key: cord-0975799-j5erjttt
authors: Chau, Nguyen Van Vinh; Nhan, Le Nguyen Thanh; Nguyet, Lam Anh; Tu, Nguyen Thi Kha; Hong, Nguyen Thi Thu; Man, Dinh Nguyen Huy; Ty, Dinh Thi Bich; Nhu, Le Nguyen Truc; Yen, Lam Minh; Khanh, Truong Huu; Quy, Du Tuan; Minh, Ngo Ngoc Quang; Ny, Nguyen Thi Han; Anderson, Danielle; Wang, Lin-Fa; van Doorn, H. Rogier; Hung, Nguyen Thanh; Thanh, Tran Tan; Thwaites, Guy; Van Tan, Le
title: Absence of SARS-CoV-2 antibodies in pre-pandemic plasma from children and adults in Vietnam
date: 2021-07-31
journal: Int J Infect Dis
DOI: 10.1016/j.ijid.2021.07.072
sha: d628caef876a5c6c0444463ff6757aa0beecae77
doc_id: 975799
cord_uid: j5erjttt

We tested pre-pandemic (2015-2019) plasma samples from 148 Vietnamese children, and 100 Vietnamese adults at high risk of zoonotic infections, for antibodies against SARS-CoV-2 nucleocapsid and spike proteins. None was positive. The data thus demonstrated that there was no evidence of prior serological cross-reactivity with SARS-CoV-2 that might explain the low numbers of COVID-19 in Vietnam. Future studies should look at pre-existing B cell and T cell memory in pre-pandemic samples in Vietnam, which might further shed light on the pathogenesis of the infection.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) emerged in late 2019, and is the cause of the ongoing coronavirus disease 2019 pandemic. Yet, according to the World Health Organization, as of the end of February 2021 countries in the Western Pacific region, including Vietnam, have so far reported only a small fraction of the global COVID-19 cases (who.int). In Vietnam, this was helped by early preparedness and proactive responses encompassing timely border closure, physical distancing (including mass wearing in public), contact tracing and testing coupled with the isolation of infected cases and their direct contacts. Yet, another hypothesis is that there may be pre-existing immunity among the population in the region through exposure to SARS-CoV-2 related viruses. Knowledge of this might help explain why the burden posed by SARS-CoV-2 and the incidence of COVID-19 cases vary significantly across the world.

It may also further shed light on the natural course of the infection.

A total of 148 young Vietnamese children with hand, foot and mouth disease (Nhan et al. 2020) , and 100 Vietnamese adults were included for analysis of antibodies responses against SARS-CoV-2. The latter was those in close contact with domestic and/or wild animals and was thus at high risk for zoonotic infections as detailed elsewhere (Nguyen et al. 2020) . Of the 148 children, 8 children had RT-PCR confirmed evidence of current infection with either human coronavirus NL63 (HCoV-NL63) (n=2) or human coronavirus OC43 (HCoV-OC43) (n=6), and one adult had HCoV-OC43 detected in an earlier sample by RT-PCR. For the 100 adult cohort participants, because they were part of a longitudinal cohort (Nguyen et al. 2020) , we used plasma samples collected at baseline and two years later. For children, we used 148 admission plasma from each participant, and convalescent plasma samples from 2 human coronavirus positive children. We included 350 plasma samples in total for the analysis. After collection, all plasma samples were divided into small aliquots and stored at 20C until analysis. We extracted information about demographics, occupation and animal contact from the metadata of the aforementioned original studies.

We measured antibodies against two main immunogens (the nucleocapsid (N) and spike (S) proteins) of SARS-CoV-2 using two well-validated sensitive and specific serological assays, namely Elecsys Anti-SARS-CoV-2 assay (Roche, Germany) (Ainsworth et al. 2020 ) and SARS-CoV-2 Surrogate Virus Neutralization Test (sVNT) (GenScript, USA) ). The former is an electrochemiluminescence immunoassay using recombinant N protein for qualitative detection of pan immunoglobulin (Ig) (including IgG) against SARS-CoV-2 in 20uL of plasma samples. The latter is a surrogate assay for measuring spike protein receptor binding domain (RBD)-targeting neutralizing antibodies (RBD-targeting NAbs) in 10uL of plasma samples ). The Elecsys assay had the sensitivity of 99.6% when validated on samples collected at 30 days post symptom onset (Ainsworth et al. 2020) , while the sVNT had the sensitivity of 98.9% when validated on samples collected at 14days post symptom onset ). These two assays detected SARS-CoV-2 antibodies in plasma samples from 11/11 Vietnamese patients with PCR-confirmed SARS-CoV-2 infection collected 2 -3 weeks after diagnosis (data not shown).

The 248 study participants came from various geographic locations in Southern Vietnam (Table 1 and 

Using in-house immunofluorescence assays, a recent study demonstrated that antibodies against spike or nucleocapsid proteins were detected in 19% (n=105) and 14.1% (n=99) pre-pandemic plasma samples collected in Tanzania and Zambia, respectively (Tso et al. 2020 ). Cross reactivity was strongly correlated with the presence of pre-existing antibodies against HCoV-NL63. Most recently, SARS-CoV-2 cross reactive antibodies, especially neutralizing antibodies, were also detected in pre-pandemic plasma from SARS-CoV-2 uninfected individuals in the UK (Ng et al. 2020) .

Human coronaviruses cause the common cold worldwide with sero-prevalence increasing with age (Dijkman et al. 2008) . Thus, it is likely that in addition to the 9 individuals of the present study with evidence of human coronavirus infection by reverse transcription polymerase chain reaction, a proportion of the study participants were also exposed to these coronaviruses some time in the past. Therefore, previous exposure to known human coronaviruses alone might not determine the observed cross reactivity in pre-pandemic plasma. Other possible contributing factors include the difference in assay performance and/or the heterogeneities between the study populations, which merits further research.

Both cellular and humoral immunities are two major components of host responses. The former was not explored in the present study due to the unavailability of peripheral blood mononuclear cells. Of note, SARS-CoV-2-reactive T cells were detected in 20 to 50% of blood collected from unexposed individuals from various geographic locations (Germany, Singapore, the Netherlands, the United States and the United Kingdom) Le Bert et al. 2020; Mateus et al. 2020 ). The correlation with protection of these preexisting immunities remains unknown.

In summary, antibodies against SARS-CoV-2 nucleocapsid or spike proteins were not detected in 350 pre-pandemic Vietnamese plasma samples. Future studies should look at pre-existing B cell and T cell memory in pre-pandemic samples, which might further shed light on the pathogenesis of the infection.

Funding Sources This study was funded by the Wellcome Trust of Great Britain (106680/B/14/Z and 204904/Z/16/Z). The serology work at Duke-NUS is supported by grants from NMRC, Singapore (STPRG-FY19-001 and COVID19RF-003). The sponsors had no role in the study design, the collection, analysis and interpretation of data; in the writing of the manuscript; and in the decision to submit the manuscript for publication.

The institutional review board of collaborating hospitals in Vietnam and the Oxford Tropical Research Ethic Committee approved the clinical study. 

Performance characteristics of five immunoassays for SARS-CoV-2: a head-tohead benchmark comparison The Lancet Infectious Diseases

Human coronavirus NL63 and 229E seroconversion in children

Targets of T Cell Responses to SARS-CoV-2 Coronavirus in Humans with COVID-19

SARS-CoV-2-specific T cell immunity in cases of COVID-19 and SARS, and uninfected controls

Selective and cross-reactive SARS-CoV-2 T cell epitopes in unexposed humans

Preexisting and de novo humoral immunity to SARS-CoV-2 in humans

Respiratory viruses in individuals with a high frequency of animal exposure in southern and highland Vietnam

Clinical, etiological and epidemiological investigations of hand, foot and mouth disease in southern Vietnam during

A SARS-CoV-2 surrogate virus neutralization test based on antibody-mediated blockage of ACE2-spike protein-protein interaction

High prevalence of pre-existing serological cross-reactivity against SARS-CoV-2 in sub-Sahara

 50 from Dong Thap province and 50 from Dak Lak province Domestic animals include dogs, chicken, pigs, cats, duck, muscovy duck, pigeons, cattle, geese, goat, rabbits, buffalo and turkeys. Wild animals include wild pigs, deers, porcupines and monkeys

) and Vinh Long (1). declare the following financial interests/personal relationships

We thank members of the VIZIONS consortium and staff from the Departments of Health and Preventative Medicine Centers (PMC) of Dak Lak and Dong Thap provinces for provision of samples, and thank VIZIONS cohort members in Dak Lak and Dong Thap provinces for their participation in the study. We are indebted to Ms Le Kim Thanh and Ms Pham Thi Hong Anh for their logistic support. We thank the patients for their participations in this study. We would also like to thank Mr Dinh Khac Hieu for his laboratory support with the ELISA analysis.