Oral Presentation Australian Society for Microbiology Annual Scientific Meeting 2019

Zika Virus Non-structural Proteins as DNA Vaccine Antigens to Elicit Cell Mediated Immunity (#15)

Ashish C Shrestha 1 , Danushka K Wijesundara 1 , Makutiro G Masavuli 1 , Zelalem A Mekonnen 1 , Eric J Gowans 1 , Branka Grubor-Bauk 1
  1. Virology Laboratory, Discipline of Surgery, Basil Hetzel Institute for Translational Health Research and The University of Adelaide, Woodville South, SA, Australia

Introduction: The causal association of Zika virus (ZIKV) with microcephaly, congenital malformations in infants and Guillain-Barré syndrome in adults highlight the need for effective vaccines. Neutralising antibodies against ZIKV structural (pre-membrane and envelope) proteins can successfully prevent the infection [1]. However, there is a concern that anti-envelope antibodies can enhance infection of dengue virus [2], which is also endemic in regions where ZIKV circulates. Therefore, we developed novel cytolytic DNA vaccines encoding non-structural proteins (NS2/NS3 and NS4/NS5) of ZIKV. These cytolytic DNA vaccines also encode a truncated form of mouse perforin (PRF), which can more effectively prime T-cell mediated immunity (CMI) in vivo compared to canonical DNA vaccines not encoding PRF [3].

Method:  Cytolytic DNA plasmids encoding ZIKV NS2 and NS3 (pNS2/NS3-PRF) and NS4 and NS5 (pNS4/NS4-PRF) (Brazil-ZKV2015 isolate) were used to prime/boost vaccinate female BALB/c mice. Empty DNA vector (pVAX-PRF) was used as a control. CMI to each vaccine-encoded ZIKV NS protein was evaluated in vivo in vaccinated mice using fluorescent target array (FTA). We have used the FTA to quantify the magnitude and avidity of CMI in vaccinated mice [4]. The assay involves challenging vaccinated mice with fluorescently bar-coded ZIKV peptide-pulsed autologous target cells, which are recognized by effector/memory CD8+ cytotoxic T-lymphocyte (CTL) cells and CD4+ T helper (Th) cells in vivo. T cell responses are assessed 18 hours later by flow cytometry analysis of FTA target cells recovered from the spleen of vaccinated mice.

Results and Conclusion: Vaccination of mice with pNS2/NS3-PRF or pNS4/NS5-PRF elicited significant CD8+ CTL and CD4+ Th responses in vivo that were mainly restricted to ZIKV NS2 and NS3, while NS4 and NS5 were poorly immunogenic. This study suggests that ZIKV NS2 and NS3 can be effective immunogens for the development of T-cell based vaccines against ZIKV. We are currently evaluating the protective efficacy of pNS2/NS3-PRF against ZIKV challenge.


  1. Tebas, P.; Roberts, C.C.; Muthumani, K.; Reuschel, E.L.; Kudchodkar, S.B.; Zaidi, F.I.; White, S.; Khan, A.S.; Racine, T.; Choi, H., et al. Safety and Immunogenicity of an Anti-Zika Virus DNA Vaccine - Preliminary Report. The New England journal of medicine 2017, 10.1056/NEJMoa1708120, doi:10.1056/NEJMoa1708120.
  2. Blackman, M.A.; Kim, I.J.; Lin, J.S.; Thomas, S.J. Challenges of Vaccine Development for Zika Virus. Viral Immunol 2018, 31, 117-123, doi:10.1089/vim.2017.0145.
  3. Shrestha, A.C.; Wijesundara, D.K.; Masavuli, M.G.; Mekonnen, Z.A.; Gowans, E.J.; Grubor-Bauk, B. Cytolytic Perforin as an Adjuvant to Enhance the Immunogenicity of DNA Vaccines. Vaccines 2019, 7, 38.
  4. Wijesundara, D.K.; Gummow, J.; Li, Y.; Yu, W.; Quah, B.J.; Ranasinghe, C.; Torresi, J.; Gowans, E.J.; Grubor-Bauk, B. Induction of Genotype Cross-Reactive, Hepatitis C Virus-Specific, Cell-Mediated Immunity in DNA-Vaccinated Mice. Journal of Virology 2018, 92, e02133-02117, doi:10.1128/JVI.02133-17.