Cyclodextrin in Vaccines: Enhancing Efficacy and Stability

Abstract

Cyclodextrins, a family of cyclic oligosaccharides, have received considerable interest in the field of pharmaceuticals due to their unique molecular structure and versatile properties. In the context of vaccines, cyclodextrins can effectively encapsulate antigens, ensuring their protection from degradation and improving their immunogenicity. Cyclodextrins offer stability advantages to vaccines by preventing the degradation of labile vaccine components during storage and transportation. Furthermore, cyclodextrins can serve as adjuvants, potentiating the immune response triggered by vaccines. Their unique structure and interaction with the immune system enhance the recognition of antigens by immune cells, leading to an improved activation of both innate and adaptive immune responses. This adjuvant effect contributes to the development of robust and long-lasting immune protection against targeted pathogens. Owing to the distinctive attributes inherent to nanoparticles, their integration into vaccine formulations has assumed an imperative role. Through the encapsulation of vaccine antigens/adjuvants within cyclodextrin nanoparticles, the potency and stability of vaccines can be notably enhanced. In particular, the capacity of amphiphilic cyclodextrins to form nanoparticles through self-assembly without surfactants or co-solvents is a captivating prospect for their application as carrier systems for antigens. In conclusion, cyclodextrins present a promising platform for enhancing the efficacy and stability of vaccines. Their ability to encapsulate antigens, stabilize labile vaccine components and act as adjuvants demonstrates their potential to revolutionize vaccine formulation and delivery. Further research and development in this field will facilitate the translation of cyclodextrin-based vaccine technologies into practical and impactful immunization strategies, ultimately benefiting global health and disease prevention.

1. Introduction

In the world of medicine, vaccines play a crucial role in preventing and controlling infectious diseases. Vaccines have historically been instrumental in reducing the global burden of diseases and saving countless lives. At present, vaccines are used not only to prevent diseases but also to treat them. The therapeutic vaccine, also known as a cancer vaccine or immunotherapy vaccine, is a type of treatment that aims to stimulate the immune system to recognize and attack cancer cells [1,2]. Unlike preventive vaccines, therapeutic vaccines are designed for people who have already had cancer. However, the development and production of effective vaccines pose several challenges, including the maintenance of vaccine stability and maximizing their efficacy. Innovative approaches and strategies to improve vaccine formulations have been continually explored to address these challenges.

One such promising approach involves the incorporation of cyclodextrins (CDs) in vaccines. CDs are cyclic oligosaccharides composed of glucopyranose units, forming a ring-like structure. They possess the unique ability to encapsulate and interact with various molecules, making them valuable in a wide range of pharmaceutical applications. Recent research has emphasized CDs’ potential to improve vaccine stability and effectiveness, sparking significant interest and investigation in the field. The instability of vaccines during storage and transportation is a significant concern. Exposure to various environmental factors, such as temperature fluctuations, light and humidity, can lead to vaccine degradation and a loss of potency.

CDs, with their encapsulating properties, can protect the vaccine components from degradation by forming inclusion complexes. These complexes can shield sensitive vaccine components from external stressors, thereby improving the stability and prolonging the shelf life of vaccines. Moreover, the efficacy of a vaccine depends on its ability to elicit a robust immune response. Vaccine antigens often encounter challenges related to their poor solubility, low bioavailability and limited interactions with the immune system [3,4,5]. CDs can enhance the solubility and bioavailability of vaccine antigens by forming inclusion complexes, enabling better recognition and uptake by immune cells. This can lead to improved antigen presentation and immune stimulation, ultimately resulting in a stronger and more effective immune response [6,7]. Furthermore, CDs offer advantages beyond stability and efficacy enhancement. In addition, some derivatives have been recognized as being of GRAS (Generally Considered Safe) status by regulatory authorities [8] and have a well-established track record of use in pharmaceutical applications. Their compatibility with various vaccine formulations and manufacturing processes further adds to their appeal as vaccine adjuvants and stabilizers.

The incorporation of nanoparticles into vaccines has become notably prominent, particularly in light of the ongoing pandemic. Nanoparticulate carrier systems offer a distinct array of advantages for vaccine formulations, particularly due to their capacity to emulate viruses, facilitated by their adaptable particle sizes. The encapsulation of delicate vaccine constituents, such as mRNA and other subunit antigens, within nanoparticles not only heightens their stability, but also enables precise transportation to the designated target sites. An additional unique attribute lies in their customizable surface modifications, which can be tailored to elicit specific immune responses [9]. CDs—polymer entities with a longstanding presence in nanoparticle formulations for diverse applications—feature prominently. CDs find utility both in direct nanoparticle preparation and in the modification of various nanoparticle carrier systems. Their distinctive cyclic structure, encompassing hydrophobic and hydrophilic segments, establishes an exceptional foundation for encapsulating and stabilizing an extensive spectrum of bioactive compounds. Upon integration into nanoparticle formulations, CDs contribute to bolstering the solubility of hydrophobic agents, affording protection against degradation and augmenting their bioavailability. Notably, CDs readily form polyplexes and nanoplexes with negatively charged biological entities like RNA and DNA, facilitating their transport sans degradation [10,11].

In this review, the potential of CDs as valuable tools in the development of stable and effective vaccines is summarized. Understanding the applications and benefits of CDs in vaccines can pave the way for improved vaccine design and contribute to the advancement of public health worldwide.

2. Brief History of Cyclodextrins

CDs have a rich history in the fields of pharmacy and biotechnology, spanning several decades. Their unique properties and versatile applications have made them valuable tools in drug delivery and formulation, as well as in various biotechnological processes.

CDs were first discovered in the late 19th century by French scientist Villiers, who observed a precipitate that formed during the enzymatic degradation of starch by Bacillus amylobacter. The Austrian scientist Schardinger proposed the structural formula and gave them their name based on their cyclic structure in 1903, when he first used the term “cyclodextrin” [8,12]. More research on the structure and characteristics of CDs was conducted in the 1950s and 1960s, which led to the realization of their full potential. It was during this time that light was shed on the complexation abilities of CDs. CDs began to acquire popularity in the pharmaceutical sector in the 1970s and 1980s. Their ability to form inclusion complexes with hydrophobic drugs opened up new possibilities for improving the solubility, stability and bioavailability of poorly soluble drugs. This breakthrough led to extensive research and development efforts focused on incorporating CDs into various drug formulations [12,13]. As CDs became more popular in pharmacy, their uses went beyond drug delivery. The extensive research and commercialization efforts have led to the availability of various CD-based products on the market [14]. In addition to pharmacy, CDs found utility in analytical chemistry, separation techniques and biotechnology. CDs are a component of gene delivery systems, protein purification and immobilization and enzyme immobilization processes in the field of biotechnology. Due to their distinctive structural features, including the hydrophobic cavity and hydrophilic outside, they are able to interact with a variety of molecules and support a variety of biological processes [15,16,17]. As mentioned in the next sections, CDs have been investigated and used for their potential to improve the stability and effectiveness of vaccinations.

The journey of CDs in pharmacy and biotechnology has been marked by continuous exploration, innovation and application development. Their unique molecular structure and versatile properties have revolutionized novel formulations, opening up new possibilities for improving therapeutic outcomes and advancing biotechnological processes. CDs are predicted to play an increasingly significant role in determining the future of biotechnological research and development.

3. Structure and Physicochemical Properties of Cyclodextrins

CDs are cyclic oligosaccharides composed of glucopyranose units linked together by alpha-1,4-glycosidic bonds, resulting in a torus-like structure. CDs are natural polymers and consist of starch (mostly maize), which is produced through degradation by the enzyme glycosyl transferase [13]. The most commonly studied CDs are α-CD, β-CD and γ-CD, which consist of six, seven and eight glucopyranose units, respectively. The physicochemical properties of CD molecules, such as the size and cavity dimensions, depend on the number of glucopyranose units (

Table 1. Physicochemical properties of natural cyclodextrins.

Physicochemical Properties	α-Cyclodextrin	β-Cyclodextrin	γ-Cyclodextrin
Number of glucopyranose unit	6	7	8
Formula	C36H60O30	C42H70O35	C48H80O40
Water solubility (25 °C, g/L)	145	18.5	232
Molar mass (Da)	972	1135	1297
Height (Å)	7.9	7.9	7.9
Inner volume (Å3)	174	262	427
Outer diameter (Å)	14.6	15.4	17.5
Inner diameter (Å)	4.7–5.3	6.0–6.5	7.5–8.3
Half-life (1M HCL, 60 °C, h)	6.2	5.4	3.0
Absorption	2–3%	1–2%	0.1%

). Their unique torus-like shape gives them certain physicochemical features that make them useful in a variety of applications [11].

Natural CDs do not hydrolyze in the small intestine after oral administration; hydrolysis occurs only in the colon [18]. CDs are metabolized more slowly than starch because β-amylase only degrades free end groups [8]. Parenteral applications of natural CDs show marked differences. The intravenous administration of β-CD causes nephrotoxicity and hemolysis. γ-CD is known to cause less than α-CD and β-CD due to its high water solubility [11]. The hydrophobic interior cavity of CDs is the most significant characteristic that distinguishes their application in biotechnological processes. In the middle of CDs, glucopyranose units form a hydrophobic cavity. Hydrophobic or poorly water-soluble substances, such as antigens and drugs, can be encapsulated in this hydrophobic inner cavity. Through hydrophobic interactions with the guest molecules, CDs create inclusion complexes. The guest molecule’s stability is greatly enhanced by this distinctive structure. The size and shape of the CD cavity determine its ability to accommodate different guest molecules. The larger γ-CD cavity can encapsulate bulkier molecules compared to the smaller α-CD and β-CD cavities. This size selectivity allows CDs to specifically interact with certain molecules and discriminate against others. CDs form inclusion complexes through host–guest complexation, where the hydrophobic guest molecule is encapsulated within the CD cavity. The formation of inclusion complexes can enhance the solubility, stability and bioavailability of guest molecules [19,20]. CDs exhibit good thermal stability, allowing them to withstand processing and manufacturing conditions. They can withstand moderate temperatures during formulation processes without the significant degradation or loss of their complexation abilities. The complexation behavior of CDs can be influenced by the pH of the medium. The inclusion complex formation may vary depending on the pH conditions, with different pH values affecting the stability and release of the guest molecule from the CD cavity [21,22]. The ability of CDs to form inclusion complexes is schematized in Figure 1.

In order to further enhance the properties and expand the applications of CDS, numerous derivatives have been synthesized through chemical modifications of the hydroxyl groups present in the natural CD structure. Some common CD derivatives include hydroxypropyl, methylated and sulfobutyl ether CD. Hydroxypropyl derivatives involve the substitution of one or more hydroxyl groups with hydroxypropyl moieties. These derivatives exhibit improved water solubility and possess enhanced encapsulation abilities for hydrophobic molecules compared to natural CDs. Methylated CD derivatives involve the methylation of hydroxyl groups, resulting in increased lipophilicity. This lipophilicity can enhance the complexation and solubility of hydrophobic compounds. Sulfobutyl ether derivatives are created by attaching sulfobutyl ether groups to the hydroxyl groups of CDS [10,23]. These derivatives are highly water-soluble and possess anionic properties, making them useful in drug solubilization, stabilization and targeted drug delivery.

4. Cyclodextrin in Vaccine Formulations

The vaccine has been the most effective disease prevention method used all over the world since it was discovered in the 18th century. Diseases that have become a global epidemic and health threat for many years, such as smallpox, have been eradicated thanks to vaccination programs [24]. The importance of the vaccine came to the fore again with the pandemic of the last century, and the World Health Organization declared an end to the “global emergency” status of SARS-CoV-2 in May 2023, thanks to the vaccines that were developed in a very short time [25]. Vaccine formulation is a complex process that involves various factors, including the design of the immunogen (antigen), vaccine type, formulation, adjuvant, dosing and administration. Different approaches are required in vaccine formulations due to the problems of conventional vaccines with poor immunogenicity, in vivo intrinsic instability, toxicity and the need for multiple administrations. At present, there is a trend towards subunit, DNA and mRNA vaccines in which a specific part of the pathogen is used, as well as conventional vaccines that use the whole attenuated or inactivated pathogen. As the antigen used in the vaccine formulation becomes smaller, the tolerability of the vaccine increases and its side effects decrease. However, as the distance from the pathogen’s structure increases, the immunogenicity decreases. New and powerful vaccine formulations are needed to protect the antigen and to strengthen the immune response. Longer-lasting immunity can be achieved by adding a carrier system and adjuvant to vaccine formulations [26,27,28].

The incorporation of CDs in vaccine formulations presents several advantageous features that have garnered increasing interest in the field of vaccine development. One significant application of CDs in vaccines is their ability to stabilize and protect labile vaccine components, such as antigens, proteins and nucleic acids. Vaccines are often sensitive to environmental factors, such as the temperature and humidity, which can lead to the degradation of crucial vaccine components during storage and transportation [29,30]. CDs form inclusion complexes with these sensitive molecules, creating a protective shield that preserves their integrity and bioactivity. This stabilization effect extends the shelf life of vaccines, ensuring their potency and efficacy even under challenging storage conditions. Furthermore, CDs offer a controlled and sustained release of the encapsulated vaccine components, ensuring a prolonged and more targeted immune response upon administration. Moreover, CDs are biocompatible and biodegradable, making them suitable candidates for vaccine adjuvants or delivery systems. CDs can function as adjuvants in vaccine formulations. CDs, particularly modified versions, can stimulate the immune system by promoting antigen presentation and enhancing the activation of immune cells [31]. This adjuvant effect contributes to the development of stronger and longer-lasting immune protection against targeted pathogens. This not only enhances the overall immunogenicity, but also allows for tailoring the immune response to specific diseases or target populations. Various studies on the use of CDs in vaccine formulations for different purposes are summarized in

Table 2. Summary of the selected studies performed in the last 5 years on the use of cyclodextrins in vaccine formulations.

Type of Cyclodextrin	Vaccine Type and Targeted Disease	Function of Cyclodextrin	Stage of Vaccine Development	References
per-fluoroalkyl-β-cyclodextrin	Protein (OVA)-based melanoma vaccine	Cyclodextrin channels to improve stability of liposome	Evaluation of efficacy with model antigen in an in vivo murine melanoma model	[32]
Hydroxypropyl-β-cyclodextrin	Protein (HA)-based Seasonal influenza vaccine	Adjuvant	Phase 1 clinical trial (Clinical trial registry: UMIN000028530)	[33]
γ-cyclodextrin	Protein (OVA)-based veterinary vaccine	Span 85 modified γ-cyclodextrin metal-organic framework as novel adjuvant	Evaluation of efficacy in immunized mice	[34]
β-cyclodextrin	mRNA (encoding OVA) vaccine	Branched PEI conjugated β-cyclodextrin as carrier system	Antigen-specific antibody detection in mice vaccinated subcutaneously, intradermally and intramuscularly	[35]
α-cyclodextrin	Combined cell vaccines (tumor whole cells +DCs) for melanoma	CpG adjuvanted-α-cyclodextrin-PEG hydrogel as carrier system	Evaluation of efficacy in an in vivo murine melanoma model	[36]
Mannosylated-β-cyclodextrin	Peptide-based and active targeted vaccine for melanoma	Specific delivery of antigen and TLR7 agonists to antigen presenting cells	Evaluation of immune response in immunized mice	[37]
Polyanionic amphiphilic β-cyclodextrin	Peptide-based vaccine for HIV	Nanoparticulate carrier system	Determination of cytokine release, DC targeting and immune response by cell culture studies	[38]

OVA Ovalbumin; HA hemagglutinin; PEI polyethyleneimine; DCs dendritic cells; CpG cytosine-phosphate-guanine; PEG polyethylene glycol; TLR7 Toll-like-receptor-7; HIV Human Immunodeficiency Virus.

.

Thanks to all of these advantages, studies on the use of CDs in vaccine formulations are rapidly advancing, reflecting the growing interest in this field. While there are currently limited examples of CD-based vaccine formulations available in the market, the number of research studies conducted in this area is expanding at an accelerated pace. Researchers are recognizing the potential benefits that CDs can offer in vaccine development, including improved stability, enhanced solubility, increased immunogenicity and targeted delivery. The continuous exploration of CDs in vaccine formulations signifies a promising avenue for optimizing vaccine efficacy and expanding the repertoire of vaccination strategies. With each passing day, new insights and advancements are being made, contributing to the rapid growth in knowledge in this field. The approved viral vector-based SARS-CoV-2 vaccine Ad26.COV2.S, developed by Johnson & Johnson, is a sterile suspension for intramuscular injection. The vaccine consists of a recombinant adenovirus type 26 (Ad26) vector expressing spike protein in a stabilized conformation. It contains citric acid monohydrate, trisodium citrate dihydrate, ethanol, 2-hydroxypropyl-β-CD (HP-β-CD), polysorbate 80, sodium chloride, sodium hydroxide and hydrochloric acid as excipients and does not contain any preservatives. It has been reported that the cyclodectrin used in the formulation acts as an adjuvant, preservative and stabilizer [39,40]. CDs were also included in the formulation of two different veterinary vaccines. The adjuvanted inactivated vaccine (Suvaxyn PCV) developed by the Pfizer company contains sulfolipo β-CD that works against porcine circovirus type 2 in piglets. The live vaccine CEVAC^® BI L containing the Massachusetts B48 strain of the Infectious Bronchitis virus and the Hitchner B1 strain of the Newcastle Disease virus, released by the Ceva-Phylaxia company, contains HP-β-CD [14].

4.1. Cyclodextrins in Vaccine Targeting

It is possible to strengthen the immune response by delivering vaccine antigens directly to certain immune cells. Targeting immune cells with vaccines plays a crucial role in initiating and directing immune responses, making immune cells attractive targets for vaccine administration. Dendritic cells (DCs), which are necessary for initiating and coordinating immune responses, are the primary cells used for this purpose. DCs capture and process antigens and present them to T cells. In this way, they activate the adaptive immune response. There are different approaches to targeting DCs with vaccines [41,42,43]. Vaccines can be formulated to deliver antigens directly to DCs using specific receptors, such as C-type lectin receptors expressed on DCs [44,45]. Antigens can be conjugated or fused with antibodies or ligands that bind to these receptors, allowing selective uptake and presentation by DCs.

CDs can be modified or functionalized to allow the targeted delivery of vaccines to specific cells, tissues or receptors. For this targeted delivery approach, the surfaces of CDs can be chemically modified to bind ligands or targeting moieties. These ligands can specifically recognize receptors or antigens expressed on the surface of specific cells or tissues. By conjugating a targeting ligand to CD, the vaccine formulation can be designed to selectively deliver vaccine components to the desired target cells. As another approach, CDs can be functionalized with antibodies or antibody fragments that specifically recognize and bind to target antigens. This approach, known as antibody-directed targeting, allows the vaccine to be delivered directly to cells that express the target antigen, such as tumor cells or specific immune cell populations [46,47,48].

Mucosal vaccines, where site-specific immunity can be achieved, are a specific type of vaccine applied to mucosal surfaces. These vaccines aim to provide local protection and prevent infection by stimulating the immune responses on mucosal surfaces where pathogens frequently enter the body. It is possible to provide targeted immunization with the mucosal vaccines that have been studied in recent years, thanks to their advantages such as allowing needle-free administration and providing wider protection by stimulating both mucosal and systemic immunity [49,50]. The use of CDs in the formulations of mucosal vaccines also provides advantages.

Although CDs are primarily known for their incorporation and stabilizing properties, they can also exhibit muco-adhesive properties. The muco-adhesive properties of CDs make them suitable for mucosal vaccine applications. By increasing muco-adhesion, CDs can increase the efficacy and bioavailability of active compounds on mucosal surfaces and provide targeted delivery to certain tissues or cells. In particular, the high affinity of CDs to mucin increases their muco-adhesive properties. Mucin is the primary component of mucus and plays a crucial role in muco-adhesion. CDs can form interactions with mucin via hydrogen bonding or electrostatic interactions. These interactions between CDs and mucin can lead to the formation of adhesive bonds that promote the muco-adhesion of CD-based formulations [18,51,52,53]. In addition, the muco-adhesive properties of CDs can be improved through structural modifications. For example, the incorporation of hydrophilic functional groups such as carboxyl, hydroxyl or amino groups into the CD molecule can enhance its mucoadhesive properties by promoting interactions with the mucus layer [52,53]. Muco-adhesive properties allow CDs to adhere to mucosal surfaces, prolonging the residence time of vaccine formulations at the target site. This prolonged contact increases the uptake of antigens by immune system cells, providing both strengthened immunity and a prolonged stimulation of the immune system with continuous antigen release. Intranasal vaccination is a versatile approach for addressing virus infection, but the nasal epithelium remains a major barrier. A cationic CD-polyethylenimine 2k conjugate complex was developed for the HIV-1 mRNA vaccine. The delivery vehicle reversibly opened tight junctions, enhancing the paracellular delivery and minimizing toxins-absorption in the nasal cavity. Strong systemic and mucosal anti-HIV immune responses, as well as cytokine production, were achieved using this approach [54]. As another nasal vaccine delivery system, the potential of HP-β-CD as a mucosal adjuvant for intranasal flu vaccines was examined. The findings demonstrated that mice given hemagglutinin split and an inactivated whole-virion influenza vaccine with HP-β-CD secreted antigen-specific IgA and IgGs in the airway mucosa and serum. Both HP-β-CD adjuvanted-flu vaccines protected the mice against the lethal influenza virus challenge. According to the study’s findings, HP-β-CD could serve as an effective mucosal adjuvant for influenza vaccinations because it significantly reduced antigen-specific IgE reactions when compared to the aluminum salt adjuvant [31].

4.2. Cyclodextrins as Stabilizers

At present, stability is one of the most important parameters to be provided in vaccines. Stabilization is essential to ensure that vaccines remain effective and safe until they are administered to individuals. The term “stabilization of vaccines” refers to the process of maintaining the integrity, efficacy and potency of vaccines throughout their storage, transport and distribution [55,56]. It includes the implementation of measures to prevent the deterioration or loss of vaccine quality due to various environmental factors, such as the temperature, light, humidity and exposure to certain chemicals. Because they are biological products, vaccines must be transported and stored at certain temperature ranges. Most vaccines need to be stored and transported in the cold chain (a system that maintains a constant temperature between 2–8 °C). The change in temperature may cause the vaccine to lose its effectiveness [57].

The next generation mRNA vaccines have certain stability requirements due to the nature of the mRNA molecule. mRNA vaccines typically require very low temperature storage to maintain their stability. For example, the Pfizer-BioNTech vaccine can be stored at around −70 °C and the Moderna vaccine at −20 °C [58]. Just like mRNA vaccines, other nucleic acid vaccines and subunit vaccines need to be protected against enzymatic activity and physiological conditions, as well as temperature, due to their natural structure. These requirements necessitate the addition of new components, such as preservatives, adjuvant and delivery systems, to vaccine formulations.

CDs can play an important role in stabilizing vaccine formulations by protecting vaccine components from degradation and improving their stability. Their hydrophobic cavities are well suited to trapping sensitive molecules, such as vaccine antigens. In this way, they can protect vaccine components such as antigens or adjuvants from degradation caused by factors such as heat, light, moisture or enzymatic activity. This protection helps to maintain the integrity and efficacy of the vaccine. In addition, CDs have the ability to encapsulate, dissolve and disperse vaccine components with limited solubility, thanks to their hydrophobic cavities [59,60,61]. This improved solubility and dispersion contribute to the prevention of aggregation and instability caused by the dissolution problems encountered in vaccine formulations. In addition to ensuring shelf stability, it is very important to ensure the stability of the vaccine in the biological environment. A well-developed vaccine formulation is very important in providing the desired immunity against chemical and enzymatic degradation in the organism. In this context, CDs encapsulate vaccine components, limiting their exposure to reactive molecules or environments and reducing the possibility of chemical reactions that could affect the vaccine stability. Moreover, enzymes in biological fluids can degrade vaccine components such as proteins or peptides [62,63]. The SARS-CoV-2 vaccine Ad26.COV2.S, developed by Johnson & Johnson, has received great attention in the context of CDs. In the Ad26.COV2.S vaccine, HP-β-CD was added as a stabilizing agent to preserve the integrity and potency of the viral vector-based vaccine.

4.3. Cyclodextrins as Immunmodulator

Vaccines are designed to stimulate the immune system by introducing antigens derived from specific pathogens and play an important role in immunomodulation (modulation or regulation of the immune system). Vaccines activate both innate and adaptive immune responses, leading to antibody production, the activation of T cells and the development of immunological memory. Vaccines promote the presentation of antigens to immune cells, particularly antigen-presenting cells (APCs). APCs capture and process vaccine antigens, presenting them to the T cells necessary for the initiation of adaptive immune responses. This antigen presentation facilitates the activation of specific immune responses against the targeted pathogen. Finally, memory cells are formed, which are immune cells that “remember” the encountered pathogens. This immunological memory is a crucial aspect of vaccines, and an effective vaccine would be expected to contribute to long-term immunity (Figure 2). Vaccines can modulate immune responses by influencing the type and magnitude of the immune responses. Vaccines can be formulated to stimulate the production of certain types of antibodies (such as neutralizing antibodies) or to activate cellular immune responses (such as cytotoxic T cell responses), depending on the desired immune outcome [64,65,66].

Vaccine adjuvants are substances that are added to vaccines to enhance the immune response generated by the vaccine antigens. They work by stimulating and modulating the immune system, resulting in a more robust and long-lasting immune response. Adjuvants amplify and prolong the immune response to vaccine antigens. They stimulate various components of the immune system, including APC, B cells and T cells [67,68]. Adjuvants can activate immune cells, promote antigen presentation and enhance the production of antibodies and memory cells, ultimately leading to a stronger and more effective immune response. They can promote the uptake and processing of antigens by APC, enhancing the presentation of antigen fragments to T cells. This step is crucial for activating specific immune responses against the targeted pathogen. Adjuvants can increase the duration of exposure to vaccine antigens. By slowing down the release or degradation of antigens, adjuvants allow for a more prolonged interaction between antigens and immune cells, leading to a sustained immune response [69,70]. Adjuvants can shape the immune response towards a desired direction. They can bias the immune response in a particular type of immunity, such as stimulating a stronger antibody response (humoral immunity) or enhancing cellular immune responses (cell-mediated immunity). Adjuvants can also promote the production of specific types of antibodies, such as neutralizing antibodies or those with enhanced effector functions.

Figure 2. Schematic representation of effect mechanism of vaccines [68].

Figure 2. Schematic representation of effect mechanism of vaccines [68].

Another feature that makes CDs attractive for use in vaccine formulations is their immunomodulatory effects, which can affect the immune response. Although not considered potent immunostimulants on their own, CDs can interact with immune cells and modulate their activation, maturation and antigen-presenting abilities. Moreover, CDs can act as adjuvants—substances that enhance the immune response to vaccines. They can stimulate the activation and maturation of APCs, such as DCs and macrophages. This leads to increased antigen presentation and the initiation of a robust immune response. CDs are known to influence T cell responses by promoting T cell activation, proliferation and differentiation. For example, HP-β-CD has been shown to induce the T-helper (Th2) immune response. In addition, HP-β-CD has also been reported to promote the production of Th2-related cytokines [71,72]. Kim et al. investigated the maturation and activation of human DCs treated with HP-β-CD. They found that HP-β-CD induced DC maturation and activation, suggesting its potential as an adjuvant [73]. CDs can also activate Toll-like receptors present on immune cells, triggering intracellular signaling pathways that promote the production of pro-inflammatory cytokines and chemokines, further enhancing immune activation [74,75]. CDs have a high affinity for cholesterol. They also show immunomodulatory and even antiviral activity as a cholesterol-depleting agent with antiviral activity and immunomodulatory effects [71,76].

In one study, a seasonal influenza vaccine (Flu-vac) adjuvanted with HP-β-CD was compared to a regular seasonal influenza vaccination (Flu-vac) for its safety and immunogenicity in healthy people. FluCyD-vac—which contains 9 g of hemagglutinin strain plus 20% w/v of HP-β-CD—and Flu-vac—which contains 15 g of hemagglutinin strain alone—are two quadrivalent split seasonal influenza vaccines. A single dosage of Flu/CyD-vac or Flu-vac was given to participants at random in a 2:1 ratio. Using hemagglutination inhibition titers and T-cell function in peripheral blood mononuclear cells after stimulation with hemagglutinin vaccination strains, the study evaluated requested and unrequested adverse events and immunological responses. Despite having 40% fewer hemagglutinin antigens than Flu-vac, FluCyD-vac was well tolerated and immunogenic in 36 healthy volunteers [33].

HP-β-CD was reported to act as an effective adjuvant for influenza vaccines. According to one study, intranasally administered HP-β-CD stimulates the transient release of IL-33 from lung alveolar epithelial type 2 cells, but not other vaccination adjuvants. When given intravenously, HP-β-CD has an adjuvant effect that is exclusively due to IL-33/ST2 signaling. The protective immunity against influenza virus infection brought on by the intranasal injection of the HP-β-CD-adjuvanted influenza split vaccine included the release of IL-33 [77]. In another influenza vaccine study, it was reported that HP-β-CD enhanced protective type-2 immunogenicity in co-administered seasonal influenza split vaccines [78].

4.4. Cyclodextrins as Nano-Sized Carrier Systems

Carrier systems, especially nanoparticles, have unique properties thanks to their particle size. The most important feature that makes the use of nanoparticulate systems in vaccine technology attractive is that nanoparticles and viruses are on the same size scale [79,80]. Like viruses, nanoparticles can be targeted to virus-targeted cells because they have a similar size distribution. Nanoparticles can mimic viruses thanks to their adjustable size and surface properties. Nanoparticles can strengthen vaccine formulations’ antigen stability and shield antigens from proteolytic degradation. Moreover, by adding cell-targeted peptides, proteins or polymers to their surfaces, active targeted antigen transport can be provided, lowering the amount of vaccine required to trigger a powerful immune response [81,82,83].

In a study aiming to understand the effects of nanoparticles on prophylactic vaccine efficacy, a pH-sensitive nanovaccine containing FSHR peptide (derived from an epitope of follicle stimulating hormone receptor) was developed. Acetalized β-CD and poly(lactic-co-glycolic acid) were used in the preparation of the carrier system. In the study, as a result of in vivo evaluations, it was shown that the nanovaccine with high antigen loading activity was more potent than Freund’s adjuvant in inducing the anti-FSHR antibody, reducing sperm count, inhibiting sperm motility and increasing the teratosperm rate. In both dose- and time-dependent ways, DCs efficiently uptake nanovaccines through endocytosis. Moreover, the intracellular trafficking of the nanovaccine is directly dependent on the pH sensitivity of the carrier materials. Moreover, in a co-culture study with T cells and activated DCs, it was shown that the nanovaccine caused the release of inflammatory cytokines [84]. The biggest problem that occurs in CD derivatives synthesized to increase the solubility of natural CDs is that the outer surface, as well as the inner surface, is hydrophilic; this situation causes a decrease in the contact of CDs with biological membranes. In order to prevent this undesirable result, amphiphilic CD derivatives are synthesized. Other reasons for synthesizing amphiphilic CDs are to make them hydrophobic by adding long aliphatic chains to the structure of CDs. It can be summarized as increasing its interaction with molecules and allowing the formation of nanoparticle systems by spontaneously coming together at a physiological pH and aqueous medium [11,85]. For this purpose, Geisshüsler et al. synthesized two different amphiphilic CD derivatives: heptakis-6-octanethio-β-CD (CD-C8) and heptakis-6-dodecanethio-β-CD (CD-C12). Ovalbumin-derived peptide-loaded amphiphilic CD nanoparticles with a size of 150 nm were prepared using the nanoprecipitation method. It was reported that mice immunized with peptide-loaded CD nanoparticles have higher frequencies of antigen-specific and cytokine-producing CD8+ T cells. Contrary to peptide-loaded CD nanoparticles, the blank CD nanoparticles did not cause changes in the CD80 or CD86 levels, and based on this result, it was stated that amphiphilic CD nanoparticles are safe to use as a carrier system in vaccines [86].

Yu et al. developed a CD-based nanoparticle delivery system for mucin-1 (MUC1), which is one of the tumor-associated antigens that is frequently used as an antigen in cancer vaccines. MUC1 peptide vaccines were developed using β-CD grafted chitosan via a host–guest interaction between the adamantane moiety and β-CD. The study aimed to use chitosan nanoparticle, another polymer known to have adjuvant properties. However, due to the low water solubility of chitosan, the MUC1 peptide was combined with adamantane through modification. Then, the solubility problem of chitosan was avoided by using the hydrophobic central cavities of β-CD, which has a high affinity for adamantane. Different vaccine formulations were developed with particle sizes between 91.28–129.0 nm, with and without adamantane. When serum samples taken from mice after vaccination were analyzed, it was stated that the levels of IgG subtypes in the groups developed as a result of different conjugations were different. It was emphasized that the CD-based host–guest interaction can regulate the type of immune responses, and the β-CD modification improves chitosan’s solubility, making it a promising carrier for vaccine construction [87].

4.5. Future Directions

In the field of future vaccine formulations, the use of CDs offers a compelling avenue of discovery, offering a range of potential applications that hold the promise of revolutionizing vaccine development, administration and efficacy. These properties of CDs—which have potential in many areas, from stability enhancement to vaccine targeting—are explained in the above sections. Although there are many different examples in the field of medicine, both in the literature and on the market, the applications and studies of CDs in the field of vaccines are limited.

Today, as new vaccine types and vaccine administration routes diversify beyond conventional vaccines, potential carriers and adjuvants, such as CDs, appear as candidates to facilitate new approaches. It is clear that in nasal and oral vaccine administration, these molecules can be highly beneficial in increasing the mucosal absorption of antigens by triggering strong local and systemic immune responses. In the evolving vaccine formulation landscape, CDs may facilitate the consolidation of combination vaccines, facilitate administration and potentially elicit synergistic immune responses. Moreover, as vaccine formats expand with the emphasis on new delivery methods, such as powders, patches or aerosols, CDs offer a stabilizing effect, ensuring that vaccine ingredients remain effective and accessible in non-traditional environments. In this context, another interesting potential for incorporating CDs into vaccine formulations is the cholesterol affinity of CDs and the associated antiviral use. When studies on infectious diseases are examined, studies on the antiviral activities of CDs and their role in preventing viral transmission are intensified. The ability of CDs to modulate cholesterol levels could be exploited for synergy in vaccine formulations alongside their antiviral potential. The application of the personalized approach to vaccines—which is also on the agenda in treatment and attracts considerable attention—is another future trend. Personalized vaccines tailored to individual patient profiles can become a reality with the customizable nature of CDs. These molecules can accommodate varying dosages of antigens or adjuvants for vaccination.

In summary, the potential applications of CDs in future vaccine formulation encompass a range of innovations, from stability improvement and targeted delivery to personalized vaccination. While research and development are essential to harnessing their full potential, these versatile molecules hold the promise of reshaping the future of vaccines, ushering in a new era of immunization strategies marked by improved efficacy, accessibility and versatility.

5. Conclusions

In conclusion, CDs offer promising prospects in vaccine formulation and development in terms of stability, solubility, immunogenicity and targeted delivery. The use of HP-β-CD in the approved SARS-CoV-2 vaccine Ad26.COV2.S has added excitement and speed to studies in this area. While the number of CD-based vaccines on the market remains limited, the rapid growth of research in this area indicates growing interest in, and acceptance of, the potential of CDs to optimize vaccine formulations. Continuing the research on CDs in vaccine development can provide valuable information and contribute to the advancement of vaccination strategies, strengthening the ability to effectively combat infectious diseases and cancer.