key: cord-0972406-ppxtfxit
authors: Melgoza‐González, Edgar A.; Hinojosa‐Trujillo, Diana; Reséndiz‐Sandoval, Mónica; Mata‐Haro, Verónica; Hernández‐Valenzuela, Sofía; García‐Vega, Melissa; Bravo‐Parra, Marlene; Arvizu‐Flores, Aldo A; Valenzuela, Olivia; Velázquez, Edgar; Soto‐Gaxiola, Alan; Gómez‐Meza, Martha B.; Pérez‐Jacobo, Fernando; Villela, Luis; Hernández, Jesús
title: Analysis of IgG, IgA and IgM antibodies against SARS‐CoV‐2 spike protein S1 in convalescent and vaccinated patients with the Pfizer‐BioNTech and CanSinoBio vaccines
date: 2021-10-25
journal: Transbound Emerg Dis
DOI: 10.1111/tbed.14344
sha: 5beb9b4a74834683ea73a3060eefded022b95356
doc_id: 972406
cord_uid: ppxtfxit

The SARS‐CoV‐2 virus was detected for the first time in December 2019 in Wuhan, China. Currently, this virus has spread around the world, and new variants have emerged. This new pandemic virus provoked the rapid development of diagnostic tools, therapies and vaccines to control this new disease called COVID‐19. Antibody detection by ELISA has been broadly used to recognize the number of persons infected with this virus or to evaluate the response of vaccinated individuals. As the pandemic spread, new questions arose, such as the prevalence of antibodies after natural infection and the response induced by the different vaccines. In Mexico, as in other countries, mRNA and viral‐vectored vaccines have been widely used among the population. In this work, we developed an indirect ELISA test to evaluate S1 antibodies in convalescent and vaccinated individuals. By using this test, we showed that IgG antibodies against the S1 protein of SARS‐CoV‐2 were detected up to 42 weeks after the onset of the symptoms, in contrast to IgA and IgM, which decreased 14 weeks after the onset of symptoms. The evaluation of the antibody response in individuals vaccinated with Pfizer‐BioNTech and CanSinoBio vaccines showed no differences 2 weeks after vaccination. However, after completing the two doses of Pfizer‐BioNTech and the one dose of CanSinoBio, a significantly higher response of IgG antibodies was observed in persons vaccinated with Pfizer‐BioNTech than in those vaccinated with CanSinoBio. In conclusion, these results confirm that after natural infection with SARS‐CoV‐2, it is possible to detect antibodies for up to 10 months. Additionally, our results showed that one dose of the CanSinoBio vaccine induces a lower response of IgG antibodies than that induced by the complete scheme of the Pfizer‐BioNTech vaccine.

In December 2019, the city of Wuhan, China, reported an outbreak of pneumonia. Later in that month, the World Health Organization declared that a novel virus was the cause of this problem, and it was initially called new coronavirus 2019 (nCoV-2019) (WHO, 12 January, 2020) . Subsequently, the International Committee on Virus Taxonomy named the virus SARS-CoV-2, as currently known ("The species

Severe acute respiratory syndrome-related coronavirus: classifying 2019-nCoV and naming it SARS-CoV-2," 2020). The global impact of this virus is undeniable. On 11 January 2020, almost 2 weeks after the first report of this pathogen, the first death was documented. On 20 January, multiple cases were reported in Japan, South Korea, and Thailand (EWHO, 2020). The first case in the United States was declared one day later (Ghinai et al., 2020) . Since then, the SARS-CoV-2 virus has been reported all across the globe. In Mexico, the first case was reported in February 2020, and since then it has continued to spread among the population. At the time this report was written (10 July 2021), approximately 2,764,852 cases were documented, including 246,910 deaths (SS-México, 2021) .

Reverse transcription polymerase chain reaction (RT-PCR) is an essential assay for diagnosing coronavirus disease 19 , especially during the active virus-shedding phase. However, 2 weeks after the onset of the symptoms, the viral loads decrease . Antibody assays have shown significant sensitivity. Zhao et al. (2020) showed that the median times for IgM and IgG seroconversion were 12 and 14 days, respectively. In contrast, demonstrated that IgM and IgG antibody seroconversion occurred on day 6. Interestingly, the analysis of seroconversion of these isotypes did not show significant differences between critical and noncritical patients . However, showed that at 2 weeks post-symptom onset, IgG was significantly higher in patients with severe disease than in those with non-severe disease. The analysis of humoral response in symptomatic and asymptomatic patients showed a significant reduction in the levels of IgG in the asymptomatic group . This study also revealed that an important percentage of asymptomatic patients were IgG negative (40%) compared with symptomatic (12%) patients who were IgG negative . Antibody responses have been evaluated against the N and S proteins , including receptor binding domain (Roy et al., 2020) and S1 (Krähling et al., 2021) . Currently, several commercial kits for the detection of antibodies against SARS-CoV-2 are available. Some studies have evaluated different commercial serological assays, and positivity rates vary among assays, probably due to the low antibody response of asymptomatic patients. In general, the conclusions were that antibody detection is suitable for 10 days after the onset of symptoms (Herroelen et al., 2020; Trabaud et al., 2020) .

Today, one crucial question in the immune response against SARS-CoV-2 is the persistence of antibodies. The first reports suggested that the persistence of IgG antibodies is up to 6 months after the onset of the symptoms . Similar results were observed by Liu et al. (2021) . In that study, IgG antibodies were detected for 6 months.

In contrast, IgM antibodies were reduced in approximately 80% of the patients. Recently, a study evaluated a cohort of 254 samples with diverse symptoms and observed that IgG titres remained detectable for 6-8 months after symptom onset (Dan et al., 2021) . However, a recent report suggests that IgG antibodies can persist for 10 months (Dudreuilh et al., 2021) . In that report, 2 of 84 patients showed IgG antibodies after 10 months.

Currently, several vaccines have been approved in different countries (Forni & Mantovani, 2021) . For COVID-19, there are mRNA-based vaccines available that have shown the highest levels of protection, followed by viral vector, protein subunit, and whole-inactivated viruses (Kim et al., 2021) . In Mexico, mRNA and viral vectors are vaccines being used to immunize the population (Vacunacovid.Gob.Mx, 2021 -CoV-2 (Stankov et al., 2021) . In contrast, the available information for CanSinoBio, a viral-vector vaccine, its limited (Zhu et al., 2020) and indicates an efficacy of 65%, which places it as one of the vaccines with the lowest efficacy (Forni & Mantovani, 2021) .

In Mexico, as in other countries, many questions have arisen about this vaccine. Our study established an indirect enzyme-linked immunosorbent assay (ELISA) to detect IgM-, IgA-, and IgG-specific antibodies against the S1 protein of SARS-CoV-2 in convalescent patients. In addition to measuring the persistence of antibodies, we evaluated the response of individuals with a history of COVID-19 or naïve individuals to COVID-19 vaccinated with either Pfizer-BioNTech or CanSinoBio.

For this study, 145 negative control serum samples were included.

Sixty-five samples were collected between 2017 and 2019 from a previous breast cancer project, and they were kindly provided by Dr. Graciela Caire-Juvera (CIAD, A.C.). Eighty samples were collected between 2015 and 2018 from a project on seroprevalence against Cryptosporidium spp., and they were kindly provided by Dr. Olivia Valenzuela (Universidad de Sonora). These serum samples were included because no SARS-CoV-2 was circulating at that time.

Serum samples from RT-PCR-positive adult patients were included.

One hundred forty-two samples were from convalescent and non- 

The S1 domain of SARS-CoV-2 includes amino acids 1-633 from the full Spike protein. The expression gene construct was designed with the deduced S1 sequence, preceded by a signal peptide and a Hist-tag (6xHist) terminal carboxyl domain (Supplementary Figure S1A ). The gene was synthesized and cloned into a pcDNA3.1(-) vector by Gen-Script (GenScript, Piscataway, New Jersey, USA), producing the expression plasmid pcDNA3.1(-)/SARS-CoV-2 S1 for a mammalian expression system.

The expression of the recombinant SARS-CoV-2 S1 protein was performed in the Expi293 Expression System following the manufacturers' instructions (Thermo Fisher Scientific, Waltham, Massachusetts, USA). Briefly, Expi293 cells were grown in Expi293 expression medium.

Then, the transfection complex was prepared by mixing 30 µg of pcDNA3.1(-)/SARS-CoV-2 S1 and 81 µg of Expifectamine (both diluted in OptiMEM-I) and incubated for 20 min at room temperature. The complex was added to a flask with 75 × 10 6 Expi293 cells in 25.5 mL of Expi293 expression medium and incubated at 37 • C and 125 rpm with 8% CO 2 . After 20 h, enhancers 1 and 2 were added to the medium and the cells were incubated for 4 more days until harvesting.

After 4 days of transfection, the culture supernatant was harvested and clarified by centrifugation at 1600 rpm for 10 min. Then, the clarified supernatant was filtered through a 0.22-µm filter followed by purifica-tion on immobilized metal affinity chromatography (IMAC) using a His-Trap HP column (Cytiva, Shrewsbury, Massachusetts, USA). The elution was carried out in the chromatograph ÄKTA prime plus (GE Healthcare Life Sciences, Marlborough, Massachusetts, USA) by a linear gradient from 0 to 100% elution buffer (500 mM imidazole), where the recombinant S1 protein was eluted in 26% elution buffer (approximately 150 mM imidazole), collecting 1 mL fractions. The total S1 protein purified was quantified by a Bradford assay, concentrated, and desalted with an Amicon Ultra15 (10 kDa cutoff) cartridge (Millipore, Burlington, Massachusetts, USA) followed by dilution in phosphate buffered saline (PBS) pH 7.4 to a final concentration of approximately 1 mg/mL. For characterization of the purified recombinant S1 protein, SDS-PAGE and western blotting were performed. Briefly, 3 µg of purified S1 was heat-denatured and electrophoresed in a 10% polyacrylamide gel under reducing conditions followed by Coomassie blue staining.

The gel was transferred to a PVDF membrane and blocked overnight with 5% nonfat milk in PBS with 0.05% of Tween 20. Afterwards, the blocked membrane was incubated with alkaline phosphataseconjugated mouse anti-polyhistidine (Sigma-Aldrich, St. Louis, Missouri, USA) for 1 h at 37 • C, and BCIP®/NBT alkaline phosphatase substrate (Sigma-Aldrich, St. Louis, Missouri, USA) was added for detection.

SARS-CoV-2 S1 protein (2 µg/mL) was used to coat Maxisorp ELISA 

Statistical comparisons between negative samples and positive con- 

The recombinant S1 protein produced in this study (633 aa) was successfully expressed in Expi293 cells. Sodium dodecyl sulphatepolyacrylamide gel electrophoresis (SDS-PAGE) Coomassie blue staining allowed visualization of the S1 protein with an approximate weight of 130 kDa and a purity over 90% (Supplementary S1B). Western blot analysis confirmed the purification of the recombinant S1 protein by identifying the carboxyl-terminal domain His-tag (Supplementary S1C). It is notable that even though the estimated molecular weight from the amino acid sequence is approximately 74 kDa, the purified protein presented a higher weight. After expression, purification and desalting, the yield of recombinant SARS-CoV-2 S1 protein expressed in Expi293 cells was 40 mg/L of cell culture harvested at day 4 posttransfection.

This study developed three indirect ELISA types to detect IgG, IgA and IgM antibodies against SARS-CoV-2 using the recombinant S1

protein as the target. First, the optimal antigen concentration for coating was evaluated and set at a concentration of 2 µg/mL (50 µL/well = 100 ng/well). Serum dilution was set at 1:100 and diluted with w/0.1% low-free milk PBST. Serum and conjugate incubations were performed for 30 min at room temperature with slight agitation.

In some experiments, the coated plates were frozen immediately at −20 • C until further use or blocked for 1 h at room temperature and kept at 4 • C for 2 weeks. No significant differences were observed when plates were either frozen or blocked and kept at 4 • C (data not shown).

After optimization, the cutoff was determined. For IgG, 145 samples with no history of SARS-CoV-2 were used as a negative control (Figure 1a) . In this case, the cutoff value represents the mean ± 2 SD and it was set at 0.150 (mean = 0.071, 2 SD = 0.079). For IgA and IgM, 88 samples with no history of SARS-CoV-2 were used as negative controls (Figure 1b,c) . The cutoff for IgA (mean ± 2 SD) was set at 0.180 (mean = 0.074, 2 SD = 0.105). For IgM, the cutoff (mean ± 2 SD) was set at 0.130 (mean = 0.048, 2 SD = 0.080). Few samples showed a value over the cutoff, suggesting high specificity. 

In addition to the persistence of the antibodies, this work evaluated the IgG, IgA and IgM antibody response in persons with a history of COVID-19 (the convalescent-COVID-19 group) and without a history of COVID-19 (naïve-COVID-19 group). One group was vaccinated with Pfizer-BioNTech and the other with CanSinoBio. Figure Finally, a comparison between the naïve COVID-19 group vaccinated with CanSinoBio and Pfizer-BioNTech is shown in Figure 6 . Two weeks after vaccination, no differences were observed in the IgG or IgA response. However, after completing the vaccination scheme for both vaccines, IgG and IgA were significantly higher (p = .0001 and p = .0004, respectively) in individuals vaccinated with Pfizer-BioNTech than in those vaccinated with CanSinoBio.

In this work, we standardized an indirect ELISA to detect IgM, IgG and IgA in sera from convalescent donors and evaluated the antibody (657-664) (Dawood et al., 2020) . In addition, approximately 90% of neutralizing antibodies are induced by the receptor-binding domain (amino acids 306-527) (Piccoli et al., 2020) . In contrast, the S2 protein is more conserved within other coronaviruses. These results confirm that the S1 protein is an excellent option for antibody detection.

In terms of yield, we obtained a higher performance than the efficiency reported for other SARS-CoV-2 antigens, such as the whole spike protein (5 mg/L) produced in the same expression system (Amanat et al., 2020) .

The specificity and sensitivity of this S1 ELISA were evaluated with respectively. For IgM, the specificity was 100.00% (95.89% -100%).

These results are similar to those observed in other studies (Krähling et al., 2021) . However, one limitation was that we were unable to evaluate the cross-reactivity against other coronaviruses. The sensitivity for IgG was 94.37% (CI, 89.20% -97.54%), and eight of 142 positive samples were negative. Interestingly, one of these negative samples was positive for IgA. The remaining seven samples were negative for IgG, IgA and IgM. All positive samples were selected from donors of convalescent plasma, and all were PCR-confirmed for COVID-19. The lack of antibodies in SARS-CoV-2-infected patients has been reported previously (Marklund et al., 2020) These values are similar to other reports (Okba et al., 2020; Suhandynata et al., 2020) . In conclusion, these results can support the accuracy of the S1 ELISA.

The dynamics of the antibody response after SARS-CoV-2 infection have been explored in several studies. Many of them have evaluated antibodies, especially IgG, in periods ranging from 6 to 8 months. In most, the results confirmed the presence of IgG. A small number of studies have evaluated the dynamics at 10 months (Dan et al., 2021; Liu et al., 2021) . Our study evaluated IgG, IgA and IgM at three different time points. The longest was at approximately 10 months and confirmed that IgG could be detected at this time point in some patients.

IgG has been used as a marker for past infection. These results confirm that in patients without vaccines, IgG can be detected until the 10th month after symptom onset. Currently, with the massive use of vaccines, the dynamics of IgG could be different. Interestingly, five of our patients received the vaccine just before the third blood sampling, and an increase in IgG antibodies was obvious. Interestingly, IgA and IgM were not detected at longer periods, in agreement with previous reports (Padoan et al., 2020) .

Today, several reports support the efficacy of Pfizer-BioNTech, in contrast to CanSinoBio. However, the available information is limited (Zhu et al., 2020) . In this study, we confirmed that hybrid immunity is induced by different vaccines, including CanSinoBio. Our results showed that vaccination of convalescent COVID-19 individuals triggered a robust response of IgG 2 weeks after vaccination. This response was observed in the groups vaccinated with

Pfizer-BioNTech and with CanSinoBio. We did not observe significant differences between the vaccines in this situation. These results demonstrated that one dose of CanSinoBio can refresh and reinforce the immunity induced by the infection, similar to other vaccines, such as Pfizer-BioNTech (Saadat et al., 2021) . However, in naïve COVID-19

persons, one dose of CanSinoBio induced a significantly lower response to IgG and IgA antibodies than vaccination with two doses of Pfizer-BioNTech. Another important observation was that at 4 weeks after a single dose of CanSinoBio, 18% of the individuals remained antibody negative. We decided to evaluate the response at 4 weeks after vaccination because previous reports showed that at that point the vaccine induced a robust IgG response (Zhu et al., 2020) . Unfortunately, we were not able to evaluate the presence of neutralizing antibodies in this study. However, several studies agreed that there is a correlation between the levels of IgG and neutralizing antibodies. Even with this limitation, it is possible to state that one dose of CanSinoBio in naïve COVID-19 individuals induces moderate immunity, which is associated with the observed moderate (65%) efficacy. It is probable that a second dose of CanSinoBio could increase immunity, as well as its efficacy.

These considerations are important because the new variants of the virus represent a challenge to our immunity.

In this study, we verified that in individuals with previous SARS-CoV-2 infection, a single dose of vaccine induces a response that can be immunologically equivalent to a full vaccine schedule in naïve individuals. Several authors have confirmed that one dose of vaccine in previously infected patients induces a strong antibody response and that naïve patients require a second dose of vaccine to induce a robust response to antibodies, in contrast to recovered individuals (Callegaro et al., 2021; Ciccone et al., 2021; Ma et al., 2020) . The results obtained in our report are in agreement with these observations. In previous reports, showed that IgA antibody responses were low compared with IgG and that IgM was lower in patients vaccinated with RNA vaccines . In agreement with these results, we observed that the IgM response was low for both vaccines. This is different from the IgM response in naturally infected patients, where

IgM is produced at higher levels. These results could suggest that proteins other than S are involved in the induction of IgM and that viral infection activates an additional immunological mechanism that produces a high response of IgM, in contrast to vaccines.

In conclusion, this study developed an indirect ELISA to detect IgG, IgA and IgM against SARS-CoV-2 spike protein S1. With this assay, it was possible to demonstrate that the persistence of antibodies continues for up to 10 months for IgG antibodies, in contrast to IgA and

IgM. Additionally, we demonstrated that in individuals with a history of COVID-19, the CanSinoBio vaccine induced a robust immune response to IgG, similar to that reported for Pfizer-BioNTech. We also showed that in persons without a previous infection of COVID-19, CanSino-Bio induced a moderate response to antibodies after 4 weeks. These results highlight the importance of more studies to improve immunity in those vaccinated with CanSinoBio who had no history of COVID-19

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The authors are grateful to Nilza Cordova and Julia Real for their valuable technical assistance. This research was funded by Consejo Nacional de Ciencia y Tecnología (CONACyT), grant number 314320.

The authors declare no conflicts of interest. 

Raw data in this study are available from the corresponding authors on request. All requests for raw and analyzed data and materials will be reviewed by the corresponding authors to verify whether the request is subject to any intellectual property or confidentiality obligations.

The authors confirm that the ethical policies of the journal, as noted on the journal's author guidelines page, have been adhered to. The study was approved by the Ethics Committee of CIAD (Decision number:CONBIOÉTICA-26-CEI-001-20200122).

Verónica Mata-Haro https://orcid.org/0000-0002-7394-4569Olivia Valenzuela https://orcid.org/0000-0002-4456-2107Fernando Pérez-Jacobo https://orcid.org/0000-0003-0295-5508Jesús Hernández https://orcid.org/0000-0002-5131-3600