Abstract
Introduction: Understanding, which factors determine the immunogenicity and immune polarizing properties of proteins, is an important prerequisite for designing better vaccines and immunotherapeutics. While extrinsic immune modulatory factors such as pathogen associated molecular patterns are well-understood, far less is known about the contribution of protein inherent features. Protein fold-stability represents such an intrinsic feature contributing to immunogenicity and immune polarization by influencing the amount of peptide-MHC II complexes (pMHCII). Here, we investigated how modulation of the fold-stability of the grass pollen allergen Phl p 6 affects its ability to stimulate immune responses and T cell polarization.Methods: MAESTRO software was used for in silico prediction of stabilizing or destabilizing point mutations. Mutated proteins were expressed in E. coli, and their thermal stability and resistance to endolysosomal proteases was determined. Resulting peptides were analyzed by mass spectrometry. The structure of the most stable mutant protein was assessed by X-ray crystallography. We evaluated the capacity of the mutants to stimulate T cell proliferation in vitro, as well as antibody responses and T cell polarization in vivo in an adjuvant-free BALB/c mouse model.Results: In comparison to wild-type protein, stabilized or destabilized mutants displayed changes in thermal stability ranging from −5 to +14°. While highly stabilized mutants were degraded very slowly, destabilization led to faster proteolytic processing in vitro. This was confirmed in BMDCs, which processed and presented the immunodominant epitope from a destabilized mutant more efficiently compared to a highly stable mutant. In vivo, stabilization resulted in a shift in immune polarization from TH2 to TH1/TH17 as indicated by higher levels of IgG2a and increased secretion of TNF-α, IFN-γ, IL-17, and IL-21.Conclusion: MAESTRO software was very efficient in detecting single point mutations that increase or reduce fold-stability. Thermal stability correlated well with the speed of proteolytic degradation and presentation of peptides on the surface of dendritic cells in vitro. This change in processing kinetics significantly influenced the polarization of T cell responses in vivo. Modulating the fold-stability of proteins thus has the potential to optimize and polarize immune responses, which opens the door to more efficient design of molecular vaccines.
Highlights
Understanding, which factors determine the immunogenicity and immune polarizing properties of proteins, is an important prerequisite for designing better vaccines and immunotherapeutics
To be able to exclude relevant T cell epitopes from the mutation process, the dominant T cell epitopes of Phl p 6 were identified by culturing splenocytes from Phl p 6-immunized mice together with 33 overlapping peptides covering its entire sequence (15mers, 3AA overlap)
Accumulating evidence suggests that the quantity and the quality of the T cell receptor (TCR) signaling induced by peptide-MHC II complexes (pMHCII) stimulation plays a crucial role in T cell polarization
Summary
Understanding, which factors determine the immunogenicity and immune polarizing properties of proteins, is an important prerequisite for designing better vaccines and immunotherapeutics. An important question for the design and understanding of molecular vaccines is how protein intrinsic factors determine the immunogenic properties of an antigen. It has been shown that TCR signaling strength is crucial for the induction of Tfh polarization [10,11,12], persistence of Foxp3+ Tregs [13, 14], and differentiation of TH17 effector cells [15] These findings have important implications for the design of novel vaccines, and for our understanding why some proteins are potent TH2 inducers (allergens), while other proteins induce TH1 responses [e.g., viral proteins [16]]
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