AU694204B2 - Methods to diagnose and treat HIV-1 infection - Google Patents

Description

WO 95/25124 PCT/US95/03236 METHODS TO DIAGNOSE AND TREAT HIV-1 INFECTION BACKGROUND OF THE INVENTION FIELD OF THE INVENTION The invention generally is related to the field of immunology and more specifically to methods to diagnose HIV-1 infection in a subject and to stimulate an immune response against HIV-1.

BACKGROUND INFORMATION As the incidence of acquired immune deficiency syndrome (AIDS) continues to increase, the number of persons infected by the type 1 human immunodeficiency virus (HIV-1) has achieved an epidemic level. In spite of the large amount of research aimed at elucidating the mechanism and pathogenesis of AIDS, simple, inexpensive methods for early diagnosis of HIV-1-infected individuals and methods for immunizing a person against HIV-1 remain unrealized.

Upon infection of an individual with HIV-1, a primary immune response is mounted against the virus. In the primary response, a small number of lymphocytes have the appropriate cell surface receptor to recognize the virus. Upon recognition, the specific lymphocyte population increases and begins secreting anti-HIV-1 antibodies into the circulation. Since only a small number of lymphocytes can initially respond to the HIV-1 infection, a latent period occurs before circulating anti- X HIV-1 antibodies are present following infection. This latent period lasts a minimum of one month and some persons can remain seronegative for several months.

I WO 95/25124 PCT/US95/03236 Following a primary immune response, specific memory cells remain in the circulation and, upon subsequent exposure to an antigen such as HIV-1, a secondary immune response is rapidly mounted. Antibodies generated in a secondary immune response can be detected in the circulation within one or two weeks of exposure to HIV-1.

Two currently used tests for HIV-1 infection depend on the presence of circulating anti-HIV-1 antibodies in an infected individual. Both tests rely on the use of an HIV-l-derived antigen, which is attached to a solid support. Although one of these tests is relatively simple and inexpensive to perform, it is only 90% specific. Thus, it can only be used as an initial screen and, if a positive result is obtained, the second test must be performed. A positive result in the second test is diagnostic of EIV-1 infection. However, the second test is expensive and is laborious to perform. In addition, since the currently used tests rely on the detection of circulating anti-HIV-1 antibodies, they are inherently limited by the latent period for generation of a primary immune response following HIV-1 infection. Thus, these assays cannot be used to detect HIV-1 infection within the first month after infection.

Efforts to provide immunologic protection against HIV-1 infection also require viral protein. Various HIV-1 proteins have been used in an attempt to generate an anti- HIV-1 immune response. However, the use of viral material in humans carries the attendant risks associated with introducing such materials into a person. In any case, these attempts at active immunization have been unsuccessful and decline of the immune system is an inexorable consequence of AIDS.

Thus, there exists a need for a simple, inexpensive diagnostic method that can identify an HIV-1i WO 95/25124 PCTIUS95/03236 3 infected individual at an early stage of infection and an effective method for stimulating an anti-HIV-1 immune response in a subject. The present invention satisfies this need and provides related advantages as well.

SUMMARY OF THE INVENTION The present invention provides a method for diagnosing HIV-1 infection in a subject by identifying the presence of anti-HIV-1 antibodies in the subject's serum that react with an autoantigen such as 70K. The method of diagnosis is particularly useful f~r diagnosing HIV-1 infection at an early time after a subject is infected.

The invention also provides a method of stimulating an immune response against HIV-1 in a subject comprising immunizing the subject with an amino acid sequence of an autoantigen that crossreacts with neutralizing epitopes present on HIV-1. The invention provides, for example, amino acid sequences of 70K that are immunologically homologous to neutralizing epitopes of HIV- 1, but not with regions of HIV-1 that mediate the deleterious effects of the virus.

The invention further provides a skin test that is useful for diagnosing a subject having an HIV-1 infection. The skin test provides a simple, inexpensive method to screen large populations of persons suspected of being infected with HIV-1.

BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 shows the amino acid sequence homology between the Ul snRNP splicing protein 70K and gpl20/41.

Portions of the amino acid sequence of are shown in linear order in bold type (Theissen et al., L i I: i WO 95/25124 PCT/US95103236 4 EMBO J. 5:3209-3217 (1986), which is incorporated herein by reference). Numbers indicate amino acid positions from the amino terminus. Immunologically homologous sequences in gpl20/41 and their amino acid positions appear above and below, respectively, the sequence of 70K. The sequence boxed at amino acid position 322 in 70K indicates the consensus binding sequences (CBS). Underlined segments A and B indicate major epitope domains of Consensus binding sequences of a family of U1 RNA binding proteins (Krainer et al., Cell 66:383-394 (1991), which is incorporated herein by reference) and the immunologically homologous sequence in V3 of HIV-1 strain IIIB. The solid box indicates the eight amino acid CBS; broken boxes indicate the invariant amino acids, G and F, and the nearly invariant amino acids, A and V. The CBS's of heterogeneous nuclear ribonucleoproteins (hnRNP's) A2 and Bl, which also are involved in splicing, are identical.

The nucleotide sequences o- 70K and gpl20/41 were obtained from GenBank and translai.d into the amino acids shown using a VAX/VMS computer.

Figure 2 illustrates the congruence of neutralizing epitopes in the V3 loop of gpl20 and immunodominant epitopes in 70K. The amino acid sequence of V3 strain IIIB (positions 303 to 338) was obtained from GenBank. The numbered solid lines 1 to 7 represent portions of the V3 sequence that reportedly induce neutralizing antibodies. Regions of V3 that are immunologically homologous to 70K are shaded. Internal to S.V3 are shown the eight amino acids of the CBS of 70K Ul snRNP A and B1 and hnRNP Al The CBS is within the immunodominant B domain of 70K. Also shown is a sequence spanning amino acid positions 239 to 248 of including the immunodominant A domain, which is homologous to amino acid positions 303 to 313 of V3.

e WO 95/25124 PCT/US95/03236 Figure 3 compares the cross-reactivity between HIV-1 antigens gpl20, V3 and gp41 and the anti-RNP antibodies as determined by ELISA. Sera were diluted 1:100 in phosphate-buffered saline/0.1% bovine serum albumin (PBS/BSA; pH Horseradish peroxidase-conjugated goat anti-human antibody (Zymed, Inc.) was used as the second antibody and o-phenyldiamine dihydrochloride was used as the substrate. Optical densities (arbitrary units at OD,o) were recorded with an automated ELISA reader. Horizontal bars indicate the mean of each serum group.

Figure 4 compares the reactivity of anti-RNP and HIV-infected sera to V3 of HIV-1 strain IIIB (closed bars) and strain MN (open bars).

HIV-infected sera 1 to 4 and a normal serum sample (NL); anti-RNP sera 1 to 7. Absolute OD 490 values varied by less than 5% between triplicates performed on the same day.

Figure 5 shows a western blot analysis of HIV-1infected sera against U1 snRNP 70K. Partially purified was isolated from rat liver nuclei by differential centrifugation and affinity chromatography on anti-RNP IgG- Sepharose as described by Douvas et al., J. Biol. Chem.

254:3608-3614 (1979), and Douvas, Proc. Natl. Acad. Sci., USA 79:5401-5405 (1982), each of which is incorporated herein by reference. Samples were fractionated by electrophoresis in a 10% polyacrylamide gel and transferred to nylon membranes for western blot analysis. HIV-infected sera, anti-RNP sera and normal sera were obtained as described in Figure 3 and diluted 1:250 for western blot analysis. Blots were developed using horseradish peroxidase-conjugated goat anti-human Ig (1:3000 dilution) as a second antibody (Tago). Lane 1 contains partially I 1 WO 95/25124 ruI ycLv iJuo 6 purified Ul snRNP 70K antigen, showing 70K and a breakdown product. Western blot strips were reacted with HIV-1-positive human sera (Lanes 2 to 11), with anti-RNP sera from MCTD patients (lanes 12 to 14), with no first antibody (lane 15) or with control human sera (lanes 16 to 18).

Figure 6 shows the deduced amino acid sequence of the 70K polypeptide (Theissen et al., 1986).

DETAILED DESCRIPTION OF THE INVENTION The present invention provides a method for diagnosing HIV-1 infection in a subject by identifying the presence of anti-HIV-1 antibodies that react with an autoantigen such as 70K, which is part of the U1 small nuclear ribonucleoprotein (snRNP) complex. The method is particularly useful in that HIV-1 infection can be detected within one to two weeks of the time of initial HIV-1 infection.

As used herein, the term "70K" refers to a particular polypeptide that is a component of the U1 snRNP complex. The deduced amino acid sequence of the entire polypeptide is shown in Figure 6 (Theissen et al., EMBO J.

5:3209-3217 (1986), which is incorporated herein by reference; GenBank accession number X04654)). The term also is used more broadly to refer to an amino acid sequence comprising a portion of the 70K polypeptide, provided that the amino acid sequence is immunologically homologous to the HIV-1 envelope glycoprotein, gpl20/41 (see Figure As used herein, the term "immunologically homologous" means that either 1) two or more sequences of at least about ten amino acids have at least 50% amino acid identity or 2) two or more core sequences of at least about i LY- WO 95/25124 PCTUS95/03236 7 four amino acids have at least 75% identity and, 3) in addition, identical amino acid sequences are present in the same order in each homolog, and 4) the amino acid sequence can crossreact with an anti-HIV-1 antibody. As used herein, the term "core sequence" means a sequence of at least about four contiguous amino acids that are contained within a longer sequence. For example, the core sequence "GYAF" (SEQ ID NO: 22) is contained within the consensus binding sequence "GYAFIEYE" (SEQ ID NO: Such amino acid sequence homologies are considered significant because the probability of such matches occurring at random are 1 in 1.6 x 106 and 1 in 1.3 x 105, respectively, and because the amino acid sequences crossreact with anti-HIV-1 antibodies. Examples of amino acid sequences of 70K that are immunologically homologous to gpl20/41 are provided in Figure 1. Amino acid sequences of other autoantigens such as the centromere protein, CENP-B, which is antigenic in scleroderma, also can be immunologically homologous to gpl20/41 (Douvas and Sobelman, Proc. Natl. Acad. Sci., USA 88:6328-6332 (1991), which is incorporated herein by reference). Immunologically homologous amino acid sequences can be identified using the methods described herein.

The 70K polypeptide has a relatively hydrophobic amino terminus and a hydrophilic carboxy terminus (Douvas and Sobelman, 1991). Three structures within the polypeptide are important for antibody recognition. These three structures include the A and B domains as well as scattered hydrophilic sequences, including the consensus ERPEEREERRR (SEQ ID NO: 23) sequence and the ERKRR (SEQ ID NO: 24) and RDRDR (SEQ ID NO: 16) motifs (see Figure Domain B encompasses a sequence of eight amino acids that is necessary and sufficient for binding to Ul RNA and is referred to herein as the "consensus binding sequence" (see Surowy et al., Mol. Cell. Biol. 9:4179-4186 (1989), which is incorporated herein by reference).

WO 95/25124 PCTIUS95/03236 8 is a component of the U1 small nuclear RNP nuclear RNA splicing complex, which also consists of a U1 RNA core and the associated polypeptides, A, B, C, D, F and G. The complex of U1 RNA and the 70K, A and C polypeptides defines the RNP antigen, which is the target of IgG anti-RNP autoantibodies in the human systemic rheumatic disorder, mixed connective tissue disease (MCTD), and related syndromes (see below). 70K is the immunodominant polypeptide for anti-RNP autoantibodies and is the target of autoimmune anti-RNP antibodies that are induced in MCTD.

As disclosed herein, autoimmune disorders constitute a paradigm for early HIV-1 infection. The autoimmune disorders such as MCTD, scleroderma and systemic lupus erythematosus (SLE) belong to the class of systemic rheumatic diseases. A common characteristic of these disorders is the presence of T cell-dependent antibody production, wherein the antibodies react to the nuclear RNA splicing particle, U1 snRNP. In addition to reacting with Ul snRNP, the anti-RNP antibodies crossreact with epitopes that are present on HIV-1 gpl20/41 and are immunologically homologous to amino acid sequences of 70K. This crossreactivity forms a basis of the present invention.

shares multiple immunologically homologous regions with the major neutralizing epitopes of gpl20/41 (Table These neutralizing epitopes also are the dominant sites for reactivity of anti-70K autoantibodies that occur in MCTD. One immunologically homologous region Sis shared between the functionally essential RNA binding site of 70K and the apex of the V3 loop, which contains protease cleavage sites, which have a role in viral infectivity (see below and Figure Another cluster of immunologically homologous regions involves the hydrophilic carboxy terminus of 70K and the epitopes in gpl20/41 (see Figure One of these epitopes (sequence 4) is a peptide P Table 1: Beneficial ind deleterious epitopis in gpt20/41: homologies to 70K and MI-C determinants.

A. Neutralizing gpl120/41 epitopes homologous to 70K Deleterious and M HC-homologous epitopes gpI120 Ret gp4l Ref Ref I. 303 TRP*NNI1RKR 1, 38 4. 645 LiErsQNQQI--KN 1. .1 6. 579 RILAVERYLKDQQLGIWGCSGKI.I.C 42 (239) TREERMERKR (80) LIEDQQQRQ 7. 644 SLEQAQIQQEKNEQELLKL 42 8. 837 EGTDRVI (MIIC 11) 11 2. 311 RlQRCrGRAr--VTlG 1, 3, 6, 5. 732 Gpr)RPEGtEEEGGEnDRDR 3,.41 9. 805 SDAKAYD)TEV (MIIC this analysis (98) PGRA 33, 38, (513) GPDGPDCPEEKORDRDRER 10. 56 GSTMGAASMITLTV (MiWC I AND 11) this analysis (322) GYAFISY 40 (408) RDRDRDR 11. 527 QELKNSAVSL (MItC 11) this analysis (460) DRDR 3. 469 RI.G(GI)MR 35 (542) RDRDR (471) GG0DM -(368) RLGGG (484) LRGOG Tbe HImv- i sequences listed in panel A in hold type wcre identified as neutralizing epitopes in the published studies given as references by each sequence. Ntimhers to the left of each sequence indicate the position of the First amino acid in the sequence. 70K homologies appear below each HIV sequence. with aa positions indicated in parentheses. Homologies were identified as described in Fig. 1. The deleterious and MH-C-homologous epitopes in panel B3 were excerpted from the studies referenced beside each of the sequences 6-8. Additional MH-C homologies in gp4l (sequences 9 and I I in the table) and in gp120 (sequence 10) were identified as described in Materials and Methods.

WO 95/25124 PCTIUS95/03236 sequence that strongly inhibits HIV-1 replication (Jiang et al., Nature 365:113 (1993)).

Although neutralizing antibodies are considered essential for immunoprotection against many viruses, their role in HIV-1 infection is still ambiguous. Primary neutralizing determinants for anti-HIV-1 antibodies cluster in three regions of gpl20/41: the V3 loop, the C4 domain and gp41 (see, for example, Moore and Ho, J. Virol. 67:863- 875 (1993). However, antibodies elicited in high titers by vaccination with HIV-1 proteins may not target the most effectively neutralizing epitopes. Moreover, monoclonal antibodies acting in synergy can enhance neutralization or can enhance HIV infection. Adding further to the complexities of understanding and therapeutically amplifying protective immunity is the role of discontinuous or conformationally sensitive epitopes, particularly in the C4 domain (see Moore and Ho, 1993).

As disclosed herein, IgG antibodies produced in patients suffering from the autoimmune disorder MCTD crossreact with HIV-1 gpl20/41. The primary antigen for MCTD patients is the RNA splic .g protein, 70K, which shares immunologically homologous regions with gpl20/41 (Figure The cross-reactivity between anti-RNP antibodies and gpl20/41 is attributable to clusters of epitopes in V3 and gp41 homologous to 70K and appears to be sequence specific.

Autoimmune T cell clones and antibodies produced by B cells exist at low levels in normal individuals. In particular, a background level of anti-RNP antibodies can be detected in normal individuals and these antibodies specifically react with 70K as shown by enzyme-linked immunosorbent assays (ELISA's) and western blot analysis (see Example II). Thus, normal individuals already have generated a primary immune response against 70K, this WO 95/25124 PCT/US95/03236 11 response being analogous to having memory immune cells that are primed to generate a secondary immune response. These memory immune cells can be stimulated by HIV-1 infection and, as a result, HIV-1 infection can be diagnosed within one to two weeks following initial HIV-1 infection by detecting the presence of a greater than normal level of circulating anti-70 antibodies in a subject.

Imrmunologically homologous regions have been identified between eight polypeptides, which are major antigens in the systemic rheumatic disorders, and several proteins involved in immune cluster viruses, including HIV-1, herpes simplex virus type 1 (HSV-1), Epstein-Barr virus (EBV) and cytomegalovirus (CMV) (Douvas and Sobelman, 1991). However, only 70K and CENP-B have significantly more homology to the immune cluster viruses than do normal proteins. In fact, not only does 70K share extensive amino acid sequence homology with gpl20/41, but there alr.) is a congruence of dominant epitopes between the two proteins that is strc.,gly predictive of mutual antibody crossreactivity (see Figure In contrast, 70K lacks the gpl20/41 epitopes that are associated with deleterious effects, including enhancement of infectivity by antibodies, anergy, immunosuppression and accelerated demise of CD4 T cells (see Table Cellular and humoral immune responses to HIV-1 that are neutralizing and, therefore, potentially protective, have multiple targets (epitopes) on gpl20/41.

SMajor neutralizing determinants for anti-HIV-1 antibodies are found in a region of conserved and variable amino acid sequences in the V3 loop of gpl20 (see Figure The conserved sequences form potential proteolytic cleavage sites, including a trypsin site, GPGR I AFVT, and a chymotrypsin-like site, GPGRAF i VT. Cleavage at these sites may be required for fusion of the viral and cellular i Vr i tea u aiea u s uay u Signature 7 I Signatory's Name enn.i s.E. D. gerty....en.ior.. V.i.ce esj.dent...Admi.n station....

n mA:ionr,1.do F.B. RICE CO. PATENT ATTORNEYS I WO 95/25124 PCT/US95103236 12 membranes and, therefore, for HIV infection of cells.

Major neutralizing epitopes also have been identified in gp41, including some discrete regions involved in viralhost cell fusion and syncytium formation.

The epitope, GRAFVTIG (SEQ ID NO: 25), which is in the V3 loop of HIV-1 strain IIIB gpl20 (V3 IIIB) is homologous to the functionally essential Ul RNA-binding site of 70K. Results obtained using ELISA assays revealed a mean reactivity of anti-RNP antibodies to V3 IIIB that is as high as that of HIV sera (see Figure Similarly, the V3 loop of HIV-1 strain MN contains the framework sequence GRAFXT (SEQ ID NO: 26; where indicates any one independently selected amino acid) and also crossreacts with anti-RNP antibodies, as do hydrophilic epitopes in gp41 homologous to the carboxy terminus of 70K. The GRAFXT (SEQ ID NO: 26) sequence also occurs in the V3 loop of HIV- 1 strains SF2 and SC.

Strong crossreactivity between HIV sera and also was observed using western blots (see Figure In contrast, antibodies from a related autoimmune disorder, Sjbgren's syndrome are neither V3 nor gp41 selective.

Thus, the substantial crossreactivity likely is due to conserved, antigenically dominant B cell epitopes having homologous counterparts in 70K and gpl20/41.

The molecular mimicry and mutual crossreactivity between 70K and gpl20/41 antigens and antibodies have significant functional, immunological and therapeutic implications. For example, amino acid positions 321 to 328 of the V3 loop and the CBS of 70K and similar Ul RNAbinding proteins contain the conserved framework sequence that is immunologically homologous to the V3 loop of HIV-1 strains IIIB and MN GRAFVT (SEQ ID NO: 27) and GRAFYT (SEQ ID NO: 28), respectively, as delineated in Figure l.B.

Thus, the lack of marked strain specificity in anti-RNP 'i gi. 1 1 WO 95/25124 PCTIUS95/03236 antibodies (Figure 4) can be attributed to their affinity for the conserved sequences.

The V3 sequence, GRAFVT (SEQ ID NO: 27), and its immunologically homologous forms in Ul RNA-binding proteins are referred to collectively as the multifunctional (mf) motif because they are important in five different biological contexts: the mf motif is a primary neutralizing determinant for antibodies in HIV infections; it is contained in the dominant epitope domain of it has an essential role in RNA splicing (essentially all anti-RNP antibodies react with domain B of 70K, which contains the mf motif, and inhibit RNP splicing); it contains proteolytic cleavage sites that may have an important function in viral and cell membrane fusion, and therefore in HIV infectivity; and it is an epitope not only for antibodies but also for T cells. Thus, the mf motif participates in interactions with an RNA molecule, an enzyme (protease), an IgG molecule and a T cell receptor.

The extended sequence, RIQRGPGRAFVTIG (SEQ ID NO: 29), the core of which is the mf motif, is an epitope domain for both CD4' T helper (Th) cells and CD8 cytotoxic T lymphocytes (CTL) and can restimulate T cells that were previously exposed to HIV-1. These results, along with the role of the mf motif in RNA splicing and the potent inhibition of splicing by anti-RNP antibodies were discussed above, indicate that the mf motif can be a common immunogen in both autoimmune disease and HIV infection.

The mf motif also can have a role in immunoregulation in these diseases. For example, the anti- V3 and anti-70K titers of an MCTD patient that has been infected with HIV-1 for seven years fluctuate in tandem while anti-gpl20 titers remain at high levels. In addition, the patient's CD4* T cell counts fluctuate in parallel with the anti-V3/70K titers. Thus, the loss of i i i

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WO 95/25124 PCTIUS95/03236 14 lymphocyte responsiveness to the mf motif may result in the demise of CD4' lymphocytes. In contrast, perpetual responsiveness to this motif occurs in MTCD patients and results in sustained autoimmunity.

The results disclosed herein indicate that an autoimmune disease such as MCTD, scleroderma or SLE can be a useful model for developing immunoprotective strategies that allow sustained, high level immunity. The autoimmune model allows, for example, the development of an optimal spectrum of antibodies. For example, in addition to the lack of specificity for deleterious HIV-1 epitopes (Table anti-RNP antibodies are harmless in MCTD and, in fact, are correlated with a better clinical prognosis. Moreover, a significant number of anti-RNP sera inhibit syncytium formation in HIV-1-infected target cells by greater than thereby demonstrating neutralizing potency. Also, can be used as a surrogate immunogen for stimulating the immune system at both the B and T cell level. Furthermore, in addition to containing the mf motif, 70K also presents multiple epitopes, which can act in concert to induce immunoresponsiveness.

The results provided herein also indicate that the autoimmune model can be useful for designing ligands based on Ul RNA and the mf motif. For example, a ligand that binds to a functionally essential site on 70K also can be useful for binding immunologically homologous sites on V3. Such ligand binding can abrogate the role of V3 in infectivity. One target for such a ligand is Ul RNA, which contains an 8-10 nucleotide sequence that binds specifically to the CBS of 70K. Ul, but not U2, can bind to gpl20 (not shown).

Because antibody production in both MCTD and HIV- 1 infection is T cell-dependent, common T cell and B cell memory-like clones can be activated from a latent state in Li jS WO 95/25124 PCTUS95103236 these two diseases. The activation of these cells can result in both activation of latent cells and production of crossreacting anti-HIV-1 antibodies. The crossreacting antibodies and the presence of activated anti-70K latent cells provide a method for diagnosing HIV-1 infection at an early stage. As used herein, the term "latent cell" means an autoimmune clone that is analogous to a memory cell, which is primed to react rapidly upon contact with a specific antigen.

A diagnostic test can be performed by contacting or CENP-B with a sample obtained from a subject suspected of being infected with HIV-1 and, therefore, of having produced crossreacting antibodies. The sample can be, for example, a tissue sample or a sample of a body fluid. The presence of anti-70K or anti-CENP-B antibodies in the serum can be determined using well known assays such as ELISA assays or western blots (see Example II). An autoantigen such as 70K can be obtained, for example, by extraction from an uninfected tissue or can chemically synthesized or produced using recombinant DNA methods, as described below.

An autoantigen such as 70K can be attached to a solid substrate such as a plastic tissue culture well and antibodies can be detected using an ELISA assay.

If 70K is obtained from tissue extracts, the 70K antigen may not be 100% pure. In this case, the preferred method of diagnosis is by western blot analysis, wherein 70K is 1 fractionated by electrophoresis and transferred to a paper or nylon support. The western blot assay allows for specific reactivity of an anti-70K antibody with a polypeptide that migrates at the expected moleculer mass of can be identified. On the otherhand, if recombinant is used as an antigen, an ELISA assay can provide sufficiently precise diagnosis.

i inexpensive diagnostic method that can identify an HIV-1i I I p I I I I I I I WO 95/25124 PCT/US95/03236 16 Early diagnosis of HIV-1 infection is desirable for many reasons. In particular, the disclosed method for early diagnosis of HIV-1 infection is useful for screening blood samples. The use of the disclosed method can identify blood samples that are obtained from a donor that was infected with HIV-1 within the prior one to two weeks of donating the blood. Other potential methods of identifying HIV-1 infected blood at this stage of infection are prohibitively expensive and, therefore, are not used as a matter of routine screening.

It can be desirable to provide a kit for performing the disclosed method of diagnosis. Such a kit can contain 70K attached to a solid support and also can contain, if desired, standard reagents such as a predetermined amount of an anti-70K antibody. Such reagents can provide a means to readily determine whether a sample obtained from a subject contains a greater than normal amount of circulating anti-70K antibody. It is recognized that a population of normal serum samples must be analyzed in order to determine the "normal" level of antibody in an person that is not infected with HIV-1. However, methods to obtain a statistically significant normal level of anti-70K antibodies are well known and routine in the art.

The invention also provides a method of stimulating an immune response against HIV-1 in a subject comprising immunizing the subject with an amino acid sequence of an autoantigen such as 70K that is a surrogate for neutralizing epitopes present on HIV-1. Amino acid sequences of 70K that cross-stimulate a protective or neutralizing immune response against HIV-1 can be identified by the immunologically homologous regions shared between these sequences in 70K and gpl20/41. Examples of I'i amino acid sequences are provided in Figures 1 and 2.

7 sareactive and, therefore, cross-stimulating amino acid *i are shown in linear order in bold type (Theissen et al.,

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1 WO 95/25124 PCTIUS95/03236 17 sequences can be identified using methods such as ELISA and western blot analysis as described herein (see Example II).

Since autoimmune T cell clones and antibodies produced by B cells exist at low levels in normal individuals, these normal individuals have latent immune cells that are primed to generate a secondary immune response. It follows that 70K can be used as a surrogate immunogen that is useful as a vaccine to provide continuous stimulation of the immune system. As used herein, the term "surrogate immunogen" means an autoantigen such as 70K or an amino acid sequence of an autoantigen that is immunologically homologous to a neutralizing epitope present on gpl20/41 and that can stimulate an immune response in a subject against HIV-1. Thus, a surrogate immunogen is an amino acid sequence that is immunologically homologous to HIV-1 and that, in addition, can stimulate an immune response.

A surrogate immunogen can be immunogenic by itself or can be attached to a carrier molecule such as bovine serum albumen or an inert carrier such that the surrogate immunogen-carrier complex can stimulate an immune response. An immune response can be stimulated in vivo or ex vivo. For example, immune cells such a T cells and B cells can be obtained from a subject and placed in a tissue culture medium. The cells can be contacted with a surrogate immunogen, which can stimulate the immune cells by inducing a primary or secondary immune response.

Anti-RNP antibodies are crossreactive with gpl20/41 and are effective in arresting the infectivity of HIV-1 in infected cells in vitro (data not shown).

Specifically, the anti-RNP antibodies can recognize the GRAFTVIG (SEQ ID NO: 25) sequence. This result indicates that a surrogate immunogen such as 70K or GRAFTVIG (SEQ ID NO: 25), for example, can be used to stimulate memory anti-

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WO 95/25124 PCTIUS95/03236 18 cells in a subject and the anti-70K antibodies can crossreact with homologous epitopes present on gpl20/41.

Use of a surrogate immunogen such as 70K is advantageous because it is an autoimmune protein that represents an enrichment of sequences that can stimulate the early shared clones. As used herein, the term "shared clones" means anti-70K latent T cells and B cells that can be rapidly activated to react with HIV-1 or can produce crossreactive anti-gpl20/41 antibodies that can neutralize HIV-1. The use of a surrogate immunogen also is advantageous in that it can provide co-amplification or synergistic amplification of shared clones with gpl20/41.

In addition, a surrogate immunogen such as 70K does not contain amino acid sequenc'zs that stimulate "harmful" antibodies that mediate the df'aberious effects associated with HIV-1 infection (see Furthermore, the use of a surrogate immunogen such as 70K precludes the introduction of viral-derived material into a subject.

An immune response against HIV-1 can be stimulated in a subject by administering a therapeutically effective amount of a surrogate immunogen, which comprises an amino acid sequence of an autoantigen such as 70K that crossreacts with neutralizing epitopes present on gpl20/41 and stimulates an immune response, and a pharmacologically acceptable carrier. Pharmaceutically acceptable carriers are well known in the art and include aqueous solutions such as physiologically buffered saline oz other solvents or vehicles such as glycols, glycerol, oils such as olive oil or injectable organic esters. As used herein, the term "therapeutically effective amount" means an amount of a surrogate immunogen that can stimulate an immune response.

The amount will vary, of course, depending, for example, on whether stimulation of the immune response is in vivo or i ex vivo or on whether the administration is a first administration or a booster administration. A as a second antibody (Tago). Lane 1 contains partially WO 95125124 PCT1US9503236 19 therapeutically effective amount can be determined using methods known in the art (see, for example, Harlow and Lane, 1988).

A composition comprising a surrogate immunogen and a pharmaceutically acceptable carrier also can contain an adjuvant if desired. Adjuvants, which include, for example, Freund's complete or incomplete adjuvant, are known in the art and commercially available (Ribi Immunochem Research, Inc.; Hamilton, MT). The addition of an adjuvant can affect the amount of surrogate immunogen that is required to obtain a therapeutically effective amount.

A pharmaceutically acceptable carrier also can contain other physiologically acceptable compounds that act, for example, to stabilize the surrogate immunogen or increase the absorption of the surrogate immunogen. Such physiologically acceptable compounds include, for example, carbohydrates, such as glucose, sucrose or dextrans, antioxidants, such as ascorbic acid or glutathione, chelating agents, low molecular weight proteins or other stabilizers or excipients. One skilled in the .art would know that the choice of a pharmaceutically acceptable carrier, including a physiologically acceptable compound, depends, for example, on the route of administration of the composition and on the particular physico-chemical characteristics of the surrogate immunogen. Various routes of administration are known in the art and include, for example, intravenous, intradermal and subcutaneous injection, oral administration and transdermal administration.

The amino acid sequences of an autoantigen such as 70K that crossreact with neutralizing antibodies can be obtained, for example, by chemical synthesis of the amino acid sequences. A particularly useful means for obtaining at least about ten amino acids have at least 50% amino acid identity or 2) two or more core sequences of at least about WO 95/25124 PCT/US95103236 sufficient amounts of an amino acid sequence such as a peptide is by the use of recombinant DNA methods, which are well known in the art (see, for example, Sambrook et al., Molecular Cloning: A laboratory manual (Cold Spring Harbor Laboratory Press 1989), which is incorporated herein by reference). For example, the polymerase chain reaction (PCR) can be used to amplify the nucleotides encoding an amino acid sequence of 70K that is immunologically homologous to gp120/41 and the amplified sequence can be cloned into an expression vector, which allows for transcription and translation of the cloned sequence. The amino acid sequence then can be isolated in relatively pure form. Methods for amplifying a nucleotide sequence and cloning and expressing the nucleotide sequence are well known in the art (see, for example, Sambrook et al., 1989; see, also, Ehrlich, PCR Technology: Principles and Application for DNA Amplification (Stockton Press 1989), which is incorporated herein by reference).

Methods for stimulating an immune response in a subject are well known in the art and described, for example, in Harlow and Lane, Antibodies: A laboratory manual (Cold Spring Harbor Laboratory Press 1988), which is incorporated herein by reference. For example, the composition can be administered intradermally, intramuscularly or intravenously. In addition, it can be advantageous to administer one or more booster immunizations. The need to administer a booster immunization can be determined experimentally by measuring the presence of anti-70K antibodies in a subject's serum using the methods described herein.

The invention also provides a skin test that is useful for diagnosing a subject having an HIV-1 infection, A composition comprising an amino acid sequence that id immunologically homologous to an epitope present on HIV-1 and a pharmacologically acceptable carrier is administered jfII^ (see Surowy et al., Mol. Cell. Biol. 9:4179-4186 (1989), which is incorporated herein by reference).

II I: 1 1 WO 95/25124 PCTIUS95/03236 21 intradermally to a subject suspected of being infected with HIV-1. A diagnosis of HIV-1 infection is made by observing evidence of an immune response at the site of intradermal injection. Such evidence, whih includes redness or swelling at the site of injection, ib indicative of a delayed-type hypersensitivity response, which, in turn, provides a positive diagnosis of HIV-1 infection.

The diagnostic skin test is performed by intradermal injection of about 0.1 ml of a composition comprising a surrogate immunogen, which is an amino acid sequence that is immunologically homologous to an epitope present on HIV-1 and can stimulate an immune response, and a pharmacologically acceptable carrier (see Example III).

The test results are evaluated by measuring the maximum extent of erythema and induration or, if desired, by biopsy. The test is particularly useful in that the subject being tested can examine the site and, in the case of a negative result, can avoid a return visit to the health care provider for evaluation of the test result.

The following examples are intended to illustrate but not limit the invention.

I EXAMPLE I Immunoloqically Homologous Regions of and HIV-1 _p120/41 This example demonstrates the high degree of homology shared between the immunodominant regions of and the neutralizing epitopes of gpl20/41 and identifies amino acid sequences of 70K that are immunologically homologous to HIV-1.

The HIV-1 strain IIIB DNA sequence (K03455) was obtained from GenBank and translated into the amino acid sequences for gpl20 (amino acid positions 1 to 511) and gp41 (512 to 856). Sequences for V3 IIIB and V3 MN were rl WO 95/25124 PCT/US95103236 22 obtained from D'Souza et al., AIDS 5:1061-1070 (1991), which is incorporated herein by reference. The DNA sequence of U1 snRNP-associated 70K was obtained from GenBank AX04654) and translated into the amino acid sequence. The 614 amino acid sequence, rather that the shorter published 438 amino acid sequence, was used for the reasons discussed by Douvas and Sobelman, 1991. The shorter sequence lacks the extreme amino terminus, which is present in the longer sequence. However, this omission does not affect the overall homology between 70K and gplO0/41.

Homologous sequences were defined as sequences of at least ten amino acids having at least 50% amino acid identity or as sequences of at least four amino acids having at least 75% identity, with identical amino acid sequences occurring in the same order in each homolog. The probabilities of such matches occurring at random are 1 in 1.6 x 106 and 1 in 1.3 x 10 s respectively. Consensus binding sequences (CBS's) were identified by visual inspection. Other regions of homology were identified by dot-matrix plot (window size 10, matches 5, score 100, ktup 2, speed 1) using the GenePro routine (Riverside Scientific; Riverside, CA) and a VAX/VMS computer.

Extensive analysis of 41 control proteins (11,743 amino .acids) also was performed to confirm the significance of the identified homologous regions (pee Douvas and Sobelman, 1991).

Regions of homology shared between 70K and gpl20/41 are shown in Figure l.A. The large number of homologous regions (25) is apparent and irnvolves 206 amino acids of 70K (33.5% of the amino acid sequence). One region of homology is the eight amino acid binding site of for U1 RNA, which is contained in domain B (see rectangle at positions 322 to 329). In fact, this eight amino acid sequence is not an exact sequence but is a

IV

WO95/25124 PCTUS95103236 b o a -u several nuclear U1 RNA binding proteins (Figure The CBS contains two invariant amino acids, G and F, and two Si i i cis A as in.

repeating RDRDR (SEQ ID NO: 16) motif, are homologous 513 in 70K and position 732 in gp41; as well as three U o i additional sequences in 70K (see Table 1, sequence WO 95/25124 PCT/US95/03236 (Pinus et al., J. Clin. Invest. 91:1987-1996 (1993)).

several nuclear Uc RvA binding proteins i (Figure The CBS contains two invariant amino acids, G andlso mF, and two nearly invariant amino acids, A and V, as indicated Although the V3 domain shown in Figure l.B. was obtained from HIV-1 IIIB, the GRAFXT (SEQ ID NO: 26) configuration also occurs in strains MN, SF and SC.

Hydrophilic sequences in 7ofK, including the repeating RDRDR (SEQ ID NO: 16) motif, are homologous 0 primarily to gp41 (Figure These regions of homology include a19 amisno acid sequence, which begins at position h513 in 70Ktand position 732 in gp4ii, th well as three additional sequences in 70K (see Table 1, sequence near the V3 apex. Figure 2 also sho1s the CBS's of U1RNA This sequence in gp4 is a major tareget of neutralizing antibodies in individuals vaccinated with recombinant (Pincus et al., J. Clin. Invest. 91:1987-1996 (1993)).

The congruence between framework sequences in the Ul RNA CBS and conserved amino acids in V3 (Figure l.B.) indicates that ligands that bind to the CBS also may bind specifically to V3 and, therefore, can be iomunologically homologous amino acid sequences. The immunologically iomologous regions shoarec between 70es arnd the V loop involve a major cluster of neutralizing determinants for HIV antibodies. Two amino acid sequences of 70K align with the V3 sequence (Figure One sequence, which spans the amino terminus of V3. In addition, the CBS from domain B (sequence a) is immunologically homologous to a sequence near the V3 apex. Figure 2 also shows the CBS's of U1 RNA binding polypeptides A and B1 (sequences b and c) o Overlapping neutralizing domains that have been 1 ideantified in V3 are shown as solid lines around the loop 2 in Figure 2. Synthetic peptides were used in direct and i competition ELISA's to showthat a broad regionof 24 amino B (qn a) is i o c h o u to a sequence near the apex. Figure 2 also shows the C BS's of U 1 1 1

A*

3 binding 1 1 poypptde A an Bi (sequences b'and c) Ovrapn netalzn doan tha hav been 1 idnife V3 ar shw as soli lie arun th loop in-~ Fiur 2. Synthetic-- pepide wer use in diec and u (see Example II). Thus, normal individuals already have jgenerated a primary immune response against 70K, this !'1 WO 9525124 PCTul)S95/03236 24 acids (line contains major neutralizing epitopes recognized by HIV-1-infected human sera (Rusche et al., Proc. Natl. Acad. Sci., USA 85:3198-3202 (1988). In addition, broadly neutralizing monoclonal antibodies identify a dominant neutralizing region (line 2; Durda et al., AIDS Res. Hum. Retroviruses 6:1115-1123 (1990)).

Furthermore, a sequence largely overlapping line 2 (line 3) reacted with and blocked neutralization by a large panel of HIV-infected sera (Broliden at al., Proc. Natl. Acad. Sci., USA 89:461-465 (1992)). Sequences delineated by lines 4 and 5 reacted with and blocked neutralization by polyclonal and monoclonal anti-HIV antibodies, respectively (Javaherian et al., Proc. Natl. Acad. Sci.., USA 86:6768- 6772 (1989); Laman et al., Virology 66:1823-1831 (1992)).

At the amino terminus of the V3 loop, the region delineated by line 6 reacted with type-specific neutralizing HIV antibodies (Kenealy et al., AIDS Res. Hum. Retroviruses 5:173-182 (1989)).

The superposition of the six lines reveals that 2C the major neutralizing V3 epitope cluster coincides with sequences in the two immunodominant domains A and B of and that immunologically homologous regions of 70K involve of the V3 loop. Fine mapping of the apex of the V3 loop using human monoclonal antibodies further emphasizes the conserved ,ature of key immunologically homologous sequences. Two short overlapping epitopes, GPGR (SEQ ID NO: 30) and GRAF (SEQ ID NO: 31; lines 7 and are present in divergent strains, including MN and IIIb, and are the targets of broadly neutralizing antibodies (Gorny et al., Proc. Natl. Acad. Sci., USA 88:32383242 (1991); Gorny et al., J. Immunol. 150:635-643 (1993)). An additional conserved segment (line 8) also is the target of broadly neutralizing antibodies and is continuous with the GPGR epitope in MN, but not in IIIB.

M a 4 I WO 95/25124 PCT/US95/03236 Discontinuous and Jonformationally sensitive epitopes are important in the neutralizing C4 cluster of epitopes that involve the CD4-binding site of gpl20, but less so in the V3 cluster. The linear V3 epitope delineated by lines 7 and which are targets of broadly neutralizing antibodies, are congruent with highly conserved amino acids in the U1 RNA-binding site of 70K and related splicing proteins. In 70K, as in other nuclear autoimmune antigens, the immu; -dominant sites also are the functionally critical sites and virtually all anti-RNP antibodies inhibit RNA splicing.

EXAMPLE II Crossreactivitv of 70K and HIV-1 qp120/41 This example demonstrates that antiserum obtained from HIV-1 iudividuals can react with 70K and anti-RNP antibodies can react with gpl20/41.

Anti-RNP sera were obtained from MCTD patients treated in the outpatient Rheumatology Clinic of the University of Southern California Health Sciences Center (Los Angeles, CA) and were confirmed positive for antinuclear antibody (ANA) as determined by immunofluorescence and positive for anti-RNP as determined by double diffusion. Six donors having a clinical diagnosis of Sjbgren's syndrome were confirmed to be SS-A/Ro antibody positive. Normal sera were obtained from institutional personnel and were confirmed ANA negative. Sera from HIV-1 infected donors were obtained from the University of Southern California Health Sciences Center and were confirmed HIV* by western blotting using a kit obtained from Organon Teknika (Durham, NC). The immunoassay results presented in Figure 3 and Figure 4 were confirmed by isolating IgG from some anti-RNP sera and some control sera by ammonium sulfate fractionation and DEAR chromatography as described 2 ouvas (1982).

1' i 1 1 i 1 I 1 Thus, the lack of marked strain specificity in anti-RNP WO95/25124 PCT/US95/03236 26 Recombinant HIV-1 gpl20 (Du Pont; Boston, MA), V3 IIIB (Sigma; St. Louis, MO) and gp41 and V3 MN (ABT; Cambridge, MA) were purchased. Partially purified 70K for western blot analysis was isolated from rat liver (Pelfreeze; Rogers, AK) using the nuclear fractionation and antibody affinity chromatography method described by Douvas et al. (1979) and Douvas (1982).

ELISA assays were performed essentially as described by Crow et al., Cell. Immunol. 121:99-112 (1989), which is incorporated herein by reference. Briefly, saturating concentrations of antigen were adsorbed to plastic microti.ter plates for 12 hr at 4 0 C, then the plates were washed and unreacted sites were blocked with 1% bovine serum albumin-phosphate-buffered saline (BSA/PBS; pH Sera were diluted 1:100 in 0.1% BSA/PBS and added to the appropriate wells. Samples were incubated at 4 °C overnight. Following incubation, horseradish peroxidase (HRP)-conjugated goat anti-human immunoglobulin (Ig) (2vmed, San Francisco, CA) was diluted 1:1000 and added to the sample. Incubation was continued for 1 hr at room temperature and bound antibody was identified using ophenyldiamine. Optical densities were determined, at 490 nm using an automated ELISA reader.

Electrophoresis was performed using polyacrylamide gels and western blot analysis was performed as described by Towbin et al., Proc. Natl. Acad. Sci., USA 76:4350-4354 (1979), which is incorporated herein by reference. Sera were diluted 1:250 for western blot analysis. Blots were developed using HRP-conjugated goat anti-human Ig (1:3000 dilution) as a second antibody (Tago; Concord, CA).

The crossreactivity of 70K and gpl20/41 that was Spredicted by the amino acid sequence homology analyses (Figures 1 and 2) was coifirmed using ELISA to compare the i' U, L~ne patient's CD4 T cell counts fluctuate in parallel with the anti-v3/70K titers. Thus, the loss of t WO 95/25124 PCTIUS95/03236 27 reactivity of twelve HIV-1-positive sera, ten anti-RNP sera, a rheumatoid control group of six Sjbgren's syndrome sera (SS) and eight normal sera (NL) to HIV-1 antigens (Figure The HIV-1 antigens used were recombinant gpl20, V3 (IIIB) and gp41.

The mean reactivity to gp120 was highest for HIV sera (0.67 OD,, 0 as compared to RNP SS (0.14) and NL (0.03) sera (Figure Similarly, HIV sera had the highest reactivity against gp41 as compared to RNP SS (0.15) and NL (0.09) sera (Figure In contrast, RNP sera had the highest reactivity (0.29) against the V3 loop as compared to HIV SS (0.16) and NL (0.09) sera (Figure The results also indicate that the reactivity of the SS group of sera are higher than normal but are essentially the same for all three HIV antigens. In addition, the SS sera react with the Ro/SSA antigen, which has no significant structural homology to gpl20/41. Thus, the SS sera demonstrate the well known general hyperreactivity of autoimmune sera, but no evidence of epitope specificity based on structural homology.

The results indicate that HIV sera have the highest reactivity to gpl20, which contains a number of additional epitopes, including those in the C4 cluster that are not homologous to 70K. In contrast, the anti-RNP sera demonstrate a greater than two-fold higher reactivity to V3 than to the entire gpl20 molecule and a reactivity to V3 that is equivalent (slightly higher) to the reactivity of HIV sera. This result is consistent with the higher concentration of immunologically homologous regions in the V3 loop that are congruent with dominant epitopes in (see Figure Anti-RNP sera also are two-fold more reactive to gp41 than to gpl20. As indicated in Figure 1, a large proportion of the gp41 immunologically homologous 1 infection is T cell-dependent, common T cell and B cell memory-like clones can be activated from a latent state in WO95/25124 PCT/US95/03236 28 regions corresponds to hydrophilic sequences and motifs such as the repeating RDRDR (SEQ ID NO: 16) sequence, which are epitopes in 70K and which account for a large proportion of the hydrophilic carboxy terminus of These include a 19 amino acid sequence, which begins at position 732 of gp41 (see, also, Table 1).

The reactivity of HIV and anti-kNP sera against the V3 loop of two divergent HIV-1 strains, IIIb and MN, also was compared by ELISA. A marked strain specificity of HIV sera for MN over IIIB was observed (Figure mean values of 0.536 and 0.144, respectively, or a 3.7-fold preference for MN). Figure 4.A. also shows the reactivities of a normal serum (NL) for IIIB (0.02) and MN The anti-RNP sera had only a 33% greater reactivity against IIIB than MN (Figure These results provide experimental support for the homology analyses, which revealed that conserved invariant sequences in the CBS of 70K and related Ul RNA-binding proteins were immunologically homologous to conserved amino acid sequences in the V3 loop (Figures 1 and 2).

Anti-RNP autoantibodies are predominantly of the IgG isotype. To determine the isotype of the crossreactive antibodies detected by ELISA assays (Figures 3 and 4), heat-inactivated sera were subjected to ammonium sulfate fractionation and ion-exchange chromatography as described by Douvas (1982). The results indicated that 95% of the reactivity in each serum examined was due to IgG antibodies and that less than 5% was due to IgM. Moreover, when comparisons shown in Figure 4 were repeated using purified IgG, the results again showed marked preference for MN by HIV sera and no substantial selectivity by the anti-RNP autoantibodies.

Western blot analysis was used to determine whether sera from HIV-infected individuals recognize I' s Lil_ ~UL LL~5L.J.J WO 95/25124 PCT/US95/03236 29 epitopes. 70K was partially purified from nuclei -as described by Douvas (1982). All ten HIV sera reacted with as well as a breakdown product of 70K (Figure Eight of the HIV sera reacted strongly with 70K and one serum (lane 7) consistently reacted more strongly than anti-RNP sera (lanes 12 to 14). The strong crossreactivity of HIV sera with 70K indicate that the T cells of HIVinfected individuals can react with a 70K epitope.

Table 1 compares the immunologically homologous regions of 70K with gpl20/41 epitopes that are associated with viral neutralization, which is a measure of immunoprotection, and epitopes that are associated with deleterious effects. Although the gpl20/41 sequences that are associated with enhancement of infection, anergy, or immunosuppression include some sequences that are homologous to class I and class II MHC molecules, none of the sequences were homologous to 70K (Table In contrast, neutralizing epitopes of gp120/41 aligned with a total of eleven non-overlapping immunologically homologous regions. In particular, the sequences 1 and 5 include two major antigenic motifs of ERKR (SEQ ID NO: 32) and RDRDR (SEQ ID NO: 16). The RDRDR (SEQ ID NO: 16) motif is contained in a long sequence that shares extensive homology to sequence 5 of gp41. Sequence 5 of gp41 is considered a dominant antigen both as a site of reactivity for HIV-infected sera and as a target of antibodies from volunteers vaccinated with The homology between the 70K CBS and sequence 2 was discussed above.

Sequence 3 of gpl20, RLGGGDMR, is immunologically homologous to three permutations of a 70K sequence, GGGDM (SEQ ID NO: 33), RLGGG (SEQ ID NO:' 34) and LRGGG (SEQ ID NO: 35). Antibodies to peptides containing this sequence are neutralizing, although at low titers.' Deletion

A

I

t i amino acid sequences are provided in Figures 1 and 2.

sreactive and, therefore, cross-stimulating amino acid l i i n WO 95/25124 PCT/US95/03236 mutation of gpl20 in the GGG triplet and beyond abolishes its ability to bind to CD4* cells (Kowalski et al., Science 237:1351-1355 (1987)). Sequence 4, a neutralizing epitope in gp41, which is immunologically homologous to a sequence near the amino terminus of 70K, also is contained in a peptide that directly inhibits HIV-1 replication (Surowy et al., 1989).

The results presented above indicate that (1) anti-RNP antibodies cross-react with the V3 loop at titers equivalent to those of HIV' sera (Figure major neutralizing epitopes in V3 coincide with dominant epitopes in 70K, including its U1 RNA CBS (Fig hydrophilic epitopes in gp41 are immunologically homologous to antigenic, hydrophilic motifs in 70K (Figure 1 and Table autoimmune disease control antibodies from Sjbgren's syndrome lack selectivity for both V3 and gp41 (Figure and HIV antibodies cross-react with (Figure EXAMPLE III Diagnostic Skin Test for HIV-1 Infection This example provides the method for performing the skin test and evaluating the results of the test.

Approximately 0.1 ml of a composition comprising an amino acid sequence that is immunologically homologous to an epitope present on HIV-1 and can stimulate an immune Sresponse and a pharmacologically acceptable carrier is injected intradermally on the flexor or dorsal surface of the forearm, about 4 inches below the elbow. Prior to injection, the sit.. should be cleansed with a solution of 70% ethyl alcohol. A disposable syringe and needle can be used for injection and a separate sterile unit is used for each person tested.

'h 1 f 1 1 1 NO: 25) for example, can be used to stimulate memory anti- WO 95/25124 PCT1US95I03236 31 The point of the needle is inserted into the most superficial layers of the skin with the needle bevel pointing upward. Injection of the composition results in the formation of a pale bleb 6-10 mm in size, which is quickly absorbed. If no bleb forms, the injection was likely delivered subcutaneously and the test should be repeated immediately at another site at least 5 cm removed.

Similarly, if the composition leaks from the injection site, the test should be repeated.

Evidence of an immune response. is determined between 48 and 72 hr after injection of the composition and at any additional times prescribed. The reaction size is calculated as one-half the sum of the perpendicular diameters. Reactions greater than 5 mm are considered "positive." Care should be taken to determine that a "negative" reaction is not erroneous due, for example, to a non-specific suppressor such as non-HIV viral infections, live virus vaccines, prior administration of corticosteroids or malnutrition.

Although the invention has been described with reference to the above examples, it should be understood that various modifications can be made without departing from the spirit of the invention. Accordingly, the invention is limited only by the following claims.

1 WO9/52 CTU9L33 j wnetner ei.mu.LaT..ju uL.. L- Iiex vivo or on whether the administration is a first 35 administration or a booster administration.

A

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WO 95/25124 PCT/US95/03236 32 SEQUENCE LISTING GENERAL INFORMATION: APPLICANT: university of southern Californi (ii) TITLE OF INVENTION: Methods to Diagnose and Treat HIV-1 Infection (iii) NUMBER OF SEQUENCES: 66 (iv) CORRESPONDENCE ADDRESS: ADDRESSEE: Campbell and Flores STREET: 4371 La Jolla Village Drive, Suite 700 CITY: San Diego STATE: California COUNTRY: USA ZIP: 92122 COMPUTER READABLE FORM: MEDIUM TYPE: Floppy disk COMPUTER: IBM PC compatible OPERATING SYSTEM: PC-DOS/MS-DOS SOFTWARE: PatentIn Release Version #1.25 (vi) CURRENT APPLICATION DATA: APPLICATION NUMBER: FILING DATE: 13-MAR-1995

CLASSIFICATION:

(viii) ATTORNEY/AGENT INFORMATION: NAME: Imbra, Richard J.

REGISTRATION NUMBER: 37,643 REFERENCE/DOCKET NUMBER: FP-SI 1394 (ix) TELECOMMUNICATION INFORMATION: TELEPHONE: (619) 535-9001 TELEFAX: (619) 535-8949 INFORMATION FOR SEQ ID NO:1: SEQUENCE CHARACTERISTICS: LENGTH: 10 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: upotide S(xi) SEQUENCE DESCRIPTION: SEQ ID NO:1: Gly Glu Arg Leu Asp Arg Arg Lys Glu Arg 1 5 f' o uDtained, for example, by chemical synthesis of the amino acid sequences. A particularly useful means for obtaining W095/25124 PCT/US95103236 33 INFORMATION FOR SEQ ID NO:2: SEQUENCE CHARACTERISTICS: LENGTH: 10 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2: Leu Ile Glu Asp Gin Gln Gin Arg Gin Arg 1 5 INFORMATION FOR SEQ ID NO:3: SEQUENCE CHARACTERISTICS: LENGTH: 9 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3: Pro Gly Arg Ala Ala ser Ser Ala Gly 1 INFORMATION FOR SEQ ID NO:4: SEQUENCE CHARACTERISTICS: LENGTH: 10 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (Xi) SEQUENCE DESCRIPTION: SEQ ID NO:4: ser Gly Leu Val Arg Ser Ser Ser Gly Arg 1 5 INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 10 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID Pro Arg Ala ser Gly Gln Thr Pro Glu Arg f. i j immunologically homologous to an epitope present on HIV-1 and a pharmacologically acceptable carrier is administered 1.! 1 1 F WO 95/25124 PCT/US95/03236 34 INFORMATION FOR SEQ ID NO:6: SEQUENCE CHARACTERISTICS: LENGTH: 10 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6: Thr A-g Clu Glu Arg Met Glu Arg Lys Arg 1 5 INFORMATION FOR SEQ ID NO:7: SEQUENCE CHARACTERISTICS: LENGTH: 11 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7: Leu Lys Met Trp Asp Pro His Asn Asp Pro Asn 1 5 INFORMATION FOR SEQ ID NO:8: SEQUENCE CHARACTERISTICS: LENGTH: 8 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (Xi) SEQUENCE DESCRIPTION: SEQ ID NO:8: Gly Tyr Ala Phe Ile Glu Tyr Glu 1 INFORMATION FOR SEQ ID NO:9: SEQUENCE CHARACTERISTICS: LENGTH: 8 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9: Pro Arg Arg Leu Gly Gly Gly Leu 1 L obtained f rom GenBank and translated into the amino acid sequences for gpl2O (amino acid positions 1 to 511) and Igp4l (512 to 856). Sequences f or V3 IIIB and V3 MN were WO095/25124 PCTIUS95/03236 INFORMATION FOR SEQ ID liO:1O: SEQUENCE CHARACTERISTICS: LENGTH: 11 amino acids TYPE: amino acid TOPOLOGY- linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID Gly Ala Asp Val Asn Ile Arg His ser Gly Arg 1 5 INFORMATION FOR SEQ ID NO:11: SEQUENCE CHARACTERISTICS: LENGTH: 10 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11: Arg Pro Gly Asp Ser Pro Leu pro His Arg 1 5 INFORMATION FOR SEQ ID NO:12: SEQUENCE CHARACTERISTICS: LENGTH: 10 amino acids TYPE: amino acid TOPOT.OGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID WO:12: Glu Arg Asp LyB Glut Arg Arg Arg Ser Arg 1 5 INFORMATION FOR SEQ ID NO:13: SEQUENCE CHARACTERISTICS: LENIGTH: 10 amino acids TYPE: amino acid TOP%'LOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:i.3: Asp Lys Asp Arg Asp Arg Lys Arg Arg Ser 1 5 rectangle at positions 322 to 329). In fact, this eight amino acid sequence i6 not an exact sequence but is a WO 95/25124 PCT/US95/03236 36 INFORMATION FOR SEQ ID NO:14: SEQUENCE CHARACTERISTICS: LENGTH: 11 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:14: Glu Glu Leu Arg Gly Gly Gly Gly Asp Met Ala 1 5 INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 19 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SLQ ID Gly Pro Asp Gly Pro Asp Gly Pro Glu Glu Lys Gly Arg Asp Arg Asp 1 5 10 Arg Glu Arg INFORMATION FOR SEQ ID NO:16: SEQUENCE CHARACTERISTICS: LENGTH: 5 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:16: Arg Asp Arg Asp Arg 1 INFORMATION FOR SEQ ID NO:17: SEQUENCE CHARACTERISTICS: LENGTH: 10 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:17: Arg Gly Gly Gly Gly Gly Gin Asp Asn Gly 1 5 in Figure 2. synthetic peptides were used in direct and competition ELISA's to show that a broad region 'of 24 amino WO 95/25 124 pC121US95/03236 37 INFORMATION FOR SEQ I0 NO418: fi) SEQUENCE CHARACTERISTICS: ()LENGTH: 8 amino acids TYPE% amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:18.: Gly Phe Gin Phe Val Thr Phe Asp 1 INFORMATION FOR SEQ ID NQ:19: SEQUENCE CHARACTERISTICS: LENGTH: 8 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULL TYPE: peptide (xi) SEQUENCE DESCRIPTION: LVEQ ID NO:19: Gly Phe Ala Phe Val Thr Phe ASP 1 INFORMATION FOR SEQ ID V0:20% SEQUENCE CHARACTERISTICS: LENGTH: 8 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: peptide, (xi) SEQUENCE DESCRIPTION: SEQ ID Gly Gin Ala Phe Val Ile Phe Lys WO 95/25124 PCT71US95/03236 38 INFORMATION FOR SEQ ID N0:21: SEQUENCE CHARACTERISTICS: LENGTH: 614 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein (Xi) SEQUENCE DESCRIPTION: SEQ ID NO:21: Met Gly Thr 7Ie Ser Gly G y Gly Gly Ser Asn Ala Ala Thr Arg Gin 1 5 10 Val Gly Cys Ala Pro ser Gly Arg Pro Ser hr Arg Pro Ser Gly Thi.

25 3 0 Ala Ile Arg Ala Arg Pro Val Ala Ser Val Lys Pro Ile Asp Giu Gly 40 Lau Ala Giu Val Arg Val Ile Giu Asp Giu Ala Ile Gly Ile Glu Gly 55 Glu Arg Lau A~p Arg Arg Lys Giu Arg Arg Arg Gln Giu Ala Lou Ile 70 75 Glu. Asp Gin Gin Gin Arg Gin Arg Arg Trp Pro Gly Lou Pro Ala Ala 90 Arg Pro Gly Arg Ala Ala ser ser Ala Gly le Gly Gly Arg Gin Gly 100 105 110 Lou Leu, Ala Arg Gly Thr Lou Trp Trp, Leu Ser Ser Gly Leu Val Arg 115 120 125 Ser Ser Ser Gly Arg Arg Asn Gin Thr Asp Val Asp Ala Pro Gly Val 130 135 140 Giu Ala Giu Ala Gly Val Val Val Ala GiU Gly LOU Pro Gin Pro Pro 145 150 155 160 Arg Ala Ser Gly Gin Tim Pro Giu Arg Gly Gly Ala Thr Arg Leu Gly 165 170 175 Lys Met Thr Gin Phe Lou Pro Pro Asn Lou Lou Ala Lou Phe Ala Pr~o 180 185 190 1*Arg Asp Pro Ile Pro Tyr Lou Pro Pro LOU GlU Lys LOu Pro His Glu 195 200 205 Lys His His Asn Gin Pro Tyr Cys Gly Ile Ala Pro Tyr Ile Arg Giu 210 23.5 .220, Phe Giu Asp Pro Arg Asp Ala Pro Pro Pro Thr Arg Ala Glua Thr Arg 225 230 235 240 Giu Glu Arg Met Glu Arg Lys Arg Arg GiU Lys Ile Giu Arg Arg Gin 245 250 255 Gin Giu Val Glu. Thr Giu Lou Lys Met Trp Asp'Pro His Asn Asp Pro 260 265 270 Afn Aia ,Gin Gly Asp Ala Phe Lys Thr -Lou Phe .Vai Ala Arg Val Asn "275 280 285 -s '~sarr.V a.,is *'sc I 7, III. ii WO 95125124 PCTUS95103236 39 Tyr Asp Thr Thr Giu ser Lys Leu Arg Arg Giu Phe GlU Val Tyr Gly 290 295 300 Pro Ile Lys Arg Ile His Met Vai Tyr ser Lys Arg Ser Gly Lys Pro 305 310 315 320 Arg Gly Tyr Ala Phe Ile Glu Tyr Giu His Giu Arg Asp Met His ser 325 330 335 Ala Tyr Lys His Ala Asp Gly Lys Lys Ile Asp Gly Arg Arg Val Leu 340 345 350 Val Asp Val Giu Arg Gly Arg Thr Val Lys Gly Trp Arg Pro Arg Arg 355 360 365 Leu Gly Gly Giy Leu Gly Gly Thr Arg Arg Gly Gly Ala Asp Val Asn 370 375 380 Ile Arg His Ser Gly Arg Asp Asp Thr ser Arg Tyr Asp Giu Arg Pro 385 390 395 400 Gly Pro Ser Pro Leu Pr,' His Arg Asp Arg Asp Arg Asp Arg Giu Arg 405 410 415 Glu Arg Arg Giu Arg ser Arg Glu Arg Asp Lys Giu Arg Giu Arg Arg 420 425 430 Arg ser Arg Ser Arg Asp ArZ Arg Arg Arg Ser Arg Ser Arg Asp Lys 435 440 445 Glu Giu Arg Arg Arg Ser Arg Glu Arg ser Lys Asp Lys Asp Arg Asp 450 455 460 Arg Lys Arg Arg Ser Ser Arg Ser Arg Giu Arg Ala Arg Arg Giu Arg 465 470 475 480 Glu Arg Lys Glu Glu Leu Arg Gly Gly Gly Gly Asp Met Ala Glu Pro 485 490 495 Ser GIu Ala Gly Asp Ala Pro Pro Asp Asp Gly Pro Pro Gly Glu Leu 500 505 510 Gly Pro Asp Gly Pro Asp Gly Pro Glu Giu Lys Gly Arg Asp Arg Asp 515 520 525 Arg Giu Arg Arg Arg Ser His Arg Ser Glu Arg Giu Arg Arg Arg Asp 530 535 540 Arg Asp Arg Asp Arg Asp Arg Asp Arg Glu His Lys Arg Gly Giu Arg 545 550 555 560 Gly Ser Glu Arg Gly Arg Asp Gl, Ala Arg Gly Gly Gly Gly Gly Gln 565 570 575 Asp Asn Gly Leu Slu Qly Leu Gly Asr Asp Ser Arg Asp Met Tyr Met 580 585 590 Glu Ser Glu Gly Gly Asp Gly Tyr Leu Ala Pro Glu Asn Gly Tyr Leu 595 600 605 'Met Giu Ala Ala Pro Glu, 610 '0 tN.LJLJL W0 L J;ASA 41 WCLO LLL0 .LLI&V "lu&wc w WO95/25124 PCT/US95/03236 INFORMATION FOR SEQ ID NO:22: SEQUENCE CHARACTERISTICS: LENGTH: 4 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:22: Gly Tyr Ala Phe 1 INFORMATION FOR SEQ ID NO:23: SEQUENCE CHARACTERISTICS: LENGTH: 11 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:23: Glu Arg Pro Glu Glu Arg Glu Glu Arg Arg Arg 1 5 INFORMATION FOR SEQ ID NO:24: SEQUENCE CHARACTERISTICS: LENGTH: 5 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:24: Glu Arg Lys Arg Arg 1 INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 8 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID Gly Arg Ala Phe Val Thr I1e Gly 1

I

a large proportion of the gp4l immunologically homologous WO 951n5124 PCT17US95/03236 rA 41 INFORMATION FOR SEQ ID NO:26: SEQUENCE CHARACTERISTICS: LENGTH: 6 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (ix) FEATURE: NAME/KEY: Peptide LOCATION: OTHER INFORMATION: /note= "Xaa =any one independently se.lected AMino acid.., (xi) SEQUENCE DESCRIPTION: SEQ ID NO:26: Gly Arg Ala Phe Xaa Thr 1 INFORMATION FOR SEQ ID NO:27: SEQUENCE CHARACTERISTICS: LENGTH: 6 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (Xi) SEQUENCE DESCRIPTION: SEQ ID NO:27: Gly Arg Ala Phe Val Thr 1 INFORMATION FOR SEQ ID NO:28: SEQUENCE CHARACTERISTICS: LENGTH: 6 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: pept-.de (Xi) SEQUENCE DESCRIPTION: SEQ ID NO:28: Gly Arg Ala Phe Tyr Thr 1 Western blot analysis was used to determine whether sera from HIV-infected individuals recognize WO 95/25124 PCTIUS95/03236 42 INFORMATION FOR SEQ ID NO:29: SEQUENCE CHARACTERISTICS: LENGTH: 14 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:29: Arg Ile Gin Arg Gly Pro Gly Arg Ala Phe Val Thr Ile Gly 1 5 INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 4 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID Gly Pro Gly Arg 1 INFORMATION FOR SEQ ID NO:31: SEQUENCE CHARACTERISTICS: LENGTH: 4 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:31: Gly Arg Ala Phe 1 INFORMATION FOR SEQ ID NO:32: SEQUENCE CHARACTERISTICS: LENGTH: 4 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:32: Glu Arg Lys Arg i1 i m P 1 q i 1 1 1 p l i l 1 1 1 are neutralizing, although at low titers. Deletion WO 95/25124 PCT/US95/03236 43 INFORMATION FOR SEQ ID NO:33: SEQUENCE CHARACTERISTICS: LENGTH: 5 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:33: Gly Gly Gly Asp Met 1 INFORMATION FOR SEQ ID NO:34: SEQUENCE CHARACTERISTICS: LENGTH: 5 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:34: Arg Leu Gly Gly Gly 1 INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 5 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID -Leu Arg Gly Gly Gly 1

P*.

each person tested.

WO 95125124 PCTIUS95/03236 44 INFORMATION FOR SEQ ID 110:36: SEQUE4NCE CHARACTERISTICS: LENGTH: 220 amino acids TYPE: amino acid TOPOLOGY. linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:36: Giy Ile Giu Gly Glu Arg Leu ASP Arg Arg Lys Giu Arg Arg Arg Gin 1510 Gin Giu Ala Le Ile Giu Asp Gin Gin Gin Arg Gin Arg Pro Gly Arg 25 Ala Ala Ser Ser Ala Giy Ile Gly Giy Arg Gin Gly Leu Len Ser Gly 40 Leu Val Arg Ser Ser Ser Gly Arg Pro Arg Ala Ser Gly Gin Thr Pro 55 Gin Arg Thr Arg Giu Gin Arg Met Giu Arg Lys Arg Len Lys Met Trp 70 75 ASP Pro His Asn Asp Pro Asn ser Lys Len Arg Arg Giu Phe Gin Vai 90 Tyr Giy Tyr Ala Phe le Giu Tyr Gin His Pro Arg Arg Len Gly Gly 100 105 110 Giy Leu Giy Gly Thr Arg Arg Gly Giy Ala Asp Val Ann Ile Arg His 115 120 125 Ser Giy Arg Arg Pro Gly Asp ser Pro Len Pro Hio Arg Asp Arg Asp 130 135 140 Arg Asp Arg Gin Arg Asp Lys Gin Arg Arg Arg ser Arg Asp Lys Asp 145 150 155 160 Arg Asp Arg Lys Arg Arg Ser Ser Arg Gin Gin Leu Arg Giy Gly Gly 165 170 175 Giy Asp Met Ala Gly Pro Asp Gly Pro Asp Giy Pro Gin Giu, Lys Giy 180 185 190 Arg Asp Arg Asp Arg Gin Arg Arg Asp Arg Asp Arg Asp Arg Asp Arg 195 200 205 Asp Arg Arg Giy Gly Giy Gly Gly Gin Asp Asn Giy 210 215 220 6 WO 95/25124 WO 9525124PCT1US95/03236 INFORMATION FOR SEQ ID NO:37: SEQUENCE CHARACTERISTICS: LENGTH: 183 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (Xi) SEQUENCE DESCRIPTION: SEQ ID IqO:37: Gly Ile Glu Giu Giu Gly GiU Arg Asp Arg Asp Arg Ser Ile I ifA

.LU

Ile Ile Pro Thr Lys Gly Arg Arg Gly 145 Gly GlU Gin Arg Lys Arg Pro.

Ile 115 Arg Met Arg Gin GiU Gly Gin LeU Glu Thr Asp Asn Asn Gin Gly 105 Gly Tyr 120 le Arg Arg Pro Giu Arg Pro Gly Ile Arg Arg Thr Leu Lys Gly Arg Leu Giy Ser Pro Gl~ Ile Giu Gly 155 Asp Arg 170 Arg Cys Arg Asn Ala Ala Leu Phe 140 le Asp Gly Asp Met Arg Asp Asn Trp 180 INFORMATION FOR SEQ ID 140:38: SEQUENCE CHARACTERISTICS: LENGTH: 5 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (Xi) SEQUENCE DESCRIPTION: SEQ ID 140:38: ser ser ser Gly Arg 1 1 WO 95/25124 PCT/US95/03236 46 INFORMATION FOR SEQ ID NO:39: SEQUENCE CHARACTERISTICS: LENGTH: 5 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:39: Thr Arg Asp Gly Gly 1 INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 6 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID Glu Arg Asp Arg Asp Arg 1 INFORMATION FOR SEQ ID NO:41: SEQUENCE CHARACTERISTICS: LENGTH: 10 amino acids TYPE: amino acid TOPOLOGY:.linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:41: Glu Arg Asp Arg Asp Ser Arg Ser Ile Arg 1 5 INFORMATION FOR SEQ ID NO:42: SEQUENCE CHARACTERISTICS: LENGTH: 10 amin, acidi TYPE: amAno acid TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:42: Glu Leu Leu Gly Arg Arg Gly Trp Glu Ala 1 5 WO 95/25124 PCTIUS95/03236 47 INFORMATION FOR SEQ ID NO:43: SEQUENCE CHARACTERISTICS: LENGTH: 12 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:43: Arg Gly Pro Gly Arg Ala Phe Val Thr Ile Gly Lys 1 5 INFORMATION FOR SEQ ID NO:44: SEQUENCE CHARACTERISTICS: LENGTH: 12 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:44: Lys Lys Arg Gly Phe Gln Phe Val Thr Phe Asp Asp 1 5 INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 12 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID Lys Lys Arg Gly Phe Ala Phe Val Thr Phe Asp Asp 1 5 INFORMATION FOR SEQ ID NO:46: SEQUENCE CHARACTERISTICS: LENGTH: 12 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:46: Lys Pro Arg Gly Tyr Ala Phe Ile Glu Tyr Glu His 1 5 i_ WO 95/25124 PCTIUS95/03236 48 INFORMATION FOR SEQ ID NO:47: SEQUENCE CHARACTERISTICS: LENGTH: 12 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:47: Lys Ala Arg Gly Gin Ala Phe Val Ile Phe Lys Glu 1 5 INFORMATION FOR SEQ ID NO:48: SEQUENCE CHARACTERISTICS: LENGTH: 12 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:48: Lys Met Arg Gly Gin Ala Phe Val Ile Phe Lys Glu 1 5 INFORMATION FOR SEQ ID NO:49: SEQUENCE CHARACTERISTICS: LENGTH: 12 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:49: Arg Pro Arg Gly Val Ala Phe Val Arg Tyr Asn Lys 1 5 INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 8 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID Arg Leu Gly Gly Gly Asp Met Arg 1 j 1 r WO 95/25124 PCT/US95/03236 49 INFORMATION FOR SEQ ID NO51: SEQUENCE CHARACTERISTICS: LENGTH: 10 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:51: Thr Arg Pro Asn Asn Asn Thr Arg Lys Arg 1 5 INFORMATION FOR SEQ ID NO:52: SEQUENCE CHARACTERISTICS: LENGTH: 14 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:52: Arg Ile Gin Arg Gly Pro Gly Arg Ala Phe val Thr Ile Gly 1 5 INFORMATION FOR SEQ ID NO:53: SEQUENCE CHARACTERISTICS: LENGTH: 4 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:53: Pro Gly Arg Ala 1 INFORMATION FOR SEQ ID NO:54: SEQUENCE CHARACTERISTICS: LENGTH: 7 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:54: Gly Tyr Ala Phe Ile Glu Tyr 1 Arg Gly Gly Gly Gly Gly Gin Asp Asfl Gly 1 5 WO 95/25124 PCTIUS95/03236 INFOPRMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 12 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID Leu Ile Giu Giu Ser Gin Asn Gin Gin eVAu Lys Asn 1 5 INFORMATION FOR SEQ ID NO:56: SEQUENCE CHARACTERISTICS: LENGTH: 9 amino acidu TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (Xi) SEQUENCE DESCRIPTION: SEQ ID NO:56: Leu Ile GiU Asp Gin Gin Gin Arg Gin 1 ()INFORMATION FOR SEQ ID NO:57: SEQUENCE CHARACTERISTICS: LENGTH: 19 amino acids TYPE: amino acid TOPOLOGY: iinear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:57: Gly Pro Asp Arg Pro Giu Giy Ile GiU GlU Giu Gly Gly Giu Arg Asp 1 5 10 Arg Asp Arg wz8 WO 95/25124 PC'fIUS9SIO3236 51 INFORMAWION FOR SEQ ID NO:58: SEQ*4ENCE CHARACTERISTICS: LENGTH: 19 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (Xi) SEQUENCE DESCRIPTION: SEQ ID NO:58: Gly Pro Asp Gly Pro Asp Gly Pro GlU GlU Lys Gly Arg Asp Arg Asp 10 Arg Glu Arg INFORMATION FOR SEQ ID) NO:59: SEQUENCE CHARACTERISTICS: LENGTH: 7 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (Xi) SECUENCE DESCRIPTION: SEQ ID NO:59: Arg Asp Arg Asp Axg Asp Arg 1 INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 4 amino acids TYPE: amino acid TOPOLOGY: linear MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID Asp Arg Asp Arg Gin GJ.u va. uiu *nr 260 Asn Ala\ Gin Gly Asp 175 WD.Ia ~ieu LAiYN D1~Iu Ajv~ w 265 270 Ala Phe Lys Thr LeU Phe-val Ala Arg Val. Asn 280 285 WO 95/25124 PCTIUS95/03236 INFORMATION FOR SEQ ID NO:61: SEQUENCE CHARACTERISTICS: LENGTH: 25 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (Xi) SEQUENCE LDESCRIPTION: SEQ ID NO: 61: Arg Ile Leu Ala Val Giu Arg Tyr Leu Lys Asp Gin Gin Leu Giy Ile 1 5 10 Trp Gly Cys Ser Giy Lys Leu Leu Cys INFORMATION FOR SEQ ID NO:62: SEQUENCE CHARACTERISTICS: LENGTH: 19 amino acids TYPE: amino acid TOPOLOGY: linear MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:62: Ser Leu Giu Gin Ala Gin Ile Glrv Gin Giu Lys Asn Glu Gin Giu Leu 1 5 10 Leu Lys Leu INFORMATION4 FOR SEQ ID NO:63: SEQUENCE CHARACTERISTICS: LENGTH: 7 amino acids TYPE: amino acid I TOPOLOGY: linear (ii) M4OLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO,,63: Giu Giy Thr Asp Arg Val Ile 1 WO 95/25124 PCT/US95/03236 53 INFORMATION FOR SEQ ID NO:64: SEQUEACE CHARACTERISTICS: LENGTH: 10 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: Q ID NO:64: Scr Asp Ala Lys Ala Tyr Asp Thr Glu Val 1 5 INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 13 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID Gly Ser Thr Met Gly Ala Ala Ser Met Thr Leu Thr V~l 1 5 INFORMATION FOR SEQ ID NO:66: SEQUENCE CHARACTERISTICS: LENGTH: 10 amino acida TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:66: Gln Glu Leu Lys Asn Ser Ala Val Set Leu 1 5

Claims (19)

1. A method of detecting a cell proliferative disorder associated with glutathione-S-transferase (GSTP1) in a subject, the method including: contacting a target nucleic acid in a sample of tissue or biological fluid with a reagent which detects GSTP1, wherein the reagent detects methylation of the promoter region of GSTP1 when the target nucleic acid is DNA, and wherein the reagent detects the level of GSTP1 RNA when the target nucleic acid is RNA; and detecting GSTPI target nucleic acid, wherein hypermethylation of the promoter of GSTP1 DNA, or decreased levels of GSTP1 RNA as compared with the level of GSTP1 RNA in a normal cell, is indicative of a GSTP1- associated cell proliferative disorder.

2. The method of claim 1, wherein the cell is an epithelial cell.

3. The method of claim 1, wherein the tissue is prostate tissue.

4. The method of claim 1, wherein the reagent is a probe. The method of claim 4, wherein the probe is detectably labelled.

6. The method of claim 5, wherein the label is selected from the group consisting of a radioisotope, a bioluminescent compound, a chemiluminescent compound, a fluorescent compound, a metal chelate, and an enzyme.

7. The method of claim 1, wherein the reagent is a restriction endonuclease.

8. The method of claim 7, wherein the restriction endonuclease is methylation sensitive.

9. The method of claim 8, wherein the restriction endonuclease is selected from the group consisting of MspI, HpaII and BssHlI. A method of treating a cell proliferative disorder associated with GSTP1 expression including administering to a subject with the disorder, a tuerapeutically effective amount of reagent which modulates GSTP1 expression.

11. The method of claim 10, wherein the cell proliferative disorder is in a urogenital tissue.

12. The method of claim 11, wherein the urogenital tissue is the prostate.

13. The method of claim 10, wherein the reagent comprises a promoter i 35 sense polynucleotide sequence.

14. The method of claitm si.3, wherein the reagent includes a structural r 7 jt i i polynucleotide sequence which encodes GSTP1. A method of gene therapy including introducing into the cells of a host subject, an expression vector comprising a nucleotide sequence encoding GSTP1.

16. The method of claim 15, wherein the reagent comprises a promoter sense polynucleotide sequence.

17. The method of claim 16, wherein the reagent includes a structural polynucleotide sequence which encodes GSTP1.

18. The method of claim 15, wherein the expression vector is introduced into the cells of the host subject in vitro and the transformed cells are then reintroduced into the subject.

19. The method of claim 15, wherein the expression vector is an RNA virus. The method of claim 19, wherein the RNA virus is a retrovirus.

21. The method of claim 15, wherein the subject is human.

22. The method of claim 1, wherein the biological fluid is selected from the group consisting of ejaculate, urine and blood.

23. The method of claim 1, wherein the GSTP1 is a member of the GSTP1 7t family. DATED this 29th day of May 1998 THE JOHN HOPKINS UNIVERSITY SCHOOL OF MEDICINE Patent Attorneys for the Aiplicant: F.B. RICE CO.' *i