Dry powder measles vaccine: Particle deposition, virus replication, and immune response in cotton rats following inhalation

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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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 <5.8 ␮m aerodynamic diameter) was determined by a modification of USP method 601 (United States Pharmacopoeia) using an Andersen Cascade Impactor (ACI) (Westech, Marietta, Georgia). The mass of particles on each stage of the ACI was determined by gravimetric methods and/or by total organic carbon analysis [17]. 2.4. Bioburden assay Yeast extract medium (BD #211320, Difco Malt Extract Broth) was prepared as a 15 g/L solution with sterile water, and autoclaved for 20 min at 121 ◦ C. To test for bioburden, this sterile growth medium was added to a sterile vial containing about 10 mg of dry powder, and vortexed until no powder particles were left visible to the eye. The sample was then placed in 30 ◦ C incubator and checked daily for 1 week for signs of growth (turbidity, cloudiness, or precipitation).

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for long-term storage of the powder at 2–8 ◦ C. After removing the lid from the blister and placing it into the disperser the powder is blown from the opened blister into the reservoir by squeezing a 160 mL capacity bulb attached to the disperser. A silicone tetrafoliate pressure release valve between the bulb and disperser limits the overpressure to 2 psi. For infants and young children under 5 years of age the mask assembly will be affixed to the patient for inhalation by free-breathing. For older children it is expected they will inhale through a breathing tube upon instruction to do so. 2.8. Administration of dry powder vaccine to cotton rats using the PuffHaler Fig. 1. Design and use of the PuffHaler dry powder inhaler. The PuffHaler has four main components: a squeeze bulb and pressure release valve to provide air pressure, a disperser containing a single dose of vaccine packaged in a moisture-proof aluminum blister pack, a reservoir to receive the dispersed powder, and a mask which is placed on the subjects face though which they inhale the dispersed powder. The system was adapted to allow use with rats by inserting the nose of the animals directly into the reservoir.

2.5. Moisture content Moisture content was measured by Karl Fischer according to United States Pharmacopoeia 29 Chapter 921 (Water Determination, Method Ic, Coulometric Titration) using a Denver Instruments Model 275KF Titration Module with a Model 260 Controller (Denver, CO). 2.6. Animals Cotton rats (80–100 g) were obtained from Harlan Laboratories, Indianapolis, IN. The animals were housed in the AALAAC accredited rodent facility at National Jewish Medical and Research Center. Rats were maintained on standard rodent chow, autoclaved water, and a standard light–dark cycle (12/12). 2.7. Dry powder inhaler A dry powder inhaler, the PuffHaler, was developed by AktivDry. The PuffHaler is shown assembled and with a blister loaded in Fig. 1. The blister lid, which is removed prior to actuation, is shown coming out of the disperser. The device is designed for pulmonary delivery of dry powders through masks placed over the nose and mouth of younger children or through a mouthpiece of subjects more than 5 years in age. It is operated without electricity, and is composed of a single-use Dosing Kit and a multiple-use squeeze bulb and burst valve assembly. For this study, rats were allowed to inhale the powders by placing their noses into the reservoir through the hole where the mask normally attaches (Fig. 1). Rats are preferential nose breathers and it is most likely that any inhaled powder landing in the respiratory tract traveled through the nasal passages. The PuffHaler emits a powder into a reservoir of approximately 160 mL capacity. The PuffHaler reservoir is made from thin, very flexible, clear plastic, which enables observation of the powder being emitted into the reservoir as well as clearance of the powder cloud from inhalation by the animals. The PuffHaler reservoir is filled with the vaccine powder by a small device called a disperser that houses an aluminum blister containing 10 mg of the powder formulation and is attached to the squeeze bulb. The aluminum blister (Alcan Ltd., Waterloo, Canada) is filled with powder using an Omnidose TT (Harro Hofliger, Almersbach, Germany) and the blister is sealed with lidding material (Alcan Ltd.) using a custommade cavity to hold the powder-loaded blisters (ATG Pharma, Oakville, Ontario). The sealed blister provides a moisture barrier

The protocol for administering dry powders to cotton rats was developed at National Jewish Medical and Research Center, where it was also reviewed and approved by the Institutional Animal Care and Use Committee (IACUC). The rats were gently restrained in conical, flexible plastic devices allowing access to the nose (DecapiCones® , Braintree Scientific, Braintree, MA). Powder was then dispersed, filling the reservoir with suspended powder, and the rat’s nose was inserted through the opening into the reservoir. In an initial study of vaccine deposition into the lungs, groups of 5 rats were allowed to breathe powder-containing air for either 1 min (1 actuation of a blister containing 10 mg of powder), 2 min (2 actuations-20 mg total) or 3 min (3 actuations-30 mg total). The rats were sacrificed immediately after completion of vaccine inhalation. In a second study, 5 rats served as negative controls, and 2 groups of 5 rats (positive controls) received SII liquid MV vaccine subcutaneously (s.c.) containing either 100 CCID50 or 1000 CCID50 . Thirty rats received identical doses of dry powder measles vaccine by inhalation using PuffHaler. Each of the thirty animals was allowed to breathe powder-containing air continuously for 4 min from the reservoir, during which time 4 actuations of 10-mg blisters of powder into the reservoir occurred at 1-min intervals, after which they were removed from the reservoir and restrainer. Five of those animals were sacrificed immediately for assessment of particle deposition, and groups of five additional animals were sacrificed after 24 h, 3, 7, 21, and 30 days for assessment of MV replication. Blood was collected from all surviving animals beginning at 7 days for assessment of antibody titer. In both the first and second studies, 30 mg portions (approximate) of lung tissue were frozen on dry ice immediately after dissection. In order to extract RNA, the portion was precisely weighed, and then disrupted in 0.5 mL of tissue lysing solution (RNAeasy kit, Qiagen, Valencia, CA) according to the manufacturer’s instructions. The RNA in the disrupted samples was extracted via chromatography, using Qiagen RNAeasy isolation columns according to the manufacturers instructions. The RNA from each sample was eluted from the columns in a volume of 30 ␮L, and the concentration determined spectrophotometrically at 260 and 280 nm. The samples were then stored at −80 ◦ C until analysis. 2.9. PCR analysis and calculation of nucleoprotein RNA content One rigorous method to normalize the content of a particular unknown RNA between samples and between experiments is to compare the unknown sample values to the concentration levels of a known “housekeeping” mRNA also measured for each sample. However, this was not feasible in the cotton rat samples, because the precise genomic sequences needed to design probe and primer sets for the housekeeping gene are unknown. Therefore, we designed a surrogate normalization algorithm based on the amount of total RNA in the tissue. One-microliter of the RNA solution from the 30 ␮L column eluent was used for each 50 ␮L amplification reaction in an ABI 7600 Real-time (RT) PCR instrument, using primer and probe sets specific

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Table 2 Example spreadsheet calculations for determination of nucleoprotein RNA copy number in tissues.. Sample

CT#

Input copy number

MV copies in 30 ␮L

MV copies in tissue

Correction for extraction efficiency

Estimate of nucleoprotein RNA in the lungs

lung 6 lung 7 lung 8

30.5 37.6 38.3

2.26 4.87 2.66

6.77E+01 1.46E+02 7.98E+01

1.73E+03 3.73E+03 2.04E+03

15.1 2.00 18.8

2.61E+04 7.46E+03 3.83E+04

for measles nucleoprotein RNA. The amplification cycle at which the fluorescent signal from the reaction exceeded twice the standard deviation of the background was reported by the software as the “threshold cycle,” or Ct. The Ct values of triplicate wells for each RNA sample were then computed back to the number of input copies via a standard regression formula of the form: ln x + 39.39 =y −1.1554

(1)

in which “x” is the reported Ct value, and y is the input copy number. The input copy number was multiplied by 30 to arrive at the amount of nucleoprotein RNA present in the entire 30 ␮L of eluent. The total number of copies extracted was then multiplied by the average weight of both intact lungs (500 mg) and divided by the exact mass of tissue piece extracted (e.g., 27.5 mg). This provides an estimate of the number of nucleoprotein RNA copies, which were in the entire right and left lungs of the rats. That number was then corrected for extraction efficiency, using Eq. (2) as a multiplication factor: 538.5 × (exact mass of tissue extracted) [RNA] yielded per ␮L × 30

(2)

This provides a factor of the amount of RNA in the lung sample at 100% extraction efficiency (measured), divided by the actual yield. An example EXCEL spreadsheet performing this calculation is shown in Table 2. 2.10. Plaque reduction neutralization (PRN) assay Neutralization of SII MV vaccine was measured in 24-well plates (BD Labware, Franklin Lakes, NJ). Briefly, Vero cells (American Type Culture Collection, Manassas, VA) were grown in DMEM containing 2% FBS, and then transferred into the 24 well plates. When the cells were 90–95% confluent in the wells, the medium was removed and replaced with the assay samples. These consisted of (1) diluted vaccine containing approximately 30 PFU (negative control); (2) vaccine exposed to anti-measles positive human serum diluted 1:1000, 1:333, or 1:100 (positive control); or (3) vaccine exposed to rat serum diluted 1:333, 1:100, or 1:33 in triplicate wells. The cultures were then incubated for 1 h at 37 ◦ C, after which they were rinsed and covered by an overlay medium containing DMEM, and carboxymethylcellulose (Sigma, St. Louis, MO). Six days after infection, the wells were rinsed, and stained with crystal violet (Sigma, St. Louis, MO) for counting of plaques.

3. Results 3.1. Vaccine formulation and manufacturing of measles dry powder vaccine The key characteristics of the measles vaccine dry powder used in this study are listed in Table 3. Development of the formulation and process used to make the powders is covered elsewhere [5,6]. Potency loss through CAN-BD drying and filling into blisters, as well as on storage at ambient temperature and 37 ◦ C, are similar to the current lyophilized vaccine. This means that current upstream manufacturing capacity is preserved when converting the product from lyophilized/reconstituted/injected to Bubble Dried/inhaled. We report powder potency in PFU’s and used CCID50 for the powder stability test (37 ◦ C for 1 week) as well as for the injected doses of reconstituted lyophilized vaccine. In our hands, these two different analytical methods have shown a ratio of about 6:1 (CCID50:PFU) with this lot of vaccine virus. This observation is not unique as it was recently reported that mumps viruses can show ratios of 0.66 to 10 depending upon the virus strain and plaque appearance [18]. 3.2. Adaptation of the delivery device for exposure of cotton rats The PuffHaler device consists of a squeeze bulb and burst valve, a disperser which holds single-dose vaccine powder containers, a disposable inflatable reservoir into which the powder is discharged, and a disposable mask as shown in Fig. 1. In order to adapt the system to allow rats to breathe the powder aerosol cloud, powder was dispersed into the reservoir. The nose of each rat (restrained in DecapiCones) was inserted directly into the reservoir and held there during the total time of exposure and allowed to inhale the aerosolized powder cloud directly from the reservoir. This system was relatively easy to use, and minimally stressful for the animals. The rats tolerated up to 40 mg of vaccine powder dispersed into the reservoir in 10 mg aliquots once a minute over a period of 4 min. However, when administering more than 40 mg of powder in a 4 min period, we observed that the nasal passages of the rats could become clogged with powder. Powder was visible on the nares of the rats following administration at all dosages. 3.3. Deposition of dry powder measles vaccine following inhalation In the initial study, deposition of vaccine powder in the lungs increased as a function of increased dose exposure, as shown in

2.11. Statistical analysis Group sizes for animal the animal studies were determined from power calculations, based on data from preliminary experiments. In order to compare viral deposition and replication among the animal subjects, PFU data were imported into GraphPad Prism (GraphPad Software, Inc., La Jolla, CA). Mean differences among the groups were first evaluated by ANOVA. Differences between control groups and test groups were tested by Dunnett’s test. Differences among test groups were tested via Tukey–Kramer analysis. For all statistical tests, a p-value of less than 0.05 (p < 0.05) was considered significant.

Table 3 Measles dry powder vaccine characteristics. Potency Stability (37 ◦ C incubation for 1 week)

441 PFU/mg Day 0: 4.45 log CCID50 /10 mg Day 7: 4.07 log CCID50 /10 mga

Particle size distribution

FPF < 5.8 ␮m = 44.9% ± 3.6% (S.D.) FPF < 3.3 ␮m = 18.9% ± 2.7% (S.D.)

Moisture content Bioburden Potency of final filled blisters

0.6% Negative 319 PFU/mg

a

Passes WHO test for measles virus potency of less than 1 log loss.

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Fig. 2. Deposition of vaccine powders in rat tissues. (a) RNA extracted from tissues as noted on the x-axis were evaluated for measles nucleoprotein sequence. The values obtained were converted to PFU equivalents based on the PFU/copy in the powder administered, and are plotted on the y-axis. Each symbol represents triplicate measurements from a single animal. (b) Lungs of animals exposed to increasing amounts of vaccine powder on the x-axis were assessed for nucleoprotein RNA content. The number of nucleoprotein copies was then converted into the mass equivalents of powder, based on the copies/mg of the administered powder. Each symbol represents the mean and standard error of the mean (SEM) of four animals.

3.4. Replication of Edmonston-Zagreb measles virus following inhalation as dry powder A key question surrounding dry powders involves reconstitution of complex biological structures in physiological milieu. Desiccated MV in the vaccine formulation was able to reconstitute in defined, buffered biological media as demonstrated by the measurement of viral titers in the PFU and CCID50 assays of the vaccine powders. However, that did not assure that the virus could reconstitute and infect cells in the respiratory tracts of animals. Therefore, we measured the levels of MV nucleoprotein RNA in the noses, lungs, livers, spleens, kidneys, and forebrains of the cotton rats at intervals up to 30 days after exposure to the vaccine powders. Viral replication was only detected in the lungs. Low levels of RNA were found associated with the nose 24 h after exposure to the powder, but after that it was not detected in the nose. No nucleoprotein RNA was detected in the brain, liver, spleen, or kidney at any time point. Immediately after exposure to the vaccine powder, the lungs of the rats contained an average of 290,000 (±32,000) copies of nucleoprotein RNA. However, nucleoprotein RNA was found to decline for the first 24 h post exposure to 47,000 (±12,000) copies as shown in Fig. 3. Nucleoprotein RNA levels then increased in the lungs, reaching a peak of 654,000 (±113,000) 7 days after exposure. Nucleoprotein RNA levels then declined again between 7 and 28 days, but remained

detectable in the lungs of 9/10 rats examined. Not only was MV able to reach the lungs of the cotton rats following exposure to dry powder, but the virus in the powder was also able to deposit, reconstitute, infect, and replicate to a certain extent within the lungs. In contrast, the nose did not appear to support replication of the virus despite the high exposure. During replication in the lungs, the EZ MV did not disseminate to the liver, spleen, kidney, or brain. 3.5. Immune response to Edmonston-Zagreb measles virus vaccine following administration as inhaled dry powder The objective of vaccination with an attenuated strain of the MV [14] is to elicit a cross protective immune response to wildtype MV. A surrogate marker of protection is the acquisition of neutralizing antibody in the serum. Therefore, we measured the ability of serum from cotton rats that received EZ MV vaccine via inhalation of powder to neutralize the plaque-forming activity of EZ MV, in comparison with animals immunized via s.c. injection of the standard commercial EZ vaccine. The sera were incubated with virus at dilutions of 1:333, 1:100, and 1:33. Fig. 4a shows the number of plaques remaining from each rat (mean ± standard error of the mean, SEM) after 30 min of incubation with the serum diluted 1:33. Vaccine powder delivery to the cotton rats caused seroconversion of 10/10 rats relative to controls. The seroconversion rate and the level of neutralizing activity of the vaccine-treated groups were significantly better than in the untreated controls (p < 0.05, ANOVA–Dunnett’s, Fig. 4c). In addition, the antibody response to

Copies of nucleoprotein mRNA in lungs

Fig. 2b. Deposition of the vaccine in the lungs was found to be reproducible in the second study in the group of rats sacrificed immediately following delivery. The equivalent of 4.6–7.8 PFU of MV RNA was recovered from the lungs of 4 of 5 rats. The fifth rat yielded 16 PFU equivalents of MV RNA, for an average of 8.3 ± 4.5 PFU recovered from the group. No MV RNA was recovered from the spleen, liver, kidney, or forebrain (including the olfactory bulb) of any of the rats at any time point. However, varying amounts were recovered from the nares, consistent with the presence of visible powder on the nares immediately after administration. No measles-specific RNA was detected in any tissue of the negative controls, which were not exposed to vaccine powder (Fig. 2a). Therefore, we concluded that vaccine could reach the lungs in a dose-dependent manner when inhaled as a dry powder with the appropriate particle size characteristics. The measles RNA contained in the powder served as an effective marker for deposition immediately after administration, and could be quantified via real time PCR following extraction from the tissue. The powder deposited only in the upper airways and lungs, as no measles RNA was detected in the forebrain (including the olfactory bulb), liver, spleen, or kidney.

1,000,000 750,000 500,000 250,000 0 0

5

10

15

20

25

30

Day after inoculation Fig. 3. Kinetics of nucleoprotein RNA in cotton rat lungs. RNA extracted from cotton rat lungs at increasing time after administration of vaccine powder was assessed for the content of measles nucleoprotein sequence. Numbers on the y-axis represent the number of copies in both lungs. Each symbol is the mean and SEM of 5 animals.

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Fig. 4. Plaque neutralization by cotton rat serum after immunization (PuffHaler). Serum was collected 28 days after immunization via inhalation of dry powder, or subcutaneous injection of 100 or 1000 CCID50 of EZ vaccine. (a) Seroconversion was observed in all 10 animals (green symbols). (b) 6/10 animals showed high levels of antibody. (c) Group means of aerosol group and negative controls were significant at 1:33 dilution, and 1:100 dilution (d). Error bars represent SEM of 5 samples. † p < 0.05 relative to negative control (ANOVA, Dunnett’s). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)

vaccine powder delivery was not significantly different than either 100 CCID50 or 1000 CCID50 of EZ MV vaccine (SII commercial vaccine) injected s.c. (Fig. 4c and d). The rats inhaled sufficient vaccine from the PuffHaler device to result in limited viral replication in the lungs, and acquisition of neutralizing antibodies in the serum after 28 days. 4. Discussion In 2003, in an initiative funded by the Bill and Melinda Gates Foundation, 14 Grand Challenges in Global Health were identified, one of which was developing needle-free delivery systems for childhood vaccines [19]. The undertaking to develop a novel dry-powder vaccine designed for inhalation, coupled with development of an inhalation device inexpensive and robust enough for mass campaigns in developing countries, has indeed been a “Grand Challenge”. The number of scientific, manufacturing, and regulatory hurdles that needed to be overcome required creative application of existing tools, and development of new ones. One new tool that was needed to develop an inhalable dry powder measles vaccine was a way to evaluate the candidate dry powder formulation and delivery device for potential efficacy. The only well accepted animal model for measles vaccination and challenge involves non-human primates, an extremely expensive and difficult model to screen large numbers of potential vaccine formulations and dry powder inhalers. In addition, the dry powder inhalers in development or commercially available are designed for adults and children who must follow the instructions for use to receive the specified dose. An important target for a dry powder

measles vaccine is infants, for whom an external powder dispersion mechanism is required, similar to the method of powder administration used in this study. Measles virus, including its attenuated vaccine strains, is known to infect and replicate within Cotton rats (S. hispidus), and in mice transgenic for human CD46, or hSLAM [8,20,21]. Therefore, these rodent species were investigated for suitability as surrogates for testing (1) the efficacy of candidate delivery devices to generate a respirable powder aerosol; (2) the ability of vaccine virus in dry powder particles to reconstitute, infect, and replicate at the surface of the airway mucosa; and (3) the ability of the delivery system and vaccine to stimulate an immune response. Our initial efforts involved delivery of vaccine powders to YACCD46 transgenic mice [20]. However, while vaccine powder could be delivered into the lungs of the mice via ad lib breathing, not enough could be delivered to reliably initiate viral replication, or an immune response. This was due to a combination of the specific activity of the vaccine powder (active virus per mg of powder), coupled with the relatively small volume of the mouse lungs. In most trials with ad lib breathing in mice, we achieved less than 1 active viral particle per animal. This limitation, coupled with relatively high doses of liquid vaccine injected s.c. required to elicit an immune response (1000–10,000 CCID50 with boost) suggested that a much more active vaccine powder per unit mass, or a more sensitive model was required. Cotton rats are also reported to support replication of vaccine strains of MV [12,22]. In our hands, we were able to detect neutralizing antibody in the blood of cotton rats after a single s.c. administration of as little as 100 CCID50 of EZ MV vaccine. There-

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fore, it was postulated that the increased sensitivity of the rats to immunization with vaccine, coupled with greater lung capacity that allows more powder to be inhaled, would make cotton rats a particularly useful small animal model for studying inhaled dry powder measles vaccines. Although the rats could potentially inhale enough powder to allow viral replication sufficient to provoke an immune response, the problem of adaptation of the device remained. The options available to disperse the powder for breathing by the rats included: (1) use a commercially available aerosol exposure chamber designed for rodents, (2) allow the rats to breathe the powder cloud generated by adapting a novel dry powder inhaler such as the PuffHaler, or (3) design a custom dry powder inhaler for rats. We chose to allow the rats to breathe powder from the PuffHaler reservoir in order to gain further information about the performance of the device when used in a manner similar to its intended use to vaccinate infants. Adaptation to rats proved to be straightforward, to the extent that placing the nose of the rats into the reservoir allowed the animals to sample the powder in much the same way that a human subject would with two exceptions: (1) the presence of a mask for human infants to breathe from, and (2) the infant’s ability to essentially inhale the entire reservoir volume in one or two breaths. We judged these differences to be acceptable, given the greater limitations of Options 1 and 3. With visual observation immediately after the dispersal of vaccine powder into the PuffHaler reservoir, we were able to determine that the particles were suspended as a cloud in the reservoir. Subsequent to administration, powder was deposited on the whiskers and nares of the rats. In addition, ad lib breathing of the reservoir contents by the animals resulted in inhalation and deposition of approximately 0.1% of the powder initially packaged in the blister packs into the lungs (Fig. 2). Powder was also likely deposited between the nares and the trachea, which were not extracted and quantified. When we examined tissues other than lung and nares for the presence of viral RNA immediately after administration of the vaccine, none was detectable. This suggests that despite the proximity of the forebrain and olfactory bulb to large amounts of the EZ MV during administration, there was no penetration of the virus to those sensitive tissues [22]. This bears mentioning because it has previously been discussed as a potential concern for aerosol delivery of live-virus vaccines [8]. The vaccine also does not seem to have been disseminated from the lungs to other sites, as we were not able to detect the viral RNA in the spleen, liver, or kidney. The initial decline in MV measured by RT-PCR during the first 24 h after administration of the vaccine was not surprising (Fig. 3). The cleared cellular lysate, from which the viral prep is made, contains high levels of nucleoprotein and other viral RNAs, which are not associated with viral particles (S. Sharma, personal communication). Exposure of these naked RNAs to the nucleases present in the airway surface fluid [23], coupled with mucociliary elevator action within the airways would be expected to destroy or expel the majority of the inhaled vaccine material. However, at least some active virus remained associated with the lungs after administration, since a dramatic increase in the number of measles nucleoprotein RNA copies occurred between 1 and 7 days after administration. This increase in viral RNA could only have occurred by infecting target cells in the lungs, followed by replication of the virus. Between 7 and 28 days after administration of the vaccine, the levels of viral RNA declined to very low levels again, suggesting an effective immune response developed, which limited further viral replication (Fig. 3). Since protective immunity against measles infection in humans can be defined as a particular level of neutralizing antibodies in the serum [15], it was important to measure whether neutralizing antibodies appeared in the blood of the vaccinated rats. Such antibodies appeared in the blood between 21 and 28 days after exposure to the

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vaccine, and were readily detectable in all of the vaccinated animals at a 1:33 dilution of serum (Fig. 4a). Six of the 10 rats vaccinated via inhalation of dry powder had significant viral-neutralizing activity even when the serum was diluted 1:100 (Fig. 4b), comparing favorably with the neutralizing activity in control rats which received up to 1000 CCID50 of the standard liquid vaccine s.c. (Fig. 4c and d). However, the number of animals in each group was small and we cannot eliminate the possibility of type 2 error. 5. Conclusions A novel dry powder inhaler, the PuffHaler, and a dry powder measles vaccine stabilized with myo-inositol replacing sorbitol were tested using a rodent model which had not previously been utilized for preclinical development of inhaled vaccines. The model proved to be sufficiently sensitive and robust to detect delivery of MV to the airways of the rats, as well as replication of MV in the lungs, followed by development of measles-specific neutralizing antibodies to levels comparable to injected delivery. Therefore, the cotton rat model is suitable for screening of dry powder vaccine formulations, and is suitable for evaluation of some aspects of dry powder delivery devices. Further development of this model is warranted. Acknowledgements Funded in part by Grant 1077 from the Foundation for the National Institutes of Health through the Grand Challenges in Global Health initiative. The authors thank the Serum Institute of India, Limited (for the bulk liquid measles vaccine samples and technical support), Lia Rebits, David McAdams, Jessica Burger, and Prof. Mark Hernandez for their technical support. References [1] Clements CJ, Nshimirimanda D, Gasasira A. Using immunization delivery strategies to accelerate progress in Africa towards achieving the Millennium Development Goals. Vaccine 2008;26:1926–33. [2] Paul Y. Oral polio vaccines and their role in polio eradication in India. Expert Rev Vaccines 2009;8:35–41. [3] Low N, Kraemer S, Schneider M, Restrepo AM. Immunogenicity and safety of aerosolized measles vaccine: systematic review and meta-analysis. Vaccine 2008;26:383–98. [4] Dilraj A, Cutts FT, de Castro JF, Wheeler JG, Brown D, Roth C, et al. Response to different measles vaccine strains given by aerosol and subcutaneous routes to schoolchildren: a randomised trial. Lancet 2000;355:798–803. [5] Cape SP, Villa JA, Huang ET, Yang TH, Carpenter JF, Sievers RE. Preparation of active proteins, vaccines and pharmaceuticals as fine powders using supercritical or near-critical fluids. Pharm Res 2008;25:1967–90. [6] Burger JL, Cape SP, Braun CS, McAdams DH, Best JA, Bhagwat P, et al. Stabilizing formulations for inhalable powders of live-attenuated measles virus vaccine. J Aerosol Med Pulm Drug Deliv 2008;21:25–34. [7] LiCalsi C, Maniaci MJ, Christenson T, Phillips E, Ward GH, Witham C. A powder formulation of measles vaccine for aerosol delivery. Vaccine 2001;19:2629–36. [8] Cutts FT, Clements CJ, Bennett JV. Alternative routes of measles immunization: a review. Biologicals 1997;25:323–38. [9] De Swart RL, LiCalsi C, Quirk AV, van Amerongen G, Nodelman V, Alcock R, et al. Measles vaccination of macaques by dry powder inhalation. Vaccine 2007;25:1183–90. [10] Simon JK, Pasetti MF, Viret JF, Mischler R, Munoz A, Lagos R, et al. A clinical study to assess the safety and immunogenicity of attenuated measles vaccine administered intranasally to healthy adults. Hum Vaccin 2007;3:54–8. [11] Wyde PR, Ambrose MW, Voss TG, Meyer HL, Gilbert BE. Measles virus replication in lungs of hispid cotton rats after intranasal inoculation. Proc Soc Exp Biol Med 1992;201:80–7. [12] Wyde PR, Stittelaar KJ, Osterhaus AD, Guzman E, Gilbert BE. Use of cotton rats for preclinical evaluation of measles vaccines. Vaccine 2000;19:42–53. [13] Niewiesk S. Current animal models: cotton rat animal model. Curr Top Microbiol Immunol 2009;330:89–110. [14] Ikic D, Juzbasic M, Beck M, Hrabar A, Cimbur-Schreiber T. Attenuation and characterisation of Edmonston-Zagreb measles virus. Ann Immunol Hung 1972;16:175–81. [15] Cohen BJ, Audet S, Andrews N, Beeler J. Plaque reduction neutralization test for measles antibodies: description of a standardised laboratory method for use in immunogenicity studies of aerosol vaccination. Vaccine 2007;26:59–66.

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