A live-attenuated Zika virus vaccine candidate induces sterilizing immunity in mouse models

Abstract

Zika virus (ZIKV) infection of pregnant women can cause a wide range of congenital abnormalities, including microcephaly, in the infant, a condition now collectively known as congenital ZIKV syndrome. A vaccine to prevent or significantly attenuate viremia in pregnant women who are residents of or travelers to epidemic or endemic regions is needed to avert congenital ZIKV syndrome, and might also help to suppress epidemic transmission. Here we report on a live-attenuated vaccine candidate that contains a 10-nucleotide deletion in the 3' untranslated region of the ZIKV genome (10-del ZIKV). The 10-del ZIKV is highly attenuated, immunogenic, and protective in type 1 interferon receptor-deficient A129 mice. Crucially, a single dose of 10-del ZIKV induced sterilizing immunity with a saturated neutralizing antibody titer, which no longer increased after challenge with an epidemic ZIKV, and completely prevented viremia. The immunized mice also developed a robust T cell response. Intracranial inoculation of 1-d-old immunocompetent CD-1 mice with 1 × 10⁴ infectious focus units (IFU) of 10-del ZIKV caused no mortality, whereas infections with 10 IFU of wild-type ZIKV were lethal. Mechanistically, the attenuated virulence of 10-del ZIKV may be due to decreased viral RNA synthesis and increased sensitivity to type-1-interferon inhibition. The attenuated 10-del ZIKV was incapable of infecting mosquitoes after oral feeding of spiked-blood meals, representing an additional safety feature. Collectively, the safety and efficacy results suggest that further development of this promising, live-attenuated ZIKV vaccine candidate is warranted.

Extended Data Figure 1.. Predicted RNA structure of 3’ UTR and sequence alignment of deleted nucleotides.

(a) Predicted RNA secondary structure of ZIKV 3’ UTR. The stem-loop structure of 3’UTR of ZIKV genome is presented as previously reported^,. The nucleotide sequence of the shaded stem-loop is shown. The deleted sequences for 10-del, 20-del, 30-del-a, and 30-del-b mutants are displayed in blue, megaton, green, and orange, respectively. (b) Sequence alignment of deleted region (nucleotide position 10,630-10,674) in 3’UTR. The 10-del nucleotides are indicated. Within the 10-del region, sequence variations are observed for early isolates (P6–740, MR766, and DAK-41525 were isolated in 1966, 1947, and 1984, respectively), while an identical sequence is observed for the strains isolated after 2010 (FSS13025, H/PF2013, PRVABC 59, Natal RGN, and ZKV2015).

Extended Data Figure 2.. IFA of viral protein expression in cells transfected with WT or 3’UTR deletion ZIKV RNA.

Vero cells were electroporated with 10 μg of genomic WT or 3’UTR deletion RNA of ZIKV. On day 2 and 3 post-transfection, IFA was performed to examine viral E protein expression using a mouse mAb (4G2) and Alexa Fluor® 488 goat anti-mouse IgG as the primary and secondary antibodies, respectively. Green and blue represent E protein and nuclei (stained with DAPI), respectively.

Extended Data Figure 3.. Stability analysis of the 3’UTR deletion ZIKVs in cell culture.

P0 viruses (derived from the culture fluids of RNA-transfected cells from Fig. 1) were continuously cultured on Vero cells for five rounds (5 days for each round of culture), resulting in P5 viruses. The P5 viruses were subjected to the following characterization. (a) Immunostaining focus assay. WT and P5 mutant viruses were analyzed using an immunostaining focus assay on Vero cells. For each mutant virus, three independent selections were performed on Vero cells. Representative images of infectious foci for each P5 mutant virus are presented. (b) Replication kinetics. Vero cells in 24-well plates (2×10⁵ cells per well) were infected with WT and P5 mutant viruses at an MOI of 0.01. Culture fluids were quantified for infectious viruses on days 1 to 5 using the immunostaining focus assay on Vero cells. (c) Adaptive mutations in P5 mutant viruses. The complete genomes of P5 mutant viruses were sequenced for each of the three independent selections. The adaptive mutations are indicated by their amino acid positions of indicated genes based on ZIKV FSS13025 strain (GenBank number KU955593.1).

Extended Data Figure 4.. Construction of mCherry ZIKV.

(a) Schematic genome of a mCherry ZIKV. A DNA fragment (encoding the first 25 amino acids of C gene, the mCherry gene, and the foot-and-mouth virus 2A protein) was in-frame fused with the open-reading-frame of ZIKV genome. (b) The mCherry expression in Vero cells transfected with mCherry ZIKV RNA. The expression of mCherry in transfected Vero cells was analyzed by a fluorescent microscopy at the indicated days post-transfection. The mCherry ZIKV was used to estimate antibody neutralization titers of mouse sera, as described in Methods.

Extended Data Figure 5.. Efficacy of immunization with 100 IFU 10-del virus.

(a) Viremia after immunization with 100 IFU of WT or 10-del ZIKV. Three-week-old A129 mice (n=5) were immunized with 100 IFU WT or 10-del virus via the S.C. route. Viremia were quantified by immunostaining focus assay from day 2 to 6. L.O.D., limit of detection. (b) Pre-challenge neutralization antibody titers. On day 28 post-immunization, mouse sera were quantified for ZIKV neutralizing antibody titers. (c) Viremia after challenge with ZIKV (Puerto Rico strain PRVABC59). On day 28 post-immunization, the mice were challenged with 1×10⁶ IFU of ZIKV via the I.P. route. Viremias were quantified by immunostaining focus assay on day 2 post-challenge.

Extended Data Figure 6.. Efficacy of immunization with 10 IFU 10-del virus.

(a) Viremia after immunization with 10 IFU of WT or 10-del ZIKV. Three-week-old A129 mice (n=5) were immunized with 10 IFU WT or 10-del virus via the S.C. route. Viremia were quantified by immunostaining focus assay from day 4 to 7. L.O.D., limit of detection. (b) Pre-challenge neutralization antibody titers. Three-week-old A129 mice (n=5) were immunized with 10 IFU 10-del ZIKV and PBS via the S.C. route. On day 28 post-immunization, mouse sera were quantified for ZIKV neutralizing antibody titers. (c) Viremia after challenge with epidemic ZIKV (Puerto Rico strain PRVABC59). On day 28 post-immunization, the mice were challenged with 1×10⁶ IFU of ZIKV via the I.P. route. On day 2 post-challenge, viremias were quantified using an immunostaining focus assay.

Extended Data Figure 7.. Comparison of viremia and efficacy of P0 and P5 10-del viruses.

(a) Viremia after immunization with 100 IFU of P0 or P5 10-del ZIKV. Three-week-old A129 mice (n=5) were immunized with 100 IFU P0 or P5 10-del virus via the S.C. route. Viremia were quantified by immunostaining focus assay from day 4 to 6. L.O.D., limit of detection. (b) Pre-challenge neutralization antibody titers. On day 28 post-immunization, mouse sera were quantified for ZIKV neutralizing antibody titers. (c) Viremia after challenge with wild-type ZIKV. On day 28 post-immunization, the mice were challenged with 1×10⁶ IFU of an epidemic strain of ZIKV (Puerto Rico strain PRVABC59) via the I.P. route. On day 2 post-challenge, viremias were quantified using an immunostaining focus assay.

Figure 1.. Characterization of the 3’UTR deletion mutants in cell culture.

(a) Sequences of the ZIKV 3’UTR deletions. (b) Immunostaining focus assay of mutant viruses. Equal amounts of RNAs (10 μg) transcribed from their corresponding infectious cDNA clones were electroporated into Vero cells. On day 4 or 5 post-transfection, culture fluids form the transfected cells were harvested and quantified for infectious viruses (defined as P0 virus ) using an immunostaining focus assay on Vero cells. (c) Replication kinetics of WT and mutant viruses. Vero cells in 24-well plates (2×10⁵ cells per well) were infected with WT and mutant viruses at an MOI of 0.01. Culture fluids were quantified for infectious viruses on days 1 to 5 using the immunostaining focus assay. (d) Replicon analysis of the 3’UTR deletions. A Renilla luciferase reporter replicon of ZIKV (top panel) was engineered with various 3’UTR deletions. Equal amounts of replicon WT and mutant RNAs (10 μg) were electroporated into Vero cells. Luciferase signals were measured at the indicated time points (bottom panel). A non-replicative replicon containing an NS5 polymerase-inactive GDD mutation was included as a negative control. The averages of three replicates are presented. Error bars represent standard deviations. RLU, relative light units. (e) Interferon-β inhibition of WT and mutant ZIKVs. Vero cells were seeded in 96-well plate (1.5×10⁴ cell per well) one day before interferon treatment and viral infection. The cells were infected at an MOI 0.05 in the presence of IFN-β (55, 167, 500, or 1,500 IU/ml). Viral infection and interferon treatment were initiated at the same time. At 48 h post-infection and interferon-β treatment, viral titers were quantified using the immunostaining focus assay on Vero cells. Percentages of viral titer inhibition are presented in log₁₀ scale. Viral titers without interferon-β treatment are set as 100%. Average results of three independent experiments are shown. Error bars represent standard deviations. Symbols ** and *** indicate P values <0.01 and <0.001, respectively.

Figure 2.. Characterization of 3’UTR mutants in the A129 mouse model.

(a) Experimental scheme. In two separate experiments, three-week old A129 mice (n=8) were immunized via the S.C. route with 1×10⁴ IFU WT and mutant viruses. The immunized mice were monitored for weight loss (b), survival (c), and viremia (d). Weight loss is indicated by percentage using the weight on the day before immunization as 100%. Viremias were quantified by an immunostaining focus assay from day 2 to 4 post-infection. (e) Pre-challenge neutralization antibody titers. On day 28 post-immunization, mouse sera were measured for neutralizing titers using a mCherry ZIKV infection assay (Extended data Fig. 4). (f) Post-challenge viremia. On day 28 post-immunization, mice were challenged with 1×10⁵ PFU parental virus (ZIKV strain FSS13025) via the I.P. route. Viremia on day 2 post-challenge was quantified using the immunostaining focus assay. (g) Post-challenge neutralization antibody titer. On day 28 post-challenge, mouse sera were quantified for neutralizing titers using the mCherry ZIKV infection assay. L.O.D.: limit of detection.

Figure 3.. T cell responses after primary infection with ZIKV WT or 10-del mutant.

A129 mice were infected with 1×10⁴ IFU WT and 10-del viruses. On day 28 post-infection, mouse spleens were harvested. Splenocytes were counted, cultured ex vivo with WT ZIKV for 24 h, and stained for markers (IFN-γ, CD3, and CD4 or CD8). The T cells were gated, and percentages of CD4⁺IFN-γ⁺ cells and CD8⁺IFN-γ⁺ are shown (a). Total number of T cell subsets per spleen is shown (b). Supernatants from the ex vivo culture were harvested on day 2 after WT ZIKV restimulation, and measured for IFN-γ (c) and IL-2 (d) production. Data are presented as means ± SEM, n = 2–4 per group. *P < 0.05 or **P < 0.01 difference between the virus- and mock-infected mice.

Figure 4.. Safety evaluation of 10-del virus.

(a) Viral loads in organs of infected A129 mice. Three-week-old A129 mice were immunized with 1×10⁴ IFU of WT and 10-del viruses. Organs from infected mice were collected and homogenized on day 6 and 10 post-infection. The amounts of viruses were quantified on Vero cells using an immunostaining focus assay. The mean results from three animals are presented. Bars denote standard errors. “?*” denotes no detectable virus. (b) Sperm count analysis of A129 mice infected with WT or 10-del mutant virus. Male A129 mice were infected with 1×10⁴ IFU of WT and 10-del viruses (n=4 per group). On day 16 p.i., epididymis was harvested for sperm count analysis. Total sperm counts and motile sperm counts were measured. One-way ANOVA test was performed to indicate statistical significance among different infection groups. n.s., not significant; ***very significant (p value <0.001); ****extremely significant (p value <0.0001). In addition, testes were harvested and weighted. No statistic difference in testis weight was detected from the WT-, 10-del-, and mock-infected mice (data not shown). (c) Comparison of neurovirulence of WT and 10-del viruses in CD1 newborn mouse. Groups of one-day-old CD1 mice (n=7–10) were injected via the I.C. route with 10 to 1×10⁴ IFU of WT or 10-del virus. (d) Mosquito infectivity assay. Aedes aegypti were fed with WT or 10-del virus on artificial blood-meals. On day 7 post-feeding, individual engorged, incubated mosquitoes were homogenized and infection was assayed by immunostaining of viral protein expression on inoculated Vero cells (see Methods for details). The number of infected mosquitos and total number of engorged mosquitoes are indicated.