Immunogenicity of adjuvanted plant-produced SARS-CoV-2 Beta spike VLP vaccine in New Zealand white rabbits.

The outbreak of the SARS-CoV-2 global pandemic heightened the pace of vaccine development with various vaccines being approved for human use in a span of 24 months. The SARS-CoV-2 trimeric spike (S) surface glycoprotein, which mediates viral entry by binding to ACE2, is a key target for vaccines and therapeutic antibodies. Plant biopharming is recognized for its scalability, speed, versatility, and low production costs and is an increasingly promising molecular pharming vaccine platform for human health. We developed Nicotiana benthamiana-produced SARS-CoV-2 virus-like particle (VLP) vaccine candidates displaying the S-protein of the Beta (B.1.351) variant of concern (VOC), which triggered cross-reactive neutralising antibodies against Delta (B.1.617.2) and Omicron (B.1.1.529) VOCs. In this study, immunogenicity of the VLPs (5 µg per dose) adjuvanted with three independent adjuvants i.e. oil-in-water based adjuvants SEPIVAC SWETM (Seppic, France) and "AS IS" (Afrigen, South Africa) as well as a slow-release synthetic oligodeoxynucleotide (ODN) adjuvant designated NADA (Disease Control Africa, South Africa) were evaluated in New Zealand white rabbits and resulted in robust neutralising antibody responses after booster vaccination, ranging from 1:5341 to as high as 1:18204. Serum neutralising antibodies elicited by the Beta variant VLP vaccine also showed cross-neutralisation against the Delta and Omicron variants with neutralising titres ranging from 1:1702 and 1:971, respectively. Collectively, these data provide support for the development of a plant-produced VLP based candidate vaccine against SARS-CoV-2 based on circulating variants of concern.

Engineering, expression in transgenic plants and characterisation of E559, a rabies virus-neutralising monoclonal antibody.

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.

Plant made anti-HIV microbicides--a field of opportunity.

HIV remains a significant global burden and without an effective vaccine, it is crucial to develop microbicides to halt the initial transmission of the virus. Several microbicides have been researched with various levels of success. Amongst these, the broadly neutralising antibodies and peptide lectins are promising in that they can immediately act on the virus and have proven efficacious in in vitro and in vivo protection studies. For the purpose of development and access by the relevant population groups, it is crucial that these microbicides be produced at low cost. For the promising protein and peptide candidate molecules, it appears that current production systems are overburdened and expensive to establish and maintain. With recent developments in vector systems for protein expression coupled with downstream protein purification technologies, plants are rapidly gaining credibility as alternative production systems. Here we evaluate the advances made in host and vector system development for plant expression as well as the progress made in expressing HIV neutralising antibodies and peptide lectins using plant-based platforms.

Biolistic-mediated transformation protocols for maize and pearl millet using pre-cultured immature zygotic embryos and embryogenic tissue.

Maize (Zea mays L.) is the most important cereal food crop in sub-Saharan Africa and Latin America, and a key feed crop in Asia, whereas pearl millet (Pennisetum glaucum (L.) R. Br.) is a staple food that supplies a major proportion of calories and protein to large segments of the populations living in the semi-arid tropical regions of Africa and Asia. The limitations of biological gene transfer with Agrobacterium tumefaciens specifically related to recalcitrant cereal crops, led to the development of alternative methods of which high-velocity microprojectiles, biolistic genetic transfer is the most successful and also the most widely employed. Agrobacterium facilitated transformation is the method of choice especially for deregulation of commercial transgenic food crop products, but biolistic-mediated transformation is still valid for proof of concept and functional genomics applications. Biolistic-mediated transformation and the production of transgenic plantlets via somatic embryogenesis of two maize strains viz. Hi-II (a laboratory strain) and M37W (a South African elite white maize genotype) as well as a pearl millet strain (842B) are described in this chapter. The stages described include: (1) proliferation of immature zygotic embryos for biolistic-mediated transformation, (2) induction and maintenance of transgenic embryogenic tissue on selection medium; (3) maturation (both morphological and physiological) of transgenic somatic embryos; and (4) germination of the somatic embryos to putative transgenic primary events. Maize and pearl millet cultures were regenerated via somatic embryogenesis as they are bipolar structures that shoot and root simultaneously. The culture media described in this chapter rarely induced or regenerated plantlets via organogenesis.