Identification of Conserved Epitopes in SARS-CoV-2 Spike and Nucleocapsid Protein

BackgroundSevere acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a novel virus that first occurred in Wuhan in December 2019. The spike glycoproteins and nucleocapsid proteins are the most common targets for the development of vaccines and antiviral drugs.ObjectiveWe herein analyze the rate of evolution along with the sequences of spike and nucleocapsid proteins in relation to the spatial locations of their epitopes, previously suggested to contribute to the immune response caused by SARS-CoV-2 infections.MethodsWe compare homologous proteins of seven human coronaviruses: HCoV-229E, HCoV-OC43, SARS-CoV, HCoV-NL63, HCoV-HKU1, MERS-CoV, and SARS-CoV-2. We then focus on the local, structural order-disorder propensity of the protein regions where the SARS-CoV-2 epitopes are located. ResultsWe show that most of nucleocapsid protein epitopes overlap the RNA-binding and dimerization domains, and some of them are characterized by a low rate of evolutions. Similarly, spike protein epitopes are preferentially located in regions that are predicted to be ordered and well- conserved, in correspondence of the heptad repeats 1 and 2. Interestingly, both the receptor-binding motif to ACE2 and the fusion peptide of spike protein are characterized by a high rate of evolution.ConclusionOur results provide evidence for conserved epitopes that might help develop broad-spectrum SARS-CoV-2 vaccines.     

A Truncated Receptor-Binding Domain of MERS-CoV Spike Protein Potently Inhibits MERS-CoV Infection and Induces Strong Neutralizing Antibody Responses: Implication for Developing Therapeutics and Vaccines

An emerging respiratory infectious disease with high mortality, Middle East respiratory syndrome (MERS), is caused by a novel coronavirus (MERS-CoV). It was first reported in 2012 in Saudi Arabia and has now spread to eight countries. Development of effective therapeutics and vaccines is crucial to save lives and halt the spread of MERS-CoV. Here, we show that a recombinant protein containing a 212-amino acid fragment (residues 377-588) in the truncated receptor-binding domain (RBD: residues 367–606) of MERS-CoV spike (S) protein fused with human IgG Fc fragment (S377-588-Fc) is highly expressed in the culture supernatant of transfected 293T cells. The purified S377-588-Fc protein efficiently binds to dipeptidyl peptidase 4 (DPP4), the receptor of MERS-CoV, and potently inhibited MERS-CoV infection, suggesting its potential to be further developed as a therapeutic modality for treating MERS-CoV infection and saving the patients’ lives. The recombinant S377-588-Fc is able to induce in the vaccinated mice strong MERS-CoV S-specific antibodies, which blocks the binding of RBD to DPP4 receptor and effectively neutralizes MERS-CoV infection. These findings indicate that this truncated RBD protein shows promise for further development as an effective and safe vaccine for the prevention of MERS-CoV infection.     

SARS-CoV-2刺突蛋白基因密码子偏好性与进化分析

新型冠状病毒(SARS-CoV-2)属于冠状病毒家族新成员,其刺突蛋白(spike protein,S-蛋白)是侵染人体并决定其入侵宿主的亲和能力及组织特异性的包膜蛋白,研究S-蛋白基因的密码子偏好性不仅有利于重组表达,也利于分析其进化规律.利用Codon W和SPSS等在线服务器及软件,首先分析SARS-CoV-2等...     

Induction of Potent Neutralizing Antibody Responses by a Designed Protein Nanoparticle Vaccine for Respiratory Syncytial Virus

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Control of SARS-CoV-2 infection after Spike DNA or Spike DNA+Protein co-immunization in rhesus macaques

The speed of development, versatility and efficacy of mRNA-based vaccines have been amply demonstrated in the case of SARS-CoV-2. DNA vaccines represent an important alternative since they induce both humoral and cellular immune responses in animal models and in human trials. We tested the immunogenicity and protective efficacy of DNA-based vaccine regimens expressing different prefusion-stabilized Wuhan-Hu-1 SARS-CoV-2 Spike antigens upon intramuscular injection followed by electroporation in rhesus macaques. Different Spike DNA vaccine regimens induced antibodies that potently neutralized SARS-CoV-2 in vitro and elicited robust T cell responses. The antibodies recognized and potently neutralized a panel of different Spike variants including Alpha, Delta, Epsilon, Eta and A.23.1, but to a lesser extent Beta and Gamma. The DNA-only vaccine regimens were compared to a regimen that included co-immunization of Spike DNA and protein in the same anatomical site, the latter of which showed significant higher antibody responses. All vaccine regimens led to control of SARS-CoV-2 intranasal/intratracheal challenge and absence of virus dissemination to the lower respiratory tract. Vaccine-induced binding and neutralizing antibody titers and antibody-dependent cellular phagocytosis inversely correlated with transient virus levels in the nasal mucosa. Importantly, the Spike DNA+Protein co-immunization regimen induced the highest binding and neutralizing antibodies and showed the strongest control against SARS-CoV-2 challenge in rhesus macaques.     

Genetic and Structural Analysis of SARS-CoV-2 Spike Protein for Universal Epitope Selection

Evaluation of immunogenic epitopes for universal vaccine development in the face of ongoing SARS-CoV-2 evolution remains a challenge. Herein, we investigate the genetic and structural conservation of an immunogenically relevant epitope (C662–C671) of spike (S) protein across SARS-CoV-2 variants to determine its potential utility as a broad-spectrum vaccine candidate against coronavirus diseases. Comparative sequence analysis, structural assessment, and molecular dynamics simulations of C662–C671 epitope were performed. Mathematical tools were employed to determine its mutational cost. We found that the amino acid sequence of C662–C671 epitope is entirely conserved across the observed major variants of SARS-CoV-2 in addition to SARS-CoV. Its conformation and accessibility are predicted to be conserved, even in the highly mutated Omicron variant. Costly mutational rate in the context of energy expenditure in genome replication and translation can explain this strict conservation. These observations may herald an approach to developing vaccine candidates for universal protection against emergent variants of coronavirus.     

Immunogenic Properties of the DNA Construct Encoding the Receptor-Binding Domain of the SARS-CoV-2 Spike Protein

Molecular Biology - The development of preventive vaccines became the first order task in the COVID-19 pandemic caused by SARS-CoV-2. This paper reports the construction of the pVAX-RBD plasmid...     

Precisely Molded Nanoparticle Displaying DENV-E Proteins Induces Robust Serotype-Specific Neutralizing Antibody Responses

Dengue virus (DENV) is the causative agent of dengue fever and dengue hemorrhagic fever. The virus is endemic in over 120 countries, causing over 350 million infections per year. Dengue vaccine development is challenging because of the need to induce simultaneous protection against four antigenically distinct DENV serotypes and evidence that, under some conditions, vaccination can enhance disease due to specific immunity to the virus. While several live-attenuated tetravalent dengue virus vaccines display partial efficacy, it has been challenging to induce balanced protective immunity to all 4 serotypes. Instead of using whole-virus formulations, we are exploring the potentials for a particulate subunit vaccine, based on DENV E-protein displayed on nanoparticles that have been precisely molded using Particle Replication in Non-wetting Template (PRINT) technology. Here we describe immunization studies with a DENV2-nanoparticle vaccine candidate. The ectodomain of DENV2-E protein was expressed as a secreted recombinant protein (sRecE), purified and adsorbed to poly (lactic-co-glycolic acid) (PLGA) nanoparticles of different sizes and shape. We show that PRINT nanoparticle adsorbed sRecE without any adjuvant induces higher IgG titers and a more potent DENV2-specific neutralizing antibody response compared to the soluble sRecE protein alone. Antigen trafficking indicate that PRINT nanoparticle display of sRecE prolongs the bio-availability of the antigen in the draining lymph nodes by creating an antigen depot. Our results demonstrate that PRINT nanoparticles are a promising platform for delivering subunit vaccines against flaviviruses such as dengue and Zika.     

The Emergence of the Spike Furin Cleavage Site in SARS-CoV-2

Compared with other SARS-related coronaviruses (SARSr-CoVs), SARS-CoV-2 possesses a unique furin cleavage site (FCS) in its spike. This has stimulated discussion pertaining to the origin of SARS-CoV-2 because the FCS has been observed to be under strong selective pressure in humans and confers the enhanced ability to infect some cell types and induce cell–cell fusion. Furthermore, scientists have demonstrated interest in studying novel cleavage sites by introducing them into SARSr-CoVs. We review what is known about the SARS-CoV-2 FCS in the context of its pathogenesis, origin, and how future wildlife coronavirus sampling may alter the interpretation of existing data.     

抗新型冠状病毒单克隆中和抗体药物研发进展*

随着新型冠状病毒肺炎(COVID-19)疫情在全球的不断蔓延,开发有效的治疗药物迫在眉睫。中和抗体作为最有希望的新型冠状病毒特异性治疗药物,已经在临床研究中展现很好的治疗效果。对抗新冠病毒单克隆中和抗体药物研发的进展、涉及的主要技术和主要临床试验结果进行了总结,以期为包括COVID-19在内的新发、突发传染病中和抗体药物研发提供参考。     

新型冠状病毒变异株VOC 202012/01的全球早期传播与刺突蛋白进化特征分析

新型冠状病毒(SARS-CoV-2)自2019年底流行至今,已进化出多个不同的亚型或分支并在全球共同传播。2020年12月14日,英国报道了一种新的SARS-CoV-2 variants of concern 202012/01(VOC 202012/01)变异株,其刺突(spike,S)蛋白累积了10个氨基酸突变,传播力增强。为分析SARS-CoV-2 VOC 202012/01变异株的全球传播与进化规律,本研究对截至到2020年12月31日的GISAID数据库中符合VOC 202012/01变异株特征的全基因组序列进行了时空分布及S蛋白进化特征分析。结果表明,VOC 202012/01变异株自2020年9月20日于英国首次出现后,在英国境内迅速蔓延,毒株数量逐月增多,并逐步扩散到全球4个大洲22个国家。在2020年9~12月传播期间,VOC 202012/01变异株的S蛋白除10个特征性位点稳定突变以外,均呈随机突变,仅有2个氨基酸突变位点存在于50条以上的序列中,行成小的分支。本文初步阐明了SARS-CoV-2 VOC 202012/01变异株的在全球早期流行中的传播与S蛋白的进化特征,为我国应对VOC 202012/01变异株的监测与防控提供科学依据。     

Nitrogen Under-and Over-supply Induces Distinct Protein Responses in Maize Xylem Sap

Xylem sap primarily transports water and mineral nutrients such as nitrogen(N)from roots to shoots in vascular plants.However,it remains largely unknown how nitrogenous compounds,especially proteins in xylem sap,respond to N under-or over-supply.We found that reducing N supply increased amino-N percentage of total N in maize(Zea mays L.)xylem sap.Proteomic analysis showed that 23 proteins in the xylem sap of maize plants,including 12 newly identified ones,differentially accumulated in response to various N supplies.Fifteen of these 23 proteins were primarily involved in general abiotic or biotic stress responses,whereas the other five proteins appeared to respond largely to N under-or over-supply,suggesting distinct protein responses in maize xylem upon N under-and over-supply.Furthermore,one putative xylanase inhibitor and two putative O-glycosyl hydrolases had preferential gene expression in shoots.     

Human Monoclonal Antibodies against Highly Conserved HR1 and HR2 Domains of the SARS-CoV Spike Protein Are More Broadly Neutralizing

Immune sera from convalescent patients have been shown to be effective in the treatment of patients infected with Severe Acute Respiratory Syndrome Virus (SARS-CoV) making passive immune therapy with human monoclonal antibodies an attractive treatment strategy for SARS. Previously, using Xenomouse (Amgen British Columbia Inc), we produced a panel of neutralizing Human monoclonal antibodies (HmAbs) that could specifically bind to the ectodomain of the SARS-CoV spike (S) glycoprotein. Some of the HmAbs were S1 domain specific, while some were not. In this study, we describe non-S1 binding neutralizing HmAbs that can specifically bind to the conserved S2 domain of the S protein. However, unlike the S1 specific HmAbs, the S2 specific HmAbs can neutralize pseudotyped viruses expressing different S proteins containing receptor binding domain sequences of various clinical isolates. These data indicate that HmAbs which bind to conserved regions of the S protein are more suitable for conferring protection against a wide range of SARS-CoV variants and have implications for generating therapeutic antibodies or subunit vaccines against other enveloped viruses.