NZ243929A - Compositions of e.coli outermembrane proteins - Google Patents

Description

New Zealand Paient Spedficaiion for Paient Number £43929 24 3 9 2 Priority Date(s): ft i Complete Specification Filed: " Class: I Publication Date: ..? ? .?£?.!??? >.0. Journal. No: ,.,. my. i NO DRAWS Patents Form No. 5 NEW ZEALAND PATENTS ACT 1953 COMPLETE SPECIFICATION IMMUNOSTIMULATION WEr BIOTECH AUSTRALIA PTY LIMITED, an Australian company of 28 Barcoo Street, Roseville, New South Wales, AUSTRALIA and ST VINCENT'S HOSPITAL SYDNEY LIMITED, an Australian company of Victoria Street, Darlinghurst, New South Wales, AUSTRALIA hereby declare the invention, for which we pray that a patent may be granted to us, and the method by which it is to be performed, to be particularly described in and by the following statement: (followed by page la) n.z. PATEMT OFFICE 12 AUG 1992 received - 10r 2439po Immunostimulation Field of the Invention The present invention relates to - novel compositions useful in increasing responsiveness of T-cells, in particular helper T-cells. More specifically, the invention relates to conpositions including the E. Coli 10 outer-membrane proteins OmpA, OmpF or TraT.

Background of the Invention certain type of T-cell, known as the helper T-cell, which bears the CD4 phenotype, is required to assist the B-cell to differentiate into a plasma cell which in turn secretes soluble antibody. Before undergoing activation and proliferation, helper T-cells, with the aid of the T-cell 20 receptor and other accessory molecules, must first recognise antigens on the surface of antigen-presenting cells (APC) such as macrophages, dendritic cells or B-cells. More recent data are consistent with the hypothesis that T-cells require two signals for their 25 activation. One signal is delivered as a result of the binding of a peptide to a Class II Major histocompatibility complex (MHC) molecule on the APC, and the subsequent interaction of this MHC-peptide complex with the T-cell receptor. Although a necessary condition, 30 T-cell receptor occupancy by the MHC-peptide and its associated biochemical consequences are not sufficient to induce T-cell activation. For most cells, a second signal or co-stimulator molecule must be provided by the APC (Lafferty, Prowse and Simeonovic, 1983; Mueller, Jenkins 35 and Schwartz, 1989). Bacterial products, such as LPS and During the development of an immune response, a 243 82- BCG, which have been shown to elicit co-stimulator activity from APC are known as costimulatory inducers (Janeway, 1990). It is suggested that costimulation inducers will augment or improve the immune response of 5 individuals suffering from Immunodeficiency syndromes which are associated, at least in part, by a lack of helper T-cells.

Acquired Immunodeficiency Syndrome (AIDS) is a debilitating disease of man characterized by high 10 morbidity and mortality of infected individuals. The disease, which is characterized by an initial infection with a lentivirus, the Human immunodeficiency virus (HIV), is diagnosable in its early stages by the presence of antibodies in serum against the HIV and/or the presence of 15 the virus in the serum of asymptomatic individuals.

Almost without exception these asymptomatic individuals go on to develop full blown AIDS with its many associated complications, which ultimately leads to death of the infected individuals.

During the course of the disease, HIV positive individuals show a progressive depletion of their helper T-cell population [CD4-positive cells (CD4+)] with an increase in the numbers of CD8 cells. Accompanying the loss of CD4 cells, infected individuals show a 25 progressive loss of their ability to mount a protective immune response to HIV, or to a number of opportunistic pathogens which may invade the infected individuals. This chronic depletion of helper (CD4+) T-cells, and the resultant impairment of cell-mediated immunity, correlates 30 closely with disease progression towards AIDS (Fahey et al, 1990; Lange et al, 1989).

In an attempt to halt the spread of AIDS amongst at-risk individuals, and to develop a cure for the treatment of infected individuals, many research groups 35 have concentrated their efforts on the development of a 24 3 S vaccine against the HIV. Thus, since the initial isolation of the HIV, some 10 years ago, many sophisticated virological and biotechnological approaches have been used in the design and production of a multitude 5 of candidate vaccines (Hu et al, 1987; Kennedy et al, 1987) . Almost without exception the various vaccine candidates have failed dismally in clinical trials. The failure of the many vaccine candidates and the inexorable progression of the disease in infected patients towards 10 full blown AIDS, has led many to the pessimistic view that the development of an effective AIDS vaccine is almost impossible. This view is not, however, shared by Desrosiers and co-workers (1989) who have recently reported promising results vaccinating rhesus monkeys with 15 inactivated HIV preparations.

One of the major problems in the development of an effective vaccine to combat already established HIV infections is that the HIV itself invades and inactivates the CD4+ helper T-cells, which are the very cells which 20 must be stimulated for an individual to mount a protective immune response. During the course of a normal immune response, activated CD4 helper cells produce cytokines such as Interleukin-2 (IL-2) which are known to drive the clonal proliferation of primed T-cells, which can 25 ultimately lead to the elimination of virally infected cells. It has been shown, however, that the addition of exogenous IL-2 to the peripheral blood mononuclear cells (PBMC) of HIV-seropositive individuals, can restore both antigen- and mitogen-driven blastogenesis in vitro (Bell 30 et al. , 1990). Similar stimulation of PBMC in vivo, leading to the production of CD4-derived IL-2, would help to maintain strong CD8-associated antiviral immunity. It would appear, therefore, that there is considerable merit in adopting therapies that lead to an increase in the + level of CD4 T-lymphocytes, particularly during the 243929 asymptomatic stage of HIV-induced disease.

Nevertheless, IL-2 which has been recommended for approval by the FDA ( Stone R.. Science. 255, 528, 1992) as an immunotherapeutic for the treatment of kidney 5 cancer, has a number of disturbing side effects including...."circulatory problems that can be as severe as heart attacks and strokes".... There is clearly a need therefore for an effective immunostimulant which is devoid of the undesirable side effects attributed to IL-2. 10 One approach to improving the prognosis of HIV-infected individuals, would be to increase either the absolute number, or the responsiveness of helper T-cells in HIV-infected individuals and thereby improve the individual's capacity to mount an immune-attack on 15 HIV-infected cells and to develop effective responses to opportunistic pathogens.

In international patent application No PCT/AU87/00107 (TO87/06590), it is disclosed that in complexes with an immunogen TraT, OmpA and OmpF act as potent immunoadjuvants in 20 immunocompetent hosts. There is, however, no disclosure in this reference that TraT, OmpA or OmpF have any ability to increase the responsiveness of helper T-cells.

The present inventors have made the surprising finding that these proteins increase the responsiveness of 25 helper T-cells from patients suffering a deficiency in helper T-cell function. Further, the present inventors have found a synergistic effect on helper T-cell responsiveness between these proteins and other antigens. Summary of the Present Invention 30 Accordingly, in a first aspect the present invention consists in a composition comprising in admixture a protein selected from the group consisting of TraT, OmpA, and OmpF and parts thereof, at least one other antigen and a pharamaceutically acceptable carrier.

In a second aspect the present invention consists in , ■ ' /' " * s o , .5 r>U. V:j'^ 9 2f 3 9 2 a composition comprising a protein selected from the group consisting of TraT, OmpA, OmpF and parts thereof, coupled to an antigen selected from the group consisting of HIV antigens, influenza virus antigens, diphtheria antigens, 5 whooping cough antigens, measles antigens, tetanus antigens, Pneumocystis antigens, Candida antigens, Toxoplasmosis antigens, Cytomegalovirus antigens, hepatitis antigens, polio antigens, combinations thereof and individual subunit proteins, peptides or polysaccharides 10 isolated from said antigens, and a pharmaceutically acceptable carrier.

The composition according to the present invention may be used in a method of increasing immune responsiveness in a patient with an immunodeficiency, the method comprising 15 administering to the patient a composition comprising an effective amount of a protein selected from the group consisting of TraT, OmpA, OmpF and parts thereof and a pharmaceutically acceptable carrier.

The present invention relates to 20 the use of a composition comprising an effective amount of a protein selected from the group consisting of TraT, OmpA, OmpF and parts thereof and a pharmaceutically acceptable carrier, diluent and/or excipient in the manufacture of a medicament for increasing immune responsiveness in a 25 patient with a deficiency in immune function.

A composition according to the present invention may be administered to a patient with a deficiency in T-cell functions. The T-cells may be helper T-cells. If the patient is deficient in helper T-cell functions the responsiveness of 30 T-cells is increased.

In a preferred embodiment of the present invention the pharmaceutically acceptable carrier is a hydrophobic depot carrier. Suitable depot carriers include alhydrogel, proteosomes and liposomes. 243 S2D In a further preferred embodiment of the present invention the at least one other antigen is selected from the group consisting of HIV antigens, influenza virus antigens, diphtheria antigens, whooping cough antigens, 5 measles antigens, tetanus antigens Pneumocystis antigens, Candida antigens, Toxoplasmosis antigens, Cytomegalovirus antigens, hepatitis antigens, polio antigens and combinations thereof and individual subunit proteins, peptides or polysaccharides isolated from said antigens. 10 It is presently preferred, however, that the at least one other antigen is a HIV antigen, diphtheria toxoid or tetanus toxoid, and most preferably a HIV antigen selected from gp41[8] peptide and V3 loop peptide.

In yet a further preferred embodiment of the present 15 invention the protein is TraT or a part thereof.

TraT, OmpF and OmpA are outer membrane proteins of Gram negative bacteria. The TraT protein is an outer membrane protein of certain strains of E.coli which is responsible for the resistance of these strains to killing 20 by serum. The OmpA and OmpF proteins also fall in the same class of proteins. These proteins may be obtained from other Gram negative bacteria such as E.coli or Salmonella species. It is, however, presently preferred that the proteins are obtained from strains of E.coli. 25 The studies presented in this invention have shown an ability to enhance the level and/or activity of helper T-cells in individuals with deficiencies in helper T-cell function which indicates that TraT, OmpF and OmpA of E. coli and parts thereof can function as costimulator 30 inducers separate from, but with similar function to, the costimulator inducers BCG and LPS, as described by Janeway (1990).

The present inventors have shown that the costimulator inducer activity of outer membrane proteins TraT and OmpA 24 3 8 2 9 of E. coli can be used to enhance the stimulation of helper T-cells, derived from HIV-positive individuals, in the presence of antigen, and specifically, peptides derived from the viral proteins or recall antigens such as 5 Diphtheria toxoid (DT) and Tetanus toxoid (TT).

In contrast to BCG and LPS, TraT, OmpA and OmpF do not produce undesirable side-effects such as endotoxic shock and granuloma formation at the injection site.

TraT, OmpF and OmpA can, therefore, be used as 10 inducers of costimulatory activity in antigen presenting cells and thereby stimulate helper T-cells in the induction of immune responses to, for instance, a number of HIV-derived antigens, and thereby overcome the CD4-positive T-cell non-responsiveness in HIV-infected individuals. 15 The clinical outcome of increased helper T-cell numbers is improved immune function which in turn will result in an increased capacity of an individual to combat opportunistic infections.

The use of these molecules would greatly improve the 20 efficacy of candidate AIDS vaccines by stimulating the production of helper T-cells. Alternatively, when used in conjunction with other antigens to which an individual has previously developed memory T-cells, these molecules will enhance the overall level of immunity of the individual. 25 The ability of these molecules to restore helper T-cell function could also be exploited to enhance helper T-cell production in immunodeficiency conditions such as those which may arise following certain types of cancer, organ transplantation and various autoimmune conditions. 30 The compositions of the present invention are prepared by mixing, preferably homogeneously mixing, TraT, OmpA or OmpF or a part of TraT, OmpA or OmpF, which part stimulates an antigen presenting cell to provide a costimulator signal for helper T-cells, with a pharmaceutically acceptable 35 carrier, diluent, and/or excipient using standard methods 243 9 2 9 of pharmaceutical preparation.

Preferably the method additionally comprises using at least one other antigen in the preparation of the pharmaceutical composition. The antigen may be an antigen 5 against which it is desirable to raise an immune response in the patient. For instance in AIDS patients HIV antigens may be used. Other antigens which might be used include influenza virus antigens, diphtheria antigens, whooping cough antigens, measles antigens Pneumocystis antigens, 10 Candida antigens, Toxoplasmosis antigens, Cytomegalovirus antigens, combinations thereof and individual subunit proteins, peptides or polysaccharides isolated from said antigens.

The TraT, OmpA and OmpF proteins which can be used in 15 accordance with the present invention may be purified from publicly available standard E. coli strains which produce these proteins.

One such strain of E. coli is ATCC 67331 which was deposited with the American Type Culture Collection of 20 12301 Parklawn Drive, Rockville MD 20852, U.S.A. on 2 March 1987. Purification of TraT, OmpF and OmpA from E. coli is described in International Patent Application No.

PCT/AU87/00107 (WO 87/06590).

Alternatively these proteins may be obtained from 25 other bacterial strains which carry recombinant DNA molecules encoding these proteins, and purified by a method appropriate to the site of production of the recombinant TraT, OmpA or OmpF protein.

Where parts of these proteins, which stimulate an 30 antigen presenting cell to provide a costimulatory activity for helper T-cells are to be used, the required parts can be identified and prepared as follows.

The intact molecule is employed to identify the receptor which binds the molecule on the antigen presenting 35 cell. The intact molecule is then digested by standard 9 P ^o92 protein digestion techniques and the parts generated are assayed for binding to the identified receptor. Those parts which can bind and stimulate production of costimulatory activity by the antigen presenting cell are 5 suitable for use in the compositions and methods of the present invention.

As will be readily understood by persons skilled in the art homologues and analogues of TraT, OmpA and OmpF could be used in the present invention with similar 10 effect. It is intended that the use of such homologues and analogues are encompassed within the scope of this application. the present invention may be any antigen against which it 15 is desirable to raise an immune response in an immunocompromised or immunosuppressed patient.

Examples include, for instance, antigens of the HIV such as gp41[8] peptide which may be of use to stimulate blastogenesis of HIV-specific lymphocytes in HIV-infected 20 patients. Other antigens might include influenza virus antigens, diphtheria antigens, whooping cough antigens, measles antigens Pneumocystis antigens, Candida antigens, Toxoplasmosis antigens, Cytomegalovirus antigens, combinations thereof and individual subunit proteins, 25 peptides or polysaccharides isolated from said antigens.

The compositions of the invention may be prepared using standard pharmaceutical techniques.

Where an antigen is to be used in the composition, it may be admixed with the costimulator inducer in the depot. 30 Alternatively, the antigen and costimulator inducer may be complexed by chemical conjugation using chemical modification and/or linking groups where required. For proteinaceous antigens, the costimulator inducer and antigen could be provided as a fusion protein, by The antigens to be used in compositions of recombinant DNA techniques. 243929 In each case, it is to be understood that the process for joining the antigen to the costimulator inducer should not destroy the desired antigenicity of the antigen or the costimulator inducer activity of the TraT, OmpA, OmpF or 5 part thereof.

The costimulator-inducer or costimulator-inducer and antigen can be formulated in a depot carrier. Where both components are to be included it is desirable to keep them together. A depot carrier is suitable to achieve this and 10 the types of depot carrier which can be used include alhydrogel, proteosomes and liposomes. The compositions are prepared by standard techniques appropriate to the carrier being used.

Where the costimulator-inducer is to be used without 15 antigen or where the costimulator-inducer is complexed or fused to the antigen, traditional carriers other than depot carriers can also be used.

The composition of the present invention is preferably administered parenterally to the patient by standard 20 techniques of parenteral administration.

Typically lOOng-lOmg of each costimulator inducer and antigen is used in each dose.

The precise dose and ratio of each costimulator inducer and antigen to be used will depend on: (i) the type 25 and nature (e.g. immunogenicity) of the antigen; (ii) the genetic background of the subject; (iii) the immunological history of the subject; and (iv) the type of immune response (e.g. humoral, lymphocyte-mediated, macrophage-mediated or granulocyte-mediated) one is seeking 30 to modify.

A skilled addressee will be able to determine the appropriate ratio of costimulator inducer to antigen by systematically varying the relative dose and proportions of costimulator to antigen until the desired immune response 35 has been achieved.

It is recognised that a number of factors will affect the determination of an appropriate dosage for a particular patient. Such factors include the age, weight, sex, general health and concurrent disease status of the 5 patient. The determination of the appropriate dose level for the particular patient is performed by standard pharmaceutical techniques.

Patients for whom the use of the compositions of the invention is envisaged are patients 10 having a deficiency in helper T-cell function such as patients suffering from disease states including autoimmune diseases, some cancers and AIDS, and patients where an immunosuppressed state is artificially induced during treatment of a particular disease state or condition, for 15 instance transplant patients and cancer patients undergoing chemotherapy or radiotherapy.

The compositions of the invention might be used to raise their helper T-cell levels in general or the inclusion of specific antigens can be desirable in order to raise helper 20 T-cell levels in order to protect the patient from specific infections which could prove fatal in their immunocompromised or immunosuppressed state.

While it is understood that the primary focus of the present invention is ccmpositions useful in the treatment of human patients, 25 the ccmpositions are equally applicable for the treatment of non-human animals. Accordingly, as used herein the term "patient" is intended to cover both non-human and human animals.

In order that the nature of the present invention may 30 be more clearly understood, preferred forms thereof will now be described with reference to the following examples. 24 3 9 2 9 EXAMPLE 1. (i) TraT augments the in vitro T-cell proliferative responses elicited by immunodominant HIV-derived synthetic peptides.

A. Selection of the HIV-derived peptide (gp41[8])- The majority of the immunodominant sequences of the Human Immunodeficiency virus type-1 (HIV-1) are coded by hypervariable gene sequences and these sequences are interspersed with regions that are highly conserved 10 amongst HIV-1 strains. Immunogenic viral proteins that show minimal strain-to-strain variation and that consistently elicit both humoral and cell-mediated immune responses may be useful components for inclusion in a subunit vaccine. In this connection, Bell and co-workers 15 (Bell et al, 1992) have studied HIV-seronegative subjects and HIV-infected individuals classified as asymptomatic (AS), as AIDS-related complex (ARC) or as AIDS. In accordance with the clinical classification system of the CDC (Centers for Disease Control, 1986), AS HIV-infected 20 individuals constituted CDC Group Il/lII; ARC patients were CDC Group IVA/IVC2, and AIDS were CDC Group IVCl/lV D. They initially determined which of three short synthetic peptides derived from the conserved sequences of the envelope gp 120 (amino acids 262-284), gp41 (aa 579-601), 25 and core pl7 (aa 106-125) regions of the HTLV-IIIg isolate, could elicit B-cell as well as T-cell responses in HIV infected individuals. Only the gp41-derived sequence was immunogenic at the B- and T-cell levels. The gp41 region was characterized further by using a series of 30 overlapping synthetic peptides derived from a conserved region of the envelope gp41 (aa 572-613). The authors subsequently identified an immunodominant dodecamer (aa 593-604; termed gp41[8]) which consistently evoked both T-blastogenic and antibody responses in asymptomatic 35 HIV-seropositive individuals to a lesser extent in ARC, 243 92 9 but not in AIDS patients.

B. The synthesis of HIV-l-derived peptides.

The two peptides, gp41[8] and V3 loop derived from the gpl20 region of HIV-1, were synthesized on an Applied 5 Biosystems No. 430A peptide synthesizer following the manufacturers instructions. The peptides were purified by chromatography on G-25 Sephadex (Pharmacia) in 10% Acetic Acid, followed by Reverse Phase HPLC on a VYDAC C-18 column using a linear gradient of 5-60% acetonitrile in 10 0.1% TFA. The sequences of the peptides synthesised are as follows: R-S-S-ap41r8]; To improve the solubility of this peptide, Arg-Ser-Ser was added to the amino terminal end of the gp41[8] sequence viz., 15 Arg-Ser-Ser-Leu-Gly-Ile-Trp-Gly-Cys-Ser-Gly-Lys-Leu-Ile-Cys, V3 loop peptide: Asn-Thr-Arg-Lys-Ser-Ile-Arg-Ile-Gln-Arg-Gly-Pro-Gly-Arg-Ala-Phe-Val-Thr-Ile-Gly-Lys-Ile-Gly-Asn.

C. Assessment of human T-cell proliferative responses. Peripheral blood mononuclear cells (PBMC) were isolated by Ficoll-Paque (Pharmacia) gradient centrifugation, and 200,000 PBMC were cultured in 0.2 ml RPMI-160 medium (Cytosystems Pty. Ltd., containing 10% human AB serum, 50 mM each of penicillin and streptomycin, 2.5 mM glutamine and 2 mM HEPES buffer solution, pH 7.4) in 96-well round-bottom microtitre plates in the presence of gp41[8] or V3 loop synthetic peptides (2^M), rIL-2 (lOu/ml), 30 Diphtheria toxoid (DT; Commonwealth Serum Laboratories, Melbourne, Australia, 1570Lf units/ml; 4 and 40 yg/ml), Tetanus toxoid (TT; Commonwealth Serum Laboratories, Melbourne, Australia, lOOLf units/ml; 5 and 50 yg/ml); TraT and OmpA (purified as described by Croft et al. 35 1991; 40 pg/ml). After 6 days of culture at 37°C 24 3 0 2 9 (with or without antigen), each culture was pulsed overnight with 50pl of H-thymidine (20yCi/ml; Amersham, U.K.), harvested on glass-fibre filter papers and counted in a liquid scintillation spectrometer (Beckman, U.S.A.).

Results were expressed as Stimulation Indices (S.I.) which were calculated as follows: S.I. = mean counts per minute fc.p.m.1 with antigen - mean c.p.m. without antigen mean c.p.m. without antigen D. Definition of TraT- and IL-2-mediated effects on qp41f8]-. V3 peptide-. Diphtheria toxoid (DT)-.or Tetanus toxoid (TT)- specific lvmphoproliteration.

The effects of TraT and 11-2 on proliferative responses (expressed as Stimulation Indices) to the various 15 HIV-derived and recall (DT and TT) antigens have been defined by using a modified version of a documented formula (Denz et al., 1985): An additive effect was defined as: iAB^ = 1 (A) + (B) A Synergistic effect was defined as: CAB) > 1 (A) + (B) and an Inhibitory effect was defined as: (ABJ < 1 (A) + (B) where (A) and (B) signify the proliferative responses to the individual agents (e.g., gp41 [8] and TraT), respectively, and (AB) is the lymphoproliferative response seen when both agents are combined and added to the same cultures. 2 4 3 9 2 9 Results The addition of TraT to cultures of PBMC from eleven asymptomatic (AS) individuals, thirteen patients with AIDS-related complex (ARC) and one AIDS-lymphoma patient, 5 augmented the T-cell proliferative response to the gp41[8] peptide to levels which were higher than that achieved by the addition of IL-2 to cultures containing the gp41[8] peptide (Table 1). In every case, with the exception of patient #110, a synergistic effect was seen in cultures 10 that had been stimulated with a mixture of TraT and gp41[8] and this effect was maximal when 40 pg of TraT was used in the cell cultures (Table 2). By contrast, a synergistic effect was observed in only three from twenty-five cases, when IL-2 was combined with gp41[8] 15 (Table 1). The effect of another outer-membrane protein, OmpA, was also tested on PBMC cultures of one asymptomatic, and one symptomatic individual, and in both cases a synergistic effect was seen when OmpA was co-cultured with gp41(8) (Table 1).

In view of the impressive results obtained with gp41[8] in the presence of TraT, it was important to determine whether TraT would augment the T-cell response to another HIV-derived peptide. The V3 loop peptide was considered a suitable candidate, as this peptide (La Rosa et al., 25 Science 249: 932, 1990), the principal neutralising determinant of HIV-1, is currently in clinical trials (Scrip. No. 1703, 26, 1992). The results in Table 3 show that in all eight cases tested TraT augmented the proliferative responses to the V3 peptide. However, an 30 inhibitory effect was observed when PBMC from the eight individuals were cultured in the presence of IL-2 and V3 peptide (Table 3). In summary, TraT was far more effective than IL-2, a lymphokine which has been trialled as an Immunotherapeutic (Rosenberg, Lotze and Mul , 1988), 35 in augmenting the T-cell responses to the HIV-derived 24 3 S peptides gp41[8] and the V3 loop. (ii) TraT augments the in vitro T-cell proliferative responses to recall antigens such as Diphtheria 5 toxoid (DT) and Tetanus toxoid (TT).

It is well established that lymphoproliferative responses to recall antigens are impaired even during the asymptomatic disease period when CD4-positive T-cell 10 numbers are often only slightly reduced (Lane et al., 1985). Since TraT has been shown to augment HIV-specific lymphoproliteration in both symptomatic and asymptomatic individuals, it was reasonable to expect that it would have a similar effect in enhancing the response to recall 15 antigens. The ability of TraT to enhance the response to recall antigens will be of clinical importance in boosting immunity and thereby enabling immunocompromised individuals to combat opportunistic infections.

In all six patients studied (#123 to #128), TraT 20 significantly augmented DT - and T T- specific proliferative responses (Table 4). This finding would make TraT an attractive immunomodulator molecule for restoring defective T-cell responses, not only to HIV-derived antigens, but also to a range of recall 25 antigens. From a clinical viewpoint, a molecule with co-stimulator inducer-like properties such as TraT would be an attractive immunotherapeutic for boosting general immunity in immunocompromised individuals. 243 92 EXAMPLE 2 Flow cytometric analysis of T-cell subset HiRi-r*ihnt-ion indicates that TraT preferentially stimulates CD4-positive helper T-cells.

The T-cell subset distribution of Peripheral blood mononuclear cells (PBMC), that had been stimulated with TraT, Interleukin-2 (IL-2) or with gp41[8], after a 6-day incubation, were analysed using immunofluorescence and flow cytometry.

The phenotypes of the T-cells in proliferating cultures of PBMC were compared with those from unstimulated cell cultures.

T-cell phenotype analysis.

Two-millilitre volumes of both stimulated and unstimulated 15 PBMC (5 x 10^ cells/ml) were cultured for 6 days under the same conditions as described in Example 1. After 6 days of culture, pelleted cells were resuspended, and the cell suspensions layered onto 1 ml Ficoll-Hypaque gradients (Pharmacia), and centrifuged at 800g for 10 20 min. Viable (as judged by Trypan blue exclusion) lymphoblastoid cells were collected from the interface and washed in Hank's Balanced Salt Solution (HBSS; Cytosystems, Pty. Ltd.) pH 7.4. Viable cells that had been isolated from unsorted cultures of PBMC were 25 phenotyped using dual combinations of fluorescent monoclonal antibodies (i.e., CD 3/4 and CD 3/8; Coulter Electronics Inc., Hialeah FL, U.S.A.). Two-thousand viable cells from each PBMC culture were assayed for surface-bound fluorescence. Using both the viable PBMC 30 count (i.e., after 6 days of culture with and without stimulation), and the relative percentages of the 2 4 3 9 2 respective T-cell subsets gated in each bitmap of 2,000 cells, the absolute numbers of T-cells from the CD4-positive and CD8-positive T-cell subsets were calculated. The formula used for the calculation of 5 subset numbers was: T-cell subset % (per 2,000 "gated" cells) x viable cell number (as determined by Trypan blue exclusion). Bitmaps were continually adjusted to encircle the majority of either stimulated or unstimulated lymphocytes. After 6 days of culture of purified PBMC, > 10 90% of viable cells were consistently found to be CD3-positive, indicating that they were predominantly T-cells.

There was a 23 to 48% increase in absolute CD4-positive T-cell numbers (based on absolute cell numbers in the 15 unstimulated cultures) when PBMC were cultured in the presence of TraT, gp41[8]), or IL-2. However, when TraT was combined with gp41[8], CD4-positive T-cells increased by 63% (#123) and 96% (#124) respectively (Table 5). By contrast, there was a somewhat lower (0 to 42%) increase 20 in CD8-positive T-cell numbers when PBMC were incubated in the presence of any of the three stimulants, and this increased to 10% (#123) and 79% (#124) when PBMC were exposed to a mixture of TraT and gp41[8] in a 6-day culture. Another notable feature of these results is that 25 after a 6-day incubation with the various stimulants, there was a significant increase in the CD4/CD8 ratio compared with the CD4/CD8 ratio measured immediately after isolation of the PBMC.

The synergistic effect between TraT and gp41[8] was even 30 more pronounced when these stimulants were tested on the PBMC from patients #125 and #126 (Table 6). In these two ARC patients, the percentage increases in CD4 cell numbers were: TraT, 58% (#125), 36% (#126); gp41[8], 0% (#125), 18% (#126); IL-2, 378% (#125), 216% (#126). However, 35 there was a dramatic increase in CD4 cell numbers when 2*392 TraT was combined with gp41[8]s 737% (#125) and 1,763% (#126) respectively. The percentage increases in CD4 were significantly higher than the corresponding increases for the CD8-positive population, i.e., 74% (#125) and 185% 5 (#126) (Table 5). The preferential increase in CD4-positive helper T-cells in cultures incubated with TraT, or with a combination of gp41[8] and TraT, suggests that TraT will boost helper T-cell numbers in vivo and thereby enable HIV-infected individuals to combat 10 opportunistic infections. Further improvement in HIV-positive individuals would be obtained by combining TraT with anti-retroviral agents such as zidovudine. Industrial Application The current invention is applicable to the preparation of 15 vaccines designed to combat immunodeficiency disorders such as AIDS and the compositions may be useful for patients suffering a deficiency in helper T-cell function in qeneral.

It will be appreciated by persons skilled in the art that numerous variations and/or modifications may be made 20 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. c\i cd hO C\J o Diagnosis TraT gp-41[8] IL-2 gp41[8] + IL-2 gp41[8] + TraT OmpA gp41[8]+OmpA 100 AIDS-lymphoma 8.0 0.6 1.2 1.0(1)* 21.5(S) nt nt 101 Mild ARC 0.6 2.2 0.4 .9(S)# 17.8(S) nt nt 102 Mild ARC 0.9 2.5 2.0 1.6(1) 7.0(S) nt nt 103 Mild ARC 3.3 3.3 2.6 l.KD 21.2(S) nt nt 104 AS 8.5 52.1 26.2 nt 163.0(S) nt nt 105 AS 26.6 66.7 27.4 nt 252.0(S) nt nt 106 AS 13.4 12.8 11.7 nt 50.5(S) nt nt 107 AS 13.1 .8 41.1 nt 48.7(S) nt nt 108 AS 3.0 2.0 4.9 nt .9(S) nt nt 109 AS 3.5 3.6 6.8 nt 28.3(S) nt nt 110 Mild ARC 7.2 .6 2.9 nt 13.1(A)** nt nt 111 Mild ARC 8.0 .5 6.3 14.0(1) 21.7(S ) nt nt 112 AS 9.0 .9 8.5 16.3(S) 39.7(S) nt nt 113 ARC 29.0 7.9 12.0 18.1(1) 48.0(S) 16.0 29.3(S) 114 AS 56.4 19.9 22.1 36.0(1) 99.3(S) 17.9 42.8(5) 115 ARC 6.9 12.2 29.1 44.1(S) 33.7(S) nt nt 116 ARC 8.0 9.0 23.5 34.8(A) 23.4(S) nt nt 117 ARC 8.0 13.1 28.5 43.8(A) 32.8(S) nt nt 118 AS 12.0 7.0 13.5 22.0(A) 34.5(S) nt nt 119 AS 9.9 3.5 8.3 11.4(A) 21.9(S) nt nt 120 Mild ARC 42.8 32.5 67.7 22.0(1) 109.7(S) nt nt 123 ARC- .4 6.3 8.5 13.8(1) 50.0(S) nt nt Kaposi's Sarcoma 124 AS .7 6.2 6.1 12.4(A) 38.2(S) nt nt 125 ARC 7.2 4.3 4.4 7.4(1) 24.1(S) nt nt 126 ARC 12.2 6.9 8.7 12.4(1) 32.9(S) nt nt *1 - Inhibitory effect ; #s - Synergistic effect; **A - Additive effect, nt « not tested For an explanation of these terms see Example 1 243 92 9 TABLE 2 Dose-Response effect of TraT on crp41 r 81 - specific T-cell proliferation Patient No. and Diagnosis Treatments 111 112 113 114 Hild ARC AS AS AS Stimulation Indices TraT - lOfjg 3 .0 4.3 7 .8 16.5 TraT - 20yg .2 7.8 18 .1 .2 TraT - 40jjg 8 .0 9.0 29 .0 56.4 TraT - 60jjg 9 .0 7.7 23 .2 42.5 TraT - 80pg .4 6.8 14 .7 .7 gp41 [ 8 ] .5 .9 7 .9 19.9 TraT (lOpg) + gp41[8] 13 .3 .9 .2 38.6 TraT (20yg) + gp4i[8] 17 .2 .3 .5 75.8 TRAT (40^g) + gp41[8] 21 .7 39.7 48 .0 99.3 TRAT (60yg) + gp4i[8] 24 .8 18.8 .8 70.0 TRAT (80^g) + gp4I[ 8 j 19 .3 17.8 24 .5 48.2 TABLE 3 o> CNJ o> hO The augmentation by TraT of V3 loop peptide-specifxc T-cell proliferative responses CNI Treatments (Stimulation Indices) Patient No.

Diagnosis TraT V3 IL-2 V3 + IL2 V3 + TraT 125 ARC 7.2 3.8 4.4 6.9(1) 18.7(S) 126 AS 12.2 .6 8.7 .6(1) 26.0(S) 127 AS 12.1 6.3 8.5 .0(1)* .9(S) 128 Mild ARC 4.6 6.7 8.1 11.8(1) 36.9(S) 129 AS 3.7 2.7 4.1 3.7(1) 21.0(S) 130 AS .5 4.5 6.7 .7(1) .9(S) 131 ARC-Kaposi1 s Sarcoma .4 4.4 6.9 .7(1) .9(S) 132 ARC 4.1 .0 .7 6.3(1) 22.6(S) * I - Inhibitory effect; S - Synergistic effect CM CM 243 9 2 9 TABLE 4 The auementation by TraT of T-cell proliferative responses to Diphtheria toxoid (DTI and Tetanus toxiud (TT) Patient No and Diagnosis 123 124 125 126 127 128 Kaposi's AS ARC** ARC AS Mild ARC Treatments Sarcoma (Stimulation Indices) TraT(40pg .4 .7 7.2 12.2 12.1 11 .2 DT(40/jg) 6.5 .3 3.3 .1 6.2 6 .1 DT(4jjg) 4.0 2.7 1.4 1.41 3.31 3 .9 TT(50yg) 4.5 .4 2.8 3.7 .5 3 .0 TT(5fig) 3.0 2.8 1.6 2.7 3.1 2 .7 TraT+DT(40jjg) 37.6(S)* 27.0(S) .9(S) 24.0(S) .9(S) • 2(S) TraT+DT(4jJg) 21.6(S) 18.7(S) 11.5(S) 21.6(S) 23.8(S) 19 • 0(S) TraT+TT (50/ig) 34.0(S) 27.5(S) 16.0(S) 29.5(S) 26.5(S) 21 • 1(S) TraT+TT(5/Jg) 21.3(S) 13.4(S) 11.2(S) 19.5(S) 21.1(S) 19 • 6(S) * Synergistic effect AS - Asymptomatic; ** ARC - AlDS-related complex 243 9 2 9 TABLE 5 Flow cytometric analysis of T-cell subset distribution of proliferating and unstimulated cells after a 6-day Incubation Patient No. and Diagnosis #123 - Kaposi's Sarcoma* #124 - Mild ARC* Absolute T-cell numbers Absolute T-cell numbers Treatment CD4 CD8 CD4 CD4 CD8 CD4 NIL 3 .04xl06 2. 7xl06 TraT 3 .7xl06 2. 28x10 gp41[8] 3 .8xl06 2. 40x10 IL-2 3 .9x10® 2. 5xl06 [8]+TraT 4 .9xl06 2. 9xl06 [8]+IL-2 4 .lxlO6 2. 4xl06 1.1 2.5xl06 2.4xl06 1.0 1.6 3.0x10® 2.5xl06 1.2 1.6 3.7xl06 2.9xl06 1.3 1.6 3.6xl06 3.4xl06 1.1 1.7 4.9xl06 4.3xl06 1.1 1.7 3.6xl06 3.8xl06 0.9 * CD4/CD8 at day 0 was #123-0.23; #124-0.52 *\ 243 0 2 TABLE 6 Flow cytometric analysis of T-cell subset distribution of proliferating and unstimulated cells after a 6-day incubation Patient No. and Diagnosis #125 - ARC #126 - ARC Absolute T-cell numbers Absolute T-cell numbers Treatment CD4 CD8 CD4 CD8 CD4 CD8 CD4 CD8 NIL 92,000 880, 000 0.10 44, 000 780, 000 0.06 TraT 145,000 1,150, 000 0.13 60, 000 1,500, 000 0.04 gp41[8] 54,000 980, 000 0.06 52, 000 550, 000 0.09 IL-2 440,000 1,160, 000 0.38 139, 000 1,130, 000 0.12 [8]+TraT 770,000 1,530, 000 0.50 820, 000 2,220, 000 0.37 [8]+IL-2 29,000 1,280, 000 0.03 68, 000 1,310, 000 0.05 * CD4/CD8 at day 0 was #125 - 0.125; #126 - 0.09 243 92 8 REFERENCES (for Inununostimulation Patent) Lafferty, K.J., S.J. Prowse, and C.J. Simeonovic. 1983. Immunobiology of tissue transplantation: A return to the 5 passenger leukocyte concept. Ann. Rev. Immunol. Is 43. Mueller, D.L., M.K. Jenkins, and R.H. Schwartz. 1989. Clonal expansion versus functional clonal inactivations a costimulatory signalling pathway determines the outcome of T cell antigen receptor occupancy. Ann. Rev. Immunol. 10 7: 445.

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Desrosiers, R., M. Wyard, T. Kodama, D.J. Ringler, R. Arthur, P.K. Sehgal, N.L. Letvin, N.W. King, and M.D. Daniel. 1989. Vaccine protection against simian immunodeficiency virus infection. P.N.A.S. 86s 6353. 35 Bell, S.J.D., R. Doherty, B. Kemp, and D.A. Cooper. 24 3 9 2 0 1990. Exogenous IL-2 can re-instate T-cell proliferative response to HIV-1 envelope and core-derived peptides. A.S.H.M. 4th National Conference on AIDS, abstract.

Bell, S.J.D., D.A. Cooper, B.E. Kemp, R.R. Doherty, and R. 5 Penny. 1992. Definition of an immunodominant T-cell epitope contained in the envelope gp41 sequence of HIV-1 Clin. Exp. Immunol. 87: 37.

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Claims (11)

- 28 - PCT/AU87/00107 (V30 87/06590). Barnes, T.M., Lehrbach, P. and Russell-Jones, G.J. "Iirununopotentiation". Stone, R. (1992). Anti-cancer drug IL-2 may finally be approved. Science. 255: 528. 2439 - 29 - X/i 4 H ^ c a CMJHS-:- WHAT^WE CLAIM IS:-

1. A composition comprising in admixture a protein selected from the group consisting of TraT, OmpA, and OmpF and parts thereof (as herein defined), at least one other antigen and a 5 pharmaceutically acceptable carrier.

2. A composition as claimed in claim 1 in which the pharmaceutically acceptable carrier is a hydrophobic depot carrier.

3. A composition as claimed in claim 1 or claim 2 in 10 which the at least one other antigen is selected from the group consisting of HIV antigens, influenza virus antigens, diphtheria antigens, whooping cough antigens, measles antigens, tetanus antigens, Pneumocystis antigens, Candida antigens, Toxoplasmosis antigens, Cytomegalovirus 15 antigens, hepatitis antigens and polio antigens, .combinations thereof and individual subunit proteins, peptides or polysaccharides isolated from said antigen.

4. A composition as claimed in claim 3 in which the at least one other antigen is a HIV antigen selected from 20 gp41[8] peptide and V3 loop peptide.

5. A composition as claimed in any one of claims 1 to 4 in which the protein is derived from E.coli.

6. A composition as claimed in any one of claims 1 to 5 in which the protein is TraT or a part thereof (as herein defined). 25

7. A composition comprising a protein selected from the group consisting of TraT, OmpA and CtapF and parts thereof (as herein defined) coupled to an antigen selected from the group consisting of HIV antigens, influenza virus antigens, diphtheria antigens, whooping cough antigens, measles antigens, 30 tetanus antigens, Pneumocystis antigens, Candida antigens, Toxoplasmosis antigens, Cytomegalovirus antigens, hepatitis antigens and polio antigens, combinations thereof and individual subunit proteins, peptides or polysaccharides isolated from said antigen, and 35 pharmaceutically acceptable carrier.

8.- A composition as claimed in claim 7 in which the antigen is selected from the group consisting of HIV antigens, diphtheria toxoid and tetanus toxoid.

9. A composition as claimed in claim 8 in which the HIV 5 antigen is gp41[8] peptide or V3 loop peptide.

10. A composition as claimed in any one of claims 7 to 9 in which the protein is TraT or a part thereof (as herein defined.

11. A composition as claimed in claim 1, substantially as herein described with reference to any one of the 10 Examples. BIOTECH AUSTRALIA PTY LIMITED and ST VINCENT'S/llOSP ITAL SYDNEY LIMITED fw3li/mo^. .By Their ifftorneys BALDWIN SON & CAREY 20 25 30