EP0824360A1 - Induzierung und steigerung des immunantwortes auf polysacchariden mittels bacterialen lipoproteinen - Google Patents

Induzierung und steigerung des immunantwortes auf polysacchariden mittels bacterialen lipoproteinen

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Publication number
EP0824360A1
EP0824360A1 EP96911776A EP96911776A EP0824360A1 EP 0824360 A1 EP0824360 A1 EP 0824360A1 EP 96911776 A EP96911776 A EP 96911776A EP 96911776 A EP96911776 A EP 96911776A EP 0824360 A1 EP0824360 A1 EP 0824360A1
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EP
European Patent Office
Prior art keywords
lipoprotein
cells
cell
polysaccharide
lipo
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP96911776A
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English (en)
French (fr)
Inventor
Clifford M. Snapper
James J. Mond
Carine Capiau
Pierre Hauser
Jean-Paul Prieels
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
GlaxoSmithKline Biologicals SA
Henry M Jackson Foundation for Advancedment of Military Medicine Inc
Original Assignee
SmithKline Beecham Biologicals SA
Henry M Jackson Foundation for Advancedment of Military Medicine Inc
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Application filed by SmithKline Beecham Biologicals SA, Henry M Jackson Foundation for Advancedment of Military Medicine Inc filed Critical SmithKline Beecham Biologicals SA
Publication of EP0824360A1 publication Critical patent/EP0824360A1/de
Withdrawn legal-status Critical Current

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/02—Bacterial antigens
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C07K14/285—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Pasteurellaceae (F), e.g. Haemophilus influenza
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/385—Haptens or antigens, bound to carriers
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
    • A61K2039/55511—Organic adjuvants
    • A61K2039/55516—Proteins; Peptides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
    • A61K2039/55588—Adjuvants of undefined constitution
    • A61K2039/55594—Adjuvants of undefined constitution from bacteria
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/60—Medicinal preparations containing antigens or antibodies characteristics by the carrier linked to the antigen
    • A61K2039/6031—Proteins
    • A61K2039/6068—Other bacterial proteins, e.g. OMP
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • the present invention relates to the use of bacterial lipoproteins in inducing humoral immunity in response to polysaccharide antigens and other T cell-independent antigens.
  • TI T cell-independent
  • PRP polyribosyl- ribitolphosphate
  • Pneumococcal capsular polysaccharides including type III
  • P. aerugino ⁇ a capsular polysaccharides including strain Fisher type 1 .
  • 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.
  • 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.
  • conjugate vaccines 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.
  • Alum does not adjuvant all antigens (for reasons not entirely clear but perhaps due to a charge effect) and both alum and other experimental adjuvants may cause inflammatory responses.
  • 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.
  • the lipoprotein of the invention is lipoprotein D.
  • FIG. 1 is a graph depicting the over 25-fol ⁇ enhancements in 3 H-TdR incorporation observed with combined ⁇ -dex + lipo-D stimulation, relative to that seen using ⁇ -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 ⁇ -dex, showing that lipo-D alone failed to stimulate significant Ig secretion but that the combined action of lipo-D and ⁇ -dex led to an over 10, 000- fold induction in Ig secretion.
  • FIG. 3 is a series of charts demonstrating that lipo-D costimulates both IgM secretion and proliferation by ⁇ -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 ⁇ -dex-activated-cells with lipo-OSPA.
  • FIG. 6 sets forth the anti-polysaccharide response (Fig. 6a) and the anti-protein response (Fig. 6b) between vaccines based on two different proteins, tetanus toxoid and lipoprotein D.
  • the present invention provides methods of inducing and enhancing the immune response to TI antigens by the coadministration of lipoproteins.
  • 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.
  • the foreign entity of interest in the present invention is the thy us 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.
  • the thymus dependent antigen (TD) requires T cell help for antibody synthesis.
  • Type 1 antigens such as bacterial lipopolysaccharides
  • 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 the surface of the B cell, a process known as membrane (m) Ig- mediated signaling.
  • 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. meningi tides, Salmonella, P. aeruginosa ucoexopolysaccharides, and P. aeruginosa (including strain Fisher type 1) .
  • PRP polyribosyl-ribitol-phosphate
  • S. Pneumonia S. Pneumonia
  • Group B Streptococcus Group B Streptococcus
  • N. meningi tides Salmonella
  • P. aeruginosa ucoexopolysaccharides and P. aeruginosa (including strain Fisher
  • Lipoproteins have been previously shown to deliver non- mlg-mediated signals to B cells. Melchers. et al.. 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.
  • 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 infl uenzae and lipoprotein-OSPA from Borrelia burgdorferi . Id. and see Son ⁇ 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 a ino 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.
  • lipoprotein D 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.
  • these molecules costimulate striking inductions of Ig secretion and marked enhancements in cellular proliferation in the absence of exogenous cytokines.
  • the lipoproteins of the claimed invention may be coadministered with the antigen in any way familiar to those of ordinary skill in the art.
  • 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 compr ' .sed of lipoproteins and the antigen of interest and/or also injection of the DNA from which these fusion proteins were derived.
  • 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 specifically incorporated herein by reference.
  • the lipoproteins may be co-administered with the vaccine in any way familiar to those of ordinary skill in the art.
  • 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.
  • 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 ⁇ g to 100 ⁇ g 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.
  • typical doses may range from 0.1 to 100 ⁇ g per inoculum and may be given at the same site as the antigen or vaccine, or at a different site of injection.
  • protein D is important in enhancing its effectiveness as a carrier molecule, at least in mice and possibly in other species as well.
  • protein D is not as effective as lipoprotein D in enhancing anti-polysaccharide responses when injected into mice as a protein-polysaccharide conjugate.
  • 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 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.
  • RPMI 1640 Biofluids, Rockville, MD
  • 10% fetal bovine serum (Sigma, St. Louis, MO)
  • L-glutamine (2 mM)
  • 2-mercaptoethanol 0.05 mM
  • penicillin 50 ⁇ g/ml
  • streptomycin 50 ⁇ g/ml
  • ⁇ -Dex was prepared by conjugation of H ⁇ a /1 (monoclonal mouse IgG2b (b allotype) anti-mouse IgD (a allotype) ) to a high molecular weight dextran (2 x 10 6 M.W.) as previously described in Pecanha, L. , et al. , J. Immunol. (1991) 146:833. Approximately 9 H ⁇ a /1 antibodies were conjugated to each dextran molecule.
  • Pam 3 Cys (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 Pam 3 Cys in 1 ml of 95% ethanol, and stored at -20°C until used.
  • Murine rIFN- ⁇ 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.
  • PE-labelled affinity- purified polyclonal goat anti-mouse IgM antibody was purchased from Southern Biotechnology Associates (Birmingham, AL) .
  • Monoclonal rat IgG2b anti-mouse Fc ⁇ RII (2.4G2) was purified from ascites.
  • B cells Preparation and culture of B cells. Enriched populations of B cells were obtained from spleen cells from which T cells were eliminated by treatment with rat anti-Thy-I, anti-CD4, and anti-CD8 monoclonal antibodies, followed by monoclonal mouse anti-rat Ig ⁇ 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.
  • B 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 10 5 cells/ml in a total volume of 200 ⁇ L (96-well plate) or 1 ml (24-well plate) at 37°C in a humidified atmosphere containing 6% C0 2 .
  • Cells were harvested (PHD cell harvester, Cambridge Technology, Watertown, MA) onto glass fiber filters and [ 3 H]TdR incorporation was determined by liquid scintillation spectrometry.
  • Ig isotype concentrations were measured by an ELISA assay.
  • concentrations of secreted IgM, IgG3, (IgGl, IgG2b, IgG2a) , and IgA in culfre 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 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.
  • IgE concentrations 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) .
  • monoclonal rat IgG2a anti-mouse IgE clone EM95
  • affinity-purified polyclonal rabbit anti-mouse IgE obtained from Dr. Ildy Katona, USUHS, Bethesda, MD
  • 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.
  • lipoproteins including lipoprotein-D (lipo-D)
  • lipoprotein-D lipoprotein-D
  • studies used heterogeneous populations of lymphoid cells typically cultured at relatively high cell density (1 x 10 6 cells/ml) .
  • 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 5 cells/ml) .
  • ⁇ -dex is an in vi tro 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.
  • 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 subopti al concentrations of ⁇ -dex with varying concentrations of lipo-D for B cell mitogenesis, as measured by 3 H-TdR incorporation. Whereas lipo-D by itself was relatively ineffective, it was markedly synergistic with ⁇ -dex for induction of proliferation.
  • Lipo-D acts directly on the B cell to costimulate proliferation and Ig secretion
  • lipo-D acts directly on the resting B cell to costimulate proliferation and Ig secretion in combination with ⁇ -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- 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.
  • lipo-D costimulated both proliferation and IgM secretion by ⁇ -dex-activated sort- purified B cells in a manner similar to that observed for the non-sorted B cell-enriched population.
  • lipo-D acts directly at the level of the B cell to mediate these effects.
  • IgM and 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 ⁇ -dex- activated B cells. As indicated in Table 1, lipo-D induced mostly IgM secretion by ⁇ -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.
  • B cells were stimulated with 5 ⁇ g/ml of lipo-D in combination with 0.3 ng/ml of ⁇ -dex and the concentrations of various Ig isotypes in culture SN were measured 6 days later by ELISA.
  • Lipo-D by itself is a relatively poor costimulator of cytokine-dependent Ig secretion
  • Ig secretion in response to ⁇ -dex activation requires the concomitant action of cytokines.
  • IL-4 + IL-5 induce large Ig secretory responses in both ⁇ -dex-activated B cells.
  • the inventors recently defined a second cytokine pathway for eliciting Ig secretory response which operates in ⁇ -dex- activated cells.
  • IL-3, GM-CSF, and IFN- ⁇ each synergize with IL-I + IL-2 for induction of Ig secretion by ⁇ -dex- activated sort-purified B cells.
  • IL-I + IL-2 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.
  • this experiment involved the addition of either IL-4 + IL-5 or IL-I + IL-2 and/or IL-3, GM- CSF, or IFN- ⁇ to lipo-D-stimulated B cell-enriched cultures and the direct comparison of Ig secretion with analogous cultures stimulated with ⁇ -dex.
  • Lipo-D is a relatively poor costimulator of cytokine-mediated I ⁇ secretion.
  • B cells were stimulated in the presence or absence of lipo-D (5 ⁇ g/ml) or ⁇ -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- ⁇ 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- ⁇ (10 U/ml).
  • Lipo-D provides key signals for induction of Ig class switching
  • lipo-OSPA lipo-OSPA from Borrelia burgdorferi
  • the causative agent of Lyme disease in humans as well as a synthetic lipoprotein consensus structure, Pam 3 Cys.
  • lipo-OSPA strongly costimulated IgM secretion by ⁇ -dex-activated-cells.
  • Pam 3 Cys also strongly costimulated mitogenesis and Ig secretion.
  • lipo-D and in contrast to previous studies, neither lipo-OSPA nor Pam 3 Cys by themselves significantly enhanced either proliferation or Ig secretion by small resting B cells but required coactivation with ⁇ -dex to mediate these affects.
  • Lipoprotein D can enhance the anti-polysaccharide response when given together with polysaccharide-protein conjugates
  • DT-Pnl4 Diphtheria toxoid pneumococcal polysaccharide 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.
  • 0.01 ⁇ g of DT-Pnl4 elicited 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.
  • mice 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.
  • Lipoprotein D can enhance anti-polysaccharide response in T-cell deficient animals
  • mice were injected with 500 ⁇ g - 1.0 mg of an anti-CD4 antibody (GK1.5, obtained from ATCC) to induce T cell depletion.
  • GK1.5 an anti-CD4 antibody
  • mice were injected with 5.0 ⁇ g of either pneumococcal polysaccharide type 14-lipoprotein D ("PN14-LPD") or PN14 alone.
  • IgGl anti- PN14 responses were measured.
  • lipoprotein D conjugates stimulated high levels of anti-PN14 response in T cell depleted mice.
  • Conjugate vaccine with lipoprotein D as a component stimulates anti-polysaccharide response in T cell depleted mice
  • 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.
  • Hib vaccines included either tetanus toxoid (TT) and lipoprotein D (LPD) as the source of T cell epitopes.
  • TT tetanus toxoid
  • LPD lipoprotein D
  • 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.
  • Figure 6a 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.
  • the PRP-LPD conjugate induced a very low anti- lipoprotein D response.
  • 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.
  • the conjugates were prepared using the CDAP activation and coupling chemistry (different PS/protein ratios) described below.
  • the conjugates were characterized in vi tro 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) .
  • 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.
  • 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 g CDAP/mg Pnl4.
  • 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.
  • 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.
  • 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.
  • 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 Lees, et al.. Vaccine, 1994, 12, 1160.
  • 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.
  • Groups of 10 OFA rats (female, 5-6 weeks old) were injected subcutaneously (200 ⁇ l) with an amount of conjugate corresponding to 2.5 ⁇ g of PS (for PRP) or ranging from 0.1 to 10 ⁇ g 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.
  • a reference serum was used and the titers were calculated in arbitrary units using the 4-parameter methods.
  • mice (outbred OF1, 6 weeks old, female mice) were injected intraperitonealiy with 10 3 CFU of S. pneumoniae bacteria type 6B, 24 hours after passive immunization with serum (100 ⁇ l 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.
  • anti-PS response 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.
  • Table 0 Evaluation of PRP conjugates obtained by the CDAP technology using TT or LPD as carrier.
  • Non immunized rats have mean liters of 0. M and 0 04 for anti-PS in Bxp. I and II respectively.
  • the bacteremia was determined after 24 hrs.
  • mice with ⁇ 10 CFU/100 ⁇ l of blood were considered protected.
  • Controls have 10 3 - 10° CFU ⁇ l of blood.
  • Table 8 Evaluation of PS-6B conjugates obtained by the CDAP technology using TT or LPD as carrier.
  • mice 5 weeks old female OFA mice were injected IP with 100 ⁇ l of serum diluted 5 fold and challenged 24 hours with 100 ⁇ l of Pn 6B strain (6/6B/52) passaged twice in mice. The mortality was recorded up to 18 days after challenge.
  • lipidation rats were injected with 0.1 to 30 ⁇ g 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 20-21. Tetanus toxoid is easily obtained from many sources known to those ordinarily skilled in the art.
  • Conjugates were prepared as discussed above at pages 20-22.
  • 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, anti- polysaccharide responses were significantly higher in mice injected with the Lipo D-conjugate as compared to those injected with the protein D conjugate.
EP96911776A 1995-04-17 1996-04-16 Induzierung und steigerung des immunantwortes auf polysacchariden mittels bacterialen lipoproteinen Withdrawn EP0824360A1 (de)

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US568342 1995-12-06
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AU2001286405B2 (en) * 2000-07-31 2007-05-10 Yale University Innate immune system-directed vaccines
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SE466259B (sv) * 1990-05-31 1992-01-20 Arne Forsgren Protein d - ett igd-bindande protein fraan haemophilus influenzae, samt anvaendning av detta foer analys, vacciner och uppreningsaendamaal
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ATE254475T1 (de) * 1993-09-22 2003-12-15 Jackson H M Found Military Med Verfahren zur aktivierung von löslichem kohlenhydraten durch verwendung von neuen cyanylierungsreagenzien, zur herstellung von immunogenischen konstrukten

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