Enhancement of Zika virus pathogenesis by preexisting antiflavivirus immunity

Abstract

Zika virus (ZIKV) is spreading rapidly into regions around the world where other flaviviruses, such as dengue virus (DENV) and West Nile virus (WNV), are endemic. Antibody-dependent enhancement has been implicated in more severe forms of flavivirus disease, but whether this also applies to ZIKV infection is unclear. Using convalescent plasma from DENV- and WNV-infected individuals, we found substantial enhancement of ZIKV infection in vitro that was mediated through immunoglobulin G engagement of Fcγ receptors. Administration of DENV- or WNV-convalescent plasma into ZIKV-susceptible mice resulted in increased morbidity-including fever, viremia, and viral loads in spinal cord and testes-and increased mortality. Antibody-dependent enhancement may explain the severe disease manifestations associated with recent ZIKV outbreaks and highlights the need to exert great caution when designing flavivirus vaccines.

Fig. 1. ZIKV binding and enhancement of infection by DENV- and WNV-immune plasma

(A and B) Plasma samples from seropositive DENV-infected (n = 141), seropositive WNV-infected (n = 146), or seronegative control (n = 15) donors were evaluated for reactivity to ZIKV E protein by ELISA (A) or enhancement of ZIKV infection of K562 cells (B). Calculations of area under the curve (AUC) based on serially diluted plasma measurements are shown. Black bars, geometric means. (C and D) Scatterplots showing the relationship between ZIKV binding and enhancement of ZIKV infection for DENV-immune (C) and WNV-immune (D) plasma. Each point represents one donor. Significance was analyzed by nonparametric unpaired Mann-Whitney U tests for (A) and (B) and by nonparametric Spearman’s rank correlation for (C) and (D) (r, correlation coefficient). **P < 0.01; ****P < 0.0001.

Fig. 2. Enhancement of ZIKV infection through IgG engagement of Fcγ receptors

(A) IgG purified from plasma pooled (n = 15 per group) from control, DENV-, or WNV-infected donors was evaluated for enhancement of ZIKV infection in K562 cells. (B) Pooled plasma samples from control, DENV-, or WNV-infected donors were evaluated for enhancement of ZIKV infection in K562 cells before and after IgG purification. (C to E) Purified IgG from control, DENV-, or WNV-seropositive donors was tested for enhancement of ZIKV infection in K562 cells in the presence or absence of Fcγ receptor binding inhibitor (FcγR BI) (C), antibodies (α) against CD32 (D), or antibodies against CD16 (E). (F) Plasma IgG from control, DENV-, or WNV-seropositive donors was incubated in the presence or absence of PNGase F before evaluation of ZIKV infection enhancement in K562 cells.The same control, DENV, and WNV IgG samples are shown in (A), (C), (D), and (E). All graphs show means ± SD.

Fig. 3. In vivo enhancement of ZIKV infection by DENV- and WNV-convalescent plasma

(A) 20 μl of PBS or plasma from control, DENV-, or WNV-infected donors was administered intraperitoneally to Stat2^−/−mice 2 hours before intradermal inoculation with 5 × 10³ plaque-forming units of ZIKV strain PRVABC59.The Kaplan-Meier survival curve is shown; significance was determined using the Mantel-Cox log-rank test and adjusted for multiple comparisons using the Bonferroni correction. Mice were monitored for (B) weight loss and (C) clinical score using a 6-point system (n = 5 per group), with a score of 7 awarded to deceased animals. Significance was determined using Student’s t test and adjusted for multiple comparisons using the Bonferroni correction. In symptom score comparisons, the day with the highest average score per group was used. Daily body temperature measurements were taken from mice receiving PBS (D) or control (E), DENV- (F), or WNV-immune plasma (G). Statistically significant differences were calculated by comparing day 3 (the day of the highest total average temperature) and day 0 for each group. *P < 0.05; **P < 0.01; ***P < 0.001. (B) and (C), means ± SEM; (D) to (G), means ± SD.

Fig. 4. In vivo ADE correlates with amplified ZIKV replication

(A) Blood viral titers of mice treated with PBS or control, DENV-, or WNV-positive donor plasma were assessed by real-time polymerase chain reaction (PCR) on days 3 and 6 postinfection. Viral titers were quantified by a plaque assay on spinal cords (B) and testes (C) isolated on day 6 postinfection from ZIKV-infected Stat2^−/−mice treated with PBS or a low dose of control, DENV-, or WNV-positive donor plasma. Paraffin-embedded spinal cords (D) and testes (E), taken on day 6 postinfection from ZIKV-infected Stat2^−/−mice treated with PBS or a low dose of control, DENV-, or WNV-positive donor plasma sections, were stained for ZIKV (green) and nuclear DAPI (4',6-diamidino-2-phenylindole; blue). Representative images are shown at 10× magnification; scale bars, 50 μm. Brains (F), ovaries (G), and eyes (H) were also evaluated for viral loads on day 6. *P < 0.05; **P < 0.01. Means ± SEM. PFU, plaque-forming unit.