Advances of Nanotechnology Toward Vaccine Development Against Animal Infectious Diseases

Vaccines are the most effective means of controlling infectious diseases. Increasingly epidemics have driven the development of vaccines for human use. However, insufficient attention has been paid to preventing animal epidemics. The spread of animal epidemics directly affects food and nutritional safety, the economy, and outbreaks of zoonotic diseases, which can eventually threaten human health. Reducing safety risks, saving production costs, adapting to mass vaccination, and improving immunogenicity are important that must be considered in animal vaccines. Nanotechnology in vaccine development provides unique advantages in meeting these challenges, both at in vitro preparation and in vivo immune response levels. In terms of in vitro vaccine formulation, nanoparticles offer the possibility to provide multiple antigen display sites, maintain antigen conformation, and improve vaccine stability, which facilitates the development of vaccines with enhanced immunogenicity and thermal stability. In terms of in vivo vaccine‐induced immunity, nanoparticles show superior immunostimulatory activity which facilitates multiple immune response processes, including antigen delivery, cellular uptake, antigen presentation, and lymphocyte activation, thus markedly augmenting vaccine‐mediated immune responses. Herein, the application of nanotechnology to vaccine development and the characteristics of animal vaccines are reviewed, aiming to provide general guidelines for the design of future vaccines against animal infectious diseases and promote harmonious coexistence of humans and animals.


Introduction
Vaccination is one of the most effective ways to combat the spread of epidemics both in humans and animals.The increasing DOI: 10.1002/adfm.202305061frequency of infectious disease epidemics has drawn attention to the development of vaccines for human use.However, the need to prevent infectious diseases in animals cannot be ignored.Animal epidemics are closely related to human livelihood (Figure 1).The spread of infectious diseases in animals causes major economic losses for the cultivation industry. [1]The outbreak of highly pathogenic avian influenza from 2014 to 2015 affected ≈50 million poultry, leading to a loss of nearly US$ 3.3 billion to the United States economy. [2]Since August 2018, ≈100 outbreaks of African swine fever have been reported in China, resulting in 916 000 pigs being culled and pork production reduced, [3] disrupting trade in the domestic pork market.Moreover, some animal infections are zoonoses and can be transmitted to humans.Zoonoses account for >60% of human infectious diseases [4] and pose a considerable threat to global health.Studies have shown that vaccination of poultry has prevented human infection with avian influenza virus. [5]Additionally, with the growing public awareness of animal health protection, the demand for vaccination of pets and wildlife is also increasing.Moreover, there are no available vaccines against certain animal infections, such as African swine fever, because candidate vaccines have not yet been approved for marketing. [6]Therefore, veterinary vaccines have a broad market impact.The global veterinary vaccine market was reported to be worth ≈US$ 7.4 billion in 2018 and is expected to grow continuously in the coming years.
Reducing safety risks, saving production costs, adapting to mass vaccination, and improving immunogenicity are fundamental problems faced in animal vaccine development (Figure 1).However, commercially available traditional vaccines and adjuvants generally have limitations that prevent them from meeting these requirements, such as cold-chain dependence, [7] adjuvantinduced adverse effects, [8] and weak immunogenicity. [9]In order to circumvent these limitations, nanotechnology as an emerging technology has revolutionized vaccine development in recent decades. [10]Compared with conventional vaccines, nanoparticles (NPs) offer more benefits owing to their unique physicochemical characteristics, [11] which provide new prospects for animal vaccine development.Various NPs have been developed for nanovaccine preparation including virus-like particles (VLPs), [12] Figure 1.Schematic diagram of the relationship between animal epidemics, animal vaccines, and human health and wellness.Animal epidemics affect human livelihood, mainly through economic losses, zoonotic transmission, companion pet health, and wildlife protection.Therefore, the prevention and control of the spread of animal epidemics cannot be ignored.As one of the most cost-effective ways to combat animal epidemics, an ideal animal vaccine should possess the features of good biosafety, low cost, suitability for mass vaccination, and good immunogenicity.
nanoemulsions, [13] carbon nanomaterials, [14] polymeric NPs, [15] and liposomes. [16]The two main contributions of NPs animal vaccine development are: 1) enhancing immunogenicity and stability of antigens at the in vitro formulation level, which contribute to reducing antigen dosage and breaking dependence on the cold chain; [17] and 2) facilitating multiple immune response processes at the in vivo immunity level, including antigen delivery, [18] cellular uptake, [19] antigen presentation, [20] and lymphocyte activation, [21] which exhibit superior adjuvant activity (Figure 2).In this review, we discuss the advantages of nanotechnology over traditional vaccines and adjuvants, summarize the contributions of nanotechnology to vaccine development from both an in vitro preparation and in vivo effectiveness perspective, and discuss the safety risks associated with the application of nanotechnology to animal vaccines, in order to provide general guidelines for future veterinary vaccine design and promote the harmonious coexistence of humans and animals.

Use of Nanotechnology to Overcome the Limitations of Traditional Vaccines and Adjuvants
Currently, inactivated and subunit vaccines are the most commonly used vaccines in animals. [22]However, inactivated vaccines have poor immunogenicity and require repeated vaccinations, whereas live-attenuated vaccines pose a risk of pathogenic reversion. [23]Although subunit vaccines have been developed to improve vaccine safety and tolerability, isolated and purified antigens are less immunogenic than the whole pathogen. [24]In recent years, nucleic acid vaccines have been widely studied as a new generation of vaccines owing to their advantages such as: safety, reproducible large-scale production, and the ability to induce humoral and cellular immune responses. [25]Nevertheless, the inefficient delivery and low transfection efficiency of naked nucleic acid pose a challenge to the development of nucleic acid vaccines. [26]The application of nanotechnology in vaccine development offers an opportunity to overcome these limitations.For example, nanoparticle-based vaccine-delivery sys-tems have been developed to protect protein or nucleic acid antigens from enzymatic degradation and help them cross various biological barriers. [24,27]The use of thermostable NPs enhances the in vitro stability of vaccines, breaking dependence on the cold chain, and prolonging the storage period. [28]Multiple antigens displayed on the surface of NPs enhance the immunogenicity of protein vaccines. [29]Therefore, nanotechnology overcomes the limitations of traditional vaccines from various perspectives, and the mechanisms behind them are discussed in later sections.
Adjuvants are widely used in veterinary vaccines to enhance the antigen-specific immune response and improve the body's defense against infection.Currently, alum-based adjuvants and oil-based emulsions such as Freund's adjuvant and incomplete Seppic adjuvants are commonly used in animal vaccine formulations to enhance vaccine potency. [30]Alum-based adjuvants, the first approved adjuvants used in vaccines, have been the only adjuvants available for more than seven decades. [31]However, aluminum adjuvants fail to induce cellular immunity and usually cause inflammation at the injection site. [9]Oil-based emulsions can prolong and enhance the immune response by slowly releasing antigens through a depot effect. [30]However, adverse reactions such as fever and granuloma formation can be induced by the poorly metabolized oil phase and the high concentration of surfactants added to the emulsions. [8]Faced with the limitations of traditional adjuvants, nanoparticle-based adjuvants have been developed to overcome these shortcomings and achieve satisfactory efficacy.For example, compared with commercial aluminum gel and Freund's adjuvant, rod-shaped aluminum NPs significantly enhance rod-like antigen delivery to immune cells and reduce local inflammation. [32]10d] Compared with Freund's adjuvant, the use of N-trimethyl chitosan NPs as an adjuvant in oral vaccines elicits a stronger Th17 response, thereby enhancing mucosal immune responses against brucellosis. [33]oreover, NPs can be used as delivery carriers for molecular adjuvants, such as toll-like receptor (TLR) agonists and cytokines, to enhance adjuvant delivery efficiency and activity. [34]Collectively, compared with traditional adjuvants, nanoparticle-based adjuvants provide more benefits and have broad application potential in animal vaccine development.
In conclusion, nanotechnology provides new prospects for developing animal vaccines to overcome the limitations of traditional vaccines and adjuvants.Nanotechnology facilitates the development of vaccines through a variety of mechanisms.In Sections 3 and 4, we summarize the contribution of nanotechnology to vaccine formulations at the in vitro preparation level and in vivo immune response level, respectively.

Contribution of Nanotechnology to Vaccine Formulations in vitro
Rational design and preparation of vaccine formulations are prerequisites for exerting immune effects in the body.At the level of in vitro formulations, an ideal vaccine requires sufficient immunogenicity to activate an immune response and stability to avoid degradation.Nanoparticulate vaccine formulations show unique advantages in these features, enabling a reduced antigen dose, extended storage period, and eliminating the cold chain, greatly reducing the cost of preparation, storage, and transportation, [17c,35] and thus contributing to the development of vaccines against animal infectious disease.This section summarizes the contribution of nanotechnology to in vitro vaccine formulations from a perspective of immunogenicity and stability.

Nanovaccine Formulations for Enhancing Immunogenicity
The accuracy and fidelity of antigen conformation in vaccine formulations are directly related to vaccine immunogenicity.Faced with the challenge of the weak immunogenicity of free antigens, NPs provide a scaffold for the repetitive display of antigens.Based on chemical binding or protein engineering, NPs allow multiple copies of antigens to be distributed on the surface in a highly ordered manner, which facilitates the maintenance of antigen conformation and provides superior immunogenicity. [36]he insertion of cobalt porphyrin-phospholipid (CoPoP) into the lipid bilayer enables polyhistidine-tagged recombinant antigens to be stably presented on the liposome surface through coordination. [37]This design allows the antigen to display in a uniformly oriented manner and maintains its conformational integrity.CoPoP liposomes enhanced the immunogenicity of malaria antigen and SARS-CoV-2 receptor-binding domain (RBD) antigen, inducing stronger antibody responses compared with other "mix-and-inject" vaccine formulations. [38]38b] Compared with chemically synthesized NPs, genetically engineered protein NPs have a more homogeneous structure and provide more abundant antigen-specific binding sites.Ferritin NPs composed of 24 subunits with a hollow core have been widely used as scaffolds for antigen display.The three-fold symmetry axis of ferritin allows the antigen to be displayed as a trimer, which closely resembles the status of the polymerized antigen on the virus surface and shows stronger immunogenicity than the monomeric antigen. [39]Several antigen epitopes, such as the influenza viral haemagglutinin (HA) spike, [39,40] enterovirus 71 epitopes, [41] foot-and-mouth disease virus (FMDV) VP1 protein, [42] and SARS-CoV-2 spike, [43] have been genetically fused to the N-or C-terminus of ferritin subunits and assembled into ferritin NPs with eight copies of a trimeric antigen presentation on the surface for nanovaccine design.
In addition to natural protein NPs, synthetic protein nanoassemblies have been computationally designed to facilitate modular multimerization of complex antigens, thereby improving vaccine valence and immunogenicity.34b] Biolayer interferometry-based binding experiments showed that S protein-I53-50 NPs have a strong binding affinity for S protein-specific monoclonal neutralizing antibodies (nAbs) isolated from individuals who have recovered from COVID-19, suggesting that the S protein epitopes were fully exposed.Similarly, SARS-CoV-2 RBD antigen was designed to be conjugated to proteinaceous NPs including ferritin NPs, mi3 NPs, and I53-50 NPs (Figure 3a). [45]Notably, the SpyTag-SpyCatcher coupling system has been applied to the design of polymerized antigen presentation on NPs, to prevent the inefficient folding of antigens with complex structures caused by genetic fusion.Compared with the soluble RBD-SpyTag monomer, all three polymerized RBD NPs are more readily recognized by RBD-specific nAbs, which significantly enhance their antigenicity (Figure 3b).Among the three RBD-conjugated NPs, RBD-mi3 NPs show the strongest binding ability to the RBD-specific receptor with a binding affinity constant (K D ) value <1.0 × 10 −12 .A modular nanovaccine Nano-B5 platform, based on the combination of in vivo selfassembly of a bacterial toxin pentamer and unnatural trimer peptides, has been constructed for efficient loading of polypeptide or polysaccharide antigens via fusion expression or proteinglycan coupling technologies. [46]Moreover, polysaccharide antigens can be synthesized autonomously by bacteria without additional artificial intervention, which ensures their natural configuration and contributes to their immunogenicity.In summary, self-assembling protein NPs offer a platform for antigen multimerization and multiple presentations, which facilitates the development of vaccines against weakly immunogenic antigens.Furthermore, multiple presentations of antigens on NPs can facilitate B-cell activation and humoral immune responses, which are discussed in Section 4.

Nanovaccine Formulations for Maintaining Stability
Maintaining vaccine stability against degradation in vitro is another challenge for vaccine formulation.Up to half of vaccine products are discarded annually owing to their poor thermal stability. [47]High temperatures destroy the special configuration of proteins and nucleic acid antigens, resulting in antigen denaturation and reducing their immunogenicity.In addition, high temperatures can trigger the demulsification of emulsion-based vaccines, causing a loss of immunogenicity.To this end, most current vaccine products require maintenance of the cold chain during storage and transportation, which is costly and inconvenient.Therefore, developing thermostable vaccines to extend the storage period and eliminate cold-chain requirements results in considerable cost savings.Nanotechnology provides new strategies for improving the thermostability of vaccines, including the preparation of lyophilized NPs and mineralization of the viral surface. [35,48]yophilization is the most common method used to improve the stability of biologics against degradation by converting their liquid form to a more stable solid form. [49]However, the lyophilization cycle generates several stressors, including ice crystal formation, increased solute concentration, and breakage of hydrogen bonds, which lead to antigen unfolding and poor reconstitution, resulting in decreased immunogenicity. [50]n addition, some adjuvants in vaccine formulations, such as aluminum and oil adjuvants, are sensitive to the freezing cycle, which can cause undesirable precipitation and phase separation. [51]Therefore, the development of lyophilized vaccine formulations still falls short of expectations.In the development of lyophilized vaccine formulations, a variety of NPs have shown superior resilience to lyophilization.The 60-mer mi3 antigen display platform via the SpyCatcher/SpyTag system shows high stability against heating, freezing, and lyophilization. [44a, 48,52] The nanoplatform is based on an aldolase from the thermophilic bacteria T. maritima, which endow high thermal resilience to the NPs.Studies have shown minimal changes in the solubility of SpyCatcher-mi3 NPs after lyophilization.Moreover, RBD-conjugated Spy-mi3 NPs showed good immunoreactivity to monoclonal antibodies (mAbs) after reconstitution following lyophilization, similar to that before lyophilization (Figure 3ce). [48]Synthetic nanocarriers can act as containers for antigens, encapsulating them and protecting them from degradation.The E. coli antigen is harbored between two polymeric layers of bilayer nanocapsules (NCs). [53]The chitosan/dextran sulfate bilayer structure improved the stability of the vaccine formulation, which was able to maintain the particle size in freeze-dried form for 12 weeks, whereas the size of the monolayer chitosan NCs nearly doubled after the freeze-drying process.mRNA-coated lipid nanoparticle (LNP) vaccines were lyophilized using sucrose and maltose cryoprotectants in a buffer. [54]54b] However, in other studies, the mRNA delivery efficacy of the reconstructed LNP formulation in vivo was lower than expected, despite the use of stabilizers to control the size. [55]A thermostable nanostructured lipid carrier (NLC) platform was  [45] Copyright 2021, The Authors, published by American Chemical Society.c-e) RBD-conjugated Spy-mi3 NPs showed high stability against lyophilization.c) Cryo-electron micrograph of RBD-SpyVLP (Scale bar 200 Å).d,e) The solubility and antigenicity to monoclonal antibodies (mAbs) of the Spy-mi3 NPs were not significantly changed before and after lyophilization.Reproduced with permission. [48]Copyright 2021, Springer Nature.f-g) His-aZn-mIM-coated NPs designed for improving the thermostability of viral vaccines.f) Schematic diagram of the nanoparticle design.g) The His-aZn-mIM-coated Ad5 NPs retained 90.8% infective potency after storage at 25 °C for 90 days, showing longer-lasting thermostability than the CaP-coated Ad5 NPs and the native Ad5 virus particles.Reproduced with permission. [28]Copyright 2022, American Chemical Society.
developed to prepare lyophilizable RNA vaccine formulations. [56]ompared with the lipid bilayer structure of LNP, NLC system contains an oil core composed of a mixture of solid and liquid lipids, which shows higher colloidal stability without considering bilayer rupture or drug leakage during the lyophilization cycle.NLC complexed with an RNA vaccine was demonstrated to be readily lyophilized and maintain stability at room temperature for 8 months.
Mineralization of viral NPs is another strategy used in nanotechnology to improve the thermostability of vaccines.The application of mineralization to viral NPs includes packaging them with calcium, silicon, and metal-organic frameworks (MOFs).Calcium-mineralized FMDV VLPs were shown to retain their immunogenicity after storage at 37 °C for 11 days in storage. [35]Notably, the mineralized peptide W6 was inserted into the VLPs, which bore abundant Asp and Glu residues and contributed to the generation of calcium phosphate (CaP) mineral shells on the VLPs.Wang et al. [57] reported a bioinspired biomimetic silicification strategy to enhance VLPs thermal tolerance by introducing a hydrated silica exterior onto the virus surface.The silica nanoclusters acted as physiochemical nano-anchors to prohibit the disassociation of the viral capsid and thus conferred inner virus heat-desiccation resistance.The silicified polio vaccine retained 90% potency in mice after 35 days of storage at room temperature.In addition to calcification and silication, MOFs have also been used as an alternative mineralization approach to improve vaccine thermostability.Specifically, zeolitic imidazolate framework-8 (ZIF-8)-based MOFs have been biomimetically mineralized onto the surface of the model antigens ovalbumin (OVA), [17b] tobacco mosaic virus, [58] adenovirus [28] and FDMV VLPs, [59] demonstrating enhanced thermal and chemical stability at higher temperatures.The ZIF-8 metal-organic coordination networks provide sufficient coordinated linkages between the virus and the nanocoating via metal-protein interactions, which favor maintaining viral protein rigidity and stabilizing the antigen conformation. [28]17b] Adenovirus type 5 (Ad5), a common vaccine vector, has been encapsulated in a histidine (His)-modified Zn-mIM coordination complex (His-aZn-mIM) to maintain its long-term conformational stability. [28]Notably, the His layer formed an ultrastable hydration shell on the surface of the NPs, which stabilized the conformational stability of the inner virus particles by prohibiting the hydration ion from penetrating the shell in biological buffers.The His-aZn-mIM-coated Ad5 NPs retained 90.8% infective potency after storage at 25 °C for 90 days, showing longer-lasting thermostability than the CaPcoated Ad5 NPs and the native Ad5 particles (Figure 3f,g).Overall, nanotechnology contributes to the maintenance of in vitro stability and immunogenicity and offers the possibility of breaking dependence on the cold chain and prolonging the storage period.

Contribution of Nanotechnology to Vaccine Immunity In Vivo
Vaccine formulations must enter the body and trigger an immune response to exert protection against infection.From the administration of vaccine via different routes, to the eventual induction of the immune response, vaccines need to go through several steps, including antigen delivery to antigen-presenting cells (APCs), antigen capture by APCs, and delivery to lymph nodes (LNs), APC activation and antigen presentation, and T-and B-cell activation, eventually inducing cellular and humoral immunity.During this process, NPs-based vaccine formulations induce superior immune responses.As carriers of antigens or antigens/adjuvants, NPs can enhance vaccine stability against enzymatic degradation, [60] control and sustain the release of antigens at the target site, [27] enhance delivery efficiency, and reduce the side effects of available adjuvants. [61]In addition, NPs can inherently facilitate activation of APCs, and act as adjuvants to enhance the immune response. [62]This section discusses the contribution of nanotechnology to vaccine-induced immunity in animals, focusing on how the NPs can affect key stages of the immune response process (Figure 4).

Antigen Delivery from Vaccination Site to APCs Across Biological Barriers
Various routes have been studied for delivery of vaccines to the body.Once the vaccine enters the body from various vaccination sites, it first needs to be delivered to APCs to initiate an immune response.Intramuscular or subcutaneous injections are the most common routes of vaccination as they allow antigens to interact with tissue-resident immune cells, such as macrophages and dendritic cells (DCs), without crossing biological barriers. [63]However, these injection methods have several limitations, particularly in veterinary vaccination.Mass vaccination requires a considerable workforce and resources, which is costly and impractical for the large-scale livestock industry.In addition, injection can induce local inflammation caused by contaminated needles [64] or the adjuvants. [65]Moreover, intramuscular or subcutaneous injections generally fail to induce cross-protection and provide little protection against variants of viruses with multiple subtypes. [66]To address these problems, a variety of needle-free administration routes, such as oral, intranasal, and immersion vaccinations, have received much attention.However, these needle-free pathways require vaccines to deliver antigens to mucosa-associated lymphoid tissue (MALT) for APC recognition across diverse biological barriers such as gastrointestinal, nasal mucosal, and skin barriers.Nanotechnologybased antigen delivery strategies can help antigens cross these biological barriers to achieving needle-free administration, which is more convenient and economical for animal vaccination (Figure 5a).

Oral Vaccine Delivery
Oral vaccination can be achieved by mixing vaccines into animal feed, which is a safe, convenient, and low-cost method for animal vaccine administration that is generally applicable to mass immunization of animals.The body's largest immune structure, the gut-associated lymphoid tissue, plays a critical role in oral immunity. [67]Antigens in the enteric cavity can be directly captured by APCs, absorbed by enterocytes, or taken up by microfold cells (M cells) in Peyer's patches (PPs), all of which can elicit an immune response. [67,68]Nevertheless, most free antigens are not resistant to the harsh environment of the gastrointestinal tract and are degraded prior to cellular internalization.Polymeric nanomaterials such as chitosan-based NPs, [33,69] poly(lactic-coglycolic acid) (PLGA) NPs, [70] polyanhydride NPs, [71] polystyrene NPs, [72] and polymer nanovesicles [73] have been used as carriers to load antigens for oral vaccine delivery.For example, Ntrimethyl chitosan (TMC) NPs are considered as a promising delivery system for oral immunization because of their stable positive charges, regardless of pH, which enhances penetration and residence time in the intestine. [33,74]In a study by Abkar et al., [33] TMC NPs were prepared for loading B. melitensis Omp31 antigen by ionic gelation.Oral immunization with TMC/Omp31 NPs prolonged the antigen release time and induced an increased mucosal immune response with higher immunoglobulin A (IgA) levels than Omp31 vaccines supplemented with incomplete Freund's adjuvant.
Owing to their unique gastrointestinal environment, pHresponsive intelligent NPs have also been used for oral vaccine delivery.It should be noted that the design of a responsive vaccine should match the evolutionary level of the immune system of the targeted species.For example, the large intestine is the optimal antigen delivery site in lower organisms, such as tilapia, which have a relatively simple gut immune system without Peyer's patches.Based on this, a pH-trypsin-sensitive bilayer poly[(methyl methacrylate)-co-(methyl acrylate)-co-(methacrylic acid)] (PMMMA)-PLGA nanoshell has been used to protect the antigen from degradation in the enzyme-abundant foregut and to control the release of inner PLGA/antigen NPs into the large intestine.The mechanism is based on the pH-increased phase transition of the nanoshell, thereby realizing the targeted absorption of the antigen in tilapia (Figure 5b,c). [27]In addition, Eudragit L100 has also been used as an enteric coating material to achieve a similar effect. [75]

Intranasal Vaccine Delivery
Compared with the oral route, antigens are less likely to be degraded during the intranasal delivery of vaccines because the enzymatic activity in the nasal cavity is lower than that in the gastrointestinal tract, [76] thus enhancing the bioavailability of degradable vaccines.In addition, the nasal mucosa provides a large, highly vascularized surface for antigen deposition.The nasal epithelium also contains a large number of M cells overlaying nasal-associated lymphoid tissue, which can facilitate antigen uptake and lymphocyte recruitment, followed by the triggering of strong local and systemic immune responses. [76,77]owever, nasal mucociliary clearance, which affects the dwell time of antigens on the nasal mucosa, is a significant limitation in the nasal administration of vaccines resulting in a limited dose being captured by APCs beneath the nasal epithelium. [78]Therefore, to prolong the residence time of antigens in the nasal cavity and enhance delivery efficiency, high-loading vaccine delivery systems based on mucoadhesive nanomaterials have attracted increasing attention in recent years.A positively charged derivative of chitosan has been constructed as NPs loaded with Newcastle disease virus to protect poultry from respiratory disease. [79]The positive charge endows the NPs with superior bio-adhesion and mucosal permeability, which enhance the delivery of the antigen through mucosal barriers and facilitate its interaction with lymphocytes.Intranasal polyanhydride NP-based vaccines induced cross-reactive mucosal immune responses with a 16-fold reduction in nasal virus shedding compared with soluble viral antigen alone and provide better protection against zoonotic influenza A viruses (IAVs) in pigs. [80]In addition to polymeric NPs, hollow, inorganic Ag@SiO2 NPs have been applied in intranasal vaccine ) and primary conditions (pH 7.4) with the presence of trypsin.c) In vivo fluorescence imaging of tilapia 36 h after oral immunization, demonstrating predominant accumulation of PMMMA-PLGA nanoparticles in the large intestine.Reproduced with permission. [27]Copyright 2016, Elsevier.d,e) Pulmonary surfactant (PS)-biomimetic liposome for pulmonary delivery of a small molecule adjuvant.d) Quantification of the cellular uptake of DiD-labeled nanoparticles with or without PS in vitro and e) in vivo fluorescence imaging of the distribution of nanoparticles in the lung after receiving DiD-labeled nanoparticles.Negatively charged nano4 localized within alveolar macrophages after intranasal administration, whereas positively charged nano5 showed diffuse staining along the alveolar surface due to electrostatic interaction with the negatively charged PS layer.34a] Copyright 2020, The American Association for the Advancement of Science.f) Quantitative analysis of chitosan-based mucoadhesive nanovaccines (CS) in fish gill tissues following immersion vaccination, demonstrating more efficient attachment of CS nanovaccines and CS-coated nanoemulsion vaccines (CS-NE) to the fish gill mucosal surface than that of killed whole-cell vaccines (WC) and nanoemulsion vaccines (NE).89c] Copyright 2019, Elsevier.
delivery, with improved bio-adhesivity achieved by modification with 3-aminopropyltrimethoxysilane (APTMS) to enhance the positive surface charge. [81]oreover, through intranasal administration, NPs of an appropriate size loaded with antigens can be delivered to the respiratory tract to be taken up by different resident cells, [82] including respiratory epithelial cells, alveolar macrophages (AMs), and other phagocytic cells, [83] to elicit an immune response.Therefore, for prophylaxis against infectious diseases caused by bacteria and viruses such as influenza viruses, whose main route of entry is the nasal mucosa or the respiratory tract, intranasal nanovaccines are capable of simulating the process of natural infection by the microorganism to induce a potent immune response and robust immune memory. [84]However, a pulmonary surfactant (PS) layer is distributed on the surface of alveoli, hindering antigen and adjuvant delivery.To overcome this obstacle, a PS-biomimetic anionic liposome was developed to promote pulmonary delivery of small-molecule adjuvants into alveolar epithelial cells.34a]

Immersion Vaccine Delivery
Immersion vaccinations are primarily used to prevent epidemics in the aquaculture industry.This method can be easily realized by immersing the fish in water containing the vaccine.This strategy is efficient and convenient, requires little labor, and is particularly suitable for mass vaccination of young fish that are difficult to treat by injection.In addition, fish epidermal mucus, secreted by epidermal mucus cells on the body surface, contains numerous immune components that play a crucial role in mucosal immunity. [85]Thus, immersion vaccination mimics the natural exposure of fish to pathogens and simultaneously targets the gills, skin, gut, and nasal mucosa to elicit extensive mucosal immune responses.Given these advantages, immersion vaccination has become an area of significant interest in fish immunization.
However, the skin and mucosal barriers restrict antigen uptake through the skin and gills, resulting in low efficiency of immersion inoculation. [86]Nanomaterials with enhanced penetrability and mucoadhesive properties have been developed as vehicles to overcome these obstacles.In order to permeate the skin barrier, single-walled carbon nanotubes (SWCNTs) have been widely used in DNA and recombinant subunit vaccine-delivery systems for bath immunization to permeate the skin barrier. [87]87c] These properties allow SWCNT-based immersion vaccines to induce a more robust immune response against viral pathogens in fish.Polylactide (PLA) NPs have also been shown to effectively cross fish mucosal barriers and serve as potential nanocarriers for immersion vaccines. [88]ecause fish skin mucus contains numerous negatively charged mucins, [85a] chitosan NPs have also been used in immersion vaccines as cationic delivery carriers to enhance the adhesion of immersion vaccines to the surfaces of these organs by electrostatic interaction. [89]89c] Additionally, liposomes loaded with immunostimulants reduce the susceptibility of zebrafish larvae to bacterial diseases, indicating that liposomes could potentially be developed as vehicles for immersion vaccines. [90]n addition, some aqueous nano-adjuvants, such as Montanide IMS 1312 VG, consisting of water-dispersed liquid NPs, have been added to vaccine formulations to improve antigen uptake via the fish surface and enhance the potency of immersion vaccination. [91]

Antigen Capture and Delivery to Lymph Nodes
APCs, mainly dendritic cells (DCs), macrophages, and B cells, play an intermediary role between innate and adaptive immune responses.After entering the body via different routes of administration, antigens need to be captured, digested, and presented by APCs to induce acquired immune responses.In addition to a few tissue-resident APCs at the vaccination site, many APCs are present in LNs, which are important sites for subsequent immune responses.Therefore, enhancing the recognition and uptake of antigens by APCs is an approach that can improve vaccine efficacy.NPs present significant advantages in this respect because of their unique physicochemical properties, and the capture efficiency of APCs can be enhanced by tuning the size, shape, and surface hydrophobicity of the NPs (Figure 6a).
The size of NPs has a crucial impact on the transportation of antigens throughout the organism and the rate of cellular uptake. [92]NPs with an average diameter of 10-100 nm can naturally pass through lymphatic vessels to the LNs to become accessible to LN-resident APCs, which can then capture the NPs to facilitate the initiation of immune responses. [93]NPs over 100 nm in diameter generally need to be internalized by DCs for transport to LNs.In studies of the size dependence of LN delivery, 20 nmsized poly(propylene-sulfide) NPs showed more effective LN passage and APC uptake than 100 nm-sized particles. [94]Similarly, 20-nm PLGA-b-PEG NPs showed superior LN targeting, retention, and paracortex penetration in mice than 40-and 100-nm NPs, which promoted antigen capture by APCs (Figure 6b,c). [95]evertheless, different nanomaterials have different optimal particle sizes for LN targeting.Regarding antigen uptake by APCs, the mechanisms mainly comprise receptor-mediated endocytosis (e.g., mannose receptors), macropinocytosis, and phagocytosis, all of which depend on the properties of the antigen. [96]Nanoscale particles show more effective internalization and activation of APCs than microparticles, and several studies have shown that sub-500 nm NPs are optimal for DC uptake. [97]For example, aluminum oxyhydroxide NPs (≈110 nm) with adsorbed antigens are more readily taken up by macrophages than microparticles (≈9 μm), and thus show stronger adjuvant activity. [98]n addition to particle size, efficient cellular uptake depends on shape, surface charge, and hydrophobicity.Antigen-loaded nanocarriers designed to be comparable to the pathogen in shape and size, enable preferential capture of antigens by APCs.For example, a rod-shaped nano-aluminum adjuvant (Al-NR) is more effective in improving bacterial vaccine potency against P. aeruginosa infection than commercial aluminum adjuvants because the shape and size of the former adjuvant are similar to those of the rod-shaped pathogens, making P. aeruginosa-adsorbed nanoaluminum adjuvant (Al-NRs) more easily recognized and internalized by APCs (Figure 6d,e). [32]enerally, NPs with positive surface charges enhance cellular uptake by electrostatic interaction with negatively charged cell membranes. [99]Therefore, positively charged nanocarriers have been loaded with negatively charged antigens or nucleic acids to achieve effective uptake by APCs and enhance immune responses. [100]Alternatively, cationic polymers have been used as coatings on negatively charged nanocarriers to improve cellular uptake.For example, surface modification of OVA-loaded PLGA NPs with cation-charged polyethyleneimine (PEI) can significantly enhance the uptake of NPs by DCs. [101]ydrophobicity plays a vital role in immune system activation.The hydrophobic portion of immunostimulatory molecules, such as pathogen-associated molecular patterns (PAMPs) and damage-associated molecular patterns, interact with the transmembrane pattern-recognition receptors (PRRs) of DCs or macrophages to achieve effective cellular uptake of pathogens and trigger downstream signaling pathways. [102]Based on this principle, hydrophobic nanomaterials are often adopted or NPs  [95] Copyright 2019, Springer Nature.d,e) NPs similar to the targeted pathogen in shape and size are more easily recognized and internalized by APCs.d) Scanning electron microscopy (SEM) images of a P. aeruginosa-adsorbed, rod-shaped nano-aluminum adjuvant (Al-NRs).e) The internalized percentage of free antigen, antigen-adsorbed Al-NRs, and antigen-adsorbed Al(OH) 3 in J774A.1 cell.Reproduced with permission. [32]Copyright 2018, American Chemical Society.f) Gold nanoparticles (Au NPs) with different hydrophobic surfaces are generated by tuning the functionalities (blue) at the ligand termini.Log P represents the calculated hydrophobic values of the headgroup.g) Surface hydrophobicity of NPs is positively correlated with the efficiency of innate immunity responses.102c] Copyright 2012, American Chemical Society.
21b,100a,102c,103] In one experiment, a series of gold NPs with different hydrophobicity was prepared and the gold NPs were exposed to splenocytes.102c] However, cytotoxicity needs to be considered.High levels of hydrophobic and cationic groups on the surface of NPs can increase cytotoxicity. [104]To enhance vaccine potency, Jambhrunkar et al. [21b] developed invaginated mesoporous carbon hollow spheres (IMHCs) as a hydrophobic nanocarrier and adjuvant.Compared with the commercial adju-vant, Quil-A, IMHCs showed enhanced uptake by macrophages, higher adjuvant activity, and better biocompatibility.
Additionally, modifying the surface of the NPs with natural receptor ligands or specific mAbs for actively targeted vaccine delivery to APCs can enhance antigen recognition and cellular uptake efficiency (Figure 7a).The most widely studied DC-and macrophage-targeting receptors for facilitating cellular uptake are lectin-like surface receptors, which mainly comprise CD205 (DEC205), CD209 (DC-SIGN), and mannose receptors. [19,105]hese molecules can bind to the sugar residues of antigens, thereby mediating cellular uptake. [106]Based on this property, Zhang et al. [87f] constructed a targeted vaccine delivery system Reproduced with permission. [19]Copyright 2014, Elsevier.d) Confocal scanning microscopy images of adhere-effects between M-cell layer and nanoparticles after the transcytosis assay using an in vitro M-cell model, indicating that nanoparticles modified with M cells-targeted ligand Ulex europaeus agglutinin (UEA) effectively bind to the M-cell layer and are transported by M cells.118b] Copyright 2014, Elsevier.
(SWCNTs-MG) by conjugating the mannosylated antigen glycoprotein (MG) to SWCNTs for rhabdovirus prevention in fish.The addition of mannose to the nanovaccine significantly enhanced its uptake by carp macrophages and immune-related tissues, followed by the induction of robust immune responses (Figure 7b).A similar targeted nanovaccine delivery system has been shown to provide the desired APC targeting to protect against grass carp reovirus. [107]PLGA nanovaccine modified with anti-CD-205 mAbs can significantly increase NPs uptake by bovine CD205 + DCs. [108]The addition of specific antibodies can improve cellular uptake compared with that of natural ligands.In addition to lectin-like receptors, TLRs, scavenger receptors, tumor necrosis factor-alpha (TNF-) family receptors (e.g., CD40), and complement receptors (e.g., CD11c) have also been exploited as targets for vaccine delivery systems. [19,109]For example, modification of PLGA NPs with anti-CD40, DEC-205, or CD11c to target the corresponding molecules on DCs elicited superior receptor binding and internalization than non-targeted NPs (Figure 7c).Nanovaccines supplemented with a TLR4 agonist, monophosphoryl lipid A (MPLA), promotes antigen internalization by canine monocyte-derived dendritic cells. [110]Additionally, modification of NPs with specific ligands can alter the NP uptake mechanism and cellular metabolism, ultimately affecting antigen presentation.TLR agonists are thought to play a vital role in APC activation, as discussed in the following section.In a study by Liu et al., [111] cationic NPs decorated with hyaluronic acid (HA) improved cellular uptake by DCs through HA-CD44 receptor-mediated endocytosis.Furthermore, HA can reduce the cytotoxicity caused by the particles' high positive charge by shielding the surface cations of the NPs and can be degraded in the endosome by hyaluronidases to maintain the endosomal escape ability of cationic NPs.
NPs inevitably adsorb numerous biomolecules, which generate a corona on their surface once exposed to the body.The effect of the protein corona on cellular uptake, distribution, transformation, and clearance of NPs has been reported in several studies. [112]The protein corona composition has a marked influence on the recognition and cellular uptake of NPs by immune cells.Saha et al. [113] reported that the complement, apolipoprotein, and coagulation factors in the protein corona promote macrophage uptake.Different types of protein corona have diverse internalization mechanisms.112a] Modifying carbon nanotubes (CNTs) with globular heads of the complement component, C1q, facilitates uptake of CNTs by macrophages and downregulates inflammation. [115]10d] Further studies are required to finely tune the protein corona components to optimize macrophage targeting for immunomodulation.
M cells, which play an essential role in mucosal immunity, are another target for vaccine delivery.M cells are found in the follicle-associated epithelium.They sample foreign particulate antigens and transcytose them without degradation of the underlying PPs, where numerous lymphocytes reside, so that the antigens can be effectively recognized by APCs, followed by the induction of an immune response. [117]Strong transcytosis by M cells makes them an attractive target for mucosal vaccine delivery.Compared with unmodified NPs, an oral nanovaccine modified with the M-cell-targeting ligand, Ulex europaeus agglutinin 1 (UEA-1), was more effectively transported by M cells to PPs and continuously retained in the PPs, generating higher levels of serum immunoglobulin G (IgG) and intestinal immunoglobulin A (IgA) in mice and piglets (Figure 7d). [118]In the study, incorporating MPLA into UEA-modified NPs further enhanced NP capture by mucosal DCs.Renu et al. [69b,71] showed that a flagellar (F) protein surface-anchored oral Salmonella nanovaccine could specifically target chicken ileal M cells and translocate to PPs to induce antigen-specific immune responses.However, active targeting strategies are not always required.Some polymer NPs, such as PLA NPs, possess natural mucosal DC-targeting properties.After mucosal administration, these NPs naturally accumulate in mucosal DCs without modification by targeting ligands.However, the specific targeting mechanism has not yet been identified. [88]In summary, efficient capture of nanovaccines by APCs is influenced by multiple factors that need to be considered from multiple perspectives.

APC Activation and Antigen Presentation
When pathogens are captured and internalized by APCs, downstream signaling pathways are activated through the recognition of PAMPs by PRRs localized on/in the APCs, resulting in upregulation of major histocompatibility complexes (MHCs) and co-stimulatory molecules, followed by antigen presentation and cytokine secretion. [119]This process is known as APC activation.PRR agonists (PRRa), particularly TLR agonists, have been widely explored as adjuvants to accelerate APC activation during vaccine development.Apart from natural ligands, synthetic smallmolecule agonists such as the TLR7 ligand imiquimod (IMQ) have gradually become a new generation of vaccine adjuvants because of their well-defined and discrete molecular structure and greater immunogenicity than large biological molecules. [120]In order to overcome the limitations of these soluble PRR agonists, such as their short half-life, rapid diffusion after administration, and uncontrolled systemic innate responses, the agonists are often loaded into/onto nanocarriers by physical encapsulation or covalent ligation to enhance the adjuvant activity. [121]ost nanocarriers inherently elicit adjuvant effects during APC activation, serving as more than a simple vaccine delivery vehicle.They can promote APC stimulation by activating NODlike receptor protein 3 (NLRP3) inflammasomes, [62a] triggering TLR-dependent pathways [62b-62d] and complement pathways, [122] inducing reactive oxygen species (ROS) generation, [123] and regulating antigen presentation.For example, NPs with a highly hydroxylated surface can activate APCs via the complement pathway. [122,124]62c] Poly--glutamic acid (-PGA) NPs activate APCs via the TLR4 and MyD88 signaling pathways. [125]Furthermore, -PGA-based NPs loaded with the TLR9 ligand, CpG oligonucleotide (ODN), can synergistically activate macrophages by stimulating multiple TLRs. [126]Therefore, incorporating PRRa into nanoparticle platforms can further promote APC activation by the synergistic adjuvant activity of the nanocarriers and agonists.However, direct immune stimulation by these NPs can trigger undesirable inflammatory responses, which should be monitored during nanovaccine development. [127]dditionally, both the antigens and PRRa need to act on the same APC for efficient immune response activation.Based on this requirement, strategies for co-delivery of antigens and PRRbased adjuvants have been developed to ensure consistent temporal and spatial delivery of antigens and adjuvants and to achieve an improved level of APC activation (Table 1).Furthermore, combined delivery of multiple PRRa via NPs has a synergistic immunostimulatory effect and generates unique immune polarization. [128]128b] Additionally, optimal PRRa density on the NP surface drives APC activation and promotes adaptive responses.This is an important parameter to consider for surface PRRa modification of NPs. [129]ntigen processing and presentation are essential for the initiation of adaptive immune responses.Only antigens presented by APCs can be recognized by T cells, and the full activation of B cells usually depends on the activation of T cells. [130]In addition, the route of antigen presentation determines the type of immune response.Generally, exogenous antigens are degraded into peptide fragments in the endo-lysosomal compartment after uptake by APCs.Next, the antigen peptide is loaded onto MHC class II (MHC-II) molecules and exported to the cell surface for CD4 + T-cell recognition.Endogenous antigens are degraded by proteasomes in the cytoplasm and transported into the endoplasmic reticulum (ER) to be presented on MHC class I (MHC-I) molecules for CD8 + T-cell recognition.CD8 + T cells, also known as cytotoxic T cells (CTLs), induce apoptosis in infected cells by releasing various cytotoxins to exert CTL responses.In several DC subsets, such as CD8 + DCs localized in LNs, exogenous antigens can also be presented via the MHC-I pathway, termed cross-presentation, which activates both CD4 + and CD8 + T cells to trigger robust immune responses. [131]The cross-presentation of exogenous antigens is mainly achieved through the cytosolic and vacuolar pathways.131a,133] Nevertheless, the exact mechanism of cross-presentation, including the origin of the recycling of MHC-I molecules, has not yet been fully elucidated. [133]xogenous antigen presentation through the MHC-I pathway is essential for triggering the robust CTL responses necessary for vaccination against parasitic or viral infections.Compared with soluble antigens, antigens in particulate form (especially at the nanoscale) are more likely to be cross-presented by APCs. [134]Various nanoparticulate vaccine delivery systems have been developed to induce both humoral and cellular immune responses via the cross-presentation pathway (Figure 8a).NPs with a positive charge are likely to escape from lysosomes via electrostatic interaction with the lysosomal membrane or through the proton-sponge effect.PLGA NPs cross the endosomal barrier to deliver cargo into the cytosol, which has been attributed to the rapid reversal of the surface charge from anionic to cationic in the acidic environment of the endo-lysosomal compartment. [135]vidence suggests that the MHC-I processing of PLGA-OVA nanospheres occurs via the vacuolar pathway. [136]It is possible that both endosomal and vacuolar routes are involved in the cross-presentation of antigen-loaded PLGA NPs.Antigen cross-presentation has been used in PLGA-based porcine vaccines. [84,137]In one study, the frequency of activated CTLs and T-cell response were significantly augmented in PLGA-KAg vaccinated pigs compared with pigs vaccinated with mock and freekilled antigen (KAg) vaccines. [84]NPs surfaces have been coated with cationic polymers or lipids, which improve cellular uptake and promote the lysosomal escape of antigens. [101,111,138]Decorating the PLGA-OVA NP surface with positively charged PEI promotes cytosolic delivery via the proton-sponge effect. [101]Moreover, OVA-specific CD8 + T cells are activated more efficiently us-ing PEI-coated PLGA NP than with PLGA NP or soluble OVA.In a study by Liu et al., [138b] cationic lipid-PLGA hybrid NPs were developed as nanovehicles to carry OVA by different means.OVAencapsulated and OVA-adsorbed/encapsulated NPs could deliver partial antigens from endosomes or lysosomes into the cytosol, indicating that the antigens can be presented via both MHC-I and MHC-II pathways.Moreover, the study suggested that antigenloading methods can affect the potency of cationic lipid-PLGAbased nanovaccines.Similarly, exposure of the cationic lipid layer of HA-modified NPs to the endosome after internalization by APCs enhances escape to the cytosol through the proton-sponge effect. [111]he acidic and reductive environment of endosomes/lysosomes (pH ≈5.0-6.5)can be advantageous for pH-responsive and reduction-sensitive NPs that have been developed to deliver loaded antigens to the cytosol and promote the MHC-I presentation pathway for CD8 + T cell activation.Polymers with pH-dependent membrane-lytic activity, such as the terpolymer DMAEMA-co-PAA-co-BMA, are commonly used as endosomal-releasing cores in the pH-responsive NP delivery strategy for cytosolic antigen delivery. [139]Furthermore, reduction-sensitive groups, such as disulfide groups, have been introduced into NPs for reversible antigen conjugation and reduction-triggered release.Addition of environmentally sensitive promoters to vaccine formulations can facilitate lysosomal escape.In one study, ammonium bicarbonate (NH 4 HCO 3 ) was co-encapsulated with OVA in NPs with a thin PLGA shell to promote pH-sensitive antigen release in DCs. [20]When the NPs entered the acidic endosomes or lysosomes, NH 4 HCO 3 interacted with protons to generate NH 3 and CO 2 , resulting in the breakage of the PLGA shell wall and rapid release of of SIINFEKL-MHC-I + CD8 + T cells in immunized mouse splenocytes using the antigenic peptide-MHC pentamer staining method, indicating that pHresponsive NPs significantly augment the frequency of antigen-specific CD8 + T cells in mice.Reproduced with permission. [20]Copyright 2015, American Chemical Society.e-g) Cross-presentation by photoinduced endosomal escape.e) Confocal scanning microscopy images of bone marrow-derived dendritic cells (BMDCs) treated with nanoparticles (EV15/ICG/MSN) without (upper panels) and with (lower panels) laser irradiation.Endosomes were stained with Lysotracker Red.NPs were labeled with DiO (green).f-g) Percentage MHC-I-and MHC-II-positive BMDCs was determined by flow cytometry; laser irradiation promoted cytosolic release and cross-presentation of antigens.Reproduced under the terms of the CC-BY license. [140]Copyright 2020, The Authors, published by Ivyspring International Publisher.
antigens.In addition, most of the released antigens escaped to the cytoplasm, and higher levels of antigen-specific CD8 + T cells were observed in mice vaccinated with pH-responsive NPs compared with controls (Figure 8b-d).
Photoinduced endosomal escape has also been applied to cross-presentation in vaccine studies.For example, indocyanine green (ICG) has been used as a photosensitizer to encapsulate bacterial extracellular vesicle-coated NPs and modulate antigen presentation in DCs. [140]When the NPs were exposed to laser irradiation, ICG converted the absorbed photons into heat, resulting in endo-lysosomal rupture and cytosolic release (Figure 8e).Proteasome activity and MHC-I protein levels were markedly enhanced in BMDCs incubated with NPs upon laser irradiation (Figure 8f,g).This was attributed to ROS production triggered by endosome rupture.
Targeting antigens to specific DC subsets capable of crosspresenting exogenous antigens is another promising strategy for enhancing CTL responses.For example, by adjusting the properties of nanocarriers and choosing appropriate vaccination routes, nanovaccines can be passaged to draining LNs to be captured by the LN-resident, cross-presenting CD8 + DCs, thereby promoting CD8 + T-cell activation. [141]The cellular uptake mechanism also influences antigen presentation pathways.Nanovaccines internalized by clathrin-mediated pathways generally follow the cross-presentation pathway. [142]Cholera toxin (CTB) has been used as an intra-adjuvant in the design of modular fusion protein nanovaccines. [46]The self-assembling NPs expose multiple CTB pentamers on the surface, which markedly enhance MHC-I presentation via monosialotetrahexosylganglioside (GM1)-dependent cytoplasm transportation for CD8 + T-cell activation.128a,134a,143] In summary, various strategies have been developed to trigger CD8 + T-cell responses by promoting antigen presentation through the MHC-I pathway.Although understanding how to improve the delivery of exogenous antigens into the cytosol for MHC-I presentation is important for optimizing nanovaccine efficacy, maintaining the classical MHC-II pathway for antigen presentation cannot be ignored, because efficient triggering of both cellular and humoral immune responses is ultimately necessary for a robust immune response.

T-Cell Activation and Cellular Immunity
T cells are essential immunocytes, involved in both humoral and cellular immune functions.Activation of naive T cells requires two signals: an antigenic stimulus and a costimulatory factor.Therefore, efficient activation of APCs by nanomaterials must result in the upregulation of cell surface markers such as MHC and co-stimulatory molecules, which then directly activate T cells.Additionally, the cross-presentation of antigens via an NP-based vaccine delivery system further facilitates T-cell activation, which is also a prerequisite for vaccine-mediated induction of CD8 + T-cell responses.
Under the action of various cytokines, activated CD8 + and CD4 + T cells proliferate and differentiate into various effector T cells, including CTLs and helper T (Th) cells, to exert specific immune effects. [144]Th cells assist in subsequent B-cell and T-cell immune responses, whereas CTLs affect cellular immunity by releasing granzyme and perforin to induce apoptosis in infected cells.The cytokines crucial for these effector T-cell responses are interleukin 2 (IL-2), IL-12, and IFN-, which promote Th1 cell responses to support cellular immunity and memory T-cell differentiation; IL-4, IL5, IL-10, and IL-13, which enhance Th2-cell responses to support humoral immunity; and IL-23, which promotes Th17 maturation to prevent bacterial and fungal infections. [144]Thus, cytokines, which play a vital and complex role in immune responses, are being developed as vaccine adjuvants to modulate the immune response and improve vaccine potency.As with PRR ligands, the use of soluble cytokines presents several challenges, such as susceptibility to degradation and diffuse distribution after administration; thus, cytokines or cytokine expression vectors are often packaged into nanocarriers.For example, a recombinant porcine IL-2 (pIL-2) plasmid loaded into a solid LNP can enhance inactivated foot-and-mouth disease vaccine potency by promoting T-cell proliferation and improving Th1 immune responses more effectively than recombinant pIL-2. [145]A gene delivery system for the co-expression of pIL-2 and a fusion IL-4/6 gene loaded into chitosan NPs increased the expression of IL-2, IL4, IL-6, and TNF- in vivo, inducing Th1-biased responses to porcine circovirus (PCV)−2 infections in piglets. [146]he same nanocarrier, loaded with a recombinant IL-23 plasmid, upregulated transcription activators and improved the immune memory response against PCV infections, acting as an effective adjuvant for PCV-2 vaccines. [147]otably, both Th1 and Th2 responses are required to trigger a comprehensive immune response.NPs-based vaccines or adjuvants can drive mixed Th1/Th2 responses in animals, overcoming the limitations of classical vaccines and adjuvants, which can solely induce a single Th-subtype-polarized response. [148]100a] Jambhrunkar et al. [21b] showed that OVA delivered by immortalized hepatocyte-like cells (IMHCS) can induce both Th1 and Th2 immune responses.Moreover, higher titers of IgG1 than IgG2a were observed in the vaccinated mice, indicating a Th2-biased response, suitable for preventing bacterial infections.A PLGA NP-based vaccine delivery system against brucellosis has been shown to induce a mixed cytokine response consistent with the profile of IgG isotype in mice, indicating the absence of an entirely Th1-biased response, which is more conducive to pathogen clearance. [149]he intracellular Th1/Th2 balance is also of great importance in regulating immunity and maintaining the balance between pro-and anti-inflammatory cytokine profiles in cells.Based on these considerations, attention is being focused on developing more effective vaccines that induce a balanced Th1/Th2 response. [150]For example, a balanced Th1 and Th2 response to parasitic infection was observed in mice vaccinated with a multiwalled CNT-based synthetic peptide nanovaccine.150c] In addition, adjuvants that stimulate natural cellular immunity, aiming to up-regulate the expression of specific cytokines via certain pathways, can also promote T-cell activation.Cyclic guanosine monophosphate-adenosine monophosphate (cGAMP), a small-molecule agonist of the stimulator of interferon genes (STING), has been widely used as a potent adjuvant in tumor vaccines to activate CD8 + T cells. [151]In a recent study, cGAMP was encapsulated in liposomes to confer adjuvant activity in an inactivated influenza vaccine.34a] Manganese (Mn 2+ ) can also promote the STING pathway by activating cGAMP synthase (cGAS) and inducing the synthesis of cGAMP. [152]10d] The MnARK nanovaccine induced robust T cells responses in mice, activating the cGAS-STING pathway in DC  and d) antigen-specific antibody production in sera after immunized with different sizes of OVA-AuNPs vaccine at 5 weeks in mice, indicating the larger sized NPs induce 5-fold enhanced antibody-mediated humoral immune responses than the smaller ones.Reproduced with permission. [156]Copyright 2019, American Chemical Society.e-g) Mosaic nanoparticle immunogen containing diverse influenza HA trimers prepared for providing supra-seasonal protection against influenza viruses.e) Negative-stain electron micrographs of the mosaic HA nanoparticles.Scale bar, 200 nm.f,g) The in vitro self-assembling HA nanoparticles afford nearly complete protection against both heterologous (H1N1 PR8) and heterosubtypic (H7N9 AN13) challenges in mice in the absence of adjuvant.Reproduced with permission. [166]Copyright 2021, Springer Nature.cells and induced 2-and 6-fold greater IFN- levels compared with controls immunized with aluminum-adjuvanted vaccine and vaccine alone, respectively.

B-Cell Activation and Humoral Immunity
Effective B-cell activation first requires the transport of intact antigens into B-cell areas of secondary lymphoid organs, where B-cell immunity is initiated subsequently. [130]Among immuneinducing sites, the LN follicle is a key target for antigen delivery to enhance the B-cell immune response.NP size affects antigen transport in the body, with sub-100 nm NPs preferentially trafficking from the injection site to LNs.After NPs traffic into the afferent lymphatic vessel, smaller NPs (≈5 nm) can directly enter LN follicles through fiber conduits, whereas larger NPs (>20 nm) are captured by subcapsular sinus (SCS24) macrophages and transported into the follicles. [153]Subsequently, NPs are either captured directly by B cells in the LN follicles or transported to the surface of follicular dendritic cells (FDCs) for B-cell recognition in a complement receptor-dependent manner (Figure 9a).
However, only a fraction of the NPs was transported into B-cell follicles, whereas the majority remained localized outside, resulting in a low level of B-cell activation.Glycosylation of the antigen sequence could promote engineered nanovaccine transportation to LNs and deposition in B-cell follicles, mediated by mannosebinding lectin. [154]Additionally, a recent study reported that subcapsular sinus (SCS) macrophages prevented OVA-loaded NPs transport to the LN follicles. [155]Adding SCS macrophage-uptake inhibitors as "reverse adjuvants" to the nanovaccine formulation allowed more NPs to reach and be retained in the LN follicles, resulting in an almost 30-fold increase in antigen-specific antibody generation compared with administration of the nanovaccine alone.This provides a new mechanism for improving the antigen delivery to lymphatic follicles.However, nonspecific inhibition of macrophage uptake can cause side effects.The size of NPs also determines their fate in B-cell follicles.Antigen-loaded gold NPs of size 50-100 nm can reside on FDC dendrites for several weeks through greater opsonization of complement C3, whereas 5-15 nm-sized NPs are taken up and cleared by FDCs within 48 h. [156]The long-term retention of antigens on FDCs is beneficial for the maintenance of germinal centers, and subsequent studies have shown that 50-100 nm-sized NPs induce a 5-fold greater antibody-mediated humoral immune response compared with smaller NPs (Figure 9b-d).
In contrast to T cells, antigen-specific B cells can directly recognize natural antigens through B-cell receptors (BCR), without MHC restriction.Once B cells bind to antigens, signaling via the BCR initiates B-cell activation. [130]NPs provide large surfaces for multiple antigen copies display, greatly facilitating BCR cross-linking and triggering a cascade of B-cell activation events, which promote the subsequent germinal center (GC) response and enhance humoral immunity. [157]Temchura et al. [158] developed biodegradable calcium phosphate NPs coated with ≈16 440 hen egg lysozyme (HEL) antigen molecules by covalent attachment.Functionalized NPs (CaP-HEL) with multivalent particulate structures were able to induce BCR-specific loss of CD62L as an early indicator of BCR cross-linking and enhanced B-cell activation efficiency 100-fold compared with the monovalent antigen.Administration of HA-displayed ferritin nanovaccines to mice induced enhanced GC reactions, resulting in higher titer antibody production and better protective efficacy than soluble HA and the licensed inactivated vaccine. [39,40]Singledose vaccination with SARS-CoV-2 spike-ferritin NPs greatly enhances neutralizing antibody responses in mice, eliciting 2-fold higher titers than those in the serum of convalescent COVID-19 patients. [43]In addition to capturing native antigens, complete activation of B cells generally requires signals from effector Th2 cells.Therefore, nano-adjuvants that promote the Th2 response are conducive to B-cell activation.Antigen-specific T-B cell interaction induces B-cell proliferation and consequent GC differentiation.The GC is an important site where B cells undergo somatic hypermutation, class-switch recombination, and affinity maturation with the help of T follicular helper cells (Tfh), eventually resulting in long-lived plasma and memory B-cell generation.The intensity and duration of GC action directly determine the potency of the humoral immune response.Polymeric nanocarriers provide a slow-delivery antigen platform that allows constant antigen exposure to GCs and is a powerful driver of the GC response. [159]159b] Faced with continuous viral mutation, the development of broad-spectrum vaccines that can provide broad protection against various divergent strains has attracted considerable attention.Repetition of highly conserved epitopes on the NP surface can induce the production of broadly neutralizing antibodies (bNAbs). [160]The highly conserved HA stem region of the IAV has been displayed on ferritin-based NPs to generate cross-reactive bNAbs against divergent influenza strains. [161]Multiple copies of an antigenically conserved B-cell epitope mimetic based on pneumococcal surface protein A (PspA) were displayed on VLPs. [162]accination of mice with these VLPs elicited cross-reactive antibody responses against at least eight genetically diverse pneumococcal strains.Furthermore, assembled NPs also provide an opportunity to display heterotypic or heterologous antigens to elicit broad antibody responses.Colocalization into the VLP of multiple HA variants containing H5, H7, and H9 protected vaccinated chickens from heterologous avian influenza invasion. [163]Mosaic NPs co-displaying diverse RBDs from distinct animal betacoronaviruses induced higher antibody titers against mismatched RBDs than homotypic SARS-CoV-2 NPs and provided crossprotection against multiple zoonotic coronaviruses. [29]21a] Mosaic antigen arrays provided an avidity advantage that preferentially activated cross-reactive B cells over mono-specific B cells, and consequently facilitated cross-reactive B cell expansion and elicited broad neutralizing antibody responses against a broad spectrum of influenza viruses spanning a 90-year period.12a] For example, PCV2 capsid protein-derived VLP was chosen to display repetitive conserved M2e epitopes of IAV. [164]In mice and pigs, the bivalent nanovaccine provided broad protection against IAVs from different species and induced high PCV2-specific antibody titers similar to those of commercial vaccines.Moreover, the study provided simultaneous prevention of IAV and PCV2, which prevented immunosuppression caused by PCV2 and offered the potential to block zoonotic IAV cross-species reassortment and transmission among humans, avians, and swine. [164]imilarly, HA and matrix (M2) proteins of IAV inserted into the capsid of infectious bursal disease virus, efficiently protected vaccinated mice against viral challenge. [165]Most recently, a novel mosaic nanoparticle immunogen containing diverse influenza HA trimers was constructed to provide supra-seasonal protection against influenza viruses. [166]The assembly process relied only on the self-assembly of antigen protein in vitro.The results showed that the mosaic nanoparticle triggered cross-reactive stem-directed neutralizing antibody responses, which afforded nearly complete protection against both heterologous (H1N1 PR8) and heterosubtypic (H7N9 AN13) challenges in mice and ferrets, even without the addition of an adjuvant.(Figure 9e-g) Overall, antigen-loaded NPs show unique advantages in promoting antigen delivery to B-cell follicles, BCR cross-linking, and cross-reactivity, resulting in high-affinity and broad-spectrum antibody production, which improves the depth and breadth of vaccine immune protection.

Biosafety Risks Associated with Nanoparticles use in Animals
Biosafety of animal vaccines is a major concern because it is closely related to animal and food safety.With the increasing use of NPs in vaccine development, the associated safety risks should be considered.The toxic effects of NPs are mainly determined by their size, composition, synthesis method, and dosage.Gold NPs exert size-dependent toxic effects on cells, including oxidative stress, autophagosome accumulation, and lysosomal impairment. [167]Moreover, naked gold NPs show aggregation and high levels of accumulation in the liver and spleen, where they cannot be metabolized and can induce irreparable toxic effects. [168]168b] The GSH-modified gold nanoclusters showed highly efficient renal clearance, with 94% of the gold metabolized after 28 days, indicating great potential for vaccine development.
Studies have reported the toxicity of silver NPs in vitro and in vivo. [169]Silver NPs can be distributed in many systems via various administration routes, which causes deposition in multiple organs, including the spleen, liver, kidney, and lungs. [169,170]The silver ions released from silver NPs, which may induce oxidative stress, mitochondrial damage, and cell apoptosis, are the main cause of their toxic effects. [171]Asgary et al. [172] reported that silver NPs significantly enhanced the antibody responses induced by inactivated rabies virus vaccines but caused severe cytotoxicity even at the lowest effective concentration.To improve the biosafety of NPs, green synthesis of silver NPs produced by E. procera has been explored.Green-synthesized silver NPs added to rabies vaccines showed adjuvant effects comparable to those of aluminum adjuvants without any toxicity in mice and dogs according to the European Pharmacopeia 8.0. [61]CNT-based nanomaterials have been widely used as vaccine delivery systems in the aquaculture industry because of their desirable penetrability. [87e,f] However, the toxicity of CNTs to both humans and animals is a concern. [173]Highly soluble, functionalized SWCNTs have the potential for developmental toxicity to newly hatched larvae; [173b] therefore, it is necessary to consider and evaluate the safety of chronic exposure to CNTs in aquatic environments.
Considering the biodegradation concerns associated with NPs, the use of degradable NPs in animal vaccines has attracted widespread attention.For example, biodegradable polymeric NPs such as chitosan, [69b] PLGA, [84] and polyanhydride [80] NPs are widely used as biodegradable and non-toxic antigen delivery systems in animals.Biodegradable MOFs, such as Zn-based MOFs, have been developed as biocompatible carriers to improve the efficiency of antigen delivery. [58]The toxicity of MOFs is closely related to the selected metal ions, organic ligands, and synthesis strategies. [174]174a] Although most studies have shown biocompatibility of MOFs used in vaccines, [58,175] some studies have shown in vitro toxicity caused by cation-induced ROS production. [176]In order to avoid the toxicity caused by the introduction of heavy ions and toxic solvents during MOF synthesis, a green preparation process for novel MOFs has been developed. [177]Cyclodextrin MOFs (CD-MOFs) have been prepared as adjuvants by green synthesis to enhance immune responses. [177]CD-MOFs display excellent biosafety in various animals, including chickens, mice, and goats, whereas they have a significant hemolytic effect on rabbits.LNPs have been used in mRNA vaccines, and clinical studies have shown low reactogenicity and good tolerability. [178]Nevertheless, the toxicity of LNPs is strongly correlated with their lipid components. [179]The application of cationic lipids can induce hepatotoxicity, splenic necrosis, and lymphocyte depletion. [180]ollectively, there is a need to consider and evaluate the potential safety risks posed by NPs during animal vaccine development.To avoid the safety risks associated with the in vivo application of vaccines, constant efforts are being made to develop biocompatible NPs, by exploring biodegradable nanomaterials and improving the synthesis process without using toxic solvents.

Conclusion and Outlook
Given the current epidemic threats and limitations of traditional vaccines, there is an urgent need to develop more effective vaccines with facile high-throughput deployment.Nanotechnology is a powerful tool for vaccine development, with broad prospects.It is the SARS-CoV-2 outbreak that pushed lipid nanoparticlebased vaccines into clinical trials.The application of nanotechnology in vaccine development offers opportunities to address concerns regarding currently available vaccines, including breaking dependence on the cold chain, enhancing the immunogenicity of antigens, reducing the side effects caused by traditional adjuvants, and providing cross-protection against multiple infectious diseases.Nanotechnology has also attracted growing attention in the field of animal vaccines to prevent zoonoses and promote animal welfare.The spread of animal infectious diseases not only causes losses to the global economy but also threatens the health of both humans and animals.IAVs, coronaviruses, rabies, and brucellosis are the most common zoonotic diseases that can be transmitted from animals to humans through zoonotic spillover, posing a threat to public health. [181]Table 2 summarizes representative cases of the application of nanotechnology in the design of vaccines against zoonotic diseases.In conclusion, nanotechnology promotes the development of animal vaccines in multiple ways.Here, we review the role of nanotechnology at the key stages of the full vaccination process, from in vitro vaccine preparation to in vivo immunity.For in vitro preparations, the rational design of vaccine formulations based on nanotechnology offers the possibility of maintaining antigen conformation, providing multiple antigen display sites, and improving vaccine stability against degradation and/or denaturation, which markedly reduces antigen dose and production costs.At the level of vaccine immunity in vivo, NPs provide superior adjuvant activity, which can enhance each stage of the immune response, from the delivery of antigens to the activation of immune cells.Notably, the in vitro stability and immunogenicity of vaccine formulations directly affect the in vivo immune response, whereas the in vivo immune response guides the in vitro optimization of vaccine.Therefore, these two levels are interdependent and both need to be considered in vaccine development.
The application of nanotechnology has enabled rapid progress in the development of vaccines for both human and animal use.However, several questions regarding nanovaccinology remain unanswered.First, given the complexity of the immune system, understanding of the specific mechanisms of nano-adjuvants is still relatively limited.Vaccine and adjuvant design relies on a comprehensive understanding of the immune system.Although enhanced immune effects induced by NPs have been widely reported, their intrinsic mechanisms have not been fully explained.For example, the spontaneous targeting of PLA-based NPs to APCs following mucosal administration, [88] differences in DC maturation due to the chiral structure of NPs, [182] different Thbiased immune responses promoted by various NPs, [21b,150c] and their underlying mechanisms are not well understood.Further understanding of the relationship between structure and function in nano-adjuvants and intracellular signaling pathways in immune cells is required. [183]Moreover, during complex immune processes, the majority of attention has been focused on the interaction of NPs with APCs to promote APC activation, whereas little attention has been paid to the direct regulation of T-cell activation.In previous studies, NPs have been shown to trigger T-cell activation mainly by promoting APC maturation and cross-presentation, [20] whereas little is known about the interaction of NPs with T cells to directly promote T-cell activation.Nanovaccines with antigen self-presentation have been developed to directly activate CD8 + T cells without complex crosspresentation in APCs, thereby inducing powerful CTL responses for cancer immunotherapy. [184]Vaccines that directly promote Tcell activation need to be developed to control viral pandemics and allow for more rapid immune protection.Second, more efforts are needed to induce long-term protective immunity for future vaccine development.Faced with the ongoing COVID-19 pandemic, the development of long-lasting protective vaccines has come into focus.The long-term protection provided by a vaccine depends on its ability to induce immune memory and resist viral mutations. [185]However, in current nanoparticle-based vaccine development, most studies have only measured antibody levels in the serum and cellular immune responses, and have not conducted a detailed assessment of immune memory induced by vaccines. [186]Studies have shown that 90-95% of antigen-specific T cells undergo apoptosis following antigen elimination, and the surviving T cells enter the memory phase to provide long-term protection against reinfection. [187]emory T cells are categorized as central memory, effector memory, and tissue-resident memory T cells, which play distinct roles in immune response to reinfection. [187]Changes in these memory phenotypes over time should be evaluated after vaccination with nanovaccines.Mass spectrometry flow cytometry based on single-cell analysis facilitates the detection of these immune phenotypes and can be widely used in future vaccine innovations. [188]Long-term antibody production depends on the generation and maintenance of long-lived plasma and memory B cells. [189]It is unknown whether vaccines in development can expand the memory pool, which requires further study of the immune mechanisms.Faced with highly mutating pathogens, the future development of conserved epitopes and polyvalent vaccines has attracted attention, and interdisciplinary studies combining nanotechnology with structural biology, reverse vaccinology, and bioinformatics are required to fully display the dominant epitopes on antigens and track lymphocyte cell clones and somatic hypermutation. [190]hird, the potentially toxic effects triggered by nanomaterialbased vaccines require additional consideration.The exploration of green synthesis, application of biodegradable materials, and development of biomimetic technology provide new opportunities for the safe application of nanotechnology in vaccines.Nevertheless, the distribution and metabolic behavior of NPs after vaccination via different routes needs to be studied further.Furthermore, the impact of nanomaterial degradation on the stability of the entrapped antigens needs to be considered.For example, acidic degradation products can be generated by polymeric materials, such as PLA and PLGA; [191] however, whether the decreased pH caused by the accumulation of these acidic products alters antigen conformation and reduces vaccine potency is unknown.In addition, pre-existing immunity to nanocarriers must be evaluated.Pre-existing immunity to adenoviruses in humans limits the effectiveness of multiple vaccinations of adenovirus vectorbased vaccines. [192]Similarly, immune responses to nanomaterials in the body should be considered when developing nanoparticle delivery systems.For example, anti-PEG antibodies have been reported after the administration of PEGylated drugs in recent studies [193] suggesting an unfavorable effect of PEG modification on NPs.Finally, matching the cost of developing an animal vaccine with the value of immunized animals is an important consideration.Although various NPs have been developed to enhance vaccine potency for animal use, the increased production costs of nanomaterials must be considered in clinical applications. [194]However, the cost of a vaccine is not only related to the materials used in vaccine research, but also to the cost of transportation, vaccine administration, antigen dose, and vaccination frequency, which require comprehensive consideration.
In conclusion, advances in nanotechnology offer new strategies for vaccine development against animal infectious diseases, but further in-depth studies are needed to address the concerns arising from the application of nanotechnology in vaccines.Combining nanotechnology with interdisciplinary research, such as the analysis of modern omics, [190b] the prediction of antigen epitopes by machine learning, [195] bioengineering strategies and other disciplines [196] will facilitate solutions to these issues in the future, and the number of safe and cost-effective nanovaccines is expected to increase.

Figure 2 .
Figure 2. Contribution of nanotechnology to vaccine from in vitro formulations to in vivo immunity.At the in vitro formulation level, nanoparticles enhance the immunogenicity and stability of antigens through nano-scaffold and nanocoating strategies.At the in vivo immunity level, nanoparticles facilitate multiple immune response processes, including antigen delivery, cellular uptake, antigen presentation, and lymphocyte activation, showing superior adjuvant activity.NPs, nanoparticles; LN, lymph node; APC, antigen-presenting cell; DC, dendritic cell; MHC-I, major histocompatibility complex I; MHC-II, major histocompatibility complex II.These figures were created using Servier Medical Art templates (https://smart.servier.com),which are licensed under a Creative Commons Attribution 3.0 Unported License: https://creativecommons.org/licenses/by/3.0/.

Figure 3 .
Figure 3. Nanovaccine formulations for enhancing immunogenicity and stability.a,b) Proteinaceous nanoparticles designed for multiple displaying polymerized RBD antigens.a) Schematic representation of RBD-conjugated NP design.b) Biolayer interferometry kinetic assays showing all three polymerized RBD NPs were more readily recognized by RBD-specific nAbs (CB6 antibody) than the RBD monomer.KD, binding affinity constant, smaller values generally indicate a stronger binding ability.Reproduced under the terms of the CC-BY license.[45]Copyright 2021, The Authors, published by American Chemical Society.c-e) RBD-conjugated Spy-mi3 NPs showed high stability against lyophilization.c) Cryo-electron micrograph of RBD-SpyVLP (Scale bar 200 Å).d,e) The solubility and antigenicity to monoclonal antibodies (mAbs) of the Spy-mi3 NPs were not significantly changed before and after lyophilization.Reproduced with permission.[48]Copyright 2021, Springer Nature.f-g) His-aZn-mIM-coated NPs designed for improving the thermostability of viral vaccines.f) Schematic diagram of the nanoparticle design.g) The His-aZn-mIM-coated Ad5 NPs retained 90.8% infective potency after storage at 25 °C for 90 days, showing longer-lasting thermostability than the CaP-coated Ad5 NPs and the native Ad5 virus particles.Reproduced with permission.[28]Copyright 2022, American Chemical Society.

Figure 4 .
Figure 4.The contribution of nanotechnology to vaccine-induced immunity in animals.Nanoparticles (NPs) introduced into vaccine formulations facilitate the key stages in the immune process from the start of vaccination of different routes to the eventual induction of the immune response, including a) at various vaccination sites via different administration routes; b) antigen capture by an antigen-presenting cell (APC); c) APC activation; d) T cell activation; and e) B cell activation, which greatly enhances vaccine potency against animal infectious diseases.DC, dendritic cell; MALT, mucosaassociated lymphoid tissues; LN, lymph node; MHC-I, major histocompatibility complex I; MHC-II, major histocompatibility complex II; Th1 cell, T helper cell type 1; Th2 cell, T helper cell type 2; BCR, B-cell receptor; GC, germinal center; Tfh cell, T follicular helper cells.

Figure 5 .
Figure 5. Antigen delivery from vaccination site to antigen-presenting cell (APC) across biological barriers.a) Nanotechnology-based antigen delivery strategies by needle-free vaccination.NPs can deliver cargoes across mucosal barriers to mucosa-associated lymphoid tissues (MALT) for antigenpresenting cell (APC) recognition.DC, dendritic cell.b,c) pH-trypsin sensitive PMMMA-PLGA nanoparticles (PTRBL/Trx-SIP) for oral vaccine design.b) Morphology of nanoparticles under acidic conditions (pH 5.5) and primary conditions (pH 7.4) with the presence of trypsin.c) In vivo fluorescence imaging of tilapia 36 h after oral immunization, demonstrating predominant accumulation of PMMMA-PLGA nanoparticles in the large intestine.Reproduced with permission.[27]Copyright 2016, Elsevier.d,e) Pulmonary surfactant (PS)-biomimetic liposome for pulmonary delivery of a small molecule adjuvant.d) Quantification of the cellular uptake of DiD-labeled nanoparticles with or without PS in vitro and e) in vivo fluorescence imaging of the distribution of nanoparticles in the lung after receiving DiD-labeled nanoparticles.Negatively charged nano4 localized within alveolar macrophages after intranasal administration, whereas positively charged nano5 showed diffuse staining along the alveolar surface due to electrostatic interaction with the negatively charged PS layer.Reproduced with permission.[34a]Copyright 2020, The American Association for the Advancement of Science.f) Quantitative analysis of chitosan-based mucoadhesive nanovaccines (CS) in fish gill tissues following immersion vaccination, demonstrating more efficient attachment of CS nanovaccines and CS-coated nanoemulsion vaccines (CS-NE) to the fish gill mucosal surface than that of killed whole-cell vaccines (WC) and nanoemulsion vaccines (NE).Reproduced with permission.[89c]Copyright 2019, Elsevier.

Figure 6 .
Figure 6.Promotion of antigen-presenting cell (APC) capture by tuning the physicochemical properties of nanoparticles (NPs).a) Size, shape, and surface properties of NPs affect antigen capture by antigen-presenting cells (APCs).b,c) NP size affects their transport and retention in lymph nodes (LNs).b) Quantification of Cy7.5-labeled PLGA-b-PEG NPs with different particle sizes trafficking over 24 h in LNs, indicating that 20 nm NPs show superior LNs retention in mice over 40 nm and 100 nm NPs.c) Colocalization of 20 nm NPs with CD11c + DCs in inguinal LN sections, as determined by confocal microscopy, indicating paracortex penetration by 20 nm NPs, with the majority taken up by DCs.Reproduced with permission.[95]Copyright 2019, Springer Nature.d,e) NPs similar to the targeted pathogen in shape and size are more easily recognized and internalized by APCs.d) Scanning electron microscopy (SEM) images of a P. aeruginosa-adsorbed, rod-shaped nano-aluminum adjuvant (Al-NRs).e) The internalized percentage of free antigen, antigen-adsorbed Al-NRs, and antigen-adsorbed Al(OH) 3 in J774A.1 cell.Reproduced with permission.[32]Copyright 2018, American Chemical Society.f) Gold nanoparticles (Au NPs) with different hydrophobic surfaces are generated by tuning the functionalities (blue) at the ligand termini.Log P represents the calculated hydrophobic values of the headgroup.g) Surface hydrophobicity of NPs is positively correlated with the efficiency of innate immunity responses.Reproduced with permission.[102c]Copyright 2012, American Chemical Society.

Figure 7 .
Figure 7. Nanoparticles (NPs) promote antigen-presenting cell (APC) capture by active targeting.a) Surface functionalization of NPs mediates targeted antigen delivery to antigen-presenting cells (APCs).b) Mean fluorescence intensity of cellular uptake of FITC-labeled nanovaccine (SWCNTs-G and SWCNTs-MG) by carp macrophages, indicating the improved cellular uptake of mannose-modified nanovaccines (SWCNT-MG).Reproduced under the terms of the CC-BY license. [87f] Copyright 2020, The Authors, published by Springer Nature.c) Targeting poly(lactic-co-glycolic acid) (PLGA) nanoparticles to CD40, DEC-205, or CD11c molecules on dendritic cells (DCs) improves binding and internalization compared with non-targeted nanoparticles.Reproduced with permission.[19]Copyright 2014, Elsevier.d) Confocal scanning microscopy images of adhere-effects between M-cell layer and nanoparticles after the transcytosis assay using an in vitro M-cell model, indicating that nanoparticles modified with M cells-targeted ligand Ulex europaeus agglutinin (UEA) effectively bind to the M-cell layer and are transported by M cells.Reproduced with permission.[118b]Copyright 2014, Elsevier.

Figure 8 .
Figure 8. Nanoparticles (NPs) promote the cross-presentation of antigens.a) Nanoparticles (NPs) promote cross-presentation of antigens in antigenpresenting cells (APCs) via lysosomal escape or direct cytosolic delivery.Lysosomal escape of antigens from NPs includes two strategies: membrane fusion and endosomal rupture.Antigens that are delivered into the cytosol can be degraded by proteasomes, followed by cross-presentation on MHC-I molecules.MHC-I, major histocompatibility complex I; MHC-II, major histocompatibility complex II; ER, endoplasmic reticulum.b-d) pH-responsive NPs for rapid antigen release in acidic endosomes and escape to the cytosol.b) Schematic illustration of the structure of pH-responsive poly(lacticco-glycolic acid; PLGA) NPs and their mechanism of action.c) In vitro release of antigens from pH-responsive PLGA NPs incubated in test media with different pH values to mimic the cytoplasm (pH 7.4), early endosomes (pH 6.5), and late endosomes/lysosomes (pH 5.0) at 37 °C.d) Numberof SIINFEKL-MHC-I + CD8 + T cells in immunized mouse splenocytes using the antigenic peptide-MHC pentamer staining method, indicating that pHresponsive NPs significantly augment the frequency of antigen-specific CD8 + T cells in mice.Reproduced with permission.[20]Copyright 2015, American Chemical Society.e-g) Cross-presentation by photoinduced endosomal escape.e) Confocal scanning microscopy images of bone marrow-derived dendritic cells (BMDCs) treated with nanoparticles (EV15/ICG/MSN) without (upper panels) and with (lower panels) laser irradiation.Endosomes were stained with Lysotracker Red.NPs were labeled with DiO (green).f-g) Percentage MHC-I-and MHC-II-positive BMDCs was determined by flow cytometry; laser irradiation promoted cytosolic release and cross-presentation of antigens.Reproduced under the terms of the CC-BY license.[140]Copyright 2020, The Authors, published by Ivyspring International Publisher.

Figure 9 .
Figure 9. Nanoparticles (NPs) promote B-cell activation.a) Schematic of Nanoparticles (NPs) transport in the lymph-node follicles and the strategy for NPs promoting B-cell activation.LN, lymph node; SCS, subcapsular sinus; M, macrophages; BCR, B cell receptor; DC, dendritic cell; FDC, follicular dendritic cell; Tfh, T follicular helper cell; GC, germinal center.Adapted with permission. [153]Copyright 2020, Springer Nature.b-d) Nanoparticle size influences antigen location and retention in lymph node follicles for B-cell responses.b) Quantification of NPs location either inside the FDC or on FDC dendrites by transmission electron microscope (TEM) images, showing that 5 nm OVA-AuNPs internalized by FDCs whereas 50−100 nm OVA-AuNPs retained on FDC dendrites.c) Numbers of germinal center B cells,and d) antigen-specific antibody production in sera after immunized with different sizes of OVA-AuNPs vaccine at 5 weeks in mice, indicating the larger sized NPs induce 5-fold enhanced antibody-mediated humoral immune responses than the smaller ones.Reproduced with permission.[156]Copyright 2019, American Chemical Society.e-g) Mosaic nanoparticle immunogen containing diverse influenza HA trimers prepared for providing supra-seasonal protection against influenza viruses.e) Negative-stain electron micrographs of the mosaic HA nanoparticles.Scale bar, 200 nm.f,g) The in vitro self-assembling HA nanoparticles afford nearly complete protection against both heterologous (H1N1 PR8) and heterosubtypic (H7N9 AN13) challenges in mice in the absence of adjuvant.Reproduced with permission.[166]Copyright 2021, Springer Nature.

Table 1 .
Co-delivery strategies of antigens and pattern-recognition-receptor agonists in veterinary vaccines.

Table 2 .
Nanoparticle-based vaccines to prevent zoonotic diseases.