CA2115424A1 - Immunostimulation - Google Patents

Abstract

The present invention provides novel compositions and methods for increasing immune responsiveness, in particular T-cell responsiveness in patients with an immunodeficiency, particularly in T-cell function. The present invention is particularly useful for increasing responsiveness of helper T-cells. The method of the present invention comprises administering to a patient a protein selected from the group consisting of TraT, OmpA, OmpF and parts thereof and a pharmaceutically acceptable carrier. Preferably, at least one other antigen is administered. The present invention should be particularly useful in the treatment of HIV positive patients.

Description

W093/03762 PCT/AU92/0~23 15~2~

Immuno~timulation Field of the Invention The present invention relates to the use of costimulator inducers to augment or to boost the immune response in patient~ with deficiencies in T-cell function, in particular helper T-cell function, and to novel compositions useful in increasing responsivene~s of T-cells, in particular helper T-cells. Nore specifically, the invention relates to the use of the E. coli outer-membrane proteins OmpA, OmpF or TraT to augment the immune response to antigens in immunocompromised individuals and to compositions including these proteins.
Background of the Invention During the development of an immune response, a ~15 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 ~oluble antibody. Before undergoing activation and proliferation, helper T-cells, with the aid of the T-cell receptor and other accessory molecules, must first recognise antigens on th~ surface of antigen-presenting cells (APC) such as macrophages, dendritic cells or B-cells. Nore recent data are consistent with the hypothesis that ~-cells require two signals for their acti~ation. One signal is delivered as a result of the binding of a peptide to a Class ~I Na~or histocompatibility complex (MHC) molecule on the APC, and the subsequent interaction of this NHC-peptide complex with the T-cell receptor. Although a necessary condition, 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 :

WOs3/03762 PCT/AU92/OW23~
2i1~24 `

BCG, which have been shown to elicit co-stimulator activity from APC are known ~s costimulatory inducers (Janeway, 1990). It is suggested that costimulation inducers will augment or improve the immune response of individuals suffering from Immunodeficiency syndromes which ~re associated, at least in p~irt, by a lack of helper T-cells.
Acquired Immunodeficiency Syndrome (AIDS) is a debilitating disease of man characterized by high 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 stageæ by the presence of antibodies in serum against the HIV and/or the presence of 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 complication~, which ultimately leads to death of the inifected individuals.
During the course of the disease, HIV positive ~: individu~ls show a progr~ssive depletion of their helper T-cell population tCD4-positive cells (CD4 )1 with an increase in the numbers of CD8 cells. Accompanying the 1088 of CD4 cells, infected individuals show a 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 closely with disease progression towards AIDS (Fahey et al, 1990; ~ange et al, 1989).
In an attempt to halt the s~read of AIDS amongst at-risk individuals, and to develop a cure for the treatment of infected individuals, many research groups have concentrated their efforts on the development of a ~:

W093/03762 PCT/AU92/~23 _ 3 _ 2~ 24 vaccine again~t the HIV. Thus, since the initial isolation of the HIV, some 10 years ago, many ~ophisticated virological and biotechnologic~l approache~
have b~en used in the de~ign and production of a multitude of candidate vaccine~ (Hu et al, 1987; ~ennedy et al, 1987). Almo~t without exception the variou~ vaccine candidate~ have failed dismally in clinical trial~. The failure of the many vaccine candidate~ and the inexorable progression of the di~ease in infected patients towards full blown AIDS, has led many to the pessimistic view that the development of an effective AIDS vaccine i8 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 ma~or problems in the development of an effective vaccine to combat already e~tablished HIV
nfections i8 that the HIV itself invades and insctivates the CD4~ helper T-cells, which are the very cells which mu~t be stimulated for an individusl to mount a protective immune response. During the course of a normal immune respon~e, activated CD4 helper cells produce cytokines ` such a8 Interleukin-2 (IL-2) which are known to drive the clonal proliferation of primed T-cell~, which can 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 (PBNC) of HIV-seropositive individuals, can restore both antigen- and mitogen-driven blastogenesis in vitro (Bell 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 antivir~l immunity. It would appear, therefore, that there is considerable merit in adopting therapies that lead to an increase in the level of CD4~ T-lymphocyte~, particul~rly during the `.

W093/03762 PCT/AU92/0~23~i~
211S~24 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 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 i5 devoid of the undesirable side effects attributed to IL-2.
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 HIV-infected cells and to develop effective responses to opportunistic pathogens.
In international patent application No PCT/AU87/00107 it is disclosed that in complexes with an immunogen TraT, OmpA and OmpF act as pvtent immunoad~uvants in immunocompetent hosts. There is, however, no disclosure in this reference that TraT, OmpA or OmpF have any ability .
to increase the responsiveness of helper ~-cells.
The present inventors have made the surprising finding that these proteins increase the responsiveness of helper ~-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 Accordingly, in a first aspect the pre~ent invention consists in a composition comprising in admixture a protein selected from the group consisting of TraT, OmpA, OmpF and parts thereof, at least one other antigen and a pharamaceutically acceptable carrier.
In a second aspect the present invention consists in `

W093/03762 PCT/AU92/~423 211~ 42~

a compo~ition comprising a protein selected from the group consisting of TraT, OmpA, Omp~ and parts thereof, coupled to an antigen selected from the group con~i~ting of HIV
antigens, influenza virus antigens, diphtheria antigens, whooping cough antigens, measles antigens, tetanus antigens, Pneumocystis antigens, Candida antigen~, Toxoplasmosis antigens, CytomegaIovirus antigens, hepatitis antigens, polio antigens, combinations thereof and individual subunit proteins, peptides or polysaccharides isolated from said antigens, and a pharmaceutically acceptable carrier.
In a third aspect the present invention consists in a method of increasing immune responsiveness in a patient with an immunodeficiency, the method comprising administering to the patient a composition comprising an effective amount of a protein selected from the group con~isting of TraT, OmpA, OmpF and parts thereof and a pharmaceutically acceptable carrier.
In a fourth aspect the present invention consists in the use of a composition comprising an effective amount of a protein ~elected from the group con~isting 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 patient with a deficiency in immune function.
In a preferred embodiment of the present invention the responsiveness of T-cells is increased and the patient has a deficiency in T-cell function.
In a further preferred embodiment of the present invention the T-cells are helper T-cel~s and the patient has a deficiency in helper T-cell functions.
In a preferred embodiment of the present invention the pharmaceutically acceptably carrier is a hydrophobic depot carrier. Suitable depot carriers include alhydrogel, proteosomes and liposomes.

W093/03762 PCT/AU9V~2 211~42~ ~

In a further preferred embodLment of the present invention the at least one other antigen is seleeted from the group consisting of HIV antiqens, influenza virus antigens, diphtheria antigens, whooping eough antigens, measles antigens, tetanus antigens Pneumoeystis antigens, Candida antigens, Toxoplasmos i8 antigen~, Cytome~alovirus antigens, hepatitis antigens, polio antigens and combination~ thereof and individual subunit proteins, peptides or polysaceharides isolated from said antigens.
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 seleeted from gp4118] peptide and V3 loop peptide.
In yet a further preferred embodiment of the present invention the protein is TraT or a part thereof.
~; Tr~T, OmpF and OmpA are outer membrane proteins of Gram negative baeteria. The TraT protein is an outer membrane protein of eertain strains of E.eoli whieh is re~ponsible for the resistanee of these strains to killing by serum. The OmpA and OmpF proteins also fall in the same elass of proteins. These proteins may be obtained from other Gram negative baeteria sueh as E.eoli or Salmonella speeies. It is, however, presently preferred that the proteins are obtsined from strains of E.eoli.
The studies presented in this invention have shown an ability to enhanee the level and/or aetivity of helper T-eells in individuals with deficiencies in helper T-eell function which indicates that TraT, OmpF and OmpA of E. eoli and parts thereof ean function as eostimulator indueers separate from, but with similar function to, the eostimulator ~ndueers BCG and LPS, as deseribed by Janeway (1990).
The present inventors have shown that the eostimulstor inducer activity of outer membrane proteins TraT and OmpA

~: :

W093/03762 PCT/AU92/0~23 ~il5~2~

of E. coli can be used to enhance the ~timulation of helper T-cell~, derived from HIV-positive individuals, ln the presence of antigen, and specifically, peptides derived from the viral proteins or recall antigen~ such as Diphtheria toxoid (DT) and Tetanus toxoid (TT).
In contra~t to BCG and LPS, TraT, OmpA and OmpF do not produce undesirable side-effects such as endotoxic shock and granuloma formation at the in~ection site.
TraT, OmpF and OmpA can, therefore, be u~ed as inducerc of co~timulatory activity in antigen presenting cell~ 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. ~-The clinical outcome of increa~ed helper T-cell numbers i8 improved immune function which in turn will re~ult in an increased capacity of an individual to combat ~`
opportun~stic infection~.
The u~e of these molecules would greatly improve the efficacy of candidate AIDS vaccine~ by stimulating the ~`~ production of helper T-cells. Alternatively, when used in con~unction with other antigens to which an individual has previously devaloped memory T-cells, these molecules will enhance the overall level of immunity of the individual.
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, orgsn transplantation and various autoimmune conditions.
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 co~timulator signal for helper T-cells, with a pharmaceutically acceptable carrier, diluent, and/or excipient using standard methods :

w093/03762 PCT/AU92/~23 211~42'~

of pharmaceutical preparation.
Preferably the method additionally compri~e~ u~ing at least one other antigen in the preparation of the pharmaceutical composition. The antigen may be an antigen 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, 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 accordance with the present invention may be purified from publicly available standard E. coli strains which produce these proteins.
One such strain of ~ coli is ATCC 67331 which was deposited with the American Type Culture Collection of 12301 Parklawn Drive, Rockville MD 20852, U.S.A. on 2 Narch 1987. Purification of TraT, OmpF and OmpA from E. coli is describQd in International Patent Application No.
PCT/AU8~/00107 (WO 87/06590).
Alternatively these proteins may be obtained from 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 antigen presenting cell to provide a co~timulatory 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 :

W093/03762 2 11 ~ ~ 2 ~PCT/AU92/~23 _ 9 _ protein digestion teehniques and the parts genersted are assayed for binding to the identified reeeptar. Those parts whieh ean bind and stimulste produetion of eostimulatory aetivity by the antigen presenting eell are suitable for use in the eomposition~ and methods of the pre~ent in~ention.
As will be readily understood by persons skilled in the art homologues and analo~ues of TraT, OmpA and OmpF
eould be used in the present invention with similar effeet. It is intended that the use of sueh homologues and analogues are eneompassed within the seope of this applieation.
The antigens to be used in eompositions and method of the present invention may be any antigen against which it is desirable to raise an immune response in an immunoeompromised or immunosuppressed patient.
Examples inelude, for instanee, antigens of the HIV
~ueh as gp41[8~ peptide whieh may be of use to stimulate blastogenesis of HIV-speeifie lymphoeyte~ in HIV-infected patient~. Other antigen~ might inelude influenza virus antigens, diphtheria antigens, whooping eough antigens, measles antigens Pneumoeystis antigen~, Candida antigens, oxoplasmoæis antigens, Cytome~alovirus antigens, eombinations thereof and individual subunit proteins, peptides or polysaceharides isolated from said antigens.
The eompositions of the invention may be prepared using standard pharmaceutieal teehniques.
Where an antigen is to be used in the composition, it `
may be admixed with the costimulator indueer in the depot.
Alternatively, the antigen and eostimulator inducer may be eomplexed by ehemical con~ugation using ehemieal modifieation and/or linking groups where required. For proteinaeeous antigens, the eostimulator indueer and antigen eould be provided as a fusion protein, by reeombinant DNA techniques.

wo9i/lo356~ 2 ~ PCT/AU92/0042~

In eaeh case, it is to be understood that the proeess for ~oining the antigen to the eostimulator indueer ~hould not de~troy the desired antigenicity of the antigen or the eo~timulator indueer aetivity of the TraT, OmpA, OmpF or part thereof.
The eostimulator-indueer or eostimulator-inducer and antigen ean 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 aehieve this and 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 eostimulator-inducer is to be used without lS antigen or where the costimulator-indueer i~ complexed or fused to the antigen, traditional carriers other than depot earrier~ ean also be used.
The eomposition of the present invention i8 preferably admini~tered parenterally to the patient by standard teehniques of parenteral administration~
Typieally 100ng-lOmg of eaeh eostimulator indueer and antigen is used in each dose.
The preeise dose and ratio of each costimulator indueer and antigen to be used will depend ons (i) the type and nature (e.g. immunogenicity) of the antigen;
(ii) the genetic background of the ~ub~ect; (iii) the immunologieal history of the sub~ect; and (iv) the type of immune response (e.g. humoral, lymphocyte-mediated, macrophage-mediated or granulocyte-mediated) one is seeking to modify.
A skilled addressee will be able to determine the appropriate ratio of costimulator inducer to antigen by systematieally varying the relative dose and proportions of eostimulator to antigen until the desired immune response has ~een aehieved.

: ~ ~ ..... ... -W093/03762 2 11 ~ 4 2 ~ PCT/AU92/00423 It is recognised that a number of factor will affect the determination of an appropriate dosage for a particular patient. Such factors include the age, weight, sex, general health and concurrent disea~e status of the patient. The determinatiQn of the appropriate dose level for the particular patient is performed by standard pharmaceuti-al techniques.
Patients for whom the use of the methods and compositions of the invention is envisaged are patients having a deficiency in helper T-cell function such a~
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 method of the invention might be used to raise their h lper T-cell levels in general or the inclusion of specific antigens can be desirable in order to raise helper 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 the treatment of human patients the present invention is 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 be more clearly understood, preferred forms thereof will now be described with reference to the following examples.

.

W093/03762 PCT/AU92/0~2~
2`~15~24 `~ ;l ExAMeLE 1.
(i) TraT augm~nts the in vitro T-cell ~roliferative re~Fonse~ elicited by immunodominant HrV-derived ~ynthetic peptides.
S A. Selection of the HIV-derived peptide (gp41t8]).
The ma~ority of the immunodominant sequences of the Human Immunodeficiency virus type-l (HIV-l) are coded by hypervariable gene sequences and these sequences are interspersed with regions that are highly conserved amongst HIV-l 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 (Bell et al, 1992) have studied HIV-seronegative sub~ects 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 individuals constituted CDC Group II/III; ARC patients were CDC Group IVA/IVC2, and AIDS were CDC Group IVCI/IV 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), and core pl7 (aa 106-125) regions of the HTLV-IIIB
isolate, could elicit B-cell as well as T-cell responses in HIV infected individuals. Only the gp41-derived sequence was immunogenic at the ~- and T-cell levels. The gp41 region was characterized further by using a series of 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 gp4118]) which consistently evoked both T-blastogenic and antibody responses in asymptomatic HIV-seropositive individuals to a lesser extent in ARC, :

W093/03762 2 ~ 2 ~ PCT/AU92/~23 but not in AIDS patients.
B. The ~ynthe~i~ of HIV-l-derived peptide~.
The two peptides, gp4118] and V3 loop derived from the gpl20 region of HIV-l, were synthesized on an Applied S 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 O.1% TFA. The sequences of the peptides synthesised are as follows:
R-S-S-op4118~: To improve the solubility of this peptide, Arg-Ser-Ser was added to the amino terminal end of the gp41181 sequence viz., 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. A~se~sment of human T-cell ~roliferative response~.
Peripheral blood mononuclear cells (PBMC) were i~olated 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 esch of penicillin and streptomycin, 2.5 mM glutamine and 2 mN HEPES buffer solution, pH 7.4) in 96-well round-bottom microtitre plates in the presence of gp41~8]
or V3 loop synthetic peptides (2yN), rIL-2 ~lOu/ml), Diphtheria toxoid (DT; Commonwealth Serum Laboratories, Melbourne, Australia, 1570Lf units/ml; 4 and 40 ~g/ml), Tetanus toxoid (TT; Commonwealth Serum Laboratories, Melbourne, Australia, 100Lf units/ml; 5 and 50 yg/ml);
TraT and OmpA (purified as described by Croft et al.
1991; 40 yg/ml). After 6 days of culture at 37C

`:

W093/03762 PCT~AU92/~23~
21~L5~24 (with or without antigen), each culture wa8 pul8ed overnight with 50yl of H-thymidine ~20~Ci/ml;
Amersham, U.g.), harvested on glass-fibre filter papers and counted in a liquid scintillation spectrometer (Beckman, U.S.A.).
Re~ult~ were expressed a~ Stimulation Indices (S.I.) which were calculated as follows:
S.l. = mean counts per minute (c.p.m.) with antigen - mean c.p.m. without antigen mean c.p.m. without antigen D. Definition of TraT- and IL-2-mediated effects on gp41~8]-. V3 peptide-. Diphtheria to~id ~pT)-.or Tetanus to~oid (TT)- specific lymphoproliferation.
The effects of TraT and Il-2 on proliferative responses (expressed as Stimulation Indices) to the various 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:

(AB) (A) ~ (B) A Synergistic effect was defined as 5 (AB~ ~ 1 (A) + (B) and an Inhibitory effect was defined as:
(AB) ~ 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.

W093/03762 211~ 12 4 PCT/AU92/0~23 The addition of TraT to eultures of PBNC from eleven asymptomatie (AS) individuals, thirteen patients with AIDS-re~ated complex (ARC) and one AIDS-lymphoma patient, S augmented the T-eell proliferative response to the gp41[8]
peptide to levels whieh were higher than that achieved by the addition of IL-2 to eultures eontaining the gp41t8]
peptide (Table 1). In every ea~e, with the exeeption of patient $110, a synergistic effeet was seen in eultures that had been stimulated with a mixture of TraT and gp4l[8] and this effeet was maximal when 40 ~g of TraT
was used in the eell eultures (Table 2). By contrast, a synergistic effect was observed in only three from twenty-five cases, when IL-2 was eombined with gp4118]
(Table 1). The effeet of another outer-membrane protein, OmpA, was alæo tested on PBMC eultures of one ::~asymptomatie, and one symptomatie individual, and in both eases a synergiætie effeet was ~een when OmpA was eo-eultured with gp41(8) (Table 1).
~: 20 In view of the impressive results obtained with gp4118] in the presenee of TraT, it was important to determine whether TraT would augment the T-eell response to another ~: HIV-derived peptide. The V3 loop peptide was eonsidered a suitable eandidate, as this peptide (La Rosa et al., Seienee 2~9: 932, 1990), the prineipal neutralising determinant of HIV-l, is eurrently in elinieal trials (Serip, No. 1703, 26, 1992). The results in Table 3 show that in all eight cases tested TraT augmented the proliferative responses to the V3 peptideO However, an inhibitory effeet was observed when PBNC from the eight individuals were eultured in the presenee of IL-2 and V3 peptide (Table 3). In summary, TraT was far more effeetive than IL-2, a lymphokine whieh haæ been trialled as an Immunotherapeutie (Rosenberg, Lotze and Mul , 1988), in augmenting the T-eell responses to the HIV-derived ~ .

PCT/AU92/0~2~;~
211542'1 peptides gp41t8] and the V3 loop.

(ii) TraT augments the in vitro T-cell prolif rative ~ onses to recall anti~ens such as Dightheria S toxoid (DT) ~nd Tetanus tQsoid (~T).

It is well esta~lished that lymphoproliferative responses to recall antigens are impaired even during the asymptomatic disease period when CD4-positive T-cell numbers are often only slightly reduced (Lane et al., 198S). Since TraT has been shown to augment HIV-specific lymphoproliferation in both symptomatic and asymptomatic individuals, it was reasonable to expect that it would have a similar effect in enhancing the response to recall 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
~ignificantly augmented DT - and T T- specific proliferative re~ponses (Table 4). This finding would make TraT an attractive immunomodulator molecule for rastoring defective T-cell responses, not only to HIV-derived antigens, but also to a range of recall 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.

`

W093/03762 ~ 4 2 ~1 PCT/AU92/~23 ~XAMPL~ 2 ElQ~Lçy$Q~çtriç-anslyuluLof T-cell subset di~tribution indicates th~t TraT preferentially ~tiDNlate~ CD4-positive helper T-cell~.

The T-cell ~ubset distribution of Peripheral blood mononuclear cells (PBMC), that had been stimulated with TraT, Interleukin-2 (IL-2) or with gp41t8], after a 6-day incubation, were analysed using immunofluorescence and f}ow cytometry.
The phenotypes of the T-cells in proliferating cultures of PBMC were compared with those from unstimulated cell cultures~

T-cell phenotype analysi~.
` `:

T~o-millilitre volumes of both stimulated and unstimulated PB~C (5 x 106 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-Hyp~que gradients (Pharmacia), and centrifuged at 800g for 10 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 phenotyped using dual combinations of fluorescent monoclonal antibodies ~ i . Q ., CD 3/4 and CD 3/8; Coulter Electronics Inc., Hialeah FL, V.S.A.). Two-thousand viable cells from each PBMC culture were assayed for surface-bound fluorescence. Using both the viable PBMC
count (i.e., after 6 days of culture with and without ~- stimulation), and the relative percentages of the ~, ~

~' WO 93/03762 PCI`/AU92/00423~.s~
2115424 ` `
_ 18 -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 subset~ were calculated. The formula used for the calculation of S subset numbers was: T-cell subset % (per 2,000 ~gated~
cells) x viable cell number (as determined by Trypan blue exclusion). Bitmaps were continually ad~usted to encircle the ma~ority of either stimulated or unstimulated lymphocytes. After 6 days of culture of purified PBMC, >
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 un~timulated cultures) when PBMC were cultured in the presence of TraT, gp4118]), or IL-2. However, when TraT
was combined with gp4118], 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 in CD8-positive T-cell numbers when PaNC 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 m~xture of TraT and gp41t8] in a 6-day ; culture. Another notable feature of these results is that 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~ wa~ even 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); gp41t8~, 0% (#125), 18% (#126); IL-2, 3~8% (#125), 216% ($126). However, there was a dramatic increase in CD4 cell numbers when ~ ~ ' W093/03762 2115 ~ 2 4 PcT/Au92/o~23 `; !

TraT was combined with gp41181: 737% (#125) and 1,763~
(#126) respectively. The percentage increase~ in CD4 were significantly higher than the corresponding increases for the CD8-positive population, i.e., 74% ($125) and 185%
(#126) (Tsble 5). The preferential increase in CD4-positive helper T-cells in cultures incubated with TraT, or with a combination of gp41t8~ and TraT, suggests that TraT will boost helper T-cell numbers in vivo and ~; thereby enable HIV-infected individuals to combat opportunistic infections. Further improvement in HIV-positive individuals would be obtained by combining TraT with anti-retroviral sgents such as zidovudine.
Industrial Application The current invention is applicable to the preparation of `~
vaccines designed to combat immunodeficiency disorders such as AIDS and to the treatment of patients ~uffering a deficiency in helper T-cell function in general.
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 ~pec~fic embodiments without departing from the spirit or scope of the invention as broadly described. The present embodiments ara, therefore, to be considered in all respects as illustrative and not restrictive.

~: ~

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WO 93/03762 211 ~ 4 2 ~ PCl`/AU92tO0423 : ! .

Do~e-Re~ponse effect_of TraT
on gp4118] - ~pecific T-cell proliferation Patient No. and Diagno~is Treatment~ 111 112 113 114 nild ARC AS AS AS

StLmulation Indices `~:
Tra$ - lOyg 3.0 4.3 7.8 16.5 TraT - 20yg 5.2 7.8 18.1 35.2 TraT - 40yg 8.0 9.0 29.0 56.4 TraT - 60yg 9.0 7.7 23.2 42.5 ~:: TraT - 80yg 5.4 6.8 14.7 25.7 ;~ gp41t8] 10.5 5 9 7.9 19.9 TraT (lOyg) + gp4118]13.3 25.9 20.2 38.6 TraT (20yg) + gp41~8]17.2 25.3 35.5 75.8 TRAT (40yg) + gp41~8]21.7 39.7 48.0 99.3 TRAT (60yg) + gp41~8]24.8 18.8 30.8 70.0 TRAT (80yg) + gp41~8]19.3 17.8 24.5 48.2 :

WO 93/03762 PCI'~AU92/0042~
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W O 93/03762 PC~r/AU92J00423 ~ 2 il542~ ~:

Th- au~-ent~tlon bv TrsT of T-cell prol~fer~t~vc re6pon~e~ to Diphtherla to~o~d lDT) ant Tetsnu~ to~ud tTT) Pat~ent No ~nd Dia~nos~

Rapos~'6 AS ~RC** ARC AS ~ild ARC
Tre~tmQnt6 S~rco~a tSt$mul~t~on Indic-s) TraTt40~g 10.4 5.7 7.2 12.2 12.1 11.2 DT(40~g) 6.5 5.3 3.3 5.1 6.2 6.1 DT(4yg~ 4.0 2.7 1.4 1.41 3.31 3.9 TT(SOyg) 4.5 5.4 2.8 3.7 5.5 3.0 TT(Syg) 3.0 2.8 1.6 2.7 3.1 2.7 TrsT+DT(40yg)37.6(S)* 27.0(S) 20.9(S) 24.0tS) 30.9(S) 30.2(S) TraS~DT(4~g)21.6~S) 18.7tS) 11.5(S) 21.6(S) 23.8(S) l9.0(S) TraT~TT (50y8) 34.0(S) 27.5(S)16.0tS) 29.5(S) 26.5(S) 21.1(S) TraTITT(5~g)21.3(S) 13.4(S) 11.2(S) l9.5(S) 21.1(S) 19.6(S) _ * S~nergistic effect AS - Asymptomat~c; ** ARC - AIDS-related comple~

WO 93/03762 PCl`/AU92/00423r. ~
21i542~

Flo~ cvto~etr~c analvsi6 of T-c-ll sub~t~ EL__t~on of prol~eratln~ and unsti~ulated cell6 afto~ a 6-d~y ~ncubatlon Pat~ent No. and D~a~no~6 ~123 - Kapo~ '8 Sarco~a* ~124 - ~ild ARC*
Ab601ute T-cell numbers Absolute T-cell numbers Treat~ent CD4 CD8 CD4 CD4 CD8 CD4 NIL 3.04xlO 2.7xlO 1.1 2.5xlO 2.4xlO 1.0 TraT 3.7xlO 2.28s106 1.6 3.0xlO . 2. 5Y10 1 . 2 gp41[8] 3.ax106 2.40~106 1.6 3.7x106 2.9x106 1.3 IL-2 3.9x106 2.5xlO 1.6 3.6x106 3.4x106 1.1 8]*TraT 4.9x106 2.9x106 1.7 4.9xlO~ 4~3ylO6 1.1 8]~IL-2 4.1~106 2.4x106 1.7 3.6~106 ~ 3.8x106 0.9 * CD4/CDa at day 0 was ~123-0.23; ~124-0.52 ~: :

W O 93/03762 211 5 4 2 -4 PC~r~AU92tO0423 TABL~ 6 ~low cvtometric analvsi6 of T-cell sub~et dl~tribut~Qn of proliferating a~d unst~mulated c~118 after ~ 6-da~ i~cub6tio~

Patlent No. and Diaenos~
#125 - ARC ~126 - ARC

Absolute T-cell numbers Absolute T-cell numbers Treatment CD4 CD8 CD4 CD4 CD8 CD4 NIL 92,0008B0,000 0.10 44,000780,000 0.06 TraT 145,0001,150,000 0.13 60,000l,S00,000 0.04 gp4118~54,000980,~00 0.06 S~,000550,000 0.09 IL-2 440,0001,160,000 0.38 139,0001,130,000 0.12 8]+TraT 770,000 1,530,000 0.50 820,000 2,220,000 0.37 [8~IL-229,0001,280,000 0.03 68,0001,310,000 O.OS

* CD41CD8 at da~ 0 vas ~125 = 0.125; tl26 - 0.09 WO 93/03762 PCI/AU92/0042~....~
211~2~ 26-REFERENCES
(for Immunostimulation Patent) Lafferty, K.J., S.J. Prowse, and C.J. Simeonovic. 1983.
Immunobiology of ti~sue transplantation: A return to the passenger leukocyte concept. ~nn. Rev. Immunol. 1: 43.
Mueller, D.L., M.K. Jenkins, and R.H. Schwartz. 1989.
Clonal expansion versus functional clonal inactivation: a costimulatory signalling pathway determines the outcome of T cell antigen receptor occupancy. Ann. Rev. Immunol.
7s 445.
Janeway, C.A. 1990. Approaching the asymptote:
Revolution and evolution in immunology. Cold Spring Harbor Symp. Ouant.~Biol. 54: 1.
Fahey, J.L., J.M.G. Taylor, R. Detels, B. Hofmann, R.
~- 15 Nelmed, P. Nishanian, and J. Giorgi. 1990. The prognostic value of cellular and serologic markers in infection with human immunodeficiency virus type 1. N.
~" EngI. J. Med. 322: 166.
Lange, J.M.A., F. de Wolf, and J. Goudsmit. 1989.
Narkers for progression in H.I.V. infection. ~1~ 3 (8uppl. 1): S153.
Hu, S-L., S.G. Xosowski, and J.M. Dalrymple. 1986.
Exprefision of AIDS virus envelope gene in recombinant vaccinia virus Nature 320: 537.
2S Kennedy, R.C., G.R. Dreesman, T.C. Chanh, R.N. Baswell, J.S. Allan, T.H. Lee, M. Essex, J.T. Sparrow, D.D. Ho, and P. Xanda. 1987. Use of a resin-bound synthetic peptide for identifying a neutralizing antigenic determinant associated with the human immunodeficiency virus envelope. J. Biol. Chem. 262: 5769.
Desrosiers, R., M. Wyard, T. Kodama, D.J. Ringler, K.
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. 86: 6353.
Bell, S.J.D., R. Doherty, B. Kemp, and D.A. Cooper.

W093/03762 2 11 5 ~ 2 I PCT/AU92/~23 1990. Exogenous IL-2 can re-instate T-cell proliferative re~ponse to HIV-l envelope and core-derived peptide8.
A.S.H.M. 4th National Conference on AIDS, ab~tract.
Bell, S.J.D., D.A. Cooper, B.E. Kemp, R.R. Doherty, and R.
S Penny. 1992. Definition of an immunodom~nant T-cell epitope contained in the envelope gp41 sequence of HIV-l Clin. Exp. Immunol. 87: 37. `
LaRosa, G.J., J.P. Davide, K. Weinhold, J.A. Waterbury, A.T. Profy, J.A. Lewis, A.J. Langlois, ~.R. Dreesman, R.N.
Bo~well, P. Shadduck, L.H. Holley, M. Karplus, D.P.
Bolognesi, T.J. Matthews, E.A. Emini, and S.D. Putney.
1990. Conserved sequence and structural elements in the HIV-l principal neutralizing determinant. Science 249:
932.
;~ 15 Ro~enberg, S.A., M.T. Lotze, and J.J. Nul , 1988. New ; approaches to the Immunotherapy of cancer using Interleukin-2. Ann. Intern. Med. 108: 853.
Lane, H.C., J.M. Depper, W.C. Greene, G. Nhalen, T.A.
aldmann, and Fauci, A.S. 1985. Qualitative analysis of immune function in patients with acquired immunodeficiency ~ syndrome. Evidence for a selective defect in soluble ; antigen recognition. N. Engl. J. Ned. 313s 79.
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W093/03762 PCT/AU92/~2~
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~: -: ~

Claims (32)

CLAIMS:-

1. A composition comprising in admixture a protein selected from the group consisting of TraT, OmpA, OmpF and parts thereof, at least one other antigen and a 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 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 antigens, hepatitis antigens, 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 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.

7. 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, 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 isolated from said antigen, and a 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
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.

11. A method of increasing immune responsiveness in a patient with an immunodeficiency, the method comprising 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.

12. A method as claimed in claim 11 in which the responsiveness of T-cells is increased in a patient with a deficiency in T-cell function.

13. A method as claimed in claim 12 in which the T-cells are helper T-cells and the patient has a deficiency in helper T-cell function.

14. A method as claimed in any one of claims 11 to 13 in which the composition further includes an effective amount of at least one other antigen.

15. A method as claimed in any one of claims 11 to 14 in which the pharmaceutically acceptable carrier is a hydrophobic depot carrier.

16. A method as claimed in any one of claims 11 to 15 in which the protein is TraT or a part thereof.

17. A method as claimed in any one of claims 14 to 16 in 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 antigens, hepatitis antigens, polio antigens, combinations thereof, and individual subunit proteins, peptides or polysaccharides isolated from said antigen.

18. A method as claimed in any one of claims 13 to 17 in which the antigen is a HIV antigen selected from gp41[8]
peptide and V3 loop a peptide.

19. A method as claimed in any one of claims 13 to 18 in which the protein and the at least one other antigen are in admixture.

20. A method as claimed in any one of claims 11 to 19 in which the patient is HIV positive.

21. A method as claimed in any one of claims 11 to 20 in which the protein is derived from E.coli.

22. 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 in the manufacture of a medicament for increasing immune responsiveness in a patient with a deficiency in immune function.

23. The use as claimed in claim 22 in which the medicament is for increasing the responsiveness of T-cells in a patient with a deficiency in T-cell function.

24. The use as claimed in claim 23 in which the T-cells are helper T-cells and the patient has a deficiency in helper T-cell function.

25. The use as claimed in any one of claims 22 to 24 in which the composition further includes an effective amount of at least one other antigen.

26. The use as claimed in any one of claims 22 to 25 in which the pharmaceutically acceptable carrier is a hydrophobic depot carrier.

27. The use as claimed in any one of claims 22 to 26 in which the protein is TraT or a part thereof.

28. The use as claimed in any one of claims 25 to 27 in which the at least one other antigen is selected from the group consisting of HIV antigens, influenza virus antigens, diphtheria antigens, whooping cough antigens, tetanus antigens, measles antigens, Pneumocystis antigens, Candida antigens, Toxoplasmosis antigens, Cytomegalovirus antigens, hepatitis antigens, polio antigens, combinations thereof and individual subunit proteins, peptides or polysaccharides isolated from said antigen.

29. The use as claimed in any one of claims 25 to 28 in which the antigen is a HIV antigen selected from gp41[8]
peptide and V3 loop peptide.

30. The use as claimed in any one of claims 25 to 29 in which the protein and the at least one other antigen are in admixture.

31. The use as claimed in any one of claims 22 to 30 in which the patient is HIV positive.

32. The use as claimed in any one of claims 22 to 31 in which the protein is derived from E. coli.