Soluble recombinant influenza vaccines.

Soluble, recombinant forms of influenza 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 > di > 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-specific. A third influenza 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 influenza 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.

A universal influenza A vaccine based on the extracellular domain of the M2 protein.

The antigenic variation of influenza virus represents a major health problem. However, the extracellular domain of the minor, virus-coded M2 protein is nearly invariant in all influenza A strains. We genetically fused this M2 domain to the hepatitis B virus core (HBc) protein to create fusion gene coding for M2HBc; this gene was efficiently expressed in Escherichia coli. Intraperitoneal or intranasal administration of purified M2HBc particles to mice provided 90-100% protection against a lethal virus challenge. The protection was mediated by antibodies, as it was transferable by serum. The enhanced immunogenicity of the M2 extracellular domain exposed on HBc particles allows broad-spectrum, long-lasting protection against influenza A infections.

Protection of mice against a lethal influenza virus challenge after immunization with yeast-derived secreted influenza virus hemagglutinin.

The A/Victoria/3/75 (H3N2-subtype) hemagglutinin (HA) gene was engineered for expression in Pichia pastoris as a soluble secreted molecule. The HA cDNA lacking the C-terminal transmembrane anchor-coding sequence was fused to the Saccharomyces cerevisiae alpha-mating factor secretion signal and placed under control of the methanol-inducible P. pastoris alcohol oxidase 1 (AOX1) promoter. Growth of transformants on methanol-containing medium resulted in the secretion of recombinant non-cleaved soluble hemagglutinin (HA0s). Remarkably, the pH of the induction medium had an important effect on the expression level, the highest level being obtained at pH 8.0. The gel filtration profile and the reactivity against a panel of different HA-conformation specific monoclonal antibodies indicated that HA0s was monomeric. Analysis of the N-linked glycans revealed a typical P. pastoris type of glycosylation, consisting of glycans with 10-12 glycosyl residues. Mice immunized with purified soluble hemagglutinin (HA0s) showed complete protection against a challenge with 10 LD50 of mouse-adapted homologous virus (X47), whereas all control mice succumbed. Heterologous challenge with X31 virus [A/Aichi/2/68 (H3N2-subtype)], resulted in significantly higher survival rates in the immunized group compared with the control group. These results, together with the safety, reliability and economic potential of P. pastoris, as well as the flexibility and fast adaptation of the expression system may allow development of an effective recombinant influenza vaccine.

An antibody which binds to the membrane-proximal end of influenza virus haemagglutinin (H3 subtype) inhibits the low-pH-induced conformational change and cell-cell fusion but does not neutralize virus.

A monoclonal antibody, LMBH6, was derived from mice which had been sequentially immunized with bromelain-cleaved haemagglutinin (BHA) from influenza virus A/Aichi/2/68, A/Victoria/3/75 and A/Philippines/2/82 (all H3N2). LMBH6 recognizes the haemagglutinin (HA) of all H3N2 influenza A strains tested, which were isolated between 1968 and 1989. HA in the low-pH-induced conformation is not recognized, and cleavage of the HA0 precursor to HA1 and HA2 is needed to obtain efficient binding. Compared to other monoclonal antibodies, binding of LMBH6 to virus and to virus-infected cells is weak, while binding to BHA is comparable. Electron microscopy demonstrates binding to the membrane proximal end of the stem structure. The antibody shows no haemagglutination-inhibition activity, but inhibits polykaryon formation and the low-pH-induced conformational change of BHA. However, LMBH6 cannot prevent infection of MDCK cells but slows the growth of virus when included in a plaque assay overlay.

pH-dependent aggregation and secretion of soluble monomeric influenza hemagglutinin.

We previously reported the expression of soluble A/Victoria/3/75 (H3N2) hemagglutinin in insect cells and the molecular and immunological structure of an aggregated fraction, only observed in cell supernatant when expression was performed at low pH [23]. Here we report that besides this aggregated a monomeric and possibly a trimeric structure is detected in cell supernatant, irrespective of the pH of the medium. Evidence is presented that the aggregated fraction is generated out of monomeric HAOs molecules due to a low intracellular pH encountered during secretion.

Recombinant neuraminidase vaccine protects against lethal influenza.

The N2 neuraminidase gene of A/Victoria/3/75 influenza virus was engineered to encode a secretable protein (NAs) by replacing the natural N-terminal membrane anchor sequence with the cleavable signal sequence of the corresponding influenza hemagglutinin gene. Soluble NAs was expressed by a baculovirus/insect cell system and accumulated in the medium at levels between 6 and 8 microgram ml-1. A combination of biochemical and standard chromatographic techniques allowed the purification of NAs to homogeneity. Cross-linking analysis indicated that NAs was partly recovered as an authentic tetrameric protein, while the remaining fraction was composed of dimeric molecules and small amounts of monomeric NAs. Purified NAs was supplemented with low-reactogenic adjuvants and used to immunize mice. After a challenge infection with a lethal dose of homologous mouse-adapted X47 influenza virus, vaccinated animals showed resistance against severe disease symptoms and were protected from lethality. Based on the results of a passive immunization experiment, it may be concluded that performed antibody plays a central role in the mechanism by which vaccination with NAs confers viral protection.

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

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.

A fairly conserved epitope on the hemagglutinin of influenza A (H3N2) virus with variable accessibility to neutralizing antibody.

A monoclonal antibody LMBH5 was derived from mice which had been immunized with A/Victoria/3/75 (H3N2)-type recombinant, secreted hemagglutinin (HA), and were subsequently challenged with a potentially lethal dose of X31 [A/Aichi/2/68 (H3N2) x A/PR/8/34 (H1N1)] virus. LMBH5 reacted strongly with the native and low-pH-induced conformations of the HA of A/Aichi (X31 strain) and A/Victoria (X47 strain), but very weakly with the native structure of the HA of A/Philippines/2/82 (X79 strain) and not at all with the HA of A/Guizhou/54/89 H3 (NIB25 strain). However, the acid-induced conformations of the latter two viruses were recognized by LMBH5. The antibody prevented infection of MDCK cells with X31 and X47, whereas X79 virus was partially neutralized by LMBH5. X31 monoclonal escape variants had single amino acid substitutions (Ser 227-->Pro) near the interface. The data obtained suggest that the neutralizing LMBH5 reacts with a fairly conserved epitope of influenza A (H3N2) virus, which as a result of antigenic drift becomes inaccessible in the native state of the HA.

Recombinant secreted haemagglutinin protects mice against a lethal challenge of influenza virus.

Balb/c mice were immunized with 2 x 2 micrograms of purified recombinant secreted haemagglutinin, derived from the A/Victoria/3/75 (H3N2) virus. In the first immunization, Ribi adjuvant was used, while for the booster injection a monophosphoryl lipid A/muramyl dipeptide combination was chosen. Mice immunized in this way were 90-100% protected against a challenge with 20 LD50 of mouse-adapted, homologous virus (strain X47). Bromelain-solubilized haemagglutinin gave only 70% protection under comparable conditions.

High-level transient expression of influenza virus proteins from a series of SV40 late and early replacement vectors.

We have constructed a collection of simian virus 40 (SV40) plasmid vectors useful for transient or constitutive expression of cDNA or genomic DNA in animal cells. Most vectors contain several unique restriction sites downstream from the SV40 late or early promoter, and are available with or without the virus-specific splicing signals. The use of these vectors for transient expression in monkey cells of X47 (H3N2) influenza hemagglutinin (HA) and matrix protein (M1) was demonstrated. Membrane-bound (HAm) as well as secreted forms of the HA glycoprotein lacking the sequence of the C-terminal anchor (HA-) have been obtained. Depending on the insert, the type of vector and the amount of transfected DNA, HA levels in COS cells [Gething and Sambrook, Nature 293 (1981) 620-625] transfected with late replacement SV40 vectors vary from 10(9) (HAm) to 10(8) (HA-) molecules per transfected cell. The maximum expression levels with early replacement vectors in COS cells are at least 50 times lower. In addition to the optimalization and the characterization of the expression of each vector-coded influenza protein, cotransfections, including vectors expressing HAm, neuraminidase (NA) and M1, were undertaken. The latter experiments did not result in a measureable amount of HAm or NA in the cell culture medium, suggesting that expression of these three structural viral proteins does not result in budding of (empty) influenza particles from the cell surface.

Complete nucleotide sequence of the influenza B/Singapore/222/79 virus hemagglutinin gene and comparison with the B/Lee/40 hemagglutinin.

The complete nucleotide sequence of the hemagglutinin (HA) gene of the human type B influenza virus B/Singapore/222/79 is presented. Comparison with the only other known sequence of a B hemagglutinin (B/Lee/40) shows that antigenic drift in type B HA genes is essentially the same as already observed within the influenza A H3 subtype, i.e., an accumulation of point mutations. The main difference is that the apparent evolution is significantly slower, most likely due to the cumulative effect of a lower occurrence in the population (slower evolution) and/or less immunological pressure. There is a striking cluster of changes at positions 127 until 137 of the HA1 subunit which may represent one of the antigenic sites of the molecule.

Antigenic drift between the haemagglutinin of the Hong Kong influenza strains A/Aichi/2/68 and A/Victoria/3/75.

A DNA copy of the gene coding for the influenza A/Aichi/2/68 haemagglutinin protein was cloned in the plasmid pBR322 and the complete nucleotide sequence determined. Comparison of this primary structure and the deduced amino acid sequence with the haemagglutinin gene and protein of strains belonging to the same (H3) subtype and to different subtypes, of both human (H2) and avian (Hav1) origin, documents further at the molecular level the two independent modes of antigenic variation of the virus--drift and shift.

Complete structure of the hemagglutinin gene from the human influenza A/Victoria/3/75 (H3N2) strain as determined from cloned DNA.

The complete sequence of a hemagglutinin (HA) gene of a recent human influenza A strain, A/Victoria/3/75, is 1768 nucleotides long and contains the information for 567 amino acids. It codes for a signal peptide of 16 amino acids, the HA1 chain of the mature hemagglutinin of 329 amino acids, a connecting region between HA1 and HA2 consisting of a single arginine residue and the HA2 portion of 221 amiino acids. The sequence is compared with the hemagglutinin of two members of other subtypes, the human H2 strain A/Jap/305/57 and the avian Hav1 strain A/FPV/Rostock/34, and with one of the same H3 subtype, A/Memphis/3/72. To align the HA1 chain of different major subtypes several deletions/insertions of single amino acids must be invoked, but two more extensive differences are found at both ends, one leading to an extension of the amino terminal sequence of HA1 and the other (four residues) occurring in the region processed away between HA1 and HA2. Comparison of the HA1 of two H3 strains suggests that drift probably depends on single base mutations, some of which change antigenic determinants. The HA2 region, which apparently is not involved in the immune response, is highly conserved even between different subtypes, and single base substitutions account for all the observed diversity. A hydrophobic segment of 24 residues is present in the same position close to the carboxyl terminus of HA2 in both Victoria and FPV, and presumably functions in implantation into the lipid bilayer. The many conserved features not only in HA2 but also in HA1 suggest a rather rigid architecture for the whole hemagglutinin molecule.

Bacteriophage MS2 RNA: a correlation between the stability of the codon: anticodon interaction and the choice of code words.

The non-random distribution of degenerate code words in Bacteriophage MS2 RNA can be explained partially by considerations of the stability of the codon-anticodon complex in prokaryotic systems. Supporting this hypothesis we note that wobble codons are positively selected in codons having G and/or C in the first two positions. In contrast, wobble codons are statistically less likely in codons composed of A and U in the first two positions. Analyses of nucleotides adjacent to 5' and 3' ends of codons indicate a nonrandom distribution as well. It is thus likely that some elements of RNA evolution are independent of the structural needs of the RNA itself and of the translated protein product.

On the possible modulating role of the isoleucine AUA-codon in bacteriophage MS2 RNA.

A set of MS2 mutants were shown to have an additional silent mutation met --> ile at position 108 of the coat protein. As transitions are more frequent than transversions one would have expected an AUA codon in this position in the mutant RNAs. As the AUA codon is one of the best candidates for a modulation role in the control of translation in E. coli, the presence of this AUA in the gene for the protein made in major amounts upon viral infection would impose serious doubt on the theory of modulation. We have directly proven by minifinger-printing of mutant RNA and further analysis of the relevant spots that, in fact, the isoleucine residue at position 108 of the coat protein gene is specified by the non-rate-limiting AUU codon, in agreement with a modulation type of control of protein synthesis.