Characterization of Adaptive Immune Responses to SARS-CoV-2 Updated Bivalent Booster

Bivalent COVID-19 vaccines that contain two mRNAs encoding Wuhan-1 and Omicron BA.4/5 spike proteins are successful in preventing infection from the original strain and Omicron variants, but the quality of adaptive immune responses is still not well documented. This study aims at characterizing adaptive immune responses to the bivalent booster vaccination in 46 healthy participants. Plasma and PBMC were collected prior and three weeks after bivalent booster. We measured anti-N, anti-S, and RBD IgM, IgA, IgG plasma titers against original, Omicron BA.1, and BA.5 variants (pending) as well as total anti-S IgG titers and surrogate Virus Neutralization capacity against the Alpha, Delta, and BA.1 variant. With spectral flow-cytometry we identified peripheral blood B-cells specific for the RBD of the S-protein of the original and BA.1 variants. T-cell-specific responses were assessed by cytokine release assay after stimulation with SARS-CoV-2 peptides from the original, BA.1, BA.4, and BA.5 variants (pending). Finally, we performed TRB and IGH repertoire studies on sorted CD4+, CD8+, CD19+ lymphocytes, to study breadth of SARS-CoV-2 specific clonotypes (pending). 27/46 participants were analyzed;9 had SARS-CoV-2 infection (COVID+), while 18 are infection naive (COVID-). In both groups, median time since last dose of SARS-CoV-2 vaccine (3rd or 4th) was 11 months. All subjects were positive for anti-S IgG prior to bivalent booster. The COVID + group displayed anti-S IgG pre-booster levels and neutralization against BA.1 higher than the COVID- group. Significant increase post-boost of total anti-S IgG and BA.1 neutralizing activity was detected in the COVID- but not in the COVID+ group;however, no difference in neutralization activity post-boost was detected between the two groups. Furthermore, the COVIDgroup showed significant increase in the frequency of CD19+ and CD27+ switched memory B-cells specific for BA.1 RBD in post-boost compared to pre-boost samples. However, post-boost frequencies of the same B-cells were higher in the COVID+ compared to the COVID- group. These preliminary findings confirm that among individual immunized with the original COVID-19 mRNAvaccine, prior COVID infection provides increased protection against SARS-CoV-2 variants. They also demonstrate that booster immunization with the bivalent vaccine induces robust adaptive immune responses against Omicron variant.[Formula presented][Formula presented]Copyright © 2023 Elsevier Inc.

Mastocytosis and COVID19 infection and vaccination: A pediatric series

Introduction: Mastocytosis encompasses a heterogeneous group of diseases characterized by an accumulation of clonal mast cells (MC) in the skin and/or internal organs, and symptoms of MC activation. This MC activation can be elucidated by several factors, including infections or vaccination. Objective(s): We present our experience with COVID infection and vaccination in a series of 133 patients with pediatric mastocytosis. Method(s): Between January 1998 and December 2022, 133 pediatric patients have been referred to our hospital owing to clinically suspected MC disorder, mainly with mastocytosis in the skin. The final diagnoses of mastocytosis were established by the presence of typical skin lesions together with an increase of MC numbers in a biopsy from lesional skin or activating KIT mutations in lesional skin tissue. Serum baseline tryptase and total immunoglobulin E levels were measured, and patients underwent a comprehensive allergy workup to confirm atopic status and history of anaphylaxis. Regarding vaccination, REMA's (Spanish Network on Mastocytosis) protocol was followed. Result(s): 13 patients with COVID infection were identified, of which 25 (56,8%) were female and 0% had symptoms of MC activation. All of them had an asymptomatic or mild course of COVID infection. None of the patients experimented MC activation symptoms during viral illness. Regarding COVID vaccination, all patients received premedication with antihistamine 60 minutes prior vaccination. No one experimented immediate reactions and only one patient (0,75%) referred worsening of MC activation symptoms (baseline pruritus, urtication and brain fog) only after the first doses, recovering without changes in his treatment (oral cromoglycate and antihistamine) in two months. Discussion(s): Although MC have been implicated in the pathogenesis of cytokine storm in COVID19, there is no clinical evidence of SARSCoV- 2-induced MC activation, perhaps related to the fact that bone marrow MC lack angiotensin-converting enzyme 2 receptors.

Covid-19: Cases of inflammatory syndrome in children surge after urgent alert

Thirty fold increase In the province of Bergamo, Italy, researchers have reported a 30 fold increased incidence of Kawasaki like disease since the start of the covid-19 outbreak. Among the covid-19 group more children had cardiac symptoms (6 out of 10), Kawasaki disease shock syndrome (5 out of 10), macrophage activation syndrome (5 out of 10), and the need for adjunctive steroid treatment (8out of 10). In the pre-covid-19 group only two of 19 children had cardiac involvement and just three required adjunctive steroid treatment. A distinct syndrome Julia Kenny, a consultant in paediatric infectious diseases and immunology at Evelina London Children's Hospital, said that the Italian findings appear consistent with cases seen in the south east of England.

SINGLE CELL MULTIOMIC PROFILING OF DISRUPTED REGULATORY NETWORKS IN COVID19 ASSOCIATED MULTISYSTEM INFLAMMATORY SYNDROME IN CHILDREN

BackgroundChildren infected with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) usually present minimal symptoms or are asymptomatic. Nevertheless, a subset of children 2-6 weeks after the initial SARS-CoV-2 infection develops a postinfectious SARS-CoV-2-related multisystem inflammatory syndrome in (MIS-C). Recently, transient expansion of TRBV11-2 T cell clonotypes in MIS-C was associated with signatures of inflammation and T cell activation, however, the underlying pathophysiology of MIS-C is not fully understood [1].ObjectivesThe purpose of our project is to characterize the complexity of cell populations and capture cellular heterogeneity to uncover the regulatory networks and interactions that are disrupted during MIS-C flare with simultaneous profiling of gene expression and open chromatin regions from the same nuclei.MethodsSamples of peripheral blood mononuclear cells from patients with MIS-C diagnosed at the University Children's Hospital, University Medical Center Ljubljana, were collected during the initial presentation before any treatment and at 6-12 months in remission. The primary aim is to identify which regulatory networks are driving inflammation in MIS-C flare, for which we are performing single cell Multiome ATAC + Gene Expression Sequencing. To enable simultaneous profiling of epigenomic landscape and gene expression from the same nuclei, we are using Chromium Next GEM Single Cell Multiome ATAC + Gene Expression kit from 10X Genomics.ResultsWe included 32 patients with MIS-C from whom we collected paired blood samples during the initial presentation before treatment and at 6-12 months in remission. In single cell multiomic experiment we included 10 patients with paired samples, with the most viable cell count prior cryopreservation. All samples that are included into multiomic single cell analysis have 75% - 99% viability prior cryopreservation. In the protocol the key is to remove remaining granulocytes causing high mitochondrial RNA burden and extensively optimize the dilution factor of lysis buffer and the length of cell lysis step in order to get intact nuclei with no significant blebbing. Afterward, the single cell ATAC libraries as well as single-cell gene expression libraries are constructed and sequenced. Data are undergoing pairwise analysis to compare the cell population heterogeneity, expression profile and open chromatin landscape in the time of the initial presentation of MIS-C and in the remission, with Cell ranger software as well as with R package scREG [2], and custom scripting. In the second step we will inspect if the resulting altered transcriptomic signature from single-cell experiment is present on larger cohort. In that regard, we will perform bulk transcriptomic profiling on all paired collected samples during the initial presentation of MIS-C before treatment and at 6-12 months in remission.ConclusionThe results of this project are expected to enlighten the underlying pathophysiology of MIS-C flare and thus support clinical decision on more targeted treatment. The identified disrupted networks during MIS-C flare could lead the way to establish an early diagnosis and improve long-term outcome, including prevention of myocardial and neuropsychological impairment. Moreover, a better understanding of the disrupted regulatory networks that are driving inflammation in MIS-C, could lead to new insights into diseases with similar clinical presentations as is Kawasaki Disease.References[1]Sacco, K., Castagnoli, R., Vakkilainen, S. et al. Immunopathological signatures in multisystem inflammatory syndrome in children and pediatric COVID-19. Nat Med 28, 1050–1062 (2022).[2]Duren, Z., Chang, F., Naqing, F. et al. Regulatory analysis of single cell multiome gene expression and chromatin accessibility data with scREG. Genome Biol 23, 114 (2022).AcknowledgementsThis research was supported by Slovenian research agency grant J3-3061 and Interreg ITA-SLO project Cattedra.Disclosure of InterestsNone Declared.

COVID-associated pernio: Mechanistic insights to an abortive, seronegative SARS-CoV-2 infection

Introduction: SARS-CoV-2 replicates primarily in the airways but generates a systemic immune response mediated by Type I interferons (IFN-I). Pernio is a rare skin manifestation of disorders characterized by excessive IFN-I signalling. Although pernio increased in incidence during the pandemic, the relationship to SARS-CoV-2 remains controversial. Because of the pivotal nature of interferons in COVID-19 outcomes, pernio offers a window to investigate the biology underlying host resiliency to SARS-CoV-2 infection. Method(s): To further assess COVID-associated pernio, we characterized clinical samples from affected patients across 4 waves of the pandemic and investigated mechanistic feasibility in a rodent model. Patients were followed longitudinally with banking of blood and tissue. Golden hamsters were mock-treated or intra-nasally infected with SARS-CoV-2 and harvested at 3-and 30-days post-infection. Result(s): In affected tissue, immunophenotyping utilizing multiplex immunohistochemistry profiled a robust IFN-1 signature characterized by plasmacytoid dendritic cell activation. Viral RNA was detectable in a subset of cases using in situ hybridization for the SARS-CoV-2 S gene transcript. Profiling of the systemic immune response did not reveal a durable type 1 interferon signature. Consistent with previous literature, antibody and T-cell specific responses to SARS-CoV-2 were not detected. Nasopharyngeal SARS-CoV-2 inoculation in hamsters resulted in rapid dissemination of viral RNA and the generation of an IFN-I response that were both detectable in the paws of infected animals. Conclusion(s): Our data support a durable local IFN signature, with direct evidence of viral SARS-CoV-2 RNA in acral skin and suggest that COVID-associated pernio results from an abortive, seronegative SARS-CoV-2 infection.

Immunotherapy: FAMILIAL CD45RA DEPLETED T CELLS TO TREAT SEVERE REFRACTORY INFECTIONS IN IMMUNOCOMPROMISED PATIENTS

Background & Aim: Immunocompromised patients are susceptible to high-risk opportunistic infections and malignant diseases. If available, most antiviral and antifungal drugs are quite toxic, relatively ineffective, and induce resistance in the long term. Methods, Results & Conclusion(s): We have previously demonstrated the safety of adoptive cell therapy for COVID-19 patients with CD45RA negative cells containing SARS-CoV-2-specific T cells from a donor, chosen based on HLA compatibility and cellular response to SARS-CoV-2 peptide pools. After finishing a Phase 2 randomized multicenter clinical trial (RELEASE, NCT04578210), we concluded that the infusion is safe, effective, accelerates lymphocyte recovery and shows hallmarks of an immune response. To use adoptive cell therapy to treat COVID-19 it would be necessary to develop a biobank of living drugs. For that, we examined the immune evolution performing a longitudinal analysis from previously SARS-CoV-2 infected and infection- naive individuals covering 21 months from infection. Cellular responses were maintained over time while humoral responses increased after vaccination but were gradually lost. Therefore, the best donors would be recovered individuals and two months after vaccination. We also evaluated the effect of dexamethasone (current standard of care treatment for COVID-19 and other infections involving lymphopenia) and Interleukin-15 (cytokine involved in T-cell maintenance and survival) on CD45RA negative. Dexamethasone did not alter cell functionality, proliferation or phenotype at a clinical-practice concentration, while interleukin-15 increased the memory T-cell and T-regulatory cell activation state, and interferon gamma release. Furthermore, we applied the adoptive passive transfer of CD45RA negative cells containing pathogen-specific memory T-cells to other infectious diseases characterized by sustained lymphopenia. We infused six immunocompromised patients with Cytomegalovirus, BK virus, Aspergillus, and Epstein-Barr virus lymphoproliferative disease. Patients experienced pathogen clearance, resolution of symptoms and lymphocyte increase. Transient microchimerism was detected in three patients. The use of CD45RA negative cells containing specific memory T cells of a third-party donor for treating severe pathogenic diseases in immunocompromised patients is feasible, safe, and effective, and has an advantage over other cell therapies such as lower costs and a less complex regulatory environment.Copyright © 2023 International Society for Cell & Gene Therapy

Prolonged T-cell activation and long COVID symptoms independently associate with severe COVID-19 at 3 months.

Coronavirus disease-19 (COVID-19) causes immune perturbations which may persist long term, and patients frequently report ongoing symptoms for months after recovery. We assessed immune activation at 3-12 months post hospital admission in 187 samples from 63 patients with mild, moderate, or severe disease and investigated whether it associates with long COVID. At 3 months, patients with severe disease displayed persistent activation of CD4+ and CD8+ T-cells, based on expression of HLA-DR, CD38, Ki67, and granzyme B, and elevated plasma levels of interleukin-4 (IL-4), IL-7, IL-17, and tumor necrosis factor-alpha (TNF-α) compared to mild and/or moderate patients. Plasma from severe patients at 3 months caused T-cells from healthy donors to upregulate IL-15Rα, suggesting that plasma factors in severe patients may increase T-cell responsiveness to IL-15-driven bystander activation. Patients with severe disease reported a higher number of long COVID symptoms which did not however correlate with cellular immune activation/pro-inflammatory cytokines after adjusting for age, sex, and disease severity. Our data suggests that long COVID and persistent immune activation may correlate independently with severe disease.

Neuromuscular disease: 2021 update.

This review highlights ten important advances in the neuromuscular disease field that were first reported in 2020. The overarching topics include (i) advances in understanding of fundamental neuromuscular biology; (ii) new / emerging diseases; (iii) advances in understanding of disease etiology and pathogenesis; (iv) diagnostic advances; and (v) therapeutic advances. Within this broad framework, the individual disease entities that are discussed in more detail include neuromuscular complications of COVID-19, supervillin-deficient myopathy, 19p13.3-linked distal myopathy, vasculitic neuropathy due to eosinophilic granulomatosis with polyangiitis, spinal muscular atrophy, idiopathic inflammatory myopathies, and transthyretin neuropathy/myopathy. In addition, the review highlights several other advances (such as the revised view of the myofibrillar architecture, new insights into molecular and cellular mechanisms of muscle regeneration, and development of new electron microscopy tools) that will likely have a significant impact on the overall neuromuscular disease field going forward.

Leukocyte Cell Population Data as Potential Markers of Covid-19 Disease Characterization

Background: The usefulness of leukocyte cell population data (CPD) is currently being investigated. In COVID-19 pandemic several reports showed the clinical importance of hematological parameters. Our study aimed to assess CPDs in Sars CoV-2 patients as new disease markers. Method(s): From February to April 2020 (1st wave) 540 and from September to December 2020 (2nd wave) 2821 patients respectively were enrolled. SARS CoV-2 infection diagnosis was carried out by Multiplex rRT-PCR from nasopharyngeal swabs. CPDs were detected by XN 2000 hematology analyzer (Sysmex Corporation). A comparison between two disease waves was performed. Additionally, C-reactive protein (CRP) and lactate dehydrogenase (LDH) were assayed. Result(s): CPDs were classified into: cell complextity, DNA/RNA content and abnormal sized cells. We detected parameters increased from the reference population for all cell types for both 1st and 2nd wave (p<0.05). However, in the 2nd vs 1st wave 5 CPDs vs 9 CPDs were found. In addition we observed higher CPD values of the 1st compared to 2nd wave: (NE-SFL) (p<0.001), (LY-Y) (p<0.0001), (LY-Z) (p<0.0001), (MO-X) (p<0.0001), (MO-Y) (p<0.0001). These findings were confirmed by the higher concentrations of CRP and LDH in the 1st vs 2nd wave: 17.3 mg/L (8.5-59.3) vs 6.3 mg/L (2.3-17.6) (p<0.001) and 241.5 IU/L (201-345) vs 195 IU/L (174-228) (p< 0.001) (median, interquartile range) respectively. Conclusion(s): CPDs showed increased cell activation in 1st wave patients confirmed by clinical and biochemical data, associated with worse clinical conditions. Results highlighted the CPDs as disease characterization markers or useful for a risk model.Copyright © 2023 Sciendo. All rights reserved.

ANALYSIS OF EPITOPE-SPECIFIC T CELLS IN SARS-CoV-2 INFECTION AND VACCINATION

Background: T cells play a critical role in the adaptive immune response to SARS-CoV-2 in both infection and vaccination. Identifying T cell epitopes and understanding how T cells recognize these epitopes can help inform future vaccine design and provide insight into T cell recognition of newly emerging variants. Here, we identified SARS-CoV-2 specific T cell epitopes, analyzed epitope-specific T cell repertoires, and characterized the potency and cross-reactivity of T cell clones across different common human coronaviruses (HCoVs). Method(s): SARS-CoV-2-specific T cell epitopes were determined by IFNgamma ELISpot using PBMC from convalescent individuals with mild/moderate disease (n=25 for Spike (S), Nucleocapsid (N) and Membrane (M)), and in vaccinated individuals (n=27 for S). Epitope-specific T cells were isolated based on activation markers following a 6-hour peptide stimulation, and scRNAseq was performed for TCR repertoire analysis. T cell lines were generated by expressing recombinant TCRs in Jurkat cells and activation was measured by CD69 upregulation. Result(s): We identified multiple immunodominant T cell epitopes across S, N and M proteins in convalescent individuals. In vaccinated individuals, we detected many of the same dominant S-specific epitopes at similar frequencies as compared to convalescent individuals. T cell responses to peptide S205 (amino acids 817-831) were observed in 56% and 59% of individuals following infection and vaccination, respectively, while 20% and 19% of individuals responded to S302 (a.a. 1205-1219) following infection and vaccination, respectively. For S205, a CD4+ T cell response, we confirmed 8 unique TCRs and determined the minimal epitope to be a 9mer (IEDLLFNKV). While TCR genes TRAV8-6*01 and TRBV30*01 were commonly utilized across the TCRs, we did identify TCRs with unique immunogenetic properties with different potencies of cross-reactivity to other HCoVs. For S302, a CD8+ T cell response, we identified two unique TCRs with different immunogenetic properties that recognized the same 9mer (YIKWPWYIW) and cross-reacted with different HCoV peptides (Figure 1). Conclusion(s): These data identify immunodominant T cell epitopes following SARS-CoV-2 infection and vaccination and provide a detailed analysis of epitope-specific TCR repertoires. The prospect of developing a vaccine that broadly protects against multiple human coronaviruses is bolstered by the identification of conserved immunodominant SARS-CoV-2 T cell epitopes that cross react with multiple other HCoVs.

Chronic HIV infection influences the immune response during acute COVID-19 and long COVID

OBJECTIVES/GOALS: Despite highly effective antiretroviral therapy, people living with HIV (PLWH) experience chronic immune activation and inflammation which may influence the progression of infections such as SARS-CoV-2. Here, we explore the immune response and clinical outcomes in HIV(+) and HIV(-) individuals experiencing acute COVID-19 and long COVID (LC). METHODS/STUDY POPULATION: We performed flow cytometric analyses on peripheral blood mononuclear cells from the following: 1) HIV(-) individuals experiencing acute COVID-19, 2) PLWH experiencing acute COVID-19, and 3) pre-COVID-19 pandemic PLWH. Additionally, we will perform similar analyses for the following: 1) PLWH experiencing LC, 2) PLWH previously infected with SARS-CoV-2 who recovered, 3) pre-COVID-19 pandemic PLWH, and 4) HIV(-) individuals experiencing LC. Flow cytometry panels include surface markers for immune cell populations, activation and exhaustion surface markers (with and without SARS-CoV-2-specific antigen stimulation), and intracellular cytokine staining. We will also analyze how chronic HIV infection and other clinical and demographic factors (e.g., age, CD4 %) impact persistent symptomatic burden. RESULTS/ANTICIPATED RESULTS: Acute COVID-19 results–Overall, PLWH had higher baseline expression of activation markers OX40 and CD137 on CD4+ and CD8+ T cells, along with increased levels of TNFa producing CD8+ T cells. Interestingly, PLWH had increased expression of exhaustion markers PD1 and TIGIT but decreased expression of TIM3 on CD4+ and CD8+ T cells. Additionally, PLWH had decreased levels of IL-2 and IFNg producing CD4+ T cells which suggests functional exhaustion. Long COVID-19 expected results–we hypothesize that the activation and inflammation seen in chronic HIV infection will lead to more immune dysregulation and subsequently worsened symptomatic burden. Additionally, we hypothesize that PLWH may have different frequencies of certain LC manifestations, such as increased rates of neurocognitive impairment. DISCUSSION/SIGNIFICANCE: Our findings suggest that chronic HIV infection influences acute immune response during SARS-CoV-2 infection, and that PLWH have variable expression of exhaustion markers which warrants further study. Additionally, our findings in the LC cohort will aid in characterizing clinical manifestations and immunologic mechanisms of LC in PLWH.

Jing Si Herbal Drink as a prospective adjunctive therapy for COVID-19 treatment: Molecular evidence and mechanisms

Background: SARS-CoV-2 has led to a sharp increase in the number of hospitalizations and deaths from pneumonia and multiorgan disease worldwide;therefore, SARS-CoV-2 has become a global health problem. Supportive therapies remain the mainstay treatments against COVID-19, such as oxygen inhalation, antiviral drugs, and antibiotics. Traditional Chinese medicine (TCM) has been shown clinically to relieve the symptoms of COVID-19 infection, and TCMs can affect the pathogenesis of SARS-CoV-2 infection in vitro. Jing Si Herbal Drink (JSHD), an eight herb formula jointly developed by Tzu Chi University and Tzu Chi Hospital, has shown potential as an adjuvant treatment for COVID-19 infection. A randomized controlled trial (RCT) of JSHD as an adjuvant treatment in patients with COVID-19 infection is underway Objectives: This article aims to explore the efficacy of the herbs in JSHD against COVID-19 infection from a mechanistic standpoint and provide a reference for the rational utilization of JSHD in the treatment of COVID-19. Method(s): We compiled evidence of the herbs in JSHD to treat COVID-19 in vivo and in vitro. Result(s): We described the efficacy and mechanism of action of the active ingredients in JSHD to treat COVID-19 based on experimental evidence. JSHD includes 5 antiviral herbs, 7 antioxidant herbs, and 7 anti-inflammatory herbs. In addition, 2 herbs inhibit the overactive immune system, 1 herb reduces cell apoptosis, and 1 herb possesses antithrombotic ability. Conclusion(s): Although experimental data have confirmed that the ingredients in JSHD are effective against COVID-19, more rigorously designed studies are required to confirm the efficacy and safety of JSHD as a COVID-19 treatment.Copyright © 2021

mRNA anti-cancer vaccine research gains momentum

Ongoing research into the use of messenger RNA (mRNA) vaccines for the treatment of cancer has been expediated by the coronavirus pandemic because similar technology was used in the development of mRNA COVID-19 vaccines. So how close are we now to the widespread clinical use of mRNA anti-cancer vaccines?.Copyright © 2023 Wiley Interface Ltd.

Role of T Lymphocyte Activation Profile in Predicting SARS-CoV-2 Severity: Experience from Tertiary Care Centre of North India.

Background: Immune dysregulation plays a key role in determining COVID-19 disease severity. We aimed to analyze the T cell activation profile in COVID - 19 cases and its predictive role in disease severity and outcome. Material & methods: This was a prospective observational pilot study from a tertiary care COVID-19 hospital. Peripheral blood samples obtained between the fifth and seventh day of COVID-19 illness, were subjected to lymphocyte subset analysis using multicolor flowcytometry using a single tube, 8 antibodies (CD45, CD19, CD3, CD4, CD8, CD38, HLADR, and CD56) analysis. Correlation between lymphocyte subset analysis and clinical profile was determined. Results: 26 patients including 11 with mild disease and 15 with severe disease were enrolled. The median age was 58 years (range: 33-81), with a male: female ratio of 1.36:1. Significant lymphopenia was observed in the severe group compared to the mild group (p < 0.02). The absolute numbers of CD3+, CD4+, CD8 + T cells, B cells, and NK cells were significantly reduced in the severe group as compared to the mild group (p < 0.05). In patients with severe disease, the proportion of CD8 + and CD4 + T cells were significantly higher than those in patients with mild disease (p = 0.0372). Using ROC analysis, a CD4:8 T cell ratio of ≥ 2.63 and an activated (CD38 + HLA-DR+) CD8 T cell proportion of > 15.85% of the total CD8 T cell population, significantly determined the severe disease category. Conclusions: Severe COVID-19 is associated with severe lymphopenia, altered CD4/CD8 ratio and markedly increased CD8 T cell activation profile. Supplementary Information: The online version contains supplementary material available at 10.1007/s12288-022-01558-6.

Novel application of [18F]DPA714 for visualizing the lower respiratory tract of SARS-CoV-2-exposed rhesus monkeys

Aim/Introduction: A hallmark of coronavirus disease 2019 (COVID-19) is lower respiratory tract infection and pneumonia. Infection of nonhuman primates mirrors mild-to-moderate human disease determined with concordant immunologic, virologic, and lung histopathologic findings in combination with imaging abnormalities. Particularly in the lower respiratory tract, medical imaging of pulmonary disease contributes uniquely to the understanding and measuring of the consequences of an infection. Advanced medical imaging, such as PET-CT, of the lungs of SARS-CoV-2-exposed nonhuman primates shows great promise in detecting and longitudinally evaluating disease in a non-invasive manner. The aim of this study was to investigate the application of [18F]DPA714 for visualizing the inflammation processes in the lungs of SARS-CoV-2-infected rhesus monkeys. Material(s) and Method(s): Four experimentally SARS-CoV-2 infected rhesus monkeys were followed for seven weeks post infection (pi) with a weekly PETCT using a MultiScan Large Field of View Extreme Resolution Research Imager (LFER) 150 (Mediso Medical Imaging Systems Ltd., Budapest, Hungary) with [18F]DPA714 as radiotracer. Following a retrospectively gated CT, an intravenous bolus of approximately 180 MBq [18F]DPA714 (molar activity: 77.4 GBq/mumol (range 39.8- 150.6 GBq/mumol, radioactivity concentration: of 277.0+/-172.8 MBq/ mL, a radiochemical purity > 98.0%) was administered. Two PET images, ten minutes each, of a single field-of-view covering the chest area, were obtained ten and thirty minutes after injection. To confirm the infection of SARS-CoV-2 , both nasal and tracheal swabs and blood samples were obtained. Result(s): All animals tested positive for SARS-CoV-2 in both swabs on multiple timepoints pi. The initial development of pulmonary lesions was already detected at the first scan 2-days pi. PET revealed an increased tracer uptake in the pulmonary lesions and mediastinal lymph nodes of all animals starting from the first scan pi onwards. However, also an increased uptake was detected in the lung tissue surrounding the lesions, which persisted until day 30 and then subsided by day 37-44 pi. In parallel, a similar pattern of increased expression of activation markers was observed on dendritic cells in blood. Conclusion(s): This study illustrates that [18F]DPA714 is a valuable radiotracer to visualize SARS-CoV-2-associated pulmonary inflammation even during mild-to-moderate disease. In addition, the inflammatory process detected in anatomically unaffected lung tissue coincided with dendritic cell activation detected in blood samples. Our data indicate that [18F]DPA714 can be applied to visualize the pulmonary tract following a SARS-CoV-2 infection in rhesus monkeys and may be translated to the clinic as diagnostic tracer for this indication too.

Peculiar Imunophenotype of Peripheral Blood Lymphocytes in Three Cases with Persistent Isolated Lymphocytosis after Covid-19 Vaccination

Introduction: Transient increase in the lymphocyte count (lymphocytosis) is a common laboratory finding caused by acute (usually viral) infections. When persistent lymphocytosis prolonged for several months is found in otherwise healthy individuals, flow cytometry immunophenotyping (FCI) of peripheral blood lymphocytes is required to rule out lymphoproliferative disorder. Here, we presented the immunophenotype analysis of lymphocytes in three cases with persistent isolated lymphocytosis after vaccination for coronavirus disease 2019 (COVID-19). Novel vaccines contain glycolipids that activate natural killer T-cells (NKT-cells), a subset of T-cells that have properties of both T-cells and NK-cells. NKT-cells normally constitute 1-2 % of all peripheral blood T-cells. Activation of NKT-cells by vaccines enchance both the innate and adaptive immune response to the vaccine. Method(s): Three patients were admitted to the Department of Clinical Hematology of the University Hospital Osijek to determine the cause of isolated lymphocytosis in differential blood count with normal leukocyte count, accidentaly detected by regular check-ups. For that purpose, FCI analysis of peripheral blood lymphocytes was requested. Approximately 4 months ago, two patients were vaccinated with mRNAbased COVID-19 vaccine and the third one with adenovirus-based vaccine. Persistence of ymphocytosis was monitored 3-4 months after it was first observed. Result(s): FCI analysis of lymphocytes in all three blood samples showed T-cell proliferation (>80 % of all lymphocytes). A high percentage of cells within the gate of all T-cells (medians 13 %, 29 % and 48 %) expressed surface markers CD2+CD3+CD5+(moderate to dim expression)CD7+CD8+CD16+56+, corresponding to immunophenotype of NKT-cells. Conclusion(s): The results showed that NKT-cells were the dominant subset of T-cells underlying lymphocytosis, regardless of the type of COVID-19 vaccine used. Also, isolated lymphocytosis after COVID-19 vaccination can persist for more than 3 months. COVID-19 vaccines contain glycolipids to facilitate vaccine entry into cells, but as shown here, they probably activate NKT-cells as well. The problem with FCI analysis of such cases is that the immunophenotype of NKT-cells does not differ from the immunophenotype of T-cell large granular lymphocytic leukemia (T-LGL) cells. Future post-vaccination lymphocytosis studies should explain mechanisms of NKT-cell activation with COVID-19 vaccines and determine exactly when to raise the alarm for a more detailed analysis of such cases.

Severe respiratory viral infections: T-cell functions diverging from immunity to inflammation.

Respiratory viral infections such as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and influenza A virus (IAV) trigger distinct clinical outcomes defined by immunity-based viral clearance or disease associated with exaggerated and prolonged inflammation. The important role of T cells in shaping both antiviral immunity and inflammation has revived interest in understanding the host-pathogen interactions that lead to the diverse functions of T cells in respiratory viral infections. Inborn deficiencies and acquired insufficiency in immunity can prolong infection and shift the immune response towards exacerbated inflammation, which results from persistent innate immune activation and bystander T-cell activation that is nonspecific to the pathogen but is often driven by cytokines. This review discusses how virus variants, exposure doses, routes of infection, host genetics, and immune history can modulate the activation and function of T cells, thus influencing clinical outcomes. Knowledge of virus-host interaction can inform strategies to prevent immune dysfunction in respiratory viral infection and help in the treatment of associated diseases.

Multilamellar Mrna Lipid Particles Induce Immunologic Reprogramming in Canine and Human Glioblastoma Patients

BACKGROUND: Although mRNA vaccines have been deployed with great success against COVID-19, unlocking this technology against glioblastoma will necessitate new lipid-nanoparticle formulations that overcome cancer tolerance and immunosuppression. OBJECTIVE/METHODS: We sought to develop a novel mRNA vaccine system to make tolerogenic tumor antigens appear more dangerous through use of unmodified nucleosides (pathogen associated molecular patterns, PAMPs) and highly cationic lipid shells that elicit a systemic damage response against cancer antigens. RESULT(S): We developed a novel vaccine formulation that increases payload packaging of tumor amplified mRNA into multilamellar (onion-shaped) particles for systemic (intravenous) administration. We demonstrate significant immunogenicity and efficacy of multilamellar RNA-NPs in syngeneic murine models for high-grade glioma (KR158b-pp65), and diffuse midline glioma (H3K27M DMG). Remarkably, RNA-NPs significantly improve median survival outcomes of DMG bearing mice beginning therapy at endpoint (Day 35 after midline intracranial implantation). Unlike prototypical mRNA vaccines that activate endosomal toll-like receptors (i.e. TLR7), multilamellar RNA-NPs elicit immunologic response predominantly through intracellular pathogen recognition receptors (RIG-I);long-term survival benefits from RNA-NPs were completely abrogated in RIG-I knockout mice. In canines (pet dogs) with spontaneous gliomas, RNA-NPs elicit massive recruitment/activation of peripheral blood mononuclear cells (PBMCs) which correlate with their trafficking into lymphoreticular organs (in follow-up murine studies). In canines receiving neoadjuvant RNA-NPs, prior to glioma biopsy, we see significant reprogramming of the glioma microenvironment with increased gene signatures for antigen processing/ presentation, interferon signaling and cytotoxicity. Upon translation into human clinical trials for glioblastoma patients (NCT04573140), RNA-NPs elicit rapid (within hours) release of cytokines (e.g. IL-1, IL-6, IL-12 TNF- alpha, interferons) and chemokines (e.g. MIP1alpha, MCP-1, IP-10), which correlate with mobilization of PBMCs and activation of dendritic cells/CD8 lymphocytes. CONCLUSION(S): First-in-human application of systemic multilamellar RNA-NP vaccines results in significant biologic effects and rapid immunologic reprogramming.

Cerebral Venous and Sinus Thrombosis with Immunological Pathogenesis: VITT and Pre-VITT

In this review, we summarise the current knowledge on vaccine-induced immune thrombotic thrombocytopenia (VITT) and new insights into its underlying pathogenesis. VITT is characterised by severe thromboses occurring 5-20 days after vaccination with an adenoviral vector-based SARS-CoV-2 vaccine (AstraZeneca or Johnson & Johnson). Thromboses typically involve the cerebral sinus and venous system. Routine laboratory analyses show thrombocytopenia and high D-dimer levels. The pathogenesis is based on immunological processes similar to those in heparin-induced thrombocytopenia. Accordingly, VITT is associated with high-titre immunoglobulin G directed against platelet factor 4 (PF4). Interaction with adenoviral vector-based vaccines leads to modifications of PF4 allowing antibody-producing cells to identify PF4. Anti-PF4 antibodies activate platelets through FcgammaIIa receptors. The detection of platelet-activating anti-PF4 antibodies confirms the diagnosis of VITT. Treatment is based on anticoagulation, which inhibits thrombin itself or thrombin formation, and high-dose intravenous immunoglobulin G, which inhibits cell activation via FcgammaIIa receptors. In severe cases, plasma exchange could also be an option. In some patients, a pre-VITT syndrome precedes VITT. Pre-VITT patients typically present with severe headache before thromboses are manifest. The early identification of a pre-VITT syndrome allows for the prevention of thrombotic complications. The specific dynamics of the immune reaction in VITT correspond to a transient, secondary immune response. Current studies address how PF4 binds to different adenoviral proteins and investigate the functional role of other vaccine components. Some of these factors contribute to the induction of a pro-inflammatory danger signal that triggers the first stage of VITT pathogenesis. In the second stage, high-titre anti-PF4 antibodies activate platelets and granulocytes. In a process called NETosis (neutrophil extracellular traps), activated granulocytes release DNA. Anti-PF4 antibodies then bind to complexes of PF4 and DNA. This enhances further cell activation via Fcgamma receptors and consequently also the formation of thrombin. At the end of the article, we comment on how the current knowledge on VITT may influence global vaccination campaigns against SARS-CoV-2 and we address how anti-PF4 antibodies may be involved in recurrent arterial and venous thromboses not associated with VITT and HIT. Copyright © 2022 Georg Thieme Verlag. All rights reserved.