One-Pot Expression and Assembly of Resurfaced Zika Virus E Domain III Nanoparticle Immunogens in Mammalian Cells

Abstract

The presentation of antigens on self-assembling nanoparticles is a promising approach for augmenting the B-cell response of vaccines. In many cases, the nanoparticle and antigen are recombinantly expressed and purified separately prior to conjugation, requiring multiple steps that may be inefficient for large-scale production. Here, we describe a genetically encodable method and "one-pot" production of nanoparticle immunogens from mammalian cells. Our approach relies on the in situ assembly of the nanoparticle-antigen conjugate, between protein nanoparticles bearing SpyCatcher003 and antigen fused to SpyTag003, using simple cotransfection in mammalian cells, which can then be purified to homogeneity in a single step. We demonstrate this method with an antigen based on Zika virus E glycoprotein domain III ("rsZDIII-2.39") and compare the biochemical properties and immunogenicity in mice relative to genetic fusions. This work provides a framework for streamlining immunogen production and raises the possibility of employing such approaches for encoding nanoparticle immunogens as nucleic acid vaccines.

1

Optimization of immunogen production involving resurfaced ZIKV EDIII 2.39 (rsZDIII-2.39) nanoparticles. (A) ZIKV-Fabs (ZV-2, red; ZV-67, green; ZV-64, blue) in complex with ZIKV EDIII (PDB: 5KVD, 5KVG, and 5KVF, respectively), which were used to develop rsZDIII-2.39, in which critical contact residues in the ABDE sheet (orange spheres) and CC′-loop (purple spheres) were masked by mutation and lateral ridge epitope (light green) preserved. These mutations were designed to dampen the immune response to the CC′-loop and ABDE sheet while amplifying the immune response to the LR region. (B) Schematic of the traditional approach to express, conjugate, and purify immunogen components (nanoparticle, blue; antigen, orange). (C) Single-pot expression and purification of the nanoparticle immunogen, potentially increasing the production efficiency.

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Engineering rsZDIII-2.39 nanoparticles via direct fusion or in situ conjugation to A. aeolicus lumazine synthase (aaLS). (A) Schematic representation of mammalian expression vectors used to produce a panel of fusion and in situ-conjugated constructs. Erythropoietin signal sequence (Epo SS) enabled the release of the translated construct into the extracellular space. The His6 tag allowed for Ni-NTA affinity purification. (B) Approaches to link the aaLS monomer and rsZDIII-2.39. Direct fusion nanoparticle constructs utilized a linker domain between aaLS and rsZDIII-2.39 referred to as “PseudoSpy”, which encoded inert SpyCatcher and SpyTag as a genetic fusion to one another (structural model modified from PDB: 4MLI). Alternatively, a quadruple GGGS linker was used (“qGGGS”). (C) Modeled structural cross section of an aaLS-rsZDIII-2.39 nanoparticle. aaLS protomers in center multicolored, SpyCatcher-SpyTag in blue-yellow, and rsZDIII-2.39 in red (PDB: 1HQK, 4MLI, and 5KVG, respectively). (D) Schematic of two-component transfection approaches to generate aaLS–aaLS0 by in situ conjugation.

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Expression and characterization of purified aaLS-rsZDIII-2.39 nanoparticle immunogens. (A) SDS-PAGE gel demonstrating that one-step Ni-NTA purification yields high purity, soluble nanoparticle immunogens. (B) Binding reactivity of ZIKV-mAb ZV-67 to purified nanoparticle immunogens determined by ELISA. (C) Predominant particle size (left Y axis, colored bars) and percent of the particle size (right Y axis, colored line with percentages represented as Xs) in purified nanoparticle immunogens, determined by dynamic light scattering. (D) Negative stain microscopy of aaLS-PseudoSpy-2.39 preparations.

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Immunogenicity of aaLS–aaLS nanoparticle immunogens adjuvanted with Addavax. (A) Mouse immunization and blood collection schedule. (B) Day 56 serum reactivity to ZIKV EDIII WT from two sets of mice for each immunization group (n = 7–10) determined by ELISA. Data graphed as mean EC₅₀ values for each animal in duplicate. (C) Day 56 FRNT neutralization titers for each immunization group. Data graphed as mean FRNT₅₀ values from serum pooled by the immunization group (n = 7–10), from three independent experiments in duplicate. (Cross-reactivity and FRNT neutralization analysis: two-way ANOVA with Tukey’s multiple comparisons test: **p < 0.01; ***p < 0.001; ****p < 0.0001).