Respiratory Syncytial Virus (RSV) infections in children: recent concepts in immunopathogenesis and diagnostic dilemma

Respiratory viral infections are important cause of morbidity and mortality in early life. The relative influence of host and viral factors possibly contribute to the disease pathogenesis. Predisposing conditions like prematurity, Low birth weight and congenital heart diseases etc. have been incriminated in the disease progression. The development of cough, wheezing, and tachypnea, usually peaking on days 4 to 5, go parallel with host cytokine responses and viral load. Various host cytokines, chemokines and molecules involved in the immune response against RSV infection might be responsible for the outcome of the disease process. Nasopharyngeal aspirates (NPAs) from children (n = 349) between 2013-2017 were subjected for IL-17A, IFN-gamma, TNF-alpha, IL-10, IL-6 levels by CBA and MMP-9 and TIMP-1 levels by ELISA. The viral load in RSV positive samples and cytokine levels were correlated with the WHO criteria for acute lower respiratory tract illness (ALRTI). RSV viral load, Pro-inflammatory cytokine (TNF-alpha) levels in severe ALRTI patients were significantly higher than the ALRTI patients [p<0.001]. Whereas Th17 cytokine (IL-17) was found to be significantly higher (p<0.05) in ALRTI patients than severe patients. MMP-9 is secreted in higher levels in severe ALRTI patients (n = 77) in comparison to Acute LRTI patients (n = 35) with an increase of thirty seven fold (p<0.001). Thus, the study highlights the role of TNF -alpha, IL-17 and Th2 cytokine biasness in the pathogenesis of RSV disease with the possible contribution of higher MMP-9/TIMP-1 ratio as a bad prognostic marker towards disease severity. To study the gene expression of autophagy and mTOR signalling pathways in RSV infected children with ALRTI. Nasopharyngeal aspirate (NPA) samples (n = 145) from children suffering from ALRTI were subjected for detection of RSV (Oct 2019 to March 2020). Semi-quantitative gene expression analysis for 5 representative genes each of mTOR signalling and autophagy pathway were performed in respiratory tract epithelial cells using 25 RSV positive cases and 10 healthy controls subjects. Autophagy gene expression analysis revealed significant upregulation in NPC1 and ATG3 autophagy genes. mTOR, AKT1 and TSC1 genes of mTOR pathway were significantly down-regulated in RSV positive patients except RICTOR gene which was significantly upregulated. Thus, survival of RSV within autophagosome might have been facilitated by upregulation of autophagy and downregulation of mTOR signalling genes. To assess the impact of SARS-CoV2 pandemic on RSV, samples were collected from children with ALRTIs admitted to emergency, PICU and indoor admissions during pre-pandemic period (October 2019 to February 2020;n = 166) and during COVID-19 Pandemic (July 2021 to July 2022;n = 189, SARS-CoV2 negative). These NP swabs were analyzed for pdm InfA H1N1, InfA H3N2, Inf B, RSV, hMPV, hBoV, hRV, PIV-2 and PIV-3 by PCR. Higher proportion of children with ALRTIs have had virus/es isolated during pre-pandemic period than during pandemic period (p<0.001). During pre-pandemic period, significantly higher proportion of children had RSV positivity (p<0.001);and significantly lower positivity for hRV (p<0.05), hMPV (p<0.05), and hBoV (p <= 0.005). The occurrence of COVID-19 pandemic has significantly impacted the frequency and pattern of detection of RSV among hospitalized children with LRTIs. RSV Fusion protein plays a critical role in the entry of the virus into the host cell by initiating the fusion of host and viral membranes. It happens to be a target of neutralizing antibodies paving the way as a vaccine candidate. Hence effort was made to introduce point mutation in hRSV fusion protein which can confer stability in its prefusion form. In-silico a stable structure of RSV fusion protein was generated making it a potential vaccine candidate. The timely diagnosis of RSV infection in this population is important for initiating therapy and instituting appropriate infection prevention measures. Serological testing is not widely used for the diagnosis of RSV. C ll Cultures including shell vial culture were used for RSV diagnosis. However, culture approaches lack sensitivity, often quite significantly, compared to nucleic acid amplification assays for the diagnosis of RSV infections. Molecular multiplex assays now offer increased sensitivity for a more accurate diagnosis. However issues with the use of these types of commercial panel assays include the requirement for substantial training, quality systems, and infrastructure to maintain and run these assays and many a times identification of viruses where the true pathogenic potential of those multiple viruses are debatable. Studies are available with laboratory- developed nucleic acid amplification test systems for the detection of RSVA and RSVB in clinical specimens either by PCRbased technologies or RT-LAMP. Gene targets of laboratory-developed molecular assays point towards M gene and the N gene in RSVA and -B with the benefits of flexibility to modify assays when targets are under evolutionary pressure to change, as well as a perceived initial low cost to carry out testing.

Three Doses of Covid-19 Vaccines in Pwh: Immunogenicity and Effects on Hiv Reservoir

Background: People with HIV (PWH) older than age 55 have an enhanced risk of complications from SARS-CoV-2 infection. It is unclear whether COVID-19 vaccines with a booster are as durable in terms of immunogenicity in this cohort or whether these vaccines can destabilize HIV reservoirs. Method(s): We prospectively studied 91 PWH on cART aged 55 or over (n=91) and 23 age-matched individuals without HIV (control group, CG) who received three doses of COVID-19 vaccines (D1-D3) over 48 weeks. Participants received combinations of BNT162b2, mRNA-1273, and ChAdOx1. Of PWH, 42 were immune responders (IR), 20 were non-responders (INR), and 3 had a low-level viremia (LLV). Total and neutralizing Abs to SARS-CoV-2 spike (S) and RBD in sera and saliva, frequency of anti-RBD/NTD memory B cells (spectral flow cytometry), S-specific T cell immunity (IFN-g, IL-2 ELISpot) and HIV reservoirs in peripheral CD4+ T cells (IPDA) were measured. Result(s): No significant differences in vaccine regimens or dosing intervals were observed between PWH and CG. Vaccines elicited equally strong anti-S IgG in PWH vs CG in serum and saliva, and RBD IgG in serum. Serum Abs peaked at 4w after D3. Week 48 serum IgG in PWH vs CG were 916 vs 919 BAU/ mL for S (p=0.624) and 706 vs 752 for RBD (p=0.198), respectively. Week 48 median saliva S IgG: 48.1% AUC of the positive control in PWH vs 95.9% for CG (p=0.384). S IgA: 3.83 vs 20.5 in PWH vs CG (p=0.039). Median neutralizing titers post-D2 were significantly lower in PWH than in CG (NT50 82.9 vs 535, p< 0.001). However, after D3, at 48w, PWH had similar titers as CG: 309 vs 269 (p=0.745), mirroring an increase in RBD/NTD-specific B cells in PWH. Anti-S T cell cytokine responses were stronger in IR PWH after D2 and D3 than in CG. Week 48 S IL-2 responses: median 135 SFC/106 PBMC vs 43.8 (p< 0.001), but only 12.5 in INR (p=0.001 vs IR). COVID-19 vaccines did not affect the size of HIV reservoir in PWH (change in median frequency of intact proviruses from baseline: 95.0 vs 90.9, p=0.952), except in three LLV PWH (mean increase 93.7% at 48w). Conclusion(s): PWH aged 55 and over show diminished neutralizing Ab responses to SARS-CoV-2 with two vaccine doses which are 'rescued' after a booster. PWH have lower S-specific IgA in saliva after vaccination which may affect protection. Enhanced S-specific T cell immunity in PWH suggests Th1 imprinting from preexistent HIV infection. COVID-19 vaccines did not destabilize the HIV reservoir in most PWH but may pose potential risk in unsuppressed viremia.

Versatile Use of Mycobacterium indicus pranii (MIP) Vaccine

Mycobacterium indicus pranii (MIP) earlier known as Mw is a soil-borne, non-pathogenic, saprophytic and rapidly growing strain of mycobacteria. MIP is approved as a vaccine/ immunomodulator for various indications including mycobacterium infections like leprosy in humans. Its administration has resulted in satisfactory clinical improvement, accelerated bacillary clearance, and increased immune responses to Mycobacterium leprae antigens, thereby shortening the full recovery time of the patients. It also shares its antigens with M.tuberculosis. In the last decade, RCTs have been done establishing immunotherapeutic properties of MIP in the treatment of leprosy, tuberculosis, warts and experimently in leishmaniasis. Through its immune inducing and cytotoxic property, it has also proved beneficial for human use especially in treating lung cancer. The beneficial role of it is also being explored in breast, cervical, oral, liver, and bladder cancers. Various studies on MIP have shown that it has immune-modulating properties in humans. The curiosity of the human mind has led to it being tried in Covid treatment trials. The results have shown that administering MIP has lowered inflammatory markers in Covid 19 patients, promising us for it to be a potential treatment option. More RCTs with a larger sample size should be done to establish this. Cytokine storm seen in bacterial sepsis is also decreased with MIP administration. Considering the encouraging results in hastening recovery in various diseases it appears that MIP is perhaps not being exploited to its fullest potential. © 2023, Hind Kusht Nivaran Sangh (Indian Leprosy Association). All rights reserved.

Encephalitis as a post-COVID-19 complication: causation or association?

Case Report: Although the coronavirus disease 2019 (COVID-19) affects the respiratory system, neurological complications in children have been reported. Neurological manifestations in children with acute COVID-19 infection are rare and range from headaches, transverse myelitis, strokes, and encephalitis which presents as a part of Multisystem Inflammatory Syndrome in Children (MIS-C). However, encephalitis presenting post-COVID-19 in the absence of MIS-C in children has not been described. Case presentation: A 9-year-old Hispanic female with no past medical history presented with altered mental status and seizures. Associated symptoms prior to seizures included worsening headaches and vomiting. Initial labs were significant for an elevated erythrocyte sedimentation rate of 32 mm/hr, C-reactive protein of 2 mg/dL, and white blood cell (WBC) count of 28 000 cells/mcl with neutrophilia. Comprehensive metabolic panel was normal. Computed tomography of the head and urine drug screen were normal. Magnetic resonance imaging of the brain demonstrated diffusion restriction in the left frontal lobe as well as mild leptomeningeal enhancement concerning for meningoencephalitis. Lumbar puncture (LP) showed pleocytosis (WBC 169 cells/mcl, 76% neutrophils), elevated glucose 77 mg/dl, normal protein 56 mg/dl, and elevated myelin basic protein indicative of a demyelinating disease. Infectious workup was significant for a positive COVID-19 immunoglobulin (Ig) G (19.66), positive Mycoplasma pneumoniae (M. pneumoniae) IgM (0.87 units/L), with an equivocal IgG (0.11 units/L). Autoimmune workup was negative. She received dexamethasone 0.15 mg/kg/dose for 1 day, followed by methylprednisolone (10 mg/kg/dose) for 3 days and oral prednisone for 5 days resulting in significant improvement. Although CSF cultures returned negative, she received a 7-day course of doxycycline for a possible coexisting M. pneumoniae infection. Repeat LP showed improving pleocytosis, and lymphocytic predominance. Discussion: In this case report, rapid neurological recovery after administration of corticosteroids in the presence of positive COVID-19 IgG and demyelinating disease was suggestive of encephalitis presenting post- COVID-19 infection. Although M. pneumoniae can present with neurological symptoms (e.g., encephalitis), repeat titers at follow-up after recovery did not show the expected 4-fold increase in IgG, making it less likely the cause of this presentation. The proposed pathophysiology of COVID-19-mediated encephalitis includes direct invasion of the nervous system, immune-mediated cytokine response, and molecular mimicry between coronaviruses and neuronal proteins causing demyelination. The mainstay treatment includes immunomodulators such as corticosteroids, Intravenous Immunoglobulin, monoclonal antibodies (eg., rituximab), or plasma exchange. Conclusion: COVID-19 infection should be considered when evaluating a patient with meningoencephalitis or post-infectious encephalitis.

Shared and Specific Systemic Cytokine Responses in Infants Hospitalized with SARS-CoV-2 versus RSV Infection

Background. Cytokines play a major role in the immune response to viral infections, contributing to viral clearance but also mediating immunopathology following infection. We sought to define and compare systemic cytokine responses in infants hospitalized with COVID-19 versus RSV infection (RSVi). Methods. Prospective study of convenience cohort of infants hospitalized with PCR confirmed SARS-CoV-2 or RSVi, as well as pre-pandemic healthy controls (HC). Blood samples were obtained at enrollment and cytokine analysis performed using a 92-cytokine inflammation panel (Olink platform). Statistical analyses were performed in R environment. Results. We enrolled 26 infants with COVID-19, 77 with RSVi, and 18 healthy infants as a comparator control group. Oxygen requirement was significantly more frequent in infants with RSVi (p=0.02), while presence of comorbidities was significantly more common in infants with COVID-19 (p=0.01). No statistical differences were identified in terms of length of stay, admission to pediatric intensive care unit, need for mechanical ventilation, and lymphocyte counts (Table 1). Principal component analysis (PCA) revealed clustering of the global cytokine profiles differentiating HC from infants with COVID-19 and RSVi (Figure 1A). Multiple comparison analysis among the three groups yielded 49 significantly different cytokines clustered in three groups. A first cluster that included cytokines such as CCL11, CCL19 and TNFSF12 were lower in both COVID-19 and RSVi compared to HC;a second cluster with CCL8, CXCL8 and CASP8 that were mildly increased in both COVID-19 and RSVi;and a third cluster that included IL6, IL17C and IFN-gamma were markedly increased in both viral groups compared with HC (ANOVA padj< 0.05) (Figure 1B). Direct comparison between COVID-19 and RSVi (padj< 0.05 and FC >1.5) identified 7 statistically different cytokines. CCL8, CXCL1, CCL20 concentrations were increased in COVID-19, while SIRT2, STAMBP, MMP10 and EIF4EBP1 concentrations were increased in RSVi (Figure 1C-D). Conclusion. Analysis of systemic cytokine profiles identified shared but also distinct cytokine responses in infants with SARS-CoV-2 and RSVi suggesting important differences in the pathogenesis of these viral infections. (Figure Presented).

Mucosal Antiviral Cytokine Responses Are Impaired in Children and Adolescents with Severe COVID-19

Background. The factors associated with severe COVID-19 in pediatric patients remain poorly understood. We sought to determine whether mucosal innate immunity cytokines were associated with disease severity in children and adolescents with acute COVID-19. Methods. Single-center, prospective study including children and adolescents < 21 years of age hospitalized because of symptomatic COVID-19 from 3/2020 to 1/ 2021, and age, sex and race matched pre-pandemic (2016-2019) healthy controls. Nasopharyngeal (NP) samples were obtained at enrollment for measurement of SARS-CoV-2 viral loads by rt-PCR and cytokine concentrations using a 92-plex inflammation/ antiviral panel (Olink). Disease severity was assessed by the need for supplemental oxygen or PICU admission, and patients classified as severe and non-severe based on these two parameters. Statistical analyses were performed in R studio and Benjamini-Hochberg applied to adjust for multiple comparisons. Results. Of the 75 children with acute COVID-19 (median IQR age: 3.4 [0.2-15.3] years), 28 (37%) were classified as severe COVID-19 (19 PICU;25 supplemental oxygen) and 47 (63%) were non-severe. Children with severe COVID-19 were predominantly male and had an underlying condition more frequently than those with non-severe disease (79% vs 49% respectively;p< 0.01, Table 1). SARS-CoV-2 viral loads were comparable between groups, yet patients with severe COVID-19 had significantly higher concentrations of C-reactive protein (p=0.04), more frequent lymphopenia (p=0.03) and cardiac involvement (p=0.04), and received COVID-19 directed therapies more commonly (p< 0.001). Comparative analyses identified 24 cytokines that were significantly different between children with acute COVID-19 versus 45 healthy controls. Of those, concentrations of IFN-gamma (p=0.004), CXCL10 (p=0.01), CXCL11 (p=0.02) and CCL19 (p=0.02) were significantly lower in children with severe versus those with non-severe COVID-19 (Fig 1). Conclusion. Mucosal concentrations of antiviral/regulatory cytokines were decreased in children with severe COVID-19. These findings suggest that impaired mucosal innate immune responses might favor SARS-CoV-2 disease progression and severity in children. (Figure Presented).

Maternal Transmission of SARS-CoV-2-specific Antibodies, but not Cytokines/Chemokines to Neonates Following Infection and Vaccination During Pregnancy

Background. Despite extensive studies of human immune responses to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection and coronavirus disease 2019 (COVID-19) vaccination, research examining protective correlates of vertical transmission following maternal exposure in pregnancy remain limited. Here, we characterized antibody and cytokine responses in maternal and cord blood following infection or vaccination at various timepoints during gestation. Methods. Spike S1 protein-specific binding antibodies and antibodies capable of blocking the interaction between the receptor binding domain (RBD) and the angiotensin converting enzyme 2 (ACE2) were measured in maternal and cord blood by ELISA. Serum concentrations of 74 cytokines/chemokines were measured by multiplex assay. Humoral responses and cytokine levels from matched maternal and fetal cord sera were compared and examined for potential correlations. Results. We observed a highly significant correlation between Spike S1-specific antibody titer and RBD-ACE2 blocking antibody activity between maternal and fetal cord serum (p < 2.2e-16, R > 0.90). Blocking antibody activity was significantly higher for mothers infected during the 3rd trimester compared to earlier trimesters;however, vaccinated mothers developed and transferred higher antibody titers with greater RBD-ACE2 blocking antibody activity to their neonates than infected mothers. Furthermore, vaccine-induced Spike S1 IgG transfer ratios (fetal cord/maternal) were significantly higher than those induced by infection (p = 0.002). Multiplex assay showed significantly elevated levels of 33 cytokines/chemokines, mainly pro-inflammatory in infected maternal serum samples, while the paired fetal cord samples exhibited an anti-inflammatory cytokine predominance. Conclusion. Our data support selective vertical transmission of potentially protective humoral responses against SARS-CoV-2, especially following vaccination in the 3rd trimester. The anti-inflammatory cytokine predominance in cord blood that persists despite maternal SARS-CoV-2 infection may offset the adverse outcomes of inflammation in pregnancy for the neonate.

Therapeutic Targeting of Inflammation and Virus Simultaneously Ameliorates Influenza Pneumonia and Protects from Morbidity and Mortality.

Influenza pneumonia is a severe complication caused by inflammation of the lungs following infection with seasonal and pandemic strains of influenza A virus (IAV), that can result in lung pathology, respiratory failure, and death. There is currently no treatment for severe disease and pneumonia caused by IAV. Antivirals are available but are only effective if treatment is initiated within 48 h of onset of symptoms. Influenza complications and mortality are often associated with high viral load and an excessive lung inflammatory cytokine response. Therefore, we simultaneously targeted the virus and inflammation. We used the antiviral oseltamivir and the anti-inflammatory drug etanercept to dampen TNF signaling after the onset of clinical signs to treat pneumonia in a mouse model of respiratory IAV infection. The combined treatment down-regulated the inflammatory cytokines TNF, IL-1ß, IL-6, and IL-12p40, and the chemokines CCL2, CCL5, and CXCL10. Consequently, combined treatment with oseltamivir and a signal transducer and activator of transcription 3 (STAT3) inhibitor effectively reduced clinical disease and lung pathology. Combined treatment using etanercept or STAT3 inhibitor and oseltamivir dampened an overlapping set of cytokines. Thus, combined therapy targeting a specific cytokine or cytokine signaling pathway and an antiviral drug provide an effective treatment strategy for ameliorating IAV pneumonia. This approach might apply to treating pneumonia caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2).

ANCA-Associated Vasculitis Following Johnson and Johnson COVID-19 Vaccine in Nepal

Introduction: Antineutrophil cytoplasmic autoantibodies (ANCA) associated vasculitis (AAV) after influenza vaccination has been previously reported, there are a very few case reports of AAV following COVID-19 vaccination. Case Description: A 52-year-old hypertensive female presented with complaints of fever, joint pain, and weakness of all limbs. She developed symptoms 12 days after getting vaccinated with Johnson and Johnson (J & J) COVID-19 vaccine in late July 2021. The fever occurred for 10 days, intermittent type, and associated with chills and rigor. The joint pain was for 3 days which was acute in onset involving small joints of hands and feet. Concurrently she also developed weakness of all four limbs with relatively more weakness in lower limb than upper limbs. She consumed alcohol occasionally. Urinalysis revealed 12 red blood cells per high-power field (HPF), 4 white blood cells/HPF, and 0.631 gm/day protein with albumin (2+). Serologic evaluation was notable for increased C-reactive protein, decreased C3 complement level, normal C4 level, positivity for p-ANCA and c-ANCA (3+). Renal ultrasound showed tiny non-obstructive calculus in both kidneys. Following this she was started on antibiotics, NSAIDS, and methylprednisolone. She was planned for kidney biopsy and was started on cyclophosphamide. Blood transfusion was done. Renal biopsy revealed necrotizing and crescentic glomerulonephritis with insignificant glomerular immune complex deposit suggesting ANCA associated glomerulonephritis in the view of history of ANCA positivity (Figure 1). She was discharged after 10 days of hospitalization. Discussion(s): AAV following adenovirus vector vaccine has not been reported previously. There have been reports of AAV after administration of influenza vaccine. So, J&J vaccines was thought to be a potential trigger for development of AAV. The temporal causal association between autoimmune manifestations like AAV and COVID-19 vaccines can be explained by hypothesized mechanisms like molecular mimicry, defective neutrophilic apoptosis, polyclonal activation, and systemic proinflammatory cytokine response.

Combinatorial analysis reveals highly coordinated early-stage immune reactions that predict later antiviral immunity in mild COVID-19 patients.

While immunopathology has been widely studied in patients with severe COVID-19, immune responses in non-hospitalized patients have remained largely elusive. We systematically analyze 484 peripheral cellular or soluble immune features in a longitudinal cohort of 63 mild and 15 hospitalized patients versus 14 asymptomatic and 26 household controls. We observe a transient increase of IP10/CXCL10 and interferon-ß levels, coordinated responses of dominant SARS-CoV-2-specific CD4 and fewer CD8 T cells, and various antigen-presenting and antibody-secreting cells in mild patients within 3 days of PCR diagnosis. The frequency of key innate immune cells and their functional marker expression are impaired in hospitalized patients at day 1 of inclusion. T cell and dendritic cell responses at day 1 are highly predictive for SARS-CoV-2-specific antibody responses after 3 weeks in mild but not hospitalized patients. Our systematic analysis reveals a combinatorial picture and trajectory of various arms of the highly coordinated early-stage immune responses in mild COVID-19 patients.

IMMUNOSUPPRESSION SECONDARY TO SEPSIS AND POSTACUTE CARE SYNDROME LEADING TO LATENT TUBERCULOSIS REACTIVATION

CASE: A 59-year-old Mexican-American man with hypertension and type II diabetes (Hemoglobin A1c 11.5) was admitted for sepsis and Acute Respiratory Distress Syndrome secondary to COVID-19 pneumonia. He was ventilator- dependent for 66 days. His clinical course was complicated by acute renal failure requiring hemodialysis, pulmonary embolism, and recurrent ventilator-associated bacterial pneumonia. He was discharged to a long-term acute care center four months after his initial presentation, but was readmitted two weeks later for abdominal pain and fever. CT abdomen revealed diffuse mesenteric nodular stranding and pelvic ascites concerning for peritoneal carcinomatosis. Biopsy of an omental nodule, however, showed necrotizing granulomatous inflammation and no malignant cells. No cultures were sent from the initial biopsy, so repeat sampling was performed and culture was positive for Mycobacterium tuberculosis complex. Treatment for active tuberculosis was initiated with subsequent recovery. IMPACT/DISCUSSION: Initial infection by tuberculosis occurs in the lungs, where alveolar macrophages encounter and phagocytose the bacteria. The macrophages initiate a cytokine response and recruit lymphocytes to form a granuloma, which segregates the infection within the host. The granuloma is then perpetually maintained by an ongoing immune response that is driven by monocytes and CD-4 T cells. Reactivation of tuberculosis occurs when the ongoing immune response is disrupted. Sepsis has profound and complex effects on the immune system, including marked inhibition of lymphocyte proliferation that leads to reduced levels of B cells, CD-4 T cells, and follicular dendritic cells. Signaling pathways are disrupted without these lymphocytes, which then leads to the dysfunction of the remaining leukocytes. Further, critically ill patients often suffer from post-intensive care unit syndrome. This syndrome is marked by persistent inflammation, which prompts an immunosuppressive response that suppresses T-cell function and leads to T-cell apoptosis. Both sepsis and post-intensive care unit syndrome predispose patients to opportunistic infection by attenuation of the usual immune response. In this particular case, the specific loss of T-cell function in both syndromes allowed this patient's latent tuberculosis to reactivate several months after his initial presentation with sepsis from COVID-19 pneumonia. This case highlights the importance of maintaining a high index of suspicion for opportunistic infection after critical illness. CONCLUSION: Sepsis and post-intensive care unit syndrome disrupted this patient's ability to maintain the immune responses that prevent the progression of latent tuberculosis infection. The diagnosis was delayed due to a lack of awareness of the profound immunosuppression that accompanies and follows critical illness. Providers must recognize these syndromes and the impact they have on immunity in order to diagnose and treat opportunistic infections in a timely manner.

Novel Agents for Treatment of Delirium: A Proposal for a Multimodal Approach

Background: Delirium, a syndrome characterized by impairment in attention and consciousness, commonly occurs in hospital setting and is associated with higher mortality and poor long-term outcomes. With precise etiology of delirium yet to be elucidated, our current understanding describes delirium as a state of multifactorial global brain dysfunction occurring in susceptible elderly and critically ill patients. (Maldonado, 2008) To treat such a multimodal disorder, we propose investigating two novel multimodal pharmacological approaches: Granulocyte-macrophage colony stimulating factor (GM-CSF) inhibitors and pro-cholinergic muscarinic-receptor agonists. Discussion: Neuroinflammation is a focus of inquiry in a number of neuropsychiatric diseases. Unlike individual cytokine (TNF, IL-6) inhibitors, GM-CSF inhibitors combat the entire inflammatory cascade implicated in delirium: blunting the cytokine response (IL-1, IL-6, TNF) to reduce inflammation, limiting chemotaxis (IL-8 inhibition), reducing cell degradation (H2O2, MMPs), and dulling the T- and B-cell response. (Patel, 2021) GC-CSF inhibitors (otilimab and TMJ2) have already been shown to be effective in and approved for the treatment of inflammatory conditions, such as Rheumatoid Arthritis, and have been effective in treatment of inflammatory processes of COVID-19. (Patel, 2021) Given the role of the neuroinflammatory cascade in delirium, we propose investigating the GM-CSF inhibitors to treat delirium. Acetylcholine dysregulation (‘anticholinergic surge’), on the other hand, has long been implicated in delirium and studies suggest some efficacy of acetylcholinesterase inhibitors in treatment of delirium. Novel schizophrenia treatment studies combine xanomeline, a M-receptor agonist, with trospium, a peripheral anticholinergic, to create a net-positive pro-cholinergic state in the brain while minimizing systemic side-effects. (Brannan, 2020) In addition to treating schizophrenia and cognitive impairment, this combination (KarXT), may be useful in reversing the anti-cholinergic state of the delirious brain. Currently in Phase III clinical trials, KarXT should be considered a viable candidate for delirium treatment, once FDA-approved. Conclusions: Pharmacological approaches to delirium have been limited to managing behavioral dysregulation and sleep-wake cycle disturbances. Presently, we are looking at two potential additional approaches to delirium treatment. One is limited to cholinergic circuitry and aims to restore AcH balance via direct M-receptor agonism. The other, based on Systems Integration Failure Hypothesis, addresses the entire inflammatory cascade via GM-CSF inhibition. As agents from both classes are either approved or are close to FDA-approval, we should consider them as candidates for delirium management. References: 1. Maldonado, J, Kapinos, G. (2008). Pathoetiological Model of Delirium, Critical care clinics. 24. 789-856, ix. 10.1016/j.ccc.2008.06.004. 2. Brannan, S, Sawchak, S, et al.(2020). Efficacy and Safety of Xanomeline, a M1/M4 Receptor Preferencing Agonist, Plus Trospium, a Peripheral Muscarinic Antagonist, in Schizophrenia. Biological Psychiatry, 87(9), S169. doi: 10.1016/j.biopsych.2020.02.446 3. Patel, S, Saxena, B., Mehta, P. (2021). Recent updates in the clinical trials of therapeutic monoclonal antibodies targeting cytokine storm for the management of COVID-19. Heliyon, 7(2), e06158. doi: 10.1016/j.heliyon.2021.e06158

RESTRICTED INFECTION of MACROPHAGES by SARS-CoV-2 INDUCES PROINFLAMMATORY RESPONSES

Background: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has been associated with immune hyperactivation and high levels of proinflammatory cytokines. Extensive lung infiltration by CD169+ inflammatory monocytes and presence of activated CD169+ alveolar macrophages suggest monocyte/macrophages are key drivers of severe morbidity and mortality. In this study, we determined whether CD169 mediated ACE2-independent SARS-CoV-2 entry and restricted viral genome replication in macrophages triggers pro-inflammatory cytokine expression. Methods: Monocyte-derived macrophages (MDMs) and PMA-differentiated THP-1 macrophages engineered to constitutively express CD169, ACE2, or CD169 and ACE2 were infected with USA-WA1/2020/SARS-CoV-2 isolate with or without Remdesivir pre-treatment. To identify mechanism of innate immune activation, nucleic acid sensing pathways were selectively depleted in CD169+ macrophages. Extent of viral genomic (gRNA) and sub-genomic (sgRNA) expression and induction of pro-inflammatory cytokines was determined by qRT-PCR and single molecule RNA FISH analysis. Viral protein expression and infectious virus particle production was determined by immunofluorescence analysis and TCID50. Results: While productive virus infection (viral protein expression and infectious virus particle release) was only observed in ACE2+ macrophages, SARS-CoV-2 N or S expression and infectious virus production was not observed in CD169+ macrophages. Co-expression of ACE2 and CD169 significantly enhanced infectious virus production and spread. Interestingly, smFISH and RT-qPCR analysis revealed CD169+ cells express cytosolic negative-strand gRNA and positive strand sgRNA. Importantly, CD169-mediated SARS-CoV-2 infection of macrophages and expression of viral mRNAs led to induction of pro-inflammatory cytokines, IL-6, TNFα, and IL-1β, despite lack of viral protein expression in CD169+ macrophages. Pre-treatment with Remdesivir blocked de novo expression of viral mRNAs and induction of inflammatory cytokines in CD169-dependent infection of macrophages. Furthermore, knockdown of cytosolic RLRs (RIG-I and MDA-5) or MAVS significantly attenuated inflammatory cytokine expression in CD169+ macrophages, confirming that nucleic acid sensing of restricted cytosolic viral mRNA expression in macrophages triggers innate immune activation. Conclusion: These results suggest that restricted SARS-CoV-2 infection of CD169+ macrophages contributes to COVID-19-associated hyperinflammatory cytokine response.

Optimization of Lipid Nanoparticles for saRNA Expression and Cellular Activation Using a Design-of-Experiment Approach.

Lipid nanoparticles (LNPs) are the leading technology for RNA delivery, given the success of the Pfizer/BioNTech and Moderna COVID-19 mRNA (mRNA) vaccines, and small interfering RNA (siRNA) therapies (patisiran). However, optimization of LNP process parameters and compositions for larger RNA payloads such as self-amplifying RNA (saRNA), which can have complex secondary structures, have not been carried out. Furthermore, the interactions between process parameters, critical quality attributes (CQAs), and function, such as protein expression and cellular activation, are not well understood. Here, we used two iterations of design of experiments (DoE) (definitive screening design and Box-Behnken design) to optimize saRNA formulations using the leading, FDA-approved ionizable lipids (MC3, ALC-0315, and SM-102). We observed that PEG is required to preserve the CQAs and that saRNA is more challenging to encapsulate and preserve than mRNA. We identified three formulations to minimize cellular activation, maximize cellular activation, or meet a CQA profile while maximizing protein expression. The significant parameters and design of the response surface modeling and multiple response optimization may be useful for designing formulations for a range of applications, such as vaccines or protein replacement therapies, for larger RNA cargoes.

Cancer patients display diminished viral RNA clearance and altered T cell responses during SARS-CoV-2 infection

Cancer patients display immunomodulation related to malignancy and anti-cancer therapies, but how these factors impact COVID-19 remains unknown. To investigate immune responses in cancer patients with COVID-19, we undertook a prospective case-control study, enrolling hospitalized solid tumor patients with acute COVID-19, as well as age-, gender-, and comorbidity-matched COVID-19 patients without cancer as controls. Using biospecimens collected during hospitalization, we performed virologic measurements as well as in-depth immunophenotyping of cellular, antibody and cytokine responses. We enrolled 17 cancer patients (cases) admitted to Yale-New Haven Hospital between March 15 and June 30, 2020 with COVID-19, as well as 17 matched non-cancer patients (controls) admitted with COVID-19. No significant differences were observed between cases and controls based on patient characteristics (age, gender, race, co-morbidities, smoking history, days from symptom onset to COVID-19 diagnosis) or outcomes (COVID-19 severity, length of hospital stay, rate of intubation or mortality). The most common primary tumor sites were lung (4/17) and gastrointestinal (4/17);all cases had received cancer-directed therapy within 6 months of COVID-19 diagnosis, with 13/17 receiving treatment less than 1 month prior to hospitalization. Three of 17 cases had received immune checkpoint inhibitor therapies. Despite having similar SARS-CoV-2 viral RNA loads at the time of COVID-19 diagnosis when compared with controls, cancer cases had increased viral RNA abundance during hospitalization, suggesting slower clearance. Antibody responses against SARS-CoV-2 were preserved in cancer cases, with cases displaying similar levels of IgM and IgG antibodies directed against SARS-CoV-2 epitopes compared to controls. Cytokine profiling revealed higher plasma levels of CCL3, IL1A and CXCL12 in cancer cases compared to controls. Using flow cytometric immunophenotyping, we found that innate immune and non-T cell adaptive immune parameters were similar between cases and controls hospitalized with COVID-19. However, among cancer cases on conventional therapies, T cell lymphopenia was more profound, and these cases demonstrated higher levels of CD8+ exhausted (CD8+CD45RA-PD1+TIM3+ ), CD8+GranzymeB+ and CD4+CD38+HLA-DR+ and CD8+CD38+HLA-DR+ activated T cells when compared with controls;interestingly, these differences were not observed in patients who had received immune checkpoint inhibition. Thus, we found reduced viral RNA clearance and specific alterations in T cell and cytokine responses in cancer patients hospitalized with COVID-19 compared with matched controls with COVID-19. This dysregulated T cell response in cancer patients, which may reflect immune modulation due to chronic antigen stimulation as well as cancer therapies, may lead to altered virologic and clinical outcomes in this population.

Group-based multi-trajectory analysis of c-reactive protein and ferritin levels in pediatric sepsis

INTRODUCTION: Interest in using bedside C-reactive protein and ferritin levels to identify patients with hyperinflammatory sepsis who might benefit from anti-inflammatory therapies piqued with the COVID-19 pandemic experience. These widely available low-cost biomarkers might be similarly useful for assessing inflammatory profiles of all critically ill children with sepsis and septic shock and eventually guiding the use of precision anti-inflammatory therapies. We hypothesized groupbased trajectories of CRP and ferritin among critically ill children with sepsis would be associated with mortality and distinct inflammatory cytokine profiles. METHODS: Children with sepsis and organ failure from 9 pediatric intensive care units were enrolled in a prospective, observational cohort. Plasma CRP (mg/dL), ferritin (ng/mL), and 29 cytokine levels were measured at two samplings during sepsis (median Day 2 and Day 5). Group-based multi-trajectory models (GBMTM) identified groups of children with distinct patterns of CRP and ferritin. RESULTS: Two hundred and fifty-five children had at least 2 CRP and ferritin measurements. Five distinct clinical multitrajectory groups were identified with significantly different median maximum organ failures (MOF) and mortality. Group 1 had normal CRP and ferritin levels (n = 8;median MOF 2.0 [interquartile range 1.0, 2.0] and 0 % mortality);Group 2 had high CRP levels that became normal, with normal ferritin levels throughout (n = 80;median MOF 2.0 [1.0, 2.0] and 5% mortality);Group 3 had high ferritin levels alone (n=16;median MOF 2.5 [2.0, 3.0] and 6.3% mortality);Group 4 had very high CRP levels, and increased ferritin levels (n = 121;median MOF 2.0 [2.0, 4.0] and 10.7% mortality);and, Group 5 had very high CRP and very high ferritin levels (n = 30;median MOF 3.0 [2.0, 4.0] and 40% mortality). Cytokine responses differed across the 5 groups, with ferritin levels associated with macrophage inflammatory protein 1 a, and CRP levels reflective of many cytokines. CONCLUSIONS: Bedside CRP and ferritin levels can be used together to compute distinct groups of children with sepsis who have different systemic inflammation cytokine responses and mortality risks potentially targetable in clinical trials evaluating specific anti-inflammatory therapies.

T cell response to influenza vaccination remains intact in adults with congenital heart disease who underwent early thymectomy

Introduction: Thymectomy is routine during surgery for congenital heart defects to access to the heart. T cells developed in the thymus play a key role in immunity. Individuals with thymectomy in infancy have altered T cell populations suggesting early immunosenescence. Hypothesis: Adults with Congenital Heart Disease (ACHD) who underwent thymectomy in the first year of life have an altered response to influenza vaccination due to T cell immunosenescence. Methods: We recruited ACHD with early thymectomy ≤ 1 year of age (ACHD-ET;n = 12), ACHD and no thymectomy (ACHD-NT;n = 8), and healthy controls (HC;n = 14). Peripheral blood was collected prior to influenza vaccine and 4 weeks following administration. Flow cytometry of T cell subsets and intracellular cytokine staining of CD4 T cells was done following in vitro stimulation with influenza viral antigen. Results: Subject's mean age was 34 ± 10.6 years with no difference between the groups. At baseline, the median (IQR) frequency of naïve CD4 T cells was 24.7% (15.9) in ACHD-ET vs. 43.6% (16.9) in HC (P=0.01). Similarly, naïve CD8 T cells were lower with 37.5% (25.7) in ACHDET vs 62.8% (22.9) in HC (P=0.02). This also resulted in a reciprocal increase in memory CD4 and CD8 T Cells in the ACHD-ET group. The ACHD-NT was not significantly different than the other groups. The frequencies of influenza antigen-specific memory CD4 T cells expressing IFN-γ and TNF-α were significantly increased in post-vaccine blood samples compared to pre-vaccine samples across all 3 groups (P<0.05). Conclusions: ACHD-ET have a smaller population of naïve T cells, suggestive of immunosenescence. Despite this they have an equivalent cytokine response suggesting that early thymectomy does not inhibit the response to vaccination in young adulthood. Our findings support the recommendation that preventative vaccination against pathogens including influenza virus and the newly emerging SARS-CoV-2 should continue to be routinely performed in ACHD.

Identification of human CD4+ T cells specific for dominant epitopes of SARS-CoV-2 Spike and Nucleocapsid proteins with therapeutic potential

Introduction: Since December 2019, COVID-19 has spread rapidly across the world, leading to a global effort to develop vaccines and treatments. Despite extensive progress, there remains a need for treatments to bolster the immune responses in infected immunocompromised individuals, such as patients after allogeneic haematopoietic stem cell transplantation. Immunological protection against COVID-19 is mediated by both shortlived neutralising antibodies and long-lasting virus-reactive T cells. Therefore, we propose that T cell therapy may augment efficacy of current treatments. For the greatest efficacy with minimal adverse effects, it is important that any cellular therapy is designed to be as specific and directed as possible. Methods: Activation of CD4+ T cells from 18 COVID-19 patients was determined by flow cytometry, both ex vivo and after in vitro restimulation with SARS-CoV-2 Spike and Nucleocapsid antigens. Immunodominant, 15-mer peptides were identified using epitope mapping. T cells clones specific for these epitopes were further chararacterised for the sensitivity and polarisation of their cytokine responses after in vitro restimulation, by ELISA and cytometric assay. Next-generation sequencing revealed fulllength, paired T Cell Receptor (TCR) αβ sequences. Results: We identified three patients with strong CD4+ T cells to SARSCoV- 2 antigens. From these patients, 81 T cell clones specific for a selection of 9 immunodominant epitopes (7 Spike and 2 Nucleocapsid epitopes) were generated. Cytokine analysis showed that the sensitivity and polarisation of T cell responses varied depending on the specific epitope. Moreover, TCRαβ sequences revealed an epitope-dependent difference in the level of clonality. Conclusions: We provide detailed information on SARS-CoV-2-specific CD4+ T cells, including their antigen-specificity, the nature of their cytokine responses and the full sequence of their TCRαβ. These cells have the potential to direct an effective immune response in COVID-19 patients. Our results form a crucial first step towards T cell therapy. Efforts are underway to develop transgenic CD4+ T cells that express the SARS-CoV- 2-specific TCRs identified.

Role of IL-13-induced Th2 inflammation in host responses to SARSCoV-2

Background: Chronic lung inflammation affects the response to respiratory viruses such as SARS-CoV-2 in airway epithelia. Based on the pattern associated with disease endotype, airway inflammation can be simplified into type 2 and type 17. Half of asthmatics have type 2 high endotype driven by IL-13/IL-4 cytokine signaling that induces goblet cell metaplasia. Other inflammatory diseases such as cystic fibrosis, chronic bronchitis, and sarcoidosis are associated with the type 17 cytokines IL-17 and TNF-α. Recent case-control studies have suggested that asthma may protect against or at least not worsen SARS-CoV-2 infection. However, the effect of inflammation on COVID-19 outcomes is unclear. Although interferons and cytokine-driven inflammation may modulate antiviral response, epithelial remodeling might also affect susceptibility to viruses.We applied a singlecell RNA-seq approach to investigate responses to SARS-CoV-2 in primary human airway epithelia treated with inflammatory cytokines. We hypothesized that IL-13-induced type 2 inflammation and IL-17-induced type 17 inflammation would respond differently to SARS-CoV-2-infected human airway epithelia and that IL-13 would protect the epithelia from SARS-CoV-2 infection through goblet cell-secreted factors Methods: We infected primary human airway epithelia (n = 3 donors) grown at the air–liquid interface with 0.1 multiplicity of infection of SARSCoV- 2 and obtained viral titers and single-cell suspensions at 6 and 72 hours after infection. The epitheliawere pretreated with IL-13 or IL-17 plus TNF-α for a short (4 days) or long (56 days) course to differentiate the early effects of cytokine response from late goblet cell metaplasia that develops over weeks. We then performed single-cell RNA-seq to analyze viral transcripts Results: We found that IL-13, but not IL17 plus TNF-α, protected epithelia from SARS-CoV-2 at 72 hours after infection. Moreover, the protection seemed to be independent of interferons because interferon-stimulated genes, induced by short IL-13 exposure, failed to protect the epithelia from viral infection. Our analysis shows that the genes with the largest expression change in long-course IL-13-treated epithelia were mediated by the appearance of goblet cells andwere goblet-specific genes. Using our single-cell RNA-seq data, we analyzed how cytokines change response in each cell type after viral infection. We found that, when cells become infected, their response is not abnormal. Conclusion: IL-13 protects human airway epithelia from SARS-CoV-2 infection in vitro;the protective mechanism may involve secreted products from goblet cells. IL-13-treated airway epithelial cells have an otherwise normal response to SARS-CoV-2. Our findings suggest that products secreted by goblet cells may have potential therapeutic applications for respiratory viral diseases.