Rational design of West Nile virus vaccine through large replacement of 3′ UTR with internal poly(A)

Abstract The genus Flavivirus comprises numerous emerging and re‐emerging arboviruses causing human illness. Vaccines are the best approach to prevent flavivirus diseases. But pathogen diversities are always one of the major hindrances for timely development of new vaccines when confronting unpredicted flavivirus outbreaks. We used West Nile virus (WNV) as a model to develop a new live‐attenuated vaccine (LAV), WNV‐poly(A), by replacing 5ʹ portion (corresponding to SL and DB domains in WNV) of 3ʹ‐UTR with internal poly(A) tract. WNV‐poly(A) not only propagated efficiently in Vero cells, but also was highly attenuated in mouse model. A single‐dose vaccination elicited robust and long‐lasting immune responses, conferring full protection against WNV challenge. Such “poly(A)” vaccine strategy may be promising for wide application in the development of flavivirus LAVs because of its general target regions in flaviviruses.


Appendix Figure S5
Appendix Figure S5. Construction of the infectious clone of WNV with internal poly(A)

insertion.
A. Flow chart of internal poly(A) insertion within infectious clone of WNV. Firstly, fragment A which covered nearly the whole NS5 gene and 6 nt at start from 3'-UTR was amplified from pACYC-WT-WNV template and inserted into pMD18T vector following transcribed into RNAs and added poly(A) tail in vitro. Then, RNAs with poly(A) tail were reverse transcribed into cDNA and subjected to fusion PCR with 3ꞌSL-sequence also amplified from pACYC-WT-WNV template, producing a long fragment named as NS5-polyA-3'SL. Finally, SpeI digested sequence of NS5-polyA-3'SL and PCR amplified long genome with T7 promoter was ligated in vitro to generate WNV-poly(A) infectious clone.
B. Left: Identification of in vitro transcriptional RNA of NS5 fragments. Right: Long DNA fragment of fusion PCR. Different lengths (1Kb, ~3Kb, 3Kb+) of DNA fragments were obtained. C.
Sequencing of different fusion DNA fragments. Poly(A) sequences were obtained when sequencing using forward or adverse primers.
A. Western Blotting assay for quantification of the levels of viral envelope protein in equal amounts (10 6 PFU) of WT and WNV-poly(A) viruses using anti-Envelope polyclonal antibody. ﹡represents non-specific signal.
B. Virulence study of WNV-poly(A). 4-week-old ICR mice (n=5/per group) were intracranially infected with different dosages of WNV-poly(A) or WT viruses and survival were monitored during 21 day-observation period. Log-rank test were used for survival analysis. ****p < 0.0001. C. Total anti-WNV IgG antibody of WNV-poly(A) and UV-inactivated WNV-poly(A). Six-weekold C57BL/6 (n=5/per group) mice were i.p immunized with 10 5 PFU of both WNV-poly(A) and UV-inactivated virus, respectively, and PBS immunized mice as the negative control. At 14 and 28 days after immunization, sera of mice were collected for ELISA assay. The data represent mean ± sd in each group and the horizontal dotted line represents the limit of detection. Student's T-tests were used to determine statistical analysis. ** p < 0.01, *** p < 0.001, ns represents not significant.

Appendix Figure S7
Appendix Figure S7. Plaque purified WNV-poly(A) for virulence and immunization study in C57BL/6 mice.
A-B. Virulence study. Four to six-week-old C57BL/6 mice (n=5 per group) were injected i.p. with 10 7 PFU of pooled WNV-poly(A), plaque purified WNV-poly(A) or PBS (negative control) in a volume of 200 μL. Viremia was quantified by plaque assay from days 1 to 3 post-infection. (The values represent mean ± sd in each group, each symbol represents a mouse. ***p< 0.001, ns represents not significant by one-way ANOVA analysis) (A) and survival was monitored daily till the end of experiment. (****p< 0.0001 by Log-rank test) (B).
C-F. Immunization study. Total anti-WNV IgG antibody (C) and neutralizing antibody titers (D) in mouse serum on the indicated days post immunization were determined. (For C and D, the values represent mean ± sd in each group and the horizontal dotted line represents the limit of detection. *p< 0.05,**p<0.01, *** p<0.001, ns represents not significant by one-way ANOVA analysis).
On day 30 post-immunization, mice were challenged i.p. with 3× 10 7 PFU of WT WNV. Viremia was quantified by plaque assay on day 2 post-challenge. (The values represent mean ± sd in each group, each symbol represents a mouse. ****p< 0.0001, ns represents not significant by one-way ANOVA analysis) (E). Survival was monitored daily for two weeks (****p< 0.0001 by Log-rank test) (F).