The immunological profile of SARS-CoV-2 infection in children is linked to clinical severity and age (preprint)

Coronavirus disease 19 (COVID-19) is clinically less severe in children, even if the large variety and degree of severity of symptoms reported in children pose a still unresolved challenge to clinicians. We performed an in-depth analysis of immunological profiles in 18 hospitalized SARS-CoV-2-infected children; results were compared to those obtained in 13 age- and sex-matched healthy controls (HC). Patients were categorized as paucisymptomatic/moderate (55.6%) or severe/critical (44.5%), according to established diagnostic criteria, and further stratified into infants (1–12 months, 39%), children (1–12 years, 44%), and adolescents (> 12 years, 17%). We assessed SARS-CoV-2-specific RBD antibodies (Ab), neutralizing antibodies (nAb) and circulating cytokines/chemokines in plasma; SARS-CoV-2-specific immune response was measured in peripheral blood mononuclear cells by gene expression and secretome analyses. Our results disclose peculiar circulating cytokine/chemokine profiles in patients sharing a similar clinical phenotype. A cluster of patients consisted of infants with severe symptoms who presented a hyperinflammatory profile, and extremely polarized antibody profiles, ranging from patients who were negative for Abs and nAbs to those who displayed very high levels of both. In a second cluster consisting of paucisymptomatic patients, a less pronounced increase of inflammatory cytokines along with an association between selected cytokines and humoral responses emerged. A third cluster still consisting of paucisymptomatic patients showed a circulating cytokine/chemokine profile which substantially overlapped with that of HC. SARS-CoV-2-stimulated production of pro-inflammatory proteins (IL-1β, IL-2, IL-4, IL-6, IL-7, IL-8, IL-17, MIP-1β, and TNF-α), as well as T lymphocytes activation and migration-specific proteins were significantly increased in SARS-CoV-2 infected children compared to HC. Our findings suggest that immune response activation is directly correlated to clinical severity and to a lesser extent to age.

Development and evaluation of low-volume tests to detect and characterise antibodies to SARS-CoV-2 (preprint)

Low-volume antibody assays can be used to track SARS-CoV-2 infection rates in settings where active testing for virus is limited and remote sampling is optimal. We developed 12 ELISAs detecting total or antibody isotypes to SARS-CoV-2 nucleocapsid, spike protein or its receptor binding domain (RBD), 3 anti-RBD isotype specific luciferase immunoprecipitation system (LIPS) assays and a novel Spike-RBD bridging LIPS total-antibody assay. We utilised pre-pandemic (n=984) and confirmed/suspected recent COVID-19 sera taken pre-vaccination rollout in 2020 (n=269). Assays measuring total antibody discriminated best between pre-pandemic and COVID-19 sera and were selected for diagnostic evaluation. In the blind evaluation, two of these assays (Spike Pan ELISA and Spike-RBD Bridging LIPS assay) demonstrated >97% specificity and >92% sensitivity for samples from COVID-19 patients taken >21 days post symptom onset or PCR test. These assays offered better sensitivity for the detection of COVID-19 cases than a commercial assay which requires 100-fold larger serum volumes. This study demonstrates that low-volume in-house antibody assays can provide good diagnostic performance, and highlights the importance of using well-characterised samples and controls for all stages of assay development and evaluation. These cost-effective assays may be particularly useful for seroprevalence studies in low and middle-income countries.

The risk of COVID-19 death is much greater and age-dependent with type I IFN autoantibodies (preprint)

SARS-CoV-2 infection fatality rate (IFR) doubles with every five years of age from childhood onward. Circulating autoantibodies neutralizing IFN-α, IFN-ω, and/or IFN-β are found in ~20% of deceased patients across age groups. In the general population, they are found in ~1% of individuals aged 20-70 years and in >4% of those >70 years old. With a sample of 1,261 deceased patients and 34,159 uninfected individuals, we estimated both IFR and relative risk of death (RRD) across age groups for individuals carrying autoantibodies neutralizing type I IFNs, relative to non-carriers. For autoantibodies neutralizing IFN-α2 or IFN-ω, the RRD was 17.0[95% CI:11.7-24.7] for individuals under 70 years old and 5.8[4.5-7.4] for individuals aged 70 and over, whereas, for autoantibodies neutralizing both molecules, the RRD was 188.3[44.8-774.4] and 7.2[5.0-10.3], respectively. IFRs increased with age, from 0.17%[0.12-0.31] for individuals <40 years old to 26.7%[20.3-35.2] for those ≥80 years old for autoantibodies neutralizing IFN-α2 or IFN-ω, and from 0.84%[0.31-8.28] to 40.5%[27.82-61.20] for the same two age groups, for autoantibodies neutralizing both molecules. Autoantibodies against type I IFNs increase IFRs, and are associated with high RRDs, particularly those neutralizing both IFN-α2 and -ω. Remarkably, IFR increases with age, whereas RRD decreases with age. Autoimmunity to type I IFNs appears to be second only to age among common predictors of COVID-19 death.

Seasonal betacoronavirus antibodies expansion post BNT161b2 vaccination associates with reduced SARS-CoV-2 VoCs neutralization (preprint)

SARS-CoV-2 vaccination is known to induce antibodies that recognize also variants of concerns (VoCs) of the virus. However, epidemiological and laboratory evidences indicate that these antibodies have a reduce neutralization ability against VoCs. We studied binding and neutralizing antibodies against the Spike RBD and S2 domains of the Wuhan-Hu-1 virus and its alpha and beta VoCs and of seasonal betacoronaviruses (HKU1 and OC43) in a cohort of 31 health care workers vaccinated with BNT162b2-Comirnaty and prospectively followed post-vaccination. The study of sequential samples collected up to 64 days post-vaccination showed that serological assays measuring IgG against Wuhan-Hu-1 antigens were a poor proxy for VoCs neutralization. In addition, in subjects who had asymptomatic or mild COVID-19 prior to vaccination the loss of nAbs following disease can be rapid and protection from re-infection post-vaccination is often no better than in naïve subjects. Interestingly, in health care workers naïve for SARS-CoV-2 infection, vaccination induced a rapid and transient reactivation of pre-existing seasonal coronaviruses IgG responses that was associated with a subsequent reduced ability to neutralize some VoCs.

Preexisting and new-onset diabetes in patients with COVID-19 pneumonia (preprint)

Objective. The aim of the current study was to compare clinical characteristics, laboratory findings and major outcomes of patients hospitalized for COVID-19 pneumonia with new-onset or preexisting diabetes. Design. A cohort of 176 adult patients with a diagnosis of pre-existing (n=112) or new-onset diabetes (n=55) and confirmed COVID-19 pneumonia was studied. Clinical outcomes and laboratory findings were analysed according to the presence of preexisting or new-onset diabetes. The time to viral clearance and the persistence of hyperglycemia were assessed during the follow-up after hospital discharge. Results. Patients with new-onset diabetes had lower BMI, significantly less comorbidities and higher levels of inflammatory markers and indicators of multi-organ injury than those with preexisting diabetes. No differences between preexisting and new onset diabetes were evident for symptoms at admission, humoral response against SARS-CoV-2 or autoantibodies to glutamic acid decarboxylase or interferon alpha-4. New-onset diabetes was independently associated with the risk of adverse clinical outcome defined as ICU admission or death (HR 2.11, 95% CI 1.34-3.31; p=0.001), even after adjustment for age, sex and other selected variables associated with COVID-19 severity. Furthermore, we documented a negative association (HR 0.661, 95% CI 0.43-1.02; p=0.063) between new-onset diabetes and the time to swab negativization. During follow-up we observed that in 30% of the patients with new-onset diabetes hyperglycemia reversed when the viral infection resolved. Conclusions. The recognition of new-onset diabetes as a specific clinical entity associated with COVID-19 pneumonia is relevant for early and appropriate patient management and close monitoring for the progression of disease severity.

Thromboembolism risk among patients with diabetes/stress hyperglycemia and COVID-19 (preprint)

A bstract Purpose Individuals with diabetes/stress hyperglycemia carry an increased risk for adverse clinical outcome in case of SARS-CoV-2 infection. The purpose of this study was to evaluate whether this risk is, at least in part, modulated by an increase of thromboembolic complications. Methods We prospectively followed 180 hospitalized patients with confirmed COVID-19 pneumonia admitted to the Internal Medicine Units of San Raffaele Hospital. Data from 11 out of 180 patients were considered incomplete and excluded from the analysis. We analysed inflammation, tissue damage biomarkers, hemostatic parameters, thrombotic events (TEs) and clinical outcome according to the presence of diabetes/stress hyperglycemia. Results Among 169 patients, 51 (30.2%) had diabetes/stress hyperglycemia. Diabetes/stress hyperglycemia and fasting blood glucose (FBG) were associated with increased inflammation and tissue damage circulating markers, higher D-dimer levels, increased prothrombin time and lower antithrombin III activity. Forty-eight venous and 10 arterial TEs were identified in 49 (29%) patients. Diabetes/stress hyperglycemia (HR 2.71, p=0.001), fasting blood glucose (HR 4.32, p<0.001) and glucose variability (HR 1.6, p < 0.009) were all associated with an increased risk of thromboembolic complication. TEs significantly increased the risk for an adverse clinical outcome only in the presence of diabetes/stress hyperglycemia (HR 3.05, p=0.01) or fasting blood glucose ≥ 7 mmol/l (HR 3.07, p=0.015). Conclusions Thromboembolism risk is higher among patients with diabetes/stress hyperglycemia and COVID-19 pneumonia and is associated to poor clinical outcome. In case of SARS-Cov-2 infection patients with diabetes/stress hyperglycemia could be considered for a more intensive prophylactic anticoagulation regimen.

Dysglyceamia after COVID-19 pneumonia: a six-month cohort study. (preprint)

Aim. The aim of this study was to understand whether the dysglyceamia associated with SARS-CoV-2 infection persists or reverts when the viral infection resolves. Methods. We analyzed fasting blood glucose (FBG) after hospital discharge in a cohort of 621 adult cases with suspected COVID-19 pneumonia. Results. At admission,, 18.8% of the patients in our cohort had pre-existing diabetes, 9.3% fasting glucose in the diabetes range without a prior diagnosis (DFG), 26% impaired fasting glucose (IFG), 44.9% normal fasting glucose (NFG), while 2% had no FBG available. FBG categories were similarly distributed in the 71 patients without confirmed COVID-19 pneumonia. During follow up (median time 6 month) FBG was available for 321 out of the 453 (70.9%) surviving patients and showed a trend to a marginal increase [from 97 (87–116) to 100 (92–114) mg/dL; p = 0.071]. Transitions between FBG categories was analysed in subjects without pre-existing diabetes (265 out of 321). We identified three groups: i) patients who maintained or improved FBG during follow-up [Group A, n = 185; from 100 (86–109) to 94 (88–99) mg/dL; p < 0.001]; ii) patients who moved from the NFG to IFG category [Group B, n = 66: from 89 (85–96) to 106 (102–113) mg/dl; p < 0.001]; iii) patients who maintained or reached DFG during follow-up [Group C, n = 14: from 114 (94–138) to 134 (126–143) mg/dl; p = 0.035]. Male sex and ICU admission during the hospitalization were more prevalent in Group C compared to Group A or B. Conclusions. Six months after the SARS-CoV-2 infection DFG was evident in only few patients who experienced severe COVID-19 pneumonia.

COVID-BioB Cohort Study: the neutralizing antibody response to SARS-CoV-2 in symptomatic COVID-19 is persistent and critical for virus control and survival. (preprint)

Understanding how antibody to SARS-CoV-2 evolve during infection may provide important insight into therapeutic approaches to prevent fatal COVID-19 illness and vaccines. Here, we profile the antibody response of 162 well-characterized COVID-19 symptomatic patients followed longitudinally for up to eight months from symptom onset. Using two newly developed assays we detect SARS-CoV-2 neutralization and antibodies binding to Spike antigens and nucleoprotein as well as to Spike S2 antigen of seasonal beta-coronaviruses, and to hemagglutinin of the H1N1 flu virus. Presence of neutralizing antibodies withing the first weeks from symptom onset correlates with time to a negative swab result (p=0.002) while lack of neutralization with an increased risk of a fatal disease outcome (HR 2.918, 95%CI 1.321-6.449; p=0.008). Neutralizing antibody titers progressively drop after 5-8 weeks but are still detectable up to 8 months in the majority of recovered patients regardless of age or co-morbidities. IgG to Spike antigens are the best correlate of neutralization. Antibody responses to seasonal coronaviruses are temporary boosted and parallel those to SARS-CoV-2 without dampening the specific response or worsening disease progression. Thus, a compromised immune response to the Spike rather than an enhanced one is a major trait of patients with critical conditions. Patients should be promptly identified and immediately start therapeutic interventions aimed at restoring their immunity.