Increasing Efficacy of Enveloped Whole-Virus Vaccines by In situ Immune-Complexing with the Natural Anti-Gal Antibody

Abstract

The appearance of variants of mutated virus in course of the Covid-19 pandemic raises concerns regarding the risk of possible formation of variants that can evade the protective immune response elicited by the single antigen S-protein gene-based vaccines. This risk may be avoided by inclusion of several antigens in vaccines, so that a variant that evades the immune response to the S-protein of SARS-CoV-2 virus will be destroyed by the protective immune response against other viral antigens. A simple way for preparing multi-antigenic enveloped-virus vaccines is using the inactivated whole-virus as vaccine. However, immunogenicity of such vaccines may be suboptimal because of poor uptake of the vaccine by antigen-presenting-cells (APC) due to electrostatic repulsion by the negative charges of sialic-acid on both the glycan-shield of the vaccinating virus and on the carbohydrate-chains (glycans) of APC. In addition, glycan-shield can mask many antigenic peptides. These effects of the glycan-shield can be reduced and immunogenicity of the vaccinating virus markedly increased by glycoengineering viral glycans for replacing sialic-acid units on glycans with α-gal epitopes (Galα1-3Galβ1-4GlcNAc-R). Vaccination of humans with inactivated whole-virus presenting α-gal epitopes (virus_α-gal) results in formation of immune-complexes with the abundant natural anti-Gal antibody that binds to viral α-gal epitopes at the vaccination site. These immune-complexes are targeted to APC for rigorous uptake due to binding of the Fc portion of immunecomplexed anti-Gal to Fcγ receptors on APC. The APC further transport the large amounts of internalized vaccinating virus to regional lymph nodes, process and present the virus antigenic peptides for the activation of many clones of virus specific helper and cytotoxic T-cells. This elicits a protective cellular and humoral immune response against multiple viral antigens and an effective immunological memory. The immune response to virus_α-gal vaccine was studied in mice producing anti-Gal and immunized with inactivated influenza-virus_α-gal. These mice demonstrated 100-fold increase in titer of the antibodies produced, a marked increase in T-cell response, and a near complete protection against challenge with a lethal dose of live influenza-virus, in comparison to a similar vaccine lacking α-gal epitopes. This glycoengineering can be achieved in vitro by enzymatic reaction with neuraminidase removing sialic-acid and with recombinant α1,3galactosyltransferase (α1,3GT) synthesizing α-gal epitopes, by engineering host-cells to contain several copies of the α1,3GT gene (GGTA1), or by transduction of this gene in a replication-defective adenovirus vector into host-cells. Theoretically, these methods for increased immunogenicity may be applicable to all enveloped viruses with N-glycans on their envelope.

Keywords: Inactivated whole-virus vaccine; enveloped virus vaccines; glycan-shield; natural anti-Gal antibody; vaccine immunogenicity; variants; α-gal epitope.

Figure 1.

Enzymatic synthesis of α-gal epitopes on the glycan-shield of enveloped viruses by α1,3galactosyltransferase (α1,3GT). Left chain- N-glycans of the complex type on the glycan-shield synthesized in the host-cell on asparagine (N) in amino acid sequences (sequon) N-X-S/T-. In most viruses these glycans are capped by sialic acid (SA). Center chain- Sialic acid is removed from the glycan by neuraminidase to expose the penultimate Galβ1–4GlcNAc-R called N-acetyllactosamine (LacNAc). In influenza virus, SA is absent from the glycan-shield. Right chain- α1,3GT links to LacNAc the galactose (Gal) provided by the sugar donor uridine diphosphate galactose (UDP-Gal), resulting in synthesis of α-gal epitopes (Galα1–3Galβ1–4GlcNAc-R) on the carbohydrate chain. These epitopes readily bind the anti-Gal antibody. Adapted with permission from ref. .

Figure 2.

Hypothesis on mechanisms for amplification of inactivated whole-virus vaccine immunogenicity by glycoengineering the glycan-shield to present α-gal epitopes (virus_α-gal). Inactivated influenza virus presenting α-gal epitopes is used as vaccine example. Step 1- Anti-Gal IgM and IgG bind to α-gal epitopes on virus_α-gal at the vaccination site and activate the complement system to form complement cleavage chemotactic peptides that recruit APC such as dendritic cells and macrophages. Step 2- Anti-Gal IgG immunocomplexed with the virus_α-gal targets it for rigorous uptake by the recruited dendritic cells and macrophages via Fc/Fcγ receptors (FcγR) interaction. Step 3- The APC transport the large amounts of internalized virus_α-gal to regional lymph nodes, process and present virus antigenic peptides on MHC molecules for the activation of many virus-specific CD8⁺ and CD4⁺ T cells. HA, hemagglutinin; NA, neuraminidase; TCR, T cell receptor. Modified with permission from “Galili U. The natural anti-Gal antibody as foe turned friend in medicine. Academic Press/ Elsevier Publishers, London, 2018, page 153”.

Figure 3.

Amplification of antibody and T cell response to PR8 influenza virus in mice immunized twice with 1μg inactivated PR8_α-gal virus (●), or with inactivated PR8 virus (○) (n=6 per group). Antibody activity in the serum was measured by ELISA with PR8 virus attached to ELISA wells as solid-phase antigen. T cell activation was analyzed by ELISPOT for IFNγ secretion, by splenocytes incubated with PR8 pulsed dendritic cells. Immune response was evaluated 14 days following the second immunization. A. Anti-PR8 IgG production in GT-KO mice. B. Anti-PR8 IgG production in WT mice. C. Anti-PR8 IgA production in GT-KO mice. D. Spots per well in ELISPOT (mean of triplicates) of secreted IFNγ. Hatched columns- splenocytes incubated with PR8 pulsed dendritic cells, open columns- Splenocytes incubated with non-pulsed dendritic cells. PR8_α-gal immunized mice (#1 to #6), PR8 immunized mice (#7 to #12). Modified from ref. , with permission.

Figure 4.

Survival and lung infection in mice immunized twice with inactivated PR8 or PR8_α-gal and challenged intranasal with a lethal dose of live PR8 virus. A. PR8 vaccine (○), or PR8_α-gal vaccine (●) (n=25/group). Survival at various days post challenge presented as proportion (%) of live mice. The survival on Day 30 was similar to Day 15. B. Measurement of virus titer as tissue culture infectious dose (TCID) in lungs of the immunized mice, 3 days post challenge (n=5/group). Cytopathic effects were determined in Madin Darby Canine Kidney (MDCK) cell monolayers cultured for 4 days (n=5/group). From ref. with permission.