An attenuated Zika virus NS4B protein mutant is a potent inducer of antiviral immune responses

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. Live attenuated vaccines are used to protect against some flavivirus infections; however, no Zika virus (ZIKV) vaccine has yet been approved for human use. Here, Tian Wang and colleagues use nonstructural protein 4B (NS4B), which is involved in virus replication and evasion of host immunity, as a basis for a potential ZIKV vaccine. Using site-directed mutagenesis, they generated mutations in the conserved N-terminal and central hydrophobic regions of this protein, and identify three single-site mutations that caused the virus to become attenuated. One mutation in particular was found to be more attenuated in an immunocompromised mouse model of ZIKV—the NS4B-C100S mutant. When compared with the wild-type virus strain and a previously mutated ZIKV strain, it induced stronger antiviral immune responses in this mouse model, including increased type I IFN and IL-6 production, and increased ZIKV-specific CD4+ and CD8+ T-cell responses, yet similar neutralizing antibody titerss. Both active and passive immunization with this mutant provided protection when mice were challenged with wild-type ZIKV, suggesting that this mutant strain may warrant further studies as a potential vaccine.