Aerosol Delivery of Palivizumab in a Neonatal Lamb Model of Respiratory Syncytial Virus Infection

(1) Background: Palivizumab has been an approved preventative monoclonal antibody for respiratory syncytial virus (RSV) infection for over two decades. However, due to its high cost and requirement for multiple intramuscular injections, its use has been limited mostly to high-income countries. Following our previous study showing the successful lung deposition of aerosolised palivizumab in lambs, this current study evaluated the “proof-of-principle” effect of aerosolised palivizumab delivered as a therapeutic to neonatal lambs following RSV infection. (2) Methods: Neonatal lambs were intranasally inoculated with RSV-A2 on day 0 (day 3 post-birth) and treated with aerosolised palivizumab 3 days later (day 3 post-inoculation). Clinical symptoms, RSV viral load and inflammatory response were measured post-inoculation. (3) Results: Aerosolised therapeutic delivery of palivizumab did not reduce RSV viral loads in the nasopharynx nor the bronchoalveolar lavage fluid, but resulted in a modest reduction in inflammatory response at day 6 post-inoculation compared with untreated lambs. (4) Conclusions: This proof-of-principle study shows some evidence of aerosolised palivizumab reducing RSV inflammation, but further studies using optimized protocols are needed in order to validate these findings.

Currently, there are no approved RSV treatments for infants.The two recently approved RSV preventatives, a long-acting monoclonal antibody (Niservimab, Sanofi, Astra Zeneca, Cambridge, UK) for newborns and a maternal vaccine (RSVPreF, Pfizer, NY, USA), have high costs which are out of the reach of many LMICs.The risk of preterm births associated with RSVPreF remains a concern and requires more data, but is currently approved for use in pregnant women at 32-36 weeks of gestation [7][8][9][10].Given their high costs, these two newly approved preventions are not likely to be available in LMICs.Palivizumab Viruses 2023, 15, 2276 2 of 9 (Synagis ® , Sobi, MA, USA) is also a monoclonal antibody which has been used for more than 20 years for RSV prophylaxis, mainly in high-income countries, but is restricted to only very high-risk infants.The requirement of repeated monthly intramuscular injections (IM) before each RSV season and high cost have been critical limitations to its broader use among infants [11].
Despite these newer RSV preventives, there remains a need to identify alternative cost-effective strategies to prevent RSV infection and severe disease in infants.Aerosol delivery represents one such approach.We previously demonstrated the deposition of aerosolised palivizumab into the lungs of lambs, suggesting that this might be a feasible approach by which to combat respiratory infections while potentially reducing the dose and cost of palivizumab using a non-invasive delivery route [12].Due to the significant similarities to humans in lung structure and function, neonatal lambs serve as a useful large animal model in which to study RSV infection [13][14][15][16][17].
This study aimed to evaluate the effect of aerosolised palivizumab when given as a treatment in a newborn lamb model of RSV infection.We hypothesised that the delivery of therapeutic aerosolised palivizumab would reduce RSV viral load and lung inflammation.

Study Design
This study was approved by the Murdoch Children's Research Institute (MCRI) Animal Ethics Committee (A895).The experiments were designed and are reported with reference to the ARRIVE (Animal Research: Reporting of in vivo Experiments) guidelines [18].
An overview of the RSV inoculation, treatment, and sampling regime for this experiment is summarised in Figure 1.A total of 25 newborn lambs were allocated to five groups (Table 1).Group 0 lambs served as uninfected, untreated controls.For intranasal RSV inoculation (Group 1 and Group 2), the human RSV-A2 strain (obtained from ATCC, VA, USA) [19] was prepared in sterile saline and delivered by gentle infusion via a syringe into each nostril (maximum volume of 2 mL/nostril, total of 102 × 10 6 PFU inoculation per lamb) on day 0 (day 3 post-birth).Group 1 lambs served as RSV-infected, untreated controls (RSV/untreated).Group 2 lambs were infected with RSV and treated with aerosolised palivizumab (RSV/palivizumab) (Synagis ® , Sobi, MA, USA) while under sedation, which was performed on day 3 post-inoculation using an Aeroneb Go ® (Aerogen, Galway, Ireland) nebuliser (15 mg/kg diluted to 10 mg/mL in sterile saline) based on our previous study [12].
Viruses 2023, 15, x FOR PEER REVIEW approved for use in pregnant women at 32-36 weeks of gestation [7][8][9][10].Given the costs, these two newly approved preventions are not likely to be available in L Palivizumab (Synagis ® , Sobi, MA, USA) is also a monoclonal antibody which ha used for more than 20 years for RSV prophylaxis, mainly in high-income countries restricted to only very high-risk infants.The requirement of repeated monthly intr cular injections (IM) before each RSV season and high cost have been critical limi to its broader use among infants [11].
Despite these newer RSV preventives, there remains a need to identify alter cost-effective strategies to prevent RSV infection and severe disease in infants.A delivery represents one such approach.We previously demonstrated the deposi aerosolised palivizumab into the lungs of lambs, suggesting that this might be a f approach by which to combat respiratory infections while potentially reducing th and cost of palivizumab using a non-invasive delivery route [12].Due to the sign similarities to humans in lung structure and function, neonatal lambs serve as a large animal model in which to study RSV infection [13][14][15][16][17].
This study aimed to evaluate the effect of aerosolised palivizumab when giv treatment in a newborn lamb model of RSV infection.We hypothesised that the d of therapeutic aerosolised palivizumab would reduce RSV viral load and lung infla tion.

Study Design
This study was approved by the Murdoch Children's Research Institute (MCR imal Ethics Committee (A895).The experiments were designed and are reported w erence to the ARRIVE (Animal Research: Reporting of in vivo Experiments) guid [18].
An overview of the RSV inoculation, treatment, and sampling regime for this iment is summarised in Figure 1.A total of 25 newborn lambs were allocated groups (Table 1).Group 0 lambs served as uninfected, untreated controls.For intr RSV inoculation (Group 1 and Group 2), the human RSV-A2 strain (obtained from VA, USA) [19] was prepared in sterile saline and delivered by gentle infusion via a s into each nostril (maximum volume of 2 mL/nostril, total of 102 × 10 6 PFU inoculati lamb) on day 0 (day 3 post-birth).Group 1 lambs served as RSV-infected, untreate trols (RSV/untreated).Group 2 lambs were infected with RSV and treated with aero palivizumab (RSV/palivizumab) (Synagis ® , Sobi, MA, USA) while under sedation, was performed on day 3 post-inoculation using an Aeroneb Go ® (Aerogen, Galwa land) nebuliser (15 mg/kg diluted to 10 mg/mL in sterile saline) based on our pr study [12].Study design and sampling timeline using an RSV lamb model.Neonatal lambs were infected with the RSV-A2 strain intranasally on day 0. Nasopharyngeal (NP) swabs, blood, and bronchoalveolar lavage fluid (BALF) were collected at baseline (day 0) and then on day 2, 4, 6, 8 and 10 post-inoculation.Lambs were euthanised either on day 6 (peak infection) or day 10 (recovery) postinoculation according to the study group (refer to Table 1).Image was created using BioRender.com.

In-Life Measures and Sample Collection for Assessment of RSV Infection
Clinical parameters were measured using a calibrated weighing scale (body weight), digital rectal thermometer (body temperature), and pulse oximetry for oxygen saturation.Nasopharyngeal (NP) swabs were collected between day 0 to day 10 post-inoculation from the posterior nasal cavity of both nostrils using a sterile neonatal flocked swab, then stored in RNALater at −80 • C. Lambs were anaesthetised with ketamine, which was administered intramuscularly, for the collection of bronchoalveolar lavage fluid (BALF) samples on days 0, 2, 6, and 10 post-inoculation.Autopsy tissue samples were collected either at day 6 (peak infection) or day 10 (resolution of infection) from all lambs.Tissues from the cranial, middle, caudal, and accessory lung lobes were fixed in 4% (w/v) paraformaldehyde for histopathological analysis.

RSV RT-qPCR
Viral RNA was extracted from NP swabs and BALF samples using a QIAamp Viral RNA Mini Kit (Qiagen, Hilden, Germany), then processed for quantitative RT-PCR using a published protocol [20].

Lung Histopathology
The slides were deparaffinized, stained with haematoxylin and eosin (H&E), and cover-slipped with Depex TM .Additional slides were stained with Alcian blue and periodic acid schiff (AB-PAS).

Light Microscopy Analysis
The stained slides were scanned at 40× magnification on ScanScope (ScanScope XT, Leica Aperio Technologies) and analysed using Imagescope v.12.1 (Leica Aperio Technologies, Nussloch, Germany) software.A histological score was given to all lambs according to the inflammation and congestion observed after H&E staining.A representative image was considered for each score to assess three fields of view of the lung regions independently.An average inflammation/congestion score was then calculated per lamb in each group to compare the effect of palivizumab using the scoring matrix below, which was adapted from [21].Validation of the analysis was carried out by an independent operator in a blinded manner.

Statistical Analysis
Clinical data (body temperature, body weight, oxygen saturation) were presented as the mean ± SEM.Viral load and histopathology data were presented as the median ± interquartile range (IQR), as appropriate.Differences between groups were analysed using the Mann-Whitney test.A p value < 0.05 was considered statistically significant.All of the results are graphically presented with GraphPad Prism version 7 (GraphPad Software, San Diego, CA, USA).
5->75% consolidation.AB-PAS stain was used for the identification of goblet cells (GC) in the lung Five cartilaginous and non-cartilaginous airways in each lung section were used fo ual GC counts under 40× magnification.A mean number of cells per 100 µm ba membrane length per lamb in each group was then calculated.

Statistical Analysis
Clinical data (body temperature, body weight, oxygen saturation) were prese the mean ± SEM.Viral load and histopathology data were presented as the medi terquartile range (IQR), as appropriate.Differences between groups were analyse the Mann-Whitney test.A p value < 0.05 was considered statistically significant.A results are graphically presented with GraphPad Prism version 7 (GraphPad So CA, USA).

Clinical Observations
No clinical signs of RSV infection were observed in any of the inoculated la to day 6 post-inoculation (Figure 2B-D).The increases in body weight were wit healthy range of 240 g-280 g per day for all lambs, with no difference between the Body temperature was within the normal range of 38-40 °C, with a recorded a

Clinical Observations
No clinical signs of RSV infection were observed in any of the inoculated lambs up to day 6 post-inoculation (Figure 2B-D).The increases in body weight were within the healthy range of 240 g-280 g per day for all lambs, with no difference between the groups.Body temperature was within the normal range of 38-40 • C, with a recorded average baseline temperature of 39 • C across all groups.Oxygen saturation, as measured by pulse oximetry, was within the normal range of 95-100% at all times, with clinically irrelevant fluctuations in lambs across all groups.

Viral Load
RSV was detected in RSV-inoculated lambs only (groups 1 and 2) in NP swabs and BALF samples (Figure 3).On day 6 post-inoculation (stable peak infection), RSV/palivizumab lambs (Group 2) had an 85.2% lower median viral load of 3.1 × 10 2 copies RNA/mL compared to the RSV/untreated lambs (Group 1), which had a median viral load of 2.1 × 10 3 copies RNA/mL, although this was not significant (Figure 3A).
fluctuations in lambs across all groups.

Viral Load
RSV was detected in RSV-inoculated lambs only (groups 1 and 2) in NP swa BALF samples (Figure 3).On day 6 post-inoculation (stable peak infection), RSV zumab lambs (Group 2) had an 85.2% lower median viral load of 3.1 × 10 2 copies RN compared to the RSV/untreated lambs (Group 1), which had a median viral load 10 3 copies RNA/mL, although this was not significant (Figure 3A).
The BALF viral load values were also evaluated for each group (Figure 3B).6, the median RSV load was similar across all groups.There were three lambs that s detectable viral loads in the RSV/untreated group (group 1) and two in the RSV zumab group (group 2).
Viral load kinetics according to NP swabs and BALF over the 10-day post-inoc period are shown in the Supplementary Materials.

Lung Histopathology
Inflammation scores were assessed for each group (Figure 4).In Figure 4A-C resentative image of lung inflammation is shown for the three groups.On day 6 tional alveolar spaces and thin alveolar walls were seen in the control (group 0) (median ± IQR of 1.3 ± 0.2), whereas the RSV/untreated (group 1a) lambs showed cantly increased (p = 0.035) congestion and lung pathologies, with thick alveolar wa reduced alveolar spaces (median ± IQR of 3.1 ± 1.4).For the RSV/palivizumab (gro lambs, the inflammation scores were significantly lower than those of the RSV/un lambs (median ± IQR of 1.7 ± 0.8; p = 0.041) (Figure 4D).AB-PAS stains were utilized to identify goblet cell (GC) mucin as a marker histopathology on day 6 post-inoculation (Figure 5).In Figure 5A-C, a representat age of cartilaginous and non-cartilaginous airways across the three groups is There was a non-significant increase in GC observed in the cartilaginous airway RSV/untreated (Group 1a) lambs (median ± IQR 2.6 ± 0.8) compared to the RSV zumab lambs in Group 2a (median ± IQR 1.9 ± 0.8; Figure 5D).The average GC cou 100 µm in the non-cartilaginous airways were similar across all groups.The BALF viral load values were also evaluated for each group (Figure 3B).On day 6, the median RSV load was similar across all groups.There were three lambs that showed detectable viral loads in the RSV/untreated group (group 1) and two in the RSV/palivizumab group (group 2).
Viral load kinetics according to NP swabs and BALF over the 10-day post-inoculation period are shown in the Supplementary Materials.

Lung Histopathology
Inflammation scores were assessed for each group (Figure 4).In Figure 4A-C, a representative image of lung inflammation is shown for the three groups.On day 6, additional alveolar spaces and thin alveolar walls were seen in the control (group 0) lambs (median ± IQR of 1.3 ± 0.2), whereas the RSV/untreated (group 1a) lambs showed significantly increased (p = 0.035) congestion and lung pathologies, with thick alveolar walls and reduced alveolar spaces (median ± IQR of 3.1 ± 1.4).For the RSV/palivizumab (group 2a) lambs, the inflammation scores were significantly lower than those of the RSV/untreated lambs (median ± IQR of 1.7 ± 0.8; p = 0.041) (Figure 4D).
AB-PAS stains were utilized to identify goblet cell (GC) mucin as a marker of RSV histopathology on day 6 post-inoculation (Figure 5).In Figure 5A-C, a representative image of cartilaginous and non-cartilaginous airways across the three groups is shown.There was a non-significant increase in GC observed in the cartilaginous airways of the RSV/untreated (Group 1a) lambs (median ± IQR 2.6 ± 0.8) compared to the RSV/palivizumab lambs in Group 2a (median ± IQR 1.9 ± 0.8; Figure 5D).The average GC counts per 100 µm in the non-cartilaginous airways were similar across all groups.

Discussion
This study examined the effect of therapeutic aerosolised palivizumab in a lamb model of RSV infection.Lambs were infected with high-dose administration of RSV to the upper airways.No clinical symptoms were observed in these lambs following RSV inoculation.On day 6 post-inoculation (i.e., at peak infection), there was no difference in viral loads between the untreated group and the group treated with aerosolised palivizumab.

Discussion
This study examined the effect of therapeutic aerosolised palivizumab in a model of RSV infection.Lambs were infected with high-dose administration of RSV upper airways.No clinical symptoms were observed in these lambs following RSV ulation.On day 6 post-inoculation (i.e., at peak infection), there was no difference in loads between the untreated group and the group treated with aerosolised palivizu

Discussion
This study examined the effect of therapeutic aerosolised palivizumab in a lamb model of RSV infection.Lambs were infected with high-dose administration of RSV to the upper airways.No clinical symptoms were observed in these lambs following RSV inoculation.On day 6 post-inoculation (i.e., at peak infection), there was no difference in viral loads between the untreated group and the group treated with aerosolised palivizumab.However, significantly reduced inflammation scores in the lungs were observed for the palivizumabtreated group compared to the untreated, RSV-infected group.The goblet cell analysis at day 6 post-inoculation showed that, around the larger cartilaginous airways, there was a trend towards reduced GC numbers that was not observed in the smaller non-cartilaginous airways.Together with our earlier report that showed good lung deposition of aerosolised palivizumab in lambs [12], this proof-of-principle study provides support for the potential feasibility of an aerosolised therapy approach to treat RSV infection.
While the lambs in our study did not exhibit severe RSV clinical symptoms, as is consistent with previous studies, we found significant differences in the inflammation/congestion scoring when comparing the treated and untreated lambs infected with RSV.In studies by Derscheid and Ackermann and Larios Mora et al., moderate-to-severe necrotic bronchitis, bronchiolitis, and syncytial formation were reported for both the RSV A2 and M37 strains [14][15][16].One explanation for this difference in clinical symptoms is that the newborn lambs in our model were not colostrum-deprived.Colostrum-deprived models would have prevented the transfer of maternal immunoglobulin to the newborn lambs, potentially making them more susceptible to severe RSV disease [13,21].We housed the newborn lambs with the ewes for the full study duration to achieve similarity to the human context.
Increased mucus production within the airway is also a known consequence of lung inflammation due to RSV infection.Mucin-producing cells or goblet cells are highly responsive in the respiratory tract, with rapid hyperplasia/metaplasia and hypersecretion in response to inflammatory mediators [22,23].Given the limited knowledge on mucin production in RSV lamb models, our study analysed goblet cell counts with two approaches: comparing the cell counts in deeper non-cartilaginous bronchioles and in upper airway cartilaginous bronchi to determine the effectiveness of aerosolised palivizumab in terms of reducing inflammation.Although not statistically significant, lower GC counts in the treated lambs compared to the untreated lambs were observed in the cartilaginous airways, but not in the non-cartilaginous airways.The cartilage seen within the lungs was restricted to the upper airways, and the relevance of palivizumab delivery in the upper airway vs. the lower airway is still unknown.Any effect of palivizumab is likely to be within the lower airways, where RSV bronchiolitis occurs.Therefore, further studies are required in order to investigate this effect.
The aerosolised delivery route is a promising approach, as it is non-invasive and directly targets the lung, providing a way to maximise the exposure of pathogens to therapeutic agents.ALX-0171, a trivalent nanobody developed for RSV treatment, was shown to have an anti-viral effect on RSV lung lesions accompanied by a reduction in disease symptoms [21].Recently, aerosolised delivery methods have been explored for SARS-CoV-2 to block transmission by targeting the upper respiratory tract [24].We previously demonstrated the pulmonary delivery of aerosolised palivizumab using a lamb model, with over 88% delivery of this mAb to all regions of the lungs, suggesting that the aerosolisation of palivizumab is likely to have an effect in the lower airways [12].Therefore, aerosolised delivery of palivizumab may be effective for treating RSV infection as well as reducing the currently required dosage, which would reduce cost and thereby improve access in low-resource settings.Further dose-sparing studies are needed in order to address this important question, as well as the potential for this approach to be applied to the newer long-acting monoclonal antibody Nirsevimab.
This study has some limitations.The sample size, while similar to other lamb studies, was relatively small and may have prevented us from detecting any beneficial effects [14].Our lack of viral load detection in the BALF specimens could be due to variability in the collection method or to not being able to collect across all lung regions, potentially missing areas of RSV infection.The optimal therapeutic dose was not determined in this study and requires further investigation.Despite these limitations, the therapeutic delivery of palivizumab may still be a useful approach to limit the severity of RSV infection.The long-acting version of palivizumab was not available when our study was designed,

Figure 1 .
Figure 1.Study design and sampling timeline using an RSV lamb model.Neonatal lambs w fected with the RSV-A2 strain intranasally on day 0. Nasopharyngeal (NP) swabs, bloo

Figure 1 .
Figure1.Study design and sampling timeline using an RSV lamb model.Neonatal lambs were infected with the RSV-A2 strain intranasally on day 0. Nasopharyngeal (NP) swabs, blood, and bronchoalveolar lavage fluid (BALF) were collected at baseline (day 0) and then on day 2, 4, 6, 8 and 10 post-inoculation.Lambs were euthanised either on day 6 (peak infection) or day 10 (recovery) postinoculation according to the study group (refer to Table1).Image was created using BioRender.com.

Viruses 2023 ,
15, x FOR PEER REVIEW