Development of recombinant COVID-19 vaccine based on CHO-produced, prefusion spike trimer and alum/CpG adjuvants

Haitao Liu, Chenliang Zhou, Jiao An, Yujiao Song, Pin Yu, Jiadai Li, Chenjian Gu, Dongdong Hu, Yuanxiang Jiang, Lingli Zhang, Chuanqi Huang, Chao Zhang, Yunqi Yang, Qianjun Zhu, Dekui Wang, Yuqiang Liu, Chenyang Miao, Xiayao Cao, Longfei Ding, Yuanfei Zhu, Hua Zhu, Linlin Bao, Lingyun Zhou, Huan Yan, Jiang Fan, Jianqing Xu, Zhongyu Hu, Youhua Xie, Jiangning Liu, Ge Liu, Haitao Liu, Chenliang Zhou, Jiao An, Yujiao Song, Pin Yu, Jiadai Li, Chenjian Gu, Dongdong Hu, Yuanxiang Jiang, Lingli Zhang, Chuanqi Huang, Chao Zhang, Yunqi Yang, Qianjun Zhu, Dekui Wang, Yuqiang Liu, Chenyang Miao, Xiayao Cao, Longfei Ding, Yuanfei Zhu, Hua Zhu, Linlin Bao, Lingyun Zhou, Huan Yan, Jiang Fan, Jianqing Xu, Zhongyu Hu, Youhua Xie, Jiangning Liu, Ge Liu

Abstract

COVID-19 pandemic has severely impacted the public health and social economy worldwide. A safe, effective, and affordable vaccine against SARS-CoV-2 infections/diseases is urgently needed. We have been developing a recombinant vaccine based on a prefusion-stabilized spike trimer of SARS-CoV-2 and formulated with aluminium hydroxide and CpG 7909. The spike protein was expressed in Chinese hamster ovary (CHO) cells, purified, and prepared as a stable formulation with the dual adjuvant. Immunogenicity studies showed that candidate vaccines elicited robust neutralizing antibody responses and substantial CD4+ T cell responses in both mice and non-human primates. And vaccine-induced neutralizing antibodies persisted at high level for at least 6 months. Challenge studies demonstrated that candidate vaccine reduced the viral loads and inflammation in the lungs of SARS-CoV-2 infected golden Syrian hamsters significantly. In addition, the vaccine-induced antibodies showed cross-neutralization activity against B.1.1.7 and B.1.351 variants. These data suggest candidate vaccine is efficacious in preventing SARS-CoV-2 infections and associated pneumonia, thereby justifying ongoing phase I/II clinical studies in China (NCT04982068 and NCT04990544).

Keywords: SARS-CoV-2; Subunit vaccine; Trimeric spike protein.

Conflict of interest statement

Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Copyright © 2021. Published by Elsevier Ltd.

Figures

Fig. 1
Fig. 1
Molecular design and characterization of SΔTM and RBD. (A) Domain architecture of the SARS-CoV-2 S protein. SS, signal sequence; NTD, N-terminal domain; RBD, receptor-binding domain; SD1, subdomain 1; SD2, subdomain 2; S1/S2, S1/S2 protease cleavage site; S2′, S2′ protease cleavage site; FP, fusion peptide; HR1, heptad repeat 1; CH, central helix; CD, connector domain; HR2, heptad repeat 2; TM, transmembrane domain; CT, cytoplasmic tail. Two recombinant SARS-CoV-2 spike antigens were designed: SΔTM (the prefusion S ectodomain with proline substitutions at residues 986 and 987 to retain S2 in the prefusion conformation, a “GGSG” substitution at the furin cleavage site, a C-terminal T4 fibritin trimerization motif), and 260-mer RBD (RBD-SD1). The structure model of S-trimer was generated by the SWISS-MODEL using homology modelling techniques (http://swissmodel.expasy.org/), and the 3D structure figures were prepared using PyMOL (www.pymol.org). (B) SDS-PAGE analysis of purified SΔTM and RBD. Molecular weight standards are indicated at the left in kDa. (C) Size-Exclusion HPLC chromatogram of purified SΔTM (shown as blue line) and a 670 kDa molecular weight standard (shown as black line). (D) and (E) Binding profiles of SΔTM and RBD to human ACE2 measured by BLI in GatorPrime. The data are shown as blue and orange lines for SΔTM and RBD, respectively, and the best fit of the data to a 1:1 binding model is shown in red. (F) Antigenicity of SΔTM and RBD measured by serially diluted HCS. HCS, human convalescent sera.
Fig. 2
Fig. 2
Immune responses in vaccinated BALB/c mice. (A) Experiment schedule. BALB/c mice were immunized twice intramuscularly at Day 0 and Day 21. Vaccine components were 5 μg SΔTM or RBD, which adjuvanted with 50 μg Alum and/or 50 μg CpG. Alum/CpG only groups were as control. On Day 35, Day 144 (only SΔTM groups) and Day 201 (only SΔTM groups), blood was collected to perform serological assays (SAs) (N = 10). On Day 35, 5 mice from each SΔTM groups were sacrificed to conduct intracellular staining (ICS) assay (N = 5). On Day 201, 5 mice from each SΔTM groups were sacrificed to conduct B cell ELISPOT (N = 5). (B) Binding antibody titers and (C) pseudovirus neutralizing antibody titers of sera collected at Day 35 from SΔTM or RBD vaccinated mice. (D) Neutralizing antibody titers of sera collected at Day 35 from SΔTM vaccinated mice detected by live virus-based neutralization assay. (E) IgG isotyping of sera collected at Day 35 from SΔTM vaccinated mice. (F) Antigen specific CD4+ T cell responses and (G) CD8+ T cell responses in SΔTM vaccinated mice were determined by ICS at Day 35. Dotted lines represent the limit of detection (LOD). Each dot represents an individual mouse. Numbers on the top of each bar represent geometric mean titers. Statistical analysis was performed using t-test with Welch’s correction. *P < 0.05; ****P < 0.0001.
Fig. 3
Fig. 3
Immune persistence in vaccinated BALB/c mice. (A) Pseudovirus neutralizing antibody responses monitored at different timepoints in mice vaccinated with different vaccine components as indicated. (B) Representative results of SΔTM-specific B cell ELISPOT. (C) SΔTM-specific memory B cells detected in each vaccinated group by ELISPOT 180 days post second immunization.
Fig. 4
Fig. 4
Immune responses in vaccinated cynomolgus monkeys. (A) Experiment schedule. Each group of Cynomolgus monkey (N-5) were immunized twice intramuscularly with different formulations of vaccines at Day 0 and Day 21. Formulation 1 contained 50 μg SΔTM, 500 μg Alum and 500 μg CpG; Formulation 2 contained 25 μg SΔTM, 500 μg Alum and 500 μg CpG; and Formulation 3 contained 50 μg SΔTM, 500 μg Alum and 250 μg CpG. 7 days before first immunization (Day −7), blood was collected from all individual monkeys to detect the baseline of both humoral and cellular immune responses. Blood was also collected at Day 14, Day 35, Day 105 and Day 189 to perform serological assays (SAs). In addition, PBMCs were isolated at Day 35 to detect cellular immune responses by intracellular cytokine staining (ICS) and cytometric bead array (CBA). (B) Binding antibody titers, (C) pseudovirus neutralizing antibody titers, and (D) live virus neutralizing antibody titers were evaluated at different timepoints as indicated. (E) Antigen specific T cell responses in vaccinated monkeys were determined by ICS at Day −7 (Pre-immune, as negative control) and Day 35 (Post-immune). (F) Th1-associated cytokines (IFN-γ, IL-2, TNF-α) and Th2-associated cytokines (IL-4, IL-5, IL-6) secreted from formulation 1 vaccine vaccinated monkeys were detected by CBA. (G) Pseudovirus neutralizing antibody responses were detected at Day −7, Day 14, Day 35, Day 105 and Day 189 to monitor the immune persistence. Dotted lines represent the limit of detection (LOD). Each dot represents an individual monkey. Numbers on the top of each bar represent geometric mean titers. Statistical analysis was performed using t-test with Welch’s correction. n.s.: no significance.
Fig. 5
Fig. 5
Immune responses and protective efficacy in vaccinated golden Syrian hamsters. (A) Experiment schedule. Hamsters from different groups (N = 10) were prime-boost immunized intramuscularly at Day 0 and Day 21. Adjuvant group vaccine contained 100 μg Alum and 100 μg CpG; RBD group contained 10 μg RBD, 100 μg Alum and 100 μg CpG, and SΔTM group contained 10 μg SΔTM, 100 μg Alum and 100 μg CpG. Blood was collected at Day 35 from each group to detect antibody responses. On Day 42, all hamsters were challenged intranasally with 105 TCID50 SARS-CoV-2. On Day 49, a subset of hamsters in each group (N = 6) was euthanized for detecting viral loads of lungs and nasal turbinates by qRT-PCR and evaluating lung histopathology by hematoxylin and eosin (H.E.) staining. (B) Binding antibody titers and (C) live virus neutralizing antibody titers of sera from each group of hamsters at Day 35. Viral loads of lungs (D) and nasal turbinates (E) determined by qRT-PCR. (F) Representative lung pathology scaled as mild, moderate or severe. Microscope images were taken at 100 × magnification. (G) Number of hamsters that displayed mild, moderate or severe lung pathology in each group. Each dot represents an individual hamster. Numbers on the top of each bar represent the value of geometric mean. Statistical analysis was performed using t-test with Welch’s correction. **P < 0.01; ****P < 0.0001.
Fig. 6
Fig. 6
Cross-reactivity with SARS-CoV-2 variants. (A) Pseudovirus neutralizing antibody responses against both wildtype and variant SARS-CoV-2 as indicated. In this experiment, B.1.617 variant contains three mutants, including L452R, D614G and E484Q. Samples were Day 35 sera of NHPs vaccinated with formulation 1, formulation 2 or formulation 3 vaccines. (B) Fold decrease in neutralization relative to wild type SARS-CoV-2. Samples were Day 35 sera of NHPs vaccinated with formulation 1 vaccine.

References

    1. Zhou P., Yang X.-L., Wang X.-G., Hu B., Zhang L., Zhang W., et al. A Pneumonia Outbreak Associated With A New Coronavirus Of Probable Bat Origin. Nature. 2020;579(7798):270–273. doi: 10.1038/s41586-020-2012-7.
    1. Zhu N.a., Zhang D., Wang W., Li X., Yang B.o., Song J., et al. A Novel Coronavirus From Patients With Pneumonia In China, 2019. N Engl J Med. 2020;382(8):727–733. doi: 10.1056/NEJMoa2001017.
    1. Mao H.H., Chao S. Advances In Vaccines. Adv Biochem Eng Biotechnol. 2020;171:155–188. doi: 10.1007/10_2019_107.
    1. Dai L., Gao G.F. Viral Targets For Vaccines Against Covid-19. Nat Rev Immunol. 2021;21(2):73–82. doi: 10.1038/s41577-020-00480-0.
    1. Shang J., Wan Y., Luo C., Ye G., Geng Q., Auerbach A., et al. Cell Entry Mechanisms Of Sars-Cov-2. Proc Natl Acad Sci U S A. 2020;117(21):11727–11734. doi: 10.1073/pnas.2003138117.
    1. Cao Y., Su B., Guo X., Sun W., Deng Y., Bao L., et al. Potent Neutralizing Antibodies Against Sars-Cov-2 Identified By High-Throughput Single-Cell Sequencing Of Convalescent Patients' B Cells. Cell. 2020;182(1):73–84.e16. doi: 10.1016/j.cell.2020.05.025.
    1. Voss W.N., Hou Y.J., Johnson N.V., Delidakis G., Kim J.E., Javanmardi K., et al. Prevalent, Protective, And Convergent Igg Recognition Of Sars-Cov-2 Non-Rbd Spike Epitopes. Science. 2021;372(6546):1108–1112. doi: 10.1126/science:abg5268.
    1. Ruckwardt T.J., Morabito K.M., Graham B.S. Immunological Lessons From Respiratory Syncytial Virus Vaccine Development. Immunity. 2019;51(3):429–442. doi: 10.1016/j.immuni.2019.08.007.
    1. Pallesen J., Wang N., Corbett K.S., Wrapp D., Kirchdoerfer R.N., Turner H.L., et al. Immunogenicity And Structures Of A Rationally Designed Prefusion Mers-Cov Spike Antigen. Proc Natl Acad Sci U S A. 2017;114(35):E7348–E7357. doi: 10.1073/pnas.1707304114.
    1. Marrack P., McKee A.S., Munks M.W. Towards An Understanding Of The Adjuvant Action Of Aluminium. Nat Rev Immunol. 2009;9(4):287–293. doi: 10.1038/nri2510.
    1. Lee S., Nguyen M.T. Recent Advances Of Vaccine Adjuvants For Infectious Diseases. Immune Netw. 2015;15:51–57. doi: 10.4110/In.2015.15.2.51.
    1. Krieg A.M. Therapeutic Potential Of Toll-Like Receptor 9 Activation. Nat Rev Drug Discov. 2006;5(6):471–484. doi: 10.1038/nrd2059.
    1. Scheiermann J., Klinman D.M. Clinical Evaluation Of Cpg Oligonucleotides As Adjuvants For Vaccines Targeting Infectious Diseases And Cancer. Vaccine. 2014;32(48):6377–6389. doi: 10.1016/j.vaccine.2014.06.065.
    1. Shirota H., Klinman D.M. Recent Progress Concerning Cpg Dna And Its Use As A Vaccine Adjuvant. Expert Rev Vaccines. 2014;13(2):299–312. doi: 10.1586/14760584.2014.863715.
    1. Hotez P.J., Corry D.B., Bottazzi M.E. Covid-19 Vaccine Design: The Janus Face Of Immune Enhancement. Nat Rev Immunol. 2020;20(6):347–348. doi: 10.1038/s41577-020-0323-4.
    1. Haynes B.F., Corey L., Fernandes P., Gilbert P.B., Hotez P.J., Rao S., et al. Prospects For A Safe Covid-19 Vaccine. Sci Transl Med. 2020;12(568) doi: 10.1126/scitranslmed.abe0948.
    1. Graham B.S. Rapid Covid-19 Vaccine Development. Science. 2020;368(6494):945–946. doi: 10.1126/science:abb8923.
    1. Zakhartchouk A.N., Sharon C., Satkunarajah M., Auperin T., Viswanathan S., Mutwiri G., et al. Immunogenicity Of A Receptor-Binding Domain Of Sars Coronavirus Spike Protein In Mice: Implications For A Subunit Vaccine. Vaccine. 2007;25(1):136–143. doi: 10.1016/j.vaccine.2006.06.084.
    1. Song Z., Bao L., Yu P., Qi F., Gong S., Wang J., et al. Sars-Cov-2 Causes A Systemically Multiple Organs Damages And Dissemination In Hamsters. Front Microbiol. 2020;11 doi: 10.3389/fmicb.2020.618891.
    1. Sia S.F., Yan L.-M., Chin A.W.H., Fung K., Choy K.-T., Wong A.Y.L., et al. Pathogenesis And Transmission Of Sars-Cov-2 In Golden Hamsters. Nature. 2020;583(7818):834–838. doi: 10.1038/s41586-020-2342-5.
    1. Van Der Lubbe, J. E. M. Et Al. Ad26.Cov2.S Protects Syrian Hamsters Against G614 Spike Variant Sars-Cov-2 And Does Not Enhance Respiratory Disease. Npj Vaccines6, 39, Doi:10.1038/S41541-021-00301-Y (2021).
    1. Imai, M. Et Al. Syrian Hamsters As A Small Animal Model For Sars-Cov-2 Infection And Countermeasure Development. Proc Natl Acad Sci U S A117, 16587-16595, Doi:10.1073/Pnas.2009799117 (2020).
    1. Starr T.N., Greaney A.J., Hilton S.K., Ellis D., Crawford K.H.D., Dingens A.S., et al. Deep Mutational Scanning Of Sars-Cov-2 Receptor Binding Domain Reveals Constraints On Folding And Ace2 Binding. Cell. 2020;182(5):1295–1310.e20. doi: 10.1016/j.cell.2020.08.012.
    1. Jangra S., et al. The E484k Mutation In The Sars-Cov-2 Spike Protein Reduces But Does Not Abolish Neutralizing Activity Of Human Convalescent And Post-Vaccination Sera. Medrxiv. 2021 doi: 10.1101/2021.01.26.21250543.
    1. Greaney A.J., Starr T.N., Gilchuk P., Zost S.J., Binshtein E., Loes A.N., et al. Complete Mapping Of Mutations To The Sars-Cov-2 Spike Receptor-Binding Domain That Escape Antibody Recognition. Cell Host Microbe. 2021;29(1):44–57.e9. doi: 10.1016/j.chom.2020.11.007.
    1. Garcia-Beltran W.F., Lam E.C., St. Denis K., Nitido A.D., Garcia Z.H., Hauser B.M., et al. Multiple Sars-Cov-2 Variants Escape Neutralization By Vaccine-Induced Humoral Immunity. Cell. 2021;184(9):2372–2383.e9. doi: 10.1016/j.cell.2021.03.013.
    1. Liang J.G., Su D., Song T.-Z., Zeng Y., Huang W., Wu J., et al. S-Trimer, A Covid-19 Subunit Vaccine Candidate, Induces Protective Immunity In Nonhuman Primates. Nat Commun. 2021;12(1) doi: 10.1038/s41467-021-21634-1.
    1. Richmond P., Hatchuel L., Dong M., Ma B., Hu B., Smolenov I., et al. Safety And Immunogenicity Of S-Trimer (Scb-2019), A Protein Subunit Vaccine Candidate For Covid-19 In Healthy Adults: A Phase 1, Randomised, Double-Blind. Placebo-Controlled Trial. Lancet. 2021;397(10275):682–694. doi: 10.1016/S0140-6736(21)00241-5.
    1. Kuo T.-Y., Lin M.-Y., Coffman R.L., Campbell J.D., Traquina P., Lin Y.-J., et al. Development Of Cpg-Adjuvanted Stable Prefusion Sars-Cov-2 Spike Antigen As A Subunit Vaccine Against Covid-19. Sci Rep. 2020;10(1) doi: 10.1038/s41598-020-77077-z.
    1. Lien C.-E., Lin Y.-J., Chen C., Lian W.-C., Kuo T.-Y., Campbell J.D., et al. Cpg-Adjuvanted Stable Prefusion Sars-Cov-2 Spike Protein Protected Hamsters From Sars-Cov-2 Challenge. Sci Rep. 2021;11(1) doi: 10.1038/s41598-021-88283-8.
    1. Halperin S.A., Van Nest G., Smith B., Abtahi S., Whiley H., Eiden J.J. A Phase I Study Of The Safety And Immunogenicity Of Recombinant Hepatitis B Surface Antigen Co-Administered With An Immunostimulatory Phosphorothioate Oligonucleotide Adjuvant. Vaccine. 2003;21(19-20):2461–2467. doi: 10.1016/S0264-410X(03)00045-8.
    1. COOPER C.L., DAVIS H.L., MORRIS M.L., EFLER S.M., ADHAMI ., KRIEG A.M., et al. Cpg 7909, An Immunostimulatory Tlr9 Agonist Oligodeoxynucleotide, As Adjuvant To Engerix-B Hbv Vaccine In Healthy Adults: A Double-Blind Phase I/Ii Study. J Clin Immunol. 2004;24(6):693–701. doi: 10.1007/s10875-004-6244-3.
    1. Arunachalam P.S., Walls A.C., Golden N., Atyeo C., Fischinger S., Li C., et al. Adjuvanting A Subunit Covid-19 Vaccine To Induce Protective Immunity. Nature. 2021;594(7862):253–258. doi: 10.1038/s41586-021-03530-2.
    1. Corbett K.S., Edwards D.K., Leist S.R., Abiona O.M., Boyoglu-Barnum S., Gillespie R.A., et al. Sars-Cov-2 Mrna Vaccine Design Enabled By Prototype Pathogen Preparedness. Nature. 2020;586(7830):567–571. doi: 10.1038/s41586-020-2622-0.
    1. Tian J.-H., Patel N., Haupt R., Zhou H., Weston S., Hammond H., et al. Sars-Cov-2 Spike Glycoprotein Vaccine Candidate Nvx-Cov2373 Immunogenicity In Baboons And Protection In Mice. Nat Commun. 2021;12(1) doi: 10.1038/s41467-020-20653-8.
    1. Zhang N.-N., Li X.-F., Deng Y.-Q., Zhao H., Huang Y.-J., Yang G., et al. A Thermostable Mrna Vaccine Against Covid-19. Cell. 2020;182(5):1271–1283.e16. doi: 10.1016/j.cell.2020.07.024.
    1. Dai L., Zheng T., Xu K., Han Y., Xu L., Huang E., et al. A Universal Design Of Betacoronavirus Vaccines Against Covid-19, Mers, And Sars. Cell. 2020;182(3):722–733.e11. doi: 10.1016/j.cell.2020.06.035.
    1. Walls A.C., Fiala B., Schäfer A., Wrenn S., Pham M.N., Murphy M., et al. Elicitation Of Potent Neutralizing Antibody Responses By Designed Protein Nanoparticle Vaccines For Sars-Cov-2. Cell. 2020;183(5):1367–1382.e17. doi: 10.1016/j.cell.2020.10.043.
    1. Abbas A.K., Burstein H.J., Bogen S.A. Determinants Of Helper T Cell-Dependent Antibody Production. Semin Immunol. 1993;5(6):441–447. doi: 10.1006/smim.1993.1050.
    1. Tan H.-X., Juno J.A., Lee W.S., Barber-Axthelm I., Kelly H.G., Wragg K.M., et al. Immunogenicity Of Prime-Boost Protein Subunit Vaccine Strategies Against Sars-Cov-2 In Mice And Macaques. Nat Commun. 2021;12(1) doi: 10.1038/s41467-021-21665-8.
    1. Yan H., Jiao H., Liu Q., Zhang Z., Xiong Q., Wang B.-J., et al. Ace2 Receptor Usage Reveals Variation In Susceptibility To Sars-Cov And Sars-Cov-2 Infection Among Bat Species. Nat Ecol Evol. 2021;5(5):600–608. doi: 10.1038/s41559-021-01407-1.

Source: PubMed

Patrocinadores y Colaboradores

Condiciones médicas

Intervenciones de drogas

3
Suscribir