JPH02502183A - Monoclonal antibodies in vaccine formulation - Google Patents

Abstract

(57) [Summary] This bulletin contains application data before electronic filing, so abstract data is not recorded.

Description

[Detailed description of the invention] Monoclonal antibodies in vaccine formulation 1, Introduction The invention stimulates an active immune response in an inoculated host or Vaccine formulations that provide protection against infection by pathogens by providing dynamic immunity It includes things. The active ingredients of the vaccine formulations of the invention are Fabs.

Idiotypes such as Fv fragments containing F(ab')2 or Fab' consisting of fragments of monoclonal antibodies or monoclonal antibodies, including . The present invention also aims at a method for preparing vaccine formulations that does not require isolation or handling of pathogens. The target is

The immunogen of the vaccines of the invention that stimulates an active immune response against pathogens is a monochrome local antibodies or their fragments, which target specific receptors of pathogens. It has a three-dimensional conformational image. Vaccines are formulated to provide short-term passive immunity monoclonal antibodies or their fragments are directed against the pathogen's receptor. Contains a conformational image of a specifically binding ligand. Any implementation The vaccine of the present invention also protects the host without exposing the host to pathogens. can provide protection.

The present invention provides a monoclonal anti-idiotide that mimics polymerizable human serum albumin. The virus retains the internal image of the hepatitis B virus surface antigen in vivo, and its immunity Illustrated by an example used to generate anti-antiidiotypes that mimic virulence. It will be done.

Vaccines traditionally render viruses harmless without destroying their immunogenicity. It is prepared by This may inactivate the pillion's infectivity. Alternatively, this can be achieved by selecting non-toxic mutants. inactivated vaccine says that formaldehyde treatment usually destroys the infectivity of pillions. In its meaning, it is a state of "death". transfer of these “killed” virus particles to the host The injection elicits an immune response that can neutralize future infections with viable virus. numerous Many problems are associated with the use of inactivated vaccines. The main problem is It is impossible to inactivate all virus particles. However, this Even if this were achieved, the killed virus would not multiply within its host, so Immunizations achieved are often short-lived and require additional immunizations. inactive The other major difficulty that arises with the use of sexualized vaccines is that sufficient to prepare a vaccine that provides the necessary quality of suitable antigens to promote antibody production. The goal is to prepare a suitable virus.

Inoculation with a weakened viable virus reverses the ability of the killed vaccine to generate vitality. generally lasts for a long time because the virus actually imitates within the host. It is a good immunogen that provides long-lasting immunity. However, how many The problem arises with live virus vaccines (there is no insufficient attenuation) . Viral attenuation is traditionally due to rapid successive passage of the virus within the external host. This is done by producing nontoxic variants. caused by live virus vaccines Some problems include undesirable contamination by accidental viruses during transit in cell culture; Avoid thermal instability of live virus and interference by wild-type virus during vaccine formulation. Contains genetic instability of a debilitated virus that produces a high degree of virulence. Ru.

An alternative to inactivated and live virus vaccines are subunit vaccines. There is a use of This is due to host infection caused by immune substances associated with pathogenic viruses alone. immunization. For example, many enveloped viruses Encoded glycoproteins or capsid proteins of nonenveloped viruses can be neutralized. It has the ability to induce antibodies. Subunit vaccines are derived from these viruses. It can be prepared by purifying the protein. One of the subunit vaccines One advantage is that the associated viral material and genetic information as well as the replication capabilities of the virus are is to be excluded. The main difficulty is that when preparing immunological doses of purified protein, Occurs when

The use of recombinant DNA technology for the production of subunit vaccines has recently become very popular. has been attracting attention. This is because proteins that can elicit a neutralizing response within the host involves the expression and molecular cloning of the coding for viral genetic information. . Viral genetic material is removed and the host is never exposed to the whole virus. Therefore, the host is spared the risk of infection. Furthermore, used in the subunit vaccine The production of large quantities of epitopes becomes possible. However, these technologies It is limited to the production of protein epitopes and is not applicable to lipid or carbohydrate antigens.

used in killed virus vaccines or subunit vaccines The immune response to the immunogen is administered in an emulsion with an adjuvant and is greatly multiplied. The mechanisms by which adjuvants increase immune responses are complex. , including the stimulation of activity occurring in the reticuloendothelial system.

However, conventional azinivite, based on mineral oil, is non-metabolizable and has a potential It cannot be used on humans because it is carcinogenic.

2.2. passive immunity In the management of certain diseases or infections, viral vaccines can provide active immunity. Instead of sterilization, use immune serum or concentrated immunoglobulins that can bind or neutralize pathogens. Administration of preformed antibodies such as brin can provide short-term protection Met. This is called passive immunity. Human immunoglobulins are usually heterogeneous Antibodies are preferred for medicine in humans because they cannot elicit an immune response.

Passive immunity generally refers to It is considered an emergency method for immediate protection. However, passive immunization is It is also regarded as an important preventive measure in viral infections. For example, human Immunoglobulin has been shown to be effective in short-term prevention of measles or hepatitis A. In contrast, the prevention of hepatitis B using normal human immunoglobulin Not very successful.

2.3. Anti-idiotypic antibodies and vaccines Recently, active immune responses within the host New types of vaccines include the use of anti-idiotypic antibodies as immunogens to attract cutin has been suggested (Medical News, JAMA Jan 2 4/31.1986, Vol. 25514, pp. 447-448). Anti-Idiot Anti-idiotype antibodies or anti-idiotypes are antigen-binding sites or variable antibodies of other antibody molecules. An antibody that is directed against a site (called an idiotype). In theory, Je A network model of idiotypic relations (Jerne, N, , et al., 1974 Ann, Immunol, (Paris) 125c: 37 3; Jerne, N., et al., 1982゜EMBO1:234) , an antibody molecule that expresses an antibody binding site (antigen binding site) for a given antigen. immunization produces a group of anti-antibodies, some of which have antibody binding sites. The auxiliary structure at the position is matched with the antigen. by subpopulations of anti-idiotypic antibodies. immunization produces a subpopulation of anti-idiotypic antibodies that bind to the initial antigen. Should. Therefore, certain antibodies that counter virus-neutralizing antibodies are must be imitated. In addition, when inoculated into the host, specific antifungal an virologic response must be elicited.

Studies with various infectious disease models, including viral and bacterial systems, have showing anti-idiotypic regulation of epidemics (data from infectious disease model systems). For a summary list of data, see Regan K. J., 1985. Curr , , Topics Microbiol, I+nuno1.119:1g, (See 19). Studies in viral systems include hepatitis B virus (Kenne dy, R,C,, 1985, Curr, Topics Mlcrobiol , Ig+muno1.119:l-13) feline leukemia virus (Uytde haag, F, G, C, M, et al., 198B, ll1unO1, Revi evs 20:93-113) and poliovirus (Regan, supra, uy tdehaag, supra). Vaccines for reovirus (Fiel ds et al., 1984. Modern Approaches to Vaccl nes, Co1d spring Harbor Laboratory-No. 258-287) and rabies vaccines (Reagan, ibid.; K. oprowskl, 1985. Vaccines 85. Co1d Sp rlng l1arbor Laboratory, pages 1151-156) Animals Anti-idiotypic antibodies, which have been studied experimentally, have been found to be lethal in mice. Used to induce immunity consisting of protection against Daivirus infection ( Ertd, Sci, USA 81:2850-2854), HB V system Immunization with anti-idiotypic antibodies in (Kennedy, ibid.).

2.4. hepatitis B Hepatitis B (HBV) is the causative agent in acute and chronic hepatitis, and 01oofnagle included as an etiologic agent in cellular tumors, J.H. , 1981, Sem1nors Liver Dlseases l Near-15 ). HBV acts homogeneously on hepatocytes, and the mechanism remains a mystery (Hans on et al., 1979.11fection and ImmunitL 26:12 5-130), bacterial hepatitis B surface antigen (HBsAg), virus and hepatocytes pol mediates HBV hepatotropism by acting as a bridge between For human albumin (polyHA) polymerized in hepatocyte membranes suggesting yHA The existence of a binding site has been reported (Robinson et al., 197B, Engl, J. Med, 295:1168-1175HIgai et al., 1 979. Gastroenterology 76:242-247HMach ida et al., 1984.　Ga5tr.

enterology 86:910-918)6 Furthermore, individuals infected with HBV The above from several authors contain autoantibodies for polyHA (Lenkei et al. et al., 1977, J. Med. Virol, 1:29-34), However, polyHA receptors on viruses or hepatocytes have not been isolated. It has never been identified either.

3. Outline of the invention The present invention includes, but is not limited to, viruses, bacteria, parasites, etc. Vaccines that provide active or passive immunity against infection by non-human pathogens This includes the following formulations: The active ingredients of the vaccines of the present invention are monoclonal From fragments of monoclonal antibodies that contain the idiotype of the antibody or molecule. These fragments are Fab, F (ab’)2, Fab’ F This includes, but is not limited to, any Fv fragment such as There isn't.

In a first embodiment of the invention, the vaccine formulation is Give the main active immunization. In these formulations, the immunogen of the vaccine is Clonal antibodies or idiotypic fragments thereof, which are pathogenic It has a three-dimensional image of the body's unique receptors. According to the method of the present invention, pathogenic The body is not used to produce monoclonal antibodies. In contrast, the specificity of the pathogen Monoclonal antibodies that mimic receptors (a) specifically bind to the receptor of a pathogen; (b) monoclonal antibodies raised against the ligand of the pathogen; The idiotype of a third antibody that sequentially limits the ligand specifically bound to Monoclonal antibodies raised against sequentially limiting idiotypes of a second antibody , or (C) a receptor for a pathogen as depicted in the cascade shown in Figure 1. It consists of other anti-idiotypic monoclonal antibodies that mimic all In many cases, monoclonal antibodies also competitively inhibit pathogen binding to the ligand. must be stopped. In a second embodiment of the invention, the vaccine formulation is , have passive immunity that confers short-term resistance to infection by pathogens. child In embodiments, the active ingredient of the vaccine is specifically directed against the receptor of the pathogen. A monoclonal antibody or an image thereof that has a conformational image of the binding ligand Consists of diotypic fragments. According to the method of the present invention, pathogens are Not used for local antibody production. Ligands that bind to specific receptors on pathogens A monoclonal antibody that mimics (a) a second antibody that is sequentially directed against the ligand; a monoclonal antibody directed against the idiotype of the antibody; (b) a monoclonal antibody directed against the idiotype of the antibody; sequentially directed against the idiotype of a fourth antibody directed against an idiotype of a second antibody that is sequentially directed against an idiotype of a third antibody; monoclonal antibodies directed against the type or (C) within the cascade shown in Figure 1. Other anti-idiots that mimic ligands that bind to pathogens, such as those depicted in It is made up of self-promoting antibodies. In all cases, monoclonal antibodies are used against pathogens. The binding of the ligand that binds to the protein must also be competitively inhibited.

Some advantages of the vaccine formulations of the present invention are as follows.

(a) Pathogenic immunogens are not required to stimulate host immunity.

(b) Pathogenic immunogens are not required for the preparation of the vaccine formulations of the present invention.

(C) A protein immunogen is cloned and prepared using recombinant DNA technology. Unlike the vaccines of the present invention, the vaccines of the present invention contain lipids, carbohydrates, glycolipids, etc. can be prepared for epitopes consisting of non-proteins such as.

(d) A “subunit” vaccine in which large amounts of monoclonal antibodies stimulate host immunity. In order to obtain a sufficient amount of immunogen in the tin formulation.

3.1. Definition The terms and abbreviations below have the following meanings.

anti-id: anti-idiotypic antibody BSA: bovine serum albumin EL I SA: Enzyme-linked immunosorbent assay F■: Variable region of antibody molecule or Original binding site.

This includes but is not limited to Fab, F(ab')2, Fab', etc. Any fragment containing the idiotype of the molecule may be used.

HA: human albumin (monomer) HBV: Hepatitis B HBsAg: Hepatitis B surface antigen Kd: Kilodalton polyHA: human albumin polymer RIA: Radioimmunoassay SDS sodium nidodecyl sulfate Tween-20: Polyoxyethylene sorbitan monolau Figure 1 shows the pathogen of anti-idiotypic antibodies that can arise from ligands that specifically bind to the receptors of It is a drawing showing a cascade. Activity in active or passive vaccine formulations of the invention The antibodies used as sexual components are shown in the figure. Anti-ligand antibodies and anti- ID2, anti-ID4, etc. mimic the receptors of pathogens and are used in vaccines prescribed for active immunization. It can be used as an immunogen in cutin. Anti-idl, anti-id3, etc. Vaccines that mimic ligands that bind specifically to the body and are formulated for passive immunization It can be used as an active ingredient in

5. Detailed description of the invention The vaccine formulation of the present invention contains viruses, bacteria, parasites, etc. It provides protection against infection of the host by a number of pathogens, including but not limited to: Main departure The active ingredient in Ming's vaccine formulations is a monoclonal antibody or molecular idiot. These fragments consist of monoclonal antibody fragments containing also includes, but is not limited to, Fab and F (ab').

Monoclonal antibodies of the invention are generated without the use of pathogens. these Antibodies or fragments thereof stimulate an active immune response or act against a pathogen. Idiotypic traits that can be used to provide short-term passive immunity in a host that It is prescribed as a quality vaccine.

To clarify the discussion, vaccine formulations of the invention conferring active or passive immunity. The invention provides a cascade of anti-idiotypic antibodies that can be used in other embodiments of the invention. This is described in a separate section below with reference to FIG. 1 which illustrates the code.

5.1. Vaccines for active immunization Many pathogens have special Binds to a surface that specifically binds to a substrate or receptor (hereinafter referred to as a ligand) (hereinafter referred to as a receptor). Usually pathogen receptors and host ligands The binding interaction between do. This attachment is usually caused by infection, multiplication, and colonization by pathogens that lead to disease within the host. It is indispensable for the development, etc.

According to the method of the invention, the host ligand is an antibody (or antibody thereof) that mimics a pathogen receptor. (see Figure 1). this antibody or its anti-antiidiotypes are used in the preparation of vaccine formulations to induces active immunity against. Alternatively, pathogen receptors or host ligands If the pathogen is unknown or unidentified, neutralizing immune responses (e.g. Anti-pathogen antibodies obtained from hosts that develop (by exposure to Can be used as a starting material to develop type cascades. In this case, the pathogen Anti-idiots of neutralizing antibodies that mimic the conformational structure of the body and induce neutralizing responses is selected as the active ingredient of the vaccine formulation.

In either embodiment, the vaccine is free of pathogens and It is prescribed without any need for treatment.

The vaccine formulation according to the invention and the method for its preparation are specific for receptors on pathogens. an antibody component directed against the idiotype of a ligand or neutralizing antibody that binds to The child idiotype site provides a conformational image and structure of the receptor on the pathogen. It is based in part on the theory that one must have. For example, the resistor shown in Figure 1 See ligand. Similarly, the idiotypes of anti-ligand antibodies are sequentially indicated. The antibody directed against the idiotype of the second antibody directed against the antigen on the pathogen must also have a conformational image for the idiotype of the target antibody. . For example, anti-idiotypes that can be produced from the ligand and/or anti-ligand See anti-id2 in Figure 1 depicting the cascade of . In the cascade Anti-ligands such as anti-id2, anti-id4, etc. can mimic the receptors of pathogens. is expected. Anti-ligand or anti-idiotypic antibodies are present on pathogens. The degree of body mimicry can be determined using competitive binding assays. For example, anti-ligand or anti-idiotype competitively inhibits pathogen binding to the ligand. It is possible to conclude that the idiotype of an antibody molecule mimics a receptor on a pathogen. I can do it.

appropriate anti-idiotypic or anti-ligand antibodies that mimic receptors on the pathogen; The body's effective dose is the amount used to stimulate an active immune response in the inoculated host. It can be formulated as an immunogen in vaccines. antibody molecule idio for the idiotype because the type mimics the conformation of the receptor on the pathogen. The resulting immune response is also directed against receptors on the pathogen. child receptors ensure attachment of the pathogen to its target, so the immune response It should neutralize the infectivity of the body.

In a preferred embodiment, use as immunogen in vaccine formulations of the invention The anti-ligand antibodies that are produced are It should be a monoclonal antibody. However, used as an immunogen In the preparation of monoclonal anti-idiotypic antibodies, intermediate antibodies in the cascade are It is not necessary for the body to have monoclonal antibodies. For example, the monochrome image in Figure 1 Null anti-id4 is an anti-ligand, anti-idl, anti-id2 and anti-id3 are monoclonal It does not have to be a null antibody.

in the production of antibody molecules by continuous cell lines using any technique used to produce antibody molecules. Can be manufactured. For example, Kohler and Mi hybridoma technology first developed by (1980, Sci. , A■, 243(4):6B-74) and human B cell hybridoma technology (Kozbor et al., 1983, Its+unology Today 4th A2) and EBV-hybridoma technology for human monoclonal antibody production (Cole et al., 1985, Monoclonal Antibodies a nd Cancer Therapy, Alan R.

More recently available such as Llss, Inc., pp. 77-96), etc. Techniques that have been developed are also within the scope of the present invention. Human clonal antibodies can be derived from any species. Although monoclonal antibodies can be used to increase the desired specificity of the immune response, Preferred for strength.

The invention is also limited to the use of whole antibody molecules as immunogens within vaccine formulations. rather than the idiotype of the antibody (e.g., the Fv portion of the antibody molecule). Any fragment of the antibody containing the antibody can be used. A fragment like this is , including but not limited to: i.e. pepsin F('ab')2 fragment, which can be obtained by treating an antibody molecule with F It can be obtained by reducing the disulfide bond of the (ab')2 fragment. Fab' fragments, as well as antibody molecules, are treated with papain and disulfide. There is a reduced ab fragment that reduces the ridge. F(ab’)2 fragment is , is preferred as an immunogen to enhance the elicitation of an immune response of desired specificity.

There are many pathogens for which vaccines can be prepared. For example, many viruses Therefore, pyrion attachment to target cells is a step in the preparation of virus to host cells and It is indispensable for the growth and subsequent proliferation. The specificity of attachment is the poliovirus and viruses such as influenza virus. for example , poliovirus attaches to primate chain cells but not to carrion cells.

This specificity may be due to the interaction of the virus with specific cell receptors in primates. It is believed. According to the invention, the receptor is an anti-ligand and/or an anti-ligand. A cascade of diotypic antibodies can be used as a ligand to be generated.

Appropriate antibodies are used in vaccine formulations as described.

Similarly, the presence of certain proteins on the surface of host cells is associated with This is necessary for the adhesion of substances. These cell surface proteins are responsible for viral hemagglutination. It appears to be a receptor for the tinin antigen. Therefore, the receptor protein is As a ligand for which anti-ligand and/or anti-idiotypic antibodies are produced can be used.

Penetration of host cells during retroviral infection is dependent on viral envelope glycoproteins. Cr1ttend and host cell receptors. en L, B, 1968. J, Natl, Cancer In5t, 41:145-153; Piraino, P. 1967, Vir.

1ogy 32 near 0Q-707: 5teck, P, T, and Rubin , H,, 1965, Vlrology 29:642-653) 0 In the present invention In other words, these host receptors are responsible for the production of anti-idiotypic cascades of antibodies. It can be used as an immunogen.

In certain embodiments of the invention, the anti-idiotypic cascade is an acquired immune system. Cell receptors related to the AIDS virus, the causative agent of AIDS syndrome (AIDS) (Gallo, R.C., .

et al,, 1984.5science 224:500: Popovie , M, et al.

19845science 224:497; Barre-3inoussi, F, et al, 1984, 5science 225:840) oThe receptor for the AIDS virus is the CD4 (T4) antigen on lymphocytes. Evidence suggests that (Dagleish et al., 1984.N ature 312 near 63-786: Klatzmann et al,, 1984. Nature 312 Near 87-768) o AIDS virus The epitope is an anti-identifier that mimics the viral protein that binds to the cellular T4 antigen. Can be used as an initial antigen for the final production of iotypic antibodies Will. One possible source of such AIDS virus epitopes is Currently available AIDS diagnostic kits, many of which have been destroyed by the AIDS virus. It contains spirion. Another source for initial immunization purposes is recombinant DNA Chemically synthesized or purified viruses produced using A technology It contains AIDS virus proteins such as those obtained from. Or again , anti-AIDS virus antibodies induced from patients are Used as a starting point for the generation of anti-idiotypic antibodies directed against the receptor be able to. Hepatitis B virus with binding site for polymerized serum albumin (HBV) adheres to the surface of hepatocytes via polymerized albumin, and then this H BV-polyalbumin complex is taken up by hepatocytes by endocytosis It is also assumed that consistent with the specificity of the virus for a particular host. Therefore, HBV particles and HBsAg particles are found in human and chimpanzee serum polyalpha Binds specifically only to bumin. According to the present invention, polymerized human albumin as a ligand for which ligand-like and/or anti-idiotypic antibodies are generated. It is used as Alternatively, a cascade of anti-idiotypic antibodies may produced using human anti-polyHA that can be obtained from infected patients. obtain. This group of suitable antibodies can be formulated as vaccines as described herein. can be done.

Many diseases or disorders caused by bacteria or parasites also affect host cell receptors. It depends on specific interactions with For example, certain disease-causing bacterial toxins Susceptibility to cholera toxin (choleragen), which is a (Moss, J. and Vaughan, M., 197 9. Ann, Rev, Blochew, 48:581-600) o -L Schierichia coli non-thermolabile enterotoxin also Sid probably uses GMI as its cellular receptor (Moss, J. and Vaughan, M., 1979. Ann, R ev, old ochem.

48:581-600). Therefore, in this embodiment of the invention, GM l as the immunized ligand against which anti-idiotypic antibodies are generated. It can be used in many ways.

5.2. Vaccines for passive immunization This embodiment of the invention is directed against the idiotype of the second antibody; For example, an antibody molecule directed against a ligand that specifically binds to a receptor on a pathogen. The idiotypic region should bear the conformational image and structure of the ligand. It is based in part on the theory that For example, see anti-id2 in Figure 1. thing. Other resistors such as resistor id3 shown in the cascade depicted in Figure 1 Diotypic antibodies also mimic ligands that specifically bind to receptors on pathogens. It is expected that they will emulate this. The degree to which an anti-idiotypic antibody mimics its ligand is , which can be confirmed in competitive binding assays. For example, anti-ideal If a competitive antibody competitively blocks the binding of a ligand to a pathogen, the antibody It can be inferred that the idiotype of the ligand mimics the ligand.

According to this embodiment of the invention, the anti-idiotypic antibody that mimics the ligand is , used to provide passive immunization for short-term protection against infection by pathogens. It can be formulated into a "vaccine" that can be administered. The anti-idiotypic antibody The antibody mimics the conformation of the receptor on the pathogen. will be combined with Binding of the antibody to the receptor inhibits the pathogen from targeting its target. adhesion and therefore prevent subsequent infection by the pathogen. Dew.

In a preferred embodiment, the antibiotic used in the vaccine formulation for passive immunization is Idiotypic antibodies can be used to provide a continuous supply and in large quantities of the antibodies. It should be a monoclonal antibody so that it can be manufactured. but However, monoclonal anti-idiotypic antibodies used in vaccine formulations In body preparation, the intermediate antibodies in the cascade are not necessarily monoclonal antibodies. There's no need. For example, monoclonal anti-Ld3 in Figure 1 provides passive immunization. Anti-ID2, anti-IDL and anti-ligan Intermediate antibodies in the cascade, such as secondary antibodies, are not necessarily monoclonal antibodies. There is no need to Monoclonal antibodies used in passive immunization vaccine formulations The body is used for the production of antibody molecules by successive cell lines in culture It may be prepared by any technique. For example, Kohler ] and the hybrid first developed by Milstein [Mf. market technology (1980, Sci, Amber, 243(4):66-74) as well as other techniques that have recently become available, such as human B cell hyperplasia. Brideoma technology (Kozbar et al., 1983, Igmunol ogy Today 4 Near 2) and E for human monoclonal antibody production BV hybridoma technology (Cole et al., 1985. Monoc lonal Antibodies and Cancer Therapy, AJan R, Li5s, Inc., pp, 77-98) etc. It is within the realm of the Ming Dynasty.

Human monoclonal antibodies have the potential to elicit an immune response directed against the antibody molecule Therefore, it is suitable for use. for passive immunization Repeated administration of monoclonal antibodies appears to reduce the risk of triggering autoimmunity. , would preferably be minimized.

The present invention provides a complete monoclonal compound as an active ingredient in vaccine formulations for passive immunization. There is no limitation to the use of null anti-idiotypic antibody molecules. stomach. Rather, any of the antibodies containing the idiotype of the antibody (eg, the Fv portion of an antibody molecule) can be used. Such a f As a fragment, F can be obtained by treating an antibody molecule with pepsin. (ab')2 fragment, disulfide bond of this F(ab-)2 fragment Reduction to reduce the F disulfide bond that can be obtained by reducing the 2Fab or Fab fragments that can be obtained by treatment with agents, Of course, it is not limited to these.

However, complete antibody molecules as active ingredients in vaccine formulations for passive immunization The use of Fv fragments of antibodies (i.e., those containing the Fc region of the molecule) would offer advantages over using . For example, monoclonal anti- Idiotypic antibodies activate complement or ADCC (antibody-dependent cytotoxicity). If the antibody molecule is of a class that mediates pathogenic harm (e.g. Binding not only prevents infection by pathogens, but also mediates their destruction. becomes. For example, if the pathogen is a bacterium or a parasite, complement activation may not occur. ADCC results in the decomposition of the bacteria or parasite. If the pathogen is present in the host If the virus is already infecting cells, antibodies are directed against the virus's receptors. will bind to infected cells expressing . These infected cells do not have complement activation It will be destroyed in the same way via ADCC.

Passive immunization can be used to treat a variety of viral or bacterial diseases. I can do it. For example, hepatitis A virus is a human immunoglobulin that provides passive immunity. It has been successfully treated using phosphorus. The human immunoglobulin of obtained from a donor who has had contact with Rus.

Monoclonal antibodies directed against the protein inhibit Au hepatitis via passive immunization. It can be used to inactivate viruses.

Many bacteria have surface antigens that confer adhesion to specific cellular receptors. There is. As provided by the present invention, these receptors have anti-idiotypic properties. It can serve as a starting immunogen in the creation of antibodies. Bacterial antigen Anti-idiotypic antibodies directed against either cellular receptors are: can be used in passive immunization. Shigella disenteriae ( Shiga Shigella), Vibrio cholerae (Vibrio cholerae), various Salmonella and Escherichia Bacteria that are enteric pathogens, such as Chia coli species, have anti-idiotypic antibodies. can be treated by passive immune mechanisms taken orally.

The following passages refer to antiligands or agents that stimulate an active immune response against a specific pathogen. be prepared using anti-idiotypic monoclonal antibodies as immunogens. The various vaccine formulations that can be used are described. infection by certain pathogens Monochrome that would provide passive immunity in short-term compromised hosts Vaccine formulations that can be prepared using null anti-idiotypic antibodies can also be stated.

Vaccine formulations that stimulate active immune responses are made using receptor- or pathogen-mimicking models. In vivo use of noclonal antibodies or their Fv fragments can be prepared by mixing in a suitable carrier. In fact, the monoclonal Antibodies or Fv fragments thereof may be used in a suitable manner to enhance the immunological response of the host. Should be prescribed with an adjuvant. A suitable adjuvant is hydroxyl Aluminum chloride, surfactants, lysolecithin, Pluronic c) Includes polyols, polyanions, and peptides, but these It is not limited to these. Corynebacterium parvum [eoryneb [acterium parvu 凰]] and BCG (Basil Calmetogheri) bacille [Calmette-Guerin]) etc. Other potentially useful adjuvants within the scope of this invention include: It is.

Alternatively, the monoclonal antibody or Fv fragment is present in liposomes. or incorporated onto liposomes, or polysaccharides and/or vaccine formulations. can be conjugated to other polymers useful for. Antibody fragments butenes, i.e., those that have antigenic properties but are unable to elicit an immune response. In some cases, this may be due to the presence of serum albumin proteins in order to render the conjugate immunogenic. can be conjugated to large carrier molecules such as polymers and the like.

Vaccines may be formulated as multivalent vaccines. For this purpose, different pathogens A mixture of different antibodies, each mimicking a receptor, are combined together in one formulation. can be mixed with

Many methods are used to introduce vaccine formulations such as those described above into the host. obtain. These include sarcastic, intramuscular, intraperitoneal, intravenous, subcutaneous and intranasal routes. However, it is of course not limited to these.

5, 3.2. passive immunization Monoclonal antibody molecules that bind to pathogens or mimic Gant May be prescribed to confer active immunity for short periods of time. The purpose of this regimen is to stimulate the immune response. The purpose of this study is to bind to and inactivate pathogens rather than to inactivate them. Moreover, no adjuvant is required in this type of preparation. Because of this, Any suitable pharmacological carrier may be used. Passive immunization poses special risks. used on an emergency basis for rapid protection of non-immunized individuals. can be

6. Reacts with HBsAg receptor for example double whitened human albumin anti-idiotypic antibodies The following examples demonstrate the polymerized human aliphatic acid that binds to HBV according to the present invention. We describe the preparation of monoclonal antibodies that mimic rubumin.

This monoclonal antibody is used in vaccine formulations provided for passive immunization. Alternatively, this monoclonal antibody can be Immunization in vaccines that elicit an active immune response as exemplified in Section 7 below. It can be used to create anti-idiotypic antibodies useful as epidemiologists. Wear. Polymerized serum albumin (polyHA) is used to treat HBsAg and liver parenchymal cells. of HBV by interacting with specific receptors expressed in both cells. It has been proposed to mediate hepatic tropism. Patients infected with HBV are poly Develop autoimmunity against HA. Take the internal image of polyHA and to a human anti-polyHA antibody that can mimic its binding to BsAg. Bipridomas that give rise to anti-id are described. Specificity of the two anti-ids , and especially their HBsAg binding ability in RIASELISA, Western demonstrated using lot and cell staining techniques. The inhibition assay was performed using HA (single human polyclonal anti-HBsAg antibodies) and two polyclonal anti-HBsAg antibodies. Only HA (polymerizable human albumin) is present in antibodies 63.14 and 70. F9 conclusion This shows that it was possible to interfere with the performance. Monoclonal anti-HBs Partial inhibition of anti-HBsAg reactivity of anti-id by Ag is likely due to steric hindrance. This is due to this.

These data indicate that anti-id shares a common three-dimensional structure with polyHA; It is a well-known method for identifying the polyHA binding site expressed on HBsAg. It is suggestive.

This discrimination indicates that the antigen is different from that detected by conventional anti-HBsAg antibodies. It is something that happens in the area.

Although the existence of hepatic receptors for polyHA has not yet been clearly demonstrated, The ability of olyHA to bind HBsAg has been documented by several researchers. There is. The question on which HBsAg peptides polyHA is localized is , already controversial (Peterson st al., 1981. J, Bioof, Chet 256:8975-6983; u-Matiu et al, 1980゜J,) 4ed, Virol, 6:175-178; Machida et al., 1984. Ga 5troentero+ogy 86:910-918) Our data are Both anti-ids react with the major 24kd peptide of the antigen as well as its glycosylated forms. This was an indication that Our anti-id also has a smaller HBsAg component. Other larger peptides, possibly representing undivided precursors, or pre-s and A protein consisting of both S genes encoding peptides was detected (1/a sser+san et al, 1982. Proc, Natl, A cad, Sc1. USA 79*4810-4814: Thung, e tal, 1982. Hepatology 2:832-835; A gustin, et al, 19835urv, Immunol, Res, 2 Near 8-83; Ionescu-Matiu et al,, 1980. J, Med, Virol, 6:175-178), these The data show that polyHA was used as a probe for the HBsAg receptor. Consistent with existing studies, polyHA receptors fragment, i.e. restricted to the pre-s region (Trevisan, et al. al., 1982. Hepatology 2: 832-835 ; Lenkei et al, 1977, J, Med, V irol, 1:29-34; Augustin et al, 198 3.5urv, l+uuno1. Res, 2nia g-83).

In short, we keep the internal image of polyHA and its HBsA Monoclonal directed against human anti-polyHA antibodies that mimic binding to g. Anti-id was produced. These anti-IDs can be used with conventional anti-HBsAg antibodies. Sites expressed on the main peptide of the antigen that would not have been detected It is used to identify Expression of polyHA receptors on HBV is relatively Our anti-i d different patients with viral hepatitis and healthy carriers who are more likely to transmit the infection. It is valuable in identifying subgroups. Finally, polyHA receptor If this is involved in the invasion of HBV into hepatocytes, the passive immune mechanism These anti-IDs are effective in blocking viral infections when used as Will. Alternatively, antibodies created against the anti-Ids described herein. Anti-idiotypic antibodies obtained can be used in active vaccine formulations. It is possible.

Ba1b/c mice were affinity-purified obtained from patients with autoimmune hepatitis. immunized with human IgG anti-polyHA. These are the first complete Freund subcutaneously in the inguinal and axillary areas using 100 μg of immunogen in adjuvant. Injected locally and while stepping. Successful additional stimulation of incomplete Freund's adjuvant and saline. After the third injection with saline, The mice were bled and the serum was assayed for anti-i using a hemagglutination inhibition assay (HIA). tested for the presence of d. Human red blood cells are Nis, N, Chun [8, N, T Hung et al. (1981, Gastroenterology 80 : 260-264). polyHA is The method of R. Lenkei et al. (1977, J. Med. Crosslinking with glutaraldehyde according to Virol, 1:29-34) It was formed by

The coated human red blood cells were prepared using aliquots of mouse serum. After preincubation, react with the immunogen in microtiter plates. It was.

6. 1. 2. Monoclonal anti-ID production Spleen cells obtained from positive mice Using 44% PEG, HAT sensitive cell line 5P210-Az-14 was fused with. Two weeks after HAT selection, hybridomas were harvested as described above. were screened for the presence of anti-id using HIA. The positive clone is immunized by expansion, secondary purification by limiting dilution, and growth in ascites. Monoclonal anti-id against original (Fab-)2 as well as against HBsAg The binding ability of the protein was tested in a solid-phase radioimmunoassay (RIA).

Here, 11 different human pancreatic proteins and one unrelated monoclonal Anti-id (MOPC173) was provided as a negative control. HBV- Obtained from concentrated supernatant of transfected 373 cell line 4.10. HBsAg obtained from the serum of patients with acute hepatitis and acute hepatitis was Purified CChrlsttxan et al., 1982.　Proc, Natl, Acad, Sci, LISA 79:1815-1818 ), immunogen (Fab =)2, HBsAg and human spleen protein (1 0 μm/ml) was incubated overnight at 4°C in the wells of a microtiter plate. was exported. 3% bovine serum alkaline in phosphate buffered saline (PBS), pH 7.2 After blocking with Bumin (BSA), the wells were filled with 50 μm anti-id. and incubated for 1 hour at 37°C. o, ooi% Tween 20 After washing with PBS containing PBS (PBS-Tween), the wells were Incubate μl of 1251-labeled rat anti-mouse chain antibody for 1 hour at 37°C. It was written. The plates were then washed with PBS-Tween and each Radioactivity in the wells was counted using a gamma counter.

In inhibition experiments, polyHA, monotypic HA and rabbit anti-HB SA g (DAKO Corporation, Sendover, California) Different concentrations were added to the microwafer prior to incubation with the monoclonal anti-id. added to the file.

6.1.4. ELISA Monoclonal anti-Ld63.14 and 70. F9 is Vol Ier ] et al.'s method (1976, Bull, World Health Organ, 53:55-65), alkaline phosphatase (type ■-T1S1 ga+a Ches+1cal Corporation, Missouri State (2000). Wells of microtiter plates were incubated overnight at 4°C. It was coated with HBsAg (10 Mg/ml) by baking. Transf Supernatant obtained from untransfected 3T3 fibroblasts served as a control. Given the. After blocking with 3% BSA in PBS, wells were filled with 50 μl of alkaline phosphatase stand wipe i d (10 Mg/ml) and 1 at 37°C. incubated for an hour. After washing with PBS-Tween, the wells were Phosphatase substrate (0, 05M carbonic acid containing 2mM MgC12 1 mg/ml Sigma substrate in buffer, pH 9.5 and in Curated. The optical density of each well is (MR600, Dynatech Laboratories Inc.) (Alexandria, Victoria) at a wavelength of 405 nm. . polyHA, monogenic HA, and monoclonal anti-HBsAg H2 5B10 (American Ce1l Type Culture Co11 ect1on Maryland 0-7 Couvir) was used as a blocking agent. It was.

63.14 and 70. The affinity of F9 was determined based on competitive ELISA. Muller [R, Muller's method (1983, in Methods I n Enzymology, vol, 92. Co1ovic.

S, P, and Laplan, N, D, (eds,), Longon e, J, J, and Vunakis, H, V,, pp, 589-6 01). In these experiments, alkaline phosphatase Joining 63. 14 and 70. F9 is pre-injected with various dilutions of non-table wipe id. After being incubated, it reacted with HBsAg. Antibody affinity determination The number (K) is calculated from the molar concentration of inhibitor that gave 50% inhibition and The molar concentration of and the amount of tracer are linked to the absence of inhibitor.

4.10 Obtained from cell supernatant and from serum of patients with acute hepatitis HBsAg was incubated on a 12% 5DS-polyacrylamide gel overnight at 10 mA. was electrophoresed. The protein was incubated with nitrocellulose filament for 3 hours at 60V. electrophoretically transferred onto a router. This filter was then treated with 1mM Tri Blocked with 5% BSA in s-HCρ buffer, pH 7.6 and monochrome Naru anti-ID and incubated overnight at 4°C.

After washing with Tris-Tween, the filter was treated with horseradish peroxidase. Goat anti-mouse IgG (BioRad Laboratories, Cali (Fornia, Suchmond), washed, and Oxidase substrate solution (0.01% 4-chloro-1-naphthol in methanol) and 0.02% hydrogen peroxide).

Monoclonal anti-HBsAg obtained from H25B10 and 3T3 fibroblasts cell extracts served as positive and negative comparison controls, respectively.

6, 1.6. cell staining HBsAg-secreting liver tumor cell line PLC/PRF15, HBv transfection cell line 4.10, the 3T3 cell line from which it was derived, and the liver tumor cell line. Standard Hep-G 2 (Amerlcan Ce1l Type Cu1ture Co11ection) were cultured on coverslips and cooled. Fixed in acetone for 5 minutes. After washing with PBS, cells were treated with monoclonal anti- Antibody 1d63.14 to 70. F9 and subsequent biotin contact. Anti-mouse IgG (Vector Laboratories, Calif. Burlingham, California). After washing with PBS, the cells Avidin-biotin-peroxidase complex (A B C, Vector L aboratories, Burlingham, California) and incubation. It was written. The peroxidase reaction is then carried out using diaminobenzidine and water peroxide. was developed using the same material as a substrate. polyHA and monoclonal anti-id MOPC173 served as positive and negative comparison controls, respectively.

6, 1.7. Radiation immunoprecipitation 4.10, PLC/PRF15, and Hep-G2 cell lines, and 3T3 Fibroblasts and human fetal liver cells were treated with [35S]methionine, 250 mC1/m 1 = 9.25 GBq - overnight, internally radiolabeled and RIP Buffer A (10mM Tris-HC, QSpH7゜4.150mM Na C, Q, 20mM EDTA, 0.02% NaN3.1%Non1det- P2O).

Aliquots of cell extract (106-10107 cp were shared on agarose beads) Conjugated anti-mouse IgG (Sigma Chemical Co., 50 μm 63.14, pre-bonded to St. Louis, Missouri) H25B10 and MOPo 173 were incubated with ascites. centrifugation Afterwards, the beads are thoroughly washed with RIPA buffer and electrophoresis sample buffer. (0,5M Tr i 5-HCfIS pH6,8,10% SDS, 2% 2-mercaptoethanol, 10% glycerol); and boiled for 5 minutes. Immunoprecipitates were prepared using 12% 5DS-polysiloxane as described above. Electrophoresed on an acrylamide gel. The gel is subsequently dried and X-OmatAR film (Eastman-Kodak, New York) Rochester) for about a week.

Ba1b/c mice retain the internal image of polyHA as mentioned above. and to induce an anti-idiotypic response that mimics its binding activity. Immunizations were made using purified human IgG specific for polymerized albumin.

Using spleen cells derived from mice that are serum positive in the RIA assay A fusion took place. Six clones derived from these fusions were tested in this assay. Four of these No. 1 hybridomas subsequently showed positive reactivity in expanded, secondarily purified, and further used as an immunogen in RIA. These were tested for their ability to bind the (Fab-)2 moiety of IgG, which had previously been tested. determined. The supernatant was simultaneously treated with HBsAg as well as 1, which served as a control. Their ability to bind to a panel of different human myeloma cells It was also tested.

Two monoclonal anti-1d63, 14 and 70. F9 is an idiot type and HBsAg, while 63.7 and 28. A is exempt Reacts weakly with the epizootic (Fab’)2 and has little or no reactivity with HBsAg. (See Table 1).

Table 1 Monoclonal anti-1d70, F9 compared to various human myeloma immunoglobulins ．． 6B, 14.63.7 and 28. A human IgG anti-polyHA (Fab ') Binding IgG1k to 2 and to HBsAg 2158±263 697±512 1392±1161228=1091979±1084108 4l 2501±290 2800±562 1980±249 110 0±710 1600±2271gG21 1595±269 1875± 1oli2 2101±342 838±140 1469=1401gG3 k 1910±268 1892±749 2100±890 1920 ±670 1336=8371gG31 1023±392 1012±5 33 1010±123 990±345 1834±9191gG4k 180g±227 2400±1087 1806±389 1341±9 02 1028'±497IgMK 1724:832 2240±f ! 28 1802±120 1512±742 1540±112IgM1 861:390 1885±891 2002±423 2004±788 1098±595IgAk 1475=243 1901±794 1671=802 1980±816 1968±712HBsAg 14 147±1275 8487-! =832 2500±280 980±20 1 1288±354 (Fab') 2 9598±382 9360±762 3010±400 4169±1021 719±438 One of mouse anti-id Samples were immobilized in sea urchins that were incubated with. Anti-id binding is 125 (2) By secondary incubation with radiolabeled anti-mouse antibody.

6, 2. 2. 63.14 and 70. by polyHA. Binding capacity of F9 prevention of Ability of polyHA to block binding of anti-id to idiotype and HBsAg Power was tested. Monoclonal antibodies 63.14 and 70. F9 is poly HA, monoclonal HA and polyclonal goat anti-HBsAg as competitive reagents was used in a competitive inhibition assay. The RIA results show that polyHA Not only monoclonal HA or goat anti-HBsAg but also monoclonal anti-id (F This shows that it does not block binding to both ab-)2 and HBsAg. there were. These data correspond to No. 1 hybrido-malo 3.14 and 70. F9 pure protein is conjugated with alkaline phosphatase and reacted with HBsAg. This was confirmed in a direct EL ISA. However, RIA and In both ELISA and ELISA, anti-HBsAg antibodies showed 63.14 and 70. F It partially blocked the binding of 9 to HBsAg. Competitive ELI SA is also 63.14 and 70. was used to evaluate the affinity of F9. child The results are shown in Table 2.

Table 2 HBsA g 63.14 measured in competitive EL I SA 63.14 9.4X10 3.4X1014, 8X101 HB s A g 70. F9 70. F9 3.8X10 capacity 6.5X 1019, 5X101 [1t] = concentration of inhibitor at 50% inhibition [Tt = concentration of tracer 6.2.3. Monoclonal anti-fd that identifies the major peptide of HBsAg To investigate the specificity of binding of monoclonal anti-Ld to the constituent peptides of HBsAg. For this purpose, the Western blot assay was used to detect purified H Monoclonal anti-1d63.14 and 70. to BsAg. F9 binding smell was executed. Normal human serum was used as a control. The next peptide is electric Separated in a running gel and transferred to a cellulose filter = a ) H25B10+HBsAg; b) 70. F9+HBsAg; c) MOPC17 3+HBsAg; d) 63.14+ normal human serum; e) 63.14+HB As sAg, a molecular weight indicator, we used the following protein: bovine albumin. ovalbumin, glyceraldehyde-3-phosphate dehydrogena -ase, carbonic anhydrase, trypsinogen, trypsin inhibitor, alpha-lac Toalbumin (Slgma Chemlcal Co., Missouri) Truis).

Human serum negative for HBsAg served as a negative control for the peptide assay. while monoclonal anti-HBsAg H25B10 and anti-id M OPC173 is anti-1d70. Positive and negative comparisons for F9 and 63.14 was used as a control. The results showed that both anti-ids interacted with the main peptide of HBsAg. This shows that the company has responded to the request. The strongest staining is in the 24kd and 29kd bands. It occurred in 63.14 and 70. F9 is also a secondary component of HBsAg particles. It also binds to larger peptides known as

6, 2.4. cell staining Binding sites for polyHA are found not only on HBV but also incidentally on hepatocytes. Therefore, we stained various hepatocyte preparations. The anti-id was tested for its ability to color. Anti-1d63.14 and 70°F9 , using immunohistochemical and immunofluorescence techniques to detect the HBsAg-secreting cell line PL. The surface and cytoplasm of C/PRF15 could be stained. However, I When they stained normal human liver sections and HBsAg-negative liver cancer cell line HepG2. No reactivity was observed. This suggests that earlier staining may result in infected PLC cells. This suggests that this was due to the presence of HBsAg in the cells. Ta. To test this, we tested HBV) Lance expressing HBsAg. using transfected 4.10 cells and 3T3 fibroblasts derived from them. there was. Both anti-IDs were monoclonal antibody H25B, which was used as a control. The pattern was similar to that of 4.10 cells, and only 4.10 cells were stained.

Furthermore, this staining was markedly blocked by polyHA and also by H25B10 partially blocked by. These data are based on the results of radioimmunoprecipitation experiments. It was confirmed that This indicates that 63.14 is radiolabeled 4.10 and PLC/PRF1 Identify proteins extracted from 5 cell lines, but not proteins extracted from other cell lines. This indicated that no identification was made. Ta precipitated by anti-id proteins, although these are bound by monoclonal anti-HBsAg, compared to Since it is not bound by the control antibody, the protein corresponding to HBsAg protein These molecular weights correspond to those of purified HBsAg protein. It is.

7. Example 2 Hepatitis B Virus Vaccine The experiments and procedures described in the following passages and the results are similar to those produced according to the present invention, i.e., produced without using HBV. These are monoclonal and polyclonal antibodies. Insofar as it binds HBsAg, this indicates that it imitates HBsAg. be.

The ability of these anti-anti-ids to bind po1yH8A further demonstrates that HBsAg These mimics, and binding sites for polyHA in viral antibodies. This is to confirm the existence of

When tested in vivo, one of these anti-anti-IDs, Gll, was found to be an active antibody. HBsAg was as effective as HBsAg in inducing immune responses.

Sera obtained from rabbits immunized with G11 and HBsAg showed that these were 2 reacts equally well against two different antigenic subtypes, and the virus particles 24kd, which is one of the proteins in HBsAg, was precipitated. It appears to be specific for the "A" determinant. These results simulate HBsAg. Viral anti-inflammatory agents are effective in mimicking and inducing specific immune responses in vivo. Isogenic monoclonal anti-IDs that are as effective as the original This shows that it can be manufactured without using epidemiological agents.

7.1. material and method 7. 1. 1. Immunization and fusion techniques Monoclonal anti-1d63.14 purified by Tin A affinity chromatography and purified by BALB/C mouse in complete Freund's adjuvant and subsequent incomplete Freund's adjuvant. It was injected intraperitoneally at a dose of 100 μg in a bunt. 1 week of second booster stimulation After a while, the mice were bled and these sera were added to 63.14 HBsAg. The ability to inhibit the binding of the enzymes was tested by ELISA. Third additional stimulus Afterwards, spleen cells obtained from positive mice were treated with 44% polyethylene glycol (B Aker Chemical Co., Phillips Bar, New Chassis was fused with the murine myeloma cell line 5P210-Az-14 using the following method. Melt The combined cells were then cultured in HAT medium for 2 weeks and the hybridomas were Tested in EL ISA using the method described for Mausch serum.

Positive hybridomas were expanded and purified secondary by limited dilution. .

7, 1.2.　ELISA 63.14 to HBsAg, monoclonal anti-HBsAg H25B10 and The ability of hybridoma supernatant to block the binding of polyHA to direct EL Tested in ISA. HBV transfected cell line 4.1 0 (Christman et al., 1982. Proc, Natl. , Acad, Sci. (USA) 79:1815). HBsAg was prepared at a concentration of 1 Mg/ml in cerium chloride buffer, pH 9.6. Adjusted to degree. The antigen and normal human serum are then added to the microtiter plate. was used to coat the tubes by incubating overnight at 4°C.

The wells were then treated with 3% bovine serum aqueous solution in 0.02M Tris, pH 7.6. blocked with ruhumin and alkaline phosphatase conjugated 63.14, H B25 B10 (Amerlcan Ce1l Type Cuttur e Co11ection, Maryland 0-/Kuvir) or po were incubated with a 1:200 dilution of lyHA. these molecules is the method of Vol Ierl et al. (197B, Bull, World d l1ealth Organ, 53:55-65) Sphatase (type ■-TSSigma Chemical Cor stoppage experiment) Regarding 63.14, HB25B10 and polyHA on hybridoma It was preincubated or co-incubated with an aliquot of clear fluid. 5 p210 cells and AIIE6 (having anti-angiotensin receptor activity) A supernatant obtained from a hybridoma (hybridoma) served as a control. 1 at 37℃ After incubating for an hour, the plates were incubated with Tris-containing 0.05% 7we Washed three times using For about 30 minutes, add alkaline phosphatase substrate (0° 0.5M carbonate buffer, p 5mg/ml p-nitrophenyl phosphate in H9,6 and 2M gCI2). The wells are then ELISA microliquid. Dynatech Laboratories Inc, Victoria The probe was scanned at 405 nm using a 400-nm probe (Alexandria, Tex.). Although the results are multiple Expressed as mean and standard deviation.

Monoclonal anti-HBsAg to anti-anti-id H25B10 and po1 To test the binding capacity of yHa, we filled the wells of a microtiter plate with carbonate It was coated with hybridoma supernatant diluted 1:1 with sodium buffer. this next developed alkaline phosphatase-conjugated HB25B10 and polyHA. 1:200 dilution (10 Mg/ml). blockin' The cleaning, cleaning and color development techniques were similar to those described above. Polyclaw Goat, human and mouse anti-HBsAg anti-anti-ID expressing hybridomas A similar method was used to assess the ability to respond. In these experiments, Alkaline phosphatase-conjugated rabbit serum IgG (Kirkegaar and Perry's Gaithersburg, Maryland), goat anti-human and Anti-mouse IgG (Siga+a, St. Louis, MO) was used as a detection test. Used as a medicine. In blocking experiments, polyclonal anti-HBsAg pre-incubated with various concentrations of id. EL I SA method is a rabbit It was used to assess the presence of antibodies against HBsAg in the serum of pigs. anti - Obtained from rabbits injected with anti-id and HBsAg, as well as from non-immune animals. Serum obtained from herons contains peroxidase-conjugated protein A (Boehri ngerMannhei■, Indianapolis, IN) and substrates. 1 mg/ml as 4-chloro-1-naphthol and 0.2% water peroxide was tested against HBsAg using In these experiments, we , two different sources of HBsAg, namely the 4.10 cell line (ayr) and Serum from infected patients (adw) was used.

7. 1. 3. Rabbit immunization procedure Rabbits were previously placed on a protein A-Sepharose column with affinity Anti-anti-id Gll purified by chromatography, and also HBs immunized with Ag. Incomplete and subsequent incomplete Freund's adjuvant Gll and HBsAg dissolved in Freund's adjuvant (0,5 mg ), with a 4-week interval between the first and second doses, on the neck and during jumping. Injected inferiorly. Serum collected after the second injection was tested using the ELISA described above. were tested for the presence of anti-HBsAg.

7-1.4. Radiation immunoprecipitation ! Mark 25I: J&HBsAg (Travenof-Genentech DiagnostiaS, Cambridge, Massachusetts; 10106cp is anti-rabbit IgG (Sigma smi) immobilized on agarose beads. 50 μm of immunized and non-immunized rabbits (St. Louis, Zurich) pre-conjugated to serum and incubated on ice for 45 minutes. The beads are then centrifuged pelleted, RIPA buffer (10mM Tris-HCIs pH7,4 , 150mM Na (Jl, 20mM EDTA.

0602% NaN3.1%Non1det-P2O). and electrophoresis sample buffer (0.5M Tri 5-HCN, pH 6.8 , 10% sodium dodecyl sulfate (SDS), 2% 2-mercaptoethano 10% glycerol) and boiled for 5 minutes. immunoprecipitation Precipitation: 12% 5DS-polyacrylamide and X-OmatAR film (Eastman-Kodak, Rochester, New York) for about a week. exposed. To confirm the specificity of radioimmunoprecipitation, we used Cooled HBsAg and Human IgG (Siga+a, Sen., Missouri) Parallel blocking experiments were conducted using the

7.2. Results: Isogenic monochromes that mimic HBsAg in eliciting immune responses Ronal anti-antiidiotype The results shown below indicate that pancreatic cells from BALB/C mice immunized with 63.14 Nine hybridomas obtained from It is capable of blocking the binding of atase conjugate 63.14 to HBsAg. This indicates that the Direct and competitive EL ISAs are Four of the loans were p.

1yHA and the virility of monoclonal and voriclonal anti-HBsAg. HBsAg can be imitated because it blocks binding to the viral antigen. This shows that it was completed. These anti-anti-IDs inhibit HB in vivo. To determine whether it is possible to induce an immune response against sAg, We injected a group of rabbits with anti-anti-id Gll or HBsAg and These sera were tested after the second administration. ELISA, radio immunoassay B and Western blot experiments show that G11 is a specific anti-HBsAg immune response. It was shown to be as effective as HBsAg in inducing HBsAg. these days Our anti-anti-ID was tested in vitro and in vivo. HBsAg can be imitated in both This indicates that it may be useful as an alternative vaccine for BV infection.

7, 2. 1. Preparation of isogenotypic monoclonal anti-anti-ID as described in Section 6 above. As mentioned above, we have developed a mouse model that mimics polyHA and binds HBsAg. Noclonal anti-1d63.14 immunized BAL B/c mice, and used to generate an anti-anti-id that retains the internal image of HBsAg. It was. Spleen cells obtained from mice expressing circulating anti-63.14 were isolated from mouse bone. It was fused with the myeloma cell line SP2/Q-Az-14. 28 hybridoma cases Nine of them are alkaline phosphatase conjugated to a similar extent as viral antigens. The binding of 63.14 to HBsAg could be blocked.

Table 3 by anti-anti-idiotypic antibodies Anti-inhibitor that blocks the binding of McA863.14 to HBsAg 63.14゜HBsAg C11,10,09±0.01HB sAg D8.1 0.08±0.01HBsAg C8,10,09±0.02HBsAg G3.2 0 ．． 06±0. OIHBsAg F7. 1 0.08±0. OIHBsAg　　　　G11. 1 0.10±0.02HBsA g D2.2 0.08±0.01HBsAg E7.1 0.09±0.02HBsAg AIIE6 0.99±0.44HB s A g HB s A g O,07±0.01a Anti-anti-id supernatant was pre-coated with HBsAg. were co-incubated with McAb 63.14 in the same wells. result is expressed as the standard deviation above the mean of duplicates (405 nm).

7, 2. 2. Monoclonal anti-anti-id that mimics HBsAg in vitro Whether these hybridomas can actually mimic HBsAg To measure these, we used monoclonal anti-HBs in an ELISA. Tested for ability to bind Ag (H25B10) as well as polyHA . As shown in Table 4, the four hybridomas are H25B10 and poly It reacted strongly with both HA and blocked the latter's binding to HBsAg. these The reactivity of these hybridomas is, in turn, due to their ability to mimic Willo antigens. derived from a different species to ensure that it is not genetically restricted. was tested against a polyclonal anti-HBsAg. selected for subsequent analysis The anti-anti-id Gll is a goat, human and mouse anti-antibody to HBsAg. can react with viral antibodies and bind to these viral antibodies. It was specifically designed to prevent

Table 4 Anti-HBsAg (H2 5B10) and C11,10,28±0.01 0.15±0.02D 8. 1 0.25±0.04 0.14±0.00GIO, 10 ,33±0.04 0.1g±0.03C8,10,47±0.23 0.22±0.02G3.2 1.24±0.04 0.5B ±0.09F7. 1 1.65±0.10 0.65±0.07 G11.1 0.97±0.15 0.90±0.09D2.2 0.53±0.14 0.31 +0.07F7. 1 0 ．． 61±0.2L O,40±0.04AIIE6 0.14± 0.08 0.04±0. OIHBsAg O,21±0.10 0.81±0.06a Enzyme-conjugated H25B10 and polyHA are on anti-anti-id that had been previously bound to the wells of a plastic 96-well plate. Reacted with clear liquid. The results are the standard deviation above the mean of the duplicates (405nm) and It is expressed as

7, 3. 3. Anti-HBsAg immunity in vivo by monoclonal anti-id Induction of epidemic response Anti-anti-id Gll induces an immune response against HBsAg in vivo It was used in a study in which students were tested on their ability to In these experiments, rabbits Gll or HB used as host and in complete Freund's adjuvant sAgO, 5 mg, was injected and boosted subcutaneously 1 month later. Second After the next immunization, rabbit serum was tested for two different concentrations of HBsAg in an ELISA. The preparations ('a dw, ay r) were tested. Inject anti-anti-id Sera obtained from rabbits immunized with HBsAg showed It was found that the anti-HBsAg activity was expressed at a titer similar to that of the anti-HBsAg activity. I was taken out. Similar reactivity and potency were observed using a commercially available radioimmunoassay (AU SRI A%Abbott Diagnostics, North Jersey, Illinois It was also detected using a basket). The specificity of these sera is then determined by radioimmunoprecipitated fractions. This was confirmed by the analytical results. Here, these are derived from serum and Showed to react with the major 24kd protein of radiolabeled HBsAg particles. It was something. It is possible that these sera can cross-react with human immunoglobulin light chains. To remove the sex, we used a specific polyclonal antibody (Sigma, Mizu St. Louis, Lee) and using ELI SA and immunoelectrophoresis techniques. , the viral particles were tested for the presence of human immunoglobulin heavy and light chains. . In these assays, there was no reactivity between rabbit serum and anti-human H and L chains. However, there was no difference between the latter and the human IgG given as a control. reactivity was observed. In addition, 24kd HBsAg protein was detected by the rabbit serum. Protein binding and precipitation was achieved by cooling HBsAg (1 μg/m1) particles. was inhibited by human IgG (100 μg/ml). Ta.

The invention is disclosed in examples that are intended merely to illustrate one aspect of the invention. The scope is not limited in any way by the manner in which it is implemented, and any functional Products and processes equivalent to are included within the scope of the present invention.

Indeed, various variations of the invention other than those shown or described herein are possible. Modifications will be apparent to those skilled in the art from the above description. like this Modifications are intended to be included within the scope of the claims.

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Claims (27)

[Claims] 1. (a) Create monoclonal antibodies against ligands that bind to pathogen receptors. made, (b) monochromes with idiotypes that mimic the conformational structure of pathogen receptors; Select a local antibody, (c) the selected monoclonal antibody is sufficient to elicit an active immune response. active immunity against pathogens by mixing in a pharmacological carrier at appropriate dosages. Methods of manufacturing vaccine formulations that provide immunization. 2. The ligand is a neutralizing antibody obtained from an animal or human exposed to the pathogen. In addition, selected monoclonal antibodies mimic the conformational structure of pathogen receptors. 2. The method according to claim 1, wherein the method comprises an anti-idiotype. 3. Ability of monoclonal antibodies to competitively block binding of pathogens to ligands The method according to claim 1 or 2, wherein the method is selected based on. 4. The selected monoclonal antibody has Fv, Fab, F(ab')2 or The method according to claim 1 or 2, further comprising a Fab′ region or a Fab′ region. Law. 5. The method according to claim 1 or 2, wherein the pathogen consists of a microorganism. 6. 3. The method according to claim 1 or 2, wherein the pathogen consists of microorganisms. 7. The method according to claim 1 or 2, wherein the pathogen consists of a parasite. 8. 3. The method according to claim 1 or 2, wherein the pathogen consists of a virus. 9. 9. The method according to claim 8, wherein the virus is hepatitis B virus. 10. The method according to claim 9, wherein the ligand is composed of polymerizable human albumin. Law. 11. (a) Antibody to the first antibody that limits the ligand that binds to the receptor of the pathogen Create idiotypic antibodies, (b) an anti-idiotype having an idiotype that mimics the conformational structure of the ligand; select specific antibodies, (c) anti-anti-idiotypic to this selected anti-idiotypic antibody; (d) idiotype that mimics the conformational structure of the pathogen's receptor; selecting an anti-anti-idiotypic antibody having (e) using selected anti-anti-idiotypic antibodies to elicit an active immune response; ability against pathogens by mixing in a pharmacological carrier in sufficient doses. Methods of manufacturing vaccine formulations that provide dynamic immunization. 12. The anti-idiotypic antibody is an antibody Fv, Fab, F(ab')2 or 12. The method of claim 11, further comprising a Fab' moiety. 13. 12. The method according to claim 11, wherein the pathogen consists of a microorganism. 14. 12. The method according to claim 11, wherein the pathogen consists of bacteria. 15. 12. The method according to claim 11, wherein the pathogen consists of a parasite. 16. 12. The method according to claim 11, wherein the pathogen consists of a virus. 17. 17. The method of claim 16, wherein the virus is hepatitis B virus. 18. 18. The ligand according to claim 17, wherein the ligand is composed of polymerizable human albumin. Method. 19. (a) Anti-identifier for antibodies that limit the ligand binding to pathogen receptors Create biotypic antibodies, (b) an anti-idiotype having an idiotype that mimics the conformational structure of the ligand; Select the specific antibody, (c) using the selected anti-idiotypic antibody to confer a passive immune response; susceptibility to pathogens by mixing in pharmacological carriers in sufficient doses. Methods of manufacturing vaccine formulations that provide dynamic immunization. 20. Anti-idiotypic antibodies competitively block the binding of ligands to pathogens. 20. The method of claim 19, wherein the method is selected based on its ability to 21. The anti-idiotypic antibody is an antibody Fv, Fab, F(ab')2 or 20. The method of claim 19, further comprising a Fab' moiety. 22. 20. The method according to claim 19, wherein the pathogen consists of a microorganism. 23. 20. The method according to claim 19, wherein the pathogen consists of bacteria. 24. 20. The method according to claim 19, wherein the pathogen consists of a parasite. 25. 20. The method according to claim 19, wherein the pathogen consists of a virus. 26. 26. The method of claim 25, wherein the virus is hepatitis B virus. 27. Anti-idiotypic antibodies maintain the conformational structure of polymerizable human albumin 27. The method according to claim 26.