Study On Immune Effect Of Receptor-binding Domains Of Clostridium Difficile Toxins

C.difficle causes more than25%antibiotic-associated diarrhea (AAD). It got barelyattention before it was found to be associated with pseudomembranous colitis (PMC) in1978. C.difficle infection (CDI) caused by a new strain belonging to ribotype027(RT027) broke out in some countries worldwide since2003, resulting in a largenumber of death. RT027infections have also been reported in China. HypervirulentRT027has serval variations that cause its increased infectious and drug resistance,which all eventually lead to higher rate of mortality and morbidity than traditionalstrains. C.difficile has been on the legal surveillance list in a lot of countries includingChina as the pathogen causing infectious diseases. It is generally considered that avaccine approach is a preferred fundamental and effective control strategy.Toxin A and toxin B are major virulence determinants of C.difficile. Immunity tothe toxins provides protection for inhibiting the action of the toxins which caneffectively prevent serious injury caused by infection. The carboxy-terminal portion ofboth toxins contains a highly repetitive structure that contains binding sites for cellsurface receptor carbohydrates. The length of RBDs or the number of repeats has adirect impact on the binding capacity.24toxinotypes and150ribotypes exsit in C.difficile, and toxins that differentstrains produce may vary. To cover protective antigen epitopes as many as possible, andto provide maximum protection, We have constructed recombinant proteins containingfull-length RBD from toxin A or toxin B(named TcdA and TcdB, respectively)ofreference strain ATCC43255(toxinotype0).We immunized mice with the recombinant TcdA and/or TcdB to test their immunogenicity and protection effectagainst toxins extracted from the reference strain and ATCC BAA1870.Part1. Extraction、purification and identification of C.difficile toxinsTo verify the differences between toxins of ATCC BAA1870and the referencestrain, we sequencd genes encoding toxins A and B. The results show that genes of bothtoxins in ATCC BAA1870are as same as other two RT027strains, R20291and CD196which have been published in Genbank. And amino acids of toxins A and B showrespectively98.15%and92.18%with the reference strain. The most conservativedomain is the N-terminal catalytic domain, and the most changes happen in RBDs.To obtain natural toxins A and B of ATCC43255and ATCC BAA1870, we havefirst precipitate the culture supernant with60%(NH4)2SO4.As a result, we got80mg/L,or86.5mg/L crude toxins. Toxins can bind to specific anti-toxin A and anti-toxin Bmouse monoclonal IgG antibodies used in Western blot, and toxins show the rightmolecular weight as reproted. We challenged mice and cells using crude toxins to testtheir toxicity. The minimal dose that casused completely lethal(MLD) in mice was360ng for ATCC43255and540ng for ATCC BAA1870. Dose that cause50%Vero toround(CD50) is70pg for ATCC43255，and90pg for ATCC BAA1870. These datas areclose to literature. We then loaded crude toxins onto a DEAE Sepharose column toseparate toxin A from toxin B. Single band of toxin A and toxin B can be identified byspecific anti-toxins antibody by Western blot and VIDAS-mini. The purify method weused in this study has simple steps than gel filtration and some ion-exchange reprotedused by others, and purification efficiency is high.Part2. RBDsbpreparation and study on their immune effects in mice modelBinding to receptors by RBDs is essential for toxicity of C.difficle toxins A and B,inhibiting the binding can therefore protect host from injury by toxins. We prepared recombinant RBDs first to be candidate antigens in this part of study. RecombinantTcdA and TcdB with purity more than90%were identified by Western blot.BALB/C mice were immunized with TcdA and/or TcdB by intramscularinjection.with aluminum hydroxide as adjuvant. More than1:102titer of specific IgGwere detected7days after the first immunization, and the titer is up to1:1057days aftera booster. The highest level came2months after the booster, and103titer1month later.C57BL/6mice were immunized with2doses that is1μg or10μg to study therelationship between the immuizing dose and antibodies level. The results show thatthere is nearly no statistically significant difference expect anti-TcdB antibodies level inTcdA10μg+TcdB10μg and TcdA1μg+TcdB1μg. We also detected the toxinneutralization titer of the serum of C57BL/6mice. The serum dilution which gives a50%reduction in toxin activity (ED50) were as follows: for1μg immuizing dose, totoxins of ATCC43255:1:4960,1:1196,1:7510; to ATCC BAA1870:1:529,1:1223,1:2310; for10μg immuizing dose, to toxins of ATCC43255:1:5987,1:1190,1:10643; toATCC BAA1870:1:1563,1:933,1:6410.To study immune effect of TcdA and TcdB, BALB/C and C57BL/6mice wereimmunized with TcdA,TcdB or TcdA+TcdB. Mice were challenged wihe natural toxinsof ATCC43255or ATCC BAA1870after two immunizations. The results show that (1)in the BALB/C model, when challenge1MLD of toxins, to ATCC43255, allimmunized mice were protected, to ATCC BAA1870, can only TcdA+TcdBimmunized mice protected100%, no other teams could protect.When challenge5MLDof toxins, to ATCC43255, all immunized mice were protected expect TcdB whichsupplied no protection; to ATCC BAA1870, no mice survived. When challenge20MLD of ATCC43255toxins, TcdA and TcdA+TcdB suvived60%and80%.(2) In theC57BL/6mice model, we studied the relationship between immunizing dose andprotection effect. For mice immunized the dose of1μg, when challenged1MLD oftoxins, to ATCC43255,all immunized mice suvived, to ATCC BAA1870,survival percents are0,100%,100%; when challenged3MLD of toxins, to ATCC43255,survival percents were100%,40%,100%; to ATCC BAA1870, no mice survived;whenchallenged5MLD of ATCC43255toxins, the rates were87.5%;0,75%;0,50%,80%survived when challenged2MLD of ATCC BAA1870toxins.For miceimmunized the dose of10μg, when challenged1MLD of toxins, to ATCC43255, allimmunized mice suvived, to ATCC BAA1870, survival percents are100%.20%,100%;when challenged3MLD of toxins, to ATCC43255, survival percents were100%,20%,100%; to ATCC BAA1870,0,0,60%; when challenged5MLD of ATCC43255toxins, the rates were75%,0,87.5%；0,0,100%survived when challenged2MLD ofATCC BAA1870toxins. Protection effect of C57BL/6is lower than that of BALB/Cwhen compare the survival rate of same immunization, the reason is not clear.With all results we have observed, both recombinant TcdA and TcdB are highlyimmunogenic for high titers of IgG were induced in immunized mice. Antibodies canneutralize toxin effectively. Animals can obtain certain protection by twoimmunizations, especially by immunizing TcdA combined with TcdB. Protection effectof ATCC BAA1870is weaker than reference strain, which suggest that vaccine whichcover combined and effective epitopes of variate toxins can provide comprehensiveimmune protection against CDI caused by different C. difficile strains.