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Author's personal copy Vaccine 29 (2011) 905–912
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Dry powder measles vaccine: Particle deposition, virus replication, and immune response in cotton rats following inhalation Kevin O. Kisich a,∗ , Michael P. Higgins b , Insun Park a , Stephen P. Cape c , Lowry Lindsay d , David J. Bennett f , Scott Winston f , Jim Searles f , Robert E. Sievers c,e,f a
Department of Immunology, National Jewish Medical and Research Center, 1400 Jackson Street, Denver, CO 80206, United States Department of Medicine, National Jewish Medical and Research Center, 1400 Jackson Street, Denver, CO 80206, United States Cooperative Institute for Research in Environmental Sciences (CIRES), 216 UCB, United States d Department of Civil, Environmental and Architectural Engineering, 428 UCB, United States e Department of Chemistry and Biochemistry, 214 UCB, University of Colorado, Boulder, CO 80309, United States f Aktiv-Dry LLC, 6060 Spine Road, Boulder, CO 80301, United States b c
a r t i c l e
i n f o
Article history: Received 23 February 2010 Received in revised form 5 October 2010 Accepted 10 October 2010 Available online 23 October 2010 Keywords: Measles vaccine Cotton rat animal model Dry powder Aerosol
a b s t r a c t A stable and high potency dry powder measles vaccine with a particle size distribution suitable for inhalation was manufactured by CO2 -Assisted Nebulization with a Bubble Dryer® (CAN-BD) process from bulk liquid Edmonston-Zagreb live attenuated measles virus vaccine supplied by the Serum Institute of India. A novel dry powder inhaler, the PuffHaler® was adapted for use in evaluating the utility of cotton rats to study the vaccine deposition, vaccine virus replication, and immune response following inhalation of the dry powder measles vaccine. Vaccine deposition in the lungs of cotton rats and subsequent viral replication was detected by measles-specific RT-PCR, and viral replication was confined to the lungs. Inhalation delivery resulted in an immune response comparable to that following injection. The cotton rat model is useful for evaluating new measles vaccine formulations and delivery devices. © 2010 Elsevier Ltd. All rights reserved.
1. Introduction Important strides have recently been made by the global health community in reducing illness and death due to measles in developing countries [1]. Development of new vaccines which are more cost effective, and easier to store and deliver during mass campaigns can aid the national health systems, World Health Organization, and various non-governmental organizations in the ultimate eradication of measles. There is strong interest by the global health community in the development of needle-free vaccination systems for use in the developing world. One example is polio vaccine, which can be administered orally, with high efficacy [2]. Another example is the delivery of traditional measles vaccines to the respiratory mucosa via liquid nebulization (reviewed by [3]). Mucosal immunization of human infants with nebulized measles vaccine during mass campaigns has been shown to be as effective as immunization via parenteral injection [4].
∗ Corresponding author at: 400 Sutton Circle, Lafayette, CO 80026, United States. Tel.: +1 720 271 9433; fax: +1 303 398 1225. E-mail addresses:
[email protected],
[email protected] (K.O. Kisich). 0264-410X/$ – see front matter © 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.vaccine.2010.10.020
An alternative to wet aerosols of traditional vaccines is to formulate the vaccines as dry powders suitable for inhalation and respiratory deposition, with mean aerodynamic particle diameters less than 5 m [5–7]. As suggested by Cutts et al. a dry powder measles vaccine is desirable as it would avoid problems associated with reconstitution including instability and possible contamination [8]. In 2007 De Swart et al. published a report of dry powder pulmonary administration of a live, attenuated measles vaccine to cynomolgus macaques [9]. Rather than have the animals breathe the dry powder aerosol directly, the animals were anesthetized and administered the vaccine through an intra-tracheal tube. Compared with animals that received measles vaccine by injection or by nebulized aerosol vaccines much lower levels of immunity were achieved in animals that received the dry powder vaccine and the authors suggested that this could possibly be improved by either a different formulation or method of administration [9]. In a Phase 1 clinical trial of intranasal administration of a liquid live-attenuated measles vaccine the vaccine was safe and well tolerated but the immune response was poor, indicating that a vaccine with a smaller particle size or a higher dose may be necessary [10]. The possibilities that a different dry powder formulation with appropriate particle size and stability, along with an improved delivery to the pulmonary system led us to devise the experiments
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K.O. Kisich et al. / Vaccine 29 (2011) 905–912
Table 1 Composition of MCVP-3. Ingredients
Source
Quantity (g/L)
Myo-inositol Gelatin (hydrolyzed) l-Alanine l-Arginine hydrochloride TC lactalbumin hydrolysate l-Histidine Tricine Ingredients from the Minimum Essential Medium (MEM) measles virus vaccine harvest fluid
Sigma–Aldrich, USA E-Merck, Germany Sigma–Aldrich, USA Sigma–Aldrich, USA Becton Dickinson Sigma–Aldrich, USA Sigma–Aldrich, USA Serum Institute of India
50 25 1 16 3.5 2 3 8.3
described herein, the objectives of which were to determine if freebreathing of a uniquely formulated aerosol dry powder measles vaccine would result in an immune response. We chose the cotton rat as our animal model. Cotton rats (Sigmodon hispidus) are the only rodents that support the replication of measles virus following intranasal inoculation [11] and have proven useful in the preclinical evaluation of measles vaccine [12,13]. Primates are considered to be the best model for studying measles vaccines [12], but with our novel dry powder formulation and method of delivery we wanted to establish a proof of concept before moving forward with testing in monkeys. In the following report, we elaborate on the details of an effort to formulate traditional Edmonston-Zagreb (EZ) live attenuated measles virus vaccine [14] as a well-characterized, dry powder that has the ability to reconstitute at the mucosal surface after inhalation and allow replication of the vaccine virus to induce an immune response. The powder is delivered with PuffHaler® , a simple, inexpensive inhalation device that does not require electricity, and has single-use patient-contact parts. In this work we have established proof of concept in cotton rats that free inhalation of a measles vaccine dry powder aerosol deposited in the nose and lungs, was able to replicate in the lungs, and induced an immune response against the vaccine strain that was comparable to injected delivery. 2. Material and methods 2.1. Vaccine formulation and manufacturing of measles dry powder vaccine A modified clarified virus pool (MCVP-3, Table 1) consisting of EZ live attenuated measles virus (MV), myo-inositol and other stabilizing excipients was prepared at the Serum Institute of India Ltd. (SII), filtered, frozen, and shipped on dry ice to Aktiv-Dry for processing into measles dry powder vaccine. Vaccine powders were prepared at the University of Colorado using the CO2 -Assisted Nebulization with a Bubble Dryer® (CAN-BD) process [5,6]. A total of 120 mL of MCVP-3 was thawed, and processed for about 4 h at an aqueous flow rate of 0.5 mL/min; the CO2 nebulizing fluid pressure was 1200 psi, the N2 drying gas flow rate was 30 L/min, and the temperature of the drying chamber during nebulization and microdroplet drying was 50 ◦ C. After completing the initial particle formation and drying process, the collected powder was subjected to 30 min of additional drying by flowing dry nitrogen at 30 L/min at 30–50 ◦ C over the bed of powder. 2.2. Bioassay of measles dry powder vaccine Potency assays were conducted in two ways: the Plaque Forming Units (PFU) assay [15] and the 50% Cell Culture Infectious Dose (CCID50 ) assay. For our modified PFU assay, cultures of low passage
Vero cells were propagated and maintained in Dulbecco’s Modified Eagle Medium (Invitrogen Corp., Carlsbad, CA, 1×-DMEM, Cat. # 11965-092) supplemented with 100 units of penicillin, 100 g of streptomycin, 0.25 g of amphotericin B (PSA) (Invitrogen Corp., Carlsbad, CA, Cat. # 15240-062), and 10% fetal bovine serum (FBS) (Hyclone, Logan, UT, Cat. # SH30070.03). One day prior to assay of the vaccine dry powders, sterile 24-well plates were seeded with enough Vero cells to achieve a 85–95% confluent cell mono-layer after 1 day at 37 ◦ C in a 5% CO2 incubator. Powder samples were reconstituted and serial dilutions prepared using DMEM supplemented with PSA and 2% FBS. Dilutions expected to give plaque counts of 8 to about 80 per well were assayed, along with positive and negative control samples. Each sample was tested in triplicate. After completion of sample transfer (200 L/well), each plate was incubated for 1 h at 37 ◦ C in a 5% CO2 incubator to allow cell infection to occur. Then, 1 mL of overlay solution (1× Modified Eagle Medium (prepared from Invitrogen, 2×-MEM, Cat. # 11935-046) supplemented with PSA, 2% FBS, and 2% carboxymethylcellulose (Sigma–Aldrich, St. Louis, MO, Cat. # C5678)) was placed in each well and incubated for 6 days to allow plaque growth. On day 6, the liquid in each well was removed and the cells stained with a crystal violet (Fisher Scientific, Pittsburgh, PA, Cat. # S93213) solution to visualize and count plaques. Potency in terms of PFU/10 mg was calculated for each powder sample using the raw plaque counts of 8 per well or greater but less than “too numerous to count” (typically about 80), the known powder sample mass, reconstitution volume, well inoculation volume, and dilution levels. For the CCID50 assay, cultures of low passage Vero cells were propagated and maintained as described above. Sterile 96-well plates were seeded with 100 L/well of Vero cell suspension (cell count of 17,000–20,000 cells/100 L) and incubated for 2–6 h to allow the cells to adhere. Powder samples were reconstituted and serial dilutions in 0.5 log steps were prepared using DMEM supplemented with PSA and 2% FBS. The dilutions expected to produce cytopathic effects (CPE) in 90–10% of the wells were selected. A 100-L aliquot from each dilution sample was transferred into each well of a set of 8 wells. Positive and negative control samples were also tested in a set of 8 wells in each 96-well plate. Samples were tested in triplicate. The assay plates were incubated for a period of 10 days at 37 ◦ C in a 5% CO2 incubator to allow cell infection and the development of CPE. On day 10, the cell monolayer in each well was observed using a light microscope, and each well was scored for the presence or absence of CPE. The Spearman–Karber method [16] was used to calculate the potency in terms of log CCID50 /10 mg from the CPE scoring and the known powder sample mass, reconstitution volume, well inoculation volume, and log dilution levels. 2.3. Particle size distribution Fine Particle Fraction (FPF < 3.3 m and
