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Broadly neutralising antibodies (bNAbs) against human immunodeficiency virus type 1 (HIV-1), such as CAP256-VRC26 are being developed for HIV prevention and treatment. These Abs carry a unique but crucial post-translational modification (PTM), namely O-sulfated tyrosine in the heavy chain complementarity determining region (CDR) H3 loop. Several studies have demonstrated that plants are suitable hosts for the generation of highly active anti-HIV-1 antibodies with the potential to engineer PTMs. Here we report the expression and characterisation of CAP256-VRC26 bNAbs with posttranslational modifications (PTM). Two variants, CAP256-VRC26 (08 and 09) were expressed in glycoengineered Nicotiana benthamiana plants. By in planta co-expression of tyrosyl protein sulfotransferase 1, we installed O-sulfated tyrosine in CDR H3 of both bNAbs. These exhibited similar structural folding to the mammalian cell produced bNAbs, but non-sulfated versions showed loss of neutralisation breadth and potency. In contrast, tyrosine sulfated versions displayed equivalent neutralising activity to mammalian produced antibodies retaining exceptional potency against some subtype C viruses. Together, the data demonstrate the enormous potential of plant-based systems for multiple posttranslational engineering and production of fully active bNAbs for application in passive immunisation or as an alternative for current HIV/AIDS antiretroviral therapy regimens.

Local manufacturing of protein-based vaccines and therapies in Africa is limited and contributes to a trade deficit, security of supply concerns and poor access to biopharmaceuticals by the poor. Plant molecular farming is a potential technology solution that has received growing adoption by African scientists attracted by the potential for the competitive cost of goods, safety and efficacy. Plant-made pharmaceutical technologies for veterinary and human vaccination and treatment of non-communicable and infectious diseases are available at different stages of development in Africa. There is also growth in the translation of these technologies to commercial operations. Africa is poised to benefit from the real-world impact of molecular farming in the next few years.

Since the demonstration of the first plant-produced proteins of medical interest, there has been significant growth and interest in the field of plant molecular farming, with plants now being considered a viable production platform for vaccines. Despite this interest and development by a few biopharmaceutical companies, plant molecular farming is yet to be embraced by ‘big pharma’. The plant system offers a faster alternative, which is a potentially more cost-effective and scalable platform for the mass production of highly complex protein vaccines, owing to the high degree of similarity between the plant and mammalian secretory pathway. Here, we identify and address bottlenecks in the use of plants for vaccine manufacturing and discuss engineering approaches that demonstrate both the utility and versatility of the plant production system as a viable biomanufacturing platform for global health. Strategies for improving the yields and quality of plant-produced vaccines, as well as the incorporation of authentic posttranslational modifications that are essential to the functionality of these highly complex protein vaccines, will also be discussed. Case-by-case examples are considered for improving the production of functional protein-based vaccines. The combination of all these strategies provides a basis for the use of cutting-edge genome editing technology to create a general plant chassis with reduced host cell proteins, which is optimised for high-level protein production of vaccines with the correct posttranslational modifications.

Endonucleases play a crucial role as reagents in laboratory research and diagnostics. Here, metagenomics was used to functionally screen a fosmid library for endonucleases. A fosmid library was constructed using metagenomic DNA isolated from soil sampled from the unique environment of the Kogelberg Nature Reserve in the Western Cape of South Africa. The principle of acquired immunity against phage infection was used to develop a plate-based screening technique for the isolation of restriction endonucleases from the library. Using next-generation sequencing and bioinformatics tools, sequence data were generated and analysed, revealing 113 novel open reading frames (ORFs) encoding putative endonuclease genes and ORFs of unknown identity and function. One endonuclease designated Endo52 was selected from the putative endonuclease ORFs and was recombinantly produced in Escherichia coli Rosetta™ (DE3) pLysS. Endo52 was purified by immobilised metal affinity chromatography and yielded 0.437 g per litre of cultivation volume. Its enzyme activity was monitored by cleaving lambda DNA and pUC19 plasmid as substrates, and it demonstrated non-specific endonuclease activity. In addition to endonuclease-like genes, the screen identified several unknown genes. These could present new phage resistance mechanisms and are an opportunity for future investigations.

Met die toenemende voorkoms van weerstandige Plasmodium stamme het die beheer van malaria-voorkoms en -mortaliteit weer op die voorgrond getree. Nuwe teikens en antimalariamiddels wat effektief is teen weerstandige malaria-parasiete word dus dringend benodig. Kalsium-afhanklike proteïenkinases (calcium dependent protein kinases – CDPKs) is betrokke by die beheer van ’n aantal biologiese prosesse in die malaria-parasiet, Plasmodium falciparum, met CDPK4 die belangrikste ensiem in hierdie klas. In hierdie studie is die struktuur van PfCDPK4 gebruik as templaat vir die soeke na nuwe malariamiddels. Die PfCDPK4 modelstruktuur is deur middel van homologiemodellering gegenereer en die stereochemiese kwaliteit gevalideer. Die molekulêre modelleringbenadering deur middel van in silico sifting teen die teiken-molekuul PfCDPK4 het ’n beskeie biblioteek van 20 000 chemiese verbindings ingesluit, asook ’n aantal aktiewe natuurprodukte en kliniesgoedgekeurde kinase-inhibeerders. In silico sifting van die Biofocus biblioteek teen PfCDPK4 het 26 verbindings opgelewer; in vitro sifting het bevestig dat drie van hierdie verbindings matig aktief is teen Plasmodium falciparum NF54, met persentasie inhibisie tussen 42% en 47%.

Aims To investigate the production of soluble Cross-Reacting Material 197 (CRM197) in Escherichia coli, a safe and effective T-cell dependent protein carrier for polysaccharides used in the manufacture and application of multivalent conjugate vaccines. Methods and Results The use of co-expression of a sulfhydryl oxidase and protein disulfide isomerase for the production of soluble CRM197 in E. coli is described. CRM197 contains two disulfide bonds, which are normally unable to form in the reducing environment of the E. coli cytoplasm. It was found that co-expression yielded soluble CRM197, at a production rate ~10% of the production of insoluble CRM197, in equivalent small-scale cultures. Structural analysis of the purified CRM197 compared to CRM197 commercially produced in cultures of recombinant Pseudomonas fluorescens indicated that the E. coli soluble protein compares favorably on all structural levels. Conclusions Sulfhydryl oxidase and protein disulfide isomerase are enzymes involved in the formation of intra-protein disulfide bonds, and can influence the tertiary structure of the protein being produced, resulting in increased solubility due to the correct folding of the protein. Their use enabled the production of soluble untagged CRM197 in E. coli, which was previously unachievable. Significance and Impact of Study Previous literature reports have shown that CRM197 can be expressed in Escherichia coli, though only in an insoluble form, or in soluble form as a fusion protein. It is currently commercially produced in cultures of recombinant Pseudomonas fluorescens. The use of a widely-used, well-characterized expression host such as E. coli, rather than P. fluorescens broadens the applicability of the production technology, and the production system described here is worthy of further investigation for scaled up manufacture of CRM197. This article is protected by copyright. All rights reserved.

Background African horse sickness (AHS) is a severe arthropod-borne viral disease of equids, with a mortality rate of up to 95% in susceptible naïve horses. Due to safety concerns with the current live, attenuated AHS vaccine, alternate safe and effective vaccination strategies such as virus-like particles (VLPs) are being investigated. Transient plant-based expression systems are a rapid and highly scalable means of producing such African horse sickness virus (AHSV) VLPs for vaccine purposes. Results In this study, we demonstrated that transient co-expression of the four AHSV capsid proteins in agroinfiltrated Nicotiana benthamiana dXT/FT plants not only allowed for the assembly of homogenous AHSV-1 VLPs but also single, double and triple chimeric VLPs, where one capsid protein originated from one AHS serotype and at least one other capsid protein originated from another AHS serotype. Following optimisation of a large scale VLP purification procedure, the safety and immunogenicity of the plant-produced, triple chimeric AHSV-6 VLPs was confirmed in horses, the target species. Conclusions We have successfully shown assembly of single and double chimeric AHSV-7 VLPs, as well as triple chimeric AHSV-6 VLPs, in Nicotiana benthamiana dXT/FT plants. Plant produced chimeric AHSV-6 VLPs were found to be safe for administration into 6 month old foals as well as capable of eliciting a weak neutralizing humoral immune response in these target animals against homologous AHSV virus.

Bluetongue (BT) is a hemorrhagic non-contagious, biting midge-transmitted disease of wild and domestic ruminants that is caused by bluetongue virus (BTV). Annual vaccination plays a pivotal role in BT disease control in endemic regions. Due to safety concerns of the current BTV multivalent live attenuated vaccine (LAV), a safe efficacious new generation subunit vaccine such as a plant-produced BT virus-like particle (VLP) vaccine is imperative. Previously, homogenous BTV serotype 8 (BTV-8) VLPs were successfully produced in Nicotiana benthamiana plants and provided protective immunity in sheep. In this study, combinations of BTV capsid proteins from more than one serotype were expressed and assembled to form chimaeric BTV-3 and BTV-4 VLPs in N. benthamiana plants. The assembled homogenous BTV-8, as well as chimaeric BTV-3 and chimaeric BTV-4 VLP serotypes, were confirmed by SDS-PAGE, Transmission Electron microscopy (TEM) and protein confirmation using liquid chromatography-mass spectrometry (LC-MS/MS) based peptide sequencing. As VP2 is the major determinant eliciting protective immunity, the percentage coverage and number of unique VP2 peptides detected in assembled chimaeric BT VLPs were used as a guide to assemble the most appropriate chimaeric combinations. Both plant-produced chimaeric BTV-3 and BTV-4 VLPs were able to induce long-lasting serotype-specific neutralizing antibodies equivalent to the monovalent LAV controls. Antibody levels remained high to the end of the trial. Combinations of homogenous and chimaeric BT VLPs have great potential as a safe, effective multivalent vaccine with the ability to distinguish between vaccinated and infected individuals (DIVA) due to the absence of non-structural proteins.

Rabies is an acute viral encephalomyelitis in warm-blooded vertebrates, caused by viruses belonging to Rhabdovirus family and genus Lyssavirus. Although rabies is categorised as a neglected disease, the rabies virus (RABV) is the most studied amongst Lyssaviruses which show nearly identical infection patterns. In efforts to improving post-exposure prophylaxis, several anti-rabies monoclonal antibodies (mAbs) targeting the glycoprotein (G protein) sites I, II, III and G5 have been characterized. To explore cross-neutralization capacity of available mAbs and discover new possible B-cell epitopes, we have analyzed all available glycoprotein sequences from Lyssaviruses with a focus on sequence variation and conservation. This information was mapped on the structure of a representative G protein. We proposed several possible cross-neutralizing B-cell epitopes (GUVTTTF, WLRTV, REECLD and EHLVVEEL) in complement to the already well-characterized antigenic sites. The research could facilitate development of novel cross-reactive mAbs against RABV and even more broad, against possibly all Lyssavirus members.

African horse sickness (AHS) is caused by multiple serotypes of the dsRNA AHSV and is a major scourge of domestic equids in Africa. While there are well established commercial live attenuated vaccines produced in South Africa, risks associated with these have encouraged attempts to develop new and safer recombinant vaccines. Previously, we reported on the immunogenicity of a plant-produced AHS serotype 5 virus-like particle (VLP) vaccine, which stimulated high titres of AHS serotype 5-specific neutralizing antibodies in guinea pigs. Here, we report a similar response to the vaccine in horses. This is the first report demonstrating the safety and immunogenicity of plant-produced AHS VLPs in horses. Electronic supplementary material The online version of this article (10.1186/s13567-018-0600-4) contains supplementary material, which is available to authorized users.

The Kogelberg Biosphere Reserve in the Cape Floral Kingdom in South Africa is known for its unique plant biodiversity. The potential presence of unique microbial and viral biodiversity associated with this unique plant biodiversity led us to explore the fynbos soil using metaviromic techniques. In this study, metaviromes of a soil community from the Kogelberg Biosphere Reserve has been characterised in detail for the first time. Metaviromic DNA was recovered from soil and sequenced by Next Generation Sequencing. The MetaVir, MG-RAST and VIROME bioinformatics pipelines were used to analyse taxonomic composition, phylogenetic and functional assessments of the sequences. Taxonomic composition revealed members of the order Caudovirales, in particular the family Siphoviridae, as prevalent in the soil samples and other compared viromes. Functional analysis and other metaviromes showed a relatively high frequency of phage-related and structural proteins. Phylogenetic analysis of PolB, PolB2, terL and T7gp17 genes indicated that many viral sequences are closely related to the order Caudovirales, while the remainder were distinct from known isolates. The use of single virome which only includes double stranded DNA viruses limits this study. Novel phage sequences were detected, presenting an opportunity for future studies aimed at targeting novel genetic resources for applied biotechnology.

Enterotoxaemia (pulpy kidney) is a common bacterial disease of sheep caused by Clostridium perfringens type D epsilon toxin. It has mortality rates of up to 30% in non-vaccinated animals. Current vaccines from whole cell cultures are expensive to manufacture and can induce local inflammatory responses in sheep. They usually have reduced immunogenicity because of the difficulty of standardising the inactivation step in vaccine manufacturing. In the current study, we evaluated the safety and potency of a recombinant plant-made epsilon toxoid protein (r-Etox) as an affordable and safer alternative vaccine for developing countries. Results of injection site reactions, rectal temperature and toxin neutralisation test in single and prime– boost inoculations of mice, guinea pigs and sheep suggest that the product is not toxic to animals and could protect sheep against enterotoxaemia.

Family VIII esterases represent a poorly characterised esterase family, with high sequence identity to class C β-lactamases, peptidases and penicillin binding proteins. This study reports on the metagenomic library screening and biochemical characterisation of a novel esterase (Est22) derived from an acidic Leachate environment. The enzyme is 423 amino acids in length and contained 22 aa signal peptide. The Est22 primary structure revealed the presence of N-terminus S-x-x-K sequence, which is also highly conserved in class C β-lactamases, peptidases as well as carboxylesterases belonging to family VIII. Phylogenetic analysis using the representative sequences from class C β-lactamases and family VIII esterases indicated that Est22 is a member of family VIII esterases. Substrate specificity profiling using p-nitrophenyl esters (C2–C16) indicated that Est22 preferred shorter chain p-nitrophenyl esters (C2–C5), a characteristic that is typical for true carboxylesterases. In addition of hydrolysing Nitrocefin, Est22 also hydrolysed first generation cephalosporin based derivatives. Detailed selectivity study using different cephalosporin based substrates indicated that Est22 selectively hydrolyse the ester bond of a cephalosporin derivatives leaving the amide bond of the β-lactam ring intact. The selective nature of Est22 makes this enzyme a potential candidate for the use in the synthesis and modification cephalosporin based molecules.

Rabies is an ancient and neglected zoonotic disease caused by the rabies virus, a neurotropic RNA virus that belongs to the Rhabdoviridae family, genus Lyssavirus. It remains an important public health problem as there are cost and health concerns imposed by the current human post exposure prophylaxis therapy. The use of monoclonal antibodies (mAbs) is therefore an attractive alternative. Rabies mostly affects people that reside in resource-limited areas where there are occasional failures in the cold-chain. These environmental changes may upset the stability of the mAbs. This study focused on mAbs 62-71-3 and E559; their structures, responses to freeze/thaw (F/T) and exposure to reactive oxygen species were therefore studied with the aid of a wide range of biophysical and in silico techniques in order to elucidate their stability and identify aggregation prone regions. E559 was found to be less stable than 62-71-3. The complementarity determining regions (CDR) contributed the most to its instability, more specifically: peptides (EIWD102)-E-99 and (92)ATSPYT(97) found in CDR3, Trp33 found in CDR1 and the oxidised Met34. The constant region "(158)SWNSGALTGHTFPAVL(175)" was also flagged by the special aggregation propensity (SAP) tool and F/T experiments to be highly prone to aggregation. The E559 peptides "(4)LQESGSVL(11) from the heavy chain and (4)LTQSPSSL(11) from the light chain, were also highly affected by F/T. These residues may serve as good candidates for mutation, in the aim to bring forward more stable therapeutic antibodies, thus paving a way to a more safe and efficacious antibody-based cocktail treatment against rabies.

Rabies is a neglected zoonotic disease that has no effective treatment after onset of illness. However the disease can be prevented effectively by prompt administration of post exposure prophylaxis which includes administration of passive immunizing antibodies (Rabies Immune Globulin, RIG). Currently, human RIG suffers from many restrictions including limited availability, batch-to batch inconsistencies and potential for contamination with blood-borne pathogens. Anti-rabies monoclonal antibodies (mAbs) have been identified as a promising alternative to RIG. Here, we applied a plant-based transient expression system to achieve rapid, high level production and efficacy of the two highly potent anti-rabies mAbs E559 and 62-71-3. Expression levels of up to 490 mg/kg of recombinant mAbs were obtained in Nicotiana benthamiana glycosylation mutants by using a viral based transient expression system. The plant-made E559 and 62-71-3, carrying human-type fucose-free N-glycans, assembled properly and were structurally sound as determined by mass spectrometry and calorimetric density measurements. Both mAbs efficiently neutralised diverse rabies virus variants in vitro. Importantly, E559 and 62-71-3 exhibited enhanced protection against rabies virus compared to human RIG in a hamster model post-exposure challenge trial. Collectively, our results provide the basis for the development of a multi-mAb based alternative to RIG.

Publisher Summary There are two distinct pathways for the conversion of nitriles to carboxylic acids in nature. The nitrilase (NTase) pathway catalyzes the direct conversion of a nitrile to its corresponding carboxylic acid, whereas the nitrile hydratase (NHase) pathway catalyzes the conversion of a nitrile to its corresponding amide, which is then converted to carboxylic acid by an amidase. A large number of NHases have been detected in a variety of microorganisms. The genus Rhodococcus dominates the collective of known mesophilic NHase-producing microorganisms, and only five thermophilic NHase-producing organisms have thus far been described. Despite significant differences in origin, stability, cofactor requirements, and catalytic characteristics between the various NHase subgroups, there is a high degree of similarity between these enzymes in terms of size and protein sequence. The chapter discusses the reaction mechanism of NHase. Three reaction models involving the metal ion as a Lewis acid have been proposed. These are direct association of nitrile to metal cofactor, nucleophilic attack by a metal-bound hydroxide ion, and nucleophilic attack by an activated water molecule.