HU210605A9 - 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

FIELD OF THE INVENTION

The present invention relates to the use of costimulation inducers to improve or enhance the immune response of patients with T-cell deficiency, in particular helper T-cell deficiency, and to novel formulations which are capable of responding to T-cells, in particular helper T-cells. can be used to increase. More particularly, the present invention relates to the use of OmpA, OmpF or TraT E.coli outer memrane proteins in immunologically compromised patients for enhancing the immune response to antigens, and to compositions containing these proteins.

BACKGROUND OF THE INVENTION

During the development of the immune response, a certain type of T cell, the so-called helper T cell bearing the CD4 phenotype, is required for the B cells to differentiate into plasma cells, which then secrete soluble antibodies. Prior to helper T cell activation and proliferation, helper T cells must first recognize antigens by antigen presenting cells (APCs), such as macrophages, dendritic cells, or other complementary molecules. on the surface of B cells. Recent data support the hypothesis that T cells require two signals to be activated. One signal is generated when a peptide binds to the Class II major histocompatibility complex (MHC) on the APC and this MHC peptide complex reacts with the T-body receptor. Although this is a necessary condition, the T cell receptor occupied by the MHC peptide and the associated biochemical consequences are not sufficient to induce T cell activation. Most cells must be provided with a second signal or costimulatory molecule by APC (Lafferty, Prowse, & Simeonovic, 1983, Mueller, Jenkins, & Schwartz, 1989). Certain bacterial products, such as LPS or BCG, which have been shown to induce constitutive activity from APC, are known as costimulation inducers (Janeway, 1990). Consimulation inducers have been hypothesized to enhance or improve immune selection in patients with immune deficiency syndromes. Immunodeficiency syndromes are associated, at least in part, with a lack of helper cells.

Acquired Immunodeficiency Syndrome (AIDS) is a debilitating disease in humans characterized by a high rate of morbidity and mortality in infected patients. The disease, initially caused by a lentiviral infection, the Human Immunodeficiency Virus (HIV), can be diagnosed at an early stage by antibodies to HIV in the serum and / or by detection of HIV in the serum of asymptomatic patients. Almost without exception, these asymptomatic patients also develop complete AIDS, with many associated complications that eventually lead to the death of the infected patient.

During the course of the disease, the helper T-cell population of HIV positive patients [CD4-positive cells (CD4 ₊ )] gradually decreases, while with the loss of CD8 ⁺ cells, infected patients gradually lose their ability to develop a protective immune response to HIV. or a number of opportunistic pathogens that can attack infected patients. This chronic depletion of helper (CD4 ⁺ ) T cells and the consequent impairment of cell-mediated immunity are closely related to the progression of the disease to AIDS (Fahey et al., 1990, Lángé et al., 1989).

In order to stop the spread of AIDS among vulnerable individuals and to develop a cure for infected individuals, several complex virology and biotechnology approaches have been tried since the isolation of several research teams to isolate HVV about 10 years ago to design a multitude of imaginable vaccines. (Hu et al., 1987, Kennedy et al., 1987). Vaccine candidates were almost invariably unsuccessful in clinical trials. The ineffectiveness of many experimental vaccines and the unstoppable progression of the disease towards full-fledged AIDS have led many to take the pessimistic view that developing an effective AIDS vaccine is nearly impossible. However, this view has not been shared by Desrosiers et al. (1989), who recently reported promising results in vaccinating Rhesus monkeys with inactivated HIV formulations.

One of the major problems in developing an effective vaccine to combat existing HIV infections is that HIV itself captures and inactivates CD4 ⁺ helper T cells, although these cells alone need to be stimulated in order for a patient to have a protective immune response. During normal immune response, activated CD4 ⁺ helper cells produce cytokines, such as interleukin-2 (IL-2), which are known to initiate clonal proliferation of activated T cells, which can eventually lead to the removal of virus-infected cells. However, it has been shown that the addition of exogenous IL-2 to peripheral blood mononuclear cells (PBMCs) in HIV-positive patients can restore either antigen or mitogen-induced blastogenesis in vitro (Bell et al., 1990). ). Similar in vivo stimulation of PBMCs, which results in the production of IL-2 from CD4, would contribute to maintaining antiviral immunity associated with strong CD8. Therefore, it would seem worthwhile to introduce therapies that would lead to elevated CD4 ⁺ Tymphocyte levels, especially in the asymptomatic phase of HIV-induced disease.

However, IL-2, which has been recommended by the FDA (Food and Drug Administration) as an immunotherapeutic drug, has been tested [R. Stone, Science, 255, 528 (1992)] has a number of disturbing side effects, such as ... "circulatory problems,

EN 210 605 A9 which can be severe heart attacks or chills' ... So, obviously, there is a need for an effective immune stimulation that has no undesirable side effects such as those attributed to IL-2.

One approach to improving the prognosis of HIV-infected patients would be to either increase the absolute number of helper T cells or improve their responsiveness in HIV-infected patients. This would enable the patient to initiate an immunological attack on HIV-infected cells and develop effective responses against opportunistic pathogens.

International Patent Application APCT / AU87 / 00107 discloses that TraT, OmpA and OmpF behave as efficient immune adjuvants in immunocompetent hosts in immunogen complexes. However, this reference does not imply that TraT, OmpA, or OmpF has any skill in improving the responsiveness of helper T cells.

We have surprisingly found that these proteins improve the responsiveness of helper T cells from patients with helper T cell deficiency. In addition, it has been found that the synergistic effect between these proteins and other antigens has a synergistic effect on helper T cell responsiveness.

Summary of the Invention

Accordingly, the present invention first provides a composition comprising TraT, OmpA or OmpF, or portions thereof, in admixture with at least one other antigen and a pharmaceutically acceptable carrier.

The invention further relates to a composition wherein TraT or the OmpA or OmpF protein or portions thereof are linked to an antigen selected from the group consisting of HIV antigens, influenza virus antigens, diphtheria antigens, pertussis bacillus antigens, measles antigens, tetanus antigens , Pneumocystis antigens, Candida antigens, Toxoplasma antigens, cytomegalovirus antigens, hepatitis antigens, polio antigens, combinations thereof, or individual subunit proteins, peptides or polysaccharides isolated from said antigens, further comprising a pharmaceutically acceptable carrier.

Thirdly, the invention relates to a method of improving the immune response of patients with immunodeficiency, comprising administering to the patient a composition comprising an effective amount of TraT or OmpA or OmpF protein or a portion thereof and a pharmaceutically acceptable contains an acceptable carrier.

A fourth aspect of the present invention is the use of a composition for improving the immune response of immunodeficient patients comprising an effective amount of TraT or OmpA or OmpF protein, or a portion thereof, together with a pharmaceutically acceptable carrier, diluent and / or excipient.

In a preferred embodiment of the invention, the T cells are helper T cells and the patient is suffering from helper T cell function.

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 a further preferred embodiment of the invention, the at least one other antigen is selected from the group consisting of HIV antigens, influenza virus antigens, diphtheria antigens, pertussis bacillus antigens, measles antigens, tetanus antigens, Pneumocystis antigens, Candida antigens, Toxoplasma antigens, hepatitis antigens, polio antigens, combinations thereof, or 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, a diphtheria toxoid, or a tetanus toxoid, and it is most preferred that the HIV antigen is selected from the gp41 [8] peptide and the V3 loop peptide.

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

Outer membrane proteins of TraT, OmpA and OmpF Gram negative bacteria. TraT protein is an outer membrane protein of certain E.coli strains, which is responsible for the resistance of these strains, ie it is not destroyed by serum. OmpA and OmpF also belong to the same protein class. These proteins can be isolated from Gram negative bacteria, such as E.coli or Salmonella. Presently, it is preferred that these proteins are prepared from E.coli strains.

Research related to the present invention has shown the possibility of raising and / or increasing the level of helper T cells in patients with helper T cell function, indicating that CostTimulation of TraT, OmpA and OmpF from E. coli or portions thereof can act as inducers independently of BCG or LPS costimulation inducers (Janeway, 1990), but have similar functions.

The present inventors have demonstrated that the costimulation induction activity of E. coli-derived TraT, OmpF, and OmpA outer membrane proteins can be used to enhance the stimulation of helper T cells from HIV-positive patients in the presence of antigen, particularly peptides or priming proteins derived from viral proteins. Thus, in the presence of diphtheria toxoid (DT) and tetanus toxoid (TT).

Unlike BCG and LPS, TraT, OmpA and OmpF have no undesirable side effects such as endotoxic shock and injection site granuloma.

TraT, OmpF and OmpA can thus be used as inducers of costimulatory activity of antigen presenting cells: they stimulate helper T cells to elicit immune responses3.

HU 210 605 A9, for example, against a number of HIV-derived antigens and thus overcoming the non-response of CD4-positive T cells in an HIV-infected patient.

The clinical consequence of the increased number of helper T cells is improved immune function, which will then result in the patient being more effective in resisting opportunistic infections.

These molecules greatly improve the effectiveness of the AIDS vaccines to be developed by stimulating the production of helper T cells. Alternatively, when used in combination with other antigens that previously produced T-cells in the patient, these molecules raise the overall level of immunity of the patient.

The ability of these molecules to repair helper T-cell function can also be used to enhance the production of helper T-cells in immune deficient states resulting from certain types of cancer, organ transplants and various autoimmune processes.

The compositions of the invention are prepared by mixing TraT, OmpA or OmpF, or a portion of Trat, OmpA or OmpF, that stimulates the antigen-presenting cell to provide a costimulatory signal to the helper T-cell, using conventional drug manufacturing techniques. , preferably homogenized with a pharmaceutically acceptable carrier, diluent and / or excipient.

Preferably, the process for preparing the pharmaceutical composition also comprises the use of at least one other antigen. This antigen may be an antigen against which an immune response is desired in the patient. For example, AIDS patients may use HIV antigens. Other useful antigens include, but are not limited to, influenza virus antigens, diphtheria antigens, pertussis bacillus antigens, measles antigens, tetanus antigens, Pneumocystis antigens, Candida antigens Toxoplasma antigens, cytomelagovirus antigens, hepatitis antigens, polio antigens, or combinations thereof, , peptides or polysaccharides.

The TraT, OmpA and OmpF proteins useful in the present invention can be purified from commonly available E. coli strains producing such proteins.

One such E.coli strain is strain ATCC 67331, deposited March 2, 1987, at the American Type Culture Collection (12301 Parklawn Drive, Rockville, MD., 20852, USA). Purification of TraT, OmpF and OmpA from E. coli is disclosed in International Patent Application PCT / AU87 / 00107 (WO 87/06590).

These proteins can also be prepared from other bacterial strains that contain recombinant DNA molecules encoding these proteins and purified by methods suitable for the production of recombinant TraT, OmpA or OmpF protein.

If portions of these proteins which stimulate antigen presenting cells to generate a stimulatory activity for helper T cells are desired, then the desired portions can be identified and prepared as follows.

The intact molecule is used to identify the receptor that binds the molecule to the antigen presenting cell. The intact molecule is then digested by standard protein digestion techniques and the resulting portions examined for binding to the identified receptor. The portions which are capable of binding and of stimulating the formation of costimulatory activity of antigen presenting cells are suitable for use in the compositions and methods of the present invention.

It will, of course, be readily appreciated that those skilled in the art can use homologous and analogous proteins to the TraT, OmpA and OmpF proteins in the present invention. The use of such homologous and analogous proteins is also within the scope of the invention.

The antigens used in the compositions and methods of the invention may be any antigen for which an immune response is desired in patients who are immunologically compromised or unable to respond.

Examples include, for example, HIV antigens, such as the gp41 [8] peptide, which could be used to stimulate blastogenesis of HIV-specific lymphocytes in HIV infected patients. Other antigens may include influenza virus antigens diphtheria antigens, pertussis bacillus antigens, measles antigens, tetanus antigens Pneumocystis antigens, hepatitis antigens, Poho antigens, Candida antigens, Toxoplasma antigens, cytomegalovirus antigens, combinations thereof, or said alpha antigens, or polysaccharides.

The compositions of the invention are prepared by standard pharmaceutical techniques.

If an antigen is to be used in the formulation, this may be combined in a depot with a costimulation inducer. Alternatively, the antigen and the costimulatory inducer may be complexed by chemical conjugation, if necessary by chemical modification and / or linking groups. In the case of proteinaceous antigens, the costimulation inducer and the antigen as fused proteins are prepared by recombinant DNA techniques.

In any event, it will be understood that a method of coupling an antigen to a costimulation inducer cannot eliminate the desired antigenicity of the antigen or the costimulatory inducer activity of TraT, OmpA or OmpF proteins or portions thereof.

The costimulation inducer or the costimulation inducer may be formulated in a carrier antigen depot. If both components are used, it is desirable to keep them together. Depot carriers are suitable for this purpose, and the types of depot carriers that can be used include Alhydrogel, proteasomes and liposomes. The formulations are prepared by conventional techniques appropriate to the vehicle used.

Where the costimulation inducer is used without antigen or where the costimulation inducer is used with the antigen

In addition to depot carriers, conventional carriers can be used in addition to the depot carriers.

Preferably, the composition of the invention is administered parenterally to the patient using standard parenteral techniques.

Generally, 100 ng to 100 mg of each costimulation inducer and antigen are used per dose.

The exact dose of inducer and antigen to be used for the costimulation to be used and their ratio will depend on: (i) the type and nature of the antigen (e.g., immunogenicity); (ii) the genetic background of the patient; (iii) the patient's immunological history; and (iv) the type of immune response (e.g., humoral, lymphocyte-mediated, macrophage-mediated, granulocyte-mediated) that we seek to modify.

One skilled in the art can determine the appropriate ratio of costimulation inducer to antigen by systematically changing the dose and ratio of costimulation inducer to antigen until the desired immune response is achieved.

It is known that many factors affect the determination of the appropriate dosage for a particular patient. Such factors include body weight, age, sex, general health of the patient, and co-morbid condition. Determining the appropriate level of data for a particular patient is accomplished using standard medical techniques.

Patients for whom the methods and compositions of the present invention are intended are patients with helper T-cell deficiency, for example, those suffering from autoimmune diseases, certain cancers and AIDS, and patients with immunodeficiency states have been artificially induced during the treatment of a particular disease or condition, such as transplant patients and cancer patients receiving chemotherapy or radiation therapy.

In general, the method of the invention may be used to increase helper T cell levels or to include specific antigens to increase helper T cell levels in order to protect the patient from specific infections that may be fatal in the immunologically compromised or immunodeficient patient.

While it is understood that the primary aspect of the invention is the treatment of sick humans, the invention can also be applied to the treatment of non-human animals. Accordingly, the term "patient" we use refers to both non-human and human subjects.

For a better understanding of the subject matter of the invention, preferred embodiments thereof are described in the following examples.

Example 1 (i) TraT Enhances T-Cell Proliferative Response Induced by Immunomodulatory HIV-Derived Synthetic Peptides in vitro

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

Most of the immunodominant sequences of human immunodeficiency virus type 1 (HIV1) are encoded by hypervariable gene sequences and are filled with stretches that are highly constant in HIV-1 strains. Immunogenic viral proteins that show minimal variation from one strain to another and which elicit both humoral and cell-mediated immune responses may be useful components of a subunit vaccine. In this context, Bell et al. (Bell et al., 1992) examined HIV-seronegative individuals and HIV-infected individuals belonging to the following group: asymptomatic = AS, AIDS-related complex = ARC) or AIDS patients. According to the clinical classification system of the CDC (Centers Forum Disease Control (1986)), HIV infected patients belonging to the AS group constitute the CDC group II /,, ARC patients belong to the CDC IVA / IVC2 group and AIDS patients belong to the IVCI / IVD group. . First, it was determined that of the three short synthetic peptides produced from the constant sequence sequences of gp120 envelope (262-284 amino acids), gp41 envelope (579-601 amino acids) and p177 core (106-125 amino acids) isolated from HTLV-III _B which can elicit B-cell and T-cell responses in HIV-infected patients. Only the gp41-derived sequence was immunogenic at both B-cell and T-cell levels. The gp41 section was further investigated by identifying a series of gp41 constant regions (amino acids 572-613; termed gp41 [8]) which consistently elicited T-blastogenic and antibody responses in asymptomatic HIV seropositive individuals. ARC patients, but not AIDS patients.

B. Synthesis of HIV-1 Peptides

The two peptides, gp41 [8] and the V3 loop peptide from the gp120 region of HIV-1, were synthesized using Applied Biosystems 430A peptide synthesizer according to the manufacturer's protocol. 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 in 0.1% trifluoroacetic acid (TFA) 5-60%. using a linear gradient of acetonitrile.

The synthesized peptides have the following sequence:

R-S-S-gp41 [8] :( To improve the readability of the peptide, an Arg-Ser-Ser residue was added to the amino terminus of the gp41 [8] sequence)

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. Determination of human T cell proliferative responses

Peripheral blood mononuclear cells (PBMCs) were isolated by Ficoll-Paque (Pharmacia) gradient centrifugation and 200,000 PBMCs were cultured in 10% human AB.

HU 210,605 A9 rum, 50-50 mM penicillin and streptomycin, 2.5 mM glutamine and 2 nM HEPES buffer (pH 7.4) in 0.2 ml RPMI160 medium (Cytosystems Pty. Ltd.), 96 wells ( round bottom microtiter plates, synthetic gp41 [8] or V3 loop peptide (2 μΜ) or rIL-2 (10 μ / ml) or diphtheria toxoid (DT; Commonwealth Serum Laboratories, Melbourne, Australia, 1570 Lf / ml; 4 and 40 μg / ml), or tetanus toxoid (TT; Commonwealth Serum Laboratories, Melbourne, Australia; 100 Lf / ml; 5 and 50 μg / ml) or TraT and OmpA (Purification according to Croft et al., 1991, 40 g / ml). Cultures were performed for 6 days at 37 ° C (with or without antigen), and cultures were pulsed overnight with 50 μΐ ³ H-thymidine (20 μΐΐΐ / ιηΐ: Amersham, UK). Cells were harvested on glass fiber filter papers and counted on a liquid scintillation spectrophotometer (Beckman, USA).

The results are expressed in the Stimulation Index (S.I.). The S.I. was calculated using the following formula:

I average with cpm antigen - mean cpm without antigen mean cpm without antigen (cpm = count per minute = cpm)

D. Definition of the effect of TraT and IL-2 on gp41 [8] ~, V3 peptide, diphtheria toxoid DT or tetanus toxoid TT-specific lymphoproliferation

The effect of TraT and IL-2 on the proliferative responses (expressed in the Stimulation Index) of various HIV-derived and stimulatory (DT and TT) antigens was determined by the formula of Denz et al. (1985):

Additive effect:

(AB) _ (A) + (B)

Synergistic effect:

Inhibitory effect:

wherein (A) and (B) represent proliferative responses to individual drugs (e.g., gp41 [8] and TraT) and (AB) the resulting lymphoproliferative response when both are combined and added to the same cultures.

Results

TraT added to PBMC cultures from 11 asymptomatic (AS) patients, 13 AIDS-related complex disease (ARC) patients, and an AIDS lymphoma patient increased the T-cell proliferative response to gp41 [8] compared to IL-2 was added to cultures containing the gp41 [8] peptide (Table 1). In all cases, except for patient 110, a synergistic effect was observed in cultures stimulated with a mixture of TraT and gp41 [8] and this effect was maximal when 40 μg of TraT protein was added to the cell cultures (Table 2). . In contrast, only three of the 25 cases showed synergistic effects when IL-2 was combined with gp41 [8] (Table 1). The effect of another membrane protein, OmpA, was also tested in asymptomatic and asymptomatic PBMC cultures, and in both cases a synergistic effect was observed when OmpA was used with gp41 [8] (Table 1).

Given the remarkable results obtained with the gp41 [8] peptide in the presence of TraT, it was important to determine whether TraT also increases T cell response with other HIV-derived peptides. The V3 loop peptide was thought to be an appropriate choice as this peptide (LaRosa et al., Science, 249, 932, 1990), the major neutralizing determinant of HIV-1, is currently in clinical trials [Scrip, No. 1703. , 26, 1992). The results summarized in Table 3 show that TraT increased the proliferative response to V3 in all eight cases tested. However, inhibitory activity was observed when PBMC from eight individuals were cultured in the presence of IL-2 and V3 peptides (Table 3). In summary, TraT was much more potent than IL-2, a lymphokine that has been tested as an immunotherapeutic agent (Rosenberg, Lotze, & Mull, 1988) for T cell given to HIV-derived gp41 [8] and V3 loop peptides. increasing responses.

(ii) TraT enhances in vitro T-cell proliferative responses induced by reminding antigens such as diphtheria toxoid (DT) and tetanus toxoid (TT) It is well known that lymphoproliferative response to reminding antigens even in the asymptomatic phase of the disease , when the number of CD4-positive T cells is often only slightly decreased (Lane et al., 1985). Because TraT has been shown to increase HIV-specific hmfoproliferation in both symptomatic and asymptomatic patients, it was expected to have a similar effect on enhancing response to priming antigens. The ability of TraT to enhance response to booster antigens may be clinically important for enhancing immunity by enabling immunologically compromised individuals to fight opportunistic infections. TraT significantly increased the DT- and TT-specific proliferative responses in all six patients (numbered 123 to 128) (Table 4). This experience would make TraT a beneficial immunomodulatory molecule that improves disabling T-cell responses not only to HIV-derived antigens, but also to many of the reminding antigens. Clinically, a costimulation is inducer-like6

A molecule with traits such as TraT may be a preferred immunotherapeutic agent for improving immunity in immunologically compromised individuals.

Example 2

Liquid cytometric analysis of T cell subgroup distribution indicates that TraT preferentially stimulates CD4-positive helper T cells with TraT protein, interleukin-2, or gp41 [8] protein for 6 days incubation with peripheral blood mononuclear cells (PBMCs). ), the distribution of the T cell subgroup was analyzed by immunofluorescence and liquid cytometry.

T cell phenotypes in PBMC proliferating cultures were compared with those found in unstimulated cell cultures.

T-cell phenotype analysis

Both stimulated and unstimulated PBMCs were cultured in 2 ml for 6 days under the same conditions as described in Example 1. After culturing for six days, the pelleted cells are resuspended. Cell suspensions were layered on 1 ml Ficoll-Hypaque gradient (Pharmacia) and centrifuged at 800 g for 10 minutes. Live lymphoblastoid cells (as determined by the trypan blue exclusion method) were harvested from the interface and washed with Hank's balanced saline (pH 7.4; Hank's Balanced Salt Solution = HBSS; Cytosystems, Pty. Ltd.). Live cells isolated from unclassified cultures of PBMC were phenotyped with a dual combination of fluorescent monoclonal antibodies (CD3 / 4 and CD3 / 8; Coulter Electronics Inc., Hialeah, FL., USA). Two thousand live cells from each PBMC culture were assayed for surface-bound fluorescence. Using the live PBMC count (after 6 days of culture with or without stimulation) and relative percentages of the corresponding T cell subgroups present in a small sample of 2,000 cells, the% of CD4 positive and CD8 positive T cell subgroups (2000 cells sampled) multiplied by live cell count (as determined by the trypan blue exclusion method). The small samples were continuously precisely adjusted to cover the majority of either stimulated or unstimulated lymphocytes. After 6 days of culturing in purified PBMC cultures, it was found that 90% of the living cells were regularly found to be CD3 positive, indicating that they were predominantly T cells.

When PBMC was cultured in the presence of TraT or gp41 [8] or IL-2, the absolute number of CD4-positive T cells increased from 23 to 48% today (based on the absolute cell number present in unstimulated cultures). However, when TraT was combined with gp41 [8] peptide, CD4-positive T-cell numbers increased to 63% (patient 123) and 96% (patient 124) (Table 5). In contrast, there was a slightly lower (0-42%) increase in CD8-positive T cell counts when PBMC was incubated in the presence of any of the three stimulants and increased to 10% (123) and 79% (124), when PBMC was exposed to a mixture of TraT + gp41 [8] for 6 days in culture. Another notable feature of these results is that when the various stimuli were incubated for 6 days, there was a significant increase in the CD4 / CD8 ratio compared to the CD4 / CD8 ratio immediately after PBMC isolation.

The synergistic effect between TraT and gp41 [8] was even more pronounced when tested in PBMCs from patients 125 and 126 (Table 6). The percent increase in CD4 cell counts in these two ARC patients was 58% (125), 36% (126); gp41 [8], 0% (125), 18% (126); IL-2, 378% (125), 216% (126). However, CD4 cell fiber increased dramatically when TraT was combined with gp41 [8] peptide: 737% (125) and 1,763% (126). The percentage increase in CD4 cells was significantly higher than the corresponding increase in the CD8 positive population: 74% (125) and 185% (126) (Table 5). The advantageous growth of CD4-positive helper T cells in cultures incubated with TraT or with the combination of gp41 [8] and TraT suggests that TraT will also increase helper T cell counts in vivo and thereby enable HIV-infected patients to to deal with opportunistic infections. Further improvement in HIV-positive patients can be achieved by combining TraT protein with anti-retroviral agents such as zidovudine.

Industrial application

The present invention is useful in the preparation of vaccines for the treatment of disorders associated with immune deficiency, such as AIDS, and for the treatment of patients with helper T cell function in general.

It will be appreciated by those skilled in the art that various modifications and / or modifications may be made to the specific embodiments of the invention without departing from the spirit or scope of the invention as generally described. Thus, the embodiments described are intended to be illustrative in all respects and are not intended to limit the scope of the invention.

Table 1

Gp41 [8] -specific T cell proliferative responses with TraT protein

Treatments (Stimulation Indexes)

Number of patient Diagnosis TraT gp41 [8] IL-2 gp-41 [8] + IL-2 gp-41 [8] + TraT OmpA gp-41 [8] + OmpA 100 AIDS lim- lymphoma 8.0 0.6 1.2 1.0 (I) * 21.5 (S) nt nt

HU 210 605 A9

Number of patient Diagnosis TraT gp41 [8] IL-2 gp-41 [8] + IL-2 gp-41 [8] + TraT OmpA gp-41 [8] + OmpA 101 Slight ARC 0.6 2.2 0.4 10.9 (S) ^# 17.8 (S) nt nt 102 Slight ARC 0.9 2.5 2.0 1.6 (I) 7.0 (S) nt nt 103 Slight ARC 3.3 3.3 2.6 1.1 (I) 21.2 (S) nt nt 104 DIG 8.5 52.1 26.2 nt 163.0 (s) nt nt 105 DIG 26.6 66.7 27.4 nt 252.0 (s) nt nt 106 DIG 13.4 12.8 11.7 nt 50.5 (S) nt nt 107 DIG 13.1 15.8 41.1 nt 48.7 (S) nt nt 108 DIG 3.0 2.0 4.9 nt 15.9 (S) nt nt 109 DIG 3.5 3.6 6.8 nt 28.3 (S) nt nt 110 Slight ARC 7.2 5.6 2.9 nt 13.1 (A) ** nt nt 111 Mild FACE 8.0 10.5 6.3 14.0 (I) 21.7 (S) nt nt 112 DIG 9.0 5.9 8.5 16.3 (S) 39.7 (S) nt nt 113 FACE 29.0 7.9 12.0 18.1 (I) 48.0 (S) 16.0 29.3 (S) 114 DIG 56.4 19.9 22.1 36.0 (I) 99.3 (S) 17.9 42.8 (S) 115 FACE 6.9 12.2 29.1 44.1 (S) 33.7 (S) nt nt 116 FACE 8.0 9.0 23.5 34.8 (A) 23.4 (S) nt nt 117 FACE 8.0 13.1 28.5 43.8 (A) 32.8 (S) nt nt 118 DIG 12.0 7.0 13.5 22.0 (A) 34.5 (S) nt nt 119 DIG 9.9 3.5 8.3 11.4 (A) 21.9 (S) nt nt 120 Slight ARC 42.8 32.5 67.7 22.0 (I) 109.7 (s) nt nt 123 ARC-Kaposi's sarcoma 10.4 6.3 8.5 13.8 (I) 50.0 (S) nt nt 124 DIG 5.7 6.2 6.1 12.4 (A) 38.2 (S) nt nt 125 FACE 7.2 4.3 4.4 7.4 (I) 24.1 (S) nt nt 126 FACE 12.2 6.9 8.7 12.4 (I) 32.9 (S) nt nt

* 1 = inhibitory effect:

#S = Synergistic effect:

nt = not tested

See Example 1 for an explanation of the terms

Table 2

Effect of TraT dose response on gp41 [8] -specific T cell proliferation

treatments Number and diagnosis of patients 111 112 113 114 Slight ARC DIG DIG DIG Stimulation Indices TraT -10 pg 3.0 4.3 7.8 16.5 TraT - 20 pg 5.2 7.8 18.1 35.2 TraT - 40 pg 8.0 9.0 29.0 56.4 TraT - 60 pg 9.0 7.7 23.2 42.5 TraT - 80 pg 5.4 6.8 14.7 25.7 gp41 [8] 10.5 5.9 7.9 19.9

HU 210 605 A9

treatments Number and diagnosis of patients 111 112 113 114 Slight ARC DIG DIG DIG Stimulation Indices TraT - 10gg + gp41 [8] 13.3 25.9 20.2 38.6 TraT - 20gg 4gp41 [8] 17.2 25.3 35.5 75.8 TraT - 40 gg + gp41 [8] 21.7 39.7 48.0 99.3 TraT - 60 gg + gp41 [8] 24.8 18.8 30.8 70.0 TraT - 80 gg + gp41 [8] 19.3 17.8 24.5 48.2

Table 3

Enhancement of V3 Loop Peptide-Specific T-Cell Proliferative Responses with TraT Protein Treatments (Stimulation Indexes)

Number of patient Diagnosis TraT V3 IL-2 V3 4IL2 V3 4 TraT 125 FACE 7.2 3.8 4.4 6.9 (I) 18.7 (S) 126 DIG 12.2 5.6 8.7 10.6 (I) 26.0 (S) 127 DIG 12.1 6.3 8.5 10.0 (I) * 30.9 (S) 127 Slight ARC 4.6 6.7 8.1 11.8 (I) 36.9 (S) 129 DIG 3.7 2.7 4.1 3.7 (I) 21.0 (S) 130 DIG 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 FACE 4.1 5.0 5.7 6.3 (I) 22.6 (S)

* 1 = inhibitory effect S = synergistic effect

Table 4

Enhancement of T-cell proliferative responses to diphtheria toxoid (DT) and tetanus toxoid (TT) by TraT protein

treatments Patient number and diagnosis 123 124 125 126 127 128 Kaposi's sarcoma DIG FACE** FACE DIG Slight ARC (Stimulation Indexes) TraT (40 gg) 10.4 5.7 7.2 12.2 12.1 11.2 DT (40gg) 6.5 5.3 3.3 5.1 6.2 6.1 DT (4gg) 4.0 2.7 1.4 1.41 3.31 3.9 TT (50g) 4.5 5.4 2.8 3.7 5.5 3.0 TT (5g) 3.0 2.8 1.6 2.7 3.1 2.7 TraT 4 DT (40gg) 37.6 (S) * 27.0 (S) 20.9 (S) 24.0 (S) 30.9 (S) 30.2 (S) TraT 4 DT (4gg) 21.6 (S) 18.7 (S) 11.5 (S) 21.6 (S) 23.8 (S) 19.0 (S) TraT 4 TT (50gg) 34.0 (S) 27.5 (S) 16.0 (S) 29.5 (S) 26.5 (S) 21.1 (S) TraT 4 TT (5gg) 1.3 (S) 13.4 (S) 11.2 (S) 19.5 (S) 21.1 (S) 19.6 (S)

* S = synergistic effect

AS = asymptomatic ** ARC = AIDS-related complex

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Table 5

Proliferation and unstimulated cells Distribution of T cell subset after 6 days incubation as determined by liquid cytometry

Patient number and diagnosis # 123-Kaposi's sarcoma * # 124-mild ARC

Treatment Absolute T cell numbers Absolute T cell numbers CD4 CD8 CD4 CD4 CD8 CD4 NIL 3.04 X 10 ⁶ 2.7 X 10 ⁶ 1.1 2.5 X 10 ⁶ 2.4 X 10 ⁶ 1.0 TraT 3.7 X 10 ⁶ 2.28 X 10 ⁶ 1.6 3.0 X 10 ⁶ 2.5 X 10 ⁶ 1.2 gp41 [8] 3.8 X 10 ⁶ 2.40 X 10 ⁶ 1.6 3.7 X 10 ⁶ 2.9 X 10 ⁶ 1.3 IL-2 3.9 X 10 ⁶ 2.5 X 10 ⁶ 1.6 3.6 X 10 ⁶ 3.4 X 10 ⁶ 1.1 [8] + TraT 4.9 X 10 ⁶ 2.9 X 10 ⁶ 1.7 4.9 X 10 ⁶ 4.3 X 10 ⁶ 1.1 [8] + IL-2 4.1 X 10 ⁶ 2.4 X 10 ⁶ 1.7 3.6 X 10 ⁶ 3.8 X 10 ⁶ 0.9

The * CD4 / CD8 ratio at day 0 was 0.23 for 123 and 0.52 for 124

Table 6

Proliferation and unstimulated cells Distribution of T cell subset after 6 days incubation as determined by liquid cytometry

Patient Number and Diagnosis # 125-ARC # 12-mild ARC

Treatment Absolute T cell numbers Absolute T cell numbers CD4 CD8 CD4CD8 CD4 CD8 CD4 CDS 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

The * CD4 / CD8 ratio at day 0 was 0.125 for 125 patients and 0.09 for 126 patients.

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Claims (32)

PATENT CLAIMS A composition comprising a mixture of a protein of the group consisting of TraT, OmpA, OmpF-bot and portions thereof, at least one other antigen, and a pharmaceutically acceptable carrier. A composition according to claim 1, wherein the pharmaceutically acceptable carrier is a hydrophobic depot carrier. A composition according to claim 1 or 2, wherein the at least one other antigen is derived from HIV antigens, influenza virus antigens, diphtheria antigens, whooping cough bacillus antigens, measles antigens, tetanus antigens, Pneumocystis antigens, Cancida antigens, Toxoplasma antigens, selected from the group consisting of hepatitis antigens, polio antigens, combinations thereof, and individual subunit proteins, peptides, and polysaccharides isolated from said antigens. The composition of claim 3, wherein the at least one other antigen is a HIV antigen selected from the group consisting of gp41 [8] peptide or V3 loop peptide. 5. One that is 1 ^ 1. A composition according to any one of claims 1 to 6, wherein the protein is derived from E. coli. 6. A composition according to any one of claims 1 to 5, wherein the protein is TraT or a portion thereof. A composition comprising a protein selected from the group consisting of TraT, OmpA, OmpF, or portions thereof which is selected from the group consisting of HIV antigens, influenza virus antigens, diphtheria antigens, pertussis bacillus antigens, measles antigens, tetanus antigens, The antigen isolated from a group consisting of Pneumocystis antigens, Candida antigens, Toxoplasma antigens, cytomegalovirus antigens, hepatitis antigens, polio antigens, combinations thereof, and individual subunit proteins, peptides and polysaccharides selected from the group consisting of a pharmaceutically acceptable antigen. A composition according to claim 7, wherein the antigen is selected from the group consisting of HIV antigens, diphtheria toxoid and tetanus toxoid. A composition according to claim 8, wherein the HIV antigen is a gp41 [8] peptide or a V3 loop peptide. 10. One of Figures 7-9. A composition according to any one of claims 1 to 5, wherein the protein is TraT or part thereof. 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 use in a method of enhancing immune responsiveness in a patient having an immune deficiency. A composition according to claim 11, wherein the responsiveness of T cells is increased in a patient suffering from a T cell deficiency. A composition according to claim 12, wherein the T cells are helper T cells and the patient is deficient in T cell function. 14. One of Figures 11-13. The composition of any one of claims 1 to 4, wherein the composition further comprises an effective amount of at least one other antigen. 15. The composition of any one of claims 1 to 4, wherein the pharmaceutically acceptable carrier is a hydrophobic depot carrier. 16. A composition according to any one of claims 1 to 5, wherein the protein is TraT or part thereof. 17. The composition according to any one of claims 1 to 6, wherein the at least one other antigen is derived from HIV antigens, influenza virus antigens, diphtheria antigens, pertussis bacillus antigens, measles antigens, tetanus antigens, Pneumocystis antigens, Candida antigens, Toxoplasma antigens, cytomig combinations and a single subunit of proteins, peptides and polysaccharides isolated from said antigens. 18. A composition according to any one of claims 1 to 6, wherein the antigen is a HIV antigen selected from the group consisting of gp41 [8] peptide and V3 loop peptide. HU 210 605 A9 19. A composition according to any one of claims 1 to 6, wherein the protein and the at least one antigen are mixed. 20. A composition according to any one of claims 1 to 6, wherein the patient is HIV positive. 21. A composition according to any one of claims 1 to 6, wherein the protein is derived from E. coli. Use of a composition comprising an effective amount of a protein selected from the group consisting of TraT, OmpA, OmpF-bot and portions thereof, and a pharmaceutically acceptable carrier in the manufacture of a composition for enhancing immune response in a patient with an immune deficiency. The use of claim 22, wherein the medicament is for increasing T cell responsiveness in a patient suffering from a T cell deficiency. The use of claim 23, wherein the T cells are helper T cells and the patient is suffering from helper T cell function deficiency. 25. A 22-24. The use according to any one of claims 1 to 4, wherein the composition further comprises an effective amount of at least one other antigen. 26. A 22-25. The use according to any one of claims 1 to 4, wherein the pharmaceutically acceptable carrier is a hydrophobic depot carrier. 27. A 22-26. The use according to any one of claims 1 to 4, wherein the protein is TraT or a portion thereof. 28. A 25-27. The use according to any one of claims 1 to 6, wherein the at least one other antigen is from HIV antigens, influenza virus antigens, diphtheria antigens, tetanus antigens, measles antigens, hepatitis B antigens, hepatitis B antigens, hepatitis B antigens, combinations thereof and a group of individual subunit proteins, peptides and polysaccharides isolated from said antigen. 29. A 25-28. The use according to any one of claims 1 to 6, wherein the antigen is a HIV antigen selected from the group consisting of gp41 [8] peptide and V3 loop peptide. 30. A 25-29. The use according to any one of claims 1 to 3, wherein the protein and the at least one antigen are mixed. 31. A 22-30. Use according to any one of claims 1 to 3, wherein the patient is HIV positive. 32. A 22-31. The use according to any one of claims 1 to 3, wherein the protein is derived from E. coli.