Establishment Of Rabbit Model For Lethal Toxin (LT) Challengeand Efficacy Evaluation Of Anti-anthrax Antibody

Anthrax caused by Bacillus anthracis is considered a serious lethal infectious disease and has a wide distribution in the world.According to the route of infection,anthrax can be divided into four types,which include cutaneous,gastrointestinal,inhalational and injectional anthrax.Without treatment,the mortality rate of inhalational anthrax approaches 90%.It is treated as the class A infectious disease in China.Anthrax spore is a potential biowarfare agent and biological bioterrorism agent.The virulence factors of this pathogen contain two main aspects:the poly-D-glutamic acid capsule and anthrax toxins.The polyglutamate capsule prevents phagocytosis of the bacterium.The three toxin components produced by B.anthracis are protective antigen?PA?,lethal factor?LF?,and edema factor?EF?.The components alone are not pathogenic.The lethal toxin?LT?composed of LF and PA and the edema toxin?ET?composed of EF and PA are the main reason which result in the death of the host.LF and EF are delivered into the cytosol and induce the toxicity via receptor binding of PA.Blocking the delivery process of PA is a critical mechanism against anthrax.Prevention and treatment are two means against anthrax.Vaccines are good choices for the prevention of anthrax.But vaccination cannot take effect in the short term.By contrast antibodies had the potential to provide emergent prevention for acute anthrax.On the other side,current therapeutic strategies against anthrax mainly include antibiotics and anti-toxins.Antibiotics can kill sensitive B.anthracis,but they can not clear anthrax toxins.Antibodies can neutralize toxins,resulting in reduced morbidity and mortality.5E11 is a humanized IgG1???monoclonal antibody?MAb?.Previously our studies have proved that 5E11 showed strong neutralizing activity on the cell and LT-challenged rat model.It is necessary to evaluate the efficacy of 5E11 on the other animal models.Rabbit models and monkey models can better simulate the situations of infected human beings compared with mouse models and rat models.But virulent strain challenge must be limited in Biosafety Level 3?BSL-3?laboratory.So we want to establish a new large animal model independent of BSL-3 to assess the efficacy of antibodies.But rabbits and monkeys are not sensitive to attenuated strains.Considering the critical effects of LT to hosts during the infection,we expect to establish a LT-challenged animal model.Rabbits are considered well-characterized anthrax animal models.Compared to monkeys,they are low cost and are easily operated.And there is no relevant report for the LT-challenged rabbit model up to now.In this new model,we want to know about the sensibility of rabbits to lethal toxin,the pathology changes following the LT challenge and some other aspects.We focus on the characteristic of LT-challenged rabbit model and compare the common points and differences between LT challenge and spore challenge.And then it can be used to evaluate the efficacy of 5E11.Firstly,we prepared enough PA and LF.Then the rabbits were i.v.challenged with PA and LF with the mass ratio of 2 to 1.And the results showed a dose of 1 mg PA+0.5mg LF could be lethal.When the dose reached to 2 mg PA+1 mg LF,it could cause 100%mortality.And the high dose of 4 mg PA+2 mg LF can limit the rabbit death time within2 or 3 days,which showed good consistency.In the experiment,clinical signs including declined activity,diarrhea,inappetence,respiratory distress,and unresponsiveness were observed in LT-challenged rabbits and are similar to those in spore-challenged rabbits.Liver,kidney and lungs obtained from rabbits challenged with 4 mg PA+2 mg LF all showed pathological injuries,including hemorrhage,necrosis,inflammatory cell infiltration and so on.These pathological injuries are similar to those in spore-challenged rabbits.No significant changes are found in temperature in rabbits.Some cytokines were analyzed in rabbit serum.An increase of IL-4 level was found in some of the rabbits.By calculation,we found that the peak concentration of PA in blood in LT-challenged rabbit is the same order of magnitude?10⁴ ng/mL?as that in spore-challenged rabbit in the medium to late infection stage in other papers.Thus we believe the established LT-challenged rabbit model can in some way simulate the situations in rabbits in the medium to late infection stage.Next we evaluated the efficacy of 5E11 with the LT-challenged rabbit model.Pharmacokinetics?PK?parameters of 5E11 in rabbits were measured.The mean elimination half-life was about 1.5 days.Prophylaxis experiment were designed according to the PK parameters.Administration of 40 mg/kg 5E11 1 or 4 days prior to challenge?4 mg PA+2 mg LF?could provide full protection.Administration of 40 mg/kg5E11 7 days prior to challenge saved no rabbit and pathological analysis showed that the liver and lungs were damaged more seriously.The results showed that the efficacy was better when 5E11 was administrated earlier.Two studies,according to the administration dosage and treatment time,were conducted to investigate 5E11 efficacy in the therapeutic study.Administration of 2.5,10 and 40 mg/kg 5E11 10 minutes following challenge of 4mg PA+2 mg LF led to survival rates of 40%?2/5?,80%?4/5?and 100%?5/5?,respectively.Administration of 40mg/kg 5E11 at 30 minutes and 1 hour postchallenge led to only 40%?2/5?and 20%?1/5?survival rate.Pathological analysis showed that the organs of rabbits with delayed treatment time were damaged more seriously than those with reduced administration dosage.Although LT-challenged rabbit model are different from spore-challenged rabbit model,the results suggest that high toxin levels in medium and late infection stage can most likely causes damage and death.Furthermore,some of the survival rabbits were re-challenged.No rabbit died and high levels of rabbit anti-PA and anti-LF polyclonal antibody were detected in rabbit serum.The re-challenged experiment showed the survival rabbits could establish self-immunity response after the first LT challenge.Considering that spore challenge is closer to natural infection,we tried to evaluate the efficacy of 5E11 with low virulent anthrax strain.DBA/2J mice were challenged with a series of doses of A16R spores.The median lethal dose?LD50?was 7.8×10³ CFU by Bliss method.Then PK parameters of 5E11 in mice were measured with ELISA.The mean elimination half-life was about 7.5 days.According to the spore-challenged mouse model,all groups of the mice were challenged with 100×LD₅₀0 spores and different groups were treated with 5E11 at different time and doses,after that the survival of the mice were observed.The results showed that administration with 5E11 one day after challenge could provide full protection and administration two or three days after challenge could reach the survival rate of 40%to 80%.In addition the results showed the survival was dose independent.Anti-toxin polyclonal antibodies were detected to prove that A16R could generate toxins and can induce self-immunity response.In summary,we established LT-challenged rabbit model by using LT to challenge rabbits and doing observations and analysis in some aspects in the experiments.We evaluated the efficacy of 5E11 with the model in prevention and treatment.The results showed 5E11 could effectively protect the host against anthrax toxins.Then we assessed the efficacy of 5E11 with A16R spore-challenged DBA/2J mouse model.5E11 also exhibited good protective efficacy.All taken into account,we can conclude that 5E11 has the potential to provide emergent prevention and treatment for acute anthrax.