结核分枝杆菌B/R菌株灭活全菌裂解液的制备及其免疫原性与安全性评价OA

This study was aimed at developing a safe and effective inactivated whole-cell lysate of a novel Mycobacterium tubercu-losis(M.tb)fusion strain(B/R strain)by using ultrasonic microbubble cavitation combined with enzymatic lysis(lysozyme/glycine),hereafter referred to as the ultrasonically-killed B/R strain(UK-B/R).Inactivation efficacy,preservation of conformational epitopes,immunogenicity,and safety were systematically evaluated.The B/R strain was inactivated with ultrasonic microbubble cavitation(30 minutes)combined with pretreatment with lysozyme-glycine(6 hours).The inactivation was verified through colony culture,flow cyto-metric analysis of FDA esterase activity,and N01/PI fluorescence staining.The effects of the inactivation process on key M.tb anti-genic proteins(ESAT6,CFP10,MPT64,and Ag85B)were assessed with western blotting.C57BL/6J mice were subcutaneously im-munized and divided into four groups:PBS,BCG,B/R,and UK-B/R.At weeks 2 and 4 post-immunization,serum cytokines(IFN-γ,TNF-α,and IL-2)and antigen-specific antibodies were measured with ELISA.The cytotoxicity of UK-B/R toward RAW 264.7 macrophages was analyzed with CCK-8 assays.Culture colony assays,bacterial esterase activity tests,and fluorescence staining to-gether confirmed the complete inactivation of the B/R strain.Western blot analysis indicated that the B/R strain retained key tuberculo-sis antigens such as ESAT6/CFP10(11 kDa),MPT64(25 kDa),and Ag85B(37 kDa)both before and after inactivation.Subcutane-ous immunization with UK-B/R induced high levels of cytokines,whose expression showed time-dependent dynamics and sex-specific differences,and elicited high levels of antigen-specific IgG in serum.Furthermore,no adverse reactions were observed in mice immunized with UK-B/R.In vitro experiments demonstrated that various doses of UK-B/R had no effects on macrophage viabil-ity.This study demonstrated that UK-B/R,prepared by ultrasonic microbubble cavitation combined with enzymatic lysis,demon-strated complete inactivation,preserved critical mycobacterial antigens,effectively activated both cellular and humoral immune re-sponses,and exhibited a favorable safety profile.Therefore,this strain has potential as a candidate inactivated whole-cell vaccine against tuberculosis.