Chikungunya Outbreak in the Republic of the Congo, 2019-Epidemiological, Virological and Entomological Findings of a South-North Multidisciplinary Taskforce Investigation.

The Republic of Congo (RoC) declared a chikungunya (CHIK) outbreak on 9 February 2019. We conducted a ONE-Human-Animal HEALTH epidemiological, virological and entomological investigation. Methods: We collected national surveillance and epidemiological data. CHIK diagnosis was based on RT-PCR and CHIKV-specific antibodies. Full CHIKV genome sequences were obtained by Sanger and MinION approaches and Bayesian tree phylogenetic analysis was performed. Mosquito larvae and 215 adult mosquitoes were collected in different villages of Kouilou and Pointe-Noire districts and estimates of Aedes (Ae.) mosquitos' CHIKV-infectious bites obtained. We found two new CHIKV sequences of the East/Central/South African (ECSA) lineage, clustering with the recent enzootic sub-clade 2, showing the A226V mutation. The RoC 2019 CHIKV strain has two novel mutations, E2-T126M and E2-H351N. Phylogenetic suggests a common origin from 2016 Angola strain, from which it diverged around 1989 (95% HPD 1985-1994). The infectious bite pattern was similar for 2017, 2018 and early 2019. One Ae. albopictus pool was RT-PCR positive. The 2019 RoC CHIKV strain seems to be recently introduced or be endemic in sylvatic cycle. Distinct from the contemporary Indian CHIKV isolates and in contrast to the original Central-African strains (transmitted by Ae. aegypti), it carries the A226V mutation, indicating an independent adaptive mutation in response to vector replacement (Ae. albopictus vs Ae. aegypti).

Severe Human Lassa Fever Is Characterized by Nonspecific T-Cell Activation and Lymphocyte Homing to Inflamed Tissues.

Lassa fever (LF) is a zoonotic viral hemorrhagic fever caused by Lassa virus (LASV), which is endemic to West African countries. Previous studies have suggested an important role for T-cell-mediated immunopathology in LF pathogenesis, but the mechanisms by which T cells influence disease severity and outcome are not well understood. Here, we present a multiparametric analysis of clinical immunology data collected during the 2017-2018 Lassa fever outbreak in Nigeria. During the acute phase of LF, we observed robust activation of the polyclonal T-cell repertoire, which included LASV-specific and antigenically unrelated T cells. However, severe and fatal LF cases were characterized by poor LASV-specific effector T-cell responses. Severe LF was also characterized by the presence of circulating T cells with homing capacity to inflamed tissues, including the gut mucosa. These findings in LF patients were recapitulated in a mouse model of LASV infection, in which mucosal exposure resulted in remarkably high lethality compared to skin exposure. Taken together, our findings indicate that poor LASV-specific T-cell responses and activation of nonspecific T cells with homing capacity to inflamed tissues are associated with severe LF.IMPORTANCE Lassa fever may cause severe disease in humans, in particular in areas of endemicity like Sierra Leone and Nigeria. Despite its public health importance, the pathophysiology of Lassa fever in humans is poorly understood. Here, we present clinical immunology data obtained in the field during the 2018 Lassa fever outbreak in Nigeria indicating that severe Lassa fever is associated with activation of T cells antigenically unrelated to Lassa virus and poor Lassa virus-specific effector T-cell responses. Mechanistically, we show that these bystander T cells express defined tissue homing signatures that suggest their recruitment to inflamed tissues and a putative role of these T cells in immunopathology. These findings open a window of opportunity to consider T-cell targeting as a potential postexposure therapeutic strategy against severe Lassa fever, a hypothesis that could be tested in relevant animal models, such as nonhuman primates.

Ebola Virus Disease Survivors Show More Efficient Antibody Immunity than Vaccinees Despite Similar Levels of Circulating Immunoglobulins.

The last seven years have seen the greatest surge of Ebola virus disease (EVD) cases in equatorial Africa, including the 2013-2016 epidemic in West Africa and the recent epidemics in the Democratic Republic of Congo (DRC). The vaccine clinical trials that took place in West Africa and the DRC, as well as follow-up studies in collaboration with EVD survivor communities, have for the first time allowed researchers to compare immune memory induced by natural infection and vaccination. These comparisons may be relevant to evaluate the putative effectiveness of vaccines and candidate medical countermeasures such as convalescent plasma transfer. In this study, we compared the long-term functionality of anti-EBOV glycoprotein (GP) antibodies from EVD survivors with that from volunteers who received the recombinant vesicular stomatitis virus vectored vaccine (rVSV-ZEBOV) during the Phase I clinical trial in Hamburg. Our study highlights important differences between EBOV vaccination and natural infection and provides a framework for comparison with other vaccine candidates.

Regulation of the Ebola Virus VP24 Protein by SUMO.

Some viruses take advantage of conjugation of ubiquitin or ubiquitin-like proteins to enhance their own replication. One example is Ebola virus, which has evolved strategies to utilize these modification pathways to regulate the viral proteins VP40 and VP35 and to counteract the host defenses. Here, we show a novel mechanism by which Ebola virus exploits the ubiquitin and SUMO pathways. Our data reveal that minor matrix protein VP24 of Ebola virus is a bona fide SUMO target. Analysis of a SUMOylation-defective VP24 mutant revealed a reduced ability to block the type I interferon (IFN) pathway and to inhibit IFN-mediated STAT1 nuclear translocation, exhibiting a weaker interaction with karyopherin 5 and significantly diminished stability. Using glutathione S-transferase (GST) pulldown assay, we found that VP24 also interacts with SUMO in a noncovalent manner through a SIM domain. Mutation of the SIM domain in VP24 resulted in a complete inability of the protein to downmodulate the IFN pathway and in the monoubiquitination of the protein. We identified SUMO deubiquitinating enzyme ubiquitin-specific-processing protease 7 (USP7) as an interactor and a negative modulator of VP24 ubiquitination. Finally, we show that mutation of one ubiquitination site in VP24 potentiates the IFN modulatory activity of the viral protein and its ability to block IFN-mediated STAT1 nuclear translocation, pointing to the ubiquitination of VP24 as a negative modulator of the VP24 activity. Altogether, these results indicate that SUMO interacts with VP24 and promotes its USP7-mediated deubiquitination, playing a key role in the interference with the innate immune response mediated by the viral protein.IMPORTANCE The Ebola virus VP24 protein plays a critical role in escape of the virus from the host innate immune response. Therefore, deciphering the molecular mechanisms modulating VP24 activity may be useful to identify potential targets amenable to therapeutics. Here, we identify the cellular proteins USP7, SUMO, and ubiquitin as novel interactors and regulators of VP24. These interactions may represent novel potential targets to design new antivirals with the ability to modulate Ebola virus replication.

Comparative pathogenesis of Ebola virus and Reston virus infection in humanized mice.

Filoviruses of the genus Ebolavirus include 6 species with marked differences in their ability to cause disease in humans. From the highly virulent Ebola virus to the seemingly nonpathogenic Reston virus, case fatality rates can range between 0% and 90%. In order to understand the molecular basis of these differences, it is imperative to establish disease models that recapitulate human disease as faithfully as possible. Nonhuman primates (NHPs) are the gold-standard models for filovirus pathogenesis, but comparative studies are skewed by the fact that Reston virus infection can be lethal for NHPs. Here we used HLA-A2-transgenic, NOD-scid-IL-2γ receptor-knockout (NSG-A2) mice reconstituted with human hematopoiesis to compare Ebola virus and Reston virus pathogenesis in a human-like environment. While markedly less pathogenic than Ebola virus, Reston virus killed 20% of infected mice, a finding that was linked to exacerbated inflammation and viral replication in the liver. In addition, the case fatality ratios of different Ebolavirus species in humans were recapitulated in the humanized mice. Our findings point to humanized mice as a putative model to test the pathogenicity of newly discovered filoviruses, and suggest that further investigations on Reston virus pathogenesis in humans are warranted.

Distinct Immunogenicity and Efficacy of Poxvirus-Based Vaccine Candidates against Ebola Virus Expressing GP and VP40 Proteins.

Zaire and Sudan ebolavirus species cause a severe disease in humans and nonhuman primates (NHPs) characterized by a high mortality rate. There are no licensed therapies or vaccines against Ebola virus disease (EVD), and the recent 2013 to 2016 outbreak in West Africa highlighted the need for EVD-specific medical countermeasures. Here, we generated and characterized head-to-head the immunogenicity and efficacy of five vaccine candidates against Zaire ebolavirus (EBOV) and Sudan ebolavirus (SUDV) based on the highly attenuated poxvirus vector modified vaccinia virus Ankara (MVA) expressing either the virus glycoprotein (GP) or GP together with the virus protein 40 (VP40) forming virus-like particles (VLPs). In a human monocytic cell line, the different MVA vectors (termed MVA-EBOVs and MVA-SUDVs) triggered robust innate immune responses, with production of beta interferon (IFN-ß), proinflammatory cytokines, and chemokines. Additionally, several innate immune cells, such as dendritic cells, neutrophils, and natural killer cells, were differentially recruited in the peritoneal cavity of mice inoculated with MVA-EBOVs. After immunization of mice with a homologous prime/boost protocol (MVA/MVA), total IgG antibodies against GP or VP40 from Zaire and Sudan ebolavirus were differentially induced by these vectors, which were mainly of the IgG1 and IgG3 isotypes. Remarkably, an MVA-EBOV construct coexpressing GP and VP40 protected chimeric mice challenged with EBOV to a greater extent than a vector expressing GP alone. These results support the consideration of MVA-EBOVs and MVA-SUDVs expressing GP and VP40 and producing VLPs as best-in-class potential vaccine candidates against EBOV and SUDV.IMPORTANCE EBOV and SUDV cause a severe hemorrhagic fever affecting humans and NHPs. Since their discovery in 1976, they have caused several sporadic epidemics, with the recent outbreak in West Africa from 2013 to 2016 being the largest and most severe, with more than 11,000 deaths being reported. Although some vaccines are in advanced clinical phases, less expensive, safer, and more effective licensed vaccines are desirable. We generated and characterized head-to-head the immunogenicity and efficacy of five novel vaccines against EBOV and SUDV based on the poxvirus MVA expressing GP or GP and VP40. The expression of GP and VP40 leads to the formation of VLPs. These MVA-EBOV and MVA-SUDV recombinants triggered robust innate and humoral immune responses in mice. Furthermore, MVA-EBOV recombinants expressing GP and VP40 induced high protection against EBOV in a mouse challenge model. Thus, MVA expressing GP and VP40 and producing VLPs is a promising vaccine candidate against EBOV and SUDV.

Novel Cross-Reactive Monoclonal Antibodies against Ebolavirus Glycoproteins Show Protection in a Murine Challenge Model.

Out of an estimated 31,100 cases since their discovery in 1976, ebolaviruses have caused approximately 13,000 deaths. The vast majority (∼11,000) of these occurred during the 2013-2016 West African epidemic. Three out of five species in the genus are known to cause Ebola Virus Disease in humans. Several monoclonal antibodies against the ebolavirus glycoprotein are currently in development as therapeutics. However, there is still a paucity of monoclonal antibodies that can cross-react between the glycoproteins of different ebolavirus species, and the mechanism of these monoclonal antibody therapeutics is still not understood in detail. Here, we generated a panel of eight murine monoclonal antibodies (MAbs) utilizing a prime-boost vaccination regimen with a Zaire ebolavirus glycoprotein expression plasmid followed by infection with a vesicular stomatitis virus expressing the Zaire ebolavirus glycoprotein. We tested the binding breadth of the resulting monoclonal antibodies using a set of recombinant surface glycoproteins from Reston, Taï Forest, Bundibugyo, Zaire, Sudan, and Marburg viruses and found two antibodies that showed pan-ebolavirus binding. An in vivo Stat2-/- mouse model was utilized to test the ability of these MAbs to protect from infection with a vesicular stomatitis virus expressing the Zaire ebolavirus glycoprotein. Several of our antibodies, including the broadly binding ones, protected mice from mortality despite lacking neutralization capability in vitro, suggesting their protection may be mediated by Fc-FcR interactions. Indeed, three antibodies displayed cellular phagocytosis and/or antibody-dependent cell-mediated cytotoxicity in vitro Our antibodies, specifically the two identified cross-reactive monoclonal antibodies (KL-2E5 and KL-2H7), might add to the understanding of anti-ebolavirus humoral immunity.IMPORTANCE This study describes the generation of a panel of novel anti-ebolavirus glycoprotein monoclonal antibodies, including two antibodies with broad cross-reactivity to all known ebolavirus species. The antibodies were raised using a heterologous DNA-viral vector prime-boost regimen, resulting in a high proportion of cross-reactive antibodies (25%). Similar vaccination regimens have been used successfully to induce broad protection against influenza viruses in humans, and our limited data indicate that this might be a useful strategy for filovirus vaccines as well. Several of our antibodies showed protective efficacy when tested in a novel murine challenge model and may be developed into future therapeutics.

Ebola virus infection kinetics in chimeric mice reveal a key role of T cells as barriers for virus dissemination.

Ebola virus (EBOV) causes severe systemic disease in humans and non-human primates characterized by high levels of viremia and virus titers in peripheral organs. The natural portals of virus entry are the mucosal surfaces and the skin where macrophages and dendritic cells (DCs) are primary EBOV targets. Due to the migratory properties of DCs, EBOV infection of these cells has been proposed as a necessary step for virus dissemination via draining lymph nodes and blood. Here we utilize chimeric mice with competent hematopoietic-driven immunity, to show that EBOV primarily infects CD11b+ DCs in non-lymphoid and lymphoid tissues, but spares the main cross-presenting CD103+ DC subset. Furthermore, depletion of CD8 and CD4 T cells resulted in loss of early control of virus replication, viremia and fatal Ebola virus disease (EVD). Thus, our findings point out at T cell function as a key determinant of EVD progress and outcome.

Increased Proinflammatory Cytokine Levels in Prolonged Arthralgia in Ross River Virus Infection.

Ross River virus, a mosquitoborne alphavirus, causes epidemic polyarthritis in Australia and the Pacific region. We analyzed serum cytokine, chemokine, and growth factor levels in travelers returning to Germany from Australia. Serum samples showed elevated concentrations in the acute phase of the illness and, more pronounced, in the long-lasting convalescent phase.

Humanized Mice Reproduce Acute and Persistent Human Adenovirus Infection.

Severe human adenovirus (HAdV) infections are an increasing threat for immunosuppressed individuals, particularly those who have received stem cell transplants. It has been previously hypothesized that severe infections might be due to reactivation of a persistent infection, but this hypothesis has been difficult to test owing to the lack of a permissive in vivo model of HAdV infection. Here we established a humanized mouse model that reproduces features of acute and persistent HAdV infection. In this model, acute infection correlated with high mortality, weight loss, liver pathology, and expression of viral proteins in several organs. In contrast, persistent infection was asymptomatic and led to establishment of HAdV-specific adaptive immunity and expression of early viral genes exclusively in the bone marrow. These findings validate the use of humanized mice to study acute and persistent HAdV infection and strongly suggest the presence of cellular reservoirs in the bone marrow.

Monocyte-derived dendritic cells enhance protection against secondary influenza challenge by controlling the switch in CD8+ T-cell immunodominance.

Influenza virus infection triggers an increase in the number of monocyte-derived dendritic cells (moDCs) in the respiratory tract, but the role of these cells during antiviral immunity is still unclear. Here we show that during influenza infection, moDCs dominate the late activation of CD8+ T cells and trigger the switch in immunodominance of the CD8+ T-cell response from acidic polymerase specificity to nucleoprotein specificity. Abrogation of monocyte recruitment or depletion of moDCs strongly compromised host resistance to secondary influenza challenge. These findings underscore a novel function of moDCs in the antiviral response to influenza virus, and have important implications for vaccine design.

Cytokine kinetics of Zika virus-infected patients from acute to reconvalescent phase.

Zika virus is an emerging mosquito-borne flavivirus currently causing large epidemics in the Pacific Ocean region and Brazil. Clinically, Zika fever resembles dengue fever, but is less severe. Whereas the clinical syndrome and laboratory diagnostic procedures have been described, little attention was paid to the immunology of the disease and its possible use for clinical follow-up of patients. Here, we investigate the role of cytokines in the pathogenesis of Zika fever in travelers returning from Asia, the Pacific, and Brazil. Polyfunctional T cell activation (Th1, Th2, Th9, and Th17 response) was seen during the acute phase characterized by respective cytokine level increases, followed by a decrease in the reconvalescent phase.

Intranasal Administration of Recombinant Influenza Vaccines in Chimeric Mouse Models to Study Mucosal Immunity.

Vaccines are one of the greatest achievements of mankind, and have saved millions of lives over the last century. Paradoxically, little is known about the physiological mechanisms that mediate immune responses to vaccines perhaps due to the overall success of vaccination, which has reduced interest into the molecular and physiological mechanisms of vaccine immunity. However, several important human pathogens including influenza virus still pose a challenge for vaccination, and may benefit from immune-based strategies. Although influenza reverse genetics has been successfully applied to the generation of live-attenuated influenza vaccines (LAIVs), the addition of molecular tools in vaccine preparations such as tracer components to follow up the kinetics of vaccination in vivo, has not been addressed. In addition, the recent generation of mouse models that allow specific depletion of leukocytes during kinetic studies has opened a window of opportunity to understand the basic immune mechanisms underlying vaccine-elicited protection. Here, we describe how the combination of reverse genetics and chimeric mouse models may help to provide new insights into how vaccines work at physiological and molecular levels, using as example a recombinant, cold-adapted, live-attenuated influenza vaccine (LAIV). We utilized laboratory-generated LAIVs harboring cell tracers as well as competitive bone marrow chimeras (BMCs) to determine the early kinetics of vaccine immunity and the main physiological mechanisms responsible for the initiation of vaccine-specific adaptive immunity. In addition, we show how this technique may facilitate gene function studies in single animals during immune responses to vaccines. We propose that this technique can be applied to improve current prophylactic strategies against pathogens for which urgent medical countermeasures are needed, for example influenza, HIV, Plasmodium, and hemorrhagic fever viruses such as Ebola virus.

Ebola virus disease in mice with transplanted human hematopoietic stem cells.

The development of treatments for Ebola virus disease (EVD) has been hampered by the lack of small-animal models that mimick human disease. Here we show that mice with transplanted human hematopoetic stem cells reproduce features typical of EVD. Infection with Ebola virus was associated with viremia, cell damage, liver steatosis, signs of hemorrhage, and high lethality. Our study provides a small-animal model with human components for the development of EVD therapies.

Mucosal polyinosinic-polycytidylic acid improves protection elicited by replicating influenza vaccines via enhanced dendritic cell function and T cell immunity.

Live-attenuated influenza vaccines (LAIVs) have the potential to generate CD8 T cell immunity that may limit the virulence of an antigenically shifted influenza strain in a population lacking protective Abs. However, current LAIVs exert limited T cell immunity restricted to the vaccine strains. One approach to improve LAIV-induced T cell responses is the use of specific adjuvants to enhance T cell priming by respiratory dendritic cells, but this hypothesis has not been addressed. In this study, we assessed the effect of the TLR3 ligand polyinosinic-polycytidylic acid (poly IC) on CD8 T cell immunity and protection elicited by LAIVs. Mucosal treatment with poly IC shortly after vaccination enhanced respiratory dendritic cell function, CD8 T cell formation, and production of neutralizing Abs. This adjuvant effect of poly IC was dependent on amplification of TLR3 signaling by nonhematopoietic radioresistant cells and enhanced mouse protection to homosubtypic, as well as heterosubtypic, virus challenge. Our findings indicate that mucosal TLR3 ligation may be used to improve CD8 T cell responses to replicating vaccines, which has implications for protection in the absence of pre-existing Ab immunity.