Finding the Goldilocks zone: modulating glycoprotein cleavage and fusogenicity optimizes the efficacy of a candidate Crimean-Congo hemorrhagic fever virus vaccine

Crimean-Congo hemorrhagic fever virus (CCHFV) is the etiologic agent of a highly lethal hemorrhagic disease spread by ticks throughout Europe, the Middle East, Africa, and Asia, for which no FDA- or EMA-approved vaccines or therapeutics exist. A lack of fundamental understanding of the molecular mechanisms of CCHFV assembly, egress, and entry has challenged the development of medical countermeasures. Herein, we investigated the molecular basis of replicative efficacy of a highly protective recombinant vesicular stomatitis virus vaccine bearing the CCHFV glycoprotein complex (rVSV-GPC). We identified key amino acid residues associated with rVSV-GPC adaptation and showed that these could be transferred to a divergent CCHFV genetic background to afford a new vaccine vector with enhanced protective efficacy against heterologous viral challenge. We show that the key amino acid substitutions enhance rVSV-GPC infectivity via multiple distinct biochemical and cell-biological mechanisms. Our mechanistic studies provide the first evidence that SKI-1 cleavage at the GP38-Gn boundary modulates the pH threshold of CCHFV membrane fusion and points to a critical role for virion-associated GP38 and GP38-Gn interaction in regulating the pre-fusion conformation of the glycoprotein spike during virion biogenesis, egress, and cell entry. IMPORTANCE: Herein, we investigated the molecular basis of replicative efficacy of a highly protective rVSV-GPC vaccine candidate. We identified key amino acid residues associated with rVSV-GPC adaptation, including two novel residues, and showed that a minimal set of these could be transferred to a divergent CCHFV genetic background to afford a new vaccine vector with enhanced protective efficacy against heterologous viral challenge. Our work provides important new information to refine VSV-vectored CCHFV vaccines, to assess which critical GPC adaptations might benefit other vaccine modalities such as subunit and RNA, and to aid in the development of vaccines against divergent orthonairoviruses. Our findings also shed new light on the basic biology of CCHFV. We show that the key amino acid substitutions enhance rVSV-GPC infectivity via multiple, distinct biochemical and cell-biological mechanisms. Most notably, we provide the first evidence that SKI-1 cleavage at the GP38-Gn boundary modulates the pH threshold of viral membrane fusion. This points to an unanticipated role for virion-associated GP38 and GP38-Gn interaction in protecting and regulating the pre-fusion conformation of GPC during CCHFV biogenesis, egress, and cell entry.