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

Evaluation of antibody titer from the combination 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 zoonotic and severe infectious disease that caused by Bacillus anthracis [1, 2]. The pathogenicity of Bacillus anthracis is depends on two important factors in the production of toxins and the formation of capsules. Bacterial toxin is made from three protein components: Protective Antigen (PA) (83 kDa), Lethal Factor (LF) (90 kDa), and Edema Factor (EF) 89 kDa [3-5]. The PA protein consists of four regions where its carboxyl terminal region (region 4 or PAD4), with binding to host cell surface receptors, causes the introduction of LF and EF into the cell. On the other hand, the LF factor, which has four regions, binds to the PA through the amino-terminal region (region 1 or LFD1) [6]. Current vaccines for human use are prepared in England and the United States based on cell extract deposition [9].The production of the new generation of anthrax vaccines has focused on various recombinant expression systems [7-11]. Also, according to studies, the production of recombinant vaccines containing PA and lethal factor can be more effective in activating the immune system[12]. Today, attempts are being made to develop vaccines that contain one or more specific components of the organism. These vaccines are called subunit vaccines and have significant advantages over traditional vaccines; On the other hand, it has created more opportunities for engineering vaccines and determining the appropriate formulation to create an immune response [13]. Due to recent advances in materials science and nanoengineering, nanoparticles have received much attention for applications in biology and medicine. 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. The purpose of this study was to purify and encapsulated of mixed antigen (LFD1 + PAD4) in PEG-PLA double-block copolymer nanospheres and to evaluate the antibody titer in the laboratory animal.
Methods: Expression of recombinant proteins The pET 28a-lfD1 and pET28a-pa4 gene construct obtained from Department of Biological science in Imam Hossein University which described elsewhere .The recombinant LFD1 and PA4 proteins expressed and purified as described elsewhere by this laboratory [14]. Encapsulation of mixed proteins and chracterization An equal mixture of LFD1 and PAD4 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 and PA4 proteins (LFD1 + PA4) Encapsulated and non Encapsulated LFD1 + PA4 proteins were injected four times (20, 15, 10, 10μg respectively) combined with the VAX-ORIENT IPA-70 Adjuvant, Pars Company, 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 AND PAD4 proteins 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 33 and 28 kDa respectively.(Fig.1,2). Encapsulation of mixed proteins and chracterization The results of PEG-PLA nanoparticles loaded with mixed proteins LFD1 and PAD4 (separately) 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) and LFD1 + PA4 (mixed) proteins. After serum isolation, ELISA was performed. The antibody titration is shown in Diagrams 2. Figure (1) Purification of LFD1 protein 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 collected, Column 5) Collected buffer E, Column 6) Protein marker Column 7) Collected MES buffer. Figure (2) Purification of PAD4 protein from Ni column: Column 1) Before Ni column, Column 2) Sample containing proteins (Flow) collected from Ni column Column 3) Buffer D collected, Column 4) Buffer E aggregate ¬ Collected, columns 5 and 6) Collected MES buffer, column 8) Protein marker. 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 -27.7 109 0.394 Diagram 1- PEG-PLA nanoparticle size distribution range containing LFD1 + PAD4 mixed protein under optimal conditions by DLS. Diagrams 2-ELISA curve of IgG antibody production after each injection of mixed protein LFD1 and PAD4 in three forms of free (A), loaded one injection (B) and loaded two injections (C) against LFD1-PA4 protein (curves are shown in the picture box).
Conclusion: In this study, we evaluate the antibody titer produced by injecting mixed proteins (LFD1 and 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 mixed antigen. Most researchers are currently working on vaccines based on recombinant protective antigen (rPA) for anthrax(19-22). The triple nature of anthrax toxin raises the expectation that other parts of the toxin, such as lethal factor and PA, will also be involved in the production of toxin-neutralizing antibodies. Various studies have shown that concomitant administration of lethal factor and PA increases the level of antibodies against PA in mice, and this effect of lethal anticoagulant adjuvant is related to its amino terminus, LFD1. This region has been used by researchers to transmit various antigens and has been shown to elicit immune responses to CD4 + and CD8 + T cells [23-24,26]. Based on the findings of this study, it can be concluded that encapsulation of combination of region 1 of lethal factor (LFD1) and region 4 of protective antigen (PAD4) in PEG-PLA diblock copolymer nanospheres can be a good candidate for anthrax vaccine.
Keywords: Bacillus anthracis. protective ag , lethal factor,polymeric nanoparticles,