Vaccine 30 (2012) 5073–5080
Contents lists available at SciVerse ScienceDirect
journal homepage: www.elsevier.com/locate/vaccine
Evaluation of synthetic infection-enhancing lipopeptides as adjuvants for a
live-attenuated canine distemper virus vaccine administered intra-nasally to
D. Tien Nguyena, Martin Ludlowb, Geert van Amerongena, Rory D. de Vriesa, Selma Yüksela,
R. Joyce Verburgha, Albert D.M.E. Osterhausa, W. Paul Duprexb, Rik L. de Swarta,∗
aDepartment of Virology, Erasmus MC, University Medical Center, P.O. Box 2040, 3000 CA Rotterdam, The Netherlands
bDepartment of Microbiology, Boston University School of Medicine and National Emerging Infectious Diseases Laboratories, Boston University, 72 East Concord Street, Boston, MA
02118, United States
a r t i c l ei n f o
Received 6 January 2012
Received in revised form 11 May 2012
Accepted 30 May 2012
Available online 21 June 2012
Live-attenuated virus vaccine
a b s t r a c t
Background: Inactivated paramyxovirus vaccines have been associated with hypersensitivity responses
virus vaccines are available, but for human respiratory syncytial virus and human metapneumovirus
development of such vaccines has proven difficult. We recently identified three synthetic bacterial
lipopeptides that enhance paramyxovirus infections in vitro, and hypothesized these could be used as
adjuvants to promote immune responses induced by live-attenuated paramyxovirus vaccines.
Methods: Here, we tested this hypothesis using a CDV vaccination and challenge model in ferrets. Three
groups of six animals were intra-nasally vaccinated with recombinant (r) CDV5804PL(CCEGFPC) in the
presence or absence of the infection-enhancing lipopeptides Pam3CSK4 or PHCSK4. The recombinant
CDV vaccine virus had previously been described to be over-attenuated in ferrets. A group of six animals
was mock-vaccinated as control. Six weeks after vaccination all animals were challenged with a lethal
dose of rCDV strain Snyder-Hill expressing the red fluorescent protein dTomato.
Results: Unexpectedly, intra-nasal vaccination of ferrets with rCDV5804PL(CCEGFPC) in the absence of
lipopeptides resulted in good immune responses and protection against lethal challenge infection. How-
ever, in animals vaccinated with lipopeptide-adjuvanted virus significantly higher vaccine virus loads
were detected in nasopharyngeal lavages and peripheral blood mononuclear cells. In addition, these ani-
mals developed significantly higher CDV neutralizing antibody titers compared to animals vaccinated
with non-adjuvanted vaccine.
Conclusions: This study demonstrates that the synthetic cationic lipopeptides Pam3CSK4 and PHCSK4
not only enhance paramyxovirus infection in vitro, but also in vivo. Given the observed enhancement of
immunogenicity their potential as adjuvants for other live-attenuated paramyxovirus vaccines should
© 2012 Elsevier Ltd. All rights reserved.
Canine distemper virus (CDV) is a member of the family
Paramyxoviridae, which also includes measles virus (MV), human
respiratory syncytial virus (HRSV) and human metapneumovirus
(HMPV). CDV is best known as a pathogen of dogs, but has a broad
host range and causes disease in different carnivore species and
in non-human primates [1,2]. CDV infection induces a systemic
disease, which is associated with immunosuppression and often
involves infection of the central nervous system [3,4].
∗Corresponding author. Tel.: +31 10 7044280; fax: +31 10 7044760.
E-mail address: firstname.lastname@example.org (R.L. de Swart).
We recently demonstrated that the synthetic bacterial lipopep-
tide Pam3-Cys-Ser-Lys4 (Pam3CSK4) enhanced in vitro infections
with HRSV, HMPV and MV . Pam3CSK4 is a synthetic tri-
palmitoylated lipopeptide and is the prototype agonist for the
heterodimeric TLR1/2 complex [6,7], and had previously been
associated with enhancement of infections with human immun-
odeficiency virus (HIV) type-1 [8–10]. Although in these studies
it was assumed that enhancement of infection was related to
TLR activation, we identified two structurally-related synthetic
lipopeptides, PHCSK4 and Pam-Cys-SK4, which did not function as
TLR ligands, but showed a similar enhancement of paramyxovirus
or HIV-1 infections as Pam3CSK4. In contrast, other lipopeptides
that did stimulate TLR1/2 responses did not enhance paramyx-
ovirus or HIV-1 infections . Enhancement of infection seemed
0264-410X/$ – see front matter © 2012 Elsevier Ltd. All rights reserved.
D.T. Nguyen et al. / Vaccine 30 (2012) 5073–5080
dependent on the cationic properties and the N-palmitoylated cys-
teine of the lipopeptides, and was mediated by increased virus
lipopeptides could be used as adjuvants for live-attenuated virus
vaccines. Formulation of an over-attenuated paramyxovirus vac-
cine with an infection-enhancing lipopeptide may enhance the
capacity of the virus to infect cells during the first round of infec-
tion. Thus the formulated vaccine could induce adequate immune
responses, whereas the risk of (serious) adverse effects for the vac-
cinee remains low.
rets were intra-nasally (IN) vaccinated with a live-attenuated CDV
vaccine virus , followed by a lethal challenge infection with a
previously described to induce sub-optimal protective immune
erated by inserting the open reading frame encoding enhanced
green fluorescent protein (EGFP) into the sequence coding for the
second hinge of the CDV large (L) protein, which is the major
component of the viral RNA-dependent RNA polymerase. Such
manipulations of morbillivirus L proteins attenuate pathogenic
viruses in a single step, whilst keeping all of the structural proteins
authentic wild-type. It was reported that the level of attenua-
tion was greater than desired, resulting in sub-optimal vaccine
virus replication in ferrets and incomplete protection following
challenge with a wild-type virus. However, in that study only six
animals were challenged and two succumbed to the infection. We
hypothesized that in the ferret model, formulation of the vaccine
vaccination efficacy after a single IN administration of the rCDV
which, in turn, would result in enhanced CDV-specific immune
responses and thus better protection.
2. Material and methods
2.1. Ethical statement
The animal protocol was approved by an independent ani-
mal experimentation ethics committee prior to the start of the
experiments. All experiments were performed in compliance with
European guidelines for animal experimentation (EU Directive
2010/63/EU). Animals were monitored daily, and animal care was
in compliance with institutional guidelines.
Twenty-four 12-month-old unvaccinated female European fer-
rets (Mustela putorius furo) were purchased from a breeding farm
in the Netherlands. Animals were tested for the presence of
method see below). A temperature probe was inserted intraperi-
toneally two weeks before the beginning of the experiments to
monitor body temperatures non-invasively. Following infection
animals were housed in groups of six in negatively pressurized,
HEPA-filtered biosafety level 3 (BSL-3) isolator cages and received
food and water ad libitum.
Lymph nodes were collected from control ferrets in unrelated
approved animal experiments prior to vaccination. These were
stored in phosphate-buffered saline (PBS) for a maximum time
of 4h, after which single cell suspensions were prepared using
cell strainers with a 100?m pore size (BD Biosciences). Cells
were resuspended in RPMI-1640 medium (Lonza) supplemented
with l-glutamine (2mM), penicillin (100U/ml), streptomycin
Sigma–Aldrich) and used directly for virus infections.
African green monkey kidney cells transfected with canine sig-
naling lymphocyte activation molecule (VeroDogSLAM cells) 
and were cultured in Dulbecco’s Modified Eagle Medium (DMEM;
Lonza) supplemented with l-glutamine, penicillin, streptomycin
and 10% (v/v) FBS.
The live-attenuated rCDV strain, rCDV5804PL(CCEGFPC), and the
challenge rCDV Snyder-Hill (SH) strain expressing the red flu-
orescent protein dTomato, rCDVSHdTom(6), had been generated
previously [11,12]. A challenge virus expressing a fluorescent pro-
tein different from EGFP was used to avoid interfering specific
T-cell responses directed against the fluorescent protein follow-
ing challenge. Virus titers were calculated by the Reed and Muench
method at 3.5×105and 3.5×10650% tissue culture infective dose
Pam3CSK4, PHCSK4, Pam-Cys-SK4, and Pam3CSP4 were a kind
gift of Dr. K.H. Wiesmüller (EMC microcollections, Germany) and
their molecular structures have been previously reported .
Stocks were prepared in distilled water at 1mg/ml.
2.6. Ex vivo CDV infection of lymphocytes
The lipopeptidesPam3CSK4,PHCSK4, Pam-Cys-SK4or
Pam3CSP4 were added to the viruses to a final concentration
of 10?g/ml, and the mixtures were incubated for 5min at 37◦C to
allow binding of the lipopeptides to the viruses. Ferret lymph node
cells were infected with rCDV5804PL(CCEGFPC) or rCDVSHdTom(6),
at a multiplicity of infection (MOI) of 0.1. One day post infection
(d.p.i.) cells were analyzed for fluorescence by flow cytometry .
2.7. In vivo CDV infection experiment
The study included four groups of six ferrets. Groups 1–3 were
vaccinated IN with 200?l (100?l in each nostril) PBS containing
3×104TCID50rCDV5804PL(CCEGFPC). In two of these groups, the
virus was mixed with lipopeptide (group 2: Pam3CSK4 or group 3:
PHCSK4, 30?g/ml). The mixture was incubated for 5min at 37◦C.
The fourth group was mock-vaccinated and served as controls in
the challenge experiment, which followed six weeks after vacci-
nation. For challenge infection, animals received rCDVSHdTom(6)
IN (3.5×105in 200?l) and intra-tracheally (IT, 3.5×104TCID50in
The total study lasted 69 days (Fig. 1). Nasopharyngeal lavages
were collected on days −1, 2, 4, 7, 11, and 14 after vaccination.
collected at the same time points and additionally on days 21, 28,
35 and 43 after vaccination. The endpoints of the study were body
weight loss (>15% in two days or >20% of total body weight [bw]),
severe respiratory or cardiac complications, or abnormal behav-
ioral changes. All remaining animals were euthanized 26 days after
onized with atipamezole (0.005ml/kgbw).
D.T. Nguyen et al. / Vaccine 30 (2012) 5073–5080
X-axis: time in days post vaccination
= collection of blood
= collection of nasopharyngeal lavage
= collection of throat swab
C2 # % +
C5 # % +
C7 # % +
C10 # % +
C12 # % +
C14 # % +
C19 # % +
C21 # % +
C23 # % +
C26 # % +
Fig. 1. Study design of in vivo experiment. Time schedule for the vaccination and challenge period. Numbers represent sampling time points in days post vaccination,
C-numbers in days post challenge infection. Sampling time points of blood, nasopharyngeal lavages and throat swabs are depicted by #, % and +, respectively.
2.8. Clinical samples
Nasopharyngeal lavages were obtained by flushing one nostril
with 1ml PBS, collecting the wash from the opposing nostril and
adjusting the total volume to 650?l. Throat swabs were collected
by brushing the back of the oro-pharyngeal cavity, after which
the brush (Medscand Medical, Cytobrush Plus cell collector) was
placed in 1.5ml virus transport medium (EMEM with Hanks’ salts,
supplemented with lactalbumin enzymatic hydrolysate [0.5g/ml],
[50U/ml], gentamicin [2.5mg/ml] and glycerol [10% (v/v)]). Sam-
ples were vortexed, after which 200?l was combined with 200?l
high pure RNA lysis buffer (Roche Diagnostics), the remainder was
used directly for virus isolations. Blood samples (approximately
700?l per animal per time point) were collected in tubes con-
taining K3EDTA as an anticoagulant. Plasma was separated from
the blood by centrifugation, heat inactivated at 56◦C for 30min
and stored at −20◦C. Peripheral blood mononuclear cells (PBMC)
were isolated from blood by density gradient centrifugation. Cells
were resuspended in 700?l complete RPMI 1640 medium, supple-
mented with l-glutamine, penicillin, streptomycin, and FBS. Cell
suspensions were used for RT-PCR (200?l), virus isolation (400?l)
or flow cytometry for direct detection of EGFP- or dTom-positive
2.9. Virus isolations
VeroDogSLAM (2×104cells/well) were seeded in flat bottom
96-wells plates one day before sampling. Clinical samples were
divided over the wells of the first column, followed by three-fold
serial dilution over the complete plate. Plates with throat and
nasopharyngeal samples were spinoculated at 1000×g for 15min.
After 1h incubation at 37◦C, supernatant was removed and DMEM
(100?l) supplemented with l-glutamine, penicillin, streptomycin,
fluorescence 5–7d.p.i., and titers expressed in TCID50/ml, which
were calculated by the Reed and Muench method.
2.10. TaqMan RT-PCR
The presence of CDV genomic RNA in nasopharyngeal lavages
was determined by RT-PCR, using primers in the nucleocap-
sid (N) gene as previously described with slight modifications
. Primers used for amplification were as follows: forward
nucleotides 2216–2229), forward 5?-GTCGGTAATCGAGGATTCG-
AGAG-3?(rCDVSHdTom(6)-N, nucleotides 2216–2229) and reverse,
rCDVSHdTom(6)-N, nucleotides 2297–2319). Sequence for the
TaqMan probe was 5?-FAM-AGCACTGAGGATTCTGGCGAAGAT-
nucleotides 2251–2274). The cycle threshold (Ct) value was
calculated automatically when the FAM-specific CDV signal was
above the background and was used to give a semi-quantitative
indication of viral loads.
Animals were euthanized by exsanguination under anesthe-
sia. Macroscopic detection of EGFP and dTom was performed at
necropsy as previously described [12,15]. Briefly, tissues were illu-
minated with custom-made blue or green lamps containing six
LEDs mounted with the appropriate excitation filters for EGFP and
were used as emission filters. Photographs were made using a
Nikon D80 SLR camera.
2.12. Virus neutralization assay
Two-fold serial dilutions of heat inactivated plasma (50?l)
in flat bottom 96 wells plates were mixed with 150TCID50of
rCDVSHdTom(6) (50?l/well) and incubated for 1h at 37◦C. Next,
2×106VeroDogSLAM cells/plate (100?l/well) were added at a
final FBS concentration of 2% (v/v). Infection was screened by flu-
orescence microscopy for 5–7 days. Neutralizing antibody titers
were calculated by the Reed and Muench method. Results are
shown as the reciprocal2log virus neutralizing (VN) antibody titer.
2.13. Statistical analysis
Ex vivo infections were performed three times, using tripli-
to the same control, differences were compared using 1-Way
ANOVA with Dunnett’s Correction for multiple comparisons. For
in vivo data, differences between the groups in body temperatures,
body weights, virus loads, or VN antibody titers were compared
with area under the curve (AUC) integrated using the trapezoidal
D.T. Nguyen et al. / Vaccine 30 (2012) 5073–5080
rule and statistically tested with Mann–Whitney U tests. Data are
expressed as mean±standard error of the mean (SEM) or geomet-
A two-sided (uncorrected) p-value<0.05 was considered statisti-
cally significant. All statistical analyses were performed with SPSS
version 17 software (SPSS, Inc., Chicago, USA).
3.1. Lipopeptide-mediated enhancement of in vitro CDV infection
of ferret lymph node cells
Three synthetic cationic bacterial lipopeptides were previously
described to enhance HRSV, HMPV, MV and HIV-1 infection in vitro
in different primary cells and cell lines . To determine if these
lipopeptides also enhanced in vitro CDV infection, ferret lymph
node cells were infected with the rationally-attenuated wild-type
stain, rCDV5804PL(CCEGFPC) , or the pathogenic CDV strain
rCDVSHdTom(6) , in the presence or absence of lipopeptide
(10?g/ml). Pam3CSK4 and PHCSK4 significantly enhanced infec-
tion with both CDV strains as compared to the medium and
lipopeptide control (Fig. 2, p<0.05) and were selected as potential
adjuvants for the rationally attenuated rCDV.
3.2. Vaccination – clinical signs
Four groups of six ferrets were IN vaccinated with rCDV with or
without lipopeptides as adjuvants (or mock-vaccinated with PBS).
Limited changes in body temperature were recorded during the
vaccination period for groups 2, 3, and 4 (see Fig. 3). Three animals
in group 1 experienced fever (>1.5◦C temperature rise) for three
of group 2 and one of group 3 also experienced subfebrile periods
of a week. However, this temperature increase was one week later
and the duration was shorter in comparison to group 1.
All ferrets lost some weight in the first few days after vaccina-
tion (Fig. 3, dashed lines). Over the next month the body weights
of the majority of the animals stabilized or increased, and no
statistically significant differences were observed between the
Fig. 2. Lipopeptide-mediated enhancement of CDV infection in ferret lymph node
cells. Two CDV strains (rCDV5804PL(CCEGFPC) or rCDVSHdTom(6)) were incubated
with four different lipopeptides (10?g/ml) for 5min at 37◦C before the virus sus-
pension was added to ferret lymph node cells. Formulation with Pam3CSK4 or
PHCSK4 resulted in significantly increased rCDV infection percentages (bars rep-
resent mean±SEM, *p<0.05 in 1-way ANOVA and post hoc Dunnett’s correction for
multiple comparisons with medium control).
One animal, vaccinated with rCDV5804PL(CCEGFPC) formulated
bacterial jaw abscess as the cause of death. A similar, more super-
ficially located abscess was observed an animal from group 4 and
in several other ferrets not included in the present study but from
the same source. Therefore, it was considered unlikely that either
the abscess or the death were related to the virus or lipopeptide.
3.3. Vaccination – virus loads and VN titers
Vaccine virus loads in nasopharyngeal lavages and PBMC were
detected by virus isolation (Fig. 4A and C) or Taqman RT-PCR
(Fig. 4B). Virus could be isolated from 7 days post vaccination
(d.p.v.) onwards, peaked at 11d.p.v., and decreased afterwards. At
14d.p.v. vaccine virus was isolated from one animal of the control
group. For the PHCSK4-adjuvanted group volume-adjusted virus
titers were significantly higher compared to the mock controls for
the whole sample period (p<0.05). For all groups viral genomes
were detected in nasopharyngeal lavages at 7d.p.v., increased
between 7 and 11d.p.v., and stabilized at the final nasopharyngeal
lavage sample day.
Vaccine virus could also be isolated from PBMC from 7d.p.v.
onwards (Fig. 4C). At 14d.p.v. virus in PBMC was detected in one
animal of groups 1 and 2, and 4 animals of group 3. Significantly
VN antibodies were first detected 11d.p.v. in n=3, n=5 and
tially until 21d.p.v. and stabilized afterwards (Fig. 4D). All ferrets
in groups 1, 2 and 3 had seroconverted by day 14. Neutraliz-
ing antibody titers of the lipopeptide-adjuvanted groups were on
average 2.3 fold higher compared to animals vaccinated with non-
adjuvanted virus (geometric mean VN titers measured during the
period 21–43d.p.v.: group 1: 113, group 2: 260, and group 3: 263).
For the complete vaccination period VN antibody titers in groups 2
and 3 were significantly higher than those of group 1 (p<0.05).
3.4. Challenge – virus loads in mock-vaccinated animals
Forty-three d.p.v., all vaccinated animals (n=17) and six mock-
vaccinated control animals were infected with rCDVSHdTom(6) by
both IN (3.5×105TCID50) and IT (3.5×104TCID50) inoculation.
During the challenge period virus isolations from throat swabs,
nasal lavages and PBMC were performed in VeroDogSLAM cells
(Fig. 5). Up to 6.3×106TCID50/ml was isolated from the throat
swabs. PBMC were analyzed in flow cytometry to determine the
percentage of infected cells. A significant viremia was observed
and up to 60% of lymphocytes were infected at the peak of
infection (7d.p.i.). Macroscopically, red fluorescence produced in
rCDVSHdTom(6)-infected cells was first observed 5d.p.i. on the
throat/lower jaw and at later time-points on other body parts, like
the conjunctivae of the eyes, rectum, skin (systemic) and footpads.
The rapid deterioration of the condition of all mock-vaccinated
animals after infection was also evidenced by fever and weight
loss (Fig. 6A and B). Recorded body temperatures showed a typical
biphasic (sub)febrile period in mock-vaccinated control animals.
One animal was euthanized ten d.p.i., the remaining animals were
euthanized at 14d.p.i.
3.5. Challenge – protective immunity in all vaccinated ferrets
IN vaccination with rCDV5804PL(CCEGFPC) protected all ferrets
against lethal challenge infection with rCDVSHdTom(6), irrespec-
tive of the formulation in lipopeptides. No significant differences
were observed in behavioral changes, body temperatures (Fig. 6A),
body weights (Fig. 6B), or virus detection. One animal of group 1
D.T. Nguyen et al. / Vaccine 30 (2012) 5073–5080
Fig. 3. Vaccination – body temperatures and body weights. Solid lines with round symbols represent the body temperatures (mean±SEM of six ferrets, left y-axis; group 3:
five ferrets). In contrast to the adjuvanted groups, the vaccinated control showed an average body temperature increase during the second week after vaccination. Dashed
lines with triangle symbols correspond to percentages of initial body weight (mean±SEM, right y-axis). Body weight losses were comparable for all groups.
not observe a strong secondary humoral immune response during
the challenge period (Fig. 6C).
Two infection-enhancing lipopeptides  were evaluated as
potential adjuvants for live-attenuated paramyxovirus vaccines
using a CDV vaccination and challenge model in ferrets. The
rationally attenuated CDV vaccine delivered by the IN route was
very effective in inducing protective immunity, indeed more so
than expected on basis of a previous study . All vaccinated
ferrets were fully protected against a high dose lethal IN and IT
challenge with a highly virulent wild-type virus, irrespective of the
presence or absence of lipopeptides. However, vaccination with
lipopeptide-formulated live-attenuated rCDV elicited statistically
significantly higher vaccine virus loads and VN antibody titers.
This is the first demonstration of the in vivo infection-enhancing
properties of Pam3CSK4 and PHCSK4, and proves their potential as
adjuvants for needle-free paramyxovirus vaccination.
Currently there are no licensed adjuvants for live-attenuated
D.T. Nguyen et al. / Vaccine 30 (2012) 5073–5080
Fig. 4. Vaccination – virus loads and neutralizing antibody titers. (A) Vaccine virus loads in nasopharyngeal lavages as measured by virus isolation. Virus loads in the
PHCSK4-adjuvanted group were statistically significantly higher compared to the non-adjuvanted group (lines represent geometric mean±GSD, *p<0.05). (B) Vaccine
virus loads in nasopharyngeal lavages as measured by Taqman RT-PCR (lines represent mean±SEM). (C) Vaccine virus loads in PBMC as measured by virus isolation. Virus
loads in the PHCSK4-adjuvanted group were statistically significantly higher compared to the non-adjuvanted group (lines represent geometric mean±GSD, *p<0.05).
(D) Virus neutralizing (VN) antibody titers were measured between 0 and 43 days post vaccination. At 11d.p.v. the first ferrets seroconverted. Ferrets vaccinated with
lipopeptide-adjuvanted CDV vaccine mounted significantly higher VN antibody titers (lines represent geometric mean±GSD, *p<0.05, AUC).
(alpha-GalCer) stimulates natural killer (NK) T-cells modulat-
ing cells of the adaptive immune response . Alpha-GalCer can
be used as an adjuvant for both live recombinant vaccines and
inactivated vaccines [17,18]. Alpha-GalCer was successfully tested
as an adjuvant for a live-attenuated influenza virus vaccine, but the
mode of action is different than the proposed lipopeptide adjuvant
The lipopeptide Pam3CSK4 has been tested as an adjuvant in
peptide vaccination studies, based on its TLR-activating properties
[19,20] being covalently conjugated to synthetic peptides contain-
ing the T- or B-lymphocyte epitopes [20,21]. It also functions as an
adjuvant in DNA and virosomal immunization studies [22,23]. The
mode of action uses the TLR activating properties of Pam3CSK4 to
elicit powerful innate immune responses, thereby modulating the
adaptive immune responses. However, we used the lipopeptides
in a fundamentally different manner, namely to enhance infectiv-
ity of a live-attenuated virus. This could increase the number of
host cells in which the virus can replicate, thereby leading to larger
Fig. 5. rCDVSHdTom(6) infection of control ferrets. Bars correspond to rCDVSHdTom(6) virus loads in throat swabs, nasopharyngeal lavages and PBMC collected from ferrets
of group 4. The black line with round white symbols shows the percentages of dTom+lymphocytes as measured by flow cytometry in PBMC (bars and symbols represent
D.T. Nguyen et al. / Vaccine 30 (2012) 5073–5080
Fig. 6. rCDVSHdTom(6) challenge infection: body temperature, body weights, and
VN serum antibody responses. (A) Symbols represent the body temperatures of the
this was more dramatic in the mock-vaccinated than in the vaccinated animals. (C)
During the challenge period almost no secondary humoral immune response was
observed. Overall, average VN antibody titers of groups 2 and 3 remained higher
compared to those of group 1 (geometric mean±GSD, *p<0.05, AUC).
amounts of viral antigen produced. In addition, the lipopeptide
could potentially decrease the percentage of non-responders when
giving a live-attenuated paramyxovirus vaccine via the intra-nasal
route, or could be used for dose-sparing of vaccines that are hard to
enhancing lipopeptides could also result in an effect equivalent to
partial vaccine virus de-attenuation. Therefore, clinical evaluation
and assessment of tolerability will be crucial.
Initially, it was planned that vaccinated ferrets would only be
cine virus was over-attenuated. However, as VN antibodies to CDV
lead to protection [4,24], and we observed high VN antibody levels
in all vaccinated ferrets, it was decided to administer a higher dose
of the virus via both the IN and IT route and thus provide a stronger
challenge for the protective immune response.
All vaccinated ferrets lost weight after challenge infection,
which was consistent with a previous study  in which
ferrets lost weight for 16 days for an unknown reason without any
lipopeptides being used. Virus isolations from all groups 9d.p.i.
(data not shown) suggest that despite the presence of protective
immunity the challenge virus may have caused a subclinical infec-
The ultimate objective is to achieve a similar effect of these
lipopeptides on a live-attenuated HRSV vaccine. Despite the high
global burden of HRSV disease, no licensed vaccines against HRSV
vaccines are these should be safe and effective at an early age in the
presence of maternal antibodies and an immature immune system
. Over the last few decades several candidate live-attenuated
RSV vaccines have been developed for IN vaccination, but is has
proven difficult to find a proper balance between attenuation and
There are significant differences in the pathogenesis of and
immune response to morbilliviruses and pneumoviruses. Morbil-
liviruses initially infect lymphocytes, macrophages and dendritic
cells in the airways [3,29]. Subsequently, the viruses cause viremia
and systemic disease. Therefore, pre-existing VN antibodies in the
bloodstream can effectively inhibit infections [4,24]. Dependent on
the CDV strain, infections in dogs and ferrets are associated with
mortality of 30–100% and in animals that survive, the resulting
immunity is normally life-long. The same is true for measles in
humans. In contrast, HRSV mainly infects respiratory epithelial cell
HRSV infections, despite high VN antibody titers in blood. Unlike
for morbilliviruses, VN antibodies to HRSV wane to pre-infection
levels within one year [30,31].
In conclusion, we have shown that the infection-enhancing
lipopeptides Pam3CSK4 and PHCSK4 can enhance CDV infection
We thank Stephen McQuaid, Queen’s University of Belfast,
United Kingdom, and all zootechnicians at the isolator facility of
the Netherlands Vaccine Institute in Bilthoven, the Netherlands,
for their contributions to this study. This study received financial
support from the VIRGO project and from MRC (grant# G0801001).
ing sources had no involvement in study design or data analysis.
 Harder TC, Osterhaus ADME. Canine distemper virus – a morbillivirus in search
of new hosts. Trends Microbiol 1997;5:120–4.
in rhesus monkeys, China. Emerg Infect Dis 2011;17:1541–3.
 Von Messling V, Milosevic D, Cattaneo R. Tropism illuminated: lymphocyte-
based pathways blazed by lethal morbillivirus through the host immune
system. Proc Natl Acad Sci U S A 2004;101:14216–421.
 BeinekeA, PuffC,Seehusen F,
immunopathology of systemic and nervous canine distemper. Vet Immunol
 Nguyen DT, De Witte L, Ludlow M, Yüksel S, Wiesmuller K-H, Geijtenbeek
TBH, et al. The synthetic bacterial lipopeptide Pam3CSK4 modulates respi-
ratory syncytial virus infection independent of TLR activation. PLoS Pathog
 Takeuchi O, Sato S, Horiuchi T, Hoshino K, Takeda K, Dong Z, et al. Cutting
edge: role of Toll-like receptor 1 in mediating immune response to microbial
lipoproteins. J Immunol 2002;169:10–4.
 Jin MS, Kim SE, Heo JY, Lee ME, Kim HM, Paik SG, et al. Crystal structure of the
TLR1-TLR2 heterodimer induced by binding of a tri-acylated lipopeptide. Cell
 De Jong MAWP, De Witte L, Oudhoff MJ, Gringhuis SI, Gallay P, Geijtenbeek
TBH. TNF-alpha and TLR agonists increase susceptibility to HIV-1 transmission
by human Langerhans cells ex vivo. J Clin Invest 2008;118:3440–52.
5080 Download full-text
D.T. Nguyen et al. / Vaccine 30 (2012) 5073–5080
 Ogawa Y, Kawamura T, Kimura T, Ito M, Blauvelt A, Shimada S. Gram-positive
bacteria enhance HIV-1 susceptibility in Langerhans cells, but not in dendritic
cells, via Toll-like receptor activation. Blood 2009;113:5157–66.
 Thibault S, Fromentin R, Tardif MR, Tremblay MJ. TLR2 and TLR4 triggering
exerts contrasting effects with regard to HIV-1 infection of human dendritic
cells and subsequent virus transfer to CD4+ T cells. Retrovirology 2009;6:42.
 Silin D, Lyubomska O, Ludlow M, Duprex WP, Rima BK. Development of a
challenge-protective vaccine concept by modifying the viral RNA-dependent
RNA polymerase of canine distemper virus. J Virol 2007;81:13649–58.
 Ludlow M, Nguyen DT, Silin D, Lyubomska O, De Vries RD, Von Messling V,
et al. Recombinant canine distemper virus strain Snyder Hill expressing green
or red fluorescent proteins causes meningoencephalitis in the ferret. J Virol
 Seki F, Ono N, Yamaguchi R, Yanagi Y. Efficient isolation of wild strains of
canine distemper virus in Vero cells expressing canine SLAM (CD150) and their
adaptability to marmoset B95a cells. J Virol 2003;77:9943–50.
 Scagliarini A, Dal Pozzo F, Gallina L, Vaccari F, Morganti L. TaqMan based real
time PCR for the quantification of canine distemper virus. Vet Res Commun
 De Swart RL, Ludlow M, De Witte L, Yanagi Y, Van Amerongen G, McQuaid S,
et al. Predominant infection of CD150+ lymphocytes and dendritic cells during
measles virus infection of macaques. PLoS Pathog 2007;3:e178.
 Van Kaer L. Alpha-Galactosylceramide therapy for autoimmune diseases:
prospects and obstacles. Nat Rev Immunol 2005;5:31–42.
 Ko SY, Ko HJ, Chang WS, Park SH, Kweon MN, Kang CY. Alpha-
Galactosylceramide can act as a nasal vaccine adjuvant inducing pro-
tective immune responses against viral infection and tumor. J Immunol
vaccine. Vaccine 2009;27:3766–74.
 BenMohamed L, Wechsler SL, Nesburn AB. Lipopeptide vaccines – yesterday,
today, and tomorrow. Lancet Infect Dis 2002;2:425–31.
 Deres K, Schild H, Wiesmuller KH, Jung G, Rammensee HG. In vivo priming
of virus-specific cytotoxic T lymphocytes with synthetic lipopeptide vaccine.
 BenMohamed L, Gras-Masse H, Tartar A, Daubersies P, Brahimi K, Bossus M,
et al. Lipopeptide immunization without adjuvant induces potent and long-
lasting B, T helper, and cytotoxic T lymphocyte responses against a malaria
liver stage antigen in mice and chimpanzees. Eur J Immunol 1997;27:1242–53.
 Jayakumar A, Castilho TM, Park E, Goldsmith-Pestana K, Blackwell JM, Mahon-
Pratt D. TLR1/2 activation during heterologous prime-boost vaccination (DNA-
(Viannia). PLoS Negl Trop Dis 2011;5:e1204.
 Stegmann T, Kamphuis T, Meijerhof T, Goud E, De Haan A, Wilschut
J. Lipopeptide-adjuvanted respiratory syncytial virus virosomes: A safe
and immunogenic non-replicating vaccine formulation. Vaccine 2010;28:
immunity in dogs and cats. J Comp Pathol 2010;142:S102–8.
 Stephensen CB, Welter J, Thaker SR, Taylor J, Tartaglia J, Paoletti E. Canine
distemper virus infection of ferrets as a model for testing morbillivirus vac-
cine strategies: NYVAC-and ALVAC-based CDV recombinants protect against
symptomatic infection. J Virol 1997;71:1506–13.
 Nair H, Nokes DJ, Gessner BD, Dherani M, Madhi SA, Singleton RJ, et al.
Global burden of acute lower respiratory infections due to respiratory syn-
cytial virus in young children: a systematic review and meta-analysis. Lancet
 Collins PL, Melero JA. Progress in understanding and controlling respira-
tory syncytial virus: still crazy after all these years. Virus Res 2011;162:
 Graham BS. Biological challenges and technological opportunities for respira-
tory syncytial virus vaccine development. Immunol Rev 2011;239:149–66.
 Lemon K, De Vries RD, Mesman AW, McQuaid S, Van Amerongen G, Yüksel S,
et al. Early target cells of measles virus after aerosol infection of non-human
primates. PLoS Pathog 2011;7:e1001263.
 Falsey AR, Walsh EE, Looney RJ, Kolassa JE, Formica MA, Criddle MC, et al.
Comparison of respiratory syncytial virus humoral immunity and response to
infection in young and elderly adults. J Med Virol 1999;59:221–6.
 Falsey AR, Singh HK, Walsh EE. Serum antibody decay in adults follow-
ing natural respiratory syncytial virus infection. J Med Virol 2006;78: