key: cord-0328215-30zru2qb
authors: Sulbaran, Guidenn; Maisonnasse, Pauline; Amen, Axelle; Guilligay, Delphine; Dereuddre-Bosquet, Nathalie; Burger, Judith A.; Poniman, Meliawati; 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: 2021-07-26
journal: bioRxiv
DOI: 10.1101/2021.07.26.453755
sha: 212c5841acea9a61f51f86501678b71354e670e8
doc_id: 328215
cord_uid: 30zru2qb

The SARS-CoV-2 pandemic causes an ongoing global health crisis, which requires efficient and safe vaccination programs. Here, we present synthetic SARS-CoV2 S glycoprotein-coated liposomes that resemble in size and surface structure virus-like particles. Soluble S glycoprotein trimers were stabilized by formaldehyde cross-linking and coated onto lipid vesicles (S-VLP). Immunization of cynomolgus macaques with S-VLPs induced high antibody titers and TH1 CD4+ biased T cell responses. Although antibody responses were initially dominated by RBD specificity, the third immunization boosted non-RBD antibody titers. Antibodies showed potent neutralization against the vaccine strain and the Alpha variant after two immunizations and robust neutralization of Beta and Gamma strains. Challenge of animals with SARS-CoV-2 protected all vaccinated animals by sterilizing immunity. Thus, the S-VLP approach is an efficient and safe vaccine candidate based on a proven classical approach for further development and clinical testing.

S-VLPs were produced for a small vaccination study of cynomolgus macaques to evaluate 141 safety, immunogenicity and elicitation of neutralizing antibodies. Four cynomolgus macaques 142 were immunized with 50 µg S-VLPs adjuvanted with monophospholipid A (MPLA) liposomes by 143 the intramuscular route at weeks 0, 4, 8 and 19 (Figure 2A) . Sera of the immunized macaques 144 were analysed for binding to native S glycoprotein (S), FA cross-linked S glycoprotein (FA-S) and 145 RBD in two weeks intervals. This revealed median S ED50 titers of 100 at week 4, 3000 at week 146 8 and 25 000 at week 12 ( Figure 2B ). Slight reductions in titers were detected against FA-S 147 ( Figure 2C ). Titers against RBD alone were also high with median ED50s of 80 at week 4, 2000 148 at week 8 and 5200 at week 12 ( Figure 2D ). This suggests that the first and second 149 immunization induced significant RBD titers, while the third immunization boosted non-RBD 150 antibodies since the week 12 S-specific titers were > 5 times higher than the RBD titers ( Figure   151 2C). A fourth immunization did not further boost antibody generation and titers at week 22 were 152 lower or comparable to week 12 titers ( Figure 2B , C, D). We conclude that S-VLP immunization 153 induces primarily RBD-specific antibodies after the first and second immunization, while the third 154 immunization increases the generation of non-RBD antibodies significantly.

Serum neutralization titers using WT pseudovirus were significant in all four animals. At

week 2 after the first immunization, a median ID50 titer of 480 was observed, which dropped 157 close to baseline at week 4, but was significantly increased at week 6, two weeks after the 158 second immunization demonstrating a median ID50 of 9,000. The ID50s then decreased to a 159 median of 6,000 at week 8 and increased to a median of 18,500 at week 11, three weeks after 160 the third immunization. At week 19, neutralization potency decreased but was still high with a 161 median of 5,200, indicating that three immunizations induced robust neutralization titers. The 162 fourth immunization boosted neutralization titers to a median ID50 of 20,000, the same level as 163 after the third immunization ( Figure 3A ).

Since antibody titers indicated the induction of high levels of RBD-specific antibodies, we 165 depleted the serum at week 11 by anti-RBD affinity chromatography resulting in no detectable 166 RBD antibodies by ELISA. RBD-specific Ab-depleted serum showed 10 to 30% neutralization 167 compared to the complete serum, indicating non-RBD specific neutralization. While RBD-specific

Ab neutralization dominated in one animal, the other three revealed 30 to 48% RBD-specific Ab 169 neutralization activity (Figure 3B) , suggesting nAb synergy to achieve the high neutralization 170 titers ( Figure 3A) .

In order to determine the extent of S-VLP vaccination induced protection, vaccinated and non-174 vaccinated animals (n=4) were infected with the primary SARS-CoV-2 isolate 175 (BetaCoV/France/IDF/0372/2020) with a total dose of 1 x 10 5 plaque forming units (pfu). Infection 176 was induced by combining intranasal (0.25 mL into each nostril) and intratracheal (4.5 mL) 177 6 routes at week 24, 5 weeks after the last immunization. Viral load in the control animal group 178 peaked in the trachea at 3 days post-exposure (dpe) with a median value of 6.0 log 10 copies/ml 179 and in the nasopharynx at day 6 pe with a median copy number of 6,6 log 10 copies/ml ( Figure   180 4A). Viral loads decreased subsequently and no virus was detected on day 10 pe in the trachea,

while some animals showed viral detection up to day 14 pe in the nasopharyngeal swabs 182 ( Figure 4A ). In the bronchoalveolar lavage (BAL), three CTRL animal out of four showed 183 detectable viral loads at day 3 pe, and two of them remained detectable at day 7 pe with mean 184 value of 5.4 and 3.6 log 10 copies/mL respectively. Rectal fluids tested positive in one animal, 185 which also had the highest tracheal and nasopharyngeal viral loads ( Figure S2 ). Viral 186 subgenomic RNA (sgRNA), which is believed to estimate the number of infected and productive 187 cells collected with the swabs or during the lavage, showed peak copy numbers between day 3/4 188 and 6 pe in the tracheal and nasopharyngeal fluids, respectively ( Figure 4B ). In the BALs, the 189 two animals presenting high genomic viral loads also showed detectable sgRNA at days 3 and 7 190 pe, with medians of 5,1 and 3.1 log 10 copies/mL respectively ( Figure 4B ). 

Similar to previous observations (Maisonnasse et al., 2020; Brouwer et al., 2021) , during 

Before exposure, Th1 type CD4 + T-cell responses were observed in all vaccinated macaques 213 following ex vivo stimulation of PBMCs with S-peptide pools (Figure 6 and S6) . None had 7 detectable anti-S CD8 + T cells ( Figure S5 ). No significant difference was observed at day 14 pe, 215 also in agreement with the absence of an anamnestic response. In contrast, the anti-S Th1 CD4+ 216 response increased post exposure for most of the control animals ( Figure S6 and S7).

We conclude that S-VLP vaccination can produce sterilizing immunity indicating that the 218 vaccination scheme is efficient to interrupt the chain of transmission. proline mutations that enhanced stability . However, this S '2P' version still 245 showed limited stability over time as reported , which may be due to cold 246 sensitivity (Edwards et al., 2021) . We overcame the problem of stability by using formaldehyde 247 cross-linking that increased the thermostability to 65°C, preserving the native S conformation 248 over extended storage time periods. Notably, formaldehyde cross-linking is widely used in 249 vaccine formulations (Eldred et al., 2006) . S stability has been since improved by engineering six 250 proline mutations (S '6P') which increased the thermostability to 50°C (Hsieh et al., 2020) and by 251 8 disulfide-bond engineering (Xiong et al., 2020) . Furthermore, ligand binding renders S more 252 stable (Rosa et al., 2021; Toelzer et al., 2020) .

Many previous studies have shown that immunogen multimerization strategies are highly 

Serum neutralization was already significant after the first immunization, but increased by 281 a factor of ~20 after the second immunization and by a factor of 3 after the third immunization 282 indicating that two immunizations with S-VLPs may suffice to confer protection. BnAb titers 283 decline within 11 weeks after the third immunization to the levels of week 8 (prior to the third 284 immunization) and increase to the median ID50 level attained after the third immunization. ID 50 285 neutralization values decline by a factor of ~4 between week 22 and week 28 after the fourth 286 immunization consistent with general Ab decline over time.

Vaccination prevented lymphopenia and lung damage in animals infected with SARS-

CoV-2 at a dose comparable (Corbett et al., 2020; Guebre-Xabier et al., 2020; Mercado et al., 289 2020; van Doremalen et al., 2020; Yu et al., 2020) or lower (Brouwer et al., 2021) than in 290 previous studies. Protection was sterilizing since no replication could be detected in the upper 291 and lower respiratory tract suggesting that vaccination with S-VLPs will prevent virus shedding 292 and transmission. Sterilizing immunity likely correlates with mucosal antibody responses that 293 protects the upper respiratory tract from infection (Isho et al., 2020a; Randad et al., 2020) .

However, we failed to detect significant IgA or IgG in nasopharyngeal fluids, which may be due to 295 the low sensitivity of the ELISA tests used.

Most of the antibodies (up to 90%) generated by vaccination are directed against RBD, 297 which is immunodominant (Piccoli et al., 2020) . RBD antibodies can be grouped into three 298 classes (Barnes et al., 2020a; Barnes et al., 2020b) and seem to be easily induced by 299 immunization as many of them are generated by few cycles of affinity maturation indicating that 300 extensive germinal center reactions are not required (Kreye et al., 2020) . Consistent with these 301 findings we show that RBD-specific antibodies are predominant after the first and second 302 immunization revealing similar S-specific and RBD-specific titers. However, after the third 303 immunization median S-specific ED50s are 3 times higher than RBD-specific ED50s four weeks 304 after the third immunization. This trend is continued after the fourth immunization which revealed 305 a 3.5 times higher median ID50 for S than for RBD five weeks post immunization. This, thus 306 demonstrates that more than two immunizations allow to expand the reactive B cell repertoire 307 that target non-RBD S epitopes.

Current variants carry the B.1 D614G mutation and have been reported to be more 309 infectious (Cai et al., 2021; Gobeil et al., 2021; Korber et al., 2020; Ozono et al., 2021; Yuan et 310 al., 2021; Zhang et al., 2021; Zhang et al., 2020b) . Although the D614G mutation alone was 311 reported to increase neutralization susceptibility (Weissman et al., 2021) , further mutations 

In summary, S-VLP vaccination represents an efficient strategy that protects macaques 327 from high dose challenge. Although the animals have been challenged only after the fourth 328 immunization, which did not boost Ab titers or neutralization titers, our neutralization data 329 suggests that the animals might have been protected after two immunizations. Furthermore, we 330 provide evidence that the third immunization boosts non-RBD antibodies which is likely important 331 to protect against different variants. This also suggests that future vaccination strategies should 332 probably boost non-RBD antibodies to compensate for the loss of neutralization targeting RBD. 2, 4, 6, 8, 11, 12, 14, 19, 21 and 22 . At week 24, all 436 animals were exposed to a total dose of 10 5 pfu of SARS-CoV-2 virus (hCoV-19/France/ 437 lDF0372/2020 strain; GISAID EpiCoV platform under accession number EPI_ISL_410720) via 438 the combination of intranasal and intra-tracheal routes (0,25 mL in each nostril and 4,5 mL in the 439 trachea, i.e., a total of 5 mL; day 0), using atropine (0.04 mg/kg) for pre-medication and ketamine

(5 mg/kg) with medetomidine (0.042 mg/kg) for anesthesia. Nasopharyngeal, tracheal and rectal 441 swabs, were collected at days 2, 3, 4, 6, 7, 10, 14 and 27 days past exposure (dpe) while blood 442 was taken at days 2, 4, 7, 10, 14 and 27 dpe. Bronchoalveolar lavages (BAL) were performed 443 using 50 mL sterile saline on 3 and 7 dpe. Chest CT was performed at 3, 7, 10 and 14 dpe in 444 anesthetized animals using tiletamine (4 mg kg -1 ) and zolazepam (4 mg kg -1 ). Blood cell counts,

haemoglobin, and haematocrit, were determined from EDTA blood using a DHX800 analyzer 446 (Beckman Coulter). in nonhuman primates. Nat Commun 12, 1346. 8, 11, 12, 19, 22, 24, 25, 28 . Median values calculated for the 4 animals are indicated.

(C) ELISA of SARS-CoV-2 FA-S-protein-specific IgG determined during the study.

(D) ELISA of SARS-CoV-2 S RBD-specific IgG determined during the study. 

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Antibody IgG titers were determined by 998 ELISA at weeks 24 (challenge), 25, 26 and 28 against (A) SARS-CoV-2 S, (B) SARS-CoV-2 FA-999 S and (C) SARS-CoV-2 S RBD

CoV-2 pseudovirus neutralization titers at week 24 (challenge) and 1, 2 and 4 weeks 1002 post exposure (weeks 25, 26, 28). The Bars show the median titers

Antigen-specific CD4 T-cell responses in S-VLP immunized cynomolgus 1007 macaques. Frequency of (A) IFNγ+, TFNα+ and IL-2+, (B), Th1 (IFN γ +/-, IL-2+/-, TNFα+)

for each immunized macaque (n = 4) at week (W)21 post-immunization (p.im.) (i.e. 1010 two weeks after the 4 th immunization, pre-exposure) and 14 days post-exposure (d

Figure 7: S-VLP vaccination induces robust neutralization of SARS CoV-2 variants

B.1.351 (Beta, SA) and P.1 (Gamma, BR) pseudovirus neutralization titers 1021 were compared to the Wuhan vaccine strain. Titers were determined using total IgG purified from 1022 sera at weeks 8 (2 immunizations)

Figure S4: S-VLP vaccination induces neutralization of SARS CoV-2 variants

Titers were determined at weeks 24 1054 (exposure) and 28 (4 weeks pe). Comparison of sera from vaccinated macaques and the control 1055 group indicated high background values at week 24 (challenge) for Beta and Gamma

IL-13+ (bottom 1073 left) and IL-17+ (bottom right) antigen-specific CD8+ T cells (CD137+) in the total CD8+ T cell 1074 population, respectively, for each immunized macaque (n = 4) at week (W)21 post-first 1075 immunization (p.im.) (i.e. two weeks after the 4 th immunization, pre-exposure) and 14 days post-1076 exposure (d

Time points in each experimental group were 1078 compared using the Wilcoxon signed rank test

Th1 (IFN γ +/-, IL-2+/-, TNFα+), (C) IL-13+and IL-17+ antigen-1086 specific CD4+ T cells (CD154+) in the total CD4+ T cell population, respectively, for each control 1087 (n=4, black) and immunized macaque (n = 4, red) at week (W)21 post-first immunization

PBMCs were stimulated overnight with medium (open symbols) or SARS-CoV-2 S overlapping 1090 peptide pools (filled symbols)

Wilcoxon signed rank test. Groups were compared using the non-parametric Mann-Whitney test