WO2002097034A2

WO2002097034A2 - Antibodies and antigens of hiv viremia controllers

Info

Publication number: WO2002097034A2
Application number: PCT/US2002/016026
Authority: WO
Inventors: Martina M. Berger; Xi Yu Jia; Jeremiah G. Tilles
Original assignee: The Regents Of The University Of California
Priority date: 2001-05-25
Filing date: 2002-05-22
Publication date: 2002-12-05
Also published as: WO2002097034A3; AU2002311968A1

Abstract

Disclosed herein are methods for identifying peptides that are serologically reactive with samples from HIV-infected subjects who control viremia (LTNP or viremia controllers) at a greater frequency and binding affinity than samples from subjects with progressive HIV disease (HIV progressors). Also disclosed are compositions and methods for using the compositions in the diagnosis, treatment, and prevention of HIV infection, AIDS or both. The compositions of the present invention include peptides that are serologically reactive with samples from viremia controllers at a greater frequency and binding affinity than samples from HIV progressors, polynucleotides encoding such polypeptides, binding agents such as antibodies, antigen presenting cells (APCs), immune system cells ((e.g.), T cells), and combinations thereof.

Description

ANTIBODIES AND ANTIGENS OF HUMAN IMMUNODEFICIENCY VIRUS (HIV) VIREMIA CONTROLLERS AND METHODS OF USING THEREOF

CROSS REFERENCE TO RELATED APPLICATIONS [01] This application claims the benefit of U.S. Provisional Patent Application Nos.

60/316,262 filed 4 September 2001, and 60/293,355 filed 25 May 2001, listing Martina M. Berger, Xi Yu Jia, Jeremiah G. Tilles, and Donald N. Formal as joint inventors, both of which are herein incorporated by reference in their entirety.

BACKGROUND OF THE INVENTION

1. FIELD OF THE INVENTION.

[02] The present invention generally relates to human immunodeficiency virus (HIV).

In particular, the present invention relates to antibodies and antigens of HTV viremia controllers and methods of making and using thereof.

2. DESCRIPTION OF THE RELATED ART.

[03] infection with the human immunodeficiency virus (HIV) generally results in high levels of viremia, gradual destruction of the host immune system, and disease progression leading up to the development of Acquired Immune Deficiency Syndrome (ADDS) about 8 to 10 years after infection. There are three stages of HTV disease progression. The first stage is an asymptomatic stage wherein an infected individual is seropositive for HTV antibodies, but does not exhibit any symptoms of HW-related disease. The second and third stages, AEDS-Related Complex (ARC) and, respectively, are symptomatic and characterized by tumors and opportunistic infections and diseases.

[04] Shortly after HIV infection a vigorous humoral response is initiated. This phase is characterized by elevated levels of circulating antibodies. Specific neutralizing antibodies are directed against the various component proteins of HTV and the initial virus is drastically reduced to levels where it is often difficult to isolate. This point marks the beginning of the disease-free phase of HIV infection with its hallmarks of normal T4 counts and high antibody activity against HTV component proteins. However, despite the presence of cellular and humoral immunity in the infected individual, the virus persists and after several years of latency will become active, often mutating to variant forms, and eventually destroying the immune system leading to full-blown AIDS. [05] Traditional methods of diagnosing H1N infection include serological tests to detect the presence of HIV antibodies and polymerase chain reaction for virus detection. However, there are drawbacks to these traditional methods. Although they confirm the absence or presence of HIV, they do not indicate the stage of disease progression. Subjects entering the symptomatic stages of disease often fail to recognize the onset of symptoms and delay seeking help. Currently there is no effective treatment for H1N- infection. No effective vaccine is presently available. Nucleoside reverse transcriptase inhibitors (NRTIs) such as zidovudine (AZT), didanosine (ddl), zalcitabine (ddC), stavudine (d4T), lamivudine (3TC), Combivir™ (AZT + 3TC), and abacavir (Ziagen™), Protease inhibitors (Pis) such as saquinavir ( ivirase™ & Fortovase™), ritonavir (Norvir™), indinavir (Crixivan®), and nelfinavir (Viracept ®) and non-nucleoside reverse transcriptase inhibitors (NNTRIS) such as nevirapin (Viramune®, delaviridine (Rescriptor®) and efavirenz (Sustiva^TM) and other pharmaceutical compounds can temporarily alleviate symptoms in AIDS patients, but have been unable to stimulate the immune system to clear the virus.

[06] Almost every individual infected with the virus will develop ADDS and die. A small number of individuals (about 5% of those infected), however, sustain very low or undetectable viral loads in the absence of anti-retroviral therapy. These individuals remain disease free, maintain normal CD4+ lymphocyte counts for many years, and are often referred to as long-term non-progressors (LTNP) or viremia controllers. See Buchbinder, et al. (1994) AIDS 8(8): 1123-8; and Harrer, et al. (1996) AIDS Res. Hum. Rexroviruses 12(7):585-924. Additionally, there is another small group of people who remain uninfected after repeated exposure (UARE) to HIV even though they develop a wide range of immune responses to the virus.

[07] Differences in co-receptor expression on host cells or differences in the infecting virus strain may account for viremia control in some individuals and strong HIN-specific cytotoxic T-cell (CTL), and helper T-cell and neutralizing antibody activities have been demonstrated in a number of LTNPs. See Patki, et al. (1996) Cell. Immunol. 169(1):40- 6; Cecilia, et α/.(1999) J. Infect. Dis. 179(6):1365-74; Fowke, et al. (2000) Immunol, and Cell Biol. 78(6):586-95; Brambilla, et al (1999) Virol. 259:349-68; Belts, et al (1999) AIDS Res. and Human Retroviruses 15(13):1219-28; Barker, et al. (1995) PNAS USA 92:11135-9; Martin, et al. (1998) Science 282(5395): 1907-11; Scala, et al. (1999) Immunol. J. 162(10):6155-61. [08] There are, however, some studies, which suggest that the immune responses in

UARE and/or LTNP differ from those of the patients who become infected or once infected progresses to AIDS. See Betts, et al. (1999) ADDS Res. and Human Retro viruses 15(13):1219-28; Cao, et al. (1995) N. Engl. J. Med 332: 201-208; Harrer, et al. (1996) ADDS Res. Hum. Retroviruses 12(7):585-924. The mechanisms leading either to prevention of H1N infection or to non-progression in people UARE and LTΝP individuals are not yet known. The elucidation of distinct immune responses in UARE and LTΝP might be particularly important both in understanding of pathogenesis of HIV and in development of an effective H1N vaccine.

[09] Unfortunately, the type of immune response, which is required to protect the host from either HIV infection or the development of ADDS, is largely unknown and the elements of the mucosal immune system, which block or at least limit entry of pathogens, are not clear yet. Since female genital and homosexual rectal transmission of human immunodeficiency virus are the main routes of HIV infection, stimulation of the mucosal immune system has been suggested to be a requirement for protection from sexually transmitted H1N-1 infection in humans.

[10] Thus, there exists a need for methods of identifying the antigens that elicit the immune mechanisms responsible for UARE and LTΝP, effective treatments and vaccines against HIV, and effective drug delivery systems for HIV vaccines.

SUMMARY OF THE INVENTION

[11] The present invention generally relates to antibodies and antigens of human immunodeficiency virus (HF ) viremia controllers.

[12] In some embodiments, the present invention relates to a purified peptide that has a higher reactivity with sera, plasma, antibodies, or a combination thereof of HIV long-term non-progressors than with sera, plasma, or antibodies of HIV progressors. Li preferred embodiments, the purified peptide of the present invention, wherein the peptide reacts with the sera, plasma, or antibodies of about 14 of 25 viremia controllers, or alternatively, 24 of 25 viremia controllers. In some embodiments, the purified peptide is serologically detected by about 50% to about 100% of viremia controllers.

[13] In some embodiments, the present invention relates to an epitope that is recognized to a greater degree by an antibody obtained from an HIV long-term non- progressor than by an antibody obtained from an HIV progressor. In some embodiments, the epitope is about 55% homologous to an H1N-1 Tat consensus sequence. In other embodiments, the epitope is about 70% homologous to an HTV-1 gp41 consensus sequence.

[14] In some embodiments, the present invention relates to a purified peptide comprising SEQ ID ΝO:l. hi some embodiments, the purified polypeptide consists of SEQ ID NO: 1.

[15] In some embodiments, the present invention relates to a purified peptide comprising SEQ ID NO:2. In some embodiments, the purified polypeptide consists of SEQ ID NO:2.

[16] In some embodiments, the present invention relates to a purified peptide having an amino acid sequence that has about 90% sequence identity to SEQ ID NO:l or SEQ ID NO:2 and exhibits a differential immunoactivity against antibodies obtained from HIV long-term non-progressors as compared with antibodies obtained from HIV progressors.

[17] hi some embodiments, the present invention relates to an immunogenic composition comprising a peptide having SEQ ID NO:l, a peptide having SEQ ID NO:2, or both.

[18] hi some embodiments, the present invention relates to a multivalent vaccine comprising a peptide having SEQ ID NO:l, a peptide having SEQ ID NO:2, or both.

[19] h other embodiments, the present invention relates to a composition comprising at least one antibody that specifically binds with or was raised against a peptide having SEQ DD NO:l, SEQ JJD NO:2, or both.

[20] hi some embodiments, the present invention relates to an assay for at least one epitope characteristic of an HIV long-term non-progressor or a UARE subject comprising biopanning a phage display library to obtain at least one target DNA; creating an expression library with the target DNA; and immunoscreenmg the expression library with a pooled sample obtained from HIV long-term non-progressors by colony lifting. In other embodiments, the assay may be used in a method of diagnosing whether an HIV infected subject is likely to be an H1N long-term non-progressor or an HTV progressor comprising detecting the presence or absence of at least one epitope identified by the assay of the present invention, wherein the presence indicates that the subject is likely to be an HTV long-term non-progressor.

[21] In some embodiments, the present invention relates to a method of treating a subject infected with HIV comprising determining whether the subject is an HTV-long term non-progressor or an HIV progressor and treating the subject according to whether the subject is an HlN-long term non-progressor or an HIV progressor.

[22] In some embodiments, the present invention relates to a method of immunizing a subject comprising administering to the subject at least one peptide of the present invention, hi other embodiments, the present invention relates to a method of passively immunizing a subject comprising administering to the subject at least one antibody that specifically binds or was raised against the peptide of the present invention.

[23] h some embodiments, the present invention relates to a kit comprising at least one purified peptide of the present invention and instructions for use. The kit may further comprise diagnostic reagents, solid supports for assays, devices for delivering the peptide to a subject, and the like.

[24] It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are intended to provide further explanation of the invention as claimed. The accompanying drawings are included to provide a further understanding of the invention and are incorporated in and constitute part of this specification, illustrate several embodiments of the invention and together with the description serve to explain the principles of the invention.

DESCRIPTION OF THE DRAWINGS [25] This invention is further understood by reference to the drawings wherein:

[26] Figure 1 illustrates, with hypothetical data, the colony lifting and differential immunoscreening procedures used to identify clones reactive with viremia controllers. (A) Petri dish; (B) viremia controllers; (C) subjects with progressive disease; and (D) uninfected control subjects. The arrows on the filter paper (B) indicate colonies that react only with plasma from viremia controllers, and the arrows on the Petri dish (A) indicate the original position of those colonies, which can then be amplified for further evaluation. Black filled circles indicate colonies that react by immunostaining with the respective plasma pool. [27] Figure 2 shows ELISA reactivity against HIVp5 (A) or HIVρ2 (B) of serum or plasma from viremia controllers, patients with progressive disease (progressors), patients with acute HIV infection, and uninfected controls. Adjusted optical density (OD) was calculated by dividing the OD of each test plasma or serum by the mean OD + 2 SD of negative control sera/plasma. Each subject's specimen was assayed two to five times in independent experiments, and the mean of each subject's adjusted OD is depicted. Adjusted OD's >1 (shown above the horizontal dotted line) are considered positive. The horizontal bars represent the median adjusted OD for each group of patients. P values refer to the difference in adjusted OD values between viremia controllers and progressors (Kruskal-Wallis test).

[28] Figure 3 provides amino acid sequence homologies of peptide HIVp5 to Tat (3 A) and HIVp2 to gp41 (3B). Shown are the amino acid sequences of HIVp5 and HIVp2 and the respective homologous amino acids (in bold) from consensus sequences of each major HIN-1 subtype (A, B, C, D, etc.), "x" indicates lack of consensus. Amino acid sequences of HFVp5 and HIVp2 peptides were derived from nucleotide sequences.

[29] Figure 4 shows serum antibody responses to synthetic Tat protein from rabbits before and after immunization with HIVp5. Bound antibodies were detected by ELISA. Each experiment was performed in triplicate, and data are shown as OD at 405nm.

DETAILED DESCRIPTION OF THE INVENTION

[30] The present invention is directed to a method for identifying peptides that are serologically reactive with samples from HlV-infected subjects who control viremia (LTNP or viremia controllers) at a greater frequency and binding affinity than samples from subjects with progressive HIV disease (HIV progressors). Additionally, the present invention is directed to compositions and methods for using the compositions in the diagnosis, treatment, and prevention of HIV infection, ADDS, or both. The compositions of the present invention include peptides that are serologically reactive with samples from viremia controllers at a greater frequency and binding affinity than samples from HIV progressors, polynucleotides encoding such polypeptides, binding agents such as antibodies, antigen presenting cells (APCs), immune system cells (e.g., T cells), and combinations thereof.

[31] Peptide display libraries provide a powerful way to study immunological responses during virus infection. See Scala, et al. (1999) Journal of Immunology 162(10):6155-6161; and Prezzi, et al. (1996) Journal oflmmunology 156(11):4504-4513, which are herein incorporated by reference. A previous study using peptide libraries composed of random nonamers displayed on filamentous phages demonstrated that HIN- 1 -specific epitopes or epitope mimics could be selected using polyclonal sera from H1N- infected individuals. See Scala, et al. (1999) Journal oflmmunology 162(10):6155-6161, which is herein incorporated by reference. Prior art assays, however, have not used phage displayed peptide libraries to characterize immune responses that differentiate two clinically distinct groups of HlN-infected patients. Thus, the present invention also provides methods for identifying and selecting immunogenic epitopes that are differentially recognized by a given clinical group of HlV-infected subjects which comprise panning with a peptide display library and differential immunoscreenmg.

[32] The methods of the present invention comprise screening random peptide libraries displayed on phages to identify peptides that react specifically with antibodies of viremia controllers but not with or to a lesser extent to antibodies of HIV progressors. Generally, magnetic beads coated with anti-human IgG are incubated with sera from viremia controllers. Then "panning" (alternatively "biopanning") is conducted by exposing the magnetic beads to a phage-displayed random peptide library, such as pDI proteins of Ml 3 phages, after which any unbound phage is washed away. Phages bound to the antibodies on the magnetic beads are eluted. The eluted phages are then amplified by infection of a suitable host, such as E. coli. Then the whole process is repeated twice. Phage DΝA containing the peptide sequence identified from the third round of biopanning is amplified by PCR, cloned and expressed using a suitable expression system, such as a PET32b vector in E. coli, as a pooled library. Then the expressed peptide library is subject to specific screening by immunocolony lifting assay using antibodies from viremia controllers, HIV progressors, and uninfected controls. The clones which react specifically with antibodies from viremia controllers are selected and amplified for further characterization.

[33] The methods of the present invention may be used to identify immunogenic epitopes that are recognized by antibodies of viremia controllers to a much lesser extent or not at all by antibodies in the sera or plasma of either HIV infected subjects, HIV progressors, or both. Alternatively, samples from UARE subjects may be used to identify immunogenic epitopes that are recognized by antibodies of UARE subjects to a much lesser extent or not at all by antibodies in the sera or plasma of HIV infected subjects, h order to identify IgA specific epitopes in UARE subjects, the method of either Example 1 or Example 2 may be modified by using magnetic beads coated with anti-human IgA and the vaginal lavage from obtained from UARE subjects.

[34] It should be noted that the methods of the present invention are not limited to HIV epitopes, but may be used to identify immunogenic epitopes that play a crucial role in controlling the viral loads in individuals infected with other viruses, such as hepatitis C, human papilloma virus, and the like. As compared to conventional methods, the panning and differential immunoscreening of the present invention is time efficient and economical. The method of the present invention allows analysis of up to 109 sequences with only a few microliters of sample.

[35] The present invention also provides peptides that are serologically reactive with samples from viremia controllers at a greater frequency and binding affinity than sera from HIV progressors. The peptides that are serologically reactive with samples from viremia controllers at a greater frequency and binding affinity than samples from HIV progressors are referred to as "LTNP" peptides. As described herein, a random peptide library combined with differential immunoscreening was used to investigate differences in serological reactivity between viremia controllers and HIV progressors. From about 2 x 10⁹ phages, 7 clones were identified that reacted on initial immunoscreening exclusively with pooled plasma samples from viremia controllers. One of the 7 clones, HTVp5, was serologically recognized by about 56% of the viremia controllers and by only about 11%) of subjects with progressive infection. A second clone, HIVp2, was serologically recognized by the majority of subjects in both groups, but with greater frequency and stronger binding by the viremia controllers. Thus, the LTNP peptides of the present invention include the peptides designated HIVp2 (SEQ DD NO:l) and HIVp5 (SEQ DD NO:2) and variants thereof.

[36] As used herein, "HIVp2" peptides have an amino acid sequence (1) set forth in

SEQ DD NO:l, (2) that has substantial identity to the sequence set forth in SEQ DD NO:l, or (3) variants thereof. HIVp2 polypeptides are differentially recognized by antibodies of viremia controllers as compared to antibodies of HIV progressors. As used herein, "HlNp5" peptides have an amino acid sequence (1) set forth in SEQ DD ΝO:2, (2) that has substantial identity to the sequence set forth in SEQ DD NO:2, or (3) variants thereof. HIVp5 polypeptides are differentially recognized by antibodies of viremia controllers as compared to antibodies of HIV progressors. Whether a polypeptide is differentially recognized by antibodies of a viremia controllers as compared to antibodies of HIV progressors, may be determined by the methods disclosed herein or other methods known in the art.

[37] The present invention also provides polynucleotides that encode LTNP peptides

(LTNP polynucleotides) and variants thereof, preferred embodiments the polynucleotides are isolated. As used herein, an "isolated" polynucleotide refers to a DNA molecule that is isolated from its native environment. An "isolated" polynucleotide may be substantially isolated away from, or purified free from, total genomic DNA of the species from which the DNA segment is obtained. An "isolated" polynucleotide may include a DNA segment that is separated from other DNA segments with which is normally or natively associated at either the 5' end, 3' end, or both. An "isolated" polynucleotide may include a DNA segment that is substantially away from other coding sequences, and does not contain large portions of unrelated coding DNA, such as large chromosomal fragments or other functional genes or amino acid coding regions. "Nucleic acid sequence", "nucleic acid molecule", and "polynucleotide" are used interchangeably to refer to an oligonucleotide, nucleotide, or polynucleotide, and fragments or portions thereof, and to DNA or RNA of genomic or synthetic origin and may be single- or double- stranded, and represent the sense or antisense strand.

[38] The polynucleotides of the present invention may include genomic sequences, extra-genomic and plasmid-encoded sequences and smaller engineered gene segments that express, or may be adapted to express, proteins, polypeptides, peptides, and the like. The polynucleotides of the present invention may be in its native form or synthetically modified. The polynucleotides of the present invention maybe single-stranded (coding or antisense) or double-stranded, and may be DNA (genomic, cDNA or synthetic) or RNA molecules. RNA molecules include mRNA molecules, which contain introns and correspond to a DNA molecule in a one-to-one manner, and mRNA molecules, which do not contain introns. Additional coding or non-coding sequences may be present within a polynucleotide of the present invention, and a polynucleotide may be linked to other molecules, support materials, or both.

[39] The polynucleotides of the present invention may comprise a native sequence, i. e. , an endogenous sequence that encodes an LTNP peptide or a portion thereof, or may comprise a variant or a biological or antigenic funct onal equivalent thereof. Polynucleotide variants may contain one or more substitutions, additions, deletions, insertions, or combinations thereof so long as the biological activity, such as immunogenicity, of the encoded polypeptide is not diminished, relative to the LTNP polypeptide. A "variant" of a polynucleotide refers to the chemical modification of the LTNP polynucleotide or the encoded LTNP peptide. Examples of chemical modifications include replacements of hydrogen by an alkyl, acyl, or amino group. A variant of an LTNP polynucleotide encodes a polypeptide that is differentially recognized by antibodies of viremia controllers as compared to antibodies of HIV progressors.

[40] The polynucleotides of the present invention, or fragments thereof, regardless of the length of the coding sequence itself, maybe combined with other DNA sequences, such as promoters, polyadenylation signals, additional restriction enzyme sites, multiple cloning sites, other coding segments, and the like, such that their overall length may vary considerably. It is therefore contemplated that a nucleic acid fragment of almost any length may be employed, with the total length preferably being limited by the ease of preparation and use in the intended recombinant DNA protocol.

[41] h other embodiments, the present invention is directed to polynucleotides that are capable of hybridizing under moderately stringent conditions to a polynucleotide that encodes a polypeptide of the present invention. Hybridization techniques are well known in the art. As used herein, "stringent conditions" refers to the "stringency" which occurs within a range from about 5 °C below the melting temperature (Tm) of the probe to about 20 °C to about 25 °C below Tm. As will be understood by those of skill in the art, the stringency of hybridization may be altered in order to identify or detect identical or related polynucleotide sequences. For purposes of illustration, suitable moderately stringent conditions for testing the hybridization of a polynucleotide of this invention with other polynucleotides include prewashing in a solution of 5X SSC, 0.5% SDS, 1.0 mM EDTA (pH 8.0); hybridizing at about 50 °C to about 65 °C, 5X SSC, overnight; followed by washing twice at about 65 °C for 20 minutes with each of 2X, 0.5X and 0.2X SSC containing 0.1% SDS.

[42] Moreover, it will be appreciated by those of ordinary skill in the art that, as a result of the degeneracy of the genetic code, there are many nucleotide sequences that encode a polypeptide as described herein. Some of these polynucleotides bear minimal homology to the nucleotide sequence of any native gene. Nonetheless, polynucleotides that vary due to differences in codon usage are specifically contemplated by the present invention. Further, alleles of the genes comprising the polynucleotide sequences provided herein are within the scope of the present invention. Alleles are endogenous genes that are altered as a result of one or more mutations, such as deletions, additions and/or substitutions of nucleotides. The resulting mRNA and protein may, but need not, have an altered structure or function. Alleles may be identified using standard techniques (such as hybridization, amplification and/or database sequence comparison). [43] A polynucleotide that encodes a polypeptide having substantial identity to either

SEQ DD NO:l or SEQ DD NO:2 can be made by introducing one or more nucleotide substitutions, insertions, or deletions into the nucleotide sequence that encodes SEQ DD NO:l or SEQ DD NO:2 such that one or more amino acid substitutions, insertions, or deletions are introduced into the encoded polypeptide. Mutations can be introduced by standard techniques, such as site-directed mutagenesis and PCR-mediated mutagenesis.

[44] h other embodiments of the present invention, the polynucleotide sequences provided herein can be used as probes or primers for nucleic acid hybridization. As such, nucleic acid segments that comprise a sequence region of at least about a 15 nucleotide long contiguous sequence that has the same sequence as, or is complementary to, a 15 nucleotide long contiguous sequence disclosed herein are contemplated. Longer contiguous identical or complementary sequences up to full-length sequences are also contemplated.

[45] The ability of such nucleic acid probes to specifically hybridize to a sequence of interest will enable them to be of use in detecting the presence of complementary sequences in a given sample. However, other uses are also envisioned, such as the use of the sequence information for the preparation of mutant species primers, or primers for use in preparing other genetic constructions. The polynucleotides that encode SEQ DD NO:l and SEQ DD NO:2 are particularly contemplated as hybridization probes in recombinant DNA and molecular biology techniques. The total size of fragment, as well as the size of the complementary sequence, will ultimately depend on the intended use or application of the particular nucleic acid segment. Smaller fragments will generally find use in hybridization embodiments, wherein the length of the contiguous complementary region maybe varied, such as between about 15 and about 100 nucleotides, but larger contiguous complementarily stretches may be used. Small polynucleotide fragments of the present invention may be readily prepared by conventional methods known in the art, for example, directly synthesizing the fragment with an automated oligonucleotide synthesizer, PCR technology, and recombinant DNA techniques. The polynucleotides of the present invention may be identified, prepared, amplified, or manipulated by conventional methods known in the art.

[46] "Amino acid sequence", "amino acid molecule", "polypeptide", "protein", and

"peptide", are used interchangeably to refer to an oligopeptide, peptide, polypeptide, or protein sequence, and fragments or portions thereof, and to naturally occurring or synthetic molecules. Polypeptides of the present invention may be of any length. Additional sequences derived from the native protein and or heterologous sequences may be present, and such sequences may possess further immunogenic or antigenic properties.

[47] As used herein an "immunogenic portion" is a portion of a protein that is recognized by a B-cell and/or T-cell surface antigen receptor. An immunogenic portion of the present invention includes at least one antigenic epitope that is differentially recognized by antibodies of viremia controllers as compared to antibodies of HIV progressors. Examples of such immunogenic portions include HIVp2 (SEQ DD NO:l), HlNp5 (SEQ DD ΝO:2), and variants thereof. Variants of HTvp2 or HIVp5 are polypeptides that exhibit a similar sequence homology and bioactivity (inimunogenicity) to HIVp2 (SEQ DD ΝO:l) or HIVp5 (SEQ DD ΝO:2). Immunogenic portions maybe identified using the methods described herein or by methods known in the art. See Paul, Fundamental Immunology, 3rd ed., 243-247 (Raven Press, 1993) and references cited therein, which are herein incorporated by reference. The terms "antigenic determinant" or "antigenic epitope" or "epitope" are used interchangeably to refer to a region of a molecule with the ability or potential to elicit, and combine with, an antibody or fragment thereof.

[ 81 The polypeptides of the present invention need not be identical to those exemplified herein so long as the subject polypeptides are able to induce an immune response against the antigenic epitope or immunogenic portion of the present invention. Thus, as used herein "variants" of the polypeptides of the present invention refer to polypeptides having insignificant changes. "Insignificant changes" refer to modifications in the amino acid sequence of a given polypeptide that do not change the biological activity, such as the immunological activity, of the polypeptide. Such insignificant changes include a methionine as the first amino acid residue at the amino terminus, conservative amino acid substitutions, deletions, or insertions, and co-translational or post-translational surface modifications such as the addition of covalently attached sugars, lipids, or combinations thereof.

[49] Examples of such variants include those wherein at least one amino acid residue of SEQ DD NO:l, SEQ DD NO:2, or SEQ DD NO:7 is replaced with an amino acid residue that does not change the immunogenic activity of a polypeptide having SEQ DD NO:l or SEQ DD NO:2 and fusion proteins comprising a polypeptide having SEQ DD NO:l or SEQ DD NO: 2 and an amino acid molecule such as an HIV core protein or fragment thereof. [50] Polypeptide "variants" of the present invention also include a polypeptides that differs from HIVp2 (SEQ DD NO: 1) or ffiVp5 (SEQ DD NO:2) by one or more substitutions, deletions, additions, insertions, or a combination thereof such that the immunogenicity of the polypeptide is not substantially diminished. For example, the ability of a variant to react with antibodies specific for HIVp2 (SEQ DD NO: 1) or HIVp5 (SEQ DD NO:2) may be enhanced or unchanged, relative to the native protein, or may be diminished by less than 50%), and preferably less than 20%, relative to the native protein. "Immunogenicity" refers to the capability of the natural, recombinant, or synthetic peptide, or any ohgopeptide thereof, to induce a specific immune response in appropriate animals or cells and to bind with specific antibodies.

[51] The variant may have "conservative" changes, wherein a substituted amino acid has similar structural or chemical properties, e.g. replacement of leucine with isoleucine. More rarely, a variant may have "nonconservative" changes, e.g. , replacement of a glycine with a tryptophan. Similar minor variations may also include amino acid deletions, insertions, or both. Guidance in determining which amino acid residues may be substituted, inserted, or deleted without abolishing biological or immunological activity may be found using computer programs well known in the art, for example, LASERGENE software. Polypeptide variants encompassed by the present invention include those exhibiting at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more identity to the polypeptides disclosed herein.

[52] The polypeptides of the present invention may also be modified to provide a variety of desired attributes, e.g., improved pharmacological characteristics, while increasing or at least retaining substantially all of the immunological activity of the antigenic epitope. By using conventional methods in the art, one of ordinary skill will be readily able to make a variety of polypeptides having substantial identity to those explicitly provided for herein, and then screen the polypeptides for stability, toxicity, and immunogenicity according to the present invention.

[53] Additionally, single amino acid substitutions, deletions, or insertions can be used to determine which residues are relatively insensitive to modification. Amino acid substitutions are preferably made between relatively neutral moieties, such as alanine, glycine, proline, and the like. Substitutions with different amino acids, of either D or L isomeric forms, or amino acid mimetics can be made. The number and types of substitutions, deletions, and insertions depend on the functional attributes that are sought such as hydrophobicity, immunogenicity, three-dimensional structure, and the like.

[54] An "amino acid mimetic" as used herein refers to a moiety other than a naturally occurring amino acid residue that conformationally and functionally serves as a suitable substitute for an amino acid residue in a polypeptide of the present invention. A moiety is a suitable substitute for an amino acid residue if it does not interfere with the ability of the peptide to elicit an immune response against the polypeptide of the present invention. Examples of amino acid mimetics include cyclohexylalanine, 3-cyclohexylpropionic acid, L-adamantyl alanine, adamantylacetic acid, and the like. See e.g. Morgan and Gainor, (1989) Ann. Repts. Med. Chem. 24:243-252, which is herein incorporated by reference.

[55] Individual amino acid residues may be incorporated in the polypeptides of the present invention with peptide bonds or peptide bond mimetics. "Peptide bond mimetics" include peptide backbone modifications of the amide nitrogen, the α-carbon, amide carbonyl, complete replacement of the amide bond, extensions, deletions or backbone crosslinks. See e.g. Spatola (1983) Chemistry and Biochemistry of Amino Acids, Peptides and Proteins, Vol. VD, Weinstein ed., which is herein incorporated by reference. The polypeptides of the present invention may include an additional methionine as the first amino acid residue on the protein amino terminus. The polypeptides may be truncated or contain co-translational or post-translational surface modifications, such as the addition of covalently attached sugars or lipids.

[56] In preferred embodiments, the polypeptides of the present invention have a substantial sequence identity to the amino acid sequence set forth in SEQ DD NO:l or SEQ DD NO:2. As used herein "sequence identity" means that two sequences are identical over a window of comparison. The percentage of sequence identity is calculated by comparing two optimally aligned sequences over the window of comparison, determining the number of positions at which the identical residues occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison (i.e., the window size), and multiplying the result by 100 to yield the percentage of sequence identity.

[57] A "conservative amino acid substitution" is one in which the amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta- branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan).

[58] The polypeptides of the present invention may comprise a signal or leader sequence at the N-terminal end of the polypeptide, which co-translationally or post- translationally directs transfer of the protein. The polypeptide may also be conjugated to a linker or other sequence for ease of synthesis, purification or identification of the polypeptide such as a histidine tag, or to enhance binding of the polypeptide to a solid support. For example, a polypeptide may be conjugated to an immunoglobulin Fc region.

[59] The polypeptides of the present invention and fragments thereof may be made by conventional methods known in the art. The polypeptides of the present invention may be manually or synthetically synthesized using conventional methods and devices known in the art. See e.g., Stewart and Young (1984) Solid Phase Peptide Synthesis, 2 ed. Pierce, Rockford, IL; Merrifield, (1963) J. Am. Chem. Soc. 85:2149-2146, which are herein incorporated by reference. Equipment for automated synthesis of polypeptides is commercially available from suppliers such as Perkin Elmer/Applied BioSystems Division (Foster City, CA), and maybe operated according to the manufacturer's instructions. The composition of the synthetic peptides may be confirmed by conventional methods in the art, such as amino acid analysis or sequencing.

[60] The polypeptides of the present invention may be purified from natural sources using conventional protein purification techniques such as reverse phase high- performance liquid chromatography (HPLC), ion-exchange or immunoaffinity chromatography, filtration or size exclusion, or electrophoresis. See e.g., Scopes (1982) Protein Purification, Springer- Verlag, NY, which is herein incorporated by reference.

[61] In some embodiments, the polypeptides of the present invention may be substantially purified. As used herein, a "purified" polypeptide means that the polypeptide is not in its native state or natural environment of which it is naturally associated. As used herein, a "substantially purified" compound refers to a compound that is removed from its native environment and is at least about 60% free, preferably about 75% free, and most preferably about 90% free from other macromolecular components with which the compound is naturally associated. A polypeptide of the present invention may be substantially purified by preparative high performance liquid chromatography or other comparable techniques available in the art. See e.g., Creighton, T. (1983) Proteins, Structures and Molecular Principles, WH Freeman and Co., New York, which is herein incorporated by reference.

[62] h other embodiments of the invention, polynucleotide sequences or fragments thereof which encode LTNP polypeptides, or fusion proteins or variants thereof, may be used in recombinant DNA molecules to direct expression of the polypeptide in appropriate host cells. Due to the inherent degeneracy of the genetic code, other DNA sequences that encode substantially the same or a functionally equivalent amino acid sequence may be produced and these sequences may be used to clone and express a given polypeptide.

[63] As will be understood by those of skill in the art, it may be advantageous in some instances to produce polypeptide-encoding nucleotide sequences possessing non-naturally occurring codons. For example, codons preferred by a particular prokaryotic or eukaryotic host can be selected to increase the rate of protein expression or to produce a recombinant RNA transcript having desirable properties, such as a half-life that is longer than that of a transcript generated from the naturally occurring sequence.

[64] Sequences encoding a desired polypeptide may be synthesized, in whole or in part, using conventional methods known in the art. See Caruthers, M.H., et al. (1980) Nucl. Acids Res. Symp. Ser. 215-223; Horn, T., et al. (1980) Nucl. Acids Res. Symp. Ser. 225-232; and Roberge, J.Y., et al. (1995) Science 269:202-204, which are herein incorporated by reference. Commercially available automated synthesizers, such as ABI 431 A Peptide Synthesizer (Perkin Elmer), may be used. Moreover, the polynucleotide sequences of the present invention can be engineered using methods generally known in the art in order to alter polypeptide encoding sequences which modify the cloning, processing, or expression of the gene product, or a combination thereof. For example, DNA shuffling by random fragmentation and PCR reassembly of gene fragments and synthetic oligonucleotides may be used to engineer the nucleotide sequences. In addition, site-directed mutagenesis may be used to insert new restriction sites, alter glycosylation patterns, change codon preference, produce splice variants, introduce mutations, and the like.

[65] In some embodiments, the polypeptide may be a fusion protein that comprises multiple polypeptides as described herein, or that comprises at least one polypeptide as described herein and an unrelated sequence, such as a known HIV protein such as tat or env. Fusion proteins may generally be prepared using standard techniques, including recombinant techniques and chemical conjugation. A fusion protein may also be engineered to contain a cleavage site located between the polypeptide-encoding sequence and the heterologous protein sequence, so that the polypeptide may be cleaved and purified away from the heterologous moiety. A fusion partner may, for example, assist in providing T helper epitopes (an immunological fusion partner), preferably T helper epitopes recognized by humans, or may assist in expressing the polypeptide (an expression enhancer) at higher yields than the native recombinant protein. Certain preferred fusion partners are both immunological and expression enhancing fusion partners. Other fusion partners may be selected so as to increase the solubility of the polypeptide or to enable the polypeptide to be targeted to desired intracellular compartments. Still further fusion partners include affinity tags, which facilitate purification of the polypeptide.

[66] A peptide linker sequence may be employed to separate the first and second polypeptide components by a distance sufficient to ensure that each polypeptide folds into its secondary and tertiary structures. Such a peptide linker sequence is incorporated into the fusion protein using standard techniques well known in the art. Suitable peptide linker sequences may be chosen based on the following factors: (1) their ability to adopt a flexible extended conformation; (2) their inability to adopt a secondary structure that could interact with functional epitopes on the first and second polypeptides; and (3) the lack of hydrophobic or charged residues that might react with the polypeptide functional epitopes. Preferred peptide linker sequences contain Gly, Asn and Ser residues. Other near neutral amino acids, such as Thr and Ala may also be used in the linker sequence. Amino acid sequences which may be usefully employed as linkers include those disclosed in Maratea, et al. (1985) Gene 40:39-46; Murphy, et al. (1986) PNAS USA 83:8258- 8262; U.S. Pat. No. 4,935,233 and U.S. Pat. No. 4,751,180. The linker sequence may generally be from 1 to about 50 amino acids in length. Linker sequences are not required when the first and second polypeptides have non-essential N-terminal amino acid regions that can be used to separate the functional domains and prevent steric interference.

[67] In order to express a polypeptide of the present invention, the nucleotide sequence encoding the polypeptide, may be inserted into a suitable expression vector, expression system, or cell and then induced to express the polypeptide by methods known in the art. Either the expression vector or the host may comprise the regulatory sequences necessary for expression of the polypeptide. Where the regulatory sequences are within the expression vector, the regulatory sequences are operatively linked to the sequence encoding the polypeptide. As used herein, "operably linked" means that the nucleotide sequence of interest is linked to at least one regulatory sequence in a manner that allows the polypeptide to be expressed in an in vitro transcription/translation system or in a host cell. Regulatory sequences include promoters, enhancers and other expression control elements (e.g., polyadenylation signals). See e.g., Goeddel (1990) Gene Expression Technology: Methods in Enzymology, Academic Press, San Diego, CA, which is herein incorporated by reference. It will be appreciated by those skilled in the art that the design of the expression vector can depend on such factors as the choice of the host cell to be transformed, the desired expression levels of the polypeptide, the compatibility of the host cell and the expressed polypeptide, and the like.

[68] The vectors can be designed for expressing the polypeptides of the present invention in prokaryotic or eukaryotic host cells such as bacterial cells, insect cells, plant cells, yeast cells, or mammalian cells. In preferred embodiments, the host cells are bacterial cells. Suitable host cells are discussed further in Goeddel supra; Baldari, et al. (1987) EMBO J. 6:229-234; Kurjan and Herskowitz (1982) Cell 30:933-943; Schultz, et al. (1987) Gene 54:113-123; Smith, et al. (1983) Mol. Cell Biol. 3:2156-2165; Lucklow and Summers (1989) Virology 170:31-39; Seed (1987) Nature 329:840; Kaufman, et al. (1987) EMBO J. 6:187-6195; Sambrook, et al. (2000) Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, all of which are herein incorporated by reference.

[69] Thus, the present invention also provides host cells comprising polynucleotides that encode the polypeptides of the present invention. Host cells include the progeny or potential progeny of the primary cell in which the polynucleotide was introduced. Because certain modifications may occur in succeeding generations due to either mutation or environmental influences, such progeny may not, in fact, be identical to the parent cell, but are still included within the scope and meaning of host cell.

[70] The present invention also provides antibodies and fragments specific for the polypeptides, polynucleotides, and fragments thereof of the present invention and compositions comprising such. A polypeptide of the present invention may be used to prepare antibodies specific for HIV by immunizing a suitable subject, e.g., rabbit, goat, mouse or other mammal with the polypeptide by conventional methods known in the art. Large quantities of neutralizing antibodies could be generated and then used in immunotherapies for treating, inhibiting or preventing HIV infection, disease progression, or ADDS by methods known in the art. The antibodies raised against the polypeptides of the present invention may be used to treat, inhibit, or prevent HIV and ADDS by providing passive immunity or by creating immunotoxic or antiviral compositions that are targeted to HTV, HIV infected cells, or both.

[71] Antibodies of the present invention may be produced by conventional methods known in the art. See e.g., Coligan (1991) Current Protocols in Immunology. Wiley/Greene, NY; and Harlow and Lane (1989) Antibodies: A Laboratory Manual, Cold Spring Harbor Press, NY; Stites, et al. (1986) Basic and Clinical Immunology. 4th ed. Lange Medical Publications, Los Altos, CA; Goding (1986) Monoclonal Antibodies: Principles and Practice. 2d ed. Academic Press, New York, NY; and Kohler and Milstein (1975) Nature 256:495-497, which are herein incorporated by reference. Therapeutic antibodies may be produced specifically for clinical use in humans by conventional methods known in the art. See Chadd, H.E. and S.M. Chamow (2001) Curr. Opin. Biotechnol. 12:188-194 and references therein, all of which are herein incorporated by reference.

[72] As used herein, "antibody" refers to immunoglobulin molecules and immunologically active fragments that comprise an antigen binding site which specifically binds an antigen, such as HIVρ2 (SEQ DD NO:l) or HJVp5 (SEQ DD NO:2). Examples of immunologically active fragments of immunoglobulin molecules include F(ab) and F(ab')2 fragments which may be generated by treating the antibody with an enzyme such as pepsin. Polyclonal and monoclonal antibodies against the polypeptides of the present invention may be made by conventional methods known in the art.

[73] The present invention also provides binding agents, including antibodies and antigen-binding fragments thereof, which specifically bind to the polypeptide of the present invention. As used herein, an antibody, or antigen-binding fragment thereof "specifically binds" the polypeptides of the present invention if the reaction is detectable with convention assay methods known in the art and reactions, if any, with unrelated proteins under similar conditions are not detectable. As used herein, "binding" refers to a noncovalent association between two separate molecules such that a complex is formed. The ability to bind may be evaluated by, for example, determining a binding constant for the fonnation of the complex using methods known in the art.

[74] Binding agents may be used to differentiate LTNP subjects and/or UARE subjects, from HIV progressors, using the assays provided herein and known in the art. Binding agents that indicate that a subject is an LTNP or an UARE subject will bind to a target antigen to generate a signal that is greater than a signal, if any, of a an HIV progressor and/or an uninfected control subject. To determine whether a binding agent satisfies this requirement, biological samples from HIV infected and uninfected subjects, as determined using standard clinical tests, may be assayed. Binding agents may be used alone or in combination to improve sensitivity or selectivity.

[75] In preferred embodiments, the binding agent is an antibody or an antigen-binding fragment thereof that is specific for the HIVp2, HIVp5, or both, and fragments thereof. Antibodies and fragments thereof may be prepared by any of a variety of techniques known to those of ordinary skill in the art. See e.g., Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, 1988. Monoclonal antibodies specific for an antigenic polypeptide of interest may be prepared, for example, using the technique of Kohler and Milstein (1976) Eur. J. Immunol. 6:511-519, and improvements thereto, which are herein incorporated by reference.

[76] Antibodies of the present invention may be coupled to one or more therapeutic agents, such as drugs, toxins, and the like. Suitable drugs include compounds that are used to treat subjects suffering from ADDS such as nucleoside reverse transcriptase inhibitors (NRTIs) such as zidovudine (AZT), didanosine (ddl), zalcitabine (ddC), stavudine (d4T), lamivudine (3TC), Combivir™ (AZT + 3TC), and abacavir (Ziagen™), Protease inhibitors (Pis) such as saquinavir (h virase™ & Fortovase™), ritonavir (Norvir™), indinavir (Crixivan®), and nelfinavir (Viracept ®) and non-nucleoside reverse transcriptase inhibitors (NNTRIS) such as nevirapin (Viramune®, delaviridine (Rescriptor®) and efavirenz (Sustiva™⁾ and other pharmaceutical compounds can temporarily alleviate symptoms in ADDS patients, and the like. Suitable toxins include ricin, abrin, diptheria toxin, cholera toxin, gelonin, Pseudomonas exotoxin, Shigella toxin, and pokeweed antiviral protein. The therapeutic agent may be coupled (e.g., covalently bonded) to the antibody either directly or indirectly. In some embodiments multiple molecules of a therapeutic agent are coupled to one antibody molecule. In some embodiments, more than one type of agent may be coupled to one antibody. [77] A linker group may be used to couple the therapeutic agent and the antibody. A linker group can function as a spacer to distance an antibody from an agent in order to avoid interference with binding capabilities. A linker group can also serve to increase the chemical reactivity of a substituent on an agent or an antibody, and thus increase the coupling efficiency. A variety of bifunctional or polyfunctional reagents, both homo- and hetero-functional (such as those described in the catalog of the Pierce Chemical Co., Rockford, D ), may be employed as the linker group. Coupling may be effected, for example, through amino groups, carboxyl groups, sulfhydryl groups or oxidized carbohydrate residues using conventional methods known in the art. Where a therapeutic agent is more potent when free from the antibody portion of the immunoconjugates of the present invention, it may be desirable to use a linker group that is cleavable during or upon internalization into a cell. A number of different cleavable linker groups are known in the art. Carriers may be used to couple the binding agent to the therapeutic agent or encapsulate the agents together. Suitable carriers include proteins such as albumins, peptides and polysaccharides such as aminodextran, and the like.

[78] The compositions of the present invention also include T cells specific for the

LTNP polypeptides of the present invention. The T cells may be prepared by conventional methods known in the art. The T cells may be stimulated with an LTNP polypeptide, LTNP polynucleotide, an antigen presenting cell (APC) that expresses the LTNP polypeptide, or a combination hereof. APCs may be transfected ex vivo or in vivo with a polynucleotide of the present invention such that the polypeptide, or an immunogenic portion thereof, is expressed on the cell surface by conventional methods known in the art. See e.g. WO 97/24447; and Mahvi, et al. (1997) Immunol. Cell Biol. 75:456-460, which are herein incorporated by reference. Stimulation is performed under conditions and for a time sufficient to permit the generation of T cells that are specific for the LTNP polypeptide. T cells are considered to be specific for the polypeptides of the present invention if the T cells specifically proliferate, secrete cytokines or kill target cells coated with the polypeptide or expressing a gene encoding the polypeptide. T cell specificity may be evaluated using any of a variety of conventional techniques.

[79] The polypeptides, polynucleotides, antibodies, or fragments thereof of the present invention may be used as an active agent in pharmaceutical compositions used to treat, prevent, inhibit, or modulate HIV infection, HIV disease progression, or ADDS. Preferred pharmaceutical compositions are those comprising at least one polypeptide, polynucleotide, antibody, or fragment thereof of the present invention in a therapeutically effective amount, and a pharmaceutically acceptable vehicle. Supplementary active agents can also be incorporated into the compositions. Suitable supplementary active agents compounds that are used to treat subjects suffering from ADDS such as nucleoside reverse transcriptase inhibitors (NRTIs) such as zidovudine (AZT), didanosine (ddl), zalcitabine (ddC), stavudine (d4T), lamivudine (3TC), Combivir™ (AZT + 3TC), and abacavir (Ziagen™), Protease inhibitors (Pis) such as saquinavir (Invirase™ & Fortovase ), ritonavir (Norvir ), indinavir (Crixivan®), and nelfinavir (Viracept ®) and non-nucleoside reverse transcriptase inhibitors (NNTRIS) such as nevirapin (Viramune®, delaviridine (Rescriptor®) and efavirenz (Sustiva™⁾ and other pharmaceutical compounds can temporarily alleviate symptoms in ADDS patients, and the like. The pharmaceutical compositions of the present mvention may also include an adjuvant. As used herein, an "adjuvant" refers to any substance which, when administered with or before the polypeptide, polynucleotide, or antibody of the present invention, aids the polypeptide, polynucleotide, or antibody in its mechanism of action.

[80] As used herein, "vehicle" and "carrier" are used interchangably to indicate any and all solvents, dispersion media, vehicles, coatings, diluents, antibacterial and antifungal agents, isotonic and absorption delaying agents, buffers, carrier solutions, suspensions, colloids, and the like. As used herein, "pharmaceutically acceptable" vehicle or carrier is intended to include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration. The use of such media and agents for pharmaceutical active substances is well known in the art. See e.g. Remington: The Science and Practice of Pharmacy. 20th ed, (2000) Lippincott Williams & Wilkins. Baltimore, MD, which is herein incorporated by reference. Except insofar as any conventional media or agent is incompatible with the active ingredient, its use in the therapeutic compositions is contemplated. Pharmaceutical carriers are preferably biocompatible, and may also be biodegradable.

[81] The compositions described herein may be administered as part of a sustained release formulation that provides a slow release of the active agent following administration. Time-delay or time-release material is known in the art and includes glyceryl monostearate or glyceryl distearate alone or with a wax, ethylcellulose, hydroxypropylmethylcellulose, methylmethacrylate, and the like. The use of such media and agents for pharmaceutically active substances is well known in the art. Likewise, the sustained release formulations may be prepared by conventional methods in the art. See e.g. Coombes et al. (1996) Vaccine 14:1429-1438, which is herein incorporated by reference. The sustained-release formulations may contain a polypeptide, polynucleotide or antibody dispersed in a carrier matrix, contained within a reservoir surrounded by a rate controlling membrane, or a combination thereof. Other delayed-release vehicles include supramolecular biovectors, which comprise a non-liquid hydrophilic core and, optionally, an external layer comprising an amphiphilic compound, such as a phospholipid. The amount of active agent contained within a sustained release formulation depends upon the site of implantation, the rate and expected duration of release and the nature of the condition to be treated or prevented.

[82] The pharmaceutical compositions of the present invention may include pharmaceutically acceptable salts, multimeric forms, prodrugs, active metabolites, precursors and salts of such metabolites of the polypeptides, polynucleotides, antibodies, or fragments thereof described herein.

[83] The term "pharmaceutically acceptable salts" refers to salt forms that are pharmacologically acceptable and substantially non-toxic to the subject being treated with the compound of the invention. Pharmaceutically acceptable salts include conventional acid-addition salts or base-addition salts formed from suitable non-toxic organic or inorganic acids and bases. Exemplary acid-addition salts include those derived from inorganic acids such as hydrochloric acid, hydrobromic acid, hydroiodic acid, sulfuric acid, sulfamic acid, phosphoric acid, and nitric acid, and those derived from organic acids such as p-toluenesulfonic acid, methanesulfonic acid, ethane-disulfonic acid, isethionic acid, oxalic acid, p-bromophenylsulfonic acid, carbonic acid, succinic acid, citric acid, benzoic acid, 2-acetoxybenzoic acid, acetic acid, phenylacetic acid, propionic acid, glycolic acid, stearic acid, lactic acid, malic acid, tartaric acid, ascorbic acid, maleic acid, hydroxymaleic acid, glutamic acid, salicylic acid, sulfanilic acid, and fumaric acid. Exemplary base-addition salts include those derived from ammonium hydroxides (e.g., a quaternary ammonium hydroxide such as tetramethylammonium hydroxide), those derived from inorganic bases such as alkali or alkaline earth-metal (e.g., sodium, potassium, lithium, calcium, or magnesium) hydroxides, and those derived from non- toxic organic bases such as basic amino acids. [84] The term "multimer" refers to multivalent or multimeric forms of active forms of the compounds of the invention. Such "multimers" may be made by linking or placing multiple copies of an active compound in close proximity to each other, e.g., using a scaffolding provided by a carrier moiety. Multimers of various dimensions (i.e., bearing varying numbers of copies of an active compound) may be tested to arrive at a multimer of optimum size with respect to receptor binding. Provision of such multivalent forms of active receptor-binding compounds with optimal spacing between the receptor-binding moieties may enhance receptor binding. See e.g., Lee et al, (1984) Biochem. 23:4255, which is herein incorporated by reference. The artisan may control the multivalency and spacing by selection of a suitable carrier moiety or linker units. Useful moieties include molecular supports comprising a multiplicity of functional groups that can be reacted with functional groups associated with the active compounds of the invention. A variety of carrier moieties may be used to build highly active multimers, including proteins such as BSA (bovine serum albumin) or HSA, peptides such as pentapeptides, decapeptides, pentadecapeptides, and the like, as well as non-biological compounds selected for their beneficial effects on absorbability, transport, and persistence within the target organism. Functional groups on the carrier moiety, such as amino, sulfhydryl, hydroxyl, and alkylamino groups, may be selected to obtain stable linkages to the compounds of the invention, optimal spacing between the immobilized compounds, and optimal biological properties.

[85] "A pharmaceutically acceptable prodrug" is a compound that may be converted under physiological conditions or by solvolysis to the specified compound or to a pharmaceutically acceptable salt of such compound. "A pharmaceutically active metabolite" is intended to mean a pharmacologically active product produced through metabolism of a specified compound or salt thereof in the body. Prodrugs and active metabolites of a compound may be identified using routine techniques known in the art. See e.g., Bertolini, G. et al, (1997) J. Med. Chem. 40:2011-2016; Shan, D. et al, J. Pharm. Sci., 86(7):765-767; Bagshawe K., (1995) Drug Dev. Res. 34:220-230; Bodor, N., (1984) Advances in Drug Res. 13:224-331; Bundgaard, H., Design of Prodrugs (Elsevier Press, 1985); and Larsen, I. K., Design and Application of Prodrugs, Drug Design and Development (Krogsgaard-Larsen et al, eds., Harwood Academic Publishers, 1991), which are herein incorporated by reference. [86] A pharmaceutical composition of the invention is formulated to be compatible with its intended route of admimstration. Examples of routes of administration include parenteral, e.g., intravenous, intradermal, subcutaneous, oral (e.g., inhalation), transdermal (topical), transmucosal, and rectal administration. Suitable pharmaceutical formulations for particular routes of administration are well known in the art. See e.g. Remington: The Science and Practice of Pharmacy. 20th ed. (2000) Lippincott Williams & Wilkins. Baltimore, MD, which is herein incorporated by reference.

[87] The compositions of the invention may be manufactured in manners generally known for preparing pharmaceutical compositions, e.g., using conventional techniques such as mixing, dissolving, granulating, dragee-making, levigating, emulsifying, encapsulating, entrapping or lyophilizing. Pharmaceutical compositions may be formulated in a conventional manner using one or more physiologically acceptable carriers, which may be selected from excipients and auxiliaries that facilitate processing of the active agents.

[88] The pharmaceutical compositions of the present invention may be provided in dosage unit forms in unit-dose or multi-dose containers, such as sealed ampoules or vials. Such containers are preferably hermetically sealed to preserve sterility of the formulation until use. In general, the compositions of the present invention may be stored as suspensions, solutions or emulsions in oily or aqueous vehicles. Alternatively, the compositions maybe stored in a freeze-dried condition requiring only the addition of a sterile liquid carrier prior to use. As used herein, "dosage unit form" refers to physically discrete units suited as unitary dosages for the subject to be treated; each unit containing a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier. The specification for the dosage unit forms of the invention are dictated by and directly dependent on the unique characteristics of the active compound and the particular therapeutic effect to be achieved, and the limitations inherent in the art of compounding such an active compound for the treatment of individuals.

[89] It will be appreciated that the actual dosages of the polynucleotides, polypeptides, antibodies, and fragments thereof used in the compositions of this invention will vary according to the particular complex being used, the particular composition formulated, the mode of admimstration, and the particular site, host, and disease being treated. Optimal dosages for a given set of conditions may be ascertained by those skilled in the art using conventional dosage-determination tests in view of experimental data. Administration of prodrugs may be dosed at weight levels that are chemically equivalent to the weight levels of the fully active forms.

[90] The biological activity of the polynucleotides, polypeptides, antibodies, fragments thereof, and compositions of the present invention may be measured by any of the methods available to those skilled in the art, including in vitro and in vivo assays and those provided herein. Other pharmacological methods may also be used to determine the efficacy of the polynucleotides, polypeptides, antibodies, fragments thereof, and compositions of the present invention as HIV vaccines and HIV and ADDS therapeutics.

[91] Toxicity and therapeutic efficacy of such compounds can be determined by standard pharmaceutical procedures in cell cultures or experimental animals. For example, the LD₅₀ (the dose lethal to 50%ι of the population), and the ED₅₀ (the dose therapeutically effective in 50% of the population) may be determined by conventional methods in the art. The dose ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed as the ratio LD₅o/ED₅o. Compounds that exhibit large therapeutic indices are preferred. While compounds that exhibit toxic side effects may be used, care should be taken to design a delivery system that targets such compounds to the site of affected tissue in order to minimize potential damage to normal and healthy cells and, thereby, reduce side effects.

[92] The data obtained from the cell culture assays and animal studies can be used in formulating a range of dosage for use in humans. The dosage of such compounds lies preferably within a range of circulating concentrations that include the ED₅₀ with little or no toxicity. The dosage may vary within this range depending upon the dosage form employed and the route of administration utilized. For any compound used in the method of the invention, the therapeutically effective dose can be estimated initially from cell culture assays. A dose may be formulated in animal models to achieve a circulating plasma concentration range that includes the IC₅₀ (i.e., the concentration of the test compound which achieves a half-maximal inhibition of symptoms) as determined in cell culture. Such information can be used to more accurately determine useful doses in humans. Levels in plasma may be measured, for example, by high performance liquid chromatography.

[93] The pharmaceutical compositions of the present invention include immunogenic compositions. The immunogenic compositions include an active immunizing agent, such as a polypeptide of the present invention, or a passive immunizing agent, such as an antibody that is raised against or specifically binds a polypeptide of the present invention. The immunogenic composition may elicit an immune response that need not be protective or the immunogenic composition may provide passive immunity. A vaccine elicits a local or systemic immune response that is protective against subsequent challenge by the immunizing agent such as the polypeptides of the present invention, or an immunologically cross-reactive agent. Accordingly, as used herein, an "immunogenic composition" can refer to vaccines as well as antibodies. A protective immune response may be complete or partial, i.e. a reduction in symptoms as compared with an unvaccinated mammal. Conventional methods in the art may be used to determine the feasibility of using the polypeptides of the present invention as an HIV vaccine. [94] Thus, the present invention provides immunogenic compositions comprising the polypeptides, polynucleotides, antibodies, or fragments thereof of the present invention that may be used to treat, prevent, or inhibit HIV infection, HIV disease progression, ADDS, or a combination thereof. In some preferred embodiments, the immunogenic compositions of the present invention are capable providing a protective immune response in a subject. Preferably, the subject is mammalian, more preferably, the subject is human. As used herein, an "immune response" refers to a humoral or cellular response caused by exposure to an antigenic substance. Thus, an immune response against HIV or "HIV immune response" refers to a humoral or cellular response in a subject that is caused by exposing the subject to an antigenic substance such as polypeptides of the present invention. A "protective immune response" against HIV refers to humoral immune responses, cellular immune responses, or both, that are sufficient to prevent or inhibit HIV infection. As used herein, an "immunogenic amount" is an amount that is sufficient to elicit an immune response in a subject and depends on a variety of factors such as the immunogenicity of the polypeptide, the manner of administration, the general state of health of the subject, and the like. The typical immunogenic amounts for initial and boosting immunization for therapeutic or prophylactic administration ranges from about 0.01 to about 0.1 mg per about 65 to about 70 kg body weight of a subject. Examples of suitable immunization protocols include initial immunization injections at time 0 and 4 or initial immunization injections at 0, 4, and 8 weeks, which initial immunization injections may be followed by further booster injections at 1 or 2 years. [95] A variety of immunostimulants may be employed in the immunogenic compositions of the present invention. For example, an immunostimulant may be included. An "immunostimulant" is any substance that enhances or potentiates an immune response (antibody and/or cell-mediated) to an exogenous antigen. Examples of immunostimulants include adjuvants, biodegradable microspheres (e.g., polylactic galactide), liposomes, cytokines, interleukins, and chemokines. Most adjuvants contain a substance designed to protect the antigen from rapid catabolism, such as aluminum hydroxide or mineral oil, and a stimulator of immune responses, such as lipid A, Bortadella pertussis or Mycobacterium tuberculosis derived proteins. Suitable adjuvants include Freund's Incomplete Adjuvant and Complete Adjuvant (Difco Laboratories, Detroit, MI); Merck Adjuvant 65 (Merck and Company, h e, Rahway, NJ); AS-2 (SmithKlineGlaxo); aluminum salts such as aluminum hydroxide gel (alum) or aluminum phosphate; salts of calcium, iron or zinc; an insoluble suspension of acylated tyrosine; acylated sugars; cationically or anionically derivatized polysaccharides; polyphosphazenes; biodegradable microspheres; monophosphoryl lipid A and quil A; and cytokines, such as GM-CSF or interleukin-2,-7, or -12, may be used as adjuvants. Other suitable adjuvants include Montanide ISA 720 (Seppic, France), SAF (Chiron, Emeryville, CA), ISCOMS (CSL), MF-59 (Chiron), the SB AS series of adjuvants, such as SBAS-2 or SBAS-4 (SmithKlineGlaxo), Detox, RC-529, other aminoaikyl glucosaminide 4-phosphates (AGPs); incomplete N-acetyl-muramyl-L-threonyl-D- isoglutamine (thr-MDP); N-acetyl-nor-muramyl-L-alanyl-D-isoglutamine (CGP 11637, nor-MDP), N-acetylmuramyl-Lalanyl-D-isoglutaminyl-L-alanine-2-( -2'-dipa-lmitoyl- sn-glycero-3-hydroxyphosphoryloxy)-ethylamine (CGP 19835 A, MTP-PE); and PJBL which comprise three components extracted from bacteria, monophosphoryl lipid A, trehalose dimycolate and cell wall skeleton (NPL+TDM+CWS) in a 2% squalene/Tween 80 emulsion. The effectiveness of an adjuvant may be determined by conventional methods in the art.

[96] The vaccines of the present invention may be prepared by conventional methods known in the art. See e.g. M. F. Powell and M. J. Newman, eds. Vaccine Design Plenum Press (NY, 1995), which is herein incorporated by reference. The immunogenic compositions of the present invention may further comprise other agents, which may be biologically active or inactive. For example, one or more immunogenic portions of other HIV antigens, such as Tat and env, either incorporated into a fusion polypeptide or as a separate compound, may be included in the compositions.

[97] A variety of delivery vehicles may be employed within pharmaceutical compositions and vaccines to facilitate production of an antigen-specific immune response that targets H1N and H1N infected cells. Suitable delivery vehicles include antigen presenting cells (APCs), such as dendritic cells, macrophages, B cells, monocytes and other cells that may be engineered to be efficient APCs. Such cells may be genetically modified to increase the capacity for presenting the antigen, to improve activation of the T cell response, maintenance of the T cell response, to have anti-tumor effects per se, to be immunologically compatible with the receiver, or a combination thereof. APCs may generally be isolated from any of a variety of biological fluids and organs, including tumor and peritumoral tissues, and may be autologous, allogeneic, syngeneic, or xenogeneic cells by methods known in the art.

[98] The present invention also provides polypeptides, polynucleotides, antibodies, or compositions of the present invention that may be provided in kits along with instructions for use. A kit comprising a pharmaceutical composition may include the pharmaceutical composition as a single dose or multiple doses. The kit may include a device for delivering the pharmaceutical composition. The device may be a multi-chambered syringe for intramuscular delivery, a microneedle or set of microneedle arrays for transdermal delivery, a small balloon for intranasal delivery, a small aerosol generating device for delivery by inhalation, or a gene-gun for mucosal delivery.

[99] The polynucleotides, polypeptides, antibodies, and compositions of the present invention may be used as active agents in therapeutic methods to treat, prevent, or inhibit HJN infection, HIV disease progression, or ADDS in a subject. Generally, these therapeutic methods comprise administering to the subject a therapeutically effective amount of at least one active agent.

[100] As used herein, a "therapeutically effective amount" refers to an amount of an active agent that may be used to treat, prevent, or inhibit HTV infection, HIV disease progression, or ADDS in a subject as compared to a control. As used herein, a "therapeutically effective amount" for preventing, or inhibiting "HIV infection" is an amount that protects a subject from obtaining detectable levels of HIV as compared to control using conventional assays in the art. As used herein a "therapeutically effective amount" for treating, preventing, or inhibiting "HIV disease progression" or "ADDS" is an amount that maintains or reduces the HIV viral load in a subject already infected with HIV as compared to a control using conventional assays in the art. Again, the skilled artisan will appreciate that certain factors may influence the dosage required to effectively treat a subject, including the severity and stage of the HJV, viral load, previous treatments, the general health and age of the subject, and the like. A therapeutically effective amount may be readily determined by conventional methods known in the art. It should be noted that treatment of a subject with a therapeutically effective amount of a polypeptide, a polynucleotide, or an antibody of the present invention can include a single treatment or a series of treatments.

[101] The polynucleotides, polypeptides, antibodies, and compositions of the present invention may be used in conjunction with other H1N and ADDS therapies, such as antiviral therapies and immunotherapies. The polynucleotides, polypeptides, antibodies, and compositions of the present invention may be administered prior to, during, after, or a combination thereof other HIV and ADDS therapies by any suitable method, including administration by intravenous, intraperitoneal, intramuscular, subcutaneous, intranasal, intradermal, anal, vaginal, topical and oral routes. The polynucleotides, polypeptides, antibodies, and compositions of the present invention maybe used in combination with or as a substitution for conventional HIV and ADDS therapies. For example, the polynucleotides, polypeptides, antibodies, and compositions of the present invention may also be used alone or in combination with a supplementary active compound such as nucleoside reverse transcriptase inhibitors (ΝRTIs) such as zidovudine (AZT), didanosine (ddl), zalcitabine (ddC), stavudine (d4T), lamivudine (3TC), Combivir™ (AZT + 3TC), and abacavir (Ziagen ), Protease inhibitors (Pis) such as saquinavir (Invirase & Fortovase™), ritonavir (Norvir™), indinavir (Crixivan®), and nelfinavir (Viracept ®) and non-nucleoside reverse transcriptase inhibitors (NNTRIS) such as nevirapin (Viramune®, delaviridine (Rescriptor®) and efavirenz (Sustiva™) and other pharmaceutical compounds can temporarily alleviate symptoms in ADDS patients, and the like.

[102] The immunotherapeutic methods of the present invention may be broadly classified as adoptive, passive, and active. Active immunotherapeutic methods rely on the in vivo stimulation of the endogenous host immune system to react against HIV with the administration of immune response-modifying agents, such as the polypeptides and polynucleotides of the present invention. Active immunotherapies of the present invention include administering at least one antigenic epitope of the present invention, a polynucleotide encoding the antigenic epitope, adjuvants, immunostimulants, and the like. In some embodiments, other HIV antigens, such as Tat and env and the like, may be administered along with the antigenic epitope of the present invention. In some preferred embodiments, the IgM antibody response in the subject being treated is activated or enhanced.

[103] Passive immunotherapeutic methods involve the delivery of agents with established HIV reactivity (such as effector cells or antibodies) that can directly or indirectly mediate anti-HTV effects and does not necessarily depend on an intact host immune system. The T cell receptors and antibody receptors specific for the polypeptides of the present invention maybe cloned, expressed, and transferred into other vectors or effector cells for adoptive immunotherapy. The polypeptides provided herein may also be used to generate antibodies or anti-idiotypic antibodies for passive immunotherapy by methods known in the art. Passive immunotherapies include administering antibodies raised against or specific for at least one antigenic epitope of the present invention or a variant thereof alone or coupled to toxins, antiviral agents, radioactive isotopes, and the like. In some embodiments, antibodies against other HIV antigens, such as those raised against or specific for Tat, env and the like, may be administered along with the antibodies raised against or specific for at least one antigenic epitope of the present invention or a variant thereof. In some embodiments, lymphokines and other types of immunostimulants maybe administered. See e.g., Bajorin et al. (1988) Proc. Annu. Meet. Am. Soc. Clin. Oncol. 7:A967, which is herein incorporated by reference.

[104] Adoptive immunotherapeutic methods of the present invention include isolating a subject's circulating lymphocytes and activating the lymphocytes by conventional methods known in the art and then administering the activated lymphocytes to a subject to be treated. Thus, in some embodiments, the present invention provides methods of enhancing the immune response in a subject comprising the steps of contacting at least one lymphocyte with at least one LTNP peptide or a variant thereof. In some embodiments, additional HIV antigenic polypeptides are contacted with the lymphocyte. Additional compounds, such as immunostimulants maybe administered to the subject.

[105] Routes and frequency of administration of the therapeutic compositions described herein, as well as dosage, will vary from individual to individual, and may be readily established using standard techniques. Preferably, between 1 and 10 doses maybe administered over a 52- week period. Preferably, 6 doses are administered, at intervals of 1 month, and booster vaccinations may be given periodically thereafter. Alternate protocols may be appropriate for individual subjects and may be readily determined by those skilled in the art.

[106] A "suitable dose" is an amount of a polynucleotide, polypeptide, antibody, or composition of the present invention that, when administered as described above, is capable of promoting an anti-HIN immune response, and is at least about 10% or more above the basal level. In general, for pharmaceutical compositions and vaccines comprising one or more polypeptides, the amount of each polypeptide present in a dose ranges from about 25 μg to about 5 mg per kg of the subject. Suitable dose sizes will vary with the size of the subject, but will typically range from about 0.1 ml to about 5 ml. Methods of using the vaccines of the present invention are also contemplated. Suitable vaccines are capable of causing an immune response that leads to an improved clinical outcome, such as more frequent remissions, complete or partial or longer disease-free survival, in vaccinated patients as compared to non-vaccinated patients.

[107] Responses to the therapeutic methods of the present invention can be monitored by measuring the HIV viral load in a subject using conventional methods in the art. Immune responses may generally be evaluated using conventional immunoassays or cytokine assays, which may be performed using samples obtained from a subject before and after treatment. Other methods known in the art for monitoring the effect of a given therapeutic method are also contemplated.

[108] As used herein, a "therapeutically effective amount" of a polynucleotide, polypeptide, antibody, or composition of the present invention is an amount which provides an observable decrease in HJN viral load, HIV disease progression, or ADDS symptoms in a subject as compared to a control. As defined herein, a therapeutically effective amount of a compound of the present invention may be readily determined by one of ordinary skill by routine methods known in the art. For example, a therapeutically effective amount of a polypeptide of the present invention ranges from about 2 to about 20 mg/kg body weight of the subject. The skilled artisan will appreciate that certain factors may influence the dosage required to effectively treat a subject, including but not limited to the severity of the disease or disorder, previous treatments, the general health and/or age of the subject, and other diseases present. [109] The therapeutic methods of the present invention may include a single treatment or a series of treatments. For example, a subject may be treated with an immunogenic composition of the invention at least once. However, the subject may be treated with the immunogenic composition from about one time per week to about once daily for a given treatment period. The length of the treatment period will depend on a variety of factors such as the disease stage and viral load. It will also be appreciated that the effective dosage of the composition used for treatment may increase or decrease over the course of a particular treatment. Changes in dosage may result and become apparent by standard diagnostic assays known in the art. In some instances chronic administration may be required.

[110] The polynucleotides, polypeptides, antibodies, and fragments thereof of the present invention may be used to detect, diagnose, or monitor HIV in a subject. Specifically, whether a subject is a viremia controller or an HIV progressor may be detected or monitored by the presence or amount of LTNP peptides of the present invention in a biological sample obtained from the subject. Thus, the polynucleotides, polypeptides, antibodies, and fragments thereof of the present invention may be used as markers to classify the subject as a viremia controller, HIV progressor, or a UARE. After the subject is classified, an HIV treatment regime may be tailored to the subject. For example, viremia controllers and UARE subjects need not be put on a rigorous antiviral treatment regime which may result in undesirable side-effects and complications.

[Ill] The binding agents provided herein generally permit detection of the level of antigen that binds to the agent in the biological sample, some embodiments, the binding agent used in the assays of the present invention is immobilized on a solid support to bind to and remove the polypeptide from the remainder of a sample. The bound polypeptide may then be detected using a detection reagent that contains a reporter group and specifically binds to the binding agent/polypeptide complex. Such detection reagents may comprise, for example, a binding agent that specifically binds to the polypeptide or an antibody or other agent that specifically binds to the binding agent, such as an anti-immunoglobulin, protein G, protein A, or a lectin. Alternatively, a competitive assay may be utilized, in which a polypeptide is labeled with a reporter group and allowed to bind to the immobilized binding agent after incubation of the binding agent with the sample. The extent to which components of the sample inhibit the binding of the labeled polypeptide to the binding agent is indicative of the reactivity of the sample with the immobilized binding agent. Suitable polypeptides for use in assays include HIVp2 (SEQ DD NO:l), HIVp5 (SEQ DD NO:2), and variants thereof to which the binding agent binds.

[112] The solid support may be any material known to those of ordinary skill in the art to which a polypeptide may be attached. For example, the solid support may be a test well in a microtiter plate or a nitrocellulose or other suitable membrane. Alternatively, the support may be a bead, fiber, or disc, such as glass, fiberglass, latex, or plastic such as polystyrene or polyvinylchloride. The support may also be a magnetic particle or a fiber optic sensor. The binding agent may be immobilized on the solid support by conventional methods known in the art. As used herein, "immobilization" refers to both noncovalent association, such as adsorption, and covalent attachment (which may be a direct linkage between the agent and functional groups on the support or may be a linkage by way of a cross-linking agent). Adsorption may be achieved by contacting the binding agent, in a suitable buffer, with the solid support for a suitable amount of time sufficient to immobilize an adequate amount of binding agent. Covalent attachment of binding agent to a solid support may be achieved by first reacting the support with a bifunctional reagent that will react with both the support and a functional group, such as a hydroxyl or amino group, on the binding agent. See e.g. Pierce hnmunotechnology Catalog and Handbook (1991) at A12-A13, which is herein incorporated by reference.

[113] The presence of antibodies that specifically react with at least one of the LTNP peptides of the present invention in a biological sample may indicate that the subject is a viremia controller. Alternatively, one may determine whether a subject is a viremia controller by detecting the level of mRNA encoding at least one of the LTNP peptides of the present invention in a biological sample. Techniques for both PCR based assays and hybridization assays are well known in the art. See e.g. Mullis et al (1987) Cold Spring Harbor Symp. Quant. Biol., 51:263; and Erlich ed., PCR Technology, Stockton Press, NY, 1989, which are herein incorporated by reference.

[114] The present invention further provides kits for use within any of the above diagnostic methods. Such kits typically comprise two or more components necessary for performing a diagnostic assay. Components may be compounds, reagents, containers and/or equipment. For example, one container within a kit may contain at least one of the LTNP peptides of the present invention. The LTNP peptide may be provided attached to a support material, as described above. One or more additional containers may enclose elements, such as reagents or buffers, to be used in the assay. Such kits may also, or alternatively, contain a detection reagent as described above that contains a reporter group suitable for direct or indirect detection of antibody binding.

[115] In some embodiments, antibodies raised against or specific for at least one of the

LTNP peptides of the present invention may be used for detecting whether an LTNP epitope is present in a given sample. The antibodies useful for diagnostic purposes may be prepared in the same manner as those described above for therapeutics. The antibodies may be used with or without modification, and may be labeled by joining them, either covalently or non-covalently, with a reporter molecule. A wide variety of reporter molecules that are known in the art may be used.

[116] In another embodiment of the invention, the polynucleotides encoding at least one of the LTNP peptides of the present invention maybe used for diagnostic purposes. The polynucleotides that may be used include oligonucleotide sequences, antisense RNA and DNA molecules, and PNAs. Hybridization with PCR probes which are capable of detecting polynucleotide sequences, including genomic sequences, encoding at least one of the LTNP peptides of the present invention, may be used for the detection of related sequences, and should preferably contain at least 50% of the nucleotides from any of the LTNP encoding sequences.

[117] Means for producing specific hybridization probes for DNAs encoding at least one of the LTNP peptides of the present invention include the cloning of nucleic acid sequences encoding at least one of the LTNP peptides of the present invention into vectors for the production of mRNA probes. Such vectors are known in the art, commercially available, and may be used to synthesize RNA probes in vitro by means of the addition of the appropriate RNA polymerases and the appropriate labeled nucleotides. Hybridization probes may be labeled by a variety of reporter groups, for example, radiolabels such as 32P or 35S, or enzymatic labels, such as alkaline phosphatase coupled to the probe via avidin/biotin coupling systems, and the like.

[118] Additional diagnostic uses for oligonucleotides designed from the sequences encoding at least one of the LTNP peptides of the present invention may involve the use of PCR. Such oligomers may be chemically synthesized, generated enzymatically, or produced from a recombinant source. Oligomers will preferably consist of two nucleotide sequences, one with sense orientation (5 ' 3 ') and another with antisense (3 ' 5 '), employed under optimized conditions for identification of a specific gene or condition. The same two oligomers, nested sets of oligomers, or even a degenerate pool of oligomers may be employed under less stringent conditions for detection and/or quantitation of closely related DNA or RNA sequences.

[119] Methods that may also be used to quantitate the expression of at least one of the

LTNP peptides of the present invention include radiolabeling or biotinylating nucleotides, co amplification of a control nucleic acid, and standard curves onto which the experimental results are interpolated. See Melby, P.C, et al. (1993) J. Immunol. Methods 159:235-244; and Duplaa, C, et al. (1993) Anal. Biochem. 229-236, which are herein incorporated by reference. The speed of quantitation of multiple samples may be accelerated by running the assay in an ELISA format where the oligomer of interest is presented in various dilutions and a spectrophotometric or colorimetric response gives rapid quantitation.

[120] h another embodiment of the invention, at least one of the LTNP peptides of the present invention can be used for screening libraries of compounds in any of a variety of drug screening techniques. The peptides employed in such screening may be free in solution, affixed to a solid support, borne on a cell surface, or located intracellularly. The formation of binding complexes, between the peptide and the agent being tested, may be measured.

[121] Another technique for drug screening that may be used provides for high throughput screening of compounds having suitable binding affinity to the protein of interest as described in published PCT application WO84/03564 and by using cells described in WO 99/15650. In another embodiment, one may use competitive drug screening assays in which neutralizing antibodies capable of binding at least one of the LTNP peptides of the present invention specifically compete with a test compound for binding the LTNP peptide.

[122] A variety of protocols for detecting and measuring the expression of polynucleotide-encoded products, using either polyclonal or monoclonal antibodies specific for the product are known in the art. See Hampton, R., et al. (1990) Serological Methods, a Laboratory Manual, APS Press, St Paul, MN; and Maddox, D. E., et al. (1983) J. Exp. Med. 158:1211-1216, which are herein incorporated by reference.

[123] A wide variety of labels and conjugation techniques are known by those skilled in the art and may be used in various nucleic acid and amino acid assays. Methods for producing labeled hybridization or PCR probes for detecting sequences related to polynucleotides include oligolabeling, nick translation, end-labeling or PCR amplification using a labeled nucleotide. Alternatively, the sequences, or any portions thereof may be cloned into a vector for the production of an mRNA probe. Such vectors are known in the art, are commercially available, and may be used to synthesize RNA probes in vitro by addition of an appropriate RNA polymerase such as T7, T3, or SP6 and labeled nucleotides. These procedures may be conducted using a variety of commercially available kits such as those available from Amersham Pharmacia Biotech, Promega and US Biochemical Corp. Suitable reporter molecules or labels, which may be used, include radionuclides, enzymes, fluorescent, chemiluminescent, or chromogenic agents as well as substrates, cofactors, inhibitors, magnetic particles, and the like.

[124] The following Examples are intended to illustrate, but not to limit the present invention.

Example 1 Identification of Epitopes of Viremia Controllers

[125] Phage display libraries and differential immunoscreening were used to identify epitopes that serologically distinguish HlV-infected patients who control viremia without therapy from those with progressive infection. Generally, the phage display libraries were made by inserting oligonucleotides that encode peptides of interest into coat protein genes of filamentous phages such that peptides are expressed and displayed on the phage surface and "panning" the phages with plasma or serum. See e.g. Rodman, et ah (1999) Human Immunol. 60(8): 631-9, which is herein incorporated by reference.

A. Serum and Plasma Samples

Serum or plasma samples (samples) were obtained from 25 viremia controllers (18 subjects with plasma HIV RNA of less than about 50 copies/ml and 7 subjects with between about 50 to about 3000 copies/ml on one or more occasions). None of the viremia controllers ever received anti-retroviral therapy. Samples were also obtained from 26 HlV-infected subjects with progressive H infection, and from 5 subjects with acute HIV infection. A subject having progressive HIV infection (progressor) was identified by having plasma HIV RNA levels of less than about 20,000 copies/ml on at least one occasion or by the need for anti-retroviral therapy. An additional 18 samples were collected from uninfected, healthy control subjects. B. Phage Display Library

[127] In order to screen for antigens that serologically differentiate viremia controllers from progressors, an M13 phage display library, Ph.D. -12 (New England Biolabs), was used. Ph.D.-12 displays about 2.7 x 10⁹ random, 12-mer peptides fused to the minor coat protein (pID) of M13 phage. Magnetic beads coated with anti-human IgG (Pierce, Rockford, EL) were incubated with pooled plasma from 4 viremia controllers (1 : 100 dilution), washed, blocked, and then incubated with the phage display library using the panning procedure described by Prezzi, et al. (1996) J. Immunol. 156(11):4504-13, which is herein incorporated by reference.

[128] Phages bound to the sample IgG were captured magnetically, and non-binding phages were washed away. Captured phages were eluted from the beads and amplified by infection of E. coli in order to increase the target phages for subsequent rounds of panning. To eliminate background and enrich the pool of phages displaying consensus- binding sequences, two additional rounds of panning were employed.

C. Cloning and Expression of Phage DNA

[129] DNA from phages captured in the third round of panning were amplified by polymerase chain reaction (PCR) using 20 μM of each dNTP, 20 pmol of forward primer

5 " -CGC-AAT-TCC-TTT-AGT-GGT-ACC-3 ' (SEQ ID NO : 3 ) and reverse primer

5 - -ACA-GTT-GGG-CCC-GGT-CGT-CTT-TCC-AGA-CGT-TAG -3 ' ( SEQ ID NO : 4 ) and 2.5 U of Taq polymerase (Perkin Elmer) in a reaction volume of 100 μl. One microliter of phage culture supernatant was added, and amplification was achieved with 25 cycles at 95°C for 30 sec, 48°C for 30 sec, and 72°C for 1 min. [130] Initially, the PCR product was ligated into the pMal-pHJ expression vector and transformed in E. coli (DH5α) by conventional methods in the art.

D. Differential Immnunostaining

[131] For differential immunostaining, transformed E. coli colonies growing in a Petri dish on LB medium supplemented with 100 μg/ml ampicillin were overlaid with nitrocellulose filter paper in triplicate to allow colony lifting. Filters containing colonies in the same distribution as those on the Petri dish were then placed in fresh medium- containing Petri dishes and incubated overnight. The filters were then blocked with 3% dry milk and 2% E. coli lysate in tris-buffered saline (TBS, pH 7.4) overnight at 4°C.

[132] Pooled samples from 4 viremia controllers, pooled samples from 4 progressors, and pooled samples from 4 healthy controls were preincubated in the same solution, 3% dry milk and 2% E. coli lysate in tris-buffered saline (TBS, pH 7.4), (1:100 dilution) for 2 hours at 37°C. Each of the three pooled samples was then incubated with one of the triplicate filters. After rinsing the filters 3 times with TBS containing 0.1% Tween 20, goat anti-human IgG conjugated with horseradish peroxidase (1:150 dilution, Calgene) was applied. Following a 2-hour incubation at 37°C, the filters were washed three times with 2X TBS containing 0.1% Tween and IX TBS alone and incubated with 1 ml of 4- chloro-1-naphtol (3mg/ml in 100% ethanol) in 9 ml TBS and 0.03% H₂O₂. The colonies that stained exclusively with samples from viremia controllers were selected for further characterization.

[133] Figure 1 the colony lifting and differential immunoscreening procedures used to identify clones reactive with viremia controllers. Specifically, nitrocellulose filter paper was laid into three new Petri dishes and colonies were transferred to the filter paper using standard methods in the art over the Petri dish (A) to lift colonies for immunostaining with pooled plasma from viremia controllers (B), subjects with progressive disease (C), and uninfected control subjects (D). The arrows on the filter paper (B) indicate colonies that react only with plasma from viremia controllers, and the arrows on the Petri dish (A) indicate the original position of those colonies, which can then be amplified for further evaluation. Black filled circles indicate colonies that react by immunostaining with the respective plasma pool.

[134] Peptides fused to the maltose protein of pMal pUJ were expressed and loaded onto a 10% sodium dodecyl sulfate-polyacrylamide gel and transferred to a nitrocellulose membrane at 80 mV for 1 hour, hnmunoblotting was done as described above for colony immunostaining using pooled samples (1:100 dilution) from viremia controllers, progressors, and controls. Sample and secondary antibody incubations were conducted for 1.5 hours at room temperature.

E. Subcloning and Expression of Fusion Proteins [135] Because the use of the pETd32 expression vector (Novagen), rather than pMal- pDI, was found to facilitate and improve protein expression and purification, some immunoblotting, as well as all of the ELISAs and DNA sequencing experiments described below were performed using pETd32. Subcloning into pET32d was accomplished by PCR amplification of DNA encoding the pMal-pDI fusion proteins using a forward primer

5 ' - CAACGC CC ATG GCA GTA CCT TTC TAT TCT CAC TCT- 3 ' (SEQ ID NO : 5 ) and a reverse primer

5 " -CCACGT AAGCTT GTC GTC TTT CCA GAC GTT AG-3 ' ( SEQ ID NO : 6 ) as described above and ligated into the Nco I/Hind DI site using conventional methods in the art. [136] The pET32d expression vectors encoding the cloned peptides were expressed in E. coli (BL21 DE3, Novagen) as fusion proteins of random peptide, Ml 3 pπi-N-terminal peptide, and thioradox protein with a histidine tag to facilitate purification. The fusion proteins were purified using the His. Bind Purification Kit (Novagen) according to the manufacturer's instructions.

F. Seroreactivity Assays

[137] Due to the higher sensitivity and the small amount of sample needed for Enzyme-

Linked Immunosorbent Assay (ELISA) compared to Western blot, the seroreactivity of an individual sample to the purified fusion proteins was determined using ELISA. About 100 ng of fusion protein, purified by HisBind Purification kit from Novagen, was coated onto ELISA plate wells by incubating overnight at 4°C in bicarbonate-binding buffer. The wells were blocked with 3% dry milk in phosphate buffered saline (PBS) and washed 5 times in PBS with 1% Tween 20. After test samples (1 :50 dilution) were added the wells and incubated for 2 hours at 37°C, the wells were washed 5 times with PBS AND 1% TWEEN. Goat anti-human IgG conjugated with horseradish peroxidase (1 :3000 dilution) was then added. After incubating for 1 hour and then washing 5 times with PBS AND 1% TWEEN, 100 μl of ready to use TMB (Sigma, St. Louis, MO) was added. The color reaction was terminated with H SO₄. The intensity of the color was measured in a microplate reader at 405 nm. Samples from 6 uninfected control subjects that consistently gave low optical density (OD) values against the fusion proteins were used as negative controls.

[138] In each ELISA assay, an adjusted OD was calculated by dividing the OD of each test plasma or serum by the mean OD + 2 standard deviations of negative control sera/plasma. Each subject's sample was independently assayed 2 to 5 times, and the mean of each subject's adjusted OD was reported. A mean adjusted OD greater than 1 was considered positive.

G. Selection of Clones

[139] After panning and immunoselection with plasma from viremia controllers, progressors, and uninfected controls, 7 clones were identified and isolated. Using Western blot with pooled subject samples, 5 clones did not differentiate between HIV- infected and uninfected individuals (data not shown) and were not analyzed further. Two clones, HIVp5 and HIVp2 were serologically recognized exclusively by the pooled sample from the viremia controllers. Individual samples at a dilution of 1 :50 were next tested by ELISA using purified fusion proteins from each of the two clones as antigen.

[140] The first clone, HlNp5, was recognized by serum or plasma from 14 of 25 viremia controllers and from only 3 of 26 progressors (p = 0.001, Fisher's exact test). Serum or plasma from none of 5 acutely infected patients and 1 of 18 uninfected controls recognized HIVp5. See Table 1 and Figure 2 A.

[141] Figure 2 shows ELISA reactivity against HIVp5 (A) or HIVp2 (B) of serum or plasma from viremia controllers, patients with progressive disease (progressors), patients with acute HIV infection, and uninfected controls. The adjusted optical density (OD) was calculated by dividing the OD of each test plasma or serum by the mean OD + 2 SD of negative control sera/plasma. Each subject's specimen was assayed two to five times in independent experiments, and the mean of each subject's adjusted OD is depicted. Adjusted OD's greater than about 1 (shown above the horizontal dotted line) are considered positive. The horizontal bars represent the median adjusted OD for each group of patients. P values refer to the difference in adjusted OD values between viremia controllers and progressors (Kruskal-Wallis test). [142] As shown, the second clone, HTVp2, was recognized by 24 of 25 viremia controllers and by 19 of 26 progressors (p = 0.05, Fisher's exact test). None of 5 acutely . infected patients and 2 of 18 uninfected subjects recognized HIVp2 (the two uninfected controls may be HTV infected subjects since they were also reactive to gp41 in an ELISA assay, but further investigation is necessary). See Table 1. Seroreactivity against HIVp2 was significantly stronger among the viremia controllers than among the progressors (median adjusted OD = 5.1 for viremia controllers and 1.6 for progressors, p = 0.00001, Kruskal Wallis test). See Figure 2B.

H. Amino Acid Sequence Analysis [143] To determine the amino acid sequences of HIVp2 and HTVp5, the pET32d expression vectors expressing the clones of interest were amplified in E. coli, and plasmid

DNA was extracted by the alkaline lysis method and further purified by DNA Clean &

Concentrator (Zymo Research, Orange, CA) according to the manufacturer's instructions.

DNA sequencing was performed at the University of California, Irvine sequence facility using the ABI prism method. [144] The amino acid sequences were derived from the nucleotide sequences. Thus, the amino acid sequence of HINp2 is:

PKDAELLAI G (SEQ ID ΝO:l) and the amino acid sequence of HTVp5 is:

S PHASPWRHPV (SEQ ID NO: 2)

[145] A BLAST search showed that HιVp5 has about 55% sequence identity with the consensus sequences of HJN-1 Tat from all major subtypes at amino acids 6-14. See Fig. 3A. A BLAST search showed that HIVp2 has about 70% sequence identity with the consensus sequences of HTN-1 gp41 from all major HTN-1 subtypes at amino acids 591- 600 (based on the numbering from strain HIV-1 JC, GenBank accession #AAC 68848). See Fig. 3B.

I. Characterization as Tat or gp41 Epitopes or Mimics [146] To determine whether HJNρ5 is an epitope or epitope mimic of HIV-1 Tat the peptide:

SSWPHASPWRHPVGGGSAC ( SEQ ID NO : 7 ) was synthesized and coupled to keyhole limpet hemocyanin (KLH) by a commercial service (Genemed Biotechnologies, Inc., CA). New Zealand white rabbits were immunized with 0.5 mg of synthesized HIVp5 3 times at 3-week intervals. Serum was collected before each immunization and 3 weeks after the last immunization. The sera of the rabbit immunized with the synthetic HJVp5 peptide exhibited anti-Tat antibody following the second and third immunization. See Fig. 4. The pre-immunization and the serum after the first immunization had no detectable anti-Tat antibody levels by ELIS A. Since the HIVp5 peptide has homology with Tat from many HIV strains, anti-HTVp5 antibodies and HTVp5 may be used in immunotherapies and diagnostics.

J. Serological Analysis ofHIVp2 Compared with gp41 [147] As shown in Table 2, further serological analysis of HIVp2 showed that is more reactive to sera of viremia controllers as compared to the sera of HIV progressors, but the reactivity of gp41 to the sera of viremia controllers and the sera of HTV progressor was the same. ^»

[148] Generally, 40%> of all HIV progressors do not react with HTNp2 but react with gp41 MΝ and/or gp41-S. These results indicate that viremia controllers often respond immunologically to Tat, whereas HIV progressors either infrequently mount an anti-Tat antibody response or lose this response over time. These results indicated that the presence of anti-Tat antibody was associated with very profound suppression of viremia and progression to ADDS.

Example 2 Alternative Method for Identifying Epitopes [149] The method described above in Example 1 provides highly stringent selection for

LTΝP specific peptides, but may not allow the identification of certain important epitopes due to the diverse background of different subjects. Thus, the method of Example 2 may be modified to provide a less stringent selection of epitopes. For example, for the planned biopanning process, three-pooled plasma each containing 4 LTNP plasma are used. Same pooled plasma is used for the first, second and third round of biopanning. The resulting third phage pool is processed further to construct 3 separate expression libraries and analyzed for differences of the immune reactivity of samples from viremia controllers as compared to samples of HIV progressors or HIV uninfected controls. The selected peptides are expressed in E. coli and further characterized using different samples from HIV progressors and viremia controllers in Westemblot and ELISA assays. Clones specific for the antibodies obtained from viremia controllers are sequenced, the corresponding peptides are synthesized and further analyzed.

To the extent necessary to understand or complete the disclosure of the present invention, all publications, patents, and patent applications mentioned herein are expressly incorporated by reference therein to the same extent as though each were individually so incorporated. Having thus described exemplary embodiments of the present invention, it should be noted by those skilled in the art that the within disclosures are exemplary only and that various other alternatives, adaptations, and modifications may be made within the scope of the present invention. Accordingly, the present invention is not limited to the specific embodiments as illustrated herein, but is only limited by the following claims.

Claims

We claim:

1. A purified peptide that has a higher reactivity with sera, plasma, antibodies, or a combination thereof of HIV long-term non-progressors than with sera, plasma, or antibodies of HIV progressors.

2. The purified peptide of claim 1 , wherein the peptide reacts with the sera, plasma, or antibodies of about 12 of 23 viremia controllers.

3. The purified peptide of claim 1 , wherein the peptide reacts with the sera, plasma, or antibodies of about 14 of 25 viremia controllers.

4. The purified peptide of claim 1 , wherein the peptide reacts with about the sera, plasma, or antibodies of about 24 of 25 viremia controllers.

5. The purified peptide of claim 1 , wherein the peptide is serologically detected by about 50% to about 100% of viremia controllers.

6. An epitope that is recognized to a greater degree by an antibody obtained from an HIV long-term non-progressor than by an antibody obtained from an HIV progressor.

7. The epitope of claim 6, wherein the epitope is about 55% homologous to an HTV- 1 Tat consensus sequence.

8. The epitope of claim 6, wherein the epitope is about 70% homologous to an HTV- 1 gp41 consensus sequence.

9. A purified peptide comprising SEQ DD NO : 1.

10. The purified peptide of claim 9, consisting of SEQ DD NO:l.

11. A purified peptide having an amino acid sequence that has about 90% sequence identity to SEQ DD NO:l or SEQ DD NO:2 and exhibits a differential immunoactivity against antibodies obtained from HIV long-term non-progressors as compared with antibodies obtained from HTV progressors.

12. A purified peptide comprising SEQ DD NO:2.

13. The purified peptide of claim 12, consisting of SEQ DD NO:2.

14. An immunogenic composition comprising a peptide having SEQ DD NO: 1, a peptide having SEQ DD NO:2, or both.

15. A multivalent vaccine comprising a peptide having SEQ DD NO: 1 , a peptide having SEQ DD NO:2, or both.

16. A composition comprising at least one antibody that specifically binds with or was raised against a peptide having SEQ DD NO:l, SEQ DD NO:2, or both.

17. An assay for at least one epitope characteristic of an HIV long-term non- progressor or a UARE subject comprising biopanning a phage display library to obtain at least one target DNA; creating an expression library with the target DNA; and immunoscreening the expression library with a pooled sample obtained from HIV long- term non-progressors by colony lifting.

18. A method of diagnosing whether an HIV infected subject is likely to be an HIV long-term non-progressor or an HIV progressor comprising detecting the presence or absence of at least one epitope identified by the assay of claim 16, wherein the presence indicates that the subject is likely to be an HIV long-term non- progressor.

19. A method of treating a subject infected with HIV comprising determining whether the subject is an HIV-long term non-progressor or an HIV progressor by the method of claim 16; and treating the subject according to whether the subject is an HIV-long term non-progressor or an HIV progressor.

20. A method of immunizing a subject comprising administering to the subject at least one peptide of claim 1.

21. A method of passively immunizing a subj ect comprising administering to the subject at least one antibody that specifically binds or was raised against the peptide of claim 1.

22. A kit comprising the purified peptide of claim 1 and instructions for use.