A Carboxy-Terminal Trimerization Domain Stabilizes Conformational Epitopes on the Stalk Domain of Soluble Recombinant Hemagglutinin Substrates

Abstract

Recently, a new class of broadly neutralizing anti-influenza virus antibodies that target the stalk domain of the viral hemagglutinin was discovered. As such, induction, isolation, characterization, and quantification of these novel antibodies has become an area of intense research and great interest. Since most of these antibodies bind to conformational epitopes, the structural integrity of hemagglutinin substrates for the detection and quantification of these antibodies is of high importance. Here we evaluate the binding of these antibodies to soluble, secreted hemagglutinins with or without a carboxy-terminal trimerization domain based on the natural trimerization domain of T4 phage fibritin. The lack of such a domain completely abolishes binding to group 1 hemagglutinins and also affects binding to group 2 hemagglutinins. Additionally, the presence of a trimerization domain positively influences soluble hemagglutinin stability during expression and purification. Our findings suggest that a carboxy-terminal trimerization domain is a necessary requirement for the structural integrity of stalk epitopes on recombinant soluble influenza virus hemagglutinin.

Expression of Functional Recombinant Hemagglutinin and Neuraminidase Proteins from the Novel H7N9 Influenza Virus Using the Baculovirus Expression System

“…In order to preserve these oligomerizations, we add a C-terminal trimerization domain to the HA-, and a N-terminal tetramerization domain to the NA constructs (Figure 1). We have shown conclusively that such a trimerization domain stabilizes functional, conserved conformational epitopes on the stalk-domain of HA 1 . The correct tetramerization of the NA might also contribute to correct folding and NA function 10 .…”

“…These domains ensure correct folding of the molecules usually assisted by the transmembrane domain, which is not present if only the ectodomain is expressed. For example, the expression of the HA ectodomain without a trimerization domain leads to oligomerization and destabilization of important neutralizing epitopes in the stalk domain 1 . Further, the stability of the proteins is decreased and degradation by insect cell and baculovirus proteases occurs.…”

“…The choice of the trimerization or tetramerization domain is less important, a number of them ranging from leucine zipper domains 22 to T4 phage foldons 11 have been tested so far and all showed satisfying results. It is important to keep in mind that multibasic cleavage sites as they occur in highly pathogenic H5N1 24 or H7N7 25 isolates (but not in H7N9) have to be removed before expression since they are cleaved by furin like proteases which are present at all times in the baculovirus expression system 1,12 .…”

“…Using these plasmids we were able to quickly construct baculoviral expression vectors for production of recombinant HA and NA in insect cells. This expression system is well established in our laboratory and has proven pivotal to many of our ongoing research projects [1][2][3][4][5][6][7][8] . Insect cells are able to correctly fold and post-translationally modify complex proteins.…”

Hemagglutinin Stalk-Based Universal Vaccine Constructs Protect against Group 2 Influenza A Viruses

“…Recombinant baculoviruses for the expression of cH4/3 (H4 globular head domain from A/duck/ Czech/56 in combination with the H3 stalk domain from Perth09), cH5/3 (H5 globular head domain from A/Viet Nam/1203/04 in combination with the stalk domain from Perth09), cH7/3 (H7 globular head from A/mallard/Alberta/24/01 on top of the H3 stalk domain of Perth09), and Perth09 HA were generated as described elsewhere (3,28) and were propagated in Sf9 cells. For expression, High Five cells were infected with recombinant baculoviruses at a multiplicity of infection of approximately 10, transferred into Fernbach flasks, and incubated at 28°C, with shaking.…”