Experimental Study Of Targeting C3d Molecular Complement Inhibitors For The Treatment Of Severe Pneumonia Caused By Influenza

Objective: In this study,we established a lethal model of influenza-induced severe pneumonia in mice by expressing and purifying two targeted complement inhibitors,C3 dScFv-DAF and C3d-ScFv-CD59 and conducted an experimental study to explore a new strategy for the treatment of severe pneumonia in influenza.Methods: Firstly,high concentrations of C3d-ScFv-DAF and C3d-ScFv-CD59 fusion proteins were purified by AKTA system expression,and their protein activities were determined by ELISA affinity assay,and the toxicity effects of the two fusion proteins on mice were identified at different concentrations;secondly,a mouse model of severe pneumonia was constructed by using specific concentrations of influenza virus nasal drip.Finally,two targeted complement inhibitors,C3d-ScFv-DAF and C3d-ScFv-CD59,were injected into the tail vein of the influenza virus-infected severe pneumonia mouse model,and the PR8 group,PBS group,DAF group(3.75μg/each),CD59 group(14.65μg/each),C3d-ScFv-CD59 group(60μg/each),C3d-ScFv-CD59 group(60μg/each),and C3d-ScFvDAF group(15μg/each),and the therapeutic effects of both were investigated by observing their body weight change,survival rate,lung pathological characteristics(HE staining),DAF and CD59 expression(immunohistochemistry),and other indicators.Results:(1)The results of ELISA experiments identified by protein activity showed that with the increase of C3d(sigma),C3d(quidel)concentration,the OD value of its binding reaction with C3d-ScFv-DAF and C3d-ScFv-CD59 showed an increasing trend,indicating that C3d-ScFv-DAF and C3d-ScFv-CD59 fusion proteins were able to bind to C3d(sigma),C3d(quidel),respectively,and both had high biological activities.(2)The toxicity experiments of C3d-ScFv-DAF and C3d-ScFv-CD59 on mice showed that the body weight of mice after administration tended to increase within the observation days and 100%survived.The results of immunohistochemistry as well as HE is staining showed no significant changes in the lung tissue structure and the appearance of tissue inflammation and pulmonary edema and hemorrhage in the mice compared to the control group(PBS group).(3)In the targeted complement therapy experiments,an influenza virus infection mouse model was successfully constructed under the existing natural environmental conditions,and the survival rate was significantly improved by treating the influenza virus infection model with different doses of C3d-ScFv-DAF and C3d-ScFv-CD59,respectively,at 15 μg/each(C3d-ScFv-DAF)and 60 μg/each(C3d-ScFv-CD59).C3 dScFv-CD59)doses,respectively;the C3d-ScFv-DAF and C3d-ScFv-CD59 treatment groups showed significantly higher survival rates,significantly reduced lung pathological damage,significantly reduced complement activation product C3 d,and targeted complement inhibitors in the lung lesion compared to the effector molecules DAF and CD59 treatment groups alone and the blank control group,respectively.regions.Conclusions:(1)By targeting the complement inhibitors C3d-ScFv-CD59 and C3d-ScFvDAF after injection had prolonged the survival rate of mice with severe influenza infection and significantly suppressed the persistent weight loss in mice.(2)The targeted complement inhibitors C3d-ScFv-CD59 and C3d-ScFv-DAF significantly inhibited the inflammatory response in the lungs of mice infected with influenza virus followed by excessive activation of complement in the lungs of mice and alleviated the inflammatory response.(3)Targeted complement inhibitors C3d-ScFv-CD59 and C3d-ScFv-DAF effectively alleviated the severe pathological damage in mouse lungs and reduced edema,hemorrhage,and inflammation in mouse lung tissues.reduced the mortality of severe pneumonia,relative to mice in the infected group.