A spatial and temporal assessment of nonresponse in the national forest inventory of the U.S.

In nearly all national forest inventories (NFI), some sample plots are unable to be measured such that nonresponse may be an issue of concern. Thus, it is of particular interest to understand the phenomenon in terms of current status and temporal change in nonresponse rates and the associated spatial distribution on the landscape. In the NFI of the USA, denial of access permission on privately owned forest land and hazardous conditions has led to an overall nonresponse rate of 9.8% with some areas exceeding 20% of plots being inaccessible. Further, it was found that nearly 50% of the areas studied were exhibiting increasing rates of nonresponse over time. Comparisons between response and nonresponse plots via remote sensing characteristics suggested there may be systematic differences in some parts of the country, which may cause bias in the sample and resulting estimates. The findings indicate that improved communication strategies with private landowners are needed to reduce nonresponse rates. Due to the unlikelihood of eliminating nonresponse entirely, methods to mitigate potential nonresponse bias should be considered for incorporation into the estimation of population parameters.

Stressed stability and protective efficacy of lead lyophilized formulations of ID93+GLA-SE tuberculosis vaccine.

With the recent exception of coronavirus disease 2019 (COVID-19), tuberculosis (TB) causes more deaths globally than any other infectious disease, and approximately 1/3 of the world's population is infected with Mycobacterium tuberculosis (Mtb). However, encouraging progress in TB vaccine development has been reported, with approximately 50% efficacy achieved in Phase 2b clinical testing of an adjuvanted subunit TB vaccine candidate. Nevertheless, current lead vaccine candidates require cold-chain transportation and storage. In addition to temperature stress, vaccines may be subject to several other stresses during storage and transport, including mechanical, photochemical, and oxidative stresses. Optimal formulations should enable vaccine configurations with enhanced stability and decreased sensitivity to physical and chemical stresses, thus reducing reliance on the cold chain and facilitating easier worldwide distribution. In this report, we describe the physicochemical stability performance of three lead thermostable formulations of the ID93 + GLA-SE TB vaccine candidate under various stress conditions. Moreover, we evaluate the impact of thermal stress on the protective efficacy of the vaccine formulations. We find that formulation composition impacts stressed stability performance, and our comprehensive evaluation enables selection of a lead single-vial lyophilized candidate containing the excipient trehalose and Tris buffer for advanced development.

Lyophilization Process Engineering and Thermostability of ID93 + GLA-SE, a Single-Vial Adjuvanted Subunit Tuberculosis Vaccine Candidate for Use in Clinical Studies.

Promising clinical efficacy results have generated considerable enthusiasm for the potential impact of adjuvant-containing subunit tuberculosis vaccines. The development of a thermostable tuberculosis vaccine formulation could have significant benefits on both the cost and feasibility of global vaccine distribution. The tuberculosis vaccine candidate ID93 + GLA-SE has reached Phase 2 clinical testing, demonstrating safety and immunogenicity as a two-vial point-of-care mixture. Earlier publications have detailed efforts to develop a lead candidate single-vial lyophilized thermostable ID93 + GLA-SE vaccine formulation. The present report describes the lyophilization process development and scale-up of the lead candidate thermostable ID93 + GLA-SE composition. The manufacture of three full-scale engineering batches was followed by one batch made and released under current Good Manufacturing Practices (cGMP). Up to 4.5 years of stability data were collected. The cGMP lyophilized ID93 + GLA-SE passed all manufacturing release test criteria and maintained stability for at least 3 months when stored at 37°C and up to 24 months when stored at 5°C. This work represents the first advancement of a thermostable adjuvant-containing subunit tuberculosis vaccine to clinical testing readiness.

Evaluation of the stability of a spray-dried tuberculosis vaccine candidate designed for dry powder respiratory delivery.

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 °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 °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 °C. Nanoemulsion size and oil content of the adjuvant system were maintained after one year at temperatures up to 40 °C, and the agonist content was maintained after one year at temperatures up to 25 °C. The antigen was completely degraded in the control formulation at seven months of storage at 40 °C; by contrast, 45% of the antigen was still present in the trehalose-trileucine formulation after one year of storage at 50 °C. Comparatively, the antigen was completely degraded in a liquid sample of the vaccine candidate after only one month of storage at 37 °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.

Microparticle encapsulation of a tuberculosis subunit vaccine candidate containing a nanoemulsion adjuvant via spray drying.

Spray drying is a technique that can be used to stabilize biopharmaceuticals, such as vaccines, within dry particles. Compared to liquid pharmaceutical products, dry powder has the potential to reduce costs associated with refrigerated storage and transportation. In this study, spray drying was investigated for processing an adjuvanted tuberculosis subunit vaccine, formulated as an oil-in-water nanoemulsion, into a dry powder composed of microparticles. Applying in-silico approaches to the development of formulation and processing conditions, successful encapsulation of the adjuvanted vaccine within amorphous microparticles was achieved in only one iteration, with high retention (>90%) of both the antigen and adjuvant system. Moisture-controlled stability studies on the powder were conducted over 26 months at temperatures up to 40 °C. Results showed that the powder was physically stable after 26 months of storage for all tested temperatures. Adjuvant system integrity was maintained at temperatures up to 25 °C after 26 months and after one month of storage at 40 °C. The spray-dried product demonstrated improved antigen thermostability when stored above refrigerated temperatures as compared to the liquid product. These results demonstrate the feasibility of spray drying as a method of encapsulating and stabilizing an adjuvanted vaccine.

Development of a formulation platform for a spray-dried, inhalable tuberculosis vaccine candidate.

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.

Development and Testing of a Spray-Dried Tuberculosis Vaccine Candidate in a Mouse Model.

Converting a vaccine into a thermostable dry powder is advantageous as it reduces the resource burden linked with the cold chain and provides flexibility in dosage and administration through different routes. Such a dry powder presentation may be especially useful in the development of a vaccine towards the respiratory infectious disease tuberculosis (TB). This study assesses the immunogenicity and protective efficacy of spray-dried ID93+GLA-SE, a promising TB vaccine candidate, against Mycobacterium tuberculosis (Mtb) in a murine model when administered via different routes. Four administration routes for the spray-dried ID93+GLA-SE were evaluated along with relevant controls-1) reconstitution and intramuscular injection, 2) reconstitution and intranasal delivery, 3) nasal dry powder delivery via inhalation, and 4) pulmonary dry powder delivery via inhalation. Dry powder intranasal and pulmonary delivery was achieved using a custom nose-only inhalation device, and optimization using representative vaccine-free powder demonstrated that approximately 10 and 44% of the maximum possible delivered dose would be delivered for intranasal delivery and pulmonary delivery, respectively. Spray-dried powder was engineered according to the different administration routes including maintaining approximately equivalent delivered doses of ID93 and GLA. Vaccine properties of the different spray-dried lots were assessed for quality control in terms of nanoemulsion droplet diameter, polydispersity index, adjuvant content, and antigen content. Our results using the Mtb mouse challenge model show that both intranasal reconstituted vaccine delivery as well as pulmonary dry powder vaccine delivery resulted in Mtb control in infected mice comparable to traditional intramuscular delivery. Improved protection in these two vaccinated groups over their respective control groups coincided with the presence of cytokine-producing T cell responses. In summary, our results provide novel vaccine formulations and delivery routes that can be harnessed to provide protection against Mtb infection.