WO2016172762A1 - Vaccin contre la schistosomiase - Google Patents

Vaccin contre la schistosomiase Download PDF

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Publication number
WO2016172762A1
WO2016172762A1 PCT/AU2016/050290 AU2016050290W WO2016172762A1 WO 2016172762 A1 WO2016172762 A1 WO 2016172762A1 AU 2016050290 W AU2016050290 W AU 2016050290W WO 2016172762 A1 WO2016172762 A1 WO 2016172762A1
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seq
isolated
mammal
nos
proteins
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PCT/AU2016/050290
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English (en)
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Alex LOUKAS
Mark Pearson
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James Cook University
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Priority claimed from AU2015901481A external-priority patent/AU2015901481A0/en
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Publication of WO2016172762A1 publication Critical patent/WO2016172762A1/fr

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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
    • A61K39/002—Protozoa antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P33/00—Antiparasitic agents
    • A61P33/10—Anthelmintics
    • A61P33/12—Schistosomicides
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/44—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from protozoa
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • the invention relates to prevention and/or treatment of schistosomiasis.
  • the present invention provides a vaccine that comprises one or more schistosome immunogenic proteins.
  • S. haematobium lay between 20 and 200 eggs daily (3), which penetrate the vessel wall and move towards the lumen of the bladder. Some of the eggs become sequestered in the tissue of the pelvic organs such as the urinary bladder, ureters, cervix, vagina, prostate gland, and seminal vesicles, where they cause chronic inflammation, pelvic pain, bleeding, and an altered cervical epithelium in women (4).
  • S. haematobium is unique among the schistosomes in its recognition as a group I carcinogen by the International Agency for Research on Cancer because of its robust association with urothelial carcinoma (5). S. haematobium infection also increases susceptibility to infection with HIV-1, progression to disease, and results in a higher likelihood of transmitting infection to others (6).
  • PZQ Praziquantel
  • An added benefit of PZQ treatment is that it mediates destruction of flukes thereby exposing antigens on the worm surface to the host immune system. This release of surface antigens induces and/or enhances parasite-specific immune responses (8), resulting in immune-mediated killing of the parasite.
  • Early studies reported modifications in T cell proliferative responses (9), whereas recent studies noted modifications in the levels and types of antibody (10-13) and cytokine responses (14-16) following PZQ treatment.
  • DIR drug-induced resistance
  • PZQ treatment introduces a large amount of adult fluke antigen directly into the bloodstream as a result of many worms dying at once (21), whereas naturally acquired resistance in the absence of PZQ treatment (PR) is stimulated by the introduction smaller quantities of adult antigen due to a more gradual worm death.
  • PR naturally acquired resistance in the absence of PZQ treatment
  • the process of PR is additionally stimulated by the release of antigens from naturally dying larval schistosomes (schistosomula) primarily through the skin and pulmonary vasculature, thus inducing different APCs and resulting in different interactions between the antigens and the immune system (22).
  • This additional stimulus does not appear to factor significantly in DIR due to the ineffectiveness of PZQ against schistosomula (7, 8).
  • SWAP where worms are homogenized and solubilized under native conditions in the absence of detergents that will solubilize the cell membranes
  • SWAP the utilization of SWAP (where worms are homogenized and solubilized under native conditions in the absence of detergents that will solubilize the cell membranes) does not result in full representation of the S. haematobium proteome.
  • numerous abundantly expressed proteins with multiple membrane spanning domains that are released from the tegument with detergents 29, 30
  • are accessible to chemical labeling on the surface of live worms (30) are recognized by sera from PR individuals, and are lead vaccine antigens against schistosomiasis (31-33).
  • a third mechanism of resistance to schistosomiasis is seen in the rhesus macaque (Macacca mulatto).
  • a broad form of the invention provides one or a plurality of isolated, immunogenic proteins obtained, or obtainable from, a schistosome such as S. haematobium, or a variant or fragment thereof, for use in preventing, treating or immunizing against a schistosome infection.
  • a schistosome such as S. haematobium, or a variant or fragment thereof
  • An aspect of the invention provides an immunogenic composition comprising one or a plurality of isolated proteins comprising respective amino acid sequences set forth in SEQ ID NOS: 1-3, or a fragment or variant thereof; one or a plurality of isolated nucleic acids encoding SEQ ID NOS: 1-3, or a fragment or variant thereof; or an antibody or antibody fragment which binds one or a plurality of isolated proteins comprising respective amino acid sequences set forth in SEQ ID NOS: 1-3, or a fragment or variant thereof.
  • the immunogenic composition is capable of eliciting a protective immune response upon administration to a mammal.
  • the immunogenic composition is a vaccine.
  • Another aspect of the invention provides a method of eliciting an immune response in a mammal, said method including the step of administering an immunogenic composition to the mammal to thereby elicit an immune response to a schistosome in the mammal, said immunogenic composition comprising one or a plurality of isolated proteins comprising respective amino acid sequences set forth in SEQ ID NOS: 1-3, or a fragment or variant thereof; one or a plurality of isolated nucleic acids encoding SEQ ID NOS: 1-3, or a fragment or variant thereof; or an antibody or antibody fragment which binds one or a plurality of isolated proteins comprising respective amino acid sequences set forth in SEQ ID NOS: 1-3, or a fragment or variant thereof.
  • the immunogenic composition elicits a protective immune response after administration to the mammal.
  • Yet another aspect of the invention provides a method of immunizing a mammal, said method including the step of administering an immunogenic composition to the mammal to thereby immunize the mammal against infection by a schistosome, said immunogenic composition comprising one or a plurality of isolated proteins comprising respective amino acid sequences set forth in SEQ ID NOS: 1-3, or a fragment or variant thereof; one or a plurality of isolated nucleic acids encoding SEQ ID NOS: 1-3, or a fragment or variant thereof; or an antibody or antibody fragment which binds one or a plurality of isolated proteins comprising respective amino acid sequences set forth in SEQ ID NOS: 1-3, or a fragment or variant thereof.
  • Still yet another aspect of the invention provides a method of preventing or treating a schistosome infection in a mammal, said method including the step of administering an immunogenic composition to the mammal to thereby prevent or treat a Schistosome infection in the mammal, said immunogenic composition comprising one or a plurality of isolated proteins one or a plurality of isolated proteins comprising respective amino acid sequences set forth in SEQ ID NOS: 1-3, or a fragment or variant thereof; one or a plurality of isolated nucleic acids encoding SEQ ID NOS: 1- 3, or a fragment or variant thereof; or an antibody or antibody fragment which binds one or a plurality of isolated proteins comprising respective amino acid sequences set forth in SEQ ID NOS: 1-3, or a fragment or variant thereof.
  • SEQ ID NOS: 1-3 are in the form of a single protein, such as a chimeric protein.
  • the chimeric protein comprises an amino acid sequence set forth in SEQ ID NO:4 or SEQ ID NO:5.
  • a further aspect of the invention provides an isolated, immunogenic chimeric protein comprising at least two of the respective amino acid sequences set forth in SEQ ID NOS: 1-3, or at least two fragments or variants of SEQ ID NOS: 1-3.
  • the isolated, immunogenic chimeric protein comprises an amino acid sequence set forth in SEQ ID NO: 4 or SEQ ID NO: 5.
  • This further aspect of the invention also provides an isolated nucleic acid encoding the isolated, immunogenic chimeric protein, a genetic construct comprising the isolated nucleic acid and/or a host cell comprising the genetic construct.
  • This further aspect of the invention also provides an antibody which binds and/or is raised against the isolated chimeric protein.
  • indefinite articles “a” and “an” may refer to one entity or a plurality of entities (e.g. proteins) and are not to be read or understood as being limited to a single entity.
  • Figure 1 Characterization of study cohort and sub-cohort used for the study described herein. *Treatment efficacy was assessed by urinalysis 6 weeks after praziquantel therapy - all subjects were egg-negative (no eggs found in any of 3 urine samples, each collected on a separate day). ⁇ Subjects remained in the endemic study area and had regular water contact for the study duration.
  • FIG. 1 Antibody responses to arrayed antigens differ in Schistosoma haematobium-infected humans before and after praziquantel treatment.
  • A IgGl .
  • B IgE. Average adjusted signal intensity values depicting the antibody response to each reactive antigen are shown for the drug-induced resistant (DIR) cohort before and after praziquantel treatment.
  • the dashed and solid lines are the respective cut-offs for IgGl (8239) and IgE (1861) reactivity, calculated as one standard deviation of the mean of the no-DNA control spots on the array.
  • Statistical analysis was performed using student's t test. *p ⁇ 0.05, **p ⁇ 0.01, ***p ⁇ 0.001.
  • IgGl antibody profiles to arrayed antigens differ between Schistosoma haematobium-infected humans who do and do not acquire drug-induced resistance after praziquantel treatment. Average adjusted signal intensity values depicting IgGl antibody responses to each reactive antigen are shown for the drug- induced resistant (DIR) and chronically infected (CI) cohorts after praziquantel treatment. Boxed antigens indicate homologues of known vaccine candidates. The dashed line is the cut-off for IgGl reactivity (8239), calculated as one standard deviation of the mean of the no-DNA control spots on the array. Statistical analysis was performed using student's t test. *p ⁇ 0.05, **p ⁇ 0.01, ***p ⁇ 0.001.
  • FIG. 4 Some arrayed antigens that induce IgGi responses in Schistosoma haematobium-infected humans who acquire drug-induced resistance after praziquantel treatment are not the targets of IgE. Average adjusted signal intensity values depicting IgGl and IgE antibody responses to each IgGi antigen reactive to post-treatment sera from drug-induced resistant (DIR) humans. The dashed and solid lines are the respective cut-offs for IgGl (8239) and IgE (1861) reactivity, calculated as one standard deviation of the mean of the no-DNA control spots on the array. Schistosoma japonicum SEA is included for comparative purposes.
  • FIG. 5 Gene transcription in the adult and egg stages of S. haematobium for all arrayed proteins inducing significantly different and reactive IgG responses to DIR post-treatment sera. Data was assembled from publicly available RNA-seq databases (Young et al, 2012.). These data were filtered for quality (PHRED score of >30) using Trimmomatic [8] and aligned to the open reading frames of the published gene set [7] using Bowtie (v2.1.0) [9] Normalised levels of gene transcription were calculated using the software package RSEM (v 1.2.11) [10] and reported as the numbers of transcripts per million reads sequenced (TPMs). The TPM value of each gene was log 2 -transformed and subjected to heat map visualisation using R.
  • FIG. 1 IgG antibody profiles to arrayed antigens differ in Schistosoma japonicum-infected, self-curing rhesus macaques during the course of infection from exposure to perfusion. Average adjusted signal intensity values depicting IgG antibody responses to each significantly reactive antigen are shown at baseline (0 weeks), 12 weeks post-infection and elimination (20 weeks post-infection). The dashed line is the cut-off for IgG reactivity (3210), calculated as one standard deviation of the mean of the no-DNA control spots on the array. Statistical analysis was performed using student's i test. *p ⁇ 0.05, **p ⁇ 0.01, ***p ⁇ 0.001. [0028] Figure 7.
  • FIG. 1 Venn Diagram depicting common IgG reactive proteins between Schistosoma haematobium- infected humans from an endemic area in Africa who acquire drug-induced resistance after praziquantel treatment (DIRs), Schistosoma japonicum-infected self-curing rhesus macaques and Schistosoma mansoni- infected humans from an endemic area of Brazil who are naturally resistant (PRs).
  • DIRs drug-induced resistance after praziquantel treatment
  • PRs naturally resistant
  • AY812195 *data from Gaze et al, 2014; % IgGi response to AY812195 is significantly different between DIRs before and after praziquantel treatment but not between DIRs and CIs post-treatment; AY814977 and Smp_124240 are the respective S. japonicum and S. mansoni orthologues of SNaKip. ⁇ we believe the sequence represented by AY815690 ("myosin-7 [S. japonicum]”) has been incorrectly annotated due to its high degree of homology with other parasite orthologues of ribosome-binding protein 1 and lack of blastP hits with any form of myosin.
  • the present invention is directed to an immunogenic composition, such as a vaccine, that is capable of preventing or treating schistosomiasis.
  • the immunogenic composition suitably comprises one or a plurality of immunogenic proteins that respectively comprise amino acid sequences set forth in one or a plurality of SEQ ID NOS: 1-3. These proteins are the targets of humoral immune responses in "drug- induced resistance" human subjects from an S. haematobin-endemic area in Africa and also in rhesus macaques that had undergone self-cure after experimental S. japonicum infection.
  • immunogenic proteins may be chimeras comprising respective amino acid sequences of two or more of SEQ ID NOS: 1-3, such as the embodiments set forth in SEQ ID NOS:4 and 5. It is therefore proposed that the isolated, immunogenic proteins disclosed herein and/or their encoding nucleic acids, may be useful in a safe and efficacious vaccine against various Schistosoma species, including but not limited to S. mansoni, S. japonicum and S. haematobium.
  • An aspect of the invention therefore provides an immunogenic composition comprising one or a plurality of isolated proteins comprising respective amino acid sequences set forth in SEQ ID NOS: 1-3, or a fragment or variant thereof; one or a plurality of isolated nucleic acids encoding SEQ ID NOS: 1-3, or a fragment or variant thereof; or an antibody which binds one or a plurality of isolated proteins comprising respective amino acid sequences set forth in SEQ ID NOS: 1-3, or a fragment or variant thereof.
  • a further aspect of the invention provides an isolated, immunogenic chimeric protein comprising at least two of the respective amino acid sequences set forth in SEQ ID NOS: 1-3, or at least two fragments or variants of SEQ ID NOS: 1-3.
  • the isolated, immunogenic chimeric protein comprises an amino acid sequence set forth in SEQ ID NO:4 or SEQ ID NO:5, or a chimeric fragment or variant thereof; or an immunogenic composition comprising said isolated immunogenic chimeric protein..
  • schistosome “schistosomal” and “schistosomiasis” refer to trematode blood flukes of the genus “Schistosoma” and/or diseases or conditions associated with, or caused by, trematodes of this genus.
  • Schistosoma species include but are not limited to S. mansoni, S. japonicum and S. haematobium.
  • Infective schistosomulae migrate through several tissues and stages to their residence in the veins.
  • Adult worms in humans reside in the mesenteric venules in various locations, which at times seem to be specific for each species. For instance, S.
  • japonicum is more frequently found in the superior mesenteric veins draining the small intestine, and S. mansoni occurs more often in the superior mesenteric veins draining the large intestine.
  • S. haematobium most often occurs in the venous plexus of bladder but it can also be found in the rectal venules.
  • Eggs are deposited in the small venules of the portal and perivesical systems. The eggs progressively move toward the lumen of the intestine (S. mansoni and S. japonicum) and of the bladder and ureters (S. haematobium). Pathology of S. mansoni and S.
  • japonicum schistosomiasis includes Katayama fever, hepatic perisinusoidal egg granulomas, Symmers' pipe stem periportal fibrosis, portal hypertension, and occasional embolic egg granulomas in brain or spinal cord.
  • Pathology of S. haematobium schistosomiasis includes hematuria, scarring, calcification, squamous cell carcinoma, and occasional embolic egg granulomas in brain or spinal cord.
  • Various mammals such as dogs, cats, rodents, pigs, horse and goats, serve as reservoirs for S. japonicum.
  • isolated material that has been removed from its natural state or otherwise been subjected to human manipulation. Isolated material may be substantially or essentially free from components that normally accompany it in its natural state, or may be manipulated so as to be in an artificial state together with components that normally accompany it in its natural state. Isolated material includes material in native and recombinant form. The term “isolated” also encompasses terms such as "enriched”, “purified” and/or “synthetic”. The term “synthetic” includes recombinant synthetic and chemical synthetic.
  • a "protein” is an amino acid polymer that may comprise natural and/or non-natural amino acids, D- or L- amino acids and/or amino acid derivatives as are well known in the art.
  • a peptide is a protein comprising no more than fifty (50) contiguous amino acids and a polypeptide is a protein that comprises more than fifty (50) contiguous amino acids.
  • a "chimeric protein” is a protein that comprises at least two different amino acid sequences that are not normally present in the same protein.
  • the at least two different amino acid sequences may be contiguous or non-contiguous in the chimeric protein.
  • the chimeric protein comprises respective amino acid sequences of two or three of SEQ ID NOS: 1-3, or of fragments of SEQ ID NOS: 1-3.
  • Particular embodiments of a chimeric protein comprise an amino acid sequence set forth in SEQ ID NO:4 or SEQ ID NO:5.
  • a protein "fragment” may be an epitope, sub-sequence, domain or other portion of a protein.
  • the fragment is an immunogenic fragment. Fragments may comprise no more than 6, 12, 15, 18, 20, 25, 30, 40, 50, 60, 78, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600 or 650 contiguous amino acids of a protein.
  • a protein "variant" may be a homolog, ortholog, allelic variant, polymorphic variant, mutant or artificially modified form of a protein disclosed herein. Artificial modification may be performed using recombinant DNA mutagenesis, chemical mutagenesis or by chemical synthesis, although without limitation thereto.
  • the protein variant may comprise an amino acid sequence at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of any one of SEQ ID NOS: 1-5.
  • the variant is immunogenic.
  • Amino acid sequence similarity and identity may be defined with reference to: GAP (Wisconsin Package, Accelerys, San Diego USA) which uses the Needleman and Wunsch algorithm; BLAST (which uses the method of Altschul et al., 1990, J. Mol. Biol. 215 405-410); or FAST A (which uses the method of Pearson & Lipman, 1988, PNAS USA 85 2444-2448), or the Smith- Waterman algorithm (Smith & Waterman, 1981, J. Mol Biol. 147 195-197).
  • GAP Garnier et al., 1990, J. Mol. Biol. 215 405-410
  • FAST A which uses the method of Pearson & Lipman, 1988, PNAS USA 85 2444-2448
  • Smith- Waterman algorithm Smith & Waterman, 1981, J. Mol Biol. 147 195-197.
  • Particular versions of BLAST include the TBLASTN program and the psi-Blast algorithm. It is preferable to maximize the number of matches and minimize the number of
  • immunogen and “immunogenic” refer to an ability or property of a composition, protein, fragment, variant, encoding nucleic acid and/or antibody to elicit an immune response to a schistosome upon administration to a mammal.
  • an immune response is meant that upon administration to a mammal, an immunogenic protein, epitope or other fragment thereof, a nucleic acid encoding same, or an antibody or antibody fragment, stimulates, provokes, induces, potentiates or otherwise elicits an immune response to a schistosome parasite in the mammal.
  • the immune response includes an antibody response.
  • a particularly preferred immune response generates specific antibodies at a titer of about >1 to about 1 x 10 6 or greater.
  • the titer is from about 1 x 10 4 or 1 x 10 5 to about 1 x 10 6 or more, such as measured by Enzyme Linked Immunosorbent Assay (ELISA) or greater than 1,000 antibody units as defined previously (Malkin et al, 2005a; 2005b).
  • the antibody response includes IgG (e.g IgGi) and/or IgE antibodies.
  • the elicited immune response includes the generation of a T cell response such as during the acquisition of drug induced resistance (DIR) to agents such as Praziquantel (PZQ).
  • DIR drug induced resistance
  • PZQ Praziquantel
  • the elicited immune response is not necessarily protective, but may reduce, alleviate or decrease one or more symptoms of schistosome infection in the mammal.
  • a decrease in schistosome burden of a least about 30% in a mammal may occur compared to a mammal that has not received the immunogenic composition.
  • the level of decrease in parasite egg production and/or worm burden could exceed 40%, as per standards set previously by the World Health Organization.
  • the immune response is a protective immune response.
  • the immunogenic composition is preferably a vaccine.
  • vaccine is meant an immunogenic composition that elicits a protective immune response.
  • vaccinate or “immunize” is meant delivery of an immunogenic composition to a mammal to thereby elicit a protective immune response in the mammal.
  • Another aspect of the invention therefore provides a method of immunizing a mammal, said method including the step of administering an immunogenic composition disclosed herein to the mammal to thereby immunize the mammal against infection by a schistosome.
  • Still yet another aspect of the invention provides a method of preventing or treating a schistosome infection in a mammal, said method including the step of administering an immunogenic composition disclosed herein to the mammal to thereby prevent or treat a schistosome infection in the mammal.
  • treating refers to a therapeutic intervention that ameliorates a sign or symptom of a schistosome infection after it has begun to develop.
  • ameliorating with reference to a schistosome infection, refers to any observable beneficial effect of the treatment.
  • the beneficial effect can be determined using any methods or standards known to the ordinarily skilled artisan.
  • preventing refers to a course of action initiated prior to the onset of a symptom, aspect, or characteristic of schistosome infection so as to prevent or reduce the symptom, aspect, or characteristic. It is to be understood that such preventing need not be absolute to be beneficial to a mammalian subject.
  • a "prophylactic” treatment is typically administered to a mammalian subject who is not infected with schistosomes, does not exhibit signs of schistosome infection or exhibits only early signs or symptoms consistent with a schistosome infection.
  • a vaccine, vaccination or method of immunization is an example of a "prophylactic" composition or treatment.
  • immunization, prevention or treatment of schistosomiasis may be facilitated by administration of immunogenic compositions comprising one or more antibodies or antibody fragments that bind and/or are raised against one or more isolated proteins, such as according to SEQ ID NOS: 1-5, or fragments or variants thereof.
  • immunogenic compositions comprising one or more antibodies or antibody fragments that bind and/or are raised against one or more isolated proteins, such as according to SEQ ID NOS: 1-5, or fragments or variants thereof.
  • Such antibodies may confer or invoke passive immunity to schistosomes upon administration to a mammal.
  • Preferred antibodies include IgG (e.g. IgGi) and IgE antibodies.
  • the antibodies may be polyclonal or monoclonal as are well understood in the art and include synthetic antibody constructs such as diabodies and triabodies.
  • Antibody fragments may include Fab, F(ab') 2 and single chain fragments such as scFv fragments. It will also be appreciated that antibodies may be recombinant antibodies and include antibodies of non-human origin that have been "humanized” to optimize administration to humans.
  • isolated proteins such as comprising an amino acid sequence according to SEQ ID NOS: 1-5, fragments or variants thereof, encoding nucleic acids and/or antibodies thereto may be present in an immunogenic composition and/or administered in combination with a pharmaceutically- acceptable carrier, diluent or excipient.
  • the pharmaceutically-acceptable carrier, diluent or excipient is suitable for administration to mammals, and more preferably, to humans.
  • pharmaceutically-acceptable carrier diluent or excipient
  • a solid or liquid filler diluent, binder or encapsulating substance that may be safely used in systemic administration.
  • a variety of carriers well known in the art may be used.
  • These carriers may be selected from a group including sugars, starches, cellulose and its derivatives, malt, gelatine, talc, calcium sulfate, vegetable oils, synthetic oils, polyols such as glycerol, alginic acid, phosphate buffered solutions, dextrose, alcohols such as ethanol, zwiterrionic detergents emulsifiers, isotonic saline and salts such as mineral acid salts including hydrochlorides, bromides and sulfates, organic acids such as acetates, propionates and malonates, and pyrogen-free water.
  • polyols such as glycerol, alginic acid, phosphate buffered solutions, dextrose, alcohols such as ethanol, zwiterrionic detergents emulsifiers, isotonic saline and salts such as mineral acid salts including hydrochlorides, bromides and sulfates, organic acids such as acetates,
  • Any safe route of administration may be employed for providing a subject with compositions comprising one or more isolated proteins such as according to SEQ ID NOS: 1-5, or fragment or variants thereof.
  • oral, rectal, parenteral, sublingual, buccal, intravenous, intra-articular, intra-muscular, intra-dermal, subcutaneous, inhalational, intra-nasal, intraocular, intraperitoneal, intracerebroventricular, transdermal, and other routes may be employed.
  • Dosage forms may include tablets, dispersions, suspensions, injections, solutions, syrups, troches, capsules, suppositories, aerosols, transdermal patches, and the like. These dosage forms may also include injecting or implanting controlled releasing devices designed specifically for this purpose or other forms of implants modified to act additionally in this fashion. Controlled release of one or more isolated proteins such as according to SEQ ID NOS: 1-5, or a fragment or variant thereof, may be effected by coating the same, for example, with hydrophobic polymers including acrylic resins, waxes, higher aliphatic alcohols, polylactic and polyglycolic acids, and certain cellulose derivatives such as hydroxypropylmethyl cellulose. In addition, the controlled release may be affected by using other polymer matrices, liposomes and/or microspheres.
  • compositions may be administered in a manner compatible with the dosage formulation, and in such amount as is pharmaceutically/therapeutically- effective.
  • the dose administered to a subject should be sufficient to effect a beneficial response (e.g., prevention or treatment of schistosomiasis) in a subject over an appropriate period of time.
  • the quantity of one or more isolated proteins comprising an amino acid sequence according to SEQ ID NOS: 1-5, or a fragment or variant thereof, to be administered may depend on the subject to be treated, inclusive of the age, sex, weight and general health condition thereof, factors that will depend on the judgement of a practitioner of ordinary skill in the art.
  • isolated proteins comprising an amino acid sequence according to SEQ ID NOS: 1-5, fragments or variants thereof, encoding nucleic acids and/or antibodies or antibody fragments, may be present in an immunogenic composition and/or administered in combination with an immunostimulatory agent.
  • An immunostimulatory agent may be an adjuvant, nucleic acid, bacterial toxin, cytokine or other immunoregulatory molecule that enhances the immunogenicity of the isolated proteins comprising an amino acid sequence according to SEQ ID NOS: 1-5, fragments or variants thereof, or encoding nucleic acids, upon administration to a mammalian subject.
  • Non-limiting examples of immunostimulatory agents include squalane and squalene (or other oils of plant or animal origin); block copolymers; detergents such as Tween®-80; Quil® A, mineral oils such as Drakeol or Marcol, vegetable oils such as peanut oil; Corynebacterium-derived adjuvants such as Corynebacterium parvum; Propionibacterium-derived adjuvants such as Propionibacterium acne; Mycobacterium bovis (Bacille Calmette and Guerin or BCG); Bordetella pertussis antigens; tetanus toxoid; diphtheria toxoid; surface active substances such as hexadecylamine, octadecylamine, octadecyl amino acid esters, lysolecithin, dimethyldioctadecylammonium bromide, N,N-dicoctadecy1-N
  • Suitable adjuvants include but are not limited to an aluminum- based adjuvant, CpG and Synthetic Lipid A.
  • Other immunological agents administrable together with the isolated nucleic acids and/or nucleic acids disclosed herein may include carrier proteins such as thyroglobulin; albumins such as human serum albumin; toxins, toxoids or any mutant cross-reactive material (CRM) of the toxin from tetanus, diphtheria, pertussis, Pseudomonas, E. coli, Staphylococcus, and Streptococcus; polyamino acids such as poly(lysine: glutamic acid); influenza; Rotavirus VP6, Parvovirus VP1 and VP2; hepatitis B virus core protein; hepatitis B virus recombinant vaccine and the like.
  • carrier proteins such as thyroglobulin
  • albumins such as human serum albumin
  • an isolated nucleic acid may encode one or a plurality of isolated protein comprising an amino acid sequence set forth in any one of SEQ ID NOS: 1-5, or a fragment thereof.
  • An aspect of the invention provides an isolated nucleic acid encoding an amino acid sequence set forth in SEQ ID NO: 4 or 5, or a chimeric fragment thereof.
  • nucleic acid may be single- or double-stranded DNA inclusive of cDNA and genomic DNA or RNA inclusive of mRNA, tRNA and inhibitory RNA (e.g interfering RNA such as siRNA), although without limitation thereto.
  • inhibitory RNA e.g interfering RNA such as siRNA
  • the isolated nucleic acid may be suitable for recombinant expression of an isolated protein disclosed herein.
  • the isolated nucleic acid may be administered to a mammalian subject to elicit an immune response to a schistosome in the mammalian subject.
  • the isolated nucleic acid may be present in a genetic construct.
  • a genetic construct may comprise the isolated nucleic acid operably linked or connected to one or more other nucleotide sequences.
  • Such nucleotide sequences may include regulatory nucleotide sequences such as promoters, enhancers, polyadenylation sequences, splice sites, translation initiation or termination sequences, antibiotic resistances genes and selection marker genes although without limitation thereto. Promoters are typically selected according to a host cell for intended expression of the encoded isolated protein, such as yeast, bacterial, insect, plant or mammalian host cells.
  • Fusion partner or epitope tag sequences may also be added, such as hexahistidine, MBP, GST, haemagglutinin, FLAG and/or c-myc sequences.
  • the genetic construct is suitably manipulated, propagated and/or expressed in a host cell engineered or manipulated to comprise the genetic construct.
  • host cells may include yeast, bacterial, insect, plant or mammalian host cells, although without limitation thereto.
  • genetic constructs may be, or comprise, viral vectors that comprise one or more regulatory nucleotide sequences from vaccinia, adenovirus, adenovirus- associated viruses (AAV), retroviruses, lentiviruses, herpes simplex virus or cytomegalovirus, although without limitation thereto.
  • AAV adenovirus-associated viruses
  • retroviruses lentiviruses
  • herpes simplex virus or cytomegalovirus although without limitation thereto.
  • Immunization with DNA or "DNA vaccination” is well known in the art and a non-limiting overview is provided in Ferraro et al, 2011, Clin. Infect. Dis. 53 296.
  • compositions and/or methods disclosed herein may be suitable for immunizing, preventing or treating one or more diseases or conditions caused by, or associated with, a schistosome infection.
  • Diseases or conditions caused by, or associated with, S. mansoni and S. japonicum schistosomiasis include Katayama fever, hepatic perisinusoidal egg granulomas, Symmers' pipe stem periportal fibrosis, portal hypertension, and occasional embolic egg granulomas in brain or spinal cord.
  • Diseases or conditions caused by, or associated with, S. haematobium schistosomiasis includes hematuria, scarring, calcification, squamous cell carcinoma, and occasional embolic egg granulomas in brain or spinal cord.
  • haematobium eggs at least one egg found in at least one of 3 urine samples, each collected on a separate day
  • urinalysis were treated with PZQ by weight (40 mg/kg) and then assessed by urinalysis at 6 weeks to confirm clearance of the infection (no eggs found in any of 3 urine samples, each collected on a separate day).
  • Individuals were followed for 18 months and maintained regular water contact throughout this period.
  • Subjects were assessed for infectivity with S. haematobium at 6 months and at the end of the study.
  • Rhesus macaques anaesthetized with ketamine hydrochloride (6 mg/kg body weight, Gutian Pharmaceutical Corporation, Fujian, China) were infected percutaneously with 600 cercariae via the shaved abdominal skin for 30 minutes. Blood was obtained by intravenous sampling prior to infection (week 0) and at 12 and 20 weeks after exposure. Elimination of infection was confirmed at week 20 by assessment of eggs per gram of feces using both the Percoll technique (45) and Kato-Katz method (46).
  • Human IgGl and IgE responses to antigens were determined by probing arrays with sera as previously described (20).
  • Macaque antibody responses were determined by probing of arrays with sera as described for human sera with the exception that a goat anti-monkey IgG- biotin (1 :500) (Sigma) secondary antibody was used.
  • Array data analysis was conducted using the "group average” method (20), where the mean signal intensity (SI) of the negative control (empty vector) spots for all sera were subtracted from the SI of each protein spot.
  • SI mean signal intensity
  • the following reactivity cutoffs (calculated as one standard deviation above the negative control spots for all groups) were used: human IgGl - 8239; human IgE - 1861; macaque IgG - 3210.
  • Statistical analyses (Student's t test) were conducted with Graphpad Prism 6 to determine significant differences between samples for a given reactive protein.
  • IgGl responses of the CI cohort to reactive proteins before and after PZQ treatment were not significantly different for any protein (data not shown).
  • IgE responses in the DIR group were significantly lower at 18 months post-PZQ treatment compared to baseline for the majority (78%) of the 18 reactive antigens ( Figure 2B).
  • Arrayed antigens that were the targets of IgE in post-treatment DIRs included AY814430 (calpain), AY812195 (extracellular superoxide dismutase [SOD]) and AY814497 (Na + /K + ATPase ⁇ subunit - SNaKlp).
  • Homologues and/or family members of known schistosome vaccine candidates such as calpain (50) (AY814430), a 28 kilodalton glutathione- s-transferase (GST) - S/z28GST (51) (AY815303) and the tetraspanins (TSP)s Sm-TSP-1 and Sm-TSP-2 (33, 52) (AY815196) were also identified.
  • Table 1 lists all of the antigens depicted in Figure 3 along with their S. haematobium orthologues as we reasoned that these were probably the native parasite antigens that our DIR and CI sera were targeting during the course of S. haematobium infection.
  • Of the 20 antigens that were targets of significantly elevated IgGl responses in post-treatment DIRs compared to CIs only 7 (35%) were targets of IgE responses that were deemed to be above the reactivity cutoff (Figure 4).
  • IgGl is one of the main drivers of the protective humoral response to schistosomiasis (23, 24), an observation supported by studies showing that key tegument vaccine antigens like Smp80 (calpain), Sm-TSP-2 and Sm29 are the targets of these responses in schistosome-resistant individuals (32, 33, 55).
  • IgE is thought to be critical in resistance to schistosomiasis, including the DIR process (25, 56, 57), but caution is warranted in development of anti-helminth vaccines that drive IgE responses due to potential anaphylactic responses in individuals who are pre- sensitized from chronic helminth infection/exposure (58).
  • Sh28GST a homologue of the arrayed immunoreactive protein AY815303 is a multi-functional enzyme present in the tegument and sub-tegument of adult (61) and larval (62) schistosomes and the current focus of vaccine trials in humans (51).
  • TSP-based vaccines have shown to be efficacious against schistosomiasis with Sm-TSP-l and Sm-TSP-2 (33, 52) and 5 23 (68, 69) conferring protection in animal challenge models.
  • IgGi-reactive proteins whose therapeutic potential has not yet been examined include mastin (AY812951) and a MARVEL domain-containing lipid- raft associated protein (AY815056).
  • Mastin is a trypsin-like serine protease and, in schistosomes, proteases of this class are known as cercarial elastases (CEs) for their role in skin degradation to facilitate penetration of the free-living cercaria into the definitive host (75).
  • CEs Cerarial elastases
  • Mastin differs in structural homology to CEs and has been assigned to a group of "non-CE" serine proteases (76).
  • mastin is unique in that it is highly upregulated in the intra-mammalian schistosomula and adult stages (60% and 150% relative to the constitutively-expressed smcoxl, respectively (76)) compared to the free-living stages of the parasite (77, 78), alluding to a fundamental parasitic function.
  • MARVEL domains have a four-transmembrane helix architecture and proteins containing these motifs associate with membrane micro-domains and have been implicated in membrane biogenesis (79).
  • the MARVEL domain-containing protein Nee 102 regulates actin organization and invasive growth of Candida albicans, with Nee 102 deletion mutants showing decreased virulence in mice (80).
  • Antigens such as mastin and the MARVEL domain protein are attractive vaccine candidates for the reasons described herein as well as the successful use of proteases (81-83) and membrane structural proteins as anti-helminth vaccines (eg: (33, 69, 84, 85)).
  • Ribosome-associated proteins were also the targets of significantly higher IgGi responses in DIRs compared to CIs post-treatment and included ribosome-binding protein 1.
  • Ribosome-associated proteins have received attention in the field of parasite immunology because of their classification as "patho-antigens" - conserved intracellular molecules capable of inducing an immunopathological response (86).
  • Patho-antigens such as acidic ribosomal protein P0 conferred protection as vaccines against the intracellular parasites Leishmania major (86) and Plasmodium yoelii (87) in mouse challenge models of infection, and antibodies to P. falciparum P0 have been detected in individuals who are immune to malaria (88).
  • ribosome-binding protein 1 The roles of these antigens, such as ribosome-binding protein 1, in the induction of anti- schistosome immunity is unclear, but it is possible that these intracellular molecules are stimulating host immune effectors through exo some-mediated pathways (recently identified in related helminths (89, 90)). It should also be noted that ribosome-binding protein 1 was one of the two antigens recognised by both S. mansoni-exposed PR subjects in Brazil and S. haematobium-exposed DIR subjects in Africa ( Figure 6), possibly highlighting a common role in different mechanisms of schistosomiasis resistance.
  • IgE responses to arrayed antigens were, for the most part, significantly weaker in post-therapy DIRs compared to pre-treatment responses, which appears to be in contrast to the positive association between IgE levels and the process of acquiring DIR status (25, 56). This could be likely for 2 reasons: (1) these earlier studies on DIR employed soluble antigen preparations to detect IgE responses, whereas the majority of arrayed proteins are membrane-associated and therefore would not have been present in buffer-soluble parasite extracts or (2) the DIR cohort, being egg negative, do not receive the IgE-inducing stimulus of egg antigens (91).
  • IgE poses somewhat of a conundrum for helminth vaccinologists due to its clear association with naturally acquired protection (22, 57), but the accompanying risk of vaccinating people with a recombinant protein that is the target of pre-existing IgE responses poses the risk of inducing atopy (58), or potentially anaphylaxis.
  • IgGi potentially protective IgGi -inducing antigens that are the targets of parasite-derived IgE in exposed individuals from further vaccine development
  • Another strategy aimed at minimising potential allergenicity of helminth proteins involves their fusion to Fey, thereby directing the chimeric protein to the negative signalling receptor Fc ⁇ RIIb expressed on pro-allergic cells (94).
  • Hotez PJ Bethony JM, Diemert DJ, Pearson M, Loukas A (2010) Developing vaccines to combat hookworm infection and intestinal schistosomiasis. Nature Rev Microbiol 8: 814-826.
  • Gazzinelli A Bethony J, Fraga LA, LoVerde PT, Correa-Oliveira R, Kloos H (2001) Exposure to Schistosoma mansoni infection in a rural area of Brazil. I: water contact. Trop Med Int Health 6: 126-135.
  • Chimeric protein comprising amino acid sequence from AY815196, AY812951 and AY810792

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Abstract

L'invention concerne une composition immunogène comprenant une ou une pluralité de protéines isolées comprenant les séquences d'acides aminés respectives présentées dans SEQ ID NOS: 1 à 3, ou un fragment ou un variant de ces dernières ; une ou une pluralité d'acides nucléiques isolés codant pour SEQ ID NOS: 1 à 3, ou un fragment ou un variant de ces derniers ; ou un anticorps qui se lie à une ou une pluralité de protéines isolées comprenant les séquences d'acides aminés respectives présentées dans SEQ ID NOS: 1 à 3, ou un fragment ou un variant de ces dernières. La protéine isolée peut être une protéine chimérique comprenant deux séquences ou plus parmi SEQ ID NOS: 1 à 3. La composition immunogène suscite, de préférence, une réponse immunitaire protectrice contre les schistosomes chez un mammifère. En conséquence, des procédés permettant de susciter une réponse immunitaire, une immunisation, la prévention et/ou le traitement d'infections par schistosome sont décrits.
PCT/AU2016/050290 2015-04-25 2016-04-22 Vaccin contre la schistosomiase WO2016172762A1 (fr)

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Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
NL2023863B1 (en) 2019-09-20 2021-05-25 Univ Griffith Protein particles and uses thereof
CN114751970A (zh) * 2022-03-30 2022-07-15 中国医学科学院病原生物学研究所 日本血吸虫抗原蛋白rSjScP15及其应用
CN114805524A (zh) * 2022-03-30 2022-07-29 中国医学科学院病原生物学研究所 日本血吸虫抗原蛋白rSjScP92及其应用
CN115043922A (zh) * 2022-03-30 2022-09-13 中国医学科学院病原生物学研究所 日本血吸虫抗原蛋白rSjScP57及其应用

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BENTLEY G. N. ET AL.: "Mapping and sequencing of acetylcholinesterase genes from the platyhelminth blood fluke Schistosoma", GENE, vol. 314, 2003, pages 103 - 112, XP004460844 *
JONES A. K. ET AL.: "Molecular characterisation of an acetylcholinesterase implicated in the regulation of glucose scavenging by the parasite Schistosoma", THE FASEB JOURNAL, vol. 16, 2002, pages 441 - 443, XP055326297 *
LIU F. ET AL.: "New perspectives on host-parasite interplay by comparative transcriptomic and proteomic analysis of Schistosoma japonicum", PLOS PATHOGENS, vol. 2, no. 4, 2006, pages e29, XP009149773 *
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Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
NL2023863B1 (en) 2019-09-20 2021-05-25 Univ Griffith Protein particles and uses thereof
CN114751970A (zh) * 2022-03-30 2022-07-15 中国医学科学院病原生物学研究所 日本血吸虫抗原蛋白rSjScP15及其应用
CN114805524A (zh) * 2022-03-30 2022-07-29 中国医学科学院病原生物学研究所 日本血吸虫抗原蛋白rSjScP92及其应用
CN115043922A (zh) * 2022-03-30 2022-09-13 中国医学科学院病原生物学研究所 日本血吸虫抗原蛋白rSjScP57及其应用
CN115043922B (zh) * 2022-03-30 2023-09-15 中国医学科学院病原生物学研究所 日本血吸虫抗原蛋白rSjScP57及其应用

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