A Semi-Synthetic Glycoconjugate Vaccine Candidate for Carbapenem-Resistant Klebsiella pneumoniae

Article abstract of DOI:10.1002/anie.201700964

Hospital-acquired infections are an increasingly serious health concern. Infections caused by carpabenem-resistant Klebsiella pneumoniae (CR-Kp) are especially problematic, with a 50 % average survival rate. CR-Kp are isolated from patients with ever greater frequency, 7 % within the EU but 62 % in Greece. At a time when antibiotics are becoming less effective, no vaccines to protect from this severe bacterial infection exist. Herein, we describe the convergent [3+3] synthesis of the hexasaccharide repeating unit from its capsular polysaccharide and related sequences. Immunization with the synthetic hexasaccharide 1 glycoconjugate resulted in high titers of cross-reactive antibodies against CR-Kp CPS in mice and rabbits. Whole-cell ELISA was used to establish the surface staining of CR-Kp strains. The antibodies raised were found to promote phagocytosis. Thus, this semi-synthetic glycoconjugate is a lead for the development of a vaccine against a rapidly progressing, deadly bacterium.

Full text of DOI:10.1002/anie.201700964

A Journal of the Gesellschaft Deutscher Chemiker
Angewandte
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Accepted Article
Title: A semi-synthetic glycoconjugate vaccine candidate for
carbapenem-resistant Klebsiella pneumoniae
Authors: Peter H. Seeberger, Claney L. Pereira, Naeem Khan, Guozhi
Xiao, Elizabeth Diago-Navarro, Katrin Reppe, Bastian Opitz,
Bettina C. Fries, and Martin Witzenrath
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To be cited as: Angew. Chem. Int. Ed. 10.1002/anie.201700964
Angew. Chem. 10.1002/ange.201700964
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10.1002/anie.201700964
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A semi-synthetic glycoconjugate vaccine candidate for
carbapenem-resistant Klebsiella pneumoniae
Peter H. Seeberger,*[1] Claney L. Pereira,*[1] Naeem Khan,[1]+ Guozhi Xiao,[1]+ Elizabeth Diago-
Navarro,[2] Katrin Reppe,[3] Bastian Opitz,[3] Bettina C. Fries[2] and Martin Witzenrath[3]
CPS and lipopolysaccharides (LPS). Active immunization with
LPS-containing vaccines holds severe risks associated with
endotoxic components such as Lipid A. [8] CPS, on the other hand,
are highly immunogenic and nontoxic. [9] A 24-valent Klebsiella
vaccine manufactured by the Swiss Serum and Vaccine Institute,
composed of CPS antigens from Kp and K. oxytoca that are
expressed by about 70% of Klebsiella strains associated with
bacteremia, proved safe and immunogenic in Phase I clinical
trials.[10] Challenges associated with glycan isolation rendered the
vaccine too costly and development ended following Phase I
clinical trials.[11]
Abstract:
Hospital acquired infections are an increasingly serious health
concern. Infections caused by carpabenem-resistant Klebsiella
pneumoniae (CR-Kp) are especially problematic, with a 50%
average survival rate. CR-Kp are isolated from patients with ever
greater frequency: 7% within the EU but 62% in Greece. At a time
when antibiotics are becoming less effective, no vaccines to
protect from this severe bacterial infection exist. Here, we
describe the convergent [3+3] synthesis of the hexasaccharide
repeating unit from its capsular polysaccharide and related
sequences. Immunization with the synthetic hexasaccharide 1
glycoconjugate resulted in high titers of cross-reactive antibodies
against CR-Kp CPS in mice and rabbits. Whole cell ELISA was
used to establish the surface staining of CR-Kp strains. The
antibodies raised were found to promote phagocytosis. Thus, this
semi-synthetic glycoconjugate is a lead for the development of a
vaccine against a rapidly progressing, deadly bacterium.
The production of well-defined glycans by chemical synthesis is
attractive as it is reliable and scalable method. Long lasting, T-cell
dependent protection from bacterial infections can be provided by
oligosaccharide antigens that are linked to a carrier protein[12] as
demonstrated by a semi-synthetic glycoconjugate vaccine against
Haemophilus influenzae type b marketed in Cuba.[13] Semi-
synthetic oligosaccharide vaccine candidates against Shigella[14]
,
meningococci[15] and streptococci[16]are currently under preclinical
evaluation.
Klebsiella pneumoniae (Kp) is the leading cause of nosocomial
respiratory and urinary tract infections as well as bacteremia,
primarily among newborns and immunocompromised patients.[1]
Carbapenem-resistant Kp (CR-Kp) are now commonly
encountered in hospitals worldwide. Outbreaks occur with
increasing frequency,[2] and these strains are highly contagious[3]
and cause high morbidity and mortality.[4] Isolates belonging to the
sequence type 258 (ST258) that expresses Kp carbapenemase
are largely responsible for the global spread of CR-Kp.[5]
Efficacious vaccination of risk groups is direly needed as
treatment options for CR-Kp are diminishing.
Successful
vaccines
against
streptococci[6]
and
meningococci[7] are based on conjugates of capsular
polysaccharides (CPS) with carrier proteins. Currently, no
vaccines for prophylactic or therapeutic use against Kp are
available. These Gram-negative pathogens are surrounded by
[1]
Dr. N. Khan,[+] Dr. G. Xiao,[+] Dr. C. L. Pereira, Prof. P. H. Seeberger
Department of Biomolecular Systems
Max Planck Institute of Colloids and Interfaces
Am Mühlenberg 1, 14424 Potsdam (Germany)
and
Institute of Chemistry and Biochemistry, Freie Universität Berlin
Arnimallee 22, 14195 Berlin (Germany)
E-mail: claney.pereira@vaxxilon.com
peter.seeberger@mpikg.mpg.de
Homepage: http://www.mpikg.mpg.de/en/bs
Dr. E. D. Navarro, Prof. B. C. Fries
[2]
Department of Medicine, Infectious Diseases Division
Albert Einstein College of Medicine and Montefiore Medical Center
1300 Morris Park Avenue, Bronx, NY 10461 (USA)
Prof. B. Opitz, Dr. K. Reppe and Prof. M. Witzenrath
Charité - Universitätsmedizin Berlin, Department of Infectious Diseases and
Pulmonary Medicine, Charitéplatz 1, 10117 Berlin, Germany
These authors contributed equally.
[3]
[+]
Supporting information for this article is given via a link at the end of the
document.
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10.1002/anie.201700964
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Figure 1. A) Hexasaccharide RU from two CR-Kp clones isolated during a 2011
hospital outbreak; B) Synthetic hexasaccharide 1 and substructures 2-6 used
for immunological studies.
rhamnosyl building block 7 afforded disaccharide 16 as the α-
anomer. A two step removal of the allyl group via the enol ether
provided disaccharide 17.[26] Glycosylation of 17 with galactosyl
trichloroacetimidate 9 proceeded smoothly at -30oC to give
trisaccharide thioglycoside 18 as a single β-anomer, which was
Clinical CR-Kp isolates responsible for a serious 2011
outbreak killing six of the 18 infected patients were found to have
identical CPS comprising a hexasaccharide repeating unit (RU)
(Figure 1 a).[17] This RU resembles CPS of other isolated
serotypes even though it is not identical. A vaccine based on
hexasaccharide antigen 1 may provide a much needed defense
against these devastating pathogens (Figure 1 b).
converted to glycosyl N-phenyltrifluoroacetimidate 19[27]
.
Herein, we report the synthesis of hexasaccharide 1 as well
as related substructures 2-6 (Figure 1 b) and their immunological
evaluation in vivo. Glycan microarray studies revealed that only
hexasaccharide 1 is recognized by monoclonal antibody (MAb)
1C9 that cross-reacts with CR-Kp CPS[20]. The glycoconjugate
hexasaccharide CRM197-1 vaccine candidate is immunogenic in
mice, and is promising for further preclinical development to
protect against CR-Kp outbreaks.
Scheme 2. Synthesis of trisaccharide building blocks 18 and 19.
An iterative glycosylation and deprotection strategy was
employed to construct trisaccharide acceptor 22, using rhamnosyl
trichloroacetimidate building block 7 (Scheme 3). Coupling of Rha
7 with the protected aminopentanol linker afforded -linked
rhamnoside; subsequent removal of the C2-acetate by treatment
with sodium methoxide provided acceptor 20. Repeating this
glycosylation-deprotection sequence twice with Rha 7 produced
trisaccharide acceptor 22.
With donors 18 and 19 as well as acceptor 22 in hand, the key
[3+3] glycosylation was investigated. Identification of effective
reaction conditions proved challenging due to the poor reactivity
and sterics of the uronic acid donor 18 and the inert C-2 hydroxyl
of the trisaccharide acceptor 22.[28-29] Ultimately, thioglycoside 18
failed
to
react
with
acceptor
22.
However,
N-
phenyltrifluoroacetimidate 19, after some optimization, reacted
o
smoothly in a mixture of toluene and diethyl ether at -70 C to
Scheme 1. Retrosynthetic analysis of hexasaccharide 1.
provide the hexasaccharide 23 (Scheme 3). The desired
configuration of the -isomer of 23 was confirmed by the coupling
constant between galacturonic acid C-1 and H-1 (1JC-H = 172 Hz;
Supporting Information).[30]
To prepare the glycans, a convergent [3+3] approach based on
three monosaccharide building blocks, Rha 7[18], GalA 8, and Gal
9[19], was selected (Scheme 1). Differentially protected GalA 8
carrying benzyl ether (Bn), allyl ether (All) and levulinyl ester (Lev)
groups at C2, C3 and C4 was to be synthesized by cyclization of
thioacetal 10, which is derived from aldehyde 11 and auxiliary 12
via aldol addition. For glycan immobilization on array surfaces and
subsequent immunization studies an aminopentanol linker is
included at the reducing end. The synthesis of differentially
The initial plan for the global deprotection of 23, relying on the
initial cleavage of acetate and methyl esters followed by
hydrogenation of the benzyl groups, did not meet with success as
only the pentasaccharide -elimination product was obtained
(data not shown). In contrast, when hydrogenolysis was carried
out first, followed by saponification, the target hexasaccharide 1
was obtained in 76 % yield (Scheme 3). NMR spectra of synthetic
hexasaccharide 1 were in good agreement with those reported for
the CPS from the CR-Kp outbreak[17] (Supporting Information).
The substructures 2-6 (Figure 1 b) were also synthesized for
epitope mapping studies (Supporting Information).
[21-23]
protected GalA building block 8 via a de novo approach
commenced with treatment of auxiliary 12 with LDA at -78 oC,
followed by addition of aldehyde 11 (Supporting Information) to
furnish aldol adduct 13 (d.r. = 10.4:1; Scheme 2).[24] Levulination
afforded fully protected thioacetal 10; treatment with
trifluoroacetic acid and anisole led to cleavage of p-
methoxybenzyl ether and concomitant cyclization produced the
desired thioglycoside 14 as a mixture of anomers (α/β = 3.9/1).
Isolation of the -product followed by methanolysis of the chiral
auxiliary under mild acidic conditions provided GalA 8.[25]
The assembly of trisaccharide donor 18 commenced by selective
removal of the C4-Lev ester of 8 upon exposure to hydrazine,
providing alcohol 15 (Scheme 2). Glycosylation of 15 with
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represented as mean ± SD (triplicate) following subtraction of background
values.
Carbohydrate based, type 2 T-independent antigens are
[31]
poorly immunogenic
and small synthetic glycans alone are
ineffective in eliciting an immune response. Attachment of
synthetic glycan antigens to carrier proteins that induce a T cell
response enhances glycan immunogenicity and triggers specific
memory responses. We used CRM197, a nontoxic mutant of
diphtheria toxin, as a carrier because it is a component of several
marketed conjugate vaccines.[32] The p-nitrophenyladipate
ester[33] derivative of hexasaccharide 1 was covalently coupled to
CRM197 to yield the CRM197-1 conjugate with an average of 7.5
hexasaccharide 1 molecules per CRM197 (Figure S2).
The immunogenicity of the CRM197-1 conjugate formulated
either with Freund’s adjuvant (FA) or human approved aluminum
hydroxide was assessed. Mice were immunized subcutaneously
and received two booster shots, one on day 14 and one on day
35 (Figure 3 A). High antibody titers were detected using glycan
microarrays one week after the final immunization (Figures 3 B
and 3 C). The antibody response was adjuvant dependent as
conjugate formulated with FA induced more cross-reactive
antibodies than the alum formulation. Antibodies against FA
formulated conjugate recognize both trisaccharides 2 and 3 while
those elicited in response to the alum formulation cross-react only
with trisaccharide 3 (Figures 3 B and 3 C). Antibody binding to
hexasaccharide 1 and trisaccharide 3 was similar for both
formulations (Figure 3 C). Interestingly, the FA adjuvanted
conjugate also induces CPS cross-reactive antibodies while the
alum formulation does not (Figure 3 D). Synthetic glycan antigens
elicited a significantly stronger antibody response in rabbits when
compared to mice.[34] Rabbits were immunized with CRM197-1
conjugate formulated with alum, an adjuvant approved for use in
humans.[35] The serological analysis using glycan microarrays and
ELISA revealed that CRM197-1 conjugate elicits a robust immune
responses against native CR-Kp CPS (Figure S3). Collectively,
these results demonstrate that the CRM197-1 conjugate is
immunogenic in mice and rabbits and produces antibodies that
are cross-reactive with native CR-Kp CPS.
Scheme 3. [3+3] Glycosylation to complete hexasaccharide 1.
The synthetic glycans along with the native CR-Kp CPS
were printed on microarray slides and incubated with the partially
protective mouse IgM monoclonal antibody (MAb) 1C9 raised
against CR-Kp CPS [20]. Mab 1C9 specifically recognizes RU
hexasaccharide 1 as no binding was observed for the other
synthetic glycans (Figure 2 A). Immunogenic structures that
induce cross-reactive immune responses to native CPS can be
determined by CPS-based inhibition studies[15]. Accordingly, the
MAb 1C9 was first incubated with CR-Kp CPS and S. pneumoniae
type 3 (ST3) CPS was used as a negative control (Figure 2 C).
MAb binding was abolished after pre-incubation with CR-Kp CPS
but not with other synthetic glycans (Figures 2 B and 2 E),
suggesting that hexasaccharide 1 effectively mimics the natural
CPS epitope. Therefore,
1
was further immunologically
characterized.
Figure 3. Mice immunized with CRM197-1 produce serum antibodies that
recognize CPS. (A) Schematic immunization schedule. Mice (three individuals
per group) were immunized on day 0 with CRM197-1 conjugate (3 µg sugar per
dose) formulated in aluminum hydroxide (alum) or FA and boosted on days 14
and 35 with the same amount of antigen subcutaneously in 100 µL volume.
Control mice received PBS with alum. (B) A representative microarray scan
following incubation of pooled sera from each group. (C and D) The MFI of
serum antibody cross-reactivity with synthetic structures and native CPS were
expressed as mean ± SD of three microarray spots.
Figure 2. CR-Kp CPS inhibits binding of MAb 1C9 to hexasaccharide 1. (A)
Glycan microarray was incubated with different amounts of MAb 1C9. Bound
antibodies were detected using goat anti-mouse IgM Alexa-flour 594 secondary
antibodies. (B) Pre-adsorption of MAb 1C9 with CR-Kp CPS (10 µg/mL)
followed by incubation with the printed array inhibits binding to 1. (C) ST3 CPS
was used as negative control. (D) Glycan microarray printing pattern. (E) Mean
Fluorescence Intensities (MFI) measured for the inhibition assay. Data are
In order for CRM197-1 to qualify as a vaccine lead,
immunological memory and anti-CPS antibody mediated
protection through challenge has to be demonstrated. CR-Kp
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strains are avirulent in mice; therefore, there is currently no
established animal challenge model available to test CRM197-
1[20]. The production of different types of antibodies is being used
to assess the immunological response in vivo. The majority of
antibodies produced by mice in response to immunization with
CRM197-1 are IgG1 as determined by glycan array analyses
(Figures 4 A and 4 C) while less IgG2a and IgG3 are observed
(Figures 4 D and 4 E). The CRM197-1 specific total serum
antibody level (IgG and IgM) is significantly higher than the PBS
group (Figures 4 F and 4 G). The strong immunological response
and antibody class switch underscores the potential of
hexasaccharide 1 as a vaccine lead against CR-Kp.
Figure 4. Hexasaccharide 1 specific serum antibody isotyping. Immunization
with CRM197-1 conjugate induces high antibody titers in mice. Mouse sera
obtained at different time points were qualitatively analyzed using glycan
microarrays for multiple antibody isotypes. The MFI of hexasaccharide 1 specific
serum (A) IgG, (B) IgM antibodies at different time points. The IgG isotype
antibodies as expressed in MFI values of (n=3) are shown in (C) IgG1, (D) IgG2a,
and (E) IgG3, respectively. Total serum antibody titers at day 49 (F) IgG and (G)
IgM. MFIs for antibody titers was plotted as mean ± SD (triplicate) and analyzed
by unpaired t-test using GraphPadPrism. p values of < 0.05 were considered
statistically significant. *P≤ 0.1, **P≤ 0.05, ***P≤ 0.01 and ****P≤ 0.001.
Figure 5. (A) Surface binding of CRM197-1 antibodies was analyzed by whole
cell ELISA. Antibody binding was assessed for CR-Kp strains 85/12, NRZ-
00319, NRZ-02497, NRZ-01099, carbapenem sensitive strains ATCC43816
(serotype K1), Kp52145 (serotype 2) and other capsular antigen expressing
strains Salmonella typhi, Legionella pneumophila, Clostridium difficile and
Streptococcus pneumoniae. Surface staining was performed with murine anti-
CRM197-1 antibodies (n=3) raised with alum and FA formulation. PBS with
alum was served as control. Anti-CRM197-1 antibodies promote the
phagocytosis of CR-Kp. The differentiated HL-60 cells were incubated with
85/12 (B and C) and NRZ-00319 (D and E) strains pretreated with anti-CRM197-
1 antibodies and control sera, and Klebsiella survival was assessed after 90 min.
Percent killing of Klebsiella were calculated based on viable Klebsiella colonies
obtained relative to no sera control. Values represent an independent
experiment done in duplicates. Values represent mean ± SD. ND, not detected.
In order for an antibody to be protective in vivo, it has to be able
to bind to Klebsiella and induce antibody dependent in vitro
phagocytosis that typically correlates with in vivo protection.[36]
Therefore, we assessed the surface binding of CRM197-1
specific antibodies to intact Klebsiella strains (both carbapenem-
resistant and sensitive). The whole cell ELISA assay suggests
that anti-CRM197-1 antibodies bind broadly to CR-Kp strains
while there was no binding observed to other capsular antigen
expressing stains (Figure 5 A).
In summary, synthetic oligosaccharides help to identify and define
glycan cell-surface epitopes as a basis for vaccine development
against bacterial infections. The synthesis of CR-Kp CPS
hexasaccharide RU 1 and related glycans 2-6 enabled glycan
microarray analyses that revealed that hexasaccharide 1 is
selectively recognized by the monoclonal antibody 1C9, which
cross-reacts with CR-Kp CPS.[17] The CRM197-1 conjugate elicits
high antibody titers in mice and rabbits and shows immunological
memory in mice, cross-reacting with native CPS. Immunization
with CRM197-1 conjugate elicits CR-Kp CPS specific antibodies
that bind the surface of intact bacteria and result in vitro
phagocytic killing of CR-Kp Klebsiella strains. The full potential of
CRM197-1 as a vaccine candidate can be evaluated only once a
suitable animal challenge model, currently being established for
CR-Kp strains, is available.
The functional relevance of the antibodies induced in response to
immunization with CRM197-1 conjugate was assessed by a
opsonophagocytic killing assay (OPKA). Differentiated HL-60
cells were incubated with CR-Kp strains 85/12 and NRZ-00319
preopsonized with anti-CRM197-1 antibodies. The phagocytic
activity of Klebsiella was assessed by plating on blood agar plates
and was expressed as percent killing. CRM197-1 conjugate
immunization elicits opsonic antibodies that exhibit phagocytic
activity against CR-Kp strains tested (Figures 5 B-E). Antibodies
promoting bacterial killing 50 % or more were considered
biologically significant. The antibody titer values for 50 % killing
for 85/12 and NRZ-00319 are 153.6 and 134.2, respectively
(Figure 5 C and 5 E). CRM197-1 conjugate produce functional
antibodies that promote uptake and killing of Klebsiella by
phagocytic cells in mice as well as in rabbits (Figure S4).
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We thank the Max Planck Society and the Körber Foundation (Award
to P.H.S.) for generous financial support. The authors thank Dr.
Allison Berger for editing the manuscript and Andreas Geissner for
help in microarray printing. This work was supported in part by the
German Research Foundation (SFB-TR 84, C3 and C6 to M.W., C8
to P.H.S., and A1 and A5 to B.O.), and by the German Ministry for
Education and Research (e:Med and CAPSyS to M.W.). The authors
gratefully thank Ivonne Pfeifer (Robert-Koch-Institute, Wernigerode,
Germany) and Martin Kaase (National Reference Laboratory for
Multidrug-Resistant GRAM-Negative Bacteria, Bochum, Germany) for
kindly providing K. pneumoniae strains 85/12, NRZ-00319, NRZ-
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Keywords:Klebsiella pneumoniae•antibiotic
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This article is protected by copyright. All rights reserved.

10.1002/anie.201700964
Angewandte Chemie International Edition
COMMUNICATION
Warrener, B. R. Sellman, J. Suzich, Q. Wang, C. K. Stover, JCI Insight
2017, 2, DOI:10.1172/jci.insight.92774.
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10.1002/anie.201700964
Angewandte Chemie International Edition
COMMUNICATION
COMMUNICATION
Resisting the Resistant. A hexasaccharide lead antigen has been identified en
route to developing a vaccine against carbapenem-resistant Klebsiella pneumoniae
(CR-Kp), an agent in hospital-acquired infections.
Peter H. Seeberger,*[1] Claney L.
Pereira,*[1] Naeem Khan,[1]+ Guozhi
Xiao,[1]+ Elizabeth Diago-Navarro,[2]
Katrin Reppe,[3] Bastian Opitz,[3]
Bettina C. Fries[2] and Martin
Witzenrath[3]
Page No. – Page No.
A semi-synthetic glycoconjugate
vaccine candidate for carbapenem-
resistant Klebsiella pneumoniae
This article is protected by copyright. All rights reserved.