The Antigenicity And Pharmacokinetics Of Staphylococcus Aureus Phage Lysin LysGH15

Staphylococcus aureus is a ubiquitous and zoonotic pathogen that causes high morbidity and mortality in a variety of diseases.Infections caused by S.aureus are a major health problem in both hospital and community settings.The treatment of these infections has become increasingly difficult because of the emergence of multidrug-resistant strains,particularly methicillin-resistant S.aureus(MRSA),during the past decade.Lysin is a kind of hydrolytic enzyme which is encoded by phage and able to lyse bacteria specifically and efficiently.The bactericidal mechanism of lysin is different from that of antibiotics.Bacteria that are resistant to antibiotics also exhibit sensitive to lysin.Hence,lysin belongs to a novel antibacterial agent.In the previous studies,we obtained a lysin named LysGH15(encoded by phage GH15)which shows broad host-range and strong lytic activity against multi-drug resistant Staphylococcal aureus in vitro and in vivo.LysGH15 shows great potential as alternative treatment strategy for infections caused by multi-drug resistant S.aureus.The three-dimensional structure and molecular function mechanism of LysGH15 have been further revealed.All these researches laid solid foundation for the development of LysGH15 as a novel antibacterial.However,the antigenicity and the pharmacokinetic of LysGH15 have not been revealed.It was the scientific question that must be resolve for the application of LysGH15.Thus,in the following work,the antigenicity and the application formulation of LysGH15 will be studied.More importantly,the pharmacokinetic features of LysGH15,including the characterizations of adsorption,distribution,metabolism and excretion in vivo,will be revealed systematically by methods of ELISA.These studies will provide theoretical and experimental basis for utilization of LysGH15 as novel polypeptide drug in the treatment of infections caused by S.aureus,especially by drug resistant S.aureus.First,we found that LysGH15 did not induce resistance in MRSA or methicillin-sensitive S.aureus(MSSA)strains after repeated treatment.AlthoughLysGH15 triggered the generation of LysGH15-specific antibodies in mice,these antibodies did not block lytic activity in vitro(nor the binding capacity of LysGH15).More importantly,when the antibody titre was highest in mice immunized with LysGH15,a single intravenous injection of LysGH15 was sufficient to protect mice against lethal infection with MRSA.These results indicated that LysGH15-specific antibodies did not affect the killing efficiency of LysGH15 against MRSA in vitro or in vivo.LysGH15 also reduced pro-inflammatory cytokines in mice with lethal infections.Furthermore,a high-dose LysGH15 injection did not cause significant adverse effects or pathological changes in the main organs of treated animals.In order to analyze the pharmacokinetics of LysGH15 in rats,this study established a sandwich enzyme-linked immunosorbent assay(ELISA).We prepare and purify the HIS-tagged LysGH15-165 rabbit polyclonal antibody as a coated antibody,and HRP-anti-HIS monoclonal antibody was used as the detection antibody to establish the double antibody sandwich method,and the method was verified.The capture antibody was(1: 800)(3.2 ?g / m L)and the detective antibody was(1: 1600)(0.6 ?g / m L).The method is parallel to the method,accuracy(intra-and inter-assay variation),accuracy(recovery)and stability.The practical working range is about0.6-300 ?g / m L,the recovery range is from 91.2 to 100.53%,and the inter-batch and inter-batch precision is ?8.31% and ?12.81%,respectively.(40.87%-77.23%),but when the lysin was stored at-80 ?,repeated freezing and thawing for 1,2,3 times.The recovery rate of the lysin was decreased.However when it stored at-80 ? for 1month,2 month and 4 months,the recovery rate was 84.18%-101.61%.The accuracy(intra – and inter – assay variation),accuracy(recovery)and stability of the method were investigated for different tissues and organs,heart,liver,spleen,lung,kidney,stomach,intestine and intramuscular injection.The pharmacokinetic parameters of the LysGH15 in the blood were given at a dose of 12.5 mg / kg of LysGH15 and buffer,and the elimination half-life of the drug in plasma t1 / 2: mean = 10.529 h;AUC(0-?):mean=345.986 mg/L*h;the maximum plasma concentration of Cmax: mean = 320.164 mg / L * h;AUC(0-?): mean =345.986 mg / L * h;L,the average residence time of MRT was 3.928 h.LysGH15 was mainly distributed in the small intestine,liver,kidney and blood,and the content of small intestine was the largest in the dose of 12.5mg / kg lysate for 1h.After 6h injection,LysGH15 mainly distributed in the kidney,stomach,small intestine,thecontent in the blood has been very small,and at 6h,the largest distribution of the organization;24 hours after administration of kidney,stomach,small intestine dose reduction,but there is still a high drug presence.The cleavage enzyme was detected at3 organs by immunohistochemistry.At 6h,the content of pyrolysis enzyme was detected in the kidney,intestine and stomach.24 h,the drug was reduced,but it could still be observed.We also measured hematology for the subchronic toxicity of the LysGH15.It was demonstrated that 4 weeks of injection,compared with the saline group,did not produce an inflammatory response.Finally,the potential for combination therapy of LysGH15 and api for topical decolonization is presented.LysGH15 and api were added to Aquaphor to form an LAA ointment.The LAA ointment simultaneously exhibited bactericidal activity on S.aureus and hemolysis inhibition activity.The bacterial colony count in the wound skin of LAA-protected mouse model of skin infection with MRSA was approximately 102CFU/mg within 18 h after treatment(and became undetectable),whereas the mean colony count in unprotected mice was approximately 106CFU/mg.LAA ointment also reduced pro-inflammatory cytokines and accelerated skin wound healing in a mouse model of skin infection with MRSA.The transcription level of alternatively activated macrophage-associated markers(arginase I and YM1)in wound tissue was facilitated by LAA ointment.These data demonstrated the potential for developing a combination therapy of LysGH15 and api as a novel class of topical antimicrobial agents that are specific to staphylococcus.