A Fusion Protein Based on the Second Subunit of Hemagglutinin of Influenza A/H2N2 Viruses Provides Cross Immunity

Abstract

Conserved fragments of the second subunit of hemagglutinin (HA2) are of great interest for the design of vaccine constructs that can provide protective immunity against influenza A viruses of different subtypes. A recombinant fusion protein, FlgMH, was constructed on the basis of flagellin and a highly conserved HA2 fragment (35-107) of influenza viruses of the subtype A/H2N2, containing B cell, CD4+ T cell, and CD8+ T cell epitopes. The native conformation of the HA2 fragment was partially preserved upon its attachment to the C-terminus of flagellin within the recombinant fusion protein FlgMH. FlgMH was shown to stimulate a mixed Th1/Th2 response of cross-reactive antibodies, which bind to influenza viruses of the first phylogenetic group (H1, H2, H5), to the target sequence as well as the induction of specific cytotoxic T cells (CD3+CD8+IFNγ+). Immunization with the recombinant protein protected animals from a lethal influenza infection. The developed FlgMH protein is a promising agent that may be included in an influenza vaccine with a wide spectrum of action which will be able to stimulate the T and B cell immune responses.

Keywords: HA2; flagellin; influenza; recombinant protein; vaccine.

Fig. 1

A – alignment of HA2 consensus sequences of influenza A viruses of different subtypes. Two highly conserved fragments with sequence identities of 78.3 and 62.5%, respectively, are shown in blue boxes. A fragment selected for inclusion in the fusion protein is underlined. B – the three-dimensional structure of the hemagglutinin molecule (trimer, a model 3WR7 from the Protein Data Bank). The HA2 fragment (35–107) of one of the monomers is shown in red. Main fragments: the small α-helix (35–58); an unstructured region (59–73); a fragment of the large α-helix containing a highly conserved fragment (74–107). C – amino acid frequency plot of a HA2(35-107) fragment of human influenza A/H2N2 hemagglutinin (in HA0 numbering), the less conserved residue (Arg416 – 58.3%) is indicated by a red arrow.

Fig. 2

A – experimental B and CD4+ T cell epitopes homologous to the HA2 consensus sequence fragment (35–107) at least by 90%; based on the search in the IEDB database. B – potential CD8+ T cell epitopes included in the HA2 fragment (35–107) for a representative set of HLA alleles; the result of an analysis using the NetCTLpan1.1 Server [19].

Fig. 3

A – map of a plasmid encoding the protein FlgMH. BamHI and HindIII are cloning sites of the gene encoding the fusion protein FlgMH into the vector pQE30. ColE1 – origin of replication; PT5 – the T5 promoter; AmpR – the β-lactamase gene, an ampicillin resistance marker; His-tag – a N-terminal histidine tag. B – a theoretical model of the three-dimensional structure of a monomer of the fusion protein FlgMH: the HA2 fragment (35–107) is shown in red; flagellin is shown in violet; the histidine tag is shown in green.

Fig. 4

A – expression of recombinant proteins in E. coli cells. 1 – molecular weight markers (Fermentas, EU) denoted in kDa; 2–4 – lysates of cells transformed with the plasmid pQE30 carrying an indicated insert; 5 – a lysate of cells transformed with the vector pQE30 without an insert. Bands of recombinant proteins are indicated by arrows. B – recombinant proteins FlgMH, Flg, and MH after chromatographic purification on a Ni-sorbent. Results of electrophoresis and Western blot using polyclonal antibodies to flagellin (6–8) and antiserum to a fragment MH (9–12) are presented.

Fig. 5

Antibody titers in mice of the experimental and control groups 2 weeks after triple immunization. A – titers of IgA antibodies, in serum and BAL, to the target antigen MH. B – GMT of IgG subclasses to the target antigen MH in mice of the experimental group. C – GMT of serum IgG to influenza viruses A/PR/8/34 (H1N1), A/Singapore/1/57 (H2N2), A/Aichi/2/68 (H3N2), and A/Kurgan/05/2005 (H5N1). The Mann-Whitney test was used to calculate the p value.

Fig. 6

IFNγ production by spleen CD8+ T-cells after re-stimulation with the G-47, G-48, and G-49 peptides and the influenza virus A/Singapore/1/57 (H2N2) in mice C57Bl/6 (A) and Balb/c (B). The spleens were taken from three mice each of the experimental and control groups 14 days after triple immunization. * – the difference from control p < 0.05 (Mann-Whitney test).

Fig. 7

Efficacy of FlgMH immunization. Two weeks post second boost mice were challenged with 2LD50 A/Singapore/ 1/57 (H2N2). Body weight (A) and survival rate (B) were monitored daily during 14 days. The p value was calculated using the Mantel-Cox test.