Aneesh Thakur is currently an Assistant Professor at University of Copenhagen, Department of Pharmacy, where he is working in the research group of Vaccine Design and Delivery. Prior to this, he was an Assistant Professor in the Department of Veterinary Microbiology at CSK Himachal Pradesh Agricultural University (2013-2016). He carried out postdoctoral research positions at University of Copenhagen (2016-2020) and Technical University of Denmark (2012-2013). He was a research scientist in the Tuberculosis Aerosol Challenge Facility at International Centre for Genetic Engineering and Biotechnology (2007-2009). He obtained his undergraduate (DVM) and master degree (MVSc) in Veterinary Microbiology and Immunology from CSK Himachal Pradesh Agricultural University (2007) and his PhD in Immunology from Technical University of Denmark (2013). His research is highly multidisciplinary and involves (i) the development of next-generation nanoparticle-based formulation strategies to enhance efficacy of subunit and nucleic acid-based vaccines and therapies and (ii) strategies for controlled modulation of pulmonary T cell immunity using adjuvants. He investigates topics at the interface of immunology, drug delivery, and nanotechnology to pursue novel vaccine designs for translational applications against infectious diseases and cancer.

Project summary

Tuberculosis (TB) caused by Mycobacterium tuberculosis (Mtb) is one of the top 10 causes of death worldwide, despite decades-long use of the Bacillus Calmette–Guérin (BCG) vaccine. Hence, a new vaccine is urgently needed to control this TB pandemic. TB typically affects the lungs, and the transmission of Mtb occurs primarily through inhalation of droplets containing bacilli from infected individuals. As the respiratory tract is the natural route of Mtb infection, mucosal immunization in the airways with highly purified recombinant Mtb antigen(s) is a very promising strategy for the development of novel TB vaccines. We have made a major breakthrough by identifying the molecular components and a formulation strategy for a thermostable, dry powder-based inhalable TB subunit vaccine candidate, H56/CAF01. The overall goal of this project is to design a vaccine and a vaccination strategy that together induces strong H56-specific humoral and cellular immunity in the lungs and systemically, which is independent of cold-chain. We hypothesize that a vaccine, which elicits strong anti-TB immunity in the lungs, can induce rapid clearance of Mtb-infected cells very early at the site of infection and hence enhance protection. However, vaccine delivery in lungs is associated with many challenges such as: (1) design of inhalable, dry powder-based vaccine formulations suitable for inhalation, (2) the specific aerodynamic size requirement of particles for deep lung deposition (1.5-4 µm), and (3) lack of devices for preclinical administration of powders. Here, we aim to target the respiratory mucosa for immunization using PreciseInhale® equipment to induce strong immune responses at mucosal sites and systemically. We hypothesize that by using a combination of dry powder technology, prime-boost mucosal immunization, and precise dosing of aerosols, we will: (1) enhance delivery of the vaccine to the deep lungs and draining lymph nodes that will facilitate antigen uptake by dendritic cells, (2) generate and maintain local and systemic memory T cells and antibodies, and (3) eliminate the need for expensive cold-chain. To test our hypothesis, we will use a heterologous prime/boost immunization regimen consisting of parenteral prime vaccination with BCG, followed by boosting with dry powder-based CAF01-adjuvanted H56 in the airways. Our recent data show that mucosal boost immunization in the lungs robustly boost parenterally primed H56-specific CD4+ Th1/Th17 and IgA responses. We have also identified spray drying conditions and stabilizing excipients, which are optimal for manufacturing of the dry powder-based vaccine. In this project, we will identify the most efficient dry powder vaccine formulation and mucosal immunization regimen for enhancing vaccine-mediated protection against TB. A thermostable and self-administrable TB vaccine that can be mass distributed, e.g., to the developing world might in the long-term have a tremendous impact on global health.

Career Development Award Proposal

My current postdoctoral research work in the lab of Prof. Camilla Foged at University of Copenhagen focuses on image-guided design of a thermostable and self-administrable subunit vaccine against TB in humans. I have developed a novel immunization strategy and novel dual-isotope (111In/67Ga) radiolabeling of the candidate TB subunit vaccine, H56/CAF01 and characterized the immunogenicity and SPECT/CT-based biodistribution of the vaccine following pulmonary delivery in mice (Front. Immunol., 2018). I also engineered a novel gadoteridol-loaded liposomal CAF01 formulation using a quality-by-design (QbD) approach for studying pulmonary delivery of this vaccine in vivo by contrast-enhanced magnetic resonance imaging (MRI). My results show that QbD-optimized CAF01-gadoteridol formulation is safe and stable in vivo and enhances MRI signal by 1.5 fold and changes T1 relaxation by ~ 30% in mice lungs (Mol. Pharm., 2019). I have analyzed the uptake of H56/CAF01 vaccine by pulmonary antigen presenting cell (APC) populations and trafficking to regional lymph nodes in combination with Mass Spectrometry (MS) imaging (Front. Immunol., in press). The results show that the prime and mucosal boost immunization of the H56/CAF01 vaccine elicits a distinct innate myeloid response and activation of APCs (Front. Immunol., in press). The MS imaging data show that the H56/CAF01 vaccine is safe following pulmonary delivery as there is no upregulation of phospholipid lysophosphatidylcholine (16:0) and sphingolipid ceramide, which are known biomarkers of inflammation and local tissue damage (Front. Immunol., in press). I have also made considerable progress in my efforts to design a dry powder H56/CAF01 vaccine formulation. Using spray drying, we have previously produced dry powder CAF01 with preserved adjuvanticity (J. Control Release, 2013) and I have recently shown that spray-dried, reconstituted H56/CAF01 induces Th1/Th17 responses in mice (Vaccine, 2018). Using a QbD approach, I have now formulated dry powder CAF01 in the presence of trehalose and dextran as the stabilizing excipients and observed optimal aerosol performance (flyability and aerosol yield) of the 2 µm sized powders by using the PreciseInhale® equipment (unpublished data). Presently, I am working to optimize these dry powder vaccine formulations and to study their aerosol performance, exposure, and in vivo pulmonary delivery through PreciseInhale® system. In a side project in the lab, I standardized lipopolysaccharide-induced mice model of inflammation. We generated proof-of-concept that lipidoid-polymer-hybrid nanoparticles (LPNs) loaded with siRNA targeted against the pro-inflammatory cytokine tumor necrosis factor (TNF-α) can reduce experimental inflammation (unpublished data). Using a QbD approach, we identified the factors of importance for spray drying of TNF-α siRNA-loaded LPNs for inhalation (Pharm. Res., 2019). We found that the dispersed micro-embedded LPNs had preserved physicochemical characteristics as well as in vitro siRNA release profile and gene silencing, as compared to the non-spray-dried LPNs (Pharm. Res., 2019). We are continuing our efforts to evaluate the aerosol performance of these powders in PreciseInhale® with the ultimate goal to demonstrate the siRNA-mediated gene silencing of dry powder formulations in animal models of chronic obstructive pulmonary disease.

My motivation for applying to the DDL Career Development Grant proposal is to make use of the experience developed in the area of drug delivery to the lungs especially the delivery of vaccines. I aim to use this grant for optimizing pulmonary delivery of the dry powder-based H56/CAF01 vaccine using dry powder technology, prime-boost immunization, and powder administration with PreciseInhale® system. Specifically, my objectives are:
1. To optimize high-quality aerosol generation from H56/CAF01 dry powder and evaluate their detailed aerosol characteristics (particle size, flyability, density, shape, solid form), which are important before in vivo exposure.
2. To optimize the in vivo exposure of dry powder vaccine formulations in mice using the PreciseInhale® system and to evaluate the immunogenicity and safety of H56 in combination with the CAF01 adjuvant using homologous parenteral and mucosal prime‐boost immunization strategies.
3. To conduct an aerosol challenge study with Mtb in mice to measure the protective efficacy and safety of the dry powder vaccine after priming with BCG, using the best immunization strategy and adjuvant combination identified under objective 2.

I trust that the support from DDL Career Development Grant will allow me to generate a novel data on the in vivo immunogenicity and protective efficacy of the dry powder H56/CAF01 vaccine using the PreciseInhale® equipment. I expect to present this data at one of the DDL conferences. This interdisciplinary project at the interface between basic and applied research has exciting translational potential. If the results are promising, the optimized vaccine formulation will be scaled up, and preclinical toxicology and stability studies will be performed. Finally, the optimized dry powder H56/CAF01 formulation and immunization strategy, as determined by the iterative studies in animal models, will be tested in a non-human primate Mtb infection model. These studies will provide crucial pre-clinical data for translating the dry powder H56/CAF01 vaccine forward into human clinical trials as an improved and cold-chain independent TB vaccine candidate.

Following this project grant, I aim to not only expand my current research but also to broaden my scientific horizons in the field of drug delivery to the lungs in general and dry powder vaccines in particular. As described, I have experience in producing dry powder-based subunit vaccine and siRNA formulations. However, we have to date been limited in our preclinical studies by the lack of a suitable device for powder administration to mice. In this project proposal, the application of PreciseInhale® system will allow us the precision dosing of mice lungs with H56/CAF01 vaccine and evaluation of the dry powder-based vaccine immunogenicity and efficacy. These studies can be translated for testing the prophylactic and therapeutic efficacy of novel dry powder-based subunit vaccine and siRNA formulations for other respiratory infections, inflammatory conditions, and cancer. For example, using the PreciseInhale® system in the near future, I plan to evaluate the in vivo therapeutic effect of dry powder TNF-α siRNA-loaded LPNs against chronic obstructive pulmonary disease (COPD). The ability to evaluate the therapeutic effect of dry powder-based biologics will potentially benefit our research group in vaccine design and delivery at the University of Copenhagen.

Finally, through sharing my research results in one of the DDL conferences, I expect to get feedback for my results so as to improve my research. It will also give me an excellent opportunity to expand my scientific network in the field of drug delivery to the lungs and possibilities of future research collaborations and knowledge transfer. Therefore, I would highly appreciate the support under the DDL Career Development Grant for my personal and professional development.