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Background The isolation of recombinant antibody fragments from displayed libraries represents a powerful alternative to the generation of IgGs using hybridoma technology. The selected antibody fragments can then be easily engineered into (multi)-tagged constructs of variable mass and complexity as well as reconstituted into Camelidae IgG-like molecules when expressed fused to Fc domains. Nevertheless, all antibody constructs depend on an oxidizing environment for correct folding and consequently still belong to the proteins difficult to express in bacteria. In such organisms they are mostly produced at low yields in the periplasmic space. Results We demonstrate that fusion constructs of recombinant antibodies in combination with multiple tags can be produced at high yields and totally functional in the cytoplasm of bacteria expressing sulfhydryl oxidase. The method was applied to structurally demanding molecules such as VHHs fused to SNAP and Fc domains and was validated using the antibody-derived reagents in a variety of immune techniques (FACS, ELISA, WB, IP, SPR, and IF). Conclusions The collected data demonstrate the feasibility of a method that establishes a totally new approach for producing rapidly and inexpensively functional Camelidae IgG-like monoclonal antibodies and antibody-based reagents containing multiple disulfide bonds and suitable for both basic research and clinical applications.

The development of safe and effective vaccines in a record time after the emergence of the Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) is a remarkable achievement, partly based on the experience gained from multiple viral outbreaks in the past decades. However, the Coronavirus Disease 2019 (COVID-19) crisis also revealed weaknesses in the global pandemic response and large gaps that remain in our knowledge of the biology of coronaviruses (CoVs) and influenza viruses, the 2 major respiratory viruses with pandemic potential. Here, we review current knowns and unknowns of influenza viruses and CoVs, and we highlight common research challenges they pose in 3 areas: the mechanisms of viral emergence and adaptation to humans, the physiological and molecular determinants of disease severity, and the development of control strategies. We outline multidisciplinary approaches and technological innovations that need to be harnessed in order to improve preparedeness to the next pandemic.

Second messenger signalling pathways are known to play a fundamental role in governing bacterial physiology and mediating rapid adaptive responses to stimuli. Despite the extensive characterisation of second messenger signalling systems such as the cyclic di-GMP (c-di-GMP) signalling system in pathogens, relatively little is known about the role of these pathways in the priority pathogen Acinetobacter baumannii . To address this, we carried out a comprehensive exploration of c-di-GMP signalling across multiple modern A. baumannii clinical isolates. We elucidate this second messenger’s regulon and its role in biofilm formation and other virulence-associated behaviours. Furthermore, we demonstrate that specific enzymes controlling c-di-GMP levels are associated with specific international clones, with PdeD being identified as the primary functional phosphodiesterase in the worldwide prevalent International Clone I. Further characterisation of PdeD revealed it as a key regulatory node, controlling host colonisation in a murine model and persistence in the hospital environment. This indicates that the physiological control exerted by PdeD represented a distinctive advantage for the global dissemination of this lineage. Overall, this work uncovers the core regulatory role of c-di-GMP in underpinning the recalcitrant pathobiology of A. baumannii and also reveals PdeD as a potential novel therapeutic target.