Antibody production in plants and green algae

Monoclonal antibodies (mAbs) have a wide range of modern applications, including research, diagnostic, therapeutic, and industrial uses. Market demand for mAbs is high and continues to grow. Although mammalian systems, which currently dominate the biomanufacturing industry, produce effective and safe recombinant mAbs, they have a limited manufacturing capacity and high costs. Bacteria, yeast, and insect cell systems are highly scalable and cost effective but vary in their ability to produce appropriate posttranslationally modified mAbs. Plants and green algae are emerging as promising production platforms because of their time and cost efficiencies, scalability, lack of mammalian pathogens, and eukaryotic posttranslational protein modification machinery. So far, plant-and algae-derived mAbs have been produced predominantly as candidate therapeutics for infectious diseases and cancer. These candidates have been extensively evaluated in animal models, and some have shown efficacy in clinical trials. Here, we review ongoing efforts to advance the production of mAbs in plants and algae

Plant-produced recombinant transmission blocking vaccine candidates to combat malaria

Malaria is one of the world’s most devastating infectious diseases, resulting in over half a million deaths annually. No licensed vaccine is currently available, although the pre-erythrocytic vaccine RTS,S is in advanced stage clinical trials and significantly reduces cases of malaria. However, RTS,S alone will be insufficient to eradicate malaria and, consequently, a variety of other vaccines targeting erythrocytic and sexual stages of the parasite’s life cycle are under development. The latter comprise transmission blocking vaccines (TBVs) that elicit antibodies against surface proteins of the parasite’s sexual stages and so prevent sexual development and transmission. Therefore, the World Health Organization has proposed TBVs as an effective means to reduce malaria transmission and serve as a key element in potentially eradicating the disease. Several TBV candidates are under development, including subunit vaccines based on the gametocyte and gamete antigens P4 8/45 and P230 and the zygote and ookinete antigens P25 and P28. These antigens have proven challenging to produce in established recombinant systems, potentially in part due to the large number of disulfide bonds. However, recently, plant-based platforms have been recruited for the production of these challenging antigens. Recombinant plants have several advantages for the production of subunit vaccines and therapeutic proteins, including cost-effectiveness, scalability, lack of harbored mammalian pathogens, and possession of the machinery for eukaryotic posttranslational protein modifications. Here, we review ongoing efforts to produce TBV candidates in plant-based expression systems

Hybrid viral vectors for vaccine and antibody production in plants

Plants have a demonstrated potential for large-scale, rapid production of recombinant proteins for diverse product applications, including subunit vaccines and monoclonal antibodies. In this field, the accent has recently shifted from the engineering of "edible" vaccines based on stable expression of target protein in transgenic or transplastomic plants to the development of purified formulated vaccines that are delivered via injection. The injectable vaccines are commonly produced using transient expression of target gene delivered into genetically unmodified plant host via viral or bacterial vectors. Most viral vectors are based on plant RNA viruses, where nonessential sequences are replaced with the gene of interest. Utilization of viral hybrids that consist of genes and regulatory elements of different virus species, or transcomplementation systems (vector/transgene) had a substantial impact on the level of target protein expression. Development and introduction of agroviral hybrid vectors that combine genetic elements of bacterial binary plasmids and plant viral vectors, and agroinfiltration as a tool of the vector delivery have resulted in significant progress in large-scale production of recombinant vaccines and monoclonal antibodies in plants. This article presents an overview of plant hybrid viral vector expression systems developed so far

Intermolecular interactions between the a-subunit and b-subunit of heat-labile enterotoxin from escherichia-coli promote holotoxin assembly and stability invivo

Cholera toxin and the related heat-labile enterotoxin (LT) produced by Escherichia coli consist of a holotoxin of one A subunit and five B subunits (AB5). Here we investigate the domains of the A subunit (EtxA) of E. coli LT which influence the events of B-subunit (EtxB) oligomerization and the formation of a stable AB5 holotoxin complex. We show that the C-terminal 14 amino acids of the A subunit comprise two functional domains that differentially affect oligomerization and holotoxin stability. Deletion of the last 14 amino acids (-14) from the A subunit resulted in a molecule that was significantly impaired in its capacity to promote the assembly of a mutant B subunit, EtxB191.5. In contrast, deletion of the last four amino acids (-4) from the A subunit gave a molecule that retained such a capacity. This suggests that C-terminal residues within the -14 to -4 region of the A subunit are important for promoting the oligomerization of EtxB. In addition, we demonstrate that the truncated A subunit lacking the last 4 amino acids was unable to form a stable AB5 holotoxin complex even though it promoted B-subunit oligomerization. This suggests that the last 4 residues of the A subunit function as an "anchoring" sequence responsible for maintaining the stability of A/B subunit interaction during holotoxin assembly. These data represent an important example of how intermolecular interactions between polypeptides in vivo can modulate the folding and assembly of a macromolecular complex

Stability and pre-formulation development of a plant-produced anthrax vaccine candidate

Second generation anthrax vaccines focus on the use of recombinant protective antigen (rPA) to elicit a strong, toxin neutralizing antibody responses in immunized subjects. The main difference between the rPA vaccines compared to the current licensed vaccine, anthrax vaccine absorbed (AVA), is the rPA vaccines are highly purified preparations of only rPA. These second generation rPA vaccines strive to elicit strong immune responses with substantially fewer doses than AVA while provoking less side effects. Many of the rPA candidates have shown to be effective in pre-clinical studies, but most of the second generation molecules have stability issues which reduce their efficacy over time. These stability issues are evident even under refrigerated conditions and thus emphasis has been directed to stabilizing the rPA molecule and determining an optimized final formulation. Stabilization of vaccines for long-term storage is a major challenge in the product development life cy cle. The effort required to identify suitable formulations can be slow and expensive. The ideal storage for stockpiled vaccines would allow the candidate to withstand years of storage at ambient temperatures. The Fraunhofer Center for Molecular Biotechnology is developing a plant-produced rPA vaccine candidate that shows instability when stored under refrigerated conditions in a solution, as is typical for rPA vaccines. Increased stability of our plant-produced rPA vaccine candidate was achieved in a spray dried powder formulation that could eliminate the need for conventional cold chain allowing greater confidence to stockpile vaccine for civilian and military biodefense

Safety and immunogenicity of a plant-produced Pfs25 virus-like particle as a transmission blocking vaccine against malaria: A Phase 1 dose-escalation study in healthy adults

Malaria continues to be one of the world’s most devastating infectious tropical diseases, and alternative strategies to prevent infection and disease spread are urgently needed. These strategies include the development of effective vaccines, such as malaria transmission blocking vaccines (TBV) directed against proteins found on the sexual stages of Plasmodium falciparum parasites present in the mosquito midgut. The Pfs25 protein, which is expressed on the surface of gametes, zygotes and ookinetes, has been a primary target for TBV development. One such vaccine strategy based on Pfs25 is a plant-produced malaria vaccine candidate engineered as a chimeric non-enveloped virus-like particle (VLP) comprising Pfs25 fused to the Alfalfa mosaic virus coat protein. This Pfs25 VLP-FhCMB vaccine candidate has been engineered and manufactured in Nicotiana benthamiana plants at pilot plant scale under current Good Manufacturing Practice guidelines. The safety, reactogenicity and immunogenicity of Pfs25 VLP-FhCMB was assessed in healthy adult volunteers. This Phase 1, dose escalation, first-in-human study was designed primarily to evaluate the safety of the purified plant-derived Pfs25 VLP combined with Alhydrogel® adjuvant. At the doses tested in this Phase 1 study, the vaccine was generally shown to be safe in healthy volunteers, with no incidence of vaccine-related serious adverse events and no evidence of any dose-limiting or dose-related toxicity, demonstrating that the plant-derived Pfs25 VLP-FhCMB vaccine had an acceptable safety and tolerability profile. In addition, although the vaccine did induce Pfs25-specific IgG in vaccinated patients in a dose dependent manner, the transmission reducing activity of the antibodies generated were weak, suggesting the need for an alternative vaccine adjuvant formulation

Purification and immunogenicity of hemagglutinin from highly pathogenic avian influenza virus H5N1 expressed in Nicotiana benthamiana

Highly pathogenic avian influenza (HPAI) H5N1 is an ongoing global health concern due to its severe sporadic outbreaks in Asia, Africa and Europe, which poses a potential pandemic threat. The development of safe and cost-effective vaccine candidates for HPAI is considered the best strategy for managing the disease and addressing the pandemic preparedness. The most potential vaccine candidate is the antigenic determinant of influenza A virus, hemagglutinin (HA). The present research was aimed at developing optimized expression in Nicotiana benthamiana and protein purification process for HA from the Malaysian isolate of H5N1 as a vaccine antigen for HPAI H5N1. Expression of HA from the Malaysian isolate of HPAI in N. benthamiana was confirmed, and more soluble protein was expressed as truncated HA, the HA1 domain over the entire ectodomain of HA. Two different purification processes were evaluated for efficiency in terms of purity and yield. Due to the reduced yield, protein degradation and length of the 3-column purification process, the 2-column method was chosen for target purification. Purified HA1 was found immunogenic in mice inducing H5 HA-specific IgG and a hemagglutination inhibition antibody. This paper offers an alternative production system of a vaccine candidate against a locally circulating HPAI, which has a regional significance

A novel plant-produced Pfs25 fusion subunit vaccine induces long-lasting transmission blocking antibody responses.

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Antibodies to plant-produced Plasmodium falciparum sexual stage protein Pfs25 exhibit transmission blocking activity

Malaria is a serious and sometimes fatal mosquito-borne disease caused by a protozoan parasite. Each year, it is estimated that over one million people are killed by malaria, yet the disease is preventable and treatable. Developing vaccines against the parasite is a critical component in the fight against malaria and these vaccines can target different stages of the pathogen's life cycle. We are targeting sexual stage proteins of P. falciparum which are found on the surface of the parasite reproductive cells present in the mosquito gut. Antibodies against these proteins block the progression of the parasite's life cycle in the mosquito, and thus block transmission to the next human host. Transmission blocking vaccines are essential to the malaria eradication program to ease the disease burden at the population level. We have successfully produced multiple versions of the Pfs25 antigen in a plant virus-based transient expression system and have evaluated these vaccine candidates in an animal model. The targets are expressed in plants at a high level, are soluble and most importantly, generate strong transmission blocking activity as determined by a standard membrane feeding assay. These data demonstrate the feasibility of expressing Plasmodium antigens in a plant-based system for the economic production of a transmission blocking vaccine against malaria