Increased immunogenicity of human immunodeficiency virus gp120 engineered to express Galalpha1-3Galbeta1-4GlcNAc-R epitopes

Abstract

The glycan shield comprised of multiple carbohydrate chains on the human immunodeficiency virus (HIV) envelope glycoprotein gp120 helps the virus to evade neutralizing antibodies. The present study describes a novel method for increasing immunogenicity of gp120 vaccine by enzymatic replacement of sialic acid on these carbohydrate chains with Galalpha1-3Galbeta1-4GlcNAc-R (alpha-gal) epitopes. These epitopes are ligands for the natural anti-Gal antibody constituting approximately 1% of immunoglobulin G in humans. We hypothesize that vaccination with gp120 expressing alpha-gal epitopes (gp120(alphagal)) results in in vivo formation of immune complexes with anti-Gal, which targets vaccines for effective uptake by antigen-presenting cells (APC), due to interaction between the Fc portion of the antibody and Fcgamma receptors on APC. This in turn results in effective transport of the vaccine to lymph nodes and effective processing and presentation of gp120 immunogenic peptides by APC for eliciting a strong anti-gp120 immune response. This hypothesis was tested in alpha-1,3-galactosyltransferase knockout mice, which produce anti-Gal. Mice immunized with gp120(alphagal) produced anti-gp120 antibodies in titers that were >100-fold higher than those measured in mice immunized with comparable amounts of gp120 and effectively neutralized HIV. T-cell response, measured by ELISPOT, was much higher in mice immunized with gp120(alphagal) than in mice immunized with gp120. It is suggested that gp120(alphagal) can serve as a platform for anti-Gal-mediated targeting of additional vaccinating HIV proteins fused to gp120(alphagal), thereby creating effective prophylactic vaccines.

FIG. 1.

Synthesis of α-gal epitopes on gp120. SA residues capping the N-linked carbohydrate chains of the complex type on gp120 (left chain) are removed by neuraminidase (middle chain). α-gal epitopes are synthesized by linking of galactosyls (Gal) from the sugar donor UDP-galactose (UDP-GAL), due to the catalytic activity of recombinant α1,3GT. These α-gal epitopes on immunizing gp120_αgal readily bind in situ the natural anti-Gal IgG molecules, thus forming immune complexes that target the vaccinating gp120_αgal to APC.

FIG. 2.

Synthesis of α-gal epitopes on gp120 by recombinant α1,3GT as demonstrated by SDS-PAGE and Western blotting. Upper gel: Coomassie staining of SDS-PAGE gel. Lower gels: Western blot stained with human anti-Gal, mouse anti-Gal, and the α-gal epitope-specific BS lectin. Note that the antibodies and the lectin bind only to gp120_αgal.

FIG. 3.

Evaluation of α-gal epitope expression on gp120_αgal. The figure shows binding of the monoclonal anti-Gal M86 to gp120, gp120_αgal, and α-gal BSA that expresses 10 synthetic α-gal epitopes, as measured by ELISA with different amounts of glycoproteins coating the ELISA wells.

FIG. 4.

Characteristics of mouse anti-Gal in comparison to human anti-Gal. A. Comparison of anti-Gal IgG activity in human sera (•) and sera of mice immunized with PKMs (○). Data are for 3 out of 30 humans and 30 KO mice with similar results. B. Classes and subclasses of anti-Gal in KO mice as measured by ELISA with α-gal BSA as solid-phase antigen. The antibody activity was measured at a serum dilution of 1:100. Data from 4 representative mice out of 15 with similar results are shown.

FIG. 5.

Elicited anti-gp120 antibodies in response to immunization with gp120 or gp120_αgal. The figure shows production of anti-gp120 antibodies in KO mice immunized twice at 2-week intervals (A to C) or in wild-type (WT) mice (D), immunized with either gp120 (○) or gp120_αgal (•). (A) 5.0 μg/vaccine; (B) 0.5 μg/vaccine; (C) 50 μg/vaccine; (D) 5.0 μg/vaccine. Note that KO mice in panels A to C immunized with gp120 produced anti-gp120 antibodies in low titers or completely lacked such antibodies, whereas a significant increase in anti-gp120 antibody production was observed in mice immunized with gp120_αgal. In contrast, no differences in anti-gp120 antibody production are detected in panel D in wild-type mice immunized with the two glycoproteins, as the wild-type mice are incapable of producing anti-Gal despite repeated PKM immunizations.

FIG. 6.

HIV neutralization activity in mice immunized with gp120_αgal or with gp120. The figure shows the titer of neutralization activity in various mice immunized twice with 5 μg gp120 (mice 1 to 6) or gp120_αgal (mice 7 to 12). Titer is defined as the reciprocal of the serum dilution displaying 50% neutralization.

FIG. 7.

ELISPOT analysis for IFN-γ secretion in mice immunized with gp120 or gp120_αgal. A. Actual wells with splenocytes from three mice, each tested in triplicate (vertical lanes) in the absence or presence of gp120-pulsed DC. B. Presentation of ELISPOT data for six mice immunized twice with 5 μg gp120 (mice 1 to 6) and six mice immunized twice with 5 μg gp120_αgal (mice 7 to 12), as the number of spots per 10⁶ splenocytes.