Monoclonal Antibodies Recognize at Least Five Epitopes on the SIV Nef Protein and Identify an in Vitro-Induced Mutation

Abstract

Eleven monoclonal antibodies (MAbs) to SIV Nef were produced and characterized. Five antibody-binding sites on SIV Nef were identified on the basis of the reactivity of the antibodies with recombinant proteins. Two of the five epitopes were defined using overlapping peptides. A further three epitopes could not be defined with peptides but all antibodies reacted in Western blot, suggesting that the epitopes were at least partially conformation dependent. Antibodies in two of the five epitope groups were further differentiated by competition analysis. The panel of MAbs described is able to distinguish between a number of recombinant Nef proteins currently under investigation in vivo in macaques. Two of the MAbs described are able to distinguish between the Nef protein from pathogenic (J5) and attenuated (C8) strains of SIV, thus providing useful tools for studying the relevance of the Nef protein in the pathogenesis of SIV infection. In FACScan analysis two of the MAbs, KK70 and KK75, were used to identify an in vitro-induced mutation in J5 Nef grown in C8166 cells. Sequence analysis of the phenotypic variants identified a mutation of the tryptophan (TGG) at amino acid 214 to a stop codon (TGA), thus truncating the Nef protein. The functional significance of this observation remains unclear but highlights the need to interpret data with caution if virus has been cultured in vitro even for a short period of time.

Neuropathology of wild-type and nef-attenuated T cell tropic simian immunodeficiency virus (SIVmac32H) and macrophage tropic neurovirulent SIVmac17E-Fr in cynomolgus macaques

“…Sections were incubated in 50 μg mL −1 proteinase K (Roche, Lewes, UK) in PBS pH 7.4 for 15 min at 37°C prior to immunolabelling with CD3, CD8 (C8/144B), CD68 (KP1), GFAP (astrocytes), CD163 (GH1/61; Santa Cruz Biotechnology Inc, California, USA) and CCR5 (3A9; Pharmingen, Oxford, UK). Sections were heated at full power (800 W) for 5 min fully immersed in Vector unmasking solution (Vector Laboratories, Peterborough, UK) previously heated to 96°C for immunolabelling of CD4 (H370, Santa Cruz, Autogen Bioclear, Wiltshire), CNPase1 (oligodendrocytes, 11-5B) (Neomarkers), FF1 (phosphorylated neurofilaments, Dr E Gardner, William Paterson University, USA) or 10 mM citrate buffer (pH 6), KK41 (SIV gp41, Kent et al 1991), KK75 (SIV Nef, Arnold et al 1999). Sections were treated using a MenaPath pressurised antigen access unit (125°C 30 s, 90°C 10 s, Access Super solution) for immunolabelling with iba-1 (microglia; Menarini Diagnostics).…”

Macaques infected long-term with attenuated simian immunodeficiency virus (SIVmac) remain resistant to wild-type challenge, despite declining cytotoxic T lymphocyte responses to an immunodominant epitope

“…Virus isolation was performed by co-cultivation with C8166 cells, either at a single concentration of 10 6 MNCs or by limiting dilution to determine cell-associated virus load, as described previously (Cranage et al, 1998). Immunofluorescent staining with the monoclonal anti-Nef antibody KK75, which binds only to the intact Nef protein that is present in the wild-type challenge virus (Arnold et al, 1999), was used to distinguish challenge virus from attenuated virus.…”

In situ hybridization and immunolabelling study of the early replication of simian immunodeficiency virus (SIVmacJ5) in vivo

“…To complement ISH, immunolabelling techniques were developed using anti-SIV envelope (KK13; Kent et al , 1991 ) and Nef (KK75; Arnold et al , 1999 ) MAbs. Attempts to use MAbs KK59 and KK61 (Kent et al , 1991 ) to detect specifically SIV Gag protein were unsuccessful, as high levels of binding were observed with these antibodies on sections of lymph node processed from uninfected macaques (data not shown).…”

“…Once the sections had cooled to room temperature, they were rinsed in TBS (20 mM Tris–HCl, pH 7·5, 225 mM NaCl) and immersed in TBS containing 5% (w/v) non-fat milk powder for 60 min at room temperature. Monoclonal antibodies (MAbs) of the IgG1 isotype to SIV Gag p17/p27 (KK59/KK64; Kent et al , 1991 ), envelope (KK13; Kent et al , 1991 ) and Nef (KK75; Arnold et al , 1999 ) were respectively diluted 1:50, 1:2 and 1:50 in TBS containing 1% (w/v) BSA, added to the section and incubated overnight at 4 °C. After extensive washing in TBS, bound antibodies were visualized by using a biotinylated universal horse anti-mouse/rabbit antibody (Vector Laboratories) and an avidin/biotinylated horseradish peroxidase complex (ABC, Vector Laboratories) using 3,3′-diaminobenzidine tetrahydrochloride (DAB) as chromogenic substrate.…”