Special issue: Vaccine Biotechnology. This Special issue edited by Reingard Grabherr and Udo Reichl includes articles on the design of cell lines for viral vaccine production, downstream processing of virus-like particles and plant-based production of vaccines. The cover shows particles of highly pathogenic viruses transmitted by mosquitoes: Chikungunya, dengue and Rift Valley fever virus. Image by Gorben Pijlman.

Arthropod-borne arboviruses form a continuous threat to human and animal health. Enveloped virus-like particles are complex assemblies of membrane and viral glycoproteins and can be produced for flaviviruses (e.g. dengue, West Nile), phleboviruses (e.g. Rift Valley fever) and alphaviruses (e.g. chikungunya). Production of eVLPs in insect cells using modern biotechnology has a realistic potential to be used for novel vaccines against arboviral diseases.

The threat of West Nile virus (WNV) epidemics demands the development and licensing of effective vaccines. Plant-made vaccines against West Nile virus are potent, safe, and economically feasible: studies demonstrate that plants provide a robust system for the production of subunit and VLP-based vaccines against WNV. These vaccines are safer and as potent as those produced by other platforms but with much lower cost, indicating that plant-based systems will offer a more favorable cost/benefit ratio for WNV vaccination programs and encourage the eventual licensure and commercialization of human vaccines against WNV.

Defective interfering particles (DIPs) are subgenomic deletion mutants that can only replicate in the presence of a standard virus. Yet, DIPs interfere with the replication of the standard virus, and coinfected cells mainly produce DIPs. This review demonstrates that DIPs not only affect viral replication, evolution and pathogenesis, but can also have an impact on the production yield and product quality of viral vaccines and vectors. Hence, the generation and/or amplification of DIPs should be avoided in virus production processes.

Several different types of influenza viruses are currently circulating in the human population and cause seasonal epidemics. These viruses constantly change and adapt to the human herd immunity which makes vaccine production difficult. In addition, novel influenza viruses from the animal reservoir cause pandemics at irregular intervals and seasonal influenza virus vaccines do not provide protection against them. This paper reviews zoonotic influenza viruses with pandemic potential and technological advances towards better vaccines that induce broad and long lasting protection from influenza virus infection.

The baculovirus expression vector system (BEVS) has become an established commercial manufacturing platform. The authors review this technology, which enables fast, flexible and scalable protein production and is increasingly being used for the production of viral vaccines and gene therapy vectors. Nine BEVS-derived products have been licensed by global regulatory authorities, paving the way for new products that are in development.

Virus-like particles are protein assemblages that mimic the structure of viruses but lack viral nucleic acids. They are produced in recombinant systems and represent a new platform for vaccines and gene vectors requiring robust purification processes. In this review, we present recent advances in the design of downstream processes and unit operations for recombinant-based virus-like particles, focusing on novel strategies and trends for reducing manufacturing costs and development times for VLP-based products.

High producer cell lines for viral vaccine production are still identified by combining genetic modification with selection pressure and screening, or by using primary cells without modifications. Nevertheless, the contents of the genetic tool box are growing. In the future, hopefully this tool box and the knowledge base on virus replication in producer cell lines will become broad and deep enough to allow the direct design of a high producer cell line for a specific virus.

Poxvirus and adenovirus are among the most promising antigen delivery vectors for prevention of infectious diseases and treatment of cancer. They induce strong humoral and robust CTL responses. Mass production of this novel class of vectored vaccine candidates to support large-scale clinical trials largely exploits advanced cell-culture and downstream processing technologies. Yet, more research and development is needed to improve the production yields and streamline the manufacturing processes and analytical technologies to take full advantage of the recent progress made in the field of cell culture engineering and manufacturing.

SAG1 is the main surface antigen of the intracellular parasite Toxoplasma gondii and a promising candidate to produce an anti-T. gondii vaccine. In the current study, the authors demonstrate that Leishmania infantum Hsp83 (LiHsp83) can enhance the expression of SAG1 antigen in plants by using LiHsp83 as a carrier protein. SAG1 retains the structural integrity to elicit significant protection in orally immunized mice. The fusion of recombinant proteins of interest to LiHsp83 is a novel strategy that presents advantages over other carrier proteins used in heterologous plant expression systems, promoting the use of chloroplast transformation technology in molecular farming.

Adenovirus are widely used in vaccine and gene therapy vector development. This work presents the generation of a human amniocyte-derived cell line (1G3) for adenovirus production, excluding the formation of replication-competent adenovirus (RCA). The cells were adapted to grow in single-cell suspension using serum-free conditions and the production of adenovirus vectors was evaluated in different culture system relevant for process development. The cell line 1G3 presented good productivities, demonstrating it to be a valid cell host alternative for adenovirus production.

Adjuvants are critical for eliciting immune responses and the subsequent generation of antibodies with high specificity. The authors evaluated mAbs produced against histidine-rich protein2 of Plasmodium falciparum using poly(N-isopropylacrylamide) as an adjuvant, and characterized the mAbs. The mAbs generated using poly(N-isopropylacrylamide) showed good sensitivity and affinity constants, indicating that this polymer adjuvant has great potential to be used with proteins/polypeptides or small polypeptides for the generation of antibodies for various high value applications.

Antibody “libraries” can be screened to discover new therapeutic and diagnostic antibodies. The authors have developed a method of producing antibody libraries in the bacterial cytoplasm that may be screened for binding to external targets. This technology allows a rapid process for discovery of antibodies that can be made inexpensively in bacteria.

CHO cells are major production hosts for recombinant biologics including monoclonal antibodies. This study demonstrates that CHO cell overexpression of heat shock protein 27 (HSP27), a molecule with cytoprotective functions, results in an increase in cumulative integrated viable cell density, leading to greater recombinant monoclonal antibody titres. The modulation of apoptosis signaling pathways and delay of caspase activities induced by overexpression of HSP27 enable the improved performance of CHO fed-batch bioreactor cultures.

Cryopreservation is essential for long-term storage of cells and tissues. This manuscript demonstrates that the recombinant protein enolase can protect liver and pancreatic cells during long-term storage at freezing temperatures, producing thawed cells with good viability, morphology and metabolic functionality. Enolase is non-toxic and more efficient than other cryoprotective agents such as DMSO, and thus has promising potential as a new, non-toxic technology for cryopreservation protocols used for clinical applications.

Time- and labor-intensive protein purification and immobilization procedures still represent a major bottleneck limiting the widespread application of enzymes in synthetic chemistry and industry. The authors exemplify a simple strategy that circumvents this problem by enabling the simultaneous purification and site-specific immobilization of Arabidopsis thaliana hydroxynitrile lyase from crude cell extracts by fusion of a family 2 carbohydrate-binding module. The resulting immobilizates enable the efficient synthesis of (R)-mandelonitrile, a chiral cyanohydrin, and facilitate easy catalyst recycling.