Saliva SARS-CoV-2 Antibody Prevalence in Children

ABSTRACT COVID-19 patients produce circulating and mucosal antibodies. In adults, specific saliva antibodies have been detected. Nonetheless, seroprevalence is routinely investigated, while little attention has been paid to mucosal antibodies. We therefore assessed SARS-CoV-2-specific antibody prevalence in serum and saliva in children in the Netherlands. We assessed SARS-CoV-2 antibody prevalence in serum and saliva of 517 children attending medical services in the Netherlands (irrespective of COVID-19 exposure) from April to October 2020. The prevalence of SARS-CoV-2 spike (S), receptor binding domain (RBD), and nucleocapsid (N)-specific IgG and IgA were evaluated with an exploratory Luminex assay in serum and saliva and with the Wantai SARS-CoV-2 RBD total antibody enzyme-linked immunosorbent assay in serum. Using the Wantai assay, the RBD-specific antibody prevalence in serum was 3.3% (95% confidence interval [CI]. 1.9 to 5.3%). With the Luminex assay, we detected heterogeneity between antibodies for S, RBD, and N antigens, as IgG and IgA prevalence ranged between 3.6 and 4.6% in serum and between 0 and 4.4% in saliva. The Luminex assay also revealed differences between serum and saliva, with SARS-CoV-2-specific IgG present in saliva but not in serum for 1.5 to 2.7% of all children. Using multiple antigen assays, the IgG prevalence for at least two out of three antigens (S, RBD, or N) in serum or saliva can be calculated as 3.8% (95% CI, 2.3 to 5.6%). Our study displays the heterogeneity of the SARS-CoV-2 antibody response in children and emphasizes the additional value of saliva antibody detection and the combined use of different antigens. IMPORTANCE Comprehending humoral immunity to SARS-CoV-2, including in children, is crucial for future public health and vaccine strategies. Others have suggested that mucosal antibody measurement could be an important and more convenient tool to evaluate humoral immunity compared to circulating antibodies. Nonetheless, seroprevalence is routinely investigated, while little attention has been paid to mucosal antibodies. We show the heterogeneity of SARS-CoV-2 antibodies, in terms of both antigen specificity and differences between circulating and mucosal antibodies, emphasizing the additional value of saliva antibody detection next to detection of antibodies in serum.

Reviewer #1 (Comments for the Author): Thank you for this paper it was very interesting. When you compare your seroprevalence rates with the national seroprevalence study it may help to expand on the fact that their study was community based rather than your hospital based study. Your sample is very different as you have recruited from a hospital population rather than a representative paediatric population. The fact that you are seeing similar results both in the community and the hospital based seroprevalence studies is interesting. I think you make a good case for saliva antigens to be used in future research especially since this is more likely to be acceptable to parents of younger children who may be put off by a blood test.
Reviewer #2 (Comments for the Author): Dear authors, Thank you very much for this interesting article. The usefulness of saliva for diagnosing SARS-CoV-2 seroprevalence in children is a key for this population age-group, and the discordances with serum are a matter of concern. However, I would like to share with you some doubts about the manuscript. Abstract: it is difficult to read, the information in the results need to be improved, i.e. lines 51 and 52 cannot begin with numbers of percentages. Introduction: why do you comment the incidence of COVID-19 in December if the study was carried out before this date? It is not needed to put this data here. When you mention HIV for first time you should write the complete name of it as "Human Immunodeficiency virus". Methods: why do you not include corticosteroids as immunomodulating drugs? Regarding underlying diseases you included most of them but not hepatic or other neurological diseases. In line 168 you defined as negative controls those with negative antigen, why not those with negative PCR? could it lead a bias in selecting the controls? When defining the ages, should I understand in line 177 that 0-1 year are children less than 12 months? Results: You say in line 186: "Most children (38.9%) did not have an immunocompromised..." Does it mean that most of the children included into the study had an immunocompromised state or underlying illness? If this is the case, you are selecting a specific population that is not representative of the general pediatric population, leading to a bias for the results. In line 199 and 201 I would like to see 95%CI as shown before in lines 198. Regarding the Wantai (+) children you selected 13/16 with symptoms, again this is not representative of pediatric population. Discussion needs to improve before accept the manuscript.
Yours sincerely, Staff Comments:

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Reviewer #1
1. Thank you for this paper it was very interesting. When you compare your seroprevalence rates with the national seroprevalence study it may help to expand on the fact that their study was community based rather than your hospital based study. Your sample is very different as you have recruited from a hospital population rather than a representative paediatric population. The fact that you are seeing similar results both in the community and the hospital based seroprevalence studies is interesting. I think you make a good case for saliva antigens to be used in future research especially since this is more likely to be acceptable to parents of younger children who may be put off by a blood test.

Response:
We thank the reviewer for their comments and their acknowledgement of the potential of saliva antibody assays for children. We agree with the reviewer that we should emphasize the similarity in prevalence of our hospital population when compared to community based studies. In the Discussion section, we elaborated on the similar antibody prevalence despite the difference in study population.

Reviewer #2
1. Abstract: it is difficult to read, the information in the results need to be improved, i.e. lines 51 and 52 cannot begin with numbers of percentages.

Response:
We appreciate the reviewer's interest in our manuscript and thank the reviewer for their comments. We have rewritten the abstract results to improve the readability of this section. (page 2 lines 51 -57) 2. Introduction: why do you comment the incidence of COVID-19 in December if the study was carried out before this date? It is not needed to put this data here.

Response:
We agree with the reviewer that the national incidence in December 2020 is not of additional value in this section. We aimed to provide the readers some background information on the national situation during recruitment of our study. We thus rephrased this sentence to better explain the context during the recruitment period. (page 4 lines 78 -79) 4. Methods: why do you not include corticosteroids as immunomodulating drugs? Regarding underlying diseases you included most of them but not hepatic or other neurological diseases.
Response: Corticosteroids were considered as immunomodulating drugs, although not reported in this section of the manuscript. We have added this information to improve the Methods section. (page 6 line 123) Children with hepatic or neurological diseases were rarely included and were always primarily categorized as endocrine/metabolic, malignancy or psychomotor retardation.

5.
In line 168 you defined as negative controls those with negative antigen, why not those with negative PCR? could it lead a bias in selecting the controls? Response: We acknowledge the sentence in line 168 was poorly phrased. We meant to indicate the presence of an assay control in the form of beads with no antigen to confirm for each individual that there is no nonspecific binding to the beads or blocking components. Additionally, we used healthy donor serum and saliva samples with antigen-coated beads to determine the assay background. We have added clarification to improve the methods section. (page 8 line 170) also to indicate the selection of the healthy donor samples.
6. When defining the ages, should I understand in line 177 that 0-1 year are children less than 12 months? Response: The youngest age group is indeed defined as children aged less than 12 months. We have rewritten the sentence to improve readability. (page 8 line 182) 7. Results: You say in line 186: "Most children (38.9%) did not have an immunocompromised..." Does it mean that most of the children included into the study had an immunocompromised state or underlying illness? If this is the case, you are selecting a specific population that is not representative of the general pediatric population, leading to a bias for the results.

Response:
We thank the reviewer for pointing out this sentence, as comorbidity was not clearly described. This sentence is rewritten to improve phrasing. (page 9 lines 190 -192) In addition, we elaborated upon the comorbidities in our population in the Discussion section, including the fact that our population is different from a community-based study. (page 12 lines 255 -258) 8. In line 199 and 201 I would like to see 95% CI as shown before in lines 198.

Response:
Following the reviewer's suggestion we have added confidence intervals for lines 204 -206. 9. Regarding the Wantai (+) children you selected 13/16 with symptoms, again this is not representative of pediatric population.
Response: This sentence aimed to explain that in this population most children with antibodies in the Wantai either reported (mild) symptoms or close COVID-19 contacts. Our study was not powered to evaluate associations between antibody positivity and symptomatology as this was not our objective. Thus we did not intend to provide a representation of the pediatric COVID-19 population, nor conclude on any associations. We have rewritten this sentence to better explain that we describe characteristics. (page 9 line 211 -212) 10. Discussion needs to improve before accept the manuscript.

Response:
We thank the reviewer for pointing this out and have made improvements to the discussion in several sections.