Isolating And Expressing Human Antibodies Of HIV On Yeast Surface Display System

Recently, it has been a great challenge for the clinical researchers to find solutions to prevent and defeat HIV (Human Immunodeficiency Virus). As HIV has the character of high variation, many obstacles have to be tackled for finding broadly neutralizing antibodies (nAbs). This work focuses on designing efficient experiment platform and solution to isolate and express the specific antibodies targeted at HIV. During our work, two issues have to be solved:how to pick the target antigen and how to display the antibody library?For the first issue, previous research works have shown that most of the viral antigen epitopes corresponding to these antibodies were located in some of the conserved sites on envelope protein gpl20/gp41of HIV by analyzing known neutralizing antibodies. Based on the observation, a unique solution has been proposed to pick the target antigen by the researchers in our group, which considers designing and synthesizing the antigen peptide according to the conserved epitopes of the virus. Specifically, this work focuses on designing target antigen on the epitope of a broad nAb,10E8-a conserved region of gp41membrane-proximal external region (MPER) and synthesizing some peptides based on it. These peptides can be modified to act as the actual antigens and used to scan the antibody library.To solve the second issue, the optimal surface display technique has to be found. This work adopts the well-known yeast surface display system. The reasons are twofold. On one hand, the yeast cell is a specific kind of eukaryotic cell, which has the inherent advantages in post-translational modifications of proteins and mammalian protein process. On the other hand, it is also much easier for the yeast cell to be cultured and screened during experiments. In addition, we orchestrate single chain fragment variable (scFv) library displayed on the surface of yeast, which makes it much easier for genetic manipulation and expression. In our experiment, A scFv library with109non-immune human antibodies scFv fragments has been displayed on yeast surface and the target genes for broadly neutralizing antibodies can be extracted.The contribution of our work is the experiment platform, which contains three major steps. First, the scFv library displayed on yeast surface is screened by two rounds of magnetic activated cell sorting (MACS) and two rounds of Fluorescence activated cell sorting(FACS). Second, we select30strains for gene sequencing randomly from the screened antibody library and receive12individual clones with different gene sequences. By identifying received clones, we get4positive clones, which have to be secreted and purified further. Last, we construct the expression vector. After expressing and verifying the full-length antibodies, the full-length antibodies still can be bound to the peptides. Experimental results show that we successfully isolate and express the antibodies specifically bound to the peptides on our experimental platform, which can isolate and engineer monoclonal antibodies against target antigen from a non-immune scFv library displayed on the surface of yeast. This solution has profound influence in developing a universal vaccine. In the future, researchers can find required peptide used to isolate broadly neutralizing antibodies by repeatedly designing and modifying the peptides. The optimal peptide can be a vaccine candidate and guide the further development of a universal vaccine.