Hemostasis defects underlying the hemorrhagic syndrome caused by mammarenaviruses in a cynomolgus macaque model.

Viral hemorrhagic fevers (HF) are a group of acute febrile diseases with high mortality rates. Although hemostatic dysfunction appears to be a major determinant of the severity of the disease, it is still unclear what pathogenic mechanisms lead to it. In clinical studies it is found that arenaviruses, such as Lassa, Machupo, and Guanarito viruses cause HF that vary in symptoms and biological alterations. In this study we aimed to characterize the hemostatic dysfunction induced by arenaviral HF to determine its implication in the severity of the disease and to elucidate the origin of this syndrome. We found that lethal infection with Machupo, Guanarito, and Lassa viruses is associated with cutaneomucosal, cerebral, digestive, and pulmonary hemorrhages. The affected animals developed a severe alteration of the coagulation system, which was concomitant with acute hepatitis, minor deficit of hepatic factor synthesis, presence of a plasmatic inhibitor of coagulation, and dysfunction of the fibrinolytic system. Despite signs of increased vascular permeability, endothelial cell infection was not a determinant factor of the hemorrhagic syndrome. There were also alterations of the primary hemostasis during lethal infection, with moderate to severe thrombocytopenia and platelet dysfunction. Finally, we show that lethal infection is accompanied by a reduced hematopoietic potential of the bone marrow. This study provides an unprecedented characterization of the hemostasis defects induced by several highly pathogenic arenaviruses.

Rapid protection induced by a single-shot Lassa vaccine in male cynomolgus monkeys.

Lassa fever hits West African countries annually in the absence of licensed vaccine to limit the burden of this viral hemorrhagic fever. We previously developed MeV-NP, a single-shot vaccine protecting cynomolgus monkeys against divergent strains one month or more than a year before Lassa virus infection. Given the limited dissemination area during outbreaks and the risk of nosocomial transmission, a vaccine inducing rapid protection could be useful to protect exposed people during outbreaks in the absence of preventive vaccination. Here, we test whether the time to protection can be reduced after immunization by challenging measles virus pre-immune male cynomolgus monkeys sixteen or eight days after a single shot of MeV-NP. None of the immunized monkeys develop disease and they rapidly control viral replication. Animals immunized eight days before the challenge are the best controllers, producing a strong CD8 T-cell response against the viral glycoprotein. A group of animals was also vaccinated one hour after the challenge, but was not protected and succumbed to the disease as the control animals. This study demonstrates that MeV-NP can induce a rapid protective immune response against Lassa fever in the presence of MeV pre-existing immunity but can likely not be used as therapeutic vaccine.

A MOPEVAC multivalent vaccine induces sterile protection against New World arenaviruses in non-human primates.

Pathogenic New World arenaviruses (NWAs) cause haemorrhagic fevers and can have high mortality rates, as shown in outbreaks in South America. Neutralizing antibodies (Abs) are critical for protection from NWAs. Having shown that the MOPEVAC vaccine, based on a hyperattenuated arenavirus, induces neutralizing Abs against Lassa fever, we hypothesized that expression of NWA glycoproteins in this platform might protect against NWAs. Cynomolgus monkeys immunized with MOPEVACMAC, targeting Machupo virus, prevented the lethality of this virus and induced partially NWA cross-reactive neutralizing Abs. We then developed the pentavalent MOPEVACNEW vaccine, expressing glycoproteins from all pathogenic South American NWAs. Immunization of cynomolgus monkeys with MOPEVACNEW induced neutralizing Abs against five NWAs, strong innate followed by adaptive immune responses as detected by transcriptomics and provided sterile protection against Machupo virus and the genetically distant Guanarito virus. MOPEVACNEW may thus be efficient to protect against existing and potentially emerging NWAs.

Pathogenesis of recent Lassa virus isolates from lineages II and VII in cynomolgus monkeys.

The area of Lassa virus (LASV) circulation is expanding, with the emergence of highly pathogenic new LASV lineages. Benin recently became an endemic country for LASV and has seen the emergence of a new LASV lineage (VII). The first two outbreaks in 2014 and 2016 showed a relatively high mortality rate compared to other outbreaks. We infected cynomolgus monkeys with two strains belonging to lineage II and lineage VII that were isolated from deceased patients during the 2016 outbreak in Benin. The lineage VII strain (L7) caused uniform mortality. Death was associated with uncontrolled viral replication, unbalanced inflammatory responses characterized by increased concentrations of pro- and anti-inflammatory mediators, and the absence of efficient immune responses, resembling the pathogenesis associated with the prototypic Josiah strain in monkeys. The lineage II strain (L2) showed apparently lower virulence than its counterpart, with a prolonged time to death and a lower mortality rate. Prolonged survival was associated with better control of viral replication, a moderate inflammatory response, and efficient T-cell responses. Transcriptomic analyses also highlighted important differences in the immune responses associated with the outcome. Both strains caused strong inflammation in several organs. Notably, meningitis and encephalitis were observed in the cerebral cortex and cerebellum in all monkeys, independently of the outcome. Due to their apparently high pathogenicity, emerging strains from lineage VII should be considered in preclinical vaccine testing. Lineage II would also be beneficial in pathogenesis studies to study the entire spectrum of Lassa fever severity.

Frontal cortical functional connectivity is impacted by anaesthesia in macaques.

A critical aspect of neuroscience is to establish whether and how brain networks evolved across primates. To date, most comparative studies have used resting-state functional magnetic resonance imaging (rs-fMRI) in anaesthetized nonhuman primates and in awake humans. However, anaesthesia strongly affects rs-fMRI signals. The present study investigated the impact of the awareness state (anaesthesia vs. awake) within the same group of macaque monkeys on the rs-fMRI functional connectivity organization of a well-characterized network in the human brain, the cingulo-frontal lateral network. Results in awake macaques show that rostral seeds in the cingulate sulcus exhibited stronger correlation strength with rostral compared to caudal lateral frontal cortical areas, while more caudal seeds displayed stronger correlation strength with caudal compared to anterior lateral frontal cortical areas. Critically, this inverse rostro-caudal functional gradient was abolished under anaesthesia. This study demonstrated a similar functional connectivity (FC) organization of the cingulo-frontal cortical network in awake macaque to that previously uncovered in the human brain pointing toward a preserved FC organization from macaque to human. However, it can only be observed in awake state suggesting that this network is sensitive to anaesthesia and warranting significant caution when comparing FC patterns across species under different states.

A single-shot Lassa vaccine induces long-term immunity and protects cynomolgus monkeys against heterologous strains.

A safe and protective Lassa virus vaccine is crucially needed in Western Africa to stem the recurrent outbreaks of Lassa virus infections in Nigeria and the emergence of Lassa virus in previously unaffected countries, such as Benin and Togo. Major challenges in developing a Lassa virus vaccine include the high diversity of circulating strains and their reemergence from 1 year to another. To address each of these challenges, we immunized cynomolgus monkeys with a measles virus vector expressing the Lassa virus glycoprotein and nucleoprotein of the prototypic Lassa virus strain Josiah (MeV-NP). To evaluate vaccine efficacy against heterologous strains of Lassa virus, we challenged the monkeys a month later with heterologous strains from lineage II or lineage VII, finding that the vaccine was protective against these strains. A second cohort of monkeys was challenged 1 year later with the homologous Josiah strain, finding that a single dose of MeV-NP was sufficient to protect all vaccinated monkeys. These studies demonstrate that MeV-NP can generate both long-lasting immune responses and responses that are able to protect against diverse strains of Lassa virus.

Apoptosis, G1 Phase Stall, and Premature Differentiation Account for Low Chimeric Competence of Human and Rhesus Monkey Naive Pluripotent Stem Cells.

After reprogramming to naive pluripotency, human pluripotent stem cells (PSCs) still exhibit very low ability to make interspecies chimeras. Whether this is because they are inherently devoid of the attributes of chimeric competency or because naive PSCs cannot colonize embryos from distant species remains to be elucidated. Here, we have used different types of mouse, human, and rhesus monkey naive PSCs and analyzed their ability to colonize rabbit and cynomolgus monkey embryos. Mouse embryonic stem cells (ESCs) remained mitotically active and efficiently colonized host embryos. In contrast, primate naive PSCs colonized host embryos with much lower efficiency. Unlike mouse ESCs, they slowed DNA replication after dissociation and, after injection into host embryos, they stalled in the G1 phase and differentiated prematurely, regardless of host species. We conclude that human and non-human primate naive PSCs do not efficiently make chimeras because they are inherently unfit to remain mitotically active during colonization.

Cestode infections in non-human primates suggest the existence of zoonotic cycles in the area surrounding the Strasbourg primatology center.

BACKGROUND: Several cases of infections due to Echinococcus multilocularis, Taenia martis and Taenia crassiceps were recently described in various species of captive non-human primates (NHPs) harbored in the Strasbourg Primate Center (SPC). Furthermore, one of the first cases of human cysticercosis due to T. martis was described in the Strasbourg region. These data suggest the existence of zoonotic cycles of tapeworm infections in the direct environment of the SPC. The aim of our study was to assess the prevalence of larval cestode infections among intermediate and definitive hosts in the close neighborhood of the center. We analyzed carnivore mammal fecal samples as well as rodent carcasses, collected inside or near the SPC, using PCR. Furthermore, we performed serology for Echinococcus spp. and Taenia spp. on NHP sera. RESULTS: We found that 14.5% (95% CI [8.6; 20.4]) of 138 carnivore feces were positive for E. multilocularis-DNA, as well as 25% (95% CI [5.5; 57.2]) of 12 rodent carcasses, and 5.1% (95% CI [1.4; 8.7]) for T. martis or T. crassiceps. Of all NHPs tested, 10.1% (95% CI [3.8; 16.4]) were seropositive for Echinococcus spp. and 8.2% (95% CI [1.3; 15.1]) for Taenia spp. CONCLUSIONS: Our data support the existence of zoonotic cycles of larval cestode infections in the direct environment of the primatology center affecting NHPs harbored in the SPC, potentially threatening the human population living in this area. Since this zoonotic risk is borne by local wildlife, and given the severity of these infections, it seems necessary to put in place measures to protect captive NHPs, and further studies to better assess the risk to human populations.

A comparison of methods to measure central and peripheral oxytocin concentrations in human and non-human primates.

Oxytocin (OT) concentration in the blood is considered to be a marker of its action in the brain. However, two problems have emerged when measuring OT level in the blood. First, it is unclear whether different methods of assessment lead to similar OT values. Second, it is unclear if plasma OT concentrations is informative on what OT does in the brain. To clarify these issues, we collected cerebrospinal fluid (CSF) from the brain ventricle of 25 patients during surgery to compare with plasma OT after simultaneous blood withdrawal. Additionally, we collected 12 CSF and blood samples from non-human primates while awake or under anaesthesia. We used four methods to assay OT concentrations: Commercial EIA with/without extraction, laboratory developed EIA with filtration and RIA with extraction. Three of these methods showed a positive correlation between plasma and CSF OT, suggesting a link between plasma and central OT, at least under specific testing conditions. However, none of the methods correlated to each other. Our results show major disagreements among methods used here to measure peripheral and brain OT and therefore they call for more caution when plasma OT is taken as a marker of central OT.