Buchmann, Kurt

Jørgensen, Louise von Gersdorff

Kania, Per

Lorenzen, Niels

Sepúlveda, Dagoberto

Stratmann, Ansgar

English

Copenhagen University Research Information System

u n i ve r s i t y  o f  co pe n h ag e n  Københavns UniversitetA recombinant vaccine targeting the parasitic ciliate Ichthyophthirius multifiliisJørgensen, Louise von Gersdorff; Kania, Per; Sepúlveda, Dagoberto; Lorenzen, Niels;Stratmann, Ansgar; Buchmann, KurtPublication date:2019Document versionOther versionCitation for published version (APA):Jørgensen, L. V. G., Kania, P., Sepúlveda, D., Lorenzen, N., Stratmann, A., & Buchmann, K. (2019). Arecombinant vaccine targeting the parasitic ciliate Ichthyophthirius multifiliis. Poster session presented at The 8thCongress of the Scandinavian-Baltic Society for Parasitology (SBSP), Copenhagen, Denmark.Download date: 03. Feb. 2020A RECOMBINANT VACCINE TARGETING THE PARASITIC CILIATE ICHTHYOPHTHIRIUS MULTIFILIISL. v. G. Jørgensen δ1, P. W. Kania1, D. A. S. Araneda2, N. Lorenzen2, A. Stratmann3 and K. Buchmann11 Laboratory of Aquatic Pathobiology at the Department of Veterinary and Animal Sciences, University of Copenhagen, Frederiksberg, Denmark2 DTU AQUA, National Institute of Aquatic Resources, the Technical University of Denmark3 W42 Industrial Biotechnology GmbH, Dortmund, GermanyAbstractNew vaccine candidates were identified targeting the one celled parasite Ichthyophthirius multifiliis, which negatively affects aquaculture freshwater fish productions all over the world. In silico selection with the use of artificial intelligence identified several potential vaccine candidates and three of these were recombinantly expressed using E. coli and insect cells. Following a vaccine trial one protein (a so-called neurohypophysial n-terminal domain protein, #10) was found to induce moderate protection against I. multifiliis in rainbow trout (Oncorhynchus mykiss). To develop a highly protective heterologous vaccine we aim to combine #10 with a protective epitope from the already known homologous protective antigen Iag52b, which is a GPI-anchored cysteine rich surface protein. To be able to produce #10 at low costs, recombinant expression has been conducted in an eukaryotic host. Purified Iag52b does not induce immunity in fish without the use of adjuvants, thus the most potentially protective epitope of Iag52 was selected in silico and coupled to a virus-like particle. This coupling enables the epitope to be presented in a virus-like conformation, which theoretically should be immunogenic to the fish. Immunization 13 wpi• Blood sampling• Estimation of parasite burden• Immobilization• ELISA4 wpi• Blood sampling• ELISA9,5 wpiChallengeExperimental vaccinationBenchmark vaccineDisrupted parasitesAlhydrogelFreund’s incomplete adjuvantSubunit experimental vaccine2 hypothetical proteins1 neurohypophysial n-terminaldomain proteinAlhydrogelFreund’s incomplete adjuvantEffect of the experimental vaccinationPichia pastoris for recombinant expressionVLP displaying  #10 proteinIag52b epitope#10 proteinVLP displaying  Iag52b epitopesImproving immunogenicity of the vaccinePlasmidsSequence: VLP fused to #10 proteinSequence: VLPfused to Iag52bepitopeVLP = Virus-Like ParticleVLPs are immunogenic and can possibly replace adjuvants The improved vaccine will contain VLPs displaying both Iag52b epitopes and #10 protein and will be injected with and without adjuvant. So far we have been able to produce VLPs with Iag52b epitopes to harvest in the supernatant from Pichia pastoris but protein #10 however is withheld within the cells. We are modifying signal sequences but If we are unable to target the protein to the supernatant, cell material will instead be used for the vaccine. The overall ambition is to develop a low cost recombinant vaccine that induce a high level of protection against the economically devastating parasitic ciliate I. multifiliis without the use of adjuvants in a wide range of fresh water fish species.U N I V E R S I T Y  O F  C O P E N H A G E N ,  F A C U L T Y  O F  H E A L T H  A N D  M E D I C A L  S C I E N C E SAn already described protective antigen from the surface of I. multifiliis, called Iag52b, has not successfully been expressed recombinantly in conventional expression systems. Therefore we have identified two potentially protective epitopes in silico for recombinant expression and fusion with VLPsParasite burdenELISA showed that the group of fish injected with the experimental vaccine and the parasite homogenate erected antibodies against one of the recombinant proteins, #10 (neurohypophysial n-terminaldomain protein)Immobilization assay conducted with live parasites incubated in plasma from vaccinated fish showed that the experimental vaccine and the parasite homogenate immobilized the parasites moderately.  The parasite burden was significantly reduced in the subunit and the parasite homogenate vaccine groups. We estimated the protection to be moderate.The presented work was supported by ParaFishControl H2020 Food, Grant/Award number: 634429and the Danish Council for Independent Research (FTP), Grant/Award number: 0602-02567BVirus-like conformationThe proteome of Ichthyophthirius multifiliiswas screened using artificial intelligence in silico and 12 vaccine candidates were further investigated. Out of these, recombinant production was achieved for 7 proteins. Following a pilot study 3 proteins were selected as vaccine candidates. A total of 190 fish weredivided into five duplicated groups with 20 fish per replicate. Each fish in the recombinant vaccine group received 150 µL of vaccine consisting of equal amounts of alhydrogel and FIA with a mixture of the recombinant proteins bound to alhydrogel. Challenge was conducted by adding a suspension of theronts (10,000 theronts per fish) to the fish tank. Filtration was removed for the first 4 hours during challenge. 

A recombinant vaccine targeting the parasitic ciliate <i>Ichthyophthirius multifiliis</i>

https://curis.ku.dk/ws/files/230389653/Poster_ISFSI_2019.pdf

A recombinant vaccine targeting the parasitic ciliate <i>Ichthyophthirius multifiliis</i>

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