A synthetic biology approach for consistent production of plant-made recombinant polyclonal antibodies against snake venom toxins

Abstract

Antivenoms developed from the plasma of hyperimmunized animals are the only effective treatment available against snakebite envenomation but shortage of supply contributes to the high morbidity and mortality toll of this tropical disease. We describe a synthetic biology approach to affordable and cost-effective antivenom production based on plant-made recombinant polyclonal antibodies (termed pluribodies). The strategy takes advantage of virus superinfection exclusion to induce the formation of somatic expression mosaics in agroinfiltrated plants, which enables the expression of complex antibody repertoires in a highly reproducible manner. Pluribodies developed using toxin-binding genetic information captured from peripheral blood lymphocytes of hyperimmunized camels recapitulated the overall binding activity of the immune response. Furthermore, an improved plant-made antivenom (plantivenom) was formulated using an in vitro selected pluribody against Bothrops asper snake venom toxins and has been shown to neutralize a wide range of toxin activities and provide protection against lethal venom doses in mice.

Keywords: molecular pharming; recombinant polyclonal antibodies; snake antivenoms.

Figure 1

Principles of superinfection exclusion. (a) N. benthamiana leaves showing somatic expression mosaics produced by three magnICON viral clones encoding DsRed, green and blue florescent proteins agroinfected simultaneously at OD ₆₀₀ = 0.01. Mosaics are observed either with red/green filters for DsRed (left) or with UV light (right). Below, detail of the mosaic in the framed leaf area. (b) NetLogo computer simulation of a triple viral co‐infection (red, green and blue) subjected to SE in a virtual area comprising 62 500 virtual cells. The virtual fitness (VF) for each clone was adjusted to 1.0 : 1.0 : 0.7 (R:G:B), and virtual OD ₆₀₀ (VOD) was adjusted to 300 (upper image) and 3000 (lower image) arbitrary units to match the natural infection on (a, d) (see Methods for detailed definitions of VF and VOD). (c) Repeated NetLogo simulations of the evolution of triple R‐G‐B infections using VOD values of 300 (up) and 3000 (down), respectively. (d) Experimental data evolution of triple R‐G‐B infections in N. benthamiana leaves were performed at two different OD ₆₀₀ and recorded daily up to day 7 postinfiltration. Evolution of clone expansion is represented as percentage of total leaf area occupied by each clone ± SD.

Figure 2

Assessment of antiserum diversity. (a) Schema of the cloning and transient expression procedures used for the production of polyclonal V_HH antibodies. (b) First and second dimension electrophoresis separation of V_HH‐His clones purified from N. benthamiana leaves using Ni‐NTA affinity columns. (c) Expression of a GFP clone as a viral subpopulation within a V_HH ‐His library. Images were taken after 10 dpi, and the average tile size was recorded and used to estimate tile density and its dependence with OD600 (right). AttB, site‐specific recombination site; SP, signal peptide; NTR, nontranslated region; Tnos, terminator of nopaline synthase.

Figure 3

Assessment of pluribody reproducibility. (a) Individual and merged 2D DIGE images of three independent hyperimmune pluribody preparations in V_HH‐His format (PIM_1, PIM_2 and PIM_3). (b) Comparison of the relative abundance of individual antibody clones in plant samples agroinfiltrated with inocula from independent WCBs derived from the same MCB. Antibody clones were identified by deep sequencing of V_HH CDR3 region. PPI_1 and PP_2 correspond to equivalent pre‐immune WCBs. PIM_1 and PIM_2 correspond to equivalent immune polyclonal WCBs. In each comparison, the same colours correspond to identical antibody clones. (c). DIGE comparison of PPI and PIM purified pluribody preparations. Centred dots represent volume ratios close to one. (d) ELISA test showing the venom‐binding activity of three nonequivalent PIM preparations each obtained from a different individual camel and analysed against each of the three venoms employed in the immunization. (e) Specificity test of PIM venom binding, comparing reactivity against Crotalus simus and cobra venoms.

Figure 4

Strategy for pluribody formulation enrichment. (a) Chromatographic profile and electrophoresis separation of Bothrops asper venom employed in antivenom pluribody enrichment, showing the four groups of toxins employed in phage display selection to reduce antigen drift. (b) Cloning procedure for V_HH variable regions from PBMC cDNA to pHEN2‐derived pSword phage display vector and from here to binary vector for plant expression adapted from magnICON deconstructed system.

Figure 5

Assessment of plantivenom functionality (A) Coomassie gel of an enriched oligoclonal plantivenom (PEO_1) production and purification steps: lane1, crude apoplastic fluid; lane 2, leaf crude extract; lane 3 control apoplastic fluid from uninfected leaf; lane 4 clarified apoplastic fluid; lane 5, protein A flow through; line 6, first wash; lanes 7 and 8, eluted plantivenom. (B) Comparison of ELISA binding activities against B. asper venom of sequential plantivenom enrichment steps. PPI, pre‐immune plantivenom; PIM, immune plantivenom; PEP, enriched polyclonal plantivenom derived from second and third phage display selection rounds. (C) Comparison of binding activities between PEP and PEO_1. (D) Antivenomic profile of PEO_1 plantivenom. Upper (a) and lower (b) panels display, respectively, chromatographic profiles of whole Costa Rican B. asper (Pacific population) venom, and the venom fraction nonimmunoretained in the immobilized PEO_1 affinity column. (E) Neutralization of B. asper venom haemorrhagic activity in vivo with PEO_1 plantivenom preparation (46.5 mg protein/mL). The figure shows the abdominal surface of mouse skin after 2‐h injection with a constant dose of full venom and different amounts of plantivenom. (a) haemorrhagic spot appearing in mice injected intradermally with 15 μg (5 MHD) of venom; (b) inhibition of 5 MHD haemorrhagic spot with an antivenom dose of 250 μL PEO_1/mg venom; (c) 500 μL PEO_1/mg venom; (d) 750 μL PEO_1/mg venom; (e) negative control (plantivenom diluted in PBS).