key: cord-0774186-hsnilx3g
authors: Sulbaran, Guidenn; Maisonnasse, Pauline; Amen, Axelle; Effantin, Gregory; Guilligay, Delphine; Dereuddre-Bosquet, Nathalie; Burger, Judith A.; Poniman, Meliawati; Grobben, Marloes; Buisson, Marlyse; Dergan Dylon, Sebastian; Naninck, Thibaut; Lemaître, Julien; Gros, Wesley; Gallouët, Anne-Sophie; Marlin, Romain; Bouillier, Camille; Contreras, Vanessa; Relouzat, Francis; Fenel, Daphna; Thepaut, Michel; Bally, Isabelle; Thielens, Nicole; Fieschi, Franck; Schoehn, Guy; van der Werf, Sylvie; van Gils, Marit J.; Sanders, Rogier W.; Poignard, Pascal; Le Grand, Roger; Weissenhorn, Winfried
title: Immunization with synthetic SARS-CoV-2 S glycoprotein virus-like particles protects Macaques from infection
date: 2022-01-24
journal: Cell Rep Med
DOI: 10.1016/j.xcrm.2022.100528
sha: 7159c200840618e27105b0ece2b45fb3525b1e26
doc_id: 774186
cord_uid: hsnilx3g

The SARS-CoV-2 pandemic causes an ongoing global health crisis. Here, we present as a vaccine candidate synthetic SARS-CoV2 S glycoprotein-coated lipid vesicles that resemble virus-like particles. Soluble S glycoprotein trimer stabilization by formaldehyde cross-linking introduces two major inter protomer cross-links which keeps all receptor binding domains in the “down” conformation. Immunization of cynomolgus macaques with S coated onto lipid vesicles (S-LV) induces high antibody titers with potent neutralizing activity against the vaccine strain, alpha, beta and gamma variants as well as TH1 CD4+ biased T cell responses. Although anti-RBD specific antibody responses are initially predominant, the third immunization boosts significant non-RBD antibody titers. Challenging of vaccinated animals with SARS-CoV-2 shows complete protection through sterilizing immunity, which correlates with the presence of nasopharyngeal anti-S IgG and IgA titers. Thus, the S-LV approach is an efficient and safe vaccine candidate based on a proven classical approach for further development and clinical testing.

Introduction 7

Serum neutralization was further tested against variants B.1.1.7 (Alpha, UK), B.1.351 (Beta, SA) 215 and P.1 (Gamma, BR). Comparing the sera of the vaccinated and the non-vaccinated group at 216 weeks 24 and 28 showed high neutralization titers for all three variants with median ID50s ranging 217 from 10.000 to 20.000, comparable to WT pseudovirus neutralization ( Figure S9) . However, since 218 the background of pre-exposure serum neutralization of the non-vaccinated challenge group was 219 relatively high (median ID50s ranging from 400 of 1100), we repeated the neutralization with 220 purified IgG from serum samples of the vaccinated group from week 8 (after 2 immunizations), 221 week 12 (3 immunizations) and weeks 24 and 28 (4 immunizations). This showed median ID50s 222 of ~4500 for WT and Alpha on week 8 (Figure 7) , comparable to WT serum neutralization ( Figure   223 3A). Lower ID50s were observed against Beta and Gamma at week 8, respectively. Neutralization 224 potency was increased after the third immunization (week 12) with median ID50s of ~5000 (WT), 225 ~8000 (Alpha), ~800 (Beta) and 1000 (Gamma). Neutralization titers did not increase after the 226 fourth immunization at week 24 and started to decrease at week 28 (Figure 7) . We conclude that 227 three immunizations provided robust protection against the variants although neutralization titers 228 maybe have been already within the protective range after two immunizations for the three variants 

Serum neutralization was already significant after the first immunization, but increased by 270 a factor of ~20 after the second immunization and by a factor of 3 after the third immunization 271 indicating that two immunizations with S-LVs may suffice to confer protection. BnAb titers decline 272 within 11 weeks after the third immunization to the levels of week 8 (prior to the third immunization) 273 and increase slightly after the fourth immunization to the median ID50 level attained after the third 274 immunization.

Vaccination prevented lymphopenia and lung damage in animals infected with SARS-CoV- indicating that extensive germinal center reactions are not required 96 . Consistent with these 288 findings we show that RBD-specific antibodies are predominant after the first and second 289 immunization as indicated by similar S-specific and RBD-specific titers. However, after the third 290 immunization median S-specific ED50s are 3 times higher than RBD-specific ED50s four weeks 291 after the third immunization. This trend is continued after the fourth immunization which revealed 292 a 3.5 times higher median ID50 for S than for RBD five weeks post immunization. This, thus 293 suggests that more than two immunizations allow to expand the reactive B cell repertoire that 294 target non-RBD S epitopes. potency compared to WT, which is slightly more potent than the median ID50 of vaccinated and 307 hospitalized patient cohorts using the same assay setup 115 .

In summary, S-LV vaccination represents an efficient strategy that protects macaques from 309 high dose challenge. Although the animals have been challenged only after the fourth 310 immunization, which did not boost Ab titers or neutralization titers, our neutralization data suggests 311 that the animals might have been protected after two immunizations. Furthermore, our data 312 suggest that the third immunization increases S-protein Ab titers more significantly than RBD- For panels A to D, differences between matched groups were compared using the Wilcoxon 444 signed-rank test (p<0.1). Data presented in A to D are from technical duplicates. 15 Medium) without FBS, supplemented with 1% P/S (penicillin at 10,000 U ml -1 and streptomycin at 507 10,000 μg ml -1 ) and 1 μg ml -1 TPCK-trypsin at 37 °C in a humidified CO2 incubator and titrated on 508 Vero E6 cells. Whole genome sequencing was performed as described 120 with no modifications 509 observed compared with the initial specimen and sequences were deposited after assembly on 510 the GISAID EpiCoV platform under accession number ID EPI_ISL_410720. (0,25 mL in each nostril and 4,5 mL in the trachea, i.e., a total of 5 mL; day 0), using atropine (0.04 536 mg/kg) for pre-medication and ketamine (5 mg/kg) with medetomidine (0.042 mg/kg) for 537 anesthesia. Nasopharyngeal, tracheal and rectal swabs, were collected at days 2, 3, 4, 6, 7, 10, 538 14 and 27 days past exposure (dpe) while blood was taken at days 2, 4, 7, 10, 14 and 27 dpe.

Bronchoalveolar lavages (BAL) were performed using 50 mL sterile saline on 3 and 7 dpe. Chest

CT was performed at 3, 7, 10 and 14 dpe in anesthetized animals using tiletamine (4 mg kg -1 ) and 541 zolazepam (4 mg kg -1 ). Blood cell counts, haemoglobin, and haematocrit, were determined from 542 EDTA blood using a DHX800 analyzer (Beckman Coulter). 

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