Novel Dry Hyaluronic Acid-Vancomycin Complex Powder for Inhalation, Useful in Pulmonary Infections Associated with Cystic Fibrosis.

Polyelectrolyte-drug complexes are interesting alternatives to improve unfavorable drug properties. Vancomycin (VAN) is an antimicrobial used in the treatment of methicillin-resistant Staphylococcus aureus pulmonary infections in patients with cystic fibrosis. It is generally administered intravenously with a high incidence of adverse side effects, which could be reduced by intrapulmonary administration. Currently, there are no commercially available inhalable formulations containing VAN. Thus, the present work focuses on the preparation and characterization of an ionic complex between hyaluronic acid (HA) and VAN with potential use in inhalable formulations. A particulate-solid HA-VAN25 complex was obtained by spray drying from an aqueous dispersion. FTIR spectroscopy and thermal analysis confirmed the ionic interaction between HA and VAN, while an amorphous diffraction pattern was observed by X-ray. The powder density, geometric size and morphology showed the suitable aerosolization and aerodynamic performance of the powder, indicating its capability of reaching the deep lung. An in vitro extended-release profile of VAN from the complex was obtained, exceeding 24 h. Microbiological assays against methicillin-resistant and -sensitive reference strains of Staphylococcus aureus showed that VAN preserves its antibacterial efficacy. In conclusion, HA-VAN25 exhibited interesting properties for the development of inhalable formulations with potential efficacy and safety advantages over conventional treatment.

Lung-to-Heart Nano-in-Micro Peptide Promotes Cardiac Recovery in a Pig Model of Chronic Heart Failure.

BACKGROUND: The lack of disease-modifying drugs is one of the major unmet needs in patients with heart failure (HF). Peptides are highly selective molecules with the potential to act directly on cardiomyocytes. However, a strategy for effective delivery of therapeutics to the heart is lacking. OBJECTIVES: In this study, the authors sought to assess tolerability and efficacy of an inhalable lung-to-heart nano-in-micro technology (LungToHeartNIM) for cardiac-specific targeting of a mimetic peptide (MP), a first-in-class for modulating impaired L-type calcium channel (LTCC) trafficking, in a clinically relevant porcine model of HF. METHODS: Heart failure with reduced ejection fraction (HFrEF) was induced in Göttingen minipigs by means of tachypacing over 6 weeks. In a setting of overt HFrEF (left ventricular ejection fraction [LVEF] 30% ± 8%), animals were randomized and treatment was started after 4 weeks of tachypacing. HFrEF animals inhaled either a dry powder composed of mannitol-based microparticles embedding biocompatible MP-loaded calcium phosphate nanoparticles (dpCaP-MP) or the LungToHeartNIM only (dpCaP without MP). Efficacy was evaluated with the use of echocardiography, invasive hemodynamics, and biomarker assessment. RESULTS: DpCaP-MP inhalation restored systolic function, as shown by an absolute LVEF increase over the treatment period of 17% ± 6%, while reversing cardiac remodeling and reducing pulmonary congestion. The effect was recapitulated ex vivo in cardiac myofibrils from treated HF animals. The treatment was well tolerated, and no adverse events occurred. CONCLUSIONS: The overall tolerability of LungToHeartNIM along with the beneficial effects of the LTCC modulator point toward a game-changing treatment for HFrEF patients, also demonstrating the effective delivery of a therapeutic peptide to the diseased heart.

Long-acting inhaled medicines: Present and future.

Inhaled medicines continue to be an essential part of treatment for respiratory diseases such as asthma, chronic obstructive pulmonary disease, and cystic fibrosis. In addition, inhalation technology, which is an active area of research and innovation to deliver medications via the lung to the bloodstream, offers potential advantages such as rapid onset of action, enhanced bioavailability, and reduced side effects for local treatments. Certain inhaled macromolecules and particles can also end up in different organs via lymphatic transport from the respiratory epithelium. While the majority of research on inhaled medicines is focused on the delivery technology, particle engineering, combination therapies, innovations in inhaler devices, and digital health technologies, researchers are also exploring new pharmaceutical technologies and strategies to prolong the duration of action of inhaled drugs. This is because, in contrast to most inhaled medicines that exert a rapid onset and short duration of action, long-acting inhaled medicines (LAIM) improve not only the patient compliance by reducing the dosing frequency, but also the effectiveness and convenience of inhaled therapies to better manage patients' conditions. This paper reviews the advances in LAIM, the pharmaceutical technologies and strategies for developing LAIM, and emerging new inhaled modalities that possess a long-acting nature and potential in the treatment and prevention of various diseases. The challenges in the development of the future LAIM are also discussed where active research and innovations are taking place.

Development of inhalation powders containing lactic acid bacteria with antimicrobial activity against Pseudomonas aeruginosa.

OBJECTIVES: The aim of the project was to develop and characterise powders containing a probiotic (Lactiplantibacillus plantarum [Lpb. plantarum], Lacticaseibacillus rhamnosus, or Lactobacillus acidophilus) to be administered to the lung for the containment of pathogen growth in patients with lung infections. METHODS: The optimised spray drying process for the powder manufacturing was able to preserve viability of the bacteria, which decreased of only one log unit and was maintained up to 30 days. RESULTS: Probiotic powders showed a high respirability (42%-50% of particles had a size < 5 µm) suitable for lung deposition and were proven safe on A549 and Calu-3 cells up to a concentration of 107 colony-forming units/mL. The Lpb. plantarum adhesion to both cell lines tested was at least 10%. Surprisingly, Lpb. plantarum powder was bactericidal at a concentration of 106 colony-forming units/mL on P. aeruginosa, whereas the other two strains were bacteriostatic. CONCLUSION: This work represents a promising starting point to consider a probiotic inhalation powder a value in keeping the growth of pathogenic microflora in check during the antibiotic inhalation therapy suspension in cystic fibrosis treatment regimen. This approach could also be advantageous for interfering competitively with pathogenic bacteria and promoting the restoration of the healthy microbiota.

Cyclosporine A micellar nasal spray characterization and antiviral action against SARS-CoV-2.

The upper airways represent the point of entrance from where Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) infection spreads to the lungs. In the present work, α-tocopheryl-polyethylene-glycol succinate (TPGS) micelles loaded with cyclosporine A (CSA) were developed for nasal administration to prevent or treat the viral infection in the very first phases. The behavior of the micelles in presence of simulated nasal mucus was investigated in terms of stability and mucopenetration rate, evidencing long-term stability and fast diffusion across the glycoproteins matrix. Moreover, the spray characteristics of the micellar formulation and deposition profile in a silicon nasal model were studied using three nasal spray devices. Results allowed to identify the nasal spray pump (BiVax, Aptar) able to provide the wider and uniform deposition of the nasal cavity. The cyclosporine A micelles antiviral activity against SARS-CoV-2 was tested on the Omicron BA.1 variant using Vero E6 cells with protocols simulating treatment before, during and after the infection of the upper airways. Complete viral inactivation was observed for the cyclosporine-loaded micelles while a very low activity was evidenced for the non-formulated drug, suggesting a synergistic activity of the drug and the formulation. In conclusion, this work showed that the developed cyclosporine A-loaded micellar formulations have the potential to be clinically effective against a wide spectrum of coronavirus variants.

A dry powder formulation for peripheral lung delivery and absorption of an anti-SARS-CoV-2 ACE2 decoy polypeptide.

One of the strategies proposed for the neutralization of SARS-CoV-2 has been to synthetize small proteins able to act as a decoy towards the virus spike protein, preventing it from entering the host cells. In this work, the incorporation of one of these proteins, LCB1, within a spray-dried formulation for inhalation was investigated. A design of experiments approach was applied to investigate the optimal condition for the manufacturing of an inhalable powder. The lead formulation, containing 6% w/w of LCB1 as well as trehalose and L-leucine as excipients, preserved the physical stability of the protein and its ability to neutralize the virus. In addition, the powder had a fine particle fraction of 58.6% and a very high extra-fine particle fraction (31.3%) which could allow a peripheral deposition in the lung. The in vivo administration of the LCB1 inhalation powder showed no significant difference in the pharmacokinetic from the liquid formulation, indicating the rapid dissolution of the microparticles and the protein capability to translocate into the plasma. Moreover, LCB1 in plasma samples still maintained the ability to neutralize the virus. In conclusion, the optimized spray drying conditions allowed to obtain an inhalation powder able to preserve the protein biological activity, rendering it suitable for a systemic prevention of the viral infection via pulmonary administration.

Metered dose inhalers in the transition to low GWP propellants: what we know and what is missing to make it happen.

INTRODUCTION: The urgency to replace the propellants currently in use with the new sustainable ones has given rise to the need for investigation and reformulation of pMDIs. AREAS COVERED: The reformulation requires in-depth knowledge of the physico-chemical characteristics of the new propellants, which impact the atomization capacity and the plume geometry. Among the investigated propellants, HFA 152a, due to its lower vapor pressure and higher surface tension compared to HFA 134a, deliver larger particles and has a higher solvent capacity toward lipophilic drugs. On the other hand, HFO 1234ze has properties more similar to HFA 134a, but showed lower reproducibility of the generated spray, indicating a possible high susceptibility to variation in the consistency of the dose delivered. In addition, the device components currently in use are compatible with the new propellants. This allowed promising preliminary results in the re-formulation of pMDIs by academia and pharma companies. However, there is little information about the clinical studies required to allow the marketing of these new products. EXPERT OPINION: Overall, studies conducted so far show that the transition is technically possible, and the main obstacle will be represented by the investment required to put the product on the market.

Nasal delivery as a strategy for the prevention and treatment of COVID-19.

INTRODUCTION: The upper respiratory tract is a major route of infection for COVID-19 and other respiratory diseases. Thus, it appears logical to exploit the nose as administration site to prevent, fight, or minimize infectious spread and treat the disease. Numerous nasal products addressing these aspects have been considered and developed for COVID-19. AREAS COVERED: This review gives a comprehensive overview of the different approaches involving nasal delivery, i.e., nasal vaccination, barrier products, and antiviral pharmacological treatments that have led to products on the market or under clinical evaluation, highlighting the peculiarities of the nose as application and absorption site and pointing at key aspects of nasal drug delivery. EXPERT OPINION: From the analysis of nasal delivery strategies to prevent or fight COVID-19, it emerges that, especially for nasal immunization, formulations appear the same as originally designed for parenteral administration, leading to suboptimal results. On the other hand, mechanical barrier and antiviral products, designed to halt or treat the infection at early stage, have been proven effective but were rarely brought to the clinics. If supported by robust and targeted product development strategies, intranasal immunization and drug delivery can represent valid and sometimes superior alternatives to more conventional parenteral and oral medications.

Aerosolized sulfated hyaluronan derivatives prolong the survival of K18 ACE2 mice infected with a lethal dose of SARS-CoV-2.

Despite several vaccines that are currently approved for human use to control the pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), there is an urgent medical need for therapeutic and prophylactic options. SARS-CoV-2 binding and entry in human cells involves interactions of its spike (S) protein with several host cell surface factors, including heparan sulfate proteoglycans (HSPGs), transmembrane protease serine 2 (TMPRSS2), and angiotensin-converting enzyme 2 (ACE2). In this paper we investigated the potential of sulphated Hyaluronic Acid (sHA), a HSPG mimicking polymer, to inhibit the binding of SARS-CoV-2 S protein to human ACE2 receptor. After the assessment of different sulfation degree of sHA backbone, a series of sHA functionalized with different hydrophobic side chains were synthesized and screened. The compound showing the highest binding affinity to the viral S protein was further characterized by surface plasmon resonance (SPR) towards ACE2 and viral S protein binding domain. Selected compounds were formulated as solutions for nebulization and, after being characterized in terms of aerosolization performance and droplet size distribution, their efficacy was assessed in vivo using the K18 human (h)ACE2 transgenic mouse model of SARS-CoV-2 infection.

Nebulizers effectiveness on pulmonary delivery of alpha-1 antitrypsin.

The nebulization of alpha-1 antitrypsin (AAT) for its administration to the lung could be an interesting alternative to parenteral infusion for patients suffering from AAT genetic deficiency (AATD). In the case of protein therapeutics, the effect of the nebulization mode and rate on protein conformation and activity must be carefully considered. In this paper two types of nebulizers, i.e., a jet and a mesh vibrating system, were used to nebulize a commercial preparation of AAT for infusion and compared. The aerosolization performance, in terms of mass distribution, respirable fraction, and drug delivery efficiency, as well as the activity and aggregation state of AAT upon in vitro nebulization were investigated. The two nebulizers demonstrated equivalent aerosolization performances, but the mesh nebulizer provided a higher efficiency in the delivery of the dose. The activity of the protein was acceptably preserved by both nebulizers and no aggregation or changes in its conformation were identified. This suggests that nebulization of AAT represents a suitable administration strategy ready to be translated to the clinical practice for delivering the protein directly to the lungs in AATD patients, either as a support therapy to parenteral administration or for subjects with a precocious diagnosis, to prevent the onset of pulmonary symptoms.

Inhalable Microparticles Embedding Biocompatible Magnetic Iron-Doped Hydroxyapatite Nanoparticles.

Recently, there has been increasing interest in developing biocompatible inhalable nanoparticle formulations, as they have enormous potential for treating and diagnosing lung disease. In this respect, here, we have studied superparamagnetic iron-doped calcium phosphate (in the form of hydroxyapatite) nanoparticles (FeCaP NPs) which were previously proved to be excellent materials for magnetic resonance imaging, drug delivery and hyperthermia-related applications. We have established that FeCaP NPs are not cytotoxic towards human lung alveolar epithelial type 1 (AT1) cells even at high doses, thus proving their safety for inhalation administration. Then, D-mannitol spray-dried microparticles embedding FeCaP NPs have been formulated, obtaining respirable dry powders. These microparticles were designed to achieve the best aerodynamic particle size distribution which is a critical condition for successful inhalation and deposition. The nanoparticle-in-microparticle approach resulted in the protection of FeCaP NPs, allowing their release upon microparticle dissolution, with dimensions and surface charge close to the original values. This work demonstrates the use of spray drying to provide an inhalable dry powder platform for the lung delivery of safe FeCaP NPs for magnetically driven applications.

An Enhanced Dissolving Cyclosporin-A Inhalable Powder Efficiently Reduces SARS-CoV-2 Infection In Vitro.

This work illustrates the development of a dry inhalation powder of cyclosporine-A for the prevention of rejection after lung transplantation and for the treatment of COVID-19. The influence of excipients on the spray-dried powder's critical quality attributes was explored. The best-performing powder in terms of dissolution time and respirability was obtained starting from a concentration of ethanol of 45% (v/v) in the feedstock solution and 20% (w/w) of mannitol. This powder showed a faster dissolution profile (Weibull dissolution time of 59.5 min) than the poorly soluble raw material (169.0 min). The powder exhibited a fine particle fraction of 66.5% and an MMAD of 2.97 µm. The inhalable powder, when tested on A549 and THP-1, did not show cytotoxic effects up to a concentration of 10 µg/mL. Furthermore, the CsA inhalation powder showed efficiency in reducing IL-6 when tested on A549/THP-1 co-culture. A reduction in the replication of SARS-CoV-2 on Vero E6 cells was observed when the CsA powder was tested adopting the post-infection or simultaneous treatment. This formulation could represent a therapeutic strategy for the prevention of lung rejection, but is also a viable approach for the inhibition of SARS-CoV-2 replication and the COVID-19 pulmonary inflammatory process.

Nano-adjuvanted dry powder vaccine for the mucosal immunization against airways pathogens.

Nasal vaccination has been shown to provide optimal protection against respiratory pathogens. However, mucosal vaccination requires the implementation of specific immunization strategies to improve its effectiveness. Nanotechnology appears a key approach to improve the effectiveness of mucosal vaccines, since several nanomaterials provide mucoadhesion, enhance mucosal permeability, control antigen release and possess adjuvant properties. Mycoplasma hyopneumoniae is the main causative agent of enzootic pneumonia in pigs, a respiratory disease responsible for considerable economic losses in the pig farming worldwide. The present work developed, characterized, and tested in vivo an innovative dry powder nasal vaccine, obtained from the deposition on a solid carrier of an inactivated antigen and a chitosan-coated nanoemulsion, as an adjuvant. The nanoemulsion was obtained through a low-energy emulsification technique, a method that allowed to achieve nano droplets in the order of 200 nm. The oil phase selected was alpha-tocopherol, sunflower oil, and poly(ethylene glycol) hydroxystearate used as non-ionic tensioactive. The aqueous phase contained chitosan, which provides a positive charge to the emulsion, conferring mucoadhesive properties and favoring interactions with inactivated M. hyopneumoniae. Finally, the nanoemulsion was layered with a mild and scalable process onto a suitable solid carrier (i.e., lactose, mannitol, or calcium carbonate) to be transformed into a solid dosage form for administration as dry powder. In the experimental study, the nasal vaccine formulation with calcium carbonate was administered to piglets and compared to intramuscular administration of a commercial vaccine and of the dry powder without antigen, aimed at evaluating the ability of IN vaccination to elicit an in vivo local immune response and a systemic immune response. Intranasal vaccination was characterized by a significantly higher immune response in the nasal mucosa at 7 days post-vaccination, elicited comparable levels of Mycoplasma-specific IFN-γ secreting cells and comparable, if not higher, responsiveness of B cells expressing IgA and IgG in peripheral blood mononuclear cells, with those detected upon a conventional intramuscular immunization. In conclusion, this study illustrates a simple and effective strategy for the development of a dry powder vaccine formulation for nasal administration which could be used as alternative to current parenteral commercial vaccines.

The impact of possible improper use on the performance in vitro of NEXThaler in comparison with Ellipta inhaler.

The correct use of dry powder inhalers by the patients is essential to ensure effective treatment and management of the disease. The purpose of the work was to assess the consequence of inhaler misuse in terms of emitted dose and aerodynamic parameters. One reservoir multidose device (Foster-NEXThaler®) and one pre-dosed device (Relvar-Ellipta®), both sharing the "open, inhale and close" procedure, were the subject of the study. NEXThaler activated at different degrees of inclination showed a consistent dose delivery for both the drugs included in the formulation (beclometasone dipropionate/formoterol fumarate). Contrary, Ellipta showed a decrease of the emitted dose for both fluticasone furoate (FluF) and vilanterol trifenatate (VT) when the device was operated facing downward (-14% at 45° and -22% at 90°). Similarly, the delivered dose of NEXThaler was unaffected by an accidental fall, while Ellipta released FluF and VT doses 50% lower than control values. The presence of the dose protector in NEXThaler offers the advantage of retaining the powder if the inhaler is subjected to incorrect manipulations. Both products proved to be reliable in double activation. Finally, simulation exhalation conditions impaired, although not significantly, the aerodynamic profile of the two products.

No-shaking and shake-fire delays affect respirable dose for suspension but not solution pMDIs.

It has long been accepted that suspension pressurized metered-dose inhalers (pMDIs) must be shaken if a correct dose is to be delivered, if not, it will usually be higher than the label claim. The purpose of this work was to investigate the influence of the device being unshaken, shaken and after a period of delay in pMDI actuation on the Fine Particle Mass (<5 µm), Extra Fine Particle Mass (<2 µm) and MMAD. Solution and suspension commercial pMDIs containing one, two or three components were used in the study. Most of the suspension pMDIs produced variable amounts of respirable size drug following the shake-fire delays tested in terms of the label claim dose. The effect was even more critical if the inhaler was not shaken and the FPM was found to be between -82 % for Symbicort and 363 % for Ventolin compared with the control values. In the case of MMAD measurements, Seretide and Serzyl inhalers showed the largest change from around 3 µm to 4.2-5.1 µm when not shaken. Conversely, the FPM and MMAD for the solution aerosols remained unchanged whether or not they were shaken or when a progressive increase in the delay in actuation after shaking was employed.

Recent Patents on Nasal Vaccines Containing Nanoadjuvants.

Vaccines are one of the greatest medical achievements of modern medicine. The nasal mucosa represents an effective route of vaccination for both mucosal immunity and peripheral, being at the same time an inductive and effector site of immunity. In this paper, the innovative and patented compositions and manufacturing procedures of nanomaterials have been studied using the peerreviewed research literature. Nanomaterials have several properties that make them unique as adjuvant for vaccines. Nanoadjuvants through the influence of antigen availability over time affect the immune response. Namely, the amount of antigen reaching the immune system or its release over prolonged periods of time can be effectively increased by nanoadjuvants. Mucosal vaccines are an interesting alternative for immunization of diseases in which pathogens access the body through these epithelia. Nanometric adjuvants are not only a viable approach to improve the efficacy of nasal vaccines but in most of the cases they represent the core of the intellectual property related to the innovative vaccine.

Curcumin and Quercetin-Loaded Lipid Nanocarriers: Development of Omega-3 Mucoadhesive Nanoemulsions for Intranasal Administration.

Curcumin (CUR) and quercetin (QU) are potential compounds for treatment of brain diseases such as neurodegenerative diseases (ND) because of their anti-inflammatory and antioxidant properties. However, low water solubility and poor bioavailability hinder their clinical use. In this context, nanotechnology arises as a strategy to overcome biopharmaceutical issues. In this work, we develop, characterize, compare, and optimize three different omega-3 (ω-3) fatty acids nanoemulsions (NEs) loaded with CUR and QU (negative, cationic, gelling) prepared by two different methods for administration by intranasal route (IN). The results showed that formulations prepared with the two proposed methods exhibited good stability and were able to incorporate a similar amount of CUR and QU. On the other side, differences in size, zeta potential, in vitro release kinetics, and permeation/retention test were observed. Considering the two preparation methods tested, high-pressure homogenization (HPH) shows advantages, and the CQ NE- obtained demonstrated potential for sustained release. Toxicity studies demonstrated that the formulations were not toxic for Caenorhabditis elegans. The developed ω-3 fatty acid NEs have shown a range of interesting properties for the treatment of brain diseases, since they have the potential to increase the nose-to-brain permeation of CUR and QU, enabling enhanced treatments efficiency.

In Vitro Evaluation of Curcumin- and Quercetin-Loaded Nanoemulsions for Intranasal Administration: Effect of Surface Charge and Viscosity.

The nose-to-brain delivery of neuroprotective natural compounds is an appealing approach for the treatment of neurodegenerative diseases. Nanoemulsions containing curcumin (CUR) and quercetin (QU) were prepared by high-pressure homogenization and characterized physicochemically and structurally. A negative (CQ_NE-), a positive (CQ_NE+), and a gel (CQ_NEgel) formulation were developed. The mean particle size of the CQ_NE- and CQ_NE+ was below 120 nm, while this increased to 240 nm for the CQ_NEgel. The formulations showed high encapsulation efficiency and protected the CUR/QU from biological/chemical degradation. Electron paramagnetic resonance spectroscopy showed that the CUR/QU were located at the interface of the oil phase in the proximity of the surfactant layer. The cytotoxicity studies showed that the formulations containing CUR/QU protected human nasal cells from the toxicity evidenced for blank NEs. No permeation across an in vitro model nasal epithelium was evidenced for CUR/QU, probably due to their poor water-solubility and instability in physiological buffers. However, the nasal cells' drug uptake showed that the total amount of CUR/QU in the cells was related to the NE characteristics (CQ_NE- > CQ_NE+ > CQ_NEgel). The method used allowed the obtainment of nanocarriers of an appropriate size for nasal administration. The treatment of the cells showed the protection of cellular viability, holding promise as an anti-inflammatory treatment able to prevent neurodegenerative diseases.

A respirable HPV-L2 dry-powder vaccine with GLA as amphiphilic lubricant and immune-adjuvant.

Vaccines not requiring cold-chain storage/distribution and suitable for needle-free delivery are urgently needed. Pulmonary administration is one of the most promising non-parenteral routes for vaccine delivery. Through a multi-component excipient and spray-drying approach, we engineered highly respirable dry-powder vaccine particles containing a three-fold repeated peptide epitope derived from human papillomavirus (HPV16) minor capsid protein L2 displayed on Pyrococcus furious thioredoxin as antigen. A key feature of our engineering approach was the use of the amphiphilic endotoxin derivative glucopyranosyl lipid A (GLA) as both a coating agent enhancing particle de-aggregation and respirability as well as a built-in immune-adjuvant. Following an extensive characterization of the in vitro aerodynamic performance, lung deposition was verified in vivo by intratracheal administration in mice of a vaccine powder containing a fluorescently labeled derivative of the antigen. This was followed by a short-term immunization study that highlighted the ability of the GLA-adjuvanted vaccine powder to induce an anti-L2 systemic immune response comparable to (or even better than) that of the subcutaneously administered liquid-form vaccine. Despite the very short-term immunization conditions employed for this preliminary vaccination experiment, the intratracheally administered dry-powder, but not the subcutaneously injected liquid-state, vaccine induced consistent HPV neutralizing responses. Overall, the present data provide proof-of-concept validation of a new formulation design to produce a dry-powder vaccine that may be easily transferred to other antigens.