CZ28294A3 - 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

The invention relates to the use of costimulatory inducers to enhance or support the immune response in patients with T cell insufficiency, particularly helper T cells, and to novel compositions suitable for enhancing the reactivity of T cells, particularly helper T cells. More particularly, the invention relates to the use of the OmpA, OmpF or TraT outer membrane proteins of E. coli to enhance the immune response to antigens in immunocompromised individuals and compositions comprising these proteins.

BACKGROUND OF THE INVENTION

During the development of an immune response, a certain type of T cell, known as a helper T cell that carries the CD4 phenotype, is required to assist in B cell differentiation into a plasma cell, which then secretes a soluble antibody. Before their activation and proliferation, T helper cells must first recognize antigens on the surface of antigen-presenting cells (APCs), such as macrophages, dendritic cells or B cells, with the help of T cell receptors and other accompanying molecules. . More recent data is consistent with the hypothesis that T cells require two signals to activate. One signal is given as a result of binding of the peptide to the Major Histocompatibility Complex (MHC) class II molecule on APC and subsequent interaction of this MHC-peptide complex with the T cell receptor. The occupation of the T cell receptor and the associated biochemical consequences, although necessary, are not sufficient to induce T cell activation. Most cells require a second signal from APC or a co-stimulator (Lafferty,

K.J., Prowse, S.J., and Simeonovic, C.J., 1983, Immunobiology of Tissue Transplantation: A Return to the Passenger Leukocyte Concept, Ann. Roar. Immunol. 1, 43, Mueller, D.L., Jenkins, M.K., and Schwartz, R.H., 1989, Clonal expansion versus functional clonal inactivation: a costimulatory signaling pathway determining the outcome of T cell antigen receptor occupancy, Ann. Roar. Immunol. 7, 445). Bacterial products such as LPS and BCG, which have been found to induce costimulatory activity in APC, are known as costimulatory inducers (Janeway,

C.A., 1990, Approaching the Asymptote: Revolution and Evolution in Immunology, Cold Spring Harbor Symp. Quant Biol. 54,

1). Costimulatory inducers are reported to increase or improve the immune response of individuals suffering from immune deficiency syndromes that are, at least in part, associated with T helper cell deficiency.

Acquired Immune Deficiency Syndrome (AIDS) is a debilitating human disease characterized by high morbidity and mortality in infected individuals. This disease, characterized by an initial infection with lentivirus, human immunodeficiency virus (HIV), is diagnosed in its early stages by the presence of anti-HIV antibodies in the serum and / or by the presence of the virus in the serum of asymptomatic individuals. Almost without exception, these asymptomatic individuals continue to develop full AIDS with numerous of their associated complications that eventually lead to the death of the infected individuals.

During disease, HIV-positive individuals show a progressive decrease in their T-cell helper population [CD4-positive cells (CD4 ⁺ )] and an increase in the number of CD8 ⁺ cells. Along with the loss of CD4 ⁺ cells, infected individuals show a progressive loss of their ability to produce a protective immune response to HIV or to numerous opportunistic pathogens that may attack infected individuals. This chronic loss of helper (CD4 ⁺ ) T cells and the resulting impairment of cell-mediated immunity closely correlates with the progression of the disease towards AIDS (Fahey, JL, Taylor, JMG, Detels, R., Hofmann, B., Melmed, R., Nishinian , P., and Giorgi, J., 1990, The Prognostic Value of Cellular and Serology Markers in Human Immunodeficiency Virus Type 1 Infection, N. Engl, J. Med., 332, 166, Lange, JMA, de Volf, F. , and Goudsmit, J., 1989, Markers for Progression in HIV Infection, AIDS 3 (suppl. 1): S153).

In an effort to stop the spread of AIDS among at-risk individuals and to develop a method of treating infected individuals, numerous research groups have focused on developing an HIV vaccine. Thus, since the initial isolation of HIV about 10 years ago, numerous sophisticated virological and biotechnological approaches have been used to design and produce a variety of possible vaccines (Hu, S-L., Kosowski,

SG, and Dalrymple, JM, 1986, Expression of AIDS virus envelope gene in recombinant vaccinia virus, Nature 320: 537, Kennedy, RC, Dreesman, GR, Chanh, TC, Baswell, RN, Allan, JS, Lee, TH, Essex , Μ., Sparrow, JT, Ho,

D.D., and Kanda, P., 1987, Use of a resin-bound synthetic peptide for identifying and neutralizing antigenic determinants associated with the human immunodeficiency virus envelope, J. Biol. Chem. 262: 5769). Almost without exception, however, these proposed vaccines have failed in clinical trials. The failure of many of the vaccines proposed and the inexorable development of the disease in infected patients towards full AIDS have led many to pessimistic view that the development of an effective AIDS vaccine is almost impossible. Desrosiers and collaborators (Desrosiers, R., Vyard, M., Kodama, T., Ringler, DJ, Arthur, K., Sehgal, PK, Letvin, NL, King, NV, and Daniel, MD, 1989 do not share this view. (Vaccine protection against simian immunodeficiency virus infection, PNAS 86: 6353), who have recently reported 4 promising results of vaccinating rhesus monkeys with inactivated HIV preparations.

One of the main problems in developing an effective vaccine to combat already existing HIV infections is that HIV itself attacks and inactivates CD4 ⁺ helper T cells, which are cells that need to be stimulated to generate a protective immune response in an individual. During the normal immune response, activated CD4 ⁺ helper cells produce cytokines such as Interleukin-2 (IL-2), which are known to induce clonal proliferation of activated T cells, which may ultimately lead to the elimination of virally infected cells. However, it has been shown that addition of exogenous IL-2 to peripheral blood mononuclear cells (PBMCs) of HIV-seropositive individuals can restore both antigenic and mitogenic induced blastogenesis in vitro (Bell, SJD, Doherty, R., Kemp, B., and Cooper , DA,

1990, Exogenous IL-2 can re-instate T-cell proliferative response to HIV-1 envelope and core-derived peptides,

ASHM 4th National Conference on AIDS, abstract). Similar stimulation of PBMCs in vivo, leading! to produce CD4-derived IL-2 would help maintain the strong anti-viral immunity associated with CD8. Thus, it would seem advantageous to use therapies which result in an increase in the level of CD4 ⁺ T cells, particularly during the asymptomatic stages of the HIV-induced disease.

However, IL-2, which has been recommended for approval by the FDA (Stone R., anti-cancer drug IL-2 may finally be approved, Science 235, 528, 1992) as an immunotherapeutic for the treatment of renal cancer, has numerous interfering side effects including .. Thus, there is a clear need for an effective immunostimulant without 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 reactivity of helper T cells in HIV-infected individuals, thereby improving the individual's ability to lead an immune attack against HIV-infected cells and develop effective responses to opportunistic pathogens.

International Patent Application No. PCT / AU87 / 00107 (Barnes, TM, Lehrbach, P., and Russell-Jones, GJ, Immunopotentiation) discloses that in complexes with an immunogen, TraT, OmpA and OmpF act as effective immunoadjuvants in immunocompetent hosts. . However, there is no mention in this reference that TraT, OmpA or OmpF had the ability to increase T-helper cell reactivity.

The present inventors have surprisingly found that these proteins increase the reactivity of T helper cells of patients suffering from T helper cell insufficiency. In addition, we have discovered a synergistic effect of these proteins and other antigens on T helper cell reactivity.

SUMMARY OF THE INVENTION

Accordingly, the present invention provides a composition comprising in admixture a protein selected from the group consisting of TraT, OmpA, OmpF and portions thereof, at least one other antigen and a pharmaceutically acceptable carrier.

A second object of the invention is a composition comprising a protein selected from the group consisting of TraT, OmpA, OmpF and portions thereof, bound to an antigen selected from the group consisting of HIV antigens, influenza virus antigens, diphtheria antigens, pertussis antigens, measles antigens, tetanus antigens, antigens Pneumocystis, Candida antigens, Toxoplasmosis antigens, Cytomegalovirus antigens, hepatitis antigens, polio antigens, combinations thereof and individual subunit proteins, peptides or polysaccharides isolated from said antigens, and a pharmaceutically acceptable carrier.

A third object of the invention is a method of increasing immune reactivity in a patient with immune deficiency comprising administering a composition comprising an effective amount of a protein selected from the group consisting of TraT, OmpA, OmpF and portions thereof, and a pharmaceutically acceptable carrier.

A fourth object of the invention is the use of a composition comprising an effective amount of a protein selected from the group consisting of TraT, OmpA, OmpF and portions thereof, and a pharmaceutically acceptable carrier, diluent and / or excipient, in the manufacture of a medicament for enhancing immune reactivity in a patient with immune deficiency.

In a preferred embodiment of the invention, the T-cell reactivity is increased and the patient has T-cell insufficiency.

In another preferred embodiment of the invention, the T cells are helper T cells and the patient has insufficient helper T cell functions.

In a preferred embodiment of the invention, the pharmaceutically acceptable carrier is a hydrophobic depot carrier. Suitable depot carriers include alhydrogel, proteosomes, and liposomes.

In another preferred embodiment of the invention said at least one other antigen is selected from the group consisting of HIV antigens, influenza virus antigens, diphtheria antigens, pertussis 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 antigens. However, it is presently preferred that the at least one other antigen is an HIV antigen, diphtheria toxoid or tetanus toxoid, and preferably an HIV antigen selected from gp41 peptide [8] and V3 loop peptide.

In yet another preferred embodiment of the invention, the protein is TraT or a portion thereof.

TraT, OmpF and OmpA are the outer membrane proteins of gram-negative bacteria. The TraT protein is an outer membrane protein of certain E. coli strains which is responsible for the resistance of these strains to serum killing. The OmpA and OmpF proteins also belong to the same family of proteins. These proteins can be obtained from other gram-negative bacteria, such as E. coli or Salmonella. However, it is presently preferred that proteins are obtained from E. coli strains.

The studies cited in the present invention have shown the possibility of increasing the level and / or activity of helper T cells in individuals with T helper deficiency, suggesting that TraT, OmpF and OmpA from E. coli and parts thereof can function as costimulatory inducers separately from the costimulatory inducers BCG and LPS described by Janeway (1990), but with a similar function.

We have shown that the costimulatory inducer activity of the outer membrane proteins TraT and OmpA of E. coli can be used to stimulate helper T cells derived from

HIV-positive individuals, in the presence of antigens and particularly peptides derived from viral proteins or recall antigens such as diphtheria toxoid (DT) and tetanus toxoid (TT).

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

TraT, OmpF and OmpA can therefore be used as inducers of costimulatory activity in antigen-containing cells, thus stimulating helper T cells in inducing immune responses to, for example, HIV-derived antigens, and thus overcoming CD4-positive T cell inactivity in HIV-infected individuals. .

The clinical outcome of an increased number of helper T cells is an improved immune function, which further results in an increased ability of the individual to fight opportunistic infections.

The use of these molecules would greatly improve the efficacy of potential AIDS vaccines by stimulating helper T cell production. Alternatively, when used in conjunction with other antigens to which an individual has previously developed memory T cells, these molecules may increase the overall level of immunity of the individual.

The ability of these molecules to restore helper T cell function could also be used to increase helper T cell production in immune deficiency states, such as those that may result from a certain type of cancer, organ transplants and various autoimmune conditions.

The compositions of the invention are prepared by mixing, preferably by homogeneous mixing of TraT, OmpA or OmpF or a portion thereof.

TraT, OmpA or OmpF, which portion stimulates an antigen-containing cell to elicit a costimulatory signal for T helper cells, with a pharmaceutically acceptable carrier, diluent and / or excipient using standard methods of preparing pharmaceutical compositions.

Preferably, the method further comprises using at least one other antigen in the preparation of a pharmaceutical composition. The antigen may be an antigen against which it is desired to elicit an immune response in a patient. For example, HIV antigens may be used in AIDS patients. Other useful antigens include influenza virus antigens, diphtheria antigens, pertussis antigens, measles antigens, Pneumocystis antigens, Canidida antigens, Toxoplasmosis antigens, Cytomegalovirus antigens, combinations thereof and individual subunit proteins, peptides or polysaccharides isolated from said antigens.

The TraT, OmpA and OmpF proteins that can be used in accordance with the invention can be purified from publicly available wild-type E. coli strains that produce these proteins.

One such E. coli strain is ATCC 67331, which was deposited March 2, 1987 in the American Type Culture Collection, 12301 Parklawn Drive, Rockville MD 20852, USA. 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 other bacterial strains containing recombinant DNA molecules encoding the proteins and purified by a method appropriate to the site of production of the recombinant protein TraT, OmpA or OmpF.

If portions of these proteins that stimulate an antigen-containing cell are to be used to elicit costimulatory T-cell stimulatory activity, the desired portions can be identified and prepared as follows.

An intact molecule is used to identify the receptor that binds the molecule to the antigen-presenting cell. This intact molecule is then digested by standard protein digestion methods and the generated portions tested for binding to the identified receptor. Those portions that can bind and can stimulate the induction of costimulatory activity by an antigen-presenting cell are suitable for use in the compositions and methods of the invention.

As one skilled in the art will appreciate, homologues and analogs of TraT, OmpA and OmpF could be used with similar effect. The use of such homologs and analogs is therefore considered to be within the scope of this application.

As antigens, any antigen against which it is desired to elicit an immune response in an immunocompromised or immunosuppressed patient can be used in the compositions and methods of the invention.

Examples of these antigens include, for example, HIV antigens, such as the gp41 peptide [8], which can be used to stimulate HIV-specific lymphocyte blastogenesis in HIV-infected patients. Other antigens may include influenza virus antigens, diphtheria antigens, pertussis 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 compositions of the invention may be prepared by standard pharmaceutical methods.

If an antigen is to be used in the composition, it may be mixed in a reservoir with a costimulatory inducer. Alternatively, the antigen and the costimulatory inducer may be complexed by chemical conjugation, optionally using chemical modification and / or linking groups. In the case of protein antigens, the costimulatory inducer and antigen may be in the form of a condensed protein obtained by recombinant DNA methods.

In any case, it is important that the process of attaching the antigen to the costimulatory inducer does not damage the desired antigenicity or costimulatory inducer activity of TraT, OmpA, OmpF or portions thereof.

The costimulatory inducer or costimulatory inducer with the antigen may be formulated into a depot carrier. If both components are to be included, it is desirable to keep them together. This can be achieved with a depot carrier, and types of suitable depot carriers include alhydrogel, proteosomes, and liposomes. The compositions are prepared by standard methods appropriate to the carrier used.

If the costimulatory inducer is to be used without the antigen or if the costimulatory inducer is complexed or coupled with the antigen, traditional carriers other than depot can be used.

The composition of the invention is preferably administered to a patient parenterally by standard methods of parenteral administration.

Most often, 100 ng-10 mg of each costimulatory inducer and antigen is used per dose.

The precise dose and costimulatory inducer to antigen ratio used will depend on: (i) the type and nature (e.g., immunogenicity) of the antigen, (ii) the subject's genetic background, (iii) the immunological history of the subject, and , macrophage-mediated or granulocyte-mediated) to be modified.

The appropriate costimulatory inducer to antigen ratio can be determined by a qualified user by systematically varying the relative dose and costimulator to antigen ratios until the desired immune response is achieved.

It is recognized that a number of factors affect the determination of the appropriate dosage for a particular patient. These factors include age, weight, sex, general health, and concomitant diseases of the patient. Determination of the appropriate dose level for a particular patient is accomplished by standard pharmaceutical methods.

Patients contemplated for use of the methods and compositions of the invention are those with helper T cell function deficiencies, such as those suffering from disease states including autoimmune diseases, certain types of cancer and AIDS, and patients who develop immunosuppression during treatment of a particular disease. or a disease state, for example, transplant patients and cancer patients, treated chemotherapeutically or radiotherapeutically.

The method of the invention could generally be used to increase their helper T cell level, or it may be desirable to have specific antigens to increase helper T cell levels and protect the patient against specific infections that could be fatal in immunocompromise or immunosuppression.

The invention is primarily directed to the treatment of human patients, but is equally applicable to other animals. Thus, the term patient includes both human and other animals.

DETAILED DESCRIPTION OF THE INVENTION

BRIEF DESCRIPTION OF THE DRAWINGS The invention is illustrated by the following examples.

Example 1 (i) TraT enhances in vitro T cell proliferative responses induced by HIV-derived immunodominant synthetic peptides

A. Selection of HIV-derived peptide (gp41 [8])

Most human immunodeficiency virus type 1 (HIV-1) immunodominant sequences are encoded by hypervariable gene sequences, and regions that are highly conserved between HIV-1 strains are scattered between these sequences. Immunogenic viral proteins that exhibit minimal variation between strains and consistently elicit both a humoral and a cell-mediated immune response may be suitable components of a subunit vaccine. In this context, Bell and coworkers (Bell, SJD, Cooper, DA, Kemp, BE, Doherty, RR, and Penny, R., 1992, studied the definition of an immunodominant T-cell epitope contained in the enve14 lope gp41 sequence of HIV- 1, Clin. Exp. Immunol. 87: 37) HIV-seronegative subjects and HIV-infected individuals, classified as asymptomatic (AS), AIDS-related complex (ARC), or AIDS. In accordance with the CDC clinical classification system (Centers for Disease Control, 1986, Classification System for Human T-Lymphotropic Virus Type-III / Lymphadenopathy-Associated Virus Infections, Morbid. Mortal. Veekly Report 35: 334), HIV-infected AS subjects form a group II / III, ARC patients are group IVA / IVC2 and AIDS patients are group IVCI / IVD. The authors first investigated which of the three short synthetic peptides derived from the conserved gp 120 envelope sequences (amino acids 262-284), gp41 (amino acids 579-601), and the p17 core (amino acids 106-125) of the isolated HTLV-IIIg regions could induce in both HIV-infected individuals, both B cell and T cell response. Only gp41-derived sequences were immunogenic at the level of BiT cells. The gp41 region was further characterized using a series of overlapping synthetic peptides derived from the conserved gp41 envelope region (amino acids 572-613). The authors then identified an immunodominant dodecamer (amino acids 593-604, called gp41 [8]) that consistently elicited both a T-blastogenic and antibody response in asymptomatic HIV-seropositive individuals, to a lesser extent in ARC patients but not in AIDS patients.

B. Synthesis of HIV-1-derived peptides

Two peptides, gp41 [8] and loop V3, derived from the gp120 region of HIV-1, were synthesized according to the manufacturer's instructions on an Applied Biosystems type 430A peptide synthesizer. The peptides were purified by chromatography on Sephadex G-25 (Pharmacia) in 10% acetic acid and then by reverse phase HPLC on a VYDAC C-18 column using a linear gradient of 5 to 60% acetonitrile in 0.1% TFA. The sequences of synthesized peptides are as follows:

R-S-S-gp41 [8]: to improve the solubility of this peptide, the Arg-Ser-Ser sequence was added to the nitrogen end of gp41 [8], ie:

Arg-Ser-Ser-Leu-Gly-Ile-Trp-Gly-Cys-Ser-Gly-Lys-Leu-Ile-Cys loop V3:

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. Evaluation of human T cell proliferative responses

Peripheral blood mononuclear cells (PBMCs) were isolated by Ficoll-Paque gradient centrifugation (Pharmacia) and 200,000 PBMCs were cultured in 0.2 ml RPMI-160 medium (Cyto systems Pty, Ltd. containing 10% human serum AB, 50 mM penicillin, 50 mM streptomycin, 2.5 mM glutamine and 2 mM HEPES buffer pH 7.4) on 96-well round bottom microtiter plates in the presence of synthetic gp41 peptides [8] or V3 loop (2 μΜ), rIL-2 (10 µl) diphtheria toxoid (DT, Commonwealth Serum Laboratories, Melbourne, Australia, 1570Lf Units / ml, 4 and 40 pg / ml), tetanus toxoid (TT, Commonwealth Serum Laboratories, Melbourne, Australia, 10OLf Units / ml, 5 and 50 pg / ml), TraT and OmpA (purified according to Croft et al., 1991, 40 pg / ml, Croft, S., Valsh, J., Lloyd, V., and Russell-Jones, GJ). After 6 days of culture at 37 ° C (with or without antigen therefrom), each culture overnight pulsed with 50 pl ^and H-thymidine (20 pCi / ml, Amersham, UK), separated using a filter paper, glass fiber and subjected to measurement on a liquid scintillation spectrometer (Beckman, USA).

Results were expressed as stimulation index (SI), calculated according to equation of mean cpm with antigen - mean cpm without antigen

SI = - medium cpm without antigen where cpm is frequency per minute.

D. Definition of TraT- and IL-2-mediated effects on lymphoproliferation specific for gp41 peptides [8],

V3, diphtheria toxoid (DT) or tetanus toxoid (TT)

The effect of TraT and IL-2 on the proliferative response (expressed as a stimulatory index) on various HIV-derived antigens and recall antigens (DT and TT) was defined by a modified version of the known formula (Denz, Η., Lechleitner, M., Marth , C., Daxenbichler, G., Gastl, G., and Braunsteiner, Η., 1985, Effect of Human Recombinant and -2 and G-Interferon on the Growth of Human Cell Lines from Solid Tumors and Hematology Malignancies, J. Interferon Res. 5: 147)

Additive effect was defined as (AB) -: - =!

(A) + (B)

The synergistic effect was defined as (AB)

->!

(A) + (B)

The inhibitory effect was defined as (AB)

- <1 (A) + (B) where (A) and (B) represent a proliferative response to individual agents (eg gp41 [8] and TraT, respectively) and (AB) is a lymphoproliferative response observed when a combination of both agents is added to the same cultures.

Results:

Addition of TraT to PBMC cultures from eleven asymptomatic (AS) individuals, thirteen ARC patients and one AIDS lymphoma patient increased T cell proliferative response to gp41 peptides [8] to a higher level than IL-2 addition to cultures containing gp41 peptide [8] (Table 1).

With the exception of patient # 110, in any case, a synergistic effect was observed in cultures that were stimulated with a mixture of TraT and gp41 [8], and this effect was maximal when 40 pg of TraT was used in cell culture (Table 2). In contrast, when combining gp41 [8] with IL-2, a synergistic effect was observed in only three out of 25 cases (Table 1). The effect of another outer membrane protein, OmpA, was also tested on PBMC cultures from one asymptomatic and one symptomatic individual, and in both cases a synergistic effect was observed when OmpA was co-cultured with gp41 [8] (Table 1).

In view of the remarkable results obtained when combining gp41 [8] with the presence of TraT, it was important to determine whether TraT increased T cell response to other HIV-derived peptides. Loop peptide V3 was chosen as a suitable candidate (La Rosa, GJ, David, JP, Veinhold, K., Vaterbury, JA, Profy, AT, Lewis, JA, Lang18 lois, AJ, Dreesman, GR, Boswell, RN, Shadduck, P., Holley, LH, Karplus, M., Bolognesi, DP, Matthews, TJ, Emini, EA, and Putney, SD, 1990, Conserved sequence and structural elements in the HIV-1 principal neutralizing determinant, Science 249: 932), which is the major neutralizing 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 the TraT tested increased the proliferative response to the V3 peptide. However, an inhibitory effect was observed when PBMCs from these eight individuals were cultured in the presence of IL-2 and V3 peptide (Table 3). In summary, TraT was much more effective than IL-2, a lymphokine that was tested as an immunotherapeutic (Rosenberg, SA, Lotze, MT, and Mul, in increasing T cell responses to HIV-derived gp41 [8] and V3 peptides). JJ, 1988, New approaches to Immunotherapy of cancer using Interleukin-2, Ann. Intern,

Copper. 108: 853).

(ii) TraT enhances T cell proliferative responses in vitro to recall antigens such as diphtheria toxoid (DT) and tetanus toxoid (TT)

It is generally accepted that the lymphoproliferative response to recall antigens deteriorates during the asymptomatic stage of the disease, where CD4-positive T cell counts are often only slightly reduced (Lane, HC, Depper, JM, Greene, VC, Vhalen, G., Valdman, TA, and Fauci, AS, 1985, Qualitative Analysis of Immune Function in Patients with Acquired Immunodeficiency Syndrome (Evidence for Selective Defect in Soluble Antigen Recognition, N. Engl. J. Med. 313: 79). Since TraT was shown to increase HIV-specific lymphoproliferation in both symptomatic and asymptomatic individuals, it was expected to have a similar effect in enhancing the 19 responses to recall antigens. The ability of TraT to enhance the response to recall antigens would be of clinical importance in boosting immunity and helping immunocompromised individuals to combat opportunistic infections. In all six patients studied (Nos. 123 to 128), TraT significantly enhanced the DTi TT-specific proliferative response (Table 4). Thanks to this finding, TraT has become an attractive immunomodulatory molecule for restoring defective T cell reactivity not only to HIV-derived antigens but also in the field of recall antigens.

In clinical terms, a molecule with costimulatory inducer-like properties, such as TraT, would be an attractive immunotherapeutic to support general immunity in immunocompromised individuals.

Example 2

Flow cytometric analysis of T cell subset distribution shows that TraT preferentially stimulates CD4-positive T helper cells

The distribution of a subset of peripheral blood mononuclear cell (PBMC) T cells stimulated with TraT, Interleukin-2 (IL-2) or gp41 [8] was analyzed by immunofluorescence and flow cytometry after an incubation day.

The phenotypes of T cells in proliferating cultures of PBMC were compared to those of unstimulated cell cultures.

T cell phenotype analysis with ml volumes of stimulated and unstimulated PBMC (5 x 10 6 cells / ml) were cultured for 6 days under the same conditions as in Example 1. After 6 days of culture, the pelleted cells were resuspended and the cell suspension was plated on 1 ml Ficoll-Hypaque gradi20 ent. (Pharmacia) and centrifuged for 10 min at 800 g. Viable (assessed by Trypan blue exclusion) lymphoblastoid cells were harvested from the interface and washed in Hank's equilibrium salt solution (HBSS, Cytosystems, Pty. Ltd.) at pH 7.4. Viable cells that were isolated from unsorted PBMC cultures were phenotyped using double combinations of fluorescent monoclonal antibodies (ie, CD 3/4 and CD 3/8, Coulter Electronics Inc, Hialeah FL, USA).

Two thousand viable cells were tested for surface bound fluorescence from each PBMC culture. Using both the number of viable PBMCs (ie after 6 days of stimulation with and without pacing) and the relative proportions of the respective T cell subsets gated in each 2000 cell bitmap, the absolute T cell counts from the subsets were calculated.

CD4-positive and CD8-positive T cells. The formula was used to calculate the number of subsets:% subset of T cells (per 2000 gated cells) x number of viable cells (determined by Trypan blue exclusion). The bitmaps were continually adjusted to include most of either stimulated or unstimulated lymphocytes. After 6 days of cultivation of purified PBMCs, and 90% of viable cells were consistently found to be CD3-ingestive, indicating that they were predominantly T cells.

When PBMCs were cultured in the presence of TraT, gp41 [8] or IL-2, there was a 23-48% increase in absolute CD4-positive T cell counts. However, when TraT was combined with gp41 [8], the number of CD4-positive T cells increased by 63% (# 123), respectively. by 96% (No 124) (Table 5). In contrast, there was a slightly lower (0-42%) increase in the number of CD8-positive T cells when PBMCs were incubated in the presence of any of the three stimulators, which increased to 10% (# 123) and 79% (# 124). ) when PBMCs were exposed to an Traen-gp41 blend [8]. Another noteworthy feature of these results is that following an en day incubation with various stimulators, there was a significant increase in the CD4 / CD8 ratio compared to the CD4 / CD8 ratio measured immediately after PBMC isolation.

The synergistic effect between TraT and gp41 [8] was even more pronounced when these stimulators were tested for PBMCs from patients # 125 and 126 (Table 6). In these two patients with ARC, the percentage increments of CD4 cell counts were: TraT - 58% (# 125), 36% (# 126), gp41 [8] - 0% (# 125), 18% (# 125). 126), IL-2 - 378% (# 125), 216% (# 126). However, there was a dramatic increase in CD4 cell counts when TraT was combined with gp41 [8]; 737% (No. 125), respectively. 1763% (No. 126). The percent CD4 increments were significantly higher than the corresponding CD8-positive population increments, ie 74% (# 125) and 185% (# 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 may increase helper T cell numbers in vivo, helping HIV-infected individuals to fight opportunistic infections . Further improvement in HIV-positive individuals can be achieved by combining TraT with antiretroviral agents such as zidovudine.

Table 1. Increase in gp41 [8] -specific proliferative response

T cells using TraT Treatment (Stimulation Index)

Patient No diagnosis Track sp41f 81 IL-2 ep4ir81 + IL-2 100 ALIGN! AIDS-lymphoma 8.0 0.6 1,2 1.0 (AND)* 101 moderate ARC 0.6 2.2 0.4 10.9 (WITH)' 102 moderate ARC 0.9 2.5 2.0 1.6 (AND) 103 moderate ARC 3.3 3.3 2.6 1.1 (AND) 104 AS 8.5 52.1 26.2 nt 105 AS 26.6 66.7 27.4 nt 106 AS 13.4 12.8 11.7 nt 107 AS 13.1 15.8 41.1 nt 108 AS 3.0 2.0 4.9 nt 109 AS 3.5 3.6 6.8 nt 110 moderate ARC 7.2 5.6 2.9 nt 111 moderate ARC 8.0 10.5 6.3 14.0 (AND) 112 AS 9.0 5.9 8.5 16.3 (WITH) 113 ARC 29.0 7.9 12.0 18.1 (AND) 114 AS 56.4 19.9 22.1 36.0 (AND) 115 ARC 6.9 12.2 29.1 44.1 (WITH) 116 ARC 8.0 9.0 23.5 34.8 (AND) 117 ARC 8.0 13.1 28.5 43.8 (AND) 118 AS 12.0 7.0 13.5 22.0 (AND) 119 AS 9.9 3.5 0.3 11.4 (AND) 120 moderate ARC 42.8 32.5 67.7 22.0 (AND) 123 ARC - Kaposi sarcoma 10.4 6.3 8.5 13.8 (AND) 124 AS 5.7 6.2 6.1 12.4 (AND) 125 ARC 7.2 4.3 4.4 7.4 (AND) 126 ARC 12.2 6.9 8.7 12.4 (AND) Patient No gp41i81 + TraT OmnA gp4ir8l + OmpA

100 ALIGN!

21.5 (S) mt mt

101 17.8 (WITH) nt nt 102 7.0 (WITH) nt nt 103 21.2 (WITH) nt nt 104 163.0 (WITH) nt nt 105 252.0 (WITH) nt nt 106 50.5 (WITH) nt nt 107 48.7 (WITH) nt nt 108 15.9 (WITH) nt nt 109 28.3 (WITH) nt nt 110 13.1 (AND)** nt nt 111 21.7 (WITH) nt nt 112 39.7 (WITH) nt nt 113 48.0 (WITH) 16.0 29.3 (WITH) 114 99.3 (WITH) 17.9 42.8 (WITH) 115 33.7 (WITH) nt nt 116 23.4 (WITH) nt nt 117 32.8 (WITH) nt nt 118 34.5 (WITH) nt nt 119 21.9 (WITH) nt nt 120 109.7 (WITH) nt nt 123 50.0 (WITH) nt nt 124 38.2 (WITH) nt nt 125 24.1 (WITH) nt nt 126 32.9 (WITH) nt nt

I - inhibitory effect ´S - synergistic effect ** A additive effect nt - not tested (the terms are explained in example 1)

Table 2. Effect of TraT on gp41 [8] -specific T cell proliferation (dose-response)

Patient No and Diagnosis

111 moderate ARC 112 AS 113 AS 114 AS treatment stimulating index TraT (10 gg) 3.0 4.3 7.8 16.5 TraT (20 gg) 5.2 7.8 18.1 35.2 TraT (40 gg) 8.0 9.0 29.0 56.4 TraT (60gg) 9.0 7.7 23.2 42.5 TraT (80gg) 5.4 6.8 14.7 25.7 gp41 [7] 10.5 5.9 7.9 19.9 TraT (10 gg) + gp41 [9] 13.3 25.9 20.2 38.6 TraT (20 gg) + gp41 [9] 17.2 25.3 35.5 75.8 TraT (40gg) + gp41 [8] 21.7 39.7 48.0 99.3 TraT (60gg) + gp41 [8] 24.8 18.8 30.8 70.0 TraT (80gg) + gp41 [8] 19.3 17.8 24.5 48.2

Table 3. Increase of V3-specific T cell proliferative response by TraT treatment (stimulation index)

Patient No diaenosis Track V3 IL-2 V3 + IL-2 V3 + TraT 125 ARC 7.2 3.8 4.4 6.9 (I) 18.7 (S) 126 AS 12.2 5.6 8.7 10.6 (I) 26.0 (S) 127 AS 12.1 6.3 8.5 10.0 (I) * 30.9 (S) 128 moderate ARC 4.6 6.7 8.1 11.8 (I) 36.9 (S) 129 AS 3.7 2.7 4.1 3.7 (I) 21.0 (S) 130 AS 5.5 4,5 6.7 5.7 (I) 30.9 (S) 131 ARC - Kaposi sarcoma 5.4 4.4 6.9 5.7 (I) 30.9 (S) 132 ARC 4.1 5.0 5.7 6.3 (I) 22.6 (S)

inhibitory effect synergistic effect

Table 4. Increase of T cell proliferative response to

Diphtheria toxoid (DT) using TraT and tetanus toxoid Patient No 123 and diagnosis 124 125 126 treatment Kaposi's sarcom AS ARC ** ARC

stimulation index

TraT (40 pg) 10.4 5.7 7.2 12.2 DT (40 pg) 6.5 5.3 3.3 5.1 DT (3 pg) 4.0 2.7 1.4 1.41 TT (50 pg) 4,5 5.4 2.8 3.7 TT (5 pg) 3.0 2.8 1.6 2.7 TraT + DT (40 pg) 37.6 (S) * 27.0 (S) 20.9 (S) 24.0 (S) TraT + DT (3 pg) 21.6 (S) 18.7 (S) 11.5 (S) 21.6 (S) TraT + TT (50 pg) 34.0 (S) 27.5 (S) 16.0 (S) 29.5 (S) TraT + TT (5 pg) 21.3 (S) 13.4 (S) 11.2 (S) 19.5 (S)

127 AS 128 moderate ARC 12.1 11.2 6.2 6.1 3.31 3.9 5.5 3.0 3.1 2.7 30.9 30.2 (S) 23.8 (S) 19.0 (S) 26.5 (S) 21.1 (S) 21.1 (S) 19.6 (S) - * synergistic effect

AS - asymptomatic ** ARC - AIDS-related complex

Table 5. Flow cytometric analysis of the distribution of proliferating and unstimulated cells of a subset of T cells after an δ day incubation

Patient No and Diagnosis

123 - Kaposi's sarcoma * 124 - mild ARC *

absolute number T cells absolute number T cells treatment CD4 CD8 CD4 CD4 CD8 CD4 NILE 3,04.10 ' ⁶ 2.7.10 ⁶ 1.1 2.5.10 ⁶ 2.4.10 ⁶ 1.0 Track 3.7.10 ⁶ 2,28.10 ⁶ 1.6 3.0.10 ⁶ 2.5.10 ⁶ 1,2 gp41 [7] 3,8.10 ⁶ 2,40.10 ⁶ 1.6 3.7.10 ⁶ 2.9.10 ⁶ 1.3 IL-2 3,9.10 ⁶ 2,5.10 ⁶ 1.6 3.6.10 ⁶ 3.4.10 ⁶ 1.1 [8] + TraT 4,9.10 ⁶ 2,9.10 ⁶ 1.7 4.9.10 ⁶ 4.3.10 ⁶ 1.1 [8J + IL-2 4.1.10 ⁶ 2.4.10 ⁶ 1.7 3.6.10 ⁶ 3.8.10 ⁶ 0.9 * CD4 / CD8 on day 0 was 0.23 µ No. 123, 0.52 for No. 124

Table 6, Flow Cytometric Analysis of Distribution of Proliferating and Unstimulated Cells of a Subset of T Cells After an Incubate Incubation

Patient No and Diagnosis

125 - ARC 126 - ARC absolute T cell count absolute T cell count

treatment CD4 CD8 CD4 / CD8 CD4 CD8 CD4 / CD8 NILE 92000 880000 0.10 44000 780000 0.06 Track 145000 1150000 0.13 60000 1500000 0.04 gp41 [7] 54000 980000 0.06 52000 550000 0.09 IL-2 440000 1160000 0.38 139000 1130000 0.12 [8] + TraT 770000 1530000 0.50 820000 2220000 0.37 [81 + IL-2] 29000 1280000 0.03 68000 1310000 0.05

CD4 / CD8 on day 0 was 0.125 at # 125, 0.09 at. 126

Industrial applicability

The invention is useful in the preparation of vaccines intended to combat immune disorders such as AIDS and to treat patients with T helper deficiency in general.

It will be apparent to those skilled in the art that the invention described in particular embodiments may be varied and / or modified in various ways without departing from the broad description or scope of the invention. The embodiments described are therefore to be considered in all respects as illustrative and not restrictive.

Claims (21)

PATENT CLAIMS A composition comprising in a mixture a protein selected from the group consisting of TraT, OmpA, OmpF and portions thereof, at least one additional antigen and a pharmaceutically acceptable carrier. The composition of claim 1, wherein the pharmaceutically acceptable carrier is a hydrophobic depot carrier. The composition of claim 1 or 2, wherein the at least one additional antigen is selected from the group consisting of HIV antigens, influenza virus antigens, diphtheria antigens, pertussis antigens, measles antigens, tetanus antigens, Pneumocystis antigens, Candida antigens, antigens Toxoplasmosis, Cytomegalovirus antigens, hepatitis antigens, polio antigens, combinations thereof and single subunit proteins, peptides or polysaccharides isolated from said antigens. A composition according to claim 3, characterized in that. wherein at least one further antigen is an HIV antigen selected from gp41 peptide [8] and V3 loop peptide. Composition according to any one of claims 1 to 4, characterized in that the protein is derived from E. coli. The composition of any one of claims 1 to 5, wherein the protein is TraT or a portion thereof. 7. A composition comprising a protein selected from the group consisting of TraT, OmpA, OmpF and portions thereof, bound to an antigen selected from the group consisting of HIV antigens, influenza virus antigens, diphtheria antigens, pertussis antigens, measles antigens, tetanus antigens. antigens, Pneumocystis antigens, Candida antigens, Toxoplasmosis antigens, Cytomegalovirus antigens, hepatitis antigens, polio antigens, combinations thereof and single subunit proteins, peptides or polysaccharides isolated from said antigen, and a pharmaceutically acceptable carrier. The composition of claim 7, wherein the antigen is selected from the group consisting of HIV antigens, diphtheria toxoid, and tetanus toxoid. The composition of claim 8, wherein the HIV antigen is gp41 peptide [8] or V3 loop peptide. A composition according to any one of claims 7 to 9, wherein the protein is TraT or a portion thereof. Use of a composition comprising an effective amount of a protein selected from the group consisting of TraT, OmpA, OmpF and portions thereof, and a pharmaceutically acceptable carrier, for the manufacture of a medicament for enhancing the immune response capacity of a patient with immune deficiency. The use of claim 11, wherein the medicament is for enhancing the ability of a T cell response in a patient with T cell insufficiency. The use of claim 12, wherein the T cells are helper T cells and the patient has a helper T cell function. The use of any one of claims 11 to 13, wherein the composition further comprises an effective amount of at least one additional antigen. Use according to any one of claims 11 to 14, wherein the pharmaceutically acceptable carrier is a hydrophobic depot carrier. Use according to any one of claims 11 to 15, wherein the protein is TraT or a portion thereof. Use according to any one of claims 14 to 16, wherein the at least one additional antigen is selected from the group consisting of HIV antigens, influenza virus antigens, diphtheria antigens, pertussis 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. The use of any one of claims 14 to 17, wherein the antigen is an HIV antigen selected from gp41 peptide [8] and loop peptide V3. The use of any one of claims 14 to 18, wherein the protein and the at least one other antigen are in admixture with each other The use of any one of claims 11 to 19, wherein the patient is HIV positive. Use according to any one of claims 11 to 20, wherein the protein is derived from E. coli.