Technical factors for the development of nasal disease vaccines

Nasal disease is a potential vaccination route, especially in the prevention of respiratory transmitted infectious diseases, nasal disease has the advantage
of less invasive and easy to store and distribute compared with injected vaccines.
Influenza nasal spray seedlings have been used stably for more than 10 years, and the first nasal spray new crown epidemic seedlings in China have also been approved for emergency use.

Structure determines nature, understanding the immune process of nasal disease first needs to understand the physiological structure of the nasal cavity, the physiological structure of the nasal cavity has been introduced in the previous "Overview of Nasal Drug Administration", this section focuses on the nasal cavity and immunization related content
.

The nasal cavity is part of the respiratory tract, long-term continuous contact with foreign substances, the nasal mucosa is also often exposed to antigens, the epidemic is also a foreign antigen, in the face of foreign antigens (epidemic seedlings), the nasal cavity will initiate the immune mechanism
.
The respiratory area of the nasal cavity is rich in lymphoid tissue, where antigens are adsorbed to the mucosal epithelium after entry, and the microfold cells (M cells) of the lymphatic follicular epithelium absorb antigen granules, move antigens to the basal end of the cell and deliver them to antigen-presenting cells (APCs), such as dendritic cells (DCs) and macrophages
.
Thereafter, DC cells migrate via afferent lymphatic vessels to nearby draining lymph nodes, where antigens are presented to naïve T and B cells
via the MHC-II complex.

Nasal associated lymphoid tissues (NALTs) are an important component of the mucosal immunity of the respiratory tract, NALTs are distributed with more M cells, is the main part of the nasal cavity to absorb macromolecular drugs and particles, but also the main site of antigen presentation in the nasal immune response, NALTs site has antigen-specific effector B and T cells, they will differentiate into plasma cells that produce aggregated IgA and IgG, and eventually differentiate into sIgA, triggering an immune response to intercept antigens and pathogens
。 Mucosal immunity also has a unique set of co-immune mechanisms, which can induce an immune response in distal mucosa, such as the mucosa of the digestive tract and genital tract, but usually the intestinal immunity activated by nasal epidemic seedlings is poor
.
See "Vaccination is like "drinking milk tea"! Inventory of the advantages and difficulties of inhaled epidemic seedlings"
.

Similar to nasal administration, the role of nasal disease seedlings needs to overcome multiple factors, 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 layer and epithelial layer of the nasal mucosa
.
To solve these problems, immune adjuvants and osmotic promoters are often required
.

At present, most of the nasal epidemic vaccines on the market are live attenuated influenza virus vaccines
.
This section presents several key factors
that should be considered when researching and developing nasal disease from the perspective of the immune process.

1.
The first step of drug administration - dosage form and delivery equipment

To complete the administration of the nasal cavity, the cooperation
of a suitable device is first required.
Although the nasal cavity has a large mucosal surface area, nasal delivery of the epidemic is limited by nasal anatomy and aerodynamics, excessive particle size is usually deposited in the anterior nose to be expelled or wiped off, and small particle size (i.
e.
, <10 μm) may bypass the nose into the lungs
.
Therefore, a suitable device is required to disperse the intranasal seedling preparation into the appropriate particle size and deliver it to the posterior area of the nose, and achieving a balance of particle size is essential
to increase the antigen exposure of the nasal mucosa.
AccuSpray is currently used for FluMist seedlings, as well as MAD Nasal from Teleflex, ViaNase from Kurve, PuffHaler from Aktiv-Dry, and Solovent from BD to provide nasal mucosal atomization solutions and devices
.

So far, all approved nasal seedlings are in liquid form because of their high solubility and potency, but liquid formulations are fluid, any liquid that exceeds the volume of the nasal cavity is expelled from the nasal cavity, and only highly soluble or low-dose antigens can be delivered
intranasally through liquid preparations.
The application of dry powder preparations requires the combination of viscous polymers such as starch and chitosan to extend residence time and improve antigen availability
in nasal lymphoid tissue.

2.
Extend the residence time of infected seedlings - adhesive

In nasal disease development, adhesives are usually used, and when the adhesive comes into contact with the mucus of the nasal epithelium, the polymer will hydrate, expand and bind to the mucus, prolonging the residence time of the infected seedlings in the nasal mucosa
.
In addition, adhesives can also temporarily slow mucociliary clearance and do not inhibit the release
of antigens from the epidemic seedlings.
Commonly used adhesive polymers include such as chitosan, starch, etc
.

Chitosan is a viscous cationic polysaccharide that disrupts tight junctions between epithelial cells, increases epithelial permeability, and contributes to the efficient absorption
of antigens.
Chitosan can also promote antigen uptake by M cells in the nasal cavity, and increase the efficacy
of the epidemic in the form of adjuvants.

Starch is a non-toxic, widely available polysaccharide consisting of
amylose and pullulan.
Once hydrated, the starch expands, forming a gel-like layer that binds
to the nasal mucosa.

3.
Promote the transmembrane penetration of epidemic seedlings - penetration accelerator

The nasal epithelial layer is covered with a layer of mucus, and nasal seedlings/antigens need to complete the penetration
of the mucus layer and epithelial cells.
The first is the mucus layer, where polyethylene glycol can act as a mucus layer penetration promoter, while mannitol dilutes the mucus
.
Therefore, the use of PEG, mannitol or other mucus penetration promoters in the formulation helps to improve the penetration
of the mucus layer by the epidemic/antigen.

The transport of antigens across the epithelial cell barrier is a critical first step
in inducing immunity.
Antigens can penetrate the epithelial layer cellularly or across cells, and osmotic promoters 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 disrupt epithelial junctions and enhance paracellular motility
.
Specifically, chitosan can promote cross-cellular uptake
of antigens by M cells in the nasal mucosa and intestinal mucosa.
The enterotoxin destroys the mucosal epithelium, enhances the uptake of antigens by DC cells, and leads to the upregulation of CXCR4 and CCR7, so that DC cells migrate to lymph nodes
.

4.
Reduce potential intracranial transport – nasal-brain transport inhibitors

An important safety issue regarding nasal disease is the transport
possibility of the disease antigen or adjuvant from the olfactory epithelium to the central nervous system (CNS).
Although the researchers analyzed 460 FluMist-related adverse events and found only one case of encephalitis, it was laboratory confirmed to be caused
by enteroviruses.
But the nasal mucosa provides a direct entry point between the external environment and the central nervous system (nasal-brain pathway), and compounds can enter the cerebrospinal fluid through the associated nerves in the
olfactory area.
Therefore, neurotropic in the development of nasal disease seedlings needs to be evaluated
in the development of nasal disease seedlings.

5.
Enhance antigen immunogenicity - epidemic adjuvant

Adjuvants that target APC antigens in the process of immunity can serve as useful immunostimulants
.
Commonly used immune adjuvants are insoluble aluminum salts and TLR agonists
.

Insoluble aluminum salts are classic FDA-approved immune adjuvants
.
Several aluminum salts are used in the currently approved seedlings in the United States, such as Alhydrogel (aluminum hydroxide), Adju-phos (aluminum phosphate) and amorphous aluminum hydroxyphosphate sulfate (AAPS).

The immunostimulatory effects of aluminum salts come from a variety of mechanisms, including slowing the spread of antigens from the site of administration, increasing inflammatory cell accumulation, activating complement, inducing the differentiation of monocytes into DCs, and antigen uptake by DCs, antigen presentation, and activation of CD4+ T cells
.

TLR agonists can act as adjuvants for mucosal immunity, specifically, TLR agonists function as pathogen-associated molecular patterns (PAMPs) by binding to pathogen recognition receptors (PRRs) on DC cells
.
Commonly used are TLR2/6 agonists, TLR3 agonists, and TLR3 agonists

1.
Nasal epidemic seedlings that have been approved for marketing

Currently, the FDA has approved only one human nasal vaccine, FluMist, produced by AstraZeneca and approved by the FDA in 2003, and is the most successful nasal disease vaccine to date, initially approved as a trivalent formulation in 2003 and a quadrivalent formulation in 2012, and has also been extended to people
aged 2 to 49 years.

Nasovac-S is a live attenuated nasal disease produced by the Serum Institute of India for the prevention
of influenza A(H1N1).
On June 8, 2018, the State Medical Products Administration approved the production registration applications of Hualan Bio Epidemic Seedling Co.
, Ltd.
and Changchun Changsheng Biotechnology Co.
, Ltd.
for the production of quadrivalent influenza virus lytic epidemic seedlings, and the quadrivalent influenza virus split disease seedlings approved for marketing contain A1, A3 and BV viruses of ordinary trivalent influenza vaccines, as well as BY influenza viruses
.

At present, the nasal disease seedlings currently undergoing clinical trials mainly include influenza epidemic (H5N1, H1N1, etc.
), human parainfluenza (type 2, type 3), respiratory syncytial virus (RSV), Streptococcus pneumoniae, Mycobacterium tuberculosis, etc
.
The use of antigens in a specific mucosal region to initiate common mucosal immune system properties that can induce reactions in different mucosal regions, and nasal vaccinations against gonorrhoea and chlamydial are also being studied
.

2.
Safety and weak immunogenicity of nasal epidemic seedlings

Intranasal influenza vaccinations are generally considered safe and effective, but there have been examples of delisting due to related adverse events, such as the 2001 delisting of Nasalglu produced by Berna Biotech due to induced Bell's palsy
.
There is also evidence that live attenuated influenza seedlings (LAIVs) enter the brain (nasal-brain pathway) through the olfactory nerve to cause encephalitis
.
Although these are theoretical studies, they cannot be ignored
in practice.

However, it is more important to note that the immunogenicity of nasal epidemic seedlings, although theoretically, the ability of nasal epidemic seedlings to induce systemic immunity and mucosal immunity will lead to greater protective immunity, but the effectiveness of nasal epidemic seedlings will be affected by the short residence time in the nasal cavity, and the movement of enzymes, mucus, nasal hair, etc.
in the nasal cavity will limit the effectiveness
of the epidemic seedlings.

Ancient pharmacies usually had the couplet 'I only wish that the people of the world would be disease-free, and would not hesitate to put medicine on the shelves', but in the face of various infectious diseases, we would not mind adding another piece of equipment
in our arsenal.
Nasal epidemic has many advantages, such as ease of administration and the potential to induce systemic and mucosal immunity, especially in today's epidemic of respiratory infectious diseases
.
However, nasal disease is a multidisciplinary field that integrates preparations, devices, and clinical research, and the development of a new nasal disease product removes the disease itself, and inhaled disease including nasal disease seedlings also requires the cooperation of drug delivery application devices
.
Not only this, but also the close cooperation of drug clinical research institutions, in the clinical trial stage, the promotion of the epidemic is also full of complex and difficult twists and turns, as well as uncertain consequences
.

The history of human existence is also the history of fighting infectious diseases, there are countless kinds of viruses in the corners of our planet that have evolved, human beings use epidemic seedlings to resist viruses and save lives in battle, we have witnessed one miracle after another, but also witnessed many gloom after listing and delisting, the development of epidemic seedlings is also in hope and disappointment to move forward, although people always say that the years are quiet, but the danger has always been there, and what we really need to do is to respect the laws of nature and science
.

References

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Mechanism and progress of nasal mucosal immune adjuvant in improving the immune effect of epidemic seedlings[J].
Journal of Chongqing Medical University, 2021,46(10):5.

GE Hua, JIANG Liyong, LIU Shu, et al.
Progress in R&D of nasal immunity and COVID-19 vaccines[J].
China Journal of New Drugs, 2022,31(9):8.

HAO Xinyan, ZHANG Yuandong, HOU Yingying, et al.
Study on chitosan-modified nanoemulsion for nasal disease seedling delivery[J].
Journal of Sichuan University:Health Sciences, 2021,52(4):6.

[4] Alu A, Chen L, Lei H, et al.
Intranasal COVID-19 vaccines: From bench to bed.
[J].
EBioMedicine, 2022,76:103841.

[5] Lobaina Mato Y.
Nasal route for vaccine and drug delivery: Features and current opportunities.
[J].
International journal of pharmaceutics, 2019,572:118813.