In vitro selection and characterisation of human anti-HIV-1 antibody fragments

• Main Author: Tang, Jie
• Published: University of Westminster 2011
• Subjects: 572.8
• Online Access: https://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.540190

Generation of neutralising antibodies with broad specificity would be one of the effective approaches to control HIV-1 spread. It is clear that a method that allows rapid generation of neutralising antibodies is needed. This project aims at developing a novel approach to rapidly access human anti-HIV-1 antibodies in vitro by using ribosome display and selection from DNA libraries of HIV-1 patients. Two single-chain antibody libraries (M325 and K530) were constructed from two HIV-1 long-term non-progressors, whose sera showed cross-neutralising activities against various HIV-1 strains across a range of clades. In each library, total RNA was extracted from blood of each donor and used to synthesise cDNA. Families of 4 κ light chains, 9 λ light chains and 8 heavy chains were generated by using RT-PCR amplification. These fragments were then assembled with all possible combinatorial pairs to form diversified repertories in the form of VL-link-VH-partial CH. Both libraries were subjected to ribosome display for in vitro selection of functional antibodies. Ribosome display is a cell-free technique used to generate proteins that can bind to an immobilised antigen. During this process, the translated proteins are associated with their mRNAs, enabling a simultaneous selection of functional proteins and their gene. The employment of ribosome display facilitated rapid screening of two large libraries against recombinant gp120 (generated from patient K530).