Novel Design Architecture for Genetic Stability of Recombinant Poliovirus: the Manipulation of G/C Contents and Their Distribution Patterns Increases the Genetic Stability of Inserts in a Poliovirus-Based RPS-Vax Vector System

Abstract

Poliovirus has been studied as a live recombinant vaccine vector because of its attractive characteristics. The genetic instability, however, has hampered recombinant polioviruses (PVs) from being developed as an appropriate vaccine. A variety of different foreign inserts were cloned directly into our poliovirus Sabin 1-based RPS-Vax vector system, resulting in the production of recombinant PVs. The genetic stability of each recombinant PV was examined during 12 rounds of consecutive passage. It was found that the genetic stability of the recombinants was not well correlated with their insert size. Instead, elevated stability was frequently observed in recombinants with inserts of high G/C contents. Furthermore, a comparative study using different constructs of the human immunodeficiency virus env gene revealed that the internal deletion of the unstable insert was seemingly caused by the presence of the adjacent A/T-rich region. The instability of these inserts was completely remedied by (i) increasing the G/C contents and (ii) replacing the local A/T-rich region with the G/C-rich codon without a change of the amino acid. This means that stability is closely associated with the G/C content and the G/C distribution pattern. To see whether these findings can be applied to the design of genetically stable recombinant PV, we have reconstructed the heteromultimeric insert based on our design architecture, including the above-mentioned G/C rules and the template/ligation-free PCR protocol. The heteromultimeric insert was very unstable, as expected, but the manipulated insert with the same amino acid sequence showed complete genetic stability, not only in vitro, but also in vivo. Even though this guideline was established with our RPS-Vax vector system, to some extent, it can also be applied to other live viral vaccine vectors.Many attempts have been made to manipulate poliovirus (PV) as a favorable vaccine vector because of its attractive characteristics of safe use, low cost, convenient administration, and long-lasting protective immunity in both mucosal and systemic immune responses, which have been established for decades. Four different strategies have been employed to date in the development of PV-based versatile vaccine vectors. Early efforts were devoted to attaching small epitopes to one of the neutralizing domains in the capsid protein (9, 10, 14, 17, 36). However, widespread application of this hybrid virion strategy was limited by the size of the epitope (smaller than 25 amino acids) that could be inserted. The second strategy was to replace one of the structural viral genes with the protein insert (8,9,12,30,34,35). Using this method, desired foreign antigens could be incorporated without size restriction; however, the recombinant virus is replication defective and requires a helper virus or capsid protein-expressing system for each cycle of propagation (12,30,34,35). The propagation-defective viral vector is attractive for its safety feature, even in the neuronal delivery of cytokine (8), but may also pos...

Enumeration and Functional Evaluation of Virus-Specific CD4 + and CD8 + T Cells in Lymphoid and Peripheral Sites of Coxsackievirus B3 Infection

“…2B), and sequencing of 15 individual clones showed that the epitopes were intact. The reason for the unusual stability of this sequence is not clear; others have proposed that a high G/C content confers stability on poliovirus inserts (29), but our data do not allow us to draw conclusions regarding the insert in rCVB3.6. However, we can conclude that the low immunogenicity of rCVB3.6 is unlikely to be caused by the rapid loss of epitope sequence.…”

A host dicer is required for defective viral RNA production and recombinant virus vector RNA instability for a positive sense RNA virus

“…Rapid recombination also appears to be an underlying cause of the instability and deletion of foreign, nonviral sequences from recombinant viral RNA vectors (6; reviewed in 7). Genome instability presents one of the most important obstacles to the use of recombinant RNA viruses as gene expression vectors for various practical applications, including gene therapy (8)(9)(10).…”

Development of oral CTL vaccine using a CTP-integrated Sabin 1 poliovirus-based vector system

“…Linearized plasmid DNA was transcribed in vitro with T7 RNA polymerase (NEB) for 1 h at 37 • C. Template DNA was eliminated after transcription with RNase-free DNase I (NEB) for 30 min at 37 • C. Transcripts were transfected into HeLa cells with DEAE-dextran process as described previously [22]. When cytopathic effects (CPEs) were observed, supernatants were transferred into fresh HeLa cell cultures to amplify progeny viruses.…”

“…Foreign inserts have been integrated and expressed from the poliovirus Mahoney-originated vector [19,21], or poliovirus Sabin 3-based vector [20]. We have constructed and reported a recombinant poliovirus Sabin 1-based vaccine vector named RPS-Vax system [22]. This vector has multiple cloning sites and an artificial 3C protease-recognition site at the N-terminal end of the Sabin 1 polyprotein.…”