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. 2018 Aug 16;92(17):e00701-18.
doi: 10.1128/JVI.00701-18. Print 2018 Sep 1.

Zika Virus Attenuation by Codon Pair Deoptimization Induces Sterilizing Immunity in Mouse Models

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Free PMC article

Zika Virus Attenuation by Codon Pair Deoptimization Induces Sterilizing Immunity in Mouse Models

Penghui Li et al. J Virol. .
Free PMC article

Abstract

Zika virus (ZIKV) infection during the large epidemics in the Americas is related to congenital abnormities or fetal demise. To date, there is no vaccine, antiviral drug, or other modality available to prevent or treat Zika virus infection. Here we designed novel live attenuated ZIKV vaccine candidates using a codon pair deoptimization strategy. Three codon pair-deoptimized ZIKVs (Min E, Min NS1, and Min E+NS1) were de novo synthesized and recovered by reverse genetics and contained large amounts of underrepresented codon pairs in the E gene and/or NS1 gene. The amino acid sequence was 100% unchanged. The codon pair-deoptimized variants had decreased replication fitness in Vero cells (Min NS1 ≫ Min E > Min E+NS1), replicated more efficiently in insect cells than in mammalian cells, and demonstrated diminished virulence in a mouse model. In particular, Min E+NS1, the most restrictive variant, induced sterilizing immunity with a robust neutralizing antibody titer, and a single immunization achieved complete protection against lethal challenge and vertical ZIKV transmission during pregnancy. More importantly, due to the numerous synonymous substitutions in the codon pair-deoptimized strains, reversion to wild-type virulence through gradual nucleotide sequence mutations is unlikely. Our results collectively demonstrate that ZIKV can be effectively attenuated by codon pair deoptimization, highlighting the potential of Min E+NS1 as a safe vaccine candidate to prevent ZIKV infections.IMPORTANCE Due to unprecedented epidemics of Zika virus (ZIKV) across the Americas and the unexpected clinical symptoms, including Guillain-Barré syndrome, microcephaly, and other birth defects in humans, there is an urgent need for ZIKV vaccine development. Here we provided the first attenuated versions of ZIKV with two important genes (E and/or NS1) that were subjected to codon pair deoptimization. Compared to parental ZIKV, the codon pair-deoptimized ZIKVs were mammal attenuated and preferred insect to mammalian cells. Min E+NS1, the most restrictive variant, induced sterilizing immunity with a robust neutralizing antibody titer and achieved complete protection against lethal challenge and vertical virus transmission during pregnancy. More importantly, the massive synonymous mutational approach made it impossible for the variant to revert to wild-type virulence. Our results have proven the feasibility of codon pair deoptimization as a strategy to develop live attenuated vaccine candidates against flaviviruses such as ZIKV, Japanese encephalitis virus, and West Nile virus.

Keywords: E protein; NS1 protein; Zika virus; codon pair bias; deoptimization; vaccines.

Figures

FIG 1
FIG 1
Construction of the infectious cDNA clone of ZIKVwt and generation of codon pair-deoptimized ZIKVs. (A) Strategy for constructing the full-length cDNA clone of ZIKVwt. Four cDNA fragments, fragments A to D, that cover the complete ZIKV genome were synthesized from viral RNA using RT-PCR and sequentially cloned into plasmid pACYC177 to form the full-length cDNA clone of ZIKV (ZIKVwt-FL). The CMV promoter, HDVr/SV40 poly(A), and the positions of relevant restriction sites are shown. (B) Gene maps of the codon pair-deoptimized ZIKVs Min E, Min NS1, and Min E+NS1. Codon pair-deoptimized genes are shown as white boxes, the WT E gene is shown as orange boxes, and the WT NS1 gene is shown as blue boxes. Restriction sites (KpnI and ClaI) used for construction are indicated.
FIG 2
FIG 2
Replication of WT and codon pair-deoptimized ZIKVs in cell culture. (A to D) Vero cells (A and B) or C6/36 cells (C and D) were infected with viruses at an MOI of 0.01. Viral loads were determined by qRT-PCR (A and C), and virus titers were measured by an immunostaining focus assay on Vero cells (B and D). (E and F) Growth properties of viruses were determined by passaging them on Vero cells at an MOI of 0.01. L.O.D., limit of detection. (G) Plaque size phenotypes of virus variants on Vero cells, visualized by immunostaining following incubation for 4 days (WT and Min NS1) and 7 days (Min E and Min E+NS1) under methylcellulose at 37°C. Data shown (A to F) are the means and standard deviations (SD) analyzed by Student's t test (two tailed) (*, P < 0.05; **, P < 0.01; ***, P < 0.001).
FIG 3
FIG 3
IFA of E protein expression in Vero cells infected with WT or codon pair-deoptimized ZIKVs. Vero cells were infected with viruses at an MOI of 0.01. At 2, 3, and 4 dpi, IFA was performed as described in Materials and Methods. All the images were captured at a ×10 magnification. Green represents E protein, and blue represents nuclei (stained with Hoechst 33258).
FIG 4
FIG 4
Attenuation of codon pair-deoptimized ZIKVs in AG6 mice. Groups of AG6 mice (4 weeks old; n = 6) were infected intraperitoneally with 102 IFU of WT or codon pair-deoptimized ZIKVs. Body weight loss (A) and survival (B) were monitored daily for 4 weeks. Mice were euthanized when they lost 25% of their initial body weight.
FIG 5
FIG 5
Immunohistochemical staining of E protein in brain sections from infected mice. AG6 mice were infected with 102 IFU of viruses (n = 3). The brain tissues from mice infected with the WT virus were collected at 7 dpi. The brain tissues from mice infected with Min E+NS1 were collected at 28 dpi.
FIG 6
FIG 6
Viral loads in organs or sera of infected AG6 mice. (A and B) Mice (n = 3) were infected with 102 IFU of viruses and euthanized at day 3, day 6, or day 7, and organ viral loads were determined at day 3 and day 7 by qRT-PCR (A) and an immunostaining focus assay (spleen) (B). (C) Serum viral loads were determined at day 3 and day 6 by qRT-PCR. Data shown (A to C) are the means ± SD analyzed by Student's t test (two tailed) (*, P < 0.05; **, P < 0.01; ***, P < 0.001).
FIG 7
FIG 7
Humoral and cellular immune responses induced by codon pair-deoptimized ZIKVs in mice. (A) Prechallenge neutralization antibody titers were measured on day 28 (day 7 for WT virus) after immunization using a standard PRNT50 assay. (B) Cellular immune responses were assessed on day 28 after immunization by IFN-γ ELISpot assays. Data shown (A and B) are the means ± SD analyzed by Student's t test (two tailed) (**, P < 0.01; n.s., not significant).
FIG 8
FIG 8
Protection efficacy of codon pair-deoptimized ZIKVs in mice. Mice were immunized with 102 IFU of codon pair-deoptimized viruses or mock vaccinated with PBS. At 4 weeks postvaccination, animals were challenged with 104 IFU of WT virus (an ∼5,500-fold-higher MLD50). (A and B) Body weight loss (A) and survival (B) were evaluated for 14 days after challenge. Mice were euthanized when they lost 25% of their initial body weight. (C) Postchallenge viremia was quantified by qRT-PCR on day 3 after challenge. (D) Postchallenge neutralization antibody titers were determined at day 14 after challenge by a standard PRNT50 assay. Data shown (C and D) are the means ± SD analyzed by Student's t test (two tailed) (*, P < 0.05; **, P < 0.01; ***, P < 0.001).
FIG 9
FIG 9
Min E+NS1 immunization protected AG6 mice during pregnancy. (A) Scheme of immunization of 4-week-old AG6 female mice with 102 IFU of Min E+NS1 or PBS. (B to D) At day 32 postimmunization, vaccinated female mice were mated with AG6 males. Pregnant mice (n = 4) were infected with 104 IFU of WT virus on E6. (B) Neutralization antibody titers were measured on day 1 before challenge, using a standard PRNT50 assay. (C) Maternal viremia on day 2 after challenge was quantified by qRT-PCR. (D) Outcome of fetuses from Min E+NS1- or PBS-vaccinated dams. a, all PBS-immunized pregnant mice died without delivery; b, maternal neutralization antibody titers for pups delivered at term to Min E+NS1-vaccinated dams were measured on the 21st day after birth. Data shown (B and C) are the means ± SD analyzed by Student's t test (two tailed) (***, P < 0.001).

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