Gregory A. Prince, 1 Amy Mathews, 1 Spencer J. Curtis, 1 and David D. Porter 2

1326 Treatment of Respiratory Syncytial Virus Bronchiolitis and Pneumonia in a Cotton Rat Model with Systemically Administered Monoclonal Antibody (Palivizumab) and Glucocorticosteroid Gregory A. Prince, 1 Amy Mathews, 1 Spencer J. Curtis, 1 and David D. Porter 2 1 Virion Systems, Rockville, Maryland; 2 Department of Pathology and Laboratory Medicine, School of Medicine, University of California, Los Angeles Parenteral treatment of an experimental respiratory syncytial virus (RSV) infection in a cotton rat model with a monoclonal antibody directed against the viral F protein resulted in the clearance of infectious virus within 24 h but had no effect on the pulmonary pathology at 24 h and only a small effect on the pulmonary pathology at 72 h. Treatment with parenteral triamcinolone acetonide dramatically reduced the pathologic lesions of viral bronchiolitis and pneumonia but resulted in the delayed clearance of the virus. The combination of the monoclonal antibody given in a single dose 72 h after infection, combined with 3 daily doses of the corticosteroid starting 72 h after infection, demonstrated both the loss of infectivity and the disappearance of lesions. No rebound of lesions or infectivity was noted. Combined antiviral and anti-inflammatory therapy for RSV disease appears promising. Respiratory syncytial virus (RSV), the leading cause of infectious pulmonary disease in infants and children worldwide (reviewed in [1]), is increasingly recognized as a cause of serious disease in adults, particularly the elderly [2] and in immunocompromised persons [3]. Vaccine development programs spanning 4 decades have yet to result in a safe and effective RSV vaccine [4]. A major breakthrough in RSV prophylaxis has been the development and licensure both of polyclonal and monoclonal immunoglobulin products for use in high-risk infants (RespiGam [RSVIg] and Synagis [palivizumab]; MedImmune, Gaithersburg, MD). Although highly effective and apparently cost-effective in the targeted population, these products have not been widely used in persons at normal risk owing to the cost. Since most hospital admissions for RSV are among normal-risk patients, there is a pressing need for an effective RSV therapeutic [5]. More than a decade has elapsed since the licensure by the US Food and Drug Administration (FDA) of ribavirin (Virazole; ICN Pharmaceuticals, Costa Mesa, CA) as an RSV therapeutic. Initial enthusiasm for this drug, however, has gradually been replaced by increasing resistance to its use, as concerns over efficacy, cost, and ease of use have grown [6]. The use of RSV immunoglobulin in a therapeutic setting was first suggested in studies in cotton rats. Immunoglobulin given to RSV-infected animals reduced pulmonary virus titers 100- Received 23 June 2000; revised 28 July 2000; electronically published 9 October 2000. Financial support: National Institutes of Health (AI-33722); Virion Systems corporate funds. Reprints or correspondence: Dr. Gregory A. Prince, Virion Systems, 9610 Medical Center Dr., Ste. 100, Rockville, MD 20850 (gprince@erols.com). The Journal of Infectious Diseases 2000;182:1326 30 2000 by the Infectious Diseases Society of America. All rights reserved. 0022-1899/2000/18205-0006$02.00 fold within 6 h [7], a level of reduction far greater than that seen with ribavirin in the same animal model [8]. This observation led to a series of therapeutic clinical trials of RSV immunoglobulin formulations, including generic human IgG given parenterally [9] or via aerosol [10, 11], RSVIg given parenterally [12, 13], and palivizumab given parenterally [14]. Although these studies showed immunoglobulin therapy to be safe, thus overturning a long-standing dogma [15], and monitored virus titers showed a reduction in immunoglobulintreated patients, none resulted in a clinically beneficial outcome. Although the goal of the clinician in treating an infectious disease has long been the elimination of the pathogen, there has been a recent and increasing awareness of the need to simultaneously treat the consequences of infection, which often consist of the host response to infection rather than to the direct tissue damage by the pathogen. We reported earlier that an antiviral immunoglobulin and a glucocorticosteroid delivered topically were highly effective in eliminating virus and reversing pulmonary histopathology in a cotton rat model of human parainfluenza virus type 3 (PIV3) [16]. We now extend these findings to treatment of RSV bronchiolitis and pneumonia, using an antiviral monoclonal antibody and a glucocorticosteroid delivered systemically. Materials and Methods Animals. Inbred cotton rats (Sigmodon hispidus) were obtained from a colony maintained at Virion Systems (Rockville, MD). The cotton rats were housed in large polycarbonate rat cages with a bedding of paper mill by-products (Care Fresh; Absorption, Bellingham, WA) and were fed a diet of rodent chow and water. The cotton rat colony was monitored for antibody to paramyxoviruses, RSV, and rodent viruses, and no such antibodies were

JID 2000;182 (November) Treatment of Bronchiolitis and Pneumonia 1327 found. The animals were, on average, 5 weeks old and weighed 60 g at the time of use. Viruses and cells. The prototype Long strain of RSV (American Type Culture Collection, Manassas, VA) was propagated in HEp-2 cells after serial plaque-purification, to reduce defectiveinterfering particles [17]. A single pool of virus containing 10 7.5 pfu/ ml was used for all experiments. Pulmonary virus titers were determined by plaque assay, as described elsewhere [18]. Drugs. Palivizumab (Synagis), a humanized monoclonal antibody directed against the F glycoprotein of RSV [19], was provided by MedImmune. Triamcinolone acetonide suspension, United States Pharmacopeia, was manufactured by Steris Laboratories (Phoenix). Methoxyflurane for anesthesia was obtained from Pitman-Moore (Mundelein, IL). Tissue preparation. After the animals were killed by carbon dioxide inhalation, the heart and lungs were removed together from the cotton rats. The 2 left lobes of the lungs were ligated around the bronchi and then were removed for virus quantitation. The 3 right lobes were then inflated through the trachea to their normal volume with 10% neutral buffered formalin. The trachea was tied with a suture, and the lungs were immersed in formalin for several days. A block was cut in a coronal orientation to show the hilar and peripheral areas of the 3 lobes on a single slide. After paraffin embedding, sections were cut at a thickness of 4 mm and were stained with hematoxylin-eosin. Histopathology scoring. Peribronchiolitis is inflammatory cells, mostly lymphocytes and macrophages, that accumulate around the periphery of small airways. Alveolitis is inflammatory cells, mostly macrophages and neutrophils, within the air spaces. Interstitial pneumonitis is thickening of the alveolar walls, associated with an influx of inflammatory cells of various types. Each of these 3 parameters was scored separately for each lung section on a scale of 0 (no inflammation) to 100 (maximum inflammation). Composite scores were also determined by calculating the arithmetic mean of the 3 individual scores for each lung section. Both the magnitude and the profile of inflammation were different in these experiments than in those that we reported because of the markedly higher dose of virus that each animal received. Experimental design. Cotton rats were anesthetized with methoxyflurane and then were inoculated intranasally with 100 ml of virus suspension: 10 6.5 pfu per animal. Three days thereafter, at the time of peak viral replication, animals were given a single intramuscular (im) injection of palivizumab (15 mg/kg). Animals receiving glucocorticosteroid were given varying im doses of triamcinolone acetonide daily beginning 3 days after challenge. Statistical analysis. Virus titers were expressed as the geometric mean SE for all animals in a group at a given time. Pulmonary parameters were expressed as the arithmetic mean SE. The 2-tailed Student s t test, using summary data, was used to determine the significance of differences between groups. Statistical analysis of composite histology scores was not done, since the data are disparate. days after infection (figure 1, group 2). A single dose of palivizumab on day 3 (figure 1, group 3) reduced virus titers 11000- fold to undetectable levels within 1 day. Despite the dramatic clearance of virus, however, palivizumab-treated animals had little improvement in pulmonary lesions (figure 2, group 3; P 1.05 on day 4; P!.05 on day 6 vs. untreated animals in group 2), as shown photographically for this and subsequent treatments in figure 3. A similar lack of or only a small effect of antibody alone was seen for alveolitis and interstitial pneumonitis (data not shown). This parallels our findings with PIV3 [16] and is consistent with the clinical trials alluded to, in which virus titers were reduced, and clinical trends were in a positive direction but without statistical significance. Triamcinolone treatment without palivizumab. The immunosuppressive effect of glucocorticosteroids is well documented and is the basis for the longstanding dogma of not using such drugs without antimicrobial coverage in patients with an infectious disease. In the current experiments, triamcinolone significantly ( P!.001 for groups 4 and 5) delayed viral clearance in a dosedependent manner (figure 1), with the highest dose resulting in the greatest impairment of clearance. However, unlike the previous experiments with PIV3 [16], the yield of RSV did not exceed that of untreated cotton rats either at 4 or 6 days after infection. Results Palivizumab treatment without triamcinolone. Infected, untreated animals developed pulmonary virus titers of 10 6 pfu/g 4 Figure 1. Comparison of respiratory syncytial virus (RSV) lung titers 4 and 6 days after infection by treatment group (9 12 animals per day per treatment group). Significant at P!.001 (*) or at P!.05 (#), compared with infected but untreated animals. Bars are SE; im, intramuscularly.

1328 Prince et al. JID 2000;182 (November) Figure 2. Peribronchiolitis scores by treatment group at 4 and 6 days after infection with respiratory syncytial virus (RSV; 9 12 animals per day per treatment group). Significant at P!.001 (*), P!.02 ( ), or P!.05(#), compared with infected but untreated animals. Bars are SE; im, intramuscularly. All 3 groups treated with triamcinolone alone had accelerated clearance of peribronchiolitis (figure 2, groups 4 6). The most dramatic improvement was in animals receiving the highest dose of triamcinolone ( P!.001 vs. group 4, with either group 2 or 3 on both days 4 and 6). Treatment with palivizumab plus triamcinolone. As we reported elsewhere regarding PIV3 pneumonia [16], cotreatment with an antiviral antibody and a glucocorticosteroid accelerated the clearance of virus at the same time that it reversed the histopathologic changes in the lungs. There was a steep steroid dose-effectiveness curve: a dose of 16 mg/kg/day (days 3 5) resulted in a highly significant improvement in peribronchiolitis (figure 2, group 7; P!.001 vs. group 2, days 4 and 6), whereas a 4-fold lower dose was effective only on day 6 (figure 2, group 8). As with other treatment modes, the patterns for alveolitis and interstitial pneumonitis were essentially the same as those for peribronchiolitis (data not shown). Is there a rebound effect? In our prior report on PIV3, we showed that animals cotreated with antibody and triamcinolone remained free of virus after cessation of therapy on day 5 but experienced a reappearance of pulmonary histopathology that approached, but did not exceed, that of infected but untreated animals [16]. Prolongation of triamcinolone therapy in that study for an additional 3 days eliminated the rebound effect. To test for the possibility of rebound in the current experimental system, we infected and treated animals as in the above experiments, administering palivizumab on day 3 and triamcinolone on days 3 5. Animals were killed at 2-day intervals through postchallenge day 14. Figure 4 demonstrates that, unlike our prior work with PIV3, there is no rebound of histopathology in RSV-infected cotton rats that were cotreated with palivizumab and triamcinolone. This figure also shows that palivizumab alone has little effect on pulmonary histopathology, which is present, even if minimal, through day 14. Effect of rechallenge. Although combined therapy of RSV infection appears to be safe, the effect of therapy on subsequent RSV infection is an issue that, in light of the failed vaccine trials of the 1960s, must be addressed. In other words, does combined therapy cause an altered immune status that results in enhanced RSV disease later? We examined this possibility by infecting cotton rats with RSV, treating them with palivizumab and triamcinolone, and then rechallenging them 3 weeks later, as we did elsewhere [16], with PIV3. The results of this experiment paralleled those of the PIV3 report, namely, that animals previously infected with RSV and given combined therapy had no evidence of enhanced histopathology after rechallenge. Indeed, as in the earlier report, these animals showed milder histologic changes than did animals undergoing either primary or secondary RSV infection (data not shown). Discussion Both the medical and the economic importance of RSV are attested to by the extensive array of candidate antiviral compounds under development [20]. The current expense of passive prophylaxis, the difficulties spanning 4 decades with vaccine development, and the likelihood that many patients will not respond to vaccination ensure the need for RSV therapies into the indefinite future. The development of such therapies, however, has been hampered by 2 factors. The first has been the difficulty in formulating an RSV antiviral that is highly effective, nontoxic, and easily administered. This difficulty appears to have been overcome by the development of palivizumab. The second factor has been the failure of any RSV antiviral compound to modulate the host inflammatory response to RSV infection, which appears to account for most of the disease manifestation. The lack of clinical efficacy of RSV immunoglobulins, either polyclonal or monoclonal, despite their acceleration of viral clearance, strongly endorses the hypothesis that an effective therapeutic regimen must down-regulate the host inflammatory response, in addition to eliminating the virus. Our prior reports documented the ability of such a combined approach to reverse pulmonary disease caused by human PIV3 and by RSV in the cotton rat model, when the antiviral component was polyclonal immunoglobulin and the combined formulation was administered intranasally [16, 21]. Although we still see the potential value in a topically administered formu-

JID 2000;182 (November) Treatment of Bronchiolitis and Pneumonia 1329 Figure 3. Lung histology in cotton rats infected with respiratory syncytial virus (RSV) 6 days earlier. Bronchiole is shown in each frame. A, Uninfected and untreated animals; B, infected and untreated animals; C, infected and treated with monoclonal antibody only animals; D, infected and treated with both monoclonal antibody and triamcinolone animals. Infected but untreated animals had severe peribronchiolitis, interstitial pneumonitis, and alveolitis. Treatment with monoclonal antibody alone had little to no effect on intensity of the lesions. Treatment with triamcinolone together with monoclonal antibody abolished the peribronchiolitis and markedly reduced the interstitial pneumonitis and alveolitis. Hematoxylineosin stain; original magnification, 120. lation, logistic considerations that involves the stability of a combined formulation and the practicality of a delivery system adaptable to infants will probably delay clinical testing and implementation of such an approach. In the meantime, an impressive track record of systemic RSV prophylaxis with the use of palivizumab has been achieved. Unlike its first-generation predecessor, RSVIg, which required intravenous infusion over several hours, palivizumab is administered within seconds via intramuscular injection. Similarly, glucocorticosteroids have an extensive track record of efficacy, safety, and simple administration via parenteral injection. Thus, the methodology described in the current report has logistic advantages of employing 2 drugs that are already licensed by the FDA and administering them in a manner already widely used. Our findings in the current study parallel those in our earlier work on PIV3 in all of the essential elements. First, they demonstrate that palivizumab, although highly effective in eliminating RSV from the experimental host, had no significant effect on pulmonary histopathology, which we consider to be the best surrogate of clinical disease in the cotton rat model. Second, we again showed that glucocorticosteroids, used alone, have a beneficial effect on pulmonary histopathology but at the expense of impaired virus clearance. Such a situation is of unknown, but potential, detriment to the patient and represents an undesirable increased risk of nosocomial spread of RSV, already a major concern in pediatric hospital settings. By combining antiviral and anti-inflammatory drugs, however, we have achieved reversal of pulmonary histopathology while, at the Figure 4. Composite pathology scores 4 14 days after respiratory syncytial virus (RSV) infection by treatment group. Nos. of animals in group are at tops of bars. Statistical evaluation was not performed on these data because of their disparate nature. MAb, monoclonal antibody. Tri, triamcinolone.

1330 Prince et al. JID 2000;182 (November) same time, overcoming the immunosuppressive effect of the glucocorticosteroid. The one point of departure of the current study from our earlier work with PIV3 is the lack of a rebound effect with RSV. It is not clear whether this is due to the different pathogen, the different potency of the antiviral immunoglobulin (monoclonal vs. polyclonal), or the different mode of administration (systemic vs. topical) of both components. Nonetheless, the lack of rebound found in the current study is encouraging, since it suggests that clinical use of the glucocorticosteroid can be limited to a period that will not result in suppression of adrenal function. The selection of triamcinolone acetonide as the anti-inflammatory component was based on our earlier screening of a variety of commonly used glucocorticosteroids [16, 21], which showed triamcinolone to be the most effective when used topically. Systemic use of triamcinolone in a pediatric setting, however, is not common and may be greeted with reluctance by some clinicians. Therefore, we have undertaken a study in which we are comparing triamcinolone with 2 other glucocorticosteroids that are commonly administered systemically by pediatricians, methyl prednisolone and dexamethasone. That study will form the basis of a separate report. References 1. Collins PL, McIntosh K, Chanock RM. Respiratory syncytial virus. In: Fields BN, Knipe DM, Howley PM, et al. Fields virology. 3d ed. Vol. 1. Philadelphia: Lippincott-Raven, 1996:1313 51. 2. Han LL, Alexander JP, Anderson LJ. Respiratory syncytial virus pneumonia among the elderly: an assessment of disease burden. J Infect Dis 1999; 179:25 30. 3. McCarthy AJ, Kingman HM, Kelly C, et al. The outcome of 26 patients with respiratory syncytial virus infection following allogeneic stem cell transplantation. Bone Marrow Transplant 1999;24:1315 22. 4. Dudas RA, Karron RA. Respiratory syncytial virus vaccines. Clin Microbiol Rev 1998;11:430 9. 5. Domachowske JB, Rosenberg HF. Respiratory syncytial virus infection: immune response, immunopathogenesis, and treatment. Clin Microbiol Rev 1999;12:298 309. 6. Ohmit SE, Moler FW, Monto AS, Khan AS. Ribavirin utilization and clinical effectiveness in children hospitalized with respiratory syncytial virus infection. J Clin Epidemiol 1996;49:963 7. 7. 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. 8. Hruska JF, Morrow PE, Suffin SC, Douglas RG. In vivo inhibition of respiratory syncytial virus by ribavirin. Antimicrob Agents Chemother 1982; 21:125 30. 9. Hemming VG, Rodriguez W, Kim HW, et al. Intravenous immunoglobulin treatment of respiratory syncytial virus infections in infants and young children. Antimicrob Agents Chemother 1987;31:1882 6. 10. Rimensberger PC, Schaad UB. Clinical experience with aerosolized immunoglobulin treatment of respiratory syncytial virus infection in infants. Pediatr Infect Dis J 1994;13:328 30. 11. Rimensberger PC, Burek-Kozlowska A, Morell A, et al. Aerosolized immunoglobulin treatment of respiratory syncytial virus infection in infants. Pediatr Infect Dis J 1996;15:209 16. 12. Rodriguez, WJ, Gruber WC, Welliver RC, et al. Respiratory syncytial virus (RSV) immune globulin intravenous therapy for RSV lower respiratory tract infection in infants and young children at high risk for severe RSV infections. Pediatrics 1997;99:454 61. 13. Rodriguez WJ, Gruber WC, Groothuis JR, et al. Respiratory syncytial virus immune globulin treatment of RSV lower respiratory tract infection in previously healthy children. Pediatrics 1997;100:937 42. 14. Malley R, DeVincenzo J, Ramilo O, et al. Reduction of respiratory syncytial virus (RSV) in tracheal aspirates in intubated infants by use of humanized monoclonal antibody to RSV F protein. J Infect Dis 1998;178:1555 61. 15. Chanock RM, Kapikian AZ, Mills J, Kim HW, Parrott RH. Influence of immunological factors in respiratory syncytial virus disease of the lower respiratory tract. Arch Environ Health 1970;21:347 55. 16. Prince GA, Porter DD. Treatment of parainfluenza virus type 3 bronchiolitis and pneumonia in a cotton rat model using topical antibody and glucocorticosteroid. J Infect Dis 1996;173:598 608. 17. Gupta CK, Leszczynski J, Gupta RK, Siber GR. Stabilization of respiratory syncytial virus (RSV) against thermal inactivation and freeze-thaw cycles for development and control of RSV vaccines and immune globulin. Vaccine 1996;14:1417 20. 18. Prince GA, Jenson AB, Horswood RL, Camargo E, Chanock RM. The pathogenesis of respiratory syncytial virus infection in cotton rats. Am J Pathol 1978;93:771 91. 19. Johnson S, Oliver C, Prince GA, et al. Development of a humanized monoclonal antibody (MEDI-493) with potent in vitro and in vivo activity against respiratory syncytial virus. J Infect Dis 1997;176:1215 24. 20. Prince GA. Respiratory syncytial virus antiviral agents. Exp Opin Ther Patents 1999;9:753 62. 21. Prince GA, Hemming VG. Method for treating infectious respiratory diseases. US patent 5,290,540, 1994.