WO2004012657A2 - Compositions contenant du beta-glucane, procedes pour produire des beta-glucanes et pour produire et utiliser des beta-glucanes et des conjugues de ces derniers comme adjuvants de vaccins - Google Patents

Compositions contenant du beta-glucane, procedes pour produire des beta-glucanes et pour produire et utiliser des beta-glucanes et des conjugues de ces derniers comme adjuvants de vaccins Download PDF

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Publication number
WO2004012657A2
WO2004012657A2 PCT/US2003/023741 US0323741W WO2004012657A2 WO 2004012657 A2 WO2004012657 A2 WO 2004012657A2 US 0323741 W US0323741 W US 0323741W WO 2004012657 A2 WO2004012657 A2 WO 2004012657A2
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Prior art keywords
glucan
beta
vaccine
molecules
cells
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PCT/US2003/023741
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English (en)
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WO2004012657A3 (fr
Inventor
Kenneth W. Hunter
Frank M. Jordan
Ruth A. Gault
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Immusonic, Inc.
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Priority to AU2003257007A priority Critical patent/AU2003257007A1/en
Publication of WO2004012657A2 publication Critical patent/WO2004012657A2/fr
Publication of WO2004012657A3 publication Critical patent/WO2004012657A3/fr

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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
    • A61K39/39—Medicinal preparations containing antigens or antibodies characterised by the immunostimulating additives, e.g. chemical adjuvants
    • 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
    • A61K2039/55555—Liposomes; Vesicles, e.g. nanoparticles; Spheres, e.g. nanospheres; Polymers
    • 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
    • A61K2039/55583—Polysaccharides

Definitions

  • the present invention relates generally to an improved method for the preparation of small particle sized glucans. More particularly, the present invention relates to the preparation of small particle sized glucans that modulate immunological activity in humans and animals. The present invention also generally relates to the immunopharmacologic upregulation of a molecule of a family of B7 molecules to effectuate a costimulatory reaction that allows for an appropriate effector cell immune response.
  • the present invention relates to the use of small particle size glucans as vaccine adjuvants.
  • Glucans are polymers of glucose. Glucans are commonly found in the cell walls of bacteria, yeast, and various plant species. A common glucan is beta (l,3)-linked glucopyranose (commonly referred to as beta glucan). Other common examples include mixtures of beta-(l ,3)- linked glucopyranose with beta-(l ,6)-linked glucopyranose. These glucans have been shown to have immunopharmacological activity in humans and animals. More particularly, beta (1,3)
  • glucan has been shown to effect some immune responses.
  • glucan particles tend to aggregate, or re-aggregate, upon hydration, or re-hydration, as the case may be, thus reducing or eliminating the desired result.
  • the re-aggregation and resistance to de-aggregation is accentuated in environments with low pH such as a human digestive tract, such as with a pH of less than 1.0.
  • a human digestive tract such as with a pH of less than 1.0.
  • the glucans aggregate, or re-aggregate into particles of greater diameter, they appear to pass through an animal or human digestive system without substantially complete absorption.
  • Fig.1 is a diagrammatic representation of a unit of beta linked glucopyranose
  • Fig. 2 is an illustration of glucan particles in a naturally hydrated state
  • Fig. 3 is an illustration of glucan particles and the effects of various methods of
  • Fig.3b is an illustration of a microscopic examination of the effects of dehydration on
  • Fig. 3c is an illustration of a microscopic examination of effects of sonic energy on
  • Fig. 4 is an illustration of tabular results of a phagocytosis assay
  • Fig.5 is an illustration of tabular results for Nitric Oxide production of a glucan activated
  • Fig. 6 is an immunofluorescence photomicrograph demonstrating the upregulation and cell surface expression of B7.2 on the mouse tumor macrophage cell line P388D 1 treated in vitro with a ⁇ -l,3-glucan-containing composition;
  • Fig.7 is an immunofluorescence photomicrograph demonstrating the upregulation and cell surface expression of B7.2 on mouse peritoneal macrophages treated in vitro with a ⁇ -1,3- glucan-containing composition
  • Fig.8 is an immunofluorescence photomicrograph demonstrating the upregulation and cell surface expression of B7.2 on mouse peritoneal macrophages treated in vivo with a ⁇ -1,3- glucan-containing composition
  • Fig. 9 is a plot of tabular results of a vaccination study in which a prototypic vaccine antigen (fluorescein isothiocyanate-labeled bovine serum albumin, or FITC-BSA) is administered intradermally to BALB/c mice in either normal saline or as a conjugate with microparticulate
  • a prototypic vaccine antigen fluorescein isothiocyanate-labeled bovine serum albumin, or FITC-BSA
  • glucan polysaccharide can be of any number "n" to produce varying chain lengths.
  • the glucan containing composition may be made by any means common in the art.
  • a common method of manufacture of a glucan is set out as follows:
  • glucan containing composition The aforementioned method of preparation of a glucan containing composition is general and it will be understood by those of skill in the art that variations on the aforementioned method will still lie within the scope of the present invention. Further, the resulting glucan composition may be of varying compositions and percentages of glucan. In a most preferred embodiment the resulting glucan solution is about 2% to 5% glucan by volume.
  • the glucan prepared above exists predominantly in the globule form. It is desirable to reduce the predominant globule size to a range of .3 - 3.0 microns in diameter, preferably to approximately 1 - 2 microns in diameter. Reducing the size of the glucan globule, for example, down to particles preferably having a range of 1 - 2 microns in diameter, may be achieved by sonication of a glucan containing composition. In a preferred embodiment a glucan containing composition is first hydrated for a period of at least twelve hours. In a most preferred embodiment the glucan containing composition is hydrated overnight in water.
  • a portion of the glucan may be containerized prior to sonication.
  • a preferred embodiment uses an ordinary tray or dish as a container.
  • the container may then be placed in an ultrasonic water bath to dissociate the large globules of glucan.
  • Experimental results have indicated that the size of the container has a direct effect on results of sonication.
  • a container is selected that allows for 10 to 5O mm space between the container walls and a sonicator probe.
  • various other embodiments of the present invention may utilize any variety of containers of varying size.
  • the container is sonicated for three-twelve (12) minute
  • the container is sonicated for one (1)- twelve (12) minute cycle in an ice bath for cooling the glucan as it is heated during sonication.
  • the short breaks in the cycling are used because the sonication of the glucan generates a considerable amount of heat and cooling of the glucan containing composition is necessary to prevent excessive heating and denaturing or degradation of the product.
  • a bench-top sonicator is a BioLogics 300 V/T/ Sonic Dismembrator.
  • a preferred embodiment utilizes the settings of the sonicator at 80% power and 80% duty cycle for 12 minute cycles with the container in an ice bath.
  • a preferred probe for use is a 19 mm (3/4") diameter titanium probe.
  • a preferred power setting for a one (1) duty cycle sonication is 192 watts for 48 seconds with a 12 second pause at an ultrasonic output of 20 kilocycles per second.
  • Other power settings may be used for sonication, however, the time and number of duty cycles may vary accordingly.
  • a commercially available sonic dismembrator may be used. Experimental results have shown that the BioLogics, Inc. sonic dismembrator, with a continuous flow chamber, indicated that about 95% of a fully hydrated glucan may be dissociated after one (1) to three (3) treatments at a flow rate of 16 rnl/min and 80% power with 12 minutes per treatment. Preferred embodiments of this method utilize one treatment to fully disassociate the glucan.
  • the glucan After sonication, the glucan remains in suspension in an aqueous state for a sufficient period of time for applications requiring suspension of the glucan such as pharmacological applications; including pharmaceutical and pharmacological applications, nutritional applications, and supplementary applications for animals, humans and plants.
  • pharmacological applications including pharmaceutical and pharmacological applications, nutritional applications, and supplementary applications for animals, humans and plants.
  • a percentage gelatin solution may be added to the glucan solution.
  • the percentage gelatin solution is a 5% gelatin solution in de-ionized water.
  • the 5% glucan solution is diluted to about a 2% glucan solution with water and the 5% gelatin solution.
  • the glucan may be sonicated as indicated above.
  • the glucan may be utilized wet or dry.
  • a most preferred method for drying the glucan of this embodiment is lyophilizing or freeze drying. A preferred method of freeze drying utilizes an ultralow freezer to freeze the glucan at -80 degrees centigrade.
  • the time required to freeze dry the glucan varies depending upon the amount of glucan, but generally will take between 1 to 2 hours. However, the length of time to freeze dry may vary.
  • the resulting glucan containing composition will dry into a friable, paper-like consistency, and upon re- hydration the glucan will typically disassociate into substantially 1 - 2 microns in diameter
  • the wet, gelatinized glucan may be added to a capsule and freeze dried. Upon re-hydration the glucan will de-aggregate into predominantly 1 - 2 microns in diameter glucan.
  • a sugar is added to a sonicated glucan, without the gelatin.
  • the sugar is maltodextrose.
  • other embodiments of the present invention may utilize both a gelatin and a sugar in the glucan.
  • the resulting glucan containing composition may then be placed in a commercially available spray drier for application.
  • a preferred sprayer is the Spray Drying Systems spray drier.
  • a fine, non- aggregated powder is formed from the spraying.
  • the resulting glucan containing composition existing substantially as a powder, may then be loaded into capsules, pills or other containers. The powder may also be stored and later re-hydrated for future use.
  • the preferred spray dryer utilizes an inlet air temperature of 110 to 170 degrees Centigrade, an outlet air temperature of 90 to 120 degrees Centigrade and a feed solids composition of 0.5 to 1.0 percent.
  • other settings and spray dryers may be used and be within the scope of the present invention varying the quality of the sprayed glucan. In fact, other settings may be required when using a different spray dryer.
  • the preferred spray dryer produces a finely sprayed glucan powder that does not re-aggregate into glucan globules.
  • Figure 2 of the drawings illustrates the morphology of beta-glucan-containing globules of various sizes commonly available on the market.
  • the glucan-containing globules are illustrated in a hydrated state. In the hydrated state, glucan aggregates into globules 7. These glucan globules consist of numerous individual and linked beta glucans. Further, as the glucan aggregates, the size of the glucan globule becomes greater.
  • Fig. 3 of the drawing provides an illustration of glucan particle size and the effects of various methods of preparation, demonstrating the reduction in size of the glucan globules upon sonication in accordance with the invention.
  • Slide C of FIG. 3 is raw glucan at about 200 time magnification. Globule 1 of raw, untreated glucan may be observed.
  • Slide F is a sample of raw glucan at about 200 times magnification that has been ground to a fine ground particle size of the glucan globule 2. It may be observed the glucan globule 2 is generally of smaller size than the glucan globule 1 of Slide C.
  • Slide B of Fig. 3 a sample of raw glucan has been sonicated with a
  • Slide A is a sample of ground glucan that has been sonicated with a BioLogics 300 V/T Sonic Dismembrator at 80% power and 80% duty cycle for 12 minute cycles in an ice bath, dried and then rehydrated viewed at about 400 times magnification. It may be observed that globule 5 is generally of a smaller globule size than globule 1 or globule 2. It may also be observed that the globule 5 is generally more dispersed than globule 1 or globule 2. It may also be observed that the glucan rehydrated after being dried to contain globule 5 has a globule size generally less
  • Slide D is a sample of ground glucan that has been sonicated with a BioLogics V/T/ Dismembrator at 80% power and 80% duty cycle for 12 minute cycles in an ice bath, dried and then rehydrated viewed at about 400 times magnification. It may be observed that the globule 6 is generally of a smaller globule size than globule 2 or globule 1.
  • Fig.3b of the drawing provides an illustration of a microscopic examination of the effects of dehydration on sonicated glucan and is illustrative of the preferred results after sonication of a glucan.
  • Slide A is a glucan suspension after subjection to three sonication treatments at a flow rate of 16 ml/min and 80% power. The results were taken after drying the glucan and then rehydrating through vortex mixing.
  • Slide B is a glucan suspension after subjection to three sonication treatments at a flow rate of 16 ml/min and 80% power. The results were taken after drying then rehydrated by grinding with a mortar and pestle in de-ionized water.
  • Slide C is a glucan, not sonicated, only suspended in a de-ionized water solution by vortex mixing.
  • Slide D is a glucan, not sonicated, only suspended in de-ionized water after grinding by a mortar and pestle.
  • Fig. 3b an illustration is provided of a microscopic examination of the effects of dehydration on sonicated glucan, the resulting glucan is most finely separated in Slide A and in Slide B after both sonication and vortex mixing.
  • Slide C is not finely separated and results in large globules because no sonication was utilized.
  • Slide D results in a more finely separated glucan than Slide C after grinding, but still results in large glucan globules in the absence of sonication.
  • Fig. 3c an illustration is provided of microscopic examination of the effects of sonication on glucan globules, and is demonstrative of the reduction in particle size of glucan globules after sonication.
  • Slide A is a 2% glucan suspension in de-ionized water after vortex mixing viewed at 10X.
  • Slide B is the identical solution of Slide A at 20X magnification.
  • Slide C is Slide A after three treatments of sonication at 16ml/min at 80% power viewed at 10X magnification.
  • Slide D is the identical solution of Slide C viewed at 40X magnification.
  • glucan globules are reduced to small particle size glucan as a result of sonication.
  • Fig. 4 provides an illustration of a phagocytosis assay and demonstrates the enhanced phagocytosis of the small particle size glucan.
  • the data for Fig. 4 an illustration of a phagocytosis assay, was generated from an assay in which phagocytosis was measured utilizing fluorescent bio-particles. This experiment was conducted to determine the glucan induced macrophage activity. An assay was performed using the ground glucan from Fig. 2, predominantly particle size 1-100 micron in diameter, and another assay was performed using
  • sonicated beta glucan from Fig.3, Slide D predominantly 1 - 2 microns in diameter, particle size glucan.
  • a bacterium, Staphylococcus aureus was labeled with a fluorescent marker, fluorescein isothiocyanate (FTTC). This dye was chosen because when viewed using fluorescent microscopy the dye emits a yellow-green light.
  • FTTC fluorescein isothiocyanate
  • the labeled cells were mixed with macrophages for about twenty minutes. After incubation, the assays were rinsed with Tryptan Bule, pH 4.4 The acidic solution quenched the fluorescence of FTTC, causing the labeled bacterium to no longer emit the yellow-green light.
  • the bacterium that have been phagocytised are protected from the quenching and emit the yellow-green light when viewed under the fluorescent microscope.
  • Escherichia coli 0111 :B4 a known activator of macrophages, a solution of glucan globules (100 ⁇ g/ml), a solution of DSM-glucan (100 ⁇ g/ml) or media. After 1 hour incubation, the stimulant was removed and replaced with growth media. Twenty-four hours post-stimulation the cells were evaluated for activation and a phagocytic index calculated as is demonstrated in Fig. 4.
  • the greater percentage phagocytosis demonstrates the enhanced activity of the macrophage and the small particle size glucan' s ability to activate the immune system.
  • Fig. 5 of the drawing there is provided an illustration of tabular results for Nitric Oxide production of glucan activated macrophage and demonstrates the enhanced production of Nitric Oxide, NO, from the untreated glucan to the sonicated glucan.
  • the data demonstrates an approximate factor of two increase in the production of NO from comparison of the untreated glucan to the treated glucan; from 275 ⁇ M to 600 ⁇ M..
  • the measurement of NO production is indicative of an oxidative burst that kills and/or destroys the ingested microbes and/or particles by the macrophage.
  • This experiment was performed by measuring NO by antigen capture enzyme immunoassay. Macrophages were stimulated for 1 hour with LPS (50 ⁇ g/ml), glucan globules
  • glucan-containing compositions are polymers of
  • glucose and they are commonly found in the cell walls of bacteria, yeast, and various plant species.
  • the glucans can be categorized according to the types of chemical linkages between
  • glucopyranose commonly referred to as ⁇ glucan.
  • Other common examples include mixtures
  • the macrophage first engulfs foreign material in a process called phagocytosis, then processes these microbial proteins into peptides that are displayed on the macrophage cell surface in association with molecules of the major histocompatibility complex (MHC).
  • MHC major histocompatibility complex
  • Immune cells called T lymphocytes have clonally-derived receptors on their surfaces, and some of these receptors are invested with the ability to bind to a particular peptide so displayed.
  • the end result is the initiation of an immune response involving humoral immunity (antibodies), cell-mediated immunity (killer cells), or both .
  • antibodies humoral immunity
  • cell-mediated immunity killer cells
  • beta glucan- containing compositions can potentiate both innate and adaptive immunity, but the exact mechanism for this enhancement is not known.
  • the macrophage and some other cell types have receptors in their surface membranes for
  • ⁇ -glucan-containing compositions When ⁇ - glucan-containing compositions interact with the cell surface glucan receptor, the macrophage is activated and becomes capable of direct and indirect killing of the invading pathogen or tumor. However, macrophage activation alone is not responsible for the immunity enhancing effect of glucan.
  • beta glucan-containing compositions that can explain its immunopotentiating effect, and this discovery is a major feature of the present invention. To understand the importance of this invention, one must understand the mechanism by which the immune response identifies a foreign substance (antigen), and develops a response that can neutralize and/or eliminate the foreign substance before it causes significant morbidity or mortality.
  • the immune system has evolved to discriminate between self and non-self.
  • Non-self could be any of the myriad of potential pathogens for which humans and animals can be hosts, like viruses, bacteria, fungi, and parasites, or the non-self (or altered self) that is represented in the myriad of cancers that can arise from normal cells in an animal or human.
  • the challenge to the immune system is to identify that which is harmful non-self, and to mount a vigorous attack on this foreign material.
  • Extraordinarily potent immune effector mechanisms have evolved, but these effector mechanisms have the potential of causing harm to the animal or human host if they become misdirected to harmless non-self.
  • the self/non-self discrimination is, the allergic and autoimmune diseases that animals and humans suffer result from errors in discrimination. It is important in the understanding of the present invention to know that the evolution of the immune system included means of avoiding, or at least minimizing, the errors in self/non-self discrimination.
  • TCR T lymphocyte receptors
  • the immature T cells migrate to the thymus where they undergo a two step selection process that first eliminates all T cells bearing receptors that fail to recognize peptides presented on the surface of antigen presenting cells (APC, like macrophages, dendritic cells, and B cells) by major histocompatibility complex (MHC) molecules. Those that pass this test are then retested for strong reactivity against self peptides, thereby eliminating the vast majority of T cells that bear receptors capable of recognizing self peptides. The end result of this selection process is a pool of circulating T cells bearing receptors that, for the most part, will only recognize foreign antigens (central tolerance).
  • APC antigen presenting cells
  • MHC major histocompatibility complex
  • peripheral tolerance a mechanism known as peripheral tolerance has evolved .
  • the understanding of peripheral tolerance is key to understanding the subject of the present invention.
  • T cells could bind to normal cells via their TCR and be induced to proliferate and differentiate into armed effector cells, these T cells could attack and kill self tissues causing disease and possibly death.
  • the immune system evolved a process whereby a naive T cell must receive two signals in order to proliferate and differentiate into an armed effector cell. The first signal is delivered by the TCR binding to its target peptide presented to it by an MHC molecule on the surface of an APC.
  • a second signal (costimulatory signal) is required before the naive T cell can proliferate and differentiate into an armed effector cell, and that second signal is delivered by the interaction of CD28 molecules on the surface of the T lymphocyte with B7 molecules (B7-1 or
  • CD80, and B7-2 or CD86 on the surface of an APC.
  • the first signal TCR:MHC- peptide
  • the second signal not only does the T cell fail to become an armed effector cell, it actually goes into an inactive state called anergy . It is believed that such anergic cells eventually undergo a slow process of programmed cell death called apoptosis. Because most cells in an animal or human do not express, nor can they be induced to express,
  • T cell bearing a TCR specific for a self peptide interacts with a normal tissue cell bearing that self peptide expressed in a cell surface MHC molecule, the T cell is made anergic, and is unable to become an effector cell capable of killing the normal cell.
  • the combination of central and peripheral tolerance eliminates most of the potentially autoreactive T cells that arise in the course of T cell development.
  • APC dendritic cells, B cells
  • B cells dendritic cells, B cells
  • the macrophage does not normally express large amounts of B7 on its surface. It has been demonstrated that surface expression of B7 is inducible, and when a macrophage interacts with certain microbial products (i.e., lipopolysaccharide or LPS), the gene coding for B7 is actively transcribed, and the macrophage begins to express B7 on its surface membrane. Only when the macrophage is activated in this way does it become an APC capable to providing both the first and second signal needed to cause
  • microbial products i.e., lipopolysaccharide or LPS
  • T cells to proliferate and differentiate into armed effector cells.
  • the pathogen gains access to the blood or tissues of the host via one of several mechanisms, and begins to proliferate causing tissue damage.
  • One of the first host defense cells to arrive on the scene is the macrophage, and the macrophage is capable of ingesting the microbes via the process of phagocytosis.
  • the pathogen Once the pathogen is ingested, it remains inside a vacuole that serves as a digestion chamber.
  • Host cell enzymes are added to the chamber and the pathogen is killed and its proteins disrupted into peptides. The peptides are loaded onto newly formed MHC molecules, and then brought to the surface of the macrophage.
  • the MHC '.peptide complex is now ready to interact with a T cell bearing the correct TCR.
  • the macrophage in order for the macrophage to be an effective APC, it must upregulate the expression of B7 genes, and begin to express large amounts of B7 costimulatory molecules on its surface. It is presently unclear just what microbial products are responsible for causing the upregulation of the B7 gene, and indeed, not all microorganisms ingested by macrophages cause the increased expression of B7 molecules. If sufficient B7 is expressed on the surface of the APC, the requisite T cells can be stimulated to become effector cells capable of killing the invading microorganisms.
  • the B7 molecule and its surface membrane expression are key to the induction of the adaptive immune response. Failure of an APC to express B7 can actually result in the loss of protective T cells by the process of anergy. Should a T cell with an TCR specific for a peptide of an invading microorganism or tumor cell encounter that peptide on an APC that is not expressing B7, that T cell may be anergized and sent down the apoptotic pathway. This problem is addressed by the present invention.
  • upregulate herein, because the term has a well-defined meaning in this art. Although there is no specific verb form “to upregulate”, nor are its derivative forms upregulate, upregulation, upregulated, and upregulating found in the standard American
  • CD40/CD40 ligand have been shown to induce long-term graft survival with the inhibition of the Thl cytokines (INF), IL-2 and IL-12 and upregulating the Th2 cytokines IL-4 and IL-10".
  • upregulating refers to increased expression of the TH2 cytokines IL-4 and IL-10.
  • upregulate by simple extension, recognizes the pharmacologic agent as an "upregulating agent".
  • upregulate by simple extension, recognizes the pharmacologic agent as an "upregulating agent".
  • a layman might choose to use the terms “increase” or “increasing” in place of “upregulate” or “upregulating” to confer the idea that there is more of a substance present as the result of the use of the agent.
  • upregulate and upregulating have clear meaning.
  • a safe and effective pharmacologic agent that could upregulate the expression of the critical B7 costimulatory molecule on APC.
  • Such an agent can be administered to an animal or human in a dosage sufficient to cause the cell surface expression of B7 on macrophages (or other APC), thereby allowing the macrophage to better serve the function of antigen presentation to T lymphocytes.
  • Macrophages have many receptors for highly conserved microbial constituents, and the interaction of the ligand on the microbe with its receptor on the macrophage has been shown, for example in the case of the LPS receptor, to cause the activation
  • Macrophages also have glucan receptors, receptors into which ⁇ l,3-glucan-
  • the ⁇ 1 ,3-glucans have been administered to animals and humans for years with
  • tumor macrophages were incubated in vitro with various amounts of ⁇ l,3-glucan-containing
  • compositions see below.
  • APC APC in providing the critical second signal necessary for inducing specific T lymphocytes to proliferate and differentiate into armed effector cells capable of protecting against harmful non-self, like pathogenic microorganisms and cancer.
  • the B7 gene has been cloned and methods described for its expression in various cell types, we do not need to clone the B7 gene and manipulate its expression in a host cell in order to augment an immune response.
  • This invention relates, generally, to a method of augmenting the immune response to a
  • compositions The invention involves the administration of the ⁇ l,3-glucan-containing
  • composition to an animal or human in a dose and by a route that serves to bring a critical amount of this material to the vicinity of macrophages or monocytes (blood, tissues, secondary lymphoid
  • the ⁇ l,3-glucan-containing composition interacts with a specific glucan receptor on
  • B7 a family of molecules called B7 (i.e., B7.1 and B7.2).
  • B7 molecules are costimulatory molecules that are critical in the provision of a second signal to specific T lymphocytes that have received a first signal through a specific cell surface receptor (TCR) that interacts with a foreign peptide in the context of an MHC molecule on the surface of the APC.
  • TCR specific cell surface receptor
  • the naive T lymphocyte When the naive T lymphocyte has received both signals, it proliferates and then differentiates into an armed effector T lymphocyte that can effectuate one of the important defense mechanisms of the immune response (i.e., cell mediated immunity or antibody-mediated immunity). Because APC like macrophages do not normally express much cell surface B7, they are not effective in
  • compositions can cause APC like macrophages to express large amounts of B7 on their surfaces, allows us to provide a pharmacological intervention to make the APC more effective in initiating the adaptive immune response, and therefore, more effective in providing protection against foreign antigens like microbes and tumors.
  • a ⁇ l,3-glucan-containing composition causes the upregulation of cell surface expression of B7 molecules on microphages.
  • mice show that macrophages and enhance their rate of bacterial phagocytosis.
  • yeast Saccharomyces cerevisiae but another source of ⁇ 1 ,3-glucan could easily be derived from
  • Example 5 Mouse tumor macrophage cell line (P388D1) treated in vitro with a ⁇ 1,3-glucan-
  • composition upregulation of B7 surface membrane expression.
  • the tumor macrophage-like cell line P388D1 (ATCC, Manassas, VA.) was grown in wells of eight-chambered microscope slides containing tissue culture media with 10% fetal bovine serum at 37°C in 5% CO 2 . Cells were stimulated with media (control cells) or media containing 100 ⁇ g/ml ⁇ 1,3-glucan-containing composition for 1 hour. During this time, it was demonstrated (data not shown) that the macrophages ingested large quantities of the ⁇ 1,3-glucan-containing particles. After incubation, the stimulant was washed away and replaced with growth media and incubated as before.
  • a ⁇ 1,3- glucan-containing composition can cause the upregulation of a B7 costimulatory molecule in this particular mouse macrophage-like tumor cell line.
  • Example 6 Mouse peritoneal macrophages treated in vitro with a ⁇ 1,3-glucan-containing
  • composition upregulation of B7 surface membrane expression.
  • mouse peritoneal macrophagse were harvested by peritoneal lavage with cold tissue culture medium.
  • the cells were placed in tissue culture medium containing 10% fetal bovine serum and placed in the wells of eight-chambered microscope slides and incubated at 37 °C and 5% CO 2 for two hours.
  • non-adherent cells were removed by washing in warm medium, and the macrophages were found to attach to the glass substrate of the microscope slide.
  • Cells were stimulated with media (control cells) or media containing 100 ⁇ g/ml ⁇ 1,3-glucan-containing composition for 1 hour. During this time, it was demonstrated (data not shown) that the macrophages ingested large quantities of the ⁇ 1,3-glucan-containing particles. After incubation, the stimulant was washed away and replaced with growth media and incubated as before. Approximately 24 hours post-stimulation, live cells were incubated with a phycoerythrin-conjugated antibody directed to mouse B7.2.
  • Example 7 Mouse peritoneal macrophages treated in vivo with a ⁇ 1,3-glucan-containing
  • composition upregulation of B7 surface membrane expression.
  • mice were injected via the intraperitoneal route with 100 ⁇ g of a ⁇
  • Example 8 Mouse peritoneal macrophages stimulated in vivo with a ⁇ 1 ,3-glucan-containing composition: enhanced phagocytosis of bacteria.
  • mice were fed 132 ⁇ g/kg of a ⁇ 1,3-glucan-containing composition daily for 18 days.
  • Peritoneal macrophages were harvested by peritoneal lavage as described in Example 6.
  • Peritoneal macrophages were plated into each well of a 8-well chambered slide. s Macrophages were allowed to adhere for approximately 4 hours followed by washing 3X with
  • Example 9 Mouse peritoneal macrophages stimulated in vivo with a ⁇ 1,3-glucan-containing composition: potentiation of immune responses.
  • mice were fed 132 ⁇ g kg of a ⁇ 1,3-glucan- containing composition daily for 19 days.
  • mice On day 2 and day 8, mice were given an injection of 1 x 10 9 sheep red blood cells (SRBC) via the intraperitonel route.
  • SRBC sheep red blood cells
  • serum anti-SRBC antibodies blood was drawn from the mice eleven days after the last injection and allowed to coagulate. After centrifugation the serum component was aspirated and used for hemagglutination assays. Serum was serially diluted from 1: to 1:20,480 in PBS in a 96-well microtiter plate.
  • mice spleens were harvested aseptically from the same mice eleven days after the last injection of SRBC. The tissue was dissociated by mincing followed by sieving through a 210 ⁇ m polypropylene mesh into growth medium. After lysis of red blood cells the lymphocytes were counted and diluted to 1 x 10 8 cells/ml for plaque-forming cell assays to determine the number of IgM secreting B cells.
  • SRBC T cell dependent antigen
  • the present invention also relates generally to a novel composition of small particle size beta glucan which contains partially deacetylated N-acetylglucosamine that provides a free amino group for vaccine conjugation and also provides stability to the microparticles, particularly during transit through the gastrointestinal system.
  • the invention also pertains to the method of manu acture of the small particle size beta glucan which contains partially
  • an adjuvant is a substance that can augment the immune response to vaccine antigens (Cox and Coulter, 1997). Adjuvants have been around since 1926 when Ramon and his colleagues (Ramon, 1926) discovered that horses given intradermal immunizations of diphtheria toxoid made higher antibody responses if they developed bacteria-laden abscesses at the injection site. However, even today we do not understand the mechanism of action of most adjuvants.
  • CFA causes such a profound inflammatory response that it cannot typically be used in humans.
  • adjuvants simply provide an inert matrix to which the vaccine antigens are attached, and while they may promote the persistence and endocytosis of vaccine antigens, do not induce to a significant extent co-stimulatory molecules
  • induction of an immune response to most carbohydrate and all protein antigens requires both antigen recognition by cognate receptors on immune cells (e.g., T cell receptors), and the simultaneous interaction of co-stimulatory ligands on APCs (e.g., B7 family glycoproteins) with their counterpart receptors (CD28) on T cells.
  • APCs e.g., B7 family glycoproteins
  • CD28 counterpart receptors
  • failure of a vaccine antigen preparation to induce the expression of co-stimulatory molecules can lead to the phenomenon of tolerance. If a vaccine induces tolerance, not only does it fail to initiate an adaptive immune response, but the specific T cells that interact with the vaccine peptides presented in the context of MHC molecules on APCs become anergic and ultimately undergo apoptosis.
  • a rational approach to enhancement of vaccination is to use an adjuvant that induces the concomitant expression of co-stimulatory molecules and/or to design antigens that induce co-stimulation directly, or to add to the vaccines and antigenic substances that induce the expression of co-stimulatory molecules.
  • the B7 family molecules are known to be potent co-stimulatory ligands that interact with CD28 receptors on T cells to generate the so-called second signal that complements the T cell receptor:MHC:peptide first signal.
  • the combined antigen-specific first signal and co-stimulatory second signal facilitates the proliferation and differentiation of T cells into armed effector cells.
  • glucan preparations evaluated for immune enhancement can be divided into soluble and particulate, and both forms have demonstrated immune enhancing properties.
  • Our procedure for making microparticulate beta-glucan preserves chitin, while eliminating most of the contaminating mannan and protein. The preservation of chitin, as explained below, is significant.
  • the present invention relates to a novel microparticulate beta-glucan that can be used as a vaccine adjuvant, to methods of manufacturing this new microparticulate beta-glucan, to conjugates of this microparticulate beta-glucan and vaccine antigens, and to pharmaceutical formulations of these conjugates useful as vaccine adjuvants in animals and humans.
  • This new microparticulate beta-glucan binds to glucan receptors on a variety of phagocytic cells and enhances their immunological functions (e.g., cytokine production), and contains around 1- 10% deacetylated N-acetylglucosamine, preferably about 4%, that provides a free arnino group for vaccine conjugation and also provides stability to the microparticles, particularly during transit through the gastrointestinal system, and has particles which are predominantly 0.3-3 microns in size and often closer to 1 - 2 micron particles that are ideally suited to phagocytosis by macrophages and dendritic cells, and causes the expression of co-stimulatory molecules on antigen presenting cells (APCs), a critical requirement for inducing an adaptive immune response.
  • the microparticulate glucan of the present invention enhances the immune response to vaccine antigens, and thus serves as a safe and effective vaccine adjuvant.
  • glucan re-aggregates some of the beneficial effect of the glucan is not achieved because the macrophage receptors are not activated as readily by large particle size or reaggregated glucan.
  • the glucan re-aggregates it appears to pass through an animal or human digestive system without substantially complete absorption.
  • microparticle beta-glucans which retain a small percentage of chitin in the form of partially deacetylated N-acetylglucosamine and which do not reaggregate but rather remain substantially in a particle size of about 0.3-3, preferably, 1 - 2 microns in diameter throughout preparation, packaging, and/or use without re-aggregation.
  • the process set forth below describes a novel method for preparing a microparticulate glucan that can be used as a vaccine adjuvant.
  • the first aspect of this invention involves the process of preparing the microparticulate glucan from a source of glucan.
  • the process of the present invention uses a less rigorous combination of alkali and acid extractions of the starting yeast material in order to preserve around 1-10% chitin preferably containing at least 4% chitin, the usefulness of which will be apparent later in this description.
  • the present invention creates particles which are predominately 0.3-3 microns in size, and preferably 1 - 2 microns in size that have a variety of advantages over larger glucan particles. It should be noted that while S.
  • glucan cerevisiae is a convenient source of glucan, other sources of beta-glucan may be substituted as long as these sources have chitin as a constituent of their cell walls, or alternatively, chitin or chitin containing compounds can be added to the beta glucan or glucan containing compounds.
  • Chitin is a polymer which naturally occurs and is found in a variety of cells in yeast, fungi, mushrooms, and may also be obtained from mutant or genetically altered cells.
  • yeast any type of yeast can be used, including mutant or genetically altered yeasts such as Saccharomyces cerevisiae, Saccharomyces delbrueckii, Saccharomyces rosei, Saccaromyces microellipsodes, Saccharomyces carlsbergensis, Saccharomyces bisporus, Saccharomyces fermentati, Saccharomyces rouxii, Schizosaccharomyces pombe, Schizophyllum commune, Sclerotium glucanium, Lentinus edodes, Kluyveromyces lactis, Kluyveromyces fragilis, Kluyveromyces poly porus, Candida albicans, Candida cloacae, Candidatropicalis, Candida utilis, Hansenula wingei, Hansenula ami, Hansenula henricii, Hansenula americana, Hansenula americansis, Hansenula capsulata, Hansenula polymorpha, Pichia
  • the initial process comprises the steps of: (a) extracting the active dry yeast one time with an alkali solution to remove alkali-soluble material; (b) extracting the alkali- insoluble material one time with acid to remove acid soluble material; c) extracting acid insoluble material from step (b) with an organic reagent to remove residual lipid.
  • the active dry yeast is added to an alkali solution such as 2-6% NaOH or other base commonly known or used in the art with stirring such as by automation about 30-60 minutes or thereabout.
  • the material is then heated to 115°C-145 at about 8.5-12 psi for about 45-75 minutes and allowed to settle for 72-96 hours.
  • this alkali extraction is generally performed only once.
  • the sediment is resuspended and washed in dH 2 O by centrifugation (e.g., 350 X g for 20 minutes).
  • the alkali insoluble solids are combined with an acid such as 4%-6 acetic acid or other acid commonly known or used in the art and heated to 85°C-100 for about 1-4 hours, then allowed to settle at
  • the acid insoluble solids are drawn off and centrifuged as above. This acid extraction is generally performed only once.
  • the compacted solid material is mixed with a peroxidating agent such as 3% H 2 O 2 and refrigerated for 3-6 hours with periodic mixing. The material is then centrifuged and the pellet washed twice with dH 2 O followed by two washes in
  • the harvested solid material is dispersed on drying
  • hexoses (glucose) with between 1-10% chitin (measured as N-acetylglucosamine). It may also
  • beta glucan and chitin containing polysaccharide compounds can be used to extract the beta glucan and chitin containing polysaccharide compounds, and extract the water insoluble residues multiple times, while obtaining a more purified beta glucan which still retains the desired chitin in the form of
  • the aggregated glucan is first hydrated in dH 2 O overnight at room temperature, and then a 1.5% suspension of the hydrated material is subjected to sonic energy via a 19 mm probe utilizing a 300 V/T Sonic Dismembrator (BioLogics, Gainesville, VA).
  • a 300 V/T Sonic Dismembrator BioLogics, Gainesville, VA.
  • the glucan suspensions are sonicated on ice for 12 minutes(for example, twelve 48 second cycles of sonication with a 12 second pause between cycles).
  • Experimental studies have shown that excessive sonication of the glucan creates heat that may denature the glucan and shortened life of the sonic probe.
  • the process of the most preferred embodiment will dissociate substantially all of the glucan aggregates to particles of about 0.3-3 microns in diameter, preferably 1 - 2 microns.
  • the sonicated glucan suspension is spray-dried using a spray dryer such Buchi 190 Mini-Spray Dryer (Buchi, Germany), or other such spray dryer or other spray drying process known or used by one skilled in the art.
  • the sonicated suspension is preferably spray dried by such a dryer with an inlet air temperature of 110-170°C, and outlet air temperature of 90-
  • sonicated glucan consists of approximately 1.81 x 10 11 microparticles in the size range of 0.3-3 microns.
  • spray dryers may be used and be within the scope of the present invention varying the quality of the sprayed glucan. In fact, other settings may be required when using a different spray dryer.
  • the preferred spray dryer produces a finely sprayed glucan powder that does not re-aggregate into glucan globules. A fine, non-aggregated powder, is formed from the spraying.
  • the resulting glucan containing composition existing substantially as a powder, may then be loaded into capsules, pills or other containers.
  • the powder may also be stored and later re-hydrated for future use. When this dried material is rehydrated in an aqueous medium, it remains substantially unaggregated.
  • the cell walls of S. cerevisiae are composed of four major components beta-l,3-(D)- glucan (ca. 40%); beta-l,6-(D)-glucan (ca. 14%); mannoprotein (ca. 41%); and N- acetylglucosamine or chitin (ca. 5%).
  • Our new process utilizes less rigorous alkali and acid extractions in order to preserve most of the chitin. Indeed, the particle size and chemical composition of our new microparticulate beta-glucan preparation is similar to that described for yeast bud scars.
  • the chitin in our formulation also provides needed chemical and mechanical stability to the microparticles.
  • microparticulate beta-glucan of the present invention also survives transit through the stomach, duodenum, and jejunum, and can be found intact in the ileum associated with M cells overlaying the Peyer's patch lymphoid tissue. Because the microparticulate beta-glucan of the present invention has between 1-10% chitin that has been partially deacetylated by the alkaline treatment and bears a reactive primary NH 2 group, a variety of chemical procedures can be used to attach any type of vaccine or vaccine antigens and other type of non-traditional molecules and components to the particles such as polypeptides for gene therapy, genes or genes involved in the treatment of cancer or tumors, and nucleic acids for gene expression.
  • the vaccines can also be administered to the subject human or other animal through any route.
  • One such procedure involves water soluble carbodiimide.
  • vaccine antigens that cannot be attached to the NH 2 functionality they can be directly attached to the glucan by a variety of chemical reactions, such as via a maleimide linkage group. Since our microparticulate beta-glucan preparation has only trace amounts of protein, we can determine the amount of vaccine protein attached using the micro-Bradford protein assay. Because the number of vaccine antigen molecules per particle may profoundly influence the immunization success, reaction conditions can be varied to prepare conjugates with various amounts of attached vaccine protein. To more precisely determine the reaction conditions needed to attach a specific number of vaccine antigen molecules per particle, vaccine antigens can be tritium-labeled and standard radioactive binding analyses can be performed.
  • this water insoluble beta-glucan preparation which provides a free amino group for conjugation and which can be used as a vaccine adjuvant, comprises microparticulate beta - (1 ,3)-glucan with or without beta -(1 ,6)-glucan side chains which do not substantially reaggregate upon drying or rehydration, at least 4% by weight partially deacetylated N- acetylglucosamine within the beta-glucan that provides a free amino group for vaccine conjugation.
  • a vaccine or an antigenic substance is then conjugated with the free amino group on the adjuvant.
  • the adjuvant with or without the conjugated vaccine or antigenic substance may be in an aqueous suspension or solid such as a tablet, or in a colloidal mixture.
  • the source of this glucan may be obtained from a yeast cell wall extract, or a fungus or mold cell extract.
  • the adjuvant preferably contains less than or about 3%-5% by weight protein and lipid, and more than or about 85%-95 % by weight glucose, and about 1 %-10% by weight chitin or partially deacetylated N-acetylglucosamine, but could also contain more or less of these constituents .
  • sugars such as maltodextrin may also be added as a filler, and gelatin may be added as a filler.
  • Other fillers used or known by one skilled in the art may also be used.
  • Example 10 In Fig. 9 of the drawing there is provided an illustration of tabular results of a vaccination study in which a prototypic vaccine antigen (fluorescein isothiocyanate-labeled bovine serum albumin, or FTTC-BSA) is administered intradermally to BALB/c mice in either normal saline or as a conjugate with microparticulate glucan (MG). In both cases, the amount of vaccine material administered to each of five mice was 40 ⁇ g. Serum was collected from each of the five mice per group on days 6, 12, and 24 after the primary immunization.
  • a prototypic vaccine antigen fluorescein isothiocyanate-labeled bovine serum albumin, or FTTC-BSA
  • MG microparticulate glucan
  • mice given FTTC-BSA vaccine in saline has antibody titers of 111 and 220 respectively, whereas the mice given the same amount of vaccine conjugated to MG had titers of 776 and 928 for days 12 and 24.
  • the antibody titers were 6.9 times and 4.2 times higher in the MG-FITC-BSA group than in the saline FTTC-BSA control group.
  • mice On day 70, both groups of mice were rei munized with the same vaccines as given for the primary immunization, and their sera sampled on days 77 and 84.
  • the IgG antibody titers to FTTC-BSA had diminished, but after immunization, the MG- FTTC-BSA group showed average antibody titers 1.6-fold higher than the saline control group.
  • the saline control group showed a reciprocal titer of 14,708, while the MG:FTTC- BSA group had average reciprocal titers of 22,735.
  • the increase seen in the MG:FTTC-BSA groups was 1.6-fold higher than in the saline control group.
  • an adjuvant can influence the immune response to a vaccine antigen (e.g., activation of APCs for enhanced phagocytosis, upregulation of MHC expression, improved antigen processing).
  • APCs for enhanced phagocytosis
  • upregulation of MHC expression improved antigen processing
  • microparticulate beta -(l,3)-glucan is a strong inducer of the expression of the B7 co-stimulatory molecules, and thus should promote the presentation of vaccine antigen to T cells.
  • a method of using microparticulate beta -(l,3)-glucan as a vaccine adjuvant which generally comprises the steps of preparing or obtaining a microparticulate beta -(l,3)-glucan composition which does not substantially reaggregate upon drying and rehydration which contains partially deacetylated N- acetlyglucosamine with a free amino group, suspending or maintaining the microparticulate beta -(l,3)-glucan composition in liquid, adding at least one vaccine or antigenic substance, conjugating the vaccine onto the free amino group, and administering the vaccine to an animal or human.
  • the glucan will be composed of a yeast cell wall extract and/or algae, and/or fungi, but there may also be other such suitable sources used or known by one skilled in the art.
  • the vaccine adjuvant may also be stored after preparation and before the vaccine or antigenic substance is added, or conversely stored after the vaccine or antigenic substance is added.
  • conjugating a vaccine to particle such as the novel beta-glucan particle of this invention.
  • the microparticulate composition may be sterile or sterilized before it is used and/or containerized and stored.
  • liquid and/or vaccine and/or antigenic substances may be added to the container before administration.
  • the adjuvant preferably contains less than or about 2%-5% by weight protein and lipid, and more than or about 85%-95 % by weight glucose, and about 1 %-10% by weight chitin or partially deacetylated N-acetylglucosamine, but could also contain more or less of these constituents.
  • sugars or salts which are used or known by one in the art may added.
  • the resultant vaccine may be administered by any route, including but not limited to intradermally or subcutaneously, intramuscularly, intraperitoneally, orally, nasally, externally, or into the lymphatic system or blood stream. If the vaccine is given intramuscularly, a depot effect may occur after vaccination.
  • the conjugated vaccine may also provide protection from protease degradation.
  • the conjugated vaccine may also target antigens to Peyer's patches for processing and presentation to T-cells. Furthermore, this method contemplates that if orally adminstered, oral tolerance may be minimized or eliminated.
  • this adjuvant such as enhancing he immunological effects of a vaccine or antigenic substance.
  • the immunological effect may be measured or quantified in terms of a serum antibody unit, which may be enhanced or increased. Further the immunological effects or response of the vaccine occur sooner in the terms of a serum antibody unit.
  • the enhancement of immunological effects wherein the serum antibody unit is measured by hemagglutination inhibition (HAT) or passive hemagglutination (PHA), and may also include the action of enhancing the immunological effects is an action of promoting IgA. In this conjugated form, co-stimulation and proliferation of antigen-specific T-cells may occur.
  • the adjuvant may be in a dry form, in an aqueous solution, or colloidal suspension.
  • the adjuvant may also be containerized and stored until use.
  • the glucan in the adjuvant may be obtained from a yeast cell wall extract, fungus or mold cell extract, or other such suitable sources known or used by one skilled in the art. Further, the glucan in the adjuvant may contain less than about 2-5% by weight protein and lipid, about 85-95 % by weight glucose, and about 1 %-10% by weight chitin or partially deacetylated N-acetylglucosamine. Other compositions may also be suitable as long as it contains an adequate amount of chitin for conjugation.
  • the adjuvant may contain fillers such as sugar, particularly, maltodextrin, and/or gelatin.
  • fillers such as sugar, particularly, maltodextrin, and/or gelatin.
  • suitable fillers known or used by one skilled in the art are also contemplated by this invention.
  • preservatives or anti- oxidants may be appropriate depending upon the type of vaccine or antigenic substance used and depending upon whether the adjuvant and or conjugated vaccines or antigenic substances are stored.
  • the adjuvant may also be premixed or incorporated into food or drink, and may have added oils, lipids, preservatives andor antioxidants, and organic solvents or inorganic solvents known or used by one skilled in the art, as necessary or desired.
  • this invention also includes the conjugates of vaccines and other antigenic substances that are attached to the free amino group of microparticulate beta -(l,3)-glucan and which stabilizes the vaccine(s) and other antigenic substances and enhances the immunologic effects of vaccine.
  • the conjugates are comprised of microparticulate beta -(l,3)-glucan with or without beta -(l,6)-glucan side chains and partially deacetylated N-acetylglucosamine within the beta-glucan that provides a free amino group for conjugation with the vaccines and other antigenic substances and which does not substantially reaggregate upon drying or rehydration and a vaccine or vaccines or an antigenic substance, wherein the vaccine or antigenic substance is conjugated with the free amino group.
  • the microparticulate beta- glucan has particles which are predominantly 0.3-3 microns in size, preferably 1 - 2 microns. Further this beta glucan contains about 2-5% protein and lipid and carbohydrate analysis reveals about 85-95% hexoses (glucose) with between 1-10% chitin (measured as N- acetylglucosamine).
  • the conjugated vaccine or antigenic substance may be administered once, or multiple times. Further, the conjugation of the vaccine or antigenic substance may be enhanced in terms of a serum antibody unit, hemagglutination inhibition (HAT), or passive hemagglutination (PHA).
  • HAT hemagglutination inhibition
  • PHA passive hemagglutination

Abstract

Selon invention, un bêta-glucane microparticulaire est utilisé comme adjuvant de vaccins pour des animaux et des êtres humains, ledit bêta-glucane se fixant aux récepteurs du glucane sur divers phagocytes pour renforcer leurs fonctions immunologiques. Lesdites particules contiennent environ 1-10 % de N-acétylglucosamine partiellement désacétylée et présentent majoritairement un diamètre de 0,3-3 microns, de préférence de 1-2 microns. Elles sont administrées pour provoquer l'expression de molécules co-stimulantes sur les cellules présentant l'antigène. Ladite microparticule régule à la hausse l'expression de la molécule co-stimulante B7 sur la base de microparticules contenant du bêta (1,3) glucane et du bêta (1,6) glucane.
PCT/US2003/023741 2002-08-01 2003-07-30 Compositions contenant du beta-glucane, procedes pour produire des beta-glucanes et pour produire et utiliser des beta-glucanes et des conjugues de ces derniers comme adjuvants de vaccins WO2004012657A2 (fr)

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WO2006032039A3 (fr) * 2004-09-17 2007-04-05 Univ Massachusetts Compositions et leurs utilisations pour deficiences d'enzymes lysosomales
WO2007109564A2 (fr) * 2006-03-17 2007-09-27 University Of Massachusetts Particules de cellules de levures en tant que véhicules de délivrance d'antigènes par voie orale
JP2008503472A (ja) * 2004-06-16 2008-02-07 ゲーリー アール． オストロフ、 薬物送達製品および方法
US8389485B2 (en) 2007-10-29 2013-03-05 University Of Massachusetts Encapsulated nanoparticles for nucleic acid delivery
WO2023161527A1 (fr) 2022-02-28 2023-08-31 Tridem Bioscience Gmbh & Co Kg Conjugué constitué de ou comprenant au moins un ss-glucane ou un mannane
WO2023161528A1 (fr) 2022-02-28 2023-08-31 Tridem Bioscience Gmbh & Co Kg Conjugué comprenant au moins un ss-glucane

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Cited By (16)

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Publication number Priority date Publication date Assignee Title
US8637045B2 (en) 2004-06-16 2014-01-28 University Of Massachusetts Therapy for lysosomal enzyme deficiencies
US9682135B2 (en) 2004-06-16 2017-06-20 University Of Massachusetts Drug delivery product and methods
JP2008503472A (ja) * 2004-06-16 2008-02-07 ゲーリー アール． オストロフ、 薬物送達製品および方法
US8007814B2 (en) 2004-06-16 2011-08-30 University Of Massachusetts Therapy for lysosomal enzyme deficiencies
US8580275B2 (en) 2004-06-16 2013-11-12 University Of Massachusetts Drug delivery product and methods
WO2006032039A3 (fr) * 2004-09-17 2007-04-05 Univ Massachusetts Compositions et leurs utilisations pour deficiences d'enzymes lysosomales
CN101052383B (zh) * 2004-09-17 2013-01-30 马萨诸塞大学 用于溶酶体酶缺乏症的组合物和其用途
FR2887457A1 (fr) * 2005-06-23 2006-12-29 Fond Bettencourt Schueller Vaccination par ciblage transcutane
WO2006136959A1 (fr) * 2005-06-23 2006-12-28 Fondation Bettencourt-Schueller Vaccination par ciblage transcutane
US8956617B2 (en) 2005-06-23 2015-02-17 Fondation Bettencourt-Schueller Vaccination by transcutaneous targeting
WO2007109564A3 (fr) * 2006-03-17 2007-12-13 Univ Massachusetts Particules de cellules de levures en tant que véhicules de délivrance d'antigènes par voie orale
WO2007109564A2 (fr) * 2006-03-17 2007-09-27 University Of Massachusetts Particules de cellules de levures en tant que véhicules de délivrance d'antigènes par voie orale
US8389485B2 (en) 2007-10-29 2013-03-05 University Of Massachusetts Encapsulated nanoparticles for nucleic acid delivery
WO2023161527A1 (fr) 2022-02-28 2023-08-31 Tridem Bioscience Gmbh & Co Kg Conjugué constitué de ou comprenant au moins un ss-glucane ou un mannane
WO2023161528A1 (fr) 2022-02-28 2023-08-31 Tridem Bioscience Gmbh & Co Kg Conjugué comprenant au moins un ss-glucane
WO2023161526A1 (fr) 2022-02-28 2023-08-31 Tridem Bioscience Gmbh & Co Kg Conjugué constitué de ou comprenant au moins un ss-glucane ou un mannane

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