Human immunodeficiency virus type 1 evades T-helper responses by exploiting antibodies that suppress antigen processing

Abstract

T-helper responses are important for controlling chronic viral infections, yet T-helper responses specific to human immunodeficiency virus type 1 (HIV-1), particularly to envelope glycoproteins, are lacking in the vast majority of HIV-infected individuals. It was previously shown that the presence of antibodies to the CD4-binding domain (CD4bd) of HIV-1 glycoprotein 120 (gp120) prevents T-helper responses to gp120, but their suppressive mechanisms were undefined (C. E. Hioe et al., J. Virol. 75:10950-10957, 2001). The present study demonstrates that gp120, when complexed to anti-CD4bd antibodies, becomes more resistant to proteolysis by lysosomal enzymes from antigen-presenting cells such that peptide epitopes are not released and presented efficiently by major histocompatibility complex class II molecules to gp120-specific CD4 T cells. Antibodies to other gp120 regions do not confer this effect. Thus, HIV may evade anti-viral T-helper responses by inducing and exploiting antibodies that conceal the virus envelope antigens from T cells.

FIG. 1.

The presence of an anti-CD4bd MAb prevented CD4 T-cell-proliferative responses to gp120 from the following HIV-1 isolates: IIIB (X4 tropic), JRFL (R5 tropic), TH14-12 (clade B primary isolate), and 93MW959 (clade C primary isolate). A fixed concentration of gp120 (8 nM) was used to form immune complexes with various concentrations of anti-CD4bd MAb 654-D (•) or anti-C5 MAbs 450-D (□) and 670-D (▵). Proliferation of CD4 T-cell lines PS02 and PS04 recognizing the different gp120 antigens was assessed by [³H]thymidine incorporation. Representative data from two or more independent experiments are shown.

FIG. 2.

The proliferative response of CD4 T-cell lines to native gp120 expressed by HIV-1 IIIB virions was prevented by the anti-CD4bd MAbs but not by the anti-C5 MAb. Proliferation of gp120-specific CD4 T-cell line PS02 was examined in response to AT2-inactivated IIIB treated with no MAb (○), anti-CD4bd MAb 559/64-D (⧫) or 654-D (•), or anti-C5 MAb 450-D (□). The stock of AT2-inactivated IIIB virions contained 2.85 μg of gp120 ml⁻¹ and was tested at 1:1,000, 1:3,000, and 1:10,000 dilutions, while each of the MAbs was used at a fixed concentration of 10 μg ml⁻¹. Net counts per million were calculated by subtraction of background (5,031 cpm for T cells incubated with APCs in medium alone without immune complexes). Representative data from two independent experiments are shown.

FIG. 3.

The binding of anti-CD4bd MAb, but not anti-C5 MAb, rendered HIV-1 gp120 more resistant to proteolysis by cathepsins. (A) Recombinant gp120 of JRFL and IIIB strains was digested by a mixture of cathepsins B, D, and L after preincubation with anti-CD4bd MAb 654-D, anti-C5 MAb 450-D, or no MAb. The amounts of gp120 remaining after proteolysis were analyzed under nonreducing conditions in SDS-PAGE and were visualized by both Coomassie blue stain and Western blot staining with sheep anti-gp120 serum. (B) The relative amounts of gp120 remaining after digestion were quantitated from the Coomassie blue-stained gel by densitometry with the 1D Image Analysis Software (Scientific Imaging Systems Eastman Kodak Company). Representative data from two independent experiments are shown.

FIG. 4.

The fragmentation patterns of gp120 and gp120 complexed with anti-gp120 MAbs after proteolytic digestion by a mixture of cathepsins B, D, and L. Recombinant gp120_IIIB was preincubated with no MAb, anti-C5 MAb 450-D, anti-CD4bd MAb 559/64-D or 654-D, anti-V3 MAb 694/98-D, anti-V2 MAb 697-D, or an irrelevant anti-parvovirus MAb 860-55D and was digested with the cathepsins. gp120 treated with no MAb and no enzymes (undigested) was also analyzed for comparison. The reaction products were run on SDS-PAGE under nonreducing or reducing conditions and were stained after Western blotting with sheep anti-gp120 serum. Representative blots of two independent experiments are shown.

FIG. 5.

(A) Proteolysis of gp120 and gp120/MAb complexes by lysosomal enzymes. Recombinant gp120_IIIB was either left untreated or were treated with MAb to C5 (450-D), CD4bd (559/64-D or 654-D), V3 (694/98-D), V2 (697-D), or parvovirus (860-55D) and were then digested with lysosomal enzymes extracted from EBV-transformed B cells. The reaction products were run on SDS-PAGE under nonreducing or reducing conditions and were stained after Western blotting with sheep anti-gp120 serum. Representative blots of at least five repeated experiments are shown. (B) Resistance of recombinant gp120_IIIB to lysosomal enzyme digestion was dependent on the amount of anti-CD4bd MAbs. gp120_IIIB (1 μg) was treated with various amounts of anti-CD4bd MAb 654-D and was digested with lysosomal enzymes. The reaction products were analyzed by Western blot as described above. (C) The release of T-cell epitopes from digested gp120 or gp120/MAb complexes was also examined by measuring the proliferative response of the gp120-specific CD4 T-cell line PS02 to fixed autologous EBV-transformed B cells pretreated with the digestion products and used as APCs. The responses of the T cells to APCs pulsed with a C2 peptide representing the T-cell epitope and with undigested gp120 were also measured for control. Representative data of two independent T-cell assays are shown.

FIG. 6.

Proteolysis of gp120 and gp120 treated with HIV-positive IgG by lysosomal enzymes. Recombinant gp120_IIIB was mixed with serum IgG from HIV-positive progressors (P1, P2, and P3), nonprogressors (NP1, NP2, and NP3), or an HIV-negative subject (NG) and then was digested with lysosomal enzymes extracted from EBV-transformed B cells. The reaction products were run on SDS-PAGE under nonreducing and reducing conditions and were stained after Western blotting with an anti-gp120 MAb. A representative blot of at least two independent experiments is shown.