SnoopLigase peptide-peptide conjugation enables modular vaccine assembly

Abstract

For many infectious diseases there is still no vaccine, even though potential protective antigens have been identified. Suitable platforms and conjugation routes are urgently needed to convert the promise of such antigens into broadly protective and scalable vaccines. Here we apply a newly established peptide-peptide ligation approach, SnoopLigase, for specific and irreversible coupling of antigens onto an oligomerization platform. SnoopLigase was engineered from a Streptococcus pneumoniae adhesin and enables isopeptide bond formation between two peptide tags: DogTag and SnoopTagJr. We expressed in bacteria DogTag linked to the self-assembling coiled-coil nanoparticle IMX313. This platform was stable over months at 37 °C when lyophilized, remaining reactive even after boiling. IMX-DogTag was efficiently coupled to two blood-stage malarial proteins (from PfEMP1 or CyRPA), with SnoopTagJr fused at the N- or C-terminus. We also showed SnoopLigase-mediated coupling of a telomerase peptide relevant to cancer immunotherapy. SnoopLigase-mediated nanoassembly enhanced the antibody response to both malaria antigens in a prime-boost model. Including or depleting SnoopLigase from the conjugate had little effect on the antibody response to the malarial antigens. SnoopLigase decoration represents a promising and accessible strategy for modular plug-and-display vaccine assembly, as well as providing opportunities for robust nanoconstruction in synthetic biology.

Figure 1

Overview of SnoopLigase-mediated vaccine assembly. IMX is fused to DogTag at its C-terminus. When expressed in E. coli, spontaneous oligomerization yields IMX−DogTag nanoparticles. SnoopLigase promotes isopeptide bond formation upon mixing of IMX-DogTag with antigen-SnoopTagJr. The RrgA C-terminal domain (Protein Data Bank 2WW8) was previously split into three parts and engineered. The reactive Lys is located on SnoopTagJr (cyan), the reactive Asp on DogTag (yellow), and the catalytic Glu on SnoopLigase (gray) (key residues are highlighted in red).

Figure 2

IMX-DogTag purification and resilience to heat and lyophilization. (a) Nanoparticle purification. IMX−DogTag was induced in E. coli (total lysate). Protein was purified by treatment with heat, glycine, PEI, (NH₄)₂SO₄, and a final ion exchange (IEX) step. Samples were analyzed by SDS-PAGE and Coomassie staining under reducing conditions except where indicated. (b) IMX-DogTag conjugation with indicated proteins. SnoopTagJr-AffiHER2, SnoopTagJr-MBP or SnoopTagJr-CIDR at 20 μM were conjugated to 10 μM IMX-DogTag using 20 μM SnoopLigase for 48 h at 4 °C. Samples were boiled in the presence of reducing agent, and analyzed by SDS-PAGE and Coomassie staining. (c) IMX-DogTag reactivity after exposure to different temperatures. IMX-DogTag was treated for 2 h at the indicated temperature and cooled for 30 min at 12 °C. Heat-treated IMX-DogTag was reacted with SnoopTagJr-AffiHER2 with each protein at 10 μM for 2 h at 4 °C. Reaction was analyzed by SDS-PAGE with Coomassie staining and the unheated sample set at 100% (mean of triplicate ± s.d.; some error bars are too small to be visible). (d) IMX-DogTag reactivity after storing lyophilized at 37 °C. IMX-DogTag was lyophilized and incubated for the indicated days at 37 °C. IMX-DogTag was then reacted with SnoopTagJr-AffiHER2 and SnoopLigase for 2 h at 4 °C, before analyzing under reducing conditions by SDS-PAGE with Coomassie staining.

Figure 3

IMX-DogTag conjugation to antigens. (a) Efficient covalent coupling to the nanoparticle by SnoopLigase. IMX-DogTag was incubated with 1.5-fold molar excess of SnoopTagJr−CIDR or CyRPA-SnoopTagJr for 48 h at 4 °C. Biotin-SnoopLigase was removed efficiently from IMX-DogTag:Antigen-SnoopTagJr conjugates after elution from Streptavidin-agarose, or a second round of Streptavidin-agarose purification (post-recapture). Samples were analyzed by reducing SDS-PAGE with Coomassie staining. (b) Hydrodynamic radius of IMX-DogTag before and after coupling to CIDR. The hydrodynamic radius (R_h) was determined by DLS for unconjugated IMX-DogTag and IMX-DogTag conjugated with SnoopTagJr-CIDR, with or without SnoopLigase removal. (c) Hydrodynamic radius of IMX-DogTag before and after coupling to CyRPA, as in (b). (d) IMX-DogTag reactivity with SnoopTagJr-Telo peptide. IMX-DogTag was reacted with SnoopTagJr-Telo in the presence of biotinylated SnoopLigase for 48 h at 4 °C and analyzed under reducing conditions by SDS-PAGE with Coomassie staining.

Figure 4

Nanoparticle immunization against CyRPA. (a) Schematic of the different immunogens used. (b) Schematic of time-points of immunization and analysis of immune responses. (c) CyRPA-specific IgG responses after nanoparticle immunization. Mice immunized with either IMX-DogTag:CyRPA-SnoopTagJr or IMX-DogTag:CyRPA-SnoopTagJr/SnoopLigase nanoparticles, CyRPA-SnoopTagJr or CyRPA (6 mice per group) at day 0, 19 and 35, were analyzed for CyRPA-specific responses at prime, boost I, and boost II by ELISA. Each dot represents an individual mouse. The horizontal line is the median. ^*p < 0.05 and ^**p < 0.01, determined using Kruskal-Wallis test (n = 6) with Dunn’s multiple comparison post-test.

Figure 5

Nanoparticle immunization against CIDR. (a) Schematic of the different immunogens used. (b) Schematic of time-points of immunization and analysis of immune responses. (c) CIDR-specific IgG responses after nanoparticle immunization. Mice immunized with either IMX-DogTag:SnoopTagJr-CIDR or IMX-DogTag:SnoopTagJr-CIDR/SnoopLigase nanoparticles or SnoopTagJr-CIDR (6 mice per group) at day 0, 13 and 32, were analyzed for CIDR-specific responses after prime, boost I, and boost II by ELISA. (d) Anti-platform response after immunization. Mice were immunized as in (c) and the IgG response to SnoopLigaseΔC or IMX-DogTag was determined by ELISA. Each dot represents an individual mouse. The horizontal line is the median. ^*p < 0.05 and ^**p < 0.01, determined using Kruskal-Wallis test (n = 6) with Dunn’s multiple comparison post-test.