Study On Recombinant Genetic Engineering Vaccine Of Botulinum Neurotoxin

Botulinum neurotoxins(BoNTs),produced by the Gram-positive,anaerobic bacterium Clostridium botulinum,are most toxic natural substances known.BoNTs are categorized into seven serotypes,which are structurally similar but antigenically distinct.Serotypes A,B,E and F can cause naturally occurring human botulism,while serotype C,D and G can cause illness in birds,cattle and horses.Among,serotype A is the most toxic.Typically,each BoNT consists of a 100 k Da heavy chain(HC)coupled with a 50 k Da light chain(LC)by a disulfide bond.The carboxyl terminus of the heavy chain(Hc)is responsible for binding to receptors on the cell surface,and the amino terminus of the heavy chain(HN)mediates translocation of the LC domain across the cell membrane.The LC of BoNT is a zinc-dependent metalloprotease that cleaves the SNARE protein that are required to form the SNARE complex,which mediates the neurotransmitter delivery,resulting in skeletal muscle paralysis,respiratory failure and eventually death.BoNT is a potential biological weapon because they have good stability and high mortality and are simple to produce.Botulism poisoning incidents occurred occasionally in recent years.Thus,prevention and treatment of botulinum neurotoxin is very significant.Vaccination is an effective strategy to prevent botulism.Early botulinum vaccines are not widely used due to their drawbacks.Therefore,the development of a new generation of botulinum vaccine is of intensively need.Tetanus toxin(Te NT)is a toxin with many similarities to BoNTs.So the BoNTs subunit vaccine was developed by the successful strategy of tetanus toxin Hc fragment as subunit vaccine.The Hc domain,which is no zinc endopeptidase activity and nontoxic,mediates the highly specific binding of BoNTs to nerve terminals at the neuromuscular junction,and is able to induce protective immune responses against natural BoNTs in animals.This important finding triggered the researchers' interest toward developing a new generation recombinant Hc subunit vaccines and nucleic acid vaccines.DNA vaccine is a novel vaccination strategy.By cloning the gene encoding certain antigen into eukaryotic expression vector,the antigen can be expressed in immunized cannot provide adequate immune-protective effects due to their limited immunogenicity in human clinical trials.So the immunogenicity and protective efficacy of naked DNA should be further improved and amplified.Dendritic cells(DCs)are professional antigen-presenting cells(APCs)that play a vital role in antigen presentation and priming immune responses.DEC205,an endocytic receptor that is abundant on the surface of DCs,can mediate high-efficiency antigen uptake,processing and presentation.In order to enhance the immunogenicity of DNA vaccine,we constructed a DC-targeted DNA vaccine(p VAX1-sc DEC-AHc)encoding the Hc domain of BoNT/A(AHc)fused with sc DEC,a single-chain Fv antibody(sc Fv)specific for the DC-restricted antigen-uptake receptor DEC205.The non-targeted vaccine vector(p VAX1-SAHc)and negative control(p VAX1)were also constructed simultaneously.animals and stimulate the body to produce immune responses and protective effects.DNA vaccines have many advantages over toxoid vaccines.The production process is simple and the products are easy to purify and store.And antigens expressed in vivo can elicit both humoral and cellular immune responses.However,DNA vaccinesIntramuscular injections of mice with the DC-targeted DNA vaccine induced stronger AHc-specific humoral and cellular immune responses compared with the non-targeted DNA vaccine immunized mice.The anti-AHc specific antibody titers of DC-targeted DNA vaccine immunized mice were increased by 2.5-fold.Our data showed that these DNA vaccines induced both AHc-specific Ig G1 and Ig G2 a antibodies,with a predominant Ig G category Ig G1,which is an indicative of a Th2-type humoral immune response.We also showed that the p VAX1-sc DEC-AHc DNA vaccine induced stronger protective efficacy against natural BoNT/A in mice than the p VAX1-SAHc vaccine.The DC-targeted DNA vaccine could provide complete protection against challenge with 103 LD50 of BoNT/A only after 30 ?g of two doses immunization,while the p VAX1-SAHc DNA vaccine provided only 12.5% protection.After three doses immunization,the p VAX1-sc DEC-AHc DNA vaccine provided 87.5% protection against challenge with 104 LD50 of BoNT/A,while the p VAX1-SAHc DNA vaccine also provided only 12.5% protection.These data suggested that the p VAX1-sc DEC-AHc-immunized vaccine had a higher protective efficacy against BoNT/A than p VAX1-SAHc.The DC-targeting effects of the DNA vaccine were studied by flow cytometry and immunohistochemistry,the results showed that the p VAX1-sc DEC-AHc vaccine led to a striking recruitment and expansion of DCs at injection site and a substantial proliferation and maturation of DCs in spleen tissues compared with the p VAX1-SAHc vaccine.These results indicated that the enhanced mechanism of p VAX1-sc DEC-AHc DNA vaccine might be the DC-targeting effects.were immunized to study the immunogenicity and protective efficacy of this antigen.The recombinant BHc-His subunit vaccine provided complete protection against challenge with 103 LD50 and 104 LD50 of BoNT/B only after 1 ?g of one and two doses immunization,respectively.Antibody assay showed that the anti-BHc serum antibody titers in BHc-His immunized mice were 1:110000 and serum neutralization titers were 3.33 IU/ml,while m BHc expressed in yeast immunized mice were only 1:70000 and 1.25 IU/ml.These findings suggested that the recombinant BHc-His antigen expressed in E.coli was efficacious in protecting mice against challenge with BoNT/B.Moreover,a recombinant BHc antigen without his tag expressed in E.coli was also efficacious in protecting mice against challenge with BoNT/B.Thus,the recombinant BHc subunit vaccine expressed in E.coli may be useful as a subunit candidate vaccine for prophylaxis against BoNT/B.In this work,we also studied the immunogenicity of soluble recombinant Hc subunit vaccine of BoNT/B expressed in Escherichia coli.The Hc gene of BoNT/B was cloned into prokaryotic expression vector p TIG-Trx,and soluble recombinant BHc-His antigen expressed in E.coli strain BL21(DE3)was purified.Balb/C miceIn summary,our findings showed that the DNA vaccine p VAX1-sc DEC-AHc targeting the AHc antigen to DCs via DEC205 receptor can induce strong humoral and cellular responses against BoNT/A,providing an alternative strategy and technology platform for the prevention of botulinum neurotoxin poisoning,as well as diseases caused by other pathogens.The study on the immunogenicity of recombinant Hc subunit vaccine of BoNT/B expressed in E.coli showed that this BHc antigen possesses excellent immunogenicity and protective efficacy,which lends it great promise to be developed as a good candidate subunit vaccine.In addition,this antigen can be used to prepare a tetravalent botulinum subunit vaccine combined with AHc,EHc and FHc antigens expressed in E.coli.