Comparative Effects of Two Neutralizing Anti-Respiratory Syncytial Virus (RSV) Monoclonal Antibodies in the RSV Murine Model: Time versus Potency

Respiratory syncytial virus (RSV) is the leading viral pathogenresponsible for bronchiolitis and pneumonia in infants and youngchildren worldwide. We have previously shown in the mouse modelthat treatment with an anti-RSV neutralizing monoclonal antibody(MAb) against the F glycoprotein of RSV, palivizumab, decreasedlung inflammation, airway obstruction, and postmethacholineairway hyperresponsiveness. MEDI-524, or Numax, is a new MAbderived from palivizumab with enhanced neutralizing activityagainst RSV. We compared the effects of these two MAbs on differentmarkers of disease severity using the murine model of RSV infection.BALB/c mice were intranasally inoculated with RSV A2. Palivizumabor MEDI-524 was administered once at either 24 h before or 48h after RSV inoculation. Regardless of the time of administration,all treated mice showed significantly decreased RSV loads inbronchoalveolar lavage samples measured by plaque assay. OnlyMEDI-524 given at –24 h significantly decreased lung RSVRNA loads on days 5 and 28 after RSV inoculation. Pulmonaryhistopathologic scores, airway obstruction, and postmethacholineairway hyperresponsiveness were significantly reduced in micetreated with MEDI-524 at 24 h before inoculation, compared withuntreated controls and the other regimens evaluated. MEDI-524was superior to palivizumab on several outcome variables ofRSV disease assessed in the mouse model: viral replication,inflammatory and clinical markers of acute disease severity,and long-term pulmonary abnormalities.

INTRODUCTION

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Introduction

Respiratory syncytial virus (RSV) is the leading cause of lowerrespiratory tract disease in infants and young children worldwide.Although certain populations, such as children with chroniclung disease, congenital heart disease, prematurity, or immunodeficiency,are at increased risk for severe RSV disease, most infants hospitalizedfor RSV infection are previously healthy and have no known riskfactors (3). Palivizumab (Pvz) (Synagis; MedImmune, Gaithersburg,MD) is a humanized neutralizing immunoglobulin G1 (IgG1) monoclonalantibody (MAb) directed against the F protein of RSV and iscurrently approved for prevention of severe RSV infection inhigh-risk children (13). Although this preventive strategy hasdramatically reduced the number of hospitalizations due to RSVin the target populations (11, 13), there are still a smallnumber of breakthrough hospitalizations. These hospitalizationsdo not appear to be related to the emergence of palivizumab-resistantmutants (10). Thus, the development of anti-RSV antibodies withmore potent neutralizing activities, longer half-lives, or morefavorable pharmacokinetic profiles and distribution characteristicshas the potential to improve outcomes in patients with RSV disease.Potential benefits could be achieved not only for acute diseasebut also for the long-term consequences of RSV infection, suchas recurrent wheezing and airway hyperresponsiveness (25, 27).

MEDI-524 (Numax) is a novel recombinant humanized IgG1 MAb derivedfrom palivizumab, with more potent anti-RSV neutralizing activity.MEDI-524 was derived by in vitro affinity maturation of themurine complementary determining regions of the heavy and lightchains of palivizumab. Like palivizumab, the specific activityof MEDI-524 is directed to a highly conserved neutralizing epitopeof the F glycoprotein. Its binding affinity is approximately70-fold greater than that of palivizumab, which is attributedto a 4-fold increase in the association rate and an approximately17-fold decrease in the dissociation rate. In vitro studiesindicate an 18-fold-increased neutralizing activity comparedwith that of palivizumab. In the cotton rat model, at equivalentserum concentrations, MEDI-524 is 50 to 100 times more potentthan palivizumab. It decreases the RSV load in the lungs and,more importantly, in the upper respiratory tract (32). A phaseIII clinical study comparing MEDI-524 to palivizumab, to determinethe safety and efficacy of MEDI-524 in reducing RSV hospitalizationin high-risk children, is currently under way.

We have previously shown in the mouse model that RSV alone inducedlong-term airway disease, defined by persistent airway hyperresponsivenessand chronic inflammatory changes in the lungs for up to 154days after infection (14). Treatment with palivizumab markedlydecreased RSV replication and was associated with significantreduction of inflammatory and clinical markers of disease severity(17). The present study was designed to compare the effectsof the two MAbs on different aspects of RSV respiratory infectionduring both the acute and chronic phases of the disease.

MATERIALS AND METHODS

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Materials and Methods

Animals. Seven-week-old female, pathogen-free BALB/c mice were purchasedfrom Charles River Laboratories (Wilmington, MA) and housedin the animal care facility of our institution in separate filtertop cages. Mice were housed in groups according to the experimentalsetup. Their virus-free status was confirmed by use of sentinelmice that were regularly tested for different pathogens as previouslydescribed (14, 17, 22). This study was approved by the InstitutionalAnimal Care and Research Advisory Committee at the Universityof Texas Southwestern Medical Center at Dallas.

Virus. Human RSV stock A2 (RSV A2) was grown and quantified on HEp-2cells and was prepared and stored as previously described (14,17). The HEp-2 cells were maintained in Eagle minimal essentialmedium (EMEM) supplemented with HEPES, glutamine, amphotericin,streptomycin, penicillin G, and 10% fetal bovine serum (10%EMEM). The virus titer was determined by a methylcellulose plaqueassay, with a lower level of detection of 1.7 log₁₀ PFU/ml.In addition, the RSV aliquot used for each individual experimentwas tested in duplicate at the time of inoculation.

RSV inoculation. Mice were anesthetized using inhaled methoxyflurane and intranasallyinoculated on day 0 with 10^7.5 PFU/ml of RSV A2 in 100 µlof 10% EMEM (17). Uninfected control animals were sham inoculatedwith 100 µl of sterile 10% EMEM. Animals were allowed30 seconds to aspirate the inoculum while held upright untilfully recovered from the anesthesia.

Experimental design and sample collection. Mice were anesthetized with an intraperitoneal injection of75 mg/kg (body weight) of ketamine and 5 mg/kg of acepromazinebefore exsanguination by cardiac puncture. Four independentexperiments were performed, and each included four to six miceper time point per treatment group. Mice were evaluated on days1, 5, 28, and 70 after inoculation. Bronchoalveolar lavage (BAL)specimens were obtained as previously described to measure RSVloads (14, 17). Previous experiments in which blood was rinsedfrom the lungs at the time of sample collection showed thatcirculating palivizumab did not have a significant impact ondecreasing the RSV loads measured in lungs and BAL samples (17).Whole-lung supernatants were also collected to determine RSVloads by plaque assay in parallel with real-time PCR (RLT-PCR)during both the acute (days 1 and 5) and chronic (days 28 and70) phases of the disease (17). A histological evaluation wasperformed with a separate set of mice, using whole-lung specimensfixed with 10% buffered formalin solution.

To compare the effects of anti-RSV MAbs on viral replicationand disease severity, mice were treated with a single dose (50mg/kg; 1.25 mg per mouse) of one of the two different neutralizingMAbs against the RSV F protein. Palivizumab was reconstitutedas previously described (17). MEDI-524 was provided by MedImmune,Inc. (Gaithersburg, MD); formulated at 102.9 mg/ml in 25 mMhistidine-HCl buffer with a pH of 6.0, it was stored at 4°Cuntil administration and was subsequently diluted in phosphate-bufferedsaline to achieve the desired concentration. Both antibodieswere administered intraperitoneally in a total volume of 100µl, either at 24 h before RSV intranasal inoculation asprophylaxis (Pvz –24 h versus MEDI-524 –24 h) orat 48 h after RSV inoculation as treatment (Pvz +48 h versusMEDI-524 +48 h). The doses of the anti-RSV MAbs used in thepresent experiments were selected on the basis of our previousstudies, which had the purpose of comparing the effects of theMAbs on markers of disease severity rather than characterizingthe pharmacokinetic properties of the MAbs in the mouse model(17). Control groups included RSV-infected untreated mice andmedium-inoculated (uninfected) mice. In previous experimentswith palivizumab, the administration of either phosphate-bufferedsaline or an IgG1 isotype-matched control antibody, MEDI-507,in RSV-infected mice as either prophylaxis (–24 h) ortreatment (+48 h) did not affect any of the parameters evaluated,including RSV loads, BAL cytokine concentrations, lung histopathology,and pulmonary function tests (17).

Pulmonary function tests. Unrestrained, whole-body plethysmography (Buxco, Troy, NY) wasused to assess the degree of airway obstruction (AO) by measuringthe baseline enhanced pause (Penh) daily during the first 2weeks in all groups of mice. Airway hyperresponsiveness (AHR)after challenge with inhaled methacholine (50 mg/ml) was measuredonce a week from day 14 until day 70 after RSV inoculation.Penh is a value without dimension that represents a functionof the ratio of peak expiratory flow to peak inspiratory flowand a function of the timing of expiration (represented by the"pause"). Penh values were calculated according to the followingformula: Penh = peak expiratory flow/peak inspiratory flow xpause. These values correlate with pulmonary airflow resistanceor obstruction (7, 14, 17, 22). Measurements were obtained starting5 min after the completion of each methacholine nebulization.Penh values measured during this period were averaged and expressedas delta Penh, which represents the difference between the maximumpostmethacholine Penh values registered and the baseline Penh.Plethysmography was performed with the groups of mice to besacrificed for sample collection at each time point and in anadditional group of mice that was followed throughout each ofthe four independent experiments.

Histopathology. Formalin-fixed lungs were paraffin embedded, sectioned, andstained with hematoxylin and eosin prior to light microscopyexamination. The histopathologic scores (HPS) were determinedby a pathologist unaware of the infection status of the animals.This HPS system assigns values from 0 to 21 and is based ongrading of five different parameters: (i) peribronchiolar andbronchial infiltrates, (ii) bronchiolar and bronchial luminalexudates, (iii) perivascular infiltrates, (iv) monocytes, and(v) parenchymal pneumonia. The higher the score, the greaterthe inflammatory changes in the lung. This scoring system hasbeen previously validated in the RSV mouse model (14, 17).

RSV quantitative culture and RLT-PCR. Eighty-percent-confluent, 2-day-old HEp-2 cells were used tomeasure viral loads in BAL specimens and lung homogenate supernatants(14, 17). Lung homogenate supernatants were processed as describedpreviously (17), and 100-µl samples were used immediatelyfor determination of viral loads by a plaque assay. The remainingsupernatant was frozen at –80°C for viral load measurementsby RLT-PCR. One sample per mouse was evaluated as a single specimen.Briefly, RSV RNA was extracted using ion-exchange minicolumns(RNeasy mini kit; QIAGEN, Valencia, CA), and cDNA was preparedby reverse transcription using 2.5 µM random hexamersfor 10 min at 22°C, 30 min at 42°C, and 5 min at 95°C.Real-time PCR was performed with a Perkin-Elmer/Applied Biosystems7700 sequence detector (Foster City, CA) using 10 µl cDNAin a total volume of 50 µl master mix with the followingrun conditions: 1 cycle of 2 min at 50°C and 10 min at 95°C,followed by 50 cycles of 15 seconds at 95°C and 60 secondsat 60°C. Known concentrations of RSV A2 were used to derivea standard curve. Standards and negative controls were run togetherwith each PCR assay. The lower limit of detection of the assayis 5 copies/ml. Quantitative RLT-PCR was used, targeting theconserved region of the RSV N gene. Forward (5'-AGA TCA ACTTCT GTC ATC CAG CAA) and reverse (5'-TTC TGC ACA TCA TAA TTAGGA GTA TCA AT) primers amplified an 85-bp region containingthe 25-mer 6-carboxyfluorescein-labeled probe (5'-CAC CAT CCAACG GAG CAC AGG AGA T), as described elsewhere (6, 31).

To further evaluate the significance of the RSV load measuredby RLT-PCR, we conducted a set of experiments comparing miceinoculated with live RSV (10^6.8 PFU), UV-inactivated RSV (UVcross-linker for 2 h; Fisher Biotech), and control medium (EMEM).Results showed that on day 5, mice inoculated with live RSVhad a median of 7.57 (range, 7.48 to 7.68) log₁₀ copies/ml,UV-inactivated RSV had a median of 2.51 (range, 2.15 to 3.01)log₁₀ copies/ml, and medium-inoculated controls had <100copies/ml (P < 0.001). Likewise, on day 42, mice inoculatedwith live RSV had a median of 3.99 (range, 3.98 to 4.01) log₁₀copies/ml, while UV-inactivated RSV had <100 copies/ml andmedium controls had <100 copies/ml. These experiments suggestthat our ability to detect RSV RNA in the chronic phase of thedisease is highly dependent on inoculation with live virus.

Statistical methods. Differences between groups were tested using the Kruskal-Wallisone-way analysis of variance on ranks, since most of the datadid not follow a normal distribution. When this test demonstrateda significant difference between groups (P < 0.05), two differentmethods of correcting for multiple comparisons were used (Dunn'sand Tukey's tests). For all statistical analyses, Sigma Stat2000 software (SPSS Science, Chicago, IL) was used.

RESULTS

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Results

Comparative effects of anti-RSV antibodies on RSV replication determined by quantitative culture and RLT-PCR. (i) Plaque assay tissue culture. As shown in our previous studies in this mouse model, viralloads in RSV-infected untreated mice significantly increasedfrom day 1 to day 5, both in BAL samples (median [25th to 75thpercentile], 2.6 [2.26 to 2.72] PFU/ml log₁₀ on day 1 versus3.05 [2.62 to 3.6] PFU/ml log₁₀ on day 5; P < 0.01) and inlung specimens (median [25th to 75th percentile], 2.56 [2.4to 3.05] on day 1 versus 3.49 [3.18 to 3.59] on day 5; P <0.01), providing evidence of active viral replication.

(a) BAL. Administration of palivizumab and MEDI-524 resulted in significantreductions of RSV loads in BAL samples at all time points evaluatedin all treatment groups. Treatment of mice before inoculation(Pvz –24 h and MEDI-524 –24 h) and after inoculationbut before the onset of clinical disease (Pvz +48 h and MEDI-524+48 h) was effective in significantly decreasing RSV loads inBAL samples compared with RSV-infected untreated controls (P< 0.001) (Fig. 1A). On days 1 and 5, the number of plaquesdetermined by quantitative culture was reduced to below thelimit of detection of the assay (1.7 PFU/ml log₁₀) only in micetreated with MEDI-524 at 24 h before or 48 h after RSV inoculation.On days 28 and 70 postinoculation, no virus was detected bythe plaque assay.

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FIG. 1. Comparative effects of anti-RSV antibodies on RSV replication determined by quantitative culture of BAL samples and lung supernatant homogenates. Mice were intranasally inoculated with RSV (10^7.5 PFU/ml) or 10% EMEM (uninfected controls) and treated with a single dose of either palivizumab or MEDI-524 at one of several different time points. On days 1 and 5 after RSV inoculation, mice were sacrificed, and BAL and lung samples from the same mice were harvested (n = 8 to 16). The RSV loads in BAL samples (A) and both BAL samples and lung specimens (B) were determined by plaque assays. (A) The treatment groups consisted of uninfected controls, RSV-infected untreated mice, and RSV-infected mice receiving either palivizumab or MEDI-524 at either –24 h (before) or +48 h (after) with respect to RSV infection. (B) Viral load was significantly higher (P < 0.05) on day 5 in lung specimens than in BAL samples for RSV-infected untreated mice and mice that received palivizumab at –24 h or +48 h of RSV infection. The values are represented as median RSV log₁₀ PFU/ml (with error bars indicating 25th to 75th percentiles) in BAL fluid (*, P < 0.001) or lung supernatant ( ${ddagger}$ , P < 0.001) by way of Kruskal-Wallis analysis of variance on ranks for comparison with RSV-infected untreated controls. VL, viral load; dashed line, lower limit of detection of the plaque assay.

(b) Lungs. Lung RSV loads were reduced by >1 log on days 1 and 5 afterinoculation in treated mice, regardless of the time of MAb administrationor the regimen used.

On day 1, both prophylactic regimens significantly decreasedlung RSV loads compared with those of RSV-infected untreatedcontrols (median [25 to 57th percentile], 2.56 [2.4 to 3.05]PFU/ml log₁₀ in RSV-infected untreated controls versus <1.7[<1.7] PFU/ml log₁₀ in MEDI-524 –24 h and versus <1.7[1.7 to 1.92] PFU/ml log₁₀ in Pvz –24 h; P < 0.001).At this time point no significant differences were found inRSV loads measured in BAL and lung samples within each groupof mice.

On day 5, which corresponds to peak viral replication in thismodel (14), RSV loads in lung specimens were significantly greaterthan in BAL samples in RSV-infected untreated mice (P < 0.01)and in mice treated with palivizumab (P < 0.05). Only MEDI-524significantly reduced RSV loads in both BAL and lung specimensto below the limit of detection of the plaque assay (Fig. 1B).

(ii) RLT-PCR. To further characterize the dynamics of RSV infection, we measuredviral loads in lung supernatants by RLT-PCR in parallel withplaque assays in two independent experiments, each includingfour mice per time point per treatment group. Following a similarpattern as for the plaque assays, RSV RNA loads measured byRLT-PCR peaked around day 5 after inoculation (5, 6). On day1, mice treated with either MEDI-524 or Pvz at –24 h hadlower RSV RNA loads, but only in one experiment was the reductionstatistically significant compared with that for the RSV-infecteduntreated mice. On day 5, MEDI-524 given 24 h before inoculationconsistently decreased RSV RNA loads, compared with RSV-infecteduntreated controls (P < 0.001) (Table 1). Palivizumab treatmentat –24 h also showed reduced RSV RNA, but the differencedid not reach statistical significance (Table 1). In contrastto RSV loads measured by plaque assay, which became undetectableby day 7 after inoculation, RSV RNA loads measured by RLT-PCRremained consistently positive for 70 days in lung supernatants.At 4 weeks after inoculation, there was a trend towards lowerRSV RNA loads in mice treated with palivizumab at –24h or +48 h. MEDI-524 given at –24 h or +48 h significantlydecreased the RSV RNA loads compared to those of RSV-infecteduntreated controls. Ten weeks after inoculation, RSV RNA wasstill detected in all RSV-infected mice, although at much lowerconcentrations. Mice treated with MEDI-524 at –24 h showeda clear trend toward lower RSV RNA loads, but the reductiondid not achieve statistical significance (Table 1). All uninfectedcontrols showed consistently negative values by RSV real-timePCR at all time points evaluated.

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TABLE 1. Comparative effect of anti-RSV neutralizing monoclonal antibodies on lung RSV-RNA loads, determined by real-time PCR

Effects of anti-RSV antibodies on lung inflammation. The RSV-infected untreated mice, compared to sham-inoculatedcontrols, showed significantly greater HPS, with dense inflammatoryinfiltrates and severe pneumonia which peaked on day 5 afterinoculation (Fig. 2). Although the acute inflammatory infiltratesgradually declined and changed over time, MEDI-524 given at–24 h significantly decreased lung inflammation duringboth the acute (day 5) and chronic phases of the disease. Atweek 10, the lung inflammatory infiltrates partially involvedthe larger vessels and airways and were composed mostly of lymphocytes,macrophages, and plasma cells. No eosinophils were observed.At this late time point (week 10), the lung HPS of mice thatreceived MEDI-524 at –24 h were comparable to those ofthe uninfected controls. In contrast, neither palivizumab givenat –24 h or +48 h nor MEDI-524 at +48 h significantlyreduced lung inflammation (Fig. 3). Since whole-lung specimenswere used to measure either RSV RNA loads or HPS, we could notdetermine correlations between RSV RNA loads and lung HPS.

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FIG. 2. Impact of treatment with anti-RSV neutralizing MAbs on lung HPS of mice infected with RSV. Mice were inoculated with RSV and treated as described in Materials and Methods. On days 5 and 70, lung specimens were obtained and subsequently fixed and stained for further evaluation by a pathologist unaware of the status of the animals. Bars represent the results of three independent experiments with 8 to 10 mice per time point in each treatment group. Treatment groups consisted of uninfected controls, RSV-infected untreated mice, and RSV-infected mice treated with either palivizumab or MEDI-524 at –24 h (before) or at +48 h (after) with respect to RSV infection. Values are shown as medians, with error bars indicating 25th to 75th percentiles. *, P of <0.001 (day 5) and P of 0.002 (day 70) by Kruskal-Wallis analysis of variance on ranks for comparison with RSV-infected untreated controls.

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FIG. 3. Effect of MEDI-524 on lung histopathology of RSV-infected mice. Lung histopathology results for days 5 and 70 postinfection of sham-inoculated controls, RSV-infected untreated controls, and RSV-infected mice treated with MEDI-524 at –24 h are shown. Hematoxylin and eosin stain. Magnification, x40.

Effects of the anti-RSV antibodies on airway obstruction and airway hyperresponsiveness. (i) Airway obstruction. In the acute phase of the disease, RSV-infected untreated mice,compared with uninfected controls, developed significant AOas defined by increased spontaneous Penh values, with two peakson days 1 and 5. The AO peak on day 5 correlated with the worstHPS and clinical signs of disease in RSV-infected mice (P <0.001; r = 0.4). The regimen of palivizumab at –24 h significantlyreduced AO on day 5 but not on day 1, while mice that receivedMEDI-524 at –24 h developed significantly less AO on bothdays 1 and 5 (P < 0.001). Mice treated with either MAb at–24 h remained clinically asymptomatic throughout thedisease course. On visual inspection, mice that received theMAbs before inoculation behaved like uninfected mice, with normalactivities, shiny coats, and general well-being. The administrationof either anti-RSV MAb 48 h after inoculation did not have anyimpact on AO (Table 2). After day 5, AO in all groups of micegradually declined over time, and Penh returned to baselinevalues by day 14.

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TABLE 2. AO in RSV-infected mice treated with different anti-RSV monoclonal antibodies

(ii) AHR. Aerosolized-methacholine challenge elicited increases in Penhin RSV-infected untreated mice, demonstrated by greater postmethacholinedelta Penh values than those of uninfected controls (14, 17).Although mice treated with all different anti-RSV MAb regimensshowed a trend toward less AHR compared with RSV-infected untreatedmice, only MEDI-524 at –24 h significantly decreased AHRat 4 and 8 weeks postinoculation (Fig. 4).

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FIG. 4. Effects of anti-RSV neutralizing antibodies on AHR. To assess AHR, mice were challenged weekly with aerosolized methacholine, and delta Penh was recorded for up to 10 weeks postinfection. Bars represent means ± standard errors of the means for 12 to 16 mice per group. *, P of 0.03 and 0.04 by one-way analysis of variance for comparison with RSV-infected untreated controls at 4 and 8 weeks after infection, respectively.

DISCUSSION

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Discussion

Despite the fact that approximately one-third (25, 27) of patientswho recovered from acute RSV bronchiolitis developed recurrentrespiratory problems, specific therapeutic options for acuteRSV infection are limited, and currently only the antiviralagent ribavirin is approved for this indication (2). The surfaceglycoproteins G and F are the major proteins involved in RSVinfection. The F glycoprotein appears more important for inductionof protective immunity and is associated with production ofhigh serum neutralizing antibody response (8); therefore, thisglycoprotein has been a major focus for therapeutic interventionin RSV disease (15, 21). Previous studies have shown that RSVloads measured shortly after hospitalization in nasal secretionsof infants with RSV bronchiolitis were an independent predictorof disease severity (4). Passive administration of anti-RSVneutralizing antibody against the F protein is safe and effectivefor prevention of severe RSV disease in animal models and humans(11, 13, 17). However, the administration of the anti-RSV MAbas treatment for established RSV infection did not modify theclinical course of the disease, despite significant reductionsof RSV loads measured in tracheal aspirates of children intubatedfor severe RSV disease (16). A double-blind, randomized, placebocontrol study assessing the safety and tolerance of intravenouspalivizumab versus placebo in children admitted to the hospitalwith RSV lower respiratory tract infection (LRTI) showed a trendfor better outcomes in the palivizumab arm (15 mg/kg) than inthe placebo recipients. It should be noted that these childrenwere treated within 3 days of the initiation of symptoms andwere not intubated (23). Since none of the aforementioned studieswere adequately powered to detect significant differences inclinical outcomes, the efficacy of palivizumab for treatmentof RSV LRTI remains unclear.

The results of the present study in the mouse model demonstratethat passive administration of two different anti-RSV neutralizingMAbs against the F glycoprotein effectively reduced RSV replicationwhen administered as either preexposure prophylaxis or earlytreatment. MEDI-524 had superior neutralizing activity againstRSV compared with palivizumab, as measured by plaque assay.It reduced RSV loads to below the limit of detection of theassay in both BAL and lung specimens, regardless of the timingof administration and despite the greater viral loads detectedin lung samples (A. Mejías, S. Chávez-Bueno, A.M. Ríos, M. Fonseca-Aten, E. Peromingo, P. Soni, K. Olsen,A. M. Gómez, D. Pfarr, H. Wu, P. A. Kiener, H. S. Jafri,and O. Ramilo, Abstr. 44th Intersci. Conf. Antimicrob. AgentsChemother., abstr. V-1687, 2004). In this model, RSV could notbe cultured after day 7 postinfection, but genomic RSV RNA wasdetected by RLT-PCR in lung homogenates for up to 10 weeks afterinoculation.

The evidence that the same or different strains of RSV can causerepeated infections throughout patients' lifetimes (29) suggeststhat RSV does not induce durable immunity. Recent studies ofanimal models have shown that RSV infection may result in "latency"or persistence in the lower respiratory tract (6, 24). The persistenceof viral RNA, regardless of the presence of specific neutralizingantibodies, has been shown not only in animal models of RSV(6, 12, 24) but also with human metapneumovirus, a new memberof the Metapneumovirus genus closely related to RSV (1). Thesignificance of the RSV RNA detected in lung specimens duringthe chronic phase of the disease is yet to be defined. It ispossible, however, that viral persistence could contribute tothe delayed effects of severe RSV disease, such as chronic airwayinflammation, by means of low-grade replication in the lowerrespiratory tract as suggested by Schwarze et al., who recoveredlow levels of infectious RSV from the lungs of mice depletedof T cells 150 days postinfection (24). Our results demonstratethat the administration of the more potent neutralizing MAb,MEDI-524, significantly decreased the number of RSV RNA copiesduring both the acute and the chronic phases of the disease.To our knowledge, this is the first demonstration that a MAbcan reduce the RSV RNA load in the lungs and that this reductionwas associated with improved markers of disease severity inRSV infection. Indeed, mice treated with MEDI-524 at –24h, compared with those treated with palivizumab, showed significantlyless lung inflammation not only on day 5, when mice showed theworst degree of pneumonia, but more importantly in the chronicphase. On day 70 after RSV infection, the lung pathology ofmice treated with MEDI-524 at –24 h was very similar tothat of the uninfected controls (HPS median [range], 0 [0 to1] in MEDI-524-treated mice at –24 h versus 0 [0, 0] inuninfected controls) (Fig. 2). The administration of the anti-RSVMAbs at 48 h after inoculation also showed a trend toward decreasedRSV RNA loads but did not modify the lung inflammation and diseasecourse. Taken together, these results indicate that the timingof administration of the MAb has a significant impact on themarkers of disease severity.

These findings are similar to previous experiences reportedfor children and animal models when palivizumab was administeredafter infection (17, 18, 20, 23) and suggest that although directviral cytopathology plays an important role in initiating RSV-induceddisease, it appears that much of the lung injury caused by thisinfection is the result of the host inflammatory response. Hence,when the MAbs were given after infection, they could not reversethe respiratory pathology that had already occurred. Despitethe enhanced neutralizing activity of MEDI-524, viral RNA wasstill detectable in both the acute and chronic phases of thedisease, although at significantly lower concentrations. Theseresults suggest that either the anti-F neutralizing antibodywas not completely effective or that in addition to the F glycoprotein,there are other mechanisms that contribute to RSV-induced disease.It appears that there are at least two types of human antibodyresponses to RSV infection: a response to a mature envelopeon virions that is neutralizing, and a response to immatureforms of envelope that is not. Overlap between the responsesis limited. Parren et al. have suggested that viral evasioncould be explained in part by the expression by the virus ofa conformationally altered mature envelope protein that is lesssusceptible to the anti-F glycoprotein neutralizing activity(19). Recently, it has been suggested that RSV may resist thehost antiviral response by modulating the type I interferonpathway by mechanisms associated with the expression of thenonstructural NS1/NS2 proteins (26). In addition, it is knownthat the RSV G glycoprotein exhibits extensive genetic diversitythat may contribute to antigenic variation of neutralizing epitopes.The G glycoprotein has also been associated with altered chemokineexpression in pulmonary leukocytes as well as altered pulmonaryleukocyte trafficking in response to infection (28, 30).

Little is known about the long-term consequences of RSV prophylaxisin high-risk children. Preliminary results from a prospectivecase-cohort multicenter study suggested that preterm infantswho received palivizumab during the first RSV season had a lowerrisk of subsequent recurrent wheezing at the 12-month follow-upcompared with those who did not receive prophylaxis (E. A. Simoes,X. Carbonell-Estrany, J. Kimpen, C. H. L. Rieger, D. Morris,P. F. Pollack, and J. R. Groothuis, 14th European RespiratorySociety Annual Congress, abstr. 1349, 2004).

Although children hospitalized for RSV LRTI after receivingpalivizumab prophylaxis had lower nasopharyngeal RSV loads thansimilar infants not receiving palivizumab, they still carriedthe virus in the nasal mucosa (9). When assessing the pulmonarydynamics in our experiments, the administration of either palivizumabor MEDI-524 as preexposure prophylaxis markedly decreased thesigns of RSV disease objectively measured with the noninvasiveplethysmograph. Both "prophylactic" groups of mice remainedasymptomatic throughout the course of disease, and the baselinePenh values were comparable to those of uninfected controls.Only MEDI-524 at –24 h was able to significantly decreasethe peak of AO on day 1. Indeed, these mice developed significantlyless long-term AHR at 4 and 8 weeks after the infection. Therewas a trend toward lower AHR in the mice that received MEDI-524at +48 h or palivizumab at –24 h and +48 h although itwas not significant. It is possible that the use of the multiple-testcomparisons for the statistical analysis could have influencedthese results. Thus, it could be argued that if the experimentincluded fewer treatment groups, possibly other MAb regimenswould have demonstrated significant reductions of AHR as well.Taken together, these results emphasize the importance of earlyevents in RSV infection in determining the long-term consequencesof the disease. Despite the fact that the new neutralizing MAbcould not totally prevent the establishment of the infection,it certainly modulated its consequences.

In summary, the present study demonstrated the superiority ofMEDI-524 over palivizumab in preventing the manifestations ofRSV disease. The greater reductions in RSV loads in the lowerrespiratory tract achieved during the acute and chronic phasesof the disease resulted in further attenuation of the lung inflammationand long-term airway disease. Studies to determine whether theanti-RSV antibodies can decrease RSV RNA loads in the respiratorytracts of children and whether those reductions are associatedwith diminished acute and long-term morbidity induced by RSVare warranted.

ACKNOWLEDGMENTS

This work was supported in part by grants by MedImmune, Inc.(to O.R. and H.J.) and the American Lung Association (to H.J.).A.M. was supported in part by the Pediatric Fellowship Awardin Viral Respiratory Infectious Diseases from MedImmune, Inc.,presented at the Pediatric Academic Societies Annual Meeting,and the RGK Foundation fellowship in Infectious Diseases atChildren's Medical Center of Dallas.

We thank Aneta Wozniakowski for technical assistance with RLT-PCRand tissue culture assays and Dave Pfarr and H. Wu at MedImmunefor preparation of the monoclonal antibodies.

FOOTNOTES

* Corresponding author. Mailing address: Department of Pediatrics, University of Texas Southwestern Medical Center, 5323 Harry Hines Boulevard, Dallas, TX 75390-9063. Phone: (214) 648-1261. Fax: (214) 648 1265. E-mail: octavio.ramilo{at}utsouthwestern.edu.

REFERENCES

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