Potential role of heterologous flavivirus immunity in preventing urban transmission of yellow fever virus

Abstract

During the recent yellow fever (YF) epidemics in Brazil, human cases were attributed to spillover infections via sylvatic mosquito transmission. Despite YF virus (YFV) transmission in major urban centers with insufficient vaccination coverage and abundant populations of the domestic vector, Aedes aegypti, there was no evidence of human-amplified transmission. Furthermore, the historic absence of YF in Asia, despite abundant Ae. aegypti and an immunologically naive human population, is unexplained. We tested the hypothesis that pre-existing, heterologous flavivirus immunity, specifically from dengue (DENV) and Zika (ZIKV) viruses, limits YFV viremia and transmission by Ae. aegypti. We infected cynomolgus macaques with DENV or ZIKV, then challenged them 6-9 months later with YFV. We then measured viremia and disease and allowed Ae. aegypti mosquitoes to feed during peak macaque viremia. Although prior heterologous immunity had variable effects on disease, DENV and ZIKV immunity consistently suppressed YFV viremia. Despite no statistical difference due to a small sample size, the suppression in viremia led to a significant reduction in Ae. aegypti infection and a lack of transmission potential. These results support the hypothesis that, in DENV- and ZIKV-endemic regions such as South America and Asia, human flavivirus immunity suppresses YFV human amplification potential, reducing the risk of urban outbreaks.

Fig. 1. DENV-2 and ZIKV-immune NHPs have modulated YFV viremia.

a Experimental design in cynomolgus macaques (Created in BioRender. Mirchandani, D. (2023) BioRender.com/t61o691). b Neutralizing antibody titer (FRNT₅₀) against DENV-2 in flavivirus-naïve and DENV-2-exposed animals, prior to YFV challenge. c Neutralizing antibody titer against ZIKV in flavivirus-naïve and ZIKV-exposed animals. Animals with homologous FRNT₅₀ titers below the limit of detection (1:20) were reclassified as seronegative (SN). YFV viremia levels in flavivirus-naïve and DENV-2-exposed (d) and ZIKV-exposed (e) animals from 2–5 DPI. Viremia levels were measured by focus-forming assays. Log₁₀ transformed viremia values were analyzed for the flavivirus-naïve i.e., control group (n = 3), DENV-2 P8-immune (n = 8), DENV-2 NGC-immune (n = 3), DENV-2 SN (n = 2), ZIKV PR-immune (n = 3), and ZIKV DakAr-immune (n = 3) groups by two-way ANOVA with Tukey’s multiple comparison test, each group was compared against the other. A significant difference in DENV-2 P8-immune and DENV-SN groups was observed at 2 (p = 0.0039), 3 (p = 0.0203), and 4 (p = 0.0398) DPI. Data presented as mean values ± SD. Statistical significance (*) color is attributed to the significant difference between that group compared to the flavivirus-naïve group. Reported p-values are based on the results of the respective post-hoc tests. *p < 0.05, **p < 0.01. Source data are provided as a Source Data file.

Fig. 2. Majority of Ae. aegypti fed on YFV-infected DENV-2 and ZIKV-immune NHPs are refractory to YFV infection.

Mosquitoes fed on infected NHPs were allowed to incubate for 14 days at 28 °C and 75% humidity. Mosquito infection rate (number of mosquito bodies infected/total number of engorged mosquitoes) is shown for mosquitoes fed on NHPs at day 3 (a) and day 4 (b) post-YFV infection. Each bar represents a cohort of mosquitoes fed on individual NHPs at respective time points. Values on top of each bar represent the number of mosquitoes tested positive/ total number of mosquitoes engorged. Log₁₀ transformed viral titers in individual mosquito bodies at day 3 (c) and day 4 (d) post-YFV infection. Mosquito infectivity and viral titers have been shown for each animal since there is a variation in viremia levels despite a similar immune status. For statistical analyzes, the data for each group was combined and compared with the outcomes for the flavivirus-naïve group by two-tailed Fisher’s exact test. Similarly, viral titers have been shown for group of mosquitoes that fed on individual NHPs. For statistical analyzes, data from each group was combined and Log₁₀ transformed values were analyzed by one-way ANOVA with Dunnett’s multiple comparison test. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. The following significant difference compared to flavivirus-naïve (n = 3) group was observed: DENV-2 SN (n = 2) p = 0.2678 and ZIKV SN (n = 1) p = 0.0132 at 3 DPI, and DENV-2 SN (n = 2) p = 0.0026 and ZIKV SN (n = 1) p = 0.3132 at 4DPI. Source data are provided as a Source Data file.

Fig. 3. Ae. aegypti fed on DENV-immune, and YFV-challenged NHPs have low infection rates.

Mosquito infectivity (%) was analyzed against NHP viremia for flavivirus-naïve and DENV-immune groups and curve-fitting using non-linear regression. Despite higher viremia at certain timepoints, most mosquitoes that fed on DENV-immune animals were resistant to infection. Source data are provided as a Source Data file.

Fig. 4. Prior flavivirus immunity does not affect the changes in clinical parameters.

Animal weights (a, b), rectal temperatures (c, d), serum alanine transaminase (ALT) (e, f), and serum aspartate aminotransferase (AST) (g, h) were measured from 1–5 and on 14 DPI and compared to baseline (day 0) values. Data were analyzed by two-way ANOVA with Dunnett’s multiple comparison test. The means of groups DENV-2 P8-immune (n = 8), DENV-2 NGC-immune (n = 3), DENV-2 SN (n = 2), ZIKV PR-immune (n = 3), and ZIKV DakAr-immune (n = 3) were compared with the flavivirus-naïve, i.e., control group (n = 3) at each time point. Flavivirus-naïve group had significantly higher AST levels compared to DENV-2 NGC-immune group (p = 0.0092) at 2DPI, and DENV-2 P8-immune group (p = 0.0043) at 3DPI. Data are presented as mean values ± SD. Statistical significance (*) color is attributed to the significant difference between that group compared to the flavivirus-naïve group. Source data are provided as a Source Data file.

Fig. 5. Prior heterologous immunity does not affect the cytokine response to YFV infection.

Log₂ fold-change in 23 different cytokines and chemokines measured in sera of FR1565 (ZIKV-exposed, seronegative animal) on days 5 and 9 post-YFV infection and compared with baseline levels (a). Log₂ fold-change in IL-1ra (b), MCP-1 (c), sCD40L (d), IL-8 (e), and VEGf (f) at 5 DPI and euthanasia timepoints (14 DPI for all animals, 9 DPI for FR1565). Data shown for 23 animals; groups from left to right divided by dotted line are as followed: Flavivirus-naïve, DENV-2-seronegative, DENV-2 P81407-immune, DENV-2 NGC-immune, ZIKV-seronegative, ZIKV PRVABC59-immune, ZIKV DakAr-immune.