Conjugation Mechanism for Pneumococcal Glycoconjugate Vaccines
Edit
This entry is adapted from the peer-reviewed paper 10.3390/bioengineering9120774

Licensed glycoconjugate vaccines are generally prepared using native or sized polysaccharides coupled to a carrier protein through random linkages along the polysaccharide chain. These polysaccharides must be chemically modified before covalent linking to a carrier protein in order to obtain a more defined polysaccharide structure that leads to a more rational design and safer vaccines. There are classic and new methods for site-selective glycopolysaccharide conjugation, either chemical or enzymatic modification of the polysaccharide length or of specific amino acid residues of the protein carrier.

pneumococcus vaccine glycoconjugate vaccines protein carbohydrate conjugation

1. Introduction

Streptococcus pneumoniae are encapsulated Gram-positive bacteria that were first isolated independently by Louis Pasteur and George Sternbern in 1880 [1][2]. This bacterium is an important human pathogen that causes pneumonia, otitis media, meningitis, and bacteremia. It is a major global cause of morbidity and mortality, especially in children, the elderly, and immuno-compromised populations, with approximately a million deaths yearly worldwide [3][4].
One of their main virulence factors is the capsular polysaccharide (CPS), which is involved in mediating direct interactions between the bacteria and its environment, protecting the pathogen from the host immune response mechanisms [5][6]. Based on their CPS, they have been classified into more than 90 different specific types according to the Danish classification system [7][8]. Recently, serotype number 100 was identified [9].
Furthermore, this pathogen mainly infects humans, first colonizing the nasopharynx, which is its only reservoir in nature. Most clinically isolated S. pneumoniae have been susceptible to penicillin, to the β-lactams group in general, and to other antimicrobial drugs. However, due to the global emergence of resistant and multidrug-resistant strains and the fact that, in many cases, the disease can progress quickly before introduction of antimicrobial treatment, pneumococcal disease is a challenge to treat [10][11].
The circulation of many different serotypes constitutes a challenge in the development of an effective vaccine due to the limited cross reactivity between each type [12][13][14]. Consequently, optimal vaccines should include the most prevalent and virulent serotypes. Pneumo-Bacterin, a serotype-specific whole-cell heat-treated vaccine, was the first vaccine against pneumococcus, recorded in 1909 in the USA [1]. The identification of pneumococcal bacteria serotypes and their establishment as critical virulence factors by Dochez and Avery in 1917 was the starting point for the use of pneumococcal polysaccharide vaccines, which are still currently used [1][15][16][17][18].
In 1929, Avery conjugated (covalently coupled) pneumococcal polysaccharides to proteins to improve immunogenicity. They used horse globulin and egg albumin as a carrier protein, and the CPS was presumably serotypes 1 and 2 [1][19]. This improvement accomplishes real clinical value many years later with the development of conjugate pneumococcal vaccines [6][14][20]. Meanwhile, only unconjugated polysaccharide vaccines were used.
Polysaccharide pneumococcal vaccines can stimulate antibody production by B cells in the absence of T helper cells. This type of response is termed thymus independent respond or simply T independent response [21]. Most of the polysaccharides cannot be processed and presented in association with Major Histocompatibility Complex (MHC) and therefore cannot be recognized by helper T cells (an exception occurs with some zwitterionic polysaccharides like S. pneumoniae CPS 1). In general, polysaccharide antigens are composed of repeated units that induce cross linking of B cell receptors (BCR), leading to the activation of the B cell. Due to the lack of T helper collaboration, antibodies are generally of low affinity and are composed mainly of IgM with limited isotype switching to IgG and IgA, and the production of memory B cells is also diminished. Consequently, T independent antigens are poor immunogens, particularly in young infants [21][22].
The need for more effective vaccines against these capsular polysaccharides led to their conjugation to immunogenic carrier proteins, resulting in conjugates that evoked a better T-cell dependent memory response. These vaccines can be processed by antigen presenting cells (APCs) and peptides from the protein component can be presented in association with the MHC and be recognized by helper T cells. The activation of T helper cells induces a better response, including antibody affinity maturation, isotype switching, and the induction of better secondary responses by B memory cells [22].
In 1980, Schneerson and coworkers, taking into account Avery’s work, developed a Haemophilus influenzae type b (Hib) conjugated vaccine using polyribosylribitol phosphate polysaccharide (PRP) conjugated to CRM-197 (a non-toxic recombinant variant of diphtheria toxin) [1][23][24]. Some years later, due to the success of this vaccine, Wyeth pharmaceuticals conjugated the CPS of the seven most common types of Streptococcus pneumoniae to CRM197, generating the first conjugated vaccine against pneumococcus (Prevnar7®) [23][25].
The introduction of conjugate pneumococcal vaccines (PCVs) in the 21st century has been widely accepted, and they are used in children in most countries. The currently approved and in-use PCVs are shown in Table 1.
Table 1. Currently used PCVs approved by FDA and/or EMA. * Displaced by PCV13. Abbreviations: TT, tetanus toxoid; CRM197, cross-reactive material 197; NTHI PD, non-typeable Haemophilus influenzae protein D; DT, diphtheria toxoid; FDA, Food and Drug Administration; EMA, European Medicines Agency; GSK GlaxoSmithKline. References [26][27][28].
Table
Vaccine Producer CPS Type Protein Conjugation Method
Prevnar PCV7 * Pfizer 4, 6B, 9V, 14,18C, 19F, 23F CRM197 Reductive amination
Prevnar PCV13 Pfizer 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, 23F CRM197 Reductive amination
Prevnar PCV20 Pfizer 1, 3, 4, 5, 6A, 6B, 7F, 8, 9 V, 10A, 11A, 12F, 14, 15B, 18C, 19A, 19F, 22F, 23F, 33F CRM197 Reductive amination
Synflorix PCV10 GSK 1, 4, 5, 6B, 7F, 9 V, 14, 18C, 19F, 23F NTHi PD,
DT, TT		 CDAP
Vaxneuvance PCV15 Merck 1, 3, 4, 5, 6A, 6B, 7F, 9 V, 14, 18C, 19F, 19A, 22F, 23F, 33F CRM197 Reductive amination
Prevnar vaccines uses CRM197 as the carrier protein, while Synflorix vaccine uses three types of protein: diphtheria toxoid (DT) for CPS type 19F, tetanus toxoid (TT) for CPS type 18C, and non-typeable Haemophilus influenzae protein D (NTHI PD) for the other eight CPS types found in the vaccine [28]. Although vaccination with PCVs includes most of the prevalent serotypes of pneumococcus, the World Health Organization (WHO) reported the emergence of non-vaccine serotype circulation and serotype replacement, leading to an increased proportion of pneumococcal disease of these serotypes in both children and adults [29][30].

2. Classic Conjugation Methods

2.1. Reductive Amination

This method has been used in most of the licensed PCVs and consists of sugar–protein conjugation through Schiff base formation between sugar-derived aldehydes and amines in proteins followed by a reduction [31][32]. The first step is the introduction of aldehyde groups along the polysaccharide chain by partial oxidation with sodium periodate. Cis-diols in the sugar ring and sialic acid residues are the most sensitives zones to generate aldehydes by periodate oxidation [27]. The carrier protein is then added to the reaction. Aldehydes from sugars are typically reacted with the lysine residues of the carrier protein. This methodology consists of the initial formation of bonds by Schiff bases between a carbonyl group in the carbohydrate and an amino group in the protein (Figure 1). The Schiff base is then specifically reduced with the weak reductant sodium cyanoborohydride to a more stable amine [27][33].
Bioengineering 09 00774 g001
Figure 1. Conjugation by Reductive Amination and CDAP.
Many variations have been employed to improve the conjugation process. Linkers can be used to insert chemical handles for conjugation to reduce steric problems. Another alternative is the use of ammonium salts to provide a reactive amine ready for coupling or the use of dihydrazide spacers. Inserted amines can be reacted directly with carboxylic groups of protein acid residues or coupled to a variety of bifunctional linkers [27].
Reductive amination has been widely used in glycoconjugate synthesis because of its simplicity, but it has important drawbacks, including a low yield, low coupling efficiency, and the risk of the partial degradation of the carbohydrate structure.
The conjugation method can influence the immunogenicity and the functionality of the antibodies induced, leading to deficient cross-reactivity with wildtype serotypes. This was demonstrated for serotype 19F and 19A when their conjugation by reductive amination and cyanylation was compared [34]. These studies showed that the conjugation of the 19F polysaccharide using reductive amination induced a new epitope that was not present in the native form of the 19F polysaccharide or using cyanylation as a conjugation method [34].
In addition, Poolman and colleagues reported that the periodate oxidation step before reductive amination could open the saccharide ring structure, which may lead to ring-opened conjugates, depending on the serotype. The authors proposed that the ring-opened polysaccharides may reclose after the conjugation process, in some cases, causing the polysaccharides to form additional new conformations, resulting in new epitopes [34].
Besides, reductive amination is often attained to a certain extent under basic conditions, beginning with the work of Jennings and Lugowski in 1981 [35]. They used these conditions because the polysialic acids in meningococcal B and C capsular polysaccharides are acid labile. To avoid possible degradation, they proposed carrying out the conjugation between the terminally oxidized polysialic acid and tetanus toxoid in phosphate buffer at pH 9.0 in the presence of sodium cyanoborohydride [35][36]. From this point, the reductive amination standard procedure was carried out in basic conditions, although Schiff’s base can be formed under either basic or acidic conditions [36]. However, Zou and coworkers observed significant degradation of the polysaccharide under basic conditions during conjugation of several pneumococcus capsular polysaccharides by reductive amination. The authors found that performing the conjugation of oxidized polysaccharides to bovine serum albumin (BSA) in slightly acidic media improved the reductive amination and prevented degradation, particularly with β-elimination-susceptible polysaccharides [36].

2.2. 1-Cyano-4-dimethylaminopyridine Tetrafluoroborate (CDAP) Cyanilation

This methodology comes from another classic strategy for the conjugation of carbohydrates and proteins, consisting of the activation of polysaccharides with cyanogen bromide (CNBr), as described by Kohn and Wilchek in 1984 [37].
The polysaccharide aliphatic hydroxyl groups are known not to be sufficiently nucleophilic to react directly with CNBr, and it is not possible to carry out the reaction at neutral pH. These conventional procedures are characterized by the use of CNBr in a strongly basic reaction medium. The activation yields are only 0.5–2%, which means the use of large amounts of CNBr, with all the concomitant health risks. Another disadvantage of these processes is that the activated polysaccharide contains both cyanate ester and imido carbonate structures in different proportions. The cyanate ester groups are hydrolyzed at a high pH, while the imido carbonates form inactive carbonates on hydrolysis at a more acidic pH.
Although CNBr is a low-cost reagent, it is very toxic and its use requires special attention. In addition, sensitive polysaccharides can be damaged by the high pH conditions required for CNBr activation [38].
Despite these disadvantages, the conventional procedure is one of the most popular methods used for the activation of polysaccharide-type matrices for protein immobilization and in affinity chromatography. As an alternative strategy, the electrophilicity of CNBr can be increased by means of a suitable “cyano-transfer” agent, which does not require the presence of a strong inorganic base in the reaction medium and thus makes it possible to avoid the base-dependent side reactions described above. These complexes are more electrophilic than CNBr and thus capable of cyanylating hydroxyl groups in their protonated form in the polysaccharide to create cyanate esters at a lower pH than in the conventional process. One of the classical cyanotransfer procedures is based on the formation of a highly reactive salt-type complex between CNBr and a cheap and easily accessible tertiary amine, TEA (CTEA). The yield of the reaction was highly improved, but CTEA is unstable and decays at temperatures above −10 °C [37][39]. In the same way, the authors found that 1-cyano-4-(dimethylamino) pyridiniumtetrafluoroborate (CDAPBF4 or CDAP) was a highly efficient activating agent for polysaccharide resins [39].
Indeed, Lees and coworkers introduced CDAP for protein–polysaccharide conjugate vaccines, using it as a cyanylating agent for the activation of soluble polysaccharides such as pneumococcal type 14 polysaccharide and others [40][41]. CDAP, which is easier to use compared to CNBr, has the advantage that it can activate polysaccharides at a lower pH than CNBr and with fewer side reactions. Unlike CNBr, CDAP-activated polysaccharides can be directly conjugated to proteins, creating a simpler process (Figure 1). Moreover, CDAP-activated polysaccharides can be functionalized with a diamine or a dihydrazide to make amino- or hydrazide-derivatized polysaccharides. Further optimization of CDAP polysaccharide activation with pH control and buffer optimization was later described [38][42].
Lees and colleagues reported some important concerns to achieve an optimum activation of the polysaccharide: the stability of CDAP, the reaction with the polysaccharide hydroxyls, and the stability of the activated polysaccharide. They demonstrated that the CDAP reaction with the polysaccharide requires for optimization the increase of the pH and performing the reaction in the cold. These conditions reduce CDAP hydrolysis, in-creasing the active reagent available for reaction, and have minimal effect on the rate of activation. The authors conclude that to increase the efficiency of activation of polysaccharides with CDAP requires a balance between these concerns [38].
Currently, the technique has widespread use in both research and licensed vaccines, but studies on optimization of CDAP chemistry remain scarce [38].

References

  1. Grabenstein, J.D.; Klugman, K.P. A Century of Pneumococcal Vaccination Research in Humans. Clin. Microbiol. Infect. 2012, 18, 15–24.
  2. Auzat, I.; Chapuy-Regaud, S.; le Bras, G.; dos Santos, D.; Ogunniyi, A.D.; le Thomas, I.; Garel, J.-R.; Paton, J.C.; Trombe, M.-C. The NADH Oxidase of Streptococcus Pneumoniae: Its Involvement in Competence and Virulence. Mol. Microbiol. 1999, 34, 1018–1028.
  3. Blasi, F.; Mantero, M.; Santus, P.; Tarsia, P. Understanding the Burden of Pneumococcal Disease in Adults. Clin. Microbiol. Infect. 2012, 18, 7–14.
  4. Kim, G.L.; Seon, S.H.; Rhee, D.K. Pneumonia and Streptococcus Pneumoniae Vaccine. Arch. Pharm. Res. 2017, 40, 885–893.
  5. Alonso, D.E.; Verheul, A.F.; Verhoef, J.; Snippe, H. Streptococcus Pneumoniae: Virulence Factors, Pathogenesis, and Vaccines. Microbiol. Rev. 1995, 59, 591–603.
  6. Morais, V.; Dee, V.; Suárez, N. Purification of Capsular Polysaccharides of Streptococcus Pneumoniae: Traditional and New Methods. Front Bioeng. Biotechnol. 2018, 6, 145.
  7. Lund, E. Polyvalent, diagnostic pneumococcus sera. Acta Pathol. Microbiol. Scand. 1963, 59, 187–190.
  8. Geno, K.A.; Gilbert, G.L.; Song, J.Y.; Skovsted, I.C.; Klugman, K.P.; Jones, C.; Konradsen, H.B.; Nahm, M.H. Pneumococcal Capsules and Their Types: Past, Present, and Future. Clin. Microbiol. Rev. 2015, 28, 871–899.
  9. Ganaie, F.; Saad, J.S.; McGee, L.; van Tonder, A.J.; Bentley, S.D.; Lo, S.W.; Gladstone, R.A.; Turner, P.; Keenan, J.D.; Breiman, R.F.; et al. A New Pneumococcal Capsule Type, 10D, Is the 100th Serotype and Has a Large Cps Fragment from an Oral Streptococcus. mBio 2020, 11, e00937-20.
  10. Kim, L.; McGee, L.; Tomczyk, S.; Beall, B. Biological and Epidemiological Features of Antibiotic-Resistant Streptococcus Pneumoniae in Pre- and Post-Conjugate Vaccine Eras: A United States Perspective. Clin. Microbiol. Rev. 2016, 29, 525–552.
  11. AUSTRIAM, R.; GOLD, J. Pneumococcal bacteremia with especial reference to bacteremic pneumococcal pneumonia. Ann. Int. Med. 1964, 60, 759–776.
  12. Tarahomjoo, S. Recent Approaches in Vaccine Development against Streptococcus Pneumoniae. J. Mol. Microbiol. Biotechnol. 2014, 24, 215–227.
  13. Cecchini, P.; Entwisle, C.; Joachim, M.; Pang, Y.; Dalton, K.A.; Hill, S.; McIlgorm, A.; Chan, W.-Y.; Brown, J.S.; Colaco, C.A.; et al. Next Generation Vaccines: Development of a Novel Streptococcus Pneumoniae Multivalent Protein Vaccine. Bio. Processing J. 2015, 14, 18–33.
  14. Morais, V.; Texeira, E.; Suarez, N. Next-Generation Whole-Cell Pneumococcal Vaccine. Vaccines 2019, 7, 151.
  15. Dochez, A.R.; Avery, O.T. The elaboration of specific soluble substance by pneumococcus during growth. J. Exp. Med. 1917, 26, 477–493.
  16. Dochez, A.R.; Avery, O.T. Soluble Substance of Pneumococcus Origin in the Blood and Urine during Lobar Pneumonia. Exp. Biol. Med. 1917, 14, 126–127.
  17. Heidelberger, M.; Avery, O.T. The soluble specific substance of pneumococcus. J. Exp. Med. 1923, 38, 73–79.
  18. Dubos, R.; Avery, O.T. Decomposition of the capsular polysaccharide of pneumococcus type III by a bacterial enzyme. J. Exp. Med. 1931, 54, 51–71.
  19. Goebel, W.F.; Avery, O.T. Chemo-Immunological studies on conjugated carbohydrate-proteins: I. The synthesis of p-aminophenol beta-glucoside, p-aminophenol beta-galactoside and their coupling with serum globulin. J. Exp. Med. 1929, 50, 521–531.
  20. Avery, O.T.; Goebel, W.F. Chemo-Immunological studies on conjugated carbohydrate-proteins: II. Immunological specificity of synthetic sugar-protein antigens. J. Exp. Med. 1929, 50, 533–550.
  21. Daniels, C.C.; Rogers, P.D.; Shelton, C.M. A Review of Pneumococcal Vaccines: Current Polysaccharide Vaccine Recommendations and Future Protein Antigens. JPPT J. Pediatr. Pharm. 2016, 2721, 27–35.
  22. Abbas Abul, K.; Lichtman, A.H.; Pillai, S. Cellular and Molecular Immunology, 9th ed.; Elsevier: Philadelphia, PA, USA, 2018; ISBN 978-0-323-47978-3.
  23. Musher, D.M.; Anderson, R.; Feldman, C. The Remarkable History of Pneumococcal Vaccination: An Ongoing Challenge. Pneumonia (Nathan) 2022, 14, 1–15.
  24. Schneerson, R.; Barrera, O.; Sutton, A.; Robbins, J.B. Preparation, Characterization, and Immunogenicity of Haemophilus Influenzae Type b Polysaccharide-Protein Conjugates. J. Exp. Med. 1980, 152, 361–376.
  25. Black, S.; Shinefield, H.; Fireman, B.; Lewis, E.; Ray, P.; Hansen, J.R.; Elvin, L.; Ensor, K.M.; Hackell, J.; Siber, G.; et al. Efficacy, Safety and Immunogenicity of Heptavalent Pneumococcal Conjugate Vaccine in Children. Northern California Kaiser Permanente Vaccine Study Center Group. Pediatr. Infect Dis. J. 2000, 19, 187–195.
  26. Del Bino, L.; Østerlid, K.E.; Wu, D.-Y.; Nonne, F.; Romano, M.R.; Codée, J.; Adamo, R. Synthetic Glycans to Improve Current Glycoconjugate Vaccines and Fight Antimicrobial Resistance. Chem. Rev. 2022, 122, 15672–15716.
  27. Berti, F.; Adamo, R. Antimicrobial Glycoconjugate Vaccines: An Overview of Classic and Modern Approaches for Protein Modification. Chem. Soc. Rev. 2018, 47, 9015–9025.
  28. Poolman, J.T.; Peeters, C.C.; van den Dobbelsteen, G.P. The History of Pneumococcal Conjugate Vaccine Development: Dose Selection. Expert Rev. Vaccines 2013, 12, 1379–1394.
  29. Weinbergera, M.D.; Malley, R.; Lipsitch, M. Serotype Replacement in Disease Following Pneumococcal Vaccination: A Discussion of the Evidence. Lancet 2012, 378, 1962–1973.
  30. WHO Pneumococcal Conjugate Vaccines in Infants and Children under 5 Years of Age: WHO Position Paper—February 2019. Wkly. Epidemiol. Rec. 2019, 94, 85–104.
  31. Gray, G.R. The Direct Coupling of Oligosaccharides to Proteins and Derivatized Gels. Arch. Biochem. Biophys 1974, 163, 426–428.
  32. Sarkar, B.; Jayaraman, N. Glycoconjugations of Biomolecules by Chemical Methods. Front Chem. 2020, 8, 570185.
  33. Laferrière, C.A.; Sood, R.K.; De Muys, J.M.; Michon, F.; Jennings, H.J. The Synthesis of Streptococcus Pneumoniae Polysaccharide-Tetanus Toxoid Conjugates and the Effect of Chain Length on Immunogenicity. Vaccine 1997, 15, 179–186.
  34. Poolman, J.; Frasch, C.; Nurkka, A.; Käyhty, H.; Biemans, R.; Schuerman, L. Impact of the Conjugation Method on the Immunogenicity of Streptococcus Pneumoniae Serotype 19F Polysaccharide in Conjugate Vaccines. Clin. Vaccine Immunol. 2011, 18, 327–336.
  35. Jennings, H.J.; Lugowski, C. Immunochemistry of Groups A, B, and C Meningococcal Polysaccharide-Tetanus Toxoid Conjugates. J. Immunol. 1981, 127, 1011–1018.
  36. Zou, W.; Williams, D.; Cox, A. Mitigating Base-Catalysed Degradation of Periodate-Oxidized Capsular Polysaccharides: Conjugation by Reductive Amination in Acidic Media. Vaccine 2019, 37, 1087–1093.
  37. Kohn, J.; Wilchek, M. The Use of Cyanogen Bromide and Other Novel Cyanylating Agents for the Activation of Polysaccharide Resins. Appl. Biochem. Biotechnol. 1984, 9, 285–305.
  38. Lees, A.; Barr, J.F.; Gebretnsae, S. Activation of Soluble Polysaccharides with 1-Cyano-4-Dimethylaminopyridine Tetrafluoroborate (CDAP) for Use in Protein–Polysaccharide Conjugate Vaccines and Immunological Reagents. Iii Optimization of CDAP Activation. Vaccines 2020, 8, 777.
  39. Kohn, J.; Wilchek, M. 1-Cyano-4-Dimethylamino Pyridinium Tetrafluoroborate as a Cyanylating Agent for the Covalent Attachment of Ligand to Polysaccharide Resins. FEBS Lett. 1983, 154, 209–210.
  40. Lees, A.; Nelson, B.L.; Mond, J.J. Activation of Soluble Polysaccharides with 1-Cyano-4-Dimethylaminopyridinium Tetrafluoroborate for Use in Protein-Polysaccharide Conjugate Vaccines and Immunological Reagents. Vaccine 1996, 14, 190–198.
  41. Suárez, N.; Massaldi, H.; Franco Fraguas, L.; Ferreira, F. Improved Conjugation and Purification Strategies for the Preparation of Protein-Polysaccharide Conjugates. J. Chromatogr. A 2008, 1213, 169–175.
  42. Lees, A.; Zhou, J. Activation and Conjugation of Soluble Polysaccharides Using 1-Cyano-4-Dimethylaminopyridine Tetrafluoroborate (CDAP). JoVE 2021, 172, e62597.
More
© Text is available under the terms and conditions of the Creative Commons Attribution (CC BY) license; additional terms may apply. By using this site, you agree to the Terms and Conditions and Privacy Policy.
Upload a video for this entry
Information
Subjects: Biotechnology & Applied Microbiology
Contributors MDPI registered users' name will be linked to their SciProfiles pages. To register with us, please refer to https://encyclopedia.pub/register :
Victor Morais
,
Norma Suarez
View Times: 769
Revisions: 2 times (View History)
Update Date: 26 Dec 2022
Table of Contents
Video Production Service
Feedback