Impact of mutations on the plant-based production of recombinant SARS-CoV-2 RBDs.

Subunit vaccines based on recombinant viral antigens are valuable interventions to fight existing and evolving viruses and can be produced at large-scale in plant-based expression systems. The recombinant viral antigens are often derived from glycosylated envelope proteins of the virus and glycosylation plays an important role for the immunogenicity by shielding protein epitopes. The receptor-binding domain (RBD) of the SARS-CoV-2 spike is a principal target for vaccine development and has been produced in plants, but the yields of recombinant RBD variants were low and the role of the N-glycosylation in RBD from different SARS-CoV-2 variants of concern is less studied. Here, we investigated the expression and glycosylation of six different RBD variants transiently expressed in leaves of Nicotiana benthamiana. All of the purified RBD variants were functional in terms of receptor binding and displayed almost full N-glycan occupancy at both glycosylation sites with predominately complex N-glycans. Despite the high structural sequence conservation of the RBD variants, we detected a variation in yield which can be attributed to lower expression and differences in unintentional proteolytic processing of the C-terminal polyhistidine tag used for purification. Glycoengineering towards a human-type complex N-glycan profile with core α1,6-fucose, showed that the reactivity of the neutralizing antibody S309 differs depending on the N-glycan profile and the RBD variant.

Harnessing the advances of genetic engineering in microalgae for the production of cannabinoids.

Cannabis is widely recognized as a medicinal plant owing to bioactive cannabinoids. However, it is still considered a narcotic plant, making it hard to be accessed. Since the biosynthetic pathway of cannabinoids is disclosed, biotechnological methods can be employed to produce cannabinoids in heterologous systems. This would pave the way toward biosynthesizing any cannabinoid compound of interest, especially minor substances that are less produced by a plant but have a high medicinal value. In this context, microalgae have attracted increasing scientific interest given their unique potential for biopharmaceutical production. In the present review, the current knowledge on cannabinoid production in different hosts is summarized and the biotechnological potential of microalgae as an emerging platform for synthetic production is put in perspective. A critical survey of genetic requirements and various transformation approaches are also discussed.

Plant-made poliovirus vaccines - Safe alternatives for global vaccination.

Human polioviruses are highly infectious viruses that are spread mainly through the fecal-oral route. Infection of the central nervous system frequently results in irreversible paralysis, a disease called poliomyelitis. Children under five years are mainly affected if they have not acquired immunity through natural infection or via vaccination. Current polio vaccines comprise the injectable inactivated polio vaccine (IPV, also called the Salk vaccine) and the live-attenuated oral polio vaccine (OPV, also called the Sabin vaccine). The main limitations of the IPV are the reduced protection at the intestinal mucosa, the site of virus replication, and the high costs for manufacturing due to use of live viruses. While the OPV is more effective and stimulates mucosal immunity, it is manufactured using live-attenuated strains that can revert into pathogenic viruses resulting in major safety concerns and vaccine-derived outbreaks. During the last fifteen years, plant-based poliovirus vaccines have been explored by several groups as a safe and low-cost alternative, and promising results in protection against challenges with viruses and induction of neutralizing antibodies have been obtained. However, low yields and a high frequency in dose administration highlight the need for improvements in polioviral antigen production. In this review, we provide insights into recent efforts to develop plant-made poliovirus candidates, with an emphasis on strategies to optimize the production of viral antigens.

An Algae-Made RBD from SARS-CoV-2 Is Immunogenic in Mice.

Despite the current advances in global vaccination against SARS-CoV-2, boosting is still required to sustain immunity in the population, and the induction of sterilizing immunity remains as a pending goal. Low-cost oral immunogens could be used as the basis for the design of affordable and easy-to-administer booster vaccines. Algae stand as promising platforms to produce immunogens at low cost, and it is possible to use them as oral delivery carriers since they are edible (not requiring complex purification and formulation processes). Herein, a Chlamydomonas-made SARS-CoV-2 RBD was evaluated as an oral immunogen in mice to explore the feasibility of developing an oral algae-based vaccine. The test immunogen was stable in freeze-dried algae biomass and able to induce, by the oral route, systemic and mucosal humoral responses against the spike protein at a similar magnitude to those induced by injected antigen plus alum adjuvant. IgG subclass analysis revealed a Th2-bias response which lasted over 4 months after the last immunization. The induced antibodies showed a similar reactivity against either Delta or Omicron variants. This study represents a step forward in the development of oral vaccines that could accelerate massive immunization.

Current Status and Perspective on the Use of Viral-Based Vectors in Eukaryotic Microalgae.

During the last two decades, microalgae have attracted increasing interest, both commercially and scientifically. Commercial potential involves utilizing valuable natural compounds, including carotenoids, polysaccharides, and polyunsaturated fatty acids, which are widely applicable in food, biofuel, and pharmaceutical industries. Conversely, scientific potential focuses on bioreactors for producing recombinant proteins and developing viable technologies to significantly increase the yield and harvest periods. Here, viral-based vectors and transient expression strategies have significantly contributed to improving plant biotechnology. We present an updated outlook covering microalgal biotechnology for pharmaceutical application, transformation techniques for generating recombinant proteins, and genetic engineering tactics for viral-based vector construction. Challenges in industrial application are also discussed.

Two strains of a novel begomovirus encoding Rep proteins with identical ß1 strands but different ß5 strands are not compatible in replication.

Geminiviruses have genomes composed of single-stranded DNA molecules and encode a rolling-circle replication (RCR) initiation protein ("Rep"), which has multiple functions. Rep binds to specific repeated DNA motifs ("iterons"), which are major determinants of virus-specific replication. The particular amino acid (aa) residues that determine the preference of a geminivirus Rep for specific iterons (i.e., the trans-acting replication "specificity determinants", or SPDs) are largely unknown, but diverse lines of evidence indicate that most of them are closely associated with the so-called RCR motif I (FLTYP), located in the first 12-19 aa residues of the protein. In this work, we characterized two strains of a novel begomovirus, rhynchosia golden mosaic Sinaloa virus (RhGMSV), that were incompatible in replication in pseudorecombination experiments. Systematic comparisons of the Rep proteins of both RhGMSV strains in the DNA-binding domain allowed the aa residues at positions 71 and 74 to be identified as the residues most likely to be responsible for differences in replication specificity. Residue 71 is part of the ß-5 strand structural element, which was predicted in previous studies to contain Rep SPDs. Since the Rep proteins encoded by both RhGMSV strains are identical in their first 24 aa residues, where other studies have mapped potential SPDs, this is the first study lending direct support to the notion that geminivirus Rep proteins contain separate SPDs in their N-terminal domain.

The potential of plant-made vaccines to fight picornavirus.

INTRODUCTION: Several Picornaviruses are pathogens that generate serious problems for human and animal health worldwide. Vaccination is an attractive approach to fight against picornaviruses. In this regard, the development of low-cost vaccines is a priority to ensure coverage; especially in developing and low-income countries. In this context, plant-made vaccines are a convenient technology since plant cells are low-cost bioreactors capable of producing complex antigens that preserve their antigenic determinants; moreover, they can serve as biocapsules to achieve oral delivery. AREAS COVERED: In the present review the advances in the development of plant-made vaccines against picornaviruses are summarized and placed in perspective. The main diseases that have been targeted using this approach include Poliovirus, Food and mouth disease virus, Hepatitis A virus, and Enterovirus 71. EXPERT OPINION: Several vaccine candidates against picornavirus have been characterized at the preclinical level; with many of them capable of inducing humoral and cellular responses that led to neutralization of pathogens when evaluated in vitro and test animal challenge assays. Plant-made vaccines are a promise to fight picornaviruses; especially in the developing world where limited resources hamper vaccination coverage. A critical analysis of the road ahead for this technology is provided.

Expression of immunogenic poliovirus Sabin type 1 VP proteins in transgenic tobacco.

One of the milestones of vaccinology is the depletion of the global impact of Poliomyelitis. The current vaccines to deal with Polio comprise the Sabin and Salk formulations. The main limitation of the former is the use of attenuated viruses that can revert into pathogenic forms, whereas the latter is more expensive and induces no protection in the intestinal tract; the site of virus replication. Genetically engineered plants cope with such limitations. In addition, they offer a low-cost alternative for production, storage and delivery of vaccines. This technology has been narrowly applied in the development of Polio vaccines. Herein, we explored the ability of tobacco cells to express the immunogenic VP1, VP2, VP3, and VP4 Polio antigens, which are relevant for vaccine development. Evidence on the expression of the plant-made Polio VPs is presented and an immunogenicity assessment proved their capacity to induce local and systemic humoral responses when administered by subcutaneous and oral routes. The plant-made VPs will be useful in the development of low-cost vaccine formulations able to induce effective mucosal immunity without the risks associated to the use of attenuated viruses; therefore there is a potential for this technology to contribute toward Polio eradication.