Rational design of anti-ZIKV peptide Z2

First, we aligned the amino-acid sequence of ZIKV E protein with that of the corresponding fragment in the stem region of DENV E protein, which represents the basis for the design of anti-DENV peptides27,28,29. We next aligned these sequences with those in the E proteins of other flaviviruses, including Japanese encephalitis virus, yellow fever virus (YFV) and West Nile virus. We found that the sequence in this region is highly conserved among flaviviruses with amino-acid sequence conservation of 64 to 82% (Fig. 1a and Supplementary Fig. 1), implying that this region may play important roles in flavivirus infection. Finally, we located this region in the 3.8 Å resolution cryo-electron microscope structure of ZIKV (Protein Data Bank: 5IRE)30, as shown in pink in Fig. 1b, and it was confirmed as the membrane-proximal stem region of ZIKV E protein (residues 421–453), and this was then used as the basis for the design and synthesis of peptide Z2 and the scrambled peptide of Z2 (Z2-scr).

Figure 1: Design of peptide inhibitor Z2. (a) Sequence alignment of stem regions from E protein of flaviviruses. JEV, Japanese encephalitis virus; WNV, West Nile virus. The % amino-acid conservation (%AA cons.) from stem region of ZIKV is shown. (b) Sequence and location of Z2 in the stem region of ZIKV E protein. The structure of E protein was generated by SWISS-MODEL software based on the 3.8 Å resolution cryo-electron microscope structure of ZIKV (Protein Data Bank: 5IRE)30. Red, domain I of ZIKV E protein; yellow, domain II; cyan, domain III; pink, peptide Z2; purple, viral membrane. Z2-scr, scrambled peptide of Z2. Full size image

Z2 inhibited ZIKV infection at early viral replication stage

To determine the antiviral activity of Z2 against ZIKV infection in BHK21 and Vero cells, we developed a rapid and sensitive colorimetric viral infection assay using Cell Counting Kit-8 (CCK8, Dojindo, Japan)31,32,33. It was reported that ZIKV infection of these cells resulted in obvious cytopathic effects (CPE)34. Using this assay, we tested the inhibitory activity of Z2 at different concentrations on infection of ZIKV strain SZ01. We found that Z2 inhibited ZIKV infection in a dose-dependent manner with a 50% inhibitory concentration (IC 50 ) value of 1.75±0.13 μM (mean±s.d., n=3) in BHK21 cells (Fig. 2a) and 3.69±0.27 μM (n=3) in Vero cells (Fig. 2b), while Z2-scr as a negative control showed no significant inhibition on ZIKV infection. We also used the plaque reduction assay and BHK21 cells to test anti-ZIKV activity. As shown in Supplementary Fig. 2, the IC 50 value is 2.61±0.46 μM (n=3), suggesting that the result derived from the plaque reduction assay is consistent with that obtained from colorimetric CCK8 assay. The ZIKV-induced CPE in BHK21 and Vero cells were visibly reduced by Z2 at 5 μM, while Z2-scr at the same concentration exhibited no inhibitory activity (Fig. 2c). In line with the data from CCK8 assay, the result from immunofluorescence staining showed that Z2 at a concentration of 10 μM almost completely blocked ZIKV E protein expression in BHK21 and Vero cells (Fig. 2d). We then evaluated the antiviral activity of Z2 against other ZIKV strains. As shown in Table 1, Z2 was also effective in inhibiting infection of ZIKV strains FLR and MR766 in BHK21 cells with IC 50 values of about 4 and 14 μM, respectively, suggesting that Z2 possesses broad inhibitory activity against ZIKV strains isolated from patients or rhesus monkeys in different regions of the world.

Figure 2: Antiviral activity of Z2 in vitro. (a) Dose-dependent inhibition of ZIKV infection by Z2 in BHK21 cells. (b) Dose-dependent inhibition of ZIKV infection by Z2 in Vero cells. (c) Reduction of ZIKV-induced CPE in BHK21 and Vero cells by Z2. Scale bar, 20 μm. (d) Immunofluorescence assay to confirm the antiviral activity of Z2 against ZIKV. ZIKV E protein stained by the anti-E mAb 4G2 (green); nuclei stained by 4,6-diamidino-2-phenylindole (blue). Scale bar, 100 μm. (e) Inhibition of DENV-2 and YFV 17D in BHK21 cells by Z2. (f) Inhibition of pseudotyped VSV and MERS-CoV in Huh7 cells by Z2. Data are means±s.d. of triplicate experiments. Full size image

Table 1 Inhibition of ZIKV infection by Z2. Full size table

Results of the time-of-addition experiment (Supplementary Fig. 3) showed that Z2 inhibited ZIKV infection as early as 2 h post infection, indicating that Z2 may inhibit infection of ZIKV before its entry into the target cell. Following this, we investigated whether Z2 could also inhibit infection by other mosquito-borne flaviviruses, such as DENV and YFV. We found that Z2 was also highly effective in inhibiting infection by DENV-2 and YFV 17D (Fig. 2e) with IC 50 values of about 4 and 5 μM, respectively, while it had no inhibition on infection by pseudotyped vesicular stomatitis virus (VSV) and Middle East Respiratory Syndrome Coronavirus (Fig. 2f). These results suggest that Z2 may have inhibitory activity against infection of a broad spectrum of flaviviruses.

Z2 bound to E protein and inactivated ZIKV virions

It was reported that DN59, a peptide derived from the stem region of the DENV E protein, could inhibit flavivirus infection by interacting with virus particles and inducing formation of pores in viral envelope membrane, resulting in the release of viral RNA genome28. Using similar approaches, we investigated whether Z2 could interact with ZIKV E protein and induce the release of RNA genome from ZIKV virions. First, we used an immunofluorescence staining assay to show that 293T cells transfected by pcDNA3.1-Env could be bound by 4G2, a mouse mAb against E protein of pan-flaviviruses35. Meanwhile, the 293T cells transfected by the empty vector pcDNA3.1 showed no binding, suggesting that ZIKV E protein did express on the 293T cells transfected with pcDNA3.1-Env (Fig. 3a). Using a flow cytometric analysis, we demonstrated that the 293T cells transfected with pcDNA3.1-Env could be stained by Z2-Cy5, whereas the 293T cells expressing no E protein showed only background staining (Fig. 3b). Similar results were obtained from experiment using ZIKV-infected BHK21 cells (Supplementary Fig. 4). These results indicate that Z2 is able to interact with E protein of ZIKV.

Figure 3: Inactivation of ZIKV by Z2. (a) Immunofluorescence staining the ZIKV E protein expressed on 293T cells by the anti-E mAb 4G2 (green). Nuclei stained by 4,6-diamidino-2-phenylindole (blue). Scale bar, 100 μm. (b) Determination of the binding of Z2 with E protein expressed on 293T cells by flow cytometry. (c) Degradation of released genomic RNA of ZIKV mediated by Z2 in an RNase digestion assay. The primers were used to detect RNA sequences in viral genome coding PrM, E protein and Cap protein, respectively. (d) Degradation of released genomic RNA of ZIKV mediated by Z2-scr. (e) The separation of genomic RNA and E protein of ZIKV treated with Z2, Z2-scr, DMSO or Triton X-100 through a sucrose density gradient assay. Per cent of total E protein in each fraction was assessed by western blot and analysed by Image J software. Per cent of total RNA genome in each fraction was measured by RT–qPCR. (f) Inactivation of ZIKV by Z2. After incubation at room temperature for 2 h, ZIKV was separated from Z2 by PEG-8000 for the measurement of the residual infectivity. Data are means±s.d. of triplicate experiments. Full size image

Next, we measured the potential release of viral genomic RNA from ZIKV using an RNase digestion assay28. As shown in Fig. 3c,d, the genomic RNA of untreated virions (0 μM Z2) or treated by Z2-scr were protected from digestion of micrococcal nuclease. However, the genomic RNA of Z2 treated virions was digested by micrococcal nuclease in a dose-responsive manner. About 70% genomic RNA of ZIKV virions treated with 50 μM Z2 was digested by micrococcal nuclease. To further confirm the release of viral genomic RNA, ZIKV virions treated with 1% dimethylsulfoxide (DMSO), 100 μM Z2 or Z2-scr in 1% DMSO, and 1% Triton X-100, respectively, was centrifuged through a sucrose density gradient as previously described28. Then the amount of genomic RNA and E protein of ZIKV in each fraction was monitored by reverse-transcription quantitative PCR (RT–qPCR) and western blot (Supplementary Figs 5 and 6), respectively, and the data of western blot was further analysed by Image J software. As shown in Fig. 3e, the genomic RNA and E protein of virions treated by 1% DMSO or Z2-scr migrated to the same fractions. For example, the peaks of both genomic RNA and E protein were located at fraction No. 6, suggesting that virions treated by 1% DMSO or Z2-scr maintained intact. However, the genomic RNA and E protein of virions treated by 1% Triton X-100 migrated to different fractions, that is, peaks of genomic RNA and E protein were in fraction No. 1 and 7, respectively, indicating that virions were completely destroyed by Triton X-100. Interestingly, the peaks of genomic RNA and E protein of virions treated with Z2 were located in fraction No. 6 and 3, respectively. These results indicate that Z2 treatment may cause pore formation in the viral membrane, resulting in the release of viral genomic RNA through the pores.

Next, we determined whether the virions treated with Z2 peptide were inactivated. After incubation of ZIKV with Z2 or Z2-scr at room temperature for 2 h, we separated the treated ZIKV virions from the unbound free peptide with PEG-8000, as previously described36 and assessed their infectivity. As shown in Fig. 3f, ZIKV virions treated with Z2 lost infectivity in a dose-dependent manner with 50% effective concentration of 2.52±0.18 μM (n=3), while ZIKV treated with Z2-scr at concentration as high as 50 μM retained 90% infectivity (Fig. 3f), suggesting that Z2-treated ZIKV virions had been inactivated.

Importantly, although Z2 could disrupt the integrity of ZIKV membranes, it showed no cytotoxic effect on cell lines (BHK21, Vero and Huh7) tested at concentration as high as 50 μM (Supplementary Fig. 7). Z2 also showed no cytotoxic effect on red blood cells isolated from mouse peripheral blood at concentration as high as 100 μM (Supplementary Fig. 8).

Z2 could cross placental barrier of pregnant ICR mice

ZIKV can cross the placental barrier and infect the fetus during pregnancy, causing an abnormal growth of the brain and head of the fetus5,37. Therefore, an effective anti-ZIKV drug should have the ability to penetrate through the placental barrier of the infected pregnant women to protect the fetus. Here we investigate whether Z2 can enter into the pregnant mouse’s organs, and penetrate through its placenta to enter into the fetus’ body. As shown in Fig. 4a and Supplementary Fig. 9a,b, the fluorescent signals of Z2-Cy5 were shown at the sites where the liver and bladder were located and the dissected organs, such as liver, kidney, spleen and heart, suggesting that Z2 is able to enter into blood circulation and organs of pregnant mice. The average radiant efficiency (p s−1 cm−2 sr−1)(μW−1 cm2) in the liver (P=0.0275, Student’s two-tailed t-test) and bladder (P=0.0426, Student’s two-tailed t test) of the Z2-treated mice was significantly higher than that in the phosphate-buffered saline (PBS)-treated mice (Fig. 4b). Then the uteruses of the pregnant mice and the fetuses in their uteruses were removed out for evaluating the distribution of Z2-Cy5. As speculated, the signal of Z2-Cy5 was seen in the uteruses (Fig. 4c) and the fetuses (Fig. 4d and Supplementary Fig. 9c). The average radiant efficiency in uteruses (P=0.0148, Student’s two-tailed t-test) and fetuses (P<0.0001, Student’s two-tailed t-test) from the Z2-Cy5 group was significantly higher than that from PBS group (Fig. 4e). In corroboration with the data in Supplementary Fig. 10, these results suggest that Z2 peptide injected intravenously or intraperitoneally (i.p.) can penetrate through the placental barrier of pregnant ICR mice and enter into the bodies of fetuses.

Figure 4: Ability of Z2 to penetrate the placental barrier of pregnant ICR mice. (a) Imaging of pregnant ICR mice treated with Z2-Cy5 or PBS by the IVIS Lumina K Series III from PerkinElmer. Mice were injected intravenously with 100 μg Z2-Cy5 (n=3) or PBS (n=3) as control (for background fluorescence measurement), followed by imaging analysis. (b) The statistical analysis of results from Fig. 4a. (c) Imaging of the uteruses from pregnant mice. (d) Imaging of the fetuses (n=9) from uteruses. (e) The statistical analysis of results from Fig. 4c,d. Data are means±s.d., *P<0.05; ***P<0.001, Student’s two-tailed t-test. Full size image

Z2 is safe for pregnant ICR mice and their fetuses

ZIKV usually causes mild symptoms, such as fever and rash, but when it infects pregnant women, it can cause congenital brain development abnormalities to fetus6,38. Therefore, the safety of an anti-ZIKV agent for pregnant women should be taken into consideration. Accordingly, we assessed the safety of Z2 in pregnant ICR mice. The results showed that body weight changes of mothers (Fig. 5a) and pups (Fig. 5b) were almost the same among the six groups (intravenous injection of Z2 at 10, 20, 40, 80 and 120 mg kg−1, respectively, or PBS), suggesting that injection of Z2 does not cause visible damage to prenatal and postpartum health of the pregnant mice, nor does it interfere with the normal growth of pups. We did not find any pups with abnormal behaviour.

Figure 5: Safety analysis of Z2 for pregnant ICR mice and fetuses. (a) Body weight changes of mothers at different prenatal and postnatal time points. Thirty-one pregnant ICR mice (E12–14) were assigned randomly to six groups and were injected intravenously with PBS (n=5), or PBS containing Z2 at escalating dose (10 mg kg−1, n=5; 20 mg kg−1, n=6; 40 mg kg−1, n=5; 80 mg kg−1, n=5; 120 mg kg−1, n=5) every day for 3 consecutive days. Data are means±s.d. (b) Body weight changes of pups at various postnatal time points. The average litter size of the offspring of PBS-treated pregnant mice was 10.4±1.5, while that of the pregnant mice treated with Z2 at dose of 10, 20, 40, 80 and 120 mg kg−1 was 10.2±1.6, 10.5±2.0, 10.4±2.1, 10.6±1.1 and 10.8±2.6, respectively. The same legend was used for Fig. 5a,b. Data are means±s.d. (c) The ALT in the sera of the pregnant mice measured by the ALT assay kit (NJJCBIO) before the first injection and 4 h, 1, 3 and 5 days after the third injection of Z2. All error bars reflect s.d. (d) The creatinine in the sera of the pregnant mice measured by the creatinine assay kit (NJJCBIO). All error bars reflect s.d. (e) Comparison of haematoxylin and eosin staining of tissues, including livers, kidneys, brains and spleens from mothers and pups in each group. Scale bar, 50 μm. Full size image

The levels of alanine aminotransferase (ALT; Fig. 5c) and creatinine (Fig. 5d) in the sera of mice in Z2- and PBS-treated groups showed no significant difference (P>0.05, Mann–Whitney test) at all time points, suggesting that injection of Z2 at high or low doses does not affect the hepatic and renal function of pregnant mice. Using enzyme-linked immunosorbent assay, we measured Z2-specific antibodies in sera of pregnant mice at 1 or 2 weeks after the third injection of Z2 or PBS, and found that Z2-specific antibodies in both Z2- and PBS-treated mice were at a level below detection (Supplementary Fig. 11). This result suggests that Z2 has poor immunogenicity.

We then compared the potential histopathological changes of mothers and pups among the six groups. As shown in Fig. 5e, the haematoxylin-and-eosin-stained sections of livers, kidneys, brains and spleens from mice treated with Z2 at different doses exhibited no pathological abnormality, when compared with those from mice treated by PBS. None of these samples showed evidence of cell degeneration, necrosis or infiltration of inflammatory factors. Overall, Z2 is safe for pregnant ICR mice and fetuses, even at the dose as high as 120 mg kg−1 of body weight, which is 11-fold higher than that providing protection against ZIKV infection in vivo.

Z2 blocked vertical transmission of ZIKV in pregnant mice

To determine whether Z2 could protect against vertical transmission of ZIKV, pregnant C57BL/6 mice were infected by ZIKV as described previously39 and were then treated with Z2 at 10 mg kg−1 of body weight (n=12) or vehicle control (n=12). The results showed that Z2 treatment could reduce viraemia in ZIKV-infected pregnant C57BL/6 mice (P=0.0141, Mann–Whitney test; Fig. 6a). At the same time, viral RNA load in placentas from Z2-treated pregnant mice was significantly lower than that from vehicle-treated mice (P=0.0029, Mann–Whitney test), and the infection rate decreased from 18/24 to 12/24 (Fig. 6b). Interestingly, Z2 treatment resulted in the decrease of infection rate of fetal head from 14/24 to 2/24 (P=0.0001, Mann–Whitney test; Fig. 6c). These results suggest that Z2 may inactivate ZIKV virions either before or after the virions have penetrated the placenta to fetus, thus reducing the infection rate of fetuses, as well as protecting against vertical transmission of ZIKV in pregnant mice.

Figure 6: Protection against vertical transmission of ZIKV in Z2-treated pregnant C57BL/6 mice. (a) Viraemia of pregnant C57BL/6 mice. Pregnant C57BL/6 mice were infected by ZIKV for 1 h and treated with Z2 or vehicle as control. At day 1 post infection, sera were collected by retro-orbital bleeding for viraemia detection. (b) Viral RNA load in placentas. Two embryos of each pregnant mouse were randomly collected and the viral RNA load in each placenta was determined by RT–qPCR. (c) Viral RNA load in fetal heads. The viral RNA load in fetal head of each collected embryo was determined by RT–qPCR. All bars reflect median values, *P<0.05; **P<0.01; ***P<0.001, Mann–Whitney test. Full size image

Z2 protected A129 or AG6 mice from lethal ZIKV challenge

Finally, the antiviral efficacy of Z2 was confirmed in the recently established A129 (type I interferon receptor-deficient)15 or AG6 (type I/II interferon receptor-deficient)40,41 mouse model for ZIKV infection. One hour after the inoculation with ZIKV i.p., A129 mice were treated with Z2 or vehicle. As shown in Fig. 7a, all the mice treated with vehicle developed neurological symptoms from 5 days post inoculation and finally displayed a 100% mortality rate at 13 days post inoculation. In contrast, treatment with Z2 protected 75% of the A129 mice from death caused by ZIKV infection, and the survivors had no neurological symptoms. Z2 treatment also significantly prolonged mean survival time (MST) from 9 days to the end of the experiment (P=0.0010, log-rank (Mantel Cox) test). The viral load in Z2-treated A129 mice at 2 days post infection (d.p.i.) was about 7-fold lower than that of vehicle-treated mice (P=0.0002, Mann–Whitney test; Fig. 7b). Similarly, treatment with Z2 protected 67% of AG6 mice from death caused by subcutaneous administration of ZIKV and significantly prolonged MST from 10 days to the end of the experiment (P=0.0048, log-rank (Mantel Cox) test; Fig. 7c). The viral load in Z2-treated AG6 mice at 2 d.p.i. was about 13-fold lower than that of vehicle-treated mice (P=0.0022, Mann–Whitney test; Fig. 7d).

Figure 7: Protective activity of Z2 against ZIKV infection in lethal mouse models. (a) Survival of ZIKV-infected A129 mice. A129 mice (4 weeks old) were infected with 1 × 105 p.f.u. of ZIKV through the intraperitoneal injection route. After 1 h, mice were treated with Z2 (n=8) at 10 mg kg−1 of body weight, and vehicle (n=8) as control. Mouse survival was observed and recorded daily until 21 d.p.i. **P<0.01, log-rank (Mantel Cox) test. (b) Viral RNA load in sera of ZIKV-infected A129 mice. At day 2 post infection, mice were retro-orbitally bled to measure viral RNA load in sera by RT–qPCR. ***P<0.001, Mann–Whitney test. (c) Survival of ZIKV-infected AG6 mice. AG6 mice (6 weeks old) were infected with 1 × 103 p.f.u. of ZIKV via a subcutaneous route in the footpad. After 1 h, mice were treated with Z2 (n=6) at 10 mg kg−1 of body weight, and vehicle (n=6) as control. Mouse survival was observed daily and recorded until 21 d.p.i. **P<0.01, log-rank (Mantel Cox) test. (d) Viral RNA load in sera of ZIKV-infected AG6 mice. At day 2 post infection, mice were retro-orbitally bled to measure viral RNA load in sera by RT–qPCR. Whiskers: 5–95 percentile. **P<0.01, Mann–Whitney test. Full size image

Twenty-four hours after inoculation with ZIKV, treatment with Z2 still could protect 33.3% of A129 mice from death (P=0.0139, log-rank (Mantel Cox) test; Supplementary Fig. 12a). Viral load in the Z2-treated A129 mice at 3 d.p.i. was about 4-fold lower than that of the vehicle-treated mice (P=0.0043, Mann–Whitney test; Supplementary Fig. 12b). Although Z2 inactivates ZIKV at the early stage of viral replication, consecutive Z2 injection after ZIKV penetration of cells could still provide some protection of the infected A129 mice, possibly by inactivating newly produced ZIKV virions and prevent their infection of more target cells.