A single amino acid substitution (R441A) in the receptor-binding domain of SARS coronavirus spike protein disrupts the antigenic structure and binding activity

The spike (S) protein of severe acute respiratory syndrome coronavirus (SARS-CoV) has two major functions: interacting with the receptor to mediate virus entry and inducing protective immunity. Coincidently, the receptor-binding domain (RBD, residues 318-510) of SAR-CoV S protein is a major antigenic site to induce neutralizing antibodies. Here, we used RBD-Fc, a fusion protein containing the RBD and human IgG1 Fc, as a model in the studies and found that a single amino acid substitution in the RBD (R441A) could abolish the immunogenicity of RBD to induce neutralizing antibodies in immunized mice and rabbits. With a panel of anti-RBD mAbs as probes, we observed that R441A substitution was able to disrupt the majority of neutralizing epitopes in the RBD, suggesting that this residue is critical for the antigenic structure responsible for inducing protective immune responses. We also demonstrated that the RBD-Fc bearing R441A mutation could not bind to soluble and cell-associated angiotensin-converting enzyme 2 (ACE2), the functional receptor for SARS-CoV and failed to block S protein-mediated pseudovirus entry, indicating that this point mutation also disrupted the receptor-binding motif (RBM) in the RBD. Taken together, these data provide direct evidence to show that a single amino acid residue at key position in the RBD can determine the major function of SARS-CoV S protein and imply for designing SARS vaccines and therapeutics.     

Receptor-binding domain of severe acute respiratory syndrome coronavirus spike protein contains multiple conformation-dependent epitopes that induce highly potent neutralizing antibodies

The spike (S) protein of severe acute respiratory syndrome associated coronavirus (SARS-CoV) is a major antigenic determinant capable of inducing protective immunity. Recently, a small fragment on the SARS-CoV S protein (residues 318-510) was characterized as a minimal receptor-binding domain (RBD), which mediates virus binding to angiotensin-converting enzyme 2, the functional receptor on susceptible cells. In this study, we demonstrated that a fusion protein containing RBD linked to human IgG1 Fc fragment (designated RBD-Fc) induced high titer of RBD-specific Abs in the immunized mice. The mouse antisera effectively neutralized infection by both SARS-CoV and SARS pseudovirus with mean 50% neutralization titers of 1/15,360 and 1/24,737, respectively. The neutralization determinants on the RBD of S protein were characterized by a panel of 27 mAbs isolated from the immunized mice. Six groups of conformation-dependent epitopes, designated as Conf I-VI, and two adjacent linear epitopes were identified by ELISA and binding competition assays. The Conf IV and Conf V mAbs significantly blocked RBD-Fc binding to angiotensin-converting enzyme 2, suggesting that their epitopes overlap with the receptor-binding sites in the S protein. Most of the mAbs (23 of 25) that recognized the conformational epitopes possessed potent neutralizing activities against SARS pseudovirus with 50% neutralizing dose ranging from 0.005 to 6.569 microg/ml. Therefore, the RBD of SARS S protein contains multiple conformational epitopes capable of inducing potent neutralizing Ab responses, and is an important target site for developing vaccines and immunotherapeutics.     

Antigenicity and immunogenicity of SARS-CoV S protein receptor-binding domain stably expressed in CHO cells

The receptor-binding domain (RBD) of SARS coronavirus (SARS-CoV) spike (S) protein contains multiple conformation-dependent epitopes that induce neutralizing antibody responses. Here we used CHO-K1 cells to establish a cell line for stable expression of a 193-mer (residues 318-510) RBD (RBD193-CHO) and determined its antigenicity and immunogenicity. We found that RBD193-CHO reacted strongly with a panel of six monoclonal antibodies recognizing various conformational and linear epitopes in RBD, suggesting that this recombinant protein maintains intact conformation and good antigenicity. Immunization of mice with RBD193-CHO resulted in induction of high titers of RBD-specific neutralizing antibodies and potent IL-4-expressing T cell responses. RBD193-CHO induced immunity that protected a majority of the vaccinated mice from SARS-CoV challenge. These results suggest that the recombinant RBD produced in an established stable cell line maintains strong immunogenicity with high potential for use as an effective and economic subunit SARS vaccine.     

Molecular and biological characterization of human monoclonal antibodies binding to the spike and nucleocapsid proteins of severe acute respiratory syndrome coronavirus

Human monoclonal antibodies (MAbs) were selected from semisynthetic antibody phage display libraries by using whole irradiated severe acute respiratory syndrome (SARS) coronavirus (CoV) virions as target. We identified eight human MAbs binding to virus and infected cells, six of which could be mapped to two SARS-CoV structural proteins: the nucleocapsid (N) and spike (S) proteins. Two MAbs reacted with N protein. One of the N protein MAbs recognized a linear epitope conserved between all published human and animal SARS-CoV isolates, and the other bound to a nonlinear N epitope. These two N MAbs did not compete for binding to SARS-CoV. Four MAbs reacted with the S glycoprotein, and three of these MAbs neutralized SARS-CoV in vitro. All three neutralizing anti-S MAbs bound a recombinant S1 fragment comprising residues 318 to 510, a region previously identified as the SARS-CoV S receptor binding domain; the nonneutralizing MAb did not. Two strongly neutralizing anti-S1 MAbs blocked the binding of a recombinant S fragment (residues 1 to 565) to SARS-CoV-susceptible Vero cells completely, whereas a poorly neutralizing S1 MAb blocked binding only partially. The MAb ability to block S1-receptor binding and the level of neutralization of the two strongly neutralizing S1 MAbs correlated with the binding affinity to the S1 domain. Finally, epitope mapping, using recombinant S fragments (residues 318 to 510) containing naturally occurring mutations, revealed the importance of residue N479 for the binding of the most potent neutralizing MAb, CR3014. The complete set of SARS-CoV MAbs described here may be useful for diagnosis, chemoprophylaxis, and therapy of SARS-CoV infection and disease.     

SARS 冠状病毒 S 蛋白受体结合结构域的表达及其表位作图

严重急性呼吸综合征 (SARS) 是一种新出现的人类传染病,该病的病原是 SARS 冠状病毒 (SARS-CoV). S 蛋白是 SARS 冠状病毒的一种主要结构蛋白,它在病毒与宿主细胞受体结合以及诱导机体产生中和抗体中起重要作用 . 研究表明 S 蛋白与受体结合的核心区域为第 318 ~ 510 氨基酸残基的片段 . 首先克隆并用 pGEX-6p-1 载体融合表达了该受体结合结构域,并且通过蛋白质印迹分析表明,该受体结合结构域融合蛋白能被 SARS 康复患者血清和 S 蛋白特异的单克隆抗体所识别 . 为了对这一区域进行抗原表位作图,进一步设计了一套 23 个覆盖受体结合结构域的长 16 个氨基酸残基的部分重叠短肽,并进行了 GST 融合表达 . 用免疫动物血清和单克隆抗体 D3D1 对 23 个融合蛋白进行蛋白质印迹和 ELISA 免疫反应性分析,结果鉴定出两个抗原表位 SRBD3(F334PSVYAWERKKISNCV349) 和表位 D3D1 (K447LRPFERDI455). 其结果对进一步分析 S 蛋白结构与功能以及诊断试剂和基因工程疫苗的研究有一定意义 .     

Receptor-binding domain of SARS-CoV spike protein induces highly potent neutralizing antibodies: implication for developing subunit vaccine

The spike (S) protein of severe acute respiratory syndrome (SARS) coronavirus (CoV), a type I transmembrane envelope glycoprotein, consists of S1 and S2 domains responsible for virus binding and fusion, respectively. The S1 contains a receptor-binding domain (RBD) that can specifically bind to angiotensin-converting enzyme 2 (ACE2), the receptor on target cells. Here we show that a recombinant fusion protein (designated RBD-Fc) containing 193-amino acid RBD (residues 318-510) and a human IgG1 Fc fragment can induce highly potent antibody responses in the immunized rabbits. The antibodies recognized RBD on S1 domain and completely inhibited SARS-CoV infection at a serum dilution of 1:10,240. Rabbit antisera effectively blocked binding of S1, which contains RBD, to ACE2. This suggests that RBD can induce highly potent neutralizing antibody responses and has potential to be developed as an effective and safe subunit vaccine for prevention of SARS.     

Identification of an antigenic determinant on the S2 domain of the severe acute respiratory syndrome coronavirus spike glycoprotein capable of inducing neutralizing antibodies

Severe acute respiratory syndrome (SARS) is a life-threatening disease caused by a newly identified coronavirus (CoV), SARS-CoV. The spike (S) glycoprotein of CoV is the major structural protein responsible for induction of host immune response and virus neutralization by antibodies. Hence, knowledge of neutralization determinants on the S protein is helpful for designing protective vaccines. To analyze the antigenic structure of the SARS-CoV S2 domain, the carboxyl-terminal half of the S protein, we first used sera from convalescent SARS patients to test the antigenicity of 12 overlapping fragments spanning the entire S2 and identified two antigenic determinants (Leu 803 to Ala 828 and Pro 1061 to Ser 1093). To determine whether neutralizing antibodies can be elicited by these two determinants, we immunized animals and found that both of them could induce the S2-specific antisera. In some animals, however, only one determinant (Leu 803 to Ala 828) was able to induce the antisera with the binding ability to the native S protein and the neutralizing activity to the SARS-CoV pseudovirus. This determinant is highly conserved across different SARS-CoV isolates. Identification of a conserved antigenic determinant on the S2 domain of the SARS-CoV S protein, which has the potential for inducing neutralizing antibodies, has implications in the development of effective vaccines against SARS-CoV.     

Cross-neutralization of human and palm civet severe acute respiratory syndrome coronaviruses by antibodies targeting the receptor-binding domain of spike protein

The spike (S) protein of severe acute respiratory syndrome coronavirus (SARS-CoV) is considered as a protective Ag for vaccine design. We previously demonstrated that the receptor-binding domain (RBD) of S protein contains multiple conformational epitopes (Conf I-VI) that confer the major target of neutralizing Abs. Here we show that the recombinant RBDs derived from the S protein sequences of Tor2, GD03, and SZ3, the representative strains of human 2002-2003 and 2003-2004 SARS-CoV and palm civet SARS-CoV, respectively, induce in the immunized mice and rabbits high titers of cross-neutralizing Abs against pseudoviruses expressing S proteins of Tor2, GD03, and SZ3. We also demonstrate that the Tor2-RBD induced-Conf I-VI mAbs can potently neutralize both human SARS-CoV strains, Tor2 and GD03. However, only the Conf IV-VI, but not Conf I-III mAbs, neutralize civet SARS-CoV strain SZ3. All these mAbs reacted significantly with each of the three RBD variants (Tor2-RBD, GD03-RBD, and SZ3-RBD) that differ at several amino acids. Regardless, the Conf I-IV and VI epitopes were completely disrupted by single-point mutation of the conserved residues in the RBD (e.g., D429A, R441A, or D454A) and the Conf III epitope was significantly affected by E452A or D463A substitution. Interestingly, the Conf V epitope, which may overlap the receptor-binding motif and induce most potent neutralizing Abs, was conserved in these mutants. These data suggest that the major neutralizing epitopes of SARS-CoV have been apparently maintained during cross-species transmission, and that RBD-based vaccines may induce broad protection against both human and animal SARS-CoV variants.     

Identification and antigenic epitope mapping of immunodominant region amino residues 510 to 672 on the spike protein of the severe acute respiratory syndrome coronavirus

The severe acute respiratory syndrome (SARS) is a newly emerging human infectious disease caused by the severe acute respiratory syndrome coronavirus (SARS-CoV). The spike (S) protein of SARS-CoV is a major virion structural protein. It plays an important role in the interaction with receptors and neutralizing antibodies. In this study, the S1 domain of the spike protein and three truncated fragments were expressed by fusion with GST in a pGEX-6p-1 vector. Western blot results demonstrated that the 510-672 fragment of the S1 domain is a linear epitope dominant region. To map the antigenic epitope of this linear epitope dominant region, a set of 16 partially overlapping fragments spanning the fragment were fused with GST and expressed. Four antigenic epitopes S1C3 (539-559), S1C4 (548-567), S1C7/8 (583-606), and S1C10/11 (607-630) were identified. Immunization of mice with each of the four antigenic epitope-fused proteins revealed that all four proteins could elicit spike protein specific antisera. All of them were able to bind to the surface domain of the whole spike protein expressed by recombinant baculovirus in insect cells. Identification of antigenic epitopes of the spike protein of SARS-CoV may provide the basis for the development of immunity-based prophylactic, therapeutic, and diagnostic clinical techniques for the severe acute respiratory syndrome.     

SARS-CoV N蛋白与人冠状病毒HCoV-OC43和HCoV-229E的交叉反应表位及特异表位的确定

为确定SARS-CoV N蛋白的特异抗原表位,对3种人冠状病毒SARS-CoV、HCoV-OC43和HCoV-229E N蛋白之间的交叉免疫反应进行了系统研究。构建了分别表达SARS-CoV、HCoV-OC43和HCoV-229E N蛋白的重组痘苗病毒,并制备了相应的小鼠免疫血清。用间接免疫荧光方法,检测了3种N蛋白的表达及其与3种冠状病毒免疫动物血清和SARS病人恢复期血清之间的反应。与此同时,用Western blot方法分析了原核表达的39个不同区段的SARS-CoV N蛋白与3种冠状病毒动物免疫血清和SARS病人恢复期血清之间的交叉反应性。免疫荧光检测结果表明,SARS-CoV、HCoV-OC43和HCoV-229E3种病毒的N蛋白在重组痘苗病毒感染的HeLa细胞中均可以特异表达;3种N蛋白之间存在明显交叉免疫反应。Western blot结果显示,SARS-CoV N蛋白的表位主要位于30~60aa、170~184aa、301~320aa和360~422aa;与HCoV-OC43的交叉反应表位主要位于30~60aa、90~120aa、204~214aa和320~360aa;与HCoV-229E的交叉反应表位主要位于30~60aa、150~160aa和301~360aa。含SARS-CoV N蛋白特异表位的重组肽N155b(60~214aa)和N185(30~214aa)只与SARS病人恢复期血清和灭活SARS-CoV免疫小鼠的血清反应,而不与灭活HCoV-OC43和HCoV-229E免疫的山羊血清产生交叉反应。上述结果为使用SARS-CoV N蛋白抗原进行特异诊断试剂的研究,提供了重要的实验依据。     

SARS-CoV S蛋白受体结合区的重组表达及免疫原性分析

为了表达SARS-CoV的S蛋白的受体结合区并对其免疫原性进行分析,用PCR方法扩增S蛋白的受体结合区基因片段,克隆至原核表达质粒pET-F32a＋并在大肠杆菌中表达,应用Western—blot鉴定表达的目的蛋白,而后以该蛋白作为诊断抗原包被酶联卡反来检测20份SARS病人血清和28份健康人血清,结果原核表达的S蛋白能够和所用的SARS病人血清反应。这提示表达的S重组蛋白具有良好的抗原性。将变性纯化的重组蛋白和复性蛋白分别皮下免疫小鼠,第三次免疫一周后收集抗血清,用ELISA测定抗体和同时测定中和抗体活性。用变性的抗原免疫的小鼠血清均无中和活性;而用复性的蛋白免疫的小鼠产生了中和抗体。实验表明,S蛋白受体结合区无线性中和表位,中和抗体的产生是由构象表位诱导的。提示该蛋白有可能应用于亚单位疫苗的研究。     

Identification of immunodominant sites on the spike protein of severe acute respiratory syndrome (SARS) coronavirus: implication for developing SARS diagnostics and vaccines

The spike (S) protein of severe acute respiratory syndrome (SARS) coronavirus (SARS-CoV) is not only responsible for receptor binding and virus fusion, but also a major Ag among the SARS-CoV proteins that induces protective Ab responses. In this study, we showed that the S protein of SARS-CoV is highly immunogenic during infection and immunizations, and contains five linear immunodominant sites (sites I to V) as determined by Pepscan analysis with a set of synthetic peptides overlapping the entire S protein sequence against the convalescent sera from SARS patients and antisera from small animals immunized with inactivated SARS-CoV. Site IV located in the middle region of the S protein (residues 528-635) is a major immunodominant epitope. The synthetic peptide S(603-634), which overlaps the site IV sequence reacted with all the convalescent sera from 42 SARS patient, but none of the 30 serum samples from healthy blood donors, suggesting its potential application as an Ag for developing SARS diagnostics. This study also provides information useful for designing SARS vaccines and understanding the SARS pathogenesis.     

Identification of two antigenic epitopes on SARS-CoV spike protein

The spike (S) protein of severe acute respiratory syndrome-coronavirus (SARS-CoV) is a major virion structural protein. It plays an important role in interaction with receptor and inducing neutralizing antibodies. In the study, six tentative antigenic epitopes (S1 S2 S3 S4 S5 S6) of the spike protein of SARS-CoV were predicted by bio-informatics analysis, and a multi-epitope chimeric gene of S1-S2-S3-S4-S5-S6 was synthesized and fused to downstream GST gene in pGEX-6p-1. The Western blotting demonstrated that SARS patient convalescent serum could recognize the recombinant fusion protein. A number of monoclonal antibodies were developed against the fusion protein. In further, the six predicted epitope genes were individually fused to GST of pGEX-6p-1 and expressed in Escherichia coli BL21, respectively. Among six fusion peptides, S5 reacted with monoclonal antibody D3C5 and S2 reacted with monoclonal antibody D3D1 against spike protein of SARS-CoV. The epitopes recognized by monoclonal antibodies D3C5 and D3D1 are linear, and correspond to 447-458 and 789-799 amino acids of spike protein of SARS-CoV, respectively. Identification of antigenic epitope of spike protein of SARS-CoV could provide the basis for the development of immunity-based prophylactic, therapeutic, and diagnostic techniques for the control of severe acute respiratory syndrome.     

Neutralizing epitopes of the SARS-CoV S-protein cluster independent of repertoire,antigen structure or mAb technology

Neutralizing antibody responses to the surface glycoproteins of enveloped viruses play an important role in immunity. Many of these glycoproteins, including the severe acute respiratory syndrome-coronavirus (SARS-CoV) spike (S) protein form trimeric units in the membrane of the native virion. There is substantial experimental and pre-clinical evidence showing that the S protein is a promising lead for vaccines and therapeutics. Previously we generated a panel of monoclonal antibodies (mAbs) to whole inactivated SARS-CoV which neutralize the virus in vitro.^1,2 Here, we define their specificity and affinity, map several of their epitopes and lastly characterise chimeric versions of them. Our data show that the neutralizing mAbs bind to the angiotensin-converting enzyme 2 (ACE2) receptor-binding domain (RBD) of the SARS S protein. Three of the chimeric mAbs retain their binding specificity while one conformational mAb, F26G19, lost its ability to bind the S protein despite high level expression. The affinity for recombinant S is maintained in all of the functional chimeric versions of the parental mAbs. Both parental mAb F26G18 and the chimeric version neutralize the TOR2 strain of SARS-CoV with essentially identical titres (2.07 and 2.47 nM, respectively). Lastly, a comparison with other neutralizing mAbs to SARS-CoV clearly shows that the dominance of a 33 amino acid residue loop of the SARS-CoV RBD is independent of repertoire, species, quaternary structure, and importantly, the technology used to derive the mAbs. In cases like this, the dominance of a compact RBD antigenic domain and the central role of the S protein in pathogenesis may inherently create immunoselection pressure on viruses to evolve more complex evasion strategies or die out of a host species. The apparent simplicity of the mechanism of SARS-CoV neutralization is in stark contrast to the complexity shown by other enveloped viruses.Key words: SARS coronavirus, monoclonal antibody, neutralizing, epitope, immunochemistry     

A single amino acid substitution in the S1 and S2 Spike protein domains determines the neutralization escape phenotype of SARS-CoV

In response to SARS-CoV infection, neutralizing antibodies are generated against the Spike (S) protein. Determination of the active regions that allow viral escape from neutralization would enable the use of these antibodies for future passive immunotherapy. We immunized mice with UV-inactivated SARS-CoV to generate three anti-S monoclonal antibodies, and established several neutralization escape mutants with S protein. We identified several amino acid substitutions, including Y442F and V601G in the S1 domain and D757N and A834V in the S2 region. In the presence of each neutralizing antibody, double mutants with substitutions in both domains exhibited a greater growth advantage than those with only one substitution. Importantly, combining two monoclonal antibodies that target different epitopes effected almost complete suppression of wild type virus replication. Thus, for effective passive immunotherapy, it is important to use neutralizing antibodies that recognize both the S1 and S2 regions.     

Natural mutations in the receptor binding domain of spike glycoprotein determine the reactivity of cross-neutralization between palm civet coronavirus and severe acute respiratory syndrome coronavirus

The severe acute respiratory syndrome (SARS) outbreak of 2002 and 2003 occurred as a result of zoonotic transmission. Coronavirus (CoV) found in naturally infected palm civet (civet-CoV) represents the closest genetic relative to SARS-CoV, but the degree and the determinants of cross-neutralization among these viruses remain to be investigated. Studies indicate that the receptor binding domain (RBD) of the SARS-CoV spike (S) glycoprotein contains major determinants for viral entry and neutralization. We aim to characterize the impact of natural mutations within the RBDs of civet-CoVs on viral entry and cross-neutralization. In this study, the S glycoprotein genes were recovered from naturally infected civets in central China (Hubei province), extending the geographic distribution of civet-CoV beyond the southeastern province of Guangdong. Moreover, pseudoviruses generated in our laboratory with four civet S genes, each with a distinct RBD, infected cells expressing human receptor angiotensin-converting enzyme 2, but with 90 to 95% less efficiency compared to that of SARS-CoV. These four civet S genes were also constructed as DNA vaccines to immunize mice. Immunized sera elicited against most civet S glycoproteins displayed potent neutralizing activities against autologous viruses but were much less efficient (50% inhibitory concentration, 20- to 40-fold) at neutralizing SARS-CoV and vice versa. Convalescence-phase sera from humans were similarly ineffective against the dominant civet pseudovirus. Our findings suggest that the design of SARS vaccine should consider not only preventing the reemergence of SARS-CoV but also providing cross-protection, thus interrupting zoonotic transmission of a group of genetically divergent civet CoVs of broad geographic origin.     

Structural basis of neutralization by a human anti-severe acute respiratory syndrome spike protein antibody, 80R

Severe acute respiratory syndrome (SARS) is a newly emerged infectious disease that caused pandemic spread in 2003. The etiological agent of SARS is a novel coronavirus (SARS-CoV). The coronaviral surface spike protein S is a type I transmembrane glycoprotein that mediates initial host binding via the cell surface receptor angiotensin-converting enzyme 2 (ACE2), as well as the subsequent membrane fusion events required for cell entry. Here we report the crystal structure of the S1 receptor binding domain (RBD) in complex with a neutralizing antibody, 80R, at 2.3 A resolution, as well as the structure of the uncomplexed S1 RBD at 2.2 A resolution. We show that the 80R-binding epitope on the S1 RBD overlaps very closely with the ACE2-binding site, providing a rationale for the strong binding and broad neutralizing ability of the antibody. We provide a structural basis for the differential effects of certain mutations in the spike protein on 80R versus ACE2 binding, including escape mutants, which should facilitate the design of immunotherapeutics to treat a future SARS outbreak. We further show that the RBD of S1 forms dimers via an extensive interface that is disrupted in receptor- and antibody-bound crystal structures, and we propose a role for the dimer in virus stability and infectivity.     

Immunogenicity difference between the SARS coronavirus and the bat SARS-like coronavirus spike (S) proteins

SARS-like coronavirus (SL-CoV) in bats have a similar genomic organization to the human SARS-CoV. Their cognate gene products are highly conserved with the exception of the N-terminal region of the S proteins, which have only 63-64% sequence identity. The N-terminal region of coronavirus S protein is responsible for virus-receptor interaction. In this study, the immunogenicity of the SL-CoV S protein (S_SL) was studied and compared with that of SARS-CoV (S_SARS). DNA immunization in mice with S_SL elicited a high titer of antibodies against HIV-pseudotyped S_SL. The sera had low cross-reactivity, but no neutralization activity, for the HIV-pseudotyped S_SARS. Studies using wild bat sera revealed that it is highly likely that the immunodominant epitopes overlap with the major neutralizing sites of the SL-CoV S protein. These results demonstrated that SL-CoV and SARS-CoV shared only a limited number of immunogenic epitopes in their S proteins and the major neutralization epitopes are substantially different. This work provides useful information for future development of differential serologic diagnosis and vaccines for coronaviruses with different S protein sequences.     

A natural mutation between SARS-CoV-2 and SARS-CoV determines neutralization by a cross-reactive antibody

Epitopes that are conserved among SARS-like coronaviruses are attractive targets for design of cross-reactive vaccines and therapeutics. CR3022 is a SARS-CoV neutralizing antibody to a highly conserved epitope on the receptor binding domain (RBD) on the spike protein that is able to cross-react with SARS-CoV-2, but with lower affinity. Using x-ray crystallography, mutagenesis, and binding experiments, we illustrate that of four amino acid differences in the CR3022 epitope between SARS-CoV-2 and SARS-CoV, a single mutation P384A fully determines the affinity difference. CR3022 does not neutralize SARS-CoV-2, but the increased affinity to SARS-CoV-2 P384A mutant now enables neutralization with a similar potency to SARS-CoV. We further investigated CR3022 interaction with the SARS-CoV spike protein by negative-stain EM and cryo-EM. Three CR3022 Fabs bind per trimer with the RBD observed in different up-conformations due to considerable flexibility of the RBD. In one of these conformations, quaternary interactions are made by CR3022 to the N-terminal domain (NTD) of an adjacent subunit. Overall, this study provides insights into antigenic variation and potential cross-neutralizing epitopes on SARS-like viruses.     

呈现新型冠状病毒RBD抗原病毒样颗粒的表达与鉴定*

目的:以乙型肝炎病毒核心抗原HBcAg为载体,构建呈现新冠病毒刺突蛋白受体结合域的病毒样颗粒,并鉴定其免疫原性,为新冠病毒疫苗的开发提供新思路。方法:在乙型肝炎病毒核心蛋白氨基酸编码序列第78和81位插入新冠病毒刺突蛋白受体结合域(RBD),并通过柔性linker(G4S)3进行连接,序列优化后将融合基因克隆到原核表达载体pET-28a(+),转化表达菌Rosetta,在自诱导培养基中诱导表达,菌体破碎后经蔗糖密度梯度离心,透析浓缩的方法纯化病毒样颗粒。SDS-PAGE、Western blot、透射电子显微镜检测和鉴定VLPs。将制备的VLPs与佐剂等比例混合经皮下免疫BALB/c小鼠,ELISA检测小鼠血清中特异性抗体,分析该HBc-RBD VLPs的免疫原性。结果:在自诱导培养基中,大肠埃希菌可表达部分可溶的VLPs,经蔗糖密度梯度离心纯化后在透射电子显微镜下可以观察到病毒样颗粒的存在。动物实验表明HBc-RBD VLPs刺激小鼠产生了特异性抗体。结论:在原核表达系统中成功表达了展示RBD抗原的VLPs,并通过小鼠实验初步验证了免疫原性,为新冠病毒疫苗的研发提供了新方向。