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.

Vaccine Type-, Age- and Past Infection-Dependence of the Humoral Response to SARS-CoV-2 Spike S Protein.

The emergence of COVID-19 has led to a worldwide challenge for the rapid development of vaccines. Several types of safe and effective vaccines have been available in a time frame never seen before. Now that several hundred million people have been vaccinated there is an opportunity to compare vaccines in terms of protection and immune response. Here, we have applied a highly sensitive multiplexed flow cytometry method to measure simultaneously IgM, IgG1 and IgA anti-spike protein antibodies generated in response to three vaccines: ChAdOx1 (Oxford-AstraZeneca), mRNA-1273 (Moderna), and BNT162b2 (Pfizer-BioNTech). We have found that mRNA vaccines (mRNA-1273 and BNT162b2) induce a stronger humoral response, both after the first and the second dose, than the adenovirus-based ChAdOx1 vaccine. We also found that, in the elderly, antibody titers negatively correlate with the age of the donor but, also, that antibody titers remain stable for at least 6 months after complete vaccination. Finally, we found that one dose of BNT162b2 is sufficient to induce the highest antibody titers in seropositive pre-vaccination donors. We hope these data will help to guide future decisions on vaccination strategies.

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.

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.