Pierre Becquart

Human fatal zaire ebola virus infection is associated with an aberrant innate immunity and with massive lymphocyte apoptosis.

Background Ebolavirus species Zaire (ZEBOV) causes highly lethal hemorrhagic fever, resulting in the death of 90% of patients within days. Most information on immune responses to ZEBOV comes from in vitro studies and animal models. The paucity of data on human immune responses to this virus is mainly due to the fact that most outbreaks occur in remote areas. Published studies in this setting, based on small numbers of samples and limited panels of immunological markers, have given somewhat different results. Methodology/Principal Findings Here, we studied a unique collection of 56 blood samples from 42 nonsurvivors and 14 survivors, obtained during the five outbreaks that occurred between 1996 and 2003 in Gabon and Republic of Congo. Using Luminex technology, we assayed 50 cytokines in all 56 samples and performed phenotypic analyses by flow cytometry. We found that fatal outcome was associated with hypersecretion of numerous proinflammatory cytokines (IL-1β, IL-1RA, IL-6, IL-8, IL-15 and IL-16), chemokines and growth factors (MIP-1α, MIP-1β, MCP-1, M-CSF, MIF, IP-10, GRO-α and eotaxin). Interestingly, no increase of IFNα2 was detected in patients. Furthermore, nonsurvivors were also characterized by very low levels of circulating cytokines produced by T lymphocytes (IL-2, IL-3, IL-4, IL-5, IL-9, IL-13) and by a significant drop of CD3+CD4+ and CD3+CD8+ peripheral cells as well as a high increase in CD95 expression on T lymphocytes. Conclusions/Significance This work, the largest study to be conducted to date in humans, showed that fatal outcome is associated with aberrant innate immune responses and with global suppression of adaptive immunity. The innate immune reaction was characterized by a “cytokine storm,” with hypersecretion of numerous proinflammatory cytokines, chemokines and growth factors, and by the noteworthy absence of antiviral IFNα2. Immunosuppression was characterized by very low levels of circulating cytokines produced by T lymphocytes and by massive loss of peripheral CD4 and CD8 lymphocytes, probably through Fas/FasL-mediated apoptosis.

Concurrent chikungunya and dengue virus infections during simultaneous outbreaks, Gabon, 2007.

An outbreak of febrile illness occurred in Gabon in 2007, with 20,000 suspected cases. Chikungunya or dengue-2 virus infections were identified in 321 patients; 8 patients had documented co-infections. Aedes albopictus was identified as the principal vector for the transmission of both viruses.

The Acute Phase of Chikungunya Virus Infection in Humans Is Associated with Strong Innate Immunity and T CD8 Cell Activation

BACKGROUNDnRapidly spreading to new regions, including the islands of the Indian Ocean, Central Africa, and Europe, Chikungunya fever is becoming a major problem of public health. Unlike other members of the alphavirus genus, immune responses to Chikungunya virus (CHIKV) have been poorly investigated.nnnMETHODSnWe conducted a large ex vivo multiplex study of 50 cytokine, chemokine, and growth factor plasma profiles in 69 acutely infected patients from the Gabonese outbreak of 2007. We also assessed a phenotypic study of T lymphocyte responses during human acute CHIKV infection.nnnRESULTSnCHIKV infection in humans elicited strong innate immunity involving the production of numerous proinflammatory mediators. Interestingly, high levels of Interferon (IFN) α were consistently found. Production of interleukin (IL) 4, IL-10, and IFN-γ suggested the engagement of the adaptive immunity. This was confirmed by flow cytometry of circulating T lymphocytes that showed a CD8+ T lymphocyte response in the early stages of the disease, and a CD4+ T lymphocyte mediated response in the later stages. For the first time to our knowledge, we found evidence of CD95-mediated apoptosis of CD4+ T lymphocytes during the first 2 days after symptoms onset, ex vivo.nnnCONCLUSIONSnTogether, our findings suggest that strong innate immunity is required to control CHIKV infection.

High Prevalence of Both Humoral and Cellular Immunity to Zaire ebolavirus among Rural Populations in Gabon

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-γ 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.

Recent Introduction and Rapid Dissemination of Chikungunya Virus and Dengue Virus Serotype 2 Associated With Human and Mosquito Coinfections in Gabon, Central Africa

BACKGROUNDnChikungunya virus (CHIKV) and Dengue virus serotype 2 (DENV-2) were recently introduced in central Africa, along with Aedes albopictus. Simultaneous outbreaks of CHIKV and DENV-2 have subsequently occurred, in Cameroon in 2006 and Gabon in 2007.nnnMETHODSnTo study the spread of the 2 viruses, we conducted active surveillance of acute febrile syndromes throughout Gabon between 2007 and 2010. Diagnostic methods included quantitative real-time reverse-transcription polymerase chain reaction, and molecular characterization was based on partial envelope gene sequences.nnnRESULTSnBetween 2007 and 2010, 4287 acutely febrile patients were investigated for CHIKV and DENV-2 infections, of whom 1567 were CHIKV-positive, 376 DENV-2-positive, and 37 coinfected. We diagnosed 153 CHIKV and 11 DENV-2 cases in 2008, and 5 CHIKV and 9 DENV-2 cases in 2009. In 2010, CHIKV and DENV-2 caused a second large simultaneous outbreak. Among 2826 acutely febrile patients examined during this outbreak, 1112 were CHIKV-positive, 288 DENV-2-positive, and 28 coinfected. Mosquitoes were collected near the homes of coinfected patients, and 1 Aedes albopictus specimen was found to be positive for both CHIKV and DENV-2.nnnCONCLUSIONSnThese findings show the rapid dissemination of CHIKV and DENV-2 within a nonimmune population in a tropical African country, probably facilitated by the spread of Aedes albopictus. This has resulted in major simultaneous outbreaks with numerous coinfections in both human and mosquito.

Association of KIR2DS1 and KIR2DS3 with fatal outcome in Ebola virus infection.

Zaïre ebolavirus (ZEBOV) infection rapidly outruns the hosts immunity and leads to death within a week. Fatal cases have been associated with an aberrant innate, proinflammatory immune response followed by a suppressed adaptive response leading to the rapid depletion of peripheral NK cells and lymphocytes. A critical role for NK cells has been suggested but not elucidated. In this genetic study, we investigated the association of KIR genotype with disease outcome by comparing genotypes of a Gabonese control population, IgG+ contacts, survivors, and fatalities of ZEBOV infection. We showed that the activating KIR2DS1 and KIR2DS3 genes associate with fatal outcome in Ebola virus infection. In addition, this study brings supplemental evidence in favor of the specificity of the IgG+ contact population. The outcome of fulminating Ebola virus infection could depend in part on the hosts inherited KIR gene repertoire. This supports a key role for KIRs in disease susceptibility to infections.

Acute dengue virus 2 infection in Gabonese patients is associated with an early innate immune response, including strong interferon alpha production

BackgroundDengue is now a leading cause of morbidity and mortality throughout the tropics. We conducted the first ex vivo study of dengue fever (DF) in African patients infected during the first Gabonese dengue virus 2 (DENV-2) outbreak in 2007, in order to investigate cytokine production, including the antiviral cytokine IFN-α, reported to be a potent inhibitor of DENV replication in vitro.MethodsLevels of 50 cytokines, chemokines and growth factors were measured in plasma from 36 patients with DENV-2 infection, and in uninfected controls, using Luminex multiplex technology. The results were interpreted according to the day of sampling after symptom onset. PBMC from six patients were also studied for T lymphocyte cell surface marker expression by flow cytometry.ResultsAcute DENV-2 infection elicited high levels of several pro-inflammatory cytokines (IL-6 and IL-17), chemokines (MIF, RANTES, IP-10 and MCP-1) and growth factors (G-CSF, GM-CSF and VEGF-A). We also observed high levels of IFN-α for the first time in adult DF patients, and CD4+ and CD8+ T cell activation at symptom onset.ConclusionAcute DENV-2 infection in African patients elicits a strong innate response involving IFN-α production, as well as an adaptive immune response.

Identification of Continuous Human B-Cell Epitopes in the VP35, VP40, Nucleoprotein and Glycoprotein of Ebola Virus

Ebola virus (EBOV) is a highly virulent human pathogen. Recovery of infected patients is associated with efficient EBOV-specific immunoglobulin G (IgG) responses, whereas fatal outcome is associated with defective humoral immunity. As B-cell epitopes on EBOV are poorly defined, we sought to identify specific epitopes in four EBOV proteins (Glycoprotein (GP), Nucleoprotein (NP), and matrix Viral Protein (VP)40 and VP35). For the first time, we tested EBOV IgG+ sera from asymptomatic individuals and symptomatic Gabonese survivors, collected during the early humoral response (seven days after the end of symptoms) and the late memory phase (7–12 years post-infection). We also tested sera from EBOV-seropositive patients who had never had clinical signs of hemorrhagic fever or who lived in non-epidemic areas (asymptomatic subjects). We found that serum from asymptomatic individuals was more strongly reactive to VP40 peptides than to GP, NP or VP35. Interestingly, anti-EBOV IgG from asymptomatic patients targeted three immunodominant regions of VP40 reported to play a crucial role in virus assembly and budding. In contrast, serum from most survivors of the three outbreaks, collected a few days after the end of symptoms, reacted mainly with GP peptides. However, in asymptomatic subjects the longest immunodominant domains were identified in GP, and analysis of the GP crystal structure revealed that these domains covered a larger surface area of the chalice bowl formed by three GP1 subunits. The B-cell epitopes we identified in the EBOV VP35, VP40, NP and GP proteins may represent important tools for understanding the humoral response to this virus and for developing new antibody-based therapeutics or detection methods.

Immunoglobulin G in Ebola Outbreak Survivors, Gabon

To the Editor: Three well-documented outbreaks of Ebola hemorrhagic fever occurred from 1996 through 2001 in Gabon in central Africa (1). All were caused by the highly pathogenic species Zaire ebolavirus, which is associated with an ≈80% case-fatality rate. The first outbreak hit Mayibout, a village in northeast Gabon in January and February 1996, causing 31 cases and 21 deaths. The first victims were children who helped carry and butcher a chimpanzee carcass found in the forest. The second outbreak lasted from October 1996 through March 1997 and occurred in the Booue region, about 150 km southwest of Mayibout, Gabon. The outbreak area was located along a trunk road and railroad track, and the infection spread to several villages around Booue, then to Libreville, the capital of Gabon, where 15 cases were recorded. The third outbreak occurred October 2001 through May 2002 in the Mekambo area, about 150 km from Mayibout in the east (2). This outbreak consisted of several independent chains of human transmission arising from infected animal carcasses, mainly chimpanzees and gorillas. It caused 65 cases and 53 deaths and coincided with major outbreaks in great apes that decimated wild populations (3,4). A total of 207 human cases were recorded during these 3 outbreaks; 149 persons died. Of the fatal and nonfatal cases 31 and 24, respectively, were confirmed by real-time reverse transcription–PCR, antigen detection, and immunoglobulin (Ig) G ELISA at Centre International de Recherches Medicales de Franceville (CIRMF) in Gabon. n nBecause of the lack of available samples from survivors, little is known about the duration of IgG antibody response. However, studies of 20 survivors convalescing after the 1995 Kikwit outbreak in the Democratic Republic of the Congo (DRC) showed that Zaire ebolavirus IgG appeared 5 to 18 days after symptom onset and persisted at least 21 months (5,6). With the exception of 2 survivors sampled 10 years after the 1976 Yambuku outbreak in DRC (7), no data are available on Zaire ebolavirus IgG persistence beyond 21 months. Low seroprevalence rates of Ebola virus or Marburg virus found in surveys of patients in outbreak areas have been attributed to seroreversion (8–10). n nTo investigate the persistence of Zaire ebolavirus IgG, we studied laboratory-confirmed survivors of the 3 outbreaks in Gabon. The study was approved by the Gabon Ministry of Health and by the traditional chief of each village, and written informed consent was obtained from each survivor. During 3 months of investigations in the different outbreak areas beginning in June 2007, we located 11, 3, and 6 survivors of the 2001 Mekambo, 1996 Booue, and 1996 Mayibout outbreaks, respectively. During home visits, the survivors underwent a brief medical consultation, malaria smears were taken, and basic medicines were provided to the villagers. We collected blood samples in EDTA tubes; plasma was separated by centrifugation in the field and stored in dry nitrogen until transfer to the CIRMF laboratory in Gabon, where it was stored at –80°C. ELISA was performed as previously described, using reagents provided by the Special Pathogens Branch, Centers for Disease Control and Prevention (Atlanta, GA, USA) (7). The optical density (OD) cut-off value (0.13) was calculated as the mean + 3 SD of adjusted OD values for 103 negative control serum samples obtained from Caucasian persons living in Europe. n nAll 20 survivors had positive test results for Zaire ebolavirus IgG (Table). The adjusted OD values at a dilution of 1:1,600 ranged from 0.3 to 3.4 in the 9 survivors of the 1996 outbreaks and from 0.7 to 3.5 in the 11 survivors of the 2001 outbreak. Adjusted OD values determined during the symptomatic period and/or a few days to 1 month after recovery were available for some survivors (Table). Specific IgG appeared by day 5 after symptom onset, increased during the symptomatic period (as shown by higher titers on day 10), peaked by day 30 (2 weeks after recovery), then declined slowly over several years. Zaire ebolavirus IgG remained detectable, often at high levels, >11 years after the infection. n n n nTable n nAdjusted OD values in patients infected with Zaire ebolavirus during 3 outbreaks in Gabon, determined by testing at days 5, 10, and/or 30 after symptom onset and again in 2007 (7 or 11 years after recovery)* n n n nThese long-lasting IgG antibody responses found in 20 survivors of 3 different Zaire ebolavirus outbreaks rule out the hypothesis that low Ebola virus (and Marburg virus) seroprevalence rates found in epidemic regions of Africa are due to rapid loss of specific IgG. Whether this immunity is sufficient to protect from recurrent infection remains undetermined. These findings show that IgG ELISA is suitable for epidemiologic and epizootiologic investigations of Ebola and that Zaire ebolavirus IgG is an excellent indicator of Zaire ebolavirus circulation in humans.

Risk Factors for Zaire ebolavirus–Specific IgG in Rural Gabonese Populations

BACKGROUNDnIn Gabon, several Ebolavirus outbreaks have occurred exclusively in the northeastern region. We conducted a large serosurvey to identify areas and populations at risk and potential demographic, clinical, and behavioral risk factors.nnnMETHODSnBlood samples and clinical and sociodemographic data were collected from 4349 adults and 362 children in a random sample of 220 villages in the 9 provinces of Gabon. An enzyme-linked immunosorbent assay was used to detect Zaire ebolavirus (ZEBOV)-specific IgG, and thin blood smears were used to detect parasites. Logistic regression was implemented using Stata software (Stata), and a probability level of <.05 was considered to be statistically significant.nnnRESULTSnThe prevalence of ZEBOV-specific IgG was 15.3% overall, increasing to 32.4% (P< .001) in forest areas. No sociodemographic risk factors were found, but the antibody prevalence increased linearly up to 20 years of age. Chronic arthralgia and amicrofilaremia were the only factors associated with ZEBOV seropositivity.nnnCONCLUSIONSnThese findings confirm the endemicity of ZEBOV in Gabon and its link to the ecosystem. Human antibody positivity would appear to be to the result of exposure to contaminated fruits.