Study On Biological Function And Immune Potency Of The Functional Domains Of Tetanus Toxin

Background:Tetanus toxin is a neuroprotein-toxin produced by Bacillus tetanus.This protein prevents the inhibitory synapses of the infected person from releasing neurotransmitters,causing hyperreflexes and rhabdomyspasms.TeNT is highly toxic,with an estimated lethal dose of less than2.5ng/kg and an untreated case fatality rate of up to 40%.Tetanus is a life-threatening disease characterized by muscle spasms.The disease is caused by a neurotoxin from the bacterium tetanus.Tetanus toxin is first produced as a single 150 kDa protein,which is cut by an endogenous protease into a toxin-active form with heavy chains（HC,100 k Da）and light chains（LC,50 k Da）.The heavy and light chains are connected by interchain disulfide bonds.The C-terminal domain of the heavy chain is responsible for binding to nerve cells and subsequently endocytosis to form vesicles,while the N-terminal domain of the heavy chain mainly assists the light chain to cross the vesicle membrane into nerve cells.The light chain molecule is a zinc-dependent protease and is linked to HC via interchain disulfide bonds until LC reaches the cytoplasm,at which point the disulfide bond is reduced and released into the cytoplasm of the neuron.Purpose:In order to study the more effective tetanus recombinant subunit vaccines and explore their protective efficacy,this study with TeNT as the object,the optimal functional domain antigen molecules of tetanus recombinant subunit vaccine were determined by systematically studying the immunological protective efficacy of its epitopes in various functional domains.In addition,since neutralization epitopes on the three major functional domains of TeNT,especially TL-HN molecules have stronger immunoprotective potency.Therefore,to further investigate more effective candidate vaccines,this study intends to prepare a recombinant genetically engineered tetanus toxoid（m TeNT）with all TeNT neutralizing epitopes,with the aim of enhancing its protective immunity.Methods:In this study,various functional domain fragments of TeNT were prepared by genetic engineering and connected to the p TIG-Trx prokaryotic expression vector.The constructed plasmid was transferred into the E.coli expression system,appropriate conditions were selected for induced expression,and eluted from recombinant protein by affinity chromatography through AKTA purification system,so as to obtain high purity of recombinant target protein.The purity and size of each recombinant protein were identified by SDS-PAGE.The binding specificity of each recombinant protein to the anti-TeNT antibody was identified by Western blot.To evaluate the biological activity of each recombinant protein,the ability of each recombinant protein to cut substrate protein VAMP was measured by SDS-PAGE and binding to nerve cell related receptors GT1b,SV 2B and SV2C was measured by ELISA.Each recombinant protein was tested for any animal toxicity,thus ensuring that it could be safely used as subunit vaccine molecules.The recombinant protein of each functional domain was mixed as antigen and aluminum adjuvant to prepare into the subunit vaccine.The mice were tested with antigen alone,combined antigen together,and dose-dependent immunity to detect the humoral immunity level,neutralizing antibody level and immune protective efficacy.Results:The functional domain TL-HN fragment had the highest immunoprotective effect in all functional domains of TeNT.The neutralization titer of TL-HN reached 10 IU/m L and 80 IU/m L,respectively,in the 1μg group and 10μg group after the second immunization.As a protective antigen,TL-HN produced the highest level of neutralization antibody and the highest neutralization titer among all the recombinant antigens measured,with significant dose difference.The ED₅₀values of TL-HN,THc and TL+THN groups were 42.249 ng,664.433 ng and 1116.589 ng,respectively,after 10³LD₅₀TeNT injection in mice immunized once,and TL-HN,after 10³LD₅₀TeNT injection in mice immunized twice.The ED₅₀of THc and TL+THN groups were 10.852 ng,31.210 ng and 124.858 ng,respectively.From the results,the dose-dependent experiments again indicate the strongest protective effect of the functional domain antigen TL-HN in several recombinant antigens.In this study,three L-HN fragments molecules（TL-HN,TL-GS-HN and TL-2A-HN）of TeNT were prepared,consisting of L and HN domains,respectively,and excluding Hc domains.The immunological significance of these functional domain fragments of TeNT was investigated in animal models.Two variants of TL-HN（TL-GS-HN and TL-2A-HN）did not enhance its protective effect,suggesting that the natural unmodified recombinant antigen TL-HN may have more neutralizing epitopes.In addition,the protective effect of TL+THN antigen was weaker than that of TL-HN antigen,indicating that the combination of the two functional domains had no synergistic effect.This suggests that it may retain some key epitopes responsible for inducing serum neutralizing antibodies.A large number of experiments have shown that TL-HN has superior protective effect compared with THc or TL and THN combined groups.By mutating three amino acids（H233A,E234A and H237A）in the TeNT light chain enzyme active region and three amino acids（R1226L,W1289L and Y1290F）in the receptor binding region,the DNA gene sequence encoding m TeNT is linked to the p TIG-Trx prokaryotic expression vector.After induced expression,the target protein with high purity was successfully obtained by affinity chromatography through AKTA purification system.Western blot assay showed that m TeNT had good binding specificity with serum antibodies of tetanus toxin in two major functional domains（enzyme active region and receptor binding region）.KM mice were intraperitoneally injected with 20μg of m TeNT and observed for a week.The toxicity test showed that the recombinant protein m TeNT decreased by at least 400,000 times,indicating that the m TeNT prepared in this study had no toxicity.In contrast to tetanus toxin,the recombinant protein m TeNT does not cleat the substrate VAMP,nor does it bind to ganglioglycine GT1b,and includes all functional domains.Thus,the immunoprotective efficacy can be evaluated in the future.The protective and dose-dependent tests showed that the recombinant antigen m TeNT had good immunoprotective efficacy.The efficacy value of recombinant antigen m TeNT ED₅₀is much lower than that of formaldehyde inactivated TT vaccine group,indicating that the full-length toxin molecular protein modified without chemical reagent has better symbolic and neutral epitope and higher immunoprotective efficacy.The recombinant antigen m TeNT is also slightly better than the ED₅₀of the combined group antigen TL-HN+THc,which further reflects the immune efficacy of the full-length toxin molecule,and also indicates that the junction of THN may not have important antigen neutralization epitopes with THc.In summary,the recombinant antigen m TeNT contains all of the antigen-neutralizing epitopes of TeNT.Conclusions:The results of this study reveal for the first time the strong protective effect of the functional L-HN segments in TeNT.The TL-HN functional domain molecules have a correct structural conformation and are enzymatically active,and thus can be used as an antigen immune molecule for screening anti-TeNT antibody molecules,and also provides new functional molecules to study the activity and conformation of TeNT.Therefore,TL-HN fragments should play an important role in immune protection against tetanus toxin,providing new research directions for the development of tetanus toxin vaccine or antibodies,and may replace THc or TT as candidate subunit vaccines for tetanus toxin.The results of this study show that the m TeNT vaccine inactivated by genetic engineering technology has the advantages of convenient production,small differences between batches,and good immunoprotective efficacy compared with the TT inactivated by formaldehyde.Therefore,the m TeNT vaccine inactivated by genetic engineering technology can be used as a candidate vaccine variety to prevent tetanus poisoning.