Efficient In Vitro and In Vivo Activity of Glyco-Engineered Plant-Produced Rabies Monoclonal Antibodies E559 and 62-71-3.

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

Plant-based production of highly potent anti-HIV antibodies with engineered posttranslational modifications

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.The Department of Science and Technology (DST), South African Medical Research Council - Strategic Health Innovation Partnership (SAMRC SHIP) and Council for Scientific and Industrial Research (CSIR).http://www.nature.com/srepam2021Plant Production and Soil ScienceProduction Animal Studie

Deconvoluted spectrum of intact, reduced 62-71-3 HC.

<p>The inset shows the zoomed-in LC region with theoretical molecular weights for LC and HC indicated. Detected N-linked glycoforms are shown. The N-glycan nomenclature used was from <a href="http://www.proglycan.com/" target="_blank">www.proglycan.com</a>.</p

Far-UV CD spectra of humanised 62-71-3 (black) and E559 (blue) mAbs.

<p>Far-UV CD spectra of humanised 62-71-3 (black) and E559 (blue) mAbs.</p

N-linked glycans on the anti-rabies mAbs.

<p>N-glycosylation profile from E559 HC and LC and from 62-71-3 HC as determined by LC-ESI-MS of glycopeptides obtained upon trypsin digestion. Numbers represent the presence of the different glyco-species in percent of total glycan. The N-glycan nomenclature used was from <a href="http://www.proglycan.com/" target="_blank">www.proglycan.com</a>.</p

SDS-PAGE analysis of purified E559 and 62-71-3 mAbs.

<p>The middle lane was a PageRuler Prestained Protein Ladder, with the sizes indicated in kDa. The numbered E559 and 62-71-3 bands (1–5) were used for further analysis.</p

MAb E559 and 62-71-3 expression levels obtained using various combinations of either PVX or TMV-based expression vectors with either the murine (m) or rice alpha amylase (r) signal peptide.

<p>MAb E559 and 62-71-3 expression levels obtained using various combinations of either PVX or TMV-based expression vectors with either the murine (m) or rice alpha amylase (r) signal peptide.</p

Hamster survival curve after infection with rabies virus and administration of PBS, RIG or mAbs.

<p>For infected and treated groups, 9 hamsters were in each group, while untreated control groups had 4 hamsters.</p

Fifty percent end-point neutralisation activity (reciprocal titre) of E559 and 62-71-3 in a modified Rapid Fluorescent Focus Inhibition Test (RFFIT).

<p>Fifty percent end-point neutralisation activity (reciprocal titre) of E559 and 62-71-3 in a modified Rapid Fluorescent Focus Inhibition Test (RFFIT).</p

Efficient In Vitro and In Vivo Activity of Glyco-Engineered Plant-Produced Rabies Monoclonal Antibodies E559 and 62-71-3 - Fig 2

<p><b>Deconvoluted spectrum of intact, reduced E559 LC (A) and intact, reduced E559 HC (B)</b>.Theoretical molecular weights for LC and HC indicated. Detected N-linked glycoforms are shown. The N-glycan nomenclature used was from <a href="http://www.proglycan.com/" target="_blank">www.proglycan.com</a>.</p