Recombinant protein HR212 targeting heptad repeat 2 domain in spike protein S2 subunit elicits broad-spectrum neutralizing antibodies against SARS-CoV-2 and its variants

Abstract

SARS-CoV-2 variants are under continuous emergence carry numerous mutations within S1 subunit in spike protein and have escaped neutralization through many currently used vaccines and antibodies. The development of next-generation vaccines is a continuing and long-term need. In our prior research, the recombinant protein vaccine HR121 targeting the heptad repeat (HR) 1 domain of S2 subunit was constructed, which could evoke highly broad-spectrum neutralizing antibodies in vivo and confer efficient protective effect on several SARS-CoV-2 variants within multiple animal models. Compared with HR1, HR2 domain shows a more conservative degree within SARS-CoV-2 and related coronaviruses. Here, we designed a recombinant protein HR212 consisting of HR2–linker1–HR1–linker2–HR2. HR212 showed a high affinity with HR1 and was functionally analogous to HR2 within fusion intermediate in S2 subunit. Immunizing rabbits using HR212-mediated high nAbs for 28 pseudotyped SARS-CoV-2 variants, like currently circulating variants, such as BA.2.86 and JN.1. Transfer of rabbit anti-HR212 sera or immunization with HR212 offered efficient protective effect on SARS-CoV-2 ancestral strain and Omicron BA.2 variant infections of Syrian golden hamsters. According to our results, HR2 domain of S2 subunit is the novel target that can be used to develop broad-spectrum vaccines to resist SARS-CoV-2 variants.

Keywords

broadly neutralizing antibodies / HR1 domain / HR2 domain / S2 subunit / SARS-CoV-2 variants

References

[1] Ai J, Zhang H, Zhang Y, et al. Omicron variant showed lower neutralizing sensitivity than other SARS-CoV-2 variants to immune sera elicited by vaccines after boost. Emerg Microbes Infec. 2022; 11(1): 337-343.

[2] Sharun K, Tiwari R, Dhama K, et al. Emerging SARS-CoV-2 variants: impact on vaccine efficacy and neutralizing antibodies. Hum Vacc Immunother. 2021; 17(10): 3491-3494.

[3] Wu F, Zhao S, Yu B, et al. A new coronavirus associated with human respiratory disease in China. Nature. 2020; 579(7798): 265-269.

[4] Zhu N, Zhang D, Wang W, et al. A novel coronavirus from patients with pneumonia in China, 2019. N Engl J Med. 2020; 382(8): 727-733.

[5] Wrapp D, Wang N, Corbett KS, et al. Cryo-EM structure of the 2019-nCoV spike in the prefusion conformation. Science. 2020; 367(6483): 1260-1263.

[6] Xing L, Xu X, Xu W, et al. A five-helix-based SARS-CoV-2 fusion inhibitor targeting heptad repeat 2 domain against SARS-CoV-2 and its variants of concern. Viruses. 2022; 14(3): 597.

[7] Liu S, Xiao G, Chen Y, et al. Interaction between heptad repeat 1 and 2 regions in spike protein of SARS-associated coronavirus: implications for virus fusogenic mechanism and identification of fusion inhibitors. Lancet. 2004; 363(9413): 938-947.

[8] Zhu Y, Yu D, Yan H, Chong H, He Y. Design of potent membrane fusion inhibitors against SARS-CoV-2, an emerging coronavirus with high Fusogenic activity. J Virol. 2020; 94(14): e00635-20.

[9] Xia S, Liu M, Wang C, et al. Inhibition of SARS-CoV-2 (previously 2019-nCoV) infection by a highly potent pan-coronavirus fusion inhibitor targeting its spike protein that harbors a high capacity to mediate membrane fusion. Cell Res. 2020; 30(4): 343-355.

[10] Xia S, Yan L, Xu W, et al. A pan-coronavirus fusion inhibitor targeting the HR1 domain of human coronavirus spike. Sci Adv. 2019; 5(4): eaav4580.

[11] Xia S, Zhu Y, Liu M, et al. Fusion mechanism of 2019-nCoV and fusion inhibitors targeting HR1 domain in spike protein. Cell Mol Immunol. 2020; 17(7): 765-767.

[12] Pang W, Lu Y, Zhao YB, et al. A variant-proof SARS-CoV-2 vaccine targeting HR1 domain in S2 subunit of spike protein. Cell Res. 2022; 32(12): 1068-1085.

[13] Guo LY, Lin S, Chen ZM, et al. Targetable elements in SARS-CoV-2 S2 subunit for the design of pan-coronavirus fusion inhibitors and vaccines. Signal Transduct Tar. 2023; 8(1): 197.

[14] Bi WW, Chen GL, Dang BB. Novel engineered SARS-CoV-2 HR1 trimer exhibits improved potency and broad-spectrum activity against SARS-CoV-2 and its variants. J Virol. 2022; 96(13): e00681-22.

[15] Pang W, Wang RR, Gao YD, et al. A novel enzyme-linked immunosorbent assay for screening HIV-1 fusion inhibitors targeting HIV-1 Gp41 core structure. J Biomol Screen. 2011; 16(2): 221-229.

[16] Harvey WT, Carabelli AM, Jackson B, et al. SARS-CoV-2 variants, spike mutations and immune escape. Nat Rev Microbiol. 2021; 19(7): 409-424.

[17] Liu J, Liu Y, Xia H, et al. BNT162b2-elicited neutralization of B.1.617 and other SARS-CoV-2 variants. Nature. 2021; 596(7871): 273-275.

[18] Supasa P, Zhou D, Dejnirattisai W, et al. Reduced neutralization of SARS-CoV-2 B.1.1.7 variant by convalescent and vaccine sera. Cell. 2021; 184(8): 2201-2211.e2207.

[19] Ravichandran S, Coyle EM, Klenow L, et al. Antibody signature induced by SARS-CoV-2 spike protein immunogens in rabbits. Sci Transl Med. 2020; 12(550): eabc3539.

[20] Yang J, Wang W, Chen Z, et al. A vaccine targeting the RBD of the S protein of SARS-CoV-2 induces protective immunity. Nature. 2020; 586(7830): 572-577.

[21] Liu L, Wang P, Nair MS, et al. Potent neutralizing antibodies against multiple epitopes on SARS-CoV-2 spike. Nature. 2020; 584(7821): 450-456.

[22] Huang Y, Yang C, Xu XF, Xu W, Liu SW. Structural and functional properties of SARS-CoV-2 spike protein: potential antivirus drug development for COVID-19. Acta Pharmacol Sin. 2020; 41(9): 1141-1149.

[23] Wang X, Xia S, Wang Q, et al. Broad-spectrum coronavirus fusion inhibitors to combat COVID-19 and other emerging coronavirus diseases. Int J Mol Sci. 2020; 21(11): 3843.

[24] Dodero-Rojas E, Onuchic JN, Whitford PC. Sterically confined rearrangements of SARS-CoV-2 Spike protein control cell invasion. Elife. 2021; 10: e70362.

[25] Yang K, Wang C, White KI, et al. Structural conservation among variants of the SARS-CoV-2 spike postfusion bundle. Proc Natl Acad Sci USA. 2022; 119(16): e2119467119.

[26] Miller MD, Geleziunas R, Bianchi E, et al. A human monoclonal antibody neutralizes diverse HIV-1 isolates by binding a critical gp41 epitope. Proc Natl Acad Sci USA. 2005; 102(41): 14759-14764.

[27] Louis JM, Nesheiwat I, Chang L, Clore GM, Bewley CA. Covalent trimers of the internal N-terminal trimeric coiled-coil of gp41 and antibodies directed against them are potent inhibitors of HIV envelope-mediated cell fusion. J Biol Chem. 2003; 278(22): 20278-20285.

[28] Bianchi E, Joyce JG, Miller MD, et al. Vaccination with peptide mimetics of the gp41 prehairpin fusion intermediate yields neutralizing antisera against HIV-1 isolates. Proc Natl Acad Sci USA. 2010; 107(23): 10655-10660.

[29] Qi Z, Pan C, Lu H, et al. A recombinant mimetics of the HIV-1 gp41 prehairpin fusion intermediate fused with human IgG Fc fragment elicits neutralizing antibody response in the vaccinated mice. Biochem Bioph Res Commun. 2010; 398(3): 506-512.

[30] Ni L, Zhao L, Gao GF, Qian Y, Tien P. The antibodies directed against N-terminal heptad-repeat peptide of hRSV fusion protein and its analog-5-Helix inhibit virus infection in vitro. Biochem Biophys Res Commun. 2005; 331(4): 1358-1364.

[31] Xu XF. Identification and new drug screening of anti-SARS-CoV-2 protein-drugs targeting the six-helix bundle structure of spike protein transmembrane subunit. Southern Medical University; 2022.

[32] Balinsky CA, Jiang L, Jani V, et al. Antibodies to S2 domain of SARS-CoV-2 spike protein in Moderna mRNA vaccinated subjects sustain antibody-dependent NK cell-mediated cell cytotoxicity against Omicron BA. 1. Front Immunol. 2023; 14: 1266829.

[33] Grant MD, Bentley K, Fielding CA, et al. Combined anti-S1 and anti-S2 antibodies from hybrid immunity elicit potent cross-variant ADCC against SARS-CoV-2. Jci Insight. 2023; 8(15): e170681.

[34] Zheng HY, Song TZ, Zheng YT. Immunobiology of COVID-19: mechanistic and therapeutic insights from animal models. Zool Res. 2024; 45(4): 747-766.

[35] Cai YF, Zhang J, Xiao TS, et al. Distinct conformational states of SARS-CoV-2 spike protein. Science. 2020; 369(6511): 1586-1592.

[36] Tian RR, Yang CX, Zhang M, et al. Lower respiratory tract samples are reliable for severe acute respiratory syndrome coronavirus 2 nucleic acid diagnosis and animal model study. Zool Res. 2021; 42(2): 161.

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