A Review on Chitosan-Based Nanovaccines Against Newcastle Disease in Poultry

Chitosan is a polysaccharide, made up of repeating units of 2-amino-2-deoxy-D-glucopyranose and 2-amino-2-D-glucopyranose that are linked by β 1–4 glycosidic bonds (Gong et al., 2022; Elbehary et al., 2023). It consists of free hydroxyl and amine groups, as depicted in Figure 2 (chitosan trimer) that increase its popularity by which the compound can be modified (Elbehary et al., 2023). Professor C. Rouget discovered chitosan in 1859 by alkali treatment of chitin and industrialization started in Japan in 1971 (Boroumand et al., 2021). Hoppe Seiler named the deacetylated chitin, chitosan (Boroumand et al., 2021). Chitosan is obtained through alkaline deacetylation of chitin, which can be extracted from the exoskeletons of insects, crustaceans, and fungi (Dmour and Islam, 2022). It is the second most abundant compound after cellulose in the world.

Figure 1.

Mechanism of action of chitosan NPs to produce an immune response

Figure 2.

Simple methods of modification of chitosan to get various types of chitosan derivatives

Ohya and Cowerkers, in 1994, first described the chitosan NPs (Gong et al., 2022). Most of the chitosan NPs range in size from 1 to 100 nm (Dmour and Islam, 2022). Chitosan NPs are produced by different methods which are ion gelation, reverse micellar, polyelectrolyte complex, emulsification solvent diffusion, and microemulsion (Boroumand et al., 2021). Ion gelation and polyelectrolyte complex are the most widely used methods for chitosan NPs preparation because they are simple to use organic solvents and have no need for high sheer force (Boroumand et al., 2021). The morphology of chitosan NPs can be seen by scanning and transmission electron microscope. Chitosan exhibits solid-form polymorphism and semicrystalline structure (Bakshi et al., 2020). Chitosan is a defense elicitor that possesses unique properties like biocompatibility, biodegradability, non-toxicity, bioactivity, and polycationic nature (Yang et al., 2020). Due to these physicochemical and biological properties, chitosan NPs become eco-friendly, have less or no harm, and have become very famous.

Routes of vaccine delivery include oral, intranasal, ocular, eye drop, intramuscular, subcutaneous, etc. Chitosan NPs are potential mucosal adjuvants, as demonstrated by most of the studies. They enhance mucosal immunity by the opening of tight junctions present in between the cells (Moine et al., 2021). So, mucosal routes of vaccine delivery preferred for chitosan adjuvanted vaccines are oral and intranasal demonstrated by most of the studies (Table 2). In the oral route, the delivery of the vaccine is faced with very acidic (1.2–3) and gentle basic (6.5–8) pH. This pH variation can do hydrolysis, deamidation, or oxidation (de Alvarenga, 2011). Various enzymes like pepsin, trypsin, chymotrypsin, pancreatic lipase, and pancreatic amylase can change the activity of agents. This is the reason that we use adjuvants for vaccine delivery. Chitosan is tremendously used for this purpose, because of its cationic nature which increases the attachment and opens tight junctions for entering the antigen to the cells (Li et al., 2018). From the intestinal lumen, antigens go to Peyer’s patches, dendritic cells, macrophages, and M cells. Nasal vaccine delivery is a very efficient method because first of all associated lymph tissues (M cells are more in number) are present in the nasal cavity and provide an important defense system (Kang et al., 2009). Secondly, the antigens have direct access because of the leaky nasal mucosa and the presence of blood vessels, lymphoid cells, and cervical lymph nodes (Prabhu and Patravale, 2014). Chitosan NPs as an adjuvant decrease the fast clearance time and also do not change the morphology of mucosal cells (Li et al., 2021).

When the chitosan NPs based vaccine is administered in the body through mucosal routes, the amino and carboxyl groups of chitosan NPs combined with the glycoprotein of the mucus which is negatively charged, hence strong electrostatic force develops that ensures maximum absorption of the antigen (Jhaveri et al., 2021; El-Qabbany et al., 2024). After entry into the cell through tight junctions they go to the mucosa-associated lymphoid tissues which are nasal-associated lymphoid tissues (NALT) in the nasal mucosa that are involved in prolonged mucociliary clearance (Biswas et al., 2024) and gut-associated lymphoid tissues (GALT Peyer’s patches) and microfold cells (M cells). In these lymphoid tissues, B cells, T cells, dendritic cells (DCs) and macrophages are present (Ivanov et al., 2018). Antigen interacts with B cells and changes them to IgA⁺ B cell, which converts to IgA⁺ plasma cell that secretes IgA antibodies and produces a mucosal immune response (Reboldi et al., 2016). On the other side, CD8+ T cells and CD4+ Th1 cells initiate the production of cytotoxic T lymphocytes and macrophages that engulf the infected cells and pathogens that are responsible for cellular-mediated immune response (Tiwari et al., 2020). Activated CD4+ and Th2 cells secrete B lymphocytes that produce antibodies to neutralize the pathogens present extracellularly to evoke a humoral immune response. This process develops memory cells that will act against the same pathogen if interacts in the future (Gourbal et al., 2018). The M cells do the successful antigen presentation and delivery from the mucosal surface (Singh et al., 2015). These are specialized cells present in the epithelium, more specific to the follicle-associated epithelium in mucosal tissues (Nakamura et al., 2018). M cells take and transfer antigens to the underlying lymphoid tissues as depicted in Figure 1. Then they transfer to B cells or dendritic cells to evoke immune responses (Qi et al., 2014; Omidian et al., 2024). So, M cells play a pivotal role in antigen delivery to produce mucosal immune response (Biswas et al., 2024). Mucosal immunity is essential as viruses enter through mucosal membranes, so it kills the virus at the point of entry (Renu and Renukaradhya, 2020).

Chitosan not only possesses biodegradability, biocompatibility, and non-toxic properties but also possesses antiviral, antibacterial, antimicrobial, and fungicidal activities (Alkhalefa et al., 2022). Chitosan NPs show an immune response by the production of interferon from macrophages (Omidian et al., 2024). One of the amino-modified chitosan NPs enhances antiviral activity by producing IFN-β (Issar and Arora, 2022). Chitosan NPs derivatives also show antiviral activity like amino-modified derivatives (Lima et al., 2022). Chitosan on the basis of separation from different sources is classified into 3 classes viz: α chitosan, β chitosan and γ chitosan. The antiviral effect of the β chitosan is depicted in Table 2. While α and β chitosan need studies to check their antiviral activity (Dilnawaz et al., 2024). There are fewer studies that show chitosan as an anti-viral agent against ND.

Chitosan is available in different formulations depending on the degree of deacetylation and molecular weight. The deacetylation range is between 66 and 95% (Elsoud et al., 2022). According to molecular weight, they are characterized as low molecular weight chitosan polymer (<150 kDa), high-molecular-weight chitosan polymer (700–1000 kDa), and moderate molecular weight chitosan polymer (in between low and high molecular weight) (Hahn et al., 2020). They are extensively studied in the medical field for drug and vaccine delivery due to their non-toxic nature, ease of preparation, and small size for efficient transference and immersion into the mucosal membranes (Desai et al., 2023). The adjuvant effects of chitosan are better than many other adjuvants like aluminum hydroxide (Dmour and Islam, 2022). It is a potential vehicle for the transfer of live vaccines in poultry because it has been reported that chitosan is involved in enhancing Th1-type immunity (Renu and Renukaradhya, 2020).

The pH of chitosan is about 6.5, which makes it soluble in mild acidic solutions and insoluble in water (Wang and Zhuang, 2022). This limits the use of chitosan for carrying antigens that are only soluble in neutral pH. For transferring these antigens, various chemical modifications are done in the structure of chitosan by using free amino and hydroxyl groups that improve the solubility (Wang et al., 2020 a). Normally there are three methods by which researchers improve the solubility but each has pros and cons. Like the deacetylation of chitin, chitosan is only soluble in mild acidic solution, thus limiting its use (Kou et al., 2021). While the chemical modification is done by adding hydroxyl group to the chitosan, this method has a limitation that this destroys the crystallinity and hydrogen bonding (Wang et al., 2020 b). In another method, chitosan degradation is done by breaking it into water-soluble molecules. This method leads to uneven distribution of molecular weight and causes difficult separation (Desai et al., 2023). These modifications do not change the biological properties of the chitosan and also add new functions according to the characteristics of the chitosan of the added compound (Figure 2). Some examples of modifications of chitosan are as follows: quaternization is the process of modification by adding a hydrophilic group, in this, we get derivatives like the N-2 hydroxypropyl and N, O carboxymethyl that increase water solubility (Freitas et al., 2020). Substitution of carboxymethyl in chitosan NPs is associated with the enhancement of immunostimulants, like N, O carboxymethyl hydroxypropyl trimethyl ammonium chloride (HTAC) chitosan and N-carboxymethyl HTAC chitosan enhance the effect of IL-6, IL-1, and tumor necrosis factors in dendritic cells (Wang et al., 2020 a). N, O carboxymethyl HTAC chitosan NPs also increase the release of MHC-II, CD11c, CD80, and CD86 which elicits excessive antigen presentation (Xu et al., 2021). N, O carboxymethyl and HTAC chitosan had potential immune effects according to Xu et al. (2021). So, it has a great impact to be used as a novel vaccine vehicle. N-2 hydroxypropyl dimethyl ammonium chloride has high water solubility as compared to chitosan (Zhao et al., 2020). When used as an adjuvant with NDV, it proved less cytotoxic, more stable, and enhanced levels of IL-2, IFN-γ, and lymphocyte proliferation (Yakubogullari et al., 2021). N-trimethyl chitosan chloride has high efficacy and solubility. That is why it is used in the transmission of peptide drugs (Zhao et al., 2020). Carboxymethyl chitosan NPs, due to their chemical properties and feasible synthesis, have been widely reported to be used in drug and vaccine delivery in various forms like nanofibers, nanofilms, hydrogels, and nanoparticulate systems. Hence, modification to the chitosan provides good solubility, targeted delivery, and mucosal adhesion (Mura et al., 2022). Some of the chitosan-derivatives-based NDV vaccines are formulated in Table 2. In this table, different studies highlight that if chitosan-based vaccines are administered, they showed better results than other routes of vaccine delivery. So, researchers in the future should focus on intranasal transmission of the vaccine adjuvants to get higher immunity levels. Moreover, among chitosan and chitosan derivatives-based vaccines, the latter proved more efficacious and specific in producing complete protection against ND. There is a need for further studies on these derivatives-based NDV vaccines so that they may be available commercially.

Chitosan nanoparticles possess antiviral properties but there is limited data available, unable to support this property of chitosan. The toxicity of chitosan has been studied in mice which is LD₅₀: approximately 16 g/kg (Wadhwa et al., 2009). Chitosan, due to mild acidic pH is unable to be used for pH-sensitive antigens or viruses. It works easily in acidic environments, like the stomach and small intestine but is unable to work properly in alkaline environments. This limitation has been the main hurdle in the commercial use of chitosan. Moreover, size, shape, and structure also limit its proper working. Particle size is the main concern that is involved in vaccinal delivery. There are several factors which are involved in determining the particle size like concentration of chitosan, molecular weight, etc. These parameters can be corrected by further and advanced studies.

Broiler farming is an important sector for producing high quality proteins at low costs (Hayajneh et al., 2024; Susalam et al., 2024). Diseases of various kinds including bacterial and viral origin are a persistent challenge for economical farming (Afzal et al., 2024). NDV causes marked economic losses in the whole world despite massive vaccination plans. There is a need for commercially available NPs based vaccines like chitosan because of the limitations of currently available vaccines. As chitosan is abundantly present, cheap and extensively experimented in medical fields, it may be commercialized in the future because we hope that we will get more prospective remarks by using NPs as nano adjuvants. Chitosan NPs vaccinology against ND will help to mitigate it potentially. Chitosan may be the potential adjuvant to produce poultry vaccines. The use of these chitosan-based NDV-loaded vaccines may potentially reduce the outbreaks of this disease. Researchers should perform in vivo studies to check the antiviral effect of chitosan NPs as other antiviral agents cause resistance. β chitosan may be used soon as a potential antiviral agent in the veterinary field.

In conclusion, we expressed that chitosan is synthesized by the ion gelation method because it is the most appropriate method depicted by recent studies. However, chitosan derivatives are wonderful NPs because of a wide range of biological properties and some additional functions that make them better than chitosan. Chitosan and chitosan derivatives-based vaccines produce higher cytokines, interleukins and interferon, showing that they are better than the commercially available live/attenuated/oil immersion vaccines. All the experimental studies done on live animals that are natural host of the disease demonstrate that chitosan NPs based vaccines produce mucosal, humoral and cell mediated immunity, indicating that these adjuvants are wonderful in producing immunity of the vaccines against the ND.