A novel antibody-dependent cellular cytotoxicity epitope in gp120 is identified by two monoclonal antibodies isolated from a long-term survivor of human immunodeficiency virus type 1 infection

Abstract

Two monoclonal antibodies (MAbs), 42F and 43F, were isolated some 14 months apart from a single long-term survivor of human immunodeficiency virus type 1 (HIV-1) infection. These MAbs were found to be indistinguishable in terms of their isotypes, specificities, affinities, and biological activities. Both 42F and 43F directed substantial antibody-dependent cellular cytotoxicity (ADCC) against cells infected with four divergent lab-adapted strains of HIV-1, but no neutralizing activity against these strains was detectable. The ability of MAbs 42F and 43F, as well as that of MAbs against two other gp120 epitopes, to direct ADCC against uninfected CD4 ؉ cells to which recombinant gp120 SF2 had been adsorbed (i.e., "innocent bystanders") was demonstrated to be less efficient by at least an order of magnitude than their ability to direct ADCC against HIV-1-infected cells. Flow cytometry analyses showed that 42F and 43F also bind to native primary isolate Envs from clades B and E expressed on cell surfaces. By direct binding and competition assays, it was demonstrated that the 42F/43F epitope lies in a domain of gp120 outside the previously described CD4-binding site and V3 loop ADCC epitope clusters. Immunoblot analysis revealed that the 42F/43F epitope is not dependent on disulfide bonds or N-linked glycans in gp120. Epitope mapping of 42F and 43F by binding to linear peptides demonstrated specificity of these MAbs for a sequence of 10 amino acids in the C5 domain comprising residues 491 to 500 (Los Alamos National Laboratory numbering for the HXB2 strain). Thus, 42F and 43F define a new ADCC epitope in gp120. Because of the relative conservation of this epitope and the fact that it appears to have been significantly immunogenic in the individual from which these MAbs were derived, it may prove to be a useful component of HIV vaccines. Furthermore, these MAbs may be used as tools to probe the potential importance of ADCC as an antiviral activity in HIV-1 infection.

Antibody-Dependent Cellular Cytotoxicity Directed against Cells Expressing Human Immunodeficiency Virus Type 1 Envelope of Primary or Laboratory-Adapted Strains by Human and Chimpanzee Monoclonal Antibodies of Different Epitope Specificities

“…Previous studies (1) demonstrated the binding of anti-C5 MAb 43F and anti-CD4 bs MAb IgG1b12 (2) to CD4 Ϫ T cells infected with recombinant vaccinia viruses expressing laboratory-adapted and primary-isolate Envs from clades B and E. Further binding studies of this type were done with additional MAbs of various epitope specificities (data not shown), and the results of these confirmed our earlier impression that, of the three vaccinia virus recombinant primaryisolate Envs tested, the clade E Env of vCB53 was most effi- a All incubations were done under the conditions given in the table using either no Ab or MAb, pooled seropositive sera at 10 Ϫ3 dilution, or 4117C or 41148D at 20 g/ml in a buffer containing 10 mM NaN 3 as described previously (1). An aliquot of washed cells from each of these incubations was then counted for viability, while the remainder were reacted with fluorescein isothiocyanateconjugated goat anti-human IgG at 4°C prior to measurement of fluorescence intensity by flow cytometry as described previously (1). Cell viability after the 4-h incubation at 37°C (final rows of table) was Ն75%, while for all other conditions, it was Ն95%.…”

“…However, in separate experiments, uncloned MN-infected cells were moderately lysed by 42F (1). Since the apparent affinity of 42F for rgp160 LAI was similar to that of 1125H (1,11), the level of specific lysis directed by 42F was slightly lower than expected against IIIB-infected cells. Nevertheless, the level of specific lysis directed by 42F against SF2and RF-infected cells, especially at lower MAb concentrations, was notably high (Fig.…”

“…However, effector cells armed with serum Abs able to direct ADCC in vitro against either innocent bystanders or HIV-1-infected cells were found at highest frequency in asymptomatic, seropositive individuals; patients with AIDS-related complex and AIDS showed progressively diminished reactivities (20). Furthermore, in a recent study (1), the ability of monoclonal Abs (MAbs) against three distinct gp120 epitopes to direct ADCC against uninfected CD4 ϩ cells to which rgp120 SF2 had been ad-sorbed (i.e., innocent bystanders) was demonstrated to be less efficient by at least an order of magnitude than their ability to direct ADCC against HIV-1-infected cells.…”

“…Consistent with this data is our recent characterization of two MAbs, 42F and 43F, isolated from a long-term survivor of HIV-1 infection (1); these MAbs directed significant levels of ADCC and defined a new, conserved ADCC epitope in the C5 domain of HIV-1 gp120. Preliminary evidence indicated that concentrations of 42F-and 43F-like Abs in the serum of the donor were in the range required to direct high levels of ADCC, and these MAbs were shown to bind both oligomeric primary-isolate and laboratoryadapted Env efficiently (1).…”

“…Following this preincubation, the labeled target-MAb mixture was added to the unlabeled target-MAb (coldtarget) mixture, attaining the cold-target cell-per-milliliter concentrations shown in the figure. The remainder of the ADCC assay was carried out as previously described(1). The error bars represent standard deviations of duplicate points.…”