key: cord-0958548-kgzc8zr5
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; Liu, Shuo; Huang, Weijin; 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: 2022-04-01
journal: Cell
DOI: 10.1016/j.cell.2022.03.044
sha: 0aee2f3e5f3b0f1e0ae9f2bd06c202deacf177b7
doc_id: 958548
cord_uid: kgzc8zr5

As the emerging variants of SARS-CoV-2 continue to drive the worldwide pandemic, there is a constant demand for vaccines that offer more effective and broad-spectrum protection. Here, we report a circular RNA (circRNA) vaccine that elicited potent neutralizing antibodies and T cell responses by expressing the trimeric RBD of the spike protein, providing robust protection against SARS-CoV-2 in both mice and rhesus macaques. Notably, the circRNA vaccine enabled higher and more durable antigen production than the 1mΨ-modified mRNA vaccine, and elicited a higher proportion of neutralizing antibodies and distinct Th1-skewed immune responses. Importantly, we found that the circRNARBD-Omicron vaccine induced effective neutralizing antibodies against the Omicron but not the Delta variant. In contrast, the circRNARBD-Delta vaccine protected against both Delta and Omicron or functioned as a booster after two doses of either native- or Delta-specific vaccination, making it a favorable choice against the current variants of concern (VOCs) of SARS-CoV-2.

with immune escape ability have appeared, the most serious of which is Omicron. By the end of 55 January, 2022, Omicron accounted for ~85% of COVID-19 cases (GISAID). Omicron carries over 56

CircRNA RBD produced functional SARS-CoV-2 RBD antigens 124

We employed the group I intron autocatalysis strategy (Wesselhoeft et al., 2018) to produce 125 circular RNAs encoding SARS-CoV-2 RBD antigens, termed circRNA RBD ( Figure 1A ). In this 126 construct, the IRES element was placed before the RBD-coding sequence to initiate its translation. 127

To enhance the immunogenicity of RBD antigens, the signal peptide sequence of human tissue 128 plasminogen activator (tPA) was fused to the N-terminus of RBD to ensure the secretion of 129 antigens (Kou et was fused to its C-terminus. This IRES-SP-RBD-Foldon sequence was then cloned into the vector 134 to construct the IVT template for producing circRNA RBD ( Figure 1A ; Table S1 ). 135

To produce high-purity circRNA RBD , we first optimized the IVT reaction to generate 136 circRNA RBD (Figure S1A ) without extra step of GTP catalysis (Wesselhoeft et al., 2018) . High-137 performance liquid chromatography (HPLC) analysis determined that the latter half of main peak 138 contained high-purity circRNA (Figures S1B and S1C). Then we successfully manufactured 139 circRNA RBD in large quantities (Figures S1D and S1E). We found that the majority of the purified 140 circRNA RBD fractions were resistant to exonuclease-RNase R, while the nicked RNA RBD were 141 almost completely degraded, indicating that purified circRNA RBD were mostly in circular format 142 ( Figure S1F ). The purity of circRNA RBD was over 90% calculated via the denaturing gel 143 electrophoresis and the following semi-quantitative analysis (Figures S1G-S1I). The 144 circularization of circRNA RBD was further verified by reverse transcription-PCR, Sanger 145 sequencing and RNase H-mediated specific cleavage (Figures S1J-S1M). 146

To test the secretory expression of RBD produced by circRNA RBD , the purified circRNA RBD 147 was transfected into HEK293T cells or NIH3T3 cells. Abundant RBD antigens in the supernatant 148 of both human and murine cells were detected by Western blot, indicating the high compatibility 149 J o u r n a l P r e -p r o o f of circRNAs ( Figure 1B) . With the help of Foldon, the circRNA RBD encoded stable homogeneous 150 RBD trimers in the supernatant, which were dissociated into monomers under reducing conditions 151 ( Figure 1C ). The concentration of RBD antigens produced by circRNA RBD reached ~1,400 ng/ml, 152 600-fold higher than those produced by its linear precursor RNA ( Figure 1D) . 153

In addition to the group I intron-based strategy, we also developed a T4 RNA ligase-based 154 method to produce circular RNAs. This method adopted the complementary pairing sequence of 155 split IRES as the splint instead of a DNA splint to generate an intramolecular RNA nick structure 156 serving as the catalytic substrate of T4 RNA ligase ( Figure S2A ; Table S2 ). Sanger sequencing 157 confirmed the precise circularization of circRNA RBD by this approach (Figure S2B) . Similarly, 158 abundant RBD antigens were detected in the supernatant at a concentration of ~1,000 ng/ml, which 159 was ~200-fold higher than those produced by its linear precursor RNA ( Figures S2C and S2D ). 160

To verify whether the secreted SARS-CoV-2 RBD antigens produced by circRNA RBD To explore whether circRNA could be leveraged to create a vaccine, we attempted to assess the 169 immunogenicity of circRNA RBD encapsulated with LNP in BALB/c mice ( Figure 1F ). The 170 circRNA RBD encapsulation efficiency was greater than 93%, with an average diameter of 100 nm 171 ( Figure 1G ). Mice were immunized through intramuscular (i.m.) injection with 10 μg or 50 μg of 172 LNP-circRNA RBD vaccines twice at a two-week interval ( Figure 1H ). The circRNA RBD elicited a 173 high level of RBD-specific IgG endpoint geometric mean titers (GMTs), reaching ~1.9×10 4 for 174 the 10 μg dose and ~5.7×10 5 for the 50 μg dose ( Figure 1I ). 175

Sera from circRNA RBD -vaccinated mice effectively neutralized SARS-CoV-2 pseudovirus 176 with a 50% neutralization titer (NT50) of ~4.5×10 3 ( Figure 1J ) and authentic SARS-CoV-2 virus 177 with an NT50 of ~7.0×10 4 ( Figure 1K ). 178 that circRNAs were more stable than mRNAs, modified or unmodified ( Figure 3B ). Importantly, 238 LNP encapsulation further enhanced the advantage of circRNA in protein production and 239 durability from both 1mΨ-mRNA and unmodified-mRNA ( Figure 3C ). Interestingly, LNP 240 encapsulation appeared to improve the antigen-encoding efficiency of unmodified mRNA to a 241 level comparable to that of 1mΨ-mRNA ( Figure 3C ). 242

We found that even after two weeks of storage at room temperature (~25 °C), the circRNA could 243 express RBD antigens without detectable loss ( Figure 3D ), highlighting its remarkable thermal 244 stability. To further evaluate the thermostability of the vaccines, the LNP-encapsulated circRNA, 245 1mΨ-mRNA and unmodified mRNA were stored at 4 °C, ~25 °C, or 37 °C for up to 28 days prior 246 to transfection. At all temperatures tested, circRNA expressed higher levels of antigens than those 247 of the other two mRNA groups ( Figures S3B-S3D ). At 4 °C, little reduction in RBD antigens 248 produced by LNP-circRNA could be detected from 1-28 days ( Figure S3B ). The stability of LNP-249 circRNA, 1mΨ-mRNA or unmodified-mRNA was clearly reduced with increasing storage 250 temperature, especially at 37 °C ( Figures S3C and S3D) . 251 Importantly, we found that the innate immune responses elicited by LNP-encapsulated RNAs 252 were comparable to those by LNP-encapsulated 1mΨ-mRNA RBD , and significantly lower than 253 those by the transfected RNAs ( Figure 3E Given that circRNA vaccines possess higher stability and antigen-encoding efficiency, we 258 wondered whether they exhibited distinctive immunogenicity compared to mRNA vaccines. We 259 compared the balance of Th1/Th2 immune responses between circRNA RBD-Delta and mRNA RBD-Delta 260 vaccines because Th2-biased immune responses might induce vaccine-associated enhanced 261 respiratory disease (VAERD) (Corbett et al., 2020a; Graham, 2020; Sahin et al., 2020) . ELISA 262 showed that the total IgG elicited by circRNA RBD-Delta was comparable to that by 1mΨ-mRNA RBD-263 Delta ( Figure 3F) , however, the ratios of IgG2a/IgG1, IgG2c/IgG1 or (IgG2a + IgG2c)/IgG1 from 264 circRNA RBD-Delta were consistently higher than those from 1mΨ-mRNA RBD Antibody-dependent enhancement (ADE) of infection by virus-specific antibodies is another 268 potential concern for vaccines that has been reported for infections by some viruses, including 269 Zika, Dengue, and coronaviruses (Dowd and Pierson, 2011 (Table S4) , and cytokine-producing T cells were quantified by 285 intracellular cytokine staining among effector memory T cells (Tem, CD44 + CD62L -) ( Figure S4 ). 286

After stimulation with peptides, CD8 + T cells producing IFN-γ, TNF-α, and IL-2 were detected in 287 mice immunized with the circRNA RBD-Delta vaccine or 1mΨ-mRNA RBD To cope with the current Omicron emergency, we tested the neutralizing capability elicited by all 297 three circRNA vaccines against the Omicron variant. The neutralizing activity against Omicron 298 elicited by either one of the three circRNA vaccines dropped 74-fold (native), 15-fold (Beta) and 299 44-fold (Delta) in comparison with the neutralizing activity against their corresponding variants 300 ( Figure 5A ). Among all three, the circRNA RBD-Delta vaccine maintained sufficient neutralizing 301 activity against Omicron ( Figure 5A) , with an NT50 of ~4.7×10 3 , while the NT50 of the 302 circRNA RBD-Beta against Omicron dropped below 5×10 2 ( Figure 5A ). Compared to the mRNA RBD-303 Delta vaccine, the circRNA RBD-Delta vaccine elicited comparable neutralizing activity against both 304

Delta and Omicron variants for mouse sera collected 2 weeks after the boost (short-term) and 7 305 weeks after the boost (long-term) ( Figures 5A-5C ). Similar to the above observations ( Figure 3M with NT50 values of ~2.5×10 3 for the 5 μg dose and ~8.6×10 3 for the 10 μg dose ( Figure 5E ). 319

However, neutralizing activity could hardly be detected against the native strain or Delta variant 320 ( Figures 5E and 5F ). or circRNA RBD-Omicron vaccine at 7 weeks after the 2 nd dose, followed by the assessment of 327 neutralizing activity at 1 week after boost ( Figure 5G ). Only circRNA RBD-Delta effectively boosted 328 the neutralizing antibodies against both Delta ( Figure 5H ) and Omicron ( Figure 5I ). In contrast, 329 the 3 rd boost with the circRNA RBD-Beta or circRNA RBD-Omicron vaccine failed to elevate the 330 neutralizing capability against Delta or Omicron ( Figures 5H and 5I ). 331

We then tested the 3 rd booster with circRNA RBD or circRNA RBD-Delta vaccine in mice previously 332 immunized with 2-dose circRNA RBD vaccines ( Figure 5J ). Both vaccines effectively boosted 333 neutralizing antibodies against both Delta ( Figure 5K ) and Omicron ( Figure 5L ). CircRNA RBD-Delta 334 appeared to be a much better booster than circRNA RBD against both Delta and Omicron variants, 335 which elevated the geometric mean NT50 from ~4×10 2 to ~3. To further assess the immunogenicity of circRNA vaccine in nonhuman primates (NHPs), groups 343 of 2-to 4-year-old rhesus macaques were immunized i.m. with 20 μg, 100 μg or 500 μg of 344 circRNA RBD vaccines, 100 μg of circRNA Ctrl , or PBS control on days 0 and 21 ( Figure 6A ). The 345 specific antibodies were measured using the rhesus macaque plasma collected at 2 weeks after the 346 boost ( Figure 6A ). The IgG endpoint GMTs reached ~2.1×10 4 (20 μg), ~1.6×10 4 (100 μg dose) 347 and ~7×10 3 (500 μg dose) for circRNA RBD vaccines, while circRNA Ctrl -or PBS-immunized rhesus 348 macaques failed to induce RBD-specific antibodies ( Figure 6B ). The pseudovirus neutralization 349 assay showed NT50 values of ~180 for the 20 μg dose, ~520 for the 100 μg dose, and ~390 for the 350 500 μg dose ( Figure 6C ). The authentic SARS-CoV-2 neutralization assay showed NT50 values 351 of ~80 for the 20 μg dose, ~120 for the 100 μg dose, and ~50 for the 500 μg dose ( Figures 6D and  352 

We then performed a cross-neutralizing assay. Both the pseudotyped and authentic SARS-354

CoV-2 neutralization assays showed that the circRNA RBD vaccine-immunized rhesus macaque 355 J o u r n a l P r e -p r o o f plasma could effectively inhibit the corresponding native strain, while the Alpha, Delta and Beta 356 variants could also be inhibited, but with reduced activity, especially against the Beta variant 357 ( Figures 6D and 6E) . 358

Peripheral blood mononuclear cells (PBMCs) were collected on the day before challenge with 359 SARS-CoV-2. The RBD-specific T cell responses in rhesus macaques were measured using 360

PBMCs stimulated with the RBD peptide pools (Table S5 ). The ELISpot assay showed evident 361 IFN-γ and IL-2 responses, but nearly undetectable IL-4 in circRNA RBD -immunized rhesus 362 macaques ( Figure 6F ), indicating a Th1-biased T cell immune response. 363 364

Five weeks after the boost dose, the immunized rhesus macaques were challenged with 1×10 6 PFU 366 of the SARS-CoV-2 native strain as described previously (Vogel et al., 2021) . The challenged 367 rhesus macaques were euthanized at 7 days post-infection (dpi), and the lung tissues underwent 368 viral load and histopathological assays. The RT-qPCR assay using primers targeting SARS-CoV-369 2 genomic RNA (N gene) indicated that the rhesus macaques immunized with 100 μg or 500 μg 370 of circRNA RBD vaccine were well protected as the viral genomic RNAs were reduced nearly 1000-371 fold compared to the control groups ( Figure 6G ). To detect the actively replicative viral loads, we 372 performed qPCR using primers targeting SARS-CoV-2 subgenomic RNA (E gene) and found that 373 rhesus macaques immunized with circRNA RBD at all three doses had nearly no detectable viral 374 subgenomic RNA in the lung tissues ( Figure 6G with circRNA encoding nAB1, nAB1-Tri, nAB2, nAB2-Tri, nAB3 and nAB3-Tri or ACE2 decoys. 689

The nAB1-Tri, nAB2-Tri and nAB3-Tri represent the trimers of nAB1, nAB2 and nAB3, 690 respectively. The luciferase value was normalized to that of the circRNA EGFP control. were reverse transcribed to cDNA, followed by PCR amplification with the specific primers shown 720 in Figure 1A . 

Further information and requests for resources and reagents should be directed to and will be 842 

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HEK293T, NIH3T3 and Huh-7 cell lines were maintained in our laboratory. The HEK293T-875 hACE2 cell line was ordered from Biodragon Inc. (#BDAA0039, Beijing, China). The A549-876 hACE2 cell line was generated in our laboratory. These mammalian cell lines were cultured in 877 Dulbecco′s Modified Eagle Medium (Corning, 10-013-CV) with 10% fetal bovine serum (FBS) 878 (BI), supplemented with 1% penicillin-streptomycin in 5% CO2 incubator at 37 °C. The Huh-7 879 cells were cultured with the methods previously described methods . 880The production of lentivirus-based SARS-CoV-2 pseudovirus and neutralization assays were 881 performed as described previously (Pinto et al., 2020) . Briefly, the SARS-CoV-2 pseudovirus was 882 produced by cotransfecting plasmids psPAX2 (6 μg), pSpike (6 μg), and pLenti-Luc-GFP (6 μg) 883 into HEK293T cells using X tremeGENE HP DNA Transfection Reagent (Roche) according to 884 the manufacturer's instructions. Forty-eight hours after transfection, the supernatants containing 885 pseudovirus particles were harvested and filtered through a 0.22-μm sterilized membrane for the 886 neutralization assay as described below. The VSV-based pseudovirus of SARS-CoV-2 and its 887 variants were described previously Du et al., 2020; Cao et al., 2021) . Authentic 888 J o u r n a l P r e -p r o o f viruses were amplified from Vero-E6 cells and concentrated by an ultrafilter system via a 300 kD 889 module (Millipore). Amplified SARS-CoV-2 was confirmed via RT-PCR, sequencing and 890 transmission electronic microscopy, and titrated via plaque assay (10 6 PFU/ml). 891 892

The 5' homology arm sequence, 3' group I intron sequence, linker-1 sequence, IRES sequence, 895 linker-2 sequence, 5' group I intron sequence and 3' homology arm sequence were PCR amplified 896 and cloned into a plasmid backbone via the Gibson assembly strategy, generating the empty 897 pcircRNA-EV backbone. Then, the SARS-CoV-2 RBD antigen, EGFP, nanobody or hACE2-898 decoy-coding sequence was PCR amplified and cloned into the pcircRNA-EV backbone, and the 899 corresponding pcircRNA plasmids were constructed for the following IVT reaction. 900 901 Production and purification of circRNA 902The production of circRNAs was performed according to previous reports (Wesselhoeft et al., 903 2018 Table S1 . 915We used split IRES strategy to produce circular RNAs by T4 RNA ligase 2 (NEB, #M0239). 916To test the potential split sites in CVB3 IRES sequence, we analyzed the second structure of IRES. 917After multiple tests and screens, we were able to determine the split site of CVB3 IRES at the 385 th 918 nucleotide to allow T4 RNA ligase method for effective circularization. Then the circular RNA 919 J o u r n a l P r e -p r o o f precursors were produced via in vitro transcription (NEB, E2040S) with added Guanosine 920 monophosphates, and the RNA precursors were ligated by T4 RNA ligase 2 for 8 h at 25 °C. 921Finally, the ligated circular products were treated with RNase R to remove the linear RNA 922 precursors. The sequences of circRNAs produced via T4 RNA ligases were provided in Table S2 . 923To further enrich the circRNAs, the purified RNase R-treated RNA was resolved with high-924 performance liquid chromatography (Agilent HPLC1260) using a 4.6 × 300 mm size-exclusion 925 HEK293T-hACE2 cells was removed, and a mixture of serum and pseudovirus was added to each 1032 well. Thirty-six to 48 hr later, the luciferase activity, which reflects the degree of SARS-CoV-2 1033 pseudovirus transfection, was measured using the Nano-Glo Luciferase Assay System (Promega). 1034The NT50 was defined as the fold dilution that achieved more than 50% inhibition of pseudovirus 1035 infection compared with the control group. 1036The sera were serially diluted using complete DMEM as the culture medium in 96-well white 1037 plates for a total of six gradients, and then the virus solution with ~1.3×10 4 TCID50 was added. 1038Complete DMEM was used as the control group. After one hour of incubation in a 5% CO2 1039 incubator at 37 °C, Huh7 cells (100 μl/well) were added to the 96-well white plates, which were 1040 adjusted to a concentration of 2×10 5 cells/ml. After 24 h of incubation in a 5% CO2 incubator at 1041 37 °C, the culture supernatant was aspirated gently to leave 100 μl in each well, and then 100 μl 1042 The splenocytes from each immunized mouse were cultured in R10 medium (RPMI 1640 1086 supplemented with 1% Pen-Strep antibiotic, 10% HI-FBS) and stimulated with RBD peptide pools 1087 (Table S4) The T cell immune responses in rhesus macaques were detected using PBMCs with commercially 1113 available Monkey IFN-γ and IL-2 ELISpot assay kits (Mabtech) and an Monkey IL-4 ELISpot 1114 assay kit (U-CyTech). The cryopreserved rhesus macaque PBMCs were thawed and cultured with 1115 prewarmed AIM-V medium. For the IFN-γ, IL-2 and IL-4 ELISpot assays, 1.0×10 5 PBMCs were 1116 stimulated with a final concentration of 1 μg/ml for each RBD peptide (Table S5 ). The test for 1117 each rhesus macaque was performed in two or three technical repetitions. Dimethyl sulfoxide 1118 (DMSO) served as an unstimulating control, and phytohemagglutinin (PHA-P, Sigma) and CELL 1119 STIMULATION COCKTAIL (Thermo Fisher) were used as positive controls. After 24 h of 1120 stimulation with RBD peptide pools, the streptavidin-HRP substrate (for IFN-γ and IL-2) or AEC 1121 substrate (IL-4) was added to the plate. The spots were counted by Beijing Dakewei Biotechnology 1122Co., Ltd. The results are background (DMSO treated group) subtracted and normalized to SFC/10 6 1123 PBMCs. 1124