The Exciting Potential of Inhalation Vaccines

Vaccines delivered intramuscularly are effective but generally do not prevent viral shedding from the respiratory system, because they do not cause mucosal immunity. Vaccines delivered via inhalation to the upper and lower respiratory tracts and lungs generate not only systemic immune responses but also mucosal immune responses that can prevent virus transmission. Recent advances in delivery technologies are helping vaccine developers overcome the challenges to mucosal delivery, leading to effective inhalation vaccines against COVID-19 and other viral diseases.

Importance of Mucosal Immunity

Vaccines injected intramuscularly can be effective at generating systemic immune responses, but in general they do not cause strong mucosal immune defenses, which can limit the effectiveness of vaccines against pathogens that infect the mucosal membranes in the respiratory, digestive, and urogenital tracts.¹ Despite being located in disparate parts of the body, these tissues are interconnected through crosstalk between mucosal compartments, leading to immunization, even at distant mucosal sites.^1,2

In this specialized adaptive and innate immune system, mucus layers form outer protective barriers, and mucosa-associated lymphoid tissues (MALTs) produce secretory immunoglobin A (IgA) antibodies (sIgA) that entrap and clear harmful microorganisms and tissue-resident memory T (T_RM) cells that recruit circulating memory T cells and produce inflammatory cytokines and chemokines.¹ Other immune cells, including phagocytic neutrophils, macrophages, dendritic cells (DCs), natural killer cells, and mast cells are also present.² Pattern recognition receptors (PRRs) expressed in various cell types can recognize pathogen-associated molecular patterns (PAMPs) expressed on viruses or damage-associated molecular patterns (DAMPs) from infected cells, causing antigen-presenting cells (APCs) to become active and resulting in production of active immune agents.^2,3

Vaccines that target mucosal layers have the potential to provide significant benefits over parenterally delivered vaccines, particularly for infectious diseases caused by pathogens that target these tissues (in the lungs and upper respiratory tract, for example), which are numerous.⁴ Mucosal delivery enables stimulation of both mucosal and systemic immune responses. In addition, by attacking harmful microorganisms at the initial site of attack, they may also prevent shedding of viral particles and thus result in reduced transmission. Furthermore, they can be delivered via oral, rectal, vaginal, nasal, intranasal, or pulmonary administration, none of which involve the need for injection. That eliminates the risk of needle-stick injuries, needle reuse (which can create contamination issues) and patient resistance due to concerns about pain.^1,5 They can thus increase patience compliance and are suitable for wide-spread vaccination campaigns.

Overall, effective vaccination of respiratory tract mucosa results in both systemic immune responses (IgG- and cell-mediated) and a strong local mucosal immune response.³

Rising Interest in Pulmonary Vaccination

Given the potential benefits of mucosal vaccination, interest in pulmonary delivery of vaccines has increased dramatically in the wake of the COVID-19 pandemic and wider awareness of the need to decrease transmission of new variants and increase global access to effective vaccines.¹ In addition, pulmonary vaccines can be administered as liquids using nebulizers or pressurized metered dose inhalers or as powders using active or passive pressurized dry powder inhalers (DPIs), creating the potential for self-administration and thus elimination of the need for healthcare infrastructure and trained personnel.

Finally, good drug absorption, fast onset of action, and high bioavailability can all be achieved when vaccines are delivered to the lungs.⁵ These important performance features can be attributed to the large alveolar surface area, thin epithelial layer, high vascularization area, low proteolytic enzyme concentration, and hepatic metabolism avoidance within lung tissue.

Inhalation is an attractive solution for the delivery of pulmonary vaccines. It not only delivers the vaccine to the lungs but also enables exposure of all regions of the respiratory tract to the vaccine.¹ There is evidence that lung T_RM cells provide stronger protective immunity than circulating T cells and that inhalation vaccination induces the generation of these cells. Inhalation vaccines also have the potential to induce “sterilizing immunity” against mucosal pathogens.²

In a recent study, researchers at McMaster University showed, using a tuberculosis vaccine, that delivery of vaccines via inhalation is more effective than delivery via nasal spray.⁶ The latter tend to deliver vaccines mainly to the nose and throat, while the former result in vaccine delivery deep into the airway.

Challenges to Vaccine Delivery via Inhalation

Despite the potential benefits of pulmonary vaccines delivered via inhalation, few such vaccines have received regulatory approval. Several mucosal vaccines have been approved for human use, most of which are delivered orally (e.g., vaccines against cholera, rotavirus, poliovirus, and salmonella).¹ One approved flu vaccine is delivered intranasally but as a nasal spray that does not achieve true pulmonary delivery.

The main challenge in delivering mucosal vaccines is the need to overcome the natural barriers presented by mucosal tissues.^1,5 These vaccines must reach the APCs without first being enzymatically degraded or otherwise cleared from the system. The level of immune response must be controlled, however, as one that is too strong can damage the lungs and contribute to disease enhancement.³ In general, formulation of inhalation vaccines is the key hurdle to developing effective products.⁷

Live attenuated viruses tend be more successful at surviving for a sufficient time. For other types of vaccines, particularly subunit vaccines that only contain virus fragments, adjuvants and special delivery systems have been employed to impart increased stability and to allow penetration of the mucosal lining.^3,5 As with any vaccine formulation, careful selection of adjuvants is essential to avoid any safety issues.

Beyond the physicochemical issues, there are also more general challenges related to the nature of inhaled vaccines.³ No animal model exists for inhaled vaccines, because animals cannot be taught to inhale vaccine candidates. In addition, human patients must perform the act of inhalation correctly to ensure that the vaccine is delivered to the lungs, which may require education.

It is interesting to note that several studies performed to evaluate the effect of the site of deposition of vaccine particles administered via inhalation seem to reveal that the location of the deposited particles is more important for systemic viral infections than it is for pathogens that infect the respiratory system.³ It is thought that the extensive, linked mucosal immune system allows for the spread of the immune response regardless of where the vaccine particles are initially deposited.

Many Types of Vaccines Developed for Inhalation Delivery

Several types of inhalation vaccines are under development.² In addition to those based on live attenuated viruses, several vaccine candidates are based on viral vectors, mainly adenoviral vectors, as well as vectors leveraging attenuated influenza virus, parainfluenza virus (PIV) 5, lentiviruses, Newcastle disease virus (NDV), and vesicular stomatitis virus (VSV). Non–viral-vectored vaccines include bacterium-vectored vaccines and nucleic acid- (messenger RNA, DNA) based vaccines.

Even though they tend to be less immunogenic, subunit vaccines formulated with various types of adjuvants are also being developed. Adjuvants under investigation include polyethyleneimine (PEI), N-[1-(2,3-Dioleoyloxy) propyl]-N,N,N-trimethylammonium chloride (DOTAP), chitosan, toxoids, cytokines, saponins, lipids, and PRR ligands, including toll-like receptor (TLR) agonists, such as synthetic bacterial DNA, IC31 (a two-component adjuvant comprising the artificial antimicrobial cationic peptide KLK, and the TLR9-stimulatory oligodeoxynucleotide ODN1a) and other CpG oligonucleotides.³

Liquid and Dry Formulation Options

Both liquid and powder versions of inhalation vaccines are possible. Like parenteral vaccines, liquid formulations may present stability issues and require cold-chain storage and transportation.¹ In some cases, stability can be enhanced by controlling the pH, osmolarity, and ionic strength of the formulated vaccines. Lyophilization before final packaging and shipment (with reconstitution at the site of administration) is another option for vaccine actives that can survive the process.

Dry powder formulations, which can also be generated via spray drying or the formation of solid dispersions, may also face stability issues, but they can generally be managed with the additional of stabilizers.¹ They typically do not require low temperatures for storage and shipment and weigh less than liquids, reducing bulk transportation costs.³ Achieving the appropriate particle size is the bigger challenge, as the right particle size and shape with a uniform particle size distribution is essential to achieving efficient release and distribution in the lungs.⁵ Thin-film freezing has been shown to be an effective method for generating powders with optimal properties.³

Several types of delivery devices have been developed for administration of inhalation vaccines. Least effective are devices are pipette-based and place droplets of the vaccine formulation into the nostrils.² Spray devices can be effective at generating appropriate particle sizes. The most commonly used technologies include pressurized Metered-dose inhalers (MDIs), dry powder inhalers (DPIs), and jet or ultrasonic nebulizers.⁵

MDIs require patients to create a certain level of flow, which not all can achieve.⁵ DPIs address this problem and deliver fine particles of the vaccine at a high flow rate. Numerous studies have shown that dry power aerosols of different types of vaccines reach the lungs with high efficiency. While passive DPIs are simple and inexpensive, active DPIs are more complex and expensive. Disposable DPIs reduce the potential for cross-contamination, prevent re-use, and enable patients to receive consistent doses.

The different types of devices can be used to deliver inhalation vaccines via intranasal, intratracheal, or nebulization routes.⁵ Intranasal delivery allows for self-administration and good drug absorption with rapid onset of action but without first-pass metabolism issues. Intratracheal delivery is generally less expensive and typically requires smaller doses but is invasive and is thus typically used only in animal studies. Nebulization automatically provides a constant flow of vaccine particles but uses large doses and tends to be expensive.

One new device receiving attention has been developed by Aerogen. The Aerogen Ultra is a vibrating mesh aerosol drug delivery technology that requires a much smaller volume of vaccine than parenteral vaccines. It was recently approved in China for use with the inhaled COVID-19 vaccine from CanSino Biologics.

Focus on Inhalation Nanovaccines

Many of the inhalation vaccines under development today have been created as nanoparticles.¹ Nanoscale materials have large surface areas, which enables the nanovaccines to penetrate the mucosal barriers. Various approaches have enabled functionalization of vaccine nanoparticles to exhibit the right balance of mucoadhesion and mucosal penetration, withstand degradation, can be targeted to specific mucosal cells such as lung epithelial cells, and can also deliver adjuvants and/or immunostimulatory agents.

Nanoparticle technologies used for delivery of mucosal vaccine candidates via nasal, oral, sublingual, and colorectal routes include lipid nanoparticles, liposomes, lipid conjugates, virus-like particles, polymeric nanoparticles, and hybrid nanoparticles.¹ These technologies are believed to be applicable for pulmonary mucosal nanovaccines. Biodegradable and biocompatible polymeric nanoparticles (PNPs) have attracted attention because they are relatively simple to prepare and can be designed with a wide range of functionality.⁵ A combination of polymers can be used to achieve the desired level of mucoadhesion and mucosal penetration while also acting as nanocarriers with adjuvant characteristics.

Novel Approaches to Inhalation Delivery

Some researchers have elected to leverage natural compounds present in the human body as delivery vehicles for inhalation vaccines. Scientists at MIT have shown that peptide vaccines with albumin-binding lipid tails can be attached to albumin proteins found in the bloodstream.⁹ They formulated a vaccine against the vaccinia virus using the adjuvant CpG and delivered it intratracheally. The immune response (memory T cells) observed was 25 times greater than that seen when the vaccine was delivered parenterally. In addition, the mice treated with the inhalation vaccine remained protected against the vaccinia virus months later, while those that received the vaccine intramuscularly did not. The researchers believe that this technology could be applied the development of vaccines that prevent tumor formation and protect against other viruses, such as HIV, influenza, and SARS-CoV-2.

Groups at the University of North Carolina–Chapel Hill and North Carolina State University, meanwhile, developed a subunit vaccine comprising receptor-binding domain (RBD) polypeptides formulated with additional immunoadjuvants in a chitosan (CS) solution.¹⁰ The vaccine, when delivered intranasally, induced mucosal immunity after one or two administrations, with responses lasting at least five months. Performance was enhanced with the addition of CpG oligonucleotides to the formulation. Notably, the vaccines were stable at room temperature for at least one month.

Researchers at the Catholic University of America took a very different approach in developing a noninfectious, bacteriophage T4-based, multicomponent, needle- and adjuvant-free mucosal vaccine based on engineered SARS-CoV-2 spike protein trimers attached to a capsid exterior and with a nucleocapsid protein in the interior.¹¹ When two doses were administered intranasally three weeks apart, both strong mucosal immunity and systemic humoral and cellular immune responses were observed. They believe that this approach could be used for the development of mucosal vaccines against other respiratory infections.

A separate group of researchers at UNC-Chapel Hill working with scientists at Duke University used lung-derived exosomes, or nanoscale vesicles, secreted from lung spheroid cells (LSC-Exo) to effectively deliver nucleotide and protein-based vaccines to the lungs via an inhaler.¹² They showed in disease models that mRNA and protein vaccines were delivered more effectively to bronchioles and deep lung tissue using the LSC-Exo vehicle than when using synthetic LNPs. A protein-based, inhalable dry powder VLP was then created by attaching the RBD of the SARS-CoV-2 spike protein to the exterior of an LSC-Exo. Notably, the exosomes can be functionalized to improve cellular targeting and efficacy, such as through the attachment of anti-inflammatory peptides.⁷ The researchers are currently working to overcome challenges associated with large-scale manufacturing.

Development of Inhaled COVID-19 Vaccines

The potential for inhalation vaccines to increase global access to COVID-19 vaccines and dramatically reduce transmission of the SARS-CoV-2 virus has created significant interest in this technology. As of early 2022, at least a dozen nasal vaccines for COVID-19 were being evaluated in clinical trials.²

In the fall, two received approval: CanSino Biologics’ Convidecia Air in China and Bharat Biotech’s iNCOVACC in India.¹³ The former is an adenovirus-vectored vaccine delivered through the mouth, the latter through the nose. Both COVID-19 vaccine candidates developed by Oxford University and Imperial College have been formulated as inhaled vaccines that are now in clinical trials.¹⁴ Researchers at McMaster University also have a multivalent, inhaled viral-vectored vaccine in early clinical trials.¹⁵

Companies are advancing inhaled COVID-19 vaccines as well. Codagenix’s live-attenuated intranasal COVID-19 vaccine CoviLiv™, developed in collaboration with the Serum Institute of India Pvt. Ltd, is being evaluated in a phase III trial that is part of the WHO’s Solidarity Trial Vaccines to support the development of second-generation COVID-19 vaccines with greater efficacy, greater protection against variants of concern, longer duration of protection, improved storage, and/or simplified delivery with needle-free administration.¹⁶ CyanVac and its subsidiary Blue Lake Biotechnology, Inc. enrolled the first participant in a phase I study of its CVXGA1 PIV5-based intranasal COVID-19 vaccine.¹⁷

Essential to Enable Widespread Application of Inhalation Vaccines

These early successes will drive further interest in the development of inhalation vaccines for pulmonary delivery. Most researchers believe, however, that there is much more to be learned. As more knowledge is gained, additional improvements will be achieved.²

More information is needed, for instance, about the mechanisms by which mucosal vaccines activate both mucosal and systemic immune responses and the roles that adjuvants play. This knowledge will lead to better design of delivery systems and adjuvants for improved inhalation vaccine performance. Cost-effective solutions for the large-scale manufacture of inhalation vaccines leveraging these advanced formulations and delivery technologies are also needed.

References

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4. Trafton, Anne. “Vaccination by inhalation.” MIT News. 19 Mar. 2021.
5. Al-Nemrawi, Nusaiba K. et al. “Polymeric Nanoparticles for Inhaled Vaccine.” Polymers. 14: 4450 (2022).
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11. Zhu, Jingen et al. “A Bacteriophage-Based, Highly Efficacious, Needle- and Adjuvant-Free, Mucosal COVID-19 Vaccine.” mBio. 13:e0182222 (2022). DOI:
12. Inhalable COVID-19 Vaccine Shows Promise in Rodent Model. UNC-Chapel Hill. 5 Jul. 2022.
13. Hamzelou, Jessica. “Two inhaled covid vaccines have been approved—but we don’t know yet how good they are.” MIT Technnology Review. 8 Sep. 2022.
14. Smout, Alistair. “Researchers trial inhaled versions of Oxford and Imperial COVID-19 vaccine candidates.” Reuters. 14 Sep. 2020.
15. Small, Fiona. “Inhaled vaccine for COVID-19: The pandemic accelerated decades of research leading to jab-free vaccine now in human testing.” The Conversation. 1 Jun. 2022
16. Codagenix Initiates Dosing in Phase 3 Efficacy Trial of Intranasal COVID-19 Vaccine as Part of WHO-Sponsored Solidarity Trial Vaccines. Codagenix. 26 Oct. 2022.
17. CyanVac and Blue Lake Biotechnology Announce First Participant Dosed in a Phase 1 Clinical Trial to Evaluate its CVXGA1 Intranasal COVID-19 Vaccine. CyanVac. 27 Sep. 2021.