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

Abstract

The 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 microg/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.

Fig. 1

Schematic of the cloning of the immunoglobulin variable region genes from the hybridoma cells and construction of the scFv gene expression vector.

Fig. 2

The nucleotide and amino acid sequence of the anti-HIV-1 gp120 scFv. (a) The variable regions of the heavy chain (V_H). (b) The variable regions of light chain (V_L). The amino acid numbering and complementarity determining regions of the V_H and V_L domains were determined by comparison with other published variable regions of immunoglobulins stored in the GenBank database.

Fig. 3

Expression and purification of recombinant scFv 102. scFv 102 was induced using IPTG and could be purified by metal affinity chromatography (IMAC). Coomassie-blue-stained SDS-polyacrylamide gel analysis revealed hyperexpression and purification of scFv102. Lane 1 — protein molecular weight standards. Lane 2, total cell extract (E. coli DE3/pET-scFv induced with IPTG); Lane 3, metal affinity column flowthrough; Lanes 4–6, column fractions from purification (50 mM imidazole wash); Lanes 7–8, purified scFv was visible in the peak fractions from the gradient (200 mM imidazole wash).

Fig. 4

Determination of the binding specificity of mAb102 and scFv102 to HIV-1 antigens. (a) The strip immunoblot assay. The same amounts of protein were incubated with each strip. (1) Molecular markers of HIV-1 antigens; (2) mAb102; (3) scFv102. Both antibodies exhibited the same binding specificity in the immunoblot assays. (b). Indirect immuno-fluorescence assays obtained by staining Hela-Env cells with: (1) mAb 102 (2) negative control antibodies (murine IgG) and (3) expressed scFv 102. Incubation of Hela cells with the same antibodies did not yield a detectable immuno-fluorescence signal (data not shown).

Fig. 5

Indirect enzyme immunoassay. ▴ mAb, scfv. The absorbance values were normalized to B/B₀ (%) according to the following formula: B/B₀ (%) = 100 A/A0, where A is the absorbance value for the sample and A₀ is the absorbance value of the positive control.

Fig. 6

Antibody neutralization assays. Neutralization of HIV-1 SF2 by scFv () and the parent mAb (▴). Neutralization titres were determined using the formula (p24control—p24test)/p24control × 100.