Design and Optimization of a Temperature-Stable Dry Powder BCG Vaccine.

PURPOSE: Loss of vaccine potency due to extreme temperature exposure during storage and transport remains a significant obstacle to the success of many vaccines, including the Bacille Calmette-Guérin (BCG) vaccine, the only vaccine available against Mycobacterium tuberculosis. BCG is a live, attenuated vaccine requiring refrigerated storage for viability. In this study, we formulated a temperature-stable BCG dry powder using the spray drying technique. METHODS: We employed a factorial design to optimize our formulation of stabilizing excipients that included L-leucine, bovine serum albumin, polyvinylpyrrolidone, mannitol, and trehalose. Powders were characterized for their particle size, yield, water retention and uptake, glass transition temperature, and aerosol performance. Three optimal powder carrier mixtures were selected from the factorial design for BCG incorporation based on their stability-promoting and powder flow characteristics. Vaccine powders were also assessed for BCG viability and in vivo immunogenicity after long-term storage. RESULTS: Live BCG was successfully spray-dried using the optimized carriers. Dry powder BCG showed no loss in viability (25°C, up to 60% relative humidity; RH) and ~2-log loss in viability (40°C, 75% RH) after one year of storage. The aerodynamic size of the powders was in the respirable range. Further, when healthy mice were immunized intradermally with reconstituted BCG powders (storage for 2 years), the vaccine retained its immunogenicity. CONCLUSION: We developed a spray-dried BCG vaccine that was viable and antigenic after long-term storage. To our knowledge, this is a first study to show room temperature stability of live BCG vaccine without any loss in viability for 12 months.

A respirable HPV-L2 dry-powder vaccine with GLA as amphiphilic lubricant and immune-adjuvant.

Vaccines not requiring cold-chain storage/distribution and suitable for needle-free delivery are urgently needed. Pulmonary administration is one of the most promising non-parenteral routes for vaccine delivery. Through a multi-component excipient and spray-drying approach, we engineered highly respirable dry-powder vaccine particles containing a three-fold repeated peptide epitope derived from human papillomavirus (HPV16) minor capsid protein L2 displayed on Pyrococcus furious thioredoxin as antigen. A key feature of our engineering approach was the use of the amphiphilic endotoxin derivative glucopyranosyl lipid A (GLA) as both a coating agent enhancing particle de-aggregation and respirability as well as a built-in immune-adjuvant. Following an extensive characterization of the in vitro aerodynamic performance, lung deposition was verified in vivo by intratracheal administration in mice of a vaccine powder containing a fluorescently labeled derivative of the antigen. This was followed by a short-term immunization study that highlighted the ability of the GLA-adjuvanted vaccine powder to induce an anti-L2 systemic immune response comparable to (or even better than) that of the subcutaneously administered liquid-form vaccine. Despite the very short-term immunization conditions employed for this preliminary vaccination experiment, the intratracheally administered dry-powder, but not the subcutaneously injected liquid-state, vaccine induced consistent HPV neutralizing responses. Overall, the present data provide proof-of-concept validation of a new formulation design to produce a dry-powder vaccine that may be easily transferred to other antigens.

Respiratory Immunization With a Whole Cell Inactivated Vaccine Induces Functional Mucosal Immunoglobulins Against Tuberculosis in Mice and Non-human Primates.

Vaccination through the natural route of infection represents an attractive immunization strategy in vaccinology. In the case of tuberculosis, vaccine delivery by the respiratory route has regained interest in recent years, showing efficacy in different animal models. In this context, respiratory vaccination triggers lung immunological mechanisms which are omitted when vaccines are administered by parenteral route. However, contribution of mucosal antibodies to vaccine- induced protection has been poorly studied. In the present study, we evaluated in mice and non-human primates (NHP) a novel whole cell inactivated vaccine (MTBVAC HK), by mucosal administration. MTBVAC HK given by intranasal route to BCG-primed mice substantially improved the protective efficacy conferred by subcutaneous BCG only. Interestingly, this improved protection was absent in mice lacking polymeric Ig receptor (pIgR), suggesting a crucial role of mucosal secretory immunoglobulins in protective immunity. Our study in NHP confirmed the ability of MTBVAC HK to trigger mucosal immunoglobulins. Importantly, in vitro assays demonstrated the functionality of these immunoglobulins to induce M. tuberculosis opsonization in the presence of human macrophages. Altogether, our results suggest that mucosal immunoglobulins can be induced by vaccination to improve protection against tuberculosis and therefore, they represent a promising target for next generation tuberculosis vaccines.

Comparison of adjuvants for a spray freeze-dried whole inactivated virus influenza vaccine for pulmonary administration.

Stable vaccines administered to the lungs by inhalation could circumvent many of the problems associated with current immunizations against respiratory infections. We earlier provided proof of concept in mice that pulmonary delivered whole inactivated virus (WIV) influenza vaccine formulated as a stable dry powder effectively elicits influenza-specific antibodies in lung and serum. Yet, mucosal IgA, considered particularly important for protection at the site of virus entry, was poorly induced. Here we investigate the suitability of various Toll-like receptor (TLR) ligands and the saponin-derived compound GPI-0100 to serve as adjuvant for influenza vaccine administered to the lungs as dry powder. The TLR ligands palmitoyl-3-cysteine-serine-lysine-4 (Pam3CSK4), monophosphoryl lipid A (MPLA) and CpG oligodeoxynucleotides (CpG ODN) as well as GPI-0100 tolerated the process of spray freeze-drying well. While Pam3CSK4 had no effect on systemic antibody titers, all the other adjuvants significantly increased influenza-specific serum and lung IgG titers. Yet, only GPI-0100 also enhanced mucosal IgA titers. Moreover, only GPI-0100-adjuvanted WIV provided partial protection against heterologous virus challenge. Pulmonary immunization with GPI-0100-adjuvanted vaccine did not induce an overt inflammatory response since influx of neutrophils and production of inflammatory cytokines were moderate and transient and lung histology was normal. Our results indicate that a GPI-0100-adjuvanted dry powder influenza vaccine is a safe and effective alternative to current parenteral vaccines.