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. 2011 Mar;85(6):3010-4.
doi: 10.1128/JVI.01241-10. Epub 2010 Dec 29.

Adjuvant-free immunization with hemagglutinin-Fc fusion proteins as an approach to influenza vaccines

Silvia Loureiro  1 , Junyuan Ren, Pongsathon Phapugrangkul, Camilo A Colaco, Christopher R Bailey, Holly Shelton, Eleonora Molesti, Nigel J Temperton, Wendy S Barclay, Ian M Jones
Affiliations

Adjuvant-free immunization with hemagglutinin-Fc fusion proteins as an approach to influenza vaccines

Silvia Loureiro et al. J Virol. 2011 Mar.

Abstract

The hemagglutinins (HAs) of human H1 and H3 influenza viruses and avian H5 influenza virus were produced as recombinant fusion proteins with the human immunoglobulin Fc domain. Recombinant HA-human immunoglobulin Fc domain (HA-HuFc) proteins were secreted from baculovirus-infected insect cells as glycosylated oligomer HAs of the anticipated molecular mass, agglutinated red blood cells, were purified on protein A, and were used to immunize mice in the absence of adjuvant. Immunogenicity was demonstrated for all subtypes, with the serum samples demonstrating subtype-specific hemagglutination inhibition, epitope specificity similar to that seen with virus infection, and neutralization. HuFc-tagged HAs are potential candidates for gene-to-vaccine approaches to influenza vaccination.

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Figures

FIG. 1.
FIG. 1.
Expression and characterization of HA-HuFc fusion proteins. (A) Baculovirus-expressed HA-HuFc was present in cell extracts (track 1) and in supernatant (track 2) and following purification (track 3). (B) Western blot analysis performed with an anti-Hu Ig conjugate showed intermediates present in cell extracts but no evidence of breakdown in the supernatant or following purification. (Tracks are as described for panel A.) (C) Velocity gradient analysis of purified HA-HuFc showed a distribution of solution molecular masses estimated to range from dimers (∼200 kDa) to hexamers (∼600 kDa). No aggregate was present in the heavier fractions. Numbers to the left in panels A and C represent molecular mass markers, with values in kilodaltons. The numbers at the top of panel C represent solution molecular mass makers taken from a parallel gradient analysis, with values in kilodaltons. Panels A and C show stained 10% SDS-PAGE gels, and panel B shows chemiluminescence. Data shown are representative of the H3 subtype and are typical of other HA subtypes.
FIG. 2.
FIG. 2.
Serum responses to HA-HuFc fusion proteins. (A) Sera from mice immunized with H1, H3, and H5 HA-HuFc proteins were used at a 1:1,000 dilution for Western blot analysis of H1, H3, and H5 HA proteins lacking the Fc tag after separation by 10% SDS-PAGE. Track C, control insect cell extract; track 1, H1 HA; track 3, H3 HA; track 5, H5 HA. Numbers to the left of the panel represent molecular mass markers, with values in kilodaltons. (B) Isotyping of the serum responses to each immunogen. Gray bar, H1; bar with diagonal stripes, H3; clear bar, H5.
FIG. 3.
FIG. 3.
Expression, purification, and immunization of H5-HuFc-based receptor binding site mutants. (A) Wild-type (WT) H5 or H5 bearing two site-directed changes as shown was expressed and purified as described in the text. Mutant yields and stability were unaltered compared to parental H5 results. The results determined by 10% SDS-PAGE analysis of the stained final purified materials are shown. Numbers to the left of the panel represent molecular mass markers (track M), with values in kilodaltons. (B) Titration of polyvalent sera by ELISA using H5-Fc. (C) Titration of polyvalent sera by ELISA using wild-type H5 lacking the Fc tag after normalization of the titer as described for panel A. In both panels, the serum sample results are depicted as follows: ⧫, H5 wild type; ▪, H5 L133V plus A138V; ▴, H5 Q196R plus S227N; and ○, normal mouse serum. The assay was done in duplicate, and the averages of the results are plotted. Duplicate data points in the assays did not differ by more than 5%.
FIG. 4.
FIG. 4.
Epitope mapping and neutralization titers of the serum response to H5-HuFc. (A) Polyclonal sera were assayed by ELISA using immobilized peptides (biotinylated peptides absorbed to streptavidin-coated plates) spanning the HA1 domain of H5 HA. Individual mouse H5 sera gave the same profiles. Mutant sera failed to give a significant reaction. Inset: the reactive peptides from the pepscan displayed on the HA1 domain of the H5 structure. The location of the sialic binding site is indicated (SA). (B) Serum neutralization data measured by an entry inhibition assay of a lentivirus vector pseudotyped with wild-type H5 from strain A/Vietnam/1194/04. Clear bar, H5 Q196R plus S227N; gray bar, H5 L133V plus A138V; black bar, H5 wild type. The data represent averages of the results of duplicate assays.
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