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.
Highlights
The increased risk of an influenza pandemic raises the need for the generation of effective vaccines that will elicit both humoral and cellular immune responses for prevention of infection in the respiratory tract and destruction of virus-infected cells in the body
We found that we could synthesize multiple ␣-Gal epitopes on the N-linked carbohydrate chains of gp120 by using recombinant ␣1,3GT that we produced in the Pichia pastoris yeast expression system [4]
At a concentration of 100 g/ml, the virus was incubated with 30 g/ml of recombinant ␣1,3GT and 1 mM UDP-Gal (UDP-galactose) as a sugar donor. This enzyme transfers galactose from UDP-Gal and links it as Gal␣1–3 to the N-acetyllactosamines (Gal14GlcNAc-R) of the multiple HA carbohydrate chains to generate ␣-Gal epitopes by a reaction that is identical to that naturally occurring within the Golgi apparatus of nonprimate mammalian cells (Fig. 1A)
Summary
The increased risk of an influenza pandemic raises the need for the generation of effective vaccines that will elicit both humoral and cellular immune responses for prevention of infection in the respiratory tract and destruction of virus-infected cells in the body. In a recent study using a unique model of ␣1,3GT knockout mice, we showed that the recombinant gp120 protein of the human immunodeficiency virus envelope increases its immunogenicity by Ͼ100-fold if the vaccine protein is engineered to express ␣-Gal epitopes [1] This increased immunogenicity is achieved because of the in vivo formation of immune complexes between anti-Gal and the vaccinating gp120 expressing the ␣-Gal epitopes. We found that we could synthesize multiple ␣-Gal epitopes on the N-linked carbohydrate chains of gp120 by using recombinant ␣1,3GT that we produced in the Pichia pastoris yeast expression system [4] This observation raised the question of whether increasing immunogenicity by anti-Gal-mediated targeting to APC may be achieved by using intact, inactivated influenza virus engineered to express multiple ␣-Gal epitopes in a vaccine
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