The global tuberculosis (TB) epidemic affected 10 million people and caused 1.3 million deaths in 2022 alone. Multidrug-resistant TB is successfully treated in less than 60% of cases by long, expensive, and aggressive treatments. Mycobacteriophages, viruses that can infect bacteria such as Mycobacterium tuberculosis-the species responsible for TB-have the potential to redefine TB prevention and treatments. However, the development of phage-based products necessitates the assessment of numerous parameters, including virulence and adsorption, to ensure their performance and quality. In this work, we characterized the virulence of three different mycobacteriophages (Fionnbharth, Muddy, and D29), alone and as cocktails, against a TB model host (Mycobacterium smegmatis) under planktonic and early-stage biofilm growth conditions. Phage D29 and cocktails containing D29 had the highest virulence under all conditions. Interestingly, phages Fionnbharth and Muddy and their combination showed higher virulence against early-stage biofilm than against the planktonic phenotype. Adsorption assays indicated that all three phages had lower adsorption efficiencies on the early-stage biofilm phenotype than on the planktonic one, suggesting a reduced availability of receptors in the former. Given that, despite these lower adsorption efficiencies, the virulence of the phages and phage cocktails was either unchanged or higher against the early-stage biofilm, this phenotype must display properties that are favorable to other steps of the infection process. These results inform us on the dynamics of mycobacteriophage infections, both alone and in cocktail formulations, under different host growth conditions, serving as a basis for the development of phage products targeting mycobacteria biofilms. IMPORTANCE:This study provides a systematic investigation of the virulence of three mycobacteriophages, Fionnbharth, Muddy, and D29, and their combinations as cocktails against Mycobacterium smegmatis. We also included considerations on the hydrodynamic conditions (shaking and not shaking) and host phenotype (planktonic and early-onset biofilm cultures) during the infection process and adsorption of the phage to the host. We showed that virulence was strongly affected by phenotype and that higher virulence shown against the early-onset biofilm phenotype was not linked to faster adsorption to the host. We also showed that phage D29 and cocktails containing this phage had the highest virulence. These results are important as they provide a framework for a better evaluation and development of phage-based treatment against mycobacterial infections.

Particle engineering via spray drying was used to develop a dry powder presentation of an adjuvanted tuberculosis vaccine candidate. This presentation utilizing a trileucine-trehalose excipient system was designed to be both thermostable and suitable for respiratory delivery. The stability of the spray-dried vaccine powder was assessed over one year at various storage temperatures (-20, 5, 25, 40, 50 degrees C) in terms of powder stability, adjuvant stability, and antigen stability. A formulation without trileucine was included as a control. The results showed that the interior particle structure and exterior particle morphology of the powder was maintained for one year at 40 degrees C, while the control case exhibited a small extent of particle fusing under the same storage conditions. Moisture content was maintained, and powder solid state remained amorphous for all storage temperatures. Aerosol performance was assessed with a commercial dry powder inhaler in combination with a human mouth-throat model. The emitted dose and lung dose were maintained for all samples after one year at temperatures up to 40 degrees C. Nanoemulsion size and oil content of the adjuvant system were maintained after one year at temperatures up to 40 degrees C, and the agonist content was maintained after one year at temperatures up to 25 degrees C. The antigen was completely degraded in the control formulation at seven months of storage at 40 degrees C; by contrast, 45% of the antigen was still present in the trehalose-trileucine formulation after one year of storage at 50 degrees C. Comparatively, the antigen was completely degraded in a liquid sample of the vaccine candidate after only one month of storage at 37 degrees C. The spray-dried trehalose-trileucine vaccine powder clearly maintained its inhalable properties after one year's storage at high temperatures and improved overall thermostability of the vaccine. (c) 2021 Elsevier Ltd. All rights reserved.

Protection against primarily respiratory infectious diseases, such as tuberculosis (TB), can likely be enhanced through mucosal immunization induced by direct delivery of vaccines to the nose or lungs. A thermostable inhalable dry powder vaccine offers further advantages, such as independence from the cold chain. In this study, we investigate the formulation for a stable, inhalable dry powder version of ID93 + GLA-SE, an adjuvanted subunit TB vaccine candidate, containing recombinant fusion protein ID93 and glucopyranosyl lipid A (GLA) in a squalene emulsion (SE) as an adjuvant system, via spray drying. The addition of leucine (20% w/w), pullulan (10%, 20% w/w), and trileucine (3%, 6% w/w) as dispersibility enhancers was investigated with trehalose as a stabilizing agent. Particle morphology and solid state, nanoemulsion droplet size, squalene and GLA content, ID93 presence, and aerosol performance were assessed for each formulation. The results showed that the addition of leucine improved aerosol performance, but increased aggregation of the emulsion droplets was demonstrated on reconstitution. Addition of pullulan preserved emulsion droplet size; however, the antigen could not be detected after reconstitution. The trehalose-trileucine excipient formulations successfully stabilized the adjuvant system, with evidence indicating retention of the antigen, in an inhalable dry powder format suitable for lung delivery.