Polysaccharide structure dictates mechanism of SEE COMMENTARY adaptive immune response to glycoconjugate vaccines

Ximei Suna,b, Giuseppe Stefanettia,c, Francesco Bertid, and Dennis L. Kaspera,1

aDepartment of Microbiology and Immunobiology, Harvard Medical School, Boston, MA 02115; bGraduate Program in Immunology, Harvard Medical School, Boston, MA 02115; cDepartment of Chemistry, University of Milan, 20133 Milan, Italy; and dTechnical R&D, GSK Vaccines, 53100 Siena, Italy

Contributed by Dennis L. Kasper, November 1, 2018 (sent for review September 25, 2018; reviewed by Peter R. Andreana and Moriya Tsuji) Glycoconjugate vaccines are among the most effective interven- from the carrier (5). These surface-presented CPSs are + tions for preventing several serious infectious diseases. Covalent able to activate a subset of CD4 T cells, designated - linkage of the bacterial capsular polysaccharide to a carrier specific T cells (Tcarbs), which regulate the adaptive immune + provides CD4 T cells with epitopes that facilitate a memory re- response to the GBSIII glycoconjugate (5–8). Similar mechanisms sponse to the polysaccharide. Classically, the mechanism responsi- have now been shown to be responsible for T helper responses to ble for antigen processing was thought to be similar to what was glycoconjugates made with the type 3 S. pneumoniae poly- known for hapten-carrier conjugates: protease digestion of the saccharide (Pn3P) (6). carrier protein in the endosome and presentation of a resulting + In the present study, we analyze the T response to gly- peptide to the T cell receptor on classical peptide-recognizing CD4 coconjugates of several other important pathogens, including T cells. Recently, an alternative mechanism has been shown to be conjugates made from Vi antigen of Salmonella Typhi (Vi), the responsible for the memory response to some glycoconjugates. Pro- H. cessing of both the protein and the polysaccharide creates glycopep- CPS of type Ib group B streptococci (GBSIb), the CPS of tides in the endosome of antigen-presenting cells. For presentation, influenzae type b (Hib), and the group C polysaccharide of N. the peptide portion of the is bound to MHCII, allowing meningitidis (MenC). We report that only MenC-specific IgG the covalently linked to activate carbohydrate-specific helper responses are not regulated by Tcarbs. This lack of Tcarb acti- + CD4 T cells (Tcarbs). Herein, we assessed whether this same mech- vation is related to the structure of the polysaccharide. De-

anism applies to conjugates prepared from other capsular polysac- polymerization of MenC polysaccharide in the acidic environment INFLAMMATION IMMUNOLOGY AND charides. All of the glycoconjugates tested induced Tcarb-dependent of the endolysosome results in marked reduction in polysaccha- responses except that made with group C ;in ride size to monomers, with a consequent failure to be recognized the latter case, only generated from the carrier protein by T cells as an independent antigen. Given that there may be were critical for helper T cell recognition. Digestion of this acid- at least two mechanisms governing T cell responses to glyco- sensitive polysaccharide, a linear homopolymer of α(2 → 9)-linked conjugates, a deeper understanding of factors influencing anti- , to the size of the monomeric unit resulted in a dominant + gen processing and presentation as well as cooperation between CD4 T cell response to peptides in the context of MHCII. Our results T and B cells in response to glycoconjugate vaccination is a key show that different mechanisms of presentation, based on the struc- factor to be considered in improving the design of the next- ture of the carbohydrate, are operative in response to different generation glycoconjugate vaccines. glycoconjugate vaccines.

glycoconjugate | vaccine | Tcarb | antigen presentation | group C Neisseria Significance meningitidis Helper T cell responses to glycoconjugate vaccines are regu- lated through mechanisms dependent upon the structure of any pathogenic bacteria express large-molecular-sized the polysaccharide. We show that three of the four important surface called capsular polysaccharides M conjugate vaccines tested induced antibody responses regulated (CPSs). Beginning in the 1980s, CPSs of bacterial targets were primarily by carbohydrate-recognizing helper T cells. However, coupled to carrier to create effective glycoconjugate the adaptive immune response to meningococcal group C (MenC) vaccines. These vaccines were more immunogenic than un- conjugate was restricted to peptide-recognizing helper T cells. conjugated polysaccharides, especially in children under 2 y of We show that MenC is degraded to a monomeric sialic acid res- age (1). Several glycoconjugate vaccines were developed and idue that cannot be recognized by T cell receptor as an inde- have played an enormous role in preventing infectious dis- pendent antigen. The structure of the saccharide constitutes a eases caused by virulent pathogens, including vaccines against critical factor in determining the processing and presentation of Haemophilus influenzae type b, , and glycoconjugate vaccines. An understanding of the mechanisms Neisseria meningitidis (2, 3). On the basis of studies of hapten- underlying the immune responses to glycoconjugates will be carrier immune responses, it had been assumed that, in glyco- crucial in the production of highly protective knowledge-based conjugate vaccines, the peptide processed from the carrier pro- vaccines. tein is presented by the major histocompatibility class II (MHCII) molecule and that this signal plays a central role in Author contributions: X.S. and D.L.K. designed research; X.S. performed research; G.S. and + activating CD4 helper T cells. The peptide-recognizing T cells F.B. contributed new reagents/analytic tools; X.S. and D.L.K. analyzed data; and X.S., G.S., in turn help B cell maturation and the formation of immuno- F.B., and D.L.K. wrote the paper. logicmemory(4).Theoriginalhypothesis was in part based Reviewers: P.R.A., University of Toledo; and M.T., Aaron Diamond AIDS Research Center. on the failure of pure polysaccharides to elicit IgM-to-IgG The authors declare no conflict of interest. class switching and substantial memory responses. Recently, we Published under the PNAS license. reported a different mechanism governing the immune responses See Commentary on page 14. to glycoconjugate vaccines, using the CPS of type III group B 1To whom correspondence should be addressed. Email: [email protected]. Streptococcus (GBSIII) as a model antigen. We showed that This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. antigenic fragments of the polysaccharide are presented on the 1073/pnas.1816401115/-/DCSupplemental. surface by MHCII molecules in the context of a covalently linked Published online December 3, 2018.

www.pnas.org/cgi/doi/10.1073/pnas.1816401115 PNAS | January 2, 2019 | vol. 116 | no. 1 | 193–198 Downloaded by guest on September 24, 2021 Results Various Glycoconjugates Induce T Helper Cells Recognizing Different Epitopes. We had previously shown that a clear biomarker for Tcarb-dependent responses was a polysaccharide-specific anti- body response after priming with a polysaccharide covalently linked to a carrier protein and boosting with the same poly- saccharide linked to a different and unrelated carrier protein. To investigate the involvement of Tcarbs in the humoral immune response to different glycoconjugates, we performed priming and boosting immunization experiments with glycoconjugates made with Vi, GBSIb, Hib, and MenC CPSs. BALB/c mice were primed at the beginning of the experiment and boosted 14 d later with different antigen combinations. One week after the boost, serum levels of polysaccharide-specific IgG were determined. For Vi, GBSIb, and Hib glycoconjugates, boosting with a gly- coconjugate containing the same polysaccharide but a heterolo- gous carrier protein induced polysaccharide-specific IgG titers of the same magnitude as those seen after priming and boosting with glycoconjugates containing the same carrier (Fig. 1A). As reported with GBSIII and Pn3P (5, 6), this result supports a Tcarb- dependent mechanism for all three of these glycoconjugates. In marked contrast to the above results, priming and boosting with MenC glycoconjugates containing heterologous carrier proteins induced significantly lower levels of MenC-specific IgG than did primary and secondary immunization with either a MenC-OVA (ovalbumin) conjugate or a MenC-CRM197 (non- toxic mutant of diphtheria toxin) conjugate. This result was confirmed with a combination of different carriers, including MenC-TT (tetanus toxoid), MenC-HEL (hen egg lysozyme), MenC-OVA, and MenC-CRM197 (Fig. 1B). We also found that MenC-specific IgM levels were similar in groups immunized with different conjugate combinations, a result suggesting a similar level of B cell activation independent of the carrier (SI Ap- pendix,Fig.S1A). To exclude the possibility that we were seeing a T cell-independent response, we treated mice with CD4-specific blocking antibody during the interval between priming and boosting with MenC-CRM197. The excellent booster IgG response observed in mice treated with isotype control antibody was sig- nificantly reduced in mice treated with anti-CD4 (SI Appendix, Fig. S1B). The failure of MenC conjugates prepared with different carrier proteins to induce a booster IgG response suggested that perhaps these glycoconjugates were not able to induce Tcarbs and that Tcarbs were not essential in regulating MenC polysaccharide- specific IgG responses.

Carrier-Specific T Cell-Mediated Adaptive Immune Response to MenC Conjugates. On the basis of the previous results, we theorized that + carrier protein/peptide-specific CD4 T cells may be the only T cell subset essential to regulate the response to MenC conju- gate vaccines (9, 10). To further test this hypothesis, we primed mice with a combination of MenC-CRM197 plus unconjugated OVA protein, and we boosted these mice with MenC-OVA. We hypothesized that priming with this combination would yield Fig. 1. Requirement for helper T cell memory induction by either carbo- MenC-specific memory B cells resulting from MenC-CRM197 as hydrate or peptide in several glycoconjugate vaccines. BALB/c mice were well as OVA-specific memory helper T cells generated from the primed (day 0) and boosted (day 14) with different glycoconjugate combi- OVA protein component. Interestingly, after boosting with nations. Polysaccharide-specific IgG antibodies were measured by ELISA in serum obtained on day 21. (A) Tcarb-dependent IgG antibody induction to MenC-OVA, mice primed with the mixture of MenC-CRM197 and OVA protein had MenC IgG levels similar to those in mice Vi, GBSIb, and Hib CPSs. Serum titers are reported as the reciprocal dilution primed and boosted with MenC-OVA. The MenC IgG levels that results in an OD of 0.5 at 405 nm. (B) Tcarb-independent IgG antibody induction to MenC. MenC-specific IgG monoclonal antibody was used as a were significantly lower in mice primed with MenC-CRM197 standard to determine the MenC IgG concentrations. n = 4–6 mice per without OVA (Fig. 2A and SI Appendix, Fig. S2 A and B). This group. All data are expressed as mean ± SEM values. *P ≤ 0.05; **P ≤ 0.01; result suggests that in MenC glycoconjugates carrier protein- ***P ≤ 0.001; ****P ≤ 0.0001; ns, not significant. activated T cells are essential in inducing a humoral immune response and antibody class switching. To further explore the requirements for cooperation between boosted them with either MenC-CRM197 or MenC and CRM197 B and T cells, we performed another series of immunization protein (either alone or physically mixed but not conjugated). experiments. We first primed mice with MenC-CRM197 and then Booster IgG responses occurred only in mice that received the

194 | www.pnas.org/cgi/doi/10.1073/pnas.1816401115 Sun et al. Downloaded by guest on September 24, 2021 and groups B and C were immunized with MenC-OVA. The resulting IgG and IgM antibody titers in the three groups are SEE COMMENTARY shown in Fig. 2C. Groups A and C had significantly higher MenC-specific IgG titers than group B, while MenC-specific IgM levels were similar in the three groups. In a previously reported experiment supporting the role of Tcarbs in the immune re- sponse to glycoconjugates (6), Middleton et al. adoptively trans- + ferred B cells and CD4 T cells from mice immunized with Pn3P-KLH (keyhole limpet hemocyanin) to recipient mice and then immunized the recipient mice with either Pn3P-KLH or Pn3P-OVA. The similar IgG levels in these two groups sup- ported a Tcarb-mediated response. In contrast to these results with the Pn3P conjugate, our observations with MenC glyco- conjugates suggested that a mechanism in which carrier protein/ peptides are essential in recruiting T cell help is operative.

Conjugation Chemistry Is Not the Critical Factor in the Induction of Tcarbs. The structures of the GBSIII and MenC glycoconjugates were very different, though in both the polysaccharides were conjugated to carrier proteins through reductive amination. Whereas the GBSIII conjugate was cross-linked by oxidative creation of aldehydes on a fraction (∼30%) of the side-chain terminal sialic acid residues of the repeating unit, the MenC conjugate was linked at a single site on the reducing end of the polysaccharide (Fig. 3A). In addition, MenC depolymerization by mild acid hydrolysis and oxidation resulted in a size of 10– 30 kDa, while GBSIII used for conjugation maintained its orig- > INFLAMMATION inal size ( 100 kDa). Thus, we hypothesized that conjugation IMMUNOLOGY AND chemistry might affect T cell recognition of carbohydrates. To increase the resemblance to the structure of a cross-linked gly- coconjugate, we conjugated MenC through the carboxylic acid to

Fig. 2. Carrier-specific T cells are essential in regulating the adaptive im- mune response to MenC conjugate. (A) BALB/c mice were primed (day 0)

with MenC-CRM197 either alone or with unconjugated OVA protein, and all groups were boosted (day 14) with MenC-OVA. MenC-specific IgG antibodies were measured by ELISA in serum obtained on day 21. (B) Design of the adoptive transfer experiments. Recipient mice received B cells from MenC- + CRM197-immunized mice and CD4 T cells from mice immunized with either MenC-CRM197 (groups A and B) or OVA (group C). One day after adoptive transfer, recipient mice were immunized with either MenC-CRM197 (group A) or MenC-OVA (groups B and C). (C) Concentrations of MenC-specific IgG and IgM in sera from recipient mice, measured by ELISA 7 d after immunization. n = 4 or 5 mice per group. All data are expressed as mean ± SEM values. ****P ≤ 0.0001; ns, not significant.

MenC-CRM197 conjugate for both primary and secondary im- munization. Similarly, priming of mice either with unconjugated MenC or CRM197 protein alone or with a mixture of unconjugated MenC and CRM197 protein did not support a robust secondary IgG response upon boosting with the MenC-CRM197 conjugate (SI Appendix,Fig.S2C). This result suggested that a conjugate is required to stimulate MenC-specific memory B cells. To directly examine the contribution of carrier-specific T cells to MenC-specific booster IgG responses, we performed adoptive transfer experiments (Fig. 2B). Donor groups of BALB/c mice Fig. 3. Effect of conjugate chemistry of MenC on selection of T helper cells. were immunized with either MenC-CRM197 or OVA, and + (A) Schematic representation of the structure of end-conjugated (Upper) splenic and lymph node B and CD4 T cells from each group and cross-linked (Lower) MenC glycoconjugates made by reductive amina- were purified. Three groups of recipient mice all received B cells tion (Upper) and carbodiimide reaction (Lower), respectively. (B) Superdex 200 elution profile of MenC-OVA from reductive amination (Left) or carbo- from the MenC-CRM19–immunized donors. Groups A and B + diimide reaction (Right). (C) Concentration of IgG antibody to MenC in BALB/c also received CD4 T cells from MenC-CRM197–immunized + mice primed (day 0) and boosted (day 14) with cross-linked MenC glyco- donors, while group C received CD4 T cells from OVA- conjugates containing either the same or a heterologous carrier protein, as immunized donors. One day after adoptive transfer, group A measured by ELISA in serum obtained on day 21. n = 4 mice per group. Data recipient mice were actively immunized with MenC-CRM197, are expressed as mean ± SEM values. **P ≤ 0.01; ****P ≤ 0.0001.

Sun et al. PNAS | January 2, 2019 | vol. 116 | no. 1 | 195 Downloaded by guest on September 24, 2021 derivatized carrier protein treated with adipic acid dihydrazide (ADH) linker (SI Appendix, Fig. S3) (11). This method activated a fraction of repeating units along the chain of MenC without reducing its molecular size (>100 kDa). The cross-linked MenC conjugate was significantly larger than the end-linked MenC conjugate, as shown by size exclusion chromatography (Fig. 3B). We performed priming and boosting immunization experi- ments with the cross-linked MenC glycoconjugates containing either the same or different carrier proteins. Mice primed and boosted with MenC glycoconjugates containing the same carrier protein—either MenC-ADH-CRM197 or MenC-ADH-TT—had strong booster MenC-specific IgG responses. However, priming and boosting with MenC conjugates containing different carrier proteins induced significantly lower titers of MenC-specific IgG (Fig. 3C). This result was similar to that observed with MenC linked at a single reducing end of the polysaccharide to either the same or different proteins (Fig. 1B). Therefore, conjugation chemistry does not seem to explain the failure of MenC glyco- conjugates to induce Tcarbs.

Processing and Presentation of MenC Polysaccharide. We previously reported that, following immunization with a glycoconjugate vaccine, a depolymerized form of GBSIII (∼10 kDa) bound to an MHCII binding peptide can be presented and the carbohydrate recognized by the T cell receptor (TCR) (5). T cell recognition of carbohydrates was also reported for zwitterionic polysaccharides (12), which presumably bind directly to MHCII by electrostatic interactions. In this study, we speculated that MenC poly- saccharide might fail to be presented in the context of MHCII on the antigen-presenting cell (APC) surface. One possible un- derlying reason could be the sensitivity to acidic hydrolysis of MenC, which is a linear homopolymer of α(2 → 9)-linked sialic acid. It is possible that the ketosidic linkage between sialic acid repeating units on MenC is hydrolyzed to an extremely small size in the endolysosome and therefore cannot be presented to or recognized by TCRs. We conducted a flow cytometry analysis to assess this possibility by determining whether antigenically active MenC carbohydrates can be presented on the APC surface. Bone marrow-derived dendritic cells (BMDCs) from wild-type mice were incubated with unconjugated or conjugated GBSIII or MenC for 18 h. The cells were collected and stained with a monoclonal antibody specific for GBSIII or MenC at 4 °C and then incubated with a fluorophore- Fig. 4. Processing and presentation of MenC. (A) Flow cytometry analysis of conjugated secondary antibody. Consistent with previously pub- BMDCs after incubation (18 h) with unconjugated GBSIII or GBSIII-TT (Left)or lished results, the control saccharide (GBSIII) was detected on the with unconjugated MenC, MenC-TT, or MenC-CRM197 (Right) followed by surface of the cells incubated with the GBSIII conjugate. How- surface staining with monoclonal antibody to GBSIII or MenC. (B)Elution ever, no BMDC surface presentation of MenC was detected after profile of lysates of Raji B cell endolysosomes after 18 h of incubation with incubation of the cells either with unconjugated MenC or with [3H]-MenC on a size exclusion HPLC column: ProSEC 300S (MW range, 1,500– – conjugated MenC-TT or MenC-CRM197 (Fig. 4A). Thus, pro- 800,000) or Aquagel-OH 20 (MW range, 100 20,000). (C) Raji B cells were cessed MenC carbohydrate was not loaded onto the APC surface, treated with either 4-OH TEMPO (a superoxide inhibitor) or bafilomycin A1 (BFA, an endosomal acidification inhibitor) for 1 h before incubation with was altered structurally, or was depolymerized to a very small size. 3 Any of these possibilities might explain why the MenC antigenic [ H]-MenC. The molecular size distributions of the endolysosomal lysates were analyzed on a size exclusion column (ProSEC 300S). SA, sialic acid. epitope could not be detected on the surface of the APC by the monoclonal antibody and why it could not be recognized by the TCR, with consequent Tcarb induction. a size similar to that of a monomer of sialic acid (Fig. 4B). We We then examined MenC polysaccharide processing in APCs. performed parallel experiments with a control polysaccharide— CPSs are taken up into the APC endosome and are depoly- polysaccharide A (PSA) from Bacteroides fragilis—that is merized by oxidative agents such as reactive oxygen species and known to be processed to a size of ∼15 kDa (12). We compared reactive nitrogen species (5, 12, 13) into smaller carbohydrates, ∼ the molecular size of the endosomally depolymerized MenC to generally 10 kDa in size. One possible explanation for lack of 3 MenC detection on the APC surface is that MenC is digested that of similarly processed [ H]-PSA on Aquagel-OH 20 (SI Ap- through oxidative depolymerization or acidic hydrolysis to an pendix, Fig. S4) and found that the size of the processed saccha- extremely small size so that it cannot be recognized by the TCR. rides differed substantially: PSA was at least 7 kDa, and MenC ∼ Accordingly, we assessed the size of the MenC saccharide within appeared to be much smaller at 200 Da. To further define the the APC endolysosome. We incubated radioactively labeled mechanisms involved in the depolymerization of MenC, we MenC {[3H]-MenC} with Raji B cells for 18 h, then isolated and incubated Raji B cells with [3H]-MenC in the presence of either lysed the endolysosome. We ran the lysate on a size-exclusion HPLC a reactive oxygen species inhibitor (4-OH TEMPO) or an column and found that MenC was substantially depolymerized to ATPase proton pump inhibitor (bafilomycin A1). Interestingly,

196 | www.pnas.org/cgi/doi/10.1073/pnas.1816401115 Sun et al. Downloaded by guest on September 24, 2021 less depolymerization of the polysaccharide was detected in the group C polysaccharide of N. meningitidis induces carrier-specific endolysosome after incubation with either inhibitor. In bafilomycin- helper T cells, not Tcarbs. Active immunization with various SEE COMMENTARY treated cells, a small peak was seen at the void volume of the vaccine constructs and adoptive transfer experiments clearly + column that was not observed with 4-OH TEMPO treatment. showed that carrier peptide-specific CD4 T cells are sufficient to When treated with a combination of the two inhibitors, a sim- induce adaptive immune antibody responses to the MenC conju- ilar upward size shift was observed (Fig. 4C). This result sug- gate. Given that the covalent linkage between the linking sialic gested that both oxidative depolymerization and acidic hydrolysis acid residue and the group on the protein generated during contribute to MenC depolymerization, with acid hydrolysis per- chemical conjugation would not be broken down in the endoly- haps being somewhat more important. sosome, it is likely that some processed sugars from MenC are still presented on the surface along with the conjugated peptide. Discussion However, our findings indicate that these sugars do not constitute Antibodies to CPSs mediate protection against encapsulated an antigenic epitope and do not sufficiently mask the peptide in bacteria (2). Several highly effective glycoconjugate vaccines the MHCII binding cleft to prevent its recognition; thus they fail have been created using a hapten-carrier protein conjugation to induce Tcarb helper responses. We did show that the MenC strategy (14, 15). It has become standard practice to couple CPSs polysaccharide is digested to a reduced size comparable to that of from bacterial targets to T cell-dependent carrier proteins to a single sialic acid residue in the endosome. Although these sialic create glycoconjugate vaccines (16–19). Immunization with gly- acids linked to the MHCII binding peptide are too small to fit in coconjugates, as opposed to pure polysaccharides, elicits T cell the TCR pocket independent of the peptide, the attachment of help for B cells that produce IgG antibodies to the polysaccharide the monosaccharide could modify the TCR specificity to the + component (2, 4). In addition to inducing polysaccharide-specific peptide. Detailed studies on CD4 T cell recognition of glyco- IgM-to-IgG switching, glycoconjugate immunization elicits both B peptides showed that the TCR makes specific contact with both cell and T cell memory responses (2). the sugar moiety and peptide residues (20–22). In the case of the Other than polysaccharides with a zwitterionic charge motif MenC conjugate, it is possible that peptide modified with a single (e.g., PSA of B. fragilis or the polysaccharide of type 1 S. pneu- sialic acid might initiate the activation of a novel T cell subset that moniae), which bind directly to MHCII through electrostatic in- collaborates as helper T cells with the dominant peptide-specific teractions, most bacterial polysaccharides fail to bind to MHCII T cells. and therefore are not presented to the TCR. As a result, most N. meningitidis is a major cause of bacterial meningitis

pure polysaccharides induce immune responses that are T cell worldwide, especially in the African meningitis belt, and has a INFLAMMATION IMMUNOLOGY AND independent. The traditional explanation for the mechanism by high associated mortality. MenC has been which glycoconjugates induce humoral immune responses is that + shown to be safe and immunogenic and to be capable of priming the carrier protein portion of the conjugate activates CD4 Tcells infants, toddlers, young children, and adults for immunologic to help carbohydrate-specific B cells produce long-lasting IgG memory (23–26). However, preliminary surveillance data in antibodies through both cognate and cytokine-mediated interac- England and Wales suggest a waning of effectiveness from 1 y tions (2, 4). The classical hypothesis of immune activation by after three-dose priming in infancy (27). A better understanding glycoconjugate vaccines suggests that only peptides generated of the mechanisms involved in the immune response to MenC from the polysaccharide-linked carrier protein can be presented to glycoconjugate immunization could lead to better vaccines with and recognized by T cells. This view, however, ignores the syn- improved efficacy. Clearly, more than one mechanism is responsi- thetic linkage of carbohydrates to proteins by strong covalent ble for the induction of immune responses to glycoconjugates. bonds that are unlikely to be broken within the endosome. Thus, It is reasonable to assume that glycoconjugate vaccine design and we previously raised the possibility of glycopeptide presentation to scheduling might be optimized if we had a fuller understanding T cells. We considered whether T cells could recognize carbohy- of the mechanisms underlying this “decision-making” process. We drates linked to another molecule (e.g., a peptide) whose binding show that, to induce more sustainable memory responses with to MHCII allows carbohydrate presentation on the APC surface. MenC glycoconjugates, booster immunization is more effective Our earlier work uncovered a key feature of the cellular and when the polysaccharide is linked to the same carrier protein molecular mechanisms underlying adaptive immune responses rather than to a heterologous protein. Our findings support a mediated by some glycoconjugate vaccines (5). We showed that + recent controlled trial conducted in the United Kingdom and glycoconjugate immunization induces CD4 T cells, designated Malta, wherein boosting children with Hib-MenC-TT vaccine af- Tcarbs, that recognize only the carbohydrate portion of the ter priming them with a single MenC-TT dose in infancy resulted glycoconjugate vaccine. Upon endosomal uptake by APCs, a in a more robust bactericidal antibody response than boosting with GBSIII glycoconjugate undergoes depolymerization, yielding a MenC-CRM ; the response elicited by the MenC boost with the glycan of reduced size (∼10 kDa) that is chemically bound to a 197 same carrier protein used for priming resulted in antibodies per- peptide (glycanp-peptide) fragment. Glycanp-peptide is displayed + sisting at 24 mo of age (28). on the surface of APCs in the context of MHCII to the CD4 Overall, our study suggests that different mechanisms are in- T cells. We successfully generated T cell clones and validated the volved in immune responses to immunization with different existence of T cells that recognize only the processed carbohy- glycoconjugates and that the structure of the polysaccharide is drate portion of the glycoconjugate—i.e., Tcarbs (8). These critical to the mechanisms used by APCs to present antigen to findings suggested that Tcarbs contribute to the protection in- + CD4 T cells. An understanding of these differences is an im- duced by the GBSIII glycoconjugate vaccine. Similar mecha- nisms involving Tcarb responses were recently reported for type portant factor in the specific design of each glycoconjugate and 3 S. pneumoniae glycoconjugates (6). optimization of the vaccination schedule. In the present study, we sought to determine whether the Materials and Methods mechanisms involved in processing and presenting GBSIII and Mice. Six-week-old female BALB/c mice were purchased from Taconic Bio- Pn3P glycoconjugates are also present for glycoconjugates of sciences. All mouse experiments were approved by the Harvard Medical Area other bacterial polysaccharides. We show that a Tcarb-dependent Standing Committee on Animals (Animal Protocol Is00000636). response is induced by glycoconjugates made with the Vi poly-

saccharide of Salmonella Typhi, the type b polysaccharide of H. Antigens. Purified MenC, the MenC-CRM197 conjugate, Vi polysaccharide, the influenzae, and the type Ib polysaccharide of group B Streptococ- Vi-CRM197 conjugate, Hib polysaccharide, the Hib-CRM197 conjugate, and cus. In contrast, we found that a glycoconjugate made with the CRM197 protein were obtained from GSK Vaccines and GSK Vaccines Institute

Sun et al. PNAS | January 2, 2019 | vol. 116 | no. 1 | 197 Downloaded by guest on September 24, 2021 for Global Health. The Hib-OMPC (outer membrane protein complex) con- MenC-TT conjugate) for 18 h at 37 °C. After incubation, cells were collected, jugate was purchased from Merck. GBSIb was isolated and purified from washed five times with PBS, and labeled at 4 °C first with either GBSIII- type Ib group B Streptococcus. Two conjugation methods were applied: specific monoclonal antibody or MenC-specific monoclonal antibody and cross-linked GBSIb and end-linked MenC conjugates were made through then with a fluorophore-labeled secondary antibody. Surface staining was reductive amination, as previously described (8); cross-linked MenC and Vi assessed by flow cytometry (MACSQuant Analyzer). conjugates were made using EDC [1-ethyl-3-(3-dimethylaminopropyl) carbodiimide], with an ADH linker to couple the carboxylic acid groups Cell Fractionation and in Vitro Processing Assays. Raji B cells (108) were cul- from the protein and the carbohydrate. tured in the presence of 1 mg of [3H]-MenC for 18 h at 37 °C. Cells were then washed five times with PBS to remove unreacted [3H]-MenC. Cell lysis was Immunizations and Antibody Responses. Groups of BALB/c mice were primed performed by passage of cells through a 27-gauge needle in 250 mM sucrose – on day 0 and boosted on day 14 by i.p. injection of the antigen of interest (4 with 10 mM Tris·HCl, pH 7.5. Differential centrifugation was then used to μ 6 g as polysaccharide content) in PBS mixed with 0.5 mg of alum hydroxide fractionate the lysed cells into endolysosome and cell-membrane fractions, gel adjuvant. At least four mice were immunized in each experiment. Mice as previously described (5, 12, 30). Endolysosomal fractions were solubilized were bled from the tail vein 1 wk after boosting immunization. Levels of by boiling for 20 min in 1% SDS and analyzed by size exclusion chroma- carbohydrate-specific antibodies in the serum were determined by solid- tography on either Agilent ProSEC 300S [MW (molecular weight) range, phase ELISA, as previously described (29). 1,500–800,000] or Aquagel-OH 20 (MW range, 100–20,000) with an UltiMate 3000 system. Adoptive Transfer. Groups of donor BALB/c mice were primed and boosted

with 4 μg of MenC-CRM197 as saccharide content given i.p. at 3-wk intervals. + Statistical Analysis. Statistical significance was determined with the ordinary Mice were killed 5 d after boosting immunization. CD4 T cells were isolated one-way ANOVA, using GraphPad Prism 7.0c. Data with P values of ≤0.05 from spleens and lymph nodes of mice immunized with either MenC-CRM197 + were considered statistically significant (*P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001; or OVA and negatively selected with a mouse CD4 T cell isolation kit (130- ****P ≤ 0.0001). 104-454, Miltenyi Biotech). B cells from mice immunized with MenC-CRM197 were isolated with a mouse B cell isolation kit (130-104-443, Miltenyi Bio- + tech). CD4 T cells (107) from mice immunized with MenC-CRM or OVA ACKNOWLEDGMENTS. We thank Dr. Lok-To Sham and Dr. Thomas G. 197 Bernhardt for assistance with the HPLC system and GSK Vaccines and GSK and B cells (107) from MenC-CRM –immunized donors were adoptively 197 Vaccines Institute for Global Health, both part of a group of companies, for transferred to recipient mice. The recipient mice were immunized 1 d after kindly providing several important reagents. This work was supported by adoptive transfer with MenC-CRM197 or MenC-OVA. Grants 5R01AI089915 and 5U19AI109764 from the National Institute of Allergy and Infectious Diseases and by funding from the European Union’s Antigen Presentation by BMDCs. BMDCs from wild-type mice were incubated Horizon 2020 Research and Innovation Programme under Marie Skłodowska with antigen (GBSIII or the GBSIII-TT conjugate, MenC or the MenC-CRM197 or Curie Grant Agreement 661138.

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