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Immunotherapy of Respiratory Syncytial Virus Infection in Cotton Rats (Sigmodonfulviventer) Using IgG in a Small-Particle Aerosol Franco M. Piazza, Susan A. Johnson, Martin G. Ottolini, H. Joel Schmidt, Miriam E. R. Darnell, Val G. Hemming, and Gregory A. Prince

Children's National Medical Center, and Department of Pediatrics, George Washington University School ofMedicine and Health Sciences, Washington, DC; Department of Pediatrics, Uniformed Services University ofthe Health Sciences, Bethesda, and Virion Systems, Inc., Rockville, Maryland

Recent studies in experimental animals have shown that serum antibody with high neutralizing activity against respiratory syncytial virus (RSV) can provide significant prophylactic and therapeutic benefits when administered either systemically [1, 2] or by topical instillation (by drops) into the airway [3, 4]. A clinical trial of intravenous IgG containing high levels of neutralizing antibodies to RSV (titer of 1:5000 in 5% solution) given to infants and children hospitalized with RSV disease showed that antibody recipients had a significant reduction in nasal shedding of the virus and improved clinical response compared with placebo recipients [5]. A recent study of safety and pharmacokinetics of intravenous IgG that contains anti-RSV antibodies in the prevention ofRSV illness in infants and children with cardiopulmonary disease showed that IgG was well tolerated and that it may prevent severe RSV disease [6]. Topical administration orIgG offers two major advantages over systemic administration. First, topically applied IgG comes into immediate contact with infected respiratory epithelial cells, whereas systemically administered IgG requires several hours to effect a reduction in pulmonary virus titers [1]. Second, studies in the cotton rat [3] and in the owl monkey [4] have shown that topical therapy requires consider-

Received 4 March 1992; revised 10 June 1992. Financial support: American Lung Association of Maryland (research grant); Research Advisory Council, Children's National Medical Center (scientific award). Reprints or correspondence: Dr. Franco M. Piazza, Department ofPulmonary Medicine, Children's National Medical Center, III Michigan Ave., N.W., Washington, DC 20010. The Journal of Infectious Diseases 1992;166:1422-4 © 1992 by The Universityof Chicago. All rights reserved. 0022-1899/92/6606-0034$01.00

ably less IgG than does systemic administration to achieve a similar reduction in RSV shedding from lung tissue. The instillation of IgG by drops into the upper and lower respiratory tract, used effectively in anesthetized cotton rats and owl monkeys previously inoculated with RSV, would be possible only in human infants and children with RSV disease who had undergone endotracheal intubation. In those not requiring intubation, aerosolized IgG, which would reach the lower respiratory tract, could represent an advantageous alternative to intravenous IgG. The purpose of this study was to determine if human IgG that contains anti-RSV antibodies, delivered in a small-particle aerosol, would be as effectiye as intranasally instilled IgG in reducing pulmonary virus in RSV -infected cotton rats.

Materials and Methods Weanling cotton rats (Sigmodon fulviventer) were obtained from a colony maintained at Virion Systems (Rockville, MD) and were housed and fed as previously described [7]. RSV strain Long (American Type Culture Collection, Rockville, MD) was grown in HEp-2 cells to prepare virus stocks containing 106 pfu/ml., A small-particle «2 ~m) ultrasonic nebulizer (Portasonic 8500D; DeVilbiss, Somerset, PA) was used to generate aerosol particles for delivery into the lungs of the cotton rats. Purified human IgG that contained anti-RSV antibodies (Sandoglobulin, lot 2.370.069.0; Sandoz, East Hanover, NJ) was used as a 5% solution. More concentrated solutions could not be used because they became frothy when nebulized. The antiRSV neutralizing antibody titer for this 5% IgG preparation, as determined by 60% plaque reduction assay [7], was 1:1113. Ribavirin (Viratek, Costa Mesa, CA) was prepared at the standard concentration of 20 mg/mL. Human serum albumin (Sandoz) was used as a 5% solution. All of these solutions were made in PBS.

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To determine whether aerosolized IgG can be used effectively in the treatment of respiratory syncytial virus (RSV) infections, cotton rats were infected intranasally with RSV and treated 3 days later with human IgG containing anti-RSV antibodies delivered in a small-particle aerosol. Pulmonary histology and virus titers were determined 24 h after IgG treatment. A single IS-min exposure to aerosolized IgG did not exacerbate pulmonary pathology and effected a SO-fold reduction in pulmonary virus titer (2.95 vs. 4.67 log\ogeometric mean pfu/g for untreated controls, P < .001), which was comparable to that effected by intranasally instilled IgG (SO mg/kg) (3.24 vs. 4.6710g lO geometric mean pfu/g for controls, P < .001). A IS-min exposure to aerosolized ribavirin (20 mg/ml.) was not effective in reducing pulmonary virus. This study suggests that aerosolized IgG could be useful in the treatment of RSV lower respiratory tract infections and that it compares favorably with ribavirin.

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Concise Communications

Results A IS-min exposure to an aerosolized 5% solution of IgG effected a 50-fold reduction in pulmonary virus (P < .00 I vs. control) and was slightly more effective than a dose of 50 mg/kg delivered by intranasal instillation by drops (P < .00 I vs. control; table 1). In the former, virus was undetectable in 19%of the animals, and in the latter, in 5%. Virus was recovered from all control animals, with pulmonary titers of 3.25.6 10glO pfu/g, A 10-min exposure to aerosolized IgG was less effective than a IS-min exposure but still caused a significant reduction in pulmonary virus (P < .05 vs. control). A 5-min exposure was not effective (table I). A single exposure to intranasally instilled 5% solution of human serum albumin or to aerosolized ribavirin (20 mg/ mL) over 15 min was not effective in reducing pulmonary virus (table 1). A IS-min exposure to aerosolized IgG did not effect a reduction in nasal virus titers. In 12 animals inoculated with RSY and treated with aerosolized IgG, the nasal virus titer was 4.69 ± 0.16 10glO geometric mean pfu/g ± SE; in 12 RSV-inoculated untreated controls, it was 4.82 ± 0.12 (P> .05). No human IgG could be detected in untreated cotton rats. In 10 animals exposed to aerosolized IgG for 15 min, the

Table 1. Therapeutic effect of aerosolized human IgG 3. days after infection with RSY.

Mode of therapy IgG Aerosol, 15 min Aerosol, 10 min Aerosol, 5 min Intranasal. 50 mg/kg HSA, intranasal, 50 rng/kg Ribavirin, aerosol, 15 min Untreated control

Geometric mean ± SE of virus titer in lungs, loglO pfu/g

No. of animals sacrificed, day 4

21 13 10 22 7 6 31

2.95 4.24 4.56 3.24 4.61 4.41 4.67

± 0.19 (P< .001) ± 0.16 (P < .05) ± 0.13 ± 0.20 (P < .00 I) ± 0.09 ± 0.09 ± 0.11

NOTE. HSA, human serum albumin. P values are shown for groups that differed from control.

serum human IgG level was 4.55 ± 1.62 geometric mean lLg/mL ± SE; in 10 animals receiving IgG by drops, it was 2.44 ± 1.55. This difference was not statistically significant. Pathology scores were determined in 4 RSY-inoculated IgG aerosol-treated (15 min) cotton rats and in 4 RSY-inoculated untreated animals. Four uninoculated untreated animals were used as negative controls. Pathologic changes in the RSY-inoculated animals consisted of mild interstitial pneumonia and minimal bronchiolitis. There was no difference in pathology scores between the IgG-treated and untreated animals; mean scores for interstitial pneumonia were 2.00 and 1.75, respectively. No exacerbation oflung pathology was observed in the IgG aerosol-treated group.

Discussion Human IgG that contained antibodies to RSY, delivered with a small-particle ultrasonic nebulizer designed for clinical use, was effective in decreasing pulmonary virus in RSYinfected cotton rats. A IS-min exposure to an aerosolized 5% solution of IgG reduced pulmonary virus titer 50-fold and was at least as effective as a dose of 50 mg/kg delivered by intranasal drops. Serum levels of human IgG, which reflect absorbed dose, were used as an indirect measure ofdelivered dose. A IS-min exposure to the IgG aerosol was associated with a serum human IgG level that was similar to that observed after direct instillation of 50 mg/kg IgG. This finding suggests that the actual amount ofigG that reaches the lung during a IS-min aerosol exposure is - 50 rug/kg, The open system of aerosol delivery used in this study does not allow direct measurement of dose. We used a nebulizer designed for clinical use rather than a rodent system to allow an easier transition to human trials. The mechanism by which IgG effects a reduction in pulmonary virus is likely due to the presence of specific anti-RSV

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Cotton rats were inoculated intranasally after anesthesia with 105 pfu of RSY Long in a volume of 0.1 mL. Three days later they were treated once with IgG by aerosol or intranasal instillation, ribavirin by aerosol, or albumin by intranasal instillation or were left untreated. Animals treated by intranasal instillation were anesthetized by methoxyflurane inhalation and given 0.1 mL ofIgG or albumin (50 mg/kg) delivered by drops. Animals treated by aerosol were anesthetized with a mixture of ketamine HCI (25 mg/kg) and acepromazine maleate (2.5 rug/kg) given intramuscularly and allowed to breathe the aerosol as it emerged from the exit orifice of the chamber lid ofthe nebulizer. Exposures of up to 15 min were used. Longer exposures were not possible due to overheating of the nebulizer. At 24 h after treatment (4 days after viral inoculation), the animals were bled for determination of human IgG levels and sacrificed by carbon dioxide intoxication. Nasal tissues and lungs were homogenized, stored at -70°C, and subsequently titrated for infectious virus by plaque assay [7]. For histology, lungs were inflated with and fixed in 10% formalin, embedded in paraffin, and cut into sections stained with hematoxylin-eosin [7]. These sections were scored blindly for pathologic changes as previously described [8]. Human IgG in cotton rat serum wasdetermined by a competitive inhibition ELISA [3]. Geometric means of virus titers and serum human IgG levels and arithmetic means of pathology scores for each treated group were compared with those ofthe untreated controls by using the two-tailed Student's t test.

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treatment of infants and children with RSV lower respiratory tract infection who have not required endotracheal intubation as well as those who have. Direct instillation oflgG into the lower airway to treat RSV disease would be practical only in patients requiring intubation. Second, aerosolized IgG circumvents the technical difficulties and potential complications of intravenous administration of relatively large volumes of fluid necessary to deliver an adequate dose ofIgG. Infants with cardiopulmonary disease are at high risk for severe RSV illness, are fluid-sensitive, and tend to have difficult intravenous access. We speculate that IgG would have several advantages over ribavirin, which is the only antiviral drug currently approved for treatment of RSV disease. Current clinical protocols for ribavirin require continuous aerosol treatment for 18-24 h/day for 3-5 days; ribavirin is not effective when used by direct instillation into the airway. In infants and children not requiring endotracheal intubation, IgG could be effectively nebulized over short periods of time, and in those undergoing endotracheal intubation, it could be nebulized or instilled directly into the lower airway. References I. Prince GA. Hemming VG. Horswood RL. Chanock RM. Immunoprophylaxis and immunotherapy of respiratory syncytial virus infection in the cotton rat. Virus Res 1985;3: 193-206. 2. Hemming VG. Prince GA, Horswood RL. et al. Studies of passive immunotherapy for infections of respiratory syncytial virus in the respiratory tract of a primate model. J Infect Dis 1985; 152:1083-6. 3. Prince GA, Hemming VG. Horswood RL. Baron PA. Chanock RM. Effectiveness of topically administered neutralizing antibodies in experimental immunotherapy of respiratory syncytial virus infection in cotton rats. J ViroI1987;61:1851-4. 4. Hemming VG. Prince GA. London WT. Baron PA, Brown R. Chanock RM. Topically administered immunoglobulin reduces respiratory syncytial virus shedding in owl monkeys. Antimicrob Agents Chemother 1988;32: 1269-70. 5. Hemming VG. Rodriguez W. KimHW. etal. Intravenous immunoglobulin treatment of respiratory syncytial virus infections in infants and young children. Antimicrob Agents Chemother 1987;31: 1882-7. 6. Groothuis JR. Levin MJ. Rodriguez W. et al. Use of intravenous gamma globulin to passively immunize high-risk children against respiratory syncytial virus: safety and pharmacokinetics. Antimicrob Agents Chemother 1991;35: 1469-73. 7. Prince GA, Jenson AB. Horswood RL. Camargo E. Chanock RM. The pathogenesis of respiratory syncytial virus infection in cotton rats. Am J PathoI1978;93:771-92. 8. Porter DO. Prince GA. Hemming VG. Porter HG. Pathogenesis of human parainfluenza virus 3 infection in two species of cotton rats: Sigmodon hispidus develops bronchiolitis. while Sigmodon fulviventer develops interstitial pneumonia. J ViroI1991;65: 103-11. 9. Prince GA, Hemming VG. Horswood RL. Baron PA, Murphy BR. Chanock RM. Mechanism of antibody-mediated viral clearance in immunotherapy of respiratory syncytial virus infection of cotton rats. J Virol 1990;64:3091-2. 10. Gilbert BE. Wyde PRo Ambrose MW. Wilson SZ, Knight V. Further studies with short duration ribavirin aerosol for the treatment of influenza virus infection in mice and respiratory syncytial virus infection in cotton rats. Antiviral Res 1992; 17:33-42.

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antibodies [9]. Treatment with IgG caused a significant reduction in virus titers, whereas treatment with human serum albumin was not effective. We have previously demonstrated that the therapeutic effect observed in antibody recipients is due to bona fide passive immunity rather than neutralization of virus in vitro following homogenization [I]. The effectiveness ofnebulized IgG was dose dependent, as it increased proportionately to the exposure time (i.e., the longer the exposure, the larger the reduction in pulmonary virus). Preparations of human IgG containing higher levels of neutralizing antibodies against RSV, such as those used in clinical trials [5], delivered in a small-particle aerosol, would be expected to be more effective than the preparation tested in this study and may allow the use of more dilute solutions of IgG and shorter exposure times. Aerosolized IgG was not effective in reducing nasal virus titer. This finding is consistent with previous studies in cotton rats and owl monkeys that showed that IgG, instilled directly into the airway, was effective in reducing pulmonary but not nasal virus in RSV-infected animals [3, 4]. An aerosol containing higher levels of anti-RSV antibodies, longer exposure to the aerosol, or multiple exposures may effect a reduction in nasal virus such as that seen when high doses of IgG are administered systemically [I, 2, 5]. The pathologic changes in the lungs of RSV-inoculated untreated animals were mild, and we were not able to assess whether aerosolized IgG could favorably affect the progression of the disease. However, our model showed that aerosolized IgG does not appear to exacerbate pulmonary pathology. The possible disadvantages of administration of IgG by small-particle aerosol include denaturation ofthe IgG during nebulization with sensitization of patients and induction of airway hyperreactivity. While the latter issue cannot currently be addressed with the cotton rat model, the degree of virus clearance seen in animals treated with aerosolized IgG suggests that denaturation, if present, is not significant. Aerosolized ribavirin administered in the same fashion as IgG, at the concentration recommended for human use (20 mg/ml.), did not effect a reduction in pulmonary virus. This observation is consistent with that of Gilbert et al. [10], who showed that multiple 30-min exposures (starting the day after RSV inoculation) to a more concentrated solution of ribavirin (60 mg/mL) delivered by small-particle aerosol were necessary to demonstrate an effect. In that study [10], the degree of reduction in pulmonary virus seen even with the extended ribavirin protocol (20 mg/ml., nebulized over 18 h/day for 3 days, starting 24 h after virus inoculation) is still two- to fourfold lower than that after a single 15-min exposure to aerosolized IgG. We plan to study the effect of repeated exposures to aerosolized IgG to determine if its effectiveness can be increased. The findings of the present study are important for the following reasons: First, aerosolized IgG may be useful in the

JID 1992; 166 (December)

Immunotherapy of respiratory syncytial virus infection in cotton rats (Sigmodon fulviventer) using IgG in a small-particle aerosol.

To determine whether aerosolized IgG can be used effectively in the treatment of respiratory syncytial virus (RSV) infections, cotton rats were infect...
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