key: cord-0913939-pygta2g9
authors: Silva, Valéria O.; Yamashiro, Rosemeire; Ahagon, Cintia M.; de Campos, Ivana B.; de Oliveira, Isabela P.; de Oliveira, Elaine L.; López‐Lopes, Giselle I. S.; Matsuda, Elaine M.; Castejon, Marcia J.; de Macedo Brígido, Luís F.
title: Inhibition of receptor‐binding domain—ACE2 interaction after two doses of Sinovac's CoronaVac or AstraZeneca/Oxford's AZD1222 SARS‐CoV‐2 vaccines
date: 2021-10-20
journal: J Med Virol
DOI: 10.1002/jmv.27396
sha: 9274f938c2222766f5dc5c9e41b5c0074f9001c4
doc_id: 913939
cord_uid: pygta2g9

Practical laboratory proxies that correlate to vaccine efficacy may facilitate trials, identify nonresponders, and inform about boosting strategies. Among clinical and laboratory markers, assays that evaluate antibodies that inhibit receptor‐binding domain (RBD) ligation to angiotensin‐converting enzyme‐2 receptor (receptor‐binding inhibition [RBI]) may provide a surrogate for viral neutralization assays. We evaluated RBI before and after a median of 34 days (interquartile range [IQR]: 33–40) of the second dose of severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) Sinovac's CoronaVac (CN) or AstraZeneca/Oxford's AZD1222 (AZ) vaccines in 166 individuals. Both vaccines elicited high inhibitory titers in most subjects, 95% (158/166), with signal inhibition above 30% and 89% (127/143) with more than fourfold increase from prevaccination titers, but titers tend to decrease over time. Both postvaccination inhibitory titers (95%, IQR 85%–97% for AZ vs. 79%, IQR 60%–96% for CN, p = 0.004) and pre/post‐titer increase (AZ 76%, IQR 51%–86% for AZ vs. 47%, IQR 24%–67% for CN, p < 0.0001) were higher among AZ vaccinees. Previous serological reactivity due to natural infection was associated with high prevaccination signal inhibition titers. The study documents a robust antibody response capable of interfering with RBD–angiotensin‐converting enzyme binding. Evaluation of SARS‐CoV‐2 infection incidence in these populations is necessary to assess its association to protection and its duration.

| 1 antibodies are key to advance a SARS-CoV-2 vaccine candidate in the regulatory testing pipeline and its importance is supported by evidence for SARS-CoV-2 6 as well as other viral pathogens. 7 T cell response is necessary to support B cell maturation and may exert a direct antiviral effect. 4 However, techniques to detect T cell response tend to be more laborious, depend on more complex technology, and have limited standardization across laboratories, so it is tempting to identify relevant antibody-based assays. A key step in the viral life cycle is the binding of domains at spike 1 protein of the virus, the receptor-binding domain (RBD) to the main cellular receptor, the angiotensin-converting enzyme 2 (ACE2) protein. 3 To evaluate the presence of antibodies able to inhibit RBD-ACE2 binding, a potential proxy of protection, we tested binding inhibition in prevaccination blood and after the second dose among individuals vaccinated with Sinovac's CoronaVac (CN) or AstraZeneca/Oxford's anti-COVID-19 vaccines.

The study is registered and approved by the Institutional ethical committee (CAAE 43250620.4.1001 .0059 and CAAE 31924420. 8 .0000.0059), and written informed consent was obtained from all subjects. Individuals were enrolled for humoral evaluation during 2020. These volunteers had one or more serological evaluations, had COVID-19 related clinical symptoms investigated with questionnaires, and SARS-CoV-2 RNA tests if symptomatic. Some asymptomatic cases with an epidemiological link to a patient with COVID-19 were also tested. Volunteers were asked to collect an additional blood sample before vaccination and after 1 month of the second dose of available SARS-CoV-2 vaccines at the time, either CN (Sinovac Life Sciences) or AZD1222 (AZ; AstraZeneca). The cohort, although mostly laboratory workers, includes relatives and health workers with direct contact with patients. However, most were not involved in direct COVID-19 care. Clinical disease followed the list of symptoms listed by WHO 8 except "altered mental state."

Confirmation of SARS-CoV-2 RNA was obtained for quantitative reverse transcription-polymerase chain reaction (RT-qPCR) from nasopharyngeal and/or oropharyngeal secretions collected with swab or saliva/gargle throat wash. 9 SARS-CoV-2 RNA was retro transcribed and amplified using available tests, in most cases, the commercial Allplex Kit (Allplex TM 2019-nCoV), but other tests, based on the Charité protocol 10 were additionally used. Positive cases had an RT-qPCR C t < 37 in one of three viral targets (e.g., E, RdRP, and N). Study cases had a serological evaluation with one or more tests, performed following the manufacturer's instructions. Tests included (i) lateral flow immunochromatographic assay (Wondfo SARS-CoV-2 antibody test; Guangzhou Wondfo Biotech Co., Ltd.), that detects immunoglobulin G (IgG) and IgM to the SARS-CoV-2 binding domain of the spike protein (S); (ii) electrochemiluminescence immunoassay (Elecsys anti-SARS-CoV-2; Roche Diagnostics) that detects total antibodies to nucleocapsid (N) antigen, and (iii) Chemiluminescent microparticle immunoassay (SARS-CoV-2 IgG; Abbott Diagnostics) that detects IgG to N antigen, and (iv) Microarray enzyme-immunoassay (SARS-CoV-2 ViraChip ® Test Kit; ViraMed Biotech AG) that detects IgG or IgA to SARS-CoV-2 spike protein 1 and 2 subdomain (S1, S2) and N domains.

Antibodies able to interfere with RBD-ACE2 interaction were measured in sera with a competitive enzyme-linked immunosorbent assay (ELISA) 

Prevaccination signal Inhibition titers, available for 143 volunteers, were generally low, with a median of 13% inhibition titers (IQR, 5%-38%). However, some cases showed high titers, with 28% of the cases (40/143) with inhibition above 30%. Higher prevaccination titers were associated with the presence of antibodies to SARS-CoV-2 (Table 1) , with seropositive cases showing higher prevaccination inhibition titers (69%, 38%-87%), as compared to seronegative individuals (7%, 3%-14%, p < 0.0001).

The presence of symptoms did not show association to binding (signal) inhibition titers, with similar prevaccination titers for seronegative asymptomatic, including individuals with no evidence of COVID-19, (8%, 4%-15%) and symptomatic cases (6%, 2%-13%, p = 0.17) as well as among symptomatic seropositive (69%, 48%-86%) or without clinical symptoms (60%, 18%-97%, p = 0.87).

Postvaccination inhibition titers, obtained at a median of 34 days (33-40) after the second dose of either vaccine product were generally high (84%, 62%-96%), with most individuals (95.2%, 148/166) with titers above minimal inhibition (30% receptorbinding inhibition [RBI] ) and 78% (130/166), with titers above 60% inhibition (two times the cutoff). If only cases with results 3-6 weeks post the second dose are evaluated, 97% (125/129) have signal inhibition titers above 30%. Considering the 143 with both pre-/post-vaccine determinations, a fourfold or higher increase in titer was observed in 127 (89%) of volunteers. Titers tended to be associated with prevaccination values, and seropositive categories (Table 1 ) had higher inhibition titers (96%, 91%-97%) than seronegative individuals (70%, 54%-87%, p > 0.0001). There was no significant association of inhibition titers to age at Spearman correlation (p = 0.1) or by comparing age quartiles (p = 0.16).

To compare RBD-ACE2 inhibition according to the vaccine used, we compared volunteers according to the serological status before vaccination. Table 2 describes these patients, showing that seronegative AZ vaccinees, having higher postvaccination inhibition titers (95%, 85%-97%), as compared to seronegative CN vaccinees (79%, 60%-96%, p = 0.004). This increase from prevaccination titers is also higher among AZ recipients, even if only cases with low prevaccination binding inhibition (signal inhibition < 30%) were analyzed (AZ 84%, 72%-88% vs. CN 57%, 43%-73%, p < 0.0001). No significant difference was observed for previously seropositive individuals.

T A B L E 2 Volunteers' characteristics according to previous COVID-19 serological status and vaccine product used (CoronaVac or AZ) As the time of postvaccination collection is longer for some CN recipients, and antibodies may wane with time, 6 we also analyzed only cases at the same time range (28-34 days, n = 73). We still observe a difference in postvaccination titers (AZ 95%, 85%-96% vs. (51%-71%) to 42% (30%-51%) binding inhibition.

Laboratory correlates of immunity, proxies of immunological protection to SARS-CoV-2 acquisition or for development of severe disease, are key for better handling of the pandemic. Although some scientists predict an end to SARS-CoV-2, 13 viral characteristics, along with the health inequalities among nations (as well as within some countries) suggest that control, rather than eradication seems a more reasonable goal. In that scenario, correlates of immunity to SARS-CoV-2 may be used to define protection and favor targeting of nonpharmacological restrictions, an intent for an early generation of antibodies tests that proved inadequate for this purpose. 8 These markers of protection are also useful in vaccine development, facilitating the selection of products for further testing or even used as secondary endpoints of vaccine studies. These markers may also help in the triage of volunteers at enrollment, as many volunteers in new trials will be previously infected and/or previously vaccinated. They may also be used to monitor the need for boosting doses and inform on better boosting strategies.

The interaction of the virus RBD to the main host protein that allows viral ligation to a permissive cell, the ACE2 molecule, is considered a key step in the viral life cycle. 6 We evaluated the titer of RBD-ACE2 binding inhibition (RBI) of pre and postvaccination sera from a small cohort of well-characterized health workers and re- Moreover, the duration and some other characteristics of antibodies generated after natural infection may differ from that of vaccine-induced receptor binding inhibition, but both vaccineinduced response and natural infection provide protection to infection in most individuals. 6, 14 Our study documented receptor binding inhibition generated by both vaccine products evaluated, AZ and CN, with the formed inducing titers higher after about 1 month of the second dose.

Although it is tempting to associate the higher postvaccination titers of AZ vaccinees with the vaccine product, it is important to highlight that the second dose of AZ was applied after 3 months, whereas CN's second dose was 21-28 days after the first dose. Some studies suggest that a longer interdose spacing may provide a more robust immunological response. 15 However, both products have been providing protection from severe disease and the actual role of antibodies in protection to current and emerging variants of SARS-Cov-2 will need prospective studies. 

Execution of laboratory tests were performed by Rosemeire Yamashiro and Marcia J. Castejon. Samples management was performed by

Statistical analysis was performed by

The manuscript was written by Valéria O. Silva and Luís F. de Macedo Brígido

Anonymized datasets generated for the current study are available from the corresponding author upon reasonable request

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Inhibition of receptor-binding domain-ACE2 interaction after two doses of Sinovac's CoronaVac or AstraZeneca/ Oxford's AZD1222 SARS-CoV-2 vaccines