A promising Staphylococcus aureus vaccine adjuvant candidate to overcome Staphylococcal infection based on innate immunity

Abstract

Staphylococcus aureus-mediated human disease ranges from minor skin infection to life threatening diseases. In the past, infections caused by this bacterium could be treated with antibiotics. However, this species has become increasingly resistant to antibiotics. Therefore, vaccines as alternative therapeutic tools is urgently required for controlling this troubles pathogen. But thus far, all vaccines in human clinical trials for preventing S. aureus infections have failed. Three major reasons for this failure can be summarized: 1) An effective antigen has not yet been identified; 2) Host protective immune responses against S. aureus are unclear; 3) Good animal model is not yet identified. The most critical challenge is that despite robust serum immunoglobulin G titers, vaccinated hosts fail to eliminate intracellular S. aureus. To solve this problem, a vaccine inducing both humoral- and cellular-immunity should be designed and developed. Based on our research experiences and recent other groups’ published data, we propose that microbial glycopolymers, which are activating host innate immunity, should be considered as a new S. aureus vaccine adjuvant. Here, our review aims at highlighting how the latest advances in carbohydrates immunobiology can guide the design and development of better S. aureus vaccines and adjuvants.

INTRODUCTION

Staphylococcus aureus is a versatile pathogen that cause a wide range of infections in health care and in the community. These bacterium-mediated human diseases range from minor skin and soft tissue infection to life threatening diseases, such as sepsis, pneumonia, osteomyelitis, septic arthritis, and bloodstream infection [1,2,3]. S. aureus infections strike people of all ages and backgrounds, but are most severe in young children, the elderly, the immunosuppressed and other individuals with major co-morbidities [4,5]. A recent major contributing factor to the high mortality rate of S. aureus infections is increasing antimicrobial resistance. For example, methicillin resistant S. aureus (MRSA)-derived bacteremia has a high mortality rate: 30% to 40% [6,7,8,9]. Further, due to the increasing emergence of antibiotic resistant strains, such as vancomycin-resistant S. aureus in clinics [10,11], the development of an effective vaccine against S. aureus infection is urgently needed [12,13,14,15]. But, until this time, all vaccines in human clinical trials for preventing S. aureus infections have failed [14,15,16].

A major difficulty in the development of a successful vaccine against S. aureus is an incomplete understanding of protective immune mechanisms and a lack of reliable biomarkers showing durable and long-term protective immunity against S. aureus infections in humans [14,15]. This is also attributed to insufficient information about the specific host immune responses related to protecting against invasive S. aureus infections [14]. Another big reason for these clinical trial failures is that S. aureus cells survive in host immune cells, such as neutrophils and macrophages [17]. Although S. aureus is generally considered an extracellular pathogen, recent Fraunholz and Sinha [18] have uncovered the possibility that it also acts as an intracellular pathogen. Yang et al. [19] explored the mechanism by which the HDAC11/IL10 axis enhances mitophagy and promotes the intracellular survival of S. aureus. Indeed, S. aureus cells were detected inside macrophages and granulocytes in this bacterium-infection animal model, indicating that antibody-mediated opsonophagocytosis cannot kill intracellular-residing S. aureus. Therefore, future vaccine development may require the identification of antigens or adjuvants that can activate cellular immunity to kill intracellular S. aureus cells.

Until now, all vaccine candidates targeted to generate high titers of opsonic antibodies against screened antigens are expected to facilitate effective antibody-mediated opsonophagocytosis, leading to bacterial clearance completely in the vaccine-immunized animals [16,20,21]. But, unexpectedly, the residual S. aureus cell numbers in the kidneys and livers of vaccine-immunized animals were not reduced upon challenge with S. aureus cells. This indicates that bacterial clearance was not induced in the immunized animals and suggests that host cellular immunity was not properly induced. These results indicate that both B-cell-mediated humoral immunity and T-cell-mediated cellular immunity are necessary to induce effective host immunity after vaccine immunization against S. aureus infections.

Since all vaccine development by global pharmaceutical companies using single antigen was halted due to failure of clinical trials, now combinations of multiple antigens are in progress to test the efficacy of these vaccine candidates [22,23]. Although some recent clinical trials have shown that vaccination with these multiple antigens induced S. aureus-specific antibodies with opsonic activity in vitro, successful clinical trials have not yet been reported.

Recent elegant basic studies in the immunology field showed us that the activation of host innate immunity is necessary to link T-cell-mediated cellular immunity [24,25]. Also, we elucidated about host innate immune responses against S. aureus infection [26]. Currently, screening of protein-derived vaccine antigens that can activate host innate immunity is not easy work and a few molecules with this activity are reported [27]. Therefore, if we can screen some suitable vaccine adjuvant molecules that enable activation of host innate immunity upon co-injection with already screened S. aureus antigens, we may expect induction of T-cell-mediated cellular immunity. To achieve this goal, we need to understand what kind of S. aureus vaccine antigens have been screened thus far. In addition, we also need to search what kinds of animal models are reasonable for S. aureus vaccine development. Further, the biochemical characteristics of the promising vaccine adjuvant should be considered based on recent published data. Finally, increased knowledges about the mode of action of already approved vaccines, adjuvants, newly developed and promising adjuvants for the development of S. aureus vaccine are necessary.

In this review, we will summarize these issues step by step based on recently published sources and our own research experiences. Especially, our review aims at highlighting how the latest advances in carbohydrates immunobiology can guide the design and development of better S. aureus vaccines and adjuvants.

SCREENED ANTIGENS FOR S. AUREUS VACCINE DEVELOPMENT

Criteria for S. aureus vaccine candidates

Several vaccines designed to target virulence factors in microorganisms have been successful. Therefore, we believed that by targeting S. aureus virulence factors we can produce effective vaccines. The screened vaccine candidate molecules of S. aureus are largely classified into 3 groups, including surface proteins, secreted proteins and surface polysaccharides (Fig. 1) [28,29]. In response to these candidates, many researchers have designed several different vaccines and tried clinical trials against S. aureus infections. But, so far, no vaccine has successfully been developed against this notorious bacterium.

Fig. 1. Criteria of selected antigens candidates for Staphylococcus aureus vaccine development. Largely 3 groups were targeted as S. aureus vaccine antigen candidates. Six different surface proteins were screened and examined as vaccine antigens. As for secreted proteins, 5 different protein families were targeted. Two CPs, CP5 and CP8, were examined in clinical trials tested as vaccine antigens.

PVL, Panton-Valentine leukocidin; CP, capsular polysaccharide.

Vaccines that failed in clinical trials

Three of the failed vaccines, Nabi’s Staph Vax vaccine, Merck’s V710 vaccine and Pfizer’s SA4Ags, were all stopped in phase III or phase IIb trial (Table 1).

Table 1. The Staphylococcus aureus vaccines that failed in the clinical trials.

Vaccine	No. of antigens	Adjuvant	Company	Failed phase	Failure reasons
Staph Vax (CP5, CP8 and r-exotoxin A)	2	-	Nabi	Phase III	Low antigen quality; No curative effect
V710 (IsdB)	1	-	Merck	Phase III	The reduction of the level of IL-2 and IL-17; The increased mortality after S. aureus infections
SA4Ag (CP5, CP8, ClfA and MntC)	4	-	Pfizer	Phase IIb	Does not reduce S. aureus infection or postoperative infection

CP, capsular polysaccharide; IsdB, iron surface determinant B; IL, interleukin; ClfA, clumping factor A; MntC, manganese transport protein C.

S. aureus capsular polysaccharides (CPs) firstly have been used in S. aureus vaccine development. Of the 12 known CPs of S. aureus, most clinical isolates harbor type-5 and -8 CP. These 2 most common CPs were combined with the exotoxin A from Pseudomonas aeruginosa in the bivalent polysaccharide vaccine candidate known as Staph VAX, developed by Nabi. S. aureus CP5 and CP8 antibodies induced specific opsonophagocytic killing by human neutrophils in vitro and showed protective effects in animals [30]. The immunogenicity of CP5 and CP8 alone is weak, so Nabi chose a recombinant exotoxin A to be injected together to improve the immunogenicity [30,31]. But, in phase III trials, the antibodies produced by this vaccine did not lead to a reduction of S. aureus bacteremia. So, the vaccine was declared as a failure and the trial stopped [31,32].

Iron surface determinant B (IsdB) is a surface protein produced by S. aureus, which is one of the Isd systems for the bacteria to obtain iron from host sources. It can capture the heme of hemoglobin [33]. Because the ability of IsdB-deficient S. aureus to acquire iron sources is weakened in mouse models [34], Merck developed a vaccine, V710, which contains purified IsdB, and then conducted clinical studies. Initially, immunization using amorphous aluminum hydroxyphosphate sulfate adjuvant showed safety and good immunogenicity [35,36] and V710-induced immunoglobulin G (IgG)-antibody reactions against IsdB showed protective effects. Despite appearing safe and effective in the early clinical studies and preclinical models, preoperative acceptance people with V710 vaccine induced the anti-IsdB antibody response, but showed worsened outcome of postoperative staphylococcal infected patients. Another finding was that postoperative patients’ death following S. aureus infections was associated with low serum IL2 and IL17a concentrations solely in V710-immunized patients [37], indicating that IL2 and IL17a may be important cytokines for eliminating S. aureus in the host [37,38].

Pfizer’s SA4Ags is a 4-antigen S. aureus vaccine, made up of recombinant surface protein clumping factor A, recombinant manganese transporter protein C, and conjugates of CP5 and CP8 [39]. In healthy people, SA4Ags was well tolerated, safe, and quickly produced high levels of antibodies in an early phase 1/2 clinical study [40]. But following a planned interim analysis, Pfizer’s phase IIb trial was discontinued due to ineffectiveness [32,41].

Vaccines that are undergoing clinical trials

Alpha-toxin, clumping factor A (ClfA), and CP5 and CP8 conjugated to tetanus toxoid vaccine made with oil adjuvant (AS03B) by GlaxoSmithKline (named as GSK2392103A), similar to Pfizer SA4Ag, completed phase I study in Belgium. But it has not been developed further [29,42,43]. GSK named as GSK3878858A further has been conducting phase I and phase II clinical trials of SA-5Ag since 2020, and recruitment is currently underway [15].

The N-terminal of the Candida albicans agglutinin-like sequence 3 protein, which is the component of the experimental vaccine NDV-3, is prepared with aluminum hydroxide (alum) as an adjuvant. The first-in-human phase I clinical trial’s goals were to compare NDV-3 to a saline control in order to assess its immunogenicity, safety, and acceptability at 2 distinct antigen levels. NDV-3 was safe and generally well-tolerated at both dose levels. IFN-γ and interleukin (IL)-17A production by peripheral blood mononuclear cells was enhanced by NDV-3. These findings promote further research into NDV-3 as a potential vaccine against Candida and S. aureus infections [44,45].

The Novartis 4-component S. aureus vaccine (4C-Staph), including 3 secreted virulence factors α-hemolysin (Hla), Esxs (EsxA and EsxB), as well as 2 surface-associated lipoproteins, FhuD2 and Csa1A was developed. They first tried to develop a 4C-Staph vaccine with alum adjuvant, which protected against multiple strains of S. aureus in 4 infection animal models (peritonitis, abscess, skin infection, and pneumonia). They then assessed the protective effects using MF59 adjuvant and Toll-like receptor (TLR) 7-dependent aluminum (T7-Alum) adjuvant. In the study with MF59 adjuvant, it was shown that co-immunization of MF59 and 4C-Staph vaccine further enhanced antigen-specific IgG titers and CD4⁺ T-cell responses compared with alum only. Combination of MF59 and 4C-Staph showed protective effect in a peritonitis model of S. aureus infection. Also, a single dose immunization of 4C-Staph/T7-Alum vaccine induced CD4⁺ effector T cells and produced IL-17A rapidly, and finally showed effective protection against S. aureus in mice. So, they concluded that 4C-Staph/T7-Alum would be very beneficial for patients scheduled for major surgery or in the intensive care unit [46,47,48].

STEBVax vaccine is a recombinant modified version from Integrated Bio-Therapeutics of staphylococcal enterotoxin B (SEB) that has 3-point mutations (L45R, Y89A, and Y94A). These modifications of this antigen were targeted to prevent the SEB from binding to human MHC class II receptors and designed to be non-toxic while keeping the majority of its immunogenicity. The phase I trial for the STEBVax vaccine finished in 2015, and it seems to be secure, immunogenic, and able to trigger functional toxin-neutralizing antibodies [49,50,51].

IBT-V02 is a heptavalent vaccine from Integrated Bio-Therapeutics that includes toxoids for toxic shock syndrome toxin 1, Panton-Valentine leukocidin (PVL) F and S subunits (LukS), leukocidin A/B (LukAB), Staphylococcal enterotoxins (SEA and SEB), and S. aureus Hla [51,52]. IBT-V02 is designed to help patients having elective surgery avoid or lessen the risk of contracting MRSA after surgery. Also, preventing recurring skin and soft tissue infections caused by S. aureus is a second indication pursued. The vaccine is currently being planned for clinical phase I.

Nabi Biopharmaceutical and the Uniformed Services University of the Health Sciences evaluated PentaStaph vaccines based on CP5, CP8, and Hla antigens supplemented with LukS and teichoic acid. This vaccine was sold to GSK in 2009 for the further study [53].

Olymvax used Hla, SEB and 3 surface proteins Staphylococcal protein A, IsdB-N2 domain (IsdB-N2) and manganese transport protein C developed a recombinant pentagenic S. aureus vaccine (rFSAV) [54,55]. The data suggest that rFSAV is a potential vaccine candidate for the prevention of S. aureus infection (Table 2).

Table 2. The Staphylococcus aureus vaccines that are undergoing clinical trials.

Vaccine	No. of antigens	Adjuvant	Company	Clinical trial number	Clinical trials
GSK2392103A	4	AS03B	GSK	NCT01160172	Phase I
GSK3878858A	5	AS03B	GSK	NCT04420221	Phase II
NDV-3 (Als3p-N)	1	Alum	NovaDigm Therapeutics	NCT03455309	Phase II
4C-Staph	4	T7-Alum	Novartis	-	Preclinical
STEBVax (IBT-V01)	1	Alum	Integrated Bio-Theraputic	NCT00974935	Phase I
IBT-V02	7	Alum	Integrated Bio-Theraputic	-	Phase I
Pentastaph	5	Alum	CSK	-	Phase II
rFSAV	5	Alum	Olymvax	NCT02804711, NCT03966040	Phase I

Als3p, agglutinin-like sequence 3 protein; Alum, aluminum hydroxide; rFSAV, recombinant pentagenic Staphylococcus aureus vaccine.

Antigens that we have chosen for S. aureus vaccine development

My PhD laboratory investigated S. aureus surface proteins as vaccine candidates. Firstly, we showed that the N2N3 domains of 4-recombinant surface proteins, such as serine-aspartate repeat-protein E, ClfA, and fibronectin binding protein (FnbpA and FnbpB), can bind to the complement factor H (fH) and reduce factor I-mediated degradation of C3b on the bacterial surface, thereby enhancing C3b-mediated opsonophagocytosis in the human blood [56]. However, since the N2N3 domains of recombinant surface proteins themselves bind fH and may disrupt the balance of the complement system, we feel that they are not suitable for S. aureus vaccine antigens. We also studied pore-forming toxins (PFTs) and found that the mixture of 4 antibodies, including anti-γ-hemolysin A (HlgA)-IgGs, anti-LukS-IgGs, anti-Hla_H35L-IgGs and anti-LukA_E323AB-IgGs, can give the broadest protection against 10 different staphylococcal PFTs. Upon immunization with the mixture of our 4 toxin/toxoid antigens into rabbits, rabbit survival rate increased. It means that the antibodies have a certain protective effect. But the bacterial abscesses could be seen on the rabbit kidneys, meaning the infection was still not completely cleared. We suggest that screening of a new adjuvant with these 4 antigens is needed to stimulate cellular immunity for the kidney abscess clearance [57].

WHICH ANIMAL MODEL IS SUITABLE FOR S. AUREUS VACCINE DEVELOPMENT?

To best understand how immunotherapeutic agents affect S. aureus infection, selection of good animal models is critical point. The most common model for staphylococcal vaccine research has been the mouse model due to the low cost and high availability. However, there are several aspects that makes the mice infection model inferior to other animal models. Among the more crucial differences is how the cardiovascular physiology differ between mice and humans and several other model animals, such as rabbits. Rabbits are known to more closely resembles human cardiovascular physiology [58].

Another key factor is the lack of functional receptors for several of the toxins produced by S. aureus. One example is the specificity of PVL toxin for human and rabbit G-protein-coupled receptors but not in mice [59]. High correlation between PVL-producing S. aureus and severe infection have been found [60,61], proving mice models to be an inferior animal model for S. aureus infection study. Further LukAB, a toxin which genes are part of the S. aureus core genome [62] has varying species specificity. LukAB shows high specificity for human cells, mild specificity towards rabbit cells and very weak specificity to mouse cells [63]. Altering CD11b to a more humanlike CD11b in mice, increases the detectable bacterial burden in liver from 30% to 86%, showing a clear difference in wild type mice, compared to mice susceptible to LukAB [64]. Another toxin that is part of the S. aureus core genome is gamma-hemolysin AB [65,66]. It can lyse human erythrocytes, macrophages, monocytes and neutrophils by binding to CXCR1, CXCE2, CCR2 and Duffy antigen receptor for chemokines [67,68]. While it can also target mouse macrophages and monocytes, it is unable to target mouse CXCR2 and lacks the ability to lyse neutrophils in mouse [67]. There are also large differences in lethality for many toxins between mice and humans, for example, lethal dose of lipopolysaccharide (LPS) in mice is more than 80,000 µg per kg, compared to only 0.01 µg per kg in humans and between 500–0.5 µg per kg in rabbits [69,70]. There are several different Superantigens (SAgs) produced by S. aureus and they cause serious illness in humans due to their way of overstimulating the host immune system, often leading to cytokine storm [71]. The ability of SAgs leading to overstimulation of the immune system and substantial increase in cytokine production seems to be different between species. Human and rabbit T-cells respond in a comparable way to SAgs while mice T-cells show low response [70,72]. The lowered T-cell response to SAgs might explain the low lethality for SAg exposure in mice. Also, SAgs are lethal to humans in as low doses as 0.0013 µg per kg and 0.05 µg per kg in 8-month year old rabbits while mice do not appear to be susceptible to SAgs in a lethal way (Table 3) [73,74].

Table 3. The comparative analysis of mouse vs. rabbit models for Staphylococcus aureus vaccine.

Comparison	Human	Rabbit	Mouse
Cardiovascular physiology	Standard reference	Highly similar	Significant difference
The specificity of PVL toxin	Specificity for GPCRs	Specificity for GPCRs	No specificity for GPCRs
The specificity of LukAB	High specificity	Mild specificity	Very weak specificity
HlgAB toxin action	Lyse human erythrocytes, macrophages, monocytes and neutrophils	-	Target macrophages and monocytes, unable to lyse neutrophils
Lethal dose of LPS	0.01 µg per kg	500–0.5 µg per kg	More than 80,000 µg per kg
Superantigens reaction	T-cells show high response	Similar to humans	T-cells show low response
Lethal dose of SAgs	0.0013 µg per kg	0.05 µg per kg (in 8-mon year old)	Nonlethal
Model applicability evaluation	-	Recommended for toxin mechanism research and vaccine evaluation	Only applicable to non-toxin dependence studies

PVL, Panton-Valentine leukocidin; GPCR, G-protein-coupled receptor; LukAB, leukocidin A/B; HlgAB, gamma-hemolysin AB; LPS, lipopolysaccharide; SAg, superantigen.

With all these differences in mind, a rabbit model appears much more promising than a mice model for S. aureus vaccine development [75]. This is mainly due to the harboring of more human-like immune responses, compared to mice, to an S. aureus infection, and different sensitivity against several virulence factors. Another newer model is mice reconstituted with a human hematopoietic system, or humanized mice, which has shown a better insight into the human responses to specific S. aureus toxins [76,77,78,79].

INNATE IMMUNITY AGAINST S. AUREUS INFECTION

Recent studies regarding innate immunity have demonstrated that microbial pattern recognition receptors (PRRs) expressed on host macrophages, dendritic cells (DCs), and mast cells and soluble pattern recognition proteins in body fluid recognize pathogen-associated molecular patterns (PAMPs) that are expressed on the bacterial cell wall surface [80,81]. These interactions result in the secretion of antimicrobial proteins and the recruitment and activation of host immune cells by inducing the secretion of inflammatory cytokines and chemokines such as TNF-α, IL-6, IL-8, IL-12 and complement-derived peptides [82,83].

For the last 2 decades, our group and other research groups studied the molecular cross-talks between staphylococcal PAMPs and PRRs using several different S. aureus mutant strains. As summarized in Fig. 2, 1) Toll-like receptor 4 complex senses the LPSs of Gram-negative bacteria. TLR-2/1 and TLR-2/6 heterodimers sense the bacterial lipoproteins and induce the production of inflammatory cytokines [84]. Previously, bacterial peptidoglycans (PGNs) and lipoteichoic acids (LTAs) have been thought of as TLR-2 ligands [85]; however, we have demonstrated that the major ligands of TLR-2 are lipoproteins [86]. The S. aureus lipoprotein diacylglyceryl transferase deletion mutant that cannot synthesize lipoproteins is unable to induce TLR-2-dependent inflammatory cytokine secretion [86,87]. 2) Mannose binding lectin (MBL)- and serum antibody-mediated host complement activations are mediated via S. aureus cell wall components. We demonstrated that S. aureus wall teichoic acid is a ligand of MBL, resulting in activation of complement system [26,88]; 3) Peptidoglycan-recognition proteins-mediated antimicrobial peptides are induced via S. aureus PGN [89]; 4) Scavenger receptor (SR)- or paired immunoglobulin-like receptor B receptor-mediated phagocytosis occurs through S. aureus LTA [90]; 5) In mammals, nucleotide-binding oligomerization domain proteins are known to recognize bacterial PGNs [91]; and 6) N-formyl peptide receptors-mediated inflammation and chemotaxis are modulated by secreted S. aureus formylated peptides [92]. But, further studies are now needed to complete our understanding of the mechanisms of molecular recognition and activation involved in these host innate immune responses against S. aureus cells.

Fig. 2. Host innate immune responses after recognition of Staphylococcus aureus cell wall components by host proteins. The host innate immune responses induced by host proteins are shown.

TLR, Toll-like receptor; FPR, formyl peptide receptor; NOD, nucleotide-binding oligomerization domain; PGRP, peptidoglycan-recognition protein; MBL, mannose binding lectin; SR, scavenger receptor; PIR-B, paired immunoglobulin-like receptor B.

Macrophages and DCs directly recognize bacterial cell surface molecules via specific membrane receptors and then phagocytose them in an opsonin-independent manner. These receptors include the SRs-A and SR-B [93]. Both receptors are known to recognize the LTA of S. aureus [94]. Interestingly, the LTA-lacking S. aureus ΔltaS mutant was found to be highly virulent when intravenously inoculated into mice, and the ΔltaS mutant produced increased bacterial titers in the blood, suggesting that LTA-mediated phagocytosis of S. aureus by macrophages is critical in protection against S. aureus blood infection [90].

In this review, since there is a limitation of space, we will only focus on vaccine adjuvant-related host innate immune responses. The general host innate immunity responses by S. aureus infection are available in the excellent recent review papers [95,96,97,98].

Since S. aureus harbors so many different virulence factors, it is not easy to screen antigens that can activate host cellular immunity. We and other groups experienced that several screened S. aureus antigens by immunization with alum adjuvant induced high levels of serum IgG titers, confirming these antigens’ capacity to robustly activate humoral immune responses by immunization with alum adjuvant. [32,41,56,57]. As described above, S. aureus can survive inside various host immune cells. Many studies provided this evidence and showed that antibody-mediated opsonophagocytosis after vaccine immunization is not enough to kill intracellularly residing S. aureus in animal models and human clinical trials [15,99]. Upon challenging of S. aureus USA300 cells, a typical MRSA strain, to vaccine-immunized animal models, such as mice and rabbits, higher number of S. aureus USA300 cells in kidney and liver were detected [56,57], indicating that proper cellular immunity was not induced although titers of serum IgGs against immunized antigens were increased by repeated immunization.

Therefore, we suggest that screening of a new vaccine adjuvant molecule that can activate host cellular innate immunity will be one of alternative way to induce host cellular immunity. Based on this hypothesis, we tried to survey which biomolecules can activate host immune cells, leading to induction of host cellular adaptive immunity, such as Th1 or Th17 immune responses. As results, glycopolymers purified from pathogens are supposed to be promising candidates for S. aureus vaccine adjuvants.

Here, we will provide published scientific evidence why microbial glycopolymers are promising adjuvants for S. aureus vaccines.

Some microbial CPs conjugated vaccines with carrier proteins induced T-cell dependent cellular immunity

First, it will be necessary to briefly understand why screening of cellular-immunity-inducing molecules is required for S. aureus vaccines adjuvant against S. aureus infection. Bacterial carbohydrate-based vaccines started 1920 with the discovery of Heidelberger and Avery [100]: CPs from Streptococcus pneumoniae are immune-reactive. This discovery stimulated to make CP-specific neutralizing antibodies, resulting in protection against pneumococcal infections [101], and the first CP-based vaccine targeting 4 S. pneumoniae serotypes was approved at 1945 [102]. But, it turned out that clinical efficacy of approved polysaccharide-based vaccines is limited since they are largely attributed to the T-cell-independent immune response [103]. But this limitation was solved by covalently conjugation with an immunogenic carrier protein [104].

The technology to make glycoconjugate antigens was a key breakthrough in the field of vaccinology by eliciting T-cell-dependent immune response, resulting in the production of antibodies of increased affinity and in the generation of carbohydrate antigen specific memory B cells. These glycoconjugate vaccines have been successfully used until this time for protection in infants, adolescent, and adults from a variety of bacterial diseases [105].

Alums and MF-59 are approved vaccine adjuvants

First, it is necessary to address the biological functions of vaccine adjuvants. Adjuvants are defined as molecules to increase innate immune responses to vaccine antigens and have been used in immunization since the 1930s. Adjuvants co-immunized with vaccine antigens are used for several purposes, such as 1) enhancement of vaccine immunogenicity, leading to antigen sparring (i.e., use of a lower dose) and reduction of immunizations required for protective immunity and restoration of the immune response in non- or less-responding individuals; 2) broadening of the host immune response to other antigens; 3) increase in the stability of the formulation [106].

The most widely used adjuvants are aluminum salts which were first used in 1926 in London. Besides aluminum salts, only a few other adjuvants have been approved for use in human vaccines. However, recent technological advances in the field led to licensing of new adjuvants, such as squalene-based oil adjuvants (MF-59 and AS03) [107,108], which is used for several vaccine products, including protein-based malaria, influenza, human papillomavirus, and varicella zoster virus vaccines [109].

Microbial carbohydrates are functioning as promising immunomodulators in host innate immunity

Several microbial carbohydrates expressed on the microbial surface are known to be able to stimulate the host innate immune response via multiple mechanisms, including interaction with specific PRRs on the surface of host antigen-presenting cells (APCs) [109]. By stimulating innate responses, microbial carbohydrates can act as an inducer for antigen-specific adaptive immunity, leading to increase of immune potential as adjuvants in many pre-clinical and clinical studies [110]. Based on these premises, we aim at highlighting the latest advances in understanding carbohydrates immunology that will help design better S. aureus vaccine adjuvants. Among many different kinds of microbial carbohydrates, we suggest that microbial glycopolymers will be promising S. aureus vaccine adjuvant candidates based on recent studies. Here, we summarize recent published data that are supporting our suggestion.

Also, some glycopolymers derived from microbes have also been used as a delivery system to encapsulate antigens and promote their uptake by APCs [106]. Based on our recent studies [56,57,111,112], we experienced that a cellular-immunity-activating adjuvant is definitely needed to develop biological effective S. aureus vaccines. Also, recently other groups’ studies strongly supported the necessity for screening of novel S. aureus vaccine adjuvants [113,114]. Here, we summarize and discuss the established concepts and the possible mechanisms of microbial glycopolymers, leading to promising S. aureus adjuvant candidates.

Dectin-1 and Dectin-2 receptors recognize microbial glycopolymers and induce activation of innate immunity

Dectin-1 and Dectin-2 receptors belonging to the C-type lectin receptor (CLR) family recognize fungal cell wall glycopolymers although some glycopolymers are known to activate TLRs [115,116].

Since these 2 Dectin receptors are expressed by host myeloid cell subsets, glycopolymer binding to Dectin receptors can activate downstream signaling, leading to the expression of multiple pro-inflammatory cytokines that impact T-cell differentiation [117,118,119]. Especially, a particulate form of some glycopolymers was known to prime Th1, Th17, and cytotoxic T lymphocyte responses [120]. Namely, the current model of Dectin-1 activation attributed that only a particulate form can activate downstream signaling by inducing clustering of Dectin-1, displacement of regulatory phosphatases CD45 and CD148, and tyrosine-protein kinase-dependent phosphorylation of the intracellular immunoreceptor tyrosine-based activation motif-like motif of Dectin-1 [121].

The most attractive point by binding of glycopolymers to Dectin-1 receptor is eliciting a Th1 response through the induction of the pro-inflammatory cytokines IL-12p40 and IL-1β [122,123], indicating that both IL-1β production and maturation through a non-canonical caspase-8-dependent inflammasome induced host protective immunity [123]. Also, fungal glycopolymer predominantly recognized innate immune cells, such as neutrophils and macrophages, induced protective Th1 responses driven by initial IL-12 production by DC [122]. The main function of IL-12 is to differentiate naïve CD4⁺ cells into Th1 cell.

But, currently we have to solve 2 issues in order to use fungal glycopolymers as vaccine adjuvants. The first unsolved one is the determination of optimal ligand structure for the activation of Dectin-1 receptor upon co-immunization with S. aureus antigens. The collaboration studies between glycobiology and immunology are necessary to determine bona fide bioactive ligand structures of fungal glycopolymers for application as a new vaccine adjuvant. The second unsolved issue is the determination of the molecular mechanisms why particulate and soluble fungal glycopolymers activate and inhibit the activation of Dectin-1 and -2 receptors, respectively. The molecular understanding of these 2 issues is essential for development of fungal glycopolymers as a S. aureus vaccine adjuvant. Although the immune-responses elicited by Dectin-1 and Dectin-2 may exhibit some degree of overlap, signaling pathways activated by Dectin-2 have been less intensively investigated compared to Dectin-1 and require further studies.

Some other CLRs including macrophage receptor (MR) and DC-SIGN are also known to recognize fungal glycopolymers [124,125,126,127]. MR recognized fungal glycopolymers was known to induce the polarization of human Th17 cells [128]. DC-SIGN also recognized a variety of fungal glycopolymers [129]. Also, DC-SIGN activated RAF-1 (serine/threonine kinase) which in turn modulated NF-κB transcriptional signal pathway [130]. In Table 4, we summuarized the known microbial glycopolymers and their receptor targets (e.g., Dectin-1, DC-SIGN) [131,132,133,134,135,136]. We can expect adjuvant activity of fungal glycopolymers if 3 receptors, MR, DC-SIGN and Dectin receptors simultaneously activates upon recognition of their specific ligand molecules. But it will be necessary to address the relative contribution of each receptor to define the mechanism of fungal glycopolymers for the availability as vaccine adjuvant.

Table 4. Summary for some microbial glycopolymers and their receptor targets.

Microbial glycopolymers	Receptor targets	Reference
Beta-glucan	Dectin-1, TLR2	[131,132]
Mannan	DC-SIGN, SIGNR1	[133]
Lipopolysaccharide	TLR4, DC-SIGN	[134]
Peptidoglycan	NOD2, TLR2	[135]
Exopolysaccharides	TLR2, TLR4, DC-SIGN	[136]

TLR, Toll-like receptor; DC, dendritic cell; NOD2, nucleotide-binding oligomerization domain 2.

As expected, 2 recent elegant studies demonstrated the molecular evidence that fungal cell wall molecules are promising vaccine adjuvant candidates [137,138]. The first study showed that conjugation of protein nanoparticles with fungal cell wall moieties is sufficient for promoting their localization to the follicular DC network of B cell follicles and enhance their immunogenicity, likely through activation of MBL-mediated complement activation [137]. The second study demonstrated that when combined with viral glycoprotein antigens with soluble Candida glycopolymer and alum, this glycopolymer-added vaccine formulation broadened epitope recognition, elicited potent antigen-specific neutralizing antibodies, and conferred protection against viral infections of the lung. Thus, these results showed that the physical properties of microbial glycopolymers determine the outcome of the host innate immune response and can be harnessed for vaccine adjuvant development [138]. Altogether, these studies highlight the promising role of microbial glycopolymers in enhancing antigen immunogenicity upon immunization as adjuvants. The further studies for additional mechanistic studies will give us opportunity to harness their potential as a novel adjuvant.

CONCLUSION AND DISCUSSION

S. aureus is a highly successful pathogen due to production of a wide variety of virulence factors and immune evasion strategies [139]. As described above, future S. aureus vaccine development requires the identification of antigens and adjuvants that can activate both humoral and cellular immunity to completely kill intracellular S. aureus cells. Because many S. aureus vaccine antigens were screened until by global pharmaceutical companies and researchers, screening of host cellular-immunity-inducing adjuvant is currently main streams for the future development of S. aureus vaccine.

Here, we summarized historical and recent advances in the use of microbial glycopolymers as vaccine adjuvants. A better understanding of the mechanism of microbial carbohydrates’ interaction with innate and adaptive immune cells will benefit the design of a new generation of glycan-based adjuvant as an innate immune-modulators to fight both longstanding and emerging infectious diseases.

Although biological significances and signaling pathways by fungal glycopolymer-mediated CLRs are still only partially understood, scientific evidence provided to us from several different sources suggest them as promising vaccine adjuvants. It is worth noting that in order to understand the structural determinants and signaling pathways of fungal glycopolymers, several experimental works are urgently needed in the future: 1) The structure of the glycopolymer adjuvants need to be optimized to maximize the participation of CLR and the production of downstream cytokines. 2) The adjuvant-antigen compatibility studies are necessary to be conducted to evaluate combined efficacy of S. aureus antigens. 3) The best animal models should be selected to test the effectiveness of combined vaccines using multiple screened S. aureus antigens and these microbial sugar polymers as new adjuvants.

Altogether, an in-depth knowledge of the innate immune response to S. aureus will be crucial to understand the pathophysiological implications of host-bacteria interaction and define novel immunotherapeutic approaches to S. aureus diseases.