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Recently, we developed a high yield production process for outer membrane particles from genetically modified bacteria, called Generalized Modules of Membrane Antigens (GMMA), and the corresponding simple two step filtration purification, enabling economic manufacture of these particles for use as vaccines. Using a Shigella sonnei strain that was genetically modified to produce penta-acylated lipopolysaccharide (LPS) with reduced endotoxicity and to maintain the virulence plasmid encoding for the immunodominant O antigen component of the LPS, scale up of the process to GMP pilot scale was straightforward and gave high yields of GMMA with required purity and consistent results. GMMA were formulated with Alhydrogel and were highly immunogenic in mice and rabbits. In mice, a single immunization containing 29 ng protein and 1.75 ng of O antigen elicited substantial anti-LPS antibody levels. As GMMA contain LPS and lipoproteins, assessing potential reactogenicity was a key aspect of vaccine development. In an in vitro monocyte activation test, GMMA from the production strain showed a 600-fold lower stimulatory activity than GMMA with unmodified LPS. Two in vivo tests confirmed the low potential for reactogenicity. We established a modified rabbit pyrogenicity test based on the European Pharmacopoeia pyrogens method but using intramuscular administration of the full human dose (100 μg of protein). The vaccine elicited an average temperature rise of 0.5°C within four hours after administration, which was considered acceptable and showed that the test is able to detect a pyrogenic response. Furthermore, a repeat dose toxicology study in rabbits using intramuscular (100 μg/dose), intranasal (80 μg/dose), and intradermal (10 μg/dose) administration routes showed good tolerability of the vaccine by all routes and supported its suitability for use in humans. The S. sonnei GMMA vaccine is now in Phase 1 dose-escalation clinical trials.

► BEVS has matured into a commercial manufacturing technology. ► A broad range of vaccines can be produced in a universal “plug and play” process. ► Vaccines can be produced at affordable prices. ► BEVS can offer sustainability of vaccine supply. The baculovirus-insect cell expression system is a well known tool for the production of complex proteins. The technology is also used for commercial manufacture of various veterinary and human vaccines. This review paper provides an overview of how this technology can be applied to produce a multitude of vaccine candidates. The key advantage of this recombinant protein manufacturing platform is that a universal “plug and play” process may be used for producing a broad range of protein-based prophylactic and therapeutic vaccines for both human and veterinary use while offering the potential for low manufacturing costs. Large scale mammalian cell culture facilities previously established for the manufacturing of monoclonal antibodies that have now become obsolete due to yield improvement could be deployed for the manufacturing of these vaccines. Alternatively, manufacturing capacity could be established in geographic regions that do not have any vaccine production capability. Dependent on health care priorities, different vaccines could be manufactured while maintaining the ability to rapidly convert to producing pandemic influenza vaccine when the need arises.

Malaria is a widespread and infectious disease that is a leading cause of death in many parts of the world. Eradication of malaria has been a major world health goal for decades, but one that still remains elusive. Other diseases have been eradicated using vaccination, but traditional vaccination methods have thus far been unsuccessful for malaria. Infection by Plasmodium species, the causative agent of malaria, is currently treated with drug-based therapies, but an increase in drug resistance has led to the need for new methods of treatment. A promising strategy for malaria treatment is to combine transmission blocking vaccines (TBVs) that prevent spread of disease with drug-based therapies to treat infected individuals. TBVs can be developed against surface protein antigens that are expressed during parasite reproduction in the mosquito. When the mosquito ingests blood from a vaccinated individual harboring the Plasmodium parasite, the antibodies generated by vaccination prevent completion of the parasites life-cycle. Animal studies have shown that immunization with Pfs48/45 results in the production of malaria transmission blocking antibodies; however, the development of this vaccine candidate has been hindered by poor expression in both prokaryotic and eukaryotic hosts. Recently, the chloroplast of Chlamydomonas reinhardtii has been used to express complex recombinant proteins. In this study, we show that the C-terminal antigenic region of the Pfs48/45 antigen can be expressed in the chloroplast of the green algae C. reinhardtii and that this recombinant protein has a conformation recognized by known transmission blocking antibodies. Production of this protein in algae has the potential to scale to the very large volumes required to meet the needs of millions at risk for contracting malaria.

•Recombinant capsid protein of hepatitis E virus as vaccine antigen.•Multiple epitope characterization using monoclonal antibodies.•Successful scale up and robust manufacturing of vaccine antigen.•Product consistency demonstrated with orthogonal analytical methods.•In-depth characterization of recombinant virus-like particles. The hepatitis E virus (HEV) vaccine, Hecolin®, was licensed in China for the prevention of HEV infection and HEV-related diseases with demonstrated safety and efficacy [1,2]. The vaccine is composed of a truncated HEV capsid protein, p239, as the sole antigen encoded by open reading frame 2 and produced using Escherichia coli platform. The production of this virus-like particle (VLP) form of the antigen was successfully scaled up 50-fold from a bench scale to a manufacturing scale. Product consistency was demonstrated using a combination of biophysical, biochemical and immunochemical methods, which revealed comparable antigen characteristics among different batches. Particle size of the nanometer scale particulate antigen and presence of key epitopes on the particle surface are two prerequisites for an efficacious VLP-based vaccine. The particle size was monitored by several different methods, which showed diameters between 20 and 30nm for the p239 particles. The thermal stability and aggregation propensity of the antigen were assessed using differential scanning calorimetry and cloud point assay under heat stress conditions. Key epitopes on the particulate antigen were analyzed using a panel of murine anti-HEV monoclonal antibodies (mAbs). The immuno reactivity to the mAbs among the different antigen lots was highly consistent when analyzed quantitatively using a surface plasmon resonance technique. Using a sandwich ELISA to probe the integrity of two different epitopes in the antigen, the specific antigenicity of multiple batches was assessed to demonstrate consistency in these critical product attributes. Overall, our findings showed that the antigen production process is robust and scalable during the manufacturing of Hecolin®.

Background Leishmaniasis is a serious health problem in some parts of the world. In spite of the many known leishmaniasis control measures, the disease has continued to increase in endemic areas, and no effective vaccine has been discovered. Methods In this study, Leishmania tarentulae was used as a living factory for the production of two LACK and KMP11 immunogenic antigens in the mice body, and safety profiles were investigated. The sequences of the KMP11 and LACK L. major antigens were synthesized in the pLEXSY-neo 2.1 plasmid and cloned into E. coli strain Top10, and after being linearized with the SwaI enzyme, they were transfected into the genome of L. tarentolae . The L. tarentolae-LACK/KMP11/EGFP in the stationary phase with CpG ODN as an adjuvant was used for vaccination in BALB/c mice. Vaccination was performed into the left footpad. Three weeks later, the booster was injected in the same manner. To examine the effectiveness of the injected vaccine, pathogenic L. major (MRHO/IR/75/ER) was injected into the right footpad of all mice three weeks following the booster vaccination. In order to assess humoral immunity, the levels of IgG1, and IgG2a antibodies before and 6 weeks after the challenge were studied in the groups. In addition, in order to investigate cellular immunity in the groups, the study measured IFN-γ, IL-5, TNF-α, IL-6 and IL-17 cytokines before, 3 weeks and 8 weeks after the challenge, and also the parasite load in the lymph node with real-time PCR. Results The lowest level of the parasitic load was observed in the G1 group (mice vaccinated with L. tarentolae-LACK/KMP11/EGFP with CpG) in comparison with other groups ( L. tarentolae -LACK/KMP11/EGFP +non-CpG (G2); L. tarentolae -EGFP + CpG (G3, control); L. tarentolae -EGFP + non-CpG (G4, control); and mice injected with PBS (G5, control). Moreover, the evaluation of immune response showed a delayed-type hypersensitivity towards Th1. Conclusions According to the results of this study, the live recombinant vaccine of L. tarentolae-LACK/KMP11/EGFP with the CpG adjuvant reduced the parasitic load and footpad induration in infected mice. The long-term effects of this vaccine can be evaluated in volunteers as a clinical trial in future planning.

During the last 25 years, the technology to produce recombinant vaccines in plant cells has evolved from modest proofs of the concept to viable technologies adopted by some companies due to significant improvements in the field. Viral-based expression strategies have importantly contributed to this success owing to high yields, short production time (which is in most cases free of tissue culture steps), and the implementation of confined processes for production under GMPs. Herein the distinct expression systems based on viral elements are analyzed. This review also presents the outlook on how these technologies have been successfully applied to the development of plant-based vaccines, some of them being in advanced stages of development. Perspectives on how viral expression systems could allow for the development of innovative oral vaccines constituted by minimally-processed plant biomass are discussed.

► We generated rotavirus subunit ΔVP8* vaccine with P[8], P[6] or P[4] specificity. ► Each ΔVP8* protein was highly soluble and was generated in high yield in E. coli. ► Each ΔVP8* protein induced high levels of neutralizing antibodies in guinea pigs. ► These ΔVP8* parenteral vaccines may be more effective in low-income countries. Two currently licensed live oral rotavirus vaccines (Rotarix® and RotaTeq®) are highly efficacious against severe rotavirus diarrhea. However, the efficacy of such vaccines in selected low-income African and Asian countries is much lower than that in middle or high-income countries. Additionally, these two vaccines have recently been associated with rare case of intussusception in vaccinated infants. We developed a novel recombinant subunit parenteral rotavirus vaccine which may be more effective in low-income countries and also avert the potential problem of intussusception. Truncated recombinant VP8* (ΔVP8*) protein of human rotavirus strain Wa P[8], DS-1 P[4] or 1076 P[6] expressed in Escherichia coli was highly soluble and was generated in high yield. Guinea pigs hyperimmunized intramuscularly with each of the ΔVP8* proteins (i.e., P[8], P[4] or P[6]) developed high levels of homotypic as well as variable levels of heterotypic neutralizing antibodies. Moreover, the selected ΔVP8* proteins when administered to mice at a clinically relevant dosage, route and schedule, elicited high levels of serum anti-VP8* IgG and/or neutralizing antibodies. Our data indicated that the ΔVP8* proteins may be a plausible additional candidate as new parenteral rotavirus vaccines.

Today, plant biotechnology relies on two processes for delivery and expression of heterologous genes in plants: stable genetic transformation and transient infection with viral vectors. Although much faster, the transient route until recently was limited because of virus’ low infectivity and its inability to carry average-size or larger transgenes. A recently developed new generation transfection technology overcomes these limitations by relying on Agrobacterium as an infective systemic agent that delivers viral replicons. This improved process is being used to simultaneously start transient gene amplification and high-level expression in all mature leaves of a plant, and such a transfection can be done on an industrial scale. This eclectic technology, called ‘magnifection’, combines advantages of three biological systems: vector efficiency and efficient systemic DNA delivery of Agrobacterium, speed and expression level/yield of a plant RNA virus, as well as posttranslational capabilities and low production costs of a plant. The proposed process allows for industrial production that does not require genetic modification of plants, that is much faster than previous methods, and that is biologically safe. Numerous applications in the area of vaccine manufacturing are being discussed.

Cholera toxin B subunit (CTB) is a component of an internationally licensed oral cholera vaccine. The protein induces neutralizing antibodies against the holotoxin, the virulence factor responsible for severe diarrhea. A field clinical trial has suggested that the addition of CTB to killed whole-cell bacteria provides superior short-term protection to whole-cell-only vaccines; however, challenges in CTB biomanufacturing (i.e., cost and scale) hamper its implementation to mass vaccination in developing countries. To provide a potential solution to this issue, we developed a rapid, robust, and scalable CTB production system in plants. In a preliminary study of expressing original CTB in transgenic Nicotiana benthamiana, the protein was N-glycosylated with plant-specific glycans. Thus, an aglycosylated CTB variant (pCTB) was created and overexpressed via a plant virus vector. Upon additional transgene engineering for retention in the endoplasmic reticulum and optimization of a secretory signal, the yield of pCTB was dramatically improved, reaching >1 g per kg of fresh leaf material. The protein was efficiently purified by simple two-step chromatography. The GM1-ganglioside binding capacity and conformational stability of pCTB were virtually identical to the bacteria-derived original B subunit, as demonstrated in competitive enzyme-linked immunosorbent assay, surface plasmon resonance, and fluorescence-based thermal shift assay. Mammalian cell surface-binding was corroborated by immunofluorescence and flow cytometry. pCTB exhibited strong oral immunogenicity in mice, inducing significant levels of CTB-specific intestinal antibodies that persisted over 6 months. Moreover, these antibodies effectively neutralized the cholera holotoxin in vitro. Taken together, these results demonstrated that pCTB has robust producibility in Nicotiana plants and retains most, if not all, of major biological activities of the original protein. This rapid and easily scalable system may enable the implementation of pCTB to mass vaccination against outbreaks, thereby providing better protection of high-risk populations in developing countries.

In the molecular farming area, transient expression approaches for pharmaceutical proteins production, mainly recombinant monoclonal antibodies and vaccines, were developed almost two decades ago and, to date, these systems basically depend on Agrobacterium-mediated delivery and virus expression machinery. We survey here the current state-of-the-art of this research field. Several vectors have been designed on the basis of DNA- and RNA-based plant virus genomes and viral vectors are used both as single- and multicomponent expression systems in different combinations depending on the protein of interest. The obvious advantages of these systems are ease of manipulation, speed, low cost and high yield of proteins. In addition, Agrobacterium-mediated expression also allows the production in plants of complex proteins assembled from subunits. Currently, the transient expression methods are preferential over any other transgenic system for the exploitation of large and unrestricted numbers of plants in a contained environment. By designing optimal constructs and related means of delivery into plant cells, the overall technology plan considers scenarios that envisage high yield of bioproducts and ease in monitoring the whole spectrum of upstream production, before entering good manufacturing practice facilities. In this way, plant-derived bioproducts show promise of high competitiveness towards classical eukaryotic cell factory systems.