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New dry powder aerosol COVID-19 vaccine shows promise against multiple virus strains

New dry powder aerosol COVID-19 vaccine shows promise against multiple virus strains


In a recent study published in the journal Nature, researchers developed and tested a novel dry powder aerosol vaccine against COVID-19. The nanoparticle-based vaccine comprises cholera toxin B subunits with the SARS-CoV-2 RBD antigens. Study findings revealed that this single-use nasal spray promotes the robust production of IgG and IgA antibodies and bolsters local T cell responses with the nasal tract and alveoli in murine and non-human primate models. The composition of the virus allows it to confer defense against both ancestral COVID-19 variants and the more recent Omicron strains. This novel vaccine could form the basis for a new generation of non-invasive vaccines against both COVID-19 and other respiratory tract infections.

Study: Inhaled SARS-CoV-2 vaccine for single-dose dry powder aerosol immunization. Image Credit: Frau aus UA / ShutterstockStudy: Inhaled SARS-CoV-2 vaccine for single-dose dry powder aerosol immunization. Image Credit: Frau aus UA / Shutterstock

COVID-19 and the merits of vaccination

The ongoing severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) caused coronavirus disease 2019 (COVID-19), one of the most devastating pandemics in recent times. It has resulted in widespread morbidity, mortality, and socioeconomic loss, claiming almost 7 million lives and leaving more than 65 million survivors with long-term symptoms (Long COVID) since the advent of its outbreak in late 2019.

Encouragingly, the rapid development of vaccines against the virus has slowed and even halted its progress in many regions. Most commercially approved anti-COVID-19 vaccines fall into the inactivated virus, messenger RNA (mRNA), protein subunit, and viral-vectored vaccine cohorts. Unfortunately, all these vaccine types share a common demerit – they are administered via intramuscular injections aimed at producing serological immunoglobulin G (IgG). While effective, these vaccines do not provide protection in the respiratory tract.

A growing body of research suggests that the respiratory tract not only forms the first site of COVID-19 infection but remains the site of the highest viral load throughout the course of the disease and (in the case of Long COVID) beyond. This shortcoming has prompted the development of new vaccine classes designed to be administered intranasally or via nebulization. Notably, research into these vaccines has demonstrated that much lower doses are required to achieve a similar level of protection as those conferred by conventional intramuscularly administered anti-virals.

However, hitherto, most developed and preliminarily approached intranasal vaccines are stored, transported, and administered as liquids or wet aerosols, presenting significant economic cold-chain costs and requiring multiple doses to reach the desired efficacy. Furthermore, most currently available vaccines are monovalent, designed to target one of a small group of SARS-CoV-2 variants. SARS-CoV-2 is a rapidly mutating virus, with thousands of strains discovered thus far. This discordance necessitates the development of novel vaccines capable of targeting multiple COVID-19 strains while requiring minimal patient follow-up.

About the study

In the present study, researchers developed and tested a novel intranasal vaccine designed to both be polyvalent (target multiple COVID-19 lineages) and retain its efficacy following lyophilization (freeze-drying). The vaccine was conceptualized to contain particles 1–4 µm in size to optimize deposition in the alveoli (deep lung) and ensure that efficacy loss was not due to exhaling particles that were too small or resulting in particles that were too large depositing in the superficial lung.

The vaccine was developed using the cholera toxin B (CTB) subunit as the nano chassis, expressed in a genetically modified Escherichia coli BL21 strain. Sodium dodecyl-sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting were used to evaluate the molecular weight of the chassis, with its size verified using transmission electron microscopy (TEM). Dynamic light scattering (DLS) analysis revealed that chassis particle size was predominantly uniform within the desired size class. R-CNP antigenicity was evaluated using surface plasmon resonance and enzyme-linked immunosorbent assays (ELISAs).

Previous studies have demonstrated that direct inhalation of nanosized particles results in poor alveoli uptake. Researchers encapsulated the CTB chassis in microcapsules of 1–4 µm diameter to address this issue.

“By fine-tuning the membrane pore size, osmotic gradient, and pore evolution time, the microspheres can be controlled for particle size, porosity, and cavity volume, which facilitated optimization of R-CNP@M to a suitable aerodynamic size and high encapsulation efficiency”

Confocal laser scanning microscopy (CL-SEM) in tandem with 3D reconstructions was used to verify R-CNP@M (R-CNP encapsulated in microcapsules) size and absorption. Following lyophilization, R-CNP@M was found to be stable with its delivery as an aerosol, resulting in a uniform fog when administered using a dry powder aerosol generator (DPAG). Encouragingly, this aerosol was found to consistently deliver the vaccine for up to five weeks following administration without any notable efficacy loss.

The formulation was tested on mice, hamsters, and non-human primates. All test subjects showed positive results with sustained vaccine delivery to the alveoli observed for up to 42 days.

“The area-under-the-curve values of R-CNP@M were improved by 3.5-fold compared with those of R-CNP, suggesting that R-CNP@M can induce continuous antigen stimulation in the lungs”

Single-cell TNA sequencing (scRNA-seq) was used to measure immune responses in CD45+ cells on the 21st day following immunization. R-CNP@M outperformed currently available intramuscular and liquid aerosol vaccines in activating antigen-presenting cells. Similarly, the proportion of activated CD8+ memory T cells was highest under the R-CNP@M regime. ELISA measurements of collected mice, hamster, and non-human primate blood revealed that “antibody titers of RBD-specific IgM, IgG, IgG1 and IgG2a in serum were more rapidly and more robustly increased by a single dose of R-CNP@M than by two doses of free R-CNP (10 μg equivalent RBD), whereas RBD-specific antibodies were undetectable in the CNP group.”


The present study presents the development of the first dry-aerosol vaccination vehicle, thereby overcoming the demerits of conventional intramuscular vaccine delivery systems and outperforming liquid-based aerosols in both economic and efficacy metrics. The nanoparticle chassis encapsulated in a 1–4 µm microcapsule was found to be stable for up to 42 days and safe in mammalian and non-human primate test subjects. The vaccine presented consistent antigen release into the alveoli for up to five weeks following administration. It resulted in the highest immunoglobulin and memory T cell activation of all tested vaccination regimes.

“The inhalable vaccination addresses a known public health issue, in that there is more enthusiasm for this type of administration than for traditional injection, and a single-dose regimen is favorable for substantially increasing the proportion of total completed vaccination recipients. Furthermore, the dry powder form of the vaccine can provide savings in storage and transportation costs, potentially supporting increased immunization coverage in remote areas. Additionally, the prospects of clinical translation are boosted by its use of a proteinaceous nanoparticle chassis and a microcapsule based on a US Food and Drug Administration-approved material.”

Most encouragingly, this vaccine was found to be both single dose and multivalent – unlike conventional vaccines, the efficacy of R-CNP@M was stable against infection by conventional and Omicron COVID-19 lineages. Furthermore, unlike previous vaccines (including most aerosol-based ones) this vaccine requires administration only once.

“Considering the flexibility of displaying antigens on the CNP chassis, we envision that our inhaled vaccine could serve as a promising multivalent platform for fighting COVID-19 and other respiratory infectious diseases.”

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