Although there have been numerous investigations into the immunogenic properties of PS vaccines, the methods for producing and purifying them on a large scale are not widely known, and the available literature on the subject is limited, consisting mainly of patents. The virulence of Haemophilus influenzae (H. influenzae) type b (Hib) is primarily due to its capsular polysaccharide (CPS), a repeating polymer of ribosyl ribitol phosphate (PRP). The process of purifying PRP commences with the inactivation of cellular components utilising agents such as phenol, formaldehyde, or thimerosal, followed by the isolation of the culture medium through centrifugation. Subsequently, the clarified supernatant is concentrated via tangential flow ultrafiltration using a membrane with a molecular weight cut-off of 50 kDa, after which the polysaccharides are precipitated with the cationic detergent cetyltrimethylammonium bromide (cetavlon), after which the resulting PRP-cetavlon complex is solubilised with calcium chloride, accompanied by multiple rounds of ethanol precipitation and desproteinization through phenol extraction and additional ethanol precipitation. Ultracentrifugation techniques are used to separate lipopolysaccharides (LPS) from the PRP (12).

Researchers in the laboratory have developed a new technique for purifying vaccines against Neisseria meningitidis (N. meningitidis) serotype C and Streptococcus pneumoniae (S. pneumoniae) serotypes 23 and 6B, the process of phenol precipitation, employed for desproteinization, and the technique of ultracentrifugation, utilised for the elimination of LPS, have been supplanted by enzymatic treatment and ultrafiltration in conjunction with a chelating agent and detergent. The newly implemented methodologies offer the advantage of omitting toxic and corrosive solvents like phenol while reducing the volume and frequency of ethanol precipitation—a reagent known for its explosive potential. Furthermore, ultracentrifuges are expensive, and the process’s operational capacity is limited by the volume each centrifuge can accommodate. Despite the potential for enzymatic treatment and tangential ultrafiltration to augment downstream processing expenses, the novel purification approach described herein stands out for its simplicity, efficiency, non-toxicity, scalability, and environmental sustainability (12). This method was later improved upon; the culture broth underwent centrifugation for cell separation, followed by modified tangential flow ultrafiltration as per Takagi et al. (13). A polyethersulfone spiral membrane with a 100 kDa nominal molecular weight cutoff was utilised for the washing of the concentrated sample using six different buffer volumes. The concentrated fraction, termed first concentration by tangential ultrafiltration—1CTUF100, was precipitated with ethanol, yielding soluble and insoluble fractions. The PS was extracted from the insoluble fraction using deionised water, with subsequent centrifugation yielding a water-soluble fraction. The pH of the water-soluble PS was adjusted and subjected to enzymatic treatments involving endonuclease and various proteases under specified conditions. The enzymatic hydrolysis was followed by diafiltration using the same conditions as the initial concentration, producing the Purified PSb—Enz+ 2CTUF100 fraction. Previous studies indicated a 68% yield of purified PS; however, the purity of PSb relative to protein was inadequate. Additional washing with detergents and chelating agents was incorporated into the initial and final purification steps to enhance purity (14).

Centrifugation techniques are commonly utilised within the pharmaceutical industry to segregate bacteria from the culture broth. Nevertheless, tangential microfiltration systems provide a cost-efficient substitute for centrifugation, rendering it an appealing choice for reducing production expenditures. Specifically, in manufacturing the Hib vaccine, the substitution of centrifugation with tangential microfiltration has the potential to lower expenses and enhance the availability of the vaccine in economically disadvantaged areas. Centrifugation incurs more significant capital costs relative to filtration. Therefore, employing a reusable tangential microfiltration system may enhance cost-effectiveness in Hib vaccine production. In PRP isolation, Hib culture broth undergoes centrifugation to eliminate bacterial cells. The PS, approximately 622 kDa, is purified from the supernatant through tangential ultrafiltration after ethanol precipitation. The selection of microfiltration systems for Hib cell separation is influenced by membrane material properties, device architecture, and process parameters, including pressure and temperature. For clarity, these factors are categorised as equipment and process parameters. First, the membrane material’s composition significantly affects recovery rates, with distinct outcomes observed for Durapore, hollow fibre, and Hydrosart membranes. Second, membrane architecture influences fouling prevention and enhances turbulence and shear rate during the culture broth’s flow. For instance, the Prostak membrane exhibited lower recovery than the Pellicon membranes despite identical material. Third, the membrane area relative to culture broth volume is critical to prevent overloading. Process duration and feasibility are also contingent on transmembrane flow, which correlates with membrane area. Process parameters are primarily regulated by retentate pressure and feed flow. Small-scale trials revealed that increased transmembrane pressure and feed flow enhance filtrate flux. It was also noted that PRP retention in membranes decreases linearly with rising TMP, while feed flux is similarly influenced. The scaling of the process from 50 cm² to 1 m² and 0.3 L to 7 L of broth demonstrated a linear relationship. Pilot-scale separation achieved approximately 87% PRP recovery, while lab-scale experiments neared 100%. In summary, tangential filtration is a compact, eco-friendly method showing promising outcomes for cell separation and PRP recovery from Hib. While further optimisation of the pilot scale process is necessary, tangential microfiltration presents a viable alternative to centrifugation (15).

The purification of capsular polysaccharides (CPS) is essential in developing vaccines against S. pneumoniae, whether composed of CPS alone or CPS linked to proteins. Although these vaccines are effective and safe, they are costly. There have been endeavours to enhance the efficiency and output of CPS purification methods and to streamline the process. Numerous publications have addressed the purification of S. pneumoniae CPS. These works typically present enhancements to initial procedures that improve purification outcomes. Following fermentation, it is common practice to isolate cell cultures via centrifugation and lyse them with sodium deoxycholate. Alternatively, some researchers introduce sodium deoxycholate post-fermentation but pre-cell collection, extracting CPS from the resulting supernatant or filtrate. A preferred method involves bacterial inactivation with thimerosal and subsequent cell removal via filtration, as detergent lysis can compromise cell membranes, releasing intracellular contaminants that complicate purification. The primary contaminants necessitating removal are proteins, nucleic acids, and cell-wall carbohydrates. The World Health Organization (WHO) stipulates that acceptable protein and nucleic acid contaminant levels are below 3% and 2%, respectively. Classical processes emphasise the necessity of contaminant removal and the drawbacks of specific toxic reagents, which introduce significant intracellular contaminants. Consequently, incorporating additional purification steps complicates the process, reducing yields and increasing costs. Innovative capsule purification methods have been evaluated to enhance yield and purity. Despite removing or substituting some laborious steps, a multistep approach remains prevalent. Recently developed purification techniques seek to improve CPS purity and yield with a more streamlined workflow. Most methods are tailored to specific CPS types but can potentially be modified for others with analogous physicochemical characteristics (16–20). While continuous culturing and chemically defined media can aid in simplification. Adjusting the composition of growth media and culture conditions to optimise CPS production is a crucial research focus (17).

Experimental investigations were conducted to assess the impact of membrane pore dimensions, filtrate flux, and solution parameters on the transmittance of both the conjugate and the unbound PS across various ultrafiltration membranes. The purification of the conjugate was executed utilising diafiltration carried out within a linearly scalable tangential flow filtration (TFF) cassette. A removal efficiency exceeding 98% of the unbound PS was achieved within a five-diavolume diafiltration procedure, representing a notable advancement compared to previously documented outcomes for the purification of analogous conjugated vaccines. These findings unequivocally illustrate the potential for ultimately employing ultrafiltration and diafiltration techniques to purify conjugated vaccine formulations (21).

A novel methodology is engineered for universal applicability across all 15 serotypes in an imminent 15-valent conjugate vaccine. A decision-making step has been integrated to ascertain the utilisation of supernatant or precipitate based on the charge of the target CPS post-CTAB precipitation. This technique amalgamates various methods, including acid and ethanol precipitation alongside HA chromatography, to attain elevated CPS purity and minimise cell wall polysaccharide (CWPS) contamination. This approach notably prioritises CWPS reduction, a critical contaminant in CPS purification, diverging from conventional studies that mainly focus on nucleic acid and protein impurities. While traditional multi-step methods tend to diminish CPS yield, this strategy markedly lessens CWPS contamination. A comparable method utilised in the production of Prevenar 13, a reference vaccine in the immunogenicity assessment, also employs CTAB precipitation; however, it differentiates by utilising NaI for CTAB precipitation, whereas this method adopts ethanol precipitation for CPS. In summary, this CPS purification methodology integrates established separation techniques, such as ultrafiltration, CTAB precipitation, and chromatography, yet confronts challenges related to CWPS contamination. Process optimisation was conducted to mitigate these issues, significantly reducing CWPS levels, which subsequently improved immunogenicity when the CPS was incorporated into a conjugate vaccine. Refinement of the primary purification processes, notably extensive ultrafiltration/diafiltration and impurity precipitation, resulted in a substantial decrease in CWPS concentrations. ELISA and OPA evaluations in animal models further indicated that enhanced CPS quality positively influenced the immunogenicity of the multivalent pneumococcal conjugate vaccine. These results underscore that the diminished CWPS contamination, attained through this optimised methodology, significantly elevates CPS quality and facilitates the advancement of innovative multivalent conjugate vaccines against pneumococcal diseases (22).

A new study investigated a novel aeration and sterilisation approach using formaldehyde or β-propiolactone (BPL) to increase soluble PS yield and inhibit bacterial lysis. Furthermore, a refined CPS purification protocol was established, utilising ultrafiltration, diafiltration, acid and alcohol precipitation, and concluding with diafiltration and lyophilisation for pure PS production. CPSs prepared via formaldehyde and BPL exhibited markedly lower residual impurities than those produced through conventional deoxycholate sterilisation. This innovative CPS preparation methodology was also shown to be scalable for PS vaccine manufacturing (23).

Efficiencies in the purification processes for S. pneumoniae, H. influenzae type b, and N. meningitidis have been achieved by minimising ethanol precipitations and discontinuing phenol. Various modifications and techniques, including protease digestion, diafiltration, O-acetylation, hydrophobic interaction chromatography, and enzymatic hydrolysis, have been suggested for meningococcal and Hib CPS, which could be considered for pneumococcal CPS purification. The choice of downstream procedures, such as ethanol precipitation and refining steps, depends on serotype variations and CPS characteristics. Nonetheless, further research is required in the realm of pneumococcal vaccine production (17).

The virulence of various N. meningitidis serogroups is attributed to their distinct PS capsules. Among the 13 identified serogroups of N. meningitidis, serogroups A and C represent the most predominant strains associated with meningococcal diseases that are preventable through conjugate vaccination. Consequently, a necessity exists for developing cost-effective multivalent meningococcal conjugate vaccines, ideally incorporating conjugates for serogroups A and C, which can be accessible to populations in low-income countries. The advancement in the synthesis of PS conjugate vaccines represents a significant leap forward in preventing bacterial infections, exemplified by meningococcal disease. Nevertheless, developing nations encounter considerable obstacles in producing these vaccines, primarily attributable to the high costs and intricate nature of the purification methodologies necessitated to achieve the stringent purity levels required for the efficacy and safety of vaccines. The absence of accessible and streamlined techniques for purifying bacterial PSs has resulted in a reliance on well-resourced and established laboratories in developed countries. Moreover, the purification process for these PSs entails the utilisation of expensive reagents, labour-intensive procedures, and often prolonged processing times, which cumulatively exacerbate the overall cost of conjugate vaccines. Researchers are concentrating on innovating expedited, economically viable, and scalable purification techniques for PS antigens to surmount these challenges. This investigation delineates optimised protocols for purifying CPSs derived from N. meningitidis serogroups A and C (MenA-PS and MenC-PS), which are essential to vaccines directed against these pathogens. By enhancing and expediting procedures such as CTAB-based precipitation and incorporating hydrophobic interaction chromatography (HIC) for MenC-PS, this methodology diminishes processing durations by more than 70% while attaining the purity benchmarks established by the WHO. The findings of this research indicate that these novel techniques are both amenable to industrial scaling and economically feasible, thereby providing a pragmatic approach to mitigating the cost and intricacy of vaccine production in resource-constrained environments, ultimately facilitating broader access to life-saving conjugate vaccines (24).

PSs are traditionally obtained through bacterial growth, harvesting, and purification steps. The conjugation of PSs to carrier proteins involves random linkage, bringing about high-molecular-weight and heterogeneous structures. Glycan-protein conjugation can be achieved through functional moieties or by derivatizing the PS with linkers. Chemical strategies such as NaIO4 oxidation and CDAP chemistry are commonly used for conjugation. Shorter OSs are preferred for manufacturing, as they facilitate conjugate production, filtration, and purification. The utilization of consistent and defined lower-molecular-weight PS groups can be accomplished by either chemical or mechanical splitting. Nevertheless, the isolation of PSs with specific lengths could impact the overall efficiency and intricacy of the procedure (11). Specific PS chemical groups may activate in certain cases before being attached to the carrier protein. One common method requires the interaction of hydroxy groups with cyanylating agents or carbonyldiimidazole to create active esters. As an alternative, amino groups can be incorporated using ammonium salts or dihydrazide spacers, which can then react with carboxylic acids of the protein or be linked to different bifunctional linkers for conjugation purposes. It is crucial to determine the optimal level of activation of the sugar chain to ensure effective conjugation without compromising the saccharide’s structural integrity. PSs can also be broken down through various techniques such as thermal or chemical hydrolysis, sonication, or homogenization, with resulting fractions subsequently sized for more precise length and easier protein conjugation ( Figure 3 ). Chemical hydrolysis has proved to be capable of developing several commercially available glycoconjugate vaccines. Reduced-size PSs are typically linked to the protein using the end-reducing sugar, enabling selective modification and more well-defined structures. Numerous licensed glycoconjugate vaccines against specific pathogens are developed using these methodologies (36). Alternative methods for delivering PS antigens and carrier proteins in conjugate vaccines have been explored, such as the Protein Capsular Matrix Vaccine (PCMV) technology that uses crosslinked polymers to trap both components. The MAPS technology replaces covalent linkage with affinity-based coupling and has shown promise in a prototype pneumococcal vaccine, with further development underway for other pathogens (11).

The capacity to manufacture significant amounts of artificial carbohydrates for vaccine development has been proven in the case of the Quimi-Hib vaccine designed to combat Hib (30) and a synthetic glycoconjugate vaccine against Shigella flexneri (S. flexneri) serotype 2a has also been tested in clinical trials (37). Synthetic glycans present a promising strategy for advancing vaccines, particularly when acquiring natural PSs is difficult. Nevertheless, the extensive application of artificial sugars is impeded by elevated production expenses and intricate processes when contrasted with natural PSs. Nonetheless, recent progress in streamlining and hastening the creation of glycans and glycoconjugates using chemoenzymatic methods, one-pot procedures, and automated solid-phase synthesis is increasing the attractiveness of carbohydrate production for industrial purposes. By combining chemical or enzymatic synthesis techniques, targeted conjugation methods, and multi-component structures, it is possible to boost the immunological effects of glycoconjugates and streamline the development of more effective and safer vaccines by gaining a deeper insight into the factors influencing their effectiveness (38). Various methods have been explored to expedite the assembly of glycans and obtain well-defined OS structures. One such method is the one-pot strategy, which allows for the amalgamation of multiple glycosylation steps into a single process, resulting in the production of target OSs in a shorter timeframe without the need for intermediate isolation or protective group manipulations. Databases containing reactivity values of building blocks have been developed to assist in the design of target OSs. Automated methods for synthesizing compounds, exemplified by the ‘Glyconeer’ synthesizer, have been created to facilitate the production of increasingly intricate and lengthy glycans. These automated systems have successfully synthesized diverse glycans, including complex structures and biologically relevant OSs. Additionally, glycan microarray and structural characterization methods have been used to identify crucial epitopes and select synthetic vaccine candidates. Synthetic glycoconjugate vaccines have been formulated using these methods, resulting in the production of antibodies with strong opsonophagocytic activity (11). The utilization of immobilized enzymes for a sequence of chemical and enzymatic processes has been applied to produce N. meningitidis serogroup X fragments ( Figure 4 ). This has resulted in creating an on-column technique that quickly produces conjugation-ready OSs of an ideal size for vaccine effectiveness. The process commences with a fully synthetic trisaccharide acceptor and a truncated immobilized form of the N. meningitidis X capsular polymerase (CsxA) (39).

The conjugation of PSs to carrier proteins can occur randomly or at predetermined sites. Selective conjugation procedures can be achieved by targeting specific residues on the protein surface or using enzymatic chemistries ( Figure 5 ). Proteins incorporating unnatural amino acids can also be used for particular conjugation. Additionally, in vivo production of glycoproteins allows for the placement of the conjugation site at specific locations on the carrier protein (23). vaccines using this method have been tested in Phase-1/2 trials (27, 28).

Outer Membrane Vesicles (OMVs) are naturally secreted by Gram-negative bacteria during their growth process. Through genetic modification, the yield of OMVs can be enhanced, leading to the formation of Generalised Modules for Membrane Antigens (GMMA). By introducing additional genetic alterations, the toxicity of GMMA can be adjusted by modifying the lipid A composition. GMMA exhibits a diverse array of antigens displayed in their natural form and structure, mirroring the surface of the bacteria. They possess an ideal size for triggering immune responses and possess inherent adjuvant properties as they contain Toll-like receptor (TLR) agonists. GMMA derived from Gram-negative bacteria with O-antigen chains on their surface are considered potential vaccine candidates, especially in regions with limited resources, where high production expenses may pose a barrier (31). Shigella sonnei (S. sonnei) GMMA has been subjected to clinical trials where it was found to be well tolerated and capable of eliciting an immune response (40, 41). GMMA and OMVs can be manipulated to express glycans or proteins from a pathogen of interest, making them a platform for PS vaccines. The use of OMVs and GMMA as carriers for vaccines is versatile and flexible and has been extensively studied for their safety profiles and self-adjuvant properties. Their large size and surface area make them effective in uptake by APCs, and they can be efficiently manufactured using high-yielding production platforms. Several groups have reported successful applications of OMVs and GMMA as carriers for various antigens (42). In a comparative study evaluating immunogenic response in mice, GMMA generated a higher production of anti-O-antigen IgG compared to glycoconjugate when administered without Alhydrogel. When both GMMA and glycoconjugate were formulated on Alhydrogel, they resulted in comparable levels of sustained anti-O-antigen IgG with bactericidal properties. While glycoconjugates are a widely accepted method for bacterial vaccines, their utilization can be expensive, especially in cases requiring multiple components. GMMA presents a promising alternative due to its similar immunogenicity and more straightforward production process, making it an attractive candidate for Shigella vaccine development (43).

Many noncovalent efforts to develop effective delivery systems combining PSs and carrier proteins have been unsuccessful. Weakly bound carrier proteins are inefficiently retrieved by BCRs, resulting in inadequate T-cell assistance for B cells. The traditional method for manufacturing licensed glycoconjugate vaccines involves chemically conjugating PSs to carrier proteins, known as ‘classical conjugates.’ PSs are typically extracted from bacterial cultures and randomly conjugated, resulting in high molecular weight, cross-linked, and heterogeneous products. In certain cases, OSs are employed, obtained through depolymerization or synthesis, allowing for more defined conjugation via reducing ends or spacers. The production of these chemically derived glycoconjugate vaccines necessitates multiple processes for both protein carriers and PSs. Recent advancements in technology for in vivo conjugation of PSs to proteins, termed ‘bioconjugates,’ have been made in the past two decades. This technology utilizes glycoengineered Escherichia coli (E. coli) strains to produce glycans and carrier proteins simultaneously, streamlining glycoconjugate production. The PS is synthesized on a lipid anchor, transferred to the periplasm, and coupled to carrier proteins by the oligosaccharyltransferase enzyme, resulting in the formation of bioconjugates. This method enhances production efficiency, reduces process steps, and ensures control over T-cell helper protein-specific peptides. Classical conjugates yield OSs of diverse sizes and random conjugation, potentially affecting protein epitopes. In contrast, bioconjugates benefit from a regulated saccharide-expressing system, leading to smaller, more homogeneous structures and allowing for precise sugar insertion without affecting the protein, typically resulting in 1 or 2 chains per protein molecule (32). The initial discovery of the N-linked glycosylation system in bacteria, specifically in Campylobacter jejuni (C. jejuni), occurred twenty years ago. Since then, various glycosylation systems in prokaryotic organisms have been identified, including O-linked glycosylation systems that do not have analogous counterparts in eukaryotic organisms. Subsequently, the introduction of glycosylation pathways into E. coli through genetic recombination gave rise to the field of bacterial glycoengineering. This emerging biotechnological tool harnesses prokaryotic glycosylation systems within E. coli as a host to produce recombinantly glycosylated proteins. Over the past decade, advancements in our understanding of prokaryotic glycosylation systems have led to the development of an improved glycoengineering toolbox. At present, glycoengineering employs two main approaches for the recombinant glycosylation of proteins, capable of generating N- or O-linkages: OTase-dependent and OTase-independent methods. The conjugate is synthesized through a single-step process wherein the saccharide antigen and the carrier protein are expressed within E. coli cells and coupled in vivo. The formation of PS takes place on undecaprenol pyrophosphate, a lipid anchor found in the cytoplasm. Subsequently, this PS is moved to the periplasmic region, where an OTase recognizes the lipid-linked reducing end sugar and then transfers the PS to an acceptor-sequon on the carrier protein. This series of steps ultimately results in the creation of the conjugate ( Figure 6 ). In bioconjugate vaccine development, three OTases have been utilized: the widely used N-linking OTase PglB, in addition to the O-linking OTases PglL and PglS (44). a study utilizing this technique demonstrated the production and analysis of bioconjugate vaccines against the two main hypervirulent Klebsiella pneumoniae (K. pneumoniae) serotypes, K1 and K2, using genetically modified E. coli cells, resulting in immunogenic and effective bioconjugates that protected mice from lethal infection caused by two hvKp strains, NTUH K-2044 and ATCC 43816 (45). Over the past ten years, the immunogenicity of bioconjugate vaccines targeting various pathogens, including Shigella dysenteriae (S. dysenteriae), S. flexneri, Staphylococcus aureus (S. aureus), E. coli, Francisella tularensis (F. tularensis), Burkholderia pseudomallei (B. pseudomallei), S. pneumoniae, Acinetobacter baumannii (A. baumannii), and K. pneumoniae, has been validated in preclinical animal studies (46–54). Some bioconjugates are currently in clinical evaluation. The S. dysenteriae vaccine, a bioconjugate, has been tested in humans with favorable safety and immune response outcomes. It generated significant immune responses against O1 PSs and functional antibodies, demonstrating the technology’s effectiveness in maintaining both sugar and protein epitopes (55). A bioconjugate vaccine for S. flexneri 2a has successfully advanced through Phase-1 and Phase-2b trials, showing immunogenicity and protection post-Shigella challenge (56, 57). Additionally, ExPEC4V, a tetravalent bioconjugate vaccine against E. coli, has exhibited promise in two Phase-1 trials, with ongoing development by Glycovaxyn and Janssen Pharmaceuticals, Inc (58). Phase-2 trial results indicated strong immunogenicity and safety at specific antigen doses (59).

Certain microorganisms, such as K. pneumoniae strain ATCC 25955, have natural advantages over model microorganisms like E. coli in terms of growth rate, protein expression, and metabolic pathways. These advantages make K. pneumoniae strain ATCC 25955 a suitable strain for glycoengineering. In a study, two glycoengineered K. pneumoniae strains with different O-antigen serotypes were constructed and successfully used to produce glycoprotein-bearing nanovaccines. This research offers a successful remedy for developing nanovaccines against K. pneumoniae and has potential implications for future synthetic biological research (60).

The effectiveness of a glycoconjugate vaccine is influenced by factors including sugar length, sugar load, and binding interactions affecting immunogenicity. A recent review concluded that optimal glycan chain length and epitope density are crucial for vaccine efficacy in glycoconjugates (61). Classical conjugates can accommodate multiple saccharide chains, while bioconjugates face challenges in incorporating similar quantities. A reduced number of saccharide chains per protein may result in diminished immunogenicity due to lower BCR crosslinking capacity. Although traditional methods are more complex than bioconjugation, bioconjugate technology still faces challenges, particularly in optimizing E. coli as a production model. Key technical challenges include enhancing the yield of oligosaccharyltransferase to ensure effective glycosylation of protein consensus sequences. Screening oligosaccharyltransferase mutants may improve saccharide occupancy and increase antigen production yield. Due to their unique properties, bioconjugate vaccines can be likened to traditional conjugates formed by linking OSs to carrier proteins at the sugar’s reducing end, with clinical trials conducted on two bioconjugate vaccines against S. pneumoniae and S. flexneri 2a, both of which have also been examined with classical conjugates in separate studies (32).

Qβ virus-like particles (VLPs) have been evaluated as a vehicle for conveying brief synthetic S. pneumoniae serotype 3 and 14 OSs via copper-catalyzed click chemistry, demonstrating their capacity to induce serotype-specific, safeguarding, and enduring IgG antibodies of nanomolar affinity in mice (62).

Gold nanoparticles have also been utilized in numerous attempts to produce glycoconjugate vaccines. These nanoparticles possess the ability to undergo functionalization due to the robust interaction between an Au atom and sulfur, thereby allowing for the incorporation of a synthetic tetrasaccharide epitope of S. pneumoniae serotype 14, a T-helper ovalbumin 323–339 peptide and D-glucose. Studies conducted on animal models have demonstrated that these glyconanoparticles can elicit a specific functional antibody response to the saccharide (63, 64). Additionally, a recent investigation employed gold nanoparticles decorated with synthetic OSs of S. pneumoniae serotypes 19F and 14, examining the impact of simultaneously displaying two carbohydrate epitopes from different bacterial serotypes on the immune response (63–65).

Nanoparticles have been the subject of extensive investigation within the realm of vaccine research; however, their function as carriers in the development of glycoconjugate vaccines has not been thoroughly examined, which restricts the comprehension of the optimal characteristics necessary for the effective induction of immune responses. The researchers concentrated on two self-assembling proteins: the Hcp1cc protein, which assembles into ring-shaped hexamers capable of oligomerizing into nanotubes, and Helicobacter pylori ferritin (Hpf), which yields spherical nanoparticles. Both proteins were conjugated with MenW OSs owing to the presence of surface lysines, resulting in elevated levels of glycosylation. Characterization methodologies, including Asymmetrical Flow Field-Flow Fractionation (AF4), validated the successful conjugation of nanoparticles. The glycoconjugated nanoparticles were administered to mice utilizing an AS01-adjuvanted regimen, with MenW-CRM197 serving as the control vaccine. The results indicated that the MenW-Ferritin conjugate elicited a more robust immune response and enhanced bactericidal activity in comparison to other MenW-nanoparticles. Importantly, the dimensions of the nanotubes did not exert a significant effect on the immune response, whereas the spherical ferritin nanoparticles exhibited the highest levels of immunogenicity. These findings are consistent with previous studies concerning inorganic nanoparticles, which demonstrate that spherical nanoparticles facilitate cellular uptake, activate APCs, and promote trafficking to lymph nodes, rendering them more efficacious for immune activation than rod-shaped counterparts. This effectiveness is posited to arise from a reduced energy expenditure required for the cellular internalization of spherical morphologies and an optimal contact area with cell membranes, thereby enhancing dendritic cell (DC) uptake and Th1 immune responses. This investigation emphasizes the promise of protein-based nanoparticles, particularly Hpf, in the advancement of glycoconjugate vaccines. In contrast to inorganic nanoparticles, protein scaffolds such as ferritin may display a variety of T-cell epitopes, potentially amplifying the immune response. These findings underscore the notion that while the shape of nanoparticles exerts a more pronounced influence than size, protein nanoparticles represent a promising strategy for developing novel glycoconjugate vaccines targeting bacterial pathogens (66).