key: cord-0770130-1bqcb184 authors: Tai, Wanbo; Chai, Benjie; Feng, Shengyong; Zhuang, Xinyu; Ma, Jun; Pang, Mujia; Pan, Lin; Yang, Zi; Tian, Mingyao; Cheng, Gong title: Development of a ferritin-based nanoparticle vaccine against the SARS-CoV-2 Omicron variant date: 2022-03-14 journal: bioRxiv DOI: 10.1101/2022.03.13.484123 sha: 35063b4d6073fc8246ffe0f957eba2c47648dd8f doc_id: 770130 cord_uid: 1bqcb184 A new SARS-CoV-2 variant named Omicron (B.1.1.529) discovered initially in South Africa has recently been proposed as a variant of concern (VOC) by the World Health Organization, because of its high transmissibility and resistance to current vaccines and therapeutic antibodies. Therefore, rapid development of vaccines against prevalent variants including Omicron is urgently needed for COVID-19 prevention. Here, we designed a self-assembling ferritin-based nanoparticle (FNP) vaccine against the SARS-CoV-2 Omicron variant. The purified Fc-RBDOmicron automatically formed a dimer depending on the nature of the Fc tag, thus assembling onto the nanoparticles by the Fc-protein A tag interaction (FNP-Fc-RBDOmicron). The results of hACE2-transgenic mice immunization showed that SARS-CoV-2 Omicron RBD-specific IgG titer induced by FNP-Fc-RBDOmicron was much higher than that by Fc-RBDOmicron. Consistently, the sera showed a higher neutralizing activity against SARS-CoV-2 Omicron BA.1 and BA.2 in the FNP-Fc-RBDOmicron immunized mice, indicating that immunization of a self-assembling ferritin-based nanoparticle vaccine offers a robust humoral immune response against Omicron variants. This study offers a great potential for the quick response of the emerging SARS-CoV-2 variants and affords versatility to develop universal vaccines against other emerging and reemerging coronaviruses in the future. The COVID-19 pandemic has had a devastating effect on global health, resulting in over 6 .0 million deaths worldwide. Continuous emergence of adaptive mutations of SARS-CoV-2 alters its pathogenicity and transmissibility, and renders its resistance to current vaccines and antiviral drugs. 1 A new variant named Omicron (B.1.1.529) discovered initially in South Africa has recently been proposed as a variant of concern (VOC) by the World Health Organization, because of its high transmissibility and resistance to current vaccines and therapeutic antibodies. 2 Therefore, rapid development of vaccines against prevalent variants including Omicron is urgently needed for COVID-19 prevention. A previous study developed a SARS-CoV-2 vaccine based on a virus-like nanoparticle (VLP) platform, in which sixty copies of a fusion protein including a receptor binding domain (RBD) with a lumazine synthase as the structural scaffold were self-assembled into a nanoparticle. 3 Immunization with this VLP-RBD conferred nearly complete protection to human ACE2 (hACE2)-transgenic mice against a high-dose SARS-CoV-2 infection. 4 Based on this framework, we further designed a self-assembling ferritin-based nanoparticle (FNP) vaccine against the SARS-CoV-2 Omicron variant. In this system, twenty-four copies of ferritin containing an N-terminal protein A tag form a structural scaffold (Fig. 1a) . 5 The RBD (residues 331aa-524aa) of the SARS-CoV-2 Omicron spike protein with an Fc tag in the C-terminus (Fc-RBDOmicron) served as an essential immunogen (Fig. 1a) . 6, 7 The purified Fc-RBDOmicron automatically formed a dimer depending on the nature of the Fc tag 8 , thus assembling onto the nanoparticles by the Fc-protein A tag interaction (Fig. 1a) . Based on this concept, the antigen of emerging SARS-CoV-2 variants can be rapidly assembled onto nanoparticles through a separating preparation and a subsequent Fc-Protein-A-tag-mediated conjugation. We expressed and purified the ferritin containing an N-terminal protein A tag in Escherichia coli (Fig. S1a) , and its purity was confirmed by SDS-PAGE (Fig. 1d ). The characterization of the self-assembling nanoparticles was analyzed by negative-stain electron microscopy (EM) (Fig. S1b ) and dynamic light scattering (DLS) (Fig. 1f) . The results indicated that the nanoparticles were spherical with a uniform diameter of 13.0±0.5 nm. We next expressed the Fc-RBDOmicron in the FreeStyle 293-F cells (Fig. S2b) . The binding affinity of Fc-RBDOmicron for hACE2 was evaluated by both enzyme linked immunosorbent assay (ELISA) (Fig. S3b ) and flow cytometry (Fig. S3c) with a dose-dependent manner. Incubation of Fc-RBDOmicron with human HEK293T cells expressing human ACE2 (hACE2/HEK293T) potently interrupted the cellular entry of SARS-CoV-2 Omicron pseudoviruses, which were generated by a SARS-CoV-2 spike-pseudotyped human immunodeficiency virus (HIV) system. 9 We next assembled the Fc-RBDOmicron onto the 24-meric FNP by mixing these two components at a 24:1 molar ratio. The Fc-RBDOmicron was capable of tightly interacting with the nanoparticles through the Fc-Protein A, measured by ELISA (Fig.1b) and surface plasmon resonance technology (SPR) (Fig.1c) . The protein complex was co-eluted and co-purified by gel filtration chromatography, and further evaluated by SDS-PAGE (Fig.1e) . The protein complex was designated as the FNP-Fc-RBDOmicron throughout this investigation. Furthermore, the FNP-Fc-RBDOmicron complex was evaluated by DLS, which confirmed the diameter of FNP-Fc-RBDOmicron being uniformly about 37.3±1.3 nm (Fig.1g ). Altogether, we generated self-assembling ferritin-based nanoparticles to develop a vaccine against the SARS-CoV-2 Omicron variant. We next evaluated the potency of the FNP-Fc-RBDOmicron to induce neutralizing antibody responses against SARS-CoV-2. To this end, we immunized hACE2-transgenic mice with either FNP-Fc-RBDOmicron or a sole Fc-RBDOmicron. The mice were further boosted with the same dose of immunogens at 2 weeks after the primary immunization. Mouse sera were collected on Day 14 after the second immunization and analyzed for antibody titers and potency to neutralize SARS-CoV-2. The SARS-CoV-2 Omicron RBD-specific IgG titer induced by FNP-Fc-RBDOmicron was 4 times higher than that by Fc-RBDOmicron (Fig. 1h) . Subsequently, the neutralizing potency in the sera of immunized animals was assessed by HIV pseudoviruses with SARS-CoV-2 Omicron BA.1 and BA.2 spikes. The sera showed a higher neutralizing activity in the FNP-Fc-RBDOmicron immunized mice than that of a sole Fc-RBDOmicron immunization (Fig.1i, 1j) . To substantiate the SARS-CoV-2-neutralizing mechanism of vaccine-induced antibodies, we examined the interactions between the SARS-CoV-2 RBD and hACE2 in the presence of the vaccinated mouse sera by flow cytometry. Although the binding of RBDOmicron to hACE2/HKE293T cells was inhibited by either FNP-Fc-RBDOmicron or Fc-RBDOmicron serum effectively in a dose-dependent manner, the former was more potent (Fig. 1k, 1l) . We next examined whether the antibodies induced (b-c) Interaction between Fc-RBDOmicron and FNP was detected by ELISA (b) and by SPR (c). An equal amount of ovalbumin served as a control. Binding affinity was detected by an Johns Hopkins Coronavirus Resource Center SARS-CoV-2 variants and vaccination Tailoring lumazine synthase assemblies for bionanotechnology Novel virus-like nanoparticle vaccine effectively protects animal model from SARS-CoV-2 infection Two-component spike nanoparticle vaccine protects macaques from SARS-CoV-2infection Characterization of the receptor-binding domain (RBD) of 2019 novel coronavirus: implication for development of RBD protein as a viral attachment inhibitor and vaccine A novel receptor-binding domain (RBD)-based mRNA vaccine against SARS-CoV-2 Viral targets for vaccines against COVID-19 Human Immunodeficiency Viruses Pseudotyped with SARS-CoV-2 Spike Proteins Infect a Broad Spectrum of Human Cell Lines through Multiple Entry Mechanisms Isolation of potent SARS-CoV-2 neutralizing antibodies and protection from disease in a small animal model HRP-labeled anti-His tag antibody. The data are presented as mean±S.E.M. (n=7). (b) Statistical significance was calculated via ordinary unpaired parametric t-test Size distribution of the FNP complex with or without Fc-RBDOmicorn was detected by DLS. (h-j) Measurement of IgG and neutralizing antibodies induced in immunized mice. The mice were immunized via intramuscular (i.m.) prime and boost at 2 weeks (10 μg per mouse, n=5) Inhibition potency of immunized sera on SARS-CoV-2 RBD-hACE2 binding in hACE2/HEK293T cells. (k) The inhibition potency was evaluated by flow cytometry. Inhibition percentage (%) was calculated by a relative fluorescence intensity Representative images of the binding inhibition by the sera (1:40) from mice immunized with PBS (left panel), Fc-RBDOmicron (middle panel), or FNP-Fc-RBDOmicron (right panel). The violet lines represented median fluorescence intensity (MFI) values. The binding between Fc-RBDOmicron and hACE2 is shown in dark red lines This work was supported by the Emergency Key Program of Guangzhou Laboratory The authors declare no competing financial interests.