Molecular and immunological characterization of soluble aggregated A/Victoria/3/75 (H3N2) influenza haemagglutinin expressed in insect cells

Abstract

A/Victoria/3/75 (H3N2)-derived cDNA coding for a secreted haemagglutinin (HA0s) was cloned into the polyhedrin promoter-based pVL1392 transfer vector, and a recombinant baculovirus was isolated. 5 to 10 micrograms/ml of secreted HA were obtained following infection of Spodoptera frugiperda-9 cells. Gel filtration revealed the presence in the cell supernatant of immunoreactive HA molecules with varying M(r). The high M(r) fraction (aHA0s) could be purified by Matrex Cellufine Sulphate and Lentil-lectin affinity chromatography, followed by Sephacryl S300 HR gel filtration. aHA0s consisted of aggregated, non-covalently linked subunits which were not cleaved into HA1 and HA2 polypeptides; aHA0s was highly susceptible to trypsin treatment and reacted with two low pH-specific monoclonal antibodies, suggesting that a HA0s consists of monomeric subunits. When the expression medium was adjusted to pH 8.5, no aHA0s was observed, suggesting that aggregation occurred in the cells due to a low intracellular pH. Balb/c mice immunized with purified aHA0s developed high, aHA0s-specific antibody titres. Despite these high titres, almost no binding to trimeric viral HA was observed, and immunized mice were not protected against a challenge with homologous mouse-adapted X47 virus. However, when virus was subjected to low pH, resulting in a profound conformational rearrangement, strong binding was observed. Moreover, binding to the low pH-treated HA of different drift variants, isolated between 1968 and 1989, occurred.

Downstream processing of Vero cell-derived human influenza A virus (H1N1) grown in serum-free medium

“…Lectin-affinity chromatography or membrane [9,25] is also employed to capture influenza virus but the potential toxicity limited their application. CSC has been applied for the capture of MDCK cell-derived influenza viruses [27] and recombinant HA [41]. The overall yields in CSC unit operation vary tremendously in different studies, from 21% [42] to 67.6% [25].…”

Single-dose monomeric HA subunit vaccine generates full protection from influenza challenge

“…Monomeric HA is the easiest form of subunit protein to produce, but has typically been ineffective in vaccine settings. 17,[43][44][45] Our ability to stimulate potent and effective single dose HA vaccines comes from the association of monomeric HA with TMV, a plant virus that in this context is acting both as a viral antigen display scaffold, and viral delivery mechanism. TMV can directly stimulate dendritic cell (DC) antigen uptake in vitro, 24 along with DC activation, migration of DCs to lymphoid organs and robust stimulation of antigen-specific B and T cells in vivo.…”

A plant-produced H1N1 trimeric hemagglutinin protects mice from a lethal influenza virus challenge

“…22 Unlike the HA molecules on the viral surface that exist as homotrimers, recombinant HA molecules tend to be expressed as either aggregates or monomers depending on the expression system and target antigen strain. 23 Mimicking the authentic trimeric HA (tHA) structure presented on the virus surface may provide a way to improve the immunogenicity of recombinant HA-based vaccines. One approach to address this is to generate VLPs using recombinant DNA technology.…”