Longitudinal positron emission tomography and postmortem analysis reveals widespread neuroinflammation in SARS-CoV-2 infected rhesus macaques.

BACKGROUND: Coronavirus disease 2019 (COVID-19) patients initially develop respiratory symptoms, but they may also suffer from neurological symptoms. People with long-lasting effects after acute infections with severe respiratory syndrome coronavirus 2 (SARS-CoV-2), i.e., post-COVID syndrome or long COVID, may experience a variety of neurological manifestations. Although we do not fully understand how SARS-CoV-2 affects the brain, neuroinflammation likely plays a role. METHODS: To investigate neuroinflammatory processes longitudinally after SARS-CoV-2 infection, four experimentally SARS-CoV-2 infected rhesus macaques were monitored for 7 weeks with 18-kDa translocator protein (TSPO) positron emission tomography (PET) using [18F]DPA714, together with computed tomography (CT). The baseline scan was compared to weekly PET-CTs obtained post-infection (pi). Brain tissue was collected following euthanasia (50 days pi) to correlate the PET signal with TSPO expression, and glial and endothelial cell markers. Expression of these markers was compared to brain tissue from uninfected animals of comparable age, allowing the examination of the contribution of these cells to the neuroinflammatory response following SARS-CoV-2 infection. RESULTS: TSPO PET revealed an increased tracer uptake throughout the brain of all infected animals already from the first scan obtained post-infection (day 2), which increased to approximately twofold until day 30 pi. Postmortem immunohistochemical analysis of the hippocampus and pons showed TSPO expression in cells expressing ionized calcium-binding adaptor molecule 1 (IBA1), glial fibrillary acidic protein (GFAP), and collagen IV. In the hippocampus of SARS-CoV-2 infected animals the TSPO+ area and number of TSPO+ cells were significantly increased compared to control animals. This increase was not cell type specific, since both the number of IBA1+TSPO+ and GFAP+TSPO+ cells was increased, as well as the TSPO+ area within collagen IV+ blood vessels. CONCLUSIONS: This study manifests [18F]DPA714 as a powerful radiotracer to visualize SARS-CoV-2 induced neuroinflammation. The increased uptake of [18F]DPA714 over time implies an active neuroinflammatory response following SARS-CoV-2 infection. This inflammatory signal coincides with an increased number of TSPO expressing cells, including glial and endothelial cells, suggesting neuroinflammation and vascular dysregulation. These results demonstrate the long-term neuroinflammatory response following a mild SARS-CoV-2 infection, which potentially precedes long-lasting neurological symptoms.

Brain Inflammation and Intracellular α-Synuclein Aggregates in Macaques after SARS-CoV-2 Infection.

SARS-CoV-2 causes acute respiratory disease, but many patients also experience neurological complications. Neuropathological changes with pronounced neuroinflammation have been described in individuals after lethal COVID-19, as well as in the CSF of hospitalized patients with neurological complications. To assess whether neuropathological changes can occur after a SARS-CoV-2 infection, leading to mild-to-moderate disease, we investigated the brains of four rhesus and four cynomolgus macaques after pulmonary disease and without overt clinical symptoms. Postmortem analysis demonstrated the infiltration of T-cells and activated microglia in the parenchyma of all infected animals, even in the absence of viral antigen or RNA. Moreover, intracellular α-synuclein aggregates were found in the brains of both macaque species. The heterogeneity of these manifestations in the brains indicates the virus' neuropathological potential and should be considered a warning for long-term health risks, following SARS-CoV-2 infection.

The Post-Acute Phase of SARS-CoV-2 Infection in Two Macaque Species Is Associated with Signs of Ongoing Virus Replication and Pathology in Pulmonary and Extrapulmonary Tissues.

The post-acute phase of SARS-CoV-2 infection was investigated in rhesus (Macaca mulatta) and cynomolgus macaques (Macaca fascicularis). During the acute phase of infection, SARS-CoV-2 was shed via the nose and throat, and viral RNA was occasionally detected in feces. This phase coincided with a transient change in systemic immune activation. Even after the alleged resolution of the infection, computed tomography (CT) and positron emission tomography (PET)-CT revealed pulmonary lesions and activated tracheobronchial lymph nodes in all animals. Post-mortem histological examination of the lung tissue revealed mostly marginal or resolving minimal lesions that were indicative of SARS-CoV-2 infection. Evidence for SARS-CoV-2-induced histopathology was also found in extrapulmonary tissue samples, such as conjunctiva, cervical, and mesenteric lymph nodes. However, 5-6 weeks after SARS-CoV-2 exposure, upon necropsy, viral RNA was still detectable in a wide range of tissue samples in 50% of the macaques and included amongst others the heart, the respiratory tract and surrounding lymph nodes, salivary gland, and conjunctiva. Subgenomic messenger RNA was detected in the lungs and tracheobronchial lymph nodes, indicative of ongoing virus replication during the post-acute phase. These results could be relevant for understanding the long-term consequences of COVID-19 in humans.

Targeted Diet Modification Reduces Multiple Sclerosis-like Disease in Adult Marmoset Monkeys from an Outbred Colony.

Experimental autoimmune encephalomyelitis (EAE) in common marmosets is a translationally relevant model of the chronic neurologic disease multiple sclerosis. Following the introduction of a new dietary supplement in our purpose-bred marmoset colony, the percentage of marmosets in which clinically evident EAE could be induced by sensitization against recombinant human myelin oligodendrocyte glycoprotein in IFA decreased from 100 to 65%. The reduced EAE susceptibility after the dietary change coincided with reduced Callitrichine herpesvirus 3 expression in the colony, an EBV-related γ1-herpesvirus associated with EAE. We then investigated, in a controlled study in marmoset twins, which disease-relevant parameters were affected by the dietary change. The selected twins had been raised on the new diet for at least 12 mo prior to the study. In twin siblings reverted to the original diet 8 wk prior to EAE induction, 100% disease prevalence (eight out of eight) was restored, whereas in siblings remaining on the new diet the EAE prevalence was 75% (six out of eight). Spinal cord demyelination, a classical hallmark of the disease, was significantly lower in new-diet monkeys than in monkeys reverted to the original diet. In new-diet monkeys, the proinflammatory T cell response to recombinant human myelin oligodendrocyte glycoprotein was significantly reduced, and RNA-sequencing revealed reduced apoptosis and enhanced myelination in the brain. Systematic typing of the marmoset gut microbiota using 16S rRNA sequencing demonstrated a unique, Bifidobacteria-dominated composition, which changed after disease induction. In conclusion, targeted dietary intervention exerts positive effects on EAE-related parameters in multiple compartments of the marmoset's gut-immune-CNS axis.

Role of microbial translocation in soluble CD14 up-regulation in HIV-, but not in HCV-, infected chimpanzees.

During human immunodeficiency virus (HIV) infection, soluble CD14 (sCD14) is up-regulated as a consequence of pathological disruption of the gut epithelial barrier, and subsequent increased microbial translocation. Also in hepatitis C virus (HCV)-infected patients with advanced liver fibrosis, increased levels of sCD14 have been reported. Since the liver plays an important role in clearance of translocated bacterial products, hepatic fibrosis may negatively affect clearance and thus contribute to higher sCD14 levels. Chimpanzees (Pan troglodytes) infected with HCV typically show no signs of liver fibrosis. Here, we have tested the hypothesis that increased levels of sCD14 occur in the absence of hepatic fibrosis or microbial translocation in chimpanzees chronically infected with HCV. sCD14 was up-regulated in both HIV/simian immunodeficiency virus (SIV)- and HCV-infected chimpanzees. In HIV/SIV-infected chimpanzees, intestinal fatty acid-binding protein, a marker for gut perturbation, lipopolysaccharide (LPS)-binding-protein and LPS core antibodies, confirm that sCD14 up-regulation was caused by increased microbial translocation. In HCV-infected chimpanzees, no evidence was found for increased microbial translocation despite up-regulation of sCD14. Additionally, the impact of liver fibrosis on microbial translocation was addressed by direct comparison of chimpanzees with a high HCV load and human patients with advanced fibrosis. These data suggest that only in a small minority of HCV patients, hepatic fibrosis corroborates microbial translocation.

Strong vaccine-induced CD8 T-cell responses have cytolytic function in a chimpanzee clearing HCV infection.

A single correlate of effective vaccine protection against chronic HCV infection has yet to be defined. In this study, we analyzed T-cell responses in four chimpanzees, immunized with core-E1-E2-NS3 and subsequently infected with HCV1b. Viral clearance was observed in one animal, while the other three became chronically infected. In the animal that cleared infection, NS3-specific CD8 T-cell responses were observed to be more potent in terms of frequency and polyfunctionality of cytokine producing cells. Unique to this animal was the presence of killing-competent CD8 T-cells, specific for NS3 1258-1272, being presented by the chimpanzee MHC class I molecule Patr-A*03∶01, and a high affinity recognition of this epitope. In the animals that became chronically infected, T-cells were able to produce cytokines against the same peptide but no cytolysis could be detected. In conclusion, in the animal that was able to clear HCV infection not only cytokine production was observed but also cytolytic potential against specific MHC class I/peptide-combinations.