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.

Reliable and fast viral detection and quantification protocols are a requirement for the advance of basic research and clinical approaches with wild type or recombinant viruses. However, available cell-based assays are either timeconsuming or require labeled viral particles, which may alter virus biology or pose safety issues in clinical applications. Since adenoviruses constitute a major healthcare burden but also, when engineered, widely used vectors in vaccination and gene and oncolytic therapies, herein we developed a genetically encoded switch-on fluorescent biosensor consisting of a cyclized Green fluorescent proteincVisensorwith an adenoviral protease cleavable site as a switch. After initial sensor optimization (35% increase in performance), whole-cell biosensors were establishedby stably expressing cVisensor in mammalian cellsand used for live-cell monitoring of adenovirus infection as the intracellular biosensor is specifically activated by the viral protease. A rapid flow cytometry-based bioassay using cVisensor cells was established 48 h postinfection, showing an estimated limit of detection of 10 5 infectious particles/mL, in-line with previously reported flow cytometry assays requiring labeled virus, and significantly faster than standard plaque-forming assays requiring up to 14 days. cVisensor was also successfully applied in the detection of HIV-1 protease activity, validating its wider potential for the detection of other viruses. Overall, this work presents a fast and easy method for detection and quantification of label-free infectious virus, allowing the establishment of new biosensing platforms for basic research in virology and biotechnological applications of recombinant virus biopharmaceuticals.

Lentiviral vectors (LVs) are excellent tools for gene transfer into mammalian cells. It is noteworthy that the first gene therapy treatment using LVs was approved for commercialization in 2017. The G glycoprotein from rhabdovirus vesicular stomatitis virus (VSV-G) is the glycoprotein most used to pseudotype LVs, due to its high efficiency in transducing several cell types and its resistance to viral vector purification and storage conditions. However, VSV-G expression induces cytotoxicity, which limits LV production to short periods. As alternative to VSV-G, γ-retrovirus glycoproteins (4070A derived, GaLV derived, and RD114 derived) have been used to pseudotype both γ-retroviral vectors (RVs) and LVs. These glycoproteins do not induce cytotoxicity, allowing the development of stable LV producer cells. Additionally, these LV pseudotypes present higher transduction efficiencies of hematopoietic stem cells when compared to VSV-G. Here, new 4070A-, RD114-TR-, and GaLV-TR-derived glycoproteins were developed with the aim of improving its cytoplasmic tail R-peptide cleavage and thus increase LV infectious titers. The new glycoproteins were tested in transient LV production using the wild-type or the less active T26S HIV-1 protease. The GaLV-TR-derived glycoproteins were able to overcome titer differences observed between LV production using wild-type and T26S protease. Additionally, these glycoproteins were even able to increase LV titers, evidencing its potential as an alternative glycoprotein to pseudotype LVs.