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Sample 4
Sample 3
Sample 2 Brisbane B
California H1N1
Reference
Perth H3N2
VaxigripOutletInlets
Sample 1
Day 7 Day 28 Day 90 Day 150
Vaxigrip
0
100
200
300
400
500
600
700
800
Vaxigrip/Matrix-M
Vaxigrip
0
100
200
300
400
500
600
700
800
Vaxigrip/Matrix-M
Vaxigrip
0
100
200
300
400
500
600
Vaxigrip/Matrix-M
Vaxigrip
-50
-100
200
150
100
50
0
250
300
350
400
450
500
550
Vaxigrip/Matrix-M
Nor
mal
ized
rel
ativ
e re
spon
se (R
U)
Mean
95%ConfidenceInterval
Outside values
Median
Lower quartile
Upper quartile
Upper adjacentvalue
Lower adjacentvalue
0
500
1000
1500
2000
2500
3000
3500
4000
4500
0 100 200 300 400 500 600
Brisbane B
California H1N1Perth H3N2
Vaxigrip
×
×
××
Time (s)
Rela
tive
resp
onse
(RU
)
Day
10
23
34
45
54
68
76
87
11
24
37
48
57
6
7
9
12
25
38
49
59
70
80
17
30
40
P4
60
72
81
1
31
42
50
61
73
84
20
32
44
53
67
74
86
0250500
0250500
0
250500
0250500
0250
500
0
250500
0250500
0250
500
0 50 100
150 0 50 100
150 0 50 100
150 0 50 100
150 0 50 100
150 0 50 100
150
Nor
mal
ized
rel
ativ
e re
spon
se (R
U)
Day
0 50 100
150 0 50 100
150 0 50 100
150 0 50 100
150 0 50 100
150 0 50 100
150
Nor
mal
ized
rel
ativ
e re
spon
se (R
U)
13
21
2
41
55
64
77
88
14
22
33
43
56
65
78
8
15
26
35
46
58
66
79
16
27
36
47
5
69
82
18
28
39
51
62
71
83
19
29
3
52
63
75
85
0250500
0250500
0
250500
0250500
0250
500
0
250500
0250500
0250
500
Hig
her
stab
ility
0
0.0005
0.0010
0.0015
0.0020
0.0025
0 50 100 150
kd (s
-1)
Day
Indivdual 14
Indivdual 15
Indivdual 41
Indivdual 56
Indivdual 65
0
1000
2000
3000
4000
5000
6000
150 350 550 750 950
Rela
tive
resp
onse
(RU
)
Time (s)
Sample
1st reagent
2nd reagent
Regenerations
-200
0
200
400
600
800
1000
1200
1400
0 50 100 150
Nor
mal
ized
rel
ativ
e re
spon
se (R
U)
Day
Individual 14
IgA IgG4 IgM IgG1 IgG2 IgG3
-100
0
100
200
300
400
500
600
0 50 100 150
Nor
mal
ized
rel
ativ
e re
spon
se (R
U)
Nor
mal
ized
rel
ativ
e re
spon
se (R
U)
Day
Individual 15
Individual 41
100
0
100
200
300
400
500
600
700
0 50 100 150
Nor
mal
ized
rel
ativ
e re
spon
se (R
U)
Day
Individual 56
-100
0
100
200
300
400
500
600
700
0 50 100 150
Nor
mal
ized
rel
ativ
e re
spon
se (R
U)
Day
Individual 65
-100
0
100
200
300
400
500
0 50 100 150
Nor
mal
ized
rel
ativ
e re
spon
se (R
U)
Day
-50
0
50
100
150
200
250
300
350
400
450
0 50 100 150
Nor
mal
ized
rel
ativ
e re
spon
se (R
U)
Day
0
50
100
150
200
250
300
0 50 100 150
Nor
mal
ized
rel
ativ
e re
spon
se (R
U)
Day
-10
0
10
20
30
40
50
60
70
0 50 100 150
Nor
mal
ized
rel
ativ
e re
spon
se (R
U)
Day
Day
-100
0
100
200
300
400
500
600
700
0 50 100 150
Nor
mal
ized
rel
ativ
e re
spon
se (R
U)
Day
-10
0
10
20
30
40
50
60
70
0 50 100 150
Nor
mal
ized
rel
ativ
e re
spon
se (R
U)
Day
-20
0
20
40
60
80
100
120
140
0 50 100 150
Nor
mal
ized
rel
ativ
e re
spon
se (R
U)
Day
0
20
40
60
80
100
120
140
160
0 50 100 150
Nor
mal
ized
rel
ativ
e re
spon
se (R
U)
Day
-200
0
200
400
600
800
1000
1200
1400
1600
0 50 100 150
Nor
mal
ized
rel
ativ
e re
spon
se (R
U)
Day
Brisbane surface California surface Perth surface Vaxigrip surface
-500
0
500
1000
1500
2000
2500
0 50 100 150
Nor
mal
ized
rel
ativ
e re
spon
se (R
U)
Day
-100
0
100
200
300
400
500
600
0 50 100 150
Nor
mal
ized
rel
ativ
e re
spon
se (R
U)
Day
-100
0
100
200
300
400
500
600
700
800
0 50 100 150
Nor
mal
ized
rel
ativ
e re
spon
se (R
U)
Day
-200
0
200
400
600
800
1000
1200
1400
0 50 100 150
Nor
mal
ized
rel
ativ
e re
spon
se (R
U)
Day
-200
0
200
400
600
800
1000
1200
1400
1600
0 50 100 150
Nor
mal
ized
rel
ativ
e re
spon
se (R
U)
Day
0
100
200
300
400
500
600
0 50 100 150
Nor
mal
ized
rel
ativ
e re
spon
se (R
U)
Day
-500
0
500
1000
1500
2000
0 50 100 150
Nor
mal
ized
rel
ativ
e re
spon
se (R
U)
Day
Day
0 50 100
150
Brisbane B
California H1N1
Perth H3N2
Vaxigrip
GE, imagination at work, and GE monogram are trademarks of General Electric Company. Biacore is a trademark of GE Healthcare companies. Vaxigrip is a trademark of Sanofi Pasteur MSD. Matrix-M is a trademark of Isconova AB. © 2013 General Electric Company – All rights reserved. First published May 2013. All goods and services are sold subject to terms and conditions of sale of the GE Healthcare Company which supplies them. A copy of these terms and conditions are available on request. Contact your GE Healthcare representative for the most current information and a copy of the terms and conditions. GE Healthcare Bio-Sciences AB, Björkgatan 30, 751 84 Uppsala, Sweden.
29-0560-05 AA 05/2013
Fig 1. Schematic figure of assay setup in Biacore 4000 with four parallel flow cells, each with immobilized Vaxigrip and three different HA. Four injections in parallel provide results for binding to Brisbane, California, Perth and Vaxigrip for four samples. The samples were diluted 10x and injected for 400s. Binding to immobilized HA and vaccine was recorded 7s after the end of the sample injection as shown in the sensorgram. The surfaces were regenerated after each sample injection.
Fig 4. Two subclass reagents were injected per cycle after the sample and binding responses were recorded.
Fig 2. Response profiles showing response over time for each individual in the two groups. All sample responses were normalized by subtraction of the response from the day-0 sample from the same individual. Normalization against negative and positive control samples was also done.
Fig 5. Isotyping results from five individuals.
Fig 3. Examples of statistical analysis of the anti-Brisbane responses from the two groups. The comparison circles display whether the group means for all pairs are significantly different from each other or not.
Fig 6. Dissociation rate of anti-Brisbane antibodies.
Efficacy of a novel adjuvant demonstrated by SPRTanja Jarhede1, Anna Moberg1, Markku Hämäläinen1, Anita Larsson1, Robert Karlsson1, Brian Lang2, and Karin Lövgren3
1GE Healthcare, Uppsala, Sweden. 2GE Healthcare, Piscataway, NJ, USA. 3Isconova, Uppsala, Sweden
IntroductionSurface plasmon resonance (SPR) biosensors are used throughout the vaccine development process from vaccine discovery and optimization to process development and quality control. This poster describes screening of serum samples to monitor and characterize the antibody response following vaccination against influenza virus with and without addition of a novel adjuvant, Matrix-M™. A clinical study with the objective of assessing the efficacy of the vaccine adjuvant was performed by Isconova. Two groups of elderly people (ages 65-75) were given seasonal influenza vaccine (Vaxigrip®). One group received Vaxigrip together with Matrix-M adjuvant and a control group received Vaxigrip without adjuvant. Samples were taken prior to vaccination (day 0), and at day 7, 28, 90 and 150 after vaccination. Biacore™ systems were used to elucidate the magnitude, diversity and binding stability of the antibodies against Hemagglutinins (HA) and the whole vaccine.
ScreeningIn order to assess the effect of the adjuvant, 550 serum samples were analyzed with Biacore 4000 for their antibody response against HA from three strains of influenza (Brisbane B, California H1N1, Perth H3N2) and Vaxigrip. The parallel configuration of Biacore 4000 allows for simultaneous screening of the binding between four different serum samples and four immobilized HA/vaccine in each cycle (Fig 1).
Screening resultsBinding to HA and Vaxigrip were plotted against time (days) after vaccination for each individual (Fig 2). Individuals showed different responses to the different HA and Vaxigrip. Most of the individuals peaked in antibody response at day 28. Some of them maintained fairly high antibody responses throughout the study, but, in most cases, the antibody responses declined after day 28.
Statistical analysis of the screening resultsThe variation within each group was very large, as expected when working with serum samples from different individuals. However, statistical analysis of the Biacore data showed that there was a significant difference in immune response between the two groups. The group that was administrated vaccine with adjuvant developed statistically more anti-Brisbane, anti-California and anti-Vaxigrip antibodies at day 7 and 28 than the group that were provided with the regular vaccine formulation (Fig 3).
IsotypingIsotype determination was done on serum from five time points and five individuals. Analysis of the generated antibodies revealed a large individual variation in isotype pattern and response level against the different HA and Vaxigrip. IgG1 was the most common subclass but high levels of IgG3, IgM, IgG2 and IgA were also observed.
1st Class/subclass reagent
2nd Class/subclass reagent
Cycle 1 Anti-IgA Anti-IgG1
Cycle 2 Anti-IgG4 Anti-IgG2
Cycle 3 Anti-IgM Anti-IgG3
StabilityThe affinity of antibodies play a key role in protection against infection. Here, the affinity is measured as the dissociation rate of antibodies from immobilized HA. A small increase in affinity, that is a decrease in dissociation rate, could be seen for all individuals at day 7 and day 28 following vaccination. However the affinities of the antibodies from these elderly people were high already before vaccination which probably could explain why no strong antibody maturation was seen in the investigated samples.
Conclusions• Measurements of anti-HA antibodies give detailed information of the immune
response in vaccinated individuals.
• The immune response can easily be characterized with respect to class and subclass distribution.
• Dissociation rates indicate binding stability changes, possibly caused by class switch, maturation or avidity, and vary between individuals.
• The parallel analysis and high degree of automation make Biacore 4000 a valuable and efficient screening and characterization tool for vaccine development.
4 samples × 4 immobilized binding partners = 16 interactions/cycle
Biacore 4000
Sensor Chip
Elderly, no adjuvant
Elderly with adjuvant
Antibodies from different individuals showed different dissociation rates. This could be due to that the individuals had developed different isotypes which have different avidity, or different affinity maturation due to different previous exposure to influenza virus.
