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WO2024250024A2 - Vaccin pour chats pour bloquer la libération et la transmission de toxoplasma oocyst - Google Patents

Vaccin pour chats pour bloquer la libération et la transmission de toxoplasma oocyst Download PDF

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Publication number
WO2024250024A2
WO2024250024A2 PCT/US2024/032292 US2024032292W WO2024250024A2 WO 2024250024 A2 WO2024250024 A2 WO 2024250024A2 US 2024032292 W US2024032292 W US 2024032292W WO 2024250024 A2 WO2024250024 A2 WO 2024250024A2
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Prior art keywords
parasite
ift88
toxoplasma gondii
gondii
srs15b
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WO2024250024A3 (fr
Inventor
Michael E. GRIGG
Viviana PSZENNY
Aline SARDINHA DA SILVA
Jitender P. Dubey
Julius LUKES
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Biologicke Centrum Av Cr V V I
US Department of Agriculture USDA
US Department of Health and Human Services
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Biologicke Centrum Av Cr V V I
US Department of Agriculture USDA
US Department of Health and Human Services
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Publication of WO2024250024A3 publication Critical patent/WO2024250024A3/fr
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/01—Preparation of mutants without inserting foreign genetic material therein; Screening processes therefor
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/66—Microorganisms or materials therefrom
    • A61K35/68—Protozoa, e.g. flagella, amoebas, sporozoans, plasmodium or toxoplasma
    • 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
    • A61K39/012—Coccidia antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/52—Bacterial cells; Fungal cells; Protozoal cells
    • A61K2039/522—Bacterial cells; Fungal cells; Protozoal cells avirulent or attenuated
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/55—Medicinal preparations containing antigens or antibodies characterised by the host/recipient, e.g. newborn with maternal antibodies
    • A61K2039/552—Veterinary vaccine
    • 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
    • A61K2039/57—Medicinal preparations containing antigens or antibodies characterised by the type of response, e.g. Th1, Th2
    • A61K2039/575—Medicinal preparations containing antigens or antibodies characterised by the type of response, e.g. Th1, Th2 humoral response

Definitions

  • This patent application claims the benefit of U.S. Provisional Patent Application No. 63/470,773 filed June 2, 2023, which is incorporated by reference.
  • STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT [0002] This invention was made with Government support under project number AI001018 by the National Institutes of Health, National Institute of Allergy and Infectious Diseases. The Government has certain rights in the invention.
  • Toxoplasmosis is a major public health concern and also economically disruptive to agriculture involving livestock. Toxoplasma gondii is the zoonotic causative agent of toxoplasmosis.
  • Toxoplasma can infect almost any cell type found in mammals and birds. It has been estimated that more than 30% of the world population over the age of six has been infected with T. gondii, with some regions having over 60% of the population test seropositive. There are multiple transmission pathways, including consumption of undercooked meat from infected animals, consumption of unwashed plants, contaminated water supplies, blood transfers, and congenital transfer. Additionally, direct or indirect transmission can occur via contact with the stool of infected felids. Leydig 771108 E-118-2023-2-PC-01 2 [0005] Transmission of Toxoplasma in nature is highly dependent on the parasite’s sexual cycle, which occurs exclusively in felids.
  • T. gondii the molecular pathways promoting sexual stage development in T. gondii are largely unknown. Transmission of T. gondii to cats is almost entirely through the consumption of infected prey. The parasite can undergo sexual reproduction in the GI tracts of cats (in other animals, reproduction occurs asexually). T. gondii generates oocysts which are expelled in the stool. These infectious oocysts containing sporozoites are extremely hardy (they are kept in weak sulfuric acid for long term storage in laboratories) and persist in the environment for many years. In moist environments, oocysts undergo sporulation.
  • sporulated oocysts can then be consumed by animals through consumption of unwashed grass or other plants grown in the infected soil, or by consumption of contaminated water.
  • the parasite will find its way to tissues and form a cyst.
  • infected cats can shed high numbers of persistent and infectious T. gondii oocysts, which are capable of causing toxoplasmosis outbreaks in both animals and people.
  • the parasite is capable of infecting anyone (healthy and immunocompromised individuals).
  • the cysts persist in the dormant phase and are present in the muscles and in the brain of infected individuals.
  • Toxoplasmosis can present with fever, swollen lymph nodes, headaches, muscle aches, and skin rash. In some cases, Toxoplasma can infect the eye. In individuals with weakened immune systems, Toxoplasma can affect the lungs or the brain. Toxoplasmosis also is a significant concern for pregnant persons. Toxoplasma can induce a miscarriage or cause medical problems which will only be apparent at birth. These problems can include hydrocephalus, eye infections, brain tissue anomalies, or an enlarged liver or spleen. Further, the infant may present with developmental issues, blindness, hearing loss, seizures, heart disorders, jaundice, or a rash.
  • Toxoplasmosis In livestock, Toxoplasmosis is typically subclinical due to control by the host immune system, and treatment often is unnecessary. In young animals whose immune systems are not robust, however, Toxoplasmosis can cause a host of issues across a range of tissues, in some cases resulting in death. T. gondii is an important cause of abortion and stillbirth in sheep, goats, Leydig 771108 E-118-2023-2-PC-01 3 cervids, and sometimes pigs.
  • TOXOVAX® is only available for use in the United Kingdom, New Zealand, France, and Ireland.
  • the invention provides a recombinant parasite, which is a member of the phyla Apicomplexa or Euglenozoa, and which comprises a genome which lacks a native gene homologous to (including identical to if within Toxoplasma gondii or homologs in species other than T. gondii) T. gondii IFT88, one or more genes from the T. gondii tandem SRS15 array (e.g., SRS15A, SRS15B, and/or SRS15C), T. gondii SRS26B, or a combination of two or more thereof.
  • T. gondii tandem SRS15 array e.g., SRS15A, SRS15B, and/or SRS15C
  • T. gondii SRS26B or a combination of two or more thereof.
  • the invention also provides a method for producing the recombinant parasite, which involves knocking out T. gondii IFT88, one or more genes from the T. gondii SRS15 array (e.g., SRS15A, SRS15B, and/or SRS15C), T. gondii SRS26B, or homologs thereof (such as in species other than T. gondii), or a combination of two or more thereof from the genome of the parasite.
  • Reagents for accomplishing this are provided, as are vaccine compositions comprising the inventive recombinant parasites and vaccine compositions comprising T. gondii IFT88, one or Leydig 771108 E-118-2023-2-PC-01 4 more genes from the T.
  • gondii SRS15 array e.g., SRS15A, SRS15B, and/or SRS15C
  • T. gondii SRS26B T. gondii, including homologs thereof (such as in species other than T. gondii), or a combination of two or more thereof.
  • Methods of vaccinating animals using such vaccine compositions also are provided.
  • the invention facilitates the generation of live, attenuated oral vaccines and/or nanoparticle oral or mucosal vaccines that can be employed by caregivers of cats worldwide to reduce the burden of Toxoplasmosis globally.
  • vaccination of stray cats in regions were T.
  • gondii transmission by oocysts is high, through the use of the inventive attenuated oral vaccines and/or nanoparticle oral or mucosal vaccines, can further reduce the transmission of T. gondii in food and water sources destined for human consumption or consumption by livestock (such as sheep, goats, cervids, pigs, and others potentially infected with parasites of the phylum Apicomplexa).
  • Figure 1 presents data concerning cumulative oocyst yield (left panel) and serology titer (middle panel) among experimental cats challenged with bradyzoites either from wildtype (“WT”) CZ1, a knock-out (“KO”) strain deficient for IFT88 (“IFT88-KO”), or IFT88-KO complemented with a heterologous allele of IFT88.
  • WT wildtype
  • KO knock-out
  • IFT88-KO a knock-out strain deficient for IFT88
  • IFT88-KO IFT88-KO complemented with a heterologous allele of IFT88.
  • the micrograph shows Toxoplasma oocysts obtained from floating fecal pellets from cats infected with WT CZ1.
  • Figure 2 presents data concerning cumulative oocyst yield (left panel) and serology titer (middle panel) among cats first challenged with bradyzoites from WT CZ1 or SRS15B-KO. Cats infected with WT CZ1 shed oocysts, but SRS15B-KO infected cats did not. Both cats seroconverted, indicating that they generated robust titers of anti-Toxoplasma-specific IgG. Immunized cats were then re-infected with WT CZ1 (representing a secondary, homologous challenge) at 6 months post first infection to assess protection from oocyst shedding.
  • the SRS15B-KO vaccinated cats were immune to secondary challenge with WT CZ1 bradyzoites and did not shed oocysts.
  • the WT CZ1 infected cats were re-infected with WT CZ1 Leydig 771108 E-118-2023-2-PC-01 5 bradyzoites, and did not shed oocysts, as expected.
  • the micrograph (right panel) shows Toxoplasma oocysts obtained from floating fecal pellets from cats infected with WT CZ1, which, as noted above, is one method used to quantify oocyst numbers defecated from cats during their patency period of secretion.
  • the invention provides a recombinant parasite (i.e., a live organism), which is a member of the phyla Apicomplexa or Euglenozoa, which comprises a genome which lacks native genes corresponding to or homologous to T. gondii IFT88, one or more genes from the T. gondii SRS15 array (e.g., SRS15A, SRS15B, and/or SRS15C), T. gondii SRS26B, or a combination of two or more thereof, or a combination of two or more thereof.
  • the inventive recombinant parasite can be an organism from any genus within these phyla, such as being a member of the genera Toxoplasma, Cryptosporidium, Neospora, or Sarcocystis. While the experiments reported herein involve a cosmopolitan Type II strain (CZ1) from the species T. gondii, the invention can be applied to other strains of T. gondii as well.
  • the inventive recombinant parasite comprises a genome, which is recombinant through being engineered to lack a native genetic sequence encoding a homolog of T. gondii IFT88, one or more genes from the T.
  • gondii SRS15 array e.g., SRS15A, SRS15B, and/or SRS15C
  • SRS26B or a combination of two or more thereof.
  • native in this context is meant that the recombinant parasite lacks a gene that naturally occurs in a wildtype organism from which the inventive parasite is derived. This is in contradistinction to organisms which may not natively possess a homolog (ortholog) of T. gondii IFT88, one or more genes from the T. gondii SRS15 array (e.g., SRS15A, SRS15B, and/or SRS15C), SRS26B, or a combination of two or more thereof. [0016] Within T.
  • the IFT88 gene encodes a protein involved in intraflagellar transport. Without wishing to be bound by theory, it is believed that due to the absence of the IFT88 gene product, male gametes of the inventive recombinant parasite cannot effectively swim, which thus prevents them from contacting and fertilizing female gametes and the subsequent production of oocysts.
  • SRS15B is a 6-CYS fold surface antigen Leydig 771108 E-118-2023-2-PC-01 6 found at the interface between the male and female gametes.
  • SRS15B is part of a tandem array of three genes that have the 6-CYS fold established in plasmodium to promote male-female gamete recognition and fertilization (see, e.g., He et al.2002. Nat Struct Biol 9:606-611. PMID: 12091874 and Tonkin et al.2013.
  • the inventive recombinant parasite is engineered to be unable to reproduce sexually, i.e., to produce oocysts by the definitive host (felines).
  • the inventive recombinant parasite is nonetheless immunogenic within host animals.
  • the inventive recombinant parasite can serve as a vaccine for cats.
  • T. gondii such can be orally or mucosally administered to a felid (e.g., a domestic or wild cat), which, as noted above, is the host animal necessary for sexual reproduction of T. gondii.
  • felids inoculated with recombinant T As demonstrated in the Examples below, felids inoculated with recombinant T.
  • IFT88 in particular is highly conserved throughout Apicomplexa as well as other parasitic protozoa in other phyla, such as the Euglenozoa (i.e. Leishmania spp).
  • IFT88 ortholog sequences have been reported for organisms such as Hammondia hammondi (HHA_207410), Eimeria spp.
  • ETH2_0205400 Sarcocystis neurona (SN3_00600560 and SN3_00600565), and Neospora caninum (XP_003879797.1), among others.
  • gondii IFT88 in other parasite members of the phyla Apicomplexa and Euglenozoa can, in certain embodiments, similarly impede the reproduction of recombinant parasites.
  • the genomes of species from these phyla also have native homologs or orthologs of one or more genes from the T.
  • gondii SRS15 array e.g., SRS15A, SRS15B, and/or SRS15C
  • T. gondii SRS26B these likewise can be targeted in such other species.
  • inventive recombinant parasites drawn from genera, such as Cryptosporidium, Eimeria, Hammondia, Neospora, and Sarcocystis, in addition to Toxoplasma similarly can serve as vaccines for use in their host animals required for sexual reproduction. [0019]
  • inventive recombinant parasite comprising a genome lacking a native homolog of T.
  • CRISPR-Cas systems employ a tracrRNA which plays a role in the maturation of crRNA.
  • the tracrRNA is partially complementary to and base pairs with a pre-crRNA forming an RNA duplex.
  • CRISPR CRISPR mediated genome editing
  • Cas9 endonuclease
  • the Cas9 nickase enzyme is co-transfected with a guide RNA (“gRNA”) to effectuate gene editing.
  • gRNA guide RNA
  • enzymes other than Cas9 such as, for example Cas12a
  • CRISPR technology is well known to persons of ordinary skill in the art, and any suitable protocol can be employed in the context of the present invention. As information concerning the genetic sequences for T.
  • gondii IFT88, SRS15 array, and SRS26B homologs is known (see, e.g., SEQ ID Nos: 6-9 and 18-20 herein), suitable gRNAs for use in targeting CRISPR-Cas9 (or other suitable CRISPR system) to knock out all or a portion of the native T. gondii genes encoding IFT88, a gene within the SRS15 array, SRS26B, or a combination of two or more thereof (including their homologs and orthologs) can readily be designed by persons of ordinary skill in the art.
  • Non-limiting examples of sequences for constructing CRISPR-Cas9 gRNAs for knocking out genes encoding IFT88, a gene within the SRS15 array, and SRS26B homologs in T. gondii are provided herein as SEQ ID Nos:10-12 and 21-14.
  • Leydig 771108 E-118-2023-2-PC-01 8 [0021]
  • other methods for gene editing can be employed as alternatives to CRISPR to generate embodiments of the inventive recombinant parasite comprising a genome lacking a native homolog of a gene encoding either T.
  • IFT88 gondii IFT88
  • SRS15 array e.g., SRS15A, SRS15B, and/or SRS15C
  • SRS26B or a combination of two or more thereof (applicable as well for embodiments in which the inventive cell lacks functional expression of IFT88, SRS15 array (e.g., SRS15A, SRS15B, and/or SRS15C), and/or SRS26B homologs).
  • Such methods include those employing transcription activator like effector nucleases (TALENs) and the use of Zinc finger proteins, for example.
  • TALENs transcription activator like effector nucleases
  • Zinc finger proteins for example.
  • standard methodology known to those of ordinary skill can be employed to generate recombinant parasite of the present invention.
  • TALENs are customized artificial restriction nucleases that can be readily constructed to target a known genetic sequence, using methods known to persons of ordinary skill in the art.
  • Zinc finger domains can be engineered using methods known to persons of ordinary skill in the art to target specific desired DNA sequences, which thus enables Zinc finger nucleases to target unique sequences within complex genomes to alter the chromosomal DNA of cells.
  • gondii SRS26B and their native homologs in other species, as set forth herein and otherwise known in the art, can facilitate the design and construction of TALENs and Zinc finger nucleases, such as targeting the same genome loci that gRNAs bind (see SEQ ID Nos:6-12, 18-19, and 23-24), suitable for generating the inventive recombinant parasite in which native genes encoding T.
  • gondii IFT88 one or more of the SRS15 array (e.g., SRS15A, SRS15B, and/or SRS15C), SRS26B, or a combination of two or more thereof, or their homologs in other species, are “knocked out.”
  • SRS15 array e.g., SRS15A, SRS15B, and/or SRS15C
  • SRS26B or a combination of two or more thereof, or their homologs in other species
  • approaches for removing functional copies of native genes encoding T. gondii IFT88, SRS15A, B, or C, SRS26B, or a combination of two or more thereof, or their native homologs in other species can alternatively be employed in some embodiments.
  • the genetic regulatory elements controlling expression of native genes encoding T can alternatively be employed in some embodiments.
  • gondii IFT88 SRS15 array (e.g., SRS15A, SRS15B, and/or SRS15C), SRS26B, or a combination of two or more thereof, or their homologs Leydig 771108 E-118-2023-2-PC-01 9 in other species, can be altered or removed from the parasite genomes to attenuate transcription, if one or both of their coding sequences is not “knocked out.”
  • the genetic manipulation can involve using RNA interference to block or reduce translation of native genes encoding T.
  • RNAi is generally technically problematic in Toxoplasma.
  • Persons of ordinary skill in the art will be able to design suitable interfering RNA sequences for attenuating (“knocking down”) the expression of genes encoding homologs or orthologs of IFT88, SRS15B and/or SRS26B in such species.
  • an aspect of the invention provides a vaccine composition comprising the inventive recombinant parasite and a carrier.
  • the recombinant parasite can exist in the form of an active cell, while in other embodiments, the parasite can exist in the form of a bradyzoite cyst.
  • the “carrier” within the inventive vaccine composition is any suitable vehicle to deliver the inventive recombinant parasite to the host animal needed for sexual reproduction of the respective species of inventive recombinant parasite.
  • the host animal will be a cat species, and the route of administration can suitably be oral or mucosal.
  • the inventive vaccine composition can be orally or mucosally admissible, such as being a capsule, tablet, or food substance or food additive, which the cat can ingest.
  • the formulation can be manufactured in accordance with standard methodology in pharmaceutics, and the carrier(s) can be selected from those ordinarily used to formulate pharmaceutical compositions (e.g., saline for injection, petrolatum).
  • pharmaceutical compositions e.g., saline for injection, petrolatum.
  • Leydig 771108 E-118-2023-2-PC-01 10 can include other excipients and agents as desired, such as stabilizing agents, preservatives, humectants, etc.
  • the formulation can include nanoparticles (such as virus-like particles).
  • the invention comprises a composition comprising a homolog of T. gondii IFT88, SRS15A, B and/or C, SRS26B protein (from T. gondii or another species), or a combination of two or more thereof, for use as a vaccine.
  • a homolog of T. gondii IFT88, SRS15A, B and/or C, SRS26B, or a combination of two or more thereof can be recombinantly synthesized as a secreted protein and then formulated with a suitable pharmaceutically acceptable carrier, as described herein. Specific to the T.
  • gondii IFT88, SRS15A, B and/or C, and SRS26B species-specific homologs, cDNA and/or their encoded transcripts are set forth as SEQ ID NOs: 1-4 and 17 below. Production of such proteins can be achieved by employing standard methodology.
  • the homolog of T. gondii IFT88, SRS15A, B and/or C, SRS26B, or a combination of two or more thereof can be immobilized or conjugated to supports within the composition, such as nanoparticles or virus-like particles (VLPSs) pseudotyped with the homolog of IFT88, SRS15A, B and/or C, SRS26B, or a combination of two or more thereof.
  • VLPSs virus-like particles
  • the invention provides a method of vaccinating an animal.
  • the animal to be vaccinated is one which is a competent host for sexual reproduction of the species of recombinant parasite from which the homolog of T. gondii IFT88, SRS15A, B and/or C, SRS26B, or a combination of two or more thereof is drawn.
  • the inventive recombinant parasite is T. gondii
  • the animal to be vaccinated suitably can be a felid.
  • the inventive method of vaccinating the animal involves administering the inventive composition to the animal in an amount and via a route suitable for infecting the animal.
  • the preferred route to administer the inventive composition to a cat is orally or mucosally.
  • the composition may be administered via other routes (e.g., via injection, transdermally, transmucosally, intranasally, etc., as noted above).
  • Leydig 771108 E-118-2023-2-PC-01 11 [0030] The amount of the inventive composition and inventive recombinant parasite delivered to the animal in accordance with the method of vaccinating the animal can vary, and it will depend on the animal and route of administration.
  • the dose (number or weight of parasites/ml or bradyzoite cysts/ml or per weight of the animal to be inoculated, nanoparticles or VLPs/ml or per weight of the animal to be inoculated, etc.) can be more closely titrated.
  • the lowest doses tested were 40 and 80 cysts (bradyzoites) per cat, but a dosage range of from about 40 to about 400 individuals per cat may be typically employed.
  • as few as a single cyst (bradyzoite) can lead to infection in cats and can serve as an effective dose.
  • the composition comprises the homolog of T.
  • SEQ ID NO:1 - SRS15B cDNA sequence ATGGCGCGGTCAGGAGGGATGCTGCAACGTGGTCGCGGTTTCAAGTCACGAGCCCGAAAGCTGATGGCAT TGTGCATGGGTGGAGTTTTGCTGTTTTCCGGTGGACAAGCTGCTGCAGAGCCTTGGCCTGAAGGTATGAA
  • gondii strain GAAGGTGAACCGAAGTCAAGACGAGTTATGTCGTCTGCCTTATCGTTCAGCTTTGCTATATCTCAACGAA ACGCGTGTATCTGTTTCATTACCTCTAACACCTTTCGTAGAGACTGGTCCCACTGTCTCTGtggtGCTCG CTACGCCATTTGTCAAGCCGAAGAAGAGGTCCGCTCGTTGCTCGAGGCCAGTGTAACCGCGGCAAACGAA GGGAAATCTCACGAAGCCCTTGGTGCCGGAATCCAAGCCGTTAAGAAAGAAAGACACCTTCGAAAACTAC TGAGGCGTCACCAACAGGAGAGACAGACCTCAACAAGCTCCGGAGAAGCGCATCTTTTACAGCGCCATGA TCCCGAGCTGACGTTCGCTGTTGTCCTCAACCTGGCGAACCGGCATCAAGTGAGATGAATGAGTAATCAA GAGGTTGAGGGGGCTGGGAGACTCAGCGTACTGAGCCCCTTCACTGTATATCTAAACTAGTCGATGTTAG GTAGAAATTTGAGTGGTC
  • the vaccine is an attenuated strain (CZ1) of the cosmopolitan Type II T. gondii parasite that commonly infects people and livestock.
  • Toxoplasma gondii Strain A clonal isolate of the cosmopolitan Type II CZ1 strain of Toxoplasma gondii was generated to be susceptible to drug selection using Mycophenolic Acid and Xanthine by a deletion in the Hypoxanthine-Xanthine-Guanine Phosphoribosyl Transferase (HXGPRT) gene. This strain was selected because it commonly infects both humans and livestock.
  • HXGPRT Hypoxanthine-Xanthine-Guanine Phosphoribosyl Transferase
  • the strain once engineered to be susceptible to drug selection using Mycophenolic Acid and Xanthine, was next rendered deficient in Non-Homologous End-Joining (NHEJ) repair by deletion of the KU80 gene to facilitate highly specific and efficient CRISPR/Cas9 mediated gene editing by double crossover homologous recombination.
  • NHEJ Non-Homologous End-Joining
  • the mutant isolate (“CZ1 ⁇ hpt ⁇ ku80”) was then tested to confirm cat-competency; it produced in excess of 10e 6 infectious oocysts upon challenge in seronegative cats.
  • SRS15B and IFT88 knockout Toxoplasma gondii The cat-competent CZ1 ⁇ hpt ⁇ ku80 mutant isolate was used to generate SRS15B and IFT88 knockout strains.
  • the SRS15B and IFT88 genes were replaced by the drug selectable marker HPT (HXGPRT – hypoxanthine-xanthine-guanine phosphoribosyl transferase) cassette (SEQ ID NO:5) that was flanked by 30 or 40 base pairs of homology (as indicated with brackets “[]” in the sequences set forth above) to SRS15B (SEQ ID NO:6) or to IFT88 (SEQ ID NO:7) to promote targeted deletion of the respective genes by CRISPR/Cas9 gene editing using guide RNAs specific to the homology flanks for each gene (SRS15B guide sequence: GGCCTGAAGGTATGAAACAT (SEQ ID NO:10) and IFT88 guide sequence: AAGGCAGGCACGGC
  • HFF cells Human Foreskin Fibroblast cells
  • HPT positive selection HPT positive selection
  • Leydig 771108 E-118-2023-2-PC-01 24 [0039] After four to six passages under MPA/X selection, the drug-selected parasite population was tested by PCR to confirm HPT insertion into, respectively, the SRS15B (SEQ ID NO:8) or IFT88 (SEQ ID NO:9) genomic locus, and then cloned.
  • SRS15B-KO and IFT88-KO parasites were identified by PCR of the integrated HPT into the open reading frame (“ORF”) using the following primers: SRS15B forward: GTGGTCGCGGTTTCAAGTC (SEQ ID NO:13); IFT88 forward: ATGCACGAGGGAATTGTTCT (SEQ ID NO:14), and HPT reverse: GTCTTCAATGGGTTTGGACG (SEQ ID NO:16).
  • This strategy generated SRS15B and IFT88 KO parasites; no wildtype (WT) parasites were selected out of both knockout populations.
  • mice were infected by intraperitoneal injection of 5,000 to 10,000 tachyzoites of either the SRS15B-KO or IFT88-KO parasites, which had been harvested from the infected-HFF culture flasks referred to above. Forty-two days post-infection, mouse brains containing cysts (bradyzoites) were harvested and used to feed the cats. After feeding the cats (infection), stool samples were collected up to 21 days post-infection and oocyst presence/absence was assessed by microscopy and/or PCR. [0041] The results of these experiments are presented in Table 1 and Figures 1 and 2.
  • Table 1 presents data representing cat infections with CZ1 and various engineered parasites deficient in IFT88 and SRS15B. Seroconversion after primary challenge was assessed by either a Modified Agglutination Test (MAT) or reactivity to recombinant SAG1 (a Toxoplasma-specific IgG ELISA).
  • MAT Modified Agglutination Test
  • SAG1 a Toxoplasma-specific IgG ELISA
  • FIG. 1 The micrograph (right panel of Figure 1) shows Toxoplasma oocysts obtained from floating fecal pellets from cats infected with WT CZ1, which is one method commonly used to quantify oocyst numbers defecated from cats during their patency period of secretion.
  • Figure 2 presents data concerning cumulative oocyst yield (left panel) and serology titer (middle panel) among cats first challenged with bradyzoites from WT CZ1 or SRS15B-KO.
  • the micrograph shows Toxoplasma oocysts obtained from floating fecal pellets from cats infected with WT CZ1, which, as noted above, is one method used to quantify oocyst numbers defecated from cats during their patency period of secretion.
  • RNA-Seq identified stage-specific merozoite transcripts, that are conserved across the Apicomplexa, and are thought to facilitate sexual competency. These include surface antigen genes related to 6-CYS proteins in Plasmodium, genes required to form flagella, a family of transcription factors (ApiAP2s), and a large family of secreted proteins expressed exclusively in merozoites (Families A-D). [0047] To identify genes that impact oocyst formation, a forward genetic signature-tag mutagenesis screen was undertaken, using CRISPR/Cas9 to generate a library of 192 T.
  • gondii strains that each possesses a unique barcode and are each deficient in a single gene predicted to be highly expressed in the parasite’s sexual stages. These knock-out strains were pooled, used to Leydig 771108 E-118-2023-2-PC-01 27 infect mice, then used to challenge cats with brain cysts from the infected mice to perform an input/output screen that can identify sexual stage-specific genes critical for oocyst formation. [0048] Using Mi-Seq analysis, three genes were thusly identified: IFT88, SRS15, and SRS26B, which failed to produce oocysts in cats.

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Abstract

L'invention concerne un parasite recombinant, qui est un membre du phylum Apicomplexa ou Euglenozoa, et qui comprend un génome dépourvu d'un gène natif codant pour une protéine IFT88, SRS15A, SRS15B, et/ou SRS15C, SRS26B de Toxoplasma gondii, un homologue natif ou un orthologue de celle-ci, ou une combinaison d'au moins deux de celles-ci. L'invention concerne également un procédé de production du parasite recombiné, qui consiste à inactiver un ou plusieurs de ces gènes dans le génome du parasite. L'invention concerne des réactifs pour atteindre cet objectif, ainsi que des compositions de vaccin comprenant les parasites recombinés de l'invention et des compositions de vaccin comprenant une protéine IFT88, SRS15A, SRS15B et/ou SRS15C, SRS26B de Toxoplasma gondii, ou un homologue ou un orthologue de celle-ci, ou une combinaison d'au moins deux de celles-ci. L'invention concerne également des méthodes de vaccination d'animaux à l'aide de telles compositions vaccinales.
PCT/US2024/032292 2023-06-02 2024-06-03 Vaccin pour chats pour bloquer la libération et la transmission de toxoplasma oocyst Pending WO2024250024A2 (fr)

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CN120290323A (zh) * 2025-06-13 2025-07-11 江西农业大学 一种Tgsrs51基因缺失的弓形虫减毒虫株及其应用

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CA2149197A1 (fr) * 1994-06-17 1995-12-18 Irene Popiel Production d'un vaccin efficace contre la forme merozoite de toxoplasma gondii par culture cellulaire
WO2020243675A2 (fr) * 2019-05-31 2020-12-03 Whitehead Institute For Biomedical Research Compositions et procédés de régulation d'une infection par toxoplasma chronique

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
HE ET AL., NAT STRUCT BIOL, vol. 9, 2002, pages 606 - 611
TONKIN ET AL., J BIOL CHEM., vol. 288, no. 18, 2013, pages 12805 - 17

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN120290323A (zh) * 2025-06-13 2025-07-11 江西农业大学 一种Tgsrs51基因缺失的弓形虫减毒虫株及其应用

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