New Routes and Opportunities for Modular Construction of Particulate Vaccines: Stick, Click, and Glue

Abstract

Vaccines based on virus-like particles (VLPs) can induce potent B cell responses. Some non-chimeric VLP-based vaccines are highly successful licensed products (e.g., hepatitis B surface antigen VLPs as a hepatitis B virus vaccine). Chimeric VLPs are designed to take advantage of the VLP framework by decorating the VLP with a different antigen. Despite decades of effort, there have been few licensed chimeric VLP vaccines. Classic approaches to create chimeric VLPs are either genetic fusion or chemical conjugation, using cross-linkers from lysine on the VLP to cysteine on the antigen. We describe the principles that make these classic approaches challenging, in particular for complex, full-length antigens bearing multiple post-translational modifications. We then review recent advances in conjugation approaches for protein-based non-enveloped VLPs or nanoparticles, to overcome such challenges. This includes the use of strong non-covalent assembly methods (stick), unnatural amino acids for bio-orthogonal chemistry (click), and spontaneous isopeptide bond formation by SpyTag/SpyCatcher (glue). Existing applications of these methods are outlined and we critically consider the key practical issues, with particular insight on Tag/Catcher plug-and-display decoration. Finally, we highlight the potential for modular particle decoration to accelerate vaccine generation and prepare for pandemic threats in human and veterinary realms.

Keywords: SpyCatcher; bioconjugation; click chemistry; malaria; synthetic biology; vaccinology; virus-like particle.

Figure 1

Chimeric virus-like particles (VLPs) and classic approaches for their decoration. (A) Importance of the particulate state for immunogenicity. (B) Chimeric VLPs leverage the multimeric nature of a scaffold for increased immunogenicity. (C) Genetic fusion for chimeric VLP assembly. The gene encoding an antigen of interest is fused to the gene encoding the multimerizing protein. Self-assembly leads to multimerization and ordered display of the antigen. Problems encountered may be (i) structural distortion of antigen or scaffold, which may lead to failed VLP assembly or induction of ineffective antibodies, or (ii) post-translational modification by the host may not be ideal for both antigen and multimerization platform. (D) Chemical cross-linking for chimeric VLP assembly. Side chains on the antigen and VLP are connected by a cross-linker, e.g., SMPH [succinimidyl 6-(β-maleimidopropionamido)hexanoate]. Problems encountered can be (i) distortion of structure of antigen or scaffold from uncontrolled conjugation, (ii) uneven decoration of VLP with antigen, and (iii) inter-particle cross-linking and subsequent impaired solubility.

Figure 2

Stick or click approaches for decoration of virus-like particle (VLP) vaccines. Non-covalent decoration of VLPs is represented by nickel-affinity or biotin/streptavidin decoration (pink). Covalent decoration of VLPs is represented in gray. We illustrate unnatural amino acid incorporation via global incorporation of azidohomoalanine in the VLP, sortase ligation, or split intein trans-splicing. The antigen is represented in gold; not to scale.

Figure 3

Plug-and-display virus-like particle (VLP) decoration. (A) Plug-and-display principle. SpyCatcher was genetically fused to the AP205 coat protein and expressed in Escherichia coli. Upon mixing, different SpyTag antigens form spontaneous isopeptide bonds, yielding decorated particles for immunization. (B) Catcher/Tag chemistry. SpyCatcher/SpyTag side chains form a covalent bond with release of water. Reaction of SnoopCatcher with SnoopTag yields a covalent bond and release of ammonia. (C) Quantitative coupling of Spy-VLPs with antigen. SpyCatcher-VLPs were incubated with SpyTag-maltose-binding protein (MBP) or the negative control SpyTagDA-MBP, before SDS-PAGE with Coomassie staining. Data from Ref. (55) with permission. (D) Improved antibody quality by Spy-VLP display. Antibody avidity (based on resistance to 8M urea) was assessed on days 35 and 56 in serum from mice immunized with Pfs25-SpyCatcher and untagged or dual SpyTag-VLPs. Data from Ref. (75) with permission. (E) Malaria transmission blocking with Spy-VLPs. Pfs25-IMX313 is a genetically fused nanoparticle. Qβ-Pfs25 is a chemically decorated VLP. Pfs25-SpyTag:SpyCatcher-AP205 is a plug-and-display conjugate. Total IgG, purified from pooled serum after immunization, was analyzed by standard membrane feeding assay. Data points represent oocysts in a mosquito, and the lines show the arithmetic mean. Data from Ref. (39) with permission. (F) Dual plug-and-display. Expression in E. coli and spontaneous multimerization yields SpyCatcher-IMX-SnoopCatcher nanoparticles. SpyTag linked to antigen 1 (Ag1) or an immunostimulatory molecule (ISM) react with SpyCatcher, while the other nanoparticle face is decorated by SnoopTag/SnoopCatcher reaction.

Figure 4

Looking to the future for modular vaccine assembly. (Top left) Stockpiling one underlying particulate scaffold against multiple diseases may facilitate cheap rapid production of vaccines, in the face of pandemics, bioterrorism, and tropical diseases. (Top right) Personalized therapeutic vaccines are coming into view through next-generation sequencing identifying cancer neo-epitopes. One may envision neo-epitopes being chemically synthesized and coupled specifically to a virus-like particle (VLP) scaffold for immunization. (Bottom left) On-site synthesis of Tag-antigens becomes possible with cell-free protein synthesis stable at ambient temperature. Subsequent coupling to pre-assembled virus-like nanoparticles may allow in-field vaccine generation, with minimal space and weight footprint for a large number of vaccine targets. (Bottom right) Stockpiling may drive down costs of VLP vaccines for animal health applications, to combat zoonoses, for pets and for agriculture.