key: cord-0953417-rb0hrhku authors: Jiménez, Daniel; Martínez-Sanz, Javier; Sainz, Talía; Calvo, Cristina; Méndez-Echevarría, Ana; Moreno, Elena; Blázquez-Gamero, Daniel; Vizcarra, Pilar; Rodríguez, Mario; Jenkins, Robert; Sánchez-Conde, Matilde; Ron, Raquel; Norman, Francesca; Moreno, Santiago; Ferrer, Manuel; Serrano-Villar, Sergio title: Differences in saliva ACE2 activity among infected and non-infected adult and pediatric population exposed to SARS-CoV-2 date: 2022-04-29 journal: J Infect DOI: 10.1016/j.jinf.2022.04.041 sha: 5c6c74e41a088cf0092225b809d10246da1dc1dc doc_id: 953417 cord_uid: rb0hrhku 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, 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 activity is lower in the pediatric population compared to adults.  Uninfected, highly exposed adults have lower ACE2 activity than those infected.  We observe an increase in ACE2 activity as the disease's severity increases. Background: Variations in the ACE2 activity in saliva could explain the striking differences of susceptibility to infection and risk of severe disease. 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, 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). activity. These findings could inform future strategies to identify at-risk individuals. Keywords: ACE2, saliva, SARS-CoV-2, susceptibility, severity Because the angiotensin-converting enzyme 2 (ACE2) is the molecular target for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) cell entry, variations in the activity of ACE2 may play a significant role in determining an individual's susceptibility to COVID-19 and disease severity 1 . ACE2 is widely expressed in different tissues, including the surface of alveolar epithelial cells and the epithelium of oral, nasal, and nasopharynx mucosa, explaining why direct person-to-person respiratory transmission is the primary means of SARS-CoV-2 transmission 2 . Reports of pathologic findings in tissue specimens of COVID-19 patients are emerging and reinforce the role of ACE2 expression and activity in disease pathogenesis 3 . ACE2 has a higher expression in the salivary gland and the oral cavity's epithelium than in the lung. Thus, saliva could be used to study the COVID-19 pathogenesis due to the ease of detecting SARS-CoV2 in these samples 4 . ACE2 expression in the lungs seems to increase with age, which might explain the higher disease severity observed in the older population with COVID-19 5 . However, it is still unclear whether ACE2 expression is a cause of increased susceptibility in older people. 6 . While children and women are assumed to have a lower incidence of infection than adult men 6,7 , the potential role of ACE2 expression has not been fully explored. Here, we hypothesized that the activity of ACE2 in saliva would correlate with lower susceptibility to SARS-CoV-2 infection and will explain, at least in part, the formidable clinical phenotype of individuals with repeated high-risk exposures to SARS-CoV-2 who did not become infected. We analyze the expression of ACE2 in saliva in different population groups across a wide age range and disease status, and we establish differences between infected people and those exposed to the virus but not infected. We analyzed data from children recruited at Hospital Universitario La Paz and adults recruited at Hospital Universitario Ramón y Cajal, between April and June 2020. We classified children as "susceptible" if they were positive for SARS-CoV-2 IgG, and as "resistant" if they had repeated high-risk exposures 8 to SARS-Cov-2 (specifically, cohabitation with parents with confirmed COVID-19), but remained IgG negative at least after eight weeks after the exposure. We considered adults as "susceptible" when Non-induced saliva samples were collected in sterile tubes and cryopreserved at -80ºC. After thawing, ACE2 activity was measured in batch through a fluorometric assay, We Table 2 ). We observed significant differences between the susceptible and resistant adult population in the adjusted model (p=0.008) but not in the pediatric population (p=0.790). Supplemental Table 3 shows the results of the adjusted multinomial model by disease severity in adults. In this work, we observe that there is a significant correlation between ACE2 activity in saliva and susceptibility to SARS-Cov-2 and COVID19 severity. 11 . Whereas no gender difference in ACE2 expression has been appreciated in salivary glands 12 , whether a differential expression of ACE2 across age explains the more benign disease observed in children remains unclear 13 . In keeping with the study hypothesis, children showed a significantly lower ACE2 activity than adults. Despite that we did not appreciate differences in resistant vs. susceptible children, the fact that all cases were either asymptomatic/mild infections or resistant could support the idea that ACE2 activity correlates with the susceptibility to SARS-CoV-2 infection. Future directions include a thorough evaluation of ACE2 activity in different anatomic compartments, and subsequent studies should further characterize the origin of this enzymatic activity by using RNA sequencing or proteomic strategies. In conclusion, 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, as well as for the development of therapeutic strategies to reduce both susceptibility and severity of COVID-19. Role of Genetic Variants and Gene Expression in the Susceptibility and Severity of COVID-19 Transmission of SARS-CoV-2: A Review of Viral, Host, and Environmental Factors Angiotensin-converting enzyme 2 (ACE2), SARS-CoV-2 and the pathophysiology of coronavirus disease 2019 (COVID-19) Oral saliva and COVID-19 SARS-CoV-2: virus dynamics and host response COVID-19 Infection and Circulating ACE2 Levels: Protective Role in Women and Children Klein Sabra L. Considering how biological sex impacts immune responses and COVID-19 outcomes Interim Operational Considerations for Public Health Management of Healthcare Workers Exposed to or with Suspected or Confirmed COVID-19: non-U.S. Healthcare Settings COVID-19: angiotensin-converting enzyme 2 (ACE2) expression and tissue susceptibility to SARS-CoV-2 infection Epithelial Cells Lining Salivary Gland Ducts Are Early Target Cells of Severe Acute Respiratory Syndrome Coronavirus Infection in the Upper Respiratory Tracts of Rhesus Macaques Angiotensin-converting enzyme 2 (ACE2) expression increases with age in patients requiring mechanical ventilation Systematic analysis of ACE2 and TMPRSS2 expression in salivary glands reveals underlying transmission mechanism caused by SARS-CoV-2 Pediatric nasal epithelial cells are less permissive to SARS-CoV-2 replication compared to adult cells We thank to all patients and healthcare workers who participated in the study. This work was supported by Instituto de Salud Carlos III (AC17/00019, PI18/00154, COV20/00349, ICI20/00058), CRUE-Supera COVID, cofinanced by the European