Title ULOGA NKG2D LIGANADA RAZLIČITOG AFINITETA U INFEKCIJI REKOMBINANTNIM MIŠJIM CITOMEGALOVIRUSIMA I RAZVOJU VIRUSNIH CJEPIVA Title (english) ROLE OF NKG2D LIGANDS OF DIFFERENT AFFINITIES IN THE RECOMBINANT MOUSE CYTOMEGALOVIRUS INFECTION AND VACCINE DEVELOPMENT Author Lea Hiršl Mentor Astrid Krmpotić (mentor)Committee member Stipan Jonjić (predsjednik povjerenstva)Committee member Goran Tešović (član povjerenstva)Committee member Pero Lučin (član povjerenstva)Committee member Astrid Krmpotić (član povjerenstva)Granter University of Rijeka Defense date and country 2019-11-19, Croatia Scientific / art field, BIOMEDICINE AND HEALTHCARE Universal decimal classificationUDC ) 612 - Physiology ThesaurusMESH - Medical Subject Headings )
Cytomegalovirus
immunology
Muromegalovirus
immunology
Cytomegalovirus Vaccines
Abstract Cilj istraživanja: NKG2D je aktivacijski receptor na stanicama NK i kostimulacijski receptor na limfocitima T. Stanični ligandi za receptor NKG2D su razvojno i tkivno diferencijalno izražene molekule s različitim afinitetom za isti receptor. Humani citomegalovirus (HCMV) je široko rasprostranjen herpesvirus koji potiče stvaranje svojstvenog odgovora limfocita T karakteriziranog akumulacijom virus-specifičnih stanica efektorskih obilježja što ga čini dobrim kandidatom za razvoj cjepiva. Jedno od nužnih karakteristika svakog živog cjepiva je da bude sigurno za primjenu pa smo u našim prethodnim istraživanjima razvili novi koncept cjepiva na modelu mišjeg CMV-a (MCMV) koji izražava ligand za receptor NKG2D, RAE-1γ (RAE-1γMCMV) umjesto njegovog virusnog inhibitora. Kako je MULT1 mišji NKG2D ligand najjačeg afiniteta za receptor, konstruirali smo rekombinantni MCMV u kojemu je gen za MULT1 umetnut na mjestu njegovog virusnog inhibitora (MULT1MCMV) i komparativnim pristupom smo ispitali imunosni odgovor i imunosnu kontrolu RAE-1γMCMV-a i MULT1MCMV-a in vivo te odredili njihovu sposobnost poticanja zaštitne imunosti.
Materijal i metode: Kako bi ispitali kontrolu i imunosni odgovor na MULT1MCMV in vivo, inficirali smo odrasle i novookoćene miševe s MCMV-om, RAE-1γMCMV-om i MULT1MCMV-om te pratili virusni titar u organima inficiranih životinja i fenotipski okarakterizirali stanice NK i CD8 limfocite T. Zaštitnu imunost CD8 limfocita T smo ispitali adoptivnim prijenosom stanica iz imunih životinja u naivne imunokompromitirane primatelje koje smo potom inficirali MCMV-om i pratili virusni titar u organima. Testom antigenske prezentacije in vitro i eksperimentima na kimerama koštane srži miševa divljeg tipa i NKG2D-/- ispitali smo ulogu kostimulacije CD8 limfocita T putem receptora NKG2D nakon infekcije s virusima koji izražavaju NKG2D ligande.
Rezultati: MULT1MCMV bio je atenuiran in vivo od strane stanica NK u odraslim i novookoćenim miševima, čak i jače od RAE-1γMCMV-a u određenim eksperimentalnim uvjetima. Unatoč atenuaciji, MUTL1MCMV je zadržao imunogeničnost i inducirao je antigen-specifičan odgovor CD8 limfocita T i protuvirusnu humoralnu imunost sličnih obilježja kao i infekcija MCMV-om i RAE-1γMCMV-om. Potvrdili smo da kostimulacijski signal putem receptora NKG2D pojačava odgovor CD8 limfocita T nakon infekcije s virusima koji izražavaju NKG2D ligande.
Zaključak: MULT1MCMV je atenuiran in vivo čak i jače od MCMV-a koji izražava NKG2D ligand slabijeg afiniteta. Također, zadržao je imunogeničnost i potiče zaštitnu stečenu imunost i kao takav pokazuje povoljna obilježja cjepiva.
Abstract (english) Objectives: NKG2D is an activating receptor on NK cells and costimulatory receptor on T cells. Cellular ligands for this receptor are differentially expressed during the development and in the tissues and have different affinity for the receptor. Human cytomegalovirus (HCMV) is a widely spread herpesvirus which induces unique T cell response characterized by accumulation of virus-specific T cells with effector properties. This feature makes this virus a good vaccine candidate. One of the critical characteristics of every live vaccine is safety, so in our previous research we developed a new approach in designing vaccine based on mouse CMV (MCMV) expressing NKG2D ligand RAE-1γ instead of its viral inhibitor. Since MULT1 is an NKG2D ligand of highest affinity for the receptor, we constructed MCMV expressing this ligand instead of its viral inhibitor (MULT1MCMV) and compared it with RAE-1γMCMV in terms of viral control, immunobiology in vivo and explored their protective capacity as a vaccine.
Material and Methods: To examine viral control and immune response to MULT1MCMV in vivo, we infected adult and newborn mice with MCMV, RAE-1γMCMV and MULT1MCMV and followed viral titer in organs of infected animals and did phenotypic analysis of NK cells and CD8 T cells. We tested protective immunity by adoptive transfer of CD8 T cells from immunized mice into naïve recipients which were subsequently infected with MCMV and analyzed viral titer in their organs. By using in vitro antigen presentation assay and bone marrow chimeras of wild type and NKG2D-/- mice we examined the role of NKG2D costimulation on CD8 T cells after infection with recombinant viruses expressing NKG2D ligands.
Results: MULT1MCMV was attenuated in vivo by NK cells in NKG2D-dependent manner in both adult and newborn mice, even more strongly than RAE1-γMCMV in certain experimental conditions. Despite attenuation, MULT1MCMV was immunogenic and induced antigen-specific CD8 T cell response and anti-viral humoral response, comparable to infection with MCMV and RAE-1γMCMV. We showed that costimulatory signal via NKG2D receptor on CD8 T cells is able to boost their response after infection with recombinant viruses expressing NKG2D ligands.
Conclusion: MULT1MCMV is attenuated in vivo even stronger than MCMV expressing NKG2D ligand of lower affinity, RAE-1γ. Moreover, it retained its immunogenicity and was able to induce protective adaptive immunity and as such shows excellent vaccine properties.
Keywords
citomegalovirus
cjepivo
imunost
MULT1
NKG2D
RAE-1γ
Keywords (english)
cytomegalovirus
vaccine
immunity
MULT1
NKG2D
R
Language croatian URN:NBN urn:nbn:hr:184:601153 Study programme Title: Biomedicine Postgraduate (doctoral) study programme Type of resource Text File origin Born digital Access conditions Access restricted to students and staff of home institution Terms of use Repository Repository of the University of Rijeka, Faculty of Medicine Created on 2020-02-20 16:31:48
