WO1998031384A1 - Methods and compositions using interleukin-13 for enhancing immune responses - Google Patents

Abstract

A method is provided for enhancing immune response to an antigen by administering to a host simultaneously or sequentially with the antigen an effective amount of Interleukin-13 or a biologically active fragment thereof.

Description

METHODS AND COMPOSITIONS USING INTERLEUKIN-13 FOR ENHANCING IMMUNE RESPONSES

This work was in part supported by National Institute of Health grant AI40379. The US government has certain rights in this invention.

Field of the Invention

The present invention relates generally to methods and compositions for increasing the immune response to antigens, particularly for vaccine use.

Background of the Invention

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.

The current paradigm of the role of T cells in the particular immune response is that CD4⁺ 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 γ (IFN-γ) 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)]. Many methods of vaccination employ 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.

Many of the most effective adjuvants 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. 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)].

Indeed, a 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

In one aspect 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 .

In another aspect, 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.

Still another aspect of this invention involves 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.

Other aspects and advantages of the present invention will be apparent upon consideration of the following detailed description of preferred embodiments thereof.

Brief Description of the Drawings

Fig. IA is a graph of stimulation index (SI) of 21 HIV-1 infected patients' PBMC (N=21) vs. the four identified conditions for adjuvanting or not adjuvanting Influenza A (flu). Paired student t-test correlations are indicated as p values.

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. 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.

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

SI vs. CD4 cell count for PBMC exposed to the T.T. antigen and IL-12.

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-γ 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-γ 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-γ 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

T.T. antigen alone or adjuvanted with IFN-γ or IL-4. Students paired t test correlations are indicated.

Fig. 8 is a graph of the effect of TNF-α on IL-12 production in PBMC of three donors, untreated or exposed to IL-13, TNF-α, anti-TNF-α, anti-TNF-α+IL- 13 and TNF-α+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 μl/mm3) donor monocyte-derived macrophages (MDM) with or without IL-13. Results of unpaired Student's t test analysis are shown for comparison between HIV-infected MDM to healthy controls (p<0.001), while significance of IL-13's induction of increased uptake in both groups is represented by an asterisk [HIV-1+ patients : paired Student's t test (pSt) p=0.001 ; HIV-1 ; pSt, p=0.015]. Also shown are results of unpaired analysis between baseline MDM uptake by healthy controls and IL-13 -treated MDMs from HIV-infected (p=0.31). 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. The data are divided into three cluster groups derived from a bivariate nonparametric analysis between baseline influenza A response (highest correlated variable to IL-13 responsiveness) and CD 4 count, independently defined by Ward's method of hierarchical clustering into a high (4220 +957, n=32), intermediate 2121 + 324, n=24) and low (891 + 304, n=28) response group to influenza A stimulation. 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/μl, 47019 c.p.m; 419 CD4 cells/μl, 58231 c.p.m.). Not shown is the linear regression analysis on the data as a whole [Influenza A (n=76, r=0.24, p=0.02, y=1974+2.1x); Influenza A + IL-13 (n=76, r=0.11, p=0.33, y=5010+1.9x); PHA (n=76, r=0.36, p=0.001, y=3088+19.9x)]

Fig. 11 A is bar graph illustrating influenza A virus T-cell memory responses with IL-13 and neutralizing IFN-γ antibody B133.3 in HIV-1 (solid bar:n=10) and HIV-1+ (open bar :n=25, 2-604 CD4 μl/mm³) donor PBMC. Data are expressed as mean (±SD) percent change in thymidine incorporation (c.p.m.) (ratio of antigen- stimulated plus IL-13 c.p.m. to antigen-stimulated 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. Isotype control responses were negative and are not shown. Asterisks indicate a significant difference (Wilcoxson Signed-Rank test (WSR), p<0.05) from values for untreated antigen stimulated (i.e., 100%). Triangles denote significant differences (WSR, p<0.05) between IL-13 and combination treatments.

Detailed Description of the Invention

The invention provides methods and compositions for stimulating immune responses in mammalian host cells, which involve delivering interleukin- 13 (IL-13) to host cells. Where reference is made herein to IL-13, one of skill in the art will understand that the 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. Similarly, one of skill in the art will understand that 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. As used herein, the term "mammal" includes humans and non-human animals. IL-13 may be obtained from commercial sources, e.g., Genzyme and

Peprotech. Alternatively, 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. Examples of 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. Examples of bacterial antigens include those derived from and/or useful against leprosy and tuberculosis, among others. Examples of parasitic antigens include those derived from and/or useful against such infections as leishmaniasis and malaria, among others. Still other composition antigens include those derived from a protozoan, e.g., T. cruzii, or against a helminth, e.g., Schistosoma.

It is further anticipated that 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. Such 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. Particularly where the tumor antigen by itself has been unsuccessful in activating an response to a particular cancer, 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. For example, 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. Alternatively, other cancer therapeutics are designed using an antigen normally not expressed on a cancer cell. For example, 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.

A. Method of the Invention

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). This "adjuvanting" effect is provided by co-administering IL-13 to a host in conjunction with a selected antigen. By "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. Alternatively, 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.

Whether delivered as protein or nucleic acid, 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. For example, as discussed below, IL-13 can adjuvant tetanus toxoid, which is not adjuvanted by other known adjuvants. Further, 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. Still further, 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. As described below, IL-13 has been found to stimulate both humoral and cell-mediated immune (CMI) responses within immunocompromised individuals. Thus, the invention provides not only a method for adjuvanting antigens in a host, but a novel method of adjuvanting antigens in a host with ongoing immune activation or disease, which is a group of hosts known to be not recommended for vaccination with any antigen.

The present invention also provides methods of adjuvantation intended to provide an additive or a synergistic effect with other adjuvants, and particularly, IL- 12. For example, 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. 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-α; see, e.g., Example 6 and Fig. 8).

Without wishing to be bound by theory, the inventor believes that 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-γ or IL-2, IL-13 is relatively stable in vivo. Furthermore, the effects of IFN-γ 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. Thus, 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. Such diseases may be broadly defined and include conditions requiring "jump starts" to both innate and adaptive immunity. For example, 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-α, which are dependent on antigen recognition and cell-mediated responses. In addition, 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 For example, 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. In particular, 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. Further, 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.

Additionally, 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-α which may be unanticipated or not controlled. For example, the induction of IL-12 may result in differential levels of IFN-γ 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-γ, 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). For example, 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). However, 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. Unlike bacterial adjuvants, IL-13 is human in origin and thus unlikely to produce any sensitization.

Furthermore, 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. The lack of effects of IL-12 in enhancing responses to tetanus toxoid, which is internalized via non-receptor mediated pathways used by small proteins [R. Montesano et al, Nature. 296:651-653 (1982)], demonstrates that IL-12's adjuvancy may be restricted and addressed more effectively by the use of IL-13. Therefore, since antigen acquisition by presenting cells is the main focus of an in situ response at the site of injection, IL-

13 effects are a more adequate support to increase the amount of antigen to be taken to draining lymph nodes than IL-12.

Thus, IL-13 is a highly useful immunostimulant and adjuvant for use in human and veterinary compositions, including vaccines. B. Compositions of the Invention

As illustrated in the examples below, 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. ϋ 474-485 (1991) which describe similar uses of 'naked DNA', all incorporated by reference herein] Alternatively, IL-13 DNA may be incorporated, or transduced, into a microorganism, if a cellular pathogen itself is to be employed as the vaccinal antigen In still another alternative, 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 that sequence in vivo This vector permits infection of vaccinate's cells and expression of the IL-13 in vivo Numerous types of vectors are known in the art for protein expression and may be designed by standard molecular biology techniques Such plasmid and viral vectors are selected from among conventional vector types including baculoviruses, yeast, bacterial and viral expression systems See, e g , Sambrook et al, Molecular Cloning A Laboratory Manual, 2d edition, Cold Spring Harbor Laboratory, New York (1989), Miller et al, Genetic Engineering, 8 277-298 (Plenum Press 1986) and references cited therein Recombinant viral vectors, such as retroviruses or adenoviruses, are preferred for delivering DNA to a cell Suitable vectors are well known to those of skill in the art and are not a limitation on the present invention

When used as an adjuvant for a selected antigen, IL-13 is desirably admixed as part of the antigen-containing composition itself Such a composition is desirably a vaccine composition which contains a suitable carrier and, optionally, other desired components. Selection of appropriate 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. Similarly, one skilled in the art 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.

One of skill in the art should be able to readily determine suitable amounts of 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. As one example, an effective amount of IL-13 is desirably between about 0.1 μg to about 0.5 mg of IL-13 protein or fragment per about 25 μg of antigen. When used in combination with another cytokine, such as IL-12, each is present in an amount between about 0.1 μg 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.

Alternatively, the immunostimulatory (i.e., adjuvanting) effect of IL-13 may be obtained by administering IL-13 separately from the vaccine composition. When separately administered, the IL-13 is desirably in the presence of 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. When 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. Optionally, 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. For purposes of convenience, the 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. Unless otherwise stated, 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.

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. Hence, 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. Where present in one or two plasmids, 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. When 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. However, the methods of this invention also encompass other antigens as described above. For example, preliminary data on two HIV- 1 infected donors show that additional antigens, such as Candida aϊbicans, result in a greater antigenic response in the presence of IL-13, or IL-13 and IL-12. Additionally, the following experiments demonstrate that IL-13 can enhance antigen responses Xo Mycobacterium tuberculosis and HIV-1 antigens. These examples do not limit the scope of the invention.

Example 1 - Protocols for Thymidine Incorporation Assays as a Measurement of Antigen Selectivity in Enhancement of Recall Responses in Healthy and HIV-1 Positive Donors 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. Such response indicates the successful interaction between antigen presenting cells (APC) and 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)]. A. Cell Preparation

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 μg/ml streptomycin and 10% fetal calf serum (FCS).

B. Cell Cultures

PBMC were cultured at 250,000/ml in a final volume of 200 μl in 96- well plates. PBMCs were treated, as specified in the following examples, with either (i) IL-12 (O. lng/ml),

(ii) IL-13 (20ng/ml),

(iii) the combination of i and ii,

(iv) IL-4 (20ng/ml),

(v) IFN-γ (50U/ml), or (vi) no treatment for 18 hours. Cytokine concentrations used were derived from titration responses as described in Example 3 (Figs. 5A to 5D).

Thereafter, none or one of the following antigens was introduced into the PBMC: (vii) Influenza A virus (PR8 138 HAU/ml),

(viii) Tetanus Toxoid (5 μg/ml; Connaught, Ontario),

(ix) KLH (5 μg/ml; Sigma) or

(x) PHA (5 μg/ml; Sigma), and PMBC were left in a humidified

37°C incubator at 5% CO₂ for 7 days. PHA stimulation was performed immediately after PBMC isolation and cells were pulsed with [H-3] thymidine at the end of day 8. C. Proliferation Assay

³H-thymidine (ICN) incorporation assay was used to measure antigen- induced proliferation within PBMC. All tests were performed by adding a total activity of 1 μCi/well ³H-thymidine for 18 hours. 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.

D. Data Analysis: stimulation index

Triplicate wells for each condition tested were averaged in calculating means. For each group of data arising from one donor, the cpm derived from cells cultured in medium alone was used to calculate stimulation index (SI). For example, the SI for a flu response would be expressed as average cpm of flu stimulation average cpm of cells in medium only. Healthy and HIV-1 patient SI values were analyzed between test groups by paired student t-test using Sigma Plot (Jandel Scientific). Linear regressions with 95% confidence intervals were calculated for SI values in HIV-1 patients in relation to CD4 levels.

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

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. Patient CD4 count levels were reported from medical records at the time of blood collection. In general, in comparison with the results in healthy individuals in Example 4, 30 HIV-1 patients had a decreased response to Influenza A (p=0.05) and Tetanus Toxoid (p=0.08) in comparison to 27 healthy individuals. A. Results were reported as Influenza A SI responses (Fig. 1 A) and

Tetanus Toxoid SI responses (Fig. IB). Positive PHA controls for each patients and cytokine tested and negative KLH controls for each patients and cytokine tested were provided (Fig. IC).

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. For example, IL-12 had a titratable increase of Influenza A SI response (p=0.0029); IL-13 had a titratable increase for Influenza A SI (p=0.0005). IL-12 and IL-13 when used in combination increased Influenza A SI (p=0.019) in HIV-1 infected donors. In contrast, Tetanus Toxoid responses in HIV-1 infected donors are differentially enhanced between IL-13 as adjuvant (5.96 SI) and the combination IL- 12+IL-13 as adjuvant (4.12 SI), p=0.0035 (Fig. IB). IL-12+IL-13 when used in combination increased Tetanus Toxoid SI (p=0.0017) in HIV-1 infected donors (Fig. IB). 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), and for 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). While IL-12 increased influenza A responses in HIV-infected individuals, Tetanus toxoid responses were not increased at either low or high CD4 count. Conversely, IL-13 increased mean responses to both influenza A and Tetanus toxoid in HIV-1 infected samples at both high and low CD4 count. Although 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. 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.

Example 3 - IL-12 AND IL-13 Titrations on Influenza A and Tetanus Toxoid Responses in HIV-1 Patients

Culture conditions and antigen stimulation were performed as described above in Example 2. 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. IL-13 had a titratable increase for Tetanus Toxoid SI (p=0.00002) in (Figs. 5B and 5D) 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

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. IL-13 enhanced Influenza A responses in healthy donors with less than 5 SI (p=0.039). IL-13 and IL-12 enhanced Influenza A responses in healthy donors with less than 5 SI (p=0.008). "N" means the number of donors.

Table I

SI: <2 <3 <4 <5

Flu SI NN:: 66 1 177 2 222 5

IL-12 0.13 0.52 0.58 0.51

IL-13 0.35 0.12 0.059 0.039

IL-12+ IL-13 0.022 0.03 0.024 0.008

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. However, 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. These adjuvant effects in healthy donors, as shown in Table I, are targeted to those with low responses to antigens, because high and low healthy responders taken together show a less evident effect of the cytokines. Cytokine enhancement of low responders within healthy donors is consistent with the cytokine effects on HIV-1 patients who are low responders as well.

Example 5 - Effects of IFN-γ and 11-4 on Antigenic Responses to Influenza A and Tetanus Toxoid in Both Healthy and HIV-1 Positive Donors

The experimental protocol of Example 1, with adjuvants (IL-4) and (IFN-γ) and antigens (Influenza A) and (Tetanus Toxoid) was employed in this experiment. Figs. 7A and 7B indicate the effects of IL-4 and IFN-γ 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.

IL-4 showed no enhancement of Influenza A responses (p=0.21), but increased Tetanus Toxoid response (p=0.056) in healthy donors. IL-4 enhanced Tetanus Toxoid (p=0.032) and Influenza A (p=0.0025) responses in HIV-1 positive patients. This data is consistent with IL-13 enhancement observations, since IL-4 and IL-13 regulate the antigen presenting cells similarly.

IFN-γ 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-α Exposure

PBMC were collected from three healthy donors and cultured for 72 hours, at which time they were treated with IL-13 (20 ng/ml), TNF-α (20 ng/ml), 10 μg/ml TNF-α neutralizing antibody (anti-TNFα; clone B 154.7), IL-13+anti-TNF-α, TNF- α+IL-13, for 24 hours prior to lipopolysaccharide (LPS) stimulation (1 μg/ml). Supernatants were collected 24 hours after stimulation and p40 IL-12 homodimer measured by radioimmunoassay. p40 homodimer secretion is representative of bioactive p70 IL-12 heterodimer secretion.

Results are reported in Fig. 8. TNF-α can inhibit IL-12 secretion. IL-13 together with TNF-α blocks this decrease. IL-13 acts to reconstitute IL-12 production (as measured by antigen responses) within hosts with high TNF-α levels, such as HIV-1 infected hosts [D. Marchia et al, Nature. 363:464-466 (1993)]. Thus, IL- 13 may enhance responses within other chronic and high TNF-α secreting conditions or acute inflammatory conditions in which TNF-α is secreted, such as after trauma, surgery or other situations.

Example 7 - Enhancement of Antigen-Presenting Function and T-cell Memory

Responses in HIV-1 -Infected cells //? Vitro by Interleukin- 13

The following experimental methods were utilized for the studies described in

Examples 8 through 12 below. A. Sample processing

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).

HIV-infected material was received with relevant clinical information gathered by study nurses. Peripheral blood mononuclear cells (PBMC) were isolated by Ficoll gradient and the adherent cell fraction was isolated as described (L. J. Montaner et al,

J. Exp. Med.. 178:743-747 (1993)]. B. Quantification of Endocytosis

Macrophages were plated in 24-well plates at 5 x 10⁵ 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 μl 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). Twenty microliters of each cell lysate was mixed with 180 μl of hrp substrate as described [R. Steinman & Z. Cohn, J. Cell Biol. 5_5: 186-204 (1972)], and immediately analyzed in a kinetic absorbance reader at 460 nm (Rainbow Reader, STL, Austria). Hrp content was determined using an hrp standard curve, correcting for any cell loss by expressing uptake values per μg of macrophage protein in lysates. For dextran uptake assays, adherent cells in 5 cm² glass cover slips (Gold Seal) were exposed to IL-13 for 48-hr and pulsed with 1 mg/ml 70Kd dextran-Texas Red (Molecular Probes, Eugene, OR) for 1 hr. washed and fixed with 4% paraformaldehyde. Images were obtained at The Wistar Institute Microscopy Facility using a rhodamine filter (514/568 nm excitation, 590 nm emission) on a Leica microscope (Deerfield, IL) with Focus Imagecorder Plus software (Foster City, CA). C. Flow Cytometry Analysis

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

(Pharmingen), or IgG2b CD83 (Immunotech). Goat anti-mouse IgG2a-FITC (Sigma, St. Louis, MO) was used as secondary antibody. Isotype controls used were IgGl-PE (Pharmingen), and IgG2b-FITC (Pharmingen). Sample FITC and PE fluorescence was analyzed with an Epics XL-MCL (Coulter Corp., Hialeah, FL) at The Wistar Institute Flow Cytometry Facility. Data was analyzed as relative mean fluorescent intensity (mean fluorescent intensity/isotype control fluorescent intensity).

D. Lymphoproliferative Assays PBMC were isolated by Ficoll gradient and plated in 96-well plates at

250,000 cells/well in RPMI-1640- 10% human AB serum (Sigma, St. Louis, MO). Each sample was divided into the following antigen stimulation groups: with or without cytokine, cytokine/unstimulated, untreated/ unstimulated and PHA (5 μg/ml, Sigma) controls. HIV-1- samples were tested in triplicate for each test condition, while HIV-1+ were tested in duplicate as a minimum for use in this study. IL-13 (20 ng/ml) was added to test wells only once at the time of PBMC isolation. Cultures were incubated at 37°C in 5% CO₂ for 18 hr. after PBMC isolation cytokine exposure. Subsequently, UV-inactivated (Fisher UVXL-1000 UV Crosslinker at 1200μW/cm2 for 15 min at 4°C) influenza A virus PR8 Hi/Ml (138 HAU/ml), tetanus toxoid (5 μg/ml, Connaught, Swistwater, PA), PPD (5 μg/ml, Connaught) or keyhole limpet hemocyanin (5 μg/ml, Sigma) was added to wells of 96-well plates and cultured for 6 additional days before an 18-hr pulse with l μCi/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.).

E. Measurement of IFN-γ-independent lymphoproliferation

Effects of anti-IFN-γ and IFN-γ on influenza A virus (flu) responses to IL-13 in HIV-1- (n=10) and HIV-1+ (n-25) PBMC were analyzed following the lymphoproliferation method described above, except for including additional conditions; IL-13 (20 ng/ml), IFN-γ (12.5 ng/ml, concentration established by titration effects, Genzyme, Cambridge, MA), IFN-γ + IL-13, mouse anti-IFN-γ IgGl B 133.3 (10 μg/ml, concentration established by titration effects), B 133.3 + IL-13, and mouse anti-human HBV IgGl control (10 μg/ml, Sigma, St. Louis, MO). Antibody B133.3 was kindly provided by G. Trinichieri (Wistar Institute, PA). F. Statistical analysis

Each group of data was analyzed for normal distribution by the Shapiro-Wilks W test (p>0.05) and all subsequent comparisons between means were two-tailed. "Significance" in text is used for differences at an alpha level of 0.05 (p<0.05). All descriptive analysis and statistical tests were performed with JMP 3.2.1 (SAS Institute Inc., Cary, NC).

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. In elucidating mechanisms of action, 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). Initial experiments of depletion of the adherent cell monolayer at the time of PBMC isolation showed that both the antigenic response and IL-13 effects were dependent on this cell population (data not shown)

Quantitative and qualitative analysis of monocyte-derived macrophages (MDM) isolated from HIV-1+ patients (CD4 count range: 113-790 cells/μl) showed a deficiency in their ability to internalize soluble horseradish peroxidase (hrp) [unpaired Student's t-test, p<0.001] or 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 To determine whether IL-13 might also enhance the expression of cell surface molecules that support antigen presentation, the effect of IL-13 on the expression of HLA-DR and co-stimulatory ligands 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 [L. Piemonti et al, Eur. Cyt. Netwk. 6:245-252 (1995); F. Chapuis et al, Eur. J. Immunol. 27:431-441 (1997)]. Early changes at 48 hr following a single IL-13 treatment were tested.

Results showed an induction of cell surface HLA-DR and CD86 expression while CD 14 was decreased; CDla, CDlb, CD80 and CD83 levels of staining remained unchanged (n-3 for all antibodies, n=7 for CD86, HLA-DR; data not shown). These observations were confirmed in 9 additional HIV-1- donors listed in 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). In the following table, 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. Paired comparison between untreated and IL-13-treated Relative Fluorescence values by Student t-test or Wilcoxson Signed-Rank test as applicable. Table 2A HLA-DR and CD86 Mean Fluorescence Intensity (MFI) regulation by IL-13 in HIV- 1- and HIV-1+ monocyte-derived macrophage.

UNTREATED

Isotype Relative

HLA-DR Control Fluorescence³

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)

HIV-l^" (n= =9)

IL-13

Isotype Relative

HLA-DR Control Fluorescence

HΓV-Γ 208.2 (82.6-480.3) 1.7 (1.6-2.9) 122.5 (42 7-225.8)

(n=10) 247.4 (106.9-277.5) 6.5 (5.8-12.2) 19.9 (15.9-33.7) HIV-1^" (n= =9)

Table 2B - CD86 Mean Fluorescence Intensity

UNTREATED

Isotype Relative

CD86 Control Fluorescence

HIV-Γ 70.8 (25.6-121.5) 1.5 (1.3-2.0) 44.2 (16.6-80.5)

(n=10) 95.9 (74.0-122.6) 2.3 (1.9-3.5) 43.7 (22.2-55.5) HIV-1^" (n^: =9)

IL-13

Isotype Relative

CD86 Control Fluorescence

HIV-Γ 302.2 (70.8-477.2) 1.6 (1.4-2.0) 199.2 (76.6-301.2)

(n=10) 323.0 (204.2-459.0) 2.99 (1.9-3.4) 125.4 (59.0-232.6) HIV-L (n= =9)

Table 2C - CD86:HLA-DR Relative Mean Fluorescence Intensity Ratio³

a. Ratios were calculated by dividing each donor's CD86 relative MFI (RMFI) over their corresponding HLA-DR RMFI values. b. Wilcoxson Signcd-Rank test between untreated and IL-13 -treated CD86:HLA-DR RMFI ratios. Having identified HLA-DR and CD86 as target molecules for early IL-13- induced changes in cell surface expression, MDMs isolated from ten HIV-1+ patients (CD4 count range 193-580 cells/μl) were tested for IL-13-induced changes in HLA- DR and CD86 expression In contrast to uninfected controls, HIV-infected MDMs showed a significant seven-fold increase in basal mean HA-DR expression (unpaired Student's t test, p=0 039) CD86 expression was comparable between groups However, if expression levels of DC are analyzed in relation to HA-DR, a 73% decrease in CD86 expression is present in HIV-infected MDM as compared to healthy controls (Wilcoxson Rank Sum, p<0 001, Table 2C) 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 To determine whether the mechanisms of increased antigen presenting function induced by IL-13 could improve recall T-cell responses, influenza A virus and/or tetanus toxoid memory responses were tested in 44 HIV-1 and 83 HIV-1+ patient PBMC These 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)]

Recall responses varied among HIV-1 -donors, with influenza A virus eliciting the strongest responses In accordance with previous results [G Shearer et al J Clin Invest , 74 496-506 (1984), H Lane & N More, N En J Med , 3 3 79-84 (1985), H W Murray et al, N Engl J Med . 313 1504-1510 (1985)], recall responses in HIV-1- infected patients were significantly decreased as compared to responses in healthy controls (Table 3A-3C) In the following tables, data is shown as median values (25%-75% quantiles) for each condition. Lower case a indicates Wilcoxson Signed-Rank test between Influenza A and Influenza A + IL-13 c.p.m.; b indicates missing PHA data points, average of 23 donor responses within the group; c indicates missing PHA data points, average of 1 1 donor responses within the group.

Table 3A - Influenza A Virus

I. II.

Cell Baseline Influenza A

Cell Baseline Influenza A +IL-13 +IL-13

HIV-Γ 1025 2210 929 5986

(n=83) (725-1789) (1040-3698) (674-1340) (1925-8425)

1844 7660 2138 11654

Hiv-r (1313-2617) (6838-9287) (1621-2621) (10019-15480)

(n=44)

III. I. vs. II."

PHA Response Signed-Rank

HIV-Γ 6221 (2152-1 1 087) p<0.001

(n=83)

42807 (35318- •57235)^b p<0.001

HIV-Γ

(n=44) Table 3B - Tetanus Toxoid (TT)

Table 3C - Keyhole Lympet Hemocyanin (KLH)

In accordance with an increase in antigen presenting function, HIV-infected

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. Negative controls performed in each donor demonstrated IL-13 did not exert mitogenic effects when used alone or induce non-specific responses following neoantigen Keyhole Lympet Heamocynin (KLH) stimulation (Tables 3A- 3C). Consistent with a CD4 expansion mediated by the adherent MDM, flow cytometry analysis confirmed a CD4 T-cell expansion and removal of the adherent cell layer at the time of PBMC isolation followed by IL-13 and antigen stimulation resulted in the loss of responses (data not shown).

Characterization of the ability of IL-13 to increase antigenic responses in HIV- infected PBMC showed no strong correlation between patient's CD4 count and IL-13 effects consistent with the data listed in Tables 4A-4B. Data in Tables 4A-4B is shown as median (25%-75% quantiles) and mean ± standard deviations for each patient groups classified by CD4 count at the time of blood isolation. N.A. denotes Not Applicable; lower case "a" indicates a paired comparison between Influenza A and Influenza A +IL-13 c.p.m. by Student t-test or Wilcoxson Signed-Rank test as applicable; lower case "b" indicates a summary of patient's actual CD4 counts within each category, data does not include 7 of 83 patient-derived samples for which no CD4 values were available; lower case "c" indicates the absence of IL-13, refer to Table 2 for reference cell baseline + IL-13 control values.

Table 4A HIV-infected lymphoproliferative responses to influenza A and IL-13 in relation to CD4 T-cell/μl

Table 4B

In general, responsiveness to IL-13 also did not indicate a dependency on anti- retroviral therapy (Tables 5A and 5B). Data in Tables 5A and 5B is shown as median (25%-75% quantiles) and mean ± standard deviations for each patient groups classified by anti-retro viral therapy reported at the time of blood isolation. Further, N.A., Not Applicable, a. Paired comparison between Influenza A and Influenza A + IL-13 c.p.m. by Student t-test or Wilcoxson Signed-Rank test as applicable. b.CD4 data is missing 7 of 83 patient-derived samples for which no CD4 values were available, c. In the absence of IL-13, refer to Tables 3A-3C for reference cell baseline + IL-13 control values.

Table 5A

Table 5B

Baseline antigenic response was the highest correlation to an IL-13 -mediated increase in antigen lymphoproliferation consistent with an increased ability to activate antigen-specific T-cells (Spearman's rho=0.75, p<0.001). To evaluate the ability of IL-13 to augment antigen responses within the lowest responder group to influenza A stimulation in the HIV-infected group, 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). The presence of three normally distributed cluster regions was independently confirmed by hierarchical clustering (Ward's method) defining high, intermediate and low baseline antigen response groups with at least two standard deviations separating each group mean (Figures 10A-10I). While IL-13 significantly increased responses in the intermediate and high groups. IL-13 effects were not significant in the low response group suggesting the presence of a refractory population to the effects of IL-13. Correlated with low responsiveness to IL-13 was a 3.5- to 4-fold lower responsiveness to PHA in 19 of 28 patients within the lowest response group. However, data from 9 of 28 patients from this latter group retaining a greater than four-fold increase in c.p.m. following PHA stimulation showed a significant 2.4-fold increase in antigenic response by IL-13 suggesting IL-13 can increase the efficiency of antigen-specific T-cell activation within cultures that otherwise would show the lowest responses. In spite of the correlation between PHA and IL-13 response in the low responder group, the PHA response was not a limiting factor to IL-13 effects in the intermediate and high response groups (i.e., no correlation). This is in agreement with the data listed in Tables 4A-4B where the

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- γ In view of the presence of an enhanced T-cell recall responses in patient's

PBMC with low CD4 count exposed to IL-13 and the potential for this cytokine to induce IFN-γ-mediated activation of MDMs via its priming for IL-12 secretion, whether the cell-mediated responses observed were independent of interferon-γ (IFN- γ) [A. D'Andrea et al, J. Exp. Med.. 181:537-546 (1995); J. Marshall et al, L Immunol. 159:5705-5714 (1997)] was tested. IFN- γ 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)]. Surprisingly, the T-cell recall response of PBMC HIV-1 to influenza A virus in the presence of IL-13 and neutralizing IFN-γ antibody B 133.3 increased above that measured with influenza A and IL-13 (Fig. 11 A), indicating that IL- 13 can activate macrophages to initiate cell-mediated responses in the absence of IFN-γ. The lack of an increase above the enhanced incorporation levels measured in the IL-13 -induced response in PBMC from HIV-1+ following neutralization of IFN-γ may reflect the deficient IFN-γ production in this test group [H.W. Murray et al, cited above]. To examine if IFN-γ negatively regulates the APC phenotype elicited by IL-13, the effects of IFN-γ on IL-13 enhancement of influenza A responses were tested.

When added exogenously together with IL-13, IFN-γ 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-γ did not inhibit antigenic responses below those in untreated controls suggesting that IFN-γ's inhibition was restricted to the IL-13 enhancing effects on MDM. IFN-γ also inhibited IL-13 -induction of endocytic uptake yet values for internalized hrp by MDMs treated with IL-13 and INF-γ were higher than those measured with IFN-γ 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-γ secretion following T-cell activation is expected.

Although the qualitative differences between IFN-γ- and IL-13 -induced antigenic responses are still to be defined, in vivo evidence supports a central role for generation of CD4 T-cell help in maintaining and augmenting antiviral CD8 responses as well as being associated with a decreased progression to AIDS [E. Rosenberg et al, Science. 278:1447-1450 (1997); M. Battegay et al, J. Virol. 68:4700-4704 (1994); M. Matloubian et al, J. Virol. 68:8056-8063 (1994)]. Therefore, the effects of IL-13 on 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)].

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-γ, 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 following studies were performed in accordance with the methods described in Examples 7 and 9. 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. A. Purified Protein Derivative (PPD). 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

B HIV-1 Protein Antigens nef & p24

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.

a CD4 count reported at the tune of blood isolation A summary of the results of these experiments reveals that IL-12 treatment recovered the ability of cultured PBMC from HIV-1 patients to incorporate tritium labeled thymidine after Influenza A (PR8 strain) stimulation, yet did not recover responses to Tetanus Toxoid. In healthy individual's PMBC no effects were observed following addition of IL-12.

Thus, 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-γ production. Since IFN-γ 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.

Unlike IL-12, 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. Further examination of recall responses in an additional eleven HIV- infected patients with confirmed diagnosis of past tuberculosis showed a 2.4-fold significant increase of lymphoproliferative responses to PPD antigen stimulation if IL- 13 were present, while an additional 23 HIV-1 -infected patients tested against HIV-1 antigens p24 and nef also showed an increase in antigenic responses following exposure to IL-13 (see Example 8). Antigen-specific effects of IL-13 were evidenced by a lack of mitogenic or non-antigen specific lymphoproliferation to cytokine alone or to stimulation with neoantigen (KLH). As observed in pinocytic assays and the ratio of CD86: HLA-DR expression, IL-13 enhancement of recall responses in HIV- infected samples reached values approximating those measured in untreated cells from healthy donors. Taken together, these results indicate 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. These mean findings are interpreted to illustrate the low frequency of memory T-cells in healthy individuals usually vaccinated against tetanus toxoid in early childhood (note the average healthy donor's age in the group is 36) and not the absence of adjuvancy effects of IL-13 for this antigen in healthy individuals.

Unexpectedly, although mitogen responses were decreased as a function of HIV- infected patient's CD4 count, no decrease was observed for IL-13 effects on influenza A, Tetanus toxoid or PPD recall responses in patients with low CD4 count (Figures 10A-I, Tables 4A-B show influenza A responses versus CD4). In general, IL-13 effects were also not dependent on antiretroviral therapy (Tables 5A-5B), yet its contribution to IL-13 -mediated enhancement of responses with low CD4 count can not be ruled out since 16 of the 20 patients with less than 200 cells/μl were receiving two or more anti-retrovirals. 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). However, 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. Additional longitudinal studies will be needed to elucidate candidate factors in pathogenesis that may be associated with this refractory response group such as high mRNA plasma viremia recently reported to be negatively correlated to CD4 T-cell responses [E. Rosenberg et al, Science. 278: 1447-1450 (1997)]. In spite of the dependency between responses, the lack of a correlation between PHA and IL-13 -mediated enhancement when present suggest these responses are determined by separate variables such as antigen-specific clones for the IL-13 -mediated enhancing effect. This is consistent with the sustained presence of an IL-13 -mediated enhancement of antigenic responses in PBMC from patients with decreased CD4 T-cells/μl and correspondingly low PHA responses (Tables 4A-B) These results distinguishes IL-13 from other proposed cytokine-based strategies for recovery of cell-mediated responses in HIV-infected PBMC such as IL- 12 whose effects were reported as restricted to higher CD4 counts [M Clerici et al, Science. 262 1721-1724 (1993), G Pantaleo, Nature Med . 3 483-486 (1997)] To further explore IL-13's mechanism of action, 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 . 182 389-400 (1996), A Lanzavecchia, Curr Opin Immunol . 8 348 (1996)] These observations identify a potentially important mechanism contributing to decreased CD4 T-cell responses in HIV-infection Consistent with an enhancement of HIV- infected MDM antigen presentation function, IL-13 increased by at least three-fold both pinocytic and CD86 expression while already elevated levels of HLA-DR were unaffected (Tables 2A-C) As a consequence of the selective induction of CD86 expression, IL-13 restored an otherwise deficient ratio of CD86 to HLA-DR expression in HIV- infected MDMs (Table 2C) T-cell activation requires co-activation by CD28 otherwise TcR-peptide-MHC class II interactions result in T- cell anergy [Y Liu & P Linsley, Curr Opin Immunol . 4 265-270 (1992), C June et al, Immunol Today. 1_5 321-331 (1994)] Moreover, CD86 has been identified to block Fas-L and TNF-α-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 . 185 55-64 (1997)] In agreement with these results, similar observations were recently reported on IL-13's selective induction of CD86 expression in HIV-infected MDM [J Marshall et al, J Immunol . 159 5705- 5714 (1997)] In determining the role of IFN-γ in IL-13's mechanisms of action as a result of the potential for IL-12 secretion following exposure to IL-13 [A. D' Andrea et al,_J. Exp. Med.. 181:537-546 (1995); J. Marshall et al, J. Immunol. 159:5705-5714 (1997)] and antigen stimulation, IL-13 was found to induce CD4 cell-mediated responses independently of IFN-γ (Figure 11). This is consistent with IL-13 effects to enhance antigen-specific T-cells in spite of decreasing CD4 count which is positively correlated with IFN-γ secretion in HIV infection [H.W. Murray et al, N. Engl J. Med.. 31_0:883-889 (1984)]. Therefore, IL-13 can activate cell-mediated immunity independently of IFNγ, yet may also complement such activation with IL-12 since its secretion is also induced by IL-13.

Taken together, PMBC culture data indicated IL-13 can increase the responses otherwise inhibited by IL-12. However, 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.

The combined adjuvanting effects of IL-13 and IL-12 were thus shown to recover antigen specific response to both IL-12 and IL-13 specific antigens. Thus these two cytokines are useful in methods as combined adjuvants in healthy as well as in HIV-1 (or other virally infected) patients. - This data is also indicative of the use of IL-13 and IL-12 in combined immunotherapy for AIDS.

IL-13 was able to enhance T cell proliferation following Tetanus Toxoid stimulation, alone or with IL-4. IFN-γ reduced T cell proliferation to Tetanus Toxoid, which is consistent with the lack of the recovery of recall response by IL-12.

While not wishing to be bound by theory, the mechanism of action responsible for these differential effects is postulated to be at antigen intake. IL-13 enhances fluid phase uptake and mannose receptor mediated uptake of antigens while IFN-γ decreases this activity. On the other hand, IL-12 induction of IFN-γ may result in an enhancement of antigen processing within antigen presenting cells allowing for exposure of epitopes not supported by IL-13.

All documents cited herein are hereby incorporated by reference. Numerous modifications and variations in practice of this invention are expected to occur to those skilled in the art. For example, antigens in addition to those explicitly mentioned are expected to be useful in the methods and composition of this invention. Such modifications are considered to be encompassed by the following claims.

SEQUENCE LISTING

(1) GENERAL INFORMATION:

(i) APPLICANT: Wistar Institute of Anatomy & Biology Montaner, Luis J.

(ii) TITLE OF INVENTION: Methods and Compositions Using Interleukin-13 for Enhancing Immune Responses

(iii) NUMBER OF SEQUENCES: 6

(iv) CORRESPONDENCE ADDRESS:

(A) ADDRESSEE: Howson and Howson

(B) STREET: Spring House Corporate Cntr., P.O. Box 457

(C) CITY: Spring House

(D) STATE: Pennsylvania

(E) COUNTRY: USA

(F) ZIP: 19477

(v) COMPUTER READABLE FORM:

(A) MEDIUM TYPE: Floppy disk

(B) COMPUTER: IBM PC compatible

(C) OPERATING SYSTEM: PC-DOS/MS-DOS

(D) SOFTWARE: Patentln Release #1.0, Version #1.30

(vi) CURRENT APPLICATION DATA:

(A) APPLICATION NUMBER: WO

(B) FILING DATE:

(C) CLASSIFICATION:

(vii) PRIOR APPLICATION DATA:

(A) APPLICATION NUMBER: US 60/036,342

(B) FILING DATE: 22-JAN-1997

(viii) ATTORNEY/AGENT INFORMATION:

(A) NAME: Kodroff, Cathy A.

(B) REGISTRATION NUMBER: 33,980

(C) REFERENCE/DOCKET NUMBER: WST76APCT

(ix) TELECOMMUNICATION INFORMATION:

(A) TELEPHONE: 215-540-9200

(B) TELEFAX: 215-540-5818

(2) INFORMATION FOR SEQ ID NO:l:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 1290 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: double

(D) TOPOLOGY: unknown

(ii) MOLECULE TYPE: cDNA ( ix ) FEATURE :

(A) NAME/KEY: CDS

(B) LOCATION: 45..440

(Xi) SEQUENCE DESCRIPTION: SEQ ID NO:l:

TTCGGCATCC GCTCCTCAAT CCTCTCCTGT TGGCACTGGG CCTC ATG GCG CTT TTG 56

Met Ala Leu Leu 1

TTG ACC ACG GTC ATT GCT CTC ACT TGC CTT GGC GGC TTT GCC TCC CCA 104 Leu Thr Thr Val lie Ala Leu Thr Cys Leu Gly Gly Phe Ala Ser Pro 5 10 15 20

GGC CCT GTG CCT CCC TCT ACA GCC CTC AGG GAG CTC ATT GAG GAG CTG 152 Gly Pro Val Pro Pro Ser Thr Ala Leu Arg Glu Leu lie Glu Glu Leu 25 30 35

GTC AAC ATC ACC CAG AAC CAG AAG GCT CCG CTC TGC AAT GGC AGC ATG 200 Val Asn lie Thr Gin Asn Gin Lys Ala Pro Leu Cys Asn Gly Ser Met 40 45 50

GTA TGG AGC ATC AAC CTG ACA GCT GGC ATG TAC TGT GCA GCC CTG GAA 248 Val Trp Ser lie Asn Leu Thr Ala Gly Met Tyr Cys Ala Ala Leu Glu 55 60 65

TCC CTG ATC AAC GTG TCA GGC TGC AGT GCC ATC GAG AAG ACC CAG AGG 296 Ser Leu lie Asn Val Ser Gly Cys Ser Ala lie Glu Lys Thr Gin Arg 70 75 80

ATG CTG AGC GGA TTC TGC CCG CAC AAG GTC TCA GCT GGG CAG TTT TCC 344 Met Leu Ser Gly Phe Cys Pro His Lys Val Ser Ala Gly Gin Phe Ser 85 90 95 100

AGC TTG CAT GTC CGA GAC ACC AAA ATC GAG GTG GCC CAG TTT GTA AAG 392 Ser Leu His Val Arg Asp Thr Lys lie Glu Val Ala Gin Phe Val Lys 105 110 115

GAC CTG CTC TTA CAT TTA AAG AAA CTT TTT CGC GAG GGA CGG TTC AAC 440 Asp Leu Leu Leu His Leu Lys Lys Leu Phe Arg Glu Gly Arg Phe Asn 120 125 130

TGAAACTTCG AAAGCATCAT TATTTGCAGA GACAGGACCT GACTATTGAA GTTGCAGATT 500

CATTTTTCTT TCTGATGTCA AAAATGTCTT GGGTAGGCGG GAAGGAGGGT TAGGGAGGGG 560

TAAAATTCCT TAGCTTAGAC CTCAGCCTGT GCTGCCCGTC TTCAGCCTAG CCGACCTCAG 620

CCTTCCCCTT GCCCAGGGCT CAGCCTGGTG GGCCTCCTCT GTCCAGGGCC CTGAGCTCGG 680

TGGACCCAGG GATGACATGT CCCTACACCC CTCCCCTGCC CTAGAGCACA CTGTAGCATT 740

ACAGTGGGTG CCCCCCTTGC CAGACATGTG GTGGGACAGG GACCCACTTC ACACACAGGC 800

AACTGAGGCA GACAGCAGCT CAGGCACACT TCTTCTTGGT CTTATTTATT ATTGTGTGTT 860 ATTTAAATGA GTGTGTTTGT CACCGTTGGG GATTGGGGAA GACTGTGGCT GCTGGCACTT 920

GGAGCCAAGG GTTCAGAGAC TCAGGGCCCC AGCACTAAAG CAGTGGACCC CAGGAGTCCC 980

TGGTAATAAG TACTGTGTAC AGAATTCTGC TACCTCACTG GGGTCCTGGG GCCTCGGAGC 1040

CTCATCCGAG GCAGGGTCAG GAGAGGGGCA GAACAGCCGC TCCTGTCTGC CAGCCAGCAG 1100

CCAGCTCTCA GCCAACGAGT AATTTATTGT TTTTCCTCGT ATTTAAATAT TAAATATGTT 1160

AGCAAAGAGT TAATATATAG AAGGGTACCT TGAACACTGG GGGAGGGGAC ATTGAACAAG 1220

TTGTTTCATT GACTATCAAA CTGAAGCCAG AAATAAAGTT GGTGACAGAT AAAAAAAAAA 1280

AAAAAAAAAA 1290

(2) INFORMATION FOR SEQ ID NO: 2:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 132 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein

(xi) SEQUENCE DESCRIPTION: SEQ ID NO : 2 :

Met Ala Leu Leu Leu Thr Thr Val lie Ala Leu Thr Cys Leu Gly Gly 1 5 10 15

Phe Ala Ser Pro Gly Pro Val Pro Pro Ser Thr Ala Leu Arg Glu Leu 20 25 30 lie Glu Glu Leu Val Asn lie Thr Gin Asn Gin Lys Ala Pro Leu Cys 35 40 45

Asn Gly Ser Met Val Trp Ser lie Asn Leu Thr Ala Gly Met Tyr Cys 50 55 _ 60

Ala Ala Leu Glu Ser Leu lie Asn Val Ser Gly Cys Ser Ala lie Glu 65 70 75 80

Lys Thr Gin Arg Met Leu Ser Gly Phe Cys Pro His Lys Val Ser Ala 85 90 95

Gly Gin Phe Ser Ser Leu His Val Arg Asp Thr Lys lie Glu Val Ala 100 105 110

Gin Phe Val Lys Asp Leu Leu Leu His Leu Lys Lys Leu Phe Arg Glu 115 120 125

Gly Arg Phe Asn 130 (2) INFORMATION FOR SEQ ID NO: 3:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 2362 base pairs (B)^~ TYPE: nucleic acid

(C) STRANDEDNESS: double

(D) TOPOLOGY: unknown

(ii) MOLECULE TYPE: cDNA

(ix) FEATURE:

(A) NAME/KEY: CDS

(B) LOCATION: 33..1016

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 3:

GAATTCCGTC GACTCTAGAG GCCCAGAGCA AG ATG TGT CAC CAG CAG TTG GTC 53

Met Cys His Gin Gin Leu Val 1 5

ATC TCT TGG TTT TCC CTG GTT TTT CTG GCA TCT CCC CTC GTG GCC ATA 101 lie Ser Trp Phe Ser Leu Val Phe Leu Ala Ser Pro Leu Val Ala lie 10 15 20

TGG GAA CTG AAG AAA GAT GTT TAT GTC GTA GAA TTG GAT TGG TAT CCG 149 Trp Glu Leu Lys Lys Asp Val Tyr Val Val Glu Leu Asp Trp Tyr Pro 25 30 35

GAT GCC CCT GGA GAA ATG GTG GTC CTC ACC TGT GAC ACC CCT GAA GAA 197 Asp Ala Pro Gly Glu Met Val Val Leu Thr Cys Asp Thr Pro Glu Glu 40 45 50 55

GAT GGT ATC ACC TGG ACC TTG GAC CAG AGC AGT GAG GTC TTA GGC TCT 245 Asp Gly lie Thr Trp Thr Leu Asp Gin Ser Ser Glu Val Leu Gly Ser 60 65 70

GGC AAA ACC CTG ACC ATC CAA GTC AAA GAG TTT GGA GAT GCT GGC CAG 293 Gly Lys Thr Leu Thr lie Gin Val Lys Glu Phe Gly Asp Ala Gly Gin 75 80 85

TAC ACC TGT CAC AAA GGA GGC GAG GTT CTA AGC CAT TCG CTC CTG CTG 341 Tyr Thr Cys His Lys Gly Gly Glu Val Leu Ser His Ser Leu Leu Leu 90 95 100

CTT CAC AAA AAG GAA GAT GGA ATT TGG TCC ACT GAT ATT TTA AAG GAC 389 Leu His Lys Lys Glu Asp Gly lie Trp Ser Thr Asp lie Leu Lys Asp 105 110 115

CAG AAA GAA CCC AAA AAT AAG ACC TTT CTA AGA TGC GAG GCC AAG AAT 437 Gin Lys Glu Pro Lys Asn Lys Thr Phe Leu Arg Cys Glu Ala Lys Asn 120 125 130 135

TAT TCT GGA CGT TTC ACC TGC TGG TGG CTG ACG ACA ATC AGT ACT GAT 485 Tyr Ser Gly Arg Phe Thr Cys Trp Trp Leu Thr Thr lie Ser Thr Asp 140 145 150 TTG ACA TTC AGT GTC AAA AGC AGC AGA GGC TCT TCT GAC CCC CAA GGG 533 Leu Thr Phe Ser Val Lys Ser Ser Arg Gly Ser Ser Asp Pro Gin Gly 155 160 165

GTG ACG TGC GGA GCT GCT ACA CTC TCT GCA GAG AGA GTC AGA GGG GAC 581 Val Thr Cys Gly Ala Ala Thr Leu Ser Ala Glu Arg Val Arg Gly Asp 170 175 180

AAC AAG GAG TAT GAG TAC TCA GTG GAG TGC CAG GAG GAC AGT GCC TGC 629 Asn Lys Glu Tyr Glu Tyr Ser Val Glu Cys Gin Glu Asp Ser Ala Cys 185 190 195

CCA GCT GCT GAG GAG AGT CTG CCC ATT GAG GTC ATG GTG GAT GCC GTT 677 Pro Ala Ala Glu Glu Ser Leu Pro He Glu Val Met Val Asp Ala Val 200 205 210 215

CAC AAG CTC AAG TAT GAA AAC TAC ACC AGC AGC TTC TTC ATC AGG GAC 725 His Lys Leu Lys Tyr Glu Asn Tyr Thr Ser Ser Phe Phe He Arg Asp 220 225 230

ATC ATC AAA CCT GAC CCA CCC AAG AAC TTG CAG CTG AAG CCA TTA AAG 773 He He Lys Pro Asp Pro Pro Lys Asn Leu Gin Leu Lys Pro Leu Lys 235 240 245

AAT TCT CGG CAG GTG GAG GTC AGC TGG GAG TAC CCT GAC ACC TGG AGT 821 Asn Ser Arg Gin Val Glu Val Ser Trp Glu Tyr Pro Asp Thr Trp Ser 250 255 260

ACT CCA CAT TCC TAC TTC TCC CTG ACA TTC TGC GTT CAG GTC CAG GGC 869 Thr Pro His Ser Tyr Phe Ser Leu Thr Phe Cys Val Gin Val Gin Gly 265 270 275

AAG AGC AAG AGA GAA AAG AAA GAT AGA GTC TTC ACG GAC AAG ACC TCA 917 Lys Ser Lys Arg Glu Lys Lys Asp Arg Val Phe Thr Asp Lys Thr Ser 280 285 290 295

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

GAC CGC TAC TAT AGC TCA TCT TGG AGC GAA TGG GCA TCT GTG CCC TGC 1013 Asp Arg Tyr Tyr Ser Ser Ser Trp Ser Glu Trp Ala Ser Val Pro Cys 315 320 325

AGT TAGGTTCTGA TCCAGGATGA AAATTTGGAG GAAAAGTGGA AGATATTAAG 1066

Ser

CAAAATGTTT AAAGACACAA CGGAATAGAC CCAAAAAGAT AATTTCTATC TGATTTGCTT 1126

TAAAACGTTT TTTTAGGATC ACAATGATAT CTTTGCTGTA TTTGTATAGT TCGATGCTAA 1186

ATGCTCATTG AAACAATCAG CTAATTTATG TATAGATTTT CCAGCTCTCA AGTTGCCATG 1246

GGCCTTCATG CTATTTAAAT ATTTAAGTAA TTTATGTATT TATTAGTATA TTACTGTTAT 1306

TTAACGTTTG TCTGCCAGGA TGTATGGAAT GTTTCATACT CTTATGACCT GATCCATCAG 1366 GATCAGTCCC TATTATGCAA AATGTGAATT TAATTTTATT TGTACTGACA ACTTTTCAAG 1426

CAAGGCTGCA AGTACATCAG TTTTATGACA ATCAGGAAGA ATGCAGTGTT CTGATACCAG 1486

TGCCATCATA CACTTGTGAT GGATGGGAAC GCAAGAGATA CTTACATGGA AACCTGACAA 1546

TGCAAACCTG TTGAGAAGAT CCAGGAGAAC AAGATGCTAG TTCCCATGTC TGTGAAGACT 1606

TCCTGGAGAT GGTGTTGATA AAGCAATTTA GGGCCACTTA CACTTCTAAG CAAGTTTAAT 1666

CTTTGGATGC CTGAATTTTA AAAGGGCTAG AAAAAAATGA TTGACCAGCC TGGGAAACAT 1726

AACAAGACCC CGTCTCTACA AAAAAAATTT AAAATTAGCC AGGCGTGGTG GCTCATGCTT 1786

GTGGTCCCAG CTGTTCAGGA GGATGAGGCA GGAGGATCTC TTGAGCCCAG GAGGTCAAGG 1846

CTATGGTGAG CCGTGATTGT GCCACTGCAT ACCAGCCTAG GTGACAGAAT GAGACCCTGT 1906

CTCAAAAAAA AAAATGATTG AAATTAAAAT TCAGCTTTAG CTTCCATGGC AGTCCTCACC 1966

CCCACCTCTC TAAAAGACAC AGGAGGATGA CACAGAAACA CCGTAAGTGT CTGGAAGGCA 2026

AAAAGATCTT AAGATTCAAG AGAGAGGACA AGTAGTTATG GCTAAGGACA TGAAATTGTC 2086

AGAATGGCAG GTGGCTTCTT AACAGCCATG TGAGAAGCAG ACAGATGCAA AGAAAATCTG 2146

GAATCCCTTT CTCATTAGCA TGAATGAACC TGATACACAA TTATGACCAG AAAATATGGC 2206

TCCATGAAGG TGCTACTTTT AAGTAATGTA TGTGCGCTCT GTAAAGTGAT TACATTTGTT 2266

TCCTGTTTGT TTATTTATTT ATTTATTTTT GCATTCTGAG GCTGAACTAA TAAAAACTCT 2326

TCTTTGTAAT CAAAAAAAAA AAAAAAAAAC TCTAGA 2362

(2) INFORMATION FOR SEQ ID NO : 4 :

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 328 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 4:

Met Cys His Gin Gin Leu Val He Ser Trp Phe Ser Leu Val Phe Leu 1 5 10 15

Ala Ser Pro Leu Val Ala He Trp Glu Leu Lys Lys Asp Val Tyr Val 20 25 30

Val Glu Leu Asp Trp Tyr Pro Asp Ala Pro Gly Glu Met Val Val Leu 35 40 45

Thr Cys Asp Thr Pro Glu Glu Asp Gly He Thr Trp Thr Leu Asp Gin 50 55 60 Ser Ser Glu Val Leu Gly Ser Gly Lys Thr Leu Thr He Gin Val Lys 65 70 75 80

Glu Phe Gly Asp Ala Gly Gin Tyr Thr Cys His Lys Gly Gly Glu Val 85 90 95

Leu Ser His Ser Leu Leu Leu Leu His Lys Lys Glu Asp Gly He Trp 100 105 110

Ser Thr Asp He Leu Lys Asp Gin Lys Glu Pro Lys Asn Lys Thr Phe 115 120 125

Leu Arg Cys Glu Ala Lys Asn Tyr Ser Gly Arg Phe Thr Cys Trp Trp 130 135 140

Leu Thr Thr He Ser Thr Asp Leu Thr Phe Ser Val Lys Ser Ser Arg 145 150 155 160

Gly Ser Ser Asp Pro Gin Gly Val Thr Cys Gly Ala Ala Thr Leu Ser 165 170 175

Ala Glu Arg Val Arg Gly Asp Asn Lys Glu Tyr Glu Tyr Ser Val Glu 180 185 190

Cys Gin Glu Asp Ser Ala Cys Pro Ala Ala Glu Glu Ser Leu Pro He 195 200 205

Glu Val Met Val Asp Ala Val His Lys Leu Lys Tyr Glu Asn Tyr Thr 210 215 220

Ser Ser Phe Phe He Arg Asp He He Lys Pro Asp Pro Pro Lys Asn 225 230 235 240

Leu Gin Leu Lys Pro Leu Lys Asn Ser Arg Gin Val Glu Val Ser Trp 245 250 255

Glu Tyr Pro Asp Thr Trp Ser Thr Pro His Ser Tyr Phe Ser Leu Thr 260 265 270

Phe Cys Val Gin Val Gin Gly Lys Ser Lys Arg Glu Lys Lys Asp Arg 275 280 ^' 285

Val Phe Thr Asp Lys Thr Ser Ala Thr Val He Cys Arg Lys Asn Ala 290 295 300

Ser He Ser Val Arg Ala Gin Asp Arg Tyr Tyr Ser Ser Ser Trp Ser 305 310 315 320

Glu Trp Ala Ser Val Pro Cys Ser 325 (2) INFORMATION FOR SEQ ID NO: 5:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 1364 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: double

(D) TOPOLOGY: unknown

(ii) MOLECULE TYPE: cDNA

(ix) FEATURE:

(A) NAME/KEY: CDS

(B) LOCATION: 101..859

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 5:

GTCACCGAGA AGCTGATGTA GAGAGAGACA CAGAAGGAGA CAGAAAGCAA GAGACCAGAG 60

TCCCGGGAAA GTCCTGCCGC GCCTCGGGAC AATTATAAAA ATG TGG CCC CCT GGG 115

Met Trp Pro Pro Gly 1 5

TCA GCC TCC CAG CCA CCG CCC TCA CCT GCC GCG GCC ACA GGT CTG CAT 163 Ser Ala Ser Gin Pro Pro Pro Ser Pro Ala Ala Ala Thr Gly Leu His 10 15 20

CCA GCG GCT CGC CCT GTG TCC CTG CAG TGC CGG CTC AGC ATG TGT CCA 211 Pro Ala Ala Arg Pro Val Ser Leu Gin Cys Arg Leu Ser Met Cys Pro 25 30 35

GCG CGC AGC CTC CTC CTT GTG GCT ACC CTG GTC CTC CTG GAC CAC CTC 259 Ala Arg Ser Leu Leu Leu Val Ala Thr Leu Val Leu Leu Asp His Leu 40 45 50

AGT TTG GCC AGA AAC CTC CCC GTG GCC ACT CCA GAC CCA GGA ATG TTC 307 Ser Leu Ala Arg Asn Leu Pro Val Ala Thr Pro Asp Pro Gly Met Phe 55 60 65

CCA TGC CTT CAC CAC TCC CAA AAC CTG CTG AGG GCC GTC AGC AAC ATG 355 Pro Cys Leu His His Ser Gin Asn Leu Leu Arg Ala Val Ser Asn Met 70 75 80 85

CTC CAG AAG GCC AGA CAA ACT CTA GAA TTT TAC CCT TGC ACT TCT GAA 403 Leu Gin Lys Ala Arg Gin Thr Leu Glu Phe Tyr Pro Cys Thr Ser Glu 90 95 100

GAG ATT GAT CAT GAA GAT ATC ACA AAA GAT AAA ACC AGC ACA GTG GAG 451 Glu He Asp His Glu Asp He Thr Lys Asp Lys Thr Ser Thr Val Glu 105 110 115

GCC TGT TTA CCA TTG GAA TTA ACC AAG AAT GAG AGT TGC CTA AAT TCC 499 Ala Cys Leu Pro Leu Glu Leu Thr Lys Asn Glu Ser Cys Leu Asn Ser 120 125 130

AGA GAG ACC TCT TTC ATA ACT AAT GGG AGT TGC CTG GCC TCC AGA AAG 547 Arg Glu Thr Ser Phe He Thr Asn Gly Ser Cys Leu Ala Ser Arg Lys 135 140 145 ACC TCT TTT ATG ATG GCC CTG TGC CTT AGT AGT ATT TAT GAA GAC TTG 595 Thr Ser Phe Met Met Ala Leu Cys Leu Ser Ser He Tyr Glu Asp Leu 150 155 160 165

AAG ATG TAC CAG GTG GAG TTC AAG ACC ATG AAT GCA AAG CTT CTG ATG 643 Lys Met Tyr Gin Val Glu Phe Lys Thr Met Asn Ala Lys Leu Leu Met 170 175 180

GAT CCT AAG AGG CAG ATC TTT CTA GAT CAA AAC ATG CTG GCA GTT ATT 691 Asp Pro Lys Arg Gin He Phe Leu Asp Gin Asn Met Leu Ala Val He 185 190 195

GAT GAG CTG ATG CAG GCC CTG AAT TTC AAC AGT GAG ACT GTG CCA CAA 739 Asp Glu Leu Met Gin Ala Leu Asn Phe Asn Ser Glu Thr Val Pro Gin 200 205 210

AAA TCC TCC CTT GAA GAA CCG GAT TTT TAT AAA ACT AAA ATC AAG CTC 787 Lys Ser Ser Leu Glu Glu Pro Asp Phe Tyr Lys Thr Lys He Lys Leu 215 220 225

TGC ATA CTT CTT CAT GCT TTC AGA ATT CGG GCA GTG ACT ATT GAT AGA 835 Cys He Leu Leu His Ala Phe Arg He Arg Ala Val Thr He Asp Arg 230 235 240 245

GTG ATG AGC TAT CTG AAT GCT TCC TAAAAAAGCG AGGTCCCTCC AAACCGTTGT 889 Val Met Ser Tyr Leu Asn Ala Ser 250

CATTTTTATA AAACTTTGAA ATGAGGAAAC TTTGATAGGA TGTGGATTAA GAACTAGGGA 949

GGGGGAAAGA AGGATGGGAC TATTACATCC ACATGATACC TCTGATCAAG TATTTTTGAC 1009

ATTTACTGTG GATAAATTGT TTTTAAGTTT TCATGAATGA ATTGCTAAGA AGGGAAAATA 1069

TCCATCCTGA AGGTGTTTTT CATTCACTTT AATAGAAGGG CAAATATTTA TAAGCTATTT 1129

CTGTACCAAA GTGTTTGTGG AAACAAACAT GTAAGCATAA CTTATTTTAA AATATTTATT 1189

TATATAACTT GGTAATCATG AAAGCATCTG AGCTAACTTA TATTTATTTA TGTTATATTT 1249

ATTAAATTAT TCATCAAGTG TATTTGAAAA ATATTTTTAA GTGTTCTAAA AATAAAAGTA 1309

TTGAATTAAA AAAAAAAAAA AAAAAAAAAA AAAAAAAAAA AAAAAAAAAA AAAAA 1364

(2) INFORMATION FOR SEQ ID NO: 6:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 253 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 6:

Met Trp Pro Pro Gly Ser Ala Ser Gin Pro Pro Pro Ser Pro Ala Ala 1 5 10 15

Ala Thr Gly Leu His Pro Ala Ala Arg Pro Val Ser Leu Gin Cys Arg 20 25 30

Leu Ser Met Cys Pro Ala Arg Ser Leu Leu Leu Val Ala Thr Leu Val 35 40 45

Leu Leu Asp His Leu Ser Leu Ala Arg Asn Leu Pro Val Ala Thr Pro 50 55 60

Asp Pro Gly Met Phe Pro Cys Leu His His Ser Gin Asn Leu Leu Arg 65 70 75 80

Ala Val Ser Asn Met Leu Gin Lys Ala Arg Gin Thr Leu Glu Phe Tyr 85 90 95

Pro Cys Thr Ser Glu Glu He Asp His Glu Asp He Thr Lys Asp Lys 100 105 110

Thr Ser Thr Val Glu Ala Cys Leu Pro Leu Glu Leu Thr Lys Asn Glu 115 120 125

Ser Cys Leu Asn Ser Arg Glu Thr Ser Phe He Thr Asn Gly Ser Cys 130 135 140

Leu Ala Ser Arg Lys Thr Ser Phe Met Met Ala Leu Cys Leu Ser Ser 145 150 155 160

He Tyr Glu Asp Leu Lys Met Tyr Gin Val Glu Phe Lys Thr Met Asn 165 170 175

Ala Lys Leu Leu Met Asp Pro Lys Arg Gin He Phe Leu Asp Gin Asn 180 185 190

Met Leu Ala Val He Asp Glu Leu Met Gin Ala Leu Asn Phe Asn Ser 195 200 . 205

Glu Thr Val Pro Gin Lys Ser Ser Leu Glu Glu Pro Asp Phe Tyr Lys 210 215 220

Thr Lys He Lys Leu Cys He Leu Leu His Ala Phe Arg He Arg Ala 225 230 235 240

Val Thr He Asp Arg Val Met Ser Tyr Leu Asn Ala Ser 245 250

Claims

WHAT IS CLAIMED IS:

1. A method for enhancing the immune response to a selected antigen in a host comprising administering to said host simultaneously or sequentially with said antigen an effective amount of Interleukin- 13 or a biologically active fragment thereof.

2. A method for enhancing the adjuvant effect of Interleukin (IL)-12 on a selected antigen in a host comprising administering to said host simultaneously or sequentially with said antigen and IL-12 an effective amount of IL-13 or a biologically active fragment thereof.

3. The method according to claim 1 or 2, wherein said antigen is of viral origin.

4. The method according to any of claims 1 to 3, wherein said antigen is influenza A.

5. The method according to any of claims 1 to 3, wherein said antigen is an HIV protein.

6. The method according to claim -5, wherein said HIV protein is selected from the group consisting of HIV-1 p24 and HIV-1 nef protein.

7. The method according to claim 1 or 2, wherein said antigen is a bacterial antigen.

8. The method according to claim 7, wherein said antigen is selected from the group consisting of tetanus toxoid and Mycobacterium tuberculosis antigens.

9. The method according to claim 1 or 2, wherein said antigen is a parasitic antigen.

10. The method according to claims 1 or 2, wherein said antigen is a cancer antigen.

11. The method according to any of claims 1 to 10, wherein said host is a healthy mammal.

12. The method according to any of claims 1 to 10, wherein said host is a pathogen-infected mammal

13. The method according to any of claims 1 to 10 wherein said host is an HIV-infected human.

14. A vaccine composition comprising a selected antigen and Interleukin- 13 or a biologically active fragment thereof.

15. The composition according to claim 14 further comprising a second adjuvant.

16. Use of Interleukin- 13 or a biologically active fragment thereof in the preparation of a medicament, wherein said Interleukin- 13 or fragment thereof is administered to a host sequentially or simultaneously with a selected antigen, and wherein said interleukin- 13 or fragment thereof enhances the immune response to said selected antigen in a host.

17. Use according to claim 16, wherein said antigen is Tetanus Toxoid.

18. Use according to claim 16, wherein said antigen is Influenza A.

19. Use according to claim 16, wherein said antigen is an HIV antigen selected from the group consisting of HIV-1 p24 and HIV-1 nef proteins.

20. Use according to claim 16, wherein said antigen is a Mycobacterium tuberculosis antigen.

21. Use according to any of claims 16 to 18, wherein said medicament further comprises Interleukin 12 or a functional fragment thereof.