Comparison of antibody responses following natural infection with Severe Acute Respiratory Syndrome Coronavirus 2 or receipt of CoronaVac or ChAdOx1 (AZD1222) vaccination in Chiang Mai, Thailand

Highlights

• •Corona Vac vaccine induces antibody responses similar to SARS-CoV-2 infection.
• •ChAdOx1 vaccine shows superior immunogenicity than CoronaVac and natural infection.
• •Neutralizing activity had a positive correlation with levels of anti-spike Abs.

Abstract

Background

In Thailand, early vaccination initiatives for SARS-CoV-2 relied on CoronaVac (Sinovac Life Sciences) and ChAdOx1 (Oxford–AstraZeneca) vaccines. However, the data of immunogenicity of these two vaccines in Thai populations is limited. This real time, head-to-head comparative study was conducted to investigate antibody (Ab) responses to SARS-CoV-2 following infection or receipt of either CoronaVac or ChAdOx1 vaccination in Chiang Mai, Thailand.

Methods

Sera was collected within two months from participants having a history of documented SARS-CoV-2 infection or at one month after the second dose of CoronaVac vaccine. Sera from participants with a history of receiving one dose of ChAdOx1 vaccination was collected twice, at one month following each vaccine dose. Neutralizing antibodies (NAbs) were assessed using the surrogate neutralization test and anti-spike protein antibodies were assessed using an in-house enzyme-linked immunosorbent assay.

Results

The prevalence of NAbs against SARS-CoV-2 was 92.1 %, 95.7 %, 64.1 % and 100 % in the infection group, CoronaVac group, ChAdOx1 group after 1st dose, and ChAdOx1 group after 2nd dose, respectively. The inhibition rate in individuals receiving two doses of ChAdOx1 vaccine (90.8%) was significantly higher than individuals who had recovered from natural infection (71.7%) or individuals who had received two doses of CoronaVac vaccine (66.7%). The prevalence of anti-spike Abs was 97.4 %, 97.8 %, 97.4 % and 100 % in the infection group, CoronaVac group, ChAdOx1 group after 1st dose, and ChAdOx1 group after 2nd dose, respectively. Significantly higher levels of anti-spike Abs were observed in the ChAdOx1 group after two doses of vaccination (1975 AU/mL) compared to those who had recovered from natural infection (468.5 AU/mL) and individuals who had received CoronaVac (554.4 AU/mL). Neutralizing activity had a statistically significant positive correlation with levels of anti-spike Abs.

Conclusions

ChAdOx1 vaccine may provide superior immunogenicity than CoronaVac and natural infection.

Introduction

Coronavirus disease (COVID-19), caused by Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) [1] was first reported in China in December 2019. It has spread worldwide and, as of June 2022, has resulted in more than 500 million infections and over 6 million deaths across 226 countries [2]. The first COVID-19 case in Thailand was reported on January 31^, 2020, [3] and the control of SARS-CoV-2 infection in Thailand continued until late 2020.

Several COVID-19 vaccines have been rolled out in early 2021 [4]. CoronaVac inactivated vaccine and viral vector ChAdOx1 vaccines were the only two available vaccines provided by the government in Thailand between March and July 2021. Two doses of CoronaVac vaccine are administered three weeks apart, whereas the recommended doses of ChAdOx1 is two doses with an interval of 12 weeks. The data of immunogenicity of these two vaccines in Thai populations was limited. This study was conducted to investigate the seropositivity and level of antibody responses to SARS-CoV-2 after infection or vaccination with two doses of homologous CoronaVac or ChAdOx1 vaccines in northern Thai populations. This is head-to head, real-time comparison of the immunogenicity of these two vaccines and infection, in the same populations.

Materials and methods

Study populations

The study populations were adults over 18 years of age, be able to provide written consent and willing to participate in the study, living in Chiangmai. They were divided into three groups based on history of SARS-CoV-2 infection or vaccination regimens: 1) individuals with a history of documented SARS-CoV-2 infection provided by Thai Ministry of Public Health (infection group), 2) those with a history of receiving two doses of CoronaVac (Sinovac Life Sciences, Beijing, China) vaccine with certificate from Thai Ministry of Public Health (CoronaVac group), 3) those with a history of one dose of Oxford–AstraZeneca ChAdOx1 (AZD1222) vaccination (ChAdOx1 group). Exclusion criteria included history of illnesses or received other vaccines within the preceding month and those under treatment with immunosuppressants such as corticosteroids. For CoronaVac and ChAdOx1 groups, they must not have a history of SARS-CoV-2 infection. Archive sera from participants collected before SARS-CoV-2 vaccination campaign were used to represent naïve populations in Chiang Mai.

This study was approved by the ethics committees of the Faculty of Medicine (MED-2564-08155) and the Research Institute for Health Sciences (13/64), Chiang Mai University. All procedures performed in studies involving human participants were in accordance with the Declaration of Helsinki Written informed consent was obtained from all participants before enrollment into the study.

Study procedures

The study was conducted from June 1, 2021, to November 8, 2021. We advertised the project through social media (Facebook and line applications) and provided a QR code for potentially interested individuals to scan and access an online pre-screening form to determine eligibility. They were asked to select a group, to provide some information on previous illnesses and vaccinations, and to select the date available for in-person screening, participation in the informed consent process, and have a blood sample collected at the Research Institute for Health Sciences, Chiang Mai University. Ten milliliters of blood from individuals with a history of documented SARS-CoV-2 infection provided by Thai Ministry of Public Health and individuals who had received two doses of CoronaVac vaccine were collected only once on the day of enrollment (corresponding to 31–69 days after discharge from hospitals or one month after 2 doses of vaccination, respectively). Individuals with one dose of ChAdOx1 vaccination had two blood samples taken, the first collected on the day of enrollment (one month after receipt of their dose) and the second at one month after the second dose, which was scheduled 12 weeks later.

Verification of SARS-CoV-2 variants

A total of 10 positive remaining nasopharyngeal and/or throat swab laboratory samples from Maharaj Nakorn Chiangmai Hospital were collected. All samples were confirmed to be SARS-CoV-2 positive by real time reverse transcription polymerase chain reaction (RT-PCR) assay. Viral RNA was extracted from 140 µL of sample using QIAamp Viral RNA Kit (QIAGEN, Germany), according to the manufacturer’s protocol. All RNA specimens underwent conventional PCR and sequencing, with amplification of the SARS-CoV-2 Spike (S) gene using a primer set specific for SARS-CoV-2 [5].

The one-step RT-PCR reactions were conducted using the SuperScript™ III One-Step RT-PCR System with Platinum™ Taq DNA Polymerase (Invitrogen, Carlsbad, CA). Briefly, the PCR reaction was performed in a total reaction volume of 25 µL, with the reaction mixture composed of 12.5 µL of 2X Reaction Mix, 0.5 µM of each primer, 1 µL of SuperScript™ III RT/Platinum™ Taq mix, and 2 µL of RNA. The amplification reaction was performed as previously described [5]. PCR products were separated on a 2 % agarose gel and visualized on an ultraviolet trans-illuminator. PCR products were gel-purified using NucleoSpin Gel and PCR Clean-up kit (Macherey-Nagel, Germany). DNA sequencing service was performed by Macrogen Inc., Seoul, Korea. Genome sequences were aligned using ClustalW, implemented via the BioEdit program (v.7.2.5) and the Geneious Prime software (v 2021.2.2) was used for nucleotide sequence assembly. Variants analysis was performed using the GISAID Initiative (CoVsurver: Mutation Analysis of hCoV-19) to identify spike protein substitutions of SARS-CoV-2 variants.

Surrogate virus neutralization assay

SARS-CoV-2 Surrogate Virus Neutralization Test (sVNT) Kit (cPass™) was purchased from Genscript (Piscataway, USA). This neutralization antibody detection kit is designed to mimic the virus-host interaction utilizing recombinant RBD of the SARS-CoV-2 spike protein to detect antibodies that block the RBD binding to the hACE2 receptor. Sera from participants were examined for nAb using this sVNT Kit according to the manufacturer’s instructions. Briefly, 60 µL of horseradish peroxidase (HRP)–RBD was pre-incubated at 37 °C for 30 min with 60 µL of 1:10 diluted serum or control. One hundred microliters of the mixture were then added to the capture plate, which was pre-coated with the hACE2 protein. The unbound HRP-RBD and HRP-RBD bound to non-neutralizing antibodies would be captured on the plate, while HRP-RBD bound to neutralization antibodies would remain in the supernatant and be removed during washing step. After washing, 100 µL of TMB substrate solution was added followed by the stop solution after a 15 min incubation period. The absorbance of the final solution was read at 450 nm on CLARIOstar® microtiter plate reader (Ortenberg, Germany).

Determination of anti-spike antibody by indirect enzyme-linked immunosorbent assay (ELISA)

Presence of antibodies specific to the spike protein were determined by in-house enzyme-linked immunosorbent assay (ELISA). Fifty microliters of 1 µg/ml spike proteins in bicarbonate buffer (pH 9.6) were added to 96-well Maxisorp ELISA immunoplates (Thermo scientific, Roskilde, Denmark). After incubation overnight at 4 °C, plates were washed with 0.05 % Tween-20 (Calbiochem, Gibbstown, USA) in phosphate buffer saline (PBS) and blocked with 100 µL 2 % skimmed milk at 37 °C for 1 hr. Fifty microliters of samples diluted 1:100 and serially diluted positive controls were added and incubated at 37 °C for 1 h. After washing, 50 µL of 1: 2000 goat anti-human IgG or IgM conjugated with horse radish peroxidase (HRP) (Invitrogen, Carlsbad, USA) was added and incubated at 37 °C for 1 h. After washing, 50 µL of tetramethylbenzidine (TMB) substrate (Life Technologies, Frederick, USA) was added and plates were incubated at room temperature for 30 min. The enzyme reaction was terminated with 0.2 M sulfuric acid and read for absorbance at 450 nm on the CLARIOstar® microtiter plate reader. Antibody levels were determined by calculation from a serial dilution of WHO international standard anti-SARS-CoV-2 immunoglobulin (NIBSC, UK) which was assigned an arbitrary unitage of 1000 BAU/mL. Cut-off value of the levels of anti-spike antibodies (Abs) was determined as the mean plus three standard deviations (mean + 3SD) observed in the control group (54.3 BAU/mL).

Sample size calculation

Preliminary data from Chulalongkorn University showing that seropositivity against CoronaVac and ChAdOx1 vaccines were approximately 99 % and 96.7 %, respectively, in Thai populations. Seroconversion rate after SARS-CoV-2 infection among Thai population was approximately 90 % [6]. We expected a similar prevalence of presence of neutralizing antibodies in each group after vaccination or infection. This corresponded to a minimum sample of 31 samples to achieve a 95 % power of detecting neutralizing antibodies in each group, based on a target significance level of 0.05. We aimed to recruit approximately 40 people in each group.

Statistical analysis

Descriptive analyses were summarized as medians with interquartile range (IQR) and range (min–max) for continuous characteristics and as frequencies and percentages for categorical variables. The Kruskal-Wallis test was used for comparing the percent inhibition of NAbs and anti-spike protein antibodies. Post hoc comparisons for the Kruskal-Wallis test was used for pairwise comparison. The correlation between neutralizing antibodies and anti-spike protein antibodies were estimated and tested using Spearman’s correlation. The p value reported were two-tailed, with an alpha level of 0.05 considered to be statistically significant. All analyses were conducted using Stata version 14 (StataCorp LP, College Station, TX, USA).

Results

Verification of SARS-CoV-2 variants

Among the ten positive SARS-CoV-2 samples submitted for testing, we identified 10 unique mutations in the S gene (H69del, V70del, Y144del, N501Y, A570D, D614G, P681H, T716I, S982A, and D1118H) compared to the hCoV-19/Wuhan/WIV04/2019 reference strain. This pattern of amino acid changes were consistent with being that of the SARS-CoV-2 Alpha strain.

Characteristics of study populations

A total of 122 participants were enrolled, including 38 with confirmed SARS-CoV-2 infection, 45 who had received two doses of CoronaVac vaccine and 39 who had received the first dose of ChAdOx1 vaccine. Thirty-five archive sera collected prior to SARS-CoV-2 vaccination were selected as controls. Table 1 shows the participant characteristics. The proportion of males in the infection, CoronaVac and ChAdOx1 groups were 47.4 %, 26.7 % and 30.8 %, respectively. There were no statistically significant differences in gender composition among the groups. The median age of the ChAdOx1 group (29 [IQR 26–42]) was significantly lower than that of the infection (38 [IQR 30–55]) and the CoronaVac (41 [IQR 32–46]) groups. There was no difference in the median height among groups but the median weight (p = 0.008) and the median body mass index (p = 0.013) were significantly higher in the infection group (W: 68 [IQR 62–75], BMI: 24.8 [IQR 22.3–27.1]) compared to those in the CoronaVac (W: 58 [IQR 51–78], BMI: 21.9 [IQR 20.7–25.4]) and the ChAdOx1 (W: 57 [IQR 51–66], BMI: 21.8 [IQR 20.1–24.6]) groups. However, the median BMI of the infection group was still in the healthy weight range (BMI 18.5–24.9).

Table 1.

Characteristics of participants in each group.

	Infection (n = 38)	CoronaVac (n = 45)	ChAdOx1 nCoV-19 (n = 39)	P-valuea
Male, n (%)	18 (47.4 %)	12 (26.7 %)	12 (30.8 %)	0.130b
Median (IQR) age, year	38 (30–55)	41 (32–46)	29 (26–42)	0.006**
Median weight, kgs	68 (62–75)	58 (51–78)	57 (51–66)	0.008**
Median height, cms	165 (159–170)	163 (157–165)	163 (157–167)	0.209
Median BMI, kg/m2	24.8 (22.3–27.1)	21.9 (20.7–25.4)	21.8 (20.1–24.6)	0.013**

** Statistically significant difference.

^aKruskal–Wallis Test.

^bFisher exact’s test.

Neutralizing antibody levels

Sera were tested for neutralizing antibody activity by using the SARS-CoV-2 Surrogate Virus Neutralization Test (sVNT) kit. As shown in Fig. 1 and Table 2, there is no neutralizing Abs in the group that had not been vaccinated (cut-off value = 30 %). Seropositivity rates for NAbs against SARS-CoV-2 in the naive control group, infection group, CoronaVac group, ChAdOx1 group after 1st dose, and ChAdOx1 group after 2nd dose were 0 %, 92.1 %, 95.7 %, 64.1 % and 100 %, respectively. The median inhibition rate of NAbs between those who had SARS-CoV-2 infection (71.7 % [IQR 49.3–84.0]) were not significantly different from those who had received two doses of CoronaVac vaccine (66.7 % [IQR 55.4–74.0]). The inhibition rate of NAbs of the infection and CoronaVac groups were significantly higher than those who received one dose of ChAdOx1 vaccine (41.7 % [IQR 25.1–71.0]), p = 0.002 and p = 0.018, respectively. After the second dose of ChAdOx1 vaccine, the median inhibition rate of NAbs (90.8 [IQR 83.6–95.4], p < 0.001) became significantly higher than the infection and CoronaVac groups. Adjusting for age and BMI, the inhibition rate of NAbs in ChAdOx1 group after 2nd dose was still significantly higher than the infection and CoronaVac groups, whereas no statistical significance was found between the infection and CoronaVac groups.

Fig. 1.

Percent inhibition of neutralizing antibodies by sera of participants using the sVNT assay. The dash line represents the cutoff at 30% inhibition. Each symbol represents one individual. The vertical lines and error bars indicate the median and interquartile range. The p values were calculated from the post hoc comparisons using the Kruskal–Wallis Test.

Table 2.

Percent inhibition of neutralizing antibodies as determined by sVNT assay.

	Control	Infection	Sinovac	AZ (1st)	AZ (2nd)	P-valuesa
Number	35	38	45	39	37
%Positive	0.0	92.1	95.7	64.1	100.0
Median (IQR) % inhibition	4.8 (0.4–8.4)	71.7 (49.3–84.0)	66.7 (55.4–74.0)	41.7 (25.1–71.0)	90.8 (83.6–95.4)	<0.001
Min to Max % inhibition	−11.9 to 19.4	12.0 to 93.7	19.6 to 96.2	−31.9 to 92.7	41.4 to 96.7

^aKruskal–Wallis Test.

Anti-spike Ab levels

Anti-spike IgG levels were determined using an in-house ELISA and calculated against WHO international standard as described in methods. Seropositivity rates for anti-spike Abs in the control group, infection group, CoronaVac group, ChAdOx1 group after 1st dose, and ChAdOx1 group after 2nd dose were 3.03 %, 97.4 %, 97.8 %, 97.4 % and 100 %, respectively (Table 3). The median anti-spike Abs in the infection group was 468.5 AU/mL [IQR 149.3–1159.4]. Corresponding figures for the CoronaVac group was 554.4 AU/mL [IQR 329.3–798.1]) and 616.3 AU/mL [IQR 307.8–887.8]) for the ChAdOx1 group after the first dose, with the differences not significantly different (Fig. 2). Significantly higher levels of anti-spike Abs were observed in the ChAdOx1 group after two doses of vaccination (median 1975.0 AU/mL [IQR 1432.0–2455.5]) compared to the infection and CoronaVac groups (p < 0.001). Age and BMI-adjusted analysis revealed that the median anti-spike Abs remained significantly higher in those who received two doses of ChAdOx1 than those who had SARS-CoV-2 infection or those who had received two doses of CoronaVac vaccine.

Table 3.

The prevalence and levels of anti-spike protein antibodies by in-house ELISA.

	Control	Infection	Sinovac	AZ (1st)	AZ (2nd)	P-valuesa
Number	33	38	45	39	37
%Positive	3.03	97.4	97.8	97.4	100
Median (AU/mL) (IQR)	26.6 (20.7–35.1)	468.5 (149.3–1159.4)	554.4 (329.3–798.1)	616.3 (307.8–887.8)	1975.0 (1432.0–2455.5)	<0.001
Min to Max	11.5–76.8	23.3–1602.9	28.9–1658.0	49.2–2365.5	228.6–3201.6

^aKruskal–Wallis Test.

Fig. 2.

Levels of anti-spike antibodies in sera of participants as determined using an in-house ELISA. Arbitrary units were calculated against the WHO international standard anti-SARS-CoV-2 immunoglobulin. The dash line representing the cutoff was calculated from the mean arbitrary units of the control group plus 2 standard deviations (54.28). Each symbol represents one individual. The vertical lines and error bars indicate the median and interquartile range. The p values were calculated from the post hoc comparisons using the Kruskal–Wallis Test.

Correlation between neutralizing activity and the levels of anti-spike Abs

Statistically significant correlation was found between neutralizing activity and the levels of anti-spike Abs in the infection group (r = 0.934, p < 0.0001), the CoronaVac group (r = 0.880, p < 0.0001), the AZ group (r = 0.824, p < 0.0001), and when data from all groups were combined (r = 0.921, p < 0.0001) (Fig. 3).

Fig. 3.

Correlation between neutralizing antibodies and anti-spike protein antibodies (r = correlation coefficient).

Discussion

We investigated antibody responses to SARS-CoV-2 in participants living in Chiang Mai during the third wave of epidemic in Thailand, as the national COVID-19 vaccination program was expanding across the country in March 2021. We found that two doses of vaccination with the CoronaVac vaccine elicited NAbs and anti-spike Abs to the same levels as natural infection. Two doses of the ChAdOx1 vaccine induced 100 % seropositivity rates for both NAbs and anti-spike Abs in all recipients, with significantly higher inhibition rate and levels compared to the infection and Corona Vac groups.

Our finding is consistent with previous studies showing that immunization with two doses of the CoronaVac vaccine elicited desirable seropositivity rate with similar levels of NAbs and anti-spike Abs compared to individuals recovered from infection [[7], [8]]. Two doses of CoronaVac have shown 94.3 % effectiveness against mild disease and 99.9 % against severe infection as defined by World Health Organization in Colombian populations [9]. Antibody responses after two doses of CoronaVac vaccination in participants presented with smoking, obesity, and comorbidities (i.e., diabetes mellitus and cardiovascular disease) is proportionally lower [[10], [11]].

The observation that in participants who received two doses of ChAdOx1 vaccine had higher levels of antibody responses than COVID-19 convalescent individuals and those who had received CoronaVac vaccine was in line with previous studies [[12], [13], [14]], as well as the positivity rates of NAbs after the first and the second doses [[15], [16]]. The effect of age and BMI on antibody responses was not found in this study. A single standard dose of ChAdOx1 vaccine showed high efficacy capable of inducing high titers of Abs which remain above baseline for at least 1 year [17], although the efficacy against asymptomatic infection was not evident [18]. Neutralizing antibody responses after two doses of ChAdOx1 vaccine in our study was higher than that observed in Japanese adults [19], possibly attributed to a different interval before the second dose (4 weeks in that study vs 12 weeks in our study) and time of blood collection. An extended interval before the second dose of ChAdOx1 vaccine resulted in higher Ab responses after the second dose than with a shorter interval [[17], [18]].

Although individuals who received two doses of CoronaVac and ChAdOx1 vaccine groups showed high anti-spike antibody and high neutralizing antibody seropositivity, the emergence of variants of concern raises questions whether the resulting immunity remains protect individuals from infection, particularly long-term. Individuals who received CoronaVac remain seropositive for total RBD Ig and anti-RBD IgG for at six months after the first dose but a decrease in the neutralizing activity was observed, particularly to the variant of concerns [20]. Two doses of CoronaVac could not neutralize newly emerging alpha, beta, delta and gamma SARS-CoV-2 variants [[21], [22], [23], [24]], suggesting that additional dosing may be required to control transmission of new variants. The ChAdOx1 induced more higher level of anti-SARS-CoV-2 RBD IgG and NAbs against all variant of concerns compared to the CoronaVac [23]. However, sera from COVID-19-convalescent patients or from recipients of the ChAdOx1 vaccine displayed low neutralizing activity against Omicron [[25], [26]]. As the virus continues to evolve over time, these data suggest that a booster dose may be necessary, especially among vulnerable populations.

This study shows head-to head, real-time comparison of the immunogenicity following infection or receipt of either CoronaVac or ChAdOx1 vaccination in Chiang Mai, Thailand. However, our study is a cross-sectional study investigating only single time point. The limitations of our study include the small sample size and the lack of information of inhibitory capability against variants of concerns. This study was undertaken before the emerging of delta and omicron variants, therefore the data may not be generalized to the current COVID situation. It should be noted that Ab responses to nucleocapsid antigens were not assessed in this study, therefore asymptomatic infection in the vaccination groups cannot be ruled out.

In conclusion, vaccination with two doses of CoronaVac vaccine induced NAbs and anti-spike Ab levels comparable to that of natural SARS-CoV-2 infection in our population. Two doses of ChAdOx1 vaccine provides higher antibody responses than two doses of CoronaVac and natural infection. A booster dose vaccination may be beneficial for individuals previously infected with SARS-CoV-2 or those who received two doses of CoronaVac.

Declaration of Competing Interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Data availability

Data will be made available on request.