Abstract
Live attenuated vaccines (LAVs) are one of the most important strategies to control flavivirus diseases. The flavivirus nonstructural (NS) 4B proteins are a critical component of both the virus replication complex and evasion of host innate immunity. Here we have used site-directed mutagenesis of residues in the highly conserved N-terminal and central hydrophobic regions of Zika virus (ZIKV) NS4B protein to identify candidate attenuating mutations. Three single-site mutants were generated, of which the NS4B-C100S mutant was more attenuated than the other two mutants (NS4B-C100A and NS4B-P36A) in two immunocompromised mouse models of fatal ZIKV disease. The ZIKV NS4B-C100S mutant triggered stronger type 1 interferons and interleukin-6 production, and higher ZIKV-specific CD4+ and CD8+ T-cell responses, but induced similar titers of neutralization antibodies compared with the parent wild-type ZIKV strain and a previously reported candidate ZIKV LAV with a 10-nucleotide deletion in 3′-UTR (ZIKV-3′UTR-Δ10). Vaccination with ZIKV NS4B-C100S protected mice from subsequent WT ZIKV challenge. Furthermore, either passive immunization with ZIKV NS4B-C100S immune sera or active immunization with ZIKV NS4B-C100S followed by the depletion of T cells affords full protection from lethal WT ZIKV challenge. In summary, our results suggest that the ZIKV NS4B-C100S mutant may serve as a candidate ZIKV LAV due to its attenuated phenotype and high immunogenicity.
Highlights
The results indicate that the stable Zika virus (ZIKV) NS4BC100S, C100A, and P36A mutants could be produced in cell culture
The introduction of ZIKV to the Americans resulted in a public health emergency and multiple platforms have been utilized in ZIKV vaccine development, including purified inactivated virus, DNA plasmid, mRNA, adenovirus vector, measles virus vector, and Live attenuated vaccines (LAVs)
Many flavivirus T-cell epitopes have been mapped in NS proteins
Summary
Zika virus (ZIKV), a flavivirus, has caused outbreaks of disease in the Americas and Caribbean in the recent years with more than one million human infections.[1,2,3] The virus can be transmitted by mosquito bites, or less frequently by sexual contact.[4,5,6] the majority of human infections are either asymptomatic or result in a self-limiting flu-like febrile illness, the virus has been associated with severe neurological diseases, such as the autoimmune disorder Guillain-Barre syndrome in adults and congenital Zika syndrome (CZS) in fetuses and infants, including microcephaly, spontaneous abortion, and intrauterine growth restriction.[7,8] Currently, no vaccines are available for human use. The protein has extensive homology between flaviviruses and is known to be involved in virus replication and evasion of host innate immunity.[11,12,13,14,15] The two highly conserved coding regions, N-terminal and central hydrophobic of NS4B protein, have been associated with these activities. We have previously identified two mutations in wild-type (WT) WNV NY99, one in each of the two domains of NS4B, using site-directed mutagenesis.[17,18] The WNV NS4B-P38G mutant had significantly reduced neuroinvasiveness[17] but triggered stronger innate cytokine and memory T-cell responses in mice than did the parent strain WT WNV NY99.19 Another mutant, WNV NS4B-C102S, in the central hydrophobic region was highly attenuated for both neuroinvasiveness and neurovirulence in mice.[18] The NS4B-P38 and NS4B-C102 residues are shared by all mosquito-borne flaviviruses. The ZIKV NS4B-C100S mutant induced more potent antiviral innate and adaptive immune responses than the parent WT ZIKV FSS13025 strain and a previously reported candidate live attenuated vaccine (LAV) strain, which provided protection against lethal WT ZIKV challenge
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