WO2005035555A1 - Hiv/siv env chimeras that promote trimerization and maintain targets of neutralizing antibodies - Google Patents
Hiv/siv env chimeras that promote trimerization and maintain targets of neutralizing antibodies Download PDFInfo
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- WO2005035555A1 WO2005035555A1 PCT/US2004/033505 US2004033505W WO2005035555A1 WO 2005035555 A1 WO2005035555 A1 WO 2005035555A1 US 2004033505 W US2004033505 W US 2004033505W WO 2005035555 A1 WO2005035555 A1 WO 2005035555A1
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- hin
- gpl40
- chimeric polypeptide
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- hiv
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Classifications
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
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- 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
- A61K39/12—Viral antigens
- A61K39/21—Retroviridae, e.g. equine infectious anemia virus
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- 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
- A61K2039/545—Medicinal preparations containing antigens or antibodies characterised by the dose, timing or administration schedule
-
- 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
- A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
- A61K2039/55511—Organic adjuvants
- A61K2039/55566—Emulsions, e.g. Freund's adjuvant, MF59
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- 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
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
-
- 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
- C12N2740/15011—Lentivirus, not HIV, e.g. FIV, SIV
- C12N2740/15022—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
-
- 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
-
- 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
- C12N2740/16134—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2760/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses negative-sense
- C12N2760/00011—Details
- C12N2760/16011—Orthomyxoviridae
- C12N2760/16211—Influenzavirus B, i.e. influenza B virus
- C12N2760/16234—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
Definitions
- Env is the only viral protein to protrude beyond the virion membrane, and it is the major viral target of the host humoral immune response.
- the oligomeric structure of Env modulates antigenicity, presumably by reducing the exposure of epitopes close to contact sites between protomers and/or by directly altering epitope conformation.
- the ability of antibody to neutralize virus is better predicted by a capacity to bind to oligomeric Env than to monomeric Env (Fouts, T. R. et al. 1997 J Virol 71:2779-2785; Fouts, T. R. et al. 1998 AIDS Res Hum Retrovir 14:591-597; Parren, P. W. H. I.
- the present invention relates to compositions and methods for making soluble, recombinant human immunodeficiency virus (HIN) envelope (Env) polypeptides that promote trimerization and maintain targets of neutralizing antibodies in which all or a portion of the ⁇ -terminal half of the gp41 ectodomain is replaced by the corresponding region of simian immunodeficiency virus (SIN).
- HIN human immunodeficiency virus
- Env human immunodeficiency virus envelope
- the invention relates as well to the nucleic acid sequences encoding the chimeric polypeptides, recombinant vectors carrying the sequences, recombinant host cells including either the sequences or vectors, and recombinant polypeptides.
- the invention further includes methods for using the isolated, recombinant polypeptides in vaccines, assays, and for use in preventive and therapeutic applications.
- Brief Description of the Drawings Fig. 1. Analysis of the oligomeric structure of HIN-l JR -FL gpl40. Lentil lectin affinity-purified gpl40 was passed through a column of Superdex 200, and individual gel filtration fractions were analyzed using biochemical and biophysical methods.
- WO 2003 0 fractions The 440/220- and 670/335-kDa markers were the dimers and monomers of ferritin and thyroglobulin, respectively.
- Fig. 4 Gel filtration analysis of variable-loop deletion mutants of HIN-l JR-FL gpl40. (A) Aliquots of gel filtration fractions were subjected to SDS-PAGE (8% polyacrylamide) and immunoblotted with an Env-specif ⁇ c antiserum and iodinated protein A.
- gpl40 was quantified by phosphor screen autoradiography, and the results were plotted as a percentage of the total gpl40-specific signal for HIN-l j R-F1 N2 (open circles) and HIV-1 JR- FL ⁇ V1/2 (open squares). Intact HIN-l JR-FL gpl40 (see Fig. 1) is also shown for comparison (closed squares). Aliquots of fractions of HIN-l JR - FL ⁇ V 1/2 gpl40 (B) and H ⁇ N-lj R-FL ⁇ N2 g ⁇ l40 (C) were treated with the cross-linker EGS (5 M final concentration) and analyzed by SDS-PAGE (5% polyacrylamide) and immunoblotted as above.
- Fig. 5 Analysis of the oligomeric structure of HIN-1/SIN gpl40 chimeras.
- A Aliquots of gel filtration fractions were subjected to SDS-PAGE (8% polyacrylamide) and immunoblotted with an Env-specific antiserum (for H-S) or the monoclonal antibodies 36D5 (anti-SIV gpl20) (Edinger, A. L. et al. 2000 J Virol 74:7922-7935) and D50 (anti- HIN-1 gp41) (Earl, P. L. et al.
- the upper inset shows B ⁇ - PAGE (4 to 12% polyacrylamide) of a pool of fractions 50 to 54 of H-S. ⁇ gpl40 revealed by Coomassie blue staining.
- the upper and lower bars show the positions of the 440- and 220-kDa marker proteins, respectively.
- the lower inset shows the effect of the presence (+) or absence (-) of an excess of soluble four-domain CD4 on the immunoprecipitation of radiolabeled H-S. ⁇ gpl40 by monoclonal antibody 17b.
- FIG. 9 Linear representation of the HIN-l Env glycoprotein.
- Fig. 10 Amino acid sequence alignment of the HJN-1 Env protein (Louwagie J. et al. 1995 J Virol 69:263-271). — , identity with the consensus sequence; • , gap in aligned sequences; X, corrected frameshift; a space, premature stop codon.
- Isolates that belong to the same subtype are grouped. *, cysteine residues; +, amino acids believed to be involved in gpl20-gp41 interaction; ⁇ , the leucine zipper motif; #, the CD4 binding site.
- Hypervariable domains (NI to N5) in the gpl20 molecule and an immunodominant domain (ID) in gp41 are delineated by solid lines.
- Predicted amino acid sequences are shown for selected regions of env for PCR-generated clones of ⁇ C-MAC-infected cells and two cellular clones of CP-MAC- infected cells and are compared with the parental BK28 sequence. Dot, amino acid identity; asterisk, in-frame stop codon; dash, a single nucleotide deletion. Regions corresponding to variable domains in external (SU) region are shown. Sequences shown include the carboxy-terminal half of SU (amino acids 301 to 527) and the entire transmembrane domain (TM) coding region.
- Env human immunodeficiency virus type 1
- SIN simian immunodeficiency virus
- gpl40 is indicated as Env lacking the transmembrane domain and cytoplasrnic tail.
- cross hatched areas represent variable domains (NI to N5) and open boxes depict conserved sequences (CI to C5).
- CI to C5 conserved sequences
- gp41 ectodomain several domains are indicated: the ⁇ -terminal fusion peptide, and the, two ectodomain helices ( ⁇ - and C-helix).
- the membrane spanning domain is represented by a black box.
- the Tyr-X-X-Leu (YXXL) endocytosis motif and two predicted helical domains (helix- 1 and -2) are shown. Amino acid numbers are indicated.
- HIV/SIN Env chimeras that promote trimerization and maintain targets of neutralizing antibodies.
- One embodiment of the invention is a chimeric polypeptide in which all or a portion of the ⁇ -terminal half of the gp41 ectodomain of human immunodeficiency virus (HIN) envelope (Env) is replaced by the corresponding region of simian immunodeficiency virus (SJN).
- HIN human immunodeficiency virus
- SJN simian immunodeficiency virus
- the use of the closely related SIN motif is expected to better mimic the authentic HIN Env trimer.
- the amount of non-HJN sequence can be increased or decreased to confer efficient trimerization while maintaining targets of neutralizing antibodies.
- the non- HJN segment of the chimera that is, the ⁇ -terminal half of the gp41 ectodomain
- "spacer" sequences not naturally occurring in HIN or SIN can be added.
- HIN type 2 (HIV-2) is closely related to SIV, thus, described herein are H ⁇ N-1/H ⁇ N- 2 Env chimeras that promote trimerization and maintain targets of neutralizing antibodies.
- This embodiment of the invention is a chimeric polypeptide in which all or a portion of the ⁇ -terminal half of the gp41 ectodomain of HIN-l Env is replaced by the corresponding region of HIV-2.
- the use of the closely related HJV-2 motif is expected to better mimic the authentic HIN-l Env trimer.
- HIN-l genetic subtypes are divided into clades.
- Fig. 10 shows an amino acid sequence alignment of HJN-1 Env protein that includes clades A-G. The consensus sequence shown in Fig. 10 shows the relatedness of the Env proteins of the different clades.
- Embodiments of the invention include chimeric polypeptides, wherein the non-HIN segment extends from amino acid Arg-571 (A at position 571) of the consensus sequence to Ser-660 (S at position 660) of the consensus sequence. SIV genetic subtypes are shown in Fig. 11 wherein the reference sequence is represented by the BK28 sequence.
- Embodiments of the invention include chimeric polypeptides, wherein the SJV segment extends from amino acid Gly-528 (G at position 528) of the reference sequence to Cys-613 (C at position 613) of the reference sequence.
- Embodiments also include chimeric polypeptides, wherein the non-HJV segment is, for example, at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64 ,65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, or 85 amino acids in length so long as the corresponding region of SIV promotes trimerization and maintains targets of neutralizing antibodies.
- the non-HJV segment is, for example, at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23,
- Env modifications aimed at improving the elicitation of neutralization antibodies include deletions of variable loops to expose underlying conserved epitopes, mutations that enhance proteolytic cleavage and introduce disulfide bonds capable of stabilizing gpl20- gp41 complexes, chemical coupling of Env and CD4 to stabilize the CD-4-induced conformation, deletion of the gp41 fusion peptide and the interhelical region to stabilize the pre-receptor-activated conformation, and hyperglycosylation to focus the humoral immune response toward known broadly neutralizing epitopes.
- Embodiments also include polynucleotides or nucleic acids that encode the chimeric polypeptides described herein.
- the polynucleotides of the invention can be made by recombinant methods, can be made synthetically, can be replicated by enzymes in in vitro (e.g., PCR) or in vivo systems (e.g., by suitable host cells, when inserted into a vector appropriate for replication within the host cells), or can be made by a combination of methods.
- the polynucleotides of the invention can include DNA and its RNA counterpart.
- nucleic acid As used herein, “nucleic acid”, “nucleic acid molecule”, “oligonucleotide” and “polynucleotide” include DNA and RNA and chemical derivatives thereof, including RNA and DNA molecules having a radioactive isotope or a chemical adduct such as a fluorophore, chromophore or biotin (which can be referred to as a "label").
- the RNA counterpart of a DNA is a polymer of ribonucleotide units, wherein the nucleotide sequence can be depicted as having the base U (uracil) at sites within a molecule where DNA has the base T (thymidine).
- Isolated nucleic acid molecules or polynucleotides can be purified from a natural source or can be made recombinantly.
- Polynucleotides referred to herein as "isolated” are polynucleotides purified to a state beyond that in which they exist in cells. They include polynucleotides obtained by methods described herein, similar methods or other suitable methods, and also include essentially pure polynucleotides produced by chemical synthesis or by combinations of biological and chemical methods, and recombinant polynucleotides that have been isolated.
- isolated as used herein for nucleic acid molecules, indicates that the molecule in question exists in a physical milieu distinct from that in which it occurs in nature.
- an isolated polynucleotide may be substantially isolated with respect to the complex cellular milieu in which it naturally occurs, and may even be purified essentially to homogeneity, for example as determined by agarose or polyacrylamide gel electrophoresis or by A 26 Q/A 28O measurements, but may also have further cofactors or molecular stabilizers (for instance, buffers or salts) added.
- the invention further comprises the chimeric polypeptides encoded by the isolated nucleic acid molecules of the invention.
- variants have silent substitutions, additions and deletions that do not alter the properties and activities of the chimeric protein.
- variants can also be modified polypeptides in which one or more amino acid residues are modified, and mutants comprising one or more modified residues. Proteins and polypeptides described herein can be assessed for their solubility and ability to trimerize using assays described herein.
- Cells that express such a chimeric polypeptide or a variant thereof can be made and maintained in culture, under conditions suitable for expression, to produce protein for isolation. These cells can be prokaryotic or eukaryotic.
- prokaryotic cells that can be used for expression include Escherichia coli, Bacillus subtilis and other bacteria.
- eukaryotic cells that can be used for expression include yeasts such as Saccharomyces cerevisiae, Schizosaccharomyces pombe, Pichia pastoris and other lower eukaryotic cells, and cells of higher eukaryotes such as those from insects and mammals.
- Suitable cells of mammalian origin include primary cells, and cell lines such as CHO, BS- C-l, HeLa, 3T3, BHK, COS, 293, and Jurkat cells.
- Suitable cells of insect origin include primary cells, and cell lines such as Sf9 and High five cells (see, e.g., Ausubel, F.M. et al., eds., Current Protocols in Molecular Biology, Greene Publishing Associates and John Wiley & Sons Inc., containing Supplements up through 1998).
- host cells that produce a recombinant chimeric polypeptide, variant, or portions thereof can be made as follows.
- a gene encoding a chimeric polypeptide described herein can be inserted into a nucleic acid vector, e.g., a DNA vector, such as a plasmid, virus or other suitable replicon (including vectors suitable for use in gene therapy, such as those derived from vaccinia virus or others, can be present in a single copy or multiple copies, or the gene can be integrated in a host cell chromosome.
- a suitable replicon or integrated gene can contain all or part of the coding sequence for the polypeptide or variant, operably linked to one or more expression control regions whereby the coding sequence is under the control of transcription signals and linked to appropriate translation signals to permit translation.
- the vector can be introduced into cells by a method appropriate to the type of host cells (e.g., transformation, electroporation, infection).
- the host cells can be maintained under appropriate conditions (e.g., in the presence of inducer, normal growth conditions, etc.). Proteins or polypeptides thus produced can be recovered (e.g., from the cells, the periplasmic space, culture medium) using suitable techniques.
- the invention also relates to isolated proteins or polypeptides encoded by nucleic acids of the present invention. Isolated proteins can be purified from a natural source or can be made recombinantly.
- Proteins or polypeptides referred to herein as “isolated” are proteins or polypeptides purified to a state beyond that in which they exist in cells and include proteins or polypeptides obtained by methods described herein, similar methods or other suitable methods, and also include essentially pure proteins or polypeptides, proteins or polypeptides produced by chemical synthesis or by combinations of biological and chemical methods, and recombinant proteins or polypeptides which are isolated.
- isolated indicates that the polypeptide in question exists in a physical milieu distinct from the cell in which its biosynthesis occurs.
- an isolated HIV/SIN chimeric polypeptide may be purified essentially to homogeneity, for example as determined by PAGE or column chromatography (for example, HPLC), but may also have further cofactors or molecular stabilizers added to the purified protein to enhance activity.
- proteins or polypeptides are isolated to a state at least about 75% pure; more preferably at least about 85% pure, and still more preferably at least about 95% pure, as determined by Coomassie blue staining of proteins on SDS-polyacrylamide gels. Chimeric or fusion polypeptides can be produced by a variety of methods.
- a chimeric polypeptide can be produced by the insertion of an env gene or portion thereof into a suitable expression vector. The resulting construct can be introduced into a suitable host cell for expression. Upon expression, chimeric polypeptide can be purified from a cell lysate or supernatant by means of a suitable affinity matrix (see e.g., Current Protocols in Molecular Biology (Ausubel, F.M. et al., eds., Vol. 2, pp. 16.4.1-16.7.8, containing supplements up through Supplement 44, 1998). Polypeptides of the invention can be recovered and purified from cell cultures by well-known methods.
- the recombinant protein can be purified by ammonium sulfate precipitation, heparin-Sepharose affinity chromatography, gel filtration chromatography and/or sucrose gradient ultracentrifugation using standard techniques. Further methods that can be used for purification of the polypeptide include ethanol precipitation, acid extraction, ion exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxylapatite chromatography and high performance liquid chromatography. Known methods for refolding protein can be used to regenerate active conformation if the polypeptide is denatured during isolation or purification.
- a method of the invention includes administering an effective amount of a chimeric polypeptide to a subject, thereby producing an immune response sufficient for ameliorating, preventing or inhibiting virus infection in the subject, h another embodiment, a method of the invention includes administering to a subject an effective amount of a polynucleotide encoding a chimeric polypeptide of the invention. In yet another embodiment, a method of the invention includes administering an effective amount of an antibody that binds to a chimeric polypeptide to a subject, thereby ameliorating, preventing or inhibiting virus infection in the subject.
- an immune response also can be produced.
- the immune response will likely be humoral in nature, although administering a polynucleotide encoding a chimeric polypeptide may induce a CTL response. It is also understood that the methods of the invention can also be used in combination with other viral therapies, as appropriate.
- the "effective amount" will be sufficient to inhibit, prevent, or ameliorate a viral infection in a subject, or will be sufficient to produce an immune response in a subject.
- an effective amount of chimeric polypeptide can be that which elicits an immune response to the polypeptide or a virus upon which the coat protein is based.
- An effective amount administered to a subject already infected with the virus can also be that which decreases viral load, or increases the number of CD4+ cells.
- An effective amount can be that which inhibits transmission of the virus from an infected subject to another (uninfected or infected).
- a CTL response to the chimeric polypeptide can be produced against a virus that contains the corresponding coat polypeptide sequence.
- the present invention also provides pharmaceutical formulations comprising the disclosed chimeric polypeptides, polynucleotides, and antibodies.
- the compositions administered to a subject will therefore be in a "pharmaceutically acceptable” or “physiologically acceptable” formulation.
- pharmaceutically acceptable and “physiologically acceptable” refer to carriers, diluents, excipients, and the like that can be administered to a subject, preferably without excessive adverse side effects (e.g., nausea, headaches, etc.).
- Such preparations for administration include sterile aqueous or non-aqueous solutions, suspensions, and emulsions.
- nonaqueous solvents are propylene glycol, polyethylene glycol, vegetable oils, such as olive oil, and injectable organic esters, such as ethyl oleate.
- Aqueous carriers include water, alcoholic/aqueous solutions, emulsions, or suspensions, including saline and buffered media.
- Vehicles include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's or fixed oils.
- Intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishers (such as those based on Ringer's dextrose), and the like.
- Preservatives and other additives may also be present, such as, for example, antimicrobial, anti-oxidants, chelating agents, and inert gases and the like.
- Various pharmaceutical formulations appropriate for administration to a subject known in the art are applicable in the methods of the invention (e.g., Remington's Pharmaceutical Sciences, 18th ed., Mack Publishing Co., Easton, PA 1990; and The Merck Index 12th ed., Merck Publishing Group, Whitehouse, NJ, 1996).
- Controlling the duration of action or controlled delivery of an administered composition can be achieved by incorporating the composition into particles or a polymeric substance, such as polyesters, polyamine acids, hydrogel, polyvinyl pyrrolidone, ethylene- vinylacetate, methylcellulose, carboxymethylcellulose, protamine sulfate or lactide/glycolide copolymers, polylactide/glycolide copolymers, or ethylenevinylacetate copolymers.
- the rate of release of the composition may be controlled by altering the concentration or composition of such macromolecules.
- Colloidal dispersion systems include macromolecule complexes, nano-capsules, microspheres, beads, and lipid-based systems, including oil-in-water emulsions, micelles, mixed micelles, and liposomes.
- the compositions administered by a method of the present invention can be administered parenterally by injection, by gradual perfusion over time, or by bolus administration (for example, in the case of passive protection against HJV infection resulting from a needlestick injury) or by a microfabricated implantable device.
- the composition can be administered via inhalation, intravenously, intraperitoneally, intramuscularly, subcutaneously, intracavity (e.g., vaginal or anal), transdermally, topically, or intravascularly.
- compositions can be administered in multiple doses.
- the doses or "effective amount" needed for treating, inhibiting, or preventing viral infection or transmission, or for inducing an immune response preferably will be sufficient to ameliorate some or all of the symptoms of the infection, although preventing progression or worsening of the infection also is a satisfactory outcome for many viral infections, including HI .
- An effective amount can readily be determined by those skilled in the art (see, for example, Ansel et al., Pharmaceutical Drug Delivery Systems, 5th ed., Lea and Febiger, 1990, Gennaro Ed.).
- An adjuvant may be included in the pharmaceutical composition to augment the immune response to a chimeric polypeptide of the invention.
- adjuvants include, but are not limited to, muramyl dipeptide, aluminum hydroxide, saponin, polyanions, amphipatic substances, bacillus Calmette-Guerin (BCG), endotoxin lipopolysaccharides, keyhole limpet hemocyanin (KLH), interleukin-2 (IL-2), and granulocyte-macrophage colony-stimulating factor (GM-CSF).
- BCG Bacillus Calmette-Guerin
- KLH keyhole limpet hemocyanin
- IL-2 interleukin-2
- GM-CSF granulocyte-macrophage colony-stimulating factor
- Such chimeric polypeptides also are useful for identifying agents that modulate the intramolecular interaction/binding of the virus coat polypeptide sequence to the co-receptor or receptor.
- methods for identifying an agent that modulates binding between a virus and a virus co-receptor or receptor and methods for identifying an agent that modulates binding between a virus and a virus co-receptor or receptor.
- a method of the invention includes contacting a chimeric polypeptide with a co-receptor or receptor polypeptide under conditions allowing the chimeric polypeptide and the co-receptor or receptor polypeptide to bind, in the presence and absence of a test agent, and detecting binding in the presence and absence of the test agent.
- a decreased amount of binding in the presence of the test agent thereby identifies an agent that inhibits interaction/binding between the virus and the virus co-receptor or receptor.
- Increased binding in the presence of the test agent thereby identifies an agent that stimulates interaction binding between the virus and the virus co-receptor or receptor.
- Candidate agents include antibodies, antivirals, a co-receptor or receptor polypeptide sequence, peptidomimetics or active fragments thereof.
- Candidate agents also encompass numerous chemical classes, including organic molecules, like small organic compounds having a molecular weight of more than 50 and less than about 2,500 daltons.
- Candidate agents comprise functional groups necessary for structural interaction with proteins, particularly hydrogen bonding, and typically include at least an amine, carbonyl, hydroxyl, or carboxyl group, preferably at least two of the functional chemical groups.
- the candidate agents often comprise cyclical carbon or heterocyclic structures, and/or aromatic or polyaromatic structures substituted with one or more of the above functional groups.
- Candidate agents are also found among biomolecules, including, but not limited to, peptides, saccharides, fatty acids steroids, purines, pyrimidines, derivatives, structural analogs, or combinations thereof.
- Candidate agents are obtained from a wide variety of sources, including libraries of synthetic or natural compounds. For example, numerous means are available for random and directed synthesis of a wide variety of organic compounds and biomolecules, including expression of randomized oligonucleotides and oligopeptides. Alternatively, libraries of natural compounds in the form of bacterial, fungal, plant, and animal extracts are available or readily produced.
- natural or synthetically produced libraries and compounds are readily modified through conventional chemical, physical, and biochemical means, and may be used to produce combinatorial libraries.
- Known pharmacological agents may be subjected to directed or random chemical modifications, such as acylation, alkylation, esterification, amidification, etc., to produce structural analogs.
- the method detects binding, one or more of the molecules may be joined to a label, where the label can directly or indirectly provide a detectable signal.
- Various labels include radioisotopes, fluorescers, chemiluminescers, enzymes, specific binding molecules, particles, e.g. magnetic particles, and the like.
- Specific binding molecules include pairs, such as biotin and streptavidin, digoxin and antidigoxin, etc.
- the complementary member would normally be labeled with a molecule that provides for detection, in accordance with known procedures.
- reagents may be included in the assay. These include reagents, like salts, neutral proteins, e.g. albumin, detergents, etc., that are used to facilitate optimal protein-protein binding and/or reduce non-specific or background interactions. Reagents that improve the efficiency of the assay, such as protease inhibitors, nuclease inhibitors, anti-microbial agents, etc., may be used.
- the components are combined in any order that provides for the requisite binding. Incubations are performed at any suitable temperature, typically between 4 ° C and 40 ° C.
- a method of the invention includes contacting a cell susceptible to virus infection with an infectious virus particle in the presence and absence of a test agent, and determining whether the test agent inhibits virus binding or infection of the cell, thereby identifying an agent that inhibits virus infection.
- the test agent is added before or after contacting the cell with the infectious virus particle.
- the method also can be performed in any suitable animal, such as a non-human primate.
- the STEM-measured masses of 622 molecules within fraction 51 showed a broad and asymmetrical distribution with a mean value of 444 kDa (3.71 protomers) and a standard deviation of 107 kDa (Fig. 1C; Table 1), consistent with the presence of at least some oligomers with more than three protomers. Given that functional native Env is a trimer, complexes of more than three protomers are presumably nonspecifically associated; they are referred to hereafter as aggregates.
- the masses of 434 molecules within fraction 56 yielded a relatively symmetrical distribution with a mean value of 280 kDa (2.34 protomers) and a standard deviation of 64 kDa (Fig. ID; Table 1), consistent with a predominantly dimeric structure.
- HIV-1 j R - FL gpl40 forms a heterogeneous range of oligomeric species including dimers, trimers, and higher-mass aggregates.
- H-S.N 51-53 403 3.36 a Number average. h
- the mass of one protomer was estimated by summing the protein mass deduced from the amino acid sequence and the carbohydrate mass (see Example 1).
- Sedimentation coefficient distribution [c(s)J analysis of sedimentation velocity data was performed to obtain model-free information about the oligomeric state of the molecules in solution.
- the distributions of HJN-1 JR. F gpl40 fractions 51, 54, and 56 showed significant separation (Fig. 2A), consistent with differences in protomer number.
- Sedimentation equilibrium unlike gel filtration, allows shape-independent mass determinations for Env (Center, R. J. et al. 2000 J Virol 74:4448-4455).
- the data sets for sedimentation equilibrium absorbance versus radial position (Fig. 2B) were analyzed by global nonlinear regression. The masses determined by this analysis, and the calculated numbers of protomers (in parentheses) are shown in Table 2.
- HIV-IA D A 51 424.2 3.86 53 344.8 3.13 56 305.0 2.77 59 253.3 2.30
- SJN Mac32H gpl40 resolved as a symmetrical and relatively sharp peak with elution mainly between 52 and 59 ml (Fig. 3 A, open circles). HIV-IA D A gpl40 resolved as a broader and asymmetrical peak between 48 and 61 ml (Fig. 3 A, solid squares), indicating greater size heterogeneity.
- Blue Native (BN)- PAGE has recently been successfully used to analyze gpl40 (Sch ⁇ lke, N. et al. 2002 J Virol 76:7760-7776) and has proved more effective than EGS cross-linking for revealing the oligomeric profile of HIV-IA D A gpl40.
- variable domains 1 and 2 reduces dimer formation and promotes aggregation
- the second variable domain (V2) can mediate dimer formation between recombinant gpl20 subunits (Center, R. J. et al. 2000 J Virol 74:4448-4455).
- HIV-IJ R The second variable domain (V2) can mediate dimer formation between recombinant gpl20 subunits
- Fractions 52 to 56 contained what appeared to be one major EGS-cross- linked species (Fig. 5B) that was shown by sedimentation equilibrium results (Table 2) to be trimeric (range, 3.47 to 3.04 protomers for fractions 52 to 55). No H-S EGS-cross- linked dimers were detected.
- BN-PAGE (4 to 12% polyacrylamide) of a pool of fractions 50 to 54 confirmed the presence of one predominant species.
- a faint band below the major band (migrating to a position between the 440- and 220-kDa markers) indicated the presence of trace amounts of dimer.
- Fractions 45 to 51 contained a very slowly migrating EGS-cross-linked species with high mass (for example, for fraction 50 the average number of protomers was 4.55), indicating that as with HIV-I JR - FL gpl40, some H- S gpl40 was aggregated.
- the sedimentation velocity profiles of H-S fractions 50 to 55 displayed considerable overlap (Fig. 5D).
- the profiles for fractions 50 and 51 showed some skewing to a higher sedimentation coefficient. Together, these observations are consistent with the formation of trimers and some aggregates.
- Scanning transmission electron microscopy (STEM) analysis of 420 individual molecules for a pool of fractions 51 and 52 (Fig.
- H-S. ⁇ gpl40 amino acids S590 to K674 to create the chimera H-S. ⁇ .
- the increase in binding of the CD4-induced monoclonal antibody 17b in the presence of CD4 demonstrates that both the CD4 binding site and the conformationally sensitive 17b epitope are intact in H-S. ⁇ gpl40 (Fig. 6A, lower inset). This indicates that the presence of the SIV gp41 -derived sequence has not compromised the folding or function of H-S. ⁇ gpl40.
- H-S. ⁇ gpl40 revealed a sharper and more symmetrical gel filtration peak than did HIV-IJR.
- F L gpl40 (Fig. 6 A, open circles compared to solid squares).
- the EGS-cross-linking profile indicated the presence of one predominant oligomeric species (Fig. 6B). This finding was supported by the substantial overlap of the sedimentation velocity profiles for fractions 50 to 55 (Fig. 6C). The sedimentation equilibrium results (Table 2) indicated that this species was a trimer, with a range for fractions 51 to 55 of 3.47 to 2.81 protomers.
- B ⁇ -PAGE (4 to 12% polyacrylamide) of a pool of fractions 50 to 54 revealed the presence of a single major band consistent with a mainly trimeric structure. A faint band below the major band (migrating to a position between the 440- and 220-kDa markers) indicated the presence of a small amount of dimer.
- the N-terminal sequence of the SIV CP - M A C gp41 replaces the corresponding HIV-1 93M W 965 gp41 sequence (amino acids A501 to M584).
- the gel filtration peak for HIV-1 MW965 gpl40 is sharper and more symmetrical than that of JR .F L gpl40 (Fig. 7A compared to Fig. 1 A, solid squares and solid line in both), indicating less oligomeric heterogeneity.
- the EGS cross-linking profile which revealed the presence of two oligomeric species (Fig.
- HIN-l The greater level of oligomeric heterogeneity of HIN-l in comparison to SIN was also shown for molecules expressed in the absence of complex-type oligosaccharides.
- the gpl40 chimeras comprising either all or just the ⁇ -terminal half of the SIN gp41 segment in an HIN-lj R- FL background showed less aggregate formation than did unaltered HIN-l g ⁇ l40.
- the fact that the reverse chimera (SIN gpl20, HIN-lj R-FL gp41) also showed less aggregate formation indicates that the HIN-l gp41 segment does not directly induce aggregation. If aggregates form from the nontrimeric pool of molecules, reducing this pool by increasing the efficiency of trimerization (by replacing the HIN-l oligomerization domain with that of SIN) may concomitantly reduce aggregate formation.
- H-S. ⁇ gpl40 This weaker affinity between helices in H-S. ⁇ gpl40 may therefore promote gpl40 trimerization and reduce the pool of normative gpl40 available for aggregate formation.
- interaction between the ⁇ - and C-terminal helices may directly result in aggregation.
- the potentially advantageous structural properties of the H-S. ⁇ chimera are fortuitous, since this construct includes several broadly neutralizing HJV-1 epitopes in the C-terminal segment of gp41 (Muster, T. et al. 1993 J Virol 67:6642-6647; Stiegler, G. et al. 2001 AIDS Res Hum Retrovir 17:1757-1765; Zwick, M.
- the non-HJN-1 segments of the H- S. ⁇ chimeras includes the fusion peptide and the ⁇ -terminal alpha-helical regions, which have been found to be poorly immunogenic (Binley, J. M. et al. 1996 AIDS Res Hum Retrovir 12:911-924; Earl, P. L. et al.
- Env modifications aiming at improved elicitation of neutralizing antibodies include deletions of variable loops to expose underlying conserved epitopes (Barnett, S. W. et al. 2001 J Virol 75:5526-5540; Cherpelis, S. et al. 2001 J Virol 75:1547-1550; Kim, Y. B. et al. 2003 Virology 305:124-137; Lu, S. et al. 1998 AIDS Res Hum Retrovir 14:151- 155; Sanders, R. W. et al.
- the recombinant vaccinia virus vBD5 (Doranz, B. J. et al. 1999 J Virol 73:10346-10358) was used to express gpl40 derived from the HIN-lj R- FL (GenBank accession number U63632).
- Recombinant vaccinia viruses expressing HIV-1 gpl40 with deletion mutations and HIN-1/SJN gpl40 chimeras were produced by standard recombinant techniques using the HJV J -FL Env-encoding plasmid pCB28 (Broder, C. C. and E. A.
- HIV-IJR.FL or SINcp-MAC Env sequence with the initial methionine of the signal peptide as 1.
- gpl40 expression was under the control of a synthetic early-late vaccinia virus promoter (Chakrabarti, S. et al. 1997 BioTechniques 23:1094-1097).
- BS-C-1 cells an African green monkey kidney cell line
- Env generally comprised 90% of the total protein in the fractions analyzed, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Coomassie staining. Similar methods were used to express and purify unmodified and chimeric gpl40 derived from clade C 93MW965 (GenBank accession number U08455). Electrophoresis, immunoblotting and chemical cross-Unking. Individual gel filtration fractions were subjected to SDS-PAGE (8% polyacrylamide) under reducing conditions and transferred to nitrocellulose membranes.
- SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis
- Cells were loaded with volumes of 120 to 135 ⁇ l of sample and measured at either 230 or 280 nm in an optical density range of approximately 0.2 to 0.4 absorbance unit (AU). Absorbance- versus-radial-position step scanning data at radial increments of 0.001 cm with 20 repeats were obtained at 10°C using three different rotor speeds between 5,000 and 9,000 rpm for each sample. A global nonlinear regression analysis was performed using the data analysis software package provided by Beckman-Coulter instruments (version 4.0 and Microcal version 4.1). The partial specific volume for each gpl40 species was calculated from the amino acid sequence and an estimated partial specific volume of 0.622 for the carbohydrate component based on an analysis of glycoproteins (Lewis, M. S. and R.
- Annular dark-field images were acquired digitally using an electron dose of approximately 10 3 e/nm 2 and an acquisition time of 100 s. Images were processed and quantified using the IMAGE program (available on the World Wide Web at rsb.info.nih.gov/nih-image/). Mass values were calibrated using tobacco mosaic virus particles contained in the same field as gpl40.
- EXAMPLE 2 We compared the antigenicity and immunogenicity of the JR-FL H-S.N gpl40 trimers with HIV JR-FL gpl20 monomers.
- Guinea pigs were immunized three times with 40 ⁇ g of protein in a Ribi-type adjuvant (monophosphoryl-lipid A plus trehalose dicorynomycolate emulsion) at 4-week intervals.
- Ribi adjuvant was used as it is predicted to maximize the presentation of conformational epitopes, and is suitable for use in small animals.
- the most potent broadly neutralizing monoclonal antibodies identified to date are known to target conformational epitopes.
- Blood samples were taken before the first immunization (to control for non-specific antibody binding and neutralization) and 2 weeks after the third immunization, when antibody levels are expected to peak. Antibody binding data for individual animals are shown in Fig. 8 A.
- the chimeric gpl40 immunogen gave a much higher antibody response than gpl20 when measured against gpl20 (Fig. 8 A) or gpl40 trimers (Fig. 8B).
- Pre-immune sera showed no significant binding to either chimeric gpl40 or gpl20.
- Neutralization titers were determined as the reciprocal of the serum dilution that neutralized 50% of the homologous virus JR-FL or a heterologous clade B virus MN. JR-FL is difficult to neutralize and titers of 1:20 or higher are considered significant.
- chimeric gpl40 trimers is a superior immunogen compared to monomeric gpl20, both quantitatively in terms of binding titer and qualitatively in terms of viral neutralization.
- EXAMPLE 3 We envision env trimers as providing a scaffold for making additional modifications to improve immunogenicity. Deletion of the second variable domain of gpl20 has been proposed to expose coreceptor binding domains which may be good targets for cross-strain neutralization due to their conserved function and therefore presumed conserved structure (Barnett, S. W. et al. 2001 J Virol 75:5526-5540).
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Abstract
The present invention relates to compositions and methods for making soluble, recombinant human immunodeficiency virus (HIV) envelope (Env) polypeptides that promote trimerization and maintain targets of neutralizing antibodies in which all or a portion of the N-terminal half of the gp41 ectodomain is replaced by the corresponding region of simian immunodeficiency virus (SIV). The invention relates as well to the nucleic acid sequences encoding the chimeric polypeptides, recombinant vectors carrying the sequences, recombinant host cells including either the sequences or vectors, and recombinant polypeptides. The invention further includes methods for using the isolated, recombinant polypeptides in vaccines, assays, and for use in preventive and therapeutic applications.
Description
NIH285.001NPC PATENT HIV/SIN EΝN CHIMERAS THAT PROMOTE TRIMERIZATIOΝ AND MAINTAIN TARGETS OF NEUTRALIZING ANTIBODIES Related Applications This application claims the benefit of US Provisional Application No. 60/510,952, filed October 10, 2003, which is hereby incorporated by reference in its entirety. Background of the Invention The human immunodeficiency virus type 1 (HIN-l) envelope protein (Env) is synthesized as a precursor molecule, g l60, which is processed via the same cellular pathway as other cell surface integral membrane proteins. Major processing steps in the endoplasmic reticulum include extensive glycosylation, disulfide bond formation, and oligomerization (Earl, P. L. et al. 1991 J Virol 65:2047-2055). Cleavage in the Golgi complex produces gpl20 and the membrane-anchored gp41, which remain associated by noncovalent interactions. Complexes of gpl20 and gp41 are transported to the cell surface, where incorporation into budding virions occurs. The Env complex is indispensable for viral infectivity; g l20 interacts with the target cell receptors CD4 and one of the chemokine receptors (most often CCR5 or CXCR4), triggering conformational changes that culminate in gp41 fusion peptide insertion into the target cell membrane and the fusion of this membrane with that of the infected cell or virion (Eckert, D. M. and P. S. Kim 2001 Annu Rev Biochem 70:777-810). Env is the only viral protein to protrude beyond the virion membrane, and it is the major viral target of the host humoral immune response. The oligomeric structure of Env modulates antigenicity, presumably by reducing the exposure of epitopes close to contact sites between protomers and/or by directly altering epitope conformation. The ability of antibody to neutralize virus is better predicted by a capacity to bind to oligomeric Env than to monomeric Env (Fouts, T. R. et al. 1997 J Virol 71:2779-2785; Fouts, T. R. et al. 1998 AIDS Res Hum Retrovir 14:591-597; Parren, P. W. H. I. et al. 1998 J Virol 72:3512-3519). Because virion-associated HIN-l Env is trimeric (Center, R. J. et al. 2002 J Virol 76: 7863- 7867), it would be desirable for an Env immunogen designed to elicit neutralizing antibodies to also' have a trimeric structure. To obtain soluble Env oligomers for testing as immunogens, recombinant techniques have been employed to express Env lacking the transmembrane domain and cytoplasrnic tail (gpl40). Since cleavage at the gpl20-gp41 junction causes the oligomeric contacts between protomers to become labile, the cleavage
sites of most gpl40s studied are inactivated by mutagenesis. Uncleaved gρl40 has been variously reported to form dimers and tetramers (Earl, P. L. et al. 1994 J Virol 68:3015- 3026), trimers and dimers (Chakrabarti, B. K. et al. 2002 J Virol 76:5357-5368; Staropoli, I. et al. 2000 JBiol Chem 275:35137-35145), dimers, trimers, and tetramers (Schύlke, N. et al. 2002 J Virol 76:7760-7776), and mainly trimers (Zhang, C. W.-H. et al. 2001 J Biol Chem 276:39577-39585) and to largely fail to form stable oligomers (Yang, X. et al. 2000 J Virol 74:5716-5725; Yang, X. et al. 2000 J Virol 74:4746-4754). Cleaved gpl40 with engineered disulfide linkages between the gpl20 and gp41 subunits was reported to form mainly monomers or oligomers with reduced stability (Binley, J. M. et al. 2000 J Virol 74:627-643; Schϋlke, N. et al. 2002 J Virol 76:7760-7776). Segue to the Invention In the present study, we used biochemical and biophysical methods to analyze uncleaved HIN-l gρl40 proteins and confirmed the formation of nontrimeric species including dimers and aggregates (defined here as any oligomer of more than three protomers). We had previously found that simian immunodeficiency virus (SIN) gpl40 formed a relatively homogeneous population of trimers (Center, R. J. et al. 2001 Proc Natl Acad Sci USA 98:14877-14882). Through the use of HiN-1/SIN gpl40 chimeras, we show here that replacement of the Ν-terminal half of the gp41 segment of HIN-l gpl40 with the corresponding region of SIN is sufficient to promote efficient trimerization. Summary of the Invention The present invention relates to compositions and methods for making soluble, recombinant human immunodeficiency virus (HIN) envelope (Env) polypeptides that promote trimerization and maintain targets of neutralizing antibodies in which all or a portion of the Ν-terminal half of the gp41 ectodomain is replaced by the corresponding region of simian immunodeficiency virus (SIN). The invention relates as well to the nucleic acid sequences encoding the chimeric polypeptides, recombinant vectors carrying the sequences, recombinant host cells including either the sequences or vectors, and recombinant polypeptides. The invention further includes methods for using the isolated, recombinant polypeptides in vaccines, assays, and for use in preventive and therapeutic applications. Brief Description of the Drawings Fig. 1. Analysis of the oligomeric structure of HIN-l JR-FL gpl40. Lentil lectin affinity-purified gpl40 was passed through a column of Superdex 200, and individual gel
filtration fractions were analyzed using biochemical and biophysical methods. (A) Aliquots of gel filtration fractions were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) (8% polyacrylamide) and immunoblotted with an Env- specific antiserum and iodinated protein A. gpl40 was quantified by phosphor screen autoradiography, and the results were plotted as a percentage of the total g l40-specific signal. The inset shows SDS-PAGE (10% polyacrylamide) of a pool of fractions 51 to 56 as revealed by Coomassie blue staining. (B) Aliquots of fractions were treated with the cross-linker ethylene glycol bis(succinimidylsuccinate) (EGS) (5 mM final concentration), analyzed by SDS-PAGE (5% polyacrylamide) and immunoblotted as above. The bar indicates the electrophoretic mobility of a 250-kDa marker protein. (C) Scanning transmission electron microscopy (STEM)-derived mass measurements of 622 individual oligomers within fraction 51. (D) STEM-derived mass measurements of 434 individual oligomers within fraction 56. Arrowheads in panels C and D indicate the expected mass of 360 kDa for trimeric gpl40. Fig. 2. Sedimentation velocity and equilibrium analysis of HIN-l JR-F gpl40. (A)
Differential sedimentation coefficient distributions, c(s), calculated from sedimentation velocity experiments for fractions 56 (solid line), 51 (dashed line), and 54 (dash-dot line). (B) Sedimentation equilibrium concentration profiles of gel filtration fractions 51 (squares), 52 (circles), 53 (triangles), and 54 (diamonds). Solid lines show the best-fit distributions after global modeling of data obtained at three different rotor speeds. For clarity, only data obtained at 6,000 rpm are shown. All plots depicted were derived from measurements at 280 nm except for the fraction 51 plot, which was derived from measurement at 230 nm. Residuals of the fitted lines to the experimental data are displayed in the lower panel. OD, optical density. Fig. 3. Gel filtration analysis of HIN-l ADA and SIN ac32H gpl40 expressed in CHO-
Lec3.2.8.1 cells. (A) Aliquots of gel filtration fractions were subjected to SDS-PAGE (8% polyacrylamide) and immunoblotted with an Env-specific antiserum and iodinated protein A. gpl40 was quantified by phosphor screen autoradiography, and the results were plotted as a percentage of the total gpl40-specific signal for HIN-l ADA (solid squares) and SINMac32H (open circles). The inset shows SDS-PAGE (10% polyacrylamide) of a pool of fractions 51, 53, 56, and 59 of HIV-IADA gpl40 as revealed by Coomassie blue staining. (B) Blue Native (BN)-PAGE (4 to 12% polyacrylamide) of the indicated HIV-IADA gpHO
„,_ „™- n _
WO 2003 0 fractions. The 440/220- and 670/335-kDa markers were the dimers and monomers of ferritin and thyroglobulin, respectively. Fig. 4. Gel filtration analysis of variable-loop deletion mutants of HIN-l JR-FL gpl40. (A) Aliquots of gel filtration fractions were subjected to SDS-PAGE (8% polyacrylamide) and immunoblotted with an Env-specifϊc antiserum and iodinated protein A. gpl40 was quantified by phosphor screen autoradiography, and the results were plotted as a percentage of the total gpl40-specific signal for HIN-l jR-F1 N2 (open circles) and HIV-1JR-FLΔV1/2 (open squares). Intact HIN-l JR-FL gpl40 (see Fig. 1) is also shown for comparison (closed squares). Aliquots of fractions of HIN-l JR-FLΔV 1/2 gpl40 (B) and HιN-ljR-FLΔN2 gρl40 (C) were treated with the cross-linker EGS (5 M final concentration) and analyzed by SDS-PAGE (5% polyacrylamide) and immunoblotted as above. The bars indicate the electrophoretic mobility of a 250-kDa marker protein. Fig. 5. Analysis of the oligomeric structure of HIN-1/SIN gpl40 chimeras. (A) Aliquots of gel filtration fractions were subjected to SDS-PAGE (8% polyacrylamide) and immunoblotted with an Env-specific antiserum (for H-S) or the monoclonal antibodies 36D5 (anti-SIV gpl20) (Edinger, A. L. et al. 2000 J Virol 74:7922-7935) and D50 (anti- HIN-1 gp41) (Earl, P. L. et al. 1997 J Virol 71:2674-2684) (for S-H) and iodinated protein A. gpl40 was quantified by phosphor screen autoradiography, and the results were plotted as a percentage of the total gpl40-specific signal for H-S (open diamonds) and S-H (open circles). Nonchimeric HIV-IJR.FL gpl40 (see Fig. 1) is also shown for comparison (solid squares). The inset shows BN-PAGE (4 to 12% polyacrylamide) of a pool of fractions 50 to 54 of H-S gpl40 revealed by Coomassie blue staining. The upper and lower bars show the positions of the 440- and 220-kDa marker proteins, respectively. Aliquots of fractions of H-S (B) and S-H (C) were treated with the cross-linker EGS (5 mM final concentration), analyzed by SDS-PAGE (5% polyacrylamide), and immunoblotted as above. The bars indicate the electrophoretic mobility of a 250-kDa marker protein. (D) Differential sedimentation coefficient distributions, c(s), calculated from sedimentation velocity experiments for the indicated fractions of H-S. (E) STEM-derived mass measurements of 420 individual oligomers within a pool of fractions 51 and 52 of H-S. The inset shows a montage of STEM images that displayed a triangular or trilobed morphology. OD, optical density. Bar, 40 nm. Fig. 6. Analysis of the oligomeric structure of the HIV-l/SIV chimera H-S.N. (A) Aliquots of gel filtration fractions were subjected to SDS-PAGE (8% polyacrylamide) and
immunoblotted with an Env-specific antiserum and iodinated protein A. gpl40 was quantified by phosphor screen autoradiography, and the results were plotted as a percentage of the total gpl40-sρecific signal for H-S.N (open circles). Nonchimeric HIN-l JR-FL gpHO (see Fig. 1) is also shown for comparison (solid squares). The upper inset shows BΝ- PAGE (4 to 12% polyacrylamide) of a pool of fractions 50 to 54 of H-S.Ν gpl40 revealed by Coomassie blue staining. The upper and lower bars show the positions of the 440- and 220-kDa marker proteins, respectively. The lower inset shows the effect of the presence (+) or absence (-) of an excess of soluble four-domain CD4 on the immunoprecipitation of radiolabeled H-S.Ν gpl40 by monoclonal antibody 17b. (B) Aliquots of gel filtration fractions of H-S.Ν gpl40 were treated with the cross-linker EGS (5 mM final concentration), analyzed by SDS-PAGE (5% polyacrylamide), and immunoblotted as above. The bar indicates the electrophoretic mobility of a 250-kDa-marker protein. (C) Differential sedimentation coefficient distributions, c(s), calculated from sedimentation velocity experiments for the indicated gel filtration fractions of H-S.Ν gpl40. (D) STEM- derived mass measurements of 265 individual oligomers within a pool of fractions 51 to 53 of H-S.Ν gpl40. (E) A montage of STEM images which displayed a triangular or trilobed morphology. OD, optical density. Bar, 40 nm. Fig. 7. Gel filtration analysis of gpl40 and the HIN-l/SIN H-S.Ν chimera derived from clade C HIV-1 MW965- (A) Aliquots of gel filtration fractions were subjected to SDS- PAGE (8% polyacrylamide) and immunoblotted with an Env-specific antiserum and iodinated protein A. g l40 was quantified by phosphor screen autoradiography, and the results were plotted as a percentage of the total gpL40-specific signal for HIV-1 3MW965 gpl40 (solid squares) and the clade C H-S.Ν chimera (open circles). Aliquots of fractions of HIN-l 93M 965 gpl40 (B) and the clade C H-S.Ν gpl40 (C) were treated with the cross- linker EGS (5 mM final concentration), analyzed by SDS-PAGE (5% polyacrylamide) and immunoblotted as above. The bars indicate the electrophoretic mobility of a 250-kDa marker protein. Fig 8. Comparison of the antigenicity and immunogenicity of JR-FL H-S.Ν gpl40 trimers with HIVJR.FL gpl20 monomers. (A) ELISA binding titers to gpl20, (B) ELISA binding titers to trimeric gpl40, (C) Neutralization titers to HJN-1 (JR-FL), and (D) Neutralization titers to HIN-l (MΝ). Fig. 9. Linear representation of the HIN-l Env glycoprotein.
Fig. 10. Amino acid sequence alignment of the HJN-1 Env protein (Louwagie J. et al. 1995 J Virol 69:263-271). — , identity with the consensus sequence; •, gap in aligned sequences; X, corrected frameshift; a space, premature stop codon. Isolates that belong to the same subtype (A to G) are grouped. *, cysteine residues; +, amino acids believed to be involved in gpl20-gp41 interaction; Λ, the leucine zipper motif; #, the CD4 binding site. Hypervariable domains (NI to N5) in the gpl20 molecule and an immunodominant domain (ID) in gp41 are delineated by solid lines. Fig. 11. Amino acid differences of the ΝC-MAC and CP-MAC env clones compared with BK28 (LaBranche, C. C. et al. 1994 J Virol 68:5509-5522; Erratum, 68:7665-7667). Predicted amino acid sequences are shown for selected regions of env for PCR-generated clones of ΝC-MAC-infected cells and two cellular clones of CP-MAC- infected cells and are compared with the parental BK28 sequence. Dot, amino acid identity; asterisk, in-frame stop codon; dash, a single nucleotide deletion. Regions corresponding to variable domains in external (SU) region are shown. Sequences shown include the carboxy-terminal half of SU (amino acids 301 to 527) and the entire transmembrane domain (TM) coding region. Detailed Description of the Preferred Embodiment The envelope proteins (Env) of human immunodeficiency virus type 1 (HJN-1) and simian immunodeficiency virus (SIN) form homo-oligomers in the endoplasmic reticulum. The oligomeric structure of Env is maintained, but is less stable, after cleavage in a Golgi compartment and transport to the surface of infected cells. Functional, virion-associated HIV-1 and SIN Env have an almost exclusively trimeric structure. In addition, a soluble form of SrV Env (gpl40) forms a nearly homogeneous population of trimers. Here, we describe the oligomeric structure of soluble, uncleaved HIV-1 gpl40 and modifications that promote a stable trimeric structure. Biochemical and biophysical analyses, including sedimentation equilibrium and scanning transmission electron microscopy, revealed that unmodified HJN-1 gpl40 purified as a heterogeneous range of oligomeric species, including dimers and aggregates. Deletion of the N2 domain alone or, especially, both the NI and N2 domains reduced di er formation but promoted aggregation rather than trimerization. Expressing gpl40 with mannose-only oligosaccharides did not eliminate heterogeneity. Replacement of the entire gp41 segment of HIN-l gpl40 or just the Ν- terminal half (85 amino acids) of this segment with the corresponding region of SIN was sufficient to confer efficient trimerization for gpl40 derived from clade B and C isolates.
Importantly, the relatively small segment of the HIN Env replaced by SIN sequences contains no known targets of neutralizing antibody. The soluble trimeric form of HJN-1 Env has application as an antigen and an immunogen. Chimeric HTV/SIV Env Polypeptides A linear representation of the HIN-l Env glycoprotein is shown in Fig. 9. The vertical arrow indicates the site of gpl60 cleavage to gpl20 and gp41. gpl40 is indicated as Env lacking the transmembrane domain and cytoplasrnic tail. In gpl20, cross hatched areas represent variable domains (NI to N5) and open boxes depict conserved sequences (CI to C5). In the gp41 ectodomain, several domains are indicated: the Ν-terminal fusion peptide, and the, two ectodomain helices (Ν- and C-helix). The membrane spanning domain is represented by a black box. In the gp41 cytoplasrnic domain, the Tyr-X-X-Leu (YXXL) endocytosis motif and two predicted helical domains (helix- 1 and -2) are shown. Amino acid numbers are indicated. Described herein are HIV/SIN Env chimeras that promote trimerization and maintain targets of neutralizing antibodies. One embodiment of the invention is a chimeric polypeptide in which all or a portion of the Ν-terminal half of the gp41 ectodomain of human immunodeficiency virus (HIN) envelope (Env) is replaced by the corresponding region of simian immunodeficiency virus (SJN). The use of the closely related SIN motif is expected to better mimic the authentic HIN Env trimer. The amount of non-HJN sequence can be increased or decreased to confer efficient trimerization while maintaining targets of neutralizing antibodies. For example, the non- HJN segment of the chimera, that is, the Ν-terminal half of the gp41 ectodomain, can extend from the fusion peptide to the interhelical region, so that the C-terminal half of the gp41 ectodomain is maintained to preserve targets of neutralizing antibodies. Alternatively, "spacer" sequences not naturally occurring in HIN or SIN can be added. HIN type 2 (HIV-2) is closely related to SIV, thus, described herein are HιN-1/HιN- 2 Env chimeras that promote trimerization and maintain targets of neutralizing antibodies. This embodiment of the invention is a chimeric polypeptide in which all or a portion of the Ν-terminal half of the gp41 ectodomain of HIN-l Env is replaced by the corresponding region of HIV-2. The use of the closely related HJV-2 motif is expected to better mimic the authentic HIN-l Env trimer. HIN-l genetic subtypes are divided into clades. Fig. 10 shows an amino acid sequence alignment of HJN-1 Env protein that includes clades A-G. The consensus
sequence shown in Fig. 10 shows the relatedness of the Env proteins of the different clades. Embodiments of the invention include chimeric polypeptides, wherein the non-HIN segment extends from amino acid Arg-571 (A at position 571) of the consensus sequence to Ser-660 (S at position 660) of the consensus sequence. SIV genetic subtypes are shown in Fig. 11 wherein the reference sequence is represented by the BK28 sequence. Embodiments of the invention include chimeric polypeptides, wherein the SJV segment extends from amino acid Gly-528 (G at position 528) of the reference sequence to Cys-613 (C at position 613) of the reference sequence. Embodiments also include chimeric polypeptides, wherein the non-HJV segment is, for example, at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64 ,65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, or 85 amino acids in length so long as the corresponding region of SIV promotes trimerization and maintains targets of neutralizing antibodies. Another aspect of the invention includes the use of the HIN/SJN Env chimeras described herein as molecular scaffolds wherein additional Env modifications aimed at improving elicitation of neutralizing antibodies are applied in a trimeric context. For example, Env modifications aimed at improving the elicitation of neutralization antibodies include deletions of variable loops to expose underlying conserved epitopes, mutations that enhance proteolytic cleavage and introduce disulfide bonds capable of stabilizing gpl20- gp41 complexes, chemical coupling of Env and CD4 to stabilize the CD-4-induced conformation, deletion of the gp41 fusion peptide and the interhelical region to stabilize the pre-receptor-activated conformation, and hyperglycosylation to focus the humoral immune response toward known broadly neutralizing epitopes.
Nucleic Acids that Encode Chimeric HIV/SIV Env Polypeptides Embodiments also include polynucleotides or nucleic acids that encode the chimeric polypeptides described herein. The polynucleotides of the invention can be made by recombinant methods, can be made synthetically, can be replicated by enzymes in in vitro (e.g., PCR) or in vivo systems (e.g., by suitable host cells, when inserted into a vector appropriate for replication within the host cells), or can be made by a combination of methods. The polynucleotides of the invention can include DNA and its RNA counterpart.
As used herein, "nucleic acid", "nucleic acid molecule", "oligonucleotide" and "polynucleotide" include DNA and RNA and chemical derivatives thereof, including RNA and DNA molecules having a radioactive isotope or a chemical adduct such as a fluorophore, chromophore or biotin (which can be referred to as a "label"). The RNA counterpart of a DNA is a polymer of ribonucleotide units, wherein the nucleotide sequence can be depicted as having the base U (uracil) at sites within a molecule where DNA has the base T (thymidine). Isolated nucleic acid molecules or polynucleotides can be purified from a natural source or can be made recombinantly. Polynucleotides referred to herein as "isolated" are polynucleotides purified to a state beyond that in which they exist in cells. They include polynucleotides obtained by methods described herein, similar methods or other suitable methods, and also include essentially pure polynucleotides produced by chemical synthesis or by combinations of biological and chemical methods, and recombinant polynucleotides that have been isolated. The term "isolated" as used herein for nucleic acid molecules, indicates that the molecule in question exists in a physical milieu distinct from that in which it occurs in nature. For example, an isolated polynucleotide may be substantially isolated with respect to the complex cellular milieu in which it naturally occurs, and may even be purified essentially to homogeneity, for example as determined by agarose or polyacrylamide gel electrophoresis or by A26Q/A28O measurements, but may also have further cofactors or molecular stabilizers (for instance, buffers or salts) added. The invention further comprises the chimeric polypeptides encoded by the isolated nucleic acid molecules of the invention. Expression and Isolation of Chimeric HIV/SIV Env Polypeptides Another aspect of the invention relates to a method of producing a chimeric polypeptide of the invention, or a variant thereof, and to expression systems and host cells containing a vector appropriate for expression of a chimeric polypeptide of the invention. In one embodiment, variants have silent substitutions, additions and deletions that do not alter the properties and activities of the chimeric protein. Variants can also be modified polypeptides in which one or more amino acid residues are modified, and mutants comprising one or more modified residues. Proteins and polypeptides described herein can be assessed for their solubility and ability to trimerize using assays described herein.
Cells that express such a chimeric polypeptide or a variant thereof can be made and maintained in culture, under conditions suitable for expression, to produce protein for isolation. These cells can be prokaryotic or eukaryotic. Examples of prokaryotic cells that can be used for expression (as "host cells"; "cell" including herein cells of tissues, cell cultures, cell strains and cell lines) include Escherichia coli, Bacillus subtilis and other bacteria. Examples of eukaryotic cells that can be used for expression include yeasts such as Saccharomyces cerevisiae, Schizosaccharomyces pombe, Pichia pastoris and other lower eukaryotic cells, and cells of higher eukaryotes such as those from insects and mammals. Suitable cells of mammalian origin include primary cells, and cell lines such as CHO, BS- C-l, HeLa, 3T3, BHK, COS, 293, and Jurkat cells. Suitable cells of insect origin include primary cells, and cell lines such as Sf9 and High five cells (see, e.g., Ausubel, F.M. et al., eds., Current Protocols in Molecular Biology, Greene Publishing Associates and John Wiley & Sons Inc., containing Supplements up through 1998). In one embodiment, host cells that produce a recombinant chimeric polypeptide, variant, or portions thereof can be made as follows. A gene encoding a chimeric polypeptide described herein can be inserted into a nucleic acid vector, e.g., a DNA vector, such as a plasmid, virus or other suitable replicon (including vectors suitable for use in gene therapy, such as those derived from vaccinia virus or others, can be present in a single copy or multiple copies, or the gene can be integrated in a host cell chromosome. A suitable replicon or integrated gene can contain all or part of the coding sequence for the polypeptide or variant, operably linked to one or more expression control regions whereby the coding sequence is under the control of transcription signals and linked to appropriate translation signals to permit translation. The vector can be introduced into cells by a method appropriate to the type of host cells (e.g., transformation, electroporation, infection). For expression from the gene, the host cells can be maintained under appropriate conditions (e.g., in the presence of inducer, normal growth conditions, etc.). Proteins or polypeptides thus produced can be recovered (e.g., from the cells, the periplasmic space, culture medium) using suitable techniques. The invention also relates to isolated proteins or polypeptides encoded by nucleic acids of the present invention. Isolated proteins can be purified from a natural source or can be made recombinantly. Proteins or polypeptides referred to herein as "isolated" are proteins or polypeptides purified to a state beyond that in which they exist in cells and include proteins or polypeptides obtained by methods described herein, similar methods or
other suitable methods, and also include essentially pure proteins or polypeptides, proteins or polypeptides produced by chemical synthesis or by combinations of biological and chemical methods, and recombinant proteins or polypeptides which are isolated. Thus, the term "isolated" as used herein, indicates that the polypeptide in question exists in a physical milieu distinct from the cell in which its biosynthesis occurs. For example, an isolated HIV/SIN chimeric polypeptide may be purified essentially to homogeneity, for example as determined by PAGE or column chromatography (for example, HPLC), but may also have further cofactors or molecular stabilizers added to the purified protein to enhance activity. In one embodiment, proteins or polypeptides are isolated to a state at least about 75% pure; more preferably at least about 85% pure, and still more preferably at least about 95% pure, as determined by Coomassie blue staining of proteins on SDS-polyacrylamide gels. Chimeric or fusion polypeptides can be produced by a variety of methods. For example, a chimeric polypeptide can be produced by the insertion of an env gene or portion thereof into a suitable expression vector. The resulting construct can be introduced into a suitable host cell for expression. Upon expression, chimeric polypeptide can be purified from a cell lysate or supernatant by means of a suitable affinity matrix (see e.g., Current Protocols in Molecular Biology (Ausubel, F.M. et al., eds., Vol. 2, pp. 16.4.1-16.7.8, containing supplements up through Supplement 44, 1998). Polypeptides of the invention can be recovered and purified from cell cultures by well-known methods. The recombinant protein can be purified by ammonium sulfate precipitation, heparin-Sepharose affinity chromatography, gel filtration chromatography and/or sucrose gradient ultracentrifugation using standard techniques. Further methods that can be used for purification of the polypeptide include ethanol precipitation, acid extraction, ion exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxylapatite chromatography and high performance liquid chromatography. Known methods for refolding protein can be used to regenerate active conformation if the polypeptide is denatured during isolation or purification. Vaccine and Therapeutic Use In accordance with the present invention, there are provided methods for inhibiting, preventing, and ameliorating a viral infection in a subject. In one embodiment, a method of the invention includes administering an effective amount of a chimeric polypeptide to a subject, thereby producing an immune response sufficient for ameliorating, preventing or
inhibiting virus infection in the subject, h another embodiment, a method of the invention includes administering to a subject an effective amount of a polynucleotide encoding a chimeric polypeptide of the invention. In yet another embodiment, a method of the invention includes administering an effective amount of an antibody that binds to a chimeric polypeptide to a subject, thereby ameliorating, preventing or inhibiting virus infection in the subject. In the methods for inhibiting, preventing, and ameliorating a viral infection in a subject in which a chimeric polypeptide or a polynucleotide encoding a chimeric polypeptide are administered, an immune response also can be produced. The immune response will likely be humoral in nature, although administering a polynucleotide encoding a chimeric polypeptide may induce a CTL response. It is also understood that the methods of the invention can also be used in combination with other viral therapies, as appropriate. The "effective amount" will be sufficient to inhibit, prevent, or ameliorate a viral infection in a subject, or will be sufficient to produce an immune response in a subject. Thus, an effective amount of chimeric polypeptide can be that which elicits an immune response to the polypeptide or a virus upon which the coat protein is based. An effective amount administered to a subject already infected with the virus can also be that which decreases viral load, or increases the number of CD4+ cells. An effective amount can be that which inhibits transmission of the virus from an infected subject to another (uninfected or infected). In the methods of the invention in which a polynucleotide sequence encoding a chimeric polypeptide is administered to a subject, a CTL response to the chimeric polypeptide can be produced against a virus that contains the corresponding coat polypeptide sequence. As the chimeric polypeptides, polynucleotides, and antibodies of the present invention will be administered to subjects, including humans, the present invention also provides pharmaceutical formulations comprising the disclosed chimeric polypeptides, polynucleotides, and antibodies. The compositions administered to a subject will therefore be in a "pharmaceutically acceptable" or "physiologically acceptable" formulation. As used herein, the terms "pharmaceutically acceptable" and "physiologically acceptable" refer to carriers, diluents, excipients, and the like that can be administered to a subject, preferably without excessive adverse side effects (e.g., nausea, headaches, etc.).
Such preparations for administration include sterile aqueous or non-aqueous solutions, suspensions, and emulsions. Examples of nonaqueous solvents are propylene glycol, polyethylene glycol, vegetable oils, such as olive oil, and injectable organic esters, such as ethyl oleate. Aqueous carriers include water, alcoholic/aqueous solutions, emulsions, or suspensions, including saline and buffered media. Vehicles include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's or fixed oils. Intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishers (such as those based on Ringer's dextrose), and the like. Preservatives and other additives may also be present, such as, for example, antimicrobial, anti-oxidants, chelating agents, and inert gases and the like. Various pharmaceutical formulations appropriate for administration to a subject known in the art are applicable in the methods of the invention (e.g., Remington's Pharmaceutical Sciences, 18th ed., Mack Publishing Co., Easton, PA 1990; and The Merck Index 12th ed., Merck Publishing Group, Whitehouse, NJ, 1996). Controlling the duration of action or controlled delivery of an administered composition can be achieved by incorporating the composition into particles or a polymeric substance, such as polyesters, polyamine acids, hydrogel, polyvinyl pyrrolidone, ethylene- vinylacetate, methylcellulose, carboxymethylcellulose, protamine sulfate or lactide/glycolide copolymers, polylactide/glycolide copolymers, or ethylenevinylacetate copolymers. The rate of release of the composition may be controlled by altering the concentration or composition of such macromolecules. Colloidal dispersion systems include macromolecule complexes, nano-capsules, microspheres, beads, and lipid-based systems, including oil-in-water emulsions, micelles, mixed micelles, and liposomes. The compositions administered by a method of the present invention can be administered parenterally by injection, by gradual perfusion over time, or by bolus administration (for example, in the case of passive protection against HJV infection resulting from a needlestick injury) or by a microfabricated implantable device. The composition can be administered via inhalation, intravenously, intraperitoneally, intramuscularly, subcutaneously, intracavity (e.g., vaginal or anal), transdermally, topically, or intravascularly. The compositions can be administered in multiple doses. The doses or "effective amount" needed for treating, inhibiting, or preventing viral infection or transmission, or for inducing an immune response, preferably will be sufficient to ameliorate some or all of the symptoms of the infection, although preventing progression or worsening of the infection also is a satisfactory outcome for many viral infections,
including HI . An effective amount can readily be determined by those skilled in the art (see, for example, Ansel et al., Pharmaceutical Drug Delivery Systems, 5th ed., Lea and Febiger, 1990, Gennaro Ed.). An adjuvant may be included in the pharmaceutical composition to augment the immune response to a chimeric polypeptide of the invention. Examples of adjuvants include, but are not limited to, muramyl dipeptide, aluminum hydroxide, saponin, polyanions, amphipatic substances, bacillus Calmette-Guerin (BCG), endotoxin lipopolysaccharides, keyhole limpet hemocyanin (KLH), interleukin-2 (IL-2), and granulocyte-macrophage colony-stimulating factor (GM-CSF). Binding Assays In the case of the chimeric polypeptides disclosed herein, such chimeric polypeptides are useful for identifying agents that modulate binding of the HIN virus to the HIV virus co-receptor or receptor. Such chimeric polypeptides also are useful for identifying agents that modulate the intramolecular interaction/binding of the virus coat polypeptide sequence to the co-receptor or receptor. Thus, in accordance with the present invention, there are provided methods for identifying an agent that modulates binding between a virus and a virus co-receptor or receptor, and methods for identifying an agent that modulates binding between a virus and a virus co-receptor or receptor. h one embodiment, a method of the invention includes contacting a chimeric polypeptide with a co-receptor or receptor polypeptide under conditions allowing the chimeric polypeptide and the co-receptor or receptor polypeptide to bind, in the presence and absence of a test agent, and detecting binding in the presence and absence of the test agent. A decreased amount of binding in the presence of the test agent thereby identifies an agent that inhibits interaction/binding between the virus and the virus co-receptor or receptor. Increased binding in the presence of the test agent thereby identifies an agent that stimulates interaction binding between the virus and the virus co-receptor or receptor. The contacting can occur in solution, solid phase, on intact cells, or in an organism, such as a non-human primate. Candidate agents include antibodies, antivirals, a co-receptor or receptor polypeptide sequence, peptidomimetics or active fragments thereof. Candidate agents also encompass numerous chemical classes, including organic molecules, like small organic compounds having a molecular weight of more than 50 and less than about 2,500 daltons.
Candidate agents comprise functional groups necessary for structural interaction with proteins, particularly hydrogen bonding, and typically include at least an amine, carbonyl, hydroxyl, or carboxyl group, preferably at least two of the functional chemical groups. The candidate agents often comprise cyclical carbon or heterocyclic structures, and/or aromatic or polyaromatic structures substituted with one or more of the above functional groups. Candidate agents are also found among biomolecules, including, but not limited to, peptides, saccharides, fatty acids steroids, purines, pyrimidines, derivatives, structural analogs, or combinations thereof. Candidate agents are obtained from a wide variety of sources, including libraries of synthetic or natural compounds. For example, numerous means are available for random and directed synthesis of a wide variety of organic compounds and biomolecules, including expression of randomized oligonucleotides and oligopeptides. Alternatively, libraries of natural compounds in the form of bacterial, fungal, plant, and animal extracts are available or readily produced. Additionally, natural or synthetically produced libraries and compounds are readily modified through conventional chemical, physical, and biochemical means, and may be used to produce combinatorial libraries. Known pharmacological agents may be subjected to directed or random chemical modifications, such as acylation, alkylation, esterification, amidification, etc., to produce structural analogs. Where the method detects binding, one or more of the molecules may be joined to a label, where the label can directly or indirectly provide a detectable signal. Various labels include radioisotopes, fluorescers, chemiluminescers, enzymes, specific binding molecules, particles, e.g. magnetic particles, and the like. Specific binding molecules include pairs, such as biotin and streptavidin, digoxin and antidigoxin, etc. For the specific binding members, the complementary member would normally be labeled with a molecule that provides for detection, in accordance with known procedures. A variety of other reagents may be included in the assay. These include reagents, like salts, neutral proteins, e.g. albumin, detergents, etc., that are used to facilitate optimal protein-protein binding and/or reduce non-specific or background interactions. Reagents that improve the efficiency of the assay, such as protease inhibitors, nuclease inhibitors, anti-microbial agents, etc., may be used. The components are combined in any order that provides for the requisite binding. Incubations are performed at any suitable temperature, typically between 4 ° C and 40 ° C. Incubation periods are selected for optimum activity, but
may also be optimized to facilitate rapid high-throughput screening. Typically, between 0.1 and 1 hour will be sufficient. An agent identified by a method of the invention described herein can be further tested for its ability to inhibit virus binding or infection of a cell in vitro or in vivo. Thus, in accordance with the present invention, there are provided methods for identifying an agent that inhibits virus infection of a cell. A method of the invention includes contacting a cell susceptible to virus infection with an infectious virus particle in the presence and absence of a test agent, and determining whether the test agent inhibits virus binding or infection of the cell, thereby identifying an agent that inhibits virus infection. In various embodiments, the test agent is added before or after contacting the cell with the infectious virus particle. The method also can be performed in any suitable animal, such as a non-human primate.
Promoting Trimerization of Soluble HIV-1 Env Through the Use of HIV-1/SIV Chimeras HIV-l R.FL gpl40 purifies as a heterogeneous range of oligomeric species Purified HIN-l JR-FL gpl40 was initially analyzed by gel filtration, which separates proteins on the basis of molecular size. HJN-ljR-FL gpl40 resolved as a broad gel filtration peak, eluting mainly between fractions 45 and 58 (Fig. 1 A). Immunoblotting of ethylene glycol bis(succinimidlysuccinate) (EGS)-cross-linked fractions showed that essentially all HIN-l gpl40 had a lower electrophoretic mobility than the monomer (Fig. IB), which had an apparent mass of slightly less than 160 kDa (based on SDS-PAGE in the absence of cross-linking, Fig. 1A inset). The presence of multiple species was apparent from the cross-linking profile. Fractions 54 to 57 contained protein migrating to a position slightly above the 250-kDa standard. This band position was similar to that which we observed for dimeric gpl20 (Center, R. J. et al. 2000 J Virol 74:4448-4455), indicating the presence of gpl40 dimers. Fractions 50 to 54 contained gpl40 migrating to a position well above that of the 250-kDa standard but below the top of the gel. The presence of very slowly SDS- PAGE-migrating cross-linked protein (fractions 46 to 51, close to the top of the gel) indicated the presence of some Env molecules with a larger number of protomers than the two apparent species described above. Immunoblotting of the non-cross-linked sample revealed some protein within fractions 65 to 69 with an SDS-PAGE migration pattern consistent with that of monomeric gp 120. STEM is a quantitative method for measuring mass based on the elastic scattering of electrons by atoms within individual molecules. The STEM-measured masses of 622 molecules within fraction 51 showed a broad and asymmetrical distribution with a mean value of 444 kDa (3.71 protomers) and a standard deviation of 107 kDa (Fig. 1C; Table 1), consistent with the presence of at least some oligomers with more than three protomers. Given that functional native Env is a trimer, complexes of more than three protomers are presumably nonspecifically associated; they are referred to hereafter as aggregates. The masses of 434 molecules within fraction 56 yielded a relatively symmetrical distribution with a mean value of 280 kDa (2.34 protomers) and a standard deviation of 64 kDa (Fig. ID; Table 1), consistent with a predominantly dimeric structure. Overall, it is clear that in contrast to the homogeneous trimeric structure of virion-derived SIN and HIN-l Env and SIN gpl40 (Center, R. J. et al. 2002 J Virol 76: 7863-7867; Center, R. J. et al. 2001 Proc NatlAcad Sci USA 98:14877-14882; Chen, B. et al. 2000 J Biol Chem 275:34946-34953),
HIV-1 jR-FL gpl40 forms a heterogeneous range of oligomeric species including dimers, trimers, and higher-mass aggregates.
TABLE 1. Masses of gp!40 species as determined by STEM1 Source of gpl40 Fraction no. Mass (kDa) No. of protomers6
56 280 2.34
H-S 51-52 361 3.07 53-54 360 3.06
H-S.N 51-53 403 3.36 a Number average. h The mass of one protomer was estimated by summing the protein mass deduced from the amino acid sequence and the carbohydrate mass (see Example 1).
Sedimentation coefficient distribution [c(s)J analysis of sedimentation velocity data was performed to obtain model-free information about the oligomeric state of the molecules in solution. The distributions of HJN-1 JR.F gpl40 fractions 51, 54, and 56 showed significant separation (Fig. 2A), consistent with differences in protomer number. Sedimentation equilibrium, unlike gel filtration, allows shape-independent mass determinations for Env (Center, R. J. et al. 2000 J Virol 74:4448-4455). The data sets for sedimentation equilibrium absorbance versus radial position (Fig. 2B) were analyzed by global nonlinear regression. The masses determined by this analysis, and the calculated numbers of protomers (in parentheses) are shown in Table 2. Protein from fractions 56 and 55 gave sedimentation equilibrium-derived mass values close to those expected for the dimer (2.26 and 2.40 protomers respectively). gpl40 from fractions 49 and 51 gave sedimentation equilibrium-derived mass values equating to 4.85 and 3.84 protomers, respectively, indicating that some of the molecules within these fractions were aggregated. Fractions from intermediate positions within the profile (fractions 52 to 54) produced sedimentation equilibrium-derived mass values converting to 3.58 to 3.14 protomers, consistent with the presence of trimers. The fact that fraction 54 (3.14 protomers) contained two EGS-cross-linked species indicates that a substantial nontrimeric component is also present within these fractions.
TABLE 2. Masses of gpl40 species as determined by sedimentation equilibrium" Source of gpl40 Fraction no. Mass (kDa) No. of protomers*
HIN-ljR-FL 49 580.4 4.85 51 459.5 3.84 52 428.8 3.58 53 402.8 3.37 54 375.4 3.14 55 286.7 2.40 56 270.3 2.26
HIV-IADA 51 424.2 3.86 53 344.8 3.13 56 305.0 2.77 59 253.3 2.30
HJN-1JR-FLΔN2 51 486.3 4.40 52 445.2 4.03 53 398.3 3.60 54 364.5 3.30 56 297.7 2.69
HΓV-1JR-FLΔVI/2 48 634.5 6.42 51 487.5 4.93
H-S 50 535.3 4.55 51 442.2 3.76 52 408.2 3.47 53 407.6 3.46 54 381.9 3.25 55 358.0 3.04
S-H 52 394.5 3.14 56 275.2 2.19 57 267.6 2.13 62 166.2 1.32
H-S.Ν 50 469.1 3.91 51 416.6 3.47 52 398.6 3.32 ■ 53 370.7 3.09 54 351.9 2.93 55 337.2 2.81
HIV-l93MW96S 50 442.1 3.46 51 417.4 3.27 52 389.5 3.05 53 349.7 2.74 54 322.1 2.52 55 306.0 2.40
H93MW965-S.Ν 50 452.6 3.55 51 417.0 3.27 52 426.2 3.34 53 420.4 3.30 54 415.0 3.25 a Weight average. bb TThhee mmaassss ooff oonnee protomer was estimated by summing the protein mass deduced from the amino acid sequence and the carbohydrate mass (see Example 1).
HIV-IADA and SIVMac32H gpl40 expressed with mannose-only oligosaccharides have different oligomeric profiles Recent studies have suggested the utility of HJV-1 and SIV g l40 with mannose- only oligosaccharides for biochemical and structural studies (Chen, B. et al. 2000 J Biol Chem 275:34946-34953; Zhang, C. W.-H. et al. 2001 J Biol Chem 276:39577-39585). We sought to determine if mannose-only HIN-l and SIN gpl40 showed the different degree of oligomeric heterogeneity that we observed for normally glycosylated gpl40s derived from HIV-1 and SJV. HJV-IADA and SιNMac32H gpl40 (Chen, B. et al. 2000 J Biol Chem 275:34946-34953; Zhang, C. W.-H. et al. 2001 J Biol Chem 276:39577-39585) were expressed in stably transfected CHO-Lec3.2.8.1 cells (Stanley, P. 1989 Mol Cell Biol 9:377-983), which have mutations blocking complex-oligosaccharide addition, resulting in all utilized Ν-linked glycosylation sites having oligosaccharides with five mannose residues (Liu, J. et al. 1996 J Biol Chem 271:33639-33646). Purified HIV-IADA and SιNMac32H gpl40s were subjected to gel filtration and compared. SJNMac32H gpl40 resolved as a symmetrical and relatively sharp peak with elution mainly between 52 and 59 ml (Fig. 3 A, open circles). HIV-IADA gpl40 resolved as a broader and asymmetrical peak between 48 and 61 ml (Fig. 3 A, solid squares), indicating greater size heterogeneity. Blue Native (BN)- PAGE has recently been successfully used to analyze gpl40 (Schύlke, N. et al. 2002 J Virol 76:7760-7776) and has proved more effective than EGS cross-linking for revealing the oligomeric profile of HIV-IADA gpl40. The range of apparent masses revealed by BN- PAGE (approximately 670 to 220 kDa) (Fig. 3B) and the range of sedimentation equilibrium-derived protomer numbers (3.86 to 2.30, Table 2) for fractions 51, 53, 56, and 59 of HIV-IADA gpl40 confirmed oligomeric heterogeneity. The presumed trimeric component (Fig. 3B, major band, fractions 53 and 56) was enriched in comparison to HFV- 1JR-FL gpl40 expressed in B-SC-1 cells. Nevertheless, HIV-IADA gpl40 is less homogeneous in terms of oligomeric structure than is SIV ao32H gpl40. Deletion of HIV-1 variable domains 1 and 2 reduces dimer formation and promotes aggregation The second variable domain (V2) can mediate dimer formation between recombinant gpl20 subunits (Center, R. J. et al. 2000 J Virol 74:4448-4455). We hypothesized that the same contact site may mediate gpl40 dimer formation and that its elimination would redirect this subset of gpl40 molecules to a pool available for trimerization. We therefore expressed HIV-IJR.FL gpl40 lacking either the N2 domain
(amino acids F156 to LI 90) (HiN-ljR-FiAN2) or the entire first and second variable domains (amino acids K120 to Q200 replaced with a GAG tripeptide) (HJN-ljR-F ΔNl/2) and analyzed the oligomeric structure as before. Note that the reduction in protomer mass due to deletion of the N2 or NI plus N2 loops means that the average number of protomers per molecule for a given fraction number will be larger than the comparable value for nondeleted gpl40 for corresponding fractions. Comparison of the gel filtration profile (Fig. 4 A) for HJN-ljR.FLΔNl/2 (open squares) to that of nondeleted gpl40 (solid squares) showed significant skewing to larger sizes, with lower percentages of protein present in fractions 54 to 57, which in the nondeleted protein contained predominantly dimers. SDS- PAGE of EGS -cross-linked samples revealed an absence of protein with a migration consistent with the dimer (close to the 250-kDa marker) and the presence of protein with very slow migration within fractions 47 to 52 (Fig. 4B). The sedimentation equilibrium- derived mass values for two peak fractions 48 and 51 convert to 6.42 and 4.93 protomers, respectively (Table 2), indicating that most HIV-IJR-FLΔVI/2 g l40 was in the form of aggregates. Deletion of the V2 domain only (Fig. 4A, open circles) had a less dramatic effect on oligomer formation. Less cross-linked protein running close to the 250-kDa marker was detected in comparison to the nondeleted form (compare Fig. 4C with Fig. IB fractions 54 to 57), and the sedimentation equilibrium results indicated a range of 3.60 to 2.69 protomers per molecule within fractions 53 to 56 (Table 2), indicating the presence of fewer dimers and more trimers. The values seen for peak fractions 51 and 52 (4.40 and 4.03 protomers, respectively) and the presence of a slowly migrating EGS-cross-linked species in fractions 48 to 52 indicated that considerable aggregate formation had occurred. It is evident from these results that deletion of the VI and V2 domains and, to a much lesser extent, deletion of the V2 domain alone markedly reduces dimer formation and promotes aggregation without greatly enhancing trimerization.
Replacing the gp41 subunit of HIV-IJR.F gpl40 with that of SIV reduces dimer formation and promotes trimerization We previously demonstrated that unmodified SIV gpl40 purified as a homogeneous population of trimers (Center, R. J. et al. 2001 Proc Natl Acad Sci USA 98:14877-14882). We therefore sought to identify the SIN Env domain responsible for this property and to use it to promote HIN-l gpl40 trimerization by using a domain exchange strategy. Previous studies have shown that the gp41 component of HIN-l and HIN-2 (closely related to SIN)
contained the major determinants of Env oligomerization (Center, R. J. et al. 1997 J Virol 71:5706-5711; Earl, P. L. and B. Moss 1993 AIDS Res Hum Retrovir 9:589-594; Poumbourios, P. et al. 1997 J Virol 71:2041-2049). We therefore genetically combined HIV-1JR.FL gpl20 (amino acids Ml to R502) and SJN gp41 (amino acids G528 to A687) to create a chimeric gpl40 (H-S). In comparison to HIV-IJR.FL gpl40, H-S gpl40 displayed a sharper and more symmetrical gel filtration peak (Fig. 5A, open diamonds compared to solid squares). Fractions 52 to 56 contained what appeared to be one major EGS-cross- linked species (Fig. 5B) that was shown by sedimentation equilibrium results (Table 2) to be trimeric (range, 3.47 to 3.04 protomers for fractions 52 to 55). No H-S EGS-cross- linked dimers were detected. BN-PAGE (4 to 12% polyacrylamide) of a pool of fractions 50 to 54 (Fig. 5A inset) confirmed the presence of one predominant species. A faint band below the major band (migrating to a position between the 440- and 220-kDa markers) indicated the presence of trace amounts of dimer. Fractions 45 to 51 contained a very slowly migrating EGS-cross-linked species with high mass (for example, for fraction 50 the average number of protomers was 4.55), indicating that as with HIV-IJR-FL gpl40, some H- S gpl40 was aggregated. The sedimentation velocity profiles of H-S fractions 50 to 55 displayed considerable overlap (Fig. 5D). The profiles for fractions 50 and 51 showed some skewing to a higher sedimentation coefficient. Together, these observations are consistent with the formation of trimers and some aggregates. Scanning transmission electron microscopy (STEM) analysis of 420 individual molecules for a pool of fractions 51 and 52 (Fig. 5E; Table 1) and 515 molecules from a pool of fractions 53 and 54 (Table 1) yielded mass values of 361 kDa (standard deviation, 70 kDa) and 360 kDa (standard deviation, 76 kDa), respectively, which convert to 3.07 and 3.06 protomers. This confirmed the trimeric nature of the major species. We observed that a portion of individual H-S gpl40 molecules visualized by STEM had a triangular or trilobed morphology (a montage of molecules observed in the pool of fractions 51 and 52 is shown in the Fig. 5E inset), which was very similar to that which we previously reported for SIV gpl40 trimers and virion-derived SJV and HJV-1 Env trimers (Center, R. J. et al. 2002 J Virol 16: 7863-7867; Center, R. J. et al. 2001 Proc Natl Acad Sci USA 98:14877-14882). Overall, the strategy of replacing the HIN-l gp41 subunit with that of SIN was successful in redirecting oligomer formation from dimerization to trimerization. A reverse chimera (S-H) composed of SIV gpl20 (Ml to R527) and HIV-IJR.FL gp41 (A503 to K674) was also tested. S-H gpl40 showed a strikingly different gel
filtration profile (Fig. 5A, open circles) from that of either H-S or HFV-1JR-FL gpl40, with a marked skewing toward smaller size. This reflected (i) a greater percentage of dimer, present in fractions 53 to 57 (Fig. 5C), with the average number of protomers in fractions 56 and 57 being 2.19 and 2.13, respectively (Table 2); and (ii) a separate peak between fractions 61 and 64. The gel filtration elution volume of this peak, which indicated a smaller size than the dimer, and the sedimentation equilibrium-derived mass value for fraction 62, equating to 1.32 protomers (Table 2), indicated that this peak was composed of monomeric gpl40. Some gpl20 derived from gpl40 cleavage was also present in this peak, as revealed by SDS-PAGE. Less aggregate formation was apparent with S-H g l40 than with HIV-IJR.FL, as evidenced by less very slowly migrating EGS-cross-linked material (compare Fig. 5C to Fig. IB). The N-terminal half of the SIV gp41 subunit is sufficient to promote trimerization Our overall aim was to obtain trimeric soluble HFV-1 for application as an antigen and an immunogen. Since the C-terminal part of the gp41 ectodomain contains the epitopes of several broadly neutralizing monoclonal antibodies (Muster, T. et al. 1993 J Virol 67:6642-6647; Stiegler, G. et al. 2001 AIDS Res Hum Retrovir 17:1757-1765; Zwick, M. B. et al. 2001 J Virol 75:10892-10905; Zwick, M. B. et al. 2001 J Virol 75:12198- 12208), we considered it desirable to include the HIN-l sequence spanning these epitopes in a new chimera. We therefore replaced the 74 C-terminal-most amino acids of H-S (SIN- derived amino acids, CP-MAC numbering from A614 to A687) with the corresponding sequence of HIV-IJR.FL (amino acids S590 to K674) to create the chimera H-S.Ν. The increase in binding of the CD4-induced monoclonal antibody 17b in the presence of CD4 demonstrates that both the CD4 binding site and the conformationally sensitive 17b epitope are intact in H-S.Ν gpl40 (Fig. 6A, lower inset). This indicates that the presence of the SIV gp41 -derived sequence has not compromised the folding or function of H-S.Ν gpl40. As with the H-S chimera, H-S.Ν gpl40 revealed a sharper and more symmetrical gel filtration peak than did HIV-IJR.FL gpl40 (Fig. 6 A, open circles compared to solid squares). The EGS-cross-linking profile indicated the presence of one predominant oligomeric species (Fig. 6B). This finding was supported by the substantial overlap of the sedimentation velocity profiles for fractions 50 to 55 (Fig. 6C). The sedimentation equilibrium results (Table 2) indicated that this species was a trimer, with a range for fractions 51 to 55 of 3.47 to 2.81 protomers. BΝ-PAGE (4 to 12% polyacrylamide) of a pool of fractions 50 to 54 (Fig. 6A upper inset) revealed the presence of a single major band
consistent with a mainly trimeric structure. A faint band below the major band (migrating to a position between the 440- and 220-kDa markers) indicated the presence of a small amount of dimer. Unexpectedly, less aggregation was detected for H-S.N gpl40 than for H-S or HIV-IJR.FL gpl40 by EGS cross-linking, with less very slowly migrating protein present in fractions 48 to 51 (compare Fig. 6B to Fig. 5B and IB). Mass measurement of STEM images of 265 individual molecules within peak fractions 51 to 53 (Fig. 6D) yielded a mean mass of 403 kDa (3.36 protomers) with a standard deviation of 85 kDa (Table 1), confirming the predominance of the trimeric species. As with H-S gpl40, some of the STEM images of H-S.N gpl40 showed a triangular or trilobed morphology (Fig. 6E). The N-terminal half of SJV gp41 is therefore sufficient to confer efficient gpl40 trimerization. To test the general applicability of the strategy used to enhance the trimerization of H-S.N gpl40, we compared an unmodified gpl40 derived from an HIV-1 clade C primary viral isolate 93MW965 and the equivalent of the H-S.N chimera in the HIV-l93MW965 background. In this chimera, the N-terminal sequence of the SIVCP-MAC gp41 (amino acids G528 to A610) replaces the corresponding HIV-193MW965 gp41 sequence (amino acids A501 to M584). The gel filtration peak for HIV-1 MW965 gpl40 is sharper and more symmetrical than that of JR.FL gpl40 (Fig. 7A compared to Fig. 1 A, solid squares and solid line in both), indicating less oligomeric heterogeneity. The EGS cross-linking profile which revealed the presence of two oligomeric species (Fig. 7B), one with more rapid electrophoretic migration (seen in fractions 53 to 56) and one with slower migration (seen mainly in fractions 50 to 54), but less of the very slowly migrating protein aggregates seen for JR-FL gpl40 (fractions 46 to 51 in Fig. IB). The sedimentation equilibrium data are consistent with the presence mainly of trimers and dimers (range, 3.46 to 2.40 protomers for fractions 50 to 55). The gel filtration profile of the clade C H-S.N gpl40 chimera is similar overall to that of the unmodified molecule but lacks the shoulder on the more slowly eluting side of the peak seen with HIV-193MW965 gpl40 (Fig. 7A, open circles compared to solid squares). The EGS-cross-linking profile reveals the presence of one predominant oligomeric species, which was shown by sedimentation equilibrium analysis to be composed of three protomers (range, 3.55 to 3.25 for fractions 50 to 54). The ability of the N-terminal half of SIV gρ41 to confer efficient gpl40 trimerization is therefore applicable to both the divergent strains tested (clades B and C).
Discussion We show here that gpl40s derived from the clade B primary isolate HIV-IJR.FL and the clade C primary isolate HrV-l9 Mw965 have a propensity to form nontrimeric oligomers including dimers and aggregates. This oligomeric heterogeneity was also observed for gpl40s derived from a T-cell-line-adapted clade B isolate (the BH8 clone of IIIB/LAI) and primary isolates of clades A and D. An attempt to enhance trimerization by expressing HIN-l R-FL gpl40 at a lower temperature (32°C), which has been shown to facilitate protein folding in some systems, did not significantly alter the oligomeric profile. These results are in contrast to the virion-associated Env of both HIV-1 and SIV and to SIV gpl40, which display a relatively homogeneous trimeric structure. Previous studies showed that recombinant, cell-associated HIV-1 gpl60 also formed a mixture of oligomeric species including dimers (Doms, R. W. et al. 1991 Adv Exp Med Biol 300:203-219; Earl, P. L. et al. 1990 Proc Natl Acad Sci USA 87:648-652), indicating that the heterogeneity we observed for HIV-1 gpl40 may be a general property of HIN-l Env. If so, it is likely that a cellular quality control mechanism prevents nontrimeric Env from being incorporated into virions, perhaps by preventing egress to the cell surface. The greater level of oligomeric heterogeneity of HIN-l in comparison to SIN was also shown for molecules expressed in the absence of complex-type oligosaccharides. Enrichment of the trimeric component of HIN-l ADA mannose-only gpl40 in . comparison to HIV-1JR-FL gpl40 with complex-type oligosaccharides present was observed. This may be due to strain-related differences rather than to differences in carbohydrate type, since expression of HIV-1 JR-FL gpl40 using the recombinant vaccinia virus described in this study in the cell line producing mannose-only oligosaccharides (CHO-Lec3.2.8.1) did not result in an enrichment of the trimeric component. Based on the ability of the HiV-lIIIB/LAI N2 loop to mediate gpl20 dimer formation (Center, R. J. et al. 2000 J Virol 74:4448-4455), we hypothesized that deletion of this domain might block gpl40 dimerization and enhance trimerization. Deletion of both the NI and N2 loops did block gpl40 dimer formation; however, most of this protein was aggregated rather than trimeric. Deletion of the N2 loop alone had a more modest effect, with a reduction of dimerization observed, but again trimer formation was not obviously enhanced. It has been reported that deletions in this region of Env expose underlying conserved, potentially neutralizing epitopes and that such deletions may therefore be advantageous in potential immunogens (Barnett, S. W. et al. 2001 J Virol 75:5526-5540; Cao, J. et al. 1997 J Virol 71:9808-9812; Cherpelis, S. et al. 2001 J Virol 75:1547-1550;
Kim, Y. B. et al. 2003 Virology 305:124-137; Sanders, R. W. et al. 2000 J Virol 74:5091- 5100; Srivastava, I. K. et al. 2003 J Virol 77:2310-2320; Stamatatos, L. and C. Cheng- Mayer 1998 J Virol 72:7840-7845; Wyatt, R. et al. 1993 J Virol 67:4557-4565). If the pronounced increase in JR-FL gpl40 aggregation with deletion of the entire N1-N2 loop structure observed here is a general rather than strain-specific effect, this particular modification may be undesirable from a structural standpoint for potential immunogens, at least in the context of soluble gpl40. Analysis of a gpl40 chimera composed of the SIN gpl20 and HIV-1JR-FL gp41 segments revealed a strong propensity of the HIN-l gp41 domain for dimer formation. Furthermore, a significant fraction of the gpl40 of this chimera failed to oligomerize altogether, indicating that the oligomeric interface may be less stable. Conversely, replacement of either all or just the Ν-terminal half of the gp41 segment of HIN-l gpl40 with the homologous region of SIN was sufficient to block dimer formation and promote trimerization. These results are consistent with previous studies demonstrating the role of the Ν-terminal section of gp41 in oligomerization (Center, R. J. et al. 1997 J Virol 71:5706- 5711; Earl, P. L. and B. Moss 1993 AIDS Res Hum Retrovir 9:589-594; Poumbourios, P. et al. 1997 J Virol 71:2041-2049) and indicate that this region plays a role not only in oligomer formation per se but also in the type of oligomers produced. The reason for the apparent differences in the gp41 oligomerization domain of HIN-l and SIN is not immediately clear. It may be speculated that the apparently reduced stability of the oligomeric contacts in pre-receptor-activated HIN-l Env trimers may facilitate the triggering of the conformational changes induced by receptor binding, and therefore may enhance fusogenicity, at the expense of efficient trimer formation. Such an explanation would imply that the separate evolutionary courses of the two viruses favored such changes in HJN-1 but not SIN. The gpl40 chimeras comprising either all or just the Ν-terminal half of the SIN gp41 segment in an HIN-ljR-FL background showed less aggregate formation than did unaltered HIN-l gρl40. The fact that the reverse chimera (SIN gpl20, HIN-ljR-FL gp41) also showed less aggregate formation indicates that the HIN-l gp41 segment does not directly induce aggregation. If aggregates form from the nontrimeric pool of molecules, reducing this pool by increasing the efficiency of trimerization (by replacing the HIN-l oligomerization domain with that of SIN) may concomitantly reduce aggregate formation. Unexpectedly, the gpl40 chimera H-S.Ν with the Ν-terminal half of SIN gρ41 in an
HIN-ljR-FL background showed less aggregate formation than did the H-S chimera where the entire gp41 domain was exchanged with SIN. It has been suggested that changes which reduce the affinity of interaction between Ν- and C-terminal alpha-helical regions of gp41 can block the foπnation of a receptor-activated conformation and therefore may stabilize the pre-receptor-activated (native) Env trimer (Liu, J. et al. 2002 J Biol Chem 277:12891- 12900; Sanders, R. W. et al. 2002 J Nirol 76:8875-8889). It may be predicted that the affinity between the Ν-terminal helix of SIN and the C-terminal helix of HIN-l (as in H- S.Ν) is weaker than the affinity between the same- virus-type helices (all HIV-1 or all SIV), as evidenced by the finding that a peptide analogue of the HIN-l C-terminal helix which potently blocked HIN-l infectivity was required at a much higher concentration to inhibit the infectivity of HIV-2 (a virus closely related to SIN) (Wild, C. T. et al. 1994 Proc Natl Acad Sci USA 91:9770-9774). This weaker affinity between helices in H-S.Ν gpl40 may therefore promote gpl40 trimerization and reduce the pool of normative gpl40 available for aggregate formation. Alternatively, interaction between the Ν- and C-terminal helices may directly result in aggregation. The potentially advantageous structural properties of the H-S.Ν chimera (efficient trimerization, reduced aggregation) are fortuitous, since this construct includes several broadly neutralizing HJV-1 epitopes in the C-terminal segment of gp41 (Muster, T. et al. 1993 J Virol 67:6642-6647; Stiegler, G. et al. 2001 AIDS Res Hum Retrovir 17:1757-1765; Zwick, M. B. et al. 2001 J Virol 75:10892-10905; Zwick, M. B. et al. 2001 J Virol 75:12198-12208) that are absent in the H-S chimera. The non-HJN-1 segments of the H- S.Ν chimeras (the Ν-terminal half of gp41) includes the fusion peptide and the Ν-terminal alpha-helical regions, which have been found to be poorly immunogenic (Binley, J. M. et al. 1996 AIDS Res Hum Retrovir 12:911-924; Earl, P. L. et al. 1997 J Virol 71:2674-2684), and part of the immunodominant epitope, which generally elicits nonneutralizing antibodies. Other approaches used to promote HIN-l gpl40 trimerization include the addition of heterologous GCΝ4- or fibrinitin-based trimerization motifs to the C terminus of gpl40 (Yang, X. et al. 2000 J Virol 74:5716-5725; Yang, X. et al. 2000 J Virol 74:4746- 4754; Yang, X. et al. 2002 J Virol 76:4634-4642). The use of the more closely related SIN motif is contemplated as mimicking of the authentic HIN-l Env trimer. There is much current interest in the possible use of soluble Env analogues to elicit neutralizing-antibody responses. Env modifications aiming at improved elicitation of neutralizing antibodies include deletions of variable loops to expose underlying conserved epitopes (Barnett, S. W.
et al. 2001 J Virol 75:5526-5540; Cherpelis, S. et al. 2001 J Virol 75:1547-1550; Kim, Y. B. et al. 2003 Virology 305:124-137; Lu, S. et al. 1998 AIDS Res Hum Retrovir 14:151- 155; Sanders, R. W. et al. 2000 J Virol 74:5091-5100), mutations to enhance proteolytic cleavage and introduce disulfide bonds capable of stabilizing gpl20-gp41 complexes (Binley, J. M. et al. 2000 J Virol 74:627-643; Binley, J. M. et al. 2002 J Virol 76:2606- 2616; Schiilke, N. et al. 2002 J Virol 76:7760-7776), chemical coupling of Env and CD4 to stabilize the CD4-induced conformation (Fouts, T. et al. 2002 Proc Natl Acad Sci USA 99:11842-11847), deletion of the gp41 fusion peptide and the interhelical region to stabilize the pre-receptor-activated conformation (Chakrabarti, B. K. et al. 2002 J Virol 76:5357- 5368), and hyperglycosylation to focus the humoral immune response toward known broadly neutralizing epitopes (Pantophlet, R. et al. 2003 J Virol 77:5889-5901). The strategy employed with the H-S.N chimera described in the present study offers an Env format allowing such modifications to be applied in a trimeric context. EXAMPLE 1 env expression, purification, and gel filtration. The recombinant vaccinia virus vBD5 (Doranz, B. J. et al. 1999 J Virol 73:10346-10358) was used to express gpl40 derived from the HIN-ljR-FL (GenBank accession number U63632). Recombinant vaccinia viruses expressing HIV-1 gpl40 with deletion mutations and HIN-1/SJN gpl40 chimeras were produced by standard recombinant techniques using the HJVJ -FL Env-encoding plasmid pCB28 (Broder, C. C. and E. A. Berger 1995 Proc Natl Acad Sci USA 92:9004- 9008) and DΝA extracted from the recombinant vaccinia virus vAEl (Edinger, A. L. et al. 2000 J Virol 74:7922-7935). DΝA extracted and amplified from the vAEl virus encoded gpl40 derived from the SIVCP-MAC isolate (LaBranche, C. C. et al. 1994 J Virol 68:5509- 5522; Erratum, 68:7665-7667) except for the following amino acid differences: S559L, L573N, T575K and I588T. The amino acid numbering used here is based on the full-length
, HIV-IJR.FL or SINcp-MAC Env sequence with the initial methionine of the signal peptide as 1. For all viruses, gpl40 expression was under the control of a synthetic early-late vaccinia virus promoter (Chakrabarti, S. et al. 1997 BioTechniques 23:1094-1097). For env expression using recombinant vaccinia viruses, BS-C-1 cells (an African green monkey kidney cell line) were infected at a multiplicity of infection of 5 and overlaid with serum- free Opti-MEM (Gibco BRL). After 1.5 to 2 days, the supernatant was centrifuged to remove cellular debris and then adjusted to 0.2% Triton® X-100 to inactivate vaccinia virus. Although the cleavage site was intact in the vaccinia virus-expressed gpl40
molecules, subsequent analysis showed that most of the secreted protein was uncleaved. Cleavage site-negative HIN-l ADA and SFVMac32H gpl40 (Chen, B. et al. 2000 J Biol Chem 275:34946-34953; Zhang, C. W.-H. et al. 2001 J Biol Chem 276:39577-39585) were expressed in stably transfected CHO-Lec3.2.8.1 cells, which yield mannose-only oligosaccharides, using serum-free Opti-MEM plus sodium butyrate (2 mM final concentration) to induce expression. All gpl40s were purified from the supernatants using lentil lectin affinity chromatography, as previously described (Center, R. j. et al. 2000 J Virol 74:4448-4455). Concentrated eluates were subjected to gel filtration chromatography using a 16/60 Superdex 200 column (Amersham Pharmacia Biotech AB) with phosphate- buffered saline as the buffer. A flow rate of 0.5 ml/min was used, and 1-ml fractions were collected. Env generally comprised 90% of the total protein in the fractions analyzed, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Coomassie staining. Similar methods were used to express and purify unmodified and chimeric gpl40 derived from clade C 93MW965 (GenBank accession number U08455). Electrophoresis, immunoblotting and chemical cross-Unking. Individual gel filtration fractions were subjected to SDS-PAGE (8% polyacrylamide) under reducing conditions and transferred to nitrocellulose membranes. After being blocked with 4% bovine serum albumin, the membranes were sequentially probed with a rabbit serum raised against HIN-l gpl40 (IIIB/LAI strain) or monoclonal antibodies and iodinated protein A. Signal was visualized and quantified by phosphor screen autoradiography using a scanner and ImageQuant software (Molecular Dynamics). Cross-linking was performed by incubating samples in the presence of ethylene glycol bis(succinimidylsuccinate) (EGS) (Pierce) at a final concentration of 5 mM for 30 min at room temperature, followed by quenching with a final concentration of 100 mM glycine prior to SDS-PAGE (5% polyacrylamide) and immunoblotting as above. Blue-native PAGE was performed as previously described (Schϋlke, Ν. et al. 2002 J Virol 76:7760-7776) using 4 to 12% Bis- Tris gels (Invitrogen). Sedimentation equilibrium and velocity. Sedimentation equilibrium and velocity analyses were performed using a Beckman-Coulter analytical eight-cell An-50 Ti rotor with an Optima XL- A/I analytical ultracentrifuge in the absorbance optical-scanning mode. Sedimentation equilibrium was used to determine the weight-average molecular weight of gpl40 within individual gel filtration fractions that were concentrated approximately eightfold (final concentration, approximately 0.25 to 0.75 mg/ml) prior to analysis. Cells
were loaded with volumes of 120 to 135 μl of sample and measured at either 230 or 280 nm in an optical density range of approximately 0.2 to 0.4 absorbance unit (AU). Absorbance- versus-radial-position step scanning data at radial increments of 0.001 cm with 20 repeats were obtained at 10°C using three different rotor speeds between 5,000 and 9,000 rpm for each sample. A global nonlinear regression analysis was performed using the data analysis software package provided by Beckman-Coulter instruments (version 4.0 and Microcal version 4.1). The partial specific volume for each gpl40 species was calculated from the amino acid sequence and an estimated partial specific volume of 0.622 for the carbohydrate component based on an analysis of glycoproteins (Lewis, M. S. and R. P. Junghans 2000 Methods Enzymol 321:136-149). Mass-spectral analysis of vaccinia virus-expressed SJNcp. MAC and HIV-IJR.FL gpl20 (cleaved from g l40) produced mass values of 99 and 93 kDa, respectively, allowing for the determination of the average carbohydrate mass for each potential Ν-linked glycosylation site (2.06 kDa for SIN and 1.739 kDa for HIN-l). These values were used to estimate the carbohydrate mass of each vaccinia virus-expressed gpl40 species. For gpl40 produced in CHO-Lec3.2.8.1 cells, the mass of oligosaccharides with five mannose groups was used to calculate the carbohydrate component, assuming utilization of all potential Ν-linked sites. Boundary sedimentation velocity analysis was performed at 20°C with rotor speeds of 25,000 or 30,000 rpm and scanning at 230 nm. The latter experiments were carried out directly after the sedimentation equilibrium analysis by gently tilting the rotor until the contents of the cells were uniformly redistributed. Sedimentation coefficient distribution analysis was performed as previously described (Schuck, P. 2000 Biophys J78:1606-1619) using Sedfit software. The data presented were subjected to maximum-entropy regularization (Schuck, P. 2000 Biophys J 78:1606-1619). This statistical treatment produced distributions consistent with the raw data within 95% confidence limits. Maximum-entropy regularization combined with the inherently heterogeneous glycosylation (and therefore mass) of Env protomers tends to merge closely spaced peaks. STEM. Scanning transmission electron microscopy (STEM) was performed as previously described (Center, R. J. et al. 2001 Proc Natl Acad Sci USA 98:14877-14882). Briefly, 5-μl aliquots of gpl40 and tobacco mosaic virus were sequentially applied to copper grid-supported carbon films. The grids were washed, plunge-frozen into liquid ethane, cryotransferred to an HB501 STEM (NG Scientific), and freeze-dried. Annular dark-field images were acquired digitally using an electron dose of approximately
103 e/nm2 and an acquisition time of 100 s. Images were processed and quantified using the IMAGE program (available on the World Wide Web at rsb.info.nih.gov/nih-image/). Mass values were calibrated using tobacco mosaic virus particles contained in the same field as gpl40. EXAMPLE 2 We compared the antigenicity and immunogenicity of the JR-FL H-S.N gpl40 trimers with HIVJR-FL gpl20 monomers. Guinea pigs were immunized three times with 40 μg of protein in a Ribi-type adjuvant (monophosphoryl-lipid A plus trehalose dicorynomycolate emulsion) at 4-week intervals. Ribi adjuvant was used as it is predicted to maximize the presentation of conformational epitopes, and is suitable for use in small animals. The most potent broadly neutralizing monoclonal antibodies identified to date are known to target conformational epitopes. Blood samples were taken before the first immunization (to control for non-specific antibody binding and neutralization) and 2 weeks after the third immunization, when antibody levels are expected to peak. Antibody binding data for individual animals are shown in Fig. 8 A. The chimeric gpl40 immunogen gave a much higher antibody response than gpl20 when measured against gpl20 (Fig. 8 A) or gpl40 trimers (Fig. 8B). Pre-immune sera showed no significant binding to either chimeric gpl40 or gpl20. Neutralization titers were determined as the reciprocal of the serum dilution that neutralized 50% of the homologous virus JR-FL or a heterologous clade B virus MN. JR-FL is difficult to neutralize and titers of 1:20 or higher are considered significant. Sera from Guinea pigs immunized with gpl20 did not display significant neutralization at the highest concentration used (1 :20), whereas sera from those immunized with gpl40 trimers had low but significant neutralizing titers (Fig. 8C). The heterologous isolate MN is more easily neutralized than JR-FL and the neutralization titers obtained with trimeric gpl40 against MN virus were considerably higher than against JR-FL (Fig. 8D). In contrast, significant neutralization with gpl20 immunogen was still not obtained against this more readily neutralized virus (Fig. 8D). In summary, these data indicate that chimeric gpl40 trimers is a superior immunogen compared to monomeric gpl20, both quantitatively in terms of binding titer and qualitatively in terms of viral neutralization. EXAMPLE 3 We envision env trimers as providing a scaffold for making additional modifications to improve immunogenicity. Deletion of the second variable domain of gpl20 has been proposed to expose coreceptor binding domains which may be good targets
for cross-strain neutralization due to their conserved function and therefore presumed conserved structure (Barnett, S. W. et al. 2001 J Virol 75:5526-5540). We have expressed and purified a chimeric gpl40 with a deleted V2 domain and demonstrated by sedimentation equilibrium that peak gel filtration fractions have an average composition of 3.3 subunits, consistent with trimeric structure. Antigenicity studies were performed to examine the ability of monoclonal antibodies with epitopes overlapping critical parts of the coreceptor binding domain to immunoprecipitate labeled chimeric gpl40. Of two such antibodies tested (48d and 17b), an average of 12-fold and 2.7-fold more V2 deleted chimeric gpl40 was immunoprecipitated respectively compared to gpl40 chimera with the V2 domain intact. These data support the notion that additional modifications can be made in the context of chimeric gpl40 trimers that will further enhance immunogenicity and the ability to elicit neutralizing antibodies.
While the present invention has been described in some detail and form for purposes of clarity and understanding, one skilled in the art will appreciate that various changes in form and detail can be made without departing from the true scope of the invention. All figures, tables, and appendices, as well as patents, applications, and publications, referred to above, are hereby incorporated by reference.
Claims
WHAT IS CLAIMED IS: 1. A chimeric polypeptide comprising a chimeric human immunodeficiency virus (HIN) envelope (Env) in which all or a portion of the Ν-terminal half of the gp41 ectodomain is replaced by the corresponding region of simian immunodeficiency virus (SIN) to promote trimerization and maintain targets of neutralizing antibodies.
2. The chimeric polypeptide of claim 1, wherein the non-HIN segment extends from the fusion peptide to the interhelical region.
3. The chimeric polypeptide of claim 1, wherein the non-HIN segment consists essentially of the Ν helix.
4. The chimeric polypeptide of claim 1, where the HIV is type 1 (HIV-1).
5. The chimeric polypeptide of claim 1, wherein the HIV is clade B or C of type 1 (HIN-l).
6. The chimeric polypeptide of claim 1 , wherein the HIN Env is g l40.
7. The chimeric polypeptide of claim 1, wherein the non-HIN segment extends from amino acid Arg-571 of the consensus sequence to Ser-660 of the consensus sequence.
8. The chimeric polypeptide of claim 1, wherein the SIN segment extends from amino acid Gly-528 of the reference sequence to Cys-613 of the reference sequence.
9. The chimeric polypeptide claim 1, wherein the non-HIN segment is at least 3, 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50 amino acids in length.
10. The chimeric polypeptide of claim 1 , wherein the N2 domain is deleted.
11. A polynucleotide that encodes a chimeric polypeptide of any of claims 1-10.
12. A vector comprising a polynucleotide of claim 11.
13. A host cell comprising a vector of claim 12.
14. A method for producing a chimeric polypeptide of any of the above claims, said method comprising maintaining a host cell of claim 13 under conditions suitable for expression of said polynucleotide, whereby said chimeric polypeptide is produced.
15. The method of claim 14, further comprising isolating the chimeric polypeptide.
16. A method for inhibiting HIN infection in a subject comprising administering to the subject an effective amount of a chimeric polypeptide of any of the above claims or the polynucleotide of claim 11, to inhibit HIN infection of a cell, thereby inhibiting virus infection.
17. The method of claim 16, wherein the subject is a human.
18. A method for producing an immune response to HIN in a subject comprising administering to the subject an effective amount of a chimeric polypeptide of any of the above claims or the polynucleotide of claim 11, to produce an immune response to the virus.
19. The method of claim 18, wherein the subject is a human.
20. A method for identifying an agent that inhibits an interaction between a HIN virus and a HIN virus co-receptor or receptor comprising: (a) contacting a chimeric polypeptide of any of the above claims with a HIN virus co-receptor or receptor under conditions allowing the chimeric polypeptide and co-receptor or receptor to bind, in the presence and absence of a test agent; and (b) detecting binding in the presence and absence of the test agent, wherein a decreased binding in the presence of the test agent thereby identifies an agent that inhibits binding between the virus and the virus co-receptor or receptor.
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| PCT/US2004/033505 WO2005035555A1 (en) | 2003-10-10 | 2004-10-12 | Hiv/siv env chimeras that promote trimerization and maintain targets of neutralizing antibodies |
Country Status (1)
| Country | Link |
|---|---|
| WO (1) | WO2005035555A1 (en) |
Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP1754715A1 (en) * | 2005-08-19 | 2007-02-21 | Bundesrepublik Deutschland vertreten durch das Bundesminsterium für Gesundheit, dieses vertr. durch das Robert-Koch-Institut | Vaccine on the basis of virus-neutralising antibodies |
| WO2010052214A3 (en) * | 2008-11-05 | 2010-07-22 | Glaxosmithkline Biologicals S.A. | Novel method |
| JP2010530356A (en) * | 2007-03-27 | 2010-09-09 | ザ リージェンツ オブ ザ ユニバーシティ オブ カリフォルニア | Acute infection HIV envelope sign |
| WO2015036061A1 (en) * | 2013-09-16 | 2015-03-19 | Glaxosmithkline Biologicals Sa | Novel compositions |
| WO2015036062A1 (en) * | 2013-09-16 | 2015-03-19 | Glaxosmithkline Biologicals Sa | Novel compositions |
| WO2014018850A3 (en) * | 2012-07-27 | 2015-07-23 | The Trustees Of The University Of Pennsylvania | Cytoplasmic tail modifications to boost surface expression and immunogenicity of envelope glycoproteins |
| EP3178842A1 (en) * | 2008-08-14 | 2017-06-14 | Los Alamos National Security, LLC | Polyvalent vaccine |
-
2004
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Cited By (14)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2007020243A2 (en) * | 2005-08-19 | 2007-02-22 | Bundesrepublik Deutschland, Vertreten Durch Das Bundesministerium Für Gesundheit, Dieses Vertreten Durch Das Robert-Koch-Institut | Vaccine based on virus-neutralizing antibodies |
| WO2007020243A3 (en) * | 2005-08-19 | 2007-08-09 | Bundesrep Deutschland | Vaccine based on virus-neutralizing antibodies |
| EP1754715A1 (en) * | 2005-08-19 | 2007-02-21 | Bundesrepublik Deutschland vertreten durch das Bundesminsterium für Gesundheit, dieses vertr. durch das Robert-Koch-Institut | Vaccine on the basis of virus-neutralising antibodies |
| US9844590B2 (en) | 2005-08-23 | 2017-12-19 | Duke University | Mosaic clade M human immunodeficiency virus type 1 (HIV-1) gag immunogens |
| US9821053B2 (en) | 2005-08-23 | 2017-11-21 | Duke University | Human Immunodeficiency Virus type 1 group M consensus and mosaic envelope glycoproteins |
| JP2010530356A (en) * | 2007-03-27 | 2010-09-09 | ザ リージェンツ オブ ザ ユニバーシティ オブ カリフォルニア | Acute infection HIV envelope sign |
| EP2129395A4 (en) * | 2007-03-27 | 2012-05-09 | Univ California | ACUTE HIV TRANSMITTED ENVELOPE SIGNATURES |
| EP3178842A1 (en) * | 2008-08-14 | 2017-06-14 | Los Alamos National Security, LLC | Polyvalent vaccine |
| WO2010052214A3 (en) * | 2008-11-05 | 2010-07-22 | Glaxosmithkline Biologicals S.A. | Novel method |
| WO2014018850A3 (en) * | 2012-07-27 | 2015-07-23 | The Trustees Of The University Of Pennsylvania | Cytoplasmic tail modifications to boost surface expression and immunogenicity of envelope glycoproteins |
| WO2015036061A1 (en) * | 2013-09-16 | 2015-03-19 | Glaxosmithkline Biologicals Sa | Novel compositions |
| BE1021327B1 (en) * | 2013-09-16 | 2015-10-29 | Glaxosmithkline Biologicals Sa | NEW COMPOSITIONS. |
| BE1021315B1 (en) * | 2013-09-16 | 2015-10-28 | Glaxosmithkline Biologicals Sa | NEW COMPOSITIONS |
| WO2015036062A1 (en) * | 2013-09-16 | 2015-03-19 | Glaxosmithkline Biologicals Sa | Novel compositions |
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