Immunization by a bacterial aerosol

Abstract

By manufacturing a single-particle system in two particulate forms (i.e., micrometer size and nanometer size), we have designed a bacterial vaccine form that exhibits improved efficacy of immunization. Microstructural properties are adapted to alter dispersive and aerosol properties independently. Dried "nanomicroparticle" vaccines possess two axes of nanoscale dimensions and a third axis of micrometer dimension; the last one permits effective micrometer-like physical dispersion, and the former provides alignment of the principal nanodimension particle axes with the direction of airflow. Particles formed with this combination of nano- and micrometer-scale dimensions possess a greater ability to aerosolize than particles of standard spherical isotropic shape and of similar geometric diameter. Here, we demonstrate effective application of this biomaterial by using the live attenuated tuberculosis vaccine bacille Calmette-Guérin (BCG). Prepared as a spray-dried nanomicroparticle aerosol, BCG vaccine exhibited high-efficiency delivery and peripheral lung targeting capacity from a low-cost and technically simple delivery system. Aerosol delivery of the BCG nanomicroparticle to normal guinea pigs subsequently challenged with virulent Mycobacterium tuberculosis significantly reduced bacterial burden and lung pathology both relative to untreated animals and to control animals immunized with the standard parenteral BCG.

Fig. 1.

Morphology and aerosol properties of M. smegmatis: l-leucine dry powder. (a) ESEM photograph of 95:5 M. smegmatis:l-leucine powder. The rod-like M. smegmatis appears associated with sphere-like leucine particles. (b) MMAD for various ratios of leucine to M. smegmatis. Horizontal red line indicates geometric size (d₅₀) measured at 2 bar for spray-dried 100% leucine at 2.3 μm.

Fig. 2.

Stability of the 95:5 leucine:BCG powder (cfu/mg) over the 12-month study at 4°C and 20% relative humidity.

Fig. 3.

Number of viable bacteria per milliliter of tissue homogenate (cfu/ml) at necropsy in lung and spleen tissues after bacterial challenge of animals immunized with the particles or BCG solution by the different routes (average ± SD, n = 6). The ranking of lung bacterial burden in terms of statistical significance is relative to untreated animals: none > s.c. solution 2 × 10⁶ cfu (P < 0.0001) > pulmonary particles 2 × 10⁵ cfu (P = 0.0002) > pulmonary particles 2 × 10⁶ cfu (P = 0.0003), and relative to animals immunized by the s.c. route: s.c. solution 2 × 10⁶ cfu > pulmonary particles 2 × 10⁵ cfu (P = 0.0002) > pulmonary particles 2 × 10⁶ cfu (P = 0.0036). The ranking of spleen bacterial burden in terms of statistical significance is relative to untreated animals: none > s.c. solution 2 × 10⁶ cfu (P = 0.0002) > pulmonary particles 2 × 10⁵ cfu (P < 0.0001) > pulmonary particles 2 × 10⁶ cfu (P < 0.0001), and relative to animals immunized by the s.c. route: s.c. solution 2 × 10⁶ cfu > pulmonary particles 2 × 10⁶ cfu (P < 0.0001).

Fig. 4.

Lung histopathology after bacterial challenge of animals immunized with the particles or BCG solution by the different routes. (A) Untreated controls. (B–D) Animals immunized with s.c. BCG solution at 2 × 10⁶ cfu (B), s.c. particles at 2 × 10⁶ cfu (C), and insufflated particles at 2 × 10⁶ cfu (D).