key: cord-0800895-kkpt78gw
authors: Tauzin, A.; Gong, S. Y.; Beaudoin-Bussieres, G.; Vezina, D.; Gasser, R.; Nault, L.; Marchitto, L.; Benlarbi, M.; Chatterjee, D.; Nayrac, M.; Laumaea, A.; Prevost, J.; Boutin, M.; Sannier, G.; Nicolas, A.; Bourassa, C.; Gendron-Lepage, G.; Medjahed, H.; Goyette, G.; Bo, Y.; Perreault, J.; Gokool, L.; Morrisseau, C.; Arlotto, P.; Bazin, R.; Dube, M.; De Serres, G.; Brousseau, N.; Richard, J.; Rovito, R.; Cote, M.; Tremblay, C.; Marchetti, G. C.; Duerr, R.; Martel-Laferriere, V.; Kaufmann, D. E.; Finzi, A.
title: Strong humoral immune responses against SARS-CoV-2 Spike after BNT162b2 mRNA vaccination with a sixteen-week interval between doses
date: 2021-09-21
journal: nan
DOI: 10.1101/2021.09.17.21263532
sha: 7dade21e0c935a8a8a5164a9f966f7cfb5a3ed9c
doc_id: 800895
cord_uid: kkpt78gw

While the standard regimen of the BNT162b2 mRNA vaccine includes two doses administered three weeks apart, some public health authorities decided to space them, raising concerns about vaccine efficacy. Here, we analyzed longitudinal humoral responses including antibody binding, Fc-mediated effector functions and neutralizing activity against the D614G strain but also variants of concern and SARS-CoV-1 in a cohort of SARS-CoV-2 naive and previously infected individuals, with an interval of sixteen weeks between the two doses. While the administration of a second dose to previously infected individuals did not significantly improve humoral responses, we observed a significant increase of humoral responses in naive individuals after the 16-weeks delayed second shot, achieving similar levels as in previously infected individuals. Our results highlight strong vaccine-elicited humoral responses with an extended interval BNT162b2 vaccination for naive individuals.

onset, especially IgG, in agreement with previous observations (Anand et al., 2021; Dan et al., 116 2021; Tauzin et al., 2021; Wang et al., 2021b) . For both groups, the first dose of vaccine induced 117 a significant increase of total immunoglobulins (Igs) recognizing the RBD or the Spike protein 118 three weeks post-vaccine (V1), with a significantly higher response for the PI group ( Figure 1B -119 I). At V2 (i.e., 12 weeks post vaccination), while anti-Spike total Ig levels remained stable, we 120 observed a decrease in anti-RBD total Ig levels in both groups, with the exception of some naïve 121 donors where we observed an increase. We did not detect Abs recognizing the N protein for 122 these donors (not shown), suggesting that they had not been infected between the two doses. 123

This increase could therefore be linked to a delayed response or affinity maturation of the 124 antibodies in the germinal center between V1 and V2. The second dose, which was administered 125 ~16 weeks after the first one, strongly boosted the induction of anti-RBD Igs in the SARS-CoV-2 126 naïve group, particularly IgG and IgA which reached higher levels, albeit not statistically 127 significant, than those measured three weeks after the first dose ( Figure 1D and E). For the PI 128 group, the second dose also led to an increase in the level of total anti-RBD Igs that reached 129 similar levels than after the first dose. Of note, the second dose in the naïve group elicited anti-130 RBD IgG levels that reached the same levels than in the PI group receiving two doses and 131 significantly higher than PI receiving only one ( Figure 1D ). Similar patterns of responses were 132 observed when we measured the level of Abs recognizing the full-length S glycoprotein ( Figure  133 1F-I). 134 135

The BNT162b2 mRNA vaccine has been developed against the original Wuhan strain. 137

However, SARS-CoV-2 is evolving, and many variants have emerged and spread rapidly 138 worldwide. Some harbor specific mutations in S that are associated with increased transmissibility 139 and/or immune evasion (Davies et tested SARS-CoV-2 variants at V0 (Figure 2A-F, S1 ). The first dose of vaccine strongly enhanced 148 the recognition of the full D614G S and all the tested variants in both groups (Figure 2A-F) . Three 149 months after the first dose, the recognition slightly decreased but not significantly. As expected, 150 the second dose strongly increased recognition of all VOC Spikes in the naïve group and reached 151 levels that where significantly higher than after the first dose. In contrast, for the PI group, the 152 second dose did not result in a better recognition than after the first dose. Of note, we observed 153 no significant differences at V3 between PI individuals who received one or two doses, despite a 154 shorter period since the last dose for PI individuals who received two doses. The recognition of 155 all VOCs was slightly lower at V3 by the naïve group compared to the PI that received two doses 156 ( increased the recognition of every VOC Spike tested, we observed that plasmas recognized the 162 different SARS-CoV-2 variants less efficiently compared to D614G S except for the B.1.1.7 S 163 ( Figure S1 ). 164

We also evaluated whether vaccination elicited Abs that were able to recognize S 165 glycoproteins from endemic human Betacoronaviruses, (HCoV-OC43 and HCoV-HKU1). 166

Interestingly, we observed that the first but not the second dose enhanced the recognition of 167

HCoV-HKU1 S in the naïve group ( Figure 2H ). Moreover, we observed that plasma from PI donors 168 better recognized HCoV-HKU1 S than plasma from naïve donors at every time point studied, 169

suggesting that natural infection induced cross reactive Abs more efficiently than vaccination. In 170 contrast, both doses did not significantly improve the recognition of HCoV-OC43 S ( Figure 2G ). 171

We then evaluated the capacity of the different plasma samples to bind S from highly 172 pathogenic human coronaviruses (SARS-CoV-1 and MERS-CoV). We observed that PI 173 individuals did not have Abs able to recognize MERS-CoV S before vaccination, in contrast to 174 SARS-CoV-1 S ( Figure 2I al., 2021). As expected, and in agreement with the absence of SARS-CoV-2 S specific antibodies 193 at baseline, no ADCC activity was observed for the naïve group before vaccination ( Figure 3A ). 194

Plasma from the PI group maintained some levels of ADCC activity before vaccination, in 195 agreement with a longitudinal study following immune responses in convalescent donors (Anand 196 et al., 2021) . Three weeks after the first dose, ADCC activity was elicited in both groups, but 197 significantly higher in the PI group. A decline in ADCC responses was observed in both groups 198 nine weeks after V1 (V2, i.e., 12 weeks post vaccination). The second dose strongly boosted 199 ADCC activity in the naïve group but remained stable for the PI groups. We noted that the levels 200 of ADCC activity were significantly higher in the PI group at all timepoints ( Figure 3A Figure 3B ). We did not detect a significant increase in neutralization in 207 plasma isolated three weeks post vaccination of the naïve group, as previously described (Tauzin 208 et al., 2021) . Interestingly, nine weeks later (V2, i.e., 12 weeks post vaccination), we observed 209 increased neutralizing activity in a few donors ( Figure 3B ). All donors presented a significant 210 increase in neutralizing activity three weeks after the second dose. Importantly, the level of 211 neutralizing activity of double vaccinated naïve individuals reached the same levels than in the PI 212 group after one or two doses. In this latter group (PI), we measured low neutralizing activity before 213 vaccination, consistent with remaining neutralizing activity in convalescent donors after several 214 . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) preprint

The copyright holder for this this version posted September 21, 2021. ; https://doi.org/10.1101/2021.09.17.21263532 doi: medRxiv preprint weeks post vaccination). The second dose boosted the neutralizing activity to the levels reached 218 three weeks after the first dose. No difference in neutralization was observed between V1 and V3 219 for PI individuals. In contrast, in naïve individuals we observed a significantly higher neutralizing 220 activity after the second dose compared to the first one ( Figure 3B ). Thus, while one dose is 221 required to reach maximum neutralization activity in PI individuals, this activity decays over time 222 and a second dose is required to bring back its maximum potential. On the other hand, naïve 223 individuals requires both doses to achieve the same level of PI vaccinated individuals. 224 225 Neutralizing activity against variants of concern 226 SARS-CoV-2 is evolving, and variants of concern are emerging globally (Davies et (Figure 4, S2) . For all the variants tested, 231 we observed a similar pattern than for the D614G S, with neutralizing Abs mainly induced after 232 the second dose in the naïve group ( Figure 4A -E). Previously-infected individuals followed a 233 different pattern. While their plasma had some levels of neutralizing activity at baseline, it gained 234 potency and breadth after the first dose. A second dose did not further enhance this activity. 235 Accordingly, we found no significant differences in neutralizing activity in plasma from PIs that 236 received one or two doses measured at V3 (i.e., week 19) . 237

We also noted that, with the exception of B.1.1.7, plasma from the PI group prior to 238 vaccination (V0) neutralized less efficiently all pseudoviral particles bearing variant Spikes 239 compared to the D614G ( Figure S2A ). Importantly, both doses boosted the neutralizing activity 240 against all variants and SARS-CoV-1 Spike at V3 ( Figure S2D ). 241 . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) preprint is used as a representative variant that is even more dissimilar to the vaccine, which was based 244 on the ancestral Wuhan strain. While only one dose was sufficient to provide SARS-CoV-1 245 neutralizing capacity in PI individuals, two were required in naïve individuals. Remarkably, plasma 246 from naïve individuals reached the same level of neutralizing activity against pseudoviral particles 247 bearing the SARS-CoV-1 Spike than PI. Thus, suggesting that the delayed boosting in naïve 248 individuals allows antibody maturation resulting in enhanced breath ( Figure 4F ). 249 250

We also analyzed the humoral responses of 11 SARS- side comparison of humoral responses with our cohort of naïve individuals that received the two 257 doses 16 weeks apart. In other words, the V3 of individuals receiving the second dose following 258 a short interval regimen was collected ~19 weeks post boost whereas the V3 of those with a long 259 interval corresponds to 3 weeks post boost ( Figure 1A and 5A). While not a perfect match, we 260 decided to compare the humoral responses of short interval vaccinated individuals with those 261 from PI that received a single dose since their V3 was collected ~19 weeks post vaccination. 262

While we observed no significant differences in total Ig recognizing the RBD or the full S ( Figure  263 5B and F), single dose PI individuals had more anti-RBD IgG and IgA than naïve donors who 264 received their two doses 3 weeks apart ( Figure 5D and E). Despite minor differences in the overall 265 amount of anti-RBD or anti-Spike Abs, we observed major differences related to their capacity to 266 recognize the full Spike. Plasma from short interval vaccinated individuals was significantly less 267 . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) preprint

The copyright holder for this this version posted September 21, 2021. ; https://doi.org/10.1101/2021.09.17.21263532 doi: medRxiv preprint efficient at recognizing the D614G S and all other S variants tested ( Figure 5J ). Major differences 268 were also observed, particularly related to their capacity to mediate ADCC ( Figure 5K ) or 269 neutralize pseudoviral particles bearing D614G or any of the variant Spike tested ( Figure 5L ). 270

Indeed, the naïve donors with a short interval between doses had a very low ID50 against all the 271 variant tested ~19 weeks after the vaccine series. No neutralizing activity against SARS-CoV-1 272 was observed ( Figure 5L ). In contrast, plasmas from PI individual who received just one dose 273 presented neutralizing activity against all the SARS-CoV-2 variants but also the SARS-CoV-1 274 pseudoviruses ( Figure 5L PI individuals who received their two doses sixteen weeks apart. 300

We observed that in the SARS-CoV-2 naïve group the BNT162b2 mRNA vaccine elicited 301 antibodies with weak neutralizing activity but strong Fc-mediated functions three weeks after the 302 first dose . These responses declined in the following weeks in the absence 303 of a boost. However, administration of the second dose sixteen weeks later strongly enhanced 304 these responses, notably neutralization against some VOCs/VOIs and even the divergent SARS-305

CoV-1. Therefore, despite initial concerns, the long interval between the doses did not result in 306 poor immune responses, in agreement with recent findings (Parry et al., 2021) . The idea behind 307 the strategy of delaying the second dose was to provide some level of immunity to a larger number 308 of individuals than if the second dose would have been saved to administer them three weeks 309 later. However, despite the immunological benefits of increasing the interval between the two 310 doses, this also increases the probability of being infected before the boost. is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) preprint

The copyright holder for this this version posted September 21, 2021. ; https://doi.org/10.1101/2021.09.17.21263532 doi: medRxiv preprint found that vaccination of these individuals resulted in the induction of strong humoral responses. 315

These responses remained relatively stable over time. We noticed that the second dose did not 316 result in a significant enhancement of these responses, even with a long interval of 16 weeks 317 between doses. Our results demonstrate that, while the second dose boosts the humoral 318 response, PI individuals reach their peak of immunity after the first dose. Altogether, these results 319 suggest that a second dose for PI individuals might be delayed beyond sixteen weeks after the 320 To end this pandemic, it will be necessary to rapidly vaccinate the world's population, 354 including in countries where vaccines are poorly available. The research community around the 355 globe rapidly generated a wealth of data related to vaccine-elicited immune responses and 356 vaccine efficacy. Globally, these results suggest that the current vaccine strategy that was initially 357 deployed could be improved. Our results suggest that modifying the interval at which the two 358 doses are administered might be an important factor to take into account. It will be important to 359 keep in mind that a fine balance needs to be achieved in order to avoid infection between the two 360 doses and at the same time provide sufficient time to elicit optimal humoral responses. 361 362 363 . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. CoV-2 S glycoproteins, with serial dilutions of plasma for 1 h at 37°C before infecting 293T-ACE2 446 cells. Neutralization half maximal inhibitory serum dilution (ID50) values were determined using a 447 normalized non-linear regression using GraphPad Prism software. Naïve and PI donors with a 448 long interval between the two doses are represented by red and black points respectively and PI 449 . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. 

. CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. ExpiFectamine 293 transfection reagent, as directed by the manufacturer (Invitrogen). One week 584 later, cells were pelleted and discarded. Supernatants were filtered using a 0.22 µm filter (Thermo 585

Fisher Scientific). The recombinant RBD proteins were purified by nickel affinity columns, as 586 directed by the manufacturer (Invitrogen). The RBD preparations were dialyzed against 587 . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. Kruskal-Wallis (V3) tests. Differences in responses against the different Spikes for the same 704 patient were measured by Friedman tests. P values < 0.05 were considered significant; 705 significance values are indicated as * p < 0.05, * * p < 0.01, * * * p < 0.001, * * * * p < 0.0001. 706

Spearman's R correlation coefficient was applied for correlations. Statistical tests were two-sided 707 and p < 0.05 was considered significant. 708 709

Edge bundling graphs were generated in undirected mode in R and RStudio using ggraph, igraph, 711 tidyverse,and RColorBrewer packages (R; R studio). Edges are only shown if p < 0.05, and nodes 712 . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. Supplemental information can be found online at … 717 . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) preprint

The copyright holder for this this version posted September 21, 2021. ; https://doi.org/10.1101/2021.09.17.21263532 doi: medRxiv preprint . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) preprint CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. Naïve and PI donors with a long interval between the two doses are represented by red and black points respectively, PI donors who received just one dose by blue points and naïve donors with a short interval between the two doses by green points. Error bars indicate means ± SEM. (* P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001; ns, non-significant). Neutralizing activity was measured by incubating pseudoviruses bearing SARS-CoV-2 S variant or SARS-CoV-1 S glycoproteins, with serial dilutions of plasma collected at V0 (A), V1 (B), V2 (C) and V3 (D) for 1 h at 37°C before infecting 293T-ACE2 cells. Neutralization half maximal inhibitory serum dilution (ID50) values were determined using a normalized non-linear regression using GraphPad Prism software. Naïve and PI donors with a long interval between the two doses are represented by red and black points respectively, PI donors who received just one dose by blue points and naïve donors with a short interval between the two doses by green points. Undetectable measures are represented as white symbols, and limits of detection are plotted. Error bars indicate means ± SEM. (* P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001; ns, non-significant).

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