Design of an HA2-based Escherichia coli expressed influenza immunogen that protects mice from pathogenic challenge

Abstract

Influenza HA is the primary target of neutralizing antibodies during infection, and its sequence undergoes genetic drift and shift in response to immune pressure. The receptor binding HA1 subunit of HA shows much higher sequence variability relative to the metastable, fusion-active HA2 subunit, presumably because neutralizing antibodies are primarily targeted against the former in natural infection. We have designed an HA2-based immunogen using a protein minimization approach that incorporates designed mutations to destabilize the low pH conformation of HA2. The resulting construct (HA6) was expressed in Escherichia coli and refolded from inclusion bodies. Biophysical studies and mutational analysis of the protein indicate that it is folded into the desired neutral pH conformation competent to bind the broadly neutralizing HA2 directed monoclonal 12D1, not the low pH conformation observed in previous studies. HA6 was highly immunogenic in mice and the mice were protected against lethal challenge by the homologous A/HK/ 68 mouse-adapted virus. An HA6-like construct from another H3 strain (A/Phil/2/82) also protected mice against A/HK/68 challenge. Regions included in HA6 are highly conserved within a subtype and are fairly well conserved within a clade. Targeting the highly conserved HA2 subunit with a bacterially produced immunogen is a vaccine strategy that may aid in pandemic preparedness.hemagglutinin | protein design | bacterial expression

Two distinct regions of HA2 glycopolypeptide of influenza virus hemagglutinin elicit cross-protective immunity against influenza

“…These results are in accordance with previous data on protective immunity induced by immunization with hA2, which was expressed by recombinant vaccinia viruses (Gocník et al, 2008) or E. coli (Bommakanti et al, 2010). In this work, we purified ehA2 according to chen et al (1995).…”

“…Many studies have been focused on the M2 protein (okuda et al, 2001;Fan et al, 2004;liu et al, 2004;DeFilette et al, 2006;hoelscher et al, 2006;DeFilette et al, 2008a,b;huleatt et al, 2008;Zhang et al, 2009;li et al, 2011;Park et al, 2011;Staneková et al, 2011), but recently also the conserved part of hA, hA2 gp, was used as an inductor of cross-protective immunity (okuno et al, 1993;horváth et al, 1998;Gocník et al, 2008;Sui et al, 2009;Bommakanti et al, 2010;Steel et al, 2010;Wang et al, 2010;Staneková et al, 2011). The latter was the subject of our interest for several years.…”

Role of stem glycans attached to haemagglutinin in the biological characteristics of H5N1 avian influenza virus

“…The removal of glycosylation sites on the HA protein can expose conserved epitopes hidden by large glycans to elicit an immune response that recognizes HA variants at a higher titre . Broadly neutralizing HA2 stemspecific monoclonal antibodies with neutralizing activity for H1 clade viruses (Ekiert et al, 2009;Sui et al, 2009) and H3 subtype viruses (Bommakanti et al, 2010;Wang et al, 2010a) have been developed. These studies indicate that epitopes in the stem of HA are conserved and accessible to antibodies.…”

Design of an Escherichia coli Expressed HIV-1 gp120 Fragment Immunogen That Binds to b12 and Induces Broad and Potent Neutralizing Antibodies

“…Two of these are ␤-turns made up of two residues only, and the other two are loops composed of repeats of the sequence GSA. Linker length was based on the C␣-C␣ distance between the residues to be connected as described previously (23). Both constructs have three disulfides, between residues 296 and 331, 378 and 445, and 385 and 418.…”