Chemical Science EDGE ARTICLE O pe n A cc es s A rt ic le . P ub lis he d on 1 0 Fe br ua ry 2 02 2. D ow nl oa de d on 4 /2 8/ 20 22 1 1: 38 :2 5 A M . T hi s ar tic le is li ce ns ed u nd er a C re at iv e C om m on s A ttr ib ut io n- N on C om m er ci al 3 .0 U np or te d L ic en ce . View Article Online View Journal | View Issue Retaining the str aGSK, Via Fiorentina 10, 53100 Siena, Italy bInstituto de Medicina Molecular, Faculdade Lisboa, Portugal. E-mail: gbernardes@medi cYusuf Hamied Department of Chemistry, U Cambridge CB2 1EW, UK. E-mail: gb453@c † Electronic supplementary information incorporation and glycoconjugation, ma and in vivo studies are reported as ESI crystallographic data in CIF or o 10.1039/d1sc01928g ‡ F. C. and A. K. contributed equally to th Cite this: Chem. Sci., 2022, 13, 2440 All publication charges for this article have been paid for by the Royal Society of Chemistry Received 6th April 2021 Accepted 21st January 2022 DOI: 10.1039/d1sc01928g rsc.li/chemical-science 2440 | Chem. Sci., 2022, 13, 2440–2 uctural integrity of disulfide bonds in diphtheria toxoid carrier protein is crucial for the effectiveness of glycoconjugate vaccine candidates† Filippo Carboni,‡a Annabel Kitowski, ‡b Charlotte Sorieul, a Daniele Veggi,a Marta C. Marques,b Davide Oldrini,a Evita Balducci,a Barbara Brogioni,a Linda Del Bino,a Alessio Corrado,a Francesca Angiolini,a Lucia Dello Iacono,a Immaculada Margarit,a Maria Rosaria Romano,a Gonçalo J. L. Bernardes *bc and Roberto Adamo *a The introduction of glycoconjugate vaccinesmarks an important point in the fight against various infectious diseases. The covalent conjugation of relevant polysaccharide antigens to immunogenic carrier proteins enables the induction of a long-lasting and robust IgG antibody response, which is not observed for pure polysaccharide vaccines. Although there has been remarkable progress in the development of glycoconjugate vaccines, many crucial parameters remain poorly understood. In particular, the influence of the conjugation site and strategy on the immunogenic properties of the final glycoconjugate vaccine is the focus of intense research. Here, we present a comparison of two cysteine selective conjugation strategies, elucidating the impact of both modifications on the structural integrity of the carrier protein, as well as on the immunogenic properties of the resulting glycoconjugate vaccine candidates. Our work suggests that conjugation chemistries impairing structurally relevant elements of the protein carrier, such as disulfide bonds, can have a dramatic effect on protein immunogenicity. Introduction Vaccines are considered as one of the most cost-effective interventions to prevent morbidity and mortality from infec- tious diseases.1 Among the different approaches to vaccine design, glycoconjugate vaccines have been proven efficacious and cost-effective in the prevention of Haemophilus inuenzae type b (Hib), Streptococcus pneumoniae (23 serotypes), Neisseria meningitidis (A, C, W135 and Y) and Salmonella typhi.2 Conjugate vaccines are obtained by the covalent linkage of bacterial poly- saccharides to immunogenic carrier proteins, and have been demonstrated to overcome the limitations frequently exhibited by unconjugated polysaccharide vaccines.3 The T-cell help . E-mail: roberto.x.adamo@gsk.com de Medicina da Universidade de Lisboa, cina.ulisboa.pt niversity of Cambridge, Lenseld Road, am.ac.uk (ESI) available: Procedures for linker ss spectrometry, X-ray crystallography . CCDC PBD ID 7O4W. For ESI and ther electronic format see DOI: is work. 449 provided by the protein epitopes of glycoconjugates imparts to the carbohydrates – which are per se T-cell independent anti- gens – the capacity to induce long-lasting and boostable IgG antibody production. Six proteins are currently used as carriers in licensed vaccines, including tetanus toxoid (TT), diphtheria toxoid (DT), Cross-Reactive Material 197 (CRM197), the outer membrane protein complex of Meningococcus B (OMPC), protein D fromH. inuenzae, and the recombinant exotoxin A of Pseudomonas aeruginosa. Carbohydrates can be linked to proteins by using a variety of approaches. Amino acid residues that are most suitable for chemical linkage to sugars are those well-exposed onto the protein surface and whose side chains have reactive functional groups, such as primary amino groups of lysines and carboxylic groups of glutamic, or aspartic acid residues.4–6 Generally, linking of a polysaccharide (PS) to a carrier protein results invariably in a random display of the carbohydrate on the protein surface, although some selectivity homogeneity can be obtained by modulating the carbohydrate to protein stoichi- ometry. A variety of factors that are associated with the conju- gation methodology (e.g. conjugation chemistry, multiple attachment versus single-point attachment of carbohydrates, presence/absence of linkers) have an impact on the chemical and biological properties of different glycoconjugate © 2022 The Author(s). Published by the Royal Society of Chemistry http://crossmark.crossref.org/dialog/?doi=10.1039/d1sc01928g&domain=pdf&date_stamp=2022-02-19 http://orcid.org/0000-0002-8001-5928 http://orcid.org/0000-0003-1999-6007 http://orcid.org/0000-0001-6594-8917 http://orcid.org/0000-0001-5228-6088 http://creativecommons.org/licenses/by-nc/3.0/ http://creativecommons.org/licenses/by-nc/3.0/ https://doi.org/10.1039/d1sc01928g https://pubs.rsc.org/en/journals/journal/SC https://pubs.rsc.org/en/journals/journal/SC?issueid=SC013008 Edge Article Chemical Science O pe n A cc es s A rt ic le . P ub lis he d on 1 0 Fe br ua ry 2 02 2. D ow nl oa de d on 4 /2 8/ 20 22 1 1: 38 :2 5 A M . T hi s ar tic le is li ce ns ed u nd er a C re at iv e C om m on s A ttr ib ut io n- N on C om m er ci al 3 .0 U np or te d L ic en ce . View Article Online constructs.6 This poses a serious limitation in the comparison of different glycoconjugates and in understanding their mech- anism of action. Therefore, there is a need for site-selective glycoconjugation methods and effort has been recently devoted to the preparation of glycoconjugates with dened attachment.7 High regioselectivity has been achieved by target- ing amino acids naturally present in the protein carrier, such as cysteine,8,9 cysteine disulde bridges10,11 or tyrosines.12–18 Alter- natively, amino acid tags for enzyme mediated conjugation19,20 or incorporation of unnatural amino acids21 have been exploi- ted. The development of site-selective conjugation strategies on naturally occurring amino acids, such as cysteine, is appealing for the simplicity of these approaches, as more complicated and time-consuming steps like sequence engineering become unnecessary.22 Selectivity of glycoconjugation is achieved by targeting either amino acids with sufficiently high reactivity during chemical reactions, or amino acid patterns within the protein structure that allow selective reactivities. In this context, disulde bridges are an optimal target for site-selective reac- tions, as they are usually present in limited numbers and, upon reduction, show nucleophilic properties that can be used for chemical reactions.23,24 Disulde modications have been successfully used for incorporation of small molecule payloads into antibodies and have attracted specic attention for the development of glycoconjugate vaccines, because of the selec- tivity that can be achieved on these groups on the carrier protein CRM197.5,25,26 CRM197 is an enzymatically inactive and nontoxic form of diphtheria toxin, which has been detoxied through a single G52E mutation. CRM197 is a well-characterized protein, whose X-ray structure has been elucidated.27 It is synthesized as a single-chain holoprotein, which comprises two domains, fragment A (catalytic domain) and fragment B (transmembrane domain). Two disulde bridges are present in the intact hol- oprotein: one bridge joins C186 to C201, linking fragment A to fragment B, while a second bridge joins C461 to C471 within fragment B.28 Polysaccharide conjugates of CRM197 are components of vaccine formulations protecting against important bacterial pathogens including Streptococcus pneu- moniae (Prevnar), Haemophilus inuenzae type b (HibTITER, Vaxem-Hib) and Neisseria meningitidis serogroup A, C, Y and W- 135 (e.g. Menveo and Menjugate).29 Recently, disulde re- bridging with acetone was developed for site-selective conju- gation of Salmonella O-antigen to CRM197 and improved immunogenicity of the protein as an antigen was observed aer graing one of the two disulde bonds with oxetane.9,30 In this study, we investigated the scope and limitations of two different site-selective modication methods on the disul- de functionalities of the carrier protein CRM197. By choosing two opposing strategies, we sought to elaborate the importance of retaining the structural integrity of a disulde functionality through acetone re-bridging or, in contrast, the effect of opening and converting a disulde bridge into two dehy- droalanine residues, on the properties of a resulting glyco- conjugate vaccine candidate. In depth structural characterization of the modied CRM197 was carried out to unravel differences between the two methods, and the X-ray © 2022 The Author(s). Published by the Royal Society of Chemistry crystallographic structure of acetone modied CRM197 was successfully resolved. Capsular polysaccharides from group B Streptococcus (GBS) serotypes Ia and III, as well as capsular polysaccharide from S. pneumoniae (Sp) serotype 14, were chosen as model antigens and the generation of immune responses aer administration of these vaccine candidates was evaluated. Results Selective glycoconjugation at C186-201 of CRM197 From the two disulde bridges present in the carrier protein CMR197, the C461–C471 bond appeared to be buried inside the protein, while the C186–201 is surface-exposed. We have already reported that selective modication of the latter disulde bridge can be achieved by partial reduction of CRM197 to release C186 and C201 in the presence of tris(2-carboxyethyl) phosphine (TCEP).9We envisaged tomodify the C186–C201 bond using two different strategies for the subsequent two-step conjugation of large polysaccharides (Scheme 1). First, re-bridging of C186– C201 with 1,3-dichloroacetone (DCA) would allow insertion of a ketone handle, which can be further modied with a bifunc- tional linker bearing an aminoxy to form an aminooxy deriva- tive, and an azide group useful for the conjugation of polysaccharides derivatized with alkyne functionalities via azido-alkyne Huisgen (3 + 2) cycloaddition (click chemistry). This method enables incorporation of a single carbohydrate moiety per protein. Also, preliminary experiments with sugar modied with aminoxy linkers proved the direct conjugation challenging, due to the size of GBS polysaccharides (MW �150– 200 kDa). The multivalent presentation of sugar antigens on a protein carrier is known to be an important factor for the efficient generation of an immunogenic response. To enable selective conjugation of a higher number of sugar molecules on the protein, a second strategy was developed, relying on the opening of C186–C201 and subsequent conversion of the free cysteine residue into dehydroalanine (Dha). In this approach, further conjugation can be achieved through thiol Michael addition on the introduced Dha groups of a bifunctional thiol linker bearing an azido moiety for the subsequent conjugation reaction to alkyne derivatized polysaccharides by click chem- istry (Scheme 1). These two methods appeared very attractive to be compared in terms of the construction of site-selective gly- coconjugates and immunogenicity of the resulting biomole- cules, since the rst strategy retains the covalent connection between cysteine residues C186 and C201, whereas the second strategy results in the opening of this disulde bridge. For the generation of CRM197-DCA, aer selective reduction of C186–C201 in the presence of tris(2-carboxyethyl) phosphine (TCEP, 12 equiv.) at pH 7.5 for three hours, the protein was incubated with 1,3-dichloroacetone (10 equiv.) for 3.5 hours yielding the modied CRM197. Size exclusion chromatography allowed the removal of small molecules and exchange of buffer to 100 mM sodium phosphate at pH 6.3 for acid catalyzed reaction with the aminoxy linker (Scheme 1). Mass spectrometry revealed virtually complete derivatization of the starting Chem. Sci., 2022, 13, 2440–2449 | 2441 http://creativecommons.org/licenses/by-nc/3.0/ http://creativecommons.org/licenses/by-nc/3.0/ https://doi.org/10.1039/d1sc01928g Scheme 1 Site-selective modification of CRM197 into CRM197-DCA or CRM197-DHA, (i) 12 equiv. TCEP, 3 h, r.t., (ii) 10 equiv. 1,3-Dichloroacetone, 3.5 h r.t., (iii) 500 equiv. Methyl 2,5-dibromopentanoate, 5 h, r.t., (iv) 600 equiv. aminoxy-PEG5-azide, 3 d, r.t., (v) TCEP, 100 equiv. bis(11-azi- doundecyl)disulfide, 18 h, r.t. Chemical Science Edge Article O pe n A cc es s A rt ic le . P ub lis he d on 1 0 Fe br ua ry 2 02 2. D ow nl oa de d on 4 /2 8/ 20 22 1 1: 38 :2 5 A M . T hi s ar tic le is li ce ns ed u nd er a C re at iv e C om m on s A ttr ib ut io n- N on C om m er ci al 3 .0 U np or te d L ic en ce . View Article Online material (Fig. 1). The impact of the installation of the DCA moiety into CRM197 on its structure was also studied by CD (Fig. 1B) and dynamic light scattering (DLS) analysis (Fig. S4, ESI†) and compared with the native protein. We found that CD and DLS spectra of CRM197-DCA were nearly identical to those of CRM197, which indicates that the secondary structure was preserved upon the chemical stapling. The CRM197-DCA was used for condensation with an excess of aminoxy-PEG5-azido linker (600 equiv.) for quantitative insertion of an azido moiety for further polysaccharide conjugation (Scheme 1), as conrmed by LC-MS analysis (Fig. S1, ESI†). Fig. 1 A) LC-MS spectra of native CRM197 and the modified CRM197-DCA Secondary structure of CRM197, CRM197-DHA and CRM197-DCA (5 mM) de buffer, pH 7.4 at 25 �C. All the values are mean values � SEM from at le content (%) from CD spectra using the BeStSel server. 2442 | Chem. Sci., 2022, 13, 2440–2449 The second strategy was intended to open the disulde bond C186–C201 in a selective manner and convert each of the two cysteine residues into the amino acid Dha. Similar to the previous approach, CRM197 was rst selectively reduced at C186–C201,9 and then introduction of Dha was achieved by treatment with 500 equiv. of methyl 2,5-dibromopentanoate over a period of 5 hours at pH 11. Residual small molecules were removed by size exclusion chromatography with 100 mM sodium phosphate at pH 6.3 for elution. The reaction with methyl 2,5-dibromopentanoate works through a bisalkylation mechanism, followed by an elimination step.21 This second part and CRM197-DHA highlight complete conversion of native protein. (B) termined by circular dichroism. CD spectra were obtained in phosphate ast two independent experiments. (C) Estimated secondary structure © 2022 The Author(s). Published by the Royal Society of Chemistry http://creativecommons.org/licenses/by-nc/3.0/ http://creativecommons.org/licenses/by-nc/3.0/ https://doi.org/10.1039/d1sc01928g Edge Article Chemical Science O pe n A cc es s A rt ic le . P ub lis he d on 1 0 Fe br ua ry 2 02 2. D ow nl oa de d on 4 /2 8/ 20 22 1 1: 38 :2 5 A M . T hi s ar tic le is li ce ns ed u nd er a C re at iv e C om m on s A ttr ib ut io n- N on C om m er ci al 3 .0 U np or te d L ic en ce . View Article Online was the critical step, as reactions at lower pH values were found to result in incomplete elimination products with the penta- noate molecule attached to the protein. A concentration of 5 mg mL�1 protein was key to achieve a high level of modication. Higher concentrations resulted in protein precipitation during the reaction. The introduction of two Dha residues (Scheme 1) and complete conversion of the starting material was conrmed by LC-MS analysis (Fig. 1A and S2, ESI†). Despite the ring opening, a minimal level of protein aggregation was observed when freshly prepared samples were used for the subsequent modications (Fig. S3, ESI†). The impact of the opening of the disulde bridge and subsequent conversion of cysteine in Dha residues on the CRM197 structure was evaluated by CD (Fig. 1B) and DLS (Fig. S4, ESI†) experiments using the native protein as the control. We found a few differences both in CD and DLS spectra corresponding probably to a less stable structure for CRM197-DHA related to the loss of the disulde bond that seems important to maintain the 3D structure of the protein. The selective modication of only C186 and C201 was further conrmed by using peptide mapping and LC-MS analysis (Fig. S5, ESI†). Importantly, the CD spectrum, which is indica- tive of helical conformation, showed that CRM197-DHA displays a more pronounced minimum at 218 nm compared to CRM197- DCA, typical of the b-sheet conformation. We estimated quan- titatively the helix content and twist angle distribution for the antiparallel and parallel b-sheets using the BeStSel server31,32 for both the modied forms (Fig. 1C). The data indicated that the CRM197 variants have lower helical content than the wild type, with the DHA variant showing a higher degree of antiparallel b- sheets that could be related to some level of aggregation. Next, the two dehydroalanine residues of CRM197-DHA were used for thiol Michael addition of the bis(11-azidoundecyl) disulde linker (Scheme 1). The successful insertion of two linker moieties was conrmed by MS experiments (ESI, Fig. 2†). This reaction provided, as for CRM197-DCA, azido functionalities ready for click chemistry with alkyne derivatized glycan antigens. X-ray crystallography of CRM197-DCA Considering the higher similarity observed between CRM197- DCA and the native form, further structural insights were ob- tained by X-ray crystallography. To this end, we solved the structure of the apo form (NAD-free) of CRM197-DCA at 2 Å resolution (data collection and renement statistics are re- ported in Table S1, ESI†). The asymmetric unit (ASU) contains one molecule of CRM197-DCA, which interacts with a second (symmetry-related) molecule by “domain swapping” (Fig. S6, ESI†), as observed in noncovalently associated dimeric native forms previously solved.25 The structure assumes the canonical Y shape of both wild type and mutant forms of diphtheriae toxin, whose arms are composed of C (Catalytic domain) and R (Receptor domain) domains while the T domain (Trans- membrane domain) is located at the base30 (Fig. S6, ESI†). The overall fold of CRM197-DCA is superimposable to the NAD-free CRM197 (PDB ID 4AE0) with an overall root mean square devi- ation (rmsd) of 1.1 Å for 454 equiv. Ca atoms (Fig. 2A). All the © 2022 The Author(s). Published by the Royal Society of Chemistry regions appear clearly overlapping except for the loops 37–50 in the C domain, 348–353 in the T domain and 516–520 in the R domain, which are in general better resolved in this CRM197- DCA structure compared to apo CRM197 (Fig. 2A). Indeed, the electron density maps of the CRM197-DCA are overall of high quality, allowing the modelling of the entire molecule apart from the region 187–198, connecting the T and C domains and quite close in space to the S–S bond C186–C201, and the resi- dues 42–44 of the catalytic loop 37–50 which appeared completely disordered in the apo CRM197 structure. Aer the rst cycles of renement, clear extra electron density nearby the S–S bond C186–C201 appeared, conrming the successful and selective insertion of a single acetone molecule (Fig. 2B and C). The distances within Ca atoms of C186 and C201, angles and length bonds values are in agreement with the geometry of Cys–acetone–Cys bridges. Moreover, the exibility of the loop accommodating the modied S–S bridge allows this acetone moiety to be solvent-exposed and potentially accessible to the conjugation of glycan antigens. In contrast, no modica- tion of the C461–C471 disulde bond was found (Fig. 2C). Remarkably, our structural data expand what previously shown by entire mass analysis,9,28 indicating the unambiguous presence of selective acetone insertion in the S–S bond C186–C201 of the CRM197-DCA and showing no major impact of this modication on the tertiary structure of the protein. Glycoconjugation with CRM197-DCA and CRM197-DHA To compare the inuence of the introduced protein modica- tions on the immunogenicity of resulting glycoconjugate constructs, GBS and Sp polysaccharide antigens were chosen for conjugation. Structurally similar polysaccharides from two different bacteria (GBS serotypes types III and Ia and Sp serotype 14) were partially de-N-acetylated and reacted by reaction with dibenzo- cyclooctyne-N-hydroxysuccinimidyl (DBCO) ester to insert a handle suited for azide–alkyne cycloaddition, which we have shown to be particularly efficient with large polysaccharides.11 To minimize the impact on the polysaccharide structure and preserve sugar epitopes, a limited number of repeating units were modied. The relative ratio of DBCO units per poly- saccharide molecule was determined by NMR spectrometry considering the ratio of the peak intensity of the aromatic protons of the linker and the H-3equatorial or H-3axial of the sialic acid for GBS PS or the H-2 of the Glc residue Sp14 PS, respec- tively (Fig. S7, ESI†). Typically one out of 20 and 40 repeating units was modied, for GBS PS and Sp14, respectively. Azide moieties were incorporated into the protein using either a PEG or an alkyl linker (Fig. 3). Based on our previous experience with strain promoted click chemistry,15–17 it was found that while the alkyl or PEG chain is immunosilent, rigid aromatic systems like DBCO are not; however this type of linker does not shi the immune response away from the carbohydrate. Therefore, although not identical, we considered them not to impact the immunogenicity outcome. Through the inserted DBCO moiety GBS types Ia and III PS and Sp type 14 were conjugated to CRM197-DCA and CRM197-DHA Chem. Sci., 2022, 13, 2440–2449 | 2443 http://creativecommons.org/licenses/by-nc/3.0/ http://creativecommons.org/licenses/by-nc/3.0/ https://doi.org/10.1039/d1sc01928g Fig. 2 (A) Superimposition between CRM197-DCA (PDB 7O4W, green smudge) and CRM197 (PDB 4AE0, cyan). The loop connecting the C and T domains (187–198) is absent in both structures, the loops 516–520 and 348–353, lacking in the CRM197, are present in the CRM197-DCAwhile the active site loop 37–50 is still partially incomplete in the CRM197-DCA structure (42–44 residues are missing). (B) Magenta and cyan boxes show the position of the two S–S bonds (C186–C201 and C461–471) in the structure of CRM197-DCA. The C186–C201 is the modified disulfide bond, carrying an acetonemoiety, whereas the C461–C471 is still an intact disulfide bridge. (C) Top, 1s 2Fo–Fc (blue mesh) electron density map of the modified S–S bridge. Bottom, superimposition of the derivatized S–S bridge (green C sticks) with the unmodified one (PDB 4AE0, cyan C sticks). The Ca atoms of C186 and C201 are highlighted as spheres. (D) 1s 2Fo–Fc (blue mesh) electron density map of the second and still intact S–S bridge (C461–C471), located at the tip of a loop in the R domain. Chemical Science Edge Article O pe n A cc es s A rt ic le . P ub lis he d on 1 0 Fe br ua ry 2 02 2. D ow nl oa de d on 4 /2 8/ 20 22 1 1: 38 :2 5 A M . T hi s ar tic le is li ce ns ed u nd er a C re at iv e C om m on s A ttr ib ut io n- N on C om m er ci al 3 .0 U np or te d L ic en ce . View Article Online (Fig. 3) at 2 : 1 polysaccharide/protein w/w ratio and a protein concentration of 2 mg mL�1. Larger amounts of sugar did not further improve the course of conjugation. Aer removal of the unconjugated polysaccharide, the level of sugar incorporation 2444 | Chem. Sci., 2022, 13, 2440–2449 was estimated by HPAEC-PAD and the protein content was assessed by colorimetric assay (bicinchoninic acid assay). The characteristics of the synthesized site-selective conjugates are summarized in Table 1. The conversion of cysteine residue from © 2022 The Author(s). Published by the Royal Society of Chemistry http://creativecommons.org/licenses/by-nc/3.0/ http://creativecommons.org/licenses/by-nc/3.0/ https://doi.org/10.1039/d1sc01928g Fig. 3 (A) Partial derivatization of polysaccharides GBSIa, GBSIII and Pn14with dibenzocyclooctyne (DBCO), (i) 1 MNaOH, 4.5 h, 70 �C, (ii) DBCO- NHS, TEA, 4 h, r.t., (iii) 0.5 M NaOH, 1 h, 70 �C, and (iv) DBCO-NHS, TEA, 3 h, r.t. (B) Glycoconjugate constructs CRM197-DCA-PSIa and CRM197- DHA-PSIa. Edge Article Chemical Science O pe n A cc es s A rt ic le . P ub lis he d on 1 0 Fe br ua ry 2 02 2. D ow nl oa de d on 4 /2 8/ 20 22 1 1: 38 :2 5 A M . T hi s ar tic le is li ce ns ed u nd er a C re at iv e C om m on s A ttr ib ut io n- N on C om m er ci al 3 .0 U np or te d L ic en ce . View Article Online C186–C201 into Dha was already shown to be more impactful on the protein 3D structure as compared to the disulde rebridging. To further assess that the structural integrity of CRM197-DCA was preserved upon glycoconjugation, CD spectra were recorded for CRM197-PSIa and CRM197-DCA-PSIa, using unmodied CRM197 and CRM197-DCA as controls. The CD spectra of the two conju- gates (which exhibited a similar size in the DLS analysis) showed that both random and selective conjugation caused a slight shi of theminimum at 218 nm to higher molar ellipticity values, with © 2022 The Author(s). Published by the Royal Society of Chemistry respect to CRM197 and CRM197-DCA, which appeared almost overlapping (Fig. S8, ESI†). In vivo results Immune responses induced by site-selective CRM197-DCA-PSIII and CRM197-DHA-PSIa conjugates were compared to the ones obtained with random conjugates where polysaccharide is attached randomly to lysines on the surface of CRM197. These Chem. Sci., 2022, 13, 2440–2449 | 2445 http://creativecommons.org/licenses/by-nc/3.0/ http://creativecommons.org/licenses/by-nc/3.0/ https://doi.org/10.1039/d1sc01928g Table 2 OPKA titers measured for sera elicited against GBS PSIa Glycoconjugate OPKA titers Post 2 Post 3 CRM-PSIaa 120; 60 125; 75 CRM-Dha-PSIaa <50 <50 CRM-PSIab 63; 62 253; 179 CRM-DCA-PSIab 104; 51 265; 143 a OPKA associated with ELISA shown in the Fig. 4A le panel. b OPKA associated with ELISA shown in the Fig. 4A right panel. Table 1 Characteristics of GBS and PN14 glycoconjugates Glycoconjugate Glycosylation ratio (w/w) Free saccharide (%) CRM197-PSIa 2.5 16 CRM197-DHA-PSIa 1.1 <6 CRM197-DCA-PSIa 4.8 11.9 CRM197-PSIIII 1.2 <2 CRM197-DCA-PSIII 1.4 Nd CRM197-PN14PS 1.0 Nd CRM197-DCA-PN14PS 1.9 <5 Chemical Science Edge Article O pe n A cc es s A rt ic le . P ub lis he d on 1 0 Fe br ua ry 2 02 2. D ow nl oa de d on 4 /2 8/ 20 22 1 1: 38 :2 5 A M . T hi s ar tic le is li ce ns ed u nd er a C re at iv e C om m on s A ttr ib ut io n- N on C om m er ci al 3 .0 U np or te d L ic en ce . View Article Online conjugates were prepared as previously reported33 and are considered viable candidates for clinical studies.34,35 In the immunization experiments, groups of ten mice received three Fig. 4 Enzyme-linked immunosorbent assay immunoglobulin G (IgG) collected after 2 and 3 vaccine doses, reported as arbitrary units (EU m intervals from 10 serum samples; *p < 0.05, 0.001 < p**** < 0.0001 immunosorbent assay immunoglobulin G (IgG) titers anti GBS PSIII (C) an doses, reported as arbitrary units (EU mL�1); bars represent the geometr 2446 | Chem. Sci., 2022, 13, 2440–2449 doses of the prepared conjugates adjuvanted with alum hydroxide. Two weeks aer the second and third vaccine doses, individual IgG titers were measured by ELISA using full-length PSIII or PSIa conjugated to Human Serum Albumin as the coating agent. The immunogenicity of the CRM197-DCA-PSIa conjugate was 3 fold higher than that of the CRM197-DHA counterpart and statistically comparable to that of the random CRM197-PSIa conjugate, although a trend to be higher for the latter was observed. The functional activity of the elicited antibodies was estimated on pooled sera by OPKA, an assay that mimics in vivo GBS killing by effector cells in the presence of complement and specic antibodies, and correlates with mouse protection.36 In agreement with the ELISA outcome, OPKA titers of the pooled sera aer the second and third doses were comparable for CRM197-DCA-PSIa and CRM197PSIa conjugates (Table 2), while they were lower for the CRM197-DHA-PSIa conjugate. In addition, the anti-protein antibodies for CRM197-DCA were comparable to those for CRM197 and more than 10-fold higher as compared to those for CRM197-DHA (Fig. 4B and ESI, Fig. S9†), possibly as a result of protein stabilization.30 It is noteworthy that the protein dose for CRM197-DCA was 2 fold lower than that for the random conjugate. Given the positive results obtained with the CRM197-DCA conjugate, we assessed whether strong immunogenicity could be elicited through this conjugation approach despite the type titers anti GBS PSIa (A) and anti CRM197 (B) in mouse serum samples L�1); bars represent the geometric mean titers with 95% confidence (Kruskal–Wallis and Dunn multiple comparisons test). Enzyme-linked d Sp PS14 (D) in mouse serum samples collected after 2 and 3 vaccine ic mean titers with 95% confidence intervals from 10 serum samples. © 2022 The Author(s). Published by the Royal Society of Chemistry http://creativecommons.org/licenses/by-nc/3.0/ http://creativecommons.org/licenses/by-nc/3.0/ https://doi.org/10.1039/d1sc01928g Edge Article Chemical Science O pe n A cc es s A rt ic le . P ub lis he d on 1 0 Fe br ua ry 2 02 2. D ow nl oa de d on 4 /2 8/ 20 22 1 1: 38 :2 5 A M . T hi s ar tic le is li ce ns ed u nd er a C re at iv e C om m on s A ttr ib ut io n- N on C om m er ci al 3 .0 U np or te d L ic en ce . View Article Online of polysaccharide used. As shown in Fig. 4C, the immune response aer two and three injections was comparable for CRM197-PSIII and the site selective CRM197-DCA-PSIII conju- gate. The functional activity elicited by site selective CRM197- DCA-PSIII (67 and 283 are average OPK titer of three different experiments from the pool of mouse sera obtained aer two and three doses, respectively) was also comparable with the one obtained by immunizing mice with the reference random conjugate (143 and 154, respectively). Finally, a site-selective CRM197-DCA conjugate of Sp14 PS, which is part of commer- cial pneumococcal vaccines,37 was prepared and tested in mice. Again, in vivo immunogenicity on 10 mice showed aer two and three vaccine doses a level of elicited specic anti-Sp14 PS antibodies comparable to those for the respective random conjugate (Fig. 4D). Discussion The rational modication of the structure of peptides and proteins offers a wide range of opportunities for the modulation of their biological activity. Glycoconjugates present in licensed vaccines are generally generated by classic lysine random conjugation of carbohy- drates on the surface of carrier protein. These conjugates have oen heterogeneous compositions, which cause batch-to-batch variability of structure and activity, oen leading to an incom- plete understanding of their mechanism of action. Site-selective conjugation is a powerful method to direct polysaccharide conjugation at predetermined sites of the protein and ensure higher batch-to-batch consistency in comparison to classic nonspecic conjugation procedures, particularly when the protein is used with the dual role of antigen and carrier. Stefanetti et al.9 demonstrated that specic site-selective single or double attachment of glycan antigens to carrier protein CRM197 is sufficient to induce high levels of anti- Salmonella typhimurium O-antigen IgG specic antibodies with serum bactericidal activity. Conjugation at the C186–C201 bond resulted in high anti O-antigen bactericidal antibody titers. Starting from this discovery, we generated two different vaccines with a dened conjugation point, by modifying the same C186–C201 disulde bridge in CRM197. In the rst approach, a DCA gra was installed in CRM197. The second strategy involved the conversion of cysteine residues in dehy- droalanine. Both methods offer the possibility to attach through condensation reaction or thiol Michael addition bifunctional linkers for click reactions with carbohydrates. Particularly, here we incorporated azido moieties, ready for strain promoted click chemistry glycoconjugation with DBCO- derivatized polysaccharides from group B Streptococcus and S. pneumoniae. In vivo data highlighted that CRM197-DCA conjugates elicited an immune response comparable to the reference random CRM197-GBS PSIa and PSIII conjugates, which are vaccine candidates under clinical development, while CRM197-DHA resulted in poor immunogenicity. Moreover, antibodies elicited by CRM197-DCA-PSIa and PSIII are functional with an OPK titer comparable to the reference vaccines. Also, CRM197-DCA © 2022 The Author(s). Published by the Royal Society of Chemistry conjugation of S. pneumoniae equally provided a strongly immunogenic conjugate. This observation converges with the work done by Mart́ınez-Sáez et al.30 according to which oxetane gra installation on protein through the regioselective disulde stapling of the protein carrier CRM197 enables stabilization of folded structures and results in an enhanced bioactivity, e.g. a signicant increase in its immunogenicity in vivo. It has been reported that the 3D structure of CRM197 is more altered by random conjugation of glycans in comparison to the one of formaldehyde treated proteins, including DT or CRM197 itself.38 The loss of the CRM197 tertiary structure with potential detrimental impact on certain conformational epitopes could partially explain the lower propensity of this protein to be subjected to immune interference in the presence of pre- existing anti-protein antibodies.38,39 Of note, the modication induced by glycoconjugation might appear more evident in CRM197 with respect to formaldehyde treated proteins because the marked unfolding caused by chemical detoxication might result in negligible further structural impact caused by glycan coupling. Our data complement this information and suggest that slight modications of the protein tertiary structure, such as the ones induced by a random reaction of surface exposed lysine residues, are compatible with a strong anti-carbohydrate immune response. Among the two cysteine directed chemistries herein tested, disulde re-bridging of CRM197 with DCA is shown to aid preservation of the 3D structure of the protein, as demonstrated by combined CD and DLS and X-ray experiments. Importantly, the crystal structure of CRM197-DCA presented herein clearly shows the selective presence of one disulde bridge at C185–C201, while the second disulde bridge (C461– C471) remains untouched, and preservation of the 3D protein structure compared to the native form. Minimal alterations of the 3D structure caused by glycoconjugation and detectable by CD did not impair its carrier properties, resulting in a robust anti-carbohydrate response. Conversely, a modication based on ring opening (CRM197- DHA) resulted in structural changes that strongly impacted the immunogenicity of the conjugated glycan. Overall this study underpins that selective protein modi- cations based on bridging of disulde bonds are optimal to preserve the structural integrity and stability, and consequently the immunogenicity of the glycoconjugates. Conclusions This work underscores the impact of protein modication on the stability and immunogenicity of glycoconjugates and high- lights disulde stapling as an effective strategy for selective protein conjugation, widely applicable to different poly- saccharides. In addition, it opens the path for the use of highly selective chemical methods for the preparation of glyco- conjugate vaccines with advantages in terms of consistency of production and characterization, and a better understanding of their immunological mechanism of action. Chem. Sci., 2022, 13, 2440–2449 | 2447 http://creativecommons.org/licenses/by-nc/3.0/ http://creativecommons.org/licenses/by-nc/3.0/ https://doi.org/10.1039/d1sc01928g Chemical Science Edge Article O pe n A cc es s A rt ic le . P ub lis he d on 1 0 Fe br ua ry 2 02 2. D ow nl oa de d on 4 /2 8/ 20 22 1 1: 38 :2 5 A M . T hi s ar tic le is li ce ns ed u nd er a C re at iv e C om m on s A ttr ib ut io n- N on C om m er ci al 3 .0 U np or te d L ic en ce . View Article Online Ethical statement Animal experiments were performed in accordance with the regulations of the Directive 2010/63/EU and GSK ethical guidelines, under the approval of the Italian Ministry of Health (Italian Legislative Decree no. 26/2014). All mice were housed under specic pathogen-free conditions at the GSK Vaccines Animal Resource Centre in compliance with the relevant guidelines. Data availability All relevant data are reported as ESI.† Author contributions FC, AK, GJLB and RA conceived the study; FC, AK, MCM, DV, LDI, EB, BB, FA, and LDB performed experimental work; FC, AK, MCM, IM, MRR, GJLB and RA analyzed results; FC, AK, GJLB and RA wrote the manuscript; all revised the manuscript. Conflicts of interest FC, DV, LDI, DO, EB, BB, FA, LDB, IM, MRR, and RA are employees of the GSK group of companies. AK was hosted in a secondment in GSK during her PhD programme. Prevnar and HibTITER are trademarks from Pzer; Vaxem-Hib, Menveo and Menjugate are trademarks from GSK. Acknowledgements This work was sponsored by GlaxoSmithKline Biologicals SA and has received funding from the European Union's Horizon 2020 research and innovation programme under the Marie Skłodowska-Curie grant agreement no. 675671. CS is recipient of the grant no. 861194 (PAVax) under the EU Horizon 2020 program. Dr Werner Pansegrau is acknowledged for support in CD experiments. We are grateful to the staff at beamlines ID23-1 of the European Synchrotron Radiation Facility (ESRF, France) for their assistance in collecting X-ray diffraction data. Notes and references 1 R. Rosini, S. Nicchi, M. Pizza and R. Rappuoli, Front. Immunol., 2020, 11, 1048. 2 R. Rappuoli, Sci. Transl. 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Proietti, A. Seubert, S. Nuti, F. Berti and M. R. Romano, Vaccine, 2016, 34(20), 2334–2341. Chem. Sci., 2022, 13, 2440–2449 | 2449 http://creativecommons.org/licenses/by-nc/3.0/ http://creativecommons.org/licenses/by-nc/3.0/ https://doi.org/10.1039/d1sc01928g Retaining the structural integrity of disulfide bonds in diphtheria toxoid carrier protein is crucial for the effectiveness of glycoconjugate vaccine... Retaining the structural integrity of disulfide bonds in diphtheria toxoid carrier protein is crucial for the effectiveness of glycoconjugate vaccine... Retaining the structural integrity of disulfide bonds in diphtheria toxoid carrier protein is crucial for the effectiveness of glycoconjugate vaccine... Retaining the structural integrity of disulfide bonds in diphtheria toxoid carrier protein is crucial for the effectiveness of glycoconjugate vaccine... Retaining the structural integrity of disulfide bonds in diphtheria toxoid carrier protein is crucial for the effectiveness of glycoconjugate vaccine... Retaining the structural integrity of disulfide bonds in diphtheria toxoid carrier protein is crucial for the effectiveness of glycoconjugate vaccine... Retaining the structural integrity of disulfide bonds in diphtheria toxoid carrier protein is crucial for the effectiveness of glycoconjugate vaccine... Retaining the structural integrity of disulfide bonds in diphtheria toxoid carrier protein is crucial for the effectiveness of glycoconjugate vaccine... Retaining the structural integrity of disulfide bonds in diphtheria toxoid carrier protein is crucial for the effectiveness of glycoconjugate vaccine... Retaining the structural integrity of disulfide bonds in diphtheria toxoid carrier protein is crucial for the effectiveness of glycoconjugate vaccine... Retaining the structural integrity of disulfide bonds in diphtheria toxoid carrier protein is crucial for the effectiveness of glycoconjugate vaccine... Retaining the structural integrity of disulfide bonds in diphtheria toxoid carrier protein is crucial for the effectiveness of glycoconjugate vaccine... Retaining the structural integrity of disulfide bonds in diphtheria toxoid carrier protein is crucial for the effectiveness of glycoconjugate vaccine... Retaining the structural integrity of disulfide bonds in diphtheria toxoid carrier protein is crucial for the effectiveness of glycoconjugate vaccine...