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A Mechanism for Glycoconjugate Vaccine Activation of the Adaptive Immune System and Its Implications for Vaccine Design

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A Mechanism for Glycoconjugate Vaccine Activation of the Adaptive Immune System and Its Implications for Vaccine Design ARTICLES A mechanism for glycoconjugate vaccine activation of the adaptive immune system and its implications for vaccine design Fikri Y Avci1,2, Xiangming Li3, Moriya Tsuji3 & Dennis L Kasper1,2 Glycoconjugate vaccines have provided enormous health benefits globally, but they have been less successful in some populations at high risk for developing disease. To identify new approaches to enhancing glycoconjugate effectiveness, we investigated molecular and cellular mechanisms governing the immune response to a prototypical glycoconjugate vaccine. We found that in antigen-presenting cells a carbohydrate epitope is generated upon endolysosomal processing of group B streptococcal type III polysaccharide coupled to a carrier protein. In conjunction with a carrier protein–derived peptide, this carbohydrate epitope binds major histocompatibility class II (MHCII) and stimulates carbohydrate-specific CD4+ T cell clones to produce interleukins 2 and 4—cytokines essential for providing T cell help to antibody-producing B cells. An archetypical glycoconjugate vaccine that we constructed to maximize the presentation of carbohydrate-specific T cell epitopes is 50–100 times more potent and substantially more protective in a neonatal mouse model of group B Streptococcus infection than a vaccine constructed by methods currently used by the vaccine industry. Our discovery of how glycoconjugates are processed resulting in presentation of carbohydrate epitopes that stimulate CD4+ T cells has key implications for glycoconjugate vaccine design that could result in greatly enhanced vaccine efficacy. Pathogenic extracellular bacteria often express high-molecular- (Supplementary Fig. 1)—overlooks the strong covalent linkage of weight capsular polysaccharides (CPSs) that coat the microbial carbohydrates to proteins in glycoconjugate vaccines that is unlikely to be surface. CPSs have been considered T cell–independent antigens1–5, broken within the endosome3,5. This current hypothesis of peptide-only primarily because, when used as vaccines, they induce specific IgM presentation has been promulgated mainly because proteins have gener- responses in wild-type and T cell–deficient mice without inducing ally been viewed as the only antigens presented by MHCII molecules to much IgM-to-IgG switching3, fail to induce a booster response (that T cells. We considered whether T cells can recognize carbohydrates if is, a secondary antibody response after recall immunization) and fail they are covalently linked to another molecule (for example, a peptide) © 2011 Nature America, Inc. All rights reserved. All rights Inc. America, Nature © 2011 to induce sustained T cell memory4. that allows MHCII to present the hydrophilic carbohydrate on the anti- The advantages of glycoconjugate vaccines over pure glycans in induc- gen-presenting cell (APC) surface. We hypothesized that T cell failure to ing immune responses are well documented5. Covalent coupling of a respond to carbohydrates (for example, bacterial CPSs) is due to failure T cell–independent CPS to a carrier protein yields a glycoconjugate that, of these molecules to bind MHCII, not to T cell inability to recognize when used to immunize mammals, elicits T cell help for B cells that pro- presented glycans. We tested this hypothesis to gain insight into the duce IgG antibodies to the polysaccharide component5–11. Thus glycocon- mechanisms involved in carbohydrate processing and presentation by jugates induce polysaccharide-specific IgM-to-IgG switching, memory MHCII and in subsequent T cell recognition of glycoconjugate vaccines. B cell development and long-lived T cell memory. Glycoconjugate An understanding of the immune mechanisms involved in glycoconju- vaccines have played an enormous part in preventing infectious dis- gate immunization is crucial in the rational design of new-generation eases caused by virulent pathogens such as Haemophilus influenzae, vaccines against emerging infections. Streptococcus pneumoniae and Neisseria meningitidis9,12. However, the immunogenicity of these glycoconjugates has been variable, and this RESULTS variability has been attributed to the structure of the particular polysac- MHCII-presented carbohydrate epitopes elicit T cell help charide in a given construct13,14. In addition, in some populations at We investigated the mechanisms underlying APC processing and high risk for developing disease, such as the elderly or immunocom- presentation of glycoconjugates consisting of the type III polysaccha- promised individuals, immunogenicity has been relatively poor5,9. The ride of group B Streptococcus (GBSIII), a typical T cell–independent current hypothesis—that, in the context of MHCII, a peptide gener- polysaccharide, coupled to a carrier protein or peptide such as oval- 15 ated from glycoconjugates is presented to and recognized by T cells bumin (OVA), tetanus toxoid (TT) or ovalbumin peptide (OVAp). 1Channing Laboratory, Brigham and Women’s Hospital, Harvard Medical School, Boston, Massachusetts, USA. 2Department of Microbiology and Immunobiology, Harvard Medical School, Boston, Massachusetts, USA. 3HIV and Malaria Vaccine Program, Aaron Diamond AIDS Research Center, Affiliate of The Rockefeller University, New York, New York, USA. Correspondence should be addressed to D.L.K. ([email protected]). Received 16 August; accepted 22 September; published online 20 November 2011; doi:10.1038/nm.2535 1602 VOLUME 17 | NUMBER 12 | DECEMBER 2011 NaTURE MEDICINE a RTICLES Figure 1 GBSIII-specific IgG secretion can + a b be stimulated by CD4 T cells recognizing P < 0.0001 12 22 carbohydrate epitopes. (a,b) Concentration of IgG P < 0.0001 ) ) 20 –1 antibody to GBSIII in BALB/c mice (four to six mice –1 10 18 g ml per group) primed (day 0) and boosted (day 14) g ml µ µ 16 with different antigen combinations, as measured 8 14 by ELISA in serum obtained on day 21. Mice 12 6 primed with III-OVA and boosted with unconjugated 10 GBSIII and mice primed with unconjugated GBSIII 4 8 and boosted with III-TT had significantly lower 6 specific IgG levels than either mice primed and 4 2 *** *** *** *** Antibody concentration ( boosted with III-OVA or mice primed with III-OVA Antibody concentration ( 2 and boosted with III-TT (***P < 0.0001). None of 0 0 GBSIII Day 0: III-OVA III-OVA III-OVAIII-OVA III-OVA III-OVA III-OVA GBSIII III-TT the mice had detectable antibodies to either GBSIII Day 0: PBS OVA GBSIII + III-OVA Day 11, 13: anti-CD4 isotype or OVA before immunization (data not shown). OVA GBSIII Day 14: III-OVA III-TT III-TT III-TT GBSIII TT + III-TT III-TT Data represent means ± s.e.m. Day 14: III-OVA III-OVA III-OVA III-OVA III-OVA TT We first examined the adaptive immune response to glycoconjugates These results strongly support recruitment of T cell help for induc- by priming mice with OVA and boosting them 2 weeks later with tion of GBSIII carbohydrate-specific secondary immune responses GBSIII conjugated to OVA (III-OVA). We compared polysaccha- via carbohydrate recognition. ride-specific IgG levels in the sera of these mice with levels in the Another possible explanation for our finding that type III sera of mice both primed and boosted with the conjugate. Priming of polysaccharide–specific antibody response could be primed with naive mice with the carrier alone did not generate a robust second- III-OVA and boosted with III-TT is that activated B cells respond ary antibody response to the polysaccharide upon boosting with the to III-TT without T cell help. We tested this possibility by boosting glycoconjugate (Fig. 1a). However, mice primed and boosted with III-OVA–primed mice with III-TT after treatment with antibody the glycoconjugate had strong IgG responses after recall vaccina- to CD4 during the interval between priming and boosting. The tion (Fig. 1a). To determine whether the inability of OVA to induce excellent booster response observed in isotype control antibody– a priming response for glycoconjugate boosting is due to a failure treated mice was abolished in CD4-specific antibody–treated mice of T cell or B cell priming, we immunized mice with an unconju- (Fig. 1b). By flow cytometry, we found that there was complete gated mixture of GBSIII and OVA, thereby providing B cells that depletion of CD4+ T cells from CD4-specific antibody–treated mice had recent experience with GBSIII and T cells that had experience before secondary vaccination (Supplementary Fig. 2c). In addi- with presentation of the peptides derived from the OVA protein, tion, mice primed with III-OVA and boosted with GBSIII, TT or and then boosted these mice with the glycoconjugate (Fig. 1a). After GBSIII plus TT had no booster response. These results led us to III-OVA recall immune stimulation, mice primed with GBSIII plus further examine the mechanisms by which CD4+ T cell recogni- OVA—unlike III-OVA–primed mice—had essentially no secondary tion of GBSIII glycoconjugate vaccines could be mediated by the antibody response to the glycan (Fig. 1a). We measured OVA-specific carbohydrate portion. IgG titers and GBSIII-specific IgG and IgM titers after only a priming dose of either GBSIII plus OVA or III-OVA. GBSIII-specific IgG was Glycoconjugate carbohydrate is processed into smaller glycans detectable only after priming of mice with III-OVA (Supplementary To investigate the molecular and cellular mechanisms involved in © 2011 Nature America, Inc. All rights reserved. All rights Inc. America, Nature © 2011 Fig. 2a). Serum levels of IgM antibody to GBSIII were similar in both immunization with GBSIII-containing glycoconjugates, we first groups of immunized mice, whether the glycan was conjugated or not examined glycoconjugate processing and presentation by APCs (Supplementary Fig. 2b), an observation suggesting equivalent levels (for example, B cells or dendritic cells). Some CPSs are taken up by of carbohydrate-specific B cell priming. After priming, approximately APC endosomes and depolymerized into smaller carbohydrates by the same level of OVA-specific IgG was measured in serum from oxidative agents such as reactive oxygen species (ROS) and reactive both groups; this result suggested that OVA-specific T cell help was nitrogen species16,17.
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