Modular assembly of immunologically-active molecules based on coiled-coils

V zadnjem stoletju so cepiva najučinkovitejši način preprečevanja smrti zaradi nalezljivih bolezni. Napredek v znanosti je ključno gonilo razvoja cepiv, zato so raziskave na področju cepiv zelo pomembne. Raziskovanje različnih pristopov načrtovanja novih cepiv je pomembno orodje za izboljšanje njihovih lastnosti. Cepiva ločimo na živa in neživa. Razlika med prvo in drugo skupino je v tem, da prva vsebuje oslabljene patogene, ki se lahko v telesu razmnožujejo, druga pa vsebuje mrtve patogene ali njihove komponente. V doktorskem delu smo se osredotočili na peptide, ki tvorijo obvite vijačnice (ang. coiled coils, CC-peptide) kot nosilce, s katerimi bi združili imunološko aktivne komponente v učinkovito ne-živo cepivo. Zaradi svoje modularne narave, so nam CC-peptidi služili kot dobri gradniki za načrtovanje nanocepiva, ki omogoča hkratno dostavo antigena in adjuvansa v tarčno celico, kar je za učinkovitost cepiva zelo pomembno. Načrtovali smo več parov stabilnih in med seboj ortogonalnih CC-peptidov. Pri tem smo upoštevali dobro definirana pravila umeščanja posamezne aminokisline znotraj CC-peptida. Z energijskega vidika je najpomembnejši položaj tisti, ki tvori hidrofobno sredico, elektrostatske interakcije med nasprotno nabitimi aminokislinskimi ostanki pa so drugi pomemben doprinos k stabilizaciji obvite vijačnice ter ključne za selektivnost parjenja. Sintetične peptide smo, po določitvi njihovih lastnosti in vitro, smo jih uporabili kot dimerizacijske domene za sestavljanje cepljenih proteinov. Na ta način smo se prepričali, ali načrtovani peptidi ohranijo svoje lastnosti v fuziji z večjimi domenami (proteini), kar je bilo ključno za nadaljnje eksperimente. Peptidna para z najustreznejšimi lastnostmi smo uporabili kot sestavna dela nanoogrodja, kjer smo načrtovane peptide izrazili v fuziji s fluorescenčnimi proteini ter s celičnimi dostavnimi domenami. Fluorescenčni protein nam je omogočal spremljanje celične dostave s pomočjo konfokalne mikroskopije ali pretočne citometrije. Ker je za cepivo poleg izbire ustreznega antigena in adjuvansa pomembna tudi tarčna dostava v celice, smo preizkusili več dostavnih domen. Po izbiri najučinkovitejše dostavne domene, smo nanoogrodje uporabili kot nanocepivo s katerim smo tarčno dostavili antigen ter adjuvans (kratek flagelin) v modelno antigen predstavitveno celico. Dokazali smo, da s CC-interakcijami posredovana dostava imunsko aktivnih molekul izboljša vnetni odziv.In the last century, vaccines have been the most effective way to prevent deaths caused by infectious diseases. Advances in science are a key driver of vaccine development, so research in the field of vaccines is very important. Exploring different approaches to designing new vaccines is important for improvement of vaccine efficacy. Vaccines are separated in two types: live-attenuated and inactivated. The difference between the two is that the first contains attenuated pathogens that can reproduce in our body, while non-living ones contain dead pathogens or their components. In this doctoral dissertation, we focused on peptides that form coiled coils (CC-peptides) as carriers with which to combine immunologically active components into an effective inactivated vaccine. Due to their modular nature, CC-peptides have served as good building blocks for the design of a nanovaccine that allows simultaneous delivery of antigen and adjuvant to the target cell, which is important for vaccine effectiveness. We designed several sets of stable and orthogonal CC-peptides. Well-defined rules for individual position within a CC peptide were followed. Most important positions are those that form the hydrophobic core. Electrostatic interactions between oppositely charged amino acid residues are another important contribution to the stabilization of the coiled helix and crucial for selectivity. After determining the properties of synthetic peptides in vitro, they were used as dimerization domains for the assembly of split proteins. Using split proteins method, we made sure the designed peptides retained their properties in fusion with larger domains (proteins), which was crucial for further experiments. The peptide pairs with most suitable properties were used as components of the nanoscaffold, where the designed peptides were expressed in fusion with fluorescent proteins and cell delivery domains. The fluorescent protein added to the nanostructure allowed us to monitor cell delivery by confocal microscopy or flow cytometry. In addition to selecting the appropriate antigen and adjuvant, target delivery into cells is also important for vaccine efficacy. We thus focused on several delivery domains. After selecting the most efficient delivery domain, the nano framework was used as a nanovaccine to target the antigen and adjuvant (short flagellin) to the model antigen-presenting cell. We have shown that CC-mediated delivery of immunologically active molecules improves the inflammatory response

Metal ion–regulated assembly of designed modular protein cages

Coiled-coil (CC) dimers are versatile, customizable building modules for the design of diverse protein architectures unknown in nature. Incorporation of dynamic self-assembly, regulated by a selected chemical signal, represents an important challenge in the construction of functional polypeptide nanostructures. Here, we engineered metal binding sites to render an orthogonal set of CC heterodimers Zn(II)-responsive as a generally applicable principle. The designed peptides assemble into CC heterodimers only in the presence of Zn(II) ions, reversibly dissociate by metal ion sequestration, and additionally act as pH switches, with low pH triggering disassembly. The developed Zn(II)-responsive CC set is used to construct programmable folding of CC-based nanostructures, from protein triangles to a two-chain bipyramidal protein cage that closes and opens depending on the metal ion. This demonstrates that dynamic self-assembly can be designed into CC-based protein cages by incorporation of metal ion–responsive CC building modules that act as conformational switches and that could also be used in other contexts

The role of the C-terminal D0 domain of flagellin in activation of Toll like receptor 5

<div><p>Flagellin is a wide-spread bacterial virulence factor sensed by the membrane-bound Toll-like receptor 5 (TLR5) and by the intracellular NAIP5/NLRC4 inflammasome receptor. TLR5 recognizes a conserved region within the D1 domain of flagellin, crucial for the interaction between subunits in the flagellum and for bacterial motility. While it is known that a deletion of the D0 domain of flagellin, which lines the interior of flagella, also completely abrogates activation of TLR5, its functional role remains unknown. Using a protein fusion strategy, we propose a role for the D0 domain in the stabilization of an active dimeric signaling complex of flagellin-TLR5 at a 2:2 stoichiometric ratio. Alanine-scanning mutagenesis of flagellin revealed a previously unidentified region of flagellin, the C-terminal D0 domain, to play a crucial role in TLR5 activation. Interestingly, we show that TLR5 recognizes the same hydrophobic motif of the D0 domain of flagellin as the intracellular NAIP5/NLRC4 inflammasome receptor. Further, we show that residues within the D0 domain play a previously unrecognized role in the evasion of TLR5 recognition by <i>Helicobacter pylori</i>. These findings demonstrate that TLR5 is able to simultaneously sense several spatially separated sites of flagellin that are essential for its functionality, hindering bacterial evasion of immune recognition. Our findings significantly contribute to the understanding of the mechanism of TLR5 activation, which plays an important role in host defense against several pathogens, but also in several diseases, such as Crohn’s disease, cystic fibrosis and rheumatoid arthritis.</p></div

The D0 domain of <i>H</i>. <i>pylori</i> flagellin contributes to the evasion of TLR5 recognition.

<p>(A) Schematic representation of recombinant flagellins composed of SaTy and HePy. The terminal 41 amino acid residues of SaTy where exchanged with those of HePy flagellin to assess the contribution of the CD0 domain to receptor evasion. A selection of less conserved amino acid residues within the HePy CD0 domain was mutated back to the SaTy counterparts to pinpoint the specific modifications allowing this evasion (SaTy-CD0(HePy)mut). (B) Circular dichroism measurements of SaTy and chimeric flagellins confirm a similar fold and exclude the possibility of deleterious effects of protein modifications on structure. Data are representative of two independent experiments. (C) Mutations of specific selected amino acid residues within the CD0 domain partially restore receptor activation. The human lung A549 cells were stimulated with recombinant SaTy flagellin or chimeric proteins. NF-κB-dependent SEAP activities were measured. (Data are representative of two independent experiments. Bars represent the means of 4 biological replicates ±s.d.; **p<0.005, nsp>0.05) See also <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006574#ppat.1006574.s005" target="_blank">S5 Fig</a>.</p

Amino acid residues in the C-terminal region of flagellin are important for bacterial motility.

<p>A flagellin deficient strain of <i>S</i>. <i>typhimurium</i> was transformed with plasmids encoding wt or mutant flagellin and inoculated on soft agar plates to assess the effect on bacterial motility. Each agar plate contains one tested mutant (right) and the wild-type control (left). Mutations are grouped into three categories with respect to their effect on motility. Data are representative of three independent experiments.</p

The D0 domain of flagellin mediates TLR5 activation through crosslinking.

<p>(A) A schematic representation of the domain organization of SaTy, SFΔD0, and a dimer of SFΔD0 tethered by peptide linkers. Numbering refers to <i>S</i>. <i>typhimurium</i> flagellin. (B) SDS-PAGE of purified recombinant proteins. (C) SFΔD0 is unable to activate TLR5 signaling, whereas activation with a tethered variant dimSFΔD0 has improved stimulation potential. (Above) Schematic representation of the role of the D0 domain in TLR5 activation. SFΔD0 (yellow) binds to but fails to activate TLR5 (cyan, magenta), whereas the linker in a dimer of SFΔD0 mimics the role of the D0 domain in crosslinking two ectodomains, therefore inducing active dimer formation. The human lung epithelial A549 cells were stimulated with SaTy flagellin, SFΔD0, or dimSFΔD0, and NF-κB-dependent SEAP activities were measured. (D) HEK293 cells transfected with a plasmid encoding hTLR5 were stimulated with increasing concentrations (0–1000 ng/ml) of recombinant proteins (SaTy, SFΔD0, or dimSFΔD0) and the NF-κB-dependent firefly and Renilla luciferase activities were measured 18 h later. (Data are representative of three (C) or two (D) independent experiments. Bars or points represent the means of 4 biological replicates ±s.d.; **p<0.005, nsp>0.05,) See also <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006574#ppat.1006574.s004" target="_blank">S4 Fig</a>.</p

A Nanoscaffolded Spike-RBD Vaccine Provides Protection against SARS-CoV-2 with Minimal Anti-Scaffold Response

The response of the adaptive immune system is augmented by multimeric presentation of a specific antigen, resembling viral particles. Several vaccines have been designed based on natural or designed protein scaffolds, which exhibited a potent adaptive immune response to antigens; however, antibodies are also generated against the scaffold, which may impair subsequent vaccination. In order to compare polypeptide scaffolds of different size and oligomerization state with respect to their efficiency, including anti-scaffold immunity, we compared several strategies of presentation of the RBD domain of the SARS-CoV-2 spike protein, an antigen aiming to generate neutralizing antibodies. A comparison of several genetic fusions of RBD to different nanoscaffolding domains (foldon, ferritin, lumazine synthase, and beta-annulus peptide) delivered as DNA plasmids demonstrated a strongly augmented immune response, with high titers of neutralizing antibodies and a robust T-cell response in mice. Antibody titers and virus neutralization were most potently enhanced by fusion to the small beta-annulus peptide scaffold, which itself triggered a minimal response in contrast to larger scaffolds. The beta-annulus fused RBD protein increased residence in lymph nodes and triggered the most potent viral neutralization in immunization by a recombinant protein. Results of the study support the use of a nanoscaffolding platform using the beta-annulus peptide for vaccine design

A Nanoscaffolded Spike-RBD Vaccine Provides Protection against SARS-CoV-2 with Minimal Anti-Scaffold Response

The response of the adaptive immune system is augmented by multimeric presentation of a specific antigen, resembling viral particles. Several vaccines have been designed based on natural or designed protein scaffolds, which exhibited a potent adaptive immune response to antigens; however, antibodies are also generated against the scaffold, which may impair subsequent vaccination. In order to compare polypeptide scaffolds of different size and oligomerization state with respect to their efficiency, including anti-scaffold immunity, we compared several strategies of presentation of the RBD domain of the SARS-CoV-2 spike protein, an antigen aiming to generate neutralizing antibodies. A comparison of several genetic fusions of RBD to different nanoscaffolding domains (foldon, ferritin, lumazine synthase, and β-annulus peptide) delivered as DNA plasmids demonstrated a strongly augmented immune response, with high titers of neutralizing antibodies and a robust T-cell response in mice. Antibody titers and virus neutralization were most potently enhanced by fusion to the small β-annulus peptide scaffold, which itself triggered a minimal response in contrast to larger scaffolds. The β-annulus fused RBD protein increased residence in lymph nodes and triggered the most potent viral neutralization in immunization by a recombinant protein. Results of the study support the use of a nanoscaffolding platform using the β-annulus peptide for vaccine design