Absence of SARS-CoV-2 antibodies in pre-pandemic plasma from children and adults in Vietnam

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 no evidence of prior serological cross-reactivity with SARS-CoV-2 that might explain the low numbers of COVID-19 in Vietnam. No pre-existing cross-reactivity might explain Vietnam success of COVID-19 control.


Introduction
SARS-CoV-2 emerged in late 2019 and is the cause of the ongoing COVID-19 pandemic. Yet, according to the World Health Organization, as of the end of February 2021, countries in the Western Pacific region, including Vietnam, have 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 mask-wearing in public), contact tracing and testing, coupled with the isolation of infected cases and their direct contacts. However, 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 ✩ Members of the Group are listed in the acknowledgements ✩✩ Running title: SARS-CoV-2 antibodies in pre-pandemic plasma * Correspondence: Le Van Tan E-mail address: tanlv@oucru.org (L.V. Tan). 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.
* * Bloody injuries while working with animals.
admission plasma from each participant and convalescent plasma samples from 2 human coronavirus positive children for the children. Thus, in total, we included 350 plasma samples in 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 2 main immunogens (the nucleocapsid (N) and spike (S) proteins) of SARS-CoV-2 using 2 wellvalidated 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) (Gen-Script, 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 20 uL of plasma samples. The latter is a surrogate assay for measuring S protein receptor-binding domain (RBD)targeting neutralizing antibodies (RBD-targeting NAbs) in 10 uL 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 ≥14 days post symptom onset  ). These 2 assays detected SARS-CoV-2 antibodies in plasma samples from 11 of 11 Vietnamese patients with PCR-confirmed SARS-CoV-2 infection collected 2-3 weeks after diagnosis (data not shown).

Results
The 248 study participants came from various geographic locations in Southern Vietnam ( Table 1 and Figure 1 ). They were all enrolled in the clinical studies between March 2013 and July 2019 (before the COVID-19 pandemic). Of the adult participants, farmers were predominant (n = 42), followed by animal slaughterers (n = 32) and animal health workers (n = 26). The 148 children all had hand foot and mouth disease, and included 89 females and 59 males; the median age was 18 months. None of the 350 plasma samples tested had detectable antibodies against N protein SARS-CoV-2. Additionally, RBD-targeting NAbs were not detected in 240 available plasma samples from the 100 adults and 38 children (including 10 plasma samples collected from 8 children positive for HCoV-NL63 or HCoV-OC43).

Discussion
Using in-house immunofluorescence assays, a recent study demonstrated that antibodies against S or N 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 seroprevalence increasing with age ( Dijkman et al., 2008 ). Thus, it is likely that in addition to the 9 individuals with RT-PCR evidence of human coronavirus infection, 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 prepandemic plasma. Other possible contributing factors include the difference in assay performance and/or the heterogeneities between the study populations, which merits further research.
Cellular and humoral immunities are 2 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 ). How these pre-existing immunities correlate with protection remains unknown.
In summary, antibodies against SARS-CoV-2 N or S 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 (10 6 6 80/B/14/Z and 204 904/Z/16/Z). The serology work at Duke-NUS is supported by grants from NMRC, Singapore (STPRG-FY19-0 01 and COVID19RF-0 03). 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.

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