Thursday, May 16, 2024

National University Singapore Scientists Develop mRNA-based Mucosal Nasal Vaccine

Scientist, academic researcher and entrepreneur Ayça Altay Benetti, Ph.D., of National University of Singapore (NUS) along with Kevin P. White and Giorgia Pastorin—both professors also at NUS and colleagues present design and characterization attributes of a novel formulation for delivery of COVID-19 mRNA vaccine targeting the nasal mucosal pathway. They are exploring alternative substances to use as a vehicle to not only efficiently deliver mRNA payloads to the nasal mucosa, but also with the ability to promote uptake by target cells such as epithelial and immune cells.

Identifying chitosan, a natural polysaccharide derived from chitin for this purpose—given its biocompatibility, biodegradability and mucoadhesive characteristics, the Singapore-based team reports on formulations achieving a mRNA encapsulation efficiency of ~80.2% with chitosan-lipid nanoparticles, as measured by the RiboGreen assay.

Reporting on transfection levels in select human cells as part of the evaluation of SARS-CoV-2 Spike (S) receptor-binding domain (RBD) expression via ELISA for the group’s vaccine formulations, the most promising vaccine formulation was also responsive to lyophilization (freeze drying or cryodesiccation)with minimal degradation of loaded mRNA, demonstrating a pathway towards a more accessible and stable vaccine.

Studying the highly experimental vaccine in in vivo mice studies, the team evaluated the systemic and local immune responses. The authors report that a nasal bronchoalveolar lavage fluid (BALF) wash evidenced sufficient levels of antibody concentrations less any problematic systemic antibody response. The academic center scientists in Singapore suggest they have developed a potential candidate for clinical development and commercialization.

Why Researchers Look into Nasal Mucosal Vaccines against COVID-19?

Nasal mucosal vaccines targeting COVID-19 are desirable for several reasons.

First, they can potentially enhance immune response at the SARS-CoV-2 pathogen entry point. COVID-19 primarily enters the body via the respiratory tract. Nasal vaccines can induce strong mucosal immune response in nasal passages and upper respiratory tract, and this happens to be the initial site of the infection.

The premise here is that such localized immunity can potentially prevent the virus from establishing an infection, thereby providing an effective first line of defense. Furthermore, this class of vaccine can potentially reduce aid reduction in transmission, meaning that when nasal mucosal vaccines generate immunity directly in the respiratory mucosa, nasal vaccines possibly can reduce viral shedding and transmission. Put another way, if SARS-CoV-2 is neutralized in the nasal passages, it is less likely to be spread to others through respiratory droplets.

Also, this vaccine method could be administered without needles, meaning it’s easier to administer. This results in a more user-friendly form of vaccine, reducing any discomfort and anxiety associated with injections. But there is more. A nasal mucosal vaccine may stimulate broader immune response, including the production of IgA antibodies, which play a key role in mucosal immunity. This can complement the systemic immunity (IgG antibodies generated by traditional intramuscular vaccines, potentially offering more comprehensive protection.

Other potential benefits of a nasal type of vaccine targeting COVID-19 include the potential for rapid deployment, improved acceptance (given its non-invasive delivery) and cross-protection against variants. On this latter point, preliminary research suggests that mucosal immunity might offer better cross-protection against various variants of the virus. Why? Well, since the nasal passages are the primary entry point for respiratory viruses, a robust mucosal immune response could help in neutralizing diverse viral strains before they cause systemic infection.

The Singapore-based team points out in their paper published in MDPI’s Vaccines, that a chitosan-based formulation represents a good possibility for an mRNA nasal vaccine. With a mucoadhesive nature and ability to transiently open tight junctions between epithelial cells, chitosan facilitates the transport of macromolecules across the nasal mucosa. In their paper, the authors note, “One of the few natural polymers with a positive charge at a slightly acidic pH, chitosan is expected to prolong the local delivery of actives at the mucosa through electrostatic interactions with negatively charged mucin.”

Based on some evidence, the authors suggest this could enable the efficient delivery of mRNA to target cells within the nasal epithelium, ultimately offering opportunity to develop COVID-19 vaccines through the intranasal route.

The authors point to the promise of using the unique mucoadhesive properties of chitosan, potentially in combination with the delivery capabilities of LNPs or liposomal vehicles. This could lead to new avenues for the development of mRNA-based nasal therapeutics. And establish in the future ways to treat COVID-19 via local immunization, along with other benefits including improved bioavailability, reduced systemic degradation, and enhanced uptake by target cells within the nasal mucosa.

But can mucosal formulations overcome some of the current limitations or challenges associated with the current COVID-19 vaccines?

Threats to cargo (mRNA) stability mainly include RNAse enzymes, which easily degrade mRNA (which is hydrolyzed at pH > 6). Even through the existing COVID-19 vaccines use LNPs to encapsulate the nucleic acid therapeutic, regardless instability still occurs in the LNP system when cationic lipids lower the pKa of ribose 2′ hydroxyl group in mRNA, which can increase RNA hydrolysis.

Need for ultra cold chain

mRNA vaccines need to be stored at far lower temperatures. If compromised by external factors resulting from storage conditions, these dosage forms will cause the premature release and degradation of the mRNA by RNase

So, to improve mRNA formulation stability requires one of two directions: either modify the mRNA itself (the cargo) or improve the drug delivery system. The study team assumes that the current methodology is safe and effective, not concerned with a growing, yet not frequently cited literature expressing concern around stability and there are safety issues linked to the instructions to produce the spike protein, although the majority medical research establishment has yet to acknowledge or accept such concerns.

The Study Background

In this study, the team evaluated the stability and efficacy of liposomal formulations, in the presence or absence of chitosan or PEG lipids loaded with linear or circular mRNA. By comparing circular RNA (cRNA) to traditional linear poly-adenylated RNA, the team studied whether the cRNA could display greater stability due to its resistance to exonucleases.

Importantly, what they refer to as optimized formulations in the investigation depend on “self-assembling systems” involving lipids complexed with mRNA, an adjustable process allowing differing ratios of cationic lipid to cholesterol or the ratios of lipid to cholesterol to PEG lipid to chitosan.


Achieving an mRNA encapsulation efficiency of ~80.2% using chitosan-lipid nanoparticles, the team employed use of RiboGreen assay for measuring results.

Using ELISA to evaluate the team’s vaccine formulations and corresponding spike protein receptor-binding domain (RBD) expression, the team reports transfection levels “in human embryonic kidney cells (HEK 293), lung carcinoma cells (A549), and dendritic cells (DC 2.4) equal to 9.9 ± 0.1 ng/mL (174.7 ± 1.1 fold change from untreated cells (UT)), 7.0 ± 0.2 ng/mL (128.1 ± 4.9 fold change from UT), and 0.9 ± 0.0 ng/mL (18.0 ± 0.1 fold change from UT), respectively.”

With their top investigational vaccine prospect amenable to lyophilization with minimal degradation of loaded mRNA, the Singapore-based team expresses enthusiasm for the prospect of a more accessible and stable vaccine.

Summarizing at least initial in vivo studies in mice with a nasal bronchoalveolar lavage fluid (BALF) wash highlighting an optimized formulation, which led to local antibody concentrations less any systemic antibody response.

They suggest, “If further improved and developed, it could potentially contribute to the management of COVID-19 through nasopharyngeal immunization strategies.”


Ear nose and throat illness was slightly elevated among Covid vaccine users

Do COVID-19 vaccines lead to otolaryngology-based adverse events, and ultimately injuries? A medical specialty which is focused on the ears, nose, and throat as well as otolaryngology-head and neck surgeries, these specialists are trained in both medicine and surgery. An otolaryngologist is often called an ear, nose, and throat doctor, or an ENT for short, and recently, specialists from the Tri-State Area published a report on “Otolaryngologic side Effects After COVID-19 Vaccination.” Led by Christina Fang, M.D., Department of Otorhinolaryngology – Head and Neck Surgery, Albert Einstein College of Medicine, Montefiore Medical Center, Bronx, New York, the team designed a r1etrospective analysis of national registry the team investigated the frequency of otolaryngologic adverse events (AEs) after COVID-19 vaccination as compared to other vaccines in a national database.

Tapping into the Food and Drug Administration's (FDA) Vaccine Adverse Event Reporting System (VAERS) database from December 2020 to May 2021, for all COVID-19 vaccination AEs, the team represented by Dr. Fang and colleagues categorized complaints as otolaryngologic and sub stratified into different anatomic components. The study authors determine reporting odds ratios (ROR) and proportional reporting ratios (PRR) were determined for AEs of clinical significance.

While the investigators identified numerous otolaryngologic symptoms, few were found to be clinically significant. Fang and team report facial paralysis, Bell's palsy, and anaphylaxis did not meet signal thresholds to be determined significant. However, signal ratios were detected for a range of issues including dysgeusia, ageusia Tinnitus and Vertigo.

The recent outcomes were reported in the journal The Laryngoscope.


The authors reported 1,280,950 AEs from vaccination potentially linked to Moderna, Pfizer-BioNTech, and Janssen. 4.9% of the total or 62,660 fell in the otolaryngologic category.

32.6% of the incidents were associated with oropharynx/larynx, 18.3% with the nasal cavity/sinuses, 17.1% with the ears/vestibular system, 10.0% with the oral cavity, and 21.9% miscellaneous.

According to Fang and colleagues, “Signal ratios reached significance levels for dysgeusia (n = 2124, PRR: 17.33, ROR: 16.36), ageusia (n = 1376, PRR: 2.81, ROR: 2.81), anosmia (n = 983, PRR: 4.01, ROR: 4.01), rhinorrhea (n = 2203, PRR: 2.99, ROR: 3.00), throat tightness (n = 3666, PRR: 4.99, ROR: 5.00), throat irritation (n = 3313, PRR: 4.51, ROR: 4.52), dysphagia (n = 2538, PRR: 2.07, ROR: 2.07), tinnitus (n = 4377, PRR: 3.97, ROR: 3.98), and vertigo (n = 2887, PRR: 3.93, ROR: 3.93).”

However, no significant signal ratios were identified for facial paralysis, Bell's palsy, anaphylaxis, sinusitis, hearing disability, and ear pain.




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