Evaluation of antibody titer from the chimeric protein of lethal factor and protective antigen of Bacillus anthracis encapsulated in PEG-PLA diblock copolymer nanospheres

Evaluation of antibody titer from the chimeric protein of lethal factor and protective antigen of Bacillus anthracis encapsulated in PEG-PLA diblock copolymer nanospheres
hassan mirhaj,^1,* hossein honari,²
1. university
2. university
Introduction: Anthrax is a particularly dangerous infectious disease that affects humans and livestock. It is characterized by intoxication, serosanguinous skin lesions, development of lymph nodes and internal organs, and may manifest itself in either a cutaneous or septic form. The pathogenic agent is Bacillus anthracis, a gram-positive, endospore-forming, rod-shaped aerobic bacterium (Schmidt et al., 2011; Ahmadi et al., 2015). Due to the production of heat resistant spores, very high mortality, easy production and distribution and also creating respiratory, gastrointestinal, cutaneous diseases and its potential use in biological warfare, B. anthracis is one of the most important fatal biologic agents (Knight, 2001; Ahmadi et al., 2015). The principal virulence factors of B. anthracis consist of an anti-phagocytic capsule composed of poly-Dglutamic acid (PGA) and a secreted bacterial toxin. The anthrax toxin, which is predominantly responsible for the etiology of anthrax, belongs to the family of bacterial binary ABtype toxins, which consist of a receptor-binding B subunit known as the protective antigen (PA) and two catalytic A subunits, i.e., the lethal factor (LF) and edema factor (EF). Protective antigen is combined with either LF or EF to form the lethal toxin (LeTx) and edema toxin (EdTx), respectively (Okinaka et al., 1999; Koehler, 2009; Schmidt et al., 2011; Honari et al., 2014). Currently, the standard approach for anthrax therapy is to kill the germinating bacilli by administering aggressive antibiotics. However, antibiotic therapy is ineffective once systematic anthrax symptoms appear because by that time, fatal concentrations of the anthrax toxin have accumulated in the patient’s body (Schneemann and Manchester, 2009) and the emergence of antibiotic-resistant strains as a result of natural evolution or intentional modiﬁcation by genetic engineering also poses a new challenge to traditional antibiotic treatment (Gilligan, 2002; Gilligan, 2004). Therefore, the development of an antitoxin for combined use with antibiotic therapy is a top priority. Current vaccines for human administration are prepared in England and the United States based on cell extract deposition (Pannifer et al., 2001). The production of the new generation of anthrax vaccines has focused on various recombinant expression systems (Gorse et al., 2006; Campbell et al., 2007; Brown et al., 2010; Bellanti et al., 2012; Reed et al., 2015). Studies have shown that antibodies generated against four regions of PA (PAD4) are capable of neutralizing anthrax toxin (McComb and Martchenko, 2016). On the other hand, PA mixed with LF has been shown to increase the specific antibody response to PA (Pezard et al., 1995; Price et al., 2001). Chimer proteins are a product of recombinant DNA technology and have attracted the attention of researchers due to their special applications. One of the most important applications of recombinant chimeric proteins is their use as vaccines and drugs [104-103]. Nanotechnology has evolved rapidly in recent years. Due to recent advances in materials science and nanoengineering, nanoparticles have received much attention for applications in biology and medicine. In medicine, nanomaterials are used in applications such as scaffolding and tissue equipment engineering, targeted drug delivery systems, cancer treatment, and clinical diagnosis and treatment. In recent years, significant efforts have been made to use nanotechnology to deliver drugs and vaccines [113]. Polymeric nanoparticles (NPs) are solid colloidal particles with diameters of one to one thousand nanometers that are composed of large molecular weight materials and can be used in therapeutic applications, as an adjuvant in a vaccine, or as a drug carrier. One of the first reports of the use of polymer nanoparticles in the treatment of cancer dates back to 1979, when the uptake of anticancer drugs into polyalkyl cyanoacrylate nanoparticles was studied. Polymers used to form nanoparticles can be synthetic or natural. These nanocarriers have been used for various applications such as drug delivery, imaging, and apoptosis diagnosis [124]. The most important advantages of using polymer nanoparticles in conducting antigen are: Continuous release of antigen Controlled release for a specified period and ….[135]. The purpose of this study was to express the lfD1-pa4 gene of B. anthracis in Escherichia coli expression strain, purify the recombinant protein,encapsulated in PEG-PLA diblock copolymer nanospheres and produce polyclonal antibody in mice and compare the encapsulated lethal factor domain 1-protective antigen 4 (LFD1-PA4) fusion protein and nonencapsulated lethal factor domain 1-protective antigen 4 (LFD1-PA4) fusion protein.
Methods: Expression of recombinant protein The recombinant LFD1 - PA4 protein expressed and purified as described elsewhere by this laboratory [18]. Encapsulation of mixed proteins and chracterization LFD1 - PA4 proteins was encapsulated by w/o/w method (15). To investigate the size and zeta potential of the nanoparticles produced, the Malvern model DLS device made in England, belonging to Baqiyatallah University of Medical Sciences, was used. Evaluation of Antibody titration of Encapsulated and non Encapsulated LFD1 -PA4 proteins (LFD1 - PA4) NonEncapsulated LFD1 -PA4 protein were injected four times (20, 15, 10, 10μg respectively) combined with the VAX-ORIENT IPA-70 Adjuvant, Pars Company, and Encapsulated LFD1 - PA4 proteins were injected one times (first day) and two time (first and twenty-eighth day) Without Adjuvant. Finally the blood samples were taken from the mice and antibody titration evaluated by ELISA [16-18].
Results: Expression and purification of the LFD1 -PAD4 protein Selected colonies were cultured in a LB medium at 37 °C, after reaching logarithmic growth, the expression induced by 1 mM IPTG at final concentration. The expression and quality was evaluated by 12% SDS-PAGE gel .The LFD1and PAD4 protein band was indicated at 48 kDa.(Fig.1). Encapsulation of mixed proteins and chracterization The results of PEG-PLA nanoparticles loaded with chimeric protein(LFD1 - PAD4) by DLS are shown in the table1 and diagram1. Evaluation of antibody titration against recombinant protein by indirect ELISA An indirect ELISA was used to analyze the antibody titration and to characterize its ability to detect antigens in each injection phase. Blood samples were randomly taken from test and control mice, one week after the second, third, and fourth injection with LFD1-PA4 (fusion) (encapsulated and nonencapsulated proteins). After serum isolation, ELISA was performed. The antibody titration is shown in Diagrams 2. Figure 1 - Purification of proteins from Ni column: Column 1) Before Ni column, Column 2) Sample containing proteins (Flow) collected from Ni column, Column 3) Buffer C collected, Column 4) Buffer D Group Collected, columns 5 and 7) collected buffer E, column 6) protein marker column 8) collected MES buffer Table (1) Investigation of physicochemical properties of nanoparticles using dynamic light diffraction پتانسیل زتا (میلی ولت) اندازه (نانومتر) PDI CONTROL -32.1 13.88 0.337 ENCAPSULATED -23.1 93 0.280 Diagram 1- PEG-PLA nanoparticle size distribution range containing LFD1 - PAD4 chimer protein under optimal Diagrams 2-ELISA curve of IgG antibody production after each injection of chimer protein LFD1 - PAD4 in three forms of free (A), loaded one injection (B) and loaded two injections (C)
Conclusion: The present vaccines against anthrax are based on PA such as anthrax vaccine adsorbed (AVA) in USA and anthrax vaccine precipitated (AVP) in Britain. The usage of the traditional AVA or the second-generation of recombinant protective antigen vaccine ((rPA)-based vaccine) is not ideal because multiple injections are required over a long period. Besides, neither AVA nor an rPA-based vaccine is suitable for the post-exposure vaccination in persons who have been freshly infected with B. anthracis (Aulinger et al., 2005; Bouzianas, 2010). Recent studies on anthrax showed that LF is important and necessary for development of effective vaccines (Hepburn et al., 2007). Moreover, production of antibodies (toxin neutralization antibody-TNA) that cause immunity in animals are based on CD4+ T cells plays a key role in production of neutralizing antibodies, class switching and maturity of lymphocytes. After reviewing similar studies in this case and considering that the domain of lethal factor 1 at the amino terminus of the protein and PA4 are naturally located at the carboxylic end of the protective antigen, finally the chimeric protein LFD1-PA4 was selected.[46,153,152,151].In this research lfd1-pa4 synthetic gene cassettes were expressed in E. coli-BL21 (DE3) PlysS cells and related proteins were purified by affinity chromatography. After encapsulation of chimeric protein in PEG-PLA diblock copolymer nanospheres , we evaluate the antibody titer produced by injecting chimer protein (LFD1- PAD4) in three forms: free (four injections), loaded (once injected) and loaded (twice injected) and calculate its amount in each injection showed that the highest antibody titer against nanoparticles loaded with chimer antigen.
Keywords: Anthrax, Antibody titer, , LFD1-PA4, polymeric nanoparticle,peg-pla