Diversity of immune responses in children highly exposed to SARS-CoV-2

Background Children are less susceptible than adults to symptomatic COVID‐19 infection, but very few studies addressed their underlying cause. Moreover, very few studies analyzed why children highly exposed to the virus remain uninfected. Methods We analyzed the serum levels of ACE2, angiotensin II, anti-spike and anti-N antibodies, cytokine profiles, and virus neutralization in a cohort of children at high risk of viral exposure, cohabiting with infected close relatives during the lockdown in Spain. Results We analyzed 40 children who were highly exposed to the virus since they lived with severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2)-infected relatives during the lockdown for several months without taking preventive measures. Of those, 26 reported mild or very mild symptoms. The induced immune response to the virus was analyzed 3 months after the household infection. Surprisingly, only 15 children had IgG anti-S (IgG+) determined by a sensitive method indicative of a past infection. The rest, negative for IgG anti-N or S in various tests, could be further subdivided, according to IgM antibodies, into those having IgM anti-S and IgM anti-N (IgG−IgMhigh) and those having only IgM anti-N (IgG−IgMlow). Interestingly, those two subgroups of children with IgM antibodies have strikingly different patterns of cytokines. The IgMhigh group had significantly higher IFN-α2 and IFN-γ levels as well as IL-10 and GM-CSF than the IgMlow group. In contrast, the IgMlow group had low levels of ACE2 in the serum. Both groups have a weaker but significant capacity to neutralize the virus in the serum than the IgG+ group. Two children were negative in all immunological antibody tests. Conclusions A significant proportion of children highly exposed to SARS-CoV-2 did not develop a classical adaptive immune response, defined by the production of IgG, despite being in close contact with infected relatives. A large proportion of those children show immunological signs compatible with innate immune responses (as secretion of natural antibodies and cytokines), and others displayed very low levels of the viral receptor ACE2 that may have protected them from the virus spreading in the body despite high and constant viral exposure.

Diversity of immune responses in children highly exposed to SARS-CoV-2.

Background: Children are less susceptible than adults to symptomatic COVID-19 infection, but very few studies addressed their underlying cause. Moreover, very few studies analyzed why children highly exposed to the virus remain uninfected. Methods: We analyzed the serum levels of ACE2, angiotensin II, anti-spike and anti-N antibodies, cytokine profiles, and virus neutralization in a cohort of children at high risk of viral exposure, cohabiting with infected close relatives during the lockdown in Spain. Results: We analyzed 40 children who were highly exposed to the virus since they lived with severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2)-infected relatives during the lockdown for several months without taking preventive measures. Of those, 26 reported mild or very mild symptoms. The induced immune response to the virus was analyzed 3 months after the household infection. Surprisingly, only 15 children had IgG anti-S (IgG+) determined by a sensitive method indicative of a past infection. The rest, negative for IgG anti-N or S in various tests, could be further subdivided, according to IgM antibodies, into those having IgM anti-S and IgM anti-N (IgG-IgMhigh) and those having only IgM anti-N (IgG-IgMlow). Interestingly, those two subgroups of children with IgM antibodies have strikingly different patterns of cytokines. The IgMhigh group had significantly higher IFN-α2 and IFN-γ levels as well as IL-10 and GM-CSF than the IgMlow group. In contrast, the IgMlow group had low levels of ACE2 in the serum. Both groups have a weaker but significant capacity to neutralize the virus in the serum than the IgG+ group. Two children were negative in all immunological antibody tests. Conclusions: A significant proportion of children highly exposed to SARS-CoV-2 did not develop a classical adaptive immune response, defined by the production of IgG, despite being in close contact with infected relatives. A large proportion of those children show immunological signs compatible with innate immune responses (as secretion of natural antibodies and cytokines), and others displayed very low levels of the viral receptor ACE2 that may have protected them from the virus spreading in the body despite high and constant viral exposure.

Differences in saliva ACE2 activity among infected and non-infected adult and pediatric population exposed to SARS-CoV-2.

BACKGROUND: Variations in the ACE2 activity in saliva could explain the striking differences of susceptibility to infection and risk of severe disease. METHODS: We analyze the activity of ACE2 in saliva in different population groups across a wide age range and disease status during April to June 2020, before SARS-CoV-2 vaccine implementation, and we establish differences between infected people and participants considered resistant (highly exposed healthcare workers and children who cohabited with parents with COVID-19 without isolation and remain IgG negative). RESULTS: We included 74 adults, of which 47 (64%) were susceptible and 27 (36%) were resistant, and 79 children, of which 41 (52%) were susceptible and 38 (48%) were resistant. Resistant adults have significantly lower ACE2 activity in saliva than susceptible adults and non-significant higher values than susceptible and resistant children. ACE2 activity is similar in the susceptible and resistant pediatric population (p = 0.527). In contrast, we observe an increase in activity as the disease's severity increases among the adult population (mild disease vs. severe disease, 39 vs. 105 FU, p = 0.039; severe disease vs. resistant, 105 vs. 31 FU, p < 0.001). CONCLUSIONS: using an enzymatic test, we show that ACE2 activity in saliva correlates with the susceptibility to SARS-Cov-2 infection and disease severity. Children and adults with low-susceptibility to SARS-Cov-2 infection showed the lowest ACE2 activity. These findings could inform future strategies to identify at-risk individuals.

ACE2 Serum Levels as Predictor of Infectability and Outcome in COVID-19.

Background: COVID-19 can generate a broad spectrum of severity and symptoms. Many studies analysed the determinants of severity but not among some types of symptoms. More importantly, very few studies analysed patients highly exposed to the virus that nonetheless remain uninfected. Methods: We analysed serum levels of ACE2, Angiotensin II and anti-Spike antibodies in 2 different cohorts at high risk of viral exposure, highly exposed but uninfected subjects, either high risk health care workers or persons cohabiting with infected close relatives and seropositive patients with symptoms. We tested the ability of the sera of these subjects to neutralize lentivirus pseudotyped with the Spike-protein. Results: We found that the serum levels of ACE2 are significantly higher in highly exposed but uninfected subjects. Moreover, sera from this seronegative persons can neutralize SARS-CoV-2 infection in cellular assays more strongly that sera from non-exposed negative controls eventhough they do not have anti-CoV-2 IgG antibodies suggesting that high levels of ACE2 in serum may somewhat protect against an active infection without generating a conventional antibody response. Finally, we show that among patients with symptoms, ACE2 levels were significantly higher in infected patients who developed cutaneous as compared with respiratory symptoms and ACE2 was also higher in those with milder symptoms. Conclusions: These findings suggest that soluble ACE2 could be used as a potential biomarker to predict SARS-CoV-2 infection risk and to discriminate COVID-19 disease subtypes.

Metabolic Snapshot of Plasma Samples Reveals New Pathways Implicated in SARS-CoV-2 Pathogenesis.

Despite the scientific and human efforts to understand COVID-19, there are questions still unanswered. Variations in the metabolic reaction to SARS-CoV-2 infection could explain the striking differences in the susceptibility to infection and the risk of severe disease. Here, we used untargeted metabolomics to examine novel metabolic pathways related to SARS-CoV-2 susceptibility and COVID-19 clinical severity using capillary electrophoresis coupled to a time-of-flight mass spectrometer (CE-TOF-MS) in plasma samples. We included 27 patients with confirmed COVID-19 and 29 healthcare workers heavily exposed to SARS-CoV-2 but with low susceptibility to infection ("nonsusceptible"). We found a total of 42 metabolites of SARS-CoV-2 susceptibility or COVID-19 clinical severity. We report the discovery of new plasma biomarkers for COVID-19 that provide mechanistic explanations for the clinical consequences of SARS-CoV-2, including mitochondrial and liver dysfunction as a consequence of hypoxemia (citrulline, citric acid, and 3-aminoisobutyric acid (BAIBA)), energy production and amino acid catabolism (phenylalanine and histidine), and endothelial dysfunction and thrombosis (citrulline, asymmetric dimethylarginine (ADMA), and 2-aminobutyric acid (2-AB)), and we found interconnections between these pathways. In summary, in this first report several metabolic pathways implicated in SARS-CoV-2 susceptibility and COVID-19 clinical progression were found by CE-MS based metabolomics that could be developed as biomarkers of COVID-19.

Role of ACE2 genetic polymorphisms in susceptibility to SARS-CoV-2 among highly exposed but non infected healthcare workers.

We aim to evaluate the role of single-nucleotide polymorphisms of the angiotensin-converting enzyme 2 in susceptibility to SARS-CoV-2 infection. We included 28 uninfected but highly exposed healthcare workers and 39 hospitalized patients with COVID-19. Thirty-five SNPs were rationally selected. Two variants were associated with increased risk of being susceptible to SARS-CoV-2: the minor A allele in the rs2106806 variant (OR 3.75 [95% CI 1.23-11.43]) and the minor T allele in the rs6629110 variant (OR 3.39 [95% CI 1.09-10.56]). Evaluating the role of genetic variants in susceptibility to SARS-CoV-2 infection could help identify more vulnerable individuals and suggest potential drug targets for COVID-19 patients.

Flow cytometry multiplexed method for the detection of neutralizing human antibodies to the native SARS-CoV-2 spike protein.

A correct identification of seropositive individuals for the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) infection is of paramount relevance to assess the degree of protection of a human population to present and future outbreaks of the COVID-19 pandemic. We describe here a sensitive and quantitative flow cytometry method using the cytometer-friendly non-adherent Jurkat T-cell line that stably expresses the full-length native spike "S" protein of SARS-CoV-2 and a truncated form of the human EGFR that serves a normalizing role. S protein and huEGFRt coding sequences are separated by a T2A self-cleaving sequence, allowing to accurately quantify the presence of anti-S immunoglobulins by calculating a score based on the ratio of fluorescence intensities obtained by double-staining with the test sera and anti-EGFR. The method allows to detect immune individuals regardless of the result of other serological tests or even repeated PCR monitoring. As examples of its use, we show that as much as 28% of the personnel working at the CBMSO in Madrid is already immune. Additionally, we show that anti-S antibodies with protective neutralizing activity are long-lasting and can be detected in sera 8 months after infection.

Effects of tocilizumab on mortality in hospitalized patients with COVID-19: a multicentre cohort study.

OBJECTIVES: Tocilizumab has been proposed as a candidate therapy for patients with severe coronavirus disease 2019 (COVID-19), especially among those with higher systemic inflammation. We investigated the association between receipt of tocilizumab and mortality in a large cohort of hospitalized patients. METHODS: In this cohort study of patients hospitalized with COVID-19 in Spain, the primary outcome was time to death and the secondary outcome time to intensive care unit (ICU) admission or death. We used inverse probability weighting to fit marginal structural models adjusted for time-varying covariates to determine the causal relationship between receipt of tocilizumab and outcome. RESULTS: Data from 1229 patients were analysed, with 261 patients (61 deaths) in the tocilizumab group and 969 patients (120 deaths) in the control group. In the adjusted marginal structural models, a significant interaction between receipt of tocilizumab and high C-reactive protein (CRP) levels was detected. Tocilizumab was associated with decreased risk of death (adjusted hazard ratio 0.34, 95% confidence interval 0.16-0.72, p 0.005) and ICU admission or death (adjusted hazard ratio 0.39, 95% confidence interval 0.19-0.80, p 0.011) among patients with baseline CRP >150 mg/L but not among those with CRP ≤150 mg/L. Exploratory subgroup analyses yielded point estimates that were consistent with these findings. CONCLUSIONS: In this large observational study, tocilizumab was associated with a lower risk of death or ICU admission or death in patients with higher CRP levels. While the results of ongoing clinical trials of tocilizumab in patients with COVID-19 will be important to establish its safety and efficacy, our findings have implications for the design of future clinical trials.

Understanding clinical decision-making during the COVID-19 pandemic: A cross-sectional worldwide survey.

BACKGROUND: The lack of evidence-based recommendations for therapeutic decisions during the early weeks of the COVID-19 pandemic creates a unique scenario of clinical decision making which is worth to analyze. We aim to identify the drivers of therapeutic aggressiveness during the first weeks of the COVID-19 pandemic. METHODS: This cross-sectional worldwide survey (conducted April 12 to 19, 2020) was aimed at physicians who managed patients diagnosed with COVID-19. Treatment preferences were collected in five different clinical scenarios. We used multilevel mixed-effects ordered logistic regression to identify variables that were associated with the use of more aggressive therapies. FINDINGS: The survey was completed by 852 physicians from 44 different specialties and 29 countries. The heterogeneity of therapeutic decisions increased as the clinical scenario worsened. Factors associated with aggressive therapeutic decisions were higher self-perceived expertise (high vs. null, OR 1.95, 95%CI 1.31-2.89), perceived quality of COVID-19 publications (high vs. null, OR 1.92, 95%CI 1.17-3.16), and female sex (OR 1.17, 95%CI 1.02-1.33). Conversely, Infectious Diseases specialty, Latin American and North American origin, lower confidence in the treatments chosen, and having published articles indexed in PubMed as the first-author were associated with the use of less aggressive therapies. INTERPRETATION: Our study provides insight into the drivers of the decision-making process during a new and extreme health emergency. Different factors including the perceived expertise and quality of publications, gender, geographic origin, medical specialty and implication in medical research influenced this process. The clinical severity attenuated the physician's tolerance for uncertainty. FUNDING: No funding was required.

Possible role of HLA class-I genotype in SARS-CoV-2 infection and progression: A pilot study in a cohort of Covid-19 Spanish patients.

Human Leukocyte Antigen (HLA) includes a large set of genes with important actions in immune response against viral infection. Numerous studies have revealed the existence of significant associations between certain HLA alleles and the susceptibility and prognosis of different infectious diseases. In this pilot study we analyse the binding affinity between 66 class I HLA alleles and SARS-CoV-2 viral peptides, and its association with the severity of the disease. A total of 45 Spanish patients with mild, moderate and severe SARS-CoV-2 infection were typed for HLA class I; after that, we analysed if an in silico model of HLA I-viral peptide binding affinity and classical HLA supertypes could be correlated to the severity of the disease. Our results suggest that patients with mild disease present Class I HLA molecules with a higher theoretical capacity for binding SARS-Cov-2 peptides and showed greater heterozygosity when comparing them with moderate and severe groups. In this regard, identifying HLA-SARS-CoV-2 peptides binding differences between individuals would help to clarify the heterogeneity of clinical responses to the disease and will also be useful to guide a personalized treatment according to its particular risk.