Expert answer:I want someone to write paper about Glycoconjugate: A,C,W,Y; carrier proteins, how sugars are obtained, conjugation chemistry.All information have to be in Neisseria meningitidis serogroup A,C,W,Y. It have to be clear information in paper. I have all resources you can use it. writing the references and the numbers of references the side of sentences”. The references have to be original article. Did not use any references just my references. I need 8 to10 pages.
a_novel_mechanism_for_glycoconjugate_vaccine_activation_of_the_10.30.17.pdf
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Nat Med. Author manuscript; available in PMC 2012 October 28.
Published in final edited form as:
Nat Med. ; 17(12): 1602–1609. doi:10.1038/nm.2535.
A novel mechanism for glycoconjugate vaccine activation of the
adaptive immune system
Fikri Y. Avci1,2, Xiangming Li3, Moriya Tsuji3, and Dennis L. Kasper1,2,*
1Channing
Laboratory, Brigham and Women’s Hospital, Boston, Massachusetts 02115, USA
2Department
Author Manuscript
of Microbiology and Immunobiology, Harvard Medical School, Boston,
Massachusetts 02115, USA
3HIV
and Malaria Vaccine Program, Aaron Diamond AIDS Research Center, Affiliate of The
Rockefeller University, New York, NY 10016
Abstract
Author Manuscript
Although glycoconjugate vaccines have provided enormous health benefits globally, they have
been less successful in significant high-risk populations. Exploring novel approaches to the
enhancement of glycoconjugate effectiveness, we investigated molecular and cellular mechanisms
governing the immune response to a prototypical glycoconjugate vaccine. 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 to 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 constructed to maximize the presentation of carbohydrate epitopes
recognized by T cells is 50–100 times more potent and significantly more protective in an animal
model of infection than is a currently used vaccine construct.
Author Manuscript
Pathogenic extracellular bacteria often express large-molecular-weight capsular
polysaccharides (CPSs), which coat the microbial surface. CPSs have been considered T
cell–independent antigens1–5 primarily because, when used as vaccines, they induce specific
IgM responses in wild-type and T cell–deficient mice without inducing significant IgM-toIgG switching3; fail to induce a booster response (i.e., a secondary antibody response after
recall immunization); and fail to induce sustained T-cell memory4.
The advantages of glycoconjugate vaccines over pure glycans in inducing immune responses
are well documented5. Covalent coupling of a T cell–independent CPS to a carrier protein
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Correspondence and request for materials should be addressed to DLK (dennis_kasper@hms.harvard.edu).
AUTHOR CONTRIBUTIONS
FYA, MT, and DLK designed the research; FYA and XL performed the research; FYA, XL, MT, and DLK analyzed the data; and
FYA and DLK wrote the paper.
COMPETING FINANCIAL INTERESTS
The authors declare no competing financial interests.
Avci et al.
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yields a glycoconjugate that, when used to immunize mammals, elicits T-cell help for B
cells that produce IgG antibodies to the polysaccharide (PS) component5–11. Thus
glycoconjugates induce PS-specific IgM-to-IgG switching, memory B-cell development,
and long-lived T-cell memory. Glycoconjugate vaccines have played an enormous role in
preventing infectious diseases caused by virulent pathogens such as Haemophilus
influenzae, Streptococcus pneumoniae, and Neisseria meningitidis9,12. However, the
immunogenicity of these glycoconjugates has been variable, and this variability has been
attributed to the structure of the particular PS in a given construct13,14. In addition, in some
high-risk populations, immunogenicity has been relatively poor5,9. The current hypothesis—
i.e., that, in the context of major histocompatibility complex class II (MHCII), a peptide
generated from glycoconjugates can be presented to and recognized by T cells15—overlooks
the strong covalent linkage of carbohydrates to proteins in glycoconjugate vaccines that is
unlikely to be broken within the endosome3,5. The current hypothesis of peptide-only
presentation has been promulgated mainly because proteins have generally been viewed as
the only antigens presented by MHCII molecules to T cells. We considered whether T cells
can recognize “T cell–independent” carbohydrates covalently linked to another molecule
(e.g., a peptide) whose binding to MHCII allows presentation of the hydrophilic
carbohydrate on the antigen-presenting cell (APC) surface. We hypothesized that T-cell
failure to respond to carbohydrates (e.g., bacterial CPSs) is due to failure of these molecules
to bind to MHCII, not to T-cell inability to recognize presented glycans. We tested this
hypothesis to gain insight into the mechanisms involved in carbohydrate processing and
presentation by MHCII and in subsequent T-cell recognition of glycoconjugate vaccines. An
understanding of the immune mechanisms involved in glycoconjugate immunization is of
paramount importance in the rational design of new-generation vaccines against emerging
infections.
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We investigated the mechanisms underlying APC processing and presentation of
glycoconjugates consisting of the type III PS of group B Streptococcus (GBSIII)—a typical
T cell–independent PS—coupled to a carrier protein/peptide such as ovalbumin (OVA),
tetanus toxoid (TT), or ovalbumin peptide (OVAp).
RESULTS
MHCII-presented carbohydrate epitopes elicit T-cell help
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The adaptive immune response to glycoconjugates (Fig. S1) was first examined by priming
mice with OVA and boosting them 2 weeks later with GBSIII conjugated to OVA (IIIOVA). We compared PS-specific IgG levels in the sera of these mice with levels in the sera
of mice both primed and boosted with the conjugate (Fig. 1a). Priming of naïve animals with
the carrier alone did not support a robust secondary antibody response to the PS upon
boosting with the glycoconjugate. However, mice primed and boosted with the
glycoconjugate had strong IgG responses after recall vaccination. To determine whether the
inability of OVA to induce a priming response for glycoconjugate boosting is due to a
failure of T-cell or B-cell priming, we immunized mice with an unconjugated mixture of
GBSIII and OVA (GBSIII+OVA), thereby providing B cells that had recent experience with
GBSIII and T cells that had experience with presentation of the peptides derived from the
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OVA protein, and then boosted these mice with the glycoconjugate (Fig. 1a). After III-OVA
recall immune stimulation, mice primed with GBSIII+OVA—unlike III-OVA-primed mice
—had essentially no secondary antibody response to the glycan (Fig. 1a). We measured
OVA-specific IgG titers and GBSIII-specific IgG and IgM titers after only a priming dose of
either GBSIII+OVA or III-OVA. GBSIII-specific IgG levels were detectable only after
priming of mice with III-OVA (Fig. S2a). Whether the glycan was conjugated or not, serum
levels of IgM antibody to GBSIII were similar in both groups of immunized mice (Fig. S2b),
an observation suggesting equivalent levels of carbohydrate-specific B-cell priming. After
priming, approximately the same level of OVA-specific IgG was measured in serum from
both groups; this result suggested that OVA-specific T-cell help was recruited after priming
with either the GBSIII+OVA mixture or the III-OVA glycoconjugate (data not shown).
Additional control groups for this experiment involved mice primed with unconjugated
GBSIII or with no antigen (PBS+ alum) and boosted with III-OVA (Figs. 1a, S2a, and S2b).
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In experiments examining whether CD4+ T-cell recognition of a carbohydrate is a major
factor in induction of the humoral immune response to glycoconjugates, BALB/c mice were
primed with III-OVA and boosted with a conjugate comprising GBSIII and TT (III-TT), and
serum levels of GBSIII-specific IgG were measured (Fig. 1b). Control groups included mice
primed and boosted with III-TT, primed and boosted with III-OVA, primed with GBSIII
(unconjugated) and boosted with III-TT, primed with III-OVA and boosted with GBSIII
(unconjugated), primed with III-OVA and boosted with GBSIII+TT, and primed with IIIOVA and boosted with TT. Boosting of III-OVA-primed mice with III-TT induced GBSIIIspecific IgG levels similar to those after priming and boosting with III-OVA (Fig. 1b).
These results strongly support recruitment of T-cell help for induction of GBSIII
carbohydrate-specific secondary immune responses via carbohydrate recognition.
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Another possible explanation is that activated B cells respond to III-TT without T-cell help.
We tested this possibility by boosting III-OVA-primed mice with III-TT after treatment with
antibody to CD4 during the interval between priming and boosting. The excellent booster
response observed in isotype control antibody–treated mice was abolished in anti-CD4treated mice (Fig. 1b). Using flow cytometry, we demonstrated the complete depletion of
CD4+ T cells by anti-CD4 treatment of splenic mononuclear cells from anti-CD4-treated
mice before secondary vaccination (Fig. S2c). In addition, mice primed with III-OVA and
boosted with GBSIII, TT, or GBSIII+TT had no booster response. These results led to
further examination of the mechanisms by which CD4+ T-cell recognition of GBSIII
glycoconjugate vaccines could be mediated by the carbohydrate portion.
Glycoconjugate carbohydrate is processed into smaller glycans
Author Manuscript
To investigate the molecular and cellular mechanisms involved in immunization with
GBSIII-containing glycoconjugates, we first examined glycoconjugate processing and
presentation by APCs (e.g., B cells, dendritic cells). Some CPSs are taken up by APC
endosomes and depolymerized into smaller carbohydrates by oxidative agents such as ROS
and reactive nitrogen species16,17. We assessed whether pure GBSIII (>100 kDa) is
depolymerized within the APC endolysosome, as reported for Bacteroides fragilis PS A17.
Radiolabeled GBSIII (Fig. S3) was incubated with Raji B cells for 18 h, endolysosomes
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were isolated and lysed, and GBSIII within the endolysosome was determined by molecular
sieve chromatography to be significantly depolymerized, with a major peak at ~10 kDa (Fig.
2a). Western blot analysis showed that endolysosome preparations contained both the
endosomal marker Rab5 and the lysosomal marker LAMP-1 (Fig. S4). Comparison of the
band density of CD19 (cell surface protein)–labeled endolysosomal fractions with the band
density of CD19-labeled, serially diluted cell surface fractions showed that endolysosomal
fractions were essentially free (≤5%) of cell surface content (data not shown).
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Since GBSIII and proteins are processed by different mechanisms in the endolysosome, we
wondered whether the carbohydrate in the context of a glycoconjugate is also processed. We
incubated Raji B cells with III-OVA, selectively radiolabeling only the PS with 3H (Fig.
S3). After 18 h of uptake and processing, [3H]III-OVA was degraded to a molecular size
similar to that of pure unconjugated GBSIII after depolymerization [processed glycan
(glycanp) ~10 kDa; Fig. 2b]. To identify the oxidative agent(s) responsible for PS processing
in III-OVA, we incubated Raji B cells with [3H]III-OVA in the presence of ROS inhibitors,
detecting inhibition of PS processing by superoxide and hydroxyl radical inhibitors (Figs.
S5a, S5b, respectively) but not by a hydroperoxide inhibitor (Fig. S5c).
Processed carbohydrates are presented on the APC surface
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To determine whether MHCII-associated processed carbohydrates (glycansp) are presented
on the APC surface, we conducted co-immunoprecipitation (co-IP), flow cytometry, and
western blot experiments. First, Raji B cells were incubated with unconjugated pure GBSIII.
Although [3H]GBSIII was endocytosed and processed to a smaller molecular size (Fig. 2a),
co-IP of Raji B-cell surface-membrane fractions with a monoclonal antibody (mAb) to
MHCII revealed no GBSIII on the cell surface in the context of MHCII (Fig. 2a). However,
co-IP of APC surface membranes with mAb to MHCII after incubation of [3H]III-OVA with
Raji B cells (Fig. S6a) or mouse splenic mononuclear cells (Fig. 2c) demonstrated surfaceassociated, endosomally processed [3H]GBSIII (glycanp). Co-IP of Raji B cells after
incubation with either [3H]III-OVA or [3H]III-TT showed the carbohydrate epitope on the
cell surface in the context of HLA-DR but not on MHCII-deficient Raji-derived RJ2.2.5
cells (Fig. S6b). Western blot analyses of cell-surface and endolysosome fractions revealed
that the Raji cell surface was essentially free of endosomal and lysosomal content (Fig. S4).
As controls for anti-HLA-DR, cell surface membrane–solubilized extracts of Raji B cells
were immunoprecipitated with antibodies to LAMP-1 (lysosomal protein) and CD19 (cell
surface protein); no radiolabeled carbohydrate was detected in immunoprecipitates (Fig.
S6a). Co-IP with mAb to HLA-DQ or HLA-DP molecules expressed by Raji B cells18 did
not significantly enhance radioactivity (p > 0.05) over that seen with mAb to LAMP-1 (not
shown). By co-IP with [3H]III-OVA as antigen, surface-associated GBSIII was sought with
anti-IA/IE on splenocytes from C57Bl/6 wild-type mice and various knockout strains (Fig.
2c). Splenocytes from wild-type and MHCI-deficient (B2mtm1Jae) mice had surface GBSIII
(~10 kDa); cells from MHCII-deficient (H2-Ab1tm1Gru) mice did not.
For flow cytometry, bone marrow–derived dendritic cells (BMDCs) from wild-type and
MHCII-deficient mice were incubated with GBSIII or III-OVA for 18 h and then labeled at
4 C with a GBSIII-specific mAb (IgG2a) followed by fluorophore-conjugated anti-mouse
Nat Med. Author manuscript; available in PMC 2012 October 28.
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secondary antibody. Only membranes of III-OVA-incubated wild-type BMDCs were
labeled with GBSIII-specific mAb (Figs. 2d, 2e).
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Endosomally processed carbohydrates are presented by the MHCII pathway only when
covalently conjugated to carrier proteins. One possible explanation is that processed
carbohydrate epitopes (glycansp) are presented by MHCII only when linked to an MHCIIbinding peptide formed in the endolysosome by proteolytic digestion of the carrier protein
(as in a glycanp-peptide conjugate). Perhaps the peptide portion of a glycanp-peptide (an
MHCII-binding peptide covalently linked to a carbohydrate T-cell epitope) binds to MHCII,
which carries the covalently linked carbohydrate to the APC surface. To assess whether
glycanp-peptides created from the complex glycoconjugate vaccine are presented by MHCII,
we conducted a western blot experiment with a glycoconjugate containing a single peptide
epitope as the carrier (Figs. S7a, S7b). In this vaccine, ovalbumin peptide epitope OVA323–
339 (a T-cell epitope of OVA19) was conjugated to GBSIII to form III-OVAp. The peptide
was N-acetylated at its N terminus and extended with four amino acids at the C terminus to
permit controlled conjugation to the PS. (OVAp can react with only one aldehyde group on
the sugar chain through its free amino group at the C-terminus lysine residue.) In
preliminary experiments (Figs. S7b, S7c), this peptide bound readily to MHCII on Raji B
cells. Modifications of the peptide’s amino acid composition did not affect its activation of
the αβ T-cell receptor (αβTCR); the modified peptide and the OVA323–339 peptide gave
similar MHCII-restricted (e.g., T-cell activation blocked by mAb to I-Ad) CD4+ T-cell
proliferative responses in a mouse assay (not shown).
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III-OVAp, pure GBSIII, and pure OVAp were each incubated with Raji B cells; cell
surface–associated contents were examined on western blots. Membranes were incubated
with antibody to HLA-DR, antibody to GBSIII (capable of high-affinity reactions with PSs
as small as 6 repeating units), or antibody to OVAp. Immune complexes containing HLADR, GBSIII, and peptide appeared in a band at ~82 kDa in membrane extracts from cells
incubated with III-OVAp but not in those from cells incubated with unconjugated GBSIII
(Fig. S7a). HLA-DR αβ dimers (self peptide–loaded or empty) were identified with mAb to
HLA-DR at ~64 kDa20,21 (Fig. S7a). The ~18-kDa difference in size (determined by protein
markers) between unloaded HLA-DR and the glycanp-peptide–HLA-DR complex represents
the approximate molecular size of the predicted glycanp-peptide, as carbohydrates mobilize
more slowly than proteins in gels22. However, the mAb to GBSIII bound nonspecifically to
the over-expressed free MHCII at ~64 kDa (Fig. S7a). To validate the nonspecificity of this
interaction, we stimulated lysed naïve Raji cells with OVAp and stained the transferred gel
with mAb to GBSIII. A light band similar in intensity to that in lanes 5 and 6 (Fig. S7a) at
64 kDa was observed (Fig. S7b). Human MHCII molecules (e.g., HLA-DR10) have been
shown to present OVAp23. We tested whether Raji B cells (whose HLA-DRB1*100101
allele encodes for HLA-DR1018,24) can present OVAp in the context of MHCII. OVAp was
detected on a western blot by antibody to OVAp (Fig. S7b), and an OVAp-biotin conjugate
was detected when Raji cells (but not RJ2.2.5 cells) incubated with OVAp-biotin were
labeled with NeutrAvidin-fluorescein conjugate in a flow cytometry experiment (Fig. S7c).
The co-IP and flow cytometry experiments (Figs. 2c–e) demonstrate that GBSIII glycanp is
presented on the cell surface in the context of MHCII only when conjugated to a carrier
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protein/peptide. Although the exact structural features of this complex must be defined by
crystallography studies, western blot analysis (Fig. S7a) suggests the possibility that a
peptide portion of a glycanp-peptide binds to MHCII, presenting glycanp on the APC surface
in the context of MHCII.
MHCII carbohydrate presentation inhibits TCR OVAp recognition
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We constructed a glycoconjugate in which the T-cell response could be directed only toward
a single carrier peptide (III-OVAp). III-OVAp, which contains a single-peptide T-cell
epitope, proved useful in this situation. Lymphocytes were obtained from OVAp-specific
TCR transgenic mice (DO11.10) and wild-type mice (BALB/c) after immunization with IIIOVAp or OVAp (2 doses, 2 weeks apart). In T-cell proliferation experiments, irradiated
splenic mononuclear cells (iAPCs) from wild-type naïve BALB/c mice were co-incubated
with CD4+ T cells from either immune BALB/c mice (Figs. 3a, 3c) or immune DO11.10
mice (Figs. 3b, 3d). The iAPC/CD4+ T-cell mixtures were stimulated in vitro with IIIOVAp (50 μg/ml), GBSIII (37.5 μg/ml; equivalent GBSIII content to that in 50 μg/ml of IIIOVAp), or OVAp (12.5 μg/ml; equivalent OVAp content to that in 50 μg/ml of III-OVAp).
As expected, unconjugated GBSIII did not stimulate proliferation of CD4+ T cells from
immunized BALB/c or DO11.10 mice. However, CD4+ T cells from III-OVAp-immunized
BALB/c mice responded better to the glycoconjugate than to the peptide alone (Fig. 3a).
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With DO11.10 CD4+ T cells, OVAp induced a strong proliferative response in III-OVApimmunized mice [stimulation index (SI) = 284.6; Fig. 3b] and OVAp-immunized mice (SI =
277.6; Fig. 3d). As anticipated, CD4+ T cells from naïve DO11.10 mice proliferated
similarly strong …
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