Reactive astrogliosis is prominent in most neurodegenerative disorders and is often associated with neuroinflammation. The molecular mechanisms regulating astrocyte-linked neuropathogenesis during injury, aging and human immunodeficiency virus (HIV)-associated neurocognitive disorders (HAND) are not fully understood. In this study, we investigated the implications of the wingless type (Wnt)/β-catenin signaling pathway in regulating astrocyte function during gliosis. First, we identified that HIV-associated inflammatory cytokines, interleukin (IL)-1β and tumor necrosis factor (TNF)-α induced mediators of the Wnt/β-catenin pathway including β-catenin and lymphoid enhancer-binding factor (LEF)-1 expression in astrocytes. Next, we investigated the regulatory role of β-catenin on primary aspects of reactive astrogliosis, including proliferation, migration and proinflammatory responses, such as IL-6. Knockdown of β-catenin impaired astrocyte proliferation and migration as shown by reduced cyclin-D1 levels, bromodeoxyuridine incorporation and wound healing. HIV-associated cytokines, IL-1β alone and in combination with TNF-α, strongly induced the expression of proinflammatory cytokines including C-C motif chemokine ligand (CCL)2, C-X-C motif chemokine ligand (CXCL)8 and IL-6; however, only IL-6 levels were regulated by β-catenin as demonstrated by knockdown and pharmacological stabilization. In this context, IL-6 levels were negatively regulated by β-catenin. To better understand this relationship, we examined the crossroads between β-catenin and nuclear factor (NF)-κB pathways. While NF-κB expression was significantly increased by IL-1β and TNF-α, NF-κB levels were not affected by β-catenin knockdown. IL-1β treatment significantly increased glycogen synthase kinase (GSK)-3β phosphorylation, which inhibits β-catenin degradation. Further, pharmacological inhibition of GSK-3β increased nuclear translocation of both β-catenin and NF-κB p65 into the nucleus in the absence of any other inflammatory stimuli. HIV+ human astrocytes show increased IL-6, β-catenin and NF-κB expression levels and are interconnected by regulatory associations during HAND. In summary, our study demonstrates that HIV-associated inflammation increases β-catenin pathway mediators to augment activated astrocyte responses including migration and proliferation, while mitigating IL-6 expression. These findings suggest that β-catenin plays an anti-inflammatory role in activated human astrocytes during neuroinflammatory pathologies, such as HAND.
Acute myeloid leukaemia (AML) is a medical emergency often presenting with hyperleucocytosis, coagulopathy and pulmonary infiltration necessitating emergent initiation of therapy. AML with concomitant severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) infection presents a unique challenge given the lack of evidence-based guidelines or historical experience. While cohort studies have shown early serological responses to SARS-CoV-2 in healthy adults,1, 2 little is known about the serological responses to infection in patients with AML and the impact of chemotherapy on this response. In the present study, we detail the clinical presentations, treatments, serological and virological responses, and outcomes of two adolescents who presented with AML and concurrent coronavirus disease 2019 (COVID-19). Two adolescents presenting with AML and COVID-19 were enrolled on an Institutional Review Board-approved protocol to collect prospective/residual specimens used for SARS-CoV-2 serological and virological testing. Nasopharyngeal (NP) real-time reverse transcription polymerase chain reaction (RT-PCR), antibody testing by enzyme-linked immunosorbent assay (ELISA), live-virus focus reduction neutralisation assay-mNG, surface plasmon resonance (SPR) assay and viral genetic sequencing were performed (Data S1). Patient details, treatment and outcome data were abstracted from medical records. Patient 1 was a 16-year-old Caucasian male with a history of classical Hodgkin lymphoma who presented with fever, cough and hyperleucocytosis [white blood count (WBC) 176 × 109/l]. His NP SARS-CoV-2 RT-PCR test was positive and peripheral blood flow cytometry (PBFC) confirmed a diagnosis of therapy-related AML. He received hydroxyurea, followed by cytarabine starting on hospital day (HD) 3. Treatment for COVID-19 included hydroxychloroquine, remdesivir and supplemental oxygen (Fig 1). On HD4, the patient had detectable immunoglobulin (Ig)M and IgG antibodies to SARS-CoV-2 (Table I). The patient did not become lymphopenic throughout his COVID-19 course and maintained detectable binding and neutralising antibodies to SARS-CoV-2. SPR demonstrated that binding antibodies to the pre-fusion conformation of S, the receptor-binding domain (RBD) and S2 subunits all peaked by HD26. The patient had detectable IgM, IgA and IgG1 titres in the final sample on HD70 (Figure S1). He cleared the virus on HD16. Bone marrow (BM) on HD20 showed rare blasts on morphology in the setting of pancytopenia, with no disease detected by flow cytometry. He received additional chemotherapy with azacitidine and gemtuzumab starting on HD26. His treatment complications included bacteraemia and perirectal abscess with Pseudomonas aeruginosa and Epstein–Barr virus (EBV) viraemia resulting in multi-organ failure and death on HD74. Patient 2 was a 19-year-old obese (body mass index of 32 kg/m2), Hispanic male who presented with fever, cough, dyspnoea and hyperleucocytosis (WBC count 67 × 109/l). His NP SARS-CoV-2 RT-PCR test upon admission was positive and PBFC confirmed a diagnosis of AML. He began induction chemotherapy with cytarabine, daunorubicin and etoposide (ADE) on HD2 and he clinically deteriorated on HD4 requiring intubation and mechanical ventilation. For treatment of COVID-19, he received convalescent plasma (CP), remdesivir, tocilizumab and therapeutic plasma exchange (Fig 1). The patient was lymphopenic by HD5 and showed no immune response to SARS-CoV-2 with absence of IgM antibodies, waning IgG (post CP) and undetectable neutralisation titres (Table I). Concurrently, the patient's NP RT-PCR remained positive with low cycle-threshold (Ct) values. Coinciding with haematological recovery on HD25, the patient demonstrated a serological response with rising IgM, IgG, neutralising antibody titres and SARS-CoV-2 RT-PCR Ct values until his first negative NP RT-PCR result on HD48. His SPR antibody profiling demonstrated class switching (Figure S1) and coincided with robust increases in binding and neutralising titres with haematological recovery. Genetic sequencing and phylogenetic analysis of his SARS-CoV-2 virus from saliva on HD23 indicated that the sequence clustered with clade 20B,3 which was relatively uncommon in Georgia at the time (Figure S2). A total of 40 intra-host single nucleotide variants (iSNVs) were identified with little variability in S and envelope genes (Figure S3 and Table SI). On HD25 his bone marrow evaluation demonstrated complete remission, but he remained critically ill and on HD48 he suffered a fatal sudden cardiac arrest secondary to sepsis. In the present report, we highlight both the therapeutic challenges in treating COVID-19 and AML concomitantly and describe the immune response in the setting of myelosuppressive chemotherapy. The viral clearance of the two patients negatively correlated with the intensity of chemotherapy given for each and likely contributed to the overall severity of COVID-19. Paediatric recommendations for the management of AML and COVID-19 are not well defined. Use of a 'mild' induction regimen (MAG) in nine patients with AML and COVID-19 in Brazil was recently described to have excellent outcomes.4 While reduced-intensity regimens are a valid approach, they preclude response-based risk assignment in AML but warrant further investigation in the setting of concomitant COVID-19. SARS-CoV-2 evolution within immunocompromised patients with prolonged virus replication has been described.5, 6 Although our investigation was limited to one time point, we did observe within-sample SARS-CoV-2 variants for Patient 2. The functional importance of these variants is unclear; however, future studies into the intra-host variation of SARS-CoV-2 genotypes are needed in the immunocompromised population. Furthermore, Ct values associated with RT-PCR testing for SARS-CoV-2 could provide indirect assessment of viral load.7 Lower median Ct values in adults with cancer correlated with higher rates of mortality.8 Patient 2's Ct values remained <25 until HD17 suggesting a high viral load. While PCR Ct values have limitations based on sample collection and instrumentation, serial measurements could be used for clinical correlation.9 Limitations of our present study include the small number of patients, which limits generalisability of the data. Co-existing factors such as underlying obesity and race may have contributed to a worse outcome in Patient 2.10, 11 In addition, absence of serological immunity at presentation and administration of CP in Patient 2 confounds interpretation of serological results. Investigation into early immune responses is warranted in this population as they may serve as prognostic indicators at the time of presentation. In conclusion, our experience demonstrates that patients with AML and COVID-19 can mount immune responses to SARS-CoV-2 even in the face of immune suppression by chemotherapy; however, the intensity of chemotherapy may play a role in the response. Longitudinal research is needed in this vulnerable population to better understand response to SARS-CoV-2 infection and now vaccination. The authors gratefully acknowledge the clinical research co-ordinators who have contributed to patient enrolment, specimen collection, chart abstraction and data collection for this study including Laila Hussaini MPH, Lisa Macoy MSN, Felicia Glover and Amber Samuel. We thank Nadine Rouphael MD and the Emory Hope Clinic Laboratory for assisting with sample processing. We thank Robert Jerris PhD and the Children's Healthcare of Atlanta (CHOA) Research Laboratory for assistance with collection and processing of residual clinical samples. Lastly, we thank the patients and their families for consenting to donate their samples and share their data to further our understanding of COVID-19 and its impact on paediatric patients with cancer. Pratik A. Patel designed the research study, performed the research, analysed the data and helped write the paper. Gabrielle Grubbs, Stacey A. Lapp and Venkata V. Edara performed the research and analysed the data. Christina A. Rostad, Evan J. Anderson and Surender Khurana designed the research study, analysed the data and helped write the paper. Claire L. Stokes and Melinda G. Pauly helped write the paper. Anne Piantadosi and Mehul S. Suthar performed the research, analysed the data and helped write the paper. Himalee S. Sabnis analysed the data and helped write the paper. All authors made substantial contributions to research design, or acquisition, analysis or interpretation of data, drafting the paper or revising it critically and approval of the submitted and final versions of the paper. Christina A. Rostad's institution has received funds to conduct clinical research unrelated to this manuscript from BioFire Inc., MedImmune, Regeneron, PaxVax, Pfizer, GSK, Merck, Novavax, Sanofi-Pasteur, Micron, Janssen and Moderna. Christina A. Rostad is co-inventor on patented respiratory syncytial virus (RSV) vaccine technology unrelated to this manuscript, which has been licensed to Meissa Vaccines, Inc. Evan J. Anderson has received personal fees from AbbVie, Pfizer and Sanofi-Pasteur for consulting, and his institution receives funds to conduct clinical research unrelated to this manuscript from MedImmune, Regeneron, PaxVax, Pfizer, GSK, Merck, Novavax, Sanofi-Pasteur, Micron, Janssen and Moderna. He also serves on a safety monitoring board for Sanofi-Pasteur and Kentucky BioProcessing, Inc. The other authors have no relevant conflicts of interest to disclose. Mehul S. Suthar's work is supported in part by grants [National Institutes of Health (NIH) P51OD011132 and 1U54CA260563-01 to Emory University) from the National Institute of Allergy and Infectious Diseases (NIAID), NIH, by the Emory Executive Vice President for Health Affairs Synergy Fund award, the Pediatric Research Alliance Center for Childhood Infections and Vaccines and Children's Healthcare of Atlanta, COVID-Catalyst-I3 Funds from the Woodruff Health Sciences Center and Emory School of Medicine, Woodruff Health Sciences Center 2020 COVID-19 CURE Award and the Vital Projects/Proteus funds. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.
Abstract Background The effects of pre-existing endemic human coronavirus (HCoV) immunity on SARS-CoV-2 serologic and clinical responses are incompletely understood. Objectives We sought to determine the effects of prior exposure to HCoV Betacoronavirus HKU1 spike protein on serologic responses to SARS-CoV-2 spike protein after intramuscular administration in mice. We also sought to understand the baseline seroprevalence of HKU1 spike antibodies in healthy children and to measure their correlation with SARS-CoV-2 binding and neutralizing antibodies in children hospitalized with acute coronavirus disease 2019 (COVID-19) or multisystem inflammatory syndrome (MIS-C). Methods Groups of 5 mice were injected intramuscularly with two doses of alum-adjuvanted HKU1 spike followed by SARS-CoV-2 spike; or the reciprocal regimen of SARS-Cov-2 spike followed by HKU1 spike. Sera collected 21 days following each injection was analyzed for IgG antibodies to HKU1 spike, SARS-CoV-2 spike, and SARS-CoV-2 neutralization. Sera from children hospitalized with acute COVID-19, MIS-C or healthy controls (n=14 per group) were analyzed for these same antibodies. Results Mice primed with SARS-CoV-2 spike and boosted with HKU1 spike developed high titers of SARS-CoV-2 binding and neutralizing antibodies; however, mice primed with HKU1 spike and boosted with SARS-CoV-2 spike were unable to mount neutralizing antibodies to SARS-CoV-2. HKU1 spike antibodies were detected in all children with acute COVID-19, MIS-C, and healthy controls. Although children with MIS-C had significantly higher HKU1 spike titers than healthy children (GMT 37239 vs. 7551, P =0.012), these titers correlated positively with both SARS-CoV-2 binding (r=0.7577, P <0.001) and neutralizing (r=0.6201, P =0.001) antibodies. Conclusions Prior murine exposure to HKU1 spike protein completely impeded the development of neutralizing antibodies to SARS-CoV-2, consistent with original antigenic sin. In contrast, the presence of HKU1 spike IgG antibodies in children with acute COVID-19 or MIS-C was not associated with diminished neutralizing antibody responses to SARS-CoV-2.
Background The effects of pre-existing endemic human coronavirus (HCoV) immunity on SARS-CoV-2 serologic and clinical responses are incompletely understood. Objectives We sought to determine the effects of prior exposure to HCoV Betacoronavirus HKU1 spike protein on serologic responses to SARS-CoV-2 spike protein after intramuscular administration in mice. We also sought to understand the baseline seroprevalence of HKU1 spike antibodies in healthy children and to measure their correlation with SARS-CoV-2 binding and neutralizing antibodies in children hospitalized with acute coronavirus disease 2019 (COVID-19) or multisystem inflammatory syndrome (MIS-C). Methods Groups of 5 mice were injected intramuscularly with two doses of alum-adjuvanted HKU1 spike followed by SARS-CoV-2 spike; or the reciprocal regimen of SARS-Cov-2 spike followed by HKU1 spike. Sera collected 21 days following each injection was analyzed for IgG antibodies to HKU1 spike, SARS-CoV-2 spike, and SARS-CoV-2 neutralization. Sera from children hospitalized with acute COVID-19, MIS-C or healthy controls (n = 14 per group) were analyzed for these same antibodies. Results Mice primed with SARS-CoV-2 spike and boosted with HKU1 spike developed high titers of SARS-CoV-2 binding and neutralizing antibodies; however, mice primed with HKU1 spike and boosted with SARS-CoV-2 spike were unable to mount neutralizing antibodies to SARS-CoV-2. HKU1 spike antibodies were detected in all children with acute COVID-19, MIS-C, and healthy controls. Although children with MIS-C had significantly higher HKU1 spike titers than healthy children (GMT 37239 vs. 7551, P = 0.012), these titers correlated positively with both SARS-CoV-2 binding (r = 0.7577, P <0.001) and neutralizing (r = 0.6201, P = 0.001) antibodies. Conclusions Prior murine exposure to HKU1 spike protein completely impeded the development of neutralizing antibodies to SARS-CoV-2, consistent with original antigenic sin. In contrast, the presence of HKU1 spike IgG antibodies in children with acute COVID-19 or MIS-C was not associated with diminished neutralizing antibody responses to SARS-CoV-2.
Astrocytes in the central nervous system (CNS) provide supportive neural functions and mediate inflammatory responses from microglia. Increasing evidence supports their critical roles in regulating brain homoeostasis in response to pro-inflammatory factors such as cytokines and pathogen/damage-associated molecular pattern molecules in infectious and neurodegenerative diseases. However, the underlying mechanisms of the trans-cellular communication are still unclear. Extracellular vesicles (EVs) can transfer a large diversity of molecules such as lipids, nucleic acids and proteins for cellular communications. The purpose of this study is to characterize the EVs cargo proteins derived from human primary astrocytes (ADEVs) under both physiological and pathophysiological conditions. ADEVs were isolated from human primary astrocytes after vehicle (CTL) or interleukin-1β (IL-1β) pre-treatment. Label-free quantitative proteomic profiling revealed a notable up-regulation of proteins including actin-associated molecules, integrins and major histocompatibility complex in IL-1β-ADEVs compared to CTL-ADEVs, which were involved in cellular metabolism and organization, cellular communication and inflammatory response. When fluorescently labelled ADEVs were added into primary cultured mouse cortical neurons, we found a significantly increased neuronal uptake of IL-1β-ADEVs compared to CTL-ADEVs. We further confirmed it is likely due to the enrichment of surface proteins in IL-1β-ADEVs, as IL-1β-ADEVs uptake by neurons was partially suppressed by a specific integrin inhibitor. Additionally, treatment of neurons with IL-1β-ADEVs also reduced neurite outgrowth, branching and neuronal firing. These findings provide insight for the molecular mechanism of the ADEVs' effects on neural uptake, neural differentiation and maturation, and its alteration in inflammatory conditions.
Testing of vaccine candidates to prevent infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in an older population is important, since increased incidences of illness and death from coronavirus disease 2019 (Covid-19) have been associated with an older age. We conducted a phase 1, dose-escalation, open-label trial of a messenger RNA vaccine, mRNA-1273, which encodes the stabilized prefusion SARS-CoV-2 spike protein (S-2P) in healthy adults. The trial was expanded to include 40 older adults, who were stratified according to age (56 to 70 years or ≥71 years). All the participants were assigned sequentially to receive two doses of either 25 μg or 100 μg of vaccine administered 28 days apart. Solicited adverse events were predominantly mild or moderate in severity and most frequently included fatigue, chills, headache, myalgia, and pain at the injection site. Such adverse events were dose-dependent and were more common after the second immunization. Binding-antibody responses increased rapidly after the first immunization. By day 57, among the participants who received the 25-μg dose, the anti-S-2P geometric mean titer (GMT) was 323,945 among those between the ages of 56 and 70 years and 1,128,391 among those who were 71 years of age or older; among the participants who received the 100-μg dose, the GMT in the two age subgroups was 1,183,066 and 3,638,522, respectively. After the second immunization, serum neutralizing activity was detected in all the participants by multiple methods. Binding- and neutralizing-antibody responses appeared to be similar to those previously reported among vaccine recipients between the ages of 18 and 55 years and were above the median of a panel of controls who had donated convalescent serum. The vaccine elicited a strong CD4 cytokine response involving type 1 helper T cells. In this small study involving older adults, adverse events associated with the mRNA-1273 vaccine were mainly mild or moderate. The 100-μg dose induced higher binding- and neutralizing-antibody titers than the 25-μg dose, which supports the use of the 100-μg dose in a phase 3 vaccine trial. (Funded by the National Institute of Allergy and Infectious Diseases and others; mRNA-1273 Study ClinicalTrials.gov number, NCT04283461.).
The Omicron variant of SARS-CoV-2 is raising concerns because of its increased transmissibility and potential for reduced susceptibility to antibody neutralization. To assess the potential risk of this variant to existing vaccines, serum samples from mRNA-1273 vaccine recipients were tested for neutralizing activity against Omicron and compared to neutralization titers against D614G and Beta in live virus and pseudovirus assays. Omicron was 41-84-fold less sensitive to neutralization than D614G and 5.3-7.4-fold less sensitive than Beta when assayed with serum samples obtained 4 weeks after 2 standard inoculations with 100 μg mRNA-1273. A 50 μg boost increased Omicron neutralization titers and may substantially reduce the risk of symptomatic vaccine breakthrough infections.
Abstract SARS-CoV-2 has caused a devastating global pandemic. The recent emergence of SARS-CoV-2 variants that are less sensitive to neutralization by convalescent sera or vaccine-induced neutralizing antibody responses has raised concerns. A second wave of SARS-CoV-2 infections in India is leading to the expansion of SARS-CoV-2 variants. The B.1.617.1 variant has rapidly spread throughout India and to several countries throughout the world. In this study, using a live virus assay, we describe the neutralizing antibody response to the B.1.617.1 variant in serum from infected and vaccinated individuals. We found that the B.1.617.1 variant is 6.8-fold more resistant to neutralization by sera from COVID-19 convalescent and Moderna and Pfizer vaccinated individuals. Despite this, a majority of the sera from convalescent individuals and all sera from vaccinated individuals were still able to neutralize the B.1.617.1 variant. This suggests that protective immunity by the mRNA vaccines tested here are likely retained against the B.1.617.1 variant. As the B.1.617.1 variant continues to evolve, it will be important to monitor how additional mutations within the spike impact antibody resistance, viral transmission and vaccine efficacy.
