Enhanced Immunogenicity of Stabilized Trimeric Soluble Influenza Hemagglutinin

Abstract

BackgroundThe recent swine-origin H1N1 pandemic illustrates the need to develop improved procedures for rapid production of influenza vaccines. One alternative to the current egg-based manufacture of influenza vaccine is to produce a hemagglutinin (HA) subunit vaccine using a recombinant expression system with the potential for high protein yields, ease of cloning new antigenic variants, and an established safety record in humans.Methodology/Principal FindingsWe generated a soluble HA (sHA), derived from the H3N2 virus A/Aichi/2/68, modified at the C-terminus with a GCN4pII trimerization repeat to stabilize the native trimeric structure of HA. When expressed in the baculovirus system, the modified sHA formed native trimers. In contrast, the unmodified sHA was found to present epitopes recognized by a low-pH conformation specific monoclonal antibody. We found that mice primed and boosted with 3 µg of trimeric sHA in the absence of adjuvants had significantly higher IgG and HAI titers than mice that received the unmodified sHA. This correlated with an increased survival and reduced body weight loss following lethal challenge with mouse-adapted A/Aichi/2/68 virus. In addition, mice receiving a single vaccination of the trimeric sHA in the absence of adjuvants had improved survival and body weight loss compared to mice vaccinated with the unmodified sHA.Conclusions/SignificanceOur data indicate that the recombinant trimeric sHA presents native trimeric epitopes while the unmodified sHA presents epitopes not exposed in the native HA molecule. The epitopes presented in the unmodified sHA constitute a “silent face” which may skew the antibody response to epitopes not accessible in live virus at neutral pH. The results demonstrate that the trimeric sHA is a more effective influenza vaccine candidate and emphasize the importance of structure-based antigen design in improving recombinant HA vaccines.

Bacterial HA1 Vaccine against Pandemic H5N1 Influenza Virus: Evidence of Oligomerization, Hemagglutination, and Cross-Protective Immunity in Ferrets

“…These earlier HA-structural studies did not describe the oligomerization signal in the HA1 globular domain identified in the present study, suggesting that in the presence of HA2 the N-terminus ␤-sheet structure is engaged in an HA1-HA2 bridge and not in HA1 oligomerization. This may explain why most recombinant HA ectodomain proteins exist as monomers and require the addition of multimerization sequences such as "foldon" at the C terminus in order to produce stable oligomeric structures (3,28,29). This was further confirmed in a recent study in our laboratory with bacterially expressed HA proteins from the novel H1N1 A/California/04/2009 comparing the composition and immunogenicity of globular HA1 to that of the HA ectodomain (1-480).…”

“…More recent publications emphasized the importance of high-MW oligomers for optimal immunogenicity of influenza virus recombinant HA proteins. Trimerization domains such as GCN4-pII and foldon were added to the C termini of HA ectodomains produced in mammalian or insect cells in order to produce stable oligomeric structures and to elicit optimal neutralizing antibody titers (3,28,29). On the other hand, our study demonstrates oligomer formation with bacterially expressed HA1 with no requirement for the addition of any foreign trimerization sequence.…”

One-step Purification of Twin-Strep-tagged Proteins and Their Complexes on Strep-Tactin Resin Cross-linked With Bis(sulfosuccinimidyl) Suberate (BS3)

“…For this purpose, we employed a water-soluble, non-cleavable, homobifunctional amine-reactive cross-linker BS3 with a history of successful use in stabilizing protein structures [10][11][12][13] . Figure 5 shows the chemical formula of BS3 and its cross-linking reaction with free epsilon amino groups of lysine in Strep-Tactin.…”

A recombinant protein containing highly conserved hemagglutinin residues 81-122 of influenza H5N1 induces strong humoral and mucosal immune responses

“…It has been reported that the immunogenicity caused by direct expression of the major vaccine antigen HA protein is often low, requiring large doses of vaccines to generate a level of seroconversion consistent with protection (25,26). Because of the importance of structure-based antigen, oligomeric HA is more efficient than monomeric HA (27). However, oligomerization is usually ensured through the addition of an extraneous sequence of unknown risk for human immunization.…”