Background Reliable high-throughput serological assays for SARS-CoV-2 antibodies are urgently needed for the effective containment of the COVID-19 pandemic, as it is of crucial importance to understand the strength and duration of immunity after infection, and to make informed decisions concerning the activation or discontinuation of physical distancing restrictions. Methods In 184 serum samples from 130 COVID-19 patients and 54 SARS-CoV-2 negative subjects, the analytical and clinical performances of four commercially available chemiluminescent assays (Abbott SARS-Cov-2 IgG, Roche Elecsys anti-SARS-CoV-2, Ortho SARS-CoV-2 total and IgG) and one enzyme-linked immunosorbent assay (Diesse ENZY-WELL SARS-CoV-2 IgG) were evaluated and compared with the neutralization activity achieved using the plaque reduction neutralization test (PRNT). Findings Precision results ranged from 0.9% to 11.8% for all assays. Elecsys anti-SARS-CoV-2 demonstrated linearity of results at concentrations within the cut-off value. Overall, sensitivity ranged from 78.5 to 87.7%, and specificity, from 97.6 to 100%. On limiting the analysis to samples collected 12 days after onset of symptoms, the sensitivity of all assays increased, the highest value (95.2%) being obtained with VITRO Anti-SARS-CoV-2 Total and Architect SARS-CoV-2 IgG. The strongest PRNT 50 correlation with antibody levels was obtained with ENZY-Well SARS-CoV-2 IgG (R 2 adj = 0.569). Interpretation The results confirmed that all immunoassays had an excellent specificity, whereas sensitivity varied across immunoassays, depending strongly on the time interval between symptoms onset and sample collection. Further studies should be conducted to achieve a stronger correlation between antibody measurement and PRNT 50 in order to obtain useful information for providing a better management of COVID-19 patients, effective passive antibody therapy, and developing a vaccine against the SARS-CoV-2 virus. Funding None.

As the global COVID-19 pandemic progresses, it is paramount to gain knowledge on adaptive immunity to SARS-CoV-2 in children to define immune correlates of protection upon immunization or infection. We analyzed anti-SARS-CoV-2 antibodies and their neutralizing activity (PRNT) in 66 COVID-19-infected children at 7 (±2) days after symptom onset. Individuals with specific humoral responses presented faster virus clearance and lower viral load associated with a reduced in vitro infectivity. We demonstrated that the frequencies of SARS-CoV-2-specific CD4 + CD40L + T cells and Spike-specific B cells were associated with the anti-SARS-CoV-2 antibodies and the magnitude of neutralizing activity. The plasma proteome confirmed the association between cellular and humoral SARS-CoV-2 immunity, and PRNT + patients show higher viral signal transduction molecules (SLAMF1, CD244, CLEC4G). This work sheds lights on cellular and humoral anti-SARS-CoV-2 responses in children, which may drive future vaccination trial endpoints and quarantine measures policies.

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COVID-19 typically manifests as a respiratory illness, but several clinical reports have described gastrointestinal symptoms. This is particularly true in children in whom gastrointestinal symptoms are frequent and viral shedding outlasts viral clearance from the respiratory system. These observations raise the question of whether the virus can replicate within the stomach. Here we generate gastric organoids from fetal, pediatric, and adult biopsies as in vitro models of SARS-CoV-2 infection. To facilitate infection, we induce reverse polarity in the gastric organoids. We find that the pediatric and late fetal gastric organoids are susceptible to infection with SARS-CoV-2, while viral replication is significantly lower in undifferentiated organoids of early fetal and adult origin. We demonstrate that adult gastric organoids are more susceptible to infection following differentiation. We perform transcriptomic analysis to reveal a moderate innate antiviral response and a lack of differentially expressed genes belonging to the interferon family. Collectively, we show that the virus can efficiently infect the gastric epithelium, suggesting that the stomach might have an active role in fecal-oral SARS-CoV-2 transmission.

BackgroundThe immune response plays a pivotal role in dictating the clinical outcome in severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-infected adults, but it is still poorly investigated in the pediatric population.MethodsOf 209 enrolled subjects, 155 patients were confirmed by PCR and/or serology as having coronavirus disease 2019 (COVID-19). Blood samples were obtained at a median of 2.8 (interquartile, 2.1–3.7) and 6.1 (5.3–7.2) months after baseline (symptom onset and/or first positive virus detection). The immune profiles of activation, senescence, exhaustion, and regulatory cells were analyzed by flow cytometry. Neutralizing antibodies (nAbs) were detected by a plaque reduction neutralization test. In available nasopharyngeal swabs at baseline, SARS-CoV-2 levels were quantified by digital droplet PCR (ddPCR).ResultsOverall, COVID-19 patients had higher levels of immune activation, exhaustion, and regulatory cells compared to non-COVID-19 subjects. Within the COVID-19 group, activated and senescent cells were higher in adults than in children and inversely correlated with the nAbs levels. Conversely, Tregs and Bregs regulatory cells were higher in COVID-19 children compared to adults and positively correlated with nAbs. Higher immune activation still persisted in adults after 6 months of infection, while children maintained higher levels of regulatory cells. SARS-CoV-2 levels did not differ among age classes.ConclusionsAdults displayed higher immune activation and lower production of anti-SARS-CoV-2 nAbs than children. The different immune response was not related to different viral load. The higher expression of regulatory cells in children may contribute to reduce the immune activation, thus leading to a greater specific response against the virus.

Objectives SARS-CoV-2 serology presents an important role in several aspects of COVID-19 pandemic. Immunoassays performances have to be accurately evaluated and correlated with neutralizing antibodies. We investigated the analytical and clinical performances of a SARS-CoV-2 RBD IgG assay, automated on a high throughput platform, and the correlation of the antibodies (Ab) levels with the plaque reduction neutralization (PRNT50) Ab titers. Methods A series of 546 samples were evaluated by SARS-CoV-2 RBD IgG assay (Snibe diagnostics), including 171 negative and 168 positive SARS-CoV-2 subjects and a further group of 207 subjects of the COVID-19 family clusters follow-up cohort. Results Assay imprecision ranged from 3.98 to 12.18% being satisfactory at low and medium levels; linearity was excellent in all the measurement range. Considering specimens collected after 14 days post symptoms onset, overall sensitivity and specificity were 99.0 and 92.5%, respectively. A total of 281 leftover samples results of the PRNT50 test were available. An elevated correlation was obtained between the SARS-CoV-2 RBD IgG assay and the PRNT50 titer at univariate (ρ=0.689) and multivariate (ρ=0.712) analyses. Conclusions SARS-CoV-2 S-RBD IgG assay shows satisfactory analytical and clinical performances, and a strong correlation with sera neutralizing activity.

Helicobacter pylori (Hp) is a Gram-negative bacterium that infects the human gastric mucosa, leading to chronic inflammation. If not eradicated with antibiotic treatment, the bacterium persists in the human stomach for decades increasing the risk to develop chronic gastritis, gastroduodenal ulcer, and gastric adenocarcinoma. The lifelong persistence of Hp in the human stomach suggests that the host response fails to clear the infection. It has been recently shown that during Hp infection phagocytic cells promote high Hp loads rather than contributing to bacterial clearance. Within these cells Hp survives in “megasomes,” large structures arising from homotypic fusion of phagosomes, but the mechanism that Hp employs to avoid phagocytic killing is not completely understood. Here, we show that Hp infection induces the downregulation of specific microRNAs involved in the regulation of transcripts codifying for inflammatory proteins. miR-4270 targets the most upregulated gene: the immune receptor CD300E, whose expression is strictly dependent on Hp infection. CD300E engagement enhances the pro-inflammatory potential of macrophages, but in parallel it affects their ability to express and expose MHC class II molecules on the plasma membrane, without altering phagocytosis. This effect compromises the possibility for effector T cells to recognize and activate the killing potential of macrophages, which, in turn would become a survival niche for the bacterium. Taken together, our data add another piece to the complicate puzzle represented by the long-life coexistence between Hp and the human host and contribute with new insights toward understanding the regulation and function of the immune receptor CD300E.

Objectives mRNA vaccines, including Comirnaty (BNT162b2 mRNA, BioNTech-Pfizer), elicit high IgG and neutralizing antibody (NAb) responses after the second dose, but the progressive decrease in serum antibodies against SARS-CoV-2 following vaccination have raised questions concerning long-term immunity, decreased antibody levels being associated with breakthrough infections after vaccination, prompting the consideration of booster doses. Methods A total number of 189 Padua University-Hospital healthcare workers (HCW) who had received a second vaccine dose were asked to collect serum samples for determining Ab at 12 (t12) and 28 (t28) days, and 6 months (t6m) after their first Comirnaty/BNT162b2 inoculation. Ab titers were measured with plaque reduction neutralization test (PRNT), and three chemiluminescent immunoassays, targeting the receptor binding domain (RBD), the trimeric Spike protein (trimeric-S), and surrogate viral neutralization tests (sVNT). Results The median percentages (interquartile range) for decrease in antibodies values 6 months after the first dose were 86.8% (67.1–92.8%) for S-RBD IgG, 82% (58.6–89.3%) for trimeric-S, 70.4% (34.5–86.4%) for VNT-Nab, 75% (50–87.5%) for PRNT50 and 75% (50–93.7%) for PRNT90. At 6 months, neither PRNT titers nor VNT-Nab and S-RBD IgG bAb levels correlated with age (p=0.078) or gender (p=0.938), while they were correlated with previous infection (p<0.001). Conclusions After 6 months, a method-independent reduction of around 90% in anti-SARS-CoV-2 antibodies was detected, while no significant differences were found between values of males and females aged between 24 and 65 years without compromised health status. Further efforts to improve analytical harmonization and standardization are needed.