The first nasal spray COVID-19 vaccine has been approved for emergency use in China and India. Here, we focus on the influencing factors and current status of the nasal spray vaccine development.

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Development of Nasal Spray Vaccines
Nasal spray vaccines are a promising route of vaccination and offer unique advantages,
especially in the prevention of infectious diseases transmitted via the respiratory tract.
Compared to injectable vaccines, nasal spray vaccines also have the advantage of being
less invasive and easier to store and distribute.
The nasal spray vaccine for influenza has been used stably for more than a decade, and
the first nasal spray COVID-19 vaccine has been approved for emergency use in China
and India. Here, we focus on the influencing factors and current status of the nasal spray
vaccine development.
Physiological Structure and Immunogenicity of
the Nasal Cavity

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The nasal cavity refers to the interior of the nose or the structure that opens exteriorly at
the nostrils. It is the entrance to the inhaled air and is the first of a series of structures that
make up the respiratory system. The nasal cavity is completely lined by the nasal mucosa,
one of the anatomical structures that form the physical barrier to the body's immune
system. These barriers provide mechanical protection against the invasion of infectious
and allergic pathogens.
Vaccines are also foreign antigens, and the nasal cavity activates immune mechanisms
when exposed to foreign antigens (vaccines). Upon entering the nasal cavity, the antigen
is trapped on the mucosal epithelium and the microfold cells (M cells) of the lymphoid
follicular epithelium then absorb the antigen particles. Subsequently, the antigen is moved
to the basal end of the cell and passed on to antigen-presenting cells (APCs), such as

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dendritic cells (DCs) and macrophages. Thereafter, DC cells migrate through afferent
lymphatic vessels to nearby draining lymph nodes where antigen is presented to naive T
and B cells via the MHC-II complex.
Nasal-associated lymphoid tissues (NALTs) are an important component of mucosal
immunity in the respiratory tract. nALTs have a high distribution of M cells and are the
main site of uptake of macromolecular drugs and particles in the nasal cavity, as well as
the main site of antigen delivery in the nasal immune response. NALTs have
antigen-specific effector B and T cells that differentiate into plasma cells that produce
aggregated IgA and IgG and eventually differentiate into sIgA, triggering an immune
response that intercepts antigens and pathogens. Mucosal immunity also has a unique
set of mechanisms for co-immunity that induces an immune response in distal mucosa,
such as the digestive and genital tract mucosa, but usually the intestinal immunity
activated by nasal spray vaccines is less effective.
Similar to nasal administration, nasal spray vaccines need to overcome multiple
constraints, such as the effect of nasal mucosal cilia clearance, the problem of short
residence time in the nasal cavity, and the obstruction of antigens by the mucus and
epithelial layers of the nasal mucosal site. The solution of these problems often also
requires the assistance of immune adjuvants and penetration enhancers.
Key Technical Factors in the Development of
Nasal Spray Vaccines
Most of the currently marketed nasal vaccines are live attenuated influenza virus vaccines.
We now present several key factors that should be considered when researching and
developing nasal spray vaccines, mainly from the perspective of the immune process.
1. First Step of Drug Delivery - Dosage Forms and Delivery
Equipment

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Successful nasal delivery first requires the cooperation of a suitable device. Although the
nasal cavity has a large mucosal surface area, intranasal delivery of vaccines is limited by
nasal anatomy and aerodynamics, where drugs with large particle sizes are usually
deposited in the anterior nose and consequently expelled or wiped off, while small particle
sizes (i.e., <10 μm) may bypass the nose and enter the lungs. Thus, a suitable device is
needed to disperse the intranasal vaccine formulation into appropriate particle sizes for
delivery to the posterior region of the nose, and achieving a balance of particle sizes is
critical to increasing antigen exposure to the nasal mucosa. FluMist vaccines currently is
delivered using the AccuSpray nasal delivery system. In addition, MAD Nasal from
Teleflex, ViaNase from Kurve, PuffHaler from Aktiv-Dry, and Solovent from BD all offer
nasal mucosal nebulization solutions.
To date, all approved nasal spray vaccines have been in liquid form because of the high
solubility and potency of liquid vaccines. However, liquid formulations are fluid and any
liquid that exceeds the nasal volume will be drained from the nasal cavity, and only highly
soluble or low dose antigens can be delivered intranasally via liquid formulations. The
application of dry powder formulations, on the other hand, requires a prolonged residence
time in conjunction with mucoadhesive polymers such as starch and chitosan to improve
antigen availability to the nasal lymphatic tissue.
2. Prolonging The Residence Time Of Vaccine – Mucoadhesives
Mucoadhesives are commonly used in the development of nasal spray vaccines. Once
the mucoadhesive agent in the vaccine comes into contact with the mucus of the nasal
epithelium, the polymer hydrates, swells and binds to the mucus, prolonging the residence
time of the vaccine in the nasal mucosa. In addition, mucoadhesives also temporarily slow
mucus cilia clearance and do not inhibit antigen release from the vaccine. Commonly
used mucoadhesive polymers include e.g. chitosan, starch, etc.
3. Promoting Transmembrane Penetration of Vaccines -
Permeation Enhancers

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The nasal epithelial layer is covered by a layer of mucus, and nasal spray
vaccines/antigens need to complete the penetration of the mucus layer and epithelial cells.
Firstly, it is necessary to penetrate the mucus layer. Polyethylene glycol (PEG) can be
used as a mucus layer penetration enhancer, while mannitol can dilute the mucus.
Therefore, the use of PEG, mannitol or other mucus penetration enhancers in the
formulation can help improve the penetration of vaccines/antigens into the mucus layer.
The transport of antigen across the epithelial cell barrier is a critical step in the induction of
immunity. Antigens can penetrate the epithelial layer through cells or across cells, and
permeation enhancers such as chitosan and bacterial toxins are often used in this process
to increase the permeability of antigens to the mucosa. Both chitosan and bacterial toxins
can disrupt epithelial junctions and enhance paracellular motility. Specifically, chitosan
promotes transcellular uptake of antigens by M cells in the nasal mucosa and intestinal
mucosa. In contrast, enterotoxins disrupt the mucosal epithelium and enhance antigen
uptake by DC cells, leading to upregulation of CXCR4 and CCR7, thereby allowing DC
cells to migrate to lymph nodes.
4. Reducing Potential Intracranial Transport – Nose-to-Brain
Transport Inhibitors
An important safety concern regarding nasal spray vaccines is the potential for transport
of vaccine antigens or adjuvants from the olfactory epithelium to the central nervous
system (CNS), although researchers analyzing 460 adverse events associated with
FluMist found only one case of encephalitis and laboratory confirmation that it was caused
by an enterovirus. However, the nasal mucosa provides a direct entry point between the
external environment and the central nervous system ( nose-to-brain pathway), and
compounds can enter the cerebrospinal fluid through the nerves associated with the
olfactory area. Therefore, neurophilicity needs to be evaluated in nasal spray vaccine
development.
5. Enhancing the Immunogenicity of Antigens - Vaccine
Adjuvants

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Adjuvants that target APC antigens in the process of immunization can be useful as
immunostimulants. Commonly used immune adjuvants are insoluble aluminum salts and
TLR agonists.
Insoluble aluminum salts are classical FDA-approved immune adjuvants. Several
aluminum salts are currently used in vaccines approved in the United States, such as
Alhydrogel (aluminum hydroxide), Adju-Phos (aluminum phosphate), and amorphous
aluminum phosphate sulfate (AAPS). The immunostimulatory effects of aluminum salts
arise from a variety of mechanisms, including slowing the spread of antigen from the site
of administration, increasing inflammatory cell accumulation, activating complement, and
inducing the differentiation of monocytes into DCs as well as uptake of antigen, antigen
delivery, and activation of CD4+ T cells by DCs.
TLR agonists can act as adjuvants for mucosal immunity; specifically, TLR agonists
function as pathogen-associated molecular patterns (PAMPs) and act by binding to
pathogen recognition receptors (PRRs) on DC cells. Commonly used are TLR2/6 agonists,
TLR3 agonists, TLR3 agonists.
Current Development Status of Nasal Spray
Vaccine
1. Approved Nasal Spray Vaccines
FluMist® (MedImmune, LLC) is the first live attenuated influenza virus intranasal
vaccine that was successfully approved and commercialized in the US and Europe (as
Fluenz®). It is a vaccine that is sprayed into the nose to help protect against influenza. It
can be used in people 2 through 49 years old.
Nasovac-S is a live attenuated nasal spray vaccine produced by the Serum Institute of
India for the prevention of influenza A (H1N1).

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The main nasal spray vaccines currently undergoing clinical trials include influenza
vaccines (H5N1, H1N1, etc.), human parainfluenza (types 2 and 3), respiratory syncytial
virus (RSV), streptococcus pneumoniae, and mycobacterium tuberculosis. Research is
also underway on nasal spray vaccines against gonorrhea and chlamydia using antigens
from one specific mucosal region to initiate common mucosal immune system properties
that can induce responses in different mucosal regions.
2. Safety and Weak Immunogenicity of Nasal Spray Vaccines
Intranasal influenza vaccines are generally considered safe and effective, but there have
been examples of withdrawals due to associated adverse events, with the withdrawal of
Nasalglu produced by Berna Biotech in 2001 due to induced Bell's palsy. There is also
evidence that live attenuated influenza vaccines (LAIVs) can cause encephalitis through
the olfactory nerve into the brain (nasal-to-brain pathway). Although these are
theoretically sound studies, they should not be ignored in practice.
More important to note, however, is the immunogenicity of nasal spray vaccines. Although
the ability of the nasal spray vaccine to induce systemic and mucosal immunity would
theoretically lead to greater protective immunity, the efficacy of the nasal spray vaccine
would be limited by the short residence time in the nose and the movement of enzymes,
mucus, and nasal hairs in the nasal cavity.
Conclusion
Nasal spray vaccines offer many advantages such as ease of administration and the
potential to induce systemic and mucosal immunity, which has great potential especially in
today's epidemic of respiratory infectious diseases. However, nasal spray vaccines are a
multidisciplinary field that integrates formulation, device, and clinical research, and the
challenges of developing a new nasal spray vaccine are numerous.

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Biopharma PEG is a leading supplier of high-quality PEG derivatives. We can
provide PEG derivatives from lab to large scale in GMP and non-GMP grade. PEG
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References:
[1] Alu A, Chen L, Lei H, et al. Intranasal COVID-19 vaccines: From bench to bed.[J].
EBioMedicine, 2022,76:103841.
[2] Lobaina Mato Y. Nasal route for vaccine and drug delivery: Features and current
opportunities.[J]. International journal of pharmaceutics, 2019,572:118813.