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

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 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. 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.

Extensive recoding of dengue virus type 2 specifically reduces replication in primate cells without gain-of-function in Aedes aegypti mosquitoes

“…This is intriguing as the ribosome travels through the entire ORF thereby encountering individual segments of codon pair deoptimized sequences. Unlike the loss of protein expression observed in CPD Influenza viruses [ 24 , 29 ] or reduced protein expression in a codon pair deoptimized poliovirus variant [ 49 ]we do not have evidence of specific loss of recoded protein expression in DENV2 syn infected cells. Importantly, neither decreased temperature ( Fig 5 ), nor addition of innate immune inhibitor TBK-1 (not shown), nor addition of Jak Inhibitor I [ 17 ] completely rescued replication of the deoptimized DENV variants.…”

“…Since then, we have initiated research into CHIKV and ZIKV. Our CPD approach for attenuating flaviviruses is being independently pursued for Yellow Fever virus [ 48 ] and Zika virus[ 49 ]. Although this work is limited to DENV2 16681, we hope to extend our method of selective deoptimization to the other three DENV serotypes, and to other mosquito-borne viruses including emerging or re-emerging threats such as Zika virus [ 50 ], yellow fever virus, and chikungunya virus [ 51 ].…”

Animal Models of Zika Virus Infection during Pregnancy

“…This approach offers the theoretical advantage of avoiding exogenous immunosuppression, but at the expense of preventing the assessment of vaccine performance against transplacental transmission. As an alternative to a transient type I IFN blockade, in an experiment with a limited number of animals, females deficient in both type I and II IFN receptor signaling ( Ifnar1 −/− Ifngr1 −/− ) were utilized to evaluate a codon pair deoptimized LAV ZIKV vaccine [ 50 ]. Pregnant Ifnar1 −/− Ifngr1 −/− dams were challenged with ZIKV six days after mating to Ifnar1 −/− Ifngr1 −/− sires, which resulted in 100% of unvaccinated dams succumbing prior to delivery, whereas all immunized dams gave birth to healthy-appearing pups.…”

A Lassa Fever Live-Attenuated Vaccine Based on Codon Deoptimization of the Viral Glycoprotein Gene

“…CD is achieved by replacing wild-type (WT) codons for those with less-preferred codons throughout a target gene without affecting the amino acid sequence of the corresponding protein (35,39). To date, several attenuated viruses, including poliovirus (40), human respiratory syncytial virus (41), foot-and-mouth disease virus (42), influenza A virus (43), and Zika virus (44) have been generated using a CD strategy.…”