key: cord-0280740-msyttvww
authors: Andreano, Emanuele; Paciello, Ida; Piccini, Giulia; Manganaro, Noemi; Pileri, Piero; Hyseni, Inesa; Leonardi, Margherita; Pantano, Elisa; Abbiento, Valentina; Benincasa, Linda; Giglioli, Ginevra; De Santi, Concetta; Fabbiani, Massimiliano; Rancan, Ilaria; Tumbarello, Mario; Montagnani, Francesca; Sala, Claudia; Montomoli, Emanuele; Rappuoli, Rino
title: Hybrid immunity improves B cell frequency, antibody potency and breadth against SARS-CoV-2 and variants of concern
date: 2021-08-12
journal: bioRxiv
DOI: 10.1101/2021.08.12.456077
sha: 29a8c165fd883e761b4bc04275065fdd5fa73697
doc_id: 280740
cord_uid: msyttvww

To understand the nature of the antibody response to SARS-CoV-2 vaccination, we analyzed at single cell level the B cell responses of five naïve and five convalescent people immunized with the BNT162b2 mRNA vaccine. Convalescents had higher frequency of spike protein specific memory B cells and by cell sorting delivered 3,532 B cells, compared with 2,352 from naïve people. Of these, 944 from naïve and 2,299 from convalescents produced monoclonal antibodies against the spike protein and 411 of them neutralized the original Wuhan SARS-CoV-2 virus. More than 75% of the monoclonal antibodies from naïve people lost their neutralization activity against the B.1.351 (beta) and B.1.1.248 (gamma) variants while this happened only for 61% of those from convalescents. The overall loss of neutralization was lower for the B.1.1.7 (alpha) and B.1.617.2 (delta) variants, however it was always significantly higher in those of naïve people. In part this was due to the IGHV2-5;IGHJ4-1 germline, which was found only in convalescents and generated potent and broadly neutralizing antibodies. Overall, vaccination of seropositive people increases the frequency of B cells encoding antibodies with high potency and that are not susceptible to escape by any of the four variants of concern. Our data suggest that people that are seropositive following infection or primary vaccination will produce antibodies with increased potency and breadth and will be able to better control SARS-CoV-2 emerging variants.

Twenty months after the beginning of the COVID-19 pandemic, with 200 million people infected, 4.2 39 million deaths, and 3.9 billion vaccines doses administered, the world is still struggling to control the 40 virus. In most developed countries vaccines have vastly reduced severe diseases, hospitalization 41 and deaths, but they have not been able to control the infections which are fueled by new and more 42 infectious variants. A large number of studies so far have shown that protection from infection is 43 linked to the production of neutralizing antibodies against the spike protein (S protein) of the virus 1-44 4 . This is a metastable, trimeric class 1 fusion glycoprotein, composed by the S1 and S2 subunits, 45 and mediates virus entry changing from a prefusion to postfusion conformation after binding to the 46 human angiotensin-converting enzyme 2 (ACE2) receptor and heparan sulfates on the host cells 5 . 47

Potent neutralizing antibodies recognize the S1 subunit of each monomer which includes the named Variants of Concern (VoCs) by the World Health Organization 11 . The delta variant is presently 58 spreading across the globe and causing big concern also in fully vaccinated populations. It is 59 therefore imperative to understand the molecular dynamics of the immune response to vaccination 60 in order to design better vaccines and vaccination policies. Several investigators have shown that 61 vaccination of convalescent people can yield neutralizing antibodies which can be up to a thousand-62 fold higher than those induced by infection or vaccination, suggesting that one way of controlling the 63 pandemic may be the induction of a hybrid immunity-like response using a third booster dose [12] [13] [14] [15] [16] . 64

Here we compared at single cell level the nature of the neutralizing antibody response against the 65 original Wuhan virus and the VoCs in naïve and convalescent subjects immunized with the 66 BNT162b2 mRNA vaccine. Naïve subjects were seronegative before vaccination while convalescent 67 donors were already seropositive before vaccination. They will be named seronegatives and 68 seropositives respectively in this work. Our data suggest that immunization of people already 69 seropositive to the virus, increases the frequency, potency and breadth of neutralizing antibodies 70 and may help to control emerging variants. 71 5 overall higher percentage of nAbs neutralizing the VoCs compared to seronegatives. The average 110 frequency of nAbs from seropositives neutralizing the alpha, beta and gamma variants was 80.6 111 (n=274), 39.4 (n=134) and 62.0% (n=211) respectively, compared to 70.4 (n=50), 22.5 (n=16) and 112 43.6% (n=31) respectively in seronegatives ( Figure 1C ; Table S2) . 113

High potency and breadth of neutralization in seropositive COVID-19 vaccinees 115

To better characterize and understand the potency and breadth of coverage of all Wuhan SARS-116

CoV-2 nAbs, we aimed to express as immunoglobulin G1 (IgG1) all the 411 nAbs previously 117 identified. We were able to recover and express 276 antibodies for further characterization, 224 118 (89.8%) from seropositives and 52 (10.2%) from seronegatives. Initially, antibodies were tested for 119 binding against the RBD, NTD and the S2-domain of the original Wuhan SARS-CoV-2 S protein. 120

Overall, no major differences were observed in nAbs that recognized the RBD and NTD with 71.2% 121 (n=37) and 79.5% (n=178) nAbs binding the RBD, while 17.3% (n=9) and 16.5% (n=37) nAbs binding 122 the NTD for seronegatives and seropositives respectively ( Figure S3 ). None of the tested nAbs 123 targeted the S2 domain. The biggest difference between groups was seen for nAbs able to bind the 124 S protein only in its trimeric conformation i.e. not able to bind single domains. This class of nAbs was 125 almost 3-fold higher in seronegatives compared to seropositives ( Figure S3 ). nAbs were then tested 126 by CPE-MN in serial dilution to evaluate their 100% inhibitory concentration (IC100) against the 127 Wuhan SARS-CoV-2 virus and the VoCs. At this stage of the study, the B.1.617.2 (delta) spread 128 globally, and we were able to obtain the live virus for our experiments. Overall, nAbs isolated from 129 seropositive vaccinees had a significantly higher potency than those isolated from seronegatives. 130

The IC100 geometric mean (GM-IC100) in seropositives was 2.87, 2.17-, 1.17-, 1.43-, and 1.92-fold 131 lower than in seronegatives for the Wuhan virus, the alpha, beta, gamma and delta VoCs respectively 132 ( Figure 2 ). In addition, a bigger fraction of nAbs from seropositives retained the ability to neutralize 133 the VoCs. Indeed, when nAbs were individually tested against all VoCs, the ability to neutralize the 134 alpha, beta, gamma and delta variants was lost by 14, 61, 61 and 29% of the antibodies from 135 seropositives versus 32, 78, 75 and 46% respectively of those from seronegatives. The overall 136 number of nAbs that lost neutralizing activity against the beta and gamma VoCs was very high, (75-137 78% in seronegatives and 61% in seropositives), while it was much lower for the alpha and delta 138 variants (32-46% for seronegatives and 14-29% for seropositives) (Figure 2 ). Finally, a major 139 difference between seronegatives and seropositives was found in the class of medium/high potency 140 nAbs (IC100 of 11-100 ng/mL and 101-1000 ng/mL) against all variants. Indeed, nAbs in these ranges The analysis of the immunoglobulin G heavy chain variable (IGHV) and joining (IGHJ) gene 147 rearrangements of 58 and 278 sequences recovered from seronegative and seropositive subjects 148 respectively, showed that they use a broad range of germlines and share the most abundant. In 149 particular, both groups predominantly used the IGHV1-69;IGHJ4-1 and IGHV3-53;IGHJ6-1, which 150

were shared by three out five subjects per each group ( Figure 3A ). In addition, the IGHV3-30;IGHJ6-151 1 and IGHV3-33;IGHJ4-1 germlines, more abundant in seronegative donors, and IGHV1-2;IGHJ6-152 1, mainly expanded in seropositive vaccinees, were also used with high frequency in both groups. 153

Only the IGHV2-5;IGHJ4-1 germline was seen to be predominantly expanded only in seropositive 154 donors ( Figure 3A ). To better characterize these predominant gene families, we evaluated their 155 neutralization potency and breadth against SARS-CoV-2 and VoCs. In this analyses we could not 156 evaluate IGHV3-33;IGHJ4-1 nAbs, as only three of these antibodies were expressed, but we 157 included the IGHV3-53 closely related family IGHV3-66;IGHJ4-1, as this family was previously 158 described to be mainly involved in SARS-CoV-2 neutralization 9,17 . A large part of nAbs deriving from 159 these predominant germlines had a very broad range of neutralization potency against the original 160

Wuhan SARS-CoV-2 virus with IC100 spanning from less than 10 to over 10,000 ng/mL ( Figure 3B To map the regions of the S protein recognized by the identified nAbs we used a competition assay 176 with four known antibodies: J08, which targets the top loop of the receptor binding motif (RBM) 3 , 177 S309, which binds the RBD but outside of the RMB region 18 , 4A8, that recognized the NTD 19 , and 178 L19, that binds the S2-domain 3 ( Figure S6 ). The nAbs identified in this study were pre-incubated with 179 the original Wuhan SARS-CoV-2 S protein and subsequently the four nAbs labeled with different 180 fluorophores were added as single mix. 50% signal reduction for one of the four fluorescently labelled 181 nAbs, was used as threshold for positive competition. The vast majority of nAbs from both 182 seronegative (50.0%; n=26) and seropositive (51.3%; n=115) vaccinees competed with J08 ( Figure  183 4A; Table S3 ). For seronegatives, the second most abundant population was composed by nAbs 184 that did not compete with any of the four fluorescently labelled nAbs (25.0%; n=13) followed by nAbs 185 targeting the NTD (17.3%; n=9). As for seropositives, the second most abundant population was 186 composed by nAbs that competed with S309 (21.4%; n=48) followed by nAbs competing with 4A8 187 (15.6%; n=35) and not-competing nAbs (11.6%; n=26). None of our nAbs did compete with the S2 188 targeting antibody L19 ( Figure 4A ; Table S3 ). nAbs competing with J08, which are likely to bind the 189 RBM, derived from several germlines, including the predominant IGHV3-53;IGHJ6-1 (10.6%; n=14), 190 IGHV1-69;IGHJ4-1 (8.3%; n=11) and IGHV1-2;IGHJ6-1 (6.8%; n=9) ( Figure 4B ). In contrast, those 191 competing with S309 derived mostly from germline IGHV2-5;IGHJ4-1 (13.7%; n=7) which were 192 isolated exclusively from seropositive vaccinees ( Figure 4C ). As for NTD-directed nAbs the non- shows that seropositive subjects respond to vaccination with more B cells producing antibodies 207 which show higher neutralization potency and are not susceptible to escape variants. In part this is 208 due to potent antibodies derived from the IGHV2-5;IGHJ4-1 germline which was only found in 209 seropositive people. The absence of this germline in naïve vaccinees is also confirmed by previous 210 studies 16 . One limitation of our study is that we did not include people that received a third booster 211 dose of vaccine, as at the time that this work was being performed no policies for a third booster 212 dose were implemented. In spite of this limitation, we believe that our conclusions are likely to be 213 extendable to people that are seropositives following primary vaccination. Indeed, the S protein 214 produced following vaccination with mRNA, viral vectors or following infection is produced in all 215 cases by the cell of the host and it is likely to be presented to the immune system in a similar manner 216 and generate a similar antibody response. This is confirmed by the fact that neutralizing antibodies 217 following infection and vaccination derive mostly from the same immunodominant germlines, i.e. 218 IGHV3-53, 3-30 and 3-66 9,10,16,17 . Our analysis suggests that a booster dose of vaccine will increase 219 the frequency of memory B cells producing potent neutralizing antibodies not susceptible to escape 220 variants and allow better control of this pandemic. Our work also shows that more than three quarters 221 of antibodies neutralizing the Wuhan virus do not neutralize at all the beta and gamma variants, while 222 the fraction of antibodies not neutralizing the alpha and delta variants is much smaller and in all 223 cases below 50%. This suggest that the beta and gamma variants were originally selected to escape 224 natural immunity, while the alpha and gamma were selected mostly for their increased infectivity and 225 ability to spread in the population 21,22 . The massive escape from predominant germlines, such as 226 IGHV3-53, 3-66, 3-30 and 1-69, and the presence of antibodies deriving from germline IGHV2-5 that 227 are completely insensitive to the existing variants, suggest that the design of vaccines that 228 preferentially promote or avoid the expansion of selected germlines can generate broad protection 229 against SARS-CoV-2 variants. Germline-targeting vaccination, which has been pioneered in the HIV 230 field 23,24 , may be a promising strategy to fight the COVID-19 pandemic. Nested PCR II was performed as above starting from 3.5 μL of unpurified PCR I product. PCR II 340 products were purified by Millipore MultiScreen® PCRμ96 plate according to manufacture 341 instructions and eluted in 30 μL of nuclease-free water (DEPC). As for TAP expression, vectors were 342 initially digested using restriction enzymes AgeI, SalI and Xho as previously described and PCR II 343 products ligated by using the Gibson Assembly NEB into 25 ng of respective human Igγ1, Igκ and 344

Igλ expression vectors 27,28 . TAP reaction was performed using 5 μL of Q5 polymerase (NEB), 5 μL 345 of GC Enhancer (NEB), 5 μL of 5X buffer,10 mM dNTPs, 0.125 µL of forward/reverse primers and 3 346 μL of ligation product, using the following cycles: 98°/2', 35 cycles 98°/10'', 61°/20'', 72°/1' and 72°/5'. 347 348 Functional repertoire analyses 349 nAbs VH and VL sequence reads were manually curated and retrieved using CLC sequence viewer 350 (Qiagen). Aberrant sequences were removed from the data set. Analyzed reads were saved in 351 FASTA format and the repertoire analyses was performed using Cloanalyst 352 

(F) The table shows the IC100 geometric mean (GM) of 416 all nAbs pulled together from each group against all SARS-CoV-2 viruses tested

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