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Although this chimeric virus was tolerated overall by volunteers, it still caused viremia and adverse effects such as fever and arthritis, suggesting that it might not be sufficiently attenuated. In this study, the VSV\/EBOV-GP vector was further modified in order to achieve attenuation while maintaining immunogenicity. All recombinant VSV constructs were propagated on VSV G protein expressing helper cells and used to immunize guinea pigs via the intramuscular route. The humoral immune response was analysed by EBOV-GP-specific fluorescence-linked immunosorbent assay, plaque reduction neutralization test and <jats:italic>in vitro<\/jats:italic> virus-spreading inhibition test that employed recombinant VSV\/EBOV-GP expressing either green fluorescent protein or secreted Nano luciferase. Most modified vector constructs induced lower levels of protective antibodies than the parental VSV\/EBOV-GP or a recombinant modified vaccinia virus Ankara vector encoding full-length EBOV-GP. However, the VSV\/EBOV-GP(F88A) mutant was at least as immunogenic as the parental vaccine virus although it was highly propagation-restricted. This finding suggests that VSV-vectored vaccines need not be propagation-competent to induce a robust humoral immune response. 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