J Virol. 2005 Jun ;79 (12):7380-8 15919893 (P
Elisabetta Bianchi, Xiaoping Liang, Paolo Ingallinella, Marco Finotto, Michael A Chastain, Jiang Fan, Tong-Ming Fu, Hong Chang Song, Melanie S Horton, Daniel C Freed, Walter Manger, Emily Wen, Li Shi, Roxana Ionescu, Colleen Price, Marc Wenger, Emilio A Emini, Riccardo Cortese, Gennaro Ciliberto, John W Shiver, Antonello Pessi
Department of Molecular & Cell Biology, IRBM P. Angeletti, Via Pontina Km 30.600, 00040 Pomezia (Rome) Italy.
Conventional influenza vaccines can prevent infection, but their efficacy depends on the degree of antigenic “match” between the strains used for vaccine preparation and those circulating in the population. A universal influenza vaccine based on invariant regions of the virus, able to provide broadly cross-reactive protection, without requiring continuous manufacturing update, would solve a major medical need. Since the temporal and geographical dominance of the influenza virus type and/or subtype (A/H3, A/H1, or B) cannot yet be predicted, a universal vaccine, like the vaccines currently in use, should include both type A and type B influenza virus components. However, while encouraging preclinical data are available for influenza A virus, no candidate universal vaccine is available for influenza B virus. We show here that a peptide conjugate vaccine, based on the highly conserved maturational cleavage site of the HA( ) precursor of the influenza B virus hemagglutinin, can elicit a protective immune response against lethal challenge with viruses belonging to either one of the representative, non-antigenically cross-reactive influenza B virus lineages. We demonstrate that protection by the HA( ) vaccine is mediated by antibodies, probably through effector mechanisms, and that a major part of the protective response targets the most conserved region of HA( ), the P1 residue of the scissile bond and the fusion peptide domain. In addition, we present preliminary evidence that the approach can be extended to influenza A virus, although the equivalent HA( ) conjugate is not as efficacious as for influenza B virus.
Mesh-terms: Amino Acid Sequence; Animals; Antibodies, Viral :: blood; Drug Design; Hemagglutinin Glycoproteins, Influenza Virus :: chemistry; Hemagglutinin Glycoproteins, Influenza Virus :: genetics; Hemagglutinin Glycoproteins, Influenza Virus :: metabolism; Humans; Influenza :: prevention & control; Influenza A Virus, Human :: immunology; Influenza B virus :: immunology; Influenza B virus :: pathogenicity; Influenza Vaccines :: administration & dosage; Influenza Vaccines :: chemistry; Influenza Vaccines :: immunology; Mice; Mice, Inbred BALB C; Models, Molecular; Molecular Sequence Data; Peptides :: chemistry; Peptides :: genetics; Peptides :: immunology; Protein Precursors :: chemistry; Protein Precursors :: genetics; Protein Precursors :: metabolism; Vaccines, Conjugate :: administration & dosage; Vaccines, Conjugate :: chemistry; Vaccines, Conjugate :: immunology;
Most cited papers:
The immunogenic and antigenic determinants of a synthetic peptide and the corresponding antigenic determinants in the parent protein have been elucidated. Four determinants have been defined by reactivity of a large panel of antipeptide monoclonal antibodies with short, overlapping peptides (7-28 amino acids), the immunizing peptide (36 amino acids), and the intact parent protein (the influenza virus hemagglutinin, HA). The majority of the antipeptide antibodies that also react strongly with the intact protein recognize one specific nine amino acid sequence. This immunodominant peptide determinant is located in the subunit interface in the HA trimeric structure. The relative inaccessibility of this site implies that antibody binding to the protein is to a more unfolded HA conformation. This antigenic determinant differs from those previously described for the hemagglutinin and clearly demonstrates the ability of synthetic peptides to generate antibodies that interact with regions of the protein not immunogenic or generally accessible when the protein is the immunogen.
Four ‘antigenic sites’ on the three-dimensional structure of the influenza haemagglutinin are identified. At least one amino acid substitution in each site seems to be required for the production of new epidemic strains between 1968 and 1975.
We chemically synthesized 20 peptides corresponding to 75% of the HA1 molecule of the influenza virus. Antibodies to the majority (18) of these peptides were capable of reacting with the hemagglutinin molecule. These 18 peptides are not confined to the known antigenic determinants of the hemagglutinin molecule, but rather are scattered throughout its three-dimensional structure. In contrast, antibody raised to intact hemagglutinin did not react with any of the 20 peptides. Taken together these results suggest that the immunogenicity of an intact protein molecule is not the sum of the immunogenicity of its pieces.
National Institute for Medical Research, London NW7 1AA, England.
Hemagglutinin (HA) is the receptor-binding and membrane fusion glycoprotein of influenza virus and the target for infectivity-neutralizing antibodies. The structures of three conformations of the ectodomain of the 1968 Hong Kong influenza virus HA have been determined by X-ray crystallography: the single-chain precursor, HA0; the metastable neutral-pH conformation found on virus, and the fusion pH-induced conformation. These structures provide a framework for designing and interpreting the results of experiments on the activity of HA in receptor binding, the generation of emerging and reemerging epidemics, and membrane fusion during viral entry. Structures of HA in complex with sialic acid receptor analogs, together with binding experiments, provide details of these low-affinity interactions in terms of the sialic acid substituents recognized and the HA residues involved in recognition. Neutralizing antibody-binding sites surround the receptor-binding pocket on the membrane-distal surface of HA, and the structures of the complexes between neutralizing monoclonal Fabs and HA indicate possible neutralization mechanisms. Cleavage of the biosynthetic precursor HA0 at a prominent loop in its structure primes HA for subsequent activation of membrane fusion at endosomal pH (Figure 1). Priming involves insertion of the fusion peptide into a charged pocket in the precursor; activation requires its extrusion towards the fusion target membrane, as the N terminus of a newly formed trimeric coiled coil, and repositioning of the C-terminal membrane anchor near the fusion peptide at the same end of a rod-shaped molecule. Comparison of this new HA conformation, which has been formed for membrane fusion, with the structures determined for other virus fusion glycoproteins suggests that these molecules are all in the fusion-activated conformation and that the juxtaposition of the membrane anchor and fusion peptide, a recurring feature, is involved in the fusion mechanism. Extension of these comparisons to the soluble N-ethyl-maleimide-sensitive factor attachment protein receptor (SNARE) protein complex of vesicle fusion allows a similar conclusion.
Cell Biology Programme, European Molecular Biology Laboratory, Postfach 102209, 69112 Heidelberg, Germany.
Sphingolipid-cholesterol rafts are microdomains in biological membranes with liquid-ordered phase properties which are implicated in membrane traffic and signalling events. We have used influenza virus haemagglutinin (HA) as a model protein to analyse the interaction of transmembrane proteins with these microdomains. Here we demonstrate that raft association is an intrinsic property encoded in the protein. Mutant HA molecules with foreign transmembrane domain (TMD) sequences lose their ability to associate with the lipid microdomains, and mutations in the HA TMD reveal a requirement for hydrophobic residues in contact with the exoplasmic leaflet of the membrane. We also provide experimental evidence that cholesterol is critically required for association of proteins with lipid rafts. Our data suggest that the binding to specific membrane domains can be encoded in transmembrane proteins and that this information will be used for polarized sorting and signal transduction processes.
We have constructed an operational antigenic map of the hemagglutinin of influenza virus A/PR/8/34, which indicates the presence of five immunodominant antigenic regions exhibiting various degrees of operational linkage. These sites have been located by the identification of changed amino acid residues in mutant viruses that are antigenically altered at each site. Comparison of the antigenic features with the three-dimensional structure of the H3 subtype hemagglutinin shows that the antigenic sites correspond to four topographically distinct regions of the surface of the protein. One of the sites is formed when two regions that are widely separated in the hemagglutinin monomer associate in the assembled trimer. The location of the sites relative to those proposed for the H3 subtype hemagglutinin suggests that carbohydrate modulates the antigenicity of specific regions of the hemagglutinin.
Proc Natl Acad Sci U S A. 1982 Feb ;79 (4):968-72 6951181 (P
A conformational change in the hemagglutinin glycoprotein of influenza virus has been observed to occur to pH values corresponding to those optimal for the membrane fusion activity of the virus. CD, electron microscopic, and sedimentation analyses show that, in the pH range 5.2-4.9, bromelain-solubilized hemagglutinin (BHA) aggregates as protein-protein rosettes and acquires the ability to bind both lipid vesicles and nonionic detergent. Trypsin treatment of BHA in the pH 5. -induced conformation indicates that aggregation is a property of the BHA2 component and that the conformation change also involves BHA1. The implications of these observations for the role of the glycoprotein in membrane fusion are discussed.
The haemagglutinin (HA) glycoproteins of influenza virus membranes are responsible for binding viruses to cells by interacting with membrane receptor molecules which contain sialic acid (for review see ref. 1). This interaction is known to vary in detailed specificity for different influenza viruses (see, for example, refs 2-4) and we have attempted to identify the sialic acid binding site of the haemagglutinin by comparing the amino acid sequences of haemagglutinins with different binding specificities. We present here evidence that haemagglutinins which differ in recognizing either NeuAc alpha 2 leads to 3Gal- or NeuAc alpha 2 leads to 6Gal- linkages in glycoproteins also differ at amino acid 226 of HA1. This residue is located in a pocket on the distal tip of the molecule, an area previously proposed from considerations of the three-dimensional structure of the haemagglutinin to be involved in receptor binding.
Proc Natl Acad Sci U S A. 1993 Dec 15;90 (24):11478-82 8265577 (P
Department of Pathology, University of Massachusetts Medical School, Worcester 01655.
Plasmid DNAs expressing influenza virus hemagglutinin glycoproteins have been tested for their ability to raise protective immunity against lethal influenza challenges of the same subtype. In trials using two inoculations of from 50 to 300 micrograms of purified DNA in saline, 67-95% of test mice and 25-63% of test chickens have been protected against a lethal influenza challenge. Parenteral routes of inoculation that achieved good protection included intramuscular and intravenous injections. Successful mucosal routes of vaccination included DNA drops administered to the nares or trachea. By far the most efficient DNA immunizations were achieved by using a gene gun to deliver DNA-coated gold beads to the epidermis. In mice, 95% protection was achieved by two immunizations with beads loaded with as little as .4 micrograms of DNA. The breadth of routes supporting successful DNA immunizations, coupled with the very small amounts of DNA required for gene-gun immunizations, highlight the potential of this remarkably simple technique for the development of subunit vaccines.
Mesh-terms: Animals; Cell Line; Chickens; DNA, Viral :: administration & dosage; DNA, Viral :: immunology; Fowl Plague :: immunology; Fowl Plague :: prevention & control; Genes, Viral; Hemagglutinin Glycoproteins, Influenza Virus; Hemagglutinins, Viral :: biosynthesis; Hemagglutinins, Viral :: genetics; Influenza :: immunology; Influenza :: prevention & control; Influenza A Virus, Avian :: genetics; Influenza A Virus, Avian :: immunology; Influenza A Virus, Human :: genetics; Influenza A Virus, Human :: immunology; Injections; Injections, Intramuscular; Injections, Intravenous; Mice; Mice, Inbred BALB C; Mucous Membrane; Restriction Mapping; Support, Non-U.S. Gov’t; Support, U.S. Gov’t, P.H.S. ; Transfection; Viral Envelope Proteins :: biosynthesis; Viral Envelope Proteins :: genetics;
Proc Natl Acad Sci U S A. 1983 Dec ;80 (23):7155-9 6580632 (P
A DNA copy of the influenza virus hemagglutinin gene, derived from influenza virus A/Jap/305/57 (H2N2) was inserted into the genome of vaccinia virus under the control of an early vaccinia virus promoter. Tissue culture cells infected with the purified recombinant virus synthesized influenza hemagglutinin, which was glycosylated and transported to the cell surface where it could be cleaved with trypsin into HA1 and HA2 subunits. Rabbits and hamsters inoculated intradermally with recombinant virus produced circulating antibodies that inhibited hemagglutination by influenza virus. Furthermore, vaccinated hamsters achieved levels of antibody similar to those obtained upon primary infection with influenza virus and were protected against respiratory infection with the A/Jap/305/57 influenza virus.
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