NZ306316A

NZ306316A - use of lipoprotein and a type 2T independent antigen to induce an immune response to the antigen

Info

Publication number: NZ306316A
Application number: NZ306316A
Authority: NZ
Inventors: Clifford M Snapper; James J Mond; Carine Capiau; Pierre Hauser; Jean-Paul Prieels
Original assignee: Smithkline Beecham Biolog S
Priority date: 1995-04-17
Filing date: 1996-04-16
Publication date: 2001-06-29
Also published as: KR19990007858A; WO1996032963A1; JPH11508225A; EP0824360A1; AU716806B2; AU5485696A; CA2218385A1

Abstract

A method of inducing or enhancing an immune response to polysaccharides or other T cell-independent antigen by the co-administration of the antigen with bacterial lipoproteins (including covalent attachment of the antigen and lipoproteins). In a preferred embodiment, the lipoprotein is lipoprotein D.

Description

<div class="application article clearfix" id="description"> * WO 96/32963 PCTAJ S96/05226 INDUCTION AND ENHANCEMENT OF T IMMUNE RESPONSE TO POLYSACCHARIDES WITH BACTERIAL LIPOPROTEINS '% Pi £ "? GOVERNMENT INTEREST The invention described herein may be manufactured, licensed and used for governmental purposes without the payment of any royalties to us thereon. This application is a continuation-in-part of application Serial No. 08/472,640, filed June 7, 1995, which is a continuation-in-part of application serial No. 08/422,830, filed April 17, 1995'. The present invention relates to the use of bacterial lipoproteins in inducing humoral immunity in response to polysaccharide antigens and other T cell-independent antigens. The cellular basis for induction of T .cell-independent (TI) humoral immunity to bacterial organisms and their antigenic constituents is largely unknown, thus hampering efforts to develop sufficient defenses against bacterial infection. This poses serious problems, given the prevalence and significance of TI antigens from bacterial organisms such as Haemophilus influenzae type b polyribosyl-ribitolphosphate (PRP), Pneumococcal capsular polysaccharides (including type III), Group B .Streptococcus serocharides, and P. aeruginosa capsular polysaccharides (including strain Fisher type 1). This dearth of knowledge aside, immunologists do know that immunity requires the stimulation of B cell proliferation as well as Ig secretion. In patent WO 95/13089, the inventors previously demonstrated that B cells activated byaS-dex, a construct that mimics the repetitive nature of the CROSS REFERENCE TO RELATED APPLICATION FIELD OF THE INVENTION BACKGROUND 1 5 MAY 2001 WO 96S2963 PCT/US96/05226 type 2 class of T cell-independent antigens, required the presence of the cytokines IL-5, IL-3, GM-CSF, and/or IFN-y to induce strong Ig secretory responses in vitro. The Ig-inducing activity of IL-3, GM-CSF, and IFN-y, but not IL-5, required costimulation with IL-2. The implication of this work is that type 2 T cell-independent antigens may similarly not be able to stimulate Ig responses in the absence of cytokines. The source of the cytokines which are required for immune responses to TI antigens is unknown but may be T cells, NK cells, monocytes, and other cytokine-producing cells. Immunocompromised patients, such as neonates, the elderly, those with HIV disease or patients undergoing chemotherapy, may not have the T cells or functional NK cells or monocytes that produce adequate amounts of cytokines for induction of optimal humoral immunity. Without additional help, these patients may not be able to mount a defense against TI antigens. Moreover, the immune response of immunocompetent normal individuals to polysaccharide or other TI antigens is, in general, of low magnitude and low avidity. This reflects the absence of recruitment of T cell derived help. To date, the most effective way of generating an immune response to polysaccharide antigens has been to conjugate T cell epitopes to the polysaccharides (i.e., conjugate vaccines). These constructs, which stimulate T cell help, also enhance the response to the polysaccharide. While these conjugate vaccines provide benefit, those in the art recognize the many disadvantages associated with their use. Even where individuals are able to mount an immune response, as for.example after vaccination with a large inoculum of antigens, that response may require the coadministration of adjuvants. The most common adjuvants used in man are aluminum compounds (phosphate and hydroxide) , such as alum. Alum, however, does not adjuvant all antigens (for reasons not entirely clear but perhaps due 2 to a charge effect) and both alum and other experimental adjuvants may cause inflammatory responses. Thus, there is a need in the art for methods to induce immunoglobulins either to antigens that by themselves may not be able to induce adequate amounts of T cell help or cytokine-derived help, as for example, polysaccharides, as well as a need in the art to enhance the immune response in individuals that lack the ability to recruit this type of help. There is also a need in the art to enhance the immune responses to those antigens where currently available adjuvants are ineffective. The present invention addresses these needs by providing a method of inducing the immune response to polysaccharides and other TI antigens by the coadministration of either microbial or synthetic lipoproteins. This coadministration includes injection of lipoproteins together with an antigen or a vaccine, or covalently attached to the antigen or the vaccine, as well as injection of the synthetically-derived active moiety of lipoproteins either together with antigen or covalently attached to the antigen. The lipoproteins of the present invention also provide a method of enhancing the immune response. In a preferred embodiment, the lipoprotein of the invention is lipoprotein D. The invention provides the use of a Type 2 T-independent antigen and a microbial lipoprotein for the preparation of a medicament for inducing an immune response to Type 2 T-independent antigens wherein the microbial lipoprotein is selected from the group consisting of lipoprotein D and lipo-OspA, or fragments thereof. The invention also provides the use of a Type 2 T-independent antigen and a microbial lipoprotein for the preparation of a medicament for enhancing an immune response to Type 2 T-independent antigens wherein the microbial lipoprotein is selected from the group consisting of lipoprotein D and lipo-OspA, or fragments thereof. The invention also provides a vaccine comprising a Type 2 T-independent antigen covalently attached to a microbial lipoprotein selected from the group consisting of lipoprotein D and lipo-OspA, or fragments thereof wherein the immune response to the Type 2 T-independent antigen is enhanced. use of a Type 2 T-independent antigen and a synthetic lipoprotein for the preparation of a medicament for jne response to Type 2 T-independent antigens SUMMARY OF THE INVENTION !© (followed by page 3a) 3 The invention further provides the use of a Type 2 T-independent antigen and a synthetic lipoprotein for the preparation of a medicament for inducing an immune response to Type 2 T-independent antigens. The invention further provides the use of a Type 2 T-independent antigen and a synthetic lipoprotein for the preparation of a medicament for enhancing an immune response to Type 2 T-independent antigens. The invention further provides a vaccine comprising a Type 2 T-independent antigen covalently attached to a synthetic lipoprotein wherein the immune response to the Type 2 T-independent antigen is enhanced. BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 is a graph depicting the over 25-fold enhancements in 3H-TdR incorporation observed with combined cc6-dex + lipo-D stimulation, relative to that seen using a8-dex alone. FIG. 2 provides two graphs on the induction of Ig secretion by small resting B cells in the absence (Expt. A) or the presence (Expt. B) of a5-dex, showing that lipo-D alone failed to stimulate significant Ig secretion but that the combined action of lipo-D and a5-dex led to an over 10,000-fold induction in Ig secretion. 'fJfV f * 3a (followed by page 4) FIG. 3 is a series of charts demonstrating that lipo-D costimulates both IgM secretion and' proliferation by a6-dex-activated sort-purified B cells in a manner similar to that observed for the non-sorted B cell-enriched population. FIG. 4 is a chart demonstrating that lipo-D costimulates IgA class switching to a degree similar to that seen with LPS. FIG. 5 is a graph depicting the costimulation of IgM secretion by a6-dex-activated-cells with lipo-OSPA. DETAILED DESCRIPTION OF THE INVENTION. The present invention provides methods of inducing and enhancing the immune response to TI antigens by the coadministration of lipoproteins. As used herein, the immune response is the body's production of immunoglobulins, or antibodies, in response to a foreign entity.• Inducing the immune response refers to establishing an immune response that did not previously exist whereas enhancing an immune response refers to optimizing or increasing a preexisting immune response. As noted above, the foreign entity of interest in the present invention is the thymus cell .(or T cell) -independent antigen or TI antigen. The TI antigen can induce an immune response by activating B cells directly without the apparent participation of T cells. Conversely, the thymus dependent antigen (TD) requires T cell help for antibody synthesis. There are two known classes of TI antigens. Type 1 antigens, such as bacterial lipopolysaccharides, may activate B cells "polyclonally," that is, regardless of the antigen specificity of the B cell. Type 2 TI antigens are characterized by their linear nature and spaced highly repetitive determinants. Such antigens bind to antigenspecific B cells by cross-linking the Ig receptors on RECTIFIED SHEET (RULE 91) WO 96/32963 PCT/US96/05226 the surface of the B cell, a process known as membrane (m)Ig-mediated signaling. Much has been learned about mlg-mediated signaling in response to TI antigens based on a polyclonal in vitro model developed by the inventors. The inventors synthesized dextran-conjugated anti-IgD antibodies (a5-dex) in order to simulate the repeating epitope nature of polysaccharides, ad-Dex cross-links mlg in a multivalent fashion and induces potent and sustained B cell signaling. a5-Dex, which stimulates proliferation by small resting B cells, fails to induce Ig secretion in the absence of exogenous cytokines, and recent studies show that TI antigens similarly cannot induce Ig secretion in the absence of such cytokines. The requirement for cytokines may hamper the treatment of those who need it most: the immunocompromised patients. Such patients, who lack functional T cells, cannot produce cytokines and thus are at risk for infection by clinically relevant TI antigens such as polysaccharides derived from Haemophilus influenzae type b polyribosyl-ribitol-phosphate (PRP) , S. Pneumonia, Group B Streptococcus, N. meningitid.es, Salmonella, P. aeruginosa mucoexopolysaccharides, and P. aeruginosa (including strain Fisher type 1). The coadministration of lipoprotein of this invention, however, results in B cell proliferation and Ig secretion even in the absence of cytokines, as set forth in detail below. Lipoproteins have been previously shown to deliver non-mlg-mediated signals to B cells. Melchers. et a"1... 49 J. Exp. Med. (1975) 142:473. Prior studies on the B cell activating properties of lipoproteins, however, employed heterogenous populations of lymphoid cells in various stages of in vivo-preactivation and cultured at relatively high cell densities which tend to facilitate interactions of B cells with other cell types. Because these cells were not fractionated according to density and, hence, prior activation state, these studies left unresolved whether lipoproteins acted directly at the level of the resting B cell, whether additional cell types WO 96/32963 PCT/US96/05226 played key roles in their action, and how lipoprotein-mediated signaling integrated functionally with other B cell stimuli, including mlg-mediated TI-2-like stimuli also present in bacterial cell walls. In contrast to these other studies, the work that led to this invention used highly-enriched and sort-purified resting murine B cells. With this specific population of B cells, the data indicate that lipoproteins must act in concert with other stimuli to induce strong proliferative and Ig secretory responses. These data are consistent with a recent study using >98% pure, small resting human B cells, in which synthetic lipopeptide significantly enhanced proliferation and Ig secretion only when acting in concert with anti-CD40 antibody. The lipoproteins of the present invention may be either of microbial origin or may be synthetic lipoproteins. The microbial lipoproteins are generally components of bacterial cell walls and include, but are not limited to, the distinct -lipoproteins that have been identified in the cell walls of different bacteria. Erdile. L. , et al. . Inf. and Imm. (1993) 61:81. The lipoproteins of the current invention may also be derived from the genes encoding them, such as lipoprotein-D from Haemophilus influenzae and lipoprotein-OSPA from Borrelia burgdorferi. Id. and see Song et al.. Infect. & Immun. (1995) 63(2):696. The lipoproteins of the present invention also include synthetic lipid moieties, as typified by Pam3Cys, that are structurally similar to the amino terminus of bacterial lipoproteins. Klein. B., et al.. Immunology (1987) 61:29. When these synthetic lipid moieties are conjugated to a small peptide, they can mimic the B cell-activating properties of these molecules. Further, removal of this lipid moiety from bacterial lipoproteins renders them non-functional. The lipoproteins of the claimed invention also include fragments or sections thereof that impart the proliferation and Ig secretion actions observed with lipoprotein D. 6 WO 96/32963 PCT/US96/05226 ™ As set forth in the Examples, in contrast to previous studies, neither lipoprotein-D, lipoprotein-OSPA, nor Pam3Cys by themselves stimulate significant proliferation or Ig secretion. However, in combination with Tl-like, multivalent antigen receptor•cross-linking, these molecules costimulate striking inductions of Ig secretion and marked enhancements in cellular proliferation in the absence of exogenous cytokines. Moreover, these act directly at the level of the B cell without a requirement for recruitment of non-B cell types. These data suggest an additional, novel pathway for induction of specific, T cell-independent humoral immunity in response to bacterial challenge. The lipoproteins of the claimed invention may be coadministered with the antigen in any way familiar to those of ordinary skill, in the art. For example, the lipoproteins may be simply co-injected with the antigen or bound directly ^ to the antigen. Any form of chemical binding, including covalent, is within the scope of this invention. A preferred -method of covalent conjugation is set forth in WO 96/29094 which is related to WO 95/08348 the so-called "CDAP" conjugation method, the disclosures of which are_specifically incorporated herein by reference. The invention also encompasses fusion proteins comprised of lipoproteins and the antigen of interest and/or also injection of the DNA from which these fusion proteins were derived. In addition to an antigen, the lipoproteins of the claimed invention may be administered with a vaccine, such as the dual conjugate vaccines of WO 96/39182 related to US 5,585,100 and the dual conjugate vaccines of WO 93/1 5760, the disclosures of which are (followed by page 8) INTELLECTUAL PROPERTY OFFICE OF N.Z. 7 WO 96/32963 PCT/US96/05226 specifically incorporated herein by reference. As with the antigen, the lipoproteins may be co-administered with the vaccine in any way familiar to those of ordinary skill in the art. As above, the lipoproteins may be simply co-injected with the vaccine or bound directly to the vaccine by, for example, the CDAP method mentioned above, although any form of chemical binding is within the scope of this invention. When used as a vaccine, the lipoproteins of the claimed invention may be administered by any method familiar to those of ordinary skill in the art, but are preferably administered by intravenous, intramuscular, intranasal, oral, and subcutaneous injections. The dosage can be readily determined by those of ordinary skill in the art, but an acceptable range is .01 yig to 100 ]ig per inoculum. Secondary booster immunizations may be given at intervals ranging from one week to many months later. Similar approaches can be used in T cell depleted animals or humans. For use of lipoprotein D as an adjuvant, typical doses may range from 0.1 to 100 jig per inoculum and may be given at the same site as the antigen or vaccine, or at a different site of injection. Additional studies have shown that lipidation of protein D is important in enhancing its effectiveness as a carrier molecule, at least in mice and possibly in other species as well. Thus, protein D is not as effective as lipoprotein D in enhancing anti-polysaccharide responses when injected into mice as a protein-polysaccharide conjugate. The invention will be further clarified by the following examples, which are intended to be purely exemplary of the invention. Example 1 Materials and Methods Mice. Female DBA/2 mice were obtained from the National Cancer Institute (Frederick, MD) and were used at 7-10 weeks of age. The experiments were conducted according to the 8 WO 96/32963 PCT/US96/05226 ^ principles set forth in the Guide for the Care and Use of Laboratory Animals, Institute of Animal Resources, National Research Council, Department of Health, Education, and Welfare Publ No. (National Institutes of Health) 78-23. Culture medium. 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 yg/ml) and streptomycin (50 pg/ml) were used for culturing cells. Reagents. <*8-Dex was prepared by conjugation of H8a/1 (monoclonal mouse IgG2b (b allotype) anti-mouse IgD (a allotype)) to a high molecular weight dextran (2 x 106 M.W.) as previously described in Pecanha. L.. et al.. J. Immunol. (1991) 146:833. Approximately 9 H5a/1 antibodies were conjugated to each dextran molecule. ^Pain3^^]>S- [2,3-Bis(palmitoyloxy)-(2-RS)-propyl]-N-palmitoyl-(R)-cysteine) was obtained from Boehringer Mannheim Biochemica. A stock solution was prepared by dissolving 1 mg of Pam3Cys in 1 ml of 95% ethanol, and stored at -20°C until used. Murine rlFN-y prepared from Chinese hamster ovary cells, was obtained from Genentech (South San Francisco, CA). Murine recombinant IL-1, IL-2, IL-4, and IL-5 were obtained from Dr. Stephanie Vogel (USUHS, 3ethesda, MD), Dr. Maurice Gately (Hoffman-La Roche, Nutley, NJ), Dr. Alan Levine (Searle, St. Louis, MO), and Dr. Richard Hodes (NIH, Bethesda, MD), respectively. Recombinant human TGF-02 was obtained from Wendy Waegell (Celtrix Pharmaceuticals, Santa Clara, CA). Recombinant murine IL-3 and GM-CSF were purchased from Pharmingen. FITC-anti-CD3e mAb (2C11) and FITC-rat IgGl anti-mouse IgA mAb were purchased from Pharmingen (San Diego, CA). PE-labelled affinity-purified polyclonal goat anti-mouse IgM antibody was purchased from Southern Biotechnology Associates (Birmingham, AL). Monoclonal rat IgG2b anti-mouse FcYRII (2.4G2) was purified from ascites. Preparation and culture of B cells. Enriched populations of B cells were obtained from spleen cells from which T cells 9 WO 96fc2963 PCT/US96/05226 were eliminated by treatment with rat anti-Thy-I, anti-CD4, and anti-CD8 monoclonal antibodies, followed by monoclonal mouse anti-rat Igic 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. Unless otherwise indicated these cells were used in the studies reported herein. Highly purified B cells were obtained by electronic cell sorting of membrane (m)IgM+CD3-cells on an EPICS Elite cytometer (Coulter Corp, Hialeah, FL) after staining T-depleted, small, resting spleen cells with FITC-anti-CD3e + PE-anti-IgM antibodies. Sorted cells were immediately reanalyzed and found to be consistently >99% B cells. Functional assays were carried out in either 96- or 24 well flat-bottom Costar plates (Costar, Cambridge, MA) . Cultured-cells were incubated at 1 x 10s cells/ml in a total volume of 200 viL (96-well plate) or 1 ml (24-well plate) at 37°C in a humidified atmosphere containing 6% C02. Measurement-of DNA synthesis. DNA synthesis was determined by 3H-TdR uptake (2uCi/well; 6.7 Ci/nmol; lmCi = 37 GBq; ICN, Irvine, CA) over a 4 hr period. Cells were harvested (PHD cell harvester, Cambridge Technology, Watertown, MA) onto glass fiber filters and [3H]TdR incorporation was determined by liquid scintillation spectrometry. Quantitation of secreted la isotvoe concentrations in culture SN. Ig isotype concentrations were measured by an ELISA assay. For determination of concentrations of secreted IgM, IgG3, (IgGl, IgG2b, IgG2a), and IgA in culture SN, Immulon 2, 96-well flat-bottomed ELISA plates (Dynatech Laboratories, Inc., Alexandria, VA) were coated with unlabelled affinity-purified polyclonal goat anti-mouse IgM, IgG3, IgG, and IgA antibodies (Southern Biotechnology 10 WO 96/32963 PCT/VS96/05226 Associates, Birmingham, AL), respectively. Plates were then washed, blocked with FBS-containing buffer, and incubated with various dilutions of culture SN and standards. After washing, plates were incubated with alkaline phosphatase-conjugated affinity-purified, polyclonal goat anti-mouse IgM, IgG3, IgGl, IgG2b, IgG2a, and IgA antibodies (Southern Biotechnology Associates) as indicated, washed again, and a fluorescent product was generated by cleavage of exogenous 4-methyl umbilliferyl phosphate (Sigma) by the plate-bound alkaline phosphatase-conjugated antibodies. For determination of IgE concentrations, a similar procedure was followed except that plates were coated with monoclonal rat IgG2a anti-mouse IgE (clone EM95) [purified from ascites, obtained from Dr. Fred Finkelman, USUHS, Bethesda, MD], followed by samples and standards, then affinity-purified polyclonal rabbit anti-mouse IgE (obtained from Dr. Ildy Katona, USUHS, Bethesda, MD) , then alkaline phosphatase-conjugated affinity-purified polyclonal goat anti-rabbit IgG (Southern Biotechnology Associates) . Fluorescence was quantitated on a 3M FluoroFAST 96 fluorometer (Mountainview, CA) and fluorescence units were converted to Ig concentrations by interpolation from standard curves that were determined with known concentrations of purified myeloma Ig. Each assay system showed no significant cross-reactivity or interference from other Ig isotypes (IgM, IgD, IgG3, IgGl, IgG2b, IgG2a, IgE, and IgA) found in the culture supernatants. Flow cytometric analysis. Cells were first incubated for 20 min with 5 pg/ml final concentration of rat IgG2b anti-FcyRII mAb (2.4G2) to prevent cytophilic binding of FITC-rat IgGl anti-mouse IgA mAb which was subsequently added at 10 yg/ml final concentration for an additional 3 0 min. All steps were carried out at 4°C. Fluorescence analysis was accomplished on a FACScan (Beeton Dickinson, Mountain View, CA) using logarithmic amplification. Only viable cells, identified on the basis of their characteristic forward and side scatter profiles and their exclusion of propidium iodide (Sigma), were analyzed. 1 1 WO 96/32963 PCT/U S96/05226 Example 2 Lipo-D by itself is an ineffective mitogen for resting B cells but is markedly synergistic with mlg signaling Previous reports indicated that lipoproteins, including lipoprotein-D (lipo-D), by themselves stimulated substantial B cell proliferation and Ig secretion. These studies used heterogeneous populations of lymphoid cells typically cultured at relatively high cell density (1 x 106 cells/ml) . Thus, they left unanswered whether additional cell types and the state of B cell activation were important parameters in mediating these lipoprotein effects. The inventors thus tested the effects of lipo-D on a highly-enriched population of small resting B cells cultured at relatively low cell density (1 x 10s cells/ml). In contrast to the findings of previous reports, resting B cells proliferated weakly or not at all in response to lipo-D, added from 0.02 to 5 yg/ml (Fig. 1). Concentrations of lipo-D up to 40 ug/ml were also ineffective (data not shown). By contrast, B cells proliferated vigorously in response to LPS (data not shown) . The inventors had previously demonstrated that a6-dex is an in vitro model for mlg-dependent TI-2 immunity as mediated by bacterial polysaccharides, and stimulates B cell proliferation, but not Ig secretion, in the absence of additional stimuli. To test the hypothesis that bacteria express constituents that could deliver both antigen-specific and non-specific stimuli directly to B cells for induction of humoral immunity, the inventors determined the effects of combined stimulation by optimal and suboptimal concentrations of a6-dex with varying concentrations of lipo-D for B cell mitogenesis, as measured by 3H-TdR incorporation. Whereas lipo-D by itself was relatively ineffective, it was markedly synergistic with a5-dex for induction of proliferation. Over 25-fold enhancements in 3H-TdR incorporation were observed with combined a5-dex + lipo-D stimulation, relative to that seen using a5-dex alone (Fig. 1). As little as 0.3 ng/ml of a6- 12 WO 96/?2963 PCT/US96/05226 dex, which by itself was. relatively ineffective at stimulating proliferation, strongly costimulated proliferation when combined with 1 ug/ml of lipo-D. By contrast, up to 3 0 jig/ml of unconjugated bivalent anti-IgD antibody failed to costimulate proliferation with lipo-D (data not shown) indicating that multivalent mlg crosslinkage was required for this effect. The inventors had previously reported that unconjugated anti-IgD antibody was nevertheless capable of activating B cells as evidenced by its ability to upregulate MHC class II molecule expression and increase B cell size. Example 3 Combined stimulation with lipo-D and a5-dex leads to a striking induction of Ig secretion in the absence of exogenous cytokines The next experiment determined whether lipo-D induced Ig secretion by small resting B cells in the presence or absence of a8-dex. Lipo-D alone (0.2-5 pg/ml, Fig. 2, Expt A; 5-2 0 ug/ml, Fig. 2, Expt B) failed to stimulate significant Ig secretion. As reported previously, a5-dex by itself was also an ineffective inducer of Ig synthesis by resting B cells. However, the combined action of lipo-D and a6-dex led to an over 10,000-fold induction in Ig secretion in the absence of exogenous cytokines (Fig. 2). 5 ug/ml of lipo-D and 0.3 ng/ml of a5-dex were found to be optimal for costimulation of Ig secretion. Higher and lower concentrations of lipo-D and ad-dex were relatively inhibitory and ineffective, respectively. Example 4 Lipo-D acts directly on the B cell to costimulate proliferation and Ig secretion To determine whether lipo-D acts directly on the resting B cell to costimulate proliferation and Ig secretion in combination with a6-dex, the inventors obtained a highly purified population of resting B cells (>99% mIgM+CD3-) through the method of electronic cell sorting of small T cell- 13 WO 96/32963 PCT/U S96/05226 depleted spleen cells stained with PE-anti-IgM + FITC-anti-CD3:. Any residual large, activated B cells were further eliminated on the basis of their characteristic forward scatter profile. As indicated in Fig. 3, lipo-D costimulated both proliferation and IgM secretion by a5-dex-activated sort-purified B cells in a manner similar to that observed for the non-sorted B cell-enriched population. Thus, lipo-D acts directly at the level of the B cell to mediate these effects. Example 5 Activation of o6-dex-stimulated B cells with lipo-D leads to predominant secretion of IgM with smaller amounts of mostly IgG3 T cell-independent humoral immune responses to bacteria often show a selective proclivity towards the production of IgM and IgG3 . This experiment determined the Ig isotypic . profile of Ig synthesized in response to lipo-D by a5-dex-activated B cells. As indicated in Table 1, lipo-D induced mostly IgM secretion by ad-dex-activated cells. The remainder of the secreted Ig was IgG (<1%), mostly IgG3. Thus, Ig isotype secretion in response to costimulation with lipo-D is similar to that obtained in B cells activated with LPS alone. Table 1 la Secretion (na/ml) IgM IgG3 IgGl IgG2b IgG2a IgE IgA Lipo-D+a5-dex 31,900 255 12 13 <6 <1 <6 Lipo-D-mediated la isotvpe production. B cells were stimulated with 5 pg/ml of lipo-D in combination with 0.3 ng/ml of a6-dex and the concentrations of various Ig isotypes in culture SN were measured 6 days later by ELISA. 1 k WO 96/32963 PCT/U S96/05226 Example 6 Lipo-D by itself is a relatively poor costimulator of cytokine-dependent Ig secretion Ig secretion in response to a6-dex activation requires the concomitant action of cytokines. Thus, IL-4 + IL-5 induce large Ig secretory responses in both a6-dex-activated B cells. The inventors recently defined a second cytokine pathway for eliciting Ig secretory response which operates in a6-dex-activated cells. Thus, IL-3, GM-CSF, and IFN-y each synergize with IL-I + IL-2 for induction of Ig secretion by a5-dex-activated sort-purified B cells. In B cell-enriched, but not sort-purified, cell cultures, IL-I + IL-2 by itself stimulates Ig secretion that is dependent upon secretion that is dependent upon the presence of AsGm-l+ non-B, non-T cells. Thus, to determine the ability of lipo-D to costimulate cytokine-dependent Ig secretion, this experiment involved the addition of either IL-4 + IL-5 or IL-I + IL-2 and/or IL-3, GM-CSF, or IFN-y to lipo-D-stimulated B cell-enriched cultures and the direct comparison of Ig secretion with analogous cultures stimulated with a8-dex. As indicated in Table 2, a5-dex strongly costimulated Ig secretion in response to both cytokine pathways described above. By contrast, lipo-D was a relatively poor costimulator of these Ig secretory responses. Specifically, IL-4 + IL-5 exhibited some Ig inducing activity in lipo-D-stimulated cells (over 8-fold compared to 1,600-fold using a6-dex). In addition the combination of IL-1 + IL-2 + IL-3 also led to an over 4-fold induction in Ig secretion by lipo-D-activated cells compared to an over 3 80-fold induction using a6-dex-activated cells. Thus, lipo-D by itself is a relatively poor costimulator of cytokine-dependent Ig secretion. 1 5 WO 96/32963 3 7US96/05226 Table 2 TBM secretion(na/ml) Medium IL-4+IL-5 IL-l+IL-2 IL-3 GM-CSF IFN-y IL-1,2+IL-3 IL-1,2+GM-CSF IL-1,2+IFN-y Medium <6 565 <6 <6 7 <6 12 <6 7 Lipo-D 420 3,625 410 1,600 600 470 1,950 450 290 a6-dex 72 117,000 5,875 27,500 18,437 16,875 350 435 130 Lipo-D is a relatively poor costimulator of cvtokine-mediated la secretion. B cells were stimulated in the presence or absence of lipo-D (5 pg/ml) or a6-dex (3 ng/ml) with or without the indicated cytokines and IgM concentrations in culture SN were measured 6 days later by ELISA. Cytokines were added at initiation of culture, except IFN-y which was added at 24 hours, at the following concentrations: IL-1 (150 U/ml) , 11-2 (150 U/ml) , IL-3 (100 U/ml) , IL-4 (3,000 U/ml) , IL-5 (150 U/ml), GM-CSF (100 U/ml), IFN-y (10 U/ml). In WO 96/27390 the inventors recently established an in vitro model for induction of high-rate IgA class switching which required the combined action of a6-dex with either LPS or CD40L in the presence of IL-4, IL-5, and the IgA switch factor, TGF-p. In this system all stimuli were required in order to obtain over 10% mlgA* cells, as assessed by flow cytometric analysis. In this experiment, the inventors determined whether lipo-D could replace LPS for costimulation of IgA class switching. In the absence of TGF-p, few if any mIgA+ cells were detected, four days after initiation of culture (Fig. 5) . Removal from culture of LPS led to a drop in %mIgA+ cells to <2% as previously reported (data not shown) . However, replacement of LPS with lipo-D led to a Example 7 Lipo-D provides key-signals for induction of Ig class switching 16 WO 96/32963 PCT/US96/05226 restoration in the IgA class switching response to over 8% mIgA+ cells. Thus, lipo-D provided a key signal for inducing IgA class switching in this system. Example 8 Costimulation of proliferation and Ig secretion by lipo-D in a6-dex-activated B cells appears to be a general property of bacterial lipoproteins A series of analogous experiments were performed using another recombinant lipoprotein, lipo-OSPA from Borrelia burgdorferi, the causative agent of Lyme disease in humans as as well as a synthetic lipoprotein consensus structure, Pam3Cys. As indicated in Figure 5, lipo-OSPA strongly costimulated IgM secretion by a5-dex-activated-cells. Further, Pam3Cys also strongly costimulated mitogenesis and Ig secretion. As with lipo-D, and in contrast to previous studies, neither lipo-OSPA nor Pam3Cys by themselves significantly enhanced either proliferation or Ig secretion by small resting B cells but required coactivation with a5-dex to mediate these affects. Thus, these data suggest that bacterial lipoproteins in general, which deliver non-specific signals to B cells, must act in concert with specific B cell activating signals such as that mediated by mlg cross-linking (multivalent antigen binding) in order to induce a strong, humoral immune response, without a requirement for recruiting ion-B cells. Example 9 Lipoprotein D can enhance the anti-polysaccharide response when given together with polysaccharide-protein conjugates Diphtheria toxoid pneumococcal polysaccharide (DT-Pnl4) was injected at various doses in the, presence or absence of lipoprotein D. The addition of lipoprotein D induced a 5-10 fold greater anti-polysaccharide response than was seen with DT-Pnl4 alone. Furthermore, while 0.01 ug of DT-Pnl4 elicited 17 WO 9032963 PCT/US96/05226 low, if any, detectable response, it induced a significant response when injected together with lipoprotein D. This demonstrates that lipoprotein D can be used as an adjuvant to enhance responses to polysaccharide antigens. Table 3 Enhancement of anti-polysaccharide response by coinjection with lipoprotein D IaG Anti-Pnl4 titer DT-Pnl4 DT-Pnl4 dose (uct) .1 .01 .1 .01 injected lipo D + + dav 28 1,318 20 10,240 498 Groups of 5 DBA/2 mice were injected when DT-Pnl4 in the presence or absence of lipoprotein D. Anti-Pnl4 ELISA were measured 28 days later. Example 10 Lipoprotein D can enhance anti-polysaccharide response in T-cell deficient animals Mice were injected with 500 yg - 1.0 mg of ail anti-CD4 antibody (GK1.5, obtained from ATCC) to induce T cell depletion. One day later, the mice were injected with 5.0 ug of either pneumococcal polysaccharide type 14-lipoprotein D ("PN14-LPD") or PN14 alone. Fourteen days later, IgGl anti-PN14 responses were measured. As set forth below in Table 4, lipoprotein D conjugates stimulated high levels of anti-PN14 response in T cell depleted mice. 1 8 WO 96/32963 PCT/US96/05226 Table 4 Conjugate vaccine with lipoprotein D as a component stimulates anti-polysaccharide response in T cell depleted mice Antigen (Vig/mouse) Anti-CD4 IgGl anti-PN14 dl4 PN14-LPD (5.0) + 5,834 PN14 (5.0) + < 10 Thus, not only does lipoprotein D enhance anti-polysaccharide responses in immunocompetent animals, but it also enhances anti-polysaccharide responses in T cell depleted animals. Accordingly, lipoprotein D may be a valuable tool to enhance anti-polysaccharide responses in T cell deficient individuals, such as those suffering from HIV. To assess the effect of the protein on the anti-polysaccharide response, different vaccines based on Haemophilus influenzae type b polyribosyl-ribitol-phosphate (PRP) were prepared. These "Hib vaccines" included either tetanus toxoid (TT) and lipoprotein D (LPD) as the source of T cell epitopes. A PRP-TT Hib vaccine was prepared using CNBr activation of the polysaccharide and PRP-TT and PRP-LPD Hib vaccines were prepared using CDAP activation of the polysaccharide. Groups of 10 female 5 week old OFA rats were immunized twice subcutaneously 4 weeks apart with 1/4 of a H.D. of the vaccines, and bleedings were taken on day 28, 42, 56, 69 and Anti-PRR'P response evaluated by ELISA (coating with tyraminated-PRR'P). A non-parametric method called "Robust" was used for the comparison of the anti-PS titres induced by different preparations. HIB 001A44 served as reference. Example 11 83. 1 9 - 20 - Table 5a depicts the anti-polysaccharide response. The lipoprotein D conjugate, the PRP-LPD, induced a comparable primary anti-PS response to that of the tetanus toxoid conjugate, PRP-TT but induced a much higher (> 10X) secondary anti-PS response than the tetanus toxoid-PS. As set forth in Table 5b, the PRP-LPD conjugate induced a very low anti-lipoprotein D response. The experiment also suggested that the anti-polysaccharide responses are similar 14 and 28 days after booster. At day 69, (41 days after the booster), the response began to decrease for some conjugates, including the lipoprotein D conjugate. In addition, to assess the effect of combined vaccines, a DTPa.HB vaccine (Diphtheria, tetanus toxoid, acellular pertussis with Hepatitis B) was combined with the conjugates. The combination did not diminish the anti-polysaccharide x response. Table 5 sets forth the anti-polysaccharide response (Table 5a) and the anti-protein response (Table 5b) between vaccines based on two different proteins, tetanus toxoid and lipoprotein D. - 21 - TABLE 5a Anti-PRR'P responses in rats immunized with different Hih vaccines combined or not with a DTP2HB Vaccine fl) Conjugate Anti-PRjRP (nacg/ml) response (2) to Type (batch no.) PS/Prot Conjugate Alone Conjugate -f DTPa HB (batch 16710) D28 D42 D56 D69 Solvent 0.05 0.11 0.15 0.17 X CNTRr Activation PRP-TT (HIB 001A44) 1/3 0.05 25 19 6.3 CDAP Activation PRP-TT (C294) 1/1 0.3 5 4.1 4.7 PRP-TT (C295) 1/2 0.6 18 16 7.S PRP-LPD (001) 1/1 1.0 222 201 54 RECTIFIED SHEET (P.ULE 9T - 22 - TABLE 5B Anti-protein responses in rats after immunization with different Hib vaccines fl) Conjugate Anti-Carrier response on Type (batch no.) PS/Prot D28 D42 D56 ■ D69 Solvent 0.03 0.02 0.01 0.01 (1.SX2) (2-3) (3.S) (3) CNBr Activation P-TT (HIB 001A44) 1/3 0.80 7.3 S.l 5.1 CDAP Activation PRP-TT (C294) 1/1 0.25 1.4 1.6 2.0 PRP-TT (C295) 1/2 0.80 17.2 9.5 8.4 PRP-LPD (001) 1/1 1A M 10.9 14 Anti-TT (values between brackets) titres were determined in sera of saline-injected rats. RECTiFiED SHEET (RULE 9? - 23 - Exampl e 1 2 The Antigenicity and Immunogenicity of Lipoprotein D Constructs In this experiment, we evaluated the effect of the conjugation of lipoprotein D conjugated to Hib PRP and S. pneumoniae PS 14 and 6B. The conjugates were prepared using the CDAP activation and coupling chemistry (different PS/protein ratios) described below. The conjugates were characterized in vitro for their antigenicity using anti-PS and anti-LPD antibodies and the amount of free PS was determined by immunoprecipitation. The immunogenicity of the conjugates was evaluated in a rat model and the protective efficacy of anti-PS antibodies induced in rats was evaluated in infant rats (protection against Hib) or in mice (protection against S. pneumoniae 6B). Materials and Methods The Hib PRP and S. pneumoniae polysaccharide 6B and 14 were extracted and purified from inactivated cell cultures. The purified material met the WHO and US specifications in terms of residual protein, nucleic acid, endotoxin, structural sugars and molecular size distribution. Haemophilus influenzae lipoprotein D was expressed in E. coli and purified using conventional column chromatography. The purity of the proteins was above 90% as assessed by different methods (SDS-PAGE, CE, HPLC). The activated Hib PRP, S. pneumoniae polysaccharide 6B or S. Pneumoniae polysaccharide 14 were conjugated to lipoprotein D or tetanus toxoid. Two methods for conjugating the polysaccharide to the lipoprotein or tetanus toxoid are disclosed herein. Coupling of LipoD to Pnl4 A. Direct conjugation using CDAP Pnl4 is in saline @ 5 mg/ml on ice. CDAP © 100 mg/ml in acetonitrile, 0.2 M TEA, 6 mg/ml lipoprotein D in 10 mM sodium phosphate, 0.2 M NaCl, 0.1% Empigen (a detergent from CalBiochem) pH 7.2, on ice. RECTIFIED SHEET (RULE 91} v - 24 - Activation and coupling are performed at 0-4°C. CDAP is slowly added to a stirred solution of Pnl4 at a ratio of 0.75 mg CDAP/mg Pnl4. At 30 seconds, the pH is raised to 10 with TEA (usually about 2x the volume of CDAP used) and maintained at pH 10 for a total of 2 minutes with TEA. After a total of 2.5 minutes, the lipoprotein D is added to the activated Ps, while mixing, at ratio of 2.5 mg protein/mg Pnl4. The pH should be in the range of 9-9.5. After one hour, the reaction is quenched by the addition of one quarter volume of 1 M glycine @ pH 8. After an overnight incubation at 4°C, the conjugate is purified by passage over an S500HR (Pharmacia) gel filtration column. The high molecular weight conjugate, containing protein and polysaccharide, is pooled and filtered through a 0.2 micron filter. Protein is determined using the Lowery assay, polysaccharide using a resorcinol assay. B. Coupling via a spacer Pnl4 is activated with CDAP as above. At 2.5 minutes, one half volume of 0.5 M adipic dihydrazide at pH 8 is added. After one hour, the solution is exhaustively dialyzed into saline. Hydrazide content is measured using TNBS, polysaccharide using a resorcinol assay. Lipoprotein is coupled to the Pnl4-hydrazide as described v by Lees. et al.. Vaccine, 1994, 12, 1160. In brief, the Pnl4-Hydrazide is iodoacetylated with iodoacetyl N-hydroxysuccinimide (SIA, Sigma). The protein is thiolated with S-Acetylthioacetyl N-hydroxysuccinimide (SATA, Sigma). Following desalting and concentration using a Centricon 30 device, the two are combined and the pH raised to 7.5 using 1/9 volume of 0.75 M HEPES, 0.5 M hydroxylamine. After an overnight reaction at 4°C, the reaction is quenched with mercaptoethanol @ 0.2 mNM for one hour, followed by iodoacetamide @ 10 mM for 10 minutes. The conjugate is purified as described above. Those skilled in the art will (RULE 91; v m • - 25 - recognize that other methods of conjugating protein to polysaccharide may also be practiced. Experimental Results 1. Tmrnmncrenicitv of conjugates in rats Groups of 10 OFA rats (female, 5-6 weeks old) were injected subcutaneously (200 fil) with an amount of conjugate corresponding to 2.5 jxg of PS (for PRP) or ranging from 0.1 to 10 fig of PS (pneumococcal PS 6B or PS 14) . The rats were boosted with the same amount of conjugates one month later and a blood sample was collected immediately before and 15 days after the booster for antibody analysis. The anti-LPD antibodies were measured by ELISA using protein D as coating antigen. Anti-PS antibodies were measured by ELISA using tyraminated PS for coating and, for anti-PS 6B or 14, x absorption of anti-CPS antibodies was performed by addition of CPS to the serum (1 mg/ml for anti-PS 14 and 5 mg/ml for anti-PS 6B) . The detection of specific antibodies was made using an anti-rat conjugate labelled to peroxidase. For all the ELISA's, a reference serum was used and the titers were calculated in arbitrary units using the 4-parameter methods. 2 . Passive protection experiments a) Protection of infant rats against Hib challenge Infant OFA rats (4-5 days) were injected intra-peritoneally (IP) with 100 fil of serum dilutions (in PBS) containing anti-PRP antibodies. Twenty-four hours later, the rats were challenged by the IP route with about 5 x 10* colony forming units (CFU) of log phase Hib, strain Eagan. After 24 hours, the rats were killed and a blood sample was drawn from each rat. The bacteremia was measured by plating serial dilutions of the blood on chocolate agar and counting the RECTIFIED SHEET (RULE 91) - 26 - colonies one day later. Rats with less than 10 CFU/100 /jlI of blood were considered protected. b) Protecting of mice against a lethal challenge bv S. pneumoniae 6B Groups of 10 mice (outbred 0F1, 6 weeks old, female mice) were injected intraperitoneally with 103 CFU of S. pneumoniae bacteria type SB, 24 hours after passive immunization with serum (100 jjlI of 1.5 dilution) containing anti-PS 6B antibodies. The control groups were injected with the serum of nonimmunized animals or animals immunized with PS 14 conjugates. The number of deaths in each group was recorded during the next 18 days. The anti-PS response (anti-PRP, anti-PS6B) is higher (at least 10 times) when LPD is used instead of TT as the protein. Antibodies induced by PRP-LPD conjugates are at l.east equivalent or even better than antibodies induced by PRP-T^-conjugates (at equivalent titer) in a passive protection model in infant rat. Full protection was observed with anti-PS 6B-LPD conjugates in a passive mouse protection test. Conclusion These results demonstrate that LPD is a superior protein as compared to TT for the different capsular PS tested (PRP, -v PS 6B) RECTIFIED SHEET (RULE 91) Tabic 6: Evnlualion of I*Itf® conjugnlcj obtnincri by (lie CIMI' (ccltnology tiiinu TT or LI*I) :u carrier. Con)ug«(ei Anlibodiei nl tl*y 42 (rat) PRP-TP C 294 C 295 PRP-LPD 001 Exp. II PRP-LPD 001 003 004 Nod immunized mis have mean liters of 0.1 I anil 0.0<l for anli-PS in Exp. I anil II respcclivcly. - 28 - Table 7: . Passive protection in infant rats challenged with Hib by serum from rats immunized with LPD-PRP conjugates Serum from rats Protection against bacteremia immunized with with 5 l^g/m 0.2 |ig/ml 1 pg/ml NaCl (control) — - 0/9 TT-PRP 8/9 0/10 0/10 LPD-PRP 003 9/9 4/10 0/8 . x LPD-PRP 004 10/10 4/8 0/10 Groups of 10 OFA rats (5 days old) were injected IP with 100 pi of serum and challenged 24 hrs later IP with 5.104 CFU/rat (Eagan strain). The bacteremia was determined after 24 hrs. Mice with <10 CFU/100 ^1 of blood were considered protected. Controls have 103 - 106 CFU jil of blood. RECTIFIED SHEET (RULE 91) Table 8: Evaluation of PS-6B conjugates obtained by the CDAP technology using TT or LPD as carrier. Conjugates Dose (Y) Antibodies at day 42 (rat) cc-PS (Y/ml) Exp. I PS 6B-TT 1 0.1 97 1 16 — 10 21 2 0.1 140 1 110 30 30 Exp. II PS6B-LPD 001 0.1 2839 1 ' 1671 10 597 002 0.1 2941 1 1831 10 1125 Exp. m PS 68-LPD 002 0.1 2027 1 272 10 2747 RECTIFIED SHEET (RULE 91) L(lhlcJ>: Evnlualion of PS-M cnnju,;nlcs oblpincd l,y ||,c CDAP kchiiutngy usin,. IT or I.PO n.< carrier ConJiijjuIti Eip. I. PS M-TT 3d m § m o CO n: rn m —' K i p. 11 "jg unconj PS M r~ PS M-LI'D m co 001 Eip. Ill PS M-LI'D 002 Aiilibodicj Al dty .(2 (rn 1) a-l'S (y/ml) 2«l M7 373 ]7 71 3911 10 55 161 2-1 103 M'l 57 / MO 231 Table 10: Passive protection in mice challenged with S. pneumonaie type 6B by serum from mice immunized with LPD-PS 6B conjugates Serum from rats immunized with N° of surviving mice at day . io 15 18 NaCl (control) 5/10 4/10 3/10 rat anti-PS 6B 7/10 6/10 6/10 rat anti-PS 6B-LPD 002 .10/10 10/10 10/10 rabbit anti-PS 14-KLH 7/10* . 3/10 3/10 ^several mice are sick 5 weeks old female OFA mice were injected IP with 100 pi of serum diluted 5 fold and challenged 24 hours with 100 pi of Pn 6B strain (6/6B/52) passaged twice in mice. The mortality was recorded up to 18 days after challenge. mil ^ - 32 - F.xamnl p i 7 To assess the effect of lipidation, rats were injected with 0.1 to 30 of a conjugate of lipoprotein D (Lipo D), protein D, or tetanus toxoid conjugated to pneumococcal polysaccharide 6B. Lipo D, Protein D, and pneumococcal polysaccharide 6B were prepared according to the methods as discussed above at pages 6 and 23. Tetanus toxoid is easily obtained from many sources known to those ordinarily skilled in the art. Conjugates were prepared as discussed above at pages 23-24. The protein D conjugates were prepared using the same methodology as that set forth for lipoprotein D conj ugates. Anti-polysaccharide responses were measured by ELISA 28 days after initial injection of the conjugate and 28 days after a booster injection. As set forth in Table 11, anti-polysaccharide responses were significantly higher in mice injected with the Lipo D-conjugate as compared to those injected with the protein D conjugate. Other embodiments of the invention will be apparent to those skilled in the art from consideration of the specification and practice of the invention disclosed herein. It is intended that the specification and examples be considered as exemplary only, with a true scope and spirit of the invention being indicated by the following claims. - 33 - * • Table 11 Immunogenicity of PS-6B Conjugates Obtained by the CDA*5 Technology Using TT, LPD or Protein D as the Protein Carrier Conjugates Dose (|ig) Antibody Titer (day 42) anti-PS ((jg/ml) Exp. I PS 6B-TT 001 0.1 97 1 ~16 10 21 0002 0.1 140 1- 110 30 30 Exp. II x PS 6B-LPD 001 0.1 2839 1 1671 10 597 0002 0.1 2941 1 1831 10 1125 Exp, m PS 6B-LPD 0002 0.1 2027 1 272 10 2747 PS 6B-PD 001 0.1 49 1 5 10 4 RECTIFIED SHEET (RULE 91) </div>

Claims

<div class="application article clearfix printTableText" id="claims"> The use of a Type 2 T-independent antigen covalently attached to a microbial lipoprotein for the preparation of a medicament for inducing an immune response to Type 2 T-independent antigens wherein the microbial lipoprotein is selected from the group consisting of lipoprotein D and lipo-OspA, or fragments thereof. The use of claim 1 wherein the lipoprotein is lipoprotein D. The use of claim 1 wherein the lipoprotein is lipo-OspA. The use of claim 1 wherein the Type 2 T-independent antigen is a polysaccharide derived from a bacteria selected from a group consisting of Haemophilus influenzae type b, S. pneumonia, Group B Streptococcus, N. meningitidis, Salmonella, and P. aeruginosa mucoexopolysaccharides. The use of Type 2 T-independent antigen covalently attached to a microbial lipoprotein for the preparation of a medicament for enhancing an immune response to Type 2 T-independent antigen wherein the microbial lipoprotein is selected from the group consisting of lipoprotein D and lipo-OspA, or fragments thereof. The use of claim 5 wherein the lipoprotein is derived from a microorganism. The use of claim 5 wherein the lipoprotein is lipoprotein D. The use of claim 5 wherein the lipoprotein is lipo-OspA. The use of claim 5 wherein the Type 2 T-independent antigen is a polysaccharide derived from a bacteria selected from a group consisting of Haemophilus influenzae type b, S. pneumonia, Group B Streptococcus, N. meningitidis, Salmonella, and P. aeruginosa mucoexopolysaccharides. A vaccine comprising a Type 2 T-independent antigen covalently attached to a microbial lipoprotein selected from the group consisting of lipoprotein D and lipo-OspA, or fragments thereof wherein the immune response to the Type 2 T-independent antigen is enhanced. 34 INTELLECTUAL PROPERTY OFFICE OF N.Z. 2 0 MAR 2001 RECEIVED 11. 12 13 14 15 16 17 18 19 20 21 22 23 24 The use of claim 10 wherein the Type 2 T-independent antigen is a polysaccharide derived from a bacteria selected from a group consisting of Haemophilus influenzae type b, S. pneumonia, Group B Streptococcus, N. meningitidis, Salmonella, and P. aeruginosa mucoexopolysaccharides. The use of a Type 2 T-independent antigen covalently attached to a synthetic lipoprotein for the preparation of a medicament for inducing an immune response to Type 2 T-independent antigens. The use of Type 2 T-independent antigen covalently attached to a synthetic lipoprotein for the preparation of a medicament for enhancing an immune response to Type 2 T-independent antigens. A vaccine comprising a Type 2 T-independent antigen covalently attached to a synthetic lipoprotein wherein the immune response to the Type 2 T-independent antigen is enhanced. The use of claim 12 wherein the lipoprotein is a lipopeptide. The use of claim 13 wherein the lipoprotein is lipopeptide. The vaccine of claim 14 wherein the lipoprotein is a lipopeptide. The use of claim 15 wherein the lipopeptide comprises a lipidated amino acid. The use of claim 16 wherein the lipopeptide comprises a lipidated amino acid. The vaccine of claim 17 wherein the lipopeptide comprises a lipidated amino acid. The use of claim 18 wherein the lipidated amino acid comprises Parri3Cys. The use of claim 19 wherein the lipidated amino acid comprises Parri3Cys. The vaccine of claim 20 wherein the lipidated amino acid comprises ParmCys. The use of a Type 2 T-independent antigen covalently attached to a microbial 35 INTELLECTUAL PROPERTY OFFICE OF N.Z. 2 0 MAR 2001 RECEIVED lipoprotein as claimed in claim 1 or claim 5 and substantially as herein described with reference to accompanying Examples 1-13. 25.A vaccine comprising a Type 2 T-independent antigen covalently attached to a microbial lipoprotein substantially as herein described with reference to accompanying Examples 10-13. 26.The use of a Type 2 T-independent antigen covalently attached to a synthetic lipoprotein as claimed in claim 12 or claim 13 and substantially as herein described with reference to the accompanying Examples 1-13. 27.A vaccine comprising a Type 2 T-independent antigen covalently attached to a synthetic lipoprotein substantially as herein described with reference to accompanying Examples 10-13. 28.The use of a Type 2 T-independent antigen covalently attached to an adjuvanting lipoprotein for the preparation of a medicament for inducing an immune response to Type 2 T-independent antigens in an immunocompromised individual. 29.The use of a Type 2 T-independent antigen covalently attached to an adjuvanting lipoprotein for the preparation of a medicament for enhancing an immune response to Type 2 T-independent antigens in an immunocompromised individual. 30.The use of claim 28 or 29 wherein the individual is T-cell deficient. 31 .The use of claim 28 or 29 wherein the individual suffers from HIV. 32.The use of any of claims 28-31 wherein the Type 2 T-independent antigen is a polysaccharide derived from a bacteria selected from a group consisting of Haemophilus influenzae type b, S. pneumonia, Group B Streptococcus, N. meningitidis, Salmonella, and P. aeruginosa mucoexopolysaccharides. 33.The use of any of clams 28-32 wherein the lipoprotein is derived from a microorganism. 34.The use of any of claims 28-33 wherein the lipoprotein is lipoprotein D. 35.The use of any of claims 28-33 wherein the lipoprotein is lipo-OspA. 36 36.The use of any of claims 28-31 wherein the lipoprotein is synthetic. 37.The use of any of claims 28-32 wherein the lipoprotein is a lipopeptide. 38.The use of claim 37 wherein the lipopeptide comprises a lipidated amino acid. 39.The use of claim 38 wherein the lipidated amino acid comprises Parri3Cys. 40.The use of any of claims 28-39 wherein the lipoprotein is covalently attached to the Type 2 T-independent antigen. 41 .A vaccine for the treatment of immunocompromised individuals comprising a Type 2 T-independent antigen covalently attached to an adjuvanting lipoprotein. 42.The vaccine of claim 41 wherein the individual to be treated is T-cell deficient. 43.The vaccine of claim 41 wherein the individual suffers from HIV. 44.The vaccine of any of claims 41-43 wherein the Type 2 T-independent antigen is a polysaccharide derived from a bacteria selected from a group consisting of Haemophilus influenzae type b, S. pneumonia, Group B Streptococcus, N. meningitidis, Salmonella, and P. aeruginosa mucoexopolysaccharides. 45.The vaccine of any of claims 41-44 wherein the lipoprotein is derived from a microorganism. 46.The vaccine of any of claims 41-45 wherein the lipoprotein is lipoprotein D. 47.The vaccine of any of claims 41-45 wherein the lipoprotein is lipo-OspA. 48.The vaccine of any of claims 41-44 wherein the lipoprotein is synethetic. 49.The vaccine of claim 48 wherein the lipoprotein is a lipopeptide. 50.The vaccine of claim 49 wherein the lipopeptide comprises a lipidated amino acid. o7 INTELLECTUAL PROPERTY OFFICE OF N.Z. I 1 5 MAY 2001 RE(SEI¥ii 51. The vaccine of claim 50 wherein the lipidated amino acid comprises Parri3Cys. 52. The use of any of claims 1-9, 12-13, 15-16, 18-19 or 28-40 wherein the medicament further comprises at least one cytokine selected from the group consisting of IL1, IL-2, IL-3, IL-4, IL-5 and GM-GSF. 53. The use of claim 53 wherein the cytokine comprises GM-CSF. 54. The use of claim 53 wherein the cytokine is selected from IL-4 and IL-5. 55. The vaccine of any of claims 10-11, 14, 17, 20, 23 and 41-52 wherein the medicament further comprises at least one cytokine selected from the group consisting of IL1, IL-2, IL-3, IL-4, IL-5 and GM-GSF. 56. The vaccine of claim 52 wherein the cytokine comprises GM-CSF. 57. The vaccine of claim 52 wherein the cytokine is selected from IL-4 and IL-5. 58. The vaccine of any of claims 41-51 or 55-57 wherein the lipoprotein is covalently attached to the Type 2 T-independent antigen. 59. The vaccine of any of claims 10-11, 14, 17, 20, 23 or 58 wherein the covalent attachment is via CDAP, 1 -cyano-4-"dimethylamino"-pyridinium tetrafluoroborate, activation of the Type 2 T-independent antigen. 60. The vaccine of claim 59 wherein the covalent attachment is via CDAP, 1-cyano-4-"dimethylamino"-pyridinium tetrafluoroborate, activation of the Type 2 T-independent antigen. 61. The use of claim 40 wherein the covalent attachment is via CDAP, 1-cyano-4-"dimethylamino"-pyridium tetrafluoroborate, activation of the Type 2 T-independent antigen. 38 INTELLECTUAL PROPERTY OFFICE OF N.Z. 2 0 MAR 2001 RECEIVED </div>