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

Protection in Macaques Immunized with HIV-1 Candidate Vaccines Can Be Predicted Using the Kinetics of Their Neutralizing Antibodies

A vaccine is needed to control the spread of human immunodeficiency virus type 1 (HIV-1). An in vitro assay that can predict the protection induced by a vaccine would facilitate the development of such a vaccine. A potential candidate would be an assay to quantify neutralization of HIV-1.We have used sera from rhesus macaques that have been immunized with HIV candidate vaccines and subsequently challenged with simian human immunodeficiency virus (SHIV). We compared neutralization assays with different formats. In experiments with the standardized and validated TZMbl assay, neutralizing antibody titers against homologous SHIV(SF162P4) pseudovirus gave a variable correlation with reductions in plasma viremia levels. The target cells used in the assays are not just passive indicators of virus infection but are actively involved in the neutralization process. When replicating virus was used with GHOST cell assays, events during the absorption phase, as well as the incubation phase, determine the level of neutralization. Sera that are associated with protection have properties that are closest to the traditional concept of neutralization: the concentration of antibody present during the absorption phase has no effect on the inactivation rate. In GHOST assays, events during the absorption phase may inactivate a fixed number, rather than a proportion, of virus so that while complete neutralization can be obtained, it can only be found at low doses particularly with isolates that are relatively resistant to neutralization.Two scenarios have the potential to predict protection by neutralizing antibodies at concentrations that can be induced by vaccination: antibodies that have properties close to the traditional concept of neutralization may protect against a range of challenge doses of neutralization sensitive HIV isolates; a window of opportunity also exists for protection against isolates that are more resistant to neutralization but only at low challenge doses

Number of NS3-peptides that induce IFNγ production by CD4 T-cells or IL-2/IFNγ dual cytokine production by CD8 T-cells.

<p>PRE, pre challenge. POST, post challenge.</p

MHC class I genotypes of the chimpanzees.

A<p>Vac3 is likely to be homozygous A*0901.</p

Antigen sensitivity of p59 specific cytokine induction.

<p>NS3-expanded T-cells from Vac1, Vac2 and Vac3, from 4 weeks prior to HCV challenge, were restimulated with a concentration range of p59 and evaluated for induction of IL-2/IFNγ expressing CD8 T-cells (left graph) and IFNγ expressing CD4 T-cells (right graph).</p

Cytolytic killing of CFSE-loaded target cells.

<p>(A) Specific lysis of NS3_1258–1272-pulsed Patr-A*03∶01 target cells (red peak) in comparison with unpulsed Patr-A*03∶01 target cells (yellow peak) by NS3-peptide-pool expanded PBMC from Vac1 isolated 10 weeks after HCV 1bJ4 challenge (blue peak). (B) No specific lysis of NS3_1258–1272-pulsed Patr-B*02∶01 target cells. (C) and (D) Control experiments showing no killing of unpulsed Patr-A*03∶01 or Patr-B*02∶01 target cells. (E,F,G) Percentage of lysis for all MHC class-I-peptide-combinations tested in Vac1, Vac2, Vac3 respectively.</p

Evaluation of peptide-specific IL-2, IFNγ and TNFα expression in CD4 and CD8 T-cells.

<p>A representative example of the analysis performed in Vac1 (left panel) and Vac2 (right panel), two weeks after the final vaccine boost, is shown. PBMC were first expanded for a 12 day period using a pool of all NS3 peptides, restimulated with individual peptides (medium alone, p59 or p64) and analyzed for induction of IFNγ, IL-2 and TNFα cytokine expression by expanded CD4 and expanded CD8 cells, using the gating strategy described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0095103#pone.0095103.s001" target="_blank">Figure S1</a>. The numbers in the quadrants, represent the percentage of positive cells calculated from the parent-population.</p

Heat map of NS3-peptide-specific T-cell responses in four immunized chimpanzees at different study time points.

<p>(A) Percentage of expanded CD4 T-cells producing IFNγ upon restimulation with individual peptides, subdivided into three categories; i.e. low response (light green; 0.001%–0.1% specific IFNγ production), intermediate (green; 0.1–1% specific response), high (dark green;>1% specific response). (B) Percentage of expanded CD8 T-cells showing IL-2/IFNγ double cytokine production upon restimulation with individual peptide, subdivided into three categories; i.e. low response (light blue; 0.01%–0.1% specific IFNγproduction), intermediate (blue; 0.1–1% specific response), high (dark blue; > 1% specific response). White areas indicate where no responses were detected. The red boxes highlight areas within NS3 that are broadly recognized. Per animal 6 time points of analysis are shown lined up beneath each other; -12) Two weeks following the 1st MVA boost, cells stimulated with NS3_vaccine, -4) Four weeks following 2^nd MVA boost, cells stimulated with NS3_vaccine, 0) Day of HCV infection, stimulation with NS3_vaccine, 0) Day of HCV infection, stimulation with NS3_challenge, and 4 and 36 weeks after challenge, stimulation with NS3_challenge. The individual peptides tested are indicated at the top of the map by the numbers 1 to 156. The red boxes represent broadly recognized regions within NS3. Total frequency (all peptide responses combined) of (C) IFNγ production by expanded CD4 T-cells and (D) IFNγ/IL2 dual cytokine production by expanded CD8 T-cells per animal. (E) Frequency of p59 specific triple positive IL2/TNFα/IFNγ producing CD8 T-cells. The numbers on the X-axis represent the study week, relative to time of challenge. * Despite several attempts, frozen cells from Vac1 from 4 weeks following HCV infection did not respond to either NS3_vaccine or NS3_challenge peptide stimulation and no expansion could be achieved. @ Due to a high IFNγ background, peptide specific CD8 responses could not be detected</p

Dose-inhibition curves for selected NS3-peptides to Patr-A*03∶01.

<p>Binding affinity of selected NS3-peptides to Patr-A*03∶01, established by peptide binding competition assay. Patr-A*0301 cells were incubated with increasing concentrations of NS3 p59 (top left), LGFGAYMSK (LGF, top right), AATLGFGAY (AAT, bottom left), or KGGLRPRAG (control, bottom right) in the presence of biotin-labeled indicator peptide. Binding of indicator peptide was quantified by incubation with europium labeled streptavidin. Indicated is reduction in percentage of europium positive cells relative to cells incubated with indicator-peptide only. The indicated IC₅₀ values (µM) are derived from the regression curves of three independent experiments in the case of p59, LGF and AAT, and of two individual experiments in the case of KGG. The IC₅₀ values were estimated using non-linear least-squares regression with the “R” platform for statistical computing.</p

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 gamma 1-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 gutimmune-CNS axis