Conditional splicing system for tight control of viral overlapping genes.

Viral genomes frequently harbor overlapping genes, complicating the development of virus-vectored vaccines and gene therapies. This study introduces a novel conditional splicing system to precisely control the expression of such overlapping genes through recombinase-mediated conditional splicing. We refined site-specific recombinase (SSR) conditional splicing systems and explored their mechanisms. The systems demonstrated exceptional inducibility (116,700-fold increase) with negligible background expression, facilitating the conditional expression of overlapping genes in adenovirus-associated virus (AAV) and human immunodeficiency virus type 1. Notably, this approach enabled the establishment of stable AAV producer cell lines, encapsulating all necessary packaging genes. Our findings underscore the potential of the SSR-conditional splicing system to significantly advance vector engineering, enhancing the efficacy and scalability of viral-vector-based therapies and vaccines. IMPORTANCE: Regulating overlapping genes is vital for gene therapy and vaccine development using viral vectors. The regulation of overlapping genes presents challenges, including cytotoxicity and impacts on vector capacity and genome stability, which restrict stable packaging cell line development and broad application. To address these challenges, we present a "loxp-splice-loxp"-based conditional splicing system, offering a novel solution for conditional expression of overlapping genes and stable cell line establishment. This system may also regulate other cytotoxic genes, representing a significant advancement in cell engineering and gene therapy as well as biomass production.

Different strategies for expression and purification of the CT26-poly-neoepitopes vaccine in Escherichia coli.

BACKGROUND: Due to the association of hypermutated colorectal cancer (CRC) with many neo-antigens, poly-neo-epitopes are attractive vaccines. The molecular features of murine CT26 are similar to those of aggressive human CRC. CT26 contains some antigenic mutations, which can provide specific immunotherapy targets. Herein, we aimed to express, and purify the previously designed hexatope containing CT26 neoepitopes, CT26-poly-neoepitopes. METHODS AND RESULTS: In the current study, expression of the CT26-poly-neoepitopes was optimized in three different Escherichia coli strains including BL21 (DE3), Origami (DE3), and SHuffle®. Furthermore, the effect of ethanol on the CT26-poly-neoepitopes expression was investigated. The highest amount of CT26-poly-neoepitopes, which included CT26-poly-neoepitopes with the uncleaved pelB signal sequence and the processed one, was achieved when BL21 containing pET-22 (CT26-poly-neoepitopes) was induced with 0.1 mM IPTG for 48 h at 22 ºC in the presence of 2% ethanol. However, 37 ºC was the optimized induction temperature for expression of the CT26-poly-neoepitopes in the absence of ethanol. To purify the CT26-poly-neoepitopes, Ni-NTA affinity chromatography under denaturing and hybrid conditions were applied. High and satisfactory CT26-poly-neoepitopes purity was achieved by the combined urea and imidazole method. CONCLUSION: The effect of ethanol on expression of the CT26-poly-neoepitopes was temperature-dependent. Furthermore, the pelB-mediated translocation of the CT26-poly-neoepitopes into the periplasm was inefficient. Moreover, higher concentration of imidazole in the washing buffer improved the CT26-poly-neoepitopes purification under hybrid condition. Overall, the immunogenicity of CT26-poly-neoepitopes expressed in BL21 under the optimum condition and purified under hybrid condition can be studied in our future in vivo study.

Next biotechnological plants for addressing global challenges: The contribution of transgenesis and new breeding techniques.

The aim of this survey is to identify and characterize new products in plant biotechnology since 2015, especially in relation to the advent of New Breeding Techniques (NBTs) such as gene editing based on the CRISPR-Cas system. Transgenic (gene transfer or gene silencing) and gene edited traits which are approved or marketed in at least one country, or which have a non-regulated status in the USA, are collected, as well as related patents worldwide. In addition, to shed light on potential innovation for Africa, field trials on the continent are examined. The compiled data are classified in application categories, including agronomic improvements, industrial use and medical use, namely production of recombinant therapeutic molecules or vaccines (including against Covid-19). The data indicate that gene editing appears to be an effective complement to 'classical' transgenesis, the use of which is not declining, rather than a replacement, a trend also observed in the patenting landscape. Nevertheless, increased use of gene editing is apparent. Compared to transgenesis, gene editing has increased the proportion of some crop species and decreased others amongst approved, non-regulated or marketed products. A similar differential trend is observed for breeding traits. Gene editing has also favored the emergence of new private companies. China, and prevalently its public sector, overwhelmingly dominates the patenting landscape, but not the approved/marketed one, which is dominated by the USA. The data point in the direction that regulatory environments will favor or discourage innovation.

Whole-Cell Vaccine Preparation: Options and Perspectives.

Vaccines are biological preparations to elicit a specific immune response in individuals against the targetted microorganisms. The use of vaccines has caused the near eradication of many critical diseases and has had an everlasting impact on public health at a relatively low cost. Most of the vaccines developed today are based on techniques which were developed a long time ago. In the beginning, vaccines were prepared from tissue fluids obtained from infected animals or people, but at present, the scenario has changed with the development of vaccines from live or killed whole microorganisms and toxins or using genetic engineering approaches. Considerable efforts have been made in vaccine development, but there are still many diseases that need attention, and new technologies are being developed in vaccinology to combat them. In this chapter, we discuss different approaches for vaccine development, including the properties and preparation of whole-cell vaccines.

Click chemistry compared to thiol chemistry for the synthesis of site-selective glycoconjugate vaccines using CRM197 as carrier protein.

Conjugation chemistry is one of the main parameters affecting immunogenicity of glycoconjugate vaccines and a rational approach toward a deeper understanding of their mechanism of action will greatly benefit from highly-defined and well-characterized structures. Herein, different conjugation methods were investigated with the aim of controlling glycosylation site and glycosylation density on the carrier protein. S. Typhimurium lipopolysaccharide O-Antigen and CRM197 carrier protein were used as models. In particular, thiol and click chemistry were examined, both involving the linkage of the terminal reducing sugar unit of the O-Antigen chain to different amino acids on the carrier protein. Thiol chemistry allowed O-Antigen conjugation only when the carrier protein was activated on the lysines and with a relative high number of linkers, while click chemistry allowed conjugate generation even when just one position on the protein was activated and to both lysine and tyrosine sites. The study highlights click chemistry as a leading approach for the synthesis of well-defined glycoconjugates, useful to investigate the relationship between conjugate design and immune response.

Engineering microalgae through chloroplast transformation to produce high-value industrial products.

The last few years have seen an ever-increasing interest in the exploitation of microalgae as an alternative platform to produce high-value products such as biofuels, industrial enzymes, therapeutic proteins, including antibodies, hormones, and vaccines. Due to some unique attractive features, engineering of the chloroplast genome provides a promising platform for the production of high-value targets because it allows manipulation of metabolic processes in ways that would be impossible, or at least prohibitively difficult through traditional approaches. Since its initial demonstration in 1988 in Chlamydomonas reinhardtii, genetic tools have been developed, which have made it possible to produce high-value molecules in different species. However, the commercial application of microalgae as production platform is hindered by many factors like poor biomass, low product yields, and costly downstream processing methodologies. In this review, we discuss the potential of microalgae to use as an alternative production platform for high-value targets using chloroplast transformation technology.

Orbitally Shaken Single-Use Bioreactor for Animal Cell Cultivation: Fed-Batch and Perfusion Mode.

Increasing the cultivation volume from small to large scale can be a rather complex and challenging process when the method of aeration and mixing is different between scales. Orbitally shaken bioreactors (OSBs) utilize the same hydrodynamic principles that define the success of smaller-scale cultures, which are developed on an orbitally shaken platform, and can simplify scale-up. Here we describe the basic working principles of scale-up in terms of the volumetric oxygen transfer coefficient (kLa) and mixing time and how to define these parameters experimentally. The scale-up process from an Erlenmeyer flask shaken on an orbital platform to an orbitally shaken single-use bioreactor (SB10-X, 12 L) is described in terms of both fed-batch and perfusion-based processes. The fed-batch process utilizes a recombinant variant of the mammalian cell line, Chinese hamster ovary (CHO), to express a biosimilar of a therapeutic monoclonal antibody. The perfusion-based process utilizes either an alternating tangential flow filtration (ATF) or a tangential flow filtration (TFF) system for cell retention to cultivate an avian cell line, AGE1.CR.pIX, for the propagation of influenza A virus, H1N1, in high cell density. Based on two example cell cultivations, processes outline the advantages that come with using an orbitally shaken bioreactor for scaling-up a process. The described methods are also applicable to other suspension cell lines.

Perfusion Control for High Cell Density Cultivation and Viral Vaccine Production.

The global demand for complex biopharmaceuticals like recombinant proteins, vaccines, or viral vectors is steadily rising. To further improve process productivity and to reduce production costs, process intensification can contribute significantly. The design and optimization of perfusion processes toward very high cell densities require careful selection of strategies for optimal perfusion rate control. In this chapter, various options are discussed to guarantee high cell-specific virus yields and to achieve virus concentrations up to 1010 virions/mL. This includes reactor volume exchange regimes and perfusion rate control based on process variables such as cell concentration and metabolite or by-product concentration. Strategies to achieve high cell densities by perfusion rate control and their experimental implementation are described in detail for pseudo-perfusion or small-scale perfusion bioreactor systems. Suspension cell lines such as MDCK, BHK-21, EB66®, and AGE1.CR.pIX® are used to exemplify production of influenza, yellow fever, Zika, and modified vaccinia Ankara virus.

SnoopLigase peptide-peptide conjugation enables modular vaccine assembly.

For many infectious diseases there is still no vaccine, even though potential protective antigens have been identified. Suitable platforms and conjugation routes are urgently needed to convert the promise of such antigens into broadly protective and scalable vaccines. Here we apply a newly established peptide-peptide ligation approach, SnoopLigase, for specific and irreversible coupling of antigens onto an oligomerization platform. SnoopLigase was engineered from a Streptococcus pneumoniae adhesin and enables isopeptide bond formation between two peptide tags: DogTag and SnoopTagJr. We expressed in bacteria DogTag linked to the self-assembling coiled-coil nanoparticle IMX313. This platform was stable over months at 37 °C when lyophilized, remaining reactive even after boiling. IMX-DogTag was efficiently coupled to two blood-stage malarial proteins (from PfEMP1 or CyRPA), with SnoopTagJr fused at the N- or C-terminus. We also showed SnoopLigase-mediated coupling of a telomerase peptide relevant to cancer immunotherapy. SnoopLigase-mediated nanoassembly enhanced the antibody response to both malaria antigens in a prime-boost model. Including or depleting SnoopLigase from the conjugate had little effect on the antibody response to the malarial antigens. SnoopLigase decoration represents a promising and accessible strategy for modular plug-and-display vaccine assembly, as well as providing opportunities for robust nanoconstruction in synthetic biology.

Recent Patents Applications in Red Biotechnology: A Mini-Review.

BACKGROUND: The different fields of biotechnology can be classified by colors, as a "rainbow" methodology. In this sense, the red biotechnology, focused on the preservation of health, has been outstanding in helping to solve this challenge through the provision of technologies, including diagnostic kits, molecular diagnostics, vaccines, innovations in cancer research, therapeutic antibodies and stem cells. OBJECTIVE: The main goal of this work is to highlight the different areas within the red Biotechnology. In this sense, we revised some patents regarding red biotechnology as examples to cover this subject. METHODS: A literature search of patents was performed from the followings Patents Database: INPI, USPTO, Esp@cenet, WIPO and Google Patents. RESULTS: Our analysis showed the following numbers from patents found: cancer research (8), diagnosis kit (9), vaccines (8), stem cells (9) and therapeutic antibodies (5), where the United States is the leader for most filled patents in Red Biotechnology. CONCLUSION: This mini-review has provided an update of some patents on Recent Patents in Red Biotechnology. As far as we know, this is the first mini-review report on Red Biotechnology based on patents.

Plant synthetic biology could drive a revolution in biofuels and medicine.

IMPACT STATEMENT: The plant synthetic biology field has exploded in the last five years, in part driven by techniques such as CRISPR and cheap DNA synthesis. This review summarizes the current state of research in plant synthetic biology, and how it is being applied to two topics: renewable fuels and chemicals, and medicine.

Emerging Technologies for Low-Cost, Rapid Vaccine Manufacture.

To stop the spread of future epidemics and meet infant vaccination demands in low- and middle-income countries, flexible, rapid and low-cost vaccine development and manufacturing technologies are required. Vaccine development platform technologies that can produce a wide range of vaccines are emerging, including: a) humanized, high-yield yeast recombinant protein vaccines; b) insect cell-baculovirus ADDomer vaccines; c) Generalized Modules for Membrane Antigens (GMMA) vaccines; d) RNA vaccines. Herein, existing and future platforms are assessed in terms of addressing challenges of scale, cost, and responsiveness. To assess the risk and feasibility of the four emerging platforms, the following six metrics are applied: 1) technology readiness; 2) technological complexity; 3) ease of scale-up; 4) flexibility for the manufacturing of a wide range of vaccines; 5) thermostability of the vaccine product at tropical ambient temperatures; and 6) speed of response from threat identification to vaccine deployment. The assessment indicated that technologies in the order of increasing feasibility and decreasing risk are the yeast platform, ADDomer platform, followed by RNA and GMMA platforms. The comparative strengths and weaknesses of each technology are discussed in detail, illustrating the associated development and manufacturing needs and priorities.

The Pragmatic Introduction and Expression of Microbial Transgenes in Plants.

Several genetic strategies have been proposed for the successful transformation and expression of microbial transgenes in model and crop plants. Here, we bring into focus the prominent applications of microbial transgenes in plants for the development of disease resistance; mitigation of stress conditions; augmentation of food quality; and use of plants as "bioreactors" for the production of recombinant proteins, industrially important enzymes, vaccines, antimicrobial compounds, and other valuable secondary metabolites. We discuss the applicable and cost-effective approaches of transgenesis in different plants, as well as the limitations thereof. We subsequently present the contemporary developments in targeted genome editing systems that have facilitated the process of genetic modification and manifested stable and consumer-friendly genetically modified plants and their products. Finally, this article presents the different approaches and demonstrates the introduction and expression of microbial transgenes for the improvement of plant resistance to pathogens and abiotic stress conditions and the production of valuable compounds, together with the promising research progress in targeted genome editing technology. We include a special discussion on the highly efficient CRISPR-Cas system helpful in microbial transgene editing in plants.

Vaccine and antibody production in plants: developments and computational tools.

Plants as bioreactors have been widely used to express efficient vaccine antigens against viral, bacterial and protozoan infections. To date, many different plant-based expression systems have been analyzed, with a growing preference for transient expression systems. Antibody expression in diverse plant species for therapeutic applications is well known, and this review provides an overview of various aspects of plant-based biopharmaceutical production. Here, we highlight conventional and gene expression technologies in plants along with some illustrative examples. In addition, the portfolio of products that are being produced and how they relate to the success of this field are discussed. Stable and transient gene expression in plants, agrofiltration and virus infection vectors are also reviewed. Further, the present report draws attention to antibody epitope prediction using computational tools, one of the crucial steps of vaccine design. Finally, regulatory issues, biosafety and public perception of this technology are also discussed.

Exploiting Bacteriophage Proteomes: The Hidden Biotechnological Potential.

Bacteriophages encode many distinct proteins for the successful infection of a bacterial host. Each protein plays a specific role in the phage replication cycle, from host recognition, through takeover of the host machinery, and up to cell lysis for progeny release. As the roles of these proteins are being revealed, more biotechnological applications can be anticipated. Phage-encoded proteins are now being explored for the control, detection, and typing of bacteria; as vehicles for drug delivery; and for vaccine development. In this review, we discuss how engineering approaches can be used to improve the natural properties of these proteins and set forth the most innovative applications that demonstrate the unlimited biotechnological potential held by phage-encoded proteins.

Investigations into the efficacy of multi-component cocaine vaccines.

Although cocaine addiction remains a serious health and societal problem in the United States, no FDA-approved treatment has been developed. Vaccines offer an exciting strategy for the treatment of cocaine addiction; however, vaccine formulations need to be optimized to improve efficacy. Herein, we examine the effectiveness of a tricomponent cocaine vaccine, defined as having its hapten (GNE) and adjuvant (cytosine-guanine oligodeoxynucleotide 1826, CpG ODN 1826) covalently linked via the immunogenic protein ovalbumin (OVA). The tricomponent vaccine (GNE-OVA-CpG 1826) and a vaccine of analogous, individual components (GNE-OVA+CpG ODN 1826) were found to similarly induce highly specific anticocaine antibody production in mice and block cocaine's stimulant effects in hyperlocomotor testing.

Transplastomic plants yield a multicomponent vaccine against cysticercosis.

Low cost vaccines against cysticercosis are needed to fight this parasitosis, especially in developing countries. Herein polycistron arrangements were designed to accomplish the simultaneous expression of multiple protective antigens from Taenia solium in the plant cell as an attractive biofactory and delivery vehicle of vaccines. Transplastomic plants carrying synthetic polycistrons were able to simultaneously express the KETc1, KETc7, KETc12, GK1, and TSOL18/HP6-Tsol antigens; which retained their antigenicity and ability to induce humoral responses in BALB/c mice. These clones may be useful for the production of low-cost cysticercosis vaccine prototypes.

Strategies in the Design and Use of Synthetic "Internal Glycan" Vaccines.

The relative structural conservation of "internal glycans" in the cell walls of pathogens suggests that they might as target epitopes less prone to variation and hence with greater potential universality as vaccine targets. Examples of such glycans include the inner core sugars of lipopolysaccharides in Gram-negative bacteria. However, due to the buried nature of such internal epitopes, this approach has been rarely adopted. Here we briefly review and compare strategic approaches and outline practical methods associated with evaluating one synergistic strategy that combines (i) blocking of the display of "external glycans" with (ii) vaccination targeted at "internal glycans."