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CAF01 adjuvant increases the protection conferred by a
commercially available influenza split vaccine in a ferret model
C.J.M. Martel a*, T.H. Jensen a, L.P. Nielsen b, E.M. Agger b, M. Blixenkrone-Møller a, P.
Andersen b, B. Aasted a
a Department
of Veterinary Disease Biology, Faculty of Life Sciences, University of Copenhagen, Stigbøjlen 7, DK-1870 Frederiksberg C, Denmark.
b Department of Infectious Disease Immunology, Statens Serum Institut, Copenhagen, Denmark.
Abstract:The immunogenicity and protective efficacy of current preventive vaccines against influenza are considered suboptimal, and the
development of novel effective influenza vaccination strategies is urgently needed. Commercially available trivalent split vaccines are known to elicit
mainly a humoral immune response, whereas the induction of cell-mediated immune responses is negligible. Recently, a cationic liposomal adjuvant
(CAF01, dimethyldioctadecylammonium/trehalose 6,6’-dibehenate) was developed. In the current study, we compared the immune response in ferrets
vaccinated with a commercially available influenza split vaccine with the same vaccine mixed with the CAF01 adjuvant and furthermore used two
recently circulating H1N1 viruses for the challenge of the animals. CAF01 improved the immunogenicity of the vaccine, increasing the influenzaspecific IgA and IgG levels as well as triggering cellular-mediated immunity, measured by flow cytometry as the production of interferon-gamma by
lymphocytes. The adjuvant also enhanced the protection conferred by the vaccine, reducing the viral load measured in nasal washes by RT-PCR. The
protection data obtained in the human relevant challenge model supports the potential of CAF01 in future influenza vaccines.
Material and methods:
•24 ferrets were immunized twice at two-weeks intervals, then challenged after a month of rest with 107 TCID of an homologous H1N1 virus.
•Vaxigrip: 8 ferrets, 2 x 80 µl commercial vaccine
•Vaccine + adjuvant group: 8 ferrets, 2 x 80 µl vaccine + 250 µl CAF01
•Control PBS group: 8 ferrets, 2 x 250 µl PBS
•Viral titers in nasal washes were measured by RT-PCR, Influenza-specific antibodies were detected in serum via ELISA using homemade rabbit antiferret IgG, PBL positive for intracellular gamma-IFN staining after PMA stimulation were analyzed by flow cytometry.
• In a similar experiment, 24 ferrets received various doses of influenza vaccine with or without CAF01 and then were challenged with a homologous
H1N1 virus.
Results and Conclusions
B.
HI titer
ge
e
ha
lle
n
of
c
ch
of
11
0
ay
ay
D
0.15 g
0.15 g + CAF01
1.5 g
1.5 g + CAF01
15 g
15 g + CAF01
Mock-vaccinated
ay
4
of
ch
al
le
ng
e
1.010 5
D
of
0
ay
D
st
im
af
te
r1
s
ee
k
w
Figure 2. Viral excretion A. Number of viral copies found
in nasal washes of infected animals measured by
quantitative RT-PCR. B. Number of copies in nasal
washes of individual infected animals at peak replication
day (day 3) C. Percentage of animals excreting the virus in
their nasal washes during the challenge period (cumulative
results of 3 similar experiments, for a total of 24 animals in
each group).
2
Figure 1. Immune response to vaccine antigens. A.
Serum IgG antibodies measured by IgG specific ELISA
B. Hemagglutination inhibition assay C. Percentage of
IFNg positive lymphocytes at week after 24 hours of
stimulation in cell culture with a recombinant H1
hemagglutinin
Number of viral copies
D.
0
e
C.
al
le
ng
6
ch
5
of
4
1.010 6
3
3
D
s
ee
k
2
w
ee
k
w
2
2
Days after challenge inoculation
1.010
ay
1
***
7
ge
Days after challenge inoculation
0
1.010 8
ha
lle
n
Day 3
***
*
of
c
Day 2
11
10 0
**
100
ay
10 1
50
1.010 9
0.15 g
0.15 g + CAF01
1.5 g
1.5 g + CAF01
15 g
15 g + CAF01
Mock-vaccinated
D
10 2
***
200
e
10
3
***
m
Time post-immunization
10 4
100
300
al
le
ng
6
W
ee
k
4
W
ee
k
2
k
W
ee
W
ee
k
0
0
10 5
Mock-vaccinated
Vaxigrip
Vaxigrip + CAF01
ch
***
1000
10
6
150
un
iz
at
io
n
2000
Mock-vaccinated
Vaxigrip
Vaxigrip + CAF01
10 7
al
le
ng
un
iz
at
io
n
e
0
D
af
te
r1
***
10 8
IgA titer in nasal washes
3000
Mock-vaccinated
Vaxigrip
Vaxigrip + CAF01
Animals excreting the virus (%)
***
Number of viral copies
4000
HI titer
m
D.
st
im
C.
s
B.
af
te
r1
Day 3 after challenge inoculation
ge
0
4
0
ha
lle
n
3
200
B.
0
st
im
Time post-immunization
2
400
50
A.
Days after challenge inoculation
*
of
c
W
1
100
0.15 g
0.15 g + CAF01
1.5 g
1.5 g + CAF01
15 g
15 g + CAF01
Mock-vaccinated
600
D
5000
150
11
10
1
*
*** ***
5000
ay
10 2
**
200
***
10000
0.15 g
0.15 g + CAF01
1.5 g
1.5 g + CAF01
15 g
15 g + CAF01
Mock-vaccinated
D
10 3
*
10000
al
le
ng
10 4
Mock-vaccinated
Vaxigrip
Vaxigrip + CAF01
ch
6
10 5
10 0
ee
k
4
W
ee
k
2
ee
k
W
W
ee
k
0
***
10 6
15000
of
***
***
Mock-vaccinated
Vaxigrip
Vaxigrip + CAF01
***
***
***
ay
5000
4000
3000
2000
1000
0
10
7
2500
2000
1500
1000
500
***
un
iz
at
io
n
***
10
**
m
Mock-vaccinated
Vaxigrip
Vaxigrip + CAF01
8
Number of viral copies
***
Number of viral copies
***
100000
80000
60000
40000
20000
IgG titer in serum
A.
IgG titers in serum
C.
A.
Figure 3. A/Brisbane/59/2007 study. A. Vaccine-specific
serum IgG titers measured in serum by ELISA. B.
Hemagglutination inhibition assay serum titers. C. Vaccinespecific IgA titers measured in nasal washes by ELISA. D.
Number of viral copies found in nasal washes of individual
infected animals at peak replication days (day 3 and 4),
measured by quantitative RT-PCR.
CAF01 enhances antibody and CMI responses of the split vaccine
significant increase of vaccine-specific IgG antibodies titers in serum (fig 1A and 1 B)
significant increase in the percentage of IFN-g positive lymphocytes (fig 1C)
CAF01-adjuvanted split vaccine confers protection against H1N1 influenza challenge
median number of viral copies 10 to 100-fold lower than vaccine only and the mock-vaccinated (fig 2A)
CAF01 allows for a reduction in vaccine dose
0.15 µg dose of vaccine adjuvanted with CAF01 reduced the viral load to the same level as observed with the unadjuvanted 15
µg dose. With the 15 µg dose, the adjuvant led to a 100-fold reduction of the viral load compared to the same dose of vaccine
without CAF01 (fig 3 D).