INSIGHTS INTO CD8 T CELL-MEDIATED PATHOLOGY DURING EXPERIMENTAL CEREBRAL MALARIA

Ph.DDOCTOR OF PHILOSOPH

Co-infection with Chikungunya virus alters trafficking of pathogenic CD8(+) T cells into the brain and prevents Plasmodium-induced neuropathology

Arboviral diseases have risen significantly over the last 40 years, increasing the risk of co‐infection with other endemic disease such as malaria. However, nothing is known about the impact arboviruses have on the host response toward heterologous pathogens during co‐infection. Here, we investigate the effects of Chikungunya virus (CHIKV ) co‐infection on the susceptibility and severity of malaria infection. Using the Plasmodium berghei ANKA (PbA) experimental cerebral malaria (ECM ) model, we show that concurrent co‐infection induced the most prominent changes in ECM manifestation. Concurrent co‐infection protected mice from ECM mortality without affecting parasite development in the blood. This protection was mediated by the alteration of parasite‐specific CD8+ T‐cell trafficking through an IFN γ‐mediated mechanism. Co‐infection with CHIKV induced higher splenic IFN γ levels that lead to high local levels of CXCL 9 and CXCL 10. This induced retention of CXCR 3‐expressing pathogenic CD8+ T cells in the spleen and prevented their migration to the brain. This then averts all downstream pathogenic events such as parasite sequestration in the brain and disruption of blood–brain barrier that prevents ECM ‐induced mortality in co‐infected mice

Linear B-cell epitopes in the spike and nucleocapsid proteins as markers of SARS-CoV-2 exposure and disease severity

BACKGROUND Given the unceasing worldwide surge in COVID-19 cases, there is an imperative need to develop highly specific and sensitive serology assays to define exposure to Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2). METHODS Pooled plasma samples from PCR positive COVID-19 patients were used to identify linear B-cell epitopes from a SARS-CoV-2 peptide library of spike (S), envelope (E), membrane (M), and nucleocapsid (N) structural proteins by peptide-based ELISA. Hit epitopes were further validated with 79 COVID-19 patients with different disease severity status, 13 seasonal human CoV, 20 recovered SARS patients and 22 healthy donors. FINDINGS Four immunodominant epitopes, S14P5, S20P2, S21P2 and N4P5, were identified on the S and N viral proteins. IgG responses to all identified epitopes displayed a strong detection profile, with N4P5 achieving the highest level of specificity (100%) and sensitivity (>96%) against SARS-CoV-2. Furthermore, the magnitude of IgG responses to S14P5, S21P2 and N4P5 were strongly associated with disease severity. INTERPRETATION IgG responses to the peptide epitopes can serve as useful indicators for the degree of immunopathology in COVID-19 patients, and function as higly specific and sensitive sero-immunosurveillance tools for recent or past SARS-CoV-2 infections. The flexibility of these epitopes to be used alone or in combination will allow for the development of improved point-of-care-tests (POCTs)

Data-Driven Analysis of COVID-19 Reveals Persistent Immune Abnormalities in Convalescent Severe Individuals

Severe SARS-CoV-2 infection can trigger uncontrolled innate and adaptive immune responses, which are commonly associated with lymphopenia and increased neutrophil counts. However, whether the immune abnormalities observed in mild to severely infected patients persist into convalescence remains unclear. Herein, comparisons were drawn between the immune responses of COVID-19 infected and convalescent adults. Strikingly, survivors of severe COVID-19 had decreased proportions of NKT and Vδ2 T cells, and increased proportions of low-density neutrophils, IgA+/CD86+/CD123+ non-classical monocytes and hyperactivated HLADR+CD38+ CD8+ T cells, and elevated levels of pro-inflammatory cytokines such as hepatocyte growth factor and vascular endothelial growth factor A, long after virus clearance. Our study suggests potential immune correlates of “long COVID-19”, and defines key cells and cytokines that delineate true and quasi-convalescent states

Role of T Cells in Chikungunya Virus Infection and Utilizing Their Potential in Anti-Viral Immunity

10.3389/fimmu.2020.00287Frontiers in Immunology1128

Activated Brain Endothelial Cells Cross-Present Malaria Antigen

<div><p>In the murine model of cerebral malaria caused by <i>P</i>. <i>berghei</i> ANKA (PbA), parasite-specific CD8⁺ T cells directly induce pathology and have long been hypothesized to kill brain endothelial cells that have internalized PbA antigen. We previously reported that brain microvessel fragments from infected mice cross-present PbA epitopes, using reporter cells transduced with epitope-specific T cell receptors. Here, we confirm that endothelial cells are the population responsible for cross-presentation <i>in vivo</i>, not pericytes or microglia. PbA antigen cross-presentation by primary brain endothelial cells <i>in vitro</i> confers susceptibility to killing by CD8⁺ T cells from infected mice. IFNγ stimulation is required for brain endothelial cross-presentation <i>in vivo</i> and <i>in vitro</i>, which occurs by a proteasome- and TAP-dependent mechanism. Parasite strains that do not induce cerebral malaria were phagocytosed and cross-presented less efficiently than PbA <i>in vitro</i>. The main source of antigen appears to be free merozoites, which were avidly phagocytosed. A human brain endothelial cell line also phagocytosed <i>P</i>. <i>falciparum</i> merozoites. Besides being the first demonstration of cross-presentation by brain endothelial cells, our results suggest that interfering with merozoite phagocytosis or antigen processing may be effective strategies for cerebral malaria intervention.</p></div

Uptake and cross-presentation efficiency of different rodent malaria parasites by MBECs <i>in vitro</i>.

<p>Mature parasite stages were isolated from the blood of mice infected with PbA, PbNK65 or Py17X by Percoll gradient centrifugation. (A) Various numbers of frozen-thawed parasites were added to IFNγ-stimulated MBECs in triplicate wells. After 24 h, the wells were washed and Pb1 cross-presentation was assayed using LR-BSL8.4a reporter cells. *<i>P</i><0.05, ***<i>P</i><0.001, ****<i>P</i><0.0001, 2-way ANOVA with Bonferroni’s post test, comparing each of the non-ECM strains against PbA at the same dose. The dotted line indicates the background spot count from MBECs without parasites. (B) Equal numbers of frozen-thawed parasites from the three strains were labeled with PKH26 and added to unstimulated or IFNγ-stimulated MBECs in quadruplicate wells. After overnight incubation, the wells were washed and imaged by confocal microscopy (6 fields per well with 40× objective) in the presence of trypan blue to quench extracellular PKH26. The number of red pixels per field that exceed a defined brightness threshold was quantified with ImageJ. Bars represent medians and interquartile ranges. ns not significant, **<i>P</i><0.01, ****<i>P</i><0.0001, Kruskal Wallis test with Dunn’s post test comparing against the IFNγ + PbA group.</p

Cytokine requirements and timing of brain microvessel cross-presentation during PbA infection.

<p>(A–C) WT mice and mice deficient for IFNγ (A), TNFα (B), and LTα (C) were infected with PbA. After 7 days, brain microvessels were isolated from the infected mice as well as naïve WT mice, and incubated overnight with LR-BSL8.4a reporter cells. The total number of blue cells (counted as blue spots by the ELISOT reader) resulting from each brain after X-gal staining was quantified. *<i>P</i><0.05, ****<i>P</i><0.0001, ns not significant, ANOVA with Bonferroni’s post-test on log-transformed numbers. (D) Brain microvessels from uninfected mice and WT mice infected with PbA 5, 6, or 7 days previously were tested for cross-presentation as above. **<i>P</i><0.01, ****<i>P</i><0.0001, ns not significant as compared to uninfected mice by ANOVA with Bonferroni’s post-test on log-transformed numbers.</p

Cross-presentation of PbA antigen by MBECs occurs by the cytosolic pathway.

<p>(A) MBEC cultures were established from WT mice as well as TAP1-deficient mice and stimulated with IFNγ. PbA mature iRBCs were added to some wells in triplicate. After 24 h, the wells were washed and cross-presentation of Pb1 was detected using LR-BSL8.4a cells and X-gal staining. ****<i>P</i><0.0001, ns not significant, ANOVA with Bonferroni’s post test on log-transformed data. Results are representative of 2 independent experiments. (B) PbA mature iRBCs were added (or not) to IFNγ-stimulated MBECs. Lactacystin (10 μM) was added to some wells 6 h later. After another 6 h, all wells were washed and assayed for Pb1 cross-presentation using LR-BSL8.4a cells. <i>n</i> = 3, *<i>P</i><0.05, **<i>P</i><0.01, ANOVA with Bonferroni’s post test on log-transformed data. Results are representative of 2 independent experiments. (C, D) PbA mature iRBCs were added to IFNγ-stimulated MBECs 1 h after either 10 μg/ml chloroquine diphosphate (C) or 10 μM cytochalasin D (D) were added to some wells. The MBECs were washed and co-incubated with LR-BSL8.4a cells 24 h later to measure Pb1 cross-presentation. <i>n</i> = 3, ***<i>P</i><0.001, ****<i>P</i><0.0001, unpaired t-test on log-transformed spot counts.</p