A randomized, double-blind, placebo-controlled study of an RNAi-based therapy directed against respiratory syncytial virus

Abstract

RNA interference (RNAi) is a natural mechanism regulating protein expression that is mediated by small interfering RNAs (siRNA). Harnessing RNAi has potential to treat human disease; however, clinical evidence for the effectiveness of this therapeutic approach is lacking. ALN-RSV01 is an siRNA directed against the mRNA of the respiratory syncytial virus (RSV) nucleocapsid (N) protein and has substantial antiviral activity in a murine model of RSV infection. We tested the antiviral activity of ALN-RSV01 in adults experimentally infected with wild-type RSV. Eighty-eight healthy subjects were enrolled into a randomized, double-blind, placebo-controlled trial. A nasal spray of ALN-RSV01 or saline placebo was administered daily for 2 days before and for 3 days after RSV inoculation. RSV was measured serially in nasal washes using several different viral assays. Intranasal ALN-RSV01 was well tolerated, exhibiting a safety profile similar to saline placebo. The proportion of culture-defined RSV infections was 71.4 and 44.2% in placebo and ALN-RSV01 recipients, respectively (P = 0.009), representing a 38% decrease in the number of infected and a 95% increase in the number of uninfected subjects. The acquisition of infection over time was significantly lower in ALN-RSV01 recipients (P = 0.007 and P = 0.03, viral culture and PCR, respectively). Multiple logistic regression analysis showed that the ALN-RSV01 antiviral effect was independent of other factors, including preexisting RSV antibody and intranasal proinflammatory cytokine concentrations. ALN-RSV01 has significant antiviral activity against human RSV infection, thus establishing a unique proof-of-concept for an RNAi therapeutic in humans and providing the basis for further evaluation in naturally infected children and adults.

RNA interference (RNAi) is a highly conserved natural cellular mechanism of posttranscriptional regulation observed in eukaryotic cell types (1), including mammalian cells (2, 3). Specifically, RNAi is mediated through the RNA-induced silencing complex (RISC) and involves the targeted cleavage of messenger RNA by the complementary, antisense strand of a double-stranded, small interfering RNA (siRNA) resulting in suppressed expression of the corresponding protein. The discovery of RNAi raises the possibility that this cellular pathway can be harnessed as a unique approach to treat human disease. This possibility has been underscored recently by many in vivo studies of RNAi as treatment in a wide range of animal models of disease, including hypercholesterolemia (4, 5), viral hepatitis (6), Huntington’s disease (7, 8), and cancers (9). However, definitive evidence is lacking from well-controlled studies for the effectiveness of an RNAi therapeutic against any human disease.

Respiratory syncytial virus (RSV) is the most common cause of infant hospitalization in the United States, producing ≈10-fold higher mortality rates than influenza in this population (10). RSV also produces significant morbidity and mortality in adult immunocompromised, debilitated, or elderly populations (11). There is no vaccine for RSV and the only approved therapy (ribavirin) is rarely used due to its potential teratogenicity, limited antiviral effect, and controversial clinical effectiveness. Although successful, RSV monoclonal antibody prevention strategies (12) to RSV reach <3% of the at-risk infant population (13). Novel therapeutic strategies for RSV are clearly needed.

RNAi therapeutics show promising effects in murine models of RSV infection (14). The siRNA, ALN-RSV01, is directed against a highly conserved region of the mRNA encoding the nucleocapsid (N) protein. The N protein functions at numerous critical steps in the RSV replication cycle including RNA polymerase function. Its knockdown would result in an antiviral effect through the mechanism of action of the siRNA reducing RSV replication itself. Additionally, its sequence is highly conserved across all known RSV clades, and it was therefore chosen to be the target for antiviral drug development in this program (15). ALN-RSV01 is completely sequence conserved compared to its target and has a robust antiviral effect in vitro against both RSV A and B subtypes. Upon intranasal delivery to the lungs of mice, ALN-RSV01 reduces lung RSV concentration by 3–4 Logs compared to mismatched siRNA controls when administered before RSV inoculation and by 2–3 Logs when given 1–2 days following inoculation (15). ALN-RSV01 was safe when administered intranasally to healthy volunteers at doses up to 150 mg daily for 5 days (16). Healthy adults can be experimentally infected with RSV, causing a self-limited upper respiratory illness (17). We therefore tested the safety and antiviral activity of intranasal ALN-RSV01 in a human wild-type RSV experimental infection model.

Results

Patients.

Eighty-eight subjects were enrolled in the study (Fig. 1); all were evaluated for safety and all 85 who received RSV inoculation were evaluated for treatment effect including the primary endpoint of proportion RSV infected. All subjects who were RSV inoculated received all study drug doses and evaluations. The characteristics of the treatment groups were well balanced at baseline (Table 1).

Fig. 1.

Study design for randomized phase II trial of intranasal ALN-RSV01 versus placebo in subjects experimentally infected with RSV. d, study day; h, hours; i.n., intranasal; RSV, inoculation with RSV, with quantity of inoculum administered to subjects in each cohort indicated in the boxes labeled “Cohort 1” and “Cohorts 2–6”; Rx, dosing with ALN-RSV01 or placebo. Cohorts 1–6 consisted of 8, 8, 18, 16, 24, and 14 subjects, respectively. Three subjects (1 active, 2 placebo) were withdrawn from cohort 6 due to food-related gastroenteritis having received one study drug dose and without receiving RSV inoculation. Thus, 88 were evaluated for safety and 85 for antiviral effect and clinical efficacy. Subjects were quarantined from day −2 through day 11. Nasal washes were obtained daily during quarantine except on days −1, 0, and 1 so as not to affect study drug or RSV inoculation.

Table 1.

Baseline characteristics of subjects

	Placebo (n = 42)	All ALN-RSV01 (n = 43)
Mean age (years)	27.7	28.0
Mean weight (kg)	76.9	79.3
Race (%)
Caucasian	64.3	67.4
Black	11.9	7.0
Other	21.4	25.6
Mean RSV inoculum (Log PFU) (SD)	4.70 (0.261)	4.70 (0.256)
Mean day −2 RSV microneutralization antibody titer (MU*) (SD)	7.25 (1.00)	6.89 (1.03)†

Efficacy evaluable population is shown. Two patients in the placebo group and one in the ALN-RSV01 group were withdrawn before RSV inoculation due to gastroenteritis caused by food poisoning.

*Microneutralization units.

^†Difference not statistically significant by Wilcoxon’s rank-sum test.

Antiviral Effect.

A significantly lower proportion of ALN-RSV01-treated subjects were infected compared to placebo (Table 2). By quantitative culture, ALN-RSV01 was associated with a 38.1% relative reduction in the number of infected subjects (P = 0.009). Antiviral effects of ALN-RSV01 were consistent, with similar magnitudes of effect demonstrated by quantitative real-time (q)RT-PCR and by spin-culture methods. When only those subjects receiving 150 mg of study drug were evaluated (cohorts 2–6), a statistically significant treatment effect was also observed. There was no demonstrable trend to suggest a decreased antiviral response occurring in the low dose (75 mg) group, but the small numbers of subjects in this group (n = 4 ALN-RSV01 recipients) would have prevented the detection of a dose-response effect.

Table 2.

Primary outcome of effects of ALN-RSV01 on ratio of subjects infected with RSV (ALN-RSV01 vs. placebo, all cohorts)

Detection Method	Status	Placebo (n = 42)	All ALN-RSV01 (n = 43)	Magnitude of effect (relative to placebo)*	P value†
Quantitative culture	Infected	30 (71.4%)	19 (44.2%)	38.1%	0.009
	Uninfected	12 (28.6%)	24 (55.8%)	95.1%
qRT-PCR	Infected	36 (85.7%)	29 (67.4%)	21.4%	0.051
	Uninfected	6 (14.3%)	14 (32.6%)	128.0%
Spin-enhanced culture	Infected	31 (73.8%)	22 (51.1%)	30.8%	0.031
	Uninfected	11 (26.2%)	21 (48.8%)	86.3%

*Calculated as (ALN-RSV01 value minus placebo value) divided by placebo value.

^†Mantel–Haenszel test.

The time course of the antiviral effect was also examined. The median incubation period in the placebo and ALN-RSV01 groups was ≈3.5 days. The acquisition of infection over time was reduced in the ALN-RSV01 group (quantitative culture, P = 0.007; qRT-PCR, P = 0.03; Fig. 2). This reduction was observed within 3–4 days after inoculation. No rebound in the acquisition of infection was observed. If the data were censored after day 8, statistical significance remained (P < 0.05 for both PCR and culture).

Fig. 2.

Reverse Kaplan–Meier estimates of RSV infection, according to treatment assignment. Reverse Kaplan–Meier curves of the percentage of subjects infected with RSV are shown for ALN-RSV01 (dashed line) and placebo (solid line), with infection determined either by (A) culture (plaque assay) (P = 0.0069) or (B) qRT-PCR (P = 0.0321). Inoculation with RSV occurred at day 0, whereas daily treatment with intranasal ALN-RSV01 or placebo continued from day −1 through day 3. This thus represents a cumulative plot of infection. When an individual subject first meets the definition of RSV infected, the line rises 1 unit vertically.

The study was not powered to detect statistically significant effects on viral load; however, consistent trends toward lower viral load in ALN-RSV01 recipients compared to placebo were evident (Fig. 3). These trends included lower viral area under the curve (AUC) and peak viral load in subjects receiving ALN-RSV01 (Fig. 3 A and B). Mean daily viral loads (Fig. 3 C and D) were also lower in the ALN-RSV01-treated subjects from days 4 to 6 (quantitative culture) or from days 4 to 7 (qRT-PCR) with a maximal difference in mean viral load of ≈1 Log PFU/mL. There were no differences in incubation period or duration of viral shedding between ALN-RSV01 and placebo and no viral rebound was observed (Fig. 3 C and D).

Fig. 3.

Quantitative viral and disease measures in subjects treated with ALN-RSV01 or placebo. (A–D) Results shown are for both infected and uninfected subjects combined in each treatment arm, with infection measured either by quantitative culture or qRT-PCR. (A and B) The bars show the mean and standard error for viral AUC (area under the curve is calculated as the sum of the areas of each trapezoid, formed by the borders of the previous daily viral load and the subsequent day’s viral load, from day 2 through day 11) and peak viral load over days 2–11 following viral inoculation on day 0. (C and D) The curves show the mean and standard error for viral load on each day from day 2 to day 11. (E–G) Results are for all subjects in each treatment arm, with individual results (dots) and mean for the group (horizontal dashed line) shown for (E) mean total symptom score, (F) mean total directed physical examination (DPE) score, and (G) mean mucus weight. The components of the symptom scoring and the DPE scoring systems are found in Tables S2 and S3. (H and I) Mean daily symptom score and standard error for ALN-RSV01 or placebo by study day, with results shown for (H) all subjects or (I) infected subjects, with infection confirmed by quantitative culture or qRT-PCR. Inoculation with RSV occurred on day 0, and the period of treatment with either ALN-RSV01 or placebo is shown by the solid horizontal bar.

To determine whether the effect of ALN-RSV01 on the primary endpoint (RSV infection) was independent, multiple logistic regression analyses were performed, incorporating the following variables: treatment assignment (ALN-RSV01 vs. placebo), RSV inoculum, pretreatment RSV microneutralization titer, and one of three time frames of the following four intranasal proinflammatory cytokine concentrations: tumor necrosis factor alpha (TNF-α), IFN-α, granulocyte colony stimulating factor (G-CSF), and interleukin-1 receptor antagonist (IL-1RA). The specific cytokines were chosen on the basis of experience in human adults. The cytokine time frames were the influence of pretreatment cytokine concentration (day −2), cytokine AUC on days 2–4 following RSV inoculation, and cytokine AUC on days 2–8. No statistically significant differences in intranasal cytokine AUC or peak cytokine concentrations occurred between ALN-RSV01 and placebo groups. The G-CSF concentrations in the ALN-RSV01 group appeared modestly elevated throughout the observation period compared to the placebo group, whereas all other cytokine concentrations showed inconsistent changes (Fig. S1). Representative logistic regression models are shown in Table 3. In all models, including those for G-CSF, ALN-RSV01 was independently associated with reduced infection (quantitative culture or qRT-PCR, P < 0.05).

Table 3.

Multiple logistic regression evaluation of effects of ALN-RSV01 on ratio of subjects infected with RSV

	Variable	P value	Odds ratio	95% CI
Model 1	ALN-RSV01 vs. placebo	0.006	0.240	0.086–0.665
	RSV inoculum	0.010	16.459	1.975–137.2
	Day −2 RSV microneutralization titer	0.139	0.694	0.427–1.126
	TNF-α AUC (days 2–4)*	0.528	1.034	0.932–1.147
Model 2	ALN-RSV01 vs. placebo	0.005	0.225	0.080–0.633
	RSV inoculum	0.009	14.281	0.935–105.416
	Day −2 RSV microneutralization titer	0.139	0.693	0.426–1.126
	G-CSF AUC (days 2–8)†	0.342	1.000	1.000–1.000

Determined by quantitative culture. CI, confidence interval.

*The immediate postinoculation and incubation periods.

^†The immediate postinoculation period through the end of the definition of infection.

Clinical Effects.

Experimental RSV infection of healthy volunteers produces only mild to moderate upper respiratory tract illness. Within this narrow spectrum of disease severity, evaluation of disease measures in subjects treated with either ALN-RSV01 or placebo showed no statistically significant differences (Fig. 3 E–G). Analysis of mean symptom scores over time in all subjects or only in those who were RSV infected indicated the most marked lowering of symptom scores in ALN-RSV01 relative to placebo occurred on days 4–7 (Fig. 3 H–I). This result corresponded to the interval when the greatest reduction in mean daily viral load occurred (Fig. 3 C and D).

Safety.

Intranasal ALN-RSV01 was well tolerated. Adverse events were well balanced in nature and severity between ALN-RSV01 and placebo (Table S1), with few moderate adverse events (all of which occurred after discharge from quarantine) and no severe or serious adverse events.

Discussion

In this randomized, double-blind, placebo-controlled trial, we demonstrate a statistically significant antiviral effect with the RNAi therapeutic, ALN-RSV01. The proportion of subjects infected as diagnosed by two different culture-based assays was significantly lower in recipients of ALN-RSV01 as compared to placebo (P < 0.05) (Table 2). Furthermore, acquisition of infection over time as diagnosed by either culture or PCR was also significantly reduced (P < 0.01 and P < 0.05, respectively) (Fig. 2). Finally, multiple logistic regression analyses demonstrated that the antiviral effect associated with ALN-RSV01 was statistically independent of other variables (Table 3). ALN-RSV01 therefore produces a demonstrable antiviral effect.

RNA interference appears to be the mechanism producing this antiviral effect. Although it has been argued that some effects of siRNAs may be through induction of cytokines rather than through RNAi itself (18, 19), there is substantial evidence that RNAi is the mechanistic basis for the ALN-RSV01 antiviral effect. First, ALN-RSV01 reduces RSV in a murine model in which mismatched siRNAs (differing from ALN-RSV01 in as few as four nucleotides) produce no anti-RSV effect (15). Second, the cleavage products resulting from RNAi-mediated silencing of the RSV N-protein transcript have been observed in the lungs of RSV-infected mice treated with ALN-RSV01 (15). Third, immunosilent siRNAs targeting the RSV N gene are active against RSV in mice whereas mismatched siRNAs with potent in vitro immunostimulatory activity do not demonstrate anti-RSV activity in mice (15). And finally, evidence from this trial indicates that ALN-RSV01 is contributing to the observed antiviral effect independent of intranasal cytokine induction. Multivariate logistic regression models show that the statistically significant ALN-RSV01 antiviral effect was independent of the other variables examined, including levels of intranasal cytokines. Indeed, in all models where a cytokine effect was evident, it was associated with increased rather than decreased infection (Table 3). This result is likely due to RSV infection itself stimulating cytokines within human respiratory secretions (20). Although it is impossible to exclude a minimal additional effect from cytokine induction, an antiviral effect of ALN-RSV01 that appears to be mediated through RNAi has been demonstrated in this study.

Our findings represent a significant advance in the development of human therapies, as they show a definitive demonstration in humans of an RNAi effect using a synthetic siRNA. This study was designed to establish this proof-of-concept. We therefore incorporated a combination of prevention and treatment modalities into the trial design. This combination was to maximize the chances of observing a statistically significant proof-of-concept antiviral effect while simultaneously minimizing the risk of experimental viral infection to subjects by reducing the number of subjects needed to be exposed to RSV. Therapeutic drug administration in clinical application would not be accomplished before viral exposure. The first administration of ALN-RSV01 on day −1 thus provided the maximal (better-than-nature) opportunity for blocking infection, which was the most statistically significant antiviral effect observed in our study. On the other hand, the duration of dosing of ALN-RSV01 was possibly suboptimal to achieve maximal reductions in viral load or RSV disease measures in infected subjects. Previous phase 1 studies of ALN-RSV01 had evaluated its safety with intranasal dosing in a 5-day dosing scheme (16). Therefore, due to regulatory considerations, we also evaluated a 5-day dosing regimen in this proof-of-concept study. ALN-RSV01 was administered for two daily doses before and for three daily doses after RSV inoculation. Thus ALN-RSV01 administration was completed 2–4 days before the occurrence of peak viral load and peak clinical symptoms of infection and in many cases was actually completed before the incubation period had ended (before initial detection of viral replication in the volunteers).

Whereas this trial was not powered to detect improvements in viral load or RSV disease measures, we did observe some relevant trends for these endpoints. The incubation period of RSV in the placebo and ALN-RSV01 groups was a median of ≈3.5 days. Thereafter, consistently lower daily viral loads were observed in ALN-RSV01 recipients. The antiviral effect appeared to continue through days 6 and 7 after inoculation, showing a maximum 1 Log PFU/mL reduction in viral load compared to placebo. The most marked lowering of symptom scores for the ALN-RSV01 group relative to placebo occurred on days 4–7, concurrent with the relative reduction in viral load. In children, lower RSV loads are associated with decreased requirements for intensive care, decreased respiratory failure, and shorter hospitalizations (21, 22). Therefore, robust reductions in RSV load are likely to translate into reduced disease severity and clinical benefit. Optimal dosing of ALN-RSV01, to be administered through times of active viral replication, may maximize therapeutic effects on viral dynamics and clinical outcomes.

Whereas our study has established the antiviral effect of intranasal ALN-RSV01 administered before and after a large inoculum of wild-type RSV, our study does not establish that ALN-RSV01 will be efficacious in naturally infected patients with established lower respiratory tract disease. This proof-of-concept trial used a nasal spray of ALN-RSV01, a form of delivery that would not be expected to reach the lower respiratory tract. Future trials in naturally infected patients would therefore likely rely on the delivery of aerosolized drug simultaneously to both the upper and the lower respiratory tract at varying time intervals following infection initiated by relatively low inoculum natural RSV exposures. An RSV antiviral may be valuable for infants, but may also be effective in older at-risk populations including immunocompromised adults such as recipients of hematopoietic and solid organ transplantation. There is a time delay in natural RSV infection: RSV infects and causes symptoms in the upper respiratory tract before subsequently moving to involve the lungs (23). In children, the nasal quantity of virus is at or near its peak 1 day after hospitalization (22, 24). In these and other studies, hospitalization occurred ∼4 days after RSV symptoms were first recognized by the parents. Thus, there is a significant window of opportunity that appears to exist between recognition of symptoms and peak viral load, affording time for the application of an antiviral during the first 3 days of symptoms to significantly reduce overall viral load. In light of the significant effect of intranasal ALN-RSV01 shown here on preventing RSV infection when administered before and early during the course of infection, aerosolized ALN-RSV01 may be able to reduce the progression of early RSV infection from the upper respiratory tract into the lower airways. Studies are actively underway to evaluate the human therapeutic potential of aerosolized ALN-RSV01 in naturally infected adult recipients of lung transplantation.

The antiviral effect of ALN-RSV01 against RSV in our experimental infection model shows that the successful delivery of an exogenous siRNA targeting a gene central to viral replication can significantly affect human infection. In addition to supporting further investigation of ALN-RSV01 as a treatment for RSV infection, our results therefore also underscore the broader potential of locally delivered siRNAs as unique antiinfective drugs against other respiratory pathogens.

Materials and Methods

Subjects.

The study enrolled healthy males ages 18–45 with low RSV serum neutralizing antibody concentrations. Exclusion criteria included asthma, smoking, fever, or symptomatic respiratory infection within the previous 2 weeks; medications for rhinitis within the previous 7 days; potential contact with people at risk for severe RSV disease; steroid use in the past month; chronic sinusitis; or PCR positivity for RSV-A or -B, influenza-A or -B, parainfluenza-1, -2, or -3, or human metapneumovirus.

Study Drug.

ALN-RSV01 is a synthetic, double-stranded oligonucleotide consisting of two partially complementary single-strand RNAs (19 paired nucleotides with an overhang of 2 unpaired thymidine nucleotides at the 3′ end) (Fig. S2). The antisense strand is complementary to a 19-nucleotide sequence (residues 3–21) of the mRNA encoding the RSV nucleocapsid N protein. ALN-RSV01 is formulated as a sterile phosphate-buffered solution diluted in normal saline before administration. ALN-RSV01 (75 or 150 mg) or placebo (sterile normal saline) was administered by nasal spray (Becton-Dickinson Accuspray), 0.5 mL/naris.

Inoculating Virus (RSV).

RSV-A (Memphis 37), from an infant hospitalized for bronchiolitis, was GMP manufactured using vero cells. The isolate was plaque picked and passaged five more times before inoculating subjects. RSV identity was confirmed by fluorescent antibody, electron microscopy, and N-gene sequencing. It was free of adventitial agents and other human pathogens. Memphis 37 was diluted in 25% sucrose immediately before inoculation of subjects by intranasal drops (0.5 mL/naris).

Study Design and Endpoints.

The study was a 1:1 randomized, placebo-controlled, double-blind, parallel-group phase II trial conducted at a single quarantine unit (Retroscreen Virology Ltd.), clinical trial identifier NCT00496821. The study’s assigned EudraCT number was 2006-006902-27. Local regulatory, institutional review board, and Ethics Committee approval (Plymouth Independent Ethics Committee) was obtained. All subjects provided written informed consent.

The study was designed to investigate whether ALN-RSV01 had antiviral activity. The primary endpoint was reduction in number of subjects becoming RSV infected. The dosing approach was adopted because it maximized the chance of observing antiviral activity by reducing infection while simultaneously minimizing the potential risk (of experimental infection and of ALN-RSV01) to the healthy volunteers.

Subjects were enrolled in six sequential cohorts (Fig. 1). In cohort 1 (n = 8), ALN-RSV01 was administered at a dose of 75 mg/day. In cohorts 2–6 (n = 80), ALN-RSV01 was administered at a dose of 150 mg/day. Subjects were admitted to the quarantine unit for 14 days and were administered the blinded study medication (ALN-RSV01 or placebo) 32 h (day −1) and 8 h (day 0) before RSV inoculation on day 0. Daily administration of study medication continued on days 1, 2, and 3. The RSV inoculum dose was quantified by culture assay in HEp-2 cells at the exact time of inoculation of first and last subjects in each cohort, to yield a mean of the overall inoculum dose for each cohort. Nasal washes (5 mL normal saline per naris) for viral assays and cytokine measurements were obtained daily on the day of admission to the quarantine unit (day −2) and on days 2–11. For assessment of upper respiratory signs and symptoms, a physician’s daily directed physical examination (DPE) (days −2–11) and a twice-daily subject-reported RSV symptom score card (days −1–11) were completed (Tables S2 and S3). Mucus weight determined in used paper tissues was calculated for each 24-h period (days −1–11). Adverse events were recorded daily through the day 28 follow-up visit.

The primary endpoint, and the endpoint for which the study was powered, was the proportion of subjects who were RSV infected. Infection was predefined as two consecutive positive RSV assays, the first occurring between postinoculation days 2 and 8, inclusive. Additional measures of antiviral effect included viral AUC (calculated as the sum of the areas of each trapezoid formed by the borders of the previous daily viral load and the present day’s viral load from day 2 through day 11) and peak viral load. Clinical efficacy endpoints included total symptom score, total DPE score, and mucus weight.

RSV and Antibody Assays.

Nasal washes were collected into cold RSV stabilization media (25), transported on ice, and placed onto HEp-2 cell monolayers within 30 min of collection. Quantitative culture in HEp-2 cell plaque assays was performed in 12-well plates using triplicate 10-fold dilutions of nasal wash as previously described (25). RSV quantitative standards (RSV-A Long ATCC VR-26) were run in parallel with each plaque assay. Nasal washes containing less than the lower limit of quantification (LLOQ) (<1.7 Log PFU/mL) were considered culture negative.

The qRT-PCR assay amplifying an N-gene sequence distinct from that of ALN-RSV01 was performed as previously described (24). Duplicate specimens were run in 96-well plates incorporating internal standards of RNA extracted from parallel aliquots containing known RSV-A Long quantity as used in the plaque assays. Results are expressed as the means of duplicates in log₁₀ plaque forming unit equivalents per milliliter (Log PFUe/mL).

Spin-enhanced culture assays were performed using confluent HEp-2 cell monolayers on coverslips within shell vials. Monolayers inoculated with 200 μL fresh nasal wash and centrifuged at 700 × g for 60 min were acetone fixed after 2 days and then evaluated for RSV by direct fluorescent antibody techniques using RSV-specific mouse monoclonal antibodies (Bartels; Trinity Biotech).

Serum RSV-neutralizing antibodies were measured by a HEp-2 cell RSV 50% microneutralization assay as previously described (26) but performed with Memphis 37 strain.

Cytokine Assays.

Nasal wash aliquots were analyzed at neat, 1:10, and 1:50 dilutions using Pierce Searchlight chemiluminescent multiplexed sandwich ELISA cytokine arrays, quantifying G-CSF (LLOQ 1.82 pg/mL), IFN-α (LLOQ 7.5 pg/mL), IL-1RA (LLOQ 4.4 pg/mL), and TNF-α (LLOQ 6.7 pg/mL).

Statistical Analysis.

Continuous values below the LLOQ were set at zero. Continuous antiviral and clinical efficacy variables were evaluated using a two-sample t test. Data not normally distributed were analyzed using the Wilcoxon rank-sum test. As the outcomes in different cohorts may have been affected by unknown factors and differences in RSV inoculum between cohorts, the primary endpoint (comparing proportions of subjects infected) was evaluated using a two-sided Cochran–Mantel–Haenszel (CMH) test controlling for cohort. Multiple logistic regression analysis (MLRA) was employed to evaluate the effects of different study variables on the antiviral effect (treatment effect). All analyses were performed by either i3 Research or Quartesian, LLC, using SAS v8.2 or higher for Windows.