Production Of Vaccine Antigens Research Articles

An inactivated SARS-CoV-2 vaccine based on a Vero cell culture-adapted high-titer virus confers cross-protection in small animals

Rapidly waning immunity against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) requires continued global access to affordable vaccines. Globally, inactivated SARS-CoV-2 vaccines have been widely used during the SARS-CoV-2 pandemic. In this proof-of-concept study we adapted an original-D614G SARS-CoV-2 virus to Vero cell culture as a strategy to enhance inactivated vaccine manufacturing productivity. A passage 60 (P60) virus showed enhanced fitness and 50-fold increased virus yield in a bioreactor compared to the original-D614G virus. It further remained susceptible to neutralization by plasma from SARS-CoV-2 vaccinated and convalescent individuals, suggesting exposure of relevant epitopes. Monovalent inactivated P60 and bivalent inactivated P60/omicron BA.1 vaccines induced neutralizing responses against original-D614G and BA.1 viruses in mice and hamsters, demonstrating that the P60 virus is a suitable vaccine antigen. Antibodies further cross-neutralized delta and BA.5 viruses. Importantly, the inactivated P60 vaccine protected hamsters against disease upon challenge with original-D614G or BA.1 virus, with minimal lung pathology and lower virus loads in the upper and lower airways. Antigenicity of the P60 virus was thus retained compared to the original virus despite the acquisition of cell culture adaptive mutations. Consequently, cell culture adaptation may be a useful approach to increase yields in inactivated vaccine antigen production.

Fluorescent-protein co-expression to select CHO cells expressing high quantities of vaccine antigens.

Cell line development for production of vaccine antigens or therapeutic proteins typically involves transfection, selection, and enrichment for high-expressing cells. Enrichment methods include minipool enrichment, antibody-based enrichment, and enrichment based on co-expressed fluorescent biosensor proteins. However, these methods have limitations regarding labor and cost intensity, the generation of antibodies and assurance of their viral safety, and potential expression-interference or signal-saturation of the co-expressed fluorescent protein. To improve the method of fluorescent-protein co-expression, expression constructs were created that constitutively express a model vaccine antigen together with one of three fluorescent proteins having translation initiation controlled by a wildtype or mutant internal ribosome entry site (IRES), for a total of six constructs. The constructs were transfected into Chinese hamster ovary cells (CHO) cells, enriched for high fluorescence, cultured, and tested in a mini bioreactor to identify the most promising construct. The fluorescent protein, Fluorescent Ubiquitination-based Cell Cycle Indicator (FUCCI) with a mutant IRES performed best and was further tested with three additional vaccine antigens. Across the four vaccine antigens, the FUCCI fluorescent protein yielded productivity enhancements, without the need for generating an antibody and assuring its viral safety. Furthermore, FUCCI protein was present in negligible quantities in the cell supernatant, indicating a low risk for contaminating drug substances or vaccine antigen.

Evaluation of Foot-and-Mouth Disease (FMD) Virus Asia1 Genotype-V as an FMD Vaccine Candidate: Study on Vaccine Antigen Production Yield and Inactivation Kinetics.

South Korea has experienced outbreaks of foot-and-mouth disease (FMD) of serotypes O and A, leading to nationwide vaccination with a bivalent vaccine. Since the FMD virus (FMDV) Asia1 group-V genotype occurred in North Korea in 2007, an Asia1/MOG/05 vaccine strain belonging to the Asia1 group-V genotype was developed using a genetic recombination method (Asia1/MOG/05-R). This study aimed to evaluate the antigen productivity and viral inactivation kinetics of Asia1/MOG/05-R to assess its commercial viability. The antigen yield of Asia1/MOG/05-R produced in flasks and bioreactors was approximately 4.0 μg/mL. Binary ethylenimine (BEI) inactivation kinetics of Asia1/MOG/05-R showed that 2 mM and 1.0 mM BEI treatment at 26 °C and 37 °C, respectively, resulted in a virus titer <10-7 TCID50/mL within 24 h, meeting the inactivation kinetics criteria. During incubation at 26 °C and 37 °C, 10% antigen loss occurred, but not due to BEI treatment. When pigs were inoculated twice with the Asia1/MOG/05-R antigen, the virus neutralization titer increased to approximately 1:1000; therefore, it can sufficiently protect against Asia1/MOG/05-R and Asia1 Shamir viruses. The Asia1/MOG/05-R will be useful as a vaccine strain for domestic antigen banks.

Efficacy of Binary Ethylenimine in the Inactivation of Foot-and-Mouth Disease Virus for Vaccine Production in South Korea.

Foot-and-mouth disease (FMD) vaccines must be produced in a biosafety level 3 facility, so the FMD virus (FMDV) must be completely inactivated after amplification. The inactivation kinetics of FMDV during vaccine antigen production were assessed by evaluating whether the viral titer dropped below 10-7 TCID50/mL within 24 h of binary ethyleneimine (BEI) treatment. This study dealt with four FMD vaccine candidate strains for the efficacy of BEI treatment at different concentrations and temperatures to determine the optimal inactivation condition of each virus. Two domestic isolates, O/SKR/Boeun/2017 (O BE) and A/SKR/Yeoncheon/2017 (A YC), and two recombinant viruses, PAK/44/2008 (O PA-2) and A22/Iraq/24/64 (A22 IRQ), were investigated. The O BE and A22 IRQ required 2 mM BEI at 26 °C and 0.5 mM BEI at 37 °C for complete inactivation. The O PA-2 and A YC required 2 mM BEI at 26 °C and 1 mM BEI at 37 °C. Crucially, the yield of FMD virus particles (146S) in the viral infection supernatant was higher (>4.0 µg/mL) than those previously reported; additionally, there was little antigen loss, even after 24 h of treatment with 3 mM BEI. Overall, it is considered economical to produce FMD vaccines using these four kinds of viruses; therefore, these candidate strains will be prioritized for the manufacture of FMD vaccines in South Korea.

Deep longitudinal multi-omics analysis of Bordetella pertussis cultivated in bioreactors highlights medium starvations and transitory metabolisms, associated to vaccine antigen biosynthesis variations and global virulence regulation.

Bordetella pertussis is the bacterial causative agent of whooping cough, a serious respiratory illness. An extensive knowledge on its virulence regulation and metabolism is a key factor to ensure pertussis vaccine manufacturing process robustness. The aim of this study was to refine our comprehension of B. pertussis physiology during in vitro cultures in bioreactors. A longitudinal multi-omics analysis was carried out over 26 h small-scale cultures of B. pertussis. Cultures were performed in batch mode and under culture conditions intending to mimic industrial processes. Putative cysteine and proline starvations were, respectively, observed at the beginning of the exponential phase (from 4 to 8 h) and during the exponential phase (18 h 45 min). As revealed by multi-omics analyses, the proline starvation induced major molecular changes, including a transient metabolism with internal stock consumption. In the meantime, growth and specific total PT, PRN, and Fim2 antigen productions were negatively affected. Interestingly, the master virulence-regulating two-component system of B. pertussis (BvgASR) was not evidenced as the sole virulence regulator in this in vitro growth condition. Indeed, novel intermediate regulators were identified as putatively involved in the expression of some virulence-activated genes (vags). Such longitudinal multi-omics analysis applied to B. pertussis culture process emerges as a powerful tool for characterization and incremental optimization of vaccine antigen production.

A recombinant baculovirus vector vaccine (BacMCP) against the infectious spleen and kidney necrosis virus (ISKNV).

The infectious spleen and kidney necrosis virus (ISKNV) is a highly lethal virus, which has brought significant losses to aquaculture. Therefore, a new vaccine against ISKNV with high efficiency, safety and convenience must be developed. While baculoviruses are more commonly used as protein expression systems for vaccine antigen production, this paper used baculovirus technology to develop a live-vector vaccine, BacMCP, which contains the coding sequence of the major capsid protein (MCP) (GenBank accession no. AF371960) of ISKNV and is driven by a CMV promoter. Real-time PCR and immunofluorescence showed that the MCP gene was successfully delivered to and expressed in fish cells and tissues inoculated with BacMCP. Immune-related gene (IgM, TGF-β, IL-1, IL-8, TNF-α) expression was induced in BacMCP-treated groups of largemouth bass compared with control groups. Specific antibodies could be detected in the serum of BacMCP injection-vaccinated largemouth bass by ELISA. After injection or immersion vaccination with BacMCP for 21 days, largemouth bass were infected with ISKNV. The immune effect of the injected immunization on fish in different sizes was evaluated. The vaccine efficacy of injection-vaccinated bass was 100% in small bass and 85.7% in large bass. The vaccine efficacy of immersion-vaccinated small bass was 77.3%. This study suggested that BacMCP can be used as a vector-based vaccine candidate to prevent the diseases caused by ISKNV infection.

Plant-Derived Human Vaccines: Recent Developments.

The idea of producing vaccines in plants originated in the late 1980s. Initially, it was contemplated that this notion could facilitate the concept of edible vaccines, making them more cost effective and easily accessible. Initial studies on edible vaccines focussed on the use of a variety of different transgenic plant host species for the production of vaccine antigens. However, adequate expression levels of antigens, the difficulties predicted with administration of consistent doses, and regulatory rules required for growth of transgenic plants gave way to the development of vaccine candidates that could be purified and administered parenterally. The field has subsequently advanced with improved expression techniques including a shift from using transgenic to transient expression of antigens, refinement of purification protocols, a deeper understanding of the biological processes and a wealth of evidence of immunogenicity and efficacy of plant-produced vaccine candidates, all contributing to the successful practice of what is now known as biopharming or plant molecular farming. The establishment of this technology has resulted in the development of many different types of vaccine candidates including subunit vaccines and various different types of nanoparticle vaccines targeting a wide variety of bacterial and viral diseases. This has brought further acceptance of plants as a suitable platform for vaccine production and in this review, we discuss the most recent advances in the production of vaccines in plants for human use.

Factors Involved in Removing the Non-Structural Protein of Foot-and-Mouth Disease Virus by Chloroform and Scale-Up Production of High-Purity Vaccine Antigens.

Foot-and-mouth disease (FMD) is an economically important and highly infectious viral disease, predominantly controlled by vaccination. The removal of non-structural proteins (NSPs) is very important in the process of FMD vaccine production, because vaccinated and naturally infected animals can be distinguished by the presence of NSP antibodies in the FMD serological surveillance. A previous study reported that 3AB protein, a representative of NSPs, was removed by chloroform treatment. Therefore, in this study, the causes of 3AB removal and factors affecting the effect of chloroform were investigated. As a result, the effectiveness of chloroform differed depending on the virus production medium and was eliminated by detergents. In addition, it was found that 3AB protein removal by chloroform is due to the transmembrane domain of the N-terminal region (59–76 amino acid domain). Further, industrial applicability was verified by applying the chloroform treatment process to scale-up FMD vaccine antigen production. A novel downstream process using ultrafiltration instead of polyethylene glycol precipitation for high-purity FMD vaccine antigen production was established. This result will contribute toward simplifying the conventional process of manufacturing FMD vaccine antigens and ultimately reducing the time and cost of vaccine production.

COVID-19 vaccine inequity, dependency, and production capability in low-income and middle-income countries: the case of Bangladesh

COVID-19 vaccine inequity, dependency, and production capability in low-income and middle-income countries: the case of Bangladesh

Investigation of the optimal medium and application strategy for foot-and-mouth disease vaccine antigen production.

For the effective production of 146S particles, which determines foot-and-mouth disease (FMD) vaccine efficacy, we aimed to identify the optimal medium that is easy-to-use, productive and economically affordable for the large-scale production of FMD vaccine. Nine combinations of cell growth media and replacement media were tested for virus propagation. Apart from the replacement strategy, we tested a simple addition strategy involving the addition of 30% v/v of fresh medium to the total spent medium using the Cellvento BHK-200 (Vento). Unlike other tested media that produced poor yields of 146S particles when the spent media were not eliminated, Vento exhibited high productivity with the 30% addition strategy. Considering its lower price and media consumption compared to those of other media that require media replacement, the 30% addition strategy of Vento is highly effective. Furthermore, owing to its simple application strategy, it makes the scale-up process easy and helps in saving the time and labour involved in spent media removal. Through the first comparative assessment of commercial media for the 146S particle recovery, this study suggests the best practical medium for the industrial-scale production of FMD vaccines.

Viral Vector Vaccines

Viral vector vaccines use harmless, non-replicating or replicating viruses to deliver genetic material for production of vaccine antigens into host cell cytoplasm. While viral vector vaccines may theoretically induce life-long immunity with low antigen concentrations, their attenuation, safety and spread to the community are of concern. Vaccines based on recombinant viral vectors can induce both humoral and cellular immune responses. Adenovirus vectors are versatile gene transfer vectors that can be easily manufactured, and which may allow simultaneous expression of multiple antigens by a single vector construct. Adenovirus vector vaccines based on the adenovirus Ad26 vector have been widely used as vaccines against Ebola and COVID19 (see Chapters 44 and 56). A common concern of using viral vector vaccines is pre-existing immunity or induction of immunity against the vector itself, but in some circumstances it has no meaningful impact and it can be resolved in several ways. Several harmless viruses are already used as vectors for innovative vaccines and many more are in research.

Production and Immunogenicity of Soluble Plant-Produced HIV-1 Subtype C Envelope gp140 Immunogens.

The development of effective vaccines is urgently needed to curb the spread of human immunodeficiency virus type 1 (HIV-1). A major focal point of current HIV vaccine research is the production of soluble envelope (Env) glycoproteins which reproduce the structure of the native gp160 trimer. These antigens are produced in mammalian cells, which requires a sophisticated infrastructure for manufacture that is mostly absent in developing countries. The production of recombinant proteins in plants is an attractive alternative for the potentially cheap and scalable production of vaccine antigens, especially for developing countries. In this study, we developed a transient expression system in Nicotiana benthamiana for the production of soluble HIV Env gp140 antigens based on two rationally selected virus isolates (CAP256 SU and Du151). The scalability of the platform was demonstrated and both affinity and size exclusion chromatography (SEC) were explored for recovery of the recombinant antigens. Rabbits immunized with lectin affinity-purified antigens developed high titres of binding antibodies, including against the V1V2 loop region, and neutralizing antibodies against Tier 1 viruses. The removal of aggregated Env species by gel filtration resulted in the elicitation of superior binding and neutralizing antibodies. Furthermore, a heterologous prime-boost regimen employing a recombinant modified vaccinia Ankara (rMVA) vaccine, followed by boosts with the SEC-purified protein, significantly improved the immunogenicity. To our knowledge, this is the first study to assess the immunogenicity of a near-full length plant-derived Env vaccine immunogen.

Transient Expression of an Immunogenic Envelope Attachment Glycoprotein of Nipah Virus in Nicotiana benthamiana

The Nipah virus is highly virulent to swine and humans. The envelope attachment glycoprotein (G) of Nipah virus plays a key role in viral entry and induction of neutralizing antibody in mammalian hosts, thus is considered a good candidate for vaccine development. Plant transient expression systems are gaining recognition as a viable alternative for the production of vaccine antigens. In this study, we expressed the Nipah virus G protein heterologously in Nicotiana benthamiana using an agroinfiltration approach. The highest expression of recombinant G protein in N. benthamiana at RNA and protein levels was detected on day 9 post-infiltration. Western blot analysis demonstrated that the purified G protein reacted specifically with rabbit anti-Nipah Virus serum, indicating its potential for vaccine use.

Safety and immunogenicity of a plant-produced recombinant monomer hemagglutinin-based influenza vaccine derived from influenza A (H1N1)pdm09 virus: A Phase 1 dose-escalation study in healthy adults

BackgroundNovel influenza viruses continue to pose a potential pandemic threat worldwide. In recent years, plants have been used to produce recombinant proteins, including subunit vaccines. A subunit influenza vaccine, HAC1, based on recombinant hemagglutinin from the 2009 pandemic A/California/04/2009 (H1N1) strain of influenza virus, has been manufactured using a plant virus-based transient expression technology in Nicotiana benthamiana plants and demonstrated to be immunogenic and safe in pre-clinical studies (Shoji et al., 2011). MethodsA first-in-human, Phase 1, single-center, randomized, placebo-controlled, single-blind, dose escalation study was conducted to investigate safety, reactogenicity and immunogenicity of an HAC1 formulation at three escalating dose levels (15μg, 45μg and 90μg) with and without Alhydrogel®, in healthy adults 18–50 years of age (inclusive). Eighty participants were randomized into six study vaccine groups, a saline placebo group and an approved monovalent H1N1 vaccine group. Recipients received two doses of vaccine or placebo (except for the monovalent H1N1 vaccine cohort, which received a single dose of vaccine, later followed by a dose of placebo). ResultsThe experimental vaccine was safe and well tolerated, and comparable to placebo and the approved monovalent H1N1 vaccine. Pain and tenderness at the injection site were the only local solicited reactions reported following vaccinations. Nearly all adverse events were mild to moderate in severity. The HAC1 vaccine was also immunogenic, with the highest seroconversion rates, based on serum hemagglutination-inhibition and virus microneutralization antibody titers, in the 90μg non-adjuvanted HAC1 vaccine group after the second vaccine dose (78% and 100%, respectively). ConclusionsThis is the first study demonstrating the safety and immunogenicity of a plant-produced subunit H1N1 influenza vaccine in healthy adults. The results support further clinical investigation of the HAC1 vaccine as well as demonstrate the feasibility of the plant-based technology for vaccine antigen production.

Influenza virosomes supplemented with GPI-0100 adjuvant: a potent vaccine formulation for antigen dose sparing

Adjuvants can stimulate vaccine-induced immune responses and can contribute decisively to antigen dose sparing when vaccine antigen production is limited, as for example during a pandemic influenza outbreak. We earlier showed that GPI-0100, a semi-synthetic saponin derivative with amphiphilic structure, significantly stimulates the immunogenicity and protective efficacy of influenza subunit vaccine administered via a systemic route. Here, we evaluated the adjuvant effect of GPI-0100 on a virosomal influenza vaccine formulation. In contrast to influenza subunit vaccine adjuvanted with GPI-0100, virosomal vaccine supplemented with the same dose of GPI-0100 provided full protection of mice against infection at the extremely low antigen dose of 2 × 8 ng hemagglutinin. Overall, adjuvanted virosomes elicited higher antibody and T-cell responses than did adjuvanted subunit vaccine. The enhanced immunogenicity of the GPI-0100-adjuvanted virosomes, particularly at low antigen doses, is possibly due to a physical association of the amphiphilic adjuvant with the virosomal membrane. These results show that a combination of GPI-0100 and a virosomal influenza vaccine formulation is highly immunogenic and allows the use of very low antigen doses without compromising the protective potential of the vaccine.

Rational Engineering of Recombinant Picornavirus Capsids to Produce Safe, Protective Vaccine Antigen

Foot-and-mouth disease remains a major plague of livestock and outbreaks are often economically catastrophic. Current inactivated virus vaccines require expensive high containment facilities for their production and maintenance of a cold-chain for their activity. We have addressed both of these major drawbacks. Firstly we have developed methods to efficiently express recombinant empty capsids. Expression constructs aimed at lowering the levels and activity of the viral protease required for the cleavage of the capsid protein precursor were used; this enabled the synthesis of empty A-serotype capsids in eukaryotic cells at levels potentially attractive to industry using both vaccinia virus and baculovirus driven expression. Secondly we have enhanced capsid stability by incorporating a rationally designed mutation, and shown by X-ray crystallography that stabilised and wild-type empty capsids have essentially the same structure as intact virus. Cattle vaccinated with recombinant capsids showed sustained virus neutralisation titres and protection from challenge 34 weeks after immunization. This approach to vaccine antigen production has several potential advantages over current technologies by reducing production costs, eliminating the risk of infectivity and enhancing the temperature stability of the product. Similar strategies that will optimize host cell viability during expression of a foreign toxic gene and/or improve capsid stability could allow the production of safe vaccines for other pathogenic picornaviruses of humans and animals.

Transient Expression of Hemagglutinin Antigen from Low Pathogenic Avian Influenza A (H7N7) in Nicotiana benthamiana

The influenza A virus is of global concern for the poultry industry, especially the H5 and H7 subtypes as they have the potential to become highly pathogenic for poultry. In this study, the hemagglutinin (HA) of a low pathogenic avian influenza virus of the H7N7 subtype isolated from a Swedish mallard Anas platyrhynchos was sequenced, characterized and transiently expressed in Nicotiana benthamiana. Recently, plant expression systems have gained interest as an alternative for the production of vaccine antigens. To examine the possibility of expressing the HA protein in N. benthamiana, a cDNA fragment encoding the HA gene was synthesized de novo, modified with a Kozak sequence, a PR1a signal peptide, a C-terminal hexahistidine (6×His) tag, and an endoplasmic retention signal (SEKDEL). The construct was cloned into a Cowpea mosaic virus (CPMV)-based vector (pEAQ-HT) and the resulting pEAQ-HT-HA plasmid, along with a vector (pJL3:p19) containing the viral gene-silencing suppressor p19 from Tomato bushy stunt virus, was agro-infiltrated into N. benthamiana. The highest gene expression of recombinant plant-produced, uncleaved HA (rHA0), as measured by quantitative real-time PCR was detected at 6 days post infiltration (dpi). Guided by the gene expression profile, rHA0 protein was extracted at 6 dpi and subsequently purified utilizing the 6×His tag and immobilized metal ion adsorption chromatography. The yield was 0.2 g purified protein per kg fresh weight of leaves. Further molecular characterizations showed that the purified rHA0 protein was N-glycosylated and its identity confirmed by liquid chromatography-tandem mass spectrometry. In addition, the purified rHA0 exhibited hemagglutination and hemagglutination inhibition activity indicating that the rHA0 shares structural and functional properties with native HA protein of H7 influenza virus. Our results indicate that rHA0 maintained its native antigenicity and specificity, providing a good source of vaccine antigen to induce immune response in poultry species.

Contribution of Yeast and Plant Research for Improving Human Health

In the last years there was an increase in the use of several organisms such as plants and yeast for medical and biotechnological purposes. Both organisms are very useful when research in humans is nonpossible or unfeasible due to ethical issues. There are several advantages of using model organisms such as the availability of genomic information, the possibility to easily make genetic manipulation and screenings, and availability of mutants in desired genes. In addition, these organisms are generally easy to grow. The conservation in many cases of various metabolic and signaling pathways and basic cellular processes, often accompanied by proteomic and metabolomic information, is also an advantage in medical research, while the presence of specific metabolic pathways may be an advantage in the production of compounds of industrial interest. The paper of P. Jeandet et al. presents a detailed review on the biosynthesis, metabolism, and production of resveratrol, a stilbenic derivative. This compound is produced in the phenylalanine/polymalonate pathway, with the stilbene synthase (STS) being the key enzyme in this process. The increased use of resveratrol as “nutraceutical” (for nutrition and pharmaceutical uses) or in plant disease control makes its production an important goal in biotechnology. Different approaches to increase the production of this compound include the manipulation of the biosynthetic pathway and the overexpression of STS and other specific genes. Besides the production of compounds of industrial interest, the use of plants for bioremediation and for the production of vaccine antigens is other important achievement in plant biotechnology related to improving human health. Some toxic compounds that can accumulate in plants are heavy metals. This is one of the biggest problems of environmental pollution and they also have severe consequences for their toxic effects on human health. The paper of Z. Zhang et al. presents a study of the accumulation of Pb and Cd in elite maize inbred lines. This study shows that there are significant differences in Pb and Cd accumulation between maize hybrid lines. So, it is possible to select low-metal accumulation lines for nutritional purposes. In addition, the study also showed the existence of maize lines with high capacity for accumulation of heavy metals that might be useful for bioremediation purposes. On the other hand, plants become an emerging alternative for the production of vaccine antigens. The use of plants has several advantages such as easy gene engineering and low cost in the production of antigens. M. Clemente et al. reviewed the strategies for the production of antigens against malaria in plants. Malaria is a major health problem worldwide due to its high mortality rate, estimated at around one million people per year. Such strategies include the production of codon-optimized surface antigens of Plasmodium, the agent responsible for the disease, and the production of dual cholera-malaria antigens in tobacco plants and bacterial-made antigens. There is also an extensive work using yeast, such as Saccharomyces cerevisiae and plants, mainly Arabidopsis thaliana, for the study of human pathologies. One example of this is the use of Saccharomyces for the study of the function of proteins of the human immunodeficiency virus (HIV) and the identification of its cellular partners common to yeast and humans. It is known that this retrovirus is the cause of AIDS, which affects more than 30 million people around the world. The paper of M. L. Andreola and S. Litvak provides a detailed review of the work done on the identification of cellular factors associated with different viral proteins, as well as the function and regulation of known enzymes encoded by the viral genome. The knowledge of the role of the viral proteins and cellular targets might allow the development of new therapeutic targets and strategies. In addition, this research could allow the discovery of new genes and proteins involved in virus proliferation and virus infection mechanisms, shortening the time necessary to develop different therapies against new viruses. Besides the use of yeast, the study of plants, such as Arabidopsis thaliana, is a valuable tool in biomedicine, in the study of several mechanisms that might act similarly in producing human diseases, since human genes and proteins have been structurally and functionally conserved in plant organisms. The paper of C. P. Spampinato and D. F. Gomez-Casati presents a review of the current knowledge of human diseases of nuclear origin such as those related to defects in DNA repairing pathways and also mitochondrial pathologies, such as Friedreich's ataxia and respiratory complexes deficiencies, leading mainly to the production of neurological diseases. In addition, several studies in yeast could also contribute to uncover the etiology and pathogenesis of several neurodegenerative diseases as reviewed in the work of Pereira et al., including Alzheimer's, Parkinson's and Huntington's disease. These papers represent an interesting compilation on the use of yeast and plants for biotechnological and biomedical purposes and the future prospects.

Production, characterisation and immunogenicity of a plant-made Plasmodium antigen—the 19 kDa C-terminal fragment of Plasmodium yoelii merozoite surface protein 1

Development of a safe, effective and affordable malaria vaccine is central to global disease control efforts. One of the most highly regarded proteins for inclusion in an asexual blood stage subunit vaccine is the 19-kDa C-terminal fragment of merozoite surface protein 1 (MSP1(19)). As production of vaccine antigens in plants can potentially overcome cost and delivery hurdles, we set out to produce MSP1(19) in plants, characterise the protein and test its immunogenicity using a mouse model. Plasmodium yoelii MSP1(19) (PyMSP1(19)) was produced in Nicotiana benthamiana using the MagnICON® deconstructed TMV-based viral vector. PyMSP1(19) yield of at least 23% total soluble protein (TSP;3-4 mg/g Fwt) were achieved using a codon-optimised construct that was targeted to the apoplast. Freeze-dried leaf powder contained at least 20 mg PyMSP1(19) per gram dry weight and the protein retained immunogenicity in this form for more than 2 years. Characterisation studies, including SDS-PAGE, mass spectrometry and circular dichroism, indicated that the plant-expressed PyMSP1(19) was similar to its Escherichia coli- and Saccharomyces cerevisiae-expressed counterparts. Purified plant-made PyMSP1(19) induced strong immune responses following intraperitoneal immunisation, although titres were lower than those induced by an equivalent dose of purified E. coli-expressed PyMSP1(19). The reason for this is uncertain but may be due to differences in the oligomerisation profile of the vaccines. The plant-made PyMSP1(19) vaccine was also found to be orally immunogenic when delivered alone or following immunisation with a PyMSP1(19) DNA vaccine. This study adds to an increasing body of research supporting the feasibility of plants as both a factory for the production of malaria antigens, and as a safe and affordable platform for oral delivery of a temperature-stable malaria vaccine.

Evaluation of two long synthetic merozoite surface protein 2 peptides as malaria vaccine candidates

Merozoite surface protein 2 (MSP2) is a promising vaccine candidate against Plasmodium falciparum blood stages. A recombinant 3D7 form of MSP2 was a subunit of Combination B, a blood stage vaccine tested in the field in Papua New Guinea. A selective effect in favour of the allelic family not represented by the vaccine argued for a MSP2 vaccine consisting of both dimorphic variants. An alternative approach to recombinant manufacture of vaccines is the production of long synthetic peptides (LSP). LSP exceeding a length of well over 100 amino acids can now be routinely synthesized. Synthetic production of vaccine antigens cuts the often time-consuming steps of protein expression and purification short. This considerably reduces the time for a candidate to reach the phase of clinical trials. Here we present the evaluation of two long synthetic peptides representing both allelic families of MSP2 as potential vaccine candidates. The constructs were well recognized by human immune sera from different locations and different age groups. Furthermore, peptide-specific antibodies in human immune sera were associated with protection from clinical malaria. The synthetic fragments share major antigenic properties with native MSP2. Immunization of mice with these antigens yielded high titre antibody responses and monoclonal antibodies recognized parasite-derived MSP2. Our results justify taking these candidate poly-peptides into further vaccine development.