WO2017109028A1 - Recombinant aspartyl protease antigen or antibody thereto for use in immunotherapy of fungal diseases - Google Patents

Recombinant aspartyl protease antigen or antibody thereto for use in immunotherapy of fungal diseases Download PDF

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Publication number
WO2017109028A1
WO2017109028A1 PCT/EP2016/082297 EP2016082297W WO2017109028A1 WO 2017109028 A1 WO2017109028 A1 WO 2017109028A1 EP 2016082297 W EP2016082297 W EP 2016082297W WO 2017109028 A1 WO2017109028 A1 WO 2017109028A1
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seq
spp
antibody
recombinant
positions
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French (fr)
Inventor
Françoise DROMER
Guilhem Janbon
Ségolène NEUVILLE
Olivier LORTHOLARY
Frédérique VERNEL-PAUILLAC
Alexandre Alanio
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Assistance Publique Hopitaux de Paris APHP
Institut Pasteur
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Assistance Publique Hopitaux de Paris APHP
Institut Pasteur
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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
    • A61K39/0002—Fungal antigens, e.g. Trichophyton, Aspergillus, Candida
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
    • C07K16/40—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against enzymes
    • 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
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • 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
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/34—Identification of a linear epitope shorter than 20 amino acid residues or of a conformational epitope defined by amino acid residues

Definitions

  • the present invention relates to the use of a recombinant aspartyl protease antigen, in particular a recombinant Cryptococcus spp. aspartyl protease Pepl protein, as vaccine for the treatment of fungal diseases.
  • the invention relates also to protective antibodies recognizing specific epitopes of said recombinant aspartyl protease antigen, in particular anti-Pepl protective monoclonal antibodies, and their use for the treatment of fungal diseases.
  • Candida infections occur can be predicted at around 300,000 worldwide per year, with a mortality of 30- 55%.
  • Invasive aspergillosis can occur in different patients groups including leukaemic cases, stem cell transplants and chronic obstructive pulmonary disease, with a mortality over 50%- even with treatment.
  • Cryptococcosis is an opportunistic infection in subjects with cellular immune defects like those infected with HIV at a late stage of their disease, or those with solid organ transplantation, chronic lymphoid leukemia, or prolonged corticosteroid treatment.
  • Disseminated meningoencephalitis is the main and most severe presentation and is always fatal unless treated.
  • the Center for Diseases Control estimates that 1 million patients are diagnosed with cryptococcosis worldwide and that more than 600,000 die each year (Park et al, AIDS, 2009, 23, 525-530).
  • the causative agent is Cryptococcus spp., an encapsulated yeast present in the environment that is acquired through inhalation.
  • the isolates responsible for infections are serotypes A, B, C or D, haploid or diploid, and mating type alpha (MATa) or a.
  • haploid C. neoformans MAT serotype A isolates represent the most prevalent clinical isolates worldwide.
  • Diagnosis relies on culture of viable yeasts from clinical specimens such as cerebrospinal fluid (CSF), blood, urine, bronchoalveolar lavages or on detection of soluble capsular polysaccharide (CPS) in body fluids such as CSF and serum (Perfect et al, Clin. Infect. Dis., 2010, 50, 291- 322).
  • CSF cerebrospinal fluid
  • CPS soluble capsular polysaccharide
  • body fluids such as CSF and serum
  • Pepl is a 438 amino acid protein of SEQ ID NO: 1 containing two putative active sites containing consensus sequences common to all aspartyl proteases ( Figure 1).
  • the inventors have found that the aspartyl protease Pepl is relatively conserved in fungi that can be recovered during infection in humans or animals; the C-terminal region is quite conserved whereas the N-terminal region (first 100 amino acids of Pepl) is variable ( Figure 2; Table I).
  • catalytic motifs are included in the consensus sequences SEQ ID NO: 4 and SEQ ID NO: 5 corresponding to positions 142 to 155 and 326 to 333 of SEQ ID NO: 1.
  • mice Using a mouse model of cryptococcosis, the inventors have shown that vaccination with rPepl resulted in the production of detectable anti-Pep 1 antibodies in mice.
  • Vaccination with rPepl on day 7 post-inoculation in mice with established cryptococcosis provided prolonged survival and led to a drastic decrease in yeasts counts in the target organs in survival mice.
  • Anti-Pep 1 monoclonal antibodies were produced by immunization of mice with rPepl protein and protective monoclonal antibodies recognizing specific peptide epitopes of Pepl protein have been identified ( Figure 1 and Figure 2).
  • the epitopes correspond to the peptides in positions 1 to 15 (peptide 1), 271 to 285 (peptide 2), 296 to 310 (peptide 3) and 346 to 360 (peptide 4) of Pepl ( Figure 1 and 2); peptide 1 is specific of Cryptococcus spp.
  • C. neoformans rPepl and homologous aspartyl proteases of other pathogenic fungi are thus useful as antigens in preventive or therapeutic vaccines (active immunotherapy) against fungal diseases.
  • the antibodies recognizing specific epitopes of said antigens which have a protective effect against fungal diseases are thus useful for the prevention and/or treatment of fungal diseases (passive immunotherapy).
  • the invention relates to a recombinant antigen or antibody thereto for use in the treatment of a fungal disease
  • antigen is chosen from:
  • a recombinant aspartyl protease protein comprising an amino acid sequence having at least 48 % identity with SEQ ID NO: 1 and comprising two catalytic motifs, SEQ ID NO: 2 and SEQ ID NO: 3, in positions corresponding to positions 145 to 149 and 327 to 331 of SEQ ID NO: 1, and further comprising at least one protective epitope, or a functional variant thereof;
  • said antibody is an antibody recognizing specifically at least one of said protective epitope(s) or a functional fragment thereof comprising at least the antibody binding region.
  • the percent amino acid sequence identity is defined as the percent of amino acid residues in a Compared Sequence that are identical to the Reference Sequence after aligning the sequences and introducing gaps if necessary, to achieve the maximum sequence identity. Alignment for purposes of determining percent amino acid sequence identity can be achieved in various ways known to a person of skill in the art, for instance using publicly available computer software. When using such software, the default parameters, e.g., for gap penalty and extension penalty, are preferably used. Sequence alignments and sequence analyses shown herein were performed using Geneious ⁇ .1.2 (Biomatters, New Zealand), ClustalW Alignment with BLOSUM 62 Matrix, Gap open cost 10, GAP extent cost 0.1.
  • antibody refers to a glycoprotein produced by lymphoid cells in response to stimulation with an immunogen. Antibodies possess the ability to react in vitro and in vivo specifically and selectively with an antigenic determinant or epitope eliciting their production or with an antigenic determinant closely related to the homologous antigen.
  • antibody is meant to encompass constructions using the binding variable region of such an antibody, and other antibody modifications.
  • the term “recognizes specifically” refers to antibodies that have a relatively high affinity to one or more epitopes of a protein of the invention, but which do not substantially recognize and bind to peptides other than the one(s) of interest.
  • the term “relatively high affinity” means a binding affinity between the antibody and the protein of interest of at least 10 "6 M, and preferably of at least about 10 "7 M and even more preferably 10 "8 M to 10 "10 M. Determination of such affinity is preferably conducted under standard competitive binding immunoassay conditions which is common knowledge to the person of ordinary skill in the art.
  • an “epitope” or antigenic determinant refers to the portion of an antigen that is recognized by an antibody.
  • the portion recognized by the antibody may be a continuous sequence (a single peptide) or a discontinuous sequence (more than one peptide).
  • a "protective effect” refers to the reduction of subsequent (preventive treatment) or established (therapeutic treatment) disease symptoms caused by a fungal infection.
  • the protective effect comprises a partial or total inhibition of the infection that causes the disease.
  • a complete inhibition of the infection corresponds to the eradication or sterilization of the infection and the resulting disease.
  • a "protective antigen or antibody” refers to an antigen or antibody having a protective effect.
  • the protective effect of the antigen is mediated by the induction of a protective immune response directed against the antigen.
  • the immune response includes a protective humoral response comprising the induction of specific protective antibodies recognizing the protective epitopes of the antigen.
  • the use of the antigen for treating the fungal disease thus refers to active immunotherapy as opposed to passive immunotherapy when the protective antibody is used for the treatment.
  • the protective effect of the antigen or antibody according to the invention can be verified in appropriate animal models of fungal diseases which are well-known in the art and illustrated in the examples of the present application.
  • the protective effect of the antigen/antibody can be determined by measuring various parameters such as the reduction of objective clinical signs of the disease, the increase of survival, and/or the decrease of fungal load in target organ(s).
  • a "protective epitope” refers to the portion of an antigen that is recognized by a protective antibody
  • fungal disease refers to a disease caused by infection with a pathogenic fungus.
  • treatment refers to a preventive or prophylactic treatment that is administered prior to infection and/or a therapeutic treatment that is administered after infection with a pathogenic fungus.
  • the present invention relates to a recombinant antigen for use in the treatment of a fungal disease
  • antigen is chosen from:
  • a) a recombinant aspartyl protease protein comprising an amino acid sequence having at least 48 % identity with SEQ ID NO: 1 and comprising two catalytic motifs, SEQ ID NO: 2 and SEQ ID NO: 3, in positions corresponding to positions 145 to 149 and 327 to 331 of SEQ ID NO: 1 and further comprising at least one protective epitope, said epitope(s) corresponding to one or more of the peptides in positions 1 to 15, 271 to 285, 296 to 310 and 346 to 360 of SEQ ID NO: 1, or a functional variant thereof; and b) a polypeptide fragment of the protein in a) comprising at least one of said protective epitope(s).
  • the recombinant antigen for the use of the invention is derived from an aspartyl protease encoded by a fungal gene homologous to C. neoformans PEP J gene. Therefore, the recombinant antigen or protein for the use of the invention is named Pe l aspartyl protease, recombinant aspartyl protease Pe l, recombinant Pepl aspartyl protease or rPepl aspartyl protease antigen or protein.
  • Fungal aspartyl protease genes are well-known in the art and available in public data bases. Fungal genes homologous to C. neoformans PEP1 can be easily identified using standard sequence analysis software.
  • the recombinant protein for the use of the invention is a Pepl aspartyl protease encoded by a fungal gene or a functional variant thereof. It is antigenic, e.g. it is recognized by antibodies specific for the aspartyl protease protein from which it is derived, in particular it is recognized by (protective) antibodies directed to the specific protective epitopes of said protein, it is also immunogenic, e.g., capable of inducing a specific protective immune response in an individual resulting in the production of detectable specific protective antibodies.
  • a functional variant is derived from a Pepl aspartyl protease by the introduction of mutations (deletion(s), insertion(s) and/or substitutions(s)) at specific positions in the sequence of the Pepl aspartyl protease.
  • a functional variant is antigenic and immunogenic.
  • the recombinant protein is a recombinant aspartyl protease of a pathogenic fungus or a functional variant thereof.
  • the pathogenic fungus may be any fungus causing a disease in humans or animals.
  • the pathogenic fungus is selected from the group consisting of: Aspergillus spp., Candida spp., Clavispora spp., Coccidioides spp., Cryptococcus spp., Exophiala spp., Fusarium spp., Histoplasma spp., Kluyveromyces spp., Mucorales, such as Lichtheimia spp., Mucor spp.
  • Rhizopus spp. Meyerozyma spp., Microsporum spp., Neosartorya spp., Paracoccidioides spp., Pneumocystis spp., Talaromyces spp., Trichophyton spp., Trichosporon spp, and other related pathogenic fungi. Aspergillus spp.
  • Aspergillus clavatus (XPJX) 1271141), Aspergillus flavus (KOC16935, XP_002381878), Aspergillus fumigatus (XP 754479), Aspergillus nidulans (XP_660507), Aspergillus niger (XP_001399855, AAA20876), Aspergillus oryzae (XP 001819842, XPJ)01825179, EIT76268), Aspergillus parasiticus (KJK63739), Aspergillus terreus (XP 001213854), and other species.
  • Candida spp. is Candida albicans (PI 0977, XP_713194, EEQ47270, KHC45840, KGQ89270, KHC38341, CAA31962), Candida dubliniensis (XP 002418645), Candida glabrata (XP_449442), Candida parapsilosis (CCE40477), Candida tropicalis (XP_002547417), and other species.
  • Clavispora spp. is Clavispora lusitaniae (XP 002615242) and other species.
  • Coccidioides spp. is Coccidioides immitis (XP 001244246), Coccidioides posadasii (AAZ92540), and other species.
  • Cryptococcus spp. is Cryptococcus gattii (serotypes B and C:
  • KIR4841 1 KIR53730, XP_003191823, KIR86636, KGB75517), Cryptococcus neoformans variety grubii (serotype A; XP__012046817), Cryptococcus neoformans variety neoformans (serotype D, XP_566887), and other species.
  • Exophiala spp. is Exophiala dermatitidis (XP__009158792), Exophiala oligosperma (KIW43340), Exophiala spinifera (KIWI 4941), Exophiala xenobiotica (XP_013321677), and other species.
  • Fusarium spp. is Fusarium fujikuroi (CCT71500), Fusarium oxysporum (EWY86405, EGU8351 1 , KNB13049), Fusarium sp. (CEG04259), and other species.
  • Histoplasma spp. is Histoplasma capsulatum (XP_001542991,
  • Kluyveromyces spp. is Kluyveromyces lactis (XP_453326), Kluyveromyces marxianus (BA041873) and other species.
  • Lichtheimia spp. is Lichtheimia corymbifera (CDH51907, CDH52330, CDH52400), Lichtheimia ramosa (CDS08210, CDS03074, CDS05348) and other species.
  • Meyerozyma spp. is Meyerozyma guilliermondii (XP_001483416) and other species.
  • Microsporum spp. is Microsporum gypseum (XP_003175087) and other species.
  • Mucor spp. is Mucor circinelloides (EPB89553, PB83353) and other species.
  • Neosartorya spp. is Neosartorya fischeri (XP_001263323) and other species.
  • Paracoccidioides spp. is Paracoccidioides brasiliensis (XP_010756433, AAP32823, EEH19972) and other species.
  • Pneumocystis spp. is Pneumocystis jirovecii (CCJ31368) and other species.
  • Rhizopus spp is Rhizopus delemar (EIE89256, EIE7741 , EIE82161), Rhizopus microsporas (CEG73869, CEG74799, CEI95549, CEG76436, CEG73870) and other species.
  • Talaromyces spp. is Talaromyces marneffei (KFX49218, XP_002143541) and other species.
  • Trichophyton sp. is Trichophyton interdigitale (EZF32786), Trichophyton rubrum (XP_003232712, EZF25931), Trichophyton tonsurans (EGD99457), Trichophyton verrucosum (XP_003020295) and other species.
  • Trichosporon spp. is Trichosporon asahii (EKD04292, XP 01 177670, EKD04292) and other species.
  • the pathogenic fungus is selected from the group consisting of: Aspergillus spp., Candida spp., Cryptococcus spp., Mucorales, Trichosporon spp, Trichophyton spp., Microsporam spp., Exophiala spp., Fusarium spp., Pneumocystis spp. and the species responsible for severe endemic mycoses, namely Coccidioides spp., Histoplasma spp., Paracoccoidioides spp., and Talaromyces spp.
  • the recombinant protein comprises an amino acid sequence having at least 60 % identity with SEQ ID NO: 1.
  • the recombinant protein comprises an amino acid sequence having at least 70 % identity with SEQ ID NO: 1.
  • Pepl aspartyl proteases of some Trichosporon spp. in particular Trichosporon asahii have at least 70 % sequence identity with SEQ ID NO: 1 (Table I).
  • the recombinant protein comprises an amino acid sequence having at least 80 % identity with SEQ ID NO: 1.
  • Pepl aspartyl proteases of Cryptococcus spp. including Cryptococcus neoformans variety grubii (serotype A), Cryptococcus neoformans variety neoformans (serotype D) and Cryptococcus gattii (serotypes B and C) Pepl aspartyl proteases have at least 80 % sequence identity with SEQ ID NO: 1 (Table I).
  • the recombinant protein is a Cryptococcus spp. protein; more preferably a Cryptococcus spp.
  • SEQ ID NO: 1 (XP__566887), SEQ ID NO: 6 (XP_012046817), SEQ ID NO: 7 (KGB75517), SEQ ID NO: 8 (KIR4841 1), SEQ ID NO: 9 (KIR53730), SEQ ID NO: 10 (KIR86636) or SEQ ID NO: 1 1 (XPJ)03191823).
  • the recombinant protein comprises an amino acid sequence having at least 90 % identity with SEQ ID NO: 1.
  • Pepl aspartyl proteases of Cryptococcus neoformans including Cryptococcus neoformans variety gmbii (serotype A) and Cryptococcus neoformans variety neoformans (serotype D)
  • Pepl aspartyl proteases have at least 90 % sequence identity with SEQ ID NO: 1 (Table I).
  • the recombinant protein is a Cryptococcus neoformans protein; more preferably a Cryptococcus neoformans protein comprising or consisting of any one of SEQ ID NO: 1 (XP_566887) and SEQ ID NO: 6 (XP_012046817); even more preferably, a recombinant protein comprising or consisting of SEQ ID NO: 1.
  • the catalytic motif of SEQ ID NO: 2 is included in a consensus sequence of SEQ ID NO: 4 and/or the catalytic motif of SEQ ID NO: 3 is included in a consensus sequence of SEQ ID NO: 5.
  • the protective epitope(s) correspond to one or more of the peptides in positions 1 to 15 (peptide 1), 271 to 285 (peptide 2), 296 to 310 (peptide 3) and 346 to 360 (peptide 4) of SEQ ID NO: 1.
  • the peptide in positions 1 to 15 (peptide 1) consists of SEQ ID NO: 12; this peptide is specific of Cryptococcus spp. and strictly conserved in Cryptococcus spp..
  • the peptide in positions 271 to 285 (peptide 2) consists of a sequence having at least 60 %, preferably at least 70 %, 80 % or 90 % identity with SEQ ID NO: 13.
  • the peptide 2 is selected from the group consisting of: SEQ ID NO: 13 and SEQ ID NO: 14.
  • the peptide in positions 296 to 310 (peptide 3) consists of a sequence having at least 60 %, preferably at least 70 %, 80 % or 90 % identity with SEQ ID NO: 15.
  • the peptide 3 consists of SEQ ID NO: 15
  • peptide 4 consists of a sequence having at least 60 %, preferably at least 70 %, 80 % or 90 % identity with SEQ ID NO: 16.
  • the peptide 4 consists of SEQ ID NO: 16.
  • the polypeptide fragment which is different from a full-length aspartyl protease Pe l protein consists of up to 400 amino acids, preferably up to 300, 250, 200, 150, 100, 90, 80, 70, 60, 50, 40, 30 or 20 amino acids of a recombinant protein as defined above.
  • the polypeptide fragment is an antigenic and immunogenic fragment of the recombinant protein.
  • the polypeptide fragment comprises advantageously at least one of the peptides 1 to 4 as defined above.
  • the present invention also relates to an antibody for use in the treatment of a fungal disease
  • said antibody binds to an antigen being chosen from:
  • a recombinant aspartyl protease protein comprising an amino acid sequence having at least 48 % identity with SEQ ID NO: 1 and comprising two catalytic motifs, SEQ ID NO: 2 and SEQ ID NO: 3, in positions corresponding to positions 145 to 149 and 327 to 331 of SEQ ID NO: 1 and further comprising at least one protective epitope, said epitope(s) corresponding to one or more of the peptides in positions 1 to 15, 271 to 285, 296 to 310 and 346 to 360 of SEQ ID NO: I, or a functional variant thereof; and
  • said antibody is an antibody recognizing specifically at least one of said protective epitope(s) or a functional fragment thereof comprising at least the antibody binding region.
  • the antibody binds to a recombinant antigen as defined above.
  • the antibody is a polyclonal or monoclonal antibody, preferably a monoclonal antibody.
  • An antibody for the use of the invention may comprise a whole antibody, an antibody fragment, a polyfunctional antibody aggregate, or in general a substance comprising one or more specific binding sites from an antibody.
  • the antibody fragment may be a fragment such as an Fv, Fab or F(ab')2 fragment or a derivative thereof, such as a single chain Fv fragment (ScFv).
  • the Fv fragment may advantageously be from a Camelidae antibody (nanobodies® or VHH).
  • the antibody or antibody fragment may be non-recombinant, recombinant or humanized.
  • the antibody may be of an immunoglobulin isotype, e.g., IgG, IgM, and so forth.
  • an aggregate, polymer, derivative and conjugate of an immunoglobulin or a fragment thereof can be used where appropriate.
  • the antibody or functional fragment recognizes specifically at least one of the peptides 1 to 4 as defined above.
  • the antibody recognizes specifically the peptides of SEQ ID NO: 12 and SEQ ID NO: 15, the peptide of SEQ ID NO: 13, or the peptides of SEQ ID NO: 13 and SEQ ID NO: 16; said antibody is preferably a monoclonal antibody.
  • the antibody is a monoclonal antibody produced by an hybridoma selected from the hybridomas deposited by the Applicant, according to the Budapest Treaty, at CNCM (Collection Nationale de Cultures de Microorganismes, 25 rue du Do Budapest oux, Paris) on November 17, 2014, under the accession number CNCM 1-4914, 1-4915 and 1-4916 or a Fv, Fab or Fab' 2 fragment thereof.
  • hybridomas are also referred to as B4-1 (CNCM 1-4914), Jl- 26 (CNCM 1-4915) and J17-14 (CNCM 1-4916). More preferably, the monoclonal antibody is produced by the hybridoma deposited at CNCM under accession number 1-4914.
  • the invention encompasses the use of several rPepl aspartyl protease antigens and/or antibodies as defined above, either as a mixture wherein the different rPepl aspartyl protease antigens and/or antibodies are used simultaneously, or as a combination wherein the different rPepl aspartyl protease antigens and/or antibodies are used separately or sequentially.
  • the rPepl aspartyl protease antigens and/or antibodies are used simultaneously, separately or sequentially with at least one immunotherapeutic agent and/or antifungal agent.
  • the immunotherapeutic agent is for example another antigen, another antibody or an immunomodulatory drug.
  • Other antigens and antibodies include in particular fungal antigens and antigens from pathogenic microorganisms (viruses, bacteria, parasites) and antibodies thereto.
  • Antifungal agents include standard antifungal drugs which are used for treating fungal diseases such as Polyenes (Amphotericin B, Candicin, Filipin, Hanycin, Natamycin, Nystatine, Rimocidin and the like), Azoles (Imidazoles, triazoles, thiazoles), AUyamines (Amorolfm, Butenafine, Naftifine, Terbinafine and the like), Echinocandins (Ajiidulafungin, Caspofungin, Micafungin and the like) and others.
  • Polyenes Amphotericin B, Candicin, Filipin, Hanycin, Natamycin, Nystatine, Rimocidin and the like
  • Azoles Imidazoles, triazoles, thiazoles
  • AUyamines Amorolfm, Butenafine, Naftifine, Terbinafine and the like
  • Echinocandins Ajiidulafungin, Caspofung
  • the rPepl aspartyl protease antigen and/or antibody are used in the form of a composition comprising a pharmaceutically acceptable vehicle, carrier and/or adjuvant.
  • Carriers and adjuvants are usually used in vaccine compositions comprising the antigen according to the invention.
  • composition may comprise a mixture of aspartyl protease antigens and/or antibodies as defined above, and eventually also at least one immunotherapeutic agent and/or antifungal agent, as defined above.
  • the pharmaceutical vehicles and carriers are those appropriate to the planned route of administration, which are well known in the art.
  • Non-limitative examples of carriers suitable for use in the composition of the invention include uni- or multi-lamellar liposomes, ISCOMS, virosomes, viral pseudoparticules, saponin micelles, saccharid (poly(lactide-co- glycolide)) or gold microspheres, and nanoparticules.
  • Non-limitative examples of adjuvants suitable for use in the composition of the invention include: CpG oligodeoxynucleotide, Freund aduvant, polyI:C (polyinosine-polycytidylic acid), oil emulsion, mineral substances, bacterial extracts, saponin, aluminium salts, monophosphoryl -lipid A and squalene.
  • a preferred composition for the use according to the invention is a vaccine composition comprising at least one rPep 1 aspartyl protease antigen of the invention, and an aluminium salt and a CpG oligodeoxynucleotide, as adjuvants.
  • the composition comprises a recombinant Pepl aspartyl protease protein having at least 80 % identity with SEQ ID NO: 1, preferably a recombinant Cryptococcus spp. aspartyl protease Pepl protein (encoded by a Cryptococcus spp. aspartyl protease Pepl gene).
  • the composition may further comprises at least one immunotherapeutic agent and/or antifungal agent, as defined above.
  • Aluminium salt includes any insoluble aluminium salt capable of enhancing and/or directing the immune response to a vaccine antigen.
  • Non-limitative examples of aluminium salts suitable for use in the composition of the invention include aluminium potassium sulfate, aluminium hydroxide, aluminium phosphate and mixtures thereof.
  • the aluminium salt is aluminium hydroxide, more preferably aluminium hydroxide gel.
  • CpG oligodeoxynucleotide includes any short single- stranded synthetic DNA molecules containing unmethylated CpG motif(s) which are recognized by Toll-like receptor 9 (TLR9) leading to strong immuno stimulatory effects.
  • the invention encompasses the use of the various types of CpG immunostimulants including Class A (Type D), Class B (Type ), Class C, Class P, and Class S CpGs.
  • the CpG ODN is a class B CpG.
  • CpG ODNs of the B-Class stimulate strong B cell and NK cell activation and cytokine production.
  • said CpG is a phosphorothioate (nuclease resistant) CpG of SEQ ID NO: 17.
  • compositions for the use according to the invention is a pharmaceutical composition comprising at least one monoclonal antibody recognizing specifically at least one of the peptides 1 to 4 according to the invention, and a pharmaceutically acceptable vehicle.
  • the antibody recognizes specifically the peptides of SEQ ID NO: 12 and SEQ ID NO: 15, the peptide of SEQ ID NO: 13, or the peptides of SEQ ID NO: 13 and SEQ ID NO: 16.
  • the antibody is a monoclonal antibody produced by an hybridoma selected from the hybridomas deposited at CNCM under accession number 1-4914, 1-4915 and 1-4916 or a Fv, Fab or Fab'2 fragment thereof. More preferably, the monoclonal antibody is produced by the hybridoma deposited at CNCM under accession number 1-4914,
  • the pharmaceutical composition comprises a mixture of monoclonal antibodies recognizing specifically at least one of the peptides 1 to 4 according to the invention, as defined above.
  • composition of the invention comprises a therapeutically effective dose of aspartyl protease antigen, and eventually CpG and aluminium salts (vaccine composition) or of antibody (pharmaceutical composition), sufficient to induce a protective effect against a fungal disease in the individual to whom it is administered.
  • composition according to the invention can be readily verified by various assays, using appropriate animal models of fungal diseases which are known to the person of ordinary skill in the art such as those described in the examples of the present Application.
  • administration of the composition according to the invention to an individual, before or after infection with a pathogenic fungus results in decreased fungal burden and prolonged survival compared to non-vaccinated individual, as shown in the examples of the present invention.
  • the effective dose is determined and adjusted depending on factors such as the composition used, the route of administration, the physical characteristics of the individual under consideration such as sex, age and weight, concurrent medication, and other factors, that those skilled in the medical arts will recognize.
  • the invention provides also a method for treating a fungal infection, comprising: administering to an individual a therapeutically effective amount of the composition as described above.
  • the composition of the present invention is generally administered according to known procedures used for vaccination and serotherapy, at dosages and for periods of time effective to induce a protective effect against the fungal disease in the individual.
  • the administration may be by injection or by oral, sublingual, intranasal, rectal or vaginal administration, inhalation, or transdermal application.
  • the injection may be subcutaneous, intramuscular, intraveinous, intraperitoneal, intradermal or else.
  • the administration of the vaccine composition is subcutaneous, intramuscular or intradermal.
  • the administration of the pharmaceutical composition is intraveinous.
  • the protective effect of the vaccine or pharmaceutical composition is obtained when the composition is administered before (preventive effect) or after (therapeutic effect) the fungal infection is diagnosed.
  • composition may be administered before or after the individual has been diagnosed with fungal infection.
  • the antigen and/or antibody composition of the invention is used for the therapeutic treatment of a fungal disease (after diagnosis of fungal infection).
  • the antigen and/or antibody composition of the invention used for the therapeutic treatment of individuals which have been previously diagnosed with a fungal infection using standard diagnosis assays which are well-known in the art.
  • the diagnosed patients may be classified in different groups depending upon the severity of the fungal disease; the patients diagnosed with a mild fungal disease based on clinical signs and/or biological marker levels will be administered a standard antifungal treatment; whereas the patients diagnosed with a severe fungal disease will be administered the antigen and/or antibody composition of the invention alone or in combination with a standard antifungal treatment.
  • the use of the antigen and/or antibody composition of the invention increases the efficiency of standard antifungal treatments.
  • the pharmaceutical composition or the vaccine composition according to the invention are administered simultaneously, separately or sequentially, with at least one immunotherapeutic and/or antifungal agent as defined above.
  • the pharmaceutical composition and the vaccine composition according to the invention are administered simultaneously, separately or sequentially, possibly in combination with at least one immunotherapeutic and/or antifungal agent as defined above.
  • the composition of the invention is used for the treatment of animals such as pets (cats, dogs and others), livestock and the like.
  • composition of the invention is used for the treatment of humans.
  • composition of the invention is used for the treatment of a fungal disease (mycosis) caused by a pathogenic fungus such as those listed above.
  • the antigen and antibody thereto are chosen so as to induce a protective effect against the fungal disease that is treated.
  • the antigen is chosen in a fungal genus close to that of the fungus causing the disease that is treated based on aspartyl protease Pepl sequence identity.
  • a Cryptococcus spp. aspartyl protease Pepl antigen is used for treating cryptococcosis as well as other fungal diseases.
  • the composition may be used for the treatment of superficial mycoses, cutaneous mycoses, subcutaneous mycoses and systemic mycoses due to primary or opportunistic pathogens.
  • Systemic mycoses are in particular candidiasis, aspergillosis, cryptococcosis, mucormycosis, fusariosis, pneumocystosis, dermatophytomycosis, trichosporonisis, and endemic mycosis such as penicilliosis maraeffei, coccidoidomycosis, histoplasmosis or paracoccidioidomycosis. Dermatophytomycosis are caused by pathogenic fungus such as Trichophyton spp. and Microsporum spp., for example.
  • the fungal disease is a systemic mycoses as defined above.
  • the fungal disease is candidiasis, aspergillosis, cryptococcosis, trichosporonosis, mucormycosis, fusariosis, pneumocystosis, penicilliosis marneffei, coccidoidomycosis, histoplasmosis or paracoccidioidomycosis; preferably candidiasis, aspergillosis, cryptococcosis, trichosporonosis, mucormycosis, fusariosis, or pneumocystosis; more preferably cryptococcosis.
  • a preferred use of the invention is the use of a recombinant Cryptococcus spp aspartyl protease Pepl protein having at least at least 80 % identity with SEQ ID NO: 1 or a monoclonal antibody thereto recognizing specifically the peptides of SEQ ID NO: 12 and SEQ ID NO: 15, the peptide of SEQ ID NO: 13, or the peptides of SEQ ID NO: 13 and SEQ ID NO; 16 for the treatment of a systemic mycoses as defined above, preferably candidiasis, aspergillosis, cryptococcosis, trichosporonosis, mucormycosis, fusariosis, pneumocystosis, penicilliosis marneffei, coccidoidomycosis, histoplasmosis or paracoccidioidomycosis; more preferably candidiasis, aspergillosis, cryptococcosis, trichosporonosis, mucormy
  • the recombinant Cryptococcus spp aspartyl protease Pe l protein comprises advantageously any one of SEQ ID NO: 1 and 6 to 1 1, preferably SEQ ID NO: 1.
  • the monoclonal antibody is produced by an hybridoma selected from the hybridomas deposited at CNCM under accession number 1-4914, 1-4915 and 1-4916 or a Fv, Fab or Fab'2 fragment thereof; even more preferably, the monoclonal antibody is produced by the hybridoma deposited at CNCM under accession number 1-4914.
  • compositions comprising at least one rPep 1 aspartyl protease antigen of the invention, and an aluminium salt and a CpG oligodeoxynucleotide, as adjuvants.
  • the composition comprises a recombinant Pepl aspartyl protease protein having at least at least 80 % identity with SEQ ID NO: 1, preferably a recombinant Cryptococcus spp. aspartyl protease Pepl protein (encoded by a Cryptococcus spp. aspartyl protease Pepl gene).
  • the composition may further comprises at least one at least one immunotherapeutic agent and/or antifungal agent, as defined above.
  • protective antibody of the invention in particular an antibody recognizing specifically at least one of the peptides 1 to 4 as defined above, preferably the peptides of SEQ ID NO: 12 and SEQ ID NO: 15, the peptide of SEQ ID NO: 13, or the peptides of SEQ ID NO: 13 and SEQ ID NO: 16; preferably a monoclonal antibody, more preferably a monoclonal antibody produced by an hybridoma selected from the hybridomas deposited at CNCM under accession number 1-4914, 1-4915 and 1-4916 or a Fv, Fab or Fab '2 fragment thereof; even more preferably, the monoclonal antibody is produced by the hybridoma deposited at CNCM under accession number 1-4914.
  • compositions comprising at least one protective antibody of the invention or a functional fragment thereof, as defined above; in particular a composition comprising a mixture of said antibodies; preferably a composition further comprising at least one immunotherapeutic and/or antifungal agent as defined above.
  • Another aspect of the invention is an antigenic polypeptide f agment as defined above.
  • the antigenic polypeptide fragment of the invention is used to generate new antibodies specific for the recombinant aspartyl protease antigen of the invention, in particular protective monoclonal antibodies.
  • Figure 1 represents Cryptococcus neoformans v. neoformans Aspartyl protease Pepl amino acid sequence (SEQ ID NO: 1).
  • the two putative active sites common to all aspartyl proteases are in bold; the consensus sequences of the active sites are in italics.
  • the sequence of the epitopes recognized by the therapeutic monoclonal antibodies Jl-26, ⁇ 7-14 and B4-1 is underlined: SEQ ID NO: 12 (Jl-26); SEQ ID NO: 13 (J17-14 and B4-1); SEQ ID NO: 15 (Jl-26); SEQ ID NO: 16 (B4-1).
  • Figure 2 represents the alignment of Cryptococcus neoformans v. neoformans aspartyl protease Pepl amino acid sequence (SEQ ID NO: 1) with that of homologous aspartyl proteases from human pathogenic fungi (SEQ ID NO: 6, 9, 1 1 , 8, 7, 10 and 21 to 91). The two putative active sites common to all aspartyl proteases and the sequences of the epitopes recognized by the therapeutic monoclonal antibodies are shown.
  • the consensus sequence is SEQ ID NO: 92.
  • Figure 3 shows the effect of prior immunization with recombinant Pe l protein (rPepl) in various adjuvants on the survival of BALB/c mice inoculated with 10 5 H99.
  • Mice 4-5 mice/groups
  • received an initial administration followed by 2 boosts with rPepl (day 0, 14 and 35), and were inoculated on day 60 and survival was recorded daily (p 0.0009, log-rank test).
  • Figure 4 illustrates the effect of prior vaccination with rPepl on survival (A) and fungal burden (B) of BALB/c male mice inoculated with C. neoformans var. neoform ns (NIH52D).
  • Figure 5 illustrates the effect of vaccination with rPepl on survival (A) and fungal burden (B and C) of BALB/c male mice inoculated with C. neoformans var. neoformans (NIH52D).
  • Mice were treated with PBS (Control), rPepl/Alum+CpG 15 days prior to (rPepl D-1 ) or 7 days (rPepl D+7) after inoculation with 10 5 yeasts (14 mice in each group). Survival (7 mice/group) was recorded daily up to 100 dpi (panel A).
  • CFU/g of brain, lungs or spleen were determined in the surviving mice (2 in each group, panel B) and in mice sacrificed 14 dpi (3 mice) and 21dpi (4 mice) (panel C). Each mouse is represented by one symbol.
  • Figure 6 illustrates the effect of vaccination with rPepl on fungal burden (A) and severity of meningitis (B) of BALB/c male mice inoculated with C. neoformans var. neoformans (NIH52D).
  • BALB/c male mice that have been inoculated with 10 5 C. neoformans var. neoformans (NIH52D)) and treated 7 days later with either adjuvant or rPepl in adjuvant (5 mice/group) were sacrificed 4 days after inoculation.
  • the fungal load (CFU/g of organ) was determined in spleen, brain and lung (A) and the score of severity of meningitis was determined blindly on sagittal section of one hemisphere (B),
  • FIG. 7 illustrates the effect of vaccination with rPepl on immune mediators after inoculation of C. neoformans var. neoformans (NIH52D).
  • BALB/c male mice that have been inoculated with 10 5 C. neoformans var. neoformans (NIH52D)) and treated 7 days later with either adjuvant or rPepl in adjuvant (5 mice/group) were sacrificed 14 days after inoculation.
  • the mediators were measured in the supernatants of brain homogenates or in serum using the Mouse-23-Plex Panel (Bio-Rad). Only the statistically significant and biologically relevant results are presented (Kruskall Wallis test).
  • Figure 9 illustrates the effect of anti-Pepl mAb B4-1 on the course of experimental cryptococcosis in BALB/c mice inoculated with 10 s NIH52D.
  • Mice were injected ip with 20 g (A) or 100 ⁇ g (B) of B4-1, 1 day before (D-l) or 1 or 7 days after (D+l or D+7) inoculation of the yeasts. Survival of mice was significantly prolonged by B4-1 at 20 g (D-l or D+7) and at 100 ⁇ g. Moreover, fungal load was decreased in all mice surviving 100 dpi (C).
  • FIG. 10 illustrates the effect of passive serotherapy with the monoclonal anti-pep 1 antibody B4-1 7 days after inoculation of C. neoformans var. neoformans (NIH52D) on survival of BALB/c male mice.
  • Mice were injected ip with PBS (Control), or Mab B4-1 at different doses 7 days after inoculation with 0 s yeasts (7 mice in each group). Survival was recorded daily up to 100 dpi (A). Survival of mice was significantly prolonged when mice were injected with the lowest dosages of the antibody.
  • Residual fungal load was determined at 100dpi in the target organs of surviving mice (1 each in the 70 and 200 ⁇ g groups, and 4 each in the 7 and 20 ⁇ g groups) (B). Of note, 2/4 mice in the 7 ⁇ g group and 3/4 mice in the 20 g group had no viable yeast detectable.
  • Figure 11 shows the effect of the various anti-Pep 1 mAbs on the course of experimental cryptococcosis in BALB/c mice inoculated with 10 5 NIH52D. Mice were injected ip with 20 ⁇ g of B4-1, Jl-26 and J17-17 at 7dpi. Survival was recorded up to 63 days post-inoculation (A) and fungal burden was assessed in surviving mice (B).
  • Figure 12 shows the effect of the various anti-Pepl mAbs on the course of experimental cryptococcosis in BALB/c mice inoculated with 10 s H99. Mice were injected ip with 20 ⁇ g of B4-1 , Jl-26 and J17-17 at 7dpi. Survival was recorded up to 54dpi.
  • Figure 13 shows the effect of anti-C. neoformans mAbs (10 ⁇ / ⁇ ) on C. neoformans growth.
  • Yeasts H99 or 52D
  • various mAbs at anti-glucuronoxylomannan mAb El, anti-Pepl mAbs (B4-1, Jl-26 or J17-14), unrelated IgGl mAb or no mAb.
  • Growth was recorded using the Bioscreen C Automated Microbiology Growth Curve Analysis System (Thermofisher).
  • the Richards non-linear regression model was used to determine lag times (Latency), slope (Slope) and maximal growth (Maximal Growth) for each condition. Each dot represents one experiment (result of triplicate wells).
  • Figure 14 shows the comparison of C. neoformans H99 phagocytosis by the murine macrophages cell line J774 in the presence of anti-Pepl mAbs (B4-1 or J 17- 14) using anti-GXM mAb (El) as the opsonin.
  • the condition tested were the antibody B4-1 or J 17- 14 (each at 0.5 ⁇ g ml) alone or added together with El (+E1) or 30 min before El (then El) at 1 ⁇ / ⁇ 1.
  • the phagocytic index obtained in the presence of anti-Pepl mAbs was normalized (ratio of the experimental condition to the phagocytic index obtained with El at 1 g ml in the same experiment).
  • Figure 15 shows the correlation between expression of PEP1 and phagocytic index relative to those of the reference strain H99 for 9 clinical isolates.
  • the phagocytic index determined at 2 hrs inversely correlated with the intracellular expression of the PEP1 genes. Bars represent means standard deviations (SD) of duplicates from 2 independent experiments for each of the 9 clinical isolates of C. neoformans var. grubii. Phagocytosis and gene expression determination were performed as explained above, with H99 as a reference strain and El as opsonin.
  • the clinical isolates have been selected in a previous study (Ala io et al. mBio 201 1).
  • Yeasts were cultured on Sabouraud agar (SA) medium and subcultured in liquid yeast extract-peptone-glucose medium (YPD, DIFCO) or in yeast nitrogen base (YNB, DIFCO) broth supplemented with 2% glucose (30°C, 150 rpm) for 22 h. Yeasts were collected by centrifugation, washed in phosphate buffered saline (PBS), enumerated with a hemacytometer and suspended at the desire concentration in the appropriate buffer or medium.
  • SA Sabouraud agar
  • YPD liquid yeast extract-peptone-glucose medium
  • YNB yeast nitrogen base
  • Alhydrogel® 2% (referred to as Alum), is an aluminium hydroxide wet gel suspension (INVIVOGEN), CpG ODN 1826 (SEQ ID NO: 17; VacciGradeTM, CAYLA). Conjugated antibodies were purchased from BIO-RAD.
  • Monoclonal antibodies were produced after fusion of spleen cells from BALB/c mice immunized with rPepl and murine myeloma cells (P3X63- Ag8653) at a 1 :4 ratio in 45% polyethylene glycol 1000.
  • Four clones were selected and mAbs purifed from ascites by ammonium sulfate precipitation. In all cases, production of anti-Pepl Abs was tested by ELISA and westernblots.
  • Antibodies to rPepl were analyzed by ELISA with rPepl coated overnight at 4°C (1 pg/ml of rPepl in carbonate buffer of a 96-well plates) (MaxiSorp®, NUNC). Saturation was done with 1% gelatin in PBS, for 1 h at 37°C and washes with 0.1% Tween® 20 in PBS (PBST). Supematants, serum samples or peptides preincubated with mAbs (100 ⁇ /well) were incubated for 2 h at 37°C or overnight at 4°C. Dilutions were made in PBST containing 0.25% gelatin (PBSTG). Specific antibodies were detected after incubation with the HRP conjugate (BIO- RAD) followed by addition of the substrate solution (SIGMAFASTTM OPD table set, S IGM A- ALDRICH) .
  • Non- verlapping and overlapping peptides (15-mer) were synthetized (> 90% purity, PROTEOGENIX) to analyze the epitopes recognized by the various mAbs.
  • Peptides were suspended at 5 mg mL in PBS (with 50 ⁇ KL DMSO if necessary) and stored at -20°C in aliquots until use.
  • Peptides at various concentrations (1250, 750, 250, 50 or 25 ⁇ ) were incubated for 1 hr at 37°C vol: vol with the various mAbs at 5 ng/mL all in PBSTG.
  • Controls consisted in bovine serum albumin (25 ⁇ ⁇ ) or buffer (negative controls) and rPepl (50 ⁇ g/mL, positive control).
  • Optical densities (OD) were measured at 492 nm in a microplate reader (LABSYSTEMS MULTISKAN RC). Results were expressed as percent inhibition.
  • mice Six week-old outbred OF1 (CHARLES RIVER) or BALB/cJRj (JANVIER) male mice were housed 7/cage in our animal facilities and received food and water ad libitum. Mice were identified individually. Infections were performed by intravenous (iv) inoculation (10 to 10 yeasts/mouse). Yeasts viability was assessed by colony forming units (CFU) enumeration onto Sabouraud agar.
  • iv intravenous
  • Yeasts viability was assessed by colony forming units (CFU) enumeration onto Sabouraud agar.
  • mice exhibit individual patterns of susceptibility to C. neoformans infection, independently of the inoculum size. Some of the mice develop acute, disseminated and rapidly lethal infections, whereas others survive for several weeks with limited chronic infection. In the BALB/c model, all mice die with a mean survival time after inoculation depending on the inoculum size. BALB/c were thus used to assess the efficacy of passive and active immunizations.
  • Yeasts H99 or 52D in stationary phase were suspended at 10 4 /ml in YPD. Growth in the presence of Mab (final concentration 0.1, 1 or 10 ⁇ ) was measured as increased in turbidity using the Bioscreen C Automated Microbiology Growth Curve Analysis System (Thermofisher) set up to record optical density every 15 min for 32 hrs. Mabs were either El anti-glucuronoxylomannan mAb (Dromer et al. Infect. Immun. 1987) or anti-Pepl mAbs (B4-1, Jl-26 or J17-14) or an unrelated mAb IgGl . Each condition was tested in triplicate wells.
  • RNA extraction was performed on the clinical isolates and H99 cells coincubated with J774 cells for 2 hrs. J774 cells were washed twice with PBS, scraped, lysed in 2 ml 0.05% SDS-ice-cold water, and vortexed, and the pellet was collected after 3 min of centrifugation at 2,000 relative centrifugal force (RCF). RTL lysis buffer (500 ⁇ ; Qiagen) and 1 : 100 ⁇ -mercaptoethanol (Sigma) were added to the C. neoformans pellets.
  • RNA extraction was performed on 350 ⁇ supernatant using the RNeasy minikit (Qiagen). RNAs were quantified and qualified using the Nanodrop spectrometer (ThermoFisher Scientific, Inc.). cDNA was generated from Turbo DNase (Ambion)-treated RNA us- ing the Transcriptor first- strand cDNA synthesis kit (Roche Diagnostics).
  • RT-PCR Quantitative reverse transcription- PCR
  • 10 ⁇ of Light- Cycler 480 SYBR green I master 10 ⁇ of Light- Cycler 480 SYBR green I master
  • 2 ⁇ of cDNA 2 ⁇ of cDNA
  • specific primers for PEPl in a LighiCycler 480 14 ⁇
  • Each cDNA was analyzed in duplicate and normalized with the corresponding GAPDH gene expression. Fold changes for each isolate were assessed compared to H99 under the same conditions. Two independent RNA extractions for each condition were analyzed blindly, and an internal calibrator consisting of the cDNA of H99 was used in each RT-PCR run as recommended.
  • Example 2 Production of recombinant aspartyi protease Pepl (rPepl) from C. neoformans var neoformans
  • a 1590 bp amplicon containing the cDNA of PEPl (SEQ ID NO: 18) was obtained with the primers PEPlBamHI (SEQ ID NO: 19) and PEPlXhoi (SEQ ID NO: 20), and subcloned into the pGEM-TE cloning vector (PROMEGA).
  • the 1.6-kb insert was then digested by BamHI and Xhol, and subcloned into the pH ATI 0/1 1/12 expression vector (ClonTech) and used to transform E. coli strain BLl .
  • Purification of the recombinant protein (rPepl) was conducted according to the manufacturer's instructions. Preservation of the immunoreactive epitope of the rPe l was assessed by immunoblotting with the pooled immune sera from survivors.
  • Example 3 Use of rPepl as vaccine against C. neoformans infection
  • Anti-Pepl mabs, especially B4-1 seem to affect the normal growth of C. neoformans H99 and 52D by altering the growth curves (increasing the latency), decreasing the maximal growth and altering the slope which is not seen or not as much with El and an unrelated mAb ( Figures 13). The effect seems more pronounced for 52D.
  • mAbs B4-1 did not modify the phagocytosis by murine macrophages in the presence of El , a monoclonal antibody specific for Cryptococcus neoformans capsular polysaccharide (Dromer et al., Infect. Immun., 1987, 55, 742-748).
  • Rhizopus delemar EIE82161 58.20%
  • Rhizopus microsporus CEG76436 55.90%
  • Cladophialophora bantiana KIW87422 54.30%
  • CDH52400 corymbifera 73.3 73.3 73.3 73.3 73.3 73.3 73.3 73.3
  • EPB89553 circinelloides 80 80 80 80 80 80 80 80 80 80
  • EPB83353 circinelloides 66.7 66.7 66.7 66.7 66.7 66.7 66.7 66.7
  • EKD04292 Trichosporon asahii 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100
  • CDH52400 corymbifera 73.3 73.3 73.3 73.3 73.3 73.3 73.3 73.3

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Abstract

The invention related to the use of a recombinant aspartyl protease antigen, in particular a recombinant Cryptococcus spp. aspartyl protease Pep1 protein or an antibody thereto recognizing specific protective epitopes for the immunotherapy of fungal diseases.

Description

Recombinant aspartyl protease antigen or antibody thereto for use in
immunotherapy of fungal diseases
The present invention relates to the use of a recombinant aspartyl protease antigen, in particular a recombinant Cryptococcus spp. aspartyl protease Pepl protein, as vaccine for the treatment of fungal diseases. The invention relates also to protective antibodies recognizing specific epitopes of said recombinant aspartyl protease antigen, in particular anti-Pepl protective monoclonal antibodies, and their use for the treatment of fungal diseases.
Fungal diseases are becoming a major public health concern and an increasing economic burden. Candidiasis, aspergillosis and cryptococcosis represent the main life-threatening fungal illnesses worldwide. Invasive Candida infections occur can be predicted at around 300,000 worldwide per year, with a mortality of 30- 55%. Invasive aspergillosis can occur in different patients groups including leukaemic cases, stem cell transplants and chronic obstructive pulmonary disease, with a mortality over 50%- even with treatment. Cryptococcosis is an opportunistic infection in subjects with cellular immune defects like those infected with HIV at a late stage of their disease, or those with solid organ transplantation, chronic lymphoid leukemia, or prolonged corticosteroid treatment. Disseminated meningoencephalitis is the main and most severe presentation and is always fatal unless treated. The Center for Diseases Control estimates that 1 million patients are diagnosed with cryptococcosis worldwide and that more than 600,000 die each year (Park et al, AIDS, 2009, 23, 525-530). The causative agent is Cryptococcus spp., an encapsulated yeast present in the environment that is acquired through inhalation. The isolates responsible for infections are serotypes A, B, C or D, haploid or diploid, and mating type alpha (MATa) or a. Overall, haploid C. neoformans MAT serotype A isolates represent the most prevalent clinical isolates worldwide. Diagnosis relies on culture of viable yeasts from clinical specimens such as cerebrospinal fluid (CSF), blood, urine, bronchoalveolar lavages or on detection of soluble capsular polysaccharide (CPS) in body fluids such as CSF and serum (Perfect et al, Clin. Infect. Dis., 2010, 50, 291- 322). Less fatal infections but which affect large numbers of people worldwide include cutaneous fungal infections, nail infections and athletes foot. Hair infection which is common in young children is predicted to affect some 200 million people worldwide. Despite antifungal treatment, invasive fungal infections such as cryptococcosis, candidiasis, and aspergillosis are still fatal.
Therefore, there is a need for adjuvant therapy for antifungal infections.
The inventors have produced a recombinant aspartyl protease of C. neoformans variety neoformans, named rPepl, from a cDNA previously cloned and sequenced but not expressed (Patent Application CA 2 354 100). Pepl is a 438 amino acid protein of SEQ ID NO: 1 containing two putative active sites containing consensus sequences common to all aspartyl proteases (Figure 1). The inventors have found that the aspartyl protease Pepl is relatively conserved in fungi that can be recovered during infection in humans or animals; the C-terminal region is quite conserved whereas the N-terminal region (first 100 amino acids of Pepl) is variable (Figure 2; Table I). All analyzed aspartyl protease sequences have two catalytic motifs, SEQ ID NO: 2 and SEQ ID NO: 3, corresponding to positions 145 to 149 and 327 to 331 of SEQ ID NO: 1. The catalytic motifs are included in the consensus sequences SEQ ID NO: 4 and SEQ ID NO: 5 corresponding to positions 142 to 155 and 326 to 333 of SEQ ID NO: 1.
Using a mouse model of cryptococcosis, the inventors have shown that vaccination with rPepl resulted in the production of detectable anti-Pep 1 antibodies in mice. Vaccination with rPepl prior to inoculation of C. neoformans resulted in decreased fungal burden and prolonged survival, with mice surviving the infection 100 days post-inoculation. Vaccination with rPepl on day 7 post-inoculation in mice with established cryptococcosis provided prolonged survival and led to a drastic decrease in yeasts counts in the target organs in survival mice.
Anti-Pep 1 monoclonal antibodies (mAbs) were produced by immunization of mice with rPepl protein and protective monoclonal antibodies recognizing specific peptide epitopes of Pepl protein have been identified (Figure 1 and Figure 2). The epitopes correspond to the peptides in positions 1 to 15 (peptide 1), 271 to 285 (peptide 2), 296 to 310 (peptide 3) and 346 to 360 (peptide 4) of Pepl (Figure 1 and 2); peptide 1 is specific of Cryptococcus spp. while the other peptide epitopes are common to fungi; peptides 3 and 4 are more conserved than peptide 2, with peptide 3 being the most conserved peptide epitope (Figure 2; Table II and III). Serotherapy with mAbs directed against different epitopes of Pe l provided prolonged survival in mice treated before infection and even in mice with an already established infection, leading to sterilization of all target organs in some cases.
C. neoformans rPepl and homologous aspartyl proteases of other pathogenic fungi (named Pepl aspartyl proteases) are thus useful as antigens in preventive or therapeutic vaccines (active immunotherapy) against fungal diseases. The antibodies recognizing specific epitopes of said antigens which have a protective effect against fungal diseases are thus useful for the prevention and/or treatment of fungal diseases (passive immunotherapy).
Therefore, the invention relates to a recombinant antigen or antibody thereto for use in the treatment of a fungal disease,
wherein said antigen is chosen from:
a) a recombinant aspartyl protease protein comprising an amino acid sequence having at least 48 % identity with SEQ ID NO: 1 and comprising two catalytic motifs, SEQ ID NO: 2 and SEQ ID NO: 3, in positions corresponding to positions 145 to 149 and 327 to 331 of SEQ ID NO: 1, and further comprising at least one protective epitope, or a functional variant thereof; and
b) a polypeptide fragment of the protein in a) comprising at least one of said protective epitope(s), and,
wherein said antibody is an antibody recognizing specifically at least one of said protective epitope(s) or a functional fragment thereof comprising at least the antibody binding region.
Definitions
In the following description:
- "a", "an", and "the" include plural referents, unless the context clearly indicates otherwise. As such, the term "a" (or "an"), "one or more" or "at least one" can be used interchangeably herein.
The percent amino acid sequence identity is defined as the percent of amino acid residues in a Compared Sequence that are identical to the Reference Sequence after aligning the sequences and introducing gaps if necessary, to achieve the maximum sequence identity. Alignment for purposes of determining percent amino acid sequence identity can be achieved in various ways known to a person of skill in the art, for instance using publicly available computer software. When using such software, the default parameters, e.g., for gap penalty and extension penalty, are preferably used. Sequence alignments and sequence analyses shown herein were performed using Geneious νό.1.2 (Biomatters, New Zealand), ClustalW Alignment with BLOSUM 62 Matrix, Gap open cost 10, GAP extent cost 0.1.
As used herein, the term "antibody" refers to a glycoprotein produced by lymphoid cells in response to stimulation with an immunogen. Antibodies possess the ability to react in vitro and in vivo specifically and selectively with an antigenic determinant or epitope eliciting their production or with an antigenic determinant closely related to the homologous antigen. The term "antibody" is meant to encompass constructions using the binding variable region of such an antibody, and other antibody modifications.
As used herein, the term "recognizes specifically" refers to antibodies that have a relatively high affinity to one or more epitopes of a protein of the invention, but which do not substantially recognize and bind to peptides other than the one(s) of interest. As used herein, the term "relatively high affinity" means a binding affinity between the antibody and the protein of interest of at least 10"6 M, and preferably of at least about 10"7 M and even more preferably 10"8 M to 10"10 M. Determination of such affinity is preferably conducted under standard competitive binding immunoassay conditions which is common knowledge to the person of ordinary skill in the art.
an "epitope" or antigenic determinant refers to the portion of an antigen that is recognized by an antibody. The portion recognized by the antibody may be a continuous sequence (a single peptide) or a discontinuous sequence (more than one peptide).
a "protective effect" refers to the reduction of subsequent (preventive treatment) or established (therapeutic treatment) disease symptoms caused by a fungal infection. The protective effect comprises a partial or total inhibition of the infection that causes the disease. A complete inhibition of the infection corresponds to the eradication or sterilization of the infection and the resulting disease.
a "protective antigen or antibody" refers to an antigen or antibody having a protective effect. The protective effect of the antigen is mediated by the induction of a protective immune response directed against the antigen. The immune response includes a protective humoral response comprising the induction of specific protective antibodies recognizing the protective epitopes of the antigen. The use of the antigen for treating the fungal disease thus refers to active immunotherapy as opposed to passive immunotherapy when the protective antibody is used for the treatment. The protective effect of the antigen or antibody according to the invention can be verified in appropriate animal models of fungal diseases which are well-known in the art and illustrated in the examples of the present application. The protective effect of the antigen/antibody can be determined by measuring various parameters such as the reduction of objective clinical signs of the disease, the increase of survival, and/or the decrease of fungal load in target organ(s).
a "protective epitope" refers to the portion of an antigen that is recognized by a protective antibody,
- "individual" or "subject" refers to an individual susceptible to infection with a pathogenic fungus.
"fungal disease" refers to a disease caused by infection with a pathogenic fungus.
unless specified otherwise, "treatment" refers to a preventive or prophylactic treatment that is administered prior to infection and/or a therapeutic treatment that is administered after infection with a pathogenic fungus.
The present invention relates to a recombinant antigen for use in the treatment of a fungal disease,
wherein said antigen is chosen from:
a) a recombinant aspartyl protease protein comprising an amino acid sequence having at least 48 % identity with SEQ ID NO: 1 and comprising two catalytic motifs, SEQ ID NO: 2 and SEQ ID NO: 3, in positions corresponding to positions 145 to 149 and 327 to 331 of SEQ ID NO: 1 and further comprising at least one protective epitope, said epitope(s) corresponding to one or more of the peptides in positions 1 to 15, 271 to 285, 296 to 310 and 346 to 360 of SEQ ID NO: 1, or a functional variant thereof; and b) a polypeptide fragment of the protein in a) comprising at least one of said protective epitope(s).
The recombinant antigen for the use of the invention is derived from an aspartyl protease encoded by a fungal gene homologous to C. neoformans PEP J gene. Therefore, the recombinant antigen or protein for the use of the invention is named Pe l aspartyl protease, recombinant aspartyl protease Pe l, recombinant Pepl aspartyl protease or rPepl aspartyl protease antigen or protein.
Fungal aspartyl protease genes are well-known in the art and available in public data bases. Fungal genes homologous to C. neoformans PEP1 can be easily identified using standard sequence analysis software. The recombinant protein for the use of the invention is a Pepl aspartyl protease encoded by a fungal gene or a functional variant thereof. It is antigenic, e.g. it is recognized by antibodies specific for the aspartyl protease protein from which it is derived, in particular it is recognized by (protective) antibodies directed to the specific protective epitopes of said protein, it is also immunogenic, e.g., capable of inducing a specific protective immune response in an individual resulting in the production of detectable specific protective antibodies. A functional variant is derived from a Pepl aspartyl protease by the introduction of mutations (deletion(s), insertion(s) and/or substitutions(s)) at specific positions in the sequence of the Pepl aspartyl protease. A functional variant is antigenic and immunogenic.
In some embodiments, the recombinant protein is a recombinant aspartyl protease of a pathogenic fungus or a functional variant thereof.
The pathogenic fungus may be any fungus causing a disease in humans or animals.
Preferably the pathogenic fungus is selected from the group consisting of: Aspergillus spp., Candida spp., Clavispora spp., Coccidioides spp., Cryptococcus spp., Exophiala spp., Fusarium spp., Histoplasma spp., Kluyveromyces spp., Mucorales, such as Lichtheimia spp., Mucor spp. and Rhizopus spp., Meyerozyma spp., Microsporum spp., Neosartorya spp., Paracoccidioides spp., Pneumocystis spp., Talaromyces spp., Trichophyton spp., Trichosporon spp, and other related pathogenic fungi. Aspergillus spp. is Aspergillus clavatus (XPJX) 1271141), Aspergillus flavus (KOC16935, XP_002381878), Aspergillus fumigatus (XP 754479), Aspergillus nidulans (XP_660507), Aspergillus niger (XP_001399855, AAA20876), Aspergillus oryzae (XP 001819842, XPJ)01825179, EIT76268), Aspergillus parasiticus (KJK63739), Aspergillus terreus (XP 001213854), and other species.
Candida spp. is Candida albicans (PI 0977, XP_713194, EEQ47270, KHC45840, KGQ89270, KHC38341, CAA31962), Candida dubliniensis (XP 002418645), Candida glabrata (XP_449442), Candida parapsilosis (CCE40477), Candida tropicalis (XP_002547417), and other species.
Clavispora spp. is Clavispora lusitaniae (XP 002615242) and other species.
Coccidioides spp. is Coccidioides immitis (XP 001244246), Coccidioides posadasii (AAZ92540), and other species.
Cryptococcus spp. is Cryptococcus gattii (serotypes B and C:
KIR4841 1 , KIR53730, XP_003191823, KIR86636, KGB75517), Cryptococcus neoformans variety grubii (serotype A; XP__012046817), Cryptococcus neoformans variety neoformans (serotype D, XP_566887), and other species.
Exophiala spp. is Exophiala dermatitidis (XP__009158792), Exophiala oligosperma (KIW43340), Exophiala spinifera (KIWI 4941), Exophiala xenobiotica (XP_013321677), and other species.
Fusarium spp. is Fusarium fujikuroi (CCT71500), Fusarium oxysporum (EWY86405, EGU8351 1 , KNB13049), Fusarium sp. (CEG04259), and other species.
Histoplasma spp. is Histoplasma capsulatum (XP_001542991,
EGC40857, EEH04825) and other species.
Kluyveromyces spp. is Kluyveromyces lactis (XP_453326), Kluyveromyces marxianus (BA041873) and other species.
Lichtheimia spp. is Lichtheimia corymbifera (CDH51907, CDH52330, CDH52400), Lichtheimia ramosa (CDS08210, CDS03074, CDS05348) and other species. Meyerozyma spp. is Meyerozyma guilliermondii (XP_001483416) and other species.
Microsporum spp. is Microsporum gypseum (XP_003175087) and other species.
Mucor spp. is Mucor circinelloides (EPB89553, PB83353) and other species.
Neosartorya spp. is Neosartorya fischeri (XP_001263323) and other species.
Paracoccidioides spp. is Paracoccidioides brasiliensis (XP_010756433, AAP32823, EEH19972) and other species.
Pneumocystis spp. is Pneumocystis jirovecii (CCJ31368) and other species.
Rhizopus spp, is Rhizopus delemar (EIE89256, EIE7741 , EIE82161), Rhizopus microsporas (CEG73869, CEG74799, CEI95549, CEG76436, CEG73870) and other species.
Talaromyces spp. is Talaromyces marneffei (KFX49218, XP_002143541) and other species.
Trichophyton sp. is Trichophyton interdigitale (EZF32786), Trichophyton rubrum (XP_003232712, EZF25931), Trichophyton tonsurans (EGD99457), Trichophyton verrucosum (XP_003020295) and other species.
Trichosporon spp. is Trichosporon asahii (EKD04292, XP 01 177670, EKD04292) and other species.
More preferably, the pathogenic fungus is selected from the group consisting of: Aspergillus spp., Candida spp., Cryptococcus spp., Mucorales, Trichosporon spp, Trichophyton spp., Microsporam spp., Exophiala spp., Fusarium spp., Pneumocystis spp. and the species responsible for severe endemic mycoses, namely Coccidioides spp., Histoplasma spp., Paracoccoidioides spp., and Talaromyces spp.
In some embodiments, the recombinant protein comprises an amino acid sequence having at least 60 % identity with SEQ ID NO: 1. Pepl aspartyl proteases of some Aspergillus spp., Talaromyces spp., luyveromyces spp., Paracoccidioides spp., Coccidioides spp., Trichosporon spp., Candida spp., Exophiala. spp., Clavispora spp., Rbizopus spp., Meyerozyma. spp., Histoplasma. spp., Lichtheimia spp., and Microsporum spp. have at least 60 % sequence identity with SEQ ID NO: 1 (Table I).
In a preferred embodiment the recombinant protein comprises an amino acid sequence having at least 70 % identity with SEQ ID NO: 1. Pepl aspartyl proteases of some Trichosporon spp. in particular Trichosporon asahii (EKD04292) have at least 70 % sequence identity with SEQ ID NO: 1 (Table I).
In another preferred embodiment, the recombinant protein comprises an amino acid sequence having at least 80 % identity with SEQ ID NO: 1. Pepl aspartyl proteases of Cryptococcus spp. including Cryptococcus neoformans variety grubii (serotype A), Cryptococcus neoformans variety neoformans (serotype D) and Cryptococcus gattii (serotypes B and C) Pepl aspartyl proteases have at least 80 % sequence identity with SEQ ID NO: 1 (Table I). Preferably, the recombinant protein is a Cryptococcus spp. protein; more preferably a Cryptococcus spp. protein comprising or consisting of any one of SEQ ID NO: 1 (XP__566887), SEQ ID NO: 6 (XP_012046817), SEQ ID NO: 7 (KGB75517), SEQ ID NO: 8 (KIR4841 1), SEQ ID NO: 9 (KIR53730), SEQ ID NO: 10 (KIR86636) or SEQ ID NO: 1 1 (XPJ)03191823).
In another preferred embodiment, the recombinant protein comprises an amino acid sequence having at least 90 % identity with SEQ ID NO: 1. Pepl aspartyl proteases of Cryptococcus neoformans including Cryptococcus neoformans variety gmbii (serotype A) and Cryptococcus neoformans variety neoformans (serotype D) Pepl aspartyl proteases have at least 90 % sequence identity with SEQ ID NO: 1 (Table I). Preferably, the recombinant protein is a Cryptococcus neoformans protein; more preferably a Cryptococcus neoformans protein comprising or consisting of any one of SEQ ID NO: 1 (XP_566887) and SEQ ID NO: 6 (XP_012046817); even more preferably, a recombinant protein comprising or consisting of SEQ ID NO: 1.
In some embodiments, the catalytic motif of SEQ ID NO: 2 is included in a consensus sequence of SEQ ID NO: 4 and/or the catalytic motif of SEQ ID NO: 3 is included in a consensus sequence of SEQ ID NO: 5. ao
In some embodiments, the protective epitope(s) correspond to one or more of the peptides in positions 1 to 15 (peptide 1), 271 to 285 (peptide 2), 296 to 310 (peptide 3) and 346 to 360 (peptide 4) of SEQ ID NO: 1.
In a preferred embodiment, the peptide in positions 1 to 15 (peptide 1) consists of SEQ ID NO: 12; this peptide is specific of Cryptococcus spp. and strictly conserved in Cryptococcus spp..
In another preferred embodiment, the peptide in positions 271 to 285 (peptide 2) consists of a sequence having at least 60 %, preferably at least 70 %, 80 % or 90 % identity with SEQ ID NO: 13. Preferably, the peptide 2 is selected from the group consisting of: SEQ ID NO: 13 and SEQ ID NO: 14.
In another preferred embodiment, the peptide in positions 296 to 310 (peptide 3) consists of a sequence having at least 60 %, preferably at least 70 %, 80 % or 90 % identity with SEQ ID NO: 15. Preferably, the peptide 3 consists of SEQ ID NO: 15
In another preferred embodiment, the peptide in positions 346 to 360
(peptide 4) consists of a sequence having at least 60 %, preferably at least 70 %, 80 % or 90 % identity with SEQ ID NO: 16. Preferably, the peptide 4 consists of SEQ ID NO: 16.
The polypeptide fragment, which is different from a full-length aspartyl protease Pe l protein consists of up to 400 amino acids, preferably up to 300, 250, 200, 150, 100, 90, 80, 70, 60, 50, 40, 30 or 20 amino acids of a recombinant protein as defined above. The polypeptide fragment is an antigenic and immunogenic fragment of the recombinant protein. The polypeptide fragment comprises advantageously at least one of the peptides 1 to 4 as defined above.
The present invention also relates to an antibody for use in the treatment of a fungal disease,
wherein said antibody binds to an antigen being chosen from:
a) a recombinant aspartyl protease protein comprising an amino acid sequence having at least 48 % identity with SEQ ID NO: 1 and comprising two catalytic motifs, SEQ ID NO: 2 and SEQ ID NO: 3, in positions corresponding to positions 145 to 149 and 327 to 331 of SEQ ID NO: 1 and further comprising at least one protective epitope, said epitope(s) corresponding to one or more of the peptides in positions 1 to 15, 271 to 285, 296 to 310 and 346 to 360 of SEQ ID NO: I, or a functional variant thereof; and
b) a polypeptide fragment of the protein in a) comprising at least one of said protective epitope(s), and,
wherein said antibody is an antibody recognizing specifically at least one of said protective epitope(s) or a functional fragment thereof comprising at least the antibody binding region.
In particular, the antibody binds to a recombinant antigen as defined above.
The antibody is a polyclonal or monoclonal antibody, preferably a monoclonal antibody.
An antibody for the use of the invention may comprise a whole antibody, an antibody fragment, a polyfunctional antibody aggregate, or in general a substance comprising one or more specific binding sites from an antibody. The antibody fragment may be a fragment such as an Fv, Fab or F(ab')2 fragment or a derivative thereof, such as a single chain Fv fragment (ScFv). The Fv fragment may advantageously be from a Camelidae antibody (nanobodies® or VHH). The antibody or antibody fragment may be non-recombinant, recombinant or humanized. The antibody may be of an immunoglobulin isotype, e.g., IgG, IgM, and so forth. In addition, an aggregate, polymer, derivative and conjugate of an immunoglobulin or a fragment thereof can be used where appropriate.
In some embodiments, the antibody or functional fragment recognizes specifically at least one of the peptides 1 to 4 as defined above.
In a preferred embodiment, the antibody recognizes specifically the peptides of SEQ ID NO: 12 and SEQ ID NO: 15, the peptide of SEQ ID NO: 13, or the peptides of SEQ ID NO: 13 and SEQ ID NO: 16; said antibody is preferably a monoclonal antibody.
In a more preferred embodiment, the antibody is a monoclonal antibody produced by an hybridoma selected from the hybridomas deposited by the Applicant, according to the Budapest Treaty, at CNCM (Collection Nationale de Cultures de Microorganismes, 25 rue du Docteur oux, Paris) on November 17, 2014, under the accession number CNCM 1-4914, 1-4915 and 1-4916 or a Fv, Fab or Fab' 2 fragment thereof. These hybridomas are also referred to as B4-1 (CNCM 1-4914), Jl- 26 (CNCM 1-4915) and J17-14 (CNCM 1-4916). More preferably, the monoclonal antibody is produced by the hybridoma deposited at CNCM under accession number 1-4914.
The invention encompasses the use of several rPepl aspartyl protease antigens and/or antibodies as defined above, either as a mixture wherein the different rPepl aspartyl protease antigens and/or antibodies are used simultaneously, or as a combination wherein the different rPepl aspartyl protease antigens and/or antibodies are used separately or sequentially.
In some embodiments, the rPepl aspartyl protease antigens and/or antibodies are used simultaneously, separately or sequentially with at least one immunotherapeutic agent and/or antifungal agent.
The immunotherapeutic agent is for example another antigen, another antibody or an immunomodulatory drug. Other antigens and antibodies include in particular fungal antigens and antigens from pathogenic microorganisms (viruses, bacteria, parasites) and antibodies thereto.
Antifungal agents include standard antifungal drugs which are used for treating fungal diseases such as Polyenes (Amphotericin B, Candicin, Filipin, Hanycin, Natamycin, Nystatine, Rimocidin and the like), Azoles (Imidazoles, triazoles, thiazoles), AUyamines (Amorolfm, Butenafine, Naftifine, Terbinafine and the like), Echinocandins (Ajiidulafungin, Caspofungin, Micafungin and the like) and others.
In some embodiments, the rPepl aspartyl protease antigen and/or antibody are used in the form of a composition comprising a pharmaceutically acceptable vehicle, carrier and/or adjuvant.
Carriers and adjuvants are usually used in vaccine compositions comprising the antigen according to the invention.
The composition may comprise a mixture of aspartyl protease antigens and/or antibodies as defined above, and eventually also at least one immunotherapeutic agent and/or antifungal agent, as defined above.
The pharmaceutical vehicles and carriers are those appropriate to the planned route of administration, which are well known in the art. Non-limitative examples of carriers suitable for use in the composition of the invention include uni- or multi-lamellar liposomes, ISCOMS, virosomes, viral pseudoparticules, saponin micelles, saccharid (poly(lactide-co- glycolide)) or gold microspheres, and nanoparticules.
Non-limitative examples of adjuvants suitable for use in the composition of the invention include: CpG oligodeoxynucleotide, Freund aduvant, polyI:C (polyinosine-polycytidylic acid), oil emulsion, mineral substances, bacterial extracts, saponin, aluminium salts, monophosphoryl -lipid A and squalene.
A preferred composition for the use according to the invention is a vaccine composition comprising at least one rPep 1 aspartyl protease antigen of the invention, and an aluminium salt and a CpG oligodeoxynucleotide, as adjuvants. In some more preferred embodiments, the composition comprises a recombinant Pepl aspartyl protease protein having at least 80 % identity with SEQ ID NO: 1, preferably a recombinant Cryptococcus spp. aspartyl protease Pepl protein (encoded by a Cryptococcus spp. aspartyl protease Pepl gene). The composition may further comprises at least one immunotherapeutic agent and/or antifungal agent, as defined above.
Aluminium salt includes any insoluble aluminium salt capable of enhancing and/or directing the immune response to a vaccine antigen.
Non-limitative examples of aluminium salts suitable for use in the composition of the invention include aluminium potassium sulfate, aluminium hydroxide, aluminium phosphate and mixtures thereof.
In some preferred embodiment, the aluminium salt is aluminium hydroxide, more preferably aluminium hydroxide gel.
CpG oligodeoxynucleotide (CpG ODN) includes any short single- stranded synthetic DNA molecules containing unmethylated CpG motif(s) which are recognized by Toll-like receptor 9 (TLR9) leading to strong immuno stimulatory effects.
The invention encompasses the use of the various types of CpG immunostimulants including Class A (Type D), Class B (Type ), Class C, Class P, and Class S CpGs.
In some preferred embodiment, the CpG ODN is a class B CpG. CpG ODNs of the B-Class stimulate strong B cell and NK cell activation and cytokine production. In some more preferred embodiment, said CpG is a phosphorothioate (nuclease resistant) CpG of SEQ ID NO: 17.
Another preferred composition for the use according to the invention is a pharmaceutical composition comprising at least one monoclonal antibody recognizing specifically at least one of the peptides 1 to 4 according to the invention, and a pharmaceutically acceptable vehicle.
In a preferred embodiment of the pharmaceutical composition, the antibody recognizes specifically the peptides of SEQ ID NO: 12 and SEQ ID NO: 15, the peptide of SEQ ID NO: 13, or the peptides of SEQ ID NO: 13 and SEQ ID NO: 16.
In a more preferred embodiment of the pharmaceutical composition, the antibody is a monoclonal antibody produced by an hybridoma selected from the hybridomas deposited at CNCM under accession number 1-4914, 1-4915 and 1-4916 or a Fv, Fab or Fab'2 fragment thereof. More preferably, the monoclonal antibody is produced by the hybridoma deposited at CNCM under accession number 1-4914,
In some preferred embodiments, the pharmaceutical composition comprises a mixture of monoclonal antibodies recognizing specifically at least one of the peptides 1 to 4 according to the invention, as defined above.
The composition of the invention comprises a therapeutically effective dose of aspartyl protease antigen, and eventually CpG and aluminium salts (vaccine composition) or of antibody (pharmaceutical composition), sufficient to induce a protective effect against a fungal disease in the individual to whom it is administered.
The protective effect of the composition according to the invention can be readily verified by various assays, using appropriate animal models of fungal diseases which are known to the person of ordinary skill in the art such as those described in the examples of the present Application. In particular, the administration of the composition according to the invention to an individual, before or after infection with a pathogenic fungus results in decreased fungal burden and prolonged survival compared to non-vaccinated individual, as shown in the examples of the present invention.
The effective dose is determined and adjusted depending on factors such as the composition used, the route of administration, the physical characteristics of the individual under consideration such as sex, age and weight, concurrent medication, and other factors, that those skilled in the medical arts will recognize.
The invention provides also a method for treating a fungal infection, comprising: administering to an individual a therapeutically effective amount of the composition as described above.
The composition of the present invention is generally administered according to known procedures used for vaccination and serotherapy, at dosages and for periods of time effective to induce a protective effect against the fungal disease in the individual. The administration may be by injection or by oral, sublingual, intranasal, rectal or vaginal administration, inhalation, or transdermal application. The injection may be subcutaneous, intramuscular, intraveinous, intraperitoneal, intradermal or else. Preferably, the administration of the vaccine composition is subcutaneous, intramuscular or intradermal. Preferably, the administration of the pharmaceutical composition is intraveinous.
The protective effect of the vaccine or pharmaceutical composition is obtained when the composition is administered before (preventive effect) or after (therapeutic effect) the fungal infection is diagnosed.
Therefore, the composition may be administered before or after the individual has been diagnosed with fungal infection.
In some embodiments, the antigen and/or antibody composition of the invention is used for the therapeutic treatment of a fungal disease (after diagnosis of fungal infection).
In a preferred embodiment, the antigen and/or antibody composition of the invention used for the therapeutic treatment of individuals which have been previously diagnosed with a fungal infection using standard diagnosis assays which are well-known in the art.
In addition, the diagnosed patients may be classified in different groups depending upon the severity of the fungal disease; the patients diagnosed with a mild fungal disease based on clinical signs and/or biological marker levels will be administered a standard antifungal treatment; whereas the patients diagnosed with a severe fungal disease will be administered the antigen and/or antibody composition of the invention alone or in combination with a standard antifungal treatment. The use of the antigen and/or antibody composition of the invention increases the efficiency of standard antifungal treatments.
In some embodiments, the pharmaceutical composition or the vaccine composition according to the invention are administered simultaneously, separately or sequentially, with at least one immunotherapeutic and/or antifungal agent as defined above.
In some other embodiments, the pharmaceutical composition and the vaccine composition according to the invention are administered simultaneously, separately or sequentially, possibly in combination with at least one immunotherapeutic and/or antifungal agent as defined above.
In some embodiments, the composition of the invention is used for the treatment of animals such as pets (cats, dogs and others), livestock and the like.
In other embodiments, the composition of the invention is used for the treatment of humans.
The composition of the invention is used for the treatment of a fungal disease (mycosis) caused by a pathogenic fungus such as those listed above. The antigen and antibody thereto are chosen so as to induce a protective effect against the fungal disease that is treated. The antigen is chosen in a fungal genus close to that of the fungus causing the disease that is treated based on aspartyl protease Pepl sequence identity. For example a Cryptococcus spp. aspartyl protease Pepl antigen is used for treating cryptococcosis as well as other fungal diseases.
The composition may be used for the treatment of superficial mycoses, cutaneous mycoses, subcutaneous mycoses and systemic mycoses due to primary or opportunistic pathogens. Systemic mycoses are in particular candidiasis, aspergillosis, cryptococcosis, mucormycosis, fusariosis, pneumocystosis, dermatophytomycosis, trichosporonisis, and endemic mycosis such as penicilliosis maraeffei, coccidoidomycosis, histoplasmosis or paracoccidioidomycosis. Dermatophytomycosis are caused by pathogenic fungus such as Trichophyton spp. and Microsporum spp., for example.
In some embodiments, the fungal disease is a systemic mycoses as defined above. In some preferred embodiments, the fungal disease is candidiasis, aspergillosis, cryptococcosis, trichosporonosis, mucormycosis, fusariosis, pneumocystosis, penicilliosis marneffei, coccidoidomycosis, histoplasmosis or paracoccidioidomycosis; preferably candidiasis, aspergillosis, cryptococcosis, trichosporonosis, mucormycosis, fusariosis, or pneumocystosis; more preferably cryptococcosis.
A preferred use of the invention is the use of a recombinant Cryptococcus spp aspartyl protease Pepl protein having at least at least 80 % identity with SEQ ID NO: 1 or a monoclonal antibody thereto recognizing specifically the peptides of SEQ ID NO: 12 and SEQ ID NO: 15, the peptide of SEQ ID NO: 13, or the peptides of SEQ ID NO: 13 and SEQ ID NO; 16 for the treatment of a systemic mycoses as defined above, preferably candidiasis, aspergillosis, cryptococcosis, trichosporonosis, mucormycosis, fusariosis, pneumocystosis, penicilliosis marneffei, coccidoidomycosis, histoplasmosis or paracoccidioidomycosis; more preferably candidiasis, aspergillosis, cryptococcosis, trichosporonosis, mucormycosis, fusariosis, or pneumocystosis; still more preferably cryptococcosis. The recombinant Cryptococcus spp aspartyl protease Pe l protein comprises advantageously any one of SEQ ID NO: 1 and 6 to 1 1, preferably SEQ ID NO: 1. Preferably, the monoclonal antibody is produced by an hybridoma selected from the hybridomas deposited at CNCM under accession number 1-4914, 1-4915 and 1-4916 or a Fv, Fab or Fab'2 fragment thereof; even more preferably, the monoclonal antibody is produced by the hybridoma deposited at CNCM under accession number 1-4914.
Another aspect of the invention is a vaccine composition comprising at least one rPep 1 aspartyl protease antigen of the invention, and an aluminium salt and a CpG oligodeoxynucleotide, as adjuvants. In some more preferred embodiments, the composition comprises a recombinant Pepl aspartyl protease protein having at least at least 80 % identity with SEQ ID NO: 1, preferably a recombinant Cryptococcus spp. aspartyl protease Pepl protein (encoded by a Cryptococcus spp. aspartyl protease Pepl gene). The composition may further comprises at least one at least one immunotherapeutic agent and/or antifungal agent, as defined above.
Another aspect of the invention is protective antibody of the invention, in particular an antibody recognizing specifically at least one of the peptides 1 to 4 as defined above, preferably the peptides of SEQ ID NO: 12 and SEQ ID NO: 15, the peptide of SEQ ID NO: 13, or the peptides of SEQ ID NO: 13 and SEQ ID NO: 16; preferably a monoclonal antibody, more preferably a monoclonal antibody produced by an hybridoma selected from the hybridomas deposited at CNCM under accession number 1-4914, 1-4915 and 1-4916 or a Fv, Fab or Fab '2 fragment thereof; even more preferably, the monoclonal antibody is produced by the hybridoma deposited at CNCM under accession number 1-4914.
Another aspect of the invention is a pharmaceutical composition comprising at least one protective antibody of the invention or a functional fragment thereof, as defined above; in particular a composition comprising a mixture of said antibodies; preferably a composition further comprising at least one immunotherapeutic and/or antifungal agent as defined above.
Another aspect of the invention is an antigenic polypeptide f agment as defined above. The antigenic polypeptide fragment of the invention is used to generate new antibodies specific for the recombinant aspartyl protease antigen of the invention, in particular protective monoclonal antibodies.
The practice of the present invention will employ, unless otherwise indicated, conventional techniques which are within the skill of the art. Such techniques are explained fully in the literature.
In addition to the above arrangements, the invention also comprises other arrangements, which will emerge from the description which follows, which refers to exemplary embodiments of the subject of the present invention, with reference to the attached drawings in which:
Figure 1 represents Cryptococcus neoformans v. neoformans Aspartyl protease Pepl amino acid sequence (SEQ ID NO: 1). The two putative active sites common to all aspartyl proteases are in bold; the consensus sequences of the active sites are in italics. The sequence of the epitopes recognized by the therapeutic monoclonal antibodies Jl-26, Π7-14 and B4-1 is underlined: SEQ ID NO: 12 (Jl-26); SEQ ID NO: 13 (J17-14 and B4-1); SEQ ID NO: 15 (Jl-26); SEQ ID NO: 16 (B4-1).
Figure 2 represents the alignment of Cryptococcus neoformans v. neoformans aspartyl protease Pepl amino acid sequence (SEQ ID NO: 1) with that of homologous aspartyl proteases from human pathogenic fungi (SEQ ID NO: 6, 9, 1 1 , 8, 7, 10 and 21 to 91). The two putative active sites common to all aspartyl proteases and the sequences of the epitopes recognized by the therapeutic monoclonal antibodies are shown. The consensus sequence is SEQ ID NO: 92.
Figure 3 shows the effect of prior immunization with recombinant Pe l protein (rPepl) in various adjuvants on the survival of BALB/c mice inoculated with 105 H99. Mice (4-5 mice/groups) received an initial administration followed by 2 boosts with rPepl (day 0, 14 and 35), and were inoculated on day 60 and survival was recorded daily (p = 0.0009, log-rank test).
Figure 4 illustrates the effect of prior vaccination with rPepl on survival (A) and fungal burden (B) of BALB/c male mice inoculated with C. neoformans var. neoform ns (NIH52D). Mice were treated with PBS (Control), adjuvant (Alum+CpG) or rPepl/Alum+CpG (day 0, 14 and 35) prior to inoculation (day 60) with 105 yeasts (14 mice in each group). Survival (7 mice/group) was recorded daily up to 100 dpi (p = 0.0019, log-rank test). CFU/g of brain, lungs or spleen were determined in mice sacrificed 7 dpi (3 mice) and 14dpi (4 mice) (p<0.0083 for brains at 14dpi, Kruskall Wallis test). Each mouse is represented by one symbol. The 4 mice surviving at 100dpi had no viable yeasts in brain, spleen and lungs.
Figure 5 illustrates the effect of vaccination with rPepl on survival (A) and fungal burden (B and C) of BALB/c male mice inoculated with C. neoformans var. neoformans (NIH52D). Mice were treated with PBS (Control), rPepl/Alum+CpG 15 days prior to (rPepl D-1 ) or 7 days (rPepl D+7) after inoculation with 105 yeasts (14 mice in each group). Survival (7 mice/group) was recorded daily up to 100 dpi (panel A). CFU/g of brain, lungs or spleen were determined in the surviving mice (2 in each group, panel B) and in mice sacrificed 14 dpi (3 mice) and 21dpi (4 mice) (panel C). Each mouse is represented by one symbol.
Figure 6 illustrates the effect of vaccination with rPepl on fungal burden (A) and severity of meningitis (B) of BALB/c male mice inoculated with C. neoformans var. neoformans (NIH52D). BALB/c male mice that have been inoculated with 105 C. neoformans var. neoformans (NIH52D)) and treated 7 days later with either adjuvant or rPepl in adjuvant (5 mice/group) were sacrificed 4 days after inoculation. The fungal load (CFU/g of organ) was determined in spleen, brain and lung (A) and the score of severity of meningitis was determined blindly on sagittal section of one hemisphere (B),
- Figure 7 illustrates the effect of vaccination with rPepl on immune mediators after inoculation of C. neoformans var. neoformans (NIH52D). BALB/c male mice that have been inoculated with 105 C. neoformans var. neoformans (NIH52D)) and treated 7 days later with either adjuvant or rPepl in adjuvant (5 mice/group) were sacrificed 14 days after inoculation. The mediators were measured in the supernatants of brain homogenates or in serum using the Mouse-23-Plex Panel (Bio-Rad). Only the statistically significant and biologically relevant results are presented (Kruskall Wallis test). *p< 0.05 ** pO.01
Figure 8 shows the effect on survival (A) and fungal burden (B, C) of vaccination with rPe l in BALB/c male mice 7 days after inoculation with C. neoformans var. grubii (H99). Mice were inoculated with 10 yeasts and treated at 7 dpi with 1 injection of PBS (Control without vaccine; wo Vacc) or rPepl/Alum+CpG (w rPepl vacc; 14 mice in each group). (A) Survival was recorded daily up to 63 dpi (7 mice/group, p= 0.010 log-rang test), and (B) fungal burden in the target organs determined for the 4 mice surviving at day 70dpi. (C) CFU/g of brain, lungs or spleen were determined at 14 (4 mice/group) and 21 dpi (3 mice/group) (non statistically different except for spleens on day 21, p=0.0495). Each mouse is represented by one symbol.
Figure 9 illustrates the effect of anti-Pepl mAb B4-1 on the course of experimental cryptococcosis in BALB/c mice inoculated with 10s NIH52D. Mice were injected ip with 20 g (A) or 100 μg (B) of B4-1, 1 day before (D-l) or 1 or 7 days after (D+l or D+7) inoculation of the yeasts. Survival of mice was significantly prolonged by B4-1 at 20 g (D-l or D+7) and at 100 μg. Moreover, fungal load was decreased in all mice surviving 100 dpi (C).
- Figure 10 illustrates the effect of passive serotherapy with the monoclonal anti-pep 1 antibody B4-1 7 days after inoculation of C. neoformans var. neoformans (NIH52D) on survival of BALB/c male mice. Mice were injected ip with PBS (Control), or Mab B4-1 at different doses 7 days after inoculation with 0s yeasts (7 mice in each group). Survival was recorded daily up to 100 dpi (A). Survival of mice was significantly prolonged when mice were injected with the lowest dosages of the antibody. Residual fungal load was determined at 100dpi in the target organs of surviving mice (1 each in the 70 and 200 μg groups, and 4 each in the 7 and 20 μg groups) (B). Of note, 2/4 mice in the 7 μg group and 3/4 mice in the 20 g group had no viable yeast detectable.
Figure 11 shows the effect of the various anti-Pep 1 mAbs on the course of experimental cryptococcosis in BALB/c mice inoculated with 105 NIH52D. Mice were injected ip with 20 μg of B4-1, Jl-26 and J17-17 at 7dpi. Survival was recorded up to 63 days post-inoculation (A) and fungal burden was assessed in surviving mice (B).
Figure 12 shows the effect of the various anti-Pepl mAbs on the course of experimental cryptococcosis in BALB/c mice inoculated with 10s H99. Mice were injected ip with 20 μg of B4-1 , Jl-26 and J17-17 at 7dpi. Survival was recorded up to 54dpi.
Figure 13 shows the effect of anti-C. neoformans mAbs (10 μ§/ητιΙ) on C. neoformans growth. Yeasts (H99 or 52D) at 104/ml in YPD were distributed in the presence of various mAbs (at
Figure imgf000022_0001
anti-glucuronoxylomannan mAb El, anti-Pepl mAbs (B4-1, Jl-26 or J17-14), unrelated IgGl mAb or no mAb. Growth was recorded using the Bioscreen C Automated Microbiology Growth Curve Analysis System (Thermofisher). The Richards non-linear regression model was used to determine lag times (Latency), slope (Slope) and maximal growth (Maximal Growth) for each condition. Each dot represents one experiment (result of triplicate wells).
Figure 14 shows the comparison of C. neoformans H99 phagocytosis by the murine macrophages cell line J774 in the presence of anti-Pepl mAbs (B4-1 or J 17- 14) using anti-GXM mAb (El) as the opsonin. The condition tested were the antibody B4-1 or J 17- 14 (each at 0.5 μg ml) alone or added together with El (+E1) or 30 min before El (then El) at 1 μ§/ηι1. The phagocytic index obtained in the presence of anti-Pepl mAbs was normalized (ratio of the experimental condition to the phagocytic index obtained with El at 1 g ml in the same experiment).
Figure 15 shows the correlation between expression of PEP1 and phagocytic index relative to those of the reference strain H99 for 9 clinical isolates. Compared to H99, the phagocytic index determined at 2 hrs inversely correlated with the intracellular expression of the PEP1 genes. Bars represent means standard deviations (SD) of duplicates from 2 independent experiments for each of the 9 clinical isolates of C. neoformans var. grubii. Phagocytosis and gene expression determination were performed as explained above, with H99 as a reference strain and El as opsonin. The clinical isolates have been selected in a previous study (Ala io et al. mBio 201 1).
There will now be described by way of example a specific mode contemplated by the Inventors. In the following description numerous specific details are set forth in order to provide a thorough understanding. It will be apparent however, to one skilled in the art, that the present invention may be practiced without limitation to these specific details. In other instances, well known methods and structures have not been described so as not to unnecessarily obscure the description.
Example 1: Material and Methods
Experimental examples described below are obtained using following materials and methods.
Strains
Reference strains of Cryptococcus, neoformans variety grubii H99 (serotype A, MATalpha) and variety neoformans NIH52D (serotype D, MATalpha) were stored in 40% glycerol at -80°C. Yeasts were cultured on Sabouraud agar (SA) medium and subcultured in liquid yeast extract-peptone-glucose medium (YPD, DIFCO) or in yeast nitrogen base (YNB, DIFCO) broth supplemented with 2% glucose (30°C, 150 rpm) for 22 h. Yeasts were collected by centrifugation, washed in phosphate buffered saline (PBS), enumerated with a hemacytometer and suspended at the desire concentration in the appropriate buffer or medium.
Reagents All reagents were purchased from SIGMA unless otherwise specified. Alhydrogel® 2%, (referred to as Alum), is an aluminium hydroxide wet gel suspension (INVIVOGEN), CpG ODN 1826 (SEQ ID NO: 17; VacciGrade™, CAYLA). Conjugated antibodies were purchased from BIO-RAD.
Production of anti-rPepl antibodies
Monoclonal antibodies were produced after fusion of spleen cells from BALB/c mice immunized with rPepl and murine myeloma cells (P3X63- Ag8653) at a 1 :4 ratio in 45% polyethylene glycol 1000. Four clones were selected and mAbs purifed from ascites by ammonium sulfate precipitation. In all cases, production of anti-Pepl Abs was tested by ELISA and westernblots.
Detection of anti-C. neoformans antibodies and characterization of the epitopes
Antibodies to rPepl were analyzed by ELISA with rPepl coated overnight at 4°C (1 pg/ml of rPepl in carbonate buffer of a 96-well plates) (MaxiSorp®, NUNC). Saturation was done with 1% gelatin in PBS, for 1 h at 37°C and washes with 0.1% Tween® 20 in PBS (PBST). Supematants, serum samples or peptides preincubated with mAbs (100 μΐ/well) were incubated for 2 h at 37°C or overnight at 4°C. Dilutions were made in PBST containing 0.25% gelatin (PBSTG). Specific antibodies were detected after incubation with the HRP conjugate (BIO- RAD) followed by addition of the substrate solution (SIGMAFAST™ OPD table set, S IGM A- ALDRICH) .
Non- verlapping and overlapping peptides (15-mer) were synthetized (> 90% purity, PROTEOGENIX) to analyze the epitopes recognized by the various mAbs. Peptides were suspended at 5 mg mL in PBS (with 50 \KL DMSO if necessary) and stored at -20°C in aliquots until use. Peptides at various concentrations (1250, 750, 250, 50 or 25 μΜ) were incubated for 1 hr at 37°C vol: vol with the various mAbs at 5 ng/mL all in PBSTG. Controls consisted in bovine serum albumin (25 μ^η ΐ) or buffer (negative controls) and rPepl (50 μg/mL, positive control). Optical densities (OD) were measured at 492 nm in a microplate reader (LABSYSTEMS MULTISKAN RC). Results were expressed as percent inhibition. Experimental infections
Six week-old outbred OF1 (CHARLES RIVER) or BALB/cJRj (JANVIER) male mice were housed 7/cage in our animal facilities and received food and water ad libitum. Mice were identified individually. Infections were performed by intravenous (iv) inoculation (10 to 10 yeasts/mouse). Yeasts viability was assessed by colony forming units (CFU) enumeration onto Sabouraud agar.
In the OF1 model, mice exhibit individual patterns of susceptibility to C. neoformans infection, independently of the inoculum size. Some of the mice develop acute, disseminated and rapidly lethal infections, whereas others survive for several weeks with limited chronic infection. In the BALB/c model, all mice die with a mean survival time after inoculation depending on the inoculum size. BALB/c were thus used to assess the efficacy of passive and active immunizations.
Outcome was evaluated either by survival or by fungal burden.
Survival was recorded daily and weight recorded weekly up to 100 day post- inoculation (dpi). In selected experiments, blood was also withdrawn weekly from the lateral tail vein and immediately used for antibody (Ab) determination (20 μΐ). For fungal burden evaluation, mice were euthanized at selected time after inoculation, blood was withdrawn by cardiac puncture (1 ml) and serum stored at -20°C until assayed. Brain, lungs and spleens were aseptically removed for CFU determination. Tenfold dilutions of the homogenates were plated in duplicate on Sabouraud- chloramphenicol agar Petri dishes and incubated at 30°C for 48 hrs. Results were expressed as loglO CFU/g of organ. All experiments were approved by the institutional committee (CETEA 02196.01).
Efficacy of the recombinant protein rPepl prior to or after challenge with C. neoformans
Preliminary experiments were done to determine the best adjuvant for rPepl immunization in 4 groups of 5 BALB/c mice receiving subcutaneously (sc) a vohvol mixture of rPepl (10 g) and one of the following (i) Freund's complete adjuvant; (ii) Alum; (iii) 10 g CpG ; (iv) a mixture of 5 μg CpG/Alum (Alum+CPG) with boosts 14 and 35 days later. Controls were injected with saline mixed with each adjuvant. Anti-rPe l Abs titers were determined initially and 7 days after the 2nd and 3rd challenge.
Subsequent experiments testing the prophylactic efficacy of rPepl used for adjuvant Alum dO and dl4, and 5μg Alum+CpG on d35. Experiments were also performed to test the efficacy of rPe l as therapeutic vaccine. In that case, immunization was done at 7dpi with lC^g rPepl or saline in 5 g Alum+CpG. Details (strain, inoculum size, day of sacrifice, number of animals) are specified in the results section. All injections were performed at the same time when endpoints included assessment of survival and fungal burden at specific time points.
Efficacy of anti-Pepl mAbs prior to or after challenge with C. neoformans
The effect of passive serotherapy with anti-rPepl mAbs was first assessed with mAb A9-4 at 20 or 100 μg in PBS injected intraperitoneally (ip) before (d-1) or after (1dpi or 7dpi) inoculation. Subsequent experiments compared the efficacy of 20 g of A9-4, B4-1, Jl-26, and J17-14 injected 7 dpi with either strain. Growth curves
Yeasts (H99 or 52D) in stationary phase were suspended at 104/ml in YPD. Growth in the presence of Mab (final concentration 0.1, 1 or 10 ^ηιΐ) was measured as increased in turbidity using the Bioscreen C Automated Microbiology Growth Curve Analysis System (Thermofisher) set up to record optical density every 15 min for 32 hrs. Mabs were either El anti-glucuronoxylomannan mAb (Dromer et al. Infect. Immun. 1987) or anti-Pepl mAbs (B4-1, Jl-26 or J17-14) or an unrelated mAb IgGl . Each condition was tested in triplicate wells. Lag times, slope of the curve and maximal growth were calculated using the Richards non-linear regression model. Each parameter was normalized using the corresponding parameter obtained on H99 alone during the same run. Experiments were repeated twice except for mabs Jl-26 and J 17- 14 that were only tested once.
Phagocytosis of C. neoformans by J774 cells
The experiments were performed as described before (Alanio et al. mBio 201 1). Briefly, J774 cell suspensions (105 in fresh medium per well of a 24- wells culture plate) were incubated at 37°C in 5% C02 for 48 h. The day of the experiment, calcofluor-stained C. neoformans suspension and mabs were added in fresh medium at the desired concentrations to the J774 cell monolayer, and incubated at 37°C and 5% CC*2 for 2 h (phagocytosis assay, C. neoformansl ll ratio, 5:1). The anti- glucuronoxylomannan antibody El (Dromer et al. Infect, Immun. 1987) was used at 1 μg/ml as opsonin in all experiments except when testing the opsonic activity of the anti-Pepl mabs alone. Several experiments were performed in order to test; (i) the effect of the various anti-Pep mabs alone at 50, 5 and 0.5μ^ηι1; (ii) the effect of the various anti-Pepl mabs at 50, 5 and O^g/rnl on the opsonic activity of El added concomitantly (+E1); or (iii) 30 min after the addition of the anti-Pepl mabs (then El).
At the end of the incubation, nonadherent extracellular yeast cells were then removed by PBS washings, and incubation was stopped to assess phagocytosis. Phagocytosis was determined after staining residual extracellular yeasts using anti-IgG-FITC, additional PBS washings, and macrophage lysis with distilled water. The samples were then centrifuged, resuspended in 1% paraformaldehyde in PBS (PFA-PBS), vortexed, and sonicated for 3 min before analysis. Flow cytometry analyses were performed using MacsQuant analyzer and MacsQuantify software 2.0 (Milteniy BioTeC) to provide absolute quantification. Samples were analyzed using FlowJo 8.7 software (Tree Star, Inc.). Aggregates were excluded by gating relevant events in the forward scatter/side scatter (FSC/SSC) contour plot. The phagocytic index (PI) was the number of events in the Calco^Sn FITCneS gate after 2 hours. Results were expressed as the ratio of PI for the clinical strains compared to the H99 parameter determined in the same run. We assessed that results obtained during the two independent experiments were reproducible. Means of replicates were then used for analyses and shown on the graphs. Expression of PEP1
RNA extraction was performed on the clinical isolates and H99 cells coincubated with J774 cells for 2 hrs. J774 cells were washed twice with PBS, scraped, lysed in 2 ml 0.05% SDS-ice-cold water, and vortexed, and the pellet was collected after 3 min of centrifugation at 2,000 relative centrifugal force (RCF). RTL lysis buffer (500 μΐ; Qiagen) and 1 : 100 β-mercaptoethanol (Sigma) were added to the C. neoformans pellets. The suspensions were transferred to Ceramique Magna Lyser green bead tubes (Roche Diagnostics), homogenized three times with the Magna Lyser instrument (30 s at 7,000 rpm), and centrifuged (3 min at 10,000 RCF). RNA extraction was performed on 350 μΐ supernatant using the RNeasy minikit (Qiagen). RNAs were quantified and qualified using the Nanodrop spectrometer (ThermoFisher Scientific, Inc.). cDNA was generated from Turbo DNase (Ambion)-treated RNA us- ing the Transcriptor first- strand cDNA synthesis kit (Roche Diagnostics). Quantitative reverse transcription- PCR (RT-PCR) using 10 μΐ of Light- Cycler 480 SYBR green I master, 2 μΐ of cDNA, and specific primers for PEPl in a LighiCycler 480 (Roche Diagnostics) consisted of a denaturation step at 95°C, 45 cycles of amplification (95°C for 5 s, 60°C for 5s, and 72°C for 5 s). Each cDNA was analyzed in duplicate and normalized with the corresponding GAPDH gene expression. Fold changes for each isolate were assessed compared to H99 under the same conditions. Two independent RNA extractions for each condition were analyzed blindly, and an internal calibrator consisting of the cDNA of H99 was used in each RT-PCR run as recommended. Example 2: Production of recombinant aspartyi protease Pepl (rPepl) from C. neoformans var neoformans
A 1590 bp amplicon containing the cDNA of PEPl (SEQ ID NO: 18) was obtained with the primers PEPlBamHI (SEQ ID NO: 19) and PEPlXhoi (SEQ ID NO: 20), and subcloned into the pGEM-TE cloning vector (PROMEGA). The 1.6-kb insert was then digested by BamHI and Xhol, and subcloned into the pH ATI 0/1 1/12 expression vector (ClonTech) and used to transform E. coli strain BLl . Purification of the recombinant protein (rPepl) was conducted according to the manufacturer's instructions. Preservation of the immunoreactive epitope of the rPe l was assessed by immunoblotting with the pooled immune sera from survivors.
Example 3: Use of rPepl as vaccine against C. neoformans infection
Preliminary experiments were done to determine the best adjuvant for rPepl immunization. All rPepl -immunized mice had strong antibody response against rPepl . Vaccination with rPepl/CpG+Alun prior to inoculation with 105 yeasts H99 was associated with the best survival compared to other adjuvants and controls (Figure 3, p=0.0009; log-rank test).
Preimmunisation with rPe l/CpG+Alum provided prolonged survival in mice inoculated with 52D (Figure 4A, p=0.0019 log-rank test) with no detectable CFU in the target organs of the 4 mice surviving 100 dpi. CFU burden between the 3 groups did not differ at 7 dpi but was significantly decreased in the vaccinated mice compared to controls at 14 dpi in brains and spleens (Figure 4B).
One sc injection of rPepl/Alum+CpG at 7dpi provided prolonged survival of mice inoculated with 10s N1H52D comparable to that afforded by 1 immunization 15 day prior to inoculation (Figure 5 A, p = 0.042, log-rank test) with drastic decreased in the fungal load in the surviving mice (Figure 5B) and a trend towards decreased fungal load at 21 dpi significant only in the brain (Figure 5C, p =
0.023 , Kriskall wallis test).
Fungal load especially in the brain (Figure 5C and Figure 6A), and severity of meningitis (Figure 6B) were significantly decreased in mice inoculated with NIH52D and treated with the therapeutic vaccine. Analysis of the inflammation mediators showed a global decrease in mice treated by the vaccine (Figure 7).
Whether the decreased fungal burden is per se responsible for this observation or whether the neutralization of Pep 1 by anti-Pep 1 antibody induced by the vaccination is responsible for the decreased inflammation remains to be determined.
The effect of one sc injection of rPepl/Alum+CpG after inoculation was confirmed on mice inoculated with 103 H99 (p=0.0004, log-rank test, Figure 8 A).
The 4 mice surviving at 63 dpi had no viable yeasts in their brains and 2/4 had sterile spleen and lungs (Figure 8B). There was a non-significant trends towards less CFU in the target organs of vaccinated compared to control mice sacrificed at 14 and 21 dpi
(Figure 8C).
Example 4: Protection of mice by passive immunization with anti-Pep 1 antibodies
Several mAbs were produced and deposited at the CNCM on Nov
17, 2014: CNCM 1-4914 (B4-1), CNCM 1-4915 (J 1-26) and CNCM 1-4916 (J17-14). Determination of the epitopes recognized by the various mAbs produced against rPepl was done by competitive ELISA and showed that three Mabs recognized different epitopes: SEQ ID NO: 12 and SEQ ID NO: 15 are recognized by 31-26; SEQ ID NO: 13 is recognized by J17-14 and B4-1 and SEQ ID NO: 16 is recognized by B4-1.
Anti-Pepl mabs, especially B4-1 seem to affect the normal growth of C. neoformans H99 and 52D by altering the growth curves (increasing the latency), decreasing the maximal growth and altering the slope which is not seen or not as much with El and an unrelated mAb (Figures 13). The effect seems more pronounced for 52D. mAbs B4-1 did not modify the phagocytosis by murine macrophages in the presence of El , a monoclonal antibody specific for Cryptococcus neoformans capsular polysaccharide (Dromer et al., Infect. Immun., 1987, 55, 742-748). None of the anti- Pepl mabs promoted the phagocytosis of H99 by J774 cells in comparison with El (Figure 14). The combination of E1+ B4-1 did not alter phagocytosis compared to El alone. The addition of El after preincubation with the anti-Pep 1 mAbs resulted in an increased phagocytosis compared to the reference condition and to concomitant incubation. This suggests the neutralization of potential anti-phagocytic activity of Pe l .
These results are confirmed by the correlation between phagocytic index and expression of PEP J in clinical isolates (Figure 15) which suggests that Pepl may have anti-phagocytic properties that the monoclonal antibodies may be able to revert as suggested by the experiments using a combination of El and anti-Pep 1 mAbs. Injection of mAb B4-1 to mice provided prolonged survival when injected at
2C^g (Figure 9A, p=0.028, log-rank test), or l OOpg 24 h prior to or 1 day or 7 days (Fig 9B, p =0.004) after inoculation with NIH52D, The dose-response experiment (Figure 10) suggests that increasing the dose (70 and 200 μg) of mab B4-1 injected does not provide better survival and may even be less effective than smaller doses (20 and 7 μg) in terms of survival and even residual fungal load since the majority of the surviving mice were even completely cured of the infection,
A comparison of the 3 mAbs after inoculation with NIH52D, showed no decrease in fungal burden at 21 dpi Prolongation of survival after inoculation with NIH52D was seen mostly with B4-1 and J17-14 (Figure 1 1A, p = 0.0662), whereas fungal burden in all surviving mice was decreased at 63 dpi except in the brain of mice inoculated with J 17- 14 (Figure 1 IB).
Injection of mAb B4-1 , J17.14 or J1.26 to mice infected with H99 (105/mouse) provided prolonged survival (Figure 12, p=0.01 82 log-rank test). Prolongation of survival was seen mostly with B4-1 (Figure 12, p = 0.01 18). Fungal burden was decreased at 54 dpi in 2 of the 3 surviving mice (1 treated with B4.1 and 1 with J 1.26). Table I: Sequence identity of Pepl with other fungal aspartyl proteases
% Fairwise
Organism GenBaak ID
Identity
Cryptococcus neoformatis XP_566887 100.00%
Cryptococcus neoformans XP 012046817 94.10%
Cryptococcus gattii XP 003191823 86.80%
Cryptococcus gattii KIR53730 86.80%
Cryptococcus gattii KIR48411 86.20%
Cryptococcus gattii KGB75 17 84.40%
Cryptococcus gattii IR86636 83.20%
Tric osporon asahii EKD04292 71.20%
Talaromyces marneffei XPJ)02143541 67.50%
Talaromyces marneffei KFX49218 66.00%
Trichosporon asahii XP 014177670 65.30% luyveromyces marxianus BA041873 65.00%
Rhinocladiella mackenziei XP 013268148 65.00%
Paracoccidioides brasiliensis AAP32823 64.90%
Coccidioides immitis XP 001244246 64.80%
Saccharomyces cerevisiae EGA76604 64.80%
Trichosporon asahii EKD04292 64.80%
Candida auris KND96412 64.70%
Coccidioides posadasii AAZ92540 64.70%
Saccharomyces cerevisiae AJV96513 64.70%
Saccharomyces cerevisiae AJW09566 64,70%
Saccharomyces cerevisiae AJW12026 64.70%
Saccharomyces cerevisiae P 015171 64.70%
Saccharomyces cerevisiae AJW20271 64.70%
Saccharomyces cerevisiae AJW18989 64.70%
Saccharomyces cerevisiae AJU25730 64.70%
Saccharomyces cerevisiae AJW16371 64.70%
Saccharomyces cerevisiae GAA26821 64.70%
Paracoccidioides brasiliensis XP 010756433 64.60%
Exophiala dermatitidis XP 009158792 64.40%
Saccharomyces cerevisiae AJV92838 64.40%
Saccharomyces cerevisiae AJV94247 64.40%
Saccharomyces cerevisiae EEU09100 64.40%
Paracoccidioides brasiliensis EEH19972 64.30%
Clavispora lusitaniae XP 002615242 64.20%
Rhizopus delemar EIE89256 64.20%
Saccharomyces cerevisiae EDZ68928 64.20%
Saccharomyces cerevisiae EHM99929 64.10%
Sporothrix schenckii ERT02753 64.10% Aspergillus oryzae XP 001819842 63.70%
Geotrichum candidum CD051223 63.50%
Meyerozyraa guilliermondii XP 001483416 63.40%
Rhizopus microsporus CEG74799 63.30%
Candida albicans CAA31962 63.20%
Debaryomyces hansenii XP 458031 62.20%
Histoplasma capsulatum EGC40857 62.10%
Histoplasma capsulatum XP 001542991 61.80%
Histoplasma capsulatum EEH04825 61.80%
Lichtheimia ramosa CDS05348 61.70%
Kluyveromyces lactis XP 453326 61.30%
Microsporum gypseum XP 003175087 61.20%
Malassezia pachyderaiatis OS12980 60.90%
Aspergillus nidulans XP 660507 60.70%
Pneumocystis jirovecii CCJ31368 58.90%
Trichoderma harzianum KKP05288 58.90%
Candida glabrata XP 449442 58.60%
Malassezia globosa XP 001729651 58.50%
Rhizopus delemar EIE82161 58.20%
Trichophyton interdigitale EZF32786 58.10%
Trichophyton tonsurans EGD99457 58.10%
Trichophyton rubrurn EZF25931 57.90%
Trichophyton rubrum XP 003232712 57.90%
Trichoderma atroviride XP 013942144 57.50%
Trichophyton verrucosum XPJ103020295 57.30%
Fusarmm sp. CEG04259 57.20%
Fusarium oxysporum EWY86405 57.10%
Fusarium oxysporum EGU83511 57.10%
Fusarium oxysporum KNB 13049 57.10%
Fusarium fujikuroi CCT71500 57.00%
Lichtheimia corymbifera CDH52400 56.60%
Candida orthopsilosis XP 003867779 56.50%
Rhizopus microsporus CEG73869 56.50%
Rhizopus microsporus CEI95549 56.50%
Candida parapsilosis CCE40477 56.10%
Emmonsia crescens KZ62546 56.10%
Rhizopus microsporus CEG76436 55.90%»
Lichtheimia ramosa CDS08210 55.80%
Rhizopus microsporus CEG73870 55.80%
Candida dubliniensis XP 002418645 55.10%
Exophiala xenobiotica XP 013321677 55.10%
Fonsecaea pedrosoi XP 013278715 55.00% Cladophialophora carrionii XP 008724614 54.80%
Candida tropicalis XP 002547417 54.70%
Candida albicans KHC38341 54.60%
Candida albicans XP 713194 54.60%
Candida albicans HC45840 54.60%
Candida albicans GQS9270 54.60%
Candida albicans P10977 54.40%
Candida albicans EEQ47270 54.40%
Exophiala oligosperma KIW43340 54.40%
Cladophialophora bantiana KIW87422 54.30%
Exophiala spinifera KIWI 4941 54.30%
Aspergillus clavatus XP 001271141 54.20%
Aspergillus niger XP__001399855 54.20%
Aspergillus niger AAA20876 54.00%
Aureobasidium pullulans KEQ89895 54.00%
Aspergillus rumigatus XP 754479 53.90%
Neosartorya fischeri XP 001263323 53.60%
Chaetomium globosum XP 001227563 52.90%
Madurella mycetomatis KOP43023 52.70%
Myceliophthora thermophila XP 003665229 52.60%
Mucor circinelloides EPB83353 52.10%
Aspergillus terreus XP 001213854 51.90%
Lichtheimia corymbifera CDH51907 51.80%
Mucor circinelloides EPB89553 51.60%
Lichtheimia ramosa CDS03074 51.20%
Rhizopus delemar EIE77417 51.10%
Aspergillus parasiticus KJK63739 50,10%
Lichtheimia corymbifera CDH52330 49.90%
Aspergillus flavus XP 002381878 49.60%
Aspergillus oryzae EIT76268 49.60%
Aspergillus flavus OC16935 49.30%
Aspergillus oryzae XP 001825179 49.30% Table TI: Sequence identity of peptide epitope 3
of Cryptococcus spp. with that of other fungi P XP
GenBank ID Organism XP S66887 012046817 I S3730 003191823 IR4S411 GB7S5I7 K1R86636
Cryptococcus
XP 566887 neoformatis 100 100 100 100 100 100 100
Cryptococcus
XP 0Ϊ 2046817 neofonnans 100 100 100 100 100 100 100 IR53730 Cryptococcus gattii 100 100 100 100 100 100 100
XP 003191823 Cryptococcus gattii 100 100 soo 100 100 100 100 IR48411 Cryptococcus gattii 100 100 100 100 100 100 100
KGB75517 Cryptococcus gattii 100 100 100 100 100 100 SOO
KIR86636 Cryptococcus gattii 100 100 100 100 100 100 100
E D04292 Trichosporon asahii 93.3 93.3 93.3 93.3 93.3 93.3 93.3
XP 014177670 Trichosporon asahii 93.3 93.3 93.3 93.3 93.3 93.3 93.3
E D04292 Trichosporon asahii 93.3 93.3 93.3 93.3 93.3 93.3 93.3 GQ89270 Candida albicans 66.7 66.7 66.7 66.7 66.7 66.7 66.7
XP 713194 Candida albicans 66.7 66.7 66.7 66.7 66.7 66.7 66.7
KHC38341 Candida albicans 66.7 66.7 66.7 66.7 66.7 66.7 66.7
EEQ47270 Candida albicans 66.7 66.7 66.7 66.7 66.7 66.7 66.7
P10977 Candida albicans 66.7 66.7 66.7 66.7 66.7 66.7 66.7
KHC458 0 Candida albicans 66.7 66.7 66.7 66.7 66.7 66.7 66.7
Candida
XP 002418645 dublinsensss 73.3 73.3 73.3 73.3 73.3 73.3 73.3
CAA31 62 Candida albicans 66.7 66.7 66.7 66.7 66.7 66.7 66.7
XP 002547417 Candida tropicalis 73.3 73.3 73.3 73.3 73.3 73.3 73.3
Candida
CCE40477 parapsilosis 73.3 73.3 73.3 73.3 73.3 73.3 73.3
Clavispora
XP 002615242 lusitaniae 66.7 66.7 66,7 66.7 66.7 66.7 66,7
Meyerozyma
XP 0014834! 6 guilliermondii 73.3 73.3 73.3 73.3 73.3 73.3 73.3
Kluyveromyces
BA041873 marxianus 73.3 73.3 73.3 73.3 73.3 73.3 73,3
XP 449442 Candida glabrata 60 60 60 60 60 60 60
XP 002381878 Aspergillus flavus 60 60 60 60 60 60 60
XP 001825179 Aspergillus oryzae 60 60 60 60 60 60 60
EIT76268 Aspergillus oryzae 60 60 60 60 60 60 60 OC 16935 Aspergillus flavus 60 60 60 60 60 60 60
Aspergillus
KJK63739 parasiticus 60 60 60 60 60 60 60
Aspergillus
XP 001271141 ciavatus 66.7 66.7 66.7 66.7 66.7 66.7 66.7
Aspergillus
XP 754479 fiimigatus 60 60 60 60 60 60 60
XP 001399855 Aspergillus niger 73.3 73.3 73.3 73.3 73.3 73.3 73,3
AAA20876 Aspergillus niger 73.3 73.3 73.3 73.3 73.3 73.3 73.3
XP 001 842 Aspergillus oryzae 66.7 66.7 66.7 66.7 66.7 66.7 66.7
XP 001213854 Aspergillus terreus 66.7 66.7 66.7 66.7 66.7 66.7 66.7
Aspergillus
XP 660507 nidulans 66.7 66.7 66.7 66.7 66.7 66.7 66.7 Talaromyces
P 00214354! rnarneffei 60 60 60 60 60 60 60
Talaromyces
KFX49218 marneffei 60 60 60 60 60 60 60
Trichophyton
BZF32786 interdigitale 60 60 60 60 60 60 60
Trichophyton
EGD99457 tonsurans 60 60 60 60 60 60 60
Trichophyton
XP 003232712 rubrum 60 60 60 60 60 60 60
Trichophyton
EZF2593 ! rubrum 60 60 60 60 60 60 60
Trichophyton
XP 003020295 verrucosum 60 60 60 60 60 60 60 icrosporum
XP 003175087 gypseum 66.7 66.7 66.7 66.7 66.7 66.7 66.7
Coccidioides
XP 001244246 imrnitis 60 60 60 60 60 60 60
Coccidioides
AAZ92540 posadasii 60 60 60 60 60 60 60
Histoplasma
EGC40S57 capsulatum. 73.3 73.3 73.3 73.3 73.3 73.3 73.3
Histoplasma
EEH04825 capsulatum 73.3 73.3 73.3 73.3 73.3 73.3 73.3
Histoplasma
XP 00154299! capsulatum 73.3 73.3 73.3 73.3 73.3 73.3 73.3
Paracoccidioides
EEH 19972 brassliensis 53.3 53.3 53.3 53.3 53.3 53.3 53.3
Paracoccidioides
XP 010756433 brasiliensis 53.3 53.3 53.3 53.3 53.3 53.3 53.3
Paracoccidioides
AAP32S23 brasiliensis 60 60 60 60 60 60 60
Exophiak
KIW43340 oligosperma 73.3 73.3 73.3 73.3 73.3 73.3 73.3
Exophiala
XP 013321677 xenobtotica 73.3 73.3 73.3 73.3 73.3 73.3 73.3 !W 14941 Exophiala spinifera 73.3 73.3 73.3 73.3 73.3 73.3 73.3
Exophiala
XP 009158792 dermatitidis 66.7 66.7 66.7 66.7 66.7 66.7 66.7
Fusarium
EGU83511 oxysporum 73.3 73.3 73.3 73.3 73.3 73.3 73.3
Fusarium
NB 13049 oxysporum 73.3 73.3 73.3 73.3 73.3 73.3 73.3
Fusarium
EWY 86405 oxysporum 73.3 73.3 73.3 73.3 73.3 73.3 73.3
CCT71500 Fusarium fojikuroi 73.3 73.3 73.3 73.3 73.3 73.3 73.3
CEG04259 Fusarium sp. 66.7 66.7 66.7 66.7 66.7 66.7 66.7
Pneumocystis
CCJ31368 jirovecii 52.6 52.6 52.6 52.6 52.6 52.6 52.6
Lichtheimia
CDH51 07 corymbifera 86.7 86.7 86.7 86.7 86.7 86.7 86.7
CDS03074 Lichtheimia ramosa 86.7 86.7 86.7 86.7 86.7 86.7 86.7
Lichtheimia
CDH52400 corymbifera 73.3 73.3 73.3 73.3 73.3 73.3 73.3
CDS08210 Lichtheimia ramosa 73.3 73.3 73.3 73.3 73.3 73.3 73.3 ucor
EPB89553 circinelloides 80 80 80 80 80 80 80
EIE8 256 Rhizopus delemar 66.7 66.7 66.7 66.7 66.7 66.7 66.7
Rhizopus
CEG74799 microsporus 66.7 66.7 66,7 66.7 66.7 66.7 66.7
Mucor
EPB83353 circinelloides 66.7 66.7 66.7 66.7 66.7 66.7 66.7
Lichtheimia
CDH52330 corymbifera SO 80 80 80 80 80 SO
CDS05348 Lichtheimia ramosa 80 80 80 80 80 80 80
Rhizopus
CEG73870 microsporus 73.3 73.3 73.3 73.3 73.3 73,3 73.3 Rhizopus
CEG73869 microsporus 73.3 73.3 73.3 73.3 73.3 73.3 73.3
Rhizopus
CEG76436 microsporus 73.3 73.3 73.3 73.3 73.3 73.3 73.3
Rhizopus
CEI95549 microsporus 73.3 73.3 73.3 73.3 73.3 73.3 73.3
EIES2161 Rhizopus delemar 66.7 66-7 66.7 66.7 66.7 66.7 66.7
E1E77417 Rhizopus delemar 53.3 53.3 53.3 53.3 53.3 53.3 53.3
Table HI: Sequence identity of peptide epitope 4 of Cryptococcus spp. with that of other fungi
P
GenBank XP 566 01204681 KIR53 XP 00319 KIR48 KGB 75 KIR86 ID Organism 887 7 730 1823 4Π 517 636
XP 56688 Cryptococcus
7 neoformans 100 100 100 100 100 100 100
XP 01204 Cryptococcus
6817 neoformans 100 100 100 100 100 100 100 1R53730 Cryptococcus gattii 100 100 100 100 100 100 100
XP 0031
1823 Cryptococcus gattii 100 100 100 100 100 100 100
K.IR484I 1 Cryptococcus gattii 100 100 100 100 100 100 100 GB75517 Cryptococcus gattii 100 100 100 100 100 100 100
KIR86636 Cryptococcus gattii 100 100 100 100 100 100 100
EKD04292 Trichosporon asahii 100 100 100 100 100 100 100
XP 01417
7670 Trichosporon asahii 100 100 100 100 100 100 100
E D04292 Trichosporon asahii 100 100 100 100 100 100 100
KGQ89270 Candida albicans 73.3 73.3 73.3 73.3 73.3 73.3 73.3
XP 71319
4 Candida albicans 73.3 73.3 73.3 73.3 73.3 73.3 73.3 HC38341 Candida albicans 73.3 73.3 73.3 73.3 73.3 73.3 73.3
EEQ47270 Candida albicans 73.3 73.3 73.3 73.3 73.3 73.3 73.3
PI 0977 Candida albicans 73.3 73.3 73.3 73.3 73.3 73,3 73.3 HC45840 Candida albicans 73.3 73.3 73.3 73.3 73.3 73.3 73.3
XP 00241
8645 Candida dubliniensis 73.3 73.3 73.3 73.3 73.3 73.3 73.3
CAA31 62 Candida albicans 73.3 73.3 73.3 73,3 73.3 73.3 73.3
XP 00254
7417 Candida tropical is 66.7 66.7 66.7 66.7 66.7 66.7 66.7
CCE40477 Candida parapsilosis 73.3 73.3 73.3 73.3 73.3 73.3 73.3
XP 00261
5242 Ciavispora lusitaniae 73.3 73.3 73.3 73.3 73.3 73.3 73,3
XP 00148 Meycrozyma
3416 guilliermondii 66.7 66.7 __, 66.7 66.7 66.7 66.7 66.7 luyveromyces
BA041873 marxianus 73.3 73.3 73.3 73.3 73.3 73.3 73.3
XP 44944
2 Candida glabrata 60 60 60 60 60 60 60
XP 00238
1 878 Aspergillus flavus 46.7 46.7 46.7 46.7 46.7 46.7 46.7
XP 00182
5179 Aspergillus oryzae 46.7 46.7 46.7 46.7 46.7 46.7 46.7
ΕΠ76268 Aspergillus oryzae 46.7 46.7 46.7 46.7 46.7 46.7 46.7 OC 16935 Aspergillus flavus 46.7 46.7 46.7 46.7 46.7 46.7 46.7
Aspergillus
KJK63739 parasiticus 46.7 46.7 46.7 46.7 46.7 46.7 46.7
XP 00127
1 141 Aspergillus clavatus 60 60 60 60 60 60 60
XP 75447 Aspergillus
9 fumigatus 53.3 53.3 53.3 53.3 53.3 53.3 53.3
XPJXH 39
9855 Aspergillus niger 73.3 73.3 73.3 73.3 73.3 73.3 73.3 AAA20876 Aspergillus nsger 73.3 73.3 73.3 73.3 73.3 73.3 73.3
XP 00Ϊ81
9842 Aspergillus oryzae 66.7 66.7 66.7 66.7 66.7 66.7 66.7
XP 0012!
3854 Aspergillus terreus 60 60 60 60 60 60 60
XP 66050
7 Aspergillus nidulans 66.7 66.7 66.7 66.7 66.7 66.7 66.7
XP 00214 Talaromyces
3541 marneffei 93.3 93.3 93.3 93,3 93.3 93.3 93.3
Talaromyces
KFX49218 marneffei 93.3 93.3 93.3 93.3 93.3 93.3 93.3
Trichophyton
EZF32786 interdigitale 80 80 80 80 80 80 80
Trichophyton
EGD99457 tonsurans 80 80 80 80 80 80 80
XP 00323
2712 Trichophyton rubrum 80 80 80 80 80 80 80
EZF2593 1 Trichophyton rubrum 80 80 80 80 80 80 80
XP 00302 Trichophyton
0295 verrucosum 80 80 80 80 SO 80 80
XP 00317 Microsporum
5087 gypseum 80 80 80 80 80 80 80
XPJW124
4246 Coccidioides immitis 93.3 93.3 93.3 93.3 93.3 93.3 93.3
Coccidioides
AAZ92540 posadasii 93.3 93.3 93.3 93.3 93.3 93.3 93.3
Histoplasma
EGC40857 capsulatum 80 80 80 80 80 80 80
Histoplasma
EEH04825 capsulatum 73.3 73.3 73.3 73.3 73,3 73.3 73.3
XP 00154 Histoplasma
2991 capsulatum 80 80 80 80 80 80 80
Paracoccidioides
EEH 19972 brasiiiensis 80 80 80 80 80 80 80
XP 01075 Paracoccidioides
6433 brasiiiensis 80 80 80 80 80 80 80
Paracoccidioides
AAP32823 brasiiiensis 80 80 80 80 80 80 80
Exophiala
KIW43340 oligospermia 66.7 66.7 66.7 66.7 66.7 66.7 66.7
XP 01332 Exophiala
1677 xenobiotica 66.7 66.7 66.7 66.7 66.7 66.7 66.7 IW14941 Exophiala spinifera 66.7 66.7 66.7 66.7 66.7 66.7 66.7
XP 00915 Exophiala
8792 dermatitidis 73.3 73.3 73.3 73.3 73.3 73.3 73.3
EGU8351 1 Fusarium oxysporum 66.7 66.7 66.7 66.7 66.7 66.7 66.7 NB 13049 Fusarium oxysporum 66.7 66.7 66.7 66.7 66.7 66.7 66.7
EWYS640
5 Fusarium oxysporum 66.7 66.7 66.7 66.7 66.7 66.7 66.7
CCT71500 Fusarium fujikuroi 66.7 66.7 66.7 66.7 66.7 66.7 66.7
CEG04259 Fusarium sp. 73.3 73.3 73.3 73.3 73.3 73.3 73.3
Pneumocystis
CCJ3 B68 jirovecii 53.3 53.3 53.3 53.3 53.3 53.3 53.3
Lichtheimia
CDH51907 corymbifera 66.7 66.7 66.7 66.7 66.7 66.7 66.7
CDS03074 Lichtheimia ramosa 66.7 66.7 66.7 66.7 66.7 66.7 66.7
Lichtheimia
CDH52400 corymbifera 73.3 73.3 73.3 73.3 73.3 73.3 73.3
CDS082I 0 Lichtheimia ramosa 80 80 80 80 80 80 80 EPB89553 Mucor circinelioides 66.7 66.7 66.7 66.7 66.7 66.7 66.7
EIE89256 Rhizopus delemar 73.3 73.3 73.3 73.3 73.3 73.3 73.3
Rhizopus
CEG74799 microsporus 66.7 66.7 66.7 66.7 66.7 66.7 66.7
EPB83353 Mucor circinelioides 73.3 73.3 73.3 73.3 73.3 73.3 73.3
Lichtheimia
CDH52330 corymbifera 60 60 60 60 60 60 60
CDS05348 Lichtheimia ramosa 60 60 60 60 60 60 60
Rhizopus
CEG73870 microsporus 46.7 46.7 46.7 46.7 46.7 46.7 46.7
Rhizopus
CEG73869 microsporus 46.7 46.7 46.7 46.7 46.7 46.7 46.7
Rhizopus
CEG76436 microsporus 40 40 40 40 40 40 40
Rhizopus
CEI95549 microsporus 46.7 46.7 46.7 46.7 46.7 46.7 46.7
EIE82161 Rhizopus delemar 53.3 53.3 53.3 53.3 53.3 53.3 53.3
EJE774 i7 Rhizopus delemar 40 40 40 40 40 40 40

Claims

1. An antibody for use in the treatment of a fungal disease,
wherein said antibody binds to an antigen being chosen from:
a) a recombinant aspartyl protease protein comprising an amino acid sequence having at least 48 % identity with SEQ ID NO: 1 and comprising two catalytic motifs, SEQ ID NO: 2 and SEQ ID NO: 3, in positions corresponding to positions 145 to 149 and 327 to 331 of SEQ ID NO: 1 and further comprising at least one protective epitope, said epitope(s) corresponding to one or more of the peptides in positions 1 to 15, 271 to 285, 296 to 310 and 346 to 360 of SEQ ID NO: 1, or a functional variant thereof; and
b) a polypeptide fragment of the protein in a) comprising at least one of said protective epitope(s), and,
wherein said antibody is an antibody recognizing specifically at least one of said protective epitope(s) or a functional fragment thereof comprising at least the antibody binding region.
2. The antibody for the use according to claim 1, wherein:
- the peptide in positions 1 to 15 consists of SEQ ID NO: 12;
- the peptide in positions 271 to 285 consists of a sequence having at least 60 % identity with SEQ ID NO: 13;
- the peptide in positions 296 to 310 consists of a sequence having at least 60 % identity with SEQ ID NO: 15; and
- the peptide in positions 346 to 3 0 consists of a sequence having at least 60 % identity with SEQ ID NO: 1 .
3. The antibody for the use according to claim 1 or claim 2, which is a monoclonal antibody recognizing specifically the peptides of SEQ ID NO: 12 and SEQ ID NO: 15, the peptide of SEQ ID NO: 13, or the peptides of SEQ ID NO:13 and SEQ ID NO: 16, or a functional fragment thereof comprising at least the antigen binding site.
4. The antibody for the use according to claim 3, which is a monoclonal antibody produced by an hybridoma selected from the hybridomas deposited at CNCM under accession number 1-4914, 1-4915 and 1-4916 or a Fv, Fab or Fab'2 fragment thereof.
5. The antibody for the use according to any one of claims 1 to 4, wherein said antibody is used simultaneously, separately or sequentially with at least one immunotherapeutic agent and/or antifungal agent.
6. The antibody for the use according to any one of claims 1 to 5, wherein said treatment of a fungal disease is a therapeutic treatment administered after diagnosis of the fungal infection.
7. The antibody for the use according to any one of claims 1 to 6, wherein said fungal disease is a superficial mycosis, a cutaneous mycosis, a subcutaneous mycosis or a systemic mycosis due to primary or opportunistic pathogenic fungi.
8. The antibody for the use for the treatment of a fungal disease according to claim 7, wherein said fungal disease is selected from the group consisting of: candidiasis, aspergillosis, cryptococcosis, trichosporonosis, mucormycosis, fusariosis, pneumocystosis, dermatophytoniycosis, penicilliosis marneffei, coccidoidomycosis, histoplasmosis, and paracoccidoidomycosis.
9. The antibody for the use for the treatment of a fungal disease according to claim 8, wherein said fungal disease is cryptococcosis.
10. A pharmaceutical composition comprising at least an antibody according to any one of claims 1 , 2, 3 and 4 or a functional fragment comprising at least the antibody binding region, and a pharmaceutically acceptable vehicle.
11. A recombinant antigen for use in the treatment of a fungal disease,
wherein said antigen is chosen from:
a) a recombinant aspartyl protease protein comprising an amino acid sequence having at least 48 % identity with SEQ ID NO: 1 and comprising two catalytic motifs, SEQ ID NO: 2 and SEQ ID NO: 3, in positions corresponding to positions 145 to 149 and 327 to 331 of SEQ ID NO: 1 and further comprising at least one protective epitope, said epitope(s) corresponding to one or more of the peptides in positions 1 to 15, 271 to 285, 296 to 310 and 346 to 360 of SEQ ID NO: 1, or a functional variant thereof; and
b) a polypeptide fragment of the protein in a) comprising at least one of said protective epitope(s) .
12. The recombinant antigen for the use according to claim 11, wherein;
- the peptide in positions 1 to 15 consists of SEQ ID NO: 12; - the peptide in positions 271 to 285 consists of a sequence having at least 60 % identity with SEQ ID NO: 13;
- the peptide in positions 296 to 310 consists of a sequence having at least 60 % identity with SEQ ID NO: 15; and
- the peptide in positions 346 to 360 consists of a sequence having at least 60 % identity with SEQ ID NO: 16.
13. The recombinant antigen for the use according to claim 1 1 or claim 12, which is a recombinant aspartyl protease protein of a pathogenic fungus selected from the group consisting of: Aspergillus spp., Candida spp., Clavispora spp., Coccidioides spp,, Cryptococcus spp., Exophiala spp., Fusarium spp., Histoplasma spp., Kluyveromyces spp., Mucorales such as Lichtheimia spp., Mucor spp. and Rhizopus spp., Meyerozyma spp., Microsporum spp., Neosartorya spp., Paracoccidioides spp., Pneumocystis spp., Talaromyces spp., Trichophyton spp. and Trichosporon spp., or a functional variant thereof.
14. The recombinant antigen for the use according to any one of claims 11 to 13, which is a recombinant aspartyl protease protein comprising an amino acid sequence having at least 60 % identity with SEQ ID NO: 1.
15. The recombinant antigen for the use according to any one of claims 11 to 14, which is a Cryptococcus spp. recombinant aspartyl protease protein comprising an amino acid sequence having at least 80 % identity with SEQ ID NO: 1.
16. The Cryptococcus spp. recombinant aspartyl protease protein for the use according to claim 15, which comprises any one of SEQ ID NO: 1 and 6 to 11.
17. The recombinant antigen for the use according to any one of claims 11 to 16, wherein said antigen is used simultaneously, separately or sequentially with at least one immunotherapeutic agent and/or antifungal agent.
18. The recombinant antigen for the use according to any one of claims 11 to 17, wherein said treatment of a fungal disease is a therapeutic treatment administered after diagnosis of the fungal infection.
19. The recombinant antigen for the use according to any one of claims 11 to 18, wherein said fungal disease is a superficial mycosis, a cutaneous mycosis, a subcutaneous mycosis or a systemic mycosis due to primary or opportunistic pathogenic fungi.
20. The recombinant antigen for the use according to any one of claims 11 to 18, wherein said fungal disease is selected from the group consisting of candidiasis, aspergillosis, cryptococcosis, trichosporonosis, mucormycosis, fusariosis, pneumocystosis, dermatophytomycosis, penicilliosis mameffei, coccidoidomycosis, histoplasmosis, and paracoccidoidomycosis, and is preferably cryptococcosis.
PCT/EP2016/082297 2015-12-22 2016-12-22 Recombinant aspartyl protease antigen or antibody thereto for use in immunotherapy of fungal diseases Ceased WO2017109028A1 (en)

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Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CA2354100A1 (en) * 2001-08-02 2003-02-02 Segolene Neuville Polynucleotide and polypeptide antigens of cryptococcus neoformans and their vaccine and diagnostic applications

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CA2354100A1 (en) * 2001-08-02 2003-02-02 Segolene Neuville Polynucleotide and polypeptide antigens of cryptococcus neoformans and their vaccine and diagnostic applications

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
BOZZA SILVIA ET AL: "Immune sensing of Aspergillus fumigatus proteins, glycolipids, and polysaccharides and the impact on Th immunity and vaccination.", JOURNAL OF IMMUNOLOGY (BALTIMORE, MD. : 1950) 15 AUG 2009, vol. 183, no. 4, 15 August 2009 (2009-08-15), pages 2407 - 2414, XP002757108, ISSN: 1550-6606 *
F DROMER ET AL: "Protection of mice against experimental cryptococcosis by anti-Cryptococcus neoformans monoclonal antibody", INFECTION AND IMMUNITY, 1 March 1987 (1987-03-01), UNITED STATES, pages 749 - 752, XP055357661, Retrieved from the Internet <URL:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC260405/pdf/iai00087-0261.pdf> *
SHARADA RAVIKUMAR ET AL: "Optimizing Outcomes in Immunocompromised Hosts: Understanding the Role of Immunotherapy in Invasive Fungal Diseases", FRONTIERS IN MICROBIOLOGY, vol. 6, 26 November 2015 (2015-11-26), XP055357679, DOI: 10.3389/fmicb.2015.01322 *

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