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Self-amplifying RNA vaccines may induce equivalent or more potent immune responses at lower doses compared to non-replicating mRNA vaccines via amplified antigen expression. In this paper, we demonstrate that 1 μg of an LNP-formulated dual-antigen self-amplifying RNA vaccine (ZIP1642), encoding both the S-RBD and N antigen, elicits considerably higher neutralizing antibody titers against Wuhan-like Beta B.1.351 and Delta B.1.617.2 SARS-CoV-2 variants compared to those of convalescent patients. In addition, ZIP1642 vaccination in mice expanded both S- and N-specific CD3 + CD4 + and CD3 + CD8 + T cells and caused a Th1 shifted cytokine response. We demonstrate that the induction of such dual antigen-targeted cell-mediated immune response may provide better protection against variants displaying highly mutated Spike proteins, as infectious viral loads of both Wuhan-like and Beta variants were decreased after challenge of ZIP1642 vaccinated hamsters. Supported by these results, we encourage redirecting focus toward the induction of multiple antigen-targeted cell-mediated immunity in addition to neutralizing antibody responses to bypass waning antibody responses and attenuate infectious breakthrough and disease severity of future SARS-CoV-2 variants., Competing Interests: Declaration of interest S.M., A.K.M.A.H., A.V., B.W., M.P., M.S., S.V., and I.S. are current employees of Ziphius Vaccines NV and may own Ziphius shares/warrants. S.M., A.K.M.A.H., S.V., I.S., and N.N.S. are inventors on a patent application claiming subject matter related to the SARS-CoV-2 saRNA vaccine candidates described herein. The Virology Unit of the Institute of Tropical Medicine, supervised by K.K.A., and the Department of Microbiology, Immunology, and Transplantation of Rega Institute for Medical Research (KUL), supervised by J.N., received compensation from Ziphius Vaccines NV to perform the neutralization assays and hamster studies, respectively. The authors declare no other competing interests., (Copyright © 2022 The Author(s). Published by Elsevier Inc. All rights reserved.)

To engineer tissues with clinically relevant dimensions by three-dimensional bioprinting, an extended vascular network with diameters ranging from the macro- to micro-scale needs to be integrated. Extrusion-based bioprinting is the most commonly applied bioprinting technique but due to the limited resolution of conventional bioprinters, the establishment of a microvascular network for the transfer of oxygen, nutrients and metabolic waste products remains challenging. To answer this need, this study assessed the potential and processability of spheroids, containing a capillary-like network, to be used as micron-sized prevascularized units for incorporation throughout the bioprinted construct. Prevascularized spheroids were generated by combining endothelial cells with fibroblasts and adipose tissue-derived mesenchymal stem cells as supporting cells. To serve as a viscous medium for the bioink-based deposition by extrusion printing, spheroids were combined with a photo-crosslinkable methacrylamide-modified gelatin (gelMA) and Irgacure 2959. The influence of gelMA encapsulation, the printing process and photo-crosslinking conditions on spheroid viability, proliferation and vascularization were analyzed by live/dead staining, immunohistochemistry, gene expression analysis and sprouting analysis. Stable spheroid-laden constructs, allowing spheroid outgrowth, were achieved by applying 10 min UV-A photo-curing (365 nm, 4 mW cm -2 ), while the construct was incubated in an additional Irgacure 2959 immersion solution. Following implantation in ovo onto a chick chorioallantoic membrane, the prevascular engineered constructs showed anastomosis with the host vasculature. This study demonstrated (a) the potential of triculture prevascularized spheroids for application as multicellular building blocks, (b) the processability of the spheroid-laden gelMA bioink by extrusion bioprinting and (c) the importance of photo-crosslinking parameters post printing, as prolonged photo-curing intervals showed to be detrimental for the angiogenic potential and complete vascularization of the construct post printing., (© 2021 IOP Publishing Ltd.)