Patent 6,849,261

HIV entry is known to require an interaction of the viral envelope glycoprotein (Env) with CD4 and cellular chemokine receptors. HIV Env protein is produced as a precursor (gp160) that is subsequently cleaved into two parts, gp120 which binds CD4 and chemokine receptors, and gp41 which is anchored in the viral membrane and mediates membrane fusion. Differential use of chemokine receptors by HIV and SIV has largely explained differences in tropism among different isolates (Berger, 1997, AIDS 11:S3-S16; Hoffman and Doms, 1998, AIDS 12:S17-S26). While a number of chemokine receptors can be utilized by HIV or SIV (Deng et al., 1997, Nature 388:296-300; Choe et al., 1996, Cell 85, 1135-1148; Rucker et al., 1997, J. Virol. 71:8999-9007; Edinger et al., 1997, Proc. Natl. Acad. Sci. USA 94:14742-14747; Liao et al., 1997, J. Exp. Med. 185:2015-2023; Farzan et al., 1997, J. Exp. Med. 186:405-411), CCR5 and CXCR4 appear to be the principal coreceptors for HIV-1 (Zhang et al., 1998, J Virol. 72:9337-9344; Zhang et al., 1998, J. Virol. 72:9337-9344.). Isolates of HIV that first establish infection target blood lymphocytes and macrophages using CCR5 (Alkhatib et al., 1996, Science 272:1955-1958; Deng et al., 1996, Nature 381:661-666; Dragic et al., 1996, Nature 381:667-673; Doranz et al., 1996, Cell 85:1149-1158), while viruses that are generally associated with progression to AIDS and can infect T cell lines in vitro use CXCR4 (Choe et al., 1996, Cell 85:1135-1148; Feng et al., 1996, Science 272:872-876; Connor et al., 1997, J. Exp. Med. 185:621-628).

Binding of Env to CD4 initiates poorly understood conformational changes enabling gp120 to bind to a chemokine receptor and leading to fusion of the viral and cellular membranes (Jones et al., 1998, J. Biol Chem. 273:404-409; Moore et al., 1994, J. Virol. 68:469-484; Wyatt, 1992, J. Virol. 66:6997-7004; Wu et al., 1996, Nature 384:179-183). Immunologic and mutagenesis approaches have indicated that these changes involve movement of V1/V2 and V3 hypervariable loops on gp120 (Moore, et al., 1994, J. Virol. 68:469-484; Wyatt et al., 1992, J. Virol. 66:6997-7004; Wu et al.,1996, Nature 384:179-183), which play a critical role in the specificity of chemokine receptor utilization (Choe et al., 1996, Cell 85:1135-1148; Cocchi et al., 1996, Nature Med 2:1244-1247; Cho et al., 1998, J. Virol. 72:2509-2515; Speck et al., 1997, J. Virol. 71:7136-7139; Ross et al., 1998, Proc. Natl. Acad. Sci. U.S.A. 95:7682-7686; Hoffman et al., 1998, Proc. Natl. Acad. Sci. U.S.A. 95:11360-11365). The recent crystallographic resolution of a gp120 core structure bound to CD4 has revealed an intervening .beta. sheet (the "bridging sheet") between the inner and outer domains of gp120 that may serve as an additional contact site for the chemokine receptor (Wyatt and Sodroski, 1998, Science 280:1884-1888; Rizzuto et al., 1998, Science 280:1949-1953).

Although CD4 is generally required for gp120 to associate with a chemokine receptor, the identification of CD4-independent isolates of HIV-1, HIV-2, and SIV has demonstrated that functional interactions with chemokine receptors can occur in the absence of CD4 interaction (Edinger et al., 1997, Proc. Natl. Acad. Sci. USA 94:14742-14747; Reeves and Schulz, 1996, J. Virol. 71:1453-1465; Endres et al., 1996, Cell 87:745-756; Dumonceaux et al., 1998, J. Virol. 72:512-519). The determinants for the CD4-independent phenotype have been mapped to the viral env gene, but the underlying mechanisms of this phenotype are unknown. It has been proposed that mutations in env may increase the exposure and/or the affinity of the chemokine receptor binding site on gp120, thus circumventing the need for CD4 (Endres et al., 1996, Cell 87:745-756).

Biochemical assays have also shown that mutated or deglycosylated recombinant gp120 can bind directly to chemokine receptors, suggesting that domains normally activated by CD4 can be artificially exposed (Hesselgesser et al., 1997, Curr. Biol. 7: 112-121; Martin et al., 1997, Science 278:1470-1473; Bandres et al., 1998, J. Virol. 72:2500-2504; Misse et al., 1998, J. Virol. 72:7280-7288). A greater understanding of the determinants responsible for CD4-independence should provide insights into the Env domains that mediate and modulate interactions of Env with chemokine receptors and that ultimately govern viral entry.

To date, the ability of HIV-1 to escape the immune system has hindered development of efficacious vaccines to this important human pathogen. Thus, there is a long-felt and unfilled need for the development of effective vaccines and therapeutic modalities for HIV-1 infection in humans. The present invention meets those needs.

BRIEF SUMMARY OF THE INVENTION

The invention includes an isolated nucleic acid encoding a CD4-independent human immunodeficiency virus-1 (HIV-1) env, or a mutant, derivative, or fragment thereof. In one aspect, the isolated nucleic acid shares at least about 98% homology with the nucleic acid having the nucleotide sequence of SEQ ID NO:4.

In another aspect, the nucleic acid is selected from the group consisting of an HIV-1/IIIBx env, and an HIV-1/IIIBx 8x (8x) env.

In yet another aspect, the nucleic acid is an HIV-1/IIIBx 8x env.

The invention also includes an isolated nucleic acid encoding a CD4-independent HIV env having the nucleotide sequence of SEQ ID NO:4.

The invention includes an isolated nucleic acid comprising a portion of a HIV-1 env gene which confers CD4 independence on at least one HIV-1 env clone.

The invention further includes a chimeric nucleic acid comprising a first portion and a second portion, the first portion encoding at least a portion of an HIV-1/IIIBx 8x env coding sequence and the second portion encoding at least a portion of an HIV-1 env coding sequence which is not an 8x env.

In one aspect, the second portion is an env coding sequence selected from the group consisting of an S10 env, an HXB2 env, a BaL env, and an IIIB env.

In another aspect, the second portion comprises a chemokine receptor binding site selected from the group consisting of a CXCR4 chemokine receptor binding site, and a CCR5 chemokine receptor binding site.

In yet another aspect, the second portion comprises a V3-loop coding sequence selected from the group consisting of a V3-loop for a CXCR4 chemokine receptor binding site, and a V3-loop for a CCR5 chemokine receptor binding site.

The invention includes an isolated HIV-1 gp120 polypeptide comprising a stably exposed chemokine coreceptor binding site.

The invention also includes an isolated polypeptide comprising an HIV-1/IIIBx 8x Env. In one aspect, the polypeptide shares at least about 98% homology with SEQ ID NO:3.

In another aspect, the isolated polypeptide comprises the amino acid sequence of SEQ ID NO:3.

The invention includes a chimeric HIV-1 Env polypeptide comprising a gp120 polypeptide wherein the chimeric polypeptide comprises a first portion comprising an HIV-1/IIIBx 8x gp120, the chimeric polypeptide further comprising a second portion comprising a gp120 from an HIV-1 other than HIV-1/IIIBx 8x.

The invention further includes a chimeric HIV-1 Env polypeptide wherein the polypeptide is CD4-independent, and further wherein the polypeptide comprises a chemokine receptor binding site selected from the group consisting of a CXCR4 chemokine receptor binding site, and a CCR5 chemokine receptor binding site.

In one aspect, the second portion comprises a V3-loop selected from the group consisting of a HXB V3-loop, an 8x V3-loop, a BaL V3-loop, a YU-2 V3-loop, and an 89.6 V3-loop.

The invention includes a composition comprising a CD4-independent HIV-1 comprising a gp120 polypeptide comprising a stably exposed chemokine receptor binding site wherein the HIV-1 is more sensitive to antibody neutralization than an otherwise identical HIV-1 which does not comprise a stably exposed chemokine receptor binding site.

The invention also includes a pharmaceutical composition comprising a CD4-independent HIV-1 Env protein, wherein the HIV-1 Env comprises at least one mutation causing the chemokine coreceptor binding site to be stably exposed.

In one aspect, the HIV-1 Env is HIV-1/IIIBx 8x.

The invention includes a vaccine comprising an immunogenic dose of a CD4-independent HIV-1 Env.

In one aspect, the HIV-1 Env is selected from the group consisting of a HIV-1 Env polypeptide, a nucleic acid encoding HIV-1 Env, and a cell expressing HIV-1 Env.

The invention includes a vector comprising an isolated nucleic acid encoding a CD4-independent human HIV-1 env, or a mutant, derivative, or fragment thereof.

The invention also includes a vector comprising an isolated nucleic acid comprising a portion of a HIV-1 env gene which confers CD4 independence on at least one HIV-1 env clone.

The invention includes a vector comprising a chimeric nucleic acid comprising a first portion and a second portion, the first portion encoding at least a portion of an HIV-1/IIIBx 8x env coding sequence and the second portion encoding at least a portion of an HIV-1 env coding sequence which is not an 8x env.

The invention includes a cell comprising an isolated nucleic acid encoding a CD4-independent human HIV-1 env, or a mutant, derivative, or fragment thereof.

The invention also includes a cell comprising an isolated nucleic acid comprising a portion of a HIV-1 env gene which confers CD4 independence on at least one HIV-1 env clone.

The invention further includes a cell comprising a chimeric nucleic acid comprising a first portion and a second portion, the first portion encoding at least a portion of an HIV-1/IIIBx 8x env coding sequence and the second portion encoding at least a portion of an HIV-1 env coding sequence which is not an 8x env.

The invention includes a cell comprising an isolated HIV-1 gp120 polypeptide comprising a stably exposed chemokine receptor binding site.

The invention also includes a cell comprising an isolated polypeptide comprising an HIV-1/IIIBx 8x Env.

The invention includes a cell comprising a chimeric HIV-1 Env polypeptide comprising a gp120 polypeptide wherein the chimeric polypeptide comprises a first portion comprising an HIV-1/IIIBx 8x gp120, the chimeric polypeptide further comprising a second portion comprising a gp120 from an HIV-1 other than HIV-1/IIIBx 8x.

The invention also includes a cell comprising chimeric HIV-1 Env polypeptide wherein the polypeptide is CD4-independent, and further wherein the polypeptide comprises a chemokine receptor binding site selected from the group consisting of a CXCR4 chemokine receptor binding site, and a CCR5 chemokine receptor binding site.

In one aspect, the second portion comprises a V3-loop selected from the group consisting of a HXB V3-loop, an 8x V3-loop, a BaL V3-loop, a YU-2 V3-loop, and an 89.6 V3-loop.

The invention includes a cell comprising a composition comprising a CD4-independent HIV-1 Env comprising a gp120 polypeptide comprising a stably exposed chemokine receptor binding site wherein the HIV-1 is more sensitive to antibody neutralization than an otherwise identical HIV-1 which does not comprise a stably exposed chemokine receptor binding site.

The invention includes a method of identifying an amino acid residue of an HIV-1 Env protein which is involved in CD4 independence. The method comprises obtaining a full-length env coding sequence from an Env clone which is CD4-independent and replacing at least a portion of the said env coding sequence with a coding sequence from an Env clone which is CD4-dependent to form a chimera, wherein when the chimera is CD4-dependent it is an indication that the portion of the env coding sequence is involved in CD4-independence, thereby identifying an amino acid residue involved in CD4-independence.

The invention also includes a method of eliciting an immune response to a HIV-1 chemokine receptor binding site in a mammal. The method comprises administering an immunogenic dose of a CD4-independent HIV-1 Env protein to a mammal, wherein the protein comprises a stably exposed chemokine receptor binding site, thereby eliciting an immune response to a HIV-1 chemokine receptor binding site in a mammal.

The invention also includes a method of identifying a compound which affects exposure of an HIV-1 gp120 chemokine receptor binding site. The method comprises contacting a cell with the compound prior to or contemporaneous with contacting the cell with a labeled gp120 with or without pre-incubation of the gp120 with soluble CD4, measuring the amount of label bound to the cell, and comparing the amount of label bound to the cells contacted with the compound to the amount of label bound to otherwise identical cells not contacted with the compound, wherein a higher or lower amount of label bound to the cells contacted with the compound compared with the amount of label bound to the otherwise identical cells not contacted with the compound, is an indication that the compound affects exposure of an HIV-1 gp120 chemokine receptor binding site.

The invention includes a method of identifying a small-molecule which inhibits binding of an HIV-1 gp120, using its chemokine receptor binding site, to a chemokine receptor. The method comprises contacting a cell with the molecule prior to or contemporaneous with contacting the cell with labeled gp120 with or without pre-incubation of said gp120 with soluble CD4, measuring the amount of label bound to the cell, and comparing the amount of label bound to the cell contacted with the molecule with the amount of label bound to an otherwise identical cell not contacted with the molecule, wherein a lower amount of label bound to the cell contacted with the molecule compared with the amount of label bound to the otherwise identical cell not contacted with the molecule, is an indication that the molecule inhibits binding of an HIV-1 gp120 using its chemokine receptor binding site to a chemokine receptor.

The invention includes a method of producing a CD4-independent chimeric HIV-1 Env clone comprising a variable chemokine receptor binding site. The method comprises replacing the hypervariable V3-loop of the CD4-independent Env clone with the V3 loop of another HIV-1, wherein the V3-loop of another HIV-1 comprises a different chemokine receptor binding site than that of the CD4-independent Env clone.

In one aspect, the CD4-independent clone is selected from the group consisting of HIV-1/IIIBx, and HIV-1/IIIBx 8x.

In another aspect, the V3-loop from another HIV-1 is selected from the group consisting of a V3-loop from HIV-1/BaL, a V3-loop from HIV-1/YU-2, a V3-loop from HIV-1/ADA, and a V3-loop from HIV-1/89.6.

The invention also includes a method of inhibiting HIV-1 gp120 binding, using its chemokine receptor binding site, to a chemokine receptor. The method comprises contacting said gp120 with a small-molecule identified by a method of identifying a compound which affects exposure of an HIV-1 gp120 chemokine receptor binding site, the method comprising contacting a cell with the compound prior to or contemporaneous with contacting the cell with a labeled gp120 with or without pre-incubation of the gp120 with soluble CD4, measuring the amount of label bound to the cell, and comparing the amount of label bound to the cells contacted with the compound to the amount of label bound to otherwise identical cells not contacted with the compound, wherein a higher or lower amount of label bound to the cells contacted with the compound compared with the amount of label bound to the otherwise identical cells not contacted with the compound, is an indication that the compound affects exposure of an HIV-1 gp120 chemokine receptor binding site, thereby inhibiting HIV-1 gp120 binding, using its chemokine receptor binding site, to a chemokine receptor.

The invention includes a method of inhibiting HIV-1 infection of a cell. The method comprises contacting the cell with a small-molecule which inhibits binding of an HIV-1 gp120 using its chemokine receptor binding site to a chemokine receptor, wherein the small-molecule is identified using a method of identifying a small-molecule which inhibits binding of an HIV-1 gp120, using its chemokine receptor binding site, to a chemokine receptor, the method comprising contacting a cell with the molecule prior to or contemporaneous with contacting the cell with labeled gp120 with or without pre-incubation of said gp120 with soluble CD4, measuring the amount of label bound to the cell, and comparing the amount of label bound to the cell contacted with the molecule with the amount of label bound to an otherwise identical cell not contacted with the molecule, wherein a lower amount of label bound to the cell contacted with the molecule compared with the amount of label bound to the otherwise identical cell not contacted with the molecule, is an indication that the molecule inhibits binding of an HIV-1 gp120 using its chemokine receptor binding site to a chemokine receptor, thereby inhibiting HIV-1 infection of a cell.

The invention includes a composition comprising a CD4-independent HIV-1 Env and at least one compound used to treat HIV infection in a pharmaceutically suitable carrier.

In one aspect, the HIV-1 Env is selected from the group consisting of a HIV-1 Env polypeptide, a nucleic acid encoding HIV-1 Env, and a cell expressing HIV-1 env.

In another aspect, the compound used to treat HIV infection is selected from the group consisting of a protease inhibitor, a reverse transcriptase nucleoside analog inhibitor, a reverse transcriptase non-nucleoside analog inhibitor, an interferon, AZT, interleukin-2, and a cytokine.

In one aspect, the HIV-1 Env is selected from the group consisting of a HIV-1 Env polypeptide, a nucleic acid encoding HIV-1 Env, and a cell expressing HIV-1 env.

In another aspect, the method further comprises administering a compound used to treat HIV infection.

In yet another aspect, the compound used to treat HIV infection is selected from the group consisting of a protease inhibitor, a reverse transcriptase nucleoside analog inhibitor, a reverse transcriptase non-nucleoside analog inhibitor, an interferon, AZT, interleukin-2, and a cytokine.

In a further aspect, the compound is administered to said human before, during or after administration of said CD4-independent HIV-1 Env.

DETAILED DESCRIPTION OF THE INVENTION

The invention is based on the discovery of a CD4-independent variant of HIV-1/IIIB, designated HIV-1/IIIBx (IIIBx), and a functional full-length env clone therefrom termed HIV-1/IIIBx.8 (8x), which allow the study of the mechanism for virus infection of host cells involving cell receptor proteins. Further, the present invention relates to the construction of chimeras comprising portions of a nucleic acid encoding 8x env covalently linked to a least one nucleic acid encoding a portion of an env from another HIV-1 virus. Thus, the chimeras are produced by combining portions of the 8x env coding sequence with portions of the env coding sequences of other virions leading to the further discovery of which portion(s) of the 8x HIV-1 env sequence is involved in CD4-independence.

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 binding site 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. Therefore, the 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.

Accordingly, understanding which portions of the Env are involved in virus binding to cell proteins and thereby mapping the protein determinants involved in HIV-1 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, 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 important human pathogen.

Moreover, production of chimeras has led to the discovery that the CD4 dependence trait and the choice of chemokine receptor are functionally dissociable traits. One skilled in the art would appreciate, based upon the disclosure provided herein, that such chimeras are useful for mapping the various structural and functional elements of the nucleic acid encoding env and the Env protein encoded thereby. Thus, by combining various portions of different viruses having different properties, e.g., CD4-dependence or independence and/or different affinities for various chemokine receptors, the various functional elements of the Env protein may be examined and identified.

In one embodiment, replacing the V3-loop portion of 8x gp120, which binds the CXCR4 chemokine receptor in the absence of CD4, with the V3-loop of HIV-1/BaL, which is a virus strain that is CD4-dependent and binds the CCR5 chemokine coreceptor, converts the chimeric gp120 8x/V3-BaL to a CCR5 binding protein which retains CD4-independence. This further demonstrates that CD4-independence exposes the chemokine receptor binding domain such that the preceding step of CD4-binding by gp120 is no longer required regardless of the choice of chemokine receptor. These data also suggest that a chemokine receptor binding site exists on the gp120 that is able to interact with genetically divergent chemokine receptors (i.e., CXCR4 and CCR5) and this site is functional and likely exposed on CD4-independent viruses.

In addition, the present invention teaches that the CD4-independent gp120 protein exists in a stable partially "triggered" state, wherein the chemokine coreceptor binding site is more exposed in the CD4-independent gp120 protein than in the CD4-dependent conformation of the HIV-1 gp120 molecule. This has the effect of rendering the CD4-independent virus more susceptible to neutralization by anti-HIV-1 antibodies from mouse, human and rabbit. Therefore, the present invention has important implications for the development of HIV-1 therapeutics since the availability of a stably exposed, highly conserved chemokine receptor binding site, which may be otherwise camouflaged to escape immune detection, should facilitate the development of a humoral and/or cellular immune response and of small-molecule inhibitors to block this virus-host protein interaction, thereby preventing HIV-1 infection.

The present invention includes an isolated nucleic acid encoding a CD4-independent HIV env coding sequence which is comprised of two components, a portion encoding gp120 and a portion encoding gp41. In one embodiment, the full-length env clone of CD4-independent HIV-1/IIIBx, i.e., 8x, has been isolated. Further, the mutations in the 8x clone were identified relative to the known env coding sequence of HXBc2 (GenBank Accession No. AF038399) (SEQ ID NO:11). However, the present invention should not be construed to be limited to a full-length env clone of the CD4-independent HIV-1/IIIBx variant. Rather, the present invention should be construed to encompass partial env clones. Indeed, the data disclosed herein demonstrate that the entire env coding sequence of 8x is not required for CD4-independence. Thus, at least one mutation present in the 8x env coding sequence confers CD4-independence to 8x, but not all mutations in the clone are required for purposes of the present invention. Further, completely separate mutations of gp120 can also confer CD4-independence.

The experiments disclosed in the Examples below disclose the isolation of a CD4-independent strain of the invention, HIV-1/IIIBx, which was able to infect both CD4⁺ SupT1 cells and CD4^- BC7 cells, a SupT1 variant, as demonstrated by a reverse transcriptase activity assay. However, the present invention is not limited solely to infection of BC7 or SupT1 cells by HIV-1. Rather, the "CD4-independence" of the present invention encompasses infection by HIV-1 of any cell type which does not express CD4. Further, as discussed previously herein, a CD4-independent HIV-1 strain may also infect cells that are CD4⁺ although CD4/gp120 interaction is not required for infection of these cells by the CD4-independent HIV-1. Moreover, a CD4-independent HIV-1 strain need not infect every CD4^- cell type. Rather, the HIV-1 strain need only be able to infect at least one CD4^- cell type while its otherwise identical parental strain from which the clone was obtained cannot infect that cell type.

Additionally, for purposes of the invention, an HIV-1 strain variant is considered CD4-independent when it is able to infect at least about 5% of the susceptible cells in culture or the level of infection is about two to three-fold compared to background levels.

It will be appreciated by one skilled in the art, based upon the disclosure provided herein, that a CD4-independent isolate of an HIV-1 strain may be obtained by passaging a CD4-dependent HIV-1 swarm initially grown in CD4₊ cells onto cells which are CD4^-. As disclosed in the experiments described in "this patent", herein, HIV-1/IIIBx was obtained by passaging virus in CD4⁺ SupT1 cells followed by passaging virus on the otherwise identical but CD4^- BC7 cells. However, the invention should not be construed to be limited to these particular cell types. Instead, the invention encompasses a variety of CD4⁺ and CD4^- cells including, but not limited to, 293, Cf2TH, CCC⁺ L^-, and QT6 cells as well as stably transfected cells (U87, HeLa, HOS) that express a recombinant chemokine receptor in the presence or absence of CD4.

In other related aspects, the invention includes vectors which contain such an isolated nucleic acid comprising at least a portion of the HIV-1 env and which isolated nucleic acid is preferably capable of directing expression of the protein encoded by the nucleic acid; and virions, proviruses, and/or cells containing such vectors.

As the present experimental examples demonstrate, the nucleic acid encoding the Env protein may be cloned into various plasmid vectors. However, the present invention should not be construed to be limited to these plasmids or to any particular vector. Instead, the present invention should be construed as encompassing a wide plethora of vectors which are readily available and/or well-known in the art. Therefore, although in one embodiment, the full-length env coding regions were amplified by PCR and cloned into the plasmid pCDNA3, and the inserts were then sub-cloned into the 3' hemigenome of pNL4-3, the present invention should not be construed to be limited to these, or to any other, specific vectors.

The isolated nucleic acid of the invention should be construed to include an RNA or a DNA sequence encoding an Env 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 also includes an isolated polypeptide comprising the amino acid sequence of HIV-1/IIIBx 8x.

The present invention also provides for analogs of proteins or peptides which comprise a gp120 protein 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, carboxylation, or biotinylation. 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.

Further, the invention should be construed to include naturally occurring variants or recombinantly derived mutants of HIV-1/IIIBx 8x env sequences, which variants or mutants render the protein encoded thereby either more, less, or just as biologically active as the full-length 8x env clone of the invention.

In addition, the present invention includes mutants or variants of 8x gp120 comprising an altered chemokine receptor binding site. As discussed previously elsewhere herein, the gp120 protein comprises a chemokine receptor binding domain which mediates gp120 binding to various cellular chemokine receptor proteins, which binding typically occurs after gp120 binding to CD4. As disclosed in the experimental results which follow this section, 8x gp120 binds to CXCR4 chemokine receptor and does not require binding to CD4 before doing so. Further, the data disclosed elsewhere herein demonstrate that introduction of a portion of a nucleic acid encoding a portion of an HIV-1/BaL gp120 into the coding sequence of 8x gp120 gives rise to a chimeric protein that no longer binds to CXCR4. Instead, the chimeric gp120 now binds CCR5. Such mutants are useful in the methods of the invention for the study of the role of gp120-chemokine receptor protein interaction in HIV-1 virus infection. The present invention should not be construed to be limited solely to a chimeric gp120 wherein a portion of the nucleic acid encoding 8x gp120 has been replaced a portion of a nucleic acid encoding BaL gp120. Instead, the present invention should be construed to include other chimeras wherein any portion or portions of the nucleic acid encoding 8x gp120 may be replaced by at least one portion of a nucleic acid encoding a gp120 from any other HIV-1 strain, preferably, those strains of HIV (or SIV) that use CCR5 as a coreceptor. Further, such portions should not be construed as being limited to any particular domain of gp120, but rather, the portion of gp120 substituted may be from any portion of the sequence encoding the protein. Therefore, the resulting chimeric nucleic acid and the protein expressed therefrom may be a chimera comprised of various gp120s from several HIV-1 strains, in any combination possible.

As more specifically set forth elsewhere herein, a mutant gp120 gene which encodes a gp120 protein comprising an insertion, deletion, or substitution, whereby amino acids residues at or near the putative chemokine receptor binding site are altered, or whereby a truncated cytoplasmic tail of Env is produced, is useful in studying the association of gp120 with a host cell chemokine receptor protein. Indeed, as disclosed in the experiments described below, several such mutants have been discovered herein. However, the invention should not be construed as being limited to only these mutants; rather, the invention encompasses other mutants, comprising deletion, substitution, and point mutations, which demonstrate altered binding to chemokine receptor protein compared with the wild type gp120 and which mutants demonstrate CD4-independence.

The invention should also be construed to include DNA encoding variants of HIV-1 Env which may or may not retain biological activity. Such variants, i.e., analogs of proteins or polypeptides of gp120, gp41 (also referred to as TM), include proteins or polypeptides which have been or may be modified using recombinant DNA technology such that the protein or polypeptide possesses additional properties which enhance its suitability for use in the methods described herein, for example, but not limited to, variants conferring enhanced stability of the exposed chemokine receptor binding site, enhanced specific binding to CD4, CXCR4, CCR5, and the like.

The present invention includes analogs of the 8x Env protein. Analogs can 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.

Preferably, the amino acid sequence of an 8x Env analog is about 70% homologous, more preferably about 80% homologous, even more preferably about 90% homologous, more preferably, about 95% homologous, and most preferably, at least about 99% homologous to the amino acid sequence of 8x env (SEQ ID NO:3) disclosed herein at FIG. 4.

The invention should not be construed as being limited solely to the DNA and amino acid sequences disclosed herein. Once armed with the present invention, it is readily apparent to one skilled in the art that other CD4-independent env clones of HIV-1 may be obtained by following the procedures described herein in the experimental details section for the isolation of the 8x env nucleic acid (SEQ ID NO:4) encoding CD4-independent Env disclosed herein.

The invention should therefore be construed to include any and all nucleic acid sequences encoding HIV-1/IIIBx 8x Env and amino acid sequences having substantial homology to the nucleic acid encoding 8x env disclosed herein (SEQ ID NO:4) and the amino acid sequence (SEQ ID NO:3) shown in FIG. 4. Preferably, DNA which is substantially homologous is about 50% homologous, more preferably about 70% homologous, even more preferably about 80% homologous and most preferably about 90% homologous to the 8x env sequence (SEQ ID NO:4) disclosed herein. Preferably, an amino acid sequence which is substantially homologous is about 50% homologous, more preferably about 70% homologous, even more preferably about 80% homologous and most preferably about 90% homologous to the 8x Env amino acid sequences (SEQ ID NO:3) shown in FIG. 4.

Any number of procedures may be used for the generation of mutant or variant forms of 8x env. For example, generation of mutant forms of 8x which are not CD4 independent was accomplished herein by introducing portions of a nucleic acid encoding env from a virus which was CD4-dependent using recombinant DNA methodology well known in the art such as, for example, as described in Sambrook et al. (1989, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, New York) and Ausubel et al. (1997, Current Protocols in Molecular Biology, Green & Wiley, New York). Mutant Env so generated is expressed and the resulting protein is assessed for its ability to bind CD4 in a real time biosensor assay such as that described herein. Mutant proteins which bind chemokine receptor protein in a CD4-independent manner were then examined by RT, fusion activity, real time binding/dissociation kinetics, and other such assays.

Procedures for the introduction of amino acid changes in a protein or polypeptide by altering the DNA sequence encoding the polypeptide are well known in the art and are also described in Sambrook et al. (1989, supra); Ausubel et al. (1997, supra).

The invention also includes an isolated nucleic acid having nucleic acid sequence which is complementary to a portion or all of the nucleic acid encoding HIV-1 Env (SEQ ID NO:4).

As used herein, the term "fragment" as applied to a nucleic acid, may ordinarily be at least about 100 nucleotides in length, typically, at least about 200 nucleotides, more typically, from about 300 to about 600 nucleotides, typically at least about 700 to about 1000 nucleotides, preferably at least about 1000 to about 1400 nucleotides, even more preferably at least about 1600 nucleotides to about 2000 nucleotides, and most preferably, the nucleic acid fragment will be greater than about 2400 nucleotides in length.

The invention further includes a cell comprising the nucleic acids of interest. The nucleic acids need not be integrated into the cell genome nor do they 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 type.

The invention also includes antibodies specific for the chemokine receptor binding site of gp120, or a portion thereof, which antibodies comprise a monoclonal antibody.

In one embodiment, the antibody is a murine monoclonal antibody to gp120 (17b) the epitope of which overlaps with the chemokine receptor binding site, as well as a murine monoclonal antibody to gp120 termed 48d (Thali et al., 1993, J. Virol. 67:3978-3988). However, the invention should not be construed as being limited solely to these antibodies but rather, should be construed to include other antibodies, as that term is defined herein, to Env, or portions thereof, which antibodies perform in a manner substantially similar to those described herein in that, inter alia, the antibodies bind to gp120 chemokine receptor binding site, and they are able to inhibit HIV-1 infection as measured by RT activity and cell fusion activity.

The invention also comprises an isolated polypeptide comprising the amino acid sequence of 8x Env protein, and mutants, variants and fragments thereof.

The peptides of the invention may be substantially pure. A substantially pure peptide is purified by following known procedures for protein purification, wherein an immunological, enzymatic or other assay is used to monitor purification at each stage in the procedure. Protein purification methods are well known in the art, and are described, for example in Deutscher et al. (1990, In: Guide to Protein Purification, Harcourt Brace Jovanovich, San Diego).

The invention should thus be construed to include nucleic acid encoding desired proteins and fragments of nucleic acid encoding desired polypeptides.

The present invention includes an isolated nucleic acid encoding a chimeric protein comprising a first portion and a second portion. In one embodiment, the chimeric nucleic acid comprises a first portion encoding 8x env and a second portion encoding an env from S10, IIIB, or HXB2. Although these chimeras were useful in mapping which regions of 8x are required for CD4-independence, the present invention should not be construed to be limited to these chimeras. Rather, the invention should be construed to encompass any chimeras in the env coding region which may be constructed comprising any portion of 8x and any HIV-1 virus strain or variant thereof.

Further, in another embodiment, the chimeras comprised a portion of the 8x env coding region and a portion of the env coding region of a CCR5-tropic HIV-1 strain, BaL. More'specifically, the embodiment comprises the 8x env clone with the nucleic acid portion encoding the V3-loop of BaL. However, the present invention should not be construed to be limited to this particular portion of the env coding region or to this particular strain of HIV-1. Rather, as previously discussed elsewhere herein, the present invention includes the substitution of any portion of the 8x env coding sequence with a portion of the env coding sequence of at least one other HIV-1 strain or variant, and any possible permutation thereof. Therefore, the chimeras, both nucleic acid and amino acid expressed therefrom, include combinations from two or more HIV-1 env coding regions of interest. Thus, armed with the disclosure provided herein, the production of an almost infinite combination of chimeras with the predicted effects disclosed herein would be clear to one skilled in the art.

The invention also includes a method of identifying an amino acid residue of an HIV-1 Env protein which is involved in CD4-independence. The method comprises producing chimeric proteins comprising at least a portion from a CD4-independent Env clone and at least a second portion from a CD4-dependent Env clone. The resulting chimera is then examined to determine the ability of the chimeric protein to mediate CD4-independent infection by various assays as disclosed elsewhere herein. As discussed previously herein, a preferred embodiment is disclosed wherein portions of the 8x env coding sequence were combined with various portions of the env coding sequences of several CD4-dependent HIV-1 strains, e.g., S10 and HxBc2. Also as noted previously 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 CD4-dependent and -independent env coding sequences may be examined to map the CD4-independent determinants. Further, the CD4-independence may be examined using a variety of assays on various mammalian cell lines also as described previously elsewhere herein.

The present invention also includes an isolated gp120 protein comprising a stably exposed chemokine receptor binding site. In one embodiment, the increased exposure of the chemokine receptor binding site was determined by measuring the real time binding kinetics of the various proteins in biosensor experiments and the enhanced neutralization of the virus by anti-HIV antibodies and by crystallographic analyses. However, the present invention should not be construed to be limited to these particular assays. Rather, other assays well-known in the art or to be developed for the study of protein-protein interactions may be used to measure the exposure of the chemokine receptor binding site of a gp120 or Env protein of interest.

The invention includes a method of eliciting an immune response to a HIV-1 chemokine receptor binding site. The method comprises administering an immunogenic dose of a CD4-independent HIV-1 Env protein to a mammal wherein the protein comprises a stably exposed chemokine receptor binding site.

In addition, the use of purified nucleic acid to generate an immune response, where the nucleic acid is in a vector (e.g., a plasmid or a virus), or where the nucleic acid comprises naked nucleic acid not associated with any other nucleic acid, is well-known in the art. For example, methods for construction of nucleic acid vaccines are described in Burger et al. (1991, J. Gen. Virol. 72:359-367), and are well-known in the art. See also Sambrook et al., 1989, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, New York; Ausubel et al., 1997, Current Protocols in Molecular Biology, Green & Wiley, New York.

Further, cells expressing the HIV-1 Env protein of choice may also be used to generate an immune response to an HIV-1 chemokine receptor binding site.

The immune response to the Env immunogen is measured by standard immunological techniques such as ELISA or Western blotting and other such techniques well-known in the art or to be developed in the future. A variety of immunoassay formats may be used to select antibodies specifically immunoreactive with a particular protein. See, e.g., Harlow and Lane (1988, Antibodies, A Laboratory Manual, Cold Spring Harbor Publications, New York) for a description of immunoassay formats and conditions that can be used to determine specific immunoreactivity.

The CD4-independent HIV-1 Env protein of the invention may be formulated in a pharmaceutical composition which is suitable for administration of the protein to a human or veterinary patient. It will be appreciated that the precise formulation and dosage amounts will vary depending upon any number of factors, including, but not limited to, the type and severity of the disease to be treated, the route of administration, the age and overall health of the individual, the nature of the Env protein, etc. However, the preparation of a pharmaceutically acceptable composition having an appropriate pH, isotonicity, stability and other characteristics is within the skill of the art. Pharmaceutical compositions are described in the art, for example, in Remington's Pharmaceutical Sciences (1985, Genaro, ed., Mack Publishing Co., Easton, Pa.).

The amount of the CD4-independent Env administered, whether it is administered as protein or as nucleic acid or as a cell expressing HIV env, is sufficient to elicit an immune response to an HIV-1 chemokine receptor binding site. The pharmaceutical compositions useful for practicing the invention may be administered to deliver a dose of between about 1 ng/kg and about 100 mg/kg of patient body weight. Suitable amounts of the CD4-independent Env protein for administration include doses which are high enough to have the desired effect without concomitant adverse effects. When the CD4-independent Env is a protein or peptide, a preferred dosage range is from about 10 to about 1000 .mu.g of protein or peptide per kg of patient body weight. When the CD4-independent Env is administered in the form of DNA encoding the same contained within a recombinant virus vector, a dosage of between about 10² and about 10¹¹ plaque forming units of virus per kg of patient body weight may be used. When naked DNA encoding the CD4-independent Env is to be administered as the pharmaceutical composition, a dosage of between about 10 .mu.g to about several mg of DNA per kg of patient body weight may be used.

In the practice of the methods of the invention, a composition containing a CD4-independent Env protein is administered to a patient in a sufficient amount to treat, prevent, or alleviate a HIV-1 infection in the individual.

One skilled in the art would appreciate, based on the disclosure provided herein, that the Env protein/nucleic acid encoding Env may be administered to a patient to prevent HIV infection by interfering with virus binding to the appropriate chemokine receptor using the virus' chemokine receptor binding site and, thereby preventing infection. Further, the Env protein/nucleic acid encoding env may also treat or alleviate the condition in a previously infected individual by augmenting the immune response in the person that could, in turn, be beneficial as an adjunct to antiretroviral pharmacologic therapy. That is, the immunogen may boost the immune response to the virus chemokine receptor binding site thereby generating antibodies which block the requisite interactions between the virus chemokine receptor binding site and the target cell chemokine receptor.

The frequency of administration of a CD4-independent Env protein to a patient will also vary depending on several factors including, but not limited to, the type and severity of the viral infection to be treated, the route of administration, the age and overall health of the individual, the nature of the Env protein, etc. It is contemplated that the frequency of administration of the Env protein to the patient may vary from about once every few months to about once a month, to about once a week, to about once per day, to about several times daily.

Pharmaceutical compositions that are useful in the methods of the invention may be administered systemically in parenteral, oral solid and liquid formulations, ophthalmic, suppository, aerosol, topical or other similar formulations. In addition to the appropriate Env protein, or nucleic acid encoding same, these pharmaceutical compositions may contain pharmaceutically-acceptable carriers and other ingredients known to enhance and facilitate drug administration. Thus such compositions may optionally contain other components, such as adjuvants, e.g., aqueous suspensions of aluminum and magnesium hydroxides, and/or other pharmaceutically acceptable carriers, such as saline. Other possible formulations, such as nanoparticles, liposomes, resealed erythrocytes, and immunologically based systems may also be used to administer the appropriate Env protein or nucleic acid encoding it to a patient according to the methods of the invention.

Preferably, the composition of the invention is administered to the human by a parenteral or intravenous route.

An Env protein and/or a nucleic acid encoding Env, may be administered in conjunction with other compounds which are used to treat HIV infection. Such compounds include, but are not limited to, protease inhibitors, reverse transcriptases inhibitors (nucleoside and non-nucleoside analogs), AZT, interferons, interleukin-2, other cytokines, and the like. The choice of which additional compound to administer will vary depending upon any number of the same types of factors that govern the selection of dosage and administration frequency of the Env protein or nucleic acid encoding same. Selection of these types of compounds for use in conjunction with an Env protein for practice of the method of the invention is well within the skill of those in the art.

The invention also includes a vaccine comprising an immunogenic dose of a CD4-independent HIV-1 Env protein. As discussed previously elsewhere herein, generation of an immune response to the virus chemokine receptor binding site should block interaction of this virus site to the host chemokine receptor ligand thereby interfering with and/or inhibiting the requisite virus/host cell interaction needed for HIV infection.

In addition, the invention includes a method of identifying a compound which affects exposure of a gp120 protein chemokine receptor binding site. The method comprises contacting a cell with the compound and comparing the amount of labeled gp120 specifically bound to the cell with the amount of labeled chemokine bound to an otherwise identical cell not contacted with the compound. In one embodiment, the gp120 of interest was ¹²⁵ I-labeled and bound to cells expressing various chemokine receptors in the presence or absence of soluble CD4. However, the present invention should not be construed to be limited to radioiodination or to any particular gp120 or to expression of only these chemokine receptors. Rather, the invention should be construed to encompass a variety of protein labels such that binding of the gp120 of interest may be quantitated. Such methods are well-known in the art and include, but are not limited to, biotinylation, and ³⁵ S-cys and ³⁵ S-met.

The invention also includes a method of identifying a small-molecule which inhibits binding of a chemokine receptor by an HIV-1 gp120 using its chemokine receptor binding site. The method comprises contacting a cell with a small-molecule prior to or contemporaneous with contacting the cell with labeled gp120 with or without preincubation of the gp120 with soluble CD4. Then, the amount of label bound to the cell is measured thereby detecting the amount of labeled gp120 bound to the cell. The amount of bound gp120 bound to a cell contacted with the small-molecule is compared to the amount of gp120 bound to a cell not contacted with the small-molecule. If a lower amount of gp120 is bound to the cell contacted with the small molecule compared to the amount of gp120 bound to the cell which was not contacted with the small-molecule, this is an indication that contacting the cell with the small-molecule inhibits binding of HIV-1 gp120 to a chemokine receptor using its chemokine receptor binding site.

One skilled in the art would appreciate, based on the disclosure provided herein, that such small-molecules are useful therapeutics inhibiting HIV-1 infection of cells in that such small-molecules would inhibit the requisite HIV-1 gp120/chemokine receptor interactions necessary for virus infection of the target cell. Further, the prior art teaches that antibodies and chemokines which specifically bind to chemokine receptors and which block gp120 binding to the chemokine receptor often also block HIV infection (Lee et al., 1999, J. Biol. Chem., in press; Olson et al., 1999, J. Virol., in press; Wu et al., 1997, J. Exp. Med.). Thus, the small-molecule inhibitors of gp120 binding to the chemokine receptor identified using the methods of the invention are useful inhibitors of HIV infection.

Further, one skilled in the art, based upon the disclosure provided herein, would appreciate that a small-molecule inhibitor of gp120 binding using its chemokine receptor binding site to a chemokine receptor identified using the methods of the invention is a useful inhibitor of a chemokine binding to and activation of its receptor. That is, the small-molecule inhibitor may be useful for inhibiting the natural function of chemokine receptors unrelated to the role of the chemokine receptors in HIV infection. Thus, a small-molecule inhibitor identified herein is a useful therapeutic having potential uses for, among other things, immune system treatments, inflammation, and development in any non-HIV infected human.

The invention includes a method of inhibiting HIV-1 gp120 binding, using its chemokine receptor binding site, to a chemokine receptor. The method comprises contacting a the gp120 with a small-molecule which inhibits binding of gp120 to a chemokine receptor where such binding is mediated by the chemokine receptor binding site of the virus gp120 protein. The small-molecule is identified as disclosed previously elsewhere herein. Contacting the gp120 with the small-molecule binding inhibitor inhibits binding of the gp120 with the cell chemokine receptor.

The invention also includes a method of inhibiting HIV-1 infection of a cell. The method comprises contacting a cell with a small-molecule identified as described previously elsewhere herein. The small-molecule so identified inhibits the binding an HIV-1 gp120 to a cell chemokine receptor mediated by the virus gp120's chemokine receptor binding site. The small-molecule, by interfering with the requisite gp120/chemokine receptor interaction(s), thereby inhibits HIV-1 infection of the cell. Indeed, it has been demonstrated previously (Lee et al., 1999, J. Biol. Chem., in press; Olson et al., 1999, J. Virol., in press; Wu et al., 1997, J. Exp. Med.) antibodies and chemokines that block gp120 binding to the chemokine receptor often also block HIV infection. Thus, the invention includes a method of inhibiting HIV-1 infection by interfering with the receptor/ligand interactions required for HIV-1 infection of a target cell using a small-molecule inhibitor of gp120 binding to the cell chemokine receptor using the gp120 chemokine receptor binding site.

The invention also includes a composition comprising a CD4-independent HIV-1 Env and at least one compound used to treat HIV infection in a pharmaceutically suitable carrier. As described elsewhere herein, the HIV-1 Env may be a HIV-1 Env polypeptide, a nucleic acid encoding HIV-1 Env, and/or a cell expressing HIV-1 env. Further, as disclosed previously elsewhere herein, the invention should be construed to encompass compounds used to treat HIV infection such as, for example but not limited to, protease inhibitors, reverse transcriptase inhibitor, reverse transcriptase inhibitors (including both nucleoside and non-nucleoside analogs), interferons, AZT, interleukin-2, and cytokines.

The invention includes a method of treating HIV-1 infection in a human. The method comprises administering an immunogenic dose of a CD4-independent HIV-1 Env to an HIV-1 infected human. Administration of such CD4-independent HIV-1 Env induces the production of antibodies to the stably exposed chemokine receptor binding site of gp120. Thus, administration of the CD4-independent HIV-1 Env causes the production of potentially neutralizing antibodies which block the gp120/chemokine receptor interaction(s) required for HIV-1 infection of the host cell. This is suggested by the fact, disclosed elsewhere herein, that the CD4-independent gp120 is more sensitive to neutralizing antibodies than otherwise identical CD4-dependent gp120 which does not comprise a stably exposed chemokine receptor binding site. Further, antibodies that block Env-chemokine receptor interactions can neutralize HIV-1 (Wu et al., 1996, Nature 384:179-183; Trkola et al., 1996, Nature 384:184-187). Thus, increased exposure of the chemokine receptor binding site will enhance the production of antibodies to this conserved region which antibodies inhibit the requisite gp120-chemokine receptor interactions. Therefore, immunizing a human with CD4-independent Env causes the production of antibodies to the stably exposed chemokine receptor binding site which antibodies block requisite Env-chemokine receptor interactions needed for infection, thereby treating HIV-1 infection in the human.

One skilled in the art would appreciate, based upon the disclosure provided herein, that the immunogenic dose of a CD4-independent HIV-1 Env may be a useful therapeutic to treat and/or alleviate the HIV-1 infection in a human both before and after exposure to the HIV-1 virus. That is, the immunogenic dose may be administered prior to, during, or after infection of a human by HIV-1. Irrespective of when it is administered, the immunogen elicits a response in the human to, inter alia, the stably exposed chemokine receptor binding site of gp120 thereby inducing a response which inhibits the binding of the virus gp120 to the chemokine receptor. This inhibition is generated in both previously infected individuals as well as uninfected persons. In the individual already infected with HIV-1, the immunogen generates an immune response in addition to any immune response already present in the individual and thus mediates a reduction in the virus load in that individual. Thus, the CD4-independent HIV-1 Env is useful as a therapeutic vaccine in a human already infected by HIV-1 virus.

As disclosed previously elsewhere herein, one skilled in the art would appreciate, based on the disclosure provided herein, that the immunogenic dose of a CD4-independent HIV-1 Env may be administered as a protein, a nucleic acid (comprising a vector or as naked DNA), and/or a cell expressing a nucleic acid encoding a CD4-independent env.

In another aspect, the method of treating HIV-1 infection in a human comprises further administering a compound used to treat HIV infection. As disclosed previously elsewhere herein, such compounds include, but are not limited to, a protease inhibitors, a reverse transcriptase inhibitor, a reverse transcriptase inhibitor (including both nucleoside and non-nucleoside analogs), an interferon, AZT, interleukin-2, and a cytokine. The compound may be administered before, during, or after the administration of the immunogenic dose of a CD4-independent HIV-1 Env.

One skilled in the art would appreciate, based upon the disclosure provided herein, that the timing of the compound relative to the immunogenic dose of a CD4-independent HIV-1 Env would depend upon the immunization regimen regarding the HIV-1 Env and the particular compound(s) administered with the Env immunogen, as well as the health and age of the patient and the severity and stage of the disease process.

The HIV-1 Env immunogen(s) and/or compounds which are identified using any of the methods described herein may be formulated and administered to a mammal for treatment and/or prevention of HIV infection as now described.

The invention encompasses the preparation and use of pharmaceutical compositions comprising a compound useful for treatment of HIV infection as an active ingredient. Such a pharmaceutical composition may consist of the active ingredient alone, as a combination of at least one active ingredient (e.g., an immunogenic dose of a CD4-independent HIV-1 Env and a compound used to treat HIV infection such as interleukin-2) in a form suitable for administration to a subject, or the pharmaceutical composition may comprise the active ingredient and one or more pharmaceutically acceptable carriers, one or more additional ingredients, or some combination of these. The active ingredient may be present in the pharmaceutical composition in the form of a physiologically acceptable ester or salt, such as in combination with a physiologically acceptable cation or anion, as is well known in the art.

As used herein, the term "pharmaceutically acceptable carrier" means a chemical composition with which the active ingredient may be combined and which, following the combination, can be used to administer the active ingredient to a subject.

As used herein, the term "physiologically acceptable" ester or salt means an ester or salt form of the active ingredient which is compatible with any other ingredients of the pharmaceutical composition, which is not deleterious to the subject to which the composition is to be administered.

The formulations of the pharmaceutical compositions described herein may be prepared by any method known or hereafter developed in the art of pharmacology. In general, such preparatory methods include the step of bringing the active ingredient into association with a carrier or one or more other accessory ingredients, and then, if necessary or desirable, shaping or packaging the product into a desired single- or multi-dose unit.

Although the descriptions of pharmaceutical compositions provided herein are principally directed to pharmaceutical compositions which are suitable for ethical administration to humans, it will be understood by the skilled artisan that such compositions are generally suitable for administration to animals of all sorts. Modification of pharmaceutical compositions suitable for administration to humans in order to render the compositions suitable for administration to various animals is well understood, and the ordinarily skilled veterinary pharmacologist can design and perform such modification with merely ordinary, if any, experimentation. Subjects to which administration of the pharmaceutical compositions of the invention is contemplated include, but are not limited to, humans and other primates, mammals including commercially relevant mammals such as non-human primates, cattle, pigs, horses, sheep, cats, and dogs, birds including commercially relevant birds such as chickens, ducks, geese, and turkeys, fish including farm-raised fish and aquarium fish, and crustaceans such as farm-raised shellfish.

Pharmaceutical compositions that are useful in the methods of the invention may be prepared, packaged, or sold in formulations suitable for oral, rectal, vaginal, parenteral, topical, pulmonary, intranasal, buccal, ophthalmic, or another route of administration. Other contemplated formulations include projected nanoparticles, liposomal preparations, resealed erythrocytes containing the active ingredient, and immunologically-based formulations.

A pharmaceutical composition of the invention may be prepared, packaged, or sold in bulk, as a single unit dose, or as a plurality of single unit doses. As used herein, a "unit dose" is discrete amount of the pharmaceutical composition comprising a predetermined amount of the active ingredient. The amount of the active ingredient is generally equal to the dosage of the active ingredient which would be administered to a subject or a convenient fraction of such a dosage such as, for example, one-half or one-third of such a dosage.

The relative amounts of the active ingredient, the pharmaceutically acceptable carrier, and any additional ingredients in a pharmaceutical composition of the invention will vary, depending upon the identity, size, and condition of the subject treated and further depending upon the route by which the composition is to be administered. By way of example, the composition may comprise between 0.1% and 100% (w/w) active ingredient.

In addition to the active ingredient, a pharmaceutical composition of the invention may further comprise one or more additional pharmaceutically active agents. Particularly contemplated additional agents include anti-emetics and scavengers such as cyanide and cyanate scavengers and AZT, protease inhibitors, reverse transcriptase inhibitors, interleukin-2, interferons, cytokines, and the like.

Controlled- or sustained-release formulations of a pharmaceutical composition of the invention may be made using conventional technology.

A formulation of a pharmaceutical composition of the invention suitable for oral administration may be prepared, packaged, or sold in the form of a discrete solid dose unit including, but not limited to, a tablet, a hard or soft capsule, a cachet, a troche, or a lozenge, each containing a predetermined amount of the active ingredient. Other formulations suitable for oral administration include, but are not limited to, a powdered or granular formulation, an aqueous or oily suspension, an aqueous or oily solution, or an emulsion.

As used herein, an "oily" liquid is one which comprises a carbon-containing liquid molecule and which exhibits a less polar character than water.

A tablet comprising the active ingredient may, for example, be made by compressing or molding the active ingredient, optionally with one or more additional ingredients. Compressed tablets may be prepared by compressing, in a suitable device, the active ingredient in a free-flowing form such as a powder or granular preparation, optionally mixed with one or more of a binder, a lubricant, an excipient, a surface active agent, and a dispersing agent. Molded tablets may be made by molding, in a suitable device, a mixture of the active ingredient, a pharmaceutically acceptable carrier, and at least sufficient liquid to moisten the mixture. Pharmaceutically acceptable excipients used in the manufacture of tablets include, but are not limited to, inert diluents, granulating and disintegrating agents, binding agents, and lubricating agents. Known dispersing agents include, but are not limited to, potato starch and sodium starch glycolate. Known surface active agents include, but are not limited to, sodium lauryl sulphate. Known diluents include, but are not limited to, calcium carbonate, sodium carbonate, lactose, microcrystalline cellulose, calcium phosphate, calcium hydrogen phosphate, and sodium phosphate. Known granulating and disintegrating agents include, but are not limited to, corn starch and alginic acid. Known binding agents include, but are not limited to, gelatin, acacia, pre-gelatinized maize starch, polyvinylpyrrolidone, and hydroxypropyl methylcellulose. Known lubricating agents include, but are not limited to, magnesium stearate, stearic acid, silica, and talc.

Tablets may be non-coated or they may be coated using known methods to achieve delayed disintegration in the gastrointestinal tract of a subject, thereby providing sustained release and absorption of the active ingredient. By way of example, a material such as glyceryl monostearate or glyceryl distearate may be used to coat tablets. Further by way of example, tablets may be coated using methods described in U.S. Pat. Nos. 4,256,108; 4,160,452; and 4,265,874 to form osmotically-controlled release tablets. Tablets may further comprise a sweetening agent, a flavoring agent, a coloring agent, a preservative, or some combination of these in order to provide pharmaceutically elegant and palatable preparation.

Hard capsules comprising the active ingredient may be made using a physiologically degradable composition, such as gelatin. Such hard capsules comprise the active ingredient, and may further comprise additional ingredients including, for example, an inert solid diluent such as calcium carbonate, calcium phosphate, or kaolin.

Soft gelatin capsules comprising the active ingredient may be made using a physiologically degradable composition, such as gelatin. Such soft capsules comprise the active ingredient, which may be mixed with water or an oil medium such as peanut oil, liquid paraffin, or olive oil.

Liquid formulations of a pharmaceutical composition of the invention which are suitable for oral administration may be prepared, packaged, and sold either in liquid form or in the form of a dry product intended for reconstitution with water or another suitable vehicle prior to use.

Liquid suspensions may be prepared using conventional methods to achieve suspension of the active ingredient in an aqueous or oily vehicle. Aqueous vehicles include, for example, water and isotonic saline. Oily vehicles include, for example, almond oil, oily esters, ethyl alcohol, vegetable oils such as arachis, olive, sesame, or coconut oil, fractionated vegetable oils, and mineral oils such as liquid paraffin. Liquid suspensions may further comprise one or more additional ingredients including, but not limited to, suspending agents, dispersing or wetting agents, emulsifying agents, demulcents, preservatives, buffers, salts, flavorings, coloring agents, and sweetening agents. Oily suspensions may further comprise a thickening agent. Known suspending agents include, but are not limited to, sorbitol syrup, hydrogenated edible fats, sodium alginate, polyvinylpyrrolidone, gum tragacanth, gum acacia, and cellulose derivatives such as sodium carboxymethylcellulose, methylcellulose, hydroxypropyl methylcellulose. Known dispersing or wetting agents include, but are not limited to, naturally-occurring phosphatides such as lecithin, condensation products of an alkylene oxide with a fatty acid, with a long chain aliphatic alcohol, with a partial ester derived from a fatty acid and a hexitol, or with a partial ester derived from a fatty acid and a hexitol anhydride (e.g. polyoxyethylene stearate, heptadecaethyleneoxycetanol, polyoxyethylene sorbitol monooleate, and polyoxyethylene sorbitan monooleate, respectively). Known emulsifying agents include, but are not limited to, lecithin and acacia. Known preservatives include, but are not limited to, methyl, ethyl, or n-propyl-para-hydroxybenzoates, ascorbic acid, and sorbic acid. Known sweetening agents include, for example, glycerol, propylene glycol, sorbitol, sucrose, and saccharin. Known thickening agents for oily suspensions include, for example, beeswax, hard paraffin, and cetyl alcohol.

Liquid solutions of the active ingredient in aqueous or oily solvents may be prepared in substantially the same manner as liquid suspensions, the primary difference being that the active ingredient is dissolved, rather than suspended in the solvent. Liquid solutions of the pharmaceutical composition of the invention may comprise each of the components described with regard to liquid suspensions, it being understood that suspending agents will not necessarily aid dissolution of the active ingredient in the solvent. Aqueous solvents include, for example, water and isotonic saline. Oily solvents include, for example, almond oil, oily esters, ethyl alcohol, vegetable oils such as arachis, olive, sesame, or coconut oil, fractionated vegetable oils, and mineral oils such as liquid paraffin.

Powdered and granular formulations of a pharmaceutical preparation of the invention may be prepared using known methods. Such formulations may be administered directly to a subject, used, for example, to form tablets, to fill capsules, or to prepare an aqueous or oily suspension or solution by addition of an aqueous or oily vehicle thereto. Each of these formulations may further comprise one or more of dispersing or wetting agent, a suspending agent, and a preservative. Additional excipients, such as fillers and sweetening, flavoring, or coloring agents, may also be included in these formulations.

A pharmaceutical composition of the invention may also be prepared, packaged, or sold in the form of oil-in-water emulsion or a water-in-oil emulsion. The oily phase may be a vegetable oil such as olive or arachis oil, a mineral oil such as liquid paraffin, or a combination of these. Such compositions may further comprise one or more emulsifying agents such as naturally occurring gums such as gum acacia or gum tragacanth, naturally-occurring phosphatides such as soybean or lecithin phosphatide, esters or partial esters derived from combinations of fatty acids and hexitol anhydrides such as sorbitan monooleate, and condensation products of such partial esters with ethylene oxide such as polyoxyethylene sorbitan monooleate. These emulsions may also contain additional ingredients including, for example, sweetening or flavoring agents.

A pharmaceutical composition of the invention may be prepared, packaged, or sold in a formulation suitable for rectal administration. Such a composition may be in the form of, for example, a suppository, a retention enema preparation, and a solution for rectal or colonic irrigation.

Suppository formulations may be made by combining the active ingredient with a non-irritating pharmaceutically acceptable excipient which is solid at ordinary room temperature (i.e., about 20^oC.) and which is liquid at the rectal temperature of the subject (i.e., about 37^oC. in a healthy human). Suitable pharmaceutically acceptable excipients include, but are not limited to, cocoa butter, polyethylene glycols, and various glycerides. Suppository formulations may further comprise various additional ingredients including, but not limited to, antioxidants and preservatives.

Retention enema preparations or solutions for rectal or colonic irrigation may be made by combining the active ingredient with a pharmaceutically acceptable liquid carrier. As is well known in the art, enema preparations may be administered using, and may be packaged within, a delivery device adapted to the rectal anatomy of the subject. Enema preparations may further comprise various additional ingredients including, but not limited to, antioxidants and preservatives.

A pharmaceutical composition of the invention may be prepared, packaged, or sold in a formulation suitable for vaginal administration. Such a composition may be in the form of, for example, a suppository, an impregnated or coated vaginally-insertable material such as a tampon, a douche preparation, or gel or cream or a solution for vaginal irrigation.

Methods for impregnating or coating a material with a chemical composition are known in the art, and include, but are not limited to methods of depositing or binding a chemical composition onto a surface, methods of incorporating a chemical composition into the structure of a material during the synthesis of the material (i.e. such as with a physiologically degradable material), and methods of absorbing an aqueous or oily solution or suspension into an absorbent material, with or without subsequent drying.

Douche preparations or solutions for vaginal irrigation may be made by combining the active ingredient with a pharmaceutically acceptable liquid carrier. As is well known in the art, douche preparations may be administered using, and may be packaged within, a delivery device adapted to the vaginal anatomy of the subject. Douche preparations may further comprise various additional ingredients including, but not limited to, antioxidants, antibiotics, antifungal agents, and preservatives.

As used herein, "parenteral administration" of a pharmaceutical composition includes any route of administration characterized by physical breaching of a tissue of a subject and administration of the pharmaceutical composition through the breach in the tissue. Parenteral administration thus includes, but is not limited to, administration of a pharmaceutical composition by injection of the composition, by application of the composition through a surgical incision, by application of the composition through a tissue-penetrating non-surgical wound, and the like. In particular, parenteral administration is contemplated to include, but is not limited to, subcutaneous, intraperitoneal, intramuscular, intrasternal injection, and kidney dialytic infusion techniques.

Formulations of a pharmaceutical composition suitable for parenteral administration comprise the active ingredient combined with a pharmaceutically acceptable carrier, such as sterile water or sterile isotonic saline. Such formulations may be prepared, packaged, or sold in a form suitable for bolus administration or for continuous administration. Injectable formulations may be prepared, packaged, or sold in unit dosage form, such as in ampules or in multi-dose containers containing a preservative. Formulations for parenteral administration include, but are not limited to, suspensions, solutions, emulsions in oily or aqueous vehicles, pastes, and implantable sustained-release or biodegradable formulations. Such formulations may further comprise one or more additional ingredients including, but not limited to, suspending, stabilizing, or dispersing agents. In one embodiment of a formulation for parenteral administration, the active ingredient is provided in dry (i.e. powder or granular) form for reconstitution with a suitable vehicle (e.g. sterile pyrogen-free water) prior to parenteral administration of the reconstituted composition.

The pharmaceutical compositions may be prepared, packaged, or sold in the form of a sterile injectable aqueous or oily suspension or solution. This suspension or solution may be formulated according to the known art, and may comprise, in addition to the active ingredient, additional ingredients such as the dispersing agents, wetting agents, or suspending agents described herein. Such sterile injectable formulations may be prepared using a non-toxic parenterally-acceptable diluent or solvent, such as water or 1,3-butane diol, for example. Other acceptable diluents and solvents include, but are not limited to, Ringer's solution, isotonic sodium chloride solution, and fixed oils such as synthetic mono- or di-glycerides. Other parentally-administrable formulations which are useful include those which comprise the active ingredient in microcrystalline form, in a liposomal preparation, or as a component of a biodegradable polymer systems. Compositions for sustained release or implantation may comprise pharmaceutically acceptable polymeric or hydrophobic materials such as an emulsion, an ion exchange resin, a sparingly soluble polymer, or a sparingly soluble salt.

Formulations suitable for topical administration include, but are not limited to, liquid or semi-liquid preparations such as liniments, lotions, oil-in-water or water-in-oil emulsions such as creams, ointments or pastes, and solutions or suspensions. Topically-administrable formulations may, for example, comprise from about 1% to about 10% (w/w) active ingredient, although the concentration of the active ingredient may be as high as the solubility limit of the active ingredient in the solvent. Formulations for topical administration may further comprise one or more of the additional ingredients described herein.

A pharmaceutical composition of the invention may be prepared, packaged, or sold in a formulation suitable for pulmonary administration via the buccal cavity. Such a formulation may comprise dry particles which comprise the active ingredient and which have a diameter in the range from about 0.5 to about 7 nanometers, and preferably from about 1 to about 6 nanometers. Such compositions are conveniently in the form of dry powders for administration using a device comprising a dry powder reservoir to which a stream of propellant may be directed to disperse the powder or using a self-propelling solvent/powder-dispensing container such as a device comprising the active ingredient dissolved or suspended in a low-boiling propellant in a sealed container. Preferably, such powders comprise particles wherein at least 98% of the particles by weight have a diameter greater than 0.5 nanometers and at least 95% of the particles by number have a diameter less than 7 nanometers. More preferably, at least 95% of the particles by weight have a diameter greater than 1 nanometer and at least 90% of the particles by number have a diameter less than 6 nanometers. Dry powder compositions preferably include a solid fine powder diluent such as sugar and are conveniently provided in a unit dose form.

Low boiling propellants generally include liquid propellants having a boiling point of below 65^oF. at atmospheric pressure. Generally the propellant may constitute 50 to 99.9% (w/w) of the composition, and the active ingredient may constitute 0.1 to 20% (w/w) of the composition. The propellant may further comprise additional ingredients such as a liquid non-ionic or solid anionic surfactant or a solid diluent (preferably having a particle size of the same order as particles comprising the active ingredient).

Pharmaceutical compositions of the invention formulated for pulmonary delivery may also provide the active ingredient in the form of droplets of a solution or suspension. Such formulations may be prepared, packaged, or sold as aqueous or dilute alcoholic solutions or suspensions, optionally sterile, comprising the active ingredient, and may conveniently be administered using any nebulization or atomization device. Such formulations may further comprise one or more additional ingredients including, but not limited to, a flavoring agent such as saccharin sodium, a volatile oil, a buffering agent, a surface active agent, or a preservative such as methylhydroxybenzoate. The droplets provided by this route of administration preferably have an average diameter in the range from about 0.1 to about 200 nanometers.

The formulations described herein as being useful for pulmonary delivery are also useful for intranasal delivery of a pharmaceutical composition of the invention.

Another formulation suitable for intranasal administration is a coarse powder comprising the active ingredient and having an average particle from about 0.2 to 500 micrometers. Such a formulation is administered in the manner in which snuff is taken i.e. by rapid inhalation through the nasal passage from a container of the powder held close to the nares.

Formulations suitable for nasal administration may, for example, comprise from about as little as 0.1% (w/w) and as much as 100% (w/w) of the active ingredient, and may further comprise one or more of the additional ingredients described herein.

A pharmaceutical composition of the invention may be prepared, packaged, or sold in a formulation suitable for buccal administration. Such formulations may, for example, be in the form of tablets or lozenges made using conventional methods, and may, for example, 0.1 to 20% (w/w) active ingredient, the balance comprising an orally dissolvable or degradable composition and, optionally, one or more of the additional ingredients described herein. Alternately, formulations suitable for buccal administration may comprise a powder or an aerosolized or atomized solution or suspension comprising the active ingredient. Such powdered, aerosolized, or aerosolized formulations, when dispersed, preferably have an average particle or droplet size in the range from about 0.1 to about 200 nanometers, and may further comprise one or more of the additional ingredients described herein.

A pharmaceutical composition of the invention may be prepared, packaged, or sold in a formulation suitable for ophthalmic administration. Such formulations may, for example, be in the form of eye drops including, for example, a 0.1-1.0% (w/w) solution or suspension of the active ingredient in an aqueous or oily liquid carrier. Such drops may further comprise buffering agents, salts, or one or more other of the additional ingredients described herein. Other ophthalmalmically-administrable formulations which are useful include those which comprise the active ingredient in microcrystalline form or in a liposomal preparation.

As used herein, "additional ingredients" include, but are not limited to, one or more of the following: excipients; surface active agents; dispersing agents; inert diluents; granulating and disintegrating agents; binding agents; lubricating agents; sweetening agents; flavoring agents; coloring agents; preservatives; physiologically degradable compositions such as gelatin; aqueous vehicles and solvents; oily vehicles and solvents; suspending agents; dispersing or wetting agents; emulsifying agents, demulcents; buffers; salts; thickening agents; fillers; emulsifying agents; antioxidants; antibiotics; antifungal agents; stabilizing agents; and pharmaceutically acceptable polymeric or hydrophobic materials. Other "additional ingredients" which may be included in the pharmaceutical compositions of the invention are known in the art and described, for example in Remington's Pharmaceutical Sciences (1985, Genaro, ed., Mack Publishing Co., Easton, Pa.), which is incorporated herein by reference.

Typically dosages of the compound of the invention which may be administered to an animal, preferably a human, range in amount from 1 .mu.g to about 100 g per kilogram of body weight of the animal. While the precise dosage administered will vary depending upon any number of factors, including but not limited to, the type of animal and type of disease state being treated, the age of the animal and the route of administration. Preferably, the dosage of the compound will vary from about 1 mg to about 10 g per kilogram of body weight of the animal. More preferably, the dosage will vary from about 10 mg to about 1 g per kilogram of body weight of the animal.

The compound may be administered to an animal as frequently as several times daily, or it may be administered less frequently, such as once a day, once a week, once every two weeks, once a month, or even less frequently, such as once every several months or even once a year or less. The frequency of the dose will be readily apparent to the skilled artisan and will depend upon any number of factors, such as, but not limited to, the type and severity of the disease being treated, the type and age of the animal, etc.

The compound used to treat HIV infection may be co-administered with the immunogenic dose of CD4-independent HIV-1 Env. Alternatively, the compound(s) may be administered an hour, a day, a week, a month, or even more, in advance of the immunogenic dose(s) of HIV-1 Env, or any permutation thereof. Further, the compound(s) may be administered an hour, a day, a week, or even more, after the immunogenic dose(s) of HIV-1 Env, or any permutation thereof. The frequency and administration regimen will be readily apparent to the skilled artisan and will depend upon any number of factors such as, but not limited to, the type and severity of the disease being treated, the age and health status of the animal, the identity of the compound or compounds being administered, the route of administration of the various compounds and HIV-1 Env, and the like.

Claim 1 of 6 Claims

What is claimed is:

1. An isolated polypeptide comprising an HIV-1/IIIBx 8x Env, said isolated polypeptide comprising the amino acid sequence of SEQ ID NO:3.

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