CVI Accepted Manuscript Posted Online 14 January 2015 Clin. Vaccine Immunol. doi:10.1128/CVI.00662-14 Copyright © 2015, American Society for Microbiology. All Rights Reserved.
1
The long-term immunogenicity of an inactivated split-virion 2009 pandemic
2
influenza A H1N1 vaccine with or without aluminium-adjuvantin mice
3 4
Wenting Xua,c, Mei Zhenga, Feng Zhoua, Ze Chena,b,*
5 6
Shanghai Institute of Biological Products, Shanghai 200052, Chinaa; College of Life
7
Sciences, Hunan Normal University, Changsha 410081, Hunan, Chinab; School of
8
Life Sciences, Fudan University, Shanghai 200433, Chinac
9 10
Running head: long-term effection of 2009 pandemic influenza vaccine
11 12
*Correspondence should be addressed to Ze Chen
13
E-mail: [email protected]; [email protected]
14
Phone/Fax: (00) 86-(0)21-62826658
15
Address: Shanghai Institute of Biological Products, Shanghai 200052, China
16
1
17
Abstract
18
In 2009, a global epidemic of influenza A (H1N1) caused the death of tens of
19
thousands of people. Vaccination is the most effective means of controlling
20
a wide-range epidemic of influenza and of reducing the mortality rate. In this study,
21
the long-term immunogenicity of A/California/7/2009 (H1N1) split vaccine was
22
observed as long as 15 months (450 days) after the immunization in mouse model.
23
Female BALB/c mice were immunized intra-peritoneally with different doses of
24
aluminum-adjuvanted vaccine. Four hundred fifty days after the immunization, the
25
mice were challenged with a lethal dose (10 × LD50) of homologous virus. The results
26
showed that the supplemented aluminum adjuvant could not only effectively enhance
27
the protective effect of the vaccine, but also reduce the immunizing dose of the
28
vaccine. In addition, the aluminum adjuvant could enhance the IgG antibody level of
29
mice immunized with H1N1 split vaccine. The IgG level was correlated to the
30
survival rate of the mice. Aluminum-adjuvanted inactivated split-virion 2009
31
pandemic influenza A H1N1 vaccine has good immunogenicity, and could provide
32
long-term protection against lethal influenza virus challenge in mice.
33 34
Keywords：2009 pandemic, influenza, split vaccine, long-term
2
35 36
Introduction Influenza is an acute respiratory disease caused by infection of the host respiratory tract by
37
influenza virus, which often spreads globally in seasonal epidemics, and can cause
38
a worldwide influenza pandemic. In 2009, a new influenza A (H1N1) virus caused the first
39
influenza pandemic of the 21st century [1,2]. By August 2010, the 2009 H1N1 influenza
40
virus had been diagnosed in the laboratories in more than 214 countries and regions around
41
the world, resulting in 18,449 deaths [3]. However that number is regarded as well below the
42
true total, mainly because many people who die of flu-related causes are not tested for the
43
disease. Working with admittedly sparse data, a research team led by the US Centers
44
for Disease Control and Prevention (CDC) has estimated the global death toll from the 2009
45
H1N1 influenza pandemic at more than 284,000, about 15 times the number of
46
laboratory-confirmed cases [4]. Vaccination is one of the most effective means of combatting
47
influenza virus infections and of reducing the mortality rate. Therefore, countermeasures
48
have been successively taken in various countries to develop a new monovalent vaccine
49
against influenza A (H1N1) for clinical trials [5,6]. The results of clinical trials show that
50
different types of monovalent vaccines, including the whole inactivated virus vaccine, split
51
vaccine, and attenuated live vaccine, have good safety, and can induce a robust immune
52
response, which conforms to the EU standards for seasonal influenza vaccine [7-10].
53
In order to prevent and control the spread of the 2009 H1N1 influenza virus in China, in
54
August 2009 we completed clinical trials across various age groups, in which all
55
subjects were immunized with the split vaccine, and the results showed that the vaccine was
56
safe and effective [11]. The clinical trials also showed that vaccination with a single dose of 3
57
influenza A (H1N1) split vaccine containing 15μg hemagglutinin (HA) could induce good
58
immune responses in humans [11]. However, nearly all of the above clinical trials only
59
reported the protective effect of vaccine in the short term, and most of the results were
60
serological test results for the subjects, 21 days after the immunization [12]. Although the
61
pandemic of influenza A (H1N1) has reached a peak and the incidence rate has been
62
decreasing, there are still a certain number of newly infected people every year, and
63
influenza A (H1N1) has still been placed in the candidate strains of seasonal influenza
64
announced by the WHO in recent years. Therefore, it is still necessary to study the
65
persistence of antibody response to the influenza A (H1N1) split vaccine. In our previous
66
clinical trials, the long-term immune protective effect of the vaccine showed good safety and
67
immunogenicity in the human population aged 18~60 years. However, the level of antibody
68
in serum decreased in the post-immunization. Vaccination with a single dose of 15μg HA
69
split vaccine could induce a protective immune response persisting for at least six months, in
70
adults only [13]. In view of this, we hope to extend the effectiveness of the vaccine by means
71
of adjuvant.
72
In
73
aluminium-hydroxide-adjuvanted influenza A (H1N1) split vaccine in mouse model was
74
observed, and it was discovered that vaccination with a single low dose of the vaccine could
75
provide protection for up to 15 months (450 days).
this
paper,
long-term
protection
provided
76 77
Materials and Methods
78
Viruses, vaccine, mice, and adjuvant 4
by
various
doses
of
the
79
A mouse-adapted A/California/7/2009NYMC X-179A (H1N1) influenza virus was used in
80
this study. After being passaged and adapted with mice as described in our previous studies
81
[14-16], the H1N1 influenza virus was frozen at -70 °C until use. All experiments with live
82
H1N1 virus were performed in a biosafety level two plus containment facility in SIBP
83
(Shanghai Institute of Biological Products Co., Ltd.).
84
The inactivated split-virion vaccine against the H1N1 (2009) virus was developed by the
85
Shanghai Institute of Biological Products, and the seed virus was prepared from the
86
reassortant vaccine virus A/California/7/2009 NYMC X-179A, as described by our previous
87
study [13].
88
Specific-pathogen-free female BALB/c mice (six–eight weeks old), were purchased from
89
Shanghai Laboratory Animal Center, China. All mice were bred in the Animal Resource
90
Center
91
specific-pathogen-free conditions. All experiments involving animals have been approved by
92
the Animal Care Committee of the Shanghai Institute of Biological Products.
93
Al(OH)3 adjuvant (Rehydragel LV) was purchased from General Chemical, and the final
94
concentration of reagent was 0.5mg/ml after addition of the vaccine, which was used after
95
being shaken for 1h at 4° C.
96
Immunization and challenge
97
Specific-pathogen-free femaleBALB/c mice (six~eight weeks old) were intra-peritoneally
98
immunized once with 200μl H1N1 split vaccine with or without adjuvant diluted by PBS.
99
The mice were divided into groups immunized solely with 45μg, 30μg, 15μg, 1.5μg, 0.15μg
at
the
Shanghai
Institute
of
Biological
5
Products
and
maintained
in
100
and 0.015μg influenza A (H1N1) virus split vaccine alone, or groups immunized in
101
combination with 0.5mg/ml Al(OH)3 adjuvant each. The mice were immunized with PBS as
102
the blank control. On the 450th day after immunization, each mouse was intra-nasally
103
challenged with 20 μl of viral suspension containing of 10 × LD50 A/California/7/2009
104
(H1N1) mouse-adapted virus strain. Survival and weight loss were monitored for 21 days.
105
Specimens
106
At different points in time after immunization, serum samples of mice in each group were
107
collected and used for IgG Ab assays. Blood samples were collected on 3, 5, 7, 10, and 14
108
days after immunization, and then once every 7 days after 14 days, once every 14 days after
109
56 days, and once every 28 days after 109 days. Three days after the challenge, five mice
110
from each group were randomly selected for sample collection. After bleeding, the
111
mice were incised ventrally along the median line from the xiphoid process to the point of
112
the chin. The trachea and lungs were taken out and washed three times by injecting with a
113
total of 2ml of PBS containing 0.1% BSA. The bronchoalveolar wash was used for virus
114
titration after removing cellular debris by centrifugation.
115
Antibody responses
116
The concentrations of IgG, IgG1 and IgG2b Abs against the H1N1 virus were measured by
117
ELISA. ELISA was performed using a series of reagents as described in our previous studies
118
[14-16]: goat anti-mouse IgG Ab (γ-chain specific) (KPL), goat anti-mouse IgG1 Ab (KPL),
119
and goat anti-mouse IgG2b Ab (KPL) conjugated with horseradish peroxidase (HRP). The
120
optical density was detected at 450 nm. Ab-positive cutoff values were set as mean + (2 × SD)
121
of unimmunized sera. An ELISA Ab titer was expressed as the highest serum dilution giving 6
122
a positive reaction [17].
123
Hemagglutination inhibition assay
124
Hemagglutination inhibition assay was used to test the virus-specific antibody in the mouse
125
serum samples. The procedure was as follows: the mouse serum and the receptor destructive
126
enzyme (sigma) were mixed at 1:4, with water bath for 16~18h at 37°C; after being
127
processed for 30min at 56°C to inactivate the receptor destructive enzyme, the mixture was
128
diluted with multiple proportions of normal saline in a 96-pore hemagglutination plate, and
129
four hemagglutination unit antigens of equivalent volume were added into each pore,
130
and were allowed to stand for 1h at room temperature; 1% chicken erythrocyte suspension of
131
equivalent volume was then added into each pore, and allowed to stand for 1h at room
132
temperature; the result was judged when the erythrocyte control pore presented with an
133
obvious button shape and fell to the pore bottom, and finally the hemagglutination inhibition
134
titer of each specimen was calculated.
135
Virus titration
136
The bronchoalveolar wash was diluted ten-fold serially, inoculated on Madin Darby canine
137
kidney (MDCK) cells, incubated at 37° C, and examined for cytopathic effect 72h later. The
138
virus titer of each specimen, expressed as the 50% tissue culture infection dose
139
(TCID50), was calculated by the Reed-Muench method. The virus titer in each experimental
140
group was represented by the mean ± SD of the virus titer per ml of specimens from five
141
mice in each group [18, 19].
142
Statistics
143
The IgG responses of test groups were evaluated by using multivariate ANOVA analysis. The 7
144
virus titers and HI titers of test groups were evaluated by using one-way ANOVA analysis.
145
The survival rates of the mice in the experimental and control groups were compared by
146
using Kaplan Meier survival analysis. If the P-value was less than 0.05, the difference was
147
considered significant.
148 149
Results
150
Immunization of H1N1 vaccine (split virion) could provide effective long-term
151
protection for mice and enhance viral clearance.
152
One hundred ninety-five mice were divided randomly into 13 groups of 15 each (Groups
153
A-M, Table 1). The mice were immunized intra-peritoneally with 45µg, 30µg, 15µg, 1.5µg,
154
0.15µg, or 0.015µg H1N1 split vaccine alone (Groups G-L), or in combination with
155
0.5mg/ml Al(OH)3 adjuvant each (Groups A-F). Also, mice were immunized with PBS as a
156
blank control (Group M). Four hundred fifty days after immunization, ten mice in each
157
immunized group were randomly selected, and then were intra-nasally challenged with a
158
lethal dose (10×LD50) of A/California/7/2009 (H1N1) virus suspension. The trachea and
159
lungs were taken out and lavaged with PBS containing 0.1% BSA three days after challenge,
160
from five mice of each group, selected randomly. The remaining five mice in each
161
group were observed for 21 days to evaluate the potential of the vaccine to protect mice
162
against the homologous influenza virus by monitoring survival rate and weight loss.
163
The results showed that the potential of the H1N1 split vaccine to protect mice against the
164
homologous virus was related to the dose of HA, and whether an adjuvant was used (Fig. 1A
165
and 1B). The protection rate of the groups immunized with the vaccine alone increased with 8
166
the immunization dose. The protection rates of mice immunized intra-peritoneally
167
solely with 45µg (Group G), 30µg (Group H), 15µg (Group I), 1.5µg (Group J), 0.15µg
168
(Group K) and 0.015µg (Group L) H1N1 split vaccine alone were 100% (5/5), 100 (5/5),
169
100% (5/5), 40% (2/5), 0% (0/5) and 0% (0/5), respectively. It could be found that the
170
protective effect on mice depends on the dose of H1N1 split vaccine. The mice immunized in
171
combination with aluminium adjuvant were completely protected from challenge with
172
homologous virus at a lethal dose. The protection rates of mice immunized with 45µg
173
(Group A), 30µg (Group B), 15µg (Group C), 1.5µg (Group D), 0.15µg (Group E) and
174
0.015µg (Group F) H1N1 split vaccine in combination with aluminum adjuvant were 100%
175
each. In addition, the protection rates of the groups immunized in combination with
176
adjuvant were higher than those of the groups immunized with a corresponding dose of
177
non-adjuvanted vaccine. The protection rate of the three groups immunized with 1.5µg,
178
0.15µg and 0.015µg adjuvanted vaccine (Groups D, E and F) was 100% (5/5) each,
179
significantly higher than that of the groups immunized with vaccine alone (Group J: 40%
180
(2/5), Group K: 0% (0/5) and Group L: 0% (0/5)) (p
1
The long-term immunogenicity of an inactivated split-virion 2009 pandemic
2
influenza A H1N1 vaccine with or without aluminium-adjuvantin mice
3 4
Wenting Xua,c, Mei Zhenga, Feng Zhoua, Ze Chena,b,*
5 6
Shanghai Institute of Biological Products, Shanghai 200052, Chinaa; College of Life
7
Sciences, Hunan Normal University, Changsha 410081, Hunan, Chinab; School of
8
Life Sciences, Fudan University, Shanghai 200433, Chinac
9 10
Running head: long-term effection of 2009 pandemic influenza vaccine
11 12
*Correspondence should be addressed to Ze Chen
13
E-mail: [email protected]; [email protected]
14
Phone/Fax: (00) 86-(0)21-62826658
15
Address: Shanghai Institute of Biological Products, Shanghai 200052, China
16
1
17
Abstract
18
In 2009, a global epidemic of influenza A (H1N1) caused the death of tens of
19
thousands of people. Vaccination is the most effective means of controlling
20
a wide-range epidemic of influenza and of reducing the mortality rate. In this study,
21
the long-term immunogenicity of A/California/7/2009 (H1N1) split vaccine was
22
observed as long as 15 months (450 days) after the immunization in mouse model.
23
Female BALB/c mice were immunized intra-peritoneally with different doses of
24
aluminum-adjuvanted vaccine. Four hundred fifty days after the immunization, the
25
mice were challenged with a lethal dose (10 × LD50) of homologous virus. The results
26
showed that the supplemented aluminum adjuvant could not only effectively enhance
27
the protective effect of the vaccine, but also reduce the immunizing dose of the
28
vaccine. In addition, the aluminum adjuvant could enhance the IgG antibody level of
29
mice immunized with H1N1 split vaccine. The IgG level was correlated to the
30
survival rate of the mice. Aluminum-adjuvanted inactivated split-virion 2009
31
pandemic influenza A H1N1 vaccine has good immunogenicity, and could provide
32
long-term protection against lethal influenza virus challenge in mice.
33 34
Keywords：2009 pandemic, influenza, split vaccine, long-term
2
35 36
Introduction Influenza is an acute respiratory disease caused by infection of the host respiratory tract by
37
influenza virus, which often spreads globally in seasonal epidemics, and can cause
38
a worldwide influenza pandemic. In 2009, a new influenza A (H1N1) virus caused the first
39
influenza pandemic of the 21st century [1,2]. By August 2010, the 2009 H1N1 influenza
40
virus had been diagnosed in the laboratories in more than 214 countries and regions around
41
the world, resulting in 18,449 deaths [3]. However that number is regarded as well below the
42
true total, mainly because many people who die of flu-related causes are not tested for the
43
disease. Working with admittedly sparse data, a research team led by the US Centers
44
for Disease Control and Prevention (CDC) has estimated the global death toll from the 2009
45
H1N1 influenza pandemic at more than 284,000, about 15 times the number of
46
laboratory-confirmed cases [4]. Vaccination is one of the most effective means of combatting
47
influenza virus infections and of reducing the mortality rate. Therefore, countermeasures
48
have been successively taken in various countries to develop a new monovalent vaccine
49
against influenza A (H1N1) for clinical trials [5,6]. The results of clinical trials show that
50
different types of monovalent vaccines, including the whole inactivated virus vaccine, split
51
vaccine, and attenuated live vaccine, have good safety, and can induce a robust immune
52
response, which conforms to the EU standards for seasonal influenza vaccine [7-10].
53
In order to prevent and control the spread of the 2009 H1N1 influenza virus in China, in
54
August 2009 we completed clinical trials across various age groups, in which all
55
subjects were immunized with the split vaccine, and the results showed that the vaccine was
56
safe and effective [11]. The clinical trials also showed that vaccination with a single dose of 3
57
influenza A (H1N1) split vaccine containing 15μg hemagglutinin (HA) could induce good
58
immune responses in humans [11]. However, nearly all of the above clinical trials only
59
reported the protective effect of vaccine in the short term, and most of the results were
60
serological test results for the subjects, 21 days after the immunization [12]. Although the
61
pandemic of influenza A (H1N1) has reached a peak and the incidence rate has been
62
decreasing, there are still a certain number of newly infected people every year, and
63
influenza A (H1N1) has still been placed in the candidate strains of seasonal influenza
64
announced by the WHO in recent years. Therefore, it is still necessary to study the
65
persistence of antibody response to the influenza A (H1N1) split vaccine. In our previous
66
clinical trials, the long-term immune protective effect of the vaccine showed good safety and
67
immunogenicity in the human population aged 18~60 years. However, the level of antibody
68
in serum decreased in the post-immunization. Vaccination with a single dose of 15μg HA
69
split vaccine could induce a protective immune response persisting for at least six months, in
70
adults only [13]. In view of this, we hope to extend the effectiveness of the vaccine by means
71
of adjuvant.
72
In
73
aluminium-hydroxide-adjuvanted influenza A (H1N1) split vaccine in mouse model was
74
observed, and it was discovered that vaccination with a single low dose of the vaccine could
75
provide protection for up to 15 months (450 days).
this
paper,
long-term
protection
provided
76 77
Materials and Methods
78
Viruses, vaccine, mice, and adjuvant 4
by
various
doses
of
the
79
A mouse-adapted A/California/7/2009NYMC X-179A (H1N1) influenza virus was used in
80
this study. After being passaged and adapted with mice as described in our previous studies
81
[14-16], the H1N1 influenza virus was frozen at -70 °C until use. All experiments with live
82
H1N1 virus were performed in a biosafety level two plus containment facility in SIBP
83
(Shanghai Institute of Biological Products Co., Ltd.).
84
The inactivated split-virion vaccine against the H1N1 (2009) virus was developed by the
85
Shanghai Institute of Biological Products, and the seed virus was prepared from the
86
reassortant vaccine virus A/California/7/2009 NYMC X-179A, as described by our previous
87
study [13].
88
Specific-pathogen-free female BALB/c mice (six–eight weeks old), were purchased from
89
Shanghai Laboratory Animal Center, China. All mice were bred in the Animal Resource
90
Center
91
specific-pathogen-free conditions. All experiments involving animals have been approved by
92
the Animal Care Committee of the Shanghai Institute of Biological Products.
93
Al(OH)3 adjuvant (Rehydragel LV) was purchased from General Chemical, and the final
94
concentration of reagent was 0.5mg/ml after addition of the vaccine, which was used after
95
being shaken for 1h at 4° C.
96
Immunization and challenge
97
Specific-pathogen-free femaleBALB/c mice (six~eight weeks old) were intra-peritoneally
98
immunized once with 200μl H1N1 split vaccine with or without adjuvant diluted by PBS.
99
The mice were divided into groups immunized solely with 45μg, 30μg, 15μg, 1.5μg, 0.15μg
at
the
Shanghai
Institute
of
Biological
5
Products
and
maintained
in
100
and 0.015μg influenza A (H1N1) virus split vaccine alone, or groups immunized in
101
combination with 0.5mg/ml Al(OH)3 adjuvant each. The mice were immunized with PBS as
102
the blank control. On the 450th day after immunization, each mouse was intra-nasally
103
challenged with 20 μl of viral suspension containing of 10 × LD50 A/California/7/2009
104
(H1N1) mouse-adapted virus strain. Survival and weight loss were monitored for 21 days.
105
Specimens
106
At different points in time after immunization, serum samples of mice in each group were
107
collected and used for IgG Ab assays. Blood samples were collected on 3, 5, 7, 10, and 14
108
days after immunization, and then once every 7 days after 14 days, once every 14 days after
109
56 days, and once every 28 days after 109 days. Three days after the challenge, five mice
110
from each group were randomly selected for sample collection. After bleeding, the
111
mice were incised ventrally along the median line from the xiphoid process to the point of
112
the chin. The trachea and lungs were taken out and washed three times by injecting with a
113
total of 2ml of PBS containing 0.1% BSA. The bronchoalveolar wash was used for virus
114
titration after removing cellular debris by centrifugation.
115
Antibody responses
116
The concentrations of IgG, IgG1 and IgG2b Abs against the H1N1 virus were measured by
117
ELISA. ELISA was performed using a series of reagents as described in our previous studies
118
[14-16]: goat anti-mouse IgG Ab (γ-chain specific) (KPL), goat anti-mouse IgG1 Ab (KPL),
119
and goat anti-mouse IgG2b Ab (KPL) conjugated with horseradish peroxidase (HRP). The
120
optical density was detected at 450 nm. Ab-positive cutoff values were set as mean + (2 × SD)
121
of unimmunized sera. An ELISA Ab titer was expressed as the highest serum dilution giving 6
122
a positive reaction [17].
123
Hemagglutination inhibition assay
124
Hemagglutination inhibition assay was used to test the virus-specific antibody in the mouse
125
serum samples. The procedure was as follows: the mouse serum and the receptor destructive
126
enzyme (sigma) were mixed at 1:4, with water bath for 16~18h at 37°C; after being
127
processed for 30min at 56°C to inactivate the receptor destructive enzyme, the mixture was
128
diluted with multiple proportions of normal saline in a 96-pore hemagglutination plate, and
129
four hemagglutination unit antigens of equivalent volume were added into each pore,
130
and were allowed to stand for 1h at room temperature; 1% chicken erythrocyte suspension of
131
equivalent volume was then added into each pore, and allowed to stand for 1h at room
132
temperature; the result was judged when the erythrocyte control pore presented with an
133
obvious button shape and fell to the pore bottom, and finally the hemagglutination inhibition
134
titer of each specimen was calculated.
135
Virus titration
136
The bronchoalveolar wash was diluted ten-fold serially, inoculated on Madin Darby canine
137
kidney (MDCK) cells, incubated at 37° C, and examined for cytopathic effect 72h later. The
138
virus titer of each specimen, expressed as the 50% tissue culture infection dose
139
(TCID50), was calculated by the Reed-Muench method. The virus titer in each experimental
140
group was represented by the mean ± SD of the virus titer per ml of specimens from five
141
mice in each group [18, 19].
142
Statistics
143
The IgG responses of test groups were evaluated by using multivariate ANOVA analysis. The 7
144
virus titers and HI titers of test groups were evaluated by using one-way ANOVA analysis.
145
The survival rates of the mice in the experimental and control groups were compared by
146
using Kaplan Meier survival analysis. If the P-value was less than 0.05, the difference was
147
considered significant.
148 149
Results
150
Immunization of H1N1 vaccine (split virion) could provide effective long-term
151
protection for mice and enhance viral clearance.
152
One hundred ninety-five mice were divided randomly into 13 groups of 15 each (Groups
153
A-M, Table 1). The mice were immunized intra-peritoneally with 45µg, 30µg, 15µg, 1.5µg,
154
0.15µg, or 0.015µg H1N1 split vaccine alone (Groups G-L), or in combination with
155
0.5mg/ml Al(OH)3 adjuvant each (Groups A-F). Also, mice were immunized with PBS as a
156
blank control (Group M). Four hundred fifty days after immunization, ten mice in each
157
immunized group were randomly selected, and then were intra-nasally challenged with a
158
lethal dose (10×LD50) of A/California/7/2009 (H1N1) virus suspension. The trachea and
159
lungs were taken out and lavaged with PBS containing 0.1% BSA three days after challenge,
160
from five mice of each group, selected randomly. The remaining five mice in each
161
group were observed for 21 days to evaluate the potential of the vaccine to protect mice
162
against the homologous influenza virus by monitoring survival rate and weight loss.
163
The results showed that the potential of the H1N1 split vaccine to protect mice against the
164
homologous virus was related to the dose of HA, and whether an adjuvant was used (Fig. 1A
165
and 1B). The protection rate of the groups immunized with the vaccine alone increased with 8
166
the immunization dose. The protection rates of mice immunized intra-peritoneally
167
solely with 45µg (Group G), 30µg (Group H), 15µg (Group I), 1.5µg (Group J), 0.15µg
168
(Group K) and 0.015µg (Group L) H1N1 split vaccine alone were 100% (5/5), 100 (5/5),
169
100% (5/5), 40% (2/5), 0% (0/5) and 0% (0/5), respectively. It could be found that the
170
protective effect on mice depends on the dose of H1N1 split vaccine. The mice immunized in
171
combination with aluminium adjuvant were completely protected from challenge with
172
homologous virus at a lethal dose. The protection rates of mice immunized with 45µg
173
(Group A), 30µg (Group B), 15µg (Group C), 1.5µg (Group D), 0.15µg (Group E) and
174
0.015µg (Group F) H1N1 split vaccine in combination with aluminum adjuvant were 100%
175
each. In addition, the protection rates of the groups immunized in combination with
176
adjuvant were higher than those of the groups immunized with a corresponding dose of
177
non-adjuvanted vaccine. The protection rate of the three groups immunized with 1.5µg,
178
0.15µg and 0.015µg adjuvanted vaccine (Groups D, E and F) was 100% (5/5) each,
179
significantly higher than that of the groups immunized with vaccine alone (Group J: 40%
180
(2/5), Group K: 0% (0/5) and Group L: 0% (0/5)) (p
