Early seasonal coronavirus seroconversion did not produce cross-protective SARS-CoV-2 antibodies

•Seropositivity to endemic human coronaviruses (HCoV) differs by age.•Seropositivity rises in early life, increases in adolescence; declines in adulthood.•No cross neutralization of SARS-CoV-2 despite high seropositivity to endemic HCoV. To the Editor, We read with interest a recent article by Aran et al. on the reduced odds of SARS-CoV-2 infection among those with a recent diagnosis of common cold.1Aran D. Beachler D.C. Lanes S. Overhage J.M. Prior presumed coronavirus infection reduces COVID-19 risk: a cohort study.J Infect. 2020; 81: 923-930Abstract Full Text Full Text PDF PubMed Scopus (23) Google Scholar Endemic human coronaviruses (HCoV) are common causes of respiratory infection and typically cause mild illnesses in young children. Most epidemiological studies of HCoV infections have focused on acute infections in young children2Principi N. Bosis S. Esposito S. Effects of coronavirus infections in children.Emerg Infect Dis. 2010; 16: 183-188Crossref PubMed Scopus (68) Google Scholar although infections in adults have been reported.3Walsh E.E. Shin J.H. Falsey A.R. Clinical impact of human coronaviruses 229E and OC43 infection in diverse adult populations.J Infect Dis. 2013; 208: 1634-1642Crossref PubMed Scopus (113) Google Scholar Natural immunity to HCoV infection is believed to wane with age, and re-infections have been reported although without confirmation by molecular epidemiology.4Edridge A.W.D. Kaczorowska J. Hoste A.C.R. et al.Seasonal coronavirus protective immunity is short-lasting.Nat Med. 2020; 26: 1691-1693Crossref PubMed Scopus (427) Google Scholar The prevalence of HCoV seropositivity among young and older age groups in the general population is not well studied. Interest in immunological responses to HCoVs has been triggered by age-related disparities in the severity and prevalence of the novel human coronavirus SARS-CoV-2. COVID-19 infection at advanced age is strongly associated with increased risk of severe disease and death while pediatric COVID-19 infection has been consistently associated with clinically milder disease phenotypes.5Warner S. Richter A. Stamataki Z. Kelly D. Understanding COVID-19: are children the key?.BMJ Paediatr Open. 2021; 5e001063Crossref PubMed Scopus (11) Google Scholar We thus hypothesized that seropositivity to endemic coronaviruses such as HCoV-OC43, NL63, 229E and HKU1 increases in young adults due to cumulative exposure to seasonal coronaviruses but declines in late adulthood, and that loss of cross-protective neutralizing antibodies may explain the increased risk of severe disease in advanced adulthood. We also hypothesized that multipositivity (seropositive to 2 or more HCoVs) may confer greater cross protection against SARS-CoV-2 compared to monopositivity. 259 de-identified banked serum samples from Singaporean institutional ethics review board approved studies collected from non-infected healthy individuals across three different age groups were studied: 139 1-year-old infants (2004–2008); 57 adolescents aged 17–21 (2018); and 63 adults aged 22-62 (2011–2013). All sera were screened for antibodies to endemic CoVs HCoV-229E, HCoV-NL63, HCoV-OC43 and HCoV-HKU1 through their binding affinity to individual HCoV-specific spike S1 protein subunit antigens (Sino Biological, Beijing, China) in enzyme-linked immunosorbent assays performed according to manufacturer's instructions. Ethics approval was obtained from the institutional review board (NHG DSRB Ref: 2020/00304). The optical density (OD) of the final solutions were measured in terms of absorbance which was proportionate to the amount of seasonal HCoV IgG present in the original sera. Anti-human serum albumin (HSA) levels were used as a baseline to determine cutoffs for seasonal HCoV IgG antibody levels present in each sample. As HSA is the most plentiful protein found in human plasma, this was regarded as the baseline and any IgG antibodies beyond this range were determined to be positively detected. A validated surrogate viral neutralization assay6Tan C.W. Chia W.N. Qin X. et al.A SARS-CoV-2 surrogate virus neutralization test based on antibody-mediated blockage of ACE2–spike protein–protein interaction.Nat Biotechnol. 2020; 38: 1073-1078Crossref PubMed Scopus (696) Google Scholar (sVNT) to epidemic HCoVs (SARS-CoV, SARS-CoV-2 and MERS-CoV) was performed on all samples to assess cross-neutralizing capabilities. There were high seropositivity rates to seasonal HCoVs overall (Fig. 1). In the infant age group, 55% of samples were seropositive, of which 35·3% were positive to OC43 followed by NL63 (30·9%), HKU1 (26·6%) and 229E (20·9%). 39·6% of these samples were multi-positive and 2·9% of samples had positive serology to all four endemic HCoVs. Among the adolescents, 96% were seropositive to at least one HCoV (Table 1). In contrast to the infant age group, NL63 was the most common HCoV identified (93%), followed by OC43 (71·9%), 229E (68·4%) and HKU1 (57·9%). A similarly high percentage (94%) of adult samples were seropositive to at least one HCoV: OC43 (68·3%) and NL63 (66·7%) were the most common, followed by 229E (49·2%) and HKU1 (46%). Among the four endemic HCoVs, seropositivity to NL63 was highest with 53·3% of total samples, followed by OC43 (51·4%), HKU1 (38·2%), and 229E (38·2%). Seropositivity to multiple HCoVs was common with 59·5% of all samples demonstrating seropositivity to two or more HCoVs. The trend of multi-positivity increased from infancy (39·6%) to adolescence (91·2%) with a slight decline in adulthood (69·8%). Surrogate viral neutralization assays against SARS-CoV-2 performed on all HCoV seropositive samples were all negative, indicating the absence of cross-neutralization to SARS-CoV-1, MERS and SARS-CoV-2.Table 1Demographics of study cohort.Infants (n = 139)n (%)Adolescents (n = 57)n (%)Adults (n = 63)n (%)GenderFemaleMale68 (48·9)71 (51·0)36 (63·2)21 (36·8)44 (69·8)19 (30·2)Percentage Positivity to each HCoVOC43NL63HKU1229E49 (35·3)43 (30·9)37 (26·6)29 (20·9)41 (71·9)53 (93·0)33 (57·9)39 (68·4)43 (68·3)42 (66·7)29 (46·0)31 (49·2)Multi-PositivityNegative to allSingle PositiveDouble PositiveTriple PositivePositive to all63 (45·3%)21 (15·1%)32 (23%)19 (13·7%)4 (2·9%)2 (3·5%)3 (5·3%)12 (21·1%)21(36·8%)19 (33·3%)4 (6·3%)15 (23·8%)18 (28·6%)10 (15·9%)16 (25·4%) Open table in a new tab Despite the high prevalence of endemic HCoV seropositivity in the general population especially in young adults and adolescents, no cross-neutralizing activity against MERS, SARS-CoV-1 and SARS-CoV-2 was detected. This suggests neutralizing antibodies from prior endemic HCoV infections are unlikely to protect against COVID-19 infection or to explain the differences in clinical phenotype between young children and older adults. The strength of this study is the large number of pre-pandemic samples from different age groups in a general population cohort. However, our study is limited by its focus on cross-neutralizing antibodies. We did not look for other potentially cross-reacting epitopes outside of the S1-spike protein so cross protection may have come through other epitopes. We were also unable to identify banked serum from teenagers or older children although we do have a group of adolescents, young adults and older individuals for comparison with infants. We also were not able to assess T-cell responses. Investigators have found that vaccination using the BNT162b vaccine in individuals who have recovered from SARS-CoV-1 infection initiated development of broad antibodies against a spectrum of sarbecoronaviruses.7Tan C.W. Chia W.N. Young B.E. et al.Pan-sarbecovirus neutralizing antibodies in BNT162b2-immunized SARS-CoV-1 survivors.N Engl J Med. 2021; 385: 1401-1406Crossref PubMed Scopus (95) Google Scholar We have demonstrated here that this is not present in those with prior seasonal coronavirus infection. Other mechanisms such as differences in innate immune responses in the elderly, or decreased levels and binding affinity of paediatric ACE-2 (the SARS-CoV-2 target receptor) in children; may thus be responsible for the age-related disparity in COVID-19 infection. Seropositivity to endemic coronaviruses across different age groups in the general population is not well reported. A pre-pandemic study from Beijing using an IFA assay found high levels of seronconversion (> 60%) after age 3, with > 70% seropositivity for the adult age group, similar to our findings.8Zhou W. Wang W. Wang H. Lu R. Tan W. First infection by all four non-severe acute respiratory syndrome human coronaviruses takes place during childhood.BMC Infect Dis. 2013; 13: 433Crossref PubMed Scopus (82) Google Scholar Other studies have concentrated on PCR or serological studies of hospitalized individuals which may not reflect the immunological history of the patients and cannot determine cross protection.9Huang A.T. Garcia-Carreras B. Hitchings M.D.T. et al.A systematic review of antibody mediated immunity to coronaviruses: kinetics, correlates of protection, and association with severity.Nat Commun. 2020; 11: 4704Crossref PubMed Scopus (495) Google Scholar,10Zar H.J. Nicol M.P. MacGinty R. et al.Antibodies to seasonal coronaviruses rarely cross-react with SARS-CoV-2: findings from an African birth cohort.Pediatr Infect Dis J. 2021; 40: e516-e519Crossref PubMed Scopus (4) Google Scholar In conclusion, we have demonstrated that seroconversion to endemic HCoV begins early in life, increases significantly in adolescence with a slight decline in seropositivity in adulthood. However, despite the high prevalence of endemic HCoV seropositivity, there was no cross neutralization against epidemic HCoVs. More research is required to determine the reasons for the increased severity of SARS-CoV-2 infection in older adults. This work was supported by the Special NUHS COVID-19 Seed Grant Call, and NMRC COVID-19 grant [MOH-000417].