key: cord-0864478-9m76jfcf
authors: Qu, Liang; Yi, Zongyi; Shen, Yong; Lin, Liangru; Chen, Feng; Xu, Yiyuan; Wu, Zeguang; Tang, Huixian; Zhang, Xiaoxue; Tian, Feng; Wang, Chunhui; Xiao, Xia; Dong, Xiaojing; Guo, Li; Lu, Shuaiyao; Yang, Chengyun; Tang, Cong; Yang, Yun; Yu, Wenhai; Wang, Junbin; Zhou, Yanan; Huang, Qing; Yisimayi, Ayijiang; Cao, Yunlong; Wang, Youchun; Zhou, Zhuo; Peng, Xiaozhong; Wang, Jianwei; Xie, Xiaoliang Sunney; Wei, Wensheng
title: Circular RNA Vaccines against SARS-CoV-2 and Emerging Variants
date: 2022-01-11
journal: bioRxiv
DOI: 10.1101/2021.03.16.435594
sha: 2c493ad6336fe84ce5e0c9f09f24da0a2be83900
doc_id: 864478
cord_uid: 9m76jfcf

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and its emerging variants of concern (VOC), such as Delta (B.1.617.2) and Omicron (B.1.1.529), has continued to drive the worldwide pandemic. Therefore, there is a high demand for vaccines with enhanced efficacy, high thermostability, superior design flexibility, and fast manufacturing speed. Here, we report a circular RNA (circRNA) vaccine that encodes the trimeric RBD of SARS-CoV-2 Spike protein. Without the need of nucleotide modification, 5’-capping or 3’-polyadenylation, circRNA could be rapidly produced via in vitro transcription and is highly thermostable whether stored in naked or lipid-nanoparticle (LNP)-encapsulated format. LNP-encapsulated circRNARBD elicited potent neutralizing antibodies and T cell responses, providing robust protection against Beta (B.1.351) and native viruses in mice and rhesus macaques, respectively. Notably, circRNA vaccine enabled higher and more durable antigen production than 1mΨ-modified mRNA vaccine, eliciting a higher proportion of neutralizing antibodies and stronger Th1-biased immune responses. Importantly, we found that circRNARBD-Omicron vaccine induced effective neutralizing antibodies against only Omicron but not Delta variant. By contrast, circRNARBD-Delta could elicit high level of neutralizing antibodies against both Delta and Omicron. Following two doses of either native- or Delta-specific vaccination, circRNARBD-Delta, but not Omicron or Beta vaccines, could effectively boost the neutralizing antibodies against both Delta and Omicron variants. These results suggest that circRNARBD-Delta is a favorable choice for vaccination to provide a broad-spectrum protection against the current variants of concern of SARS-CoV-2.

chromatography (HPLC) showed that the RNase R treatment purged a significant amount of the

To test the secretory expression of RBD antigens produced by circRNA RBD , the purified 123 circRNA RBD was transfected into human HEK293T cells or murine NIH3T3 cells. Abundant RBD 124 antigens in the supernatant of both human and murine cells was detected by Western blot, 125

indicating the high compatibility of circRNAs (Fig. 1b) . The concentration of RBD antigens 126 produced by circRNA RBD reached ~1,400 ng/mL, 600-fold higher than its linear precursor RNA 127 (Fig. 1c) . 128

Besides the Group I ribozyme autocatalysis strategy, we developed an alternative method to 129 generate circRNA RBD using T4 RNA ligase (Extended Data Fig. 3a) . Similarly, abundant RBD 130 antigens have been detected in the supernatant at a concentration of ~1,000 ng/mL concentration, 131 which is ~200-fold higher than that produced by its linear precursor RNA (Extended Data Fig.  132 3b, c). 133

To verify whether the secreted SARS-CoV-2 RBD antigens produced by circRNA RBD were 134 functional, the supernatants of circRNA RBD -transfected cells were used in a competition assay 135 using hACE2-overexpressing HEK293 cells (HEK293T-ACE2) and SARS-CoV-2 pseudovirus 136 harboring an EGFP reporter 56 . The secreted SARS-CoV-2 RBD antigens could effectively block 137 SARS-CoV-2 pseudovirus infection (Fig. 1d) . Altogether, circRNA RBD showed robust protein 138 expression, suggesting its potential as a novel vaccine platform. 139 140

To explore whether circRNA could be leveraged to create a new type of vaccine, we attempted to 143 assess the immunogenicity of circRNA RBD encapsulated with lipid nanoparticles in BALB/c mice 144 (Fig. 1e) . The circRNA RBD encapsulation efficiency was greater than 93%, with an average 145 diameter of 100 nm (Fig. 1f) . Mice were immunized with LNP-circRNA RBD through intramuscular 146 (i.m.) injection twice at a two-week interval, at doses of 10 µg or 50 µg per mouse, with empty 147 antibodies was measured at two-or five-weeks post the boost dose. The circRNA RBD elicited high

To further evaluate the protective efficacy of circRNA RBD-Beta vaccine in vivo, we employed the 180 Beta variant for authentic virus challenge experiments. Consistent with a recent report 57,58 , the 181 Beta variant could infect wild-type BALB/c mice and replicate in their lungs (Extended Data Fig.  182 5c), likely due to the mutations in Spike protein such as K417N, E484Q, and N501Y 57,59 . Seven 183 weeks post the boost dose, the RBD-Beta-specific IgG endpoint GMT was still around 1.2×10 4 184 (Extended Data Fig. 5d) , with significant neutralizing activity against RBD-Beta antigens 185 (Extended Data Fig. 5e ). Each immunized mouse was then intranasally infected with 5×10 4 PFU 186 of Beta virus (7 weeks post the boost dose). The lung tissues were collected three days after the 187 challenge for the detection of viral RNAs. Viral loads in the lungs of vaccinated mice were 188 significantly lower compared with the placebo group (Fig. 1m) . Consistently, only the mice in the 189 placebo group underwent weight loss (Fig. 1n) . These results indicated that the circRNA vaccine could effectively protect the mice against SARS-CoV-2 Beta. 191 Considering that the 50 µg of circRNA RBD elicited a higher level of neutralizing antibodies than 192 10 µg, we postulated that the LNP delivery might have great impact on the efficacy of circular 193 RNA vaccine. After multiple tests, we were able to significantly lower the vaccine dose using one 194 of the commercial formulas (Precision Nanosystems). 10 µg of circRNA could induce neutralizing 195 antibodies at a level comparable to 50 µg (Extended Data Fig. 6 ). We thus switched our choice 196 of LNP for the rest of our experiments. 197 with the NT50 of ~1.4×10 5 for the 10 µg dose (Fig. 2c) .

was evidently reduced with the increase of storage temperature, especially at 37°C (Extended based vaccines, circRNA vaccine exhibited higher stability than mRNA vaccines (Extended Data 240 Fig. 7a-c) . BNT162b2 (Pfizer/BioNTech), both of which contain modified 1mΨ modification. Given that 246 circRNA vaccine possesses higher stability and antigen-encoding efficiency, we wonder whether 247 it exhibited superior immunogenicity to mRNA vaccine. We first compared the balance of Th1 and 248

Th2 immune responses between circRNA RBD-Delta and mRNA RBD-Delta vaccines because Th2-biased 249 immune responses might induce vaccine-associated enhanced respiratory disease (VAERD) 27,32,64 . 250 ELISA assay showed that the total IgG elicited by circRNA RBD-Delta was comparable to that by 251 mRNA RBD-Delta (Fig. 3e) , however, the ratio of IgG2a/IgG1, IgG2c/IgG1 or (IgG2a + IgG2c)/IgG1 252 from circRNA RBD-Delta was higher than from mRNA RBD-Delta vaccine ( Fig. 3f , g, Extended Data 253 Fig. 8a, b) , indicating that circular RNA vaccines tended to induce stronger Th1-biased immune 254 responses. 255

Antibody-mediated enhancement (ADE) of infection by virus-specific antibodies is another 256 potential concern for vaccines, which has been reported for infections by some viruses including 257 Zika, Dengue virus, and coronaviruses 65-69 . Previous research has reported that the virus-binding 258 antibodies without neutralizing activity elicited by infection or vaccination possibly caused the 259 ADE effects, especially for those viruses with different serotypes 70,71 . Therefore, we compared the 260 ratio of neutralizing to binding antibodies between circRNA and mRNA vaccines. Although 261 circRNA RBD-Delta exhibited equal neutralizing capability compared to mRNA RBD-Delta (Fig. 3h-j) , 262 the former induced a higher proportion of neutralizing antibodies at all doses tested (0.5 µg, 2.5 263 µg, and 5 µg) in mice (Fig. 3k) . Owing to this unique feature, the circRNA vaccine might have 264 certain advantage to circumvent potential ADE effects and tolerate viral mutations. 265 circRNA RBD-Delta vaccine elicited strong T cell immune responses 4a-c). Similar to the above observations ( Fig. 3h-k) , circRNA RBD-Delta vaccine also elicited higher 297 proportion of neutralizing antibodies against Omicron variant than 1mΨ-mRNA RBD-Delta vaccine at 298 both 2 weeks post boost (short-term) and 7 weeks post boost (long-term) (Extended Data Fig.  299 11a-d), indicating the potential superiority of circRNA vaccine against the circulating variants of 300 SARS-CoV-2, likely owing to its high proportion of neutralizing antibody (Fig. 3h-k and  301 Extended Data Fig. 11a-d) . GMTs of ~4.7×10 4 for 5 µg dose and ~2.2×10 5 for 10 µg dose (Fig. 4d) , yielding evident 310 neutralizing activities against Omicron with the NT50 of ~2.5×10 3 for the 5 µg dose and ~8.6×10 3 311 for the 10 µg dose (Fig. 4e) . However, neutralizing activity could hardly be detected against native 312 strain or Delta variant (Fig. 4e, f) . booster with circRNA RBD-Beta , circRNA RBD-Delta or circRNA RBD-Omicron vaccine at 7 weeks post the 319 2 nd dose, followed by the assessment of neutralizing activity against the Omicron variant at 1 week 320 post the 3 rd boost (Fig. 4g) . Only circRNA RBD-Delta effectively boosted the neutralizing antibodies 321 against both Delta (Fig. 4h) and Omicron (Fig. 4i) . On the contrary, the 3 rd boost with the 322 circRNA RBD-Beta or circRNA RBD-Omicron vaccine failed to elevate the neutralizing capability against 323 Omicron (Fig. 4h, i) .

previously immunized with 2 doses of circRNA RBD vaccines (Fig. 4j) . Both vaccines could circRNA RBD-Delta appeared to be a much better booster than circRNA RBD against both Delta and 328

Omicron variants, which elevated the geometric mean NT50 from ~4×10 2 to ~3.2×10 4 against 329

Omicron variant (Fig. 4k, l) . 330

Taken together, these results suggest that circRNA RBD-Delta might be a favorable choice for 331 vaccination to provide a broad-spectrum protection against the current VOC of SARS-CoV-2. 332 However, the 3 rd booster with Omicron-specific vaccines might not be an appropriate strategy 333 against the current Delta and Omicron emergency in the real world. To further assess the immunogenicity of circRNA vaccine in non-human primates (NHPs), groups 338 of 2~4-year-old rhesus macaques were immunized i.m. with 20 µg, 100 µg or 500 µg of 339 circRNA RBD vaccines, 100 µg of circRNA Ctrl , or PBS control on Days 0 and 21 (Fig. 5a) . The total 340 RBD-specific IgG binding and neutralizing antibodies were measured using the plasma of rhesus 341 macaques at two weeks post the boost dose (Fig. 5a) . The IgG endpoint GMTs reached ~2.1×10 4 342 (20 µg), ~1.6×10 4 (100 µg dose) and ~7×10 3 (500 µg dose) for circRNA RBD vaccines, while 343 circRNA Ctrl or PBS-immunized rhesus macaques failed to induce RBD-specific antibodies (Fig.  344   5b) . The SARS-CoV-2 pseudovirus neutralization assay showed the NT50 of ~180 for 20 µg dose, 345 ~520 for 100 µg dose, and ~390 for 500 µg dose (Fig. 5c) . The authentic SARS-CoV-2 346 neutralization assay showed the NT50 of ~80 for 20 µg dose, ~120 for 100 µg dose, and ~50 for 347 500 µg dose (Fig. 5d, e) . These results indicated that the 100 µg dose could elicit maximal level 348 of neutralizing antibodies (Fig. 5c, d) . 349 We then performed the cross-neutralizing assay using the plasma samples from the immunized 350 rhesus macaques. Both the pseudotyped and authentic SARS-CoV-2 neutralization assay showed 351 that the circRNA RBD vaccine-immunized rhesus macaque plasma could effectively inhibit the 352 corresponding native strain, while Alpha, Delta and Beta variants could also be inhibited, but with rhesus macaques were measured using the PBMCs stimulated with the corresponding RBD peptide 357 pools (Supplementary Table 2 ). ELISpot assay showed evident IFN-γ and IL-2 responses, but 358 nearly undetectable IL-4 in circRNA RBD -immunized rhesus macaques (Fig. 5f) Five weeks post the boost dose, the immunized rhesus macaques were challenged with 1×10 6 363 plaque forming units of SARS-CoV-2 native strain via intranasal and intratracheal routes, as 364 described previously 33 . The challenged rhesus macaques were euthanized at 7 dpi, and the lung 365 tissues were underwent viral load and histopathological assays. The RT-qPCR assay using primers 366 targeting SARS-CoV-2 genomic RNA (N protein region) indicated that the rhesus macaques 367 immunized with 100 µg or 500 µg of circRNA RBD vaccine were well protected as the viral genomic 368 RNA copies were reduced nearly 1000-fold compared to the control groups (Fig. 5g) . To detect 369 the actively replicative viral loads, we performed qPCR using primers targeting SARS-CoV-2 sub-370 genomic RNA (E protein region), and found that circRNA RBD -immunized rhesus macaques of all 371 three doses had nearly no detectable viral sub-genomic RNA in the lung tissues (Fig. 5g) . 372

Further histopathological examination demonstrated that circRNA RBD -immunized rhesus 373 macaques of all doses were well protected because only very mild pneumonia was observed, 374 especially in the two high-dose groups (Fig. 5h) . In contrast, severe pneumonia symptoms were 375 observed in the lungs of two control groups, as exemplified by the local pulmonary septal 376 thickening, moderate hemorrhage in the pulmonary septals, a large number of scattered dust cells, 377 and massive inflammation cells infiltration (Fig. 5h) . Pathological score further confirmed that 378 circRNA RBD immunization significantly protected the rhesus macaques from SARS-CoV-2 379 infection ( Fig. 5i) , likely resulting from a synergy between the humoral immune responses and T 380 cell responses elicited by vaccination (Fig. 5j) . 381 immune activation, body weight, body temperature, and blood routine examination. No severe 386 clinical adverse effects were observed following the priming dose or the 2 nd boost. ELISA results 387 showed that circRNA RBD vaccines induced high levels of IL-6 and MCP-1 (Extended Data Fig.  388 12a, b), while the TNF-α, IL-1β, and IFN-α were nearly undetectable (Extended Data Fig. 12c -389 e). Body temperatures of both immunized rhesus macaques and controls were within the normal 390 range, which have been continuously monitored for 3 days after prime and boost (Extended Data 391 Besides vaccine, circRNA could be re-purposed for therapeutics when used to express other 397 proteins or peptides, such as enzymes for rare diseases and antibodies for infectious diseases or 398 cancer. Here, we attempted to test the therapeutic potential of circRNAs by expressing the SARS-399

CoV-2 neutralizing antibodies. It has been reported that SARS-CoV-2 neutralizing nanobodies or 400 hACE2 decoys could inhibit the SARS-CoV-2 infection 73-75 . This prompted us to leverage the 401 circRNA platform to express SARS-CoV-2 neutralizing nanobodies, including nAB1, nAB1-Tri, 402 nAB2, nAB2-Tri, nAB3, and nAB3-Tri 73,74 , together with hACE2 decoys 75 (Extended Data Fig.  403 14a). Pseudovirus neutralization assay showed that supernatants of HEK293T cells transfected 404 with circRNA nAB or circRNA hACE2 decoys could effectively inhibit wild SARS-CoV-2 S-protein 405 based pseudovirus infection (Extended Data Fig. 14b) . Omicron variants of concern 76-79 . The Omicron variant has been reported to escape most of SARS-418

CoV-2 neutralizing antibodies and the sera from vaccinees or convalescent patients 4-8 . Our study 419 established a circular RNA vaccination strategy to elicit effective neutralizing antibodies and T 420 cell immune responses against SARS-CoV-2 and its emerging variants ( Fig. 4 and Fig. 5) . 421

As reported, most effective neutralizing antibodies recognize the RBD region of Spike 422 protein 73,74,80-83 , and targeting RBD may induce fewer non-neutralizing antibodies 29-32,84 . Given 423 that RBD trimers bind hACE2 better than monomeric counterparts 54 and have been shown to 424 enhance humoral immune response 32,54,85 , we chose to express RBD trimers via circRNA as the 425 immunogen. The circRNA-encoded RBD trimmers were functional (Fig. 1d) and indeed induced 426 sustained high-level neutralizing antibodies and specific T cell immune responses against SARS-427

CoV-2 and variants in both mice and rhesus macaques (Fig. 2, Fig. 5 and Extended Data Fig. 10) . and Omicron variants than mRNA RBD-Delta vaccine (Fig. 3h-k and Extended Data Fig. 11) , 436 suggesting that the circRNA vaccine platform might have superiority to cope with the potential 437 we developed could not cross-protect Delta variant (Fig. 4d-f ) suggests that the immunogenicity 445 of Omicron RBD was less effective and largely different from Delta RBD 3 . 446

A recent preprint reported that vaccinees who received two doses of SARS-CoV-2 vaccine 447 exhibited enhanced neutralizing antibodies against Delta variant after infected by Omicron, 448 implying that Omicron vaccine might provide broad-spectrum protection against other variants 86 . 449

Our result argues against such possibility because our Omicron-specific vaccine failed to cross 450 protect Delta variant (Fig. 4d-f) , or boost two-dose of Delta vaccine (Fig. 4h, i) . On the contrary, 451 circRNA RBD-Delta vaccine appeared to produce antigens possessing high immunogenicity and 452 consequently elicit high level of neutralizing antibodies against Delta (Fig. 2) . Our Delta-specific 453 vaccination could cross protect all other variants including Omicron ( Fig. 2 and Fig. 4a-c) , and it 454 could also be used as an effective booster following two-dose original SARS-CoV-2 vaccines (Fig.  455 4k, l). It is hopeful but remains to be further tested whether circRNA RBD-Delta vaccine could be 456 causing severe clinical signs of illness (Fig. 5, Extended Data Fig. 12a-f and Extended Data Fig.  462 13a-e). 463

In this study, we also tested the therapeutic potential of circRNAs that encode SARS-CoV-2-464 specific neutralizing nanobodies 73,74,80-83 or hACE2 decoys 88,89 , which could effectively neutralize 465 the SARS-CoV-2 pseudovirus (Extended Data Fig. 14a-c) . responses were shown as spots per 10 6 PBMCs detected in IFN-γ and IL-2 ELISpot assay. Fig. 1 | PCR analysis to verify the precise circularization of circRNA. a, The  608 agarose gel electrophoresis result of PCR analysis. Linear RNA precusor and circRNA RBD were 609 reverse transcription to cDNA, followed by PCR amplification with specific primers shown in Fig  610   1a 

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Spike proteins. We thank J. Yan (Institute of Microbiology

We thank the Laboratory Animal Centre of Peking University for 984 the feeding of mice. We thank the HPLC Core at the National Center for Protein Sciences at Peking

University (Beijing), particularly H. Li and G. Li for technical help. We thank the flow cytometry

Wang for technical help. This project was supported by funds from National

Municipal Science & Technology Commission (Z181100001318009); the National Science 990 Foundation of China

); the National Science 992 Foundation of China (31870893); the National Major Science & Technology Project for Control 993 and Prevention of Major Infectious Diseases in China

Cell culture. HEK293T, NIH3T3 and Huh-7 cell lines were maintained in our laboratory. The 1013 HEK293T-hACE2 cell line was ordered from Biodragon Inc. (#BDAA0039

These mammalian cell lines were cultured 1015 in Dulbecco′s Modified Eagle Medium (Corning, 10-013-CV) with 10% fetal bovine serum 1016 (FBS) (BI), supplemented with 1% penicillin

1018 1019 circRNA transfection in vitro. For the circRNA transfection in HEK293T or NIH3T3 cells, 3×10 5 1020 cells per well were seeded in 12-well plates. 2 µg of circRNAs were transfected into the HEK293T 1021 or NIH3T3 cells, using Lipofectamine MessengerMax (Invitrogen, LMRNA003) according to the 1022 manufacturer's instructions

Quantitative determination of SARS-CoV-2 Spike RBD expression in vitro. Quantification of 1026 RBD expression in cell culture supernatants was performed with a commercial SARS

Spike RBD Protein ELISA kit (ABclonal, #RK04135) according to the manufacturer's instruction

The animal 1039 experiments were approved by Peking University Laboratory Animal Center (Beijing), and 1040 undertaken in accordance with the National Institute of Health Guide for Care and Use of

Chinese 1043 Academy of Medical Sciences. All the animal experiments with SARS-CoV-2 challenge were 1044 reviewed and approved by the Committee on the Ethics of Animal Experiments of Institute of 1045 Pathogen Biology

For mouse vaccination, groups of 6-8 week-old female BLAB/c mice were intramuscularly 1047 immunized with LNP-circRNA RBD or Placebo (Empty LNP) in 100 µL using a 1 mL sterile syringe, 1048 and 2 or 3 weeks later a second dose was immunized to boost the immune responses. The sera of 1049 immunized mice were collected to detect the SARS-CoV-2-specific IgG endpoint GMTs and 1050 neutralizing antibodies as described below

All the immunized mouse serum samples 1053 were heat-inactivated at 56°C for 30 min before use. The SARS-CoV-2-specific IgG antibody 1054 endpoint GMT was measured by ELISA. Briefly, serial 3-fold dilutions (in 1% BSA) of heat-1055 inactivated sera, starting at 1:100, were added to the 96-well plates (100 µL/well; Costar) coated 1056 with recombinant SARS-CoV-2 Spike or RBD antigens

BSA for 60 min at 37°C. Then, after three washes with wash buffer, the Horseradish peroxidase

Then the plates were washed for 3 times with wash 1060 buffer and added with TMB substrates (100 µL/well) followed by incubation for 15-20 min. And SARS-CoV-2 Surrogate Virus Neutralization Assay. The neutralizing activity of mouse serum dilutions of heat-inactivated sera, starting at 1:10, were incubated with HRP-conjugated RBD 1070 solutions at 37°C for half an hour, and then the mixtures were added into 96-well plates pre-coated 1071 with human ACE2 (hACE2) proteins and incubated for 15 min at 37°C. After washing the TMB 1072 substrates and

The inhibition rates of serum samples were calculated according to the 1074 following formula. The 50% neutralization geometric mean titer (NT50) was determined using 1075 four-parameter nonlinear regression in Prism

Inhibition rate = (1-OD value of sample/OD value of negative control) × 100%

Pseudovirus-based neutralization assay. The production of lentivirus-based SARS-CoV-2 1079 pseudovirus and neutralization assay were performed as described previously 94 . Briefly, the 1080 SARS-CoV-2 pseudovirus were produced by co-transfecting plasmids psPAX2 (6 µg)

µg) into HEK293T cells using X tremeGENE HP DNA Transfection

Roche) according to the manufacturer′s instructions. 48 hr post transfection, the 1083 supernatants containing pseudovirus particles were harvested and filtered through a 0.22-µm 1084 sterilized membrane for the neutralization assay as described below

For the determination of NT50 of immunized mouse serum, the HEK293T-hACE2 cells were 1086 seeded in 96-well plates (50,000 cells/well) and incubated for approximate 24 hr until reaching 1087 over 90% confluent, preparing for pseudovirus infection. The mouse serum was 3-fold diluted, 1088 starting at 1:40, and incubated with the SARS-CoV-2 pseudovirus

Then the 1090 supernatant of HEK293T-hACE2 cells were removed and the mixer of serum and pseudovirus 1091 were added to each well. 36-48 hr later, the luciferase activity, which reflecting the degree of SARS-CoV-2 pseudovirus transfection, was measured using the Nano-Glo Luciferase Assay

The NT50 was defined as the fold-dilution, which emitted an exceeding 50% 1094 inhibition of pseudovirus infection in comparison with the control group

Relative luciferase units 1104 (RLU) were normalized to corresponding DMEM control group, and the NT50 were determined 1105 by a four

For the neutralization assay of circRNA nAB or circRNA ACE2 decoys , the HEK293T-hACE2 cells 1107 were seeded in 96-well plates (50,000 cells/well) and incubated for approximate 24 hr until 1108 reaching over 90% confluent. The pseudovirus were pre-incubated with the supernatant of the 1109 circRNA nAB or circRNA ACE2 decoys transfected cells at 37°C for 60 min, and then added to cells in 1110 the 96-well plates

hr after transduction. Luciferase activity was measured using the Nano-Glo Luciferase Assay

The relative luminescence units were normalized to cells infected with 1113 supernatant of cell transfected with the circRNA EGFP

to generate a mixture containing ~2,000 PFU/well of viruses (MOI = 1119 0.1), followed by an incubation at 37°C for 1 hr. Then the virus/sera mixtures were added to 24-1120 well plates of A549-ACE2 cells, supplemented with 100 µL of DMEM containing 10% FBS in 1121 each well. The supernatant and cell pellet precipitation were then collected, and the viral load was mice were recorded daily

All animal procedures were approved by the Institutional Animal Care 1163 and Use Committee of Institute of Medical Biology, Chinese Academy of Medical Science. Rhesus 1164 macaques were monitored at least twice daily throughout the experiment. Commercial monkey 1165 chow, treats and fruit were provided daily by trained personnel

For the vaccination of rhesus macaque, 1168 groups of 2~4-year-old rhesus macaques were immunized with LNP-circRNA RBD (20 µg

LNP-circRNA Ctrl (circRNA without the RBD-encoding sequence

300 µL (>300 µL in 500 µg dose group) via intramuscular injection 1171 in the quadriceps muscle (prime: left, boost: right) twice at a three-week interval. The plasma of 1172 immunized rhesus macaques were collected at 0, 1 and 14 days post the prime

Amplified

SARS-CoV-2 were confirmed via RT-PCR, sequencing and transmission electronic microscopy, 1179 and titrated via plaque assay

The T cell immune responses in rhesus macaques were detected using the PBMCs 1182 with commercially available NHP IFN-γ and IL-2 ELISpot assay kits (Mabtech), and NHP

The cryopreserved rhesus macaques PBMCs were thawed and 1184 cultured with pre-warmed AIM-V media. For IFN-γ, IL-2 and IL-4 ELISpot assay, 1.0×10 5 SARS-CoV-2 challenge in rhesus macaques

ml) routes. The plasma of rhesus macaques was collected and vital 1197 clinical signs were recorded on 0, 1, 3, 5 and 7 days post virus challenge

After the dissection, a general 1202 examination of the main organs was performed. The lung tissues were harvested, fixed in 10% 1203 neutral formalin buffer and embedded in paraffin. 2 µm tissue sections were prepared. Slides were 1204 stained with Hematoxylin and Eosin (H&E). The slide images were collected by using Pannoramic 1205 DESK and analyzed with Caseviewer C.V 2.3 and Image-Pro Plus 6.0. Histopathological analysis 1206 of tissue slides was

The plasma of rhesus monkeys were isolated 24 hr post prime or boost and 1209 diluted in 5-fold or 10-fold. All plasma samples were detected using following ELISA kits

TNF-α (Abcam, ab252354), IL-1211 1β (Cloud-cline Corp, SEA563Si96T) and IFN-α (Chenglin, AD0081Mk) according to the 1212 manufacturer

An unpaired two-sided Student's t-test was performed for comparison as indicated in References

Cross-neutralization of SARS-CoV-2 by a human monoclonal SARS-CoV antibody