Development and Expression of Subunit Vaccines Against Viruses in Plants.

Various systems exist for the robust production of recombinant proteins. However, only a few systems are optimal for human vaccine protein production. Plant-based transient protein expression systems offer an advantageous alternative to costly mammalian cell culture-based systems and can perform posttranslational modifications due to the presence of an endomembrane system that is largely similar to that of the animal cell. Technological advances in expression vectors for transient expression in the last two decades have produced new plant expression systems with the flexibility and speed that cannot be matched by those based on mammalian or insect cell culture. The rapid and high-level protein production capability of transient expression systems makes them the optimal system to quickly and versatilely develop and produce vaccines against viruses such as 2019-nCoV that have sudden and unpredictable outbreaks. Here, expression of antiviral subunit vaccines in Nicotiana benthamiana plants via transient expression is demonstrated.

Functional characterization of a plant-produced infectious bursal disease virus antigen fused to the constant region of avian IgY immunoglobulins.

Infectious bursal disease virus (IBDV) is the cause of an economically important highly contagious disease of poultry, and vaccines are regarded as the most beneficial interventions for its prevention. In this study, plants were used to produce a recombinant chimeric IBDV antigen for the formulation of an innovative subunit vaccine. The fusion protein (PD-FcY) was designed to combine the immunodominant projection domain (PD) of the viral structural protein VP2 with the constant region of avian IgY (FcY), which was selected to enhance antigen uptake by avian immune cells. The gene construct encoding the fusion protein was transiently expressed in Nicotiana benthamiana plants and an extraction/purification protocol was set up, allowing to reduce the contamination by undesired plant compounds/proteins. Mass spectrometry analysis of the purified protein revealed that the glycosylation pattern of the FcY portion was similar to that observed in native IgY, while in vitro assays demonstrated the ability of PD-FcY to bind to the avian immunoglobulin receptor CHIR-AB1. Preliminary immunization studies proved that PD-FcY was able to induce the production of protective anti-IBDV-VP2 antibodies in chickens. In conclusion, the proposed fusion strategy holds promises for the development of innovative low-cost subunit vaccines for the prevention of avian viral diseases.

A Plant-Produced Candidate Subunit Vaccine Reduces Shedding of Enterohemorrhagic Escherichia coli in Ruminants.

Enterohemorrhagic Escherichia coli (EHEC) are commonly present in the gastrointestinal tract of cattle and cause serious infectious disease in humans. Immunizing cattle against EHEC is a promising strategy to decrease the risk of food contamination; however, veterinary vaccines against EHEC such as Econiche have not been widely adopted by the agricultural industry, and have been discontinued, prompting the need for more cost-effective EHEC vaccines. The objective of this project is to develop a platform to produce plant-made antigens for oral vaccination of ruminants against EHEC. Five recombinant proteins were designed as vaccine candidates and expressed transiently in Nicotiana benthamiana and transplastomically in Nicotiana tabacum. Three of these EHEC proteins, NleA, Stx2b, and a fusion of EspA accumulated when transiently expressed. Transient protein accumulation was the highest when EHEC proteins were fused to an elastin-like polypeptide (ELP) tag. In the transplastomic lines, EspA accumulated up to 479 mg kg-1 in lyophilized leaf material. Sheep that were administered leaf tissue containing recombinant EspA shed less E. coli O157:H7 when challenged, as compared to control animals. These results suggest that plant-made, transgenic EspA has the potential to reduce EHEC shedding in ruminants.

Assessment of the potential utility of different regions of Streptococcus uberis adhesion molecule (SUAM) for mastitis subunit vaccine development.

Streptococcus uberis is one of the most prevalent pathogens causing clinical and subclinical mastitis worldwide. Among bacterial factors involved in intramammary infections caused by this organism, S. uberis adhesion molecule (SUAM) is one of the main virulence factors identified. This molecule is involved in S. uberis internalization to mammary epithelial cells through lactoferrin (Lf) binding. The objective of this study was to evaluate SUAM properties as a potential subunit vaccine component for prevention of S. uberis mastitis. B epitope prediction analysis of SUAM sequence was used to identify potentially immunogenic regions. Since these regions were detected all along the gene, this criterion did not allow selecting a specific region as a potential immunogen. Hence, four fractions of SUAM (-1fr, 2fr, 3fr and 4fr), comprising most of the protein, were cloned and expressed. Every fraction elicited a humoral immune response in mice as predicted by bioinformatics analysis. SUAM-1fr generated antibodies with the highest recognition ability towards SUAM native protein. Moreover, antibodies against SUAM-1fr produced the highest proportion of internalization inhibition of S. uberis to mammary epithelial cells. In conclusion, SUAM immunogenic and functionally relevant regions were identified and allowed to propose SUAM-1fr as a potential candidate for a subunit vaccine for S. uberis mastitis prevention.

The current state of therapeutic and T cell-based vaccines against human papillomaviruses.

Human papillomavirus (HPV) is known to be a necessary factor for many gynecologic malignancies and is also associated with a subset of head and neck malignancies. This knowledge has created the opportunity to control these HPV-associated cancers through vaccination. However, despite the availability of prophylactic HPV vaccines, HPV infections remain extremely common worldwide. In addition, while prophylactic HPV vaccines have been effective in preventing infection, they are ineffective at clearing pre-existing HPV infections. Thus, there is an urgent need for therapeutic and T cell-based vaccines to treat existing HPV infections and HPV-associated lesions and cancers. Unlike prophylactic vaccines, which generate neutralizing antibodies, therapeutic, and T cell-based vaccines enhance cell-mediated immunity against HPV antigens. Our review will cover various therapeutic and T cell-based vaccines in development for the treatment of HPV-associated diseases. Furthermore, we review the strategies to enhance the efficacy of therapeutic vaccines and the latest clinical trials on therapeutic and T cell-based HPV vaccines.

Developments in L2-based human papillomavirus (HPV) vaccines.

Infections with sexually transmitted high-risk Human Papillomavirus (hrHPV), of which there are at least 15 genotypes, are responsible for a tremendous disease burden by causing cervical, and subsets of other ano-genital and oro-pharyngeal carcinomas, together representing 5% of all cancer cases worldwide. HPV subunit vaccines consisting of virus-like particles (VLP) self-assembled from major capsid protein L1 plus adjuvant have been licensed. Prophylactic vaccinations with the 2-valent (HPV16/18), 4-valent (HPV6/11/16/18), or 9-valent (HPV6/11/16/18/31/33/45/52/58) vaccine induce high-titer neutralizing antibodies restricted to the vaccine types that cause up to 90% of cervical carcinomas, a subset of other ano-genital and oro-pharyngeal cancers and 90% of benign ano-genital warts (condylomata). The complexity of manufacturing multivalent L1-VLP vaccines limits the number of included VLP types and thus the vaccines' spectrum of protection, leaving a panel of oncogenic mucosal HPV unaddressed. In addition, current vaccines do not protect against cutaneous HPV types causing benign skin warts, or against beta-papillomavirus (betaPV) types implicated in the development of non-melanoma skin cancer (NMSC) in immunosuppressed patients. In contrast with L1-VLP, the minor capsid protein L2 contains type-common epitopes that induce low-titer yet broadly cross-neutralizing antibodies to heterologous PV types and provide cross-protection in animal challenge models. Efforts to increase the low immunogenicity of L2 (poly)-peptides and thereby to develop broader-spectrum HPV vaccines are the focus of this review.

CONSTRUCTION AND EXPRESSION OF DERMATOPHAGOIDES PTERONYSSINUS GROUP 1 MAJOR ALLERGEN T CELL FUSION EPITOPE PEPTIDE VACCINE VECTOR BASED ON THE MHC II PATHWAY.

UNLABELLED: Backgound and aims: Dermatophagoides peteronyssinus is one of the important house dust mites responsible for allergic asthma that can be tentatively managed by specific immunotherapy. The present study was to construct a vector encoding T-cell epitopes of major allergen group 1 of Dermatophagoides pteronyssinus as a vaccine delivered by MHC class II pathway. METHODS: the nucleotide sequences of the 3 target genes were synthesized, including TAT, IhC and the recombinant fragment of Der p 1 encoding 3 T-cell epitopes. After amplification of the 3 target fragments by PCR and digestion with corresponding restriction endonucleases, the recombinant gene TAT-IhC-Der p 1-3T was ligated using T4 DNA ligase and inserted into the prokaryotic expression vector pET28a(+) to construct the recombinant plasmid pET- 28a(+)-TAT-IhC-Der p 1-3T, which was confirmed by digestion with restriction endonucleases and sequencing. The recombinant vector was transformed into E. coli strain BL21 (DE3) and induced with IPTG, and the induced protein TAT-IhC-Der p1-3T was detected by SDS-PAGE. After purification, the recombinant protein was confirmed by Western blotting and its allergenicity tested using IgE-binding assay. RESULTS: the recombinant plasmid pET-28a-TAT-IhCDer p1-3T was successfully constructed as confirmed by restriction endonuclease digestion and sequencing, and the expression of the recombinant protein TAT-IhC-Der p1-3T was induced in E. coli. Western blotting verified successfull purification of the target protein, which showed a stronger IgE-binding ability than Der p1. CONCLUSION: we successfully constructed the recombinant expression vector pET-28a-TAT-IhC-Der p1-3T expressing a T-cell epitope vaccine delivered by MHC II pathway with strong IgE-binding ability, which provides a basis for further study on specific immunotherapy via MHC class II pathway.

Antecedentes y objetivo: el Dermatophagoides peteronyssinus es uno de los principales ácaros del polvo doméstico responsables del asma alérgica que se pueden administrar provisionalmente para una inmunoterapia específica. El presente estudio busca construir un vector que codifique epítopos de células T del grupo de alérgenos principal, el Grupo 1 de Dermatophagoides pteronyssinus como una vacuna suministrada mediante la vía MHC de clase II. Métodos: se sintetizaron las secuencias de nucleótidos de los 3 genes objetivo, incluyendo TAT, IhC y el fragmento recombinante de Der p 1 encargado de codificar 3 epítopos de célula T. Después de la amplificación de los 3 fragmentos objetivo por PCR y digestión con endonucleasas de restricción correspondientes, el gen recombinante TAT-IhC-Der p 1-3T se ligó usando T4 DNA ligasa y se insertó en el vector de expresión procariota pET28a (+) para construir el plásmido recombinante pET 28a (+)-TAT-IHC-Der p 1-3T, que se confirmó por digestión con endonucleasas de restricción y secuenciación. El vector recombinante se transformó en E. coli cepa BL21 (DE3) y se indujo con IPTG, y la proteína inducida TATIHC- Der p1-3T se detectó mediante SDS-PAGE. Después de la purificación, la proteina recombinante se confirmó por análisis de inmunotransferencia (Western blot) y se probó su alergenicidad usando el ensayo de unión a IgE. Resultados: el plásmido recombinante pET-28a-TATIHCDer p1-3T se construyó con éxito, se confirmó por digestión con endonucleasas de restricción y la secuenciación y la expresión de la proteína recombinante TAT-IHCDer p1-3T fue inducida en E. coli. Purificación con éxito verificada mediante Western blot de la proteína objetivo, que mostró una capacidad de unión a IgE más fuerte que Der p1. Conclusión: hemos construido con éxito el vector de expresión recombinante pET-28a-TAT-IHC-Der p1-3T que expresa una vacuna de epítopo de células T administrada por vía MHC II con fuerte capacidad de union a IgE. Este trabajo proporciona una base para seguir estudiando la inmunoterapia específica mediante la vía MHC de clase II.

Desarrollo de una vacuna mejorada de la subunidad del virus de la diarrea viral bovina basada en la glicoproteína E2 fusionada con un anticuerpo de cadena única que se dirige a las células presentadoras de antígenos / Development of an enhanced bovine viral diarrhea virus subunit vaccine based on E2 glycoprotein fused to a single chain antibody which targets to antigen-presenting cells

Bovine viral diarrhea virus (BVDV) is an important cause of economic losses worldwide. E2 is an immunodominant protein and a promising candidate to develop subunit vaccines. To improve its immunogenicity, a truncated E2 (tE2) was fused to a single chain antibody named APCH, which targets to antigen-presenting cells. APCH-tE2 and tE2 proteins were expressed in the baculovirus system and their immunogenicity was firstly compared in guinea pigs. APCH-tE2 vaccine was the best one to evoke a humoral response, and for this reason, it was selected for a cattle vaccination experiment. All the bovines immunized with 1.5Ag of APCH-tE2 developed high levels of neutralizing antibodies against BVDV up to a year post-immunization, demonstrating its significant potential as a subunit vaccine. This novel vaccine is undergoing scale-up and was transferred to the private sector. Nowadays, it is being evaluated for registration as the first Argentinean subunit vaccine for cattle

El virus de la diarrea viral bovina (BVDV) es causante de importantes pérdidas económicas a nivel mundial. La proteína E2 es la inmunodominante del virus y es la candidata para desarrollar vacunas de subunidad. Para mejorar su inmunogenicidad, una versión truncada de la E2 (tE2) se fusionó a un anticuerpo de cadena simple (APCH), que se dirige a las células presentadoras de antígeno. Se expresaron las proteínas APCH-tE2 y tE2 en el sistema de baculovirus y su inmunogenicidad fue evaluada y comparada en cobayos; la proteína APCH-tE2 fue la que indujo la mejor respuesta humoral. Por dicha razón se la evaluó en bovinos utilizando 1,5µg de antígeno. Los animales presentaron altos títulos de anticuerpos neutralizantes contra BVDV hasta un año posinmunización. Esta nueva vacuna está en proceso de escalado y se transfirió al sector privado. Actualmente se está evaluando para su registro como la primera vacuna argentina de subunidad para bovinos

Recombinant protein subunit vaccine synthesis in microbes: a role for yeast?

OBJECTIVES: Recombinant protein subunit vaccines are formulated using protein antigens that have been synthesized in heterologous host cells. Several host cells are available for this purpose, ranging from Escherichia coli to mammalian cell lines. This article highlights the benefits of using yeast as the recombinant host. KEY FINDINGS: The yeast species, Saccharomyces cerevisiae and Pichia pastoris, have been used to optimize the functional yields of potential antigens for the development of subunit vaccines against a wide range of diseases caused by bacteria and viruses. Saccharomyces cerevisiae has also been used in the manufacture of 11 approved vaccines against hepatitis B virus and one against human papillomavirus; in both cases, the recombinant protein forms highly immunogenic virus-like particles. SUMMARY: Advances in our understanding of how a yeast cell responds to the metabolic load of producing recombinant proteins will allow us to identify host strains that have improved yield properties and enable the synthesis of more challenging antigens that cannot be produced in other systems. Yeasts therefore have the potential to become important host organisms for the production of recombinant antigens that can be used in the manufacture of subunit vaccines or in new vaccine development.

[High-efficiency expression of a receptor-binding domain of SARS-CoV spike protein in tobacco chloroplasts].

Chloroplast-based expression system is promising for the hyper-expression of plant-derived recombinant therapeutic proteins and vaccines. To verify the feasibility of obtaining high-level expression of the SARS subunit vaccine and to provide a suitable plant-derived vaccine production platform against the severe acute respiratory syndrome coronavirus (SARS-CoV), a 193-amino acid fragment of SARS CoV spike protein receptor-binding domain (RBD), fused with the peptide vector cholera toxin B subunit (CTB), was expressed in tobacco chloroplasts. Codon-optimized CTB-RBD sequence was integrated into the chloroplast genome and homoplasmy was obtained, as confirmed by PCR and Southern blot analysis. Western blot showed expression of the recombinant fusion protein mostly in soluble monomeric form. Quantification of the recombinant fusion protein CTB-RBD was conducted by ELISA analysis from the transplastomic leaves at different developmental stages, attachment positions and time points in a day and the different expression levels of the CTB-RBD were observed with the highest expression of 10.2% total soluble protein obtained from mature transplastomic leaves. Taken together, our results demonstrate the feasibility of highly expressing SARS subunit vaccine RBD, indicating its potential in subsequent development of a plant-derived recombinant subunit vaccine and reagents production for antibody detection in SARS serological tests.

Cysteine peptidases as schistosomiasis vaccines with inbuilt adjuvanticity.

Schistosomiasis is caused by several worm species of the genus Schistosoma and afflicts up to 600 million people in 74 tropical and sub-tropical countries in the developing world. Present disease control depends on treatment with the only available drug praziquantel. No vaccine exists despite the intense search for molecular candidates and adjuvant formulations over the last three decades. Cysteine peptidases such as papain and Der p 1 are well known environmental allergens that sensitize the immune system driving potent Th2-responses. Recently, we showed that the administration of active papain to mice induced significant protection (P<0.02, 50%) against an experimental challenge infection with Schistosoma mansoni. Since schistosomes express and secrete papain-like cysteine peptidases we reasoned that these could be employed as vaccines with inbuilt adjuvanticity to protect against these parasites. Here we demonstrate that sub-cutaneous injection of functionally active S. mansoni cathepsin B1 (SmCB1), or a cathepsin L from a related parasite Fasciola hepatica (FhCL1), elicits highly significant (P<0.0001) protection (up to 73%) against an experimental challenge worm infection. Protection and reduction in worm egg burden were further increased (up to 83%) when the cysteine peptidases were combined with other S. mansoni vaccine candidates, glyceraldehyde 3-phosphate dehydrogenase (SG3PDH) and peroxiredoxin (PRX-MAP), without the need to add chemical adjuvants. These studies demonstrate the capacity of helminth cysteine peptidases to behave simultaneously as immunogens and adjuvants, and offer an innovative approach towards developing schistosomiasis vaccines.

Transient expression of VP2 in Nicotiana benthamiana and its use as a plant-based vaccine against infectious bursal disease virus.

Infectious Bursal Disease Virus (IBDV) is the etiological agent of an immunosuppressive and highly contagious disease that affects young birds. This disease causes important economic losses in the poultry industry worldwide. The VP2 protein has been used for the development of subunit vaccines in a variety of heterologous platforms. In this context, the aim of this study was to investigate VP2 expression and immunogenicity using an experimental plant-based vaccine against IBDV. We determined that the agroinfiltration of N. benthamiana leaves allowed the production of VP2 with no apparent change on its conformational epitopes. Chickens intramuscularly immunized in a dose/boost scheme with crude concentrated extracts developed a specific humoral response with viral neutralizing ability. Given these results, it seems plausible for a plant-based vaccine to have a niche in the veterinary field. Thus, plants can be an adequate system of choice to produce immunogens against IBDV.

Production of a subunit vaccine candidate against porcine post-weaning diarrhea in high-biomass transplastomic tobacco.

Post-weaning diarrhea (PWD) in piglets is a major problem in piggeries worldwide and results in severe economic losses. Infection with Enterotoxigenic Escherichia coli (ETEC) is the key culprit for the PWD disease. F4 fimbriae of ETEC are highly stable proteinaceous polymers, mainly composed of the major structural subunit FaeG, with a capacity to evoke mucosal immune responses, thus demonstrating a potential to act as an oral vaccine against ETEC-induced porcine PWD. In this study we used a transplastomic approach in tobacco to produce a recombinant variant of the FaeG protein, rFaeG(ntd/dsc), engineered for expression as a stable monomer by N-terminal deletion and donor strand-complementation (ntd/dsc). The generated transplastomic tobacco plants accumulated up to 2.0 g rFaeG(ntd/dsc) per 1 kg fresh leaf tissue (more than 1% of dry leaf tissue) and showed normal phenotype indistinguishable from wild type untransformed plants. We determined that chloroplast-produced rFaeG(ntd/dsc) protein retained the key properties of an oral vaccine, i.e. binding to porcine intestinal F4 receptors (F4R), and inhibition of the F4-possessing (F4+) ETEC attachment to F4R. Additionally, the plant biomass matrix was shown to delay degradation of the chloroplast-produced rFaeG(ntd/dsc) in gastrointestinal conditions, demonstrating a potential to function as a shelter-vehicle for vaccine delivery. These results suggest that transplastomic plants expressing the rFaeG(ntd/dsc) protein could be used for production and, possibly, delivery of an oral vaccine against porcine F4+ ETEC infections. Our findings therefore present a feasible approach for developing an oral vaccination strategy against porcine PWD.

Emerging antibody products and Nicotiana manufacturing.

Antibody based products are not widely available to address multiple global health challenges due to high costs, limited manufacturing capacity, and long manufacturing lead times. Nicotiana-based manufacturing of antibody products may now begin to address these challenges as a result of revolutionary advances in transient expression and altered glycosylation pathways. This review provides examples of emerging antibody-based products (mucosal and systemic) that could be competitive and commercially viable when the attributes of Nicotiana-based manufacturing (large scale, versatile, rapid, low cost) are utilized.

Preparation of a peptide vaccine against GnRH by a bioprocess system based on asparaginase.

GnRH is a promising target in hormone-dependent cancer immunotherapy. In our previous study, we have designed and purified a peptide vaccine GhM (GnRH3-hinge-MVP) by use of the bioprocess system based on asparaginase. Active immunization with GhM in the presence of CFA/IFA evoked strong humoral response. In this study, the motif NRLLLTG with high affinity to nanoparticle carrier VLP HBcDelta-SBD was fused to the C terminus of GhM to form a new peptide vaccine GhMNR (GnRH3-hinge-MVP-NRLLLTG). The fusion protein ansB-C-GhMNR was controlled by vigorous T7lac promotor and expressed effectively as inclusion bodies after induction by lactose and then purified by means of cell disruption, washing and cold ethanol fractionation. After hydrolyzed for 72 h, GhMNR was liberated from the fusion partner ansB-C and purified by CM52 cation exchange chromatography. These results suggested that the bioprocess system is suitable for large-scale expression and purification of the peptide vaccine GhMNR, and even some other proteins or peptides which may be important for industrial or laboratory purposes.

Yeast-expressed classical swine fever virus glycoprotein E2 induces a protective immune response.

Classical swine fever (CSF) is an economically important swine disease worldwide. The glycoprotein E2 of classical swine fever virus (CSFV) is a viral antigen that can induce a protective immune response against CSF. A recombinant E2 protein was constructed using the yeast Pichia pastoris expression system and evaluated for its vaccine efficacy. The yeast-expressed E2 (yE2) was shown to have N-linked glycosylation and to form homodimer molecules. Four 6-week-old specified-pathogen-free (SPF) piglets were intramuscularly immunized with yE2 twice at 3-week intervals. All yE2-vaccinated pigs could mount an anamnestic response after booster vaccination with neutralizing antibody titers ranging from 1:96 to 1:768. Neutralizing antibody titers at 10 weeks post booster vaccination ranged from 1:16 to 1:64. At this time, the pigs were subjected to challenge infection with a dose of 1x10(5)TCID(50) (50% tissue culture infective dose) virulent CSFV strain. At 1 week post challenge infection, all of the yE2-immunized pigs were alive and without symptoms or signs of CSF. Neutralizing antibody titers at this time ranged from 1:4,800 to 1:12,800 and even to 1:51,200 one week later. In contrast, the control pigs continuously exhibited signs of CSF and had to be euthanized because of severe clinical symptoms at 6 days post challenge infection. All of the yE2-vaccinated pigs were E(rns) antibody negative and had seroconverted against E(rns) by post challenge day 11, suggesting that yE2 is a potential DIVA (differentiating infected from vaccinated animals) vaccine. The yeast-expressed E2 protein retains correct immunogenicity and is able to induce a protective immune response against CSFV infection.

Evaluation of efficacy of mammalian and baculovirus expressed E2 subunit vaccine candidates to bovine viral diarrhoea virus.

Bovine viral diarrhoea virus (BVDV) is a worldwide pathogen of cattle causing a wide spectrum of clinical disease. The major envelope glycoprotein of BVDV, E2, induces the production of neutralising antibodies. In this study we compared the protection afforded to cattle after BVDV challenge by two separate E2 vaccine candidates produced by different heterologous protein expression systems. E2 antigen was expressed using the baculovirus expression system (brE2) and a mammalian cell expression system (mrE2). In the first vaccination study the quantity of recombinant protein expressed by the two systems differed. Vaccination of cattle with a higher dose of brE2 or low dose mrE2 gave comparable protection from viral challenge. Immunised animals showed no pyrexia and reduced leucopaenia which contrasted to the unvaccinated controls. In addition virus shedding from the nasal mucosa was decreased in the vaccinated groups and strong humoral responses were evident post-challenge. However, the efficacy of the brE2 vaccine was greatly diminished when a reduced dose was tested, indicating the importance of assessing the type of expression system used in antigen production.

Generation of plant-derived recombinant DTP subunit vaccine.

The current diphtheria-tetanus-pertussis (DTP) pediatric vaccine is produced from the corresponding pathogenic bacteria Corynebacterium diphtheriae, Clostridium tetani and Bordetella pertussis; five injected doses of DTaP (acellular) vaccine are required for every child in the standard US vaccination schedule. Because the vaccine is derived from native live sources, adverse effects are possible and production is complex and costly. To address issues of safety, ease of renewability and expense, we used recombinant technology in an effort to develop a subunit DPT vaccine derived in non-pathogenic plant expression systems. Expression of diphtheria toxin (DT), tetanus fragment-C (TetC) and the non-toxic S1 subunit of pertussis toxin (PTX S1) antigenic proteins in soluble form in low-alkaloid tobacco plants and carrot cell cultures allowed efficient downstream purification to levels suitable for intramuscular injection in BALB/c mice. At working concentrations of 5mug per dose, these preparations induced high levels of antigen-specific IgGs in mouse sera. Our results clearly support the feasibility of producing recombinant pediatric vaccine components in plants.

Display of peptides on the surface of tobacco mosaic virus particles.

In this review, we focus on the potential that tobacco mosaic virus (TMV) has as a carrier for immunogenic epitopes, and the factors that must be considered in order to bring products based on this platform to the market. Large Scale Biology Corporation developed facile and scaleable methods for manufacture of candidate peptide display vaccines based on TMV. We describe how rational design of peptide vaccines can improve the manufacturability of particular TMV products. We also discuss downstream processing and purification of the vaccine products, with particular attention to the metrics that a product must attain in order to meet criteria for regulatory approval as injectable biologics.

Multiepitope peptide-loaded virus-like particles as a vaccine against hepatitis B virus-related hepatocellular carcinoma.

UNLABELLED: To develop a hepatitis B virus (HBV) therapeutic vaccine that can induce a broad but specific immune response and significant antitumor effects both in vivo and in vitro, we inserted HBV X protein (HBx)-derived epitopes HBx(52-60), HBx(92-100), and HBx(115-123); a novel subdominant cytolytic T lymphocyte (CTL) epitope HBx(140-148); and the universal T helper epitope pan human leukocyte antigen DR-binding epitope into HBV core protein to form multiepitope peptide-loaded virus-like particles (VLPs). CTL responses against epitope-loaded VLPs were elicited by priming with VLP-pulsed dendritic cells in both HLA-A*0201 transgenic (Tg) mice and peripheral blood lymphocytes from HLA-A2(+)/HBx(+) HBV-infected hepatocellular carcinoma (HCC) patients. The multiepitope peptide-loaded VLPs demonstrated significantly higher immunogenicity in Tg mice than any single responsive epitope. Significant antitumor effects were demonstrated both with primary cultured autologous HCC cells in vitro and tumor-bearing Tg mice in vivo in an HLA-A2-restricted and epitope-specific fashion. CONCLUSION: The significant antitumor effects both in vivo and in vitro demonstrate the potential of multiepitope peptide-loaded VLPs as a vaccine against HCC.