Structure-activity relationship of lipid core peptide-based Group A Streptococcus vaccine candidates

Article abstract of DOI:10.1016/j.bmc.2016.03.063

Infection with Group A Streptococcus (GAS) can result in a range of different illnesses, some of which are fatal. Currently, our efforts to develop a vaccine against GAS focuses on the lipid core peptide (LCP) system, a subunit vaccine containing a lipoamino acid (LAA) moiety which allows the stimulation of systemic antibody activity. In the present study, a peptide (J14) representing the B-cell epitope from the GAS M protein was incorporated alongside a universal T-helper epitope (P25) in four LCP constructs of different spatial orientation or LAA lengths. Through structure-activity studies, it was discovered that while the alteration of the LCP orientation had a weaker effect on immunostimulation, increasing the LAA side chain length within the construct increased antibody responses in murine models. Furthermore, the mice immunised with the lead LCP construct were also able to maintain antibody activity throughout the course of five months. These findings highlight the importance of LAA moieties in the development of intranasal peptide vaccines and confirmed that its side chain length has an effect on the immunogenicity of the structure.

Full text of DOI:10.1016/j.bmc.2016.03.063

Bioorganic & Medicinal Chemistry xxx (2016) xxx–xxx
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Structure–activity relationship of lipid core peptide-based Group A
Streptococcus vaccine candidates
Amy Chan a, Waleed M. Hussein a,b, Khairunnisa Abdul Ghaffar a, Nirmal Marasini a, Ahmed Mostafa a,b
,
Sharareh Eskandari a, Michael R. Batzloff c, Michael F. Good c, Mariusz Skwarczynski a, Istvan Toth a,d,e,
⇑
a School of Chemistry and Molecular Biosciences, The University of Queensland, Brisbane 4072, Australia
b Faculty of Pharmacy, Helwan University, Ein Helwan, Helwan, Egypt
c Institute for Glycomics, Griffith University, Gold Coast 4215, Australia
d School of Pharmacy, The University of Queensland, Brisbane 4072, Australia
e Institute for Molecular Bioscience, The University of Queensland, Brisbane 4072, Australia
a r t i c l e i n f o
a b s t r a c t
Article history:
Infection with Group A Streptococcus (GAS) can result in a range of different illnesses, some of which are
fatal. Currently, our efforts to develop a vaccine against GAS focuses on the lipid core peptide (LCP) sys-
tem, a subunit vaccine containing a lipoamino acid (LAA) moiety which allows the stimulation of sys-
temic antibody activity. In the present study, a peptide (J14) representing the B-cell epitope from the
GAS M protein was incorporated alongside a universal T-helper epitope (P25) in four LCP constructs of
different spatial orientation or LAA lengths. Through structure–activity studies, it was discovered that
while the alteration of the LCP orientation had a weaker effect on immunostimulation, increasing the
LAA side chain length within the construct increased antibody responses in murine models.
Furthermore, the mice immunised with the lead LCP construct were also able to maintain antibody activ-
ity throughout the course of five months. These findings highlight the importance of LAA moieties in the
development of intranasal peptide vaccines and confirmed that its side chain length has an effect on the
immunogenicity of the structure.
Received 11 February 2016
Revised 22 March 2016
Accepted 30 March 2016
Available online xxxx
Keywords:
Group A Streptococcus
Peptide vaccine
Lipopeptides
Adjuvant
Copper-catalysed azide–alkyne
cycloaddition
Ó 2016 Elsevier Ltd. All rights reserved.
1. Introduction
interest: the N-terminus and the C-repeat region.5 The N-terminus
is hypervariable and frequently undergoes genetic recombination,
Group A Streptococcus (Streptococcus pyogenes or GAS) is a
Gram positive bacteria that can cause illnesses ranging from
benign pharyngitis to invasive diseases like necrotising fasciitis
and toxic shock syndrome. GAS-associated illnesses represent a
global health problem, causing more than 600 million new cases
of infection each year and resulting in a minimum of 500,000
deaths.1,2 Delayed or inadequate treatment of GAS infections may
also result in the development of post-infectious complications
such as rheumatic fever (RF) and rheumatic heart disease (RHD),
which are responsible for the majority of GAS-related mortality.3
This highlights the need for the development of an effective pro-
phylactic vaccine against GAS.
resulting in a rapid turnover of strains.6–8 Due to this, antibodies
induced against the N-terminus are serotype-specific and are only
protective against particular strains of GAS, implicating the need to
tailor vaccines for GAS endemic to different regions.9 Conversely,
the C-repeat region of the M-protein is highly conserved.10
Epitopes derived from this region can overcome the restrictions
regarding serotype specificity, allowing for the development of a
vaccine that is protective against all GAS strains.
One such promising peptide epitope is p145 (LRRDLDAS-
REAKKQVEKALE), which has been shown to generate protective
antibodies capable of opsonising multiple serotypes of GAS.10–14
However, immunisation with p145 poses a risk of generating T-cell
autoimmune responses due to its sequence similarity to the
human cardiac myosin.15 Further research has identified J14
(KQAEDKVKASREAKKQVEKALEQLEDKVK), a minimal peptide epi-
tope that excludes the potentially deleterious T-cell sequences.10
J14 is composed of 14 amino acids from p145 (sequence in bold)
enclosed between two non-streptococcal sequences designed to
The development of a vaccine against GAS has primarily focused
on the M-protein, a major virulence factor ubiquitous to all GAS
strains.4 Two areas of the M-protein present a particular research
⇑
Corresponding author at present address: School of Chemistry and Molecular
Biosciences, The University of Queensland, St Lucia, QLD 4072, Australia. Tel.: +61 7
33469892 (O); fax: +61 7 33654273.
maintain the
a-helical conformation of a native epitope in the pro-
tein. It has been demonstrated that J14, when combined with the
E-mail address: i.toth@uq.edu.au (I. Toth).
http://dx.doi.org/10.1016/j.bmc.2016.03.063
0968-0896/Ó 2016 Elsevier Ltd. All rights reserved.
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2
A. Chan et al. / Bioorg. Med. Chem. xxx (2016) xxx–xxx
appropriate delivery system, can confer protective B-cell antibod-
ies in mice against heterologous GAS strains.10,16,17
As a short peptides, J14 is immunologically inert on its own.
Therefore, it is coupled with a self-adjuvanting moiety in a delivery
system called a lipid core peptide (LCP). LCPs contain immune
immunological evaluation was performed upon intranasal admin-
istration in mice. Both spatial orientation of epitopes and
lipophilicity of LAAs were found to influence humoral immune
responses generated by LCP 1–4.
stimulating lipoamino acids (LAAs) which are a-amino acids with
2. Materials
a long alkyl side-chain. The number of LAAs incorporated into
the LCP, and the length of their side-chains, can be easily modi-
fied.18 LAAs have demonstrated potent adjuvanting activity by
activating toll-like receptor 2 (TLR2) on antigen presenting cells
(APCs), leading to the induction of an immune response.19
Protected
L-amino acids were purchased from Mimotopes
(Melbourne, Australia) or Novabiochem (Laufelfingen, Switzerland).
pMBHA resin was obtained from Peptide International Inc.
(Kentucky, USA). Trifluoroacetic acid (TFA) was purchased from
Merck (Kilsyth, Australia). 1-(1H-Benzotriazol-1-yl)-1,1,3,3-
tetramethyluronium hexafluorophosphate (HBTU) was obtained
from Mimotopes. HPLC grade acetonitrile (MeCN) and N,N-
dimethylformamide (DMF) were purchased from Ajax Finechem
(Sydney, Australia). All other reagents were obtained from Sigma–
Aldrich (Castle Hill, NSW, Australia). Microwave-assisted Fmoc-
SPPS was performed on a CEM Discovery reactor (CME Corporation,
Matthews, NC, USA). Microwave-assisted Boc-SPPS was carried out
using a CEM Discovery automated peptide synthesiser. HF cleavage
was achieved with an AKel-F HF apparatus (Peptide Institute, Osaka,
Japan). ESI-MS was performed on a Perkin-Elmer-Sciex API3000
with Analyst 1.4 software (Applied Biosystems/MDS Sciex, Toronto,
Canada). Analytical RP-HPLC was performed on an Agilent instru-
ment. Separation was achieved by running gradient mode of solvent
B (MeCN/H2O/TFA; 90:10:0.1) over solvent A (H2O/TFA; 100:0.1) on
The LCP system is able to induce protective immunity in both
the mucosal and systemic immune compartments.19,20 This per-
mits delivery of LCP vaccines via the intranasal route. As GAS typ-
ically resides on the mucosal epithelium of the upper respiratory
tract, intranasal vaccination benefits by its ability to stimulate both
IgA and IgG antibodies.21,22 IgA, the primary antibody on mucosal
surfaces, inhibits GAS adhesion and colonisation, thus preventing
bacterial dissemination that could lead to systemic infection.21,23
However, IgA is inadequate as an opsonin and as an activator of
the complement system.21 Under circumstances where bacteria
does disseminate into the bloodstream, IgG, which is a powerful
opsonin and activator of the complement system, can facilitate
clearance of the bacteria.21,24 The LCP system also features a lysine
(K) moiety (or moieties) from which different vaccine components
can be attached.25 These components can be placed and rearranged
at the
a-amino, a-carboxyl, and e-amino functional groups of the
a Vydac analytical C4-column (214TP54; 10
Vydac analytical C18-column (218TP54; 10
l
m, 4.6 Â 250 mm) or a
central lysine. This allows for the design of LCP systems with differ-
ent molecular geometries but containing the same epitopes.
Conventionally, a typical GAS LCP vaccine consists of C12
l
m, 4.6 Â 250 mm).
Purification was performed on a preparative RP-HPLC using a
Waters Delta 600 system with a 10.0 ml/min flow rate. Compounds
were detected at 230 nm and separation was achieved with
solvent B and solvent A on a C4-column (214TP1022; 10 mm,
22 Â 250 mm). DLS (dynamic light scattering) measurements were
taken on a Nanosizer instrument (Zetasizer Nano Series ZS, Malvern
Instruments, Worcestershire, UK) using the Zetasizer 6.2 software.
Particle-imaging was achieved with a JEM-1010 transmission
electron microscope (JEOL Ltd., Tokyo, Japan).
(2-amino-D,L-dodecanoic acid) LAAs attached on the a-carboxyl
side-chain of the lysine core via glycine spacers, and peptide
epitopes (usually B-cell epitopes) attached to the lysine amine
moieties.26 In recent designs of vaccine candidates against GAS, a
T-helper (Th) epitope (P25) was included on the N-terminus to
confer immunity in heterologous populations, two copies of serine
(S) were included as spacer, and the C12 LAA was replaced with the
more lipophilic C16 LAA (2-amino-D,L
-hexadecanoic acid).26,27 The
newly designed LCP was able to stimulate IgG antibody production
upon intranasal delivery.17,28 These alterations suggest that
structural changes to an LCP have the potential to affect its
immunological properties.
Herein, LCP vaccine candidates (Fig. 1) were designed to incor-
porate the same J14 B-cell epitope and P25 Th-epitope, but to differ
in lipopeptide structure (LAA side-chain length) or spatial orienta-
tion. Following LCP synthesis and self-assembly into nanoparticles,
3. Methods
3.1. Synthesis of LAAs with Dde-protective group (Dde-C16 and
Dde-C20)
C16 (2-amino-
D,L-hexadecanoic acid) and C20 (2-amino-D,L-
eicosanoic acid) were synthesised following similar methods
Figure 1. Structures of synthetic LCP 1–4. The LCPs incorporated a GAS B-cell epitope (J14: KQAEDKVKASREAKKQVEKALEQLEDKVK), a universal Th-epitope (P25:
KLIPNASLIENCTKAEL), and LAAs with differing side-chain length (C16 or C20). Ac = acetyl group; S = serine; K = lysine.
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A. Chan et al. / Bioorg. Med. Chem. xxx (2016) xxx–xxx
3
described before.29 Sodium ethoxide solution was prepared by dis-
solving sodium in an ethanol solution, to which diethyl acetomido-
malonate and 1-bromo-tetradecane or 1-bromo-octadecane was
added to produce C16 or C20, respectively. The mixture was
refluxed for 4 days and the resultant precipitate was filtered and
washed with cold water. The solid was dried then dissolved in
DMF and fuming 37% HCl and refluxed again for 4 days. Ammonia
solution was added to yield precipitate, which was filtered and
washed with cold water and acetone. The final product was dried
until powdery.
Dde-OH (N-(4,4-dimethyl-2,6-dioxycyclohexylidene)ethyl alco-
hol) was synthesised following previously described methods.30
Then, Dde-OH (1.1 equiv) was dissolved in ethanol, to which tri-
ethylamine (3 equiv) and C16 (1 equiv), or C20 (1 equiv), was
added. The mixture was refluxed for 2 days and ethanol was evap-
orated. Ethyl acetate was added, and 5% HCl and brine solution
were used to wash the organic phase. The organic layers were col-
lected and filtered by manual short flash chromatography using a
silica column. The solution was evaporated and stored at À20 °C
to afford yellow crystals. The crystals were washed with cold
MeCN, filtered, and dried until powdery. The resulting Dde-C16
and Dde-C20 were analysed by 1H NMR (300 MHz, CDCl3) to pro-
duce spectra identical with those reported previously.30
Two serine moieties and a lysine residue were incorporated using
standard Fmoc-SPPS. Amino acid activation was achieved by dis-
solving Fmoc-amino acids (4.2 equiv) in 0.5 M HATU (4.0 equiv)
in DMF, followed by the addition of DIPEA (4.2 equiv) The coupling
cycle consisted of Fmoc deprotection with 20% piperidine in DMF
(twice, 10 min and 20 min), a 1 min DMF flow-wash, followed by
couplings with preactivated Fmoc-amino acids (2 Â 1 h). Pentynoic
acid (4.2 equiv) was coupled to the N-terminus of the sequence by
using HATU (4 equiv) and DIPEA (4.2 equiv) at room temperature
(2 Â 1 h), followed by washing with DMF, DCM, MeOH, and drying
under vacuum. The peptide was cleaved from the resin in a solu-
tion of neat TFA triisopropylsilane/water (95:2.5:2.5) for 4 h. The
cleaved peptide was precipitated, filtered, and washed with ice-
cold Et2O, dissolved in acetonitrile/water 90:10 and lyophilised.
The resulting crude product (9) was purified by RP-HPLC.
Peptide 9 yield: 24%. Purity: >95%. Molecular weight: 906.3. ESI-
MS: [M+H]+ m/z 906.9 (calcd: 907.3). Rt = 33 min (35–75% solvent
B, 60 min, C4-column).
3.4. Synthesis of peptides 10 and 11
The two azido derivative peptides 10 and 11 (Scheme 2) were
synthesised according to the general procedure by microwave-
assisted Fmoc-SPPS with 2 Â 5 min couplings for each Fmoc-amino
acid (4.2 equiv) using HATU (4.0 equiv) and DIPEA (4.2 equiv). The
temperature was set at 70 °C (20 W, 10 min) for all amino acids,
except for Cys and His, which were coupled at 50 °C (20 W,
10 min). For His, Cys, and Arg, initial coupling was performed at
rt for 3 min before microwave-assisted couplings.31 Each amino
acid coupling cycle consisted of Fmoc-deprotection with 20%
piperidine in DMF at 70 °C (twice, 2.5 min and 5 min), 2 min
DMF flow wash, followed by 10 min coupling with the pre-acti-
vated amino acid. In the case of Fmoc-deprotection for Asp, 0.1 M
HOBT in 20% piperidine/DMF was used. Upon completion of syn-
thesis, the resin was washed with DMF, DCM, MeOH, and vacuum
dried. The peptide was cleaved from the resin by stirring in a solu-
tion of TFA (99%)/triisopropylsilane/water (95:2.5:2.5) for 4 h. The
crude products (10 and 11) were purified by RP-HPLC.
3.2. Synthesis of LCP 1 and LCP 2
LCP 1 was synthesised by coupling Dde-C16 onto pMBHA resin
(0.45 mmol/g; 0.2 mmol scale) by using HBTU (4.0 equiv) and
DIPEA (6.2 equiv) in DMF (2 Â 1 h). Removal of the Dde-protecting
group was performed by treatments with 2% hydrazine in DMF
(2 Â 30 min). The remaining LCP sequence was synthesised by
microwave-assisted Boc-SPPS. Amino acid activation was achieved
by dissolving Boc-amino acids (4.2 equiv) in 0.5 M HBTU
(4.0 equiv) in DMF, followed by the addition of DIPEA (6.2 equiv).
The amino acid coupling cycle consisted of Boc-deprotection with
neat TFA at room temperature (2 Â 1 min), 2 min DMF flow wash,
followed by 10 min coupling with the pre-activated amino acid.
The temperature was set at 70 °C (35 W, 10 min) for all amino
acids, except for Cys and His, which were coupled at 50 °C (35 W,
10 min). N-terminal acetylation and final cleavage from resin was
performed as reported previously.29 The lyophilised products were
purified by RP-HPLC. The collected fractions were analysed by
ESI-MS and RP-HPLC. Where appropriate, fractions were combined
and lyophilised to yield pure product.
Peptide 10 yield: 6%. Purity: >95%. Molecular weight: 5446.2.
ESI-MS: [M+4H]4+ m/z 1362.1 (calcd: 1362.6), [M+5H]5+ m/z
1090.1 (calc: 1090.3), [M+6H]6+ m/z 908.8 (calcd: 908.7),
For the synthesis of LCP 2, Dde-C20 was coupled onto pMBHA
resin (0.45 mmol/g; 0.2 mmol scale). The remaining procedures
followed that of the synthesis of LCP 1, as mentioned above.
LCP 1 yield: 12.5%. Purity: 90%. Molecular weight: 6086.4. ESI-
MS: [M+4H]4+ m/z 1523.4 (calcd: 1522.6), [M+5H]5+ m/z 1218.4
(calcd: 1218.3), [M+6H]6+ m/z 1015.7 (calcd: 1015.3), [M+7H]7+
m/z 870.6 (calcd: 870.4), [M+9H]9+ m/z 677.5 (calcd: 677.3), [M
+10H]10+ m/z 608.8 (calcd: 609.5). Rt = 25.0 min (0–100% solvent
B, 40 min, C4-column).
LCP 2 Yield: 18.5%. Purity:>99%. Molecular weight: 6198.5. ESI-
MS: [M+5H]5+ m/z 1241.1 (calcd: 1240.7), [M+6H]6+ m/z 1034.7
(calcd: 1034.1), [M+7H]7+ m/z 886.6 (calcd: 886.5), [M+9H]9+ m/z
690.4 (calcd: 689.7). Rt = 29.0 min (0–100% solvent B, 40 min, C4-
column).
3.3. Synthesis of alkyne derivative 9
The synthesis of alkyne derivative 9 was performed with man-
ual Fmoc-SPPS (Scheme 1). Dde-C16 was coupled onto rink amide
MBHA resin (0.2 mmol scale) by using HATU (4.0 equiv) and DIPEA
(4.2 equiv) in DMF (2 Â 1 h). The Dde-protecting group was
removed by treatment with 2% hydrazine in DMF (8 Â 30 min).
Scheme 1. Synthesis of alkyne derivative 9.
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4
A. Chan et al. / Bioorg. Med. Chem. xxx (2016) xxx–xxx
measurement. The average size of the particles was calculated by
the Zetasizer 6.2 software (Malvern Instruments, UK).
3.7. Transmission electron microscopy
Samples of the LCP 1–4 in PBS used for the Nanosizer experi-
ments were placed on carbon-coated 200 mesh grids. Excess liquid
was removed with filter paper after 3 min. A JEM-1010 transmis-
sion electron microscope (JEOL Ltd, Japan) operating at 80 kV
was used to take photographs of the compounds.
3.8. Intranasal immunisation of mice
Eight week-old female outbred Swiss mice were used in this
study (Griffith University, Gold Coast, Australia). All animal proto-
cols used were approved by the Animal Ethics Committee (Univer-
sity of Queensland) in accordance with National Health and
Medical Research Council (NHMRC) of Australia guidelines. Access
to food and clean water was provided ad libitum.
Scheme 2. Synthesis of LCP 3 and 4 (J14: KQAEDKVKASREAKKQVEKALEQLEDKVK),
P25: KLIPNASLIENCTKAEL)).
Cohorts of 5 mice were administered 60
lg of LCP dissolved in
[M+7H]7+ m/z 779.0 (calcd: 779.0), [M+8H]8+ m/z 691.9 (calcd:
681.8). Rt = 23.8 min (25–45% solvent B, 20 min, C18-column).
Peptide 11 yield: 12%. Purity: >95%. Molecular weight: 5446.2.
ESI-MS: [M+3H]3+ m/z 1816.3 (calcd: 1816.4), [M+4H]4+ m/z
1362.9 (calcd: 1362.6), [M+5H]5+ m/z 1090.2 (calcd: 1090.3).
Rt = 24.3 min (25–45% solvent B, 20 min, C18-column).
30
l
L (15 L/nare) of sterile PBS following anaesthetisation with
l
1:1:10 mixture of ketamine/xylazine/water (Griffith University,
Gold Coast, Australia). Similarly, a negative control group was
administered 30
of J14-K-P25 with 10
30 L). Additionally, one group was administered 30
l
L of PBS and a positive control received 30
g of CTB mixed with PBS (total volume of
g of uncon-
lg
l
l
l
jugated P25+J14+CTB. Boosters of the same dosage were given on
days 21 and 42 following primary immunisation.
3.5. Synthesis of LCP 3 and LCP 4
For the synthesis of LCP 3, a mixture of azide derivative (10)
(0.4 lmol, 1 equiv) and lipoalkyne (9) (2.2 lmol, 5 equiv) was dis-
3.9. Serum collection
solved in DMF/DMSO (1:0.6), and a piece of copper wire was added
(Scheme 2). Most of air in the reaction mixture was removed by
quick nitrogen bubbling. The reaction mixture was covered and
protected from light with aluminium foil and stirred at 50 °C under
nitrogen. The progress of the reaction was monitored by analytical
HPLC (C4-column) and ESI-MS until the peptide 10 was completely
consumed after 5 h. The reaction mixture was added to a buffer
(6 M guanidine, 50 mM sodium phosphate, 20% acetonitrile,
5 mM EDTA, ꢀpH 7.3) containing TCEP (tris(2-carboxyethyl)phos-
phine) and stirred for 1 h. The reaction mixture was purified using
a semi-preparative HPLC. After lyophilisation, the pure LCP 3 was
obtained as an amorphous white powder.
Serum was collected on days 20, 41 and 60, and at five months
post-primary immunisation (day 155). 10 lL of blood was col-
lected from the tail artery of each mouse, and serum was extracted
by centrifugation.
3.10. Determination of antibody titres
An ELISA was used to measured J14-specific murine serum IgG
as described elsewhere.10 Serial two-fold dilutions of samples were
produced in 0.5 % skim milk/PBS-Tween 20 buffer, starting at the
concentration of 1:100 for sera samples. A titre was defined as
the highest dilution that gave an optical density >5 Â SDs above
the mean optical density of control wells containing mouse sera
collected before immunisation. Statistical significance (p <0.05)
was determined using a one-way ANOVA with Tukey post-hoc test.
Horseradish peroxidase-conjugated antimouse IgG1 and IgG2a
were used as secondary antibodies.
For the synthesis of LCP 4, a mixture of azide derivative (11)
(0.3 lmol, 1 equiv) and lipoalkyne (9) (2.2 lmol, 7 equiv) was dis-
solved in DMF/DMSO (1:0.6), and a piece of copper wire was added
(Scheme 2). The remaining procedures follow those of the synthe-
sis of LCP 3, as mentioned above.
LCP 3 yield: 43%. Purity: >97%. Molecular weight: 6352.5.
ESI-MS: [M+6H]6+ m/z 1059.7 (calcd: 1059.8), [M+7H]7+ m/z
908.5 (calcd: 908.5). Rt = 27.8 min (30–70% solvent B, 60 min,
C4-column).
4. Results and discussion
In our attempts to optimise delivery systems for peptide-based
GAS vaccines, three novel analogues of lead vaccine candidate LCP
LCP 4 yield: 72%. Purity: >97%. Molecular weight: 6352.5.
ESI-MS: [M+6H]6+ m/z 1059.7 (calcd: 1059.8), [M+7H]7+ m/z
908.5 (calcd: 908.5), [M+8H]8+ m/z 795.0 (calcd: 795.1), [M+9H]9+
m/z 706.7 (calcd: 706.8). Rt = 23.0 min (30–70% solvent B, 60 min,
C4-column).
1 were designed (Fig. 1). LCP 2 incorporated C20 LAAs (2-amino-
-eicosanoic acid), a longer version of C16 LAAs presented in LCP 1.
LCP 3 and 4 differ in special orientation from 1 and 2; both have
C16 LAAs on the -amino group of the central lysine, attached
via a triazole linkage. In LCP 3, P25 was attached on the -amino
side-chain and J14 on the -carboxyl side-chain, while in LCP 4,
D,
L
e
a
3.6. Measurement of LCP particle size
a
these epitopes replaced their positions. In all compounds, the
self-adjuvanting lipopeptide moiety was attached to the branching
lysine via the lipopeptide N-terminal serine, and therefore, all LCPs
possessed C-terminal LAAs in amide form.
LCP compounds were dissolved in PBS (phosphate buffered sal-
ine) (0.2 mg/mL). The particle size of each LCP compound at 25 °C
was measured in capillary cuvettes using a Nanosizer (Zetasizer
Nano Series ZS, Malvern Instruments, UK). Measurements were
taken with a scattering angle of 173°. Correlation times were set
at 10 s per run, and a total of 5 runs were made for each
LCP 1 and 2 were synthesised using standard SPPS while LCP 3
and
4 were produced with the help of a copper-catalysed
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A. Chan et al. / Bioorg. Med. Chem. xxx (2016) xxx–xxx
5
Table 1
they can be absorbed through mucosal surfaces and thus, whether
they can be delivered effectively as intranasal vaccines.20 Their size
can also affect how they are recognised and processed by APCs, as
well as their trafficking in the lymphatic system, and in turn,
affecting the immune response that follows.37,38 The size distribu-
tion of the particles formed by LCP 1–4 was measured by DLS
(Table 1). Particles were also visualised by transmission electron
microscopy (TEM) (Fig. 2). With the exception of LCP 4, all the com-
pounds formed particles with a homogenous distribution. LCP 4
formed the largest nanoparticles with the highest variability in size
(8–25 d nm). However, this size difference is negligible and is unli-
kely to cause differences in immunogenicity of the compounds.
At sizes smaller than 100 d nm, LCP 1–4 are all optimal for
absorption through the mucosal epithelium and for entering
lymph vessels via gaps between epithelial cells.39 This permits easy
access for the particles to the systemic immune system. As all the
compounds in this study formed particles of similar sizes, it was
therefore assumed that any immunological difference between
the compounds was caused by factors other than their size.
Outbred Swiss mice were used for the immunological study of
LCP 1–4. Following the initial intranasal immunisation and two
boosters of LCP compounds, blood serum was collected to deter-
mine J14-specific IgG antibody titres by enzyme-linked
immunosorbent assay (ELISA). All LCP groups elicited significant
Average diameter of the LCP particles (d nm) dissolved in PBS (0.2 mg/mL)
Compound
Size by DLS (d nm)
Size by TEM (d nm)
LCP 1
LCP 2
LCP 3
LCP 4
9.2 0.3
11.1 0.6
10.0 1.0
13.9 3.0
7–11
8–12
8–15
8–25
azide-alkyne cycloaddition (CuAAC) reaction. At first, the alkyne
derivative 9 was synthesised by Fmoc-SPPS (Scheme 1). Couplings
of Dde-C16 (5) to the Rink amide resin was achieved with the help
of HATU and DIPEA in DMF (2 Â 1 h). The Dde-protecting group of
5 was removed by using 2% hydrazine in DMF (8 Â 30 min) to give
6, then the process was repeated to afford lipopeptide-resin 7. The
serines were also coupled using the HATU/DIPEA method. To
increase the solubility of 9, a lysine residue was introduced into
the lipopeptide sequence. Finally, pentynoic acid was coupled to
the N-terminus end of the peptide to give the alkyne lipopeptide
(8) that was cleaved with 95% of TFA to give 9 in 24% yield
(Scheme 1). The CuAAC was applied to conjugate the azide deriva-
tives 10 and 11 with the lipopeptide 9 in the presence of Cu wire32
at 50 °C (Scheme 2). The reaction mixture was added to a guani-
dine buffer solution containing TCEP to reduce the disulphide bond
formed during the click reaction. The final constructs LCP 3 and 4
were purified and obtained in 43% and 16% yields, respectively.
As LCPs are amphiphilic compounds, they can self-assemble
into particles of distinct sizes in aqueous solutions.33–36 The size
of the particles they form is important in determining whether
IgG titres (Fig. 3), with LCP
2 inducing the highest titres
(p <0.0001 vs the negative control group PBS). LCP 2 titres were
also significantly higher than those of LCP 3 and 4, and the positive
control (J14-K-P25 + CTB). LCP 2, having LAAs with longer side-
chains than LCP 1, also induced titres higher than those of LCP 1.
Figure 2. Transmission electron microscopy photographs of the investigated compounds in 2 mg/mL concentration dissolved in PBS: (A) LCP 1. (B) LCP 2. (C) LCP 3. (D) LCP 4.
Bar = 200 nm.
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6
A. Chan et al. / Bioorg. Med. Chem. xxx (2016) xxx–xxx
However, this difference was not statistically significant as the
induced immune response differed greatly between individual
mice.
As no human-approved mucosal adjuvant exists for the delivery
of peptides, CTB (which is restricted to animal use) represents a
standard adjuvant to compare against the investigated com-
pounds.40–42 Thus, LCP 2 is not only more efficient at inducing
IgG production, it is also expected to be a safer alternative for vac-
cines adjuvanted with CTB. Furthermore, it should be noted that
there was no significant difference in induced titres between mice
immunised with Th-epitope and B-cell epitope that were conju-
gated (J14-K-P25 + CTB), and mice immunised with Th-epitope
and B-cell epitope that were not conjugated (P25 + J14 + CTB). This
contrasts with some reports suggesting that such conjugation is
crucial for the induction of immune responses.43
LCP 2 was compared to LCP 1 to determine the effect of varying
the LAA length on compound immunogenicity. Despite the lack of
significance in the current data, it does appear that LCP 2 produced
higher IgG titres than LCP 1. This suggests that an increase in LAA
length may also increase the immunogenicity of the compound.
LCP 4 and 5 were compared to LCP 1 to determine the effects of
spatial orientation on compound immunogenicity. Neither LCP 3
nor 4 induced immune responses that were statistically different
from LCP 1. This suggests that, with the same LAA length, the
spatial orientation of the LCP has a rather limited effect on its
immunogenicity.
Overall, LCP 2 stimulated the highest short-term systemic
immune response. In order to further characterise the J14-specific
immune response elicited by LCP 2, IgG antibody isotypes were
evaluated by ELISA and compared to LCP 1 (Fig. 4). In the murine
system, IgG1 is predominantly induced by Th2 activity and IgG2a
induced by Th1 activity.44 Both IgG1 and IgG2a activity were
observed after immunisation with LCP 1, as well as with LCP 2,
suggesting a mixed Th1/Th2 response. Notably, LCP 2 was able to
generate a significantly greater Th1 response than LCP 1. This
may be more desirable as Th1-type cytokines are responsible for
Figure 4. J14-specific serum IgG isotype titre (log10) at day 60 post-primary
intranasal immunisation of Swiss mice (n = 5/group). Error bars represent standard
deviation.
stimulating phagocytes and promoting pro-inflammatory
responses against intracellular pathogens, which may facilitate
faster bacterial clearance.45 Furthermore, the high Th2 activity
prevents a Th1-dominated response that may cause host tissue
damage.46 In comparison, the J14-K-P25 + CTB positive control
mounted a Th2 response only.
Serum samples were obtained from the LCP 2 group at 155 days
post-primary immunisation (with the last booster given on day 42)
to determine whether this compound was capable of generating
long-term J14-specific antibody response (Fig. 5). It was found that
vaccination with LCP 2 maintained high IgG titres after five months
(p <0.05 vs the negative control PBS), which was similar to those
induced by the adjuvanted control (J14-K-P25 + CTB).
Figure 3. J14-specific IgG titres (log10) elicited in response to vaccination of Swiss
mice (n = 5/group) at day 60. Mice received a primary immunisation on day 0
Figure 5. J14-specific IgG titres (log10) elicited in response to vaccination of Swiss
mice (n = 5/group) after five months (155 days). Mice received a primary immu-
nisation on day 0 followed by two boosters on days 21 and 42. Statistical analysis
was performed by one-way ANOVA followed by the Tukey post-hoc test (ns,
p <0.05; ⁄, p <0.01; ⁄⁄, p <0.001; ⁄⁄⁄, p <0.0001; ⁄⁄⁄⁄).
followed by two boosters on days 21 and 42. Statistical analysis was performed by
⁄⁄
one-way ANOVA followed by the Tukey post-hoc test (ns, p <0.05; ⁄, p <0.01;
p < 0.001; ⁄⁄⁄, p <0.0001; ⁄⁄⁄⁄).
,
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A. Chan et al. / Bioorg. Med. Chem. xxx (2016) xxx–xxx
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5. Conclusion
Recent trends in GAS vaccine development have focused on the
application of peptide-based antigens, mainly from M-protein. For
this purpose, we have studied a multicomponent LCP construct
incorporating minimal peptide epitopes, Th-epitopes, and LAA.
The adjustable design of the LCP allowed the alteration of its spa-
tial orientation and the length of incorporated LAAs. This study
confirmed that the lipid length and position of vaccine components
has an effect on the immune response in vivo in terms of IgG anti-
body production. The leading vaccine candidate (LCP 2) in this
study featured a C-terminal C20 LAA moiety attached via a lysine
residue to P25 on the N-terminus, and J14 on the lysine e-amino
side-chain. LCP 2 was able to induce a strong mixed IgG1/IgG2a
immune response, even after five months post-primary immunisa-
tion. This compound outperformed the other constructs involved
in this study and its induced IgG response was even higher than
those induced by the classical adjuvant CTB.
Acknowledgements
The authors acknowledge the facilities and the scientific and
technical assistance of the Australian Microscopy & Microanalysis
Research Facility at the Centre for Microscopy and Microanalysis,
The University of Queensland. This work was supported by the
National Health and Medical Research Council of Australia
(NHMRC 496600).
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