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.     

Potential of plants to produce recombinant protein products

Plants have great potential as photosynthetic factories to produce pharmaceutically important and commercially valuable biomedicines and industrial proteins at low cost. The U.S. Food and Drug Administration (U.S. FDA) has approved the drug Elelyso (taliglucerase alfa) produced by carrot cells for treatment of type 1 Gaucher’s disease in 2012. The commercial potential of biomedicines produced by molecular farming has dramatically improved due to the success of an experimental drug called ZMapp, which has immunological activity in Ebola patients. A cocktail of three monoclonal antibodies was produced in tobacco (Nicotiana benthamiana) plants (Chen and Davis 2016). At present, very few drugs made by this technology have been approved by worldwide authorities such as the U.S. FDA. However, plants have been proposed as a novel paradigm for commercial production of proteins over the next decade. In recent years, leading researchers on molecular farming have given more priority to the area of animal-free therapeutic proteins such as parenteral and oral vaccines. Although plant-based platforms have considerable advantages over traditional systems such as bacterial and animal systems, there are several obstacles to commercial-scale production, especially with regards to improving the quality and quantity of plant-produced biologics and industrial materials. One of the biggest barriers to commercialization of this technology is the intense scrutiny of these new plant varieties by regulatory agencies and the public as well as the high costs associated with their regulatory approval.     

Role of commensal relationships on the spatial structure of a surface-attached microbial consortium

A flow cell-grown model consortium consisting of two organisms, Burkholderia sp. LB400 and Pseudomonas sp. B13(FR1), was studied. These bacteria have the potential to interact metabolically because Pseudomonas sp. B13(FR1) can metabolize chlorobenzoate produced by Burkholderia sp. LB400 when grown on chlorobiphenyl. The expected metabolic interactions in the consortium were demonstrated by high performance liquid chromatography (HPLC) analysis. The spatial structure of the consortium was studied by fluorescent in situ rRNA hybridization and scanning confocal laser microscopy. When the consortium was fed with medium containing a low concentration of chlorobiphenyl, microcolonies consisting of associated Burkholderia sp. LB400 and Pseudomonas sp. B13(FR1) bacteria were formed, and separate Pseudomonas sp. B13(FR1) microcolonies were evidently not formed. When the consortium was fed citrate, which can be metabolized by both species, the two species formed separate microcolonies. The structure development in the consortium was studied online using a gfp -tagged Pseudomonas sp. B13(FR1) derivative. After a shift in carbon source from citrate to a low concentration of chlorobiphenyl, movement of the Pseudomonas sp. B13(FR1) bacteria led to a change in the spatial structure of the consortium from the unassociated form towards the associated form within a few days. Experiments involving a gfp -based Pseudomonas sp. B13(FR1) growth activity reporter strain indicated that chlorobenzoate supporting growth of Pseudomonas sp. B13(FR1) is located close to the Burkholderia sp. LB400 microcolonies in chlorobiphenyl-grown consortia.     

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.     

Lentivirus vectors pseudotyped with filoviral envelope glycoproteins transduce airway epithelia from the apical surface independently of folate receptor alpha

The practical application of gene therapy as a treatment for cystic fibrosis is limited by poor gene transfer efficiency with vectors applied to the apical surface of airway epithelia. Recently, folate receptor alpha (FR alpha), a glycosylphosphatidylinositol-linked surface protein, was reported to be a cellular receptor for the filoviruses. We found that polarized human airway epithelia expressed abundant FR alpha on their apical surface. In an attempt to target these apical receptors, we pseudotyped feline immunodeficiency virus (FIV)-based vectors by using envelope glycoproteins (GPs) from the filoviruses Marburg virus and Ebola virus. Importantly, primary cultures of well-differentiated human airway epithelia were transduced when filovirus GP-pseudotyped FIV was applied to the apical surface. Furthermore, by deleting a heavily O-glycosylated extracellular domain of the Ebola GP, we improved the titer of concentrated vector severalfold. To investigate the folate receptor dependence of gene transfer with the filovirus pseudotypes, we compared gene transfer efficiency in immortalized airway epithelium cell lines and primary cultures. By utilizing phosphatidylinositol-specific phospholipase C (PI-PLC) treatment and FR alpha-blocking antibodies, we demonstrated FR alpha-dependent and -independent entry by filovirus glycoprotein-pseudotyped FIV-based vectors in airway epithelia. Of particular interest, entry independent of FR alpha was observed in primary cultures of human airway epithelia. Understanding viral vector binding and entry pathways is fundamental for developing cystic fibrosis gene therapy applications.     

埃博拉病毒检测与防治技术在中国的专利申请状况分析

近年来,埃博拉病毒(EBOV)因其在非洲造成的严峻疫情而引起了广泛关注。本文对涉及埃博拉病毒检测与防治技术方面的在中国申请的国内外专利申请数量、年代分布、技术发展状况等信息进行分析,对国内埃博拉病毒检测、免疫、治疗相关专利技术进行简要总结,并对目前热点关注药物(jk-05,ZMapp,VSV-EBOV)专利申请信息进行比较概括,结果表明,我国申请人在提出的相关专利申请数量以及技术多样性上与国外申请人存在差距,为国内科研人员提供了相关技术领域参考。     

Lessons from the Ebola epidemics and their applications for COVID‐19 pandemic response in sub‐Saharan Africa

COVID‐19, caused by a novel coronavirus named SARS‐CoV‐2, was identified in December 2019, in Wuhan, China. It was first confirmed in sub‐Saharan Africa in Nigeria on 27 February 2020 and has since spread quickly to all sub‐Saharan African countries, causing more than 111,309 confirmed cases and 2,498 deaths as of 03 June 2020. The lessons learned during the recent Ebola virus disease (EVD) outbreaks in some sub‐Saharan African countries were expected to shape and influence the region’s responses to COVID‐19 pandemic. However, some of the challenges associated with the management of the EVD outbreaks persist and create obstacles for the effective management of the COVID‐19 pandemic. This article describes the commonalities between the EVD epidemics and COVID‐19 pandemic, with a view to draw on lessons learned to effectively tackle the ongoing pandemic. Key successes, failures and lessons learned from previous EVD outbreaks are discussed. Recommendations on how these lessons can be translated to strengthen the COVID‐19 response in sub‐Saharan Africa are provided.     

Isolation of a methanogenic bacterium, Methanosarcina sp. strain FR, for its ability to degrade high concentration of perchloroethylene.

Tetrachloroethylene (PCE) is a toxic compound essentially used as a degreasing and dry-cleaning solvent. A methanogenic and sulfate-reducing consortium that dechlorinates and mineralizes high concentrations of PCE was derived from anaerobically digested sludge obtained from a waste water treatment plant (Bourg-en-Bresse, France). A methanogenic bacterium, strain FR, was isolated from this acclimated consortium. On the basis of morphological and physiological characteristics, strain FR was classified in the genus of Methanosarcina. Phylogeny analysis with the 16S rRNA gene sequence revealed that strain FR is highly related to Methanosarcina mazei and Methanosarcina frisia (99.6 and 99.5% identity, respectively). High concentrations (50-87 microM) of PCE were completely dechlorinated by strain FR cultures at the rate of 76 nM-mg protein(-1).day(-1). PCE dechlorination produced a nonidentified compound. The tracer experiments with [13C]PCE revealed that the product was nonchlorinated. Dechlorination of PCE to trichloroethylene was still active in the presence of boiled cell extract of the strain FR. However, no further dechlorination was observed. This result suggests that a cofactor rather than an enzymatic system is responsible for the first dechlorination of PCE. Dechlorination-active fractions purified from cell extracts on a XAD-4 column revealed the presence of F(420), F(430), and cobamides cofactors. This is the first report of the isolation of a methanogenic bacterium with the ability to dechlorinate high concentrations of PCE to a nonchlorinated product.     

Effects of jasmonic acid, branching and girdling on carbon and nitrogen transport in poplar

? Here, we examined the impact of jasmonate (JA) treatment, branching and phloem girdling on 13C and 1?N import, invertase activity and polyphenol accumulation in juvenile tissues of unbranched and branched hybrid poplar saplings (Populus nigra?×?P. deltoides). ? The import of 13C to juvenile tissues was positively correlated with invertase activity at the treatment site and enhanced by JA. Both invertase activity and 13C import were greater in shorter, younger branches and smaller, younger leaves. By contrast, JA treatments, branching and girdling had little or no impact on 1?N import. ? In poplar saplings with multiple lateral branches, we observed almost no 13C movement from subtending source leaves into lateral branches above them, with or without JA treatment. The presence of potentially competing branches, treated with JA or not, girdled or not, had no impact on carbohydrate (CHO) import or polyphenol accumulation in target branches. ? We conclude that poplar branches comprise modules that are relatively independent from each other and from the stem below in terms of CHO movement, carbon-based defence production and response to elicitors. By contrast, branches are closely linked modules in terms of nitrogen movement. This should produce trees that are highly heterogeneous in quality for herbivores.     

Generation of reference cell lines,media, and a process platform for CHO cell biomanufacturing

Due to the favorable attributes of Chinese hamster ovary (CHO) cells for therapeutic proteins and antibodies biomanufacturing, companies generate proprietary cells with desirable phenotypes. One key attribute is the ability to stably express multi-gram per liter titers in chemically defined media. Cell, media, and feed diversity has limited community efforts to translate knowledge. Moreover, academic, and nonprofit researchers generally cannot study “industrially relevant” CHO cells due to limited public availability, and the time and knowledge required to generate such cells. To address these issues, a university-industrial consortium (Advanced Mammalian Biomanufacturing Innovation Center, AMBIC) has acquired two CHO “reference cell lines” from different lineages that express monoclonal antibodies. These reference cell lines have relevant production titers, key performance outcomes confirmed by multiple laboratories, and a detailed technology transfer protocol. In commercial media, titers over 2 g/L are reached. Fed-batch cultivation data from shake flask and scaled-down bioreactors is presented. Using productivity as the primary attribute, two academic sites aligned with tight reproducibility at each site. Further, a chemically defined media formulation was developed and evaluated in parallel to the commercial media. The goal of this work is to provide a universal, industrially relevant CHO culture platform to accelerate biomanufacturing innovation.     

Antibody-dependent enhancement of Ebola virus infection

Most strains of Ebola virus cause a rapidly fatal hemorrhagic disease in humans, yet there are still no biologic explanations that adequately account for the extreme virulence of these emerging pathogens. Here we show that Ebola Zaire virus infection in humans induces antibodies that enhance viral infectivity. Plasma or serum from convalescing patients enhanced the infection of primate kidney cells by the Zaire virus, and this enhancement was mediated by antibodies to the viral glycoprotein and by complement component C1q. Our results suggest a novel mechanism of antibody-dependent enhancement of Ebola virus infection, one that would account for the dire outcome of Ebola outbreaks in human populations.     

Protection from Ebola virus mediated by cytotoxic T lymphocytes specific for the viral nucleoprotein

Cytotoxic T lymphocytes (CTLs) are proposed to be critical for protection from intracellular pathogens such as Ebola virus. However, there have been no demonstrations that protection against Ebola virus is mediated by Ebola virus-specific CTLs. Here, we report that C57BL/6 mice vaccinated with Venezuelan equine encephalitis virus replicons encoding the Ebola virus nucleoprotein (NP) survived lethal challenge with Ebola virus. Vaccination induced both antibodies to the NP and a major histocompatibility complex class I-restricted CTL response to an 11-amino-acid sequence in the amino-terminal portion of the Ebola virus NP. Passive transfer of polyclonal NP-specific antiserum did not protect recipient mice. In contrast, adoptive transfer of CTLs specific for the Ebola virus NP protected unvaccinated mice from lethal Ebola virus challenge. The protective CTLs were CD8(+), restricted to the D(b) class I molecule, and recognized an epitope within amino acids 43 to 53 (VYQVNNLEEIC) in the Ebola virus NP. The demonstration that CTLs can prevent lethal Ebola virus infection affects vaccine development in that protective cellular immune responses may be required for optimal protection from Ebola virus.     

Utilizing targeted integration CHO pools to potentially accelerate the GMP manufacturing of monoclonal and bispecific antibodies

Monoclonal antibodies (mAbs) are effective therapeutic agents against many acute infectious diseases including COVID-19, Ebola, RSV, Clostridium difficile, and Anthrax. mAbs can therefore help combat a future pandemic. Unfortunately, mAb development typically takes years, limiting its potential to save lives during a pandemic. Therefore “pandemic mAb” timelines need to be shortened. One acceleration tool is “deferred cloning” and leverages new Chinese hamster ovary (CHO) technology based on targeted gene integration (TI). CHO pools, instead of CHO clones, can be used for Phase I/II clinical material production. A final CHO clone (producing the mAb with a similar product quality profile and preferably with a higher titer) can then be used for Phase III trials and commercial manufacturing. This substitution reduces timelines by ~3 months. We evaluated our novel CHO TI platform to enable deferred cloning. We created four unique CHO pools expressing three unique mAbs (mAb1, mAb2, and mAb3), and a bispecific mAb (BsAb1). We then performed single-cell cloning for mAb1 and mAb2, identifying three high-expressing clones from each pool. CHO pools and clones were inoculated side-by-side in ambr15 bioreactors. CHO pools yielded mAb titers as high as 10.4 g/L (mAb3) and 7.1 g/L (BsAb1). Subcloning yielded CHO clones expressing higher titers relative to the CHO pools while yielding similar product quality profiles. Finally, we showed that CHO TI pools were stable by performing a 3-month cell aging study. In summary, our CHO TI platform can increase the speed to clinic for a future “pandemic mAb.”     

Enhancement of sialylation on humanized IgG-like bispecific antibody by overexpression of α2,6-sialyltransferase derived from Chinese hamster ovary cells

Improvement of glycosylation is one of the most important topics in the industrial production of therapeutic antibodies. We have focused on terminal sialylation with alpha-2,6 linkage, which is crucial for anti-inflammatory activity. In the present study, we have successfully cloned cDNA of beta-galactosyl alpha-2,6 sialyltransferase (ST6Gal I) derived from Chinese hamster ovary (CHO) cells regardless of reports that stated this was not endogenously expressed in CHO cells. After expressing cloned ST6Gal I in Escherichia coli, the transferase activity was confirmed by HPLC and lectin binding assay. Then, we applied ST6Gal I to alpha-2,6 sialylation of the recombinant antibody; the ST6Gal I expression vector was transfected into the CHO cell line producing a bispecific antibody. The N-glycosylation pattern of the antibody was estimated by HPLC and sialidase digestion. About 70% of the total N-linked oligosaccharide was alpha-2,6 sialylated in the transfected cell line whereas no sialylation was observed in the non-transfected cell line. The improvement of sialylation would be of practical importance for the industrial production of therapeutic antibodies.     

Evaluating Subcriticality during the Ebola Epidemic in West Africa

The 2014–2015 Ebola outbreak is the largest and most widespread to date. In order to estimate ongoing transmission in the affected countries, we estimated the weekly average number of secondary cases caused by one individual infected with Ebola throughout the infectious period for each affected West African country using a stochastic hidden Markov model fitted to case data from the World Health Organization. If the average number of infections caused by one Ebola infection is less than 1.0, the epidemic is subcritical and cannot sustain itself. The epidemics in Liberia and Sierra Leone have approached subcriticality at some point during the epidemic; the epidemic in Guinea is ongoing with no evidence that it is subcritical. Response efforts to control the epidemic should continue in order to eliminate Ebola cases in West Africa.     

Accelerating genome editing in CHO cells using CRISPR Cas9 and CRISPy,a web‐based target finding tool

Chinese hamster ovary (CHO) cells are widely used in the biopharmaceutical industry as a host for the production of complex pharmaceutical proteins. Thus genome engineering of CHO cells for improved product quality and yield is of great interest. Here, we demonstrate for the first time the efficacy of the CRISPR Cas9 technology in CHO cells by generating site‐specific gene disruptions in COSMC and FUT8, both of which encode proteins involved in glycosylation. The tested single guide RNAs (sgRNAs) created an indel frequency up to 47.3% in COSMC, while an indel frequency up to 99.7% in FUT8 was achieved by applying lectin selection. All eight sgRNAs examined in this study resulted in relatively high indel frequencies, demonstrating that the Cas9 system is a robust and efficient genome‐editing methodology in CHO cells. Deep sequencing revealed that 85% of the indels created by Cas9 resulted in frameshift mutations at the target sites, with a strong preference for single base indels. Finally, we have developed a user‐friendly bioinformatics tool, named “CRISPy” for rapid identification of sgRNA target sequences in the CHO‐K1 genome. The CRISPy tool identified 1,970,449 CRISPR targets divided into 27,553 genes and lists the number of off‐target sites in the genome. In conclusion, the proven functionality of Cas9 to edit CHO genomes combined with our CRISPy database have the potential to accelerate genome editing and synthetic biology efforts in CHO cells. Biotechnol. Bioeng. 2014; 111: 1604–1616. © 2014 The Authors. Biotechnology and Bioengineering Published by Wiley Periodicals, Inc.     

Proteomics for everyday use: activities of the HUPO Brain Proteome Project during the 5th HUPO World Congress

Long Beach hosted this year's annual congress of the Human Proteome Organisation (HUPO). In addition to the numerous sessions, talks and poster presentations organized by HUPO itself, several events were arranged by the HUPO initiatives. The Brain Proteome Project (HUPO BPP) was very active, initiating three pre-congress workshops: (i) the kick-off meeting of the EU-funded ProDaC consortium (Proteomics Data Collection) that is aiming at the bioinformatics Standardization in the proteomics field; (ii) the workshop "Standardization Issues in Proteomics: Perspectives from Vendors" giving an overview about the lessons learned by proteomics industrial partners; (iii) the 6th HUPO BPP Workshop "New Proteomics Approaches for further HUPO BPP Studies" offering new concepts for brain-related proteomics studies.     

Metabolic flux analysis of CHO cells at growth and non-growth phases using isotopic tracers and mass spectrometry

Chinese hamster ovary (CHO) cells are the main platform for production of biotherapeutics in the biopharmaceutical industry. However, relatively little is known about the metabolism of CHO cells in cell culture. In this work, metabolism of CHO cells was studied at the growth phase and early stationary phase using isotopic tracers and mass spectrometry. CHO cells were grown in fed-batch culture over a period of six days. On days 2 and 4, [1,2-¹³C] glucose was introduced and the labeling of intracellular metabolites was measured by gas chromatography-mass spectrometry (GC–MS) at 6, 12 and 24 h following the introduction of tracer. Intracellular metabolic fluxes were quantified from measured extracellular rates and ¹³C-labeling dynamics of intracellular metabolites using non-stationary ¹³C-metabolic flux analysis (¹³C-MFA). The flux results revealed significant rewiring of intracellular metabolic fluxes in the transition from growth to non-growth, including changes in energy metabolism, redox metabolism, oxidative pentose phosphate pathway and anaplerosis. At the exponential phase, CHO cell metabolism was characterized by a high flux of glycolysis from glucose to lactate, anaplerosis from pyruvate to oxaloacetate and from glutamate to α-ketoglutarate, and cataplerosis though malic enzyme. At the stationary phase, the flux map was characterized by a reduced flux of glycolysis, net lactate uptake, oxidative pentose phosphate pathway flux, and reduced rate of anaplerosis. The fluxes of pyruvate dehydrogenase and TCA cycle were similar at the exponential and stationary phase. The results presented here provide a solid foundation for future studies of CHO cell metabolism for applications such as cell line development and medium optimization for high-titer production of recombinant proteins.     

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.