US20120128684A1 - Conserved Hemagglutinin Epitope, Antibodies to the Epitope and Methods of Use - Google Patents
Conserved Hemagglutinin Epitope, Antibodies to the Epitope and Methods of Use Download PDFInfo
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- US20120128684A1 US20120128684A1 US13/061,026 US200913061026A US2012128684A1 US 20120128684 A1 US20120128684 A1 US 20120128684A1 US 200913061026 A US200913061026 A US 200913061026A US 2012128684 A1 US2012128684 A1 US 2012128684A1
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- hemagglutinin
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- influenza
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Classifications
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- C—CHEMISTRY; METALLURGY
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- C07K16/08—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses
- C07K16/10—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses from RNA viruses
- C07K16/1018—Orthomyxoviridae, e.g. influenza virus
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- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/12—Antivirals
- A61P31/14—Antivirals for RNA viruses
- A61P31/16—Antivirals for RNA viruses for influenza or rhinoviruses
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P37/00—Drugs for immunological or allergic disorders
- A61P37/02—Immunomodulators
- A61P37/04—Immunostimulants
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
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- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
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- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2299/00—Coordinates from 3D structures of peptides, e.g. proteins or enzymes
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
- C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
- C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
- C07K2319/40—Fusion polypeptide containing a tag for immunodetection, or an epitope for immunisation
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2760/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses negative-sense
- C12N2760/00011—Details
- C12N2760/16011—Orthomyxoviridae
- C12N2760/16022—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2760/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses negative-sense
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Definitions
- the disclosed invention is generally in the field of influenza and specifically in the area of antibodies and immunogens for the treatments for influenza.
- Seasonal influenza A is a scourge of the young and old, killing more than 250,000 worldwide each year, while creating an economic burden for millions (W.H.O. web site who.int/mediacentre/factsheets/2003/fs211/en/. World Health Organization factsheet 211: influenza (2003)).
- Pandemic influenza which occurs when a new virus emerges and infects people globally that have little or no immunity, represents a grave threat to human health: for example, the 1918 “Spanish Flu” pandemic caused an estimated 50 million deaths (Webster, 1918 Spanish influenza: the secrets remain elusive. Proc Natl Acad Sci USA 96, 1164-6 (1999); de Wit & Fouchier, Emerging influenza. J Clin Virol 41, 1-6 (2008)).
- Neuraminidase inhibitors especially oseltamavir (Tamiflu) remain the primary antiviral treatment, but they have limited efficacy if administered late in infection, and widespread use is likely to result in the emergence of resistant viral strains (de Jong et al., Oseltamivir resistance during treatment of influenza A (H5N1) infection. N Engl J Med 353, 2667-72 (2005); W.H.O. Clinical management of human infection with avian influenza A (H5N1) virus. web site who.int/csr/disease/avian_influenza/guidelines/ClinicalManagement07.pdf).
- Xaa 1 is K, Q, R, N, L, G, F, H or Y
- Xaa 2 is S or T
- Xaa 3 is Q or P
- Xaa 4 is F, V, I, M, L, or T
- Xaa 5 is I, T, S, N, Q, D, or A
- Xaa 6 is I, V, M, or L
- Xaa 7 is N, S, T, or D
- Xaa 8 is I, F, V, A, or T.
- nucleic acids encoding the immunogens of the invention and composition comprising the nucleic acids.
- the invention further comprises a method preventing a disease or disorder caused by an influenza virus by administering to person at risk of suffering from said disease or disorder an immunogen composition described herein.
- the method includes further administering an anti-viral drug, a viral entry inhibitor or a viral attachment inhibitor.
- the anti-viral drug is a neuraminidase inhibitor, a HA inhibitor, a sialic acid inhibitor or an M2 ion channel.
- the M2 ion channel inhibitor is amantadine or, rimantadine.
- the neuraminidase inhibitor zanamivir, or oseltamivir phosphate.
- the head region of the hemagglutinin can be modified by removing or replacing glycosylation sites. In some forms, the head region of the hemagglutinin can be modified by adding glycosylation sites. In some forms, the head region of the hemagglutinin can be modified by removing all or a portion of the head region.
- the epitope can comprise both the HA1 and HA2.
- the epitope can be non-linear.
- the epitope can comprise the amino acid position 18, 38, 40, 291 of the HA1 polypeptide and the amino acid at position 18, 19, 20, 21, 38, 41, 42, 45, 49, 52, 53 and 56 of the HA2 polypeptide.
- compositions and methods are further based upon the discovery of a protocol for generating broadly neutralizing human antibodies that target a highly conserved epitope in the stem region of HA.
- a method of producing an isolated antibody that specifically binds a pathogenic enveloped virus by exposing a single chain or Fab expression library to a membrane fusion protein of the virus, identifying an antibody in the library that specifically binds said protein; and isolating the antibody from the library.
- the fusion protein can be immobilized on a solid surface, e.g. plastic.
- the fusion protein can have modified glycosylations compared to a wild type fusion protein.
- the fusion can be produced in a non-mammalian cell, such as an insect cell.
- the fusion protein can be, for example, a trimeric hemagglutinin (HA) protein.
- a method of vaccinating a subject against pathogenic enveloped virus such as an influenza virus by administering to the subject, for example, a membrane fusion protein (e.g., a trimeric hemagglutinin (HA) protein coated) or embedded in a biologically compatible matrix.
- a membrane fusion protein e.g., a trimeric hemagglutinin (HA) protein coated
- HA hemagglutinin
- the fusion protein can have modified glycosylations compared to a wild type fusion protein.
- the invention further provides a method of screening a compound for binding to an F10 antibody by contacting said F10 antibody with a compound of interest and detecting a compound-antibody complex. Also included in the invention are the compound identified by the method and their use as immunogens.
- FIGS. 1A to 1D show in vitro binding and neutralization of anti-H5 antibodies.
- the 10 Abs were converted to soluble scFv-Fcs (scFv linked to Hinge, CH2 and CH3 domains of human IgG1) and evaluated for binding to trimeric H5-TH04 or monomeric HA1 of H5-TH04 coated on an ELISA plate.
- the H5 scFv-Fcs recognizes trimeric H5 but not HA1.
- An antibody raised against HA1 (“2A”) recognized both.
- B Neutralization of H5-TH04-pseudotyped viruses (virus-like particles with HIV-1 only cores that display H5 on their surface). % neutralization at 2 concentrations is shown with standard deviation (s.d.) bars.
- the mAb 80R (Sui et al.
- FIGS. 2A to 2F show prophylactic and therapeutic efficacy of anti-H5 neutralizing mAbs (“nAbs”) in mice.
- nAbs anti-H5 neutralizing mAbs
- a and B Prophylactic efficacy. % survival of mice treated with anti-H5 nAbs or control mAb 1 h before lethal challenge by i.n. inoculation with (A) H5-VN04 or (B) H5-HK97 viruses.
- C-F Therapeutic efficacy. Mice were inoculated with H5-VN04 and injected with nAbs at 24, 48, 72 h post-inoculation (hpi) (C, E and F) or with H5-HK97 at 24 hpi (D).
- FIGS. 3A and 3B show neutralization mechanism.
- A nAbs do not inhibit cell-binding of full-length HA from H5-TH04-pseudotyped HIV-1 viruses. None of the 3 nAb-treated viruses inhibited cell binding; mouse anti-H5 mAb, 17A2.1.2, and ferret anti-H5N1 serum, which inhibit haemagglutination, were used as positive controls; anti-SARS spike protein (“80R”) and anti-HA1 (“2A”) were used as negative controls. Vertical bars represent s.d.
- B All 3 nAbs inhibit cell fusion.
- HeLa cells were transfected with H5-TH04-expressing plasmid and expose a pH 5.0 buffer for 4 mins in the presence or absence of nAbs.
- Syncytia formation induced by brief exposure to pH 5.0 was completely inhibited by D8, F10 and A66, at 20 ⁇ g ml ⁇ 1 ( ⁇ 0.13 ⁇ M), whereas controls (“80R” and anti-HA1 mAb (“2A”)) at the same concentration had no effect.
- FIGS. 4A to 4D show structure of the H5-F10 complex.
- HA1 is depicted by the light colored ribbon diagram at the top of the figure
- HA2 is depicted by the 5 ⁇ helices and 4 ⁇ strands at the bottom of the figure closest to the membrane as well as the 3 long ⁇ helices in the middle of the figure approximately 5.5 cm high
- the ⁇ A-helix of HA2 is represented by 3 vertical ⁇ helices approximately 2 cm high (one is hidden toward the back of the complex)
- the “fusion peptide” (FP) is a thin tubular structure that wraps horizontally around the bottom of the 3 long ⁇ helices of HA2 (identified by an arrow labeled Fusion Protein)
- F10 VH and VL
- One monomer has HA1 depicted by the light-shaded region on the far left of the figure and HA2 depicted by the dark colored region without any residues labeled; the path of FP through the epitope is outlined, while mutations not affecting binding are the lightly shaded region labeled with 50 2 , 54 2 , 57 2 , and 61 2 (see FIG. 4D ).
- the fusion peptides (FP and FP') are labeled in both monomers.
- HA2 epitope residues are labeled 18 2 19 2 20 2 , 21 2 , 38 2 , 41 2 , 42 2 , 45 2 , 49 2 , 52 2 , 53 2 , and 56 2 and HA1 epitope residues are labeled 18 1 , 38 1 , 39 1 , 40 1 , 292 1 and 291 1 .
- the position of buried residue H111 2 is shown as a black ball labeled “H”.
- D Binding of the 3 nAbs to H5 mutants in the ⁇ A helix. Note the very similar response to all mutants tested. Mutations were made either to alanine or to the corresponding H7 residue.
- 293T cells were transiently transfected with mutants; 24 hours after transfection, nAbs or ferret anti-H5N1 serum were used to stain the transfected cells.
- Mean Fluorescent Intensity (MFI) was normalized against ferret anti-serum (100%) to account for different expression levels.
- FIG. 5 shows sequence conservation in HA Groups, Clusters and Subtypes at the F10 epitope. Circles below residue numbers indicate estimated contribution to the binding energy at each position. Positions 19, 21 and 45 of HA2 show a strong contribution. Position 18 of HA1 and positions 18, 20, 41, 49, 52, 53 and 56 of HA2 show intermediate contribution. Positions 38 and 291 of HA1 and positions 38 and 42 of HA2 show neutral contribution. Residues without a circle are not directly involved in the epitope but are discussed in the text. Many residues are conserved among groups, clusters and/or subtypes.
- Residues 18, 20, 21, 41, 42, 44-46, 48, 49, 51-53, 55 and 56 are all highly conversed for all clusters/groups except H12 at residues 48 and 49 and H14 and H3 at residue 49.
- Group 1 has cluster/subtype specific residues at 17 of HA1 and 38 and 111 of HA2.
- Subtype H3 and cluster H7 are identical at HA1 residue 38 and cluster H1b and subtypes H8 and H12 and cluster H3 are similar at HA1 residue 292.
- Group 2 is identical at residue 17 of HA1 and Group 1 cluster H9 is identical at residue 18 of HA1.
- Group 1 cluster H1a and Group 2 are similar at residue 18 of HA1 as well as clusters H1a and H9 at residue 38 of HA1.
- Subtypes H1 and H6 and cluster H1b are identical at residue 291 of HA1.
- Cluster H1a, subtype H8 and cluster H7 are identical at residue 19 of HA2.
- Cluster H3 is identical at residue 38 of HA2 and while residue 111 of HA2 is also identical within the cluster.
- Cluster H1b and cluster H7 are identical at residues 18 and 292 of HA1, respectively.
- Cluster H1b at residue 38 of HA1 and subtypes H2 and H5 and cluster H9 at residue 291 of HA1 are similar.
- Cluster H7 is identical at residues 38 of HA2 and 111 of HA2, respectively.
- H1-OH83 A/Ohio/83 (H1N1)
- H1-PR34 A/Puerto Rico/8/34 (H1N1)
- H1-SC1918 ((A/South Carolina/1/1918 (H1N1))
- H1-WSN33 A/WSN/1933 (H1N1)
- H2-AA60 A/Ann Arbor/6/60 (H2N2)
- H2-JP57 A/Japan/305/57(H2N2)
- H3-SY97 A/Sydney/5/97(H3N2)
- H6-HK99 A/quail/Hong Kong/1721-30/99(H6N1)
- H6-NY98 A/chicken/New York/14677-13/1998 (H6N2)
- H7-FP34 A/FPV/Rostock/34 (H7N1)
- H8-ON68 A/turkey/Ontario/6118/68
- H9-HK(G9)97 A/chicken/H
- FIG. 7 shows 3-dimensional comparison of the F10 epitope in Group 1 and Group 2 HAs.
- H1, H5 and H9 Group 1
- PB codes 1RU7, 21BX and 1JSD H3 and H7
- PB codes 1MQL and 1TI8 H1, H5 and H9
- Y is in Group 1
- H is in Group 2.
- the H/H/Q in the lower left are in Group 1 and the two H in the upper right are in Group 2.
- the two vertical W are in Group 2 while the three tilted W are in Group 1.
- the N are in Group 2 and the H are in Group 1.
- the H are in Group 1 and the T are in Group 2.
- RMS differences for pair-wise overlays are 0.56 ⁇ 0.11 ⁇ (observed range, Group 1); 0.75 ⁇ (Group 2); and 1.21 ⁇ 0.12 ⁇ between groups.
- Consistent differences between phylogenetic groups include the orientation of W21 2 and alternative side-chain directions at 18 1 and 38 1 , which are linked to the packing of buried His111 2 (the putative pH trigger in Group 1; absent in Group 2); and the burial of the larger tyrosine (Group 1) versus histidine (the putative pH trigger in Group 2) at 17 1 .
- N38 1 is glycosylated in 4 members of the Group 2 clusters. Other epitope residues are indicated by numbered circles.
- FIG. 8 shows phylogenetic relationships and sequence comparison among HA subtypes.
- Four clusters of HA subtypes are (1) Cluster H1: H2, H5, H1, H6, H13, H16, and H11; (2) Cluster H9: H8, H12, and H9; (3) Cluster H3: H4, H14, and H3; and (4) Cluster H7: H15, H7, and H10.
- Within Cluster H1 are two subclusters: subcluster H1a: H2, H5, H1, and H6; and subcluster H1b: H13, H16, and H11.
- H1 A/South Carolina/1/1918
- H2 A/Japan/305/1957
- H3 A/Aichi/2/1968
- H4 A/duck/Czechoslovakia/56
- H5 A/VietNam1203/2004
- H6 A/chicken/California/431/00
- H7 A/Netherland/219/03
- H8 A/turkey/Ontario/6118/68
- H9 A/swine/HK/9/98
- H10 A/chicken/Germany/N49
- H11 A/duck/England/56
- H12 A/duck/Alberta/60/76
- H13 A/gull/Maryland/704/77
- H14 A/mallard/Astrakhan/263/1982
- H15 A/shearwater/West Australia/2576/79
- H16 A/black-headed gull/Sweden/2/99
- FIG. 12 shows viral titers in lung, spleen and brain of mice treated with anti-H5 nAbs after H5-VN04 challenge.
- Viral titers were determined in lung, brain, and spleen collected at 96 hpi. Data are displayed in box-and-whiskers form in which the box extends from the 25th to the 75th percentile, with a horizontal line at the median. Whiskers above and below the box indicate the extreme values. Results of Student T-test statistic analysis are noted with a single star (*) for p ⁇ 0.05, and double stars (**) for p ⁇ 0.01. The arrows crossing the Y axis indicate the detection limit of the titration.
- H1-PR34 A/Puerto Rico/8/34 (H1N1)
- H1-WSN33 (A/WSN/1933 (H1N1));
- H9-HK(G9)97 A/chicken/HongKong/G9/97 (H9N2));
- Neutralization titers (0.1 mg ml ⁇ 1 Ab stock solution) of nAb F10 against H1N1-PR34, two swine flu 2009 H1N1 strains, and a control H3N2 virus. 80R is the negative control. Vertical bars and whiskers represent the lowest and highest neutralization titer (2 values of are shown on the y-axis) of 2-3 independent experiments.
- Influenza virus remains a constant public health threat, owing to its ability to evade immune surveillance through rapid genetic drift and reassortment.
- Influenza A is a negative-sense, single-stranded RNA virus, with an eight-segment genome encoding 10 proteins. It belongs to the family Orthomyxoviridae which includes the genera of influenza virus A, B and C as defined by the antigenicity of the nucleocapsid and matrix proteins. Generally, influenza A virus is associated with more severe disease in humans. Influenza A virus is further subtyped by two surface proteins, hemagglutinin (HA) which attaches the virion to the host cell for cell entry, and neuraminidase (NA) which facilitates the spread of the progeny virus by cleaving the host sialic acid attached to the progeny virus or cell surface.
- HA hemagglutinin
- NA neuraminidase
- HA subtypes and 9 NA subtypes which make up all subtypes of influenza A viruses by various combinations of HA and NA. All combinations of the 16 HA and 9 NA virus subtypes are found in water fowl. Of the hundreds of strains of avian influenza A viruses, only four are known to have caused human infections: H5N1, H7N3, H7N7 and H9N2. In general, human infection with these viruses has resulted in mild symptoms and very little severe illness: there has been only one fatal case of pneumonia caused by H7N7. However, the exception is the highly pathogenic H5N1 virus, for which there is no natural immunity in humans.
- RNA polymerase and the selective pressure of host immunity can lead to the accumulation of mutations and change in surface antigenicity of these proteins. This antigenic change is called antigenic drift.
- shuffling of gene segments can occur if two different subtypes of influenza A virus infect the same cell. For example, if a human H3N2 virus and an avian H5N1 virus co-infect a human or other member of a mammalian species, such an event can produce a novel H5N2. This novel virus can then be efficiently transmitted from human to human because all of most of the gene segments come from the human virus.
- Influenza virus hemagglutinin is the most variable antigen of influenza virus, and is responsible for virus entry into cells. It is synthesized as a trimeric precursor polypeptide HA0 which is post-translationally cleaved to two polypeptides HA1 and HA2 linked by a single disulphide bond. The HA1 chain of HA is responsible for the attachment of virus to the cell surface. HA2 mediates the fusion of viral and cell membranes in endosomes, allowing the release of the ribonucleoprotein complex into the cytoplasm. In contrast to HAL the HA2 molecule represents a relatively conserved part of HA.
- Both the HA1 and HA2 chains of HA are immunogenic and antibodies reactive with both chains have been demonstrated after natural infection in humans. While antibodies specific to HA1 are mostly neutralizing, different mechanism of virus neutralization by HA1 specific Mabs in vitro have been described including blocking the receptor site on HA1, intracellular inhibition of virus-cell fusion, or simultaneous attachment inhibition and virus-cell fusion inhibition, depending on antibody concentration. Although less well studied, inhibition of cell fusion by anti-HA2 antibodies has been reported.
- the HA molecule of the H3 subtype was characterized by sequencing the HA of antigenic drift variants and escape mutants, and the antigenic epitopes were mapped on the molecule's three-dimensional structure. Since then, the antigenic sites on H1, H2 and H5 of an avian pathogenic virus were mapped on the three-dimensional structures of H3. After the outbreak of H5N1 infection in humans in Hong Kong in 1997 and the isolation of H9N2 virus from human cases in 1999, the X-ray structures of both proteins were solved. However, antigenic drift of the 1997 swine isolate (A/Duck/Singapore/3/97) that was used to solve the structure, and more recently isolated highly pathogenic strains, is significant. Indeed, there are 28 minor changes and two potentially major changes between the swine isolate (A/Duck/Singapore/3/97) and the HPAI H5N1 strain (A/Vietnam1203/04).
- the clade 2 viruses were found in viruses isolated exclusively from birds in China, Indonesia, Japan, and South Korea.
- the most recent epidemiologic studies analyzed 82 H5N1 viruses isolated from poultry throughout Indonesia and Vietnam, as well as 11 human isolates from southern Vietnam together with sequence data available in public databases, to address questions relevant to virus introduction, endemicity and evolution (Stevens, J. et al. Structure of the uncleaved human H1 hemagglutinin from the extinct 1918 influenza virus. Science 303, 1866-70 (2004)).
- Phylogenetic analysis showed that all viruses from Indonesia form a distinct sublineage of H5N1 genotype Z viruses, suggesting that this outbreak likely originated from a single introduction via spread throughout the country during the past two years.
- Continued virus activities in Indonesia were attributed to transmission via poultry movement within the country, rather than through repeated introductions by bird migration.
- H5N1 viruses have evolved over time into geographically distinct groups within each country.
- HA from A/Vietnam1203/4 was solved. Comparison of its amino acid sequences with the HA genes from HPAI 2004 and 2005 isolates from clade 1 and 2 viruses identified 13 positions of antigenic variation that are mainly clustered around the receptor binding domain, while the rest are within the vestigual esterase domain. Regions of antigenic variation have been identified in H1 and H3 serotypes ( FIG. 13A ). For H1, these sites are designated Sa, Sb, Ca and Cb while for H3, sites are designated A, B, C and D.
- Escape mutants of H5 HAs can be clustered into three epitopes; site 1: an exposed loop (HA1 140-145) that overlaps antigenic sites A of H3 and Ca2 of H 2 ; site 2: HA1 residues 156 and 157 that corresponds to antigenic site B in H3 serotypes; and 3) HA1 129-133, which is restricted to the Sa site in H1 HAs and H9 serotypes.
- site 1 an exposed loop (HA1 140-145) that overlaps antigenic sites A of H3 and Ca2 of H 2
- site 2 HA1 residues 156 and 157 that corresponds to antigenic site B in H3 serotypes
- 3) HA1 129-133 which is restricted to the Sa site in H1 HAs and H9 serotypes.
- nAbs that utilizes the same VH germline gene, IGHV1-69*01, and encodes a CDR3 loop containing a tyrosine at an equivalent position to Y102, from a non-immune library. This indicates that broad anti-HA cross-immunity pre-exists in the H5-naive population, possibly due to previous exposure to H1, and, for library donors born before 1968, H2 subtypes.
- the antibody can have a VH CDR1 sequence that is the same as the VH CDR1 sequence of antibody D7, D8, F10, G17, H40 or A66 or of the consensus VH sequence SEQ ID NO:1. In some forms, the antibody can have a VH sequence that is the same as the VH sequence of antibody D7, D8, F10, G17, H40 or A66 or of the consensus VH sequence SEQ ID NO:1. In some forms, the antibody can have a VL sequence that is the same as the VL sequence of antibody D7, D8, F10, G17, H40, A66, D80, E88, E90, or H98 or of the consensus VL sequence SEQ ID NO:2.
- hemagglutinins are different forms. These forms of hemagglutinins are useful in the preparation of vaccines for influenza.
- the stem region of influenza hemagglutinin in the neutral pH conformation in isolation from other components of influenza virus is disclosed.
- influenza hemagglutinin in the neutral pH conformation in isolation from other components of influenza virus wherein the head region of the hemagglutinin is modified to reduce the antigenicity of the head region.
- the head region of the hemagglutinin can be modified by removing or replacing glycosylation sites.
- the disclosed antibodies and disclosed hemagglutinins can produce an immune reaction in a subject, i.e. immunogenic.
- the disclosed antibodies and disclosed hemagglutinins are formulated in compositions suitable for the use as a vaccine.
- the disclosed hemagglutinins are used for active immunization of a subject to induce an immune reaction in a subject.
- the disclosed antibodies are used for the passive immunization of a subject to provide immediate protection from an influenza infection.
- antibody and “antibodies” are used herein in a broad sense and includes both polyclonal and monoclonal antibodies.
- immunoglobulin molecules also included in the term “antibodies” are fragments or polymers of those immunoglobulin molecules, and human or humanized versions of immunoglobulin molecules or fragments thereof, and can be chosen for their ability to interact with hemagglutinin.
- antibody refers to immunoglobulin molecules and immunologically active portions of immunoglobulin (Ig) molecules, i.e., molecules that contain an antigen binding site that specifically binds (immunoreacts with) an antigen.
- Ig immunoglobulin
- the antibodies can be tested for their desired activity using the in vitro assays described herein, or by analogous methods, after which their in vivo therapeutic and/or prophylactic activities are tested according to known clinical testing methods.
- the three hypervariable regions of a light chain and the three hypervariable regions of a heavy chain are disposed relative to each other in three dimensional space to form an antigen-binding surface.
- the antigen-binding surface is complementary to the three-dimensional surface of a bound antigen, and the three hypervariable regions of each of the heavy and light chains are referred to as “complementarity-determining regions,” or “CDRs.”
- Antibodies can be purified by well-known techniques, such as affinity chromatography using protein A or protein G, which provide primarily the IgG fraction of immune serum. Subsequently, or alternatively, the specific antigen which is the target of the immunoglobulin sought, or an epitope thereof, can be immobilized on a column to purify the immune specific antibody by immunoaffinity chromatography. Purification of immunoglobulins is discussed, for example, by D. Wilkinson (The Engineer, published by The Engineer, Inc., Philadelphia Pa., Vol. 14, No. 8 (Apr. 17, 2000), pp. 25-28).
- mAb monoclonal antibody
- mAb composition refers to an antibody obtained from a substantially homogeneous population of antibodies, i.e., the individual antibodies within the population are identical except for possible naturally occurring mutations that may be present in a small subset of the antibody molecules.
- “monoclonal antibody” can refer to a population of antibody molecules that contain only one molecular species of antibody molecule consisting of a unique light chain gene product and a unique heavy chain gene product.
- the complementarity determining regions (CDRs) of the monoclonal antibody can be identical in all the molecules of the population.
- mAbs contain an antigen binding site capable of immunoreacting with a particular epitope of the antigen characterized by a unique binding affinity for it.
- DNA-based immunization can be used, wherein DNA encoding the epitope or antigen of interest (hemagglutinin, for example) expressed either alone or as a fusion protein with human IgG1 or an epitope tag is injected into the host animal according to methods known in the art (e.g., Kilpatrick K E, et al. Hybridoma.
- An alternate approach to immunizations with either purified protein or DNA is to use antigen expressed in baculovirus.
- the advantages to this system include ease of generation, high levels of expression, and post-translational modifications that are highly similar to those seen in mammalian systems.
- Use of this system involves expressing the epitope or antigen of interest (hemagglutinin, for example) as fusion proteins with a signal sequence fragment.
- the antigen can be produced by inserting a gene fragment in-frame between the signal sequence and the mature protein domain of hemagglutinin (or an engineered portion of hemagglutinin) nucleotide sequence. This results in the display of the foreign proteins on the surface of the virion. This method allows immunization with whole virus, eliminating the need for purification of target antigens.
- peripheral blood lymphocytes are used in methods of producing monoclonal antibodies if cells of human origin are desired, or spleen cells or lymph node cells are used if non-human mammalian sources are desired.
- the lymphocytes can then be fused with an immortalized cell line using a suitable fusing agent, such as polyethylene glycol, to form a hybridoma cell (Goding, “Monoclonal Antibodies: Principles and Practice” Academic Press, (1986) pp. 59-103).
- Immortalized cell lines are usually transformed mammalian cells, including myeloma cells of rodent, bovine, equine, and human origin. Usually, rat or mouse myeloma cell lines are employed.
- the hybridoma cells can be cultured in a suitable culture medium that can, for example, contain one or more substances that inhibit the growth or survival of the unfused, immortalized cells.
- a suitable culture medium that can, for example, contain one or more substances that inhibit the growth or survival of the unfused, immortalized cells.
- the culture medium for the hybridomas typically will include hypoxanthine, aminopterin, and thymidine (“HAT medium”), which substances prevent the growth of HGPRT-deficient cells.
- Useful immortalized cell lines are those that fuse efficiently, support stable high level expression of antibody by the selected antibody-producing cells, and are sensitive to a medium such as HAT medium.
- murine myeloma lines which can be obtained, for instance, from the Salk Institute Cell Distribution Center, San Diego, Calif. and the American Type Culture Collection, Rockville, Md.
- Human myeloma and mouse-human heteromyeloma cell lines also have been described for the production of human monoclonal antibodies (Kozbor, J. Immunol., 133:3001 (1984); Brodeur et al., “Monoclonal Antibody Production Techniques and Applications” Marcel Dekker, Inc., New York, (1987) pp. 51-63).
- the culture medium in which the hybridoma cells are cultured can then be assayed for the presence of monoclonal antibodies directed against hemagglutinin stem region.
- the binding specificity of monoclonal antibodies produced by the hybridoma cells can be determined by immunoprecipitation or by an in vitro binding assay, such as radioimmunoassay (RIA) or enzyme-linked immunoabsorbent assay (ELISA).
- RIA radioimmunoassay
- ELISA enzyme-linked immunoabsorbent assay
- RIA radioimmunoassay
- ELISA enzyme-linked immunoabsorbent assay
- the clones can be subcloned by limiting dilution or FACS sorting procedures and grown by standard methods. Suitable culture media for this purpose include, for example, Dulbecco's Modified Eagle's Medium and RPMI-1640 medium. Alternatively, the hybridoma cells can be grown in vivo as ascites in a mammal.
- the monoclonal antibodies secreted by the subclones can be isolated or purified from the culture medium or ascites fluid by conventional immunoglobulin purification procedures such as, for example, protein A-Sepharose, protein G, hydroxylapatite chromatography, gel electrophoresis, dialysis, or affinity chromatography.
- In vitro methods are also suitable for preparing monovalent antibodies.
- Digestion of antibodies to produce fragments thereof, particularly, Fab fragments can be accomplished using routine techniques known in the art. For instance, digestion can be performed using papain. Examples of papain digestion are described in WO 94/29348 published Dec. 22, 1994 and U.S. Pat. No. 4,342,566.
- Papain digestion of antibodies typically produces two identical antigen binding fragments, called Fab fragments, each with a single antigen binding site, and a residual Fc fragment. Pepsin treatment yields a fragment that has two antigen combining sites and is still capable of cross-linking antigen.
- the fragments can also include insertions, deletions, substitutions, or other selected modifications of particular regions or specific amino acids residues, provided the activity of the antibody or antibody fragment is not significantly altered or impaired compared to the non-modified antibody or antibody fragment. These modifications can provide for some additional property, such as to remove/add amino acids capable of disulfide bonding, to increase its bio-longevity, to alter its secretory characteristics, etc.
- the antibody or antibody fragment should possess a bioactive property, such as specific binding to its cognate antigen.
- Functional or active regions of the antibody or antibody fragment can be identified by mutagenesis of a specific region of the protein, followed by expression and testing of the expressed polypeptide.
- the immunizing agent can include the protein antigen, a fragment thereof or a fusion protein thereof.
- peripheral blood lymphocytes can be used if cells of human origin are desired, or spleen cells or lymph node cells can be used if non-human mammalian sources are desired.
- the lymphocytes can then be fused with an immortalized cell line using a suitable fusing agent, such as polyethylene glycol, to form a hybridoma cell (Goding, Monoclonal Antibodies: Principles and Practice , Academic Press, (1986) pp. 59-103).
- Immortalized cell lines are usually transformed mammalian cells, particularly myeloma cells of rodent, bovine and human origin.
- rat or mouse myeloma cell lines are employed.
- the hybridoma cells can be cultured in a suitable culture medium that can contain one or more substances that inhibit the growth or survival of the unfused, immortalized cells.
- a suitable culture medium that can contain one or more substances that inhibit the growth or survival of the unfused, immortalized cells.
- the culture medium for the hybridomas can include hypoxanthine, aminopterin, and thymidine (“HAT medium”), which substances prevent the growth of HGPRT-deficient cells.
- the culture medium in which the hybridoma cells are cultured can then be assayed for the presence of monoclonal antibodies directed against the antigen.
- the binding specificity of monoclonal antibodies produced by the hybridoma cells can be determined by immunoprecipitation or by an in vitro binding assay, such as radioimmunoassay (RIA) or enzyme-linked immunoabsorbent assay (ELISA). Such techniques and assays are known in the art.
- the binding affinity of the monoclonal antibody can, for example, be determined by, for example, the Scatchard analysis of Munson and Pollard, Anal. Biochem., 107:220 (1980).
- the clones can be subcloned by limiting dilution procedures and grown by standard methods. (See Goding, Monoclonal Antibodies: Principles and Practice , Academic Press, (1986) pp. 59-103). Suitable culture media for this purpose include, for example, Dulbecco's Modified Eagle's Medium and RPMI-1640 medium. Alternatively, the hybridoma cells can be grown in vivo as ascites in a mammal.
- the monoclonal antibodies secreted by the subclones can be isolated or purified from the culture medium or ascites fluid by conventional immunoglobulin purification procedures such as, for example, protein A-Sepharose, hydroxylapatite chromatography, gel electrophoresis, dialysis, or affinity chromatography.
- Monoclonal antibodies can also be made by recombinant DNA methods, such as those described in U.S. Pat. No. 4,816,567.
- DNA encoding the disclosed monoclonal antibodies can be readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that are capable of binding specifically to genes encoding the heavy and light chains of murine antibodies).
- the disclosed hybridoma cells can serve as a source of such DNA.
- Human monoclonal antibodies can be utilized and can be produced by using human hybridomas (see Cote, et al., 1983. Proc Natl Acad Sci USA 80: 2026-2030) or by transforming human B-cells with Epstein Barr Virus in vitro (see Cole, et al., 1985 In: M ONOCLONAL A NTIBODIES AND C ANCER T HERAPY , Alan R. Liss, Inc., pp. 77-96).
- human antibodies can also be produced using additional techniques, including phage display libraries (See Hoogenboom and Winter, J. Mol. Biol., 227:381 (1991); Marks et al., J. Mol. Biol., 222:581 (1991)).
- human antibodies can be made by introducing human immunoglobulin loci into transgenic animals, e.g., mice in which the endogenous immunoglobulin genes have been partially or completely inactivated. Upon challenge, human antibody production is observed, which closely resembles that seen in humans in all respects, including gene rearrangement, assembly, and antibody repertoire. This approach is described, for example, in U.S. Pat. Nos.
- Human antibodies can additionally be produced using transgenic nonhuman animals which are modified so as to produce fully human antibodies rather than the animal's endogenous antibodies in response to challenge by an antigen.
- transgenic nonhuman animals which are modified so as to produce fully human antibodies rather than the animal's endogenous antibodies in response to challenge by an antigen.
- the endogenous genes encoding the heavy and light immunoglobulin chains in the nonhuman host have been incapacitated, and active loci encoding human heavy and light chain immunoglobulins are inserted into the host's genome.
- the human genes can be incorporated, for example, using yeast artificial chromosomes containing the requisite human DNA segments. An animal which provides all the desired modifications can then be obtained as progeny by crossbreeding intermediate transgenic animals containing fewer than the full complement of the modifications.
- a useful form of such a nonhuman animal include, for example, a mouse, and is termed the XenomouseTM as disclosed in PCT publications WO 96/33735 and WO 96/34096.
- This animal produces B cells which secrete fully human immunoglobulins.
- the antibodies can be obtained directly from the animal after immunization with an immunogen of interest, as, for example, a preparation of a polyclonal antibody, or alternatively from immortalized B cells derived from the animal, such as hybridomas producing monoclonal antibodies.
- the genes encoding the immunoglobulins with human variable regions can be recovered and expressed to obtain the antibodies directly, or can be further modified to obtain analogs of antibodies such as, for example, single chain Fv (scFv) molecules.
- scFv single chain Fv
- U.S. Pat. No. 5,916,771 One method for producing an antibody of interest, such as a human antibody, is disclosed in U.S. Pat. No. 5,916,771.
- This method includes introducing an expression vector that contains a nucleotide sequence encoding a heavy chain into one mammalian host cell in culture, introducing an expression vector containing a nucleotide sequence encoding a light chain into another mammalian host cell, and fusing the two cells to form a hybrid cell.
- the hybrid cell expresses an antibody containing the heavy chain and the light chain.
- Retroviral vectors include moloney murine leukemia viruses.
- DNA viral vectors are an example. These vectors include pox vectors such as orthopox or avipox vectors, herpesvirus vectors such as a herpes simplex I virus (HSV) vector (see Geller, A. I. et al., J. Neurochem, 64:487 (1995); Lim, F., et al., in DNA Cloning: Mammalian Systems, D. Glover, Ed. (Oxford Univ. Press, Oxford England) (1995); Geller, A. I. et al., Proc Natl. Acad. Sci.: U.S.A.
- HSV herpes simplex I virus
- the vector can be employed to target essentially any desired target cell.
- stereotaxic injection can be used to direct the vectors (e.g. adenovirus, HSV) to a desired location.
- the particles can be delivered by intracerebroventricular (icy) infusion using a minipump infusion system, such as a SynchroMed Infusion System.
- icy intracerebroventricular
- a method based on bulk flow, termed convection, has also proven effective at delivering large molecules to extended areas of the brain and can be useful in delivering the vector to the target cell.
- convection A method based on bulk flow, termed convection, has also proven effective at delivering large molecules to extended areas of the brain and can be useful in delivering the vector to the target cell.
- Other methods that can be used include catheters, intravenous, parenteral, intraperitoneal and subcutaneous injection, and oral or other known routes of administration.
- antibody encompasses, but is not limited to, whole immunoglobulin (i.e., an intact antibody) of any class.
- Native antibodies are usually heterotetrameric glycoproteins, composed of two identical light (L) chains and two identical heavy (H) chains.
- L light
- H heavy
- each light chain is linked to a heavy chain by one covalent disulfide bond, while the number of disulfide linkages varies between the heavy chains of different immunoglobulin isotypes.
- Each heavy and light chain also has regularly spaced intrachain disulfide bridges.
- Each heavy chain has at one end a variable domain (V(H)) followed by a number of constant domains.
- V(H) variable domain
- Techniques can also be adapted for the production of single-chain antibodies specific to an epitope or antigen of interest (hemagglutinin, for example) (see e.g., U.S. Pat. No. 4,946,778).
- methods can be adapted for the construction of F(ab) expression libraries (see e.g., Huse, et al., 1989 Science 246: 1275-1281) to allow rapid and effective identification of monoclonal F(ab) fragments with the desired specificity for a protein or derivatives, fragments, analogs or homologs thereof.
- One method of producing proteins comprising the antibodies is to link two or more peptides or polypeptides together by protein chemistry techniques.
- peptides or polypeptides can be chemically synthesized using currently available laboratory equipment using either Fmoc (9-fluorenylmethyloxycarbonyl) or Boc (tert-butyloxycarbonoyl) chemistry. (Applied Biosystems, Inc., Foster City, Calif.).
- Fmoc 9-fluorenylmethyloxycarbonyl
- Boc tert-butyloxycarbonoyl
- V HH the heavy chain variable region
- V H the heavy chain variable region
- the main differences between camelid V HH variable regions and those derived from conventional antibodies (V H ) include (a) more hydrophobic amino acids in the light chain contact surface of V H as compared to the corresponding region in V HH , (b) a longer CDR3 in V HH , and (c) the frequent occurrence of a disulfide bond between CDR1 and CDR3 in V HH . immunoglobulin molecule.
- an for use in the disclosed methods can lack at least a portion of a C H2 domain (e.g., all or part of a C H2 domain).
- a C H2 domain e.g., all or part of a C H2 domain.
- these domains e.g., the heavy chain portions
- these domains can be modified such that they vary in amino acid sequence from the naturally occurring immunoglobulin molecule.
- the disclosed antibodies (such as, for example, HA stem antibodies, and epitope- or antigen-binding fragments, variants, or derivatives thereof), the heavy chain portions of one polypeptide chain of a multimer can be identical to those on a second polypeptide chain of the multimer.
- heavy chain portion-containing monomers need not be identical.
- each monomer can comprise a different target binding site, forming, for example, a bispecific antibody.
- the heavy chain portions of an antibody can be derived from different immunoglobulin molecules.
- a heavy chain portion of an antibody can comprise a C H1 domain derived from an IgG1 molecule and a hinge region derived from an IgG3 molecule.
- a heavy chain portion can comprise a hinge region derived, in part, from an IgG1 molecule and, in part, from an IgG3 molecule.
- a heavy chain portion can comprise a chimeric hinge derived, in part, from an IgG1 molecule and, in part, from an IgG4 molecule.
- the term “light chain portion” includes amino acid sequences derived from an immunoglobulin light chain.
- the light chain portion can comprise at least one of a V L or C L domain.
- the disclosed antibodies can be described or specified in terms of the epitope(s) or portion(s) of an antigen, for example, a target protein or polypeptide (a hemagglutinin composition, hemagglutinin fragment, or hemagglutinin stem portion, for example) that they recognize or specifically bind.
- a target protein or polypeptide a hemagglutinin composition, hemagglutinin fragment, or hemagglutinin stem portion, for example
- epitope includes any protein determinant capable of specific binding to an immunoglobulin, an scFv, or a T-cell receptor.
- Epitopic determinants usually consist of chemically active surface groupings of molecules such as amino acids or sugar side chains and usually have specific three dimensional structural characteristics, as well as specific charge characteristics. For example, antibodies can be raised against N-terminal or C-terminal peptides of a polypeptide.
- a target polypeptide can comprise a single epitope, but typically comprises at least two epitopes, and can include any number of epitopes, depending on the size, conformation, and type of antigen.
- an “epitope” on a target polypeptide can be or include non-polypeptide elements, e.g., an “epitope” can include a carbohydrate side chain.
- the minimum size of a peptide or polypeptide epitope for an antibody is thought to be about four to five amino acids.
- Peptide or polypeptide epitopes can contain, for example, at least seven, at least nine, or between at least about 15 to about 30 amino acids. Since a CDR can recognize an antigenic peptide or polypeptide in its tertiary form, the amino acids comprising an epitope need not be contiguous, and in some cases, may not even be on the same peptide chain.
- the HA stem region epitope described herein fall into this latter category.
- An epitope recognized by antibodies can contain a sequence of at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 15, at least 20, at least 25, or between about 15 to about 30 contiguous or non-contiguous amino acids of the target protein or polypeptide.
- immunological binding refers to the non-covalent interactions of the type which occur between an immunoglobulin molecule and an antigen for which the immunoglobulin is specific.
- the strength, or affinity of immunological binding interactions can be expressed in terms of the dissociation constant (K d ) of the interaction, wherein a smaller K d represents a greater affinity.
- Immunological binding properties of selected polypeptides can be quantified using methods well known in the art. One such method entails measuring the rates of antigen-binding site/antigen complex formation and dissociation, wherein those rates depend on the concentrations of the complex partners, the affinity of the interaction, and geometric parameters that equally influence the rate in both directions.
- both the “on rate constant” (K on ) and the “off rate constant” (K off ) can be determined by calculation of the concentrations and the actual rates of association and dissociation (See Nature 361:186-87 (1993)).
- the ratio of K off /K on enables the cancellation of all parameters not related to affinity, and is equal to the dissociation constant K d (See, generally, Davies et al. (1990) Annual Rev Biochem 59:439-473).
- “specifically binds” or “immunoreacts with” is meant that the antibody reacts with one or more antigenic determinants of the desired antigen and does not react with other polypeptides.
- “specifically binds” generally means that an antibody binds to an epitope via its antigen binding domain, and that the binding entails some complementarity between the antigen binding domain and the epitope. According to this definition, an antibody is said to “specifically bind” to an epitope when it binds to that epitope, via its antigen binding domain more readily than it would bind to a random, unrelated epitope.
- the term “specificity” is used herein to qualify the relative affinity by which a certain antibody binds to a certain epitope.
- antibody “A” can be deemed to have a higher specificity for a given epitope than antibody “B,” or antibody “A” can be said to bind to epitope “C” with a higher specificity than it has for related epitope “D.”
- preferentially binds it is meant that the antibody specifically binds to an epitope more readily than it would bind to a related, similar, homologous, or analogous epitope.
- an antibody which “preferentially binds” to a given epitope would more likely bind to that epitope than to a related epitope, even though such an antibody may cross-react with the related epitope.
- an antibody can be considered to bind a first epitope preferentially if it binds said first epitope with a dissociation constant (K D ) that is less than the antibody's K D for the second epitope.
- K D dissociation constant
- an antibody can be considered to bind a first antigen preferentially if it binds the first epitope with an affinity that is at least one order of magnitude less than the antibody's K D for the second epitope.
- an antibody can be considered to bind a first epitope preferentially if it binds the first epitope with an affinity that is at least two orders of magnitude less than the antibody's K D for the second epitope.
- an antibody can be considered to bind a first epitope preferentially if it binds the first epitope with an affinity that is at least two orders of magnitude less than the antibody's k(off) for the second epitope.
- An antibody can be said to bind a target polypeptide with an off rate (k(off)) of less than or equal to 5 ⁇ 10 ⁇ 2 sec ⁇ 1 , 10 ⁇ 2 sec ⁇ 1 , 5 ⁇ 10 ⁇ 3 sec ⁇ 1 or 10 ⁇ 3 sec ⁇ 1 .
- An antibody can be said to bind a target polypeptide with an off rate (k(off)), for example, less than or equal to 5 ⁇ 10 ⁇ 4 sec ⁇ 1 , 10 ⁇ 4 sec ⁇ 1 , 5 ⁇ 10 ⁇ 5 sec ⁇ 1 , or 10 ⁇ 5 sec ⁇ 1 , 5 ⁇ 10 ⁇ 6 sec ⁇ 1 , 10 ⁇ 6 sec ⁇ 1 , 5 ⁇ 10 ⁇ 7 sec ⁇ 1 or 10 ⁇ 7 sec ⁇ 1 .
- k(off) off rate
- An antibody or antigen-binding fragment, variant, or derivative disclosed herein can be said to bind a target polypeptide with an on rate (k(on)) of greater than or equal to 10 3 M ⁇ 1 sec ⁇ 1 , 5 ⁇ 10 3 M ⁇ 1 sec ⁇ 1 , 10 4 M ⁇ 1 sec ⁇ 1 or 5 ⁇ 10 4 M ⁇ 1 sec ⁇ 1 .
- An antibody can be said to bind a target polypeptide with an on rate (k(on)) greater than or equal to 10 5 M ⁇ 1 sec ⁇ 1 , 5 ⁇ 10 5 M ⁇ 1 sec ⁇ 1 10 6 M ⁇ 1 sec ⁇ 1 , or 5 ⁇ 10 6 M ⁇ 1 sec ⁇ 1 or 10 7 M ⁇ 1 sec ⁇ 1 .
- the term “affinity” refers to a measure of the strength of the binding of an individual epitope with the CDR of an immunoglobulin molecule. See, e.g., Harlow et al., Antibodies: A Laboratory Manual, (Cold Spring Harbor Laboratory Press, 2nd ed. 1988) at pages 27-28.
- the term “avidity” refers to the overall stability of the complex between a population of immunoglobulins and an antigen, that is, the functional combining strength of an immunoglobulin mixture with the antigen. See, e.g., Harlow at pages 29-34.
- Avidity is related to both the affinity of individual immunoglobulin molecules in the population with specific epitopes, and also the valencies of the immunoglobulins and the antigen. For example, the interaction between a bivalent monoclonal antibody and an antigen with a highly repeating epitope structure, such as a polymer, would be one of high avidity.
- Antibodies can also be described or specified in terms of their cross-reactivity.
- cross-reactivity refers to the ability of an antibody, specific for one antigen, to react with a second antigen; a measure of relatedness between two different antigenic substances.
- an antibody is cross reactive if it binds to an epitope other than the one that induced its formation.
- the cross reactive epitope generally contains many of the same complementary structural features as the inducing epitope, and in some cases, may actually fit better than the original.
- certain antibodies have some degree of cross-reactivity, in that they bind related, but non-identical epitopes, e.g., epitopes with at least 95%, at least 90%, at least 85%, at least 80%, at least 75%, at least 70%, at least 65%, at least 60%, at least 55%, and at least 50% identity (as calculated using methods known in the art and described herein) to a reference epitope.
- epitopes e.g., epitopes with at least 95%, at least 90%, at least 85%, at least 80%, at least 75%, at least 70%, at least 65%, at least 60%, at least 55%, and at least 50% identity (as calculated using methods known in the art and described herein) to a reference epitope.
- An antibody can be said to have little or no cross-reactivity if it does not bind epitopes with less than 95%, less than 90%, less than 85%, less than 80%, less than 75%, less than 70%, less than 65%, less than 60%, less than 55%, and less than 50% identity (as calculated using methods known in the art and described herein) to a reference epitope.
- An antibody can be deemed “highly specific” for a certain epitope, if it does not bind any other analog, ortholog, or homolog of that epitope.
- Antibodies can also be described or specified in terms of their binding affinity to a protein, polypeptide, epitope, antigen, etc.
- binding affinities include those with a dissociation constant or Kd less than 5 ⁇ 10 ⁇ 2 M, 10 ⁇ 2 M, 5 ⁇ 10 ⁇ 3 M, 10 ⁇ 3 M, 5 ⁇ 10 ⁇ 4 M, 10 ⁇ 4 M, 5 ⁇ 10 ⁇ 5 M, 10 ⁇ 5 M, 5 ⁇ 10 ⁇ 6 M, 10 ⁇ 6 M, 5 ⁇ 10 ⁇ 7 M, 10 ⁇ 7 M, 5 ⁇ 10 ⁇ 8 M, 10 ⁇ 8 M, 5 ⁇ 10 ⁇ 9 M, 10 ⁇ 9 M, 5 ⁇ 10 ⁇ 10 M, 10 ⁇ 10 M, 5 ⁇ 10 ⁇ 11 M, 10 ⁇ 11 M, 5 ⁇ 10 ⁇ 12 M, 10 ⁇ 12 M, 5 ⁇ 10 ⁇ 13 M, 10 ⁇ 13 M, 5 ⁇ 10 ⁇ 14 M, 10 ⁇ 14 M, 5 ⁇ 10 ⁇ 15 M, or 10 ⁇ 15 M.
- Antibodies can be “multispecific,” e.g., bispecific, trispecific or of greater multispecificity, meaning that it recognizes and binds to two or more different epitopes present on one or more different antigens (e.g., proteins) at the same time. Thus, whether an antibody is “monospecfic” or “multispecific,” e.g., “bispecific,” refers to the number of different epitopes with which an antibody reacts. Multispecific antibodies can be specific for different epitopes of a target polypeptide or can be specific for a target polypeptide as well as for a heterologous epitope, such as a heterologous polypeptide or solid support material.
- properly folded hemagglutinin or properly folder hemagglutinin stem region refers hemagglutinin (or hemagglutinin region) that has a native conformation.
- a properly folded hemagglutinin or properly folded hemagglutinin stem region is, for example, one to which antibody F10 can bind, specifically bind, preferentially bind, etc.
- in-frame fusion refers to the joining of two or more polynucleotide open reading frames (ORFs) to form a continuous longer ORF, in a manner that maintains the correct translational reading frame of the original ORFs.
- a recombinant fusion protein is a single protein containing two ore more segments that correspond to polypeptides encoded by the original ORFs (which segments are not normally so joined in nature.) Although the reading frame is thus made continuous throughout the fused segments, the segments can be physically or spatially separated by, for example, in-frame linker sequence.
- polynucleotides encoding the CDRs of an immunoglobulin variable region can be fused, in-frame, but be separated by a polynucleotide encoding at least one immunoglobulin framework region or additional CDR regions, as long as the “fused” CDRs are co-translated as part of a continuous polypeptide.
- a “linear sequence” or a “sequence” is an order of amino acids in a polypeptide in an amino to carboxyl terminal direction in which residues that neighbor each other in the sequence are contiguous in the primary structure of the polypeptide.
- expression refers to a process by which a gene produces a biochemical, for example, an RNA or polypeptide.
- the process includes any manifestation of the functional presence of the gene within the cell including, without limitation, gene knockdown as well as both transient expression and stable expression. It includes without limitation transcription of the gene into messenger RNA (mRNA), transfer RNA (tRNA), small hairpin RNA (shRNA), small interfering RNA (siRNA) or any other RNA product, and the translation of such mRNA into polypeptide(s). If the final desired product is a biochemical, expression includes the creation of that biochemical and any precursors.
- mRNA messenger RNA
- tRNA transfer RNA
- shRNA small hairpin RNA
- siRNA small interfering RNA
- a gene product can be either a nucleic acid, e.g., a messenger RNA produced by transcription of a gene, or a polypeptide which is translated from a transcript.
- Gene products described herein further include nucleic acids with post transcriptional modifications, e.g., polyadenylation, or polypeptides with post translational modifications, e.g., methylation, glycosylation, the addition of lipids, association with other protein subunits, proteolytic cleavage, and the like.
- any of the disclosed antibodies or other described antibodies can be included or excluded from any group or genus of antibodies and from use in any method, including the disclosed methods.
- hemagglutinin compositions are useful as immunogens, e.g. vaccines, to stimulate a subjects immune response to influenza virus.
- hemagglutinin compositions that comprise, for example, the hemagglutinin stem region, either as part of a full hemagglutinin complex, as part of a trimeric ectodomain of hemagglutinin, as part of the extracellular portion of hemagglutinin, or in isolation from other parts of hemagglutinin.
- the hemagglutinin composition can be immobilized on a surface.
- the hemagglutinin composition can be the stem region of hemagglutinin in the neutral pH conformation in isolation from the head region of hemagglutinin, thereby identifying antibodies of interest.
- the hemagglutinin composition can be hemagglutinin in the neutral pH conformation in isolation from other components of influenza virus, where the head region of the hemagglutinin is modified to reduce the antigenicity of the head region. Because the stem region is formed in trimeric hemagglutinin, hemagglutinin compositions that include the stem region of trimeric hemagglutinin are particularly useful.
- the hemagglutinin composition includes an isolated peptide (natural or synthetic) that includes the F10 epitope, an F10 epitope unit and any mimotope thereof.
- An F10 epitope or an F10 epitope unit for the purposes of the present invention is a portion of an antigen molecule, e.g. hemagglutinin, which is delineated by the area of interration with the F10 antibody.
- the meaning of mimotope is defined as an entity which is sufficiently similar to the native F10 epitope so as to be capable of being recognized by antibodies which recognize the native F10 epitope; (Gheysen, H. M., et al., 1986, Synthetic peptides as antigens.
- the hemagglutinin compositions are immunogen compositions.
- the immunogen compositions according to the invention contain an epitope or epitope unit recognized by a protective monoclonal antibody having the specificity for the stem region of hemagglutinin protein of an influenza virus.
- a protective monoclonal antibody having the specificity for the stem region of hemagglutinin protein of an influenza virus includes monoclonal antibody D7, D8, F10, G17, H40, A66, D80, E88, E90, or H98 disclosed herein or a monoclonal antibody that competes with the binding of monoclonal antibody D7, D8, F10, G17, H40, A66, D80, E88, E90, or H98 to the HA protein.
- the antibody binds both the HA1 and HA2 peptide.
- the hemagglutinin composition is conjugated (e.g. a chimeric peptide) having one or more peptides or peptide fragments linked to a backbone where peptides or peptide fragments are spatially positioned relative to each other so that they together form a non-linear sequence which mimics the tertiary structure of an F10 epitope. wherein said conjugate competes with the binding of monoclonal antibody F10 to the HA protein.
- the backbone is a peptide backbone wherein peptides corresponding to segments of native hemagglutinin are coupled to form an epitope mimicking an the F10 epitope.
- the peptide backbone mimics the structure of the native hemagglutin protein such that peptides are coupled to for form an epitope mimicking an the F10 epitope.
- the backbone is a non-peptide backbone having two or more attachement points onto which peptides are coupled. See, WO9738011, the contents of which are incorporated by reference.
- the peptides or peptide fragments that make up the F10 epitope include one or more of the following peptides
- Xaa 1 is S, T, F H or Y, and
- Xaa 2 is H, Y, M, L or Q;
- Xaa 1 is H, Q, Y, S, D, N or T,
- Xaa 2 is Q, E, K, I, V, M, E, R or T;
- Xaa 1 is I, V, M, or L;
- Xaa 3 is G or A
- Xaa 4 is W, R, or G; or
- Xaa 1 is K, Q, R, N, L, G, F, H or Y,
- Xaa 2 is S or T
- Xaa 3 is Q or P
- Xaa 0 is F, V, I, M, L, or T,
- Xaa 5 is I, T, S, N, Q, D, or A,
- Xaa 6 is I, V, M, or L
- Xaa 7 is N, S, T, or D
- Xaa 8 is I, F, V, A, or T;
- Xaa 1 is K, Q, R, N, L, G, F, H or Y,
- Xaa 2 is S or T
- Xaa 3 is Q or P
- Xaa 4 is F, V, I, M, L, or T,
- Xaa 5 is I, T, S, N, Q, D, or A,
- Xaa 7 is N, S, T, or D,
- Cyclized peptides are often found to possess superior immunogenic activity compared to linear peptide immunogens.
- Linear peptide immunogens comprising three or more core sequences are found to bind with the terminal sequences only, while cyclization allows binding by all core sequences present in the peptide immunogens.
- Various methods for producing cyclic peptides have been described. One involves solution or liquid phase peptide synthesis, where amino acid residues in solution are linked by peptide bonds, with reactive groups not involved in the peptide bond formation, such as the amino group of the N-terminal residue, the carboxy group of the C-terminal residue, sulfhydryl groups on cysteine residues and similar or other reactive groups in the amino acid side chains, protected by suitable protecting groups.
- Head-to-tail (backbone) peptide cyclization has been used to rigidify structure and improve in vivo stability of small bioactive peptides (see Camarero and Muir, J. Am. Chem. Soc., 121:5597-5598 (1999)).
- An important consequence of peptide cyclization is retention of biological activity and/or the identification of new classes of pharmacological agents.
- a chemical cross-linking approach was used to prepare a backbone cyclized version of bovine pancreatic trypsin inhibitor (Goldenburg and Creighton, J. Mol. Biol., 165:407-413 (1983)).
- Other approaches include chemical (Camarero et al., Angew. Chem. Int.
- the hemagglutinin composition used in the vaccinal strategy according to the present invention can also be obtained using genetic engineering methods.
- the one skilled in the art can refer to the known sequence of the phage insert that expresses a specific epitope unit of an immunogenic polypeptide mimic of the invention and also to the general literature to determine the appropriate codons that can be used to synthesize the desired peptide.
- the expression of the polynucleotide that encodes the immunogenic polypeptide mimic of interest may be optimized, according to the organism in which the sequence has to be expressed and the specific codon usage of this organism (mammal, plant, bacteria, etc.).
- the general codon usages can be found in European patent application No. EP 0 359 472 (Mycogen).
- the composition comprises a polynucleotide coding for the immunogenic polypeptide or oligomeric peptide of pharmaceutical interest.
- a specific embodiment of comprises the in vivo production of a hemagglutinin composition for example in an oligomeric form by the introduction of the genetic information in the mammal organism, specifically in the patient organism.
- This genetic information can be introduced in vitro in a cell that has been previously extracted from the organism, the modified cell being subsequently reintroduced in the said organism directly in vivo into the appropriate tissue.
- the method for delivering the corresponding protein or peptide to the interior of a cell of a vertebrate in vivo comprises the step of introducing a preparation comprising a pharmaceutically acceptable injectable carrier and a polynucleotide operatively coding for the polypeptide into the interstitial space of a tissue comprising the cell, whereby the polynucleotide is taken up into the interior of the cell and has a pharmaceutical effect.
- the invention provides a hemagglutinin composition
- a hemagglutinin composition comprising a polynucleotide operatively coding for the ypeptide of interest or one of its above-described peptides in solution in a physiologically acceptable injectable carrier and suitable for introduction interstitially into a tissue to cause cells of the tissue to express the said protein or polypeptide.
- the polynucleotide operatively coding for the hemagglutinin composition, mimic or oligomeric peptide can be a vector comprising the genomic DNA or the complementary DNA (cDNA) coding for the corresponding protein or its protein derivative and a promoter sequence allowing the expression of the genomic DNA or the complementary DNA in the desired eukaryotic cells, such as vertebrate cells, specifically mammalian cells.
- the vector component of a therapeutic composition according to the present invention is advantageously a plasmid, a part of which is of viral or bacterial origin, which carries a viral or a bacterial origin of replication and a gene allowing its selection, such as an antibiotic resistance gene.
- the DNA to be introduced is complexed with DEAE-dextran (Pagano et al., 1967, J. Virol., 1:891) or with nuclear proteins (Kaneda et al., 1989, Science, 243:375), with lipids (Felgner et al., 1987, Proc. Natl. Acad. Sci., 84:7413), or encapsulated within liposomes (Fraley et al., 1980, J. Biol. Chem., 255:10431).
- the therapeutic polynucleotide and vector according to the present invention can advantageously be administered in the form of a gel that facilitates transfection into the cells.
- a gel composition can be a complex of poly-L-lysine and lactose as described by Midoux (1993, Nucleic Acids Research, 21:871-878) or also poloxamer 407 as described by Pastore (1994, Circulation, 90:1-517).
- the therapeutic polynucleotide and vector according to the invention can also be suspended in a buffer solution or be associated with liposomes.
- the polynucleotide in another embodiment, can be introduced in vitro into a host cell, preferably in a host cell previously harvested from the patient to be treated, and more preferably a somatic cell such as a muscle cell.
- a somatic cell such as a muscle cell.
- the cell that has been transformed with the vaccinal nucleotide coding for the immunogenic polypeptide of the invention is implanted back into the patient in order to deliver the recombinant protein within the body either locally or systemically.
- the present invention also concerns an immunogenic composition comprising a polynucleotide or an expression vector as described hereinabove in combination with a pharmaceutically acceptable vehicle allowing its administration to the human or other animal.
- a further embodiment of the invention comprises a vaccine composition comprising a polynucleotide or a vector as described above in combination with a pharmaceutically acceptable vehicle allowing its administration to the human or the animal.
- a hemagglutinin composition e.g. immunogen according to the invention via a reactive group of an amino acid residue of the peptide immunogen to a protein/polypeptide carrier having one or more functional groups.
- the protein/polypeptide carrier may be human serum albumin, keyhole limpet hemocyanin (KLH), immunoglobulin molecules, thyroglobulin, ovalbumin, influenza hemagglutinin, PAN-DR binding peptide (PADRE polypeptide), malaria circumsporozite (CS) protein, hepatitis B surface antigen (HBSAg 19-28 , Heat Shock Protein (HSP) 65, Bacillus Calmette - Guerin (BCG), cholera toxin, cholera toxin mutants with reduced toxicity, diphtheria toxin, CRM 197 protein that is cross-reactive with diphtheria toxin, recombinant Streptococcal C5a peptidase, Streptococcus pyogenes ORF1224, Streptococcus pyogenes ORF1664, Streptococcus pyogenes ORF 2452, Chlamydia pneumoniae ORF T367
- the hemagglutinin composition e.g. immunogen can be used as vaccines to generate an anti-influenza HA-mediated immune response in order to prevent influenza infections.
- Synthetic peptides require both stabilization and adjuvantation for the induction of an effective immune response in vivo.
- Various methods have been employed to protect synthetic peptide immunogens against degradation in vitro and in vivo, mediated by various processes including chemical and physical pathways. (Manning M C, et al. Pharmaceutical Research, 1989, 6:903-918).
- FCA Freund's complete adjuvant
- Adjuvants are well known in the art (Vaccine Design—The Subunit and Adjuvant Approach, 1995, Pharmaceutical Biotechnology, Volume 6, Eds. Powell, M. F., and Newman, M. J., Plenum Press, New York and London, ISBN 0-306-44867-X).
- Preferred adjuvants for use with immunogens of the present invention include aluminium or calcium salts (hydroxide or phosphate).
- Adjuvants may be selected from GM-CSF, 529 SE, IL-12, aluminum phosphate, aluminum hydroxide, Mycobacterium tuberculosis, Bordetella pertussis , bacterial lipopolysaccharides, aminoalkyl glucosane phosphate compounds, MPLTM (3-O-deacylated monophosphoryl lipid A), a polypeptide, Quil A, STIMULONTM QS-21, a pertussis toxin (PT), an E.
- coli heat-labile toxin LT
- IL-1alpha IL-1 ⁇
- IL-2 IL-4
- IL-5 IL-6
- IL-7 IL-8
- IL-10 IL-13
- IL-14 IL-15
- IL-16 IL-17
- IL-18 interferon-alpha
- interferon-B interferon-gamma
- G-CSF G-CSF
- TNF-alpha TNF-alpha
- cholera toxins and mutants thereof are also useful as adjuvants, including those described in published International Patent Application No. WO 00/18434 (wherein the glutamic acid at amino acid position 29 is replaced by another amino acid (other than aspartic acid, preferably a histidine). Similar CT toxins or mutants are described in published International Patent Application number WO 02/098368 (wherein the isoleucine at amino acid position 16 is replaced by another amino acid, either alone or in combination with the replacement of the serine at amino acid position 68 by another amino acid; and/or wherein the valine at amino acid position 72 is replaced by another amino acid).
- CT toxins are described in published International Patent Application number WO 02/098369 (wherein the arginine at amino acid position 25 is replaced by another amino acid; and/or an amino acid is inserted at amino acid position 49; and/or two amino acids are inserted at amino acid position 35 and 36).
- a carrier or depot system is required for effective long-term immunogenic responses.
- Notable examples include adsorbing the immunogen onto a mineral salt or gel.
- encapsulating a peptide immunogen within a polymeric matrix (monolithic matrix) or gel, or layering a polymeric material around a peptide immunogen (core-shell) may be an effective strategy.
- an immunogen may be incorporated in a liposome or vesicular type of formulation, with the immunogen either embedded in the lipid matrix or physically entrapped in the internal aqueous phase.
- Another strategy may employ a mineral-based, vegetable-based or animal-based oil, with an aqueous solution of the immunogen in various proportions, to prepare a water-in-oil (w/o)-emulsion or a water-in-oil-in-water (w/o/w)-double emulsion.
- w/o water-in-oil
- w/o/w water-in-oil-in-water
- the disclosed hemagglutinin compositions can produce an immune reaction in a subject.
- the subject can produce an immune response that prevents or reduces the severity of an influenza infection.
- the immune response can be reactive to influenza viruses within a subtype.
- the immune response can be reactive to influenza viruses in each subtype within a cluster.
- the immune response can be reactive to influenza viruses in each cluster within a group.
- the immune response can be reactive to all influenza viruses in each subtype within a group.
- the immune response can be reactive to influenza viruses within group 1.
- hemagglutininin compositions comprising, for example, hemagglutinin, a trimeric ectodomain of hemagglutinin, the extracellular portion of hemagglutinin, a trimeric stem region of hemagglutinin lacking all or a portion or portions of the head structure, or the hemagglutinin stem region in isolation from other parts of hemagglutinin.
- hemagglutininin compositions comprising, for example, hemagglutinin, a trimeric ectodomain of hemagglutinin, the extracellular portion of hemagglutinin, or a trimeric stem region of hemagglutinin lacking a portion or portions of the head structure where the head region of the hemagglutinin is modified by removing and/or replacing one or more glycosylation sites.
- hemagglutininin compositions comprising, for example, hemagglutinin, a trimeric ectodomain of hemagglutinin, the extracellular portion of hemagglutinin, or a trimeric stem region of hemagglutinin lacking a portion or portions of the head structure where the head region of the hemagglutinin is modified by adding one or more glycosylation sites.
- hemagglutininin compositions comprising, for example, hemagglutinin, a trimeric ectodomain of hemagglutinin, the extracellular portion of hemagglutinin, or a trimeric stem region of hemagglutinin lacking a portion or portions of the head structure where the head region of the hemagglutinin is modified by removing and/or replacing one or more glycosylation sites and by adding one or more glycosylation sites.
- the head region of the hemagglutinin can be modified by removing all or a portion of the head region.
- some or all of the amino acids of the head regions in a trimeric ectodomain of hemagglutinin can be removed to leave portions of the head region of loops between the stem region portions of the trimer.
- compositions comprising hemagglutinin composition bound or immobilized on a substrate or surface. Binding, attachment, and/or immobilization of proteins to substrates, solid supports an surfaces is a well established art and those of skill in the art can use any known techniques, chemistries and materials to bind hemagglutinin and hemagglutinin compositions to any suitable material.
- Hemagglutinin and hemagglutinin compositions immobilized on a solid support can also be used, for example, to generate an immune response in a subject.
- beads, microparticles or nanoparticles with hemagglutinin composition bound to the surface can be administered to subjects.
- immobilized hemagglutinin can be bound by antibodies specific for the hemagglutinin stem region
- Such compositions can be used to generate an immune response in a subject.
- Such an immune response can produce antibodies and immune system components that can inhibit influenza virus.
- a influenza protein e.g., HA or neuramindase
- a derivative, fragment, analog, homolog or ortholog thereof can be utilized as an immunogen in the generation of antibodies that immunospecifically bind these protein components.
- the disclosed hemagglutinin compositions are an example.
- Solid supports or surface are solid-state substrates, compositions, surfaces and/or supports with which molecules, such as hemagglutinin, hemagglutinin compositions and/or antibodies, can be associated.
- Molecules can be associated with solid supports directly or indirectly.
- molecules can be bound to the surface of a solid support or associated with capture agents (e.g., compounds or molecules that bind an analyte) immobilized on solid supports.
- An array is a solid support to which multiple molecules have been associated in an array, grid, or other organized pattern.
- Solid-state substrates for use in solid supports can include any solid material with which components can be associated, directly or indirectly. This includes materials such as acrylamide, agarose, cellulose, nitrocellulose, glass, gold, polystyrene, polyethylene vinyl acetate, polypropylene, polymethacrylate, polyethylene, polyethylene oxide, polysilicates, polycarbonates, teflon, fluorocarbons, nylon, silicon rubber, polyanhydrides, polyglycolic acid, polylactic acid, polyorthoesters, functionalized silane, polypropylfumerate, collagen, glycosaminoglycans, and polyamino acids.
- materials such as acrylamide, agarose, cellulose, nitrocellulose, glass, gold, polystyrene, polyethylene vinyl acetate, polypropylene, polymethacrylate, polyethylene, polyethylene oxide, polysilicates, polycarbonates, teflon, fluorocarbons, nylon, silicon rubber, polyanhydrides
- Solid-state substrates can have any useful form including thin film, membrane, bottles, dishes, fibers, woven fibers, shaped polymers, particles, beads, microparticles, or a combination.
- Solid-state substrates and solid supports can be porous or non-porous.
- a chip is a polygonal (e.g., rectangular, square, triangular, circular) small piece of material.
- Useful forms for solid-state substrates are plates, dishes, thin films, beads, or chips.
- a useful form for a solid-state substrate is a microtiter dish. In some embodiments, a multiwell glass slide can be employed.
- An array can include a plurality of molecules immobilized at identified or predefined locations on the solid support.
- Each predefined location on the solid support generally has one type of component (that is, all the components at that location are the same).
- multiple types of components can be immobilized in the same predefined location on a solid support. Each location will have multiple copies of the given components. The spatial separation of different components on the solid support allows separate detection and identification.
- solid support be a single unit or structure.
- a set of molecules can be distributed over any number of solid supports.
- each component can be immobilized in a separate reaction tube or container, or on separate beads or microparticles.
- Each of the components immobilized on the solid support can be located in a different predefined region of the solid support.
- the different locations can be different reaction chambers.
- Each of the different predefined regions can be physically separated from each other of the different regions.
- the distance between the different predefined regions of the solid support can be either fixed or variable.
- each of the components can be arranged at fixed distances from each other, while components associated with beads will not be in a fixed spatial relationship.
- the use of multiple solid support units for example, multiple beads) will result in variable distances.
- Components can be associated or immobilized on a solid support at any density. Components can be immobilized to the solid support at a density exceeding 400 different components per cubic centimeter.
- Arrays of components can have any number of components. For example, an array can have at least 1,000 different components immobilized on the solid support, at least 10,000 different components immobilized on the solid support, at least 100,000 different components immobilized on the solid support, or at least 1,000,000 different components immobilized on the solid support.
- kits for identifying HA stem antibodies the kit comprising one or more of the antibodies D7, D8, F10, G17, H40, A66, H98, D80, E90, and E8.
- the method involves mixing or bringing into contact compositions or components or reagents
- performing the method creates a number of different mixtures. For example, if the method includes 3 mixing steps, after each one of these steps a unique mixture is formed if the steps are performed separately. In addition, a mixture is formed at the completion of all of the steps regardless of how the steps were performed.
- the present disclosure contemplates these mixtures, obtained by the performance of the disclosed methods as well as mixtures containing any disclosed reagent, composition, or component, for example, disclosed herein.
- Data structures used in, generated by, or generated from, the disclosed method.
- Data structures generally are any form of data, information, and/or objects collected, organized, stored, and/or embodied in a composition or medium.
- An antibody titre stored in electronic form, such as in RAM or on a storage disk, is a type of data structure.
- the hemagglutinin composition is conjugated (e.g. a chimeric peptide) having one or more peptides or peptide fragments linked to a backbone where peptides or peptide fragments are spatially positioned relative to each other so that they together form a non-linear sequence which mimics the tertiary structure of an F10 epitope. wherein said conjugate competes with the binding of monoclonal antibody F10 to the HA protein.
- the backbone is a peptide backbone wherein peptides corresponding to segments of native hemagglutinin are coupled to form an epitope mimicking an the F10 epitope.
- Xaa 2 is H, Y, M, L or Q;
- Xaa 4 is W, R, or G; or
- Xaa 1 is K, Q, R, N, L, G, F, H or Y,
- Xaa 2 is S or T
- Xaa 3 is Q or P
- Xaa 4 is F, V, I, M, L, or T,
- Xaa 5 is I, T, S, N, Q, D, or A,
- Xaa 6 is I, V, M, or L
- Xaa 7 is N, S, T, or D
- Xaa 8 is I, F, V, A, or T;
- Xaa 1 is K, Q, R, N, L, G, F, H or Y,
- Xaa 2 is S or T
- Xaa 3 is Q or P
- Xaa 4 is F, V, I, M, L, or T,
- Xaa 7 is N, S, T, or D,
- a method comprising screening antibodies reactive to hemagglutinin for binding to the stem region of influenza hemagglutinin in the neutral pH conformation in isolation from the head region of hemagglutinin, thereby identifying antibodies of interest.
- a method comprising screening antibodies reactive to hemagglutinin for binding to influenza hemagglutinin in the neutral pH conformation in isolation from other components of influenza virus, wherein the head region of the hemagglutinin is modified to reduce the antigenicity of the head region, thereby identifying antibodies of interest.
- the head region of the hemagglutinin can be modified by removing or replacing glycosylation sites. In some forms, the head region of the hemagglutinin can be modified by adding glycosylation sites. In some forms, the head region of the hemagglutinin can be modified by removing all or a portion of the head region.
- HA stem antibodies antibodies that can be used, identified or produced in the disclosed methods.
- antibodies that bind to the stem region of influenza hemagglutinin in the neutral pH conformation (“HA stem antibodies”).
- antibodies that bind the epitope of influenza hemagglutinin in the neutral pH conformation defined by amino acid residues 18, 38, 39, 40 and 291 of HA1 and 18, 19, 20, 21, 38, 41, 42, 45, 49, 52, 53, and 56 of HA2.
- antibodies that bind to every subtype within an influenza virus group are disclosed.
- the antibody can compete with antibody F10 for binding to hemagglutinin.
- the antibody can have a VH CDR2 sequence that is the same as the VH CDR2 sequence of antibody D7, D8, F10, G17, H40 or A66 or of the consensus VH sequence SEQ ID NO:1.
- the antibody can have a VH CDR3 sequence that is the same as the VH CDR3 sequence of antibody D7, D8, F10, G17, H40 or A66 or of the consensus VH sequence SEQ ID NO:1.
- the antibody can have a VH CDR1 sequence that is the same as the VH CDR1 sequence of antibody D7, D8, F10, G17, H40 or A66 or of the consensus VH sequence SEQ ID NO:1. In some forms, the antibody can have a VH sequence that is the same as the VH sequence of antibody D7, D8, F10, G17, H40 or A66 or of the consensus VH sequence SEQ ID NO:1. In some forms, the antibody can have a VL sequence that is the same as the VL sequence of antibody D7, D8, F10, G17, H40, A66, D80, E88, E90, or H98 or of the consensus VL sequence SEQ ID NO:2.
- the antibody can have any combination of the VH FR1, VH CDR1, VH FR2, VH CDR2, VH FR3, VH CDR3, and VH FR4 sequences of antibodies D7, D8, F10, G17, H40 and A66 and the consensus VH sequence SEQ ID NO:1, and any combination of the VL FR1, VL CDR1, VL FR2, VL CDR2, VL FR3, VL CDR3, and VL FR4 sequences of antibodies D7, D8, F10, G17, H40, A66, D80, E88, E90, and H98 and the consensus VL sequence SEQ ID NO:2.
- the antibody can prevent or inhibit virus-host membrane fusion.
- the antibody can prevent or inhibit cell fusion mediated by cell surface-expressed influenza hemagglutinin.
- the disclosed antibodies, the disclosed hemagglutinins, and the disclosed methods can produce an immune reaction in a subject.
- the subject can produce an immune response that prevents or reduces the severity of an influenza infection.
- the immune response can be reactive to influenza viruses within a subtype.
- the immune response can be reactive to influenza viruses in each subtype within a cluster.
- the immune response can be reactive to influenza viruses in each cluster within a group.
- the immune response can be reactive to all influenza viruses in each subtype within a group.
- the immune response can be reactive to influenza viruses within group 1.
- the disclosed methods can further comprise screening the antibodies of interest for competing with antibody F10 for binding to hemagglutinin, thereby identifying F10-competing antibodies.
- the hemagglutinin can be hemagglutinin from a group 2 influenza virus.
- the hemagglutinin can be hemagglutinin from a group 1 influenza virus.
- the disclosed methods can further comprising producing the identified antibodies. Also disclosed are antibodies produced by the disclosed methods. Also disclosed are antibodies identified by the disclosed methods.
- the disclosed methods can further comprise screening a compound of interest, e.g., an F10 mimitope composition for competing with antibody F10 for binding to hemagglutinin, thereby identifying F10 mimitopes.
- the F10 mimitopes are useful as immunogens.
- the hemagglutinin can be hemagglutinin from a group 2 influenza virus.
- the hemagglutinin can be hemagglutinin from a group 1 influenza virus.
- F10 mimitopes identified by the disclosed methods.
- the disclosed antibodies and/or hemagglutinin compositions can be administered to a subject alone or in combination prior to and/or following exposure or possible exposure of the subject to influenza virus.
- the disclosed antibodies and/or hemagglutinin compositions can be administered to a subject prior to entering an area of infection or suspected infection, prior to coming into the presence of infected subjects or subjects suspected of infection, after entering an area of infection or suspected infection, after coming into the presence of infected subjects or subjects suspected of infection, prior to and after entering an area of infection or suspected infection, or prior to and after coming into the presence of infected subjects or subjects suspected of infection.
- immune response refers to the development of a humoral (antibody mediated) and/or a cellular (mediated by antigen-specific T cells or their secretion products) response directed against a target protein or polypeptide in a subject.
- a response can be an active response induced by administration of immunogen (such as the peptide immunogens described herein) or a passive response induced by administration of antibody or primed T-cells.
- a cellular immune response is elicited by the presentation of polypeptide epitopes in association with Class I or Class II MHC molecules, to activate antigen-specific CD4 + T helper cells and/or CD8 + cytotoxic T cells.
- the response may also involve activation of monocytes, macrophages, NK, cells, basophils, dendritic cells, astrocytes, microglia cells, eosinophils or other components of innate immunity.
- active immunity refers to any immunity conferred upon a subject by administration of an antigen, e.g. the disclosed hemagglutinin compositions
- Passive immunity refers to any immunity conferred upon a subject without administration of an antigen. “Passive immunity” therefore includes, but is not limited to, administration of a an antibody, such as the disclosed antibodies and, for example, a HA stem antibody, or, for example, a replicating display vehicle which includes an immunological portion of an antibody presented on its surface to a subject. Although replication of such a vehicle is active, the immune response is passive from the standpoint of the subject.
- Passive immunization has proven to be an effective and safe strategy for the prevention and treatment of viral diseases (See Keller et al., Clin. Microbiol. Rev. 13:602-14 (2000); Casadevall, Nat. Biotechnol. 20:114 (2002); Shibata et al., Nat. Med. 5:204-10 (1999); and Igarashi et al., Nat. Med. 5:211-16 (1999), each of which are incorporated herein by reference)).
- Passive immunization using neutralizing human monoclonal antibodies can provide an immediate treatment strategy for emergency prophylaxis and treatment of influenza such as bird flu as an alternative or in combination with the disclosed vaccines and drugs.
- Subunit vaccines offer significant advantages over conventional immunogens. They avoid the safety hazards inherent in production, distribution, and delivery of conventional killed or attenuated whole-pathogen vaccines. Furthermore, they can be rationally designed to include only confirmed protective epitopes, thereby avoiding suppressive T epitopes (see Steward et al., J. Virol. 69:7668 (1995)) or immunodominant B epitopes that subvert the immune system by inducing futile, non-protective responses (e.g. “decoy” epitopes). (See Garrity et al., J. Immunol. 159:279 (1997)).
- the subunit vaccine comprises the F10 epitope
- Methods for the screening of antibodies that possess the desired specificity include, but are not limited to, enzyme linked immunosorbent assay (ELISA) and other immunologically mediated techniques known within the art.
- ELISA enzyme linked immunosorbent assay
- Antibodies directed against a influenza virus protein such as HA can be used in methods known within the art relating to the localization and/or quantitation of a influenza virus protein (e.g., for use in measuring levels of the influenza virus protein within appropriate physiological samples, for use in diagnostic methods, for use in imaging the protein, and the like).
- antibodies specific to an influenza virus protein, or derivative, fragment, analog or homolog thereof, that contain the antibody derived antigen binding domain can be utilized as pharmacologically active compounds (referred to herein as “therapeutics”).
- An antibody specific for an influenza virus protein can be used to isolate an influenza virus polypeptide by standard techniques, such as immunoaffinity, chromatography or immunoprecipitation.
- Antibodies directed against an influenza virus protein (or a fragment thereof) can be used diagnostically to monitor protein levels in tissue as part of a clinical testing procedure, e.g., to, for example, determine the efficacy of a given treatment regimen. Detection can be facilitated by coupling (i.e., physically linking) the antibody to a detectable substance.
- detectable substances include various enzymes, prosthetic groups, fluorescent materials, luminescent materials, bioluminescent materials, and radioactive materials.
- suitable enzymes include horseradish peroxidase, alkaline phosphatase, ⁇ -galactosidase, or acetylcholinesterase;
- suitable prosthetic group complexes include streptavidin/biotin and avidin/biotin;
- suitable fluorescent materials include umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin;
- an example of a luminescent material includes luminol;
- examples of bioluminescent materials include luciferase, luciferin, and aequorin, and examples of suitable radioactive material include 125 I, 131 I, 35 S or 3 H.
- the disclosed antibodies can be used as therapeutic agents. Such agents can generally be employed to treat or prevent an influenza virus-related disease or pathology (e.g., bird flu) in a subject.
- An antibody preparation such as, for example, one having high specificity and high affinity for its target antigen, can be administered to the subject and will generally have an effect due to its binding with the target.
- Administration of the antibody can abrogate or inhibit or interfere with the internalization of the virus into a cell. In this case, the antibody binds to the target and masks a binding site of the naturally occurring ligand, thereby blocking fusion the virus to the cell membrane inhibiting internalization of the virus.
- a therapeutically effective amount of an antibody or hemagglutinin composition relates generally to the amount needed to achieve a therapeutic objective. As noted elsewhere herein, this can be a binding interaction between the antibody and its target antigen that, in certain cases, interferes with the functioning of the target.
- the amount required to be administered will furthermore depend on the binding affinity of the antibody for its specific antigen, and will also depend on the rate at which an administered antibody is depleted from the free volume other subject to which it is administered.
- Common ranges for therapeutically effective dosing of an antibody or antibody fragment can be, by way of nonlimiting example, from about 0.1 mg/kg body weight to about 50 mg/kg body weight. Common dosing frequencies can range, for example, from twice daily to once a week.
- Antibodies specifically binding an influenza virus protein or a fragment thereof, as well as other molecules identified by the screening assays disclosed herein, can be administered for the treatment of an influenza virus-related disorders in the form of pharmaceutical compositions.
- Principles and considerations involved in preparing such compositions, as well as guidance in the choice of components are provided, for example, in Remington: The Science And Practice Of Pharmacy 19th ed. (Alfonso R. Gennaro, et al., editors) Mack Pub. Co., Easton, Pa., 1995; Drug Absorption Enhancement: Concepts, Possibilities, Limitations, And Trends, Harwood Academic Publishers, Langhorne, Pa., 1994; and Peptide And Protein Drug Delivery (Advances In Parenteral Sciences, Vol. 4), 1991, M. Dekker, New York.
- antibody fragments are used, it can be useful to use the smallest inhibitory fragment that specifically binds to the binding domain of the target protein.
- peptide molecules can be designed that retain the ability to bind the target protein sequence.
- Such peptides can be synthesized chemically and/or produced by recombinant DNA technology (See, e.g., Marasco et al., Proc. Natl. Acad. Sci. USA, 90: 7889-7893 (1993)).
- the formulation can also contain more than one active compound as necessary for the particular indication being treated, such as, for example, those with complementary activities that do not adversely affect each other.
- the composition can comprise an agent that enhances its function, such as, for example, a cytotoxic agent, cytokine, chemotherapeutic agent, or growth-inhibitory agent.
- cytotoxic agent such as, for example, a cytotoxic agent, cytokine, chemotherapeutic agent, or growth-inhibitory agent.
- Such molecules are suitably present in combination in amounts that are effective for the purpose intended.
- the active ingredients can also be entrapped in microcapsules prepared, for example, by coacervation techniques or by interfacial polymerization, for example, hydroxymethylcellulose or gelatin-microcapsules and poly-(methylmethacrylate) microcapsules, respectively, in colloidal drug delivery systems (for example, liposomes, albumin microspheres, microemulsions, nano-particles, and nanocapsules) or in macro emulsions.
- colloidal drug delivery systems for example, liposomes, albumin microspheres, microemulsions, nano-particles, and nanocapsules
- the formulations to be used for in vivo administration must be sterile. This can readily be accomplished, for example, by filtration through sterile filtration membranes.
- sustained-release preparations can be prepared. Suitable examples of sustained-release preparations include semipermeable matrices of solid hydrophobic polymers containing the antibody, which matrices are in the form of shaped articles, e.g., films, or microcapsules. Examples of sustained-release matrices include polyesters, hydrogels (for example, poly(2-hydroxyethyl-methacrylate), or poly(vinylalcohol)), polylactides (U.S. Pat. No.
- copolymers of L-glutamic acid and ⁇ ethyl-L-glutamate copolymers of L-glutamic acid and ⁇ ethyl-L-glutamate, non-degradable ethylene-vinyl acetate, degradable lactic acid-glycolic acid copolymers such as the LUPRON DEPOTTM (injectable microspheres composed of lactic acid-glycolic acid copolymer and leuprolide acetate), and poly-D-( ⁇ )-3-hydroxybutyric acid. While polymers such as ethylene-vinyl acetate and lactic acid-glycolic acid enable release of molecules for over 100 days, certain hydrogels release proteins for shorter time periods.
- the disclosed antibodies can be used as agents for detecting the presence of an influenza virus (or a protein or a protein fragment thereof) in a sample.
- the antibody can contain a detectable label.
- Antibodies can be, for example, polyclonal or monoclonal. An intact antibody, or a fragment thereof (e.g., F ab , scFv, or F (ab)2 ) can be used.
- the term “labeled”, with regard to the probe or antibody is intended to encompass direct labeling of the probe or antibody by coupling (i.e., physically linking) a detectable substance to the probe or antibody, as well as indirect labeling of the probe or antibody by reactivity with another reagent that is directly labeled.
- in vitro techniques for detection of an analyte mRNA include Northern hybridizations and in situ hybridizations.
- In vitro techniques for detection of an analyte protein include enzyme linked immunosorbent assays (ELISAs), Western blots, immunoprecipitations, and immunofluorescence.
- In vitro techniques for detection of an analyte genomic DNA include Southern hybridizations. Procedures for conducting immunoassays are described, for example in “ELISA: Theory and Practice: Methods in Molecular Biology”, Vol. 42, J. R. Crowther (Ed.) Human Press, Totowa, N.J., 1995; “Immunoassay”, E. Diamandis and T.
- in vivo techniques for detection of an analyte protein include introducing into a subject a labeled anti-analyte protein antibody.
- the antibody can be labeled with a radioactive marker whose presence and location in a subject can be detected by standard imaging techniques.
- adjuvant refers to a compound that, when administered in conjunction with an antigen, augments the immune response to the antigen, but when administered alone does not generate an immune response to the antigen.
- adjuvants can augment an immune response by several mechanisms including lymphocyte recruitment, stimulation of B and/or T cells, and stimulation of macrophages.
- the preparation can be administered to a subject or organism per se, or in a pharmaceutical composition where it is mixed with suitable carriers or excipients.
- a “pharmaceutical composition” refers to a preparation of one or more of the active ingredients described herein with other chemical components such as physiologically suitable carriers and excipients.
- the purpose of a pharmaceutical composition is to facilitate administration of a compound to a subject or organism.
- physiologically acceptable carrier and “pharmaceutically acceptable carrier” which can be interchangeably used refer to a carrier or a diluent that does not cause significant irritation to a subject or organism and does not abrogate the biological activity and properties of the administered compound.
- An adjuvant is included under these phrases.
- excipient refers to an inert substance added to a pharmaceutical composition to further facilitate administration of an active ingredient.
- excipients include calcium carbonate, calcium phosphate, various sugars and types of starch, cellulose derivatives, gelatin, vegetable oils and polyethylene glycols.
- Suitable routes of administration can, for example, include oral, rectal, transmucosal, especially transnasal, intestinal or parenteral delivery, including intramuscular, subcutaneous and intramedullary injections as well as intrathecal, direct intraventricular, intravenous, intraperitoneal, intranasal, or intraocular injections. Alternately, one can administer a preparation in a local rather than systemic manner.
- compositions can be manufactured by processes well known in the art, e.g., by means of conventional mixing, dissolving, granulating, dragee-making, levigating, emulsifying, encapsulating, entrapping or lyophilizing processes.
- compositions for use in the disclosed methods thus can be formulated in conventional manner using one or more physiologically acceptable carriers comprising excipients and auxiliaries, which facilitate processing of the active ingredients into preparations which, can be used pharmaceutically. Proper formulation is dependent upon the route of administration chosen.
- the active ingredients can be formulated in aqueous solutions, preferably in physiologically compatible buffers such as Hank's solution, Ringer's solution, or physiological salt buffer.
- physiologically compatible buffers such as Hank's solution, Ringer's solution, or physiological salt buffer.
- penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art.
- the compounds can be formulated readily by combining the active compounds with pharmaceutically acceptable carriers well known in the art.
- Such carriers enable the compounds to be formulated as tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions, and the like, for oral ingestion by a patient.
- Pharmacological preparations for oral use can be made using a solid excipient, optionally grinding the resulting mixture, and processing the mixture of granules, after adding suitable auxiliaries if desired, to obtain tablets or dragee cores.
- Suitable excipients are, in particular, fillers such as sugars, including lactose, sucrose, mannitol, or sorbitol; cellulose preparations such as, for example, maize starch, wheat starch, rice starch, potato starch, gelatin, gum tragacanth, methyl cellulose, hydroxypropylmethyl-cellulose, sodium carbomethylcellulose; and/or physiologically acceptable polymers such as polyvinylpyrrolidone (PVP).
- disintegrating agents can be added, such as cross-linked polyvinyl pyrrolidone, agar, or alginic acid or a salt thereof such as sodium alginate.
- Dragee cores are provided with suitable coatings.
- suitable coatings For this purpose, concentrated sugar solutions can be used which may optionally contain gum arabic, talc, polyvinyl pyrrolidone, carbopol gel, polyethylene glycol, titanium dioxide, lacquer solutions and suitable organic solvents or solvent mixtures.
- Dyestuffs or pigments can be added to the tablets or dragee coatings for identification or to characterize different combinations of active compound doses.
- compositions which can be used orally, include push-fit capsules made of gelatin as well as soft, sealed capsules made of gelatin and a plasticizer, such as glycerol or sorbitol.
- the push-fit capsules can contain the active ingredients in admixture with filler such as lactose, binders such as starches, lubricants such as talc or magnesium stearate and, optionally, stabilizers.
- the active ingredients can be dissolved or suspended in suitable liquids, such as fatty oils, liquid paraffin, or liquid polyethylene glycols.
- stabilizers can be added. All formulations for oral administration should be in dosages suitable for the chosen route of administration.
- compositions can take the form of tablets or lozenges formulated in conventional manner.
- the active ingredients for use in the disclosed methods can be conveniently delivered in the form of an aerosol spray presentation from a pressurized pack or a nebulizer with the use of a suitable propellant, e.g., dichlorodifluoromethane, trichlorofluoromethane, dichloro-tetrafluoroethane or carbon dioxide.
- a suitable propellant e.g., dichlorodifluoromethane, trichlorofluoromethane, dichloro-tetrafluoroethane or carbon dioxide.
- the dosage unit can be determined by providing a valve to deliver a metered amount.
- Capsules and cartridges of, e.g., gelatin for use in a dispenser can be formulated containing a powder mix of the compound and a suitable powder base such as lactose or starch.
- compositions described herein can be formulated for parenteral administration, e.g., by bolus injection or continuous infusion.
- Formulations for injection can be presented in unit dosage form, e.g., in ampoules or in multidose containers with optionally, an added preservative.
- the compositions can be suspensions, solutions or emulsions in oily or aqueous vehicles, and can contain formulatory agents such as suspending, stabilizing and/or dispersing agents.
- compositions for parenteral administration include aqueous solutions of the active preparation in water-soluble form.
- suspensions of the active ingredients can be prepared as appropriate oily or water based injection suspensions.
- Suitable lipophilic solvents or vehicles include fatty oils such as sesame oil, or synthetic fatty acids esters such as ethyl oleate, triglycerides or liposomes.
- Aqueous injection suspensions can contain substances, which increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol or dextran.
- the suspension can also contain suitable stabilizers or agents which increase the solubility of the active ingredients to allow for the preparation of highly concentrated solutions.
- compositions such as suppositories or retention enemas, using, e.g., conventional suppository bases such as cocoa butter or other glycerides.
- compositions for use in the disclosed methods include compositions wherein the active ingredients are contained in an amount effective to achieve the intended purpose. More specifically, a therapeutically effective amount means an amount of active ingredients effective to prevent, alleviate or ameliorate symptoms of disease or prolong the survival of the subject being treated.
- the therapeutically effective amount or dose can be estimated initially from in vitro and cell culture assays.
- a dose can be formulated in animal models to achieve a desired circulating antibody concentration or titer. Such information can be used to more accurately determine useful doses in humans.
- Toxicity and therapeutic efficacy of the active ingredients described herein can be determined by standard pharmaceutical procedures in vitro, in cell cultures or experimental animals.
- the data obtained from these in vitro and cell culture assays and animal studies can be used in formulating a range of dosage for use in human.
- the dosage may vary depending upon the dosage form employed and the route of administration utilized.
- the exact formulation, route of administration and dosage can be chosen by the individual physician in view of the patient's condition. (See e.g., Fingl et al in The Pharmacological Basis of Therapeutics, Ch. 1 p. 1. (1975)).
- Dosage amount and interval can be adjusted individually to provide plasma of antibodies which are sufficient to prevent or reduce viral entry (minimal effective concentration, MEC).
- MEC minimum effective concentration
- the MEC will vary for each preparation, but can be estimated from in vitro data. Dosages necessary to achieve the MEC will depend on individual characteristics and route of administration. Binding assays can be used to determine plasma concentrations.
- Dosage intervals can also be determined using the MEC value. Preparations should be administered using a regimen, which maintains plasma levels above the MEC for example, 10-90% of the time, preferable between 30-90% of the time and most preferably 50-90%.
- dosing can be of a single or a plurality of administrations, with course of treatment lasting from several days to several weeks or until cure is effected or diminution of the disease state is achieved.
- compositions to be administered will, of course, be dependent on the subject being treated, the severity of the affliction, the manner of administration, the judgment of the prescribing physician, etc.
- compositions suitable for administration can be incorporated into pharmaceutical compositions suitable for administration.
- Such compositions typically comprise the antibody or agent and a pharmaceutically acceptable carrier.
- pharmaceutically acceptable carrier is intended to include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration. Suitable carriers are described in the most recent edition of Remington's Pharmaceutical Sciences, a standard reference text in the field, which is incorporated herein by reference.
- Such carriers or diluents include, but are not limited to, water, saline, ringer's solutions, dextrose solution, and 5% human serum albumin.
- Liposomes and non-aqueous vehicles such as fixed oils can also be used.
- the use of such media and agents for pharmaceutically active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active compound, use thereof in the compositions is contemplated. Supplementary active compounds can also be incorporated into the compositions.
- a pharmaceutical composition can be formulated to be compatible with its intended route of administration.
- routes of administration include parenteral, e.g., intravenous, intradermal, subcutaneous, oral (e.g., inhalation), transdermal (i.e., topical), transmucosal, and rectal administration.
- Solutions or suspensions used for parenteral, intradermal, or subcutaneous application can include the following components: a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerine, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid (EDTA); buffers such as acetates, citrates or phosphates, and agents for the adjustment of tonicity such as sodium chloride or dextrose.
- the pH can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide.
- the parenteral preparation can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic.
- compositions suitable for injectable use include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion.
- suitable carriers include physiological saline, bacteriostatic water, Cremophor EL ⁇ (BASF, Parsippany, N.J.) or phosphate buffered saline (PBS).
- the composition must be sterile and should be fluid to the extent that easy syringeability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi.
- the carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof.
- the proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants.
- Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like.
- isotonic agents for example, sugars, polyalcohols such as manitol, sorbitol, sodium chloride in the composition.
- Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent which delays absorption, for example, aluminum monostearate and gelatin.
- Sterile injectable solutions can be prepared by incorporating the active compound in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filtered sterilization.
- dispersions are prepared by incorporating the active compound into a sterile vehicle that contains a basic dispersion medium and the required other ingredients from those enumerated above.
- methods of preparation are vacuum drying and freeze drying that yields a powder of the active ingredient plus any additional desired ingredient from a previously sterile filtered solution thereof.
- Oral compositions generally include an inert diluent or an edible carrier. They can be enclosed in gelatin capsules or compressed into tablets. For the purpose of oral therapeutic administration, the active compound can be incorporated with excipients and used in the form of tablets, troches, or capsules. Oral compositions can also be prepared using a fluid carrier for use as a mouthwash, wherein the compound in the fluid carrier is applied orally and swished and expectorated or swallowed. Pharmaceutically compatible binding agents, and/or adjuvant materials can be included as part of the composition.
- the tablets, pills, capsules, troches and the like can contain any of the following ingredients, or compounds of a similar nature: a binder such as microcrystalline cellulose, gum tragacanth or gelatin; an excipient such as starch or lactose, a disintegrating agent such as alginic acid, Primogel, or corn starch; a lubricant such as magnesium stearate or Sterotes; a glidant such as colloidal silicon dioxide; a sweetening agent such as sucrose or saccharin; or a flavoring agent such as peppermint, methyl salicylate, or orange flavoring.
- a binder such as microcrystalline cellulose, gum tragacanth or gelatin
- an excipient such as starch or lactose, a disintegrating agent such as alginic acid, Primogel, or corn starch
- a lubricant such as magnesium stearate or Sterotes
- a glidant such as colloidal silicon dioxide
- the compounds are delivered in the form of an aerosol spray from pressured container or dispenser which contains a suitable propellant, e.g., a gas such as carbon dioxide, or a nebulizer.
- a suitable propellant e.g., a gas such as carbon dioxide, or a nebulizer.
- Systemic administration can also be by transmucosal or transdermal means.
- penetrants appropriate to the barrier to be permeated are used in the formulation.
- penetrants are generally known in the art, and include, for example, for transmucosal administration, detergents, bile salts, and fusidic acid derivatives.
- Transmucosal administration can be accomplished through the use of nasal sprays or suppositories.
- the active compounds can be formulated into ointments, salves, gels, or creams as generally known in the art.
- the compounds can also be prepared in the form of suppositories (e.g., with conventional suppository bases such as cocoa butter and other glycerides) or retention enemas for rectal delivery.
- the active compounds can be prepared with carriers that will protect the compound against rapid elimination from the body, such as a controlled release formulation, including implants and microencapsulated delivery systems.
- a controlled release formulation including implants and microencapsulated delivery systems.
- Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Methods for preparation of such formulations will be apparent to those skilled in the art.
- the materials can also be obtained commercially from Alza Corporation and Nova Pharmaceuticals, Inc.
- Liposomal suspensions (including liposomes targeted to infected cells with monoclonal antibodies to viral antigens) can also be used as pharmaceutically acceptable carriers. These can be prepared according to methods known to those skilled in the art, for example, as described in U.S. Pat. No. 4,522,811.
- Dosage unit form refers to physically discrete units suited as unitary dosages for the subject to be treated; each unit containing a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier.
- the specification for the dosage unit forms can be dictated by and directly dependent on the unique characteristics of the active compound and the particular therapeutic effect to be achieved, and the limitations inherent in the art of compounding such an active compound for the treatment of individuals.
- compositions can be included in a container, pack, or dispenser together with instructions for administration.
- compositions can, if desired, be presented in a pack or dispenser device, such as an FDA approved kit, which may contain one or more unit dosage forms containing the active ingredient.
- the pack can, for example, comprise metal or plastic foil, such as a blister pack.
- the pack or dispenser device can be accompanied by instructions for administration.
- the pack or dispenser can also be accommodated by a notice associated with the container in a form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceuticals, which notice is reflective of approval by the agency of the form of the compositions or human or veterinary administration. Such notice, for example, may be of labeling approved by the U.S. Food and Drug Administration for prescription drugs or of an approved product insert.
- Compositions comprising a preparation formulated in a compatible pharmaceutical carrier can also be prepared, placed in an appropriate container, and labeled for treatment of an indicated condition, as if further detailed above.
- the term “activity” as used herein refers to a measurable result of the interaction of molecules. Some exemplary methods of measuring these activities are provided herein.
- the term “modulate” as used herein refers to the ability of a compound to change an activity in some measurable way as compared to an appropriate control. As a result of the presence of compounds in the assays, activities can increase or decrease as compared to controls in the absence of these compounds. Preferably, an increase in activity is at least 25%, more preferably at least 50%, most preferably at least 100% compared to the level of activity in the absence of the compound. Similarly, a decrease in activity is preferably at least 25%, more preferably at least 50%, most preferably at least 100% compared to the level of activity in the absence of the compound.
- a compound that increases a known activity is an “agonist”.
- One that decreases, or prevents, a known activity is an “antagonist”.
- inhibitor means to reduce or decrease in activity or expression. This can be a complete inhibition or activity or expression, or a partial inhibition. Inhibition can be compared to a control or to a standard level. Inhibition can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98,
- monitoring refers to any method in the art by which an activity can be measured.
- providing refers to any means of adding a compound or molecule to something known in the art. Examples of providing can include the use of pipettes, pipettemen, syringes, needles, tubing, guns, etc. This can be manual or automated. It can include transfection by any mean or any other means of providing nucleic acids to dishes, cells, tissue, cell-free systems and can be in vitro or in vivo.
- preventing refers to administering a compound prior to the onset of clinical symptoms of a disease or conditions so as to prevent a physical manifestation of aberrations associated with the disease or condition.
- treatment is meant the medical management of a patient with the intent to cure, ameliorate, stabilize, or prevent a disease, pathological condition, or disorder.
- active treatment that is, treatment directed specifically toward the improvement of a disease, pathological condition, or disorder
- causal treatment that is, treatment directed toward removal of the cause of the associated disease, pathological condition, or disorder.
- palliative treatment that is, treatment designed for the relief of symptoms rather than the curing of the disease, pathological condition, or disorder
- preventative treatment that is, treatment directed to minimizing or partially or completely inhibiting the development of the associated disease, pathological condition, or disorder
- supportive treatment that is, treatment employed to supplement another specific therapy directed toward the improvement of the associated disease, pathological condition, or disorder.
- in need of treatment refers to a judgment made by a caregiver (e.g. physician, nurse, nurse practitioner, or individual in the case of humans; veterinarian in the case of animals, including non-human mammals) that a subject requires or will benefit from treatment. This judgment is made based on a variety of factors that are in the realm of a care giver's expertise, but that include the knowledge that the subject is ill, or will be ill, as the result of a condition that is treatable by the compounds of the invention.
- a caregiver e.g. physician, nurse, nurse practitioner, or individual in the case of humans; veterinarian in the case of animals, including non-human mammals
- subject includes, but is not limited to, animals, plants, bacteria, viruses, parasites and any other organism or entity that has nucleic acid.
- the subject may be a vertebrate, more specifically a mammal (e.g., a human, horse, pig, rabbit, dog, sheep, goat, non-human primate, cow, cat, guinea pig or rodent), a fish, a bird or a reptile or an amphibian.
- the subject may to an invertebrate, more specifically an arthropod (e.g., insects and crustaceans).
- arthropod e.g., insects and crustaceans.
- the term does not denote a particular age or sex. Thus, adult and newborn subjects, as well as fetuses, whether male or female, are intended to be covered.
- a patient refers to a subject afflicted with a disease or disorder.
- the term “patient” includes human and veterinary subjects.
- a subject is a subject that has or can have endometriosis and/or endometriosis cells.
- the terms “higher,” “increases,” “elevates,” or “elevation” refer to increases above basal levels, e.g., as compared to a control.
- the terms “low,” “lower,” “reduces,” or “reduction” refer to decreases below basal levels, e.g., as compared to a control.
- assays for screening candidate or test compounds which modulate the interaction between the influenza virus and the cell membrane.
- assays for screening candidate or test compounds that compete for binding of hemaglutinin to the antibodies disclosed herein.
- Compound identified in these methods are useful as immunogens for influenza virus.
- a “small molecule” as used herein, is meant to refer to a composition that has a molecular weight of less than about 5 kD, such as, for example, less than about 4 kD.
- Small molecules can be, e.g., nucleic acids, peptides, polypeptides, peptidomimetics, carbohydrates, lipids or other organic or inorganic molecules.
- Libraries of chemical and/or biological mixtures, such as fungal, bacterial, or algal extracts, are known in the art and can be screened with any of the known or disclosed assays.
- At least one HA protein is provided, which is exposed to at least one neutralizing monoclonal antibody. Formation of an antibody-antigen complex is detected, and one or more candidate compounds are introduced to the complex. If the antibody-antigen complex is disrupted following introduction of the one or more candidate compounds, the candidate compounds is useful to treat a an influenza virus-related disease or disorder, e.g. bird flu or swine flu.
- the at least one influenza virus protein can be provided as an influenza virus molecule.
- Determining the ability of the test compound to interfere with or disrupt the antibody-antigen complex can be accomplished, for example, by coupling the test compound with a radioisotope or enzymatic label such that binding of the test compound to the antigen or biologically-active portion thereof can be determined by detecting the labeled compound in a complex.
- test compounds can be labeled with 125 I, 35 S, 14 C, or 3 H, either directly or indirectly, and the radioisotope detected by direct counting of radioemission or by scintillation counting.
- test compounds can be enzymatically-labeled with, for example, horseradish peroxidase, alkaline phosphatase, or luciferase, and the enzymatic label detected by determination of conversion of an appropriate substrate to product.
- the assay can comprise contacting an antibody-antigen complex with a test compound, and determining the ability of the test compound to interact with the antigen or otherwise disrupt the existing antibody-antigen complex. In some forms, determining the ability of the test compound to interact with the antigen and/or disrupt the antibody-antigen complex can comprise determining the ability of the test compound to preferentially bind to the antigen or a biologically-active portion thereof, as compared to the antibody.
- the assay can comprise contacting an antibody-antigen complex with a test compound and determining the ability of the test compound to modulate the antibody-antigen complex. Determining the ability of the test compound to modulate the antibody-antigen complex can be accomplished, for example, by determining the ability of the antigen to bind to or interact with the antibody, in the presence of the test compound.
- solubilizing agents include non-ionic detergents such as n-octylglucoside, n-dodecylglucoside, n-dodecylmaltoside, octanoyl-N-methylglucamide, decanoyl-N-methylglucamide, Triton® X-100, Triton® X-114, Thesit®, Isotridecypoly(ethylene glycol ether) n , N-dodecyl-N,N-dimethyl-3-ammonio-1-propane sulfonate, 3-(3-cholamidopropyl)dimethylamminiol-1-propane sulfonate (CHAPS), or 3-(3-cholamidopropyl)dimethylamminiol-2-hydroxy-1-propane sulfonate (CHAPSO).
- non-ionic detergents such as n-octylglucoside, n
- a fusion protein can be provided that adds a domain that allows one or both of the proteins to be bound to a matrix.
- GST-antibody fusion proteins or GST-antigen fusion proteins can be adsorbed onto glutathione sepharose beads (Sigma Chemical, St.
- the test compound for example, Louis, Mo. or glutathione derivatized microtiter plates, that are then combined with the test compound, and the mixture is incubated under conditions conducive to complex formation (e.g., at physiological conditions for salt and pH). Following incubation, the beads or microtiter plate wells are washed to remove any unbound components, the matrix immobilized in the case of beads, complex determined either directly or indirectly. Alternatively, the complexes can be dissociated from the matrix, and the level of antibody-antigen complex formation can be determined using standard techniques.
- either the antibody or the antigen can be immobilized utilizing conjugation of biotin and streptavidin.
- Biotinylated antibody or antigen molecules can be prepared from biotin-NHS (N-hydroxy-succinimide) using techniques well-known within the art (e.g., biotinylation kit, Pierce Chemicals, Rockford, Ill.), and immobilized in the wells of streptavidin-coated 96 well plates (Pierce Chemical).
- antibodies reactive with the antibody or antigen of interest can be derivatized to the wells of the plate, and unbound antibody or antigen trapped in the wells by antibody conjugation.
- Methods for detecting such complexes include immunodetection of complexes using such other antibodies reactive with the antibody or antigen.
- novel agents identified by any of the disclosed screening assays and uses thereof for treatments as described herein.
- Antibodies can be detected by appropriate assays, e.g., conventional types of immunoassays.
- an assay can be performed in which a influenza protein (e.g., HAL HA 2 or neurominidase) or fragment thereof is affixed to a solid phase. Incubation can be maintained for a sufficient period of time to allow the antibody in the sample to bind to the immobilized polypeptide on the solid phase. After this first incubation, the solid phase can be separated from the sample. The solid phase can be washed to remove unbound materials and interfering substances such as non-specific proteins which may also be present in the sample.
- a influenza protein e.g., HAL HA 2 or neurominidase
- Incubation can be maintained for a sufficient period of time to allow the antibody in the sample to bind to the immobilized polypeptide on the solid phase. After this first incubation, the solid phase can be separated from the sample. The solid phase can be washed to remove unbound materials and interfering substances such
- the solid phase containing the antibody of interest bound to the immobilized polypeptide can subsequently be incubated with a second, labeled antibody or antibody bound to a coupling agent such as biotin or avidin.
- This second antibody can be another anti-influenza antibody or another antibody.
- Labels for antibodies are well-known in the art and include radionuclides, enzymes (e.g. maleate dehydrogenase, horseradish peroxidase, glucose oxidase, catalase), fluors (fluorescein isothiocyanate, rhodamine, phycocyanin, fluorescarmine), biotin, and the like.
- the labeled antibodies are incubated with the solid and the label bound to the solid phase is measured. These and other immunoassays can be easily performed by those of ordinary skill in the art.
- An exemplary method for detecting the presence or absence of a influenza virus (in a biological sample, for example) can involve obtaining a biological sample from a test subject and contacting the biological sample with a labeled monoclonal or scFv antibody such that the presence of the influenza virus is detected in the biological sample.
- the term “labeled”, with regard to the probe or antibody, is intended to encompass direct labeling of the probe or antibody by coupling (i.e., physically linking) a detectable substance to the probe or antibody, as well as indirect labeling of the probe or antibody by reactivity with another reagent that is directly labeled.
- indirect labeling include detection of a primary antibody using a fluorescently-labeled secondary antibody and end-labeling of a DNA probe with biotin such that it can be detected with fluorescently-labeled streptavidin.
- biological sample is intended to include tissues, cells and biological fluids isolated from a subject, as well as tissues, cells and fluids present within a subject.
- the detection method can be used to detect an influenza virus in a biological sample in vitro as well as in vivo.
- in vitro techniques for detection of an influenza virus include enzyme linked immunosorbent assays (ELISAs), Western blots, immunoprecipitations, and immunofluorescence.
- in vivo techniques for detection of an influenza virus include introducing into a subject a labeled anti-influenza virus antibody.
- the antibody can be labeled with a radioactive marker whose presence and location in a subject can be detected by standard imaging techniques.
- the biological sample can contain protein molecules from the test subject.
- the biological sample can be, for example, a peripheral blood leukocyte sample isolated by conventional means from a subject.
- kits for detecting the presence of an influenza virus in a biological sample can comprise: a labeled compound or agent capable of detecting an influenza virus (e.g., an anti-influenza scFv or monoclonal antibody) in a biological sample; means, materials, and/or a system for determining the amount of an influenza virus in the sample; and means, materials, and/or a system for comparing the amount of an influenza virus in the sample with a standard.
- the compound or agent can be packaged in a suitable container.
- the kit can further comprise instructions for using the kit to detect an influenza virus in a sample.
- Chimeras with specific epitopes engrafted into the CDR3 loop have been used to induce humoral responses to either HIV-1 gp120 V3-loop or the first extracellular domain (D1) of human CD4 receptor (See Lanza et al., Proc. Natl. Acad. Sci. USA 90:11683-87 (1993); Zaghouani et al., Proc. Natl. Acad. Sci. USA 92:631-35 (1995)).
- the immune sera were able to prevent infection of CD4 SupT1 cells by HIV-1MN (anti-gp120 V3C) or inhibit syncytia formation (anti-CD4-D1).
- DNA vaccines are stable, can provide the antigen an opportunity to be naturally processed, and can induce a longer-lasting response. Although a very attractive immunization strategy, DNA vaccines often have very limited potency to induce immune responses. Poor uptake of injected DNA by professional APCs, such as dendritic cells (DCs), may be the main cause of such limitation. Combined with the antigen-Ig chimera vaccines, a promising new DNA vaccine strategy based on the enhancement of APC antigen presentation has been reported (see Casares, et al., Viral Immunol. 10:129-36 (1997); Gerloni et al., Nat. Biotech. 15:876-81 (1997); Gerloni et al., DNA Cell Biol. 16:611-25 (1997); You et al., Cancer Res. 61:3704-11 (2001)), which takes advantage of the presence of Fc receptors (Fc ⁇ Rs) on the surface of DCs.
- Fc ⁇ Rs Fc receptors
- DNA vaccine encoding an antigen (Ag)-Ig chimera.
- Ag-Ig fusion proteins can be expressed and secreted by the cells taking up the DNA molecules.
- the secreted Ag-Ig fusion proteins while inducing B-cell responses, can be captured and internalized by interaction of the Fc fragment with Fc ⁇ Rs on DC surface, which will promote efficient antigen presentation and greatly enhance antigen-specific immune responses.
- DNA encoding antigen-Ig chimeras carrying a functional anti-MHC II specific scFv region gene can also target the immunogens to all three types of APCs.
- the immune responses could be further boosted with use of the same protein antigens generated in vitro (i.e., “prime and boost”), if necessary.
- primary and boost protein antigens generated in vitro
- specific cellular and humoral immune responses against infection of influenza virus were accomplished through intramuscular (i.m.) injection of a DNA vaccine (See Casares et al., Viral. Immunol. 10:129-36 (1997)).
- cytokines can be administered in conjunction with ancillary immunoregulatory agents.
- cytokines including, but not limited to, IL-2, modified IL-2 (Cys125 ⁇ Ser125), GM-CSF, IL-12, ⁇ -interferon, IP-10, MIP1 ⁇ , and RANTES.
- the disclosed vaccines have superior immunoprotective and immunotherapeutic properties over other anti-viral vaccines.
- a method of immunization e.g., inducing an immune response, of a subject.
- a subject can be immunized by administration to the subject a composition containing a membrane fusion protein of a pathogenic enveloped virus.
- the fusion protein can be coated or embedded in a biologically compatible matrix.
- the fusion protein can be glycosylated, e.g. can contain a carbohydrate moiety.
- the carbohydrate moiety can be in the form of a monosaccharide, disaccharide(s). oligosaccharide(s), polysaccharide(s), or their derivatives (e.g. sulfo- or phospho-substituted).
- the carbohydrate can be linear or branched.
- the carbohydrate moiety can be N-linked or O-linked to a polypeptide. N-linked glycosylation is to the amide nitrogen of asparagine side chains and O-linked glycosylation is to the hydroxy oxygen of serine and threonine side chains.
- the carbohydrate moiety can be endogenous to the subject being vaccinated. Alternatively, the carbohydrate moiety can be exogenous to the subject being vaccinated.
- the carbohydrate moieties can be carbohydrate moieties that are not typically expressed on polypeptides of the subject being vaccinated.
- the carbohydrate moieties can be plant-specific carbohydrates. Plant specific carbohydrate moieties include for example N-linked glycan having a core bound ⁇ 1,3 fucose or a core bound ⁇ 1,2 xylose.
- the carbohydrate moieties can be carbohydrate moieties that are expressed on polypeptides or lipids of the subject being vaccinate. For example many host cells have been genetically engineered to produce human proteins with human-like sugar attachments.
- the fusion protein can be a trimeric hemagglutinin protein.
- the hemagglutinin protein can be produced in a non-mammalian cell such as a plant cell.
- Enveloped viruses include for example, epstein-barr virus, herpes simplex virus, type 1 and 2, human cytomegalovirus, human herpesvirus, type 8, varicella zoster virus, hepatitis B virus, hepatitis C virus, human immunodeficiency virus, influenza virus, measles virus, mumps virus, parainfluenza virus, respiratory syncytial virus, rabies virus, and rubella virus.
- Infections can be diagnosed and or monitored, typically by a physician using standard methodologies
- a subject requiring immunization can be identified by methods know in the art.
- subjects can be immunized as outlined in the CDC's General Recommendation on Immunization (51(RR02) pp 1-36) Cancer is diagnosed for example by physical exam, biopsy, blood test, or x-ray.
- the subject can be e.g., any mammal, e.g., a human, a primate, mouse, rat, dog, cat, cow, horse, pig, a fish or a bird.
- the treatment ican be administered prior to diagnosis of the infection. Alternatively, treatment can be administered after diagnosis. Efficaciousness of treatment can be determined in association with any known method for diagnosing or treating the particular disorder or infection. Alleviation of one or more symptoms of the disorder indicates that the compound confers a clinical benefit.
- a vaccine candidate targeting humoral immunity can fulfill at least three criteria to be successful: provoke a strong antibody response (“immunogenicity”); a significant fraction of the antibodies it provokes must cross-react with the pathogen (“immunogenic fitness”); and the antibodies it provokes must be protective. While immunogenicity can often be enhanced using adjuvants or carriers, immunogenic fitness and the ability to induce protection (as evidenced by neutralization) are intrinsic properties of an antigen which will ultimately determine the success of that antigen as a vaccine component.
- Immunogenic fitness is defined as the fraction of antibodies induced by an antigen that cross-react with the pathogen (See Matthews et al., J. Immunol. 169:837 (2002)). It is distinct from immunogenicity, which is gauged by the titer of all of the antibodies induced by an antigen, including those antibodies that do not cross-react with the pathogen. Inadequate immunogenic fitness has probably contributed to the disappointing track record of peptide vaccines to date. Peptides that bind with high affinity to antibodies and provoke high antibody titers frequently lack adequate immunogenic fitness, and, therefore, they fail as potential vaccine components. Therefore, it can be useful to include immunogenic fitness as one of the criteria for selecting influenza vaccine candidates.
- a common explanation for poor immunogenic fitness is the conformational flexibility of most short peptides. Specifically, a flexible peptide may bind well to antibodies from patients, and elicit substantial antibody titers in na ⁇ ve subjects. However, if the peptide has a large repertoire of conformations, a preponderance of the antibodies it induces in na ⁇ ve subjects may fail to cross-react with the corresponding native epitope on intact pathogen.
- APFs Like short peptides, some APFs may be highly flexible and, therefore may fail as vaccine components.
- the most immunogenically fit APFs are likely to consist of self-folding protein subdomains that are intrinsically constrained outside the context of the whole protein.
- immunogenic fitness is primarily a property of the APF itself, and not of the responding immune system, immunogenic fitness can be evaluated in an animal model (e.g. in mice) even though ultimately the APF will have to perform in humans.
- the immunogenic fitness achieved by APFs can be evaluated by immunosorption of anti-APF sera with purified spike or membrane protein, in a procedure analogous to that described in Matthews et al., J. Immunol. 169:837 (2002).
- IgG is purified from sera collected from mice that have been immunized.
- Purified, biotinylated proteins (as appropriate, depending on the particular APF with which the mice were immunized) can be mixed with the mouse IgG and incubated. Streptavidin-coated sepharose beads can then be added in sufficient quantity to capture all of the biotinylated protein, along with any bound IgG.
- streptavidin-coated beads are removed by centrifugation at 13,000 rpm in a microcentrifuge, leaving IgG that has been depleted of antibodies directed against the protein, respectively. Mock immunoabsorptions can be performed in parallel in the same way, except that biotinylated BSA will be substituted for influenza protein as a mock absorbent.
- the absorbed antibodies and the mock-absorbed antibodies can be titered side-by-side in ELISA against the immunizing APF.
- the antigen for these ELISAs can be purified APF-GST fusion proteins.
- the antigen for these ELISAs can be APF-Fc fusion proteins secreted by mammalian cells and purified with protein A. The percentage decrease in the anti-APF titer of absorbed antibodies compared with the mock-absorbed antibodies can provide a measure of the immunogenic fitness of the APF.
- diseases or disorders include but are not limited to, e.g., bird flu.
- hemagglutininin compositions, scFv and/or monoclonal antibody D7, D8, F10, G17, H40, A66, D80, E88, E90, and H98 can be administered in therapeutically effective amounts.
- two or more anti-influenza antibodies are co-administered.
- Subjects at risk for an influenza virus-related diseases or disorders include patients who have come into contact with an infected person or who have been exposed to the influenza virus in some other way. Administration of a prophylactic agent can occur prior to the manifestation of symptoms characteristic of the influenza virus-related disease or disorder, such that a disease or disorder is prevented or, alternatively, delayed in its progression.
- the appropriate agent can be determined based on screening assays described herein.
- the agent to be administered can be a scFv or monoclonal antibody that neutralizes an influenza virus that has been identified according to the known or disclosed methods.
- the method involves administering an agent (e.g., an agent identified by a screening assay described herein and/or an scFv antibody or monoclonal antibody identified according to known or disclosed methods), or combination of agents that neutralize the influenza to a patient suffering from the disease or disorder.
- an agent e.g., an agent identified by a screening assay described herein and/or an scFv antibody or monoclonal antibody identified according to known or disclosed methods
- two or more antibodies such as D7, D8, F10, G17, H40, A66, D80, E88, E90, and H98 that bind to the same epitope of the HA protein.
- nAbs high-affinity neutralizing mAbs
- nAbs high-affinity neutralizing mAbs
- Sequence and structural analysis of all 16 HA subtypes points to the existence of just two variants of this epitope, corresponding to the two classic phylogenetic groupings of HA (Groups 1 and 2).
- H5-F10 complexes were formed by incubating the two purified components with an excess of F10, and isolated by Superdex 200 in TBS buffer. Peak fractions were pooled and concentrated to ⁇ 11 mg ml ⁇ 1 . The integrity of the H5 trimer was examined using Gel filtration and SDS-PAGE. Crystals grew at 22° C. by equilibrating equal volumes of protein and reservoir solution (12.5% PEG 1K (w/v), 25% ethylene glycol (w/v), 100 mM Tris, pH 8.5) using the hanging drop vapor diffusion technique.
- Diffraction data were collected from crystals flash-frozen at 100K in the reservoir buffer at the Stanford Synchrotron Radiation Laboratory beam-line 9.2, set at a wavelength of 1.0 ⁇ , and processed with XDS (Kabsch, Automatic processing of rotation diffraction data from crystals of initially unknown symmetry and cell constants. Journal of Applied Crystallography 26, 795-800 (1993)) and HKL2000 (Otwinowski & Minor, Processing of X-ray diffraction data collected in oscillation mode. in Methods in Enzymology, Volume 276 : Macromolecular Crystallography, Part A (eds. Carter Jr. & Sweet) 307-326 (Academic Press, New York, 1997)).
- the structure was solved at 3.2 ⁇ resolution by molecular replacement with PHASER using the structures of H5 (A/Vietnam/1194/04; PDB code 21BX) and a homology model of F10 based on the structure of SARS nAb 80R (PDB code 2GHW) (Hwang et al., Structural basis of neutralization by a human anti-severe acute respiratory syndrome spike protein antibody, 80R. J Biol Chem 281, 34610-6 (2006); Rodriguez et al., Homology modeling, model and software evaluation: three related resources. Bioinformatics 14, 523-8 (1998)) as starting models.
- the asymmetric unit contains two H5 trimers and three F10 molecules per trimer, and was refined using REFMAC5 (Murshudov et al., Refinement of macromolecular structures by the maximum-likelihood method. Acta Crystallogr D Biol Crystallogr 53, 240-55 (1997)) with simulated annealing in CNS (Murshudov et al. (1997)) and manual rebuilding with Coot (Emsley & Cowtan, Coot: model-building tools for molecular graphics. Acta Crystallogr D Biol Crystallogr 60, 2126-32 (2004)) and Xtalview (McRee, A visual protein crystallographic software system for X11/Xview.
- REFMAC5 Refinement of macromolecular structures by the maximum-likelihood method. Acta Crystallogr D Biol Crystallogr 53, 240-55 (1997)) with simulated annealing in CNS (Murshudov et al. (1997)) and manual rebuilding with Coo
- the final maps are of high quality, and key features such as the F10 CDR loops and interfacial residues are unambiguous and consistent in the 6 copies.
- the final model includes 503/503/503/497/497/497 residues for the 6 independent copies of H5, 235/235/236/233/234/234 residues for the 6 F10scFvs, 24 N-acetyl-D-glucosamine and 6 ⁇ -D-mannose units, but no water molecules.
- the R FREE is 0.29 with excellent geometry as assessed with PROCHECK (Laskowski et al., PROCHECK: a program to check the stereochemical quality of protein structures. Journal of Applied Crystallography 26, 283-291 (1993)) and Rampage (Table 1): percentage of residues in favored, allowed and outlier regions are 90.0%, 9.5%, and 0.5%, respectively.
- Recombinant trimeric H5-VN04 ectodomain was produced for crystallization studies (see below) except that furin co-infection to ensure complete activation was not employed. Abs were identified by two-rounds of selection of a 27 billion member human scFv phage display library against recombinant trimeric H5 immobilized on Immunotube (Nunc), followed by ELISA screening. Ten unique anti-H5 Abs were identified by sequence analysis of 97 H5-positive clones out of 392 clones screened.
- H5-VN04, H5-IN05 or A/Netherland/219/03 (H7N7) (H7-NL03) viruses (10,000 pfu) were incubated with anti-H5 scFv-Fcs at three different concentrations (1, 10 or 100 ⁇ g mL ⁇ 1 ) at 37° C. for 30 mins.
- the virus-Ab mixture was diluted logarithmically and transferred onto MDCK cell monolayers in 12-well plates and incubated at 37° C. for 1 h. Cells were then washed and overlaid with agar. After 4 days of incubation, the overlay was discarded, and plaques visualized by crystal violet staining
- 0.5 ⁇ 10 6 293T cells were incubated with H5-TH04-pseudotyped HIV viruses ( ⁇ 500 ng of p24) in the presence of anti-H5 nAbs, control mAbs, or in the absence of antibodies, in PBS buffer containing 0.5% (w/v) BSA and 0.02% (w/v) NaN 3 at 4° C. After 1 h incubation, cells were spun down. Supernatants were collected and tested for p24 levels using an HIV-1 p24 CA capture ELISA kit (NCI, Frederick, NIH) to quantify unbound virus. Cells were then washed once or twice and lysed to quantify the cell-bound virus using the same method.
- HeLa cells ⁇ 90% confluent in six-well plates, were transfected with pcDNA3.1-H5-TH04 plasmid (3 ⁇ g total DNA per well) using lipofectamine 2000 (Invitrogen). After ⁇ 30 hours of transfection, the culture medium was supplemented with 1 ml of anti-H5 or control mAbs for 1-2 hours, and cells were then washed and incubated with low-pH fusion buffer (150 mM NaCl+10 mM HEPES, adjusted to pH 5.0) for 4-5 mins.
- low-pH fusion buffer 150 mM NaCl+10 mM HEPES, adjusted to pH 5.0
- mice Female 8-10 weeks old BALB/c mice were used in all experiments. Mice were weighed on the day of virus challenge and then daily for 2 weeks. Body weight was used as the clinical endpoint; mice with body weight loss ⁇ 25% of pre-infection values were euthanized. Animal studies were conducted per approved Institutional Animal Care and Use Committee protocols.
- mice were first inoculated i.n. with 10 MLD 50 of VN04. At 24, 48 and 72 hours after H5-VN04 infection, one group of mice received i.p injections of 15 mg kg ⁇ 1 body weight of one of the three nAbs or control (80R).
- HA1 is an N-terminal fragment of HA of H5N1 A/Thailand/2(SP-33)/2004 (H5-TH04), residues 11 to 325 (H3 numbering).
- the gene was codon-optimized and expressed as fusion protein with a C-terminal 9 amino-acids tag (C9-tag:GTETSQVAPA).
- C9-tag:GTETSQVAPA C-terminal 9 amino-acids tag
- the fusion protein HA1-C9 was expressed in 293T cells transiently and the secreted proteins in supernatant were harvested 48 hours after transfection and purified from the supernatant by affinity chromatography using Protein A Sepharose that coupled covalently with anti-C9 antibody 1D4 (National Cell Culture Center).
- the method to produce HA0 protein of H5-VN04 is the same as described below for crystallization of H5-F10 complex but without the baculovirus coinfection for furin-cleavage.
- All the mutants of pcDNA3.1-H5-TH04 were constructed by the QuikChange method (Stratagene, La Jolla, Calif.). Various full-length wild type HA and HA mutants expressing plasmids of H1, H5 or H7 were transfected transiently into 293T cells. 24 hours after transfection, cells were harvested for immunostaining Anti-H5 or control mAb 80R (Sui et al., Potent neutralization of severe acute respiratory syndrome (SARS) coronavirus by a human mAb to S1 protein that blocks receptor association.
- SARS severe acute respiratory syndrome
- Phage-scFvs of individual clones were produced as described previously (Sui et al., Potent neutralization of severe acute respiratory syndrome (SARS) coronavirus by a human mAb to S1 protein that blocks receptor association. Proc Natl Acad Sci USA 101, 2536-41 (2004)).
- scFv-Fcs and whole human IgG1 were produced as described previously (Sui et al., Potent neutralization of severe acute respiratory syndrome (SARS) coronavirus by a human mAb to S1 protein that blocks receptor association. Proc Natl Acad Sci USA 101, 2536-41 (2004); Gould et al., Protective and therapeutic capacity of human single-chain Fv-Fc fusion proteins against West Nile virus.
- scFv-Fcs or IgG1s were expressed in 293T or 293F cells (Invitrogen) by transient transfection and purified by protein A sepharose affinity chromatography.
- 0.2 ⁇ g of pure H5 HA proteins was coated onto 96-well Maxisorb ELISA plate (Nunc, NY) at 2 ⁇ g/mL in PBS at 4° C. overnight. The plate was washed with PBS for 3 times to remove uncoated proteins.
- 1 ⁇ g/mL of anti-H5 scFv-Fcs followed by HRP-anti-human IgG1 were used to detect the binding of anti-H5 scFv-Fcs to H5 HA proteins.
- H5 HA mAbs binding to recombinant H5-VN04 HA0 trimer were performed on a Biacore T100 (Biacore, Sweden) at 25° C.
- Anti-human IgG Fc antibody (Biacore) was covalently attached to individual flow cell surfaces of a CM4 sensor chip by amine-coupling using the amine coupling kit (Biacore).
- HA mAbs were captured onto anti-human IgG Fc surfaces (flow rate of 10 ⁇ l/min in HBS buffer (Biacore)) to ensure that mAb-H5 binding occurred as a homogenous 1:1 Langmuir interaction.
- H5 was injected over each flow cell at a flow rate of 30 ⁇ l/min in HBS buffer, and at concentrations ranging from to 0.31 to 20 nM.
- a buffer injection served as a negative control.
- All experiments contained an additional anti-human IgG Fc antibody control surface that served to account for changes in the buffer refractive index and to test for potential nonspecific interactions between H5 and anti-human IgG Fc.
- surfaces were regenerated with 3 M MgCl 2 solution.
- the association rates (ka), dissociation rates (kd), and affinity constants (K D ) were calculated using Biacore T100 evaluation software. The quality of each fit was based on the agreement between experimental data and the calculated fits, where the Chi 2 values were below 1.0. Surface densities of mAbs against H5 were optimized to minimize mass transfer and avoid any contribution of avidity effects. All ka, kd, K D values reported here represent the mean and standard error of three experiments.
- H5N1/PR8 Subbarao et al., Evaluation of a genetically modified reassortant H5N1 influenza A virus vaccine candidate generated by plasmid-based reverse genetics. Virology 305, 192-200 (2003)
- H5-VN04 and H1-PR34 viruses were mixed with Log 2 antibody dilutions in PBS and incubated at 20-22° C. for 30 minutes. A 0.5% suspension of turkey erythrocytes was added to each well and the mixture incubated for 30 min at RT before visual scoring for haemagglutination activity.
- H5-VN04 HA gene was expressed in insect cells as a fusion protein by adapting the protocol described previously (Stevens et al., Structure and receptor specificity of the hemagglutinin from an H5N1 influenza virus. Science 312, 404-10 (2006)).
- This construct contains a C-terminal trimerizing ‘foldon’ sequence from the bacteriophage T4 fibritin to stabilize the trimeric structure, followed by a thrombin site and a His 6 tag.
- the cDNA of the fusion protein was cloned into the baculovirus transfer vector, pAcGP67A (BD Biosciences, Bedford, Mass.), to allow for efficient secretion of recombinant protein.
- sf9 cells were co-infected with baculovirus stocks of HA0 and furin at an empirically derived ratio.
- the furin cDNA was a gift from Dr. Robert Fuller (University of Michigan).
- the cells were spun down and the supernatant was incubated with Ni-NTA beads (Qiagen Inc., Valencia, Calif.).
- the beads were washed with TBS buffer (10 mM Tris.HCl, 80 mM NaCl, pH8.0) with 10 mM imidazole, and eluted with TBS with 250 mM Imidazole.
- the eluted H5 protein was dialyzed against TBS buffer and further purified by ion-exchange using Mono Q HR10/10 column
- H5-F10 complexes were formed by mixing the two purified components, and isolated by Superdex 200 in TBS buffer ( FIG. 9B ). Peak fractions were pooled and concentrated to ⁇ 11 mg/ml. The integrity of the H5 trimer was examined using Gel filtration (Superdex 200 column) and SDS-PAGE. Crystals grew by the hanging drop vapor diffusion method at 22° C. Two ⁇ L of H5-F10 were mixed with an equal volume of 12.5% PEG 1K, 25% ethylene glycol, 100 mM Tris, pH 8.5. Crystals were flash-frozen in liquid nitrogen prior to data collection.
- X-ray diffraction data were collected at the Stanford Synchrotron Radiation Laboratory (SSRL) beam-lines 7.1 and 9.2. Data were processed with XDS (Kabsch, Automatic processing of rotation diffraction data from crystals of initially unknown symmetry and cell constants. J. Appl Cryst 26, 795-800 (1993)) and the HKL2000 package (Otwinowski & Minor, in Macromolecular Crystallography, part A (ed. C. W. Carter, J. R. M. S.) 307-326. (Academic Press (New York), 1997)).
- SSRL Stanford Synchrotron Radiation Laboratory
- the structure of the H5-F10 complex was determined by molecular replacement with PHASER using the structure of H5 (A/Vietnam/1194/04; PDB code 21BX) and the scFv structure of SARS nAb 80R (PDB code 2 GHW) as search models.
- the scFv structure homology model was build with WHATIF (Rodriguez et al., Homology modeling, model and software evaluation: three related resources. CABIOS 14, 523-528 (1998)).
- the asymmetric unit contains two H5 trimers and six F10 molecules.
- H5N1 epidemics involve viruses derived from a single lineage of H5 HA. Within this lineage, four distinct clades have been identified as major threats to public health (W.H.O. web site who.int/csr/disease/avian influenza/guidelines/summaryH520070403.pdf (2007); W.H.O. Evolution of H5N1 avian influenza viruses in Asia. Emerg Infect Dis 11, 1515-21 (2005)).
- Table 2 shows framework regions 1-4 (FR1-4) and complementarity-determining regions 1-3 (CDR1-3) for both the VH and VL are shown.
- FR and CDR regions are defined using the Kabat database.
- the VH and VL gene names are shown on the right (using the IMGT database). Dots denote identity with the consensus sequence, whereas hyphens denote gaps.
- Six different VH and 10 different VL genes were found. Some antibodies share the same VH gene. Five out of the six different VH belong to one gene family, IGHV1-69*01.
- the VL genes are more diverse than the VH genes, three out of the 10 VL are ⁇ chain. Highlighted residues for F10 are critical for binding to H5, and their conservation is indicated.
- VH sequences from top to bottom: SEQ ID NO:1 (consensus) and SEQ ID NOs:3-9.
- nAbs cross-competed with each other in a competition ELISA ( FIG. 10 ), indicating that they share a common epitope.
- three of the nAbs (D8, F10 and A66) were converted into full-length human IgG1s for further studies; all three IgG1s bound to recombinant H5-VN04 with high affinity (Kd ⁇ 100-200 pM) and very slow dissociation rates (kd ⁇ 10 ⁇ 4 s ⁇ 1 ) ( FIG. 11 ).
- mice were treated with IgG1s before (prophylactically) or after (therapeutically) lethal viral challenge. Prophylaxis using 10 mg kg ⁇ 1 of IgG1s effectively protected (80-100%) mice when challenged with a high lethal dose of H5-VN04 (Clade 1) or A/HongKong/483/97 (H5-HK97) (Clade 0) ( FIGS. 2A and 2B ).
- anti-HA Abs can neutralize infection is by blocking the initial binding of HA to its cellular receptor (sialic acid) or by interfering with the subsequent step of HA-mediated virus-host membrane fusion, which occurs in acidic endosomes (Skehel & Wiley, Receptor binding and membrane fusion in virus entry: the influenza hemagglutinin. Annu Rev Biochem 69, 531-69 (2000); Kida et al., Interference with a conformational change in the haemagglutinin molecule of influenza virus by antibodies as a possible neutralization mechanism. Vaccine 3, 219-22 (1985)). It was found that none of the nAbs inhibited virus binding to cells ( FIG. 3A ) or hemagglutination of red blood cells. However, it was shown, using a model system of cell fusion, that the nAbs potently inhibited membrane fusion ( FIG. 3B ).
- Cleavage leads to the partial burial of the “fusion peptide” (the first ⁇ 21 residues of each HA2) into the stem (Skehel & Wiley (2000); Ha et al., H5 avian and H9 swine influenza virus haemagglutinin structures: possible origin of influenza subtypes. Embo J 21, 865-75 (2002)), which also contributes to the formation of each of three hydrophobic “pockets” located below the large trimeric receptor-binding head.
- one F10 nAb binds into each pocket, burying ⁇ 1500 ⁇ 2 of protein surface. Only the heavy chain (VH) participates directly in binding, utilizing all three of its complementarity-determining regions (CDRs).
- the light chain (VL) points out into solution, and makes only non-specific contacts with the distal end of the oligosaccharide of glycosylated residue Asn33 1 from a neighboring monomer.
- the epitope on H5 encompasses the entire pocket, which is formed by the HA2 fusion peptide, flanked by elements of HA1 on one side and helix ⁇ A of HA2 on the other.
- CDR-H2 adopts the “type 2” conformation (Chothia et al., Structural repertoire of the human VH segments. J Mol Biol 227, 799-817 (1992)), which is relatively rare in human Abs.
- Phe55 lies across a flat hydrophobic surface formed by the main-chain of the fusion peptide, residues 18 2 -21 2 ; it also makes favorable orthogonal aromatic interactions (Samanta et al., Packing of aromatic rings against tryptophan residues in proteins.
- a large hydrophobic residue at the neighboring position 103 supports the side-chain conformation of Tyr102; and (iii) the CDR-H1 loop is characterized by small hydrophobic/polar side-chains (notably Val27, Thr 28 and Ser31) such that CDR-H1 fits snugly beneath the HA head while packing against helix ⁇ A.
- a somatic mutation of conserved Gly26 >Glu generates a non-canonical conformation for H1, with Thr27 pointing outward and making contact with H5.
- N-terminal hairpin (residues 129 2 and M30 2 ) from HA2 of the counterclockwise neighbor packs against the other side of helix ⁇ A at this point, wrapping around its fusion peptide and further locking it into place ( FIGS. 4A and 4C ).
- F10 stabilizes the fusion peptide of more than one subunit.
- the FR3 residue at position 72 is the major determinant of the choice between two distinct conformations of the H2 loop (Chothia et al. (1992)).
- CDR-H2 derived from germline V1-69
- position 55 is always Phe
- position 54 is always hydrophobic (M/I/L/V).
- CDR-H3 always has a Tyr predicted to lie at the tip of the CDR3 loop (conserved at the 6th position).
- the sixth VH gene that was isolated is derived from the germline gene, VH1-2; its H2 loop has the same length as VH1-69, but by virtue of a change from Ala to Arg at position 72 (Chothia et al. (1992)) it is predicted to adopt a distinct conformation (“type 3”), which presents loop residues 3 and 4 to the antigen (rather than residues 4 and 5 in type 2 loops).
- type 3 a distinct conformation
- the specific somatic mutation at position 4 from Asn to Met presumably promotes H5 binding. It is not possible to predict the structure of the larger H3 loop, but a tyrosine is located at the center of the loop that may play an analogous role to that in VH1-69.
- the F10-H5 crystal structure indicates a common mechanism of H5 virus neutralization for the discovered nAbs. They make no contact with the receptor-binding sites in the head and so do not inhibit cell attachment. Rather, they lock the fusion peptide and helix ⁇ A in place, thereby preventing the large structural reorganizations that are required for membrane fusion (Stevens et al., Structure and receptor specificity of the hemagglutinin from an H5N1 influenza virus. Science 312, 404-10 (2006); Skehel & Wiley (2000); Stevens et al., Structure of the uncleaved human H1 hemagglutinin from the extinct 1918 influenza virus.
- Anti-H5 nAbs Bind and Neutralize a Broad Range of Group 1 Viruses In Vitro and In Vivo
- top ‘( )/’ (amino acid variant(s)) / amino acid consensus at the position; bottom ‘( )/’, (number of amino acid variants)/ number of consensus amino acids.
- Non-highlighted amino acids are 100% conserved or variants are observed ⁇ 5 times at those positions for subtypes H4, H6, H9, H10, H11.
- Histidines H17 (HA1) and H111 (HA2) that can play a role in pH-trigger are in bold underline.
- Group 1 viruses which contain 10 of the 16 subtypes, are further classified into 3 “clusters”, H1a, H1b, and H9 (Russell et al., H1 and H7 influenza haemagglutinin structures extend a structural classification of haemagglutinin subtypes. Virology 325, 287-96 (2004); Fouchier et al., Characterization of a novel influenza A virus hemagglutinin subtype (H16) obtained from black-headed gulls. J Virol 79, 2814-22 (2005)) ( FIG. 5 ).
- the IgG1s also neutralized H5-, H1-, H2-, H6- and H11-pseudotyped virus infections ( FIG. 6A ).
- F10-IgG1 also neutralized H5N1, H1N1, H2N2, H6N1, H6N2, H8N4, and H9N2 influenza viruses ( FIG. 6B ).
- none of the nAbs neutralized Group 2 viruses, e.g. H3N2 ( FIG. 6B ).
- these nAbs recognize an epitope on HA that is conserved among H5 clades as well as in all members of Group 1 viruses.
- the epitope can be divided into 3 elements: (i) at its center, the sequence of the N-terminal segment of HA2-fusion peptide residues 18 2 -21 2 —is conserved across all HA subtypes (note that the side-chain at position 19 2 does not participate in binding); (ii) a downstream segment of HA2 adopts part of the ⁇ A helix (residues 39 2 -56 2 ), which is nearly invariant; the only significant difference is a Thr to Gln change at position 49 2 in the untested H9 cluster subtype, H12. Thr49 2 lies at the periphery of the epitope and makes one long H-bond (3.5 ⁇ ) to Ser31.
- the remarkable transformation to the fusogenic state includes repacking of the central helices of three HA2 protomers to form a new triple-helical bundle, in which residues 34-37 form an N-terminal cap, as well as the creation of C-terminal arms that extend to the N-terminus of the new bundle (Chen et al., N- and C-terminal residues combine in the fusion-pH influenza hemagglutinin HA(2) subunit to form an N cap that terminates the triple-stranded coiled coil. Proc Natl Acad Sci USA 96, 8967-72 (1999)). It is straightforward to model the locations of the F10 epitope residues in this model of the fusogenic state.
- VN/04 (H5N1) virus was propagated in MDCK cells for 72 h in the presence of 40 ⁇ g ml ⁇ 1 of each of the 3 nAbs as well as a murine Ab, 22F, that targets the receptor-binding head. Following three in vitro passages, a mutant VN04 virus (K193E) that was resistant to 22F was isolated. In contrast, no viruses resistant to any of the 3 IgG1s (D8, F10, or A66) were identified. While these experiments cannot prove that escape mutants with unimpaired viral fitness will never arise, they clearly support the notion that the pocket is more refractory than epitopes in the head.
- nAbs can be identified using the disclosed methods, or other of the disclosed HA stem antibodies that are engineered to have even broader spectrum reactivity (Sui et al., Broadening of neutralization activity to directly block a dominant antibody-driven SARS-coronavirus evolution pathway. PLoS Pathog 4, e1000197 (2008)) can be used.
- nAbs isolated against influenza A virus Prior to the present study, the vast majority of nAbs isolated against influenza A virus have targeted the receptor-binding head and lacked broad cross-neutralizing activity.
- a murine nAb termed C179 (Okuno et al., A common neutralizing epitope conserved between the hemagglutinins of influenza A virus H1 and H2 strains. J Virol 67, 2552-8 (1993)
- VH1-69 is the predominant gene utilized by a group of CD4-induced (“CD41”) nAbs raised against the HIV-1 surface glycoprotein, gp120, where the “pocket” is part of a conserved co-receptor binding site that is only exposed transiently upon binding to its primary receptor, CD4 (Huang et al. (2004)).
- an antibody raised against the HIV gp41 trimeric “inner-core” fusion protein intermediate utilizes the hydrophobic tip of its VH1-69 CDR-H2 loop to insert into a conserved hydrophobic pocket that blocks further assembly to the fusion-competent 6-helix structure (Luftig et al., Structural basis for HIV-1 neutralization by a gp41 fusion intermediate-directed antibody. Nat Struct Mol Biol 13, 740-7 (2006)).
- VH1-69 In vivo, B cells carrying the VH1-69 gene are the primary mediators of innate defense against HCV infection, generating antibodies against its membrane fusion glycoprotein, E2 (Chan et al., V(H)1-69 gene is preferentially used by hepatitis C virus-associated B cell lymphomas and by normal B cells responding to the E2 viral antigen. Blood 97, 1023-6 (2001)), although the epitope and mode of action have not been determined.
- VH1-69 is not the only germline that is suitable for achieving neutralization in a similar manner.
- the broad-spectrum nAbs described herein are not generated/expanded during successive rounds of influenza infection and repeated vaccination. Only the disclosed methods have been effective to produce such antibodies consistently and repeatedly. It is unlikely that the F10 epitope provokes self-tolerance mechanism(s) via auto-antigen mimicry (Scherer et al., Difficulties in eliciting broadly neutralizing anti-HIV antibodies are not explained by cardiolipin autoreactivity. Aids 21, 2131-9 (2007)). Rather, an immunodominant Ab response to the highly-exposed globular head can overwhelm the Ab response to the F10-epitope.
- Ranges may be expressed herein as from “about” one particular value, and/or to “about” another particular value. When such a range is expressed, also specifically contemplated and considered disclosed is the range from the one particular value and/or to the other particular value unless the context specifically indicates otherwise. Similarly, when values are expressed as approximations, by use of the antecedent “about,” it will be understood that the particular value forms another, specifically contemplated embodiment that should be considered disclosed unless the context specifically indicates otherwise. It will be further understood that the endpoints of each of the ranges are significant both in relation to the other endpoint, and independently of the other endpoint unless the context specifically indicates otherwise.
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Cited By (12)
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US20130064843A1 (en) * | 2011-06-03 | 2013-03-14 | Dana-Farber Cancer Institute, Inc. | Identification of conserved peptide blocks in homologous polypeptides |
US8877200B2 (en) | 2012-05-10 | 2014-11-04 | Visterra, Inc. | HA binding agents |
WO2015051010A1 (fr) * | 2013-10-02 | 2015-04-09 | Medimmune, Llc | Anticorps anti-grippe a neutralisants et leurs utilisations |
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US12024552B2 (en) | 2020-08-14 | 2024-07-02 | Visterra, Inc. | Ha binding agents |
Also Published As
Publication number | Publication date |
---|---|
WO2010027818A3 (fr) | 2010-07-01 |
WO2010027818A2 (fr) | 2010-03-11 |
US20190062408A1 (en) | 2019-02-28 |
US20210221873A1 (en) | 2021-07-22 |
EP4071169A2 (fr) | 2022-10-12 |
EP2328928A2 (fr) | 2011-06-08 |
EP3301116A1 (fr) | 2018-04-04 |
CA2736799A1 (fr) | 2010-03-11 |
US10774134B2 (en) | 2020-09-15 |
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