Assembly of Human Papillomavirus 16 L1 Protein in Nicotiana benthamiana Chloroplasts into Highly Immunogenic Virus-Like Particles.

Infection with human papillomavirus (HPV) can cause cervical cancers in women, and vaccination against the virus is one of most effective ways to prevent these cancers. Two vaccines made of virus-like particles (VLPs) of HPV L1 proteins are currently commercially available. However, these HPV vaccines are highly expensive, and thus not affordable for women living in developing countries. Therefore, great demand exists to produce a cost-effective vaccine. Here, we investigate the production of self-assembled HPV16 VLPs in plants. We generated a chimeric protein composed of N-terminal 79 amino acid residues of RbcS as a long-transit peptide to target chloroplasts, the SUMO domain, and HPV16 L1 proteins. The chimeric gene was expressed in plants with chloroplast-targeted bdSENP1, a protein that specifically recognizes the SUMO domain and cleaves its cleavage site. This co-expression of bdSENP1 led to the release of HPV16 L1 from the chimeric proteins without any extra amino acid residues. HPV16 L1 purified by heparin chromatography formed VLPs that mimicked native virions. Moreover, the plant-produced HPV16 L1 VLPs elicited strong immune responses in mice without adjuvants. Thus, we demonstrated the cost-effective production of HPV16 VLPs in plants. Supplementary Information: The online version contains supplementary material available at 10.1007/s12374-023-09393-6.

In Vivo Removal of N-Terminal Fusion Domains From Recombinant Target Proteins Produced in Nicotiana benthamiana.

Plants show great potential for producing recombinant proteins in a cost-effective manner. Many strategies have therefore been employed to express high levels of recombinant proteins in plants. Although foreign domains are fused to target proteins for high expression or as an affinity tag for purification, the retention of foreign domains on a target protein may be undesirable, especially for biomedical purposes. Thus, their removal is often crucial at a certain time point after translation. Here, we developed a new strategy to produce target proteins without foreign domains. This involved in vivo removal of foreign domains fused to the N-terminus by the small ubiquitin-related modifier (SUMO) domain/SUMO-specific protease system. This strategy was tested successfully by generating a recombinant gene, BiP:p38:bdSUMO : His:hLIF, that produced human leukemia inhibitory factor (hLIF) fused to p38, a coat protein of the Turnip crinkle virus; the inclusion of p38 increased levels of protein expression. The recombinant protein was expressed at high levels in the leaf tissue of Nicotiana benthamiana. Coexpression of bdSENP1, a SUMO-specific protease, proteolytically released His:hLIF from the full-length recombinant protein in the endoplasmic reticulum of N. benthamiana leaf cells. His:hLIF was purified from leaf extracts via Ni2+-NTA affinity purification resulting in a yield of 32.49 mg/kg, and the N-terminal 5-residues were verified by amino acid sequencing. Plant-produced His:hLIF was able to maintain the pluripotency of mouse embryonic stem cells. This technique thus provides a novel method of removing foreign domains from a target protein in planta.

Production of recombinant proteins through sequestration in chloroplasts: a strategy based on nuclear transformation and post-translational protein import.

Recently, plants have emerged as a lucrative alternative system for the production of recombinant proteins, as recombinant proteins produced in plants are safer and cheaper than those produced in bacteria and animal cell-based production systems. To obtain high yields in plants, recombinant proteins are produced in chloroplasts using different strategies. The first strategy is based on chloroplast transformation, followed by gene expression and translation in chloroplasts. This has proven to be a powerful approach for the production of proteins at high levels. The second approach is based on nuclear transformation, followed by post-translational import of proteins from the cytosol into chloroplasts. In the nuclear transformation approach, foreign genes are stably integrated into the nuclear genome or transiently expressed in the nucleus by non-integrating T-DNA. Although this approach also has great potential for protein production at high levels, it has not been thoroughly investigated. In this review, we focus on nuclear transformation-based protein expression and its subsequent sequestration in chloroplasts, and summarize the different strategies used for high-level production of recombinant proteins. We also discuss future directions for further improvements in protein production in chloroplasts through nuclear transformation-based gene expression.

Herbal Remedies for Coccidiosis Control: A Review of Plants, Compounds, and Anticoccidial Actions.

Coccidiosis is the bane of the poultry industry causing considerable economic loss. Eimeria species are known as protozoan parasites to cause morbidity and death in poultry. In addition to anticoccidial chemicals and vaccines, natural products are emerging as an alternative and complementary way to control avian coccidiosis. In this review, we update recent advances in the use of anticoccidial phytoextracts and phytocompounds, which cover 32 plants and 40 phytocompounds, following a database search in PubMed, Web of Science, and Google Scholar. Four plant products commercially available for coccidiosis are included and discussed. We also highlight the chemical and biological properties of the plants and compounds as related to coccidiosis control. Emphasis is placed on the modes of action of the anticoccidial plants and compounds such as interference with the life cycle of Eimeria, regulation of host immunity to Eimeria, growth regulation of gut bacteria, and/or multiple mechanisms. Biological actions, mechanisms, and prophylactic/therapeutic potential of the compounds and extracts of plant origin in coccidiosis are summarized and discussed.

Field trial of medicinal plant, Bidens pilosa, against eimeriosis in broilers.

Eimeriosis is a severe protozoan disease in poultry. Because of increasing concern about drug residue and drug resistance with the use of anticoccidial drugs, natural products are emerging as an alternative and complementary approach to control avian eimeriosis. Our previous publication showed that feed supplemented with B. pilosa (BP) was effective at combating chicken eimeriosis in experimental settings. However, its efficacy against chicken eimeriosis under field conditions is not known. Here, we investigated the efficacy of BP against eimeriosis on an organic chicken farm. We found that feed supplemented with BP, at the dose of 0.025% of feed or more, significantly reduced Eimeria infection. This treatment increased body weight gain and reduced feed conversion ratio, leading to superior growth performance. It lowered morbidity/mortality rate, decreased oocysts per gram of feces and gut pathology and augmented the anticoccidial index. Collectively, these data demonstrated the potential of BP to control chicken eimeriosis on chicken farms. BP can, therefore, be used as an effective means to control eimeriosis.

A transgenic plant cell-suspension system for expression of epitopes on chimeric Bamboo mosaic virus particles.

We describe a novel strategy to produce vaccine antigens using a plant cell-suspension culture system in lieu of the conventional bacterial or animal cell-culture systems. We generated transgenic cell-suspension cultures from Nicotiana benthamiana leaves carrying wild-type or chimeric Bamboo mosaic virus (BaMV) expression constructs encoding the viral protein 1 (VP1) epitope of foot-and-mouth disease virus (FMDV). Antigens accumulated to high levels in BdT38 and BdT19 transgenic cell lines co-expressing silencing suppressor protein P38 or P19. BaMV chimeric virus particles (CVPs) were subsequently purified from the respective cell lines (1.5 and 2.1 mg CVPs/20 g fresh weight of suspended biomass, respectively), and the resulting CVPs displayed VP1 epitope on the surfaces. Guinea pigs vaccinated with purified CVPs produced humoral antibodies. This study represents an important advance in the large-scale production of immunopeptide vaccines in a cost-effective manner using a plant cell-suspension culture system.