Recombinant rabbit single‐chain antibodies bind to the catalytic and C‐terminal domains of HIV‐1 integrase protein and strongly inhibit HIV‐1 replication

Abstract

The human immunodeficiency virus type 1 (HIV-1) integrase (IN) protein plays an important role during the early stages of the retroviral life cycle and therefore is an attractive target for therapeutic intervention. We immunized rabbits with HIV-1 IN protein and developed a combinatorial single-chain variable fragment (scFv) library against IN. Five different scFv antibodies with high binding activity and specificity for IN were identified. These scFvs recognize the catalytic and C-terminal domains of IN and block the strand-transfer process. Cells expressing anti-IN–scFvs were highly resistant to HIV-1 replication due to an inhibition of the integration process itself. These results provide proof-of-concept that rabbit anti-IN–scFv intrabodies can be designed to block the early stages of HIV-1 replication without causing cellular toxicity. Therefore, these anti-IN–scFvs may be useful agents for “intracellular immunization”-based gene therapy strategies. Furthermore, because of their epitope binding characteristics, these scFvs can be used also as new tools to study the structure and function of HIV-1 IN protein.

From rabbit antibody repertoires to rabbit monoclonal antibodies

“…The first rabbit antibody library selected by phage display was reported by Ridder et al , 143 using a scFv format as in subsequent independent studies. 39, 40, 144, 145, 146, 147, 148, 149, 150, 151 Rabbit antibody libraries in Fab format followed in short succession. 152, 153 Due to the higher expression levels of human compared to rabbit constant domains in bacteria, a chimeric rabbit/human Fab format consisting of rabbit variable domains VL and VH recombinantly fused to human constant domains CL and CH1, respectively, proved particularly successful for the selection of rabbit mAbs by phage display 19, 154, 155, 156, 157, 158, 159, 160 and their subsequent humanization (Figure 5).…”

Antigenic response to CT‐P13 and infliximab originator in inflammatory bowel disease patients shows similar epitope recognition

“…For biopanning experiments, 14 samples of anti‐CT‐P13 and 10 samples of anti‐infliximab patient sera were applied to the wells of Nunc polystyrene 96‐well microtitre plates (Nunc, Denmark) . Plates were incubated at room temperature for 2 hours to allow antibody binding before washing with 0.05% PBS‐Tween 20 (v/v).…”

“…Phages displaying infliximab peptides required for analysis in phage ELISA were prepared from individual bacterial clones as described elsewhere . Wild‐type VCMS13 helper phages were included in each assay as a control for background antibody binding.…”

“…cDNA was synthesised using the SuperScript Pre‐amplification System (Thermo Scientific, USA) and used as a template for PCR amplification of the human light and heavy gene fragments. A phage library displaying human Fab antibodies was constructed in pComb3X according to published protocols with minor modifications . Biopanning of the Fab library was carried out on purified CT‐P13 (Celltrion, South Korea) using standard protocols .…”

“…A phage library displaying human Fab antibodies was constructed in pComb3X according to published protocols with minor modifications . Biopanning of the Fab library was carried out on purified CT‐P13 (Celltrion, South Korea) using standard protocols . For phage ELISA, single colonies of anti‐CTP13 Fab clones were used to grow phage‐Fabs in three 96‐well culture plates followed by superinfection with diluted VCSM13 helper phage.…”