key: cord-0752278-maqo773b
authors: Horndler, L.; Delgado, P.; Romero-Pinedo, S.; Quesada, M.; Balabanov, I.; Laguna-Goya, R.; Almendro-Vazquez, P.; Llamas, M. A.; Fresno, M.; Paz-Artal, E.; van Santen, H. M.; Alvarez, S.; Olmo, A.; Alarcon, B.
title: DECREASED BREADTH OF THE ANTIBODY RESPONSE TO THE SPIKE PROTEIN OF SARS-CoV-2 AFTER VACCINATION
date: 2021-08-14
journal: nan
DOI: 10.1101/2021.08.12.21261952
sha: fd786496a294cb0bf1837f6895f1e41ef8e84f04
doc_id: 752278
cord_uid: maqo773b

The rapid development of vaccines to prevent infection by SARS-CoV-2 virus causing COVID-19 makes necessary to compare the capacity of the different vaccines in terms of development of a protective humoral response. Here, we have used a highly sensitive and reliable flow cytometry method to measure the titers of antibodies of the IgG1 isotype in blood of volunteers after receiving one or two doses of the vaccines being administered in Spain. We took advantage of the multiplexed capacity of the method to measure simultaneously the reactivity of antibodies with the S protein of the original strain Wuhan-1 and the variant B.1.1.7 (Alpha). We found significant differences in the titer of anti-S antibodies produced after a first dose of the vaccines ChAdOx1 nCov-19/AstraZeneca, mRNA-1273/Moderna, BNT162b2/Pfizer-BioNTech and Ad26.COV.S/Janssen. Most important, we found a relative reduction in the reactivity of the sera with the B.1.1.7 versus the Wuhan-1 variant after the second boosting immunization. These data allow to make a comparison of different vaccines in terms of anti-S antibody generation and cast doubts about the convenience of repeatedly immunizing with the same S protein sequence.

Vaccines to prevent the worst effects of infections by SARS-CoV-2 virus causing COVID-19 have been rapidly developed. This unprecedented fast development of vaccines has allowed a significant reduction in the number of deaths caused by COVID-19 as well as in the number of patients admitted to the intensive care units of hospitals. In Spain, the immunity reached by the widespread vaccination, and immunity due to previous infections, is likely responsible for the low number of deaths caused by the so-called the fifth-wave by the end of July 2021, in spite reaching a number of reported infections similar to that of previous waves in the fall of 2020 and beginning of year 2021 that caused a much higher number of deaths [1] [2] [3] [4] , although reports of rare cases of thrombotic thrombocytopenia associated to both the ChAd and Ad26 vaccines have been published 5, 6 . However, the emergence of variants of concern (VOC) of SARS-CoV-2, specifically the Delta variant (B.1.617) with a very high transmission capacity 7 is threatening the vaccination strategies in Europe and the USA, given the fact that the efficacy of vaccines against the Delta variant is reduced 8 , and fully vaccinated people can become infected and spread the virus efficiently to others 9 . The possibility of giving a third boosting vaccination to immunosuppressed patients to increase the titers of antibodies to the spike (S) protein is being considered 10 perpetuity.

preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in

The copyright holder for this this version posted August 14, 2021. ;  https://doi.org/10.1101/2021.08.12.21261952 doi: medRxiv preprint infect vaccinated individuals seems to correlate with its ability to escape neutralization by antibodies produced in response to vaccines 7 .

Serological tests are usually established by detecting the presence of viral antigenspecific IgG or IgM in the serum of individuals using recombinant fragments of the S or N proteins and tests based on ELISA or lateral flow assay 11, 12 . A disadvantage of those tests is that neutralizing antibodies are not directed against the N protein and that recombinant fragments of the S protein miss the quaternary structure of the protein trimer, which is the native form of the spike protein in the viral envelope. Therefore, part of the neutralizing antibodies directed against the native S trimer could be missed in serological tests based on the expression of recombinant proteins. Recently, we have described a flow cytometry (FC) test that makes use of the cell line of hematopoietic origin Jurkat to express the native S protein of SARS-CoV-2 together with a truncated form of the human EGF receptor (huEGFRt) as a normalizing tool 13 .

The method allows the accurate determination of seropositivity and the measurement of antibody titers in sera from post-convalescent patients 13 . Here, we have used this FC method to compare the production of antibodies against the S protein in response to the prime immunization and the booster immunization of the four most common vaccines being used in Spain. In addition, we have used the multiplexed capacity of the method to simultaneously measure the reactivity of sera from vaccinated individuals with the S protein of the original Wuhan-1 isolate versus the B.1.1.7 (Alpha) mutant as an example of VOC. Our results help to stir the debate about the convenience of a third dose of the same vaccines.

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We previously used a lentiviral vector to express the full spike "S" protein of SARS-CoV2 with the original Wuhan-1 sequence, followed by a truncated human EGFR protein (huEGFRt) linked by a T2A self-cleaving sequence in transduced cells 13 . This system allows expression of the two proteins from a monocystronic mRNA. We produced transducing supernatants to express the construct in the human leukemic cell line Jurkat. Taking advantage of the fact that the expression of S protein is coupled to that of huEGFRt, we calculated a ratio of mean fluorescence intensities of antibodies against S versus an antibody against huEGFR as direct estimation of the relative quantity of immunoglobulins against the S protein in sera or other fluids from different donors.

In order to detect simultaneously the presence of antibodies against the S protein of the B.1.1.7 VOC of SARS-CoV-2, we used a similar strategy, cloning the S sequence of the variant ( (Fig. 1A) . Simultaneously, we could gate on the EGFR+ cells to calculate the ratio of [fluorescence anti-S/fluorescence anti-EGFR] as a measurement of the titer of antibodies of the IgG1 isotype against the S protein of the Wuhan-1 variant. Figure 1A shows examples of how the plots of IgG1 anti-S and either GFP or anti-EGFR appear for a positive control serum and a negative control serum. Using this procedure, we titrated sera from donors that were diagnosed as infected either with the original Wuhan-1 or the B.1.1.7 variants. For each serum dilution, the S/GFP ratio (B.1.1.7 variant) and the S/EGFR ratio (Wuhan-1 strain) All rights reserved. No reuse allowed without permission. perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in The copyright holder for this this version posted August 14, 2021. ; https://doi.org/10.1101/2021.08.12.21261952 doi: medRxiv preprint were calculated and plotted. A lineal regression curve was fitted to the data and the result is shown in Fig. 1B . The sera of donors that were infected with the Wuhan-1 strain reacted almost as intensely with the B.1.1.7 variant as with the Wuhan-1 strain, and vice versa. However, the reactivity of the sera from patients recovered of infection with B.1.1.7 was slightly better with Jurkat-SB117 than with Jurkat-Swuhan. This was reflected in the fitting lines for those sera in the S/EGFR MFI (x-axis) versus the S/GFP MFI (y-axis) plot to be slightly above those for sera from patients recovered from infection with the Wuhan-1 strain (Fig. 1B) . This fact was also reflected by plotting the slopes of the fitting lines (Fig. 1C) . These results suggest that most of the antibodies against the S protein generated by patients recovered from infection reacted with both strains of SARS-CoV-2. Nonetheless, there were also differences in line with the sequence divergence of both strains (Fig. EV1) . These data suggested that we could make use of the reactivity of sera with Jurkat-Swuhan and Jurkat-SB117 to determine the relative capacity of the anti-S antibodies to bind to each of the two variants.

Using this system, we decided to carry out a retrospective study to compare the generation of anti-S antibodies reactive with the Wuhan-1 and the B.1.1.7 variants in response to the most common vaccines being employed at present in Spain and compare this response with that of patients recovered from infection by SARS-CoV-2 during the first wave of the pandemics (https://covid19.who.int/region/euro/country/es) between March and May 2020 (patients 2020, Fig. 2 and Table 1 ) and with that of patients recovered from infection during the fourth wave of the pandemics between March and May 2021 (patients 2021, Fig. 2 and Table   1 ). Patients 2020 were likely infected by the Wuhan-1 strain and patients 2021 were likely to be infected by the B.1.1.7 strain, given its preponderance at that time (https://theconversation.com/the-uk-variant-is-likely-deadlier-more-infectious-and-becomingdominant-but-the-vaccines-still-work-well-against-it-156951). We first compared sera from volunteers that received the first dose of the vaccine 3-4 weeks earlier. As shown in Fig perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in The copyright holder for this this version posted August 14, 2021. ; https://doi.org/10.1101/2021.08.12.21261952 doi: medRxiv preprint BNT162b2/Pfizer-BioNTech (BNT) presented significantly higher titers of IgG1 anti-S antibodies of the Wuhan-1 strain than Patients 2020. However, donors that had been immunized with the ChAdOx1 nCov-19/AstraZeneca-Oxford University vaccine (ChAd) generated lower titers of antibodies against the Wuhan-1 strain than Patients 2020 and Patients 2021 and also than donors immunized with the Moderna and BNT vaccines ( Fig. 2A) . Although the number of These data also show that the relative reactivity of sera from Patients 2021 and even Patients 2020 with the B.1.1.7 VOC was better than that of sera from all vaccinees.

We next aimed to measure the effect of the second (booster) dose of vaccine on the generation of IgG1 anti-S antibodies against the two SARS-CoV-2 variants. Compared to the titer generated after the first, priming, dose, the second immunization with Moderna and BNT did not induce a significant increase in the reactivity with the Wuhan-1 (Fig. 3A) or B.1.1.7 (Fig. 3B) variants. By contrast, a significant increase in the recognition of both variants occurred for volunteers vaccinated with the second dose of ChAd, compared to those that had received only one dose of the same vaccine ( Fig. 3A and 3B ). Nevertheless, sera from volunteers who had received two doses of BNT, but not those who received two doses of Moderna, still reacted All rights reserved. No reuse allowed without permission. perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in We next interrogated if the loss of relative reactivity against B.1.1.7 was also produced in patients recovered after the first wave in 2020 who received one dose of either BNT or Moderna (Table 1 ). Compared to the titer pre-vaccination, the priming dose of vaccine caused an increased reactivity of IgG1 antibodies against the Wuhan-1 variant and also, although at a lower extent, against the B.1.1.7 variant (Fig. 4B ). Immunization of individuals who were already immune due to prior infection by SARS-CoV-2 resulted in a relative loss of reactivity with the B.1.1.7 variant, when analyzed in a per-individual base (Fig. 4C ).

The effect of vaccination on the relative recognition of the S protein of the B.1.1.7 variant versus that of the Wuhan-1 strain was also followed in sera from volunteers of nursing All rights reserved. No reuse allowed without permission. perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in The copyright holder for this this version posted August 14, 2021. ; https://doi.org/10.1101/2021.08.12.21261952 doi: medRxiv preprint homes who had `recovered after infection in 2020 and 2021 and were later vaccinated with BNT (Table 1) . Serum samples were taken just before vaccination, 15 days after the first dose, 21 days after the first dose, 1 week after the second dose, and 4 weeks after the second dose of the BNT vaccine. The first dose of BNT resulted in a significant increase in antibody reactivity against the Wuhan-1 and B.1.1.7 variants 21 days after vaccination ( Fig. 5A and 5B) .

Interestingly, and compared with samples taken at day 21 after the first priming dose, the second dose of vaccine did not cause a significant increase in serum reactivity either 1 week or 4 weeks after the booster immunization ( Fig. 5A and 5B ). Most important, vaccination provoked a relative loss in the capacity of recognition of the B.1.1.7 variant starting as early as 15 days after the first dose of BNT and worsening since then (Fig. 5C ).

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In this study we have measured the production of antibodies of the IgG1 isotype to the spike protein of SARS-CoV-2 in response to the four different vaccines most frequently used in Spain.

We have compared those data with antibodies present in sera from patients recovered from Although antibodies made in response to vaccines based on the original Wuhan-1 strain sequence do also bind the B.1.1.7 variant, suggesting that most epitopes are conserved between both sequences, there is a relative loss of reactivity with the B.1.1.7 variant compared to the Wuhan-1 strain occurring upon administration of the booster dose of vaccine. This is somehow expected since repeated immunization with the same antigen sequence leads to the generation of higher affinity antibodies that fit better the epitopes of the immunogen. This increase in affinity has the negative side effect of reducing the "breadth" of the antibodies, that is, their capacity to bind to epitopes that differ slightly from those of the immunogen. Such effect has All rights reserved. No reuse allowed without permission. perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in The copyright holder for this this version posted August 14, 2021. ; https://doi.org/10.1101/2021.08.12.21261952 doi: medRxiv preprint been observed before, for instance, as a result of vaccination with inactivated influenza virus isolated in immunization campaigns, that result in an efficient neutralization of that particular seasonal variant of influenza but results in reduced capacity to neutralize other variants 14 perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in The copyright holder for this this version posted August 14, 2021. ; https://doi.org/10.1101/2021.08.12.21261952 doi: medRxiv preprint ncov/science/science-briefs/fully-vaccinated-people.html). At present, we are generating a Jurkat-S cell line expressing the Delta variant to confirm that our findings on loss of antibody breadth upon vaccination for the Alpha variant also hold true for the Delta VOC. The generation of this cell line will allow to measure the reactivity of antibodies against the trimeric, native, form of the S protein, something that the serological tests based on the use of recombinant proteins do not allow.

While we are awaiting for the results with Jurkat-S expressing the Delta VOC, our results with the Alpha variant suggest that third doses of the present vaccines to the general population might not be the best approach to increase the immunity to the emerging VOCs. In our opinion, a third dose should be limited to the population that has been demonstrated to have been poorly responsive to the first two prime and boost doses.

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The human T-cell line Jurkat clone E6-1 was acquired from ATCC (TIB-152) and was maintained in complete RPMI 1640 supplemented with 5% fetal bovine serum (FBS, Sigma) in a 5% CO2 incubator. Human embryonic kidney HEK293T cells (ATCC CRL-3216) were maintained in DMEM supplemented with 10% FBS in a 5% CO2 incubator. All cell lines were routinely tested for the absence of mycoplasma.

The generation of Jurkat-S cells expressing the spice protein of the Wuhan-1 (Jurkat-Swuhan) variant has been previously described 13 For transduction, lentiviral-transducing supernatants were produced from transfected packaging HEK-293T cells as described previously 18 . Briefly, lentiviruses were obtained by cotransfecting plasmids pCMV-dR (gag/pol) and using the JetPEI transfection reagent (Polyplus Transfection). Viral supernatants were obtained after 24 and 48 hours of transfection. Polybrene perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in The copyright holder for this this version posted August 14, 2021. ; https://doi.org/10.1101/2021.08.12.21261952 doi: medRxiv preprint

A total of 700 human sera were tested. A first set of 50 sera from Empireo obtained in the year 2020 after the first wave of the COVID-19 pandemics. Serum donors filled in a questionnaire to allow their clinical classification according to the following parameters: Asymptomatic, no symptoms; Mild, 3 or more of the following symptoms: non-productive cough, hyperthermia, headache, odynophagia, dyspnea, asthenia, myalgia, ageusia, anosmia, cutaneous involvement;

Moderate, 3 or more of the above symptoms plus gastrointestinal symptoms, or more than 3 of the above for 7 or more days; Moderate-Severe, 3 or more of the above symptoms plus pneumonia; Severe, pneumonia requiring hospitalization and intubation. A second set of 52 serum samples were selected from the study "Immune response dynamics as predictor of 

Unpaired two-tailed Student t-tests were used to compare statistical significance between two groups of MFI values that followed a normal distribution and one-way ANOVA tests for column analysis of different groups. All data was analyzed using the GraphPad Prism 7 software.

Serum samples were received coded from the providers and the experimentalists were blinded to their nature until all data analysis was finalized. Sample analysis was carried out in duplicate or triplicate and all experiments were repeated a minimum of two times.

This study includes no data deposited in external repositories. Jurkat-S cells are available for academic research upon request to B. Alarcón. All rights reserved. No reuse allowed without permission. perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in (C) Box and whiskers plot of all datapoints corresponding to the slopes of the fitting lines calculated in (B). A two-tailed t test was used to assess significance. **, p<0.01. All rights reserved. No reuse allowed without permission. perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in The copyright holder for this this version posted August 14, 2021. ; https://doi.org/10.1101/2021.08.12.21261952 doi: medRxiv preprint perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in A *** *** **** * All rights reserved. No reuse allowed without permission.

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preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

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The copyright holder for this this version posted August 14, 2021. ; https://doi.org/10.1101/2021.08.12.21261952 doi: medRxiv preprint

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We are indebted to Valentina Blanco and Tania Gómez for their expert technical assistance. We thank all volunteers of the CBM, school teachers, "Hospital 12 deOctubre" and "Hospital Universitario de la Princesa" for generously participating in the study.