Dry-powder pulmonary insufﬂation in the mouse for application
to vaccine or drug studies
, Cassandra L. Krone
, Shelley Dickerson
, Elizabeth Howerth
Willem Andreas Germishuizen
, Yun-Ling Wong
, David Edwards
Barry R. Bloom
, Mary K. Hondalus
Department of Infectious Diseases, University of Georgia, College of Veterinary Medicine, Athens, GA 30602, USA
Department of Immunology and Infectious Diseases, Harvard School of Public Health, Boston, MA 02115, USA
Medicine in Need South Africa, Medical Research Council Building Number 1, Soutpansberg Road, Pretoria 0002, South Africa
Harvard University School of Engineering and Applied Sciences, Cambridge, MA 02138, USA
Received 15 March 2009
Received in revised form
22 June 2009
Accepted 6 July 2009
Pulmonary delivery of substances in small animal models is often useful for experimental testing of
various vaccine and drug candidates. One of the most challenging elements to such protocols is the
efﬁcient disposition of test materials in the lungs of mice. Herein we detail a means to deliver dry
powders of an inhalant live-attenuated Mycobacterium bovis Bacille Calmette-Guerin (BCG) vaccine
against Mycobacterium tuberculosis to the lungs of mice. The direct delivery methodology is quick, safe,
and allows for repeated pulmonary insufﬂation of substances if boosting is desired. This model system
could be easily adapted for use with other dry-powder vaccine and drug candidates.
Ó 2009 Elsevier Ltd. All rights reserved.
The increasing attention now directed towards pulmonary
delivery of vaccines centers around their abilities to recruit the local
immune responses of the bronchopulmonary mucosa in addition to
the systemic immune response.
Because drug and vaccine
candidates require extensive testing in animal models prior to their
approval for use in humans, experiments must mimic closely the
intended formulation and delivery method of the ﬁnal product. Our
interest in developing a means to deliver dry powders to the lungs
of mice arises from our long-term goal of creating an inhaled
tuberculosis vaccine that obviates the need for needle delivery for
use in people. The current and only tuberculosis vaccine, Myco-
bacterium bovis BCG, is delivered by intradermal injection. BCG is
the most widely used vaccine in the world and its safety in most
populations is well documented.
Unfortunately however, the
efﬁcacy of BCG to prevent pulmonary tuberculosis in adults varies
widely between countries and trials.
Thus, there is intense
interest in developing a better tuberculosis vaccine.
The mouse is a well established and often used model system for
assessing the immunogenicity and efﬁcacy of vaccines, including
tuberculosis candidate vaccines. Using the latter model system,
several groups have recently demonstrated enhanced efﬁcacy of
BCG when administered via the intranasal route,
the notion that the route of immunization is important. To
administer a spray-dried BCG microparticle powder formulation
via the respiratory route to mice, we used tracheal intubation with
insufﬂation which provided a means to directly deposit the vaccine
in the lungs thereby insuring accuracy of dosing and overcoming
the potential pharyngeal and upper airway losses inherent in
intranasal delivery. Published protocols for intubation of the mouse
trachea often involve blind intubation, invasive surgery or the
requirement of the investigator to ﬁrst fashion the equipment
necessary for the procedure.
Blind intubation or invasive
surgical procedures allowing tracheal access require extensive skill
and time of the investigator, and increase chances of harm to the
animal. In addition, the recovery time and risks to the animal
following surgical procedures create limitations in studies that
require multiple dosing via the respiratory route. Though home-
made equipment may prove cost-effective, commercially available
equipment adapted for the intubation procedure allows for greater
accuracy and reproducibility of the procedure itself, and avoids
losses in time and wasted materials during the equipment
Corresponding author. Tel.: þ1 706 542 5778; fax: þ1 706 542 5771.
E-mail address: email@example.com (M.K. Hondalus).
Authors contributed equally to the work.
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journal homepage: http://intl.elsevierhealth.com/journals/tube
1472-9792/$ – see front matter Ó 2009 Elsevier Ltd. All rights reserved.
Tuberculosis 89 (2009) 371–377