This invention provides a DNA which upon transcription produces an RNA encoding a modified HIV-1 gp140 polypeptide, which polypeptide upon cleavage produces a modified gp120 and a modified ectodomain of gp41 which together form a complex exhibiting enhanced binding to HIV-1 neutralizing antibodies and reduced binding to HIV-1 non-neutralizing antibodies, wherein the modifications comprise an A492C mutation in gp120 and a T596C mutation in gp41, said mutations being numbered by reference to the HIV-1 isolate JR-FL, and resulting in a disulfide bond between gp120 and ectodomain gp41 which stabilizes the otherwise non-covalent gp120-gp41 interaction.

Description of the Invention

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.

In one embodiment of the above the viral surface protein is HIV-1 gp120 or a modified form of gp120 which has modified immunogenicity relative to wild type gp120. In one embodiment, 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. In one embodiment, the vaccine comprises a therapeutically effective amount of the nucleic acid. In another embodiment, the vaccine comprises a therapeutically effective amount of the protein encoded by the above nucleic acid. In another embodiment, 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 subject.

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').sub.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. In one embodiment, the antibody is derivatized. In another embodiment, 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. In a preferred embodiment, 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: (A) contacting an appropriate concentration of the compound with the mutant viral envelope protein encoded by the recombinant nucleic acid of the invention under conditions permitting binding of the compound to said protein; (B) contacting the resulting complex with a reporter molecule under conditions that permit binding of the reporter molecule to the mutant viral envelope protein; (C) measuring the amount of bound reporter molecule; and (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: (a) contacting an appropriate concentration of the compound with a host cell viral receptor or molecular mimic thereof under conditions that permit binding of the compound and receptor or receptor mimic; (b) contacting the resulting complex with the mutant viral envelope protein encoded by the recombinant nucleic acid of the invention under conditions that permit binding of the envelope protein and receptor or receptor mimic in the absence of the compound; (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. In one embodiment, wherein the viral infection is HIV-1 infection. In the preferred embodiment, 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.

DETAILED DESCRIPTION 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 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.

As used herein, "enhance 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. Alternatively or in combination, 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. Alternatively, 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. In such known viral envelope proteins, the viral surface and transmembrane proteins are physically joined by a covalent bond but are not known to form a complex, as illustrated in FIG. 1 (see Original Patent).

One embodiment of the above virus is a lentivirus. In one embodiment, 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 (Clades A, B, C, D E, F, G and H) or outlying subtype (Group O). Additional types, subtypes or classes of HIV may be discovered and used in this invention. In one embodiment, the human immunodeficiency virus is a primary isolate. In one embodiment, the human immunodeficiency virus is HIV-1.sub.JR-FL. In another embodiment the human immunodeficiency virus is HIV-1.sub.DH123. In another embodiment the human immunodeficiency virus is HIV-1.sub.Gun-1. In another embodiment the human immunodeficiency virus is HIV-1.sub.89.6. In another embodiment the human immunodeficiency virus is HIV-1.sub.HXB2.

HIV-1.sub.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.sub.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.

HIV-1.sub.DH123 is a clone of a virus originally isolated from the peripheral mononuclear cells (PBMCs) of a patient with AIDS (Shibata et al., J. Virol 69:4453, 1995). HIV-1.sub.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.

HIV-1.sub.Gun-1 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-1.sub.Gun-1 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.sub.89.6 is a cloned virus originally isolated from a patient with AIDS (Collman et al, J. Virol. 66: 7517, 1992). HIV-1.sub.89.6 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.sub.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.

Although 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 gp120 or a modified form of gp120 which has modified immunogenicity relative to wild type gp120. In one embodiment, the modified gp120 molecule is characterized by the absence of one or more variable loops present in wild type gp120. In one embodiment, the variable loop comprises V1, V2, or V3. In one embodiment, the modified gp120 molecule is characterized by the absence or presence of one or more canonical glycosylation sites not present in wild type gp120. In one embodiment, one or more canonical glycosylation sites are absent from the V1V2 region of the gp120 molecule.

In one embodiment, the transmembrane protein is gp41 or a modified form of gp41 which has modified immunogenicity relative to wildtype gp41. In one embodiment, the transmembrane protein is full-length gp41. In another embodiment, the transmembrane protein contains the ectodomain and membrane anchoring sequence of gp41 but lacks a portion or all of the gp41 cytoplasmic sequences. In one embodiment, the transmembrane protein is the gp41 ectodomain. In one embodiment, the transmembrane protein is modified by deletion or insertion of one or more canonical glycosylation sites.

One embodiment of the above viral surface protein is gp120 or a derivative thereof. In one embodiment, the gp120 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 V1, V2, V3 or a combination thereof. In another embodiment, the gp120 molecule has been modified by the deletion or insertion of one or more canonical glycosylation sites. The region of gp120 from which the canonical glycosylation sites are deleted includes but is not limited to the V1V2 region of the gp120 molecule.

The V1, V2 and V3 variable loop sequences for HIV-1.sub.JR-FL are illustrated in FIG. 10 (see Original Patent). The amino acid sequences in these variable loops will vary for other HIV isolates but will be located in homologous regions of the gp120 envelope glycoprotein.

As used herein, "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. In addition, Ser or Thr residues not present in such sequences to which a carbohydrate can be linked through an O-linkage are "canonical glycosylation sites." In the later case of a "canonical glycosylation site," 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.

When used in the context of gp41, "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 immunodominant epitope, a gp41 fusion protein, and gp41 labeled with an affinity ligand or other detectable marker.

As used herein, "ectodomain" means the extracellular region or portion thereof exclusive of the transmembrane spanning and cytoplasmic regions.

In one embodiment, 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.

In one embodiment, one or more amino acids which are adjacent to or which contain an atom within 5 Angstroms of an introduced cysteine are mutated to a noncysteine residue.

As used herein, "adjacent to" means immediately preceding or following in the primary sequence of the protein.

As used herein, "mutated" means that which is different from the wild-type.

As used herein, "noncysteine residue" means an amino acid other than cysteine.

In one embodiment, one or more cysteines in gp120 or modified form of gp120 are disulfide linked to one or more cysteines in gp41 or modified form of gp41.

In one embodiment, a cysteine in the C5 region of gp120 or modified form of gp120 is disulfide linked to a cysteine in the ectodomain of gp41 or modified form. In one embodiment, the disulfide bond is formed between a cysteine introduced by an A492C mutation in gp120 or modified form of gp120 and an T596C mutation in gp41 or modified form of gp41.

As used herein, "C5 region" means the fifth conserved sequence of amino acids in the gp120 glycoprotein. The C5 region includes the carboxy-terminal amino acids. In HIV-1.sub.JR-FL gp120, the unmodified C5 region consists of the amino acids GGGDMRDNWRRSELYKYKVVKIEPLGVAPTKAKRRVVQRE (SEQ ID NO:1). Amino acid residues 462-500 of the sequence set forth in FIG. 3A (see Original Patent) have this sequence. In other HIV isolates, the C5 region will comprise a homologous carboxy-terminal sequence of amino acids of similar length.

As used herein, "A492C mutation" refers to a point mutation of amino acid 492 in HIV-1.sub.JR-FL gp120 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.sub.NL4-3 the corresponding amino acid is A499 (Genbank Accession # 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.

As used herein, "T596C mutation" refers to a point mutation of amino acid 596 in HIV-1.sub.JR-FL gp41 from threonine to cysteine.

Because of the sequence variability of HIV, this amino acid will not be at position 596 in all other HIV isolates. For example, in HIV-1.sub.NL4-3 the corresponding amino acid is T603 (Genbank Accession # 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.

In another embodiment, a cysteine in the C1 region of gp120 is disulfide linked to a cysteine in the ectodomain of gp41.

As used herein, "C1 region" means the first conserved sequence of amino acids in the mature gp120 glycoprotein. The C1 region includes the amino-terminal amino acids. In HIV.sub.JR-FL, the C1 region consists of the amino acids VEKLWVTVYYGVPVWKEATTTLFCASDAKAYDTEVHNVWATHACVPTDPNPQEVVLENVT EHFNMWKNNMVEQMQEDIISLWDQSLKPCVKLTPLCVTLN (SED ID NO:2). Amino acid resides 30-130 of the sequence set forth in FIG. 3A have this sequence. In other HIV isolates, the C1 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

One skilled in the art would know how to make the 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, .lamda. phage or YAC containing the above nucleic acid molecule. In one embodiment, 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.

In accordance with the invention, numerous vector systems for expression of the mutant glycoprotein may be employed. For example, 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. Additionally, 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 cDNA 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 N-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).

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.

Once the expression vector or DNA sequence containing the constructs has been prepared for expression, 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. In the case of 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.

In accordance with the claimed invention, 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 293; baby hamster kidney cells (BHK); Chinese hamster ovary-cells-DHFR.sup.+ (CHO); Chinese hamster ovary-cells DHFR.sup.- (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.

Other 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.

Methods and conditions for purifying mutant envelope proteins from the culture media are provided in the invention, but it should be recognized that these procedures can be varied or optimized as is well known to those skilled in the art.

This invention provides a host cell containing the above vector. In one embodiment, the cell is a eukaryotic cell. In another embodiment, the cell is a bacterial cell.

This invention provides a vaccine which comprises the above isolated nucleic acid. In one embodiment, the vaccine comprises a therapeutically effective amount of the nucleic acid. In another embodiment, the vaccine comprises a therapeutically effective amount of the protein encoded by the above nucleic acid. In another embodiment, the vaccine comprises a combination of the recombinant nucleic acid molecule and the mutant viral envelope protein.

Numerous adjuvants have been developed to enhance the immunogenicity of protein and/or nucleic acid vaccines. As used herein, 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). In a preferred embodiment, 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. Methods of combining adjuvants with antigens are well known to those skilled in the art.

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. It is also known to those skilled in the art that 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.

As used herein, 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.

As used herein, "therapeutically effective amount" refers to a dose and dosing schedule sufficient to slow, stop or reverse the progression of a viral disorder. In a preferred embodiment, 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.

As used herein, "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.

As used herein, "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.

Depending on the nature of the vaccine and size of the subject, the dose of the vaccine can range from about 1 .mu.g to about 10 mg. In the preferred embodiment, the dose is about 300 .mu.g.

As used herein, "virally 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. In the preferred embodiment, the target cell is a bodily cell from a human subject.

As used herein, "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.

As used herein "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.

As used herein, "the subject becoming infected with a virus" means the invasion of the subject's own cells by the virus.

As used herein, "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.

As used herein "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. In the preferred embodiment, the virus is HIV.

As used herein "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.

As used herein, "exposure to the virus" means contact with the virus such that infection could result.

As used herein, "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.

In one embodiment, the mutant viral envelope protein is linked to at least one other protein or protein fragment to form a fusion protein.

This invention provides a virus-like particle which comprises the transmembrane protein and surface protein complex of the subject invention. In one embodiment, the virus-like particle comprises an immunodeficiency virus structural protein. In one embodiment, the structural protein is the gag protein.

As used herein, "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. As used herein, 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.

Advantages of 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 gp120/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.

As used herein, "mutant" means that which is not wild-type. As used herein, "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.

In one embodiment, 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').sub.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. In one embodiment, the antibody is derivatized. In another embodiment, 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.

As used herein "oligomer" means a complex of 2 or more subunits.

As used herein, "CDR" or complementarity determining region means a highly variable sequence of amino acids in the variable domain of an antibody.

As used herein, 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.

As used herein, "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 IgG1, IgG2, IgG3, IgG4, IgA, IgE and IgM molecules. A "humanized" antibody would retain a similar antigenic specificity as the original antibody.

One skilled in the art would know how to make the humanized antibodies of the subject invention. Various publications, several of which are hereby incorporated by reference into this application, also describe how to make humanized antibodies. For example, the methods described in U.S. Pat. No. 4,816,567 comprise the production of chimeric antibodies having a variable region of one antibody and a constant region of another antibody.

U.S. Pat. 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. Specifically, site directed mutagenesis is used to graft the CDRs onto the framework.

Other approaches for humanizing an antibody are described in U.S. Pat. Nos. 5,585,089 and 5,693,761 and WO 90/07861 which describe methods for producing humanized immunoglobulins. These have one or more CDRs and possible additional amino acids from a donor immunoglobulin and a framework region from an accepting human immunoglobulin. These patents describe a method to increase the affinity of an antibody for the desired antigen. Some amino acids in the framework are chosen to be the same as the amino acids at those positions in the donor rather than in the acceptor. Specifically, these patents describe the preparation of a humanized antibody that binds to a receptor by combining the CDRs of a mouse monoclonal antibody with human immunoglobulin framework and constant regions. Human framework regions can be chosen to maximize homology with the mouse sequence. A computer model can be used to identify amino acids in the framework region which are likely to interact with the CDRs or the specific antigen and then mouse amino acids can be used at these positions to create the humanized antibody.

The above U.S. Pat. Nos. 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.

In one embodiment of the above antibodies, the viral envelope protein is derived from HIV-1.

As used herein "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.

This invention provides an isolated nucleic acid molecule encoding the above antibody. 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. In a preferred embodiment, the virus is HIV.

As used herein, "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. In a preferred embodiment, 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. In one embodiment, the viral envelope protein is derived from HIV-1.

As used herein, "agent" includes but is not limited to small organic molecules, antibodies, polypeptides, and polynucleotides.

As used herein, "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:

a. contacting an appropriate concentration of the compound with the mutant viral envelope protein encoded by the recombinant nucleic acid of the invention under conditions permitting binding of the compound to said protein;

b. contacting the resulting complex with a reporter molecule under conditions that permit binding of the reporter molecule to the mutant viral envelope protein;

c. measuring the amount of bound reporter molecule; and

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.

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.).

As used herein "reporter molecule" means a molecule which when bound to mutant envelope proteins can be detected. Such molecules include but are not limited to radio-labeled or fluorescently-labeled molecules, enzyme-linked molecules, biotinylated molecules or similarly affinity tagged molecules, or molecules which are reactive with antibodies or other agents that are so labeled.

As used herein "measuring" can be done by any of the methods known to those skilled in the art. These include but are not limited to fluorometric, calorimetric, radiometric or surface plasmon resonance methods.

In one embodiment, the reporter molecule is an antibody or derivative thereof. In one embodiment, the virus is HIV-1. In one embodiment, the reporter molecule comprises one or more host cell viral receptors or molecular mimics thereof.

As used herein "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:

a. contacting an appropriate concentration of the compound with a host cell viral receptor or molecular mimic thereof under conditions that permit binding of the compound and receptor or receptor mimic;

b. contacting the resulting complex with the mutant viral envelope protein encoded by the recombinant nucleic acid of the invention under conditions that permit binding of the envelope protein and receptor or receptor mimic in the absence of the compound;

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.

In one embodiment of the above method, the virus is HIV-1. In one embodiment, the host cell viral receptor is CD4, CCR5, CXCR4 or combinations or molecular mimics thereof.

As used herein "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 gp120 envelope glycoprotein. CD4 also comprises portions of the CD4 extracellular domain capable of binding to the HIV-1 gp120 envelope glycoprotein.

As used herein, "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. As used herein, CCR5 includes extracellular portions of CCR5 capable of binding the HIV-1 envelope protein.

As used herein, "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 0.01-0.1M 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. In one embodiment, the viral infection is HIV infection. In the preferred embodiment, 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. In one embodiment, the stability of the complex is enhanced by introducing at least one disulfide bond between the transmembrane protein and the surface protein. In one embodiment, 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. In one embodiment, an amino acid residue in the surface protein is mutated to a cysteine residue, resulting in the formation of a disulfide bond between the transmembrane protein and surface protein. In one embodiment an amino acid residue in the transmembrane protein is mutated to a cysteine residue, and an amino acid residue in the surface protein is mutated to a cysteine residue, resulting in the formation of a disulfide bond between the transmembrane protein and surface protein.

In one embodiment, immunodeficiency virus is a human immunodeficiency virus. The human immunodeficiency virus includes but is not limited to the JR-FL strain. The surface protein includes but is not limited to gp120. An amino acid residue of the C1 region of gp120 may be mutated. An amino acid residue of the C5 region of gp120 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; I482; P484; G486; A488; P489; A492; and E500. The gp120 amino acid residues are also set forth in FIG. 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 FIG. 3B (see Original Patent).

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.

Numerous immunoassay formats that are known to the skilled artisan are appropriate for this diagnostic application. For example, 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.

As an example, 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. In a preferred embodiment, the subject is a human.

This invention will be better understood from the Experimental Details which follow. However, one skilled in the art will readily appreciate that the specific methods and results discussed are merely illustrative of the invention as described more fully in the claims which follow thereafter.

Claim 1 of 52 Claims

1. A DNA which upon transcription produces an RNA encoding a modified gp140 polypeptide of an HIV-1 isolate, which polypeptide upon cleavage produces a modified gp120 and a modified gp41 ectodomain which together form a complex, said complex (i) exhibiting enhanced binding to HIV-1 neutralizing antibodies and reduced binding to HIV-1 non-neutralizing antibodies, wherein the modified gp120 comprises an A492C mutation and the modified gp41 ectodomain comprises a T596C mutation, said mutations being numbered by reference to HIV-1 JR-FL, and (ii) permitting the formation of a disulfide bond between the modified gp120 and the modified gp41 ectodomain which stabilizes the complex.

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