Early immune responses accompanying human asymptomatic Ebola infections

Abstract

In a recent study we identified certain asymptomatic individuals infected by Ebola virus (EBOV) who mounted specific IgG and early and strong inflammatory responses. Here, we further characterized the primary immune response to EBOV during the course of asymptomatic infection in humans. Inflammatory responses occurred in temporal association with anti-inflammatory phase composed by soluble antagonist IL-1RA, circulating TNF receptors, IL-10 and cortisol. At the end of the inflammatory process, mRNA expression of T-cell cytokines (IL-2 and IL-4) and activation markers (CD28, CD40L and CTLA4) was up-regulated, strongly suggesting T-cell activation. This T-cell activation was followed by EBOV-specific IgG responses (mainly IgG3 ang IgG1), and by marked and sustained up-regulation of IFN gamma, FasL and perforin mRNA expression, suggesting activation of cytotoxic cells. The terminal down-regulation of these latter markers coincided with the release of the apoptotic marker 41/7 NMP in blood and with the disappearance of viral RNA from PBMC, suggesting that infected cells are eliminated by cytotoxic mechanisms. Finally, RT-PCR analysis of TCR-V beta repertoire usage showed that TCR-V beta 12 mRNA was never expressed during the infection. Taken together, these findings improve our understanding about immune response during human asymptomatic Ebola infection, and throw new light on protection against Ebola virus.

Fig. 1

Agarose gel electrophoresis of PCR DNA products from nested RT-PCR during the course of infection in 3 asymptomatic individuals. PBMC were collected from asymptomatic subjects 7, 9, 16 and 23 days after initial exposure to a sick patient. To control for possible RNA or DNA contamination, reactions without cDNA, and 10 negative controls were run in parallel (not shown).

Fig. 2

Anti-inflammatory response. Naturally circulating antagonists (IL-1RA, sTNFRI, sTNFRII in ng/ml), IL-10 (in pg/ml), and plasma cortisol (in ng/ml) were measured as described in Methods. Individual values are shown 7, 9, 16 and 23 days following initial exposure to the virus. Each asymptomatic individual is represented by a point.

Fig. 3

T-cell responses. RNA expression of Th1 and Th2 cytokines (IL-2 and IL-4) and, T-cell activation markers (CD28, CD40L and CTLA4) was analysed as described in Methods. The results (ethidium bromide staining after electrophoresis on agarose gel) show the kinetics of mRNA expression in three asymptomatic individuals, 7, 9, 15 and 23 following initial exposure, and in two endemic controls.

Fig. 4

Cytotoxic responses. (a) RNA expression of cytotoxicity markers (perforin, Fas, FasL, IFNγ and CD8) was analysed as described in Methods. The results (ethidium bromide staining after electrophoresis on agarose gel) show the kinetics of mRNA expression in three asymptomatic individuals, 7, 9, 15 and 23 following initial exposure, and in two endemic controls (b) Plasma 41/7 levels (Units/ml) were measured with an ELISA method. Each asymptomatic subject is represented by a point.

Fig. 5

Analysis of the T-cell antigen receptor-Vβ repertoire during asymptomatic Ebola infection by RT-PCR analysis. Agarose gel electrophoresis of PCR products for various (n = 20) combinations of Vβ gene family specific and Cβ oligonucleotide primers. Results show the kinetics of mRNA expression of six TCR-Vβ species in three asymptomatic individuals, 7, 9, 15 and 23 days following initial exposure, and in two endemic controls.