Research Of Humanized Antibody Against Human Cytomegalovirus

Goal and Significance Human cytomegalovirus is a wide-spread virus which is found in 1956 by Smith and named in 1964. It is the fifth member of herpes virus family with double linear DNA chain. HCMV is an opportunistic pathogen that establishes life-long latent infection without clinical disease in immunocompetent individuals, but can cause severe illness in newborns, transplant recipients, and patients with HIV. So that research of prophylaxis and therapy of human cytomegalovirus infection are paid more and more attention. The life cycle of human cytomegalovirus repeats as adhesion, penetration, replication and release. The virus evolve a serial of mechanism to escape from immune system and keep latent infection. The adhesion is the start point in virus infection and diffusion. It is a very essential step to block adhesion and penetration in virus infection. Glycoprotein B is abundant in envelope membrane of HCMV more than 55%. Glycoprotein B consist of two parts as gp55 and gp116. Glycoprotein B play an important role in virus adhesion and penetration. Neutralizing antibody against glycoprotein B can block penetration and diffusion of virus under electron microscope. There are three linear epitopes showing antigenicity of gB, two of which are target of neutralizing antibody. Antigen determinant 1, covering between amino acid residues 552 and 635, was identified at first. More than 50% of the subjects infected with HCMV are positive to this domain in serum IgG test. And about 30% is positive to antigen determinant 2, which is located between amino acid residue 50 and 86. Data above show that glycoprotein B is an important target in prophylaxis and therapy of HCMV infection. Drugs currently used in clinic include ganciclovir, valganciclovir and cidofovir et al, most of which are analog or derivates of nucleotide. These drugs prevent replication of virus and host cell at the same time, which are shown side effects obviously. Thus, more specific drugs are now emerging. With the development of gene engineering, humanized antibody becomes one of promising drug candidate in prophylaxis and therapy of virus infection. Materials and Protocols In this study, two predicted epitopes P1 and P2 were used as target, which are located in antigen determinant 1 and 2, respectively. Lymphocytes are collected from peripheral blood of subjects who were positive in serum test of HCMV IgG by ELISA. RNA was abstracted separately from lymphocytes of positive subjects. The gene fragments of antibody heavy and light chain variable region were amplified by RT-PCR. Scfv was obtained from overlap PCR. Phage display antibody library was constructed, and then panning the library with P1 and P2 was carried out. The positive clones were selected, affinity inspection of which was followed by ELISA , dot-blot and western-blot. Finally, those with affinity and specificity to targets P1 and P2 were sequenced. Main results 1. Peripheral blood samples from 216 individuals were collected. There were 159 positive samples and 57 negative samples in serum test of HCMV IgG. 2. RNA integrality was kept well shown in denaturing gel electrophoresis. ?-actin was used as the control of reverse transcription process. Amplified band of ?-actin was observed clearly, indicating the success in RT-PCR. The code fragments of antibody heavy and light chain variable region RT-PCR were observed about 400bp and 350bp, respectively. After acquirement of scfv fragment by overlap PCR, phage display antibody library, size about 4.5*108, was constructed. 3. Five genotype of glycoprotein B were searched in GenBank. After sequence alighment by Cluster program, consensus region was identified. B cell epitope of glycoprotein B of AD169 strain was predicted with combination of the information of hydrophobicity, accessibility, antigenicity, second structure and date shown in reference. Two epitope P1 TEECQLPSLKIFIAG(607-621) and P2 NETIYNTTLKYGDVV (70-84) were designed and synthesized. 4. There were 44 posotive phage antibody against P1 but no positive results to P2. After sequenced, five strains of phage antibody, named P1-24,P1-25,P1-26,P1-28 and P1-29 respectively, were obtained. Four strains of phage antibody, capable of binding both P1 epitope and whole glycoprotein B molecule, were identified. DNA sequence were submitted to GenBank. The accession number are DQ001147,DQ001148,DQ001149,DQ001150. Discussion From monoclonal antibody to phage display antibody library and to transgene antibody technology, Antibody provided a powerful engine in development of modern medical science. Monoclonal antibody combinate the specifity of B cell and infinitive proliferation of lymphoma cell; phage display antibody library provide access to get antibody diversity in vitro; transgeneantibody can make humanized antibody in animal. All three pathway have its advantages and disadvantages. The central problem in antibody therapy is humanization in order to reduce its antigenicity to human. Antivirus is a important field in antibody application. Humanized antibody is in the ascendant and is being paid more and more attention. Block of virus adhesion is the prior way in prophylaxis and therapy of virus infection. We have obtained 4 strains of humanized antibody against epitope P1 of HCMV gB in this research, which lays solid basis to develop drug candidate against human cytomegalovirus in the future.