The adult retina is organized into three cellular layers--an outer photoreceptor, a middle interneuron and an inner retinal ganglion cell (RGC) layer. Although the retinal pigment epithelium (RPE) and Müller cells are important in the establishment and maintenance of this organization, the signals involved are unknown. Here we show that Sonic hedgehog signaling from RGCs is required for the normal laminar organization in the vertebrate retina.

Zika and chikungunya viruses have caused major epidemics and are transmitted by Aedes aegypti and/or Aedes albopictus mosquitoes. The “Sementis Copenhagen Vector” (SCV) system is a recently developed vaccinia-based, multiplication-defective, vaccine vector technology that allows manufacture in modified CHO cells. Herein we describe a single-vector construct SCV vaccine that encodes the structural polyprotein cassettes of both Zika and chikungunya viruses from different loci. A single vaccination of mice induces neutralizing antibodies to both viruses in wild-type and IFNAR−/− mice and protects against (i) chikungunya virus viremia and arthritis in wild-type mice, (ii) Zika virus viremia and fetal/placental infection in female IFNAR−/− mice, and (iii) Zika virus viremia and testes infection and pathology in male IFNAR−/− mice. To our knowledge this represents the first single-vector construct, multi-pathogen vaccine encoding large polyproteins, and offers both simplified manufacturing and formulation, and reduced “shot burden” for these often co-circulating arboviruses.

Chorioallantois vaccinia virus Ankara (CVA) is the parental virus of modified vaccinia virus Ankara (MVA), which was derived from CVA by more than 570 passages in chicken embryo fibroblasts (CEF). MVA became severely host-cell-restricted to avian cells and has strongly diminished virulence in mammalian hosts, while maintaining good immunogenicity. We determined the complete coding sequence of the parental CVA and mapped the exact positions of the six major deletions that emerged in the MVA genome. All six major deletions occurred in regions of the CVA genome where one or more truncated or fragmented open reading frames (ORFs) pre-existed. The CVA genome contains 229 ORFs of which 51 are fragments of full-length orthopoxvirus (OPV) genes, including fragmented orthologues of C9L and M1L (encoding two well-conserved ankyrin-like proteins), A39R (encoding a semaphorin-like protein) and A55R (encoding a kelch-like protein). Phylogenetic analysis demonstrated that MVA was most closely related to CVA, followed by the vaccinia virus (VACV) strain DUKE, a patient-derived isolate of the Dryvax vaccine virus. Loss or mutation of genes outside the six major deletions are assumed to contribute to the restricted host range phenotype of MVA. In support of this notion, deletions, insertions and non-synonymous mutations were found in 122 of the 195 ORFs remaining in MVA when compared with their CVA counterparts. Thus, detailed knowledge of the CVA genomic sequence is a prerequisite to further dissect the genetic basis of the MVA host range phenotype as well as the particular immunological properties of MVA.

Vaccinia-based systems have been extensively explored for the development of recombinant vaccines. Herein we describe an innovative vaccinia virus (VACV)-derived vaccine platform technology termed Sementis Copenhagen Vector (SCV), which was rendered multiplication-defective by targeted deletion of the essential viral assembly gene D13L. A SCV cell substrate line was developed for SCV vaccine production by engineering CHO cells to express D13 and the VACV host-range factor CP77, because CHO cells are routinely used for manufacture of biologics. To illustrate the utility of the platform technology, a SCV vaccine against chikungunya virus (SCV-CHIK) was developed and shown to be multiplication-defective in a range of human cell lines and in immunocompromised mice. A single vaccination of mice with SCV-CHIK induced antibody responses specific for chikungunya virus (CHIKV) that were similar to those raised following vaccination with a replication-competent VACV-CHIK and able to neutralize CHIKV. Vaccination also provided protection against CHIKV challenge, preventing both viremia and arthritis. Moreover, SCV retained capacity as an effective mouse smallpox vaccine. In summary, SCV represents a new and safe vaccine platform technology that can be manufactured in modified CHO cells, with pre-clinical evaluation illustrating utility for CHIKV vaccine design and construction.