key: cord-0268571-2t1zzigc
authors: Kimura, Izumi; Yamasoba, Daichi; Tamura, Tomokazu; Nao, Naganori; Oda, Yoshitaka; Mitoma, Shuya; Ito, Jumpei; Nasser, Hesham; Zahradnik, Jiri; Uriu, Keiya; Fujita, Shigeru; Kosugi, Yusuke; Wang, Lei; Tsuda, Masumi; Kishimoto, Mai; Ito, Hayato; Suzuki, Rigel; Shimizu, Ryo; Begum, MST Monira; Yoshimatsu, Kumiko; Sasaki, Jiei; Sasaki-Tabata, Kaori; Yamamoto, Yuki; Nagamoto, Tetsuharu; Kanamune, Jun; Kobiyama, Kouji; Asakura, Hiroyuki; Nagashima, Mami; Sadamasu, Kenji; Yoshimura, Kazuhisa; Kuramochi, Jin; Schreiber, Gideon; Ishii, Ken J; Hashiguchi, Takao; Ikeda, Terumasa; Saito, Akatsuki; Fukuhara, Takasuke; Tanaka, Shinya; Matsuno, Keita; Sato, Kei
title: Virological characteristics of the novel SARS-CoV-2 Omicron variants including BA.2.12.1, BA.4 and BA.5
date: 2022-05-26
journal: bioRxiv
DOI: 10.1101/2022.05.26.493539
sha: a8d5a2ced0136bff1701a41772f7f5811167f8dd
doc_id: 268571
cord_uid: 2t1zzigc

After the global spread of SARS-CoV-2 Omicron BA.2 lineage, some BA.2-related variants that acquire mutations in the L452 residue of spike protein, such as BA.2.9.1 and BA.2.13 (L452M), BA.2.12.1 (L452Q), and BA.2.11, BA.4 and BA.5 (L452R), emerged in multiple countries. Our statistical analysis showed that the effective reproduction numbers of these L452R/M/Q-bearing BA.2-related Omicron variants are greater than that of the original BA.2. Neutralization experiments revealed that the immunity induced by BA.1 and BA.2 infections is less effective against BA.4/5. Cell culture experiments showed that BA.2.12.1 and BA.4/5 replicate more efficiently in human alveolar epithelial cells than BA.2, and particularly, BA.4/5 is more fusogenic than BA.2. Furthermore, infection experiments using hamsters indicated that BA.4/5 is more pathogenic than BA.2. Altogether, our multiscale investigations suggest that the risk of L452R/M/Q-bearing BA.2-related Omicron variants, particularly BA.4 and BA.5, to global health is potentially greater than that of original BA.2. Highlights Spike L452R/Q/M mutations increase the effective reproduction number of BA.2 BA.4/5 is resistant to the immunity induced by BA.1 and BA.2 infections BA.2.12.1 and BA.4/5 more efficiently spread in human lung cells than BA.2 BA.4/5 is more pathogenic than BA.2 in hamsters

were also resistant to these antisera (Figure 2A) . In the case of the 16 sera 203 infected with BA.1 from who were 2-dose or 3-dose vaccinated convalescents 204 (i.e., BA.1 breakthrough infection) (Table S5) , the sensitivity of BA.2.9.1 and 205 BA.2.11 to these antisera was comparable to that of BA.2 ( Figure 2B) , and 206 BA.2.12.1 was significantly (1.3-fold) more sensitive than BA.2 ( Figure 2B ; P = 207 0.021 by the Wilcoxon signed-rank test). In contrast, BA.4/5 was significantly 208

(2.3-fold) more resistant to BA.1 breakthrough infection sera than BA.2 ( Figure  209 2B; P < 0.0001 by the Wilcoxon signed-rank test), which is consistent with a 210 recent study (Khan et al., 2022) . The assay using the BA.2 S derivatives bearing 211 individual mutations of BA.4/5 and the BA.1 breakthrough infection sera showed 212 that the F486V mutation confers resistance, while the insertion of HV69-70del 213 and R493Q mutations made the pseudovirus more sensitive (Figure 2B ). 214 Interestingly, the HV69-70del mutation is present in BA. Nevertheless, our data suggest that BA.4/5 is relatively more resistant to the 220 immunity induced by BA.1 breakthrough infection than BA.2, and this resistance 221 is attributed to the BA.4/5-specific F486V mutation. 222 We next tested the 16 sera infected with BA.2 from 8 convalescents 223 who were not vaccinated and 1, 4 and 3 convalescents who were 1-dose, 224 2-dose, and 3-dose vaccinated convalescents, respectively ( Table S5) . As 225 shown in Figure 2C , BA.2 convalescent sera did not exhibit antiviral effects 226 against all variants tested including the D614G-bearing ancestral B.1.1. 227

Although the BA.2 convalescent sera after breakthrough infection exhibited a 228 stronger antiviral effect compared to the BA.2 convalescent sera without 229 vaccination, BA.2 was 3.0-fold more resistant than B.1.1 (Figure 2D shown in Figure 3A , all BA. and notably, the infectivity of BA.4/5 pseudovirus was 18.3-fold higher than that 250 of BA.2 pseudovirus ( Figure 3A) . The BA.2 derivatives bearing L452Q, 251

HV69-70del and F486V mutations exhibited increased infectivity ( Figure 3A) . 252 These results suggest that multiple mutations in the BA. S-GFP) were significantly larger than that of rBA. subcutaneous oxygen saturation (SpO 2 ) ( Figure 5A) . Notably, the body weights 343 of rBA.2.12.1-infected and rBA.4/5-infected hamsters were significantly lower 344 than that of rBA.2-infected hamsters ( Figure 5A) . Additionally, the Rpef value of 345 rBA.4/5-infected hamsters was significantly lower than that of rBA.2-infected 346 hamsters ( Figure 5A) . These data suggest that the L452R/Q-bearing 347 BA.2-related Omicron variants, particularly BA.4/5, exhibit higher pathogenicity 348 than BA.2. 349

To analyze viral spread in the respiratory organs of infected hamsters, 350 the viral RNA load and nucleocapsid (N) expression were assessed by 351

RT-qPCR analysis of viral RNA and immunohistochemistry (IHC), respectively. 352

As shown in Figure 5B , the viral RNA loads in the lung hilum of rBA.2.12.1-and 353

rBA.4/5-infected hamsters were significantly higher than that of rBA.2-infected 354 hamsters. Intriguingly, the viral RNA loads in the oral swab ( Figure 5B , top) and 355 lung periphery (Figure 5B, Figure 5B, bottom) . The higher level of viral load in the lung 361 periphery of rBA.4/5-infected hamsters than rBA.2-infected hamsters was also 362 supported by the level of infectious viruses in these regions ( Figure 5C ). These 363 results suggest that rBA.4/5 more efficiently spread in the lung of infected 364 hamsters than rBA.2. 365

To address the possibility that rBA.4/5 spreads more efficiently than the 366 BA. To investigate the pathogenicity of BA.2.12.1 and BA.4/5, the right lungs of 382 infected hamsters were collected at 1, 3, and 5 d.p.i. and subjected to 383 histopathological analysis ( Figure 5F ) and hematoxylin and eosin (H&E) 384 staining ( Figure 5G ) ( Jumpei Ito and Kei Sato wrote the original manuscript. 539

All authors reviewed and proofread the manuscript. 540

The Genotype to Phenotype Japan (G2P-Japan) Consortium contributed to the 541 project administration. 542 543

Conflict of interest 544

The authors declare that no competing interests exist. 545 546

Acknowledgments 547

We would like to thank all members belonging to The Genotype to Phenotype 548 Japan (G2P-Japan) Consortium. We thank Dr. Kenzo Tokunaga (National 549

Institute for Infectious Diseases, Japan) and Dr. Jin Gohda (The University of 550

Tokyo, Japan) and Dr. Hisashi Arase (Osaka University) for providing reagents. 551

We also thank National Institute for Infectious Diseases, Japan, for providing a 552 clinical isolate of Omicron BA.2 (GISAID ID: EPI_ISL_9595859). We gratefully 553 acknowledge the laboratories responsible for obtaining the specimens and the 554 laboratories where genetic sequence data were generated and shared via the 555 See also Figure S1 and Tables S1-S4. Assays with each serum sample were performed in triplicate to determine the 873 50% neutralization titer (NT50). Each dot represents one NT50 value, and the 874 geometric mean and 95% CI are shown. The numbers indicate the fold changes 875 of resistance versus each antigenic variant. The horizontal dashed line indicates 876 the detection limit (120-fold). Statistically significant differences between BA.2 877 and other variants (*, P < 0.05) were determined by two-sided Wilcoxon 878 signed-rank tests. Information on the vaccinated/convalescent donors is 879 summarized in Table S5 . 880 See also Table S5 . shown in Figure S3C . 959 Data are presented as the average (A and B, See also Figure S3 . 971 and deletions) present > 1% sequences were detected and referred to as the 1217 "common amino acid mutations". According to the profile of the common amino 1218 acid mutations, a common amino acid haplotype, a set of common amino acid 1219 mutations present in each sequence, was determined for all BA.2 sequences. 1220

Finally, up to 20 sequences were randomly sampled from each unique common 1221 amino acid haplotype. As outgroup sequences, the oldest isolate of B.1.1 1222 obtained in the UK (EPI_ISL_466615) and the oldest five BA.1 and BA.3 1223 sequences sampled from South Africa after December 1, 2022, were used. The 1224 ML tree was constructed by the procedure described above. In the final set, 1225 8,029 BA.2 sequences were included. Outgroup sequences are not displayed in 1226 Figure 1B . identified the branches connecting the parental-state (L) nodes and the 1238 mutated-sate (R, Q, or M) nodes (red branches in Figure S1C ). In these 1239 branches, it is expected that the mutation acquisitions in the S L452 residue 1240 occurred. Finally, we counted the descendant sequences of respective branches 1241

where the mutations in the S L452 were likely acquired. If the number of 1242 descendants is ≥ 10, we defined these descendant sequences as a common 1243 ancestry group of the BA.2 variants, which bears a mutation at position 452 in S. 1244

Information of the common ancestry group is summarized in Table S2 .

Modeling the epidemic dynamics of SARS-CoV-2 lineages 1247

To quantify the spread rate of each SARS- counted, and the count matrix was constructed as an input for the statistical 1263 model below. 1264 We constructed a Bayesian statistical model to represent relative 1265 lineage growth dynamics with multinomial logistic regression, as described in our 1266

previous study 

Cat# ant-bl-1) and 1% PS. 1142 Vero cells [an African green monkey (Chlorocebus sabaeus) kidney cell line

Sigma-Aldrich, Cat# M4655-500ML) containing 10% FBS and 1145 1% PS. VeroE6/TMPRSS2 cells (VeroE6 cells stably expressing human 1146 TMPRSS2; JCRB Cell Bank, JCRB1819) (Matsuyama et al., 2020) were 1147 maintained in DMEM

G418 (1 mg/ml; Nacalai Tesque, Cat# G8168-10ML) and 1% PS

HTB-55) were maintained in 1150 EMEM

Human airway 1158 and alveolar epithelial cells derived from human induced pluripotent stem cells 1159 (iPSCs) were manufactured according to established protocols as described 1160 below (see "Preparation of human airway and alveolar epithelial cells from 1161 human iPSCs" section) and provided by HiLung Inc. 1162 1163 METHOD DETAILS 1164 Viral genome sequencing 1165 Viral genome sequencing was performed as previously described

Paired-end 76-bp sequencing was performed using a 1172 MiSeq system (Illumina) with MiSeq reagent kit v3 (Illumina, Cat# MS-102-3001)

2018) and 1174 subsequently mapped to the viral genome sequences of a lineage A isolate 1175

At 24 hours posttransfection, target cells were 1457 detached and cocultured with effector cells in a 1:2 ratio. At 9 h post-coculture, 1458 cells were fixed with 4% paraformaldehyde in PBS (Nacalai Tesque, Cat# 1459 09154-85) for 15 minutes at room temperature. Nuclei were stained with 1460 Hoechst 33342 (Thermo Fisher Scientific, Cat# H3570). The coverslips were 1461 mounted on glass slides using Fluoromount-G

The size of syncytium (GFP-positive area) was measured using Fiji software 1464 v2.2.0 (ImageJ) as previously described

To generate 1471 the lineage A-based GFP-expressing chimeric recombinant SARS-CoV-2 1472 (rBA.2.9.1 S-GFP, rBA.2.11 S-GFP, rBA.2.12.1 S-GFP and rBA.4/5 S-GFP) 1473 (Figure 4A, left), 7 DNA fragments (fragments 1-7) encoding the partial genome 1474 of SARS-CoV-2 (strain WK-521

To prepare the fragment 8 encoding the S genes 1483 of BA.2.9.1, BA.2.11, BA.2.12.1 and BA.4/5, we inserted additional mutations 1484 into the fragment 8 plasmid encoding the BA.2 S (Yamasoba et al., 2022a) by 1485 site-directed overlap extension PCR using the primers listed in Table S6. 1486 Nucleotide sequences were determined by a DNA sequencing service (Fasmac), 1487 and the sequence data were analyzed by Sequencher software v5

RNA was extracted 1492 from the cells infected with a clinical isolate of BA.2 (GISAID ID: 1493 EPI_ISL_9595859) and cDNA was synthesized as described above (see "Viral 1494 genome sequencing" section). The two DNA fragments correspond to the 1495 fragments 1-7 and 9 were prepared by RT-PCR using PrimeSTAR GXL DNA 1496 polymerase (Takara, Cat# R050A) using the primers listed in Table S6. The 1497 fragments 8 bearing the S genes of BA

To produce recombinant SARS-CoV-2 (seed viruses), the CPER 1501 products were transfected into HEK293-C34 cells using TransIT-LT1 (Takara, 1502 Cat# MIR2300) according to the manufacturer's protocol. At one day 1503 posttransfection, the culture medium was replaced with DMEM

To remove the CPER products (i.e., SARS-CoV-2-related DNA), 1 ml of 1508 the seed virus was treated with 2 μ l TURBO DNase (Thermo Fisher Scientific, 1509 Cat# AM2238) and incubated at 37°C for 1 hour. Complete removal of the CPER 1510 products from the seed virus was verified by PCR. The working virus stock was 1511 prepared using the seed virus as described below

SARS-CoV-2 preparation and titration 1515 The working virus stocks of chimeric recombinant SARS-CoV-2 were prepared 1516 and titrated as previously described

In brief, 20 μ l of the 1518 seed virus was inoculated into VeroE6/TMPRSS2 cells (5,000,000 cells in a 1519 T-75 flask)

At 3 d.p.i., the culture medium was harvested and centrifuged, and the 1522 supernatants were collected as the working virus stock

The titer of the prepared working virus was measured as the 50% 1524 tissue culture infectious dose (TCID 50 ). Briefly, one day before infection

000 cells) were seeded into a 96-well plate. Serially 1526 diluted virus stocks were inoculated into the cells and incubated at 37°C for 4 1527 days. The cells were observed under microscopy to judge the CPE appearance

The value of TCID 50 /ml was calculated with the Reed-Muench method (Reed 1529 and Muench

Cat# 52906) and viral genome sequences were analyzed as described 1533 above (see "Viral genome sequencing" section)

TCID 50 ) at 37°C for 1 hour. 1544 Mounting solution containing 3% FBS and 1.5% carboxymethyl cellulose (Wako, 1545 Cat# 039-01335) was overlaid, followed by incubation at 37°C. At 3 d.p.i., the 1546 culture medium was removed, and the cells were washed with PBS three times 1547 and fixed with 4% paraformaldehyde phosphate (Nacalai Tesque, Cat# 1548 09154-85). The fixed cells were washed with tap water, dried, and stained with 1549 staining solution [0.1% methylene blue (Nacalai Tesque, Cat# 22412-14) in 1550 water] for 30 minutes. The stained cells were washed with tap water and dried

000 cells) were seeded into a 96-well plate. SARS-CoV-2 [1,000 TCID 50 for 1556 Vero cells (Figures 4D and 4F); 100 TCID 50 for VeroE6/TMPRSS2 cells 1557 (Figures 4E and 4G)] was inoculated and incubated at 37°C for 1 hour. The 1558 infected cells were washed, and 180 µl culture medium was added. The culture 1559 supernatant (10 µl) was harvested at the indicated timepoints and used for 1560 RT-qPCR to quantify the viral RNA copy number (see "RT-qPCR" section 1561 below) In the infection experiment using human iPSC-derived airway and × RNA lysis buffer

40% glycerol, 0.8 U/μl recombinant RNase 1577 inhibitor (Takara, Cat# 2313B)] and incubated at room temperature for 10 m

5 μ l) was used 1579 as the template for real-time RT-PCR performed according to the manufacturer's 1580 protocol using One Step TB Green PrimeScript PLUS RT-PCR kit (Takara, Cat# 1581 RR096A) and the following primers: Forward N, 5'-AGC CTC TTC TCG TTC 1582 CTC ATC AC-3'; and Reverse N, 5'-CCG CCA TTG CCA GCC ATT C-3'. The 1583 viral RNA copy number was standardized with a SARS-CoV-2 direct detection 1584 RT-qPCR kit (Takara, Cat# RC300A). Fluorescent signals were acquired using 1585 QuantStudio 3 Real-Time PCR system

Baseline 1594 body weights were measured before infection. For the virus infection 1595 experiments, hamsters were anaesthetized by intramuscular injection of a 1596 mixture of either 0.15 mg/kg medetomidine hydrochloride (Domitor ® , Nippon 1597 Zenyaku Kogyo), 2.0 mg/kg midazolam (FUJIFILM Wako Chemicals

1658 hyperplasia of type II pneumocytes, and the area of the hyperplasia of large type 1659 II pneumocytes were evaluated by certified pathologists and the degree of these 1660 pathological findings were arbitrarily scored using four-tiered system as 0 1661 (negative), 1 (weak), 2 (moderate), and 3 (severe). The "large type II 1662 pneumocytes" are the hyperplasia of type II pneumocytes exhibiting more than 1663 10-μm-diameter nucleus. We described "large type II pneumocytes" as one of 1664 the remarkable histopathological features reacting SARS-CoV-2 infection in our 1665 previous studies

Total histology score is the sum of these five indices

four hamsters infected with each virus were sacrificed at 5 d.p.i., and 1669 all four right lung lobes, including upper (anterior/cranial), middle, lower 1670 (posterior/caudal), and accessory lobes, were sectioned along with their bronchi. 1671 The tissue sections were stained by H&E, and the digital microscopic images 1672 were incorporated into virtual slides using NDP.scan software v3.2.4 1673 (Hamamatsu Photonics). The inflammatory area including type II pneumocyte 1674 hyperplasia in the

QUANTIFICATION AND STATISTICAL ANALYSIS 1678 Statistical significance was tested using a two-sided Mann-Whitney U-test, a 1679 two-sided Student's t-test or a two-sided paired t-test unless otherwise noted. 1680 The tests above were performed using Prism 9 software v9

5A-5C, 5F, and 1683 S2C), a multiple regression analysis including experimental conditions (i.e., the 1684 types of infected viruses) as explanatory variables and timepoints as qualitative 1685 control variables was performed to evaluate the difference between 1686 experimental conditions thorough all timepoints. The initial time point was 1687 removed from the analysis

Subsequently, familywise error rates (FWERs) were calculated by the Holm 1689 method. These analyses were performed in R v4

In Figures 5D, 5G and S3, photographs shown are the representative 1691 areas of at least two independent experiments by using four hamsters at each 1692 timepoint

In A and C, assays were performed in quadruplicate, and the presented data are 1024 expressed as the average ± SD. In A and D, each dot indicates the result of an 1025 individual replicate. 1026In C, statistically significant differences between BA.2 and other variants across 1027 timepoints were determined by multiple regression. FWERs calculated using the 1028Holm method are indicated in the figures. 1029In D, statistically significant differences between BA.2 and other variants (*, P < 1030 0.05) were determined by two-sided Mann-Whitney U tests. 1031 1032 Figure S3 . Phylogenetic and comparative genome analyses 1181To construct an ML tree of Omicron lineages (BA.1-BA.5) sampled from South 1182Africa (shown in Figure 1A ), the genome sequence data of SARS-CoV-2 and its 1183 metadata were downloaded from the GISAID database ( To classify the BA.2 variants bearing mutations at the S L452 residue, 1206we constructed an ML tree of BA.2 variants including those bearing mutations at 1207 the S L452 residue (shown in Figure 1B) (Figures 1E and S1D) were harvested and centrifuged. The pseudoviruses were stored at -80°C until 1358 use. 1359Neutralization assay (Figure 2 ) was prepared as previously described 1360 (Kimura et value, which indicates the amount of p24 HIV-1 antigen) was inoculated into 1384 HOS-ACE2/TMPRSS2 cells, HEK293-ACE2 cells or HEK293-ACE2/TMPRSS2 1385 and viral infectivity was measured as described above (see "Neutralization 1386 assay" section). To analyze the effect of TMPRSS2 for pseudovirus infectivity 1387 (Figure S2A) , the fold change of the values of HEK293-ACE2/TMPRSS2 to 1388 HEK293-ACE2 was calculated. 1389 1390Yeast surface display 1391Yeast surface display ( Figure 3B ) was performed as previously described as 1392previously SARS-CoV-2 S-based fusion assay (Figures 3D and S2C) Coculture experiment (Figure S2D ) was performed as previously described 1446 . This assay utilizes a dual split 1447 protein (DSP) encoding Renilla luciferase and GFP genes; the respective split 1448proteins, DSP 8-11 and DSP 1-7 , are expressed in effector and target cells by 1449transfection. Briefly, one day before transfection, effector cells (i.e., 1450S-expressing cells) were seeded on the poly-L-lysine (Sigma, Cat# P4832) 1451 coated coverslips put in a 12-well plate, and target cells were prepared at a 1452 alveolar epithelial cells (Figures 4H and 4I Histopathological scoring 1654Histopathological scoring (Figure 5F ) was performed as previously described 1655 (Saito et