WP1: Decoding the adjuvants activity of LNPs
In the first WP we assessed the adjuvants activity of different types of LNPs by performing single cell sequencing on LNP+ mature DCs in the spleen and determining their maturation program. This was complemented with functional assays to analyze the capacity of LNP+ DCs to steer the adaptive immune response toward immunity or tolerance against non-immunogenic cargo incorporated in the LNPs.
This revealed that:
1) eLNPs are not perceived as “danger” by DCs and their uptake leads to induction of a homeostatic not immunogenic maturation program
2) The cargo and not the lipids determine the adjuvanticity of LNPs
3) LNPs can be exploited as vehicles to induce either immunity or tolerance against incorporated antigens
4) The immunogenicity of the cargo needs to be carefully assessed, especially when incorporated in the LNP: synthetic peptides are non-immunogenic while most mRNA purifications are immunogenic unless care is taken to remove all dsRNA contaminants
In addition, we designed a DC maturation tool box, a set of tools that can be used to assess the immunogenicity of newly developed LNP formulations. This comprises:
1) a flow cytometry panel for mice and humans to deconvolute homeostatic from immunogenic mature DCs. This is the first time such a panel has been designed and is meanwhile widely adapted by other labs
2) a transcriptional profiling strategy to monitor the DC maturation program. This can be defined by qPCR or by sequencing methods. We developed an open access web-based tool that allows researchers to enter their sequencing data of mature DCs in an algorithm that will assign the DC maturation state along a homeostatic-immunogenic spectrum and perform a meta-analysis
WP2: Tolerogenic potential of LNP vaccines
In WP2 we aimed to address whether LNPs can be used to induce tolerance against allergens and/or autoimmune disease. As a proof-of-concept, we used the house dust mite (HDM) protocol, a well-established model for allergic airway inflammation and asthma.
The major bottleneck appeared to be the incorporation of the antigen in the LNPs.
Originally we proposed to incorporate the main HDM antigens, Derp1 and Derp2, as synthetic peptides in the LNPs, as we knew this yielded non-immunogenic antigens. However, to improve the outcome of the response we switched to whole proteins, produced recombinantly either in E.coli or P.pastoris. We took special precautions to keep the LPS during the purification procedure as low as possible. Still, even the sligthest presence of LPS (which was inevitable) yielded in immunogenic DC maturation, showing that incorporation in LNPs amplifies any TLR contaminants.
Therefore, we turned our attention towards mRNA antigen incorporation, as was used for the original mRNA-LNPs of BioNTech. We applied different strategies to reduce the dsDNA content, both by using a mutated version of the T7 RNA polymerase and by applying additional columns to remove the dsDNA. The effects on DC maturation in vivo were assessed by using the strategies from WP1.
The optimized mRNA purification strategy had success and yielded the desired non-immunogenic profile. We are currently applying similar strategies for Derp1 and Derp constructs to assess their potential as a tool to induce tolerance and improving astma outcomes.
