Establishment and Application of Stable Cell Lines Expressing Mpox Virus Surface Protein A35R and E8L

doi:10.13560/j.cnki.biotech.bull.1985.2024-0366

Abstract

Abstract:

【Objective】 A stable cell line expressing the MPXV（Mpox virus）surface proteins A35R or E8L was established and utilized for qualitative or quantitative analysis of the binding activities of antibodies or molecules associated with A35R or E8L on the cell surface.【Method】 Lentivirus vectors coding for A35R or E8L were constructed using gene recombination technology, and A35R and E8L were expressed and localized on the cell surface of engineered HEK-293T cell lines that were transduced by lentivirus carrying A35R or E8L. The expressions and localizations of A35R and E8L in the engineered cells were detected using Western Blot and immunofluorescence, and the binding affinity of the engineered cells to polyclonal antibodies was analyzed by flow cytometry. Finally, monoclonal cell lines were screened from the engineered cell lines and applied in the quantitative analysis of the binding activity of commercial anti-A35R or anti-E8L monoclonal antibodies.【Result】 Stable HEK-293T cell lines expressing A35R or E8L antigens on the surface were successfully established. Four monoclonal cell lines were selected through monoclonal screening, among which two stably expressed A35R（A35-2C10 and A35-4E3）and two stably expressed E8L（E8-6 and E8-8）. These monoclonal cell lines showed good fit in quantitative analysis of the binding activity of anti-A35R or E8L mAb.【Conclusion】 Cell lines with stable expressions of MPXV A35R or E8L may serve as tools and can be applied to assess the binding activities of neutralizing antibodies, nucleic acid aptamers, or other molecules associated with A35R or E8L in qualitative or quantitative analysis.

Key words: Mpox virus, A35R, E8L, lentivirus transduction, stable cell line

JIN Zhe-dong, LI Hui-yi, BAO Wen-xin, CUI Cai-xia, YUAN Yun-sheng. Establishment and Application of Stable Cell Lines Expressing Mpox Virus Surface Protein A35R and E8L[J]. Biotechnology Bulletin, 2024, 40(10): 315-322.

Figures/Tables 6

Fig. 1 Map of recombinant A35R and E8L lentiviral expression vectors

Fig. 2 Detection of A35R（22.2 kD）and E8L（38.3 kD）proteins expressed in HEK-293T cells by Western Blot For A35R, anti-His antibody was used as the primary antibody, HRP linked rabbit anti-mouse IgG was used as the secondary antibody. For E8L, anti-E8L antibody was used as the primary antibody, HRP linked goat anti-human IgG was used as the secondary antibody

Fig. 3 Detection of expression of A35R (A) and E8L (B) in HEK-293T cells by immunofluorescence Object lens: 40×

Fig. 4 Binding affinity analysis of 293T-A35（A）and 293T-E8L（B）with anti-serum by flow cytometry Serum from non-immunized mice was used as negative control, all serum was diluted at 1∶300, the same below

Fig. 5 Binding affinity analysis of A35-2C10（A）, A35-4E3（B）and E8-6（C）and E8-8（D）with anti-serum by flow cytometry

Fig. 6 Binding affinity analysis of A35-4E3 with anti-A35R mAb（A）and E8-8 with anti-E8L mAb（B）by flow cytometry The initial concentration of antibodies was 50 μg/mL and tripling dilution. The dashed line indicates EC50

References 20

[1]	McCollum AM, Damon IK. Human monkeypox[J]. Clin Infect Dis, 2014, 58(2): 260-267. doi: 10.1093/cid/cit703 pmid: 24158414
[2]	Cabanillas B, Murdaca G, Guemari A, et al. A compilation answering 50 questions on monkeypox virus and the current monkeypox outbreak[J]. Allergy, 2023, 78(3): 639-662. doi: 10.1111/all.15633 pmid: 36587287
[3]	Lum FM, Torres-Ruesta A, Tay MZ, et al. Monkeypox: disease epidemiology, host immunity and clinical interventions[J]. Nat Rev Immunol, 2022, 22(10): 597-613.
[4]	Kumar N, Acharya A, Gendelman HE, et al. The 2022 outbreak and the pathobiology of the monkeypox virus[J]. J Autoimmun, 2022, 131: 102855.
[5]	Heraud JM, Edghill-Smith Y, Ayala V, et al. Subunit recombinant vaccine protects against monkeypox[J]. J Immunol, 2006, 177(4): 2552-2564.
[6]	Li MJ, Ren ZN, Wang YL, et al. Three neutralizing mAbs induced by MPXV A29L protein recognizing different epitopes act synergistically against orthopoxvirus[J]. Emerg Microbes Infect, 2023, 12(2): 2223669.
[7]	Tamir H, Noy-Porat T, Melamed S, et al. Synergistic effect of two human-like monoclonal antibodies confers protection against orthopoxvirus infection[J]. Nat Commun, 2024, 15(1): 3265. doi: 10.1038/s41467-024-47328-y pmid: 38627363
[8]	Hu H, Zhang ZY, Wang RY, et al. BGC823 cell line with the stable expression of iRFP720 retains its primary properties with promising fluorescence imaging ability[J]. DNA Cell Biol, 2020, 39(5): 900-908. doi: 10.1089/dna.2019.5057 pmid: 32096664
[9]	Xiao XY, Chen H, Paerhati P, et al. Establishment and application of engineered NIH 3T3 cell line with stable human RAGE expression[J]. Pharm Fronts, 2020, 2(1): e23-e27.
[10]	Yong SEF, Ng OT, Ho ZJM, et al. Imported monkeypox, Singapore[J]. Emerg Infect Dis, 2020, 26(8): 1826-1830. doi: 10.3201/eid2608.191387 pmid: 32338590
[11]	Tang D, Liu XK, Lu J, et al. Recombinant proteins A29L, M1R, A35R, and B6R vaccination protects mice from mpox virus challenge[J]. Front Immunol, 2023, 14: 1203410.
[12]	Hou FJ, Zhang YT, Liu XH, et al. mRNA vaccines encoding fusion proteins of monkeypox virus antigens protect mice from vaccinia virus challenge[J]. Nat Commun, 2023, 14(1): 5925. doi: 10.1038/s41467-023-41628-5 pmid: 37739969
[13]	King LB, West BR, Moyer CL, et al. Cross-reactive neutralizing human survivor monoclonal antibody BDBV223 targets the ebolavirus stalk[J]. Nat Commun, 2019, 10(1): 1788. doi: 10.1038/s41467-019-09732-7 pmid: 30996276
[14]	Ma H, Guo YY, Tang HN, et al. Broad ultra-potent neutralization of SARS-CoV-2 variants by monoclonal antibodies specific to the tip of RBD[J]. Cell Discov, 2022, 8(1): 16. doi: 10.1038/s41421-022-00381-7 pmid: 35169121
[15]	Matho MH, de Val N, Miller GM, et al. Murine anti-vaccinia virus D8 antibodies target different epitopes and differ in their ability to block D8 binding to CS-E[J]. PLoS Pathog, 2014, 10(12): e1004495.
[16]	Ward BM, Weisberg AS, Moss B. Mapping and functional analysis of interaction sites within the cytoplasmic domains of the vaccinia virus A33R and A36R envelope proteins[J]. J Virol, 2003, 77(7): 4113-4126. pmid: 12634370
[17]	Gilchuk I, Gilchuk P, Sapparapu G, et al. Cross-neutralizing and protective human antibody specificities to poxvirus infections[J]. Cell, 2016, 167(3): 684-694.e9. doi: S0092-8674(16)31334-4 pmid: 27768891
[18]	Milone MC, O'Doherty U. Clinical use of lentiviral vectors[J]. Leukemia, 2018, 32(7): 1529-1541. doi: 10.1038/s41375-018-0106-0 pmid: 29654266
[19]	Wang XY, Ma CC, Rodríguez Labrada R, et al. Recent advances in lentiviral vectors for gene therapy[J]. Sci China Life Sci, 2021, 64(11): 1842-1857. doi: 10.1007/s11427-021-1952-5 pmid: 34708326
[20]	Shearer RF, Saunders DN. Experimental design for stable genetic manipulation in mammalian cell lines: lentivirus and alternatives[J]. Genes Cells, 2015, 20(1): 1-10. doi: 10.1111/gtc.12183 pmid: 25307957