Study On Recombinant Human Antibodies And Funcational Epitopes

Antibody phage display is the most common way to generate human antibodies and has already yielded thousands of recombinant antibodies for research, diagnostic and therapy. Phage display tenchonoly can dsiplay the scFv or Fab antibodies on filamentous phage by fusing the antibody of interest on to gene Ⅲ of filamentous phage. After panning, it allows the selection of human antibodies and the corresponding genes from human antibody gene libraries. Here, we describe methods for the construction and selection of human scFv gene libraries for rabies virus and SFTS bunyavirus (SFTSV).1. Study on recombinant human antibody cocktail against rabies virus.Rabies is a zoonotic viral disease caused by rabies virus with mortality rate of almost 100% after onset. Rabies virus is enveloped Rhabdoviridae Lyssavirus and has 5 types of structural proteins, of which the glycoprotein (G) is closely related to virulence and pathogenicity of the virus, and can induce the production of neutralizing antibodies with major neutralizing epitopes Ⅰ,Ⅱ and Ⅲ. According to the World Health Organization recommendation, rabies can be prevented by post-exposure prophylaxis, through the combined administration of a rabies vaccine and rabies immune globulin (RIG). However, the existing usage of human rabies immune globulin (HRIG) and equine rabies immune globulin (ERIG) has been limited for several years, so the anti-rabies virus monoclonal antibodies (mAbs) neutralizes efficiently with high safety, low-cost, mass production, so it may be able to replace RIG for rabies post-exposure prophylaxis.In this study, the lymphocytes from the blood of vaccinated donors were isolated and the cDNAs was synthesized with using primer oligo-dT. The antibody variable genes of the heavy and light chain were amplified by a set of human IgG scFv antibody primers. The gel purified light chains and heavy chains were cloned into phagmid vector pHAL14, and a phage antibody library was constructed. After phage panning and selection using purified rabies virus,5 human scFv antibodies (RV1C3、 RV2G12、RV1C5、RV1A11、RV2F5) against the rabies virus glycoprotein were obtained. The five full human IgG antibodies were epressed in 293T cells with the VH/VK vectors, and further tested by SDS-PAGE、ELISA and IFA. The affinity of the 5 IgG were up to 10-9M through the identification of non-competitive ELISA. The rapid fluorescent focus inhibition test (RFFIT) showed the 5 IgG have a high neutralizing activity to rabies virus aG strains but not to CVS strains. The epitope was further mapped by competitive ELSA and site-directed mutations. The results showed that the relationship between 5 antibodieswere against antigenic site Ⅱ of rabies virus glycoprotein.Human mAb CR4098, as an anti-rabies virus neutralizing antibody, was determined against the conformational antigenic site Ⅲ (residues 330-338). We further chose scFv phage display technology to optimize CR4098 with chain shuffling. After phage panning and selection using purified rabies virus,14 human scFv antibodies against the rabies virus glycoprotein were obtained. Further tested showed that RV3A5 has a high affinity of 2.8 X 10"9M and high neutralizing activity to rabies virus both aG strains and CVS strains. The epitope was further mapped by competitive ELSA and site-directed mutations. The results showed that RV3A5 was against site Ⅲ.We chose three monoclonal antibodies CR57, RV08 and RV3A5, which were against antigenic site Ⅰ,Ⅱ,Ⅲ of rabies virus glycoprotein individually, tested the postexposure prophylaxis acitivity. After challenged with rabies virus CVS strains, Syrian hamsters were treated with mAbs. The result showed that all three antibodies can protect animals from rabies virus, revealing a good activity for postexposure prohpylaxis.In summary, we generatedhuman scFv antibodies against rabies virus using phage antibody library. Epitope mapping and functional study showed a candidate for the antibody cocktail against rabies virus. Study on human recombinant antibody has major implications for replacing RIG for rabies post-exposure prophylaxis.2. Critical Epitopes in the Nucleocapsid Protein of SFTS Virus Recognized by a Panel of SFTS Patients Derived Human Monoclonal AntibodiesSFTS bunyavirus is a newly discovered pathogen to cause severe fever with thrombocytopenia syndrome (SFTS) in human. Successful control of SFTS virus epidemic requires better understanding of antigen target in humoral immune responses to the new bunyavirus infection.We have generated a combinatorial Fab antibody phage library from two SFTS patients recovered from SFTS bunyavirus (SFTSV) infection in Shandong province of China. To date,94 unique human antibodies have been generated and characterized from over 1200 Fab antibody clones obtained by screening the library with SFTS purified virions. All of those antibodies recognized the nucleocapsid (N) protein of SFTSV while none of them were reactive to the viral glycoproteins Gn or Gc. In addition, over screening 1000 mouse monoclonal antibody (MAbs) clones derived from SFTSV virions immunization,462 clones reacted with N protein, while only 16 clones were reactive to glycoprotein. Furthermore, epitope mapping of SFTSV N protein was performed through molecμlar simμlation, site mutation and competitive ELISA, and we found that at least 4 distinct antigenic epitopes on N protein were recognized by those human and mouse MAbs; the first 10 amino acids at N terminal and last 5 amino acids at C terminal of N protein were critical for the antigenic function of the protein, in particμlar mutation of Glu10 to Ala abolished or significantly reduced the binding activity of nearly most SFTS patients derived MAbs.The large number of human recombinant MAbs derived from recovered SFTS patient dominantly recognized the viral N protein indicated that the nucleoprotein was an important antigen in humoral immune responses to new bunyavirus infection, and the amino acids positioned N terminus was critical for antigenicity of N protein as a dominant Epitope.