Characterization of humoral immune responses against SARS-CoV-2 accessory proteins in infected patients and mouse model

Yuming Li; Yanhong Tang; Xiaoqian Wang; Airu Zhu; Dongdong Liu; Yiyun He; Hu Guo; Jie Zheng; Xinzhuo Liu; Fengyu Chi; Yanqun Wang; Zhen Zhuang; Zhaoyong Zhang; Donglan Liu; Zhao Chen; Fang Li; Wei Ran; Kuai Yu; Dong Wang; Liyan Wen; Jianfen Zhuo; Yanjun Zhang; Yin Xi; Jingxian Zhao; Jincun Zhao; Jing Sun

doi:10.1016/j.virs.2024.04.005

June 2024

Citation: Yuming Li, Yanhong Tang, Xiaoqian Wang, Airu Zhu, Dongdong Liu, Yiyun He, Hu Guo, Jie Zheng, Xinzhuo Liu, Fengyu Chi, Yanqun Wang, Zhen Zhuang, Zhaoyong Zhang, Donglan Liu, Zhao Chen, Fang Li, Wei Ran, Kuai Yu, Dong Wang, Liyan Wen, Jianfen Zhuo, Yanjun Zhang, Yin Xi, Jingxian Zhao, Jincun Zhao, Jing Sun. Characterization of humoral immune responses against SARS-CoV-2 accessory proteins in infected patients and mouse model .VIROLOGICA SINICA, 2024, 39(3) : 414-421. http://dx.doi.org/10.1016/j.virs.2024.04.005

Characterization of humoral immune responses against SARS-CoV-2 accessory proteins in infected patients and mouse model

Yuming Li ^a,b,c ,
Yanhong Tang ^c,d ,
Xiaoqian Wang ^a,b ,
Airu Zhu ^c ,
Dongdong Liu ^c ,
Yiyun He ^c ,
Hu Guo ^c ,
Jie Zheng ^c ,
Xinzhuo Liu ^a,b ,
Fengyu Chi ^a,b ,
Yanqun Wang ^c ,
Zhen Zhuang ^c ,
Zhaoyong Zhang ^c ,
Donglan Liu ^c ,
Zhao Chen ^c ,
Fang Li ^c ,
Wei Ran ^c ,
Kuai Yu ^c ,
Dong Wang ^c ,
Liyan Wen ^c ,
Jianfen Zhuo ^c ,
Yanjun Zhang ^c ,
Yin Xi ^{c
,,} ,
Jingxian Zhao ^{c,e
,,} ,
Jincun Zhao ^{c,e,f,g
,,} ,
Jing Sun ^{c
,,}

a. School of Public Health, Shandong First Medical University & Shandong Academy of Medical Sciences, Ji'nan, 250117, China;
b. Key Laboratory of Emerging Infectious Diseases in Universities of Shandong, Shandong First Medical University & Shandong Academy of Medical Sciences, Ji'nan, 250117, China;
c. State Key Laboratory of Respiratory Disease, National Clinical Research Center for Respiratory Disease, National Center for Respiratory Medicine, Guangzhou Institute of Respiratory Health, The First Affiliated Hospital of Guangzhou Medical University, Guangzhou, Guangdong, 510182, China;
d. Hunan Provincial People's Hospital, The First Affiliated Hospital of Hunan Normal University, Hunan Normal University, Changsha, 410005, China;
e. Guangzhou National Laboratory, Guangzhou, Guangdong, 510005, China;
f. Shanghai Institute for Advanced Immunochemical Studies, School of Life Science and Technology, Shanghai Tech University, Shanghai, 201210, China;
g. Institute for Hepatology, National Clinical Research Center for Infectious Disease, Shenzhen Third People's Hospital, The Second Affiliated Hospital, School of Medicine, Southern University of Science and Technology, Shenzhen, 518005, China

Corresponding author: Yin Xi, xiyin86@163.com
Jingxian Zhao, zhaojingxian@gird.cn
Jincun Zhao, zhaojincun@gird.cn
Jing Sun, sj-ji@163.com
Received Date: 05 November 2023
Accepted Date: 19 April 2024
Available online: 26 April 2024

Abstract

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of COVID-19, encodes several accessory proteins that have been shown to play crucial roles in regulating the innate immune response. However, their expressions in infected cells and immunogenicity in infected humans and mice are still not fully understood. This study utilized various techniques such as luciferase immunoprecipitation system (LIPS), immunofluorescence assay (IFA), and western blot (WB) to detect accessory protein-specific antibodies in sera of COVID-19 patients. Specific antibodies to proteins 3a, 3b, 7b, 8 and 9c can be detected by LIPS, but only protein 3a antibody was detected by IFA or WB. Antibodies against proteins 3a and 7b were only detected in ICU patients, which may serve as a marker for predicting disease progression. Further, we investigated the expression of accessory proteins in SARS-CoV-2-infected cells and identified the expressions of proteins 3a, 6, 7a, 8, and 9b. We also analyzed their ability to induce antibodies in immunized mice and found that only proteins 3a, 6, 7a, 8, 9b and 9c were able to induce measurable antibody productions, but these antibodies lacked neutralizing activities and did not protect mice from SARS-CoV-2 infection. Our findings validate the expression of SARS-CoV-2 accessory proteins and elucidate their humoral immune response, providing a basis for protein detection assays and their role in pathogenesis.
- SARS-CoV-2
- , Accessory protein
- , Humoral immune responses
- , COVID-19
- , Mouse model

Electronic Supplementary Material

10.1016j.virs.2024.04.005-ESM.docx
References
1. Akerstrom, S., Tan, Y.J.,Mirazimi, A., 2006. Amino acids 15-28 in the ectodomain of SARS coronavirus 3a protein induces neutralizing antibodies. FEBS Lett., 580, 3799-3803.
2. Arshad, N., Laurent-Rolle, M., Ahmed, W.S., Hsu, J.C., Mitchell, S.M., Pawlak, J., Sengupta, D., Biswas, K.H.,Cresswell, P., 2022. SARS-CoV-2 accessory proteins ORF7a and ORF3a use distinct mechanisms to downregulate MHC-I surface expression. bioRxiv, 10.1101/2022.05.17.492198.
3. Bacher, P., Rosati, E., Esser, D., Martini, G.R., Saggau, C., Schiminsky, E., Dargvainiene, J., Schroder, I., Wieters, I., Khodamoradi, Y., Eberhardt, F., Vehreschild, M., Neb, H., Sonntagbauer, M., Conrad, C., Tran, F., Rosenstiel, P., Markewitz, R., Wandinger, K.P., Augustin, M., Rybniker, J., Kochanek, M., Leypoldt, F., Cornely, O.A., Koehler, P., Franke, A.,Scheffold, A., 2020. Low-avidity CD4(+) T cell responses to SARS-CoV-2 in unexposed individuals and humans with severe COVID-19. Immunity, 53, 1258-1271.e1255.
4. Blanco-Melo, D., Nilsson-Payant, B.E., Liu, W.C., Uhl, S., Hoagland, D., Moeller, R., Jordan, T.X., Oishi, K., Panis, M., Sachs, D., Wang, T.T., Schwartz, R.E., Lim, J.K., Albrecht, R.A.,Tenoever, B.R., 2020. Imbalanced host response to SARS-CoV-2 drives development of COVID-19. Cell, 181, 1036-1045.e1039.
5. Bojkova, D., Klann, K., Koch, B., Widera, M., Krause, D., Ciesek, S., Cinatl, J.,Munch, C., 2020. Proteomics of SARS-CoV-2-infected host cells reveals therapy targets. Nature, 583, 469-472.
6. Burbelo, P.D., Ching, K.H., Klimavicz, C.M.,Iadarola, M.J., 2009. Antibody profiling by Luciferase Immunoprecipitation Systems (LIPS). J. Vis. Exp., 10.3791/1549.
7. Deming, D., Sheahan, T., Heise, M., Yount, B., Davis, N., Sims, A., Suthar, M., Harkema, J., Whitmore, A., Pickles, R., West, A., Donaldson, E., Curtis, K., Johnston, R.,Baric, R., 2006. Vaccine efficacy in senescent mice challenged with recombinant SARS-CoV bearing epidemic and zoonotic spike variants. PLoS Med., 3, e525.
8. Gordon, D.E., Jang, G.M., Bouhaddou, M., Xu, J., Obernier, K., White, K.M., O'Meara, M.J., Rezelj, V.V., Guo, J.Z., Swaney, D.L., et al., 2020. A SARS-CoV-2 protein interaction map reveals targets for drug repurposing. Nature, 583, 459-468.
9. Hachim, A., Gu, H., Kavian, O., Kwan, M.Y., Chan, W.H., Yau, Y.S., Chiu, S.S., Tsang, O.T., Hui, D.S., Ma, F., Lau, E.H., Cheng, S.M., Poon, L.L., Peiris, J.M., Valkenburg, S.A.,Kavian, N., 2021. The SARS-CoV-2 antibody landscape is lower in magnitude for structural proteins, diversified for accessory proteins and stable long-term in children. medRxiv, 10.1101/2021.01.03.21249180.
10. Hachim, A., Gu, H., Kavian, O., Mori, M., Kwan, M.Y.W., Chan, W.H., Yau, Y.S., Chiu, S.S., Tsang, O.T.Y., Hui, D.S.C., Mok, C.K.P., Ma, F.N.L., Lau, E.H.Y., Amarasinghe, G.K., Qavi, A.J., Cheng, S.M.S., Poon, L.L.M., Peiris, J.S.M., Valkenburg, S.A.,Kavian, N., 2022. SARS-CoV-2 accessory proteins reveal distinct serological signatures in children. Nat. Commun., 13, 2951.
11. Hachim, A., Kavian, N., Cohen, C.A., Chin, A.W.H., Chu, D.K.W., Mok, C.K.P., Tsang, O.T.Y., Yeung, Y.C., Perera, R., Poon, L.L.M., Peiris, J.S.M.,Valkenburg, S.A., 2020. ORF8 and ORF3b antibodies are accurate serological markers of early and late SARS-CoV-2 infection. Nat. Immunol., 21, 1293-1301.
12. Han, L., Zhuang, M.W., Deng, J., Zheng, Y., Zhang, J., Nan, M.L., Zhang, X.J., Gao, C.,Wang, P.H., 2021. SARS-CoV-2 ORF9b antagonizes type I and III interferons by targeting multiple components of the RIG-I/MDA-5-MAVS, TLR3-TRIF, and cGAS-STING signaling pathways. J. Med. Virol., 93, 5376-5389.
13. Huang, C., Ito, N., Tseng, C.T.,Makino, S., 2006. Severe acute respiratory syndrome coronavirus 7a accessory protein is a viral structural protein. J. Virol., 80, 7287-7294.
14. Huang, C., Wang, Y., Li, X., Ren, L., Zhao, J., Hu, Y., Zhang, L., Fan, G., Xu, J., Gu, X., Cheng, Z., Yu, T., Xia, J., Wei, Y., Wu, W., Xie, X., Yin, W., Li, H., Liu, M., Xiao, Y., Gao, H., Guo, L., Xie, J., Wang, G., Jiang, R., Gao, Z., Jin, Q., Wang, J.,Cao, B., 2020. Clinical features of patients infected with 2019 novel coronavirus in Wuhan, China. Lancet, 395, 497-506.
15. Ito, N., Mossel, E.C., Narayanan, K., Popov, V.L., Huang, C., Inoue, T., Peters, C.J.,Makino, S., 2005. Severe acute respiratory syndrome coronavirus 3a protein is a viral structural protein. J. Virol., 79, 3182-3186.
16. Jiang, H.W., Zhang, H.N., Meng, Q.F., Xie, J., Li, Y., Chen, H., Zheng, Y.X., Wang, X.N., Qi, H., Zhang, J., Wang, P.H., Han, Z.G.,Tao, S.C., 2020. SARS-CoV-2 Orf9b suppresses type I interferon responses by targeting TOM70. Cell. Mol. Immunol., 17, 998-1000.
17. Kim, D., Lee, J.Y., Yang, J.S., Kim, J.W., Kim, V.N.,Chang, H., 2020. The architecture of SARS-CoV-2 transcriptome. Cell, 181, 914-921.e910.
18. Konno, Y., Kimura, I., Uriu, K., Fukushi, M., Irie, T., Koyanagi, Y., Sauter, D., Gifford, R.J., Nakagawa, S.,Sato, K., 2020. SARS-CoV-2 ORF3b is a potent interferon antagonist whose activity is increased by a naturally occurring elongation variant. Cell Rep., 32, 108185.
19. Lam, J.Y., Yuen, C.K., Ip, J.D., Wong, W.M., To, K.K., Yuen, K.Y.,Kok, K.H., 2020. Loss of orf3b in the circulating SARS-CoV-2 strains. Emerg. Microbes Infect., 9, 2685-2696.
20. Li, J.Y., Liao, C.H., Wang, Q., Tan, Y.J., Luo, R., Qiu, Y.,Ge, X.Y., 2020. The ORF6, ORF8 and nucleocapsid proteins of SARS-CoV-2 inhibit type I interferon signaling pathway. Virus Res., 286, 198074.
21. Li, Y., Xu, Z., Lei, Q., Lai, D.Y., Hou, H., Jiang, H.W., Zheng, Y.X., Wang, X.N., Wu, J., Ma, M.L., Zhang, B., Chen, H., Yu, C., Xue, J.B., Zhang, H.N., Qi, H., Guo, S.J., Zhang, Y., Lin, X., Yao, Z., Sheng, H., Sun, Z., Wang, F., Fan, X.,Tao, S.C., 2021. Antibody landscape against SARS-CoV-2 reveals significant differences between non-structural/accessory and structural proteins. Cell Rep., 36, 109391.
22. Liu, T., Jia, P., Fang, B.,Zhao, Z., 2020. Differential expression of viral transcripts from single-cell RNA sequencing of moderate and severe COVID-19 patients and its implications for case severity. Front. Microbiol., 11, 603509.
23. Matsuoka, K., Imahashi, N., Ohno, M., Ode, H., Nakata, Y., Kubota, M., Sugimoto, A., Imahashi, M., Yokomaku, Y.,Iwatani, Y., 2022. SARS-CoV-2 accessory protein ORF8 is secreted extracellularly as a glycoprotein homodimer. J. Biol. Chem., 298, 101724.
24. Minakshi, R., Padhan, K., Rani, M., Khan, N., Ahmad, F.,Jameel, S., 2009. The SARS Coronavirus 3a protein causes endoplasmic reticulum stress and induces ligand-independent downregulation of the type 1 interferon receptor. PLoS One, 4, e8342.
25. Miorin, L., Kehrer, T., Sanchez-Aparicio, M.T., Zhang, K., Cohen, P., Patel, R.S., Cupic, A., Makio, T., Mei, M., Moreno, E., Danziger, O., White, K.M., Rathnasinghe, R., Uccellini, M., Gao, S., Aydillo, T., Mena, I., Yin, X., Martin-Sancho, L., Krogan, N.J., Chanda, S.K., Schotsaert, M., Wozniak, R.W., Ren, Y., Rosenberg, B.R., Fontoura, B.M.A.,Garcia-Sastre, A., 2020. SARS-CoV-2 Orf6 hijacks Nup98 to block STAT nuclear import and antagonize interferon signaling. Proc. Natl. Acad. Sci. U. S. A., 117, 28344-28354.
26. Pushko, P., Parker, M., Ludwig, G.V., Davis, N.L., Johnston, R.E.,Smith, J.F., 1997. Replicon-helper systems from attenuated Venezuelan equine encephalitis virus: expression of heterologous genes in vitro and immunization against heterologous pathogens in vivo. Virology, 239, 389-401.
27. Qin, C., Zhou, L., Hu, Z., Zhang, S., Yang, S., Tao, Y., Xie, C., Ma, K., Shang, K., Wang, W.,Tian, D.S., 2020. Dysregulation of immune response in patients with coronavirus 2019 (COVID-19) in Wuhan, China. Clin. Infect. Dis., 71, 762-768.
28. Ren, Y., Shu, T., Wu, D., Mu, J., Wang, C., Huang, M., Han, Y., Zhang, X.Y., Zhou, W., Qiu, Y.,Zhou, X., 2020. The ORF3a protein of SARS-CoV-2 induces apoptosis in cells. Cell. Mol. Immunol., 17, 881-883.
29. Sharma, K., Akerstrom, S., Sharma, A.K., Chow, V.T., Teow, S., Abrenica, B., Booth, S.A., Booth, T.F., Mirazimi, A.,Lal, S.K., 2011. SARS-CoV 9b protein diffuses into nucleus, undergoes active Crm1 mediated nucleocytoplasmic export and triggers apoptosis when retained in the nucleus. PLoS One, 6, e19436.
30. Sun, J., Zhuang, Z., Zheng, J., Li, K., Wong, R.L., Liu, D., Huang, J., He, J., Zhu, A., Zhao, J., Li, X., Xi, Y., Chen, R., Alshukairi, A.N., Chen, Z., Zhang, Z., Chen, C., Huang, X., Li, F., Lai, X., Chen, D., Wen, L., Zhuo, J., Zhang, Y., Wang, Y., Huang, S., Dai, J., Shi, Y., Zheng, K., Leidinger, M.R., Chen, J., Li, Y., Zhong, N., Meyerholz, D.K., Mccray, P.B., Jr., Perlman, S.,Zhao, J., 2020. Generation of a broadly useful model for COVID-19 pathogenesis, vaccination, and treatment. Cell, 182, 734-743.e735.
31. Tan, M., Liu, Y., Zhou, R., Deng, X., Li, F., Liang, K.,Shi, Y., 2020. Immunopathological characteristics of coronavirus disease 2019 cases in Guangzhou, China. Immunology, 160, 261-268.
32. Tang, Z., Yu, P., Guo, Q., Chen, M., Lei, Y., Zhou, L., Mai, W., Chen, L., Deng, M., Kong, W., Niu, C., Xiong, X., Li, W., Chen, C., Lai, C., Wang, Q., Li, B.,Ji, T., 2023. Clinical characteristics and host immunity responses of SARS-CoV-2 Omicron variant BA.2 with deletion of ORF7a, ORF7b and ORF8. Virol. J., 20, 106.
33. Wang, G., Guan, J., Li, G., Wu, F., Yang, Q., Huang, C., Shao, J., Xu, L., Guo, Z., Zhou, Q., Zhu, H.,Chen, Z., 2021. Effect of ORF7 of SARS-CoV-2 on the chemotaxis of monocytes and neutrophils in vitro. Dis. Markers, 2021, 6803510.
34. Wu, A., Peng, Y., Huang, B., Ding, X., Wang, X., Niu, P., Meng, J., Zhu, Z., Zhang, Z., Wang, J., Sheng, J., Quan, L., Xia, Z., Tan, W., Cheng, G., Jiang, T., 2020. Genome composition and divergence of the novel coronavirus (2019-nCoV) originating in China. Cell Host Microbe 27, 325–328.
35. Wu, F., Zhao, S., Yu, B., Chen, Y.M., Wang, W., Song, Z.G., Hu, Y., Tao, Z.W., Tian, J.H., Pei, Y.Y., Yuan, M.L., Zhang, Y.L., Dai, F.H., Liu, Y., Wang, Q.M., Zheng, J.J., Xu, L., Holmes, E.C.,Zhang, Y.Z., 2020. A new coronavirus associated with human respiratory disease in China. Nature, 579, 265-269.
36. Wu, J., Shi, Y., Pan, X., Wu, S., Hou, R., Zhang, Y., Zhong, T., Tang, H., Du, W., Wang, L., Wo, J., Mu, J., Qiu, Y., Yang, K., Zhang, L.K., Ye, B.C.,Qi, N., 2021. SARS-CoV-2 ORF9b inhibits RIG-I-MAVS antiviral signaling by interrupting K63-linked ubiquitination of NEMO. Cell Rep., 34, 108761.
37. Yang, R., Zhao, Q., Rao, J., Zeng, F., Yuan, S., Ji, M., Sun, X., Li, J., Yang, J., Cui, J., Jin, Z., Liu, L.,Liu, Z., 2021. SARS-CoV-2 accessory protein ORF7b mediates tumor necrosis factor-α-induced apoptosis in cells. Front. Microbiol., 12, 654709.
38. Zhang, J., Cruz-Cosme, R., Zhuang, M.W., Liu, D., Liu, Y., Teng, S., Wang, P.H.,Tang, Q., 2020. A systemic and molecular study of subcellular localization of SARS-CoV-2 proteins. Signal Transduct. Target. Ther., 5, 269.
39. Zhang, Y., Chen, Y., Li, Y., Huang, F., Luo, B., Yuan, Y., Xia, B., Ma, X., Yang, T., Yu, F., Liu, J., Liu, B., Song, Z., Chen, J., Yan, S., Wu, L., Pan, T., Zhang, X., Li, R., Huang, W., He, X., Xiao, F., Zhang, J.,Zhang, H., 2021. The ORF8 protein of SARS-CoV-2 mediates immune evasion through down-regulating MHC-Ι. Proc. Natl. Acad. Sci. U. S. A., 118.
40. Zhao, J., Zhao, J., Mangalam, A.K., Channappanavar, R., Fett, C., Meyerholz, D.K., Agnihothram, S., Baric, R.S., David, C.S.,Perlman, S., 2016. Airway memory CD4(+) T cells mediate protective immunity against emerging respiratory coronaviruses. Immunity, 44, 1379-1391.
Proportional views