Development of novel NOD2 agonists and dual NOD2 and TLR7 agonists as vaccine adjuvants : doctoral dissertation

Guzelj, Samo (author), Jakopin, Žiga (mentor)

Vaccination, one of the most effective medical interventions ever introduced, primes the immune system to respond to future infections, dramatically reducing the morbidity and mortality of many infectious diseases. The success of vaccination often depends on the careful selection of adjuvants, immunopotentiating formulations or compounds that elicit a stronger and more durable immune response to the administered antigen. Yet, despite their importance in the development of modern vaccines, few new vaccine adjuvants have entered the clinic in recent decades. In this work, we expand the library of potential vaccine adjuvant candidates by targeting the innate immune pattern recognition receptors nucleotide-binding oligomerization containing protein 2 (NOD2) and Toll-like receptor 7 (TLR7). First, we optimised the structure of a previously reported desmuramylpeptide NOD2 agonist using ligand-based design approaches. We identified key structural features required to achieve immunostimulatory activities in vitro and in vivo. This led to significantly improved adjuvant activity in vivo and the discovery of the first desmuramylpeptide with NOD2 agonist activity in the single digit nanomolar range. Second, we construct a homology model of human NOD2 using a recently reported crystal structure of rabbit NOD2 as the template and apply a hybrid docking/pharmacophore modelling method in search of novel NOD2-modulating scaffolds. Surprisingly, although our structure-based in silico approach did not yield any NOD2 agonists, it did identify two novel NOD2 antagonist scaffolds, which represent valuable new leads suitable for further optimisation. Finally, we extend the adjuvant potential of NOD2 agonists by covalent conjugation with TLR7 agonists. The resulting conjugated co-drugs induced potent Th1/Th17-type T cell immune responses in vitro and Th1 humoral immune responses in vivo. Our study has therefore shown that conjugates can generate robust higher-order interactions that individual pattern recognition receptor agonists simply cannot access.

• Language: English
• Work type: Dissertation
• Typology: 2.08 - Doctoral Dissertation
• Organization: FFA - Faculty of Pharmacy
• Place of publishing: Ljubljana
• Publisher: [S. Guzelj]
• Year: 2022
• Number of pages: 392 str.
• PID: 20.500.12556/RUL-141974
• UDC: 615.375+631.541(043.3)
• COBISS.SI-ID: 120062211
• Publication date in RUL: 13.10.2022

Secondary language

• Language: Slovenian
• Title: Razvoj novih agonistov NOD2 in dvojnih agonistov NOD2 in TLR7 kot novih adjuvansov za cepiva

Abstract

Cepljenje, ena najučinkovitejših medicinskih intervencij, ki so jih kdaj uvedli, pripravi imunski sistem na odziv proti prihodnjim okužbam, kar dramatično zmanjša obolevnost in umrljivost številnih nalezljivih bolezni. Uspeh cepljenja je pogosto odvisen od skrbne izbire adjuvansov, formulacij ali spojin, ki izzovejo močnejši in trajnejši imunski odziv na aplicirani antigen. Kljub njihovemu izjemnemu pomenu pri razvoju sodobnih cepiv, pa je v zadnjih desetletjih v kliniko vstopilo le nekaj novih adjuvansov. V tej doktorski disertaciji smo knjižnico potencialnih adjuvansov razširili s ciljanjem nukleotid-vezočo oligomerizacijsko domeno vsebujočega proteina 2 (NOD2) in Toll-u podobnega receptorja 7 (TLR7), dveh receptorjev za prepoznavo vzorcev prirojene imunosti. V prvem delu smo z načrtovanjem na osnovi liganda optimizirali strukturo predhodno odkritega dezmuramilpeptidnega NOD2 agonista. Identificirali smo ključne strukturne značilnosti, potrebne za doseganje imunostimulatorne aktivnosti in vitro in in vivo. To je privedlo do znatnega izboljšanja adjuvantne aktivnosti in vivo in do odkritja prvega dezmuramilpeptida z NOD2 agonistično aktivnostjo v enomestnem nanomolarnem območju. V drugem delu smo v iskanju novih modulatorjev NOD2 na podlagi nedavno odkrite kristalne strukture zajčjega NOD2 zgradili homologni model človeškega NOD2. Ta je služil kot osnova za uporabo hibridne metode sidranja in farmakofornega modeliranja. Presenetljivo naš strukturno podprti in silico pristop ni privedel do odkritja novih NOD2 agonistov, je pa vodil do identifikacije dveh novih skeletov z NOD2 antagonistično aktivnostjo, ki predstavljata dobro osnovo za nadaljnjo optimizacijo. V zadnjem delu smo adjuvantni potencial NOD2 agonistov razširili s kovalentno konjugacijo z agonisti TLR7. Nastala konjugirana so-zdravila so inducirala močne imunske odzive tipa Th1/Th17 in vitro in humoralne Th1 imunske odzive in vivo. Naša študija je pokazala, da lahko s konjugati dostopamo do robustnih interakcij višjega reda, ki so nedostopne posameznim agonistov receptorjev za prepoznavo vzorcev.

• Keywords: konjugati, dezmuramilpeptidi, imunska sinergija, imunomodulacija, NOD2, receptorji za prepoznavo vzorcev, TLR7, adjuvansi za cepiva