Phosphorylcholine-carbohydrate-protein conjugates efficiently induce hapten-specific antibodies which recognize both Streptococcus pneumoniae and Neisseria meningitidis: A potential multitarget vaccine against respiratory infections

Article abstract of DOI:10.1021/jm040783p

Phosphorylcholine (ChoP) is commonly expressed at the surface of pathogens of the respiratory tract, including Streptococcus pneumoniae and Neisseria meningitidis. We designed a synthetic hapten comprising ChoP and part of its native carrier structure in S. pneumoniae, i.e. N-acetyl-D-galactosamine (GalNAc). Protein conjugates of this hapten induced GalNAc-ChoP-specific antibodies which recognized ChoP on both S. pneumoniae and N. meningitidis. GalNAc-ChoP could therefore lead to the rational design of a novel multipurpose vaccine against respiratory infections.

Full text of DOI:10.1021/jm040783p

3916
J . Med. Chem. 2004, 47, 3916-3919
Letters
tion of the respiratory epithelium, as well as in the
inflammatory process leading to the invasion of the host.
Through ChoP, H. influenzae and S. pneumoniae are
able to bind to the platelet activating factor receptor
(PAF-receptor), thus mimicking endogenous processes
of cellular signaling.6,7 Furthermore, several proteins
of S. pneumoniae, which are important for its virulence,
are attached to the cell wall via ChoP.8 ChoP also
appears to be responsible for the triggering of innate
immune reactions against H. influenzae, mediated by
the C-reactive protein (CRP), which is the natural ligand
for ChoP in blood and acts as a complement-binding
opsonin.9
P h osp h or ylch olin e-Ca r boh yd r a te-
P r otein Con ju ga tes Efficien tly In d u ce
Ha p ten -Sp ecific An tibod ies Wh ich
Recogn ize Both Str eptococcu s
pn eu m on ia e a n d Neisser ia m en in gitid is:
A P oten tia l Mu ltita r get Va ccin e a ga in st
Resp ir a tor y In fection s
Sylvie Bay,*,§ Vale´rie Huteau,§,|
Maria-Leticia Zarantonelli,‡ Rene´ Pires,‡
J oe¨l Ughetto-Monfrin,§ Muhamed-Kheir Taha,‡
Patrick England, and Pierre Lafaye†
Therefore, ChoP is an attractive target for the devel-
opment of immunotherapies directed against these
major bacterial infections of the respiratory tract.10 It
has been shown previously that ChoP-specific T15-
idiotype antibodies, despite their low affinity, are pro-
tective when used in passive immunizations.11,12 Like-
wise, the injection of ChoP coupled to a protein carrier
induced the production of high-affinity specific anti-
bodies in mice.13 Moreover, intranasal14,15 or parenter-
al16 immunization experiments with a similar ChoP-
protein conjugate protected mice against a lethal chal-
lenge with S. pneumoniae.
However, the epitope recognized by the induced
antibodies is not always limited to ChoP but also
includes the covalent link used for the coupling with
the carrier.13 In the first studies, ChoP-protein conju-
gates used for the immunizations contained a diazo-
phenyl linker between ChoP and the tyrosine and
histidine residues of the protein carrier, resulting in
immunodominant responses directed against aromatic
rings. To minimize this irrelevant immune response,
efforts have been made to replace the aromatic rings
by an aliphatic linker. The resulting immunogen pro-
vided total protection of Xid mice against a lethal
challenge with S. pneumoniae, whereas the diazophenyl-
containing conjugate did not.17,18
Unite´ de Chimie Organique URA CNRS 2128, Unite´ de
Ge´ne´tique et Biochimie du De´veloppement, Unite´ des
Neisseria, and Plate-forme de Biophysique des
Macromole´cules et de leurs Interactions, Institut Pasteur,
Paris, France
Received February 9, 2004
Abstr a ct: Phosphorylcholine (ChoP) is commonly expressed
at the surface of pathogens of the respiratory tract, including
Streptococcus pneumoniae and Neisseria meningitidis. We
designed a synthetic hapten comprising ChoP and part of its
native carrier structure in S. pneumoniae, i.e. N-acetyl-D-
galactosamine (GalNAc). Protein conjugates of this hapten
induced GalNAc-ChoP-specific antibodies which recognized
ChoP on both S. pneumoniae and N. meningitidis. GalNAc-
ChoP could therefore lead to the rational design of a novel
multipurpose vaccine against respiratory infections.
Neisseria meningitidis and Streptococcus pneumoniae
are major causative bacterial agents of invasive respira-
tory infections and meningitis. These bacterial species
are genetically and antigenically variable, and therefore
currently available vaccines are far from satisfactory.1,2
Moreover, an increasing number of S. pneumoniae
strains are resistant to various antibiotics, and the
emergence of N. meningitidis strains with diminished
susceptibility to â-lactams becomes a matter of concern.3
Antibody-based therapies could therefore gain renewed
interest for the prophylaxis and treatment of these
respiratory infections.4
Phosphorylcholine (ChoP) is frequently incorporated
in the surface antigens of several prokaryotes (Haemo-
philus, Streptococcus, Neisseria, Mycoplasma, Salmo-
nella, and Pseudomonas) and eukaryotes (pathogenic
helminths and nematodes) (for review, see ref 5).
In respiratory infections, ChoP is thought to be
directly involved in the steps of adhesion and coloniza-
These results suggest that the nature of the linker
between ChoP and the protein carrier can be critical
for the induction of high affinity protective antibodies
intended for antibacterial therapies.
According to the pathogen, ChoP is coupled to a
variety of bacterial cell structures. In the case of N.
meningitidis, ChoP is linked to the glycoproteins of the
pili, but its molecular carrier (saccharide or amino acid)
has not yet been clearly identified.19,20 In H. influenzae,
ChoP is grafted to the surface lipopolysaccharides.21 In
S. pneumoniae, ChoP is part of the C-polysaccharide
(teichoic acid) and F-antigen (lipoteichoic acid).22,23
Despite this diversity in the native macromolecular
backbone, it appears that, at least for H. influenzae and
S. pneumoniae, ChoP is presented in position 6 of an
hexose or an hexosamine, respectively. Whether this
carbohydrate part of the epitope is involved in the
pathogenic process remains an open question.
* Corresponding author: Unite´ de Chimie Organique, Institut
Pasteur, 28 rue du Dr Roux, 75724 Paris Cedex 15; tel: (33) 01-45-
68-83-97; fax: (33) 01-45-68-84-04; e-mail: sbay@pasteur.fr.
§ Unite´ de Chimie Organique URA CNRS 2128.
† Unite´ de Ge´ne´tique et Biochimie du De´veloppement.
‡ Unite´ des Neisseria.
Plate-forme de Biophysique des Macromole´cules et de leurs
Interactions.
| Present address: PF7, Synthe`se d’oligonucle´otides longs a` haut
de´bit, Institut Pasteur, Paris, France.
10.1021/jm040783p CCC: $27.50 © 2004 American Chemical Society
Published on Web 07/07/2004

Letters
J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 16 3917
Sch em e 1. Synthesis of GalNAc-ChoPa
deprotections/protections gave the 6-OH key intermedi-
ate 8 (nine steps, overall yield 14%).
The ChoP was then coupled at the position 6 of the
GalNAc residue with the phosphoramidite method. By
using 31P NMR, we could follow the completion of the
different reactions, and the three following steps were
sequentially performed one-pot. 8 was reacted with
chloro 2-cyanoethyl (N,N-diisopropyl)phosphoramidite
in the presence of DIEA. After disappearance of the 31P
signal assigned to the starting material (δ 182.41 ppm)
and concomitant appearance of the new phosphor-
amidite signals (δ 150.63 and 150.21 ppm), phos-
phitylation was performed by adding choline tosylate
to the reaction mixture together with tetrazole. Reaction
was completed after 24 h as shown by 31P NMR. The
resulting new 31P signals account for the two phosphite
triester diastereoisomers (δ 141.62 and 141.41 ppm).
After oxidation, the formation of the phosphotriester
diastereoisomers 9 was indicated by new 31P signals in
the spectrum (δ -1.96 and -2.03 ppm) (three steps,
overall yield 31%).
a
Reagents and conditions: (a) 30% HBr in CH3COOH, RT, 1 h
30 min (99%); (b) Zn, NMI, dry AcOEt, reflux, 2 h; (c) (NH4)2-
Ce(NO3)6, NaN3, CH3CN, -25 °C, 3 h (two steps, 44%); (d) LiBr,
dry CH3CN, RT, 3 h (99%); (e) HO-(CH2)6-NHZ, AgOTf, collidine,
dry CH2Cl2, -40 °C, 15 h; (f) NaBH4, H3BO3, NiCl2, EtOH, RT, 1
h 30 min; (g) Ac2O, EtOH, RT, 1 h (three steps, 54%); (h) MeONa,
MeOH, RT, 10 min; (i) DMTrCl, Pyr, RT, 2 h; (j) Ac2O, Pyr, RT, 3
h; (k) 2% ABS in CH2Cl2/MeOH:7/3, 0 °C, 2 min (four steps, 60%);
(l) chloro 2-cyanoethyl (N,N-diisopropyl)phosphoramidite, DIEA,
dry CH3CN, RT, 20 min; (m) Choline+ Tos-, tetrazole, CH3CN,
RT, 24 h; (n) I2/Pyr/THF/H2O, CH3CN, RT, 15 min (three steps,
31%); (o) MeONa, MeOH, RT, 15 min; (p) H2, Pd/C, EtOH, RT, 1
h (two steps, 55%).
The carbohydrate moiety, the cyanoethyl group, and
the linker were deprotected to afford the hapten 11 (two
steps, overall yield 55%).
Syn th esis of th e Ga lNAc-Ch oP -P r otein Con ju -
ga tes. The conjugation of 11 to the tetanus toxoid
protein (TT) or the Alpaga Serum Albumin (ASA)
through activation with the EDC/SulfoNHS method
yielded the expected GalNAc-ChoP-protein conjugates.
The ChoP:protein ratio of the conjugates TT-GalNAc-
ChoP and ASA-GalNAc-ChoP were estimated at,
respectively, 17:1 and 29:1 by a microphosphate assay.26
In d u ction of Ga lNAc-Ch oP -Sp ecific An tibod ies
Wh ich Recogn ize S. pn eu m on ia e a n d N. m en in -
gitid is. Biozzi mice were immunized either with TT-
GalNAc-ChoP or with ASA-GalNAc-ChoP, and the
immune sera were tested for reactivity with the parental
immunogen after boost injections. Each animal devel-
oped either a strong anti-TT-GalNAc-ChoP response
or a strong ASA-GalNAc-ChoP response (data not
shown). For each immunogen, further experiments were
performed with the serum showing the highest reactiv-
ity. The specificity of the serum for ChoP was assessed
in inhibition assays using TT, ASA, GalNAc-ChoP,
GalNAc alone, or p-nitro-phenyl-ChoP. The 50% inhibi-
tion concentrations (IC50) are shown in Table 1. The
results suggest that immunization with both antigens
generates antibodies specific for ChoP. Moreover, these
antibodies have a stronger avidity toward GalNAc-
ChoP (IC50 ) 1 × 10-6 M for TT-GalNAc-ChoP and
IC50 ) 0.15 × 10-6 M for ASA-GalNAc-ChoP) than
toward ChoP alone (IC50 ) 20 × 10-6 M for TT-
GalNAc-ChoP and IC50 ) 7 × 10-6 M for ASA-
GalNAc-ChoP).
In this study, we aimed at raising high-affinity
antibodies against ChoP in its bacterial context in order
to target several pathogens of the respiratory tract. By
mimicking the S. pneumoniae model, we synthesized
two carbohydrate-ChoP (GalNAc-ChoP) protein con-
jugates, and we showed that these immunogens induce
hapten-specific antibodies which recognize two major
bacterial pathogens of the respiratory tract: a Gram-
positive bacterium, S. pneumoniae, and a Gram-nega-
tive bacterium, N. meningitidis.
Syn th esis of th e Ba cter ia l Ha p ten Ga lNAc-
Ch oP (Sch em e 1). In S. pneumoniae, one or two ChoP
molecules are linked at the position 6 of the N-acetyl-
D-galactosamine residues within the repeating unit of
the C-polysaccharide [-6)-â-D-Glcp-(1-3)-R-AATp-(1-4)-
R-D-GalpNAc-(1-3)-â-D-GalpNAc-(1-1)-D-ribitol-5-P-
(O-] (AAT ) 2-acetamido-4-amino-2,4,6-trideoxy-D-
galactose).22,23 Fragments of this repeating unit have
been synthesized for structural24 and immunological25
studies. However, none of them bear a ChoP residue.
To mimic the bacterial environment of the ChoP, we
designed a synthetic antigen comprising both ChoP and
part of its native carrier structure, i.e. the 6-substituted
N-acetyl-D-galactosamine residue.
The synthesis is summarized in Scheme 1. Starting
from 1,2,3,4,6-penta-O-acetyl-â-D-galactopyranoside 1,
a succession of bromination, reductive dehalogenation,
azidonitration, bromination, Koenigs-Knorr reaction
with 6-(benzyloxycarbonyl)hexanol linker, reduction-
acetylation of the azido group, and then selective
To further characterize the quality of the murine anti-
ChoP antibody response, immune sera were tested
Ta ble 1. ELISA Binding Profile of Immune Sera to Coated S. pneumoniaea
competitor, IC50 (M)
immunogen
TT
ASA
GalNAc
GalNAc-ChoP
p-nitrophenyl-ChoP
TT-GalNAc-ChoP
ASA-GalNAc-ChoP
>10-4
ND
NDb
>10-4
>10-4
1 × 10-6
20 × 10-6
>10-4
0.15 × 10-6
7 × 10-6
a
The specificity of the sera for ChoP was assessed by measuring the 50% inhibition concentration (IC50) using different antigens carrying
b
or not ChoP. ND: not determined.

3918 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 16
Letters
This study demonstrates the ability of ChoP-carbo-
hydrate-protein conjugates to raise a strong hapten-
specific antibody response against two phylogenetically
unrelated bacteria, S. pneumoniae and N. meningitidis,
that express ChoP at their surface. The efficacy of the
immune response is currently investigated in animal
models.
Taken together, our results highlight the potential of
the ChoP-carbohydrate epitope as a model antigen for
the rational design of a wide spectrum vaccine which
could confer cross-protection against several pathogens
of the respiratory tract. An additional advantage of such
ChoP-carbohydrate conjugates vaccine is their safety.
Indeed, since they closely mimic the native bacterial
antigen, they should induce more specific immune
response and prevent potential cross-reactivities with
phosphatidylcholine-bearing macromolecules of the host.
F igu r e 1. ELISA measurement of heat-inactivated S. pneu-
moniae cells reactivity for murine anti-TT-GalNAc-ChoP ([)
and ASA-GalNAc-ChoP (9) immune sera. (] and 0): cor-
responding sera of nonimmunized animals.
Ack n ow led gm en t. We are grateful to J .-M. Alonso
for helpful discussions, constant support, and for critical
reading of the manuscript. We thank M. Leduc for
valuable advice and preparation of heat-inactivated S.
pneumoniae. This work was supported by a grant from
the Institut Pasteur (Programme Transversal de Re-
cherche no. 67).
Su p p or tin g In for m a tion Ava ila ble: Experimental pro-
cedures for the preparation of 6-11 and of the protein
conjugates TT-GalNAc-ChoP and ASA-GalNAc-ChoP. Char-
acterization data for compounds 6â, 7, 8, and 11. Methods for
immunizations and ELISA. This material is available free of
charge via the Internet at http://pubs.acs.org.
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