WO2001000648A1 - Proteines d'enveloppe virale stabilisee et utilisations - Google Patents

Proteines d'enveloppe virale stabilisee et utilisations Download PDF

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Publication number
WO2001000648A1
WO2001000648A1 PCT/US2000/017267 US0017267W WO0100648A1 WO 2001000648 A1 WO2001000648 A1 WO 2001000648A1 US 0017267 W US0017267 W US 0017267W WO 0100648 A1 WO0100648 A1 WO 0100648A1
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protein
viral
nucleic acid
hiv
antibody
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PCT/US2000/017267
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English (en)
Inventor
James M. Binley
Norbert Schuelke
William C. Olson
Paul J. Maddon
John P. Moore
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Progenics Pharmaceuticals, Inc.
Aaron Diamond Aids Research Centre
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Application filed by Progenics Pharmaceuticals, Inc., Aaron Diamond Aids Research Centre filed Critical Progenics Pharmaceuticals, Inc.
Priority to EP00944801A priority Critical patent/EP1198468A4/fr
Priority to CA002370517A priority patent/CA2370517A1/fr
Priority to JP2001507055A priority patent/JP2003509013A/ja
Priority to AU58842/00A priority patent/AU782123B2/en
Publication of WO2001000648A1 publication Critical patent/WO2001000648A1/fr
Priority to HK02107688.0A priority patent/HK1046911A1/zh
Priority to AU2005220250A priority patent/AU2005220250B2/en

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/08—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses
    • C07K16/10—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses from RNA viruses
    • C07K16/1036—Retroviridae, e.g. leukemia viruses
    • C07K16/1045—Lentiviridae, e.g. HIV, FIV, SIV
    • C07K16/1063—Lentiviridae, e.g. HIV, FIV, SIV env, e.g. gp41, gp110/120, gp160, V3, PND, CD4 binding site
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • A61P31/18—Antivirals for RNA viruses for HIV
    • 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
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/53—DNA (RNA) vaccination
    • 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

Definitions

  • the human immunodeficiency virus is the agent that causes AIDS, a lethal disease characterized by deterioration of the immune system.
  • the initial phase of the HIV replicative cycle involves the attachment of the virus to susceptible host cells followed by fusion of viral and cellular membranes. These events are mediated by the exterior viral envelope glycoproteins, which are first synthesized as a fusion-incompetent precursor envelope glycoprotein (env) known as gpl ⁇ O.
  • env envelope glycoprotein
  • the gpl60 glycoprotein is endoproteolytically processed to the mature envelope glycoproteins gpl20 and gp41, which are noncovalently associated on the surface of the virus.
  • the gpl20 surface protein contains the high affinity binding site for human CD4, the primary receptor for HIV, as well as domains that interact with fusion coreceptors, such as the chemokine receptors CCR5 and CXCR4.
  • the gp41 protein spans the viral membrane and contains at its amino-terminus a sequence of amino acids important for the fusion of viral and cellular membranes.
  • the HIV envelope glycoproteins assemble as noncovalent oligomers, almost certainly trimers, of gpl20/gp41 on the virus surface. The detailed events of viral entry remain poorly understood but involve gpl20 binding first CD4 then a fusogenic chemokine receptor, followed by gp41-mediated virus-cell fusion.
  • HIV envelope glycoproteins provide important targets for HIV vaccine development. Although most HIV- infected individuals mount a robust antibody (Ab) response to the envelope glycoproteins, most anti-gpl20 and anti-gp41 Abs produced during natural infection bind weakly or not at all to virions and are thus functionally ineffective. These Abs are probably elicited and affinity matured against "viral debris" comprising gpl20 monomers or improperly processed oligomers released from virions or infected cells. (Burton and Montefiori, AIDS, 11 [Suppl A] : 587, 1997)
  • T cell lines T cell lines (TCLA viruses) . These vaccines have consistently elicited Abs which neutralize the homologous strain of virus and some additional TCLA viruses. However, the Abs do not potently neutralize primary HIV-1 isolates ( ascola et al . , J. Infec . Dis. 173:340, 1996). Compared with
  • TCLA strains the more clinically relevant primary isolates typically possess a different cellular tropism, show a different pattern of coreceptor usage, and have reduced sensitivity to neutralization by soluble CD4 and Abs. These differences primarily map to the viral envelope glycoproteins
  • a second, more subtle problem is that the structure of key gpl20 epitopes can be affected by oligomerization.
  • a classic example is provided by the epitope for the broadly neutralizing human MAb IgGlbl2 (Burton et al . Science
  • gp41 MAbs A third example of the problems caused by the native structure of the HIV-1 envelope glycoproteins is provided by gp41 MAbs . Only a single gp41 MAb (2F5) is known to have strong neutralizing activity against primary viruses (Trkola et al., J Virol, 69: 6609, 1995), and among those tested, 2F5 alone is thought to recognize an intact, gpl20-gp41 complex (Sattentau et al , Virology 206: 713, 1995).
  • gp41 MAbs that bind to virions or virus-infected cells probably react with fusion- incompetent gp41 structures from which gpl20 has dissociated. Since the most stable form of gp41 is this post -fusion configuration ( eissenhorm et al , Nature, 387: 426, 1997), it can be supposed that most anti-gp41 Abs are raised (during natural infection or after gpl60 vaccination) to an irrelevant gp41 structure that is not present on the pre-fusion form.
  • MAbs broadly reactive human monoclonal antibodies
  • Mab IgGlbl2 blocks gpl20-CD4 binding
  • a second (2G12; Trkola et al . J Virol 70: 1100, 1996) acts mostly by steric hindrance of virus-cell attachment
  • 2F5 acts by directly compromising the fusion reaction itself.
  • Critical to understanding the neutralization capacity of these MAbs is the recognition that they react preferentially with the fusion-competent, oligomeric forms of the envelope glycoproteins, as found on the surfaces of virions and virus- infected cells.
  • Neutralizing anti-HIV Abs are capable of binding infectious virus while non-neutralizing Abs are not (Fouts et al AIDS Res Human Retrovir. 14: 591, 1998).
  • Neutralizing Abs also have the potential to clear infectious virus through effector functions, such as complement-mediated virolysis. Modifying the antigenic structure of the HIV envelope glycoproteins HIV-1 has evolved sophisticated mechanisms to shield key neutralization sites from the humoral immune response, and in principle these mechanisms can be "disabled" in a vaccine.
  • V3 loop which for TCLA viruses in particular is an immunodominant epitope that directs the antibody response away from more broadly conserved neutralization epitopes.
  • HIV-1 is also protected from humoral immunity by the extensive glycosylation of gpl20. When glycosylation sites were deleted from the V1/V2 loops of SIV gpl20, not only was a neutralization-sensitive virus created, but the immunogenicity of the mutant virus was increased so that a better immune response was raised to the wild-type virus (Reitter et al , Nat Med 4:679, 1998). Similarly, removing the V1/V2 loops from HIV-1 gpl20 renders the conserved regions underneath more vulnerable to Abs (Cao et al, J. Virol. 71: 9808, 1997), although it is not yet known whether this will translate into improved immunogenicity.
  • Gpl60 The full-length gpl60 molecule often aggregates when expressed as a recombinant protein, at least in part because it contains the hydrophobic transmembrane domain.
  • One such molecule is derived from a natural mutation that prevents the processing of the gpl60 precursor to gpl20/gp41 (VanCott et al J Virol 71: 4319, 1997).
  • the gpl60 precursor does not mediate virus-cell fusion and is a poor mimic of fusion- competent gpl20/gp41.
  • recombinant gpl60 molecules offered no advantages over gpl20 monomers (Gorse et al . , Vaccine 16: 493, 1998).
  • the protein lacks the transmembrane domain and the long, intracytoplasmic tail of gp41, but retains the regions important for virus entry and the induction of neutralizing Abs.
  • the secreted protein contains full-length gpl20 covalently linked through a peptide bond to the ectodomain of gp41.
  • the protein migrates in SDS -PAGE as a single species with an apparent molecular mass of approximately 140 kilodaltons (kDa) under both reducing and nonreducing conditions.
  • the protein forms higher molecular weight noncovalent oligomers, likely through interactions mediated by the gp41 moieties.
  • uncleaved gpl40 molecule does not adopt the same conformation as native gpl20-gp41. These include observations described herein and from the finding that uncleaved gpl20-gp41 complexes do not avidly bind fusion co-receptors (R. Doms, personal communication) . Furthermore, a gpl40 protein of this type was unable to efficiently select for neutralizing MAbs when used to pan a phage-display library, whereas virions were efficient (Parren et al, J Virol. 70:9046, 1996). We refer to the uncleaved gpl20-gp41 ectodomain material as gpl40UNC.
  • gpl40NON Cleavable but uncleaved gp!40
  • gpl60 is cleaved into gpl20 and gp41 by a cellular endoprotease of the furin family.
  • Mammalian cells have a finite capacity to cleave gpl20 from gp41, as we show below.
  • the envelope glycoproteins can saturate the endogenous furin enzymes and be secreted in precursor form. Since these molecules are potentially cleavable, we refer to them as gpl40NON.
  • gpl40 ⁇ O ⁇ migrates in SDS-PAGE with an apparent molecular mass of approximately 140 kDa under both reducing and nonreducing conditions. As shown below, gpl40NON appears to possess the same non-native topology as gpl40UNC.
  • gpl40CUT refers to full-length gpl20 and ectodomain gp41 fully processed and capable of forming oligomers as found on virions.
  • the noncovalent interactions between gpl20 and gp41 are sufficiently long- lived for the virus to bind and initiate fusion with new target cells, a process which is likely completed within minutes during natural infection.
  • the association has, however, to date proven too labile for the production of significant quantities of cleaved gpl40s in near homogenous form.
  • Such conditions might include elevated temperatures in the range of 37-60 °C and/or low concentrations of detergents or chaotropic agents.
  • the envelope proteins from such viruses could be subcloned into the pPPI4 expression vector and analyzed for stability using our methods as well.
  • stable heterodimers have been successfully created by introducing complementary "knob” and "hole” mutations in the binding partners (Atwell et al . , J. Mol. Biol. 4:26, 1997).
  • SU-TM stabilization can also be achieved by means of one or more introduced disulfide bonds.
  • mammalian retroviruses only the lentiviruses such as HIV have non-covalent associations between the surface (SU) and transmembrane (TM) glycoproteins.
  • TM transmembrane
  • the type C and type D retroviruses all have an inter-subunit disulfide bond.
  • the ectodomains of retroviral TM glycoproteins have a broadly common structure, one universal feature being the presence of a small, Cys-Cys bonded loop approximately central in the ectodomain.
  • gp41 and other lentiviral TM glycoproteins lack the third cysteine, the structural homologies suggest that one could be inserted in the vicinity of the short central loop structure. Thus there is strong mutagenic evidence that the first and last conserved regions of gpl20 (Cl and C5 domains) are probable contact sites for gp41.
  • the subject invention provides isolated nucleic acid molecules that encode mutant viral surface and transmembrane proteins in stabilized, antigenically authentic forms. This invention describes the design and synthesis of the stabilized viral proteins. Importantly, when appropriate methods are used to effect the stabilization, the viral proteins adopt conformations with desirable features.
  • the subject invention further provides protein- or nucleic acid- based vaccines comprising mutant viral envelope proteins, antibodies isolated or identified using mutant viral envelope proteins, pharmaceutical compositions comprising these vaccines or antibodies, and methods of using these compositions to treat or prevent infections from viruses such as HIV.
  • the invention describes applications of the mutant viral proteins to identify whether a compound is capable of inhibiting a virus, and compounds identified in this manner. Summary of the Invention
  • This invention provides an isolated nucleic acid which comprises a nucleotide segment having a sequence encoding a viral envelope protein comprising a viral surface protein and a corresponding viral transmembrane protein wherein the viral envelope protein contains one or more mutations in amino acid sequence that enhance the stability of the complex formed between the viral surface protein and the viral transmembrane protein.
  • This invention provides an isolated nucleic acid which comprises a nucleotide segment having a sequence encoding a mutant viral envelope protein which differs from the corresponding wild type viral envelope protein sequence in at least one amino acid which upon proteolysis yields a complex comprising a surface protein and a transmembrane protein which has enhanced stability relative to the corresponding complex obtained from the wild type envelope protein.
  • the viral surface protein is HIV-1 gpl20 or a modified form of gpl20 which has modified immunogenicity relative to wild type gpl20.
  • the transmembrane protein is HIV-1 gp41 or a modified form of gp41 which has modified immunogenicity relative to wild type gp41.
  • This invention provides a vaccine which comprises the above isolated nucleic acid.
  • the vaccine comprises a therapeutically effective amount of the nucleic acid.
  • the vaccine comprises a therapeutically effective amount of the protein encoded by the above nucleic acid.
  • the vaccine comprises a combination of the recombinant nucleic acid molecule and the mutant viral envelope protein.
  • This invention provides a method of treating a viral disease which comprises immunizing a virally infected subject with the above vaccines or a combination thereof, thereby treating the subj ect .
  • This invention provides a vaccine which comprises a prophylactically effective amount of the above isolated nucleic acid.
  • This invention provides a vaccine which comprises a prophylactically effective amount of the protein encoded by the above isolated nucleic acid.
  • This invention provides a method of reducing the likelihood of a subject becoming infected with a virus comprising administering the above vaccines or a combination thereof, thereby reducing the likelihood of the subject becoming infected with the virus.
  • This invention provides the above vaccine which comprises but is not limited to the following: a recombinant subunit protein, a DNA plasmid, an RNA molecule, a replicating viral vector, a non-replicating viral vector, or a combination thereof.
  • This invention provides a method of reducing the severity of a viral disease in a subject comprising administering the above vaccine or a combination thereof, prior to exposure of the subject to the virus, thereby reducing the severity of the viral disease in the subject upon subsequent exposure to the virus.
  • This invention provides a mutant viral envelope protein which differs from the corresponding wild type protein in at least one amino acid which upon proteolysis yields a complex comprising a surface protein and a transmembrane protein which has enhanced stability relative to the corresponding complex obtained from the wild type envelope protein.
  • This invention provides a complex comprising a viral surface protein and a viral transmembrane protein which has enhanced stability relative to the corresponding complex obtained from the wildtype envelope protein, yielded by the proteolysis of a mutant viral envelope protein with a sequence which differs from the corresponding wild type protein sequence in at least one amino acid.
  • This invention provides a mutant viral envelope protein which is encoded by the above nucleic acid molecule.
  • This invention provides a vaccine which comprises a therapeutically effective amount of the above protein or complex.
  • This invention also provides a vaccine which comprises a prophylactically effective amount of the above protein or complex.
  • This invention provides a method of stimulating or enhancing in a subject production of antibodies which recognize the above protein or complex.
  • This invention provides a method of stimulating or enhancing in a subject the production of cytotoxic T lymphocytes which recognize the above protein.
  • This invention provides an antibody capable of specifically binding to the above mutant protein.
  • This invention also provides an antibody which is capable of specifically binding to the above mutant protein or complex but not to the wild type protein or complex.
  • This invention provides an antibody, antibody chain or fragment thereof identified using the viral envelope protein encoded by the above recombinant nucleic acid molecule.
  • the antibody may be of the IgM, IgA, IgE or IgG class or subclasses thereof .
  • the above antibody fragment includes but is not limited to Fab, Fab', (Fab') 2, Fv and single chain antibodies.
  • This invention provides an isolated antibody light chain of the above antibody, or fragment or oligomer thereof.
  • This invention also provides an isolated antibody heavy chain of the above antibody, or fragment or oligomer thereof.
  • This invention also provides one or more CDR regions of the above antibody.
  • the antibody is derivatized.
  • the antibody is a human antibody.
  • the antibody includes but is not limited to monoclonal antibodies and polyclonal antibodies. In one embodiment, antibody is humanized.
  • This invention provides an isolated nucleic acid molecule encoding the above antibody.
  • This invention provides a method of reducing the likelihood of a virally exposed subject from becoming infected with the virus comprising administering the above antibody or the above isolated nucleic acid, thereby reducing the likelihood of the subject from becoming infected with the virus.
  • This invention provides a method of treating a subject infected with a virus comprising administering the above antibody or the above isolated nucleic acid, thereby treating the subject.
  • the virus is HIV.
  • This invention provides an agent capable of binding the mutant viral envelope protein encoded by the above recombinant nucleic acid molecule. In one embodiment, the agent inhibits viral infection.
  • This invention provides a method for determining whether a compound is capable of inhibiting a viral infection comprising :
  • step (D) comparing the amount of bound reporter molecule in step (C) with the amount determined in the absence of the compound, a decrease in the amount indicating that the compound is capable of inhibiting infection by the virus, thereby determining whether a compound is capable of inhibiting a viral infection.
  • This invention provides a method for determining whether a compound is capable of inhibiting a viral infection which comprises:
  • step (c) measuring the amount of binding of envelope protein to receptor or receptor mimic; (d) comparing the amount of binding determined in step (c) with the amount determined in the absence of the compound, a decrease in the amount indicating that the compound is capable of inhibiting infection by the virus, thereby determining whether a compound is capable of inhibiting a viral infection.
  • This invention further provides a simple method for determining whether a subject has produced antibodies capable of blocking the infectivity of a virus.
  • This invention provides the above method wherein the compound was not previously known.
  • This invention provides a compound determined to be capable of inhibiting a viral infection by the above methods .
  • This invention provides a pharmaceutical composition comprising an amount of the compound effective to inhibit viral infection determined by the above methods to be capable of inhibiting viral infection and a pharmaceutically acceptable carrier.
  • the viral infection is HIV-1 infection.
  • the virus is HIV.
  • This invention provides a mutant viral envelope protein which differs from the corresponding wild type protein in at least one amino acid which yields a complex comprising a surface protein and a transmembrane protein which has enhanced stability relative to the corresponding complex obtained from the wild type envelope protein, wherein the surface protein and transmembrane protein are encoded by different nucleic acids.
  • This invention provides a complex comprising a viral surface protein and a viral transmembrane protein which has enhanced stability relative to the corresponding complex obtained from the wildtype envelope protein, yielded by the proteolysis of a mutant viral envelope protein with a sequence which differs from the corresponding wild type protein sequence in at least one amino acid, wherein the surface protein and transmembrane protein are encoded by different nucleic acids.
  • This invention provides an antibody which binds to the above protein or above complex but does not cross react with the individual monomeric surface protein or the individual monomeric transmembrane protein. This invention provides the above antibody capable of binding to the HIV- 1 virus.
  • the cartoons depict: i) Monomeric gpl20; ii) Full-length recombinant gpl60 iii) Proteolytically unprocessed gpl40 trimer with the peptide bond maintained between gpl20 and gp41 (gpl40UNC or gpl40NON) ; iv) The SOS gpl40 protein, a proteolytically processed gpl40 stabilized by an intermolecular disulfide bond; v) Native, virion- associated gpl20-gp41 trimer.
  • the shading of the gpl40UNC protein (iii) indicates the major antibody-accessible regions that are poorly, or not, exposed on the SOS gpl40 protein or on the native gpl20-gp41 trimer.
  • the open boxes at the C-terminus of gpl20 and the N-terminus of gp41 indicate the regions that are mutated in the gpl40UNC protein to eliminate the cleavage site between gpl20 and gp41.
  • the 35S-labelled envelope glycoproteins secreted from transfected 293T cells were immunoprecipitated with anti- gpl20 and anti-gp41 MAbs, then analyzed by SDS-PAGE.
  • the MAbs used were either 2G12 (anti-gpl20 C3-V4 region) or F91 (anti-gpl20 CD4 binding site region) .
  • the positions of the two cysteine substitutions in each protein are noted above the lanes.
  • the gpl40WT protein is shown in lane 15. All proteins were expressed in the presence of co- transfected furin, except for the gpl40WT protein.
  • the efficiency of intermolecular disulfide bond formation is dependent upon the positions of the cysteine substitutions
  • the 35S-labelled envelope glycoproteins secreted from 293T cells co-transfected with furin and the various gpl40 mutants were immunoprecipitated with the anti-gpl20 MAb 2G12, then analyzed by SDS-PAGE.
  • the intensities of the 140kDa and 120kDa bands were determined by densitometry and the gpl40/gpl40+gpl20 ratio was calculated and recorded.
  • the extent of shading is proportional to the magnitude of the gpl40/gpl40+gpl20 ratio.
  • 293T cells were transfected with plasmids expressing gpl40 proteins and, when indicated, a furin-expressing plasmid.
  • the secreted, 35S-labelled glycoproteins were immunoprecipitated with the indicated MAbs and analyzed by SDS-PAGE under reducing (+DTT) or nonreducing conditions .
  • 293T cells were transfected with plasmids expressing wild type or mutant gpl40s in the presence or absence of exogenous furin as indicated. 35S-labeled supematants were prepared and analyzed by radioimmunoprecipitation with MAb 2G12 as described above. Lane 1: SOS gpl40 protein. Lane 2: gpl40WT plus furin. Lane 3: gpl40WT without furin. (A) HIV-1 DH123. (B) HIV-1 HxB2
  • Figure 10 Amino acid sequences of the glycoproteins with various deletions in the variable regions.
  • the deleted wild-type sequences are shown in the white shade and include the following: ⁇ V1 : D132-K152; ⁇ V2 : F156-I191; ⁇ V1V2 ' : D132- K152 and F156-I191; ⁇ V1V2*: V126-S192; ⁇ V3 : N296-Q324 Figure 11
  • Nucleotide (A) and amino acid (B) sequences for HIV-1 JR _ FL SOS gpl40 The amino acid numbering system corresponds to that for wild-type JR-FL (Genbank Accession #U63632) .
  • the cysteine mutations are indicated in underlined bold type face.
  • the amino acid numbering system corresponds to that for wild-type JR-FL (Genbank Accession #U63632) .
  • the cysteine mutations are indicated in underlined bold type face.
  • the amino acid numbering system corresponds to that for wild-type JR-FL (Genbank Accession #U63632) .
  • the cysteine mutations are indicated in underlined bold type face.
  • This invention provides an isolated nucleic acid which comprises a nucleotide segment having a sequence encoding a viral envelope protein comprising a viral surface protein and a corresponding viral transmembrane protein wherein the viral envelope protein contains one or more mutations in amino acid sequence that enhance the stability of the complex formed between the viral surface protein and transmembrane protein.
  • This invention provides an isolated nucleic acid which comprises a nucleotide segment having a sequence encoding a mutant viral envelope protein which differs from the corresponding wild type viral envelope protein sequence in at least one amino acid which upon proteolysis yields a complex comprising a surface protein and a transmembrane protein which has enhanced stability relative to the corresponding complex obtained from the wild type envelope protein.
  • inhibit the stability means make more long-lived or resistant to dissociation.
  • the interaction may be stabilized by the introduction of disulfide bonds, salt bridges, hydrogen bonds, hydrophobic interactions, favorable van der Waals contacts, a linker peptide or a combination thereof.
  • the stabilizing interactions may be introduced by recombinant methods.
  • stabilized viral envelope proteins may be obtained by selection methods such as exposing a virus to conditions known to destabilize the interaction between the surface and transmembrane envelope proteins, and then selecting for resistant viruses. This process may be repeated one or more times until one obtains viral envelope proteins with the desired stability.
  • one may screen isolates for naturally occurring mutations that enhance the stability of the interaction between the surface and transmembrane proteins, relative to the stability observed for prototypic wild type viral envelope proteins.
  • the invention does not encompass known viral proteins wherein the endoproteolytic processing of the precursor envelope protein to separate surface and transmembrane proteins is prevented by expressing the protein in the absence of sufficient quantities of the endoprotease or by mutating the endoproteolytic cleavage site in the absence of additional mutations, such as the addition of a linker peptide.
  • the viral surface and transmembrane proteins are physically joined by a covalent bond but are not known to form a complex, as illustrated in Figure 1.
  • the virus is a lentivirus.
  • the virus is the simian immunodeficiency virus.
  • Another embodiment of the above virus is the human immunodeficiency virus (HIV) .
  • the virus may be either of the two known types of HIV (HIV-1 or HIV-2) .
  • the HIV-1 virus may represent any of the known major subtypes
  • the human immunodeficiency virus is a primary isolate.
  • the human immunodeficiency virus is HIV-1 JR _ FL.
  • the human immunodeficiency virus is HIV-1 DH123
  • the human immunodeficiency virus is HIV-l ⁇ . ⁇
  • the human immunodeficiency virus is HIV-1 89 6
  • the human immunodeficiency virus is
  • HIV-1 HXB2 HIV-1 HXB2 .
  • HIV-1 JR _ FL is a strain that was originally isolated from the brain tissue of an AIDS patient taken at autopsy and co- cultured with lectin-activated normal human PBMCs (O'Brien et al, Nature, 348: 69, 1990) HIV-1 JR _ FL is known to utilize CCR5 as a fusion coreceptor and has the ability to replicate in phytohemagglutinin (PHA) -stimulated PBMCs and blood-derived macrophages but does not replicate efficiently in most immortalized T cell lines.
  • PHA phytohemagglutinin
  • HIV-1 DH123 is a clone of a virus originally isolated from the peripheral mononuclear cells (PBMCs) of a pateint with AIDS (Shibata et al . , J. Virol 69:4453, 1995). HIV-1 DH123 is known to utilize both CCR5 and CXCR4 as fusion coreceptors and has the ability to replicate in PHA- stimulated PBMCs, blood-derived macrophages and immortalized T cell lines.
  • PBMCs peripheral mononuclear cells
  • HlV-l cur ,. ! is a cloned virus originally isolated from the peripheral blood mononuclear cells of a hemophilia B patient with AIDS (Takeuchi et al . , Jpn J Cancer Res 78:11 1987) .
  • HIV-I G ⁇ ,. ! is known to utilize both CCR5 and CXCR4 as fusion coreceptors and has the ability to replicate in PHA-stimulated PBMCs, blood-derived macrophages and immortalized T cell lines.
  • HIV-1 89 6 is a cloned virus originally isolated from a patient with AIDS (Collman et al , J. Virol. 66: 7517, 1992) .
  • HIV-1 896 is known to utilize both CCR5 and CXCR4 as fusion coreceptors and has the ability to replicate in PHA-stimulated PBMCs, blood-derived macrophages and immortalized T cell lines.
  • HIV-1 HXB2 is a TCLA virus that is known to utilize CXCR4 as a fusion coreceptor and has the ability to replicate in PHA-stimulated PBMCs and immortalized T cell lines but not blood derived macrophages .
  • the above strains are used herein to generate the mutant viral envelope proteins of the subject invention, other HIV-1 strains could be substituted in their place as is well known to those skilled in the art.
  • One embodiment of the above viral surface protein is gpl20 or a modified form of gpl20 which has modified immunogenicity relative to wild type gpl20.
  • the modified gpl20 molecule is characterized by the absence of one or more variable loops present in wild type gpl20.
  • the variable loop comprises VI, V2 , or V3.
  • the modified gpl20 molecule is characterized by the absence or presence of one or more canonical glycosylation sites not present in wild type gpl20.
  • one or more canonical glycosylation sites are absent from the V1V2 region of the gpl20 molecule.
  • the transmembrane protein is gp41 or a modified form of gp41 which has modified immunogenicity relative to wildtype gp41.
  • the transmembrane protein is full-length gp41.
  • the transmembrane protein contains the ectodomain and membrane anchoring sequence of gp41 but lacks a portion or all of the gp41 cytoplasmic sequences.
  • the transmembrane protein is the gp41 ectodomain.
  • the transmembrane protein is modified by deletion or insertion of one or more canonical glycosylation sites.
  • the above viral surface protein is gpl20 or a derivative thereof.
  • the gpl20 molecule has been modified by the deletion or truncation of one or more variable loop sequences .
  • the variable loop sequences include but are not limited to VI, V2 , V3 or a combination thereof.
  • the gpl20 molecule has been modified by the deletion or insertion of one or more canonical glycosylation sites.
  • the region of gpl20 from which the canonical glycosylation sites are deleted includes but is not limited to the V1V2 region of the gpl20 molecule.
  • variable loop sequences for HIV-1 JR _ FL are illustrated in Figure 10.
  • the amino acid sequences in these variable loops will vary for other HIV isolates but will be located in homologous regions of the gpl20 envelope glycoprotein.
  • canonical glycosylation site includes but is not limited to an Asn-X-Ser or Asn-X-Thr sequence of amino acids that defines a site for N-linkage of a carbohydrate.
  • Ser or Thr residues not present in such sequences to which a carbohydrate can be linked through an O-linkage are “canonical glycosylation sites.”
  • a mutation of the Ser and Thr residue to an amino acid other than a serine or threonine will remove the site of O- linked glycosylation.
  • “derivatives” include but are not limited to the gp41 ectodomain, gp41 modified by deletion or insertion of one or more glycosylation sites, gp41 modified so as to eliminate or mask the well- known imunodominant epitope, a gp41 fusion protein, and gp41 labeled with an affinity ligand or other detectable marker .
  • ectodomain means the extracellular region or portion thereof exclusive of the transmembrane spanning and cytoplasmic regions .
  • the stabilization of the mutant viral envelope protein is achieved by the introduction of one or more cysteine-cysteine bonds between the surface and transmembrane proteins.
  • one or more amino acids which are o adjacent to or which contain an atom within 5 Angstroms of an introduced cysteine are mutated to a noncysteine residue .
  • adjacent to means immediately preceding or following in the primary sequence of the protein.
  • mutated means that which is different from the wild-type.
  • cyste residue means an amino acid other than cysteine.
  • one or more cysteines in gpl20 or modified form of gpl20 are disulfide linked to one or more cysteines in gp41 or modified form of gp41.
  • a cysteine in the C5 region of gpl20 or modified form of gpl20 is disulfide linked to a cysteine in the ectodomain of gp41 or modified form.
  • the disulfide bond is formed between a cysteine introduced by an A492C mutation in gpl20 or modified form of gpl20 and an T596C mutation in gp41 or modified form of gp41.
  • C5 region means the fifth conserved sequence of amino acids in the gpl20 glycoprotein.
  • the C5 region includes the carboxy-terminal amino acids.
  • the unmodified C5 region consists of the amino acids GGGDMRDNWRSELYKYKWKIEPLGVAPTKAKRRWQRE. Amino acid residues 462-500 of the sequence set forth in figure 3A have this sequence.
  • the C5 region will comprise a homologous carboxy-terminal sequence of amino acids of similar length.
  • A492C mutation refers to a point mutation of amino acid 492 in HIV-1 JR . FL gpl20 from alanine to cysteine. Because of the sequence variability of HIV, this amino acid will not be at position 492 in all other HIV isolates. For example, in HIV-1 NL4 _ 3 the corresponding amino acid is A499 (Genbank Accesion # AAA44992) . It may also be a homologous amino acid other than alanine or cysteine. This invention encompasses cysteine mutations in such amino acids, which can be readily identified in other HIV isolates by those skilled in the art.
  • T596C mutation refers to a point mutation of amino acid 596 in HIV-1 JR _ FL gp41 from threonine to cysteine.
  • this amino acid will not be at position 596 in all other HIV isolates.
  • the corresponding amino acid is T603 (Genbank Accesion # AAA44992) . It may also be a homologous amino acid other than threonine or cysteine. This invention encompasses cysteine mutations in such amino acids, which can be readily identified in other HIV isolates by those skilled in the art.
  • a cysteine in the Cl region of gpl20 is disulfide linked to a cysteine in the ectodomain of gp41.
  • Cl region means the first conserved sequence of amino acids in the mature gpl20 glycoprotein.
  • the Cl region includes the amino-terminal amino acids.
  • the Cl region consists of the amino acids VEKLWVTVYYGVPVWKEATTTLFCASDAKAYDTEVHNVWATHACVPTDPN PQEWLENVTEHFNMWKNNMVEQMQEDIISLWDQSLKPCVKLTPLCVTLN. Amino acid resides 30-130 of the sequence set forth in in figure 3A have this sequence.
  • the Cl region will comprise a homologous amino-terminal sequence of amino acids of similar length.
  • W44C and P600C mutations are as defined above for A492 and T596 mutations. Because of the sequence variability of HIV, W44 and P600 will not be at positions 44 and 600 in all HIV isolates. In other HIV isolates, homologous, non-cysteine amino acids may also be present in the place of the tryptophan and proline. This invention encompasses cysteine mutations in such amino acids, which can be readily identified in other HIV isolates by those skilled in the art.
  • the above isolated nucleic acid includes but is not limited to cDNA, genomic DNA, and RNA
  • nucleic acid which encode mutant viral envelope proteins wherein the interaction between the viral surface and transmembrane proteins has been stabilized. Furthermore, one skilled in the art would know how to use these recombinant nucleic acid molecules to obtain the proteins encoded thereby, and practice the therapeutic and prophylactic methods of using same, as described herein for the recombinant nucleic acid molecule which encode mutant viral envelope proteins .
  • the invention provides a replicable vector comprising the above nucleic acid.
  • This invention also provides a plasmid, cosmid, ⁇ phage or YAC containing the above nucleic acid molecule.
  • the plasmid is designated PPI4.
  • the invention is not limited to the PPI4 plasmid and may include other plasmids known to those skilled in the art.
  • vector systems for expression of the mutant glycoprotein may be employed.
  • one class of vectors utilizes DNA elements which are derived from animal viruses such as bovine papilloma virus, polyoma virus, adenovirus, vaccinia virus, baculovirus, retroviruses (RSV, MMTV or MoMLV) , Semliki Forest virus or SV40 virus.
  • cells which have stably integrated the DNA into their chromosomes may be selected by introducing one or more markers which allow for the selection of transfected host cells.
  • the marker may provide, for example, prototropy to an auxotrophic host, biocide resistance, (e.g., antibiotics) or resistance to heavy metals such as copper or the like.
  • the selectable marker gene can be either directly linked to the DNA sequences to be expressed, or introduced into the same cell by cotransformation. Additional elements may also be needed for optimal synthesis of mRNA . These elements may include splice signals, as well as transcriptional promoters, enhancers, and termination signals.
  • the cD ⁇ A expression vectors incorporating such elements include those described by (Okayama and Berg, Mol Cell Biol 3:280, 1983) .
  • the vectors used in the subject invention are designed to express high levels of mutant viral envelope proteins in cultured eukaryotic cells as well as efficiently secrete these proteins into the culture medium.
  • the targeting of the mutant envelope glycoproteins into the culture medium is accomplished by fusing in- frame to the mature ⁇ - terminus of the mutant envelope glycoprotein a suitable signal sequence such as that derived from the genomic open reading frame of the tissue plasminogen activator (tPA) .
  • tPA tissue plasminogen activator
  • the mutant envelope protein may be produced by a) transfecting a mammalian cell with an expression vector for producing mutant envelope glycoprotein; b) culturing the resulting transfected mammalian cell under conditions such that mutant envelope protein is produced; and c) recovering the mutant envelope protein so produced.
  • the expression vectors may be transfected or introduced into an appropriate mammalian cell host.
  • Various techniques may be employed to achieve this, such as, for example, protoplast fusion, calcium phosphate precipitation, electroporation, retroviral transduction, or other conventional techniques.
  • protoplast fusion the cells are grown in media and screened for the appropriate activity.
  • Expression of the gene encoding a mutant envelope protein results in production of the mutant protein. Methods and conditions for culturing the resulting transfected cells and for recovering the mutant envelope protein so produced are well known to those skilled in the art, and may be varied or optimized depending upon the specific expression vector and mammalian host cell employed.
  • the preferred host cells for expressing the mutant envelope protein of this invention are mammalian cell lines.
  • Mammalian cell lines include, for example, monkey kidney CV1 line transformed by SV40 (COS-7) ; human embryonic kidney line
  • DXB11 monkey kidney cells (CV1) ; African green monkey kidney cells (VERO-76) ; human cervical carcinoma cells (HELA) ; canine kidney cells (MDCK) ; human lung cells (W138) ; human liver cells (Hep G2 ) ; mouse mammary tumor (MMT 060562) ; mouse cell line (C127) ; and myeloma cell lines .
  • eukaryotic expression systems utilizing non- mammalian vector/cell line combinations can be used to produce the mutant envelope proteins. These include, but are not limited to, baculovirus vector/insect cell expression systems and yeast shuttle vector/yeast cell expression systems.
  • This invention provides a host cell containing the above vector.
  • the cell is a eukaryotic cell.
  • the cell is a bacterial cell.
  • This invention provides a vaccine which comprises the above isolated nucleic acid.
  • the vaccine comprises a therapeutically effective amount of the nucleic acid.
  • the vaccine comprises a therapeutically effective amount of the protein encoded by the above nucleic acid.
  • the vaccine comprises a combination of the recombinant nucleic acid molecule and the mutant viral envelope protein.
  • adjuvants suitable for use with protein- based vaccines include, but are not limited to, alum, Freund's incomplete adjuvant (FIA) , Saponin, Quil A, QS21, Ribi Detox, Monophosphoryl lipid A (MPL) , and nonionic block copolymers such as L-121 (Pluronic; Syntex SAF) .
  • the adjuvant is alum, especially in the form of a thixotropic, viscous, and homogenous aluminum hydroxide gel.
  • the vaccine of the subject invention may be administered as an oil in water emulsion.
  • the adjuvant may be in particulate form.
  • the antigen may be incorporated into biodegradable particles composed of poly-lactide-co-glycolide (PLG) or similar polymeric material. Such biodegradable particles are known to provide sustained release of the immunogen and thereby stimulate long-lasting immune responses to the immunogen.
  • Other particulate adjuvants include but are not limited to a micellular mixture of Quil A and cholesterol known as immunostimulating complexes (ISCOMs) and aluminum or iron oxide beads. Methods for combining antigens and particulate adjuvants are well known to those skilled in the art.
  • cytotoxic T lymphocyte and other cellular immune responses are elicited when protein-based immunogens are formulated and administered with appropriate adjuvants, such as ISCOMs and micron- sized polymeric or metal oxide particles .
  • suitable adjuvants for nucleic acid based vaccines include, but are not limited to, Quil A, interleukin-12 delivered in purified protein or nucleic acid form, short bacterial immunostimulatory nucleotide sequence such as CpG containing motifs, interleukin-2/Ig fusion proteins delivered in purified protein or nucleic acid form, oil in water micro-emulsions such as MF59, polymeric microparticles , cationic liposomes, monophosphoryl lipid A (MPL) , immunomodulators such as Ubenimex, and genetically detoxified toxins such as E. coli heat labile toxin and cholera toxin from Vibrio.
  • Such adjuvants and methods of combining adjuvants with antigens are well known to those skilled in the art.
  • a “therapeutically effective amount” of the mutant envelope protein may be determined according to methods known to those skilled in the art .
  • terapéuticaally effective amount refers to a dose and dosing schedule sufficient to slow, stop or reverse the progression of a viral disorder.
  • the virus is HIV.
  • This invention provides a method of treating a viral disease which comprises immunizing a virally infected subject with the above vaccines or a combination thereof, thereby treating the subject.
  • treating means either slowing, stopping or reversing the progression of a viral disorder. In the preferred embodiment, “treating” means reversing the progression to the point of eliminating the disorder. As used herein, “treating” also means the reduction of the number of viral infections, reduction of the number of infectious viral particles, reduction of the number of virally infected cells, or the amelioration of symptoms associated with the virus .
  • immunizing means administering a primary dose of the vaccine to a subject, followed after a suitable period of time by one or more subsequent administrations of the vaccine, so as to generate in the subject an immune response against the vaccine.
  • a suitable period of time between administrations of the vaccine may readily be determined by one skilled in the art, and is usually on the order of several weeks to months .
  • the dose of the vaccine can range from about l ⁇ g to about lOmg. In the preferred embodiment, the dose is about 300 ⁇ g.
  • viral infected means the introduction of viral genetic information into a target cell, such as by fusion of the target cell membrane with the virus or infected cell.
  • the target may be a bodily cell of a subject.
  • the target cell is a bodily cell from a human subject.
  • subject means any animal or artificially modified animal capable of becoming infected with the virus. Artificially modified animals include, but are not limited to, SCID mice with human immune systems. The animals include but are not limited to mice, rats, dogs, guinea pigs, ferrets, rabbits, and primates. In the preferred embodiment, the subject is a human.
  • This invention provides a vaccine which comprises a prophylactically effective amount of the above isolated nucleic acid.
  • This invention provides a vaccine which comprises a prophylactically effective amount of the protein encoded by the above isolated nucleic acid.
  • a prophylactically effective amount of the vaccine may be determined according to methods well known to those skilled in the art.
  • prophylactically effective amount refers to a dose and dosing schedule sufficient to reduce the likelihood of a subject becoming infected or to lessen the severity of the disease in subjects who do become infected.
  • This invention provides a method of reducing the likelihood of a subject becoming infected with a virus comprising administering the above vaccines or a combination thereof, thereby reducing the likelihood of the subject becoming infected with the virus.
  • the subject becoming infected with a virus means the invasion of the subject's own cells by the virus .
  • reducing the likelihood of a subject's becoming infected with a virus means reducing the likelihood of the subject's becoming infected with the virus by at least two-fold. For example, if a subject has a 1% chance of becoming infected with the virus, a two- fold reduction in the likelihood of the subject's becoming infected with the virus would result in the subject's having a 0.5% chance of becoming infected with the virus. In the preferred embodiment of this invention, reducing the likelihood of the subject's becoming infected with the virus means reducing the likelihood of the subject's becoming infected with the virus by at least ten-fold.
  • administering may be effected or performed using any of the methods known to one skilled in the art.
  • the methods may comprise intravenous, intramuscular, oral, intranasal, transdermal or subcutaneous means .
  • This invention provides the above vaccine which comprises but is not limited to the following: a recombinant subunit protein, a DNA plasmid, an RNA molecule, a replicating viral vector, a non-replicating viral vector, or a combination thereof.
  • This invention provides a method of reducing the severity of a viral disease in a subject comprising administering the above vaccine or a combination thereof, prior to exposure of the subject to the virus, thereby reducing the severity of the viral disease in the subject upon subsequent exposure to the virus.
  • the virus is HIV.
  • reducing the severity of a viral disease in a subject means slowing the progression of and/or lessening the symptoms of the viral disease. It also means decreasing the potential of the subject to transmit the virus to an uninfected subject.
  • exposure to the virus means contact with the virus such that infection could result.
  • subsequent exposure means an exposure after one or more immunizations.
  • This invention provides a mutant viral envelope protein which differs from the corresponding wild type protein in at least one amino acid which upon proteolysis yields a complex comprising a surface protein and a transmembrane protein which has enhanced stability relative to the corresponding complex obtained from the wild type envelope protein.
  • This invention provides a complex comprising a viral surface protein and a viral transmembrane protein which has enhanced stability relative to the corresponding complex obtained from the wildtype envelope protein, yielded by the proteolysis of a mutant viral envelope protein with a sequence which differs from the corresponding wild type protein sequence in at least one amino acid.
  • This invention provides a viral envelope protein comprising a viral surface protein and a corresponding viral transmembrane protein wherein the viral envelope protein contains one or more mutations in amino acid sequence that enhance the stability of the complex formed between the viral surface protein and transmembrane protein.
  • This invention provides a complex comprising a viral surface protein and a corresponding viral transmembrane protein of a viral envelope protein wherein the viral envelope protein contains one or more mutations in amino acid sequence that enhance the stability of the complex formed between the viral surface protein and transmembrane protein.
  • This invention provides a mutant viral envelope protein which is encoded by the above nucleic acid molecule.
  • the mutant viral envelope protein is linked to at least one other protein or protein fragment to form a fusion protein.
  • virus-like particle which comprises the transmembrane protein and surface protein complex of the subject invention.
  • the virus-like particle comprises an immunodeficiency virus structural protein.
  • the structural protein is the gag protein.
  • virus-like particles or VLPs are particle which are non-infectious in any host, nonreplicating in any host, which do not contain all of the protein components of live virus particles.
  • VLPs of the subject invention contain the disulfide-stabilized complex of the subject invention and a structural protein, such as HIV-1 gag, needed to form membrane-enveloped virus-like particles.
  • VLPs include (1) their particulate and multivalent nature, which is immunostimulatory, and (2) their ability to present the disulfide-stabilized envelope glycoproteins in a near-native, membrane-associated form.
  • VLPs are produced by co-expressing the viral proteins (e.g., HIV-1 gpl20/gp41 and gag) in the same cell. This can be achieved by any of several means of heterologous gene expression that are well-known to those skilled in the art, such as transfection of appropriate expression vector (s) encoding the viral proteins, infection of cells with one or more recombinant viruses (e.g., vaccinia) that encode the VLP proteins, or retroviral transduction of the cells. A combination of such approaches can also be used.
  • the VLPs can be produced either in vitro or in vivo.
  • VLPs can be produced in purified form by methods that are well-known to the skilled artisan, including centrifugation, as on sucrose or other layering substance, and by chromatography.
  • mutant means that which is not wild- type.
  • linked refers but is not limited to fusion proteins formed by recombinant methods and chemical cross links. Suitable chemical cross links are well known to those skilled in the art.
  • the protein is purified by one of the methods known to one skilled in the art.
  • This invention provides a vaccine which comprises a therapeutically effective amount of the above protein or complex.
  • This invention also provides a vaccine which comprises a prophylactically effective amount of the above protein or complex.
  • This invention provides a method of stimulating or enhancing in a subject production of antibodies which recognize the above protein or complex.
  • This invention provides a method of stimulating or enhancing in a subject the production of cytotoxic T lymphocytes which recognize the above protein.
  • This invention provides an antibody capable of specifically binding to the above mutant protein.
  • This invention also provides an antibody which is capable of specifically binding to the above mutant protein or complex but not to the wild type protein or complex.
  • This invention provides an antibody, antibody chain or fragment thereof identified using the viral envelope protein encoded by the above recombinant nucleic acid molecule.
  • the antibody may be of the IgM, IgA, IgE or IgG class or subclasses thereof.
  • the above antibody fragment includes but is not limited to Fab, Fab', (Fab')2, Fv and single chain antibodies. This invention provides a labeled antibody.
  • This invention provides an isolated antibody light chain of the above antibody, or fragment or oligomer thereof.
  • This invention also provides an isolated antibody heavy chain of the above antibody, or fragment or oligomer thereof.
  • This invention also provides one or more CDR regions of the above antibody.
  • the antibody is derivatized.
  • the antibody is a human antibody.
  • the antibody includes but is not limited to monoclonal antibodies and polyclonal antibodies. In one embodiment, antibody is humanized.
  • oligomer means a complex of 2 or more subunits .
  • CDR complementarity determining region
  • a "derivatized" antibody is one that has been modified.
  • Methods of derivatization include but are not limited to the addition of a fluorescent moiety, a radionuclide, a toxin, an enzyme or an affinity ligand such as biotin.
  • humanized describes antibodies wherein some, most or all of the amino acids outside the CDR regions are replaced with corresponding amino acids derived from human immunoglobulin molecules. In one embodiment of the humanized forms of the antibodies, some, most or all of the amino acids outside the CDR regions have been replaced with amino acids from human immunoglobulin molecules but where some, most or all amino acids within one or more CDR regions are unchanged. Small additions, deletions, insertions, substitutions or modifications of amino acids are permissible as long as they would not abrogate the ability of the antibody to bind a given antigen. Suitable human immunoglobulin molecules would include IgGl, IgG2 , IgG3 , IgG4, IgA, IgE and IgM molecules. A "humanized” antibody would retain a similar antigenic specificity as the original antibody.
  • United States Patent No. 5,225,539 describes another approach for the production of a humanized antibody.
  • This patent describes the use of recombinant DNA technology to produce a humanized antibody wherein the CDRs of a variable region of one immunoglobulin are replaced with the CDRs from an immunoglobulin with a different specificity such that the humanized antibody would recognize the desired target but would not be recognized in a significant way by the human subject's immune system.
  • site directed mutagenesis is used to graft the CDRs onto the framework.
  • the above patents 5,585,089 and 5,693,761, and WO 90/07861 also propose four possible criteria which may used in designing the humanized antibodies.
  • the first proposal was that for an acceptor, use a framework from a particular human immunoglobulin that is unusually homologous to the donor immunoglobulin to be humanized, or use a consensus framework from many human antibodies.
  • the second proposal was that if an amino acid in the framework of the human immunoglobulin is unusual and the donor amino acid at that position is typical for human sequences, then the donor amino acid rather than the acceptor may be selected.
  • the third proposal was that in the positions immediately adjacent to the 3 CDRs in the humanized immunoglobulin chain, the donor amino acid rather than the acceptor amino acid may be selected.
  • the fourth proposal was to use the donor amino acid reside at the framework positions at which the amino acid is predicted to have a side chain atom within 3A of the CDRs in a three dimensional model of the antibody and is predicted to be capable of interacting with the CDRs.
  • the above methods are merely illustrative of some of the methods that one skilled in the art could employ to make humanized antibodies.
  • the viral envelope protein is derived from HIV-1.
  • derived means obtained in whole or in part from HIV in the form of genomic sequences, primary isolates, molecular clones, consensus sequences and encompasses chimeras, and sequences modified by means such as truncations and point mutations.
  • the nucleic acid molecule includes but is not limited to RNA, genomic DNA and cDNA.
  • This invention provides a method of reducing the likelihood of a virally exposed subject from becoming infected with the virus comprising administering the above antibody or the above isolated nucleic acid, thereby reducing the likelihood of the subject from becoming infected with the virus.
  • the virus is HIV.
  • reducing the likelihood means a smaller chance than would exist in a control situation without administration of the nucleic acid, protein or antibody.
  • This invention provides a method of treating a subject infected with a virus comprising administering the above antibody or the above isolated nucleic acid, thereby treating the subject.
  • the virus is HIV.
  • This invention provides an agent capable of binding the mutant viral envelope protein encoded by the above recombinant nucleic acid molecule.
  • the agent inhibits viral infection.
  • the viral envelope protein is derived from HIV-1.
  • agent includes but is not limited to small organic molecules, antibodies, polypeptides, and polynucleotides .
  • inhibits viral infection means reduces the amount of viral genetic information introduced into a target cell population as compared to the amount that would be introduced without said composition.
  • This invention provides a method for determining whether a compound is capable of inhibiting a viral infection comprising :
  • Methods such as surface plasmon resonance may also be used to measure the direct binding of the compound to the mutant viral envelope protein using commercially available instruments, methods and reagents (Biacore, Piscataway, N. J. ) .
  • reporter molecule means a molecule which when bound to mutant envelope proteins can be detected.
  • molecules include but are not limited to radio- labeled or fluorescently-labeled molecules, enzyme-linked molecules, biotmylated molecules or similarly affinity tagged molecules, or molecules which are reactive with antibodies or other agents that are so labeled.
  • measuring can be done by any of the methods known to those skilled the art. These include but are not limited to fluorometric, colorimetnc, radiomet ⁇ c or surface plasmon resonance methods.
  • the reporter molecule is an antibody or derivative thereof.
  • the virus is HIV-1.
  • the reporter molecule comprises one or more host cell viral receptors or molecular mimics thereof .
  • molecular mimics means a molecule with similar binding specificity.
  • This invention provides a method for determining whether a compound is capable of inhibiting a viral infection which comprises:
  • step (c) comparing the amount of binding determined in step (c) with the amount determined in the absence of the compound, a decrease in the amount indicating that the compound is capable of inhibiting infection by the virus, thereby determining whether a compound is capable of inhibiting a v i r a l i n f e c t i o n .
  • the virus is HIV-1.
  • the host cell viral receptor is CD4 , CCR5, CXCR4 or combinations or molecular mimics thereof.
  • CD4 means the mature, native, membrane- bound CD4 protein comprising a cytoplasmic domain, a hydrophobic transmembrane domain, and an extracellular domain which binds to the HIV-1 gpl20 envelope glycoprotein. CD4 also comprises portions of the CD4 extracellular domain capable of binding to the HIV-1 gpl20 envelope glycoprotein.
  • CCR5 is a chemokine receptor which binds members of the C-C group of chemokines and whose amino acid sequence comprises that provided in Genbank Accession Number 1705896 and related polymorphic variants.
  • CCR5 includes extracellular portions of CCR5 capable of binding the HIV-1 envelope protein.
  • CXCR4 is a chemokine receptor which binds members of the C-X-C group of chemokines and whose amino acid sequence comprises that provided in Genbank Accession Number 400654 and related polymorphic variants. As used herein, CXCR4 includes extracellular portions of CXCR4 capable of binding the HIV-1 envelope protein.
  • This invention provides a compound isolated using the above methods.
  • Pharmaceutically acceptable carriers are well known to those skilled in the art and include but are not limited to 0.01-O.lM and preferably 0.05M phosphate buffer, phosphate-buffered saline, or 0.9% saline. Additionally, such pharmaceutically acceptable carriers may include but are not limited to aqueous or non-aqueous solutions, suspensions, and emulsions. Examples of non-aqueous 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, saline and buffered media.
  • Parenteral 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, antimicrobials, antioxidants, chelating agents, inert gases and the like.
  • This invention provides a compound determined to be capable of inhibiting a viral infection by the above methods .
  • This invention provides a pharmaceutical composition comprising an amount of the compound effective to inhibit viral infection determined by the above methods to be capable of inhibiting viral infection and a pharmaceutically acceptable carrier.
  • the viral infection is HIV infection.
  • the viral infection is HIV-1 infection.
  • This invention provides a mutant complex comprising an immunodeficiency virus surface protein and an immunodeficiency virus transmembrane protein, wherein the mutant complex contains one or more mutations in amino acid sequence that enhance the stability of the complex formed between the viral surface protein and transmembrane protein, compared to the stability of the wildtype complex.
  • the stability of the complex is enhanced by introducing at least one disulfide bond between the transmembrane protein and the surface protein.
  • an amino acid residue in the transmembrane protein is mutated to a cysteine residue, resulting in the formation of a disulfide bond between the transmembrane protein and surface protein.
  • an amino acid residue in the surface protein protein is mutated to a cysteine residue, resulting in the formation of a disulfide bond between the transmembrane protein and surface protein.
  • an amino acid residue in the transmembrane protein is mutated to a cysteine residue
  • an amino acid residue in the surface protein protein is mutated to a cysteine residue, resulting resulting in the formation of a disulfide bond between the transmembrane protein and surface protein.
  • immunodeficienecy virus is a human imunodeficiency virus.
  • the human imunodeficiency virus includes but is not limited to the JR-FL strain.
  • the surface protein includes but is not limited to gpl20.
  • An amino acid residue of the Cl region of gpl20 may be mutated.
  • An amino acid residue of the C5 region of gpl20 may be mutated.
  • the amino acids residues which may be mutated include but are not limited to the following amino acid residues: V35; Y39, W44; G462; 1482; P484; G486; A488; P489; A492; and E500.
  • the gpl20 amino acid residues are also set forth in Figure 3A.
  • the transmembrane protein includes but is not limited to gp41.
  • An amino acid in the ectodomain of gp41 may be mutated.
  • the amino acids residues which may be mutated include but are not limited to the following amino acid residues: D580; W587; T596; V599; and P600.
  • the gp41 amino acid residues are also set forth in Figure 3B.
  • This invention provides a mutant viral envelope protein which differs from the corresponding wild type protein in at least one amino acid which yields a complex comprising a surface protein and a transmembrane protein which has enhanced stability relative to the corresponding complex obtained from the wild type envelope protein, wherein the surface protein and transmembrane protein are encoded by different nucleic acids.
  • This invention provides a complex comprising a viral surface protein and a viral transmembrane protein which has enhanced stability relative to the corresponding complex obtained from the wildtype envelope protein, yielded by the proteolysis of a mutant viral envelope protein with a sequence which differs from the corresponding wild type protein sequence in at least one amino acid, wherein the surface protein and transmembrane protein are encoded by different nucleic acids.
  • This invention provides a nucleic acid which encodes a mutant surface protein wherein the surface protein is complexed with its corresponding transmembrane protein and will have enhanced stability.
  • This invention provides a nucleic acid which encodes a mutant transmembrane protein wherein the transmembrane protein is complexed with its corresponding surface protein and will have enhanced stability.
  • This invention provides an antibody which binds to the above protein or above complex but does not cross react with the individual monomeric surface protein or the individual monomeric transmembrane protein.
  • This invention provides the above antibody capable of binding to the virus.
  • This invention provides a protein comprising at least a portion of a viral envelope protein which differs from the corresponding wild type protein in at least one amino acid which yields a complex comprising a surface protein and a transmembrane protein which has enhanced stability relative to the corresponding complex obtained from the wild type envelope protein, wherein the portion of the protein results in enhanced stability.
  • This invention provides a portion of the above protein, wherein the portion results in enhanced immunogenicity in comparison to the corresponding wild type portion.
  • This invention further provides a simple method for determining whether a subject has produced antibodies capable of blocking the infectivity of a virus.
  • This diagnostic test comprises examining the ability of the antibodies to bind to the stabilized viral envelope protein. As shown herein, such binding is indicative of the antibodies' ability to neutralize the virus. In contrast, binding of antibodies to non-stabilized, monomeric forms of viral envelope proteins is not predictive of the antibodies ' ability to bind and block the infectivity of infectious virus (Fouts et al . , J. Virol. 71:2779, 1997). The method offers the practical advantage of circumventing the need to use infectious virus .
  • an enzyme-linked immunosorbent assay (ELISA) format could be used wherein in the mutant virus envelope glycoprotein is directly or biospecifically captured onto the well of a microtiter plate. After wash and/or blocking steps as needed, test samples are added to the plate in a range of concentrations .
  • the antibodies can be added in a variety of forms, including but not limited to serum, plasma, and a purified immunoglobulin fraction. Following suitable incubation and wash steps, bound antibodies can be detected, such as by the addition of an enzyme-linked reporter antibody that is specific for the subject's antibodies.
  • Suitable enzymes include horse radish peroxidase and alkaline phosphatase, for which numerous immunoconjugates and colorimetric substrates are commercially available.
  • the binding of the test antibodies can be compared with that of a known monoclonal or polyclonal antibody standard assayed in parallel. In this example, high level antibody binding would indicate high neutralizing activity.
  • the diagnostic test could be used to determine .if a vaccine elicited a protective antibody response in a subject, the presence of a protective response indicating that the subject was successfully immunized and the lack of such response suggesting that further immunizations are necessary.
  • the subject is a human.
  • the plasmid designated PPI4-tPA-gpl20 JR _ FL was deposited pursuant to, and in satisfaction of, the requirements of the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure with the American Type Culture Collection (ATCC) , 12301 Parklawn Drive, Rockville, Maryland 20852 under ATCC Accession Nos. 75431.
  • the plasmid was deposited with ATCC on March 12, 1993.
  • This eukaryotic shuttle vector contains the cytomegalovirus major immediate-early (CMV MIE) promoter/enhancer linked to the full-length HIV-1 envelope gene whose signal sequence was replaced with that derived from tissue plasminogen activator.
  • CMV MIE cytomegalovirus major immediate-early
  • a stop codon has been placed at the gpl20 C-terminus to prevent translation of gp41 sequences, which are present in the vector.
  • the vector also contains an ampicillin resistance gene, an SV40 origin of replication and a DHFR gene whose transcription is driven by the ⁇ -globin promoter.
  • Mabs to gp41 epitopes included 7B2 to epitope cluster 1 (kindly provided by Jim Robinson, Tulane University) ; 25C2 to the fusion peptide region (Buchacher et al . AIDS Res. Human Retrov. 10:359, 1994) ; 2F5 to a neutralizing epitope encompassing residues 665-690 (Munster et al . J. Virol. 68:4031, 1994).
  • the tetrameric CD4-IgG2 has been described previously (Allaway et al . AIDS Res. Human Retrovir. 11:533, 1995).
  • Anti-HIV Abs were obtained from commercial sources, from the NIH AIDS Reagent Program, or from the inventor. Where indicated, the Abs were biotinylated with NHS-biotin (Pierce, Rockford, IL) according to the manufacturer's instructions .
  • Monomeric gpl20 JR . FL was produced in CHO cells stably transfected with the PPI4-tPA-gpl20 JR-FL plasmid as described (U.S. Patents 5,866,163 and 5,869,624). Soluble CD4 was purchased from Bartels Corporation (Issaquah, WA) .
  • gp!40WT Wild- type gp!40s
  • the gpl40 coding sequences were amplified using the polymerase chain reaction (PCR) from full-length molecular clones of the HIV-1 isolates JR-FL, DH123, Gun-1, 89.6, NL4-3 and HxB2.
  • the 5' primer used was designated Kpnlenv (5 ' -GTCTATTATGGGGTACCTGTGTGGAA
  • Patents #5886163 and 5869624) Ligations of insert and vector were carried out overnight at room temperature. DH5 ⁇ F'Q10 bacteria were transformed with 1/20 of each ligation. Colonies were screened directly by PCR to determine if they were transformed with vector containing the insert . DNA from three positive clones of each construct were purified using a plasmid preparation kit (Qiagen, Valencia, CA) and both strands of the entire gpl60 were sequenced.
  • pPPI4-gpl40WT JR _ FL and pPPI4- gpl40WT DH123 refer to vectors expressing wild-type, cleavable gpl40s derived from HIV-1 JR _ FL and HIV-1 DH123 , respectively .
  • crpl40UNC A gpl20-gp41 cleavage site mutant of JR-FL gpl40 was generated by substitutions within the REKR motif at the gpl20 C-terminus, as described previously (Earl et al., Proc. Natl. Acad. Sci. USA 87:648, 1990).
  • deletions were made by site-directed mutagenesis using the mutagenic primers 5'140M (5 ' -CTACGACTTCGTCTCCGCCTT CGACTACGGGGAATAGGAGCTGTGTTCCTTGGGTTCTTG-3 ' ) and 3'gpl40M (sequence conjunction with Kpnlenv and BstBlenv 5'- TCGAAGGCGGAGACGAAGTCGTAGCCGCAGTGCCTTGGTGG GTGCTACTCCTAATGGTTC-3 ' ) .
  • the PCR product was digested with Kpnl and BstBl and subcloned into pPPI4 as described above.
  • Loop-deleted gpl20s and ⁇ pl40s PPI4 -based plasmids expressing variable loop-deleted forms of gpl20 and gpl40 proteins were prepared using the splicing by overlap extension method as described previously (Binley et al . , AIDS Res. Human Retrovir. 14:191, 1998).
  • a Gly-Ala-Gly spacer is used to replace D132-K152 ( ⁇ Vl) , F156-I191 ( ⁇ V2) , or T300-G320
  • the numbering system corresponds to that for the JR-FL clone of HIV-1 (Genbank Accession # U63632) .
  • This fragment was cloned into a plasmid lacking the sequences for the V2 loop using the Kpnl and BamHl restriction sites.
  • the resulting plasmid was designated ⁇ V1V2 ' and contained a Gly-Ala-Gly sequences in place of both D132- K152 and F156-I191.
  • Envs lacking the VI, V2 and V3 loops were generated in a similar way using a fragment generated by PCR on a ⁇ V3 template with primers 3JV2-B (5'-
  • the fragment was cloned into ⁇ V1V2 ' , using BamHl and BstBl.
  • the resulting env construct was named ⁇ V1V2'V3.
  • the glycoproteins encoded by the ⁇ V1V2 ' and ⁇ V1V2'V3 plasmids encode a short sequence of amino acids spanning C125 to C130. These sequences were removed using mutagenic primers that replace T127-I191 with a Gly-Ala- Gly sequence.
  • Glycosylation si te mutants Canonical N- linked glycosylation sites were eliminated at positions 357 and 398 on gpl20 by point mutations of asparagine to glutamine . These changes were made on templates encoding both wild-type and loop-deleted HIV envelope proteins.
  • Disulf ide-stabilized gpl40s The indicated amino acids in gpl20 and gp41 were mutated in pairs to cysteines by site- directed mutagenesis using the Quickchange kit
  • HIV envelope proteins were transiently expressed in adherent 293T cells, a human embryonic kidney cell line (ATCC Cat. # CRL-1573) transfected with the SV40 large T antigen, which promotes high level replication of plasmids such as PPI4 that contain the SV40 origin.
  • 293T cells were grown in Dulbecco's minimum essential medium (DMEM; Life Technologies, Gaithersburg, MD) containing 10% fetal bovine serum supplemented with L-glutamine, penicillin, and streptomycin. Cells were plated in a 10 cm dish and transfected with 10 ⁇ g of purified PPI4 plasmid using the calcium phosphate precipitation method.
  • DMEM Dulbecco's minimum essential medium
  • cells were supplied fresh DMEM containing 0.2% bovine serum albumin along with L-glutamine, penicillin and streptomycin.
  • the medium also contained 35 S-labeled cysteine and methionine (200 ⁇ Ci/ plate) .
  • the cells were cotransfected with 10 ⁇ g of a pcDNA3.1 expression vector (Invitrogen, Carlsbad, CA) encoding the gene for human furin .
  • the concentration of gpl20 and gpl40 proteins in 293T cell supe atants was measured by ELISA (Binley et al . J. Virol 71:2799, 1997). Briefly, Immulon II ELISA plates (Dynatech Laboratories, Inc.) were coated for 16-20 hr at 4 °C with a polyclonal sheep antibody that recognizes the carboxytermmal sequence of gpl20 (APTKAKRRWQREKR) . The plate was washed with tris buffered saline (TBS) and then blocked with 2% nonfat milk in TBS. Cell supematants
  • AP activity is measured using the AMPAK kit (DAKO) according to the manufacturer's instructions.
  • unlabeled Abs were used in combination with protein G-agarose (Pierce, Rockford, IL) .
  • the beads were washed three times with RIPA buffer containing 1% Nonidet- P40 (NP40) detergent.
  • Bound proteins were eluted by heating at 100 °C for 5 min with SDS-PAGE sample buffer containing 0.05 M tris-HCl, 10% glycerol , 2% sodium dodecyl sulfate (SDS), 0.001% bromophenol blue, and where indicated, lOOmM dithiothreitol (DTT) .
  • Samples were loaded on an 8% polyacrylamide gel and run at 200V for 1 hour.
  • RIPA assay was performed using the anti-gpl20 MAb 2G12. As indicated in Figure 2, furin eliminated production of gpl40NON but had no effect on gpl40UNC. Similar results were obtained in RIPAs performed using other anti-gpl20 MAbs (data not shown) .
  • the mobility of the SOS gpl40 mutant on SDS-PAGE is identical to that of the gpl40NON protein, in which the gpl20 and gp41ECTO moieties are linked by a peptide bond.
  • the gpl40 band derived from the SOS mutant is not quite as sharp as that from the gpl40NON protein, but it is less diffuse than the gpl40 bands obtained from any of the other double-cysteine mutants (Fig. 4) . This suggests that the SOS mutant is efficiently processed.
  • the complete nucleic acid and amino acid sequences of the JR-FL SOS gpl40 mutant are provided in Figure 13.
  • Disulfide-stabilized gpl40 is not the only env species present in the 293T cell supematants. Discemable amounts of free gpl20 are also present. This implies that the disulfide bond between gpl20 and the gp41 ectodomain forms with imperfect efficiency. Although the free gpl20 can be removed by the purification methods described below, attempts were made to further reduce or eliminate its production. To this end, additional amino acid substitutions were made near the inserted cysteines. In addition, the position of the cysteine in gpl20 was varied. We retained the gp41 cysteine at residue 596, as in the SOS gpl40 protein, because this position seemed to be the one at which intermolecular disulfide bond formation was most favored.
  • quadruple-cysteine mutant (W44C/A492C/P600C/T596C) was poorly expressed, implying that there was a processing or folding problem (Fig.7) . Poor expression was also observed with two more quadruple-cysteine mutants (W44C/K491C/P600C/T596C and (W44C/K493C/P600C/T596C) (Fig.7) .
  • cells stably transfected with furin could be created so as to ensure adequate levels of furin in all cells expressing the SOS gpl40 proteins.
  • furin and the gpl40 proteins could be coexpressed from a single plasmid.
  • K491 and K493 could be mutated to non-alanine residues singly or as a pair.
  • other gpl20 and/or gp41 amino acids in the vicinity of the introduced cysteines could be mutated as well.
  • the SOS gpl40 protein Compared to gpl40NON, the SOS gpl40 protein has several antigenic differences that we believe are desirable for a protein intended to mimic the structure of the virion- associated gpl20-gp41 complex. These are summarized below.
  • the SOS gpl40 protein binds strongly to the potently neutralizing MAbs IgGlbl2 and 2G12, and also to the CD4-IgG2 molecule (Fig.8a). Although the RIPA methodology is not sufficiently quantitative to allow a precise determination of relative affinities, the reactivities of these MAbs and of the CD4-IgG2 molecule with the SOS gpl40 protein appear to be substantially greater than with the gpl40NON and gpl20 proteins (Fig.8a) . Clearly, the SOS gpl40 protein has an intact CD4 -binding site. V3 loop epitopes are also accessible on the SOS gpl40 protein, shown by its reactivity with MAbs 19b and 83.1 (Fig. 8a) .
  • SOS gpl40 protein possess the same static conformation and conformational freedom as virus-associated gpl20-gp41.
  • the gpl40NON protein bound 17b constitutively, and although there was some induction of the 17b epitope upon soluble CD4 binding, this was less than occurred with the
  • CD4-inducible epitope on gpl20 is that recognized by MAb A32 (Moore et al . J. Virol. 70:1863,
  • the neutralizing anti-gp41 MAb 2F5 bound efficiently to the SOS gpl40 protein, but not to the gpl40NON protein.
  • the 2F5 epitope is the only region of gp41 thought to be well exposed in the context of native gpl20-gp41 complexes (Sattentau et al . Virology 206: 713, 1995) . Its ability to bind 2F5 is again consistent with the adoption by the SOS gpl40 protein of a configuration similar to that of the native trimer.
  • the antigenic properties of the SOS gpl40 protein were compared with those of the W44C/T596C gpl40 mutant.
  • the W44C/T596C gpl40 reacted well with the 2G12 MAb, it bound CD4-IgG2 and IgGlbl2 relatively poorly.
  • the contrast between the properties of the W44C/T596C gpl40 protein and the SOS gpl40 protein demonstrates that the positioning of the intermolecular disulfide bonds has a significant influence on the antigenic structure of the resulting gpl40 molecule .
  • the 140kDa proteins of gpl40WT and gpl40UNC reacted strongly with non-neutralizing anti-gpl20 and anti-gp41 MAbs such as G3-519 and 7B2.
  • the epitope recognized by MAb 17B was constitutively exposed rather than CD4 -inducible (Fig. 8e) .
  • gpl40 proteins derived from the R5 HIV-1 isolate JR-FL we generated double-cysteine mutants of gpl40's from other HIV-1 strains. These include the R5X4 virus DH123 and the X4 virus HxB2. In each case, the cysteines were introduced at the residues equivalent to alanine-492 and threonine- 596 of JR-FL. The resulting SOS proteins were transiently expressed in 293T cells and analyzed by RIPA to ascertain their assembly, processing and antigenticit . As indicated in Fig. 9, 140 kDa material is formed efficiently in the DH123 and HxB2 SOS proteins, demonstrating that our methods can successfully stabilize the envelope proteins of diverse viral isolates.
  • variable loop and glycosylation site mutations provide a means to better expose underlying conserved neutralization epitopes
  • A492C/T596C JR-FL gpl40 mutants were created for each of the ⁇ Vl, ⁇ V2, ⁇ V3 , ⁇ VlVI*, and ⁇ V1V2*V3 molecules described above.
  • glycosylation site mutants were also synthesized by N ⁇ Q point mutations of amino acids 357 and 398.
  • triply loop deleted gpl40s including adjusting the location (s) of one or more introduced cysteines, adding additional pairs of cysteines, modifying amino acids adjacent to the introduced cysteines, and modifying the manner in which the loops are deleted.
  • triply loop deleted gpl40s derived from other HIV isolates may be more readily stabilized by cysteines introduced at residues homologous to 496/592.
  • Milligram quantities of high quality HIV-1 envelope glycoproteins are produced in CHO cells stably transfected with PPI4 envelope-expressing plasmids (U.S. Patent 5,886,163 and 5,869,624).
  • the PPI4 expression vector contains the dhfr gene under the control of the ⁇ -globin promoter. Selection in nucleoside-free media of dhfr + clones is followed by gene amplification using stepwise increases in methotrexate concentrations.
  • the cytomegalovirus (CMV) promoter drives high level expression of the heterologous gene, and the tissue plasminogen activator signal sequence ensures efficient protein secretion.
  • a high level of gpl20 expression and secretion is obtained only upon inclusion of the complete 5 ' non-coding sequences of the CMV MIE gene up to and including the initiating ATG codon.
  • recombinant CHO cells are seeded into roller bottles in selective media and grown to confluency. Reduced serum-containing media is then used for the production phase, when supematants are harvested twice weekly.
  • a purification process comprising lectin affinity, ion exchange, and/or gel filtration chromatographies is carried out under non-denaturing conditions.
  • Purified recombinant HIV-1 envelope proteins are formulated in suitable adjuvants (e . g. , Alum or Ribi).
  • suitable adjuvants e . g. , Alum or Ribi
  • Detox For alum, formulation is achieved by combining the mutant HIV-1 envelope glycoprotein (in phosphate buffered saline, normal saline or similar vehicle) with preformed aluminum hydroxide gel (Pierce, Rockford, IL) at a final concentration of approximately 500 ⁇ g/mL aluminum. The antigen is allowed to adsorb onto the alum gel for two hours at room temperature.
  • preformed aluminum hydroxide gel Pierford, IL
  • Guinea pigs or other animals are immunized 5 times, at monthly intervals, with approximately 100 ⁇ g of formulated antigen, by subcutaneous intramuscular or intraperitoneal routes.
  • Sera from immunized animals are collected at biweekly intervals and tested for reactivity with HIV-1 envelope proteins in ELISA as described above and for neutralizing activity in well established HIV-1 infectivity assays (Trkola et al J. Virol 72: 1876, 1998).
  • Vaccine candidates that elicit the highest levels of HIV-1 neutralizing Abs can be tested for immunogenicity and efficacy in preventing or treating infection in SHIV- macaque or other non-human primate models of HIV infection, as described below.
  • the subunit vaccines could be used alone or in combination with other vaccine components, such as those designed to elicit a protective cellular immune response.
  • the HIV-1 envelope proteins also may be administered in complex with one or more cellular HIV receptors, such as CD4 , CCR5 , and CXCR4.
  • CD4 cellular HIV receptors
  • CCR5 CCR5
  • CXCR4 cellular HIV receptors
  • the binding of soluble CD4 exposes formerly cryptic conserved neutralization epitopes on the stabilized HIV-1 envelope protein.
  • Antibodies raised to these or other neoepitopes could possess significant antiviral activity.
  • interaction of CD4-env complexes with fusion coreceptors such as CCR5 and CXCR4 is thought to trigger additional conformational changes in env required for HIV fusion.
  • Trivalent complexes comprising the stabilized env, CD4 , and coreceptor could thus adopt additional fusion intermediary conformations, some of which are thought to be sufficiently long-lived for therapeutic and possibly immunologic interventions (Kilby et al. Nat. Med. 4:1302, 1998).
  • Methods for preparing and administering env-CD4 and env-CD4 -coreceptor complexes are well-known to the skilled artisan (LaCasse et al . , Science 283:357, 1999; Kang et al . , J. Virol., 68:5854, 1994; Gershoni et al . , FASEB J. 7:1185, 1993).
  • a protocol for determining the immunogenicity of nucleic acid-based vaccines encoding stabilized HIV-1 envelope proteins encoding the stabilized HIV-1 envelope proteins
  • PCR techniques are used to subclone the nucleic acid into a DNA vaccine plasmid vector such as pVAXl available from Invitrogen (catalog #V260-20) .
  • PVAXl was developed according to specifications in the FDA document "Points to Consider on Plasmid DNA Vaccines for Preventive Infectious Disease Indications" published on December 22, 1996.
  • PVAXl has the following features: Eukaryotic DNA sequences are limited to those required for expression in order to minimize the possibility of chromosomal integration, Kanamycin is used to select the vector in E.coli because ampicillin has been reported to cause an allergic response in some individuals, Expression levels of recombinant proteins from pVAXl is comparable to those achieved with its parent vector, pc DNA3.1, and the small size of pVAXl and the variety of unique cloning sites amplify subcloning of even very large DNA fragments.
  • the codon optimization strategy could strive to increase the number of CpG motifs, which are known to increase the immunogencity of DNA vaccines (Klinman et al . , J. Immunol. 158:3635, 1997).
  • env processing into gpl20-gp41 may be facilitated by the heterologous expression of furin introduced on the same or separate expression vectors.
  • the insert containing plasmid can be administered to the animals by such means as direct injection or using gene gun techniques. Such methods are known to those skilled in the art .
  • Rhesus macaques are individually inoculated with five approximately lmg doses of the nucleic acid.
  • the doses are delivered at four week intervals.
  • Each dose is administered intramuscularly.
  • the doses are delivered at four week intervals.
  • the animals receive a single immunization at two separate sites with 2mg of nucleic acid with or without 300 ⁇ g of mutant HIV-1 envelope glycoprotein. This series may be followed by one or more subsequent recombinant protein subunit booster immunizations.
  • the animals are bled at intervals of two to four weeks. Serum samples are prepared from each bleed to assay for the development of specific antibodies as described in the subsequent sections.
  • RNA samples have been created and characterized for infectivity in Rhesus monkeys.
  • the Rhesus monkeys are injected intravenously with a pre-titered dose of virus sufficient to infect greater than 9/10 animals.
  • SHIV infection is determined by two assays. ELISA detection of SIV p27 antigen in monkey sera is determined using a commercially available kit (Coulter) . Similarly, Western blot detection of anti-gag antibodies is performed using a commercially available kit (Cambridge Biotech) .

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Abstract

La présente invention concerne un acide nucléique isolé qui comprend un segment nucléotidique possédant une séquence codante pour une protéine d'enveloppe virale, laquelle comprend une protéine de surface virale et une protéine transmembranaire virale correspondante. Cette protéine d'enveloppe virale contient une ou plusieurs mutations en séquence d'acides aminés qui renforce la stabilité du complexe formé entre la protéine d'enveloppe virale et une protéine transmembranaire virale correspondante. Cette invention concerne aussi une protéine d'enveloppe virale qui comprend une protéine de surface virale et une protéine transmembranaire virale correspondante. Cette protéine d'enveloppe virale contient une ou plusieurs mutations en séquence d'acides aminées qui renforce la stabilité du complexe formé entre la protéine d'enveloppe virale et la protéine transmembranaire virale. Cette invention concerne aussi des méthodes de traitement de l'infection par le VIH-1.
PCT/US2000/017267 1999-06-25 2000-06-23 Proteines d'enveloppe virale stabilisee et utilisations WO2001000648A1 (fr)

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EP1463521A2 (fr) * 2001-12-17 2004-10-06 THE GOVERNMENT OF THE UNITED STATES OF AMERICA, as represented by THE SECRETARY, DEPARTMENT OF HEALTH AND HUMAN SERVICES Inhibiteur de gp41
EP1496938A2 (fr) * 2002-04-05 2005-01-19 Progenics Pharmaceuticals, Inc. Glycoproteines de l'enveloppe du virus d'immunodeficience humaine liees a des particules, compositions et procedes associes
EP1633308A2 (fr) * 2003-06-12 2006-03-15 Vaxgen, Inc. Glycoproteines d'enveloppe du vih-1 a structure disulfure inhabituelle
US7397450B2 (en) 2003-09-16 2008-07-08 Samsung Sdi Co., Ltd. Image display and display panel thereof
US7479553B2 (en) 1999-06-25 2009-01-20 Progenics Pharmaceuticals, Inc. Nucleic acids encoding mutant disulfide bond-stabilized human immunodeficiency virus type 1 (HIV-1) gp140 envelope glycoproteins
US7939083B2 (en) 2006-10-23 2011-05-10 Progenics Pharmaceuticals Inc. Soluble, stabilized, proteolytically cleaved, trimeric HIV-1 gp140 proteins comprising modifications in the N-terminus of the gp41 ectodomain
US11932669B2 (en) 2018-10-17 2024-03-19 Glaxosmithkline Biologicals Sa Modified cytomegalovirus proteins and stabilized complexes

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US7479553B2 (en) 1999-06-25 2009-01-20 Progenics Pharmaceuticals, Inc. Nucleic acids encoding mutant disulfide bond-stabilized human immunodeficiency virus type 1 (HIV-1) gp140 envelope glycoproteins
US7592014B2 (en) 1999-06-25 2009-09-22 Progenics Pharmaceuticals Inc. Stabilized viral envelope proteins and uses thereof
EP1461079A2 (fr) * 2001-09-06 2004-09-29 Progenics Pharmaceuticals, Inc. Mutants de glycoproteines d'enveloppe du vih et leurs utilisations
EP1461079A4 (fr) * 2001-09-06 2006-09-20 Progenics Pharm Inc Mutants de glycoproteines d'enveloppe du vih et leurs utilisations
AU2002335709B8 (en) * 2001-09-06 2008-12-18 Cornell Research Foundation, Inc. Human Immunodeficiency Virus Envelope Clycoprotein Mutants and Uses Thereof
EP1463521A2 (fr) * 2001-12-17 2004-10-06 THE GOVERNMENT OF THE UNITED STATES OF AMERICA, as represented by THE SECRETARY, DEPARTMENT OF HEALTH AND HUMAN SERVICES Inhibiteur de gp41
EP1463521A4 (fr) * 2001-12-17 2009-06-24 Us Gov Health & Human Serv Inhibiteur de gp41
EP1496938A2 (fr) * 2002-04-05 2005-01-19 Progenics Pharmaceuticals, Inc. Glycoproteines de l'enveloppe du virus d'immunodeficience humaine liees a des particules, compositions et procedes associes
EP1496938A4 (fr) * 2002-04-05 2006-10-04 Progenics Pharm Inc Glycoproteines de l'enveloppe du virus d'immunodeficience humaine liees a des particules, compositions et procedes associes
EP1633308A2 (fr) * 2003-06-12 2006-03-15 Vaxgen, Inc. Glycoproteines d'enveloppe du vih-1 a structure disulfure inhabituelle
EP1633308A4 (fr) * 2003-06-12 2008-06-25 Vaxgen Inc Glycoproteines d'enveloppe du vih-1 a structure disulfure inhabituelle
US7397450B2 (en) 2003-09-16 2008-07-08 Samsung Sdi Co., Ltd. Image display and display panel thereof
US7939083B2 (en) 2006-10-23 2011-05-10 Progenics Pharmaceuticals Inc. Soluble, stabilized, proteolytically cleaved, trimeric HIV-1 gp140 proteins comprising modifications in the N-terminus of the gp41 ectodomain
US11932669B2 (en) 2018-10-17 2024-03-19 Glaxosmithkline Biologicals Sa Modified cytomegalovirus proteins and stabilized complexes

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