Aktivacija mišjih dendritičnih celic MutuDC1940 s konjugiranim nitastim bakteriofagom f88KE

Bizjak, Ela (avtor), Bratkovič, Tomaž (mentor), Gnidovec, Klemen (komentor)

Cepiva imajo ključno vlogo pri preprečevanju širjenja nalezljivih bolezni. Prvi korak pri delovanju cepiv je aktivacija antigen predstavitvenih celic oz. njihovih glavnih predstavnic, dendritičnih celic. Njihova glavna naloga je vzorčenje okolice in odziv na molekularne vzorce, ki izhajajo iz patogenov, kot so na primer lipopolisaharidi in z motivi CpG bogata DNA. Po vzorčenju dendritične celice privzete antigene predstavijo efektorskim celicam imunskega sistema in spodbudijo njihovo aktivacijo, kar zahteva neposreden celični stik s specifičnimi označevalci pripadnosti in signalizacijo s citokini. Če je odziv, ki ga določen antigen sproži, dovolj močen, pride do tvorbe imunskega spomina, ki zagotavlja imunost ob ponovni okužbi z istim patogenom. Zaradi vloge dendritičnih celic pri razvoju imunosti, je mnogo raziskav usmerjenih v razvoj cepiv, ki bi antigene dostavila neposredno do dendritičnih celic. Nitasti bakteriofagi so zaradi svojih lastnosti primerni kandidati za nosilce antigenov in za specifično ciljanje celic. Njihova zgradba je enostavna, dobro razvita pa je tudi tehnologija predstavitve na bakteriofagu. So tudi imunogeni, saj v genomu nosijo nemetilirane motive CpG in – ker jih pomnožujemo v po Gramu negativni bakteriji Escherichia coli – so v proteinskem plašču prisotni sledovi lipopolisaharidov. Razvili smo konstrukt nitastega bakteriofaga f88KE, ki v fuziji s strukturnim proteinom p3 izraža peptid SnoopTag. To nam je omogočilo konjugacijo virusnih delcev z do 5 kopijami fuzijskega proteina, sestavljenega iz nanotelesa, usmerjenega proti receptorju CLEC9A, in proteina SnoopCatcher, kar omogoča specifično ciljanje dendritičnih celic. Rekombinantne bakteriofage smo pred poskusih na mišjih dendritičnih celicah očistili lipopolisaharidov. Aktivacijo dendritičnih celic smo ovrednotili z metodo qPCR, kjer smo kvantificirali izražanje genov, značilnih za aktivirane celice. Ob uporabi različnih koncentracij bakteriofaga smo dosegli različno moč aktivacije. Aktivacija je vidna že eno uro po dodatku bakteriofagov, vztraja pa vsaj 18 ur. Ugotovili smo, da ciljanje dendritičnih celic preko receptorja CLEC9A zmanjša moč celične aktivacije, vendar je ta še vedno prisotna.

• Jezik: Slovenski jezik
• Ključne besede: cepiva, nitasti bakteriofagi, dendritične celice, CLEC9A, qPCR
• Vrsta gradiva: Magistrsko delo/naloga
• Tipologija: 2.09 - Magistrsko delo
• Organizacija: FFA - Fakulteta za farmacijo
• Leto izida: 2025
• PID: 20.500.12556/RUL-176532
• COBISS.SI-ID: 260007171
• Datum objave v RUL: 03.12.2025

Sekundarni jezik

• Jezik: Angleški jezik
• Naslov: Activation of mouse dendritic cells MutuDC1940 with decorated filamentous bacteriophage f88KE

Izvleček

Vaccines play a crucial role in preventing the spread of infectious diseases. The first step in the action of vaccines is the activation of antigen-presenting cells, or their main representatives, dendritic cells. Their primary task is to sample the environment and respond to molecular patterns derived from pathogens, such as lipopolysaccharides or CpG-rich DNA. After sampling, dendritic cells present the internalized antigens to effector cells of the immune system and stimulate their activation, which requires direct cell–cell contact with specific surface markers and cytokine signalling. If the response triggered by a given antigen is strong enough, immunological memory is formed, which ensures immunity upon re-infection with the same pathogen. Because of the role of dendritic cells in the development of immunity, many studies focus on designing vaccines that directly target dendritic cells. Filamentous bacteriophages, due to their properties, are suitable candidates for antigen carriers that enable specific cell targeting. Their structure is simple, and phage display technology is also well developed. They are immunogenic, since their genome contains unmethylated CpG motifs and – since they are produced in Gram negative bacteria Escherichia coli – their protein coating includes traces of lipopolysaccharides. We developed a construct of the filamentous bacteriophage f88KE that, in fusion with protein p3 expresses SnoopTag peptide. This facilitated the conjugation of viral particles with as many as 5 copies of fusion protein, composed of SnoopCatcher protein and a nanobody, targeted to the receptor CLEC9A, which allows specific targeting of dendritic cells. Recombinant bacteriophages were purified of lipopolysaccharides before they were used on murine dendritic cells. We evaluated dendritic cell activation using qPCR, where we quantified expression of genes characteristic of activated cells. By applying different concentrations of bacteriophage, we achieved varying strengths of activation. Activation is detectable as early as one hour after induction begins and persists for at least 18 hours. We discovered that targeting dendritic cells via the CLEC9A receptor reduces the strength of cellular activation, but the activation remains present.

• Ključne besede: vaccines, filamentous bacteriophage, dendritic cells, CLEC9A, qPCR