Tsinghua Science and Technology Tsinghua University, China ISSN: 1007-0212 Vol. 6, Num. 3, 2001, pp. 257-259

Tsinghua Science and Technology, Vol. 6, No. 3, August 2001 pp. 257-259

Monoclonal Anti-CD4 Antibody MT310 Binds HIV-1 gp120  Binding Site on CD4*

QIN Li , ZHU Mei, Gert Riethmuller†,  Peter Rieber† CHEN Yinghua**

Laboratory of Immunology, Research Center for Medical Science, Department of Biological  Sciences and Biotechnology, Tsinghua University, Beijing 100084, China;
† Institute of Immunology, University of Munich, Germany

* Supported  by Munich University in Germany and the National Key Basic Research Specific Funds of China (No. G1999075607).
** To  whom correspondence should be addressed, E-mail: chenyh@mail.tsinghua.edu.cn

Received: 2000-11-21

Code Number: ts01077

Abstract:  

Tests  show the monoclonal anti-CD4 antibody (mAb) MT310 recognizes the gp120-binding site on CD4 as part of its mechanism for strongly inhibiting human immunodeficiency virus type 1 (HIV-1) infection of CD4⁺ T cells. In competition tests, mAb MT310 and mAb Leu3a (an anti-CD4 mAb recognizing the gp120-binding site) all inhibited gp120-binding to CD4⁺ T lymphocytes, while mAb MT405 did not. This result suggests that MT310, like Leu3a, recognizes the gp120-binding site on CD4. To further confirm whether MT310 recognizes the gp120-binding site on CD4, we prepared rabbit anti-idiotypic antisera (Ab2) against MT310 (Ab1). The anti-idiotypic antisera against MT310 inhibited binding of MT310 and Leu3a to human CD4⁺ T lymphocytes, but did not block binding of MT151 with the second domain of CD4, while rabbit anti-idiotypic antisera to MT151 could block binding of itself to these cells, but could not inhibit the binding of MT310 and Leu3a, further indicating that MT310 recognized the gp120-binding site on CD4.

Key  words:  anti-idiotypic antibody; epitope analysis; anti-CD4 antibody

Introduction   

Human immunodeficiency virus type 1 (HIV-1), after binding by the envelope protein gp120 to its cellular receptor CD4, enters susceptible cells through pH-independent fusion[1, 2]. The site which HIV-1 gp120 binds is located in the first domain on the CD4 molecule[3-5]. The anti-CD4 mAb Leu3a recognizes the gp120 binding site on CD4[6]  and the anti-CD4 mAb MT310 strongly inhibits HIV-1 infection of CD4⁺ T cells, while the anti-CD4 mAb MT151 weakly inhibits infection [4,7,8] . mAb MT405 recognizes an epitope on the second domain of CD4, so it cannot inhibit HIV-1 infection of CD4⁺ T cells (our unpublished data).

Idiotype specificity was used to classify three types of anti-idiotypic antibodies[9]. Anti-idiotypic antibody 2b (Ab2b) binds to the antigen-binding site of the first antibody (Ab1) and may functionally and even structurally mimic the antigen. Ab2b inhibits the binding of the antigen to Ab1. Anti-idiotypic antibody 2g binds within or close to the antigen-binding site of Ab1 and inhibits antigen-binding to Ab1, but does not mimic the antigen. Anti-idiotypic antibody 2a(Ab2a) binds to idiotypes outside the antigen-binding region of Ab1 and therefore cannot inhibit the binding of the antigen to Ab1 and does not mimic the antigen[9]. This paper analyzes the specificity of monoclonal anti-CD4 antibody MT310.

1 Materials and Methods   

1.1 Cells

Peripheral blood mononuclear cells (PBMC) were isolated from healthy volunteer donors using Ficoll (Pharmacia). Preparations were analyzed as lymphocyte population by the lymphocyte-gate in the flow cytometry (fluorescence activated cell sorter, FACS)-analysis.

1.2 Antibodies

The anti-CD4 murine mAb MT310 (IgGl) and MT151 (IgG2a) were obtained from the Institute of Immunology, University of Munich (Germany). The biotinylated mAbs were obtained from Prof. Rieber (Institute of Immunology, University of Munich). FITC-conjugated Streptavidin was obtained from Sigma (Germany). The biotinylated monoclonal murine anti-gp120 antibody was obtained from Du Pont (USA).

1.3 Generation of anti-idiotypic antisera

Each white rabbit in one group (n=3) was immunized with  200 mg  of MT310 or MT151 in complete Freund's adjuvant (CFA) (1 : 1 ratio) subcutanously. Boosters were given with 200 mg of both mAbs in incomplete Freund's adjuvant (IFA) at 2-week intervals. The anti-idiotypic antisera were isolated on the fifth day after the third booster. Preimmune sera were collected before immunization.

1.4 Inhibition of mAb binding to CD4⁺  

T-lymphocytes in FACS-analysis Biotinylated anti-CD4 mAbs were initially incubated with rabbit anti-idiotypical antisera for 15 min at room temperature, with the mixture then added to human peripheral blood mononuclear cells (5x10⁵). After incubation for 30 min at 4 °C, the cells were washed with phosphate-buffered saline (PBS) three times, then incubated with  50 mL  FITC-conjugated Streptavidin (1 : 50 dilution with PBS) for 30 min at  4 °C . After washing with PBS, the cells were fixed with 1% paraformaldehyde in PBS and analysed on an FACScan (Becton Dickinson, Califonia, USA) using the lymphocyte gate. For inhibition of gp120-binding to CD4⁺ lymphocytes, the cells were first incubated with the anti-CD4 mAbs.

2 Results and Discussion  

The monoclonal anti-CD4 antibody MT310 strongly inhibited HIV-1 infection of CD4⁺ T cells. To identify whether MT310 recognizes the gp120-binding site on CD4, we compared the inhibition potencies of mAb MT310 and mAb Leu3a (an anti-CD4 mAb recognizing the gp120-binding site) in competition tests. Both mAbs inhibited gp120-binding to CD4⁺ T lymphocytes, while mAb MT405 did not (Fig.1). This result suggests that MT310 like Leu3a recognizes the gp120-binding site on CD4. To further confirm whether MT310 recognizes the gp120-binding site on CD4, we prepared rabbit anti-idiotypic antisera (Ab2) against MT310 (Ab1). A blocking test showed that the rabbit anti-idiotypic antisera to MT310 inhibited binding of MT310 and Leu3a to human CD⁺4 T lymphocytes (Figs.2(b) and (e)), but did not block binding of MT151 to these cells (Fig.2(h)), indicating that the anti-idiotypic antisera to MT310 recognized the antigen-binding site of Leu3a and, therefore, inhibited the binding of Leu3a to CD4 molecules. The rabbit anti-idiotypic antisera to MT151 inhibited the binding of itself to CD4⁺ T lymphocytes (Fig.2(i)), but not the binding of MT310 and Leu3a to these cells (Figs.2(c) and (f)), suggesting that MT151 binds another epitope on CD4. The normal rabbit serum as negative control showed no inhibition of the binding (Figs.2(a), (d), and (g)). These results indicate that MT310 recognizes the gp120-binding site on the first domain of CD4. The idiotype specificity showed that the anti-idiotypic antisera to MT310 contained the anti-idiotypic antibody Ab2b, because Ab2b can inhibit the binding of Ab1(MT310) to the antigen(CD4)[9]. The experimental results demonstrated that the anti-idiotypic antisera to MT310 inhibited the binding of Leu3a to CD4 (CD4-positive T-lymphocytes), indicating that MT310 and Leu3a have a similar antigen binding site and recognize a similar epitope on CD4.

References

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