WO2014091244A1 - Compositions immunogènes destinées à être utilisées dans une distribution d'aérosol - Google Patents

Compositions immunogènes destinées à être utilisées dans une distribution d'aérosol Download PDF

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WO2014091244A1
WO2014091244A1 PCT/GB2013/053290 GB2013053290W WO2014091244A1 WO 2014091244 A1 WO2014091244 A1 WO 2014091244A1 GB 2013053290 W GB2013053290 W GB 2013053290W WO 2014091244 A1 WO2014091244 A1 WO 2014091244A1
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antigen
immunogenic composition
vaccination
aerosol
cells
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English (en)
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Helen Mcshane
Arian V.S. HILL
Iman SATTI
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Isis Innovation Limited
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/02—Bacterial antigens
    • A61K39/04—Mycobacterium, e.g. Mycobacterium tuberculosis
    • 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
    • A61K2039/525—Virus
    • A61K2039/5254—Virus avirulent or attenuated
    • 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
    • A61K2039/525—Virus
    • A61K2039/5256—Virus expressing foreign proteins
    • 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
    • A61K2039/54—Medicinal preparations containing antigens or antibodies characterised by the route of administration
    • A61K2039/541—Mucosal route
    • A61K2039/543—Mucosal route intranasal
    • 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
    • A61K2039/54—Medicinal preparations containing antigens or antibodies characterised by the route of administration
    • A61K2039/541—Mucosal route
    • A61K2039/544—Mucosal route to the airways
    • 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
    • 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
    • C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
    • C12N2710/00011—Details
    • C12N2710/24011—Poxviridae
    • C12N2710/24111—Orthopoxvirus, e.g. vaccinia virus, variola
    • C12N2710/24141—Use of virus, viral particle or viral elements as a vector
    • C12N2710/24143—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector
    • 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 present invention relates to immunogenic compositions for use in aerosol delivery to the respiratory tract of a human.
  • the compositions comprise an expression vector, which comprise one or more exogenous nucleotide sequences from one or more pathogens.
  • the compositions are able to elicit a protective immune response and, in particular, elicit both a local mucosal and systemic immune response, which is protective.
  • Subunit vaccines present an antigen to the immune system without introducing a whole infectious organism unless the organism is a viral vector, where the vector is a whole organism.
  • One such method involves the administration of a specific, isolated protein from an infectious organism. However, this technique often induces only a weak immune response and the isolated proteins may have a different three-dimensional structure than the protein in its normal context, resulting in the production of antibodies that may not recognize the infectious organism.
  • antigens such that stronger, protective immune responses can be elicited.
  • the present invention provides an immunogenic composition for use in aerosol delivery to the respiratory tract of a human subject comprising a viral expression vector, wherein said viral expression vector comprises at least one exogenous nucleotide sequence, encoding one or more epitopes or a protein or polypeptide antigen, derived from a pathogen, and wherein said immunogenic composition elicits both a local, mucosal, immune response and a systemic immune response against the one or more protein or polypeptide antigens.
  • the antigen is a pathogen-derived antigen.
  • the pathogen is selected from the group consisting of bacteria, viruses, prions, fungi, protozoa and helminths.
  • the antigen is derived from the group consisting of M. tuberculosis, Plasmodium sp, influenza virus, HSV, HIV, Hepatitis B or C virus, Cytomegalovirus, Human papilloma virus, chlamydia species, malaria parasites, leishmania parasites or any mycobacterial species.
  • Preferred antigens include TRAP, MSP- 1 , AMA- 1 , RH5, Pfs25 and CSP from Plasmodium, influenza virus antigens and ESAT6, TBI 0.4 85 A and 85B antigens from Mycobacterium tuberculosis.
  • Particularly preferred antigens include Ag85 A from
  • NP nucleoprotein
  • Ml matrix protein 1
  • the term 'antigen' encompasses one or more epitopes from an antigen and includes the parent antigen, and fragments and variants thereof. These fragments and variants retain essentially the same biological activity or function as the parent antigen. Preferably, they retain or improve upon the antigenicity and/or immunogenicity of the parent antigen.
  • antigenic is taken to mean that the protein or polypeptide is capable of being used to raise antibodies or T cells or indeed is capable of inducing an antibody or T cell response in a subject.
  • Immunogenic is taken to mean that the protein or polypeptide is capable of eliciting a potent and preferably a protective immune response in a subject.
  • the protein or polypeptide may be capable of generating an antibody response and a non-antibody based immune response.
  • fragments of the antigens comprise at least n consecutive amino acids from the sequence of the parent antigen, wherein n is preferably at least, or more than, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 57, 58, 59, 60, 70, 80, 90 or 100.
  • the fragments preferably include one or more epitopic regions from the parent antigen. Indeed, the fragment may comprise or consist of an epitope from the parent antigen. Alternatively, the fragment may be sufficiently similar to such regions to retain their antigenic/immunogenic properties.
  • the antigens discussed herein include variants such as derivatives, analogues, homologues or functional equivalents of the parent antigen. Particularly preferred are derivatives, analogues, homologues or functional equivalents having an amino acid sequence similar to that of the parent antigen, in which one or more amino acid residues are substituted, deleted or added in any combination. Preferably, these variants retain an antigenic determinant or epitope in common with the parent antigen.
  • the derivatives, analogues, homologues, and functional equivalents have an amino acid sequence substantially identical to amino acid sequence of the parent antigen.
  • the exogeneous nucleotide sequence may encode more than one antigen.
  • the viral vector may be designed to express the one or more antigen genes as an epitope string.
  • the epitopes in a string of multiple epitopes are linked together without intervening sequences such that unnecessary nucleic acid and/or amino acid material is avoided.
  • the creation of the epitope string is preferably achieved using a recombinant DNA construct that encodes the amino acid sequence of the epitope string, with the DNA encoding the one or more epitopes in the same reading frame.
  • An exemplary antigen, TIPeGFP comprises an epitope string which includes the following epitopes: E6FP, SIV-gag, PyCD4 and Py3.
  • the antigens may be expressed as separate polypeptides.
  • One or more of the antigens or antigen genes may be truncated at the C-terminus and/or the N-terminus. This may facilitate cloning and construction of the vectored vaccine and/or enhance the immunogenicity or antigenicity of the antigen.
  • Methods for truncation will be known to those of skill in the art. For example, various well-known techniques of genetic engineering can be used to selectively delete the encoding nucleic acid sequence at either end of the antigen gene, and then insert the desired coding sequence into the viral vector. For example, truncations of the candidate protein are created using 3' and/or 5' exonuclease strategies selectively to erode the 3' and/or 5' ends of the encoding nucleic acid, respectively.
  • the wild type gene sequence is truncated such that the expressed antigen is truncated by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more amino acids relative to the parent antigen.
  • the antigen gene is truncated by 10 - 20 amino acids at the C- terminus relative to the wild type antigen. More preferably, the antigen gene is truncated by 13 - 18 amino acids, most preferably by 15 amino acids at the C- terminus relative to the wild type antigen.
  • the Ag85 A antigen is C-terminally truncated in this manner.
  • One or more of the antigen genes may also comprise a leader sequence.
  • the leader sequence may affect processing of the primary transcript to mRNA, translation efficiency, mRNA stability, and may enhance expression and/or immunogenicity of the antigen.
  • the leader sequence is tissue plasminogen activator (tPA).
  • tPA tissue plasminogen activator
  • the tPA leader sequence is positioned N-terminal to the one or more antigens.
  • the leader sequence such as the tPA leaders sequence may be linked to the sequence of the antigen via a peptide linker.
  • Peptide linkers are generally from 2 to about 50 amino acids in length, and can have any sequence, provided that it does not form a secondary structure that would interfere with domain folding of the fusion protein.
  • One or more of the antigen genes may comprise a marker such as the Green Fluorescent Protein (GFP) marker to facilitate detection of the expressed product of the inserted gene sequence.
  • One or more of the antigen genes may comprise a nucleic acid sequence encoding a tag polypeptide that is covalently linked to the antigen upon translation.
  • the tag polypeptide is selected from the group consisting of a PK tag, a FLAG tag, a MYC tag, a polyhistidine tag or any tag that can be detected by a monoclonal antibody.
  • the nucleic acid sequence encoding the tag polypeptide may be positioned such that, following translation, the tag is located at the C-terminus or the N-terminus of the expressed antigen or may be internal to the expressed antigen.
  • the tag is located at the C-terminus of the expressed antigen.
  • one or more of the antigen genes encode a PK tag.
  • a tag of this type may facilitate detection of antigen expression and clones expressing the antigen, and/or enhance the immunogenicity or antigenicity of the antigen.
  • the exogeneous nucleotide sequence is preferably operably linked to regulatory sequences necessary to drive translation, transcription and/or expression of the exogeneous nucleotide sequence /transgene in a host cell, for example a mammalian cell.
  • operably linked means that the regulatory sequences are contiguous with the nucleic acid sequences they regulate or that said regulatory sequences act in trans, or at a distance, to control the regulated nucleic acid sequence.
  • regulatory sequences include appropriate expression control sequences such as transcription initiation, termination, enhancer and promoter sequences, efficient RNA processing signals, such as splicing and polyadenylation signals, sequences that enhance translation efficiency and protein stability and sequences promote protein secretion. Additionally they may contain sequences for repression of transgene expression, for example during production in cell lines expression a transactivating receptor. Promoters and other regulatory sequences which control expression of a nucleic acid have been identified and are known in the art.
  • the promoter is selected from the group consisting of human CMV promoters, simian CMV promoters, murine CMV promoters, ubiquitin, the EF1 promoter, frog EF1 promoter, actin and other mammalian promoters. Most preferred are human CMV promoters and in particular the human CMV major immediate early promoter.
  • human CMV promoters and in particular the human CMV major immediate early promoter.
  • any internal promoter may be used, for example the p7.5 promoter or the Fl 1 promoter and many others are known.
  • strong synthetic promoters may be used such as the SSP sequence.
  • the exogeneous nucleotide sequence(s) of interest may be introduced into the viral vector as part of a cassette.
  • cassette refers to a nucleic acid molecule comprising at least one nucleotide sequence to be expressed, along with its transcriptional
  • any method known to one of skill in the art could be used to construct, modify or derive said cassette, for example PCR mutagenesis, In-Fusion ® , recombineering, Gateway ® cloning, site-specific recombination or topoisomerase cloning.
  • the expression control sequences preferably include the viral elements necessary for replication and virion encapsidation.
  • the elements flank the exogeneous nucleotide sequence.
  • viral vectors examples include Vaccinia virus vectors or Adenovirus vectors.
  • the viral vectors can be of human or simian origin.
  • the viral vector is non-replicating or replication-impaired.
  • non-replicating or “replication-impaired” means not capable of replicating to any significant extent in the majority of normal mammalian cells, preferably normal human cells. It is preferred that the viral vector is incapable of causing a productive infection or disease in the human patient. However, the viral vector is preferably capable of stimulating an immune response. Viruses which are non-replicating or replication-impaired may have become so naturally, i.e. they may be isolated as such from nature. Alternatively, the viruses may be rendered non-replicating or replication-impaired artificially, e.g. by breeding in vitro or by genetic manipulation. For example, a gene which is critical for replication may be functionally deleted.
  • the immunogenic and/or antigenic compositions as described herein may be prophylactic (to prevent infection), post-exposure (to treat after infection but before disease) or therapeutic (to treat disease).
  • the composition is prophylactic or post-exposure.
  • the composition is a vaccine.
  • compositions described herein can be administered in combination with adjuvants or other treatments.
  • the compositions could be administered with one or more additional active agents.
  • they can be coadministered with one or more antimicrobial compounds, such as suitable antimicrobial compounds including antituberculous chemotherapeutics such as rifampicin, isoniazid, ethambutol and pyrizinamide.
  • Suitable carriers and/or diluents are well known in the art and include pharmaceutical grade starch, mannitol, lactose, magnesium stearate, sodium saccharin, talcum, cellulose, glucose, sucrose, (or other sugar), magnesium carbonate, gelatin, oil, alcohol, detergents, emulsifiers or water (preferably sterile).
  • Suitable adjuvants for human use are well known in the art and include incomplete Freund's adjuvant, Freund's adjuvant with MDP (muramyldipeptide), alum (aluminium hydroxide), alum plus Bordatella pertussis and immune stimulatory complexes (ISCOMs, typically a matrix of Quil A containing viral proteins), MF59 (Novartis Vaccines, Siena) and matrix M (Isconova, Uppsala).
  • adjuvants may be encoded in the viral vector rather than mixed with the vector. Examples of encoded adjuvants include CD74 (invariant chain), IMX- 313 which is homologous to a fragment of human C4-binding protein, and co-stimulatory molecules such as 4-1BBL.
  • Aerosol formulations typically comprise a solution or fine suspension of the active substance in a physiologically acceptable aqueous or non-aqueous solvent and are usually presented in single or multidose quantities in sterile form in a sealed container, which can take the form of a cartridge or refill for use with an atomising device.
  • the sealed container may be a unitary dispensing device such as a single dose nasal inhaler or an aerosol dispenser fitted with a metering valve, which is intended for disposal once the contents of the container have been exhausted.
  • the dosage form comprises an aerosol dispenser, it will contain a pharmaceutically acceptable propellant.
  • the aerosol dosage forms can also take the form of a pump-atomiser.
  • the compositions are administered as aerosols by means of a nebuliser.
  • a nebuliser is a hand held device which may be held and / or operated by the subject being immunised.
  • One such nebuliser is that supplied by Omron, under the name MicroAir U22, which is a commercially available battery-operated hand-held lightweight ultrasonic mesh nebuliser (Omron, 2012).
  • This device is silent and generates a mist with a particle size of between 2 and 4 ⁇ from fluid placed within the nebuliser chamber.
  • a patient inhales the aerosol through a mouthpiece at their normal relaxed respiratory rate and tidal volume.
  • the composition is suitably diluted into a total volume of 1 mL with 0.9% saline which aids nebulisation.
  • Other examples of nebulisers are well known to the skilled person.
  • the composition is preferably pyrogen-free. It is preferably buffered e.g. at between pH 6 and pH 8, generally around pH 7. Preferably, the composition is substantially isotonic with humans.
  • compositions described herein deliver an immunogenically effective amount of the viral vector to a patient.
  • immunogenically effective amount means that the administration of that amount to an individual, either as a single dose or as a series of doses, is effective for prevention or treatment of a disease or condition.
  • this phrase means that a sufficient amount of the viral vector is delivered to the patient over a suitable timeframe such that a sufficient amount of the antigen is produced by the patient's cells to stimulate an immune response which is effective for prevention or treatment of a disease or condition.
  • a pharmaceutically effective dose comprises 1 x 10 to 1 x 10 viral particles.
  • the dose will be at the lower end of this range, typically 1-10 x 10 plaque forming units whilst for Adenovirus vectors it will tend to be towards the upper end of the range.
  • the treatment and prevention of many diseases are associated with the maintenance of a strong cell-mediated response to infection involving both CD4+ and CD8+ T cells and the ability to respond with Thl-type cytokines, particularly IFN- ⁇ , TNF-a, IL-2 and IL-17.
  • Thl-type cytokines particularly IFN- ⁇ , TNF-a, IL-2 and IL-17.
  • many subunit vaccine platforms effectively generate human immunity, the generation of robust cell-mediated immune responses, particularly CD4+ and CD8+ T cell immune responses, has been much more challenging.
  • the viral vectors compositions as discussed herein stimulate both cellular and humoral immune responses against the encoded antigen.
  • the aerosol administration is surprisingly found to induce stronger systemic (blood) T cell responses of particular types, e.g. interferon-gamma- secreting CD8 T cell responses, than intradermal administration. This induction of very potent blood T cell responses by use of an aerosol route was unexpected and has considerable utility for vaccination.
  • IL-17-secreting T cells have been found to be of importance in protective immunity to tuberculosis and several other bacterial and non-bacterial infectious diseases, but little has been known about how to induce these effectively in humans.
  • the induction of substantial IL-17-secreting T cells in humans in both blood and mucosal surfaces here is therefore an important advance.
  • CD8+ T cells are known to be important in protective immunity in humans to both infectious pathogens and cancer. Examples of the former include influenza, HIV, HCV and malaria and examples of the latter include melanoma, prostate cancer, breast cancer and colon cancer.
  • a means of improving on CD8 T cell induction systemically (here in blood) by using aerosol administration of viral vectors therefore has clear utility for both prophylactic and therapeutic immunisation. It is also desirable to induce a memory immune response. Memory immune responses are classically attributed to the reactivation of long-lived, antigen-specific T lymphocytes that arise directly from differentiated effector T cells and persist in a uniformly quiescent state.
  • Memory T cells have been shown to be heterogeneous and to comprise at least two subsets, endowed with different migratory capacity and effector function; effector memory T cells (TEM) and central memory T cells (CTM).
  • TEM resemble the effector cells generated in the primary response in that they lack the lymph node-homing receptors L-selectin and CCR7 and express receptors for migration into inflamed tissues. Upon re-encounter with antigen, these TEM can rapidly produce IFN- ⁇ or IL-4 or release pre- stored perform.
  • TCM express L- selectin and CCR7 and lack immediate effector function. These cells have a low activation threshold and, upon restimulation in secondary lymphoid organs, proliferate and differentiate to effectors.
  • Figure 1 shows a comparison of Ex- vivo summed 85 A peptide pool PBMC Elispot responses From subjects vaccinated with MVA85A vs the aerosol or intra dermal route;
  • Figure 2 shows a comparison of the BAL ICS Antigen 85A CD4+ T cell responses between two human patient groups vaccinated by the aerosol or intradermal route with the MVA85A vaccine.
  • Levels of Antigen-85A specific CD4+ T cells containing interferon gamma (IFNy), tumour necrosis factor (TNF), interleukin (IL) 2 and IL17 were all higher in the
  • bronchoalveolar lavage fluid in subjects immunised by the aerosol route than in subjects immunised by the intradermal route. All these cytokines are considered important in vaccine- induced protection against TB.
  • Figure 3 shows a comparison of the Whole blood ICS Antigen 85 A CD4+ T cell responses between two human patient groups vaccinated by the aerosol or intradermal route with the MVA85A vaccine.
  • Levels of Antigen-85A specific CD4+ T cells containing interferon gamma (IFNy), interleukin (IL) 2 and IL17 were all higher in whole blood in subjects immunised by the aerosol route than in subjects immunised by the intradermal route. All these cytokines are considered important in vaccine-induced protection against TB.
  • IFNy interferon gamma
  • IL 2 interleukin 2
  • IL17 interleukin 17
  • Figure 4 shows a comparison of several multifunctional CD4+ T cell subsets detected in BAL after aerosol or intra dermal vaccination with MVA85A. Higher levels of several multifunctional T cell subsets considered important in protection against M.tb were higher in the BAL after aerosol than intradermal vaccination
  • Figure 5 shows a comparison of several multifunctional CD4+ T cell subsets detected in Whole blood after aerosol or intra dermal vaccination with MVA85A. Higher levels of several multifunctional T cell subsets considered important in protection against M.tb were higher in the blood after aerosol than intradermal vaccination
  • Figure 6 shows a comparison of the BAL and whole blood Antigen 85A CD8+ T cell responses between two groups of human subjects vaccinated by the aerosol or intradermal route with the MVA85A vaccine. Seven days after vaccination, levels of IFNy+ CD8+ T cells were higher in the BAL than in whole blood after both aerosol and intradermal
  • Figure 7 shows a comparison of anti- Vaccinia IgG levels measured in the serum samples between two human patient groups vaccinated by the aerosol or intradermal route with the MVA85A vaccine.
  • Figure 8 shows a comparison of serum IgG levels measured in the serum samples between two human patient groups vaccinated by the aerosol or intradermal route with the MVA85A vaccine.
  • Figure 9 shows a comparison of various vaccination strategies on CFU in lungs and spleen of mice. Boosting BCG with intranasal ChAdOxl .85A followed by intranasal MVA85A is significantly more protective than BCG alone in mice.
  • Example 1 Immunogenicitv of a candidate TB vaccine, MVA85A, delivered by aerosol to the lungs of macaques
  • Tuberculosis is a re-emerging disease.
  • the only available vaccine, BCG is delivered intradermally and confers highly variable efficacy against pulmonary disease.
  • Murine studies suggest immunisation delivered directly to the respiratory mucosa may be a more effective route of vaccination.
  • Tuberculosis (TB) in humans is caused by infection with Mycobacterium tuberculosis (M. tb), and is one of the leading global causes of death from a single infectious agent, with an estimated 8.8 million new cases worldwide and 1.77 million deaths in 201 1.
  • the only licensed vaccine against TB is Bacille Calmette Guerin (BCG), a live attenuated strain of Mycobacterium bovis, which was first introduced in 1921 and remains in routine use around the globe. During this time it has been administered to several billion people, predominantly delivered by intradermal injection. Whilst BCG is well tolerated, it is not effective in all populations, does not prevent infection in high burden settings, and is contra- indicated in immunocompromised patient s .
  • BCG Bacille Calmette Guerin
  • Modified vaccinia Ankara is a safe, replication-defective, viral vector, capable of inducing both cellular and antibody immunity to target antigens with protective potential.
  • the most clinically advanced "boost" candidate is the viral-vectored subunit TB vaccine, MVA85A, which expresses the highly conserved, immunodominant mycobacterial antigen, 85A.
  • both BCG and MVA85A have been administered by intra-dermal (i.d.) injection, a route of inoculation that has been shown to induce strong mycobacterial-specific immune responses in the periphery.
  • i.d. intra-dermal
  • MALT mucosal associated lymphoid tissue
  • Vaccine delivery directly to the lung by aerosol also brings the advantages associated with needle-free delivery that include speed and simplicity of immunisation, which allows application by non-medical personnel and, as delivery is non invasive and pain free, it brings great social acceptance.
  • the route of vaccine delivery determines the location of antigen-specific cells and thus the protective efficacy of the vaccine.
  • Evidence from animal studies suggests that the prevalence of antigen-specific cells in the airway lumen is important for optimal protection against TB.
  • Replication-deficient vaccinia and adenoviral vectors encoding antigen 85A demonstrated better protection when delivered to the murine respiratory mucosa than when administered systemically.
  • IFN- ⁇ tumour necrosis factor alpha
  • IL-2 interleukin 2
  • tissue samples were thawed and homogenised in 2ml 2% FCS DMEM (Invitrogen, D6546) medium using GentleMacs M tubes on a Dispomix (programme 14), followed by 2 cycles of snap freeze/thaw in dry ice/isopropanol to release any viral particles from within the organ tissue.
  • Samples were spun to remove as much tissue debris as possible before being plated out on sub-confluent low passage (P3) CEF cells in a 96 well plate and incubated for 96 hours. Supernatant was then plated onto a second set of CEF cells in poly-lysine coated 96 well plates to allow further viral replication for 2 days before fixing cells with methanol.
  • P3 sub-confluent low passage
  • the animals used in this study were rhesus macaques of Indian origin obtained from an established UK breeding colony. All animals were 4.5 to 5 years old at the time of vaccination and naive in terms of prior exposure to mycobacterial antigens (M tb infection or environmental mycobacteria) as demonstrated by a negative tuberculin test whilst in their original breeding colony and by the IFN- ⁇ based PrimagamTM test kit (Biocor, CSL, USA) just prior to study start. Monkeys were housed according to the Home Office (UK) Code of Practice for the Housing and Care of Animals Used in Scientific Procedures (1989) and the National Committee for Refinement, Reduction and Replacement (NC3Rs) Guidelines on Primate Accommodation, Care and Use, August 2006.
  • UK Home Office
  • MVA85A 100 ⁇ (1 x 10 pfu) MVA85A.
  • Four animals (Group B) were exposed to an aerosol containing MVA85A created by nebulisation of 10 8 pfu in 1 ml sterile PBS using an Omron MicroAir mesh nebuliser (Omron Healthcare UK Ltd, Milton Keynes, UK) and delivered to the nose of each sedated primate via a modified paediatric anaesthesia mask.
  • the primary purpose of the study was to assess tolerability using the following readouts.
  • Animal behaviour was observed daily throughout the study for contra-indicators such as depression, withdrawal from the group, aggression, reduced food and water intake, changes in respiration rate, or cough.
  • Animals were sedated every two weeks to measure weight, body temperature, blood haemoglobin levels and erythrocyte sedimentation rate (ESR), and to collect blood samples for immunology.
  • Blood cell haemoglobin was measured using a HaemaCue haemoglobinometer (Haemacue Ltd, Dronfield, UK) and ESR was measured using the Sediplast system (Guest Medical, Edenbridge, UK). Chest X-rays were collected prior to BCG vaccination, 2 weeks prior to vaccination with MVA85A, and 1 and 8 weeks after MVA85A vaccination.
  • BAL samples were collected using a bronchoscope (All-Scope XE30 4mm flexible bronchoscope, VES, Southend on Sea, Essex. SS2 5BZ ) on two occasions four weeks apart prior to BCG vaccination and at weeks 4 and 8 after BCG vaccination and 1, 3, and 7 weeks after the MVA85A booster vaccination.
  • a bronchoscope All-Scope XE30 4mm flexible bronchoscope, VES, Southend on Sea, Essex. SS2 5BZ .
  • IFN- ⁇ Interferon-gamma
  • PBMC Peripheral blood mononuclear cells
  • PBMC Peripheral blood mononuclear cells
  • Ficoll-Hypaque Plus GE Healthcare, Buckinghamshire, United Kingdom density gradient separation using standard procedures.
  • An IFN- ⁇ ELISpot assay was used to estimate the numbers and IFN- ⁇ production capacity of mycobacteria-specific T cells in PBMC using a human/monkey IFN- ⁇ kit (MabTech, Nacka.
  • PBMCs were cultured with 10 ⁇ g/ml PPD (SSI, Copenhagen, Denmark), or a pool containing overlapping 15mer peptides spanning Ag85A (Peptide Protein Research Ltd, Wickham, UK), or without antigen, in triplicate, and incubated for 18 hours.
  • Phorbol 12-myristate (Sigma- Aldrich Dorset, UK) (100 ng/ml) and ionomycin (CN Biosciences, Nottingham, UK) (1 ⁇ g/ml) were used as a positive control.
  • spots were developed according to the manufacturer's instructions. Determinations from triplicate tests were averaged. Data were analysed by subtracting the mean number of spots in the cells and medium-only control wells from the mean counts of spots in wells with cells and antigen or peptide pools.
  • PBMCs were thawed, washed, re-suspended in medium (RIO) consisting of RPMI 1640 supplemented with L-glutamine (2 mM), penicillin (50 U/ml)/streptomycin (50 ⁇ g/ml) and 10% heat-inactivated foetal bovine serum with 1 U/ml of DNase (Sigma, Poole, UK) and incubated at 37°C for 2 hours.
  • RIO medium
  • BAL cells were isolated from BAL fluid by centrifugation (400g for 5 minutes) and assessed directly following isolation. Cell concentrations were adjusted to 1 x 10 6 cells/ml in R10 and stimulated with 10 ⁇ g/ml PPD (SSI, Copenhagen, Denmark). Intracellular cytokine staining to evaluate the production of the cytokines, IFNy, TNFa, and IL-2, was performed as previously described
  • Cytokine- secreting T cells were identified using a forward scatter-height (FSC-H) versus side scatter- area (SSC-A) dot plot to identify the lymphocyte population, to which appropriate gating strategies were applied to exclude doublet events, non- viable cells, monocytes (CD14 + ) and B cells (CD20 + ) prior to sequential gating through CD3 + , CD8 " and CD4 + versus IFNy, and CD3 + , CD8 + and CD4 " versus IFNy histograms.
  • FSC-H forward scatter-height
  • SSC-A side scatter- area
  • Anti- vector IgG titres were measured in serum samples by vaccinia-specific direct ELISA assay.
  • Nunc maxisorb 96 well plates were coated with inactivated Lister strain vaccinia (Autogen Bioclear). After blocking, samples were diluted 1/25 in assay diluent/blocking buffer (PBS + 5% skimmed milk powder + 0.1% tween-20) and added in duplicate.
  • assay diluent/blocking buffer PBS + 5% skimmed milk powder + 0.1% tween-20
  • a sample dilution series was created by 4 fold serial dilution.
  • a reference curve was created by inclusion of a polyclonal anti- vaccinia virus human immunoglobulin (VIG) control sample (NR-650, Biodefense and Emerging infections research resource repositories, USA), serial diluted in parallel.
  • VIP polyclonal anti- vaccinia virus human immunoglobulin
  • Serum and VIG control samples were incubated at room temperature for 2 hours. After washing, plates were incubated for 2 hours with anti-monkey IgG peroxidise (Insight Biotechnologies), washed, and then developed using ABTS peroxidise substrate system (Insight Biotechnologies). Absorbance at 405nm was then measured using a Versamax plate reader with Softmax Pro software. A VIG control curve was plotted for each plate and the inflection point used to interpret the serum sample IgG titre.
  • Vaccination did not lead to perturbations in any of the clinical parameters measured. Chest radiographs collected through the study demonstrated normal pulmonary structure which remained unchanged post- vaccination. Seven of the eight animals developed local reactions at the site of BCG vaccination 2 and 4 weeks post- vaccination, with reactions still visible in 3 animals at the end of the study. Intradermal vaccination with MVA85A induced reactions at the vaccination site one week post-immunisation in all 4 animals which resolved 3 weeks later. At the end of the study, 9 weeks after vaccination with MVA85A, gross pathology and histological assessments did not identify any adverse effects induced by vaccination. Overall, aerosol vaccination was well tolerated by all animals and caused no adverse effects.
  • MVA85A vaccination and single CD8+ T cell populations were only detected sporadically and at low frequencies.
  • BCG vaccination induced significant increases in both the proportion of PPD-specific CD4+ T cells in the BAL capable of producing more than one cytokine and the frequency of specific triple, dual, & single positive CD4+ T-cells from 4 weeks after BCG vaccination compared to pre-vaccination levels.
  • the frequency of PPD-specific CD4 T cell populations peaked 1 week after vaccination with MVA85A (week 13), then declined.
  • Vaccination induced a number of differences in population frequency and proportion although none significantly different.
  • An increase in the proportion of double (TNFa+IFNy+) and triple positive (TNFa+IFNy+IL-2+) PPD-specific cells compared to pre-vaccination levels was detected 4 weeks after BCG vaccination and was maintained throughout the study.
  • MVA85A vaccination a higher frequency of PPD-specific single positive IFNy-producing CD4 T cells, as well as a larger proportion of double (TNFa+IFNy+) and triple positive (TNFa+IFNy+IL-2+) PPD-specific cells, was seen in the group that received MVA85A by aerosol compared to the intradermally vaccinated group (Figure 3D).
  • the frequencies of PPD and Ag85A-specific IFNy secreting cells were measured by ELIspot in a range of lymphoid tissues (spleen, bone marrow lung, hilar, and axillary inguinal lymph nodes) collected at necropsy.
  • Responses detected in the tissues from animals that received the MVA85A by aerosol were comparable to those vaccinated intradermally (data not shown).
  • the median response in the lung of animals vaccinated by aerosol was approximately 6 fold higher than the response in the peripheral blood.
  • the response in the peripheral blood of the animals vaccinated intradermally was almost 6 fold higher than in the lung. Evaluation of the humoral response to the vaccinia vaccine vector induced by each route of vaccination in peripheral blood.
  • Vector-specific IgG responses were detected in the serum collected from animals after intradermal vaccination with MVA85A. Responses were first detected 2 weeks after vaccination with the peak increase in response from pre- vaccination levels detected 4 weeks post vaccination. In contrast, an anti-vaccinia antibody response was not detected in the serum from animals after aerosol vaccination with MVA85A. ( Figure 7)
  • Live viruses such as vaccinia and adenovirus are widely used as delivery systems in the development of new vaccines; however, their use can be limited by pre-existing immunity induced either by prior natural infection or by previous vaccination. Approaches that could circumvent previous immunity would be extremely useful in the development of improved vaccine strategies.
  • an antibody response to the MVA vector was detected in the serum of animals that received the MVA85A by intradermal injection, but not in the serum of animals which were vaccinated with MVA85A by aerosol. This suggests that delivery of vaccines to the lung provides an immunisation strategy which limits the induction of systemic anti-vector immunity and provides the potential for multiple vaccinations with poxvirus based strategies using mucosal followed by systemic delivery.
  • Example 2 Human study of aerosol delivery of MVA85A vaccine as compared to intradermal vaccination
  • Inhaled aerosol delivery was performed using the MicroAir U22 nebuliser (Omron) which is a commercially available battery-operated hand-held lightweight (140 g including batteries) ultrasonic mesh nebuliser (Omron, 2012). This device is silent and generates a mist with a particle size of between 2 and 4 ⁇ from fluid placed within the nebuliser chamber.
  • MicroAir U22 nebuliser (Omron) which is a commercially available battery-operated hand-held lightweight (140 g including batteries) ultrasonic mesh nebuliser (Omron, 2012). This device is silent and generates a mist with a particle size of between 2 and 4 ⁇ from fluid placed within the nebuliser chamber.
  • PBMC Peripheral Blood Mononuclear Cells
  • Insert- and vector-specific humoral antibody (IgG, IgA and IgM) responses were assessed by Enzyme Linked Immunosorbent Assay (ELISA) in serum samples.
  • Nunc Immunoplates were coated with r85A or MVA and incubated overnight for adsorption. The next morning samples were prepared by diluting test serum 1:10 in Casein blocking buffer. Coating solution was discarded, plates washed 6 times with PBS/Tween and blocked with 200ul of Casein per well for 1 hour at room temperature. Blocking solution was discarded; samples were added at 50ul per well and incubated at room temperature for 2 hours. Contents were discarded and plates washed 6 times with PBS/Tween.
  • Aerosol administration induces significantly higher levels of antigen 85A specific T cells in the BAL containing IFNy, IL2, TNF and IL17, compared with intradermal administration (Fig 2) 3. Aerosol administration induces higher levels of antigen 85 A specific CD4+ T cells containing IL2, TNF and IL17 in the blood, compared to intradermal vaccination. (Fig 3)
  • Aerosol administration induces higher levels of Antigen 85A specific multifunctional CD4+ T cells in the BAL compared with intradermal administration ( Figure 4)
  • Aerosol administration induces higher levels of Antigen 85A specific multifunctional CD4+ T cells in the blood compared with intradermal administration ( Figure 5)
  • Aerosol administration induces higher levels of antigen 85A specific CD8+ T cells in the blood than intradermal administration. ( Figure 6)
  • cytokines are made by CD4+ T cells from BAL and WB of volunteers vaccinated intradermally or by inhalation of MVA-85A.
  • ChAdOX1.85A was constructed as described in Dicks et al (A Novel Chimpanzee
  • Adenovirus Vector with Low Human Seroprevalence Improved Systems for Vector
  • mice Six-week old female Balb/c mice were allocated to the following groups:
  • CM - No BCG prime followed by a first boost with ChAdOxl .85 A, and then a second boost with MVA.85A
  • mice were vaccinated with BCG-Pasteur intradermally ⁇ Id.) at 4xl0 5 cfu/dose. All groups were boosted 10 weeks after BCG priming. Both MVA.85A and ChAdOxl.85 A were administered intranasally, 25 ⁇ 1/ ⁇ 8 ⁇ 1, at 5x10 pfu and 1x10 ifu respectively.
  • the BCM and BMC group had a four week interval between the first boost and second boost. Four weeks after the last immunisation (apart from the BCG group who had 16 weeks between the last vaccination and challenge), mice were challenged with M.tb.
  • mice were challenged with 50-100 cfu of aerosolised M.tuberculosis Erdman K01 strain using Biaera AeroMP- controlled nebuliser. Four weeks after challenge lungs and spleens were removed, homogenised, and plated on Middlebrook 7H11 plates with glycerol and enriched with OADC. Statistical test used: Mann- Whitney, *p ⁇ 0.05.

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Abstract

La présente invention concerne des compositions immunogènes destinées à être utilisées dans une distribution d'aérosol pour voies respiratoires humaines. Les compositions comportent un vecteur d'expression, qui comporte une ou plusieurs séquences nucléotidiques exogènes à partir d'un ou de plusieurs agents pathogènes. Les compositions sont capables de déclencher une réponse immunitaire de protection et, en particulier, de déclencher une réponse immunitaire à la fois systémique et muqueuse locale, qui est protectrice.
PCT/GB2013/053290 2012-12-14 2013-12-13 Compositions immunogènes destinées à être utilisées dans une distribution d'aérosol WO2014091244A1 (fr)

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Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2009117134A2 (fr) * 2008-03-21 2009-09-24 National Institutes Of Health Vaccins génétiques aérolisés et procédés d'utilisation
EP2497493A1 (fr) * 2009-11-02 2012-09-12 Mie University Vaccin antituberculeux pulvérisable pour administration transnasale comprenant un vecteur de paramyxovirus

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2009117134A2 (fr) * 2008-03-21 2009-09-24 National Institutes Of Health Vaccins génétiques aérolisés et procédés d'utilisation
US20110081377A1 (en) 2008-03-21 2011-04-07 The United States, as represented by the Secretary Department of Health and Human Services Aerosolized genetic vaccines and methods of use
EP2497493A1 (fr) * 2009-11-02 2012-09-12 Mie University Vaccin antituberculeux pulvérisable pour administration transnasale comprenant un vecteur de paramyxovirus

Non-Patent Citations (13)

* Cited by examiner, † Cited by third party
Title
A. D. WHITE ET AL: "Evaluation of the Safety and Immunogenicity of a Candidate Tuberculosis Vaccine, MVA85A, Delivered by Aerosol to the Lungs of Macaques", CLINICAL AND VACCINE IMMUNOLOGY, vol. 20, no. 5, 27 February 2013 (2013-02-27), pages 663 - 672, XP055104036, ISSN: 1556-6811, DOI: 10.1128/CVI.00690-12 *
BIVAS-BENITA M ET AL: "Non-invasive pulmonary aerosol delivery in mice by the endotracheal route", EUROPEAN JOURNAL OF PHARMACEUTICS AND BIOPHARMACEUTICS, ELSEVIER SCIENCE PUBLISHERS B.V., AMSTERDAM, NL, vol. 61, no. 3, 1 October 2005 (2005-10-01), pages 214 - 218, XP027805094, ISSN: 0939-6411, [retrieved on 20051001] *
CORBANIE E A ET AL: "DEPOSITION OF DIFFERENTLY SIZED AIRBORNE MICROSPHERES IN THE RESPIRATORY TRACT OF CHICKENS", AVIAN PATHOLOGY, HUNTINGDON, CAMBS, GB, vol. 35, no. 6, 1 December 2006 (2006-12-01), pages 475 - 485, XP009076790, ISSN: 0307-9457, DOI: 10.1080/03079450601028845 *
EMILY K FORBES ET AL: "Multifunctional, high-level cytokine-producing Th1 cells in the lung, but not spleen, correlate with protection against Mycobacterium tuberculosis aerosol challenge in mice", JOURNAL OF IMMUNOLOGY (BALTIMORE, MD. : 1950), 1 October 2008 (2008-10-01), United States, pages 4955, XP055103957, Retrieved from the Internet <URL:http://www.jimmunol.org/cgi/content/abstract/181/7/4955> [retrieved on 20140224] *
GAUTAM A ET AL: "Enhanced gene expression in mouse lung after PEI-DNA aerosol delivery", MOLECULAR THERAPY, NATURE PUBLISHING GROUP, GB, vol. 2, no. 1, 1 July 2000 (2000-07-01), pages 63 - 70, XP002246051, ISSN: 1525-0016, DOI: 10.1006/MTHE.2000.0087 *
GOONETILLEKE NILU P ET AL: "Enhanced immunogenicity and protective efficacy against Mycobacterium tuberculosis of bacille Calmette-Guerin vaccine using mucosal administration and boosting with a recombinant modified vaccinia virus Ankara", THE JOURNAL OF IMMUNOLOGY, THE AMERICAN ASSOCIATION OF IMMUNOLOGISTS, US, vol. 171, no. 3, 1 August 2003 (2003-08-01), pages 1602 - 1609, XP009129402, ISSN: 0022-1767 *
HELEN MCSHANE: "75th stories: Helen McShane and a new vaccine for tuberculosis | Wellcome Trust Blog", 22 June 2011 (2011-06-22), XP055104063, Retrieved from the Internet <URL:http://blog.wellcome.ac.uk/2011/06/22/75th-stories-helen-mcshane-and-a-new-vaccine-for-tuberculosis/> [retrieved on 20140224] *
M. CORBETT ET AL: "Aerosol immunization with NYVAC and MVA vectored vaccines is safe, simple, and immunogenic", PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES, vol. 105, no. 6, 12 February 2008 (2008-02-12), pages 2046 - 2051, XP055103873, ISSN: 0027-8424, DOI: 10.1073/pnas.0705191105 *
PATHAN ET AL., VACCINE, vol. 30, 2012, pages 5616 - 24
RADOSEVIC KATARINA ET AL: "Protective immune responses to a recombinant adenovirus type 35 tuberculosis vaccine in two mouse strains: CD4 and CD8 T-cell epitope mapping and role of gamma interferon", INFECTION AND IMMUNITY, AMERICAN SOCIETY FOR MICROBIOLOGY, US, vol. 751-4, 9-12, 14,, no. 8, 1 August 2007 (2007-08-01), pages 4105 - 4115, XP002544475, ISSN: 0019-9567, DOI: 10.1128/IAI.00004-07 *
ROMANO M ET AL: "Priming but not boosting with plasmid DNA encoding mycolyl-transferase Ag85A from Mycobacterium tuberculosis increases the survival time of Mycobacterium bovis BCG vaccinated mice against low dose intravenous challenge with M. tuberculosis H37Rv", VACCINE, ELSEVIER LTD, GB, vol. 24, no. 16, 12 April 2006 (2006-04-12), pages 3353 - 3364, XP028010625, ISSN: 0264-410X, [retrieved on 20060412], DOI: 10.1016/J.VACCINE.2005.12.066 *
SANTOSUOSSO MICHAEL ET AL: "Mechanisms of mucosal and parenteral tuberculosis vaccinations: adenoviral-based mucosal immunization preferentially elicits sustained accumulation of immune protective CD4 and CD8 T cells within the airway lumen", THE JOURNAL OF IMMUNOLOGY, THE AMERICAN ASSOCIATION OF IMMUNOLOGISTS, US, vol. 174, no. 12, 15 June 2005 (2005-06-15), pages 7986 - 7994, XP002544946, ISSN: 0022-1767 *
WANG JUN ET AL: "Single mucosal, but not parenteral, immunization with recombinant adenoviral-based vaccine provides potent protection from pulmonary tuberculosis", THE JOURNAL OF IMMUNOLOGY, THE AMERICAN ASSOCIATION OF IMMUNOLOGISTS, US, vol. 173, no. 10, 15 November 2004 (2004-11-15), pages 6357 - 6365, XP002329297, ISSN: 0022-1767 *

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