Introduction

Vaccinations can be categorized as either 'live' or 'inactivated'. Live vaccines induce immunity through a transient disease brought about by a recreating live organism; for instance, the smallpox vaccine was innovated by Jenner in 1796 ( 1 ). Even though low-cost and straightforward to create, they convey the real dangers related to live microbes ( 2 ). Inactivated vaccines that incorporate killed life forms and secluded non-reproducing sub-cell parts induce a lower level and shorter duration of immunity than that evoked by live vaccines ( 3 ). The objective of immunization is to elicit an immune response toward the controlled antigen, thereby providing long-term protection against infectious diseases ( 4 ). Adding an adjuvant is often necessary to achieve that goal by killing attenuated vaccines instead of live ones. The word adjuvant, which means to help or enhance, comes from the Latin adjuvate ( 5 ). The idea of adjuvants emerged during the 1920s, starting with perceptions, for example, those who noticed the horses that fostered a canker by the immunization location of diphtheria toxoid created greater specific titers of antibody ( 6 ). They thereafter found that an ulcer created by the injection of inconsequential substances alongside the diphtheria pathogen enhanced the immune reaction against the toxoid ( 7 ). Mineral salts, microbial products, emulsions, saponins, cytokines, polymers, microparticles, and liposomes are just a few of the many different kinds of chemicals that have been evaluated as adjuvants ( 8 ). Vaccine adjuvants have been generally classified into delivery methods and immuno-stimulatory adjuvants based on their hypothesized modes of action. Previously, it was believed that delivery systems generally functioned by acting as a depot, whereas immuno-stimulatory adjuvants activated innate immune system cells. However, since there is now proof that certain delivery mechanisms might trigger innate immunity, this classification is no longer valid ( 9 ).

2. Adjuvants

The aluminium-based compounds (basically aluminum phosphate or hydroxide) remain the dominating Adjuvants used by humans ( 10 ). Freund's was the evolution of a mixture of mineral oil and water, comprising Mycobacteria, which was eliminated, consequently making it perhaps one of the most powerful recognized adjuvants, Freund's complete adjuvant (FCA) ( 11 ). Despite being the highest quality adjuvant, FCA results in extreme local responses and is excessively harmful in humans. The water-oil emulsion without adjuvants, a term for Mycobacteria, which is less harmful, has been used in individuals' vaccination ( 12 ). Bacterial parts are in many cases considered potent immune activators, albeit regularly connected with harmfulness, for instance, bacterial DNA that contains CpG (cytosine-guanine dinucleotides), which is perhaps among the strongest cell adjuvants ( 13 ). Stimulating the immune system, CpG is an unmethylated dinucleotide of cytosine and guanine present in bacterial DNA; however, it is missing in mammalian DNA ( 14 ). A few 100 normal plus manufactured substances have been identified as having adjuvant effectiveness ( 15 ). Although huge quantities are distinctly more potent than alum, toxicity is perhaps the most significant barrier to introducing these adjuvants that are used by humans ( 16 ).

3. The role of adjuvants in the development of vaccines

Adjuvants can be utilized to work on the immune reactions to administer antigens in different routes ( 17):

• Adjuvants can raise the immunogenicity of weak antigens

• Upgrade the rapidity as well as the period of the immunological reaction

• Adjust antibody avidity, specificity, isotype, or subclass apportionment.

• Induce cell-mediated immunity (CMI)

• Advance the stimulation of mucosal immunity

• Upgrade the immune reactions in aged or immune young individuals

• Reduce the antigenic dose, which can help in reducing the total cost.

• Assistance to conquer antigen rivalry in mixed inoculations

Most adjuvants' activity techniques remain inadequately perceived, obstructing the level-headed advancement of new adjuvants. Vaccination frequently enacts a series of cascading reactions; also, the essential impact of adjuvants is frequently challenging to observe in vivo ( 18 ).

In any case, on the off chance that one acknowledges the recently proposed geological idea of immune reactivity, wherein antigens that are not delivered at the nearby lymph nodes, they will not stimulate immunological reactions ( 19 ).

It becomes more straightforward to suggest mechanistic explanations of the significant impacts of specific adjuvants. If antigens that do not arrive at lymphatic nodes are not able to incite reactions, then any adjuvant that improves the conveyance of the antigen in the direction of the node might upgrade the reaction ( 20 ).

Antigen conveyance might remain improved at more than one method: adjuvant might increase cell leakage addicted to the insertion location, Consequently, additional cells be available just before absorb antigen, it might straightforwardly advance the take-up of antigen into antigen presenting cells (APC), or it might straightforwardly add to the conveyance of antigen to the regional lymph nodes ( 21 ). The primary APC engaged with antigen capture are the dendritic cells (DC), which are undeveloped. The process is carried out through 'sentinels' to circle via peripheral tissues ( 22 ).

Next, after introducing antigen and cell stimulation, DCs undergo development and move to the lymph nodes, where they have an extraordinary ability to introduce antigens to naïve T cells ( 23 ).

4. Different kinds of adjuvants

To mount an efficacious immunological reaction, B cells are required to produce about 20,000 plasma cells, and also T-lymphocytes are expected to induce a cell-based reaction ( 24 ). Further, current adjuvants can produce adequate B cells yet insufficient CD8 T cells. The coupling of adjuvants is necessary to induce sufficient T cell reactions ( 25 ).

A. Damage-Associated Molecular Patterns-Type Adjuvants

1. Aluminium Salts (Alum) adjuvants

Detailed the adjuvant action of combinations made of aluminum using an interruption of alum-hastened diphtheria toxoid (DT). Aluminium adjuvants, typically called 'alum', represent the most commonly used adjuvants worldwide in humans and animals around the world. However, the mechanism by which alum hides the excitement of the immune system remains obscure ( 26 ). The adjuvant action of aluminium salts is credited to either soluble or insoluble (particulate) aluminium, or as a consolidated reaction of the two types of aluminium. Alum, the most widely recognized adjuvant in non-living vaccines, has a history of practical use in human vaccination, where it advances antibody-mediated defensive immunity ( 27 ). Nonetheless, alum is a needy inducer of cell-mediated immune reactions. However, it excites Th2 one-sided reactions, delivering an alum incapable of defensive reactions constrained by Th1-mediated immunity ( 28 ). This constraint has provoked examination concerning elective adjuvants, and due to its utilization in endorsed vaccines, alum usually serves as a benchmark for assessing novel immunostimulants. Alhydroge is a mercantile aluminium hydroxide gel supported by the FDA for human use, and underlying investigations have uncovered the aluminium stage as crystalline aluminium oxyhydroxide ( 29 ).

2. Saponin adjuvants

Saponins are a diverse group of naturally occurring active compounds found in plants and efficient compounds in Fungi belonging to over 100 families, including endophytic terrestrial and marine fungi. Basically, saponins comprise a steroid aglycone or triterpene known as sapogenin, by means of at least a single sugar bond connected on the way to it ( 30 ). The available steroidal saponins are fundamentally triterpenoid, whereas monocotyledons are found in dicotyledons. Saponins display foamy and emulsification features because of the amphiphilic character of their architecture, which includes hydrophilic sugar chains and hydrophobic aglycones ( 31 ). The combination of a hydrophilic (water-soluble) sugar base and a hydrophobic (fat-soluble) sapogenin is responsible for the saponin foaming limit. One saponin was adjuvanted ( 32 ), an authorized vaccine supported by the FDA in 2017. The recombinant zoster vaccine (RZV, Shingrix, GlaxoSmithKline) contains AS01B, a saponin-based adjuvant ( 33 ).

Figure 1.Innate and adaptive immunity, as well as the aluminum adjuvant armory. (a) A variety of possible agonists of the immune cascade are produced when the vaccine formulation is diluted into the muscle interstitial fluid (MIF), including: (1) Al3+(aq); (2) free antigen (AG); (3) particulate adjuvant (ADJ); (4) ADJ with associated AG; (5) AG-Al complex; (6) MIF ligand-Al complex; (7) ADJ with associated MIF ligand; (8) MIF ligand-AG complex; (9) particulate iron (as adjuvant contaminant), either free or with adsorbed Al/AG and the resulting reactive oxygen species (ROS); (10) ADJ with associated MIF ligand-AG complex; (11) ADJ with associated MIF ligand-Al complex. Biomolecules like ATP, albumin, transferrin, citrate, and fibrinogen may be MIF ligands. (b) A variety of cell types are affected by the array of agonists, including the resident muscle tissue (which may result in necrotic and/or apoptotic cell death) and infiltrating innate cells like monocytes (which may differentiate into dendritic cells due to AlADJ), granulocytes (which may cause AlADJ-induced eosinophilia that directly affects B cells), macrophages (which are known to remain near the injection site for extended periods of time and may be identified by AlADJ inclusions), and dendritic cells (DC). The latter could be the main APC that presents antigens. (c) Aggressors and innate cells can interact in a variety of ways, such as (i) AG2, AG-Al complex5, MIF ligand-AG complex8, and Al3+(aq)1 binding to toll-like receptors (TLRs); (ii) AG-ADJ4 binding to multiple TLRs; (iii) phagocytosis of ADJ3, AG-ADJ4, MIF ligand-ADJ7, MIF ligand-Al complex-ADJ11, and MIF ligand-AG complex-ADJ10; (iv) direct1 or indirect6 binding of Al3+(aq) by membrane receptors and extracellular (lipid membrane) or intracellular (nucleus) ROS9. (d) APCs trigger adaptive immunity by (a) releasing chemokines and cytokines (green saucers) like IL-1b and IL-18 either independently or dependently on the Nalp3 inflammasome; (b) presenting AG to the T cell receptor via MHC in conjunction with co-stimulatory molecules; and (c) directly acting on B/T cells with ADJ and/or Al3+(aq). The figures' parenthetical numbers are indicated by the superscripts (10).

Figure 2.Adjuvants containing quillaja saponins, such as QS-21, affect dendritic cells (DCs) and T cells. T cell: A costimulatory signal is sent to the T cell via the aldehyde group on QS-21, forming an imine with an e-amino group from a T cell surface receptor, most likely CD2. This signal takes the place of the one produced by contacts between the CD80 (B7-1 ligand) on the DC and the CD28 receptor on the T cell. At the level of tyrosyl phosphorylation of the mitogen-activated protein (MAP) kinase (ERK2), this signal converges with T cell receptor (TCR)-mediated signaling. This, in conjunction with modifications in the cells' K+ and Na+ transport, stimulates T cell activation biased towards Th1 immunity, which leads to the secretion of Th1 cytokines. DC: Quillaja saponins affect DCs, although not through a receptor. Endocytosis is how exogenous protein antigens (Ag) and QS-21 enter DCs. QS-21 breaks down the endosomal membrane, allowing the antigens to escape early to be processed further into peptides inside the cell. The vacuolar pathway loads properly degraded antigens into MHCI, while the cytosolic pathway transports antigens that require more processing to the proteasome for cleavage. These peptides, or cleaved proteins, are carried to the endoplasmic reticulum (ER) and then loaded into MHCI following further processing. After binding to MHCI, peptides from the vacuolar or cytosolic routes are presented to naïve CD8+ T cells on the DC surface through a process known as cross-presentation, which results in the production of cytotoxic T lymphocytes (CTLs). Based on Rhodes and Marciani (25).

3. Emulsion adjuvant

Emulsion adjuvants, such as MF59 and AS03, have been used for over 20 years as key parts of authorized vaccines. An emulsion combines at least two regularly immiscible fluids, stabilized by a surfactant. Significant clinical experience of efficiency and a well-established safety profile, alongside the facility of industrialization, have positioned emulsion adjuvants as one of the main stages for improving pandemic vaccines ( 34 ). Emulsion adjuvants consider antigen dose saving, faster immune reactions, and boosted quality and amount of adaptive immune reactions. The systems of upgraded immune responses are clear and usually described by the production of an "immunocompetent environment" at the location of injection, trailed by the stimulation of solid and durable germinal center reactions in the draining lymph nodes ( 35 ).

Subsequently, emulsion adjuvants prompt particular immunological responses, with a blended Th1/Th2 cell reaction, extensive plasma cells, an extended collection of memory B cells, and high titers of cross-killing polyfunctional antibodies against viral variations ( 36 ). Due to these different properties, emulsion adjuvants were included for pandemic influenza vaccines conveyed during the 2009 H1N1 flu pandemic, are still remembered for occasional flu vaccines, and are currently at the forefront of the advancement of vaccines against emerging SARS-CoV-2 pandemic variations ( 37 ).

In an oil-in-water emulsion, the oil is the dispersed phase in water. The most commonly involved oil in human adjuvants is squalene, a naturally occurring particle in plants and animals, including humans, in whom it is essential for producing cholesterol, steroid hormones, and vitamin D ( 38 ).

B. Pathogen-associated molecular patterns (PAMPs) adjuvants

Numerous adjuvants are basically composed of bacterial materials. They are intended to target particular PRRs, such as toll-like receptors (TLRs) ( 39 ). Accordingly, they stimulate dendritic cells and macrophages and animate the development of significant cytokines like IL-1 and IL-12. Depending on the particular bacterial material, they might improve either Th1 or Th2 reactions ( 40 ).

Lipopolysaccharide adjuvants

Lipopolysaccharides are a predominant part of the Gram-negative bacterial external layer, and they are considered a potent inducer of the innate immune system, as well as a significant basis for adaptive immune reaction due to bacterial infections ( 41 ). This adjuvant action may benefit after immunization through bacteria-inferred vaccines that typically comprise LPS, and then subsequently LPS or particles obtained after it are added to clarified injection antigens. Be that as it may, the disadvantage of the strength from the biological efficacy of LPS is its capacity to enhance the vaccine's ability to react ( 42 ). Modulation of the LPS construction permits setting off of a suitable immune reaction required in a vaccine in contradiction to a specific microorganism, while simultaneously reducing its harmfulness ( 43 ).

Fig. 3.The likely way that oil-in-water emulsions work. The oil-in-water emulsion produces an immunologically active milieu at the injection site after intramuscular injection. Chemokines cause a huge number of innate immune cells, including monocytes, dendritic cells, eosinophils, and neutrophils, to be drawn to the draining lymph nodes. Th1 and Th2 type cytokines and particular antibodies are produced when T and B cells are activated by DCs harboring antigenic signals within the draining lymph nodes. After entering the bloodstream, the particular cytokines and antibodies start to have a protective effect (30).

Figure 4.When an LPS-stimulated macrophage (middle) secretes mediators, it triggers further secretory stimuli and physiologic consequences from a variety of cell types. The effects of tumor necrosis factor (TNF), interleukin (IL)-l, IL-2, IL-4, IL-5, and IL-6, growth factor (TGF-~), and interferon-y (IFNy) on the proliferation and differentiation of T and B lymphocytes—two essential components in the antigen presentation process—are of particular interest for adjuvant development for vaccines. updated and informed by new information (40).

C. Cytokines as adjuvants

Different cytokines have been demonstrated to be compelling immunological adjuvants in various model systems, improving defense actuated by viral, bacterial, and parasitic vaccines, and raising parameters of immunity in tumour vaccination models and in clinical experimental ( 44 ). While, as a rule, cytokine adjuvanticity is not so strong as that shown by the best trial adjuvants, for example, saponin and Freund's, it can equal that of the adjuvants as of now considered for human use, and there are numerous potential courses to progress ( 45 ). The utilization of cytokines might be considered a decision of which immune parameters are promoted to improve defensive impacts further and reduce the adverse consequences of vaccines ( 46 ).

Figure 5.T lymphocyte activation and differentiation. Dendritic cells (DCs) absorb an antigen that has been administered by a vaccine (stage 1) or an infectious disease. When conserved elements of pathogens attach to pattern recognition receptors (PRRs), they trigger the release of inflammatory cytokines and the production of co-stimulatory molecules, activating DCs. Adjuvants like aluminum salts, which also trigger inflammatory pathways, are added to vaccines that lack intrinsic adjuvants. DCs break down the antigen into peptides, which are then returned to the cell surface on MHC molecules and delivered to CD8+ and CD4+ T cells. MHC class I molecules present the antigen to CD8+ T cells, while MHC class II molecules (stage 2) present it to CD4+ T cells. To be completely activated, T cells also need signals from inflammatory cytokines and co-stimulatory molecules. Cell growth (stage 3) and effector cell differentiation (stage 4) are the outcomes of activation. The production of cytokines by CD4+ T cells can stimulate innate immune cells, including neutrophils and macrophages, to eliminate infections. By producing the soluble and cell-surface mediators needed for the synthesis of high-affinity class-switched antibodies, activated CD4+ T cells can also support B cells. Effector CD8+ T cells can either activate other cell types by producing inflammatory cytokines or kill infected cells by releasing cytotoxic granules (43).

5. Evaluating the Adjuvant Vaccines Safety

The protection of a few adjuvant constituents is assessed within the framework of the vaccine. That is, whereas in the preclinical phase of vaccine advancement, every part is evaluated separately. Most protection assessments in place of slight vaccination focus on the finished product ( 47 ).

Following licensure, the assessment of vaccination protection continues indefinitely after it starts in a lab ( 48 ).

Before being administered to humans, vaccination candidates undergo a severe analysis process in animal models to identify signs of systemic or local injuriousness that could indicate a possible human safety concern ( 49 ).

Before the significant dose in humans, tests are carried out in animal models whenever possible to evaluate the impact of giving various doses, vaccine quality, immunogenicity, and preventive effectiveness. Preclinical testing for reproductive and developmental toxicity is complete if the vaccination is dedicated to women of childbearing age. Period I (first in people) injection revisions are typically minimal and involve healthy adults ( 50 ).

These studies usually use techniques like dosage controlling harmfulness, which is predetermined using animal testing outcomes, and staggered enrolment to minimize possible protection hazards. The candidate vaccine is tested in period II and III studies on many patients, including the target group, while protection, immunogenicity, and/or effectiveness endpoints are being assessed simultaneously. It is possible to deploy independent statistics observing groups who monitor protection results without blinding anyone ( 51 ).

Randomized controlled trials are the most widely used pre-licensure study design. They have a higher possibility of identifying vaccine adverse events than controls. However, their statistical influence to distinguish possibly exceptional (1:10,000 to <1:000 doses) from very exceptional (<1:10,000) adverse events is limited.Techniques like combining protection data from several studies with comparable designs should boost the ability to identify those few adverse occurrences ( 52 ).

Vaccines have a somewhat extensive safety record when certified, making it possible to comprehend the vaccine's protection profile. Nonetheless, throughout the vaccination life cycle, new information regarding the safety hazards is constantly being discovered ( 53 ).

A new vaccination can only be licensed if its expected benefits in avoiding disease are shown to obviously outbalance any possible hazards to the inhabitants it is intended for; this is known, for example, as the benefit-risk proportion. Regardless of whether a unique adjuvant is included in the preparation, the benefit-to-risk ratio of all vaccines is continuously evaluated ( 54 ).

Conclusions

A definitive objective of immunization is to create robust and long-lasting immunity against infections. Such defensive immunity may be induced by utilizing vaccine details that include proper antigens plus adjuvants. Adjuvants are significant parts of vaccines and may influence the results of vaccination. The preceding methodologies of vaccine detailing by adjuvants centred on one type of adjuvant, like alum or emulsions. Nonetheless, novel vaccine aims need the enlistment of obvious CMI, notwithstanding the high titers of antibodies. Thus, novel immunostimulant adjuvants in vaccine details are required to animate powerful immune reactions containing humoral immunity plus CMI. With extraordinary headway in adjuvant field exploration over the past twenty years, vaccinologists are now able to choose a suitable adjuvant from traditional adjuvants, immunostimulants, or blends thereof to enhance a vaccine efficacy.

Conflicts of interest

The authors declare that there is no conflict of interest.

References

1. Bloom, D. E., Canning, D.,&amp; Weston, M. (2011). The value of vaccination. Advances in Experimental Medicine and Biology, 697, 1–8. DOI: 10.1007/978-1-4419-7185-2_1.DOI
2. Sheikh, S., Biundo, E., Courcier, S., Damm, O., Launay, O., Maes, E., …&amp; Begg, N. (2018). A report on the status of vaccination in Europe. Vaccine, 36(33), 4979–4992. DOI: 10.1016/j.vaccine.2018.06.044.DOI
3. Giubilini, A. (2021). Vaccination ethics. British Medical Bulletin, 137(1), 4–12. DOI: 10.1093/bmb/ldaa036.DOI
4. MacDonald, N. E. (2015). Vaccine hesitancy: Definition, scope and determinants. Vaccine, 33(34), 4161–4164. DOI: 10.1016/j.vaccine.2015.04.036.DOI
5. McKee, A. S.,&amp; Marrack, P. (2017). Old and new adjuvants. Current Opinion in Immunology, 47, 44–51. DOI: 10.1016/j.coi.2017.06.005.DOI
6. HogenEsch, H., O&#039;Hagan, D. T.,&amp; Fox, C. B. (2018). Optimizing the utilization of aluminum adjuvants in vaccines: you might just get what you want. npj Vaccines, 3(1), 51. DOI: 10.1038/s41541-018-0089-x.DOI
7. Pulendran, B., Arunachalam, P. S.,&amp; O&#039;Hagan, D. T. (2021). Emerging concepts in the science of vaccine adjuvants. Nature Reviews Drug Discovery, 20(6), 454–475. DOI: 10.1038/s41573-021-00163-y.DOI
8. Reed, S. G., Orr, M. T.,&amp; Fox, C. B. (2013). Key roles of adjuvants in modern vaccines. Nature Medicine, 19(12), 1597–1608. DOI: 10.1038/nm.3409.DOI
9. Sun, B., Yu, S., Zhao, D., Guo, S., Wang, X.,&amp; Zhao, K. (2018). Polysaccharides as vaccine adjuvants. Vaccine, 36(35), 5226–5234. DOI: 10.1016/j.vaccine.2018.07.040.DOI
10. Zhao, T., Cai, Y., Jiang, Y., He, X., Wei, Y., Yu, Y.,&amp; Tian, X. (2023). Vaccine adjuvants: mechanisms and platforms. Signal Transduction and Targeted Therapy, 8(1), 283. DOI: 10.1038/s41392-023-01557-7.DOI
11. Facciolà, A., Visalli, G., Laganà, A.,&amp; Di Pietro, A. (2022). An overview of vaccine adjuvants: current evidence and future perspectives. Vaccines, 10(5), 819. DOI: 10.3390/vaccines10050819..DOI
12. Shi, S., Zhu, H., Xia, X., Liang, Z., Ma, X.,&amp; Sun, B. (2019). Vaccine adjuvants: Understanding the structure and mechanism of adjuvanticity. Vaccine, 37(24), 3167–3178. DOI: 10.1016/j.vaccine.2019.04.055.DOI
13. Temizoz, B., Kuroda, E.,&amp; Ishii, K. J. (2016). Vaccine adjuvants as potential cancer immunotherapeutics. International Immunology, 28(7), 329–338. DOI: 10.1093/intimm/dxw015.DOI
14. Temizoz, B., Kuroda, E.,&amp; Ishii, K. J. (2016). Vaccine adjuvants as potential cancer immunotherapeutics. International Immunology, 28(7), 329–338. DOI: 10.1093/intimm/dxw015.DOI
15. Brito, L. A., O&#039;Hagan, D. T., et al. (2016). Overview of vaccine adjuvants: Introduction, history, and current status. Vaccine Adjuvants: Methods and Protocols (pp. 1–13). DOI: 10.1007/978-1-4939-6445-1_1.DOI
16. Bonam, S. R., Partidos, C. D., Halmuthur, S. K. M.,&amp; Muller, S. (2017). An overview of novel adjuvants designed for improving vaccine efficacy. Trends in Pharmacological Sciences, 38(9), 771–793. DOI: 10.1016/j.tips.2017.06.002.DOI
17. Petrovsky, N. (2015). Comparative safety of vaccine adjuvants: a summary of current evidence and future needs. Drug Safety, 38(11), 1059–1074. DOI: 10.1007/s40264-015-0350-4.DOI
18. Del Giudice, G., Rappuoli, R.,&amp; Didierlaurent, A. M. (2018). Correlates of adjuvanticity: A review on adjuvants in licensed vaccines. Seminars in Immunology, 39, 14–21. DOI: 10.1016/j.smim.2018.05.001.DOI
19. Lee, S.,&amp; Nguyen, M. T. (2015). Recent advances of vaccine adjuvants for infectious diseases. Immune Network, 15(2), 51–57. DOI: 10.4110/in.2015.15.2.51.DOI
20. Moyer, T. J., Zmolek, A. C.,&amp; Irvine, D. J. (2016). Beyond antigens and adjuvants: formulating future vaccines. The Journal of Clinical Investigation, 126(3), 799–808. DOI: 10.1172/JCI81083.DOI
21. Nooraei, S., Sarkar Lotfabadi, A., Akbarzadehmoallemkolaei, M.,&amp; Rezaei, N. (2023). Immunogenicity of different adjuvants and nano-adjuvants in veterinary vaccines: a comprehensive review. Vaccines, 11(2), 453. DOI: 10.3390/vaccines11020453.DOI
22. Marciani, D. J. (2018). Elucidating the mechanisms of action of saponin-derived adjuvants. Trends in Pharmacological Sciences, 39(6), 573–585. DOI: 10.1016/j.tips.2018.03.005.DOI
23. Wang, P. (2021). Natural and synthetic saponins as vaccine adjuvants. Vaccines, 9(3), 222. DOI: 10.3390/vaccines9030222.DOI
24. Reed, J., Orme, A., El-Demerdash, A., Owen, C., Martin, L. B. B., Misra, R. C., ...&amp; Osbourn, A. (2023). Elucidation of the pathway for biosynthesis of saponin adjuvants from the soapbark tree. Science, 379(6638), 1252–1264. DOI: 10.1126/science.adf3727.DOI
25. Fernandez-Tejada, A., Tan, D. S.,&amp; Gin, D. Y. (2016). Developing improved vaccine adjuvants based on the saponin natural product QS-21 through chemical synthesis. Accounts of Chemical Research, 49(9), 1741–1756. DOI: 10.1021/acs.accounts.6b00242.DOI
26. O&#039;Hagan, D. T., van der Most, R., Lodaya, R. N., Coccia, M.,&amp; Lofano, G. (2021). &quot;World in motion&quot; – emulsion adjuvants rising to meet the pandemic challenges. NPJ Vaccines, 6(1), 158. DOI: 10.1038/s41541-021-00418-0.DOI
27. Huang, Z., Gong, H., Sun, Q., Yang, J., Yan, X.,&amp; Xu, F. (2024). Research progress on emulsion vaccine adjuvants. Heliyon, 10(3), e24662. DOI: 10.1016/j.heliyon.2024.e24662.DOI
28. Brito, L. A., Malyala, P.,&amp; O&#039;Hagan, D. T. (2013). Vaccine adjuvant formulations: a pharmaceutical perspective. Seminars in Immunology, 25(2), 130–145. DOI: 10.1016/j.smim.2013.05.007.DOI
29. Nooraei, S., Sarkar Lotfabadi, A., Akbarzadehmoallemkolaei, M.,&amp; Rezaei, N. (2023). Immunogenicity of different adjuvants and nano-adjuvants in veterinary vaccines: a comprehensive review. Vaccines, 11(2), 453. DOI: 10.3390/vaccines11020453.DOI
30. Moyer, T. J., Zmolek, A. C.,&amp; Irvine, D. J. (2016). Beyond antigens and adjuvants: formulating future vaccines. Journal of Clinical Investigation, 126(3), 799–808. DOI: 10.1172/JCI81083.DOI
31. Sun, J., Remmele Jr, R. L.,&amp; Sanyal, G. (2017). Analytical characterization of an oil-in-water adjuvant emulsion. AAPS PharmSciTech, 18(5), 1595–1604. DOI: 10.1208/s12249-016-0626-8.DOI
32. van Doorn, E., Liu, H., Huckriede, A.,&amp; Hak, E. (2016). Safety and tolerability evaluation of the use of Montanide ISA™51 as vaccine adjuvant: A systematic review. Human Vaccines&amp; Immunotherapeutics, 12(1), 159–169. DOI: 10.1080/21645515.2015.1071455.DOI
33. Di Pasquale, A., Bonanni, P., Garçon, N., Stanberry, L. R., El-Hodhod, M.,&amp; Da Silva, F. T. (2016). Vaccine safety evaluation: Practical aspects in assessing benefits and risks. Vaccine, 34(52), 6672–6680. DOI: 10.1016/j.vaccine.2016.10.039.DOI
34. Stassijns, J., Bollaerts, K., Baay, M.,&amp; Verstraeten, T. (2016). A systematic review and meta-analysis on the safety of newly adjuvanted vaccines among children. Vaccine, 34(6), 714–722. DOI: 10.1016/j.vaccine.2015.12.024.DOI
35. MF59-adjuvanted quadrivalent influenza vaccine in children (Phase III trial). The Lancet Respiratory Medicine, (Year), pages. DOI: 10.1016/S2213-2600(18)30108-5.DOI
36. Garçon, N.,&amp; Di Pasquale, A. (2017). From discovery to licensure, the Adjuvant System story. Human Vaccines&amp; Immunotherapeutics, 13(1), 19–33. DOI: 10.1080/21645515.2016.1225635.DOI
37. Brito, L. A., Malyala, P.,&amp; O&#039;Hagan, D. T. (2013). Vaccine adjuvant formulations: a pharmaceutical perspective. Seminars in Immunology, 25(2), 130–145. DOI: 10.1016/j.smim.2013.05.007.DOI
38. Reactions Weekly. (2016). Newly adjuvanted vaccines in children: no safety concerns. DOI: 10.1007/s40278-016-13777-8.DOI
39. Flarend, R. E., Hem, S. L., White, J. L., Elmore, D., Suckow, M. A., Rudy, A. C.,&amp; Dandashli, E. A. (1997). In vivo absorption of aluminum-containing vaccine adjuvants using ²⁶Al. Vaccine, 15(12), 1314–1318. DOI: 10.1016/S0264-410X(97)00041-8.DOI
40. Talbot, H. K., Rock, M. T., Johnson, C., Tussey, L., Kavita, U., Shanker, A., Shaw, A. R.,&amp; Taylor, D. N. (2010). Immunopotentiation of trivalent influenza vaccine when given with VAX102, a recombinant influenza M2e vaccine fused to the TLR5 ligand flagellin. PLoS ONE, 5(6), e14442. DOI: 10.1371/journal.pone.0014442.DOI
41. Stassijns, J., Bollaerts, K., Baay, M.,&amp; Verstraeten, T. (2016) A systematic review and meta-analysis on the safety of newly adjuvanted vaccines among children. Vaccine, 34(6), 714–722. DOI: 10.1016/j.vaccine.2015.12.024.DOI
42. Trombetta, C. M., Gianchecchi, E.,&amp; Montomoli, E. (2018 Influenza vaccines: Evaluation of the safety profile. Human Vaccines&amp; Immunotherapeutics, 14(3), 657–670. DOI: 10.1080/21645515.2017.1423153.DOI
43. O&#039;Hagan, D. T., Friedland, L. R., Hanon, E.,&amp; Didierlaurent, A. M. (2017) towards an evidence-based approach for the development of adjuvanted vaccines. Current Opinion in Immunology, 47, 93–102. DOI: 10.1016/j.coi.2017.07.010.DOI
44. Batista-Duharte, A., Martínez, D. T.,&amp; Carlos, I. Z. (2018) Efficacy and safety of immunological adjuvants. Where is the cut-off? Biomedicine&amp; Pharmacotherapy, 105, 616–624. DOI: 10.1016/j.biopha.2018.06.026.DOI
45. Asgary, V., Shoari, A., Baghbani-Arani, F., Sadat Shandiz, S. A., Khosravy, M. S., Janani, A., …&amp; Cohan, R. A. (2016) Green synthesis and evaluation of silver nanoparticles as an adjuvant in the rabies veterinary vaccine. International Journal of Nanomedicine, 11, 3597–3605. DOI: 10.2147/IJN.S109098.DOI
46. Danielsson, R.,&amp; Eriksson, H. (2021) Aluminium adjuvants in vaccines – A way to modulate the immune response. Seminars in Cell&amp; Developmental Biology, 115, 3–9. DOI: 10.1016/j.semcdb.2020.12.008.DOI
47. Powell, B. S., Andrianov, A. K.,&amp; Fusco, P. C. (2015) Polyionic vaccine adjuvants: another look at aluminum salts and polyelectrolytes. Clinical and Experimental Vaccine Research, 4(1), 23–45. DOI: 10.7774/cevr.2015.4.1.23.DOI
48. Post-Hoc Analysis of Potential Correlates of Protection of a Recombinant SARS-CoV-2 Spike Protein Extracellular Domain Vaccine Formulated with Advax-CpG55.2-Adjuvant. International Journal of Molecular Sciences, 25(17), 9459. DOI: 10.3390/ijms25179459.DOI
49. In vivo absorption of aluminum-containing vaccine adjuvants using ²⁶Al. Vaccine, 15(12), 1314–1318. DOI: 10.1016/S0264-410X(97)00041-8.DOI
50. Laera, D., HogenEsch, H.,&amp; O&#039;Hagan, D. T. (2023). Aluminum Adjuvants—&#039;Back to the Future&#039;. Pharmaceutics, 15(7), 1884. DOI.10.3390/pharmaceutics15071884.DOI
51. Wang, P. (2021). Natural and synthetic saponins as vaccine adjuvants. Vaccines, 9(3), 222. DOI: 10.3390/vaccines9030222.DOI
52. Zariri, A.,&amp; van der Ley, P. (2015). Biosynthetically engineered lipopolysaccharide as vaccine adjuvant. Expert Review of Vaccines, 14(6), 861–876. DOI: 10.1586/14760584.2015.1026808.DOI
53. Zhang, J., Miao, J., Han, X., Lu, Y., Deng, B., Lv, F., ...&amp; Hou, J. (2018). Development of a novel oil-in-water emulsion and evaluation of its potential adjuvant function in a swine influenza vaccine in mice. BMC Veterinary Research, 14(1), 415. DOI: 10.1186/s12917-018-1719-2.DOI
54. Zhao, T., Cai, Y., Jiang, Y., He, X., Wei, Y., Yu, Y.,&amp; Tian, X. (2023). Vaccine adjuvants: mechanisms and platforms. Signal Transduction and Targeted Therapy, 8(1), 283. DOI: 10.1038/s41392-023-01557-7.DOI