Chimeric fab fragments of antibodies to recombinant Ebola virus glycoprotein

Previously, we have determined the nucleotide and amino acid sequences of the variable domains of three mouse monoclonal antibodies specific to the individual epitopes of the Ebola virus glycoprotein: GPE118 (IgG), GPE325 (IgM) and GPE534 (IgG) [1]. In the present paper, chimeric Fab fragments of Fab118, Fab325, and Fab534 antibodies were obtained based on the variable domains of murine antibodies by attaching CH1 and CL constant regions of human kappa-IgG1 to them. The recombinant chimeric Fab fragments were synthesized in the heterologous expression system Escherichia coli, isolated and purified using metal chelate affinity chromatography. The immunochemical properties of the obtained Fab fragments were studied by immunoblotting techniques as well as indirect and competitive ELISA using recombinant Ebola virus proteins: EBOV rGPdTM (recombinant glycoprotein of Ebola hemorrhagic fever virus without the transmembrane domain), NP (nucleoprotein) and VP40 (structural protein). The identity of recombinant chimeric Fab fragments, as well as their specificity to the recombinant glycoprotein of Ebola hemorrhagic fever virus (EBOV GP) was proved. The results of indirect ELISA evidence the absence of immunological cross-reactivity to NP and VP40 proteins of Ebola virus. The dissociation constants of the antigen-antibody complex K _d equal to 5.0, 1.0 and 1.0 nM for Fab118, Fab325 and Fab534, respectively, were determined; they indicate high affinity of the obtained experimental samples to EBOV GP. The epitope specificity of Fab fragments was studied using a panel of commercial neutralizing antibodies. It was found that all studied antibodies to EBOV GP are targeted to different epitopes, while the epitopes of the recombinant chimeric Fab fragments and original murine monoclonal antibodies (mAbs) coincide. All the obtained and studied mAbs to EBOV GP are specific to epitopes that coincide or overlap the epitopes of three commercial neutralizing mAbs to Ebola virus: epitopes Fab118 and Fab325 overlap the epitope of the known commercial mAb h13F6; Fab325 epitope also overlaps mAb c6D8 epitope; Fab534 epitope is located near mAb KZ52 conformational epitope, in the formation of which amino acid residues of GP1 and GP2 domains of EBOV GP are involved.     

Prediction of Epitope-Based Peptides for Vaccine Development from Coat Proteins GP2 and VP24 of Ebola Virus Using Immunoinformatics

This study aims to design epitope-based peptides for the utility of vaccine development by targeting Glycoprotein 2 (GP2) and Viral Protein 24 (VP24) of the Ebola virus (EBOV) that, respectively, facilitate attachment and fusion of EBOV with host cells. Using various databases and tools, immune parameters of conserved sequences from GP2 and VP24 proteins of different strains of EBOV were tested to predict probable epitopes. Binding analyses of the peptides with major histocompatibility complex (MHC) class I and class II molecules, population coverage, and linear B cell epitope prediction were peroformed. Predicted peptides interacted with multiple MHC alleles and illustrated maximal population coverage for both GP2 and VP24 proteins, respectively. The predicted class-I nonamers, FLYDRLAST, LFLRATTEL and NYNGLLSSI were found to cover the maximum number of MHC I alleles and showed interactions with binding energies of ?7.8, ?8.5 and ?7.7 kcal/mol respectively. Highest scoring class II MHC binding peptides were EGAFFLYDRLASTVI and SPLWALRVILAAGIQ with binding energies of ?6.2 and -5.6 kcal/mol. Putative B cell epitopes were also found on 4 conserved regions in GP2 and two conserved regions in VP24. Our in silico analysis suggests that the predicted epitopes could be a better choice as universal vaccine component against EBOV irrespective of different strains and should be subjected to in vitro and in vivo analyses for further research and development.     

Involvement of viral envelope GP2 in Ebola virus entry into cells expressing the macrophage galactose-type C-type lectin

Ebola virus (EBOV) infection is initiated by the interaction of the viral surface envelope glycoprotein (GP) with the binding sites on target cells. Differences in the mortality among different species of the Ebola viruses, i.e., Zaire ebolavirus (ZEBOV) and Reston ebolavirus (REBOV), correspond to the in vitro infectivity of the pseudo-typed virus constructed with the GPs in cells expressing macrophage galactose-type calcium-type lectin (MGL/CD301). Through mutagenesis of GP2, the transmembrane-anchored subunit of GP, we found that residues 502–527 of the GP2 sequence determined the different infectivity between VSV-ZEBOV GP and -REBOV GP in MGL/CD301-expressing cells and a histidine residue at position 516 of ZEBOV GP2 appeared essential in the differential infectivity. These findings may provide a clue to clarify a molecular basis of different pathogenicity among EBOV species.     

Characterization of monoclonal antibodies against waterfowl parvoviruses VP3 protein

Background

Ebola viruses (EBOVs) cause severe hemorrhagic fever with a high mortality rate. At present, there are no licensed vaccines or efficient therapies to combat EBOV infection. Previous studies have shown that both humoral and cellular immune responses are crucial for controlling Ebola infection. CD8⁺ T cells play an important role in mediating vaccine-induced protective immunity. The objective of this study was to identify H-2^d-specific T cell epitopes in EBOV glycoproteins (GPs).

Results

Computer-assisted algorithms were used to predict H-2^d-specific T cell epitopes in two species of EBOV (Sudan and Zaire) GP. The predicted peptides were synthesized and identified in BALB/c mice immunized with replication-deficient adenovirus vectors expressing the EBOV GP. Enzyme-linked immunospot assays and intracellular cytokine staining showed that the peptides RPHTPQFLF (Sudan EBOV), GPCAGDFAF and LYDRLASTV (Zaire EBOV) could stimulate splenoctyes in immunized mice to produce large amounts of interferon-gamma.

Conclusion

Three peptides within the GPs of two EBOV strains were identified as T cell epitopes. The identification of these epitopes should facilitate the evaluation of vaccines based on the Ebola virus glycoprotein in a BALB/c mouse model.     

埃博拉病毒疫苗研究进展

埃博拉病毒是一种可引起人和非人灵长类动物出血热传染病的最为致命的烈性病毒,致死率可达90%。2014年在西非爆发的埃博拉疫情引起了全世界的关注。疫苗接种是预防和控制传染病最为常规和有效的方法,尽管目前还没有正式获得批准上市的埃博拉病毒疫苗,但是已有多个尚处于研究阶段的疫苗在非人灵长类动物上取得了很好的保护效果,并有几个已进入临床Ⅰ期试验阶段,有望尽快用于本次埃博拉疫情的防控。本文对目前处于研究阶段的多个类型的埃博拉病毒疫苗进行了综述,为相关研究人员提供参考。     

Identification of Ebola virus microRNAs and their putative pathological function

Ebola virus(EBOV),a member of the filovirus family,is an enveloped negative-sense RNA virus that causes lethal infections in humans and primates.Recently,more than 1000 people have been killed by the Ebola virus disease in Africa,yet no specific treatment or diagnostic tests for EBOV are available.In this study,we identified two putative viral microRNA precursors(pre-miRNAs)and three putative mature microRNAs(miRNAs)derived from the EBOV genome.The production of the EBOV miRNAs was further validated in HEK293T cells transfected with a pcDNA6.2-GW/EmGFP-EBOV-pre-miRNA plasmid,indicating that EBOV miRNAs can be produced through the cellular miRNA processing machinery.We also predicted the potential target genes of these EBOV miRNAs and their possible biological functions.Overall,this study reports for the first time that EBOV may produce miRNAs,which could serve as non-invasive biomarkers for the diagnosis and prognosis of EBOV infection and as therapeutic targets for Ebola viral infection treatment.     

Inactivated or live-attenuated bivalent vaccines that confer protection against rabies and Ebola viruses

The search for a safe and efficacious vaccine for Ebola virus continues, as no current vaccine candidate is nearing licensure. We have developed (i) replication-competent, (ii) replication-deficient, and (iii) chemically inactivated rabies virus (RABV) vaccines expressing Zaire Ebola virus (ZEBOV) glycoprotein (GP) by a reverse genetics system based on the SAD B19 RABV wildlife vaccine. ZEBOV GP is efficiently expressed by these vaccine candidates and is incorporated into virions. The vaccine candidates were avirulent after inoculation of adult mice, and viruses with a deletion in the RABV glycoprotein had greatly reduced neurovirulence after intracerebral inoculation in suckling mice. Immunization with live or inactivated RABV vaccines expressing ZEBOV GP induced humoral immunity against each virus and conferred protection from both lethal RABV and EBOV challenge in mice. The bivalent RABV/ZEBOV vaccines described here have several distinct advantages that may speed the development of inactivated vaccines for use in humans and potentially live or inactivated vaccines for use in nonhuman primates at risk of EBOV infection in endemic areas.     

Anti-Ebola therapies based on monoclonal antibodies: current state and challenges ahead

The 2014 Ebola outbreak, the largest recorded, took us largely unprepared, with no available vaccine or specific treatment. In this context, the World Health Organization declared that the humanitarian use of experimental therapies against Ebola Virus (EBOV) is ethical. In particular, an experimental treatment consisting of a cocktail of three monoclonal antibodies (mAbs) produced in tobacco plants and specifically directed to the EBOV glycoprotein (GP) was tested in humans, apparently with good results. Several mAbs with high affinity to the GP have been described. This review discusses our current knowledge on this topic. Particular emphasis is devoted to those mAbs that have been assayed in animal models or humans as possible therapies against Ebola. Engineering aspects and challenges for the production of anti-Ebola mAbs are also briefly discussed; current platforms for the design and production of full-length mAbs are cumbersome and costly.     

Antigenic Subversion: A Novel Mechanism of Host Immune Evasion by Ebola Virus

In addition to its surface glycoprotein (GP_1,2), Ebola virus (EBOV) directs the production of large quantities of a truncated glycoprotein isoform (sGP) that is secreted into the extracellular space. The generation of secreted antigens has been studied in several viruses and suggested as a mechanism of host immune evasion through absorption of antibodies and interference with antibody-mediated clearance. However such a role has not been conclusively determined for the Ebola virus sGP. In this study, we immunized mice with DNA constructs expressing GP_1,2 and/or sGP, and demonstrate that sGP can efficiently compete for anti-GP₁₂ antibodies, but only from mice that have been immunized by sGP. We term this phenomenon “antigenic subversion”, and propose a model whereby sGP redirects the host antibody response to focus on epitopes which it shares with membrane-bound GP_1,2, thereby allowing it to absorb anti-GP_1,2 antibodies. Unexpectedly, we found that sGP can also subvert a previously immunized host''s anti-GP_1,2 response resulting in strong cross-reactivity with sGP. This finding is particularly relevant to EBOV vaccinology since it underscores the importance of eliciting robust immunity that is sufficient to rapidly clear an infection before antigenic subversion can occur. Antigenic subversion represents a novel virus escape strategy that likely helps EBOV evade host immunity, and may represent an important obstacle to EBOV vaccine design.     

Antibody Derived Peptides for Detection of Ebola Virus Glycoprotein

Background

Current Ebola virus (EBOV) detection methods are costly and impractical for epidemic scenarios. Different immune-based assays have been reported for the detection and quantification of Ebola virus (EBOV) proteins. In particular, several monoclonal antibodies (mAbs) have been described that bind the capsid glycoprotein (GP) of EBOV GP. However, the currently available platforms for the design and production of full-length mAbs are cumbersome and costly. The use of antibody fragments, rather than full-length antibodies, might represent a cost-effective alternative for the development of diagnostic and possibly even therapeutic alternatives for EBOV.

Methods/Principal Findings

We report the design and expression of three recombinant anti-GP mAb fragments in Escherichia coli cultures. These fragments contained the heavy and light variable portions of the three well-studied anti-GP full-length mAbs 13C6, 13F6, and KZ52, and are consequently named scFv-13C6, scFv-13F6, and Fab-KZ52, respectively. All three fragments exhibited specific anti-GP binding activity in ELISA experiments comparable to that of full-length anti-GP antibodies (i.e., the same order of magnitude) and they are easily and economically produced in bacterial cultures.

Conclusion/Significance

Antibody fragments might represent a useful, effective, and low cost alternative to full-length antibodies in Ebola related capture and diagnostics applications.     

埃博拉病毒小分子抑制剂的研究进展

目前尚没有可靠的埃博拉病毒(Ebola virus,EBOV)疫苗和特异性治疗药物.2014年埃博拉病毒病在西非的爆发和肆虐警醒人类,需要加快对该病的防控研究.近几年,在EBOV小分子抑制剂的研究方面取得了较好的进展,有的已进入临床试验阶段.小分子化合物通常是针对病毒致病作用的某种机制而设计,是一个很有发展前途的研究领域.本文从抑制EBOV和其他病毒在生活周期中的穿入细胞、复制和出芽等方面综述EBOV小分子抑制剂的研究进展.     

Development of a cAdVax-based bivalent ebola virus vaccine that induces immune responses against both the Sudan and Zaire species of Ebola virus

Ebola virus (EBOV) causes a severe hemorrhagic fever for which there are currently no vaccines or effective treatments. While lethal human outbreaks have so far been restricted to sub-Saharan Africa, the potential exploitation of EBOV as a biological weapon cannot be ignored. Two species of EBOV, Sudan ebolavirus (SEBOV) and Zaire ebolavirus (ZEBOV), have been responsible for all of the deadly human outbreaks resulting from this virus. Therefore, it is important to develop a vaccine that can prevent infection by both lethal species. Here, we describe the bivalent cAdVaxE(GPs/z) vaccine, which includes the SEBOV glycoprotein (GP) and ZEBOV GP genes together in a single complex adenovirus-based vaccine (cAdVax) vector. Vaccination of mice with the bivalent cAdVaxE(GPs/z) vaccine led to efficient induction of EBOV-specific antibody and cell-mediated immune responses to both species of EBOV. In addition, the cAdVax technology demonstrated induction of a 100% protective immune response in mice, as all vaccinated C57BL/6 and BALB/c mice survived challenge with a lethal dose of ZEBOV (30,000 times the 50% lethal dose). This study demonstrates the potential efficacy of a bivalent EBOV vaccine based on a cAdVax vaccine vector design.     

Zoonotic risk factors associated with seroprevalence of Ebola virus GP antibodies in the absence of diagnosed Ebola virus disease in the Democratic Republic of Congo

BackgroundEbola virus (EBOV) is a zoonotic filovirus spread through exposure to infected bodily fluids of a human or animal. Though EBOV is capable of causing severe disease, referred to as Ebola Virus Disease (EVD), individuals who have never been diagnosed with confirmed, probable or suspected EVD can have detectable EBOV antigen-specific antibodies in their blood. This study aims to identify risk factors associated with detectable antibody levels in the absence of an EVD diagnosis.MethodologyData was collected from September 2015 to August 2017 from 1,366 consenting individuals across four study sites in the DRC (Boende, Kabondo-Dianda, Kikwit, and Yambuku). Seroreactivity was determined to EBOV GP IgG using Zaire Ebola Virus Glycoprotein (EBOV GP antigen) ELISA kits (Alpha Diagnostic International, Inc.) in Kinshasa, DRC; any result above 4.7 units/mL was considered seroreactive. Among the respondents, 113 (8.3%) were considered seroreactive. Several zoonotic exposures were associated with EBOV seroreactivity after controlling for age, sex, healthcare worker status, location, and history of contact with an EVD case, namely: ever having contact with bats, ever having contact with rodents, and ever eating non-human primate meat. Contact with monkeys or non-human primates was not associated with seroreactivity.ConclusionsThis analysis suggests that some zoonotic exposures that have been linked to EVD outbreaks can also be associated with EBOV GP seroreactivity in the absence of diagnosed EVD. Future investigations should seek to clarify the relationships between zoonotic exposures, seroreactivity, asymptomatic infection, and EVD.     

Expression of an immunogenic Ebola immune complex in Nicotiana benthamiana

Filoviruses (Ebola and Marburg viruses) cause severe and often fatal haemorrhagic fever in humans and non‐human primates. The US Centers for Disease Control identifies Ebola and Marburg viruses as ‘category A’ pathogens (defined as posing a risk to national security as bioterrorism agents), which has lead to a search for vaccines that could prevent the disease. Because the use of such vaccines would be in the service of public health, the cost of production is an important component of their development. The use of plant biotechnology is one possible way to cost‐effectively produce subunit vaccines. In this work, a geminiviral replicon system was used to produce an Ebola immune complex (EIC) in Nicotiana benthamiana. Ebola glycoprotein (GP1) was fused at the C‐terminus of the heavy chain of humanized 6D8 IgG monoclonal antibody, which specifically binds to a linear epitope on GP1. Co‐expression of the GP1‐heavy chain fusion and the 6D8 light chain using a geminiviral vector in leaves of N. benthamiana produced assembled immunoglobulin, which was purified by ammonium sulphate precipitation and protein G affinity chromatography. Immune complex formation was confirmed by assays to show that the recombinant protein bound the complement factor C1q. Size measurements of purified recombinant protein by dynamic light scattering and size‐exclusion chromatography also indicated complex formation. Subcutaneous immunization of BALB/C mice with purified EIC resulted in anti‐Ebola virus antibody production at levels comparable to those obtained with a GP1 virus‐like particle. These results show excellent potential for a plant‐expressed EIC as a human vaccine.     

Evaluation of the European tick‐borne encephalitis vaccine against Omsk hemorrhagic fever virus

This study focused on the antigenic cross‐reactivity between tick‐borne encephalitis virus (TBEV) and Omsk hemorrhagic fever virus (OHFV) to assess the efficacy of the commercial TBE vaccine against OHFV infection. Neutralization tests performed on sera from OHFV‐ and TBEV‐infected mice showed that neutralizing antibodies are cross‐protective. The geometric mean titers of antibodies against TBEV and OHFV from TBEV‐infected mice were similar. However, the titers of anti‐TBEV antibodies in OHFV‐infected mice were significantly lower than those of anti‐OHFV antibodies in the same animals. In mouse vaccination and challenge tests, the TBE vaccine provided 100% protection against OHFV infection. Eighty‐six percent of vaccinees seroconverted against OHFV following complete vaccination, and the geometric mean titers of neutralizing antibodies against OHFV were comparable to those against TBEV. These data suggest that the TBE vaccine can prevent OHFV infection.     

Differential expression of the Ebola virus GP(1,2) protein and its fragments in E. coli

Bacterial expression platforms are frequently used for the expression and production of different recombinant proteins. The full length Ebola virus (EBOV) GP(1,2) gene and subfragments of the GP(1) gene were cloned in a bacterial expression vector as a C-terminal His(6) fusion protein. Surprisingly, the full length EBOV GP(1,2) gene could not be expressed in Escherichia coli. The subfragments of GP(1) were only expressed in small amounts with the exception of one small fragment (subfragment D) which was expressed at very high levels as inclusion bodies. This was seen even in the in vitro translation system with no expression of full length GP(1,2), GP(1) subfragments A and C and low level expression of subfragment B. Only the subfragment D showed high level of expression. In E. coli (Top10), the recombinant GP(1) subfragment D protein was expressed exclusively as an insoluble approximately 25 kDa His(6) fusion protein, which is the expected size for a non-glycosylated recombinant protein. The IMAC purified and refolded non-glycosylated protein was used to immunize mice for the development of monoclonal anti-EBOV antibodies which successfully yielded several monoclonal antibodies with different specificities. The monoclonal and polyclonal antiserum derived from the animals immunized with this recombinant GP(1) subfragment D protein was found to specifically recognize the full length glycosylated EBOV GP(1,2) protein expressed in mammalian 293T cells, thus, demonstrating the immunogenicity of the recombinant subfragment.     

Evidence for distinct mechanisms of small molecule inhibitors of filovirus entry

Many small molecules have been identified as entry inhibitors of filoviruses. However, a lack of understanding of the mechanism of action for these molecules limits further their development as anti-filoviral agents. Here we provide evidence that toremifene and other small molecule entry inhibitors have at least three distinctive mechanisms of action and lay the groundwork for future development of anti-filoviral agents. The three mechanisms identified here include: (1) direct binding to the internal fusion loop region of Ebola virus glycoprotein (GP); (2) the HR2 domain is likely the main binding site for Marburg virus GP inhibitors and a secondary binding site for some EBOV GP inhibitors; (3) lysosome trapping of GP inhibitors increases drug exposure in the lysosome and further improves the viral inhibition. Importantly, small molecules targeting different domains on GP are synergistic in inhibiting EBOV entry suggesting these two mechanisms of action are distinct. Our findings provide important mechanistic insights into filovirus entry and rational drug design for future antiviral development.     

埃博拉病毒疫苗研究进展及相关专利浅析

通过非专利数据库的文献检索,总结了埃博拉病毒（EBOV）研究的最新进展,通过专利文献数据库文献检索分析EBOV疫苗的专利申请基本情况、发展趋势和技术点。结果发现专利申请量与疫情的爆发有一定的相关性;专利申请人多为政府主导的机构或非营利性机构;最新的专利申请均关注GP蛋白对病毒侵染的重要性,针对GP蛋白的抗体和疫苗正在开发中,其中Mapp生物制药公司的产品“ZMapp”作为试验性生物药物目前效果最佳。