AU716806B2 - Induction and enhancement of the immune response to polysaccharides with bacterial lipoproteins - Google Patents

Description

WO 96/32963 PCT/US96/05226 INDUCTION AND ENHANCEMENT OF THE IMMUNE RESPONSE TO POLYSACCHARIDES WITH BACTERIAL LIPOPROTEINS GOVERNMENT INTEREST The invention described herein may be manufactured, licensed and used for governmental purposes without the payment of any royalties to us thereon.

CROSS REFERENCE TO RELATED APPLICATION 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.

FIELD OF THE INVENTION 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.

BACKGROUND

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 polyribosylribitolphosphate (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 related patent application Serial 08/315,492, filed September 30, 1994, the inventors previously demonstrated that B cells activated by a6-dex, a construct that mimics the repetitive nature of the WO 96/32963 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 Iginducing activity of IL-3, GM-CSF, and IFN-y, but not 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 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 WO 96/32963 PCT/US96/05226 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.

SUMMARY OF THE INVENTION 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.

BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 is a graph depicting the over 25-fol' enhancements in 3 H-TdR incorporation observed with combined a6-dex lipo-D stimulation, relative to that seen using a6-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 a6-dex, showing that lipo-D alone failed to stimulate significant Ig secretion but that the combined action of lipo-D and a6-dex led to an over 10,000fold induction in Ig secretion.

4 FIG. 3 is a series of charts demonstrating that lipo-D costimulates both IgM secretion and-proliferation by a6-dexactivated 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 ECTIFIED SHEET (RULE 91) WO 96/32963 PCT/US96/05226 the surface of the B cell, a process known as membrane (m)Igmediated signaling.

Much has been learned about mIg-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 (a6-dex) in order to simulate the repeating epitope nature of polysaccharides. a6- Dex cross-links mIg in a multivalent fashion and induces potent and sustained B cell signaling. a6-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. meningitides, Salmonella, P. aeruginosa mucoexopolysaccharides, and P.

aeruginosa (including strain Fisher type 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 nonmig-mediated signals to B cells. Melchers, et 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 vivopreactivation 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 mig-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 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 cel.l walls of different bacteria. Erdile, 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 Pam 3 Cys, that are structurally similar to the amino terminus of bacterial lipoproteins. Klein. 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.

WO 96/32963 PCT/US96/05226 As set forth in the Examples, in contrast to previous studies, neither lipoprotein-D, lipoprotein-OSPA, nor Pam 3 Cys by themselves stimulate significant proliferation or Ig secretion. However, in combination with TI-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 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-in-part of application Serial No. 07/124,491, filed September 23, 1993, 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 application Serial No.

08/468,060, filed June 6, 1995 (a continuation-in-part of application Serial No. 08/402,565, filed March 13, 1995) and the dual conjugate vaccines of application Serial No.

08/444,727, filed May 19, 1995 (a continuation of 08/055,163, filed February 10, 1993), the disclosures of which are 8 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 coinjected 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 So* can be readily determined by those of ordinary skill in the 15 art, but an acceptable range is .01pg to 100pg per o inoculum. Secondary booster immunizations may be given at intervals ranging from one week to many months later.

:00* Similar approaches can be used in T cell depleted animals or humans.

20 For use of lipoprotein D as an adjuvant, typical doses may range from 0.1 to 100pg 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 30 conjugate.

Throughout the description and claims of this specification, the word "comprise" and variations of the word, such as "comprising" and "comprises", means "including but not limited to", and is not intended to exclude other additives, components, integers or steps.

The invention will be further clarified by the as following examples, which are intended to be purely H:\ARymer\Keep\Speci\VS\54856.96.doc 21/10/99 8aexemplary 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 0**e 0O 9 0 0 000 0 0 0 00 0 00 0 0*00 0 9009 C S 0*0 0 @00S 0* 0O 9 00 S. S

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00 0 S S H:\ARymer\Keep\Speci\VS\54856.96.doc 21/10/99 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 pg/ml) and streptomycin (50 pg/ml) were used for culturing cells.

Reagents. a6-Dex was prepared by conjugation of H6a/l (monoclonal mouse IgG2b (b allotype) anti-mouse IgD (a allotype)) to a high molecular weight dextran (2 x 106 as previously described in Pecanha, et al., J.

Immunol. (1991) 146:833. Approximately 9 H6a/1 antibodies were conjugated to each dextran molecule. Pam 3 Cys 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 Pam 3 Cys in 1 ml of ethanol, and stored at -20 0 C until used. Murine rIFN-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, Bethesda, 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 affinitypurified 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 WO 96/32963 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 Igx and complement. Cells were fractionated on the basis of their density over discontinuous Percoll gradients (Pharmacia, Piscataway, NJ) consisting of 70, 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 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 Tdepleted, 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). Culturedcells were incubated at 1 x 10 s cells/ml in a total volume of 200 pL (96-well plate) or 1 ml (24-well plate) at 37 0 C in a humidified atmosphere containing 6% CO 2 Measurement of DNA synthesis. DNA synthesis was determined by 3 H-TdR uptake (2uCi/well; 6.7 Ci/nmol; ImCi 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.

Ouantitation of secreted Ia isotype concentrations in culture SN. Ig isotype concentrations were measured by an ELISA assay. For determination of concentrations of secreted IgM, IgG3, (IgG1, IgG2b, IgG2a), and IgA in cultre 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 WO 96/32963 PCT/US96/05226 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, IgG1, 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, IgG1, IgG2b, IgG2a, IgE, and IgA) found in the culture supernatants.

Flow cytometric analysis. Cells were first incubated for min with 5 ig/ml final concentration of rat IgG2b anti- FcyRII mAb (2.4G2) to prevent cytophilic binding of FITC-rat IgG1 anti-mouse IgA mAb which was subsequently added at ug/ml final concentration for an additional 30 min. All steps were carried out at 4 0 C. Fluorescence analysis was accomplished on a FACScan (Becton 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.

WO 96/32963 PCTfUS96/05226 Example 2 Lipo-D by itself is an ineffective mitogen for resting B cells but is markedly synergistic with mIg 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 10 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 pg/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 mig-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 'H-TdR incorporation. Whereas lipo-D by itself was relatively ineffective, it was markedly synergistic with a6-dex for induction of proliferation. Over enhancements in 3 H-TdR incorporation were observed with combined a6-dex lipo-D stimulation, relative to that seen using a6-dex alone (Fig. As little as 0.3 ng/ml of a6- WO 96/32963 PCT/US96/05226 dex, which by itself was relatively ineffective at stimulating proliferation, strongly costimulated proliferation when combined with 1 pg/ml of lipo-D. By contrast, up to 30 pg/ml of unconjugated bivalent anti-IgD antibody failed to costimulate proliferation with lipo-D (data not shown) indicating that multivalent mIg 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 a6-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 a6-dex. Lipo-D alone (0.2-5 pg/ml, Fig. 2, Expt A; 5-20 pg/ml, Fig. 2, Expt B) failed to stimulate significant Ig secretion. As reported previously, a6-dex by itself was also an ineffective inducer of Ig synthesis by resting B cells.

However, the combined action of lipo-D and ab-dex led to an over 10,000-fold induction in Ig secretion in the absence of exogenous cytokines (Fig. 5 pg/ml of lipo-D and 0.3 ng/ml of a6-dex were found to be optimal for costimulation of Ig secretion. Higher and lower concentrations of lipo-D and a6dex 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 mIgM+CD3-) through the method of electronic cell sorting of small T cell- WO 96/32963 PCT/US96/05226 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 a8-dex-activated sortpurified 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 Activation of a6-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 a8-dexactivated B cells. As indicated in Table 1, lipo-D induced mostly IgM secretion by a6-dex-activated cells. The remainder of the secreted Ig was IgG 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 Ia Secretion (na/ml) IgM IgG3 IgG1 IgG2b IgG2a IgE IgA Lipo-D+a6-dex 31,900 255 12 13 <6 <1 <6 Lipo-D-mediated Iq isotype 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.

WO 96/32963 PCT/US96/05226 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-dexactivated cells. Thus, IL-3, GM-CSF, and IFN-y each synergize with IL-I IL-2 for induction of Ig secretion by a6-dexactivated 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 a6-dex. As indicated in Table 2, a6dex 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 380-fold induction using a6-dex-activated cells. Thus, lipo-D by itself is a relatively poor costimulator of cytokine-dependent Ig secretion.

WO 96/32963 PCTUS96/05226 Table 2 IBM secretion(nq/ml) Medium Lipo-D a6-dex Medium <6 420 72 565 3,625 117,000 IL-1+IL-2 <6 410 5,875 IL-3 <6 1,600 350 GM-CSF 7 600 435 IFN-y <6 470 130 IL-1,2+IL-3 12 1,950 27,500 IL-1,2+GM-CSF <6 450 18,437 IL-1,2+IFN-y 7 290 16,875 Lipo-D is a relatively poor costimulator of cytokine-mediated Iq 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), (150 U/ml), GM-CSF (100 U/ml), IFN-y (10 U/ml).

Example 7 Lipo-D provides key signals for induction of Ig class switching In related application Serial No. 08/400,322, filed March 8, 1995, 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 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% mIgA' 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. Removal from culture of LPS led to a drop in %mIgA+ cells to as previously reported (data not shown). However, replacement of LPS with lipo-D led to a 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, Pam 3 Cys. As indicated in Figure 5, lipo-OSPA strongly costimulated IgM secretion by a6-dex-activated-cells.

Further, Pam 3 Cys also strongly costimulated mitogenesis and Ig secretion. As with lipo-D, and in contrast to previous studies, neither lipo-OSPA nor PamnCys by themselves significantly enhanced either proliferation or Ig secretion by small resting B cells but required coactivation with a6-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 mIg 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 WO 96 3 2963 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 IqG Anti-Pnl4 titer dose (uq) iniected lipo D day 28 DT-Pn 1 4 .1 1,318 .01 DT-Pnl4 .1 10,240 .01 498 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 Lipoprotein D can enhance anti-polysaccharide response in T-cell deficient animals Mice were injected with 500 pg 1.0 mg of an anti-CD4 antibody (GK1.5, obtained from ATCC) to induce T cell depletion. One day later, the mice were injected with 5.0 pg of either pneumococcal polysaccharide type 1 4 -lipoprotein

D

("PN14-LPD") or PN14 alone. Fourteen days later, IgG1 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.

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 Anti-CD4 IgGl anti-PN14 (pg/mouse) 7 d14 PN14-LPD 5,834 PN14 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.

Example 11 To assess the effect of the protein on the antipolysaccharide 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 83.

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.

19 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 antilipoprotein D response.

The experiment also suggested that the antipolysaccharide 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 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 Anti-PRRP responses in rats im-nunized with different Hib vaccines combined or not with a DTPaH-B Vaccine (1) Conjuglate Anti-PRRP (nacg/rnl) response (2) to Conjugate Alone Conjugate DTPa HB (batch Type (batch no.) PSfProt D28 D4 ID6 Solvent 0.05 0-.11 0.15 0.17 CN\Br Activation PRP-TT (HIB 001A AA) /3 0.05 25 19 6.3 CDAP Activation PR.P-TT (C294) 1/1 0.3 5 4. 4.7 PRP-TT (C295) 1/2 0.6 18 16 7.8 PRP-LPD (001) 1/1 1.0 22 201 54r RECTIFIED SH-EET (R.ULE 91' 22 TABLE Anti-protein responses in rats after immunization with different Hib vaccines (1) Conjugate Anti-Carrier response on Type (batch no.) PS/Prot D28 D42 D56 f D69 Solvent 0.03 0.02 0.01 0.01 (3) CNBr Activation P-TT (HIB 001A44) 1/3 0.80 7.3 8.1 5,1 CDAP Activation PRP-TT (C294) 1/1 0.25 1.4 1.6 PRP-TT (C295) 1/2 0.80 17.2 9.5 8.4 PRP-LPD (001) 1/1 1.6 8.4 10.9 14 Anti-TT (values between injected rats.

brackets) titres were determined in sera of saline- RECTIFiEC SHEET (PULE 91' 23 Pxample 12 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 NaC, 0.1% Empigen (a detergent from .CalBiochem) pH 7.2, on ice.

RECTIFIED SHEET (RULE 91) 24 Activation and coupling are performed at 0-4 0 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 0 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 by TLes. et al., Vaccine, 1994, 12, 1160. In brief, the Pnl4- Hydrazide is iodoacetylated with iodoacetyl Nhydroxysuccinimide (SIA, Sigma). The protein is thiolated with S-Acetylthioacetyl N-hydroxysuccinimide (SATA, Sigma).

Following desalting and concentration using a Centricon 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 0 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 'c,77K! r: c- F ITPL.E 91 25 recognize that other methods of conjugating protein to polysaccharide may also be practiced.

Experimental Results 1. Immunogenicity of conjugates in rats Groups of 10 OFA rats (female, 5-6 weeks old) were injected subcutaneously (200 zl) with an amount of conjugate corresponding to 2.5 gg of PS (for PRP) or ranging from 0.1 to ig 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, 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 exeriments a) Protection of infant rats against Hib challenge Infant OFA rats (4-5 days) were injected intraperitoneally (IP) with 100 il 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 104 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 il of blood were considered protected.

b) Protecting of mice against a lethal challenge by S. pneumoniae 6B Groups of 10 mice (outbred OF1, 6 weeks old, female mice) were injected intraperitoneally with 103 CFU of S. pneumoniae bacteria type 6B, 24 hours after passive immunization with serum (100 il 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 least equivalent or even better than antibodies induced by PRP-TT 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, SPS 6B) .ECTIFIED SHEET (RULE 91) Tnib1lc.: Evalna lion of 11R1P conjugatecs obtained by (the CDI)Al lechitology using~ T'll or LlI') ans cairrier.

PRP-T11 C 294 C 295

PRPI-LPID

001 Exp. 11

P'RP-LPD

001 00) 004 Antibodi at day 42 (rat) a-PS (yin'!) 222 2,18 101 200 Non immunized rats havc nican tilcrs of 0.11 and 0.04I for artli-IPS in ,Ex p. I and 11 respectively.

28 Table 7: Passive protection in infant rats challenged with Hib by serum from rats immunized with LPD-PRP conjugates Serum from rats immunized with Protection against bacteremia with 1pg/m 0.2 g/ml 1 p g/ml S 0/9 NaCl (control)

TT-PRP

LPD-PRP 003 LPD-PRP 004 8/9 9/9 10/10 0/10 4/10 4/8 0/10 0/8 0/10 Groups of 10 OFA rats (5 days old) were injected IP with 100 pl of serum and challenged 24 hrs later IP with 5.10' CFU/rat (Eagan strain).

The bacteremia was determined after 24 hrs.

Mice with <10 CFU/100 pl of blood were considered protected.

Controls have 10 3 106 CFU pl of blood.

RECTIFIED SHEET (RULE 91) -29 Evaluation o-fPS-6B conjugates obtained by the CDAP technology or LPD as carrier.

Table 8: using TT' Conjugates Dose Antibodies at day 42- (rat) aX-PS (.Y/ml) Exp. I PS 6B-TT 1 Exp. II PS6'B-LPD 001 002 Exp. II PS 68-LPD 002 97 16 21 140 110 283 9 167 1 597 2941l 1831 1125 2027 272 2747 PECTIFIED SHEE":T (RULE 91) Lra.&dz.J: Evai alioll of VS. I 1 con~juignics obirilicci by ic CIA P Icchitology usiig VI' or I .ID is c~irrier.

ColiJugiales Atibutlies itt day 11i (rjtl) ri-PS (Y/ii1l) Elp. 1.

PS 14-*T 241 373 37 71 161 24 Il'4 Fip. 11 unconj PS 14 I'S 14-LPOD 001 Exp. III I'S 14-LP'D 002 57 146 231 31 Table Fassive protection in mice challenged with S. pneumcnaie type 6B by serum from mice immunized with LPD-PS 6B conjugates Serum from rats N° of surviving mice at day immunized with 10 15 18 NaCI (control) 5/10 4/10 3/10 rat anti-PS 6B 7/10 6/10 6/10 rat anti-PS 6B-LPD 10/10 10/10 10/10 002 *several mice are sick weeks old female OFA mice were injected IP with 100 pl of serum diluted fold and challenged 24 hours with 100 pl of Pn 6B strain (6/6B/52) passaged twice in mice. The mortality was recorded up to 18 days after challenge.

\M ID 32 To assess the effect of lipidation, rats were injected with 0.1 to 30 4g 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 conjugates.

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, antipolysaccharide 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.

X p -33 Table 11I Immunogenicity of PS-6B Conjugates Obtained by the CDAP Technology Using TT, LPD or Protein D as the Protein Carrier Conjugates Dose (pag) Antibody Titer (day 42).

anti-PS (pg/mi) PS 6B-TT 0002 ExtD. 11 PS 6B-LPD 2839 1671 597 2941 1831 1125 0002 PS 6B-LPD 0002 PS 6B-PD 001 2027 272 2747 RECTIFIED SHEET (RULE 91)

Claims (29)

1. Method for inducing a humoral immune response to a Type 2 T-independent antigen comprising co-administering a Type 2 T-independent antigen and an adjuvanting microbial lipoprotein containing a lipid moiety, wherein the lipoprotein is selected from the group consisting of lipoprotein D and lipo-OspA, or fragments thereof.

2. The method 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 S: 15 mucoexopolysaccharides.

3. Method for enhancing a humoral immune response to Type 2 T-independent antigen comprising co-administering a SType 2 T-independent antigen and an adjuvanting microbial 20 lipoprotein containing a lipoprotein, wherein the lipoprotein is selected from the group consisting of lipoprotein D and lipo-OspA, or fragments thereof.

4. The method of claim 3, wherein the Type 2 T- S 25 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 method of any claims 1-4, wherein the lipoprotein is directly or indirectly conjugated to the Type 2 T-independent antigen.

6. A vaccine comprising a Type 2 T-independent antigen covalently attached to an adjuvanting microbial lipoprotein containing a lipid moiety, wherein the H:\ARymer\Keep\Speci\VS\54856.96.doc 21/10/99 35 lipoprotein is selected from the group consisting of lipoprotein D and lipo-OspA, or fragments thereof, and wherein a humoral immune response to the Type 2 T- independent antigen is induced.

7. The vaccine of claim 6 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.

8. A vaccine comprising a Type 2 T-independent antigen covalently attached to an adjuvanting microbial S 15 lipoprotein containing a lipid moiety, wherein the Soe lipoprotein is selected from the group consisting of lipoprotein D and lipo-OspA, or fragments thereof, and wherein a humoral immune response to the Type 2 T- independent antigen is enhanced.

9. The vaccine of claim 8 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. S 25 meningitidis, Salmonella, and P. aeruginosa mucoexopolysaccharides.

10. Method of inducing a humoral immune response to Type 2 T-independent antigens comprising co-administering a 30 Type 2 T-independent antigen and a synthetic lipoprotein. o.

11. Method of enhancing a humoral immune response to Type 2 T-independent antigens comprising co-administering a Type 2 T-independent antigen and a synthetic lipoprotein.

12. The method of claim 10 or claim 11, wherein the lipoprotein is a lipopeptide. H:\ARymer\Keep\Speci\vs\54856.96.doc 21/10/99 36

13. The method of claim 12, wherein the lipopeptide comprises a lipidated amino acid.

14. The method of claim 13, wherein the lipidated amino acid comprises Pam 3 Cys.

The method of any claim 10-14, wherein the lipoprotein is directly or indirectly conjugated to the Type 2 T-independent antigen.

16. A vaccine comprising a Type 2 T-independent antigen directly or indirectly covalently attached to a synthetic lipoprotein wherein the humoral immune response 15 to the Type 2 T-independent antigen is enhanced.

17. The vaccine of claim 16, wherein the lipoprotein is a lipopeptide. 20

18. The vaccine of claim 17, wherein the lipopeptide comprises a lipidated amino acid. S*

19. The vaccine of claim 18, wherein the lipidated amino acid comprises Pam 3 Cys.

20. The use of a Type 2 T-independent antigen and an adjuvanting microbial lipoprotein for the preparation of a medicament for inducing a humoral immune response to Type 2 T-independent antigens, wherein the adjuvanting microbial 30 lipoprotein contains a lipid moiety and is selected from the group consisting of lipoprotein D and lipo-OspA, or fragments thereof.

21. The use of claim 20, 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. H:\ARymer\Keep\Speci\VS\54856.96.doc 21/10/99 37 meningitidis, Salmonella, and P. aeruginosa mucoexopolysaccharides.

22. The use of a Type 2 T-independent antigen and an adjuvanting microbial lipoprotein for the preparation of a medicament for enhancing a humoral immune response to Type 2 T-independent antigens, wherein the adjuvanting microbial lipoprotein contains a lipid moiety and is selected from the group consisting of lipoprotein D and lipo-OspA, or fragments thereof.

23. The use of claim 22, wherein the Type 2 T- independent antigen is a polysaccharide derived from a :bacteria selected from a group consisting of Haemophilus S 15 influenzae type b, S. pneumonia, Group B Streptococcus, N. Smeningitidis, Salmonella, and P. aeruginosa mucoexopolysaccharides.

24. The use of a Type 2 T-independent antigen and a 20 synthetic lipoprotein for the preparation of a medicament for enhancing the humoral immune response to Type 2 T- independent antigens.

The use of a Type 2 T-independent antigen and a 25 synthetic lipoprotein for the preparation of a medicament for inducing a humoral immune response to Type 2 T- independent antigens.

26. The use of any of claims 20-25, wherein the lipoprotein is a lipopeptide.

27. The use of claim 26, wherein the lipopeptide comprises a lipidated amino acid.

28. The use of claim 27, wherein the lipidated amino acid comprises Pam 3 Cys. H:\ARymer\Keep\Speci\VS\54856.96.doc 22/10/99 38

29. The use of any of claims 20-28, wherein the lipoprotein is directly or indirectly conjugated to the Type 2 T-independent antigen. Dated this 21st day of October 1999 HENRY M. JACKSON FOUNDATION FOR THE ADVANCEMENT OF MILITARY MEDICINE and SMITHKLINE BEECHAM BIOLOGICALS By their Patent Attorneys GRIFFITH HACK Fellows Institute of Patent and Trade Mark Attorneys of Australia. SO W *0 0 H:\ARymer\Keep\Speci\vs\54856.96.doc 21/10/99