EP1858547A1 - Compositions immunogènes - Google Patents

Compositions immunogènes

Info

Publication number
EP1858547A1
EP1858547A1 EP05782989A EP05782989A EP1858547A1 EP 1858547 A1 EP1858547 A1 EP 1858547A1 EP 05782989 A EP05782989 A EP 05782989A EP 05782989 A EP05782989 A EP 05782989A EP 1858547 A1 EP1858547 A1 EP 1858547A1
Authority
EP
European Patent Office
Prior art keywords
antigen
pharmaceutical composition
gene vector
microspheres
immune response
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP05782989A
Other languages
German (de)
English (en)
Inventor
José Maciel RODRIGUES, Jr.
Karla De Melo Lima
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Nanocore Biotecnologia SA
Original Assignee
Nanocore Biotecnologia SA
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Filing date
Publication date
Application filed by Nanocore Biotecnologia SA filed Critical Nanocore Biotecnologia SA
Publication of EP1858547A1 publication Critical patent/EP1858547A1/fr
Withdrawn legal-status Critical Current

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Classifications

    • 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
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/53—DNA (RNA) vaccination
    • 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
    • A61K2039/545—Medicinal preparations containing antigens or antibodies characterised by the dose, timing or administration schedule
    • 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

Definitions

  • the present invention is related to a novel formulation of immunogenic composition.
  • This composition contains antigens in different forms and an adjuvant, both entrapped in biodegradable microspheres. Principles of the invention:
  • biodegradable polymers such as lactic acid polymers (PLA) and co-polymers of lactic and glycolic acid (PLGA) with different characteristics of degradation allowed the development of new drug and vaccine delivery systems.
  • PLA lactic acid polymers
  • PLGA co-polymers of lactic and glycolic acid
  • microspheres prepared with these co ⁇ polymers in the development of vaccines has allowed reaching different immunogenic compositions with optimization of antigen presentation to the immune system.
  • This technique has been applied to protect the antigen and to form a depot in the site of administration resulting in the recruitment of immune competent cells.
  • Particles lower than 10 micrometers are selectively taken up by antigen presenting cells, which can optimize the immune response. Particles with higher diameters can be desirable in order to slowly release the antigen in the site of administration.
  • antigen may be a purified subunit, a protein or peptide, and a gene vector like plasmid DNA.
  • mice and guinea pigs have failed to be effective in large animals, probably due to the need of optimization of formulation, schedule, dose and also the antigenic composition.
  • the patent BR-PI0103887 describes the use of a vaccine obtained from microspheres formulation containing at least one antigen, for example a gene vector or a purified protein, associated to an adjuvant.
  • the protein model was a mycobacterial 65kDa heat shock protein, hsp-65, entrapped in its recombinant form or in the form of a gene vector that codify it.
  • the gene vector or the purified protein is presented entrapped into PLGA microspheres formulations associated to an adjuvant, for example the trehalose dymicolate (TDM) .
  • TDM trehalose dymicolate
  • the formulation described in the patent BRPI0103887 can be used in the prevention or treatment infectious diseases where the induction of protective immune response is dependent of the production of pro-inflammatory cytokines such as gamma-interferon.
  • the duration of protection resulted from vaccination is associated to the residence time of the antigen in the body. In some vaccination schedules it is necessary the administration of boosters in order to sustain the protective immune response.
  • the booster has the function of expanding and maintain the immune response initially stimulated.
  • prime-boost protocols involve the use of DNA vaccines or purified subunits during the initial phase, using live carriers, such as recombinant virus or bacteria expressing the desired antigen, in the boosters.
  • the prime-boost strategy involves the use of different vaccines, each one codifying the same antigen, administered in days or weeks intervals.
  • Prime boost vaccines power up in people. Wat. Med., 9 (6) : 642-643; Cheevers, W.P., Snekvik, K.R., Trujillo, J.D., Kumpula-McWhirter, N.M., Pretty, On Top K.J., Knowles, D.P. (2003) Prime-boost vaccination with plasmid DNA encoding caprine-arthritis encephalitis lentivirus env and viral SU suppresses challenge virus and development of arthritis.
  • HCV hepatitis C virus
  • the prime-boost strategy has been evaluated combining the priming with plasmid DNA and the maintenance of the immune response with attenuated BCG or recombinant protein (McShane, H., Brookes, R., Gilbert, S.C, Hill, A.V. (2001) Enhanced immunogenicity of CD4(+) t-cell responses and protective efficacy of a DNA-modified vaccinia virus Ankara prime- boost vaccination regimen for murine tuberculosis.
  • a DNA prime-live vaccine boost strategy in mice can augment IFN-gamma responses to mycobacterial antigens but does not increase the protective efficacy of two attenuated strains of Mycobacterium bovis against bovine tuberculosis. Immunology;108 (4) -.548-555) .
  • the patent BR-PI0103887 additionally describes that PLGA microspheres using lactide and glycolide co-polymers co-encapsulating DNA-hsp65 and the adjuvant TDM are able to protect mice from the challenge with a virulent strain of Mycobacterium tuberculosis after a single dose. More details on this vaccine are presented by Lima et al. (Lima, K.M., Santos, S.A., Lima, V.M., Coelho-Castelo, A.A., ' PCT/BR2005/000179
  • This formulation allowed the reduction of the number of injections and also the dose of DNA, after verifying that the entrapment of DNA in microspheres protects DNA from degradation by nucleases in vivo.
  • the evidence of the functionality of plasmid DNA allows concluding that the DNA is released in the cytosol and it is able to reach the cell nucleus.
  • the patent describes the use of DNA or recombinant protein entrapped into PLGA microspheres in association with trehalose dymicolate. Otherwise, the patent does not describe the concomitant use of protein and DNA associated in the same formulation.
  • the polymers composed by lactic and glycolic monomers present an essential condition for the development of single-shot vaccines exploring the prime- boost concept, containing protein and DNA in the same formulation.
  • Figure 1 illustrates the kinetic of recombinant protein release from PLGA 50:50 (A) and PLGA 75:25 (B) microspheres.
  • Figure 2 illustrates the cytokine profile in calves after vaccination with DNA-hsp65 in different formulations.
  • Figures 3a and 3b illustrate the antibody profiles in calves after vaccination with DNA-hsp65 in different formulations: (a) IgGl, (b) IgG2a. Detailed Description of the invention
  • This present invention refers to the formulation of novel immunogenic compositions containing antigens in different forms entrapped in biodegradable microspheres containing adjuvants.
  • This present invention describes an immunogenic composition that includes in a single dose the association of microspheres containing one antigen in the form of DNA vaccine and a second antigen in the form of subunit protein.
  • This composition was able to induce a specific immune response of ThI pattern which was sustained over the evaluated period.
  • the DNA is entrapped in PLGA microspheres with higher hydrophilic degree allowing its release from microspheres in a period inferior to 30 days, before total protein release.
  • the protein was entrapped in higher hydrophobic degree PLGA microspheres presenting kinetic release in a period superior to 30 days.
  • the mixture of both formulations permits, after a single dose administration, the faster release of DNA from microspheres stimulating the immune system to trigger a response characterized by the production of gamma- interferon and IgG2a subtype against the antigen.
  • the posterior release of the antigen in the protein form, entrapped in slow release polymers, has the ability to induce the expansion of the subpopulations primed by the DNA acting as a booster.
  • this formulation allows jointing the prime and booster doses in the same formulation allowing the reduction of costs, more successful vaccination programs and increased coverage.
  • This ability to induce an immune response can be attributed to the stimulation of CD4+ cells that produce gamma-interferon and cytotoxic CD8+ cells.
  • the process of entrapment of hsp65 and/or DNA-hsp65 into microspheres does not compromise its ability to induce the immune response.
  • This invention presents an immunogenic composition, which is composed by, at least one gene vector and one protein, associated or not to an adjuvant, entrapped into biodegradable microspheres with an average diameter inferior to 20 micrometers, preferentially with an average diameter comprised between 0.5 and 10 micrometers.
  • the composition can be alternatively composed by one protein and a gene vector and an adjuvant.
  • the antigens are preferentially antigens from parasites and other pathogenic agents including viral antigens, intra and extra cellular bacterial antigens.
  • the adjuvants are preferentially those that stimulate pro-inflammatory cytokines.
  • the antigens of the present invention may be entrapped in PLGA microspheres with rate of PLA and PGA monomers varying from 00 to 100 and 100 to 00.
  • the plasmid that codify the Mycobacterium protein such as the gene that codify the hsp65 and also the protein itself may be entrapped in the purified or recombinant form, or peptides derived of the same protein or yet antigens that can stimulate the immune response taking profit of the prime boost technology.
  • the formulation can also contain trehalose dymicolate entrapped in the same formulation of PLGA microspheres.
  • Theses systems containing trehalose dymicolate and the plasmid DNA containing the gene that codify the hsp ⁇ protein and also the hsp ⁇ protein, can be used as vaccine against tuberculosis. Additionally this formulation can be also applied in the therapy of tuberculosis when the disease is already installed or in diseases that requires the induction of a ThI pattern to be controlled.
  • microencapsulation of a gene vector, antigen and adjuvant can be obtained as the following protocols:
  • Protocol A (i) dissolution of polymeric material composed by lactic and glycolic acids derivatives (relation in % of weight ranging from 100:00 to 00:100) into an immiscible or partially immiscible organic solvent in water.
  • concentration of polymer in this solution varies from 0.2 a 10%.
  • Methylene chloride, ethyl acetate and Chloroform among others, are examples of organic solvents used in this process;
  • solubilization of lipophilic antigen and adjuvant in the polymer solution (iii) addition of the solution obtained at (ii) into an aqueous phase constituted by a polyol, eg.
  • Polyvinyl alcohol to obtain an oil-in-water emulsion; (iv) agitation of the emulsion in an appropriate velocity (200 a 1000 rpm) allowing the formation of small microspheres with diameter smaller than 10 micrometer, preferentially ranging from 0.5 to 5 micrometer and e; (v) solvent evaporation to form a microspheres suspension and microspheres recovery optionally by centrifugation or filtration; (vi) Addition of a' second antigen (soluble or dispersed into the aqueous phase) and water elimination by liophilization or other drying method.
  • Protocolo B (i) dissolution of polymeric material composed by lactic and glycolic acids derivatives (relation in % of weight ranging from 100:00 to 00:100) into an immiscible or partially immiscible organic solvent in water.
  • concentration of polymer in this solution varies from 0.2 a 10%.
  • Methylene chloride, ethyl acetate and Chloroform among others, are examples of organic solvents used in this process.
  • Lipophilic substances as antigens and adjuvants should be added in this solution; (ii) solubilization of genetic vector, antigen and/or adjuvant with hydrophilic nature into an aqueous phase which volume ranges from 0.1 to ImL; (iii) addition of the solution obtained at (ii) into the polymeric solution obtained at (i) and agitation with -rotation ranging from 500 to 15,000 rpm, to form an water-in-oil emulsion or dispersion; (iv) addition of the water-in-oil emulsion or dispersion to an emulsifing aqueous phase, constituted by a polyol, eg.
  • Polyvinyl alcohol to obtain an water-in-oil-in-water double emulsion; (iv) agitation of the emulsion in an appropriate velocity allowing the formation of small microspheres with diameter smaller than 10 micrometer, preferentially ranging from 0.5 to 5 micrometer and e; (v) solvent evaporation to form a microspheres suspension and microspheres recovery optionally by centrifugation or filtration; (vi) and water elimination by liophilization or other drying method.
  • the Table II summarizes the entrapment rate and average diameter of microspheres containing protein, plasmid DNA and trehalose dimicolate.
  • the pCDNA3-hsp65 construct was derived from the pCDNA3 vector (Invitrogen®, Carlsbad, CA, USA) , previously digested with Bam HI and Not I (Gibco BRL, Gaithersburg, MD, USA); a 3.3 kb fragment, corresponding to the M. leprae hsp65 gene and the CMV intron A, was then inserted in the vector. Plasmid pCDNA3 without the hsp65 gene was used as control. DH5 ⁇ E.
  • coli transformed with plasmid pCDNA3 or the plasmid containing the hsp ⁇ 5 gene (pCDNA3-hsp65) was cultured in LB liquid medium (Gibco BRL, Gaithersburg, MD, 2005/000179
  • E. coli BL21 transformed with the plasmid containing the mycobacterial hsp65 gene was cultured in LB medium containing ampicillin (100 ⁇ g/ ⁇ L) .
  • the bacterial growing was monitored by spectrophotometry at ⁇ 600nm in a Shimadzu UV-I650 spectrophotometer. When the OD reached the value of 0.6, the culture was induced with 0.1M of IPTG (Gibco, BRL, Gaithersburg, MD, USA) and incubated at 30°C under agitation for 4 hours. The cells were harvested centrifugation, suspended in citrate buffer and disrupted by sonication.
  • Microspheres were obtained by the double emulsion/solvent evaporation technique. Briefly, 30 ml dichloromethane solution containing 400 mg of polymer PLGA 50:50 or PLGA 75:25 (Resomerfrom Boehringer Ingelheim, Ingelheim, Germany) was emulsified with 0.3 ml of an inner aqueous phase containing 5 mg of DNA (pCDNA3 or pCDNA3- hsp65) or 1 mg of recombinant hsp65 protein using a T25 Ultraturrax homogenizer (IKA® - Labortechnik, Germany) to produce a primary water-in-oil emulsion.
  • pCDNA3 or pCDNA3- hsp65 DNA
  • recombinant hsp65 protein recombinant hsp65 protein
  • Microspheres were obtained by the double emulsion/solvent evaporation technique. Briefly, 30 ml dichloromethane solution containing 400 mg of polymer PLGA 50:50 or PLGA 75:25 (Resomerfrom Boehringer Ingelheim, Ingelheim, Germany) was emulsified with 0.3 ml of an inner aqueous phase containing 5 mg of DNA (pCDNA3 or pCDNA3-hsp65) or 1 mg of recombinant hsp65 protein using a T25 Ultraturrax homogenizer (IKA® - Labortechnik, Germany) to produce a primary water-in-oil emulsion.
  • pCDNA3 or pCDNA3-hsp65 DNA
  • recombinant hsp65 protein recombinant hsp65 protein
  • the recombinant hsp65 protein was initially entrapped into PLGA 50:50 or PLGA 75:25 microspheres.
  • the kinetics of protein release from microspheres was evaluated by ressuspending 30 mg of protein-loaded microspheres in 3 mL of PBS containing sodium azide (0.05% w/v) .
  • the suspension was maintained at 37 0 C under constant agitation at 200 rpm.
  • samples of the supernatant (0.1 mL) were collected and replaced with fresh buffer.
  • the protein concentration in the supernatant was determined by using the Comassie Reagent.
  • the evaluation of the in vitro protein release kinetic from these particles allowed the selection of the more suitable formulation able to sustain the immune response.
  • a new vaccine composed by PLGA 75:25 microspheres containing recombinant protein plus PLGA 50:50 microspheres containing the plasmid (DNA-hsp65) and an adjuvant was able to elicit a sustained immune response. This effect is better observed when the protein is associated to plymers which present molar ratio greater that 50% in lactic acid, preferentialy PLGA 75:25 or PLGA 85:15.
  • Polymer with 50% of lactic acid (PLGA 50:50) presented a fast protein release profile, releasing 80% of entrapped protein over five days.
  • PLGA 75:25 microspheres were able to sustain the protein release over a longer time interval. This effect could be attributed to the lower hydrophilic characteristic of PLGA 75:25 which are associated to less glycolic acid in polymeric chain.
  • EXAMPLE 6 Evaluation of immune response in vaccinated cattle.
  • Nelore - Red Angus- Senepol crossbred calves were divided in four groups of 3 animals. Background levels of in vitro IFN- ⁇ response against mammalian and avian tuberculin PPD were, determined prior to they purchase and non-responding animals were selected.
  • the animals were immunized by intramuscular injection of the following formulations: 3 doses of naked DNA-hsp ⁇ 5 in saline administered in a 2 weeks time interval/ a single dose of microspheres-based formulations.
  • the 2 microspheres formulation evaluated were DNA-hsp65 plus TDM-loaded microspheres and the prime-boost microspheres as described on Table I.
  • the cellular immune response was evaluated by cell proliferation and interferon gamma secretion using ELISPOT.
  • the humoral immune response was evaluated by the measurement of anti-hsp65 specific antibody in serum. Since the encapsulation of antigen into PLGA microspheres allows the development of controlled release delivery systems, where we can design the release profile of the encapsulated material, we evaluated the use of a vaccine formulation based on a mixture of two different PLGA microspheres, composed by faster and slower release profiles, containing DNA encoding hsp65 and the recombinant hsp65 protein respectively, aiming at DNA priming and protein boost after a single dose vaccination.
  • Prime-Boost formulation was able to induce high levels of anti-hsp65 specific antibodies. The serum levels of these specific antibodies remaining high 90 days after vaccination while the DNA-Me formulation was not able to sustain the antibodies levels in the same fashion. Production of IFN- ⁇ was significant in mice vaccinated with DNA-Me and Prime-Boost formulations being that Prime-Boost vaccinated mice sustained the high levels of this cytokine during all period evaluated. Moreover, when the same Prime- Boost formulation was applied to cattle, we showed for the first time, that a DNA vaccine was able to elicit humoral and cellular specific immune response after a single-dose administration in large animals ( Figures 2 and 3) .

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  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Mycology (AREA)
  • Epidemiology (AREA)
  • Immunology (AREA)
  • Medicinal Chemistry (AREA)
  • Microbiology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Pharmacology & Pharmacy (AREA)
  • General Chemical & Material Sciences (AREA)
  • Animal Behavior & Ethology (AREA)
  • General Health & Medical Sciences (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
  • Medicinal Preparation (AREA)

Abstract

L'invention concerne une composition pharmaceutique capable d'induire une réponse immunitaire durable et dont la formulation est composée de microsphères biodégradables polymères contenant un vecteur antigénique et un vecteur génique, éventuellement associé à un adjuvant et dont la formulation est capable de libérer le vecteur génique pendant une période inférieure à la libération de l'antigène. La libération du matériau piégé se prolonge pendant une période qui n'est pas inférieure à 30 jours. La composition est capable d'induire une réponse immunitaire, cellulaire et humorale spécifique après administration d'une ou de plusieurs doses.
EP05782989A 2004-09-03 2005-09-05 Compositions immunogènes Withdrawn EP1858547A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
BRPI0404983 BRPI0404983A (pt) 2004-09-03 2004-09-03 composições imunogênicas
PCT/BR2005/000179 WO2006024122A1 (fr) 2004-09-03 2005-09-05 Compositions immunogenes

Publications (1)

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EP1858547A1 true EP1858547A1 (fr) 2007-11-28

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EP05782989A Withdrawn EP1858547A1 (fr) 2004-09-03 2005-09-05 Compositions immunogènes

Country Status (5)

Country Link
EP (1) EP1858547A1 (fr)
AU (1) AU2005279641A1 (fr)
BR (1) BRPI0404983A (fr)
WO (1) WO2006024122A1 (fr)
ZA (1) ZA200702752B (fr)

Family Cites Families (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6461643B2 (en) * 1993-04-22 2002-10-08 Emisphere Technologies, Inc. Oral drug delivery compositions and methods
WO1999016466A2 (fr) * 1997-09-29 1999-04-08 Beth Israel Deaconess Medical Center Compositions de vaccin et procedes servant a augmenter l'efficacite d'un vaccin
WO2000004876A2 (fr) * 1998-07-23 2000-02-03 Johns Hopkins University School Of Medicine Liberation controlee de substances bioactives
US6998393B2 (en) * 2000-06-23 2006-02-14 Biopharm Solutions, Inc. Aquespheres, their preparation and uses thereof
CA2476589C (fr) * 2002-02-27 2014-02-25 Pharmain, Ltd. Compositions pour l'administration de substances therapeutiques et analogues, et procedes de fabrication et d'utilisation desdites compositions

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
See references of WO2006024122A1 *

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Publication number Publication date
AU2005279641A1 (en) 2006-03-09
WO2006024122A1 (fr) 2006-03-09
ZA200702752B (en) 2008-09-25
BRPI0404983A (pt) 2006-05-02

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