3.1. Vaxjo Statistics
Vaxjo is a web-based relational database and analysis system based on a classical three-tier architecture design [15
]. Currently, Vaxjo has included 103 vaccine adjuvants. In total, 384 vaccines have been detected to use these vaccine adjuvants. Occasionally, more than one vaccine adjuvant is used in a single vaccine. These 384 vaccines are developed against infections of 76 pathogens, such as Bacillus anthracis
spp. (brucellosis), influenza virus, and Plasmodium
spp. (malaria). Vaxjo also stores 15 vaccine adjuvants that have been used for vaccine development against many cancers and allergies.
3.2. Analyses of Vaxjo Vaccine Adjuvants Based on Vaccine Adjuvant Types
With the establishment of the Vaxjo vaccine adjuvant database, we were able to systematically analyze the vaccine adjuvants and their usages in vaccine development. shows the types of vaccine adjuvants and lists those adjuvants which have five or more associated vaccines. The adjuvant types are based on the derivation of the adjuvants or the composition of the adjuvants. The largest numbers of vaccine adjuvants are found in microorganism-derived adjuvants (18), synthetic adjuvants (28), and mineral salt adjuvants (13) (). If the numbers of vaccines using different adjuvants are considered, vaccine adjuvants based on mineral salts, emulsions, and microorganism derivatives have been used in 151, 115, and 73 vaccines, respectively (). Aluminum hydroxide is the most common adjuvant found, with 62 associated vaccines collected in VIOLIN. Freund's complete and incomplete adjuvants are also commonly used with each being associated with 42 vaccines. Although 28 synthetic adjuvants are curated in Vaxjo, only 28 vaccines listed in VIOLIN use these synthetic adjuvants. Only two of these synthetic vaccine adjuvants collected in Vaxjo are not associated with any vaccines in VIOLIN.
Curated vaccine adjuvants used in at least five vaccines curated in VIOLIN.
Those commonly used vaccine adjuvants and their associated vaccines are introduced below.
3.2.1. Salt-Based Vaccine Adjuvants
Mineral salt adjuvants are commonly used as aluminum adjuvants that are approved for use in licensed vaccines. lists the 13 mineral salt adjuvants in Vaxjo and displays their properties, including the number of associated vaccines to each adjuvant, mechanism of action, route of administration, and whether or not the adjuvant is used in licensed vaccines. Of the mineral salt adjuvants in Vaxjo, 10 are licensed for use in vaccines either in Europe or the United States. Most mineral salt adjuvants act as vehicles for antigens rather than as immunostimulants ().
Mineral salts, specifically aluminum salts, have been used as adjuvants for decades and are therefore common in vaccine formulations [3
]. Alum salts have a depot effect, allowing the antigen to persist in the body so the immune system can react to the antigen and facilitate uptake into antigen-presenting cells (APCs). Aluminum also enhances the antigenicity of some vaccines such as diphtheria and tetanus toxoids [18
]. Aluminum-adsorbed diphtheria and tetanus toxoids are more effective than plain fluid toxoids in primary immunization of children. However, there is little difference between plain and adsorbed toxoids for booster immunization [19
]. Immune responses to proteins adjuvanted with alum tend to be a T helper cell type 2 (Th2) response [20
]. The Chapter 21 of the US Code of Federal Regulations (610.15(a)) limits the amount of aluminum in human vaccines to 0.85
mg/dose. The majority are parenterally used, rather than mucosally. The amount of an aluminum adjuvant in vaccines currently licensed in the US ranges from 0.125 to 0.85
]. In more than six decades, aluminum vaccine adjuvants usually do not induce serious adverse effects. However, they often induce local reactions such as redness, swelling, and tenderness at the injection site. Occasionally, aluminum adjuvants were found to be associated with severe local reactions such as erythema, subcutaneous nodules, and contact hypersensitivity [19
3.2.2. Emulsion-Based Vaccine Adjuvants
Emulsion based adjuvants are oil-in-water or water-in-oil emulsions, which work to enable slow release of an antigen at the injection site. Fourteen emulsion-based vaccine adjuvants have been curated and stored in Vaxjo (). With the exception of the nanoemulsion vaccine adjuvant [21
], all the emulsion adjuvants in Vaxjo are delivered via the parenteral route. Nanoemulsion vaccine adjuvant is delivered mucosally [21
]. Water-in-oil emulsion adjuvants turn to cause high levels of reactogenicity. Oil-in-water emulsion adjuvants turn to have a low reactogenicity profile. MF59 and AS03 are two licensed oil-in-water emulsion adjuvant used in humans. MF59 is not toxic and prepared with a low content of squalene (4.3% w/w), a biodegradable oil naturally found in plants and animals including humans. In humans, squalene is an intermediate organic compound in the steroid hormone biosynthetic pathway and is a direct synthetic precursor to cholesterol. MF59 induces low reactogenicity at the site of injection. MF59 is able to induce fast priming of antigen-specific CD4+ T-cell responses and to induce strong and long-lasting memory T- and B-cell responses [22
]. MF59 has been licensed in more than 20 countries for more than 14 years. It has been used in many licensed vaccines such as Fluad, Focetria, and Aflunov. AS03 (for “Adjuvant System 03”) is another squalene-based immunologic adjuvant used in various vaccine products by GlaxoSmithKline (GSK). AS03 is used, for example, in GSK's A/H1N1 pandemic flu vaccine Arepanrix H1N1.
3.2.3. Microorganism-Based Vaccine Adjuvants
Microorganism-based vaccine adjuvants are adjuvants that are derived from microorganisms. Bacteria have an abundance of capacity for stimulating the immune system, and the adjuvants derived from bacteria are abundant. As whole bacteria are generally too toxic to use as an adjuvant, many bacterium-derived vaccine adjuvants are derived from parts of bacteria [1
]. displays the properties of the 18 microorganism-based adjuvants in Vaxjo. Only two of the microorganism-based adjuvants are licensed: MPL licensed in the United States as part of the AS04 adjuvant formulation [23
], and Bordetella pertussis
component vaccine adjuvant [24
] that is used in several licensed vaccines. Microorganism-derived adjuvants cholera toxin and MPL are used in 17 and 10 vaccines, respectively. Many microorganism-derived adjuvants are based on microbial nucleic acids. For example, CpG DNA is the unmethylated CpG dinucleotides in what is called motifs, which are abundant in microbial DNA, but not in the DNA of vertebrates [25
]. CpG DNA is immunostimulants that have the ability to activate Th1 immunity [26
All but one microorganism-derived adjuvants act as immunostimulants as their mechanism of action. Adjuvants derived from the toxins of bacteria are used to stimulate mucosal immunity, which is important for protecting against many pathogens that are contracted mucosally [27
]. The route of administration is able to reveal what type of immunity the adjuvant can stimulate, with eight microorganism-derived adjuvants being mucosally administered (generally intranasally). These are adjuvants which can help to stimulate mucosal immunity which is often critical in inducing protection from pathogens that are contracted mucosally. Six of the adjuvants are administered both parenterally and mucosally, and five are parenterally administered ().
3.2.4. Other Vaccine Adjuvants
The other six adjuvant types are synthetic adjuvants, cytokine adjuvants, particulate antigen delivery systems, carbohydrate vaccine adjuvants, tensoactive compounds, and combination vaccine adjuvants ().
Synthetic adjuvants are synthetically made compounds that mostly are utilized as immunostimulants and occasionally act as carriers. There are 28 synthetic adjuvants in Vaxjo. Some are derived to mimic bacterial components (). For example, muramyl dipeptide (MDP) is formulated to imitate a component found in the cell walls of mycobacteria used in Freund's complete adjuvant [28
]. In addition, many synthetic MDP analogs are used as vaccine adjuvants. Administration routes are diverse, as there are parenterally, mucosally, and also topically administered synthetic vaccine adjuvants.
Cytokines, signaling molecules of the immune system, are also used as adjuvants to elicit a specific immune response [23
]. There are nine cytokines as adjuvants in Vaxjo (). For example, granulocyte-macrophage colony-stimulating factor (GM-CSF), a cytokine that activates mature granulocytes and macrophages, is being used as an adjuvant for Hepatitis B in immunocompromised patients [29
]. The GM-CSF adjuvant is also used in vaccines for HIV and cancer [30
]. Currently, none of cytokine-based adjuvants are licensed.
Particulate antigen delivery systems are adjuvants that have a depot effect for the antigen and include liposomes, polymeric microspheres, virus-like particles, and immunostimulating complexes (ISCOMs) [13
]. There are nine particulate antigen delivery systems in Vaxjo. These adjuvants act as vehicles for the antigen, while often also containing immunostimulating compounds. They are parenteral adjuvants. Liposome-based adjuvants consist of spheres formed by lipid layers that can encapsulate antigens and thus act both as a vehicle and an immunostimulant [1
]. Six liposome-based vaccine adjuvants are listed in Vaxjo. MTP-PE liposomes are liposomes that contain a synthetic analog of MDP, MTP-PE. It is used in vaccines for hepatitis B and herpes simplex virus [32
Carbohydrate adjuvants are adjuvants composed of complex carbohydrates of natural origin that stimulate the immune system. The main sources of these carbohydrates are plants and fungi [1
]. For example, gamma inulin is a carbohydrate derived from the root of plants of the Compositae
family. Gamma inulin is an activator of humoral and cellular immunity as well as the alternate complement pathway [1
]. Two carbohydrate adjuvants are curated in Vaxjo (). They are generally parenterally administered and are immunostimulating adjuvants.
Tensoactive compounds are surfactants or active surface agents. They are immunostimulating adjuvants generally derived from plants, namely, Quillaja saponaria.
There are five tensoactive adjuvants in Vaxjo (). Most saponins are considered too toxic for human use, although Quil A has been used in animals [1
]. The saponins act as a vehicle/immunostimulant and are parenterally administered.
Combination vaccine adjuvants include two or more different adjuvants that are used in combination. Examples of combination adjuvants include adjuvants such as AS04 and Algammulin. There are six such adjuvants in Vaxjo (). These adjuvants combine different mechanisms of action from two or more adjuvants [13
]. For example, AS04, a licensed vaccine in the United States, is a combination of aluminum and MPL [34
]. Alum and MPL complement one another: Alum provides signals required for generating long-lived memory cells, whereas MPL enhances the differentiation of cytotoxic T lymphocytes (CTL) [35
3.3. Analysis of Vaccine Adjuvants Based on the Pathogen Types
displays adjuvanted vaccines by pathogen/disease types and vaccine types. Of the pathogens/diseases in VIOLIN, 46.2% have vaccines that use adjuvants. Gram-positive pathogens and parasites are associated with a high percentage of adjuvanted vaccines, with 79% and 71% of species containing adjuvanted vaccines, respectively. Meanwhile, 45.8% of Gram-negative pathogen vaccines use adjuvants. Of 1,982 viral vaccines in VIOLIN, only 4% are adjuvanted. For the cancer and allergy vaccines in VIOLIN, 20.83% use adjuvants. 13% of all the vaccines in VIOLIN are adjuvanted.
Vaccines with adjuvants by vaccine-targeted pathogens or diseases.
To develop vaccines for a specific pathogen, various strategies, including different vaccine adjuvants, may have been used. The choice of using different vaccine adjuvants may be dependent on the type of protective immunity required for the specific pathogen. As an example of illustrating these points, here we summarize and compare all vaccine adjuvants used in development of Brucella
, a Gram-negative intracellular pathogen that causes brucellosis in humans, is a disease for which there is no licensed human vaccine. Although humoral immune response against Brucella
lipopolysaccharides (LPS) provides partial protection, the cell-mediated immunity plays a major role in protection against virulent Brucella
]. In VIOLIN, 45 Brucella
vaccines are curated. Of these 45 Brucella
vaccines, nine use adjuvants (). Among these nine vaccines, six are subunit vaccines [38
]. This phenomenon follows the trend of subunit vaccines being the majority of vaccines that use adjuvants. Among three of these six subunit vaccines that use Freund's adjuvants, two of them use complete Freund's adjuvant for the primary immunization and incomplete Freund's adjuvant for the boost vaccination [40
]. Three other vaccines are not categorized as subunit vaccines [43
]. Among them is a vaccine that uses a DNA vaccine for priming and uses a recombinant protein adjuvanted by incomplete Freund's adjuvant for boosting [45
]. The NPAP Brucella
vaccine is a killed vaccine that uses Freund's incomplete adjuvant [43
]. The recombinant O. anthropi
Cu/Zn SOD is live vaccine that uses with CpG DNA adjuvant [44
]. The CpG DNA adjuvant is able to bias the immune response to the Brucella
antigen towards a cell-mediated immunity. The recombinant O. anthropi
Cu/Zn SOD alone without the CpG DNA adjuvant is not able to induce protection, indicating the importance of the cell-mediated immunity in the protection induction [44
Brucella vaccines with adjuvants in VIOLIN.
Although most subunit vaccines use adjuvants, some subunit vaccines do not use adjuvants. For example, two of these Brucella
subunit vaccines contain Brucella
lipopolysaccharide (LPS) as a primary antigen that can also perform as an adjuvant-like function by inducing cell-mediated immunity [46
]. The recombinant chimera vaccine BLSOmp31 includes a known Brucella
Omp31-derived protective epitope fused to Brucella
lumazine synthase (BLS) [48
]. The BLS is highly immunogenic and functions as a scaffold protein and a carrier for the foreign peptide [49
3.4. Analysis of Vaccine Adjuvants Based on Vaccine Types
The majority of adjuvanted vaccines, with 245 out of 384 vaccines (63.8%), are subunit vaccines. Other types of adjuvanted vaccines stored in VIOLIN include toxoid vaccines (8%), killed vaccines (7%), DNA vaccines (4%), and conjugate vaccines (3%). Forty-three vaccines in VIOLIN contain more than one adjuvant (http://www.violinet.org/vaxjo/stat2.php
). In many cases, a primary vaccination uses one adjuvant, and then a boost immunization uses another adjuvant. Alternatively, one vaccine includes more than one adjuvant for one vaccination.
A subunit vaccine is composed of a purified protein(s) or other antigenic determinant(s) from a disease-causing organism. A subunit vaccine does not include pathogen nucleic acids. The subunits have less risk of causing adverse reactions. Subunit vaccines usually consist of specific proteins targeted to generate protection; however, they are rarely immunogenic on their own to trigger a response that will generate protection [50
]. The very nature of subunit vaccines generates a need for adjuvantation. The vast majority of vaccines using adjuvants are subunit vaccines, representing 63.8% of the adjuvanted vaccines in VIOLIN (). In Vaxjo, 53 of the vaccine adjuvants are used in subunit vaccines. Most of these adjuvants have not frequently been used and occur only in one or two vaccines. A few are used more frequently, such as aluminum hydroxide, aluminum phosphate, Alhydrogel, Freund's complete and incomplete adjuvants, and cholera toxin.
The most frequently adjuvanted killed vaccines, with 19 out of 26, are viral vaccines (). Twelve adjuvants have been used in killed vaccines, with the most frequently used being aluminum hydroxide. The list of the US licensed vaccines that contain aluminum adjuvants include Anthrax vaccine, Hepatitis A and B vaccines, Rabies vaccine, Human papillomavirus vaccine, Pneumococcal conjugate vaccine, DTP (diphtheria-tetanus-pertussis vaccine) [19
]. However, licensed human inactivated Polio Virus (IPV) vaccine, measles, mumps and rubella vaccine (MMR), varicella vaccine, and influenza vaccines do not contain any aluminum salt vaccine adjuvant [50
]. Some of the other adjuvants used in killed vaccines in research are QuilA, aluminum phosphate, AS03, AS04, AF03, and Ribi vaccine adjuvant.
A toxoid vaccine consists of a toxoid (also called inactivated toxin), which is a processed toxin that has been treated by chemical means, heat, or irradiation and is no longer capable of causing disease. Toxoid vaccines, which are similar to subunit vaccines, are also usually adjuvanted, typically with aluminum. There are nine adjuvants associated with 32 toxoid vaccines (), which include aluminum adjuvants, squalene, and nanoemulsion vaccine adjuvant. The most frequently used is aluminum phosphate, which is used in nine toxoid vaccines.
A conjugate vaccine is vaccine that conjugates/links antigens to the molecules that form the outer coat of disease-causing bacteria to promote an immune response. Conjugate vaccines are not often adjuvanted, as they are usually immunogenic enough to not need an adjuvant. However, there are seven adjuvants in Vaxjo that are used in 13 conjugate vaccines (), including aluminum hydroxide, DDA, Ribi vaccine adjuvant, and MPL.
Live vaccines generally do not need adjuvants as they are rather immunogenic on their own. However, there are five vaccine adjuvants in Vaxjo that are used in four live attenuated vaccines (). These include aluminum hydroxide, Freund's complete and incomplete adjuvants, DHEA vaccine adjuvant, and Arlacel A. These three vaccines are for protection against Venezuelan equine encephalitis virus (VEE), cancer, smallpox, and E. maxima. For these vaccines, the adjuvant was given prior to immunization with the live attenuated vaccine.
Similar to live attenuated vaccines, DNA vaccines are not often adjuvanted. As for DNA vaccines, there are also eight adjuvants used that are in Vaxjo. There are 14 DNA vaccines that use adjuvants use GM-CSF, Resiquimod, and DDA among others ().
3.5. Ontology-Based Vaccine Adjuvant Representation for Exchange, Transfer, and Download of Vaccine Adjuvant Data
The basic information of vaccine adjuvants in Vaxjo is now stored in the Vaccine Ontology (VO; http://www.violinet.org/vaccineontology/
]. A biomedical ontology is a set of terms and relations that represent entities in a biomedical domain and how they relate to each other. Ontology terms are associated with documentation and definitions, which are, ideally, expressed in formal logic in order to support automated reasoning [51
]. VO is a community-based ontology in the vaccine domain. It is developed based on the Web Ontology Language (OWL) format (http://www.w3.org/TR/owl-ref/
), which can be processed by many existing software programs and used for Semantic Web applications. The storage of vaccine adjuvants in VO allows development of new software programs to integrate the vaccine adjuvant data with other biomedical data and support further bioinformatics analyses.
provides an example of how a VO vaccine adjuvant term “aluminum vaccine adjuvant” is shown in VO. The top level terms “entity → continuant → independent continuant → material entity” come from the Basic Formal Ontology (BFO: http://www.ifomis.org/bfo/
), which is a top ontology that covers the universal entities. A continuant (e.g., a person or a quality) is an entity that exists in full at any time. An independent continuant (e.g., a person which is a material entity, but not a quality) is a continuant that bears its quality and is independent of other entities. All the other terms in the hierarchy shown in are VO-specific terms. As a part of a vaccine, a vaccine additive (e.g., preservative and adjuvant) is a material entity that is added to the immunogen of a vaccine. An aluminum vaccine adjuvant is a mineral salt vaccine adjuvant, a common vaccine adjuvant type (). indicates all vaccines that use this aluminum vaccine adjuvant (including all it child terms). One advantage of this OWL format is that it can be parsed and read by many computer programs. The machine readable feature supports automated reasoning.
Figure 3 Modeling and application of vaccine adjuvants in the Vaccine Ontology (VO). (a) The hierarchical structure of vaccine adjuvants in VO. (b) All the vaccines that use this adjuvant. The usage information is obtained by a SPARQL query based on the logical (more ...)
3.6. Vaxjo Data Query and Display
The manually curated and precomputed Vaxjo data can be efficiently queried and visualized as demonstrated in . The vaccine adjuvant can be queried by specifying one or multiple criteria: (1) a characteristic of a vaccine adjuvant, including name, alternative name, adjuvant VO ID, description, components, and storage, (2) vaccine type, and (3) pathogen (). All query hits are first displayed on a web table containing basic adjuvant information (). Individual vaccine adjuvant can further be selected by a user to show more detailed information (). The associated vaccines are viewed by clicking on the number of associated vaccines ().
Figure 4 An example of searching a vaccine adjuvant and its related information in Vaxjo. (a) The keyword “aluminum” is queried in Vaxjo. (b) The adjuvants resulting from the Vaxjo keyword search. (c) The Vaxjo page obtained by clicking on “Aluminum (more ...)