Soluble recombinant influenza vaccines

Abstract

Soluble, recombinant forms of in£uenza A virus haemagglutinin and neuraminidase have been produced in cells of lower eukaryotes, and shown in a mouse model to induce complete protective immunity against a lethal virus challenge. Soluble neuraminidase, produced in a baculovirus system, consisted of tetramers, dimers and monomers. Only the tetramers were enzymatically active. The immunogenicity decreased very considerably in the order tetra 4 di 4 mono. Therefore, we fused the head part of the neuraminidase gene to a tetramerizing leucine zipper sequence; the resulting product was enzymatically active, tetrameric neuraminidase. The protective immunity induced by this engineered neuraminidase, however, remained fairly strain-speci¢c. A third in£uenza A virus protein, the M2 protein, has only 23 amino acids exposed on the outer membrane surface. This extracellular part, M2e, has been remarkably conserved in all human in£uenza A strains since 1933. By fusing the M2e sequence to hepatitis B virus core protein, we could obtain highly immunogenic particles that induced complete, strain-independent, long-lasting protection in mice against a lethal viral challenge. Native M2 is a tetrameric protein and this conformation of the M2e part can also be mimicked by fusing this sequence to a tetramerizing leucine zipper. The potential of the resulting protein as a vaccine candidate remains to be evaluated.

Prevention of influenza virus shedding and protection from lethal H1N1 challenge using a consensus 2009 H1N1 HA and NA adenovirus vector vaccine

“…Immunity to NA reduced clinical symptoms of disease in both mice and ferrets. This finding is in agreement with other reports that vaccination against both targets results in a broader protective immunity against influenza infection than vaccination against HA alone [29,30]. …”

A Generic System for the Expression and Purification of Soluble and Stable Influenza Neuraminidase

“…These results suggest that the replacement of the NA stalk by more stable and rigid domains that are known to form parallel homotetrameric structures could promote greater NA stability. This approach was first formulated by Fiers et al for influenza NA [20] and its feasibility to promote homotetramerization has been shown for proteins such as the influenza M2 [31] or human p53 [32] . These publications made use of the yeast transcription factor GCN4 as a heterologous domain to promote tetramerization.…”

“…Thrombin) has overcome the problem of non-specific cleavage they still could render the stalk-less NA head prone to dissociation into non-active NA monomers or dimers. The generic expression system outlined in the present publication aims to overcome these obstacles by a combination of the melittin signalling peptide (MSP) secretion signal [18] , FLAG tag [19] and the use of two artificial tetramerization domains from yeast (GCN4-pLI, [20] , [21] ) and the deep sea archae bacterium Staphylothermus marinus (Tetrabrachion, [22] ). The latter has been shown to be stable at temperatures up to 130°C making it a potentially good choice to stabilize a tetrameric active form of secreted soluble NA [23] .…”

M2e-tetramer-specific memory CD4 T cells are broadly protective against influenza infection

“…[28][29][30][31][32][33] M2 is expressed in low copy numbers on the virion, but infected target cells abundantly express M2 on their surface where it is accessible for antibodies. 30,32,34,35 M2e-specific antibodies are not neutralizing. Rather, the protective mechanism of action for M2e-specific IgG antibodies depends on FcgR expression on alveolar macrophages and antibody-dependent cellular cytotoxicity or phagocytosis, which is similar to how anti-HA stem IgG antibodies protect.…”