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 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.

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 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.

Materials and Methods

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 group was 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 reverse transcriptase-polymerase chain reaction (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. We used plasma samples collected at baseline and 2 years later for the adult cohort because they were part of a longitudinal cohort (Nguyen et al. 2020). We used 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 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) (Tan et al. 2020). 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 (Tan et al. 2020). 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 (Tan et al. 2020). 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.

Table 1

Demographics and animal contacts of the study participants

Variables	Children, N=148	Adults, N=100
Age#	18 (1 - 153)	44 (21 – 72)
Female/male	89/59 (1.5)	32/68 (0.47)
Occupation, n (%)
Farmers	NA	42 (42%)
Slaughters	NA	32 (32%)
Animal health workers	NA	26 (26%)
Geographic locations of study participants
HCMC (Ho Chi Minh City)	62 (41.89%)	0
Other provinces*	86 (58.11%)	100 (100%)Ψ
Collection period	June, 2015 – July, 2019	March, 2013 – September, 2016
Activity/event at risk of zoonotic infection
Raising domestic animals	NA	63 (63%)
Raising wild animals	NA	16 (16%)
Bitten by animals	NA	19 (19%)
Bloody injuries⁎⁎	NA	42 (42%)

Note toTable 1:#in months, median (range) for children and in years, median (range) for adults

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.

Other provinces include An Giang (8), Ba Ria Vung Tau (2), Bac Lieu (1), Ben Tre (4), Binh Duong (4), Binh Phuoc (2), Binh Thuan (1), Ca Mau (1), Can Tho (3), Dong Nai (3), Dong Thap (2), Kien Giang (2), Lam Dong (1), Long An (15), Quang Ngai (1), Soc Trang (1), Tay Ninh (13), Tien Giang (18), Tra Vinh (3) and Vinh Long (1). The geographic locations of these provinces can be found in Figure 1.

Bloody injuries while working with animals.

Figure 1

Map showing the geographic distributions of the study participants. (Inset map; https://mapchart.net).

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 un-infected 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 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.

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) (Grifoni et al. 2020; 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.

Conflict of Interest

None

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.

Ethical Approval

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

OUCRU COVID-19 Research Group

Hospital for Tropical Diseases, Ho Chi Minh City, Vietnam: Nguyen Van Vinh Chau, Nguyen Thanh Dung, Le Manh Hung, Huynh Thi Loan, Nguyen Thanh Truong, Nguyen Thanh Phong, Dinh Nguyen Huy Man, Nguyen Van Hao, Duong Bich Thuy, Nghiem My Ngoc, Nguyen Phu Huong Lan, Pham Thi Ngoc Thoa, Tran Nguyen Phuong Thao, Tran Thi Lan Phuong, Le Thi Tam Uyen, Tran Thi Thanh Tam, Bui Thi Ton That, Huynh Kim Nhung, Ngo Tan Tai, Tran Nguyen Hoang Tu, Vo Trong Vuong, Dinh Thi Bich Ty, Le Thi Dung, Thai Lam Uyen, Nguyen Thi My Tien, Ho Thi Thu Thao, Nguyen Ngoc Thao, Huynh Ngoc Thien Vuong, Pham Ngoc Phuong Thao, Phan Minh Phuong

Oxford University Clinical Research Unit, Ho Chi Minh City, Vietnam: Dong Thi Hoai Tam, Evelyne Kestelyn, Donovan Joseph, Ronald Geskus, Guy Thwaites, H. Rogier van Doorn, Ho Van Hien, Huynh Le Anh Huy, Huynh Ngan Ha, Huynh Xuan Yen, Jennifer Van Nuil, Jeremy Day, Joseph Donovan, Katrina Lawson, Lam Anh Nguyet, Lam Minh Yen, Le Nguyen Truc Nhu, Le Thanh Hoang Nhat, Le Van Tan, Sonia Lewycka Odette, Louise Thwaites, Maia Rabaa, Marc Choisy, Mary Chambers, Motiur Rahman, Ngo Thi Hoa, Nguyen Thanh Thuy Nhien, Nguyen Thi Han Ny, Nguyen Thi Kim Tuyen, Nguyen Thi Phuong Dung, Nguyen Thi Thu Hong, Nguyen Xuan Truong, Phan Nguyen Quoc Khanh, Phung Le Kim Yen, Sophie Yacoub, Thomas Kesteman, Nguyen Thuy Thuong, Tran Tan Thanh, Tran Tinh Hien, Vu Thi Ty Hang