Mastitis in Mice

“…Even though the effectiveness of endolysins has been demonstrated in vivo, there is a lack of research regarding their safety and toxicity. Nevertheless, safety studies carried out with endolysins Cpl-1 and PaI supported the safety of these compounds, since there was no significant pro-inflammatory response, hypersensitivity or allergic reaction after injecting 0.3 mg per mouse (15 mg/kg) of each protein (Harhala et al, 2018). Moreover, toxicity in mammals is not a worrying issue since it has been proven that intravenous administration of endolysin SAL-200 (highly similar to LysRODI) showed no signs of toxicity in rodents after single-and repeateddose experiments (Jun et al, 2014).…”

Bacteriophage-Derived Endolysins Applied as Potent Biocontrol Agents to Enhance Food Safety

“…Obviously, the safety and stability of endolysins must be considered ahead of their application to food products and in food processing facility systems. Therefore, studies exist in which the safety of the endolysins is reported; for example, after endolysin SAL-1, Cpl-1, and Pal injections, animals did not show mortality or signs of toxicity [54,55]. There are several studies of endolysins those introduced the potential for food applications ( Table 1).…”

“…As detailed here, numerous studies describe the characterization of new endolysins with activities against foodborne pathogens-not only in vitro, but also when applied to food and utensils. Moreover, a few studies have revealed the safety of these endolysins in relation to human health [54,55]. Taken together, endolysins are promising biocontrol agents that have the potential to be used in a variety of raw and processed foods as well as in food-producing facilities.…”

Advanced engineering of third-generation lysins and formulation strategies for clinical applications

“…the lysin of interest) in silico and screen it for potential immunogenicity using mapping tools. (Jado et al 2003;Loeffler et al 2003;Harhala et al 2018 Upon detection of potential immunogenic regions, amino acids can be changed to reduce immunogenicity with minimal changes in protein conformation. Both aspects were incorporated into a single protein design tool, called EpiSweep (Parker et al 2013).…”

“…This branch of lysin engineering is only in its infancy and includes engineering efforts to reduce unwanted effects upon clinical administration. Examples of unwanted effects may include immune responses to the lysin upon (systemic) administration, a limited halflife, and proteolysis on the site of infection due to the presence of inflammatory proteases (Agren et al 2000;Jado et al 2003;Harhala et al 2018). These engineering approaches include both protein engineering and biochemical modifications through covalent attachment of specific groups.…”