Joana Pinto da Silva

In vivo assessment of bacterial lysate-loaded locust bean gum microparticles aimed at respiratory disease prevention upon inhalation

Background and Significance

Respiratory infections continue to pose significant health challenges globally, with diseases such as Covid-19, pneumonia, and COPD-related infections causing substantial morbidity and mortality [1]. Although bacterial lysates have been used as immunomodulators for preventing respiratory tract infections due to their ability to stimulate the immune system, their clinical significance remains uncertain [2]. Contradictory trial results and the European Medicines Agency's (EMA) recent recommendations limit their usage primarily to cases of recurrent respiratory infections [3]. To overcome the posed limitations of lack of efficacy in respiratory infections, we propose the development of an innovative inhalable formulation of bacterial lysates to provide enhanced protection and improve the therapeutic outcomes.

Bacterial lysates (BL) consist of extracts of pathogenic bacteria, such as S. aureus, S. pyogenes, and K. pneumoniae, which are responsible for various respiratory infections [2]. Currently, they are predominantly administered orally; however, their low efficacy in inducing mucosal immunity limits their protective effect against lung infections. An alternative approach involves delivering the antigens directly to the lungs via inhalation, allowing for targeted interaction with mucosa-associated lymphoid tissues (MALT) in the bronchial zone (BALT), thereby enhancing local immune responses [4].
Our proposed solution leverages locust bean gum (LBG), a galactomannan known for its high affinity towards macrophages, as an antigen carrier for the inhalable formulation [5,6]. By combining BL with LBG, we developed an innovative approach for the inhalation of BL, with the intention to elicit an immune response in the site of entry of pathogens.

Output summary

The results obtained thus far indicate that BL were successfully microencapsulated by spray-drying using LBG as the matrix material, achieving an association efficiency of 81%. MP exhibited suitable morphology, and those with an LBG:BL ratio of 10:0.2 (w/w) demonstrated optimal aerodynamic properties for lung deposition (MMAD of 4.6 µm and FPF of 29%). Serum agglutination and immunochromatography assays confirmed the presence of native antigens in BL-loaded MP formulations, indicating that spray-drying did not compromise antigen integrity. In vitro release studies showed gradual BL release, reaching approximately 80% within 6 h. Mucoadhesive properties of LBG-based MP were demonstrated, potentially prolonging lung residence time and, potentially enhancing antigen uptake by antigen-presenting cells (APCs), improving the immune response. Furthermore, the formulated inhalable MP demonstrated no adverse effects on respiratory cell viability, suggesting a favorable safety profile. This career development proposal is particularly dedicated to the last phase of the project, which is expected to comprise the in vivo proof of concept.

Work performed

Thus far, extensive work has been performed and presented in several conferences including DDL, with a poster presentation at the DDL2022 [7], and an oral presentation in last year’s DDL [8]. Those communications showcased the development and optimization of inhalable LBG-based microparticles loaded with BL. MP characterization studies evaluated aerodynamic properties for lung deposition, as well as MP morphology and mucoadhesive properties. Furthermore, the performed analysis included evaluation of antigen integrity after processing and antigen release profile.
Current studies are evaluating MP safety and antigen presenting cell targeting. This includes analysing the cytocompatibility of MP with respiratory cell lines A549, Calu-3 and THP-1, as well as in vitro uptake of MP by antigen presenting cell models such as differentiated THP-1 cells (dTHP-1). The results already obtained provide a positive outlook and indicate the potential success of the application. However, to provide a proof-of-concept , in vivo studies are now required.

Proposal goal

Evaluate in vivo protective efficacy by using an animal model to compare the elicited immune response (mucosal and systemic) and the real protection against respiratory challenge following inhaled vs. oral immunization with BL-loaded LBG MP.

Detailed Description of Research Design

Wistar rats will be used in this study, performed in collaboration with Dr. Manuela Gaspar from iMed.ULisboa and following ethical guidelines. Based on power analyses derived from comparable studies, seven groups consisting of six rats per group will participate in the experiment [9, 10]. The studies will follow a rigorous experimental design, including multiple study groups:
Group 1 (Test - Inhalation): Rats in this group will receive a first dose of BL-loaded LBG MP by inhalation using a nose-only inhalation device optimized for the assay [6] on day 0 and a booster dose on day 14.
Group 2 (Control - Naïve): These rats will serve as naïve controls and will not undergo any treatment.
Group 3 (Positive Control - Oral): Rats in this group will receive commercially available BL orally (by gavage) on days 0 and 14, serving as a reference standard for comparative purposes.
Group 4 (Control – LBG MP): Rats in this group will receive LBG MP formulated without BL on days 0 and 14, to understand the effects caused by the empty MP.
Immune Response Evaluation:
At day 28, all rats will be euthanized using CO2 inhalation. Serum will be collected to determine the levels of IgG1 and IgG2a against Streptococcus pyogenes M protein using ELISA tests employing previously isolated antigens. Bronchoalveolar lavage (BAL) procedures will also be conducted to assess the lung mucosal immune response. Specific IgA concentrations will be measured in BAL fluids, while cell counts for macrophages, neutrophils, and eosinophils will be determined.
Challenge Experiment:
Groups 5 (Test - Inhalation + Challenge) and 6 (Positive Control - Oral + Challenge) will be challenged with a lethal dose of Streptococcus pyogenes on day 28 [11], after receiving the vaccine doses in days 0 and 14 as for groups 1 and 3. Rat survival rates will be recorded and compared to observe any protective effects conferred by the pre-existing immunity developed through either inhalation or oral administration. Group 7 (Control - Infected but Not Immunized) will serve as a negative control, with death expected within two days post infection.
One week after the challenge, surviving rats from Groups 5 and 6 will be sacrificed, and lung tissue samples will be taken for histopathology examination and bacterial counts in lung homogenates [12]. Throughout the study, animal weights will be recorded weekly, and signs of pain, distress, or morbidity will be closely monitored.

Research team and facilities

The proposed studies will be conducted at iMed.ULisboa (Faculty of Pharmacy, University of Lisbon) in collaboration with Dr. Manuela Gaspar, who has certification from Direção Geral de Alimentação e Veterinária (DGAV), and facilities for conducting animal experimentation studies according to European regulations. This collaborative environment will foster the applicant’s development as a scientist and provide essential networking opportunities.

Expected Outcomes and Impact

This project aims to demonstrate the feasibility and advantages of a novel inhaled bacterial lysate immunization strategy. Success will support further pre-clinical studies and offers potential for translation into improved prevention for respiratory infections. The research findings are to be disseminated through presentations at scientific conferences and meetings. Emphasis will be placed on international meetings relevant to pulmonary drug delivery and vaccine development. Specifically, we intend to present the obtained results at the Drug Delivery to the Lungs conference.

Applicant Career Development

This project provides interdisciplinary training in formulation development, biological evaluation, and immunology, enhancing the applicant's skills as a pharmaceutical scientist and vaccine development researcher. Throughout the course of the in vivo study, the applicant will acquire diverse skillsets spanning other aspects of the development of a pharmaceutical formulation not addressed so far, including animal experimentation, immunology, and histopathology, with the corresponding data analysis and scientific communication. Particularly, the understanding of immunology will expand exponentially with the analyzes of systemic and mucosal immune responses, performance of bronchoalveolar lavage procedures and ELISA assays, and dissect cellular responses. Moreover, the applicant will hone crucial skills in ethical animal handling, model selection, immunization protocol execution (both inhalation and oral methods), challenge experiments, and operating nose-only inhalation device.
Upon completion of this project, the applicant will emerge as a versatile pharmaceutical scientist, uniquely positioned to excel in various roles related to vaccine development and mucosal immunology. Some possible career trajectories would include becoming a researcher in respiratory drug delivery, working in academia or industry on the development of novel inhalation therapies. There would be opportunities to become a formulation scientist, specializing in the creation and optimization of inhalable formulations for a variety of respiratory conditions. An additional path would be exploring respiratory therapy research, independently investigating mechanisms of the respiratory system, and designing innovative inhalation-based treatment strategies. With the acquisition of some particular skills in the field of immunization, the applicant might also seek opportunities in the development of inhaled vaccines.

References:

[1] Forum of International Respiratory Societies. The global impact of respiratory disease. Third Edition. European Respiratory Society, 2021. Accessed 22 September, 2021. firsnet.org/images/publications/FIRS_Master_09202021.pdf
[2] S.C. Kearney, M. Dziekiewicz, W. Feleszko, Immunoregulatory and immunostimulatory responses of bacterial lysates in respiratory infections and asthma, Annals of Allergy, Asthma & Immunology, 114 (2015) 364-369.
[3] EMA, Bacterial lysate medicines for respiratory conditions to be used only for prevention of recurrent infections, in, European Medicines Agency, 2019.
[4] M. Hellfritzsch, R. Scherließ, Mucosal vaccination via the respiratory tract, Pharmaceutics, 11 (2019) 375.
[5] S. Rodrigues, A. Alves, J. Cavaco, J. Pontes, F. Guerreiro, A. Rosa da Costa, F. Buttini, A. Grenha, Dual antibiotherapy of tuberculosis mediated by inhalable locust bean gum microparticles, International Journal of Pharmaceutics, 529 (2017) 433-441.
[6] A. Grenha, A.D. Alves, F. Guerreiro, J. Pinho, S. Simões, A.J. Almeida, M.M. Gaspar, Inhalable locust bean gum microparticles co-associating isoniazid and rifabutin: Therapeutic assessment in a murine model of tuberculosis infection, European Journal of Pharmaceutics and Biopharmaceutics, 147 (2020) 38-44.
[7] J. Pinto-da-Silva, J. Cruz, A. Grenha, Locust bean gum microparticles as carriers for lung delivery of bacterial lysates, Drug Delivery to the Lungs Conference 2022, Edinburgh, Scotland, 2022, pp. 544-547.
[8] J. Pinto-da-Silva, M. Berzosa, A. Delgado-López, C. Gamazo, A. Grenha, Repurposing bacterial lysates: an inhalation approach for respiratory infection management, Drug Delivery to the Lungs Conference 2023, Edinburgh, Scotland, 2023, pp. 106-109.
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[11] S. Roberts, J.R. Scott, L.K. Husmann, C.A. Zurawski, Murine models of Streptococcus pyogenes infection, Current protocols in microbiology, Chapter 9 (2006) Unit 9D.5.
[12] D. Sanchez-Guzman, P. Le Guen, B. Villeret, N. Sola, R. Le Borgne, A. Guyard, A. Kemmel, B. Crestani, J.-M. Sallenave, I. Garcia-Verdugo, Silver nanoparticle-adjuvanted vaccine protects against lethal influenza infection through inducing BALT and IgA-mediated mucosal immunity, Biomaterials, 217 (2019) 119308.