A Plant-Derived Antigen-Antibody Complex Induces Anti-Cancer Immune Responses by Forming a Large Quaternary Structure.

The antigen-antibody complex (AAC) has novel functions for immunomodulation, encouraging the application of diverse quaternary protein structures for vaccination. In this study, GA733 antigen and anti-GA733 antibody proteins were both co-expressed to obtain the AAC protein structures in a F1 plant obtained by crossing the plants expressing each protein. In F1 plant, the antigen and antibody assembled to form a large quaternary circular ACC structure (~30 nm). The large quaternary protein structures induced immune response to produce anticancer immunoglobulins G (IgGs) that are specific to the corresponding antigens in mouse. The serum containing the anticancer IgGs inhibited the human colorectal cancer cell growth in the xenograft nude mouse. Taken together, antigens and antibodies can be assembled to form AAC protein structures in plants. Plant crossing represents an alternative strategy for the formation of AAC vaccines that efficiently increases anticancer antibody production.

Expression and In Vitro Function of Anti-Breast Cancer Llama-Based Single Domain Antibody VHH Expressed in Tobacco Plants.

Overexpression of human epidermal growth factor receptor type 2 (HER2) is considered as a prognostic factor of breast cancer, which is positively associated with recurrence when cancer metastasizes to the lymph nodes. Here, we expressed the single variable domain on a heavy chain (VHH) form of anti-HER2 camelid single domain antibody in tobacco plants and compared its in vitro anticancer activities with the anti-HER2 full size antibody. The gene expression cassette containing anti-HER2 camelid single domain antibody VHH fused to human IgG Fc region with KDEL endoplasmic reticulum (ER) (VHH-FcK) was transferred into the tobacco plant via the Agrobacterium-mediated transformation. The transformants were screened with polymerase chain reaction and Western blot analyses. Enzyme-linked immunosorbent assay (ELISA) confirmed the binding of the purified anti-HER2 VHH-FcK to the HER2-positive breast cancer cell line, SK-BR-3. Migration assay results confirmed anticancer activity of the plant-derived anticancer camelid single chain antibody. Taken together, we confirmed the possibility of using anti-HER2 VHH-FcK as a therapeutic anticancer agent, which can be expressed and assembled and purified from a plant expression system as an alternative antibody production system.

A synthetic biology approach for consistent production of plant-made recombinant polyclonal antibodies against snake venom toxins.

Antivenoms developed from the plasma of hyperimmunized animals are the only effective treatment available against snakebite envenomation but shortage of supply contributes to the high morbidity and mortality toll of this tropical disease. We describe a synthetic biology approach to affordable and cost-effective antivenom production based on plant-made recombinant polyclonal antibodies (termed pluribodies). The strategy takes advantage of virus superinfection exclusion to induce the formation of somatic expression mosaics in agroinfiltrated plants, which enables the expression of complex antibody repertoires in a highly reproducible manner. Pluribodies developed using toxin-binding genetic information captured from peripheral blood lymphocytes of hyperimmunized camels recapitulated the overall binding activity of the immune response. Furthermore, an improved plant-made antivenom (plantivenom) was formulated using an in vitro selected pluribody against Bothrops asper snake venom toxins and has been shown to neutralize a wide range of toxin activities and provide protection against lethal venom doses in mice.

Antibody-mediated modulation of cytokinins in tobacco: organ-specific changes in cytokinin homeostasis.

Cytokinins comprise a group of phytohormones with an organ-specific mode of action. Although the mechanisms controlling the complex networks of cytokinin metabolism are partially known, the role of individual cytokinin types in the maintenance of cytokinin homeostasis remains unclear. Utilizing the overproduction of single-chain Fv antibodies selected for their ability to bind trans-zeatin riboside and targeted to the endoplasmic reticulum, we post-synthetically modulated cytokinin ribosides, the proposed transport forms of cytokinins. We observed asymmetric activity of cytokinin biosynthetic genes and cytokinin distribution in wild-type tobacco seedlings with higher cytokinin abundance in the root than in the shoot. Antibody-mediated modulation of cytokinin ribosides further enhanced the relative cytokinin abundance in the roots and induced cytokinin-related phenotypes in an organ-specific manner. The activity of cytokinin oxidase/dehydrogenase in the roots was strongly up-regulated in response to antibody-mediated formation of the cytokinin pool in the endoplasmic reticulum. However, we only detected a slight decrease in the root cytokinin levels. In contrast, a significant decrease of cytokinins occurred in the shoot. We suggest the roots as the main site of cytokinin biosynthesis in tobacco seedlings. Conversely, cytokinin levels in the shoot seem to depend largely on long-range transport of cytokinin ribosides from the root and their subsequent metabolic activation.

Enhanced transport of plant-produced rabies single-chain antibody-RVG peptide fusion protein across an in cellulo blood-brain barrier device.

The biomedical applications of antibody engineering are developing rapidly and have been expanded to plant expression platforms. In this study, we have generated a novel antibody molecule in planta for targeted delivery across the blood-brain barrier (BBB). Rabies virus (RABV) is a neurotropic virus for which there is no effective treatment after entry into the central nervous system. This study investigated the use of a RABV glycoprotein peptide sequence to assist delivery of a rabies neutralizing single-chain antibody (ScFv) across an in cellulo model of human BBB. The 29 amino acid rabies virus peptide (RVG) recognizes the nicotinic acetylcholine receptor (nAchR) at neuromuscular junctions and the BBB. ScFv and ScFv-RVG fusion proteins were produced in Nicotiana benthamiana by transient expression. Both molecules were successfully expressed and purified, but the ScFv expression level was significantly higher than that of ScFv-RVG fusion. Both ScFv and ScFv-RVG fusion molecules had potent neutralization activity against RABVin cellulo. The ScFv-RVG fusion demonstrated increased binding to nAchR and entry into neuronal cells, compared to ScFv alone. Additionally, a human brain endothelial cell line BBB model was used to demonstrate that plant-produced ScFv-RVGP fusion could translocate across the cells. This study indicates that the plant-produced ScFv-RVGP fusion protein was able to cross the in celluloBBB and neutralize RABV.

Rapid Transient Production of a Monoclonal Antibody Neutralizing the Porcine Epidemic Diarrhea Virus (PEDV) in Nicotiana benthamiana and Lactuca sativa.

Porcine epidemic diarrhea virus (PEDV) causes acute diarrhea, vomiting, dehydration, weight loss, and high mortality rate in neonatal piglets. Porcine epidemic diarrhea (PED) has been reported in Europe, America, and Asia including Thailand. The disease causes substantial losses to the swine industry in many countries. Presently, there is no effective PEDV vaccine available. In this study, we developed a plant-produced monoclonal antibody (mAb) 2C10 as a prophylactic candidate to prevent the PEDV infection. Recently, plant expression systems have gained interest as an alternative for the production of antibodies because of many advantages, such as low production cost, lack of human and animal pathogen, large scalability, etc. The 2C10 mAb was transiently expressed in Nicotiana benthamiana and lettuce using geminiviral vector. After purification by protein A affinity chromatography, the antibody was tested for the binding and neutralizing activity against PEDV. Our result showed that the plant produced 2C10 mAb can bind to the virus and also inhibit PEDV infection in vitro. These results show excellent potential for a plant-expressed 2C10 as a PEDV prophylaxis and a diagnostic for PEDV infection.

Development of Antibody Therapeutics against Flaviviruses.

Recent outbreaks of Zika virus (ZIKV) highlight the urgent need to develop efficacious interventions against flaviviruses, many of which cause devastating epidemics around the world. Monoclonal antibodies (mAb) have been at the forefront of treatment for cancer and a wide array of other diseases due to their specificity and potency. While mammalian cell-produced mAbs have shown promise as therapeutic candidates against several flaviviruses, their eventual approval for human application still faces several challenges including their potential risk of predisposing treated patients to more severe secondary infection by a heterologous flavivirus through antibody-dependent enhancement (ADE). The high cost associated with mAb production in mammalian cell cultures also poses a challenge for the feasible application of these drugs to the developing world where the majority of flavivirus infection occurs. Here, we review the current therapeutic mAb candidates against various flaviviruses including West Nile (WNV), Dengue virus (DENV), and ZIKV. The progress of using plants for developing safer and more economical mAb therapeutics against flaviviruses is discussed within the context of their expression, characterization, downstream processing, neutralization, and in vivo efficacy. The progress of using plant glycoengineering to address ADE, the major impediment of flavivirus therapeutic development, is highlighted. These advancements suggest that plant-based systems are excellent alternatives for addressing the remaining challenges of mAb therapeutic development against flavivirus and may facilitate the eventual commercialization of these drug candidates.

Production of an active anti-CD20-hIL-2 immunocytokine in Nicotiana benthamiana.

Anti-CD20 murine or chimeric antibodies (Abs) have been used to treat non-Hodgkin lymphomas (NHLs) and other diseases characterized by overactive or dysfunctional B cells. Anti-CD20 Abs demonstrated to be effective in inducing regression of B-cell lymphomas, although in many cases patients relapse following treatment. A promising approach to improve the outcome of mAb therapy is the use of anti-CD20 antibodies to deliver cytokines to the tumour microenvironment. In particular, IL-2-based immunocytokines have shown enhanced antitumour activity in several preclinical studies. Here, we report on the engineering of an anti-CD20-human interleukin-2 (hIL-2) immunocytokine (2B8-Fc-hIL2) based on the C2B8 mAb (Rituximab) and the resulting ectopic expression in Nicotiana benthamiana. The scFv-Fc-engineered immunocytokine is fully assembled in plants with minor degradation products as assessed by SDS-PAGE and gel filtration. Purification yields using protein-A affinity chromatography were in the range of 15-20 mg/kg of fresh leaf weight (FW). Glycopeptide analysis confirmed the presence of a highly homogeneous plant-type glycosylation. 2B8-Fc-hIL2 and the cognate 2B8-Fc antibody, devoid of hIL-2, were assayed by flow cytometry on Daudi cells revealing a CD20 binding activity comparable to that of Rituximab and were effective in eliciting antibody-dependent cell-mediated cytotoxicity of human PBMC versus Daudi cells, demonstrating their functional integrity. In 2B8-Fc-hIL2, IL-2 accessibility and biological activity were verified by flow cytometry and cell proliferation assay. To our knowledge, this is the first example of a recombinant immunocytokine based on the therapeutic Rituximab antibody scaffold, whose expression in plants may be a valuable tool for NHLs treatment.

Apoplastic venom allergen-like proteins of cyst nematodes modulate the activation of basal plant innate immunity by cell surface receptors.

Despite causing considerable damage to host tissue during the onset of parasitism, nematodes establish remarkably persistent infections in both animals and plants. It is thought that an elaborate repertoire of effector proteins in nematode secretions suppresses damage-triggered immune responses of the host. However, the nature and mode of action of most immunomodulatory compounds in nematode secretions are not well understood. Here, we show that venom allergen-like proteins of plant-parasitic nematodes selectively suppress host immunity mediated by surface-localized immune receptors. Venom allergen-like proteins are uniquely conserved in secretions of all animal- and plant-parasitic nematodes studied to date, but their role during the onset of parasitism has thus far remained elusive. Knocking-down the expression of the venom allergen-like protein Gr-VAP1 severely hampered the infectivity of the potato cyst nematode Globodera rostochiensis. By contrast, heterologous expression of Gr-VAP1 and two other venom allergen-like proteins from the beet cyst nematode Heterodera schachtii in plants resulted in the loss of basal immunity to multiple unrelated pathogens. The modulation of basal immunity by ectopic venom allergen-like proteins in Arabidopsis thaliana involved extracellular protease-based host defenses and non-photochemical quenching in chloroplasts. Non-photochemical quenching regulates the initiation of the defense-related programmed cell death, the onset of which was commonly suppressed by venom allergen-like proteins from G. rostochiensis, H. schachtii, and the root-knot nematode Meloidogyne incognita. Surprisingly, these venom allergen-like proteins only affected the programmed cell death mediated by surface-localized immune receptors. Furthermore, the delivery of venom allergen-like proteins into host tissue coincides with the enzymatic breakdown of plant cell walls by migratory nematodes. We, therefore, conclude that parasitic nematodes most likely utilize venom allergen-like proteins to suppress the activation of defenses by immunogenic breakdown products in damaged host tissue.

Tobacco seeds as efficient production platform for a biologically active anti-HBsAg monoclonal antibody.

The use of plants as heterologous hosts is one of the most promising technologies for manufacturing valuable recombinant proteins. Plant seeds, in particular, constitute ideal production platforms for long-term applications requiring a steady supply of starting material, as they combine the general advantages of plants as bioreactors with the possibility of biomass storage for long periods in a relatively small volume, thus allowing manufacturers to decouple upstream and downstream processing. In the present work we have used transgenic tobacco seeds to produce large amounts of a functionally active mouse monoclonal antibody against the Hepatitis B Virus surface antigen, fused to a KDEL endoplasmic reticulum retrieval motif, under control of regulatory sequences from common bean (Phaseolus vulgaris) seed storage proteins. The antibody accumulated to levels of 6.5 mg/g of seed in the T3 generation, and was purified by Protein A affinity chromatography combined with SEC-HPLC. N-glycan analysis indicated that, despite the KDEL signal, the seed-derived plantibody bore both high-mannose and complex-type sugars that indicate partial passage through the Golgi compartment, although its performance in the immunoaffinity purification of HBsAg was unaffected. An analysis discussing the industrial feasibility of replacing the currently used tobacco leaf-derived plantibody with this seed-derived variant is also presented.

Delayed treatment of Ebola virus infection with plant-derived monoclonal antibodies provides protection in rhesus macaques.

Filovirus infections can cause a severe and often fatal disease in humans and nonhuman primates, including great apes. Here, three anti-Ebola virus mouse/human chimeric mAbs (c13C6, h-13F6, and c6D8) were produced in Chinese hamster ovary and in whole plant (Nicotiana benthamiana) cells. In pilot experiments testing a mixture of the three mAbs (MB-003), we found that MB-003 produced in both manufacturing systems protected rhesus macaques from lethal challenge when administered 1 h postinfection. In a pivotal follow-up experiment, we found significant protection (P < 0.05) when MB-003 treatment began 24 or 48 h postinfection (four of six survived vs. zero of two controls). In all experiments, surviving animals that received MB-003 experienced little to no viremia and had few, if any, of the clinical symptoms observed in the controls. The results represent successful postexposure in vivo efficacy by a mAb mixture and suggest that this immunoprotectant should be further pursued as a postexposure and potential therapeutic for Ebola virus exposure.

Generation of a phage-display library of single-domain camelid VH H antibodies directed against Chlamydomonas reinhardtii antigens, and characterization of VH Hs binding cell-surface antigens.

Single-domain antibodies (sdAbs) are powerful tools for the detection, quantification, purification and subcellular localization of proteins of interest in biological research. We have generated camelid (Lama pacos) heavy chain-only variable VH domain (VH H) libraries against antigens in total cell lysates from Chlamydomonas reinhardtii. The sdAbs in the sera from immunized animals and VH H antibody domains isolated from the library show specificity to C. reinhardtii and lack of reactivity to antigens from four other algae: Chlorella variabilis, Coccomyxa subellipsoidea, Nannochloropsis oceanica and Thalassiosira pseudonana. Antibodies were produced against a diverse representation of antigens as evidenced by sera ELISA and protein-blot analyses. A phage-display library consisting of the VH H region contained at least 10(6) individual transformants, and thus should represent a wide range of C. reinhardtii antigens. The utility of the phage library was demonstrated by using live C. reinhardtii cells to pan for VH H clones with specific recognition of cell-surface epitopes. The lead candidate VH H clones (designated B11 and H10) bound to C. reinhardtii with EC50 values ≤ 0.5 nm. Treatment of cells with VH H B11 fused to the mCherry or green fluorescent proteins allowed brilliant and specific staining of the C. reinhardtii cell wall and analysis of cell-wall genesis during cell division. Such high-complexity VH H antibody libraries for algae will be valuable tools for algal researchers and biotechnologists.

Structural and functional characterization of an anti-West Nile virus monoclonal antibody and its single-chain variant produced in glycoengineered plants.

Previously, our group engineered a plant-derived monoclonal antibody (MAb pE16) that efficiently treated West Nile virus (WNV) infection in mice. In this study, we developed a pE16 variant consisting of a single-chain variable fragment (scFv) fused to the heavy chain constant domains (CH) of human IgG (pE16scFv-CH). pE16 and pE16scFv-CH were expressed and assembled efficiently in Nicotiana benthamiana ∆XF plants, a glycosylation mutant lacking plant-specific N-glycan residues. Glycan analysis revealed that ∆XF plant-derived pE16scFv-CH (∆XFpE16scFv-CH) and pE16 (∆XFpE16) both displayed a mammalian glycosylation profile. ∆XFpE16 and ∆XFpE16scFv-CH demonstrated equivalent antigen-binding affinity and kinetics, and slightly enhanced neutralization of WNV in vitro compared with the parent mammalian cell-produced E16 (mE16). A single dose of ∆XFpE16 or ∆XFpE16scFv-CH protected mice against WNV-induced mortality even 4 days after infection at equivalent rates as mE16. This study provides a detailed tandem comparison of the expression, structure and function of a therapeutic MAb and its single-chain variant produced in glycoengineered plants. Moreover, it demonstrates the development of anti-WNV MAb therapeutic variants that are equivalent in efficacy to pE16, simpler to produce, and likely safer to use as therapeutics due to their mammalian N-glycosylation. This platform may lead to a more robust and cost-effective production of antibody-based therapeutics against WNV infection and other infectious, inflammatory or neoplastic diseases.

Comparative in vitro and experimental in vivo studies of the anti-epidermal growth factor receptor antibody nimotuzumab and its aglycosylated form produced in transgenic tobacco plants.

A broad variety of foreign genes can be expressed in transgenic plants, which offer the opportunity for large-scale production of pharmaceutical proteins, such as therapeutic antibodies. Nimotuzumab is a humanized anti-epidermal growth factor receptor (EGFR) recombinant IgG1 antibody approved in different countries for the treatment of head and neck squamous cell carcinoma, paediatric and adult glioma, and nasopharyngeal and oesophageal cancers. Because the antitumour mechanism of nimotuzumab is mainly attributed to its ability to interrupt the signal transduction cascade triggered by EGF/EGFR interaction, we have hypothesized that an aglycosylated form of this antibody, produced by mutating the N(297) position in the IgG(1) Fc region gene, would have similar biochemical and biological properties as the mammalian-cell-produced glycosylated counterpart. In this paper, we report the production and characterization of an aglycosylated form of nimotuzumab in transgenic tobacco plants. The comparison of the plantibody and nimotuzumab in terms of recognition of human EGFR, effect on tyrosine phosphorylation and proliferation in cells in response to EGF, competition with radiolabelled EGF for EGFR, affinity measurements of Fab fragments, pharmacokinetic and biodistribution behaviours in rats and antitumour effects in nude mice bearing human A431 tumours showed that both antibody forms have very similar in vitro and in vivo properties. Our results support the idea that the production of aglycosylated forms of some therapeutic antibodies in transgenic plants is a feasible approach when facing scaling strategies for anticancer immunoglobulins.

Oral immunotherapy with transgenic rice seed containing destructed Japanese cedar pollen allergens, Cry j 1 and Cry j 2, against Japanese cedar pollinosis.

Transgenic rice accumulating the modified major Japanese cedar pollen allergens, Cryptomeria japonica 1 (Cry j 1) and Cryptomeria japonica 2 (Cry j 2), which were deconstructed by fragmentation and shuffling, respectively, in the edible part of the seed was generated by transformation of a good-tasting rice variety, 'Koshihikari'. These modified cedar pollen antigens were deposited in ER-derived protein bodies (PB-I), which are suitable for delivery to the mucosal immune system in gut-associated lymphoid tissue when orally administered because antigens bioencapsulated in PB-I are resistant against hydrolysis by intestinal enzymes and harsh environments. Mice fed transgenic seeds daily for three weeks and then challenged with crude cedar pollen allergen showed marked suppression of allergen-specific CD4(+) T-cell proliferation, IgE and IgG levels compared with mice fed nontransgenic rice seeds. As clinical symptoms of pollinosis, sneezing frequency and infiltration of inflammatory cells such as eosinophils and neutrophils were also significantly reduced in the nasal tissue. These results imply that oral administration of transgenic rice seeds containing the structurally disrupted Cry j 1 and Cry j 2 antigens, serving as universal antigens, is a promising approach for specific immunoprophylaxis against Japanese cedar pollinosis.

Oral delivery of bioencapsulated exendin-4 expressed in chloroplasts lowers blood glucose level in mice and stimulates insulin secretion in beta-TC6 cells.

Glucagon-like peptide (GLP-1) increases insulin secretion but is rapidly degraded (half-life: 2 min in circulation). GLP-1 analogue, exenatide (Byetta) has a longer half-life (3.3-4 h) with potent insulinotropic effects but requires cold storage, daily abdominal injections with short shelf life. Because patients with diabetes take >60 000 injections in their life time, alternative delivery methods are highly desired. Exenatide is ideal for oral delivery because insulinotropism is glucose dependent, with reduced risk of hypoglycaemia even at higher doses. Therefore, exendin-4 (EX4) was expressed as a cholera toxin B subunit (CTB)-fusion protein in tobacco chloroplasts to facilitate bioencapsulation within plant cells and transmucosal delivery in the gut via GM1 receptors present in the intestinal epithelium. The transgene integration was confirmed by PCR and Southern blot analysis. Expression level of CTB-EX4 reached up to 14.3% of total leaf protein (TLP). Lyophilization of leaf material increased therapeutic protein concentration by 12- to 24-fold, extended their shelf life up to 15 months when stored at room temperature and eliminated microbes present in fresh leaves. The pentameric structure, disulphide bonds and functionality of CTB-EX4 were well preserved in lyophilized materials. Chloroplast-derived CTB-EX4 showed increased insulin secretion similar to the commercial EX4 in beta-TC6, a mouse pancreatic cell line. Even when 5000-fold excess dose of CTB-EX4 was orally delivered, it stimulated insulin secretion similar to the intraperitoneal injection of commercial EX4 but did not cause hypoglycaemia in mice. Oral delivery of the bioencapsulated EX4 should eliminate injections, increase patient compliance/convenience and significantly lower their cost.

Utility of the P19 suppressor of gene-silencing protein for production of therapeutic antibodies in Nicotiana expression hosts.

To study how the P19 suppressor of gene-silencing protein can be used effectively for the production of therapeutic glycoproteins, the following factors were examined: the genetic elements used for expressing recombinant proteins; the effect of different P19 concentrations; compatibility of P19 with various Nicotiana tabacum cultivars for transgenic expression; the glycan profile of a recombinant therapeutic glycoprotein co-expressed with P19 in an RNAi-based glycomodified Nicotiana benthamiana expression host. The coding sequences for the heavy and light chains of trastuzumab were cloned into five plant expression vectors (102-106) containing different 5' and 3' UTRs, designated as vector sets 102-106 mAb. The P19 protein of Tomato bushy stunt virus (TBSV) was also cloned into vector 103, which contained the Cauliflower mosaic virus (CaMV) 35S promoter and 5'UTR together with the terminator region of the nopaline synthase gene of Agrobacterium. Transient expression of the antibody vectors resulted in different levels of trastuzumab accumulation, the highest being 105 and 106 mAb at about 1% of TSP. P19 increased the concentration of trastuzumab approximately 15-fold (to about 2.3% of TSP) when co-expressed with 103 mAb but did not affect antibody levels with vectors 102 and 106 mAb. When 103 mAb was expressed together with P19 in different N. tabacum cultivars, all except Little Crittenden showed a marked discolouring of the infiltrated areas of the leaf and decreased antibody expression. Co-expression of P19 also abolished antibody accumulation in crosses between N. tabacum cv. I-64 and Little Crittenden, indicating a dominant mode of inheritance for the observed P19-induced responses.

Purification of the therapeutic antibody trastuzumab from genetically modified plants using safflower Protein A-oleosin oilbody technology.

Production of therapeutic monoclonal antibodies using genetically modified plants may provide low cost, high scalability and product safety; however, antibody purification from plants presents a challenge due to the large quantities of biomass that need to be processed. Protein A column chromatography is widely used in the pharmaceutical industry for antibody purification, but its application is limited by cost, scalability and column fouling problems when purifying plant-derived antibodies. Protein A-oleosin oilbodies (Protein A-OB), expressed in transgenic safflower seeds, are relatively inexpensive to produce and provide a new approach for the capture of monoclonal antibodies from plants. When Protein A-OB is mixed with crude extracts from plants engineered to express therapeutic antibodies, the Protein A-OB captures the antibody in the oilbody phase while impurities remain in the aqueous phase. This is followed by repeated partitioning of oilbody phase against an aqueous phase via centrifugation to remove impurities before purified antibody is eluted from the oilbodies. We have developed this purification process to recover trastuzumab, an anti-HER2 monoclonal antibody used for therapy against specific breast-cancers that over express HER2 (human epidermal growth factor receptor 2), from transiently infected Nicotiana benthamiana. Protein A-OB overcomes the fouling problem associated with traditional Protein A chromatography, allowing for the development of an inexpensive, scalable and novel high-resolution method for the capture of antibodies based on simple mixing and phase separation.

Chlamydomonas reinhardtii as a viable platform for the production of recombinant proteins: current status and perspectives.

Chlamydomonas reinhardtii has many advantages compared with traditional systems for the molecular farming of recombinant proteins. These include low production costs, rapid scalability at pilot level, absence of human pathogens and the ability to fold and assemble complex proteins accurately. Currently, the successful expression of several proteins with pharmaceutical relevance has been reported from the nuclear and the chloroplastic genome of this alga, demonstrating its usefulness for biotechnological applications. However, several factors affect the level of recombinant protein expression in Chlamydomonas such as enhancer elements, codon dependency, sensitivity to proteases and transformation-associated genotypic modification. The present review outlines a number of strategies to increase protein yields and summarizes recent achievements in algal protein production including biopharmaceuticals such as vaccines, antibodies, hormones and enzymes with implications on health-related approaches. The current status of bioreactor developments for algal culture and the challenges of scale-up and optimization processes are also discussed.

Chloroplast-derived vaccines against human diseases: achievements, challenges and scopes.

Infectious diseases represent a continuously growing menace that has severe impact on health of the people worldwide, particularly in the developing countries. Therefore, novel prevention and treatment strategies are urgently needed to reduce the rate of these diseases in humans. For this reason, different options can be considered for the production of affordable vaccines. Plants have been proved as an alternative expression system for various compounds of biological importance. Particularly, plastid genetic engineering can be potentially used as a tool for cost-effective vaccine production. Antigenic proteins from different viruses and bacteria have been expressed in plastids. Initial immunological studies of chloroplast-derived vaccines have yielded promising results in animal models. However, because of certain limitations, these vaccines face many challenges on production and application level. Adaptations to the novel approaches are needed, which comprise codon usage and choice of proven expression cassettes for the optimal yield of expressed proteins, use of inducible systems, marker gene removal, selection of specific antigens with high immunogenicity and development of tissue culture systems for edible crops to prove the concept of low-cost edible vaccines. As various aspects of plant-based vaccines have been discussed in recent reviews, here we will focus on certain aspects of chloroplast transformation related to vaccine production against human diseases.