Human IgG Fc promotes expression, secretion and immunogenicity of enterovirus 71 VP1 protein

In transfected 293T cells, codon-optimized enterovirus 71 (EV71) VP1 is secreted in all four tested configurations (wt-VP1, tPA-VP1 with an N-terminal human tissue plasminogen activator leader, tPA-VP1-dimer, and tPA-VP1-hFc with a C-terminal human IgG Fc fragment), as measured by ELISA of culture supernatants. Fusing the tPA leader to VP1 increases VP1 protein expression relative to wt-VP1.

Fusing human IgG Fc (hFc) to tPA-VP1 (tPA-VP1-hFc, "VP1-hFc") gives the highest EV71 VP1 protein level among wt-VP1, tPA-VP1, tPA-VP1-dimer and VP1-hFc, both in lysates and in concentrated supernatants (40 µL concentrated to 1 mL) of 293T cells transfected with equal amounts of each construct and probed by Western blot with an anti-VP1 monoclonal antibody.

In 293T cell Western blots, wt-VP1 and tPA-VP1 are detected in concentrated supernatants as well as lysates, although they are not detected in unconcentrated supernatants, whereas tPA-VP1-dimer and VP1-hFc supernatants are detected without concentration. Intracellular VP1 species run at about 35 kDa (wt-VP1), 38 kDa (tPA-VP1), 71 kDa (tPA-VP1-dimer) and 62 kDa (VP1-hFc), and the secreted forms migrate larger than the intracellular forms, which is attributed to glycosylation.

In BALB/c mice immunized intramuscularly with 100 µg of DNA followed by electroporation (100 V, 60 ms, 60 Hz) at weeks 0, 2, 4 and 8, the VP1-specific serum antibody response to the tPA-VP1 DNA vaccine exceeds that to wt-VP1, and the tPA-VP1-hFc vaccine gives the highest antibody levels of the four constructs. The antibody response peaks after the fourth immunization.

The tPA-VP1-dimer DNA vaccine, which encodes two tandem copies of EV71 VP1, expresses and secretes VP1 at higher levels than monomeric tPA-VP1 in 293T cells yet elicits approximately no detectable VP1-specific antibody in BALB/c mice. The paper suggests the dimer may fold in a way that obscures epitopes needed for secretion and immune response initiation.

In Western blots against VP1 produced by 293T cells, sera from mice immunized with the tPA-VP1-hFc DNA vaccine recognize VP1 in the supernatants of both tPA-VP1-dimer-transfected and tPA-VP1-hFc-transfected cells, whereas sera from mice immunized with the other VP1 constructs do not recognize VP1 in those supernatants.

A VP1 fusion to mouse IgG Fc (tPA-VP1-mFc) expresses at significantly lower levels and fails to mount an effective immune response. A mouse IgG Fc fusion also cannot be used in mouse immunization assays that use an anti-mouse secondary antibody, because the mouse Fc fragment reacts with that antibody.

Human IgG Fc fused to EV71 VP1 allows protein A affinity capture of tPA-VP1-hFc from 293T culture supernatant (200 mL, bound in phosphate buffer pH 7.0 and eluted with 0.1 M citric acid pH 4.0). The yield is low: VP1 is not detectable by SDS-PAGE in the supernatant or in unconcentrated purified fractions, only a few VP1 proteins are visible after concentration to 1 mL (0.16 µg/µL), and the amount is not sufficient for structural analysis, although ELISA and Western blot signal of the purified protein scales with the dilution of the original supernatant.

In 293T cells transfected with tPA-VP1-hFc, anti-human IgG Fc Western blots detect more VP1-hFc protein in lysates than in supernatants. The paper suggests that VP1-hFc may be hydrolyzed into fragments that cannot be purified by protein A, which would indicate that the linkers between the tPA leader, VP1 and hFc are unstable and that proteolysis-resistant linkers should be tested.