Controlled Modular Multivalent Presentation of the CD40 Ligand on P22 Virus-like Particles Leads to Tunable Amplification of CD40 Signaling

Abstract

Ligands of the tumor necrosis factor superfamily (TNFSF) are appealing targets for immunotherapy research due to their integral involvement in stimulation or restriction of immune responses. TNFSF-targeted therapies are currently being developed to combat immunologically based diseases and cancer. A crucial determinant of effective TNFSF receptor binding and signaling is the trimeric quaternary structure of the ligand. Additionally, ligand multivalency is essential to propagate strong signaling in effector cells. Thus, designing a synthetic platform to display trimeric TNFSF ligands in a multivalent manner is necessary to further the understanding of ligand-receptor interactions. Viral nanocages have architectures that are amenable to genetic and chemical modifications of both their interior and exterior surfaces. Notably, the exterior surface of virus-like particles can be utilized as a platform for the modular multivalent presentation of target proteins. In this study, we build on previous efforts exploring the bacteriophage P22 virus-like particle for the exterior multivalent modular display of a potent immune-stimulating TNFSF protein, CD40 ligand (CD40L). Using a cell-based reporter system, we quantify the effects of tunable avidity on CD40 signaling by CD40L displayed on the surface of P22 nanocages. Multivalent presentation of CD40L resulted in a 53.6-fold decrease of the half maximal effective concentration (EC₅₀) compared to free CD40L, indicating higher potency. Our results emphasize the power of using P22-based biomimetics to study ligand-receptor interactions within their proper structural context, which may contribute to the development of effective immune modulators.

Keywords: CD40; CD40L; P22; TNFSF; VLP; biomimetics; multivalency; signaling.

Figure 1.

CD40L is at the gateway between the innate and adaptive immune systems. Antigen is processed by an antigen-presenting cell (APC), such as a B cell or a dendritic cell (DC). Peptides derived from the antigen are presented via major histocompatibility complex (MHC) II cell surface proteins, which engage a specific CD4+ T cell through its T cell receptor (TCR). The CD40L–CD40 costimulatory interaction triggers the APC to mature and become activated, leading to an effective adaptive immune response.

Figure 2.

Strategy for multivalent DechCD40L presentation mediated by Dec binding to P22. Dec is genetically fused to hCD40L resulting in a soluble fusion protein, DechCD40L, which self-trimerizes and can be bound to P22 by simply mixing. The multivalent nature of the bound cargo allows for cross-linking of CD40 receptors on the surface of effector cells.

Figure 3.

Design of DechCD40L. N-terminus of the soluble portion of the human CD40L was fused to a flexible linker (GSSGS) preceded by Dec and a 6x histidine tag. Expressed DechCD40L self-assembles into homotrimers.

Figure 4.

DechCD40L can be purified as a soluble product and readily binds to P22. DechCD40L or Dec was mixed with P22 for 30 min at a ratio of 240 trimers per capsid followed by ultracentrifugation. Proteins successfully bound to P22 were detected by denaturing SDS polyacrylamide gel electrophoresis and Western blot analysis. (A) SDS-PAGE of nickel-affinity purified DechCD40L displaying a major band for DechCD40L near the expected molecular weight of 32.7 kDa. (B) SDS-PAGE of DechCD40L or Dec bound to P22. Arrows point to bands at the expected molecular weights for DechCD40L (32.7 kDa), P22 coat protein (CP, 47 kDa), and Dec (14.1 kDa). (C) Western blot analysis of DechCD40L bound to P22 compared to Dec bound to P22. The left blot is developed with an anti-P22 polyclonal mixture. The right blot is developed with an anti-Dec polyclonal mixture. (D) Purified DechCD40L developed with monoclonal anti-hCD40L. Soluble hCD40L is included for reference.

Figure 5.

DechCD40L binds expanded P22 particles at the expected quasi threefold and true threefold sites. Additional density (red) can be seen at the anticipated Dec-binding sites when cryo-EM reconstructions of (A) expanded P22 particles were compared to (B) DechCD40L-decorated P22 particles. This added density has a threefold symmetric base with a central pillar extending away from the capsid surface and terminating in a poorly resolved globular head.

Figure 6.

Partial decoration of P22 with Dec. SEC-MALS was utilized to analyze Dec-P22 with multiple stoichiometries of the Dec trimer to P22. (A) Measured molecular weight of the particle versus number of Dec trimers. (B) Number of input Dec trimers plotted against the number of output Dec trimers derived from the molecular weight shows linear correlation (r = 0.64) between 0 and 80 trimers and no additional increase in molecular weight at greater than 80 Dec trimers.

Figure 7.

Controlled multivalent display of DechCD40L exhibits tunable signaling. Normalized cellular response is reported as percent activation compared to the maximum response at 1.5 nM DechCD40L-P22. Error bars indicate the standard error of the mean. (A) Concentration-dependent response was observed and shown to be strongest for DechCD40L displayed on P22 compared to unbound DechCD40L, hCD40L, and an admixture of P22 and hCD40L. Lines indicate Hill function fits. (B) Concentration of DechCD40L was held constant, while the amount of P22 varied leading to a range from low multivalency state (VLP oversaturated) to DechCD40L oversaturated (VLP starved), with the optimal stoichiometry (60 trimers/capsid) resulting in fully decorated particles and no unbound ligand. (C) Varying the amounts of P22 in constant 9 nM DechCD40L or hCD40L showed changes in signaling intensity only for DechCD40L. Lines are interpolations between points. (D) Percent activation of DechCD40L-P22 over the control hCD40L/P22 admixture at each ratio. Varying the background CD40L concentration reveals that the effect of changing CD40L valency is most notable at 3 and 9 nM. Lines are interpolations between points.