HK1008950A - The gc1q receptor, hiv-1 gp120 region binding thereto, and related peptides and targeting antibodies - Google Patents

Description

gC1q receptor, HIV-1gp120 region binding thereto and related peptides and targeting antibodies

Technical Field

The present invention relates to (1) peptides that bind to HIV-1gp120 and are based on the gC1q receptor (gC1q-R), and antibodies directed against these peptides; (2) HIV-1gp 120-related peptides that bind to gC1q-R, and antibodies directed against these peptides.

Background

C1q is a component of the C1 complex of the classical complement pathway (R.B.Sim and K.B.M.Reid, today's immunology 1991; 12: 307-311), C1q has a wide variety of biological functions, including the initiation of the complement cascade for opsonization and cytolysis, and the mediation of several different functions depending on the cell type expressing the C1q receptor. C1q enhances FcR and CR1 mediated phagocytosis in monocytes/macrophages (D.A. Bobak et al, J.Eur. Immunol, 1988; 18: 2001. J. 2007; D.A. Bobak et al, J.Immunol 1987; 138: 1150. 1156), stimulates B cells to produce immunoglobulins (K.R. Young et al, J.Immunol 1991; 146: 3356. sup. 3364), activates platelets to express. alpha.IIb/. beta.₃Integrins, P-selectins and procoagulants (procoagulant) (E.I.B.Peershke et al, J.Immunol 1993; 178: 579-587; E.I.B.Peershke et al, J.Immunol 1994; 152: 5896-5901), activate tumor toxicity of macrophages (R.W.Leu et al, J.Immunol 1990; 144: 2281-2286), and exert an antiproliferative effect on T cell growth (A.Chen et al, J.Immunol 1994; 153: 1430-1440).

A33 kilodalton (kD) receptor, designated gC1q-R, has recently been identified, cloned and sequenced, which binds to the globular head of the C1q molecule (B.Ghebrehit et al, J.Immunol 1994; 179: 1809-1821; E.I.B.Peerschke et al, J.Immunol 1994; 152: 5896-5901; A.Chen et al, J.Immunol 1994; 153: 1430-1440). Another 60kD receptor, designated cC1q-R, binds to the amino-terminal collagen-like (collagen-like) region of C1q (B.Ghebrehit et al, Behring Inst.Mitt.1989; 84: 204-215; A.Chen et al, J. Immunol 1994; 153: 1430-1440). This receptor has been found to be present in many different cell types, e.g., B cells, T cells, monocytes/macrophages, neutrophils, eosinophils, fibroblasts, platelets, endothelial cells, hepatocytes, nerve cells and smooth muscle cells, based on detection of gC1q-R mRNA via Polymerase Chain Reaction (PCR) amplification and detection of expression of gC1q-R protein via immunochemical methods. However, gC1q-R is not known to bind to HIV-1gp120 and neutralize HIV-1 infectivity.

The CD4 antigen, which is predominantly expressed on the surface of helper/inducer T cells, has been known to be the major receptor for HIV-1gp120 (P.J.Maddon et al, cell 1986; 47: 333-385; J.S.McDougal et al, science 1986; 231: 382-385), with the exception of CD4⁺T cells, HIV-1, may also bind to and infect other cell types, such as monocytes/macrophages, B cells, colon epithelial cells and glial cells, which express undetectable or minimal levels of cell surface CD 4. Several other receptors have been suggested to be associated with HIV-1 infected target cells, such as galactosylceramide (Gal-Cer) on the surface of human colonic epithelial cells, Schwann cells and oligodendrocytes (N.Yahl et al, virology 1994; 204: 550-_H3 gene product (molecular weight 950kD) (L. Berberian et al, science 1993; 261: 1588-.

The present invention was accomplished in a study intended to discover cell-binding proteins or receptors for HIV-1gp120 on non-CD 4 expressing cells. For this purpose, lysates of CEM-SS cells from P.J.Nara (AIDS Res. hum. retroviruses 1987; 3: 283- & 302), which are T cells expressing high levels of cell surface CD4, and of DAKIKIKIKIKIKIKI cells from the American type culture Collection (Rockville, Maryland), which are CD4 negative B cells, were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and the separated proteins were transferred onto nitrocellulose membranes for Western immunoblot analysis, and it was found that recombinant gp120 or HIV-1-infected cell-derived gp120 reacted with one of the 32-33kD protein bands of the CEM-SS and DAKIKIKIKIKIKI cell lysates, which was clearly separated from the 55kD protein band reacted with the anti-human CD4 monoclonal antibody.

To purify this novel gp 120-binding protein for N-terminal amino acid sequencing identification, large amounts of DAKIKIKI Cell lysates were prepared and this gp 120-binding protein was partially purified by preparative electrophoresis using Prep Cell Model 491(Bio-Rad laboratories, Hercules, Calif.). The samples were then subjected to two-dimensional gel electrophoresis and blotted onto polyvinylidene fluoride (PVDF) membranes for Western immunoblot analysis to identify gp 120-reactive protein spots and N-terminal amino acid sequencing. The first 15N-terminal amino acid sequence of the gp 120-reactive protein has been shown to be identical to the first 15N-terminal amino acid sequence of a protein previously identified as p32 (MW 32kD), p32 was co-purified with the human pre-mRNA splicing factor as reported by A.R. Krainer et al (cell 1991; 66: 383-394) and B.Honore et al (Gene 1993; 134: 282-287). Further experiments revealed that the DAKIKIKIKI cells could be stained with rabbit anti-p 32 immunoglobulin, indicating that p32 is present on the surface of DAKIKIKIKIKI cells, and that rabbit anti-p 32 immunoglobulin was produced by using pure p32 expressed in E.coli. DAKIKIKI cells have also been shown to bind recombinant HIV-1gp120, although the cells do not express CD4 on their surface, which can be detected by immunofluorescence. Taken together, these findings demonstrate that p32 is another cell surface binding protein of HIV-1gp120, followed by the demonstration that p32 has the same sequence as gC1q-R (SEQ ID NO: 1) (B.B.Ghebrehiwet et al, J.Immunol. 1994; 179: 1809-1821).

The precursor or preproprotein of gC1q-R contains 282 amino acids (a.a.), and the functional mature protein contains 209 amino acids, this mature protein containing the amino acid sequence of SEQ ID NO: 1 (leucine) to amino acid 74 (glutamine) to amino acid 282 (glutamine). Mature gC1q-R is highly charged and acidic, having 3 potential N-glycosylation sites at amino acids 114, 136 and 223, and therefore, the apparent molecular weight of gC1q-R in SDS-PAGE is 32kD, rather than 24.3kD as calculated from amino acid composition. Since the mature protein has only one cysteine at position 186, there are no intrachain disulfide bonds in the protein. gC1q-R is found in many cell types, such as B cells, T cells, monocytes/macrophages, eosinophils, neutrophils, platelets, endothelial cells, fibroblasts, and hepatocytes.

Since gC1q-R is associated with a variety of immunological and physiological functions, the interaction between gC1q-R and HIV-1gp120 may result in some immunological and physiological dysfunction exhibited by HIV-1 infected individuals. It may also result in the known ability of HIV-1 to infect a variety of non-CD 4 expressing human cells in different tissues and organs. Manual intervention of the interaction between gC1q-R and HIV-1gp120 represents a new strategy for combating HIV-1 disease, several inventions based on such manual intervention are discussed below.

Summary of The Invention

The present invention includes immunogens and peptides based on a binding site for gC1q-R to HIV-1gp120, and immunogens and peptides based on a binding site for HIV-1gp120 to gC1 q-R. The gC1q-R binding site sequence for gp120 is shown in SEQ ID NO: 2, the sequence of the HIV-1gp120 binding site for gC1q-R is shown in SEQ ID NO: 3, respectively. The invention also includes antibodies and binding molecules directed against all of these immunogens and peptides and induces the endogenous production of such antibodies. Other aspects and embodiments of the invention are further described below.

Brief description of the drawings

FIG. 1 is a restriction map of vector pT7-7 for expressing the full-length mature form of gC1q-R (1-209a.a.) and 3 truncated forms thereof in E.coli, gC1q-R (delta 1-57) stands for gC1q-R (58-209a.a.), gC1q-R (C) for gC1q-R (95-209a.a.), and gC1q-R (N) for (1-94 a.a.).

FIG. 2 shows the binding of HIV-1gp120 to gC1q-R as determined by ELISA, the Y-axis representing the reactivity of HIV-1gp120 with gC1q-R as OD at 450nm, and the X-axis being the concentration of HIV-1gp120 in solution.

FIG. 3A shows the neutralization of HIV-1IIIB infectivity by gC1q-R in CEM-SS cells; where Vn is the average number of syncytia in duplicate test wells and Vo is the average number of syncytia in duplicate control wells.

FIG. 3B shows the neutralization of infectivity of HIV-1MN by gC1q-R in CEM-SS cells; where Vn is the average number of syncytia in duplicate test wells and Vo is the average number of syncytia in duplicate control wells.

FIG. 3C shows neutralization of infectivity of HIV-1RF by gC1q-R in CEM-SS cells; where Vn is the average number of syncytia in duplicate test wells and Vo is the average number of syncytia in duplicate control wells.

FIG. 4 shows GC1q-R on HIV-1IIIB infected H9 cells and CD4⁺-inhibition of the fusion of Hela cells.

FIG. 5 shows the binding of E.coli expressed purified gC1q-R to HIV-1IIIB infected H9 cells as determined by flow cytometry.

FIG. 6 shows the effect of C1q on HIV-1gp120 binding to gC1q-R in ELISA, the line drawn with filled circles represents C1q, the line drawn with filled squares represents gC1q-R, the Y axis represents the reactivity of gp120 with gC1q-R expressed as OD at 450nm, and the X axis is the concentration of C1q and gC1q-R in solution.

FIG. 7 shows the reactivity of HIV-1gp120 as a solid phase antigen in ELISA with various forms of E.coli expressed gC1q-R, the line drawn with solid squares representing the full-length mature form of gC1q-R (1-209 a.a.); the line drawn with filled circles represents truncated gC1q-R (58-209a.a.), i.e., gC1q-R with the deletion of the N-terminal 57 amino acids; the line drawn with filled triangles represents the N-terminal construct, gC1q-R (1-94 a.a.); the line drawn with filled inverted triangles represents the C-terminal construct, gC1q-R (95-209 a.a.). The Y-axis represents the reactivity of HIV-1gp120 with various forms of gC1q-R expressed as OD at 450nm, and the X-axis is the concentration of gp120 in solution.

FIG. 8 shows the binding of antibody 99-12-1 to gC1q-R peptide LM12DN, the line drawn with filled squares representing peptide LM12DN, the line drawn with filled circles representing peptide TD18EE, the Y-axis representing the reactivity of 99-12-1 with the peptide expressed as OD at 450nm, and the X-axis representing the concentration of 99-12-1 in solution.

FIG. 9 shows the competition of anti-gC 1q-R monoclonal antibody 99-12-1 for gC1q-R binding to HIV-1gp 120.

FIG. 10 shows competition of anti-HIV-1 gp120 antibodies G3-299 and 6205 for HIV-1gp120 binding to gC1 q-R. Lines drawn with filled circles represent the anti-HIV-1 gp 120C 4 regions G3-299; the line drawn with filled squares represents the polyclonal sheep antibody 6205, which is specific for the anti-HIV-1 gp 120C 5 region peptide (number of amino acid residues HXB 2R: 497-511, SEQ ID NO: 10); the line drawn with solid triangles represents recombinant soluble CD4(rsCD 4).

FIG. 11 shows the reactivity of gC1q-R with HIV-1gp 120C 4 region peptides RC12LT, TG12NN and RC16 GG. The line drawn with solid triangles represents RC16 GG; the line drawn with a filled circle represents TG12 NN; the line drawn with a solid square represents RC12 LT. The Y-axis represents the reactivity of gC1q-R with the peptide expressed as OD at 450nm, and the X-axis represents the concentration of gC1q-R in solution.

The A.gC1q-R peptides of the invention

The complete nucleotide sequence and deduced amino acid sequence of gC1q-R is shown in SEQ ID NO: 1, recombinant gC1q-R expressed in E.coli and used for immunization and other examples described below is a polypeptide sequence starting from SEQ ID NO: 1, the complete C-terminal stretch of the 74 th amino acid residue leucine, this stretch representing the identified soluble mature protein of gC1 q-R. The N-terminal segment may represent a hydrophobic membrane anchor in the preproprotein.

Comprises the amino acid sequence of SEQ ID NO: 1 was identified as binding to the active binding site of gp120 based on competitive inhibition of gp120 binding to gC1q-R by the anti-gC 1q-R monoclonal antibody 99-12-1 (which binds the peptide of SEQ ID NO: 2) discussed in example 10 below.

The active binding site for the gC1q-R binding to gp120 can be expressed as a separate peptide (hereinafter "gp 120 binding site"), or as any longer peptide comprising the gp120 binding site (e.g., a peptide representing the entire gC1q-R sequence) (see example 1 below), or the gp120 binding site or such longer peptide can be linked to another carrier protein such as Keyhole Limpet Hemocyanin (KLH) or another carrier molecule that enhances its immunogenicity, or the gp120 binding site can be conjugated to a peptide or molecule that can generate an immunogenic conformation to enhance its immunogenicity, or a peptide that is structurally or immunologically equivalent to the gp120 binding site can be used for the same purpose (all such peptides are hereinafter referred to as "gp 120 binding site peptides").

The gp120 binding site peptide can be used in a diagnostic assay to detect or quantify HIV-1gp120, HIV-1 virions, or HIV-1 infected cells, using standard assay procedures, such as enzyme-linked immunosorbent assays (ELISAs). In an ELISA, the gp120 binding site peptide can be immobilized on an inert solid phase substrate or magnetic beads, either directly or indirectly via a cross-linking agent or specific binding agent. The biological fluid sample is then incubated with the coated substrate, free HIV-1gp120 molecules or HIV-1 virions carrying gp120 molecules that react with the gp120 binding site peptide will bind to the substrate, and the bound HIV-1gp120 molecules or HIV-1 virions can then be detected with a monoclonal or polyclonal anti-HIV-1 antibody, which can then be reacted with an enzyme-linked secondary detection antibody for quantification based on color reactions. Alternatively, captured gp120 or viral particles can be detected by other means such as fluorescence, chemiluminescence, radioactive counting or PCR.

The gp120 binding site peptides can also be used to detect and quantify HIV-1 infected cells in patient blood samples or other samples by direct or indirect immunochemical procedures. For example, in one such assay, infected cells are contacted with gp120 binding site peptides and then labeled with antibodies that bind thereto, which detection antibodies can be conjugated directly or indirectly to a fluorescent probe, and then immunofluorescence is detected by observing the cells under a fluorescent microscope or by flow cytometry. Alternatively, the gp120 binding site peptide may be directly labeled with an enzyme, radionuclide, fluorescent probe or biotin, and the labeled peptide bound to the cells may be subsequently detected by a corresponding well-known method.

gp120 binding site peptides or any derivative product such as conjugates with toxins (e.g., ricin A, diphtheria toxin, Pseudomonas aeruginosa exotoxin A, pokeweed antiviral protein), energetic radionuclides (e.g., iodine-131, indium-111, and yttrium-90), cytotoxic drugs (e.g., doxorubicin), cell membrane active molecules (e.g., phospholipases, complements, and saponins), or as microcarriers (e.g., liposomes) can be used to treat HIV-1 disease or prevent HIV-1 infection. It has been demonstrated that peptides corresponding to the complete sequence of the mature protein of gC1q-R neutralize the in vitro infectivity of several divergent evolutionary strains of HIV-1 and inhibit the formation of syncytia between CD4 positive cells and cells infected with HIV-1 IIIB. See examples 5 and 6 below. Administration of the gp120 binding site peptide is expected to bind gp120 and neutralize HIV-1, as well as prevent HIV-1 from infecting other cells via cell-cell fusion (syncytial formation). Such administration may be as a treatment for HIV-1 disease or may be prophylactic administration to prevent or inhibit HIV-1 infection immediately prior to or after exposure to HIV-1. In prophylactic applications, it can be given to high risk groups as a precautionary measure, such as intravenous drug users and nursing workers. Alternatively, it can be used after needle stick injury to prevent infection, or immediately before or after birth to prevent maternal-fetal HIV-1 transmission.

The gp120 binding site peptide may be conjugated to a solid phase matrix, and this modified matrix may be used in vitro (extracorporeally) to remove free HIV-1 viral particles or to remove HIV-1 infected cells from HIV-1 infected individuals to reduce viral load (load). B. Antibodies to gC1q-R peptide

Monoclonal or polyclonal antibodies against gC1q-R can be readily prepared using well known techniques described in example 2 below, and such monoclonal antibodies can be generated using monovalent or multivalent gC1q-R or gp120 binding site peptides as immunogens. The gC1q-R peptide (gp120 binding site peptide) can also be used to screen hybridomas after immunization and fusion. Another approach is to use a gp120 binding site peptide or equivalent peptide that binds a relevant portion of the gp 120C 4 region in combination with one or more protein carriers to produce an immunogen, preferably KLH, tetanus toxoid or bacille calmette-guerin (BCG), and also recombinant protein antigens such as those from vaccinia virus, hepatitis B virus, adenovirus or influenza virus. These vectors can be chemically conjugated to gp120 binding site peptides, or the complete vector-peptide can be expressed by recombinant host cells. Polyclonal antibodies can be raised from animals (e.g., rodents, sheep, goats, guinea pigs, rabbits, and non-human primates) using a gp120 binding site peptide or equivalent peptide as an immunogen (see example 3 below).

Monoclonal or polyclonal antibodies specific for the gp120 binding site of gC1q-R can also be generated by immunizing animals with DNA immunogens such as deoxyoligonucleotides encoding gp120 binding site peptides (J.J.Donnelly et al, J.Immunol. 1994; 176: 145-152). Specific oligonucleotides encoding gC1q-R may be constructed with other expression vectors (e.g., vaccinia virus, hepatitis B virus, cytomegalovirus, retroviruses, or adenoviruses) for enhanced expression and gene targeting. Alternatively, direct intramuscular injection, mechanical procedures such as transfection of epidermis with high-speed gold particles coated with DNA, or ex vivo (ex vivo) transfection by chemical (e.g. calcium phosphate) or electrical methods may be employed to insert oligonucleotides into host cells for expression of antigens to induce specific antibody responses.

Such monoclonal or polyclonal antibodies have many uses and can be used to detect cells expressing the gC1q-R receptor, such as B cells, T cells, monocytes/macrophages, neutrophils, eosinophils, fibroblasts, platelets, and endothelial and smooth muscle cells, by using the above-described immunofluorescent assay or other methods. Monoclonal antibodies 99-12-1 which react with gC1q-R have been shown to compete with gp120 for binding to gC1q-R (see the experiment described in example 11 below), and thus, may also be used as a therapy for HIV-1 disease, or to prevent or inhibit HIV-1 infection if administered prophylactically, either before or immediately after exposure to HIV-1.

If used for the treatment of HIV-1 diseases or the prevention of human infections, they are preferably used in the form of chimeric, humanized or human antibodies. Chimeric antibodies are produced using recombinant methods well known in the art, having animal variable regions and human constant regions. Humanized antibodies have a higher degree of human peptide sequence than chimeric antibodies, in which only the Complementarity Determining Regions (CDRs) responsible for antigen binding and specificity are of animal origin and have an amino acid sequence corresponding to an animal antibody, and almost all the rest of the antibody molecule is of human origin and corresponds to the amino acid sequence of a human antibody, see l.riechmann et al, nature 1988; 332: 323-327; winter, U.S. Pat. No. 5,225,539; queen et al, International patent WO 90/07861.

Human antibodies can be made by several different routes, including the production of fragments of human antibodies (V) using human immunoglobulin expression libraries (Stratagene Corp., La Jolla, California)_H、V_L、F_VFd, Fab or F (ab')₂) And these fragments are constructed as fully human antibodies using techniques similar to those used to produce chimeric antibodies. Human antibodies can also be produced in transgenic mice carrying the human immunoglobulin genome, produced by Genpharm International (Mountain View, California). Immunization of animals with a gp120 binding site peptide allows human antibodies against the gp120 binding site peptide to be found in the immunized recipient of the immunogen. Hybridoma or EBV transformed B cell lines can be developed from donor B cells. Human antibodies can also be produced by the combinatorial library method (T.A. Collet et al, USA national science advances 1992; 89: 10026-.

Alternatively, a single peptide chain binding molecule can be prepared in which the Fv regions of the heavy and light chains are joined (J.S. Huston et al, national science evolution 1983; 85: 5879-. All fully or partially human antibodies are less immunogenic than fully murine monoclonal antibodies, and fragments or single chain antibodies are also less immunogenic, and therefore, all of these types of antibodies do not readily elicit an immune or allergic response. Therefore, they are more suitable than whole animal antibodies for in vivo administration in humans, particularly when repeated or prolonged administration is required.

Antibodies against the gp120 binding site peptide can be injected into animals (e.g., mice) to generate anti-idiotypic monoclonal antibodies that can mimic the original antigen and thus can be used as immunogens to generate antibodies against the gp120 binding site peptide, or to block HIV-1 infection as gC1 q-R. HIV-1gp120 peptide binding to gC1q-R

A recombinant peptide having the sequence of amino acid residues 444-459 of the gp120 of HIV-1 strain HXB2R (SEQ ID NO: 3), a peptide fragment located within the region designated C4 of gp120 (C.K. Leonard et al, J. Biochem. 1990; 265: 10373-10382), hereinafter referred to as the "gC 1q-R binding site peptide", was demonstrated to bind gC1q-R by the method of example 13 described below. This peptide, or a longer peptide containing this peptide or an equivalent peptide with the ability to bind gC1q-R, can be used as an immunogen to generate monoclonal or polyclonal antibodies that target this gp120 region.

As described above, the peptide having SEQ ID NO: 3 or a longer peptide containing such a peptide or an equivalent peptide having the ability to bind gC1q-R, or other immunogens based on such a peptide (e.g., immunogens prepared by conjugating such peptides to a preferred carrier protein such as KLH, tetanus toxoid or BCG, or immunogens prepared by using such peptides as part of an immunogenic structure such as a defective or attenuated hepatitis b virus, adenovirus or influenza virus) may be used as vaccines against HIV-1. These vectors can be chemically conjugated to gp120 binding site peptides, or the complete vector-peptide can be expressed from recombinant host cells. When used as vaccines, they will either produce antibodies endogenously which will bind to the gC1q-R binding site of gp120 and neutralize HIV-1, or they will bind to HIV-1 infected cells to inhibit viral transmission through cell-cell fusion or kill infected cells through antibody-dependent cellular cytotoxicity (ADCC) or complement-mediated cytolysis (CMC).

Monoclonal or polyclonal antibodies specific for the binding site of gp120 binding to gC1q-R can also be generated by immunizing animals with deoxyoligonucleotides encoding gC1q-R binding site peptides as DNA immunogens (J.J.Donnelly et al, J.Immunol. 1994; 176: 145-152). Specific oligonucleotides encoding this portion of gp120 can be constructed with other expression vectors (e.g., vaccinia virus, hepatitis B virus, cytomegalovirus, retroviruses, or adenoviruses) to enhance expression and gene targeting. Alternatively, direct intramuscular injection, mechanical procedures such as transfection of epidermis with high-speed gold particles coated with DNA, or ex vivo (ex vivo) transfection by chemical (e.g. calcium phosphate) or electrical methods may be employed to insert oligonucleotides into host cells for expression of antigens to induce specific antibody responses. Antibodies to HIV-1gp120 peptides

SEQ ID NO: 3 can be generated using conventional techniques by immunizing an animal (e.g., a rodent or non-human primate) with gp120 or such a peptide or an immunogen as described above. Polyclonal antibodies can also be generated using well known techniques as described above.

Monoclonal or polyclonal antibodies to the gC1q-R binding site peptide (in the C4 region of gp 120) can also be produced in animals or humans as described above using oligonucleotides encoding this or longer regions as DNA immunogens or vaccines.

These monoclonal or polyclonal antibodies can be used to detect or quantify HIV-1gp120, HIV-1 virions, or HIV-1 infected cells in a diagnostic assay that can employ standard assays such as ELISA. ELISA was performed in a similar manner as described above for the gp120 binding site peptides except that anti-HIV-1 gp120 antibodies were immobilized on an inert solid phase substrate or magnetic beads instead of these peptides. The biological fluid sample is then incubated with these coated substrates, to which HIV-1gp120 or HIV-1 viral particles carrying gp120 molecules, reactive with antibodies, will bind. The bound virus particles or gp120 are then detected with other monoclonal or polyclonal anti-HIV-1 antibodies, which can then be reacted with an enzyme-linked secondary detection antibody for quantification based on color reactions. Alternatively, the captured gp120 or HIV-1 virions can be detected by other methods, such as fluorescence, chemiluminescence, or PCR.

These antibodies can also be used to detect and quantify HIV-1 infected cells in blood samples by direct or indirect immunofluorescence procedures, such as the procedures described above for detecting gp120 binding site peptides. These antibodies may also be labeled directly with an enzyme, a radionuclide, a fluorescent probe, or biotin, and the labeled antibody bound to the cells may be subsequently detected by known methods.

Antibodies directed against this gC1q-R binding site of the C4 region of the HIV-1gp120 region may also potentially be used for the treatment of HIV-1 disease or the prevention of HIV-1 infection. They can be used alone or in combination with other anti-HIV-1 neutralizing antibodies, recombinant soluble CD4, antiretroviral drugs or cytokines. These antibodies are expected to be neutralizing in that once bound to gp120, they inhibit gp120 binding to gC1q-R thereby preventing subsequent infection of the target cell. When used to treat or prevent HIV-1 infection, these antibodies may be in the form of chimeric, humanized or human antibodies, and the methods for producing these forms of antibodies are described above. Alternatively, transgenic mice bearing the human immunoglobulin genome can be immunized with the gC1q-R binding site peptide to produce human antibodies, or human antibodies against the gp 120C 4 region can be found in HIV-1 infected individuals or in vaccinees vaccinated with the gC1q-R binding site peptide. Hybridoma or EBV transformed B cell lines can be developed from the B cells of these donors. Human antibodies can also be generated by the combinatorial library method (T.A. Collet et al, national science advancement 1992; 89: 10026-.

Monoclonal or polyclonal antibodies may be used in the form of conjugates or microcarriers (e.g., liposomes) for targeting cytotoxic agents to kill infected cells, and conjugated agents may be, for example, toxins, cytotoxic drugs, membrane-activating enzymes or chemicals, and energetic radionuclides.

Antibodies directed against the gC1q-R binding site on gp120 can be conjugated to solid phase matrices and these modified matrices can be used in vitro to remove free HIV-1 viral particles or to remove HIV-1 infected cells from HIV-1 infected individuals to reduce viral load.

Antibodies directed against the gC1q-R binding site on gp120 can be modified by binding to another antibody with a different specificity (e.g.against a cell surface marker or the HIV-1 antigen), such bispecific antibodies can be generated by chemical conjugation (S.A. Kostelny et al, J. Immunol 1992; 148: 1547-1553; A.Mabondzo et al, J. Infect. 1992; 166: 93-99) or fusion of two hybridomas (S.M. Chamow et al, J. Immunol 1994; 153: 4268-4280).

Antibodies to the gC1q-R binding site peptide can be injected into an animal (e.g., a mouse) to produce anti-idiotypic monoclonal antibodies that can mimic the original antigen used to produce the target antibody, and thus can be used as immunogens to produce antibodies to the gC1q-R binding site.

Examples of the invention and illustrations of its applicability are described below.

Example 1 expression of gC1q-R protein in E.coli

A. RNA isolation and cDNA preparation: from 5X 10 by RNA-ZOL extraction according to the manufacturer's recommendations (Biotecx, Houston, Texas)⁶Total RNA was isolated from individual DAKIKIKI cells. First strands of cDNA were prepared using 10. mu.g RNA as template in a reverse transcription reaction mixture containing 50mM Tris-HCl (pH8.3) and 20 units of RNASIN (Promega, Madison, Wisconsin), 0.5mM each of dATP, dTTP, dCTP, dGTP, 10. mu.M oligo dT and 2 units of AMV reverse transcriptase (Gibco BRL, Gaithersburg, Maryland). The reaction was carried out at 42 ℃ for 1 hour.

PCR amplification of the gC1q-R cDNA encoding the mature full-length gC1q-R protein (leucine 74 to glutamine 282 of SEQ ID NO: 1):

the sequences of the two primers used for PCR were derived from the gC1q-R cDNA gene (A.R. Krainer et al, cell 1991; 66: 383-394), primer 1 with an NdeI restriction site and primer 2 with a PstI restriction site (primer 1, SEQ ID NO: 4 TACATATGCTGCACACCGACGGAGAC; primer 2)SEQ ID NO: 5 GCCCTGCAGCATCTGTCTGCTCTA). In 50mM KCl, 10mM Tris-HCl (pH8.3), 1.5mM MgCl₂0.01% gelatin, 0.2mM each dATP, dTTP, dCTP, dGTP, 0.5mM each primer, 2. mu.l of the reverse transcription reaction mixture and 1 unit Taq DNA polymerase (biochemicals USA, Cleveland, Ohio) were subjected to PCR, which was performed on GeneAmp9600(Perkin Elmer, Norwalk, Connecticut) for 40 cycles at 94 ℃ (1 minute), 55 ℃ (2 minutes) and 72 ℃ (2 minutes).

C. Constructing a gC1q-R expression vector and expressing the full-length mature recombinant protein in Escherichia coli:

the gC1q-R cDNA fragment was cloned into pT7-7 vector (Biochemical Co., USA) (FIG. 1) by NdeI and PstI double digestion, and the host E.coli was BL 21. The cloned gene was under the control of a strong T7 promoter and expression was induced by 1mM IPTG.

D. Expression of the truncated gC1q-R recombinant protein:

recombinant gC1q-R with a truncated N-terminal portion was generated as shown in FIG. 1 and designated gC1q-R (C). The gene fragment encoding the N-terminal part was removed from pT7-7/gC1q-R expression vector by restriction enzyme digestion with EcoRI and BstXI. gC1q-R (C) comprises SEQ ID NO: 1 phenylalanine at position 168 to glutamine at position 282. The ends of the vector were filled in with T4 DNA polymerase and religated with T4 DNA ligase. The peptide gC1q-R (C) was expressed in BL21 cells in the same manner as full-length gC1 q-R.

Recombinant gC1q-R with a truncated C-terminal portion was generated as shown in FIG. 1, designated gC1q-R (N.gC1q-R (N)) containing a peptide fragment between leucine 74 to aspartic acid 167 of SEQ ID NO: 1. the gene fragment encoding the C-terminal portion was removed from pT7-7/gC1q-R expression vector by BstXI and PstI restriction enzyme digestion the ends of the vector were filled in with T4 DNA polymerase and religated with T4 DNA ligase. peptide gC1q-R (N) was expressed in BL21 cells in the same manner as full-length gC1 q-R.

Recombinant gC1q-R with an N-terminal 57 amino acid truncation was generated as shown in FIG. 1 and was designated gC1q-R (. delta.1-57). gC1q-R (. delta.1-57) contains the amino acid sequence of SEQ ID NO: 1 from valine at position 131 to glutamine at position 282. The gene fragment was first synthesized by PCR using the full-length gC1q-R cDNA as template, two primers primer 2, SEQ ID NO: 5 and primer 3 of sequence AAGAATTCCGGTCACTTTCAACATT (SEQ ID NO: 6), the reaction conditions for PCR were the same as described above except that the amplification cycles were reduced to 25. The PCR-amplified DNA fragment was cloned into EcoRI and PstI sites of pT7-7 vector. The peptide gC1q-R (. delta.1-57) was expressed in BL21 cells in the same manner as full-length gC1 q-R.

E. Production and purification of gC1q-R from E.coli:

coli expressing gC1q-R was cultured and harvested, sonicated in Tris buffer, the lysate was centrifuged and the supernatant was dialyzed against a buffer containing 20mM HEPES, 0.1M KCl, 0.5% glycerol, 0.2mM EDTA and 1mM dithiothreitol pH 8.0. The dialyzed sample was loaded onto a pre-equilibrated Fast Q ion exchange column (Pharmacia Biotechnology, Piscataway, New Jersey). Bound proteins were eluted with a buffer containing 20mM HEPES and 1MKCl pH 8.0. Fractions tested positive for gC1q-R by SDS-PAGE and Western immunoblot analysis to determine reactivity with recombinant gp120 were collected. The pooled fractions were further purified on a Sephacryl 200 gel filtration column (Pharmacia Biotechnology). The purity of the final material was determined by SDS-PAGE and its binding activity to gp120 was determined by ELISA (see example 4 below). EXAMPLE 2 preparation of monoclonal antibodies against gC1q-R peptide

Male BALB/cJ mice (Jackson Laboratories, Bar Harbor, Maine) of 12 weeks age were injected subcutaneously with 100. mu.g of pure gC1q-R expressed in E.coli in Freund's complete adjuvant (Difco Laboratories, Detroit, Michigan) in 200. mu.l PBS. One month later, mice were injected subcutaneously with 100 μ g of gC1q-R in Freund's incomplete adjuvant. One month later mice were again injected subcutaneously with 100 μ g of the same antigen in Freund's incomplete adjuvant and three days later sacrificed. For each fusion, a single cell suspension was prepared from the spleen of the immunized mice and used for fusion with Sp2/0 myeloma cells. Will be 5X 10⁸Sp2/0 cells and 5X 10 cells⁸Spleen cellFusion was performed in medium containing 50% polyethylene glycol (molecular weight 1450) (Kodak, Rochester, New York) and 5% dimethylsulfoxide (Sigma Chemical co., st. louis, Missouri). The cells were then adjusted to 1.5X 10 per 200. mu.l suspension in Iscove's medium (Gibco, Grand Island, New York) supplemented with 10% fetal bovine serum, 100 units/ml penicillin, 100. mu.g/ml streptomycin, 0.1mM hypoxanthine, 0.4. mu.M aminopterin and 16. mu.M thymidine⁵And (4) spleen cells. 200 microliters of cell suspension was added to each well of approximately 20 96-well microplates. After about 10 days of culture, the supernatant was applied at 50X 10 per well³Individual DAKIKI cells were screened by ELISA on Immulon 1 micro-assay plates (Dynatech Laboratories, Alexandria, Virginia) pre-coated. DAKIKIKI cells were determined to abundantly express gC1q-R on the surface. Briefly, 200 μ l of 5% BLOTTO (skim milk powder) in Phosphate Buffered Saline (PBS) was added to the wells of the micro-assay plate for DAKIKIKIKI cell coating to block non-specific sites. The wells were washed 1 hour later with buffer PBST (PBS with 0.05% Tween 20). 50 microliters of culture supernatant was collected from each fusion well and mixed with 50 μ l of BLOTTO and then added to each well of the micro-test plate. After 1 hour incubation, wells were washed with PBST. Murine antibodies bound to the cells were then detected by reaction with goat anti-mouse IgG conjugated with horseradish peroxidase (HRP) diluted 1: 1000 in BLOTTO (Jackson ImmunoResearch laboratories, West Grove, Pennsylvania). Peroxidase substrate solution containing 1.1% 3 ', 3', 5 ', 5' tetramethylbenzidine (Sigma) and 0.0003% hydrogen peroxide was added to the wells to develop color. Add 50. mu.l of 2M H per well₂SO₄The reaction was terminated. The Optical Density (OD) of the reaction mixture at 450nm was read out with a BioTek ELISA reader (BioTek Instruments, Winooski, Vermont).

Culture supernatants from those positive wells were also tested for reactivity with recombinant gC1q-R in ELISA. Briefly, 50. mu.l of E.coli-expressed gC1q-R at a concentration of 1. mu.g/ml was added to the wells of Immulon 2(Dynatech Laboratories) microtest plates overnight at room temperature. After flicking the plate to remove the coating solution, 200. mu.l of BLOTTO was added to each well while incubating for 1 hour to block non-specific sites. The wells were then washed with PBST and bound murine antibody was detected as described above. Those positive wells were cloned by limiting dilution of the cells and these clones were again tested for reactivity with gC1q-R in ELISA. Selected hybridomas were cultured in spinner flasks and culture supernatants were collected to purify antibodies by protein a affinity chromatography. One such generated monoclonal antibody 99-12-1 directed against gC1q-R was tested to determine if it competed with HIV-1gp120 for binding to gC1q-R (see example 11 below). EXAMPLE 3 preparation of polyclonal antibodies against gC1q-R

Two male New Zealand white rabbits, about 15 weeks old, were immunized subcutaneously with 100. mu.g of E.coli-expressed pure gC1q-R (Difco laboratories) in Freund's complete adjuvant. They were re-injected two weeks later with the same amount of antigen in Freund's incomplete adjuvant. The same immunization was repeated two weeks later. Sera were collected from immunized animals and tested for reactivity with gC1q-R in ELISA as described above except bound antibodies were detected using HRP-conjugated donkey anti-rabbit IgG (Jackson ImmunoResearch Laboratories, Inc.) diluted 1: 2000 in BLOTTO. Serum was collected using animals with higher serological response. Polyclonal rabbit anti-gC 1q-R immunoglobulins were purified by affinity column using gC1q-R coupled Affigel 102(BioRad Laboratories). Example 4 binding of HIV-1IIIB gp120 to gC1q-R expressed in E.coli in ELISA

The wells of Immulon 2 plates were coated with 100. mu.l of E.coli-expressed gC1q-R (5. mu.g/ml in 0.1M sodium acetate buffer pH6) and incubated overnight at room temperature. The wells were treated with blocking buffer PBSTB (PBST containing 2% bovine serum albumin) for 1 hour at room temperature and washed with PBST, 100. mu.l of baculovirus expressed HIV-1IIIB gp120 (Biotech USA, Cambridge, Mass.) was added in duplicate to the respective wells (from 2. mu.g/ml to 0.016. mu.g/ml) and reacted at room temperature for 1 hour, followed by washing as described previously. Bound gp120 was detected with HRP-conjugated anti-HIV-1 gp 120V 3 domain mouse monoclonal antibody BAT123 diluted 1: 2000. The antibody was tested to react with SEQ ID NO: 7 (HXB2R strain amino acid residue number: 308-322) and does not interfere with the binding between gC1q-R and gp 120. To each holeTo this was added 100. mu.l of the diluted conjugate and reacted at room temperature for 1 hour, then the wells were washed and 200. mu.l of peroxidase substrate solution was added to each well to develop color for 30 minutes. By adding 50. mu.l of 2M H₂SO₄The reaction was stopped and the OD was measured at 450nm with a BioTek ELISA reader. Specific reactivity was obtained by subtracting the OD of the test wells with gp120 added to the OD of the control wells without gp120 added.

The results are shown in FIG. 2, where it can be seen that the binding of gp120 to a constant amount of immobilized recombinant gC1q-R increases in an S-type fashion as the concentration of gp120 increases, showing dose-dependent binding of gp120 to gC1 q-R. Since the anti-gp 120V 3 region antibody BAT123 still reacts with gp120 binding to gC1q-R, it was demonstrated that binding of gC1q-R to gp120 does not involve the amino acid sequence as shown in SEQ ID NO: region V3 of gp120 shown in FIG. 7. Example 5 HIV-1 neutralization assay

To test the inhibitory activity of gC1q-R on HIV-1 infectivity, a syncytial formation microanalysis was carried out using CEM-SS cells as target cells, as described by P.L. Nara et al (AIDS Res., human retrovirus 1987; 3: 283-302). Briefly, 50. mu.l of diluted E.coli-expressed gC1q-R was mixed with 50. mu.l of viral culture supernatant containing 200 HIV-1IIIB, HIV-1MN, or HIV-1RF syncytia-forming units (SFU) and incubated at room temperature for 1 hour. Adding the mixture to a solution containing 5X 10⁴Subjecting a culture of DEAE-dextran-treated CEM-SS cells in a microculture well containing 5% CO₂Maintaining at 37 deg.C for 3-4 days. Syncytia were counted under an inverted microscope. Neutralizing activity with IC₅₀Expressed, it is defined as the concentration required to achieve 50% inhibition of infection (i.e. Vn/Vo 50%), where Vn is the SFU in the test wells and Vo is the SFU in the control wells without test antibody.

The results are shown in FIGS. 3A, 3B and 3C, and it can be seen that gC1q-R neutralizes the IC of HIV-1IIIB, MN and RF₅₀4.3, 13.5 and 1.65. mu.g/ml, respectively. These neutralization data suggest that gC1q-R can inhibit infectivity of a wide range of HIV-1 extracts. Example 6 analysis of inhibitory Activity on Syntotome formation

H9 cells infected with HIV-1 and expressionHeLa cells of CD4 investigated the effect of gC1q-R on HIV-1 transmission through cell-cell fusion, which upon contact fused and formed syncytia in culture. HeLa is a human cancer cell line, HeLa-CD4⁺Contained within its genome was CD4 DNA introduced by transfection, which expressed CD4 antigen on its cell surface. The procedure used in this study was similar to that described in p.j.madden et al, cell 1986; 47: 333-348 are similar to those described above. Briefly, HeLa-CD4⁺The cells were cultured at 1X 10⁵Individual cells/well were plated on 24-well micro-culture plates and the plates were incubated for 36 hours when the monolayer of epithelial cells was nearly confluent. HeLa-CD4 when sub-confluent⁺Adding 1X 10 to the cells⁴In infected H9 cells, the cells were in contact and fused, forming multinucleated giant cells (syncytia) after 8 hours. Those syncytia with more than 5 nuclei are readily identified and counted, providing a quantitative measure of syncytia formation. When investigating the effect of pure gC1q-R expressed by E.coli on the formation of syncytia, varying concentrations of gC1q-R were mixed with infected H9 cells and added to HeLa-CD4⁺In the cell. The final medium volume in each well was 1 ml. At 37 ℃ in 5% CO₂After 8 hours of incubation, the wells were washed with RPMI-1640 medium, fixed with methanol, air dried and stained with 0.1% methylene blue in methanol. Cells were examined at 100-fold magnification to determine the number of syncytia (containing more than 5 nuclei) as an average over 4 randomly selected regions. The percent inhibition of syncytia formation was calculated as the reduction in the number of syncytia in the wells to which gC1q-R was added compared to the control.

The results shown in FIG. 4 demonstrate that recombinant gC1q-R inhibits HIV-1 IIIB-infected H9 cells and CD4⁺Fusion between HeLa cells, IC thereof₅₀21. mu.g/ml. Example 7 binding of gC1q-R to HIV-1IIIB infected H9 cells as measured by flow cytometry

The binding between pure gC1q-R expressed by E.coli and gp120 expressed on the surface of HIV-1 IIIB-infected H9 cells was analyzed by indirect immunofluorescence flow cytometry. 50 microliters of infected cells (10X 10) were added⁶Individual cells/ml PBSB (phosphate buffered saline pH with 1% bovine serum albumin)7.4)) were mixed on ice for 30 minutes with the series of concentrations of pure gC1q-R expressed by E.coli. The cells were then washed 1 time in the same buffer, centrifuged at 300 Xg for 5 minutes, the cell pellet resuspended in 50. mu.l of the same buffer and reacted on ice for 30 minutes with the addition of 50. mu.l of affinity purified rabbit anti-gC 1q-R at a concentration of 5. mu.g/ml. Cells were then washed as before and 50. mu.l of fluorescein conjugated donkey anti-rabbit IgG (Jackson ImmunoResearch laboratories) diluted 1: 40 was added and placed on ice for 30 minutes. The cells were subsequently washed, fixed in 1% oligoaldehyde and analyzed with a Coulter EPIC cytoanalyzer (Coulter Electronics, Hialeah, Florida). In the control, only rabbit anti-gC 1q-R and/or fluorescein conjugated donkey anti-rabbit IgG was used. Specific cell staining was calculated by subtracting the percent cell binding of the control from the percent cell binding of the test, which was set at 10%. Uninfected H9 cells were also tested as a negative control for binding studies.

The results show that recombinant gC1q-R binds to HIV-1 IIIB-infected H9 cells in a dose-dependent manner (FIG. 5), but not to uninfected H9 cells. Example 8 Effect of C1q on binding of HIV-1gp120 to gC1q-R in ELISA

To test whether the natural ligand C1q shares a binding site with gp120 for binding to gC1q-R, the effect of C1q on the binding of HIV-1gp120 to gC1q-R was investigated by ELISA. Immulon 2 wells were coated with 50. mu.l of E.coli expressed pure gC1q-R at a concentration of 5. mu.g/ml in 0.1M sodium acetate buffer and incubated overnight at room temperature, after which the wells were blocked with 200. mu.l of blocking/dilution buffer PBSTB for 1 hour at room temperature. After washing the wells with PBST, 50. mu.l of baculovirus-expressed HIV-1IIIB gp120 was added to each well at a concentration of 0.2. mu.g/ml in the presence and absence of gC1q-R expressed in E.coli or C1q purified from human serum (Quidel, San Diego, California) serially diluted with blocking/dilution buffer, and the wells were washed after incubation at room temperature for 1 hour. Mu.l of HRP-conjugated monoclonal antibody BAT123 diluted 1: 2000 in blocking/dilution buffer was added to each well and incubated for 1 hour at room temperature, after which the plate was washed and developed by adding peroxidase substrate solution as described above. Specific reactivity was obtained by subtracting the OD of the test wells with gp120 plus the OD of the control wells without gp 120.

The results are shown in FIG. 6, where the solid dashed circle represents C1q and the solid dashed square represents gC1 q-R. C1q did not compete with gp120 for binding to gC1q-R, whereas gC1q-R competed as expected. The results show that the binding site of gp120 on gC1q-R is different from the binding site of C1q on gC1q-R, and they do not interfere with each other. This is a very important finding, because the presence of large amounts of C1q in the blood does not interfere with the binding of gC1q-R to HIV-1gp120 on virions or on infected cells when gC1q-R is administered to HIV-1 infected individuals or to HIV-1 exposed humans. In addition, the ability of gC1q-R to bind both C1q and gp120 is significant and beneficial because gC1q-R may mediate the activation of the classical complement pathway to lyse HIV-1 virions and HIV-1 infected cells. Example 9 reactivity of different recombinant constructs of HIV-1gp120 with gC1q-R

To locate sites on gC1q-R for HIV-1gp120 binding, gC1q-R recombinant constructs of various lengths were prepared for ELISA. The wells of Immulon 2 were coated with 100. mu.l of pure full length gC1q-R (1-209a.a.), gC1q-R (58-209a.a.), gC1q-R (1-94a.a.), or gC1q-R (95-209a.a.), expressed in E.coli at a concentration of 5. mu.g/ml in 0.1M sodium acetate buffer (pH6) and incubated overnight at room temperature, the wells were treated with blocking/dilution buffer PBSTB for 1 hour at room temperature and washed with PBST, 100. mu.l of serially diluted HIV-1IIIB gp120 (Biotech Co., USA) (from 2. mu.g/ml to 0.016. mu.g/ml) of baculovirus was added in duplicate to the respective wells, reacted for 1 hour at room temperature, and then the wells were washed as before. Bound gp120 was detected by addition of 100. mu.l of HRP-conjugated monoclonal antibody BAT123 diluted 1: 2000, incubated at room temperature for 1 hour, followed by washing of the wells and development for 30 minutes by addition of 200. mu.l of peroxidase substrate solution to each well. 50 μ l of 2M H was added₂SO₄The reaction was stopped and OD was measured at 450nm with a BioTek ELISA reader. Specific reactivity was obtained by subtracting the OD of the test wells with gp120 plus the OD of the control wells without gp 120.

The results are shown in FIG. 7, in which the solid square-dashed line represents the full length gC1q-R (1-209a.a.), the solid circular-dashed line represents gC1q-R (58-209a.a.), the solid triangle represents gC1q-R (1-94a.a.), and the solid inverted triangle represents gC1q-R (95-209 a.a.). gp120 binds to gC1q-R (1-209a.a.), gC1q-R (58-209a.a.), and gC1q-R (95-209a.a.), but not gC1q-R (1-94 a.a.). Thus, the results suggest that the site on gC1q-R for binding to gp120 is located in its C-terminal portion, an observation consistent with the results shown in FIG. 6, i.e., the gp120 binding site is distinct from the C1q binding site, and the C1q binding site is located in the N-terminal peptide fragment of gC1q-R, as shown in SEQ ID NO: 8 (B.Ghebrehiwet et al, journal of Experimental medicine 1994; 179: 1809-1821). This peptide fragment is equivalent to SEQ ID NO: 1, amino acid residues 76-93. Example 10 peptide mapping to locate the functional domain on gC1q-R for binding to 99-12-1

To locate the epitope on gC1q-R that binds 99-12-1, a multineedle peptide Synthesis technique (H.M. Geysen et al, science 1987; 235: 1184-1190) was used. 41 12-mer peptides were independently synthesized in a series on plastic pins of a 96-well micro-test plate matrix, the 41 peptides including the entire gC1q-R sequence (adjacent peptides overlapped with each other by 7 amino acids). The set of peptides was commercially synthesized by Chiron Mimotopes pty.ltd., Clayton, Victoria, australia.

The procedure for epitope mapping using this multi-needle peptide system is similar to the manufacturer's instructions. Briefly, the needles were first treated with a pre-coating buffer containing 2% bovine serum albumin and 0.1% Tween 20 in PBS for 1 hour at room temperature, and then inserted into wells of a 96-well micro-test plate containing antibody 99-12-1 at a concentration of 2. mu.g/ml in the pre-coating buffer, and incubated for 1 hour at room temperature. The needles were washed in PBST (3 times for 10 minutes each) and then incubated for 1 hour at room temperature in wells of a 96-well micro-test plate containing 100. mu.l HRP-conjugated goat anti-mouse IgG (Fc) (Jackson ImmunoResearch laboratories) at a 1: 4000 dilution. After washing the needle as described above, the needle was placed in a solution containing 2, 2' -azino-bis [ 3-ethylbenzothiazoline-b-sulfonic acid]Diammonium (ABTS) and H₂O₂Peroxidase substrate solution (Kirkegaard)&Perrylaboratories Inc., Gaithersburg, Maryland) for development at room temperature for 30 minutes. Reading of plates by BioTek ELISA reader at 405nm vs. 492nmBackground absorption. The colored wells indicate the reactivity of the gC1q-R derived peptide with 99-12-1 in these wells.

The results of epitope mapping showed that 99-12-1 was similar to SEQ ID NO: 2.

To confirm the binding epitope of 99-12-1, the epitope of SEQ ID NO: 2, the ability of the peptide LM12DN to bind to 99-12-1. Using the previously identified SEQ id no: peptide 8 (peptide TD18EE) served as a negative control. Peptides LM12DN and TD18EE were synthesized by an automated peptide synthesizer (Applied Biosystems, Inc., Foster City, California) using the 9-fluorenylmethyloxycarbonyl synthesis program recommended by the manufacturer. Wells of Immulon 2 microtest plates were coated with 50 μ l of 1 μ g/ml peptide LM12DN or TD18EE in a peptide ELISA, and coated overnight at room temperature. After removing the coating solution by gentle shaking of the plate, 200. mu.l PBSTB was added to each well to saturate the non-specific sites for 1 hour at room temperature, and then the wells were washed with PBST. To the wells, 50 microliters of 99-12-1 serially diluted in PBSTB was added in duplicate, incubated at room temperature for 1 hour, and then the plates were washed again. Mu.l of HRP-conjugated goat anti-mouse IgG (Fc) (Jackson ImmunoResearch laboratories) diluted 1: 2000 were added to each well and incubated at room temperature for 1 hour. The wells were then washed and developed by addition of peroxidase substrate as described above. The plate was read at 450nm using an ELISA reader and the OD of the control well was subtracted from the OD of the test well to which 99-12-1 was added to obtain specific reactivity.

The results are shown in FIG. 8, where the solid squares and dashes represent peptide LM12DN and the solid circles and dashes represent peptide TD18 EE. 99-12-1 binds to peptide LM12DN (SEQ ID NO: 2) in a dose-dependent manner, but 99-12-1 does not bind to peptide TD18EE (SEQ ID NO: 8). Example 11 Competition of anti-gC 1q-R monoclonal antibody 99-12-1 for HIV-1gp120 binding to gC1q-R in ELISA

The wells of the Immulon 2 micro-assay plate were coated overnight at room temperature with 50. mu.l of pure gC1q-R expressed in 5. mu.g/ml E.coli in 0.1M sodium acetate buffer (pH6), and then the wells were blocked with 200. mu.l blocking/dilution buffer PBSTB for 1 hour at room temperature, followed by washing with PBST. To each well 50. mu.l of baculovirus expressed HIV-1IIIB gp120 at a concentration of 0.2. mu.g/ml was added in the presence and absence of 99-12-1 serially diluted with blocking/dilution buffer, and the wells were washed after incubation for 1 hour at room temperature. Mu.l of HRP-conjugated monoclonal antibody BAT123 diluted 1: 2000 in blocking/dilution buffer was added to each well and incubated for 1 hour at room temperature, after which the plate was washed and developed by adding peroxidase substrate solution as described above. Inhibition of gp120 binding to gC1q-R by 99-12-1 was calculated as the difference in OD of wells with and without gp 120.

FIG. 9 shows that 99-12-1 inhibits gp120 binding to gC1q-R in a dose-dependent manner, suggesting that 99-12-1 binds to a site on gC1q-R that is critical for gp120 interaction. This site was located as described in example 10 in SEQ ID NO: 2. example 12 Competition of antibodies G3-299 and 6205 for binding of HIV-1gp120 to gC1q-R in an ELISA

To facilitate identification of the gC1q-R binding site on HIV-1gp120, a competition ELISA was designed to examine the effect of various anti-HIV-1 gp120 antibodies and recombinant soluble CD4(rsCD4) on the binding of gp120p120 to gC1 q-R.

The wells of the Immulon 2 micro-assay plate were coated overnight at room temperature with 50. mu.l of pure gC1q-R expressed in 5. mu.g/ml E.coli in 0.1M sodium acetate buffer (pH6), and then blocked with 200. mu.l blocking/dilution buffer PBSTB for 1 hour at room temperature. To each well 50. mu.l of baculovirus-expressed HIV-1IIIB gp120 at a concentration of 0.2. mu.g/ml was added in the presence and absence of anti-HIV-1 gp120 antibody and rsCD4(Biogen, Boston, Massachusetts) serially diluted in blocking/dilution buffer and the wells were incubated for 1 hour at room temperature. The anti-HIV-1 gp120 antibodies tested included: mouse monoclonal antibody G3-299 (binding to the C4 region (amino acid residue number: 423-437 of HXB2R strain) as shown in SEQ ID NO: 9), sheep anti-HIV-1 gp 120C 5 region antibody, 6205 (binding to the sequence of amino acid residue number 497-511 of HXB2R strain as shown in SEQ ID NO: 10), and BAT085 (binding to the V2 region of amino acid residue number 169-183 of HXB2R strain as shown in SEQ ID NO: 11). The properties of G3-299 and BAT085 have been described previously (N.C.Sun et al, J. Virol 1989; 63: 3579-3585; M.S.C.Fung et al, J. Virol 1992; 66: 848-856). Antibody 6205 was purchased from International Enzymes, Fallbrook, California. After washing the plates, 50. mu.l of HRP-conjugated monoclonal antibody BAT123 diluted 1: 2000 in blocking/dilution buffer was added to each well and incubated at room temperature for 1 hour, after which the plates were washed and developed by adding peroxidase substrate solution as described above. Inhibition of gp120 binding to gC1q-R by anti-HIV-1 gp120 antibody or 4sCD4 was calculated as the difference between the OD of wells with and without competitor.

The results are shown in FIG. 10, where the solid dashes represent regions G3-299 of anti-gp 120C 4, the solid dashes represent regions 6205 of polyclonal sheep anti-gp 120C 5, and the solid dashes represent rsCD 4. It can be seen that G3-299 competes very efficiently for gp120 binding to gC1q-R, 6205 competes moderately, but not rsCD 4. Antibody BAT085 against V2 region also did not inhibit binding. Taken together, the results of example 4 and example 12 suggest that the gC1q-R binding site is located in the vicinity of the C4 and C5 regions, which are distinct from the CD4 binding site. This important finding suggests that gC1q-R and rsCD4 may be used in combination to treat HIV-1 infection, thus extending the effectiveness of preventing infection of CD 4-positive target cells and CD 4-negative target cells. Example 13 peptide mapping to locate a functional domain on HIV-1gp120 that binds gC1q-R

To determine the peptide epitope on HIV-1gp120 that binds to the gC1q-R domain, the multi-needle peptide synthesis technique described above was again used. 94 12-mer peptides were synthesized by the Chiron Mimotopes peptide system on plastic pins in a 96-well microtiter plate matrix, the 94 peptides including the complete HIV-1MN gp120 sequence (adjacent peptides overlap each other by 7 amino acids), and the amino acid sequence of HIV-1MN gp120 was based on the LosAlamos national laboratory database (G.Myers et al, human retrovirus and AIDS, 1992). The procedure for locating epitopes is similar to that described in example 10 above.

Pure gC1q-R expressed in E.coli at 5. mu.g/ml was reacted with the peptide on the plastic needle. After washing the needles, bound gC1q-R was detected by reaction with affinity purified rabbit anti-gC 1q-R immunoglobulin for 1 hour at room temperature. The needles were washed as before and reacted with HRP-conjugated donkey anti-rabbit igg (jackson ImmunoResearch laboratories). The color reaction procedure was as described previously.

The gC1q-R binding epitope on HIV-1gp120 was confirmed using overlapping synthetic peptides covering the binding region as envelope antigens in ELISA. The HIV-1 HXB2R gp120 overlapping peptide RC16GG (amino acid residue number: 444-459, shown in SEQ ID NO: 3), RC12LT (amino acid residue number: 444-455, shown in SEQ ID NO: 12), and TG12NN (amino acid residue number: 450-461, shown in SEQ ID NO: 13) were purchased from biotech USA. Wells of Immulon 2 micro-assay plates were coated with 100 μ l of 2 μ g/ml of the corresponding peptide overnight at room temperature, and then treated with 200 μ l of blocking/dilution buffer PBSTB for 1 hour at room temperature before washing the wells with PBST. Pure gC1q-R expressed in E.coli was added in duplicate to wells at different concentrations, reacted at room temperature for 1 hour, and then the plates were washed as before. To each well was added 100. mu.l of affinity purified rabbit anti-gC 1q-R immunoglobulin at 2. mu.g/ml, reacted at room temperature for 1 hour, and then the plate was washed. Add 100. mu.l diluted HRP conjugated donkey anti-rabbit IgG, react for 1 hour at room temperature, wash plate. The peroxidase substrate solution was added as described above to carry out a color development reaction.

The results are shown in FIG. 11, where the solid dashed square line represents RC12LT, the solid dashed circle line represents TG12NN, and the solid dashed triangle line represents RC16 GG. It can be seen that only RC16GG reacted with gC1q-R, a result consistent with the fact that the gC1q-R binding site on gp120 is located in SEQ ID NO: 3, i.e., in the C4 region of gp 120. As shown IN example 12, antibodies G3-299 can effectively compete for gp120 binding to gC1q-R, probably because the binding site for G3-299 (SEQ IN NO: 9) is IN close proximity to the binding site for gC1q-R (SEQ IN NO: 3).

It is understood that the terms, expressions and examples described herein are merely exemplary and not limiting, and that the scope of the invention is defined only by the claims and includes all equivalents.

Sequence listing (1) general information: (i) the applicant: michael S C von, belch N C grandson, seili R Y grandson, jinyu, the name of the invention (ii): gC1q receptor, HIV-1gp120 region bound to it and the sequence numbers of the related peptides and targeting antibody (iii): 13(iv) communication address:

(A) the addressee: tanox Biosystems, Inc.

(B) Street: 10301 Stella Link Rd.

(C) City: houston

(D) State: state of Texas

(E) The state is as follows: united states of America

(F) And (4) post code: 77025(v) computer readable form:

(A) type of medium: 3.5 inch floppy disk

(B) A computer: IBM PS/2

(C) Operating the system: DOS 3.30

(D) Software: wordperfect 5.1(vi) current application data:

(A) application No.:

(B) application date:

(C) and (4) classification: (vii) prior application for information:

(A) application No.: 08/410,360

(B) Application date: 24/03/1995(viii) attorney/attorney information:

(A) name: mirabel, Eric P.

(B) Registration number: 31,211

(C) Case number/document number: TNX95-1-PCT (ix) telecommunication information:

(A) telephone: (713)664-2288

(B) Faxing: (713)664-8914(2) SEQ ID NO: 1, information: (i) sequence features;

(A) length: 1138 nucleotides

(B) Type (2): nucleic acids

(C) Chain property: double chain

(D) Topological structure: linear (ix) sequence description: SEQ ID NO: 1CCGGCGGCGC CTCAGGTCGC GGGGCGCCTA GGCCTGGGTT 40GTCCTTTGCA TCTGCACGTG TTCGCAGTCG TTTCCGCG 78ATG CTG CCT CTG CTG CGC TGC GTG CCC CGT GTG CTG GGC TCC TCC 123Met Leu Pro Leu Leu Arg Cys Val Pro Arg Val Leu Gly Ser Ser

5 10 15GTC GCC GGC CTC CGC GCT GCC GCG CCC GCC TCG CCT TTC CGG CAG 168Val Ala Gly Leu Arg Ala Ala Ala Pro Ala Ser Pro Phe Arg Gln

20 25 30CTC CTG CAG CCG GCA CCC CGG CTG TGC ACC CGG CCC TTC GGG CTG 213Leu Leu Gln Pro Ala Pro Arg Leu Cys Thr Arg Pro Phe Gly Leu

35 40 45CTC AGC GTG CGC GCA GGT TCC GAG CGG CGG CCG GGC CTC CTG CGG 258Leu Ser Val Arg Ala Gly Ser Glu Arg Arg Pro Gly Leu Leu Arg

50 55 60CCT CGC GGA CCC TGC GCC TGT GGC TGT GGC TGC GGC TCG CTG CAC 303Pro Arg Gly Pro Cys Ala Cys Gly Cys Gly Cys Gly Ser Leu His

65 70 75ACC GAC GGA GAC AAA GCT TTT GTT GAT TTC CTG AGT GAT GAA ATT 348Thr Asp Gly Asp Lys Ala Phe Val Asp Phe Leu Ser Asp Glu Ile

80 85 90AAG GAG GAA AGA AAA ATT CAG AAG CAT AAA ACC CTC CCT AAG ATG 393Lys Glu Glu Arg Lys Ile Gln Lys His Lys Thr Leu Pro Lys Met

95 100 105TCT GGA GGT TGG GAG CTG GAA CTG AAT GGG ACA GAA GCG AAA TTA 438Ser Gly Gly Trp Glu Leu Glu Leu Asn Gly Thr Glu Ala Lys Leu

110 115 120GTG CGG AAA GTT GCC GGG GAA AAA ATC ACG GTC ACT TTC AAC ATT 483Val Arg Lys Val Ala Gly Glu Lys Ile Thr Val Thr Phe Asn Ile

125 130 135AAC AAC AGC ATC CCA CCA ACA TTT GAT GGT GAG GAG GAA CCC TCG 528Asn Asn Ser Ile Pro Pro Thr Phe Asp Gly Glu Glu Glu Pro Ser

140 145 150CAA GGG CAG AAG GTT GAA GAA CAG GAG CCT GAA CTG ACA TCA ACT 573Gln Gly Gln Lys Val Glu Glu Gln Glu Pro Glu Leu Thr Ser Thr

155 160 165CCC AAT TTC GTG GTT GAA GTT ATA AAG AAT GAT GAT GGC AAG AAG 618Pro Asn Phe Val Val Glu Val Ile Lys Asn Asp Asp Gly Lys Lys

170 175 180GCC CTT GTG TTG GAC TGT CAT TAT CCA GAG GAT GAG GTT GGA CAA 663Ala Leu Val Leu Asp Cys His Tyr Pro Glu Asp Glu Val Gly Gln

185 190 195GAA GAC GAG GCT GAG AGT GAC ATC TTC TCT ATC AGG GAA GTT AGC 708Glu Asp Glu Ala Glu Ser Asp Ile Phe Ser Ile Arg Glu Val Ser

200 205 210TTT CAG TCC ACT GGC GAG TCT GAA TGG AAG GAT ACT AAT TAT ACA 753Phe Gln Ser Thr Gly Glu Ser Glu Trp Lys Asp Thr Asn Tyr Thr

215 220 225CTC AAC ACA GAT TCC TTG GAC TGG GCC TTA TAT GAC CAC CTA ATG 798Leu Asn Thr Asp Ser Leu Asp Trp Ala Leu Tyr Asp His Leu Met

230 235 240GAT TTC CTT GCC GAC CGA GGG GTG GAC AAC ACT TTT GCA GAT GAG 843Asp Phe Leu Ala Asp Arg Gly Val Asp Asn Thr Phe Ala Asp Glu

245 250 255CTG GTG GAG CTC AGC ACA GCC CTG GAG CAC CAG GAG TAC ATT ACT 888Leu Val Glu Leu Ser Thr Ala Leu Glu His Gln Glu Tyr Ile Thr

260 265 270TTT CTT GAA GAC CTC AAG AGT TTT GTC AAG AGC CAG 924Phe Leu Glu Asp Leu Lys Ser Phe Val Lys Ser Gln

275280 TAGAGCAGAC AGATGCTGAA AGCCATAGTT TCATGGCAGG 964CTTTGGCCAG TGAACAAATC CTACTCTGAA GCTAGACATG 1004TGCTTTGAAA TGATTATCAT CCTAATATCA TGGGGGAAAA 1044AATACCAAAT TTAAATTATA TGTTTTGTGT TCTCATTTAT 1084TATCATTTTT TTCTGTACAA TCTATTATTT CTAGATTTTT 1124GTATAACATG ATAG 1138 (2) SEQ ID NO: 2, information:

(i) sequence features;

(A) length: 12 amino acids

(B) Type (2): amino acids

(D) Topological structure: linearity

(ix) Description of the sequence: SEQ ID NO: 2Leu Met Asp Phe Leu Ala Asp Arg Gly Val Asp Asn

510 (2) SEQ ID NO: 3, information:

(i) sequence features;

(A) length: 16 amino acids

(B) Type (2): amino acids

(D) Topological structure: linearity

(ix) Description of the sequence: SEQ ID NO: 3Arg Cys Ser Ser Asn Ile Thr Gly Leu Leu Leu Thr Arg Asp Gly Gly

51015 (2) SEQ ID NO: 4:

(i) sequence features;

(A) length: 26 nucleotides

(B) Type (2): nucleic acids

(C) Chain property: double chain

(D) Topological structure: linearity

(ix) Description of the sequence: SEQ ID NO: 4TACATATGCT GCACACCGAC GGAGAC 26(2) SEQ ID NO: 5, information:

(i) sequence features;

(A) length: 24 nucleotides

(B) Type (2): nucleic acids

(C) Chain property: double chain

(D) Topological structure: linearity

(ix) Description of the sequence: SEQ ID NO: 5GCCCTGCAGC ATCTGTCTGC TCTA 24(2) SEQ ID NO: 6:

(i) sequence features;

(A) length: 25 nucleotides

(B) Type (2): nucleic acids

(C) Chain property: double chain

(D) Topological structure: linearity

(ix) Description of the sequence: SEQ ID NO: 6AAGAATTCCG GTCACTTTCA ACATT 25(2) SEQ ID NO: 7, information:

(i) sequence features;

(A) length: 15 amino acids

(B) Type (2): amino acids

(D) Topological structure: linearity

(ix) Description of the sequence: SEQ ID NO: 7Arg Ile Gln Arg Gly Pro Gly Arg Ala Phe Val Thr Ile Gly Lys

51015 (2) SEQ ID NO: information of 8:

(i) sequence features;

(A) length: 18 amino acids

(B) Type (2): amino acids

(D) Topological structure: linearity

(ix) Description of the sequence: SEQ ID NO: 8Thr Asp Gly Asp Lys Ala Phe Val Asp Phe Leu Ser Asp Glu Ile LysGlu

51015 (2) SEQ ID NO: 9, information:

(i) sequence features;

(A) length: 15 amino acids

(B) Type (2): amino acids

(D) Topological structure: linearity

(ix) Description of the sequence: SEQ ID NO: 9Ile Ile Asn Met Trp Gln Lys Val Gly Lys Ala Met Tyr Ala Pro

51015 (2) SEQ ID NO: 10, information:

(i) sequence features;

(A) length: 15 amino acids

(B) Type (2): amino acids

(D) Topological structure: linearity

(ix) Description of the sequence: SEQ ID NO: 10Ala Pro Thr Lys Ala Lys Arg Arg Val Val Gln Arg Glu Lys Arg

51015 (2) SEQ ID NO: 11, information:

(i) sequence features;

(A) length: 15 amino acids

(B) Type (2): amino acids

(D) Topological structure: linearity

(ix) Description of the sequence: SEQ ID NO: 11Val Gln Lys Glu Tyr Ala Phe Phe Tyr Lys Leu Asp Ile Ile Pro

51015 (2) SEQ ID NO: 12, information: (i) sequence features;

(A) length: 12 amino acids

(B) Type (2): amino acids

(D) Topological structure: linear (ix) sequence description: SEQ ID NO: 12Arg Cys Ser Ser Asn Ile Thr Gly Leu Leu Leu Thr

510 (2) SEQ ID NO: 13, information: (i) sequence features;

(A) length: 12 amino acids

(B) Type (2): amino acids

(D) Topological structure: linear (ix) sequence description: SEQ ID NO: 13Thr Gly Leu Leu Leu Thr Arg Asp Gly Gly Asn Asn

5 10

Claims (14)

1. A polypeptide having the sequence of SEQ ID NO: 2.

2. The peptide of claim 1 conjugated to a carrier that enhances its immunogenicity.

3. The peptide of claim 2 wherein said carrier is KLH.

4. Encoding the amino acid sequence of SEQ ID NO: 2.

5. The oligonucleotide of claim 4, which is a deoxyoligonucleotide.

6. A binding molecule or antibody targeting the peptide of claim 1.

7. The binding molecule or antibody of claim 6 which is a monoclonal antibody.

8. Monoclonal antibody 99-12-1.

9. A cell line producing monoclonal antibody 99-12-1.

10. A polypeptide having the sequence of SEQ ID NO: 3.

11. The peptide of claim 10 conjugated to a carrier that enhances its immunogenicity.

12. The peptide of claim 10 wherein said carrier is KLH.

13. Encoding the amino acid sequence of SEQ ID NO: 3.

14. The oligonucleotide of claim 13, which is a deoxyoligonucleotide.