Engineering and functional evaluation of a single-chain antibody against HIV-1 external glycoprotein gp120

Abstract

SummaryThe HIV-1 envelope glycoprotein surface subunit gp120 is an attractive target for molecular intervention. This is because anti-HIV-1 gp120 neutralizing antibodies display the potential ability to inhibit HIV-1 infection. The present investigation describes the construction of a genetically engineered single chain antibody (scFv102) against HIV-1 gp120, its expression and functional evaluation. The parental hybridoma cell line (102) produces an immunoglobulin directed against the conserved CD4-binding region of gp120. cDNAs encoding the variable regions of the heavy (V H ) and light (V L ) chains were prepared by reverse transcription PCR and linked together with an oligonucleotide encoding a linker peptide (Gly 4 Ser) 3 to produce a single chain antibody gene. The resulting DNA construct was cloned into a prokaryotic expression vector (pET28) and recombinant scFv102 was expressed in Eserichia coli as an insoluble protein. The denatured scFv102 was refolded and purified by immobilized metal ion affinity chromatography. Purified scFv102 had the same specificity as the intact IgG in immuno-blotting assays and immuno-fluorescence (IF) detection, but ELISA analyses demonstrated the affinity of scFv102 to be 5-fold lower than that of the parental monoclonal antibody. In neutralization assays, scFv102 at concentrations lower than 40 m g/ml exhibited efficient interference with viral replication and inhibition of viral infection (90%) across a range of primary isolates of subtype B HIV-1. These results suggest that the constructed anti-HIV-1 gp120 scFv102 has good biological activity and can potentially be used for in vitro diagnostic and in vivo therapeutic applications.

Comparative Modeling: The State of the Art and Protein Drug Target Structure Prediction

“…Antibodies represent a special case [106] for which the relationship between the sequence and structure of the functional site is well defined. This has allowed the rational design of new antibodies with different biochemical properties, which have had broad application in therapy and research [107][108][109][110][111]. More complex is the prediction of enzyme structures to be subsequently used in the design of enzyme inhibitors.…”

Neutralizing human recombinant antibodies against herpes simplex virus type 1 glycoproteins B from a phage-displayed scFv antibody library

“…Thus far, several neutralizing scFv antibodies have been produced against a number of viruses including scFv antibodies against human immunodeficiency virus type 1 (HIV-1) [18], scFvs to human cytomegalovirus [19] and scFv against influenza A virus H5N1 subtype [20] that can inhibit viral infections in vitro. Nejatollahi et al described a neutralizing scFv-phage antibody against glycoprotein D of HSV-1 with neutralizing effect of 76%, which was capable of neutralizing HSV-1 and inhibiting virus entry to host cell [21].…”

Expression of Human Immunodeficiency Virus Type 1 Neutralizing Antibody Fragments Using Human VaginalLactobacillus

“… 15 Numerous scFvs and dAb variants and camel-derived antibodies have now been identified that retain the broadly neutralizing anti-HIV activity. 16–22 Among these broadly neutralizing antibody fragments are ScFv-m9, 23 , 24 dAb-m36, 25 , 26 and dAb-36.4 variant 19 that bind CD4i regions and facilitate neutralization of a broad range of HIV isolates.…”