IAI Accepts, published online ahead of print on 13 October 2014 Infect. Immun. doi:10.1128/IAI.02334-14 Copyright © 2014, American Society for Microbiology. All Rights Reserved.
1
Mucosal immunization with a live attenuated vaccine SPY1 induces humoral and
2
Th2-Th17-Treg cellular immunity and protects against pneumococcal infection
3
Xiuyu Xu1, 2, Hong Wang1, Yusi Liu1, Yiping Wang1, Lingbing Zeng1, Kaifeng Wu1,
4
Jianmin Wang1, Feng Ma1, Wenchun Xu1, Yibing Yin1, and Xuemei Zhang1*
5
1 Department of Laboratory Medicine, Key Laboratory of Diagnostic Medicine
6
(Ministry of Education), Chongqing Medical University, Chongqing, People’s
7
Republic of China
8
2 Department of Laboratory Medicine, The First Affiliated Hospital of Chongqing
9
Medical University, Chongqing, People’s Republic of China
10
Abstract
11
Mucosal immunization with attenuated vaccine can protect against pneumococcal
12
invasion infection, but the function was unknown. Our study found that mucosal
13
delivery with the live attenuated vaccine SPY1 strain can confer T cells and B cells
14
dependent protection against pneumococcal colonization and invasive infection, yet
15
it’s still unclear which cell subsets contribute to the protection and their roles in
16
pneumococcal colonization and invasion remain elusive. Adoptive transfer of
17
anti-SPY1 antibody conferred protection to naïve μMT mice and immune T cells were
18
indispensable to protection examined in nude mice. A critical role of IL-17A in
19
colonization was demonstrated in mice lacking IL-17A and vaccine-specific Th2 * Corresponding authors at: Mailing address: Department of Laboratory Medicine, Key Laboratory of Diagnostic Medicine (Ministry of Education), Chongqing Medical University, 1 Yixueyuan Road, Yuzhong District, Chongqing 400016, People’s Republic of China. Tel.: +86 23 68485216; fax: +86 23 68485005. E-mail addresses: [email protected] (X. Zhang). 1
20
immune subset was necessary for systemic protection. Of note, we found that SPY1
21
could stimulate immunoregulatory response and SPY1-elicited regulatory T cells
22
participated in protection against colonization and lethal infection. The data presented
23
here aid our understanding of how live attenuated strains are able to function as
24
effective vaccines, and may be contribute to a more comprehensive evaluation of live
25
vaccines and other mucosal vaccines.
26
Key words:
27
Streptococcus pneumoniae; live attenuated vaccine; Th2; Th17; regulatory T cell
28
Running title: SPY1 induces humoral and Th2-Th17-Treg immunity
29
Introduction
30
Vaccination is an indispensable strategy to prevent infection caused by Streptococcus
31
pneumonia (S. pneumoniae), which is estimated to cause mortality rate of more than
32
50 deaths in every 1000 births in children under 5 years of age in some countries (1).
33
The commercially available 23-valent polysaccharide vaccine contains the most
34
common types causing pneumococcal infection and is effective in adults but fails to
35
protect children of less than 2 years of age, who are most vulnerable to pneumococcal
36
infection. The recent extensive introduction of conjugated capsular polysaccharide
37
vaccine (PCV) has drastically decreased the child morbidity and mortality caused by
38
strains of S. pneumoniae expressing capsular serotypes included in the vaccine,
39
however, the serotypes coverage of PCV is limited and growing evidences showed
40
that PCV could induce selective pressure and gradual replacement with nonvaccine 2
41
types (serotype replacement) (2, 3). The conjugated vaccine is also very expensive
42
and is complex in design, making its application in the low income countries having
43
the highest burden of S. pneumoniae infections more difficult (4).
44
As a consequence of these shortcomings with the commercially available S.
45
pneumoniae vaccines, other approaches have been explored, including protein
46
antigens vaccines, killed whole cell S. pneumoniae, or attenuated live S. pneumoniae
47
vaccines (5, 6). The wide range of antigenic molecules present in live attenuated
48
vaccines promising the immune responses they induce are likely to be multiple and
49
powerful and may also more closely mimic those obtained in natural infection
50
compared to immune responses to a sub-component or killed bacterial vaccine (7).
51
Some of live bacterial vaccines have been clinically used, including the BCG (8) and
52
vaccines for preventing typhoid (9). Hence live vaccines could be potentially useful
53
against S. pneumoniae, and there are some reports about live attenuated vaccine
54
candidates. Roche et al. proved a live unencapsulated attenuated S. pneumoniae
55
strains elicited effective systemic and mucosal protection (10), and Richards et al.
56
demonstrated that prior nasopharyngeal colonization with a pneumolysin deficient
57
pneumococcal mutant resulted in serotype independent protection against invasive
58
pneumoccoal infection (11). More recently, serotype cross protection induced by a
59
bacterial lysis deficient pneumococcal mutant was reported in systemic and mucosal
60
infection mice model (12).
61
Streptococcus pneumoniae strain SPY1 is a capsule-negative mutant obtained
62
accidently when we were studying the molecular function of SPD 1672 gene which 3
63
was deleted by allelic replacement with erm cassette from D39 strain using the
64
method we previously described (13). The SPY1 vaccine strain formed small,
65
rough-like colonies, in sharp contrast with the big and smooth colonies of wild type
66
D39 strain. In addition to variation in SPD 1672 gene, several mutations outside the
67
SPD 1672 gene including mutation in the promoter of the cps locus were identified in
68
SPY1 by whole genome sequencing, which contributed to the significant attenuation
69
of SPY1 (14). In the previous study, we have found that intranasal immunization with
70
SPY1 can reduce colonization and protect against otherwise-lethal challenge of
71
homologous pneumococcal strains in a serotype-independent manner (14, 15).
72
However, the immunity mechanisms mediating the protection elicited by SPY1 were
73
not clear. Many researches have shown that the protection induced by living vaccine
74
not only depends on the effect of B cells, but also on the effect of CD4 + T cells (16,
75
17). And the vaccine-specific antibody has been recognized to participate in the
76
protection. In the previous study, we have found that SPY1 can induce the foundation
77
of IFN-γ, IL-17A, IL-10 and IL-4 in BALB/c mice. But, the subtypes of CD4 + T
78
cells (Th1, Th2, of Th17 and Treg cells) have been shown playing different role in the
79
process of resistance to S. pneumoniae infection. IL-17A has been reported to play
80
different roles in local and lethal pneumococcal infections (16, 17). Also, there was no
81
article reporting the function of Treg cells in pneumococcal vaccine induced
82
protection.
83
In this study, mechanisms mediating the protection elicited by SPY1 were
84
evaluated in immune deficient mice model. Vaccine-specific protection is mediated by 4
85
humoral and T cell immunity together. Th17-mediated phagocytes infiltration and Th2
86
immune subset are responsible for protection against colonization and lethal
87
pneumococcal challenge, respectively. We showed, for the first time, that
88
SPY1-specific T regulatory cells were protective in the mucosal and systemic
89
protection against pneumococcal infection. Together, these findings demonstrate the
90
immune mechanisms involved in protection elicited by mucosal vaccination of SPY1,
91
may contribute to a more comprehensive evaluation of living vaccines and other
92
mucosal vaccines.
93 94
Materials and methods
95
Mice. C57BL/6 mice, BALB/c mice and nude BALB/c mice (4-6 weeks, female)
96
were purchased from Chongqing Medical University, Chongqing, China. B cell
97
deficient BALB/c mice (μMT mice) were obtained from Chinese Academy of
98
Sciences, China. IFN-γ deficient C57BL/6 mice (B6.129S7-Ifngtm1Ts/J) and IL-4
99
deficient C57BL/6 mice (B6.129P2-Il4tm1Cgn/J) were purchased from The Jackson
100
Laboratories. IL-17A deficient C57BL/6 mice were obtained from Nankai University,
101
China. Mice were kept under specific-pathogen-free conditions at the animal facilities
102
of Chongqing Medical University during the time of the experiments. All the animal
103
experiments were done in accordance with the Institutional Animal Care and Use
104
Committee’s guidelines of Chongqing Medical University.
105
Bacterial strains and immunogens. S. pneumoniae strain NCTC 7466 (D39,
106
serotype 2) was obtained from the National Collection of Type Cultures (NCTC, 5
107
London, UK). S. pneumoniae strain TIGR4 (serotype 4) was obtained from the
108
American Type Culture Collection (ATCC, Manassas, USA). S. pneumoniae clinical
109
isolates CMCC 31693 (serotype 19F), CMCC 31614 (serotype 14), CMCC 31207
110
(serotype 6B), CMCC 31436 (serotype 3) and CMCC 31203 (serotype 3) were
111
obtained from the National Center for Medical Culture Collections (CMCC, Beijing,
112
China). SPY1 is a novel live attenuated S. pneumoniae which could be used as
113
vaccine (14, 15). All S.pneumoniae strains were grown on Columbia sheep blood agar
114
plates or in C plus Y medium at 37°C in 5% CO2.
115
Intranasal immunization and challenge. SPY1 was grown at 37°C in 5% CO2 in C
116
plus Y medium to approximately 2×108 CFU/ml. The cells were collected by
117
centrifugation, washed twice, and then resuspended in sterile PBS. The final vaccine
118
mixture for routine immunization contained 1×108 CFU of SPY1 and 1μg of adjuvant
119
cholera toxin (CT, Sigma-Aldrich) per 20 μl dose. SPY1 was demonstrated to be
120
rapidly cleared from the nasopharynx within 24 hours post intranasal vaccination in
121
BALB/c mice, and no SPY1 colonies were found in mice lungs 12 hours post
122
immunization (14). Similar colonization results were observed in wild C57BL/6 mice,
123
as well as in the mutant mice. C57BL/6 mice were anesthetized with ethyl ether and
124
received vaccine or adjuvant alone intranasally for four times at one week interval.
125
The positive control group comprised mice that received intraperitoneal injection of
126
PPV23 (Chengdu Institute of Biological Products, China) for three times (5 μg per
127
serotype) at one-week interval. For colonization model, two weeks after the last
128
vaccination, mice were anesthetized and challenged with 1×108 CFU of pneumococcal 6
129
strain CMCC 31693 (serotype 19F) and strain TIGR4, respectively. Mice were
130
sacrificed and CFUs in nasal washes and lung homogenates were determined as
131
previously described 72 hours post challenge (18). For lethal intranasal challenge
132
model, C57BL/6 mice were anesthetized and then inoculated intranasally with either
133
pneumococcal strain NCTC 7466 (D39, serotype 2, 5×107 CFU), strain CMCC 31436
134
(serotype 3, 1.5×108 CFU), strain CMCC 31203 (serotype 3, 4×108 CFU), strain
135
CMCC 31207 (serotype 6B, 5×108 CFU) or strain CMCC 31614 (serotype 14, 4×108
136
CFU) in 20 μl of PBS. Survival was monitored for 21 days.
137
Adoptive transfer. Passive/adoptive transfer was performed as described previously
138
(19). Briefly, serum (200 μl) or approximately 1×108 whole spleen cells from
139
immunized or control mice were intraperitoneally transferred to μMT mice or nude
140
mice, respectively. Then, about 24 hours later, the mice were intranasally infected
141
with 5×107 CFU of strain NCTC 7466 (D39, serotype 2) or 1×108 CFU of strain
142
CMCC 31693 (serotype 19F), respectively. Survival rates as well as CFUs in the
143
upper respiratory tract were observed as described above.
144
Killing assays. Phagocytic killings for S. pneumoniae TIGR4 by vaccine-specific
145
antibodies and splenocyte supernatants using differentiated HL-60 cells (promyelotic
146
leukemia cells, CCL240; American Type Culture Collection, Rockville, MD) (20)
147
were performed as described previously (20, 21). Pneumococcal strain TIGR4 (1×103
148
CFU/ 20 μl) were primed with 20 μl of SPY1-specific serum at 37℃ and 5% CO2 for
149
15 min. Following this incubation period, sterile baby rabbit serum as complement
150
resource (10 μl) was added and washed differentiated HL-60 cells (4×105 cells/ 40 μl) 7
151
was added to each well. The assay plate was incubated at 37°C for 45 min with
152
horizontal shaking (220 rpm). After this incubation period, the incubations were
153
diluted and the dilutions were plated onto blood agar plates. To evaluate killing
154
enhanced by vaccine-specific cellular immunity, the neutrophils were added into 24
155
well plates, cultured for 4 hours in 50% conditioned media (from vaccinated or naive
156
splenocytes exposed to killed SPY1 for 3 days) plus 50% complete media (DMEM +
157
10% FBS). The conditioned media was then aspirated, and the pneumococcal strain
158
TIGR4 was added to the wells in fresh DMEM plus 10% fetal bovine serum. Bacteria
159
were added to the wells at a ratio of 10:1 cells to bacteria. The cells were incubated at
160
37℃ for 1 h and after this incubation period, mixtures were diluted and the dilutions
161
were plated onto blood agar plates. Killing was defined as the percent reduction CFUs
162
in wells containing co-cultures of phagocytes cells and bacteria compared to wells just
163
containing bacteria.
164
Cytokines measurement. One week after the final immunization, after washing and
165
removal of red blood cells by hemolysis, splenocytes from immunized or control mice
166
were plated into 24-well tissue culture plates (2×106 cells/well) in 1 ml of DMEM
167
culture medium with 10% fetal calf-serum (Hyclone). Following 72-hour stimulation
168
with concanavalin A (5 mg/ml, Sigma) or 70% ethanol-killed SPY1 (equivalent to 107
169
CFU/ml), levels of IFN-γ, IL-4, IL-10 and IL-17A in the culture supernatants were
170
detected
171
recommendations. Samples were diluted when required.
172
P17 peptide challenge. P17 peptide (KRIWFIPRSSWYERA, purity＞95% by HPLC)
by
ELISA
kits
(Biolegend)
8
according
to
the
manufacturer’s
173
was synthesized by GL Biochem (Shanghai, China). The procedure of intraperitoneal
174
injection of P17 to down-regulate Treg cells was performed as described previously
175
(22, 23).
176
Flow cytometry. Mouse lung cell suspensions, at 2×108 cells/ml were incubated with
177
purified anti-Fc receptor blocking antibody (anti-CD16/CD32; eBioscience) before
178
addition of the specific antibodies. A combination of FITC anti-mouse Ly-6G
179
antibody (clone RB6-8C5; BD Pharmingen) and PE anti-mouse CD-11b antibody
180
(clone M1/70; BD Pharmingen) was used for neutrophils and macrophages detection.
181
For Treg cells detection, cells were stained with anti-mouse CD4-FITC (clone RM4-5;
182
eBioscience), anti-mouse CD25-APC (clone PC61.5; eBioscience) followed by
183
anti-mouse FoxP3-APC (clone FJK-16s; eBioscience) according to the manufacturer
184
instructions. Then samples were analyzed using a Becton Dickinson FACScalibur
185
flow cytometer.
186
Statistical analysis. Data were compared using Student’s t-test or the Mann-Whitney
187
U-test. Survival rates were analyzed with the Log Rank test. Differences with P
1
Mucosal immunization with a live attenuated vaccine SPY1 induces humoral and
2
Th2-Th17-Treg cellular immunity and protects against pneumococcal infection
3
Xiuyu Xu1, 2, Hong Wang1, Yusi Liu1, Yiping Wang1, Lingbing Zeng1, Kaifeng Wu1,
4
Jianmin Wang1, Feng Ma1, Wenchun Xu1, Yibing Yin1, and Xuemei Zhang1*
5
1 Department of Laboratory Medicine, Key Laboratory of Diagnostic Medicine
6
(Ministry of Education), Chongqing Medical University, Chongqing, People’s
7
Republic of China
8
2 Department of Laboratory Medicine, The First Affiliated Hospital of Chongqing
9
Medical University, Chongqing, People’s Republic of China
10
Abstract
11
Mucosal immunization with attenuated vaccine can protect against pneumococcal
12
invasion infection, but the function was unknown. Our study found that mucosal
13
delivery with the live attenuated vaccine SPY1 strain can confer T cells and B cells
14
dependent protection against pneumococcal colonization and invasive infection, yet
15
it’s still unclear which cell subsets contribute to the protection and their roles in
16
pneumococcal colonization and invasion remain elusive. Adoptive transfer of
17
anti-SPY1 antibody conferred protection to naïve μMT mice and immune T cells were
18
indispensable to protection examined in nude mice. A critical role of IL-17A in
19
colonization was demonstrated in mice lacking IL-17A and vaccine-specific Th2 * Corresponding authors at: Mailing address: Department of Laboratory Medicine, Key Laboratory of Diagnostic Medicine (Ministry of Education), Chongqing Medical University, 1 Yixueyuan Road, Yuzhong District, Chongqing 400016, People’s Republic of China. Tel.: +86 23 68485216; fax: +86 23 68485005. E-mail addresses: [email protected] (X. Zhang). 1
20
immune subset was necessary for systemic protection. Of note, we found that SPY1
21
could stimulate immunoregulatory response and SPY1-elicited regulatory T cells
22
participated in protection against colonization and lethal infection. The data presented
23
here aid our understanding of how live attenuated strains are able to function as
24
effective vaccines, and may be contribute to a more comprehensive evaluation of live
25
vaccines and other mucosal vaccines.
26
Key words:
27
Streptococcus pneumoniae; live attenuated vaccine; Th2; Th17; regulatory T cell
28
Running title: SPY1 induces humoral and Th2-Th17-Treg immunity
29
Introduction
30
Vaccination is an indispensable strategy to prevent infection caused by Streptococcus
31
pneumonia (S. pneumoniae), which is estimated to cause mortality rate of more than
32
50 deaths in every 1000 births in children under 5 years of age in some countries (1).
33
The commercially available 23-valent polysaccharide vaccine contains the most
34
common types causing pneumococcal infection and is effective in adults but fails to
35
protect children of less than 2 years of age, who are most vulnerable to pneumococcal
36
infection. The recent extensive introduction of conjugated capsular polysaccharide
37
vaccine (PCV) has drastically decreased the child morbidity and mortality caused by
38
strains of S. pneumoniae expressing capsular serotypes included in the vaccine,
39
however, the serotypes coverage of PCV is limited and growing evidences showed
40
that PCV could induce selective pressure and gradual replacement with nonvaccine 2
41
types (serotype replacement) (2, 3). The conjugated vaccine is also very expensive
42
and is complex in design, making its application in the low income countries having
43
the highest burden of S. pneumoniae infections more difficult (4).
44
As a consequence of these shortcomings with the commercially available S.
45
pneumoniae vaccines, other approaches have been explored, including protein
46
antigens vaccines, killed whole cell S. pneumoniae, or attenuated live S. pneumoniae
47
vaccines (5, 6). The wide range of antigenic molecules present in live attenuated
48
vaccines promising the immune responses they induce are likely to be multiple and
49
powerful and may also more closely mimic those obtained in natural infection
50
compared to immune responses to a sub-component or killed bacterial vaccine (7).
51
Some of live bacterial vaccines have been clinically used, including the BCG (8) and
52
vaccines for preventing typhoid (9). Hence live vaccines could be potentially useful
53
against S. pneumoniae, and there are some reports about live attenuated vaccine
54
candidates. Roche et al. proved a live unencapsulated attenuated S. pneumoniae
55
strains elicited effective systemic and mucosal protection (10), and Richards et al.
56
demonstrated that prior nasopharyngeal colonization with a pneumolysin deficient
57
pneumococcal mutant resulted in serotype independent protection against invasive
58
pneumoccoal infection (11). More recently, serotype cross protection induced by a
59
bacterial lysis deficient pneumococcal mutant was reported in systemic and mucosal
60
infection mice model (12).
61
Streptococcus pneumoniae strain SPY1 is a capsule-negative mutant obtained
62
accidently when we were studying the molecular function of SPD 1672 gene which 3
63
was deleted by allelic replacement with erm cassette from D39 strain using the
64
method we previously described (13). The SPY1 vaccine strain formed small,
65
rough-like colonies, in sharp contrast with the big and smooth colonies of wild type
66
D39 strain. In addition to variation in SPD 1672 gene, several mutations outside the
67
SPD 1672 gene including mutation in the promoter of the cps locus were identified in
68
SPY1 by whole genome sequencing, which contributed to the significant attenuation
69
of SPY1 (14). In the previous study, we have found that intranasal immunization with
70
SPY1 can reduce colonization and protect against otherwise-lethal challenge of
71
homologous pneumococcal strains in a serotype-independent manner (14, 15).
72
However, the immunity mechanisms mediating the protection elicited by SPY1 were
73
not clear. Many researches have shown that the protection induced by living vaccine
74
not only depends on the effect of B cells, but also on the effect of CD4 + T cells (16,
75
17). And the vaccine-specific antibody has been recognized to participate in the
76
protection. In the previous study, we have found that SPY1 can induce the foundation
77
of IFN-γ, IL-17A, IL-10 and IL-4 in BALB/c mice. But, the subtypes of CD4 + T
78
cells (Th1, Th2, of Th17 and Treg cells) have been shown playing different role in the
79
process of resistance to S. pneumoniae infection. IL-17A has been reported to play
80
different roles in local and lethal pneumococcal infections (16, 17). Also, there was no
81
article reporting the function of Treg cells in pneumococcal vaccine induced
82
protection.
83
In this study, mechanisms mediating the protection elicited by SPY1 were
84
evaluated in immune deficient mice model. Vaccine-specific protection is mediated by 4
85
humoral and T cell immunity together. Th17-mediated phagocytes infiltration and Th2
86
immune subset are responsible for protection against colonization and lethal
87
pneumococcal challenge, respectively. We showed, for the first time, that
88
SPY1-specific T regulatory cells were protective in the mucosal and systemic
89
protection against pneumococcal infection. Together, these findings demonstrate the
90
immune mechanisms involved in protection elicited by mucosal vaccination of SPY1,
91
may contribute to a more comprehensive evaluation of living vaccines and other
92
mucosal vaccines.
93 94
Materials and methods
95
Mice. C57BL/6 mice, BALB/c mice and nude BALB/c mice (4-6 weeks, female)
96
were purchased from Chongqing Medical University, Chongqing, China. B cell
97
deficient BALB/c mice (μMT mice) were obtained from Chinese Academy of
98
Sciences, China. IFN-γ deficient C57BL/6 mice (B6.129S7-Ifngtm1Ts/J) and IL-4
99
deficient C57BL/6 mice (B6.129P2-Il4tm1Cgn/J) were purchased from The Jackson
100
Laboratories. IL-17A deficient C57BL/6 mice were obtained from Nankai University,
101
China. Mice were kept under specific-pathogen-free conditions at the animal facilities
102
of Chongqing Medical University during the time of the experiments. All the animal
103
experiments were done in accordance with the Institutional Animal Care and Use
104
Committee’s guidelines of Chongqing Medical University.
105
Bacterial strains and immunogens. S. pneumoniae strain NCTC 7466 (D39,
106
serotype 2) was obtained from the National Collection of Type Cultures (NCTC, 5
107
London, UK). S. pneumoniae strain TIGR4 (serotype 4) was obtained from the
108
American Type Culture Collection (ATCC, Manassas, USA). S. pneumoniae clinical
109
isolates CMCC 31693 (serotype 19F), CMCC 31614 (serotype 14), CMCC 31207
110
(serotype 6B), CMCC 31436 (serotype 3) and CMCC 31203 (serotype 3) were
111
obtained from the National Center for Medical Culture Collections (CMCC, Beijing,
112
China). SPY1 is a novel live attenuated S. pneumoniae which could be used as
113
vaccine (14, 15). All S.pneumoniae strains were grown on Columbia sheep blood agar
114
plates or in C plus Y medium at 37°C in 5% CO2.
115
Intranasal immunization and challenge. SPY1 was grown at 37°C in 5% CO2 in C
116
plus Y medium to approximately 2×108 CFU/ml. The cells were collected by
117
centrifugation, washed twice, and then resuspended in sterile PBS. The final vaccine
118
mixture for routine immunization contained 1×108 CFU of SPY1 and 1μg of adjuvant
119
cholera toxin (CT, Sigma-Aldrich) per 20 μl dose. SPY1 was demonstrated to be
120
rapidly cleared from the nasopharynx within 24 hours post intranasal vaccination in
121
BALB/c mice, and no SPY1 colonies were found in mice lungs 12 hours post
122
immunization (14). Similar colonization results were observed in wild C57BL/6 mice,
123
as well as in the mutant mice. C57BL/6 mice were anesthetized with ethyl ether and
124
received vaccine or adjuvant alone intranasally for four times at one week interval.
125
The positive control group comprised mice that received intraperitoneal injection of
126
PPV23 (Chengdu Institute of Biological Products, China) for three times (5 μg per
127
serotype) at one-week interval. For colonization model, two weeks after the last
128
vaccination, mice were anesthetized and challenged with 1×108 CFU of pneumococcal 6
129
strain CMCC 31693 (serotype 19F) and strain TIGR4, respectively. Mice were
130
sacrificed and CFUs in nasal washes and lung homogenates were determined as
131
previously described 72 hours post challenge (18). For lethal intranasal challenge
132
model, C57BL/6 mice were anesthetized and then inoculated intranasally with either
133
pneumococcal strain NCTC 7466 (D39, serotype 2, 5×107 CFU), strain CMCC 31436
134
(serotype 3, 1.5×108 CFU), strain CMCC 31203 (serotype 3, 4×108 CFU), strain
135
CMCC 31207 (serotype 6B, 5×108 CFU) or strain CMCC 31614 (serotype 14, 4×108
136
CFU) in 20 μl of PBS. Survival was monitored for 21 days.
137
Adoptive transfer. Passive/adoptive transfer was performed as described previously
138
(19). Briefly, serum (200 μl) or approximately 1×108 whole spleen cells from
139
immunized or control mice were intraperitoneally transferred to μMT mice or nude
140
mice, respectively. Then, about 24 hours later, the mice were intranasally infected
141
with 5×107 CFU of strain NCTC 7466 (D39, serotype 2) or 1×108 CFU of strain
142
CMCC 31693 (serotype 19F), respectively. Survival rates as well as CFUs in the
143
upper respiratory tract were observed as described above.
144
Killing assays. Phagocytic killings for S. pneumoniae TIGR4 by vaccine-specific
145
antibodies and splenocyte supernatants using differentiated HL-60 cells (promyelotic
146
leukemia cells, CCL240; American Type Culture Collection, Rockville, MD) (20)
147
were performed as described previously (20, 21). Pneumococcal strain TIGR4 (1×103
148
CFU/ 20 μl) were primed with 20 μl of SPY1-specific serum at 37℃ and 5% CO2 for
149
15 min. Following this incubation period, sterile baby rabbit serum as complement
150
resource (10 μl) was added and washed differentiated HL-60 cells (4×105 cells/ 40 μl) 7
151
was added to each well. The assay plate was incubated at 37°C for 45 min with
152
horizontal shaking (220 rpm). After this incubation period, the incubations were
153
diluted and the dilutions were plated onto blood agar plates. To evaluate killing
154
enhanced by vaccine-specific cellular immunity, the neutrophils were added into 24
155
well plates, cultured for 4 hours in 50% conditioned media (from vaccinated or naive
156
splenocytes exposed to killed SPY1 for 3 days) plus 50% complete media (DMEM +
157
10% FBS). The conditioned media was then aspirated, and the pneumococcal strain
158
TIGR4 was added to the wells in fresh DMEM plus 10% fetal bovine serum. Bacteria
159
were added to the wells at a ratio of 10:1 cells to bacteria. The cells were incubated at
160
37℃ for 1 h and after this incubation period, mixtures were diluted and the dilutions
161
were plated onto blood agar plates. Killing was defined as the percent reduction CFUs
162
in wells containing co-cultures of phagocytes cells and bacteria compared to wells just
163
containing bacteria.
164
Cytokines measurement. One week after the final immunization, after washing and
165
removal of red blood cells by hemolysis, splenocytes from immunized or control mice
166
were plated into 24-well tissue culture plates (2×106 cells/well) in 1 ml of DMEM
167
culture medium with 10% fetal calf-serum (Hyclone). Following 72-hour stimulation
168
with concanavalin A (5 mg/ml, Sigma) or 70% ethanol-killed SPY1 (equivalent to 107
169
CFU/ml), levels of IFN-γ, IL-4, IL-10 and IL-17A in the culture supernatants were
170
detected
171
recommendations. Samples were diluted when required.
172
P17 peptide challenge. P17 peptide (KRIWFIPRSSWYERA, purity＞95% by HPLC)
by
ELISA
kits
(Biolegend)
8
according
to
the
manufacturer’s
173
was synthesized by GL Biochem (Shanghai, China). The procedure of intraperitoneal
174
injection of P17 to down-regulate Treg cells was performed as described previously
175
(22, 23).
176
Flow cytometry. Mouse lung cell suspensions, at 2×108 cells/ml were incubated with
177
purified anti-Fc receptor blocking antibody (anti-CD16/CD32; eBioscience) before
178
addition of the specific antibodies. A combination of FITC anti-mouse Ly-6G
179
antibody (clone RB6-8C5; BD Pharmingen) and PE anti-mouse CD-11b antibody
180
(clone M1/70; BD Pharmingen) was used for neutrophils and macrophages detection.
181
For Treg cells detection, cells were stained with anti-mouse CD4-FITC (clone RM4-5;
182
eBioscience), anti-mouse CD25-APC (clone PC61.5; eBioscience) followed by
183
anti-mouse FoxP3-APC (clone FJK-16s; eBioscience) according to the manufacturer
184
instructions. Then samples were analyzed using a Becton Dickinson FACScalibur
185
flow cytometer.
186
Statistical analysis. Data were compared using Student’s t-test or the Mann-Whitney
187
U-test. Survival rates were analyzed with the Log Rank test. Differences with P
