Exploring Viral Diversity in a Unique South African Soil Habitat

Abstract 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

Evaluation of plant-produced Clostridium perfringens type D epsilon toxoid in a vaccine against enterotoxaemia in sheep

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.Onderstepoort Biological Products (OBP), the CSIR Parliamentary Grant and the Technology Innovation Agency (TIA) of the Republic of South Africa.http://www.ojvr.orgam2017Plant Scienc

The study of degradation mechanisms of glyco-engineered plant produced anti-rabies monoclonal antibodies E559 and 62-71-3

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

Engineering enhanced thermostability into the Geobacillus pallidus nitrile hydratase

Nitrile hydratases (NHases) are important biocatalysts for the enzymatic conversion of nitriles to industrially-important amides such as acrylamide and nicotinamide. Although thermostability in this enzyme class is generally low, there is not sufficient understanding of its basis for rational enzyme design. The gene expressing the Co-type NHase from the moderate thermophile, Geobacillus pallidus RAPc8 (NRRL B-59396), was subjected to random mutagenesis. Four mutants were selected that were 3 to 15-fold more thermostable than the wild-type NHase, resulting in a 3.4–7.6 ​kJ/mol increase in the activation energy of thermal inactivation at 63 ​°C. High resolution X-ray crystal structures (1.15–1.80 ​Å) were obtained of the wild-type and four mutant enzymes. Mutant 9E, with a resolution of 1.15 ​Å, is the highest resolution crystal structure obtained for a nitrile hydratase to date. Structural comparisons between the wild-type and mutant enzymes illustrated the importance of salt bridges and hydrogen bonds in enhancing NHase thermostability. These additional interactions variously improved thermostability by increased intra- and inter-subunit interactions, preventing cooperative unfolding of α-helices and stabilising loop regions. Some hydrogen bonds were mediated via a water molecule, specifically highlighting the significance of structured water molecules in protein thermostability. Although knowledge of the mutant structures makes it possible to rationalize their behaviour, it would have been challenging to predict in advance that these mutants would be stabilising.The Royal Society (UK) and the National Research Foundation (South Africa) in the form of a Collaborative Research Project grant; a UK Global Challenge Research Fund grant: START- Synchrotron Techniques for African Research and Technology.https://www.sciencedirect.com/journal/current-research-in-structural-biologyhj2023BiochemistryGeneticsMicrobiology and Plant Patholog

Transient proteolysis reduction of Nicotiana benthamiana-produced CAP256 broadly neutralizing antibodies using CRISPR/Cas9

The hypersensitive response is elicited by Agrobacterium infiltration of Nicotiana benthamiana, including the induction and accumulation of pathogenesis-related proteins, such as proteases. This includes the induction of the expression of several cysteine proteases from the C1 (papain-like cysteine protease) and C13 (legumain-like cysteine protease) families. This study demonstrates the role of cysteine proteases: NbVPE-1a, NbVPE-1b, and NbCysP6 in the proteolytic degradation of Nicotiana benthamiana (glycosylation mutant 1XTFT)-produced anti-human immunodeficiency virus broadly neutralizing antibody, CAP256-VRC26.25. Three putative cysteine protease cleavage sites were identified in the fragment crystallizable region. We further demonstrate the transient coexpression of CAP256-VRC26.25 with CRISPR/Cas9-mediated genome editing vectors targeting the NbVPE-1a, NbVPE-1b, and NbCysP6 genes which resulted in a decrease in CAP256-VRC26.25 degradation. No dierences in structural features were observed between the human embryonic kidney 293 (HEK293)-produced and 1XTFT broadly neutralizing antibodies produced with and without the coexpression of genome-editing vectors. Furthermore, despite the presence of proteolytically degraded fragments of plant-produced CAP256-VRC26.25 without the coexpression of genome editing vectors, no influence on the in vitro functional activity was detected. Collectively, we demonstrate an innovative in planta strategy for improving the quality of the CAP256 antibodies through the transient expression of the CRISPR/Cas9 vectors.The Department of Science and Innovation (DSI), South African Medical Research Council– Strategic Health Innovation Partnership (SAMRC SHIP), National Research Foundation (NRF), Council for Scientific and Industrial Research (CSIR), and the CSIR: Young Researcher Establishment Fund.http://www.frontiersin.org/Plant_Sciencedm2022Plant Production and Soil Scienc

Engineering, expression in transgenic plants and characterisation of e559, a rabies virus-neutralising monoclonal antibody.

Rabies post-exposure prophylaxis (PEP) currently comprises administration of rabies vaccine together with rabies immunoglobulin (RIG) of either equine or human origin. In the developing world, RIG preparations are expensive, often in short supply, and of variable efficacy. Therefore, we are seeking to develop a monoclonal antibody cocktail to replace RIG. Here, we describe the cloning, engineering and production in plants of a candidate monoclonal antibody (E559) for inclusion in such a cocktail. The murine constant domains of E559 were replaced with human IgG1κ constant domains and the resulting chimeric mouse-human genes were cloned into plant expression vectors for stable nuclear transformation of Nicotiana tabacum. The plant-expressed, chimeric antibody was purified and biochemically characterized, was demonstrated to neutralize rabies virus in a fluorescent antibody virus neutralization assay, and conferred protection in a hamster challenge model

Investigating the role of a yeast membrane protein, HSP30 in tolerance to ethanol stress

Bibliography: leaves 59-64.One of the contributors of the widespread interest the yeast Saccharomyces cerevisiae has received is its ability to yield and tolerate high levels of ethanol. S. cerevisiae is able to grow and remain viable in growth media containing ethanol concentrations as high as 19.8% (w/v), a level that is toxic to many other microorganisms. Since production of ethanol is a normal event in the growth cycyle of S. cerevisiae, this organisms has evolved a number of mechanisms to cope with deleterious effects of ethanol. These include induction of heat shock proteins (HSPs). Among these, HSP30 is particularly interesting in that it is the only stress-induced protein known to be instrinsically bound to the yeast plasma membrane. Another ethanol induced HSP; HSP12 has previously been shown to have a peripheral plasma membrane localisation. It has further been shown that HSP12 protects liposomes against damage by ethanol. This study was initially aimed at investigating whether there is co-operation between HSP30 and HSP12 in this membrane protection role

Structure, enzymology and genetic engineering of Bacillus sp. RAPc8 nitrile hydratase

Philosophiae Doctor - PhDMicrobial nitrile hydratases (NHases) are important industrial enzymes that catalyse the conversion of nitriles to the corresponding amides. A thermostable, cobalt-type Bacillus sp. RAPc8 NHase was previously cloned and expressed in E. coli. In this study, the primary aim was to determine the molecular structure of Bacillus sp. RAPc8 NHase. The heterotetrameric enzyme was purified to near homogeneity using heatpurification, hydrophobic interaction chromatography and ion exchange chromatography. Purified NHase was crystallised using the hanging-drop vapourdiffusion method. Crystals produced in the presence of 30% PEG 400, 0.1M MES pH 6.5 and 0.1M magnesium chloride were selected for X-ray diffraction studies. These crystals diffracted well, with diffraction spots visible beyond 2.4Å, with little mosaicity. At 2.5Å, the data were 93% complete. The crystal structure of Bacillus sp. RAPc8 NHase was solved via molecular replacement using the crystal structure of Pseudonocardia thermophila NHase as a search model. The final refined structure had good refinement statistics and geometry. The overall fold was very similar to that of previously determined NHase structures. Bacillus sp. RAPc8 NHase was most similar to Bacillus smithii NHase (0.355År.m.s.d.) and least similar to Rhodococcus sp. R312 NHase (1.191Å r.m.s.d.). One cobalt atom per heterodimer was bound to a typical NHase metal-binding motif, with post-translationally modified cysteine residues among the ligands to the metal. The substrate-binding and catalytic cavity of Bacillus sp. RAPc8 NHase was identified and described in detail. Surface representation of the structure revealed an extended, curved solvent accessible channel with access to bulk solvent from two locations in the heterodimer. The amino-acid residues forming the channel were identified and the geometric dimensions measured. Enzyme inhibition kinetics indicated that benzonitrile was a potent uncompetitive inhibitor of NHase. This information was used to aid the genetic engineering of aromatic substrate specificity into Bacillus sp. RAPc8 NHase. Site-directed mutants of NHase were prepared using the Quickchange mutagenesis procedure. Mutant W76G showed a two to three fold decrease in benzonitrile inhibition compared with the wild-type. Analysis of the substrate channel of this mutant NHase showed an 11% increase in volume and a 20% increase in inner surface area compared to that of the wild-type NHase. Due to the lack of other significant differences between the two structures (an r.m.s.d. of only 0.101Å was observed), this difference was thought to be responsible for the decrease in benzonitrile inhibition. A structure-modelling based approach for assessing the likely structural differences that may result as a result of a specific mutation was suggested and tested. This approach may be of value in future mutagenesis work.South Afric

Engineering Approaches in Plant Molecular Farming for Global Health

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

Engineering Approaches in Plant Molecular Farming for Global Health

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