key: cord-1005377-s8a3fcmz authors: Cotugno, Nicola; Ruggiero, Alessandra; Pascucci, Giuseppe Rubens; Bonfante, Francesco; Petrara, Maria Raffaella; Pighi, Chiara; Cifaldi, Loredana; Zangari, Paola; Bernardi, Stefania; Cursi, Laura; Santilli, Veronica; Manno, Emma Concetta; Amodio, Donato; Linardos, Giulia; Piccioni, Livia; Barbieri, Maria Antonietta; Perrotta, Daniela; Campana, Andrea; Donà, Daniele; Giaquinto, Carlo; Concato, Carlo; Brodin, Petter; Rossi, Paolo; De Rossi, Anita; Palma, Paolo title: Virological and immunological features of SARS‐COV‐2 infected children with distinct symptomatology date: 2021-07-16 journal: Pediatr Allergy Immunol DOI: 10.1111/pai.13585 sha: 59d020259037235cb19706fef28c7548d5f4afd8 doc_id: 1005377 cord_uid: s8a3fcmz BACKGROUND: Although SARS‐CoV‐2 immunizations have started in most countries, children are not currently included in the vaccination programs; thus, it remains crucial to define their anti‐SARS‐CoV‐2 immune response in order to minimize the risk for other epidemic waves. This study sought to provide a description of the virology ad anti‐SARS‐CoV‐2 immunity in children with distinct symptomatology. METHODS: Between March and July 2020, we recruited 15 SARS‐CoV‐2 asymptomatic (AS) and 51 symptomatic (SY) children, stratified according to WHO clinical classification. We measured SARS‐CoV‐2 viral load using ddPCR and qPCR in longitudinally collected nasopharyngeal swab samples. To define anti‐SARS‐CoV‐2 antibodies, we measured neutralization activity and total IgG load (DiaSorin). We also evaluated antigen‐specific B and CD8+T cells, using a labeled S1+S2 protein and ICAM expression, respectively. Plasma protein profiling was performed with Olink. RESULTS: Virological profiling showed that AS patients had lower viral load at diagnosis (p = .004) and faster virus clearance (p = .0002) compared with SY patients. Anti‐SARS‐CoV‐2 humoral and cellular response did not appear to be associated with the presence of symptoms. AS and SY patients showed similar titers of SARS‐CoV‐2 IgG, levels of neutralizing activity, and frequency of Ag‐specific B and CD8+ T cells, whereas pro‐inflammatory plasma protein profile was found to be associated with symptomatology. CONCLUSION: We demonstrated the development of anti‐SARS‐CoV‐2 humoral and cellular response with any regard to symptomatology, suggesting the ability of both SY and AS patients to contribute toward herd immunity. The virological profiling of AS patients suggested that they have lower virus load associated with faster virus clearance. While SARS-CoV-2 immunization programs have started in most countries, the achievement of herd immunity still seems far ahead. Indeed, due to different restrictive measures among countries and the lack of vaccination programs for children, the risk for second epidemic waves and health system overburden remains high. Children infected by SARS-CoV-2 usually present with a milder course of disease compared with COVID-19 adults, with a consistent proportion of being fully asymptomatic (AS). 1 Pathogenic reasons underlying such differences have been poorly defined. 2 Children play a crucial role in SARS-CoV-2 transmission and in epidemic waves, especially in school settings. [3] [4] [5] Test and trace interventions, implemented by government policies for epidemic control, may fail in the pediatric population, where AS patients range between 5% and 16% [6] [7] [8] [9] and where a consistent proportion remain undiagnosed. 10 13 on mitigating symptoms in children still needs to be antigen-specific B and CD8+T cells, using a labeled S1+S2 protein and ICAM expression, respectively. Plasma protein profiling was performed with Olink. Results: Virological profiling showed that AS patients had lower viral load at diagnosis (p = .004) and faster virus clearance (p = .0002) compared with SY patients. Anti-SARS-CoV-2 humoral and cellular response did not appear to be associated with the presence of symptoms. AS and SY patients showed similar titers of SARS-CoV-2 IgG, levels of neutralizing activity, and frequency of Ag-specific B and CD8+ T cells, whereas proinflammatory plasma protein profile was found to be associated with symptomatology. We demonstrated the development of anti-SARS-CoV-2 humoral and cellular response with any regard to symptomatology, suggesting the ability of both SY and AS patients to contribute toward herd immunity. The virological profiling of AS patients suggested that they have lower virus load associated with faster virus clearance. Ag-specific cellular response, asymptomatic patients, neutralization humoral activity, SARS-CoV-2, symptomatic patients defined. In addition, the magnitude of the inflammatory phase associated with viral infection in severe cases 14 may represent a distinctive feature of AS children compared with SY children. In the present work, we attempt to define virological and immunological characteristics of 15 AS patients compared with 51 SY patients in order to define their ability to produce anti-SARS-CoV-2 immunity. AS and SY patients were further compared with 11 SARS-CoV-2-negative (CoV-2-) patients that were enrolled for suspicion of COVID-19, but that tested negative to both nasopharyngeal swab and serology. Sixty-six SARS-CoV-2-infected children (CoV-2+) and 11 SARS-CoV-2-negative controls (CoV-2) were enrolled from March to April 2020 at Bambino Gesù Children's Hospital in Rome for the CACTUS (Immunological studies in Children AffeCTed by COVID and acUte reSpiratory diseases). The study was approved by local ethical committee, and written informed consent was obtained from all participants or legal guardians. Age, gender, and clinical and routine laboratory characteristics are described in Table 1 . Inclusion criteria for positive cases were detection of SARS-CoV-2 in nasopharyngeal (NP) swab using SARS-CoV-2 real-time reverse transcriptase-polymerase chain reaction (RT-PCR) tests (GeneXpert, Cepheid, Sunnyvale, CA; 250 copies/mL sensitivity and 100% specificity). Serology was performed as additional confirmatory test using LIAISON® SARS-CoV-2 S1/S2 IgG test (DiaSorin, Stillwater, MN, USA). CoV-2+ patients were stratified according to WHO clinical classification (https://www.who.int/ publi catio ns/i/item/WHO-2019-nCoV-clini cal-2021-1) as follows: (i) asymptomatic CoV-2+ (AS) patients without any symptoms despite confirmed SARS-CoV-2 infection that were summoned to the hospital since they belonged to the same nuclear family of symptomatic patients; and (ii) symptomatic CoV-2+ (SY) patients. Full list and timing of symptoms are specified in Tables S1 and S2. All SARS-CoV-2-infected children were admitted to the hospital. We also included 11 SARS-CoV-2-negative children with suspected SARS-CoV-2 infection, which was excluded by: (i) two consecutive nasopharyngeal swabs, performed at enrollment and after 24 h; and (ii) SARS-CoV-2 serology, performed at discharge (approximately 7-10 days after admission). Prior to therapy initiation, venous blood was collected in EDTA tubes and processed within 2 h. Plasma was isolated from blood and stored at −80°C. Peripheral blood mononuclear cells (PBMCs) were isolated by Ficoll and cryopreserved in liquid nitrogen. PBMCs and plasma used for the "acute"-phase analysis were collected on the same day of first positive SARS-CoV-2 PCR. NP swab-preserving media were stored at −80°C until use. Virological analysis was performed on NP collected at diagnosis and every 48 h up to virus clearance. Serologic analysis was performed on vials collected at diagnosis and after 10-14 days (herein referred to as "post-acute phase"). Nasopharyngeal swabs were collected by using flocked swabs in liquid-based collection and transport systems. Total nucleic acids were purified from 200 µl NP swab-preserving media and eluted in a final volume of 100 µl. Copies of SARS-CoV-2 were quantified by a home-made multiplex quantitative assay based on One-Step TA B L E 1 Continuous data were presented as mean (SD) calculated on the total number of patients, unless otherwise stated. RT-ddPCR, as previously described. [15] [16] [17] Each sample was run at least in duplicate. Results were expressed as SARS-CoV-2 copies/5 µl. Nasopharyngeal swabs were longitudinally collected from CoV- Focus-forming assay was performed as previously described. 17 Focus-forming units per ml (FFU/ml) were counted after acquisition of pictures at a high resolution of 4,800 × 9,400 dpi, on a flatbed scanner. A high-throughput PRNT method was developed and validated inhouse, as described before. 17 The serum neutralization titer was defined as the reciprocal of the highest dilution resulting in a reduction of the control plaque count >50% (PRNT50). We considered a titer of 1:10 as the seropositive threshold. This method was validated in both adults and children using pre-pandemic serum (2017-2018) isolated from age-and gender-matched individuals. 18,19 Ag-specific B cells were analyzed used a S1+S2 Spike SARS-CoV-2 PE-labeled protein as described before. 17, [20] [21] [22] Gating strategy is shown in Figure 2B . Data analyses were performed using Kaluza and included in the Appendix S1. PBMC timing of collection from first SARS-CoV-2-positive PCR is now reported in Table S2 . Following a previously validated method, 23 ICAM-1-Fc multimers were labeled in-house with polyclonal anti-human Fc-PE F(ab′)2 fragments. Full labeling and staining protocol details can be found in the Appendix S1. Pre-COVID-19 era (2017-2018) PBMC samples from healthy age-and gender-matched controls were used to set the gate for the Ag CD8 T cells. Proteins in plasma were analyzed through a multiplexed proximity ligation as described in detail before. 24 Further details are given in the Appendix S1. Statistical analyses were performed using R software (version 3.6.2) and GraphPad Prism 6 (GraphPad Software, Inc., San Diego, CA In order to define virological differences according to the symptoms, To assess whether asymptomatic children presented similar ability to induce protective and neutralizing humoral response, we quan- We also investigated SARS-CoV-2-specific cellular immunity in peripheral blood prior to any therapy initiation. We studied Ag-specific B cells gated on switched memory B cells (CD10-CD19+CD27+IgD-) (gating strategy for B-cell populations in Figure S1 ), using and in-house fluorescently labeled probe expressing S1+S2 SARS-CoV-2 proteins (gating strategy in Figure 2B ). Ag-specific B cells were detectable in both AS and SY patients in similar levels ( Figure 2C) . Also, the analysis of maturational subsets Figure S1F ). We further assessed the frequency of Ag-specific CD8+T cells at diagnosis in both groups as previously described 23 (gating strategy in Figure 2D ). The frequency of CD8+Ag-specific T cells was similar between AS and SY children in terms of both frequency ( Figure 2E ) and absolute counts (not shown). As expected, Ag-specific CD8+T cells showed an enrichment within the memory subsets with 33% effector memory (CD45RA-CCR7−) and 31% central memory (CD45RA-CCR7+) ( Figure S2A ). No differences were found between the groups in terms of distribution of Ag-specific T cell in the maturational subsets ( Figure S2B ). The cytotoxic potential of ICAM+CD8+T cells was measured after SARS-CoV-2 peptide in vitro stimulation by intracellular production of IL-2, TNF-alpha, and IFN-gamma (gating strategy in Figure S2 ). Boolean analysis showed that TNF-alpha-positive and TNF-alpha and IL-2 bifunctional ICAM+CD8+T cells were significantly higher in AS vs SY (p = 0.003 and p = 0.004, respectively) ( Figure 2F ) patients, suggesting how these cells in AS patients maintain an effective antiviral cytotoxic response. No differences in terms of frequency of total CD8+ nor maturational subsets were found among the groups ( Figure S1 ). We further evaluated natural killer (NK) frequency and distribution to define whether innate immune determinants could distinguish between patients presenting with distinct symptomatology. In line with a recent report on adult patients, 25 our analysis did not show any significant difference in the frequency of total NK cells between SY compared with asymptomatic AS CoV-2+ patients ( Figure S3 ). Our analysis further showed no difference in terms of CD56bright and CD56dim frequency. We deepened the characterization of AS and SY patients by investigating their immunological profiles at admission, using two Olink panels focused on both immune response and inflammation. PCA suggested that proteomic data could only partially define differences between AS and SY ( Figure 3A) This study provides a description of the virological and immunologi- To define the cytokine profiles of these patients and its association with clinical course of SARS-CoV-2 infection, we analyzed plasma proteome. In a recent work, 12 we demonstrated that severe COVID-19 manifestations such as the multisystem inflammatory syndrome (MIS-C) were characterized by a specific cytokine storm with unique features as compared to mild COVID-19 and Kawasaki disease. Conversely, in the present patients' cohort, which lacked of severe cases such as MIS-C, we find that proteomics could only marginally discriminate between AS and SY patients, as found in adults. 33 Further, in our cohort either NK cell frequency or CD56+CD16+ distribution was able to discriminate between AS and SY patients. In line with this, Ramaswamy et al 34 found that NK and CD8+ Tcell phenotype characteristics alone are not sufficient to define the highly symptomatic/severe cases of SARS-CoV-2 (eg, MIS-C), but a deepen study on cytotoxicity genes is required. This study presents some limitations. First, we could only include a small group of the AS patients, albeit this is a reflection of the clinical reality, which could not be resolved considering that the recruitment included only hospital admissions and not home-assisting surveillance. As previously stated, the infection onset cannot be clearly defined especially in AS children, and this could affect the viral load at diagnosis. Further longitudinal studies on larger cohorts with quantitative correlates of viral dynamics and an adult COVID-19 cohort for comparison would be crucial to confirm our observations. In conclusion, this study demonstrated that AS patients has lower viral load and associated in vitro infectivity in upper respiratory tract compared with SY children. Development of both humoral and cell-mediated immunity is not associated with symptomatology, suggesting that importantly AS patients contribute to achieve herd We would like to thank all patients and guardians who participated in the study and all the CACTUS study nurses team of the COVID-19 Center of "Bambino Gesù Children's Hospital." We also thank our laboratory manager Ilaria Pepponi for her key support and Jennifer Faudella and Giulia Neccia for their administrative assistance. The authors have no conflict of interest to declare. Conceptualization (equal); Data curation (equal); Funding acquisition (supporting) Writing-original draft (lead) Alessandra Ruggiero: Conceptualization (equal) Data curation (lead) Writing-original draft (lead) Writing-review & editing (lead) Giuseppe Rubens Pascucci: Data curation (equal). Francesco Bonfante: Data curation (supporting) Maria Raffaella Petrara: Methodology (equal) Formal analysis (supporting) Loredana Cifaldi: Data curation (equal) Writing-review & editing (support Chiara Medri: Methodology (supporting). Veronica Santilli: Data curation (equal) Donato Amodio: Data curation (equal) Daniele Donà: Data curation (equal). Carlo Giaquinto: Data curation Cactus Study Team: Data curation (equal); Project administration (supporting). Carlo Concato: Data curation (supporting) Petter Brodin: Methodology (supporting) Supervision (supporting) Writing-review & editing (supporting). Paolo Rossi: Resources (supporting) Supervision (supporting) Anita De Rossi: Funding acquisition (supporting) Supervision (supporting) Writingreview & editing (supporting). Paolo Palma: Conceptualization (lead) Funding acquisition (supporting) Supervision (lead) Writing-review & editing (lead) A retrospective cohort study of 12,306 pediatric COVID-19 patients in the United States Why is COVID-19 so mild in children? Determining the optimal strategy for reopening schools, the impact of test and trace interventions, and the risk of occurrence of a second COVID-19 epidemic wave in the UK: a modelling study The effect of school closures and reopening strategies on COVID-19 infection dynamics in the San Francisco Bay Area: a cross-sectional survey and modeling analysis. medRxiv Comprehensive and safe school strategy during COVID-19 pandemic Coronavirus Infection in Pediatric Emergency Departments Research, G. 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