Title: A nanobody recognizes a unique conserved epitope and potently neutralizes against the SARS-CoV-2 omicron variants.

The SARS-CoV2 Omicron variant sub-lineages spread rapidly worldwide, mostly due to their immune-evasive properties. This has put a significant part of the population at risk for severe disease and underscores the need for effective anti-SARS-CoV-2 agents against emergent strains in vulnerable patients. Camelid nanobodies are attractive therapeutic candidates due to their high stability, ease of large-scale production and potential for delivery via inhalation. Here, we characterize the RBD-specific nanobody W25 and show superior neutralization activity towards Omicron sub-lineages in comparison to all other SARS-CoV2 variants. Structure analysis of W25 in complex with the SARS-CoV2 spike glycoprotein shows that W25 engages an RBD epitope not covered by any of the antibodies previously approved for emergency use. In vivo evaluation of W25 prophylactic and therapeutic treatments across multiple SARS-CoV-2 variant infection models, together with W25 biodistribution analysis in mice, demonstrates favorable pre-clinical properties. Together, these data endorse W25 for further clinical development.

Lipid Nanoparticles as Promising Carriers for mRNA Vaccines for Viral Lung Infections.

In recent years, there has been an increase in deaths due to infectious diseases, most notably in the context of viral respiratory pathogens. Consequently, the focus has shifted in the search for new therapies, with attention being drawn to the use of nanoparticles in mRNA vaccines for targeted delivery to improve the efficacy of these vaccines. Notably, mRNA vaccine technologies denote as a new era in vaccination due to their rapid, potentially inexpensive, and scalable development. Although they do not pose a risk of integration into the genome and are not produced from infectious elements, they do pose challenges, including exposing naked mRNAs to extracellular endonucleases. Therefore, with the development of nanotechnology, we can further improve their efficacy. Nanoparticles, with their nanometer dimensions, move more freely in the body and, due to their small size, have unique physical and chemical properties. The best candidates for vaccine mRNA transfer are lipid nanoparticles (LNPs), which are stable and biocompatible and contain four components: cationic lipids, ionizable lipids, polyethylene glycols (PEGs), and cholesterol, which are used to facilitate cytoplasmic mRNA delivery. In this article, the components and delivery system of mRNA-LNP vaccines against viral lung infections such as influenza, coronavirus, and respiratory syncytial virus are reviewed. Moreover, we provide a succinct overview of current challenges and potential future directions in the field.

Exhaled patient derived aerosol dispersion during awake tracheal intubation with concurrent high flow nasal therapy.

Awake Tracheal Intubation (ATI) can be performed in cases where there is potential for difficult airway management. It is considered an aerosol generating procedure and is a source of concern to healthcare workers due to the risk of transmission of airborne viral infections, such as SARS-CoV-2. At present, there is a lack of data on the quantities, size distributions and spread of aerosol particles generated during such procedures. This was a volunteer observational study which took place in an operating room of a university teaching hospital. Optical particle sizers were used to provide real time aerosol characterisation during a simulated ATI performed with concurrent high-flow nasal oxygen therapy. The particle sizers were positioned at locations that represented the different locations of clinical staff in an operating room during an ATI. The greatest concentration of patient derived aerosol particles was within 0.5-1.0 m of the subject and along their midline, 2242 #/cm3. As the distance, both radial and longitudinal, from the subject increased, the concentration decreased towards ambient levels, 36.9 ± 5.1 #/cm3. Patient derived aerosol particles < 5 µm in diameter remained entrained in the exhaled aerosol plume and fell to the floor or onto the subject. Patient derived particles > 5 µm in diameter broke away from the exhaled plume and spread radially throughout the operating room. Irrespective of distance and ventilation status, full airborne protective equipment should be worn by all staff when ATI is being performed on patients with suspected viral respiratory infections.

In vitro model for investigating aerosol dispersion in a simulated COVID-19 patient during high-flow nasal cannula treatment.

The use of high-flow nasal cannula in the treatment of COVID-19 infected patients has proven to be a valuable treatment option to improve oxygenation. Early in the pandemic, there were concerns for the degree of risk of disease transmission to health care workers utilizing these treatments that are considered aerosol generating procedures. This study developed an in vitro model to examine the release of simulated patient-derived bioaerosol with and without high-flow nasal cannula at gas flow rates of 30 and 50 L/min. Aerosol dispersion was evaluated at 30 and 90 cm distances. Reduction of transmission risk was assessed using a surgical facemask on the manikin. Results indicated that the use of a facemask facilitated a 94-95% reduction in exhaled aerosol concentration at 30 cm and 22-60% reduction for 90 cm distance across both gas flow rates. This bench study confirms that this in vitro model can be used as a tool to assess the risk of disease transmission during aerosol generating procedures in a simulated patient and to test factors to mitigate the risk.

Development of Inhalable ATRA-Loaded PLGA Nanoparticles as Host-Directed Immunotherapy against Tuberculosis.

Developing new effective treatment strategies to overcome the rise in multi-drug resistant tuberculosis cases (MDR-TB) represents a global challenge. A host-directed therapy (HDT), acting on the host immune response rather than Mtb directly, could address these resistance issues. We developed an HDT for targeted TB treatment, using All Trans Retinoic Acid (ATRA)-loaded nanoparticles (NPs) that are suitable for nebulization. Efficacy studies conducted on THP-1 differentiated cells infected with the H37Ra avirulent Mycobacterium tuberculosis (Mtb) strain, have shown a dose-dependent reduction in H37Ra growth as determined by the BACT/ALERT® system. Confocal microscopy images showed efficient and extensive cellular delivery of ATRA-PLGA NPs into THP-1-derived macrophages. A commercially available vibrating mesh nebulizer was used to generate nanoparticle-loaded droplets with a mass median aerodynamic diameter of 2.13 µm as measured by cascade impaction, and a volumetric median diameter of 4.09 µm as measured by laser diffraction. In an adult breathing simulation experiment, 65.1% of the ATRA PLGA-NP dose was inhaled. This targeted inhaled HDT could offer a new adjunctive TB treatment option that could enhance current dosage regimens leading to better patient prognosis and a decreasing incidence of MDR-TB.

Advances and applications of dextran-based nanomaterials targeting inflammatory respiratory diseases

Dextran, a hydrophilic polysaccharide consists essentially of α-1,6 linked glucopyranoside residues that form the parent chain, along with α-1,2/3/4 linked residues that constitute its side chain. A considerable biocompatibility, stability under mildly acidic and basic conditions, solubility in water, non-immunogenicity, and presence of chemically modifiable –OH groups make dextran an ideal candidate for development of drug delivery vehicles and excipients. The presence of α-1,6 linkages in the parent chain provides enhanced chain mobility that determines the aqueous solubility of dextran, while its metabolism by the digestive enzymes to generate physiologically harmless degradation products validates its biocompatibility. Native dextran can be tuned for the development of pH-sensitive delivery systems by chemical modification that ensure an optimal drug concentration at the target site, and lowered dosing frequency that may ensure an overall improved patient compliance. The physicochemical properties of dextran can be changed by performing a chemical modification predominantly at the –OH group to obtain ester, ether, acetal, and dialdehyde of dextran. The review presented by us is a comprehensive account of the chemical modification strategies for native dextran and their clinical applications in containing pulmonary diseases. Furthermore, the presented review highlights the importance of nanomaterials derived from chemically modified dextran for the management of an optimal respiratory health by containing the inflammatory respiratory diseases.

Porcine Respiratory Coronavirus as a Model for Acute Respiratory Coronavirus Disease

In the light of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic, we have developed a porcine respiratory coronavirus (PRCV) model for in depth mechanistic evaluation of the pathogenesis, virology and immune responses of this important family of viruses. Pigs are a large animal with similar physiology and immunology to humans and are a natural host for PRCV. Four PRCV strains were investigated and shown to induce different degrees of lung pathology. Importantly, although all four strains replicated equally well in porcine cell lines in vitro and in the upper respiratory tract in vivo, PRCV strains causing more severe lung pathology were also able to replicate in ex vivo tracheal organ cultures as well as in vivo in the trachea and lung. The time course of infection of PRCV 135, which caused the most severe pulmonary pathology, was investigated. Virus was shed from the upper respiratory tract until day 10 post infection, with infection of the respiratory mucosa, as well as olfactory and sustentacular cells, providing an excellent model to study upper respiratory tract disease in addition to the commonly known lower respiratory tract disease from PRCV. Infected animals made antibody and T cell responses that cross reacted with the four PRCV strains and Transmissible Gastroenteritis Virus. The antibody response was reproduced in vitro in organ cultures. Comparison of mechanisms of infection and immune control in pigs infected with PRCVs of differing pathogenicity with human data from SARS-CoV-2 infection and from our in vitro organ cultures, will enable key events in coronavirus infection and disease pathogenesis to be identified.

Fugitive Aerosol Therapy Emissions during Mechanical Ventilation: In Vitro Assessment of the Effect of Tidal Volume and Use of Protective Filters

Background: During mechanical ventilation of a patient requiring ventilatory support, bystanders could potentially be exposed to aerosolised drug. Methods: Fugitive drug aerosol emissions during simulated adult mechanical ventilation was assessed on a dual limb circuit. Tidal volume was set at 270 mL and 820 mL. The use of a protective filter on the exhalation port of the mechanical ventilator was assessed. Results: Higher fugitive aerosol mass concentrations in the local environment were associated with larger tidal volume (0.077 (0.073, 0.091) mg m–3 at Vt = 820 mL vs. 0.062 (0.056, 0.065) mg m–3 at Vt = 270 mL) when no protective filter was used. The range of mass median aerodynamic diameters recorded was from 0.93 to 2.96 µm. When a filter was placed on the exhalation port of the mechanical ventilator, no fugitive emissions were recorded. Conclusion: This study confirms that an appropriate filtration protocol mitigates the risk of fugitive emissions being released when patients undergo aerosol therapy during mechanical ventilation. A larger tidal volume resulted in higher fugitive aerosol mass.

Aggregates Associated with Instability of Antibodies during Aerosolization Induce Adverse Immunological Effects.

BACKGROUND: Immunogenicity refers to the inherent ability of a molecule to stimulate an immune response. Aggregates are one of the major risk factors for the undesired immunogenicity of therapeutic antibodies (Ab) and may ultimately result in immune-mediated adverse effects. For Ab delivered by inhalation, it is necessary to consider the interaction between aggregates resulting from the instability of the Ab during aerosolization and the lung mucosa. The aim of this study was to determine the impact of aggregates produced during aerosolization of therapeutic Ab on the immune system. METHODS: Human and murine immunoglobulin G (IgG) were aerosolized using a clinically-relevant nebulizer and their immunogenic potency was assessed, both in vitro using a standard human monocyte-derived dendritic cell (MoDC) reporter assay and in vivo in immune cells in the airway compartment, lung parenchyma and spleen of healthy C57BL/6 mice after pulmonary administration. RESULTS: IgG aggregates, produced during nebulization, induced a dose-dependent activation of MoDC characterized by the enhanced production of cytokines and expression of co-stimulatory markers. Interestingly, in vivo administration of high amounts of nebulization-mediated IgG aggregates resulted in a profound and sustained local and systemic depletion of immune cells, which was attributable to cell death. This cytotoxic effect was observed when nebulized IgG was administered locally in the airways as compared to a systemic administration but was mitigated by improving IgG stability during nebulization, through the addition of polysorbates to the formulation. CONCLUSION: Although inhalation delivery represents an attractive alternative route for delivering Ab to treat respiratory infections, our findings indicate that it is critical to prevent IgG aggregation during the nebulization process to avoid pro-inflammatory and cytotoxic effects. The optimization of Ab formulation can mitigate adverse effects induced by nebulization.

Aerosol release, distribution, and prevention during aerosol therapy: a simulated model for infection control.

Aerosol therapy is used to deliver medical therapeutics directly to the airways to treat respiratory conditions. A potential consequence of this form of treatment is the release of fugitive aerosols, both patient derived and medical, into the environment and the subsequent exposure of caregivers and bystanders to potential viral infections. This study examined the release of these fugitive aerosols during a standard aerosol therapy to a simulated adult patient. An aerosol holding chamber and mouthpiece were connected to a representative head model and breathing simulator. A combination of laser and Schlieren imaging was used to non-invasively visualize the release and dispersion of fugitive aerosol particles. Time-varying aerosol particle number concentrations and size distributions were measured with optical particle sizers at clinically relevant positions to the simulated patient. The influence of breathing pattern, normal and distressed, supplemental air flow, at 0.2 and 6 LPM, and the addition of a bacterial filter to the exhalation port of the mouthpiece were assessed. Images showed large quantities of fugitive aerosols emitted from the unfiltered mouthpiece. The images and particle counter data show that the addition of a bacterial filter limited the release of these fugitive aerosols, with the peak fugitive aerosol concentrations decreasing by 47.3-83.3%, depending on distance from the simulated patient. The addition of a bacterial filter to the mouthpiece significantly reduces the levels of fugitive aerosols emitted during a simulated aerosol therapy, p≤ .05, and would greatly aid in reducing healthcare worker and bystander exposure to potentially harmful fugitive aerosols.

Cellular Immunotherapy and the Lung.

The new era of cellular immunotherapies has provided state-of-the-art and efficient strategies for the prevention and treatment of cancer and infectious diseases. Cellular immunotherapies are at the forefront of innovative medical care, including adoptive T cell therapies, cancer vaccines, NK cell therapies, and immune checkpoint inhibitors. The focus of this review is on cellular immunotherapies and their application in the lung, as respiratory diseases remain one of the main causes of death worldwide. The ongoing global pandemic has shed a new light on respiratory viruses, with a key area of concern being how to combat and control their infections. The focus of cellular immunotherapies has largely been on treating cancer and has had major successes in the past few years. However, recent preclinical and clinical studies using these immunotherapies for respiratory viral infections demonstrate promising potential. Therefore, in this review we explore the use of multiple cellular immunotherapies in treating viral respiratory infections, along with investigating several routes of administration with an emphasis on inhaled immunotherapies.

Evaluation of Aerosol Drug Delivery Options during Adult Mechanical Ventilation in the COVID-19 Era.

Drug delivery devices used for aerosol therapy during mechanical ventilation to ease the symptoms of respiratory diseases provide beneficial treatment but can also pose challenges. Reflecting the significant changes in global guidance around aerosol usage and lung-protective ventilation strategies, seen in response to the COVID-19 pandemic, for the first time, we describe the drug delivery performance of commonly used devices under these conditions. Here, vibrating mesh nebuliser (VMN), jet nebuliser (JN) and pressurised metered-dose inhaler (pMDI) performance was assessed during simulated adult mechanical ventilation. Both standard test breathing patterns and those representatives of low tidal volume (LTV) ventilation with concurrent active and passive humidification were investigated. Drug delivery using a VMN was significantly greater than that with a JN and pMDI for both standard and LTV ventilation. Humidification type did not affect the delivered dose across all device types for standard ventilation. Significant variability in the pMDI dosing was evident, depending on the timing of actuation and the adapter type used. pMDI actuation synchronised with inspiration resulted in a higher delivered drug dose. The type of adapter used for pMDI actuation influenced drug delivery, with the highest dose observed using the CombiHaler.

An in vitro visual study of fugitive aerosols released during aerosol therapy to an invasively ventilated simulated patient.

COVID-19 can cause serious respiratory complications resulting in the need for invasive ventilatory support and concurrent aerosol therapy. Aerosol therapy is considered a high risk procedure for the transmission of patient derived infectious aerosol droplets. Critical-care workers are considered to be at a high risk of inhaling such infectious droplets. The objective of this work was to use noninvasive optical methods to visualize the potential release of aerosol droplets during aerosol therapy in a model of an invasively ventilated adult patient. The noninvasive Schlieren imaging technique was used to visualize the movement of air and aerosol. Three different aerosol delivery devices: (i) a pressurized metered dose inhaler (pMDI), (ii) a compressed air driven jet nebulizer (JN), and (iii) a vibrating mesh nebulizer (VMN), were used to deliver an aerosolized therapeutic at two different positions: (i) on the inspiratory limb at the wye and (ii) on the patient side of the wye, between the wye and endotracheal tube, to a simulated intubated adult patient. Irrespective of position, there was a significant release of air and aerosol from the ventilator circuit during aerosol delivery with the pMDI and the compressed air driven JN. There was no such release when aerosol therapy was delivered with a closed-circuit VMN. Selection of aerosol delivery device is a major determining factor in the release of infectious patient derived bioaerosol from an invasively mechanically ventilated patient receiving aerosol therapy.

Revolutionizing polymer-based nanoparticle-linked vaccines for targeting respiratory viruses: A perspective.

Viral respiratory tract infections have significantly impacted global health as well as socio-economic growth. Respiratory viruses such as the influenza virus, respiratory syncytial virus (RSV), and the recent SARS-CoV-2 infection (COVID-19) typically infect the upper respiratory tract by entry through the respiratory mucosa before reaching the lower respiratory tract, resulting in respiratory disease. Generally, vaccination is the primary method in preventing virus pathogenicity and it has been shown to remarkably reduce the burden of various infectious diseases. Nevertheless, the efficacy of conventional vaccines may be hindered by certain limitations, prompting the need to develop novel vaccine delivery vehicles to immunize against various strains of respiratory viruses and to mitigate the risk of a pandemic. In this review, we provide an insight into how polymer-based nanoparticles can be integrated with the development of vaccines to effectively enhance immune responses for combating viral respiratory tract infections.

In vitro evaluation of disposable transport ventilators with combination aerosol therapy.

BACKGROUND: The COVID-19 pandemic has highlighted the need for alternative short-term, reliable means to aid in the treatment of patients requiring ventilatory support. Concurrent aerosol drug delivery is often prescribed to such patients. As such, this study examines one such short-term option, the disposable gas-powered transport ventilator to effectively deliver aerosol therapy. Factors such as aerosol generator type, patient breathing pattern, humidification and nebuliser position within the respiratory circuit were also examined. METHODS: Aerosol drug delivery characterisation was undertaken using two different disposable transport ventilators (DTVs). Two different nebuliser types, a closed circuit vibrating mesh nebuliser (VMN) and an open circuit jet nebuliser (JN), at different locations in a respiratory circuit, proximal and distal to an endotracheal tube (ETT), with and without passive humidification, were evaluated in simulated adult and paediatric patients. RESULTS: Placement of a nebuliser proximal to the ETT (VMN: 25.19%-34.15% and JN: 3.14%-8.92%), and the addition of a heat and moisture exchange filter (VMN: 32.37%-40.43% and JN: 5.60%-9.91%) resulted in the largest potential lung dose in the adult patient model. Irrespective of nebuliser position and humidification in the respiratory circuit, use of the VMN resulted in the largest potential lung dose (%). A similar trend was recorded in the paediatric model data, where the largest potential lung dose was recorded with both nebuliser types placed proximal to the ETT (VMN: 8.12%-10.89% and JN: 2.15%-3.82%). However, the addition of a heat and moisture exchange filter had no statistically significant effect on the potential lung dose (%) a paediatric patient would receive (p>>0.05). CONCLUSIONS: This study demonstrates that transport ventilators, such as DTVs, can be used concurrently with aerosol generators to effectively deliver aerosolised medication in both adult and paediatric patients.

Nebuliser Type Influences Both Patient-Derived Bioaerosol Emissions and Ventilation Parameters during Mechanical Ventilation.

COVID-19 may lead to serious respiratory complications which may necessitate ventilatory support. There is concern surrounding potential release of patient-derived bioaerosol during nebuliser drug refill, which could impact the health of caregivers. Consequently, mesh nebulisers have been recommended by various clinical practice guidelines. Currently, there is a lack of empirical data describing the potential for release of patient-derived bioaerosol during drug refill. This study examined the release of simulated patient-derived bioaerosol, and the effect on positive end expiratory pressure during nebuliser refill during mechanical ventilation of a simulated patient. During jet nebuliser refill, the positive end expiratory pressure decreased from 4.5 to 0 cm H2O. No loss in pressure was noted during vibrating mesh nebuliser refill. A median particle number concentration of 710 particles cm-3 above ambient was detected when refilling the jet nebuliser in comparison to no increase above ambient detected when using the vibrating mesh nebuliser. The jet nebuliser with the endotracheal tube clamped resulted in 60 particles cm-3 above ambient levels. This study confirms that choice of nebuliser impacts both the potential for patient-derived bioaerosol release and the ability to maintain ventilator circuit pressures and validates the recommended use of mesh nebulisers during mechanical ventilation.

Defining a Regulatory Strategy for ATMP/Aerosol Delivery Device Combinations in the Treatment of Respiratory Disease.

Advanced Therapeutic Medicinal Products (ATMP) are a heterogenous group of investigational medicinal products at the forefront of innovative therapies with direct applicability in respiratory diseases. ATMPs include, but are not limited to, stem cells, their secretome, or extracellular vesicles, and each have shown some potential when delivered topically within the lung. This review focuses on that subset of ATMPs. One key mode of delivery that has enabling potential in ATMP validation is aerosol-mediated delivery. The selection of the most appropriate aerosol generator technology is influenced by several key factors, including formulation, patient type, patient intervention, and healthcare economics. The aerosol-mediated delivery of ATMPs has shown promise for the treatment of both chronic and acute respiratory disease in pre-clinical and clinical trials; however, in order for these ATMP device combinations to translate from the bench through to commercialization, they must meet the requirements set out by the various global regulatory bodies. In this review, we detail the potential for ATMP utility in the lungs and propose the nebulization of ATMPs as a viable route of administration in certain circumstances. Further, we provide insight to the current regulatory guidance for nascent ATMP device combination product development within the EU and US.

State of the Art Review of Cell Therapy in the Treatment of Lung Disease, and the Potential for Aerosol Delivery.

Respiratory and pulmonary diseases are among the leading causes of death globally. Despite tremendous advancements, there are no effective pharmacological therapies capable of curing diseases such as COPD (chronic obstructive pulmonary disease), ARDS (acute respiratory distress syndrome), and COVID-19. Novel and innovative therapies such as advanced therapy medicinal products (ATMPs) are still in early development. However, they have exhibited significant potential preclinically and clinically. There are several longitudinal studies published, primarily focusing on the use of cell therapies for respiratory diseases due to their anti-inflammatory and reparative properties, thereby hinting that they have the capability of reducing mortality and improving the quality of life for patients. The primary objective of this paper is to set out a state of the art review on the use of aerosolized MSCs and their potential to treat these incurable diseases. This review will examine selected respiratory and pulmonary diseases, present an overview of the therapeutic potential of cell therapy and finally provide insight into potential routes of administration, with a focus on aerosol-mediated ATMP delivery.