key: cord-0748110-s3uirmf5
authors: Wu, Yangtao; Wang, Shaojuan; Zhang, Yali; Yuan, Lunzhi; Zheng, Qingbing; Wei, Min; Shi, Yang; Wang, Zikang; Ma, Jian; Wang, Kai; Nie, Meifeng; Xiao, Jin; Huang, Zehong; Chen, Peiwen; Guo, Huilin; Lan, Miaolin; Xu, Jingjing; Hou, Wangheng; Hong, Yunda; Chen, Dabing; Sun, Hui; Xiong, Hualong; Zhou, Ming; Liu, Che; Guo, Wenjie; Guo, Huiyu; Gao, Jiahua; Li, Zhixiong; Zhang, Haitao; Wang, Xinrui; Li, Shaowei; Cheng, Tong; Zhao, Qinjian; Chen, Yixin; Wu, Ting; Zhang, Tianying; Zhang, Jun; Cao, Hua; Zhu, Huachen; Yuan, Quan; Guan, Yi; Xia, Ningshao
title: Lineage-mosaic and mutation-patched spike proteins for broad-spectrum COVID-19 vaccine
date: 2022-01-26
journal: bioRxiv
DOI: 10.1101/2022.01.25.477789
sha: 4a9c5dbd3ad8d9aef2459c06f11a59eb7e221fce
doc_id: 748110
cord_uid: s3uirmf5

The widespread SARS-CoV-2 in humans results in the continuous emergence of new variants. Recently emerged Omicron variant with multiple spike mutations sharply increases the risk of breakthrough infection or reinfection, highlighting the urgent need for new vaccines with broad-spectrum antigenic coverage. Using inter-lineage chimera and mutation patch strategies, we engineered a recombinant monomeric spike variant (STFK1628x), which showed high immunogenicity and mutually complementary antigenicity to its prototypic form (STFK). In hamsters, a bivalent vaccine comprised of STFK and STFK1628x elicited high titers of broad-spectrum antibodies to neutralize all 14 circulating SARS-CoV-2 variants, including Omicron; and fully protected vaccinees from intranasal SARS-CoV-2 challenges of either the ancestral strain or immune-evasive Beta variant. Strikingly, the vaccination of hamsters with the bivalent vaccine completely blocked the within-cage virus transmission to unvaccinated sentinels, for either the ancestral SARS-CoV-2 or Beta variant. Thus, our study provides new insights and antigen candidates for developing next-generation COVID-19 vaccines.

aspects, such as viral infectivity, pathogenicity, and antigenicity. Critically, the 29 amino-acid substitutions in the antigenic sites of the spike protein may enable 1 viruses to escape from naturally acquired and vaccine-induced immunity (1). 2 Among the variants currently identified as variants of concern (VOCs) or 3 variants of interest (VOIs), many were able to cause immune escape. The Beta 4 (B.1.351) variant, first identified in South Africa, was found to cause a 6.5-8.6-5 fold decrease in nAb titers raised by existing mRNA vaccines (2). Besides, the In this study, using inter-lineage chimera and mutation patch strategies, we yield of homogeneous trimeric proteins for vaccine production. Therefore, we 20 tried to design and produce monomeric spike proteins for the COVID-19 21 vaccine to address these issues. Although numerous high-resolution structures 22 of SARS-CoV-2 spike trimers were reported, the detailed structure of the S2 C-23 terminus, particularly for those after amino acid (aa) 1146, was not resolved. 24 We tested eight constructs of furin site mutated spike ectodomain with 25 progressively truncated C-terminus in Chinese hamster ovary (CHO) cells. 26 Interestingly, the C-terminal truncation to various positions between aa1152 and 27 aa1192 resulted in higher expression levels and purification yields than the 28 construct encompassing the entire ectodomain (S1208) (Fig. 1A) . In addition, 29 the C-terminal truncated spike proteins presented comparable, even better 1 ACE2 binding activity to the trimeric StriFK protein (17) ( fig. S1A ). To minimize 2 the potential epitope loss associated with C-terminal truncation, we finally 3 chose the construct of S1192 encompassing aa 1-1192 (hereafter designated 4 STFK) as an immunogen candidate for further study. As expected, the STFK 5 was presented in monomeric form, as evidenced by the SEC-HPLC and native-6 PAGE analyses (Fig. 1B) . In contrast to the trimeric StriFK, the STFK elicited 7 significantly higher nAb titers against the pseudotyped virus (PsV) in mice at 8 weeks 1 (P = 0.002) and 2 (P =0.028) after the 1 st prime vaccine dose, 9 suggesting the advantage of the STFK for induction of rapid humoral response.

After the 2 nd dose immunization, both STFK and StriFK-based vaccines 11 generated comparable nAb titers (Fig. 1C) . 12 To determine the structural basis for the excellent immunogenicity of the STFK, 13 we resolved the cryo-electron microscopy (cryo-EM) structure of the STFK in 14 complex with three previously reported nAbs 36H6, 83H7, and 85F7 (18, 19). 15 Following the previous classifications of the nAbs targeting epitopes (Class I-V) 16 (20, 21), the 36H6, 83H7, and 85F7 were categorized into Class II, V, and III, 17 according to their binding modes, respectively ( fig. S2 and S3) . The 3.81Å 18 resolution structure of the immune-complex confirmed the monomeric form of 19 the STFK, which could interact with three antigen-binding fragments (Fabs) of 20 nAbs simultaneously (Fig. 1D, fig. S2A , and Table S1 ). The STFK is 21 structurally similar to the monomeric form dissociated from a spike trimer (22).

Due to the conformational flexibility in the monomeric form, the S2 subunit was 23 not visualized in the reconstruction. However, in contrast to the trimeric spike, 24 the STFK presents a more exposed RBD and NTD, thereby making the nAb 25 epitopes more accessible and may contribute to its advantage for eliciting rapid 26 nAb response.

Next, we evaluated the dose-dependent immunogenicity of the STFK-based 28 vaccine with the FH002C adjuvant in the BALB/c mice, rhesus monkeys, and 29 golden hamsters. In our previous study, the FH002C, a risedronate-modified 1 new adjuvant, showed potent immunostimulatory effects for hormonal and 2 cellular immune responses (17). In BALB/c mice, STFK vaccinations generated 3 a dose-dependent response for the anti-spike IgG, anti-RBD IgG ( fig. S1B) , 4 and neutralizing antibodies at 0.01 to 10 μg dose levels (Fig. 1E) . Two injections 5 of STFK at a dose level as low as 0.1 μg induced a potent nAb response 6 showing geometric mean titers (GMT) of 3.9 log10 against the PsV and 362 7 against the authentic virus (200 TCID50) that were 3.5-and 2.3-fold higher than 8 that of the NIBSC 20/136 anti-SARS-CoV-2 standard (1,000 IU/mL) in the 9 corresponding assays (Fig. 1E) . In rhesus monkeys, STFK vaccinations at 1 or 10 15 μg dose levels also elicited strong humoral immune responses ( Fig. 1F and   11 fig. S1C), as shown that immunized animals presented high nAb titers against 12 either PsV (GMT=4.1 and 4.5 log10 for 1 and 15 μg groups, respectively) or 13 authentic virus (GMT=588 and 1,351 for 1 and 15 μg groups, respectively). In 25 We investigated the impacts of 14 VOC/VOI variants on nAbs raised by the 26 prototypic STFK in animals. Notably, all sera from ten monkeys and eight 27 hamsters at week-2 after 2-dose vaccinations showed detectable nAbs against 28 all PsVs bearing VOC/VOI spike variants, including the newly emerged 29 Omicron (Fig. 1H) . 1H and 1I) . For Alpha, Delta, Epsilon (B.1.429), 6 and Lambda (C.37) variants, the nAb titers only slightly changed a (<2-fold). 7 Moreover, sera from immunized hamsters presented highly similar (R 2 =0.889, 8 P < 0.001) cross-neutralizing profiles to that of monkeys (Fig. 1I) . These results 9 are consistent with findings in humans that the E484K-harboring variants and 10 the Omicron may markedly evade nAbs raised by the prototypic spike.

Following the approach as graphically depicted in Fig. 2A , we aimed to develop 12 a new STFK antigen providing complementary antigenic coverage to the 13 prototypic protein to address the concerns for the evasive variants ( Fig. 2A) . 14 As the Mu and Omicron variants had not emerged when our experiment started, 15 we firstly tested mutated STFK antigens based on the spikes of Beta 16 (STFK1351), Gamma (STFK1128), and B.1.620 (STFK1620) variants ( fig. S4 ).

Compared to those immunized with STFK, hamsters vaccinated with 18 STFK1351, STFK1128, and STFK1620 showed 1.0-3.0×, 1.3-6.2×, and 1.7- STFK1628x also exhibited higher nAb titers against most VOC/VOI variants 18 ( Fig. 2B and fig. S7B ). These data supported the STFK1628x as a promising 19 antigen candidate for the updated COVID-19 vaccine. As our previous study 20 suggested that the aa 439-448 was another hot-spot region in addition to aa484 21 (19), we further made two modified STFK1628x versions, designated 22 STFK1628y and STFK1628z, that included N440K and G446V, respectively 23 ( Fig. 2B and fig. S7B ). The STFK1628y and STFK1628z displayed distinct . S7C ). Following these data, we 29 formulated a bivalent vaccine using the STFK1628x and the prototypic STFK 1 at a mass ratio of 1:1. To most of the VOC/VOI variants, hamsters immunized 2 with the bivalent vaccine showed significantly (P < 0.05) increased nAb levels 3 to that elicited by the prototypic antigen in neutralizing the D614G virus (~4.0 4 log10) ( Fig. 2B and 2C) . Strikingly, the bivalent vaccine yielded a nAb GMT of 5 2,130 (ID50 range: 9,61 to 4,763) to the highly immune-evasive Omicron, which 6 was about 36-fold higher than the NIBSC 20/136 immunoglobulin standard 7 (ID50=60 to Omicron). Taken together, STFK plus STFK1628x provided a full-8 spectrum neutralization coverage to all VOC/VOI variants. 9 We also obtained a 3.88 Å cryo-EM structure of the STFK1628x in complexed 10 with three nAbs 83H7, 85F7, and 2B4 (Fig. 2D, fig. S2B , and Table S1 ). As the 11 T478K abolishes the activity of 36H6 nAb, we replaced the 36H6 with a class 12 IV mAb of 2B4 with cross-SARS-CoV-1/2 neutralization potency ( fig. S3D ) (18).

As expected, the STKF1628x presented a similar structure to STFK but showed 14 distinguished densities on the mutation sites, such as 417, 452, 478, 484, and 15 501, corresponding to its alternative antigenic profile (Fig. 2E) . 16 The bivalent vaccine protects hamsters against intranasal SARS-CoV-2 17 challenges 18 To assess the ability of the STFK-based vaccine to mediate protection against 19 SARS-CoV-2, we intranasally challenged hamsters that received STFK, 20 STFK1628x, or bivalent vaccines (Fig. 3A) . For either challenge with the respectively, but none in the vaccinated groups ( Fig. 3D and 3E) . 28 At 7 dpi, the median viral RNA levels of control hamsters challenged by the 29 prototypic virus were 7.26 (range 5.24-7.78) log10 in the lung, 6.88 (range 6.12-1 7.32) log10 in the nasal turbinate, and 6.29 (range 5.04-6.67) log10 copies/mL in 2 the trachea ( Fig. 3F and fig. S8A ). By contrast, hamsters that received 3 vaccinations of either STFK, STFK1628x, or the bivalent version showed 4 significant (P < 0.01 for each comparison) viral RNA reductions by >5.0 log10, 5 2.0-3.0 log10, 3.0-4.0 log10 copies/mL in tissues of the lung, nasal turbinate, and 6 trachea, respectively ( Fig. 3F and fig. S8A ). To protect the prototypic virus 7 challenge, the three vaccine candidates appeared with comparable efficacy (P > In summary, our study provides a new way to design new antigens for next-20 generation COVID-19 vaccines aiming to confer broad-spectrum protection. selections and single-cell clonings. Polyhistidine-tagged proteins (S1152 to 18 S1208) purified from culture supernatants were collected on day 7 after 19 transfection using Ni Sepharose 6FF (Cytiva). The tag-free STFK proteins were 20 purified by using Q-FF Sepharose ion-exchange chromatography (Cytiva).

Recombinant human ACE2 (human Fc tag, rACE2) protein also was produced 22 in ExpiCHO-S cells and purified by protein-A affinity chromatography column 23 (Cytiva) as previously described (17). The SEC-HPLC analysis shown in Fig. 1B was performed using a TSK-GEL 6 G3000PWXL column on an HPLC system (Waters Alliance) and conducted as 7 described previously(17). Immunized mice were sacrificed on day 7 after immunization to collect 26 splenocytes for further assay. The total electron dose was set to 60 e − Å −2 , and the exposure time was 4.5 s. The initial models of nAbs were generated from homology modeling by Accelrys naïve sentinels for one day (Fig. 4A) . The daily diet was limited to 7 g per 100 7 g of body weight to prevent animals from overeating. All hamsters were 

A. G. Wrobel et al., Antibody-mediated disruption of the SARS-CoV-2 spike 1 glycoprotein. Nat Commun 11, 5337 (2020 progressive truncations from the C terminus of the furin site with mutated spike 5 ectodomain in CHO cells. S1208, aa 1-1208; S1200, aa 1-1200; S1192, aa 1-6 1192; S1184, aa 1-1184; S1176, aa 1-1176; S1168, aa 1-1168; S1160, aa 1- comparisons. Asterisks indicate statistical significance (****P < 0.0001; ***P < 5 0.001; **P < 0.01; *P < 0.05; ns, not significant). later, each 2 index hamsters were cohoused with 4 naïve sentinels for one day 7 (separated by a double-layer ventilated fence in the same cage). Sentinel 8 hamsters (n=8) were followed for 7-day and then euthanized for tissue analyses. Kruskal-Wallis test (D, E) were used for intergroup statistical comparisons, and 8 asterisks indicate statistical significance (****P < 0.0001; ***P < 0.001; **P < 9 0.01; *P < 0.05; ns, not significant). Uncorrected Kruskal-Wallis test (B, D) or Mann-Whitney U test (C, E) were used 12 for intergroup statistical comparisons. Asterisks indicate statistical significance 13 (****P < 0.0001; ***P < 0.001; **P < 0.01; *P < 0.05; ns, not significant). Wallis tests were used for statistical comparison. Asterisks indicate statistical 12 significance (****P < 0.0001; ***P < 0.001; **P < 0.01; *P < 0.05; ns, not 13 significant). Kruskal-Wallis tests were used for intergroup statistical comparison. Asterisks 5 indicate statistical significance (****P < 0.0001; ***P < 0.001; **P < 0.01; *P < 6 0.05; ns, not significant). 

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