Niche Of Antigenic HIV-1 Broadly Neutralizing Antibodies PGT135 Structure And Recognition

Human Immunodeficiency Virus (HIV) has two major types HIV-1and HIV-2. HIV infection is largely caused by HIV-1and more than ninety percent of the infected persons will develop into AIDS in10to20years. During infection, HIV-1can easily escape from the monitoring of human immune system through rapid and continuous sequence evolution. Designing efficacious HIV vaccine is a dream for human but so far it has been difficult to elicit broadly neutralizing antibodies (bnAbs) by vaccination. Despite great advances have been made in controlling HIV-1infection with antiretroviral drug treatment, a safe and efficacious HIV vaccine has yet to be developed. Broadly neutralizing antibodies can only be produced in tiny fraction of infected individual during the natural infection. VRC01class antibodies, the PT series antibodies and PGT series antibodies are the good examples and they are able to neutralize significant proportion of epidemic HIV-1strains in the world. However, these bnAbs are usually generated after several months and even years of infection,making them useless in clearing the latent virus established early during infection. Through structure and function studies of the bnAbs and the HIV-1envelope, we learn a great deal about bnAbs and their conserved epitopes on the HIV-1envelope, which may help us to rationally design vaccines. In addition, bnAbs can be applied to treat and prevent HIV-1infection through passive immunization.In this thesis, I conducted a series of studies on one of HIV-1bnAbs, PGT135. Gene encoding PGT135was optimized based on human codon usage and large scale production and purification of PGT135was conducted in293T cells. We also performed crystal structure analysis of PGT135. We analyzed sensitivity of Chinese epidemic strains of HIV-1to PGT135neutralization. To map the epitope of PGT135, we used random antigen fragment library and random mutant library of HIV-1envelope displayed on the yeast surface display. Through staining and sorting by fluorescence-activated cell sorting (FACS), mutant antigen fragments that lost binding to PGT135were selected and used to build pseudovirus for identification of key residues of PGT135epitope. Finally, I also screened the nonimmune human scFv yeast library with the fragments recognized by PGT135in a hope to select PGT135like bnAbs against HIV-1.I successfully solved the crystal structure of PGT135. Like many other bnAbs, PGT135had long CDRH3, and it was even longer than VRC01class antibodies. The long CDRH3is believed to be useful in recognizing shielded glycosylation sites. I also found that PGT135can neutralize most of the Chinese epidemic strains of HIV-1, suggesting most of the Chinese HIV-1had the PGT135epitope. Using the random antigen fragment library on the yeast surface, I found the epitope of PGT135was localized between the V3loop and V4loop of HIV-1envelope glycoprotein gp120. I went further to fine mapping epitope recognized by PGT135with random mutant library on the yeast surface and identified several key residues that could significantly changed viral sensitivity to PGT135neutralization. In particular, I found that deleting single glycosylation site could completely abolish viral sensitivity to PGT135neutralization. Finally, by screening the nonimmune human scFv yeast library with PGT135recognized fragment, I obtained some antibodies that were able to bind to the PGT135epitope.I believe that results obtained in my thesis will help us to better understand the structure and function of bnAb PGT135at atomic levels, which will provide critical reference for the development of therapeutic and preventative measures against HIV-1infection.