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 ΔXTFT)-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 differences in structural features were observed between the human embryonic kidney 293 (HEK293)-produced and ΔXTFT 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.

A plant-biotechnology approach for producing highly potent anti-HIV antibodies for antiretroviral therapy consideration.

Despite a reduction in global HIV prevalence the development of a pipeline of new therapeutics or pre-exposure prophylaxis to control the HIV/AIDS epidemic are of high priority. Antibody-based therapies offer several advantages and have been shown to prevent HIV-infection. Plant-based production is efficient for several biologics, including antibodies. We provide a short review on the work by Singh et al., 2020 who demonstrated the transient production of potent CAP256-VRC26 broadly neutralizing antibodies. These antibodies have engineered posttranslational modifications, namely N-glycosylation in the fragment crystallizable region and O-sulfation of tyrosine residues in the complementary-determining region H3 loop. The glycoengineered Nicotiana benthamiana mutant (ΔXTFT) was used, with glycosylating structures lacking ß1,2-xylose and/or α1,3-fucose residues, which is critical for enhanced effector activity. The CAP256-VRC26 antibody lineage targets the first and second variable region of the HIV-1 gp120 envelope glycoprotein. The high potency of this lineage is mediated by a protruding O-sulfated tyrosine in the CDR H3 loop. Nicotiana benthamiana lacks human tyrosyl protein sulfotransferase 1, the enzyme responsible for tyrosine O-sulfation. The transient coexpression of the CAP256-VRC26 antibodies with tyrosyl protein sulfotransferase 1 in planta had restored the efficacy of these antibodies through the incorporation of the O-sulfation modification. This approach demonstrates the strategic incorporation of posttranslational modifications in production systems, which may have not been previously considered. These plant-produced CAP256-VRC26 antibodies have therapeutic as well as topical and systemic pre-exposure prophylaxis potential in enabling the empowerment of young girls and women given that gender inequalities remain a major driver of the epidemic.

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.

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.

Molecular farming for therapies and vaccines in Africa.

Local manufacturing of protein-based vaccines and therapies in Africa is limited and contributes to a trade deficit, security of supply concerns and poor access to biopharmaceuticals by the poor. Plant molecular farming is a potential technology solution that has received growing adoption by African scientists attracted by the potential for the competitive cost of goods, safety and efficacy. Plant-made pharmaceutical technologies for veterinary and human vaccination and treatment of non-communicable and infectious diseases are available at different stages of development in Africa. There is also growth in the translation of these technologies to commercial operations. Africa is poised to benefit from the real-world impact of molecular farming in the next few years.