Bispecific Antibody Directed Against Both Virus And Membrane Bound Complement Regulator Enhances Virus Lysis By Complement

Complement is one of the first line of the host defence to be faced and countered by viruses as they struggle to establish an infection. As an important innate immune system, and as an important arm of the humoral immune response, the complement system is immediately ready to target and eliminate virus particles ,to lysis those virions that have lipoprotein membranes,or to prevent it from entering host cells,or to marker them for destruction by other branch of the immune response. At the same time, the host normal tissue are protected from damaging by complement through recognizing the regulators of complement activation (RCA) expressed on self cells. In general, microorganisms lack mammalian RCA and thus cannot restrict complement deposition and attack. Nevertheless, the immune system and viruses have substantially evolved together and viruses demonstrate many stratiges for subverting immune response. For enveloped virus , for instance HIV and EEV can incorporate host RCA into their envelope by budding through the plasma membrane, and these protect the virion against host complement attack. The subversion strategies may be the targets of antiviral therapy and therefore well worth exploring. So it is interesting to know whether target block mRCA on virus envelope could improve the the anti-virus efficacy of complement ?To answer this question, a bispecific ,trifunctional antibody constructs which can not only target block virus incorporated RCA, but also can induce complement activation by it's Fc fragment were designed and constructed andIVthe role of this kind of bispecific antibody in virus neutralization was studied .1. To test our idea, Human Immunodeficient Virus (HIV) and Enveloped Extracellular Virus (EEV) of Vaccinia Virus (VV) were selected in our study because of their complex immune evasion stratiges, their threaten to humans, and because both these two kinds of virus can escape complement attack by incorporating host RCA into their envelope. First, the expression of CD55,CD59 on human T cell line MT4,the host cell of HIV, and on human epithelia Hela cell, the host cell of EEV was detected with FACS. Then the incorporation of CDS5 and CD59 on EEV envelope were detected by immunoblot analysis. Results showed that both moleculars expressed on MT4 and Hela cells. Host cellular protein CD55, CD59 are incorporated into EEV out membrane as showed in immunoblotting assay. Finally, in order to investigate whether these two virus are resistant to complement attack, and whether the resistance were mediated by incorporating cellular complement regulators of CD55, CD59 into their out membrane, complement lysis assay and plaque reduction assay were carried out respectively to find the lysis or neutralization activity of complement to HIV and EEV under the condition of with or without the CD55 and CD59 blocking antibody. Results showed that the lysis or neutralization activity of complement to HIV and EEV were significantly enhanced provided that the CD55 and CD59 blocking antibody were added. From these results we can conclude that host cellular protein CD55, CD59 are incorporated into EEV and HIV out membrane and mediated virus resisitance to complement attack. At the same time, the results suggest that virus incorporated CD55 and CD59 can be used as the target in the strategy aimed at target blocking RCA on virions and enhancing the anti virus effects of complement.2. The specificity and affinity and antibody are the key points of antibody based immunotherapy for target andtibody binding and for expected effects.The results of the first part showed that both CD55 and CD59 neutralizing antibody can enhance the lysis or neutralization activity of complement. However, since RCA are distributed widely, techniques that can mediate target RCA blocking on virions are necessary. Fortunately, recently widely used bispecific antibody technique provided us a good model for target RCA blocking for it's dual antigen binding activity and for the availab...