Amplifying immunogenicity of prospective Covid-19 vaccines by glycoengineering the coronavirus glycan-shield to present α-gal epitopes

Abstract

The many carbohydrate chains on Covid-19 coronavirus SARS-CoV-2 and its S-protein form a glycan-shield that masks antigenic peptides and decreases uptake of inactivated virus or S-protein vaccines by APC. Studies on inactivated influenza virus and recombinant gp120 of HIV vaccines indicate that glycoengineering of glycan-shields to present α-gal epitopes (Galα1-3Galβ1-4GlcNAc-R) enables harnessing of the natural anti-Gal antibody for amplifying vaccine efficacy, as evaluated in mice producing anti-Gal. The α-gal epitope is the ligand for the natural anti-Gal antibody which constitutes ~1% of immunoglobulins in humans. Upon administration of vaccines presenting α-gal epitopes, anti-Gal binds to these epitopes at the vaccination site and forms immune complexes with the vaccines. These immune complexes are targeted for extensive uptake by APC as a result of binding of the Fc portion of immunocomplexed anti-Gal to Fc receptors on APC. This anti-Gal mediated effective uptake of vaccines by APC results in 10-200-fold higher anti-viral immune response and in 8-fold higher survival rate following challenge with a lethal dose of live influenza virus, than same vaccines lacking α-gal epitopes. It is suggested that glycoengineering of carbohydrate chains on the glycan-shield of inactivated SARS-CoV-2 or on S-protein vaccines, for presenting α-gal epitopes, will have similar amplifying effects on vaccine efficacy. α-Gal epitope synthesis on coronavirus vaccines can be achieved with recombinant α1,3galactosyltransferase, replication of the virus in cells with high α1,3galactosyltransferase activity as a result of stable transfection of cells with several copies of the α1,3galactosyltransferase gene (GGTA1), or by transduction of host cells with replication defective adenovirus containing this gene. In addition, recombinant S-protein presenting multiple α-gal epitopes on the glycan-shield may be produced in glycoengineered yeast or bacteria expression systems containing the corresponding glycosyltransferases. Prospective Covid-19 vaccines presenting α-gal epitopes may provide better protection than vaccines lacking this epitope because of increased uptake by APC.

Keywords: Covid-19 vaccine; Glycan shield; S-protein; SARS-CoV-2; anti-Gal; α-gal epitopes.

Fig. 1

Glycoengineering of α-gal epitopes on Spike (S)-protein of SARS-CoV-2 virus. Left chain- Carbohydrate chains (glycans) of the complex type on viral envelope glycoproteins are synthesized on asparagine (N) in amino acid sequences of N-X-S/T-. Based on the information about a similar glycan shield of HIV-gp120, it is probable that many of these carbohydrate chains of the S-protein are capped by sialic acid (SA). Center chain- Sialic acid is removed from the carbohydrate chain by neuraminidase to expose the penultimate Galβ1-4GlcNAc-R, called N-acetyllactosamine (LacNAc). Right chain- Incubation of inactivated virus or soluble S-protein (split or subunit vaccine) carrying the desialylated glycan, with recombinant α1,3galactosyltransferase (rα1,3GT) and with UDP-Gal results in synthesis of α-gal epitopes (Galα1-3Galβ1-4GlcNAc-R) on the glycans in the same reaction as synthesis of these epitopes on glycoproteins and glycolipids within mammalian cells. These epitopes readily bind the natural anti-Gal antibody at the vaccination site and form immune complexes that are targeted for extensive uptake by APC.

Fig. 2

Anti-Gal mediated targeting of SARS-CoV-2 S-proteintoAPC. A. The negative charges of sialic acids (SA) on the glycan-shield of the S-protein and on APC surface glycoproteins and glycolipids generate electrostatic repulsion (ζ [zeta]-potential) between the SARS-CoV-2 inactivated virus or S-protein vaccines and the APC. This repulsion decreases the uptake by random endocytosis of the negatively charged vaccine into the APC. B. Synthesis of α-gal epitopes on the glycan-shield of S-protein (S-protein_αgal) eliminates the electrostatic repulsion and enables binding of the natural anti-Gal IgG antibody to the S-protein_αgal and formation of anti-Gal/S-protein_αgal immune complexes. These immune complexes are effectively targeted for binding to the Fcγ receptors (FcγR) on APC, further resulting in extensive active uptake of the vaccine by APC. Modified from .

Fig. 3

Suggested mechanism for amplification of SARS-CoV-2_αgal vaccine immunogenicity by anti-Gal mediated targeting to APC. Inactivated SARS-CoV-2 presenting α-gal epitopes (SARS-CoV-2_αgal) is used as a vaccine example. Step 1- Anti-Gal IgM and IgG bind to α-gal epitopes on the vaccinating virus at the vaccination site, activate the complement system which generates complement cleavage chemotactic peptides that recruit APC such as dendritic cells and macrophages. Step 2- Anti-Gal IgG coating the virus targets it for active extensive uptake by the recruited dendritic cells and macrophages, via Fc/Fcγ receptors (FcγR) interaction. Step 3- These APC transport the internalized virus vaccine to the regional lymph nodes and process the virus antigens. Within the lymph nodes, the APC present the immunogenic virus peptides on class I and class II MHC molecules for the activation of SARS-CoV-2 specific CD8 + and CD4 + T cells, respectively. TCR- T cell receptor. Modified from .

Fig. 4

Protection against intranasal infection by a lethal dose of PR8 influenza virus in mice immunized with inactivated PR8 or PR8_αgal virus. A. Survival of mice immunized twice with 1 μg inactivated PR8 vaccine (○) in GT-KO mice; PR8_αgal (●) vaccine in GT-KO mice; or with PR8_αgal (△) vaccine in wild-type mice. The mice were challenged with 2000 PFU of live PR8 in 50 μl (n = 25 per group). Survival data are presented as % of live mice at various days following the challenge. Survival results on Day 30 were similar to those on Day 15 post challenge. B. PR8 virus titers in lungs of GT-KO mice, 3 days after challenge with live virus. The virus titers were assayed in supernatants of lung homogenates from the immunized mice, by hemagglutination of chicken red blood cells (n = 5 per group). Modified from .

Fig. 5

In vitro demonstration of anti-Gal mediated uptake of human B lymphoma cells by autologous APC. Human fresh lymphoma cells (B cell lymphoma) were glycoengineered to present α-gal epitopes by incubation with neuraminidase, recombinant α1,3galactosyltranferase and UDP-Gal (as illustrated in Fig. 1). Lymphoma cells presenting α-gal epitopes or lacking this epitope (i.e. original cells) were incubated with autologous anti-Gal for 30 min, subsequently, for 2 h at 37 °C with autologous macrophages or dendritic cells, then washed and subjected to staining. Arrowheads mark nuclei of the APC. Note the uptake of 9 lymphoma cells presenting α-gal epitopes by the macrophage and one lymphoma cell by the dendritic cell. No uptake of lymphoma cells lacking α-gal epitopes was observed (May Grünwald Giemsa staining, ×1000). Adapted with permission from Manches et al., 2005 .