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.
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.
Customer demands for fresh salads are increasing, but leafy green vegetables have also been linked to food‐borne illness due to pathogens such as Escherichia coli O157:H7. As a safety measure, consumers often wash leafy vegetables in water before consumption. In this study, we analyzed the efficiency of household washing to reduce the bacterial content. Romaine lettuce and ready‐to‐eat mixed salad were washed several times in flowing water at different rates and by immersing the leaves in water. Lettuce was also inoculated with E. coli before washing. Only washing in a high flow rate (8 L/min) resulted in statistically significant reductions (p < .05), “Total aerobic count” was reduced by 80%, and Enterobacteriaceae count was reduced by 68% after the first rinse. The number of contaminating E. coli was not significantly reduced. The dominating part of the culturable microbiota of the washed lettuce was identified by rRNA 16S sequencing of randomly picked colonies. The majority belonged to Pseudomonadaceae, but isolates from Enterobacteriaceae and Staphylococcaceaceae were also frequently found. This study shows the inefficiency of tap water washing methods available for the consumer when it comes to removal of bacteria from lettuce. Even after washing, the lettuce contained high levels of bacteria that in a high dose and under certain circumstances may constitute a health risk.
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.
SummaryGriffithsin (GRFT) is an antiviral lectin, originally derived from a red alga, which is currently being investigated as a topical microbicide to prevent transmission of human immunodeficiency virus (HIV). Targeting GRFT to the apoplast for production in Nicotiana benthamiana resulted in necrotic symptoms associated with a hypersensitive response (HR)‐like cell death, accompanied by H2O2 generation and increased PR1 expression. Mannose‐binding lectins surfactant protein D (SP‐D), cyanovirin‐N (CV‐N) and human mannose‐binding lectin (hMBL) also induce salicylic acid (SA)‐dependent HR‐like cell death in N. benthamiana, and this effect is mediated by the lectin's glycan binding activity. We found that secreted GRFT interacts with an endogenous glycoprotein, α‐xylosidase (XYL1), which is involved in cell wall organization. The necrotic effect could be mitigated by overexpression of Arabidopsis XYL1, and by co‐expression of SA‐degrading enzyme NahG, providing strategies for enhancing expression of oligomannose‐binding lectins in plants.
The polB gene encodes DNA polymerase II in Escherichia coli. The nucleotide sequence shows an open reading frame of 2,304 nucleotides coding for a protein of 88 kD. The protein initiation signal is preceded by a lexA box lying 2 nucleotides from the termination signal of araD, and begins with GUG 75 nucleotides after the termination of araD. The polB gene and the araD gene are transcribed in the same direction. Initiation of protein synthesis was confirmed by peptide sequence. We have also demonstrated that the polB sequence is lacking in some strains. We conclude that DNA polymerase II is not a required protein in the cell. Sequence comparisons show that DNA polymerase II is an alpha-like DNA polymerase.
An esterase, designated EstTs1, was identified and characterized from a genomic library of Thermus scotoductus SA-01 (ATCC 700910). The library was screened in Escherichia coli for lipolytic activity on tributyrin agar plates. A 1.7-kb DNA fragment from a lipolytic positive clone was sequenced and two open reading frames (ORFs) were identified. A 774-bp ORF, designated EstTs1 with an estimated molecular mass of 28.6 kDa, and a 693-bp ORF, designated EstTs2 with an estimated molecular mass of 25.6 kDa, were identified. These two ORFs appear to form part of an operon. Sequence analysis showed that both proteins contained the G-X-S-X-G signature sequence motif present in most esterases and lipases. The deduced amino sequence of EstTs1 was found to display significant sequence identity with putative hydrolase proteins from both Thermus aquaticus Y51MC23 and Thermus thermophilus HB27. Similarly, EstTs2, also displayed significant homology to a second putative hydrolase protein present in the same two organisms. The cloning and characterization of these two ORFs from T. aquaticus Y51MC23 and T. thermophilus strain HB27 encoding putative hydrolase genes have not been reported. E. coli cells harbouring EstTs1 on a multicopy vector produced a clearing zone on tributyrin agar plates, whereas no enzymatic activity was observed for E. coli harbouring EstTs2 on a multicopy vector. EstTs1 displayed optimum activity at pH 7 and 80 degrees C with a half life of 48 h at 70 degrees C.
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