Here we show the possibility that the L452R-mediated resistance to cilgavimab is attributed to the steric hindrance between cilgavimab and its binding interface around the spike

Here we show the possibility that the L452R-mediated resistance to cilgavimab is attributed to the steric hindrance between cilgavimab and its binding interface around the spike. Acknowledgments We would like to thank all members of The Genotype to Phenotype Japan (G2P-Japan) Consortium (Keita Matsuno, Naganori Nao, Hirofumi Sawa, Shinya Tanaka, Masumi Tsuda, Lei Wang, Yoshikata Oda, Marie Kato, Zannatul Ferdous, Hiromi Mouri, Ken-ji Shishido, Takasuke Fukuhara, Tomokazu Tamura, Rigel Suzuki, Saori Suzuki, Hayato Ito, Jumpei Ito, Naoko Misawa, Keiya Uriu, Pan Lin, Mai Suganami, Mika Chiba, Ryo Yoshimura, Kyoko Yasuda, Keiko Iida, Naomi Ohsumi, So Nakagawa, Jiaqi Wu, Akifumi Takaori-Kondo, Kotaro Rabbit polyclonal to PELI1 Shirakawa, Kayoko Nagata, Yasuhiro Kazuma, Ryosuke Nomura, Yoshihito Horisawa, Yusuke Tashiro, Yugo Kawai, Takashi Irie, Ryoko Kawabata, Terumasa Ikeda, Hesham Nasser, Ryo Shimizu, MST Monira Begum, Otowa Takahashi, Kimiko Ichihara, Takamasa Ueno, Chihiro Motozono, Mako Toyoda, Akatsuki Saito, Maya Shofa, Yuki Shibatani, Tomoko Nishiuchi, Kazuo Takayama, Rina Hashimoto, Sayaka Deguchi, Yukio Watanabe, Ayaka Sakamoto, Naoko Yasuhara, Takao Hashiguchi, Tateki Suzuki, Kanako Kimura, Jiei Sasaki, Yukari Nakajima, Hisano Yajima, Kaori Tabata). Funding Statement This study was supported in part by AMED Research Program on Emerging and Re-emerging Infectious Diseases 20fk0108146 (to K.S.), and 21fk0108494 (to K.S.); AMED Research Program on HIV/AIDS 21fk0410039 (to K.S.); JST CREST JPMJCR20H4 (to K.S.); JSPS Core-to-Core Program JPJSCCA20190008 (A. contamination Isosilybin A and COVID-19 treatment, a variety of therapeutic monoclonal antibodies (mAbs) have been developed [1]. These therapeutic mAbs bind to the spike protein of SARS-CoV-2 and impair viral entry into the cells [1]. However, substitutions in the viral spike protein potentially affect the efficacy of therapeutic mAbs [1,2,3]. SARS-CoV-2 has considerably diversified during the pandemic, and as of July 2022, the Omicron BA.2 variant is the most dominant variant worldwide [4]. Compared with an ancestral Wuhan/Hu-1/2019 reference strain [5], the Omicron BA.2 variant possesses 29 substitutions in the spike protein [4] and exhibits robust resistance to multiple therapeutic mAbs [6]. Recently, new Omicron sublineages, such as BA.4, and BA.5, emerged in multiple countries and have begun to outcompete BA.2 [7]. The spike proteins of BA.4 and BA.5 are identical, and the BA.4/5 spike bears four substitutions compared with the BA.2 spike. Importantly, we have recently exhibited that BA.4/5 is resistant to cilgavimab, a therapeutic mAb, and the resistance to cilgavimab of the BA.4/5 spike is attributed to the L452R substitution [8]. Cilgavimab (AZD1061) is used with Tixagevimab (AZD8895) as the antibody cocktail Evusheld (AZD7442) for COVID-19 treatment [9]. Previous reports show that this epitopes of cilgavimab are the residues 440-452 and 490-500 of SARS-CoV-2 spike protein, and these two regions are included in the receptor-binding domain name (RBD) [9,10]. Interestingly, although cilgavimab neutralizes a variety of variants of concern and variants of interest, the Epsilon variant (B.1.429) is relatively resistant to cilgavimab [9,11]. Since the common substitution in the Epsilon and BA.4/5 spikes is L452R, it is suggested that this L452R is crucial for the resistance to cilgavimab. However, how the spike L452R substitution renders resistance to cilgavimab remains unclear. 2. Materials and Methods 2.1. Isosilybin A Cell Culture HEK293T cells (a human embryonic kidney cell line; ATCC CRL-3216) and HOS-ACE2/TMPRSS2 cells (kindly provided by Dr. Kenzo Tokunaga) [12], a derivative of HOS cells (a human osteosarcoma cell line; ATCC CRL-1543) stably expressing human ACE2 and TMPRSS2, were maintained in Dulbeccos altered Eagles medium (high glucose) (Wako, Cat# 044-29765) made up of 10% fetal bovine serum (Sigma-Aldrich Cat# 172012-500ML), 100 models penicillin, and 100 g/mL streptomycin (Sigma-Aldrich, Cat# P4333-100ML). 2.2. Plasmid Construction A plasmid expressing cilgavimab was prepared in our previous study [8]. Plasmids expressing the SARS-CoV-2 spike proteins of Omicron BA.2 and Omicron BA.2 L452R were prepared in our previous studies [4,8]. Plasmids expressing the spike proteins of Omicron BA.2 L452A and BA.2 L452K were generated by site-directed overlap extension PCR using pC-SARS2-S BA.2 [4] as the template and the following primers: pC-S_L452A-F, CAA CTA CAA CTA CGC CTA CAG ACT GTT CA, pC-S_L452A-R, TGA ACA GTC TGT AGG CGT AGT TGT AGT TG, pC-S_L452K-F, GGA GGC AAC TAC AAC TAC AAG TAC AGA CTG TTC AGG AAG AG and pC-S_L452K-R, CTC TTC CTG AAC AGT CTG TAC TTG TAG TTG TAG TTG CCT Isosilybin A CC. The resulting PCR fragment was subcloned into the KpnI-NotI site of the pCAGGS vector using an In-Fusion? HD Cloning Kit (Takara, Cat# Z9650N). Nucleotide sequences were determined by DNA sequencing services (Eurofins), and the sequence data were analyzed by Sequencher v5.1 software (Gene Codes Corporation). 2.3. Preparation of Monoclonal Antibodies Cilgavimab was prepared as previously described [4,8]. Briefly, the pCAGGS vectors made up of the sequences encoding the immunoglobulin heavy and light chains were cotransfected into HEK293T cells at a 1:1 ratio using PEI Max (Polysciences, Cat# 24765-1). The culture medium was refreshed with Dulbeccos altered Eagles medium (low glucose) (Wako, Cat# 041-29775) made up of 10% fetal bovine serum without antibiotics. At 96 h posttransfection, the culture medium was harvested, and the antibody was purified using a NAb protein A plus spin kit (Thermo Fisher Scientific, Cat# 89948) according to the manufacturers protocol. 2.4. Neutralization Assay Pseudoviruses were prepared as previously described.