Hib Vaccines: Past, Present, and Future Perspectives

2.1. CPS Vaccines and Adjuvants

The idea of using the bacterial CPS in vaccines dates back to 1930s and involves inducing polysaccharide-specific antibodies to protect against pathogenic bacteria [39–41]. The CPS of Hib, polyribosylribitol phosphate (PRP, Figure 1), has several significant characters, such as immunogenicity in humans, the same antigenic properties in all strains of type b, and stimulation of anti-Hib antibodies. In 1985 purified PRP was certified for use in a vaccine in the United States. However, rapid reduction of antibodies stimulated by purified PRP was the main drawback, as it offered no immunological memory [42]. The solution was to give more injections as boosters that took dropping levels of antibodies back up to postvaccination levels, but this failed to generate either strong response nor immunological memory, as injection of the booster did not stimulate IgM class switching to IgG [43]. In Finland, the vaccine was very successful in experiments in children 18 months and older, while in the United States postmarketing surveillance showed fluctuating efficiency (from 69% to 88%) [44]. However, the level of ≥0.15 μg/mL of anti-PRP in unvaccinated children and level of ≥1.0 μg/mL in vaccinated children were considered protective against Hib infections, as suggested by many studies [45].

CPS-specific receptors on the surfaces of native B cells (BCRs) recognize the multivalency and large size of CPSs. Upon immunization with pure CPS, these BCRs bind to the CPSs and the BCR-CPS complex stimulates the B cell to induce and secrete IgM antibodies against CPS. In the event of bacterial infection following CPS immunization, IgM antibodies bind to the CPS expressed on the surface of the particular pathogenic bacteria and help eradicate that pathogen [11, 46].

The compounds that are able to enhance and/or regulate the antigen's immunogenicity are called adjuvants and represent a varied collection of compounds. Put simply, an adjuvant is a vaccine assistant (adjuvare is a Latin word meaning “to help”) and is a synonym of immunostimulant. It can be a combination of one or more compounds that act and function differently: that is, a molecule, carrier or depot, immunomodulator, and/or immunostimulant. The adjuvant industry has grown to meet demand for enhancing the immunogenicity of insufficient vaccines. The two main basic mechanisms by which adjuvants act are enhancing antigen presentation to the immune elements and stimulating immunity so that a stronger or wider response is reached. By these means, immunogenicity can be improved and/or doses of the vaccines can be decreased to a level that has no negative effect on immunogenicity [47, 48].

Animals, plants, and bacteria are sources of widespread immunostimulatory constituents of several adjuvants that function as stimulators of immunological components. One highly effective method of adjuvanticity is the conjugation of inactivated toxoids to purified PRP. This method has been used successfully in anti-Hib, anti-N. meningitidis, and anti-Streptococcus pneumoniae conjugate vaccines. Compounds such as alum and calcium phosphate are the only mineral salts authorized for use as adjuvants with vaccines in the United States since the 1920s. The primary mineral salt adjuvant for use in humans is alum, either as aluminum hydroxide or as aluminum phosphate. It acts by creating a depot spot, such that mild and constant release of the antigen improves presentation to immunological components [47, 48].

Studies have investigated the role of innate cells in adjusting adaptive immunity. Numerous antigen presenting cell (APC) signals are needed to start T-helper responses. First, a signal is activated by presentation of a certain peptide by class II molecules to the T cell receptor (TCR). Without the contribution of an additional signal, abortive and anergy responses are stimulated; a costimulatory second signal is required, via receptor-ligand contacts among APC/T antigens. It appears that another (preceding) signal, the zero signal, is required to stimulate APCs and orient subsequent Th responses, for example, throughout IL12 for Th1 responses. Zero signals are typically stimulated by detection of pathogen-associated molecular patterns (PAMPs) through pathogen recognition receptors (PRR), involving toll-like receptors (TLRs). APCs class I presentation for CD8 stimulation may be controlled by these signals. Promotion of CD4 cells that secrete interferon gamma (IFNg) by Th1 adjuvants is not enough to activate CD8 cytotoxic T cells, which also need class I antigen presentation [48]. These outcomes closely connect native immunity and the following adaptive immunity. The essential signals and steps needed to stimulate T and B cell responses suggest that signals of innate immunity also control the quality (not only the degree) of adaptive immunity. For example, innate cells permit adaptive T and B cells to differentiate and grow, and Th1 polarization is observed throughout IL12 secretion [48]. Adjuvants may be able to perform on any of these signals. Affecting exactly costimulatory molecules (second signal) throughout antibodies, chemokines, or cytokines is an attractive option [49], but it may lead to unfocused overreaction status [48].

While earlier development of adjuvants generated effective products, there is still a need to create new generations, rationally designed based on recent discoveries in immunology, especially innate immunity. The safe and optimal preparation of adjuvants is the most challenging task in the field. Little work has been dedicated to adjuvants with free PRP or with glycoconjugate vaccines, compared to protein vaccines. However, a number of researchers have examined the capacity of several molecules, especially TLR agonists, to increase the immunogenicity of conjugated or free carbohydrate antigens [48]. Free PRP vaccines against Hib are no longer used in the United States [50].

2.2. Conjugate Vaccines and Adjuvants

Most proteins need participation of T cells to stimulate antibody synthesis and consequently are considered T cell dependent (TD) antigens [51]. These proteins promote increased response of antibodies in the booster vaccine and encourage class switching from IgM to IgG via the involvement of T-helper cells in the immune process. Moreover, subsets of B-memory cells are generated from B cells in TD immunity, resulting in the establishment of memory against a specific antigen. This is accomplished by covalently connecting PRP to protein carriers, which results in production of Hib glycoconjugate vaccines [52]. By this kind of conjugation, the humoral immune response is induced with features of TD immunity reactions, such as affinity maturation, memory, and, crucially, the presence of immunogenicity in children aged more than 2 months [30]. Development of protein-conjugated polysaccharide vaccines has been accelerated by increasing antibiotic resistance and the need to stop Hib infections in high-risk populations [12].

In 1931, Avery and Goebel invented the improvement of polysaccharide immunogenicity by the tool of conjugation to proteins. Nevertheless, this was not widely practiced until the invention of polysaccharide conjugate vaccines. In the 1970s through 1980s, the application of bacterial polysaccharides and proteins for stimulating immunity increased. Conjugate Hib vaccines are created by Hib CPS (polyribosylribitol phosphate; 5-d-ribitol-(1→1)-β-d-ribose-3-phosphate; PRP) attached to proteins, with excellent safety and distinctly different structures and conformations [53–55]. The first invented and approved conjugate vaccine in the United States was created from PRP conjugated to diphtheria toxoid (PRP-D), which was withdrawn from the market soon after introduction of more effective vaccines using meningococcal outer membrane protein (PRP-OMP), CRM₁₉₇ (PRP-CRM) protein carriers, or tetanus toxoid (PRP-T) [56].

The structure of polysaccharides and carrier proteins properties largely determine the immunogenicity of the conjugates, and the techniques used in the conjugation process can play a central effect. The conjugation process should be uncomplicated and mild and cause little alteration to the constituents, such that it does not damage important epitopes on either the protein or the PS, cause undesired depolymerization of the PS, or introduce any harmful epitopes. While a number of conjugation chemistries are available for the synthesis of PS-protein conjugates [57], only a few have been used in licensed vaccines. For proteins, surface-exposed amines (e.g., ε amines of lysine residues) and carboxyls (e.g., the side chains of carboxyl of aspartic and glutamic acid residues) are the major groups used for conjugation [58, 59]. Prior to the conjugation process, fermentation/isolation techniques of Hib CPS are used to produce Hib conjugate vaccines on a large scale [60].

Chemical synthesis of the Hib saccharide antigen was a breakthrough in conjugated vaccines, which led to development of a novel type of Hib conjugate vaccine. This generally includes the formulation of the oligosaccharide moiety by chemical or enzymatic methods, which represents the immunological specificity for the vaccine and then attachment of this to an immunogenic protein by a linker. Chemical synthesis of these complex oligosaccharides is difficult and requires a suitable mixture of techniques that allow appropriate treatments by applicable glycosyl donors, acceptors, protecting groups, and coupling reactions settings. This option has come to be accessible due to notable growth of enzymatic and chemical oligosaccharides preparation [61–64]. Currently, the synthetic Quimi-Hib vaccine is approved for use in the National Immunization Program in Cuba [64]. Figure 2 summarizes the types of currently used Hib vaccines according to the type of PRP.

Glycoconjugate syntheses include the usage of well-defined or random activation sites in the polysaccharide as possible attachment sites for proteins. The choice of method for activation is mainly governed by the molecular size of the polysaccharide. Large polysaccharides are frequently arbitrarily activated, while selective activation at the reducing end is typical for oligosaccharides [12]. Molecular differences between conjugation methods do not affect their clinical performance but do influence strategies for quality control [57].

The conjugation process can be divided into the following steps (Figure 3): (I) preparing the carbohydrate, (II) preparing the protein carrier, (III) performing the conjugation, and (IV) finishing. Some conjugation schemes combine several steps, whereas in others a particular step may be unnecessary [58].

Preparation of the Carbohydrate

1. Sizing. Native CPSs have molecular sizes in the millions of Daltons, and solutions tend to be viscous. Reducing the molecular size of the PSs decreases their viscosity and makes the solutions easier to handle.

2. Activation. Carbohydrates have hydroxyl groups, which are relatively inactive and must be translated into a more reactive form (functional group). The activated PS can be either immediately conjugated to the protein or further functionalized. The positions of the activated groups determine the positions of the linkages on the PS.

3. Functionalization. This procedure adds reactive chemical groups to the carbohydrate or converts the activated PS into a more stable form while maintaining its reactivity. Functionalization can include the addition of a spacer molecule between the carbohydrate and the reactive group and in some cases is a multistep process [58].

Protein Carrier Preparation. Amines (the ε amines of lysines) and carboxyls (glutamic and aspartic acids) can be applied to link directly to the activated or functionalized carbohydrate. However, some protocols rely on the addition of chemical groups that are more reactive and/or more specific in their reaction with the activated or functionalized carbohydrate than carboxyls or amines. Functionalization of the protein can also provide a spacer molecule such that the reactive groups on the protein are more accessible to the carbohydrate [58].

Conjugation. Conditions that promote conjugation-high concentrations of protein and PS and high numbers of reactive groups bring the risk of excessive cross-linking when both the protein and the PS have multiple reactive groups. Also of concern is the need to confirm uniform mixing and reaction, which can be challenging on a production scale due to the high viscosity of the PSs. Careful control over factors relevant to the particular chemistry is crucial for successful conjugation. These factors include pH, temperature, the ratio of protein to PS, and the concentration of each. The type of linkage achieved depends on the chemistry and may be reversible or irreversible [58].

Finishing

1. Quenching. Quenching inactivates any residual groups to prevent further cross-linking. This step is commonly completed with monovalent blocking reagents, such as ethanolamine or glycine.

2. Locking. A locking (or blocking) step makes the conjugation linkage fundamentally irreversible.

3. Purification. The conjugate must be purified to eliminate the conjugation reagents and to guarantee low levels of unconjugated carbohydrate and protein. Unconjugated PS has been shown to decrease the immune response to the conjugate [65, 66].

Two models for carbohydrate-protein conjugation have been established: the single ended model (terminal amination-single method) and cross-linked lattice matrix (dual amination method) [12, 57].

3.1. Conjugation Development

The classic antigen presentation hypothesis for glycoconjugate vaccines suggests that helper CD4⁺ T cells identify a peptide originated from the carrier protein [129]. According to the traditional pattern, the glycoconjugate binds to the surface of a B cell whose specific job is to make antibodies to the polysaccharide component. The B cell deals with the protein portion of the glycoconjugate and exhibits a peptide from the covalently linked carrier protein in the setting of the major histocompatibility complex class II (MHC II) to the αβ-TCR of CD4⁺ T-helper cells. Stimulation of T cells by peptide-MHC II complexes and other costimulatory molecules leads to production of the cytokine interleukin 4 (IL-4) by the T cell, which in sequence encourages cognate B cell maturation and subsequent creation of carbohydrate-specific antibodies, with associated class switching of immunoglobulin to IgG and memory responses. This hypothesis was originally based on the theory that only protein antigens can be presented to and identified by T cells. Initial research on hapten-carrier conjugates (i.e., small molecular weight noncarbohydrate molecules connected to carrier proteins) suggested that peptide presentation is the triggering tool for glycoconjugate induced T cell activation [130].

A new study [129] strongly suggests that carbohydrate-recognizing T cells, not peptide-recognizing T cells, are the principal T cell population driving the T cell-mediated adaptive immune response. It shows that carbohydrates, which were previously thought to be “T cell-independent” antigens, can be precisely distinguished by T cells, as they are conjugated with carrier peptides that permit their presentation by MHC II molecules on the surface of APCs. These conclusions are different from the classical understanding that T cells can only recognize peptides [129]. A model new-generation glycoconjugate vaccine [129] has enriched carbohydrate T cell epitopes compared to a glycoconjugate vaccine made by traditional conjugation methods. This model vaccine is 50–100 times more immunogenic than classical vaccines. This type of carbohydrate-recognition T cell may lead to significantly better vaccines against infectious diseases. Elucidation of the T cell epitopes of glycoconjugate vaccines would allow us to imitate those epitopes and enhance vaccines by joining carbohydrate epitopes at a frequency and density that enhances immunogenicity and protection through APCs proficient processing and presentation and highly specific T cell recognition [131]. The zwitterionic polysaccharides are a class of complex carbohydrates that activate T cells [131, 132]. Figure 7 summarizes the most important approaches to the improvement of glycoconjugate vaccines.

The conjugation of biomolecules to metal nanoclusters and nanoemulsions has opened new opportunities in the design and synthesis of multifunctional and multimodal built systems for biomedical uses. Gold nanoparticles have been widely studied for their low toxicity, relative inertness, easy handling, and easy chemistry of surface control. The surface of gold can be shaped in a controlled way with various ligands through thiol chemistry, producing multifunctional and multivalent nanoparticles [101, 133].

Carrier proteins are fundamental components of classical conjugate vaccines. Peptides offer covalently bound carbohydrate epitopes to the TCR of CD4⁺ T cells [134]. Thus, optimization of the MHC II-binding peptide concentration in a conjugate vaccine would theoretically enhance the immunogenicity of the conjugate vaccine by increasing the number of carbohydrates that can be presented on MHC II proteins. The best approach to accomplish this is to connect polysaccharides to MHC II-binding peptides in place of the intact proteins. This would allow the presentation of significantly greater amounts of peptide-bound carbohydrate epitopes to T cells, compared to when intact proteins are. In fact, many studies have shown that conjugating peptides to polysaccharides creates highly effective conjugate vaccines [134–137]. In one study, three polypeptides consisting of strings of 6, 10, or 19 human MHC II-binding peptides from several antigenic proteins were created [138]. In each construct, the MHC II-binding peptides were separated by the lysine-glycine spacer to give flexibility to the polypeptide and to permit the conjugation of the glycan to the amino side chains of the lysine spacer. The 19-valent polypeptide was then conjugated with CPSs of N. meningitides serogroups A, C, W-135, and Y [139]. In immunization tests, the polypeptide-containing glycoconjugate vaccines had greater immunogenicity than glycoconjugates that contained CRM₁₉₇ as carrier protein. Compared to CRM₁₉₇-based conjugates, smaller quantities of polypeptide-based conjugates stimulated a higher bactericidal antibody titer against all four meningococcal polysaccharides. This strategy illustrates the use of MHC II-binding polypeptides as carrier molecules to create greatly effective glycoconjugate vaccines. The main improvement enabled by this strategy is that these synthetic polypeptide carriers can be identified by most of the human MHCII haplotypes. Furthermore, peptide carriers can be expressed in E. coli, which makes them easy to produce for production of glycoconjugate vaccines [11].

An important step in conjugation chemistry is to improve conjugation efficiencies to levels at which residual unconjugated components, particularly free PSs, do not affect the stimulation of protective immune responses. The use of an effective, mild conjugation chemistry would allow for higher yields of vaccine. For example, reaching more than 90% efficiency of coupling of the PS may allow for a simplified purification process. It would also be needed for development of conjugate vaccines that do not require refrigeration [58].

Current improvements in carbohydrate synthesis must simplify the enzymatic and chemical preparation of well-characterized and well-defined homogenous carbohydrate antigens for further development of glycoconjugate vaccines. Several carbohydrate antigens analogs that are stable metabolically, such as S-glycosides and C-glycosides, may enhance the immunogenicity and antigenicity of the glycoconjugate vaccines. Moreover, such analogs should expand our knowledge of antibody carbohydrate identification, processing and presentation of antigens, and immune system stimulation. Such improved knowledge of glycoimmunology would facilitate the creation of the ideal glycoconjugate vaccine for a particular disease or infection. Synthetic C-glycoside analogs of tumor-combined antigens, which are metabolically stable, nonhydrolysable carbohydrates, would similarly enhance batch-to-batch consistency in vaccine manufacturing and bypass the cold storage that is generally needed to preserve and guarantee effectiveness of existing carbohydrate-based vaccines [12].

Recently, a study reported highly immunogenic PRP-TT conjugates generated from shorter chain PRP, compared to both their high-molecular-weight counterparts from the same laboratory and licensed vaccines. The authors have optimized methods to prepare more immunogenic low-molecular-weight PRP-TT conjugates in a reproducible manner. The higher reactivity of hydrazide groups compared to the lysine ε-amino group resulted in a shorter conjugation time and an optimal yield. The conjugates thus created are immunogenic in rats. A hallmark of this study is the development of highly immunogenic PRP-TT conjugates using short-chain PRP, which forms the basis for further development of oligosaccharide conjugates as successful vaccine candidates alone or in combination [140].

3.2. Conjugation through DNA Biotechnology

Generally, chemical approaches that allow for well-controlled conjugation reactions between distinct-length glycans and proteins [141–143] are vital for designing highly immunogenic vaccines. To date, the most effective vaccines used for protection from invasive pathogenic bacterial infection are the glycoconjugate vaccines, mainly consisting of bacterial capsular polysaccharides coupling to a desired protein carrier. Within the pathogenic bacterial strains there is effective single CPS component and/or multiple CPS from different strains. The later condition makes glycoconjugate vaccine production a very hard process, since each strain requires extraction, hydrolysis, chemical activation, and conjugation to a carrier protein. To avoid this hard situation for conserved and/or nonconserved CPS of different bacterial pathogen, the use of bioglycoconjugates machinery of innovative E. coli will substantially simplify the production of glycoconjugate vaccines.

DNA biotechnology has revolutionized vaccinology and generated new methods for vaccine discovery, such as antigenome technology, reverse vaccinology, surfome analysis, genetics vaccinology, and immunoproteomics to uncover innovative immunogenic antigens [144]. Genetically engineered vaccines are expected to be a major type of future vaccines due to their high safety level, improved immunogenicity, and decreased reactogenicity. However, absence of posttranslational modifications (PTMs) machinery in E. coli limits its use for the production of recombinant biopharmaceuticals and/or biosimilars [145–152]. Various posttranslational modifications including glycosylation and phosphorylation, which are critical for functional activity, do not take place in E. coli [149–153]. Protein glycan coupling technology (PGCT) is a strategy that has been used to move toward the target of making structurally defined, homogenous glycoconjugate vaccines with a well-controlled conjugation reaction [154–156]. N-linked glycosylation of proteins is one of the most important posttranslational modifications in eukaryotes. Kowarik et al. identified a novel N-linked glycosylation pathway in the bacterium Campylobacter jejuni and also showed the successful transfer of functionally active N-glycosylation pathway into E. coli [157]. PGCT uses the bacterial glycosyltransferase enzyme Pg1B, an oligotransferase expressed in Campylobacter jejuni, to enzymatically link a bacterial polysaccharide with a carrier protein. Pg1B specifically recognizes a 2-acetamido-containing a reducing end sugar and glycosidically links it to a carrier protein that has a specific sequence identified by the Pg1B enzyme for N-linked glycosidation. The enzymatic conjugation, and glycan and protein biosynthesis, occurs in an E. coli host strain. The target glycan, protein, and Pg1B are cloned and expressed in E. coli. This technology removes the necessity for multistep production and purification of the glycan and protein and several steps of chemical conjugation. Consequently, the process of creating glycoconjugate vaccines is shortened and vaccine production is enhanced. Furthermore, current chemical conjugation strategies require alteration of the polysaccharides or proteins (e.g., random oxidation of the sugar chain), which may change the natural epitopes and lead to generation of less-effective conjugates. Additionally, enzymatic conjugation uses highly specific substrates with distinct binding domains, and as a result, the conjugation outcomes are homogenous and structurally defined. Finally, PGCT avoids the need for pathogenic bacteria from which polysaccharides and/or proteins are isolated, as both glycan and protein synthesis and glycoconjugate assembly take place completely in an E. coli system [156].

Although the bacterial N-glycan structure is not similar to that seen in eukaryotes, engineering of glycosylation pathway of C. jejuni into E. coli has not paved the way for expressing and production of glycosylated protein E. coli [158, 159], but the glycoconjugate vaccines too [154, 156, 160–164], which will avoid the chemical conjugation vaccines producers for the harsh capsular polysaccharide-protein conjugation chemical reactions. The coming challenge for synthesis of glycosylated proteins in the engineering E. coli is the robotic machinery for secretory and extracellular N-linked glycoproteins production [165].