High prevalence of both humoral and cellular immunity to Zaire ebolavirus among rural populations in Gabon

Abstract

To better understand Zaire ebolavirus (ZEBOV) circulation and transmission to humans, we conducted a large serological survey of rural populations in Gabon, a country characterized by both epidemic and non epidemic regions. The survey lasted three years and covered 4,349 individuals from 220 randomly selected villages, representing 10.7% of all villages in Gabon. Using a sensitive and specific ELISA method, we found a ZEBOV-specific IgG seroprevalence of 15.3% overall, the highest ever reported. The seroprevalence rate was significantly higher in forested areas (19.4%) than in other ecosystems, namely grassland (12.4%), savannah (10.5%), and lakeland (2.7%). No other risk factors for seropositivity were found. The specificity of anti-ZEBOV IgG was confirmed by Western blot in 138 individuals, and CD8 T cells from seven IgG+ individuals were shown to produce IFN-gamma after ZEBOV stimulation. Together, these findings show that a large fraction of the human population living in forested areas of Gabon has both humoral and cellular immunity to ZEBOV. In the absence of identified risk factors, the high prevalence of "immune" persons suggests a common source of human exposure such as fruits contaminated by bat saliva. These findings provide significant new insights into ZEBOV circulation and human exposure, and raise important questions as to the human pathogenicity of ZEBOV and the existence of natural protective immunization.

Figure 1. Map of Gabonese villages (red circles) included in the survey, according to the ecological region.

Villages where children <16 years were specifically included in the study are indicated by yellow circles. The overall prevalence of ZEBOV-specific IgG in each ecological region is shown.

Figure 2. Prevalence of ZEBOV-specific IgG in villages within each ecological region.

Each circle represents a group of two or three neighboring villages.

Figure 3. Prevalence of ZEBOV-specific IgG according to age in the north-eastern region of Gabon (green).

The overall prevalence of ZEBOV-specific IgG according to age is shown in brown.

Figure 4. Western blot analysis of ZEBOV-specific IgG from two symptomatic individuals who recovered (T+), one negative endemic control, and 10 IgG+ asymptomatic individuals.

Figure 5. ZEBOV-specific memory T cell analysis by flow cytometry.

IFN-γ production by CD3+CD8+ T lymphocytes was evaluated by intracellular flow cytometry on PBMC stimulated with mock supernatant (green histograms) or heat-inactivated ZEBOV culture supernatant (iZEBOV, red histograms). Analysis performed 2 and 3 days after stimulation of PBMC from seven IgG+ asymptomatic individuals (A), four negative controls and three laboratory-confirmed survivors of the 2001–2002 outbreak in Gabon (B). No significant responses were observed in CD4+ T lymphocytes.

Figure 6. ZEBOV-specific memory T cell analysis by flow cytometry.

IFN-γ production by CD8+ T lymphocytes was evaluated by intracellular flow cytometry on PBMC stimulated with mock supernatant (green histograms) or heat-inactivated ZEBOV culture supernatant (iZEBOV, red histograms). Analyses performed 2, 3, 5 and 7 days after initial iZEBOV or mock stimulation (arrow) of PBMC from three IgG+ asymptomatic individuals. A second round of stimulation was performed on day 6. Results for one IgG+ individual are shown.