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WO1998031384A1 - Procedes et compositions utilisant l'interleukine-13 dans l'amelioration de reponses immunes - Google Patents

Procedes et compositions utilisant l'interleukine-13 dans l'amelioration de reponses immunes Download PDF

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Publication number
WO1998031384A1
WO1998031384A1 PCT/US1998/001500 US9801500W WO9831384A1 WO 1998031384 A1 WO1998031384 A1 WO 1998031384A1 US 9801500 W US9801500 W US 9801500W WO 9831384 A1 WO9831384 A1 WO 9831384A1
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antigen
hiv
leu
responses
ser
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PCT/US1998/001500
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WO1998031384A9 (fr
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Luis J. Montaner
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The Wistar Institute Of Anatomy & Biology
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Publication of WO1998031384A9 publication Critical patent/WO1998031384A9/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/52—Cytokines; Lymphokines; Interferons
    • C07K14/54—Interleukins [IL]
    • C07K14/5437—IL-13
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/39—Medicinal preparations containing antigens or antibodies characterised by the immunostimulating additives, e.g. chemical adjuvants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
    • A61K2039/55511—Organic adjuvants
    • A61K2039/55522—Cytokines; Lymphokines; Interferons
    • A61K2039/55527—Interleukins
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
    • A61K2039/55511—Organic adjuvants
    • A61K2039/55522—Cytokines; Lymphokines; Interferons
    • A61K2039/55527—Interleukins
    • A61K2039/55538—IL-12
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • the present invention relates generally to methods and compositions for increasing the immune response to antigens, particularly for vaccine use.
  • the elicitation of protective immunity by vaccination i.e., the deliberate presentation of an antigen to a healthy or immunocompromised host, is dependent on the capacity of the antigen to elicit the appropriate immune response.
  • Whether such immune responses are cell-mediated or humoral is determined by the nature of the T cells that develop after immunization. For example, many bacterial, protozoal and intracellular parasitic and viral infections appear to require a strong cell-mediated immune response for protection.
  • Other pathogens, such as helminths primarily respond to a humoral, or antibody, response.
  • T cells can be separated into subsets on the basis of the repertoire of cytokines produced and that the distinct cytokine profile observed in these cells determines their function.
  • This T cell model includes two major subsets: T H1 cells that produce IL-2 and interferon ⁇ (IFN- ⁇ ) and mediate cellular immune responses, and T H2 cells that produce Interleukin-4 (IL-4), Interleukin-5 (IL-5), and Interleukin-10 (IL-10) and augment humoral immune responses [T. R. Mosmann et al, J. Immunol. 126:2348 (1986)].
  • adjuvants that is, substances which enhance the immune response when administered together with an antigen. See such texts as "The Theory and Practical Application of Adjuvants", Duncan E. S. Steward- Tull, eds., John Wiley and Sons, Ltd. (1994) for further discussion of conventional adjuvants and method for their use.
  • the ability of an adjuvant to induce and increase a specific type of immune response and the identification of that ability is a key factor in the selection of particular adjuvants for vaccine use with an antigen of a particular pathogen.
  • Typical adjuvants include water and oil emulsions, e.g., Freund's adjuvant, and chemical compounds such as aluminum hydroxide or alum. At present, alum is the only adjuvant approved in the United States for human vaccines.
  • bacteria or their products include bacteria or their products, e.g., microorganisms such as the attenuated strain of ' Mycobacterium bovis, bacillus Calmette-Guerin (BCG); microorganism components, e.g., alum-precipitated diphtheria toxoid, bacterial lipopolysaccharide (LPS) and endotoxins.
  • BCG Bacillus Calmette-Guerin
  • LPS bacterial lipopolysaccharide
  • Many bacteria or their products, lipopolysaccharide, Staphylococcus aureus, Mycobacterium tuberculosis, and C. parvu stimulate IL-12 production by macrophages [A. D'Andrea et al, J. Exp. Med., 176: 1387 (1992)].
  • cytokine such as Interleukin-12 has been shown to have adjuvanting properties on certain antigens. See, e.g., United States Patent No. 5,571,515, issued November 5, 1996, and references described therein.
  • Interleukin-13 another cytokine, and its DNA and protein sequences have been previously described. See, e.g., International Patent Application WO94/04680, published March 3, 1994. IL-13 was described as useful for diagnostic methods and therapeutic applications, particularly for conditions exhibiting abnormal expression of IL-13 or for conditions where activated B cell growth is required. IL-13 is presented in the literature as a "weaker” version of Interleukin-4, as a B-cell stimulant and deactivator of macrophage function. It is known that IL-13 has no receptor on T- cells. See, e.g., G. Zurawski et al, Immunol. Today. 15: 19-26 (1994). There exists a need in the art for additional adjuvants which are useful in stimulating a mammal's immune response, and which are suitable for use in pharmaceutical compositions, such as vaccines. Summary of the Invention
  • the present invention provides a method for stimulating or enhancing immune response in a mammalian host.
  • the method comprises administering an effective amount of Interleukin-13 or a biologically active fragment thereof (IL-13) to the host simultaneously or sequentially with a selected antigen .
  • IL-13 Interleukin-13 or a biologically active fragment thereof
  • the invention provides a method for enhancing the adjuvant effect of Interleukin-12 (IL-12) on a selected antigen in a host by co-administering to the host an effective amount of IL-13.
  • IL-12 Interleukin-12
  • Still another aspect of this invention involves a vaccine composition
  • a vaccine composition comprising a selected antigen and an effective adjuvanting amount of IL-13, the vaccine capable of enhancing presentation of said antigen to the host's immune system.
  • the composition may further comprise a second adjuvant, such as IL-12.
  • Fig. IB is a similar graph of SI of 18 HIV-1 infected patients' PBMC vs. the four identified conditions for adjuvanting or not adjuvanting Tetanus Toxoid (T.T.).
  • Fig. IC is a graph of SI of 35 HIV-1 infected patients' PBMC vs. exposure to keyhole limpet hemocyanin (KLH) or phytohemagglutinen (PHA) as negative and positive controls, respectively.
  • KLH keyhole limpet hemocyanin
  • PHA phytohemagglutinen
  • Fig. 2 A is a graph of flu antigen response in HIV-1 infected patients, plotting SI vs. CD4 cell count for PBMC exposed only to the flu antigen but otherwise untreated.
  • Fig. 2B is a graph of flu antigen response in HIV-1 infected patients, plotting SI vs. CD4 cell count for PBMC exposed to the flu antigen and IL-12.
  • Fig. 2C is a graph similar to Fig. 2B, but in which PBMC were exposed to the flu antigen and IL-13.
  • Fig. 2D is a graph similar to Fig. 2C, but in which PBMC were exposed to the flu antigen and a combination antigen of IL-13 and IL-12 (IL-13+IL-12).
  • Fig. 3A is a graph of T.T. antigen response in HIV-1 infected patients, plotting SI vs. CD4 cell count for PBMC exposed only to the T.T. antigen but otherwise untreated.
  • Fig. 3B is a graph of T.T. antigen response in HIV-1 infected patients, plotting
  • Fig. 3C is a graph similar to Fig. 3B, but in which PBMC were exposed to the T.T. antigen and IL-13.
  • Fig. 3D is a graph similar to Fig. 3C, but in which PBMC were exposed to the T.T. antigen and IL-13+IL-12.
  • Fig. 4A is a graph showing the distribution of SI over CD4 count for PHA responses in HIV patients.
  • Fig. 4B is a graph showing the distribution of SI vs. CD4 counts for KLH responses without adjuvant.
  • Fig. 4C is a graph showing the distribution of SI vs. CD4 counts for KLH responses with IL-12.
  • Fig. 4D is a graph showing the distribution of SI vs. CD4 counts for KLH responses with IL-13.
  • Fig. 4E is a graph showing the distribution of SI vs. CD4 counts for KLH responses with IL- 13 +IL- 12.
  • Fig. 5 A is a graph showing the T.T. responses of HIV patients, plotting SI vs. indicated concentrations of IL-12.
  • Fig. 5B is a graph showing the T.T. responses of HIV patients, plotting SI vs. indicated concentrations of IL-13.
  • Fig. 5C is a graph showing the flu antigen responses of HIV patients, plotting SI vs. indicated concentrations of IL-12.
  • Fig. 5D is a graph showing the flu antigen responses of HIV patients, plotting SI vs. indicated concentrations of IL-13.
  • Fig. 6 A is a graph of SI of healthy patient PBMC vs. the four identified conditions for adjuvanting or not adjuvanting flu. Students paired t test correlations are indicated.
  • Fig. 6B is a graph of SI of 12 healthy patient PBMC vs. the four identified conditions for adjuvanting or not adjuvanting T.T. Students paired t test correlations are indicated.
  • Fig. 7A is a graph of SI of healthy patient PBMC vs. exposure to flu antigen alone or adjuvanted with IFN- ⁇ or IL-4. Students paired t test correlations are indicated.
  • Fig. 7B is a graph of SI of 8 healthy patient PBMC vs. exposure to T.T. antigen alone or adjuvanted with IFN- ⁇ or IL-4. Students paired t test correlations are indicated.
  • Fig. 7C is a graph of SI of 8 HIV-1 infected patients' PBMC vs. exposure to flu antigen alone or adjuvanted with IFN- ⁇ or IL-4. Students paired t test correlations are indicated.
  • Fig. 7D is a graph of SI of 5 HIV-1 infected patients' PBMC vs. exposure to
  • Fig. 8 is a graph of the effect of TNF- ⁇ on IL-12 production in PBMC of three donors, untreated or exposed to IL-13, TNF- ⁇ , anti-TNF- ⁇ , anti-TNF- ⁇ +IL- 13 and TNF- ⁇ +IL-13.
  • Fig. 9 is a graph illustrating amount of horseradish peroxidase uptake by HIV- 1 (right) and HIV-1+ (left, 1 13-790 CD4 ⁇ l/mm3) donor monocyte-derived macrophages (MDM) with or without IL-13.
  • Figs. 10A-10I illustrates data demonstrating that IL-13 enhances low responders to influenza A stimulation in HIV-infected PBMC which are responsive to PHA.
  • FIG. 10 A- IOC Shown are linear regression analysis with 95% confidence intervals between patient CD4 count for the three cluster groups [Fig. 10 A- IOC; high (H); Figs. 10D-10F, intermediate (I); Figs. 10G-10I, low (L)].
  • Each antigenic response group is shown with their corresponding antigen + IL-13 and PHA responses.
  • Seven patients were not included in linear regression analysis due to lack of CD4 information; each point in scatter plots represents the mean of duplicate or triplicate measurements and the PHA scatter plot for the Influenza A (H) cluster is shown with a reduced y axis not showing two outlier responses (340 CD4 cells/ ⁇ l, 47019 c.p.m; 419 CD4 cells/ ⁇ l, 58231 c.p.m.).
  • Fig. 1 IB is a bar graph illustrating Influenza A virus T-cell memory responses with IL-12 and IFN-y tested in parallel in the same donors as in Fig. 11 A.
  • the invention provides methods and compositions for stimulating immune responses in mammalian host cells, which involve delivering interleukin- 13 (IL-13) to host cells.
  • IL-13 interleukin- 13
  • IL-13 protein, fragments thereof, as well as fusion proteins containing IL-13 or fragments thereof, which fragments and fusion proteins have IL- 13 biological function may be used in the methods and compositions of the invention.
  • Such fragments may be obtained by conventional methods of fragmenting. Any fragment may be readily assessed for IL-13 activity by testing in an assay, such as those described in WO 94/04680, which is incorporated by reference.
  • nucleic acid sequences encoding full-length IL- 13, IL-13 fragments, or IL-13 fusion proteins as defined above may be readily utilized in these methods and compositions.
  • These methods and compositions of the invention which utilize IL-13 have been found not only to adjuvant selected antigens, but also to stimulate immune responses in both healthy, virus-infected, and immunocompromised mammalian hosts, with and without other additional adjuvants.
  • the term "mammal” includes humans and non-human animals.
  • IL-13 may be obtained from commercial sources, e.g., Genzyme and
  • IL-13 may be obtained using a variety of known synthetic and recombinant techniques.
  • the nucleic acid sequences and amino acids of human Interleukin- 13 (IL-13) are provided in International Patent Application WO 94/04680, published March 3, 1994, which is incorporated by reference herein. For purposes of convenience, these sequences are reproduced herein as SEQ ID NO: 1 and 2.
  • the IL- 13 encoding nucleic acid sequences useful in the invention include "naked DNA", which is defined herein as substantially pure DNA which is not associated with protein, lipid, carbohydrate or contained within a cell or an artificial delivery system such as a liposome.
  • the nucleic acid sequences useful in the invention also encompass vectors encoding IL-13 and/or IL-13 fragments or fusion proteins under the control of suitable regulatory control sequences which direct expression thereof in the target host cells.
  • the antigens selected for the methods and compositions of the invention are not a limitation on this invention.
  • the antigen may be, without limitation, a whole cell, a virus, a protein, a protein subunit or fragment.
  • viral antigens which may be enhanced by adjuvantation with IL-13, alone or in combination with IL- 12, include, without limitation, those derived from and/or useful in treatment or prevention of HIV, Hepatitis A, Hepatitis B, Hepatitis C, rabies virus, polio virus, influenza virus, meningitis virus, measles virus, mumps virus, rubella, pertussis, encephalitis virus, papilloma virus, yellow fever virus, respiratory syncytial virus, parvovirus, chikungunya virus, haemorrhagic fever viruses, Klebsiella, and Herpes viruses, particularly, varicella, cytomegalovirus and Epstein-Barr virus.
  • bacterial antigens include those derived from and/or useful against leprosy and tuberculosis, among others.
  • parasitic antigens include those derived from and/or useful against such infections as leishmaniasis and malaria, among others.
  • composition antigens include those derived from a protozoan, e.g., T. cruzii, or against a helminth, e.g., Schistosoma.
  • IL-13 can be used as an adjuvant in so-called therapeutic vaccines for certain cancers and solid tumors, and infectious diseases including, without limitation, malaria, and HIV.
  • a therapeutic vaccine is used in a manner similar to that disclosed above for its use as an adjuvant for vaccines containing antigens of a pathogenic microorganism or virus.
  • the use of IL-13 as an adjuvant in a cancer vaccine or therapeutic is encompassed by the present invention.
  • Cancer vaccines typically include an antigen expressed on and isolated from a cancer cell or a cancer cell transfected with, and capable of expressing, a selected antigen.
  • any purified tumor antigen may be co-administered with IL-13 as described above for pathogenic vaccines. Identification of relevant cancer antigens will permit the development of such vaccines.
  • other cancer therapeutics are designed using an antigen normally not expressed on a cancer cell.
  • a selected antigen may be transfected into the cancer cell and the transfected cell itself, expressing the antigen, is used as the vaccine or therapeutic.
  • the methods and compositions of the present invention are useful for a variety of medical and veterinary uses, as will be readily apparent from the discussion below.
  • the present invention provides novel methods of stimulating the immune response of a mammalian host, and is particularly well suited to enhance the immune response to a selected molecule (e.g., an antigen).
  • a selected molecule e.g., an antigen
  • This "adjuvanting" effect is provided by co-administering IL-13 to a host in conjunction with a selected antigen.
  • co-administration as used herein is meant delivering to the host cell or tissue an effective adjuvanting amount of IL-13 simultaneously with the antigen (e.g., in the same composition formulation) or sequentially with the antigen (e.g., either before or after antigen administration).
  • IL-13 proteins or protein fragments may be delivered to the host cells by a variety of conventional means, as described in detail in the discussion of compositions below.
  • nucleic acid sequences which direct expression of IL-13 proteins or protein fragments may be used to infect or transfect a host cell, either in the form of "naked DNA” or via plasmid or viral vectors.
  • Such vectors are well known to those of skill in the art.
  • the IL-13 may be separately formulated and co-administered with an active agent (e.g., a selected antigen). It may be desirable to administer the IL-13 composition substantially contemporaneously with the antigenic composition (within about 24 hours). Alternatively, it may be desirable to administer the IL-13 composition between 1 day and about 1 week before or after administration of the antigenic composition. However, this time frame may be readily adjusted, i.e., extended, by of skill in the art based on such factors as the condition of the veterinary or human patient, the type of treatment which the patient is undergoing, other therapies being received by the patient, and may also take into consideration such factors as the half- life of the antigenic composition and IL-13. As an alternative to separate formulations, IL-13 may be formulated as part of a pharmaceutical (vaccinal or therapeutic) composition containing the selected antigen. Exemplary compositions useful in the methods of the invention are described in more detail below.
  • IL-13 has been found to be capable of adjuvanting antigens which are inhibited or not responsive to the presence of other adjuvants.
  • IL-13 can adjuvant tetanus toxoid, which is not adjuvanted by other known adjuvants.
  • the adjuvanting activity of IL-13 is demonstrated in healthy hosts, e.g., healthy humans, or in infected hosts, e.g., in human immunodeficiency virus-infected humans.
  • IL-13 can adjuvant influenza A, Mycobacterium antigens and HIV-1 antigens in HIV-infected individuals at end- stage disease demonstrating a property of IL-13 not present with other adjuvants such as IL-12.
  • IL-13 has been found to stimulate both humoral and cell-mediated immune (CMI) responses within immunocompromised individuals.
  • CMI cell-mediated immune
  • the present invention also provides methods of adjuvantation intended to provide an additive or a synergistic effect with other adjuvants, and particularly, IL- 12.
  • IL-13 increases the immune response to tetanus toxoid or influenza A, when administered in combination with IL-12 in healthy mammals (see Fig. 6B; and Table I below).
  • Such an adjuvanted vaccine composition demonstrates an enhanced immune response as evidenced by restricted IL-13 effects on antigen presenting cells (APC) together with IL-12's action of a greater elicitation of cytotoxic T lymphocytes (CTLs) and activated phagocytes by its direct effects on T cell responses.
  • APC antigen presenting cells
  • CTLs cytotoxic T lymphocytes
  • This proliferative effect may exhibit some resistance to chemotherapeutics and thus provide another therapeutic agent and regimen for cancer treatment or in the stimulation of the immune response in the environment of inflamed tissues.
  • the present invention's ability to induce protective immune responses within inflamed tissues or chronic states of immune activation is believed to be due to IL-13's effects in recovering immune responsiveness by acting directly against agents that inhibit de novo responses such as sustained levels of high tumor necrosis factor (TNF- ⁇ ; see, e.g., Example 6 and Fig. 8).
  • TNF- ⁇ tumor necrosis factor
  • IL- 13 functions by enhancing the presentation of the antigen with which it is co- administered in the host cell and increases the range of immune recognition to antigens, as compared to other adjuvants. Because the induction of IL-13 is a key component to presentation of the antigen, the use of IL-13 as an adjuvant may be preferable to known adjuvants. For example, unlike adjuvants such as IFN- ⁇ or IL-2, IL-13 is relatively stable in vivo. Furthermore, the effects of IFN- ⁇ or IL-2 are either negative or absent in affecting antigen uptake by APCs, a central function of the APC which determines the extent of the in situ response.
  • the method of this invention may be useful in adjuvanting a number of antigens in both healthy and immunocompromised mammals.
  • These antigens may be useful in vaccines against diseases requiring CMI stimulation for effective protection.
  • diseases may be broadly defined and include conditions requiring "jump starts" to both innate and adaptive immunity.
  • IL-13 (with or without IL-12) is expected to be particularly useful in compositions for treatment of AIDS, cancer, chronic illnesses or other conditions associated with high TNF- ⁇ , which are dependent on antigen recognition and cell-mediated responses.
  • IL-13 in the present method is expected to be similarly used in adjuvanting antigens administered to reduce the morbidity and mortality associated with infections following trauma or as post- operative prophylactic medication in conjunction with antibiotics
  • IL-13 in the present method is expected to be similarly used in adjuvanting antigens administered to reduce the morbidity and mortality associated with infections following trauma or as post- operative prophylactic medication in conjunction with antibiotics
  • decreased immune function following trauma associated with diseases caused by intracellular or extracellular parasites, certain bacterium, protozoan, helminths and viruses are most likely to benefit from an enhanced antigen recognition for CMI and protection.
  • IL-13 has certain advantages over known adjuvants.
  • IL- 13 is advantageous over alum for use in human vaccines.
  • Alum induces T H2 helper cells rather than the response inherent to the antigen given as observed with IL-13, and thus, alum adjuvanted vaccines may be ineffectual for those pathogenic microorganisms against which a T H1 response is most effective.
  • IL-13 is effective in adjuvanting immune responses to Influenza A in both healthy and immunocompromised mammals, while Alum was ineffective in increasing immune responses against this antigen [J. Immunol., 100: 1139-40 (1968)].
  • Alum has also been reported to be a deficient adjuvant for small-size antigens, such as peptides [Immunol.. 6 _: 1-6 (1987)] due to the limited denaturation caused by the process of alum absorbance. Further, alum has been suggested to induce a greater proteolysis of antigens which would act against antigens such as peptides requiring little or no processing ["Immunological Adjuvants and vaccines", eds. G. Gregoriadis et al, Plenum Publ., New York, pp 35-51 (1989)]. The use of IL-13 administered together with small-size antigens would not have such limitations.
  • IL-13 is superior to bacterial adjuvants, such as mycobacteria or its derivatives (i.e., complete Freunds Adjuvant or Muramyl Dipeptides) which may induce pro-inflammatory cytokines including IL-12 and TNF- ⁇ which may be unanticipated or not controlled.
  • mycobacteria or its derivatives i.e., complete Freunds Adjuvant or Muramyl Dipeptides
  • the induction of IL-12 may result in differential levels of IFN- ⁇ secretion as determined by the degree of IL- 12 receptors on T-cells (variable depending on host immune activation state) which may inhibit further antigen uptake by antigen presenting cells.
  • IFN- ⁇ induced by IL- 12 in activated T cells, does not act to enhance antigen recognition of exogenously administered antigens in either healthy or immunocompromised mammals (see, Figs. 7A through 7D).
  • responses to tetanus toxoid adjuvanted by IL-13 in immunocompromised individuals are superior to those produced in such patients when tetanus toxoid is adjuvanted with IL-12 (Fig. IB).
  • the significant enhancement observed with IL-13 and IL-12 when used in combination in both healthy or immunocompromised humans (Fig. 1 ; Table I) provides evidence that IL- 13 acts together with stimulants of IL-12, such as muramyl dipeptides or derivatives.
  • IL-13 is human in origin and thus unlikely to produce any sensitization.
  • IL-13 is superior to IL-12 as an adjuvant by its direct action on the APC.
  • IL-12 may be a more effective determinant to the character of the T-cell immune response, but its activity is dependent on the presence of activated T- cells.
  • the inventors have observed that IL-13 is superior to IL-12 in allowing small protein acquisition by immune cells in stimulating immune responses within human peripheral blood cells. More desirably, IL-13's action to stimulate antigen presentation by its associated induction of CD86 and pinocytic update demonstrate a direct regulation of antigen presenting function not induced by IL-12.
  • IL-13 is a highly useful immunostimulant and adjuvant for use in human and veterinary compositions, including vaccines.
  • IL-13 is administered in accordance with the method of the invention as an effective adjuvant alone or, optionally, in conjunction with another adjuvant, e.g. IL-12.
  • IL-13 may be delivered in protein form or expressed in the target host cell following infection or transfection of nucleic acid sequences encoding IL-13. As stated above, 'naked DNA' may be used to express the IL-13 protein or peptide fragment in the target host cell [See, e g , J Cohen, Science. 259 1691-1692 (March 19, 1993), E Fynan et al, Proc Natl Acad Sci , 90 11478-11482 (Dec 1993), J A Wolff et al, Biotechniques.
  • IL-13 DNA may be incorporated, or transduced, into a microorganism, if a cellular pathogen itself is to be employed as the vaccinal antigen
  • IL-13 DNA may be administered as part of a vector or as a cassette containing the IL-13 DNA sequences operatively linked to a promoter sequence See, e g , International Patent Application WO94/01139, published January 20, 1994, incorporated herein by reference Briefly, the DNA encoding the IL-13 protein or desired fragment thereof may be inserted into a nucleic acid cassette This cassette may be engineered to contain, in addition to the IL-13 sequence to be expressed, other optional flanking sequences which enable its insertion into a vector This cassette may then be inserted into an appropriate DNA vector downstream of a promoter, an mRNA leader sequence, an initiation site and other regulatory sequences capable of directing the replication and expression of
  • IL-13 When used as an adjuvant for a selected antigen, IL-13 is desirably admixed as part of the antigen-containing composition itself
  • a composition is desirably a vaccine composition which contains a suitable carrier and, optionally, other desired components.
  • suitable carriers e.g., phosphate buffered saline and the like, are well within the skill of those in the art and are not a limitation on the present invention.
  • IL-13 may readily select appropriate stabilizers, preservatives, and the like for inclusion in the composition.
  • IL-13 is suited to be administered by the same route as the vaccinal antigen. Any route of administration may be employed for the administration of this vaccine, e.g., subcutaneous, intraperitoneal, oral, intramuscular, intranasal and the like.
  • IL-13 to adjuvant particular vaccines or other immunogenic compositions. Such amounts will depend upon the condition for which the vaccine or other composition is designed to treat or prevent, the nature of the antigen, the dosage amounts of the antigen as well as the species and physical and medical conditions (e.g., general healthy, weight, etc.) of the vaccinate.
  • an effective amount of IL-13 is desirably between about 0.1 ⁇ g to about 0.5 mg of IL-13 protein or fragment per about 25 ⁇ g of antigen.
  • another cytokine such as IL-12
  • each is present in an amount between about 0.1 ⁇ g to about 0.5 mg.
  • the IL-13 may be administered as a protein, or sufficient amounts of nucleic acid may be administered to achieve expression of these levels of IL-13.
  • the immunostimulatory (i.e., adjuvanting) effect of IL-13 may be obtained by administering IL-13 separately from the vaccine composition.
  • the IL-13 is desirably in the presence of a suitable carrier, such as saline, a liposomal delivery system or the like.
  • a suitable carrier such as saline, a liposomal delivery system or the like.
  • the amount of the IL- 13 used in this mode of vaccination is similar to the ranges identified above when IL- 13 is part of the vaccine composition.
  • the IL-13 may be administered contemporaneously with the vaccine composition, either simultaneously therewith, or before or after the vaccine antigen administration. If the IL-13 is administered before the vaccine composition, it is desirable to administer it one or more days before the vaccine.
  • IL-13 activity in conditioning antigen presenting cells to secrete higher levels of IL-12 by its presence 24 hours before the antigen would specifically highlight this ability to generate both an increased immune and IL-12 response [A. D' Andrea et al, J. Exp. Med.. 181:537-546 (1995)], the latter being an added benefit.
  • IL-13 is administered as a separate component from the vaccine, it is desirably administered by the same route as the vaccinal antigen, e.g., subcutaneous route, or any other route as selected by a physician.
  • IL-13 may be used as an adjuvant in combination with IL-12 or a biologically active fragment thereof.
  • Interleukin- 12 (IL-12), originally called natural killer cell stimulatory factor, is a heterodimeric cytokine described, for example, in M. Kobayashi et al, J. Exp. Med., 1709:827 (1989).
  • the expression and isolation of IL- 12 protein in recombinant host cells is described in detail in International Patent Application WO90/05147, published May 17, 1990. See, also, US Patent 5,457,038.
  • Biologically active fragments of IL-12 may be readily obtained using standard techniques (e.g., fragmentation, chemical synthesis, and the like) which are tested for biological activity using such standard assays such as those described in WO9-/05147 and US Patent 5,457,038.
  • sequences of the 40 kd subunit of human IL-12 is provided herein as SEQ ID NO: 3 and 4 and the sequences of the 30-35 kd subunit of IL-12 is provided herein as SEQ ID NO: 5 and 6.
  • discussion of IL-12 refers to the heterodimer.
  • Recombinant human and murine IL-12 are available commercially from sources, such as Genetics Institute, Inc., Cambridge, Massachusetts. See also the amounts used in the examples below.
  • the adjuvanting amount for any particular antigen will be readily defined by balancing the efficacy and toxicity of the IL-13 and antigen combination.
  • IL-13 nucleic acid sequences When IL-13 nucleic acid sequences are used as an adjuvant, these sequences may be operably linked to DNA sequences which encode, IL-2, IL-12 and/or the antigen.
  • the vector or cassette, as described above, encoding the IL-13 DNA sequences may additionally include sequences encoding IL-12 and/or the antigen. Each of these sequences may be operatively linked to the promoter sequence of the vector or cassette. Alternatively, 'naked DNA' encoding the antigen may be in a separate plasmid.
  • the naked DNA encoding the antigen and/or IL-13 upon introduction into the host cells, permits the infection of vaccinate's cells and expression of both IL-13 and the antigen in vivo.
  • IL-13 nucleic acid sequences are employed as the adjuvant either as 'naked DNA' operatively linked to a selected promoter sequence or transduced into a strain of the pathogenic microorganism, rather than the protein itself, the amounts of DNA to be delivered and the routes of delivery may parallel the IL- 13 protein amounts and delivery described above and may also be determined readily by one of skill in the art. Similarly the amounts of the antigen as DNA would be selected by one of skill in the art.
  • the following examples illustrate the methods of the present invention for enhancing the immune response to such antigens, as tetanus toxoid, Influenza A, Mycobacterium tuberculosis, and HIV-1 p24 and nef proteins.
  • the methods of this invention also encompass other antigens as described above.
  • additional antigens such as Candida a ⁇ bicans
  • IL-13 can enhance antigen responses Xo Mycobacterium tuberculosis and HIV-1 antigens.
  • Thymidine incorporation assays are used as an indication of cell proliferation and activation.
  • Introduction of a soluble antigen to a peripheral blood mononuclear cell (PBMC) culture generally results in higher thymidine incorporation, if a response to the antigen is present.
  • PBMC peripheral blood mononuclear cell
  • APC antigen presenting cells
  • memory T cells thus called a recall response assay.
  • a recovery or enhanced memory response is directly related to the efficiency of antigen presentation as well as the frequency of memory cells.
  • Antigen presentation is a determining step in the initiation of immune responses to pathogens, cancer or vaccination. This assay has been generally used as an indirect measure of immune responsiveness in vivo [J. Immunol. Meth.. 182: 177-184 (1995); Science. 262: 1721-1724 (1993)].
  • Peripheral blood was collected from 27 healthy and 30 HIV-1 positive donors.
  • Peripheral blood mononuclear cells (PBMC) were isolated on Ficoll-Paque gradients. PBMC were washed at least three times in phosphate-buffered saline (PBS) and resuspended in RPMI medium supplemented with 100 ⁇ g/ml streptomycin and 10% fetal calf serum (FCS).
  • PBS phosphate-buffered saline
  • FCS fetal calf serum
  • PBMC peripheral blood mononuclear cells
  • HIV-1 patient material was lysed in a buffer containing 0.5 M NaCl, 0.1% SDS, lOmM EDTA, 1% NP40, 1% Tween 20 before nuclei were harvested.
  • Nuclei were harvested using an automatic multi-well harvester (Gast, Benton Harbor, MI) onto fiberglass filter paper (Packard, Meriden, CT), air dried and assessed for H-3 activity using a Matrix direct Beta Counter 9600 (Packard, Meriden, CT). Data was generated as count per minute (cpm). All results are the average of triplicates tests.
  • SI stimulation index
  • Example 2 The Effects of IL-12. IL-13 and" Their Combination on Antigenic Responses to Influenza A and Tetanus Toxoid in HIV-1 Patients
  • Example 2 This experiment was performed using the protocols of Example 1 on variously grouped HIV-1 infected patients: one group receiving flu antigen only; a second group receiving flu antigen and IL-12; a third group receiving flu antigen and IL-13, a fourth group receiving flu antigen plus IL-12 and IL-13; a fifth group receiving
  • Tetanus Toxoid only a sixth group receiving Tetanus Toxoid and IL-12; a seventh group receiving Tetanus Toxoid and IL-13, and the eighth group receiving Tetanus Toxoid plus IL-12 and IL-13.
  • Results were reported as Influenza A SI responses (Fig. 1 A) and
  • Fig. IA The data in Fig. IA showed that Influenza A responses in HIV-1 infected donors are not differentially enhanced between IL-12, IL-13 or their combination.
  • B. Results were also reported as distribution of SI over CD4 count for
  • PBMC exposed only to the flu antigen but otherwise untreated Fig. 2 A
  • flu + IL-12 Fig. 2B
  • flu + IL-13 Fig. 2C
  • flu + 1L-13+IL-12 Fig. 2D
  • PBMC exposed only to the Tetanus Toxoid antigen Fig. 3A
  • T.T. + IL-12 Fig. 3B
  • T.T. + IL-13 Fig. 3C
  • T.T. + IL-13+IL-12 Fig. 3D
  • IL-13 increased mean responses to both influenza A and Tetanus toxoid in HIV-1 infected samples at both high and low CD4 count.
  • IL-13 + IL-12 increased the mean response to Influenza A and tetanus toxoid in HIV-1 patients, the combination was not as effective at high CD4 count suggesting that IL-12 has an inhibitory effect on the IL-13 -mediated enhancement.
  • C. Results were also reported as SI for KLH responses following exposure to the three adjuvant formulations, and PHA responses in relation to HIV-1 patient CD4 number. Distribution of SI over CD4 count for PHA responses is reported in Fig. 4A and KLH responses with or without the adjuvants in Figs. 4B through 4E. Fig.
  • FIG. 4 A illustrates that PHA responses in HIV-1 patients are positively correlated to CD4 level.
  • Figs. 4B through 4E illustrate that IL-12, IL-13 or IL- 13+IL-12 showed no non-specific proliferation effects in HIV-1 patients stimulated with KLH.
  • IL-12 concentrations used in this experiment were: 0.01 ng/ml, 0.1 ng/ml, 0.5 ng/ml, 1 ng/ml, 5 ng/ml and 10 ng/ml.
  • IL-13 concentrations used were 6.25 ng/ml, 12.5 ng/ml, 25 ng/ml, 50 ng/ml and 100 ng/ml.
  • Titrations of IL-12 and IL-13 on PBMCs stimulated with Tetanus Toxoid are shown in Figs. 5 A and 5B.
  • Titration of IL-12 and IL-13 on PBMCs stimulated with Influenza A are shown in Figs. 5C and 5D.
  • HIV-1 patient CD4 level is indicated for each titration.
  • IL-12 had no effect on Tetanus Toxoid SI response (Figs. 5 A and 5C) in HIV-1 infected donors.
  • Example 4 The Effects of IL-12. IL-13 and Their Combination on Antigenic Responses to Influenza A and Tetanus Toxoid in Healthy Donors
  • Example 2 The experiment described in Example 2 was repeated in this example except that healthy donor PBMC were used.
  • Table I shows the results of paired Student t- test analysis between cytokine treated and untreated donor responses classified based on their baseline response to influenza A stimulation. Each column outlines the number of donors with baseline antigenic responses lower than a specified stimulation index and the effects of cytokine regulation on these samples. Therefore, results show how the combination of IL-13 and IL-13 was more effective in increasing antigenic responses than either cytokine used alone.
  • the results with IL-12 showed no enhancement of Influenza A responses in healthy donors.
  • N means the number of donors.
  • Figs. 6A and 6B also show Influenza A SI responses and Tetanus Toxoid SI responses, respectively.
  • Tetanus Toxoid responses after cytokine treatment in healthy individuals showed mean differences consistent with results in HIV-1 infected donors.
  • IL-12 showed no indication of enhanced responses with Tetanus toxoid as observed in HIV-1 infected individuals, suggesting a greater dependence on IL-13 for adjuvancy activity in healthy donors.
  • Fig. 6B shows enhanced mean enhancement with IL-13+IL-12 administration in Tetanus Toxoid responses and increased significance of the enhanced Influenza A responses in healthy donors with a stimulation index of less than 5.
  • Example 5 Effects of IFN- ⁇ and 11-4 on Antigenic Responses to Influenza A and Tetanus Toxoid in Both Healthy and HIV-1 Positive Donors
  • Figs. 7A and 7B indicate the effects of IL-4 and IFN- ⁇ on Tetanus Toxoid responses for healthy patients and on Influenza A responses for healthy patients.
  • Figs. 7C and 7D indicate the effects on HIV-1 infected patients.
  • the horizontal bar in each data group identifies the mean value with standard error annotation. Paired Student t- test results are shown as p values between groups.
  • IFN- ⁇ had no effect on Influenza A or Tetanus Toxoid responses in healthy or HIV-1 positive individuals.
  • Example 6 Effect of IL-13 after TNF- ⁇ Exposure
  • PBMC peripheral blood mononuclear cells
  • TNF- ⁇ can inhibit IL-12 secretion.
  • IL-13 together with TNF- ⁇ blocks this decrease.
  • IL-13 acts to reconstitute IL-12 production (as measured by antigen responses) within hosts with high TNF- ⁇ levels, such as HIV-1 infected hosts [D. Marchia et al, Nature. 363:464-466 (1993)].
  • IL- 13 may enhance responses within other chronic and high TNF- ⁇ secreting conditions or acute inflammatory conditions in which TNF- ⁇ is secreted, such as after trauma, surgery or other situations.
  • Venous peripheral blood samples were provided following donor signed consent by The Wistar Institute's phlebotomy unit (HIV-1-, 500 ml each) and by the Philadelphia Field Initiating Group for HIV-1 Trials (HIV-1+, 12-20 ml each).
  • PBMC peripheral blood mononuclear cells
  • Macrophages were plated in 24-well plates at 5 x 10 5 cells/well and cultured in RMPI 1640 10% human AB serum (Sigma, St. Louis, MO) with or without IL-13 (20 ng/ml, R&D, Minneapolis, MN) for 48 hours. Cells were subsequently incubated for 60 min with 1 mg/ml horseradish peroxidase (hrp, Sigma), washed and lysed in 100 ⁇ l of 0.05% Triton X-100 (Berringer Mannheim, Indianapolis, IN). Total protein in cell lysates was determined by Dc protein assay (Bio-Rad laboratories, Hercules, CA).
  • Adherent cells were prepared -and treated with IL-13 as described in the hrp assay above.
  • IL-13 -treated (20 ng/ml) and untreated HIV-1- samples were stained with either IgGl CD14-FITC (Biosource International, Camarillo, CA), IgG2a CDla (Immunotech, Westbrook, ME), IgG2a CDlb (Immunotech), IgGl CD86-PE (Pharmingen, San Diego, CA), IgGl CD80 (Immunotech), IgG2b HLA-DR-FITC
  • PBMC peripheral blood mononuclear cells
  • UV-inactivated (Fisher UVXL-1000 UV Crosslinker at 1200 ⁇ W/cm2 for 15 min at 4°C) influenza A virus PR8 Hi/Ml (138 HAU/ml), tetanus toxoid (5 ⁇ g/ml, Connaught, Swistwater, PA), PPD (5 ⁇ g/ml, Connaught) or keyhole limpet hemocyanin (5 ⁇ g/ml, Sigma) was added to wells of 96-well plates and cultured for 6 additional days before an 18-hr pulse with l ⁇ Ci/well of tritiated thymidine (Amersham, Arlington Heights, IL).
  • PBMC in each well were lysed and harvested nuclei analyzed with a Direct Beta Counter 9600 (Packard, Meriden, CT). Intra-assay variability was below 34% of mean response. Data was analyzed as stimulation index (antigen c.p.m.) and percentage change (antigen+IL- 13 c.p.m./antigen c.p.m.).
  • Example 8 - IL-13 increases deficient macrophage pinocytic function in HIV infection
  • Monocytes and macrophages play an important role in immunosurveillance and regulation of immune responses to pathogens by their constitutive ability to acquire antigen for presentation to T-cells by both fluid phase and receptor-mediated pinocytosis.
  • the data provided herein shows that IL-13 can improve recall CD4 T- cell-mediated responses in vitro at all stages of HIV disease.
  • the lack of any direct regulation of T-cell function by IL-13 lead to the examination of IL-13 effects in supporting antigen-specific T-cell activation by its regulation of accessory cell function in monocyte-derived macrophages (MDM).
  • MDM monocyte-derived macrophages
  • MDM monocyte-derived macrophages isolated from HIV-1+ patients
  • CD4 count range 113-790 cells/ ⁇ l
  • dextran-Texas Red tracers as compared with HIV-1- donors
  • Fig. 9 Deficient pinocytic function in HIV patients was not associated to the patient's CD4 count or anti-retroviral therapy.
  • HIV-1+ MDMs exposed to IL-13 significantly increased their hrp uptake to a level that was not different from that measured in untreated MDMs from HIV-1 donors. Confocal microscopy with a second pinocytic tracer, dextran-Texas Red, qualitatively confirmed the hrp results at the single cell level.
  • Example 9 - IL-13 enhances expression of T-cell co-activation molecule CD86
  • IL-13 might also enhance the expression of cell surface molecules that support antigen presentation
  • CD80 and CD86 as well as other molecules (CDla, CDlb, CD14, and CD83) associated with IL-13 effects on long-term cultures for monocyte-derived dendritic cell differentiation
  • Tables 2A-2C where IL-13 -induced changes in relative mean fluorescence intensity (RMFI) for HLA-DR (1.4-fold increase) and CD86 (3.5-fold increase).
  • RMFI relative mean fluorescence intensity
  • data is shown as median values (25%-75% quantiles) for each condition; N.A., indicates not applicable.
  • Relative Fluorescence values were calculated by dividing MFI of HLA-DR or CD86 by their corresponding isotype control MFI.
  • HIV-1 + 181.6 (76.8-340.8) 2.0 (1.5-2.8) 95 1 (23.1-197 7) (n 10) 111.2 (81.3-197.5) 6.3 (5.5-12.7) 17.3 (9.4-25.7)
  • Ratios were calculated by dividing each donor's CD86 relative MFI (RMFI) over their corresponding HLA-DR RMFI values.
  • RMFI relative MFI
  • HLA-DR and CD86 as target molecules for early IL-13- induced changes in cell surface expression
  • MDMs isolated from ten HIV-1+ patients were tested for IL-13-induced changes in HLA- DR and CD86 expression
  • CD86 expression was comparable between groups
  • IL-13 effects on HIV- infected MDMs were restricted to a four-fold induction of CD86 expression which resulted in a ratio of CD86 to HLA-DR expression that was not significantly different to that measured in HIV-uninfected controls
  • Example 10 - IL-13 induced CD4 T-cell memory responses at all stages of HIV- infection
  • influenza A virus and/or tetanus toxoid memory responses were tested in 44 HIV-1 and 83 HIV-1+ patient PBMC
  • antigens were selected based on their prevalence in the general population and the recommended re-exposure to both antigens in HIV-1+ individuals through vaccination [Centers for Disease Control and Prevention Recommendations of the Advisory Committee on Immunization Practices (ACIP) use of vaccines and immune globulins in persons with altered immunocompetence [Morb Mortal Wkly Rep . 42 (RR-4) 1-18 (1993)]
  • PBMC exposed to IL-13 significantly increased Influenza A and tetanus toxoid T-cell expansion as compared to baseline antigenic responses (Tables 3A-3C).
  • Higher recall responses to Tetanus toxoid in the HIV-1+ group in the presence of IL-13 are interpreted to reflect a re-exposure to Tetanus toxoid antigen by re-vaccination of this patient group.
  • KLH Keyhole Lympet Heamocynin
  • baseline antigenic response was analyzed by nonparametric bivariate density scatterplot analysis (CD4 count vs. c.p.m.) identifying three hierarchical response cluster regions not restricted by CD4 count (scatter plot not shown).
  • PHA response as a function of CD4 count did not predict for a lower responsiveness to IL-13. Comparable analysis of responses to IL-13 and influenza A in the uninfected population did not identify a refractory population.
  • Example 1 1 - IL-13 induces cell-mediated responses independent of IFN- ⁇ In view of the presence of an enhanced T-cell recall responses in patient's
  • IFN- ⁇ secretion is positively correlated to CD4 count and has been proposed as a key cytokine able to promote cell-mediated responses by its effects on macrophage activation and antigen presentation such as increased cell surface expression of HLA-DR, CD80 and CD86 [H.W. Murray et al, N. Engl J. Med.. 310:883-889 (1984); A. Freeman et al, Cejl Immunol. 137:429-437 (1991)].
  • the T-cell recall response of PBMC HIV-1 to influenza A virus in the presence of IL-13 and neutralizing IFN- ⁇ antibody B 133.3 increased above that measured with influenza A and IL-13 (Fig.
  • IFN- ⁇ When added exogenously together with IL-13, IFN- ⁇ inhibited IL-13- mediated effects in both HIV-1- and HIV-1+ PBMC indicating a differential regulation of macrophage activation and antigen presentation between cytokines (Fig. 1 IB). However, IFN- ⁇ did not inhibit antigenic responses below those in untreated controls suggesting that IFN- ⁇ 's inhibition was restricted to the IL-13 enhancing effects on MDM. IFN- ⁇ also inhibited IL-13 -induction of endocytic uptake yet values for internalized hrp by MDMs treated with IL-13 and INF- ⁇ were higher than those measured with IFN- ⁇ alone (data not shown). The latter suggests both cytokines may jointly regulate macrophage function, consistent with IL-13's enhancement of recall responses in HIV-1-uninfected controls where no deficiency of IFN- ⁇ secretion following T-cell activation is expected.
  • IL-13 antigen presentation as a cytokine secreted by both naive and memory T-cells (type-1 and type-2) may contribute to maintain CD4 T-cell responses and antiviral resistance [R. De Waal Malefyt et al, Internat. Immunol. 7: 1405-1416 (1995); T. Jung et al, Eur. J. Immunol. 26:571-577 (1996)].
  • IL-13 The potential for IL-13 to augment type-1 cell-mediated immune function in vivo [I. Flesch et al, Internat. Immunol. 9:467-474 (1997)] together with its effects on healthy and HIV-1+ patient cells by enhancing pinocytic uptake, IL-12 secretion, CD86 co-activation potential, the capacity of stimulating CD4 cell-mediated responses in early and late stages of HIV infection independently of IFN- ⁇ , and its direct antiviral effects on HIV-1 -infected macrophages which account for a large proportion of viral expression during opportunistic infections, suggests a potential adjuvant property benefit for this cytokine as immunotherapy in combination with anti- retrovirals in HIV-1 infection, as well as in healthy individuals.
  • Example 12 - IL-13 Enhances T-cell Memory Responses to Mycobacterium Tuberculosis and HIV-1 Antigens
  • the purified protein derivative of Example 12A was obtained from Connaught Laboratories.
  • the HIV-1 proteins of Example 12B were obtained from the National Institutes of Health (NIH) AIDS reagent repository.
  • Mycobacterium tuberculosis IL-13 enhances peripheral blood mononuclear cell responses to PPD antigen stimulation. Activation of memory responses was measured in eleven HIV- infected donors with a confirmed past diagnosis of tuberculosis infection. Shown are the counts per minute data as medians (25%-75% quantiles) for each group.
  • Table 6A Purified Protein Derivative
  • IL-13 enhances peripheral blood mononuclear cell responses to HIV-1 antigens in HI V- 1 -infected individuals Activation of memory responses was measured in 23 HIV-infected donors Shown in Table 6B are the counts per minute data as medians (25%-75% quantiles) for each group.
  • IL-12 does support proliferative responses to antigens such as flu in HIV-infected samples, yet displays antigen specificity by not recovering recall responses to Tetanus Toxoid.
  • No direct IL-12 effects are described on APC function other than stimulating T cell or NK cell IFN- ⁇ production. Since IFN- ⁇ can act to decrease fluid phase uptake by APC, IL-12 may thus be restricted in its effects to enhance antigen specific activity in healthy and HIV-1 patients.
  • IL-13 was effective in increasing CD4 T cell activation at all stages of disease and for several antigens tested. Associated with IL-13 effects on antigen presentation function was an enhancement of antigen-specific T-cell activation in both HIV-infected and uninfected PBMC exposed to a single treatment of IL-13 (Tables 3A-3C). Decreased recall responses to Influenza A and Tetanus toxoid in HIV-1 patient PBMC as compared to healthy controls were increased 2.8- to 3 -fold by IL-13.
  • IL-13 enhancement of recall responses in HIV- infected samples reached values approximating those measured in untreated cells from healthy donors.
  • IL-13 can efficiently enhance responses to the level of uninfected donors yet does not restore the level or responsiveness to cytokine in uninfected donors.
  • IL-13 effects in healthy controls also supported an increase in antigenic response to influenza A, while responses to tetanus toxoid were increased within four individual donors but not when analyzing the group of 18 as a whole.
  • Basal antigenic response was found as the highest correlated variable to IL-13 enhancement of lymphoproliferative responses in both healthy and HIV-infected PBMCs.
  • Hierarchical analysis of influenza A responses in both healthy and HIV-infected PBMC revealed IL-13 was able to increase antigenic responses in PBMC whose baseline antigenic response would have otherwise been classified as the lowest in the group ( Figures 10A-I).
  • IL-13 had no effect on 19 of 83 HIV-infected PBMC with both low and high CD4 count that were also refractory to mitogen-mediated T-cell activation.
  • IL-13's effects on endocytic uptake and cell surface expression of molecules associated with antigen presentation were both tested with the effects of IL-13 on antigen-specific T-cell activation HIV- infected patient's MDMs showed a highly significant deficiency in pinocytic tracer uptake indicating a general decreased ability by these cells to acquire antigen since both routes of entry for hrp and dextran endocytic tracers (fluid phase and mannose receptor-mediated) represent mayor pathways of antigen intake by APC [F Sallusto et al, J Exp Med .
  • CD86 has been identified to block Fas-L and TNF- ⁇ -mediated mechanisms of T-cell death [L Lu et al, J Immunol , 158 5676-5684 (1997), R -H Lin et al, J Immunol , 158 598-603 (1997)], both of which have been reported to account for the lack of T-cell activation in HIV infection [A Badley et al, J Exp Med .
  • IL-13 can activate cell-mediated immunity independently of IFN ⁇ , yet may also complement such activation with IL-12 since its secretion is also induced by IL-13.
  • IL-13 can increase the responses otherwise inhibited by IL-12.
  • IL-12 acts to decrease the level of enhancement observed with IL-13 alone, albeit still higher than untreated control.
  • Combined IL- 13 and IL- 12 results in immune responses to antigens such as Tetanus Toxoid otherwise inhibited by IL-12 alone.
  • the benefit of using IL-12 and IL-13 alone when vaccinating against antigens such as Tetanus Toxoid is based on 1L-I2's ability to stimulate type-1 immunity.
  • IL-13 was able to enhance T cell proliferation following Tetanus Toxoid stimulation, alone or with IL-4.
  • IFN- ⁇ reduced T cell proliferation to Tetanus Toxoid, which is consistent with the lack of the recovery of recall response by IL-12.
  • IL-13 enhances fluid phase uptake and mannose receptor mediated uptake of antigens while IFN- ⁇ decreases this activity.
  • IL-12 induction of IFN- ⁇ may result in an enhancement of antigen processing within antigen presenting cells allowing for exposure of epitopes not supported by IL-13.
  • GCC ACG GTC ATC TGC CGC AAA AAT GCC AGC ATT AGC GTG CGG GCC CAG 965 Ala Thr Val He Cys Arg Lys Asn Ala Ser He Ser Val Arg Ala Gin 300 305 310
  • MOLECULE TYPE protein

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Abstract

L'invention concerne un procédé visant à améliorer la réponse immune à un antigène en administrant à un hôte, simultanément ou séquentiellement, pourvu de l'antigène, une quantité efficace d'interleukine-13 ou d'un fragment biologiquement actif de celle-ci.
PCT/US1998/001500 1997-01-22 1998-01-22 Procedes et compositions utilisant l'interleukine-13 dans l'amelioration de reponses immunes WO1998031384A1 (fr)

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US7585500B2 (en) 2004-11-17 2009-09-08 Amgen Inc. Fully human monoclonal antibodies to IL-13

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Publication number Priority date Publication date Assignee Title
EP1410798A3 (fr) * 1999-01-08 2004-10-13 Maxim Pharmaceuticals, Inc. Traitement et prevention des dommages cellulaires induits par des metabolites reactives d'oxygene
US7585500B2 (en) 2004-11-17 2009-09-08 Amgen Inc. Fully human monoclonal antibodies to IL-13
US7994302B2 (en) 2004-11-17 2011-08-09 Amgen Inc. Fully human monoclonal antibodies to IL-13

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