The four R-spondin (Rspo) proteins are secreted agonists of Wnt signalling in vertebrates, functioning in embryogenesis and adult stem cell biology. Through ubiquitination and degradation of Wnt receptors, the transmembrane E3 ubiquitin ligase ZNRF3 and related RNF43 antagonize Wnt signalling. Rspo ligands have been reported to inhibit the ligase activity through direct interaction with ZNRF3 and RNF43. Here we report multiple crystal structures of the ZNRF3 ectodomain (ZNRF3 ecto ), a signalling-competent Furin1-Furin2 (Fu1-Fu2) fragment of Rspo2 (Rspo2 Fu1-Fu2 ), and Rspo2 Fu1-Fu2 in complex with ZNRF3 ecto , or RNF43 ecto . A prominent loop in Fu1 clamps into equivalent grooves in the ZNRF3 ecto and RNF43 ecto surface. Rspo binding enhances dimerization of ZNRF3 ecto but not of RNF43 ecto .Comparison of the four Rspo proteins, mutants and chimeras in biophysical and cellular assays shows that their signalling potency depends on their ability to recruit ZNRF3 or RNF43 via Fu1 into a complex with LGR receptors, which interact with Rspo via Fu2.

Objectives Vaccination is considered the most successful health intervention; yet incomplete immunisation coverage continues to risk outbreaks of vaccine preventable diseases worldwide. Vaccination coverage improvement through a singledose prime-boost technology would revolutionise modern vaccinology, impacting on disease prevalence, significantly benefiting health care and lowering economic burden of disease. Key findings Over the past 30 years, there have been efforts to develop a singledose delayed release vaccine technology that could replace the repeated primeboost immunisations required for many current vaccines. Biocompatible polymers have been employed to encapsulate model vaccines for delayed delivery in vivo, using either continuous or pulsed release. Biomaterial considerations, safety aspects, particle characteristics and immunological aspects of this approach are discussed in detail. Summary Despite many studies showing the feasibility of vaccine encapsulation for single-dose prime-boost administration, none have been translated into convincing utility in animal models or human trials. Further development of the encapsulation technology, through optimising the particle composition, formulation, antigen loading efficacy and stability, could lead to the application of this important approach in vaccine deployment. If successful, this would provide a solution to better global vaccination coverage through a reduction in the number of immunisations needed to achieve protection against infectious diseases. This review provides an overview of single-dose vaccination in the context of today's vaccine needs and is derived from a body of literature that has not been reviewed for over a decade.