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  • Open Access

Differential glycosylation of envelope gp120 affects reactivity with HIV-1 specific antibodies

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BMC Infectious Diseases201414 (Suppl 2) :P68

https://doi.org/10.1186/1471-2334-14-S2-P68

  • Published:

Keywords

  • Human Immunodeficiency Virus
  • Antigenic Determinant
  • Envelope Glycoprotein
  • Partial Removal
  • Cytometric Bead Array

Introduction

Cellular entry of human immunodeficiency virus type 1 (HIV-1) depends on envelope glycoprotein (gp120/gp41) interactions with host-cell receptors. Approximately one-half of molecular mass of gp120 consists of N-glycans which may act as antigenic determinants as well as a shield against immune recognition. We have shown that glycosylation of subtype B HIV-1 gp120 varies according to the producing cell type and the differential glycosylation affects reactivities with serum antibodies of persons infected with HIV-1 subtype B. Here we studied reactivities of above proteins with panel of monoclonal gp120-specific broadly neutralizing antibodies and sera from persons infected with HIV-1 subtype A/C.

Materials and methods

Recombinant gp120 produced in different cell lines (HEK 293T, Jurkat, RD, HepG2, and CHO) were tested as a native and after partial removal of N-glycans by PNGase F under native conditions. Several methods (ELISA, Cytometric Bead Array - CBA, SDS-PAGE with western blot, and dot blot) were used for determination and comparison of reactivities with monoclonal gp120-specific antibodies (268-D IV, F425 B4e8, 257-D IV, 447-52D, 19b, 2G12, and b12) or with sera of HIV-1 subtype A/C-infected persons.

Results

After partial removal of N-glycans from gp120, the reactivity of most monoclonal antibodies increased, as did the reactivities of sera from HIV-1-infected persons. The largest increase in binding of polyclonal antibodies after PNGase F treatment was found for gp120 expressed in HEK 293T and CHO cell lines, common producers of recombinant proteins for vaccination purposes. Conversely, the gp120 produced by T cells (Jurkat) displayed the least increase in reactivity after partial deglycosylation.

Conclusion

Changes in reactivity of selected monoclonal antibodies with native and PNGase F-treated proteins indicated that some glycans were resistant to deglycosylation and that this characteristic was dependent on producer cell type. Furthermore, CBA allowed more sensitive detection (about 40-times) of gp120-specific antibodies compared to ELISA.

Supported by CZ.1.07/2.3.00/20.0164 European Social Fund, UAB CFAR Developmental Grant (P30AI027767) and a Pilot Grant from UAB School of Medicine.

Authors’ Affiliations

(1)
Department of Immunology, Palacky University, Olomouc, Czech Republic
(2)
University of Alabama, Birmingham, Alabama, USA
(3)
Clinic of the University of Munich, Munich, Germany
(4)
NIMR-Mbeya Medical Research Program, Mbeya, Tanzania

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