key: cord-0278116-49grxm8t authors: Cho, Alice; Muecksch, Frauke; Wang, Zijun; Tanfous, Tarek Ben; DaSilva, Justin; Raspe, Raphael; Johnson, Brianna; Bednarski, Eva; Ramos, Victor; Schaefer-Babajew, Dennis; Shimeliovich, Irina; Dizon, Juan; Yao, Kai-Hui; Schmidt, Fabian; Millard, Katrina G.; Turroja, Martina; Jankovic, Mila; Oliveira, Thiago Y.; Gazumyan, Anna; Gaebler, Christian; Caskey, Marina; Hatziioannou, Theodora; Bieniasz, Paul D.; Nussenzweig, Michel C. title: Antibody evolution to SARS-CoV-2 after single-dose Ad26.COV2.S vaccine date: 2022-04-01 journal: bioRxiv DOI: 10.1101/2022.03.31.486548 sha: 6ce7092d7d989867db73d152b6386c8d28a8ce97 doc_id: 278116 cord_uid: 49grxm8t The single dose Ad.26.COV.2 (Janssen) vaccine elicits lower levels of neutralizing antibodies and shows more limited efficacy in protection against infection than either of the available mRNA vaccines. In addition, the Ad.26.COV.2 has been less effective in protection against severe disease during the Omicron surge. Here, we examined the memory B cell response to single dose Ad.26.COV.2 vaccination. Compared to mRNA vaccines, Ad.26.COV.2 recipients had significantly lower numbers of RBD-specific memory B cells 1.5 or 6 months after vaccination. Memory antibodies elicited by both vaccine types show comparable neutralizing potency against SARS-CoV-2 and Delta. However, the number of memory cells producing Omicron neutralizing antibodies was somewhat lower after Ad.26.COV.2 than mRNA vaccination. The data help explain why boosting Ad.26.COV.2 vaccine recipients with mRNA vaccines is effective, and why the Janssen vaccine appears to have been less protective against severe disease during the Omicron surge than the mRNA vaccine. One-Sentence Summary Ad.26.COV.2 vaccine results in lower quantity but comparable quality of protective memory B cells compared to mRNA vaccines. 8 192 anti-RBD monoclonal antibodies were expressed and tested for binding by ELISA. 93% 160 (n=179) bound to the Wuhan-Hu-1 RBD, indicating the high efficiency RBD-specific memory B 161 cell isolation (table S3) . At the initial time point, the geometric mean ELISA half-maximal 162 concentration (EC50) of the monoclonal antibodies obtained from Janssen vaccinees was 163 significantly higher than individuals receiving a single dose of an mRNA vaccine (p=0.0001, 164 Fig. 3a, (9) ). However, the EC50 of RBD-binding antibodies elicited by the Janssen vaccine 165 improved over time such that the antibodies elicited by the two vaccines had comparable EC50s 166 after 5-6 months (Fig. 3a) . 167 EC50s represent an indirect measure of affinity. To directly examine anti-RBD antibody affinity 169 we performed biolayer interferometry (BLI) experiments on a subset of the antibodies (n=33 170 from 1.5 and 6 months, each). Affinity was significantly higher among antibodies elicited by the 171 Janssen vaccine compared to those obtained after the mRNA prime and 2 nd dose (p<0.0001, and 172 p=0.03, respectively, Fig. 3b, (9) ). For both vaccine platforms, antibody affinity improved over 173 time, reaching equivalent levels at the 5-6-month time point (Fig. 3b) . 174 175 All 179 RBD-binding antibodies were tested for neutralization (84 and 95 antibodies isolated 176 after 1.5 and 6 months, respectively). Compared to the mRNA prime, memory antibodies elicited 177 by the Janssen vaccine were significantly more potent against viruses pseudotyped with the 178 Wuhan-Hu-1 RBD (IC50 140 vs 421 ng/ml, p=0.0002, Fig. 3c ). However, the neutralizing 179 activity of the anti-RBD memory antibodies elicited by mRNA vaccination improved after the 180 second dose, and the two vaccines generated antibodies of equivalent potency after 5-6 months 181 (IC50 152 vs. 156, p>0.99, Fig 3c, (9) ). To examine the repertoire of NTD-specific memory B cells elicited by the Janssen vaccine, we 184 expressed 60 and 20 antibodies obtained 1.5 and 6 months after vaccination, respectively (table 185 S4). 59 bound to NTD with relatively poor EC50s that did not improve over time (Fig. 3d, table 186 S4). When tested for neutralizing activity against Wuhan-Hu-1-pseudotyped virus, only 4 of the 187 59 NTD-binding monoclonal antibodies showed neutralizing activity, with no change over time 188 (Fig. 3e) . Thus, the overall frequency of memory B cells producing neutralizing anti-NTD 189 antibodies is significantly lower than those producing anti-RBD (Fig. 3f) . We conclude that anti-190 NTD memory antibodies are likely to make a more modest contribution to protection against 191 subsequent viral challenge than their anti-RBD counterparts. antibodies, that block ACE2 binding directly, tend to be more potent, Class 3 and 4 target more 198 conserved regions and can be broader (8, 10, 12, 21) . To define the epitopes recognized by anti-199 RBD memory antibodies elicited by the Janssen vaccine, we performed BLI competition 200 experiments. A preformed antibody-RBD-complex was exposed to a second antibody targeting 201 one of four classes of structurally defined epitopes (11, 20) (C105 as Class 1; C144 as Class 2; 202 C135 as Class 3; and C118 as Class 1/4). We examined 33 random RBD-binding antibodies 203 obtained from the 1.5-and 6-month time points each, including 18 of 33 with IC50s lower than 204 1000 ng/mL. In contrast to the antibodies elicited after a single dose of an mRNA vaccine that 205 primarily target Class 1 and 2 epitopes, Class 3 and 1/4 specific antibodies dominated the 206 repertoire 1.5 months after Janssen vaccination (p= 0.016, Fig. 4a ). This difference is particularly 207 striking when considering neutralizing as opposed to non-neutralizing antibodies ( Fig. 4b and c) . 208 However, shifts in the repertoire of the mRNA vaccinees over time alleviated these differences 209 ( Fig. 4a and b, (8, 9) ). 210 211 The neutralizing breadth of memory antibodies obtained from convalescent individuals increased 213 significantly after 5 months (10, 12, 21). Memory antibodies elicited by mRNA vaccination 214 show more modest improvement over the same period of time (9), which is further increased by 215 a 3 rd dose (8). To determine how neutralizing breadth evolves after Janssen vaccination we 216 analyzed a panel of 34 randomly selected Wuhan-Hu-1-neutralizing antibodies from Janssen 217 vaccinees (n=16 at 1.5 months, and n=18 at 6 months). Neutralizing activity was measured 218 against SARS-CoV-2 pseudoviruses carrying amino acid substitutions found in variants of 219 concern. Neutralizing breadth improved significantly in Janssen vaccinees against pseudoviruses 220 containing single amino acid substitutions found in different SARS-CoV-2 variants (K417N, 221 N440K, and A475V, Fig. 5a, fig. S4a and b) . These mutations typically alter the binding and 222 neutralization properties of Class 1 and 3 antibodies (21). 223 224 A larger panel of randomly selected antibodies (n=71) with IC50s below 1000 ng/mL was tested 225 for neutralizing activity against viruses pseudotyped with Wuhan-Hu-1, Delta, and Omicron 226 RBDs ( Fig. 5b and fig. S4c ). In contrast to natural infection and mRNA vaccination there was no 227 improvement in neutralizing activity against Delta or Omicron between 1.5 and 6 months after 228 Janssen vaccination. Nevertheless, 86% of the 6-month memory antibodies tested neutralized 229 Delta and 31% neutralized Omicron ( fig. S4c ). Thus, 6 months after vaccination the memory B 230 cell compartment in Ad26.COV2.S recipients is smaller in size than the RBD-specific memory 231 B cell compartment in mRNA vaccinees but contains cells with the ability to produce antibodies 232 with comparable activity against Delta and Omicron. 233 234 Administration of a single dose of the Ad26.COV2.S vaccine results in less effective protection 236 against infection than mRNA vaccination, and also affords lower levels of protection against 237 severe disease and hospitalization from COVID-19 (4, 6, 23). The difference in protective 238 efficacy from infection between the 2 vaccine modalities has been attributed to significantly 239 lower levels of circulating neutralizing antibodies elicited by the Janssen vaccine (14, 24). We 240 find that 5-6 months after vaccination there is a 2.5-fold difference in the number of memory B 241 cells produced by the 2 vaccine modalities. A third mRNA dose further magnifies the difference 242 to nearly 6 fold (8). Nevertheless, the antibodies encoded by the individual memory cells show 243 similar levels of activity against Wuhan-Hu-1, and Delta, and Omicron BA.1. The ability of 244 these cells to respond rapidly to viral challenge may account in part for the partial protection Hu-1, and Delta at both, 1.5 and 6 months after vaccination. Activity against Omicron was lower 264 after Ad26.COV2.S but the difference was not statistically significant. 265 266 Class 1 and 2 antibodies develop early after infection or mRNA immunization and are generally 267 more potent than class 3 and 4, because they interfere directly with the interaction between the 268 SARS-CoV-2 RBD and its cellular receptor ACE2 (10, 20, 21). However, this renders Class 1 269 and 2 antibodies highly sensitive to amino acid substitutions within the ACE2 binding ridge of 270 the RBD found in many SARS-CoV-2 variants (10, 21). The epitopes targeted by Class 3 and 4 271 are generally more conserved and antibodies binding to these epitopes may be more broadly Heatmap ranging from 0.1-1000 ng/ml in white to red. The E484K, K417N/E484K/N501Y and 286 L452R/T478K substitution, as well as the deletions/substitutions corresponding to viral variants 287 were incorporated into a spike protein that also includes the R683G substitution, which disrupts 288 the furin cleavage site and increases particle infectivity. Neutralizing activity against mutant 289 pseudoviruses were compared to a wildtype (WT) SARS-CoV-2 spike sequence (NC_045512), 290 carrying R683G where appropriate. **** *** Structure-based Design of Prefusion-stabilized SARS-CoV-2 Spikes Effectiveness of Ad26.COV2.S Vaccine vs BNT162b2 Vaccine for COVID-295 Comparative Effectiveness of Moderna Effectiveness of Covid-19 Vaccines over a 9 Differential Kinetics of Immune Responses Elicited by Covid-19 Final Analysis of Efficacy and Safety of Single-Dose Ad26 Durability of Protection against COVID-19 Breakthrough Infections Severe Disease by Vaccines in the United States. medRxiv Increased Potency and Breadth of SARS-CoV-2 Neutralizing 310 Antibodies After a Third mRNA Vaccine Dose. bioRxiv Anti-SARS-CoV-2 receptor-binding domain antibody evolution after mRNA 312 vaccination Naturally enhanced neutralizing breadth against SARS-CoV-2 one year 314 after infection Convergent antibody responses to SARS-CoV-2 in convalescent 316 individuals Evolution of antibody immunity to SARS-CoV-2 Durable Humoral and Cellular Immune Responses 8 Months after 320 Divergent SARS CoV-2 Omicron-reactive T-and B cell 322 responses in COVID-19 vaccine recipients Immunogenicity and Reactogenicity of Vaccine Boosters after 324 mRNA vaccine-elicited antibodies to SARS-CoV-2 and circulating variants. 326 Vaccines elicit highly conserved cellular immunity to SARS-CoV-2 Omicron. 328 Generation of High Quality Memory B Cells Conserved Neutralizing Epitopes on the N-Terminal Domain of Variant 332 SARS-CoV-2 Spike Proteins. bioRxiv **** **** **** **** **** Participants were healthy volunteers who had previously received one dose of the Janssen 33 were carried out using the software iRIS by iMedRIS (v. 11.02). All participants provided 46 written informed consent before participation in the study and the study was conducted in 47 Tween-20 (Sigma-Aldrich)) and incubated with 170 μl per well blocking buffer (1× PBS with 66 2% BSA and 0.05% Tween-20 (Sigma)) for 1 hour at room temperature. Immediately after 67 blocking, monoclonal antibodies or plasma samples were added in PBS and incubated for 1 hour 68 at room temperature. Plasma samples were assayed at a 1:66 starting dilution and 10 additional 69 threefold serial dilutions. Monoclonal antibodies were tested at 10 μg/ml starting concentration samples from healthy donors were used for validation (for more details, please see (11)). For 84 monoclonal antibodies, the ELISA half-maximal concentration (EC50) was determined using 85 four-parameter nonlinear regression (GraphPad Prism V9.1). EC50s above 1000 ng/mL for RBD-86 binding were considered non-binders; EC50s above 10000 ng/mL for NTD-binding were 87 considered non-binders. 88 89 The mammalian expression vector encoding the Receptor Binding-Domain (RBD) of SARS-91CoV-2 (GenBank MN985325.1; Spike (S) protein residues 319-539) was previously described 92 (46). Mammalian expression vector encoding the SARS-CoV-2 Wuhan-Hu-1 NTD (GenBank 93 MN985325.1; S protein residues 14-307) was previously described (19) . The E484K, K417N/E484K/N501Y and L452R/T478K substitution, as well as the 110 deletions/substitutions corresponding to variants of concern listed above, were incorporated into 111 a spike protein that also includes the R683G substitution, which disrupts the furin cleavage site 112 and increases particle infectivity. Neutralizing activity against mutant pseudoviruses were 113 paired, and clonotypes were assigned based on their V and J genes using in-house R and Perl 202 scripts. All scripts and the data used to process antibody sequences are publicly available on 203 GitHub (https://github.com/stratust/igpipeline/tree/igpipeline2_timepoint_v2). 204The frequency distributions of human V genes in anti-SARS-CoV-2 antibodies from this study 205 was compared to 131,284,220 IgH and IgL sequences generated by (50) and downloaded from 206 cAb-Rep (51), a database of human shared BCR clonotypes available at https://cab-207rep.c2b2.columbia.edu/. Based on the 150 distinct V genes that make up the 1099 analyzed 208 sequences from Ig repertoire of the 6 participants present in this study, we selected the IgH and 209IgL sequences from the database that are partially coded by the same V genes and counted them 210 according to the constant region. The frequencies shown in fig. S3 JR215 32 22 24 1000 32 1000 39 42 41 1000 38 1000 JR086 55 23 41 45 30 130 1000 1000 31 1000 125 1000 JR233 16 6 1000 4 9 11 158 13 33 188 1000 122 JR174 344 148 160 70 272 147 214 473 246 63 692 146 JR210 961 96 1000 164 390 290 125 466 504 52 519 1000 JR039 25 17 20 25 21 35 27 20 35 24 17 213 JR173 27 20 17 28 24 32 13 20 30 14 21 1000 JR088 72 78 46 71 90 137 79 70 185 57 95 1000 JR216 96 103 74 99 116 214 131 91 283 190 103 1000 JR212 132 84 64 114 63 83 105 150 183 77 156 1000 JR177 5 2 3 1 3 3 1000 82 3 1000 9 1000 JR042 24 13 14 146 18 31 22 41 633 1000 9