A Cinderella story: how the vacuolar proteases Pep4 and Prb1 do more than cleaning up the cell’s mass degradation processes –

To mitigate the massive COVID-19 burden caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), several vaccination campaigns were initiated. We performed a single-center observational trial to monitor the mid- (3 months) and long-term (10 months) adaptive immune response and to document breakthrough infections (BTI) in healthcare workers (n = 84) upon BNT162b2 vaccination in a real-world setting. Firstly, serology was determined through immunoassays. Secondly, antibody functionality was analyzed via in vitro binding inhibition and pseudovirus neutralization and circulating receptor-binding domain (RBD)-specific B cells were assessed. Moreover, the induction of SARS-CoV-2-specific T cells was investigated by an interferon-γ release assay combined with flowcytometric profiling of activated CD4⁺ and CD8⁺ T cells. Within individuals that did not experience BTI (n = 62), vaccine-induced humoral and cellular immune responses were not correlated. Interestingly, waning over time was more pronounced within humoral compared to cellular immunity. In particular, 45 of these 62 subjects no longer displayed functional neutralization against the delta variant of concern (VoC) at long-term follow-up. Noteworthily, we reported a high incidence of symptomatic BTI cases (17.11%) caused by alpha and delta VoCs, although vaccine-induced immunity was only slightly reduced compared to subjects without BTI at mid-term follow-up.

With this study, we have validated the use of the APEX2 tag to define the localization of proteins in the model yeast S. cerevisiae . As such, FIB-SEM can identify the exact 3D location of a protein of interest in the cell with nanometer-scale resolution. Such detailed imaging could provide essential information on the elucidation of various biological processes. APEX2, which adds electron density to a fused protein of interest upon addition of the substrate DAB, originally was used in mammalian studies. With this study, we expand its use to protein localization studies in one of the most important models in molecular biology.

The live-attenuated yellow fever 17D strain is a potent vaccine and viral vector. Its manufacture is based on embryonated chicken eggs or adherent Vero cells. Both processes are unsuitable for rapid and scalable supply. Here, we introduce a high-throughput workflow to identify suspension cells that are fit for the high-yield production of live YF17D-based vaccines in an intensified upstream process. The use of an automated parallel ambr15 microbioreactor system for screening and process optimization has led to the identification of two promising cell lines (AGE1.CR.pIX and HEK_Dyn) and the establishment of optimized production conditions, which have resulted in a >100-fold increase in virus titers compared to the current state of the art using adherent Vero cells. The process can readily be scaled up from the microbioreactor scale (15 mL) to 1 L stirred tank bioreactors. The viruses produced are genetically stable and maintain their favorable safety and immunogenicity profile, as demonstrated by the absence of neurovirulence in suckling BALB/c mice and consistent seroprotection in AG129 mice. In conclusion, the presented workflow allows for the rapid establishment of a robust, scalable, and high-yield process for the production of live-attenuated orthoflavivirus vaccines, which outperforms current standards. The approach described here can serve as a model for the development of scalable processes and the optimization of yields for other virus-based vaccines that face challenges in meeting growing demands.

Background/Objectives: Chimpanzee adenoviral-vectored vaccines have proven to be both safe and effective, with a manufacturing and distribution pipeline capable of rapid global supply, as demonstrated during the COVID-19 pandemic. Yellow fever is a mosquito-borne viral hemorrhagic disease endemic in parts of Africa and Latin America, and although an effective live attenuated vaccine exists, its use is limited by safety and eligibility restrictions. Moreover, large outbreaks continue to expose critical challenges, such as an insufficient vaccine supply, reliance on fractional dosing, and slow and difficult-to-scale manufacturing processes. Here, we report the design, development and in vivo immunogenicity of multiple yellow fever virus (YFV) antigen constructs based on the pre-membrane (prM) and envelope (E) proteins—with or without the transmembrane domain (TM or ΔTM)—delivered using the ChAdOx1 adenoviral vector. Methods: Four ChAdOx1 YF vaccines were developed, and immunogenicity was evaluated. The efficacy of the full-length YF envelope vaccine was also tested in Balb/c mice. Results/Conclusions: In contrast to previously described orthoflavivirus vaccines on the same platform, the full-length antigen elicited superior immunogenicity and conferred protection against intracranial challenge with the YF17D virus in mice. Notably, this protection was comparable to that induced by the licensed YF17D vaccine, highlighting the promise of this platform as a next-generation yellow fever vaccine candidate.