The present invention relates to replication competent variants of mammalian immunodeficiency virus comprising mutations and/or deletions of the V3 hypervariable loop and compensatory mutations, as well as methods for producing such variants. The invention also relates to V3-loop deletion mammalian immunodeficiency virus mutants that have compensatory mutations, deletions of the V1/V2 loops, or both. The invention further relates to isolated Env, gp120 polypeptides, and gp41 polypeptides comprising novel mutations useful in conjunction with, or separate from, a virus of the invention, as well as nucleic acids encoding the same.

Description of the Invention

The present invention relates to novel methods for producing novel mammalian immunodeficiency virus envelope proteins ("Envs") that conserve functional domains required for entry and/or replication while removing hypervariable loops and exposing core epitopes important for virus entry into cells and thereby providing useful constructs for development of therapeutic modalities relating to development of neutralizing antibodies.

The invention also relates to novel Env polypeptides (e.g., Env, gp120, gp41, and the like), and nucleic acids encoding the same, wherein V1 and V2 have been deleted therefrom, and further where the V3 region, or a substantial portion thereof, has also been removed from the polypeptide. Surprisingly, and despite contrary teachings thereto in the art, the data disclosed herein demonstrate, for the first time, that an Env lacking V1, V2, and at least a substantial portion of V3, or even the entire V3 region, can exhibit detectable function, including, but not limited to, binding with a ligand on a cell, fusion of the Env with the cell, and even replication competence, among other functions. These results are unprecedented and the data disclosed herein demonstrate that novel virus constructs, where hypervariable regions, including V3, have been removed, can be used as potential therapeutics to develop, among other things, useful virus neutralizing antibodies and compounds, such as small molecules, peptidomimetics and such, to inhibit virus infection. This is because the skilled artisan, armed with the teachings provided herein, would realize that the novel polypeptides, and nucleic acids encoding them, provide useful tools for elucidating the requisite interaction(s) between the virus Env and host cell surface ligands and further provide methods for developing and identifying molecules (such as, but not limited to, antibodies, small molecules, peptidomimetics, and others) that can inhibit these interactions thereby preventing infection or inhibiting further infection processes.

For example, and in no way limiting the invention to this, or any other, particular virus construct, it has been shown in the present invention, using an HIV-2 isolate known for its CD4-independent use of CXCR4 and CCR5 and its high affinity binding to CXCR4 as an exemplary system, that variants can be adapted for replication with truncated or even absent V3 loops. Prior to this finding, V3 has been considered and essential for viral entry as a result of its well-documented interactions with cellular chemokine receptors. The data disclosed herein demonstrate that adaptations enabling viruses to replicate in the absence of hypervariable loops entail novel compensatory mutations in gp120 and/or in gp41 that were selected during long term propagation in vitro. In one aspect of the invention, high efficiency replication has been achieved with Envs lacking V1/V2 and all but the first and last 6 amino acids of V3 flanking the disulfide bond forming the loop, termed .DELTA.V3(6,6) and yielding a "gp12o" of only about 75 kD compared to full-length gp120 of about 120 kD in size. Therefore, critical protein function(s) have been remarkably conserved despite a reduction in the size of the polypeptide of almost 40%. Surprisingly, deletion of all but two amino acids flanking the disulfide bond, termed .DELTA.V3(1,1), still maintained the fusogenicity of the construct while removing most, if not all, of V1/V2, and V3 regions. These remarkable accomplishments were achieved despite the widely held belief in the art that these mutants could not be produced because the V3 region was essential to Env function.

Thus, in one aspect, the present invention provides, for the first time, that HIVs can replicate without V3 (as well as V1/V2) while maintaining essential functional domains for cell binding, fusion and/or entry. Without wishing to be bound by any particular theory, the data disclosed herein support an evolutionary model suggesting that Envs of modern lentiviruses evolved from a primordial core protein, and that hypervariable loops were subsequently acquired not only to facilitate chemokine receptor utilization and to mediate specificity, but also to enable these viruses to replicate in the face of coevolving host immune responses. The data disclosed herein demonstrate for the first time, that functional "core" Env can be produced. This is an important breakthrough because such functional core particles, wherein potential neutralizing antibody-eliciting epitopes are exposed and presented in a useful context of a functional molecule, can be used to develop potentially therapeutic virus neutralizing antibodies to these important human pathogens. Given the current state of the art regarding the generation of broadly neutralizing antibodies, the minimized, functional Envs of the invention are useful for generating novel immune responses and provide a major achievement in the development of useful treatments for these devastating human pathogens.

In addition to vaccine potential, the V3-truncated or V3-deleted viruses of the present invention exhibit novel functional properties useful for development of various non-vaccine-based therapeutics. For example, although they can utilize CXCR4, mammalian immunodeficiency viruses of the invention show greater dependence on the CXCR4 N-terminus, in marked contrast to other X4 tropic strains, which utilize primarily the extracellular loops (ECL). Consistent with this, they become resistant to the CXCR4 inhibitor AMD3100, which is thought to interact with the extracellular loops of the receptor. This activity may reveal a mechanism by which HIV can acquire resistance to both CCR5 and CXCR4 inhibitors and thus provide an important system for design and development of therapeutics that prevent virus acquisition of such resistance. Moreover, replication competent, V3-truncated/deleted viruses of the invention can also utilize CCR5 to infect cells, and this property indicates that this dual-tropism in the absence of V3 is based on involvement of a conserved interaction between the bridging sheet domain on the Env core with a motif shared on the N-termini of CXCR4 and CCR5. These data demonstrate potential new drug targets for treatment of viral infection and provide useful tools for development of novel therapeutics relating to inhibiting these interactions now identified for the first time herein.

The invention includes a replication-competent derivative of a mammalian immunodeficiency virus that lacks in its entirety hypervariable loops V1/V2 and V3. As an example, although by no means limiting the invention in any way, .DELTA.V1/V2; .DELTA.V3(6,6), which has a 12 amino acid V3 remnant, and p16.9.DELTA.V3(1,1) which contains no V3 loop, but still has V1/V2, were produced using a HIV-2/VCP backbone. The data shows that combinations of these viruses generate .DELTA.V1/V2; .DELTA.V3(1,1) (i.e., a "loopless" replication competent "core"). The findings set forth herein with HIV-2/vcp Env represent proof of concept that these variable loops can be deleted while preserving functional integrity of the viral Env and suggests that similar approaches are translatable to other HIV-1, HIV-2, and SIV strains because of the high degree of structural conservation of the core Env among these viruses. Thus, the skilled artisan would appreciated, based upon the disclosure provided herein, that the present invention includes replication-competent variants of mammalian immunodeficiency viruses, including, but not limited to, SIV, HIV-1 and HIV-2, and the present invention is in no way limited to any particular mammalian immunodeficiency virus. Thus, the present invention encompasses an Env protein (i.e., gp120 and gp41) where the V3 region is substantially deleted, and where the loop-deleted Env retains detectable biological activity and/or function when compared to full-length Env. That is, the variant Env retains detectable activity in that it binds with a chemokine receptor, mediates Env fusion with a cell, and when incorporated into a virus, permits a virus to establish and infection that spreads cell to cell, and/or there is detectable virus replication in a cell. The skilled artisan would appreciate, based upon the disclosure provided herein, that the invention encompasses adaptive changes in gp41, since mutations in gp41 also mediate the retention and/or restoration of protein function upon truncation of the V3 region of gp120.

The invention is based, in part, on the discovery of a variant of HIV-2, termed VCP, that can utilize both CXCR4 and CCR5 as primary receptors without a need for CD4 triggering, can further comprise a truncation of V3 and yet retain detectable biological activity. While CD4-independence is not a requisite feature of the novel viruses and polypeptides of the invention, the minimal gp120 components required for infectivity were demonstrated herein by making deletions of hypervariable loops V1/V2 and V3 on an infectious molecular clone of VCP. Remarkably, a virus containing deletion of approximately 65% deletion of the V3 loop (leaving only the first 6 and last 6 amino acids on either side of the disulfide bond and termed .DELTA.V3(6,6)), was shown to be replication competent on SupT1 cells. This finding demonstrated for the first time that a full V3 is not required for infectivity and allowed the identification of determinants of gp120 required for virus infection of host cells involving cell receptor proteins.

Further, the present invention relates to a "combination deleted" virus, termed .DELTA.V1/V2; .DELTA.V3(6,6), that produced a gp120 of only about 70 kD. This combination deleted virus was also found to be replication competent. Thus, mammalian immunodeficiency viruses produced by deleting portions of the V3 hypervariable loop are useful for discovery of the gp120 and gp41-based determinants of fusogenicity and replication of such viruses.

The data disclosed herein suggest that changes in both gp120 and gp41 are required for virus ability to replicate in the absence of the V3 loop. This has been demonstrated for VCP and the data suggest that this can be readily applied to other viruses, including, HIV-1 and SIV. Thus, the invention involves mutations to both gp120 and gp41, preferably, about two mutations in gp120 and about two mutations in gp41 are required for the phenotype of being able to replicate without V3.

CD4-independence is important in that it is an indicator that the chemokine binding site of gp120 is stably exposed on the virus envelope and is capable of binding to the cellular chemokine receptor binding protein without prior binding of the gp120 to CD4. Typically, the chemokine receptor binding site on gp120 is hidden until such binding to CD4 causes a conformational change exposing the site and resulting in a "triggered" conformation capable of binding to the chemokine receptor protein on the host cell. CD4-independence (CD4i) is an apparent indicator for increased exposure of the chemokine coreceptor binding site for the host cell chemokine receptor, which is in some cases also associated with an increased affinity that appears to render binding of CD4 by the virus gp120 unnecessary for fusion. A virus gp120 that can bind a chemokine receptor with such affinity that the V3 region can be deleted and the gp120 can still mediate binding with the cell, fusion of the Env with the cell, and/or replication, even where CD4 binding is required, is encompassed in the present invention. The interaction of gp120 with chemokine receptors involves at least two steps: the binding of the V3 loop to extracellular loops of the chemokine receptor (principally the second extracellular loop), and the binding of the bridging sheet ("BS") of gp120 with the chemokine receptor amino terminus. The data disclosed herein suggest that that viruses with a sufficiently strong interaction of the BS with the chemokine receptor can better tolerate loss of the V3 loop. A "favorable" interaction of the BS with the chemokine amino terminus can be reflected in CD4-independence, dual tropism or (most notably) Envs that are resistant to inhibitors that act on the extracellular loops. Thus, HIV-2 VCP with deletions of V3 that could no longer interact with ECL2, became resistant to the CXCR4 inhibitor AMD3100. Thus, based upon the disclosure provided herein, a property that can be utilized in the screening of HIV envelope glycoproteins for the ability to tolerate a V3 deletion is relative resistance to AMD3100.

CD4-independent gp120 represents a stable intermediate configuration which may be used to, inter alia, identify the protein determinants involved in gp120 binding to a chemokine receptor protein, produce neutralizing antibodies capable of recognizing the gp120 chemokine receptor binding site, and to identify small-molecule inhibitors which can block gp120/chemokine receptor binding.

Moreover, production of gp120 hypervariable loop-deleted mutants has led to the discovery that a "core" domain of gp120, lacking some or all of the V1/V2 and V3 loop amino acids, is responsible for the fusogenicity and replication competence of the virus.

Accordingly, understanding which portions of the Env are involved in virus binding to cell proteins and thereby functionally mapping the protein determinant(s) that mediate immunodeficiency virus binding to host cell receptors is important in the development of effective antiviral vaccines to viral protein domains crucial for virus infection. Such domains are believed to be highly conserved but somehow "camouflaged" from the immune system such that a protective immune response is not mounted to such protein domains. Therefore, for example, identification of these protein domains and the ability to present them to the immune system such that an immune response is generated to HIV-1 is an important goal of vaccine development to this, and other important human pathogenic immunodeficiency viruses.

I. Isolated Nucleic Acids

The present invention includes an isolated nucleic acid encoding a mammalian immunodeficiency virus gp120 polypeptide, or a fragment thereof, wherein the nucleic acid encodes a variant of gp120 that comprises a deletion of hypervariable loop 1 (V1), a deletion of hypervariable loop 2 (V2) (hereinafter referred to as a "deletion of V1/V2"), and a substantial deletion of hypervariable loop 3 (V3). In an embodiment of the invention, a nucleic acid shares at least about 90% identity with at least one nucleic acid having the sequence of SEQ ID NO:8, SEQ ID NO:14, SEQ ID NO:20 and SEQ ID NO:26. Preferably, the nucleic acid is about 95% homologous, and most preferably, about 99% homologous to at least one sequence of SEQ ID NO:8, SEQ ID NO:14, SEQ ID NO:20 and SEQ ID NO:26, disclosed herein. Even more preferably, the nucleic acid is at least one sequence of SEQ ID NO:8, SEQ ID NO:14, SEQ ID NO:20 and SEQ ID NO:26.

Thus, the invention encompasses an isolated nucleic acid encoding a mammalian immunodeficiency virus glycoprotein (gp) 120 polypeptide, or a mutant, derivative, or fragment thereof, wherein the gp120 polypeptide comprises a deletion of hypervariable loop 3 (V3), and further comprises a compensatory mutation. This is because, as demonstrated by the data disclosed herein, the present invention provides deletion mutants of gp120 wherein the V3 region is deleted/truncated while retaining biological function of the gp120 peptide. Such biological activity includes, but is not limited to, detectable binding with a chemokine receptor, detectable fusogenic activity, and detectable virus replication competence using a variety of assays either well-known in the art, disclosed herein, as well as assays to be developed in the future. This is remarkable in that prior art dogma was that the V3 was essential for peptide function and that deletion of this region obliterated such biological activity so that V3-deletion mutant comprising detectable function could not be generated.

Therefore, the present invention demonstrates that despite prior art teachings to the contrary, functional V3-deletion mutants can be produced, as amply exemplified by the mutants disclosed herein. Further, the data disclosed herein demonstrate certain features and characteristics useful for identification of potential modifiable virus Env, gp120, and gp41 peptides that can be used, according to the methods disclosed elsewhere herein, to produce deletion mutants of the invention. These mutants are important potential therapeutics since such deletion mutants represent functional "core" components that can be used to examine virus interaction with host cell components, identify novel compounds that can inhibit such interactions, and for development of neutralizing antibodies as well as vaccines for the generation thereof.

While the present invention is exemplified herein by development of HIV-2 deletion mutants, the teachings provided herein can be readily adapted to development of similar mutants in other mammalian immunodeficiency viruses, including, but not limited to, HIV-1 and SIV. This is due, in part, to the high degree of amino acid homology in the Env proteins of these viruses, including high homology in the gp120 across these viruses as demonstrated diagrammatically in FIG. 20 (see Original Patent) comparing the amino acid sequences of HIV-2 and SIVmac239. Further, the teachings of the present invention have already been extended to HIV-1 as demonstrated by data establishing a functional V3-deletion mutant of HIV-1 "580". Therefore, one skilled in the art, based upon the disclosure provided herein, would appreciate that the present invention is not limited to any particular mammalian immunodeficiency virus, but encompasses various such viruses including, but not limited to, simian immunodeficiency virus (SIV), human immunodeficiency virus type 1 (HIV-1), and human immunodeficiency virus type 2 (HIV-2).

The invention relates to a nucleic acid encoding a V3-deleted/truncated gp120 where the deletion includes a deletion of V3 is selected from about amino acid residue number 303 to amino acid residue number 324 (.DELTA.V3(6,6)) and a deletion from about amino acid residue number 298 to amino acid residue number 331 (.DELTA.V3(1,1)). These deletions are mapped relative to the amino acid sequence of the parental HIV-2/vcp gp120 as provided in SEQ ID NO:5. Therefore, the invention encompasses deletions that remove all but a single amino acid adjacent to the cysteines that form the loop to deletions that leave no more than six amino acids adjacent to each of the cysteines.

One skilled in the art would appreciate, once provided with the nucleic and amino acid sequences of the various mutants of the invention, as well as with those sequences of the parental virus, that the deletions of the amino acids of interest correspond with a deletion of the nucleotides encoding the pertinent amino acid residues deleted. For instance, while in no way limiting the invention to this particular deletion, a deletion of V3 of HIV-2/VCP gp120 termed (.DELTA.V3(1,1)), which deletes from about amino acid residue number 298 to amino acid residue number 331 relative to the amino acid sequence of HIV-2/vcp gp120 (SEQ ID NO:5) corresponds to a deletion from about nucleotide number 894 to nucleotide number 1032 relative to the nucleic acid encoding such gp120 (SEQ ID NO:2). Thus, each mutation specified according to a deletion of certain amino acids can be readily matched to the corresponding nucleotides encoding such amino acids to determine the corresponding deletion at the nucleic acid level of the nucleic acid encoding the gp120 peptide at issue.

The invention encompasses V-3 deletion mutants where the V1/V2 region of the gp120 is also deleted/truncated. Such double deletion mutants comprising deletion of both V1/V2 and V3 are exemplified by clone p16.5, clone p16.7, and clone 8c.3, but the invention is not limited to these or any particular mutants as would be appreciated by the artisan armed with the teachings provided herein.

The invention includes a compensatory mutation that mediates or is associated with prevention of loss of detectable virus function. While not limited to any particular compensatory mutation, such mutations in gp120 can include the following: an amino acid substitution from isoleucine to valine at amino acid residue number 55, an amino acid substitution from asparagine to aspartic acid at amino acid residue number 79, an amino acid substitution from phenylalanine to serine at amino acid residue number 94, an amino acid substitution from aspartic acid to glycine at amino acid residue number 142, an amino acid substitution from threonine to isoleucine at amino acid residue number 160, an amino acid substitution from alanine to threonine at amino acid residue number 173, an amino acid substitution from threonine to lysine at amino acid residue number 202, an amino acid substitution from glutamic acid to lysine at amino acid residue number 203, an amino acid substitution from threonine to isoleucine at amino acid residue number 231, an amino acid substitution from alanine to threonine at amino acid residue number 267, an amino acid substitution from asparagine to aspartic acid at amino acid residue number 279, an amino acid substitution from asparagine to aspartic acid at amino acid residue number 280, an amino acid substitution from glutamic acid to lysine at amino acid residue number 334, an amino acid substitution from glutamic acid to lysine at amino acid residue number 340, an amino acid substitution from asparagine to aspartic acid at amino acid residue number 391, an amino acid substitution from threonine to alanine at amino acid residue number 393, an amino acid substitution from glutamine to arginine at amino acid residue number 399, an amino acid substitution from valine to isoleucine at amino acid residue number 405, an amino acid substitution from valine to isoleucine at amino acid residue number 429, an amino acid substitution from glutamic acid to valine at amino acid residue number 437, an amino acid substitution from threonine to alanine at amino acid residue number 439, and an amino acid substitution from glycine to alanine at amino acid residue number 666. The amino acid residue position of these mutations is provided relative to the amino acid sequence of parental HIV-2/vcp gp120 (SEQ ID NO:5), which does not comprise a hypervariable region deletion.

This is because as more fully discussed elsewhere herein, certain mutations in gp120 and/or gp41 "compensate" for any loss of function resulting from truncation or deletion of a hypervariable region of gp120 such that the combination of at least one compensatory mutation, and more preferably, at least two compensatory mutations, in at least one of gp120 and gp41, can restore and/or preserve a biological function of gp120 once a substation, or all, of the V3 region is deleted from the protein.

Certain combinations of compensatory mutations are disclosed herein, and these include, but are not limited to, a gp120 comprising a .DELTA.V3(6,6) deletion and further wherein the compensatory mutation is at least one amino acid substitution selected from the group consisting of an amino acid substitution from isoleucine to valine at amino acid residue number 55, an amino acid substitution from asparagine to aspartic acid at amino acid residue number 79, an amino acid substitution from threonine to lysine at amino acid residue number 202, an amino acid substitution from threonine to isoleucine at amino acid residue number 231, an amino acid substitution from alanine to threonine at amino acid residue number 267, and an amino acid substitution from asparagine to aspartic acid at amino acid residue number 391, where the amino acid residue number is relative to the amino acid sequence of parental HIV-2/vcp gp120 as provided in SEQ ID NO:5. This particular combination of V3-deletion and compensatory mutations is exemplified in the p16.5 clone, but the invention is not limited to these mutations, or to this particular combination thereof. While some combinations can be preferred, other combinations of these and additional mutations are encompassed in the invention where the methods of the invention provide useful assays for isolating and identifying additional compensatory mutations and combinations thereof, which preserve/restore biological function following deletion of a hypervariable region of gp120.

Additional preferred combinations of V-3 deletion mutations and compensatory mutations include, but are not limited to, .DELTA.V3(6,6) deletion and compensatory mutations comprising an amino acid substitution from isoleucine to valine at amino acid residue number 55, an amino acid substitution from asparagine to aspartic acid at amino acid residue number 79, an amino acid substitution from phenylalanine to serine at amino acid residue number 94, an amino acid substitution from asparagine to aspartic acid at amino acid residue number 280, and an amino acid substitution from asparagine to aspartic acid at amino acid residue number 391, wherein the amino acid residue number of the compensatory mutation is relative to the amino acid sequence of parental HIV-2/vcp gp120 as provided in SEQ ID NO:5. This particular combination of V-3 deletion and compensatory mutations is exemplified by the gp120 p16.7 clone (SEQ ID NO:17), but the invention is not limited to this clone or to this particular combination of mutations.

Likewise, the invention encompasses a gp120 mutant comprising a .DELTA.V3(6,6) deletion and further comprising an amino acid substitution from threonine to alanine at amino acid residue number 393, and an amino acid substitution from valine to isoleucine at amino acid residue number 429, wherein the amino acid residue number of the compensatory mutation is relative to the amino acid sequence of parental HIV-2/vcp gp120 as provided in SEQ ID NO:5. This particular combination is exemplified by the p16.9 clone, but as stated previously elsewhere herein, the present invention is not limited to this particular clone, these particular compensatory mutations, or the particular combination set forth herein. Rather, the invention includes additional compensatory mutations identified and produced according to the teachings provided herein, and any combination thereof.

Further, the invention encompasses a gp120 mutant comprising a .DELTA.V3(1,1) deletion and further comprising a compensatory mutation such as an amino acid substitution from alanine to threonine at amino acid residue number 173, an amino acid substitution from glutamic acid to lysine at amino acid residue number 203, an amino acid substitution from threonine to alanine at amino acid residue number 393, an amino acid substitution from glutamine to arginine at amino acid residue number 405, an amino acid substitution from valine to isoleucine at amino acid residue number 429, an amino acid substitution from threonine to alanine at amino acid residue number 439, and an amino acid substitution from glycine to alanine at amino acid residue number 666, wherein the amino acid residue number of the compensatory mutation is relative to the amino acid sequence of parental HIV-2/vcp gp120 as provided in SEQ ID NO:5. The amino acid sequence of the 8c.3 clone is depicted in FIG. 19C (SEQ ID NO:29 (see Original Patent)) and the nucleic acid sequence encoding this clone is depicted in FIG. 19D (SEQ ID NO:26 (see Original Patent)). This particular combination of V-3 deletion and compensatory mutations is exemplified herein by HIV-2 clone 8c.3, but the invention is in no way limited to this clone.

The invention includes an isolated nucleic acid encoding a mammalian immunodeficiency virus glycoprotein (gp) 120 polypeptide, or a mutant, derivative, or fragment thereof, wherein the gp120 polypeptide comprises a deletion of hypervariable loop 3 (V3), a deletion of hypervariable loops V1/V2, and further comprises a compensatory mutation and where the nucleic acid sequence of the nucleic acid encoding the gp120 is selected from the group consisting of the sequence of SEQ ID NO:8, the sequence of SEQ ID NO:14, and the sequence of SEQ ID NO:26. Further, the V3 deletion encompasses a deletion from about amino acid residue number 303 to amino acid residue number 324 (.DELTA.V3(6,6)), and a deletion from about amino acid residue number 298 to amino acid residue number 331 (.DELTA.V3(1,1)), relative to the amino acid sequence of HIV-2/vcp gp120 as provided in SEQ ID NO:5. The invention also encompasses a nucleic acid that is, preferably, at least about 95% homologous, more preferably, 99% homologous, and even more preferably, is the sequence of at least one of SEQ ID NO:8, the sequence of SEQ ID NO:14, and the sequence of SEQ ID NO:26.

The invention encompasses an isolated nucleic acid encoding a mammalian immunodeficiency virus glycoprotein (gp) 120 polypeptide, or a mutant, derivative, or fragment thereof, wherein the gp120 polypeptide comprises a .DELTA.V3(6,6) deletion, and further comprises a compensatory mutation wherein the nucleic acid sequence of the nucleic acid comprises the sequence of SEQ ID NO:20. That is because, as exemplified by HIV-2 clone p16.9 disclosed herein, a mutant of the invention can include a V-3 deletion mutant where V1/V2 region of gp120 is not deleted.

The invention further relates to an isolated nucleic acid encoding a gp120 V-3 deletion variant of the invention, wherein the sequence of the nucleic acid is at least one sequence selected from the group consisting of SEQ ID NO:8, SEQ ID NO:14, SEQ ID NO:20, and SEQ ID NO:26.

The invention encompasses an isolated nucleic acid encoding a gp120 V-3 deletion variant of the invention, wherein the amino acid sequence of the gp120 polypeptide encoded by the nucleic acid is selected from the group consisting of the amino acid sequence of SEQ ID NO:11, the amino acid sequence of SEQ ID NO:17, the amino acid sequence of SEQ ID NO:23, and the amino acid sequence of SEQ ID NO:29. Preferably, the amino acid sequence encoded by the nucleic acid is at least 95% homologous with, more preferably, at least about 99% homologous with, and even more preferably, the sequence is at least one of the amino acid sequence of SEQ ID NO:11, the amino acid sequence of SEQ ID NO:17, the amino acid sequence of SEQ ID NO:23, and the amino acid sequence of SEQ ID NO:29.

One skilled in the art would appreciate, based upon the disclosure provided herein, that similar gp120 variant homologs exist and/or may be created in mammalian immunodeficiency viruses and can be readily identified and isolated using the methods described herein using the sequence data disclosed herein regarding the. HIV-2 .DELTA.V1/V2; .DELTA.V3(6,6), HIV-2 .DELTA.V1/V2; .DELTA.V3(1,1) HIV-2 .DELTA.V3(6,6) and HIV-2 .DELTA.V3(1,1) gp120 deletion mutants. Thus, the present invention encompasses additional gp120 variants that can be readily identified based upon the disclosure provided herein.

An isolated nucleic acid of the invention should be construed to include an RNA or a DNA sequence encoding a gp120 variant protein of the invention, and any modified forms thereof, including chemical modifications of the DNA or RNA which render the nucleotide sequence more stable when it is cell free or when it is associated with a cell. Chemical modifications of nucleotides may also be used to enhance the efficiency with which a nucleotide sequence is taken up by a cell or the efficiency with which it is expressed in a cell. Any and all combinations of modifications of the nucleotide sequences are contemplated in the present invention.

The present invention should not be construed as being limited solely to the nucleic and amino acid sequences disclosed herein. Once armed with the present invention, it is readily apparent to one skilled in the art that other nucleic acids encoding gp120 variant proteins such as those present in other mammalian immunodeficiency viruses (e.g., HIV-1, SIV) can be obtained by using the sequence information disclosed herein for human HIV-2 gp120 variant nucleic acids encoding human HIV-2 gp120 variant polypeptides as disclosed herein as would be understood by one skilled in the art. Methods for isolating a nucleic acid based on a known sequence are well-known in the art (e.g., screening of genomic or cDNA libraries), and are not described herein.

Further, any number of procedures may be used for the generation of mutant, derivative or variant forms of a gp120 variant using recombinant DNA methodology well known in the art. A wide plethora of techniques is available to the skilled artisan to produce muteins of interest and to select those with desired properties.

Techniques to introduce random mutations into DNA sequences are well known in the art, and include PCR mutagenesis, saturation mutagenesis, and degenerate oligonucleotide approaches. See Sambrook and Russell (2001, Molecular Cloning, A Laboratory Approach, Cold Spring Harbor Press, Cold Spring Harbor, N.Y.) and Ausubel et al. (2002, Current Protocols in Molecular Biology, John Wiley & Sons, NY).

As described in detail elsewhere herein, the present invention also features a nucleic acid encoding a mutant, derivative or variant of a gp120 polypeptide, wherein the gp120 polypeptide comprises at least one compensatory mutation. By way of a non-limiting example, in response to the deletion of a stabilizing domain from a polypeptide sequence, one or more amino acid mutations may be induced in the remaining polypeptide sequence in order to stabilize the truncated polypeptide. Further, a compensatory mutation encompasses where a deletion in one region of a polypeptide would otherwise result in a loss of a biological activity or function, but a mutation in another region of the polypeptide can detectably preserve or restore the loss of biological activity of function.

A compensatory mutation useful in the present invention includes, but is not limited to, an amino acid mutation, insertion, or deletion in an Env protein, wherein an amino acid mutation, insertion, or deletion arises, is induced, or is designed such that the resulting gp120 has the property of being fusogenic, supporting replication competence of a mammalian immunodeficiency virus comprising such gp120, or both. As discussed in greater detail elsewhere herein, a compensatory mutation useful in the present invention may arise or be induced in a gp120.

Further, the skilled artisan, based upon the disclosure provided herein, would appreciate that any discussion relating to a compensatory mutation that preserves or restores function despite a truncation of gp120 includes a mutation in gp41. This is because binding of gp120 to chemokine receptors, typically though interactions of the bridging sheet ("BS") with the chemokine receptor amino terminus and the V3 loop with the ECLs, transmits a signal to gp41 that causes it to initiate the fusion reaction. Thus, one way to compensate for the loss of a V3 loop can be through changes in gp41 that facilitate transmission of this signal, i.e., a "hair triggered" Envelope protein), and such mutations are therefore encompassed in the invention.

In the present invention, a "second change" that can induce or require the need for a compensatory mutation comprises a deletion of one or more hypervariable loops of a gp120. "Deletion of a hypervariable loop" of a gp120 comprises deletion of one or more amino acid residues in a hypervariable loop of the gp120, and is described in greater detail elsewhere herein. For example, "deletion of the V1/V2 loop" of a gp120 can range from the removal of a single nucleic acid triplet (codon) encoding the V1 loop region of a gp120 such that a single amino acid of the gp120 V1/V2 loop is not coded and is missing from the polypeptide where the reading frame for the rest of the sequence is maintained and the remaining amino acid residues following the deletion are produced. The deletion of the V1/V2 region can range to where all the nucleotides encoding amino acids on either sides of the disulfide bonds at amino acid residues number 110 to amino acid residue number 193 are deleted, resulting in a total deletion of the V1/V2 loop from a gp120. Such a deletion of V1/V2 is illustrated in FIGS. 1B and 1E (see Original Patent) using HIV-2/VCP for illustrative purposes only.

It would be understood by the skilled artisan, armed with the teachings provided herein, that reference to a "V1/V2" region encompasses the hypervariable loop V1 and V2 regions of a gp120 peptide since the loops of SIV and HIV-2 comprise more cysteines in this region such that it is well-known in the art that certain hypervariable region loops are not clearly divided into V1 and V2. The important feature of the invention is that truncation of V1/V2 at the base of the region can be readily applied to HIV-1, HIV-2 and SIV and it is not necessary to consider V1 and V2 regions separately for purposes of the present invention.

More specifically, one skilled in the art would appreciate, based upon the disclosure provided herein, that for HIV-2, the V1/V2 region includes from about amino acid residue number 110 to about amino acid residue number 194 relative to the amino acid sequence of SEQ ID NO:5 (full-length HIV-2/VCP gp120), corresponding to from about nucleotide number 330 to about nucleotide number 582 relative to the nucleic acid sequence of SEQ ID NO:2 (n.a. sequence of HIV-2/VCP gp120). Further, the V3 region comprises from about amino acid residue number 298 to about amino acid residue number 329 relative to the amino acid sequence of SEQ ID NO:5 (full-length HIV-2/VCP gp120), corresponding to from about nucleotide number 894 to about nucleotide number 1032 relative to the nucleic acid sequence of SEQ ID NO:2 (n.a. sequence of HIV-2/VCP gp120). Moreover, the HIV-2 V4 region comprises from about amino acid residue number 392 to about amino acid residue number 411 relative to the amino acid sequence of SEQ ID NO:5 (full-length HIV-2/VCP gp120), corresponding to from about nucleotide number 1176 to about nucleotide number 1233 relative to the nucleic acid sequence of SEQ ID NO:2 (n.a. sequence of HIV-2/VCP gp120).

For SIV, using SIVmac251 for illustrative purposes, the skilled artisan would understand, based upon the disclosure provided herein, that the V1/V2 region includes from about amino acid residue number 110 to about amino acid residue number 211 relative to the amino acid sequence of full-length SIVmac251 gp120 (FIG. 20 (see Original Patent)), corresponding to from about nucleotide number 330 to about nucleotide number 633 relative to the nucleic acid sequence of the nucleic acid sequence of full-length SIVmac251 gp120 which is known in the art. Further, the V3 region comprises from about amino acid residue number 315 to about amino acid residue number 344 relative to the amino acid sequence of full-length SIVmac251 gp120, corresponding to from about nucleotide number 945 to about nucleotide number 1032 relative to the nucleic acid sequence of full-length SIVmac251 gp120. Moreover the SIV V4 region comprises from about amino acid residue number 406 to about amino acid residue number 432 relative to the amino acid sequence of full-length SIVmac251 gp120, corresponding to from about nucleotide number 1218 to about nucleotide number 1296 relative to the nucleic acid sequence of the nucleic acid sequence of full-length SIVmac251 gp120.

For HIV-1, using HIV-1/HXB c2 by way of non-limiting example, the skilled artisan would understand, based upon the disclosure provided herein, that the V1/V2 region includes from about amino acid residue number 128 to about amino acid residue number 194 relative to the amino acid sequence of full-length HIV-1/HXB c2 gp120, corresponding to from about nucleotide number 384 to about nucleotide number 582 relative to the nucleic acid sequence of the nucleic acid sequence of full-length HIV-1/HXB c2 gp120, which are both well-known in the art. Further, the V3 region comprises from about amino acid residue number 298 to about amino acid residue number 329 relative to the amino acid sequence of full-length HIV-1/HXB c2 gp120, corresponding to from about nucleotide number 894 to about nucleotide number 987 relative to the nucleic acid sequence of the nucleic acid sequence of full-length HIV-1/HXB c2 gp120. Moreover the HIV-1 V4 region comprises from about amino acid residue number 387 to about amino acid residue number 416 relative to the amino acid sequence of full-length HIV-1 gp120, corresponding to from about nucleotide number 1161 to about nucleotide number 1248 relative to the nucleic acid sequence of the nucleic acid sequence of full-length HIV-1/HXB c2 gp120.

Thus, the skilled artisan, based upon the disclosure provided herein, would readily understand which portion(s) of gp120 should be deleted to produce a deletion mutant of the invention. Once armed with the amino and nucleic acids which comprise the hypervariable region of interest, one skilled in the art could readily produce a desired mutation thereby deleting any amino acid, or acids, of interest, including the aforementioned amino acid residues and the corresponding nucleotides encoding them. The amino acids comprising the various hypervariable regions of a wide plethora of mammalian immunodeficiency virus gp120 are well known in the art, as are the nucleic acids encoding those amino acids, and these sequences are therefore not discussed further herein.

Likewise, the various amino and nucleic acid sequences, as well as the functional domains and structural regions of a wide plethora of pg41 peptides are well known in the art and are therefore not discussed further herein since the skilled artisan would readily understand, based upon the disclosure provided herein, which amino acids and/or nucleic acids to mutagenize and to produce the mutant peptides of the invention.

Deletion of an amino acid from a hypervariable loop of a gp120 protein can include deletion of one or more amino acids responsible for the structure, function, or both, of the hypervariable loop. Further, deletion of an amino acid from a hypervariable loop of a gp120 protein can include deletion of one or more amino acids responsible for interaction of the hypervariable loop with other hypervariable loops, with core regions of the gp120, or with other Env proteins. The structure and function of the hypervariable loops of gp120 of mammalian immunodeficiency viruses, including, but not limited to HIV-1, HIV-2, and SIV, are known in the art and will not be discussed herein. Similarly, methods of deleting nucleotides of interest to produce deletions of interest of certain amino acid residues of a polypeptide are well known in the art and are not discussed further herein. Techniques for selective mutagenesis to produce deletions of interest are well known in the art and are available to the routineer such that they need not be set forth. The invention is not limited in any way to any particular method for producing the relevant deletion mutants and encompasses such methods as are known in the art or which are developed in the future.

In one aspect of the invention, a deletion mutation is produced in a gp120 by a deletion of the nucleic acid sequence encoding at least one amino acid of hypervariable loop 1 ("the V1 loop"). In another aspect, a deletion mutation is induced in a gp120 by a deletion of the nucleic acid sequence encoding at least one amino acid of the V2 loop. In yet another aspect, a deletion mutation is induced in a gp120 by a deletion of the nucleic acid sequence encoding at least one amino acid of the V3 loop. In another aspect of the invention, a deletion mutation is induced in a gp120 by a deletion of the nucleic acid sequence encoding at least one amino acid of the V4 loop.

In yet another aspect of the invention, a deletion mutation is induced in a gp120 by a deletion of the nucleic acid sequence encoding an entire hypervariable loop of gp120. In one embodiment, the deletion of a nucleic acid sequence encoding an entire hypervariable loop of gp120 results in the deletion of the entire V1 loop. In another embodiment, the deletion of a nucleic acid sequence encoding an entire hypervariable loop of gp120 results in the deletion of the entire V2 loop. In another embodiment of the invention, the deletion of a nucleic acid sequence encoding an entire hypervariable loop of gp120 results in the deletion of the entire V3 loop. In yet another embodiment, the deletion of a nucleic acid sequence encoding an entire hypervariable loop of gp120 results in the deletion of the entire V4 loop.

The present invention also features a nucleic acid encoding a gp120, wherein a mutation is induced by deletion of more than one hypervariable loop of a gp120. By way of a non-limiting example, a compensatory mutation may be induced in a gp120 comprising a deletion of the entire V1 loop, the entire V2 loop, and a substantial portion of the V3 loop of the gp120. By way of another example, a compensatory mutation may be introduced into a gp120 by deletion of the V1/V2 loops. By way of a further non-limiting example, a compensatory mutation may be induced in a gp120 by deletion of only the V3 hypervariable loop.

The skilled artisan would appreciate, once armed with the teachings provided herein, that an Env containing a V3 deletion was inserted into a replication competent clone of HIV-2/VCP and electroporated into SupT1 cells. Virus produced by these cells was then serially passaged on SupT1 and, following several rounds of infection, viruses were isolated that demonstrated increased infectivity. However, the invention is not limited to these methods for producing a replication-competent clone, as other methods would be understood to be included in the invention by one skilled in the art provided with the disclosure provided herein.

Envs were cloned from these viruses, sequenced, and were evaluated in cell to cell fusion assays. Differences that were identified in the adapted Env have been interpreted as being "compensatory mutations" (i.e., they impart increased infectivity to a parental loop-deleted Env). The following shows compensatory mutations that were observed in the serial passaging of HIV-2/VCP containing V3(6,6) deletion. This adapted Env was further mutated to V3(1,1) and when introduced into a virus and the process repeated, different mutations were observed as follows -- see Original Patent.

When armed with the disclosure provided herein, the skilled artisan will understand that multiple variations of hypervariable loop deletions can be used in any combination with an additional compensatory mutation in a nucleic acid encoding a gp120 polypeptide. Further, the present disclosure provides ample guidance for the skilled artisan to select either a portion or the entirety of a hypervariable loop for deletion, and for the skilled artisan to select multiple hypervariable loops for deletion, as well as for the production and selection of at least one compensatory deletion that detectably preserves or restores a gp120-mediated function or activity.

The present invention also includes a nucleic acid encoding a gp120 variant wherein the nucleic acid encoding a tag polypeptide is covalently linked thereto. That is, the invention encompasses a chimeric nucleic acid wherein the nucleic acid sequences encoding a tag polypeptide is covalently linked to the nucleic acid encoding at least one of HIV-2 .DELTA.V1/V2; .DELTA.V3(6,6), HIV-2 .DELTA.V1/V2; .DELTA.V3(1,1), HIV-2 .DELTA.V3(6,6) and HIV-2 .DELTA.V3(1,1). Such tag polypeptides are well known in the art and include, for instance, green fluorescent protein (GFP), myc, myc-pyruvate kinase (myc-PK), His6, maltose biding protein (MBP), an influenza virus hemagglutinin tag polypeptide, a flag tag polypeptide (FLAG), and a glutathione-S-transferase (GST) tag polypeptide. However, the invention should in no way be construed to be limited to the nucleic acids encoding the above-listed tag polypeptides. Rather, any nucleic acid sequence encoding a polypeptide which may function in a manner substantially similar to these tag polypeptides should be construed to be included in the present invention.

The nucleic acid comprising a nucleic acid encoding a tag polypeptide can be used to localize a gp120 variant within a cell, a tissue, and/or a whole organism (e.g., a mammalian embryo), and to study the role(s) of a gp120 variant in a cell or animal. Further, addition of a tag polypeptide facilitates isolation and purification of the "tagged" protein such that the proteins of the invention can be produced and purified readily.

As described in detail above with respect to compensatory mutations in nucleic acids encoding gp120 polypeptides, the present invention also provides for a compensatory mutation that can be induced in a nucleic acid encoding a gp41 polypeptide. A compensatory mutation of the invention in a gp41 can be selected for that detectably preserves or restores a virus activity or function despite the presence of a hypervariable loop deletion of gp120, as discussed in greater detail elsewhere herein.

A gp41 compensatory mutation useful in the present invention includes, but is not limited to, an amino acid mutation, insertion, or deletion in a gp41 protein, wherein an amino acid mutation, insertion, or deletion arises, is induced, or is designed such that the resulting gp41 has the property of being fusogenic, supporting replication competence of a mammalian immunodeficiency virus comprising such gp41, or both, where the gp120 of the virus comprises deletion of at least one hypervariable region, more preferably, where the gp120 deletion is a V3 deletion, and even more preferably, where the gp120 deletion is a deletion of V1, V2, and a substantial portion of V3, and most preferably, where the gp120 deletion is deletion of V1, V2, and V3.

The present invention includes an isolated nucleic acid encoding a mammalian immunodeficiency virus gp41 polypeptide, or a fragment thereof, wherein the nucleic acid encodes a variant of gp41 that comprises a compensatory mutation where the compensatory mutation comprises deletion comprising a truncation of the cytoplasmic domain. In an embodiment of the invention, a nucleic acid shares at least about 90% identity with at least one nucleic acid having the sequence of gp41 .DELTA.733, gp41 .DELTA.753 and gp41 .DELTA.764. Preferably, the nucleic acid is about 95% homologous, and most preferably, about 99% homologous to at least one of a nucleic acid encoding a truncated gp41 comprising the amino acid sequence disclosed herein where the truncation is set forth relative to the full-length sequence of parental HIV-2/VCP g41 (SEQ ID NO:6).

The invention relates to an isolated nucleic acid encoding a mammalian immunodeficiency virus gp41 polypeptide, wherein the gp41 polypeptide comprises a compensatory mutation. This is because, as more fully-discussed elsewhere herein, such compensatory mutation can surprisingly preserve and/or restore detectable biological function following deletion/truncation of a V3 region of gp120.

The invention includes an isolated nucleic acid comprising a nucleic acid sequence of SEQ ID NO:9, SEQ ID NO:15, SEQ ID NO:21, and SEQ ID NO:27. However, the invention is no way limited to these, or any other, particular nucleic acid sequences as other mutants comprising these and other compensatory mutations can be readily produced, identified and isolated following the novel teachings provided herein.

The amino acid sequence of the gp41 polypeptide encoded by the nucleic acid of the invention includes, but is not limited to, the amino acid sequence of SEQ ID NO:12, the amino acid sequence of SEQ ID NO:18, the amino acid sequence of SEQ ID NO:24, and the amino acid sequence of SEQ ID NO:30. While not limited to these particular amino acid sequences, the skilled artisan would appreciate that changes in the nucleotide sequence of the nucleic acid encoding the gp41 peptide of the invention which do not alter the amino acid sequence of the gp41 due to the degeneracy of the genetic code, are clearly encompassed by the present invention.

The invention encompasses a nucleic acid encoding a gp41 polypeptide of the invention, where the compensatory mutation in gp41 is a truncation of the cytoplasmic domain. The truncation can include, but is not limited to, truncation at amino acid residue number 733, truncation at amino acid residue number 753, and truncation at amino acid residue number 764, wherein the amino acid residue number of the truncation is provided in reference to the amino acid sequence of HIV-2/vcp gp41 (SEQ ID NO:6).

Further, the invention encompasses a nucleic acid encoding a gp41 of the invention where the compensatory mutation is at least one mutation selected from the group consisting of an amino acid substitution from leucine to valine at amino acid residue number 518, an amino acid substitution from alanine to threonine at amino acid residue number 529, an amino acid substitution from isoleucine to valine at amino acid residue number 531, an amino acid substitution from alanine to threonine at amino acid residue number 561, and an amino acid substitution from alanine to threonine at amino acid residue number 673, wherein the amino acid residue number of the compensatory mutation is relative to the amino acid sequence of HIV-2/vcp gp41 (SEQ ID NO:6). While these mutations are preferred, the invention is not limited in any way to these, or any other, particular compensatory mutations in gp41, or combinations thereof.

The present invention includes an isolated nucleic acid encoding mammalian immunodeficiency virus gp41 polypeptide, or a fragment thereof, wherein the nucleic acid comprises at least one compensatory mutation selected from the group consisting of a mutation that encodes a substitution of leucine to valine at amino acid residue number 518, and a mutation that encodes a substitution of an alanine to a threonine at amino acid residue number 529, relative to the amino acid sequence of SEQ ID NO:6 (HIV-2/VCP gp41). This particular mutant is exemplified by gp41 obtained from HIV-2 clone p16.5 and the sequence is depicted in FIG. 16 (SEQ ID NO:12 (see Original Patent)).

The present invention includes an isolated nucleic acid encoding mammalian immunodeficiency virus gp41 polypeptide, or a fragment thereof, wherein the nucleic acid comprises at least one compensatory mutation selected from the group consisting of a mutation that encodes a substitution of leucine to valine at amino acid residue number 518, a mutation that encodes a substitution of an alanine to a threonine at amino acid residue number 529, and an amino acid substitution from isoleucine to valine at amino acid residue number 531, relative to the amino acid sequence of SEQ ID NO:6 (HIV-2/VCP gp41). This particular mutant is exemplified by gp41 obtained from HIV-2 clone p16.7 and the sequence is depicted in FIG. 17 (SEQ ID NO:18 (see Original Patent)).

The present invention includes an isolated nucleic acid encoding mammalian immunodeficiency virus gp41 polypeptide, or a fragment thereof, wherein the nucleic acid comprises at least one compensatory mutation selected from the group consisting of a mutation that encodes a substitution of leucine to valine at amino acid residue number 518, and an amino acid substitution from alanine to threonine at amino acid residue number 561, relative to the amino acid sequence of SEQ ID NO:6 (HIV-2/VCP gp41). This particular mutant is exemplified by gp41 obtained from HIV-2 clone p16.9 and the amino acid sequence is depicted in FIG. 18 (SEQ ID NO:24 (see Original Patent)).

The present invention includes an isolated nucleic acid encoding mammalian immunodeficiency virus gp41 polypeptide, or a fragment thereof, wherein the nucleic acid comprises at least one compensatory mutation as depicted in the amino acid sequence set out in FIG. 19E (SEQ ID NO:30 (see Original Patent)), which shows the amino acid sequence of gp41 obtained from clone 8c.3. The nucleic acid encoding this clone comprises the nucleic acid sequence depicted in FIG. 19F (SEQ ID NO:27).

As noted previously with respect to various mutants of gp120, the present invention is not limited in any way to these, or any other, gp41 mutants comprising compensatory mutations, or combinations thereof. Rather, the gp41 mutants described herein serve illustrative purposes and demonstrate that using the methods disclosed herein these and additional mutants of the invention can be readily produced and isolated by the skilled artisan once armed with the disclosure provided herein.

The present invention includes an isolated nucleic acid encoding mammalian immunodeficiency virus gp41 polypeptide, or a fragment thereof, wherein the nucleic acid shares greater than about 90% homology with at least one of SEQ ID NO:9, SEQ ID NO:15, SEQ ID NO:21, and SEQ ID NO:27. Preferably, the nucleic acid is about 95% homologous, and most preferably, about 99% homologous to at least one of SEQ ID NO:9, SEQ ID NO:15, SEQ ID NO:21, and SEQ ID NO:27. Even more preferably, the nucleic acid is at least one of SEQ ID NO:9, SEQ ID NO:15, SEQ ID NO:21, and SEQ ID NO:27.

One skilled in the art would appreciate, based upon the disclosure provided herein, that similar gp41 variant homologs exist and/or may be created in mammalian immunodeficiency viruses and can be readily identified and isolated using the methods described herein using the sequence data and the selection strategy and assays disclosed herein regarding the .DELTA.733, .DELTA.753, .DELTA.764 gp41 deletion mutants. Thus, the present invention encompasses additional gp41 variants that can be readily identified based upon the disclosure provided herein.

An isolated nucleic acid of the invention should be construed to include an RNA or a DNA sequence encoding a gp41 variant protein of the invention, and any modified forms thereof, including chemical modifications of the DNA or RNA which render the nucleotide sequence more stable when it is cell free or when it is associated with a cell. Chemical modifications of nucleotides may also be used to enhance the efficiency with which a nucleotide sequence is taken up by a cell or the efficiency with which it is expressed in a cell. Any and all combinations of modifications of the nucleotide sequences are contemplated in the present invention.

The present invention should not be construed as being limited solely to the nucleic and amino acid sequences disclosed herein. Once armed with the present invention, it is readily apparent to one skilled in the art that other nucleic acids encoding gp41 variant proteins such as those present in other mammalian immunodeficiency viruses (e.g., HIV-1, SIV) can be obtained by using the sequence information disclosed herein for human HIV-2 gp41 variant nucleic acids encoding human HIV-2 gp41 variant polypeptides as disclosed herein as would be understood by one skilled in the art. Methods for isolating a nucleic acid based on a known sequence are well-known in the art (e.g., screening of genomic or cDNA libraries), and are not described herein.

Further, any number of procedures may be used for the generation of mutant, derivative or variant forms of a gp41 variant using recombinant DNA methodology well known in the art. A wide plethora of techniques is available to the skilled artisan to produce muteins of interest and to select those with desired properties.

The present invention also includes a nucleic acid encoding a gp41 variant wherein the nucleic acid encoding a tag polypeptide is covalently linked thereto. That is, the invention encompasses a chimeric nucleic acid wherein the nucleic acid sequences encoding a tag polypeptide is covalently linked to the nucleic acid encoding at least one of HIV-2 .DELTA.733 gp41, HIV-2 .DELTA.753 gp41, HIV-2 .DELTA.764 gp41, gp41 encoded by a nucleic acid comprising at least one sequence of SEQ ID NO:9, SEQ ID NO:15, SEQ ID NO:21, and SEQ ID NO:27. Such tag polypeptides are well known in the art and include, for instance, green fluorescent protein (GFP), myc, myc-pyruvate kinase (myc-PK), His6, maltose biding protein (MBP), an influenza virus hemagglutinin tag polypeptide, a flag tag polypeptide (FLAG), and a glutathione-S-transferase (GST) tag polypeptide. However, the invention should in no way be construed to be limited to the nucleic acids encoding the above-listed tag polypeptides. Rather, any nucleic acid sequence encoding a polypeptide which may function in a manner substantially similar to these tag polypeptides should be construed to be included in the present invention.

II. Isolated Polypeptides

The invention also includes an isolated mammalian immunodeficiency virus gp120 polypeptide. Preferably, the isolated polypeptide is about 95% homologous, more preferably, about 99% homologous, to at least one amino acid sequence of SEQ ID NO:11, SEQ ID NO:17, SEQ ID NO:23 and SEQ ID NO:29. More preferably, the isolated polypeptide is at least one of an amino acid sequence of SEQ ID NO:11, SEQ ID NO:17, SEQ ID NO:23 and SEQ ID NO:29.

The skilled artisan would appreciate, based upon the disclosure provided herein, that the mammalian immunodeficiency virus includes, but is not limited to, human and simian virus, such as, but not limited to, SIV, HIV-1 and HIV-2.

The invention includes a mammalian immunodeficiency virus gp120 polypeptide comprising a deletion of V1 and V2, and further comprising a deletion of V3. The skilled artisan would understand, once armed with the teachings provided herein, that the deletion is one that deletes all but the first and last amino acid of the V1/V2 loop. The deletion of V3 can range from one that deletes all but the first and last 6 amino acids of the V3 loop, to one that contains only the first and the last amino acid. (i.e., in the HIV-2/VCP sequence a deletion of a single amino acid residue from the residues from about amino acid residue number 110 to amino acid residue number 194 of gp120), to a deletion of the entire V3 region (i.e., a deletion of from about amino acid residue number 298 to amino acid residue number 331).

The invention includes an isolated gp120 polypeptide, where the deletion of V3 can be a deletion of from about amino acid residue number 303 to amino acid residue number 324 (.DELTA.V3(6,6)) relative to the amino acid sequence of HIV-2/vcp gp120 as provided in SEQ ID NO:5, and a deletion from about amino acid residue number 298 to amino acid residue number 331 (.DELTA.V3(1,1)) relative to the amino acid sequence of HIV-2/vcp gp120 as provided in SEQ ID NO:5. And the gp120 polypeptide can further comprise a deletion of the V1/V2 region. This is because, as more fully disclosed elsewhere, such V-loop deletion peptides are useful for elucidating the structure and function of otherwise obscured or inaccessible domains of gp120 and also provide important potential immunogens for generation of neutralizing antibodies and for the development of novel therapeutics for immunodeficiency virus related diseases.

As disclosed previously elsewhere herein, the invention includes a gp120 mutant comprising at least one compensatory mutation. Such compensatory mutations include, but are not limited to, an amino acid substitution from isoleucine to valine at amino acid residue number 55, an amino acid substitution from asparagine to aspartic acid at amino acid residue number 79, an amino acid substitution from phenylalanine to serine at amino acid residue number 94, an amino acid substitution from aspartic acid to glycine at amino acid residue number 142, an amino acid substitution from threonine to isoleucine at amino acid residue number 160, an amino acid substitution from alanine to threonine at amino acid residue number 173, an amino acid substitution from threonine to lysine at amino acid residue number 202, an amino acid substitution from glutamic acid to lysine at amino acid residue number 203, an amino acid substitution from threonine to isoleucine at amino acid residue number 231, an amino acid substitution from alanine to threonine at amino acid residue number 267, an amino acid substitution from asparagine to aspartic acid at amino acid residue number 279, an amino acid substitution from asparagine to aspartic acid at amino acid residue number 280, an amino acid substitution from glutamic acid to lysine at amino acid residue number 334, an amino acid substitution from glutamic acid to lysine at amino acid residue number 340, an amino acid substitution from asparagine to aspartic acid at amino acid residue number 391, an amino acid substitution from threonine to alanine at amino acid residue number 393, an amino acid substitution from valine to isoleucine at amino acid residue number 399, an amino acid substitution from glutamine to arginine at amino acid residue number 405, an amino acid substitution from valine to isoleucine at amino acid residue number 429, an amino acid substitution from glutamic acid to valine at amino acid residue number 437, an amino acid substitution from threonine to alanine at amino acid residue number 439, and an amino acid substitution from glycine to alanine at amino acid residue number 666, wherein the amino acid residue number of the compensatory mutation is relative to the amino acid sequence of parental HIV-2/vcp gp120 as provided in SEQ ID NO:5.

The data disclosed herein demonstrate that these mutations are associated with and can potentially mediate the preservation and/or restoration of detectable biological acitivity to gp120 following deletion/truncation of the V3 region of the protein.

Additionally, the invention encompasses a gp120 where the V3 deletion is .DELTA.V3(6,6) and further wherein the compensatory mutation is at least one of an amino acid substitution selected from the group consisting of an amino acid substitution from isoleucine to valine at amino acid residue number 55, an amino acid substitution from asparagine to aspartic acid at amino acid residue number 79, an amino acid substitution from threonine to lysine at amino acid residue number 202, an amino acid substitution from threonine to isoleucine at amino acid residue number 231, an amino acid substitution from alanine to threonine at amino acid residue number 267, and an amino acid substitution from asparagine to aspartic acid at amino acid residue number 391, wherein the amino acid residue number of the compensatory mutation is relative to the amino acid sequence of parental HIV-2/vcp gp120 as provided in SEQ ID NO:5. Such combination of V3 deletion and compensatory mutations is exemplified by the HIV-2 p16.5 clone gp120. The amino acid sequence of this clone is depicted in FIG. 22C (SEQ ID NO:11 (see Original Patent)).

Likewise, the invention encompasses a gp120 polypeptide where the V3 deletion is .DELTA.V3(6,6) and where the compensatory mutation is at least one of an amino acid substitution selected from the group consisting of an amino acid substitution from isoleucine to valine at amino acid residue number 55, an amino acid substitution from asparagine to aspartic acid at amino acid residue number 79, an amino acid substitution from phenylalanine to serine at amino acid residue number 94, an amino acid substitution from asparagine to aspartic acid at amino acid residue number 280, and an amino acid substitution from asparagine to aspartic acid at amino acid residue number 391, wherein the amino acid residue number of the compensatory mutation is relative to the amino acid sequence of parental HIV-2/vcp gp120 as provided in SEQ ID NO:5. Such combination of V3 deletion and compensatory mutations is exemplified by the HIV-2 p16.7 clone gp120. The amino acid sequence of this clone is depicted in FIG. 23C (SEQ ID NO:17).

The invention encompasses an isolated gp120 polypeptide where the V3 deletion is .DELTA.V3(6,6) and further where the compensatory mutation is at least one of an amino acid substitution selected from the group consisting of an amino acid substitution from threonine to alanine at amino acid residue number 393, and an amino acid substitution from valine to isoleucine at amino acid residue number 429, wherein the amino acid residue number of the compensatory mutation is relative to the amino acid sequence of parental HIV-2/vcp gp120 as provided in SEQ ID NO:5. Such combination of V3 deletion and compensatory mutations is exemplified by the HIV-2 p16.9 clone gp120. The amino acid sequence of this clone is depicted in FIG. 24C (SEQ ID NO:23).

The invention also includes an isolated gp120 polypeptide where the V3 deletion is .DELTA.V3(1,1) and further where the compensatory mutation is at least one of an amino acid substitution selected from the group consisting of an amino acid substitution from alanine to threonine at amino acid residue number 173, an amino acid substitution from glutamic acid to lysine at amino acid residue number 203, an amino acid substitution from threonine to alanine at amino acid residue number 393, an amino acid substitution from glutamine to arginine at amino acid residue number 405, an amino acid substitution from valine to isoleucine at amino acid residue number 429, an amino acid substitution from threonine to alanine at amino acid residue number 439, and an amino acid substitution from glycine to alanine at amino acid residue number 666, wherein the amino acid residue number of the compensatory mutation is relative to the amino acid sequence of parental HIV-2/vcp gp120 as provided in SEQ ID NO:5. Such combination of V3 deletion and compensatory mutations is exemplified by the HIV-2 8c.3 clone gp120. The amino acid sequence of this clone is depicted in FIG. 19C (SEQ ID NO:29).

As more fully discussed elsewhere herein, these various clones of HIV-2 are set forth herein for illustrative purposes only. The present invention is not limited in any way to these, or any other, particular combinations of V3 deletions and compensatory mutations.

The invention encompasses a n isolated gp120 polypeptide, or a mutant, derivative, or fragment thereof, comprising a deletion of hypervariable loop 3 (V3), a deletion of hypervariable loops V1/V2, and further comprising a compensatory mutation wherein the amino acid sequence of the gp120 polypeptide is selected from the group consisting of the sequence of SEQ ID NO:11, the sequence of SEQ ID NO:17, and the sequence of SEQ ID NO:29. Also, the invention includes an isolated gp120 polypeptide, or a mutant, derivative, or fragment thereof, wherein the gp120 polypeptide comprises a deletion of hypervariable loop 3 (V3), and further comprises a compensatory mutation wherein the amino acid sequence of the gp120 polypeptide comprises the sequence of SEQ ID NO:23, as exemplified, for illustrative purposes only, but HIV-2 gp120 p16.9 clone.

The present invention also provides for analogs of proteins or peptides which comprise a mammalian immunodeficiency virus gp120 polypeptide as disclosed herein. Analogs may differ from naturally occurring proteins or peptides by conservative amino acid sequence differences or by modifications which do not affect sequence, or by both. For example, conservative amino acid changes may be made, which although they alter the primary sequence of the protein or peptide, do not normally alter its function. Conservative amino acid substitutions typically include substitutions within the following groups: glycine, alanine; valine, isoleucine, leucine; aspartic acid, glutamic acid; asparagine, glutamine; serine, threonine; lysine, arginine; phenylalanine, tyrosine. Modifications (which do not normally alter primary sequence) include in vivo, or in vitro, chemical derivatization of polypeptides, e.g., acetylation, or carboxylation. Also included are modifications of glycosylation, e.g., those made by modifying the glycosylation patterns of a polypeptide during its synthesis and processing or in further processing steps; e.g., by exposing the polypeptide to enzymes which affect glycosylation, e.g., mammalian glycosylating or deglycosylating enzymes. Also embraced are sequences which have phosphorylated amino acid residues, e.g., phosphotyrosine, phosphoserine, or phosphothreonine.

Also included are polypeptides which have been modified using ordinary molecular biological techniques so as to improve their resistance to proteolytic degradation or to optimize solubility properties or to render them more suitable as a therapeutic agent. Analogs of such polypeptides include those containing residues other than naturally occurring L-amino acids, e.g., D-amino acids or non-naturally occurring synthetic amino acids. The peptides of the invention are not limited to products of any of the specific exemplary processes listed herein.

The present invention should also be construed to encompass "mutants," "derivatives," and "variants" of the peptides of the invention (or of the DNA encoding the same) which mutants, derivatives and variants are mammalian immunodeficiency virus gp120 peptides which are altered in one or more amino acids (or, when referring to the nucleotide sequence encoding the same, are altered in one or more base pairs) such that the resulting peptide (or DNA) is not identical to the sequences recited herein, but has the same biological property as the gp120 variant peptides disclosed herein, in that the peptide has biological/biochemical properties of a mammalian immunodeficiency virus gp120 polypeptide of the present invention (e.g., despite deletion of all or a substantial portion of the V3 region, the polypeptide specifically binds with its ligand chemokine coreceptor, it can mediate detectable fusion with the host cell, and/or the polypeptide can mediate detectable replication competence of the virus).

The skilled artisan would understand, based upon the disclosure provided herein, that gp120 biological activity encompasses, but is not limited to, the ability of a molecule to specifically interact with a cellular chemokine coreceptor, to mediate detectable fusogenicity, and/or to mediate detectable virus replication in a cell.

Further, the invention should be construed to include naturally occurring variants or recombinantly derived mutants of gp120 variant sequences, which variants or mutants render the protein encoded thereby either more, less, or just as biologically active as the sequences of the invention.

The nucleic acids disclosed herein, and peptides encoded thereby, are useful tools for elucidating the function(s) of a gp120 molecule in a cell. Further, nucleic and amino acids comprising a mammalian gp120 polypeptide of the invention are useful diagnostics which can be used, for example, to identify a compound that affects gp120 function or expression, which compound is a potential drug candidate for a disease, disorder or condition associated with, or mediated by, mammalian immunodeficiency virus infection. The nucleic acids, the proteins encoded thereby, or both, can be administered to a cell, tissue, or mammal to increase or decrease expression or function of gp120 as disclosed herein, in the cell, tissue or mammal to which it is administered. This can be beneficial for the cell, tissue, and/or mammal in situations where the presence of gp120, or variant thereof, on the surface of a mammalian immunodeficiency virus in the cell, tissue or mammal mediates a disease or condition associated with gp120 interaction with one or more cellular cytokine receptors.

That is, the data disclosed herein demonstrate for the first time that core regions of the gp120 protein are responsible, at least in part, for immunodeficiency virus entry into a cell. Thus, these gp120 molecules are important targets for the production of potential therapeutics. Further, the data suggest that specific segments and amino acid residues of gp120 are non-essential for immunodeficiency virus entry into a cell. Production of the gp120 polypeptides of the invention in a cell provide sufficient quantities of the polypeptide to be used, for instance, in an assay to assess the role of various determinants in chemokine coreceptor binding and also to identify a compound that affects such binding, which is a potential useful therapeutic to inhibit the binding and thereby prevent and/or treat virus invention, but the invention is not limited to these, or any other particular use of such polypeptides.

The invention also includes an isolated mammalian immunodeficiency virus gp41 polypeptide comprising a compensatory mutation. Preferably, the isolated mammalian immunodeficiency virus gp41 polypeptide is shares greater than about 90% identity with a polypeptide having the amino acid sequence of at least one of SEQ ID NO:12, SEQ ID NO:18, SEQ ID NO:24, and SEQ ID NO:30. Preferably, the isolated polypeptide is about 95% homologous, and most preferably, about 99% homologous to at least one of SEQ ID NO:12, SEQ ID NO:18, SEQ ID NO:24, and SEQ ID NO:30. Most preferably, the amino acid sequence of the gp41 polypeptide is at least one of the sequence of SEQ ID NO:12, SEQ ID NO:18, SEQ ID NO:24, and SEQ ID NO:30.

The invention also encompasses an isolated mammalian immunodeficiency virus gp41 polypeptide comprising a truncation of the cytoplasmic domain where the gp41 polypeptide is at least one of HIV-2 gp41 .DELTA.733, HIV-2 gp41 .DELTA.753 and HIV-2 gp41 .DELTA.764, where the truncation is located at the indicated amino acid residue number relative to the amino acid sequence of full-length parental HIV-2/VCP gp41 (SEQ ID NO:6).

The invention encompasses a gp41 polypeptide comprising at least one compensatory mutation selected from the following: an amino acid substitution from leucine to valine at amino acid residue number 518, an amino acid substitution from alanine to threonine at amino acid residue number 529, an amino acid substitution from isoleucine to valine at amino acid residue number 531, an amino acid substitution from alanine to threonine at amino acid residue number 561, and an amino acid substitution from alanine to threonine at amino acid residue number 673, wherein the amino acid residue number of the compensatory mutation is relative to the amino acid sequence of HIV-2/vcp gp41 (SEQ ID NO:6).

The invention further includes an isolated mammalian immunodeficiency virus gp41 polypeptide comprising a compensatory mutation where, preferably, the gp41 polypeptide is shares greater than about 90% identity with a polypeptide having the amino acid sequence of at least one of SEQ ID NO:12 (gp41 of p16.5 clone, shown in FIG. 22E), SEQ ID NO:18 (gp41 p16.7 clone shown on FIG. 23E), SEQ ID NO:24 (gp41 p16.9 clone depicted in FIG. 24E), and SEQ ID NO:30 (clone 8c.3 gp41 depicted in FIG. 19E). Preferably, the isolated polypeptide is about 90% homologous, more preferably, about 95% homologous, and most preferably, about 99% homologous to at least one of SEQ ID NO:12, SEQ ID NO:18, SEQ ID NO:24, and SEQ ID NO:30. More preferably, the isolated polypeptide comprising a mammalian immunodeficiency virus gp41 variant is at least one of HIV-2 gp41 p16.5, HIV-2 gp41 p16.7, HIV-2 gp41 p16.9, and HIV-2 gp41 p16.7. Most preferably, the isolated polypeptide comprising a mammalian gp41 variant is at least one of SEQ ID NO:12, SEQ ID NO:18, SEQ ID NO:24, and SEQ ID NO:30.

The invention also includes an isolated human immunodeficiency virus gp41 polypeptide comprising at least one compensatory mutation selected from the group consisting of an amino acid substitution from leucine to valine at amino acid residue number 518, an amino acid substitution from alanine to threonine at amino acid residue number 529, and an amino acid substitution from alanine to threonine at amino acid residue number 561. This is the combination of mutations as depicted in FIG. 16, setting forth the amino acid sequence of HIV-2/VCP gp41 obtained from p16.5 clone. The invention also includes a gp41 comprising at least one compensatory mutation as follows: an amino acid substitution from leucine to valine at amino acid residue number 518, an amino acid substitution from alanine to threonine at amino acid residue 529, and an amino acid substitution from isoleucine to valine at amino acid residue 531. This combination of mutations is depicting in FIG. 17, setting forth the amino acid sequence of HIV-2/VCP gp41 obtained from p16.7 clone. Additionally, the invention includes a gp41 comprising at least one compensatory mutation as follows: an amino acid substitution from leucine to valine at amino acid residue number 518, an amino acid substitution from alanine to threonine at amino acid residue 561, and an amino acid substitution from alanine to threonine at amino acid residue 673. This combination of mutations is depicted in FIG. 18, showing amino acid sequence and illustrating the conformation of HIV-2/VCP gp41 obtained from p16.9 clone. Clone 8c.3 comprises a gp41 (SEQ ID NO:30) comprising certain compensatory mutations when compared with parental HIV-2/VCP gp41 (SEQ ID NO:6).

As noted previously elsewhere herein, the present invention is in no way limited to these, or any other, particular compensatory mutations, or combinations thereof. Thus, one skilled in the art would appreciate, based upon the disclosure provided herein, that the present invention is not limited to these particular gp41 compensatory mutations, nor to compensatory mutations limited solely to truncation of the cytoplasmic domain of gp41. Nor is the present invention limited to these particular truncation mutations in the cytoplasmic domain of gp41. This is because the skilled artisan, armed with the teachings provided herein, could readily identify and isolate additional compensatory mutations of gp41 that detectably preserve and/or restore gp120 function and/or activity upon deletion of all, or part, of gp120 V3 by following the teachings set forth herein.

The present invention should also be construed to encompass "mutants," "derivatives," and "variants" of the peptides of the invention (or of the DNA encoding the same) which mutants, derivatives and variants are mammalian immunodeficiency virus gp41 peptides which are altered in one or more amino acids (or, when referring to the nucleotide sequence encoding the same, are altered in one or more base pairs) such that the resulting peptide (or DNA) is not identical to the sequences recited herein, but has the same biological property as the gp41 variant peptides disclosed herein, in that the peptide has biological/biochemical properties of a mammalian immunodeficiency virus gp120 polypeptide of the present invention (e.g., the gp120 can specifically bind a chemokine coreceptor, mediates detectable fusogenicity, and/or can mediate detectable virus replication in a cell).

The present invention should not be construed as being limited solely to the polypeptides disclosed herein. Once armed with the present invention, it is readily apparent to one skilled in the art that other gp120 and gp41 variant proteins such as those present in other mammalian immunodeficiency viruses (e.g., HIV-1, SIV) can be obtained by using the sequence information and the extensive teachings disclosed herein for human HIV-2 gp120 and HIV-2 gp41 variant polypeptides, respectively, as disclosed herein and as would be understood by one skilled in the art. Methods for isolating a polypeptide based on a known sequence are well-known in the art (e.g., affinity chromatography), and are not described herein. Further, as will be understood by the skilled artisan in light of the disclosure provided herein, gp120 and gp41 variant proteins such as those present in other mammalian immunodeficiency viruses (e.g., HIV-1, SIV) would be useful in the present invention due to similarities in sequence, structure, and function of such proteins to the polypeptides of the present invention. Therefore, using the methods and techniques disclosed herein, additional gp120 and/or gp41 mutants can be readily produced, characterized and isolated which possess the requisite characteristics disclosed herein in that they can, among other things, comprise a complete or substantial deletion of V3 and can nevertheless demonstrate detectable binding with a chemokine coreceptor, fuse with a cell, and/or demonstrate detectable replication in a cell.

III. Vectors

In other related aspects, the invention includes an isolated nucleic acid encoding a mammalian immunodeficiency virus gp120 as disclosed previously elsewhere herein operably linked to a nucleic acid specifying a promoter/regulatory sequence such that the nucleic acid is preferably capable of directing expression of the protein encoded by the nucleic acid. Thus, the invention encompasses expression vectors and methods for the introduction of exogenous DNA into cells with concomitant expression of the exogenous DNA in the cells such as those described, for example, in Sambrook et al. (1989, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York), and in Ausubel et al. (1997, Current Protocols in Molecular Biology, John Wiley & Sons, New York).

That is, the invention encompasses an isolated nucleic acid encoding a mammalian immunodeficiency virus glycoprotein gp120 polypeptide, wherein the gp120 comprises a deletion of V1, a deletion of V2, and further comprises a substantial deletion of V3, where the nucleic acid is operably linked to a nucleic acid specifying a promoter/regulatory sequence.

Similarly, the invention encompasses an isolated nucleic acid encoding a mammalian immunodeficiency virus glycoprotein gp41 polypeptide, wherein the gp41 comprises a compensatory mutation, including, but not limited to a truncation of the cytoplasmic domain of the gp41, where the nucleic acid is operably linked to a nucleic acid specifying a promoter/regulatory sequence.

Expression of the afore-mentioned gp120 and/or gp41, either alone or fused to a detectable tag polypeptide, in cells which either do not normally express the polypeptide, or which do not express the polypeptide fused with a tag polypeptide, can be accomplished by generating a plasmid, viral, or other type of vector comprising the desired nucleic acid operably linked to a promoter/regulatory sequence which serves to drive expression of the protein, with or without tag, in cells in which the vector is introduced. Many promoter/regulatory sequences useful for driving constitutive expression of a nucleic acid of interest are available in the art and include, but are not limited to, for example, the cytomegalovirus immediate early promoter enhancer sequence, the SV40 early promoter, as well as the Rous sarcoma virus promoter, and the like.

Moreover, inducible and tissue specific expression of the nucleic acid encoding the gp120 and/or gp41 of the present invention can be accomplished by placing the nucleic acid encoding WNK, with or without a tag, under the control of an inducible or tissue specific promoter/regulatory sequence. Examples of tissue specific or inducible promoter/regulatory sequences which are useful for his purpose include, but are not limited to the MMTV LTR inducible promoter, and the SV40 late enhancer/promoter. In addition, promoters which are well known in the art which are induced in response to inducing agents such as metals, glucocorticoids, and the like, are also contemplated in the invention. Thus, it will be appreciated that the invention includes the use of any promoter/regulatory sequence, which is either known or unknown, and which is capable of driving expression of the desired protein operably linked thereto.

The invention includes methods of inhibiting expression, translation, and/or activity in a cell of gp120 and/or gp41 of the invention, as well as methods relating to increasing expression, protein level, and/or activity of the gp120 and/or gp41 of the invention since both decreasing and increasing gp120 and/or gp41 expression and/or activity can be useful in providing effective therapeutics and/or diagnostic reagents.

Selection of any particular plasmid vector or other DNA vector is not a limiting factor in this invention and a wide variety of vectors is well-known in the art. Further, it is well within the skill of the artisan to choose particular promoter/regulatory sequences and operably link those promoter/regulatory sequences to a DNA sequence encoding a desired polypeptide. Such technology is well known in the art and is described, for example, in Sambrook et al. (1989, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, N.Y.), and in Ausubel et al. (1997, Current Protocols in Molecular Biology, John Wiley & Sons, New York).

The invention thus includes a vector comprising an isolated nucleic acid encoding a mammalian immunodeficiency virus gp120 and/or gp41 of the invention as disclosed elsewhere herein. The incorporation of a desired nucleic acid into a vector and the choice of vectors is well-known in the art as described in, for example, Sambrook et al. (1989, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, N.Y.), and in Ausubel et al. (1997, Current Protocols in Molecular Biology, John Wiley & Sons, New York).

The invention also includes cells, viruses, proviruses, and the like, containing such vectors. Methods for producing cells comprising vectors and/or exogenous nucleic acids are well-known in the art, and is detailed in, for example, Sambrook et al. (1989, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York), and in Ausubel et al. (1997, Current Protocols in Molecular Biology, John Wiley & Sons, New York).

The nucleic acids encoding a gp120 and/or gp41 of the invention can be cloned into various plasmid vectors. However, the present invention should not be construed to be limited to plasmids, or to any particular vector. Instead, the present invention encompasses a wide plethora of vectors which are readily available and/or well-known in the art, or as will be developed in the future. One skilled in the art would understand, once provided with the nucleic and amino acid sequences of the present invention, as well as the various teachings provided herein, that a wide plethora of molecular biology techniques can be applied to producing various recombinant constructs which can be used in a variety of techniques as are well-known in the art.

IV. Recombinant Cells

The invention includes a recombinant cell comprising, inter alia, an isolated nucleic acid encoding a mammalian immunodeficiency virus gp120 polypeptide, wherein the polypeptide comprises a deletion of a V1, deletion of V2, and further comprises a substantial deletion of V3, or a complete deletion thereof. The invention also encompasses an antisense nucleic acid complementary thereto, a nucleic acid encoding an antibody that specifically binds a gp120 polypeptide encoded by that nucleic acid, and the like. In one aspect, the recombinant cell can be transiently transfected with a plasmid encoding a portion of the nucleic acid encoding the gp120 V3 deletion polypeptide. The nucleic acid need not be integrated into the cell genome nor does it need to be expressed in the cell. Moreover, the cell may be a prokaryotic or a eukaryotic cell and the invention should not be construed to be limited to any particular cell line or cell type. Such cells include, but are not limited to, bacterial cells, yeast, insect cells, mammalian cells, and the like.

The invention should be construed to include any cell type into which a nucleic acid encoding a mammalian immunodeficiency virus gp120 polypeptide (a transgene) is introduced, including, without limitation, a prokaryotic cell and a eukaryotic cell comprising an isolated nucleic acid encoding the mammalian gp120 polypeptide of the invention.

The invention also encompasses a recombinant cell where an endogenous target nucleic acid gp120 variant is activated by introduction of an exogenous activating nucleic acid into the cell such that the endogenous target nucleic acid is expressed and/or the gp120 polypeptide is produced. Such techniques of gene activation are well-known in the art and are described, for example, in U.S. Pat. No. 6,270,989, among many others.

When the cell is a eukaryotic cell, the cell may be any eukaryotic cell which, when the transgene of the invention is introduced therein, and the protein encoded by the desired gene is no longer expressed therefrom, a benefit is obtained. Such a benefit may include the fact that there has been provided a system in which lack of expression of the desired gene can be studied in vitro in the laboratory or in a mammal in which the cell resides, a system wherein cells comprising the introduced gene deletion can be used as research, diagnostic and therapeutic tools, and a system wherein animal models are generated which are useful for the development of new diagnostic and therapeutic tools for selected disease states in a mammal including, for example, Acquired Immune Deficiency Syndrome, or any other disease, disorder or condition mediated by gp120 interaction with a cellular chemokine receptor, and the like.

Alternatively, the invention includes a eukaryotic cell which, when the transgene of the invention is introduced therein, and the protein encoded by the desired gene is expressed therefrom where it was not previously present or expressed in the cell or where it is now expressed at a level or under circumstances different than that before the transgene was introduced, a benefit is obtained. Such a benefit may include the fact that there has been provided a system in the expression of the desired gene can be studied in vitro in the laboratory or in a mammal in which the cell resides, a system wherein cells comprising the introduced gene can be used as research, diagnostic and therapeutic tools, and a system wherein animal models are generated which are useful for the development of new diagnostic and therapeutic tools for selected disease states in a mammal.

Further, expression in a cell of an immunodeficiency virus gp120, comprising a deletion of the V3 region of the protein can provide a target for an immune response against that cell now bearing the gp120 of the invention. That is, by expressing a gp120 of the invention in which the lack of at least one hypervariable region can expose certain epitopes that are otherwise "camouflaged" by various hypervariable regions in an unmodified virus, the cell can be targeted for an immune response such that expression of the polypeptides of the invention can provide a therapeutic method whereby infected cells can be targeted by the immune system.

Additionally, a cell expressing an isolated nucleic acid encoding a gp120 polypeptide of the invention can be used to provide the gp120 polypeptide to a cell, tissue, or whole animal where a higher level of gp120 variant can be useful to treat or alleviate a disease, disorder or condition wherein soluble gp120 can alleviate such a disease, disorder or condition. Therefore, the invention includes a cell expressing a gp120 polypeptide comprising a substantial, or complete, deletion of the V3 such as, but not limited to, HIV-2 .DELTA.V1/V2; .DELTA.V3(6,6) gp120; HIV-2 .DELTA.V1/V2; .DELTA.V3(1,1) gp120; HIV-2 .DELTA.V3(6,6) gp120; and HIV-2 .DELTA.V3(1,1) gp120, to increase or induce gp120 variant activity, where increasing gp120 variant protein level and/or activity can be useful to treat or alleviate a disease, disorder or condition, since increasing soluble gp120 V3 deletion polypeptide can, for instance, inhibit the binding of virus-bound gp120 to a cellular chemokine receptor and inhibit viral entry into the cell.

Methods and compositions useful for maintaining mammalian cells in culture are well known in the art, wherein the mammalian cells are obtained from a mammal including, but not limited to, a rat and a human.

The recombinant cell of the invention can be used to study the effect of qualitative and quantitative alterations in the level of gp120 polypeptide comprising a substantial deletion of V3 on a cell, including the effect of decreased viral entry into the cell. This is because the fact that HIV-2 virus gp120, and variants thereof comprising core gp120 structures and sequences, have now been demonstrated to mediate CD4-independent entry into a cell, wherein viral entry is correlated with, among other things, Acquired Immune Deficiency Syndrome. Further, the recombinant cell can be used to produce a gp120 polypeptide of the invention for use for therapeutic and/or diagnostic purposes. That is, a recombinant cell expressing a gp120 V3 deletion polypeptide of the invention can be used to, among other things, produce large amounts of purified and isolated gp120 polypeptide that can in turn be administered to treat or alleviate a disease, disorder or condition associated with or caused by an increased or inappropriate level of viral-associated gp120 polypeptide.

Alternatively, recombinant cells expressing a gp120 V3 deletion polypeptide of the invention can be administered in ex vivo and in vivo therapies where administering the recombinant cells thereby administers the protein to a cell, a tissue, and/or an animal. Additionally, the recombinant cells are useful for the discovery of processes affected and/or mediated by gp120 polypeptide core components and/or gp120 determinants that are exposed after CD4 binding. Thus, the recombinant cell of the invention may be used to study the effects of elevated or decreased gp120 where the polypeptide comprises a deletion of the V3 region, and further comprises deletions of V1 and V2 regions as well.

The invention further includes a recombinant cell comprising an isolated nucleic acid encoding a mammalian immunodeficiency virus gp41 polypeptide, wherein the polypeptide comprises a compensatory mutation. The invention encompasses a nucleic acid encoding such a gp41 polypeptide, where the compensatory mutation is truncation of the cytoplasmic domain (CD) of the peptide. The invention also encompasses an antisense nucleic acid complementary thereto, a nucleic acid encoding an antibody that specifically binds a gp41 polypeptide encoded by that nucleic acid, and the like.

In one aspect, the recombinant cell can be transiently transfected with a plasmid encoding a portion of the nucleic acid encoding the gp41 compensatory mutation polypeptide. The nucleic acid need not be integrated into the cell genome nor does it need to be expressed in the cell. Moreover, the cell may be a prokaryotic or a eukaryotic cell and the invention should not be construed to be limited to any particular cell line or cell type. Such cells include, but are not limited to, bacterial cells, yeast, insect cells, mammalian cells, and the like.

The invention should be construed to include any cell type into which a nucleic acid encoding a mammalian immunodeficiency virus gp41 polypeptide (a transgene) is introduced, including, without limitation, a prokaryotic cell and a eukaryotic cell comprising an isolated nucleic acid encoding the mammalian gp41 polypeptide of the invention.

The invention also encompasses a recombinant cell where an endogenous target nucleic acid gp41 comprising a compensatory mutation is activated by introduction of an exogenous activating nucleic acid into the cell such that the endogenous target nucleic acid is expressed and/or the gp41 polypeptide is produced. Such techniques of gene activation are well-known in the art and are described, for example, in U.S. Pat. No. 6,270,989, among many others.

When the cell is a eukaryotic cell, the cell may be any eukaryotic cell which, when the transgene of the invention is introduced therein, and the protein encoded by the desired gene is no longer expressed therefrom, a benefit is obtained. Such a benefit may include the fact that there has been provided a system in which lack of expression of the desired gene can be studied in vitro in the laboratory or in a mammal in which the cell resides, a system wherein cells comprising the introduced gene deletion can be used as research, diagnostic and therapeutic tools, and a system wherein animal models are generated which are useful for the development of new diagnostic and therapeutic tools for selected disease states in a mammal including, for example, Acquired Immune Deficiency Syndrome, or any other disease, disorder or condition mediated by gp41, including fusion with a cell membrane, and the like.

Additionally, a cell expressing an isolated nucleic acid encoding a gp41 polypeptide of the invention can be used to provide the gp41 polypeptide to a cell, tissue, or whole animal where a higher level of gp41 variant can be useful to treat or alleviate a disease, disorder or condition wherein gp41 can alleviate such a disease, disorder or condition. Therefore, the invention includes a cell expressing a gp41 polypeptide comprising a compensatory mutation such as, but not limited to, truncation of the CD. Such mutations include, but are not limited to, gp41 .DELTA.733 (SEQ ID NO:22), gp41 .DELTA.753 (SEQ ID NO:23), and gp41 .DELTA.764 (SEQ ID NO:25), where truncation of CD of the gp41 polypeptide increased fusogenicity of the virus.

V. Antibodies

The invention also includes an antibody that specifically binds a mammalian immunodeficiency virus gp120, wherein the polypeptide comprises a substantial deletion of V3. The invention further includes an antibody that binds the gp120 wherein the polypeptide further comprises deletion of V1 and V2 as well.

One skilled in the art would understand, based upon the disclosure provided herein, that an antibody that specifically binds a gp120 polypeptide of the invention binds a polypeptide such as, but not limited to, HIV-2 .DELTA.V1/V2; .DELTA.V3(6,6) gp120, HIV-2 .DELTA.V1/V2; .DELTA.V3(1,1) gp120, HIV-2 .DELTA.V3(6,6) gp120 or HIV-2 .DELTA.V3(1,1) gp120, or an immunogenic portion thereof. In one embodiment, the antibody is directed to: HIV-2 .DELTA.V1/V2; .DELTA.V3(6,6) gp120, comprising the amino acid sequence of SEQ ID NO:2, HIV-2 .DELTA.V1/V2; .DELTA.V3(1,1) gp120, comprising the amino acid sequence of SEQ ID NO:4, HIV-2 .DELTA.V3(6,6) gp120, comprising the amino acid sequence of SEQ ID NO:2a, and HIV-2 .DELTA.V3(1,1) gp120, comprising the amino acid sequence of SEQ ID NO:4a.

The invention encompasses a wide plethora of antibodies, including, but not limited to, polyclonal and monoclonal antibodies, among many others. Polyclonal antibodies are generated by immunizing rabbits according to standard immunological techniques well-known in the art (see, e.g., Harlow et al., 1988, In: Antibodies, A Laboratory Manual, Cold Spring Harbor, N.Y.; and Wilson et al., 2001, Science 293: 1107-1112). Such techniques include immunizing an animal with a chimeric protein comprising a portion of another protein such as a maltose binding protein or glutathione (GSH) tag polypeptide portion, and/or a moiety such that the gp120 variant portion is rendered immunogenic (e.g., gp120 variant conjugated with keyhole limpet hemocyanin, KLH) and a portion comprising the respective gp120 variant amino acid residues. The chimeric proteins are produced by cloning the appropriate nucleic acids encoding a gp120 variant (e.g., SEQ ID NO:8, SEQ ID NO:14, SEQ ID NO:20, and SEQ ID NO:26) or a gp41 variant (e.g., SEQ ID NO:3, SEQ ID NO:9, SEQ ID NO:15 and SEQ ID NO:27) or a variant Env (e.g., SEQ ID NO:7, SEQ ID NO:13, SEQ ID NO:19 and SEQ ID NO:25) into a plasmid vector suitable for this purpose, such as but not limited to, pMAL-2 or pCMX.

However, the invention should not be construed as being limited solely to these antibodies or to these portions of the protein antigens. Rather, the invention should be construed to include other antibodies, as that term is defined elsewhere herein, to a gp120 variant of the invention, or portions thereof. Further, the present invention should be construed to encompass antibodies, inter alia, that bind to a gp120 variant and they are able to bind a gp120 variant present on Western blots, in immunohistochemical staining of tissues thereby localizing a gp120 variant in the tissues, and in immunofluorescence microscopy of a cell transiently transfected with a nucleic acid encoding at least a portion of a gp120 variant.

Moreover, the invention encompasses an antibody that specifically binds with a gp41 polypeptide comprising a compensatory mutation, and, more preferably, where the compensatory mutation comprises truncation of the CD of the polypeptide. Further, the present invention should be construed to encompass antibodies, inter alia, that bind to a gp41 of the invention and they are able to bind the gp41 of the invention when present on Western blots, in immunohistochemical staining of tissues thereby localizing a gp41 of the invention in a cell, a tissue, and any sample, and in immunofluorescence microscopy of a cell transiently transfected with a nucleic acid encoding at least a portion of the relevant gp41.

One skilled in the art would appreciate, based upon the disclosure provided herein, that the antibody can specifically bind with any portion of the protein and the full-length protein can be used to generate antibodies specific therefor. However, the present invention is not limited to using the full-length protein as an immunogen. Rather, the present invention includes using an immunogenic portion of the protein to produce an antibody that specifically binds with a mammalian immunodeficiency virus gp41 variant. That is, the invention includes immunizing an animal using an immunogenic portion, or antigenic determinant, of the gp120 variant protein.

The antibodies can be produced by immunizing an animal such as, but not limited to, a rabbit or a mouse, with a protein of the invention, or a portion thereof, or by immunizing an animal using a protein comprising at least a portion of a gp120 polypeptide of the invention, or a fusion protein including a tag polypeptide portion comprising, for example, a maltose binding protein tag polypeptide portion, covalently linked with a portion comprising the appropriate gp120 variant amino acid residues. One skilled in the art would appreciate, based upon the disclosure provided herein, that smaller fragments of these proteins can also be used to produce antibodies that specifically bind an gp120 variant.

One skilled in the art would appreciate, based upon the disclosure provided herein, that various portions of an isolated bp120 variant polypeptide can be used to generate antibodies to either conserved regions of a gp120 variant or to non-conserved regions of the polypeptide. As disclosed elsewhere herein, gp120 comprises various conserved domains, including core domains that have been shown herein to be responsible for gp120-containing virus into a cell.

Once armed with the sequence of gp120 of the invention, and the detailed analysis localizing the various conserved and non-conserved domains of the protein and their potential function(s), the skilled artisan would understand, based upon the disclosure provided herein, how to obtain antibodies specific for the various portions of a gp120 variant polypeptide using methods well-known in the art or to be developed in the future.

Further, the skilled artisan, based upon the disclosure provided herein, would appreciate that the non-conserved regions of a protein of interest can be more immunogenic than the highly conserved regions which are conserved among various organisms. Further, immunization using a non-conserved immunogenic portion can produce antibodies specific for the non-conserved region thereby producing antibodies that do not cross-react with other proteins which can share one or more conserved portions. Thus, one skilled in the art would appreciate, based upon the disclosure provided herein, that the non-conserved regions of each gp120 molecule can be used to produce antibodies that are specific only for that gp120 variant and do not cross-react non-specifically with other gp120 variants or with other proteins. More specifically, the skilled artisan, once armed with the teachings provided herein, would readily appreciate that antibodies can be produced that react with HIV-2 .DELTA.V1/V2; .DELTA.V(6,6) gp120, but not with HIV-2 .DELTA.V1/V2; .DELTA.V(1,1) gp120, and vice-a-versa.

Alternatively, the skilled artisan would also understand, based upon the disclosure provided herein, that antibodies developed using a region that is conserved among one or more gp120 molecules can be used to produce antibodies that react specifically with one or more gp120 molecule(s). That is, once armed with the sequences disclosed herein, one skilled in the art could readily prepare, using methods well-known in the art, antibodies that specifically bind with HIV-2 .DELTA.V1/V2; .DELTA.V(6,6) gp120 and with HIV-2 .DELTA.V1/V2; .DELTA.V(1,1) gp120. Methods for producing antibodies that specifically bind with a conserved protein domain which may otherwise be less immunogenic than other portions of the protein are well-known in the art and have been discussed previously, and include, but are not limited to, conjugating the protein fragment of interest to a molecule (e.g., keyhole limpet hemocyanin, and the like), thereby rendering the protein domain immunogenic, or by the use of adjuvants (e.g., Freund's complete and/or incomplete adjuvant, and the like), or both. Thus, the invention encompasses antibodies that recognize at least one gp120 variant and antibodies that specifically bind with more than one gp120 variant, including antibodies that specifically bind with all gp120 variants of the invention.

The teachings provided herein can be applied with equal force to development of antibodies of interest that specifically bind with the gp41 and Env polypeptides of the invention.

One skilled in the art would appreciate, based upon the disclosure provided herein, which portions of a gp120 variant are less homologous with other proteins sharing conserved domains. However, the present invention is not limited to any particular domain; instead, the skilled artisan would understand that other non-conserved regions of the gp120 variant proteins of the invention can be used to produce the antibodies of the invention as disclosed herein.

Therefore, the skilled artisan would appreciate, based upon the disclosure provided herein, that the present invention encompasses antibodies that neutralize and/or inhibit gp120 variant activity (e.g., by inhibiting necessary gp120 variant/cytokine receptor protein/protein interactions) which antibodies can recognize one or more gp120 variants, including, but not limited to, HIV-2 .DELTA.V1/V2; .DELTA.V(6,6) gp120 and with HIV-2 .DELTA.V1/V2; .DELTA.V(1,1) gp120.

One skilled in the art would also understand, based upon the disclosure provided herein, that it may be advantageous to inhibit the activity of one type of gp120 variant molecule without affecting the activity of other gp120 variants or other gp120 molecules. For example, it may be beneficial to inhibit HIV-2 .DELTA.V1/V2; .DELTA.V(6,6) gp120 activity, while not inhibiting the activity of HIV-2 .DELTA.V1/V2; .DELTA.V(1,1) gp120, or wildtype parental gp120. Thus, whether inhibition of gp120 activity is achieved using antibodies or other techniques, one skilled in the art would appreciate, based upon the disclosure provided herein, that the present invention encompasses selectively affecting one or more gp120 molecules and, in certain cases, the invention encompasses inhibiting the activity of all gp120 molecules. Whether one or more gp120 molecule should be affected can be readily determined by the skilled artisan based on which disease, disorder or condition is being treated, and the specific cell and/or tissue being targeted.

The invention should not be construed as being limited solely to the antibodies disclosed herein or to any particular immunogenic portion of the proteins of the invention. Rather, the invention should be construed to include other antibodies, as that term is defined elsewhere herein, to gp120 polypeptide comprising a substantial deletion of V3, or portions thereof, or to proteins sharing greater than 90% homology with a polypeptide having the amino acid sequence of at least one of SEQ ID NO:11, SEQ ID NO:17, SEQ ID NO:23, and SEQ ID NO:29. Preferably, the polypeptide is about 95% homologous, and more preferably, about 99% homologous to at least one of SEQ ID NO:11, SEQ ID NO:17, SEQ ID NO:23, and SEQ ID NO:29. More preferably, the polypeptide that specifically binds with an antibody specific for mammalian gp120 variant is at least one of SEQ ID NO:11, SEQ ID NO:17, SEQ ID NO:23, and SEQ ID NO:29.

The invention should not be construed as being limited solely to the antibodies disclosed herein or to any particular immunogenic portion of the proteins of the invention. Rather, the invention should be construed to include other antibodies, as that term is defined elsewhere herein, to gp41 polypeptide comprising at least one compensatory mutation, or portions thereof, or to proteins sharing greater than 90% homology with a polypeptide having the amino acid sequence of at least one of SEQ ID NO:12, SEQ ID NO:18, SEQ ID NO:24, and SEQ ID NO:30. Preferably, the polypeptide is about 95% homologous, and more preferably, about 99% homologous to at least one of SEQ ID NO:12, SEQ ID NO:18, SEQ ID NO:24, and SEQ ID NO:30. More preferably, the polypeptide that specifically binds with an antibody specific for mammalian gp120 variant is at least one of SEQ ID NO:12, SEQ ID NO:18, SEQ ID NO:24, and SEQ ID NO:30.

The invention should not be construed as being limited solely to the antibodies disclosed herein or to any particular immunogenic portion of the proteins of the invention. Rather, the invention should be construed to include other antibodies, as that term is defined elsewhere herein, to Env polypeptide comprising a substantial deletion of V3, or portions thereof, and further comprising at least one compensatory mutation, or to proteins sharing greater than 90% homology with a polypeptide having the amino acid sequence of at least one of SEQ ID NO:10, SEQ ID NO:16, SEQ ID NO:22, and SEQ ID NO:28. Preferably, the polypeptide is about 95% homologous, and more preferably, about 99% homologous to at least one of SEQ ID NO:10, SEQ ID NO:16, SEQ ID NO:22, and SEQ ID NO:28. More preferably, the polypeptide that specifically binds with an antibody specific for mammalian gp120 variant is at least one of SEQ ID NO:10, SEQ ID NO:16, SEQ ID NO:22, and SEQ ID NO:28.

The invention encompasses polyclonal, monoclonal, synthetic antibodies, and the like. One skilled in the art would understand, based upon the disclosure provided herein, that the crucial feature of the antibody of the invention is that the antibody bind specifically with a gp120 variant. That is, the antibody of the invention recognizes a gp120 variant, or a fragment thereof (e.g., an immunogenic portion or antigenic determinant thereof), on Western blots, in immunostaining of cells, and immunoprecipitates a gp120 variant using standard methods well-known in the art.

One skilled in the art would appreciate, based upon the disclosure provided herein, that the antibodies can be used to localize the relevant protein in a cell and to study the role(s) of the antigen recognized thereby in cell processes. Moreover, the antibodies can be used to detect and or measure the amount of protein present in a biological sample using well-known methods such as, but not limited to, Western blotting and enzyme-linked immunosorbent assay (ELISA). Moreover, the antibodies can be used to immunoprecipitate and/or immuno-affinity purify their cognate antigen using methods well-known in the art. In addition, the antibody can be used to decrease the level of a gp120 variant in a cell thereby inhibiting the effect(s) of gp120 variant in a cell. Thus, by administering the antibody to a cell or to the tissues of an animal or to the animal itself, the required gp120 variant/cytokine receptor protein/protein interactions are therefore inhibited such that the effects of gp120 variant-mediated activity are also inhibited. One skilled in the art would understand, based upon the disclosure provided herein, that detectable effects upon inhibiting gp120 variant/cytokine receptor protein/protein interaction and/or activity using an anti-gp120 variant antibody can include, but are not limited to, decreased interaction of virus-bound gp120 with a cytokine receptor (such as CCR5 and CXCR4), decreased entry into a cell of a virus having gp120 as part of the Env, decreased fusogenicity of a virus having gp120 as part of the Env, decreased apparent replication competence of a virus having gp120 as part of the Env, and the like.

One skilled in the art would appreciate, based upon the disclosure provided herein, that the invention encompasses administering an antibody that specifically binds with a gp120 variant orally, parenterally, or both, to inhibit gp120 variant function in enabling entry into a cell of a virus having gp120 as part of the Env.

The generation of polyclonal antibodies is accomplished by inoculating the desired animal with the antigen and isolating antibodies which specifically bind the antigen therefrom using standard antibody production methods such as those described in, for example, Harlow et al. (1988, In: Antibodies, A Laboratory Manual, Cold Spring Harbor, N.Y.).

Monoclonal antibodies directed against full length or peptide fragments of a protein or peptide may be prepared using any well known monoclonal antibody preparation procedures, such as those described, for example, in Harlow et al. (1988, In: Antibodies, A Laboratory Manual, Cold Spring Harbor, N.Y.) and in Tuszynski et al. (1988, Blood, 72:109-115). Quantities of the desired peptide may also be synthesized using chemical synthesis technology. Alternatively, DNA encoding the desired peptide may be cloned and expressed from an appropriate promoter sequence in cells suitable for the generation of large quantities of peptide. Monoclonal antibodies directed against the peptide are generated from mice immunized with the peptide using standard procedures as referenced herein.

Nucleic acid encoding the monoclonal antibody obtained using the procedures described herein may be cloned and sequenced using technology which is available in the art, and is described, for example, in Wright et al. (1992, Critical Rev. Immunol. 12:125-168), and the references cited therein.

Further, the antibody of the invention may be "humanized" using the technology described in, for example, Wright et al. (1992, Critical Rev. Immunol. 12:125-168), and in the references cited therein, and in Gu et al. (1997, Thrombosis and Hematocyst 77:755-759), and other methods of humanizing antibodies well-known in the art or to be developed.

To generate a phage antibody library, a cDNA library is first obtained from mRNA which is isolated from cells, e.g., the hybridoma, which express the desired protein to be expressed on the phage surface, e.g., the desired antibody. cDNA copies of the mRNA are produced using reverse transcriptase. cDNA which specifies immunoglobulin fragments are obtained by PCR and the resulting DNA is cloned into a suitable bacteriophage vector to generate a bacteriophage DNA library comprising DNA specifying immunoglobulin genes. The procedures for making a bacteriophage library comprising heterologous DNA are well known in the art and are described, for example, in Sambrook et al., (1989, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York).

Bacteriophage which encode the desired antibody, may be engineered such that the protein is displayed on the surface thereof in such a manner that it is available for binding to its corresponding binding protein, e.g., the antigen against which the antibody is directed. Thus, when bacteriophage which express a specific antibody are incubated in the presence of a cell which expresses the corresponding antigen, the bacteriophage will bind to the cell. Bacteriophage which do not express the antibody will not bind to the cell. Such panning techniques are well known in the art and are described for example, in Wright et al. (1992, Critical Rev. Immunol. 12:125-168).

Processes such as those described above, have been developed for the production of human antibodies using M13 bacteriophage display (Burton et al., 1994, Adv. Immunol. 57:191-280). Essentially, a cDNA library is generated from mRNA obtained from a population of antibody-producing cells. The mRNA encodes rearranged immunoglobulin genes and thus, the cDNA encodes the same. Amplified cDNA is cloned into M13 expression vectors creating a library of phage which express human Fab fragments on their surface. Phage which display the antibody of interest are selected by antigen binding and are propagated in bacteria to produce soluble human Fab immunoglobulin. Thus, in contrast to conventional monoclonal antibody synthesis, this procedure immortalizes DNA encoding human immunoglobulin rather than cells which express human immunoglobulin.

The procedures just presented describe the generation of phage which encode the Fab portion of an antibody molecule. However, the invention should not be construed to be limited solely to the generation of phage encoding Fab antibodies. Rather, phage which encode single chain antibodies (scFv/phage antibody libraries) are also included in the invention. Fab molecules comprise the entire Ig light chain, that is, they comprise both the variable and constant region of the light chain, but include only the variable region and first constant region domain (CH1) of the heavy chain. Single chain antibody molecules comprise a single chain of protein comprising the Ig Fv fragment. An Ig Fv fragment includes only the variable regions of the heavy and light chains of the antibody, having no constant region contained therein. Phage libraries comprising scFv DNA may be generated following the procedures described in Marks et al. (1991, J. Mol. Biol. 222:581-597). Panning of phage so generated for the isolation of a desired antibody is conducted in a manner similar to that described for phage libraries comprising Fab DNA.

The invention should also be construed to include synthetic phage display libraries in which the heavy and light chain variable regions may be synthesized such that they include nearly all possible specificities (Barbas, 1995, Nature Medicine 1:837-839; de Kruif et al. 1995, J. Mol. Biol. 248:97-105).

Further, the invention encompasses production of an antibody that specifically binds with a mammalian immunodeficiency virus gp41 polypeptide, wherein the polypeptide comprises a compensatory mutation, more specifically, the compensatory mutation is a truncation of the CD of the protein. As discussed previously with regard to gp120, similar methods can be applied to the gp41 polypeptide of the invention. Because certain important epitopes of the gp41 are exposed due to truncation of the cytoplasmic domain, as evidenced by the increased fusogenicity of the gp41 polypeptide of the invention compared with wild type gp41, development of an antibody directed against such a polypeptide can provide a method for producing antibodies that specifically bind with important functional epitopes of gp41 and can provide important diagnostic and therapeutic tools relating to gp41-mediated entry of the virus into a cell.

VI. Methods and Compositions Relating to Mammalian Immunodeficiency Viruses Containing Hypervariable Loop Mutations

The present invention features compositions and methods related to mammalian immunodeficiency viruses comprising one or more amino acid mutations in at least one of hypervariable loops V1, V2, V3 and V4, whereby such mutation does not result in loss of fusogenicity and/or replication competence. Deletion mutation of the hypervariable loops of gp120 and mutations relating to compensatory mutation of gp41, including truncation of the cytoplasmic domain of the polypeptide, are set forth more fully previously elsewhere herein and are therefore referred to herein without further discussion.

The present invention encompasses a composition comprising a mammalian immunodeficiency virus gp120 polypeptide, wherein the polypeptide comprises a substantial, or complete, deletion of the V3 region. Methods of making the desired deletion, as well as assays for selecting the deletion mutants of interest, that is, those mutants having the desired quality (e.g., where detectable chemokine receptor binding, fusogenicity and/or replication competence are maintained despite deletion of all, or part, of the V3 region), are described in great detail elsewhere herein.

The composition further comprises a deletion of V1 and a deletion of V2, such that most of the hypervariable regions of the gp120 are absent from the polypeptide. Surprisingly, the data disclosed herein demonstrate that even though the gp120 comprises these deletions, the polypeptide retains the ability to mediate detectable binding with a chemokine receptor, fuse with a cell, and/or virus replication competence is preserved. As more fully disclosed elsewhere herein, such compositions are useful in that they provide a "core" polypeptide, with little or no hypervariable regions to camouflage various domains of the polypeptide that are important for function. Thus, the skilled artisan would appreciate, based upon the disclosure provided herein, that the compositions of the invention can be used for, among many other things, identifying and studying the functional domains of gp120, as well for the development of useful therapeutics based on inhibiting such functions and for the development of useful immune-based methods, including vaccine development, for inhibiting and/or preventing virus infection. This is because, as more fully discussed elsewhere, exposure of the core functional domains of gp120 can provide a useful immunogen for development of a neutralizing antibody that can inhibit requisite virus function mediated by such core domain(s) of gp120.

The invention also encompasses a composition comprising a gp120 as discussed previously, and further comprising a gp41 polypeptide. Further, the gp41 polypeptide can comprise a compensatory mutation, such as, but not limited to, truncation of the cytoplasmic domain of the gp41 polypeptide. Such compositions are useful as noted previously, for the study and identification of virus domains and mechanisms required for virus infection. Further, the compositions are useful for the development of useful therapeutics based on inhibition of core functions and the development of a virus neutralizing antibody that specifically binds with the polypeptides of the compositions of the invention.

The invention encompasses an isolated mammalian immunodeficiency virus wherein the virus comprises a gp120 comprising a substantial deletion of V3 where the virus retains detectable function, such as, but not limited to, chemokine receptor binding, fusogenicity and replication competence, compared with an otherwise identical virus not comprising the mutation deletion of V3. One skilled in the art would appreciate, once armed with the teachings provided herein, that the virus can further comprise a deletion of V1 and a deletion of V2. Such viruses are useful for the study of function of the various protein domains that remain after deletions of the hypervariable region(s). Moreover, as more fully discussed elsewhere herein, the virus can be used to produce a useful neutralizing antibody, as well as to identify a useful compound that can inhibit virus function required for infection. The skilled artisan would understand that the mammalian human immunodeficiency virus includes, but is not limited to, SIV, HIV-1 and HIV-2, among others.

The invention further includes the an isolated mammalian immunodeficiency virus comprising a gp120 wherein the gp120 comprises a substantial deletion of V3, wherein the virus further comprises a gp41. Additionally, the invention comprises a virus where the gp41 further comprises a compensatory mutation. This virus is useful not only for the study and identification of gp120 domains that mediate virus function needed for infection, but also for the development of useful therapeutics such as, but not limited to, a neutralizing antibody and a compound that can inhibit the function of gp120 thereby preventing or inhibiting virus infection.

As described elsewhere herein, a compensatory mutation enables a mammalian immunodeficiency virus to remain fusogenic, to remain replication competent, or to become highly cytopathic, despite at least one other mutation in a virus polypeptide that would otherwise reduce the level of that function/characteristic of the virus. Thus, compensatory mutation enables a virus containing a deletion of one or more hypervariable loops to remain replication competent and highly infectious. That is, a compensatory mutation "compensates" for the effect of the other mutation.

As discussed in detail elsewhere herein a deletion of the gp120 V1 loop may comprise the deletion of at least one amino acid naturally present in the loop. In another embodiment, a deletion of the gp120 V1 loop may comprise deletion of the entire V1 loop. As discussed elsewhere herein, any gp120 hypervariable loop (i.e., V1, V2, V3 or V4) may be deleted for the purposes of the present invention. Further, any combination of hypervariable loop deletion may be used in the present invention for the purpose of producing an isolated mammalian immunodeficiency virus comprising a mutation in a gp120 protein where at least a substantial portion of V3 is deleted, where the virus can further comprise a gp41 protein, where gp41 comprises a compensatory mutation. For example, an isolated mammalian immunodeficiency virus of the invention can be produced by deletion of the gp120 V1/V2 loops in their entirety, in addition to substantial deletion of the gp120 V3 loop, wherein despite such loop deletions, the gp120 retains detectable function (e.g., binding of a chemokine receptor, fusogenicity, and replication competence). As described elsewhere herein, isolated virus containing compensatory mutations may be obtained by serially passaging virus onto CD4.sup.+ cell lines, among other methods.

Another embodiment of the invention provides an isolated mammalian immunodeficiency virus, wherein deletion of the gp120 V1/V2 loops in their entirety, in addition to partial deletion of the gp120 V3 loop, and where the virus further comprises at least one compensatory mutation in the gp41 protein of the virus. Yet another embodiment of the invention provides an isolated mammalian immunodeficiency virus, wherein deletion of the gp120 V1/V2 loops in their entirety, in addition to partial deletion of the gp120 V3 loop, is used to produce a gp41 comprising at least one compensatory mutation.

The invention includes a method of producing a replication-competent mammalian immunodeficiency virus comprising deletion of at least one hypervariable V3 loop of gp120. The invention further includes a method where the virus further comprises a compensatory mutation. As discussed in detail elsewhere herein, a compensatory mutation in the virus comprising a loop-deleted gp120 polypeptide provides a mammalian immunodeficiency virus with increased or enhanced fusogenic property, replication competence, or both, compared with an otherwise identical virus not comprising the compensatory mutation.

In one aspect of the invention, a compensatory mutation is induced in a gp120 polypeptide by deletion of the entirety of hypervariable loops V1 and V2, along with a partial deletion of hypervariable loop V3, such that only the first six and the last six amino acids of the V3 loop remain. This mutation resulted in gp120 and/or gp41 that retained detectable function, and where the polypeptides comprised mutations including, in gp120: 55 I/V, 79 N/D, 202 T/K, 231 T/I, 280 N/D, 391 N/D, 429 V/I, and in gp41: 518 L/V, 529 A/T, 561 A/T.

In another aspect of the invention, a mutation is induced in a gp120 polypeptide by deletion of the entirety of hypervariable loops V1 and V2, along with a partial deletion of hypervariable loop V3, such that only the first and the last amino acids of the V3 loop remain. This mutation resulted in gp120 and/or gp41 that retained detectable function, and where the polypeptides comprised mutations including a mutation in gp120 such as, but not limited to, 142 D/G, 160 T/I, 203 E/K, 279 N/D, 334 E/K, 340 E/K, 399 V/I, 437 E/V.

In order to produce a compensatory mutation, an infectious molecular clone of HIV-2/VCP was used to create a gp120 polypeptide by deletion of the entirety of hypervariable loops V1 and V2, along with a partial deletion of hypervariable loop V3, as discussed in greater detail in the Experimental Examples.

It will be appreciated by one skilled in the art, based upon the disclosure provided herein, that, for example, an isolate of an HIV-2 strain containing compensatory mutations in gp120, gp41, or both gp120 and gp41 may be obtained by serially passaging a clone of HIV-2/VCP comprising deletions in V1/V2 and V3 hypervariable loops in CD4.sup.+ cells and screening for highly cytopathic variants. Methods of serially passaging and screening cells are well known in the art. For example, as disclosed in U.S. patent application No. 2003/0091594A1, incorporated herein by reference in its entirety, HIV-1/ IIIBx was obtained by passaging virus in CD4.sup.+ SupT1 cells followed by passaging virus on the otherwise identical but CD4.sup.- BC7 cells. However, the present invention should not be construed to be limited to these particular cell types. Instead, the invention encompasses a variety of CD4.sup.+ and CD4.sup.- cells including, but not limited to, 293, Cf2TH, CCC.sup.+L.sup.-, and QT6 cells as well as stably transfected cells (U87, HeLa, HOS), or any other cell either known in the art or to be developed in the future. One skilled in the art, armed with the teachings set forth herein, could readily determine what cell could be used in the methods of the invention.

The invention also includes a method of identifying an amino acid residue of an gp120 protein which is a compensatory mutation. The method comprises producing gp120 proteins comprising a total deletion of the V1/V2 hypervariable loops and a partial deletion of the V3 hypervariable loop, wherein the remaining V3 loop contains only about the first six and the last six amino acid residues of the native HIV-2 V3 loop. The resulting gp120 loop deletion mutant is then examined to determine the ability of the loop-deleted protein to generate functional virus using various assays, including, but not limited to, cell fusion assays and to generate replication-competent virus by various assays as disclosed elsewhere herein.

As discussed elsewhere herein, a preferred embodiment is disclosed wherein portions of the gp120 protein acquire mutations such that highly cytopathic viral variants emerge. Also as noted elsewhere herein, the present invention is not limited to these particular combinations or to these particular strains. Rather, one skilled in the art would appreciate, based on the disclosure provided herein, that any combination of gp120 loop-deleted variants may be examined to produce and identify useful compensatory mutations in gp120, gp41, or both, and to identify viruses comprising such compensatory mutations, where the virus is functional in cell fusion assays and that is replication-competent. Further, the effect of compensatory mutations that arise using methods of the present invention may be examined using a variety of assays using a wide plethora of mammalian cell lines as described elsewhere herein.

Claim 1 of 7 Claims

1. An isolated nucleic acid encoding a mammalian immunodeficiency virus glycoprotein (gp) 120 polypeptide, wherein said gp120 polypeptide comprises a deletion of hypervariable loop 3 (V3), and further comprises a compensatory mutation, wherein said compensatory mutation is an amino acid substitution from threonine to alanine at amino acid residue number 391, wherein the amino acid residue number of said compensatory mutation is relative to the amino acid sequence of parental HIV-2/vcp gp120 as provided in SEQ ID NO:5.

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