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. 2007 Sep;81(17):9131-41.
doi: 10.1128/JVI.00647-07. Epub 2007 Jul 3.

Immunogenicity of influenza virus vaccine is increased by anti-gal-mediated targeting to antigen-presenting cells

Ussama M Abdel-Motal  1 , Heath M Guay, Kim Wigglesworth, Raymond M Welsh, Uri Galili
Affiliations

Immunogenicity of influenza virus vaccine is increased by anti-gal-mediated targeting to antigen-presenting cells

Ussama M Abdel-Motal et al. J Virol. 2007 Sep.

Abstract

This study describes a method for increasing the immunogenicity of influenza virus vaccines by exploiting the natural anti-Gal antibody to effectively target vaccines to antigen-presenting cells (APC). This method is based on enzymatic engineering of carbohydrate chains on virus envelope hemagglutinin to carry the alpha-Gal epitope (Gal alpha 1-3Gal beta 1-4GlcNAc-R). This epitope interacts with anti-Gal, the most abundant antibody in humans (1% of immunoglobulins). Influenza virus vaccine expressing alpha-Gal epitopes is opsonized in situ by anti-Gal immunoglobulin G. The Fc portion of opsonizing anti-Gal interacts with Fc gamma receptors on APC and induces effective uptake of the vaccine virus by APC. APC internalizes the opsonized virus to transport it to draining lymph nodes for stimulation of influenza virus-specific T cells, thereby eliciting a protective immune response. The efficacy of such an influenza vaccine was demonstrated in alpha 1,3galactosyltransferase (alpha 1,3GT) knockout mice, which produce anti-Gal, using the influenza virus strain A/Puerto Rico/8/34-H1N1 (PR8). Synthesis of alpha-Gal epitopes on carbohydrate chains of PR8 virus (PR8(alpha gal)) was catalyzed by recombinant alpha1,3GT, the glycosylation enzyme that synthesizes alpha-Gal epitopes in cells of nonprimate mammals. Mice immunized with PR8(alpha gal) displayed much higher numbers of PR8-specific CD8(+) and CD4(+) T cells (determined by intracellular cytokine staining and enzyme-linked immunospot assay) and produced anti-PR8 antibodies with much higher titers than mice immunized with PR8 lacking alpha-Gal epitopes. Mice immunized with PR8(alpha gal) also displayed a much higher level of protection than PR8 immunized mice after being challenged with lethal doses of live PR8 virus. We suggest that a similar method for increasing immunogenicity may be applicable to avian influenza vaccines.

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Figures

FIG. 1.
FIG. 1.
Synthesis of α-Gal epitopes on influenza virus HA. (A) Illustration of enzymatic synthesis. α-Gal epitopes (Galα1-3Galβ1-4GlcNAc-R) were synthesized on the N (Asn)-linked carbohydrate chains on HA by the linking of galactosyls (Gal) from the sugar donor, UDP-GAL, to N-acetyllactosamine (Galβ1-4GlcNAc-R) residues as a result of the catalytic activity of recombinant α1,3GT. (B) Expression of α-Gal epitopes on PR8αgal, as shown by Western blots stained with serum anti-Gal purified from KO mouse serum and with monoclonal anti-Gal M86. Note that both anti-Gal antibodies bind only to HA1 from PR8αgal and not to the HA1 from unprocessed PR8. (C) α-Gal epitope expression levels on PR8αgal virus attached to ELISA wells and studied for binding of the monoclonal anti-Gal M86. PR8αgal virus treated with 0.2% formalin for 20 h had the same level of binding as PR8αgal virus generated by incubation of PR8 with active recombinant α1,3GT. The data shown are representative of three independent experiments with similar results. ▵, unprocessed PR8 virus; □, PR8 virus incubated with inactivated recombinant α1,3GT; ○, PR8αgal virus generated by incubation of PR8 with active recombinant α1,3GT.
FIG. 2.
FIG. 2.
Analysis by ELISPOT assay of IFN-γ secretion levels in mice immunized with PR8 or PR8αgal. Lymphocytes from six mice immunized with inactivated PR8αgal virus (#1 to #6) or inactivated PR8 (#7 to #12) were obtained 14 days after the second immunization and incubated with cells of the dendritic cell line DC2.4, prepulsed with inactivated PR8, and subjected to ELISPOT (hatched columns). Data for lymphocytes incubated with DC that were not prepulsed with PR8 are presented as empty columns. The data are presented as means ± standard deviations of the results for triplicate wells.
FIG. 3.
FIG. 3.
Intracellular staining of IFN-γ in CD8+ (A) and CD4+ (B) T cells in PR8αgal- or PR8-immunized mice. ICS analysis of levels of CD8+ (A, including two left panels) and CD4+ (B, including two right panels) T cells in PR8αgal (mice no. 1 to 6)- or PR8 (mice no. 7 to 12)-immunized mice. Lymphocytes were obtained as described in the legend to Fig. 2 and four-color stained for CD3+, CD8+, CD4+ membrane marker, and intracellular IFN-γ. Gated CD3+/CD8+- or CD3+/CD4+-positive events were analyzed for IFN-γ production. The percentage of CD8+ or CD4+ T cells with intracellular IFN-γ is indicated in the upper right quadrant for each mouse. Note that four of the six mice immunized with PR8αgal (#1 to #4) had much higher proportions of IFN-γ-producing CD8+ and CD4+ T cells than mice immunized with PR8.
FIG. 4.
FIG. 4.
Production of anti-PR8 antibodies in mice immunized twice with 1 μg inactivated PR8αgal (•) or with inactivated PR8 (○) in Ribi adjuvant and measured by ELISA with PR8 virus as a solid-phase antigen. (A) Anti-PR8 IgG response in KO mice. (B) Anti-PR8 IgG response in WT mice. (C) Anti-PR8 IgA response in KO mice (n = 6 per group). The two KO mice in panels A and C with the lowest levels of response (•) are mice no. 5 and 6 in Fig. 2 and 3 above.
FIG. 5.
FIG. 5.
HI activity in KO mice immunized with PR8αgal or PR8. Results are presented as reciprocals of the serum dilutions that displayed HI activity. Note that the levels of HI activity in mice immunized twice with 1 μg PR8αgal is much higher than that in the sera of mice immunized with 1 μg PR8 (with the exception of mice no. 5 and 6).
FIG. 6.
FIG. 6.
Survival rates and analyses of lungs from mice immunized twice with inactivated PR8 or PR8αgal and receiving intranasal challenge with live PR8. (A) PR8 (○)- or PR8αgal (•)-immunized mice were challenged with 2,000 PFU of live PR8 in 50-μl solutions (n = 25/group). Survival data are presented as percentages of live mice at different time points postchallenge. The survival data for day 30 were similar to those for day 15 postchallenge. (B) Analysis of virus titers as the TCID in lungs of immunized mice 3 days postchallenge (n = 5/group). The supernatants of lung homogenates were incubated in serial 10-fold dilutions in 96-well plates with MDCK cell monolayers. The levels of cytopathic effects were scored after 96 h. (C) Analysis of virus titer by hemagglutination in lungs of immunized mice 3 days postchallenge (n = 5/group). (D) Levels of anti-PR8 IgA antibodies in lungs of immunized mice 3 days post challenge (n = 5/group). •, PR8αgal-immunized mice; ○, PR8-immunized mice (n = 5 mice/group).
FIG. 7.
FIG. 7.
Anti-Gal-mediated targeting of vaccinating PR8αgal virus to APC. Inactivated PR8αgal virus with α-Gal epitopes (red diamonds) was injected as a vaccine. Anti-Gal binds to the α-Gal epitopes on the virus and opsonizes it. The Fc portion of anti-Gal interacts with FcγR on APC and induces uptake of the vaccine by the APC. The internalized virus undergoes processing in the endocytic vesicles and the cytoplasm. The viral immunogenic peptides are presented on MHC class I molecules for activation of CD8+ CTL precursors (Tc cells) and on MHC class II molecules for activation of helper T cells (Th cells). Signal 2 provided by the APC also facilitates this activation. Activated Th cells provide help for the antibody response by B cells and for CTL activation. Activated Tc cells differentiate into CTL, which kill virus-infected cells. TCR, T-cell receptor.
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