EP0485380A1 - Immuntestverfahren unter verwendung von mikrowellen - Google Patents

Immuntestverfahren unter verwendung von mikrowellen

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Publication number
EP0485380A1
EP0485380A1 EP90902980A EP90902980A EP0485380A1 EP 0485380 A1 EP0485380 A1 EP 0485380A1 EP 90902980 A EP90902980 A EP 90902980A EP 90902980 A EP90902980 A EP 90902980A EP 0485380 A1 EP0485380 A1 EP 0485380A1
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EP
European Patent Office
Prior art keywords
microwave
epitope
antigen
resistant
entity
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP90902980A
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English (en)
French (fr)
Other versions
EP0485380A4 (de
Inventor
Peter Gregory Bundesen
Dennis Brian Rylatt
Carmel Judith Hillyard
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Agen Ltd
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Agen Ltd
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Publication date
Application filed by Agen Ltd filed Critical Agen Ltd
Priority claimed from AU50949/90A external-priority patent/AU640634C/en
Publication of EP0485380A4 publication Critical patent/EP0485380A4/de
Publication of EP0485380A1 publication Critical patent/EP0485380A1/de
Withdrawn legal-status Critical Current

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K41/00—Medicinal preparations obtained by treating materials with wave energy or particle radiation ; Therapies using these preparations
    • A61K41/10—Inactivation or decontamination of a medicinal preparation prior to administration to an animal or a person
    • A61K41/13—Inactivation or decontamination of a medicinal preparation prior to administration to an animal or a person by ultrasonic waves
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/43504—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates
    • C07K14/43536—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates from worms
    • C07K14/4354—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates from worms from nematodes
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/08—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses
    • C07K16/10—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses from RNA viruses
    • C07K16/1036—Retroviridae, e.g. leukemia viruses
    • C07K16/1045—Lentiviridae, e.g. HIV, FIV, SIV
    • C07K16/1054—Lentiviridae, e.g. HIV, FIV, SIV gag-pol, e.g. p17, p24
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/08—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses
    • C07K16/10—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses from RNA viruses
    • C07K16/1036—Retroviridae, e.g. leukemia viruses
    • C07K16/1045—Lentiviridae, e.g. HIV, FIV, SIV
    • C07K16/1063—Lentiviridae, e.g. HIV, FIV, SIV env, e.g. gp41, gp110/120, gp160, V3, PND, CD4 binding site
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/73—Inducing cell death, e.g. apoptosis, necrosis or inhibition of cell proliferation
    • C07K2317/732—Antibody-dependent cellular cytotoxicity [ADCC]

Definitions

  • the present invention relates to antigens having microwave-resistant epitopes, and the role of such antigens, and of antibodies to such epitopes, in the diagnosis or treatment of a disease or condition of the body.
  • a successful infectious agent is one which modulates the host's immune response to its advantage such that an immunocompetent host responds with an immune response that is not harmful to the infectious agent.
  • the antigens which cause the predominant immune response, particularly the antibody response are antigens that are not involved in key metabolic or biological processes fundamental to the survival of the infectious agent in the host.
  • the antigens which cause the predominant immune response camouflage key infectious agent antigens thus protecting them from the host's immune response.
  • the infectious agent may present antigens which the host recognizes as "self” and consequently does not respond to; such antigens camouflage epitopes borne by antigens essential to the survival of the infectious agent.
  • the HIV-1 virus is known to have antigenie sites on its surface and to leave viral antigens on infected cell surfaces, which mirror normal cellular receptor molecules (e.g., the glycoprotein gp120, binds to the normal CD4 receptor). These epitopes are exposed to the infected host's immune system but are apparently seen as "self” and an antibody response is not usually generated.
  • mirror normal cellular receptor molecules e.g., the glycoprotein gp120
  • U.S. 4,658,022 (Molecular Diagnostics) teaches denaturing a protein by chemical or physical means to destroy conformational epitopes and expose or enhance the exposure of linear epitopes. Antibodies are prepared against these treatment-resistant epitopes.
  • the methods of denaturation include "heat, sonication, high or low pH” or treatment with a “chaotropic reagent.” (Col. 8, lines 8-13). There is no reference to denaturation by radiation.
  • Milich U.S. 4,683,136 teaches use of a denaturing agent to destroy a conformational epitope, specifically, of a Hepatitis B virus antigen.
  • the modified antigen is then used on an assay for sample antibodies which react with a conformation-independent determinant.
  • Vaccine use of microwaved antigens is known. Injection of mice with microwaved B16 melanoma cell membranes, prior to challenge with B16 melanoma, increased the subjects' survival time as compared with mice immunized with untreated cell membranes. Douss, et al., C.R. SOC. Biol., 179:615-24 (1985). Gray, U.S. 3,660,234 attenuated a virus by microwave treatment.
  • Microwave irradiation can have a heating effect. Heat has been used to selectively denature epitopes. Hansen, W088/09934 describes a CEA immunoassay in which the sample is heated to 90°C and then assayed using monoclonal antibodies which bind to heat-stable CEA epitopes. Anacker, et al., Infection and Immunity, 55:825 (1987) reports that Rickettsia
  • rickettsi surface proteins exhibit both heat-sensitive and heat-resistant epitopes.
  • Monoclonal antibodies specific for one type or the other have been developed. Only antibodies against heat-sensitive epitopes had a protective effect on immunized mice. "Conceivably, the heat-resistant epitopes are exposed by denaturing influences, such as heat, and are not immunoaccessible to any appreciable extent on the surfaces of normal infectious rickettsiae.” (p. 827). See also, Leye and Walker, Infection and Immunity, 56:2587-93 (1988); Sakomoto, et al., 761 Bakt. Hyg. A., 259: 507-19 (1985). However, microwave treatment does not need to heat the subject. See Chen, U.S. 4,327,180.
  • Microwaves have been used to fix cells without antigen loss. See Leong and Milios, J. Pathol., 148:183-7 (1986); Patterson, Jr. and Bulard, Stain Technology, 55:71 (1980); and Hopgood, et al., Histochemical J., 16:1171-91 (1984).
  • the object of this invention relates to the use of microwave radiation in the preparation of antibodies against
  • immunorecessive ("masiced") epitopes of an antigen associated with an infectious agent, and especially against the epitope of an antigen essential to the survival or pathogenicity of the agent.
  • Such antibodies may be used to detect the circulating antigen having such epitopes and thereby detect the presence of the infectious agent.
  • the agent is more reliably detected in this manner, since the normally immunorecessive epitopes are less likely to be altered by strain-to-strain variations or blocked by the presence of circulating antibodies generated against the immunodominant epitopes.
  • the antibodies may also be used in immunotherapy.
  • the microwaved antigens or the microwave-resistant epitopes thereof, are prepared and used to detect whether the body has mustered an immune response against such epitopes, to raise antibodies against such antigens and epitopes for diagnostic or therapeutic use, and for direct immunization of patients to stimulate an immune response.
  • a process for altering the immunological characteristic of an antigenie entity which comprises irradiating an antigenic entity having at least one microwave-resistant epitope and at least one microwave-labile epitope with microwave radiation of an intensity, wavelength and duration effective to reduce the antigenicity of said microwave-labile epitope while retaining said microwave-resistant epitope, thereby yielding an altered antigenic entity.
  • a process for altering the immunological characteristic of an antigenic entity which comprises irradiating an antigenic entity having at least one microwave-resistant epitope with microwave radiation of an intensity, wavelength and duration effective to produce a altered antigenic entity retaining said microwave-resistant epitope and providing increased
  • an antibody specific for a natively immunorecessive, microwave-resistant epitope of a naturally occurring antigen is provided.
  • a process for preparing an antibody specific for a microwave-resistant epitope which comprises immunizing an animal with a microwave-heated antigenic entity, said entity natively having at least one
  • microwave-resistant epitope the antigenicity of said microwave treated antigenic entity differing from that of said entity prior to microwave treatment.
  • a non-natural ly occurring antigen characterized by the presence of a microwave resistant epitope common to a naturally occurring antigen and the absence of a microwave-labile epitope common to the naturally- occurring antigen, said naturally occurring antigen being associated with an infectious agent.
  • an immunoassay for the detection in a sample of an antigen associated with an infectious agent, wherein an antibody reagent which recognizes said antigen is incubated with the sample or a portion thereof, the improvement consisting of providing an antibody reagent specific for a
  • microwave-resistant epitope of such antigen and of subjecting the sample of a portion thereof to microwave-treatment prior to incubating it with said antibody.
  • an immunoassay for the detection in a sample of an antibody produced in a subject in response to an antigen associated with an infectious agent, wherein an antigenic reagent recognized by said antibody is incubated with the sample or a portion thereof, the improvement consisting or providing a first antigenic reagent which presents essentially only microwave-resistant epitopes of said antigen.
  • a therapeutic or prophylactic composition comprising an antigen according to the first, second or fifth embodiments or an an antibody according to the third embodiment and a pharmaceutically acceptable carrier.
  • a ninth embodiment of this invention there is provided a method of inducing immunity in a host to a condition associated with an antigenic entity comprising administering to said host an immunity inducing amount of a composition according to the eighth embodiment.
  • a method for the in vitro detection and diagnosis of an antigenic entity having at least one microwave-resistant epitope in an animal or human suspected of having the antigenic entity comprises:
  • an eleventh embodiment of this invention there is provided a method for the in vitro detection and diagnosis of an antigenic entity having at least one microwave-resistant epitope and at least one microwave-labile epitope in an animal or human suspected of having the antigenic entity, which method comprises:
  • a method for the in vitro detection and diagnosis of an antigenic entity having at least one microwave-resistant epitope in an animal or human suspected of having the antigenic entity comprises:
  • an anti-idiotypic antibody specific for an antibody according to the invention is provided.
  • a fourteenth embodiment of this invention there is provided a diagnostic kit for the detection of an antigenic entity having a microwave radiation resistant epitope or antibodies to the antigenic entity, comprising:
  • a diagnostic kit for the detection of an antigenic entity having a microwave radiation resistant epitope comprising:
  • the diagnostic kits of the invention also include:
  • an antigenic entity having a microwave radiation resistant epitope having a microwave radiation resistant epitope
  • Monoclonal antibodies of the invention are typically produced by bybridomas formed by fusion of cells from a mouse myeloma line and spleen cells from a mouse prior immunized with an altered antigenic entity of the invention. More specifically, mice are immunized with an altered antigenic entity of the invention and monoclonal antibodies are produced according to the method of Bundesen et al., 1984, Vet. Immunol, and Immunopath. the contents of which are incorporated herein by cross-reference.
  • Anti-idiotypic monoclonal antibodies of the invention are typically prepared from a monoclonal antibody of the invention (prepared from a hybridoma cell line) by:
  • Antibodies can be raised in either the natural host or an alternative animal or tissue culture system which are important to the life cycle of the infectious agent.
  • Fig. 1 is a flow diagram of the particular combination of steps used in processes of the invention for the development of the antibodies and subsequent vaccines and kits.
  • An antigen is any molecule that can generate or be induced to generate an immune response in an appropriate animal. It may be a polypeptide, protein, glycoprotein or a carbohydrate, but is not
  • An epitope is the structural feature of an antigen which is recognized fay an antibody. This feature, in the case of a polypeptide, may be formed by a continuous sequence of amino acids, in which case the epitope is said to be linear or sequential, or by the three-dimensional folding of the polypeptide so that portions distant in sequence are brought into close proximity, whereupon the determinant is said to be
  • a "cryptic" epitope may be considered an extreme case of immunorecessivity.
  • the present invention is generally directed to the use of microwave radiation, with or without a heating effect, to render an immunorecessive epitope of an antigen more immunologically accessible.
  • Non-essential epitopes are believed to be more likely to be immunodominant and microwave labile.
  • Epitopes essential to the survival of the infectious agent are more likely to be immunorecessive and microwave resistant. Our reasoning is as follows:
  • the body defends itself against infectious agents by immune
  • Table 2 provides examples of antigens associated with some of the agents of Table 1.
  • the monoclonal antibody is preferably but not essentially of the same species as the recipient when the recipient is immunocompetent (e.g., in the case of a human subject, the monoclonal antibody is preferably humanized or of human origin).
  • the recipient of an antigen or antibody according to this invention may be a human, a nonhuman mammal, a bird, a fish, a reptile or an amphibian, i.e., any vertebrate.
  • the recipient is a human or a farm animal (e.g., cow, pig, horse, goat, sheep), pet (e.g., cat, dog) or research (e.g., mouse, rat, guinea pig, ape, chimpanzee, rabbit) animal.
  • the antigens to be irradiated may be obtained from nature or prepared synthetically.
  • a protein antigen may be isolated from its natural source, synthesized directly by joining amino acids, or produced by transforming a host cell with a recombinant DNA molecule which bears an expressible gene encoding the protein, or a molecule cleavable to obtain that protein.
  • microwave-resistant epitopes of an antigen it is also possible to dispense with isolating and irradiating the whole antigen, and instead prepare smaller molecules bearing the desired microwave-resistant epitopes. If these molecules are too small to induce an immune response, they may be conjugated to an immunogenic carrier. In the case of a peptide antigen, the conjugate may be expressed by recombinant DNA techniques as a fusion of the desired hapten and carrier protein.
  • the antigens may be subjected to other physical or chemical treatments, before or after exposure to microwaves, which affect their immunological characteristics. For example, they may be cleaved with a pfoteolytic agent, or partially denatured by heat,
  • soni cation high or low pH, chaotropic agents, non-MW radiation, and the like.
  • the antigens, when irradiated, may still be associated with the infectious agent or infected cell, or they may be in an isolated form.
  • microwaves are defined as electromagnetic waves having a wavelength primarily in the range of 0.1 to 30 cm (and thus frequencies of 1 to 300 GHz).
  • the antigens are irradiated for 30 to 180 seconds with intensities of 400 to 700 watts at a frequency of 2.450 GHz.
  • Microwave radiation may be produced by backward wave oscillators, klystrons or other suitable devices.
  • Microwave treatment heats its subject by transferring energy to water, which in turn can transfer energy to adjacent antigen molecules; such heating will affect some epitopes.
  • a heating effect is not desired, the subject is microwaved in the presence of a heat sink such as a volume of water contained in a suitable vessel such as a glass beaker.
  • microwave-resistant antigen refers to (1) natural antigens containing only microwave-resistant epitopes, (2) antigens initially having both microwave-resistant and microwave-labile epitopes which have been irradiated to degrade the latter, and (3) synthetic antigens constructed so as to present only the
  • microwave-resistant epitopes of a corresponding natural antigen should not be taken in an absolute sense, e.g., depending on the duration and intensity of the MW exposure, the percentage of molecules in which a microwave-labile epitope is destroyed will vary. Similarly, an antibody may be said to be “specific” for a MW-resistant epitope even though it has some cross-reactivity with other epitopes.
  • Polyclonal antibodies to microwave resistant epitopes present on the surface of the infectious agent or on the surface of a cell infected with the infectious agent can be prepared by immunizing an appropriate animal using standard immunization procedures and collecting serum. Human polyclonal antibodies may be recovered from the sera of infected
  • Antibodies which recognize irradiated and non-irradiated antigen can be purified from the serum by conventional affinity purification techniques.
  • Monoclonal antibodies to microwave resistant epitopes present on the surface of the infectious agent or on the surface of a cell infected with the infectious agent can be prepared by immunizing mice or rats or human lymphoid cells in tissue culture with microwave irradiated antigen. In the murine system this would be performed by obtai ning spl een cel l s from the immunized animal . The spl een cel ls are then fused with myeloma cells to produce hybridomas. The hybridomas are screened and cloned to select those hybridomas producing antibodies which recognize irradiated and
  • non-irradiated antigen Screening of the hybridomas is performed using .both irradiated and non-irradiated antigen preferably.
  • these antibodies are used, for passive immune treatment of a host. Passive immunotherapy for AIDS patients with
  • Monoclonal antibodies because of their specificity are preferred. These monoclonal antibodies may be able to mediate natural host immune responses, such as antibody dependent cellular cytotoxicity (ADCC) or complement mediated lysis. Alternatively, they may be conjugated to a chemotherapeutic agent or toxin, such as ricin.
  • ADCC antibody dependent cellular cytotoxicity
  • This invention provides a process for preparing antibodies against an immunorecessive epitope of an infectious agent or antigen, comprising:
  • the process includes screening the specific antibodies to select antibodies to microwave radiation resistant epitopes present on the surface of the infectious agent or on the surface of a cell infected with the infectious agent and accessible to a humoral, cell-mediated or a combination of humoral and cell-mediated immune response in a host.
  • the antibody may be associated with a therapeutic agent or chemotherapeutic agent (such as ricin) effective against the infectious agent or infected cells and used to direct the therapeutic or chemotherapeutic agent to kill or immobilise the infectious agent or infected cells.
  • the antibody may be a human, murine, bovine, porcine, rabbit, goat or other species of monoclonal antibody, or a chimeric antibody in which the constant and variable regious are derived from different species (e.g., human constant and murine variable).
  • the antibodies are used diagnostically for the detection of antigens which bear microwave-resistant epitope.
  • a sample believed to contain the antigens of interest is subjected to microwave treatment, with or without preliminary separation or modification steps, and assayed using the antibodies as reagents.
  • Any assay format conventional in the immunological arts may be used, including both sandwich, competitive assays.
  • the antibody may be insolubilized directly or indirectly, covalently or noncovalently, on an insoluble support, or it may be labeled with a convenient label, such as a
  • radioisotope fluorophore or enzyme. It may also be immobilized on an agglutinable particle of a kind conventionally used in agglutination assays, such as a latex bead.
  • the samples to be assayed using the reagents of this invention may be biological samples, such as tissues and biological fluids (e.g., blood, urine, milk, etc.) or nonbiological samples such as soil, water, or food.
  • biological samples such as tissues and biological fluids (e.g., blood, urine, milk, etc.) or nonbiological samples such as soil, water, or food.
  • the antibodies are also useful in identifying those microwave resistant epitopes that may be present in or on the surface of both the infectious organism and normal cells of the infected host.
  • a host immune response to such epitopes can lead to autoimmune disease processes.
  • the antibodies are also useful in the purification of the microwave-resistant antigens and the characterization of their microwave-resistant epitopes.
  • the antibodies are associated with an affinity chromatography support.
  • the epitopes may be localized by preparing fragments of the antigen (either by proteolysis of the whole antigen or by direct production of the fragment) and testing different fragments for affinity to the antibodies of this invention.
  • the amino acid sequence data may also be used to predict antigenic sites for further analysis.
  • synthetic analogues may be designed which present the desired microwave resistant, natively immunorecessive epitopes in a more immunologically accessible form than in the native antigen.
  • the antigens of this invention have a variety of uses. First, they may be used in labeled or insolubilized form in a diagnostic kit for the detection of circulating antibodies reacting with microwave resistant epitopes present on both native and treated antigen which is not seen by the normal immune response in a host which allows detection of whether the host has been vaccinated against an infectious agent.
  • a diagnostic kit for the detection of circulating antibodies reacting with microwave resistant epitopes present on both native and treated antigen which is not seen by the normal immune response in a host which allows detection of whether the host has been vaccinated against an infectious agent.
  • Such a kit comprises antigens having microwave resistant epitopes and the corresponding monoclonal antibodies or affinity purified polyclonal antibodies as positive control.
  • the kit may additionally include native antigens to permit detection of the normal antibody response and thereby allow
  • the antigens may be used therapeutically.
  • By rendering an epitope immunologically accessible and raising monoclonal antibodies against such epitopes it is possible to identify candidate epitopes for the provision of recombinant vaccines comprising the epitope(s) that have been demonstrated to be unique to the infectious organism and able to mediate a protective immune response, while omitting those epitope(s) that have been demonstrated to be normal host cell autoantigen(s) that have the potential to produce autoimmune disease.
  • the epitope may also be provided in the form of the infectious agent or antigen itself after treatment to render the relevant epitope immunologically accessible or may be provided in combination with other epitopes of the native infectious agent or antigen, to provide a vaccine.
  • care is preferably taken to eliminate microwave resistant epitopes that are common to both the infectious organism and the host, in order to preclude the possibility of generating an autoimmune response when using such a vaccine.
  • Anti-idiotypic antibodies may be used for the same purposes as antigens.
  • Anti-idiotypic antibodies which mimic the microwave resistant epitopes present on the surface of the infectious agent or on the surface of a cell infected with the infectious agent can be prepared by immunizing an immunocompetent host with said monoclonal antibodies; harvesting antibodies against said monoclonal antibodies from said host; and screening the harvested antibodies to detect those antibodies which are
  • the vaccines of the present invention may be (a) a
  • microwave-treated infection-related natural antigen that has been treated to remove epitopes common to both the infectious organism and the host, (b) a synthetic or recombinant antigen presenting a microwave resistant epitope of a unique natural infection-related antigen that is not a shared autoantigen of the host, or (c) an anti-idiotypic antibody raised against an antibody specific for a unique microwave-resistant epitope of an infection-related antigen that is not a shared autoantigen of the host.
  • the antigen lacks one or more masking epitopes of an analogous antigen of the infectious agent of interest, so that the immune response to other epitopes of the antigen is enhanced.
  • a plurality of antigens presenting the same or different microwave-resistant epitopes, may be administered to achieve the desired immune response.
  • the immunization agent may include an adjuvant such as aluminum hydroxide.
  • Microwave-resistant, infection-specific antigens may be of particular value in the treatment of viral infections associated with immunoparalysis and/or autoimmune reactions.
  • HIV-1 is now generally accepted as being the etiologic agent in the development of acquired immune deficiency syndrome (AIDS). It is suspected that other disease states displaying autoimmune phenomena where there is an involvement of anti-lymphocyte antibodies (Juvenile Rheumatoid Arthritis, JRA; Systemic Lupus Erythematosus, SLE), or auto-antibodies to other cell types (Type 1 Diabetes Mellitus; Multiple Sclerosis, MS; Graves Disease; Myasthenia Gravis) may also have a viral aetology.
  • ADCC antibody-dependent cellular cytotoxicity
  • Viral infections are a known cause of transient iymphocytotoxic antibodies which can result in T lymphocytopenia, which usually resolves during recovery from the infection (De Horatius).
  • T lymphocytopenia which usually resolves during recovery from the infection (De Horatius).
  • auto-antibodies to lymphocytes as well as other cell types have also been reported (Kloster, et al. 1984; Dorsett, et al., 1985; Kiprov, et al. 1985; Williams, et al. 1984; Tomar, et al. 1985).
  • Cytotoxic complement fixing IgG anti-lymphocyte antibodies are present in
  • AIDS patients Up to 50% of ARC patients have also been found to have anti-lymphocyte antibodies. These anti-lymphocyte antibodies have been variously identified as specific for the helper/inducer OKT4, OKT 11 positive T cells, or reactive with all T cell sub-sets and B cells. Some 18% of AIDS patients have also been found to have autoantibodies that react with red blood cells (Toy, et al. 1985) and autoantibody mediated thrombocytopenia has been found to be a major hematological abnormality associated with AIDS (Strieker, et al. 1985).
  • HIV specific cytotoxic T lymphocytes capable of killing infected cells, have been widely reported and are believed to be an important mechanism for host defense against infection. HIV-1 specific cytotoxic T cells have, also been found in infected individuals that are able to preferentially kill either autologous B cells expressing recombinant HIV-1 vaccinia virus antigens or autologous HIV-1 infected macrophages and thus indicate that the MHC may be important in restricting the cytotoxic response. Although the major response appears to be against HIV-1 env products, there is also a demonstrable but lesser gag specific cytolytic response (Walker, et al., 1987).
  • gag-specific cytotoxic lymphocytes were found in 13 out of 15 healthy HIV-1 seropositive patients using a recombinant vaccinia virus to express the gag protein of HIV-1. Further, cytotoxic lymphocytes from one patient recognized a 15 amino acid peptide in p24 in association with HLA-B27.
  • the finding of gag specific cytolytic T cells in HIV-1 infections is not surprising as a number of other viral infections (e.g., influenza) have also been shown to elicit cytotoxic T cell responses to internal viral structural proteins and the gag gene encoded target proteins were
  • gag specific cytotoxic T cell responses exert a beneficial role for the duration of the disease; they may in fact perform a harmful role by mediating autoimmune cytotoxicity.
  • Type 1 MAb react with crossreacting p24 epitopes that are present on the surface of both HIV-1 infected and uninfected peripheral blood lymphocytes, and red blood cells.
  • Type 1 monoclonal antibodies H69.67.2D4
  • Type 2 MAb react with p24 epitopes that are only expressed on HIV-1 infected lymphocytes and are absent from normal uninfected peripheral blood lymphocytes or other normal cells.
  • immunorecessive epitopes are finally able to generate an immune response and this mechanism would particularly facilitate the production of antibodies to crossreacting virus/self autoantigens.
  • Monoclonal antibodies have been generated that bind to a
  • autoantibodies show remarkable poly-specificity; not only do they react with DNA, but with phospholipids such as cardiolipin and proteins such as vimentin. Some 66% of SLE patients have anti-cardiolipin antibodies.
  • peripheral nerve cells could also, with further immune processing of cell bound antibody or antibody/antigen complexes, generate cytotoxic T cells reactive to these viral/self epitopes expressed on normal uninfected cell populations. Further, some of these antibodies could be enhancing and thus provide an alternative mechanism for viral infection in non-CD4 bearing cell types. Thus this overall process would provide a simple unifying mechanism to explain the totality of HIV-1 pathogenesis and should allow rational and effective strategies for immune modulation of the disease. Importantly the identification of unique HIV-1 gag epitopes expressed only on the surface of infected cells, could pave the way for the construction of safe and effective vaccines that would only contain the unique epitopes and eliminate crossreactive epitopes. Further, the Type 2 murine
  • monoclonal antibodies that react with the unique gag epitopes may provide an effective means of passive immunotherapy in AIDS patients.
  • Type II monoclonal antibodies to microwave resistant epitopes may also be present in stable regions of envelope antigens, such as the region of gp41 with the amino acid sequence RILAVERYLKDQQLLGIWQCSGK, as demonstrated by the Mab H69.67.2A2.26.
  • gag and env HTLV-1 related sequences were detected in peripheral blood mononuclear cells from MS patients and antibodies that react with the gag p24 HTLV-1 antigen have also been found in the serum and cerebrospinal fluid. Consequently, it is highly likely that crossreacting p24 epitopes will be found on the normal cell surface of oligodendrocytes and that such epitopes would mediate an autoimmune response.
  • antibodies developed against epitopes of microwaved antigens that are present on both the untreated and microwaved antigen may be used for the development of diagnostic kits for applications such as the detection of circulating heartworm antigen in dog plasma without the need to process the circulating antigen in order to remove or destroy the autologous antibody that would block test antibody from binding.
  • diagnostic kits for applications such as the detection of circulating heartworm antigen in dog plasma without the need to process the circulating antigen in order to remove or destroy the autologous antibody that would block test antibody from binding.
  • P. immitis material was then centrifuged for 15 min at 1,000 g at room temperature to pellet the particul ate material and the supernatant containing soluble P. immitis antigens was carefully removed and stored. Immunization
  • mice Female BALB/c mice were immunized with 2 subcutaneous injections of 10 ⁇ g (protein) microwaved D. immitis antigen in Freunds complete
  • the spleens were removed aseptically from 3 immunized mice and the pooled spleen cells were fused with NS-1 myeloma cells (Bundesen et al., 1984, Vet. Immunol, and Immunopath.). Selected crude clones chosen on the basis of the screening assay were subcloned by limiting dilution and subsequently a number of MAb secreting hybridoma cell lines were finally chosen which recognized both irradiated and non-irradiated D. immitis antigen for expansion to mass culture and for the production of ascitic fluids.
  • the hybridoma cell lines D44.8.3B4/64 and D46.8.4D4/23, which bind to different epitopes on both microwaved and unmicrowaved heartworm antigen were produced and selected in this manner.
  • mice Female BALB/c mice were given an intraperitoneal injection of 0.5 ml 2, 10, 14, tetramethylepentadecane (Pristane, Aldrich Chemical Corp., Milwaukee, Wisconsin) 14 days prior to the injection of 2 x 10 viable hybridoma cells and ascites fluids were collected from the mice 12 to 14 days after injection of the ceils.
  • the ascitic fluid was clarified by centrifugation and MAb were recovered by precipitation with 45% ammonium sulphate and stored at either 4°C or -70°C in phosphate buffered saline (PBS) containing 0.01% sodium azide.
  • PBS phosphate buffered saline
  • test antibodies In order to detect circulating antigen derived from an infectious agent in a blood, serum or plasma sample in the presence of an anti-infectious agent host antibody response, test antibodies must either see epitopes not recognized by the host antibody response, or recognize new epitopes in an antigen that has been modified in some manner.
  • the epitope may be stable to the modification process but any host antibody binding to that epitope will be stripped off and denatured, allowing test monoclonal antibody to bind.
  • the process of microwaving antigen allows the production of monoclonal antibodies that can be applied to these types of tests.
  • Serum or plasma samples are diluted 1/5 in phosphate buffered saline (PBS) and microwaved for 90 seconds on the high setting in a domestic microwave oven.
  • PBS phosphate buffered saline
  • the microwaved samples are then reacted with the wells of 96 well microtitre plates coated with the monoclonal antibody, which was given the reference code D46.8.4D4/23, for 60 minutes at room temperature, followed by 3 washes with PBS/Tween (PBST).
  • PBS phosphate buffered saline
  • a process for preparing antibodies against immunorecessive epitopes of HIV which are expressed on the surface of HIV and/or expressed on the surface of a host cell infected with HIV such as HIV infected lymphocytes is given below.
  • Appropriate immunizing antigens which can be prepared or isolated by known techniques include:
  • At least one of (a)-(e) is treated with microwave radiation (eg 1ml of a one mg per ml protein solution of (b) + (c) in a Nunc Cryotube microwaved for 60 - 120 seconds at 680 watts at 2450MHz, using a heat sink such as a container of 250ml water to maintain the temperature of the sample below 56 -60°C so as to be below protein denaturing temperature) to render immunorecessive epitopes of the antigen immunologically accessible.
  • microwave radiation eg 1ml of a one mg per ml protein solution of (b) + (c) in a Nunc Cryotube microwaved for 60 - 120 seconds at 680 watts at 2450MHz, using a heat sink such as a container of 250ml water to maintain the temperature of the sample below 56 -60°C so as to be below protein denaturing temperature
  • Antibodies are raised against the immunologically accessible epitopes on the microwave treated antigens by known techniques (e.g
  • the antibodies are screened by immunoassay (e.g., enzyme immunoassay or radioimmunoassay) to select specific antibodies to microwave radiation resistant epitopes present on native HIV antigens and microwaved HIV antigens (e.g. gp120, gp41 and p24.
  • immunoassay e.g., enzyme immunoassay or radioimmunoassay
  • specific antibodies are further screened to select antibodies to the microwave radiation resistant epitopes which are not recognized by the normal human immune response in HIV infected individuals.
  • the antibodies resulting from this further screening can be used in the construction of immunodiagnostic kits (e.g., enzyme immunoassay or radioimmunoassay or red cell agglutination assay) for the detection of circulating HIV antigens (e.g., gp120, gp41 and p24) in infected individuals.
  • immunodiagnostic kits e.g., enzyme immunoassay or radioimmunoassay or red cell agglutination assay
  • HIV antigens e.g., gp120, gp41 and p24
  • the antibodies to the microwave radiation resistant epitopes are screened further to select antibodies to unique HIV epitopes present on the surface of a cell infected with HIV and accessible to a humoral,
  • the antibodies obtained from these latter screenings can further be used to identify the specific epitopes and construct either peptide or recombinant antigens for use as a vaccine.
  • these peptide or recombinant antigens would only contain unique HIV epitopes and exclude HIV and normal cell surface crossreacting epitopes, to prevent the possibility of stimulating an autoimmune response as a consequence of immunization.
  • the final vaccine formulation can include a combination of native epitopes and modified epitopes (e . g . , native p17 , p24, gp41 and gp120 and microwave treated HIV antigen).
  • such native HIV antigens would also be treated in a manner to eliminate the previously mentioned HIV/normal cell crossreacting epitopes.
  • the antibodies reacting with the unique HIV epitopes can be used to produce antiidiotypic antibodies which can be used as a vaccine.
  • the peptide or recombinant antigens or the antiidiotypic antigens may be used singly, in combination or alternatively in combination with other elements derived from the virus (e.g., microwaved HIV, microwaved HIV components or microwaved recombinant HIV antigens) to provide composite vaccines.
  • viruses e.g., microwaved HIV, microwaved HIV components or microwaved recombinant HIV antigens
  • the unique antibodies selected in the last screening step or antiidiotypic antibodies can be used to construct immunodiagnostic tests which will discriminate between HIV immunized and HIV infected
  • microwave modified antigen could be used to discriminate between HIV infected patients and vaccinated patients. Such tests would have application in blood bank screening.
  • anti idiotypic antibodies which are monoclonal any of the above described antibodies could be polyclonal antibodies, produced from suitable experimental animal species or murine or human monoclonal antibodies.
  • mice Three groups of mice were immunized in the following manner:
  • Group A Six female BALB/c mice were each immunized twice with
  • mice Four days before the mice were killed and their spleens removed for fusion, a final intraperitoneal injection was given consisting of 10 ⁇ g of antigen In 0.5 ml PBS.
  • Group B Six female BALB/c mice were each immunized twice with
  • intraperitoneal injection was given consisting of 5 ⁇ g of each antigen in 0.5 ml PBS.
  • Group C Six female BALB/c mice were each immunized twice with
  • mice emulsified with 0.05 ml FCA, at weekly intervals. Four days before the mice were killed and their spleens removed for fusion, a final
  • intraperitoneal injection was given consisting of 5 ⁇ g of each antigen in 0.5 ml PBS.
  • the spleen was removed aseptically from one mouse from each of the 3 immunization groups and pooled.
  • a single cell suspension was prepared and the pooled spleen cells were fused with NS-1 myeloma cells (Bundesen et al, 1984, Vet. Immunol, and Immunopath.).
  • Selected crude clones chosen on the basis of the screening assay were subcloned by limiting dilution and subsequently a number of MAb secreting hybridoma cell lines were finally chosen for expansion to mass culture and for the production of ascitic fluids.
  • Monoclonal antibodies in accordance with the invention can be utilized for recognition of epitopes for production of synthetic or recombinant vaccines.
  • Monoclonal antibodies displaying this type of response were classified as Type 2 antibodies and were able to mediate in vitro antibody dependent cellular cytotoxic (ADCC) reactions on HIV-1 infected cells (Table 6).
  • Other selected monoclonal antibodies from this panel recognized crossreacting p24 epitopes that were present on both HIV-1 infected cells and normal human cell types e.g. red blood cells, platelets, peripheral blood lymphocytes and brain and nerve tissue. Such antibodies were classified as Type 1 antibodies and were able to mediate ADCC
  • monoclonal antibodies will identify viral antigens expressed only on the surface of infected cells (Type 2 MAb) and some of these monoclonal antibodies will be able to mediate ADCC and/or C' dependent lysis reactions.
  • anti-viral peptide or recombinant vaccines that contain viral epitopes that are only expressed on the surface of infected cells from which the specific cell surface crossreacting epitopes have been identified and el iminated .
  • a "safe" vaccine may be produced that will protect against disease, rather than enhancing infection or causing autoimmune disease or immune paralysis.
  • a third method is proposed for the immune modulation of the
  • autoimmune disease or immune paralysis by treatment with antiidiotypic antibodies to the specific crossreactive MAb which could act to switch off the autoimmune antibody response.
  • recombinant antigens or peptides identified by the specific crossreactive monoclonal antibodies could also be used to down-regulate the autoimmune response. This approach could be employed for HIV-1 and other retroviruses (including those recently found to be associated with MS, SLE, etc.).
  • the methods of the invention are useful in the production of
  • monoclonal antibodies against an antigen or an epitope of an antigen which is immunorecessive are also useful in the production of anti-idiotypic antibodies raised against these monoclonal antibodies.
  • the antibodies of the invention can be used in diagnostic kits, therapeutically or for vaccination purposes.
  • Monoclonal antibodies in accordance with the invention can be utilised for recognition of epitopes for production of synthetic or recombinant vaccines.
  • HIV e.g. HIV, Rubella, Rotaviruses, e.g. Neisseria gonorrhoeae.

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Title
BIOLOGICAL ABSTRACTS, vol. 60, no. 8, 15th October 1975, pages 4681-4682, abstract no. 43607, Philadelphia, PA, US; J. MUEHLFELD et al.: "Studies on the effect of ultrasound and enzyme treatment on antigenic properties of human red cells: I. Effects of ultrasound", & BLUT 30(5): 349-352. 1975. *
BIOLOGICAL ABSTRACTS, vol. 85, no. 5, 1988, page AB-643, abstract no. 49037, Philadelphia, PA, US; T. STIGBRAND et al.: "A hidden antigenic determinant on membrane-bound human placental alkaline phosphatase", & TUMOR BIOL. 8(1): 34-44. 1987. *
INFECTION AND IMMUNITY, vol. 55, no. 3, March 1987, pages 825-827, American Society for Microbiology, Washington, US; R.L. ANACKER et al.: "Neutralizing activity of monoclonal antibodies to heat-sensitive and heat-resistant epitopes of Rickettsia rickettsii surface proteins" *
See also references of WO9008782A1 *

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