WO1988006040A1 - Monoclonal antibodies in vaccine formulations - Google Patents

Abstract

Vaccine formulations which provide for active or passive immunity against infection by pathogens. The active component of the vaccines of the present invention comprises a monoclonal antibody or a fragment of the monoclonal antibody which contains the idiotype of the molecule. Vaccine formulations which provide for active immunization of a host against a particular pathogen, are prepared using monoclonal antibodies or idiotypic fragments that bear the conformational image of a specific receptor of the pathogen. Vaccine formulations which provide for passive immunity are prepared from anti-idiotypic antibodies or idiotypic fragments thereof that bear the conformational image of a ligand that binds specifically to a receptor of the pathogen. In either approach, the pathogen is not required to prepare the antibody which comprises the active component of the vaccine formulation.

Description

MONOCLONAL ANTIBODIES IN VACCINE FORMULATIONS

1. INTRODUCTION The present invention involves vaccine formulations which provide for protection against infection by pathogens, either by stimulating an active immune response in a vaccinated host or by conferring short-term passive immunity. The active ingredients of the vaccine formulations of the present invention comprise monoclonal antibodies or fragments of monoclonal antibodies which contain the idiotype of the molecule, such as any Fv fragment including the Fab, F(ab')_, or Fab' and the like. The invention is also directed to methods for preparing such vaccine formulations which do not require isolating or handling the pathogen.

The immunogen of the vaccines of the invention which stimulate an active immune response against a pathogen comprises a monoclonal antibody or a fragment thereof, which bears the conformational image of a specific receptor of the pathogen. Where the vaccine is formulated to confer short- term passive immunity, the monoclonal antibody, or fragment thereof, bears the conformational image of a ligand which specifically binds to a receptor on the pathogen. In either embodiment, the vaccines of the present invention can confer protection to a host without exposing the host to the pathogen.

The invention is demonstrated by way of examples in which monoclonal anti-idiotypes which mimic polymeric human serum albumin were used to generate anti-anti-idiotypes which bear the internal image of hepatitis B virus surface antigen and mimic its immunogenicity in vivo.

2. BACKGROUND OF THE INVENTION

2.1. VACCINES THAT ELICIT AN ACTIVE IMMUNE RESPONSE

Vaccines are traditionally prepared by rendering viruses harmless without destroying their immunogenicity. This is achieved either by inactivating the infectivity of the virion, or by selecting an avirulent mutant. Inactivated vaccines are "dead" in the sense that the infectivity of the virions has been destroyed, usually by 5 treatment with ormaldehyde. Injection of these "killed" virus particles into a host will then elicit an immune response capable of neutralizing a future infection with live virus. A number of problems are associated with the use of inactivated vaccines. One major concern is the 0 failure to inactivate all the virus particles. However, even when this is accomplished, because the killed viruses do not multiply in their host, the immunity achieved is often short lived and additional immunizations are required. Another major difficulty encountered in using inactivated

15 vaccines lies in producing enough virus in order to prepare a vaccine which provides the necessary quantity of the relevant antigen to promote systemic antibody production in the host.

Vaccination with attenuated live virus overcomes some

__n of the problems associated with the killed vaccine preparations. Attenuated viruses have essentially lost their disease producing ability and generally are good immunogens that provide for long lasting immunity because the attenuated virus actually replicates in the host. However, several problems are associated with live virus

25 vaccines - not the least of which is insufficient attenuation. Attenuation of a virus is traditionally accomplished by rapid serial passage of the virus in a foreign host to produce an avirulent mutant. Some of the problems associated with the live virus vaccines include genetic instability of the attenuated virus which results in a greater degree of virulence than is desirable, contamination by adventitious virus during the passages in cell culture, interference by wild type virus and heat __ lability of the live virus in the vaccine formulation. An. alternative to inactivated and live virus vaccines, is the use of a subunit vaccine. This involves immunization of the host solely with the relevant immunogenic material of the pathogenic virus. For example, virus encoded glycoproteins of many of the enveloped viruses or the capsid proteins of non-enveloped viruses are capable of eliciting neutralizing antibodies. Subunit vaccines may be prepared by purifying these proteins from the viruses. One advantage of subunit vaccines is that irrelevant virus material, and the genetic information as well as the replication machinery of the virus are excluded. A major difficulty is encountered in producing the purified proteins in immunogenic amounts.

The use of recombinant DNA technology for the production of subunit vaccines has recently received much attention. This involves the molecular cloning and expression of the viral genetic information coding for proteins which can elicit a neutralizing response in the host. The viral genetic material is excluded and the host is never exposed to the whole virus, therefore, the host stands no risk of becoming infected. Moreover, the production of larger quantities of the epitopes used in the subunit vaccine is made possible. These techniques, however, are limited to the production of proteinaceous epitopes, and so far have not been applicable to lipid or carbohydrate antigens.

The immunological response to immunogens used in the killed virus vaccines or subunit vaccines can be greatly enhanced if they are administered in an emulsion with adjuvants. The mechanism by which adjuvants increase an immune response are complex, involving the stimulation of activities associated with the reticuloendothelial system.

However, conventional adjuvants which are based on mineral oils are not accepted for use in man because they are nonmetabolizable and are potentially carcinogenic. 2.2. PASSIVE IMMUNITY In the management of certain diseases or infections, instead of actively immunizing with viral vaccines, it has been possible to confer short-term protection by the 5 administration of preformed antibody, such as immune serum or concentrated immunoglobulin which can bind to and neutralize the pathogen; this is called passive immunization. Human immunoglobulin is usually preferred in human medicine because a heterologous antibody could provoke 0 an immune response.

Passive immunization is generally regarded as an emergency procedure for the immediate protection of unimmunized individuals exposed to special risks. However, passive immunization is also regarded as an important 5 prophylactic measure in several viral infections. For example, human immunoglobulin has proven effective in the short-term prophylaxis of measles and hepatitis A; by contrast, prevention of hepatitis B using human normal immunoglobulin has not been so successful.

20

2.3. ANTI-IDIOTYPIC ANTIBODIES AND VACCINES

Recently, a new type of vaccine has been suggested which involves the use of anti-idiotype antibodies as the im unogen which elicits an active immune response in the

„ host (see. Medical News, JAMA Jan 24/31, 1986, Vol. 255(4): 25

447-448) . Anti-idiotypic antibodies or anti-idiotypes are antibodies directed against the antigen-combining region or variable region (called the idiotype) of another antibody molecule. In theory, based on Jerne's network model of idiotypic relationships (Jerne, N.K., 1974, Ann. Immunol.

(Paris) 125c:373; Jerne, N.K. , et al. , 1982, EMBO 1:234), immunization with an antibody molecule expressing a paratope

(antigen-combining site) for a given antigen should produce a group of anti-antibodies, some of which share with the antigen a complementary structure to the paratope. 35 Immunization with a subpopulation of the anti-idiotypic antibodies should in turn produce a subpopulation of anti- idiotypic antibodies that bind the initial antigen. Thus certain anti-idiotypes directed against a virus-neutralizing antibody should mimic the virus, and when inoculated into a host should induce a specific antiviral response.

Work with a variety of infectious disease models, including viral and bacterial systems, has demonstrated anti- idiotypic modulation of specific immunity (for a summary listing of data from infectious disease model systems, see Reagan, K.J. , 1985, Curr. Topics Microbiol. Immunol. 119:18, 19). Investigation in viral systems has included Hepatitis B Virus (Kennedy, R.C., 1985, Curr. Topics Microbiol. Immunol. 119:1-13), feline leukemia virus, (Uytdehaag, F.G.C.M., et al., 1986, Immunol. Reviews 20:93-113), and poliovirus (Reagan, supr ; Uytdehaag, supra) . Vaccines for reovirus (Fields et al., 1984, in Modern Approaches to Vaccines, Cold Spring Harbor Laboratory, pp. 285-287) and for rabies virus (Reagan, supra; Koprowski, 1985, _in Vaccines85, Cold Spring Harbor Laboratory, pp. 151-156) have been investigated in experimental animals. Anti-idiotypic antibodies have been used to induce immunity comprising protection against a lethal Sendai virus infection in mice (Ertl, H.C.J. and Finberg, R.W., 1984, Proc. Natl. Acad. Sci. USA 81:2850- 2854) . Immunization with anti-idiotypic antibodies in the HBV system led to the induction of an anti-HBsAg response (Kennedy, supra) .

2.4. HEPATITIS B VIRUS

Hepatitis B virus (HBV) is an etiologic agent in both acute and chronic hepatitis, and has been implicated as an etiologic agent in hepatocellular carcinoma (Hoofnagle,

J.H., 1981, Seminars Liver Diseases 1:7-15). HBV displays a tropism for hepatocytes, the mechanism of which is still an enigma (Hanson, et al., 1979, Infection and Immunity, 26: 125-130) . Recently, reports of the presence of binding sites for polymerized human albumin (polyHA) on both the hepatitis B surface antigen (HBsAg) and the liver cell membrane suggested that polyHA could mediate the liver tropism of HBV, by acting as a bridge between virus and hepatocytes (Robinson et al., 1976, N. Engl. J. Med. 295: 1168-1175; Imai et al., 1979, Gastroenterology 76:242-247; Machida et al., 1984, Gastroenterology 86: 910-918). Moreover, sera from individuals infected with HBV contain autoaπtibodies specific for polyHA (Lenkei et al., 1977, J. Med. Virol. 1: 29-34). However, polyHA receptors on the virus or on hepatocytes have never been isolated nor identified.

3. SUMMARY OF THE INVENTION

The present invention involves vaccine formulations which provide for active or passive immunity against infection by pathogens, including but not limited to viruses, bacteria, parasites, etc. The active component of the vaccines of the present invention comprises a monoclonal antibody or a fragment of the monoclonal antibody which contains the idiotype of the molecule; these fragments include but are not limited to any Fv fragment such as the

Fab, F(ab')_, Fab' fragments and the like. ^

In one embodiment of the invention, the vaccine formulations provide for active immunization of a host against a particular pathogen. In these formulations, the immunogen of thi vaccine comprises a monoclonal antibody, or an idiotypic fragment thereof, which bears the conformational image of a specific receptor of the pathogen.

In accordance with the method of the invention, the pathogen is not utilized in the production of the monoclonal antibody. By contrast, the monoclonal antibodies which mimic a specific receptor of the pathogen may comprise: (a) a monoclonal antibody generated against a ligand that binds specifically to the receptor of the pathogen; (b) a monoclonal antibody generated against the idiotype of a second antibody which, in turn, defines the idiotype of a

5 third antibody which, in turn, defines a ligand that binds specifically to the receptor of the pathogen; or (c) any other anti-idiotypic monoclonal antibody which mimics the receptor of the pathogen as depicted in the cascade shown in

FIG. 1. In all cases the monoclonal antibody should also

10 competitively inhibit the binding of the pathogen to the ligand.

In a second embodiment of the present invention the vaccine formulations may provide for passive immunity to confer short-term resistance to infection by a pathogen. In

15 this embodiment, the active ingredient of the vaccine comprises a monoclonal antibody or idiotypic fragment thereof which bears the conformational image of a ligand that binds specifically to a receptor of the pathogen. In accordance with the method of the invention, the pathogen is

_ not utilized in the production of the monoclonal antibodies.

The monoclonal antibodies which mimic a ligand that binds to a specific receptor of the pathogen may comprise: (a) a monoclonal antibody directed against the idiotype of a second antibody which, in turn, is directed against the

__ ligand; (b) a monoclonal antibody directed against the 2b idiotype of a second antibody which, in turn, is directed against the idiotype of a third antibody which, in turn, is directed against the idiotype of a fourth antibody which is directed against the ligand; or (c) any other anti-idiotypic antibody which mimics the ligand that binds to the pathogen as depicted in the cascade shown in FIG. 1. In all cases the monoclonal antibody should also competitively inhibit the binding of the ligand to the pathogen.

Some of the advantages of the vaccine formulations of the present invention include the following: (a) a pathogenic immunogen is not required to stimulate the immunity of the host;

(b) a pathogenic immunogen is not required for the -preparation of the vaccine formulations of the present invention;

(c) unlike vaccines prepared using recombinant DNA techniques to clone proteinaceous immunogens, the vaccines of the present invention can be prepared against epitopes that comprise non-proteins such as lipids, carbohydrates, or glycolipids, etc.; and

(d) large quantities of the monoclonal antibodies can be obtained to provide for a sufficient amount of immunogen in a "subunit" vaccine formulation which stimulates the host immunity.

3.1. DEFINITIONS

The following terms and abbreviations will have the meanings indicated:

Anti-id = anti-idiotype antibody

BSA = bovine serum albumin

ELISA ,= enzyme-linked immunoadsorption assay

Fv = the variable region or antigen-combining site of an antibody molecule. This may be any fragment which contains the idiotype of the molecule including but not limited to the Fab, F(ab')₂, Fab', and the like.

HA = human albumin (monomeric)

HBV = hepatitis B virus

HBsAg = hepatitis B surface antigen

Kd = kilodalton

PEG = polyethylene glycol polyHA = polymeric human albumin

RIA = radioimmunoassay

SDS = sodium dodecylsulfate TWEEN-20 = polyoxyethylene sorbitan onolaurate

4. DESCRIPTION OF THE FIGURES FIG. 1 is a diagrammatic representation of the cascade ₅ of anti-idiotypic antibodies that can be generated from a ligand which specifically binds to a receptor of a pathogen. The antibodies that can be used as the active ingredient in either the active or passive vaccine formulations of the present invention are indicated in the figure. The anti¬ ng ligand antibody and anti-id2, anti-id4, etc. mimic the receptor of the pathogen and can be used as the immunogen in the vaccines formulated for active immunization. The anti- idl, anti-id3, etc. mimic the ligand which binds specifically to the receptor of the pathogen, and can be ₁₅ used as the active ingredient in the vaccines formulated for passive immunization.

5. DETAILED DESCRIPTION OF THE INVENTION The vaccine formulations of the invention can provide for protection against infection of a host by a number of pathogens, including but not limited to viruses, bacteria, parasites, etc. The active ingredient of the vaccine formulations of the present invention comprise monoclonal antibodies or fragments of the monoclonal antibody which contain the idiotypic region of the antibody molecule; these include, but are not limited to the fragments which include the Fv region, such as the Fab, F(ab')_, Fab' fragments and the like. The monoclonal antibodies of the present invention are generated without utilizing the pathogen. These antibodies, or fragments thereof, are formulated as a vaccine which, depending upon the nature of the idiotype, can be used either to stimulate an active immune response or to confer short-term passive immunity in a host against a pathogen. 5 For purposes of clarity of discussion, the vaccine formulations of the present invention which provide for either active or passive immunity are described in separate sections below, and with reference to FIG. 1 which diagrammatically represents the cascade of anti-idiotypic antibodies that could be used in different embodiments of the invention.

5.1. VACCINES FOR ACTIVE IMMUNIZATION A number of pathogens possess binding sites

(hereinafter referred to as receptors) on their surfaces that specifically bind to particular substrates or receptors

(hereinafter referred to as ligands) that are present in the host which is the target for infection by the pathogen. ₅ Usually, the binding interaction between the pathogen receptor and the host's ligand enables the attachment of the pathogen to a target host cell surface. This attachment is generally a prerequisite for successful infection, multiplication, colonization, etc. by the pathogen which

„ leads to illness in the host. 0

In accordance with the method of the invention, the host ligand may be used to generate an antibody (or its anti-anti—idiotype) which mimics the pathogen receptor (see

FIG. 1) . This antibody, or its anti-anti-idiotype, may be used in vaccine formulations to induce active immunity against the pathogen. Alternatively, where the pathogen receptor or host ligand is unknown or uncharacterized, anti-pathogen antibody obtained from a host who has developed a neutralizing immune response (e.g. , through exposure to the pathogen) may be used as the starting material to develop the anti-idiotype cascade. In this case, the anti-idiotype of the neutralizing antibody which mimics the conformational structure of the receptor of the pathogen and induces a neutralizing response is selected as the active ingredient of the vaccine formulation. In either 5 embodiment, a vaccine can be formulated without using or handling the pathogen.

The vaccine formulations and their method of preparation in accordance with the invention is based, in part, upon the theory that the idiotypic region of an antibody molecule directed against the idiotype of a neutralizing antibody or a ligand which binds specifically to a receptor on a pathogen should bear the conformational image and structure of the receptor on the pathogen; for example, see anti-ligand in FIG. 1. Similarly, an antibody directed against the idiotype of a second antibody which, in turn, is directed against the idiotype of the anti-ligand antibody should also bear the conformational image of the receptor on the pathogen; for example, see anti-id2 in FIG. 1 which diagrammatically depicts the cascade of anti- idiotypes that can be generated from the ligand and/or the anti-ligand. The anti-ligand as well as anti-id2, anti-id4, etc. in the cascade would be expected to mimic the receptor of the pathogen. The degree to which the anti-ligand antibody or anti-idiotypic antibody mimics the receptor on the pathogen can be ascertained in a competitive binding assay. For example, if the anti-ligand, or the anti- idiotype, competitively inhibits the binding of the pathogen to the ligand, then one can conclude that the idiotype of the antibody molecule mimics the receptor on the pathogen. An effective dose of the anti-ligand antibody, or the appropriate anti-idiotypic antibody which mimics the receptor on the pathogen, may be formulated as an immunogen in a vaccine which is used to stimulate an active immune response in a vaccinated host. Because the idiotype of the antibody molecule mimics the conformation of the receptor on the pathogen, the resulting immune response directed against the idiotype will also be directed against the receptor on the pathogen. Since this receptor is responsible for the attachment of the pathogen to its target, the immune response should neutralize the infectivity of the pathogen.

In the preferred embodiment, the anti-ligand antibodies which are used as the immunogen in the vaccine formulations of the present invention should be monoclonal antibodies in order to insure a continuous supply and in order to be able to produce the antibody in large quantities. However, in preparing the monoclonal anti-idiotypic antibodies which are used as immunogens, the intermediate antibodies of the cascade need not be monoclonal antibodies. For example, if monoclonal anti-id4 in FIG. 1 is to be formulated in a vaccine that stimulates active immunity, then the anti- ligand, and anti-idl, anti-id2, and anti-id3 need not be monoclonal antibodies. The monoclonal antibodies which are used as immunogens can be prepared using any technique which provides for the production of antibody molecules by continuous cell lines in culture. For example, the hybridoma technique originally developed by Kohler and Milstein (1980, Sci. Am. 243(4):

66-74) as well as other techniques which have more recently become available, such as the human B-cell hybridoma technique (Kozbor et al., 1983, Immunology Today 4: 72) and the EBV-hybridoma technique to produce human monoclonal antibodies (Cole et al., 1985, Monoclonal Antibodies and

Cancer Therapy, Alan R. Liss, Inc., pp. 77-96) and the like are within the scope of the present invention. Human monoclonal antibodies are preferred as immunogens in order to enhance the desired specificity of the immune response, although monoclonal antibodies originating from any species may be used.

The invention is not limited to the use of whole antibody molecules as the immunogen in the vaccine formulations, rather, any fragment of the antibody which contains the idiotype of the antibody (e.g. , the Fv portion of the antibody molecule) could be used. Such fragments include but are not limited to: the F(ab')_ fragment which can be generated by treating the antibody molecule with pepsin; the Fab' fragments which can be generated by reducing the disulfide bridges of the F(ab')_ fragment; and the 2Fab or Fab fragments which can be generated by treating the antibody molecule with papain and a reducing agent to reduce the disulfide bridges. The F(ab') fragments would be preferred as immunogen, in order to enhance the induction of an immune response of the desired specificity. The pathogens for which the vaccines can be prepared are numerous. For example, in many viruses, attachment of virions to a target cell is a prerequisite for penetration of the virus into the host cell and subsequent multiplication. Specificity of attachment has been demonstrated for viruses such as poliovirus and influenza virus. Poliovirus, for instance, will attach to primate but not to rodent cells. This specificity is believed to be due to the interaction of the virus with a specific cellular receptor of the primate. According to the invention, the receptor can be used as the ligand from which anti-ligand and/or the cascade of anti-idiotypic antibodies could be generated. The appropriate antibodies could be used in a vaccine formulation as described.

Similarly, the presence of certain proteins on the surface of the host cell is required for the attachment of influenza virus. These cell-surface proteins appear to be receptors for the viral hemagglutinin antigen. Accordingly, the receptor proteins can be used as the ligand from which the anti-ligand and/or cascade of anti-idiotypic antibodies are generated. The appropriate antibodies can be formulated in a vaccine as described.

Penetration of the host cell during retrovirus infection has been shown to be dependent upon a specific interaction between the viral envelope glycoprotein and a host cellular receptor (Crittenden, L.B., 1968, J. Natl. Cancer Inst. 41: 145-153; Piraino, F., 1967, Virology 32:700-707; Steck, F.T., and Rubin, H. , 1965, Virology :642-653). In the present invention, these host receptors can be used as the immunogen for generation of the anti- idiotypic cascade of antibodies.

In a particular embodiment of the invention, an anti- idiotypic cascade can be used to produce antibodies to the cellular receptor for the AIDS virus, the etiological agent of Acquired Immune Deficiency Syndrome (AIDS) (Gallo, R.C., et al. , 1984, Science 224:500; Popovic, M. , et al., 1984, Science 224:497; Barre-Sinoussi, F. , et al., 1983, Science 220: 868; Levy, J.A., et al., 1984, Science 225: 840). Evidence suggests that the receptor for the AIDS virus is the CD4 (T4) antigen on lymphocytes (Dagleish et al., 1984, Nature 312:763-766; Klatzmann et al. , 1984, Nature 312:767-

768) . AIDS virus epitopes may be used as the initial antigens for the eventual production of anti-idiotypic antibodies that mimic the viral protein which binds the cellular T4 antigen. One possible source of such AIDS virus

_ epitopes are the currently available diagnostic kits for

AIDS, many of which contain disrupted AIDS virus virions.

Other sources for initial immunization purposes include AIDS virus proteins which are produced by recombinant DNA techniques, chemically synthesized, obtained from purified virus, etc. Alternatively, anti-AIDS virus antibody derived from patients could be used as the starting point for generation of anti-idiotypic antibodies directed against the cellular receptor for the AIDS virus.

It has also been postulated that hepatitis B virus

(HBV) which has binding sites for polymerized serum albumin, attaches to the surface of hepatocytes via the polymerized albumin; the HBV-polyalbumin complex is then taken into the liver cells by endocytosis. Coincident with the specificity of the virus for a particular host, HBV particles and HBsAg particles only bind specifically to human and chimpanzee 5 serum polyalbumin. According to the invention, the polymerized human albumin is used as the ligand from which the anti-ligand and/or anti-idiotypic antibodies are generated. Alternatively, a cascade of anti-idiotypic 5 antibodies can be generated using the human anti-polyHA which can be obtained from patients infected with HBV. The appropriate antibodies can be formulated as a vaccine as described herein.

Many diseases or disorders of bacterial or parasitic

10 origin are also dependent upon specific interactions with host cellular receptors. For example, sensitivity to cholera toxin (choleragen) , a disease-producing bacterial toxin, is dependent upon the presence of its receptor, ganglioside G_M_, in the host cell plasma membrane (Moss, J.

1₅ and Vaughan, M. , 1979, Ann. Rev. Biochem. 48:581-600). E. coli heat-labile enterotoxin also utilizes a host cell ganglioside, probably G_M1 as its cellular receptor (Moss, J., and Vaughan, M. , 1979, Ann. Rev. Biochem. 48: 581-600). Thus, in this embodiment of the invention, G_M_ may be used as the immunizing ligand from which anti-idiotypic antibodies are generated.

5.2. VACCINES FOR PASSIVE IMMUNIZATION

This embodiment of the invention is based, in part, upon the theory that the idiotypic region of an antibody 25 molecule directed against the idiotype of a second antibody which, in turn, is directed against a ligand which binds specifically to a receptor on a pathogen should bear the conformational image and structure of the ligand; for

_Λ example, see anti-idl in FIG. 1. Other anti-idiotypic 30 antibodies, such as anti-id3, indicated in the cascade depicted in FIG. 1 would also be expected to mimic the ligand which binds specifically to the receptor on the pathogen. The degree to which the anti-idiotypic antibody mimics the ligand can be ascertained in a competitive wO binding assay. For example, if the anti-idiotypic antibody competitively inhibits the binding of the ligand to the pathogen, then one can conclude that the idiotype of the antibody molecule mimics the ligand. According to this embodiment of the invention, the anti-idiotypic antibody which mimics the ligand is formulated in a "vaccine" that can be used to provide passive immunization for short-term protection against infection by the pathogen. Because the idiotype of the anti-idiotypic antibody mimics the conformation of the ligand, the antibody will bind to the receptor on the pathogen. Binding of the antibody to the receptor will interfere with attachment of the pathogen to its target and, therefore, will prevent subsequent infection by the pathogen.

In the preferred embodiment, the anti-idiotypic antibodies used in the vaccine formulations for passive immunization should be monoclonal antibodies in order to insure a continuous supply and in order to be able to produce the antibody in large quantities. However, in preparing the monoclonal anti-idiotypic antibodies used in the vaccine formulations, the intermediate antibodies of the cascade need not be monoclonal antibodies. For example, if monoclonal anti-id3 in FIG. 1 is to be formulated to provide for passive immunization, then the intermediate antibodies of the cascade such as anti-id2, anti-idl, and the anti- ligand antibody need not be monoclonal antibodies. The monoclonal antibodies used in the vaccine formulations for passive immunization can be prepared by any technique that provides for the production of antibody molecules by continuous cell lines in culture. For example, the hybridoma technique originally developed by Kohler and Milstein (1980, Sci. Amer. 243(4): 66-74) as well as other techniques which have recently become available, such as the human B-cell hybridoma technique (Kozbar et al., 1983, I munology Today 4: 72) and the EBV-hybridoma technique to produce human monoclonal antibodies (Cole et al., 1985, Monclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc. , pp. 77-96) and the like are within the scope of the present 5 invention.

Human monoclonal antibodies are preferred for use, since they would reduce the possibility of eliciting an immune response directed against the antibody molecule. Repeated administrations of the monoclonal antibody for 10 passive immunization would preferably be minimized so as to reduce the risk of inducing auto-immunity.

The invention is not limited to the use of whole monoclonal anti-idiotypic antibody molecules as the active ingredient in vaccine formulations for passive immunity. 15 Rather, any fragment of the antibody which contains the idiotype of the antibody (e.g. , the Fv portion of the antibody molecule) could be used. Such fragments include but are not limited to: the F(ab')_ fragment which can be generated by treating the antibody molecule with pepsin; the Fab' fragments which can be generated by reducing the

20 disulfide bridges of the F(ab')_ fragments; and the 2Fab or Fab fragments which can be generated by treating the antibody molecule with papain and a reducing agent to reduce the disulfide bridges.

However, the use of a whole antibody molecule (i.e. ,

___.0 one that includes the Fc region of the molecule) as the active ingredient of a vaccine formulation for passive immunity may offer advantages over using an Fv fragment of the antiboⁿdy. For example, if the monoclonal anti-idiotypic antibody is of a class that activates complement or mediates

ADCC (antibody dependent cellular cytotoxicity) , the binding of the antibody molecule to the pathogen receptor would not merely prevent infection by the pathogen, but would mediate the destruction of the pathogen. For instance, if the pathogen is a bacteria or a parasite, complement activation 35 or ADCC could result in lysis of the bacteria or the parasite. Where the pathogen is a virus which has already infected some cells in the host, the antibody would bind to the infected cells which express the viral receptor. These infected cells would similarly be destroyed via complement activation or ADCC.

Passive immunization can be used to treat a variety of viral or bacterial diseases. For example. Hepatitis A virus has successfully been treated with human immunoglobulin to provide passive immunity. This human immunoglobulin is obtained from donors who have had contact with the virus. According to this embodiment of the invention, monoclonal antibodies directed against envelope proteins of HAV could be used to inactivate the hepatitis A virus via passive immunization.

Many bacteria have surface antigens that promote adherence to specific cellular receptors. As provided for by the present invention, these receptors could serve as the initiating immunogen in the generation of anti-idiotypic antibodies. Anti-idiotypic antibodies directed against either the bacterial antigen or cellular receptor can then be used in passive immunization. Bacteria which are intestinal pathogens such as Shigella dysenteriae, Vibrio cholerae, various Salmonella and E. coli species and others can be treated by passive immunization schemes in which the anti-idiotypic antibody is taken orally.

5.3. FORMULATION OF THE VACCINES The subsections below describe various vaccine formulations that can be prepared using the anti-ligand, or anti-idiotypic monoclonal antibodies as immunogens that will stimulate an active immune response against a particular pathogen. Also described are vaccine formulations that can be prepared using a monoclonal anti-idiotypic antibody which will provide for passive immunity in a host exposed to short-term risks of infection by a particular pathogen.

5.3.1. ACTIVE IMMUNIZATION The vaccine formulations which stimulate an active immune response can be prepared by mixing the monoclonal antibody, or an Fv fragment thereof, which mimics the receptor or the pathogen in a carrier suitable for use in vivo. In fact, the monoclonal antibody or its Fv fragment should be formulated with a suitable adjuvant in order to enhance the iπimunological response of the host. Suitable adjuvants include, but are not limited to aluminum hydroxide, surface active substances, lysolecithin, pluronic polyols, polyanions, and peptides. Other potentially useful adjuvants in humans such as corynebacterium parvum and BCG

(bacille Cal ette-Guerin) are within the scope of the present invention.

Alternatively, the monoclonal antibody or the Fv fragment could be incorporated into or on liposomes or conjugated to polysaccharides and/or other polymers that are useful in vaccine formulations. Where the antibody fragment is a hapten, i.e. , antigenic but not capable of eliciting an immune response, it may be conjugated to a large carrier molecule, such as protein serum albumin and the like to confer immunogenicity to the complex.

The vaccine could be formulated as a multivalent vaccine. To this end, a mixture of different antibodies, each of which mimics the receptor of a different pathogen can be mixed together in one formulation.

Many methods may be used to introduce the vaccine formulations described above into a host; these include but are not limited to intrader al, intramuscular, intraperitoneal, intravenous, subcutaneous and intranasal routes. 5.3.2. PASSIVE IMMUNIZATION The monoclonal antibody molecules which mimic the ligand that binds to the pathogen can be formulated to confer short-term passive immunity to the host. Adjuvants 5 are not needed in this type of preparation because the object of this formulation is not to stimulate an immune response, but rather to bind to and inactivate the pathogen. Thus, any suitable pharmaceutical carrier may be used. Passive immunization could be used on an emergency basis for 0 immediate protection of unimmunized individuals exposed to special risks.

6. EXAMPLE: ANTI-IDIOTYPIC ANTIBODIES REACTIVE WITH HBsAg RECEPTOR FOR POLYMERIC HUMAN ALBUMIN

The examples below describe the preparation of a

15 monoclonal antibody that mimics polymeric human albumin which, according to the present invention, is a ligand that binds to HBV. This monoclonal antibody could be used in a vaccine formulation that provides for passive immunity; alternatively, the monoclonal antibody could be used to

_ ^A?Ω" generate an anti.-i.di.otypic anti.body that i.s useful as an immunogen in a vaccine that elicits an active immune response as demonstrated in Section 7 infra.

It has been suggested that polymeric human albumin (polyHA) mediates the liver tropism of HBV by interacting

²⁵ with specific receptors expressed on both HBsAg and hepatocyte. Patients infected with HBV develop an autoimmunity to polyHA. Hybridomas producing anti-id to the human anti-polyHA antibody, which bear the internal image of polyHA and were able to mimic its binding to HBsAg are

³⁰ described. The specificity of two anti-id, and in particular their ability to bind HBsAg, was demonstrated using RIA, ELISA, western blot and cell staining techniques.

Inhibition assays showed that only polyHA (polymeric human albumin) could block the binding activity of antibodies 35 63.14 and 70.F9, as compared to HA (monomeric human albumin) and two polyclonal anti-HBsAg antibodies. A partial inhibition of the anti-HBsAg reactivity of the anti-id by a monoclonal aήti-HBsAg is probably due to steric hindrance..' 5 These data suggest that the anti-id share a common three dimensional structure with polyHA, and recognize a polyHA binding site expressed on HBsAg. This recognition occurs on a domain of the antigen different from that detected by conventional anti-HBsAg antibodies.

1₀ While the existence of a liver receptor for polyHA has never been clearly demonstrated, the ability of polyHA to bind HBsAg has been documented by several investigators. The question of which HBsAg peptide polyHA is localized on is still controversial (Peterson et al., 1981, J. Biol.

15 Chem. 256: 6975-6983; Ionescu-Matiu et al., 1980, J. Med.

Virol. 6: 175-178; Machida et al., 1984, Gastroenterology

86: 910-918). Our data showed that both anti-id react with the major 24 kd peptide of the antigen as well as with its glycosylated form. Our anti-id also detected other larger peptides which probably represent uncleaved precursors of the smaller HBsAg components, or proteins comprising both the pre-s and s gene encoded peptides (Wasserman et al. ,

1982, Proc. Natl. Acad. Sci. USA 79:4810-4814; Thung, et al., 1981, Gastroenterology 80: 260-264; Trevisan, et al.,

__ 1982, Hepatology 2: 832-835; Augustin, et al., 1983 Surv. 25

Immunol. Res. 2: 78-83; Inoescu-Matiu, et al., 1980, J. Med. Virol. 6: 175-178) . These data are consistent with previous studies in which polyHA was used as a probe for the HBsAg receptor, but are at variance with recent observations which suggested that the polyHA receptor might be restricted to a small fragment of HBsAG, namely the pre-s region (Trevisan, et al., 1982, Hepatology 2: 832-835; Lenkei et al., 1977, J. Med. Virol. 1: 29-34; Augustin, et al., 1983, Surv. Immunol. Res. 2: 78-83) .

35 In sum, we have produced monoclonal anti-id directed against a human anti-polyHA antibody which bear the internal image of polyHA and mimic its binding to HBsAg. These anti-id recognize a site expressed on the major peptides of

5 the antigen which seems not to be detected by conventional anti-HBsAg antibodies. Since the expression of the polyHA receptor on HBV appears to correlate with a higher viral replication and infectivity, our anti-id can be valuable in identifying different subsets of patients with viral 0 hepatitis and the healthy carriers who are more likely to transmit the infection. Finally, if the polyHA receptor is involved in the penetration of HBV into the liver cells these anti-id when used in a passive immunization scheme might be effective in preventing the viral infection. 5 Alternatively, an anti-idiotypic antibody generated against the anti-id described herein could be used in an active vaccine formulation.

6.1. MATERIALS AND METHODS 0

6.1.1. IMMUNIZATION

Balb/c mice were immunized with an affinity purified human IgG anti-polyHA obtained from a patient with autoimmune hepatitis. They were at first injected subcutaneously in the inguinal and axillary regions and in the footpads with 100 micrograms of the immunogen in complete Freund's adjuvant. The successive boosts were given intraperitoneally using incomplete Freund's adjuvant and saline. After the third injection in saline, mice were bled and the serum tested for the presence of anti-id using a hemagglutination inhibition assay (HIA) . Human red blood cells were coated with polyHA according to S.N. Thung et al.

(1981, Gastroenterology 80: 260-264) . PolyHA was generated by cross-linking with glutaraldehyde according to R. Lenkei

__ et al. (1977, J. Med. Virol. 1: 29-34). The coated human 35 red blood cells were reacted with the immunogen in a microtiter plate after preincubation with an aliquot of the mice sera.

5 6.1.2. PRODUCTION OF MONOCLONAL ANTI-ID

Spleen cells obtained from the^ positive mice were fused with the HAT-sensitive cell line SP2/0-AZ-14, using PEG 44%. After two weeks of HAT selection, hybridomas were screened for the presence of anti-id with the HIA, as previously 0 described. The positive clones were expanded, subcloned by limiting dilutions and grown in ascites.

6.1.3. RADIOIMMUNOASSAY The ability of the monoclonal anti-id to bind the

1 (Fab')₂ of the immunogen as well as HBsAg was tested in a solid phase RIA, in which 11 different human myeloma proteins and an unrelated monoclonal anti-id (MOPC 173) served as negative control. HBsAg obtained from the concentrated supernatant of the HBV-transfected 3T3 cell

„_n line 4.10 and from the serum of a patient with acute hepatitis was purified on a cesium chloride gradient (Christman et al., 1982, Proc. Natl. Acad. Sci. USA 79: 1815-1818). The (Fab')_ of the immunogen, HBsAg and the human myeloma proteins (10 micrograms/ml) were incubated in the wells of a microtiter plate overnight at 4^βC. After blocking with 3% bovine serum albumin (BSA) in phosphate buffer saline (PBS), pH 7.2, the wells were incubated with 50 ul of anti-id for 1 hour at 37°C. After washing with PBS containing 0.01% Tween-20 (PBS-Tween) , the wells were incubated with 50 ul of 125I-labeled rat anti-mouse K chain antibody for 1 hour at 37^βC. The plates were then washed with PBS-Tween and the radioactivity of each well was counted in a gamma-counter.

In the inhibition experiments different concentrations of polyHA, monomeric HA, and of a rabbit anti-HBsAg (DAKO 35 Corporation, St. Barbara, CA) were added to the microwells before incubating with the monoclonal anti-id.

6.1.4. ELISA Monoclonal anti-id 63.14 and 70.F9 were conjugated with alkaline phosphatase (type VII-T, Sigma Chemical Corporation, St. Louis, MO) according to Voller et al. (1976, Bull. World Health Organ. 53: 55-65). The wells of a microtiter plate were coated with HBsAg (10 micrograms/ml) , by incubating overnight at 4^βC. Supernatants obtained from the untransfected 3T3 fibroblasts served as control. After blocking with 3% BSA in PBS, wells were incubated with 50 ul of alkaline phosphatase-conjugated anti-id (10 ug/ml) for 1 hour at 37^βC. After washing with PBS-Tween, wells were incubated with the phosphatase substrate (1 mg/ml Sigma substrate in 0.05 M carbonate buffer, pH 9.5, containing 2 mM MgCl_) . The optical density of each well was determined at a wavelength of 405 nm using an ELISA microreader (MR600,

Dynatech Laboratories Inc., Alexandria, VI). PolyHA, monomeric HA and the monoclonal anti-HBsAg H25B10 (American

Cell Type Culture Collection, Rockville, MD) were used as blocking agents.

The affinity of 63.14 and 70.F9 was calculated following the methods of R. Muller (1983, in Methods in

Enzymology, vol. 92, Colowick, S.P. and Kaplan, N.D. (eds.),

Longone, J.J. and Vunakis, H.V. , pp. 589-601), on the basis of a competitive ELISA. In these experiments alkaline phosphatase-conjugated 63.14 and 70.F9 were reacted with

HBsAg after preincubation with different dilutions of the unconjugated anti-id. The antibodies affinity constant (k) was calculated from the molar concentration of the inhibitor giving 50% inhibition, the molar concentration of the tracer and the amount of tracer bound in the absence of the inhibitor. 6.1.5. WESTERN BLOT Purified HBsAg, obtained from the 4.10 cell supernatant and from the serum of a patient with acute hepatitis, were electrophoresed on a 12% SDS-polyaery1amide gel, overnight 5 at 10 A. The proteins were then electrophoretically transferred to a nitrocellulose filter for 3 hours at 60 V. The filter was then blocked with 5% BSA in 1 mM Tris-HCl buffer, pH 7.6, and incubated with the monoclonal anti-id overnight at 4°C. 0 After washing with Tris-Tween, the filter was incubated with horseradish peroxidase-σonjugated goat anti-mouse IgG (BioRad Laboratories, Richmond, CA) , washed, and reacted with the peroxidase substrate solution (0.01% 4-chloro-l- napthol in methanol and 0.02% hydrogen peroxide). The 1 monoclonal anti-HBsAg H25B10 and cellular extract obtained from 3T3 fibroblasts served, respectively, as positive and negative controls.

6.1.6. CELL STAINING -_ The HBsAg-secreting hepatoma cell line PLC/PRF/5, the HBV-transfected cell line 4.10, the 3T3 cell line from which it was derived, and the hepatoma cell line Hep-G2 (American Cell Type Culture Collection) were grown on coverslips and fixed in cold acetone for 5 minutes. After washing with PBS, the cells were incubated with the monoclonal anti-id 63.14 or 70.F9, and subsequently with a biotin-conjugated horse anti-mouse IgG (Vector Laboratories, Burlingame, CA) . After washing with PBS, the cells were incubated with the avidin-biotin-peroxidase complex (ABC, Vector Laboratories, Burlingame, CA) . The peroxidase reaction was then developed using diaminobenzidine and hydrogen peroxide as substrate. PolyHA and the monoclonal antibody H25B10 served as blocking agents. H25B10 and the monoclonal anti-id MOPC 173 served respectively as positive and negative controls. 5 6.1.7. RADIOIMMUNOPRECIPITATION 4.10, PLC/PRF/5, Hep-G2 cell lines, as well as the 3T3 fibroblasts and human fetal liver cells were internally radiolabeled with [ 35S]methionme, 250 mCi/ml=9.25 GBq, overnight, and then lysed using RIPA buffer (10 mM Tris-HCl, pH 7.4, 150 mM NaCl, 20 mM EDTA, 0.02% NaN , 1% Nonidet- P40) . Aliquots of the cellular extract (10 6-107 cpm) were incubated with 50 μl of 63.14, H25B10 and, MOPC 173 ascites, previously bound to anti-mouse IgG covalently linked to agarose beads (Sigma Chemical Co., St. Louis, MO). After centrifugation, the beads were washed extensively with RIPA buffer incubated in electrophoresis sample buffer (0.5 M Tris-HCl, pH 6.8, 10% SDS, 2% 2-mercaptoethanol, 10% glycerol) and boiled for 5 minutes. Immunoprecipitates were then electrophoresed on a 12% SDS-polyacrylamide gel as described above. The gel was then dried and exposed to a X-Omat AR film (Eastman-Kodak, Rochester, NY) , for about a week.

6.2. RESULTS

6.2.1. MONOCLONAL ANTI-ID BIND THE IDIOTYPE AND HBsAg

Balb/c mice were immunized with purified human IgG specific for human polymeric albumin in order to induce an anti-idiotypic response which would bear the internal image of polyHA and mimic its binding activity as described.

Fusions were done using spleen cells derived from mice whose sera were positive in the RIA assay. Six clones derived from these fusions subsequently showed positive reactivity in this assay; four of these hybrido as were then expanded, subcloned and further analyzed for their ability to bind the

(Fab')₂ portion of the IgG, which was used as the immunogen in a RIA. The supernatants were simultaneously tested for their ability to bind HBsAg as well as a panel of eleven different human myeloma proteins which served as controls. Two of the monoclonal anti-id, 63.14 and 70.F9, reacted specifically with both the idiotype and HBsAg, while 63.7 and 28.A reacted weakly with the (Fab')₂ of the immunogen and poorly or not at all with HBsAg (see Table I) .

TABLE I

Binding of Monoclonal Anti-id 70.F9, 63.14, 63.7 and 28.A to the (Fab')-, of Human IgG Anti-polyHA, and to HBsAg as Compared to Different Human Myeloma Immunoglobulins

Human

Protein Radioactive Counts Bound to the Human Protein

70.F9 63.14 63.7 28.A MOPC173

IgGlk 2158+263 697+512 1392+116 1228+109 1979+108

IgG2k 2501+290 2800+562 1980+249 1100+710 1600+227

IgG21 1595+269 1875+1082 2101+342 838+140 1469+140

IgG3k 1910+266 1892+749 2100+890 1920+670 1336+837

IgG31 1023+392 1012+533 1010+123 990+345 1634+919

IgG4k 1808+227 2400+1087 1806+389 1341+902 1028+497

IgMk 1724+832 2240+826 1802+120 1512+742 ^• 1540+112

IgMl 861+390 1665+891 2002+423 2004+788 1096+595

IgAk 1475+243 1901+794 1671+802 1980+816 1968+71

HBsAg 14147+1275 6487+832 2500+280 980+201 1288+35

(Fab') 9598+382 9360+762 3010+400 4169+1021 719+43 anti-po3 .yHA

Binding was assayed in a solid phase RIA. The human proteins tested were immobilized in sample wells which were incubated with one of the mouse anti-ids indicated. Binding of₅the anti-id was determined by a second incubation with I- radiolabled anti-mouse antibody. The plates were washed and counted in a gamma-counter. Ascites derived from the mouse myeloma cell line MOPC 173 was used as a negative control. Results are expressed as mean and standard deviation of triplicates. 6.2.2. POLY-HA INHIBITS BINDING

ACTIVITY OF 63.14 AND 70.F9

The ability of polyHA to inhibit the binding of the anti-id to the idiotype and HBsAg was tested. Monoclonal antibodies 63.14 and 70.F9 were used in a competitive inhibition assay in which polyHA, monomeric HA and a polyclonal goat anti-HBsAg were used as competing agents. T results of the RIA demonstrated that only polyHA but not monomeric HA nor the goat anti-HBsAg blocks the binding of t monoclonal anti-id to both the (Fab')_ and HBsAg. These dat

10 were confirmed in a direct ELISA in which the purified proteins of hybridomas 63.14 and 70.F9 were conjugated with alkaline phosphatase and reacted with HBsAg. However in bot RIA and ELISA the anti-HBsAg antibodies partially block the binding of 63.14 and 70.F9 to HBsAg. A competitive ELISA wa

15 also used to evaluate the affinity of 63.14 and 70.F9, which is shown in Table II.

TABLE II

20 AFFINITY K VALUE OF 63.14 AND 70.F9, AS MEASURED IN A COMPETITIVE ELISA

Antigen Inhibitor Tracer [It] [Tt] K(l/M)

HBsAg 63.14 63.14 9.4xl0~⁸ 3.9xl0~⁸ g

254.8x10

HBsAg 70.F9 70.F9 3.8xlθ^"8 6.5xl0~⁸

9.5X10⁸

[It] = concentration of inhibitor at 50% inhibition; [Tt] = concentration of tracer.

30

6.2.3. MONOCLONAL ANTI-ID RECOGNIZE THE MAJOR PEPTIDE OF HBsAg

In order to examine the specificity of the binding of th monoclonal anti-id to the constituent peptides of HBsAg, a

35 western blot analysis was performed of the binding of monoclonal anti-id 63.14 and 70.F9 to purified HBsAg obtained from the serum of a patient with acute hepatitis. Normal human serum served as controls. The following peptides were separated on electrophoretic gels and transferred to nitrocellulose filters: a) H25B10 + HBsAg; b) 70.F9 + HBsAg; c) MOPC173 + HBsAg; d) 63.14 + Normal human serum; e) 63.14 + HBsAg. As molecular weight markers we used the following proteins: bovine albumin, ovalbumin, glyceraldehyde-3- phosphate dehydrogenase, carbonic anhydrase, trypsinogen, trypsin inhibitor, alpha-lactalbumin (Sigma Chemical Co., St. Louis, MO) .

Human serum negative for HBsAg served as a negative control for the peptide analysis while the monoclonal anti- HBsAg H25B10, and anti-id MOPC173 were used as positive and negative controls for the anti-id 70.F9 and 63.14. The results demonstrated that both anti-id reacted with the major peptides of HBsAg. The most intense staining occurred with the 24 and 29 kd bands, but 63.14 and 70.F9 also bound larger peptides known to be minor components of the HBsAg particle.

6.2.4. CELLULAR STAINING

Since it was postulated that binding sites for polyHA were expressed not only on HBV, but concomitantly on hepatocytes, we tested the anti-id for their ability to stain different liver cell preparations. Anti-id 63.14 and 70.F9 were able to stain the surface and cytoplasm of the HBsAg- secreting cell line PLC/PRF/5, using immunohistochemical and immunofluorescent techniques. However, when we stained norma liver sections and the HBsAg-negative hepatoma cell line Hep

G2 there was no reactivity. This suggested that the earlier staining had been due to the presence of HBsAg on the infecte

PLC cells. In order to test this, we used the HBV-transfecte

4.10 cells which expressed HBsAg, as well as the 3T3 fibroblasts from which it had been derived. Both anti-id stained only the 4.10 cells with a pattern similar to that o the monoclonal antibody H25B10, which served as a control. Moreover, the staining was specifically inhibited by polyHA and partially by H25B10. These data were confirmed by the results of radioimmunoprecipitation experiments, which showe that 63.14 recognizes proteins extracted from radiolabeled 4.10 and PLC/PRF/5 cells but not from the other cell lines. The proteins precipitated by the anti-Id corresponded to HBs proteins since they were bound by the monoclonal anti-HBsAg and not by the control antibody, and their molecular weight corresponded to that of purified HBsAg proteins.

7. EXAMPLE: HEPATITIS B VIRUS VACCINE

The experiments and results described in the subsections below demonstrate that monoclonal antibodies made in accordance with the invention i.e. , without the use of HBV, mimic HbsAg in as much as they bind to monoclonal and polyclonal anti-HBsAg. The ability of these anti-anti-Id to bind polyHSA further confirms their mimicry of HBsAg, and the existence on the viral antigen of a binding site for polyHA.

When tested ij vivo, one of these anti-anti-ids, Gil, was as effective as HBsAG in inducing an active anti-HBsAg immune response. Sera obtained from rabbits immunized with Gil and

HBsAg appeared to be specific for the common "a" determinant of HBsAg, since they reacted equally well against two different antigen subtypes and precipitated the 24-kD a protein of the viral particles. These results demonstrate that without the use of a viral immunogen, a syngeneic monoclonal anti-anti-Id was produced which mimics HBsAg and i as effective as viral antigens in eliciting a specific immune response in vivo. 7.1. MATERIALS AND METHODS

7.1.1. IMMUNIZATION AND FUSION PROCEDURES Monoclonal anti-Id 63.14 was purified by protein A affinity chromatography and injected intraperitoneally in BALB/c mice at a dose of lOOμg in complete and subsequently incomplete Freund's adjuvant. A week after the second boost mice were bled and their sera tested by ELISA for the ability to inhibit the binding of 63.14 to HBsAg. After the third boost, spleen cells derived from positive mice were fused wit the mouse myeloma cell line SP2/0-AZ-14, using 44% polyethylene glycol (Baker Chemical Co., Phillipsburg, NJ) . The fused cells were then grown in HAT medium for 2 weeks, an hybridomas were tested in ELISA using the method described fo the mice sera. Positive hybridomas were expanded and subcloned by limiting dilution.

7.1.2. ELISA The ability of hybridoma supernatants to inhibit the binding of 63.14, monoclonal anti-HBsAg H25B10, and polyHA to HBsAg was tested in a direct ELISA. HBsAg obtained from the supernatant of the HBV-transfected cell line 4.10 (Christman et al., 1982, Proc. Natl. Acad. Sci. (USA) 79:1815), was purified on a cesium chloride gradient and adjusted to a concentration of 1 μg/ml in 0.05 M sodium carbonate buffer, p 9.6. The antigen as well as normal human serum were then use to coat the wells of a microtiter plate by incubating overnight at 4^βC. The wells were then blocked with 3% bovine serum albumin in 0.02 M Tris, pH 7.6, and incubated with a 1:200 dilution of alkaline phosphatase-conjugated 63.14, HB25B10 (American Cell Type Culture Collection, Rockville, MD or polyHA. These molecules were conjugated with alkaline phosphatase (type VII-T, Sigma Chemical Co., St. Louis, MO) according to Voller et al. (1976, Bull WHO 53:55). For inhibition experiments 63.14, HB25B10 and polyHA were preincubated or coincubated with an aliquot of hybridomas supernatant. Supernatant derived from the Sp2/0 cells as we as AIIE6 (hybridoma with anti-angiotensin II receptor activity) served as controls. After a 1 hour incubation at 37°C, the plates were washed three times with Tris containin 0.05% Tween-20 (Tris-Tween) , and incubated with the alkaline phosphatase substrate (5 mg/ml p-nitrophenyl phosphate in 0. M sodium carbonate buffer, pH 9.6, and 0.2 M MgCl₂) for abou 30 minutes. The wells were then scanned at 405 n with an ELISA microreader (Dynatech Laboratories, Alexandria, VA) . Results were expressed as mean and standard deviation of duplicates.

To test the ability of monoclonal anti-HBsAg H25B10 and polyHA to bind the anti-anti-Id, we coated the wells of a microtiter plate with the hybridoma supernatants diluted 1:1 with sodium carbonate buffer. These were then incubated wit

1:200 diluton of alkaline phoshatase-conjugated H25B10 and polyHA (10 μg/ml) . The blocking, washing and color development procedures were the same as described above. A similar procedure was used to assess the ability of polyclona goat, human and mouse anti-HBsAg to react with anti-anti-Id- expressing hybridomas. In these experiments, alkaline phosphatase conjugated rabbit anti-goat IgG (Kirkegaard and

Perry, Gaithersburg, MD) , goat anti-human and anti-mouse IgG

(Sigma, St. Louis, MO) were used as detecting reagents. In the inhibition experiments the polyclonal anti-HBsAg were preincubated with different concentrations of anti-anti-Id.

An ELISA procedure was used to evaluate the presence of antibodies to HBsAg in the rabbit serum. Serum obtained from rabbits injected with anti-anti-Id and HBsAg, as well as from nonimmune rabbits were tested against HBsAg using peroxidase- conjugated protein A (Boehringer Mannheim, Indianopolis, IN) , and 1 mg/ml 4-chloro-l-naphthol and 0.2% hydrogen peroxide as substrate. In these experiments we used two different sources of HBsAg: 'the-4.10. cell line (ayr) and the serum of an infected patient (adw) .

7.1.3. RABBIT IMMUNIZATION PROCEDURE Rabbits were immunized with anti-anti-Id Gil, previously purified by affinity chromatography on a protein A-Sepharose column, and with HBsAg. Gil and HBsAg (0.5 mg) , dissolved in complete and subsequently in incomplete Freund's adjuvant, were injected subcutaneously in the neck and footpads with an 0 interval of 4 weeks between the first and second boost. Seru collected after the second injection was tested for the presence of anti-HBsAg using the ELISA described above.

7.1.4. RADIOIMMUNOPRECIPITATION 1 125I-labeled HBsAg (Travenol-Genentech Diagnostics,

Cambridge, MA; 10 cpm) was incubated with 50 μl of immune an nonimmune rabbit serum previously bound to anti-rabbit IgG linked to agarose beads (Sigma, St. Louis, MO) for 45 min on ice. The beads were then pelleted by centrifugation, washed extensively with RIPA buffer (10 mM Tris-HCl, pH 7.4, 150 mM

NaCl, 20 mM EDTA, 0.02% NaN_, 1% Nonidet-P40) , resuspended in electrophoresis sample buffer (0.5 M Tris-HCl, pH 6.8, 10% sodium dodecyl sulfate (SDS) , 2% 2-mercaptoethanol, 10%

Glycerol) and boiled for 5 minutes. Immunoprecipitates were

__ electrophoresed on a 12% SDS-polyacrylamide gel. The gel was 2b then dried and exposed to a X-Omat AR film (Eastman-Kodak, Rochester, NY) for about a week. To confirm the specificity of the radioimmunoprecipitation we performed in parallel inhibition experiments using cold HBsAg and human IgG (Sigma, St. Louis, MO) as competing agents.

35 7.2. RESULTS: SYNGENEIC MONOCLONAL ANTI-ANTI IDIOTYPES WHICH MIMIC HBsAg IN THE INDUCTION OF IMMUNE RESPONSIVENESS

The results described below demonstrate that nine hybridomas obtained from spleen cells of BALB/c mice immuniz with 63.14 were isolated, which were_^able to inhibit the binding of alkaline phosphatase-conjugated 63.14 to HBsAg. Both direct and competition ELISA showed that 4 of these clones were able to mimic HBsAg since they reacted with poly and inhibited the binding of monclonal and polyclonal anti- HBsAg to the viral antigen. To determine whether these anti anti-Id could induce an immune response against HBsAg in viv we injected a series of rabbits with anti-anti-Id Gil or HBs and tested their sera after the second boost. ELISA, radioimmunoassay and Western blot experiments showed that Gil was effective as HBsAg in inducing a specific anti-HBsAg immune response. These data indicate that our anti-anti-Id can mimic HBsAg both in vitro and ^n vivo and may be useful a alternative vaccine for HBV infection.

7.2.1. PREPARATION OF SYNGENEIC MONOCLONAL ANTI-ANTI-ID Mouse monoclonal anti-Id 63.14, which mimics polyHA and binds HBsAg described in Section 6 et seq. , su ra, was used t immunize BALB/c mice and produce anti-anti-Id which bear the internal image of HBsAg. Spleen cells obtained from mice expressing circulatig anti-63.14 were fused with the mouse myeloma cell line SP2/0-Az-14. Nine out of 28 hybridomas wer able to inhibit the binding of alkaline phosphatase-conjugate 63.14 to HBsAg to the same extent as the viral antigen (Table III) . TABLE III

INHIBITION OF McAB 63.14 BINDING TO HBsAg BY ANTI-ANTI-IDIOTYPIC ANTIBODIES

Antigen Inhibitor 63.14^a

HBsAg Cll.1 0. ,09 + 0.01

HBsAg D8.1 0. ,08 + 0.01

HBsAg G10.1 0, ,10 + 0.02

HBsAg C8.1 0. .09 + 0.02

HBsAg G3.2 0. .06 + 0.01

HBsAg F7.1 0. ,08 + 0.01

HBsAg Gil.1 0. .10 + 0.02

HBsAg D2.2 0. .08 + 0.01

HBsAg E7.1 0. ,09 + 0.02

HBsAg AIIE6 0. ,99 + 0.44 HBsAg HBsAg 0. .07 + 0.01

a Anti-anti-id supernatants were coinσubated with McAb 63.14 in wells previously coated with HBsAg. Results are expressed as mean + SD (A 405 nm) of duplicates.

7.2.2. MONOCLONAL ANTI-ANTI-ID MIMIC HBsAG In Vitro

To determine whether these hybridomas were indeed able t mimic HBsAg, we tested them for the ability to bind a monoclonal anti-HBsAg (H25B10) , as well as polyHA in ELISA.

As shown in Table IV, 4 hybridomas strongly reacted with both H25B10 and polyHA, and inhibited the binding of the latter to HBsAg. The reactivity of these hybridomas was then tested against polyclonal anti-HBsAg derived from different species to ensure that their ability to mimic the viral antigen was not genetically restricted. Anti-anti-Id Gil, which was selected for further analysis, was able to react with goat, human and mouse antiserum to HBsAg, and specifically inhibite their binding to the viral antigen. TABLE IV

BINDING OF ANTI-HBsAg (H25B10) AND PolyHA TO ANTI-ANTI-ID) SUPERNATANTS AS MEASURED BY ELISA

Anti-Anti-Id H25B10^a PolyHA³ b

Cll.l 0.28 + 0.01 0.15 + 0.02

D8.1 0.25 + 0.04 0.14 + 0.00

G10.1 0.33 + 0.04 0.18 + 0.03

C8.1 0.47 + 0.23 0.22 + 0.02

G3.2 1.24 + 0.04 0.56 + 0.09 F7.1 1.65 + 0.10 0.65 + 0.07

Gll.l 0.97 + 0.15 0.90 + 0.09

D2.2 0.53 + 0.14 0.31 + 0.07

E7.1 0.61 + 0.21 0.40 + 0.04

⁵ AIIE6 0.14 + 0.08 0.04 + 0.01

HBsAg 1.21 + 0.10 0.81 + 0.06

Enzyme-conjugated H25B10 and polyHA were reacted with anti-anti-Id supernatants previously bound to the wells o a plastic 96-well plate. Results are expressed as mean + SD (A 405 nm) of duplicates.

7.3.3. MONOCLONAL ANTI-ANTI-ID INDUCE

AN ANTI-HBsAq IMMUNE RESPONSE IN VIVO

Anti-anti-Id, Gil was used in studies testing its abilit to induce an immune response against HBsAg in vivo. In these experiments rabbits were used as the hosts and were injected with 0.5 mg of Gil or HBsAg in complete Freund's adjuvant and boosted subcutaneously 1 month later. After the second immunization the rabbits' sera were tested in ELISA against two different preparations of HBsAg (adw, ayr) . Serum obtained from rabbits injected with anti-anti-Id was found to express anti-HBsAg activity, with a titer almost as high as the showed by rabbits immunized with HBsAg. Similat reactivity and titer were also detected using a commercial radioimmunoassay (AUSRIA, Abbott Diagnostics, North Chicago, IL) . The specificity of these sera was then confirmed by the results of a radioimmunoprecipitation analysis, where they showed to react with the major 24-kDa protein of radiolabeled serum-derived HBsAg particles. To rule out the possibility that these sera could cross-react with human immunoglobulin light chains, contained in the radiolabled HBsAg preparation and co-migrating with the HBsAg peptide, we tested the viral particles for the presence of human immunoglobulin heavy and light chains using specific polyclonal antibodies (Sigma, St. Louis, MO) and ELISA and immunoelectrophoresis techniques. N reactivity was observed in these assays between the rabbits sera and antihuman heavy and light chains, but only between the latter and a human IgG which served as control. Moreover binding and precipitation of the 24-kDa HBsAg protein by the rabbits sera was inhibited by cold HBsAg (1 μg/ml) particles but not by human IgG (100 μg/sΩ.) .

The present invention is not to be limited in scope by the embodiment disclosed in the examples which is intended as a single illustration of one aspect of the invention, and any functionally equivalent products or processes are within the scope of this invention. Indeed, various modifications of th invention in addition to those shown and described herein wil become apparent to those skilled in the art from foregoing description. Such modifications are intended to fall within the scope of the appended claims.

Claims

WHAT IS CLAIMED IS:

1. A method for the production of a vaccine formulatio that provides for active immunization against a pathogen, comprising:

(a) generating monoclonal antibodies against a ligand that binds to a receptor of the pathogen;

(b) selecting a monoclonal antibody whose idiotype mimics the conformational structur of the receptor of the pathogen; and

(c) mixing the selected monoclonal antibody in pharmaceutical carrier at a dose sufficient to elicit an active immune response.

2. The method of claim 1 in which the ligand comprises neutralizing antibody obtained from an animal or human expos to the pathogen, and the selected monoclonal antibody comprises an anti-idiotype that mimics the conformational structure of the receptor of the pathogen.

3. The method of claim 1 or 2 in which the monoclonal antibody is selected based upon its ability to competitively inhibit the binding of the pathogen to the ligand.

4. The method of claim 1 or 2 in which the selected monoclonal antibody further comprises an Fv, Fab, F(ab') , or Fab' portion of the antibody.

5. The method of claim 1 or 2 in which the pathogen comprises a microorganism.

6. The method of claim 1 or 2 in which the pathogen comprises a bacterium.

7. The method of claim 1 or 2 in which the pathogen comprises a parasite.

8. The method of claim 1 or 2 in which the pathogen comprises a virus.

9. The method of claim 8 in which the virus comprises Hepatitis B Virus.

10. The method of claim 9 in which the ligand comprises polymeric human albumin.

11. A method for the production of a vaccine formulatio that provides for active immunization against a pathogen, comprising:

(a) generating an anti-idiotypic antibody agains a first antibody that defines a ligand which binds to a receptor of the pathogen;

(b) selecting the anti-idiotypic antibody whose idiotype mimics the conformational structure of the ligand;

(c) generating an anti-anti-idiotypic antibody against the selected anti-idiotypic antibody;

(d) selecting the anti-anti-idiotypic antibody whose idiotype mimics the conformational structure of the receptor of the pathogen; and

(e) mixing the selected anti-anti-idiotypic antibody in a pharmaceutical carrier at a dose sufficient to elicit an active immune response.

12. The method of claim 11 in which the anti-idiotypic antibody further comprises an Fv, Fab, F(ab')- or Fab' portio of the antibody.

13. The method of claim 11 in which the pathogen comprises a microorganism.

14. The method of claim 11 in which the pathogen comprises a bacterium.

15. The method of claim 11 in which the pathogen comprises a parasite.

16. The mehthod of claim 11 in which the pathogen comprises a virus.

17. The method of claim 16 in which the virus comprise Hepatitis B Virus.

18. The method of claim 17 in which the ligand compris polymeric human albumin.

19. A method for the prouction of a vaccine formulatio that provides for passive immunization against a pathogen, comprising:

(a) generating an anti-idiotypic antibody again an antibody that defines a ligand which bin to a receptor of the pathogen;

(b) selecting the anti-idiotype antibody whose idiotype mimics the conformational structur of the ligand; and

(c) mixing the selected anti-idiotypic antibody in a pharmaceutical carrier at a dose sufficient to confer passive immunity agains the pathogen.

20. The method according to claim 19 in which the anti- idiotypic antibody is selected based upon its ability to competitively inhibit the binding of the ligand to the pathogen.

21. The method of claim 19 in which the anti-idiotypic antibody further comprises an Fv, Fab, F(ab')₂, or Fab' portion of the antibody.

22. The method of claim 19 in which the pathogen comprises a microorganism.

23. The method of claim 19 in which the pathogen comprises a bacterium.

24. The method of claim 19 in which the pathogen comprises a parasite.

25. The method of claim 19 in which the pathogen comprises a virus.

26. The method of claim 25 in which the virus comprises Hepatitis B virus.

27. The method of claim 26 in which the anti-idiotypic antibody bears the conformational structure of polymeric huma albumin.