|Publication number||USRE40786 E1|
|Application number||US 09/586,747|
|Publication date||23 Jun 2009|
|Filing date||2 Jun 2000|
|Priority date||16 Mar 1984|
|Publication number||09586747, 586747, US RE40786 E1, US RE40786E1, US-E1-RE40786, USRE40786 E1, USRE40786E1|
|Inventors||Paul R. Burnett, John E. van Hamont, Robert H. Reid, Jean A. Setterstrom, Thomas C. Van Cott, Deborah L. Birx|
|Original Assignee||The United States Of America As Represented By The Secretary Of The Army|
|Export Citation||BiBTeX, EndNote, RefMan|
|Patent Citations (50), Non-Patent Citations (21), Classifications (19), Legal Events (3)|
|External Links: USPTO, USPTO Assignment, Espacenet|
This application is a continuation-in-part of U.S. patent application Ser. No. 08/242,960, filed May 16, 1994, pending; which in turn is a continuation-in-part of U.S. patent application Ser. No. 07/867,301 filed Apr. 10, 1992, now U.S. Pat. No. 5,417,986 which in turn is a continuation-in-part of U.S. patent application Ser. No. 07/805,721 filed Nov. 21, 1991; now abandoned, which in turn is a continuation-in-part of U.S. patent application Ser. No. 07/690,485 filed Apr. 24, 1991, now abandoned; which in turn is a continuation-in-part of U.S. patent application Ser. No. 07/521,945 filed May 11, 1990, now abandoned. Additionally, this application is a continuation-in-part of U.S. patent application Ser. No. 08/446,149 filed May 22, 1995, pending; which in turn is a continuation of U.S. patent application Ser. No. 06,590,308 filed Mar. 16, 1984, now abandoned.
The invention descried herein may be manufactured, licensed and used by or for governmental purposes without the payment of any royalties to us thereon.
This invention relates to parenteral and mucosal vaccines against diseases cause by intracellular pathogens using antigens encapsulated within biodegradable-biocompatible microspheres(matrix).
Most infections by viruses and other intracellular pathogens are countered in the human host by a combination of humoral (antibody) and cellular (lymphocyte and phagocyte) immune effectors. Although the precise identity of immune effectors capable of protecting the host against some chronic intracellular pathogens (e.g. HIV-1) remains unknown, attempts to develop preventive and therapeutic vaccines still focus on the induction of appropriate humoral and cellular immune responses. Furthermore, since most human viral pathogens (including HIV-1) are transmitted across mucosal surfaces, it is important that vaccines induce such responses locally (at the mucosal surface) as well as systemically and that they are durable for long-term protection.
The issues of durability and mucosal immunogenicity have been previously addressed by encapsulating vaccine antigens in appropriately-sized biodegradable, biocompatible microspheres made of lactide/glycolide copolymer (the same materials used in resorbable sutures). It has been shown that such microspheres can be made to release their load in a controlled manner over a prolonged period of time and can facilitate the interaction of their contents with the local immune system when administrated mucosally.
In the case of HIV-1 infection, there is insufficient information at this time regrading the virus and its interactions with the human immune system to permit the rational design of a preventive vaccine. However, it has been noted that many candidate HIV vaccines tested to date fail to elicit antibodies capable of neutralizing wild-type HIV-1 or binding to native HIV-1 proteins, fail to induce a substantial population of effector cells capable of destroying HIV-1-infected cells, and fail to induce significant responses at mucosal surfaces. A possible approach to overcoming these problems (applicable to both HIV-1 and other chronic intracellular pathogens) is to identify a native protein, accessible to the immune system on the surface of both free virus and infected cells, and present it to the immune system (systemic and mucosal) encapsulated in microspheres to protect and augment its immunogenicity.
This invention relates to a novel pharmaceutical composition, a microcapsule/sphere formulation, which comprises an antigen encapsulated within a biodegradable polymeric matrix, such as poly(DL-lactide co glycolide) (PLG), wherein the molecular weight of the PLG is about 4,000 to 100,000 daltons and wherein the relative ratio between the lactide and glycolide component of the PLG is within the range of 52:48 to 0:100, and its use, as a vaccine, in the effective induction of antiviral immune responses comprising both virus-specific cytotoxic T lymphocytes and antibodies reactive against native viral antigens. In the practice of this invention, applicants found that when a complex (oligomeric) native envelope protein of HIV-1 was encapsulated in PLG microspheres, it retained its native antigenicity and function upon its release in vitro. Furthermore, when used as a vaccine in animals, this product elicited HIV-specific cytotoxic T lymphocytes and antibodies reactive with native (oligomeric) HIV-1 envelope protein.
The following examples illustrate the invention:
Materials and Methods
Immunogens, Non-CD4-binding, baculo-expressed, recombinant gp 160IIIB (rgp 160) was obtained from MicroGeneSys (Meriden, Conn.). CD4-binding, oligomeric gp 160 CDC451 (o-gp 160) was obtained from Advanced BioScience Laboratories (Kensington, Md.).
Microencapsulation of immunogens: PLG microspheres ranging from 1 nanometer to 20 um in diameter and containing a 0.5 to 1.0% antigen core load were prepared by a solvent extractive method. 0.5 to 5.0% by weight antigen core load could also be used. The solvent extraction method involves dissolving the viral antigen and sucrose (1:4 ratio w:w) in 1 ml of deionized water. This solution is flash frozen and lyophilized. The resulting antigen-loaded sucrose particles are resuspended in acetonitrile and mixed into PLG copolymer dissolved in acetonitrile. This antigen-polymer mixture is then emulsifyed into heavy mineral oil, transferred into heptane and mixed for 30 min to extract the oil and acetonitrile from the nascent spheres. The spheres are harvested by centrifugation, washed three times in heptane and dried overnight under vacuum. Microsphere size was determined by both light and scanning electron microscopy. The antigen core load was determined by quantitative amino acid analysis of the microspheres following complete hydrolysis in 6N hydrochloric acid.
Analysis of immunogen spontaneously released from microspheres in vitro by binding to soluble CD4 and recognition by HIV-positive patient serum. PLG microspheres loaded with native (oligomeric) gp 160 were suspended in phosphate-buffered saline, pH 7.4 (PBS), incubated at 37 C. for 3 h, and then at 4 C overnight. The microspheres were then sedimented by centrifugation (2 min at 200×g), the supernatants harvested, and the released gp 160 assayed for binding to CD4 and recognition by HIV-positive patient serum by surface plasmon resonance (described below). A sample of the stock protein used for microencapsulation was assayed for comparison.
Immunization of animals. HIV-seronegative, 8-10 week old NZW rabbits were immunized intramuscularly with rgp 160- or o-gp 160-loaded PLG microspheres suspended in PBS or with alum-adjuvanted rgp 160 in PBS. Groups receiving rgp 160-loaded microspheres (n=2) were primed with 50 ug of immunogen on day 0 and boosted with 25 ug on day 42. Groups receiving o-gp 160-loaded microspheres (n=3) were primed with 70 ug of immunogen on day 0 and boosted with 35 ug on day 56. Groups receiving alum-adjuvanted rgp 160 (n=2) got 85 ug of immunogen on days 0, 7, and 28.
BALB/c mice were immunized subcutaneously with rgp 160-loaded PLG microspheres suspend in PBS or with alum-adjuvanted rgp 160 in PBS. The mice in all groups (n=4) received 10 ug of immunogen on days 0 and 21.
Determination of the ratio of antibody binding to “native”/denatured rgp 120IIIB measured by surface plasmon resonance (SPR). Real-time binding interactions between ligand (gp 120 covalently linked to a biosensor matrix) and ligate (Abs in solution) were measured using surface plasmon resonance (BIAcore, Pharmacia Biosensor, Piscataway, N.J.). “Native”rgp 120(IIIB) (Genentech, South San Francisco, Calif.) or reduced, carboxymethylated (denatured) rgp 120(IIIB) (Genentech) was covalently linked to the biosensor dextran matrix. Sera and mAbs were diluted in HBS running buffer (10 mM HEPES, 150 mM NaCl, 3.4 mM EDTA, 0.05% (BIAcore) surfactant P20, pH 7.4) and injected through the dextran matrices at a flow rate of 5 ul/min. The binding value of each serum or mAb was measured in resonance units (RU), and the “native”/denatured gp 120 ratios were determined by dividing the corresponding RU values and correcting for small differences in matrix concentration. Control included an HIV-positive patient serum and mAb 1c1.
Assessment of HIV-specific cell-mediated immunity in immunized mice by secondary CTL assay. The spleens of BALB/c mice immunized on days 0 and 21 were harvested and single cell suspensions prepared aseptically in complete RPMI medium on day 35. The cells were then pooled within experimental groups (n=4), underlay with ficoll, centrifuged 30 min at 450×g (RT), washed, and resuspended in complete RPMI medium. Following a 1 h stimulation with peptide p18 (1 uM) at 37° C., the cell suspensions were diluted with complete RPMI supplemented with 2ME (1:1000) and transferred to flasks for a 6 day incubation at 37° C. After 2 days, recombinant IL-2 (10 u/ml) was added to all flasks. On day 6, P815 target cells were pulsed with peptide p18 (1 uM) or with nothing (control) in PBS supplemented with 0.1% BSA. 3×10A6 target cells were labelled with 300 uCi of 51Cr, washed, and plated out with the effector cells at effector:traget (E:T) ratios of 45:1, 15:1, 5:1, and 1.7:1. After a 6 h incubation at 37° C., the supernatants were harvested and counted, and % specific lysis was calculated.
Comparison of the native (oligomeric) gp 160 prior to microencapsulation and following spontaneous release from PLG microspheres showed the two to be essentially indistinguishable in terms of their binding to CD4 and recognition by HIV-positive patient serum. (Table 1). This retention of conformation-dependent binding shows that structure of the antigen is not appreciably altered by the microencapsulation process.
Materials and Methods
This experiment was similar to that described in Example 1 except for the method of microencapsulation employed.
Microencapsulation of immunogens: PLG microspheres ranging from 1 to 15 um in diameter and containing a 0.5 to 1.0% antigen core load were prepared by a solvent evaporation method. The solvent evaporation method involves emulsifying the viral antigen dissolved in deionized water into poly(DL-lactide-co-glycolide) polymer dissolved in methylene chloride. This emulsion is mixed into 0.9% polyvinyl alcohol and stirred. After 10 min of stirring, 0.35 l of water is added and gentle mixing is continued for 1.5 h. The resulting spheres are harvested by centrifugation, washed three times in distilled water, and dried overnight under vacuum. Microsphere size was determined by both light and scanning electron microscopy. The antigen core load was determined by quantitative amino acid analysis of the microspheres following complete hydrolysis in 6N hydrochloric acid.
Analysis of spontaneously released antigen showed it to retain its CD4 binding capacity. Its native antigenicity (recognition by the serum of an HIV-positive patient) was only slightly less than that of the antigen prior to encapsulation and following spontaneous release from microspheres produced by a solvent extraction method (Table 1).
The results of immunizing animals with either non-native (denatured) or native oligomeric gp 160 in PLG microspheres produced by a solvent evaporation method were essentially indistinguishable from those obtained using microspheres produced by a solvent extraction method (example 1). Microencapsulated antigen induced significantly greater CTL activity than antigen administered in a conventional alum-adjuvanted formulation. Furthermore, preservation of the structure of PLG-microencapsulated antigens is supported by the findings of preferential binding of antibodies elicited by microspheres loaded with denatured antigen to denatured gp 120 (
BIA (released o-gp160)
Capture o-gp160-451 (stock vs microsphere-released)
on tvc 391 fc3/fc4 sCD4 (4 mg/m)
1 ul/min flow rate foe o-gp160 ini.: 5 ul/min for all others
HIV+/sCD4 (RU ratio)
HIV+ pool 1:100
HIV+ pool 1:100
In view of the above it will be seen that the objects of the invention are achieved. As various changes could be made in the above materials and methods without departing from the scope of the invention, it is intended that all matter contained in the above description shall be interpreted as illustrative and not limiting.
|Cited Patent||Filing date||Publication date||Applicant||Title|
|US3540444||15 Jan 1968||17 Nov 1970||Scherer Corp R P||Plastic ampoule for use with hypodermic injector|
|US3773919||8 Oct 1970||20 Nov 1973||Du Pont||Polylactide-drug mixtures|
|US3788315||20 Apr 1971||29 Jan 1974||S Laurens||Disposable cutaneous transjector|
|US4166800||31 Jul 1978||4 Sep 1979||Sandoz, Inc.||Processes for preparation of microspheres|
|US4384975||13 Jun 1980||24 May 1983||Sandoz, Inc.||Process for preparation of microspheres|
|US4389330 *||6 Oct 1980||21 Jun 1983||Stolle Research And Development Corporation||Microencapsulation process|
|US4530840||29 Jul 1982||23 Jul 1985||The Stolle Research And Development Corporation||Injectable, long-acting microparticle formulation for the delivery of anti-inflammatory agents|
|US4542025||30 Apr 1984||17 Sep 1985||The Stolle Research And Development Corporation||Injectable, long-acting microparticle formulation for the delivery of anti-inflammatory agents|
|US4585482||25 May 1984||29 Apr 1986||Southern Research Institute||Long-acting biocidal compositions and method therefor|
|US4622244||23 Aug 1984||11 Nov 1986||The Washington University||Process for preparation of microcapsules|
|US4637905||11 Apr 1985||20 Jan 1987||Batelle Development Corporation||Process of preparing microcapsules of lactides or lactide copolymers with glycolides and/or ε-caprolactones|
|US4675189||8 Feb 1985||23 Jun 1987||Syntex (U.S.A.) Inc.||Microencapsulation of water soluble active polypeptides|
|US4798786||6 May 1982||17 Jan 1989||Stolle Research And Development Corporation||Living cells encapsulated in crosslinked protein|
|US4835139||19 May 1987||30 May 1989||Debiopharm S.A.||Process for increasing the antagonistic effect of peptidic compounds on hormone-dependent diseases|
|US4863735||2 Oct 1986||5 Sep 1989||Massachusetts Institute Of Technology||Biodegradable polymeric drug delivery system with adjuvant activity|
|US4897268||3 Aug 1987||30 Jan 1990||Southern Research Institute||Drug delivery system and method of making the same|
|US4938763||3 Oct 1988||3 Jul 1990||Dunn Richard L||Biodegradable in-situ forming implants and methods of producing the same|
|US4941880||12 Dec 1988||17 Jul 1990||Bioject, Inc.||Pre-filled ampule and non-invasive hypodermic injection device assembly|
|US5000886||26 May 1987||19 Mar 1991||American Cyanamid Company||Silicone-hardened pharmaceutical microcapsules and process of making the same|
|US5019096||14 Oct 1988||28 May 1991||Trustees Of Columbia University In The City Of New York||Infection-resistant compositions, medical devices and surfaces and methods for preparing and using same|
|US5059187||4 May 1990||22 Oct 1991||Dey Laboratories, Inc.||Method for the cleansing of wounds using an aerosol container having liquid wound cleansing solution|
|US5064413||9 Nov 1989||12 Nov 1991||Bioject, Inc.||Needleless hypodermic injection device|
|US5075109||18 Mar 1988||24 Dec 1991||Southern Research Institute||Method of potentiating an immune response|
|US5102872||12 Aug 1988||7 Apr 1992||Cetus Corporation||Controlled-release formulations of interleukin-2|
|US5129825||10 Oct 1989||14 Jul 1992||Discko John Jr||Dental syringe and capsule for use therewith|
|US5133701||15 Oct 1990||28 Jul 1992||Sang In Han||Disposable pressure wound irrigation device|
|US5236355||5 Oct 1990||17 Aug 1993||American Cyanamid Company||Apparatus for the treatment of periodontal disease|
|US5278202||23 Dec 1991||11 Jan 1994||Atrix Laboratories, Inc.||Biodegradable in-situ forming implants and methods of producing the same|
|US5290494||16 Jul 1992||1 Mar 1994||Board Of Regents, The University Of Texas System||Process of making a resorbable implantation device|
|US5360610||15 Mar 1993||1 Nov 1994||Southern Research Institute||Method for stimulating nerve fiber growth|
|US5384133||29 Jun 1993||24 Jan 1995||Innovata Biomed Limited||Pharmaceutical formulations comprising microcapsules|
|US5407609||17 May 1993||18 Apr 1995||Southern Research Institute||Microencapsulation process and products therefrom|
|US5417986||10 Apr 1992||23 May 1995||The United States Of America As Represented By The Secretary Of The Army||Vaccines against diseases caused by enteropathogenic organisms using antigens encapsulated within biodegradable-biocompatible microspheres|
|US5429822||13 Mar 1992||4 Jul 1995||Cambridge Scientific, Inc.||Biodegradable bursting release system|
|US5500228||26 Nov 1990||19 Mar 1996||American Cyanamid Company||Phase separation-microencapsulated pharmaceuticals compositions useful for alleviating dental disease|
|US5538739||18 Jan 1991||23 Jul 1996||Sandoz Ltd.||Sustained release formulations of water soluble peptides|
|US5639480||6 Jun 1995||17 Jun 1997||Sandoz Ltd.||Sustained release formulations of water soluble peptides|
|US5643605||2 Jun 1995||1 Jul 1997||Genentech, Inc.||Methods and compositions for microencapsulation of adjuvants|
|US5648096||22 Jun 1994||15 Jul 1997||Schwarz Pharma Ag||Process for the production of microcapsules|
|US5650173||3 Oct 1996||22 Jul 1997||Alkermes Controlled Therapeutics Inc. Ii||Preparation of biodegradable microparticles containing a biologically active agent|
|US5688530||6 Jun 1995||18 Nov 1997||Novartis Ag||Sustained release formulations of water soluble peptides|
|US5693343||16 May 1994||2 Dec 1997||The United States Of America As Represented By The Secretary Of The Army||Microparticle carriers of maximal uptake capacity by both M cells and non-M cells|
|US5762965||9 Feb 1996||9 Jun 1998||The United States Of America As Represented By The Secretary Of The Army||Vaccines against intracellular pathogens using antigens encapsulated within biodegradble-biocompatible microspheres|
|US5811128||7 Sep 1993||22 Sep 1998||Southern Research Institute||Method for oral or rectal delivery of microencapsulated vaccines and compositions therefor|
|US5814344||6 Jun 1995||29 Sep 1998||Southern Research Institute||Method for delivering bioactive agents into and through the mucosally associated lymphoid tissues and controlling their release|
|US5820883||6 Jun 1995||13 Oct 1998||Southern Research Institute||Method for delivering bioactive agents into and through the mucosally-associated lymphoid tissues and controlling their release|
|US5853763||6 Jun 1995||29 Dec 1998||Southern Research Institute||Method for delivering bioactive agents into and through the mucosally-associated lymphoid tissue and controlling their release|
|EP0052510B2||17 Nov 1981||19 Oct 1994||Syntex (U.S.A.) Inc.||Microencapsulation of water soluble polypeptides|
|WO1992022654A1 *||10 Jun 1992||23 Dec 1992||Microgenesys, Inc.||Vaccine and treatment method of human immunodeficiency virus infection|
|WO1995011010A1 *||13 Oct 1994||27 Apr 1995||Genentech, Inc.||Methods and compositions for microencapsulation of antigens for use as vaccines|
|1||Biotechnology News, Aug. 22, 1997, vol. 17, No. 20, Topical DNA vaccine elicits immune response.|
|2||Brown, A hypothetical model of the foreign antigen blinding site of Class II histocompatibility molecules, Nature, vol. 332, Apr. 28, 1988, pp. 845-850.|
|3||Brown, Wonder Drugs' Losing Healing Aura, The Washing Post, Jun. 26, 1995, A section.|
|4||Cassels, et al., Analysis of Escherichia coli Colonization Factor Antigen I Linear B-Cell Epitopes, as Determined by Primate Responses, following Protein Sequence Verification, Infection and Immunity, Jun. 1992, pp. 2174-2181, vol. 60, No. 6.|
|5||Evans, et al. Purification and Characterization of the CFR/1 Antigen of Enterotoxigenic Escherichia coli, Infection and Immunity, Aug. 1979, pp. 738-748, vol. 25.|
|6||Gilding, Biodegradable polymers for use in surgery-polyglycolic/poly (ac c acid) homo-and copolymers: 1, Polymer, vol. 20, Dec. 1979, pp. 1459-1464.|
|7||Hall, et al., Purification and Analysis of Colonization Factor Antigen 1, Coli Surface Antigen 1, and Coli Surface ANtigen 3 Fimbriae from Enterotoxigenic Escherichis Coli, Journal of Bacteriology, Nov. 1989, pp. 6372-6374, vol. 171, No. 11.|
|8||Jeyanthi, et al., Novel, Burst Free Programmable Biodegradable Microspheres For Controlled Release of Polypeptides, Proceedings Int. Symp. control Release Bioact. Mater. (1996) pp. 351-362/.|
|9||Jordi, et al., Analysis of the first two genes of the CSI fimbrial operon in human enterotoxigenic Escherichia coli of serotype 0139: H28, FEMS Microbiology Letters 80, (1991) pp. 265-270.|
|10||Karjalainen, et al., Molecular Cloning and Nucleotide Sequence of the Colonization Factor Antigen I Gene of Escherichia coli, Infection and Immunity, Apr. 1989, pp. 1126-1130, vol. 57.|
|11||Maister, First Oral AIDS Vaccine Trials Near, BioWorld Today, Tuesday, Apr. 19, 1994, p. 4.|
|12||McConnel, et al., Antigenic homology within human enterotoxigenic Esherichia coli fimbrial colonization factor antigens: CFA/I, coli-surface-associated antigens (CS)1, CS2, CS4 and CS17, FEMS Microbiology Letters 61 (1989) 105-108.|
|13||Perez-Casal, et al., Gene Encoding the Major Subunit of CS1 Pili of Human Enterotoxigenic Escherichia Coli, Infection and Immunity, Nov., 1990, pp. 3594-3600, vol. 58, No. 11.|
|14||Rognan, et al., Molecular Modeling of an Antigenic Complex Between a Viral Peptide and a Class I Major Histocompatibility Glycoprotein, Proteins Structure, Function and Genetics 13 70-85 (1992).|
|15||Romagnoli, et al. Peptide-MHC Interaction: A Rational Approach to Vaccine Design, Inter, RE. Immunol. 6, 1990, 00 61-73.|
|16||Setterstrom, Controlled Release of Antibiotics From biodegradable Microcapsules For Wound Infection Control, Chemical Abstracts, 1983, pp. 215-226.|
|17||Tan, et al., Mapping the Antigenic Epitopes of Human Dihydrofolate Reductase by Systematic Synthesis of Peptides on solid Supports, The Journal of Biological Chemistry, vol. 265, No. 14, Issue of May 15, pp. 8022-8026 (1990).|
|18||Van der Zee, Efficient mapping and characterization of a T cell epitope by the simultaneous synthesis of multiple peptides, Eur. J. Immunol. 1989, 19: 43-47.|
|19||Wang, et al., Influence of formulation methods on the in vitro controlled release of protein from poly (ester) microspheres Journal of Controlled Release, 17 (1991) 23-32.|
|20||Yan, Characterization and morphological analysis of protein-loaded poly(lactide-co-glycolide) microparticles prepared by watewr-in-oil-in-water emulsion technique, Journal of Controlled Release, 32 (1994) 231-241.|
|21||Yeh, A novel emulsification-solvent extraction technique for production of protein loaded biodegradable microparticles for vaccine and drug delivery, Journal of Controlled Release 33 (1995) 437-445.|
|U.S. Classification||424/499, 424/455, 424/488, 424/426, 424/486, 424/422|
|International Classification||A61K9/16, A61K9/00, A61K38/00, C07K14/245, C07K14/195, A61K9/50|
|Cooperative Classification||A61K9/5031, C07K14/245, A61K38/00, A61K9/1647|
|European Classification||C07K14/245, A61K9/16H6D4, A61K9/50H6D|
|20 Feb 2009||AS||Assignment|
Owner name: ARMY, UNITED STATES, MARYLAND
Free format text: ASSIGNMENT OF ASSIGNORS INTEREST;ASSIGNORS:BURNETT, PAUL R.;VAN HAMONT, JOHN E.;REID, ROBERT H.;ANDOTHERS;REEL/FRAME:022293/0134;SIGNING DATES FROM 19960715 TO 19960823
|11 Jan 2010||REMI||Maintenance fee reminder mailed|
|6 Jun 2010||LAPS||Lapse for failure to pay maintenance fees|