Characterization of plant produced VHH antibodies against cobra venom toxins for antivenom therapy.

Cobra (Naja kaouthia) venom contains many toxins including α-neurotoxin (αNTX) and phospholipase A2 (PLA2), which can cause neurodegeneration, respiratory failure, and even death. The traditional antivenom derived from animal serum faces many challenges and limitations. Heavy-chain-only antibodies (HCAb), fusing VHH with human IgG Fc region, offer advantages in tissue penetration, antigen binding, and extended half-life. This research involved the construction and transient expression of two types of VHH-FC which are specific to α-Neurotoxin (VHH-αNTX-FC) and Phospholipase A2 (VHH-PLA2-FC) in Nicotiana benthamiana leaves. The recombinant HCAbs were incubated for up to six days to optimize expression levels followed by purification by affinity chromatography and characterization using LC/Q-TOF mass spectrometry (MS). Purified proteins demonstrated over 92 % sequence coverage and an average mass of around 82 kDa with a high-mannose N-glycan profile. An antigen binding assay showed that the VHH-αNTX-Fc has a greater ability to bind to crude venom than VHH-PLA2-Fc.

Efficiency Comparative Approach of Plant-Produced Monoclonal Antibodies against Rabies Virus Infection.

Rabies encephalitis is a fatal zoonotic viral disease caused by the neurotropic rabies virus. It remains a major public health concern as it causes almost 100% fatality and has no effective medication after the onset of the disease. However, this illness is preventable with the timely administration of effective post-exposure prophylaxis (PEP) consisting of the rabies vaccine and passive immune globulins (HRIG and ERIG). Recently, conventional PEP has been shown to have many limitations, resulting in little support for these expensive and heterologous globulins. Monoclonal antibody (mAb) production via recombinant technology in animal and human cell cultures, as well as a plant-based platform, was introduced to overcome the costly and high-tech constraints of former preparations. We used transient expression technology to produce two mAbs against the rabies virus in Nicotiana benthamiana and compared their viral neutralizing activity in vitro and in vivo. The expression levels of selective mAbs E559 and 62-71-3 in plants were estimated to be 17.3 mg/kg and 28.6 mg/kg in fresh weight, respectively. The plant-produced mAbs effectively neutralized the challenge virus CVS-11 strain in a cell-based RFFIT. In addition, the combination of these two mAbs in a cocktail protected hamsters from rabies virus infection more effectively than standard HRIG and ERIG. This study suggests that the plant-produced rabies antibody cocktail has promising potential as an alternative biological to polyclonal RIG in rabies PEP.

Plant-Made Antibodies: Properties and Therapeutic Applications.

BACKGROUND: A cost-effective plant platform for therapeutic monoclonal antibody production is both flexible and scalable. Plant cells have mechanisms for protein synthesis and posttranslational modification, including glycosylation, similar to those in animal cells. However, plants produce less complex and diverse Asn-attached glycans compared to animal cells and contain plant-specific residues. Nevertheless, plant-made antibodies (PMAbs) could be advantageous compared to those produced in animal cells due to the absence of a risk of contamination from nucleic acids or proteins of animal origin. OBJECTIVE: In this review, the various platforms of PMAbs production are described, and the widely used transient expression system based on Agrobacterium-mediated delivery of genetic material into plant cells is discussed in detail. RESULTS: We examined the features of and approaches to humanizing the Asn-linked glycan of PMAbs. The prospects for PMAbs in the prevention and treatment of human infectious diseases have been illustrated by promising results with PMAbs against human immunodeficiency virus, rotavirus infection, human respiratory syncytial virus, rabies, anthrax and Ebola virus. The pre-clinical and clinical trials of PMAbs against different types of cancer, including lymphoma and breast cancer, are addressed. CONCLUSION: PMAb biosafety assessments in patients suggest that it has no side effects, although this does not completely remove concerns about the potential immunogenicity of some plant glycans in humans. Several PMAbs at various developmental stages have been proposed. Promise for the clinical use of PMAbs is aimed at the treatment of viral and bacterial infections as well as in anti-cancer treatment.