Designed Virus-like Particles as Novel Modular Vaccine Delivery System for Synthetic Peptide Antigens
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Beschreibung
This thesis focuses on the synthetic design of lipopeptide-based nanoparticles and their applications as vaccine delivery system for synthetic peptide antigens. The use of synthetic peptides mimicking surface antigens of pathogens offer several advantages compared to classical vaccination strategies using either killed or recombinant viruses. However, the problem encountered with small molecules such as synthetic peptides is their low immunogenicity thus requiring a macromolecular carrier for targeting immune cells efficiently. Synthetic virus-like particles (SVLPs) have been shown to offer a promising approach to vaccine design as they tend to mimic the size and shape of viruses and are optimal for targeting lymphoid dentritic cells (DCs). SVLPs consist of self-assembling helical lipopeptide bundles, which spontaneously self-associate into homogenous nanoparticles (20- 25 nm in diameter) in aqueous solution. After particle assembly, peptide-antigens conjugated to the lipopeptide are displayed on the nanoparticle’s surface in a multivalent fashion, able to stimulate immune cells and trigger adaptive immune responses.
The first part of this work aimed at expanding the scope for such kinds of SVLPs by design of synthetic lipopeptides bearing signaling molecules of microbial origin, so called pathogenassociated molecular patterns (PAMPs). The PAMPs should allow specific stimulation of immune cells by targeting their pattern recognition receptors (PRRs). Toll-like receptors (TLRs), widely expressed by antigen presenting cells (APCs) are an important group of these receptors and able to induce immune responses by sensing different types of microbial invaders. For example, bacterial cell wall components such as the lipid anchors Pam2Cys and Pam3Cys have been found to target TLR1/2 and were shown to boost the immune response in terms of activating DCs and enhancing the number of responding T cells. To another class of PRRs belong the C-type lectin receptors (CLRs) which are capable of binding carbohydrates of microbial glycoproteins. Although the role of CLRs in terms of immune activation is not yet fully understood, it is presumed that they are involved in antigen internalization and presentation. The Mannose Receptor (MR) and DC-SIGN are prominent members of this family.
By combining recent knowledge on the function of TLRs, CTLs and DCs in terms of antigen uptake, processing and signaling, attractive vaccine formulations were designed to target antigens to DCs in order to shape antigen-specific immune responses. Pam2Cys- and Pam3Cys-peptide conjugates were synthesized to evaluate their potential as immunopotentiating delivery systems. Therefore, a model system based on earlier SVLP designs comprising a de novo designed isoleucine zipper peptide, able to form a stable coiled coil, fused to a T helper cell epitope derived from the circumsporozoite protein (CSP) of the malaria parasite P. falciparum was chosen. A linear peptide known to elicit P. falciparum cross-reactive antibodies served as model antigen. Structural properties of SVLPs formed from these Pam2Cys- and Pam3Cys-peptide conjugates were studied using biophysical methods, such as dynamic light scattering, analytical ultracentrifugation and transmission electron microscopy. Lipopeptide building blocks were found to self-assemble into stable, homogeneous particles, with a mean diameter in the 20–30 nm size range as already seen with previous investigations using phospholipid-containing lipopeptides. The immunological profile of SVLPs and their self-adjuvant function were tested in rodent models and were analyzed by enzyme-linked immunosorbent assays. The immune responses in mice and rabbits were comparable to those elicited by SVLPs comprising phospholipid-peptide conjugates and were characterized by a strong booster effect on IgG titers and significant avidity improvements following the second immunization. Antibody responses generated by self-adjuvanting SVLPs were at least comparable to those elicited by other virus-like particles. The results demonstrated that SVLPs are robust nanostructures, amenable to engineering, and represent an attractive antigen-delivery vehicle for synthetic vaccine design without the need for an adjuvant.
Further studies involved the design and exploration of glycolipopeptide-SVLPs based on the above mentioned model system. Previously designed SVLPs based on self-adjuvanting lipopeptides were refined through incorporation of additional glyco-structures to target MR and DC-SIGN on cell-surfaces of APCs. For this purpose, two distinct glycolipopeptide species were tailored: one comprising three consecutive monosaccharide units and the other comprising one disaccharide unit, by incorporating α-D-mannopyranosylserine or α-1,2-bismannosylglutamate residues, respectively. The sugar moieties were expected to be exposed on the outer surface of the derived SVLPs in a multivalent manner. Biophysical studies showed that the attachment of the sugar residues had no negative effect regarding shape and particle sizes of the SVLPs. However, regarding the immunogenicity of CTLtargetingSVLPs in terms of antibody production in rodent models it was demonstrated that (bis)mannosylated SVLPs quantitatively and qualitatively enhance the immune response. Even at lower SVLP dosages, they are able to induce a stronger antigen-specific antibody response of higher avidity. These studies provide important initial steps towards a selfadjuvanting vaccine carrier, which could be employed to efficiently present antigens to DCs, even at low doses. Exploiting this approach to design disease-specific SVLPs may open doors for novel vaccine candidates.
One such approach towards a fully synthetic HIV-1 vaccine candidate was investigated in the second part of this thesis. Starting from available crystal structures of the V3 epitope of HIV-1 gp120 bound to neutralizing antibodies, the objective was to translate this structural information into immunogens that could elicit cross-reactive neutralizing antibodies in vivo.The first step involved the synthesis of cyclic V3-loop-mimicking immunogens, based on template-bound β-hairpin mimetics. The V3 loop sequences from gp120 were transplanted onto a hairpin-stabilizing D-Pro-L-Pro template in order to stabilize the conformations of the antibody-V3 loop complexes. The conformational properties of the V3-loop mimetics were evaluated by solution NMR spectroscopy. Structure calculations using distance restraints from NOESY measurements and a simulated annealing protocol were performed in collaboration with Dr. K. Möhle. It was shown that these synthetic V3-loop peptides were able to accurately mimic the distinct conformations of the V3-antibody complexes in aqueous solution.
The next step was to evaluate whether one of these mimetics could stimulate a V3-specific immune response. The focus was put on one mimetic V3MN-IY1 which closely mimics the tructure of a linear V3 peptide bound to the neutralizing mAb F425-B4e8. First, it was shown that the constrained V3-mimetic exhibits enhanced antigenicity (affinity for mAb F425-B4e8) compared to a linear V3 peptide, which suggested that the V3-mimetic with a constrained conformation might also be of higher immunogenic relevance for eliciting V3-specific neutralizing antibodies. Next, V3MN-IY1 was tested as an immunogen by coupling it to a lipopeptide carrier which can self-assemble into SVLPs. Sera obtained from immunizations of New Zealand white rabbits with V3MN-IY1-SVLPs were analyzed by ELISA in terms of antibody responses and were tested for their ability to cross-react with gp120 and with envelope spikes on native HIV-1. A luciferase reporter gene assay with molecularly cloned Env-pseudotyped viruses was performed in cooperation with Dr. P. Rusert and Prof. A. Trkola (Institute of Virology, University of Zurich) to test if viral infectivity can be inhibited in vitro by the elicited antibodies. In addition to several wild-type strains, genetically divergent mutant strains containing a deleted V1V2 loop region, which has been shown to shield the V3 loop in native spikes on the viral surface, were tested. The results showed that the V3MN-IY1 mimetic was successful in eliciting a strong specific humoral immune response, but with anti-V3MN-IY1 antibodies only able to induce neutralizing activity against one sensitive wild-type laboratory strain. However, most of the mutants lacking the shielding effect of the V1V2 loops were neutralized, showing that the V3MN-IY1 mimetic was able to elicit antibodies with cross-neutralizing activity but their accessibility in the contex of native viral spikes is impeded by the V1V2 region.
Overall, this study demonstrated the enormous effect that conformational restriction has onthe antigenicity and presumably also on the immunogenicity of short, synthetic peptides. Constrained peptides provide a means for focusing antibodies on defined epitopes and in combination with SVLPs provide a promising strategy for synthetic vaccine research.
Autor*in
Tina Riedel
Themen in »Designed Virus-like Particles as Novel Modular Vaccine Delivery System for Synthetic Peptide Antigens«
Impfstoffträger Synthetic virus-like particles Synthetische, virusähnliche Partikel lipopeptide-based nanoparticles synthetic peptide antigens synthetische Peptid-Antigene synthetischen Nanopartikeln auf Lipopeptid-Basis vaccine delivery system