WO2005018666A1 - Polypeptide multimers having antiviral activity - Google Patents

Polypeptide multimers having antiviral activity Download PDF

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Publication number
WO2005018666A1
WO2005018666A1 PCT/US2003/025295 US0325295W WO2005018666A1 WO 2005018666 A1 WO2005018666 A1 WO 2005018666A1 US 0325295 W US0325295 W US 0325295W WO 2005018666 A1 WO2005018666 A1 WO 2005018666A1
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heptad
seq
polypeptide
amino acid
monomer
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PCT/US2003/025295
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French (fr)
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Carol D. Weiss
Yong He
Russell Vassell
Eve De Rosny
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The Government Of The United States Of America, As Represented By The Department Of Health And Human Services
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Priority to PCT/US2003/025295 priority Critical patent/WO2005018666A1/en
Priority to AU2003265420A priority patent/AU2003265420A1/en
Publication of WO2005018666A1 publication Critical patent/WO2005018666A1/en

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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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • 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
    • C07K16/1036—Retroviridae, e.g. leukemia viruses
    • C07K16/1045—Lentiviridae, e.g. HIV, FIV, SIV
    • C07K16/1063—Lentiviridae, e.g. HIV, FIV, SIV env, e.g. gp41, gp110/120, gp160, V3, PND, CD4 binding site
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56983—Viruses
    • G01N33/56988—HIV or HTLV
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/64—Medicinal preparations containing antigens or antibodies characterised by the architecture of the carrier-antigen complex, e.g. repetition of carrier-antigen units
    • A61K2039/645—Dendrimers; Multiple antigen peptides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • 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
    • C12N2740/00—Reverse transcribing RNA viruses
    • C12N2740/00011—Details
    • C12N2740/10011—Retroviridae
    • C12N2740/16011—Human Immunodeficiency Virus, HIV
    • C12N2740/16111—Human Immunodeficiency Virus, HIV concerning HIV env
    • C12N2740/16122—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/005—Assays involving biological materials from specific organisms or of a specific nature from viruses
    • G01N2333/08—RNA viruses
    • G01N2333/15—Retroviridae, e.g. bovine leukaemia virus, feline leukaemia virus, feline leukaemia virus, human T-cell leukaemia-lymphoma virus
    • G01N2333/155—Lentiviridae, e.g. visna-maedi virus, equine infectious virus, FIV, SIV
    • G01N2333/16—HIV-1, HIV-2
    • G01N2333/162—HIV-1, HIV-2 env, e.g. gp160, gp110/120, gp41, V3, peptid T, DC4-Binding site

Definitions

  • the envelope glycoprotein (Env) of human immunodeficiency virus (HIV) mediates fusion of the viral membrane to the target cell membrane resulting in delivery of the viral nucleocapsid into the cytoplasm of the target cell.
  • the Env glycoprotein is synthesized as 160 kiloDalton glycoprotein precursor that is cleaved post translationally into gpl20 and the gp41 transmembrane protein in the Golgi complex.
  • the gpl20 and gp41 polypeptides are noncovalently associated following cleavage of the precursor. It is thought that gp41 mediates cell membrane fusion critical to viral entry. Chan, D. C, and P. S.
  • HIV transmembrane protein gp41 contains two heptad repeat regions, N hejptad and C heptad, which are highly conserved. It is believed that the heptad repeat regions in gp41 self assemble into a six-helix bundle structure(or trimer of hairpins) that likely represents a fusion-active conformation of gp41 that forms after receptor binding and is critical for membrane fusion.
  • This structure consists of a triple stranded coiled coil formed by a trimer of N heptads. The C heptads pack in the grooves of the coiled coil in an antiparallel manner.
  • the gp41 Env polypeptide is a molecule that has conserved regions that form a constrained structure important for the infectivity of the virus.
  • the N heptad regions of gp41 are conserved among many HIV strains; and form a coiled coil structure that is an important mediator of membrane fusion.
  • polypeptide multimers comprised of at least one monomer with a ratio of N heptads to C heptads of at least about 2: 1 and that have antiviral activity and compositions comprising the polypeptide multimers and methods for inhibiting HIV and/or SIV infectivity with the compositions.
  • the polypeptide multimer comprises at least one monomer comprising a first and second N heptad, a C heptad, and a first and second linker moiety, wherein the first and second N heptad and C heptad are each connected to one another by the first or second linker moieties.
  • the monomer forms a homodimer or homotrimer in solution.
  • the N and C heptads may be connected to one another in different arrangements but each heptad is separated from the other by a linker moiety.
  • the first N heptad is connected to the C heptad by the first linker moiety and the second N heptad is connected to the C heptad by the second linker moiety.
  • the first N heptad is connected to the second N heptad by the first linker moiety and the second N heptad is connected to the C heptad by the second linker moiety.
  • the C heptad is connected to the first N heptad by the first linker moiety and the first N heptad is connected to the second N heptad by the second linker moiety.
  • the present invention is also directed to a method of raising a broadly neutralizing antibody response to HIV by administering to a mammal a composition including one or more novel peptides and proteins, herein referred to as conjugates or monomers, that mimic fusion-active transmembrane protein structures.
  • conjugates are formed from two or more amino acid sequences that comprise: (a) one or more amino acid sequences that are capable of forming a stable coiled-coil solution structure corresponding to or mimicking the heptad repeat region of gp41 (N-helical domain); and (b) one or more amino acid sequences that correspond to, or mimic, an amino acid sequence of the transmembrane-proximal amphipathic - helical segment of gp41 (C-helical domain); wherein said one or more sequences (a) and (b) are alternately linked to one another via a bond, such as a peptide bond (amide linkage) or by an amino acid linking sequence consisting of about 2 to about 25 amino acids.
  • a bond such as a peptide bond (amide linkage) or by an amino acid linking sequence consisting of about 2 to about 25 amino acids.
  • conjugates or monomers are preferably recombinantly produced.
  • one or more of these conjugates or monomer folds and assembles in solution into a structure corresponding to, or mimicking, the gp41 core six helix bundle.
  • one or more of the monomers folds and assembles into structures that may not solely form a six-helix bundle.
  • the invention contemplates that the monomers may form other structures such as shown in Figure 1.
  • the polypeptide multimer comprises at least one monomer comprising a first N heptad, wherein the first N heptad comprises i) a polypeptide having an amino acid sequence of SEQ ID NO: 1, or ii) a polypeptide having at least 75% sequence identity to SEQ ID NO: 1 and preferably, is capable of forming a coiled coil structure in solution or when in contact with another N and/or C heptad.
  • the N heptad forms a coiled coil structure with one or more N heptads or when it contacts a C heptad.
  • the C heptad comprises a polypeptide having an amino acid sequence of SEQ ID NO: 76 or a polypeptide having at least about 65% sequence identity to SEQ LD NO: 76 and is capable of forming an amphipathic helical structure in solution.
  • the second N heptad comprises i) a polypeptide having an amino acid sequence of SEQ LD NO: 1, or ii) a polypeptide having at least 75% sequence identity to SEQ ID NO: 1 and is capable of forming a coiled coil structure in solution.
  • the first and second N heptad have the same amino acid sequence.
  • the polypeptide multimer comprises at least one monomer comprising a polypeptide having i) an amino acid sequence of SEQ ID NO:87 or SEQ LD NO:88 or SEQ LD NO:89 or SEQ ID NO:90 or ii) an amino acid sequence that at least about 75% sequence identity to SEQ ID NO:87 or SEQ ID NO:90 or ii) an amino acid sequence that at least about 75% sequence identity to SEQ ID NO:87 or SEQ ID
  • polypeptide multimer comprises a) at least one monomer comprising: i) a first N heptad and second N heptad; ii) a first and second linker moiety; and iii) a C heptad, wherein each heptad is separated from one another by the first or second linker moiety; and b) a third N heptad capable of forming a coiled coil structure in solution; wherein the monomer and the third N heptad form a 4 helix bundle in solution.
  • polypeptide multimer is a trimer comprising three monomers, each monomer comprising: i) a first N heptad and a second N heptad; ii) a first and second linker moiety; iii) a C heptad; and iv) a peptide; wherein each heptad is separated from one another by the first or second linker moiety; wherein the peptide is located at the C terminal end of the monomer; and wherein the monomer forms a homotrimer in solution.
  • HIV or SIV infection is inhibited by administering to a subject an antiviral effective amount of a composition comprising multimers of the invention.
  • the antiviral compositions administered to the subject comprise a polypeptide multimer comprising at least one monomer comprising a polypeptide having i) an amino acid sequence of SEQ LD NO: 87 or SEQ ID NO:88 or SEQ ID NO:89 or SEQ ID NO:90 or ii) an amino acid sequence that at least about 75% sequence identity to SEQ LD NO:87 or SEQ LD NO:88 or SEQ ID NO:89 or SEQ ID NO:90 and preferably, is capable of forming a homodimer of homotrimer in solution.
  • the polypeptide multimer comprises at least one monomer comprising a polypeptide having i) an amino acid sequence of SEQ ID NO:87 or SEQ ID NO:88 or SEQ LD NO:89 or SEQ ID NO:90 or ii) an amino acid sequence that at least about 75% sequence identity to SEQ ID NO:87 or SEQ LD NO:88 or SEQ ID NO:89 or SEQ LD NO:90 and preferably is capable of forming a homodimer of homotrimer in solution.
  • antibodies that specifically bind the polypeptide multimers of the invention and compositions comprising the antibodies.
  • the antibodies may be monoclonal or polyclonal.
  • the antibodies may be useful therapeutically.
  • the antibodies specifically bind a polypeptide multimer comprising at least one monomer comprising a polypeptide having i) an amino acid sequence of SEQ ID NO:87 or SEQ LD NO:88 or SEQ ID NO:89 or SEQ ID NO: 90 or ii) an amino acid sequence that at least about 75% sequence identity to SEQ ID NO:87 or SEQ LD NO:88 or SEQ LD NO:89 or SEQ ID NO:90 and preferably is capable of forming a homodimer of homotrimer in solution.
  • polypeptides, immunogens, antibodies, and compositions thereof of the invention can be administered in combination with other agents useful in the treatment of HIV infection, SIV infection, AIDS, or ALDS-related complex (ARC).
  • the polypeptides, immunogens, antibodies, and compositions thereof can be admimstered prior to, during, or after a period of actual or potential exposure to HIV and/or SIV.
  • Figure 1 A schematic diagram of the predicted structure of multimers formed by oligomerization of 3-helix heptad repeat (3HR) monomers.
  • the multimers form stabilized coiled coil structures that mimic gp41 fusion intermediates.
  • a monomer may fold in solution and form a 4-helix bundle in the presence of free N heptad.
  • a homodimer may fold in solution and form a five-helix bundle connected to an uncomplexed N heptad.
  • a homotrimer may fold in solution and form a six-helix bundle connected to a trimer of N heptads.
  • FIG. 2 The secondary structure of the heteromultimers formed by the recombinant monomers in solution was analyzed by circular dichroism (CD) spectroscopy in the far-uv spectral region (190-250 nm). CD spectra for the 3HR- NT homodimer and 3HR-Avi homotrimer show that both the dimer and trimer have significant helical structure.
  • the homodimer complex was estimated to be about 85% helical.
  • the homotrimer complex was estimated to be about 76% helical.
  • FIG. 3 The stability of the polypeptide multimers was determined by the apparent melting temperature (2m) of each of the forms of polypeptide multimers.
  • the T of the 3HR-HA homodimer and 3HR-Avi homotrimer were determined by CD spectroscopy.
  • the signal at 220 nm was used to monitor the unfolding of the polypeptide multimers as the temperature was increased.
  • a 10°C step was used during the thermal melts and the temperature range was 0.5 to 90.5°C.
  • the Tax of the homodimer was approximately 80°C, suggesting a very compact bundle.
  • FIG. 4 The novel 3HR polypeptides selectively bind to receptor-activated gp41.
  • immunoprecipitation assay were performed using intact Env-express cells in the presence or absence of soluble CD4 receptor (sCD4).
  • 3HR-gp41 complexes were immunoprecipitated with anti-HA and anti-Avi monoclonal antibodies as described in Furuta et al., 1998, Nature Struct. Biol, 5:276-279.
  • the immunoprecipitated complexes were separated by SDS-Page and immunoblotted with anti-gp41 monoclonal antibody.
  • 3HR-HA formed a complex with and immunoprecipitated gp41 only in the presence of sCD4 (lane 4). No complex was seen in the absence of receptor (lane 3).
  • FIG. 5 Specific binding of the novel 3HR polypeptides to receptor-activated gp41 inhibited HIV-1 Env mediated HIV infectivity.
  • 3HR-His and 3HR-NT U87-CD4-CXCR4 cells were inoculated with Env-expressing luciferase reporter p24 pseudoviruses and incubated for 48 h. The cells were then lysed and inhibition ofHIV infectivity was determined by measuring luciferase activity.
  • 3HR-His and 3HR-NT each displayed a dose-dependent inhibitory effect with an IC 50 of approximately 30nM.
  • Anti-3HR sera prepared against the homodimer immunoprecipitates both receptor-activated and non-receptor activated forms of gp41 from cell surfaces. Rabbits were immunized with homodimers comprising 3HR-NT. Sera from rabbit 782 was diluted 1 :50 in the immunoprecipitation assay. Sera from rabbits 783 and 784 was diluted 1 : 100 in the immunoprecipitation assay. Sera from all three rabbits immunoprecipitated both receptor-activated gp41 and non-receptor activated gp41 from cell surfaces. See lanes 3 and 4, 7 and 8, 11 and 12 in Figure 6.
  • FIG. 7 Polyclonal antisera to 3HR-NT homodimers inhibited syncytia formation at both 31°C and 37°C.
  • the effector cells in the assays expressed LAI Env.
  • the IC 50 of the polyclonal antibodies at 37°C ranged from 24 ⁇ g/ml to 32 ⁇ g/ml, dependent upon the particular rabbit from which the antisera was obtained.
  • the IC 50 for the polyclonal antibodies preincubated with the target and effector cells at 31.5°C ranged from 4.4 ⁇ g/ml to 7 ⁇ g/ml.
  • Figure 8 Polyclonal antisera to 3HR-NT homodimers inhibited syncytia formation between effector cells expressing JR-FL Env or 89.6 Env and target cells at 37°C.
  • the IC50 for the polyclonal antibodies was 21 ⁇ g/ml when the effector cells expressed 89.6 Env and 8 ⁇ g/ml when the effector cells expressed JR-FL Env.
  • Figure 9 Illustrates the construction of conjugates (monomers) derived from repeating gp41 fragments and then subsequent folding and interaction to form immunologically relevant epitopes.
  • N heptad refers to a polypeptide having at least one 7 amino acid heptad repeat region and preferably, is capable of forming a coiled coil structure in solution or when it contacts a N and/or C heptad.
  • the N heptad comprises at least 4 heptad repeat regions, hi one embodiment, the N heptad region comprises at least about 28 to 55 amino acids of the N heptad repeat region of the extracellular domain of gp41.
  • the N heptad may be synthetic or recombinant.
  • the N heptad may be selected from any strain or isolate of HIV or SIV or selected from any of the known N heptad peptides that have antiviral activity.
  • the N heptad may be derived from any strain of HIV or SIV by insertional, substitutional, and/or deletional changes of an N heptad sequence selected from any strain or isolate of HIV or STV or from any known N heptad peptides with the proviso the N heptad so derived is capable of forming a coiled coil structure in solution or when it contacts a N and/or C heptad.
  • C heptad refers to a polypeptide having at least 7 amino acids and preferably, is capable of forming an amphipathic a helical structure when it contacts a N heptad coiled coil.
  • the C heptad comprises at least a 4 heptad repeat region, h one embodiment, the C heptad comprises at least about 24 to 56 amino acids of extracellular C heptad domain of gp41.
  • the C heptad may be synthetic or recombinant.
  • the C heptad may be selected from any strain or isolate of HIV or SIV or selected from any of the known C heptad peptides that have antiviral activity.
  • the C heptad may be derived from any strain of HIV or SIV by insertional, substitutional, and/or deletional changes of a C heptad selected from any strain of HIV or SIV or any known C heptad with the proviso the C heptad is capable of forming an amphipathic a helical structure when it contacts a N heptad coiled coil.
  • the phrase "heptad repeat" or "heptad repeat region” refers to a common protein motif having a 4-3 repeat of amino acids, and is often associated with alpha helical secondary structure and coiled coil structure.
  • the heptad repeat region can be represented by the following sequence: a-b-c-d-e-f-g where a and d are each hydrophobic amino acids.
  • a is V, I, L, M, F, W, C, X, S, Q or T and d is V, I, L, M, F, W, C, Y, S, Q or T and b, c, e, f, g can be any naturally occurring amino acid. More preferably, a and d are V, I or L (Wild et al., 1992, Proc Natl. Acad. Sci., 89-10537-10541).
  • linker moiety refers to a moiety that separates and/or connects heptads from one another.
  • the linker moiety is flexible, allowing the monomer to fold so that N heptads can form a coiled coil structure and associate with other N heptads and/or the C heptad.
  • the linker comprises a peptide from about 2 to about 36 amino acid residues, preferably from 2 to about 16 amino acid residues.
  • the linker comprises a native sequence linker or derivative of a native sequence linker.
  • Native sequence linkers useful in the invention include, but are not limited to, peptides comprising the full-length native sequence linker, or a truncated sequence, containing one or more amino acid substitutions, insertions, and/or deletions.
  • Preferred peptides comprise amino acid residues glycine and serine, and/or glycine and cysteine.
  • linker moieties include, but are not limited to: (GGGGS) X (SEQ LD NO: 77) wherein x is 1 to 5 (WO 00/40616); (GGC) X where X is 1 to 5 (WO 00/40616); GGSGG (SEQ ID NO: 78) (Root et al., 2001, Science, 291:884-888); GSSGG (SEQ ID NO: 79) (Root et al., 2001, Science, 291:884-888); SGGRGG (SEQ ID NO: 80) which is trypsin cleavable (-GGR-) (Tan et al., 1997, Proc. Natl. Acad. Sci.
  • the linker moiety can optionally include one or more amino acids that provide for proteolytic cleavage.
  • the term "antibody” is used in the broadest sense and specifically covers monoclonal antibodies and polyclonal antibodies.
  • the term "homodimer” as used herein means a polypeptide multimer comprising two polypeptide monomers wherein each of the monomers comprise the same arrangement of N heptads and C heptads. For example, each monomer has a first N heptad separated by a first linker moiety followed by a C heptad followed by a second linker moiety followed by the second N heptad.
  • the monomers comprising the homodimer each have the same amino acid sequence and are capable of forming a coiled coil structure in solution.
  • the term "homotrimer” as used herein means a polypeptide multimer comprising three polypeptide monomers wherein each monomer comprises the same arrangement of N heptads and C heptads.
  • each monomer comprises a first N heptad separated by a first linker moiety followed by a C heptad followed by a second linker moiety followed by the second N heptad.
  • the monomers comprising the homotrimer are capable of forming a coiled coil structure in solution.
  • the term "monoclonal antibody” as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations that may be present in minor amounts. Monoclonal antibodies are highly specific, being directed against a single antigenic site. Furthermore, in contrast to conventional polyclonal antibody preparations that typically include different antibodies directed against different epitopes, each monoclonal antibody is directed against a single epitope on the antigen.
  • the modifier "monoclonal” indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method.
  • the monoclonal antibodies of the present invention may be made by the hybridoma method first described by Kohler et al, Nature 256:495 (1975), or may be made by recombinant DNA methods (see, e.g., U.S. Patent No. 4,816,567).
  • the "monoclonal antibodies” may also be isolated from phage antibody libraries using the techniques described in Clackson et al, Nature 352:624-628 (1991) and Marks et al, J. Mol Biol. 222:581-597 (1991), for example.
  • the term "therapeutically effective amount” refers to an amount of a drug effective to treat a disease or disorder in a mammal.
  • therapeutically effective amount is a dose, which provides some therapeutic benefit on administration, including, in the context of the invention, reduced viral activity or viral load in a patient, or inhibition of virus replication.
  • treating refers to both therapeutic treatment and prophylactic or preventative measures. Those in need of treatment include those already infected with HIV as well as those in which HIV infection is to be prevented. “Treatment” refers to both therapeutic treatment and prophylactic or preventative measures.
  • a nucleic acid is "operably linked,” as used herein, when it is placed into a functional relationship with another nucleic acid sequence.
  • DNA for a presequence or secretory leader is operably linked to DNA for a antibody if it is expressed as a preprotein that participates in the secretion of the antibody; a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the sequence; or a ribosome binding site is operably linked to a coding sequence if it is positioned so as to facilitate translation.
  • "operably linked" means that the DNA sequences being linked are contiguous, and, in the case of a secretory leader, contiguous and in reading phase. However, enhancers do not have to be contiguous. Linking is accomplished by ligation at convenient restriction sites.
  • Polynucleotide means a sequence of nucleic acids that encode a polypeptide of the invention.
  • Peptide sequences defined herein are represented by one-letter symbols for amino acid residues as follows: A alanine L leucine R arginine K lysine N asparagine M methionine D aspartic acid F phenylalanine C cysteine P proline Q glutamine S serine E glutamic acid T threonine G glycine w tryptophan H histidine Y tyrosine I isoleucine V valine "Percent (%) amino acid sequence identity” means the percentage of amino acid residues in a polypeptide that are identical with amino acids in a reference polypeptide, after aligning the sequence and introducing gaps, if necessary to achieve the maximum sequence identity, and not considering any conservative substitutions as part of the sequence identity.
  • % amino acid sequence identity of a given amino acid sequence A to, with, or against a given amino acid sequence B is calculated as follows:
  • % amino acid sequence identity may also be determined using the sequence comparison program such as ALIGN 2 or NCBI-BLAST2 (Altschul et al., 1997, Nucleic Acids Res., 25:3389-3402).
  • NCBI-BLAST2 sequence comparison program may be downloaded from http://www-ncbi-nlm-nih-gov or otherwise obtained from the National Institute of Health, Bethesda, MD.
  • N heptads are highly conserved among all strains of HIV and SIV.
  • the six- helix bundle formed by coiled coil N heptads is a conserved structure that can readily lose function upon loss of that coiled coil structure.
  • the conserved nature of the N heptad makes it an attractive target for membrane fusion inhibitors.
  • Polypeptide monomers and multimers have been constructed that have a greater ratio of N heptads to C heptads. A greater N heptad to C heptad ratio means more hydrophobic faces of the N heptads are exposed to the solvent and are more accessible.
  • N heptads may provide for enhanced antiviral effect and expose additional immunogenic epitopes.
  • an increase in exposure of the hydrophobic N heptads to solvent may also present problems of solubility, aggregation, and stability of the monomer or multimers. It was unexpected that stable multimers could be formed from a monomer with a ratio of N heptads to C heptads of at least about 2:1.
  • a ratio of N to C heptads of at least about 2:1 allows the construction of monomers, dimers, and/or trimers that correspond to or mimic gp41 fusion intermediates.
  • the ratio of N to C heptads is about 3:1.
  • A. Polypeptide Monomers comprised of at least one monomer with a ratio of N heptads to C heptads of at least about 2:1.
  • the multimer comprises at least one monomer comprising a first and second N heptad, a C heptad, and a first and second linker moiety wherein each heptad is connected to another heptad by the first or second linker moiety and wherein the monomer is capable of forming a homodimer or homotrimer in solution.
  • the first N heptad is connected to the C heptad by the first linker moiety and the second N heptad is connected to the C heptad by the second linker moiety.
  • the first N heptad is connected to the second N heptad by the first linker moiety and the second N heptad is connected to the C heptad by the second linker moiety.
  • the C heptad is connected to the first N heptad by the first linker moiety and the first N heptad is connected to the second N heptad by the second linker moiety.
  • the C-terminal end of the first N heptad and the N-terminal end of the C heptad are separated by the first linker moiety, and the C-terminal end of the C heptad and the N-terminal end of the second N heptad are separated by the second linker moiety.
  • the C-terminal end of the first N heptad and the N-terminal end of the second N heptad are separated by the first linker moiety.
  • the C-terminal end of the second N heptad and the N-terminal end of the C heptad are separated by the second linker moiety.
  • N heptads The N heptad is a polypeptide that has at least one 7 amino acid heptad repeat region , and preferably is capable of forming a coiled coil structure in solution or when in contact with another N and/or C heptad.
  • Polypeptides capable of forming a coiled coil structure generally have at least one 7 amino acid heptad repeat region where the first and fourth amino acids are hydrophobic.
  • the 7 amino acid polypeptide comprises: a-b-c-d-e-f-g; wherein a is V, I, L, M, F, W, C, Y, S, Q or T and d is V, I, L, M, F, W, C, Y, S, Q or T.
  • b, c, e, f, g are any of the naturally occurring amino acids.
  • a and d are V, I or L.
  • the N heptad comprises at least 4 heptad repeat regions.
  • the N heptad region comprises at least 4 heptad repeat regions of the extracellular domain of gp41.
  • the N heptad region of gp41 includes amino acids 542-592 as described and shown in Jiang et al, 2002, Current Pharmaceutical Design, 8:563-580.
  • the N heptads may be selected from any strain of HIV or SIV. Representative examples of N heptads from different HIV strains include, but are not limited to, the following peptides. Many other sequences are known and described at www/hiv-web/lanl/gov. All sequences are listed from N-terminus to C- terminus.
  • Subtype B Isolate LAI ARQLLSGIVQQQQNNLLRAIEAQQHLLQLTVWGIKQLQARILAVERYL KDQQLLGI (SEQ LD NO: 2) SG ⁇ VQQQNNLLRAIEAQQHLLQLTVWGIKQLQARILAVERYLKDQ (SEQ ID NO: 3) SG ⁇ VQQQNNLLRA ⁇ EAQQHLLQLTVWGIKQLQARIL (SEQ ED NO: 4) NNLLRALEAQQHLLQLTVWGL QLQARILAVERYLKDQ (SEQ ID NO: 5)
  • Subtype B Isolate ADA SG ⁇ VQQQNNLLRAIEAQQHLLQLTVWGIKQLQARVLALERYLRDQ (SEQ LD NO: 6) SGrVQQQNNLLRALEAQQHLLQLTVWGIKQLQARVL (SEQ ID NO: 7) NNLLRALEAQQHLLQLTVWGIKQLQARVLALERYLRDQ (SEQ LD NO: 8)
  • Subtype B Isolate JRFL SG ⁇ VQQQNNLLRALEAQQRMLQLTVWGIKQLQARVLAVERYLGDQ
  • Subtype G Isolate FI. HH8793
  • NILLRALEAQQHLLQLSrWGIKQLQAKVLAiERYLRDQ Group O Isolate: CM. ANT70C KGrVQQQDNLLRAIQAQQQLLRLSxWGIRQLRARLLALETLLQNQ (SEQ ID NO: 36) KG ⁇ VQQQDNLLRAIQAQQQLLRLSXWGIRQLRARL (SEQ LD NO: 37) DNLLRAIQAQQQLLRLSXWGIRQLRARLLALETLLQNQ (SEQ ID NO: 38)
  • CONSENSUS-BC128 sGIvqQQnnLlrAieaQqhllqLTVwGiKQLqarvLaverYLkdX (SEQ ID NO: 93)
  • CONSENSUS-F2 (5 SGIVQQQsNLLKAJEAQQHLLQLTVWGIKQLQARiLAVERYLKDQ (SEQ ID NO: 97)
  • CONSENSUS-Hffl SGLVQQQSNLLRAIQAqQHMLQLTVWGiKQLQARVLAVERYLkDQQ (SEQ LD NO: 99)
  • CONSENSUS-03B SGTVQQQNNLLRAIEAQQHLLQLTVWGIKQLQARVLAvERYLKDQ (SEQ ID NO: 103)
  • CONSENSUS-04C3 sGrVQQQSNLLRALEAQQhLLrLTVWGiKQLQARVLALESYLkDQ (SEQ ID NO: 104)
  • the N heptad has the following sequence: aa# 546 SGIVQQQNNLLRAIEAQQHLLQLTVWGIKQLQAR aa# 579 (SEQ ED
  • amino acid positions correspond to amino acid positions 546 to 579 of the N heptad sequence of gp41 from HXB 2 isolate of HIV.
  • the first and fourth position of each heptad repeat region are underlined.
  • Two examples of useful peptides include the peptide P-17, which has the formula, from amino terminus to carboxy terminus, of: NH 2 NNLLRAIEAQQHLLQLTVWGIKQLQARILAVERYLKDQ-COOH
  • peptides are optionally coupled to a larger carrier protein, or optionally include a terminal protecting group at the N- and/or C-termini.
  • Useful peptides further include peptides corresponding to P- 17 or P- 15 that include one or more, preferably 1 to 10 conservative substitutions, as described below.
  • the N heptad sequences in a monomer can vary because amino acid sequences of native gp41 of different strains or isolates can vary in sequence as described by the representative sequences shown above.
  • the N heptad sequence can be any one of the consensus sequences that have been identified.
  • the N heptad may be derived from any strain of HLV or SIV by insertional, substitutional, and/or deletional changes of an N heptad sequence selected from any strain or isolate of HIV or SIV from any known N heptad peptides with the proviso the N heptad so derived is capable of forming a coiled coil structure in solution or when in contact with another N and/or C heptad.
  • N heptad sequences can also be derived from any native sequence by changing amino acids. Amino acid positions and the type of substitutions that can be made at these positions can be identified by aligning and analyzing known sequences and/or conforming retention of coiled coil structures using known algorithms.
  • Alignment and comparison of all known N heptad sequences provides one method for identifying those amino acid positions that can tolerate amino acid substitutions. Aligning and comparing all of the known N heptad sequences of HIV isolates indicates that they have about 75% sequence identity to SEQ ID NO: 1 when aligned with amino acid positions 546 to 579.
  • amino acid residues that participate in the formation and/or stabilization of the coiled coil structure formed by the N heptad in solution are not substituted.
  • amino acid residues at positions a and d of the heptad repeat region(s) are not substituted, hi addition, amino acid residues that form a hydrophobic pocket are preferably not substituted.
  • the residues that form this hydrophobic pocket include amino acid residues LLQLTVWGIKQLQAR (SEQ ID NO: 111). Chan et al., 1997, Cell, 89:263-273. This sequence corresponds approximately to amino acid positions 565 - 579 of isolate HBX 2 .
  • the hydrophobic pocket may vary in sequence in different isolates, but corresponds to about amino acid positions 565-579 of isolate HBX 2 .
  • the amino acid positions corresponding to GIVQQQ (amino acid positions 547 to 552) are not substituted. Comparison of the alignment of sequences shows that certain positions in the heptad repeats tolerate amino acid substitution.
  • amino substitutions are made at the second amino acid position from the N terminus or at least one of the last two C terminal amino acids of a heptad repeat region or both.
  • the positions that tolerate amino acid substitution comprise substitutions at at least one amino position 546, 553, 557, 560, 564, 565, 567, 574, or 577, or mixtures thereof corresponding to amino acid positions of N heptad region of HBX 2 isolate of HIV.
  • An amino acid substitution made at these positions preferably increases the hydrophobic character of the heptad repeat region.
  • N heptad variants with amino acid substitutions that are capable of forming a coiled coil structure can also be identified using any one of a number of algorithms that predict coiled coil structure such as the Socket program at http://www-biols-susx-ac- uk/Biochem/Woolfson/html/coiledcoils/socket.
  • amino acid substitution with cysteines provides for the formation of disulfide bonds.
  • a disulfide bonds may be employed to stabilize the 4-helix bundle, homodimer or homotrimer for example, for in vivo use.
  • cysteine substitutions are made at the N terminal amino acids, C terminal amino acids, or both of an N heptad.
  • the solubility of N heptads may be improved by mutating positions in the heptad repeat region that do not directly interact with N or C heptad interactions.
  • the solubility of the N heptad is improved by mutating the amino acid residues at positions b and c of the heptad repeat region.
  • the solubility of an N heptad may also be improved by fusing the N heptad with another helix with greater solubility forming a chimeric helix.
  • the solubility of an N heptad may also be improved by substituting amino acids with cysteine residues providing for the formation of disulfide bonds, as described above, that stabilize the 4-helix bundle, homodimer, homotrimer.
  • the N heptads useful in the invention include polypeptide variants that have at least about 75% amino acid sequence identity to SEQ ID NO: 1 when aligned to amino acid positions corresponding to amino acid positions 546 to 579, and preferably, are capable of forming a coiled coil structure when in contact with another N and/or C heptad.
  • N heptads that are useful in the invention also include polypeptide variants that have at least about 75% sequence identity, more preferably have any one of % sequence identity between 75% to 100% sequence identity (eg. 16%, 11%, 78%, etc.), more preferably 80% sequence identity, more preferably 85% sequence identity, more preferably 90% sequence identity, more preferably 95% sequence identity to SEQ ID NO: 1.
  • the polypeptide variants do not have amino acid residue substitutions at positions a and d of the N heptad repeat region(s) or at the amino acid positions that form the hydrophobic pocket such as amino acids that correspond to amino acids 565 - 579 of gp41 of HBX 2 .
  • monomers of the invention may include truncated first and/or second N heptads, which exhibit the ability to form a coiled coil structure in solution.
  • Such truncated N heptads may comprise peptides having at least one 7 amino acids heptad repeat region, more preferably between about 7 and 55 amino acid residues.
  • truncated N heptads are those that preferably lack the first N terminal or the last C terminal heptad repeat region. Truncation of the N heptad regions have been described in Dwyer et al, 2003, Biochemistry, 42:4945.
  • Truncations can occur at the N and/or C terminal end of the N heptad region. At the N terminal end of the N heptad, about 13 amino acids, more preferably 6 amino acids, can be removed with minimal effect on stability of the N heptad in solution. Examples include the peptides P- 15
  • the C terminal end of an N heptad can be truncated up to 11 amino acids and the N terminal end of the N heptad can be truncated up to 4 amino acids as in SEQ ID NO: 1.
  • the C terminal end of the N heptad includes the amino acid positions or residues that include the hydrophobic pocket, i.e. amino acid positions corresponding to amino acid positions
  • the first and second N heptads of the monomer can comprise the same amino acid sequence or a related amino acid sequence.
  • N heptad regions can be selected from different strains or isolates and combined to form a single monomer.
  • the first and second N heptads may have an amino acid sequence of SEQ ID NO: 1 ; the first N heptad may have an amino acid sequence of SEQ ED NO: 1 and the second N heptad may have an amino acid sequence having at least 75 percent identity with SEQ DD NO: 1 and is capable of forming a coiled coil structure in solution; or the first N heptad may have an amino acid sequence having at least 75 percent identity with SEQ DD NO: 1 and is capable of forming a coiled coil structure in solution and the second N heptad may have an amino acid sequence of SEQ ID NO: 1.
  • C Heptad refers to a polypeptide having at least one 7 amino acid heptad repeat region and preferably, that is capable of forming an amphipathic helical structure when it contacts a N heptad coiled coil.
  • Polypeptides capable of forming an amphipathic helical structure generally have at least 7 amino acids where the first and fourth amino acids are hydrophobic.
  • the 7 amino acid polypeptide comprises: a-b-c-d-e-f-g; wherein a is V, I, L, M, F, W, C, Y, S, Q or T and d is V, I, L, M, F, W, C, Y, S, Q, D, E or T.
  • the amino acids comprising positions b, c, e, f, and g are any of the naturally occurring amino acids.
  • a is W, S or N and d is W, Y, L, F, Q, E or D.
  • the C heptad comprises at least 4 heptad repeat regions of the extracellular domain of gp41 of HEV.
  • the C heptad sequence includes amino acids 623 to 663 of gp41.
  • the C heptad may be selected from any strain of HEV or SEV. Representative examples of C heptads from different HEV strains include, but are not limited to, the following peptides. Many other sequences are known and described at www/hiv-web/lanl gov. All sequences are listed from N-terminus to C- terminus.
  • HEV-1 Group M Subtype B Isolate: LAI WNNMTWMEWDREENNYTSLEBSLffiESQNQQEKNEQELLELDKWAS LWNWF NITNW (SEQ ED NO: 39) WMEWDREENNYTSLIHSLEeESQNQQEKNEQELLELDKWASLWNWF (SEQ ED NO: 40) WMEWDREENNYTSLEHSLEEESQNQQEKNEQELL (SEQ ED NO: 41)
  • Subtype B Isolate ADA
  • Subtype G Isolate FI. HH8793
  • Group N Isolate CM. YBF30 WQQWDEKVRNYSGVEFGLEEQAQEQQNTNEKSLLELDQWDSLWSW F (SEQ ED NO: 70) WQQWDEKVRNYSGVIFGLIEQAQEQQNTNEKSLL (SEQ ID NO: 71) YSGVEFGLEEQAQEQQNTNEKSLLELDQWDSLWSWF (SEQ ED NO: 72)
  • Group O Isolate CM. ANT70C WQEWDRQISMSSTIYEEIQKAQVQQEQNEKKLLELDEWASIWNWL (SEQ ED NO: 73) WQEWDRQISNISSTIYEEIQKAQVQQEQNEKKLL (SEQ ED NO: 74) ISSTJYEEIQKAQVQQEQNEKKLLELDEWASIWNWL (SEQ ED NO: 75)
  • CONSENSUS-1K6 WieWeReldNYTqtlYTLlEESQiiQQEKNEQdLLXLDKWASLWnWFdlsn WL (SEQ ED NO: 130)
  • CONSENSUS-12(3) WMqWEKELXNYSXelYRLEEeSQnQQEKNEQELLALDKWASLWnWFD ISnWL (SEQ LD NO: 131)
  • the C heptad has a sequence of: aa# 628 W £EWDREINNYTSLIHSLffiESQNQQEK aa# 655 (SEQ DD NO. 76) position a d a d a d a d
  • This sequence corresponds to amino acid positions 628 to 655 in gp41 of
  • HEV isolate HBX 2 The first and fourth position of each heptad repeat region are underlined.
  • useful peptides for this aspect of the invention include the peptide P-18 which corresponds to a portion of the transmembrane protein gp41 from the HEV- 1 LA i isolate, and has the 36 amino acid sequence reading from amino to carboxy terminus): NH 2 -YTSLEHSLffiESQNQQEKNEQELLELDKWASLWNWF-COOH
  • peptides are optionally coupled to a larger carrier protein.
  • Useful peptides further include peptides corresponding to P-18 or P-16 that include one or more, preferably 1 to 10 conservative substitutions, as described below.
  • the peptides of this aspect of the invention may include truncations of the P-18 and P- 16, as long as the truncations is capable of forming a six helix bundle when mixed with P-17.
  • the C heptad sequences in a monomer can vary because amino acid sequences of native gp41 of different strains or isolates can vary in sequence as described and shown above.
  • the C heptad sequence can be any one of the consensus sequences that have been identified.
  • the C heptad may be derived from any strain of HEV or SEV by insertional, substitutional, and/or deletional changes of a C heptad sequence selected from any strain or isolate of HEV or SEV or from any known C heptad peptides with the proviso the C heptad so derived is capable of forming an amphipathic alpha helical structure when it contacts a N heptad coiled coil.
  • C heptad sequences can also be derived from any native sequence by changing amino acids.
  • Amino acid positions and the type of substitutions that can be made at these positions can be identified by aligning and analyzing known sequences and/or conforming retention of helical structures using known algorithms. Alignment and comparison of all known C heptad sequences provides one method for identifying those amino acid positions that can tolerate amino acid substitutions. Aligning and comparing all of the known N heptad sequences of HEV isolates indicates that they have about 65% sequence identity to SEQ ED NO: 76 when aligned to amino acid positions 628 to 655 of SEQ ID NO: 76. Preferably amino acid residues that participate in the formation and/or stabilization of the amphipathic alpha helical structure formed by the C heptad in solution are not substituted.
  • amino acid residues at positions a and d of the heptad repeat region(s) are not substituted.
  • variants are not substituted at positions that interact with the hydrophobic pocket of the N heptad such as those amino acid positions that correspond to Trp 628, Trp 631 and He 635 of SEQ DD NO:76.
  • the C heptads also useful in the invention include C heptad variants that have at least about 65% amino acid sequence identity to SEQ ID NO: 76 and are capable of forming amphipathic a helical structure when in contact with a N heptad.
  • Useful C heptads also include C heptad variants having at least 65% amino acid sequence identity to SEQ ED NO: 76, more preferably any % identity between 65% and 100% sequence identity (eg. 65%, 66%, 61%, 68% etc.), more preferably 70% sequence identity, more preferably 75% sequence identity, more preferably 80% sequence identity, more preferably 85% sequence identity, more preferably 90% sequence identity, and more preferably 95% sequence identity to SEQ ED NO: 76.
  • amino acid residues that participate in the formation and/or stabilization of the amphipathic alpha helical structure formed by the C heptad are not substituted.
  • amino acid residues at positions a and d of the heptad repeat region(s) are not substituted.
  • variants are not substituted at positions that interact with the hydrophobic pocket of the N heptad such as those amino acid positions that correspond to Trp 628, Trp 631 and He 635.
  • the substitutions are at the second or third N terminal amino acid position of the heptad repeat region or the last two C terminal amino acid positions of the heptad repeat region.
  • Amino acids at those positions can be substituted with amino acids that have uncharged polar R groups or charged polar R groups.
  • amino acid positions are substituted with glutamic acid, threonine, aspartic acid, or lysine.
  • the C heptad can tolerate more charged and hydrophillic amino acid substitutions at positions other than position a and d of the heptad repeat regions.
  • amino acid substitutions can be made at amino acid positions 629, 630, 633, 634, 636, 640, 641, 648, or 651 mixtures thereof corresponding to amino acid positions in the C heptad region of gp41 of HBX 2 isolate of HEV.
  • Polypeptides comprising variant C heptads capable of forming amphipathic helical structure can also be predicted using algorithms such as
  • monomers of the invention may include truncated C heptads, which exhibit the ability to form an amphipathic, helical structure when in contact with a N heptad.
  • Such truncated C heptads may comprise peptides at least about one 7 amino acid heptad repeat region, more preferably, between about 28 and 56 amino acids.
  • truncated C heptads include the peptides P-16 (WMEWBP ⁇ INNYTSLfflSLffiESQNQQEKNEQELL (SEQ DD NO: 133) and P-18 (YTSLfflSLIEESQNQQEKNEQELLELDKWASLWNWF (SEQ ID NO: 134) which are truncated forms of the C helical domain of HEV-I LAI gp41 where the peptides are derived by truncating the C heptad region at both the N terminal and C terminal ends.
  • Preferred truncations are those that lack N terminal heptad repeat region or the last C terminal heptad repeat region.
  • the C heptad is truncated 5 amino acids at the N terminal end and 8 amino acids at the C terminal end as compared to the C heptad sequence of amino acids 623 to 663 of gp41.
  • C heptads useful in the invention also include truncations that may include, but are not limited to, peptides comprising the full-length, or a truncated sequence, containing one or more amino acid substitutions, insertions, and/or deletions.
  • C heptads are not truncated at positions that interact with the hydrophobic pocket of the N heptad corresponding to amino acid positions Trp 628, Trp 631 and He 635 of SEQ ED NO:76.
  • the first and second linker moieties are agents or molecules that are flexible and allow the N heptads to fold and form a coiled coil structure.
  • the first and/or second linker moiety may contain one or more sites that are cleavable by chemical or enzymatic means.
  • the polypeptide multimer comprises at least one monomer comprising a first and second N heptad, a C heptad, and a first and second linker moiety.
  • the first N heptad is connected to the C heptad by the first linker moiety and the second N heptad is connected to the C heptad by the second linker moiety.
  • first N heptad is connected to the second N heptad by the first linker moiety and the second N heptad is connected to the C heptad by the second linker moiety.
  • the C heptad is connected to the first N heptad by the first linker moiety and the first N heptad is connected to the second N heptad by the second linker moiety.
  • the linker moiety is preferably a peptide.
  • the first and/or second linker moiety may comprise a peptide with the same or different amino acid sequences.
  • Each linker moiety may comprise an amino acid sequence from about 2 to about 36 amino acid residues, preferably from 2 to about 16 amino acid residues.
  • Native sequence gp41 has a linker region that connects the N and C heptad.
  • the native linker region is about 31 amino acids long.
  • the first and/or second linker may comprise a native sequence linker, which corresponds to amino acids 592 to 623 of gp41.
  • Native sequence linkers can be selected or derived from any of the known linker regions.
  • Linker moieties useful in the invention may include, but are not limited to, peptides comprising the full-length native sequence linker, or a truncated sequence, containing one or more amino acid substitutions, insertions, and/or deletions. The linker moiety is flexible, allowing the monomer to fold.
  • Preferred peptides comprise amino acid residues glycine and serine, and/or glycine and cysteine.
  • linker moieties include, but are not limited to: (GGGGS) X (SEQ DD NO: 77) wherein x is 1 to 5 (WO 00/40616); (GGC) X where X is 1 to 5 (WO 00/40616); GGSGG (SEQ ED NO: 78) (Root et al, 2001, Science, 291:884- 888); GSSGG (SEQ ID NO: 79) (Root et al., 2001, Science, 291:884-888); and SGGRGG (SEQ ID NO: 80) which is trypsin cleavable (-GGR-) (Tan et al., 1997, Proc. Natl. Acad. Sci. USA, 94: 12303-12308); and GGSSGG (SEQ ID NO: 81).
  • linkers designed for flexibility are also known (Wung et al., 1997, J. Immunol. Methods, 204:33-41), such as the Gly-Ala 3 (SEQ ID NO: 82) (Holmes et al., 1996, Protein Pept. Lett., 3:415-422) or Gly 4 Ser 3 linkers (SEQ ID NO: 83) (Micheal et al., 1996, Immunotechnology, 2:47-57) and to incorporate sites for specific proteolysis (Lucic et al., 1998, Australia J. Biotechnol, 61-108; Mathews, D.J. and Wells, J.A., 1993, Science, 26:1113-1117.
  • the linker moiety may also comprise additional functional features including, but not limited to, antigenic epitopes and sites for specific proteolysis.
  • the linker may incorporate a trypsin cleavable site.
  • the first linker moiety of the monomer is trypsin cleavable (-GGR-).
  • the first linker moiety has an amino acid sequence of SEQ DD NO: 80.
  • the monomer may also comprise an additional peptide that, preferably, provides for ease of purification or detection.
  • additional peptides include, but are not limited to, peptide tags such as histidine 6 (His), haemoglutinin protein (HA), avidin (Avi), biotin, c-Myc, VSV-G, HSV, V5, or FLAGTM.
  • His histidine 6
  • HA haemoglutinin protein
  • Avi avidin
  • biotin c-Myc
  • VSV-G histidine 6
  • HSV haemoglutinin protein
  • V5 anti-HA monoclonal antibodies
  • FLAGTM FLAGTM
  • a HA peptide tag for example, may be linked to the C-terminus of the second N heptad of the monomer to facilitate detection of the monomer by anti-HA monoclonal antibodies.
  • An Avi tag for example, may be linked to the second N heptad of the monomer to facilitate biotinylation of the
  • the additional peptide is preferably not another N and/or C heptad.
  • a monomer may comprise more than one peptide tag.
  • a His peptide tag for example, may be linked to the C-terminus of a HA peptide tag to facilitate purification. The His peptide tag binds to a Ni-NTA column. Once purified, the His peptide tag may be cleaved by a protease at a protease site located between the HA and His peptide tags.
  • the monomer comprises a HA, His, or Avi peptide tag.
  • the HA tag comprises an amino acid sequence of SEQ ID NO: 84.
  • the His tag comprises an amino acid sequence of SEQ ED NO: 85.
  • the Avi tag comprises an amino acid sequence of SEQ ED NO: 86.
  • the monomer comprising an additional peptide has an amino acid sequence of SEQ ED NO: 87, SEQ ED NO: 88, SEQ ID NO: 89, or SEQ DD NO: 90.
  • a monomer of the invention has an amino acid sequence: 3-helix HR no tag (3-HR-NT) MSGEVQQQNNLLRAIEAQQHLLQLTVWGIKQLQARSGGRGGWME WDREINNYTSLmSLIEESQNQQEKGGSSGGSGEVQQQNNLLRAEEAQ QHLLQLTVWGIKQLQAR (SEQ ED NO: 87) 3-helix HR haemoglutinin tag (3-HR-HA) MSGJVQQQNNLLRAIEAQQHLLQLTVWGEKQLQARSGGRGGWME WDREINNYTSLIHSLIEESQNQQEKGGSS FGSGrVQQQNNLLRAEEAQ OHLLOLTVWG ⁇ OLOARGffiGRYPYDVPDYAGPG (SEQ DD NO: 88) 3-helix HR histidine 6 tag (3-HR-HA-His) MSGEVQQQNNLLRAIEAQ
  • a monomer of the invention has an amino acid sequence: 3-helix HR avidin tag (3-HR-Avi) MSGEVQQQNNLLRAIEAQQHLLQLTVWGEKQLQARSGGRGGWME WDREINNYTSLIHSLIEESQNQQEK(7G r SS ?G !
  • a monomer or conjugate of the invention can comprise: (1) three tandem repeating units consisting of P-17-linker-P-18 (P-17- linker-P- 18-linker-P- 17-linker-P- 18-linker-P- 17-linker-P- 18), (2) P-17-linker-P-18-linker-P-17, (3) P-18-linker-P-17-linker-P-18, (4) P-17-linker-P-17, (5) three tandem repeating units consisting of P-15-linker-P-16 (P-15- linker-P- 16-linker-P- 15 -linker- P- 16-linker-P- 15 -linker-P- 16, (6) P-15-linker-P-16-linker-P-15, (7) P-16-linker-P-15-linker-P-16, or (8) P-16
  • Preferred amino acid residues include glycine and serine, for example (GGGGS) X , (SEQ ID NO: 77) wherein x is 1, 2, 3, 4, or 5, or glycine and cysteine, for example (GGC) y , where y is 1, 2, 3, 4, or 5.
  • GGC glycine and cysteine
  • P-15 and P-17 are interchangeable and P-16 and P-18 are interchangeable.
  • Representative structures and formation of the six helix bundles are shown in Figure 9.
  • the monomers of the invention may also include variants of a polypeptide with an amino acid sequence of SEQ ID NO: 87, SEQ ID NO: 88, SEQ ID NO: 89 or SEQ ID NO: 90.
  • Variants include amino acid substitutions, deletions and insertions.
  • N heptad and C heptad regions of the monomer can be varied as long as these regions when combined maintain an helical or coiled coil structure.
  • N heptad regions typically have about 1 to 4 amino acid substitutions and in one case has up to about 9 amino acid substitutions when known N heptad sequences are compared to SEQ ID NO: 1.
  • known C heptad sequences are aligned and compared, they, typically, have about five to nine amino acid substitutions compared to SEQ DD NO: 76.
  • the monomer may have a multiple substitutions in one or more of the N heptad and/or C heptad region sequences or in the linker regions.
  • each N heptad and C heptad has up to 9 amino acid substitutions, this would result in about 75 % sequence identity to any of SEQ ID NO: 87, SEQ ID NO: 88, SEQ ED NO: 89 or SEQ ED NO: 90.
  • each N heptad has about 4 amino acid substitutions and each C heptad has up to 9 amino acid substitutions, this would result in a polypeptide that has about 85% sequence identity to any of SEQ ED NO: 87, SEQ ED NO: 88, SEQ ED NO: 89 or SEQ ED NO: 90.
  • Variants of the monomers preferably have at least about 75% sequence identity, more preferably any % identity from about 75% to 100% (eg. 75%, 76%, 77%, 78%, etc.), more preferably 80% sequence identity, more preferably 85% sequence identity, more preferably 90% sequence identity, and more preferably 95% sequence identity to a monomer with an amino acid sequence of SEQ ED NO: 87, SEQ ED NO: 88, SEQ ED NO: 89 or SEQ ED NO: 90.
  • Variants preferably can form a stable coiled coil structure in solution.
  • Variants also, preferably, include the hydrophobic pocket region of the N heptad corresponding to amino acids 565-579 of gp41, for example, SEQ ED NO : 111.
  • C heptads are not truncated or substituted at positions that interact with the hydrophobic pocket such as positions that correspond to Trp 628, Trp 631 and He 635 in SEQ ED NO:76.
  • the monomer of the invention may include amino acid insertions that consist of single amino acid residues or stretches of residues ranging from 2 to 15 amino acids in length. One or more insertions may be introduced into the peptide, peptide fragment, analog and/or homolog.
  • the monomers of the invention may include amino acid deletions of the full length peptide, analog, and/or homolog. Such deletions consist of the removal of one or more amino acids from the full-length polypeptide sequence, with the lower limit length of the resulting peptide sequence being about 7 amino acids. Such deletions may involve a single contiguous portion or greater than one discrete portion of the peptide sequences.
  • peptides comprising the sequences described above may be synthesized with additional chemical groups present at their amino and/or carboxy termini, such that, the stability, bioavailability, and/or immunogenic activity of the peptides is enhanced.
  • hydrophobic groups such as carbobenzoxy, dansyl, or t-butyloxycarbonyl groups, may be added to the peptides' amino termini.
  • an acetyl group or a 9-fluorenylmethoxy- carbonyl group may be placed at the peptides' amino termini.
  • the hydrophobic group t-butyloxycarbonyl, or an amido group may be added to the peptides' carboxy termini.
  • the monomer is capable of forming number of different multimers by the folding of the N heptad regions into a coiled coil structure and association of the C heptad with the N heptad coiled coil structure.
  • a schematic diagram of the predicted structures of multimers is shown in Figure 1.
  • a homotrimer may form a six-helix bundle connected to a trimer of N heptads.
  • the monomer would be able to fold into stable multimers in solution because the ratio of N heptads to C heptads of at least about 2:1 in the monomer made it likely the hydrophobic N heptad would be more exposed to the solvent.
  • An increase in exposure of the hydrophobic N heptads to solvent may present problems of solubility, aggregation, and stability of the monomer or multimers.
  • stable multimers could be formed from a monomer with a ratio of N heptads to C heptads of at least about 2:1. Homodimers or trimers can readily be formed by monomers in aqueous solution and in absence of other polypeptides comprising N or C heptad regions.
  • the homodimers and/or homotimers are formed in aqueous solution.
  • the aqueous solution employed may have a pH or about 4.5 or less.
  • the pH of the solution is closer to a pH of 7.0 there may be tendency for some aggregation to occur depending on the concentration of the monomer. Even though some aggregation may occur, a portion of the mixture remains as a homodimer or homotrimer.
  • Lower concentrations of the homodimer or homotrimer eg.
  • the homodimer has a molecular weight of about 10,000 to 40,000 daltons, or more preferably about 20,000 to 30,000 daltons.
  • the molecular weight of the homodimer can vary depending on the presence or absence of an additional peptide such as a peptide tag.
  • the homodimer is comprised of two monomers, each having an amino acid sequence of SEQ ED NO: 87, and has a molecular weight of about 24,300 daltons as determined by sedimentation equilibrium.
  • the homotrimer has a molecular weight of about 30,000 daltons to 52,000 daltons, or more preferably about 35,000 to 45,000 daltons.
  • the homotrimer is comprised of three monomers each having a sequence of SEQ ID NO: 90, and has a molecular weight of about 39,400 daltons as determined by sedimentation equilibrium.
  • Stability of the polypeptide multimer can be determined by the melting temperature and/or helicity of the polypeptide multimer.
  • the apparent melting temperature (T m ) may be determined by circular dichroism spectroscopy or other methods known in the art. Wild et al., 1992, Proc. Natl. Acad. Sci., 89-10537- 10541.
  • a multimer of the invention has a T m of at about 60 to 90°C, more preferably 70 to 90°C, or even more preferably 80 to 90°C.
  • the homodimer has a T m of about 80°C.
  • a multimer of the invention has helical structure. Helicity of the multimer may be determined by circular dichroism spectroscopy in the far-uv spectral region (190-250nm) or by other methods known in the art. Wild et al., 1992, Proc. Natl. Acad Sci., 89-10537-10541. Preferably, the multimer has a helicity of about 60 % or greater, more preferably any of 60 to 90%.
  • a 4 helix bundle may be formed comprising a monomer and a free N heptad. The monomer comprises a first and second N heptad and a C heptad.
  • the monomer comprises a first and second N heptad connected by a C heptad via linker moieties.
  • a 4 helix bundle is formed when the monomer is combined with a free N heptad polypeptide in solution.
  • the monomer comprises an amino acid sequence of SEQ DD NO: 87 and the N heptad comprises a sequence of SEQ DD NO: 1.
  • the multimer has a ratio of N heptads to C heptads of about 3:1.
  • a homodimer comprising 2 monomers, each monomer comprising an amino acid sequence: MSGEVQQNNLLRAEEAQQHLLQLTVWG ⁇ QLQARSGGRGGWMEWD REENNYTSLFFLSLEEESQNQQEKGGSSGGSGEVQQQNNLLRAIEAQQHLLQLT VWGIKQLQAR (SEQ JD NO: 87)
  • the homodimer is preferably formed in acidic solution and has a molecular weight of about 24,300 daltons as determined by sedimentation equilibrium, and has a Tm of about 85°C and has a helicity of about 85%.
  • the coiled coil structure may also be a homotrimer.
  • the homotrimer may form a six-helix bundle with a three-helix tail.
  • monomers comprising the homotrimer comprise an additional peptide at the C terminal end of about 18 amino acids in length.
  • the additional peptide is the avidin tag.
  • Another embodiment is a homotrimer comprising 3 monomers, each monomer comprising an amino acid sequence of: MSGEVQQQNNLLRAIEAQQHLLQLTVWGI QLQARSGGRGGWMEW
  • a polypeptide multimer of the invention has biological function.
  • the multimer has antiviral and/or immunogenic activity.
  • Antiviral activity includes, but is not limited to, binding receptor activated gp41, inhibiting HEV-1 Env mediated envelope fusion, and/or inhibiting HIV infectivity.
  • a multimer with antiviral activity binds receptor-activated gp41 in vitro and/or in vivo. Binding of the multimer to receptor-activated gp41 may be assayed by immunoprecipitation (Furuta et al., 1998, Nat. Struc. Biol, 5:276-279; He et al., 2003, J. Virol, 77:1666- 1671) or other methods known in the art.
  • a multimer comprising at least one monomer comprising an amino acid sequence SEQ ED NO: 87 or an amino acid sequence having at least 75% percent identity to SEQ ED NO:87 binds receptor activated gp41.
  • a multimer that binds receptor-activated gp41 inhibits HEV-1 Env mediated HEV infectivity in vitro and/or in vivo. Inhibition of HEV infectivity may be assessed by an infectivity assay (Weng, Y. and Weiss, C, 1998, J. Virol, 72:9676-9682; see Example 2) or other methods known in the art.
  • a multimer comprising at least one monomer comprising SEQ ED NO: 87 or an amino acid sequence having at least 75 percent identity to SEQ ID NO: 87 inhibits HEV infectivity.
  • a multimer of the invention is immunogenic.
  • Immunogenicity of the multimer may be assessed, for example, by small animal immunization and ELISA (de Rosny et al., 2001, J. Virol, 75:8859-8863) or other methods well known in the art.
  • antibodies to the multimer bind receptor- activated gp41.
  • antibodies to the multimer inhibit HEV-1 mediated membrane fusion and/or HEV infectivity.
  • polypeptide multimers of the invention are produced by synthetic and recombinant methods. Accordingly, a second aspect of the invention relates to polynucleotides encoding the polypeptide multimers, recombinant vectors, and host cells containing the recombinant vectors, as well as methods of making such vectors and host cells by recombinant methods.
  • the monomers of the invention may be synthesized or prepared by techniques well known in the art. See, for example, Creighton, Proteins: Structures and Molecular Principles, W. H. Freeman & Co., New York, NY (1983), which is incorporated herein by reference in its entirety.
  • Nucleotide sequences for N and C heptads are known and readily available, for example, on the Internet at www/hiv- web/lanl/gov. Short peptides, for example, can be synthesized as a solid support or in solution. Longer peptides may be made using recombinant DNA techniques. The nucleotide sequences encoding the peptides of the invention may be synthesized, and/or cloned, and expressed according to techniques well known to those of ordinary skill in the art. See, for example, Sambrook, et al., Molecular Cloning, A
  • the polynucleotides may be produced by standard recombinant methods known in the art, such as polymerase chain reaction (Sambrook, et al., 1989, Molecular Cloning, A Laboratory Manual, Vols. 1-3, Cold Spring Harbor Press, Cold Spring Harbor, NY.
  • the peptide constructs may be assembled from polymerase chain reaction cassettes sequentially cloned into a vector containing a selectable marker for propagation in a host.
  • markers include dihydrofolate reductase or neomycin resistance for eukaryotic cell culture and tetracycline or ampicillin resistance genes for culturing in E. coli and other bacteria.
  • Several bacterial expression vectors have been developed specifically for the expression of small proteins.
  • Plasmid pAED4 confers ampicillin resistance.
  • the nucleotide sequence and map of the pAED4 vector is known and readily available (Doering and Matsudaira, 1996, Biochem., 35:12677-12685.
  • Plasmid pTCLE-G2C, based on pAED4, is another example of a suitable expression vector (Calderone et al., 1996, J. Mol Biol, 262:407-412).
  • Representative examples of appropriate hosts include, but are not limited to, bacterial cells such as E.
  • the polynucleotide should be operably linked to an appropriate promoter, such as the T7 promoter in plasmid pAED4. Other suitable promoters are known in the art.
  • the expression constructs may further contain sites for transcription initiation, transcription termination, and a ribosome binding site for translation.
  • the coding portion of the mature polypeptide expressed by the constructs preferably includes a translation initiating codon at the beginning and a termination codon (UAA, UGA, or UAG) appropriately positioned at the end of the polypeptide to be translated.
  • Introduction of the recombinant vector into the host cell can be effected by calcium phosphate transfection, DEAE-dextran mediated transfection, cationic lipid- mediated transfection, electroporation, transduction, infection, or other methods.
  • the polypeptide may be expressed in a modified form, such as a fusion protein, and may include secretion signals and/or additional heterologous functional regions.
  • a region of additional amino acids may be added to the N- terminus or C-terminus of the polypeptide to facilitate detection or purification, or improve persistence in the host cell during, for example, purification or subsequent handling and storage.
  • additional amino acids include peptide tags that may be added to the polypeptide to facilitate detection and/or purification.
  • Such peptide tags include, but are not limited to, His, HA, Avi, biotin, c-Myc, VSV-G, HSV, V5, or FLAGTM.
  • the polypeptide can be recovered and purified from recombinant cell cultures by methods known in the art, including ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxylapatite chromatography, and lectin chromatography. In an embodiment, high performance liquid chromatography (HPLC) is employed for purification.
  • HPLC high performance liquid chromatography
  • polypeptide multimers of the present invention are useful, for example, as antiviral agents, immunogens, oligomerization domains for making bispecific molecules, for purifying anti-HEV antibodies from sera, and for identifying and/or purifying therapeutic anti-HEV or anti-SEV monoclonal antibodies, and in a method to screen for small molecule inhibitors of HEV or SEV membrane fusion.
  • antiviral compositions The multimers of the invention can be employed in antiviral compositions.
  • HEV infectivity is inhibited, for example, by contacting the virus with an effective inhibitory amount of a composition of the invention.
  • HEV is inhibited by administering to a subject an antiviral effective amount of a composition of the invention
  • the composition administered to the subject is a dimer or trimer comprising at least one monomer comprising a first N heptad capable of forming a coiled coil structure in solution; a C heptad; and a second N heptad that can form a coiled coil structure with the first N heptad wherein each heptad is connected to another heptad by a first or second linker moiety.
  • the first N heptad is linked to the C heptad by a first linker moiety and the C heptad is linked to the second N heptad by a second linker moiety.
  • the monomer has a sequence of SEQ ED NO: 87 or SEQ ED NO: 90 or the monomer has at least 75% sequence identity to SEQ ED NO: 87 or SEQ ED NO: 90.
  • the polypeptide is preferably administered in combination with a physiological acceptable carrier.
  • the polypeptide of the invention may be admimstered in combination with other antiviral agents, immunomodulators, antibiotics or vaccines.
  • compositions of the invention can be administered orally or parentally, including subcutaneous injection, intravenous, intramuscular, intrasternal or infusion techniques, in dosage unit formulations containing conventional non-toxic pharmaceutically acceptable carriers, adjuvants or vehicles.
  • Compositions of the invention can be in the form of suspensions or tablets suitable for oral administration or sterile injectable preparations, such as sterile injectable aqueous or oleagenous suspensions.
  • sterile injectable aqueous or oleagenous suspensions for oral administration as a suspension, the compositions can be prepared according to techniques well-known in the art of formulation.
  • the compositions can contain microcrystalline cellulose for imparting bulk, alginic acid or sodium alginate as a suspending agent, methylcellulose as a viscosity enhancer, and sweeteners or flavoring agents.
  • the compositions can contain microcrystalline cellulose, starch, magnesium stearate and lactose or other excipients, binders, extenders, disintegrants, diluents and lubricants known in the art.
  • the compositions can be formulated according to techniques well-known in the art, using suitable dispersing or wetting and suspending agents, such as sterile oils, including synthetic mono- or diglycerides, and fatty acids, including oleic acid.
  • suitable dispersing or wetting and suspending agents such as sterile oils, including synthetic mono- or diglycerides, and fatty acids, including oleic acid.
  • suitable dispersing or wetting and suspending agents such as sterile oils, including synthetic mono- or diglycerides, and fatty acids, including oleic acid.
  • the amounts to be administered for any particular treatment protocol can readily be determined by one skilled in the art without undue experimentation. See for example the HEV clinical trials database at on the Internet at www/hiv- web/lan
  • Dosage levels of approximately 100 ⁇ g to approximately 100 mg of a composition of the invention two times per day are useful as an antiviral treatment or prevention HEV infection.
  • dosages of about 3 mg to about 100 mg of composition are administered intravenously to a subject.
  • the specific dosage level and frequency for any particular subject will be varied and will depend upon a variety of factors, including the activity of the specific compound the metabolic stability and length of action of that compound, the age, body weight, general health, sex, and diet of the subject, mode of administration, rate of excretion, drug combination, and severity of the particular condition.
  • the polypeptides of the invention can be administered in combination with other agents useful in the treatment of HEV infection, ADDS or AEDS-related complex (ARC).
  • the compound of the invention can be administered in combination with effective amounts of an antiviral, immunomodulator, anti- infective, or vaccine.
  • the compound of the invention can be administered prior to, during, or after a period of actual or potential exposure to HEV.
  • b. Immunogenic Compositions The polypeptide multimers of the present invention can be employed in compositions useful to prepare antibodies to gp41 fusion intermediates and/or induce active immunity towards antigens in subjects.
  • HEV infectivity is inhibited by administering to a subject an immunogenic effective amount of a composition of the invention.
  • compositions of the invention may be useful in multicomponent vaccines such as epitope vaccines.
  • the compositions may also be used to immunize an animal to prepare antibodies that may be useful therapeutically, or in purification and detection of HEV strains, or in screening of inhibitors of gp41 mediated fusion.
  • the subjects are preferably mammals, and more preferably humans.
  • the administration of the vaccine may be for either a prophylactic or therapeutic purpose.
  • the vaccine (s) are provided in advance of any symptoms of HEV infection, or in advance of any known exposure to HEV.
  • the prophylactic administration of the vaccine (s) serves to prevent or attenuate any subsequent infection.
  • the vaccine (s) When provided therapeutically, the vaccine (s) is provided upon or after the detection of symptoms which indicate that an animal may be infected with HEV, or upon or after exposure to the virus.
  • the therapeutic administration of the vaccine (s) serves to attenuate any actual infection, for example as measured by improving the symptoms of a subject, or by reducing the level of viral replication.
  • the vaccines may be provided either prior to the onset of infection (so as to prevent or attenuate an anticipated infection) or after the initiation of an actual infection.
  • the effective amount of vaccine is dependent on the route of administration.
  • the vaccine can be administered by subcutaneous, intramuscular, intraperitoneal, or intravenous route. Subcutaneous or intramuscular routes of administration are prefened.
  • Dosage levels of about 100 ⁇ g to about 1 mg of a composition administered intramuscularly to a subject are useful as a vaccine. In an embodiment, about 500 ⁇ g to about 1 mg of the vaccine compositions is administered intramuscularly to a subject.
  • the amounts to be administered for any particular treatment protocol can readily be determined by one skilled in the art without undue experimentation. See for example the HEV clinical trials database on the Internet at http ://www/hiv-web/lanl/go v.
  • the vaccines of the present invention may be employed in such forms as capsules, liquid solutions, suspensions or elixirs for oral administration, or sterile liquid forms such as solutions or suspensions, with optional physiologically acceptable carriers, excipients or stabilizers. Acceptable carriers, excipients, or stabilizers are nontoxic to recipients at the dosages and concentrations employed. Any inert carrier may be used.
  • the inert carrier is saline, phosphate-buffered saline, or any inert carrier in which the vaccine has suitable solubility properties.
  • the vaccine may be in the form of a single dose preparation or in multi-dose flasks which can be used for mass vaccinations programs.
  • the vaccines of the present invention may further comprise adjuvants which enhance production of HEV-specific antibodies.
  • adjuvants include, but are not limited to, various oil formulations such as Freund's complete adjuvant (CFA), stearyl tyrosine (ST, see U. S. Patent No.
  • the dipeptide known as MDP, saponins and saponin derivatives, such as Quil A and QS-21, aluminum hydroxide, and lymphatic cytokine.
  • the adjuvant alum (aluminum hydroxide) or
  • the vaccine may be absorbed onto the aluminum hydroxide from which it is slowly released after injection.
  • an adjuvant will aid in maintaining the secondary and quaternary structure of the immunogens.
  • the vaccine may also be entrapped in microcapsules (including, but not limited to, liposomes, albumin microspheres, microemulsions, nano-particles and nanocapsules) in colloidal drug delivery systems or in macroemulsions. Such techniques are disclosed in Remington 's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980). hi an embodiment, the vaccine is encapsulated within liposomes according to Fullerton, U. S. Patent No.
  • polypeptide multimers of the invention are combined with other immunogens that are used to vaccinate animals.
  • the invention relates to a method of inducing an immune response in a subject comprising admimstering to the subject a composition of the invention in an amount effective to induce an immune response.
  • the composition of the invention may be coadmimstered with effective amounts of other immunogens as to generate multiple immune responses in the subject.
  • Antibodies The polypeptide multimers of the invention are immunogenic and elicit anti- multimer antibodies.
  • the antibodies have many uses, including purifying the polypeptide multimers of the invention, detecting HEV, screening for inhibitors of membrane fusion, and inhibiting and/or neutralizing HEV.
  • the antibodies may be polyclonal or monoclonal antibodies.
  • the antibody may be used to detect HEV in a fluid or tissue from a subject.
  • the antibody typically will be labeled with a detectable moiety including, but not limited to, a fluorescent label, a radioisotope, or an enzyme-substrate label.
  • the label may be indirectly conjugated with the antibody.
  • the antibody can be conjugated with biotin and any of the three broad categories of labels mentioned above can be conjugated with avidin, or vice versa.
  • Biotin binds selectively to avidin and thus, the label can be conjugated with the antibody in this indirect manner.
  • the antibody is conjugated with a small hapten (e.g., digoxin) and one of the different types of labels mentioned above is conjugated with an anti-hapten antibody (e.g., anti-digoxin antibody).
  • the antibody does not need to be labeled.
  • the antibody is detected using a labeled antibody that binds to the first antibody.
  • antibodies to the multimers of the invention inhibit Env mediated membrane fusion. Assaying for inhibition of syncytia formation is useful for determining inhibition of fusion.
  • Inhibition of syncytia formation by anti-multimer antibodies may be assessed by effector cell/target cell conjugates (Golding et al., 2002, J. Virol, 76:6780-6790) or other methods well known in the art.
  • antibodies that specifically bind a polypeptide multimer comprising SEQ ID NO:l inhibit syncitia formation.
  • Antibody neutralization may be assessed by HEV infectivity assay (Golding et al., 2002, J Virol, 76:6780-6790; de Rosny et al., 2001, J. Virol, 75:8859-8863) or other methods well known in the art.
  • antibodies that specifically bind a multimer comprising at least one monomer comprising an amino acid sequence of SEQ ED NO: 87 and inhibits membrane fusion are useful in inhibiting HEV infectivity.
  • the antibodies may be polyclonal or monoclonal. Because neutralizing antibodies in polyclonal sera may not be high titered, it may be necessary to purify and concentrate the fraction of antibodies that contain neutralizing activity, as had been shown by Louis et al., 2003, JBC, 278:20278. Monoclonal neutralizing antibodies could also be generated by the 3-HR constructs.
  • the antibodies are preferably admimstered in combination with any of the pharmaceutically acceptable carriers.
  • the antibodies of the invention may be administered in combination with other antiviral agents, immunomodulators, antibiotics or vaccines.
  • the amount of antibody administered for any particular treatment protocol can readily be determined by one skilled in the art without undue experimentation.
  • the appropriate dosage of antibody will depend on the type of disease to be treated, the severity and course of the disease, whether the antibody is administered for preventive or therapeutic purposes, previous therapy, the patient's clinical history and response to the antibody, and the discretion of the attending physician.
  • the antibody is suitably administered to the patient at one time or over a series of treatments.
  • ⁇ g/kg to 15 mg/kg (e.g., 0.1-20mg/kg) of antibody is an initial candidate dosage for administration to a subject, whether, for example, by one or more separate administrations, or by continuous infusion.
  • a typical daily dosage might range from about 1 ⁇ g/kg to 100 mg/kg or more, depending on the factors mentioned above.
  • the treatment is sustained until a desired suppression of disease symptoms occurs.
  • other dosage regimens may be useful. The progress of this therapy is easily monitored by conventional techniques and assays.
  • Therapeutic antibody compositions generally are placed into a container having a sterile access port, for example, an intravenous solution bag or vial having a stopper pierceable by a hypodermic injection needle.
  • the antibodies of the invention can be administered in combination with other agents useful in the treatment of HEV infection, AEDS or AEDS-related complex (ARC).
  • the antibodies can be admimstered in combination with effective amounts of an antiviral, immunomodulator, anti-infective, or vaccine.
  • the antibodies can be administered prior to, during, or after a period of actual or potential exposure to HEV.
  • Polyclonal antibodies to a polypeptide multimer of the invention are preferably raised in animals by multiple subcutaneous (sc) or intraperitoneal (ip) injections of the relevant antigen and an adjuvant.
  • a protein that is immunogenic in the species to be immunized e.g., keyhole limpet hemocyanin, serum albumin, bovine thyroglobulin, or soybean trypsin inhibitor
  • Animals are immunized against the antigen, immunogenic conjugates, or derivatives by combining, e.g., 100 ⁇ g or 5 ⁇ g of the protein or conjugate (for rabbits or mice, respectively) with 3 volumes of Freund's complete adjuvant and injecting the solution intradermally at multiple sites.
  • the animals are boosted with 1/2 to 1/10 the original amount of peptide or conjugate in Freund's complete adjuvant by subcutaneous injection at multiple sites.
  • Seven to 14 days later the animals are bled and the serum is assayed for antibody titer. Animals are boosted until the titer plateaus.
  • the animal is boosted with the conjugate of the same antigen, but conjugated to a different protein and/or through a different cross-linking reagent.
  • Conjugates also can be made in recombinant cell culture as protein fusions.
  • aggregating agents such as alum are suitably used to enhance the immune response.
  • the animals are immumzed with a recombinant adenovirus vector expressing a polypeptide multimer of the invention followed by booster immunizations with multimers of the invention.
  • the polyclonal antibodies generated by the immunizations undergo an initial screen for virus inhibition. Antiviral activity is evaluated in both cell-cell fusion and neutralization assays. Both assays are carried out according to protocols well known in the art (Wild et al., 1992, Proc. Natl. Acad. Sci, 89:10537-10541; Wild et al.,
  • antibodies are selected that can neutralize or inhibit membrane fusion for a variety of different viral isolates.
  • the polyclonal antibodies are also screened by enzyme-linked immunoabsorbent assay (ELISA) to characterize binding.
  • ELISA enzyme-linked immunoabsorbent assay
  • the antigen panel includes all experimental immunogens. Animals with sera samples that test positive for binding to one or more experimental immunogens are candidates for use in monoclonal antibody production. The criteria for selection for monoclonal antibody production is based on a number of factors including, but not limited to, neutralizing antibody titers and, in the absence of neutralization, binding patterns against the panel of structured HEV immunogens.
  • Monoclonal antibodies Monoclonal antibodies to a polypeptide multimer of the invention may be made using the hybridoma method first described by Kohler et al, Nature, 256:495
  • lymphocytes may be immunized in vitro. Lymphocytes then are fused with myeloma cells using a suitable fusing agent, such as polyethylene glycol, to form a hybridoma cell (Goding, Monoclonal Antibodies: Principles and Practice, pp.
  • the hybridoma cells are than seeded and grown in a suitable culture medium that preferably contains one or more substances that inhibit the growth or survival of the unfused, parental myeloma cells.
  • a suitable culture medium that preferably contains one or more substances that inhibit the growth or survival of the unfused, parental myeloma 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.
  • Preferred myeloma cells are those that fuse efficiently, support stable high- level production of antibody by the selected antibody-producing cells, and are sensitive to a medium such as HAT medium.
  • prefened myeloma cell lines are murine myeloma lines, such as those derived from MOPC-21 and MPC-11 mouse tumors available from the Salk Institute Cell Distribution Center, San Diego, California USA, and SP-2 or X63-Ag8-653 cells available from the American Type Culture Collection, Rockville, Maryland USA.
  • 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, pp. 51-63 (Marcel Dekker, Inc., New York, 1987)).
  • Culture medium in which hybridoma cells are growing is assayed for production of monoclonal antibodies directed against the antigen and HEV Env.
  • the binding specificity of monoclonal antibodies produced by hybridoma cells is determined by immunoprecipitation or enzyme-linked immunoabsorbent assay (ELISA).
  • the clones may be subcloned by limiting dilution procedures and grown by standard methods (Goding, Monoclonal Antibodies: Principles and Practice, pp. 59-103 (Academic Press, 1986)). Suitable culture media for this purpose include, for example, D-MEM or RPMI-1640 medium.
  • the hybridoma cells may be grown in vivo as ascites tumors m an animal.
  • the monoclonal antibodies secreted by the subclones are suitably separated from the culture medium, ascites fluid, or serum by conventional immunoglobuhn purification procedures such as, for example, protein A-Sepharose, hydroxylapatite chromatography, gel electrophoresis, dialysis, or affinity chromatography.
  • the monoclonal antibodies are characterized for specificity, anti-viral activity, and inhibition of membrane fusion from a variety of HEV isolates as described previously.
  • the polypeptide monomer and multimers of the invention also have other uses.
  • the polypeptide monomers and/or multimers may be useful in oligomerization of other heterologous polypeptides, or can be used to screen for small molecules or antibodies that can inhibit membrane fusion.
  • the monomers of the invention comprise two N heptads and a C heptad.
  • the two N heptads of the monomer form a very stable coiled coil structure and the C heptad associates with the coiled coil structure.
  • the monomer preferably forms homodimer or homotrimer.
  • a first heterologous peptide or polypeptide can be connected to the N terminal end of the first N heptad and a second heterologous polypeptide can be connected to the C terminal end of the second N heptad either before or after a peptide tag or linker moiety.
  • the oligomerization via action of the monomer can provide for oligomerization of the heterologous polypeptide or polypeptides. Oligomerization of polypeptides may be useful, for example, to form multifunctional molecules such as bispecific molecules forming two different ligand binding domains.
  • polypeptide monomers and multimers of the invention may be useful in a method to screen for inhibitors of HEV membrane fusion.
  • a method involves a competitive inhibition assay.
  • the dimer or trimer of the invention is incubated with the potential inhibitor and agents that bind to the dimer or trimer can be identified. Once such agents are identified, they can be evaluated for inhibition of membrane fusion as described in Golding et al., 2002, J. Virol, 76:6780-6790 and/or for inhibition of viral infectivity. The ability of the inhibitor to inhibit membrane fusion in the presence or the trimer is measured.
  • those inhibitors that exhibit an IC 50 of about 13 nM to 19 nM may be useful as an inhibitor of HEV mediated membrane fusion.
  • the inhibitors screened may include small molecules such as peptide mimetics, peptide variants of N and C heptads, and antibodies to gp41 or gp41 fusion intermediate or to the dimer or trimer.
  • the method involves identifying agents that bind the polypeptide monomers and/or multimers of the invention and evaluating agents that bind the 3HR multimers or monomers for HEV fusion or infectivity inhibiting activity.
  • Example 1 Production and Characterization of 3-HR polypeptide
  • the heptad repeat regions, N heptad and C heptad, of HEV transmembrane protein gp41 are highly conserved.
  • the heptad repeat regions in gp41 are thought to self assemble into a six-bundle structure that plays a role in viral entry by mediating membrane fusion.
  • This structure consists of triple stranded coiled coil core layer (N heptad) and external layer with 3 helices (C heptad)that pack in the grooves of the coiled coil in an antiparallel manner.
  • Peptides corresponding to these heptad repeats are potent inhibitors of HEV infection.
  • novel polypeptides utilizing combinations of N heptad and C heptad regions of gp41 and characterized the structure and function of these novel polypeptides.
  • the novel 3 -HR polypeptide comprises two N heptad regions separated by a C heptad region.
  • N and C heptad regions are those of gp41 from isolate HXB2.
  • the sequence of gp41 of this isolate can be found at Los Alamos National Laboratory HEV Sequence Database, accessible via the Internet at http://hiv-web. lanl.gov/content/hiv-db/mainpage.html.
  • the N- terminal N heptad region is connected to the C heptad region by a linker having the amino acid sequence SGGRGG (Tan et al., 1997, Proc. Natl. Acad. Sci, 94:12303- 12308).
  • the C Heptad region is connected to a C terminal N-heptad region by a linker GGSSGG (Root et al., 2001, Science, 291:884-888).
  • Some of the embodiments for the novel polypeptide also comprise an additional sequence that may provide for ease of purification or detection.
  • Those additional sequences can include peptide tags, such as histidine 6 (His), haemoglutinin protein (HA), avidin (Avi), biotin, c-Myc, VSV-G, HSV, V5, or FLAGTM.
  • the N-to-C linker is bolded
  • the C-to-N linker is bold italicized
  • the peptide tag is underlined.
  • 3-HR-HA a HA peptide tag was added to the C terminal end of 3HR polypeptide to facilitate detection of the polypeptide by anti-HA monoclonal antibodies.
  • 3HR-His a His peptide tag was added to the C terminal end of the HA peptide tag to facilitate purification. The His peptide tag binds to a Ni-NTA column.
  • the His peptide tag may be cleaved by thrombin at the radiolytic site located between the HA and His peptide tags (Coleman et al., 1981, Methods Enzymol, 80:341-361), and the HA tag may be cleaved by factor Xa at the radiolytic site located between 3HR and HA (Travy et al, 1992, Methods Enzymol, 215:300-360).
  • avidin was added to the C terminal end of 3HR polypeptide to facilitate biotinylation of the polypeptide (Cull, M.G., 2000, Methods Enzymol, 326:430-440).
  • BL21(DE3) Novagen, Madison, WI
  • T7 polymerase expression system Studier et al, 1990 Methods Enzymol, 185:60-89.
  • Transformed BL21(DE3) cells were grown in Luria-Bertani medium to an optical density (at 600 nm) to 0.6 before induction with isopropyl-b-D-thiogalacopyranoside (EPTG) (0.5 mM) for 4 hours.
  • EPTG isopropyl-b-D-thiogalacopyranoside
  • Bacterial pellets were resuspended in 50 ml/L cell culture of lysis buffer (50 mM Tris-HCl, pH 8.0, 50 mM NaCl) supplemented lOmMEDTA and with complete EDTA free protease inhibitor tablets (Roche, Indianapolis, IN). The cells were lysed by French press and pelleted at 20,000 x g for 30 minutes. The pelleted inclusion bodies were washed with the 50 ml /L cell culture of lysis buffer supplemented with 0.2 mg/ml lysozyme, 1 mg/ml deoxycholate, and 1 mM EDTA (wash buffer).
  • lysis buffer 50 mM Tris-HCl, pH 8.0, 50 mM NaCl
  • complete EDTA free protease inhibitor tablets Roche, Indianapolis, IN.
  • the pelleted inclusion bodies were washed with the 50 ml /L cell culture of lysis buffer supplemented with 0.2 mg/ml lyso
  • the pellet was then washed three times with 25 ml/ L cell culture of wash buffer without lysozyme followed by three washes with 25 ml/L cell culture of lysis buffer and two washes with 25 ml/ L cell culture of deionized water.
  • the proteins were dissolved in 50 ml/ L cell culture of 10% acetic acid, then purified by reverse-phase HPLC, using a C-18 preparative column (Delta-Pak, Waters) and a linear gradient of acetonitrile containing 0.1% (v/v) trifuoroacetic acid. The identities of polypeptides were confirmed by mass spectrometry.
  • 3-HR polypeptide As shown in Figure 1, 3-HR may form two distinct stabilized coiled coil structures: a dimer or a trimer.
  • the peptides were analyzed by ultracentrifugation and circular dichroism (CD) spectroscopy as described by Wild et al., 1992, Proc. Nat. Acad. Sci, 89:10537-10541.
  • the molecular weight of dimers or trimers of the 3-HR polypeptide were directly determined by sedimentation equilibrium.
  • Analytical ultracentrifugation was carried out using an Optima XL-I analytical ultracentrifuge (Beckman Coulter, Fullerton, CA). Absorption optics, an An-60 Ti rotor and standard double-sector centerpiece cells were used.
  • the predicted mass of the dimers and trimers was calculated from the amino acid sequence of the polypeptide multimers. hi all cases, the initiating N-terminal methionine had been processed. 2 The experimental mass of the dimers and trimers was determined by sedimentation equilibrium ultracentrifugation. The molecular weight of 3HR homodimers and homotrimers were directly determined by sedimentation equilibrium and the results are summarized in Table 1. 3HR-NT, 3HR-HA, and 3HR-HA-His polypeptides are all dimeric at pH 3.0.
  • the molecular weight of the 3HR-NT homodimer as determined by sedimentation equilibrium was 24,400 daltons, which is in agreement with the molecular weight of the homodimer of 24,300 daltons calculated from the sequence of the polypeptide multimer. At pH values greater than 4.5, some aggregation may occur.
  • the sedimentation equilibrium data in Table 1 shows that 3HR-Avi is trimeric at pH 3.0.
  • the predicted molecular weight of the 3HR-Avi homotrimer calculated from the amino acid sequence of the polypeptide multimer was 42,480 daltons.
  • the results of the sedimentation equilibrium show a molecular weight of 39,400 at pH 3.0. At pH values greater than 4.5, some aggregation occurs.
  • the molecular weight at pH 7.0 was >51 ,000.
  • the signal at 220 nm was used to monitor unfolding as the temperature was increased.
  • a 10°C step was used during the thermal melts and the temperature range was 0.5 to 90.5° C.Chan et al., 1998, Proc. Natl. Acad. Sci, 95:15613-15617.
  • the results are shown in Figure 3.
  • the T m of the 3-HR-HA dimer is approximately 80 °C, suggesting a very stable bundle.
  • the homogeneity of the polypeptide multimers was high at acidic pH values where accurate molecular weight determinations were made and secondary structures determined.
  • Polypeptide multimers comprising 3HR-NT and 3HR-HA appear to be dimeric whereas multimers comprising 3HR-Avi appear to be trimeric.
  • the dimeric or trimeric association status does not affect the overall helical nature, both association states consist of about 76% helix (data not shown).
  • Similar analysis of the CD-spectra of the HEV - and SEV - gp41 ectodomains determines about 80% helix (Wingfield et al., 1997, Protein Science, 6:1653-1660).
  • the CD spectrum of 3-HR is typical of an alpha-helix with minima at 208 and 222 nm ( Figure 2).
  • DMEM Dulbecco's minimal essential medium
  • FCS fetal calf serum
  • penicillin and streptomycin Hepes, sodium pyruvate, nonessential amino acids, and 2mM glutamine.
  • FCS fetal calf serum
  • the cells were incubated at 37°C in a CO 2 incubator until the cells were 30 to 40% confluent.
  • FuGENE 6TM transfection reagent (Roche Diagnostics, Indianapolis, EN) was diluted 1:11 in Optimem-1 (Gibco), mixed gently, and incubated at room temperature for 5 min.
  • Optimem-1 Optimem-1
  • 3 ⁇ g of Env expression plasmid pSM-WT and 1.5 ⁇ g of pRev DNA were added to 100 ⁇ l of Optimem-1 and mixed.
  • the diluted FuGENE 6TM transfection reagent was then added directly to the plasmid mixture, the tube was gently tapped to mix the contents, and incubated at room temperature for 15 min to allow DNA-liposome complexes to form.
  • the membranes were probed with anti-gpl20 polyclonal goat serum (Env 2-3; kindly provided by Kathelyn Steimer, Chiron Corp., Emeryville, Calif.) at 1:5,000 in 5% milk- phosphate-buffered saline, washed, reprobed with peroxidase-conjugated anti-goat antiserum (Sigma, St. Louis, Mo.), and washed before detection by chemiluminescence and autoradiography. Weng, Y. and Weiss, C, 1998, J. Virol, 72:9676-9682.
  • Env-expressing luciferase reporter p24 pseudoviruses were prepared by cotransfecting approximately 3 x IO 6 293T cells in 10-cm culture dishes with 5 ⁇ g of Env expression plasmid pSM-WT and 5 ⁇ g Env-deficient viral reporter vector pNL43-Luc-R ⁇ E ⁇ by the FuGENE 6 method as described in Example 2. Supematants containing p24 pseudoviruses were collected 48 h post transfection and frozen at 80°C. The quantity of virus in the transfected supematants was determined by measuring p24 using a commercial enzyme-linked immunosorbent assay (Coulter, Westbrook, Maine).
  • gpl20 expression 250 ng of p24 pseudovirus was lysed with NP- 40 lysis buffer containing 1 ⁇ g of soluble CD4-Ig G (kindly provided by Genentech, South San Francisco, Calif.). The sample was incubated in the lysis buffer for 1 h at room temperature with mixing. Approximately 25 ⁇ l of a 25% suspension of protein A-agarose (GIBCO BRL) was then added to the sample, and the sample was incubated for an additional hour at room temperature with mixing. The precipitates were then washed three times with lysis buffer, separated by SDS-PAGE (4 to 12% NuPAGE gels), and immunoblotted with polyclonal gpl20 antiserum, as described in Example 2.
  • NP- 40 lysis buffer containing 1 ⁇ g of soluble CD4-Ig G (kindly provided by Genentech, South San Francisco, Calif.). The sample was incubated in the lysis buffer for 1 h at room temperature with mixing. Approximately 25 ⁇ l of a 25% suspension
  • U87- CD4-CXCR4 cells were plated the day before infection in 48-well dishes in DMEM containing 10% FCS, 2 mM glutamine, l ⁇ antibiotics, 1 mM sodium pyruvate, and l nonessential amino acids (DMEM + ) and incubated at 37°C.
  • DMEM + fetal calf serum
  • the U87-CD4-CXCR4 cells were inoculated with 80 ng of ⁇ 24 pseudovirus in 300 ⁇ l of DMEM + containing 2.4 ⁇ g of Polybrene and serially diluted 3-HR polypeptides.
  • 3HR-HA-His and 3HR-Avi also inhibit HEV-1 infectivity in a dose-dependent manner (Table 2).
  • 3HR-HA-His had an IC 50 of 44 nM and 3HR-Avi had an IC 50 of 90 nM.
  • Table 2 Inhibition of HIV-1 Infectivity
  • 3-HR polypeptides were purified as described in Example 1. New England White rabbits were immunized subcutaneously with 200 ⁇ g of 3HR polypeptide in complete Fruend's adjuvant at 4-week intervals, de Rosny et al., 2001, J. Virol, 75:8859- 8863. Following the fourth immunization, blood was collected from the rabbit's marginal ear vein, centrifuged, and the serum fraction was collected. IgG was purified from the serum fraction using a protein A column and the specificity of the purified polyclonal antibodies for 3HR polypeptide was verified by ELISA.
  • Sera from rabbits 783 and 784 was diluted 1 : 100 in the immunoprecipitation assay.
  • Sera from all three rabbits immunoprecipitated both receptor-activated gp41 and non-receptor activated gp41 from cell surfaces in the assay.
  • TF228 cells Jonak et al., 1993, AIDS Res. Hum. Retroviruses, 9:23-32
  • PM1 cells Liso et al., 1995, J. Virol, 69:3712-3720
  • the number of syncytia was than scored as described in Golding et al., 1992, Aids Res. Hum.
  • Retroviruses 8:1607-1612. Syncytia were counted visually using a microscope. Three of more fused cells were scored as a syncytia.
  • polyclonal rabbit antibodies to 3HR-NT homodimers inhibited syncytium formation. Purified total IgG from the rabbit anti-3HR-NT sera inhibited syncytia formation when added to the effector and target cells at 37°C.
  • the IC 50 of the polyclonal antibodies at 37°C ranged from 24 ⁇ g/ml to 32 ⁇ g/ml, dependent upon the particular rabbit from which the antisera was obtained. See Figure 7 and Table 3.
  • the polyclonal sera bind gp41 from at least 3 different Env from different isolates of HEV.
  • the polyclonal antibodies inhibited syncytia formation between effector cells expressing LAI Env, 89.6 Env, or JR-FL Env and target cells. See Figures 7 and 8.
  • the syncytia inhibitory activity of the polyclonal antibodies was greater when effector cells expressed JR-FL Env or 89.6 Env. See Figure 8.
  • the inhibitory activity of the polyclonal antibodies to 3HR-NT homodimers in viral infectivity and syncytia formation assays is summarized in Table 3. These results show that polyclonal antibodies to 3HR-NT homodimer could inliibit syncytia formation at either 31°C or 37°C with a fairly low IC 50 , ranging from 4.4 ⁇ g/ml to 32 ⁇ g/ml. However, the polyclonal antisera to homodimers was not able to inhibit infectivity of the virus in the assays utilized.
  • polyclonal antisera as developed in rabbits may not have sufficiently high titer of high affinity antibodies that may be needed toinhibit the more efficient process of virus-cell fusion, as compared to the slower process of syncytia (multi-cell) fusions. This is consistent with others who have shown that it was necessary to concentrate and purify high affinity antibodies from polyclonal antisera to see inhibition of syncytia formation. Louis et al., 2003, J. Biol. Chem., 218:20218-20285. The polyclonal antisera specific for 3HR-NT homodimers may be used to isolate therapeutic antibodies and characterize the epitopes that bind to such antibodies. All publications (including patents and patent applications) cited herein are hereby incorporated in their entirety by reference.

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Abstract

One aspect of the invention relates to polypeptide multimers that have antiviral and immunogenic activity and compositions and methods for inhibiting viral infectivity. The polypeptide multimers are comprised of at least one monomer with a ratio of N heptads to C heptads of at least about 2:1. Another aspect of the invention relates to antibodies that specifically bind the polypeptide multimers and compositions and methods for inhibiting viral infectivity.

Description

Polypeptide Multimers Having Antiviral Activity
This application is a Continuation- i-Part of United States patent application Serial No. 09/408,336 filed January 7, 2000 which application claims priority under 35 USC §119(e) to United States Patent Application Serial No. 60/115,404 filed January 8, 1999.
STATEMENT OF RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSORED RESEARCH AND DEVELOPMENT Part of the work performed during the development of this invention utilized United States government funds under an NLH Intramural AIDS Targeted Antiviral Program. The United States government has certain rights in the invention. FIELD OF THE INVENTION One aspect of the invention relates to polypeptide multimers that have antiviral and immunogenic activity and compositions and methods for inhibiting viral infectivity. Another aspect of the invention relates to antibodies that specifically bind the polypeptide multimers of the invention and compositions comprising the antibodies and methods for inhibiting viral infectivity.
BACKGROUND OF THE INVENTION The envelope glycoprotein (Env) of human immunodeficiency virus (HIV) mediates fusion of the viral membrane to the target cell membrane resulting in delivery of the viral nucleocapsid into the cytoplasm of the target cell. The Env glycoprotein is synthesized as 160 kiloDalton glycoprotein precursor that is cleaved post translationally into gpl20 and the gp41 transmembrane protein in the Golgi complex. The gpl20 and gp41 polypeptides are noncovalently associated following cleavage of the precursor. It is thought that gp41 mediates cell membrane fusion critical to viral entry. Chan, D. C, and P. S. Kim, 1998, Cell, 93:681-684. HIV transmembrane protein gp41 contains two heptad repeat regions, N hejptad and C heptad, which are highly conserved. It is believed that the heptad repeat regions in gp41 self assemble into a six-helix bundle structure(or trimer of hairpins) that likely represents a fusion-active conformation of gp41 that forms after receptor binding and is critical for membrane fusion. This structure consists of a triple stranded coiled coil formed by a trimer of N heptads. The C heptads pack in the grooves of the coiled coil in an antiparallel manner. Chan et al., 1997, Cell, 89:263-273; Weissenhorn et al., 1997, Nature, 387:426-430; Tan et al, 1997, Proc. Natl. Acad. Sci. USA, 94:12303-12308. The importance of the six-helix bundle in membrane fusion and virus entry led to the hypothesis that the six-helix bundle and/or its components might serve as targets for potential membrane fusion inhibitors. Peptides corresponding to N or C heptad repeats and polypeptides that specifically bind these heptad repeats are potent inhibitors of HIV infection. Root et al., 2001, Science, 291 :884-888; Wild et al, 1994, Proc. Natl. Acad. Sci., 91:9770-97 '4; Jiang et al, 1993, Nature, 365:113; Wild et al., 1992, Proc. Natl. Acad. Sci., 89:10537-10541. These inhibitors of HIV appear to block virus entry by forming a complex with gp41 fusion intermediates that interfere with the formation of the gp41 six-helix bundle. Furuta et al., 1998, Nat. Struct. Biol, 5-276-279; Chan et al., 1998, Cell, 93:681-684; He et al, 2003, J. Virol. , 77:1666-1671. Inhibition of six-helix bundle formation by N and C heptads suggest that fusion-active conformations of gp41 are accessible, at least transiently, during membrane fusion. A great deal of effort is being directed to the design and testing of agents that inhibit HIV infectivity and can be used to develop vaccine components including those that target gp41 fusion intermediates. Effective inhibitors of HIV infectivity and/or vaccine components are still needed.
SUMMARY OF THE INVENTION Molecules that are conserved among many HIV or SJV strains and that have structural and/or functional constraints that minimize the development of mutations serve as valuable targets for development of antiviral agents and vaccine components. The gp41 Env polypeptide is a molecule that has conserved regions that form a constrained structure important for the infectivity of the virus. The N heptad regions of gp41 are conserved among many HIV strains; and form a coiled coil structure that is an important mediator of membrane fusion. We designed novel polypeptide multimers utilizing combinations of N and C heptad regions of gp41 that may mimic gp41 fusion intermediates. These novel polypeptide multimers are useful, interalia, as antiviral agents. One aspect of the invention is polypeptide multimers comprised of at least one monomer with a ratio of N heptads to C heptads of at least about 2: 1 and that have antiviral activity and compositions comprising the polypeptide multimers and methods for inhibiting HIV and/or SIV infectivity with the compositions. The polypeptide multimer comprises at least one monomer comprising a first and second N heptad, a C heptad, and a first and second linker moiety, wherein the first and second N heptad and C heptad are each connected to one another by the first or second linker moieties. The monomer forms a homodimer or homotrimer in solution. The N and C heptads may be connected to one another in different arrangements but each heptad is separated from the other by a linker moiety. In one embodiment, the first N heptad is connected to the C heptad by the first linker moiety and the second N heptad is connected to the C heptad by the second linker moiety. In another embodiment, the first N heptad is connected to the second N heptad by the first linker moiety and the second N heptad is connected to the C heptad by the second linker moiety. In another embodiment, the C heptad is connected to the first N heptad by the first linker moiety and the first N heptad is connected to the second N heptad by the second linker moiety. The present invention is also directed to a method of raising a broadly neutralizing antibody response to HIV by administering to a mammal a composition including one or more novel peptides and proteins, herein referred to as conjugates or monomers, that mimic fusion-active transmembrane protein structures. These conjugates are formed from two or more amino acid sequences that comprise: (a) one or more amino acid sequences that are capable of forming a stable coiled-coil solution structure corresponding to or mimicking the heptad repeat region of gp41 (N-helical domain); and (b) one or more amino acid sequences that correspond to, or mimic, an amino acid sequence of the transmembrane-proximal amphipathic - helical segment of gp41 (C-helical domain); wherein said one or more sequences (a) and (b) are alternately linked to one another via a bond, such as a peptide bond (amide linkage) or by an amino acid linking sequence consisting of about 2 to about 25 amino acids. These conjugates or monomers are preferably recombinantly produced. In a preferred embodiment of this aspect of the invention, one or more of these conjugates or monomer folds and assembles in solution into a structure corresponding to, or mimicking, the gp41 core six helix bundle. In other embodiments, one or more of the monomers folds and assembles into structures that may not solely form a six-helix bundle. The invention contemplates that the monomers may form other structures such as shown in Figure 1. In another embodiment, the polypeptide multimer comprises at least one monomer comprising a first N heptad, wherein the first N heptad comprises i) a polypeptide having an amino acid sequence of SEQ ID NO: 1, or ii) a polypeptide having at least 75% sequence identity to SEQ ID NO: 1 and preferably, is capable of forming a coiled coil structure in solution or when in contact with another N and/or C heptad. In a further embodiment, the N heptad forms a coiled coil structure with one or more N heptads or when it contacts a C heptad. In another embodiment, the C heptad comprises a polypeptide having an amino acid sequence of SEQ ID NO: 76 or a polypeptide having at least about 65% sequence identity to SEQ LD NO: 76 and is capable of forming an amphipathic helical structure in solution. In yet a third embodiment, the second N heptad comprises i) a polypeptide having an amino acid sequence of SEQ LD NO: 1, or ii) a polypeptide having at least 75% sequence identity to SEQ ID NO: 1 and is capable of forming a coiled coil structure in solution. In a preferred embodiment, the first and second N heptad have the same amino acid sequence. In a further embodiment, the polypeptide multimer comprises at least one monomer comprising a polypeptide having i) an amino acid sequence of SEQ ID NO:87 or SEQ LD NO:88 or SEQ LD NO:89 or SEQ ID NO:90 or ii) an amino acid sequence that at least about 75% sequence identity to SEQ ID NO:87 or SEQ ID
NO:88 or SEQ LD NO:89 or SEQ LD NO:90 and is capable of forming a homodimer or homotrimer in solution. Another embodiment of the polypeptide multimer comprises a) at least one monomer comprising: i) a first N heptad and second N heptad; ii) a first and second linker moiety; and iii) a C heptad, wherein each heptad is separated from one another by the first or second linker moiety; and b) a third N heptad capable of forming a coiled coil structure in solution; wherein the monomer and the third N heptad form a 4 helix bundle in solution. Another embodiment of the polypeptide multimer is a trimer comprising three monomers, each monomer comprising: i) a first N heptad and a second N heptad; ii) a first and second linker moiety; iii) a C heptad; and iv) a peptide; wherein each heptad is separated from one another by the first or second linker moiety; wherein the peptide is located at the C terminal end of the monomer; and wherein the monomer forms a homotrimer in solution. hi another embodiment, HIV or SIV infection is inhibited by administering to a subject an antiviral effective amount of a composition comprising multimers of the invention. In a further embodiment, the antiviral compositions administered to the subject comprise a polypeptide multimer comprising at least one monomer comprising a polypeptide having i) an amino acid sequence of SEQ LD NO: 87 or SEQ ID NO:88 or SEQ ID NO:89 or SEQ ID NO:90 or ii) an amino acid sequence that at least about 75% sequence identity to SEQ LD NO:87 or SEQ LD NO:88 or SEQ ID NO:89 or SEQ ID NO:90 and preferably, is capable of forming a homodimer of homotrimer in solution. Another aspect of the invention is immunogenic polypeptide multimers that correspond to or mimic gp41 fusion intermediates in membrane fusion and compositions comprising the immunogenic polypeptides and methods for eliciting an immune response with the composition. In one embodiment, the polypeptide multimer comprises at least one monomer comprising a polypeptide having i) an amino acid sequence of SEQ ID NO:87 or SEQ ID NO:88 or SEQ LD NO:89 or SEQ ID NO:90 or ii) an amino acid sequence that at least about 75% sequence identity to SEQ ID NO:87 or SEQ LD NO:88 or SEQ ID NO:89 or SEQ LD NO:90 and preferably is capable of forming a homodimer of homotrimer in solution. Another aspect of the invention is antibodies that specifically bind the polypeptide multimers of the invention and compositions comprising the antibodies. The antibodies may be monoclonal or polyclonal. The antibodies may be useful therapeutically. In an embodiment, the antibodies specifically bind a polypeptide multimer comprising at least one monomer comprising a polypeptide having i) an amino acid sequence of SEQ ID NO:87 or SEQ LD NO:88 or SEQ ID NO:89 or SEQ ID NO: 90 or ii) an amino acid sequence that at least about 75% sequence identity to SEQ ID NO:87 or SEQ LD NO:88 or SEQ LD NO:89 or SEQ ID NO:90 and preferably is capable of forming a homodimer of homotrimer in solution. The polypeptides, immunogens, antibodies, and compositions thereof of the invention can be administered in combination with other agents useful in the treatment of HIV infection, SIV infection, AIDS, or ALDS-related complex (ARC). The polypeptides, immunogens, antibodies, and compositions thereof can be admimstered prior to, during, or after a period of actual or potential exposure to HIV and/or SIV.
BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1. A schematic diagram of the predicted structure of multimers formed by oligomerization of 3-helix heptad repeat (3HR) monomers. The multimers form stabilized coiled coil structures that mimic gp41 fusion intermediates. A monomer may fold in solution and form a 4-helix bundle in the presence of free N heptad. A homodimer may fold in solution and form a five-helix bundle connected to an uncomplexed N heptad. A homotrimer may fold in solution and form a six-helix bundle connected to a trimer of N heptads.
Figure 2. The secondary structure of the heteromultimers formed by the recombinant monomers in solution was analyzed by circular dichroism (CD) spectroscopy in the far-uv spectral region (190-250 nm). CD spectra for the 3HR- NT homodimer and 3HR-Avi homotrimer show that both the dimer and trimer have significant helical structure. The homodimer complex was estimated to be about 85% helical. The homotrimer complex was estimated to be about 76% helical.
Figure 3. The stability of the polypeptide multimers was determined by the apparent melting temperature (2m) of each of the forms of polypeptide multimers. The T of the 3HR-HA homodimer and 3HR-Avi homotrimer were determined by CD spectroscopy. The signal at 220 nm was used to monitor the unfolding of the polypeptide multimers as the temperature was increased. A 10°C step was used during the thermal melts and the temperature range was 0.5 to 90.5°C. The Tax of the homodimer was approximately 80°C, suggesting a very compact bundle.
Figure 4. The novel 3HR polypeptides selectively bind to receptor-activated gp41. To assess 3HR-HA binding to Env, immunoprecipitation assay were performed using intact Env-express cells in the presence or absence of soluble CD4 receptor (sCD4). 3HR-gp41 complexes were immunoprecipitated with anti-HA and anti-Avi monoclonal antibodies as described in Furuta et al., 1998, Nature Struct. Biol, 5:276-279. The immunoprecipitated complexes were separated by SDS-Page and immunoblotted with anti-gp41 monoclonal antibody. 3HR-HA formed a complex with and immunoprecipitated gp41 only in the presence of sCD4 (lane 4). No complex was seen in the absence of receptor (lane 3).
Figure 5. Specific binding of the novel 3HR polypeptides to receptor-activated gp41 inhibited HIV-1 Env mediated HIV infectivity. To assess inhibition of HIV infectivity by 3HR-His and 3HR-NT, U87-CD4-CXCR4 cells were inoculated with Env-expressing luciferase reporter p24 pseudoviruses and incubated for 48 h. The cells were then lysed and inhibition ofHIV infectivity was determined by measuring luciferase activity. As shown in Figure 6, 3HR-His and 3HR-NT each displayed a dose-dependent inhibitory effect with an IC50 of approximately 30nM.
Figure 6. Anti-3HR sera prepared against the homodimer immunoprecipitates both receptor-activated and non-receptor activated forms of gp41 from cell surfaces. Rabbits were immunized with homodimers comprising 3HR-NT. Sera from rabbit 782 was diluted 1 :50 in the immunoprecipitation assay. Sera from rabbits 783 and 784 was diluted 1 : 100 in the immunoprecipitation assay. Sera from all three rabbits immunoprecipitated both receptor-activated gp41 and non-receptor activated gp41 from cell surfaces. See lanes 3 and 4, 7 and 8, 11 and 12 in Figure 6.
Figure 7. Polyclonal antisera to 3HR-NT homodimers inhibited syncytia formation at both 31°C and 37°C. The effector cells in the assays expressed LAI Env. The IC50 of the polyclonal antibodies at 37°C ranged from 24 μg/ml to 32 μg/ml, dependent upon the particular rabbit from which the antisera was obtained. The IC50 for the polyclonal antibodies preincubated with the target and effector cells at 31.5°C ranged from 4.4 μg/ml to 7 μg/ml.
Figure 8. Polyclonal antisera to 3HR-NT homodimers inhibited syncytia formation between effector cells expressing JR-FL Env or 89.6 Env and target cells at 37°C. The IC50 for the polyclonal antibodies was 21 μg/ml when the effector cells expressed 89.6 Env and 8 μg/ml when the effector cells expressed JR-FL Env.
Figure 9. Illustrates the construction of conjugates (monomers) derived from repeating gp41 fragments and then subsequent folding and interaction to form immunologically relevant epitopes.
DETAILED DESCRIPTION OF THE INVENTION I. Definitions As used herein, the term "N heptad" as refers to a polypeptide having at least one 7 amino acid heptad repeat region and preferably, is capable of forming a coiled coil structure in solution or when it contacts a N and/or C heptad. Preferably, the N heptad comprises at least 4 heptad repeat regions, hi one embodiment, the N heptad region comprises at least about 28 to 55 amino acids of the N heptad repeat region of the extracellular domain of gp41. The N heptad may be synthetic or recombinant. The N heptad may be selected from any strain or isolate of HIV or SIV or selected from any of the known N heptad peptides that have antiviral activity. The N heptad may be derived from any strain of HIV or SIV by insertional, substitutional, and/or deletional changes of an N heptad sequence selected from any strain or isolate of HIV or STV or from any known N heptad peptides with the proviso the N heptad so derived is capable of forming a coiled coil structure in solution or when it contacts a N and/or C heptad. As used herein, the term "C heptad" as used herein refers to a polypeptide having at least 7 amino acids and preferably, is capable of forming an amphipathic a helical structure when it contacts a N heptad coiled coil. Preferably, the C heptad comprises at least a 4 heptad repeat region, h one embodiment, the C heptad comprises at least about 24 to 56 amino acids of extracellular C heptad domain of gp41. The C heptad may be synthetic or recombinant. The C heptad may be selected from any strain or isolate of HIV or SIV or selected from any of the known C heptad peptides that have antiviral activity. The C heptad may be derived from any strain of HIV or SIV by insertional, substitutional, and/or deletional changes of a C heptad selected from any strain of HIV or SIV or any known C heptad with the proviso the C heptad is capable of forming an amphipathic a helical structure when it contacts a N heptad coiled coil. As used herein, the phrase "heptad repeat" or "heptad repeat region" refers to a common protein motif having a 4-3 repeat of amino acids, and is often associated with alpha helical secondary structure and coiled coil structure. The heptad repeat region can be represented by the following sequence: a-b-c-d-e-f-g where a and d are each hydrophobic amino acids. Preferably, a is V, I, L, M, F, W, C, X, S, Q or T and d is V, I, L, M, F, W, C, Y, S, Q or T and b, c, e, f, g can be any naturally occurring amino acid. More preferably, a and d are V, I or L (Wild et al., 1992, Proc Natl. Acad. Sci., 89-10537-10541). As used herein, the term "linker moiety" or "linker" refers to a moiety that separates and/or connects heptads from one another. The linker moiety is flexible, allowing the monomer to fold so that N heptads can form a coiled coil structure and associate with other N heptads and/or the C heptad. In one embodiment, the linker comprises a peptide from about 2 to about 36 amino acid residues, preferably from 2 to about 16 amino acid residues. In another embodiment, the linker comprises a native sequence linker or derivative of a native sequence linker. Native sequence linkers useful in the invention include, but are not limited to, peptides comprising the full-length native sequence linker, or a truncated sequence, containing one or more amino acid substitutions, insertions, and/or deletions. Preferred peptides comprise amino acid residues glycine and serine, and/or glycine and cysteine. Examples of linker moieties include, but are not limited to: (GGGGS)X (SEQ LD NO: 77) wherein x is 1 to 5 (WO 00/40616); (GGC)X where X is 1 to 5 (WO 00/40616); GGSGG (SEQ ID NO: 78) (Root et al., 2001, Science, 291:884-888); GSSGG (SEQ ID NO: 79) (Root et al., 2001, Science, 291:884-888); SGGRGG (SEQ ID NO: 80) which is trypsin cleavable (-GGR-) (Tan et al., 1997, Proc. Natl. Acad. Sci. USA, 94:12303-12308); and SSGGSS (SEQ ID NO: 81). The linker moiety can optionally include one or more amino acids that provide for proteolytic cleavage. The term "antibody" is used in the broadest sense and specifically covers monoclonal antibodies and polyclonal antibodies. The term "homodimer" as used herein means a polypeptide multimer comprising two polypeptide monomers wherein each of the monomers comprise the same arrangement of N heptads and C heptads. For example, each monomer has a first N heptad separated by a first linker moiety followed by a C heptad followed by a second linker moiety followed by the second N heptad. Preferably, the monomers comprising the homodimer each have the same amino acid sequence and are capable of forming a coiled coil structure in solution. The term "homotrimer" as used herein means a polypeptide multimer comprising three polypeptide monomers wherein each monomer comprises the same arrangement of N heptads and C heptads. For example, each monomer comprises a first N heptad separated by a first linker moiety followed by a C heptad followed by a second linker moiety followed by the second N heptad. Preferably the monomers comprising the homotrimer are capable of forming a coiled coil structure in solution. The term "monoclonal antibody" as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations that may be present in minor amounts. Monoclonal antibodies are highly specific, being directed against a single antigenic site. Furthermore, in contrast to conventional polyclonal antibody preparations that typically include different antibodies directed against different epitopes, each monoclonal antibody is directed against a single epitope on the antigen. The modifier "monoclonal" indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method. For example, the monoclonal antibodies of the present invention may be made by the hybridoma method first described by Kohler et al, Nature 256:495 (1975), or may be made by recombinant DNA methods (see, e.g., U.S. Patent No. 4,816,567). The "monoclonal antibodies" may also be isolated from phage antibody libraries using the techniques described in Clackson et al, Nature 352:624-628 (1991) and Marks et al, J. Mol Biol. 222:581-597 (1991), for example. The term "therapeutically effective amount" refers to an amount of a drug effective to treat a disease or disorder in a mammal. With respect to HIV, therapeutically effective amount is a dose, which provides some therapeutic benefit on administration, including, in the context of the invention, reduced viral activity or viral load in a patient, or inhibition of virus replication. As used herein, the term "treating" refers to both therapeutic treatment and prophylactic or preventative measures. Those in need of treatment include those already infected with HIV as well as those in which HIV infection is to be prevented. "Treatment" refers to both therapeutic treatment and prophylactic or preventative measures. A nucleic acid is "operably linked," as used herein, when it is placed into a functional relationship with another nucleic acid sequence. For example, DNA for a presequence or secretory leader is operably linked to DNA for a antibody if it is expressed as a preprotein that participates in the secretion of the antibody; a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the sequence; or a ribosome binding site is operably linked to a coding sequence if it is positioned so as to facilitate translation. Generally, "operably linked" means that the DNA sequences being linked are contiguous, and, in the case of a secretory leader, contiguous and in reading phase. However, enhancers do not have to be contiguous. Linking is accomplished by ligation at convenient restriction sites. If such sites do not exist, the synthetic oligonucleotide adaptors or linkers are used in accordance with conventional practice. Polynucleotide means a sequence of nucleic acids that encode a polypeptide of the invention. Peptide sequences defined herein are represented by one-letter symbols for amino acid residues as follows: A alanine L leucine R arginine K lysine N asparagine M methionine D aspartic acid F phenylalanine C cysteine P proline Q glutamine S serine E glutamic acid T threonine G glycine w tryptophan H histidine Y tyrosine I isoleucine V valine "Percent (%) amino acid sequence identity" means the percentage of amino acid residues in a polypeptide that are identical with amino acids in a reference polypeptide, after aligning the sequence and introducing gaps, if necessary to achieve the maximum sequence identity, and not considering any conservative substitutions as part of the sequence identity. For purposes herein, the % amino acid sequence identity of a given amino acid sequence A to, with, or against a given amino acid sequence B (which can alternatively be phrased as a given amino acid sequence A that has or comprises a certain % amino acid sequence identity to, with, or against a given amino acid sequence B) is calculated as follows:
100 times the fraction X/Y where X is the number of amino acid residues scored as identical matches by the sequence alignment , and where Y is the total number of amino acid residues in B. It will be appreciated that where the length of amino acid sequence A is not equal to the length of amino acid sequence B, the % amino acid sequence identity of A to B will not equal the % amino acid sequence identity of B to A. The % amino acid sequence identity may also be determined using the sequence comparison program such as ALIGN 2 or NCBI-BLAST2 (Altschul et al., 1997, Nucleic Acids Res., 25:3389-3402). The NCBI-BLAST2 sequence comparison program may be downloaded from http://www-ncbi-nlm-nih-gov or otherwise obtained from the National Institute of Health, Bethesda, MD. NCBI- BLAST2 uses several search parameters, wherein all of those search parameters are set to default values including, for example, unmask = yes, strand = all, expected occurrences = 10, minimum low complexity length = 15/5, multi-pass e- value = 0.01, constant for multi-pass = 25, dropoff for final gapped alignment = 25 and scoring matrix = BLOSUM62.
II. Modes for Carrying Out the Invention N heptads are highly conserved among all strains of HIV and SIV. The six- helix bundle formed by coiled coil N heptads is a conserved structure that can readily lose function upon loss of that coiled coil structure. The conserved nature of the N heptad makes it an attractive target for membrane fusion inhibitors. Polypeptide monomers and multimers have been constructed that have a greater ratio of N heptads to C heptads. A greater N heptad to C heptad ratio means more hydrophobic faces of the N heptads are exposed to the solvent and are more accessible. An increase in accessibility to N heptads may provide for enhanced antiviral effect and expose additional immunogenic epitopes. However, an increase in exposure of the hydrophobic N heptads to solvent may also present problems of solubility, aggregation, and stability of the monomer or multimers. It was unexpected that stable multimers could be formed from a monomer with a ratio of N heptads to C heptads of at least about 2:1. A ratio of N to C heptads of at least about 2:1 allows the construction of monomers, dimers, and/or trimers that correspond to or mimic gp41 fusion intermediates. In an embodiment, the ratio of N to C heptads is about 3:1. Immunization with these monomers and multimers generate antibodies that preferentially target gp41 fusion intermediates that occur during Env fusion. A. Polypeptide Monomers One aspect of the invention relates to novel polypeptide multimers comprised of at least one monomer with a ratio of N heptads to C heptads of at least about 2:1. The multimer comprises at least one monomer comprising a first and second N heptad, a C heptad, and a first and second linker moiety wherein each heptad is connected to another heptad by the first or second linker moiety and wherein the monomer is capable of forming a homodimer or homotrimer in solution. In one embodiment, the first N heptad is connected to the C heptad by the first linker moiety and the second N heptad is connected to the C heptad by the second linker moiety. In another embodiment, the first N heptad is connected to the second N heptad by the first linker moiety and the second N heptad is connected to the C heptad by the second linker moiety. In another embodiment, the C heptad is connected to the first N heptad by the first linker moiety and the first N heptad is connected to the second N heptad by the second linker moiety. In a further embodiment, the C-terminal end of the first N heptad and the N-terminal end of the C heptad are separated by the first linker moiety, and the C-terminal end of the C heptad and the N-terminal end of the second N heptad are separated by the second linker moiety. In another embodiment, the C-terminal end of the first N heptad and the N-terminal end of the second N heptad are separated by the first linker moiety. The C-terminal end of the second N heptad and the N-terminal end of the C heptad are separated by the second linker moiety. In another embodiment, the C-terminal end of the C heptad and the N-terminal end of the first N heptad are separated by the first linker moiety. The C-terminal end of the first N heptad and the N-terminal end of the second N heptad are separated by the second linker moiety. i) N heptads The N heptad is a polypeptide that has at least one 7 amino acid heptad repeat region , and preferably is capable of forming a coiled coil structure in solution or when in contact with another N and/or C heptad. Polypeptides capable of forming a coiled coil structure generally have at least one 7 amino acid heptad repeat region where the first and fourth amino acids are hydrophobic. In one embodiment, the 7 amino acid polypeptide comprises: a-b-c-d-e-f-g; wherein a is V, I, L, M, F, W, C, Y, S, Q or T and d is V, I, L, M, F, W, C, Y, S, Q or T. b, c, e, f, g are any of the naturally occurring amino acids. In another embodiment, a and d are V, I or L. Preferably, the N heptad comprises at least 4 heptad repeat regions. In another embodiment, the N heptad region comprises at least 4 heptad repeat regions of the extracellular domain of gp41. The N heptad region of gp41 includes amino acids 542-592 as described and shown in Jiang et al, 2002, Current Pharmaceutical Design, 8:563-580. The N heptads may be selected from any strain of HIV or SIV. Representative examples of N heptads from different HIV strains include, but are not limited to, the following peptides. Many other sequences are known and described at www/hiv-web/lanl/gov. All sequences are listed from N-terminus to C- terminus.
HIV-1 Group M: Subtype B Isolate: LAI ARQLLSGIVQQQNNLLRAIEAQQHLLQLTVWGIKQLQARILAVERYL KDQQLLGI (SEQ LD NO: 2) SGΓVQQQNNLLRAIEAQQHLLQLTVWGIKQLQARILAVERYLKDQ (SEQ ID NO: 3) SGΓVQQQNNLLRAΓEAQQHLLQLTVWGIKQLQARIL (SEQ ED NO: 4) NNLLRALEAQQHLLQLTVWGL QLQARILAVERYLKDQ (SEQ ID NO: 5)
Subtype B Isolate: ADA SGΓVQQQNNLLRAIEAQQHLLQLTVWGIKQLQARVLALERYLRDQ (SEQ LD NO: 6) SGrVQQQNNLLRALEAQQHLLQLTVWGIKQLQARVL (SEQ ID NO: 7) NNLLRALEAQQHLLQLTVWGIKQLQARVLALERYLRDQ (SEQ LD NO: 8) Subtype B Isolate: JRFL SGΓVQQQNNLLRALEAQQRMLQLTVWGIKQLQARVLAVERYLGDQ
(SEQ ID NO: 9)
SGΓVQQQNNLLRALEAQQRMLQLTVWGIKQLQARVL (SEQ ID NO:
10) NNLLRAIEAQQRMLQLTVWGIKQLQARVLAVERYLGDQ (SEQ
ID NO: 11)
Subtype B Isolate: 89.6
SGΓVQQQNNLLRAIEAQQHMLQLTVWGIKQLQARVLALERYLRDQ
(SEQ LD NO: 12)
SGΓVQQQNNLLRAEAQQHMLQLTVWGIKQLQARVL (SEQ LD NO: 13) NNLLRAIEAQQHMLQLTVWGLKQLQARVLALERYLRDQ (SEQ ID
NO: 14)
Subtype C Isolate: BU910812
SGIVQQQSNLLRAIEAQQHMLQLTVWGIKQLQARVLAIERYLRDQ (SEQ LD NO: 15)
SGJVQQQSNLLRAIEAQQHMLQLTVWGIKQLQARVL (SEQ ID NO: 16) SNLLRAIEAQQHMLQLTVWGLKQLQARVLAIERYLRDQ (SEQ LD NO: 17)
Subtype D Isolate: 92UG024D
SGΓVQQQNNLLRAIEAQQHLLQLTVWGIKQLQARVLAVESYLKDQ
(SEQ ID NO: 18)
SGΓVQQQNNLLRAIEAQQHLLQLTVWGIKQLQARVL (SEQ LD NO: 19) NNLLRAIEAQQHLLQLTVWGIKQLQARVLAVESYLKDQ (SEQ LD NO: 20)
Subtype F Isolate: BZ163A
SGTVQQQSNLLRAIEAQQHLLQLTVWGIKQLQARVLAVERYLQDQ
(SEQ LD NO: 21) SGΓVQQQSNLLRAIEAQQHLLQLTVWGIKQLQARVL (SEQ ID NO: 22)
SNLLRAΓEAQQHLLQLTVWGΠ QLQARVLAVERYLQDQ (SEQ LD
NO: 23)
Subtype G Isolate: FI. HH8793
SGΓVQQQSNLLRAIEAQQHLLQLTVWGLKQLQARVLALERYLRDQ
(SEQ ID NO: 24)
SGIVQQQSNLLRAΓEAQQHLLQLTVWGIKQLQARVL (SEQ ID NO: 25) SNLLRALEAQQHLLQLTVWGIKQLQARVLALERYLRDQ (SEQ LD NO: 26)
Subtype H Isolate: BE. VI997
SGΓVQQQSNLLRAIQAQQHMLQLTVWGVKQLQARVLAVERYLKDQ
(SEQ LD NO: 27) SGΓVQQQSNLLRAIQAQQHMLQLTVWGVKQLQARVL (SEQ LD NO:
28)
SNLLRAIQAQQHMLQLTVWGVKQLQARVLAVERYLKDQ (SEQ ID NO: 29)
Subtype J Isolate: SE. SE92809
SGΓVQQQSNLLKABEAQQHLLKLTVWGLKQLQARVLAVERYLKDQ
(SEQ LD NO: 30)
SGTVQQQSNLLKAIEAQQHLLKLTVWGIKQLQARVL (SEQ ID NO: 31) SNLLKAIEAQQHLLKLTVWGIKQLQARVLAVERYLKDQ (SEQ LD NO: 32)
Group N Isolate: CM. YBF30
SGIVQQQNILLRAffiAQQHLLQLSIWGIKQLQAKVLAiERYLRDQ (SEQ LD NO: 33) SGIVQQQNILLRAffiAQQHLLQLSIWGIKQLQAKVL (SEQ LD NO: 34)
NILLRALEAQQHLLQLSrWGIKQLQAKVLAiERYLRDQ (SEQ LD NO: 35) Group O Isolate: CM. ANT70C KGrVQQQDNLLRAIQAQQQLLRLSxWGIRQLRARLLALETLLQNQ (SEQ ID NO: 36) KGΓVQQQDNLLRAIQAQQQLLRLSXWGIRQLRARL (SEQ LD NO: 37) DNLLRAIQAQQQLLRLSXWGIRQLRARLLALETLLQNQ (SEQ ID NO: 38)
CONSENSUS-A-Alf2n sgLVQQQsNLLrAIeaQQhlLkLTVwGIKQLQArvlAvErYLkDQ (SEQ ID NO: 91)
CONSENSUS-A2(3 sGLVQQQSNLLkAIEAQQhlLkLTVWGIKQLQARVLAlERYLqDQ (SEQ ID NO: 92)
CONSENSUS-BC128 sGIvqQQnnLlrAieaQqhllqLTVwGiKQLqarvLaverYLkdX (SEQ ID NO: 93)
CONSENSUS-Cf5n SgiVQQQsXNLLrAffiaqQHmLQLtVWGIKqLqtrvLaiERyLkdQ (SEQ LD
NO: 94)
CONSENSUS-DC^ sGIVqQQnNLLrAIeAQQHlLQLTVWGIKQLQARilAVErYLkDQ (SEQ ID NO: 95)
CONSENSUS-FU5) SglVQQQnNLLrAIEAQQHlLQLTVWGIKQLQARVLAVERYLkDQ (SEQ ID NO: 96)
CONSENSUS-F2(5 SGIVQQQsNLLKAJEAQQHLLQLTVWGIKQLQARiLAVERYLKDQ (SEQ ID NO: 97) CONSENSUS-G(9 SGIVQQQsNLLRAffiaQQHLLQLTVwGIKQLqaRvLAvERyLkDQ (SEQ ID NO: 98) CONSENSUS-Hffl SGLVQQQSNLLRAIQAqQHMLQLTVWGiKQLQARVLAVERYLkDQQ (SEQ LD NO: 99)
CONSENSUS-OαO KGIVQQQDnLLRAIQaQQqLLRLSvWGIRQLRARLlALETLiQNQ (SEQ
BD NO: 100)
CONSENSUS-0U33 sGrVQQQSNLLRaLEAqqHlLQLTVWGIKqLqARvLAverYLKdQ (SEQ LD NO: 101)
CONSENSUS-02(16 SGIVQQQsNLLXAIEAQQHlLkLTVWGπ QLQARVLALErYLrDQ (SEQ ID NO: 102)
CONSENSUS-03B SGTVQQQNNLLRAIEAQQHLLQLTVWGIKQLQARVLAvERYLKDQ (SEQ ID NO: 103) CONSENSUS-04C3 sGrVQQQSNLLRALEAQQhLLrLTVWGiKQLQARVLALESYLkDQ (SEQ ID NO: 104)
CONSENSUS-06(5) SGTVqQQSNLLRAEAQQHLLQLTVWGIKQLQARvLAvERYLkDQ
(SEQ LD NO: 105)
CONSENSUS-08f4 SGIVQQQSNLLRAffiAQQHmLQLTVWGIKQLQTRVLAffiRYLKDQ (SEQ LD NO: 106)
CONSENSUS- 10(3) SGIVQQQNNLLrAIEAQQHlLQLTVWGIKQLQARvLAVESYLKDQ
(SEQ ID NO: 107)
CONSENSUS- 11(6) SGIVQQQsrιLLkAIeaQQXlLKLTVWGIKQLQARVLAvErYLkDq (SEQ ID NO: 108)
CONSENSUS-12(3) SGIVQQQsNLLrAIEAQQHlLQLTVWGIKQLQARVLAVErYLKDQ (SEQ ID NO: 109)
CONSENSUS-CPZ(5) sGTVQQQnNLLrAIEaQQHLLQLSvWGiKQLQARvLAVErYLXDQ (SEQ ID NO: 110) In one embodiment, the N heptad has the following sequence: aa# 546 SGIVQQQNNLLRAIEAQQHLLQLTVWGIKQLQAR aa# 579 (SEQ ED
NO:l) position d a d a d a d a d The amino acid positions correspond to amino acid positions 546 to 579 of the N heptad sequence of gp41 from HXB2 isolate of HIV. The first and fourth position of each heptad repeat region are underlined. Two examples of useful peptides include the peptide P-17, which has the formula, from amino terminus to carboxy terminus, of: NH2NNLLRAIEAQQHLLQLTVWGIKQLQARILAVERYLKDQ-COOH
(SEQ LD NO: 135) and the peptide P-15, which has the formula, from amino terminus to carboxy terminus, of: NH2SGrVQQQNNLLRAffiAQQHLLQLTVWGIKQLQARIL-COOH (SEQ ID NO: 136)
These peptides are optionally coupled to a larger carrier protein, or optionally include a terminal protecting group at the N- and/or C-termini. Useful peptides further include peptides corresponding to P- 17 or P- 15 that include one or more, preferably 1 to 10 conservative substitutions, as described below. The N heptad sequences in a monomer can vary because amino acid sequences of native gp41 of different strains or isolates can vary in sequence as described by the representative sequences shown above. The N heptad sequence can be any one of the consensus sequences that have been identified. The N heptad may be derived from any strain of HLV or SIV by insertional, substitutional, and/or deletional changes of an N heptad sequence selected from any strain or isolate of HIV or SIV from any known N heptad peptides with the proviso the N heptad so derived is capable of forming a coiled coil structure in solution or when in contact with another N and/or C heptad. N heptad sequences can also be derived from any native sequence by changing amino acids. Amino acid positions and the type of substitutions that can be made at these positions can be identified by aligning and analyzing known sequences and/or conforming retention of coiled coil structures using known algorithms. Alignment and comparison of all known N heptad sequences provides one method for identifying those amino acid positions that can tolerate amino acid substitutions. Aligning and comparing all of the known N heptad sequences of HIV isolates indicates that they have about 75% sequence identity to SEQ ID NO: 1 when aligned with amino acid positions 546 to 579. Preferably, amino acid residues that participate in the formation and/or stabilization of the coiled coil structure formed by the N heptad in solution are not substituted. Preferably amino acid residues at positions a and d of the heptad repeat region(s) are not substituted, hi addition, amino acid residues that form a hydrophobic pocket are preferably not substituted. In one embodiment, the residues that form this hydrophobic pocket include amino acid residues LLQLTVWGIKQLQAR (SEQ ID NO: 111). Chan et al., 1997, Cell, 89:263-273. This sequence corresponds approximately to amino acid positions 565 - 579 of isolate HBX2. The hydrophobic pocket may vary in sequence in different isolates, but corresponds to about amino acid positions 565-579 of isolate HBX2. hi another embodiment, the amino acid positions corresponding to GIVQQQ (SEQ ID NO: 112) (amino acid positions 547 to 552) are not substituted. Comparison of the alignment of sequences shows that certain positions in the heptad repeats tolerate amino acid substitution. Preferably, amino substitutions are made at the second amino acid position from the N terminus or at least one of the last two C terminal amino acids of a heptad repeat region or both. In an embodiment, the positions that tolerate amino acid substitution comprise substitutions at at least one amino position 546, 553, 557, 560, 564, 565, 567, 574, or 577, or mixtures thereof corresponding to amino acid positions of N heptad region of HBX2 isolate of HIV. An amino acid substitution made at these positions preferably increases the hydrophobic character of the heptad repeat region. N heptad variants with amino acid substitutions that are capable of forming a coiled coil structure can also be identified using any one of a number of algorithms that predict coiled coil structure such as the Socket program at http://www-biols-susx-ac- uk/Biochem/Woolfson/html/coiledcoils/socket. - Optionally, amino acid substitution with cysteines provides for the formation of disulfide bonds. In one embodiment, a disulfide bonds may be employed to stabilize the 4-helix bundle, homodimer or homotrimer for example, for in vivo use. Preferably, cysteine substitutions are made at the N terminal amino acids, C terminal amino acids, or both of an N heptad. The solubility of N heptads may be improved by mutating positions in the heptad repeat region that do not directly interact with N or C heptad interactions. In an embodiment, the solubility of the N heptad is improved by mutating the amino acid residues at positions b and c of the heptad repeat region. The solubility of an N heptad may also be improved by fusing the N heptad with another helix with greater solubility forming a chimeric helix. The solubility of an N heptad may also be improved by substituting amino acids with cysteine residues providing for the formation of disulfide bonds, as described above, that stabilize the 4-helix bundle, homodimer, homotrimer. The N heptads useful in the invention include polypeptide variants that have at least about 75% amino acid sequence identity to SEQ ID NO: 1 when aligned to amino acid positions corresponding to amino acid positions 546 to 579, and preferably, are capable of forming a coiled coil structure when in contact with another N and/or C heptad. N heptads that are useful in the invention also include polypeptide variants that have at least about 75% sequence identity, more preferably have any one of % sequence identity between 75% to 100% sequence identity (eg. 16%, 11%, 78%, etc.), more preferably 80% sequence identity, more preferably 85% sequence identity, more preferably 90% sequence identity, more preferably 95% sequence identity to SEQ ID NO: 1. Preferably, the polypeptide variants do not have amino acid residue substitutions at positions a and d of the N heptad repeat region(s) or at the amino acid positions that form the hydrophobic pocket such as amino acids that correspond to amino acids 565 - 579 of gp41 of HBX2. In addition to full length N heptads, monomers of the invention may include truncated first and/or second N heptads, which exhibit the ability to form a coiled coil structure in solution. Such truncated N heptads may comprise peptides having at least one 7 amino acids heptad repeat region, more preferably between about 7 and 55 amino acid residues. For example, truncated N heptads are those that preferably lack the first N terminal or the last C terminal heptad repeat region. Truncation of the N heptad regions have been described in Dwyer et al, 2003, Biochemistry, 42:4945. Truncations can occur at the N and/or C terminal end of the N heptad region. At the N terminal end of the N heptad, about 13 amino acids, more preferably 6 amino acids, can be removed with minimal effect on stability of the N heptad in solution. Examples include the peptides P- 15
(SGΓVQQQNNLLRAIEAQQHLLQLTVWGKQLQARIL (SEQ ED NO: 135) and P-17 (NNLLRAffiAQQHLLQLTVWGIKQLQARILAVERYLKDQ (SEQ LD NO: 136) which are truncated forms of the N helical domain of HJV-ILAI gp41 where the peptides are derived by truncating the N heptad region at both the N terminal and C terminal ends. At the C terminal end ofthe N heptad, there seems to be less tolerance for truncation, hi one embodiment, the C terminal end of an N heptad can be truncated up to 11 amino acids and the N terminal end of the N heptad can be truncated up to 4 amino acids as in SEQ ID NO: 1. Preferably, the C terminal end of the N heptad includes the amino acid positions or residues that include the hydrophobic pocket, i.e. amino acid positions corresponding to amino acid positions
565 -579 in the gp41 of HBX2. The first and second N heptads of the monomer can comprise the same amino acid sequence or a related amino acid sequence. For example, N heptad regions can be selected from different strains or isolates and combined to form a single monomer. In one embodiment, the first and second N heptads may have an amino acid sequence of SEQ ID NO: 1 ; the first N heptad may have an amino acid sequence of SEQ ED NO: 1 and the second N heptad may have an amino acid sequence having at least 75 percent identity with SEQ DD NO: 1 and is capable of forming a coiled coil structure in solution; or the first N heptad may have an amino acid sequence having at least 75 percent identity with SEQ DD NO: 1 and is capable of forming a coiled coil structure in solution and the second N heptad may have an amino acid sequence of SEQ ID NO: 1.
ii) C Heptads The C heptad refers to a polypeptide having at least one 7 amino acid heptad repeat region and preferably, that is capable of forming an amphipathic helical structure when it contacts a N heptad coiled coil. Polypeptides capable of forming an amphipathic helical structure generally have at least 7 amino acids where the first and fourth amino acids are hydrophobic. hi one embodiment, the 7 amino acid polypeptide comprises: a-b-c-d-e-f-g; wherein a is V, I, L, M, F, W, C, Y, S, Q or T and d is V, I, L, M, F, W, C, Y, S, Q, D, E or T. The amino acids comprising positions b, c, e, f, and g are any of the naturally occurring amino acids. In a preferred embodiment, a is W, S or N and d is W, Y, L, F, Q, E or D. Preferably, the C heptad comprises at least 4 heptad repeat regions of the extracellular domain of gp41 of HEV. In one embodiment, the C heptad sequence includes amino acids 623 to 663 of gp41. The C heptad may be selected from any strain of HEV or SEV. Representative examples of C heptads from different HEV strains include, but are not limited to, the following peptides. Many other sequences are known and described at www/hiv-web/lanl gov. All sequences are listed from N-terminus to C- terminus.
HEV-1 Group M: Subtype B Isolate: LAI WNNMTWMEWDREENNYTSLEBSLffiESQNQQEKNEQELLELDKWAS LWNWF NITNW (SEQ ED NO: 39) WMEWDREENNYTSLIHSLEeESQNQQEKNEQELLELDKWASLWNWF (SEQ ED NO: 40) WMEWDREENNYTSLEHSLEEESQNQQEKNEQELL (SEQ ED NO: 41)
YTSLIHSLffiESQNQQEKNEQELLELDKWASLWNWF
(SEQ DD NO: 42)
Subtype B Isolate: ADA
WMEWEREIENYTGLIYTLLEESQNQQEKNEQDLLALDKWASLWNWF (SEQ LD NO: 43)
WMEWEREIENYTGLIYTLIEESQNQQEKNEQDLL (SEQ ED NO: 44) YTGLIYTLIEESQNQQEKNEQDLLALDKWASLWNWF (SEQ ED NO: 45)
Subtype B Isolate: JRFL
WMEWEREEDNYTSEEYTLEEESQNQQEKNEQELLELDKWASLWNWF (SEQ ED NO: 46) WMEWEREEDNYTSEIYTLEEESQNQQEKNEQELL (SEQ ED NO: 47)
YTSEIYTLIEESQNQQEKNEQELLELDKWASLWNWF (SEQ ED NO: 48)
Subtype B Isolate: 89. 6
WMEWΈREΠDNYTDYIYDLLEKSQTQQEKNEKELLELDKWASLWNW F
(SEQ ED NO: 49)
WMEWEREΠDNYTDYEYDLLEKSQTQQEKNEKELL (SEQ ED NO: 50) YTDYIYDLLEKSQTQQEKNEKELLELDKWASLWNWF (SEQ ED NO:
51)
Subtype C Isolate: BU910812
WIQWDREISNYTGIEYRLLEESQNQQENNEKDLLALDKWQNLWSWF
(SEQ ED NO: 52)
WIQWDREISNYTGIIYRLLEESQNQQENNEKDLL (SEQ ED NO: 53) YTGIIYRLLEESQNQQENNEKDLLALDKWQNLWSWF (SEQ ED NO:
54)
Subtype D Isolate: 92UG024D
WMEWEREISNYTGLIYDLffiESQIQQEKNEKDLLELDKWASLWNWF (SEQ ED NO: 55)
WMEWEREISNYTGLIYDLEEESQIQQEKNEKDLL (SEQ DD NO: 56) YTGLIYDLEEESQIQQEKNEKDLLELDKWASLWNWF (SEQ ED NO: 57)
Subtype F Isolate: BZ163A
WMEWQKEISNYSNEVYRLEKSQNQQEKNEQGLLALDKWASLWNW F
(SEQ ED NO: 58)
WMEWQKEISNYSNEVYRLEBKSQNQQEKNEQGLL (SEQ ED NO: 59) YSNEVYRLIEKSQNQQEKNEQGLLALDKWASLWNWF (SEQ ED NO:
60)
Subtype G Isolate: FI. HH8793
WIQWDREISNYTQQIYSLIEESQNQQEKNEQDLLALDNWASLWTWF (SEQ ED NO: 61)
WIQWDREISNYTQQIYSLIEESQNQQEKNEQDLL (SEQ ED NO: 62) YTQQIYSLIEESQNQQEKNEQDLLALDNWASLWTWF (SEQ ED NO: 63)
Subtype H Isolate: BE. VI997
WMEWDRQEDNYTEVIYRLLELSQTQQEQNEQDLLALDKWDSLWNW
F
(SEQ LD NO: 64)
WMEWDRQEDNYTEVIYRLLELSQTQQEQNEQDLL (SEQ ED NO: 65) YTEVIYRLLELSQTQQEQNEQDLLALDKWDSLWNWF (SEQ ID NO:
66)
Subtype J Isolate: SE. SE92809
WIQWEREENNYTGΠYSLEEEAQNQQENNEKDLLALDKWTNLWNWF N
(SEQ ED NO: 67)
WIQWEREENNYTGΠYSLEEEAQNQQENNEKDLL (SEQ ED NO: 68) YTGΠYSLEEAQNQQENNEKDLLALDKWTNLWNWFN (SEQ ED NO: 69)
Group N Isolate: CM. YBF30 WQQWDEKVRNYSGVEFGLEEQAQEQQNTNEKSLLELDQWDSLWSW F (SEQ ED NO: 70) WQQWDEKVRNYSGVIFGLIEQAQEQQNTNEKSLL (SEQ ID NO: 71) YSGVEFGLEEQAQEQQNTNEKSLLELDQWDSLWSWF (SEQ ED NO: 72)
Group O Isolate: CM. ANT70C WQEWDRQISMSSTIYEEIQKAQVQQEQNEKKLLELDEWASIWNWL (SEQ ED NO: 73) WQEWDRQISNISSTIYEEIQKAQVQQEQNEKKLL (SEQ ED NO: 74) ISSTJYEEIQKAQVQQEQNEKKLLELDEWASIWNWL (SEQ ED NO: 75)
CONSENSUS-A-AK21) wlqwdkEisnyTXilYnLieesQnqQEkNeqdLlaLDKwanLwnwfdlsnWL (SEQ ED NO: 113)
CONSENSUS-A2(3) WLQWDkEIsnYTnliYXLLEESQNQQEKNEQDLLALDKWAXLWnWFX ItXWL (SEQ ED NO: 114) CONSENSUS-B(128) wmewereidnytsliytLieesqnqQekNeqeLleldkwaslwnwf (SEQ ID NO: 115)
CONSENSUS-C(51 WmqwdrEisnYtntiYrLLedSqnqQekNekdLLaldswqnLWnWF (SEQ DD NO: 116)
CONSENSUS-D(17) WmeWErEIdnYTgliYsLiEeSQtQQEKNEqeLLeLDkWASlWNWF (SEQ DD NO: 117) CONSENSUS-FK5) WMeWEKEisNYSXXIYrLIEeSQnQQEkNEQELLaLDKWaSLWnWFDisn WL (SEQ ED NO: 118)
CONSENSUS-F2(5) WmqWEKEIsNYTXTIYrLEEXAQNQQEkNEQXLLALDKWDnLWsWFsI TnWL (SEQ ED NO: 119) CONSENSUS-G(9) WieWeREisNYTqqIYsliEeSQnQQEKNEQDLLALDkWasLWXWF (SEQ LD NO: 120)
CONSENSUS-HB) WMEWDkQEXNYTeelYRLLEvSQtQQEkNEQDLLALDKWasLWnWF
(SEQ ED NO: 121)
CONSENSUS-O(IO) WQeWDqqisNiSsilydEIqkAQvQQEqNekklLELDEWASiwNWl (SEQ HD NO: 122)
CONSENSUS-0K33) wieWerEIsnyTnqlYeiLTeSQnQQdrNEkdLLeLdkWasLWnWFdltnWL (SEQ ED NO: 123)
CONSENSUS-02(16) WlQWdkEisNYTdilYXLieesQNqQekNEqdLLALDkWasLWnWfdltXWL (SEQ ED NO: 124) CONSENSUS-03(3) WMEWERELMNYTGLEYNLIEESQNQQEKNEQEiLALDKWASLWnWF DISKWL (SEQ ED NO: 125)
CONSENSUS-04(3) WLQWDKEINNYTqliYXLLeEsQnQQEKNEQDLLAlDKWAnLWnWFXI sXWL (SEQ ED NO: 126)
CONSENSUS-06(5) WleWDRELriNYTQQIYsLIEeSQXQQEKNEQdLLALDKWasLWsWFDIs
NWL (SEQ ED NO: 127)
CONSENSUS-08(4) WMQWDKEISNYTNTIYRLLEDSQNQQERNEKDLLALDSWKNLWSW FDITNWL (SEQ ED NO: 128)
CONSENSUS- 10(3) WMeWErEEDNYTGLiYsLLEESQXQQEKNEqELLqLDKWASLWnWFSI TnWL (SEQ LD NO: 129)
CONSENSUS-1K6) WieWeReldNYTqtlYTLlEESQiiQQEKNEQdLLXLDKWASLWnWFdlsn WL (SEQ ED NO: 130) CONSENSUS-12(3) WMqWEKELXNYSXelYRLEEeSQnQQEKNEQELLALDKWASLWnWFD ISnWL (SEQ LD NO: 131)
CONSENSUS-CPZ(5) WQXWDkXVkNysGvIfslieqAqeQQntNekXLlELDqWsSLWnWFdITqWL
(SEQ ED NO: 132)
In one embodiment, the C heptad has a sequence of: aa# 628 W £EWDREINNYTSLIHSLffiESQNQQEK aa# 655 (SEQ DD NO. 76) position a d a d a d a d
This sequence corresponds to amino acid positions 628 to 655 in gp41 of
HEV isolate HBX2. The first and fourth position of each heptad repeat region are underlined. Examples of useful peptides for this aspect of the invention include the peptide P-18 which corresponds to a portion of the transmembrane protein gp41 from the HEV- 1 LAi isolate, and has the 36 amino acid sequence reading from amino to carboxy terminus): NH2-YTSLEHSLffiESQNQQEKNEQELLELDKWASLWNWF-COOH
(SEQ LD NO: 133); and the peptide P-16, which has the following amino acid sequence (reading from amino to carboxy terminus): NH2-WMEWDRELNNYTSLIHSLDEESQNQQEKNEQELL-COOH (SEQ DD NO: 134).
These peptides are optionally coupled to a larger carrier protein. Useful peptides further include peptides corresponding to P-18 or P-16 that include one or more, preferably 1 to 10 conservative substitutions, as described below. In addition to the full-length P-18, 36-mer and the full length P-16, the peptides of this aspect of the invention may include truncations of the P-18 and P- 16, as long as the truncations is capable of forming a six helix bundle when mixed with P-17. The C heptad sequences in a monomer can vary because amino acid sequences of native gp41 of different strains or isolates can vary in sequence as described and shown above. The C heptad sequence can be any one of the consensus sequences that have been identified. The C heptad may be derived from any strain of HEV or SEV by insertional, substitutional, and/or deletional changes of a C heptad sequence selected from any strain or isolate of HEV or SEV or from any known C heptad peptides with the proviso the C heptad so derived is capable of forming an amphipathic alpha helical structure when it contacts a N heptad coiled coil. C heptad sequences can also be derived from any native sequence by changing amino acids. Amino acid positions and the type of substitutions that can be made at these positions can be identified by aligning and analyzing known sequences and/or conforming retention of helical structures using known algorithms. Alignment and comparison of all known C heptad sequences provides one method for identifying those amino acid positions that can tolerate amino acid substitutions. Aligning and comparing all of the known N heptad sequences of HEV isolates indicates that they have about 65% sequence identity to SEQ ED NO: 76 when aligned to amino acid positions 628 to 655 of SEQ ID NO: 76. Preferably amino acid residues that participate in the formation and/or stabilization of the amphipathic alpha helical structure formed by the C heptad in solution are not substituted. Preferably amino acid residues at positions a and d of the heptad repeat region(s) are not substituted. Preferably, variants are not substituted at positions that interact with the hydrophobic pocket of the N heptad such as those amino acid positions that correspond to Trp 628, Trp 631 and He 635 of SEQ DD NO:76. The C heptads also useful in the invention include C heptad variants that have at least about 65% amino acid sequence identity to SEQ ID NO: 76 and are capable of forming amphipathic a helical structure when in contact with a N heptad. Useful C heptads also include C heptad variants having at least 65% amino acid sequence identity to SEQ ED NO: 76, more preferably any % identity between 65% and 100% sequence identity (eg. 65%, 66%, 61%, 68% etc.), more preferably 70% sequence identity, more preferably 75% sequence identity, more preferably 80% sequence identity, more preferably 85% sequence identity, more preferably 90% sequence identity, and more preferably 95% sequence identity to SEQ ED NO: 76. Preferably amino acid residues that participate in the formation and/or stabilization of the amphipathic alpha helical structure formed by the C heptad are not substituted. Preferably amino acid residues at positions a and d of the heptad repeat region(s) are not substituted. Preferably, variants are not substituted at positions that interact with the hydrophobic pocket of the N heptad such as those amino acid positions that correspond to Trp 628, Trp 631 and He 635. Preferably, the substitutions are at the second or third N terminal amino acid position of the heptad repeat region or the last two C terminal amino acid positions of the heptad repeat region. Amino acids at those positions can be substituted with amino acids that have uncharged polar R groups or charged polar R groups.
Preferably, the amino acid positions are substituted with glutamic acid, threonine, aspartic acid, or lysine. The C heptad can tolerate more charged and hydrophillic amino acid substitutions at positions other than position a and d of the heptad repeat regions. In some embodiments, amino acid substitutions can be made at amino acid positions 629, 630, 633, 634, 636, 640, 641, 648, or 651 mixtures thereof corresponding to amino acid positions in the C heptad region of gp41 of HBX2 isolate of HEV. Polypeptides comprising variant C heptads capable of forming amphipathic helical structure can also be predicted using algorithms such as
PredictProtein available on the Internet at cubic-bioc-columbia-edu/predictprotein. In addition to full-length C heptads, monomers of the invention may include truncated C heptads, which exhibit the ability to form an amphipathic, helical structure when in contact with a N heptad. Such truncated C heptads may comprise peptides at least about one 7 amino acid heptad repeat region, more preferably, between about 28 and 56 amino acids. Examples of truncated C heptads include the peptides P-16 (WMEWBP^INNYTSLfflSLffiESQNQQEKNEQELL (SEQ DD NO: 133) and P-18 (YTSLfflSLIEESQNQQEKNEQELLELDKWASLWNWF (SEQ ID NO: 134) which are truncated forms of the C helical domain of HEV-ILAI gp41 where the peptides are derived by truncating the C heptad region at both the N terminal and C terminal ends. Preferred truncations are those that lack N terminal heptad repeat region or the last C terminal heptad repeat region. In one embodiment, the C heptad is truncated 5 amino acids at the N terminal end and 8 amino acids at the C terminal end as compared to the C heptad sequence of amino acids 623 to 663 of gp41. C heptads useful in the invention also include truncations that may include, but are not limited to, peptides comprising the full-length, or a truncated sequence, containing one or more amino acid substitutions, insertions, and/or deletions. Preferably, C heptads are not truncated at positions that interact with the hydrophobic pocket of the N heptad corresponding to amino acid positions Trp 628, Trp 631 and He 635 of SEQ ED NO:76.
iii) Linker moieties The first and second linker moieties are agents or molecules that are flexible and allow the N heptads to fold and form a coiled coil structure. Optionally, the first and/or second linker moiety may contain one or more sites that are cleavable by chemical or enzymatic means. The polypeptide multimer comprises at least one monomer comprising a first and second N heptad, a C heptad, and a first and second linker moiety. In one embodiment, the first N heptad is connected to the C heptad by the first linker moiety and the second N heptad is connected to the C heptad by the second linker moiety. In another embodiment, the first N heptad is connected to the second N heptad by the first linker moiety and the second N heptad is connected to the C heptad by the second linker moiety. In another embodiment, the C heptad is connected to the first N heptad by the first linker moiety and the first N heptad is connected to the second N heptad by the second linker moiety. The linker moiety is preferably a peptide. The first and/or second linker moiety may comprise a peptide with the same or different amino acid sequences. Each linker moiety may comprise an amino acid sequence from about 2 to about 36 amino acid residues, preferably from 2 to about 16 amino acid residues. Native sequence gp41 has a linker region that connects the N and C heptad. The native linker region is about 31 amino acids long. The first and/or second linker may comprise a native sequence linker, which corresponds to amino acids 592 to 623 of gp41. Native sequence linkers can be selected or derived from any of the known linker regions. Linker moieties useful in the invention may include, but are not limited to, peptides comprising the full-length native sequence linker, or a truncated sequence, containing one or more amino acid substitutions, insertions, and/or deletions. The linker moiety is flexible, allowing the monomer to fold. Preferred peptides comprise amino acid residues glycine and serine, and/or glycine and cysteine. Examples of linker moieties include, but are not limited to: (GGGGS)X (SEQ DD NO: 77) wherein x is 1 to 5 (WO 00/40616); (GGC)X where X is 1 to 5 (WO 00/40616); GGSGG (SEQ ED NO: 78) (Root et al, 2001, Science, 291:884- 888); GSSGG (SEQ ID NO: 79) (Root et al., 2001, Science, 291:884-888); and SGGRGG (SEQ ID NO: 80) which is trypsin cleavable (-GGR-) (Tan et al., 1997, Proc. Natl. Acad. Sci. USA, 94: 12303-12308); and GGSSGG (SEQ ID NO: 81).
Other linkers designed for flexibility are also known (Wung et al., 1997, J. Immunol. Methods, 204:33-41), such as the Gly-Ala3 (SEQ ID NO: 82) (Holmes et al., 1996, Protein Pept. Lett., 3:415-422) or Gly4Ser3 linkers (SEQ ID NO: 83) (Micheal et al., 1996, Immunotechnology, 2:47-57) and to incorporate sites for specific proteolysis (Lucic et al., 1998, Australia J. Biotechnol, 61-108; Mathews, D.J. and Wells, J.A., 1993, Science, 26:1113-1117. The linker moiety may also comprise additional functional features including, but not limited to, antigenic epitopes and sites for specific proteolysis. For example, the linker may incorporate a trypsin cleavable site. In one embodiment, the first linker moiety of the monomer is trypsin cleavable (-GGR-). In a further embodiment, the first linker moiety has an amino acid sequence of SEQ DD NO: 80.
iv) Peptide The monomer may also comprise an additional peptide that, preferably, provides for ease of purification or detection. These additional peptides include, but are not limited to, peptide tags such as histidine6 (His), haemoglutinin protein (HA), avidin (Avi), biotin, c-Myc, VSV-G, HSV, V5, or FLAG™. A HA peptide tag, for example, may be linked to the C-terminus of the second N heptad of the monomer to facilitate detection of the monomer by anti-HA monoclonal antibodies. An Avi tag, for example, may be linked to the second N heptad of the monomer to facilitate biotinylation of the monomer. The additional peptide is preferably not another N and/or C heptad. A monomer may comprise more than one peptide tag. A His peptide tag, for example, may be linked to the C-terminus of a HA peptide tag to facilitate purification. The His peptide tag binds to a Ni-NTA column. Once purified, the His peptide tag may be cleaved by a protease at a protease site located between the HA and His peptide tags. In one embodiment, the monomer comprises a HA, His, or Avi peptide tag.
In a further embodiment the HA tag comprises an amino acid sequence of SEQ ID NO: 84. In a further embodiment, the His tag comprises an amino acid sequence of SEQ ED NO: 85. In a further embodiment, the Avi tag comprises an amino acid sequence of SEQ ED NO: 86. hi yet another embodiment, the monomer comprising an additional peptide has an amino acid sequence of SEQ ED NO: 87, SEQ ED NO: 88, SEQ ID NO: 89, or SEQ DD NO: 90.
v) Monomers In one embodiment, a monomer of the invention has an amino acid sequence: 3-helix HR no tag (3-HR-NT) MSGEVQQQNNLLRAIEAQQHLLQLTVWGIKQLQARSGGRGGWME WDREINNYTSLmSLIEESQNQQEKGGSSGGSGEVQQQNNLLRAEEAQ QHLLQLTVWGIKQLQAR (SEQ ED NO: 87) 3-helix HR haemoglutinin tag (3-HR-HA) MSGJVQQQNNLLRAIEAQQHLLQLTVWGEKQLQARSGGRGGWME WDREINNYTSLIHSLIEESQNQQEKGGSS FGSGrVQQQNNLLRAEEAQ OHLLOLTVWGπ OLOARGffiGRYPYDVPDYAGPG (SEQ DD NO: 88) 3-helix HR histidine6 tag (3-HR-HA-His) MSGEVQQQNNLLRAIEAQQHLLQLTVWGIKQLQARSGGRGGWME WDREINNYTSLfflSLIEESQNQQEKGGSSCFGSGrVQQQNNLLRAEEAQ OHLLQLTVWGIKQLQARGGffiGRYPYDVPDYAGPGGLVPRGSHHHH HH (SEQ ED NO: 89)
En another embodiment, a monomer of the invention has an amino acid sequence: 3-helix HR avidin tag (3-HR-Avi) MSGEVQQQNNLLRAIEAQQHLLQLTVWGEKQLQARSGGRGGWME WDREINNYTSLIHSLIEESQNQQEK(7GrSS ?G!SGEVQQQNNLLRAIEAQ OHLLQLTVWGIKOLQARGGSGLNDEFEAOKIEWHE (SEQ ED NO: 90) fri another embodiment, a monomer or conjugate of the invention can comprise: (1) three tandem repeating units consisting of P-17-linker-P-18 (P-17- linker-P- 18-linker-P- 17-linker-P- 18-linker-P- 17-linker-P- 18), (2) P-17-linker-P-18-linker-P-17, (3) P-18-linker-P-17-linker-P-18, (4) P-17-linker-P-17, (5) three tandem repeating units consisting of P-15-linker-P-16 (P-15- linker-P- 16-linker-P- 15 -linker- P- 16-linker-P- 15 -linker-P- 16, (6) P-15-linker-P-16-linker-P-15, (7) P-16-linker-P-15-linker-P-16, or (8) P-16-linker-P-15; wherein each linker is an amino acid sequence, which may be the same or different, of from about 2 to about 25, preferably 2 to about 16 amino acid residues. Preferred amino acid residues include glycine and serine, for example (GGGGS)X, (SEQ ID NO: 77) wherein x is 1, 2, 3, 4, or 5, or glycine and cysteine, for example (GGC)y, where y is 1, 2, 3, 4, or 5. hi any of the described constructs, P-15 and P-17 are interchangeable and P-16 and P-18 are interchangeable. Representative structures and formation of the six helix bundles are shown in Figure 9. vi) Variants The monomers of the invention may also include variants of a polypeptide with an amino acid sequence of SEQ ID NO: 87, SEQ ID NO: 88, SEQ ID NO: 89 or SEQ ID NO: 90. Variants include amino acid substitutions, deletions and insertions. As described previously, the N heptad and C heptad regions of the monomer can be varied as long as these regions when combined maintain an helical or coiled coil structure. For example, typically, N heptad regions have about 1 to 4 amino acid substitutions and in one case has up to about 9 amino acid substitutions when known N heptad sequences are compared to SEQ ID NO: 1. When known C heptad sequences are aligned and compared, they, typically, have about five to nine amino acid substitutions compared to SEQ DD NO: 76. The monomer may have a multiple substitutions in one or more of the N heptad and/or C heptad region sequences or in the linker regions. In an embodiment where each N heptad and C heptad has up to 9 amino acid substitutions, this would result in about 75 % sequence identity to any of SEQ ID NO: 87, SEQ ID NO: 88, SEQ ED NO: 89 or SEQ ED NO: 90. In another embodiment, where each N heptad has about 4 amino acid substitutions and each C heptad has up to 9 amino acid substitutions, this would result in a polypeptide that has about 85% sequence identity to any of SEQ ED NO: 87, SEQ ED NO: 88, SEQ ED NO: 89 or SEQ ED NO: 90. Variants of the monomers preferably have at least about 75% sequence identity, more preferably any % identity from about 75% to 100% (eg. 75%, 76%, 77%, 78%, etc.), more preferably 80% sequence identity, more preferably 85% sequence identity, more preferably 90% sequence identity, and more preferably 95% sequence identity to a monomer with an amino acid sequence of SEQ ED NO: 87, SEQ ED NO: 88, SEQ ED NO: 89 or SEQ ED NO: 90. Variants preferably can form a stable coiled coil structure in solution. Variants also, preferably, include the hydrophobic pocket region of the N heptad corresponding to amino acids 565-579 of gp41, for example, SEQ ED NO : 111. Preferably, C heptads are not truncated or substituted at positions that interact with the hydrophobic pocket such as positions that correspond to Trp 628, Trp 631 and He 635 in SEQ ED NO:76. The monomer of the invention may include amino acid insertions that consist of single amino acid residues or stretches of residues ranging from 2 to 15 amino acids in length. One or more insertions may be introduced into the peptide, peptide fragment, analog and/or homolog. The monomers of the invention may include amino acid deletions of the full length peptide, analog, and/or homolog. Such deletions consist of the removal of one or more amino acids from the full-length polypeptide sequence, with the lower limit length of the resulting peptide sequence being about 7 amino acids. Such deletions may involve a single contiguous portion or greater than one discrete portion of the peptide sequences In yet another embodiment of the invention, peptides comprising the sequences described above may be synthesized with additional chemical groups present at their amino and/or carboxy termini, such that, the stability, bioavailability, and/or immunogenic activity of the peptides is enhanced. For example, hydrophobic groups such as carbobenzoxy, dansyl, or t-butyloxycarbonyl groups, may be added to the peptides' amino termini. Likewise, an acetyl group or a 9-fluorenylmethoxy- carbonyl group may be placed at the peptides' amino termini. Additionally, the hydrophobic group t-butyloxycarbonyl, or an amido group may be added to the peptides' carboxy termini.
B. Polypeptide Multimers The monomer is capable of forming number of different multimers by the folding of the N heptad regions into a coiled coil structure and association of the C heptad with the N heptad coiled coil structure. A schematic diagram of the predicted structures of multimers is shown in Figure 1. A monomer folds in solution and may form a 4-helix bundle in the presence of free N heptad. A homodimer folds and may form a 5-helix bundle connected to an uncomplexed N heptad. A homotrimer may form a six-helix bundle connected to a trimer of N heptads. It was unexpected that the monomer would be able to fold into stable multimers in solution because the ratio of N heptads to C heptads of at least about 2:1 in the monomer made it likely the hydrophobic N heptad would be more exposed to the solvent. An increase in exposure of the hydrophobic N heptads to solvent may present problems of solubility, aggregation, and stability of the monomer or multimers. It was unexpected that stable multimers could be formed from a monomer with a ratio of N heptads to C heptads of at least about 2:1. Homodimers or trimers can readily be formed by monomers in aqueous solution and in absence of other polypeptides comprising N or C heptad regions. These structures form novel structures that can mimic gp41 fusion intermediates that form upon membrane fusion. The homodimers and/or homotimers are formed in aqueous solution. When it is desirable to have a homogeneous population of the homodimer or homotrimer (e.g. for characterization of the structure), the aqueous solution employed may have a pH or about 4.5 or less. When the pH of the solution is closer to a pH of 7.0 there may be tendency for some aggregation to occur depending on the concentration of the monomer. Even though some aggregation may occur, a portion of the mixture remains as a homodimer or homotrimer. Lower concentrations of the homodimer or homotrimer (eg. μg/ml range) are more homogeneous at pHs greater than 4.5. The homodimer has a molecular weight of about 10,000 to 40,000 daltons, or more preferably about 20,000 to 30,000 daltons. The molecular weight of the homodimer can vary depending on the presence or absence of an additional peptide such as a peptide tag. In one embodiment, the homodimer is comprised of two monomers, each having an amino acid sequence of SEQ ED NO: 87, and has a molecular weight of about 24,300 daltons as determined by sedimentation equilibrium. The homotrimer has a molecular weight of about 30,000 daltons to 52,000 daltons, or more preferably about 35,000 to 45,000 daltons. In one embodiment, the homotrimer is comprised of three monomers each having a sequence of SEQ ID NO: 90, and has a molecular weight of about 39,400 daltons as determined by sedimentation equilibrium. Stability of the polypeptide multimer can be determined by the melting temperature and/or helicity of the polypeptide multimer. The apparent melting temperature (Tm) may be determined by circular dichroism spectroscopy or other methods known in the art. Wild et al., 1992, Proc. Natl. Acad. Sci., 89-10537- 10541. A multimer of the invention has a Tm of at about 60 to 90°C, more preferably 70 to 90°C, or even more preferably 80 to 90°C. In one embodiment, the homodimer has a Tm of about 80°C. A multimer of the invention has helical structure. Helicity of the multimer may be determined by circular dichroism spectroscopy in the far-uv spectral region (190-250nm) or by other methods known in the art. Wild et al., 1992, Proc. Natl. Acad Sci., 89-10537-10541. Preferably, the multimer has a helicity of about 60 % or greater, more preferably any of 60 to 90%. In one embodiment, a 4 helix bundle may be formed comprising a monomer and a free N heptad. The monomer comprises a first and second N heptad and a C heptad. In an embodiment, the monomer comprises a first and second N heptad connected by a C heptad via linker moieties. A 4 helix bundle is formed when the monomer is combined with a free N heptad polypeptide in solution. In one embodiment, the monomer comprises an amino acid sequence of SEQ DD NO: 87 and the N heptad comprises a sequence of SEQ DD NO: 1. The multimer has a ratio of N heptads to C heptads of about 3:1. In another embodiment, a homodimer is formed comprising 2 monomers, each monomer comprising an amino acid sequence: MSGEVQQNNLLRAEEAQQHLLQLTVWGΠ QLQARSGGRGGWMEWD REENNYTSLFFLSLEEESQNQQEKGGSSGGSGEVQQQNNLLRAIEAQQHLLQLT VWGIKQLQAR (SEQ JD NO: 87)
The homodimer is preferably formed in acidic solution and has a molecular weight of about 24,300 daltons as determined by sedimentation equilibrium, and has a Tm of about 85°C and has a helicity of about 85%. The coiled coil structure may also be a homotrimer. The homotrimer may form a six-helix bundle with a three-helix tail. Preferably, monomers comprising the homotrimer comprise an additional peptide at the C terminal end of about 18 amino acids in length. In one embodiment, the additional peptide is the avidin tag. Another embodiment, is a homotrimer comprising 3 monomers, each monomer comprising an amino acid sequence of: MSGEVQQQNNLLRAIEAQQHLLQLTVWGI QLQARSGGRGGWMEW
DREENNYTSLIHSLIEESQNQQEKGGSSGGSGEVQQNNLLRAIEAQQHLLQLT VWGKOLOARGGSGLNDIFEAOKIEWHE (SEQ JD NO: 90). Monomers as described herein could also be combined with other N and/or C heptad containing polypeptides to form heteromultimers. A polypeptide multimer of the invention has biological function. Preferably, the multimer has antiviral and/or immunogenic activity. Antiviral activity includes, but is not limited to, binding receptor activated gp41, inhibiting HEV-1 Env mediated envelope fusion, and/or inhibiting HIV infectivity. A multimer with antiviral activity binds receptor-activated gp41 in vitro and/or in vivo. Binding of the multimer to receptor-activated gp41 may be assayed by immunoprecipitation (Furuta et al., 1998, Nat. Struc. Biol, 5:276-279; He et al., 2003, J. Virol, 77:1666- 1671) or other methods known in the art. In one embodiment, a multimer comprising at least one monomer comprising an amino acid sequence SEQ ED NO: 87 or an amino acid sequence having at least 75% percent identity to SEQ ED NO:87 binds receptor activated gp41. Preferably, a multimer that binds receptor-activated gp41 inhibits HEV-1 Env mediated HEV infectivity in vitro and/or in vivo. Inhibition of HEV infectivity may be assessed by an infectivity assay (Weng, Y. and Weiss, C, 1998, J. Virol, 72:9676-9682; see Example 2) or other methods known in the art. In one embodiment, a multimer comprising at least one monomer comprising SEQ ED NO: 87 or an amino acid sequence having at least 75 percent identity to SEQ ID NO: 87 inhibits HEV infectivity. Preferably, a multimer of the invention is immunogenic. Immunogenicity of the multimer may be assessed, for example, by small animal immunization and ELISA (de Rosny et al., 2001, J. Virol, 75:8859-8863) or other methods well known in the art. In one embodiment, antibodies to the multimer bind receptor- activated gp41. In another embodiment, antibodies to the multimer inhibit HEV-1 mediated membrane fusion and/or HEV infectivity.
C. Vectors, Host Cells, and Recombinant Methods The polypeptide multimers of the invention are produced by synthetic and recombinant methods. Accordingly, a second aspect of the invention relates to polynucleotides encoding the polypeptide multimers, recombinant vectors, and host cells containing the recombinant vectors, as well as methods of making such vectors and host cells by recombinant methods. The monomers of the invention may be synthesized or prepared by techniques well known in the art. See, for example, Creighton, Proteins: Structures and Molecular Principles, W. H. Freeman & Co., New York, NY (1983), which is incorporated herein by reference in its entirety. Nucleotide sequences for N and C heptads are known and readily available, for example, on the Internet at www/hiv- web/lanl/gov. Short peptides, for example, can be synthesized as a solid support or in solution. Longer peptides may be made using recombinant DNA techniques. The nucleotide sequences encoding the peptides of the invention may be synthesized, and/or cloned, and expressed according to techniques well known to those of ordinary skill in the art. See, for example, Sambrook, et al., Molecular Cloning, A
Laboratory Manual, Vols. 1-3, Cold Spring Harbor Press, Cold Spring Harbor, NY
(1989). The polynucleotides may be produced by standard recombinant methods known in the art, such as polymerase chain reaction (Sambrook, et al., 1989, Molecular Cloning, A Laboratory Manual, Vols. 1-3, Cold Spring Harbor Press, Cold Spring Harbor, NY. The peptide constructs may be assembled from polymerase chain reaction cassettes sequentially cloned into a vector containing a selectable marker for propagation in a host. Such markers include dihydrofolate reductase or neomycin resistance for eukaryotic cell culture and tetracycline or ampicillin resistance genes for culturing in E. coli and other bacteria. Several bacterial expression vectors have been developed specifically for the expression of small proteins. Plasmid pAED4, for example, confers ampicillin resistance. The nucleotide sequence and map of the pAED4 vector is known and readily available (Doering and Matsudaira, 1996, Biochem., 35:12677-12685. Plasmid pTCLE-G2C, based on pAED4, is another example of a suitable expression vector (Calderone et al., 1996, J. Mol Biol, 262:407-412). Representative examples of appropriate hosts include, but are not limited to, bacterial cells such as E. coli, Streptomyces and Salmonella typherium, fungal cells such as yeast; insect cells such as Drosophilia S2 and Spodoptera Sf9, animal cells such as CHO, COS, and Bowes melanoma cells, and plant cells. Appropriate culture medium and conditions for the above-described host cells are known in the art. The polynucleotide should be operably linked to an appropriate promoter, such as the T7 promoter in plasmid pAED4. Other suitable promoters are known in the art. The expression constructs may further contain sites for transcription initiation, transcription termination, and a ribosome binding site for translation. The coding portion of the mature polypeptide expressed by the constructs preferably includes a translation initiating codon at the beginning and a termination codon (UAA, UGA, or UAG) appropriately positioned at the end of the polypeptide to be translated. Introduction of the recombinant vector into the host cell can be effected by calcium phosphate transfection, DEAE-dextran mediated transfection, cationic lipid- mediated transfection, electroporation, transduction, infection, or other methods.
Such methods are described in standard laboratory manuals such as Sambrook, et al.,
1989, Molecular Cloning, A Laboratory Manual, Vols. 1-3, Cold Spring Harbor
Press, Cold Spring Harbor, NY or Davis et al., 1986, Basic Methods in Molecular
Biology. Commercial transfection reagents, such as Lipofectamine (Invitrogen, Carlsbad, CA) and FuGENE 6™ (Roche Diagnostics, Indianapolis, IN), are also available. The polypeptide may be expressed in a modified form, such as a fusion protein, and may include secretion signals and/or additional heterologous functional regions. For example, a region of additional amino acids may be added to the N- terminus or C-terminus of the polypeptide to facilitate detection or purification, or improve persistence in the host cell during, for example, purification or subsequent handling and storage. Examples of additional amino acids include peptide tags that may be added to the polypeptide to facilitate detection and/or purification. Such peptide tags include, but are not limited to, His, HA, Avi, biotin, c-Myc, VSV-G, HSV, V5, or FLAG™. The polypeptide can be recovered and purified from recombinant cell cultures by methods known in the art, including ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxylapatite chromatography, and lectin chromatography. In an embodiment, high performance liquid chromatography (HPLC) is employed for purification. D. Compositions and Methods of Using Polypeptide Multimers i. Polypeptide multimers The polypeptide multimers of the present invention are useful, for example, as antiviral agents, immunogens, oligomerization domains for making bispecific molecules, for purifying anti-HEV antibodies from sera, and for identifying and/or purifying therapeutic anti-HEV or anti-SEV monoclonal antibodies, and in a method to screen for small molecule inhibitors of HEV or SEV membrane fusion. a. Antiviral compositions The multimers of the invention can be employed in antiviral compositions.
HEV infectivity is inhibited, for example, by contacting the virus with an effective inhibitory amount of a composition of the invention. In another embodiment, HEV is inhibited by administering to a subject an antiviral effective amount of a composition of the invention, hi a further embodiment, the composition administered to the subject is a dimer or trimer comprising at least one monomer comprising a first N heptad capable of forming a coiled coil structure in solution; a C heptad; and a second N heptad that can form a coiled coil structure with the first N heptad wherein each heptad is connected to another heptad by a first or second linker moiety. En one embodiment, the first N heptad is linked to the C heptad by a first linker moiety and the C heptad is linked to the second N heptad by a second linker moiety. En one embodiment, the monomer has a sequence of SEQ ED NO: 87 or SEQ ED NO: 90 or the monomer has at least 75% sequence identity to SEQ ED NO: 87 or SEQ ED NO: 90. The polypeptide is preferably administered in combination with a physiological acceptable carrier. The polypeptide of the invention may be admimstered in combination with other antiviral agents, immunomodulators, antibiotics or vaccines. The polypeptides of the invention can be administered orally or parentally, including subcutaneous injection, intravenous, intramuscular, intrasternal or infusion techniques, in dosage unit formulations containing conventional non-toxic pharmaceutically acceptable carriers, adjuvants or vehicles. Compositions of the invention can be in the form of suspensions or tablets suitable for oral administration or sterile injectable preparations, such as sterile injectable aqueous or oleagenous suspensions. For oral administration as a suspension, the compositions can be prepared according to techniques well-known in the art of formulation. The compositions can contain microcrystalline cellulose for imparting bulk, alginic acid or sodium alginate as a suspending agent, methylcellulose as a viscosity enhancer, and sweeteners or flavoring agents. As immediate release tablets, the compositions can contain microcrystalline cellulose, starch, magnesium stearate and lactose or other excipients, binders, extenders, disintegrants, diluents and lubricants known in the art. For administration as injectable solutions or suspensions, the compositions can be formulated according to techniques well-known in the art, using suitable dispersing or wetting and suspending agents, such as sterile oils, including synthetic mono- or diglycerides, and fatty acids, including oleic acid. The amounts to be administered for any particular treatment protocol can readily be determined by one skilled in the art without undue experimentation. See for example the HEV clinical trials database at on the Internet at www/hiv- web/lanl/gov. Dosage levels of approximately 100 μg to approximately 100 mg of a composition of the invention two times per day are useful as an antiviral treatment or prevention HEV infection. In one embodiment, dosages of about 3 mg to about 100 mg of composition are administered intravenously to a subject. Kilby et al., 1998, Nat. Med., 4:1302-1307. The specific dosage level and frequency for any particular subject will be varied and will depend upon a variety of factors, including the activity of the specific compound the metabolic stability and length of action of that compound, the age, body weight, general health, sex, and diet of the subject, mode of administration, rate of excretion, drug combination, and severity of the particular condition. The polypeptides of the invention can be administered in combination with other agents useful in the treatment of HEV infection, ADDS or AEDS-related complex (ARC). For example, the compound of the invention can be administered in combination with effective amounts of an antiviral, immunomodulator, anti- infective, or vaccine. The compound of the invention can be administered prior to, during, or after a period of actual or potential exposure to HEV. b. Immunogenic Compositions The polypeptide multimers of the present invention can be employed in compositions useful to prepare antibodies to gp41 fusion intermediates and/or induce active immunity towards antigens in subjects. In one embodiment, HEV infectivity is inhibited by administering to a subject an immunogenic effective amount of a composition of the invention. The compositions of the invention may be useful in multicomponent vaccines such as epitope vaccines. The compositions may also be used to immunize an animal to prepare antibodies that may be useful therapeutically, or in purification and detection of HEV strains, or in screening of inhibitors of gp41 mediated fusion. The subjects are preferably mammals, and more preferably humans. The administration of the vaccine may be for either a prophylactic or therapeutic purpose. When provided prophylactically, the vaccine (s) are provided in advance of any symptoms of HEV infection, or in advance of any known exposure to HEV. The prophylactic administration of the vaccine (s) serves to prevent or attenuate any subsequent infection. When provided therapeutically, the vaccine (s) is provided upon or after the detection of symptoms which indicate that an animal may be infected with HEV, or upon or after exposure to the virus. The therapeutic administration of the vaccine (s) serves to attenuate any actual infection, for example as measured by improving the symptoms of a subject, or by reducing the level of viral replication. Thus, the vaccines, may be provided either prior to the onset of infection (so as to prevent or attenuate an anticipated infection) or after the initiation of an actual infection. The effective amount of vaccine is dependent on the route of administration. The vaccine can be administered by subcutaneous, intramuscular, intraperitoneal, or intravenous route. Subcutaneous or intramuscular routes of administration are prefened. Dosage levels of about 100 μg to about 1 mg of a composition administered intramuscularly to a subject are useful as a vaccine. In an embodiment, about 500 μg to about 1 mg of the vaccine compositions is administered intramuscularly to a subject. The amounts to be administered for any particular treatment protocol can readily be determined by one skilled in the art without undue experimentation. See for example the HEV clinical trials database on the Internet at http ://www/hiv-web/lanl/go v. Methods for preparing and using vaccines are well known in the art (Remington's Pharmaceutical Sciences, Osol, ed., Mack Publishing Co., Easton, PA (1980); New Trends and Developments in Vaccines, Voller et al., eds., University Park Press, Baltimore, MD (1978)). The vaccines of the present invention may be employed in such forms as capsules, liquid solutions, suspensions or elixirs for oral administration, or sterile liquid forms such as solutions or suspensions, with optional physiologically acceptable carriers, excipients or stabilizers. Acceptable carriers, excipients, or stabilizers are nontoxic to recipients at the dosages and concentrations employed. Any inert carrier may be used. In an embodiment, the inert carrier is saline, phosphate-buffered saline, or any inert carrier in which the vaccine has suitable solubility properties. The vaccine may be in the form of a single dose preparation or in multi-dose flasks which can be used for mass vaccinations programs. The vaccines of the present invention may further comprise adjuvants which enhance production of HEV-specific antibodies. Such adjuvants include, but are not limited to, various oil formulations such as Freund's complete adjuvant (CFA), stearyl tyrosine (ST, see U. S. Patent No. the dipeptide known as MDP, saponins and saponin derivatives, such as Quil A and QS-21, aluminum hydroxide, and lymphatic cytokine. In an embodiment, the adjuvant alum (aluminum hydroxide) or
ST is be used for administration to a human. The vaccine may be absorbed onto the aluminum hydroxide from which it is slowly released after injection. Preferably, an adjuvant will aid in maintaining the secondary and quaternary structure of the immunogens. The vaccine may also be entrapped in microcapsules (including, but not limited to, liposomes, albumin microspheres, microemulsions, nano-particles and nanocapsules) in colloidal drug delivery systems or in macroemulsions. Such techniques are disclosed in Remington 's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980). hi an embodiment, the vaccine is encapsulated within liposomes according to Fullerton, U. S. Patent No. 4,235,877, or mixed with liposomes or lipid mixtures to provide an environment similar to the cell surface environment. In another embodiment, one or more polypeptide multimers of the invention are combined with other immunogens that are used to vaccinate animals. In another embodiment, the invention relates to a method of inducing an immune response in a subject comprising admimstering to the subject a composition of the invention in an amount effective to induce an immune response. Optionally, the composition of the invention may be coadmimstered with effective amounts of other immunogens as to generate multiple immune responses in the subject. c. Antibodies The polypeptide multimers of the invention are immunogenic and elicit anti- multimer antibodies. The antibodies have many uses, including purifying the polypeptide multimers of the invention, detecting HEV, screening for inhibitors of membrane fusion, and inhibiting and/or neutralizing HEV. The antibodies may be polyclonal or monoclonal antibodies. The antibody may be used to detect HEV in a fluid or tissue from a subject. The antibody typically will be labeled with a detectable moiety including, but not limited to, a fluorescent label, a radioisotope, or an enzyme-substrate label. The label may be indirectly conjugated with the antibody. For example, the antibody can be conjugated with biotin and any of the three broad categories of labels mentioned above can be conjugated with avidin, or vice versa. Biotin binds selectively to avidin and thus, the label can be conjugated with the antibody in this indirect manner. Alternatively, to achieve indirect conjugation of the label with the antibody, the antibody is conjugated with a small hapten (e.g., digoxin) and one of the different types of labels mentioned above is conjugated with an anti-hapten antibody (e.g., anti-digoxin antibody). In another embodiment of the invention, the antibody does not need to be labeled. The antibody is detected using a labeled antibody that binds to the first antibody. Preferably, antibodies to the multimers of the invention inhibit Env mediated membrane fusion. Assaying for inhibition of syncytia formation is useful for determining inhibition of fusion. Inhibition of syncytia formation by anti-multimer antibodies may be assessed by effector cell/target cell conjugates (Golding et al., 2002, J. Virol, 76:6780-6790) or other methods well known in the art. In an embodiment, antibodies that specifically bind a polypeptide multimer comprising SEQ ID NO:l inhibit syncitia formation. Antibody neutralization may be assessed by HEV infectivity assay (Golding et al., 2002, J Virol, 76:6780-6790; de Rosny et al., 2001, J. Virol, 75:8859-8863) or other methods well known in the art. h an embodiment, antibodies that specifically bind a multimer comprising at least one monomer comprising an amino acid sequence of SEQ ED NO: 87 and inhibits membrane fusion. Neutralizing antibodies are useful in inhibiting HEV infectivity. The antibodies may be polyclonal or monoclonal. Because neutralizing antibodies in polyclonal sera may not be high titered, it may be necessary to purify and concentrate the fraction of antibodies that contain neutralizing activity, as had been shown by Louis et al., 2003, JBC, 278:20278. Monoclonal neutralizing antibodies could also be generated by the 3-HR constructs. The antibodies are preferably admimstered in combination with any of the pharmaceutically acceptable carriers. The antibodies of the invention may be administered in combination with other antiviral agents, immunomodulators, antibiotics or vaccines. The amount of antibody administered for any particular treatment protocol can readily be determined by one skilled in the art without undue experimentation. For the prevention or treatment of HEV, the appropriate dosage of antibody will depend on the type of disease to be treated, the severity and course of the disease, whether the antibody is administered for preventive or therapeutic purposes, previous therapy, the patient's clinical history and response to the antibody, and the discretion of the attending physician. The antibody is suitably administered to the patient at one time or over a series of treatments. Depending on the type and severity of HEV infection, about 1 μg/kg to 15 mg/kg (e.g., 0.1-20mg/kg) of antibody is an initial candidate dosage for administration to a subject, whether, for example, by one or more separate administrations, or by continuous infusion. See for example the HEV clinical trials database at on the Internet at http://www/hiv- web/lanl/gov. A typical daily dosage might range from about 1 μg/kg to 100 mg/kg or more, depending on the factors mentioned above. For repeated administrations over several days or longer, depending on the condition, the treatment is sustained until a desired suppression of disease symptoms occurs. However, other dosage regimens may be useful. The progress of this therapy is easily monitored by conventional techniques and assays. Therapeutic antibody compositions generally are placed into a container having a sterile access port, for example, an intravenous solution bag or vial having a stopper pierceable by a hypodermic injection needle. The antibodies of the invention can be administered in combination with other agents useful in the treatment of HEV infection, AEDS or AEDS-related complex (ARC). For example, the antibodies can be admimstered in combination with effective amounts of an antiviral, immunomodulator, anti-infective, or vaccine. The antibodies can be administered prior to, during, or after a period of actual or potential exposure to HEV.
E. Production of Antibodies i. Polyclonal antibodies Polyclonal antibodies to a polypeptide multimer of the invention are preferably raised in animals by multiple subcutaneous (sc) or intraperitoneal (ip) injections of the relevant antigen and an adjuvant. It may be useful to conjugate the relevant antigen to a protein that is immunogenic in the species to be immunized, e.g., keyhole limpet hemocyanin, serum albumin, bovine thyroglobulin, or soybean trypsin inhibitor using a bifunctional or derivatizing agent, for example, maleimidobenzoyl sulfosuccinimide ester (conjugation through cysteine residues), N-hydroxysuccinimide (through lysine residues), glutaraldehyde, succinic anhydride, SOCl2, or R1N=C=NR, where R and R1 are different alkyl groups. Animals are immunized against the antigen, immunogenic conjugates, or derivatives by combining, e.g., 100 μg or 5 μg of the protein or conjugate (for rabbits or mice, respectively) with 3 volumes of Freund's complete adjuvant and injecting the solution intradermally at multiple sites. One month later the animals are boosted with 1/2 to 1/10 the original amount of peptide or conjugate in Freund's complete adjuvant by subcutaneous injection at multiple sites. Seven to 14 days later the animals are bled and the serum is assayed for antibody titer. Animals are boosted until the titer plateaus. Preferably, the animal is boosted with the conjugate of the same antigen, but conjugated to a different protein and/or through a different cross-linking reagent. Conjugates also can be made in recombinant cell culture as protein fusions. Also, aggregating agents such as alum are suitably used to enhance the immune response. In an alternative embodiment, the animals are immumzed with a recombinant adenovirus vector expressing a polypeptide multimer of the invention followed by booster immunizations with multimers of the invention. The polyclonal antibodies generated by the immunizations undergo an initial screen for virus inhibition. Antiviral activity is evaluated in both cell-cell fusion and neutralization assays. Both assays are carried out according to protocols well known in the art (Wild et al., 1992, Proc. Natl. Acad. Sci, 89:10537-10541; Wild et al.,
1994, Proc. Natl. Acad. Sci, 91:12676-12680; Wild et al, 1994; Proc. Natl. Acad.
Sci, 91:9110-9114; see also Example 3). Preferably, antibodies are selected that can neutralize or inhibit membrane fusion for a variety of different viral isolates. The polyclonal antibodies are also screened by enzyme-linked immunoabsorbent assay (ELISA) to characterize binding. The antigen panel includes all experimental immunogens. Animals with sera samples that test positive for binding to one or more experimental immunogens are candidates for use in monoclonal antibody production. The criteria for selection for monoclonal antibody production is based on a number of factors including, but not limited to, neutralizing antibody titers and, in the absence of neutralization, binding patterns against the panel of structured HEV immunogens. Cross-competition experiments using other mapped Mabs, human sera and peptides can also be performed. ii. Monoclonal antibodies Monoclonal antibodies to a polypeptide multimer of the invention may be made using the hybridoma method first described by Kohler et al, Nature, 256:495
(1975), or may be made by recombinant DNA methods (U.S. Patent No. 4,816,567). hi the hybridoma method, a mouse or other appropriate host animal, such as a hamster or macaque monkey, is immunized as hereinabove described to elicit lymphocytes that produce or are capable of producing antibodies that will specifically bind to the polypeptide multimer used for immunization. Alternatively, lymphocytes may be immunized in vitro. Lymphocytes then are fused with myeloma cells using a suitable fusing agent, such as polyethylene glycol, to form a hybridoma cell (Goding, Monoclonal Antibodies: Principles and Practice, pp. 59- 103 (Academic Press, 1986)). The hybridoma cells are than seeded and grown in a suitable culture medium that preferably contains one or more substances that inhibit the growth or survival of the unfused, parental myeloma cells. For example, if the parental myeloma cells lack the enzyme hypoxanthine guanine phosphoribosyl transferase (HGPRT or HPRT), the culture medium for the hybridomas typically will include hypoxanthine, aminopterin, and thymidine (HAT medium), which substances prevent the growth of HGPRT-deficient cells. Preferred myeloma cells are those that fuse efficiently, support stable high- level production of antibody by the selected antibody-producing cells, and are sensitive to a medium such as HAT medium. Among these, prefened myeloma cell lines are murine myeloma lines, such as those derived from MOPC-21 and MPC-11 mouse tumors available from the Salk Institute Cell Distribution Center, San Diego, California USA, and SP-2 or X63-Ag8-653 cells available from the American Type Culture Collection, Rockville, Maryland USA. 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, pp. 51-63 (Marcel Dekker, Inc., New York, 1987)). Culture medium in which hybridoma cells are growing is assayed for production of monoclonal antibodies directed against the antigen and HEV Env. Preferably, the binding specificity of monoclonal antibodies produced by hybridoma cells is determined by immunoprecipitation or enzyme-linked immunoabsorbent assay (ELISA). After hybridoma cells are identified that produce antibodies of the desired specificity, affinity, and/or activity, the clones may be subcloned by limiting dilution procedures and grown by standard methods (Goding, Monoclonal Antibodies: Principles and Practice, pp. 59-103 (Academic Press, 1986)). Suitable culture media for this purpose include, for example, D-MEM or RPMI-1640 medium. In addition, the hybridoma cells may be grown in vivo as ascites tumors m an animal. The monoclonal antibodies secreted by the subclones are suitably separated from the culture medium, ascites fluid, or serum by conventional immunoglobuhn purification procedures such as, for example, protein A-Sepharose, hydroxylapatite chromatography, gel electrophoresis, dialysis, or affinity chromatography. The monoclonal antibodies are characterized for specificity, anti-viral activity, and inhibition of membrane fusion from a variety of HEV isolates as described previously.
F. Other Uses and Methods The polypeptide monomer and multimers of the invention also have other uses. The polypeptide monomers and/or multimers may be useful in oligomerization of other heterologous polypeptides, or can be used to screen for small molecules or antibodies that can inhibit membrane fusion. The monomers of the invention comprise two N heptads and a C heptad. The two N heptads of the monomer form a very stable coiled coil structure and the C heptad associates with the coiled coil structure. The monomer preferably forms homodimer or homotrimer. The ability of the monomer to oligomerize allows for the use of a monomer as a means to oligomerize a heterologous peptide or polypeptide. In one embodiment, a first heterologous peptide or polypeptide can be connected to the N terminal end of the first N heptad and a second heterologous polypeptide can be connected to the C terminal end of the second N heptad either before or after a peptide tag or linker moiety. The oligomerization via action of the monomer can provide for oligomerization of the heterologous polypeptide or polypeptides. Oligomerization of polypeptides may be useful, for example, to form multifunctional molecules such as bispecific molecules forming two different ligand binding domains. In addition, the polypeptide monomers and multimers of the invention may be useful in a method to screen for inhibitors of HEV membrane fusion. In one embodiment, a method involves a competitive inhibition assay. In this embodiment, the dimer or trimer of the invention is incubated with the potential inhibitor and agents that bind to the dimer or trimer can be identified. Once such agents are identified, they can be evaluated for inhibition of membrane fusion as described in Golding et al., 2002, J. Virol, 76:6780-6790 and/or for inhibition of viral infectivity. The ability of the inhibitor to inhibit membrane fusion in the presence or the trimer is measured. Preferably, those inhibitors that exhibit an IC50 of about 13 nM to 19 nM may be useful as an inhibitor of HEV mediated membrane fusion. The inhibitors screened may include small molecules such as peptide mimetics, peptide variants of N and C heptads, and antibodies to gp41 or gp41 fusion intermediate or to the dimer or trimer. In another embodiment, the method involves identifying agents that bind the polypeptide monomers and/or multimers of the invention and evaluating agents that bind the 3HR multimers or monomers for HEV fusion or infectivity inhibiting activity.
EXAMPLES The invention is illustrated by the following Examples, which serve to exemplify the embodiments, and are not intended to limit the invention in any way. Example 1 Production and Characterization of 3-HR polypeptide The heptad repeat regions, N heptad and C heptad, of HEV transmembrane protein gp41 are highly conserved. The heptad repeat regions in gp41 are thought to self assemble into a six-bundle structure that plays a role in viral entry by mediating membrane fusion. This structure consists of triple stranded coiled coil core layer (N heptad) and external layer with 3 helices (C heptad)that pack in the grooves of the coiled coil in an antiparallel manner. Peptides corresponding to these heptad repeats are potent inhibitors of HEV infection. We designed novel polypeptides utilizing combinations of N heptad and C heptad regions of gp41 and characterized the structure and function of these novel polypeptides. The novel polypeptides are useful as antiviral agents. Construction of Expression Vector Design of the 3-helix polypeptide ( hereinafter "3-HR" polypeptide)was based on the N34(L6)C28 six-helix bundle crystal structure. Tan et al., 1997, Proc.
Nat. Acad. Sci, 94:12303-12308. The novel 3 -HR polypeptide comprises two N heptad regions separated by a C heptad region. A schematic representation of the 3-
HR polypeptide is shown in Figure 1. The N and C heptad regions are those of gp41 from isolate HXB2. The sequence of gp41 of this isolate can be found at Los Alamos National Laboratory HEV Sequence Database, accessible via the Internet at http://hiv-web. lanl.gov/content/hiv-db/mainpage.html. In one embodiment, the N- terminal N heptad region is connected to the C heptad region by a linker having the amino acid sequence SGGRGG (Tan et al., 1997, Proc. Natl. Acad. Sci, 94:12303- 12308). The C Heptad region is connected to a C terminal N-heptad region by a linker GGSSGG (Root et al., 2001, Science, 291:884-888). Some of the embodiments for the novel polypeptide also comprise an additional sequence that may provide for ease of purification or detection. Those additional sequences can include peptide tags, such as histidine6 (His), haemoglutinin protein (HA), avidin (Avi), biotin, c-Myc, VSV-G, HSV, V5, or FLAG™. Four peptide constructs, 3-HR-NT, 3-HR-His, 3-HR-HA, and 3-HR-Avi were assembled from polymerase chain reaction cassettes sequentially cloned into pAED4, a T7 expression vector that confers ampicillin resistance. Doering and Matsudaira, 1996, Biochem. 35:12677-12685. The nucleotide sequence and map of the pAED4 vector is known and is readily available. The amino acid sequences of the three-helix constructs are:
3-helix HR no tag (3-HR-NT) MSGEVQQQNNLLRAIEAQQHLLQLTVWGΠ QLQARSGGRGGWME WDREINNYTSLFFLSLIEESQNQQEKGGSSGGSGLVQQQNNLLRAIEAQ QHLLQLTVWGΠ QLQAR (SEQ ED NO: 87) 3-helix HR haemoglutinin tag (3-HR-HA) MSGEVQQQNNLLRAIEAQQHLLQLTVWGΠ QLQARSGGRGGWME WDRELNNYTSLFFLSLIEESQNQQEKGGSSGGSGIVQQQNNLLRAEEAQ QHLLQLTVWGIKOLOARGIEGRYPYDVPDYAGPG (SEQ ED NO: 88)
3-helix HR histidine6 tag (3-HR-HA-His) MSGEVQQQNNLLRAIEAQQHLLQLTVWGIKQLQARSGGRGGWME WDREENNYTSLEBSLIEESQNQQEKCrGSSGGSGEVQQQNNLLRAIEAQ QHLLQLTVWGIKOLQARGGffiGRYPYD DYAGPGGL RGSHHHH HH (SEQ π NO: 89) 3-helix HR avidin tag (3-HR-Avi) MSGΓVQQQNNLLRAIEAQQHLLQLTVWGIKQLQARSGGRGGWME WDRELNNYTSLIHSLIEESQNQQEKGGSSCFGSGRVQQQNNLLRAIEAQ OHLLOLTVWGIKOLOARGGSGLNDIFEAOKIEWHE (SEQ ED NO: 90)
En the sequences, the N-to-C linker is bolded, the C-to-N linker is bold italicized, and the peptide tag is underlined. In 3-HR-HA, a HA peptide tag was added to the C terminal end of 3HR polypeptide to facilitate detection of the polypeptide by anti-HA monoclonal antibodies. In 3HR-His, a His peptide tag was added to the C terminal end of the HA peptide tag to facilitate purification. The His peptide tag binds to a Ni-NTA column. Once purified, the His peptide tag may be cleaved by thrombin at the radiolytic site located between the HA and His peptide tags (Coleman et al., 1981, Methods Enzymol, 80:341-361), and the HA tag may be cleaved by factor Xa at the radiolytic site located between 3HR and HA (Travy et al, 1992, Methods Enzymol, 215:300-360). In 3HR-Avi, avidin was added to the C terminal end of 3HR polypeptide to facilitate biotinylation of the polypeptide (Cull, M.G., 2000, Methods Enzymol, 326:430-440).
Expression and Purification of 3-HR polypeptide Recombinant 3-HR polypeptides were expressed in Escherichia coli
BL21(DE3) (Novagen, Madison, WI) with the T7 polymerase expression system. Studier et al, 1990 Methods Enzymol, 185:60-89. Transformed BL21(DE3) cells were grown in Luria-Bertani medium to an optical density (at 600 nm) to 0.6 before induction with isopropyl-b-D-thiogalacopyranoside (EPTG) (0.5 mM) for 4 hours. Bacterial pellets were resuspended in 50 ml/L cell culture of lysis buffer (50 mM Tris-HCl, pH 8.0, 50 mM NaCl) supplemented lOmMEDTA and with complete EDTA free protease inhibitor tablets (Roche, Indianapolis, IN). The cells were lysed by French press and pelleted at 20,000 x g for 30 minutes. The pelleted inclusion bodies were washed with the 50 ml /L cell culture of lysis buffer supplemented with 0.2 mg/ml lysozyme, 1 mg/ml deoxycholate, and 1 mM EDTA (wash buffer). The pellet was then washed three times with 25 ml/ L cell culture of wash buffer without lysozyme followed by three washes with 25 ml/L cell culture of lysis buffer and two washes with 25 ml/ L cell culture of deionized water. The proteins were dissolved in 50 ml/ L cell culture of 10% acetic acid, then purified by reverse-phase HPLC, using a C-18 preparative column (Delta-Pak, Waters) and a linear gradient of acetonitrile containing 0.1% (v/v) trifuoroacetic acid. The identities of polypeptides were confirmed by mass spectrometry. The proteins were dissolved in 20 mM TRIS-HCL buffer, pH 8.0, with 8 M urea to 1.5 mg/ml then refolded by dialysis against citrate-phosphate buffer (30.7 mM citric acid, 38.6 mM dibasic sodium phosphate, pH 4.0, three times, each> 3 h at 4°C) or Tris-HCL buffered saline (TBS). The protein concentration was adjusted to 1 mg/ml with citrate-phosphate buffer. Characterization of 3-HR polypeptide As shown in Figure 1, 3-HR may form two distinct stabilized coiled coil structures: a dimer or a trimer. To determine the secondary structure of recombinant 3-HR polypeptides, the peptides were analyzed by ultracentrifugation and circular dichroism (CD) spectroscopy as described by Wild et al., 1992, Proc. Nat. Acad. Sci, 89:10537-10541. The molecular weight of dimers or trimers of the 3-HR polypeptide were directly determined by sedimentation equilibrium. Analytical ultracentrifugation was carried out using an Optima XL-I analytical ultracentrifuge (Beckman Coulter, Fullerton, CA). Absorption optics, an An-60 Ti rotor and standard double-sector centerpiece cells were used. Equilibrium measurements on the peptides were at 20°C and concentration profiles were recorded after 16-20 hours at 17,000 rpm. Baselines were established by over-speeding at 45,000 rpm for 4 hours. Data (the average of five scans collected using a radial step size of 0.001 cm) were analyzed using the standard Optima XL-I data analysis software (Beckman Coulter, Fullerton, CA). Protein partial specific volumes were calculated from amino acid compositions (Cohn, E. J. and Edsall, J. T., 1943, Proteins, Amino Acids and Peptides Van Nostrand-Reinhold, Princeton, NJ.). Values of 0.731, 0.728, 0.725 and 0.729 mL/g were used for 3HR-NT, 3HR-HA, 3HR-HA-His, and 3HR-Avi, respectively. Solvent density was estimated as described in Laue et al., 1992, Analytical Ultracentrifugation in Biochemistry and Polymer Science, Harding, S. E., Rowe, A. J., and Horton, J. C, Eds., Royal Society for Chemistry, Cambridge, United Kingdom, pp. 90-125.
Figure imgf000056_0001
The predicted mass of the dimers and trimers was calculated from the amino acid sequence of the polypeptide multimers. hi all cases, the initiating N-terminal methionine had been processed. 2 The experimental mass of the dimers and trimers was determined by sedimentation equilibrium ultracentrifugation. The molecular weight of 3HR homodimers and homotrimers were directly determined by sedimentation equilibrium and the results are summarized in Table 1. 3HR-NT, 3HR-HA, and 3HR-HA-His polypeptides are all dimeric at pH 3.0. The molecular weight of the 3HR-NT homodimer as determined by sedimentation equilibrium was 24,400 daltons, which is in agreement with the molecular weight of the homodimer of 24,300 daltons calculated from the sequence of the polypeptide multimer. At pH values greater than 4.5, some aggregation may occur. The sedimentation equilibrium data in Table 1 shows that 3HR-Avi is trimeric at pH 3.0. The predicted molecular weight of the 3HR-Avi homotrimer calculated from the amino acid sequence of the polypeptide multimer was 42,480 daltons. The results of the sedimentation equilibrium show a molecular weight of 39,400 at pH 3.0. At pH values greater than 4.5, some aggregation occurs. The molecular weight at pH 7.0 was >51 ,000. The alpha helical content of dimeric 3HR- NT and trimeric 3HR-Avi polypeptides was similar contributing approximately 75% to the overall structures (Figure 2). Secondary structure and thermal stability of the 3-HR-HA dimer and 3-HR- Avi trimer were analyzed by CD spectroscopy in the "far-uv" spectral region (190- 250 nm). CD measurements were performed in PBS (50 mM sodium phosphate/150 mM sodium chloride, pH 7.0). Wild et al., 1992, Proc. Nat. Acad. Sci, 89:10537- 10541. Spectra were recorded at 20 °C using a Jasco J-720 spectropolarimeter (Jasco Inc., Easton, MD). Measurements in the far (260-180 nm) ultraviolet region were made using either 0.01 or 0.02-cm path length cells. A 1 -nm bandwidth was used. The protein solutions were about 0.5 mg ml"1. The results are shown in Figure 2. CD spectra for the 3-HR dimer show that the dimer has significant helical structure. The dimer complex is estimated to be at least 85% helical. The dimer complex is also highly stable. Stability of the dimers and trimers can also be determined by determining the apparent melting temperature of the each of the forms of the 3-HR polypeptide. The apparent melting temperature (rm) of each complex was determined using Jasco J- 720 specfropolarimeter (Jasco Inc., Easton, MD). The signal at 220 nm was used to monitor unfolding as the temperature was increased. A 10°C step was used during the thermal melts and the temperature range was 0.5 to 90.5° C.Chan et al., 1998, Proc. Natl. Acad. Sci, 95:15613-15617. The results are shown in Figure 3. The Tm of the 3-HR-HA dimer is approximately 80 °C, suggesting a very stable bundle. The homogeneity of the polypeptide multimers was high at acidic pH values where accurate molecular weight determinations were made and secondary structures determined. This is comparable to the authentic HEV gp41 ectodomain where at pH 3.5 the structure of the ectodomain was determined by NMR and shown to be comparable to that determined by X-ray crystallography (Caffrey at al., 1998, EMBO J. 17:4572-4584). Also analogous to the authentic gp41 ectodomain, is the formation of some higher aggregates at neutral pH values, in the case of HIV gp41 the aggregates can consist of between 7-70 trimers (Caffrey et al., 2000, J. Biol. Chem. 275:19877-19882.). Polypeptide multimers comprising 3HR-NT and 3HR-HA appear to be dimeric whereas multimers comprising 3HR-Avi appear to be trimeric. The dimeric or trimeric association status does not affect the overall helical nature, both association states consist of about 76% helix (data not shown). Similar analysis of the CD-spectra of the HEV - and SEV - gp41 ectodomains determines about 80% helix (Wingfield et al., 1997, Protein Science, 6:1653-1660). The CD spectrum of 3-HR is typical of an alpha-helix with minima at 208 and 222 nm (Figure 2). Upon thermal denaturation there is a loss of the 222 nm peak and this can be used to monitor thermal denaturation. The unfolding of 3-HR occurs with a transition midpoint (apparent Tm) of about 80°C indicating a stable structure (Figure 3). The HEV gρ41 has a Tm (> 90°C) (unpublished data) and other trimeric versions of the ectodomain also melt in this temperature range (Lu et al, 1995). We have designed a novel polypeptide comprising two N and one C heptad region from gp41. Four different constructs were prepared including 3HR-NT, 3HR- His, and 3HR-HA, and 3HR-Avi. The polypeptides were expressed in E. coli and isolated from inclusion bodies as described previously. Refolding of isolated polypeptides was accomplished by dialyzing against citrate phosphate buffer. Characterization of the molecular weight of the polypeptides shows the formation of a number of species including a dimer and a trimer. The dimer has an observed molecular weight of 24,400 daltons as determined by sedimentation equilibrium, which is consistent with the expected molecular weight of 24,300 daltons. The trimer has an observed molecular weight of 39,400 daltons. Dimer formation was seen when a HA peptide tag was added to the C terminal end of 3HR. Trimer formation was seen when an avidin peptide tag was added to the C terminal end of3HR. Analysis of the dimers by CD and calculation of Tm showed that the dimer was of substantial helical character and was extremely stable. A Tm temperature about 80°C indicates a highly stable structure. The results of these studies show that the dimer is likely to have the structure as shown schematically in Figure 1. Analysis of the trimer by CD spectroscopy showed that the trimer was of substantial helical character and extremely stable. The results of these studies show that the trimer is likely to have the structure as shown schematically in Figure 1.
EXAMPLE 2 Novel 3-HR Polypeptides Inhibit HIV-1 Env Mediated HIV Infectivity Binding of gpl20 surface subunit of Env to cellular receptors triggers conformational changes that lead to membrane fusion mediated by gp41. Receptor activated gp41 is important for viral entry into cells because it mediates membrane fusion and pore formation. We studied whether the 3-HR polypeptide constructs could bind to receptor activated gp41 and inhibit HEV-1 Env mediated HEV infectivity.
Envelope Glycoprotein Expression
Immunoprecipitation assays were performed using intact Env-expressing cells.
Approximately 2.5 x 10s COS-7 or 1 x IO6 293T cells per well were plated in Dulbecco's minimal essential medium (DMEM; Gibco) supplemented with 10% fetal calf serum (FCS), penicillin and streptomycin, Hepes, sodium pyruvate, nonessential amino acids, and 2mM glutamine. in a 6-well plate (Falcon, Franklin Lakes, N.J.). The cells were incubated at 37°C in a CO2 incubator until the cells were 30 to 40% confluent. In a small sterile tube, FuGENE 6™ transfection reagent (Roche Diagnostics, Indianapolis, EN) was diluted 1:11 in Optimem-1 (Gibco), mixed gently, and incubated at room temperature for 5 min. En a small sterile tube, 3 μg of Env expression plasmid pSM-WT and 1.5 μg of pRev DNA were added to 100 μl of Optimem-1 and mixed. The diluted FuGENE 6™ transfection reagent was then added directly to the plasmid mixture, the tube was gently tapped to mix the contents, and incubated at room temperature for 15 min to allow DNA-liposome complexes to form. Two hundred μl of the DNA-liposome solution was added dropwise to the plate and the cells were incubated at 37°C for 48 h before harvesting for immunoblotting or immunoprecipitation assays. Envelope expression in transfected cells was confirmed by immunoblotting.
Weng, Y. and Weiss, C, 1998, J. Virol, 72:9676-9682. Transfected cells were lysed with 0.1 ml of 1% Nonidet P-40 (NP-40)-150 mM NaCl-100 mM Tris (pH 8.0) buffer (lysis buffer). Approximately 10 μl of the clarified lysate was separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) (4 to 12% NuPAGE gels; NOVEX, SanDiego, Calif.) and transfened to an ECL nitrocellulose membrane (Amersham, Arlington Heights, 111.). The membranes were probed with anti-gpl20 polyclonal goat serum (Env 2-3; kindly provided by Kathelyn Steimer, Chiron Corp., Emeryville, Calif.) at 1:5,000 in 5% milk- phosphate-buffered saline, washed, reprobed with peroxidase-conjugated anti-goat antiserum (Sigma, St. Louis, Mo.), and washed before detection by chemiluminescence and autoradiography. Weng, Y. and Weiss, C, 1998, J. Virol, 72:9676-9682. Immunoprecipitation Assay To assess 3-HR binding to Env, 3 x IO6 intact Env-expressing 293T cells were incubated in the presence or absence of 8 μg of soluble CD4 (sCD4; kindly provided by Ray Sweet, SmitliKline Beecham) and/or 20 μg of 3-helix-HA HR per ml at 37 °C for 1 h. de Rosny et al, 2001, J. Virol, 75: 8859-8863. The cells were washed three times with PBS, incubated in DMEM containing 10 μg per ml with anti-HA monoclonal antibody (12CA5, Boehringer Mannheim) at 25°C for 3 h. Furuta et al., 1998, Nat. Struc. Biol, 5:276-279. Following incubation, the cells were washed four times in PBS and lysed with 1 ml of lysis buffer (1% Triton X- 100, 150 mM ΝaCl, and 50 mM Tris-Cl pH 7.4). The clarified supematants were incubated with 25 μl of Protein A-agarose (Gibco BRL) at 4°C overnight before washing with lysis buffer three times, immunoprecipitated complexes were separated by SDS-PAGE, immunoblotted with anti-gp41 monoclonal antibody, such as Chessie 8 (obtained from ΝEH AEDS Research and Reference Reagent Program) and by chemiluminescence and autoradiography. Weng, Y. and Weiss, C, 1998, J. Virol, 72:9676-9682. As shown in Figure 4, 3-HR specifically binds receptor-activated gp41. 3- HR-HA formed a complex with and immunoprecipitated intact gp41 only in the presence of sCD4. See Figure 5, lane 4. No complex was seen in the absence of receptor. See Figure 4, lane 3.
Infectivity Assay The development of novel inhibitors of HEV infectivity is important. We assessed whether the specific binding of the novel 3-HR polypeptides to receptor- activated gp41 inhibits HEV-1 Env mediated HEV infectivity. Env-expressing p24 pseudovirus were generated as described by Weng, Y. and Weiss, C, 1998, J. Virol, 72:9676-9682. Env-expressing luciferase reporter p24 pseudoviruses were prepared by cotransfecting approximately 3 x IO6 293T cells in 10-cm culture dishes with 5 μg of Env expression plasmid pSM-WT and 5 μg Env-deficient viral reporter vector pNL43-Luc-R~E~ by the FuGENE 6 method as described in Example 2. Supematants containing p24 pseudoviruses were collected 48 h post transfection and frozen at 80°C. The quantity of virus in the transfected supematants was determined by measuring p24 using a commercial enzyme-linked immunosorbent assay (Coulter, Westbrook, Maine). Expression of gp41 and gpl20 by p24 pseudo virus was confirmed by immunoblotting. Weng, Y. and Weiss, C, 1998, J Virol, 72:9676-9682. To detect gp41 expression, 500 ng of p24 pseudovirus was centrifuged at 20,000 x g for 3 h at 4°C The pellet was then lysed with 50 μl of lysis buffer. Half of the lysate was applied to SDS-PAGE gels (10% NuPAGE gels), immunoblotted with anti-gp41 monoclonal antibody, such as Chessie 8 antibody (obtained from National Institutes of Health AEDS Research and Reference Reagent Program), and detected by chemiluminescence and autoradiography. To detect gpl20 expression, 250 ng of p24 pseudovirus was lysed with NP- 40 lysis buffer containing 1 μg of soluble CD4-Ig G (kindly provided by Genentech, South San Francisco, Calif.). The sample was incubated in the lysis buffer for 1 h at room temperature with mixing. Approximately 25 μl of a 25% suspension of protein A-agarose (GIBCO BRL) was then added to the sample, and the sample was incubated for an additional hour at room temperature with mixing. The precipitates were then washed three times with lysis buffer, separated by SDS-PAGE (4 to 12% NuPAGE gels), and immunoblotted with polyclonal gpl20 antiserum, as described in Example 2. To assess inhibition of HEV infectivity by 3-HR polypeptides, 3.5 x IO4 U87- CD4-CXCR4 cells were plated the day before infection in 48-well dishes in DMEM containing 10% FCS, 2 mM glutamine, lχ antibiotics, 1 mM sodium pyruvate, and l nonessential amino acids (DMEM+) and incubated at 37°C. On the day of infection, the U87-CD4-CXCR4 cells were inoculated with 80 ng of ρ24 pseudovirus in 300 μl of DMEM+ containing 2.4 μg of Polybrene and serially diluted 3-HR polypeptides. After 8 h of incubation, the inoculum was replaced with 0.5 ml of fresh DMEM"1" containing 15% FCS, and the cultures were incubated for an additional 40 h at 37°C. The cells were then lysed, and luciferase activity was measured with the luciferase assay kit (Promega, Madison, Wis.) and a LumiCount luminometer (Packard, Meriden, Conn.). As shown in Figure 5 and summarized in Table 2, 3HR polypeptides inhibit HEV-1 infectivity. 3HR-His and 3-HR NT each display a dose-dependent inhibitory effect with an IC50 of approximately 30 nM (Figure 5). 3HR-HA-His and 3HR-Avi also inhibit HEV-1 infectivity in a dose-dependent manner (Table 2). 3HR-HA-His had an IC50 of 44 nM and 3HR-Avi had an IC50 of 90 nM. Table 2. Inhibition of HIV-1 Infectivity
Figure imgf000062_0001
EXAMPLE 3 Antibodies to Novel 3-HR Polypeptides Conserved structures involved in fusion of viral and cellular membranes are transiently exposed during conformational changes leading to fusion. Our novel 3- HR dimer or trimer may represent or mimic a fusion-active conformation of gp41 that occurs during hairpin formation. We assessed whether antibodies to the novel 3-HR polypeptides could inhibit HEV-1 Env mediated envelope fusion and/or HEV infectivity.
Animal Immunization Dimers or trimers of 3-HR were used as immunogens. The 3-HR polypeptides were purified as described in Example 1. New England White rabbits were immunized subcutaneously with 200 μg of 3HR polypeptide in complete Fruend's adjuvant at 4-week intervals, de Rosny et al., 2001, J. Virol, 75:8859- 8863. Following the fourth immunization, blood was collected from the rabbit's marginal ear vein, centrifuged, and the serum fraction was collected. IgG was purified from the serum fraction using a protein A column and the specificity of the purified polyclonal antibodies for 3HR polypeptide was verified by ELISA.
Immunoprecipitation Assay To assess whether the antibodies could bind native or receptor-activated gp41, we performed immunoprecipitation assays using intact Env-expression cells that were incubated with antisera in the presence or absence of sCD4.
Approximately 1 x IO7293T cells where transfected with pSM-WT using FuGENE™ 6 transformation reagent as described in Example 2. The transfected 293T cells were incubated with antisera in the presence or absence of 8 μg sCD4 per ml at 37°C for 1 h and than washed twice with phosphate buffered saline. The cells were than immunoprecipitated as described in Example 2. As shown in Figure 6, polyclonal rabbit antibodies to 3HR homodimer immunoprecipitated receptor-activated gp41. Rabbits were immunized with homodimers comprising 3HR-NT. Sera from rabbit 782 was diluted 1 :50 in the immunoprecipitation assay. Sera from rabbits 783 and 784 was diluted 1 : 100 in the immunoprecipitation assay. Sera from all three rabbits immunoprecipitated both receptor-activated gp41 and non-receptor activated gp41 from cell surfaces in the assay.
Syncytium Formation To assess inhibition of syncytia formation by rabbit polyclonal antisera to 3HR-NT homodimers, TF228 cells (Jonak et al., 1993, AIDS Res. Hum. Retroviruses, 9:23-32) and PM1 cells (Lusso et al., 1995, J. Virol, 69:3712-3720) were cocultured in the absence or presence of serial diluted antisera rabbit antisera for 5 h at 37°C or for 1 h at31.5°C and then transfened to 37°C for an additional 4h. The number of syncytia was than scored as described in Golding et al., 1992, Aids Res. Hum. Retroviruses, 8:1607-1612. Syncytia were counted visually using a microscope. Three of more fused cells were scored as a syncytia. As shown in Figure 7 and summarized in Table 3, polyclonal rabbit antibodies to 3HR-NT homodimers inhibited syncytium formation. Purified total IgG from the rabbit anti-3HR-NT sera inhibited syncytia formation when added to the effector and target cells at 37°C. The IC50 of the polyclonal antibodies at 37°C ranged from 24 μg/ml to 32 μg/ml, dependent upon the particular rabbit from which the antisera was obtained. See Figure 7 and Table 3. Preincubation of the effector and target cells at 31.5°C with the polyclonal antibodies enhanced inhibition of syncytia formation. The IC50 for the polyclonal anti-3HR-NT homodimer antibodies preincubated with the target and effector cells at 31.5°C ranged from 4.4 μg/ml to 7 μg/ml. See Figure 7 and Table 3. Table 3. Anti-3HR-NT Antibody Inhibition
Figure imgf000064_0001
As shown in Figures 7 and 8, the polyclonal sera bind gp41 from at least 3 different Env from different isolates of HEV. At 37°C, the polyclonal antibodies inhibited syncytia formation between effector cells expressing LAI Env, 89.6 Env, or JR-FL Env and target cells. See Figures 7 and 8. The syncytia inhibitory activity of the polyclonal antibodies was greater when effector cells expressed JR-FL Env or 89.6 Env. See Figure 8. These results demonstrate the broad inhibitory activity of the polyclonal antibodies. These results show that polyclonal antibodies to 3HR-NT homodimer inliibit syncytia formation at either 31°C or 37°C with a fairly low IC50. The polyclonal antibodies were able to bind to receptor-activated intermediate fusion structures of gp41 in spite of the close proximity of the effector cell and target cell membranes. HIV-1 Neutralization Assay Infectivity data in Table 3 was obtained using the single-round infectivity assay using pseudotyped- Env and the luciferase reporter gene in replication defective HEV as described in Weng et al.cited supra and as described in Example 2. The inhibitory activity of the polyclonal antibodies to 3HR-NT homodimers in viral infectivity and syncytia formation assays is summarized in Table 3. These results show that polyclonal antibodies to 3HR-NT homodimer could inliibit syncytia formation at either 31°C or 37°C with a fairly low IC50, ranging from 4.4 μg/ml to 32 μg/ml. However, the polyclonal antisera to homodimers was not able to inhibit infectivity of the virus in the assays utilized. This may be due to the fact that the polyclonal antisera as developed in rabbits may not have sufficiently high titer of high affinity antibodies that may be needed toinhibit the more efficient process of virus-cell fusion, as compared to the slower process of syncytia (multi-cell) fusions. This is consistent with others who have shown that it was necessary to concentrate and purify high affinity antibodies from polyclonal antisera to see inhibition of syncytia formation. Louis et al., 2003, J. Biol. Chem., 218:20218-20285. The polyclonal antisera specific for 3HR-NT homodimers may be used to isolate therapeutic antibodies and characterize the epitopes that bind to such antibodies. All publications (including patents and patent applications) cited herein are hereby incorporated in their entirety by reference.

Claims

WHAT IS CLAIMED IS:
1. A polypeptide multimer comprising: at least one monomer comprising: a) a first N heptad and second N heptad; b) a first and second linker moiety; and c) a C heptad; wherein each heptad is separated from one another by the first or second linker moieties, and wherein the monomer is capable of forming a homodimer or homotrimer in a solution.
2. The polypeptide multimer of claim 1 , wherein the first and second N heptads each have a coiled coil structure.
3. The polypeptide multimer of claim 1 , wherein the C heptad is capable of forming an amphipathic alpha helical structure in solution.
4. The polypeptide multimer of claim 1 , wherein the first and second N heptads form a coiled coil structure.
5. The polypeptide multimer of claim 1, wherein C-terminus of the first N heptad is connected to the C heptad by the first linker moiety and C-terminus of the C heptad is connected to the second N heptad by the second linker moiety.
6. The polypeptide multimer of claim 1 , wherein the C-terminal end of the C heptad is connected to the first N heptad by the first linker moiety and a C- terminal end of the first N heptad is connected to the second N heptad by the second linker moiety.
7. The polypeptide multimer of claim 1, wherein a C-terminal end of the first N heptad is connected to the second N heptad by the first linker moiety and C- terminal end of the second N heptad is connected to the C heptad by the second linker moiety.
8. A polypeptide multimer comprising at least one monomer comprising: a) a first N heptad capable of forming a coiled coil structure in solution; b) a first linker moiety; c) a C heptad capable of forming an amphipathic helical structure in solution; d) a second linker moiety; and e) a second N heptad capable of forming a coiled coil structure in solution; wherein the first N heptad is connected to the C heptad by the first linker moiety and the C heptad is connected to the second N heptad by the second linker moiety; and wherein the monomer is capable of forming a homodimer or homotrimer in solution.
9. The polypeptide multimer according to any of claims 1 -8, wherein the first N heptad comprises i) a polypeptide having an amino acid sequence of SEQ ED NO: 1, or ii) a polypeptide having at least 75% sequence identity to SEQ ID NO: 1 and is capable of forming a coiled coil structure in solution.
10. The polypeptide multimer according to any of claims 1 -9, wherein the C heptad comprises a polypeptide having an amino acid sequence of SEQ ID NO: 76 or a polypeptide having at least about 65% sequence identity to SEQ ID NO: 76 and is capable of forming an amphipathic helical structure when in contact with a N heptad.
11. The polypeptide multimer according to any of claims 1-10, wherein the second N heptad comprises i) a polypeptide having an amino acid sequence of SEQ ID NO: 1, or ii) a polypeptide having at least 75% sequence identity to SEQ ID NO: 1 and is capable of forming a coiled coil structure in solution.
12. The polypeptide multimer of any of claims 1-11, wherein the first N heptad and second N heptad of the monomer have the same amino acid sequence.
13. The polypeptide multimer of any of claims 1-12, wherein the first linker moiety of the monomers is a peptide with an amino acid sequence of SEQ ED NO: 80.
14. The polypeptide multimer of any of claims 1-13, wherein the second linker moiety is a peptide with an amino acid sequence of SEQ ED NO: 81.
15. The polypeptide multimer of any of claims 1-14, wherein the monomer further comprises a peptide linked to the C-terminus of the second N heptad.
16. The polypeptide multimer of claim 15, wherein the peptide is selected from the group consisting of histidine6 tag, haemoglutinin protein, avidin tag, biotin, c-Myc, VSV-G, HSV, V5, or FLAG™ and mixtures thereof.
17. The polypeptide multimer of any of claims 1-16, wherein the monomer comprises a polypeptide having an amino acid sequence of SEQ ID NO:
87 or an amino acid sequence that has at least about 75% sequence identity to SEQ ED NO: 87 and is capable of forming a homodimer in solution.
18. The polypeptide multimer of any of claims 1-16, wherein the monomer comprises a polypeptide having an amino acid sequence of SEQ LD NO:
88 or an amino acid sequence that has at least 75 % sequence identity to SEQ ID NO: 88 and is capable of forming a homodimer in solution.
19. The polypeptide multimer of any of claims 1-16, wherein the monomer comprises a polypeptide having an amino acid sequence of SEQ ID NO:
89 or an amino acid sequence that has at least 75 % sequence identity to SEQ ID NO: 88 and is capable of forming a homodimer in solution.
20. The polypeptide multimer of any of claims 1-16, wherein the monomer comprises a polypeptide having an amino acid of SEQ ID NO: 90 or an amino acid sequence that has at least 75 % sequence identity to SEQ ED NO: 88 and is capable of forming a homotrimer in solution.
21. The polypeptide multimer of any of claims 1 -20, wherein the multimer binds receptor activated gp41.
22. The polypeptide multimer of claim 21 , wherein the multimer inhibits the fusion of HEV to mammalian cell membrane.
23. The polypeptide multimer of claim 21 , wherein the multimer inhibits HEV infectivity.
24. The polypeptide multimer of any of claims 1-23, wherein the monomer further comprises a peptide of about 18 amino acids linked to the C- terminal end of the monomer.
25. The polypeptide multimer of claim 24, wherein the monomer forms a homotrimer in solution.
26. The polypeptide multimer of claim 25, wherein the trimer has a molecular weight of about 39,400 daltons in a solution with a pH of about 4.5 or less.
27. The polypeptide multimer of any of claims 1-26, wherein the monomer forms a homodimer.
28. The polypeptide multimer according to claim 27, wherein the dimer has a molecular weight of about 24,300 daltons in a solution with a pH of about 4.5 or less.
29. The polypeptide multimer according to claim 27, that has a TM of about 85°C.
30. A composition comprising a multimer of any of claims 1-29 and a physiologically acceptable carrier.
31. A method of inhibiting HEV or SEV infection comprising administering to a subject an antiviral effective amount of the composition of claim 30.
32. A method of inhibiting HEV or SEV infection comprising administering to a patient in need thereof the immunogenic effective amount of the composition of claim 30.
33. A polypeptide multimer comprising: a) at least one monomer comprising: i) a first N heptad and second N heptad; ii) a first and second linker moiety; and iii) a C heptad, wherein each heptad is separated from one another by the first or second linker moiety; and b) a third N heptad capable of forming an coiled coil structure in solution; wherein the monomer and the third N heptad form a 4 helix bundle in solution.
34. An antibody that specifically binds the multimer of any one of claims 1-30 and claim 33.
35. A monoclonal antibody that specifically binds the multimer of any of claims 1-30 and claim 33.
36. A polynucleotide encoding the multimer of any one of claims 1-30 and claim 33.
37. A vector comprising the polynucleotide of claim 36.
38. An isolated host cell comprising the vector of claim 37.
39. A polypeptide monomer comprising an amino acid sequence of SEQ ED NO: 87 or an amino acid sequence having at least about 75% sequence identity to SEQ DD NO: 87 and is capable of forming a homodimer in solution.
40. A polypeptide monomer comprising an amino acid sequence of SEQ D NO: 88 or an amino acid sequence having at least about 75% sequence identity to SEQ DD NO: 88 and is capable of forming a homodimer in solution.
41. A polypeptide monomer comprising an amino acid sequence of SEQ ED NO: 89 or an amino acid sequence having at least about 75% sequence identity to
SEQ ED NO: 89 and is capable of forming a homodimer in solution.
42. A polypeptide monomer comprising an amino acid sequence of SEQ DD NO: 90 or an amino acid sequence having at least about 75% sequence identity to SEQ DD NO: 90 and is capable of forming a homotrimer in solution.
43. A trimer comprising three monomers, each monomer comprising: a) a first N heptad and a second heptad; b) a first and second linker moiety; c) a C heptad; and d) a peptide; wherein each heptad is separated from one another by the first or second linker moiety, and the peptide is located at the C-terminal end of the monomer; wherein the monomer forms a homotrimer in solution.
44. The trimer according to claim 43, wherein the peptide is at least 18 amino acids long.
45. The trimer according to claim 44, wherein the peptide is avidin.
46. A method of screening for an inhibitor that binds to a polypeptide multimer comprising: a) contacting a homodimer or homotrimer of claim 1 with a candidate inhibitor; b) determining whether the inhibitor can bind to the homodimer or homotrimer; and c) eluting the bound inhibitor and analyzing the inhibitor for inhibition of gp41 mediated membrane fusion.
PCT/US2003/025295 2003-08-14 2003-08-14 Polypeptide multimers having antiviral activity WO2005018666A1 (en)

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CN100339396C (en) * 2005-04-18 2007-09-26 中国科学院微生物研究所 Multi-helix protein for inhibiting membrane virus infection, its coding gene and use
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