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WO2009125332A1 - New and safe procedure for immunization against infectious diseases agents - Google Patents

  • ️Thu Oct 15 2009

WO2009125332A1 - New and safe procedure for immunization against infectious diseases agents - Google Patents

New and safe procedure for immunization against infectious diseases agents Download PDF

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Publication number
WO2009125332A1
WO2009125332A1 PCT/IB2009/051437 IB2009051437W WO2009125332A1 WO 2009125332 A1 WO2009125332 A1 WO 2009125332A1 IB 2009051437 W IB2009051437 W IB 2009051437W WO 2009125332 A1 WO2009125332 A1 WO 2009125332A1 Authority
WO
WIPO (PCT)
Prior art keywords
cells
bao
antigens
cell
viruses
Prior art date
2008-04-07
Application number
PCT/IB2009/051437
Other languages
French (fr)
Inventor
Peter Bromley
Jean-Jacques Sunier
Original Assignee
Peter Bromley
Jean-Jacques Sunier
Priority date (The priority date 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 date listed.)
2008-04-07
Filing date
2009-04-06
Publication date
2009-10-15
2009-04-06 Application filed by Peter Bromley, Jean-Jacques Sunier filed Critical Peter Bromley
2009-10-15 Publication of WO2009125332A1 publication Critical patent/WO2009125332A1/en

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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
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/067—Hepatocytes
    • 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
    • A61K2039/515—Animal cells
    • A61K2039/5156—Animal cells expressing foreign proteins
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/0019—Injectable compositions; Intramuscular, intravenous, arterial, subcutaneous administration; Compositions to be administered through the skin in an invasive manner
    • A61K9/0024—Solid, semi-solid or solidifying implants, which are implanted or injected in body tissue
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2510/00—Genetically modified cells
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2510/00—Genetically modified cells
    • C12N2510/04—Immortalised cells
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2830/00—Vector systems having a special element relevant for transcription
    • C12N2830/001—Vector systems having a special element relevant for transcription controllable enhancer/promoter combination
    • C12N2830/002—Vector systems having a special element relevant for transcription controllable enhancer/promoter combination inducible enhancer/promoter combination, e.g. hypoxia, iron, transcription factor
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • the swine influenza (flu) vaccine was associated with a severe paralytic illness called Guillain-Barre Syndrome (GBS).
  • GBS Guillain-Barre Syndrome
  • Some live virus vaccines such as the chickenpox vaccine, can cause mild versions of the disease they protect against, but this is usually only a serious problem if the patient has a severely compromised immune system.
  • Subunit vaccines raised against purified antigens of infectious agents have had some success, but a problem with this approach is that purifying the infectious agent, isolating the surface and other antigens, purifying these proteins etc, frequently changes the presentation form of these antigens when subsequently injected into patients.
  • the technology of this invention totally avoids these drawbacks; the subunit vaccines are produced by the cells in the Bio- artificial organs (BAO), are secreted in their natural format, with all associated post- translational modifications, ensuring an effective immune response.
  • BAO Bio- artificial organs
  • This invention ensures that infectious organism antigens capable of eliciting neutralizing antibody formation, are correctly formed, and presented to the immune system in an optimal manner. This invention avoids alterations to such sub-unit vaccines that result from purification procedures and storage prior to employment.
  • This new vaccination procedure of this invention can be applied to essentially all vaccination needs today, these include childhood infections, tropical diseases and cancer vaccines, and nosocomial infections.
  • childhood vaccines that can be treated using this invention include, but are not limited to:
  • Measles + Mumps + Rubella MMR II or Priorix
  • Pneumococcal Prevenar
  • the BCG vaccine to prevent tuberculosis is no longer given routinely to school-age children. Instead, the vaccine is now only recommended for infants and children at high risk of the disease.
  • Further applications include vaccination against essentially any organism that presents a health risk, and can be produced using this invention; for instance the antigens of organisms specific to certain parts of the world, such as tropical diseases, and cancer-associated vaccines, such as by the human papilloma viruses.
  • a major and growing health risk is that of hospital-acquired infections, which encompass almost all clinically evident infections that do not originate from a patient's original admitting diagnosis. Within hours after admission, a patient's flora begins to acquire characteristics of the surrounding bacterial pool. Most infections that become clinically evident after 48 hours of hospitalization are considered hospital-acquired. Infections that occur after the patient's discharge from the hospital can be considered to have a nosocomial origin if the organisms were acquired during the hospital stay.
  • Iatrogenic risk factors include pathogens that are present on medical personnel hands, invasive procedures (e.g., intubation, indwelling vascular lines, urine catheterization), and antibiotic use and prophylaxis.
  • Organizational risk factors include contaminated air-conditioning systems, contaminated water systems, and staffing and physical layout of the facility (e.g., nurse-to-patient ratio, open beds close together).
  • Patient risk factors include the severity of illness, underlying immunocompromised state, and length of stay.
  • NIS Surveillance System of the Centers for Disease Control and Prevention performed a survey from October 1986 to April 1998. They ranked hospital wards according to their association with central-line bloodstream infections. The highest rates of infection occurred in the burn, neonatal, and pediatric departments. Nosocomial infections are estimated to more than double the mortality and morbidity risks of any admitted patient, and they probably result in about 90,000 deaths a year in the United States alone.
  • Nosocomial infections are caused by viral, bacterial, and fungal pathogens. These pathogens should be investigated in all febrile patients who are admitted for a nonfebrile illness.
  • Viruses are the leading etiologies of nosocomial infections in pediatric patients (responsible for up to 14% of Hospital Associated Infections (HAI) with identifiable pathogens).
  • HAI Hospital Associated Infections
  • Bacterial and fungal infections are less common. However they are significantly associated with more morbidity and mortality. Most patients who are infected with nosocomial bacterial and fungal pathogens have a predisposition caused by invasive supportive measures such as intubation and the placement of intravascular lines and urinary catheters. Fungal infections more likely to arise from the patient's own flora; occasionally, they are caused by contaminated solutions (e.g., those used in parenteral nutrition).
  • Nosocomial etiologies in bloodstream infections include the following: o Coagulase-negative staphylococci, 40% o Enterococci, 11.2% o Fungi, 9.65% o Staphylococcus aureus, 9.3% o Enterobacter species, 6.2% o Pseudomonads, 4.9% o Acinetobacter baumannii with substantial antimicrobial resistance - Reported with increasing frequency
  • Urinary Tract Infections include the following: o Gram-negative enterics, 50% o Fungi, 25% o Enterococci, 10%
  • Nosocomial etiologies in surgical-site infections include the following:
  • antibiotics Besides increased drug resistance, high-dose and prolonged antimicrobial therapy can eliminate helpful bacterial flora and predispose people to infection.
  • a common adverse effect of antibiotics is diarrhea, which can lead to loss of essential vitamins and minerals, especially vitamin K, magnesium, and zinc.
  • Other adverse effects of antibiotic therapy include vitamin deficiencies, seizures, allergic shock (in people who are allergic to antibiotics), autoimmune disease, decreased platelets, kidney injury, drug/drug interaction.
  • the invention features the use of encapsulated cells in a BAO that have been genetically modified to produce antigens of the most dangerous viral, bacterial and fungal infective agents, and this under the control of a heat-inducible promoter. Such expression control allows the optimization of the production of an immune reaction against these antigens.
  • "Boosting" by repeated production of antigens is simply provided by this invention, and is achieved by repeating the heat induction cycles of protein production at defined times.
  • BAO may be used to supply biologically active molecules for the treatment or prevention of neurodegenerative conditions such as Huntington's disease, Parkinson's disease, Alzheimer's disease, and Acquired Immune Deficiency Syndrome-related dementia. Additionally, lymphokines and cytokines may also be supplied by BAOs to modulate the host immune system. Other biologically active molecules which may be provided by bioartificial organs include, catecholamines, endorphins, enkephalins, and other opioid or non-opioid peptides that are useful for treating pain. Enzymatic deficiencies may also be treated by using BAOs. Alternatively, the biologically active molecule may remove or eliminate deleterious molecules from the host. For example, a BAO may contain cells which produce a biologically active molecule that can be used to "scavenge" cholesterol from a host.
  • BAOs are known. See, e.g., Aebischer (U.S. Pat. No. 5,158,881), Dionne et al. (WO 92/03327), Mandel et al. (WO 91/00119), Aebischer (WO 93/00128). BAOs also include extravascular diffusion chambers, intravascular diffusion chambers, intravascular ultrafiltration chambers, and microcapsules. See, e.g., Lim et al., Science 210:908-910 (1980); Sun, A.M., Methods in Enzymology 137: 575-579 (1988); Dunleavy et al. (WO 93/03901) and Chick et al. (U.S. Pat. No. 5,002,661).
  • differentiated, non-dividing cells may be preferred over dividing cells for use in BAOs because they allow for the optimal production of the desired biologically active molecule.
  • differentiated, non-dividing cells produce a greater quantity of a desired therapeutic protein than dividing cells because the expression of differentiation specific genes and cell division are thought to be antagonistic processes. Wollheim, "Establishment and Culture of Insulin- Secreting beta-cell lines," Methods in Enzymology, 192, p. 223-235 (1990). Cellular replication capacity decreases as cells differentiate. In many cases, proliferation and differentiation are mutually exclusive. Gonos, "Oncogenes in Cellular Immortalisation and Differentiation," 13, Anticancer Research, p. 1117 (1993).
  • differentiated tissue is advantageous because the functional properties of tissue desired for incorporation into a BAO have most often been defined by the properties of differentiated tissue in vivo.
  • Another advantage to the use of differentiated, non-dividing cells is that the cell number within the BAO will remain relatively constant. This, in turn, leads to more predictable results and stable dosage for the recipient host.
  • differentiated cells are better suited for use in BAOs which encapsulate more than one cell type secreting biologically active molecules. In such BAOs, if dividing cells are used, different cell types may grow at different rates, resulting in the overgrowth of one cell type. By using differentiated, non-dividing cells, the relative proportions of two or more synergistic cell types can be more readily controlled.
  • necrotic tissue may also release excess cellular proteins which unnecessarily flood the host with xeno-proteins or other factors which are detrimental to the surviving cells, e.g., factors which elicit a macrophage or other immune response.
  • Controlling the cell number and cell location within the BAO also provides the advantage of facilitating optimization of the BAO membrane and other device parameters to the particular encapsulated cell type. This is because the required device characteristics are more readily determined for a fixed cell population than for a dividing cell population in the BAO. Additionally, long term delivery of biologically active molecules can be achieved.
  • the invention addresses the such problems by providing methods and compositions for controlling the distribution of cells (i.e. cell number or cell location in the BAO, or both) when encapsulated in a BAO.
  • the methods and compositions of this invention include (1) methods and compositions for modification of the cells that are encapsulated within the BAO and (2) methods and compositions for modifying the growth surfaces within the BAO.
  • Methods and compositions for cellular manipulation include genetic alteration of the cells with a gene which encodes a product that influences cell proliferation or differentiation.
  • the treatment may comprise providing a chemical compound or growth factor which inhibits proliferation or induces differentiation.
  • the treatment may comprise removing from the growth medium a chemical compound or growth factor which stimulates proliferation or inhibits differentiation.
  • the treatment may be before or after encapsulation in the BAO, preferably before encapsulation. Additionally, cell proliferation may be controlled by irradiation.
  • Methods and compositions for growth surface modification include coating at least one growth surface within the BAO with one or more extracellular matrix molecules ("ECM").
  • ECMs may be coated directly onto the luminal surface or any inner support within the BAO, or onto microsphere carriers ("microcarriers").
  • Cells or cell-seeded microcarriers may additionally be suspended in a matrix material that physically inhibits cell proliferation. Further, the matrix material may be derivatized with chemical or peptide derivatives.
  • a growth surface of the BAO can be modified by chemical treatment to inhibit cell attachment or to enhance cell attachment to the BAO's luminal surface.
  • the growth surface can be modified by addition of an inert scaffold prior to cell loading. The scaffold physically inhibits cell outgrowth and provides additional sites for cell attachment. It is to be understood that the various methods and compositions for cell modification and for growth surface modification are not mutually exclusive and may be used in combination.
  • This invention relates to a biocompatible, immuno-isolatory, implantable vehicle.
  • the instant vehicle is suitable for isolating biologically active cells or substances from the body's protective mechanisms following implantation into an individual.
  • the instant vehicle is comprised of (a) a core which contains isolated cells, either suspended in a liquid medium or immobilized within a hydrogel matrix, and (b) a surrounding or peripheral region ("jacket") of permselective matrix or membrane which does not contain isolated cells, which is biocompatible, and which is sufficient to protect the isolated cells in the core from immunological attack.
  • This immuno-isolatory vehicle is useful (a) to deliver a wide range of biologically active moieties, including high molecular weight products, to an individual in need of them, and/or (b) to provide needed metabolic functions to an individual, such as the removal of harmful substances.
  • the instant vehicle is contains a multiplicity of cells, such that implantation of a few or a single vehicle is sufficient to provide an effective amount of the needed substance or function to an individual.
  • a further advantage offered by the instant vehicle is practicality of retrieval.
  • An immuno-isolatory vehicle of the present invention is implanted into the individual (referred to as the recipient), using known techniques or methods and selected for the particular immuno- isolatory vehicle and site of implantation. Once implanted, cells isolated within the biocompatible immuno-isolatory vehicle produce the desired moieties or perform the desired function(s). If moieties are released by the isolated cells, they pass through the surrounding or peripheral permselective membrane or hydrogel matrix into the recipient's body.
  • They also relate a method of isolating cells within a biocompatible, immuno-isolatory implantable vehicle, thereby protecting the cells within the vehicle from immunological attack after being implanted into an individual.
  • some low molecular weight mediators of the immune responses e.g. cytokines
  • the isolated cells are protected from attack by the recipient's immune system and from potentially deleterious inflammatory responses from the tissues which surround the implanted vehicle.
  • the isolated cells are maintained in a suitable local environment. In this manner, needed substances or metabolic functions can be delivered to the recipient even for extended periods of time, and without the need to treat the recipient with potentially dangerous immunosuppressive drugs.
  • the vehicle is formed by co-extruding from a nested-bore extrusion nozzle materials which form the core and surrounding or peripheral regions, under conditions sufficient to gel, harden, or cast the matrix or membrane precursor(s) of the surrounding or peripheral region (and of the core region).
  • a particular advantage of this co-extrusion embodiment is that the cells in the core are isolated from the moment of formation of the vehicle, ensuring that the core materials do not become contaminated or adulterated during handling of the vehicle prior to implantation.
  • a further advantage of the co-extrusion process is that it ensures that the surrounding or peripheral region is free of cells and other core materials.
  • the permeability and biocompatibility characteristics of the surrounding or peripheral region are determined by both the matrix or membrane precursor materials used, and the conditions under which the matrix or membrane is formed.
  • the immuno-isolatory vehicle is formed stepwise.
  • the immuno-isolatory vehicle being made includes a hydrogel core containing the isolated cells
  • the core can be formed initially, and the surrounding or peripheral matrix or membrane can be assembled or applied subsequently.
  • the surrounding or peripheral matrix or membrane can be preformed, and then filled with the preformed isolated-cell containing core material or with materials which will form the core (i.e., core precursor materials).
  • the vehicle is sealed in such a manner that the core materials are completely enclosed. If a core precursor material is used, the vehicle is then exposed to conditions which result in formation of the core.
  • This present invention differs from the above mentioned BAO technologies in that the encapsulated cells contain gene constructs in which the antigens, including but not limited to those of the most dangerous bacterial and fungal nosocomial infective agents, have been placed under the expression control of a heat-inducible promoter.
  • cells encapsulated in a BAO are induced to produce and to secrete the selected antigens including but not limited to those of the most dangerous bacterial and fungal nosocomial infective agents. Placing their genes under hsp-70B expression control allows these antigens to be produced by the effect of local heat applied over the site of implantation of the BAO. DESCRIPTION OF THE PREFERRED EMBODIMENTS
  • Preferred antigens of this invention include but are not limited to those involved in chidhood infections, tropical diseases, cancer vaccines and nosocomial diseases, and include those of the following organisms:
  • Nosocomial etiologies in bloodstream infections include the following: Coagulase-negative staphylococci, Enterococci, Fungi, Staphylococcus aureus, Enterobacter species, Pseudomonads, Acinetobacter baumannii with substantial antimicrobial resistance - Reported with increasing frequency.
  • Nosocomial etiologies in UTI include Gram-negative enterics, Fungi, Enterococci,
  • Nosocomial etiologies in surgical-site infections include S aureus, Pseudomonads, Coagulase- negative staphylococci, Enterococci, fungi, Enterobacter species, and Escherichia coli.
  • Viruses are the leading etiologies of nosocomial infections in pediatric patients (responsible for up to 14% of hospital acquired diseases with identifiable pathogens). Neutralization-inducing antigens of these viruses are included in preferred embodiments of this invention.
  • This invention can also be employed to vaccinate against tropical diseases; African trypanosomiasis, Dengue fever, Leishmaniasis, Malaria, Schistosomiasis, Tuberculosis, Chagas disease, Leprosy, Lymphatic filariasis, and Onchocerciasis
  • This invention also provides a safer vaccination approach to viral diseases such as influenza, human papilloma viruses and the AIDS viruses.
  • a subunit vaccine has been shown to be effective in immunization
  • this invention provides a safe and convenient approach; in addition, vaccination against multiple infectious agents can be performed with no increase in risk.
  • the immune response to a protective antigen can be influenced by the location of the antigen. Although good immune responses have been observed for antigens retained in the cytoplasm or secreted into the periplasm of Gram-negative vaccines (Roberts, M., Chatfield, S.N., and Dougan, G. 1994. Salmonella as carriers of heterologous antigens. In Novel delivery systems for oral vaccines. D. T. O'Hagan, editor. CRC Press.
  • Hess, J. et al.2000 Protection against murine tuberculosis by an attenuated recombinant Salmonella typhimurium vaccine strain that secretes the 30-kDa antigen of Mycobacterium bovis BCG. FEMS Immunol. Med. Microbiol. 27:283-289) or homologous (Hess, J. et al.2000. Secretion of different listeriolysin cognates by recombinant attenuated Salmonella typhimurium: superior efficacy of haemolytic over non-haemolytic constructs after oral vaccination.
  • Microbes Infect. 2:1799-1806 secretion mechanisms can further enhance the level and type of immune response induced.
  • type III secretion apparatus of Salmonella and Yersinia antigens with T cell epitopes can be delivered into the cytoplasm of antigen-presenting cells within the immunized eukaryotic host, resulting in a CD 8 -restricted CTL response (R ⁇ ssmann, H. et all 998. Delivery of epitopes by the Salmonella Type III secretion system for vaccine development. Science. 281 :565-568).
  • SCOTS Selective Capture of Transcribed Sequences
  • a rapidly developing approach is the use of DNA vaccines.
  • the gene for a pathogen protein is introduced into human cells and is then expressed to produce the protein inside the body.
  • the DNA vaccination method There are many advantages to the DNA vaccination method. For example, it is much cheaper to produce and distribute large amounts of DNA than it is to produce and distribute large amounts of protein. Also, the same strategy can be used to tackle virtually any pathogen, so multiple vaccinations are possible.
  • Technical hurdles that need to be overcome include finding efficient ways of getting the DNA into human cells, making sure the gene is expressed once it is inside the cell, and making sure the DNA does not integrate into the genome and disrupt our own genes.
  • DNA vaccines in clinical and pre-clinical trials, including vaccines for HIV, herpes, hepatitis and influenza.
  • This invention describes a new approach to vaccination, whereby cells are genetically modified to produce viral, bacterial and fungal antigens, in a heat-inducible manner; these cells are introduced into the patient's body as an encapsulated BAO.
  • the organisms selected in this invention are those associated with nosocomial diseases, although the invention is not in any way limited to such organisms.
  • any organism that presents a health risk can be produced using this invention, for instance the antigens of organisms specific to certain parts of the world, such as tropical diseases, and cancer-associated infections such as by the human papilloma viruses.
  • This invention completely avoids the risks associated with attenuated organism based vaccines, and also avoids the complications encountered with DNA vaccines.
  • a variety of cell types can be employed in this invention, from animal or human origin.
  • a preferred embodiment is to have cells that do not divide extensively once introduced into the BAO.
  • Many transformed cells or cell lines are most advantageously isolated within a vehicle having a liquid core comprising a nutrient medium, optionally containing a liquid source of additional factors to sustain cell viability and function, such as fetal bovine serum.
  • the term "cells" means cells in any form, including but not limited to cells retained in tissue, cell clusters, and individually isolated cells.
  • the vehicle employed in the current invention may be prepared of sufficient size to deliver an immune response from a single or just a few (less than 10) implanted and easily retrievable vehicles.
  • the core may be composed of a matrix formed by a hydrogel which stabilizes the position of the cells in cell clumps.
  • hydrogel herein refers to a three dimensional network of cross-linked hydrophilic polymers. The network is in the form of a gel substantially composed of water, preferably but not limited to gels being greater than 90% water.
  • Cross-linked hydrogels can also be considered solids because they do not flow or deform without appreciable applied shear stress, compositions which form hydrogels fall into three classes for the purposes of this application.
  • the first class carries a net negative charge and is typified by alginate.
  • the second class carries a net positive charge and is typified by extracellular matrix components such as collagen and laminin. Examples of commercially available extracellular matrix components include Matrigel.TM. and Vitrogen.TM..
  • the third class is net neutral in charge.
  • An example of a net neutral hydrogel is highly crosslinked polyethylene oxide, or polyvinyalcohol.
  • One particularly advantageous use of hydrogel cores pertains to the encapsulation of actively dividing cells.
  • Alginate or other hydrogels may be included in suspensions of actively dividing cells to be encapsulated. Following encapsulation and generation of the gel, the encapsulated cells are somewhat immobilized within the gel and new cells produced during cell division stay localized near the parent cell. In this manner clusters of cells are produced within the core.
  • Stable human hepatocyte cell lines include, but are not limited to Hep G2, Hep 3B, C3A, C3 A/Hep G2, Chang cells, Fa2N4, HuH7, Hepa RG, LH 86, NKNT-3, OUMS-29, TTNT- 16-3 as well as cell lines immortalised using the human telemorase reverse transcriptase gene via a retroviral vector.
  • Secretory cells of the immune system include, but are not limited to DAUDI (Burkitt lymphoma cells), JURKAT and Molt 4 (human acute T cell leukaemia cell lines), and K562 human chronic myelogenous leukaemia cells).
  • Cell lines are derived from a reversibly immortalized human hepatocyte line such as TTNT- 16-3. Briefly, human hepatocytes are transduced with a recombinant retroviral vector (SSR#197) (Westerman KA, Leboulch P: Reversible immortalization of mammalian cells mediated by retroviral transfer and site-specific recombination. Proc Natl Acad Sci U S A93 :8971 -8976,1996) containing cDNAs expressing hTERT, for immortalization, and enhanced green fluorescent protein (EGFP), for a selection marker, flanked by a pair of recombination target loxPs.
  • SSR#197 Westerman KA, Leboulch P: Reversible immortalization of mammalian cells mediated by retroviral transfer and site-specific recombination. Proc Natl Acad Sci U S A93 :8971 -8976,1996) containing cDNAs expressing hTERT
  • EGFP-positive immortalized clones Two days after three rounds of SSR#197 transduction, cells are sorted using a FACSCalibur system (BD Biosciences Immunocytochemistry Systems, San Jose, CA) for recovering EGFP-positive cell populations.
  • FACSCalibur system BD Biosciences Immunocytochemistry Systems, San Jose, CA
  • One of the EGFP-positive immortalized clones is selected on the basis of its hepatocyte-specif ⁇ c gene expression profile and negative tumorigenesis.
  • These cells are transduced with a plasmid encoding a tamoxifen-inducible Cre recombinase fused with paired mutant estrogen receptor ligand-binding domains (MerCreMer) under the control of the CAG promoter (cytomegalovirus IE enhancer, chicken ⁇ -actin promoter, and ⁇ -globin splicing acceptor) with a puromycin resistance gene.
  • the resultant puromycin-resistant clones grow steadily in tissue culture with serum-free ISE- RPMI medium (Nakabayashi H, Taketa K, Miyano K, Yamane T, Sato J: Growth of human hepatoma cell lines with differentiated functions in chemically defined medium.
  • An expression vector is constructed which allows for the selection of stable transfectants by selection for the zeocin antibiotic (Cayla) in both prokaryotic and eukaryotic cells.
  • the Zeocin resistance gene is obtained as a restriction digest fragment from the pZeoSV plasmid (Invitrogen) and is ligated to a fragment containing a bacterial origin of replication obtained by PCR amplification from pUC19 (New England Biolabs). This ligation mixture is then used to transform competent E. coli cells and the presence of the desired recombinant plasmid (pUC-Zeo) is selected for on Zeocin-containing bacterial plates.
  • a synthetic poly(A) sequence obtained as a restriction fragment from a digest of pGL3-Basic (Promega) is ligated into pUC-Zeo upstream of the HSP70B promoter, and the desired recombinant (pUC-ZeoA) will selected for Zeocin resistance.
  • the HSP70B driven expression cassette drives the expression of genes for the antigens of this invention. These expression cassettes can be transfected into for instance JURKAT cells, as an example of secretory cells of the immune system.

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Abstract

A biological artificial organ (BAO) containing eukaryotic cells, said eukaryotic cells a) being transformed in a stable fashion with genes encoding viral, bacterial or fungal antigens b) such genes being placed under the operational expression control of an heat-inducible promoter c) the BAO being inserted under the skin of the patient to be immunized, in a retractable fashion d) the cells inside the BAO being induced to express and secrete the said antigens using local heat e) this induction being repeated where necessary to induce an efficient neutralizing antibody response against said antigens.

Description

NEW AND SAFE PROCEDURE FOR IMMUNIZATION AGAINST INFECTIOUS DISEASES AGENTS

BACKGROUND OF THE INVENTION

Most vaccines cause some side effects, but they are usually minor and short-lived like low- grade fever and soreness at the injection site. Serious vaccine reactions, causing disability, hospitalization, or death—are extremely rare but they do happen.

Like any medicine, vaccines carry a small risk of serious harm such as severe allergic reaction. But experts point out that the risk of being harmed by a vaccine is much lower than the risk that comes with infectious diseases.

For example, in 1976, the swine influenza (flu) vaccine was associated with a severe paralytic illness called Guillain-Barre Syndrome (GBS). According to the CDCs vaccine information sheet on the influenza vaccine, "if there is a risk of GBS from current influenza vaccines, it is estimated at 1 or 2 cases per million persons vaccinated, much less than the risk of severe influenza, which can be prevented by vaccination." Each year, flu causes tens of thousands of deaths, mostly among older people. Most people who get the influenza vaccine have no serious problem from it.

Some live virus vaccines, such as the chickenpox vaccine, can cause mild versions of the disease they protect against, but this is usually only a serious problem if the patient has a severely compromised immune system.

Subunit vaccines raised against purified antigens of infectious agents have had some success, but a problem with this approach is that purifying the infectious agent, isolating the surface and other antigens, purifying these proteins etc, frequently changes the presentation form of these antigens when subsequently injected into patients. The technology of this invention totally avoids these drawbacks; the subunit vaccines are produced by the cells in the Bio- artificial organs (BAO), are secreted in their natural format, with all associated post- translational modifications, ensuring an effective immune response.

This invention ensures that infectious organism antigens capable of eliciting neutralizing antibody formation, are correctly formed, and presented to the immune system in an optimal manner. This invention avoids alterations to such sub-unit vaccines that result from purification procedures and storage prior to employment.

This new vaccination procedure of this invention can be applied to essentially all vaccination needs today, these include childhood infections, tropical diseases and cancer vaccines, and nosocomial infections. Examples of childhood vaccines that can be treated using this invention include, but are not limited to:

Diphtheria + Tetanus + Pertussis + Polio + Hib (Pediacel or Infanrix- One IPV+Hib) injection

Pneumococcal (Prevenar) One injection

At three months

Name of vaccine How is it given?

Diphtheria + Tetanus + Pertussis + Polio + Hib (Pediacel or Infanrix- One injection IPV+Hib)

Meningitis C (Meningitec, Menjugate or NeisVac-C) One injection

At four months

Name of vaccine How is it given?

Diphtheria + Tetanus + Pertussis + Polio + Hib (Pediacel or Infanrix- One injection IPV+Hib)

Meningitis C (Meningitec, Menjugate or NeisVac-C) One injection

Pneumococcal (Prevenar) One injection

Around 12 months

Name of vaccine How is it given?

Meningitis C + Hib (Menitorix) One injection

Around 13 months

Name of vaccine How is it given?

Measles + Mumps + Rubella (MMR II or Priorix) One injection Pneumococcal (Prevenar) One injection

At 3-5 years (usually before child starts school) Name of vaccine How is it given?

Diphtheria + Tetanus + Pertussis + Polio (Repevax or Infanrix-IPV) One injection Measles + Mumps + Rubella (MMR II or Priorix) One injection

School leavers (13-18 years)

Name of vaccine How is it given?

Diphtheria + Tetanus + Polio (Revaxis) One injection

The BCG vaccine to prevent tuberculosis is no longer given routinely to school-age children. Instead, the vaccine is now only recommended for infants and children at high risk of the disease.

Further applications include vaccination against essentially any organism that presents a health risk, and can be produced using this invention; for instance the antigens of organisms specific to certain parts of the world, such as tropical diseases, and cancer-associated vaccines, such as by the human papilloma viruses.

A major and growing health risk is that of hospital-acquired infections, which encompass almost all clinically evident infections that do not originate from a patient's original admitting diagnosis. Within hours after admission, a patient's flora begins to acquire characteristics of the surrounding bacterial pool. Most infections that become clinically evident after 48 hours of hospitalization are considered hospital-acquired. Infections that occur after the patient's discharge from the hospital can be considered to have a nosocomial origin if the organisms were acquired during the hospital stay.

Within hours of admission, colonies of hospital strains of bacteria develop in the patient's skin, respiratory tract, and genitourinary tract. Risks factors for the invasion of colonizing pathogens can be categorized into 3 areas: iatrogenic, organizational, and patient related.

• Iatrogenic risk factors include pathogens that are present on medical personnel hands, invasive procedures (e.g., intubation, indwelling vascular lines, urine catheterization), and antibiotic use and prophylaxis.

• Organizational risk factors include contaminated air-conditioning systems, contaminated water systems, and staffing and physical layout of the facility (e.g., nurse-to-patient ratio, open beds close together).

• Patient risk factors include the severity of illness, underlying immunocompromised state, and length of stay.

• Nosocomial infections are estimated to occur in 5% of all acute care hospitalizations. In the USA, the estimated incidence is more than 2 million cases per year, resulting in an added expenditure in excess of $4.5 billion. The National Nosocomial Infections

• Surveillance (NNIS) System of the Centers for Disease Control and Prevention performed a survey from October 1986 to April 1998. They ranked hospital wards according to their association with central-line bloodstream infections. The highest rates of infection occurred in the burn, neonatal, and pediatric departments. Nosocomial infections are estimated to more than double the mortality and morbidity risks of any admitted patient, and they probably result in about 90,000 deaths a year in the United States alone.

• Nosocomial infections are caused by viral, bacterial, and fungal pathogens. These pathogens should be investigated in all febrile patients who are admitted for a nonfebrile illness.

• During their hospital stay, many patients acquire viral respiratory infections in the winter (e.g. influenza, parainfluenza, respiratory syncytial viruses), rotaviral infections in winter, or enteroviral infections in the summer. Viruses are the leading etiologies of nosocomial infections in pediatric patients (responsible for up to 14% of Hospital Associated Infections (HAI) with identifiable pathogens).

• Bacterial and fungal infections are less common. However they are significantly associated with more morbidity and mortality. Most patients who are infected with nosocomial bacterial and fungal pathogens have a predisposition caused by invasive supportive measures such as intubation and the placement of intravascular lines and urinary catheters. Fungal infections more likely to arise from the patient's own flora; occasionally, they are caused by contaminated solutions (e.g., those used in parenteral nutrition). For instance, among 6,290 pediatric patients surveyed between 1992-1997 in a US study, the incidence of nosocomial invasive bacterial and fungal infections were as follows: o Bloodstream infections, 28% o Ventilator-associated pneumonia, 21% o Urinary tract infection (UTI), 15% o Lower respiratory infection, 12% o Gastrointestinal, skin, soft tissue, and cardiovascular infections, 10% o Surgical-site infections, 7% o Ear, nose, and throat infections, 7%

• Nosocomial etiologies in bloodstream infections include the following: o Coagulase-negative staphylococci, 40% o Enterococci, 11.2% o Fungi, 9.65% o Staphylococcus aureus, 9.3% o Enterobacter species, 6.2% o Pseudomonads, 4.9% o Acinetobacter baumannii with substantial antimicrobial resistance - Reported with increasing frequency

• Nosocomial etiologies in Urinary Tract Infections (UTI) include the following: o Gram-negative enterics, 50% o Fungi, 25% o Enterococci, 10%

• Nosocomial etiologies in surgical-site infections include the following:

• o S aureus, 20% o Pseudomonads, 16% o Coagulase-negative staphylococci, 15% o Enterococci, fungi, Enterobacter species, and Escherichia coli, less than 10% each

Although there is essentially no treatment for most viral infections once contracted, bacterial infections in a hospital setting are generally treated with high dose antibiotics for extended periods of time.

Besides increased drug resistance, high-dose and prolonged antimicrobial therapy can eliminate helpful bacterial flora and predispose people to infection. A common adverse effect of antibiotics is diarrhea, which can lead to loss of essential vitamins and minerals, especially vitamin K, magnesium, and zinc. Other adverse effects of antibiotic therapy include vitamin deficiencies, seizures, allergic shock (in people who are allergic to antibiotics), autoimmune disease, decreased platelets, kidney injury, drug/drug interaction.

Since the morbidity risk of each of these infective elements is not the same, immunization against the most dangerous bacterial and fungal nosocomial infective agents would be a major improvement in hospital safety for patients and would greatly reduce the morbidity associated with nosocomial infections.

SUMMARY OF THE INVENTION

The invention features the use of encapsulated cells in a BAO that have been genetically modified to produce antigens of the most dangerous viral, bacterial and fungal infective agents, and this under the control of a heat-inducible promoter. Such expression control allows the optimization of the production of an immune reaction against these antigens. "Boosting" by repeated production of antigens is simply provided by this invention, and is achieved by repeating the heat induction cycles of protein production at defined times.

BAO may be used to supply biologically active molecules for the treatment or prevention of neurodegenerative conditions such as Huntington's disease, Parkinson's disease, Alzheimer's disease, and Acquired Immune Deficiency Syndrome-related dementia. Additionally, lymphokines and cytokines may also be supplied by BAOs to modulate the host immune system. Other biologically active molecules which may be provided by bioartificial organs include, catecholamines, endorphins, enkephalins, and other opioid or non-opioid peptides that are useful for treating pain. Enzymatic deficiencies may also be treated by using BAOs. Alternatively, the biologically active molecule may remove or eliminate deleterious molecules from the host. For example, a BAO may contain cells which produce a biologically active molecule that can be used to "scavenge" cholesterol from a host.

Various "macrocapsule" BAOs are known. See, e.g., Aebischer (U.S. Pat. No. 5,158,881), Dionne et al. (WO 92/03327), Mandel et al. (WO 91/00119), Aebischer (WO 93/00128). BAOs also include extravascular diffusion chambers, intravascular diffusion chambers, intravascular ultrafiltration chambers, and microcapsules. See, e.g., Lim et al., Science 210:908-910 (1980); Sun, A.M., Methods in Enzymology 137: 575-579 (1988); Dunleavy et al. (WO 93/03901) and Chick et al. (U.S. Pat. No. 5,002,661).

Because the cells encapsulated in the BAO provide the needed metabolic function, it is desirable that those cells optimally supply the biologically active molecule that effects that function. Typically, differentiated, non-dividing cells may be preferred over dividing cells for use in BAOs because they allow for the optimal production of the desired biologically active molecule. For example, many differentiated, non-dividing cells produce a greater quantity of a desired therapeutic protein than dividing cells because the expression of differentiation specific genes and cell division are thought to be antagonistic processes. Wollheim, "Establishment and Culture of Insulin- Secreting beta-cell lines," Methods in Enzymology, 192, p. 223-235 (1990). Cellular replication capacity decreases as cells differentiate. In many cases, proliferation and differentiation are mutually exclusive. Gonos, "Oncogenes in Cellular Immortalisation and Differentiation," 13, Anticancer Research, p. 1117 (1993).

The use of differentiated tissue is advantageous because the functional properties of tissue desired for incorporation into a BAO have most often been defined by the properties of differentiated tissue in vivo. Another advantage to the use of differentiated, non-dividing cells is that the cell number within the BAO will remain relatively constant. This, in turn, leads to more predictable results and stable dosage for the recipient host. Additionally, differentiated cells are better suited for use in BAOs which encapsulate more than one cell type secreting biologically active molecules. In such BAOs, if dividing cells are used, different cell types may grow at different rates, resulting in the overgrowth of one cell type. By using differentiated, non-dividing cells, the relative proportions of two or more synergistic cell types can be more readily controlled.

There is another problem associated with encapsulating cells in general. A variety of cell types have cell adherent properties such that cells tend to adhere to each other and form dense agglomerations or aggregates, especially if there is no adequate substrate available for the cells. Such cell clusters may develop central necrotic regions due to the relative inaccessibility of nutrients and oxygen to cells embedded in the core, or due to the build up of toxic products within the core. The necrotic tissue may also release excess cellular proteins which unnecessarily flood the host with xeno-proteins or other factors which are detrimental to the surviving cells, e.g., factors which elicit a macrophage or other immune response. This problem may be exacerbated when cells are encapsulated in a BAO with a semi-permeable membrane jacket because of diffusional constraints across the membrane. Often less oxygen and fewer host supplied nutrients are available within the BAO. In addition, waste products may accumulate in the BAO. These dense cellular masses can form slowly into dense colonies of cell growth or form rapidly, upon the re-association of freshly-dispersed cells or tissue mediated by cell-surface adhesion proteins. Cells or tissues with a high metabolic activity may be particularly susceptible to the effects of oxygen or nutrient deprivation, and die shortly after becoming embedded in the centre of a large cell cluster. Many endocrine tissues, which normally are sustained by dense capillary beds, exhibit this behaviour; islets of Langerhans appear to be particularly sensitive when encapsulated.

There is a need to have a method and composition for controlling the growth of encapsulated cells which combines the various advantages of both proliferating cells and differentiated, non-dividing cells. Aebischer et al., US4892538 provides methods and compositions whereby cells can be proliferated and expanded indefinitely in vitro and where the balance between proliferation and differentiation can be controlled when the cells are encapsulated within the BAO so that the device performs in the desired manner. This thus allows regulation of the cell number within the BAO and may therefore provide improved regulation of the output level of protein release the capsule. This invention also provides methods for controlling the growth of cells by controlling cell location within the BAO, thereby reducing the formation of undesirable necrotic cell cores in the BAO. Controlling the cell number and cell location within the BAO also provides the advantage of facilitating optimization of the BAO membrane and other device parameters to the particular encapsulated cell type. This is because the required device characteristics are more readily determined for a fixed cell population than for a dividing cell population in the BAO. Additionally, long term delivery of biologically active molecules can be achieved.

Schinstine et al., ZA9505721 describes the production of such implantable cell systems, as well as methods for optimizing such systems. The invention addresses the such problems by providing methods and compositions for controlling the distribution of cells (i.e. cell number or cell location in the BAO, or both) when encapsulated in a BAO. The methods and compositions of this invention include (1) methods and compositions for modification of the cells that are encapsulated within the BAO and (2) methods and compositions for modifying the growth surfaces within the BAO.

Methods and compositions for cellular manipulation include genetic alteration of the cells with a gene which encodes a product that influences cell proliferation or differentiation. The treatment may comprise providing a chemical compound or growth factor which inhibits proliferation or induces differentiation. Alternatively, the treatment may comprise removing from the growth medium a chemical compound or growth factor which stimulates proliferation or inhibits differentiation. The treatment may be before or after encapsulation in the BAO, preferably before encapsulation. Additionally, cell proliferation may be controlled by irradiation.

Methods and compositions for growth surface modification include coating at least one growth surface within the BAO with one or more extracellular matrix molecules ("ECM"). The ECMs may be coated directly onto the luminal surface or any inner support within the BAO, or onto microsphere carriers ("microcarriers"). Cells or cell-seeded microcarriers may additionally be suspended in a matrix material that physically inhibits cell proliferation. Further, the matrix material may be derivatized with chemical or peptide derivatives.

In addition, a growth surface of the BAO can be modified by chemical treatment to inhibit cell attachment or to enhance cell attachment to the BAO's luminal surface. Further, the growth surface can be modified by addition of an inert scaffold prior to cell loading. The scaffold physically inhibits cell outgrowth and provides additional sites for cell attachment. It is to be understood that the various methods and compositions for cell modification and for growth surface modification are not mutually exclusive and may be used in combination.

Dionne et al., US6322804 have further improved the BAO approach described above. This invention relates to a biocompatible, immuno-isolatory, implantable vehicle. The instant vehicle is suitable for isolating biologically active cells or substances from the body's protective mechanisms following implantation into an individual. The instant vehicle is comprised of (a) a core which contains isolated cells, either suspended in a liquid medium or immobilized within a hydrogel matrix, and (b) a surrounding or peripheral region ("jacket") of permselective matrix or membrane which does not contain isolated cells, which is biocompatible, and which is sufficient to protect the isolated cells in the core from immunological attack.

This immuno-isolatory vehicle is useful (a) to deliver a wide range of biologically active moieties, including high molecular weight products, to an individual in need of them, and/or (b) to provide needed metabolic functions to an individual, such as the removal of harmful substances. The instant vehicle is contains a multiplicity of cells, such that implantation of a few or a single vehicle is sufficient to provide an effective amount of the needed substance or function to an individual. A further advantage offered by the instant vehicle is practicality of retrieval.

They relate a method of delivering a biologically active moiety or altering a metabolic or immunologic function in an individual in need of the moiety or altered metabolic function. An immuno-isolatory vehicle of the present invention is implanted into the individual (referred to as the recipient), using known techniques or methods and selected for the particular immuno- isolatory vehicle and site of implantation. Once implanted, cells isolated within the biocompatible immuno-isolatory vehicle produce the desired moieties or perform the desired function(s). If moieties are released by the isolated cells, they pass through the surrounding or peripheral permselective membrane or hydrogel matrix into the recipient's body.

They also relate a method of isolating cells within a biocompatible, immuno-isolatory implantable vehicle, thereby protecting the cells within the vehicle from immunological attack after being implanted into an individual. Although some low molecular weight mediators of the immune responses (e.g. cytokines) may be permeable to the membrane, in most cases local or circulating levels of these substances are not high enough to have detrimental effects. The isolated cells are protected from attack by the recipient's immune system and from potentially deleterious inflammatory responses from the tissues which surround the implanted vehicle. In the core of the vehicle, the isolated cells are maintained in a suitable local environment. In this manner, needed substances or metabolic functions can be delivered to the recipient even for extended periods of time, and without the need to treat the recipient with potentially dangerous immunosuppressive drugs.

This relates further to a method of making a biocompatible immuno-isolatory vehicle. In a first embodiment, the vehicle is formed by co-extruding from a nested-bore extrusion nozzle materials which form the core and surrounding or peripheral regions, under conditions sufficient to gel, harden, or cast the matrix or membrane precursor(s) of the surrounding or peripheral region (and of the core region). A particular advantage of this co-extrusion embodiment is that the cells in the core are isolated from the moment of formation of the vehicle, ensuring that the core materials do not become contaminated or adulterated during handling of the vehicle prior to implantation. A further advantage of the co-extrusion process is that it ensures that the surrounding or peripheral region is free of cells and other core materials. The permeability and biocompatibility characteristics of the surrounding or peripheral region are determined by both the matrix or membrane precursor materials used, and the conditions under which the matrix or membrane is formed.

In another embodiment of the described method, the immuno-isolatory vehicle is formed stepwise. For example, if the immuno-isolatory vehicle being made includes a hydrogel core containing the isolated cells, the core can be formed initially, and the surrounding or peripheral matrix or membrane can be assembled or applied subsequently. Conversely, the surrounding or peripheral matrix or membrane can be preformed, and then filled with the preformed isolated-cell containing core material or with materials which will form the core (i.e., core precursor materials). The vehicle is sealed in such a manner that the core materials are completely enclosed. If a core precursor material is used, the vehicle is then exposed to conditions which result in formation of the core.

This present invention differs from the above mentioned BAO technologies in that the encapsulated cells contain gene constructs in which the antigens, including but not limited to those of the most dangerous bacterial and fungal nosocomial infective agents, have been placed under the expression control of a heat-inducible promoter.

We have previously explored the applicability of stress/heat shock promoter-based inducible gene expression as compared to constitutive gene expression systems, in a number of applications, such as therapeutic protein production in vitro (US patent 5,646,010 granted July 8, 1997) and in vivo (Dreano et al, Biotechnology, November 1988, 1340-1343. Antibody formation against heat-induced gene products expressed in animals.) and in toxicity assessment in vitro (Fischbach et al., Cell Biology and Toxicology, 9, 177-188, 1993).

Induction of the human growth hormone gene placed under human hsp-70 promoter control in mouse cells: A quantitative indicator of metal toxicity). Further we have previously demonstrated that a marker gene, human growth hormone (hGH) under human hsp-70 expression control, previously integrated into a mouse cell together with a ras oncogene, subsequently injected into nude mice, could be inducibly expressed when the mice were subjected to a whole body hyperthermia (Dreano et al., Biotechnology, November 1988, 1340-1343. Antibody formation against heat-induced gene products expressed in animals.).

In this invention, cells encapsulated in a BAO, are induced to produce and to secrete the selected antigens including but not limited to those of the most dangerous bacterial and fungal nosocomial infective agents. Placing their genes under hsp-70B expression control allows these antigens to be produced by the effect of local heat applied over the site of implantation of the BAO. DESCRIPTION OF THE PREFERRED EMBODIMENTS

The prevention of infectious diseases by vaccination has a long and successful history. Edward Jenner carried out the first vaccination in 1796 by injecting a young boy with cowpox. This conferred protection against a subsequent infection with the deadly smallpox virus. Through concerted worldwide vaccination campaigns, smallpox has now been eliminated.

Most of the vaccines in use today are based on similar principles to Jenner's original vaccine - they are live but attenuated (disabled) bacteria or viruses which cause the body to mount a protective immune response against the target pathogen. Examples include the measles, mumps, rubella and tuberculosis vaccines. Other current vaccines are 'killed vaccines' - the pathogen itself is killed so it is no longer infectious but it can still stimulate the immune system.

Preferred antigens

Preferred antigens of this invention include but are not limited to those involved in chidhood infections, tropical diseases, cancer vaccines and nosocomial diseases, and include those of the following organisms:

Nosocomial etiologies in bloodstream infections include the following: Coagulase-negative staphylococci, Enterococci, Fungi, Staphylococcus aureus, Enterobacter species, Pseudomonads, Acinetobacter baumannii with substantial antimicrobial resistance - Reported with increasing frequency.

Nosocomial etiologies in UTI include Gram-negative enterics, Fungi, Enterococci,

Nosocomial etiologies in surgical-site infections include S aureus, Pseudomonads, Coagulase- negative staphylococci, Enterococci, fungi, Enterobacter species, and Escherichia coli.

During their hospital stay, many patients also acquire viral respiratory infections in the winter (e.g. influenza, parainfluenza, respiratory syncytial viruses), rotaviral infections in winter, or enteroviral infections in the summer. Viruses are the leading etiologies of nosocomial infections in pediatric patients (responsible for up to 14% of hospital acquired diseases with identifiable pathogens). Neutralization-inducing antigens of these viruses are included in preferred embodiments of this invention.

This invention can also be employed to vaccinate against tropical diseases; African trypanosomiasis, Dengue fever, Leishmaniasis, Malaria, Schistosomiasis, Tuberculosis, Chagas disease, Leprosy, Lymphatic filariasis, and Onchocerciasis

Less common, but often serious diseases include Oropouche virus, Lobomycosis, West Nile disease, Labrea fever, Rocio disease, Mapucho hemorrhagic fever, Trachoma, Guinea worm, and Chikungunya.

This invention also provides a safer vaccination approach to viral diseases such as influenza, human papilloma viruses and the AIDS viruses. Wherever a subunit vaccine has been shown to be effective in immunization, this invention provides a safe and convenient approach; in addition, vaccination against multiple infectious agents can be performed with no increase in risk. The immune response to a protective antigen can be influenced by the location of the antigen. Although good immune responses have been observed for antigens retained in the cytoplasm or secreted into the periplasm of Gram-negative vaccines (Roberts, M., Chatfield, S.N., and Dougan, G. 1994. Salmonella as carriers of heterologous antigens. In Novel delivery systems for oral vaccines. D. T. O'Hagan, editor. CRC Press. Boca Raton, Florida, USA. 27-58.), placement on the surface or secretion into the supernatant fluid making use of heterologous (Hess, J. et al.2000. Protection against murine tuberculosis by an attenuated recombinant Salmonella typhimurium vaccine strain that secretes the 30-kDa antigen of Mycobacterium bovis BCG. FEMS Immunol. Med. Microbiol. 27:283-289) or homologous (Hess, J. et al.2000. Secretion of different listeriolysin cognates by recombinant attenuated Salmonella typhimurium: superior efficacy of haemolytic over non-haemolytic constructs after oral vaccination. Microbes Infect. 2:1799-1806) secretion mechanisms can further enhance the level and type of immune response induced. In addition, by employing the type III secretion apparatus of Salmonella and Yersinia, antigens with T cell epitopes can be delivered into the cytoplasm of antigen-presenting cells within the immunized eukaryotic host, resulting in a CD 8 -restricted CTL response (Rϋssmann, H. et all 998. Delivery of epitopes by the Salmonella Type III secretion system for vaccine development. Science. 281 :565-568).

Identification of protective antigens of a pathogen to express in a recombinant attenuated bacterial vaccine is not an easy task. Presence of antibodies in a host surviving infection can offer clues. However, a high-level antibody response does not always correlate with protection. Of course, if one can obtain an mAb to an antigen that can passively protect against the pathogen, that is a good sign that that antigen is likely to induce a protective response. Similarly, the identification of antigen-specific T cell populations whose passive transfer can induce protective immunity will also identify candidate antigens for vaccine development. Evidence that inability to express an antigen due to mutation is associated with avirulence is another useful criteria for antigen selection especially if the antigen is surface- localized or secreted.

Many protective antigens gain access to antigen-presenting pathways, and one can employ genomics mining with algorithms to identify subsets of proteins that are secreted by various secretion pathways or are surface-localized. For example, antigens delivered by the type III secretion system of various pathogens such as Salmonella and Yersinia are delivered to the cytoplasm of host cells to result in CD8-restricted CTL responses (12). This result may also be true for proteins secreted by other pathogens such as Mycobacterium species, if these proteins are secreted into the cytoplasm of cells within the infected individual. Also, if a surface antigen from one pathogen is found to induce protective immunity, one can search by computer for homologs in other pathogens that can be evaluated for their ability to induce protective immune responses.

There is a possibility that given protective antigen is synthesized only in vivo, with the gene encoding it induced by a heat shock. Genetic screens such as In Vivo Expression Technology (IVET) (Mahan, M.J., Slauch, J.M., and Mekalanos, J.J. 1993. Selection of bacterial virulence genes that are specifically induced in host tissues. Science. 259:686-688), Signature-Tagged Mutagenesis (STM) (Hensel, M. et all 995. Simultaneous identification of bacterial virulence genes by negative selection. Science. 269:400-403), and Selective Capture of Transcribed Sequences (SCOTS) (Graham, J.E., and Clark-Curtiss, J.E. 1999. Identification of Mycobacterium tuberculosis RNAs synthesized in response to phagocytosis by human macrophages by selective capture of transcribed sequences (SCOTS). Proc. Natl. Acad. Sci. USA. 96:11554-11559) can be useful in identifying in vivo-expressed genes encoding proteins essential for survival of the pathogen in vivo. Proteins constitutively synthesized by recombinant clones of genes uniquely expressed in vivo can then be used to recognize either antibodies or lymphocyte populations responding to that antigen. Proteomic analysis, quantifying proteins synthesized when the pathogen is in an in vivo compartment or in cells in culture, can identify proteins synthesized in the greatest amounts. Such proteins have a higher likelihood of inducing immune responses may or may not be protective.

Unfortunately, vaccines against all common diseases cannot be made using the above methods and other approaches are needed. One successful strategy is the use of subunit vaccines, where the gene for one specific protein on the pathogen is expressed, and the protein used as the vaccine. The current hepatitis B and influenza vaccines are protein subunits.

A rapidly developing approach is the use of DNA vaccines. Here, the gene for a pathogen protein is introduced into human cells and is then expressed to produce the protein inside the body. There are many advantages to the DNA vaccination method. For example, it is much cheaper to produce and distribute large amounts of DNA than it is to produce and distribute large amounts of protein. Also, the same strategy can be used to tackle virtually any pathogen, so multiple vaccinations are possible. Technical hurdles that need to be overcome include finding efficient ways of getting the DNA into human cells, making sure the gene is expressed once it is inside the cell, and making sure the DNA does not integrate into the genome and disrupt our own genes. There are many DNA vaccines in clinical and pre-clinical trials, including vaccines for HIV, herpes, hepatitis and influenza.

This invention describes a new approach to vaccination, whereby cells are genetically modified to produce viral, bacterial and fungal antigens, in a heat-inducible manner; these cells are introduced into the patient's body as an encapsulated BAO. The organisms selected in this invention are those associated with nosocomial diseases, although the invention is not in any way limited to such organisms. Essentially any organism that presents a health risk can be produced using this invention, for instance the antigens of organisms specific to certain parts of the world, such as tropical diseases, and cancer-associated infections such as by the human papilloma viruses. This invention completely avoids the risks associated with attenuated organism based vaccines, and also avoids the complications encountered with DNA vaccines.

Preferred Cells

A variety of cell types can be employed in this invention, from animal or human origin. In this invention, a preferred embodiment is to have cells that do not divide extensively once introduced into the BAO. Many transformed cells or cell lines are most advantageously isolated within a vehicle having a liquid core comprising a nutrient medium, optionally containing a liquid source of additional factors to sustain cell viability and function, such as fetal bovine serum. Unless otherwise specified, the term "cells" means cells in any form, including but not limited to cells retained in tissue, cell clusters, and individually isolated cells.

Implants of the vehicle and contents thereof retain functionality for greater than three months in vivo. In addition, the vehicle employed in the current invention may be prepared of sufficient size to deliver an immune response from a single or just a few (less than 10) implanted and easily retrievable vehicles. In Dione et al., US6322804, the core may be composed of a matrix formed by a hydrogel which stabilizes the position of the cells in cell clumps. The term "hydrogel" herein refers to a three dimensional network of cross-linked hydrophilic polymers. The network is in the form of a gel substantially composed of water, preferably but not limited to gels being greater than 90% water. Cross-linked hydrogels can also be considered solids because they do not flow or deform without appreciable applied shear stress, compositions which form hydrogels fall into three classes for the purposes of this application. The first class carries a net negative charge and is typified by alginate. The second class carries a net positive charge and is typified by extracellular matrix components such as collagen and laminin. Examples of commercially available extracellular matrix components include Matrigel.TM. and Vitrogen.TM.. The third class is net neutral in charge. An example of a net neutral hydrogel is highly crosslinked polyethylene oxide, or polyvinyalcohol. One particularly advantageous use of hydrogel cores pertains to the encapsulation of actively dividing cells. Alginate or other hydrogels may be included in suspensions of actively dividing cells to be encapsulated. Following encapsulation and generation of the gel, the encapsulated cells are somewhat immobilized within the gel and new cells produced during cell division stay localized near the parent cell. In this manner clusters of cells are produced within the core.

In a preferred embodiment of this invention, cells that are efficient in secretion of proteins such as those derived from hepatocytes of cells of the immune system, are advantageously employed. Stable human hepatocyte cell lines include, but are not limited to Hep G2, Hep 3B, C3A, C3 A/Hep G2, Chang cells, Fa2N4, HuH7, Hepa RG, LH 86, NKNT-3, OUMS-29, TTNT- 16-3 as well as cell lines immortalised using the human telemorase reverse transcriptase gene via a retroviral vector. Secretory cells of the immune system include, but are not limited to DAUDI (Burkitt lymphoma cells), JURKAT and Molt 4 (human acute T cell leukaemia cell lines), and K562 human chronic myelogenous leukaemia cells).

EXAMPLE 1.

Cell lines are derived from a reversibly immortalized human hepatocyte line such as TTNT- 16-3. Briefly, human hepatocytes are transduced with a recombinant retroviral vector (SSR#197) (Westerman KA, Leboulch P: Reversible immortalization of mammalian cells mediated by retroviral transfer and site-specific recombination. Proc Natl Acad Sci U S A93 :8971 -8976,1996) containing cDNAs expressing hTERT, for immortalization, and enhanced green fluorescent protein (EGFP), for a selection marker, flanked by a pair of recombination target loxPs. Two days after three rounds of SSR#197 transduction, cells are sorted using a FACSCalibur system (BD Biosciences Immunocytochemistry Systems, San Jose, CA) for recovering EGFP-positive cell populations. One of the EGFP-positive immortalized clones is selected on the basis of its hepatocyte-specifϊc gene expression profile and negative tumorigenesis. These cells are transduced with a plasmid encoding a tamoxifen-inducible Cre recombinase fused with paired mutant estrogen receptor ligand-binding domains (MerCreMer) under the control of the CAG promoter (cytomegalovirus IE enhancer, chicken β-actin promoter, and β-globin splicing acceptor) with a puromycin resistance gene. The resultant puromycin-resistant clones grow steadily in tissue culture with serum-free ISE- RPMI medium (Nakabayashi H, Taketa K, Miyano K, Yamane T, Sato J: Growth of human hepatoma cell lines with differentiated functions in chemically defined medium. Cancer Res42 :3858 -3863,1982). These cells are further transduced with a plasmid vector expressing the antigens of this invention and zeocin resistance genes under the control of the rat L-PK promoter. After transfection, cells were cultured with zeocin-containing ISE-RPMI medium, and zeocin-resistant cells are cloned by limiting dilution. EXAMPLE 2.

The method of EXAMPLE 1., where the inserted antigen is derived from the surface of Enterococcus faecalis.

EXAMPLE 3.

The method of EXAMPLE 1., where the inserted antigen is derived from the surface of respiratory syncytial viruses.

EXAMPLE 4.

The method of EXAMPLE 1., where the inserted antigen is derived from the surface of Aspergillus cadida.

EXAMPLE 5.

An expression vector is constructed which allows for the selection of stable transfectants by selection for the zeocin antibiotic (Cayla) in both prokaryotic and eukaryotic cells. The Zeocin resistance gene is obtained as a restriction digest fragment from the pZeoSV plasmid (Invitrogen) and is ligated to a fragment containing a bacterial origin of replication obtained by PCR amplification from pUC19 (New England Biolabs). This ligation mixture is then used to transform competent E. coli cells and the presence of the desired recombinant plasmid (pUC-Zeo) is selected for on Zeocin-containing bacterial plates. A synthetic poly(A) sequence obtained as a restriction fragment from a digest of pGL3-Basic (Promega) is ligated into pUC-Zeo upstream of the HSP70B promoter, and the desired recombinant (pUC-ZeoA) will selected for Zeocin resistance. The HSP70B driven expression cassette drives the expression of genes for the antigens of this invention. These expression cassettes can be transfected into for instance JURKAT cells, as an example of secretory cells of the immune system.

EXAMPLE 6.

The method of EXAMPLE 5., where the inserted antigen is derived from the surface of Enterococcus faecalis.

EXAMPLE 7.

The method of EXAMPLE 5., where the inserted antigen is derived from the surface of respiratory syncytial viruses.

EXAMPLE 8.

The method of EXAMPLE 5., where the inserted antigen is derived from the surface of Aspergillus cadida.

Claims

Claim 1. A biological artificial organ (BAO) contain eukaryotic cells, said eukaryotic cells a) being transformed in a stable fashion with genes encoding viral, bacterial or fungal antigens b) such genes being placed under the operational expression control of an heat-inducible promoter c) the BAO being inserted under the skin of the patient to be immunized, in a retractable fashion d) the cells inside the BAO being induced to express and secrete the said antigens using local heat e) this induction being repeated where necessary to induce an efficient neutralizing antibody response against said antigens.

Claim 2. The method of Claim 1, where the BAO is an implantable immuno-isolatory product, the immuno-isolatory vehicle for providing a biologically active cell source comprising:

(a) a core comprising living eukaryotic cells dispersed in a biocompatible matrix either suspended in a liquid medium or immobilized within a hydrogel or extracellular matrix, said cells being capable of secreting a selected biologically active products providing a selected biological function to an individual; and

(b) an external diffusional surface jacket surrounding said core formed of a biocompatible hydrogel material free of said cells projecting externally thereof, said jacket having a molecular weight cut-off below the molecular weight of substances essential for immunological rejection of the cells but permitting passage of substances between the individual and core through said jacket required to provide said biological product or function, said core and external jacket forming an interface substantially free of direct ionic bonding and of an intermediate linking layer.

Claim 3. The method of Claim 1, where the cell employed is a human secretory cell type, including but not limited to, hepatocytes, kidney cells, and cell lines derived from these.

Claim 4. The method of Claim 3, where the genes employed are those of infectious agents including viruses, bacteria and fungus.

Claim 5. The method of Claim 3 in which the genes employed encode surface antigens of viruses, including but limited to influenza, parainfluenza, respiratory syncytial viruses, human papilloma viruses, rotaviral, and enteroviral species.

Claim 6. The method of Claim 3 in which the genes employed encode surface antigens of bacteria, including but not limited to, Coagulase-negative staphylococci, Enterococci, Enterobacteriaceae, Staphylococcus aureus, Enterobacter species, Pseudomonads, and Acinetobacter baumannii, Klebsiella pneumoniae, P. aeruginosa,

Claim 7. The method of Claim 3 in which the genes employed encode surface antigens of infectious fungi, including but not limited to Aspergillus cadida and Aspergillus nonalbians, filamentous fungi, e.g. Fusarium Acremonian, Paecilomyces,

Pseudallescheria boydii and Scedosporiam, demaliceous filamentous fungi, e.g. Biopolaris species, Cladophialophora bantiana, Dactylania gallopava, Exophilia species and Alternaria species, and yeast-like pathogens, e.g. Trichosporan species, Malassezia species and Rhodotonula rubra.

Claim 8. The method of Claim 3 in which the genes employed encode surface antigens of childhood disease vectors including, but not limited to Diphtheria, Tetanus, Pertussis, Polio, Meningitis C, Measles, Mumps and Rubella.

Claim 9. The method of Claim 3 in which the genes employed encode surface antigens of tropical disease agents, including, but not limited to Cholera, Haemophilus Influenzae Type B, Meningitis, Hepatitis B, Japanese Encephalitis, Meningococcal Disease, Poliomyelitis, Tuberculosis, Chickenpox, Diptheria, Tetanus, Pertussis, Hepatitis A, Measles, Mumps, Rabies, Typhoid Fever, and Yellow Fever.

Claim 10. The method of Claim 3 in which the genes employed encode surface antigens of viruses associated with the development of cancer, including but not limited to human Papilloma viruses.

Claim 11. The method of Claim 3 in which the genes employed encode surface antigens of Human Immunodeficiency viruses, including but not limited to the AIDS viruses.

Claim 12. The method of Claim 2, where the heat induction, raising the temperature of the BAO and the cells it contains from 27 to 42 degrees centigrade for 15 to 30 minutes, one or more times over a period of 1 to six months.

Claiml3. The method of Claim 2 where the BAO can be removed from the patient once immunity against infective pathogens is obtained.

Claim 14, sources of heat to be applied to the site of installation of the BAO, permitting the heat increase as in Claim 1.

Claim 15, sources of heat as in Claim 14, where the heat is provides by electrical, electromagnetic, induction or radiation means.

PCT/IB2009/051437 2008-04-07 2009-04-06 New and safe procedure for immunization against infectious diseases agents WO2009125332A1 (en)

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