Application Research Of Tuberculosis Peptides Vaccine Loaded Chitosan Nanoparticles

Tuberculosis(TB)is a chronic wasting infectious disease caused by the Mycobacterium tuberculosis complex(MTC),and its morbidity and mortality are at the forefront of various infectious diseases.The only vaccine currently used for clinical prevention of tuberculosis is BCG vaccine(attenuated live M.tuberculosis vaccine,BCG).In the clinical application process,it was found that inoculation of BCG vaccine can only partially protect young children,and the protection decreases with age.No significant protection for adults.With the escalation of tuberculosis in recent years and the emergence of new epidemic features,there is an urgent need for new and effective tuberculosis vaccines.The tuberculosis peptides vaccine is prepared by chemically synthesizing peptides according to the amino acid sequence of a certain epitope known or predicted in a particular pathogen antigen gene.However,due to changes in the structural characteristics of synthetic peptides,short half-life,and small epitope peptides molecules,the immunogenicity is weak and it is difficult to produce an effective immune response.Thus,finding suitable carriers and adjuvants to produce an effective immune response of peptides is the main bottleneck in the current research on tuberculosis peptides vaccines.Chitosan(chitosan,CS)has the advantages of wide source,low price,safe and non-toxic,good tissue compatibility,can improve systemic and local immune response,etc.As a potential peptide vaccine adjuvant,it has become a hotspot of the vaccine adjuvant research within the scope in the world.This project attempts to use chitosan to construct nanocarriers,load tuberculosis polypeptides,enhance the immunogenicity of the polypeptides,and prolong the release time of the polypeptides in the body,to more effectively stimulate the body’s specific immune response.The specific research content of this topic includes:screening and in vitro synthesis of potential protective tuberculosis antigen polypeptides.Synthesis of chitosan-deoxycholic acid amphiphilic conjugate(Chit DC),and structural characterization of Chit-DC conjugate by ultraviolet spectrum(UV),infrared spectrum(IR)and nuclear magnetic resonance(~1H NMR),The peptides-loaded nanoparticles(Chit-DC-Peptides)were prepared by self-assembly,and the micro-morphology and particle size of Chit-DC nanoparticles and Chit-DC-Peptides nanoparticles were observed by high-resolution transmission electron microscope(TEM).Synthetic chitosan nanoparticles(CS-NPs)and peptides-loaded nanoparticles(Peptides-CS-NPs)were prepared by ion crosslinking method.Scanning electron microscope(SEM)was used to observe the microscopic morphology and particle size of the nanoparticles before and after peptides loading.The dynamic light scattering method(DLS)was used to measure the hydrodynamic diameter,and the Zeta potential method was used to measure the change in potential before and after the polypeptides was loaded.Fluorescence spectrophotometry was used to determine the content of peptides before and after loading,calculate the loading rate,drug loading and study the release rule of peptides in vitro.Finally,the ion-crosslinking method was used to trial-produce tuberculosis peptides nanoparticle vaccine.Use trial vaccines for animal experiments in mice.Five control groups were set:blank group,PBS group,free peptides group,peptides Freund’s incomplete adjuvant group,and peptides nanoparticle vaccine group.After immunizing mice subcutaneously three times every two weeks,the mice in each group were tracked serum Ig G antibody changes.The results showed that two tuberculosis antigen peptides were successfully screened and synthesized:Rv0180c:GLDKNKFDIRVVSPDEARRLY and Rv0227c:VSGTIVKETERANHYFARDP.The results of ultraviolet spectroscopy(UV),infrared spectroscopy(IR)and nuclear magnetic resonance analysis(1H NMR)showed that deoxycholic acid was coupled with chitosan to form a Chit-DC conjugate.TEM results show that the diameter of Chit-DC particles is 30-50 nm,and the Chit-DC-Peptides nanoparticles prepared by self-assembly method are spherical,and the diameter is between 30-100 nm.In the ion cross-linking method,the average particle size and PDI of the blank CS-NPs measured by dynamic light scattering method were(148.13±2.24)nm and 0.197±0.013 respectively;placed at 4℃for 28 d,the particle size did not increase significantly.The average particle size and PDI of Peptides-CS-NPs were(186.93±8.80)nm and 0.254±0.014,respectively.The potential of nanoparticles before and after peptides loading was(+18.00±0.89)m V and(+12.07±1.68)m V measured by Zeta potentiometry.The potential of nanoparticles before and after peptides loading was(+18.00±0.89)m V and(+12.07±1.68)m V measured by Zeta potentiometry.The loading rate of nanocarriers and the amount of loaded peptides were(45.20±2.95)%and(12.92±1.12)%,And antigen peptides in polypeptide-loaded nanoparticles reached equilibrium after about 48 h,and the cumulative release rate of peptides was 56.6%.Finally,selecting ion-crosslinking method to prepare carrier-loaded peptides trial vaccine.After immunizing mice with trial vaccines,the ELISA results showed that the peptides nanoparticle vaccine group was significantly different from the blank group,PBS group,free peptides group,and peptides Freund’s incomplete adjuvant group(P<0.01).These results indicate that the prepared chitosan nanocarrier delivery effectively played an adjuvant role,laying a foundation for the development of new tuberculosis vaccines.