The Immunological Profile of SARS-CoV-2 Infection in Children Is Linked to Clinical Severity and Age.

Coronavirus disease 19 (COVID-19) is clinically less severe in children, even if the wide variety and degree of severity of symptoms reported in children pose a still-unresolved challenge for clinicians. We performed an in-depth analysis of the immunological profiles of 18 hospitalized SARS-CoV-2-infected children, whose results were compared to those obtained from 13 age- and sex-matched healthy controls (HC). The patients were categorized as paucisymptomatic/moderate (55.6%) or severe/critical (44.5%) according to established diagnostic criteria and further stratified into the categories of infants (1-12 months), children (1-12 years), and adolescents (>12 years). We assessed SARS-CoV-2-specific RBD antibodies (Ab), neutralizing antibodies (nAb), and circulating cytokines/chemokines in the plasma, and the SARS-CoV-2-specific immune response was measured in PBMCs by gene expression and secretome analyses. Our results showed peculiar circulating cytokine/chemokine profiles among patients sharing a similar clinical phenotype. A cluster of patients consisting of infants with severe symptoms presented hyperinflammatory profiles, together with extremely polarized antibody profiles. In a second cluster consisting of paucisymptomatic patients, a less pronounced increase in the level of inflammatory cytokines, together with an association between the selected cytokines and humoral responses, was observed. A third cluster, again consisting of paucisymptomatic patients, showed a circulating cytokine/chemokine profile which overlapped with that of the HC. The SARS-CoV-2-stimulated production of pro-inflammatory proteins, T lymphocyte activation, and migration-specific proteins, were significantly increased in SARS-CoV-2-infected children compared to the HC. Our findings suggest that immune response activation in the course of SARS-CoV-2 infection in children is directly correlated with clinical severity and, to a lesser extent, age.

SARS-CoV-2 vertical transmission during the first trimester of pregnancy in asymptomatic women.

OBJECTIVES: It is now well established that in utero vertical SARS-CoV-2 transmission can occur during the late third trimester. However, little is known about other gestational ages. Recently, an increased risk of early miscarriage was reported in pregnant women who were SARS-CoV-2-positive. The objective of the current study was to evaluate the putative SARS-CoV-2 vertical transmission during the first trimester of pregnancy. DESIGN: This is an observational study on pregnant women who were SARS-CoV-2-positive during the first trimester. Fetal and syncytiotrophoblastic specimens were collected by hysterosuction from 17 pregnant women who were SARS-CoV-2-positive and voluntarily terminated the pregnancy between week 8 and 12. We investigated the viral vertical transmission using SARS-CoV-2 RNA detection in the fetus and syncytiotrophoblast by two different techniques. RESULTS: The results suggest that SARS-CoV-2 vertical transmission is indeed possible during the first trimester in asymptomatic women. Although maternal viremia was never detected, roughly 30% of the fetuses and 17% of the syncytiotrophoblasts were found to be SARS-CoV-2-positive. CONCLUSION: Indeed, SARS-CoV-2 can spread to the fetus through the syncytiotrophoblast. Concerningly, this happens in asymptomatic pregnant women as well. Possible long-term detrimental consequences on fetal development still need to be assessed. This should be taken into consideration in the management of pregnant women by implementing preventive strategies.

Pregnant Women Develop a Specific Immunological Long-Lived Memory Against SARS-COV-2.

It is well established that pregnancy induces deep changes in the immune system. This is part of the physiological adaptation of the female organism to the pregnancy and the immunological tolerance toward the fetus. Indeed, over the three trimesters, the suppressive T regulatory lymphocytes are progressively more represented, while the expression of co-stimulatory molecules decreases overtime. Such adaptations relate to an increased risk of infections and progression to severe disease in pregnant women, potentially resulting in an altered generation of long-lived specific immunological memory of infection contracted during pregnancy. How potent is the immune response against SARS-CoV-2 in infected pregnant women and how long the specific SARS-CoV-2 immunity might last need to be urgently addressed, especially considering the current vaccinal campaign. To address these questions, we analyzed the long-term immunological response upon SARS-CoV-2 infection in pregnant women from delivery to a six-months follow-up. In particular, we investigated the specific antibody production, T cell memory subsets, and inflammation profile. Results show that 80% developed an anti-SARS-CoV-2-specific IgG response, comparable with the general population. While IgG were present only in 50% of the asymptomatic subjects, the antibody production was elicited by infection in all the mild-to-critical patients. The specific T-cell memory subsets rebalanced over-time, and the pro-inflammatory profile triggered by specific SARS-CoV-2 stimulation faded away. These results shed light on SARS-CoV-2-specific immunity in pregnant women; understanding the immunological dynamics of the immune system in response to SARS-CoV-2 is essential for defining proper obstetric management of pregnant women and fine tune gender-specific vaccinal plans.

Cyclosporine A Inhibits Viral Infection and Release as Well as Cytokine Production in Lung Cells by Three SARS-CoV-2 Variants.

In December 2019, a new severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) started spreading worldwide causing the coronavirus disease 2019 (COVID-19) pandemic. The hyperactivation of the immune system has been proposed to account for disease severity and death in COVID-19 patients. Despite several approaches having been tested, no therapeutic protocol has been approved. Given that Cyclosporine A (CsA) is well-known to exert a strong antiviral activity on several viral strains and an anti-inflammatory role in different organs with relevant benefits in diverse pathological contexts, we tested its effects on SARS-CoV-2 infection of lung cells. We found that treatment with CsA either before or after infection of CaLu3 cells by three SARS-CoV-2 variants: (i) reduces the expression of both viral RNA and proteins in infected cells; (ii) decreases the number of progeny virions released by infected cells; (iii) dampens the virus-triggered synthesis of cytokines (including IL-6, IL-8, IL1α and TNF-α) that are involved in cytokine storm in patients. Altogether, these data provide a rationale for CsA repositioning for the treatment of severe COVID-19 patients. IMPORTANCE SARS-CoV-2 is the most recently identified member of the betacoronavirus genus responsible for the COVID-19 pandemic. Repurposing of available drugs has been a "quick and dirty" approach to try to reduce mortality and severe symptoms in affected patients initially, and can still represent an undeniable and valuable approach to face COVID-19 as the continuous appearance and rapid diffusion of more "aggressive"/transmissible variants, capable of eluding antibody neutralization, challenges the effectiveness of some anti-SARS-CoV-2 vaccines. Here, we tested a known antiviral and anti-inflammatory drug, Cyclosporine A (CsA), and found that it dampens viral infection and cytokine release from lung cells upon exposure to three different SARS-CoV-2 variants. Knock down of the main intracellular target of CsA, Cyclophilin A, does not phenocopy the drug inhibition of viral infection. Altogether, these findings shed new light on the cellular mechanisms of SARS-CoV-2 infection and provide the rationale for CsA repositioning to treat severe COVID-19 patients.

Immunological Characterization of HIV and SARS-CoV-2 Coinfected Young Individuals.

While the risk of SARS-CoV-2 infection and/or COVID-19 disease progression in the general population has been largely assessed, its impact on HIV-positive individuals remains unclear. We present clinical and immunological data collected in a cohort of HIV-infected young individuals during the first wave of COVID-19 pandemic. SARS-CoV-2 RNA, virus-specific antibodies, as well as the expression of factors involved in the anti-viral immune response were analyzed. Moreover, we set up an in vitro coinfection assay to study the mechanisms correlated to the coinfection process. Our results did not show any increased risk of severe COVID-19 in HIV-positive young individuals. In those subjects who contracted SARS-CoV-2 infection, an increase in IL-10 expression and production was observed. Furthermore, in the in vitro coinfection assay, we revealed a reduction in SARS-CoV-2 replication associated to an upregulation of IL-10. We speculate that IL-10 could play a crucial role in the course of SARS-CoV-2 infection in HIV-positive individuals. These results might help defining clinical management of HIV/SARS-CoV-2 co-infected young individuals, or putative indications for vaccination schedules in this population.

Unlikely SARS-CoV-2 Transmission During Vaginal Delivery.

Pregnant women display a higher risk of progression to disease and higher viral loads during infections due to their more permissive, tolerogenic immune system. However, only few studies have focused on SARS-CoV-2 intrapartum vertical transmission via vaginal secretions or faeces. The aim of this study was to investigate the presence of the virus in vaginal, rectal and blood specimens from pregnant women characterized by different COVID-19 disease severity. We enrolled 56 SARS-CoV-2-positive pregnant women, of which 46 (82%) were in the third trimester of pregnancy, 6 (10%) in the second and 4 (7%) in the first. QPCR was performed to detect the virus in vaginal and rectal swabs and in plasma samples. SARS-CoV-2 was detected in 27% of rectal swabs of pregnant women in the third trimester, while no virus particles were detected in vaginal swabs of the same patients. Furthermore, only 4% plasma samples tested positive to SARS-CoV-2. No virus was detected in newborn's nasopharyngeal swabs. Despite the low number of subjects enrolled, our data suggest that, while theoretically possible, intrapartum vaginal or orofecal SARS-CoV-2 transmission seems to be unlikely.

MiRNA Profiling in Plasma and Placenta of SARS-CoV-2-Infected Pregnant Women.

MicroRNAs are gene expression regulators associated with several human pathologies, including those generated by viral infections. Their role in SARS-CoV-2 infection and COVID-19 has been investigated and reviewed in many informative studies; however, a thorough miRNA outline in SARS-CoV-2-infected pregnant women (SIPW), at both systemic and placental levels, is missing. To fill this gap, blood and placenta biopsies collected at delivery from 15 asymptomatic SIPW were immediately analysed for: miRNA expression (n = 84) (QPCR array), antiviral/immune mRNA target expression (n = 74) (QGene) and cytokine/chemokines production (n = 27) (Multiplex ELISA). By comparing these results with those obtained from six uninfected pregnant women (UPW), we observed that, following SARS-CoV-2 infection, the transcriptomic profile of pregnant women is significantly altered in different anatomical districts, even in the absence of clinical symptoms and vertical transmission. This characteristic combination of miRNA and antiviral/immune factors seems to control both the infection and the dysfunctional immune reaction, thus representing a positive correlate of protection and a potential therapeutic target against SARS-CoV-2.

SARS-CoV-2 Entry: At the Crossroads of CD147 and ACE2.

In late 2019, the betacoronavirus SARS-CoV-2 was identified as the viral agent responsible for the coronavirus disease 2019 (COVID-19) pandemic. Coronaviruses Spike proteins are responsible for their ability to interact with host membrane receptors and different proteins have been identified as SARS-CoV-2 interactors, among which Angiotensin-converting enzyme 2 (ACE2), and Basigin2/EMMPRIN/CD147 (CD147). CD147 plays an important role in human immunodeficiency virus type 1, hepatitis C virus, hepatitis B virus, Kaposi's sarcoma-associated herpesvirus, and severe acute respiratory syndrome coronavirus infections. In particular, SARS-CoV recognizes the CD147 receptor expressed on the surface of host cells by its nucleocapsid protein binding to cyclophilin A (CyPA), a ligand for CD147. However, the involvement of CD147 in SARS-CoV-2 infection is still debated. Interference with both the function (blocking antibody) and the expression (knock down) of CD147 showed that this receptor partakes in SARS-CoV-2 infection and provided additional clues on the underlying mechanism: CD147 binding to CyPA does not play a role; CD147 regulates ACE2 levels and both receptors are affected by virus infection. Altogether, these findings suggest that CD147 is involved in SARS-CoV-2 tropism and represents a possible therapeutic target to challenge COVID-19.

CovidArray: A Microarray-Based Assay with High Sensitivity for the Detection of Sars-Cov-2 in Nasopharyngeal Swabs.

A new coronavirus (SARS-CoV-2) caused the current coronavirus disease (Covid-19) epidemic. Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) is used as the gold standard for clinical detection of SARS-CoV-2. Under ideal conditions, RT-qPCR Covid-19 assays have analytical sensitivity and specificity greater than 95%. However, when the sample panel is enlarged including asymptomatic individuals, the sensitivity decreases and false negatives are reported. Moreover, RT-qPCR requires up to 3-6 h with most of the time involved in RNA extraction from swab samples. We introduce CovidArray, a microarray-based assay, to detect SARS-CoV-2 markers N1 and N2 in the nasopharyngeal swabs. The method is based on solid-phase hybridization of fluorescently-labeled amplicons upon RNA extraction and reverse transcription. This approach combines the physical-optical properties of the silicon substrate with the surface chemistry used to coat the substrate to obtain a diagnostic tool of great sensitivity. Furthermore, we used an innovative approach, RNAGEM, to extract and purify viral RNA in less than 15 min. We correctly assigned 12 nasopharyngeal swabs, previously analyzed by RT-qPCR. Thanks to the CovidArray sensitivity we were able to identify a false-negative sample. CovidArray is the first DNA microarray-based assay to detect viral genes in the swabs. Its high sensitivity and the innovative viral RNA extraction by RNAGEM allows the reduction of both the amount of false-negative results and the total analysis time to about 2 h.

UV-C irradiation is highly effective in inactivating SARS-CoV-2 replication.

The potential virucidal effects of UV-C irradiation on SARS-CoV-2 were experimentally evaluated for different illumination doses and virus concentrations (1000, 5, 0.05 MOI). At a virus density comparable to that observed in SARS-CoV-2 infection, an UV-C dose of just 3.7 mJ/cm2 was sufficient to achieve a more than 3-log inactivation without any sign of viral replication. Moreover, a complete inactivation at all viral concentrations was observed with 16.9 mJ/cm2. These results could explain the epidemiological trends of COVID-19 and are important for the development of novel sterilizing methods to contain SARS-CoV-2 infection.

Analysis of SARS-CoV-2 vertical transmission during pregnancy.

The impact of SARS-CoV-2 infection during gestation remains unclear. Here, we analyse the viral genome on maternal and newborns nasopharyngeal swabs, vaginal swabs, maternal and umbilical cord plasma, placenta and umbilical cord biopsies, amniotic fluids and milk from 31 mothers with SARS-CoV-2 infection. In addition, we also test specific anti-SARS-CoV-2 antibodies and expression of genes involved in inflammatory responses in placentas, and in maternal and umbilical cord plasma. We detect SARS-CoV-2 genome in one umbilical cord blood and in two at-term placentas, in one vaginal mucosa and in one milk specimen. Furthermore, we report the presence of specific anti-SARS-CoV-2 IgM and IgG antibodies in one umbilical cord blood and in one milk specimen. Finally, in the three documented cases of vertical transmission, SARS-CoV-2 infection was accompanied by a strong inflammatory response. Together, these data support the hypothesis that in utero SARS-CoV-2 vertical transmission, while low, is possible. These results might help defining proper obstetric management of COVID-19 pregnant women, or putative indications for mode and timing of delivery.