No viable virus remained detectable in the A438. RabG uses the chimpanzee adenovirus serotype 68 (AdC68) backbone previously shown to achieve pre-exposure protection against rabies in non-human primates. ChAdOx2 differs from AdC68 in that it contains the human adenovirus serotype 5 (AdHu5) E4 orf6/7 region in place of the AdC68 equivalents, enhancing ease of manufacturing in ODM-203 cell lines which provide AdHu5 E1 proteins still receive PEP, but this post-PrEP ODM-203 PEP is an abbreviated and much less expensive course over one or two visits, without RIG [9]. Importantly, data suggest that PrEP relative to PEP-based strategies in many contexts, particularly if the cost of PrEP is beneath USD 4 per child [8, 18]. Child-population-wide PrEP is thus an attractive intervention in areas in which Expanded Programme on Immunization (EPI) vaccines are delivered but which have otherwise limited capacity for reliable urgent PEP or for control of rabies-transmitting animals. This role for mass PrEP has been recognised, for example, in the Peruvian Amazon: in this setting, vampire bat rabies is problematic and difficult to control, access to PEP is limited, and a PrEP programme appears to have been successful [15]. Here, we have set out to develop a tool intended to enable cost-effective PrEP against rabies within routine population-wide immunization programmes. The immunological mechanism of vaccine-induced immunity against rabies is well characterised. A virus neutralizing antibody (VNA) titer exceeding 0.5 international units per milliliter (IU/mL) is accepted as a marker of adequate immunization [19]. This threshold is thought by many to signify clinical efficacy: indeed, in animal challenge studies, 100% protection is achieved at 0.1C0.2 IU/mL, with EIF4EBP1 incomplete but substantial protection at even lower titers [20]. Simian adenovirus-vectored vaccines are an attractive platform technology for induction of antibody responses, circumventing the problem of pre-existing anti-vector antibody to human adenovirus ODM-203 serotypes and readily manufacturable at large scale and low cost [21, 22]. A chimpanzee adenovirus serotype 68 (AdC68)-vectored rabies vaccine and the ability of a single low dose of this vaccine to achieve long-lasting protection against rabies challenge in non-human macaques has been reported previously [23, 24]. We now describe the development of a closely-related simian-adenovirus-vectored rabies vaccine, ChAdOx2 RabG, which is suitable for good manufacturing practice (GMP)-compliant production. We also describe additional approaches which may make this particularly suitable for use in low-income settings, namely thermostabilisation of the vaccine and dose-sparing adjuvantation. ChAdOx2 RabG may prove to be suitable for PrEP in highly rabies-endemic settings which have adequate infrastructure to achieve appreciable levels of childhood immunization coverage [25] but which currently lack capacity for reliable dog vaccination or PEP. Methods Plasmid and adenovirus production Plasmid pC68 010-Rabgp comprising the E1- and E3-deleted AdC68 genome with the full-length ERA strain rabies virus glycoprotein coding sequence under a human cytomegalovirus immediate-early (HCMV-IE) promoter was constructed based on a virus obtained from ATCC (ATCC VR-594, Genbank accession: “type”:”entrez-nucleotide”,”attrs”:”text”:”FJ025918.1″,”term_id”:”219522365″,”term_text”:”FJ025918.1″FJ025918.1). The wildtype AdC68 was propagated in HEK 293 cells and purified by CsCl gradient centrifugation, followed by viral genomic DNA purification as described [26]. To generate the E1-deleted AdC68 molecular clone, the 5 right inverted terminal repeat (ITR) was amplified by PCR and cloned into the pNEB193 vector. Using restriction enzyme sites that are unique in assembly but not necessarily unique to the full AdC68 genome, approximately 2.6 Kb of the E1 region between SnaBI and NdeI sites (from 455bp to 3028bp) were removed and replaced with a linker which contains the rare enzyme sites of I-CeuI and PI-SceI. The resultant was the pC68 000 plasmid. To delete the E3 domain, a 3.6 kb fragment was excised using AvrII and NruI (from 27793bp to.