COMPOSITIONS AND METHOD FOR STIMULATING ANTIBODY RELEASE BY B LYMPHOCYTES

CROSS-REFERENCE TO RELATED APPLICATION

This application is a continuation-in-part of prior pending application Serial No. 08/467,146, filed June 6, 1995, which is a continuation of application Serial No. 08/315,492, filed September 30, 1994, and which is a continuation-in-part of application Serial No. 08/150,510. filed November 10, 1993, hereby incoφorated b - reference.

GOVERNMENT INTEREST

The invention described herein may be manufactured, licensed, and used for governmental purposes without the payment of any royalties to the inventors or assignee.

FIELD OF THE INVENTION

This invention relates to compositions comprising granulocyte-macrophage colony stimulating factor (GM-CSF), interleukin-2 (IL-2), interleukin-3 (IL-3), and a T cell stimulating peptide, such as a universal T cell epitope (TCE), either alone or in combination, and compositions of such factors, either alone or in combination, along with interferon-γ (IFN-γ). The compositions are useful for stimulating the release of antibodies by mammalian B lymphocytes. This invention also relates to an in vitro assay system for identifying compositions that stimulate the release of antibodies by B lymphocytes.

Stimulation of antibody release by B lymphocytes is useful in the battle to prevent, treat, and/or ameliorate the deleterious effects of infection and disease. This usefulness extends to adjuvants for bolstering mammalian immune responses under normal conditions and under immunosuppressed or immunocompromised conditions. The novel compositions can also be used in conjunction with other immunotherapies to bolster the human immune system.

BACKGROUND OF THE INVENTION

The human immune system comprises numerous different types of cells having overlapping functions which together act to protect the human body against sickness and

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disease. The cells ofthe immune system have complex multiple functions and interconnecting relationships.

GM-CSF, IL-2, IL-3, and IFN-γ are all cytokines. "Cytokines" are a class of compounds that regulate responses of cells ofthe immune system, such as B and T lymphocyte cells ("B cells" and "T cells") and natural killer ("NK") ceils. A "cytokine" is a generic term for a non-antibody protein released by certain cell populations on contact with an inducer and which acts as an intercellular mediator. A "lymphokine" is a soluble substance released by sensitized lymphocytes on contact with specific antigen or other stimuli which helps effect cellular or humoral immunity.

The terms "cytokine" and "lymphokine" have become interchangeable. In an attempt to simplify the nomenclature of these compounds, a group of participants at the Second International Lymphokine Workshop held in 1979 proposed the term "interleukin," abbreviated "IL," to develop a uniform system of nomenclature based on the ability ofthe proteins to act as communication signals between different populations of leukocytes.

To date, 21 different cytokines, most but not all of which are produced by T cells, have been identified. Each has a distinct molecular configuration and performs a different task. A number ofthe known cytokines have been show .o nave a demonstrable a ^: vity on B cells. In vitro, the lymphokines IL-1, IL-2, IL-4, IL-5, IL-6, IL-10, IFN-γ, an. ι GF-β (transforming growth factor β) have been shown to enhance B cell proliferation, immunoglobulin secretion, or to otherwise play a role in influencing the subclass of secreted Ig. Depending on the system being studied, addition of either one or a number of the above lymphokines has been shown to increase in vivo antibody production or to alter the isotype (i.e., IgG, IgM, IgE, IgA, etc.) of secreted antibody. Among the lymphokines reported to influence B cell proliferation include IL-1, IL-2, IL-4, and IL-10, and those reported to influence B cell differentiation and Ig secretion include IL-2, IL-5, IL-6. TGF-β. and IFN-γ .

None of the reported cytokines which enhance Ig secretion in vitro have been shown to play a prominent role in vivo. Thus, infusion of monoclonal antibodies specific for IL-2. IL-5, IL-6, or IFN-γ does not significantly suppress antigen-stimulated antibody

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production. This suggests that, under physiologic conditions, B cell differentiation depends solely on direct T cell interaction, or that other as yet unknown cytokines mediate this step.

While immune responses to antigens that stimulate T cell activation (the so-called T dependent antigens or "TD antigens") could rely on direct T cell interactions with B cells to effect Ig secretion, this is not the case with antigens that are unable to induce T cell activation. Antigens that are T cell independent ("TI antigens") induce high levels of antibody production in the absence of direct or even indirect T cell help. Thus, the events that regulate B cell differentiation and immunoglobulin secretion to TI antigens must rely on other as yet undefined pathways. Since B cell differentiation leading to immunoglobulin secretion is the final event which underlies a competent humoral antibody system, defining the events or cytokines which regulate this step is invaluable in designing methods for amplifying or suppressing an immune response.

To facilitate a quick appreciation ofthe invention, the following provides a brief description ofthe primary known functions of immunoglobulin, antibodies, lymphocytes, B cells, T cells, and NK cells as background. Also provided is a brief summary ofthe known activities of he cytokines reported to influence B cell proliferation or antibody secretion, namely IL-1, IL-2, IL-4, IL-5, IL-6, IL-10, TGF-β, and IFN-γ. A summary ofthe known activities of GM-CSF and IL-3 is also provided. Reference materials include Fundamental Immunology. Second Edition, William E. Paul, M.D., ed. (Raven Press. New York 1989); Fundamental Immunology. Third Edition, William E. Paul, M.D.. ed. (Raven Press, New York 1993); Interferon: Principles and Medical Applications. S. Baron et al., eds. (The University of Texas Medical Branch at Galveston, Galveston, Texas 1992); The Cvtokine Handbook. Angus Thomson, ed. (Academic Press Inc., San Diego, CA 1992); and The Cytokine Handbook. Second Edition, Angus Thomson, ed. (Academic Press Inc., San Diego, CA 1994), all of which are specifically incorporated by reference.

Mammals, including man, are confronted on a daily basis with a myriad of organisms. A major component ofthe immune system and playing an essential role in protecting the host against infection by these organisms, is the humoral antibody. Antibodies are protein molecules, also known as immunoglobulins. which have exquisite specificity for the foreign particle which stimulates their production. For example,

systemic infection with "bacteria A" will induce antibodies that bind with a high avidity to "bacteria A" but not to "bacteria B." Similarly, "bacteria B" will induce anti-"bacteria B" antibodies that do not cross-react with "bacteria A."

Immunoglobulin (Ig) is a class of structurally related proteins consisting of two pairs of polypeptide chains, one pair of light (L) [low molecular weight] Kchains (K or λ), and one pair of heavy (H) chains (γ, α, μ, δ, and e), all four linked together by disulfide bonds. Both H and L chains have regions that contribute to the binding of antigen and that are highly variable from one Ig molecule to another. In addition, H and L chains contain regions that are nonvariable or constant. On the basis of the structural and antigenic properties of the H chains, Ig's are classified as IgG, IgA, IgM, IgD, and IgE isotypes. Subclasses of IgG's, based on differences in the H chains, are referred to as IgGl, etc.

Lymphocytes are white blood cells formed in lymphatic tissues throughout the body, such as lymph nodes, spleen, thymus, tonsils, Peyer's patches (small intestine tissue), and sometimes in bone marrow. Individual lymphocytes are specialized in that they are committed to respond to a limited group of structurally related antigens. This commitment, which exists prior to the first contact ofthe immune system with a given antigen, is expressed by the presence of antigen-specific receptors (i.e., immunoglobulin) on the lymphocyte membrane. The ability of an organism to respond to virtually any antigen is achieved by the existence of a very large number of different clones of lymphocytes, each bearing receptors specific for distinct antigens. In consequence, lymphocytes are an enormously heterogeneous collection of cells.

Lymphocytes differ from one another not only in the specificity of their receptors but also in their functional properties. Two broad classes of lymphocytes are recognized: the B lymphocytes and the T lymphocytes. In addition to these two classes, lymphoid cells that mediate certain "nonspecific" cytotoxic responses are known. These include natural killer (NK) cells.

B lymphocytes, also known as "B cells," are a type of lymphocyte that derive from hematopoietic stem cells by a complex set of differentiation events that are only partially understood. B cells are precursors of antibody-secreting cells and thus are responsible for the production of immunoglobulins. The cell-surface receptor of B cells is an antibody or

immunoglobulin (lg) molecule specialized for expression on the cell surface. Newly differentiated B cells initially express surface Ig solely ofthe IgM class. Associated with maturation of a B cell is the appearance of other immunoglobulin isotypes on the surface of the B cell.

To release antibody in response to cytokines, the B cells must first be activated. There are many ways to activate B cells, including cross-linkage of membrane Ig molecules by the antigen (cross-linkage-dependent B cell activation), direct encounter with T cells ' (helper T cells or helper T cell-associated molecules, such as, for example, CD40 ligand), or encounter with mitogens. In such encounters, the antigen presents epitopes recognized by the B cell's cell-surface Ig.

Because each B cell bears multiple membrane Ig molecules with identical variable regions, optimal membrane-Ig mediated cross-linkage activation is achieved by a high level of cross-linkage ofthe cell-surface receptors, which requires that the antigen present more than one copy ofthe epitope that the cell-surface Ig recognizes. Although many simple protein antigens do not have this potential, such a requirement is fulfilled by polysaccharides and other antigens with repeating epitopes, such as surfaces of microbes and DNA. Among these antigens are the capsular polysaccharides of many medically important microorganisms, such as pneumococci, streptococci, and meningococci.

There are much data to show that cross-linkage of membrane Ig can also lead to elimination or inactivation of B cells. In general, it is believed that certain types of receptor cross-linkage events, if they occur in the absence of specific stimulatory signals, lead to inactivation rather than activation. The highly repetitive epitopes expressed on polysaccharides may lead to activation in the absence of co-stimulation, possibly because of the magnitude ofthe receptor-mediated stimulation.

T lymphocytes, or "T cells," are thymocyte derived, of immunological importance that is long-lived (months to years), and are responsible for cell-mediated immunity. T cells consist of functionally different populations, known as "helper T cells." "suppressor T cells," and "killer T cells." T cells involved in delayed hypersensitivity and related immune phenomena are also known.

Natural killer cells, or "NK cells," are lymphoid cells that mediate certain "nonspecific" cytotoxic responses. Such nonspecific cytotoxic responses kill certain forms of tumor cells using recognition systems that are different from those used by T or B cells. Killing of one cell type by another through contact interaction constitutes a major effector arm of self-defense of the immune system.

In addition to these cells, other compounds, the cytokines, play a significant role in protecting a host. One group of cytokines is the interleukins.

IL-1 is primarily an inflammatory cytokine, whereas IL-2 and other cytokines are primarily growth factors for lymphocytes. IL-1 is a polypeptide hormone synthesized by monocytes. During inflammation, injury, immunological challenge, or infection. IL-1 is produced and, because of its multiple biological properties, this cytokine appears to affect the pathogenesis ofthe disease. In animals, IL-1 is a potent inducer of hypotension and shock. IL-1 acts on the hypothalamus to induce fever and directly on skeletal muscle to promote protein catabolism

IL-2, also known as T cell growth factor, is a lymphokine and polypeptide hormone produced by both T helper and suppressor lymphocytes. This cytokine has direct effects on the growth and differentiation of T cells, B cells, NK cells, lymphokine-activated killer (LAK) cells, monocytes, macrophages, and oligodendrocytes.

IL-3, also known as multicolony stimulating factor, acts on numerous target cells within the hemopoietic system. This cytokine has the broadest target specificity of any of the haematopoietic growth factors (HPGFs), and can stimulate the generation and differentiation of hemopoietic stem cells (i.e., precursors of blood cells), which give rise to macrophages, neutrophils, eosinophils, basophils, mast cells, megakaryocytes, and erythroid cells.

The relationship between IL-3 and B cells was unclear prior to the invention. In fact, as of 1994, it was believed that the range of target cells of IL-3 did not include cells committed to the T- and B-lymphoid lineages, and there was no compelling evidence that IL-3 had a significant, direct effect on B-cell development. J. W. Schrader, "Chapter 5: Interleukin-3," The Cytokine Handbook. 2nd Ed., Angus Thomson, ed., page 84 (Academic Press, New York, 1994).

Secretion of LL-3 by B cells has not been reported, although IL-3 is synthesized by T cells and mast cells. Several reports demonstrated that IL-3 could induce a modest enhancement of Ig secretion by human B cells activated with SAC (a polyclonal activator) and IL-2. For example, Xia et al., "Human Recombinant IL-3 is a Growth Factor for Normal B Cells," J. of Immunology. 148. 491-497 (1992), reported that IL-3 enhanced the proliferation of a population of cells enriched in B cells. Similarly, Tadmori et al., "Human Recombinant IL-3 Stimulates B Cell Differentiation," J. of Immunol.. 142. 1950-1955 (1989), reported that IL-3 stimulated IgG secretion from tonsillar cells containing B cells or in a population of peripheral blood-derived enriched B cells activated by bacterial antigen. In addition. Matsumoto et al., "Induction of IgE Synthesis in Anti-IgM-Activated Nonatopic Human B Cells by Recombinant Interleukin-3," Int. Arch. Allergy Appln. Immunol.. 89_, 24-30 (1989), reported that human recombinant IL-3 augmented IgE synthesis by normal B cells or mixtures of T and B lymphocytes, and that IL-1, IL-2, IL-5, IL-6, GM-CSF, G-CSF, M-CSF, and IFN-γ failed to induce IgE synthesis. Matsumoto et al. also note that they could not conclusively identify IL-3 as the factor in the T cell supernatant responsible for inducing IgE synthesis because the activity could not be reversed by the addition of anti-IL-3 antibody.

These results were attributed to an IL-3-mediated enhancement in cell growth. In these studies, B cells were not electronically sorted and thus were not highly purified. Thus, the Ig enhancing effect may reflect the action of IL-3 on many contaminating non-B, non-T cells in the population, and it is therefore not possible to determine from these experiments whether IL-3 was acting directly on the B cell. Further, in prior experiments, B cells were not fractionated according to size. Thus, a possible role for the prior activational state ofthe B cell was not addressed.

In further contrast to the present invention, Kimoto et al., "Recombinant Murine IL-3 Fails to Stimulate T or B Lymphopoiesis In Vivo, But Enhances Immune Responses To T Cell-Dependent Antigens," J. of Immunology. 140. 1889-1894 (1988), reported that mice bearing osmotic minipumps loaded with murine recombinant IL-3 showed no increase in the lymphoid organs ofthe total number of B and T cells. Furthermore, Kimoto et al. suggested that IL-3 does not act directly on lymphocytes or their precursors, but may

potentiate the humoral immune response to T cell-dependent antigens, presumably by acting on accessory cells.

IL-4 is a glycoprotein also known as B cell stimulating factor 1 (BSF-1 ) and B cell differentiation factor. It functions to co-stimulate B cell growth, Ig class switching, T cell growth and differentiation, macrophage activation, regulate mast cell growth, and to co-stimulate hematopoietic precursor cells.

IL-5, also known as B cell growth factor II (BGF-II), T cell replacing factor, IgA-enhancing factor, and eosinophil colony stimulating factor, is a glycoprotein produced by T lymphocytes and mast cells. This cytokine has the dual functions of a colony stimulating factor, as well as promoting the differentiation of eosinophilic colonies in bone marrow. IL-5 induces specific in vitro antibody production by B cells primed with antigen in vivo. While IL-5 serves as a differentiation factor in vitro, it does not appear to act as a differentiation factor in vivo.

IL-6, also known BSF-II, hybridoma/plasmacytoma growth factor, interferon β2, and hepatocyte stimulatory factor, is a glycoprotein produced by both lymphoid and nonlymphoid cells. This cytokine regulates immune responses, acute-phase reactions, and hemopoiesis. IL-6 acts on B cell lines at the mRNA level and induces biosynthesis of secretory-type Ig. In addition to IL-5, IL-6 has also been shown under very restricted conditions to function as a differentiation factor. All other known T cell or macrophage derived factors that have been tested cannot induce activated B cells to secrete Ig in the absence of added growth factors.

IL-10, also known as cytokine synthesis inhibitory factor, is produced by T cells, macrophages, and other cell types. This cytokine inhibits several macrophage functions, including cytokine synthesis and some microbial activities, in addition to enhancing or stimulating mast cells and B cells. IL-10 causes strong proliferation of human B cells activated by anti-CD40 antibodies or cross-linking ofthe antigen receptor.

In addition to the interleukins, other cytokines have been characterized. Colony stimulating factors (CSFs) are a group of factors primarily concerned with hematopoiesis. They are defined as proteins which stimulate the clonal growth of bone-marrow cells in vitro.

Granulocyte-macrophage colony stimulating factor (GM-CSF) is a glycoprotein growth factor that modulates the growth or differentiation of hemopoietic cells. This growth factor can be produced by a number of different cells under different circumstances, including T cells, macrophages. endothelial cells, stromal cells, fibroblasts. mast cells, and others. The major actions of GM-CSF involve the regulation of survival, differentiation, and proliferative and functional activities in granulocyte-macrophage populations. There are no reports prior to the invention indicating that GM-CSF can stimulate the release of antibody by B cells.

Finally, another class of cytokines that function in the body's immune system is interferons (IFNs). IFNs are major contributors to the first line of antiviral defense by inhibiting virus replication, in addition to exerting many other important effects on cells. IFNs do not act directly to protect cells from infection. Rather, they stimulate production of a protein in neighboring cells that stops the growth ofthe virus, thus protecting the cells from infection.

IFNs are classified into three groups, alpha, beta, and gamma, based on the cells of origin and method of induction. The production of IFN-α and IFN-β is not a specialized cell function, and probably all cells ofthe organism are capable of producing these IFNs.

In contrast to IFN-α and IFN-β synthesis, which can occur in any cell, production of IFN-γ is a function of T cells and NK cells. All IFN-γ inducers activate T cells either in a polyclonal (mitogens or antibodies) or in a clonally restricted, antigen-specific manner. Human IFN-γ promotes proliferation of activated human B cells and, in cultures of human B cells, can act synergistically with IL-2 to enhance immunoglobulin light-chain synthesis.

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The brief discussion describing functions of various human immune cells, and the known activities of IL-1, IL-2. IL-3, IL-4. IL-5, IL-6, IL-10, GM-CSF, and IFN-γ exemplifies the extreme diversity of the human immune system. Despite this level of knowledge, however, there is no complete understanding of the intricacies of he immune system. Tremendous gaps remain. For example, it is not possible to make general statements about the properties of cytokines, except that they act as intercellular mediators by regulating responses of cells ofthe immune system. Thus, there remains a need in the

art for a greater understanding ofthe immune system and for the provision of additional and superior methods of treating immune disorders.

SUMMARY OF THE INVENTION

The present invention fulfills a need in the art for new and improved immunotherapies. The novel compositions and methods, employing IL-2, IL-3, GM-CSF, a universal TCE, and IFN-γ, enable improved and new treatments for immune disorders, as well as adjuvants for current immunotherapies.

This invention is directed to a composition of GM-CSF and IL-2 or IL-3, either alone or in combination, present in an amount effective for the stimulation of antibody release by B cells. Another object ofthe invention is directed to compositions of GM-CSF and IL-2 or IL-3, either alone or in combination, present in an amount effective for the stimulation of antibody release by B cells, wherein the resultant complex is conjugated to a protein, and wherein that complex is additionally conjugated to a polysaccharide. The invention also embodies combinations of other lymphokines that can enhance immune reactivity by increasing the responsiveness of cells participating in the immune response, as established by T or B cell proliferation in vitro or by in vitro antibody formation. Molecularly engineered fragments of GM-CSF, IL-2 or IL-3 that retain GM-CSF, IL-2 or IL-3 activity, respectively, can also be employed in the invention.

Another object of this invention is a composition of GM-CSF, IL-2 and IL-3, either alone or in combination, along with IFN-γ, all of which are present in amounts effective for the stimulation of antibody release by B cells.

Another object ofthe invention is a composition comprising a universal TCE non-antigen specific conjugated to a protein, wherein that complex may aiso be further conjugated to a polysaccharide. In another embodiment, a universal TCE is directly conjugated to a polysaccharide.

A further object ofthe invention is a pharmaceutical composition comprising the novel compositions and a pharmaceutically acceptable carrier.

The use ofthe novel compositions comprising GM-CSF, IL-2, IL-3, IFN-γ, and the universal TCE to stimulate the release of antibody by B cells is also encompassed by the invention. A method of stimulating the release of antibody by B cells can be used to

bolster mammalian immune responses to, for example, vaccination under conditions of normal or immunocompromised conditions.

A further object of this invention is the use ofthe novel compositions as adjuvants for vaccines. For example, many vaccines are currently administered intravenously or intramuscularly to allow a rapid stimulation of immune cells present in the blood system. By combining the novel compositions, either as a fusion protein covalently linked to a carrier molecule, admixed, or any other combination, with a vaccine to be administered, the magnitude of the antibody response can be increased both at the systemic and local levels.

Another object of this invention is neutralization of GM-CSF, IL-3, and IFN-γ under conditions where the production of antibody is pathogenic, such as in autoimmune disorders.

The compositions can also be used to optimize monoclonal antibody production in vitro or in vivo. For example, an animal can be sensitized with antigen and the compositions in vivo. Alternatively, in vitro stimulation or sensitization of lymphocytes to produce antibody can be enhanced in the presence of the novel compositions. This is particularly useful for the production of human monoclonal antibodies.

This invention is also directed to a novel assay system that allows the identification of compositions useful for stimulating antibody release by B cells. This assay system, which mimics in vivo antibody stimulation, comprises dextran-conjugated anti-Ig antibodies and highly purified B cells. The anti-Ig-dextran conjugate effectively and polyclonally activates the B cells via membrane Ig by a mechanism comparable to activation of B cells induced by antigen in vivo.

Other objects and advantages ofthe present invention will be set forth in part in the description which follows, and in part will be obvious from this description, or may be learned from the practice of this invention. The accompanying drawings and tables, which are incoφorated in and constitute a part of this specification, illustrate and, together with this description, serve to explain the principle ofthe invention.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 : IgM secretion (ng/ml) by B cells in various mediums was measured. Culture mediums are listed along the left side ofthe chart. The compositions tested showed the highest IgM secretion in medium comprising dextran-conjugated anti-IgD antibody, IL-1 , and IL-2. To this medium was added a supernatant from a cell culture described in the parent application, RA5-SN, and GM-CSF or LL-3. A control composition was also measured. The RA5-SN supernatant, having both GM-CSF and IL-3, showed significant IgM secretion by B cells as compared to other tested compositions.

Figure 2: B cells were activated with dextran-conjugated anti-IgD antibodies

(3 ng/ml) plus IL-1 (150 U/ml) + IL-2 (150 U/ml) in the presence or absence of varying concentrations of IL-3 or GM-CSF. IgM secretion was measured by ELISA 6 days after initiation of culture.

Figure 3: IgM secretion (ng/ml) by activated B cells was measured with the addition of varying concentrations of GM-CSF and IL-3. Maximum IgM secretion of about 4650 ng/ml was obtained with the addition of GM-CSF at 10 U/ml and IL-3 at about 3 U/ml.

Figure 4: B cells were activated with dextran-conjugated anti-IgD antibodies (3 ng/ml) plus IL-1 (150 U/ml) + IL-2 (150 U/ml) in the presence or absence of IL-3 (10 U/ml) or GM-CSF (10 U/ml). In addition, anti-IL-3 (10 μg/ml) and/or anti-GM-CSF (10 μg/ml) antibodies were added. A control with no added anti-IL-3 or anti-GM-CSF was also prepared. IgM concentrations were measured by ELISA 6 days after initiation of culture.

Figure 5: IgM secretion (ng/ml) by activated B cells was measured for dextran-conjugated anti-IgD antibody-activated B cells in AF7 supernatant. IgM secretion was measured in various mediums comprising two or more of the following: IL-1 , LL-2, αIL-3, and αGM-CSF.

Figure 6: B cells were activated with dextran-conjugated anti-IgD antibodies (3 ng/mfi plus IL-1 (150 U/ml) + IL-2 (150 U/ml) in the presence or absence of IL-3

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(100 U/ml) and/or GM-CSF (100 U/ml). Viable cells (those that excluded trypan blue) were enumerated 4 days after initiation of culture using a hemocytometer. IgM concentrations in replicate cultures were measured by ELISA 6 days after initiation of culture. Data is represented as mean +/- standard error ofthe mean of duplicate cultures.

Figure 7: B cells were activated with dextran-conjugated anti-IgD antibodies (3 ng/ml) plus IL-1 (150 U/ml) + IL-2 (150 U/ml) in the presence or absence of LL-3 (100 U/ml) + GM-CSF (100 U/ml). IgM concentrations in replicate cultures were measured by ELISA for a control composition, a composition to which IFN-γ was added at 0 h, and for a composition to which IFN-γ was added at 24 h. Data is represented as mean +/- standard error ofthe mean of duplicate cultures.

Figure 8: Schematic of yeast expression vector

Figure 9: Coomassie blue stained gel containing protein A-IL2 (PA-IL2) product prior to and after purification on nickel agarose column and sizing gel.

Figure 10: Immunoblot of protein A-IL2 (PA-IL2, blotted with anti-IL2 (left hand gel) and anti-protein A (PA) (right hand gel). IL2 and PA are run as controls; protein A runs at a higher molecular weight than fusion product because fusion was made with a truncated version of protein A.

Figure 11: Functional activity of IL2 in fusion product. 5x103 HT2 cells, a CTLL line responsive to IL2 was with IL2 or protein A-IL2 and thymidine incoφoration was measured 48 hours later.

Figure 12: Size exclusion HPLC (Beckman SEC 2OO0) column profile of: A)-purified protein A-IL2 (PA-IL2) fusion product B)-non purified concentrated supernatants containing PA-IL2 from protease free yeast. No purification was done on the supernatants, demonstrating that a "cleaner" product with less lower m.w breakdown products was obtained in protease free transfected yeast.

Figure 13: Purified protein D (lanes 1 and 2) and molecularly engineered universal

TCE-protein D (TCE-protein D, lane 4) and protein A (as a control, lane 5)

were run on SDS PAGE and stain by Coomassie blue (upper panel). A second gel was immunoblotted with anti-protein D (lower panel). The figure shows the proteins and TCE-protein D were purified to homogeneity. DESCRIPTION OF PREFERRED EMBODIMENTS The invention describes compositions of cytokines which individually and in combination lead to 100 fold enhancement in antibody secretion by B cells. The compositions comprise IL-2, IL-3 and GM-CSF, either alone or in combination, in an amount effective for stimulating the release of antibody by B cells. Also encompassed by the invention are compositions comprising IL-2, IL-3 and GM-CSF, either alone or in combination, and IFN-γ, all present in amounts effective for stimulating the release of antibody by B cells. More preferably, the compositions additionally comprise CD40 ligand (CD40L), or at least one other cytokine, or a combination thereof. Also more preferably, the compositions additionally comprise CD40 ligand (CD40L), a universal TCE, or at least one other cytokine, or a combination thereof. By universal TCE, is meant a non-antigen specific, strongly T cell stimulatory peptide, possessing no B cell epitopes. Strongly T cell stimulatory means able to potently stimulate T cells as reflected in T cell proliferation assays known to those ordinarily skilled in the art. By peptide is meant a molecule of about 8 to 20 amino acid residues, preferably from 1 1 to 18 residues. Non-antigen specific means that the peptide stimulates T cells across a wide variety of antigenic specificities and of divers genetic backgrounds. Possessing no B cell epitopes means that the epitopes are not recognized by B cells and, therefore, will not induce antibody formation specific to the epitope. The peptide may be chemically conjugated to a protein or a fusion protein may be constructed according to the methods well known to those ordinarily skilled in the art. The peptide may be conjugated to a polysaccharide according to methods known to those ordinarily skilled in the art, including CDAP.

Most preferably, the compositions comprise GM-CSF, IL-3, or a combination thereof, IFN-γ, CD40L, and IL-1 + IL-2. In another most preferable embodiment, the compositions comprise GM-CSF, IL-3, or a combination thereof, IFN-γ, CD40L, universal TCE and IL-1 + LL-2. Also most preferably, the compositions comprise GM-CSF, IL-3,

IFN-γ, CD40L, universal TCE, and IL-1 + IL-2, or a combination of some or all ofthe above conjugated to a protein and further conjugated to a polysaccharide.

The enhancement in Ig secretion mediated by IL-2 or IL-3 or GM-CSF typically ranges between 10-50 fold, and the combination of IL-3 and GM-CSF induces enhancement of up to 100 fold. Preferably, GM-CSF and IL-3 are present at from about

1 to about 10 U/ml, in vitro. In vivo amounts are scaled up accordingly, as is well known in the art. More preferably, GM-CSF and IL-3 are present at from about 10 up to about

100 U/ml, and in particular, at about 100 U/ml.

IDENTIFICATION OF IL-3, GM-CSF, AND IFN-γ AS B CELL STIMULATORY AGENTS

Prior to the invention, it was not known that GM-CSF or IL-3 act directly on the B cell to stimulate the release of antibody, or that GM-CSF and IL-3 act synergistically and directly on the B cell to stimulate the release of antibody. Moreover, GM-CSF has not been previously implicated in directly regulating mature B cell function. It was also not known that addition of IFN-γ 24 hours after stimulation could by itself stimulate optimal antibody secretion, nor that it could further enhance the activity of IL-3 and GM-CSF, either alone or in combination.

The composition described in parent application Serial No. 08/150,510 comprised IL-3 and GM-CSF. Early experiments determined that the Ig secretory response of electronically sorted highly purified B cells was significantly lower than B cell enriched populations that contained small numbers of "non-T, non B" cells. Based on these findings, the first application showed that NK cells and/or NK cell-derived cytokines could enhance Ig secretion in anti-Ig-dextran stimulated B cells.

The parent application also disclosed supernatants derived from T cell clones (TH1 or TH2) that enhanced Ig secretion by B cells. Experiments determined that this enhancement was not due to IL-1, IL-2, IL-5, IL-6, or IL-10. Suφrisingly, it was discovered that the differentiation-inducing activity was due to the presence of GM-CSF and IL-3. When GM-CSF and IL-3 were added to anti-Ig-dextran stimulated cells, Ig secretion was induced. Conversely, when anti-GM-CSF and or anti-IL-3 were added, the stimulatory activity of the T cell supernatants was diminished.

It was also found that Ig secretion could be further enhanced by the addition of IL-2. the addition of IFN-γ 24 hours after culture, or the addition of IFN-γ + IL-2. Maximum levels of Ig secretion were induced when IL-2, 1L-3, and GM-CSF were added to anti-Ig-dextran stimulated cells at the onset of culture, and IFN-γ was added one day later (24 hr period after stimulation with anti-Ig-dextran). IFN-γ added immediately following B cell activation does not enhance the stimulatory effect, or minimally enhances the stimulatory effect, of IL-3 or GM-CSF. Thus, the timing of the addition of IFN-γ is important for further enhancing the release of antibody by B cells. This discovery has not been reported prior to the invention.

To determine whether the activity of LL-3 or GM-CSF, or IL-3 + GM-CSF, could account for the differentiating activity of the THl or TH2 derived supematant, the effect of adding neutralizing quantities of anti-IL-3 or anti-GM-CSF antibody on Ig secretion was analyzed. While each antibody mediated significant suppression of Ig secretion, the combination of anti-IL-3 and anti-GM-CSF induced a greater than 80% suppression of Ig secretion in anti-Ig-dextran stimulated cells in the presence of THl- or TH2-derived supematant.

The findings that IL-3 and GM-CSF can enhance Ig secretion by B cells, and that anti-IL-3 + anti IL-GM-CSF can suppress Ig secretion, were completely unexpected. Moreover, it was also suφrising that IFN-γ added 24 hours after the onset of culture enhanced the stimulatory effect of IL-3 and GM-CSF. These findings have not been previously reported.

PHARMACEUTICAL COMPOSITIONS

Pharmaceutical compositions are also encompassed by the invention. Such compositions comprise an effective amount of IL-3 and GM-CSF, either alone or in combination, and a pharmaceutically acceptable carrier. Also encompassed by the invention are pharmaceutical compositions comprising an effective amount of IL-3 and GM-CSF, either alone or in combination, and an effective amount of IFN-γ. along with a pharmaceutically acceptable carrier.

Treatment comprises administering the pharmaceutical composition by intravenous., intraperitoneal. intracoφoreal injection, intra-articular, intraventricular, intrathecal.

intramuscuiar, subcutaneous, intranasally. intravaginally, orally, or by any other suitable method of administration. The composition may also be given locally, such as by injection to the particular area, either intramuscularly or subcutaneously.

Any pharmaceutically acceptable carrier can be employed for GM-CSF. IL-3, and IFN-γ. Carriers can be sterile liquids, such as water, oils, including petroleum oil, animal oil, vegetable oil, peanut oil, soybean oil, mineral oil, sesame oil, and the like. With intravenous administration, water is a preferred carrier. Saline solutions, aqueous dextrose, and glycerol solutions can also be employed as liquid carriers, particularly for injectable solutions. Suitable pharmaceutical carriers are described in Remington's Pharmaceutical Sciences, 18th Edition (A. Gennaro, ed.. Mack Pub., Easton, Pa., 1990), incoφorated by reference.

Vaccine adjuvants comprising the compositions

This invention also encompasses vaccine adjuvants comprising the compositions of the invention. Many vaccines are currently administered intravenously or intramuscularly to allow a rapid stimulation of immune cells present in the blood system. By combining the novel compositions with a drug to be administered, the magnitude ofthe antibody response is increased, both locally and systemically.

In the in vitro assay, the anti-IgM or anti-IgD antibody when presented in a multivalent form, such as dextran, acts efficiently to activate all B cells. This high level of activation, coupled with the use of highly purified B cells, allows the identification of compounds that stimulate the release of antibody by B cells. However, this response is not desired in vivo. In a patient, the goal is to activate only a small number of B cells having receptors specific for the antigen. The specific antigen ofthe vaccine acts to cross-link specific B cell receptors. In contrast, the in vitro model employing anti-Ig-dextran acts to cross-link all antigen receptors.

Preferably, when used as a vaccine adjuvant, the compositions of the invention are conjugated to a multivalent carrier molecule, such as dextran or a capsular polysaccharide of a bacteria. Pneumococci, streptococci, and meningococci capsular polysaccharides are preferred. GM-CSF, IL-2, or IL-3, molecularly engineered fragments of GM-CSF. IL-2, or IL-3 that retain GM-CSF or IL-3 activity, or a combination thereof, can be independently

conjugated to the multivalent carrier. Altematively, GM-CSF, IL-2, and IL-3 can be fused together or fused to another protein to form a fusion protein, which can also be bound to the multivalent carrier. Other vaccine variations will be apparent to one of skill in the art.

This application encompasses the use of anti-cytokine-cytokine complexes which allow for the slow but prolonged delivery ofthe cytokine. The complexes can be administered as a mixture with the antigen of a vaccine, or the complexes can be bound to the antigen of a vaccine.

In still another embodiment, the vaccine adjuvant can comprise CD40L, one or more cytokines other than GM-CSF, IL-3. or IFN-γ, or a combination thereof. CD40L and the one or more cytokines, or a universal TCE can also be bound to the multivalent carrier.

Compositions used in a conjugate vaccine

To form a conjugate vaccine, the antigen of the vaccine and the compositions ofthe invention can be covalently conjugated to a multivalent carrier molecule, such as dextran cr a capsular polysaccharide of a bacteria. Pneumococci, streptococci, and meningococci capsular polysaccharides are preferred.

The antigen is a peptide or protein specific for the disease to be vaccinated against.

To further optimize the humoral immune response upon administration of the vaccine. CD40 or at least one other cytokine, or a universal TCE, or a combination thereof, can be conjugated to the multivalent carrier.

Table III shows exemplary vaccines employing the compositions ofthe invention. As noted in the Table, several ofthe vaccines are conjugate vaccines. Methods of conjugation are well known to those of ordinary skill in the art, and include the heteroligation techniques of Brunswick et al., J. Immunol. 140:3364 (1988); Wong, S.S., Chemistry of Protein C ^niusates and Crosslinking. CRC Press, Boston (1991); and Brenkeley et al., "Brief Purvey of Methods for Preparing Protein Conjugates With Dyes, Haptens and Cross-Linking Agents," Bioconjugate Chemistry. 3_, No. 1 (Jan. 1992), specifically incoφorated by reference. A preferred method of covalent conjugation is set forth in application Serial No. 08/482,661, filed June 7, 1995, which is a continuation-in- part of application Serial No. 08/408,717, filed March 22, 1995, which is a continuation-h- part of application Serial No. 07/124.491, filed September 23, 1993, the so-called "CDAP"

conjugation method, the disclosures of which are specifically incoφorated herein by reference.

METHODS OF USING THE COMPOSITIONS

A further object of this invention is the use of the compositions comprising IL-2, IL-3 and GM-CSF, either alone or in combination, and compositions comprising IL-2, IL-3 and GM-CSF, either alone or in combination, along with IFN-γ, to stimulate the release of antibody by B cells.

Suitable hosts for treatment include any suitable mammal. Preferred hosts are humans, including neonates, adults, and immunodeficient patients.

The compositions can be administered employing any suitable administration method. Preferable methods of administering the compositions include subcutaneously, intravenously, nasally, mucosal routes, orally, intramuscularly, or a combination thereof.

The dosage ofthe compounds employed varies depending upon age, individual differences, symptoms, mode of administration, etc., and can be readily determined by one of skill in the art. Exemplary dosages of GM-CSF and IL-3 are given in J. Nemunaitis, "Granulocyte-macrophage-colony-stimulating factor: a review from preclinical development to clinical application," Transfusion. 33. 70-83 (1993); Lieschke et al., "Granulocyte Colony-Stimulating Factor and Granulocyte-Macrophage Colony-Stimulating Factor (First of Two Parts)," The N. Eng. J. of Med.. 327. 28-35 (1992); Lieschke et al., "Granulocyte Colony-Stimulating Factor and Granulocyte-Macrophage Colony- Stimulating Factor (Second of Two Parts)," The N. Eng. J. of Med.. 327. 99-106 (1992); Schulz et al., "Adjuvant Therapy with Recombinant Interleukin-3 and Granulocyte-Macrophage Colony-Stimulating Factor," Pharmac. Ther.. 52, 85-94 (1992); Hoelzer et al., "Interleukin 3 Alone and in Combination with GM-CSF in the Treatment of Patients with Neoplastic Disease," Seminars in Hematology. 28. suppl. 2 (April), 17-24 (1991). NEUTRALIZING METHODS OF USING THE COMPOSITIONS

Another object of this invention is methods of neutralizing the activity of GM-CSF, IL-3, and IFN-γ under conditions where the production of antibody is pathogenic, such as in autoimmune disorders, such as lupus, systemic lupus erythematosus (SLE), idiopathic thrombocytopenic puφura (ITP), vasculitis, Graves' disease, allergic reactions, etc.

For the construction of a neutralizing vaccine, an antibody against the cytokine is made. Those of ordinary skill in the art are familiar with the well established methods of obtaining specific antibody. The antibody is then administered to a patient. The antibody can be bound to a carrier to increase the half-life ofthe antibody.

METHODS OF USING THE COMPOSITIONS TO OPTIMIZE MONOCLONAL ANTIBODY PRODUCTION

The compositions can also be used to optimize monoclonal antibody production in vitro or in vivo. For example, an animal can be sensitized by injecting a solution containing the antigen of interest and the compositions in vivo. Because the compositions stimulate the release of antibody by B cells, the administration of the compositions in conjunction with the specific antigen will optimize the production of antibody against the specific antigen.

Those of ordinary skill in the art would be familiar with techniques for such immunization, as well as the dosages ofthe antigen and compositions needed to elicit the antibody and to stimulate its release in light ofthe teachings in this specification.

Altematively, in vitro stimulation or sensitization of lymphocytes to produce antibody can be enhanced in the presence ofthe novel compositions. The teachings for such stimulation and sensitization are well within the routine skill of those in the art, as is the determination ofthe appropriate amounts of antigen and compositions to apply in light ofthe teachings in this specification. This method is particularly useful for the production of human monoclonal antibodies.

ASSAY SYSTEM FOR IDENTIFYING COMPOSITIONS USEFUL FOR STIMULATING THE RELEASE OF ANTIBODY BY B CELLS

A further embodiment ofthe invention is the development of a novel in vitro assay system that mimics in vivo antibody stimulation. The assay system allows the identificaticin of compositions that stimulate the release of antibodies by B cells.

Because resting B cells do not release antibody, they must first be activated before antibody release can be measured. In the present invention, anti-IgM or anti-IgD antibody is covalently conjugated to high molecular weight dextran (i.e., MW = 2.0 x IO ⁶ ) to create a multivalent antigen on a polysaccharide carrier, as set forth in Snapper et al.. "Comparative

In Vitro Analysis of Proliferation, Ig Secretion, and Ig Class Switching by Murine Marginal Zone and Follicular B Cells," J. of Immunology. 150. 2737-2745 (1993), specifically incoφorated by reference. This procedure converts the bivalent anti-Ig molecule into an extremely stimulatory multivalent conjugate which can induce persistent and repetitive signaling via B cell membrane Ig, even at picomolar concentrations. The anti-Ig-dextran conjugate stimulates high levels of B cell proliferation at concentrations as low as 1 pg/ml, a concentration 10,000 fold lower than that stimulated by unconjugated anti-Ig. The activation of B cells occurs irrespective of antigen specificity. Anti-Ig-dextran does not stimulate the release of antibody by resting B cells in the absence of cytokines. With the addition of a cytokine that stimulates the release of antibody by B cells, high levels of Ig secretion are observed.

Previously, it was known that activation of B cells through the antigen receptor can be optimally achieved using an anti-immunoglobulin (Ig) antibody conjugated to a large molecular weight polysaccharide, e.g., dextran. However, it was not possible to screen for compositions that stimulate the release of antibody by B cells because, previously, the B cells employed in the assays were not highly purified. Contaminating cells in the B cell supematant often secreted sufficient amounts of cytokines that could stimulate the release of antibody by B cells. Therefore, it was not possible to determine whether the stimulatory effect of a tested composition was due to the added composition or to a contaminant.

In contrast, the present invention employs highly purified B cells in the assay system. Preferably, the B cells are electronically sorted to produce highly purified B cells. With the use of highly purified B cells, it is possible to measure the stimulatory effects of substances on the release of antibody by B cells activated through multivalent mlg cross-linking. This system was not described prior to the invention. In fact, it was not possible to test compositions for the sole characteristic of stimulatory activity on the release of antibody by B cells prior to the discovery of this assay system.

* * * * *

The unexpected effect of the present invention is demonstrated in the following experiments and is depicted in Figures 1-12.

Having generally described the invention, a more complete understanding can be obtained by reference to specific examples, which are provided herein for puφoses of illustration only and are not intended to be limiting. Example 1

This example shows that the B cell antibody stimulatory activity ofthe cell supematant reported in the parent application was not due to one known cytokine.

Materials and Methods: Female DBA/2 mice were obtained from the National Cancer Institute (Frederick, MD) and were used at 7-10 weeks of age. Culture medium used was RPMI 1640 (Biofluids, Rockville, MD) supplemented with 10% fetal bovine serum (Sigma, St. Louis, MO), L-glutamine (2 mM), 2-mercaptoethanol (0.05 mM), penicillin (50 μg/ml) and streptomycin (50 μg/ml).

Dextran-conjugated anti-IgD antibody was prepared by conjugation of Hδ 1 (monoclonal mouse IgG2b (b allotype)) anti-mouse IgD (a allotype) to a high molecular weight dextran (2 x 10 ⁶ MW). Approximately 9 dextran-conjugated anti-IgD antibodies were conjugated to each dextran molecule. FITC-anti-CD3e mAb (2C11) was purchased from Pharmingen (San Diego, CA).

PE-labeled affinity-purified polyclonal goat anti-mouse IgM antibody was purchased from Southern Biotechnology Associates (Birmingham, AL). Murine recombinant IL-1 and IL-2 were kind gifts from Dr. Stephanie Vogel (USUHS. Bethesda, MD) and Dr. Maurice Gately (Hoffman-La Roche, Nutley, NJ), respectively. Recombinan murine IL-3 and GM-CSF were purchased from Pharmingen. Polyclonal goat anti-mouse IL-3 and GM-CSF antisera were both obtained from R & D Systems (Minneapolis. MN).

Functional assays were carried out in 96-well flat-bottom Costar plates (Costar, Cambridge, MA). Cultured cells were incubated at 1 x 10 ⁵ cells/ml in a total volume of 200 μL at 37°C in a humidified atmosphere containing 6% C0 ₂ .

Polyclonal IgM concentrations were measured by ELISA. Quantitation was achieved by comparison with IgM standard curves employed in every assay.

Preparation and culture of B cells: Enriched populations of B cells were obtained from spleen cells. T cells were eliminated by treatment with rat anti-Thy-1, anti-CD4, and anti-CD8 monoclonal antibodies, followed by monoclonal mouse anti-rat Igk and

complement. Cells were fractionated on the basis of their density over discontinuous Percoll gradients (Pharmacia, Piscataway, NJ) consisting of 70, 65, 60, and 50% Percoll solutions (with densities of 1.086, 1.081, 1.074, and 1.062 g/ml, respectively). The high density (small, resting) cells were collected from the 70 to 65% interface and consisted of -90% B cells. Highly purified B cells were then obtained by electronic cell sorting of membrane (m)IgM+CD3- cells on an EPICS Elite cytometer (Coulter Coφ., Hialeah, FL) after staining with FITC-anti-CD3e + PE-anti-IgM antibodies. Sorted cells were immediately re-analyzed and found to be consistently >99% B cells. These cells were used in all experiments.

THl mouse cell lines were cloned and used for the experiments. As described in the parent application, a composition which stimulated the release of antibody by B cells was isolated. To determine the identity ofthe components ofthe composition, the activity of all known cytokines was systematically suppressed by adding monoclonal antibodies specific for IL-1, IL-2, IL-3, etc., to the composition, and then measuring activity.

Because the composition comprised two independent B cell antibody release stimulators, IL-3 and GM-CSF, experiments suppressing each known cytokine one by one did not result in completely suppressing the measured activity. Example 2

This example determines the relative stimulatory effects of a supematant of a T cell culture identified in the parent application, GM-CSF, and IL-3.

IgM secretion (ng/ml) by B cells in various mediums was measured. Culture mediums are listed along the left side Fig. 1. Culture mediums were anti-IgD-dextran and IL-1 + IL-2; anti-IgD-dextran and IL-4; soluble CD40 ligand and IL-1 + IL-2; soluble CD40 ligand and IL-4, membrane CD40 ligand; membrane CD40 ligand and IL-1 + IL-2; and membrane CD40 and IL-4. The compositions tested were a supematant of a cell culture described in the parent application, RA5-SN. GM-CSF, and IL-3. A control composition was also tested for each medium.

As set forth in Fig. 1 , compositions tested showed the highest IgM secretion in medium comprising dextran-conjugated anti-IgD antibody, IL-1. and IL-2. The RA5-SN

supematant, having both GM-CSF and IL-3, showed significant IgM secretion by B cells as compared to other tested compositions. Example 3

This example shows that IL-3 and GM-CSF stimulate Ig secretion by activated B cells.

B cells were activated with dextran-conjugated anti-IgD antibodies (3 ng/ml) plus IL-1 (150 U/ml) + IL-2 (150 U/ml) in the presence or absence of varying concentrations of IL-3 or GM-CSF. IgM secretion was measured by ELISA 6 days after initiation of culture.

Electronically sort-purified resting B cells proliferated but failed to secrete Ig in response to activation by dextran-conjugated anti-IgD antibody or dextran-conjugated anti-IgD antibody plus IL-1 + IL-2. As reported in the parent application, supernatants obtained from anti-CD3-activated CD4+ THl and TH2 clones induce strong Ig secretory responses by B cells stimulated with dextran-conjugated anti-IgD antibody plus IL-1 + IL-2. This stimulation is IL-4 and IL-5-independent.

Because THl and TH2 clones share the capacity to secrete IL-3, GM-CSF, and TNF-α upon activation, these cytokines were tested for their ability to induce Ig secretion.

Cytokines were titered in log increments from 0.1 to 1000 U/ml final concentration. TNF-α had no effect on lg synthesis. In contrast, IL-3 and GM-CSF stimulated significant enhancements in Ig secretion over that observed with dextran-conjugated anti-IgD antibody plus IL- 1 + IL-2 alone (Fig. 2). Both IL-3 and GM-CSF stimulated optimal Ig secretory responses at 100 U/ml, producing a 19- and 9-fold enhancement, respectively. Significant, though lower, levels of induction of Ig synthesis in response to IL-3 or GM-CSF were still observed at 1-10 U/ml.

In multiple experiments the degree of enhancement in Ig secretion mediated by IL-3 and GM-CSF typically ranged between 10-50 fold (Fig. 3). No significant differences were observed between IL-3 and GM-CSF for either dose response or optimal level of induction of Ig secretion.

Example 4

This example demonstrates that anti-IL-3 and anti-GM-CSF antibodies specifically inhibit the induction of Ig secretion in response to IL-3 and GM-CSF, respectively.

B cells were activated with dextran-conjugated anti-IgD antibodies (3 ng/ml) plus IL-1 (150 U/ml) + IL-2 (150 U/ml) in the presence or absence of IL-3 (10 U/ml) or GM-CSF (10 U/ml). In addition, anti-IL-3 (10 μg/ml) and or anti-GM-CSF (10 μg/ml) antibodies were added. A control with no added anti-IL-3 or anti-GM-CSF was also prepared. IgM concentrations were measured by ELISA 6 days after initiation of culture.

Neutralizing anti-IL-3 and anti-GM-CSF antibodies specifically and completely inhibited the respective Ig-inducing activities of IL-3 and GM-CSF (Fig. 4). These antibodies, either alone or in combination, had no effect on the Ig secretory response to dextran-conjugated anti-IgD antibody + IL-5 activation (data not shown). Results also demonstrated that the combination of anti-IL-3 and anti-GM-CSF antibodies could significantly reduce the IL-4 + IL-5-independent Ig-inducing activities of supernatants from either a CD4 ⁺ THl or TH2 clone. It was also demonstrated that IL-3 and GM-CSF could not induce Ig secretion by B cells activated through the CD40-mediated signalling pathway. Example 5

This example demonstrates that IL-3 and GM-CSF induce Ig secretion by B cells activated with dextran-conjugated, but not unconjugated, anti-IgD antibodies. This example also shows that IL-3 and GM-CSF act synergistically with IL-1 + IL-2.

Multivalent, but not bivalent, mlg cross-linkage co-stimulates cytokine-mediated Ig secretion and class switching. In this example, dextran-conjugated anti-IgD (HδVl mAb) was compared with unconjugated anti-IgD (HδVl mAb) for co-stimulation of Ig secretion in response to IL-3 or GM-CSF.

B cells were first cultured with AF7 supematant, αIL-4, and αIL-5. For AF7 supematant, KLH-specific. la ^b -restricted, CD4+ T cell clone, derived from C57BL/6 mice, was established and maintained by standard methodologies. AF7 is a TH2 clone on the basis of its production of IL-4 and lack of production of IL-2 and IFN-γ.

Cytokine-containing supematant was obtained from cultures of AF7 cells in the following manner: Tissue culture wells were incubated with anti-CD3 mAb (2C1 1) at

1 Oμg/ml in PBS for 3 hr at 37°C and then washed 3x in fresh PBS. AF7 cells, which were allowed to retum to their resting state, 7 days after stimulation with antigen, APCs, and IL-2, were added to anti-CD3 -coated plates at 1 x IO ⁶ cells/ml for various times, upon which cell-free supernatants were obtained and either stored at -20°C or 4°C. In the latter case, supematant was used in cellular assays within 1-2 weeks of having been harvested.

B cells were then cultured with dextran conjugated (HδVl-dex, 3 ng/ml) or unconjugated (Hδ71, 30 μg/ml) anti-IgD antibodies in the presence or absence of IL-1 (150 U/ml) + IL-2 (150 U/ml). A control composition did not contain conjugated or unconjugated dextran. IL-3 (100 ml) and or GM-CSF (100 U/ml) were added, and control compositions with no addition of IL-3 or GM-CSF were prepared (Fig. 5). IgM concentrations were measured by ELISA 6 days after initiation of culture.

In contrast to conjugated dextran (HδVl-dex), unconjugated dextran (Hδ l) was ineffective as a co-stimulator of Ig secretion in the presence of IL-3 or GM-CSF (Table 1). These results were obtained despite the ability of unconjugated dextran to induce early B cell activation events, such as increases in cell size and MHC class II induction. IL-3 or GM-CSF alone were unable to induce resting B cells to secrete detectable Ig (Table 1). However, addition of IL-3 and GM-CSF to cultures of dextran-conjugated anti-IgD antibody-activated cells, in the absence of exogenous IL-1 + IL-2, led to 7.4- and 5.4-fold inductions, respectively, of Ig secretion relative to stimulation by dextran-conjugated anti-IgD antibody alone. The addition of IL-1 + IL-2 to cells activated with dextran-conjugated anti-IgD antibody alone did not further increase Ig secretion.

The combination of IL-3 or GM-CSF with IL-1 + IL-2 led to 48-fold and 75-fold enhancements in Ig secretion relative to that observed for B cell cultures activated with dextran-conjugated anti-IgD antibody plus IL-1 + IL-2 alone. Thus, IL-1 + IL-2 is strongly synergistic with IL-3 or GM-CSF for induction of Ig secretion by dextran-conjugated anti-IgD antibody-activated resting B cells.

TABLE I

Medium Level of Stimulation

HδVl-dex 65

IL-3 <24

GM-CSF <24

HδVl-dex + IL-3 480

HδVl-dex + GM-CSF 350

HδVl-dex + IL-1 + IL-2 57

HδVl-dex + IL-1 + IL-2 + IL-3 2750

HδVl-dex + IL-1 + IL-2 + GM-CSF 4250

HδV-1 <24

HδV-l + IL-l + IL-2 <24

HδV-l + IL-l + IL-2 + IL-3 <24

HδV-1 + IL-1 + IL-2 + GM-CSF <24

Example 6

This example demonstrates that both IL-3 and GM-CSF act primarily through stimulation of B cell differentiation to Ig secretion.

To determine the mechanism by which IL-3 and GM-CSF enhance Ig secretion, the effects of IL-3 and GM-CSF on cell outgrowth as compared to Ig secretion were determined.

B cells were activated with dextran-conjugated anti-IgD antibodies (3 ng/ml) plus IL-1 (150 U/ml) + IL-2 (150 U/ml) in the presence or absence of IL-3 (100 U/ml) and/or GM-CSF (100 U/ml). Viable cells (those that excluded trypan blue) were enumerated 4 days after initiation of culture using a hemocytometer. IgM concentrations in replicate cultures were measured by ELISA 6 days after initiation of culture. Data is represented as mean +/- standard error of the mean of duplicate cultures.

IL-3 stimulated a significant but less than 2-fold enhancement in viable cell outgrowth, whereas GM-CSF had no significant effect on the degree of cell expansion (Fig. 6). Studies assessing DNA synthesis on the basis of ³ H-thymidine incoφoration are consistent with these observations.

The induction of Ig secretion of replicate cultures in response to IL-3 and GM-CSF was 13.8-fold and 13.6-fold, respectively. These results indicate that the primary effect of IL-3 and GM-CSF is to stimulate a strong increase in the average amount of Ig secreted per cell, and not enhancement in the total of number of cells present in culture. Thus, the data strongly suggest that IL-3 and GM-CSF are differentiation factors for resting B cells activated through multivalent antigen receptor cross-linking. The combined action of IL- and GM-CSF, utilizing optimal doses of each cytokine, consistently led to greater than additive Ig secretory responses (Fig. 6). These data indicate that IL-3 and GM-CSF act synergistically. A similar degree of synergism was observed with the combined use of IL-3 and GM-CSF on cells activated with dextran-conjugated anti-IgD antibody in the absence of IL-1 + IL-2 (data not shown). Example 7

This example demonstrates the stimulatory effect on antibody secretion of B cells when CD40L is added to the B cell composition.

B cells were activated with dextran-conjugated anti-IgD antibodies (3 ng/ml or 0.3 ng/ml) plus IL-1 (150 U/ml) + IL-2 (150 U/ml). IgM concentrations in replicate cultures were measured by ELISA for a control composition, for a composition with IL-3 + GM-CSF, and for a composition with IL-3 + GM-CSF + CD40 ligand (CD40L) (Table II). IL-3 and GM-CSF were present at 100 U/ml. CD40L was in the form of a recombinant soluble CD8-CD40 ligand fusion protein which was added at a final concentration of 10 μg/ml. The fusion protein was a kind gift of Dr. M. Kehry, Boehringer Ingelheim Pharmaceuticals, Inc., Ridgefield, CT.

TABLE II

IgM Secretion (ng/ml)

IL-1 + IL-2 Medium IL-3 + GM-CSF IL-3 + GM-CSF + CD40L

+ medium <12 <12 1 15

+ anti-IgD-dex 1,060 21,250 61,250

(3 ng/ml)

+ anti-IgD-dex 37 3,575 42,125

(0.3 ng/ml)

The most significant results (61,250 ng/ml) were obtained with anti-IgD-dex at 3 ng/ml and the composition consisting of IL-3 + GM-CSF + CD40L. However, even at low levels ofthe multivalent activator (anti-IgD-dex at 0.3 ng/ml), significant IgM secretion was obtained (42,125 ng/ml). Thus, even at low levels of conjugate, extremely potent antibody release responses can be obtained by including CD40L on a carrier. Example 8

This example demonstrates the stimulatory effect on antibody secretion of B cells when IFN-γ is added 24 hours after stimulation of the B cells.

B cells were activated with dextran-conjugated anti-IgD antibodies (3 ng/ml) plus IL-1 (150 U/ml) + IL-2 (150 U/ml) in the presence or absence of IL-3 (100 U/ml) + GM-CSF (100 U/ml). IgM concentrations in replicate cultures were measured by ELISA for a control composition, a composition to which IFN-γ was added at 0 h, and for a composition to which IFN-γ was added at 24 h. Data is represented as mean +/- standard error of the mean of duplicate cultures (Figure 7).

The composition consisting of anti-IgD-dex-activated B cells and IL-1 + IL-2 showed minimal IgM secretion with no added INF-γ (885 ng/ml) and minimal secretion with IFN-γ added at 0 hours (975 ng/ml). When IFN-γ was added 24 hours after stimulation of the B cells, antibody secretion jumped to 11,250 ng/ml.

Even more significant results were obtained with a composition consisting of anti-IgD-dex-activated B cells, IL-1 + IL-2, and IL-3 + GM-CSF. In medium without

IFN-γ, antibody secretion was measured at 21,250 ng/ml. Suφrisingly, the addition of IFN-γ at 0 hours suppressed antibody secretion to 7,250 ng/ml. However, with the addition of IFN-γ 24 hours after stimulation ofthe B cells, a synergistic reaction occurred, producing antibody secretion of almost 200,000 ng/ml. Example 9

This example provides exemplary vaccines and vaccine adjuvants employing the compositions of the invention.

Table III shows exemplary vaccines employing the compositions ofthe invention. As noted in the Table, several ofthe vaccines are conjugate vaccines. Methods of conjugation are well known to those of ordinary skill in the art, and include the heteroligation techniques of Brunswick et al., J. Immunol.. 140:3364 (1988); Wong, S.S., Chemistry of Protein Conjugates and Crosslinking. CRC Press, Boston (1991); and Brenkeley et al., "Brief Survey of Methods for Preparing Protein Conjugates With Dyes, Haptens and Cross-Linking Agents," Bioconjugate Chemistry. 3_, No. 1 (Jan. 1992), specifically incoφorated by reference.

The multivalent carrier replaces the multivalent conjugate (i.e., anti-IgD-dex) employed in vitro. Such a carrier can be, for example, a polysaccharide such as dextran, or a capsular polysaccharide from a bacteria, such as pneumococci, streptococci, or meningococci. The polysaccharide may or may not be medically relevant depending on the use envisioned for the vaccine.

The cytokine can be GM-CSF, IL-3, IFN-γ, or a combination thereof.

This example is not intended to be limiting, and other types of vaccines will be apparent to those skilled in the art from consideration ofthe specification and practice of the invention.

TABLE III Vaccine Structure and Components

1. cytokine ^* + antigen (admixed); coadministration in aqueous solution, slow release particles, adjuvants, etc.

2. cytokine + antigen + multivalent carrier; the cytokine, antigen, or both can be directly conjugated to the carrier, i.e.: cytokine cytokine antigen

I antigen | |

I 1 : or I + antigen; or | + cytokine multivalent carrier

3. cytokine-antigen; direct conjugation via covalent bonding

4. cytokine-antigen (covalent bonding) bound to a multivalent carrier, i.e.: antigen cytokine multivalent carrier

5. cytokine-antigen (fusion protein)

6. cytokine-antigen (fusion protein) bound to a multivalent carrier

7. peptide-cytokine (fusion protein) + antigen

8. peptide-cytokine (fusion protein) + antigen, with the fusion protein, antigen, or both bound to a multivalent carrier

9. cytokine-multivalent carrier, i.e.:

IL-3

GM-CSF IL-3 GM-CSF

.J L_ ; or J multivalent multivalent carrier carrier

10. antibody complex (i.e., IL-3 + anti-IL-3) + antigen

1 1. antibody complex (i.e., IL-3 + anti-IL-3) + antigen + multivalent carrier (the cytokine, antigen, or both can be conjugated to the carrier)

12. anti-cytokine antibody; this is a neutralizing vaccine

13. anti-cytokine antibody + multivalent carrier; this is a neutralizing vaccine

14. Vaccine examples 1 through 13 can be further modified by the addition of CD40L, either admixed or bound to the multivalent carrier

15. Vaccine examples 1 through 14 can be further modified by the addition of one or more cytokines other than GM-CSF, IL-3, or INF-γ, such as IL-1 + IL-2, either admixed or bound to the multivalent carrier

^' The cytokine can be GM-CSF, IL-3, IFN-γ, or a combination thereof.

* * * * * *

In sum, the experimental results show that IL-3 and or GM-CSF stimulate the release of antibody by B cells and that, in combination, these cytokines act synergistically to stimulate the release of antibody by B cells. This induction is specifically inhibited by anti-IL-3 and anti-GM-CSF antibodies, respectively. The stimulatory effect of IFN-γ added 24 hours after culture is also shown.

The effects of IL-3 and GM-CSF are enhanced by multivalent antigen receptor cross-linkage, as mediated by dextran-conjugated anti-Ig antibody in the experimental model. The effects of IL-3 and GM-CSF may also be enhanced with antigen that does not induce high levels of membrane Ig cross-linking. Although both dextran-conjugated anti-Ig antibody and IL-1 + IL-2 are required for optimal IL-3- and GM-CSF-mediated Ig secretion, both IL-3 and GM-CSF stimulate a modest Ig secretory response by cells activated with dextran-conjugated anti-Ig antibody alone. This is the first report demonstrating the ability of IL-3 and GM-CSF to act directly as differentiation factors for B cells.

In addition, while maximal antibody secretion is obtained with the use of GM-CSF, IL-3, IL-1 + IL-3, and IFN-γ added 24 hours after culture, a modest increase in antibody secretion was also observed with the sole addition of IFN-γ 24 hours after culture.

Example 10

This example shows the preparation of cytokine-protein fusion products.

A. Plasmid Construction. The YEpFlag-1 expression plasmid (Kodak Scientific Imaging systems) was used for the cloning and expression of the fusion proteins. The YEpFlag-1 vector contains: an ADH2 promoter for regulated expression in yeast, tryptophan marker for selection in yeast, and α factor for protein secretion in yeast. The vector also contains a multiple cloning site, the gene for ampicillin resistance and a Flag peptide sequence. The insertion ofthe fusion protein DNA within the Kpnl and BamHI restriction sites removed the Flag peptide sequence and 14 bp's off of the a Factor peptide sequence. To facilitate purification ofthe fusion protein 6 histidine residues were added to its C terminal end, thus enabling binding to and acid elution from a Ni-NTA resin. This method is superior to Flag dependent purification since it does not require subsequent enzymatic removal ofthe Flag peptide and provides for larger quantity purification. The 14 bp's removed from the 3' end ofthe Alpha Factor extracellular secretion peptide sequence were replaced by PCR.

Prior to constmction ofthe fusion proteins, individual proteins were first separately cloned. Protein D, pneumococcal surface protein A (protein A) and pneumolysin were cloned according to the methods described in Son et al.. Infect, and Immun., Vol. 63, No. 2, 696-699 (Feb. 1995); Langermann et al.. J. Exp. Med., Vol. 180, pp 2277-2286 (Dec. 1994); and Walker et al.. Infect, and Immun., Vol. 55, No. 5, pp 1184-1 189 (May 1987). The fusion proteins were constmcted with the cytokine on the N terminus end linked by 6 glycine residues to the antigenic protein containing a 6 histidine tail on the C terminus end.

B. IL-2, GM-CSF Plasmid Construction. Human IL-2 or murine GM-CSF cDNA containing plasmids were used as a template for PCR. The 5' primer contained a Kpnl restriction site and the removed alpha factor bp's, and the first 18 bases for the N-termini ofthe protein. The 3' primer contained the last 18 bases for the C-termini of the protein, three glycine codons between the cytokine sequence and the Smal restriction site as a linker. The PCR products and the YEpFlag plasmid were digested with Kpnl and Smal restriction endonucleases and ligated. E. Coli DH 10B competent cells were transformed

with the ligation mixture and ampicillin resistant clones were selected and plasmid DNA was purified from several clones. The presence of IL-2 and GMCSF genes was determined by PCR. The sequences were checked by using the Applied BioSystems DNA sequencing system and the recombinant plasmids were identified as pYIL2 and pYGM.

C. Pneumococcal surface protein A (PA), Protein D, and Pneumolysin (Ply) plasmid construction. Genomic DNA from S. pneumonia Rxl was used as the template for both PA and Ply. The plasmid (pHIC 348) with cloned gent for Immunoglobulin D-Binding Protein (PD) oi Haemophilus influenzae was used as a template for PCR to obtain PD-gene fragment with convenient restriction sites at the ends (Sma I and BamHI). 3' primer contained 6 condons for His and stop-condon. The His tail at the C-end of protein serves for purification using Ni-NTA resin (Qiagen). PCR product was cloned between Sma I and Bam HI sites of p YEP FLAG-1 expression vector (Kodak Scientific Imaging System), which provides expression and secretion in yeast culture. Plasmid, containing correct PD-gene copy was identified as p YPD. The gene for PA was truncated to 888 bp and codes for that part of PA which retains its B cell antigenic epitopes as well as epitopes for T-cell activation (obtained from Dr. Larry McDaniel, Univ. of Alabama). The PCR fragment contains a Smal-restriction site, 3 glycine codons as a linker between the components ofthe fusion protein, PA sequence (Ply sequence), 6 histidine codons (His 6 sequence to be used in protein purification) a stop codon and BamHI restriction site at the 3' termini. The PCR products were cloned into YEpFlagl between the Smal and BamHI sites. After transformation clones were selected by Am pr. The presence ofthe PA or Ply gene was detected by PCR. The plasmids were designated pYPA and Ply, and the accuracy of cloned sequence was verified by sequence analysis on the Applied Biosystems Model 373A DNA sequencing system.

D. Fusion-Protein Plasmid Construction. The Smal/BamHI fragment containing the sequences for PA and PLY were cut from their respective plasmids and ligated with either pYIL2 or pYGM digested with Smal and BamHI. The new fusion protein expression plasmids were sequenced and named pIL2PA (IL-2+PA), pIL2Ply (IL-2+PIy) pGMPA (GMCSF+PA), pGMPly (GMCSF+Ply).

E. Cloning of the single sequence for Pan DR epitope peptide (PDREP)- " universal" epitope for MHC class II-restricted T cells. One of the PDREP's, as described in the article of Alexander et al.. Immunity, Vol 1 , 751-761 (1994), has a stmcture aKFVAAWTLKAAa. In our constructs this PDREP, which forms part of fusion protein, has the same stmcture, but L-alanine residues at the ends replace D-alanine.

To produce fragments of DNA containing the sequence of PDREP, a mixture of the two complementary synthetic oligonucleotides was heated to 90° and slowly cooled down. Where necessary, 5' ends ofthe oligonucleotides were phosphorylated before annealing.

Agarose-gel electrophoresis showed the presence of double-stranded fragment (~60 b.p.). Sense strand is 53b and anti-sense is 57b. At the 5'termini the fragment had cohesive end corresponding with the Kpnl restriction site, a sequence for restoration ofthe α leader peptide codons which was excluded at the time of cloning, 39 b.p. for PDREP and blunt 3' termini. This fragment was ligated with PYPA digested with Kpnl and Smal. DNA from ampicillin resistant clones is analyzed by PCR and by sequencing.

To produce a triple PDREP sequence DNA fragment, a double sequence PDREP fragment of 78 bases was produced by annealing the respective synthetic oligonucleotides. After a few steps of ligation, we obtained a constract, containing between the Kpnl and the BamHI sites, a full size α leader peptide sequence, three 39 b.p. sequences for PDREP and a PA truncated gene.

F. Yeast Transformation. Starter cultures of BJ 3505 Tryptophan(-) Yeast were cultured at 30° overnight from frozen stocks in 25 ml YPD media. 100 ml fresh YPD media was inoculated from the ovemight culture and incubated ovemight to an O.D of .3-.5 at 280 nm. The yeast and fusion proteins plasmid were then prepared for electroporation according to published protocols. Electroporation was performed using the BioRad Gene pulser set at 1 ,5KeV, 200 ohms, 25 uF. Electroporated yeast was plated on selective media plates with transformed colonies appearing in 3-5 days.

G. Isolation of Fusion Protein Expressing Yeast. Fusion protein secreting clones were selected by growing transformed colonies on expression plates. Expression plates were prepared by placing nitrocellulose and then a cellulose acetate filter on top of expression media agar plates. After colonies were grown on the plates for 3-5 days the

nitrocellulose was removed and immunoblotted. Membranes were washed/blocked in lx TBS tween 20 (TBS20) for 2 hrs and then incubated for 1 hr with the respective anti- protein antibody. Membranes were washed for 1 hr in lx TBS20 and subsequently incubated for 1 hr in rabbit anti-mouse IgG antibody and then washed for an additional hr in lx TBS20. Membranes were incubated for 1 hr in alkaline phosphatase labeled goat anti- rabbit lg, washed for 1 hr in TBS20 and then developed with BCIP. Colonies that were positive for protein secretion were selected and used to inoculate 25 ml of expansion media which was incubated ovemight and then frozen in 60% glycerol.

H. Batch Fusion Protein Production. 25 ml starter cultures of transformed yeast were prepared from frozen stocks. 10 ml from the starter culture were added to 1 liter of expansion media, incubated at 30° C shaking at 240 revolutions/minute. Cultures were harvested 3 days after inoculation, supematant was sterile filtered after collection by centrifugation at 2000 rev/min. The media was then concentrated 5 times and dialyzed with 10 times the volume of 2x PBS using an amicron stirred cell.

I. Purification. Fusion proteins containing 6 residues of histidine were purified by incubating the concentrated media with 1 ml. Ni-NTA resin (Qiagen) per liter original culture volume for 2 hr on a rotating shak r. The media and Ni-NTA were added to a small 10 cm. column and the Ni-NTA was washed with 1 liter l PBS. The protein was eluted using 2.5 M sodium acetate pH 4.5.

Fractions were collected and the O.D. was measured at 280 nm to determine relative protein concentrations; fractions with O.D. greater than 0.1 were pooled and dialyzed against lx PBS pH 7.4. Media samples from the original media, concentrated media, and purified protein were electrophoresed on an SDS denaturing gel and then either stained with comassie blue or immunoblotted. Gels for immunoblots were transferred to nitrocellulose using Pharmacia's NovaBlot system, and immunoblotting was performed as described above. Controls for protein identification were media from yeast with pYEP plasmid without fusion protein, and positive controls for IL-2 and PSP-A. Protein bands on gels stained with coomassie blue were compared to molecular weight markers to estimate fusion protein size. Recombinant proteins without a histidine tag were isolated by ion exchange on an 5100HR column. Purities were assayed by SDS PAGE and

immunob lotting for each component ofthe fusion protein. Samples may also be examined by analytical HPLC on a Beckman Ultraspherigel 2000 gel filtration column. The final products were tested for cytokine activity.

J. Conjugation of protein or cytokine protein to polysaccharide.

Pnl4 (SmithKline or ATCC) is derivatized with amines using l-cyano-4- dimethylaminopyridinium tetrafluoroborate (CDAP), as set forth above. In brief, 1 ml of Pnl4 (10 mg/ml in water) was activated by the addition of 30 ul of CDAP (100 mg/ml in acetonitrile), followed 30 seconds later by 30 ul of 0.2 M triethylamine (TEA). At 2 min, 0.5 ml of 0.5 M hexanediamine in 0.75 M HEPES, pH 7.5 was added. 1.5 hr later, the product is desalted on P6 cartridge (BioRad), equilibrated with saline, concentrated by ultrafiltration and desalted again. The amine content was assayed by the method of Vidal et aL, J. Imm. Methods, 1986, 86, 155 and the polysaccharide by the method of Monsigny et l, Anal. Chem. 1988, 175, 525.

Protein was made up at 5-20 mg/ml in 0.15 M HEPES, pH 7.5 and thiolated with a 20 fold molar excess of N-Succinimidyl S-acetylthioacetate (SATA, ProChem, Rockford, IL) from a 0.1 M stock in DMF. After 2 hr incubation in the dark at room temp, the sample was desalted using a P6 cartridge (BioRad), equilibrated with 10 mM NaAc. 0.1 M NaCl, 2 mM EDTA, pH 5 and concentrated to 10-20 mg/ml using a Centricon 30 device (Amicon).

The NH2-Pnl4 was iodoacetylated with 10 ul of 0.1 M Nhydroxysuccinimidyl iodoacetate (SI A, ProChem, Rockford. IL) per mg Pnl4 and after 2 hr desalted and concentrated as above.

The thiolated protein and iodoacetylated Ps was combined and the reaction begun by the addition of 1/9 volume of 0.5 M hydroxyiamine in 0.75 M HEPES, pH 7.5. After an overnight reaction, the reaction was quenched by making the solution 0.2 mM in mercaptoethanol for 1 hr, followed by a 10 min incubation with 10 mM iodoacetamide. The conjugate was passed over a S400HR gel filtration column, equilibrated with PBS. The high molecular weight material was pooled and sterile filtered by passage through a Millex GV filter (Millipore). The conjugate was assayed for protein using the BioRad protein assay and for polysaccharide by the method of Monsigny et al., noted above.

K. Conjugation of "universal" T cell epitope to polysaccharide.

The universal T cell epitope peptides containing a N terminal cysteine were reduced with DTT, desalted on a G10 column, equilibrated with 10 mM sodium acetate, pH 5 and reacted with iodoacetylated polysaccharide in HEPES buffer, pH 7.5. Altematively, a one step process may be used. Peptides were synthesized with an N terminal hydrazide and directly reacted with CDAP-activated polysaccharide at pH 5. By either method, after an ovemight reaction, the conjugate may be dialyzed into saline and sterile filtered. The peptide was quantitated from its absorbance spectra and the polysaccharide assayed by the method of Monsigny et al.

L. Determination of enhancement of anti-protein and anti-polysaccharide antibody responses

Mice were injected s.c. with .01-10 μg of protein, protein-cytokine or protein- cytokine-polysaccharide, and anti-protein and anti polysaccharide responses determined 14. 28 and 52 days later. Antibody responses were determined by ELISA. The enhanced anti- protein and anti-polysaccharide responses obtained are shown in Table IV.

Table IV

INDUCTION OF ENHANCED ANTI-PROTEIN AND ANTI-POLYSACCHARIDE RESPONSES WITH PspA-IL2-Pnl4 VACCINE CONSTRUCT

ELISA TITERS vaccine constmct dose (μg) IgGl anti -protein A IgGl anti-Pnl4

Protein A-IL2 5 >40,000

.5 10,721 —

.05 <10 —

Protein A-IL2-Pn 14 5 4,690 9,708

.5 24,495 >40,000

.05 17,845 >40,000

Pnl4 <10 <10

Protein A <10

M. Determination of T Cell Proliferation in Vitro in Response to Vaccine Constructs Engineered with Universal T Cell Epitope and Conjugated Pneumococcal Polysaccharide type 14.

T cells from draining lymph nodes were purified from mice having received control or experimental vaccines. Cells were cultured at 2.5 x IO ⁴ - 2 x lOVwell with 5 x IO ⁴ mitomycin C treated spleen cells as a source of presenting cells. The results are shown in Table V.

Table V

T-Cell proliferation in vitro in response to vaccine constructs engineered with universal T cell epitope (TCE)

imunizing Antigen Antigen in vitro

(0.5 μg/mpuse) (μg/ml)

TCE TCE PD/TCE

Thymidine incoφoration (cpm) media 1,400 1,400

10 18,000 21,000

1 18,000 15,000

0.10 13,000 7,500

0.01 7,000 3,500

PD-TCE media 1,500 1,500

10 24,000 28,000

1 22,000 17,000

0.10 12,000 9,300

0.01 6,000 4,000

PD-TCE-PN14 media 2,000 1,000

10 7,000 14,000

1 5,000 7,000

0.10 3,000 5,000

0.01 3,000 3,000

Mice were immunized with universal T cell epitope (TCE), protein D-TCE, or protein D-TCE-pneumococcal polysaccharide type 14 in CFA. Ten days later draining lymph nodes were separated and cultured with the various stimuli. Thymidine incoφoration was determined 4 days later. T cell proliferation was somewhat lower in the group immunizec. with PD-TCE-Pnl4 probably as a consequence ofthe chemical conjugation of PD-TCE to the polysaccharide, which may have affected a number of important epitopes.

Having now fully described the invention, it will be apparent to one of ordinary skill in the art that many changes and modifications can be made thereto without departing from the spirit or scope of the invention as set forth herein. The appended claims are not intended to be limiting.