key: cord-102364-t5bt2eb4
authors: Yao, Dehui; Lao, Fang; Zhang, Zeyi; Liu, Yan; Cheng, Jianwei; Ding, Fengjiao; Wang, Xiaofei; Xi, Lun; Wang, Chuang; Yan, Xichong; Zhang, Rongkun; Ouyang, Fangxing; Ding, Hui; Ke, Tianyi
title: Human H-ferritin presenting RBM of spike glycoprotein as potential vaccine of SARS-CoV-2
date: 2020-06-08
journal: bioRxiv
DOI: 10.1101/2020.05.25.115618
sha: 
doc_id: 102364
cord_uid: t5bt2eb4

The outbreak of COVID-19 has so far inflicted millions of people all around the world and will have a long lasting effect on every aspect of everyone’s life. Yet there is no effective approved treatment for the disease. In an effort of utilizing human ferritin as nanoplatform for drug delivery, we engineered a fusion protein by presenting receptor-binding motif (RBM) of SARS-CoV-2 virus spike glycoprotein on the N-terminus of ferritin subunits. The designed fusion protein with a cage-like structure, similar to that of corona virus, is a potential anti-SARS-CoV-2 vaccine. We hereby show the construction, preparation, and characterization of the fusion protein RBM-HFtn. Our initial affinity study confirmed its biological activity towards ACE2 receptor which suggests its mode of action against SARS-CoV-2 could be either through vaccine therapy or blocking the cellular entry of virus as antagonist of ACE2 receptor.

The pandemic coronavirus disease (COVID-19) by SARS-CoV-2 virus has caused tremendous suffering to tens of millions of people around the world. Even though quite a few clinical studies involving different approaches are undergoing for the treatment of the disease, there is no effective cure yet up to date. Vaccines therefore is urgently needed for the preventing further spread of the COVID-19.

The corona virus, SARS-CoV-2, consists of a large RNA genome, four structural proteins, 16 nonstructural proteins, and some accessory proteins. The four structural proteins include spike, envelope, membrane, and nucleocapsid proteins, of which the spike glycoprotein is of particular interest for it is a popular vaccine target for corona virus.

Antibodies targeting the spike glycoprotein of SARS-CoV and MERS-CoV, especially its receptor-binding domain (RBD), was found to efficiently neutralize virus infection [1, 2] .

Antibodies from SARS-CoV and SARS-CoV-2 patients however showed limited cross neutralization, in spite of the high sequence similarity between two viruses [3] . Other vaccine approaches include the production of live attenuated whole virion vaccines, inactivated whole virion vaccines, recombinant protein vaccines, and mRNA based vaccines [4] . SARS-CoV-2 was found to enter cells through binding of the host cellular receptor angiotensin-converting enzyme 2 (ACE2) via its spike glycoprotein soon after its outbreak in China [3] . Later the cryo-EM revealed the structure of SARS-CoV-2 S-RBD complexed with its receptor human ACE2 [5] [6] [7] [8] . The S1 subunit of spike glycoprotein undergoes a hinge-like conformational transition from "down" conformation to "up" conformation before binding to ACE2 [8] . The receptor-binding motif (RBM, 72 amino acid in total) of spike glycoprotein in close contact of ACE2 receptor was identified for its sequence between 437 -508 [9] , and this RBM was consistent with the identified S-RBD [8] . This conformational transition state has become the target for antibody-mediated neutralization, and the atomic-level understanding of this transitional state and the identification of S-RBM would facilitate the vaccine design and development against SARS-CoV-2.

Ferritin is a 24-mer protein assembly consisting of heavy chain (21 kD) and light chain (19 kD) . It has a cage-like structure in a way similar to SARS-CoV-2. Because of its unique structure, ferritin is a promising nanoplatform for antigen presentation and immune stimulation [10] [11] [12] [13] . Its spherical architecture has an outer diameter of 12 nm, suitable for rapid tissue penetration and draining to lymph node [14] . In this work, we engineered a human ferritin heavy chain (HFtn) by fusing and presenting the RBM of its spike glycoprotein as potential vaccine of SARS-CoV-2.

Human ACE2(his tag) (10108-H08H) ，ACE2 rabbit Antibody (10108-RP01)， goat anti-Rabbit/HRP secondary antibody(SSA004, and SARS-CoV-2 (2019-nCoV) and RBD of spike glycoprotein (mFC tag)(40592-V05H) were purchased from Sino Biological (Beijing, China). 

The refolding solution was concentrated and buffer exchanged to 25 mM Tris-HCl buffer (pH 8.0) with a Millipore lab scale tangential flow filter (TFF) system. After buffer exchange, RBM-HFtn was found to have a typical soluble ferritin structure. The fusion protein with its right space structure was further purified by using anion exchange chromatography (flow through mode), followed with a size-exclusion chromatography to remove aggregates and other low molecule impurities.

The physico-chemical properties of fusion RBM-HFtn were analyzed by SEC-HPLC overnight. After blocking with 5% BSA, ACE2 protein (concentration 0.5 μg/ml) was allowed to bind to the surface coated proteins for 2 h at 37C, followed by washing with washing buffer three times. Anti-ACE2 antibody (dilution 1:1000) was incubated for 1.5 hours at 37C followed by washing for three times. Secondary antibody/HRP (1:5000 dilution) in 5% BSA was incubated 37C for 0.5 h followed by washing for three times. The TMB substrate was added and incubated for 30 min, and then the absorbance was read at 650 nm wavelength.

Ferritin heavy chain protein has found many biological applications in nanomedicines and molecular diagnostics [15] [16] [17] . Lately, a few constructs based on ferritin have been developed as antivirus and anticancer vaccines [10, 11, 13] . Because ferritin heavy chain protein is derived from a natural occurring protein in human, itself has low immunogenicity.

Its 24-mer assembly cage-like structure has four different symmetries, six 4-fold axes, eight 3-fold axes, twelve 2-fold axes, and twenty-four C3-C4 interfaces. By presenting a fusion protein at the 3-fold axes, ferritin was able to display eight trimers of fused protein, resulting in enhanced immunogenicity of protein on display [18] . In this work we engineered a human ferritin heavy chain fused with the RBM of spike glycoprotein of SARS-CoV-2 at its Nterminus with (GGGGS)3 short peptide linker ( Figure 1A ). We chose a short polypeptide sequence (72 amino acid) of spike glycoprotein RBM (S-RBM) based on latest 3-D cryo-EM studies of spike glycoprotein-ACE2 complex [8, 9] . Because this sequence doesn't appear to have a stable 3-D structure, we engineered it at the N-terminus, so that the fused S-RBM subunits are distant from each other and won't affect the formation 24-mer assembly. For the same reason, we chose a minimal size of RBD so that it is properly displayed on the surface of ferritin. In spite of those considerations, the RBM-HFtn fusion protein was found mainly in the inclusion bodies, instead of in soluble form.

Hypothetically, the designed RBM-HFtn fusion protein may involve in two different pathways against virus infection. First and most importantly, the RBM-HFtn may act as vaccine against SARS-CoV-2, and antibodies responsive to spike glycoprotein RBM may subsequently neutralize SARS-CoV-2, followed by removal of virus by immune system ( Figure 1B, upper pathway) . Two antibodies found in COVID-19 patients were shown to block the binding between virus S-protein RBD and cellular receptor ACE2 [19] . Their therapeutic effect was validated in mouse model by reducing virus titer in infected lungs [19] . Those findings supported our hypothesis that by properly presenting S-protein RBD/RBM, the designed RBM-HFtn has great potential as an anti-SARS-CoV-2 vaccine, thus inducing the production of antivirus antibodies. The second pathway, more obvious and direct, is to block the cellular entry of SARS-CoV-2 by preoccupy the ACE2 receptor with RBM-HFtn and suppress the virus proliferation ( Figure 1B , lower pathway). The first pathway is more effective for prevention of virus infection. The second pathway is valuable for treatment after virus infection as antagonist of ACE2 receptor.

The expressed RBM-HFtn was found in the form of inclusion bodies in the bacteria lysis precipitates (Supplementary Figure S2) 

RBD of Spike glycoprotein (mFc tag) and RBM-HFtn of varied concentration in 100 L was incubated in MaxiSorp plate and ACE2 was allowed to bind to the surface coated proteins.

Their binding was detected by ACE2 antibody followed by its secondary antibody. In this indirect ELISA, the maximum binding intensity correlates with the binding site on surface.

As indicated by the results in Figure 3 , S-RBD showed higher plateau than RBM-HFtn, suggesting more binding sites are available in S-RBD coated wells than in RBM-HFtn coated wells. The EC50s of the binding between ACE2 and S-RBD, RBM-HFtn were estimated to be 60.66 nM and 23.36 nM respectively. Apparently RBM-HFtn has higher binding affinity than S-protein in binding to their same ACE2 receptor. The apparent higher binding affinity may attribute to the cage-like structure of RBM-HFtn, which presents multiple copies (24 copies) of RBM on the surface. This result suggested that the RBM is properly presented on surface of heavy chain human ferritin and is recognizable by the ACE2 receptor. Its potential as SARS-CoV-2 vaccine and as antagonist of ACE2 receptor is being further studied in animal experiments.

The receptor-binding motif (RBM) of SARS-CoV-2, a 72-amino acid polypeptide, was fused with N-terminus of human heavy chain ferritin (HFtn) through a proper linker. The constructed RBM-HFtn was found in inclusion bodies of bacterial lysis and was able to refold by gradual dialysis. The purified RBM-HFtn was found to be in good purity, expected size and morphology. Its biological activity towards ACE2 receptor was confirmed by ELISA and the fusion protein RBM-HFtn bodes well as potential vaccine and therapeutics against SARS-CoV-2 as ACE2 receptor antagonist.

T. Ke holds ownership interest (including patents) in Kunshan Xinyunda Biotech Co., Ltd.

No potential conflicts of interests were disclosed by the other authors. 

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