WO2000021568A1

WO2000021568A1 - Fibrin glue as a biological adjuvant

Info

Publication number: WO2000021568A1
Application number: PCT/AU1999/000869
Authority: WO
Inventors: Jerry Kanellos; Teresa Marion Martinelli; Grace Demaria; Neil Goss
Original assignee: Csl Limited
Priority date: 1998-10-12
Filing date: 1999-10-11
Publication date: 2000-04-20
Also published as: AUPP646798A0

Abstract

The present invention relates to compositions for enhancing the immune response to one or more antigenic determinants in a host which comprise the antigenic determinant in admixture with fibrin and/or fibrinogen or a derivative or metabolite thereof; and/or one or more catalyst(s) of fibrin glue formation. The present invention also relates to methods for enhancing an immune response to an antigenic determinant comprising administering the antigenic determinant to a host simultaneously with fibrin and/or fibrinogen such that a fibrin glue matrix is formed at the site of administration.

Description

Fibrin glue as a biological adjuvant

Field of the Invention

The present invention relates to a composition and method for enhancing an immune response to an antigenic determinant.

Background of the Invention

Immunologic adjuvants have been used to augment the immune response to antigens since 1925 when Ramon first demonstrated the ability to specifically increase the antitoxin response to tetanus and diphtheria toxin (Ramon, 1925). With the addition of tapioca, metal salts, oil and pyogenic bacteria to the vaccine, he produced levels of immunity greater than those generated by the vaccine toxoids when injected alone.

Over the years, adjuvants have received much attention. This is due to the development of purified, subunit and synthetic vaccines which are themselves poor immunogens and thus require the help of adjuvants to evoke the immune response. Adjuvants are also used in conventional vaccines to elicit an early, high and long-lasting immune response. Furthermore when used, less antigen is needed to evoke the immune response and thus reduces vaccine production costs. An immunologic adjuvant can thus be defined as any substance which when incorporated into a vaccine formulation acts generally to accelerate, prolong, or enhance the quality of specific immune responses to vaccine antigens (Nogel, 1995). By manipulating different adjuvants and vaccine preparations, humoral and cellular-mediated immunity (HI and Civil) and antibody class and subclass can be selected for in some instances.

The potential advantages gained by using vaccine adjuvants include:

• enhancement of immunogenicity of weaker immunogens

• reduction of the amount of antigen or the frequency of booster immunisations needed to provide adequate protection • improvement of efficacy for vaccines used in newborns (Baker et al.,

1988), the aged ( bawuike et al., 1990), and in immunocompromised individuals (Hibbard et al., 1989)

• promotion of T cell proliferation and cell-mediated immunity (Grun et al., 1989) particularly stimulation of major histocompatability complex (MHC) class I-restricted CD8⁺ cytotoxic T lymphocyte

(CTL) responses. Adjuvants have diverse mechanisms of action and must be chosen for use with a particular antigen based on the immune responses required for that antigen. Summarised by Chedid (1985), the various modes of adjuvant action include: • the formation of a depot of antigen at the site of inoculation which is slowly released over time

• the presentation of antigen to immunocompetent cells, and

• the production of different lymphokines such as interleukins and tumour necrosis factor (T F). Immunologic adjuvant can be classified by their mechanisms of action and physical or chemical properties. There are five categories of adjuvants currently undergoing development and testing for use with human vaccines. These include:

• Gel-type adjuvants - Alum (aluminium hydroxide/phosphate) • Calcium phosphate

• Bacterial adjuvants - Muramyl dipeptides (MDPs)

Monophosphoryl Lipid A (MPL)

B. pertussis

C. tetanii Lipopolysaccharide (LPS)

• Particulate adjuvants - Immunostimulating complexes

(ISCOMS™)

Liposomes

Biodegradable microspheres • Oil emulsion and emulsifier-based adjuvants -

Incomplete Freund's Adjuvant (IF A) Freund's Complete Adjuvant (FCA) Syntex Adjuvant Formation (SAF) Saponins Squalene

• Synthetic adjuvants - Nonionic block copolymers

Muramyl peptide analogues Synthetic lipid A Synthetic polynucleotide The most common adjuvants for human use today are aluminium hydroxide and aluminium phosphate. Calcium phosphate and oil emulsions have also been used in some human vaccinations.

Apart from the intended stimulation of the immune response, adjuvants may also result in some unwanted side effects such as local inflammation or the induction of granulomas (Gupta et al., 1993). In recent years, there has been a search to find adjuvants with the ability to potentiate the immune response with minimal or no side effects. Edelman (1980), has listed a number of criteria to ensure the safety of adjuvanted vaccines. In addition to safety regarding local and systemic reactions, autoimmune diseases, hypersensitivity reactions, carcinogenicity and teratogenicity etc., an ideal vaccine adjuvant would be chemically defined so that it can be manufactured consistently. The preparation would elicit a protective immune response when combined with weakly immunogenic antigen, be effective in infants and young children, elicit a more persistent response of high quality, be stable with regard to adjuvanticity and toxicity and demonstrate no interaction with the antigen. Finally, it would be biodegradable and non-immunogenic by itself. An adjuvant which meets most or all of these objectives is therefore highly desirable. Fibrin glue is used in various surgical procedures to either supplement or replace the use of sutures. It consists of two protein components, fibrinogen and thrombin which, when mixed together in the presence of calcium chloride mimic the final stages of coagulation to form a clot. When applied to a wound which is bleeding diffusely, the fibrin clot acts as a haemostatic agent to stem the flow of blood.

Ideally, the fibrinogen concentrate should contain factor XIII whose concentration governs the elasticity of the clot and may also contain an antifibrinolytic agent which serves to inhibit fibrinolysis of the clot.

The fibrin clot is formed when fibrinogen is proteolytically cleaved and converted to a fibrin monomer. Factor XIII is also activated by thrombin in the presence of calcium ions and converts fibrin monomers into fibrin polymers. These fibrin polymers then cross link to form a fibrin glue clot. The action of Factor XIII on the clot structure has also been shown to delay clot lysis and increase clot stability. The rate at which the clot forms is dependent upon the concentration of thrombin mixed with fibrinogen. Being an enzyme dependent reaction, the higher the temperature (up to 37°C) the faster the clot formation rate. The tensile strength of the clot is dependent upon the concentration of fibrinogen used.

Recent publications describe the use of fibrin glue for the delivery of therapeutic agents. For example, U.S. Patent 4,983,393 discloses a composition for use as an intra-vaginal insert comprising agarose, agar, saline solution glycosaminoglycans, collagen, fibrin and an enzyme. Further, U.S. Patent 3,089,815 discloses an injectable pharmaceutical preparation composed of fibrinogen and thrombin.

Summary of the Invention

The present inventors have now found that antigenic determinants which are poor immunogens, or which are substantially non-immunogenic, may be incorporated into a fibrin glue matrix which in turn acts as an adjuvant to evoke an immune response to the antigen. This finding is contrary to the teachings of the prior art that fibrin glue may be used for the delivery and slow release of a medicinal compound where the inducement of an immune response to the medicinal compound is to be avoided.

The present invention therefore provides a new adjuvant, fibrin glue, which has significant advantages over other known adjuvants. In particular, fibrin glue is produced by a naturally occurring process and is biodegradable and toxicity free. Furthermore, the fibrin glue matrix may be manipulated by changing the clot matrix structure and thus increasing the immunogenicity of the antigen. When used herein the term "fibrin glue" refers to the insoluble matrix formed by the cross-linking of fibrin polymers in the presence of calcium ions. The fibrin glue may be formed from fibrinogen, or a derivative or metabolite thereof, fibrin (soluble monomers or polymers) and/or complexes thereof derived from biological tissue or fluid which forms a fibrin matrix. Alternatively, the fibrin glue may be formed from fibrinogen, or a derivative or metabolite thereof, or fibrin, produced by recombinant DNA technology.

The fibrin glue may also be formed by the interaction of fibrinogen and a catalyst of fibrin glue formation (such as thrombin and/or Factor XIII). As will be appreciated by those skilled in the art, fibrinogen is proteolytically cleaved in the presence of a catalyst (such as thrombin) and converted to a fibrin monomer. The fibrin monomers may then form polymers which may cross-link to form a fibrin glue matrix. The cross-linking of fibrin polymers may be enhanced by the presence of a catalyst such as Factor XTH. The catalyst of fibrin glue formation may be derived from blood plasma, cryoprecipitate or other plasma fractions containing fibrinogen or thrombin. Alternatively, the catalyst may be produced by recombinant DNA technology. The antigenic determinant of interest may be administered to a site in the form of a composition comprising the compound, fibrin and/or fibrinogen or a derivative or metabolite thereof and optionally a catalyst of fibrin glue formation. Alternatively, the antigen may be administered to a site in the form of a composition comprising the compound and one or more catalysts of fibrin formation. The fibrin glue matrix may then be formed by simultaneously administering fibrin and/or fibrinogen or a derivative or metabolite thereof to the site. Accordingly, in a first aspect the present invention provides a composition for enhancing the immune response to one or more antigenic determinants in a host which includes the antigenic determinant in admixture with

(i) fibrin and/or fibrinogen or a derivative or metabolite thereof; and

(ii) optionally one or more catalyst(s) of fibrin glue formation. In a second aspect the present invention provides a composition for enhancing the immune response to one or more antigenic determinants in a host which includes the antigenic determinant in admixture with one or more catalyst(s) of fibrin glue formation.

A composition of present invention may include fibrin in the form of soluble fibrin monomers or polymers. These fibrin monomers or polymers are capable of forming a fibrin glue matrix after administration of the composition to the host. The rate at which the fibrin glue matrix forms may be controlled, for example, by the adjusting the level of catalyst(s) and/or the number of catalysts included in the composition.

Alternatively, the fibrin in the composition may be in the form of a fibrin glue matrix. In a preferred embodiment, the fibrin glue matrix is encapsulated within a biocompatible coating. The encapsulated fibrin glue matrix is preferably in a form which is suitable for solid dose delivery of the composition to biological tissues such as mucosal, dermal, ocular, subcutaneous, intradermal or pulmonary tissue. The encapsulated matrix may be in the form, for example, of beads, granules, microspheres, threads, cylinders, disks or films. The encapsulated matrix may also be in the from of a transdermal "patch" or an implantable device. Examples of other composition forms which are suitable for solid dose delivery are described in WO 96/03978, the entire contents of which are incorporated herein by reference.

In a preferred embodiment of the present invention, the catalyst(s) of fibrin glue formation is thrombin and/or Factor XIII. In a more preferred embodiment, the catalyst is thrombin.

In a further preferred embodiment of the present invention, the composition further includes Ca⁺⁺ ions or a source of Ca⁺⁺ ions. Suitable sources of calcium ions include a CaCl₂ solution, CaCl₂ finely ground, poorly soluble calcium compounds such as encapsulated calcium CaCO₃ and calcium priopionate.

A wide variety of antigens are suitable for use in compositions of the first and second aspects of the present invention. Preferably, the antigen is poorly immunogenic in the absence of an adjuvant. More preferably, the antigen is substantially non-immunogenic in the absence of an adjuvant. A composition of the present invention may include two or more antigenic determinants. These antigenic determinants may be conjugated together in a single molecule.

Preferred antigenic determinants mimic the immunological stimulus associated with natural infection and include candidate proteins, sub units or peptide fragments or synthetic peptide fragments chemically manufactured or constructed by recombinant technologies selected from the group consisting of viruses, bacterial toxins, parasitic antigens and hormones. Examples include the following: Viruses: antigens for influenza viruses and for several picornaviruses, a group of human and animal pathogens which include poliovirus and foot and mouth disease virus. Other examples include Hepatitis B virus surface antigen, hepatitis A virus determinants, and envelope proteins from HT N-III, LAN and HIV. Bacterial Toxins: antigenic determinants, toxoids, fragments or peptides of cholera toxin, heat liable toxin of E. coli, bacterial diarrhoea, diptheria toxin, petussis antigens, Streptococcus M and Gonococcal pilin. Parasites: antigenic determinants, fragments and peptides of the parasites such as Plasmodium sp., Toxoplasma sp. and Pneumocystis sp. including P. falciparum, P. malariae, P. ovale and P. vivax. Hormones such as human chronic gonadotropin and hypothalamic luteinizing hormone releasing hormone (LHRH), fragments, peptides and peptide conjugates. This list is for example only and is not intended to be exclusive of other candidate antigens not listed above. Examples of suitable antigens are described in Steward and Howard (1987) and Carelli et al (1982), the entire contents of which are incorporated herein by reference.

In further preferred embodiments of the compositions of the present invention, the fibrin, fibrinogen and/or catalyst(s) are derived from the same genus as the host. For example, if the composition is to be administered to a human, it is preferred that the fibrin, fibrinogen and/or catalyst is derived from a human.

In further preferred embodiments of the compositions of the present invention, the compositions also include agents which increase the stability of the fibrin glue matrix. For example, the compositions may include one or more antifibrinolytic agents. The antifibrinolytic agents may be selected from aprotin, ε aminocaproic acid and tranexamic acid [trans-4- (aminomethyl)cyclohexane carboxylic acid], alpha 2 antiplasmin, alpha 2 macroglobulin and alpha 1 antitrypsin. In a further preferred embodiment of the first and second aspects of the present invention, the composition further includes an immunostimulator. The immunostimulator may be selected from a non-toxic derivative of MDP, such as murabutide, interleukins, such as interleukins 1, 2, 4 - 7 and 12, and interferons, especially γ-interferon, levamisole hydrochloride, and colony stimulating factors (CSF) such as granulocyte CSF and granulocyte macrophage CSF.

In further preferred embodiments of the compositions of the present invention, the antigen is conjugated, either chemically or genetically, to the fibrin, fibrinogen (or derivative or metabolite thereof) or one or more catalyst molecules, or a carrier protein.

In a third aspect the present invention provides a kit for enhancing the immune response to one or more antigenic determinants in a host, which includes:

(i) fibrin and/or fibrinogen or a derivative or metabolite thereof; and (ii) the antigenic determinant such that in normal use of the kit, the fibrin and/or fibrinogen or derivative or metabolite thereof, and antigenic determinant are administered simultaneously to the host such that a fibrin glue matrix is formed at the site of administration and the antigenic determinant is incorporated within the matrix.

In a preferred embodiment of the third aspects of the present invention, the kit further includes one or more catalyst(s) of fibrin glue formation. Preferably, the catalyst(s) of fibrin glue formation is thrombin and/or Factor XIII. In a more preferred embodiment, the catalyst is thrombin. In a further preferred embodiment of the third aspect of the present invention, the kit further includes an immunostimulator. The immunostimulator may be selected from a non-toxic derivative of MDP, such as murabutide, interleukins, such as interleukins 1, 2, 4 - 7 and 12, and interferons, especially γ-interferon, levamisole hydrochloride, and colony stimulating factors (CSF) such as granulocyte CSF and granulocyte macrophage CSF.

In further preferred embodiments of the third aspect of the present invention, the fibrin, fibrinogen and/or catalyst(s) are derived from the same genus as the host. For example, if the components of the kit are to be administered to a human, it is preferred that the fibrin, fibrinogen and/or catalyst is derived from a human.

In further preferred embodiments of the third aspect of the present invention, the kit further includes one or more agents which increase the stability of the fibrin glue matrix. For example, the kit may also include one or more antifibrinolytic agents. The antifibrinolytic agent may be selected from aprotin, ε aminocaproic acid and tranexamic acid [trans-4- (aminomethyl)cyclohexane carboxylic acid], alpha 2 antiplasmin, alpha 2 macroglobulin and alpha 1 antitrypsin.

In a further preferred embodiment of the third aspect of the present invention, the kit further includes Ca⁺⁺ ions, or a source of Ca⁺⁺ ions.

In further preferred embodiments of the third aspect of the present invention, the antigenic determinant is conjugated, either chemically or genetically, to the fibrin, fibrinogen (or derivative or metabolite thereof) or one or more catalyst molecules, or a carrier protein. In a fourth aspect the present invention provides a method of enhancing an immune response to one or more antigenic determinants in a host which includes administering the antigenic determinant simultaneously with fibrin and/or fibrinogen or a derivative or metabolite thereof such that a fibrin glue matrix is formed at the site of administration and the antigenic determinant is incorporated into the matrix. In a preferred embodiment of the fourth aspect of the present invention, the method further includes administering one or more catalyst(s) of fibrin glue formation. Preferably, the catalyst of fibrin glue formation is thrombin and/or Factor XIII. In a more preferred embodiment, the catalyst is thrombin. In a further preferred embodiment of the fourth aspect of the present invention, the method further includes administering Ca⁺⁺ ions or a source of Ca⁺⁺ ions.

In a further preferred embodiment of the fourth aspect of the present invention, the method further includes administering one or more agents which increase the stability of the fibrin glue matrix. Agents which increase the stability of the fibrin glue matrix include, for example, antifibrinolytic agents. The antifibrinolytic agents may be selected from aprotin, ε aminocaproic acid and tranexamic acid [trans-4-(aminomethyl)cyclohexane carboxylic acid], alpha 2 antiplasmin, alpha 2 macroglobulin and alpha 1 antitrypsin.

In a fifth aspect the present invention provides a method of enhancing an immune response to one or more antigenic determinants in a host which includes administering to the host a fibrin glue matrix wherein the antigenic determinant is incorporated within the matrix. In a preferred embodiment of the fifth aspect of the present invention, the fibrin glue matrix is encapsulated within a biocompatible coating for solid dose delivery to biological tissue such as mucosal, dermal, ocular, subcutaneous, intradermal or pulmonary tissue.

In a further preferred embodiment of the fourth and fifth aspects of the present invention, the method further includes administering an immunostimulator. The immunostimulator may be selected from a non-toxic derivative of MDP, such as murabutide, interleukins, such as interleukins 1, 2, 4 - 7 and 12, and interferons, especially γ-interferon, levamisole hydrochloride, and colony stimulating factors (CSF) such as granulocyte CSF and granulocyte macrophage CSF. In further preferred embodiments of the fourth and fifth aspects of the present invention, the fibrin, fibrinogen and/or thrombin are derived from the same genus as the host. For example, if the host is a human, it is preferred that the fibrin, fibrinogen and/or thrombin are derived from a human. In a further preferred embodiment of the fourth and fifth aspects of the present invention, the method involves a further "booster" immunisation. The "booster" immunisation may involve administration of the antigen alone or the antigen in combination with fibrin or fibrinogen (or a derivative or metabolite thereof) and/or a catalyst of fibrin glue formation. In further preferred embodiments of the fourth and fifth aspects of the present invention, the antigenic determinant is conjugated, either chemically or genetically, to the fibrin, fibrinogen or catalyst molecules, or a carrier protein.

In preferred embodiments of the fourth aspect of the present invention, the mode of administration is parenteral, eg. by injection, either subcutaneously, or intramuscularly, pulmonary formulations, or transdermal applications. Alternatively, the substances may be administered orally or by suppository. Dosage treatment may be a single dose schedule or a multiple dose schedule. Throughout this specification the word "comprise", or variations such as "comprises" or "comprising", will be understood to imply the inclusion of a stated element, integer or step, or group of elements, integers or steps, but not the exclusion of any other element, integer or step, or group of elements, integers or steps. In order that the nature of the present invention may be more fully understood, preferred forms thereof will be described in the following non- limiting figures and examples.

Brief Description of the Figures Figure 1: Mean titres of anti-BSA antibodies in sera from mice immunised with either fibrin glue alone, BSA alone, BSA + FIA, BSA + fibrin glue, BSA + thrombin or BSA + fibrinogen.

Figure 2: Anti-LHRH geomean titres of groups of mice (post boosting). Figure 3: Anti-DT geomen titres of groups of mice (post boosting). Detailed Description of the Invention

EXAMPLE 1- FIBRIN GLUE AS AN ADJUVANT FOR BSA Materials and Methods Groups of mice were injected with antigen formulated with fibrin glue.

No antigen control mice were injected with fibrin glue not incorporating antigen. Positive adjuvant control mice were injected with antigen emulsified in FIA. No adjuvant control mice were injected with antigen + fibrinogen, antigen + thrombin or antigen alone. Sera from appropriate groups of mice were tested by ELISA for the presence of antibodies to antigen. Reconstitution of thrombin

One vial of thrombin was reconstituted in 2.5ml WFI to give a concentration of 1000 IU/ml. Preparation of BSA for immunisation with either fibrin glue or thrombin alone

BSA (2 mg/ml) was diluted 1:1 with thrombin (1000 IU/ml) to give concentrations of 1 mg/ml BSA and 500 IU/ml thrombin. Preparation of BSA for immunisation without fibrin glue or FIA BSA (2 mg/ml) was diluted 1:3 with WFI to give a final concentration of 500 μg/ml. Preparation of BSA for injection with FIA

BSA (2 mg/ml) was diluted 1:1 with WFI to give a concentration of 1 mg/ml. Preparation of BSA for injection with fibrinogen alone

BSA (2 mg/ml) was diluted 1:1 with WFI to give a concentration of 1 mg/ml. This mixture was then diluted 1:1 with fibrinogen (70 mg/ml) to give final concentrations of 500 μg/ml BSA and 35 mg/ml fibrinogen.

Immunisations

Adult female BALB/c mice were divided into eleven groups (4 mice per group) and immunised according to the protocol in Table 1 as described below. Table 1: Experimental Immunisation protocol

Administration of immunogen in fibrin glue

Two syringes, one containing immunogen diluted in thrombin and the other containing fibrinogen (70 mg/ml), were connected to a dual flow (syringe) adaptor attached to a 23 gauge needle. This ensured delivery of equal volumes of immunogen/thrombin and fibrinogen to the injection site. Approximately 100 μl from each syringe was administered simultaneously by one subcutaneous injection into the ventral flank of each mouse. Upon injection, thrombin cleaved fibrinogen to fibrin and resulted in the formation of a fibrin glue clot containing the immunogen. After injection, mice were left to recover for one minute before returning to cage. Administration of fibrin glue alone

Group 3 mice received fibrin glue without immunogen (no antigen control). Thrombin (1000 IU/ml) was diluted 1:1 with WFI to give a concentration of 500 IU/ml. Thrombin (500 IU/ml) (100 - 150 μl) was injected simultaneously with fibrinogen (70 mg/ml) (100 - 150 μl) using dual flow syringe adaptor system described earlier. The final concentrations of thrombin and fibrinogen injected were 250 IU/ml and 35 mg/ml, respectively. Administration of immunogen in FIA

Immunogen was emulsified with an equal volume of FIA (positive adjuvant control). Two syringes, one containing immunogen and one containing FIA, were connected to a three-way stopcock and mixed until an emulsion was formed. The immunogen/FIA emulsion was then administered subcutaneously, into the ventral flank at two different sites (100 μl per site) using a 26 gauge needle and syringe. Administration of immunogen alone or with either thrombin or fibrinogen

Immunogen (negative adjuvant control) was administered subcutaneously into the ventral flank at two different sites (100 μl per site) using a 26 gauge needle and syringe. Blood Collection

Mice were bled by retro-orbital plexus puncture on days 11, 14, 21, 28 and 60. The blood was allowed to clot at room temperature and incubated at 4°C overnight. Sera was separated and stored at -30°C until testing. Screening of mouse sera by ELISA Sera from mice immunised with BSA (groups 1, 4, 6, 8 and 10) were screened for the presence of antibodies to BSA by ELISA. Mice from group 3 (fibrin glue alone) were also screened for antibodies to BSA by ELISA. Sera from mice injected with fibrin glue were screened by ELISA for the presence of antibodies to thrombin and to fibrinogen. ELISA procedure for screening of Anti-BSA Antibodies

Microtitre plates were coated with 100 μl/well of 2% BSA diluted in PBS and left to incubate overnight at 4°C. Unbound antigen was removed by washing with PBS/0.1% Tween 20 (wash buffer) in ELP-40 plate washer (3 cycle wash). Blocking buffer, 0.1% casein/PBS, (200 μl/well) was added to each well and incubated for 1 hour at 37°C. Plates were washed 3 times. PBS (100 μl/well) was added to all wells in rows B - H. Anti-BSA antisera diluted 1:50 in PBS (200 μl), was added to the appropriate wells of row A and serially diluted two-fold from 1:50 to 1:3,200. A conjugate control (PBS alone) was included on each plate. Plates were incubated 1 hour at room temperature. Plates were washed 3 times, as before, prior to the addition of 100 μl/well horseradish peroxidase-conjugated anti-mouse Ig (1:20,000 in PBS). Plates were then incubated for 1 hour at room temperature. Plates were washed 3 times, and bound antibody detected by incubation of the wells with 100 μl substrate (1:1 mixture of TMB peroxidase substrate and peroxidase solution B) for 10 minutes at room temperature. The reaction was stopped with the addition of 100 μl/well 1 M orthophosphoric acid. The reaction was measured spectrophotometrically at 450 nm using a microplate reader, using Softmax Pro software. ELISA procedure for screening of Anti-Thrombin or Anti-Fibrinogen Antibodies

Microtitre plates were coated with 100 μl/well of either 5 IU/ml thrombin or 100 μg/ml fibrinogen diluted in PBS and left to incubate overnight at 4°C. Unbound antigen was removed by washing with PBS/0.1% Tween 20 (wash buffer) in ELP-40 plate washer (3 cycle wash). Blocking buffer, PBS/0.1% Tween 20, (200 μl/well) was added to each well and incubated for 1 hour at 37°C. Plates were washed 3 times as before. PBS (100 μl/well) was added to wells in rows B, C, D, F, G and H. Nothing was added to rows A or E. Sera, anti-thrombin and anti-fibrinogen, diluted 1:50 in PBS (125 μl), was added to the appropriate wells of row A or E and serially diluted five-fold from 1:50 to 1:12,500. A conjugate control (PBS alone), positive and negative control were included on each plate. Polyclonal antibodies, rabbit anti-human thrombin IgG (thrombin ELISA positive control) and rabbit anti-human Protein C Ig (thrombin ELISA negative control) were diluted 1:500 in PBS (100 μl), and added to the appropriate wells. Monoclonal antibodies, anti-human fibrinogen (F-4639) fibrinogen ELISA positive control and anti-haemocyanin (H2143) fibrinogen ELISA negative control were diluted 1:12,500 in PBS (100 μl), and added to the appropriate wells. Plates were incubated for 1 hour at room temperature. Plates were washed 3 times prior to the addition of 100 μl/well horseradish peroxidase-conjugated rabbit anti-mouse Ig (1:20,000 in PBS) to wells containing diluted mouse sera and fibrinogen controls and 100 μl/well horseradish peroxidase-conjugated sheep anti-rabbit Ig (1:5,000 in PBS) to wells containing thrombin controls. Plates were then incubated for 1 hour at room temperature. Plates were washed 3 times and bound antibody detected by incubation of the wells with 100 μl substrate (1:1 mixture of TMB peroxidase substrate and peroxidase solution B) for 10 minutes at room temperature. The reaction was stopped with the addition of 100 μl/well 1 M orthophosphoric acid. The reaction was measured spectrophotometrically at 450 nm using a microplate reader, using Softmax Pro software. Titre Calculation

Antibody titres for anti-BSA sera were determined as the reciprocal of the highest dilution giving Absorbance 450 nm greater than 3 standard deviations above the mean of NMS (1:200). Antibody titres for anti-thrombin and anti-fibrinogen sera were determined as the reciprocal of the highest dilution giving Absorbance 450 nm greater than 3 standard deviations above the mean of NMS (1:50).

Results

BSA + Fibrin Glue immunised mice

Sera of mice immunised with fibrin glue containing BSA, and screened for anti-BSA antibodies, demonstrated antibody (mean) titres of 2400 at day 11. Antibody production then peaked at day 14, with titres in excess of 3200, before decreasing to 1000, 500 and 137.5 for days 21, 28 and 60, respectively (Table 2). This antibody profile is demonstrated in Figure 1 and compared against all groups of immunised mice. BSA + FIA immunised mice

Sera of mice immunised with FIA containing BSA, and screened for anti-BSA antibodies, demonstrated antibody (mean) titres in excess of 3200 for days 11, 14, 21, 18 and 60 (Table 2). This antibody profile is demonstrated in Figure 1 and compared against all groups of immunised mice. Other groups of immunised mice Sera of mice immunised with either fibrin glue alone, BSA alone, BSA

+ thrombin or BSA + fibrinogen, did not demonstrate antibody titres above normal mouse sera (all days) when screened for anti-BSA antibodies, suggesting no response above background (Table 2 and Figure 1).

Table 2: Median titre and titre range for experimental fibrin glue mice screened for anti-BSA antibodies

Any serum sample with titre less than or equal to 200 is equivalent to NMS.

Screening for anti-thrombin antibodies

Sera of mice immunised either BSA + fibrin glue, BSA + thrombin, or fibrin glue alone, did not demonstrate antibody titres above normal mouse sera (days 14, 28 and 60) when screened for anti-thrombin antibodies, suggesting no response above background (Table 3).

Table 3: Mean titre and titre range for experimental fibrin glue mice screened for anti-thrombin antibodies

Any serum sample with titre less than or equal to 50 is equivalent to NMS.

Screening for anti-fibrinogen antibodies

Sera of mice immunised with BSA + fibrin glue, BSA + fibrinogen, and Fibrin Glue alone, demonstrated antibodies towards fibrinogen at day 11 and continued through to day 60 (Table 4). No direct comparison of titres within and between groups can be made due to there being only 4 mice per group and that sera was not serially diluted two-fold. Variation can therefore occur since the range between five-fold dilutions is so great.

Table 4: Mean titre and titre range for experimental fibrin glue mice screened for anti-fibrinogen antibodies

Any servun sample with titre less than or equal to 50 is equivalent to NMS.

# Serum from 2 mice screened only due to serum shortage of Mice no. 3 and 4.

* Serum from 3 mice screened only due to serum shortage of Mouse no. 4.

Discussion

The way in which an antigen interacts with cells of the immune system is an important determinant of the type of response that is evoked. This interaction can be influenced by increasing the biological and immunological half-life of the antigen by holding it at its site of deposition and prolonging its adsorbtion. FIA accomplishes this by formation of an antigen-containing depot at the site of injection. This depot facilitates the slow release of antigen and the stimulation of antibody-producing cells.

Antibody production towards BSA was enhanced by incorporation of the protein (BSA) within fibrin glue or FIA. This was evident since no response was observed for the protein (BSA) alone.

The prolonged antibody response, evident by titres in excess of 3200 throughout days 11-60, demonstrated that FIA was able to hold the BSA protein within the emulsion and facilitate slow release more efficiently than the fibrin glue matrix.

Fibrin glue was able to enhance antibody production to the levels exhibited by FIA at day 14. However, this response was then observed to decrease with time. This correlates with the amount of time the fibrin glue clot persisted post administration. The presence of the clot was visualised up to day 14 in all mice receiving fibrin glue injections. No clot was observed at day 21, suggesting clot degradation. In order for fibrin glue to be as efficacious as FIA, prolonged protein release is required. This may be achieved by increasing the stability of the fibrin glue matrix which would thus delay its degradation. Protein embedded on the exterior surface of the matrix would trigger an immune response while protein embedded deep within the matrix would only be exposed after matrix degradation over a prolonged period of time.

In this preliminary study, it appears that fibrin glue is an efficacious vehicle for the slow release of BSA, although not the same extent as FIA. This is verified in that an immune response was observed for fibrin glue incorporating BSA within its matrix. Antigen injected into mice alone was not immunogenic. This suggests that both components of the glue, thrombin and fibrinogen, are required to form a clot and antibody production and that the proposed mechanism of action is by depot formation and the slow release of antigen. Furthermore, the absence of an anti-BSA immune response towards BSA + thrombin, or BSA + fibrinogen reinforces the fact that an immune response is only seen when the BSA and both components are combined together to form a clot.

An immune response was not mounted towards thrombin. All groups immunised with thrombin, i.e. BSA + fibrin glue, BSA + thrombin and fibrin glue alone, produced titres comparable to titres observed for normal mouse sera.

Fibrinogen was able to evoke an immune response in all groups injected with it, i.e. BSA + fibrin glue, BSA + fibrinogen and fibrin glue alone. This response could still be demonstrated at day 60. The anti-fibrinogen immune response is not enhanced by the fibrin glue matrix as is the anti-BSA response. This is evident because all mice injected with fibrinogen produced similar anti-fibrinogen immune responses, suggesting that the human fibrinogen is significantly different from mouse fibrinogen. Thus, the use of human fibrinogen in a mouse model may have evoked the observed anti-fibrinogen antibody response due to sequence or conformational differences between human fibrinogen and mouse fibrinogen. Further improvements to this study may include prolonging the release of protein from the fibrin glue matrix. This can be improved by increasing the stability of the fibrin glue clot, possibly by addition of antifibrinolytic agents, such as aprotinin. This would slow down degradation of the clot and slow release of the protein. This fibrin glue matrix would therefore be maintained at the site of injection longer.

The response to protein incorporated with fibrin glue may also be enhanced and prolonged by: • performing booster immunisations. Further administration of the protein alone or of the protein contained within the fibrin glue matrix, may cause immune components essential for an anti-protein response, to be continually triggered. • addition of immunostimulators. Immunostimulators are defined as any substance which act generally to accelerate, prolong, or enhance the quality of specific immune responses to particular antigens. Thus, incorporation of an immunostimulator, for example non- toxic derivatives of MDP (such as murabutide), into the fibrin glue/antigen formulation may prolong degradation and/or enhance the immune response. Conclusion

Antigens which are poor immunogens, or which are substantially non- immunogenic, may be incorporated into a fibrin glue matrix which in turn acts as an adjuvant to evoke an immune response to the antigen. Results presented herein show that protein incorporated within the fibrin glue matrix resulted in an increased anti-protein response while protein injected alone was unable to mount an immune response. This suggests that fibrin glue may be used as a potential adjuvant for enhancement of the immune response towards that antigen. As an adjuvant, fibrin glue may be improved by increasing the stability of the clot. Furthermore, addition of antifibrinolytic agents and immunostimulators to the fibrin glue/antigen formulation would also enhance responses observed in this preliminary study.

EXAMPLE 2 - EFFECT OF FIBRIN GLUE ON THE IMMUNOGENICITY OF LHRH

LHRH is a non-immunogenic 10-mer peptide. In this example, LHRH was conjugated to diphtheria toxoid (DT) (approximately 62 kDa in molecular weight), which acts as a carrier. The conjugated peptide was combined with iscoms™ to give an effective vaccine formulation. Conjugate (peptide-DT) alone does not stimulate an immune response. Groups of 6-8 week old male BALB/c mice (n=10) were immunised by subcutaneous injection with antigens (LHRH, LHRH + iscoms™, LHRH-DT & LHRH-DT + iscoms™) formulated within a fibrin glue matrix as described below. Antigen control mice (LHRH-DT) were injected with antigen alone (i.e. without fibrin glue or iscoms™). No antigen control mice were injected with fibrin glue alone (no antigen). Positive adjuvant control mice were injected with LHRH-DT + iscoms™.

Materials and Methods

Groups of mice were immunised as outlined in Table 5. Table 5: Ex erimental desi n summar

Upon immunisation, appropriate groups of mice received one or a combination of 2 μg or 10 μg LHRH, 10 μg or 50 μg LHRH-DT, 12 μg iscoms™ and 200 μL of fibrin glue. For fibrin glue immunogens, the antigen was mixed into the thrombin component prior to forming the glue. Reconstitution of thrombin One vial of thrombin was reconstituted in 2.5 mL of Water for Injection

BP (WFI) to give a concentration of 1000 IU/mL. Reconstitution of LHRH peptide

6.88 mg of LHRH peptide (69.1 % active) was reconstituted in 1 mL of WFI to give a final concentration of 4.75 mg/rnL active peptide. Preparation of antigen in thrombin

LHRH (groups 1 and 2) was mixed with PBS to 40 μg/mL or 200 μg/mL, as outlined in Table 6. This was then mixed with an equal volume of thrombin to result in concentrations of 20 μg/mL or 100 μg/mL antigen and 500 IU/mL thrombin. LHRH-DT (groups 3 and 4) was mixed with PBS to 200 μg/mL or 1000 μg/mL, as outlined in Table 6. This was then mixed with an equal volume of thrombin to result in concentrations of 100 μg/mL or 500 μg/mL antigen and 500 IU/mL thrombin Preparation of antigen + iscoms ™in thrombin

LHRH (groups 5 and 6) and iscomatrix™ were diluted with PBS to final concentrations of 40 μg/mL or 200 μg/mL antigen and 240 μg/mL iscoms™, as specified in Table 6. These were then mixed with an equal volume of thrombin to result in concentrations of 20 μg/mL or 100 μg/mL antigen, 120 μg/mL iscoms™ and 500 IU/mL thrombin.

LHRH-DT (groups 7 and 8) and iscomatrix™ were diluted with PBS to final concentrations of 200 μg/mL or 1000 μg/mL antigen and 240 μg/mL iscoms™, as specified in Table 6. These were then mixed with an equal volume of thrombin to result in concentrations of 100 μg/mL or 500 μg/mL antigen, 120 μg/mL iscoms™ and 500 IU/mL thrombin. Preparation of thrombin alone

No antigen control immunogen (group 9) was prepared by mixing thrombin 1:1 with 40 mM CaCl₂ to result in a concentration of 500 IU/mL thrombin (Table 6). Preparation of antigen in iscoms ™

LHRH-DT and iscomatrix™ (groups 10 and 11) were diluted with PBS to final concentrations of 50 μg/mL or 250 μg/mL antigen and 60 μg/mL iscoms™, according to Table 7. Preparation of immunogen alone LHRH-DT (groups 12 and 13) were diluted with PBS to final concentrations of 50 μg/mL or 250 μg/mL, as outlined in Table 8.

Table 6: Preparation of diluted immunogen in thrombin

CD cl- e-t- O o ∞ it

^•concentration of LHRH-DT used for groups 3, 4, 7 & 8 post boost immunogen preparation. Volumes are italicised.

Table 7: Preparation of diluted immunogen in iscoms

Administration of fibrin glue (Groups 1-9)

Two syringes, one containing thrombin ± antigen/iscoms™ (refer Table 6) and the other containing fibrinogen (50-70 mg/mL), were connected to a dual flow (syringe) adaptor attached to a 22 gauge needle. This served to deliver equal volumes of thrombin ± antigen and fibrinogen to the injection site. 100 μl from each syringe was administered simultaneously by one subcutaneous injection into the ventral flank of each mouse on day 0 such that the final dose of LHRH was 2 μg or 10 μg in fibrin glue, LHRH-DT was 10 μg or 50 μg in fibrin glue and iscoms™ was 12 μg in fibrin glue. Upon injection, thrombin cleaved fibrinogen to form fibrin and thus resulted in the formation of a fibrin glue clot containing the immunogen. After injection, mice were left to recover for one minute before returning to the cage. A secondary (boost) injection was also performed at day 21. Administration of antigen in iscoms™ (Groups 10-11) The immunogen/iscom™ solution was administered subcutaneously, into the ventral flank at one site (200 μl) using a 26 gauge needle and syringe such that the final dose of LHRH-DT was 10 μg or 50 μg and iscoms™ was 12 μg. Booster injections were performed at day 21. Administration of antigen alone (Group 7) Diluted LHRH-DT (see Table 8) was administered subcutaneously, into the ventral flank at one site (200 μl) using a 26 gauge needle and syringe such that the final dose of antigen was 10 μg or 50 μg. Booster injections were performed at day 21. Immunisation schedule/blood collection All mice were immunised at day 0 and bled by retro-orbital plexus puncture on days 7 and 14. The blood was allowed to clot at room temperature and incubated at 4°C for several hours. Sera was then separated and tested for antibodies by ELISA as outlined in Section 4.4. Mice were then boosted at day 21 and bled 1 week, 2 weeks, 4 weeks, 5 weeks and 9 weeks post boost. The blood and sera were treated as above and tested for antibodies to ELISA as outlined below. Screening of mouse sera by ELISA

Sera from mice in groups 1-13 were screened for the presence of antibodies to LHRH by ELISA. Sera from mice from groups 3, 4, 7, 8, 9, 10, 11, 12 and 13 were screened for the presence of antibodies to DT by ELISA. Antigen ELISA procedure

The sera of mice immunised with LHRH ± DT were screened by ELISA for the presence of antibodies to LHRH and DT. This was performed as outlined below. Microtitre plates were coated with 100 μl/well of either LHRH (10 μg/mL) or DT (10 μg/mL) diluted in coating buffer. They were then incubated overnight at 4°C, washed 3 times with Tris buffered saline (TBS)/0.05% Tween 20 and blocked using 150 μl/well 2% skim milk powder (blotto). Plates were then incubated for 30 min. at 37°C and washed as above. Anti- LHRH sera and anti-LHRH-DT sera were diluted 1:200 in blotto. Diluted sera (200 μl/well) was then added to appropriate wells of Row A and serial doubling dilutions were performed, in blotto, to Row H resulting in a final volume of 100 μl/well. Anti-LHRH (1:200) and anti-DT (1:1000) positive control sera was also included on each plate. The plates were then incubated for 1 hour at 37°C, washed as above and developed using 100 μl/well TMB Peroxidase substrate system. The plates were incubated for 15 min. at room temperature before stopping the reaction with 50 μl/well 2 M sulphuric acid. The absorbance was then measured at 450 nm and 620 nm. Statistical methods and calculations KC junior software determined a titre for each dilution of sera that fell within the standard curve. The standard curve was generated from positive control sera that had been assigned a titre. The mean titre was then determined for each mouse. These were then used to calculate the geomean titre for each group of mice. Results

Anti-LHRH response

No antibody response was detected in groups of mice following primary immunisation after bleeds on days 7 and 14. Antibody responses were only detected in groups of mice post secondary (boost) immunisation. Therefore only data generated from post boost bleeds are discussed below. Antibody responses towards LHRH were not observed in mice immunised with LHRH + fibrin glue (2 μg/dose or 10 μg/dose; groups 1 & 2), LHRH (2 μg/dose or 10 μg/dose) + iscoms™ + fibrin glue (groups 5 & 6), fibrin glue alone (group 9) or LHRH-DT alone (10 μg/dose or 50 μg/dose; groups 12 & 13) (see Figure 2). Mice immunised with LHRH-DT + fibrin glue (10 μg/dose or 50 μg/dose; groups 3 & 4) demonstrated antibody responses towards LHRH. Mice immunised with the lower dosage of LHRH-DT (10 μg/dose) demonstrated a sustained anti-LHRH geomean titre until 5 weeks post boost. The anti-LHRH geomean titre was greater for group 4 mice, which received an increased dosage of LHRH-DT (50 μg/dose). This anti-LHRH geomean titre increased gradually over time, until 5 weeks post boost before falling (see Figure 2).

Mice immunised with LHRH-DT (10 μg/dose or 50 μg/dose) + iscoms™ + fibrin glue, also generated antibody responses towards LHRH. The anti- LHRH geomean titre was greater in mice immunised with the increased dosage of LHRH-DT (50 μg/dose; group 8). The anti-LHRH response of both groups was observed to increase over the 5 week post boost period before falling. Furthermore the intensity of this response was greater than that observed for groups of mice immunised with LHRH-DT + fibrin glue (10 μg/dose or 50 μg/dose; groups 3 & 4) (see Figure 2).

Positive control groups 10 & 11 (immunised with LHRH-DT (10 μg/dose or 50 μg/dose) + iscoms™) demonstrated strong anti-LHRH responses. The anti-LHRH geomean titre was greater in mice immunised with the increased dosage of LHRH-DT (50 μg/dose; group 11), suggesting that the response may be dose dependent. The anti-LHRH geomean titre for both groups decreased over time. The intensity of these responses was greater than those observed for mice immunised with LHRH-DT ± iscoms™ + fibrin glue (groups 7 & 8) (see Figure 2). Anti-DT response

No anti-DT response was observed for groups of mice immunised with LHRH peptide ± iscoms™ + fibrin glue (groups 1, 2, 5 & 6), fibrin glue alone (group 9) or LHRH-DT alone (groups 12 & 13). One mouse in group 13 demonstrated an anti-DT response at 2 weeks post boost. This response was attributed to assay error since no response was seen at 1 week or 4 weeks post boost. The anti-DT response in groups 3 & 4 mice was negligible (see Figure 3).

Mice in groups 7 and 8, immunised with LHRH-DT (10 μg/dose or 50 μg/dose) + iscoms™ + fibrin glue, demonstrated antibody responses towards DT. As the dose increased, the anti-DT geomean titre also increased. This response seemed to peak at 1 week post boost irrespective of the dose administered, before decreasing and remaining constant over time (see Figure 3).

Antibody responses towards DT were observed in mice immunised with LHRH-DT (10 μg/dose or 50 μg/dose) + iscoms™ (groups 10 & 11). These responses followed the same profile over time irrespective of the dose of LHRH-DT administered. The anti-DT geomean titre increased until 2 weeks post boost before decreasing over time. The anti-DT response for groups 10 and 11 mice was much greater than responses observed for mice immunised with LHRH-DT + iscoms™ + fibrin glue (groups 7 & 8) (see Figure 3).

Discussion

The LHRH molecule is a small non-immunogenic peptide. The conjugated peptide, LHRH-DT, is also non immunogenic on its own, despite the presence of the DT carrier. In this experiment, fibrin glue was observed to be acting as a biological adjuvant. A vaccine adjuvant can be defined as any substance which when incorporated into a vaccine formulation acts generally to accelerate, prolong, or enhance the quality of specific immune responses to vaccine antigens (Vogel, 1995). Mice immunised with LHRH-DT + fibrin glue generated an immune response towards LHRH whereas mice immunised with the peptide alone (LHRH-DT) did not. The results detailed in this report have demonstrated that fibrin glue has enhanced the quality of specific immune responses to LHRH.

Iscoms™ are non-covalently bound complexes of Quil A adjuvant, cholesterol and amphipathis antigen, which stimulate humoral and cell mediated immunity (Takahashi et al., 1990; Hoglund et al., 1989; Morein et al., 1990). The addition of iscoms™ into the fibrin glue + LHRH-DT formulation enhanced the antibody response to the LHRH peptide compared to the fibrin glue + LHRH-DT formulation. The immune response towards LHRH generated by mice immunised with the positive control, LHRH-DT + iscoms™, was greater than that observed for LHRH-DT + iscoms™ + fibrin glue. This may be due to the fact that not all of the conjugated peptide in the fibrin glue formulation is available to the immune system. The anti-LHRH response in mice immunised with LHRH-DT ± iscoms™

+ fibrin glue was observed to increase over time compared to anti-LHRH responses in mice immunised with LHRH-DT + iscoms™ (which decreased over time). This demonstrates that the conjugated peptide was held within the fibrin glue matrix and released slowly over time as the clot was resorbed. This pattern of release may have been continually triggering the immune system.

The anti-DT response for mice immunised with LHRH-DT + iscoms™ is much greater than responses observed for mice immunised with LHRH-DT + iscoms™ + fibrin glue. This suggests that the fibrin glue was holding the DT within the matrix and preventing the immune system from generating an overwhelming response towards it. The fibrin glue was thus acting beneficially to prevent immunodominance from occurring. Immunodominance occurs when an overwhelming immune response is generated by a particular antigen and thus masks any weaker immune response generated by another antigen. Data generated from this experiment has reinforced observations from previous experiments that fibrin glue can be utilised as a biological adjuvant. It may also have great potential for use in a single dose vaccine formulation. By combining the fibrin glue vaccine formulation with the existing formulation, the primary immune response may be triggered immediately by free antigen, whilst the secondary immune response will be continually triggered by slow release of antigen from the fibrin glue clot. This response may be long lived depending on resorption time of the clot. This can be influenced by altering the matrix structure of the clot by changing the concentrations of fibrinogen and thrombin or by the addition of antifibrinolytic agents. Conclusion

In conclusion, fibrin glue was shown to facilitate the slow release of proteins incorporated within its matrix into the immune system and thus act as a biological adjuvant. LHRH-DT incorporated within the fibrin glue matrix resulted in an increased anti-LHRH-DT response while LHRH-DT injected alone was unable to mount an immune response. This suggests that fibrin glue may be used as a potential adjuvant for the slow release of antigen and enhancement of the immune response towards the antigen. As an adjuvant and vehicle for the slow release of protein antigens, fibrin glue may be improved by increasing the stability of the clot. Furthermore, addition of antifibrinolytic agents and immunostimulators to the fibrin glue/antigen formulation would also enhance the immune responses observed in this preliminary study.

It will be appreciated by persons skilled in the art that numerous variations and/or modifications may be made to the invention as shown in the specific embodiments without departing from the spirit or scope of the invention as broadly described. The present embodiments are, therefore, to be considered in all respects as illustrative and not restrictive. References

Baker, P.J., Hiernaux, J.R., Fauntleroy, M.B., Stashak, P.W., Prescott, B., Cantrell, J.L., Rudbach, J.A. (1988). Ability of monophosphoryl lipid A to augment the antibody response of young mice. Infect. Immun. 56: 3064- 3066.

Carelli, C, Audibert, F., Gaillard, J. and Chedid, L. (1982). Immunological castration of male mice by a totally synthetic vaccine administered in saline. Proc. Natl. Acad. Sci. USA. 79: 5392-5395.

Chedid, L. (1985). Adjuvants of immunity. Ann. Immunol. 136D: 283-291.

Edelman, R. (1980). Vaccine adjuvants. Rev. Infect. Dis. 2: 370-383.

Grun, J.L., Maurer, P.H. (1989). Different T helper cell subsets elicited in mice utilizing two different adjuvant vehicles: the role of endogenous interleuking 1 in proliferative responses. Cell. Immunol. 121: 134-145.

Gupta, R.K., Relyveld, E.H., Lindblad, E.B., Bizzini, B., Ben-Efraim, S., Gupta C.K. (1993). Adjuvants - a balance between toxicity and adjuvanticity. Vaccine. 11: 293-306.

Hibberd, P.L., Rubin, R.H. (1989). Immunization strategies for the immunocompromised host: The need for immunadjuvants. Ann. Intern. Med. 110: 955-956.

Hoglund, S., Dalsgaard, K., Lovgren, K., Sundquist, B., Osterhaus, A., Morein, B. (1989). ISCOMs and immunostimulation with viral antigens. In: Subcelluar Biochemistry, Vol 15. Plenum, New York, pp 39-68.

Mbawuike, I.N., Wyde, P.R., Anderson, P.M. (1990). Enhancement of the protective efficacy of inactivated influenza virus vaccine in aged mice by IL-2 liposomes. Vaccine 8: 347-352. Morein, B., Fossum, C, Lovgren, K., Hoglund, S. (1990). The ISCOM - a modern approach to vaccines. Semin. Virol. 1: 49-54.

Ramon, G. (1925). Sur l'augmentation anormale de l'antitoxine ches les cheyaux producteurs de serum antidiptherique. Bull. Soc. Centr. Med. Vet. 101: 227-234.

Stewart, M.W., and Howard, C.R. (1987) Synthetic peptides: a next generation of vaccines. Immunology Today 8: 51-58.

Takahashi, H., Takashita, T., Morein, B., Putney, S., Germain, R.N., Berzofsky, J.A. (1990). Induction of CD8+ cytotoxic T cells by immunisation with pruified HTV-l envelope protein in ISCOMS. Nature. 344: 873-875.

Vogel, F.R. (1995). Immunolgic adjuvants for modern vaccine formulations. Ann-N-Y-Acad-Sci. 754: 153-60.

Claims

Claims:

1. A composition for enhancing the immune response to at least one antigenic determinant in a host, the composition comprising at least one antigenic determinant in admixture with

(i) fibrin and/or fibrinogen or a derivative or metabolite thereof; and (ii) optionally at least one catalyst of fibrin glue formation.

2. A composition for enhancing the immune response to at least one antigenic determinant in a host, the composition comprising at least one antigenic determinant in admixture with at least one catalyst of fibrin glue formation.

3. A composition according to claim 1 or claim 2 in which the catalyst of fibrin glue formation is thrombin and/or Factor XIII.

4. A composition according to any one of claims 1 to 3 which further comprises Ca⁺⁺ ions or a source of Ca⁺⁺ ions.

5. A composition according to any one of claims 1 to 4 in which the at least one antigenic determinant is substantially non-immunogenic in the absence of an adjuvant.

6. A composition according to any one of claims 1 to 5 in which the at least one antigenic determinant is derived from a substance selected from the group consisting of viruses, bacterial toxins, parasitic antigens and hormones.

7. A composition according to any one of claims 1 to 6 in which the fibrin, fibrinogen and/or catalyst is derived from a human.

8. A composition according to any one of claims 1 to 7 in which the composition further comprises at least one agent which increases the stability of the fibrin glue matrix.

9. A composition according to claim 8 in which the at least one agent which increases the stability of the fibrin glue matrix is an antifibrinolytic agent.

10. A composition according to claim 9 in which the antifibrinolytic agent is selected from the group consisting of aprotin, ε aminocaproic acid and tranexamic acid [trans-4-(aminomethyl)cyclohexane carboxylic acid], alpha 2 antiplasmin, alpha 2 macroglobulin and alpha 1 antitrypsin.

11. A composition according to any one of claims 1 to 10 in which the composition further comprises an immunostimulator.

12. A composition according to claim 11 in which the immunostimulator is selected from the group consisting of a non-toxic derivative of MDP, interleukins, interferons, levamisole hydrochloride, and colony stimulating factors (CSFs).

13. A composition according to any one of claims 1 to 12 in which the at least one antigenic determinant is conjugated, either chemically or genetically, to the fibrin, fibrinogen (or derivative or metabolite thereof) or one or more catalyst molecules, or a carrier protein.

14. A kit for enhancing the immune response to at least one antigenic determinant in a host, the kit comprising:

(i) fibrin and/or fibrinogen or a derivative or metabolite thereof; and

(ii) at least one antigenic determinant such that in normal use of the kit, the fibrin and/or fibrinogen or derivative or metabolite thereof, and the at least one antigenic determinant are administered simultaneously to the host such that a fibrin glue matrix is formed at the site of administration and the at least one antigenic determinant is incorporated within the matrix.

15. A kit according to claim 14 which further comprises at least one catalyst of fibrin glue formation.

16. A kit according to claim 15 in which the at least one catalyst of fibrin glue formation is thrombin and/or Factor XIII.

17. A kit according to any one of claims 14 to 16 in which the at least one antigenic determinant is derived from a substance selected from the group consisting of viruses, bacterial toxins, parasitic antigens and hormones.

18. A kit according to any one of claims 14 to 17 which further comprises an immunostimulator.

19. A kit according to claim 18 in which the immunostimulator is selected from the group consisting of a non-toxic derivative of MDP, interleukins, interferons, levamisole hydrochloride, and colony stimulating factors (CSF).

20. A kit according to any one of claims 14 to 19 in which the fibrin, fibrinogen and/or catalyst is derived from a human.

21. A kit according to any one of claims 14 to 20 in which the kit further comprises at least one agent which increases the stability of the fibrin glue matrix.

22. A kit according to claim 21 in which the at least one agent which increases the stability of the fibrin glue matrix is an antifibrinolytic agent.

23. A kit according to claim 22 in which the antifibrinolytic agent is selected from the group consisting of aprotin, ε aminocaproic acid and tranexamic acid [trans-4-(aminomethyl)cyclohexane carboxylic acid], alpha 2 antiplasmin, alpha 2 macroglobulin and alpha 1 antitrypsin.

24. A kit according to any one of claims 14 to 23 in which the kit further comprises Ca⁺⁺ ions, or a source of Ca⁺⁺ ions.

25. A kit according to any one of claims 14 to 24 in which the at least one antigenic determinant is conjugated, either chemically or genetically, to the fibrin, fibrinogen (or derivative or metabolite thereof) or one or more catalyst molecules, or a carrier protein.

26. A method for enhancing an immune response to at least one antigenic determinant in a host, the method comprising administering the at least one antigenic determinant simultaneously with fibrin and/or fibrinogen or a derivative or metabolite thereof such that a fibrin glue matrix is formed at the site of administration and the at least one antigenic determinant is incorporated within the matrix.

27. A method according to claim 26 which further comprises administering at least one catalyst of fibrin glue formation at the site of administration.

28. A method according to claim 27 in which the at least one catalyst of fibrin glue formation is thrombin and/or Factor XIII.

29. A method according to any one of claims 26 to 28 in which the at least one antigenic determinant is derived from a substance selected from the group consisting of viruses, bacterial toxins, parasitic antigens and hormones.

30. A method according to any one of claims 26 to 29 which further comprises administering Ca⁺⁺ ions or a source of Ca⁺⁺ ions.

31. A method according to any one of claims 26 to 30 which further comprises administering at least one agent which increases the stability of the fibrin glue matrix at the site of administration.

32. A method according to claim 31 in which the at least one agent which increases the stability of the fibrin glue matrix is an antifibrinolytic agent.

33. A method according to claim 32 in which the antifibrinolytic agent is selected from aprotin, ε aminocaproic acid and tranexamic acid [trans-4- (aminomethyl)cyclohexane carboxylic acid], alpha 2 antiplasmin, alpha 2 macroglobulin and alpha 1 antitrypsin.

34. A method for enhancing an immune response to at least one antigenic determinant in a host, the method comprising administering to the host a fibrin glue matrix wherein the at least one antigenic determinant is incorporated within the matrix.

35. A method according to claim 34 in which the fibrin glue matrix is encapsulated within a biocompatible coating for solid dose delivery to biological tissue such as mucosal, dermal, ocular, subcutaneous, intradermal or pulmonary tissue.

36. A method according to claim 34 or 35 in which the fibrin glue matrix further comprises an immunostimulator.

37. A method according to claim 36 in which the immunostimulator is selected from a non-toxic derivative of MDP, interleukins, interferons, levamisole hydrochloride, and colony stimulating factors (CSFs).

38. A method according to claim 37 in which the fibrin, fibrinogen and/or thrombin are derived from a human.

39. A method according to any one of claims 26 to 38 in which the at least one antigenic determinant is conjugated, either chemically or genetically, to the fibrin, fibrinogen or catalyst molecules, or a carrier protein.