Screening And Function Study Of Human Monoclonal Antibodies Against Tetanus Toxin

Tetanus is a serious disease characterized by painful muscle contractions,particularly of jaw and neck muscles,and therefore is also commonly known as ?lockjaw?,and is caused by a neuron toxin secreted by Clostridium tetani.Although inactivated tetanus toxin(toxoid)as vaccine is available for prevention of tetanus,tetanus is still endemic in low-and middle-income countries and continues to be a significant cause of death and disability in unvaccinated populations.For pregnant women giving birth and neonatal infants,tetanus results in extremely high mortality rates.Repeated vaccination is needed in every ten years for maintaining effective immunity.However,such boosting vaccination has been often neglected.Thus,tetanus can occur in individuals without sufficient immunity exposed to C.tetani as results of wounds contaminated with dirt,feces,soil or saliva and other conditions.To prevent tetanus in the event of tetanus-prone wounds,such as wounds with fracture,deep penetrating wounds,bite wounds,in individuals at risk after exposure to C.tetani as results of tetanus-prone injury,tetanus antitoxin treatment is needed.Equine tetanus antitoxin has been no longer used in industrial contries and is not included in the WHO list of essential medicines as there is a risk of serum sickness and hypersensitivity.Although human immunoglobulin is superior to equine tetanus antitoxin in efficacy and safety in spite of its own potential risk of transmitting known and unknown blood-born pathogens,it is often difficult to obtain because of its high demand for raw materials and short supply in China.Therefore,there is an urgent need to replace horse and human serum-derived immunoglobulins with a highly effective and safe anti-tetanus therapeutic monoclonal antibody(mAb)to meet such an unmet need for prevention and treatment of tetanus infection.tetanus toxin consists of a 50 kD amino terminal light(L)chain and a 100 kD carboxy terminal heavy chain(H)chain linked by a disulfide bone.There are A,B and C three functional fregments.A is the active part of the toxin and has the activity of metalloproteinase.Fragment B is the N-terminal translocation domain responsible for toxin transport.Fragment C contains domains that bind to the receptor and is not neurotoxic.The A and C fragments are currently considered to be potential targets for neutralizing toxins and are aimed for developing neutralizing antibodies.Therefore,we used complete toxins to screen anti-tetanus antibodies,explored the contribution of different regions of the toxin as target in neutralization,and further defined the target and mechanism of tetanus neutralization,and provided a theoretical basis for vaccine development.In this study,a total of 32 tantus toxin specific Mabs have been isolated from a vaccinatede human volunteer by using the native fully human monoclonal antibody(Mab)technology platform,Among these Mabs,5 of them were identified to be specific to tetanus toxin C fragment(TTC).All of these 5 antibodies has high affinity for tetanus toxin and recognize 3 different epitopes on the C fragment.One of 5 antibodies,TT0067,has high titers of protective neutralization activity in mouse TT challenge model.Mab TT0067 at single dose of 50 ul injection at the concentrations ranging from 50 ?g/ml-5.56 ?g/ml completely protected mice from challenge with live toxin at dose of 20 LD50.The potency of the antibody can be translated as < 3 mg of Mab TT0067 being a single vial of 250 international unit(IU)of commercialy available human tetanus toxin specific immunoglobulin.Analysis of the X-ray co-crystal structure of TT0067 with TTC protein was carried out to understand the mechanism of action by Which Mab TT0067 neutralizes tetanus toxin.Results revealed that Mab TT0067 directly occupied the W pocket of one of the receptor binding sites for TeNT,resulting in the blocking of the binding of TTC to ganglioside on the surface of the host cells to neutralize the TeNT.This study confirmed that TTC as the neutralizing epitope and the feasibility of developing TTC as vaccine for induction of protective neutralizing antibody.Study on the C fragment mutants each with single point mutations of the critical amino acid residues identified by co-crystal structure further confirmed that amino acid residues W1289?H1293?D1296 are critically important for the binding of Mab TT0067 to the the C fragment.Taken together,we have identified a potent protective TeNT neutralizating native human monoclonal antibody with high potential for development as therapeutic antibody for prevention and treatment of tetanus infection,revealed at atomic level the mechanism of action by Mab TT0067 and the critical neutralization epitope on the C fragment of TeNT,and provided the critical information for development of to the C frgment of TeNT as a better and safer tetanus vaccine.