Elsevier

Biomaterials

Volume 172, July 2018, Pages 105-115
Biomaterials

Engineered hybrid spider silk particles as delivery system for peptide vaccines

https://doi.org/10.1016/j.biomaterials.2018.04.008Get rights and content

Abstract

The generation of strong T-cell immunity is one of the main challenges for the development of successful vaccines against cancer and major infectious diseases. Here we have engineered spider silk particles as delivery system for a peptide-based vaccination that leads to effective priming of cytotoxic T-cells. The recombinant spider silk protein eADF4(C16) was fused to the antigenic peptide from ovalbumin, either without linker or with a cathepsin cleavable peptide linker. Particles prepared from the hybrid proteins were taken up by dendritic cells, which are essential for T-cell priming, and successfully activated cytotoxic T-cells, without signs of immunotoxicity or unspecific immunostimulatory activity. Upon subcutaneous injection in mice, the particles were taken up by dendritic cells and accumulated in the lymph nodes, where immune responses are generated. Particles from hybrid proteins containing a cathepsin-cleavable linker induced a strong antigen-specific proliferation of cytotoxic T-cells in vivo, even in the absence of a vaccine adjuvant. We thus demonstrate the efficacy of a new vaccine strategy using a protein-based all-in-one vaccination system, where spider silk particles serve as carriers with an incorporated peptide antigen. Our study further suggests that engineered spider silk-based vaccines are extremely stable, easy to manufacture, and readily customizable.

Introduction

Vaccination prevents the spread of many deadly infectious diseases, and it is considered to be one of the most effective interventions for global health ever developed [1]. The protective effect of vaccines is essentially due to B lymphocyte production of neutralizing antibodies against infectious agents or toxins. Although in many infectious diseases this humoral immunity is of major importance, in some cases cellular immunity is essential for protection [2]. Indeed, cytotoxic T-cells, the main effector cells of cellular immunity, are required for the development of successful immune responses against chronic infections such as HIV, malaria or tuberculosis [3]. Furthermore, recent studies have shown that T-cell-mediated immunity can lead to cancer regression in patients, even in the case of advanced disease [4]. Unfortunately, most current vaccination strategies do not induce effective cytotoxic T-cell responses and the development of vaccines that promote cellular immunity thus remains a major challenge [5].

The initiation of cytotoxic T-cell responses requires the priming of naïve CD8+ T-cells by professional antigen-presenting cells such as dendritic cells [6,7]. Dendritic cells are located in many tissues and constantly sample their surroundings. When these cells encounter microbes or particulate matter, or indeed vaccination-delivered antigens, these are engulfed and processed in order to cross-present antigenic peptides to T-cells via major histocompatibility complex class I molecules (MHC I) [8,9]. To induce effective immunity, immature dendritic cells must be activated by specific maturation signals [10]. For this reason, in addition to microbial antigens, most vaccines include adjuvants that stimulate dendritic cell maturation and thus the generation of cytotoxic T-cell immunity [11,12]. The maturation of dendritic cells occurs during their migration from peripheral tissues to the lymph nodes [13]. In the lymph nodes, CD8+ T-cells are primed by direct contact with mature dendritic cells presenting their cognate antigen [14]. Primed T-cells proliferate in the lymph nodes and differentiate to effector cytotoxic T-cells that patrol the body to kill infected or tumoral cells.

The elucidation of the amino acid sequences of microbial or tumor-associated antigens has led to the development of peptide vaccination [15]. Vaccines comprised of antigenic peptides instead of full-length proteins activate only the cytotoxic T-cells that recognize disease-specific epitopes, thus limiting the risk of unspecific autoimmune toxicity. However, peptide vaccination has shown limited success due to several factors. First, peptides are rapidly degraded by proteases: the MelanA/MART-1 tumor antigenic peptide for instance is degraded in 22 s in plasma [16]. Second, peptides can bind to MHC I not only on antigen-presenting cells but on all nucleated cells, thus promoting tolerance rather than immunity [17,18]. Furthermore, to be effective the vaccine peptides must form a stable complex with MHC I molecules during dendritic cell migration to the lymph node [19,20], which takes approximatively 18 h [21]. To meet these challenges, antigens can be protected by particulate carriers. Particle delivery systems can limit peptide degradation, improve uptake of antigen by dendritic cells [22] and ensure that the antigen is processed intracellularly before loading onto MHC I [23].

For the design of particle-based delivery systems, antigen can be coupled to the particles by different methods [24]. The antigen may be adsorbed to the surface of the particle, as is the case with aluminum-based formulations [25]. However, binding by non-covalent interactions may result in rapid antigen release from the carrier upon changes in pH. The antigen can also be encapsulated in a polymer matrix during manufacturing, but this may damage protein-based antigens due to the relatively harsh conditions commonly applied during particle preparation [26]. Chemical conjugation of the antigen to the carrier bears the risk of losing certain epitopes during conjugation [27]. To circumvent these difficulties, we chose to use a protein-based carrier and directly incorporated the antigenic peptide into the sequence of the carrier protein derived from spider silk. Spider silk protein is a promising material, because previous studies demonstrated that the protein itself is poorly immunogenic and does not cause inflammatory reactions in rats [28,29]. Indeed, in addition to their clear advantage in terms of biocompatibility and biodegradability, spider silk particles allow for solvent-free synthesis and steam sterilization [30,31].

Section snippets

Preparation of spider silk hybrid proteins

eADF4(C16) is based on 16 repeats of the consensus sequence of spidroin ADF4 of the European garden spider (Araneus diadematus) (C-module: GSSAAAAAAAASGPGGY GPENQGPSGPGGYGPGGPG), and a T7-tag fused to the amino terminus for detection purposes [32]. OVA257-264 peptide (SIINFEKL) was fused to the eADF4(C16) by either using a cathepsin B cleavable linker (GFLG) or a cathepsin S cleavable linker (PMGLP). Fusions were made using tags and DNA sequences of the tags were inserted into the cloning

Results and discussion

The aim of this study was the development and formulation of a novel particulate delivery system to improve the induction of cytotoxic T-cell responses following peptide vaccination. To design a particulate peptide vaccine based on spider silk proteins, a MHC I-restricted peptide from the model antigen ovalbumin, OVA257-264, was fused to the C-terminus of a previously engineered spider silk protein, eADF4(C16) [32], using a cathepsin-cleavable linker. Four different recombinantly engineered

Conclusions

Cellular immunity is of major importance for protection against certain infectious diseases and in cancer. Here, we demonstrate the ability of engineered spider silk particles to function as peptide vaccine delivery system and to induce cytotoxic T-cell proliferation in the draining lymph nodes of mice. Importantly, this effect was seen even in the absence of adjuvant, although the particles alone were not pro-inflammatory and did not induce unspecific immune responses. Our results also

Competing interest statement

The authors confirm that there are no known conflicts of interest associated with this publication and there has been no significant financial support for this work that could have influenced its outcome.

Data availability statement

The raw/processed data required to reproduce these findings cannot be shared at this time due to a pending patent application.

Acknowledgements

This work was supported by a grant from the Federal Ministry of Education and Research, Germany (grant number: 13N11341 GW; grant number: 13N11340 TS), the National Center of Competence in Research (NCCR) for Bio-Inspired Materials to CB and the Swiss National Science Foundation grants 156372 and 156871 to CB. We thank C. Minke (Ludwig-Maximilians-University Munich, Department of Chemistry) for assistance with scanning electron microscopy and A. Oberson and J.Widmer (both from University of

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