Enhancing vaccine effectiveness in the elderly to counter antibiotic resistance: The potential of adjuvants via pattern recognition receptors

ABSTRACT

Vaccines are an effective way to prevent the emergence and spread of antibiotic resistance by preventing diseases and establishing herd immunity. However, the reduced effectiveness of vaccines in the elderly due to immunosenescence is one of the significant contributors to the increasing antibiotic resistance. To counteract this decline and enhance vaccine effectiveness in the elderly, adjuvants play a pivotal role. Adjuvants are designed to augment the effectiveness of vaccines by activating the innate immune system, particularly through pattern recognition receptors on antigen-presenting cells. To improve vaccine effectiveness in the elderly using adjuvants, it is imperative to select the appropriate adjuvants based on an understanding of immunosenescence and the mechanisms of adjuvant functions. This review demonstrates the phenomenon of immunosenescence and explores various types of adjuvants, including their mechanisms and their potential in improving vaccine effectiveness for the elderly, thereby contributing to developing more effective vaccines for this vulnerable demographic.

KEYWORDS:

• Vaccines
• adjuvants
• elderly
• effectiveness
• pattern recognition receptors

Introduction

Antibiotic resistance and its burden

The ‘2019 Antibiotic Resistance Threats Report’ from the Centers for Disease Control and Prevention of the USA reported that in 2019 alone, at least 1.27 million people worldwide were killed by Superbugs infections. In the USA, there were more than 2.8 million Superbug infections, killing more than 35,000 people in 2019. “Superbugs” refer to bacteria that are resistant to antibiotics.¹ The UK government has also predicted that if antibiotic-resistant bacteria are not controlled, 10 million people could be killed annually by antibiotic-resistant bacteria, resulting in an associated economic loss of US$100 trillion by 2050.² According to the World Health Organization (WHO)’s priority list of antibiotics resistant bacteria, it is mainly Gram-negative bacteria such as carbapenem-resistant Acinetobacter baumannii and carbapenem-resistant Pseudomonas aeruginosa, but Gram-positive bacteria such as vancomycin-resistant Enterococcus faecium, methicillin-resistant, and vancomycin resistant Staphylococcus aureus is also causing problems.³ Antibiotic resistance evolution has been primarily attributed to the overuse of antibiotics, and it can be easily transferred to other bacteria, thereby threatening global public health.^4,5 In addressing antibiotic resistance, the urgent development of new antibiotics is necessary; however, it is a challenging task. Specifically, the speed at which antibiotic resistance emerges and disseminates surpasses the pace at which new antibiotics are developed.⁶ This implies that diverse strategies must be explored to effectively prevent the occurrence and transmission of antibiotic resistance.

Vaccines as potential arms against antibiotic resistance

Vaccines play a pivotal role in preventing the emergence of antibiotic resistance and curbing its transmission.^7,8 Vaccination can prevent specific bacterial infections, thereby reducing the need for antibiotics in infection treatment.^9–11 Beyond bacterial infections, virus vaccines can prevent viral infections, consequently lessening the need for antibiotics in both the prevention and treatment of secondary bacterial infections.^6,8 Additionally, vaccines exert an indirect influence by diminishing resistant infections in unvaccinated populations through the establishment of herd immunity.¹² Furthermore, vaccines offer long-term protective immunity without inducing resistance, thereby reducing reliance on antibiotics.^13,14 All these contributions of vaccines against antibiotic resistance are documented in the WHO-led value attribution framework.^15,16

Low vaccine effectiveness by immunosenescence in the elderly

With the increase in life expectancy, there has been a significant rise in the proportion of the elderly population. High-income countries are experiencing a rapid aging of the population, and it is expected that between 2015 and 2050, the global population over 60 y old will nearly double from 12% to 22%.¹⁷ This demographic shift has negative implications for both public health and antibiotic resistance, particularly considering the diminished effectiveness of vaccines in the elderly.^18–22 In a study led by Kim C. et al., vaccination was found to be effective in reducing the burden of antibiotic resistance in infections caused by Mycobacterium tuberculosis (Mtb), Streptococcus pneumoniae, and Haemophilus influenzae type B (Hib). Notably, they demonstrated that the reduced effectiveness of vaccines in the elderly contributes to a lesser alleviation of the burden of antibiotic resistance through vaccination in this demographic.¹¹ The compromised vaccine effectiveness in the elderly is associated with an increase in antibiotic use.^23,24 For instance, tuberculosis, caused by Mtb, demands extensive antibiotic use for treatment.²⁵ Tuberculosis treatment necessitates continuous administration of antibiotics, significantly elevating the risk of antibiotic resistance and its potential spread.^11,26 While vaccination has the potential to mitigate the need for antibiotics in treating tuberculosis, the currently approved tuberculosis vaccine (Bacille Calmette-Guerin, BCG) only exhibits a preventive effect in childhood, not in the elderly.^18,25,27 To address the excessive use of antibiotics in the elderly due to tuberculosis,^25,28 strategies to enhance the effectiveness of the tuberculosis vaccine in this demographic must be explored.^26–29 Moreover, many studies have also reported a decrease in viral vaccine effectiveness in the elderly, such as vaccines for SARS-CoV-2 and influenza, which contribute to a high risk of antibiotic resistance by compelling antibiotic use for the prevention and treatment of secondary bacterial infections.^23,24,26

The decline in vaccine effectiveness in the elderly is attributed to immunosenescence – a phenomenon characterized by a diminished immune response, typically observed in individuals over 60 y of age.^19–22 Immunosenescence induces an ineffective immune response to new antigens, reducing the effectiveness of vaccines. The utilization of adjuvants is considered the most effective approach to enhance vaccine effectiveness in the elderly population.^22–30–32 Therefore, extensive research is underway on adjuvants as a strategy to overcome immunosenescence in the elderly and enhance vaccine effectiveness.^19,20,32

Scopes and aims

This review addresses the phenomenon of immune senescence, which reduces the effectiveness of vaccines in the elderly. Furthermore, we will explore adjuvants that can be integrated into vaccines for the elderly to improve their effectiveness by counteracting immune senescence. While numerous adjuvants are currently under development for various vaccine applications, including those targeting young adults, neonates, and cancer, this review exclusively focuses on adjuvants with the potential to enhance the effectiveness of vaccines for infectious diseases in the elderly population. This review primarily discusses results observed in humans, and findings exclusively from animal experiments were specified by a statement indicating the kind of laboratory animals. A comprehensive understanding of immune aging and the mechanisms by which adjuvants function can form the basis for enhancing the effectiveness of existing vaccines and developing new ones. This endeavor ultimately contributes to preventing the emergence and spread of antibiotic resistance by bolstering the effectiveness of vaccines in the elderly.

Immunosenescence

As life expectancy increases, the proportion of adults over 60 y old in the population is growing.¹⁷ The elderly population is vulnerable to various diseases, imposing a substantial burden on the healthcare system. Representative infectious diseases in the elderly include seasonal influenza, pneumococcal infection, and the reactivation of the varicella-zoster virus.²² The high susceptibility of the elderly to infectious diseases results in an increased use of antibiotics, posing a significant challenge in preventing the emergence of antibiotic resistance.^18–22 Vaccination is an effective way to address this issue. Nonetheless, the effectiveness of vaccines is compromised by immunosenescence in the elderly. Therefore, it is imperative to explore ways to increase the effectiveness of vaccines in the elderly, and to achieve this, an understanding of immunosenescence is crucial.^22,33

Immunosenescence is characterized by the diminished ability to respond effectively to either new or previously encountered pathogens, due to a range of declines in immune functions associated with aging.²² Immunosenescence is induced by various causes such as alcohol, smoking, pollution, tissue damage, persistent antigen stimulation, cellular senescence, and epigenetic remodeling.^34–38 Antigens, introduced via pathogen infection or vaccination, are recognized by the innate immune system, initiating the activation of adaptive immunity through antigen presentation. Following this, memory immunity is established for previously encountered antigens. However, in the elderly, efficient immune responses are hampered by persistently low levels of nonspecific induced inflammatory responses. This phenomenon, called ‘inflammaging,’ describes the tendency of aging organisms to experience distinct pro-inflammatory states due to increased levels of pro-inflammatory markers in cells and tissues, and was first introduced by Claudio Franceschi in 2000.^39,40 Furthermore, reduced functionality of B and T lymphocytes, and the formation of memory cells with diminished function could hamper efficient immune responses.^41,42 This immunosenescence renders individuals more susceptible to infections and diminishes vaccine effectiveness.^35,43 Immunosenescence shares numerous functional and phenotypic features with T cell exhaustion. However, given their independent regulatory mechanisms and distinct developmental signatures, this section addresses only the characteristics of immunosenescence that contribute to reduced vaccine effectiveness.^44,45 Numerous adjuvants employ a strategy targeting professional antigen-presenting cells (APCs), such as dendritic cells (DCs), to enhance vaccine effectiveness.^32,46,47 In light of this approach, this section provides a description of immunosenescence of human, with a specific focus on DCs, while also addressing the fundamental immunosenescence of T and B cells.

Immunosenescence of innate immunity

The innate immune system serves as the first line of defense, responding nonspecifically to pathogens or vaccine antigens. Among the various roles of innate immunity, critical functions related to vaccination include the induction and regulation of immune responses through antigen recognition, antigen presentation, and the secretion of various cytokines.⁴⁸ Representative characteristics of immunosenescence in innate immunity include inflammaging due to chronically elevated levels of pro-inflammatory cytokines, diminished activity of antigen recognition, and decreased ability to prime T cells.^21,34,35

Inflammaging is characterized by an antigen-nonspecific inflammatory response, driven by an elevation in pro-inflammatory cytokines such as basal unstimulated levels of interleukin (IL)-6 and tumor necrosis factor (TNF)-α in the elderly.^49,50 Importantly, this increase in pro-inflammatory cytokines is not a response to antigens of innate immunity. Instead, it is induced by pro-inflammatory senescence-associated secretory phenotype factors, including IL-1, IL-6, IL-8, IL-13, IL-18, metalloproteinases, miRNAs, ROS, metabolites, and extracellular vesicles. These factors are linked to cell stress caused by chronic infectious diseases and senescence.^38,51 The activities of nucleotide-binding oligomerization domain (NOD)-like receptor family pyrin domain-containing 3 (NLRP3) increases with aging, and this has been reported as one of the causes of inflammaging in humans.⁵² NLRP3 is a type of pattern recognition receptors (PRRs) in the innate immune system that recognizes pathogen-associated molecular patterns (PAMPs) and danger-associated molecular patterns (DAMPs). Upon activation, NLRP3 triggers the activation of caspase 1, leading to the release of inflammatory cytokines.^53,54 It has been reported that NLRP3 signals increase with aging in both humans and mice.^53–55 Additionally, it has been reported that dysbiosis of the gut microbiome in the elderly has detrimental effects on intestinal permeability and the translocation of bacterial components into the bloodstream, contributing to sustained inflammation.⁵⁶ These alterations in bacterial components in the bloodstream have been confirmed to cause continuous damage to lung tissue, leading to chronic inflammation.^49,57 The increased basal level of TNF-α during inflammaging accelerates the differentiation of premature monocytes in the bone marrow. Consequently, immature monocytes secrete more TNF-α upon antigen stimulation.⁵⁸ It is established that the elevated basal TNF-α level in an inflammaging environment suppresses CD28 expression in CD8⁺ T cells⁵⁹ and diminishes the activity of B cells.⁶⁰ These effects ultimately contribute to a reduction in vaccine effectiveness in the elderly.^49,60

DCs play a crucial role in recognizing antigens and presenting them to the adaptive immune system, thereby initiating T cell immune responses.⁴⁸ The immunosenescence characteristics of DCs remain a topic of debate.⁶¹ While the total cell number of DCs in older adults generally shows no significant change compared to young adults, studies showed different results concerning the decline in the number of circulating plasmacytoid DCs or myeloid DCs.^50,61–66 In addition, DCs isolated from elderly individuals exhibit decreased expression of toll-like receptor (TLR) 1, TLR3, and TLR8, pivotal for cognitive function.^50,62–65 Consequently, the antigen recognition ability and activation level of DCs are diminished. In contrast, there is no observed change in the expression levels of TLR2 and TLR4 with age.^64,66 It has been reported that the expression of TLR7 and TLR9 in DCs is reduced in older adults.^62,64,67 However, for TLR9, it remains unclear whether the expression levels are decreased or conserved.^62,63,67 The compromised TLR function leads to a reduction in the production of type I and III interferons (IFN), interleukins (IL-6, IL-10, IL-12), and TNF-α in DCs.^62,64 For instance, in response to vaccination with Fluzone®, an influenza vaccine, DCs from older adults exhibited lower levels of IL-6, IL-12, and TNF-α expression than DCs from young adults, resulting in lower hemagglutination antibody inhibition (HAI) titers.⁶⁴ In addition, a non-human primate study showed that a protein vaccine using TLR7 and TLR8 ligands as adjuvants induces a high frequency of multifunctional T cell responses producing multiple cytokines simultaneously, indicating that DC activation by TLRs is critical for vaccine efficacy.⁶⁸

DCs isolated from the elderly exhibit reduced responses of co-stimulators (CD80, and CD86) following antigen uptake compared to DCs from young adults.^21,69 Aged DCs also exhibit decreased migratory level to lymph nodes after antigen uptake, leading to an impaired capacity to present antigens to T cell.^31,70 These reduced antigen presentation functions of DCs results in impaired T cell priming and activation.^21,63 It’s worth noting that DCs isolated from older individuals tend to induce the development of aberrant effector CD8⁺ memory T cells (CD45RA⁻ CCR7⁻), which differs from the responses induced by DCs from young adults.⁷¹ Furthermore, the diminished activation of CD4⁺ T cells contributes to reduced levels of T cell-dependent antibody responses, leading to less effective antibody production.⁷² Inflammaging, characterized by pro-inflammatory cytokines that are unrelated to TLR activation by antigens, along with the decreased function of DCs, collectively contribute to the negative impact on immune responses and vaccine hyporesponsiveness in the elderly (Figure 1).

Figure 1. Immunosenescence affects various immune cell functions in response to infection or vaccination. Functions that are reduced due to immunosenescence are indicated by red downward arrows, increased functions are represented by red upward arrows, conserved functions are denoted by green circles with a “C” inside, and functions with unclear changes are marked by black circles with question marks. Inflammaging, characterized by heightened basal inflammation, leads to decreased activation of immune cells. Specifically, CD28 expression on T cells decreases due to the inflammaging. While many functions of dendritic cells (DCs) decline with immunosenescence, the functions of TLR2 and TLR4 are conserved, and NLRP3 functions are increased. Reduced expression and signaling of TLRs, exposure to chronic inflammation, and decreased co-stimulator expression levels of DCs impact the impaired functions of B and T cells. T cells show reduced functionality due to immunosenescence. Decreased co-stimulator expression on DCs and reduced CD28 expression on T cells result in diminished T cell activation. An increased TCR threshold leads to reduced TCR signaling, and a decreased TCR repertoire contributes to the decline in T cell function. A reduced BCR repertoire and decreased BCR signaling negatively affect antigen recognition and differentiation functions of B cells. In particular, decreased CD40L expression on CD4 T cells and reduced CD40 expression on B cells induce diminished activation of B cells, leading to a reduction in antibody production in response to vaccines. These functional declines in DCs, B, and T cells, result in reduced immune responses, making the elderly more susceptible to infections and diminishing the effectiveness of vaccines. Abbreviations: DCs, dendritic cells; TLR, toll-like receptor; NLRP3, nucleotide-binding oligomerization domain (NOD)-like receptor family pyrin domain-containing 3; TCR, T cell receptor; BCR, B cell receptor.

Immunosenescence of T cells

T cells are pivotal components of cellular immunity within the adaptive immune system, primarily categorized into CD4+ and CD8⁺ T cells. CD4⁺ T cells coordinate immune responses by secreting cytokines, while CD8⁺ T cells differentiate into cytotoxic T lymphocytes (CTLs), responsible for eliminating infected cells and producing proinflammatory cytokines in response to infections.^73,74 Both CD4⁺ and CD8⁺ T cells are the main targets for vaccines due to their crucial role in defending against infections.³⁰ Consequently, the decline in T cell function observed in the elderly significantly contributes to the reduction in vaccine effectiveness.^22,30,75

One of the characteristics of immunosenescence of T cells is the loss of surface CD28 expression.^76,77 As a co-stimulator molecule, CD28-mediated signaling is essential for responses of naïve T cells; in the absence of this signal, antigen recognition by the T cell receptors (TCRs) is insufficient for T cell activation.^73,74 CD8⁺CD28^null T cells exhibit reduced activity of perforin and granzyme, essential enzymes for CTL function.^51,78 CD4⁺CD28^null T cells lack the display of CD40L and demonstrate inadequate capacity to promote B cell proliferation and antibody production.^75,79 The high frequency of the CD8⁺CD28^null phenotype within the T cell subpopulation isolated from influenza vaccine non-responders, often observed in the elderly, suggests a decline in vaccine effectiveness attributable to the reduction in CD28 expression on T cells.⁷⁸

Another prominent characteristic of T cell immunosenescence is the reduction in the number of naïve T cells and an increase in the number of differentiated memory T cells.^76,80 Hematopoietic stem cells (HSCs) are responsible for generating both T and B cells.⁸¹ With aging, HSCs exhibit a bias toward the myeloid cell lineage, leading to decreased lymphoid cell generation.⁸¹ Additionally, the thymus undergoes degeneration (atrophy) as individuals age.⁸² These factors may contribute to the reduction in the number of naïve T cells. Studies have reported that homeostatic proliferation of CD4⁺ T cells is insufficient in the elderly, and CD8⁺ T cells experience significant reduction with aging.⁸³ On the other hands, despite changes in HSCs, the number of T cell precursors in the bone marrow does not significantly decrease. Furthermore, thymus atrophy is largely completed in adulthood, and the number of naïve T cells in young adults does not substantially differ from that in adolescence.^75,83–86 These findings suggest that the alteration of HSCs to the myeloid cell lineage and thymus involution may not be the only causes of the decrease in the number of naïve T cells.^75,84,85 Recently, a decrease in the expression of Bim, the proapoptotic molecule, has been identified as a factor contributing to the decline in the number of naïve T cells. Immunosenescent CD4⁺ T cells exhibit reduced Bim expression, extending the longevity of naïve CD4⁺ T cells and leading to the phenomenon known as ‘semi-memory.’ This indicates that naïve CD4⁺ T cells have entered the differentiation pathway, resulting in a decrease in the number of naïve T cells.⁸⁷

The reduction in the number of naive T cells leads to a decrease in the TCR repertoire.^30,35,84 Healthy adults have been shown to possess 2–5 times more TCR repertoire compared to aged individuals.^86,88 The aging-induced limited TCR repertoire reduces the variety of TCRs available to recognize and respond to different pathogens. With this limited repertoire, the immune system’s adaptability to novel threats might weaken, potentially hindering its capacity to mount robust and effective responses against various pathogens. Consequently, this age-related constraint in TCR diversity could impact overall immune function, raising concerns about the body’s resilience against a broad range of infection risks. While the exact mechanisms behind this reduction in TCR repertoire are not entirely clear, studies using mice have confirmed that a limited TCR repertoire can contribute to reduced immune responses to vaccine antigens.⁸⁹ Immunosenescence also leads to a decrease in TCR signaling in T cells.^89,90 One contributing factor is an increase in the threshold for low TCR signaling. In CD4⁺ T cells affected by immunosenescence, there is an elevated intracellular concentration of dual-specific phosphatase (DUSP) 6, which dampens the activation of the extracellular signal-regulated kinases (ERKs). These diminished ERK responses result in an increase in the TCR threshold, making it more challenging for TCR stimulation to generate a productive signal. This, in turn, reduces TCR signaling and impairs the function of CD4⁺ T cells.⁹⁰ Furthermore, some senescent-like T cells express natural killer (NK) cell receptors with reduced TCR function. These NK cell receptors expressing T cells can lead to significant tissue damage, functioning via NK cell receptors rather than specific killing through TCRs.^91,92 This alteration in TCR repertoire and signaling, along with the emergence of T cells with NK cell receptor-like activity, has negative implications for immune responses, including responses to vaccines, in the elderly population.

The response of regulatory T cells (Tregs) also plays a critical role in immune regulation. Tregs are responsible for returning immune responses to their basal levels after pathogens are cleared, and they also help regulate excessive immune responses and induce immune tolerance.^41,42 However, it has been reported that the number of Tregs gradually increases with age, and their activity decreases.^93–95 This Treg dysfunction results in the ineffective secretion of IL-10, contributing to another cause of inflammaging.^93–95 After the clearance of pathogens or the completion of the immune response to vaccine antigens, memory T cells are formed. Memory T cells generated during young adulthood are reported to maintain their function even as individuals age.^75,87 However, memory T cells generated in old age experience a decline in function due to immunosenescence.^{34,35,37,80,96} In the case of memory CD4⁺ T cells generated during old age, an increase in phosphorylated AMPK is observed, leading to elevated levels of DUSP4. This results in reduced expression of ERK and c-Jun N-terminal kinase (JNK), leading to decreased expression of CD40L, CD28, and cytokines. Consequently, memory CD4⁺ cells fail to adequately activate B cells, leading to a reduction in vaccine effectiveness.^75,79 The changes in memory T cell function due to immunosenescence are more significant in CD8⁺ T cells.^75,77 Changes due to immunosenescence result in reduced CD28 expression in memory CD8⁺ cells, leading to decreased T cell activity.^37,96 Overall, the impact of immunosenescence on naive, memory effector, and regulatory T cells eventually leads to diminished vaccine effectiveness in the elderly^43,97 (Figure 1).

Immunosenescence of B cells

In the context of the immune response to vaccines, the production of antibodies plays a critical role. Antibodies neutralize pathogens and prevent diseases; therefore, the level of antibody production has been used as an indicator of protection.^98,99 However, in the elderly, studies have shown lower levels of antibody production and shorter antibody retention following vaccination compared to young adults.^100,101 This reduced antibody response in the elderly has been well-reported in seasonal influenza vaccines,^33,101 pneumococcal polysaccharide vaccines,^100,102 and the zoster vaccine.^103,104 Boosting vaccines are one way to enhance vaccine effectiveness; however, even the use of booster vaccines has shown decreased immune responses in the elderly when compared to young adults.¹⁰⁵ The reduced vaccine effectiveness in the elderly is closely associated with the immunosenescence of B cells.

Immunosenescence in B cells involves a decrease in the number of naïve B cells and a reduction in the B cell receptor (BCR) repertoire. As individuals age, there is a transition of HSCs in the bone marrow toward the myeloid cell lineage, resulting in a decrease in the production of naïve B cells.¹⁰⁶ However, despite this transition, elderly individuals display similar frequencies of plasma cells and memory B cells in response to vaccination when compared to young adults.¹⁰⁶ These findings are consistent with the previous study indicating that the frequencies of pro-B, pre-B, and immature B cells remain relatively stable between the ages of 24 and 88.¹⁰⁷ This highlights the need for a more comprehensive understanding of the mechanisms underlying the decline in naïve B cells with age. In addition, in the elderly population aged 89 and above, spectratype analysis has confirmed a reduction in the BCR repertoire. This reduction is considered a limiting factor in the immune response of B cells to antigens, similar to the impact of changes in T cell receptors.¹⁰⁸ The specific mechanisms for the diminishment observed in B cells are not yet fully understood.³⁰

In comparison to young adults, the elderly cannot induce a sufficient antibody response to vaccination. B cells recognize antigens through BCRs and differentiate into plasma cells to produce antibodies (IgM and IgD). Subsequently, with interactions with follicular helper T cells, antibody class switching occurs, leading to the production of more potent antibodies such as IgG, IgE, and IgA.^73,74 B cells isolated from the elderly display reduced expression of B lymphocyte-induced maturation protein (Blimp)-1, a master regulator of plasma cells, in response to antigen stimulation, indicating a decreased ability of B cells in the elderly to differentiate into plasma cells.¹⁰⁶ Immunosenescent B cells also exhibit reduced responsiveness to BCRs, resulting in decreased B cell activation in response to antigens.^109,110 These immunosenescence phenomena of B cells are considered to be the culprit of the diminished antibody response to vaccination in the elderly, even though older people show a similar frequency of plasma cells and memory B cells compared to young adults when exposed to novel or previously encountered antigens^106,109,110

Unlike DCs, B cells are thought to maintain the function of TLR7 even with aging.^109–111 Both BCR and TLR7 signals, in conjunction with IFN-γ, induce the expression of T-bet in B cells.¹¹² T-bet expression is required for T-dependent class switching to IgG2.^113,114 In research where B cells isolated from the elderly were stimulated with a TLR7 agonist, these cells exhibited increased T-bet expression, similar to young adults.¹¹¹ Normal function of TLR9 in B cells is also observed in the elderly.^109–111 TLR9 signaling is necessary for class switching to IgG2 and plays a crucial role in the proliferation of naïve B cells and their antigen-presenting ability.^115,116 However, many of these roles in plasma cell differentiation and antibody class switching are interconnected with BCR signaling, suggesting that the antibody class switching ability of elderly individuals has decreased due to reduced BCR signals.^{109,110,113,114,117}

In the elderly, memory B cells are observed at levels similar to those in young adults. However, with aging, the frequencies and numbers of switched memory B cells, responsible for driving rapid secondary antibody responses upon re-exposure to the same antigen, decrease. This decrease is accompanied by an increase in the numbers of IgM memory and IgD⁻ CD27⁻ B cells.^106,118 These results once again signify a defect in the class-switching ability of B cells. Despite the reduced ability of B cells to undergo class-switching and differentiate into plasma cells, the responses of B cells in the elderly to new antigens tend to rely on preexisting immune cells. High-throughput parallel sequencing studies of influenza vaccine responses in the elderly revealed that they rely on responses driven by somatic mutations in the memory repertoire rather than new antibody sequences generated by naive B cells.¹¹⁹ This immune response from the preexisting repertoire can also diminish vaccine effectiveness. In conclusion, the decrease in the number of naive B cells, reduction in BCR repertoire, decreased differentiation into plasma cells, and reduced class-switching ability collectively contribute to the reduced vaccine effectiveness in the elderly (Figure 1).

Adjuvants addressing enhancement of vaccine effectiveness in the elderly

The innate immune system recognizes PAMPs, which are conserved molecules on pathogens, and DAMPs released from damaged cells in response to pathological conditions.^41,42 The recognition of PAMPs and DAMPs by the innate immune system is mediated by PRRs.^120,121 Activation of innate immunity through PRRs is essential, as it leads to antigen presentation and plays a significant role in the immune response to vaccines.¹²² Adjuvants used to enhance vaccine effectiveness aim to activate PRRs on APCs within the innate immune system, thereby promoting effective adaptive immune responses to vaccination.^122,123 Therefore, when designing vaccines that exhibit high effectiveness in the elderly, it would be beneficial to use adjuvants that target PRRs on APCs whose function and expression have not diminished due to immunosenescence. Research on the decreased function of innate immunity due to immunosenescence has reported that some of TLRs and NLRP3 maintain their function and expression levels even with aging. Moreover, several recent studies on adjuvants are focusing on targeting TLRs and NLRP3 (Table 1). In this section, we discuss major adjuvants targeting TLRs and NLRP3 that have been reported to retain their functionality in the elderly, based on their activation pathway.

Table 1. The number of kinds of adjuvants used in vaccines currently under developments for major diseases affecting the elderly (Varicella-zoster virus, VZV; Influenza A; Streptococcus pneumoniae, S. pneumoniae) [22], emerging antibiotic resistance threats (Clostridioides difficile, C. difficile; Neisseria gonorrhoeae, N. gonorrhoeae; Klebsiella pneumoniae, K. pneumoniae; Shigella sp.) [26], and pathogens under active vaccine research (Respiratory Syncytial Virus; RSV, Hepatitis B virus, HBV, Mycobacterium tuberculosis, Mtb; SARS-CoV-2) [29, 47, 281] are classified based on their target PRRs. The numbers listed in the table represent the number of types of adjuvants classified based on the targeting Toll-like receptors and immune profiles. Please note that there was no information available on adjuvant developments associated with Acinetobacter baumannii, Candida auris, and Enterobacteriaceae, despite their listing as emerging antibiotic resistance threats by the Centers for Disease Control and Prevention. (https://vac.niaid.nih.gov/, last accessed on January 20, 2024).

		Target PRRs												Immune profiles (Th responses)
		3	4	7	9	7/8	2/4	4/7	4/7/8	4/NLR	NLR	O	U	1	2	1/2	17	1/17	1/2/17	U
VZV	Pre		1										1	1		1
	Clinic		1											1
Influenza A	Pre	1	8	1		5		1	1			1	2	8		9		1	2
	Clinic	1	1		1		1				1	2	2	3		4		1	1
S. pneumoniae	Pre												1			1
	Clinic											1							1
RSV	Pre	1												1
	Clinic		1		1								1	2		1
HBV	Pre											1				1
	Clinic	1			1							2		1		2			1
Mtb	Pre		1									1	1	1		1	1
	Clinic		4		1	2						1	1	6		2		1
SARS-CoV-2	Pre		8		1	2			1		1		4	5		9		1		2
	Clinic				1	3				1			5	6		3		1
N. gonorrhoeae	Pre
	Clinic		2											2
C. difficile	Pre											1		1
	Clinic											1							1
S. aureus	Pre		1													1
	Clinic											1				1
K. pneumoniae	Pre											1							1
	Clinic
Shigella sp.	Pre
	Clinic											1							1

Activation pathways for TLRs and NLRP3

TLRs that are expressed on APCs play a crucial role in recognizing conserved PAMPs, leading to the activation of APCs and subsequent induction of adaptive immunity through the activation of innate immunity.^{41,42,120–122} Various adjuvants targeting TLRs have shown effective results in enhancing vaccine effectiveness.^124,125 TLR1, TLR2, TLR4, TLR5, and TLR6 are expressed on the cell surface. They primarily recognize microbe-derived lipopeptides, peptidoglycan, lipopolysaccharides (LPS), and flagellin, leading to inflammatory responses.¹²⁶ On the other hand, TLR3, TLR7, TLR8, and TLR9 are expressed in intracellular vesicles. They recognize double-stranded RNA (dsRNA), single-stranded RNA (ssRNA), and CpG DNA, inducing Type I IFN responses and inflammatory reactions.¹²⁷ Type I IFN promotes the differentiation of Th1 cells and facilitates antigen cross-presentation to CD8⁺ T cells¹²⁸ (Figure 2).

Figure 2. TLR activation pathways. TLRs play a crucial role in the recognition of microbial components and the initiation of immune responses. TLR1, TLR2, TLR4, TLR5, and TLR6 are expressed on the cell surface, where they primarily recognize microbe-derived lipopeptides, peptidoglycan, lipopolysaccharide (LPS), and flagellin, leading to inflammatory responses. TLR1 and TLR6 form heterodimers with TLR2 to execute their functions. TLR3, TLR7, TLR8, and TLR9 are located in intracellular vesicles, where they recognize dsRNA, ssRNA, and CpG DNA. MyD88 is a common signaling molecule utilized by all TLRs except TLR3. TLR3 uses TRIF as its signal transducer. TLR4 signaling molecule changes from MyD88 to TRIF once TLR4 relocates to endosomes. Upon binding to TLR ligands, MyD88 becomes activated, leading to the formation of the myddosome. Subsequently, the TAK1 complex and IKK complex are activated, which induces NF-κB. Activation of MyD88 by TLR2 results in the activation of MAPKs, leading to the activation of ERK. This, in turn, results in the induction of cFos protein and the activation of the transcription factor AP1. Activation of MyD88 by TLR7, TLR8, and TLR9 ligand binding can directly induce IRF7. TLR3 and TLR4 activation through TRIF leads to the induction of TRAF6 and TRAF3. TRAF3 activates TBK1 and IKKi and induces the transcription factor IRF3. Additionally, TRAF6 can activate TAK1 and IKK. Activation of various TLRs results in the activation of specific transcription factors, which influence the immune profile. Transcription factor AP1 promotes Th2-biased immune responses by stimulating the production of anti-inflammatory cytokines. NF-κB promotes the secretion of pro-inflammatory cytokines, inducing Th1-biased immune responses. Transcription factors IRF3 and IRF7 promote the secretion of type I IFN cytokines, along with the activation of CTL, contributing to Th1-biased immune responses. Abbreviations: TLR, toll-like receptor; ssRNA, double-stranded RNA; MyD88, myeloid differentiation factor 88; TRIF, TIR domain-containing adapter-inducing interferon-β; TAK1, transforming growth factor-β-activated kinase 1; IKK, IκB kinase; NF-κB, nuclear factor kappa-light-chain-enhancer of activated B cells; MAPKs, mitogen-activated protein kinases; ERK, extracellular signal-regulated kinase; AP1, dimeric transcription factor complex activator protein-1; IRF, induce interferon regulatory factor; TRAF, TNF receptor-associated factor; TBK, TANK-binding kinase; CTL, cytotoxic T lymphocytes.

TLR2 forms heterodimers with either TLR1 or TLR6 to recognize lipopeptides. TLR5, which is expressed on the cell surface, detects flagellin, and TLR4 senses LPS.^120,121 Upon ligand binding, TLR2/1, TLR2/6, TLR4, and TLR5 activate myeloid differentiation factor 88 (MyD88), which subsequently assembles Myddosome composed of MyD88, IL-1 receptor-associated kinases (IRAK) 4, and IRAK1/2.¹²⁶ Myddosome activates the transforming growth factor-β-activated kinase (TAK) 1 complex, which, in turn, activates the IκB kinase (IKK) complex, leading to the activation of the transcription factor, nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB).^120,129,130 NF-κB promotes the secretion of pro-inflammatory cytokines and induces Th1 immune responses.^46,120,131 The activation of MyD88 by TLR2 ligand binding leads to the activation of mitogen-activated protein kinases (MAPKs). However, it is not clear whether the activation of MAPKs by TLR2 is connected to the pathways of TAK1 or IKK.^120,132,133 Nonetheless, it is established that MyD88 activation by TLR2 ligand binding results in the activation of MAPKs, subsequently leading to the activation of ERK. This, in turn, triggers the production of cFos protein and induces the transcription factor activator protein 1 (AP1),^{46,120,126,134} leading to a decrease in IL-12 and the promotion of IL-10 expression.^120,133,134

TLR7, TLR8, and TLR9, upon binding nucleic acid ligands, activate MyD88 and subsequently form Myddosome. They activate NF-κB through a pathway similar to TLR2/1, TLR2/6, and TLR5.¹³⁵ Additionally, the Myddosome generated by TLR7, TLR8, and TLR9 signaling can directly interact with interferon regulatory factor (IRF) 7, leading to the activation of IRF7. IRF7 promotes the induction of Type I IFN.^46,120,131 Activation through TLR7, TLR8, and TLR9 results in the increased secretion of pro-inflammatory cytokines and Type I IFN, leading to Th1 immune responses and cytotoxic CD8⁺ T cell immune responses.^{46,120,127,128,131}

MyD88 is a common signaling molecule used by all the TLRs except TLR3. TLR3 utilizes toll-interleukin-1 receptor domain-containing adapter-inducing interferon-β (TRIF) as its signal transducer.¹²⁰ Furthermore, TLR4 signaling transitions from MyD88 to TRIF once TLR4 relocates to endosomes.¹³² The engagement of TRIF through ligand binding of TLR3 and TLR4 recruits TNF receptor-associated factor (TRAF) 6 and TRAF3. TRAF6 activates the TAK1 complex and IKK complex, subsequently activating NF-κB. Furthermore, TRAF6 derived from TLR3 signaling is known to activate induce IRF7.^136,137 TRAF3 activates TRAF-Associated NF-κB Activator-binding kinase (TBK) 1 and IKKi, and these kinases, through phosphorylation, activate IRF3.^46,120,133 IRF3 promotes the production of Type I IFN, thereby inducing Th1 immune responses and cytotoxic CD8⁺ T cell immune responses.^{46,120,127,128,131}

NOD-like receptors (NLRs) are cytosolic receptors that recognize PAMPs and DAMPs, thereby activating innate immunity.^53,54 Three important NLRs are nucleotide-binding oligomerization domain (NOD) 1, NOD2, and NLRP3. Among these, NLRP3, upon binding to its ligand, assembles the NLRP3 inflammasome, which, in turn, activates caspase-1. The activated caspase-1 converts pro-inflammatory cytokines into biologically active cytokines, thereby promoting inflammatory immune responses.⁴⁶ Overall, NLRP3 activation also stimulates the expression of MHC II in APCs and induces the secretion of IL-1β and IL-18, leading to Type 2 immune responses.^138,139

Lipopolysaccharide

LPS are endotoxins found on the outer membrane of Gram-negative bacteria. They activate immune cells by binding to TLR4 and myeloid differentiation proteins 2. Immune cells stimulated by LPS secrete inflammatory cytokines, leading to innate and Th1-biased immune responses.¹⁴⁰ LPS itself has inherent toxicity due to its endotoxic nature. To use it as an adjuvant, modifications are needed to maintain its immunostimulating effects while reducing its toxicity. Notable examples of modified LPS adjuvants include monophosphoryl lipid A (MPLA or MPL; MPL is a clinical-grade version of MPLA manufactured by GlaxoSmithKline) and glucopyranosyl lipid A (GLA).¹⁴⁰ Both MPLA and GLA stimulate TLR4, enhancing the effectiveness of vaccines. TLR4 expression is known to be similar in APCs from both the elderly and young adults, making them promising candidates for vaccine adjuvants in the elderly.^64,66

MPLA is an agonist of TLR4, which activates APCs and promotes Th1 immune responses through NF-κB by inducing the secretion of pro-inflammatory cytokines.^140,141 While MPLA showed significantly less toxic than LPS in the mouse model, it still possesses low-level toxicity.¹⁴² Interestingly, when MPLA is combined with liposomes and used as an adjuvant, it exhibits immunostimulating effects without toxicity.¹⁴³ MPLA is currently approved as a human vaccine adjuvant in the US and Europe. It is used in combination with aluminum in adjuvants for hepatitis B vaccines (Fendrix®) and human papillomavirus vaccines (Cervarix®), leading to enhanced antibody production and the activation of antigen-specific T cells.¹⁴⁴

GLA is an alternative to MPLA and serves as a synthetic LPS mimic. GLA primarily activates TLR4 in APCs, leading to the induction of Th1 immune responses.^145,146 GLA comes in various formulations, including GLA-aqueous nanosuspension (GLA-AF), GLA-stable emulsion (GLA-SE), GLA-liposome (GLA-LS), and GLA-aluminum hydroxide (GLA-alum).¹⁴⁰ Among these, GLA-SE, which is a form of squalene oil-in-water emulsion, has been extensively researched.⁴⁷ GLA-SE is known for inducing Th1 responses as well as a balanced IgG1/IgG2 response. It also promotes the expression of IFN-γ by NK cells and the formation of memory CD8⁺ T cells in the mouse model.^145,146 GLA-SE induced Th1-type CD4⁺ T cell responses and generated higher titers of Th1-type antibodies in H5N1 subunit vaccine experiments using mice and ferrets.¹⁴⁷ Coler et al. reported that GLA-SE induces the production of neutralizing antibodies against various influenza viruses in mice and non-human primates as a part of influenza vaccines.¹⁴⁸ Notably, GLA-SE promotes the expression of antigen-specific IFN-γ, leading to a significantly increased IFN-γ:IL-10 ratio (in mice).¹⁴⁸ Considering that GLA-SE activates APCs through TLR4 and induces IFN-γ production,¹⁴⁹ GLA-SE appears to have great potential as an adjuvant for elderly vaccines. Additionally, while GLA-SE did not yield significant prevention results in elderly individuals in respiratory syncytial virus vaccine experiments,¹⁵⁰ it has shown improved vaccine effectiveness along with enhanced APC function in the elderly, reaffirming its potential as an adjuvant for vaccines designed for the elderly.⁴⁷

Aluminum

Aluminum was introduced as an adjuvant in human vaccines in the 1930s, particularly in diphtheria and tetanus vaccines, and has demonstrated an excellent safety profile.^151,152 The most widely used forms of aluminum in vaccines are aluminum hydroxide and aluminum phosphate.¹⁵³ Aluminum, as a mineral-based adjuvant, serves as a short-term antigen depot, allowing for the slow release of antigens into the immune system.¹⁵⁴ This enables sufficient antigen exposure for APCs, thereby enhancing vaccine effectiveness. Additionally, aluminum is known to act as an immunostimulant by activating NLRP3 in APCs, further augmenting the immune response to vaccines.¹⁵⁵

The mechanism by which aluminum activates NLRP3 is a subject of debate. However, through studies using mice, it can be believed that when cells at the injection site die, host DNA or uric acid is released, and these DAMPs are thought to activate NLRP3 in APCs.^156,157 NLRP3 activation results in the secretion of IL-1β, IL-18, and IL-33, leading to the induction of Th2 immune responses.^{154,155,158,159} However, aluminum has a very low capacity to induce cell-mediated immunity (CMI) and is non-biodegradable, potentially causing nervous system disorders when administered to patients with renal failure.^160,161 Despite these concerns, aluminum remains widely used as an adjuvant in many vaccines, including those for hepatitis A, Influenza A, and meningococcal Group 3.^155,158 Considering that aluminum might activate APCs via the NLRP3 pathway and that NLRP3 function is enhanced by immunosenescence,^52–54 aluminum has a high potential as a vaccine adjuvant for the elderly. However, the actual use of aluminum adjuvants alone does not seem to effectively induce immune responses in the elderly.⁴⁷ This underscores the need for further research on the mechanisms of action of aluminum, as well as the immunosenescence mechanisms in DCs.

CpG ODN

CpG ODN, synthetic oligodeoxynucleotides (ODNs), is a single-stranded DNA molecule that includes unmethylated cyclic di-GMP (CpG) motifs, structurally resembling bacterial DNA.^162,163 CpG ODNs interact with TLR9 on immune cells, including DCs and B cells, thereby enhancing innate immune responses. TLR9 activation by CpG ODN leads to the secretion of Type I IFN and pro-inflammatory cytokines, ultimately resulting in the stimulation of humoral immunity, Th1-type cellular responses, and the production of cytotoxic T cells.^163,164 Additionally, CpG ODNs promote the expression of CD40 and CD80 on DCs and facilitate the secretion of IFN-γ by NK cells.^165,166 Among CpG ODNs, only CpG ODN 1018 is approved by the FDA as an adjuvant for human vaccines. It has been evaluated as an adjuvant for SARS-CoV-2 vaccines, leading to the induction of Th1 immune responses and the generation of elevated levels of neutralizing antibodies in mice.¹⁶⁷ Furthermore, the Hepatitis B virus vaccine (Heplisav-B®) that incorporates CpG ODN 1018 as an adjuvant has demonstrated diverse effects on the immune system, triggering B cell proliferation and the production of immunoglobulins. It also stimulates the secretion of various cytokines such as IL-6, IL-12, and IL-18, along with increased secretion of IFN-γ by NK cells. Notably, the inclusion of CpG ODN 1018 significantly enhances both the proportion and intensity of individuals achieving seroprotection. Enhanced immune responses, such as seroprotection rates, have also been confirmed even in the elderly, and it has been approved by the FDA for commercial use.^168,169

The alteration of TLR9 expression due to immunosenescence remains a subject of debate in humans.^62–64,67 Some studies have reported decreased TLR9 expression on DCs in the context of immunosenescence,⁶⁷ while others have suggested that TLR9 may decrease only slightly. Furthermore, it’s been proposed that TLR9 might not decrease in plasmacytoid DCs as a result of immune aging.^62–64 Therefore, there is a possibility that CpG ODN can activate APCs through TLR9 in aged DCs. Furthermore, CpG ODN promotes the expression of co-stimulators in DCs, while aged DCs exhibit reduced expression of co-stimulators.^21,69 These characteristics of CpG ODN suggest a high potential for its use as an adjuvant in vaccines for the elderly. Another interesting point is that CpG ODN can activate TLR9 in B cells. It’s worth noting that TLR9 maintains its expression even in aging B cells.^109–111 TLR9 plays a crucial role in various aspects of B cell function, including B cell activation, plasma cell differentiation, antibody class switching, and antibody production.^{109–110-112–117} Furthermore, it is assumed that inducing the expansion of CD27⁺ IgM memory B cells via the TLR9 activation of B cells may partially alleviate the defect in class-switching attributed to CD27⁻ B cells in immunosenescence.¹⁷⁰ Therefore, CpG ODN can activate TLR9 in both APCs including B cells, potentially enhancing vaccine effectiveness in the elderly population.

In addition, CpG can also enhance the efficacy of vaccines when used in conjugation with other adjuvants.^47,155,171 Recently, the combination of aluminum and CpG 1018 as adjuvants for a recombinant protein vaccine against SARS-CoV-2 has received FDA approval for human use.^167,171 Interestingly, mice and hamsters vaccinated with the SARS-CoV-2 spiking protein using the combination adjuvant of aluminum and CpG 1018 induced Th1-dominant responses, resulting in high levels of neutralizing antibodies, broad-spectrum protection, and a high ratio of IFN-γ/IL-4, despite the presence of aluminum.^167,172,173

Saponins

Saponins are natural substances containing glycosides of steroids, steroid alkaloids, and triterpene compounds. They act on cell membranes and enhance immune responses.¹⁷⁴ Extracts from Quillaja saponaria, such as Quil-A, and their derivatives, are highly regarded as adjuvants.¹⁷⁵ Among these, one fraction of Quil-A, QS-21, is actively researched as a vaccine adjuvant.¹⁷⁵ Despite numerous studies proposing the mechanism of QS-21 through mouse experiments, the exact mechanism has not been clearly established; however, it has been found that it does not act by binding to TLR2 and TLR4 of DCs.¹⁷⁶ Several studies using mice have suggested that the mechanism of action of QS-21 involves the activation of NLRP3 by the carbohydrate region of QS-21.^177,178 Activated NF-κB by NLRP3 in APC leads to the secretion of pro-inflammatory cytokines such as IL-1β, IL-6, IL-12, and IL-18, promoting Th1, Th17, and CTL responses.^{175,177,179–181} The study using mice has also shown that QS-21 not only promotes Th1 immune responses but also Th2 immune responses, resulting in the production of IgG1 and an improved Th1/Th2 balance.¹⁸² In addition to its immunostimulating effects, QS-21 has been found to enhance antigen uptake due to its high affinity for endosomal membrane cholesterol.^177,178 This high cholesterol affinity induced endosomal escape of antigens following uptake, thereby promoting cross-presentation and CTL responses.^178,182 Moreover, it was reported that QS-21 enters APC cells directly through cholesterol-dependent endocytosis, and its high affinity for endosomal membrane cholesterol destabilizes lysosomes within APCs, leading to inflammasome activation and an increased immune response to vaccines.¹⁸³ Research utilizing synthetic QS-21 has demonstrated its ability to promote antigen trafficking by APCs from the injection site to the draining lymph nodes in mice.¹⁷⁶ However, whether natural extract QS-21 has the same effect remains to be confirmed.¹⁸² QS-21, with its various vaccine-enhancing effects, is often used in combination with other delivery systems because it does not have a direct antigen depot effect.^184,185 Notably, an adjuvant form frequently used in combination with QS-21 is AS01 (containing MPLA and QS-21 in liposomes). AS01 has been utilized in the development of vaccines for malaria (Mosquirix®),¹⁸⁶ tuberculosis,¹⁸⁷ and zoster (Shingrix®),²⁰ showing enhanced cellular immunity and high levels of antibody production. The immunostimulating abilities of saponin analogs like QS-21 suggest their high potential as vaccine adjuvants for the elderly, particularly since they appear to function through pathways other than TLRs, which are known to be diminished by immunosenescence.^{175,177–179,183} Furthermore, the abilities of QS-21 to induce a combination of Th1, Th2, and Th17 immune responses and promote CTL activation through cross-presentation are significant advantages.^{175,177–182} However, the mechanisms behind the immunostimulating effects of QS-21, including other saponin analogs, are not yet fully established.^63,180,182

Matrix-M is an adjuvant composed of new saponin analogues extracted from Quillaja saponaria, forming nanoparticles of 40 nm with cholesterol and phospholipids.¹⁸⁵ Matrix-M has been reported to share a mechanism similar to other saponin analogues, eliciting Th1-dominant immune responses with a Th1/Th2 balance and enhancing immune cell trafficking. Matrix-M is known to activate innate immune cells through the inflammasome pathway and activate CD8⁺ T cells through antigen cross-presentation.^183,185,188 The ability of Matrix-M to heighten immune responses allows the reduction in antigen amount without compromising vaccine efficacy.¹⁸⁸ It also exhibits high stability at 2–3°C,^184,185,189 and mouse experiments have substantiated its capacity to augment CD86 expression in DCs.¹⁹⁰ Moreover, in mouse experiments employing Matrix-M-adjuvanted influenza HA recombinant protein vaccination, there was reported augmentation in the numbers of B cells and DCs in draining lymph nodes.¹⁸⁴ These findings suggest the potential of Matrix-M as an adjuvant that could enhance vaccine efficacy in the elderly by partially mitigating the reduced expression of co-stimulators in DCs and the decreased number of B cells caused by immunosenescence. This adjuvant has been used successfully in the Novavax Covid and influenza vaccines.^191,192

MF59

MF59 is an oil-in-water emulsion adjuvant composed of squalene oil, Tween 80, and Span 85.¹⁹³ MF59 has been widely used as an adjuvant since its approval for use in influenza vaccines by the FDA in 1992.¹⁹⁴ MF59 functions as an antigen delivery system, allowing antigens to interact with APCs for an extended period, thereby increasing vaccine effectiveness.^193,195 Additionally, MF59 has been found to acts as an immunostimulant by activating PRRs, thus enhancing the immune response to antigens through the activation of innate immunity in mice.¹⁹⁶ Through these mechanisms, MF59 activates macrophages and DCs, promoting the secretion of chemokines such as C-C motif chemokine ligand (CCL) 2, CCL4, CCL5, and C-X-C motif chemokine ligand (CXCL) 8. The secretion of these chemokines leads to the migration of more immune cells to the vaccination site, increasing the exposure of antigens to more immunocytes.^194,197 The precise mechanism of action of MF59 is not yet fully understood; however, it is known to induce endogenous danger signals to PRRs and activate APCs through the NLRP3-independent apoptosis-associated speck-like protein containing a CARD (ASC) activation pathway and TLR-independent MyD88 activation pathway. Activated APCs promote the secretion of IL-1β and IL-18, leading to the induction of a Th2-biased immune response and weak Th1 responses.^155,198,199 This implies that MF59 may have the potential to activate APCs even when TLRs are functionally reduced due to immunosenescence.^52–54 Furthermore, MF59, by promoting the secretion of chemokines such as CCL2, CCL4, CCL5, and CXCL8, facilitates the migration of APCs to lymph nodes, mitigating the reduced migratory levels of APCs associated with immunosenescence.^{31,70,194,197} Notably, the largest systematic review and meta-analysis have confirmed that MF59 can enhance antibody titers in individuals aged 60 and older when used as an adjuvant in influenza vaccines.²⁰⁰ Additionally, it has been reported to augment vaccine effectiveness in preventing influenza-related medical encounters in individuals aged 65 and older.^47,200

Combination adjuvants

Each adjuvant functions through its unique mechanisms to enhance vaccine immunity. Therefore, researches aiming to combine these adjuvants to achieve a synergistic effect and further enhance vaccine effectiveness are actively underway. A prominent adjuvant combination system is the adjuvant systems (AS) developed by GlaxoSmithKline. AS combines delivery systems and multi-immunostimulants. To date, AS01, AS02, AS03, and AS04 have been developed. Among these, AS01, AS03, and AS04 are currently used in commercial vaccines.²⁰¹

AS01 is a saponin-liposome adjuvant system composed of MPLA, a TLR4 agonist, and QS-21.²⁰² Within AS01, the liposome serves as a delivery system, protecting antigens from degradation and extending their bioavailability. This enables APCs to recognize more antigens and keeps the antigen exposed to the immune system for a longer duration.²⁰² On the other hand, AS02 is a water-in-oil emulsion adjuvant that combines MPLA and QS-21.²⁰¹ MPLA stimulates APCs via TLR4, promoting the expression of cytokines and co-stimulatory molecules, as well as the secretion of IL-1β and IL-18, leading to the induction of Th1 responses.^140,141 Additionally, QS-21 induces antigen endosomal escape within APCs, promoting CTL responses and antigen-specific antibody responses.²⁰² Furthermore, studies in mice have reported a synergistic effect of MPLA and QS-21 in stimulating the secretion of IFN-γ by APCs.²⁰²

The components of AS01 and AS02, MPLA and QS-21, are known to act through pathways that have not been reported to associated with immunosenescence.^{64,66,177,178,180,181} AS02 is a water-in-oil emulsion, which is also known to enhance both humoral and cellular immune responses.²⁰³ AS01, in particular, has been approved by the FDA and has demonstrated its potential as an adjuvant for vaccines in the elderly in previous studies,^20,187 Notably, the Shingles vaccine (Shingrix®) for Herpes zoster, which can be problematic in the elderly, uses AS01 as an adjuvant and has been approved by the FDA.^103,204

AS03 is a squalene-based oil-in-water emulsion adjuvant that contains DL-α-tocopherol and Tween 80.¹⁵⁵ The mechanism of vaccine effectiveness for AS03 is similar to that of MF59. AS03 is an emulsion that slowly releases antigens to APCs, allowing them to be exposed to immune system for an extended period.¹⁵⁵ Additionally, mouse experiments showed that squalene’s immunostimulating effects activate the NLRP3-independent ASC pathway and the TLR-independent MyD88 pathway, enhancing the immune response to the vaccine.¹⁵³ In addition to squalene, α-tocopherol also possesses immunostimulating effects by promoting the secretion of CCL2, CCL3, IL-6, and CXCL1 in APCs, leading to the recruitment of more immune cells to the injection site. This, in turn, promotes the migration of APCs to the draining lymph nodes, countering the reduction in migratory levels due to immunosenescence in humans.^31,70,205 Through these effects, AS03 induces Th2-biased immune responses with weak Th1 responses.^155,206 AS03 is currently used as an adjuvant in approved influenza vaccines in the United States and Europe and has been recently explored as an adjuvant for SARS-CoV-2 vaccines.²⁰⁷

AS04 is an aluminum-based adjuvant that contains the TLR4 agonist MPLA.^144,208 Aluminum components promote the activation of NLRP3, leading to the secretion of IL-1β and IL-18 and the induction of Th2-biased immune responses.^{154,155,158,159} Additionally, MPLA activates NF-κB, inducing Th1 immune responses.^140,141,201 The combined effects of aluminum and MPLA result in AS04 inducing balanced Th1/Th2 immune responses.²⁰¹ Therefore, AS04 appears to function effectively in immunosenescence conditions via TLR4 and NLRP3, making it a promising adjuvant for vaccines targeting the elderly. AS04 is currently used as an adjuvant in human papillomavirus vaccines (Cervarix®) and hepatitis B virus vaccines (Fendrix®).¹⁴⁴

Delivery system as adjuvants

A vaccine delivery system includes carrier materials designed to protect vaccine antigens and facilitate their transport to target immune cells. The central objectives of a delivery system are to extend the bioavailability of the antigen, ensure prolonged exposure to APCs, and facilitate antigen uptake by APCs,^171,209 thus enhancing vaccine effectiveness and reducing the required antigen dose.^171,209 To ensure the optimal use of a delivery system for each vaccine, it’s crucial to consider aspects such as prolonging antigen bioavailability, enhancing APC uptake, facilitating lymph node trafficking, and promoting antigen presentation.⁴⁶ Notably, liposomes and virus-like particles (VLP), which are prominent among various delivery systems, are briefly described, along with adjuvants that can address immunosenescence.^{46,171,210–212}

Liposomes are spherical vesicles composed of a hollow phospholipid bilayer artificial membrane.^210,213 These phospholipids have a hydrophilic head and a hydrophobic tail, which make them ideal for encapsulating and delivering antigens, rendering them valuable as an adjuvant system.²⁰² Water-soluble antigens are encapsulated within the aqueous inner side of liposomes, while lipophilic antigens are situated in the lipid bilayer.²¹⁴ Liposomes serve to prevent antigen degradation and extend bioavailability, enabling APCs to recognize more antigens, consequently enhancing the vaccine’s effectiveness.²⁰² The adjuvant properties of liposomes depend on factors such as the type of encapsulated antigens and the lipid composition of the bilayer, leading to the investigation of various liposomal formulations as potential adjuvants.²¹⁴ As adjuvants, liposomes are known to induce robust humoral and cellular immune responses²¹⁴ and facilitate antigen cross-presentation²¹⁵ in animal models. Cationic adjuvant formulation, CFA01 (or CAF01), is a typical nano-scaled liposome containing N,N’-demethyl-N,N’-dioctadecylammonium (DDA) with the synthetic mycobacterial immunomodulator α,α’-trehalose 6,6’-dibehenate (TDB) inserted into the lipid bilayers.²¹⁴ Through mouse experiments, TDB is found to enhance vaccine effectiveness by interacting with the c-type lectin Mincle on APCs²¹⁴ and promoting Th1 and Th17 immune responses.^216,217 Additionally, mRNA vaccines delivered by lipid-based nanoparticles promote follicular T cell and germinal center formation.^218–220 The ability to induce the formation of germinal centers and stimulate immune memory is essential, especially in populations where immune responses might naturally decline with age. Recently, SARS-CoV-2 mRNA-1273 using this lipid-based nanoparticle has proven immunogenicity and safety in elderly people.²²¹ Although the formation of follicular T cell and germinal center formation have been not described directly, the SARS-CoV-2 mRNA-1273 vaccine induced high levels of binding and neutralizing antibodies in older adults, with time- and dose-dependent trends²²¹ and responses similar to those in younger adults.²²²

VLPs are a delivery system of virus-derived structures, which have a size of 20–100 nm and the form of native virus particles.^212,223 The nano-size of VLPs is optimal for facilitating the movement of antigens to lymphatic vessels and lymph nodes, and their resemblance to native virus structures makes them readily taken up by APCs.²²⁴ VLPs can be manufactured through various expression systems, including mammal cell lines, plants, bacteria, yeast, and insect cell lines. However, achieving human-optimized post-translational modifications for optimal immunogenicity and preventing contamination, such as by LPS, can be challenging.²²⁵ Nevertheless, VLPs have been confirmed to enhance the effectiveness of vaccines as adjuvants in animal experiments. VLPs, with their self-assembled structural capsid proteins closely resembling native viruses and efficient display of vaccine antigens on their outer surface, effectively enhance uptake by APCs, thereby improving vaccine effectiveness.^212,225 Furthermore, VLPs can express multivalent antigenic epitopes on their surface, allowing for the effective activation of B cells through extensive cross-linking of BCRs.²²⁶ Currently, VLPs are utilized as adjuvants in vaccines such as Cervarix® and Gardasil® for human papillomavirus and Sci-B-Vac^TM for hepatitis viruses, which are available on the market.^{46,171,211,212,227}

Safety and reactogenicity

The adjuvants described in this review are reported to act through the activation of PRRs. Such adjuvants, by activating the innate immune system, can induce not only adjuvant-associated local reactogenicity but also life-threatening systemic reactogenicity.²²⁸ Aluminum can induce IL-1 and Th2 responses, leading to increased IgE production, which may trigger allergy and anaphylaxis (in animal models).^229–232 Additionally, an increase in aluminum concentration can impact the brain and bone tissue, leading to neurological syndrome and dialysis-associated dementia in humans.^233,234 Squalene-based adjuvants such as MF59, AS01, and AS02 are known to have high levels of reactogenicity.²²⁸ Generally, autoimmune responses are a major concern for adverse reactions caused by these oil emulsion adjuvants, both in humans and animals.^235–237 In the elderly population, the utilization of MF59 as an adjuvant for influenza recombinant protein vaccine resulted in a higher incidence of local adverse effects (redness, swelling, and pain) and systemic adverse effects (headache and malaise) in comparison to virosomal vaccines.²³⁸ AS04 also exhibited local and systemic reactogenicity in healthy individuals aged 14–40.²³⁹ QS-21 demonstrates reactogenicity, leading to hemolysis in vitro,²⁴⁰ and is recognized for inducing pain at the injection site when employed as an adjuvant.²⁴¹ Nevertheless, this reactogenicity has been documented to diminish when QS-21 is utilized in combination with a delivery system of cholesterol or liposomes.^202,242 LPS has inherent reactogenicity and can disrupt the immune tolerance balance by inducing potent IL-17.²⁴³ Therefore, MPLA and GLA have been developed to reduce such reactogenicity.¹⁴⁰

The clinical symptoms due to the reactogenicity of these adjuvants are mild in most cases. Local toxicity such as pain, redness, inflammation, swelling, abscesses, and nodules at the vaccination site typically resolve within a few days.²²⁸ Additionally, adjusting the dosage of adjuvants can reduce the likelihood of reactogenicity.^244,245 Nonetheless, it is crucial to meticulously weigh the reactogenicity of adjuvants against the impact of bolstering vaccine effectiveness.²⁴⁶ Notably, understanding of adjuvant reactogenicity and safety in the elderly is still poor. Given the potential variability in adjuvant reactogenicity based on health status and age,^228,247 with a tendency for heightened concerns in the elderly population,²⁴⁸ further research is needed on adjuvant-associated reactogenicity in the elderly.

Adjuvants that required further studies

TLR2 forms heterodimers with TLR1 and TLR6, serving as PRRs that specifically recognize bacterial lipopeptides. Upon ligand binding, MyD88 is activated, leading to the activation of NF-κB and subsequent promotion of the secretion of pro-inflammatory cytokines.^120,134 In addition, activation of the IKK complex and MAPKs results in the inhibition of IL-12 expression and the promotion of IL-10 expression, mediated through the transcription factor AP1^120,134 (Figure 2). Through these mechanisms, TLR2 agonists have the potential to induce both Th1 and Th2 responses, making them valuable candidates for adjuvants. PAM2CSK4, PAM3CSK4, and macrophage-activating lipopeptide 2 (MALP2) are the most extensively studied TLR2 agonists.²⁴⁹ MALP2, in particular, possesses the unique ability to directly activate B cells and has gained significant attention as a novel immunostimulant for vaccine adjuvants.²⁵⁰ PorB has been reported to induce cross-presentation to APCs,²⁵¹ and when used as an adjuvant, to present antigens via MyD88 signaling, to induce germinal center formation and antibody production, and to strengthen the follicular DCs network in mouse model.^252,253 The expression of TLR2 in aged DCs is similar to that in young adults’ DCs.^64,66 Therefore, TLR2 agonists show promise as adjuvants for vaccines in the elderly. However, most studies on TLR2 agonists have focused on anticancer.^249,254 To fully harness TLR2 agonists as adjuvants for vaccines against infectious diseases in the elderly, further research is warranted.

TLR5 is a cell surface-expressed PRR responsible for recognizing bacterial flagellin. TLR5 activates NF-κB through the MyD88 pathway, promoting the secretion of pro-inflammatory cytokines and inducing Th1 immune responses.^162,255 The phenomenon of immunosenescence affecting TLR5 on APCs remains unclear. Previous studies have presented conflicting findings, with some reporting an increase in TLR5 signaling in the elderly, leading to an increase in the secretion of IL-8.¹³¹ while others have indicated a decrease in TLR5 expression levels and a reduction in cytokine secretion related to TLR5.⁶⁴ Nevertheless, experiments involving intranasal vaccination of mice with flagellin as an adjuvant have demonstrated the production of IgG and IgA, along with IFN-γ and CD8⁺ cell immune responses.²⁵⁶ Furthermore, administering an influenza vaccine with a flagellin adjuvant to individuals aged 65 and above resulted in high antibody levels and seroprotection.²⁵⁷ However, caution must be taken with the high reactivity of flagellin.^258,259 In the phase 1 clinical study of ‘VaxInnate’s Universal Flu Vaccine,’ it was established that doses of 0.3 and 1.0 μg exhibited safety and provoked an immune response. Conversely, the administration of higher doses, specifically 3 and 10 μg, was correlated with the onset of flu-like symptoms. A separate investigation by Treanor et al. in human subjects aligned with these findings, indicating that doses exceeding 3 μg led to the emergence of moderately severe systemic symptoms, coupled with significant elevations in serum C-reactive protein levels.²⁶⁰ Considering the history of influenza vaccine causing Bell’s palsy with intranasal vaccination, administration of flagellin via intranasal route indicates the need for caution. In addition to flagellin, other TLR5 agonists like Mobilan and Entolimod are under investigation, although their primary applications are in anticancer vaccines.¹⁶² Therefore, further research is necessary to explore the potential use of TLR5 agonists as adjuvants for vaccines in the elderly.

Montanides are water-in-oil adjuvants composed of squalene, with Montanides ISA51 as a representative example.²⁶¹ Although the mechanism of action of Montanides remains largely unknown, it has been reported to enhance antigen uptake by influencing cell membranes.¹⁷¹ Furthermore, there is speculation that Montanides ISA51 activates immune cells through the signaling of DAMPs at the injection sites,^122,262 suggesting its potential as an adjuvant for vaccines in the elderly. While systemic adverse effects such as headache and nausea, as well as local adverse effects such as swelling and injection site pain,²⁶³ have been reported, Montanides ISA51 has been found to have the ability to effectively induce humoral and cellular immune responses through Th1/Th2 cytokines.^{122,262–264} This highlights its potential for utilization as a vaccine adjuvant in the future.⁴⁷

Conclusions and perspectives

Enhancing vaccine effectiveness in the elderly can not only reduce the burden on the public health system but also prevent the development and transmission of antibiotic resistance through preventing diseases and minimizing the use of antibiotics.^9–11 Strategies to improve vaccine effectiveness in the elderly include antigen discovery, improvements in vaccine programs (such as adjusting antigen dosage, modifying administration routes, and optimizing booster schedules), and the incorporation of adjuvants.²⁶⁵ However, antigen discovery has its limitations, and even with booster vaccines in the elderly, effectiveness might not reach the levels seen in young adults. Therefore, the use of adjuvants could be the most dramatic means to increase vaccine effectiveness.³²

When selecting and developing adjuvants to enhance vaccine effectiveness in the elderly, it is crucial to consider both immunosenescence and the mechanisms of adjuvants. However, it’s important to note that research results have provided varying findings, highlighting the need for further investigation. For instance, there is an ongoing debate about whether the expression and functionality of TLR5 and TLR9 are diminished as a result of immunosenescence.^{62–64,67,131} Nevertheless, research utilizing adjuvants targeting TLR5 and TLR9 has demonstrated excellent effectiveness even in the elderly population.^167,257

The safety of adjuvants is also a crucial consideration in the selection of adjuvants to enhance vaccine efficacy in the elderly.²⁴⁶ Various adjuvants have distinct reactogenicity, and this can lead to different adverse effects based on health status and age.^228,247 Especially, as the elderly are known to be more vulnerable to such reactogenicity compared to other age groups, safety should be the foremost consideration when designing adjuvants for vaccines in the elderly.^238,246,248 Generally, even for previously approved adjuvants, a new evaluation of vaccine safety is required when used with different types of antigens. A new safety assessment is also necessary when combining previously approved adjuvants.²⁶⁶ Notably, the safety assessment of an adjuvanted vaccine ultimately based on the final vaccine formulation, as adjuvants are not deemed active ingredients for approval.²⁶⁷ The evaluation of vaccine safety entails comprehensive research on the impact of incorporating adjuvants into vaccines, encompassing the assessment of harmful effects in vaccine recipients based on the mechanisms of action of adjuvants and antigens.²⁶⁷ There has been significant effort to establish standard methodologies for evaluating the safety of vaccines and adjuvants.²⁶⁸ The WHO, the Food and Drug Administration of the USA, and the European Medicines Agency Evaluation of Medicines for Human Use provide guidelines regarding the safety of adjuvants and vaccines.^{266,268–272} However, the criteria for toxicity assessment are not yet clear, and research on the reactogenicity of adjuvants in the elderly is still limited.^267,268 Hence, it is imperative to acknowledge that regulatory toxicity requirements are evolving and may be revised in the future, based on scientific advancements.

It is also necessary to conduct studies that directly compare the effectiveness of different adjuvants in humans.²³⁸ There may be limitations in applying animal experimental results related to immunosenescence to humans.³⁶ Because, there are fundamental immunological differences between mice and humans, such as expression/diversity and response of PRRs, distinct differentiation signals for CD4⁺ Th-subtypes, and a large number of variances in cell surface marker and/or receptor–ligand specificity that dictate migration, homing and function of lymphocytes and other immune cells.²⁷³ Furthermore, humans have a much longer life expectancy than mice, which means the memory T cell pool must be maintained for a substantially greater time. The longevity of human memory T cell populations may face limitations that might not be present in murine experiments.^274,275 The human studies cited in this review showed the results of effectiveness by comparing them to the non-adjuvant group. Therefore, it was challenging to directly compare the effects of various vaccine adjuvants for the same antigen. However, due to economic constraints, conducting direct comparisons of adjuvant effects at the clinical stage using the same antigen in vaccines with different adjuvants is practically close to impossible. An alternative, more feasible approach would be actively verifying adjuvant effects through various meta-analyses.^{47,248,276,277}

Despite these challenges, research on adjuvants is actively ongoing (Table 1). Numerous studies have demonstrated that adjuvants can indeed enhance vaccine effectiveness in the elderly. In particular, the induction of innate immunity through various combinations of adjuvants has shown promising results in further increasing vaccine effectiveness. In future research, the development of new adjuvants and studies focused on enhancing immune responses by combining existing adjuvants should continue. However, it’s important to note that an excessive combination of adjuvants may lead to excessive immune responses, potentially reducing vaccine effectiveness and raising the cost of vaccine production. To develop the optimal adjuvant formulation, it is necessary to identify the ideal adjuvant targets for each vaccination, adjust adjuvant dosages, modify emulsion formulations, and implement new delivery systems.

The augmentation of vaccine effectiveness in the elderly, achieved by the mitigation of immunosenescence through adjuvants, can be realized through diverse pathways. For example, the reduction in the number of naive B and T cells, a representative phenomenon of immunosenescence, might be indirectly mitigated by the elevation of immune cell migration to draining lymph nodes and the augmentation of immune cell trafficking to the vaccination site, facilitated by adjuvants.^{31,70,185,188,194} Moreover, the activation of APCs and alterations in the cytokine milieu resulting from adjuvant action can impact the interplay between APCs and B and T cells. This, in turn, influences the proliferation and differentiation of B and T cells, the generation of B and T memory cells, and the formation and function of Tregs.^{170,278–280} Consequently, comprehending the mechanisms for surmounting immunosenescence and enhancing vaccine effectiveness in the elderly through adjuvants necessitates a multifaceted approach. In addition, research on adjuvants targeting not only APCs but also T cells and non-conventional T cell lineages beyond CD4⁺ and CD8⁺ T cells, such as MAITs, CD1, and γ/δ T cells, could contribute to enhancing vaccine effectiveness in the elderly.²⁸¹

In this review, our primary focus has been on adjuvants targeting some PRRs, which have been reported to conserve their functionality even in aged APCs. Many adjuvants indeed target PRRs, and in most cases, they have demonstrated the induction of Th1 or Th2 immune responses (Table 2). In the future, there is a need for research on adjuvants that can not only induce Th1 and Th2 responses but also promote Th17 responses, which can strongly stimulate mucosal immunity.^282,283 For example, although not addressed in this review paper, the double mutant labile toxin (dmLT) adjuvant holds promise as a vaccine adjuvant for the elderly, capable of eliciting both mucosal and systemic immunity, along with Th17 responses.^284–287 In addition, the substantial germinal center responses induced by mRNA vaccines present a potential strategy to alleviate the decline in naive B cells and the dysfunction of memory B cells in the elderly.^219,220,288 Certainly, thoughtful consideration from diverse perspectives is imperative in the development or selection of adjuvants for vaccines targeting the elderly in the future.

Table 2. The characteristics of adjuvants and their application in clinical development against infectious diseases.

Adjuvants	Characteristics	Clinical trial developments
		Diseases	NCT No.	Phase
MPLA	Activating TLR4 to induce Th1 responses. Promoting APCs to secret pro-inflammatory cytokines and to express co-stimulatory molecules Approved in the USA and Europe	SARS-CoV-2	NCT05005559	3
		SARS-CoV-2	NCT05370040	1
		Influenza A virus/Season flu	NCT03945825	1
		Influenza A virus/pandemic flu	NCT03038776	1
		Hepatitis B virus	NCT01951677	2
		Campylobacter jejuni	NCT05500417	1
GLA-SE	Alternative to MPLA, synthetic LPS mimic Form of squalene oil-in-water emulsion Activating TLR4 to induce Th1 responses Prolongating the interaction between antigens and APCs Promoting APCs to secret pro-inflammatory cytokines Promoting NK-cells to secret IFN-γ Increasing IFN-γ:IL-10 ratio Effectiveness and safety Balanced IgG1/IgG2 Inducing memory CD8^+ T cells	Influenza A virus	NCT01991561	1
		Respiratory syncytial virus	NCT02508194	2
		Mycobacterium tuberculosis	NCT03806686	2
		Mycobacterium tuberculosis	NCT01599897	1
		Mycobacterium leprae	NCT03302897	1
		ETEC	NCT03729219	2
		Schistosoma	NCT01154049	1
Aluminun	Mineral-based emulsion Activating NLRP3 to induce Th2 responses Prolongating the interaction between antigens and APCs Promoting APCs to secret pro-inflammatory cytokines Excellent safety profiles Low capacity to induce CMI	SARS-CoV-2	NCT05197712	1
		Respiratory syncytial virus	NCT02247726	3
		Human immunodeficiency virus	NCT04915768	1
		Human immunodeficiency virus	NCT00006327	3
CpG ODN	Single-stranded DNA molecule resembling bacterial DNA Activating TLR9 to induce Th1 responses Promoting APCs to secret pro-inflammatory cytokines and type I IFN and to express co-stimulatory molecules Promoting NK-cells to secret IFN-γ Inducing CTL Activating B cells via binding to TLR9 on B cells Hepatitis B virus (Heplisave-B®)	SARS-CoV-2	NCT04944368	2
		SARS-CoV-2	NCT05175625	2
		Influenza A virus	NCT03038776	1
		Hepatitis B virus	NCT01951677	1
QS-21	Fraction of saponin Activating NLRP3 to induce Th1/Th2 and Th17 responses Promoting APCs to secret pro-inflammatory cytokines Promoting endosomal escape of antigens, thereby inducing cross-presentation and CTL responses Promoting APC to uptake antigens and to migrate to draining lymph nodes	Influenza A virus	NCT01897701	1
Matrix -M	Saponin analogues forming nanoparticles with cholesterol and phospholipids Inducing the rapid recruitment and activation of APCs and leukocytes Activating of innate immune cells by the inflammasome pathway Inducing a strong Th1-dominant immune response with Th1/Th2 balanced (multifunctional CD4^+ T cells producing IFN-γ, IL-2, or TNF-α) Inducing CD8^+ T cell responses through antigen cross-presentation	Malaria	NCT05252845
		Respiratory Syncytial Viruses	NCT03026348
		Influenza A virus	NCT04120194
		SARS-CoV-2	NCT05112848
		SARS-CoV-2	NCT05112848
		SARS-CoV-2	NCT04611802
MF59	Oil-in-water emulation composed of squalene, Tween 80, and Span 80 Activating NLRP3 to induce Th1/Th2 responses Prolongating the interaction between antigens and APCs Promoting APCs to secret CCL2, CCL4, CCL5, CLXCL8, and pro-inflammatory cytokine Promoting APC to migrate to draining lymph nodes Promoting more immune cells to migrate to the vaccine injection site Good at safety and effectiveness Influenza (Focetria® and Celtura®)	SARS-CoV-2	NCT04495933	1
		Seasonal influenza	NCT04576702	2
AS01	Liposomal adjuvant containing MPLA and QS-21 (oil-in-water) Activating TLR4 and NLRP3 to induce Th1/Th2 and Th17 responses Prolongating the interaction between antigens and APCs Promoting APCs to secret pro-inflammatory cytokines Promoting APCs to present antigen IFN-γ secretion of APC by synergic effects of QS-21 and MPLA Promoting endosomal escape of antigens, thereby inducing cross presentation and CTL responses Promoting NK cells to secret IFN-γ Potential as an adjuvant for vaccines in the elderly Malaria (Mosqirix®), Herpes zoster (Shingrix®)	Malaria	NCT00866619	3
AS03	Squalene-based emulsion comprised DL-α-tocopherol and Tween 80 (oil-in-water) Activating NLRP3 to induce Th2 with weak Th1 Prolongating the interaction between antigens and APCs Promoting APCs to secret CCL2, CCL3, and CXCL1 Inducing IL-6 secretion in the immune system Promoting APC to migrate to draining lymph nodes	SARS-CoV-2	NCT04742738	2
AS04	Aluminum-based emulsion containing MPLA Activating NLRP3 and TLR4 to induce Th1/Th2 responses Prolongating the interaction between antigens and APCs Promoting APCs to secret pro-inflammatory cytokines Strong antibody production HBV (Fendix®) and Human papillomavirus (Cervarix®)	Influenza A virus	NCT00616928	3
		Human papilloma virus	NCT00128661	3

Determining the optimal adjuvants for enhancing vaccine efficacy in the elderly poses challenges. The effects of adjuvants and vaccine effectiveness synergize with an efficient antigen system, eliciting an immune response capable of defending against the target disease.^267,268 The efficacy of a particular adjuvant may vary based on the types of antigens employed. According to the development of various vaccine platforms, the significance of adjuvants has gained prominence. Notably, antigens of recombinant proteins, peptides, plasmid DNA, mRNA, and miRNA alone may fall short in inducing vaccine immunogenicity, underscoring the heightened importance of adjuvants in eliciting robust vaccine responses.²⁸⁹ Developing new adjuvants and optimizing existing ones through combinations and dosages holds the potential to enhance vaccine effectiveness in the elderly population. These efforts can contribute to disease prevention and a reduction in antibiotic resistance, ultimately benefiting public health.

Abbreviations

AP1	=	Activator Protein 1
APCs	=	antigen-presenting cells
AS	=	adjuvant systems
ASC	=	apoptosis-associated speck-like protein containing a CARD
BCR	=	B cell receptor
Blimp	=	B lymphocyte-induced maturation protein
CCL	=	C-C motif chemokine ligand
CMI	=	cell-mediated immunity
CTLs	=	cytotoxic T lymphocytes
CXCL	=	C-X-C motif chemokine ligand
DAMPs	=	danger-associated molecular patterns
DCs	=	dendritic cell
DDA	=	N, N’-demethyl-N, N’-dioctadecylammonium
dsRNA	=	double-stranded RNA
DUSP	=	dual-specific phosphatase
ERKs	=	extracellular signal-regulated kinases
GLA	=	glucopyranosyl lipid A
HSCs	=	hematopoietic stem cells
IFN	=	interferons
IKK	=	IκB kinase
IL	=	interleukin
IRAK	=	IL-1 receptor-associated kinases
IRF	=	induce interferon regulatory factor
JNK	=	c-Jun N-terminal kinase
LPS	=	lipopolysaccharides
MALP2	=	macrophage-activating lipopeptide 2
MAPKs	=	mitogen-activated protein kinases
MPLA	=	monophosphoryl lipid A
Mtb	=	Mycobacterium tuberculosis
MyD88	=	myeloid differentiation factor 88
NF-κB	=	nuclear factor kappa-light-chain-enhancer of activated B cells
NK	=	natural killer
NLRP3	=	nucleotide-binding oligomerization domain (NOD)-like receptor family pyrin domain-containing 3
NOD	=	nucleotide-binding Oligomerization Domain
ODNs	=	oligodeoxynucleotides
PAMPs	=	pathogen-associated molecular patterns
PRRs	=	pattern recognition receptors
ssRNA	=	single-stranded RNA
TAK1	=	transforming growth factor-β-activated kinase
TBK	=	TRAF-Associated NF-κB Activator-binding kinase
TCRs	=	T cell receptors
TDB	=	α, α’-trehalose 6, 6’-dibehenate
TNF	=	tumor necrosis factor
TRAF	=	TNF receptor-associated factor
Tregs	=	regulatory T cells
TRIF	=	toll-interleukin-1 receptor domain-containing adapter-inducing interferon-β
VLP	=	virus-like particles
WHO	=	World Health Organization

Acknowledgments

This work supported by a grant (22202MFDS173) from Ministry of Food and Drug Safety in 2022, and the Korean Health Technology R&D Project through the Korea Health Industry Development Institute (KHIDI), funded by the Ministry of Health and Welfare, Republic of Korea (HV22C0079 and HV23C0090), and the Gyeongsang National University Fund for Professors on Sabbatical Leave, 2023. The funders had no role in decision to publish, or preparation of the manuscript. MID (Medical Illustration & Design), a part of the Medical Research Support Services of Yonsei University College of Medicine, for providing excellent support with medical illustration.

Disclosure statement

No potential conflict of interest was reported by the author(s).

Additional information

Funding

The work was supported by the Gyeongsang National University Fund for Professors on Sabbatical Leave [2023]; Korean Health Technology R&D Project through the Korea Health Industry Development Institute (KHIDI) funded by the Ministry of Health and Welfare [HV22C0079 and HV23C0090]; Ministry of Food and Drug Safety in 2022 [22202MFDS173].