Production Of Recombinant Antigens Research Articles

Plant production of recombinant antigens containing the receptor binding domain (RBD) of two SARS-CoV-2 variants.

The aim of this work was to rapidly produce in plats two recombinant antigens (RBDw-Fc and RBDo-Fc) containing the receptor binding domain (RBD) of the spike (S) protein from SARS-CoV-2 variants Wuhan and Omicron as fusion proteins to the Fc portion of a murine IgG2a antibody constant region (Fc). The two recombinant antigens were expressed in Nicotiana benthamiana plants, engineered to avoid the addition of N-linked plant-typical sugars, through vacuum agroinfiltration and showed comparable purification yields (about 35mg/kg leaf fresh weight). Their Western blotting and Coomassie staining evidenced the occurrence of major in planta proteolysis in the region between the RBD and Fc, which was particularly evident in RBDw-Fc, the only antigen bearing the HRV 3C cysteine protease recognition site. The two RBD N-linked glycosylation sites showed very homogeneous profiles free from plant-typical sugars, with the most abundant glycoform represented by the complex sugar GlcNAc4Man3. Both antigens were specifically recognised in Western Blot analysis by the anti-SARS-CoV-2 human neutralizing monoclonal antibody J08-MUT and RBDw-Fc was successfully used in competitive ELISA experiments for binding to the angiotensin-converting enzyme 2 receptor to verify the neutralizing capacity of the serum from vaccinated patients. Both SARS-Cov-2 antigens fused to a murine Fc region were rapidly and functionally produced in plants with potential applications in diagnostics.

Detection of Coxiella antibodies in ruminants using a SucB recombinant antigen.

The detection of Coxiella burnetii in ruminants remains challenging despite the use of new technology and the accumulation of novel knowledge. Serology tools, the primary methods of infection surveillance in veterinary medicine, have limitations. We used recombinant antigen production to develop an ELISA based on the SucB protein, one of the major immunodominant antigens described in humans and laboratory animals. We produced the antigen successfully in an Escherichia coli heterologous system, confirmed by sequencing and mass spectrometry, and seen as a band of ~50 kDa in SDS-PAGE and on western blot analysis. We compared the performance of the recombinant ELISA with a commercial ELISA. We observed agreement of 83.5% and a substantial Cohen κ value of 0.67 in our pilot study.

Plant-Based Vaccines: Antigen Design, Diversity, and Strategies for High Level Production.

Vaccines for human use have conventionally been developed by the production of (1) microbial pathogens in eggs or mammalian cells that are then inactivated, or (2) by the production of pathogen proteins in mammalian and insect cells that are purified for vaccine formulation, as well as, more recently, (3) by using RNA or DNA fragments from pathogens. Another approach for recombinant antigen production in the last three decades has been the use of plants as biofactories. Only have few plant-produced vaccines been evaluated in clinical trials to fight against diseases, of which COVID-19 vaccines are the most recent to be FDA approved. In silico tools have accelerated vaccine design, which, combined with transitory antigen expression in plants, has led to the testing of promising prototypes in pre-clinical and clinical trials. Therefore, this review deals with a description of immunoinformatic tools and plant genetic engineering technologies used for antigen design (virus-like particles (VLP), subunit vaccines, VLP chimeras) and the main strategies for high antigen production levels. These key topics for plant-made vaccine development are discussed and perspectives are provided.

Vaccine effect of recombinant single-chain hemagglutinin protein as an antigen

Vaccination is one of the most effective interventions for preventing the spread of influenza viruses at the population level. Currently most influenza vaccines are produced by using embryonated chicken eggs, but alternative methods that achieve more rapid large-scale production are highly desirable for vaccines against both pandemic and seasonal influenza viruses. The use of recombinant hemagglutinin (HA), a key virus surface protein, as an antigen is an attractive candidate alternative approach, because of the potential for high protein yields and the ease of cloning new antigenic variants. Although fusion of HA with trimerization domains is needed to stabilize the trimeric structure and enhance the immunogenicity of the recombinant HA protein, whether the trimerization domains are immunogenic must be considered. Here, we generated recombinant multimeric HA without trimerization domains by using a short peptide linker, termed a single-chain HA (scHA), and evaluated scHAs as potential antigens for generating vaccines against influenza virus. Using mammalian cells, we succeeded in making three types of recombinant scHAs—two dimeric scHAs and a trimeric scHA. After immunization with aluminium salts in mice, one of the dimeric scHAs induced the greatest HA-specific IgG response among the scHAs and protected against virus challenge as strongly as the typically used trimeric HA containing a trimerization domain. We did not observe IgGs specific for the short peptide linker in mice immunized with the dimeric scHA, although IgGs specific for the trimerization domain occurred in mice immunized with the trimeric HA containing that domain. Furthermore, changing to another adjuvant did not diminish the utility of the dimeric scHA. These results suggest the potential usefulness of dimeric scHA as a vaccine antigen. We believe that single-chain antigens may represent new alternatives for production of recombinant antigen–based vaccines.

Simple production of hydrophobin-fused domain III of dengue envelope protein and induction of neutralizing antibodies against the homotypic serotype of dengue virus.

Hydrophobin-fused domain III of dengue envelope proteins serotypes 1 and 2 were expressed in Rachiplusia nu larvae and purified by aqueous two-phase system. This biotechnological approach of hydrophobin-fused proteins, which allowed obtaining 97.7µg/larva of fusion protein DomIII serotype 1 and 61.4µg/larva of fusion protein DomIII serotype 2, represents an integrated strategy for simple production of recombinant antigens. Purified fusion proteins induced serotype-specific neutralizing antibodies without cross-reaction against other serotypes and arboviruses after mouse immunization. hydrophobin-fused domain III of dengue envelope protein could be a promising strategy for easy and low-cost production of components of a tetravalent sub-unit vaccine against dengue.

Priority use cases for antibody-detecting assays of recent malaria exposure as tools to achieve and sustain malaria elimination.

Measurement of malaria specific antibody responses represents a practical and informative method for malaria control programs to assess recent exposure to infection. Technical advances in recombinant antigen production, serological screening platforms, and analytical methods have enabled the identification of several target antigens for laboratory based and point-of-contact tests. Questions remain as to how these serological assays can best be integrated into malaria surveillance activities to inform programmatic decision-making. This report synthesizes discussions from a convening at Institut Pasteur in Paris in June 2017 aimed at defining practical and informative use cases for serology applications and highlights five programmatic uses for serological assays including: documenting the absence of transmission; stratification of transmission; measuring the effect of interventions; informing a decentralized immediate response; and testing and treating P. vivax hypnozoite carriers.

Recombinant LipL32 Protein Developed Using a Synthetic Gene Detects Leptospira-specific Antibodies in Human Serum Samples.

Synthetic biology is emerging as a viable alternative for the production of recombinant antigens for diagnostic applications. It offers a safe alternative for the synthesis of antigenic principles derived from organisms that pose a high biological risk. Here, we describe an enzyme-linked immunosorbent assay (ELISA) using the synthetic recombinant LipL32 (rLipL32) protein expressed in Escherichia coli for the detection of Leptospira-specific antibodies in human serum samples. The rLipL32-based ELISA was compared with a microscopic agglutination test (MAT), which is currently used as the gold standard for the diagnosis of leptospirosis. Our results showed that all the MAT-positive serum samples were positive for Leptospira-specific IgG in an ELISA, while 65% (n = 13) of these samples were also positive for Leptospira-specific IgM. In the MAT-negative serum samples, 80% and 55% of the samples were detected as negative by an ELISA for Leptospira-specific IgM and IgG, respectively. An ELISA using the synthetic rLipL32 antigen was able to distinguish Leptospira-specific IgM (sensitivity 65% and specificity 80%) and IgG (sensitivity 100% and specificity 55%) in human serum samples and has the potential to serve as a rapid diagnostic test for leptospirosis.

Fluorescence Microsphere Immunoassay for Detection of Antibodies to Porcine Reproductive and Respiratory Syndrome Virus and Porcine Circovirus type 2 using Protein A, Protein G, and Protein A/G

The purpose of this study was the development of multiplex fluorescence microsphere immunoassay (FMIA) for the detection of porcine reproductive and respiratory syndrome virus (PRRSV) and porcine circovirus type 2 (PCV2) specific IgG antibodies by incorporation of non-species-specific conjugates Protein A, G, and A/G in place of the secondary antibody. A total of 205 serum samples obtained from pigs experimentally infected with PRRSV and/or PCV2 were tested. For the production of recombinant antigens, PRRSV nucleoprotein (N) and PCV2 capsid protein (CP) were expressed in Escherichia coli and purified on a nickel affinity column. The purified proteins were covalently coupled to carboxylated microsphere beads. Four target antigens were assembled into a single multiplex and tested against sera from swine infected or co-infected with PRRSV or PCV2. Protein A, G, and A/G have been tested in place of secondary conjugate porcine IgG. All conjugates were capable of detecting antibody and the detection of IgG responses against PRRSV and PCV2 antigen targets were varied with A> A/G> G.In the absence of species specific reagents the incorporation of Protein A, G, and A/G provide a suitable substitutes.

Identification of Antigenic Proteins from Lichtheimia corymbifera for Farmer's Lung Disease Diagnosis.

The use of recombinant antigens has been shown to improve both the sensitivity and the standardization of the serological diagnosis of Farmer’s lung disease (FLD). The aim of this study was to complete the panel of recombinant antigens available for FLD serodiagnosis with antigens of Lichtheimia corymbifera, known to be involved in FLD. L. corymbifera proteins were thus separated by 2D electrophoresis and subjected to western blotting with sera from 7 patients with FLD and 9 healthy exposed controls (HEC). FLD-associated immunoreactive proteins were identified by mass spectrometry based on a protein database specifically created for this study and subsequently produced as recombinant antigens. The ability of recombinant antigens to discriminate patients with FLD from controls was assessed by ELISA performed with sera from FLD patients (n = 41) and controls (n = 43) recruited from five university hospital pneumology departments of France and Switzerland. Forty-one FLD-associated immunoreactive proteins from L. corymbifera were identified. Six of them were produced as recombinant antigens. With a sensitivity and specificity of 81.4 and 77.3% respectively, dihydrolipoyl dehydrogenase was the most effective antigen for discriminating FLD patients from HEC. ELISA performed with the putative proteasome subunit alpha type as an antigen was especially specific (88.6%) and could thus be used for FLD confirmation. The production of recombinant antigens from L. corymbifera represents an additional step towards the development of a standardized ELISA kit for FLD diagnosis.

Expression of Trypanosoma brucei gambiense Antigens in Leishmania tarentolae. Potential for Use in Rapid Serodiagnostic Tests (RDTs).

The development of rapid serodiagnostic tests for sleeping sickness and other diseases caused by kinetoplastids relies on the affordable production of parasite-specific recombinant antigens. Here, we describe the production of recombinant antigens from Trypanosoma brucei gambiense (T.b. gambiense) in the related species Leishmania tarentolae (L. tarentolae), and compare their diagnostic sensitivity and specificity to native antigens currently used in diagnostic kits against a panel of human sera. A number of T.b. gambiense protein antigen candidates were chosen for recombinant expression in L. tarentolae based on current diagnostics in field use and recent findings on immunodiagnostic antigens found by proteomic profiling. In particular, the extracellular domains of invariant surface glycoprotein 65 (ISG65), variant surface glycoproteins VSG LiTat 1.3 and VSG LiTat 1.5 were fused with C-terminal histidine tags and expressed as soluble proteins in the medium of cultured, recombinant L. tarentolae. Using affinity chromatography, on average 10 mg/L of recombinant protein was purified from cultures and subsequently tested against a panel of sera from sleeping sickness patients from controls, i.e. persons without sleeping sickness living in HAT endemic countries. The evaluation on sera from 172 T.b. gambiense human African trypanosomiasis (HAT) patients and from 119 controls showed very high diagnostic potential of the two recombinant VSG and the rISG65 fragments with areas under the curve between 0.97 and 0.98 compared to 0.98 and 0.99 with native VSG LiTat 1.3 and VSG LiTat 1.5 (statistically not different). Evaluation on sera from 78 T.b. rhodesiense HAT patients and from 100 controls showed an acceptable diagnostic potential of rISG65 with an area under the curve of 0.83. These results indicate that a combination of these recombinant antigens has the potential to be used in next generation rapid serodiagnostic tests. In addition, the L. tarentolae expression system enables simple, cheap and efficient production of recombinant kinetoplatid proteins for use in diagnostic, vaccine and drug discovery research that does not rely on animal use to generate materials.

Viral vaccines and their manufacturing cell substrates: New trends and designs in modern vaccinology.

Vaccination is one of the most effective interventions in global health. The worldwide vaccination programs significantly reduced the number of deaths caused by infectious agents. A successful example was the eradication of smallpox in 1979 after two centuries of vaccination campaigns. Since the first variolation administrations until today, the knowledge on immunology has increased substantially. This knowledge combined with the introduction of cell culture and DNA recombinant technologies revolutionized vaccine design. This review will focus on vaccines against human viral pathogens, recent developments on vaccine design and cell substrates used for their manufacture. While the production of attenuated and inactivated vaccines requires the use of the respective permissible cell substrates, the production of recombinant antigens, virus‐like particles, vectored vaccines and chimeric vaccines requires the use – and often the development – of specific cell lines. Indeed, the development of novel modern viral vaccine designs combined with, the stringent safety requirements for manufacture, and the better understanding on animal cell metabolism and physiology are increasing the awareness on the importance of cell line development and engineering areas. A new era of modern vaccinology is arriving, offering an extensive toolbox to materialize novel and creative ideas in vaccine design and its manufacture.

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

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. 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. 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.

Optimization of culture medium for cell growth and expression of 648 antigen from Leishmania infantum chagasi in recombinant Escherichia coli M15

Visceral leishmaniasis is a potentially fatal disease caused by Leishmania infantum chagasi in the New World. Anti-leishmanial treatment is problematic because anti-leishmanial drugs are toxic and costly and drug resistance is emerging. Moreover, there is no effective and safe vaccine approved for human use against any form of leishmaniasis. It seems likely that both a subunit vaccine and an optimized diagnostic test would benefit from the production of recombinant antigens. However, for recombinant protein production, the composition of the cell growth medium must be carefully formulated and monitored, because it may have significant metabolic effects on both the cells and protein expression. Thus, the aim of this study was to optimize the culture medium for cell growth and the expression of the 648 antigen from Leishmania i. chagasi in recombinant Escherichia coli M15. The induction allowed the expression of the 648 antigen; this was confirmed by electrophoresis, which showed a protein with a molecular mass of 24 kDa. The results showed that the higher cell concentration obtained in TB medium can be explained by the larger number of components such as amino acids and vitamin precursors contained in this complex medium, as well as glycerol as a carbon and energy source. Acetic acid excretion, detected at the end of fermentation, resulted in an inhibitory effect for both cell growth and protein production, because this was above the concentration limit of 0.9 g L−1.

Transient Expression of Foot and Mouth Disease Virus (FMDV)Coat Protein in Tobacco (Nicotiana tabacom) via Agroinfiltration

Background: Transient and stable transformation of host plants are the common techniques to produce transgenic plants. However, the main drawback of stable transformation is the fact that it takes quite a long time to produce a transgenic line. While, transient gene expression is a quick method to produce recombinant proteins in plants. Objective: The main goal of the present study was to evaluate efficient agroinfiltration as an efficient and rapid method for production of recombinant antigen of FMDV. Materials and Methods: Tobacco leaves were transformed via agroinfiltration using a needle-free syringe. Presence of the gene cassette was verified by polymerase chain reaction (PCR). Expression of the foreign gene was evaluated using Real Time PCR, protein dot blot and enzyme-linked immunosorbent assay (ELISA). Results: PCR analysis confirmed successful transformation of plant leaves. Expression of foreign protein was confirmed at both transcription and translation levels. Results of Real Time PCR assay indicated that the foreign gene was transcribed in transformed leaves. ELISA results showed that the foreign gene was expressed in the transformed leaves in high level. Conclusion: Here, the efficacy of agroinfiltration for transient expression of FMDV coat protein in tobacco was illustrated. Accordingly, transient agroinfilteration expedites the process of recombinant antigens expression in plant tissues.

Recombinant antigens expressed in Pichia pastoris for the diagnosis of sleeping sickness caused by Trypanosoma brucei gambiense.

BackgroundScreening tests for gambiense sleeping sickness, such as the CATT/T. b. gambiense and a recently developed lateral flow tests, are hitherto based on native variant surface glycoproteins (VSGs), namely LiTat 1.3 and LiTat 1.5, purified from highly virulent trypanosome strains grown in rodents.Methodology/Principal FindingsWe have expressed SUMO (small ubiquitin-like modifier) fusion proteins of the immunogenic N-terminal part of these antigens in the yeast Pichia pastoris. The secreted recombinant proteins were affinity purified with yields up to 10 mg per liter cell culture.Conclusions/SignificanceThe diagnostic potential of each separate antigen and a mixture of both antigens was confirmed in ELISA on sera from 88 HAT patients and 74 endemic non-HAT controls. Replacement of native antigens in the screening tests for sleeping sickness by recombinant proteins will eliminate both the infection risk for the laboratory staff during antigen production and the need for laboratory animals. Upscaling production of recombinant antigens, e.g. in biofermentors, is straightforward thus leading to improved standardisation of antigen production and reduced production costs, which on their turn will increase the availability and affordability of the diagnostic tests needed for the elimination of gambiense HAT.

A novel adjuvant-free H fusion system for the production of recombinant immunogens in Escherichia coli

The production of recombinant antigens in Escherichia coli and specific polyclonal antibodies for diagnosis and therapy is still a challenge for world-wide researchers. Several different strategies have been explored to improve both antigen and antibody production, all of them depending on a successful expression and immunogenicity of the antigen. Gene fusion technology attempted to address these challenges: fusion partners have been applied to optimize recombinant antigen production in E. coli, and to increase protein immunogenicity. Taking a 12-kDa surface adhesion antigen from Cryptosporidium parvum (CP12) by example, the novel H fusion partner was presented in this work as an attractive option for the development of recombinant immunogens and its adjuvant-free immunization. The H tag (of only 1 kDa) efficiently triggered a CP12-specific immune response, and it also improved the immunization procedure without requiring co-administration of adjuvants. Moreover, polyclonal antibodies raised against the HCP12 fusion antigen detected native antigen structures displayed on the surface of C. parvum oocysts. The H tag proved to be an advanced strategy and promising technology for the diagnosis and therapy of C. parvum infections in animals and humans, allowing a rapid and simple recombinant production of the CP12 antigen.

Dengue virus tetra-epitope peptide expressed in lettuce chloroplasts for potential use in dengue diagnosis.

Dengue virus causes about 100 million cases of dengue disease per year in the world. Laboratory diagnosis is done mainly by serological techniques, which in many cases use crude virus extracts that may cause cross-reactions to other flaviviruses. These undesirable cross-reactions can be reduced or eliminated by using recombinant proteins based on restricted epitopes. Aiming to decrease flaviviral cross-reactions and non-specific interactions in dengue serological assays, a plant expression system was chosen for recombinant antigen production as a reliable and inexpensive dengue diagnostic tool. In the present report, the lettuce plastid transformation system was applied to achieve efficient and stable tetra-epitope peptide antigen production, and its reactivity was evaluated. For this purpose, one putative epitope at positions 34 to 57 of E protein within the junction site of domains I and II of dengue virus (DENV) 1 to 4 serotypes linked by glycine linkers was expressed in lettuce chloroplasts. The potential immunoreactivity for the four DENV serotypes was evaluated using sera from patients of positive and negative dengue cases. Results indicated an overall sensitivity of 71.7% and specificity of 100%. No cross-reactions with the sera of yellow fever-positive or healthy individuals vaccinated against yellow fever were observed. This novel approach may provide an alternative system for the large-scale production of dengue recombinant antigens useful for serodiagnosis.

The human potential of a recombinant pandemic influenza vaccine produced in tobacco plants

Rapid production of influenza vaccine antigen is an important challenge when a new pandemic occurs. Production of recombinant antigens in plants is a quick, cost effective and up scalable new strategy for influenza vaccine production. In this study, we have characterized a recombinant influenza haemagglutinin antigen (HAC1) that was derived from the 2009 pandemic H1N1 (pdmH1N1) virus and expressed in tobacco plants. Volunteers vaccinated with the 2009 pdmH1N1 oil-in-water adjuvanted vaccine provided serum and lymphocyte samples that were used to study the immunogenic properties of the HAC1 antigen in vitro. By 7 d post vaccination, the vaccine fulfilled the licensing criteria for antibody responses to the HA detected by haemagglutination inhibition and single radial hemolysis. By ELISA and ELISPOT analysis we showed that HAC1 was recognized by specific serum antibodies and antibody secreting cells, respectively. We conducted a kinetic analysis and found a peak of serum HAC1 specific antibody response between day 14 and 21 post vaccination by ELISA. We also detected elevated production of IL-2 and IFNγ and low frequencies of CD4+ T cells producing single or multiple Th1 cytokines after stimulating PBMCs (peripheral blood mononuclear cells) with the HAC1 antigen in vitro. This indicates that the antigen can interact with T cells, although confirming that an effective adjuvant would be required to improve the T-cell stimulation of plant based vaccines. We conclude that the tobacco derived recombinant HAC1 antigen is a promising vaccine candidate recognized by both B and T cells.

Recombinant antigen production for assays of intradermoreaction for diagnosis and surveillance of tuberculosis

The goal of the present work was to develop reagents with potential for tuberculosis diagnosis. Genetic sequences of Mycobacterium tuberculosis secretion antigens were amplified by PCR, cloned into the Gateway(®) system, and expressed in Escherichia coli. The recombinant M. tuberculosis proteins were purified by metal affinity chromatography and preparative gel SDS-PAGE electrophoresis followed by electroelution and removal of endotoxins using Triton X-114. In total, seven recombinant proteins were obtained (ESAT-6, CFP10, TB10.3, TB10.4, MTSP11, MPT70, and MPT83). Delayed hypersensitivity reactions (DHR) was evaluated in Cavia porcellus and compared to the response using a standard purified protein derivative (PPD). All seven recombinant proteins produced a positive induration reaction in an intradermal test in guinea pigs previously sensitized with M. tuberculosis. When applied together, at a concentration of each recombinant protein 0.04 mg/mL, the intradermoreaction in C. porcellus was significantly higher than that obtained by standard PPD (p-value=0.00386).

In planta production of plant-derived and non-plant-derived adjuvants

Recombinant antigen production in plants is a safe and economically sound strategy for vaccine development, particularly for oral/mucosal vaccination, but subunit vaccines usually suffer from weak immunogenicity and require adjuvants that escort the antigens, target them to relevant sites and/or activate antigen-presenting cells for elicitation of protective immunity. Genetic fusions of antigens with bacterial adjuvants as the B subunit of the cholera toxin have been successful in inducing protective immunity of plant-made vaccines. In addition, several plant compounds, mainly plant defensive molecules as lectins and saponins, have shown strong adjuvant activities. The molecular diversity of the plant kingdom offers a vast source of non-bacterial compounds with adjuvant activity, which can be assayed in emerging plant manufacturing systems for the design of new plant vaccine formulations.