Evaluation Of Protective Effects Of Antivenom Induced By AaHⅣ And Its Domains From Agkistrodon Acutus

1. BackgroundAdministration of antivenom is the only effective way to neutralize the toxicity ofsnake venoms, and is strongly recommended by World Health Orgernization. To date, all ofthe antivenoms around the world are manufactured by hyperimmunizing horse or sheep.However, snake venom is a complex mixture of proteins and polypeptides, some toxiccomponents have strong antigenicity, but no or very weak immunogenicity. And some havestrong immunogenicity, but no or very weak antigenicity. Despite the purity of theseantivenoms are much higher than them used to be,severe adverse reactions, such asanaphylactic shock and serum sickness, associated with antivenom administrationsometimes unexpectedly occur. Because it is not only an animal-derived immunoglobulin,but also the low quality of immunogen.An ideal immunogen should comprise all of the important toxic components which canexcite body immunity, and the induced antivenom can neutralize all of the toxicity of thewhole snake venom.Agkistrodon acutus, a kind of Viperidae, is widely distributed in thesouthern of China. It has a strong toxicity and large quantity venom. In the rural area,agkistrodon acutus bite cause a critical healthy problem. Some patients disabled or lose theirlives without antiserum administration. Snake venom metalloproteinases(SVMPs) is seen asthe component, which is the most abundant and hemorrhagic toxin of Viperid crude venom.AaHⅣ is a type Ⅳ SVMPs of Agkistrodon acutus venom,which comprise the wholemetalloproteinase domain(the catalytic domain), disintegrin-like domain and Cysteine-richdomain(the non-catalytic domain). It has a strong hemorrhage activity, and could inhibitaggregation of platelet which is induced by collagen.2. Objective and MethodTo produce an ideal antigen used for manufacture a high quanlity antiserum, we planedto：1）purify hemorrhagic toxin AaHIV form Agkistrodon acutus crude venom through affinity chromatography and hydrophobic chromatography techeniques. Then, immunizemice with AaHIV, crude venom and PBS and equal volume of complete Freund’s adjuvantor incomplete Freund’s adjuvant. At the end of immunization, separate antiserum ofimmunized mice and evaluate antibody titer by indirect ELISA. Analyse theimmunoprotection difference between antiserums the crude venom and AaHⅣ induced.2)respectively express the catalytic domain and non-catalytic domain of AaHⅣ byprokaryotic expression systems. Then, induce antiserum by immunizing mice with AaHIV,the catalytic domain, the non-catalytic domain, PBS and equal volume of complete Freund’sadjuvant or incomplete Freund’s adjuvant. At the end of immunization, antibody titer areevaluated by indirect ELISA. Analyse the immunoprotection difference among antiserumsthe two fragments and AaHⅣ induced.3. Results①Purification ofAaH Ⅳ.After anion affinity chromatography, hydrophobic interaction chromatography anddesalting step, We successfully acquired purified hemorrhagin Ⅳ(AaHⅣ).According toBliss method, the LD50of AaHⅣ was10.07μg/g.②Expression of the catalytic domain and the non-catalytic domainWe got the cDNA sequence of the catalytic domain and the non-catalytic domain bygene synthesis. And expressed them respectively through prokaryotic expressionsystem(pET28a(+) and BL21(DE3)).③Antibody titersImmunized Kunming mice with Agkistrodon acutus crude venom form Jiangxi, AaHⅣ,catalytic domain fragment and non-catalytic domain fragment, PBS. Two booster weregiven by the same route. Indirect ELISA demonstrated that all of the groups except PBSgroup had high titers IgG antibodies.④The neutralization test5μl antiserum from immunized mice was mixtured with1μg crude venom, andincubated37℃for1hour.The antiserum/crude venom mixed solution was subcutaneousinjected intoback skin of mice. After24hours, the hemorrhagic area of the crude venomimmunized group was（1.50±3.67）mm2, the AaH Ⅳ immunized group was（44.83±19.48）mm2, and the PBS group was（134.83±21.97）mm2（n=6）.According to one way-ANOVA, there was statistical difference between the AaHⅣ immunized group and thecrude venomimmunized group（P＜0.001），also between the AaHⅣ immunized group and the PBSgroup（P=0.001）.5μl antiserum from immunized mice was mixtured with1μg AaHⅣ, and incubated37℃for1hour.The same method above mentioned was used to measure the hemorrhagicarea of the AaHⅣ immunized group was (2.83±4.92)mm2, the catalytic domain immunizedgroup was (5.67±6.74) mm2, the non-catalytic domain immunized group was (16.50±11.04)mm2, and the PBS group was (52.50±16.94) mm2（n=6）. Statistical difference existedbetween the AaHⅣ immunized group and the non-catalytic domain immunized group（P＜0.05），but the AaHⅣ immunized group and the catalytic domain immunized group（P＞0.05）, all of the three groups were respectively different from the PBS group（P＜0.001）.⑤The challenge experimentGroups of immunized mice were intraperitoneal injected with Agkistrodon acutuscrude venom2LD50. After24hours, the numbers of dead mice of the Agkistrodon acutuscrude venom group was1, the AaH Ⅳg roupwas3, and the PBS group was0(n=10).According to Fisher’s Exact test, there was no mortality difference between the crudevenom group and the AaH Ⅳg roup(p=0.58). Statistical difference existed between the crudevenom group and the PBS group（p＜0.01）, also between the AaH Ⅳgroup and the PBSgroup（p＜0.01）.Groups of immunized mice were challenged by intraperitoneal injection with AaHⅣ2LD50. After24hours, the numbers of survive mice from catalytic domain group was8, thenon-catalytic domain group was2, the AaHⅣ group was9, and the PBS group was0(n=10).There was no mortality difference between the catalytic domain group and the AaHⅣgroup(p＞0.05).But statistical difference existed between the catalytic domain and thenon-catalytic domain group(p＜0.05)，the non-catalytic domain group and the AaH Ⅳgroup（p＜0.01).4. Conclusion①A aHⅣ could be one of the immunogens which were used to prepare an adequateantiserum, since its distinct immunogenicity and protective effects against crude venom tomice.②The catalytic domain of AaHⅣ could induce body immunity and provide protection, as well as AaH Ⅳ did. It could be a part of the immunogens which were used to prepareantiserum.