JPH11508225A - Induction and enhancement of immune responses to polysaccharides using bacterial lipoproteins - Google Patents

Abstract

(57) Abstract: The present invention provides an immune response to these antigens by administering polysaccharides or other T cell-independent antigens together with bacterial lipoproteins (including covalent binding of antigens and lipoproteins). Methods for inducing or enhancing are provided. In a preferred embodiment, the lipoprotein of the invention is lipoprotein D.

Description

DETAILED DESCRIPTION OF THE INVENTION      Induction and enhancement of immune responses to polysaccharides using bacterial lipoproteins                                Government rights   The invention described herein does not require any royalty payment to the inventors. Manufactured, licensed, and used for government purposes.                            Cross-reference of related applications   This application is a continuation-in-part of Patent Application No. 08 / 422,830, filed April 17, 1995. The request was a continuation-in-part application of Patent Application No. 08 / 472,640 filed on June 7, 1995. is there.                                Field of the invention   The present invention provides humoral immunity against polysaccharide antigens and other T-cell independent antigens. The use of bacterial lipoproteins in inducing a response.                                Background of the Invention   T cell-independent (TI) humoral immunity in response to bacteria and their antigenic components Little is known about the cytological basis for induction, and therefore Efforts to develop adequate defense against infection are hampered. Haemophilus influenzae (Haemo philus influenzae) b-type polyribosyl ribitol phosphate (PRP), pneumococcus (Pneum ococcus), capsular polysaccharides (including type III), group B streptococcus (Streptococcus) cellophora Serocharides, and capsular polysaccharides of P. aeruginosa (Fisher) (Including Fisher type 1 strains). With consideration, this is a serious problem.   Despite the lack of knowledge, immunologists suggest that immune responses include B cell proliferation and I I understand that stimulation by secretion of g is necessary. Filed on September 30, 1994 In related patent application Ser.No. 08 / 315,492, we have previously described T cell-independent Activated by αδ-dex, a construct that mimics the repeatability of the type 2 class of antigenic antigens To induce a strong Ig secretion response in vitro Demonstrates the need for the presence of Cain IL-5, IL-3, GM-CSF, and / or IFN-γ Revealed. The Ig-inducing activities of IL-3, GM-CSF, and IFN-γ, excluding IL-5, were Intense was needed. This study shows that type 2 T cell-independent antigens Cain May not be able to stimulate an Ig response in the absence of You.   The source of cytokines required for an immune response to TI antigens is unknown, Probably T cells, NK cells, monocytes, and other cytokine-producing cells it is conceivable that. Exemptions for newborns, the elderly, patients with HIV disease or chemotherapy Patients with immunocompromised can induce optimal humoral immunity T cells or functional NK cells or monocytes that produce the appropriate amount of cytokines for May not have. Without external help, these patients would have You may not be able to defend against it.   Furthermore, immunization of normal immunocompetent individuals against polysaccharides or other TI antigens Responses are generally small and have low avidity. It can be helped by T cells Reflects that there is no. To date, an immune response has been raised against polysaccharide antigens. The most effective way to rub is to conjugate the T cell epitope to the polysaccharide (i.e. Conjugate vaccine). These constructs that stimulate T cell support It also enhances the response to polysaccharides. These conjugate vaccines offer benefits On the other hand, those skilled in the art are also aware of the many disadvantages arising in their use.   When an individual can initiate an immune response, such as after vaccination with a large dose of antigen Even so, the response may require co-administration of an adjuvant. Hi The most common adjuvants used in aluminum are aluminum such as alum Compounds (phosphates and hydroxides). However, alum is all It is not an original adjuvant (the reason is not completely understood, Alum and other experimental adjuvants May cause an inflammatory response.   Thus, alone, a suitable amount of T-cell or cytokine-derived Immunoglobulin against antigens that may not be able to induce Not only is there a need in the art for a method of guiding There is also a need in the art for ways to enhance the immune response in unprotected individuals It is. If the currently available adjuvants are ineffective, There is also a need in the art for methods to enhance the resulting immune response.                                 Summary of the Invention   The present invention provides for co-administration of either microbial lipoproteins or synthetic lipoproteins. For inducing an immune response against polysaccharides and other TI antigens Address these needs by providing This co-administration Injecting lipoproteins with an antigen or vaccine, or Can be injected covalently linked to a vaccine and synthetically made lipoproteins Injection with the antigen or covalently linked to the antigen. I will. Methods for enhancing an immune response with the lipoproteins of the present invention are also provided. In a preferred embodiment, the lipoprotein of the invention is lipoprotein D.                              BRIEF DESCRIPTION OF THE FIGURES   Fig. 1 shows the results obtained when the combination of αδ-dex and lipo D was stimulated and αδ-dex alone. Compared to,^ThreeIt is a graph which shows that the uptake of H-TdR was enhanced 25 times or more.   FIG. 2 shows a small pause B in the absence (Experiment A) or presence (Experiment B) of αδ-dex. 2 is two graphs showing induction of Ig secretion by cells, in which only lipo D has significant Ig. Secretion was not stimulated, but the effect of the combination of lipo D and αδ-dex Indicates that the induction of Ig secretion was 10,000-fold or more.   FIG. 3 shows that lipoD was expressed in αδ-d as well as in the unfractionated B cell-rich population. Co-stimulates both IgM secretion and proliferation by ex-activated and fractionated B cells. FIG.   Figure 4 shows that lipoD co-stimulates IgA class to the same extent as with LPS. FIG.   FIG. 5 shows costimulation of IgM secretion by αδ-dex-activated cells with lipo-OSPA. FIG.                              Detailed description of the invention   The present invention provides a method for inducing and enhancing an immune response against Provide a way to strengthen. As used herein, an immune response is a response to a foreign substance. In vivo production of immunoglobulins or antibodies. Induction of an immune response Is to establish an immune response that was not present in the Optimizing or increasing the immune response that has existed before.   As described above, the foreign substance of interest in the present invention includes thymocytes (or T cells). ) Independent or TI antigen. TI antigens, even if T cells are not explicitly involved, An immune response can be induced by directly activating B cells. Conversely, Thymus-dependent antigen (TD) requires the help of T cells for antibody synthesis.   It is known that there are two classes of TI antigens. Such as bacterial lipopolysaccharides Type 1 antigens make B cells "polyclonal", that is, the antigen specificity of B cells Independent of activation. Type 2 TI antigens have a linear nature and And highly repetitive determinants that exist at regular intervals. This type of Antigens crosslink Ig receptors to the surface of B cells as membrane (m) Ig-mediated signaling It binds to antigen-specific B cells by a known process.   Based on the polyclonal in vitro model developed by the present inventors, Much is known about mIg-mediated signaling in responding responses . In order to stimulate the repetitive epitope properties of polysaccharides, the present inventors An anti-IgD antibody (αδ-dex) conjugated to kisstran was synthesized. αδ-dex is as high as mIg Crosslinks valency and induces strong and continuous B cell signaling. Small resting B cells Αδ-dex, which stimulates the proliferation of cells, secretes Ig in the absence of foreign cytokines. Cannot be induced, and recent studies indicate that TI antigens It has been shown that Ig secretion cannot be induced in the presence.   The need for cytokines depends on the people who need it most, May interfere with the treatment of certain patients. These patients who lack functional T cells Unable to produce Cain, which caused Haemophilus i nfluenzae) b-type polyribosyl ribitol phosphate (PRP), S. pneumoniae (S. Pneumo nia), group B Streptococcus, meningococcus (N. meningitides), Salmonella (Salmonella), mucoexopolysaccharide of P. aeruginosa, and P. aeruginosa (P. a eruginosa) (including one Fisher type strain) polysaccharides, Risk of infection with clinically relevant TI antigens. However, according to the present invention, Co-administration of the poprotein allows for the absence of cytokines, as detailed below. Even so, it results in B cell proliferation and Ig secretion.   Lipoproteins have previously been shown to transmit non-mIg-mediated signals to B cells. ing. Melchers et al., 49J. Exp. Med. (1975) 142: 473. However However, previous studies on the properties of lipoproteins that activate B cells Of different stages of lymphocytes pre-activated Quality cell populations, B cells interact with other cell types There was a tendency. These cells are fractionated by density prior to activation. Lipoproteins were not directly expressed at resting B cell levels in these studies. Whether they interact with each other and whether another cell type plays a significant role in their action , And lipoprotein-mediated signaling is also present in bacterial cell walls With other B cell stimuli, including TI-2-like stimuli via The question of whether they have been merged remains unclear.   In contrast to these other studies, studies that lead to the present invention are highly enriched and fractionated. Fractionated and purified murine resting B cells were used. Data using this specific population of B cells Lipoproteins may induce other growth factors to induce a strong proliferation and Ig secretion response. It has been shown to work in concert with the fierce. This data shows that synthetic lipopeptides Significantly increases proliferation and Ig secretion only when acting in concert with anti-CD40 antibodies In a recent experiment using small resting human B cells with a purity of 98% or more. This is consistent with the results obtained.   The lipoprotein of the present invention may be either a microorganism-derived lipoprotein or a synthetic lipoprotein. Good. Microbial lipoproteins are generally components of the cell wall of bacteria, Includes, but is not limited to, different lipoproteins identified in the fungal cell wall No. Erdile, L. et al., Inf. and Imm. (1993) 61:81. In addition, the present invention Lipoprotein is lipoprotein D from Haemophilus influenzae And lipoprotein-O from Borrelia burgdorferi They may be derived from genes encoding them, such as SPA. Id. And song (Song) et al., Infect. & Immun. (1995) 63 (2): 696.   The lipoproteins of the present invention have structural similarities to the amino terminus of bacterial lipoproteins. Pam_ThreeSynthetic lipid moieties such as Cys are also included. Klein, B. Et al., Immunology (1987) 61:29. Complex these synthetic lipid moieties into small peptides Can mimic the properties of these molecules to activate B cells. Can You. Furthermore, removing this lipid moiety from bacterial lipoproteins results in loss of function. I will.   The lipoproteins claimed in the present invention include the growth and I observed in lipoprotein D. Also included are those fragments or portions thereof that confer g-secretion ability.   As shown in the examples, in contrast to previous studies, lipoprotein D, lipoprotein Quality-OSPA, or Pam_ThreeSignificant growth or Ig secretion alone in any of the Cys Do not stimulate. However, in combination with TI-like, multivalently crosslinked antigen receptors These molecules strongly induce Ig secretion in the absence of foreign cytokines. And significantly enhances cell proliferation. In addition, they require a non-B cell type And acts directly at the B cell level. From these data, Another novel, induction of specific T cell-independent humoral immunity in response to bacterial invasion It is suggested that there is a route.   The lipoproteins claimed in the present invention can be prepared by any method familiar to those skilled in the art. Therefore, it can be co-administered with the antigen. For example, lipoproteins simply co- Or may be injected directly linked to the antigen. Sharing Any form of chemical bond, including union, is included within the scope of the present invention. Preference for covalent bonds The new method is a continuation-in-part of Patent Application No. 07 / 124,491, filed September 23, 1993. Request for a continuation-in-part of Patent Application No. 18 / 408,717 filed March 22, 1995 No. 08 / 482,661, filed June 7, 1995. A so-called “CDAP” conjugation method, the disclosure of which is specifically incorporated herein by reference. Be included. The present invention also relates to a fusion protein comprising a lipoprotein and an antigen of interest and And / or injection of DNA encoding the fusion proteins.   In addition to the antigen, the claimed lipoprotein of the present invention was filed on June 6, 1995. Patent Application No. 08 / 468,060 (Patent Application No. 08 / 402,565 filed March 13, 1995) Conjugated vaccine) and May 1995 Patent Application No. 08 / 444,727 filed on Jan. 19, 1993 (patent application filed on Feb. 10, 1993) Application No. 08 / 055,163) (dual conjugate vaccine) ne) can be administered together with a vaccine, the disclosure of which is specifically described herein. Incorporated as a reference. Like antigens, lipoproteins are familiar to those skilled in the art. To It can be administered with the vaccine in any number of ways. As mentioned above, lipoproteins May simply be injected with the vaccine or any form of chemical bonding But directly bound to the vaccine by, for example, the CDAP method described above. May be.   When used as a vaccine, the lipoproteins claimed in the present invention will be familiar to those skilled in the art. It can be administered by any known method, but is preferably intravascular, intramuscular, It is administered by intranasal, oral and subcutaneous injection. The dosage is easily determined by one skilled in the art. The acceptable range is 0.01 μg to 100 μg per inoculation. Secondary addition Antigen stimulation may be given at intervals ranging from one week to several months. T cell depletion Similar approaches can be used for products or humans.   To use lipoprotein D as an adjuvant, a typical dose is 0.1 to 100 μg / inoculation, at the same site or Administered to a different site than the firing.   Further studies indicate that lipidation of protein D may not be effective as a carrier molecule. Has been shown to be important in enhancing the fruit But it could be the same. Therefore, as a protein-polysaccharide complex, D is as effective as lipoprotein D in enhancing anti-polysaccharide response when injected Not a target.   The following examples further illustrate the invention, but do not limit the scope of the invention. It is intended as an illustration of the invention.                                  Example 1                               Materials and methods   mouse  DBA / 2 female mice were transferred to the National Cancer Institute (Fre derick, MD) and used at 7-10 weeks of age. Experiments are described in “Care and Use of Laboratory Animals” Guide for the Care and Use of Laboratory Animals, Institute of Animal Resources, National Research Council, Department of Health, Ed ucation, and Welfare Publ No. (National Institutes of Health) 78- Performed according to the principles that have been done.   Culture medium  10% fetal bovine serum (Sigma, St. Louis, MO), L-glutamine (2 mM), 2-mercaptoethanol (0.05 mM), penicillin (50 μg / ml) and strep RPMI 1640 (Biofluids, Roc) supplemented with tomycin (50 μg / ml) The cells were cultured using kville, MD).   reagent  αδ-dex is described in “Pecanha, L. et al., J. Immunol. (1991) 146: 83. Hδa / 1 (mouse IgG2b (b allotype) anti-mouse IgD (a Allotype) monoclonal antibody) with high molecular weight dextran (2 x 10⁶M.W.) Prepared. Pam_ThreeCys (S- [2,3-bis (palmityroxy)-(2-RS) -p Ropyl] -N-palmitoyl- (R) -cysteine) is Boehringer Mannheim Biochem. Mica (Boehringer Mnnheim Biochemica). Stock solution is 1mg Pam_ThreeCys Was prepared in 1 ml of 95% ethanol and stored at -20 ° C until use. Cha Mus musculus rIFN-γ prepared from Ines hamster egg cells was (Genentech) (South San Francisco, CA). Mus musculus recombinant IL-1 , IL-2, IL-4, and IL-5 are available from Dr. Stephanie Vogel (U. SUHS, Bethesda, MD), Dr. Maurice Gately (Hoffman-La Roche, Nutley, NJ), Dr. Allan Levine (Searle, St. Louis, MO) ), And Richard Hodes (NIH, Bethesda, MD). I got each. Recombinant human TGF-β2 is available from Wendy Wargell ) (Celtrix Pharmaceuticals, Santa Clara, CA). Recombinant house mouse MI IL-3 and GM-CSF were purchased from Pharmingenn. FITC-anti-C D3e mAB (2C11) and FITC-rat IgG1 anti-mouse IgA mAb were purchased from Pharmingen (Ph armingen) (San Diego, CA). Affinity purified goat labeled with PE Anti-mouse IgM polyclonal antibody is available from Southern Biotechnology Associates (South hern Biotechnology Associates) (Birmingham, AL). Rat IgG2b The anti-mouse FcRII monoclonal antibody (2.4G2) was purified from ascites.   B Cell preparation and culture  B cell-rich cell populations include anti-Thy-I, anti-CD4, and After treatment with anti-CD8 monoclonal antibody, mouse anti-rat Igk monoclonal antibody and And spleen cells from which T cells had been removed by treatment with complement. 70, 65 cells , 60 and 50% Percoll solutions (specific gravity 1.086, 1.081, 1.074, and 1.062 g / ml) (Pharmacia, Pisc) at away, NJ). 70% of cells with high specific gravity (small, resting) When collected from the 65% interface, up to 90% were B cells. Unless otherwise indicated Thus, these cells were used in the studies reported herein. Highly purified B Cells were stained with TTC-free, small resting spleen cells with FITC-anti-CD3e + PE-anti-IgM antibody. After coloring, the EPICS Elite cytometer (Coulter Corp. Hialeah , FL) by electrocellular fractionation of membrane (m) IgM + CD3 cells. Fractionate The cells were immediately re-analysed and found to be more than 99% B cells. Functional Analysis was performed on 96- or 24-well flat-bottomed Costar plates (Costar, C ambridge, MA). The total amount of cultured cells was 200 μl (96-well plate) or 1 ml (2 4 hole plate), 1 × 10^Five6% CO in cells / ml_TwoIncubate at 37 ° C in moist air containing Privation.   DNA Measurement of synthesis  DNA synthesis spans 4 hours^ThreeH-TdR (2 μCi / well; 6.7 Ci / nmol 1mCi = 37GBq; determined by incorporation of ICN, Irvine, CA). Cells to grasfa Harvested on an Inverter filter (PHD Cell Harvester, Cambridge Technology (Cambri dge Technology), Watertown, MA), [^ThreeLiquid scintillation for uptake of [H] -TdR Determined by spectrometry.   Determination of secretory Ig isotype concentration in culture supernatant  Ig isotype concentration is ELISA solution It was measured by precipitation. Secreted IgM, IgG3, (IgG1, IgG2b, IgG2a) and I in the culture supernatant 96-well flat bottom ELISA plate (Dinatech Laboratories (Dynatech Laboratories, Inc.), Alexandria, VA) Goat anti-mouse IgM, IgG3, IgG, and IgA polyclonal antibodies (Southern Biotech) Knology Associates (Southern Biotechnology Associates), Birmingham, A L). Next, wash the plate and block with a buffer containing FBS. And incubate with various diluted culture supernatants and standards did. After washing, the plate was complexed with alkaline phosphatase according to the instructions, Affinity purified goat anti-mouse IgM, IgG3, IgG1, IgG2b, IgG2a, and IgA poly Clonal antibodies (Southern Biotechnolo gy Associates)), wash again, and bind to the plate. The antibody conjugated to the combined alkaline phosphatase allows Birif Cleavage of the ferric phosphate (Sigma) produces a fluorescent product. Determination of IgE concentration The same procedure was followed, but the plate was used for rat IgG2a anti-mouse IgE monoclonal Body (clone EM95) [purified from ascites, Dr. Fred Finkelma n), obtained from USUHS, Bethesda, MD]. Tandard, then affinity purified rabbit anti-mouse IgE polyclonal anti Body (Dr. Ildy Katona, USUHS, Bethesda, MD) followed by Arca Affinity purified goat anti-rabbit IgG polyclones complexed with rephosphatase Null antibodies (Southern Biotechnology Associates ociates)). Quantification of fluorescence was performed using 3M FluoroFAST 96 full Purified myeloma Ig of known concentration, performed with an odometer (Mountainview, CA) The fluorescence units were converted to Ig concentrations by extrapolation of the calibration curve determined using. For each analysis Other Ig isotypes (IgM, IgD, IgG3, IgG1, IgG2b, IgG2a, and IgE No significant cross-reactivity with or interference with IgA) is found in the culture supernatant. Was not.   Flow cytometry analysis  Cells were first purified from rat IgG2b anti-Fcγ at a final concentration of 5 μg / ml. Incubation with the RII mAb (2.4G2) for 20 minutes, then the final concentration Cell affinity binding to FITC-rat IgG1 anti-mouse IgA mAb added at 10 μg / ml for 30 minutes Hindered. All steps were performed at 4 ° C. Fluorescence analysis was performed by FACScan (Becton Dickinson ( Becton Dickinson), Mountain View, CA), logarithmic amplif ication). Forward and side scatter properties and propidium iodide Survival, identified based on the release of propidium iodide (Sigma) Only possible cells were analyzed.                                  Example 2          Lipo-D alone is ineffective in mitosis of resting B cells,                   Shows significant synergy with mIg signaling   According to previous reports, lipoproteins, including lipoprotein-D (lipo-D) It has been shown to stimulate B cell proliferation and Ig secretion. In these studies, Heterogeneous aggregates of damper cells, especially at high densities (1 x 10⁶Cells / ml) Vesicles were used. Thus, in these studies, activation of other cell types and B cells ,this Whether it is an important parameter mediating the effects of these lipoproteins Not. For this reason we have relatively low cell concentrations (1 × 10^FiveCells / ml) To test the effect of lipo-D on a population of cultured, highly enriched small resting B cells. Was. In contrast to previous reports, addition of lipo-D at 0.02-5 μg / ml resulted in resting B cells. Vesicle growth was weak or not seen at all (FIG. 1). Lipo-D concentration to 40 μg / ml Again, there was no effect (data not shown). In contrast, B cells respond to LPS And proliferated actively (data not shown).   We have previously shown that αδ-dex is mediated by bacterial polysaccharides and that other stimuli exist. Stimulates B cell proliferation but not Ig secretion in the absence of mIg It has been shown to be an in vitro model of dependent TI-2 immunity. Bacteria are antigen-specific Express humoral immunity by expressing components that give intense and non-specific stimuli directly to B cells Investigating the hypothesis that they are provoking, we consider the optimal and suboptimal Threading of B cells when the concentration of lipo-D is varied at an appropriate αδ-dex concentration division(^ThreeThe effect of combined stimulation on H-TdR incorporation (as measured by H-TdR incorporation) was determined. Lipo -D by itself is relatively ineffective, but significantly synergizes with αδ-dex to induce proliferation. Led. Compared with αδ-dex alone, αδ-dex and lipo-D stimulation were combined In case, more than 25 times^ThreeH-TdR incorporation was observed (FIG. 1). Proliferation bite by itself Only 0.3 ng / ml αδ-dex with relatively weak intense effect combined with 1 μg / ml lipo-D When co-stimulated, proliferation was strongly stimulated. In contrast, 30 μg / ml uncomplexed 2 No co-stimulation of proliferation occurred with the multivalent anti-IgD antibody and Lipo-D (data shown). This indicates that crosslinking with polyvalent mIg is necessary for this effect. Departure First, uncomplexed anti-IgD antibodies increased the expression of MHC class II molecules. Regulates and increases B cell size, which again activates B cells. He reported that he had no ability.                                  Example 3              By stimulation combining lipo-D and αδ-dex,          Strong induction of Ig secretion occurs in the absence of foreign cytokines   In the following experiments, lipo-D was expressed on resting B cells in the presence or absence of αδ-dex. It was determined whether or not to induce Ig secretion. Lipo-D alone (0.2-5 μg / ml, FIG. Experiment A No significant stimulation of Ig secretion occurred in 5-20 μg / ml, FIG. 2, experiment B). Already As reported, αδ-dex is itself effective in inducing Ig secretion by resting B cells. Fruit is weak. However, when lipo-D and αδ-dex act in combination, Induction of more than 10,000-fold Ig secretion occurs in the absence of foreign cytokines (FIG. 2). Five μg / ml lipo-D and 0.3ng / ml αδ-dex may be optimal for costimulation of Ig secretion all right. If the concentrations of lipo-D and αδ-dex are raised or lowered further, Became inhibitory and ineffective, respectively.                                  Example 4        Lipo-D acts directly on B cells to costimulate their proliferation and Ig secretion   When Lipo-D in combination with αδ-dex co-stimulates growth and Ig secretion, In order to determine whether they are acting directly on arrested B cells, we have highly refined them. Prepared B cell aggregates (> 99% mIgM + CD3-) were replaced with PE-anti-Ig spleen cells lacking small T cells. The cells were stained with M + FITC-anti-CD3 and obtained by a method of electrically fractionating cells. Mixed with this Large, activated B cells have a forward scatter profile Further removed based on As shown in FIG. 3, lipo-D was activated by αδ-dex Observe proliferation and IgM secretion of fractionated and purified B cells in unfractionated B cell enriched aggregates Co-stimulated as was done. Therefore, lipo-D directly affects this level of B cells. To mediate these effects.                                  Example 5              When B cells stimulated with αδ-dex are activated with lipo-D,         Predominant secretion of IgM, small secretion of mainly IgG3   The T cell-dependent humoral immune response to bacteria is responsible for IgM and IgG3 production. Tend to be selective. In this experiment, B cells activated with αδ-dex Determined the Ig isotype characteristics of Ig synthesized in response to lipo-D. Table 1 As shown in the figure, lipo-D induces mainly IgM secretion in cells activated with αδ-dex. Lead. The other secreted Ig was IgG (1% or less), mainly IgG3. Yo Therefore, secretion of Ig isotype in response to costimulation by lipo-D is active only with LPS Similar results to those obtained with transformed B cells.   Production of Ig isotype by Lipo-D  B cells were treated with 5 μg / ml lipo-D at 0.3 ng / ml α Stimulate in combination with δ-dex to increase the concentration of various Ig isotypes in the culture supernatant for 6 days Later, it was measured by ELISA.                                  Example 6              Lipo-D alone has the effect of cytokine-dependent Ig secretion.                        Weak effect as costimulator   Secretion of Ig in response to activation by αδ-dex requires cytokine synergy. It is important. Thus, IL-4 + IL-5 has a strong Ig content in B cells activated with αδ-dex. Induces a renal response. The present inventors have recently reported that Ig components working in cells activated with αδ-dex A second cytokine pathway leading to a renal response was determined. Therefore, IL-3, GM-CSF , And IFN-γ acted as adjuvants for IL-I + IL-2, respectively, and were activated by αδ-dex Induce Ig secretion by fractionated and purified B cells. B cells enriched, not fractionated In cell culture, IL-I + IL-2 by itself depends on the presence of AsGm-1 + non-B non-T cells Stimulates Ig secretion, depending on secretion. Therefore, lipo-D is cytokine-dependent To determine the ability to co-stimulate high Ig secretion, this experiment used IL-4 + IL-5 or IL-I + Stimulate IL-2 and / or IL-3, GM-CSF, or IFN-γ with lipo-D Ig secretion was stimulated with αδ-dex. Compared directly with As shown in Table 2, αδ-dex is a component of both cytokine pathways described above. In response, Ig secretion was strongly co-stimulated. In contrast, lipo-D responds to these Ig secretions. The effect was relatively weak as a co-stimulator. In particular, IL-4 + IL-5 is lipo-D The stimulated cells showed considerable Ig-inducing activity (1600 when using αδ-dex). Eight times more than double. Furthermore, in the combination of II-3 + IL-2 + IL-3, activated by lipo-D Ig secretion by activated cells is more than 380 times that of cells activated with αδ-dex Was four times as strong as the induction of Therefore, lipo-D is itself a cytokine The effect of dependent Ig secretion as a costimulator is relatively weak.   Lipo-D is a relatively weak costimulator of cytokine-mediated Ig secretion  B cells In the presence or absence of Lipo-D (5 μg / ml) or αδ-dex (3 ng / ml) Stimulated with or without the available cytokines, and the IgM concentration in the culture supernatant after 6 days Was measured by the ELISA method. Cytokine early in culture, IFN-γ only 24 hours later Were added at the following concentrations: IL-1 (150 U / ml), IL-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-γ (10 U / ml).                                  Example 7     Lipo-D provides a key signal to induce Ig class switching   In a related patent application No. 08 / 400,322 filed March 8, 1995, the present inventors Recently developed αδ-dex in the presence of IL-4, IL-5 and the IgA switch factor TGF-β. High-efficiency IgA class switch that requires the combined action of LPS or CD40L An in vitro model for induction of switching was established. In this system, the flow More than 10% mIgA as assessed by cytometric analysis⁺To get cells Required all stimulation. In this experiment, we found that the IgA class We examined whether lipo-D could replace LPS in co-stimulation of switches. TGF-β 4 days after the start of culture, mIgA⁺Few cells are detected (FIG. 5). Removal of LPS from cultures resulted in mIgA as previously reported.⁺cell Percent of Stages fell below 2% (data not shown). However, the LPS By replacing with Po-D, mIgA⁺cell Returned to more than 8%. Thus, in this system, lipo-D is an IgA class switch. A major signal was provided to induce switching.                                  Example 8        Proliferation by lipo-D in B cells activated with αδ-dex and      Co-stimulation of Ig secretion appears to be a general property of bacterial lipoproteins   Another recombinant lipoprotein, Borrelia burgdor feri) -derived lipo-OSPA, causative agent of human rye disease and common structure of synthetic lipoproteins Pam_ThreeA series of similar experiments were performed using Cys. As shown in FIG. 5, Lipo-OS PA strongly co-stimulated IgM secretion in cells activated with αδ-dex. further , Pam_ThreeCys also strongly stimulated mitogenesis and Ig secretion. Like Lipo-D, And in contrast to previous studies, Lipo-OSPA and Pam_ThreeCys both, alone Neither cell proliferation nor Ig secretion by small resting B cells was significantly enhanced. Co-stimulation with αδ-dex was required to mediate. Therefore, these data Induces a strong humoral immune response without the need for ion-B cell recruitment To guide, bacterial lipoproteins that transduce non-specific signals to B cells Generally, specific B cells as mediated by mIg cross-linking (multivalent antigen binding) It is suggested that it must work together with the activation signal.                                  Example 9                  Lipoprotein D with polysaccharide-protein complex           Can enhance the anti-polysaccharide reaction   Diphtheria toxoid pneumococcal polysaccharide (DT-Pn14) Various doses were injected in the presence or absence of white matter D. Lipoprotein D , The anti-polysaccharide reaction is 5 to 10 times larger than that of DT-Pn14 alone. Was induced. In addition, 0.01 μg of DT-Pn14 induced little detectable response. However, injection with lipoprotein D induced a significant response. this child Is a lipoprotein D adjuvant to enhance the response to polysaccharide antigens Indicates that it can be used as   DT-Pn14 was administered to 5 DBA / 2 mice in each group in the presence or absence of lipoprotein D. Injected. Anti-Pn14 ELISA was measured after 28 days.                                 Example 10            Lipoprotein D activates anti-polysaccharide responses in T-cell deficient animals                            Can be augmented   To induce T cell deletion, mice were challenged with 500 μg-1.0 mg of anti-CD4 antibody (GK1 .5, obtained from ATCC). One day later, the mice received 5.0 μg of pneumococci. Injected either polysaccharide type 14 lipoprotein D ("PN14-LPD") or PN14 alone . Fourteen days later, the IgG1 anti-PN14 response was measured. As shown in Table 4 below, White matter D complex stimulates high levels of anti-PN14 response in T cell-deficient mice Was. Thus, lipoprotein D enhances the anti-polysaccharide response in immunocompetent animals. In addition to enhancing the anti-polysaccharide response in T-cell deficient animals. But Thus, lipoprotein D can be expressed in anti-polysaccharide response in T-cell deficient individuals such as HIV patients. To It can be a valuable tool for enhancement.                                 Example 11   To assess the effect of the protein on anti-polysaccharide responses, influenza b Bacteria (Haemophilus influenzae type b) polyribosyl-ribitol phosphate (polyribos Different vaccines based on yl-ribitol-phosphate (PRP) were prepared. These "Hi b vaccines include tetanus toxoid (TT) or Contained either lipoprotein D (LPD). One PRP-TT Hib vaccine is Prepared using polysaccharide CNBr activation, PRP-TT and PRP-LPD Hib vaccines It was prepared using CDAP activation of polysaccharide.   Ten 5-week-old female OFA rats, 10 rats per group, were given twice at 4-week intervals twice a quarter of H.D. Immunized by subcutaneous injection of vaccine, 28 days, 42 days, 56 days, 69 days Blood was collected on day 83.   The anti-PRR'P response was evaluated by ELISA (coated with tyramined PRR'P). A non-parametric method called 'Robust' is used with different preparations. Used to compare induced anti-PS titers. HIB 001A44 was used as a control .   Table 5a shows the anti-polysaccharide response. PRP-LPD, a lipoprotein D complex, Induced a primary anti-polysaccharide response comparable to that of PRP-TT, a wind toxoid complex. P-LPD is very high (more than 10 times) secondary anti-tetanus toxoid polysaccharide A polysaccharide reaction was induced. As shown in Table 5b, the PRP-LPD complex was very low The anti-lipoprotein D response was induced.   Also from this experiment, the anti-polysaccharide response was comparable on days 14 and 28 after booster immunization. It was suggested that there is. On day 69 (41 days after the boost), lipoprotein The response began to decline for some complexes, including the quality D complex.   In addition, the DTPa.HB vaccine (Zif Fineness due to terrier (Diphtheria), tetanus toxoid (tetanus toxoid), hepatitis B Acellular pertussis with Hepatitis B) combined with the complex . The combination did not reduce the anti-polysaccharide response.   Table 5 is based on two different proteins, tetanus toxoid and lipoprotein D The anti-polysaccharide response (Table 5a) and the anti-protein response (Table 5b) between vaccines are shown. Anti-TT titer (value in parenthesis) was measured using serum of rats injected with saline .                                 Example 12                  Antigenic and immunogenic properties of lipoprotein D constructs   In this example, Hib PRP and S. pneumoniae PS14 and The effect of the complex in which lipoprotein D was complexed to 6B and 6B was evaluated.   The complex utilizes the activation and coupling effects of CDAP as described below. (At different polysaccharide / protein ratios). The conjugate is treated with an anti-PS antibody and an anti-PS antibody. The antigenicity was examined in vitro using the LPD antibody and the free PS The amount was determined by immunoprecipitation. The immunogenicity of the complex is evaluated in a rat model However, the protective effect of the anti-PS antibody induced in rats indicates that immature rats (Hib Protection) or mice (for protection against S. pneumoniae 6B) Case). Materials and methods   Hib PRP and S. pneumoniae polysaccharides 6B and 14 are inactivated The cells were extracted and purified from the cultured cells. The purified substance is the remaining protein, nucleic acid, WHO and US specifications in terms of toxins, structural sugars and molecular weight distribution Was consistent with the book.   Haemophilus influenzae lipoprotein D is expressed in cerebral fungi And purified using conventional column chromatography. The purity of the protein may vary. It was 90% or more when evaluated by the following methods (SDS-PAGE, CE, HPLC).   Activated Hib PRP, S. pneumoniae polysaccharide 6B or pneumonia chain Complexes S. pneumoniae polysaccharide 14 with lipoprotein D or tetanus toxoid I let it. Two methods for conjugating polysaccharides to lipoproteins or tetanus toxoids A method is disclosed herein. Coupling of Lipo D to Pn14 A. Direct compounding using CDAP   Pn14 is adjusted to 5 mg / ml with physiological saline, and cooled with ice. CDAP in acetonitrile 100 mg / ml, 0.2 M TEA, lipoprotein D 6 mg / ml are 10 mM sodium phosphate, 0.2 M NaC l, 0.1% Empigen (surfactant obtained from CalBiochem) And pH adjusted to 7.2 and cooled with ice.   Activation and coupling are performed at 0-4 ° C. CDAP is a stirred solution of Pn14 To the mixture at a ratio of 0.75 mg CDAP / mg Pn14. After 30 seconds, adjust the pH to 10 with TEA. (Usually about twice the volume of CDAP used), and a total of 2 minutes with TEA at pH10. To maintain.   2.5 minutes later, lipoprotein D was activated with stirring at a ratio of 2.5 mg protein / mg Pn14 while activating PS. Add to The pH is maintained in the range 9-9.5.   After 1 hour, the reaction is stopped by adding 1/4 volume of 1 M glycine, pH 8.0. After overnight incubation at 4 ° C, the complex was filtered on S500HR (Pharmacia) gel. Purify by passing through an excess column.   Pool high molecular weight conjugates including proteins and polysaccharides, 0.2 microfilter Filter with a filter. Protein is quantified by the Lowry method, and polysaccharides are Quantify by resorcinol analysis. B. Coupling via spacer   Pn14 is activated by CDAP as described above. After 2.5 minutes, 1/2 volume of 0.5 M adipindihi Drazide (pH 8.0) is added. After 1 hour, the solution is completely dialyzed in saline I do.   Hydrazide content is measured using TNBS, polysaccharide is measured by resorcinol analysis I do.   Lipoproteins were prepared according to the method of Lees et al. (Vaccine, 1994, 12, 1160). Attached to n14-hydrazide. Briefly, Pn14-hydrazide is converted to iodine Iodoa using acetyl N-hydroxysuccinimide (SIA, Sigma) Cetylized. The protein is S-acetylthioacetyl N-hydroxysuccinimide (SATA, Sigma) (Sigma). Using Centricon 30 device After desalting and concentrating, both were mixed and 1/9 volume of 0.75 M HEPES, 0.5 M The pH is raised to 7.5 using roxylamine. After reacting at 4 ° C overnight, The reaction was incubated for 1 hour in 0.2 mM mercaptoethanol followed by 1 hour. Incubate for 10 minutes in 0 mM iodoacetamide and stop. Complex Is purified as described above. For those skilled in the art, to complex proteins to polysaccharides It will be appreciated that other methods may also be implemented. Experimental result                    1.Immunogenicity of the complex in rats   To each group of 10 OFA rats (female, 5 to 6 weeks old), 2.5 μg of polysaccharide equivalent (PRP 0.1) to 10 μg (for pneumococcal PS6B or PS14) An amount of the conjugate was injected subcutaneously (200 μl). Rats are additionally exempted after 1 month with the same amount of complex Blood was collected for antibody analysis and immediately before and 15 days after the boost. . Anti-LPD antibody was measured by ELISA using protein D as coating antigen . Anti-PS antibody is measured by ELISA using tyramine PS as coating antigen And adding CPS to the serum to measure anti-PS6B or anti-PS14 (1 mg / ml for anti-PS14, 5 mg / ml for anti-PS6B) The anti-CPS antibody was adsorbed. Special Differential antibodies were detected using peroxidase-labeled anti-rat conjugate. All EL In ISA, a reference serum is used, and the titer is determined in arbitrary units using a 4-parameter method. Calculated.                             2.Passive defense test                  a)Hib Protection of young rats against challenge   Immature OFA rats (4-5 days old) were diluted with serum (in PBS) containing anti-PRP antibody. 00 μl was injected intraperitoneally (IP). After 24 hours, about 5 × 10^FourColony forming unit (CFU) Intraperitoneal challenge to rats using exponentially growing Hib and Eagan strains Emitted. Twenty-four hours later, the rats were sacrificed and blood samples were taken from each rat. Bacterial feeling Staining is performed by seeding serial dilutions of blood on chocolate agar and counting colonies one day later. It was measured by: Rats with less than 10 CFU per 100 μl blood are protected It was determined that it was done. b)Protection of mice against lethal challenge by S. pneumoniae 6B   Serum containing anti-PS 6B antibody was administered to 10 mice (outbred OFI, 6 weeks old, female) in each group. (1.5-fold diluted serum, 100 μl), 24 hours after passive immunization,^ThreeCFU pneumonia Bacteria (S. pneumoniae) type 6B were injected intraperitoneally. The control group contains serum from non-immunized animals. Or sera of animals immunized with the PS14 conjugate were injected. The number of deaths in each group Recorded for 18 days.   When LPD is used instead of TT as the protein, anti-PS reaction (anti-PRP, anti-PS6B) Is higher (at least 10 times). In a passive defense model of young rats , Antibodies induced by PRP-LPD complex are induced by PRP-TT complex At least equal or better than antibody (when prepared to equal titer) .   In the passive mouse protection test, complete protection was observed with the anti-PS6B-LPD conjugate. Conclusion   From these results, for the different capsular polysaccharides tested (PRP, PS 6B), LPD , TT. Twenty-four hours after intraperitoneal injection of 100 μl of serum into 10 OFA rats (5 days old) of each group 5 × 10^FourChallenge was induced by intraperitoneal route in CFU / rat (Eagan strain). Bacterial infection was measured after 24 hours. Mice with less than 10 CFU in 100 μl of blood were considered protected. The control was 10 in 1 μl of blood.^Three~Ten⁶CFU. ^*Some mice are sick Five-week-old female OFA mice were injected intraperitoneally with 100 μl of 5-fold diluted serum, and The challenge was induced 24 hours later with 100 μl of the Pn6B strain (6 / 6B / 52) passed twice. Mortality was recorded up to 18 days after challenge.                                Example 13   To examine the effects of lipidation, rats were given lipoprotein D (lipo- D), binds protein D or tetanus toxoid to pneumococcal polysaccharide 6B The conjugate was injected between 0.1 and 30 μg. Lipo-D, protein D, and pneumococcus (pn eumococcal) polysaccharide 6B was prepared according to the method described on pages 5 and 20 above. Prepared. Tetanus toxoid is readily available from many sources known to those skilled in the art. Is obtained. The conjugate was prepared according to the method described on pages 20-21 above. Was. The protein D complex is prepared by the same method used to prepare the lipoprotein D complex. Prepared.   Anti-polysaccharide response was observed 28 days after the first injection of the conjugate and 28 days after the booster injection Next, it was measured by ELISA. As shown in Table 11, the mice injected with the protein D complex Mice injected with lipo-D conjugate had significantly higher anti-polysaccharide responses compared to mice it was high.   Other aspects of the invention take into consideration the specification and examples of the invention disclosed herein. This will be apparent to those skilled in the art. The description and examples It is considered merely exemplary, and the scope and spirit of the present invention will be determined by the following claims. It is shown.

────────────────────────────────────────────────── ─── Continuation of front page    (31) Priority claim number 08 / 568,342 (32) Priority date December 6, 1995 (33) Priority country United States (US) (81) Designated countries EP (AT, BE, CH, DE, DK, ES, FI, FR, GB, GR, IE, IT, L U, MC, NL, PT, SE), OA (BF, BJ, CF) , CG, CI, CM, GA, GN, ML, MR, NE, SN, TD, TG), AP (KE, LS, MW, SD, S Z, UG), UA (AM, AZ, BY, KG, KZ, MD , RU, TJ, TM), AL, AM, AT, AU, AZ , BB, BG, BR, BY, CA, CH, CN, CZ, DE, DK, EE, ES, FI, GB, GE, HU, I S, JP, KE, KG, KP, KR, KZ, LK, LR , LS, LT, LU, LV, MD, MG, MK, MN, MW, MX, NO, NZ, PL, PT, RO, RU, S D, SE, SG, SI, SK, TJ, TM, TR, TT , UA, UG, UZ, VN (72) Inventor Snapper Clifford M.             United States Potoma, Maryland             Cook Ivy Mount Terrace 7904 (72) Inventor Mondo James Jay.             United States Potoma, Maryland             Green Leaf Avenue             11804 (72) Inventor Kapiau Karin             Mons Elvisocal Bee, Belgium             −7050 Aimonz Cross De Hauts               11 (72) Inventor Hauser Puerre             Chaumont Gieste, Belgium             Bee-1325 Lou Inchee Blue 1E             I (72) Inventor Priels Jean-Paul             Brussels B-1200 Belgium             Venue des Fabrieres 7

Claims (1)

[Claims] 1. Administering to the individual, including administering the antigen and co-administering the lipoprotein. For inducing an immune response against polysaccharides or other T-cell independent antigens . 2. The method according to claim 1, wherein the lipoprotein is derived from a microorganism. 3. 3. The method according to claim 2, wherein the lipoprotein is lipoprotein D. 4. The method according to claim 1, wherein the lipoprotein is a synthetic lipoprotein. 5. Antigen is Haemophilus influenzae type b, pneumonia S. pneumonia, Group B Streptococcus, N. meni ngitidis), Salmonella and P. aeruginosa mucoexo A polysaccharide derived from one bacterium selected from the group consisting of polysaccharides. The method of claim 1. 6. Administering an antigen and co-administering a lipoprotein, Is a method for enhancing an immune response to other T-cell independent antigens. 7. The method according to claim 6, wherein the lipoprotein is derived from a microorganism. 8. The method according to claim 6, wherein the lipoprotein is lipoprotein D. 9. 7. The method according to claim 6, wherein the lipoprotein is a synthetic lipoprotein. 10. The antigen is Haemophilus influenzae type b, Pneumonia Streptococcus (S. pneumonia), Group B Streptococcus, meningococcus (N.me ningitidis), Salmonella and P. aeruginosa mucoeki Is a polysaccharide derived from a bacterium selected from the group consisting of polysaccharides Item 7. The method according to Item 6. 11. A vaccine comprising a clinically relevant polysaccharide covalently linked to lipoprotein D. 12. Polysaccharides are Haemophilus influenzae type b, pneumonia Streptococcus (S. pneumonia), Group B Streptococcus, N. meningitidis (N. meningitidis), Salmonella and P. aeruginosa mucoe The bacterium is derived from one bacterium selected from the group consisting of xopolysaccharides. Vaccine. 13. 12. The lipoprotein D is directly covalently linked to a polysaccharide. of Method for producing vaccine. 14． 14. The method of claim 13, wherein the covalent bond is due to CDAP activation of the polysaccharide. Law.