IAI Accepts, published online ahead of print on 27 May 2014 Infect. Immun. doi:10.1128/IAI.01847-14 Copyright © 2014, American Society for Microbiology. All Rights Reserved.
1
Burkholderia pseudomallei capsular polysaccharide conjugates provide protection
2
against acute melioidosis
3 4
Andrew E. Scott1#, Mary N. Burtnick2, Margaret G. M. Stokes1, Adam O. Whelan1, E. Diane
5
Williamson1, Timothy P. Atkins1, Joann L. Prior1, Paul J. Brett2
6 7
1 Defence Science and Technology Laboratory, Porton Down, Salisbury, UK.
8
2 Department of Microbiology and Immunology, University of South Alabama, Mobile,
9
Alabama, USA
10 11
Correspondence
12
Fax: +44 01980 614307. Tel: +44 01980 613800. E-mail: [email protected]
13 14
Running title
15
Immunization against melioidosis
16 17 18 19 20 21 22 23 24 25
26
Abstract
27
Burkholderia pseudomallei, the etiologic agent of melioidosis, is a CDC Tier 1 Select Agent
28
that causes severe disease in both humans and animals. Diagnosis and treatment of
29
melioidosis can be challenging, and in the absence of optimal chemotherapeutic intervention
30
acute disease is frequently fatal. Melioidosis is an emerging infectious disease for which no
31
licensed vaccines currently exist. Due to the potential malicious use of B. pseudomallei, as
32
well as its impact on public health in endemic regions, there is significant interest in
33
developing vaccines for immunization against this disease. In the present study, type-A O-
34
polysaccharide (OPS) and manno-heptose capsular polysaccharide (CPS) antigens were
35
isolated from non-pathogenic, Select Agent-excluded strains of B. pseudomallei and
36
covalently linked to carrier proteins. Using these conjugates (OPS2B1 and CPS2B1) it was
37
demonstrated that, although high titer IgG responses against the OPS or CPS components of
38
the glycoconjugates could be raised in BALB/c mice, only those animals immunized with
39
CPS2B1 were protected against an intraperitoneal challenge of B. pseudomallei. Extending
40
upon these studies, it was also demonstrated that when the mice were immunized with a
41
combination of CPS2B1 and recombinant B. pseudomallei LolC, rather than with CPS2B1 or
42
LolC individually, they exhibited higher survival rates when challenged with a lethal dose of
43
B. pseudomallei. Collectively, these results suggest that CPS-based glycoconjugates are
44
promising candidates for the development of subunit vaccines for immunization against
45
melioidosis.
46 47
© Crown Copyright 2014. Published with the permission of the Defence Science and
48
Technology Laboratory on behalf of the controller of HMSO.
49 50
51
Introduction
52
Burkholderia pseudomallei is a Gram-negative, facultative intracellular pathogen that causes
53
melioidosis, a serious and often fatal disease in humans and animals (1). While the incidence
54
of melioidosis is particularly high in Southeast Asia and northern Australia, recent reports
55
have expanded the endemic region to include India and Brazil. Sporadic cases of melioidosis
56
have also been reported in various parts of the Caribbean, Central and South America, the
57
Middle East and Africa (2, 3). B. pseudomallei can be isolated from environmental niches
58
such as rice paddies, still or stagnant waters and moist soils which predominate in the tropics
59
(4), and it is believed that these habitats are the primary reservoirs from which susceptible
60
hosts acquire infections. Routes of infection include inhalation, ingestion and percutaneous
61
inoculation (5).
62
Melioidosis has a broad clinical spectrum and may manifest as acute localized
63
infections, acute pulmonary infections and fulminating septicemias or as chronic disease (6,
64
7). Remarkably, 20% of all community-acquired septicemias in northeast Thailand and 32%
65
of community-acquired bacteremic pneumonias in northern Australia are due to B.
66
pseudomallei infections (8, 9). Because of the non-specific presentation, the lack of rapid
67
diagnostic tests and the intrinsic resistance of B. pseudomallei to commonly used antibiotics,
68
diagnosis and treatment of melioidosis can be challenging. In the absence of optimal
69
chemotherapeutic intervention, mortality rates associated with acute human melioidosis
70
remain unacceptably high (40 - 50% in northeast Thailand and 19% in Australia (9, 10)). At
71
present, there are no human vaccines available for immunization against this emerging
72
infectious disease (for recent reviews see (11-13)). Due to the high risk of aerosol infection,
73
the severe course of disease and the potential for malicious use, B. pseudomallei is currently
74
classified as a CDC Tier 1 Select Agent.
75
Several studies have demonstrated that B. pseudomallei expresses a number of factors
76
that are required for virulence in animal models of infection. Included amongst these are the
77
Bsa type III secretion system, the VirAG two-component regulatory system and the cluster 1
78
type VI secretion system (for recent reviews see (5, 14)). Additionally, studies in our
79
laboratories and others have shown that the O-polysaccharide (OPS) component of B.
80
pseudomallei lipopolysaccharides (LPS) and the manno-heptose capsular polysaccharide
81
(CPS) are both virulence determinants (15-17) and protective antigens (18). Consequently,
82
these carbohydrate moieties have become important components of the various subunit
83
vaccines that are currently being developed for immunization against melioidosis (19, 20).
84
Unlike other Gram-negative pathogens, B. pseudomallei isolates appear to express
85
only a limited repertoire of OPS (21) and CPS antigens (16, 22). At present, the significance
86
of these observations with regards to virulence and evasion of host immune responses
87
remains to be fully determined. Nevertheless, these attributes bode well from a vaccine
88
development standpoint. Relevant to the current study, a number of studies have shown that
89
monoclonal and polyclonal antibodies specific for OPS and CPS can be used to passively
90
immunize mice against lethal challenges of B. pseudomallei (18, 20, 23-26). Such findings
91
confirm the protective capacity of these surface exposed antigens and support the rationale
92
for exploring the use of OPS and CPS antigens to actively immunize against melioidosis.
93
Immunologically, antigens can be classified as either T cell-dependent (TD) or T cell-
94
independent Type 1 or Type 2 (TI-1 or TI-2). Carbohydrates such as capsular antigens and O-
95
polysaccharides are generally considered to be TI-2 antigens (27). Typically, high molecular
96
weight TI-2 antigens such as capsular polysaccharides are immunogenic due to their ability to
97
crosslink multiple surface immunoglobulin molecules present on antigen-specific B cells
98
(28), but without the involvement of T helper (Th) cells, TI-2 antigens induce poor
99
immunological memory, and only limited affinity maturation and isotype switching (29).
100
Additionally, without dosing at frequent intervals antibody levels often decline. Efforts to
101
overcome the poor immunogenicity of many clinically relevant polysaccharides have led to
102
the development of glycoconjugate vaccines (30, 31), a number of which are currently
103
licensed for human use (32). Covalent linkage of polysaccharides to carrier proteins promotes
104
Th cell involvement which improves immunological memory (33) and increases isotype
105
switching. The affinities of the antibodies elicited by glycoconjugates may also be higher
106
than those produced by polysaccharides alone (29).
107
In the present study, B. pseudomallei OPS- and CPS-based glycoconjugates were
108
constructed and evaluated their immunogenic potential and protective capacities using a
109
murine model of melioidosis. Collectively, the results suggest that CPS-based
110
glycoconjugates are promising candidates for the development of subunit vaccines for
111
immunization against melioidosis.
112 113
Materials and methods
114
Strain and growth conditions
115
The bacteria used in this study were B. pseudomallei strains K96243 (34), RR2808 (ΔpurM,
116
ΔwcbB) (35) and RR2683 (ΔpurM, ΔrmlD) (19) and E. coli BL21 Star(DE3)(pLysS)
117
(pCRT7/NT-TOPO-LolC) (36). B. pseudomallei strains RR2808 and RR2683 are derivatives
118
of the adenine auxotroph Bp82 (37), a Select Agent excluded ΔpurM derivative of strain
119
1026b. Strain RR2808 (CPS mutant; source of OPS) and RR2683 (OPS mutant; source of
120
CPS) enable the production of highly purified polysaccharides without the requirement for
121
BSL3 facilities. B. pseudomallei K96243 and E. coli BL21 were cultured in L-broth and on
122
L-agar at 37°C. B. pseudomallei RR2808 and RR2683 were cultured in L-broth and on L-
123
agar supplemented with thiamine (5μg/mL) and adenine (100μg/mL) at 37°C. Bacterial
124
stocks were maintained at –80°C as 20% glycerol suspensions. Wild type B. pseudomallei
125
K96243 was handled at Advisory Committee for Dangerous Pathogens (ACDP) containment
126
level 3.
127 128
For animal challenges, B. pseudomallei K96243 was inoculated from a glycerol stock into
129
100ml L-broth and incubated for 24 hours at 37°C with shaking (180 rpm). The OD590 was
130
adjusted to 0.4 (corresponding to approximately 4 x 108 CFU / ml) and diluted in L-broth to
131
the correct concentration for challenge.
132 133
OPS and CPS purification
134
Broth in 2 L baffled Erlenmeyer flasks was inoculated with B. pseudomallei RR2808 or
135
RR2683 and incubated overnight at 37°C with vigorous shaking. Cell pellets were obtained
136
by centrifugation and extracted using a modified hot aqueous-phenol procedure (38). Purified
137
OPS and CPS antigens were then obtained essentially as previously described (19, 39).
138 139
Glycoconjugate synthesis
140
The OPS2B1 and CPS2B1 glycoconjugates used in this study were synthesized using
141
reductive amination chemistry as previously described (19, 39). Briefly, purified OPS or CPS
142
samples were solubilized at 5 mg/ml in PBS and added to a small amber vials. To each ml of
143
the solutions was added 6 mg (~30 mM) of sodium meta-periodate (NaIO4; Pierce). Once the
144
crystals had dissolved by gentle agitation, the reaction mixtures were gently stirred at room
145
temperature for 40 minutes. To remove any excess oxidizing agent, the reaction mixtures
146
were applied to a Zeba Desalt Spin Columns (Pierce) equilibrated with PBS and the eluates
147
collected. To facilitate conjugation of the OPS or CPS antigens to cationized bovine serum
148
albumin (cBSA; Pierce), the activated polysaccharides were added to small amber vials. To
149
each ml of the OPS or CPS solutions was added 0.5 ml of the carrier proteins (5 mg/ml in
150
PBS). After mixing by gentle agitation, 10μl aliquots of a sodium cyanoborohydride stock (1
151
M NaBH3CN in 10 mM NaOH) were added to each ml of the conjugation mixtures and the
152
reactions were gently stirred at room temperature for 4 days. Following this, 10μl aliquots of
153
a sodium borohydride stock (1M NaBH4 in 10mM NaOH) were added to each ml of the
154
conjugation mixtures and the reactions were stirred for 40 minutes. The conjugate reactions
155
were then brought to 5ml with ultrapure water, dialyzed against distilled water and then
156
lyophilized. The resulting preparations were re-suspended in ultrapure water as 1mg/ml
157
stocks and stored at -20°C until required for use. BCA assays were used to quantitate the
158
protein concentrations of the glycoconjugate stocks (the remaining masses of which were
159
assumed to be polysaccharide).
160 161
LolC protein preparation
162
LolC protein (encoded by BPSL2277 in B. pseudomallei K96243) was purified from E. coli
163
BL21 Star(DE3)(pLysS) (pCRT7/NT-TOPO-LolC) as previously described (36). Briefly,
164
cells were grown to mid-log phase at 37°C, induced by the addition of 1mM IPTG and cooled
165
to 20°C for overnight incubation. Following cell disruption by sonication, LolC was loaded
166
onto HisTrap FF columns (GE Healthcare) and eluted in buffer (40mM Tris-Cl, 750 mM
167
NaCl, 1% glucose, 5% glycerol, pH 7.5) using imidazole in steps to 500mM. The recovered
168
material was dialysed against PBS and protein purity assessed using SDS-PAGE and
169
concentration determined using a BCA assay.
170 171
SDS-PAGE and Western immunoblotting
172
Glycoconjugate samples were analysed using SDS-PAGE and Western immunoblotting as
173
previously described (19, 39). Briefly, samples solubilized in 1X SDS-PAGE sample buffer
174
and heated to 100°C for 5 minutes prior to electrophoresis on 12% Precise gels (Pierce).
175
Proteins were visualized via staining with Coomassie Blue R-250. For Western immunoblot
176
analyses, the glycoconjugate samples and controls were separated on the same 12% gels and
177
electrophoretically transferred to nitrocellulose membranes, before hybridization to either B.
178
pseudomallei OPS-specific mAb (Pp-PS-W) or a CPS-specific mAb (3C5).
179 180
Animal studies
181
All investigations involving animals were carried out according to the requirements of the
182
UK Animal (Scientific Procedures) Act 1986. Studies were performed using 6 – 8 week old
183
female BALB/c mice (Charles River) randomly allocated into cages of five upon arrival.
184
Mice were held under a 12 hour light/dark cycle with free access to food and water and were
185
implanted with a microchip to allow tracking of individual mice. After challenge with B.
186
pseudomallei, animals were handled under ACDP containment level 3 conditions within a
187
rigid-wall half-suit isolator. Mice were checked at least twice daily following challenge and
188
clinical signs for each mouse recorded. Upon reaching pre-determined humane end-points,
189
mice were culled via cervical dislocation.
190
Mice were immunized subcutaneously (s.c.) on days 0, 21 and 35 with the various
191
immunogens. All immunogens were formulated in Alhydrogel 2% (500 μg/mouse; Brenntag)
192
plus ODN 2006 (CpG; 20 μg/mouse; InvivoGen). The mice received 5µg per dose of OPS or
193
CPS as a conjugate, 5µg per dose of unconjugated OPS or CPS and 10µg per dose of cBSA
194
or LolC. Challenges with B. pseudomallei K96243 were delivered at day 70 via the intra-
195
peritoneal route (i.p.; the MLD at day 35 for B. pseudomallei K96243 via this route was
196
calculated to be 744 CFU (unpublished)). For organ bacterial enumeration, animals were
197
culled and organs removed. These were then mashed through 40μm sieves into PBS, serially
198
diluted and plated onto L-agar.
199
200
Analysis of antibody responses
201
Approximately 0.1ml of blood was collected from the tail veins of mice two weeks after the
202
final boost was administered. After clotting at 4°C, the serum was removed and stored at -
203
20°C until required for use. Responses directed against the OPS, CPS or LolC antigens were
204
assessed by ELISA essentially as previously described (40). A reading of twice background
205
or above was considered positive and the titer was determined to be the reciprocal of the final
206
positive dilution.
207 208
Statistical analysis
209
All graphs were produced using the program GraphPad PRISM V5.0. Survival data was
210
analysed using a log rank (Mantel-Cox) test. Bacterial burden data was transformed to the
211
logarithm of 10 and compared using a Mann-Whitney U-test. Antibody data was transformed
212
to the logarithm of 10 and compared using a Mann-Whitney U-test.
213 214
Results
215
Glycoconjugate synthesis
216
Polysaccharide-based glycoconjugates represent a rational approach for immunizing against
217
melioidosis. However, until recently isolating OPS and CPS antigens to develop these
218
vaccine candidates has been hampered by the fact that B. pseudomallei is a Select Agent
219
requiring specialized handling and containment practices. To address this issue, B.
220
pseudomallei RR2808 and RR2683 (derivatives of the Select Agent-excluded strain Bp82, a
221
ΔpurM derivative of 1026b) were created to enable us to produce these polysaccharides in a
222
safer and more cost-effective manner without the requirement for BSL-3 containment (19,
223
35).
224
To facilitate the construction of the glycoconjugates described in this study, the OPS
225
and CPS antigens purified from RR2808 and RR2683 were chemically activated with sodium
226
periodate and covalently linked to cBSA via reductive amination to produce OPS2B1 and
227
CPS2B1 (Figure 1A). Following conjugation, the samples were examined by SDS-PAGE.
228
Results of these analyses demonstrated that in both instances the polysaccharides had
229
covalently linked to the protein carriers as indicated by the shifts in molecular weights of the
230
glycoconjugates relative to the unconjugated cBSA controls (Figure 1B). Additionally,
231
Western immunoblotting confirmed that the structural integrity/antigenicity of the OPS and
232
CPS moieties remained intact following chemical activation and linkage to the protein
233
carriers based upon their reactivity with the Pp-PS-W or 3C5 mAbs (data not shown). Further
234
analysis of the constructs revealed that OPS2B1 and CPS2B1 contained 60% OPS or 53%
235
CPS (w/w) respectively.
236 237
Immunogenicity of the glycoconjugates
238
To compare the immunogenicity of the glycoconjugates, groups of BALB/c mice were
239
immunized with OPS2B1 or CPS2B1. For control purposes, groups of mice were also
240
immunized with a matching amount of antigen composed of mixed but unconjugated
241
polysaccharide and cBSA. Two weeks after the final boost, serum was obtained from the
242
mice and the LPS and CPS specific IgG titers were determined by ELISA (Figure 2). In both
243
cases, the glycoconjugates induced significantly higher antigen-specific IgG titers than the
244
unconjugated controls (p
1
Burkholderia pseudomallei capsular polysaccharide conjugates provide protection
2
against acute melioidosis
3 4
Andrew E. Scott1#, Mary N. Burtnick2, Margaret G. M. Stokes1, Adam O. Whelan1, E. Diane
5
Williamson1, Timothy P. Atkins1, Joann L. Prior1, Paul J. Brett2
6 7
1 Defence Science and Technology Laboratory, Porton Down, Salisbury, UK.
8
2 Department of Microbiology and Immunology, University of South Alabama, Mobile,
9
Alabama, USA
10 11
Correspondence
12
Fax: +44 01980 614307. Tel: +44 01980 613800. E-mail: [email protected]
13 14
Running title
15
Immunization against melioidosis
16 17 18 19 20 21 22 23 24 25
26
Abstract
27
Burkholderia pseudomallei, the etiologic agent of melioidosis, is a CDC Tier 1 Select Agent
28
that causes severe disease in both humans and animals. Diagnosis and treatment of
29
melioidosis can be challenging, and in the absence of optimal chemotherapeutic intervention
30
acute disease is frequently fatal. Melioidosis is an emerging infectious disease for which no
31
licensed vaccines currently exist. Due to the potential malicious use of B. pseudomallei, as
32
well as its impact on public health in endemic regions, there is significant interest in
33
developing vaccines for immunization against this disease. In the present study, type-A O-
34
polysaccharide (OPS) and manno-heptose capsular polysaccharide (CPS) antigens were
35
isolated from non-pathogenic, Select Agent-excluded strains of B. pseudomallei and
36
covalently linked to carrier proteins. Using these conjugates (OPS2B1 and CPS2B1) it was
37
demonstrated that, although high titer IgG responses against the OPS or CPS components of
38
the glycoconjugates could be raised in BALB/c mice, only those animals immunized with
39
CPS2B1 were protected against an intraperitoneal challenge of B. pseudomallei. Extending
40
upon these studies, it was also demonstrated that when the mice were immunized with a
41
combination of CPS2B1 and recombinant B. pseudomallei LolC, rather than with CPS2B1 or
42
LolC individually, they exhibited higher survival rates when challenged with a lethal dose of
43
B. pseudomallei. Collectively, these results suggest that CPS-based glycoconjugates are
44
promising candidates for the development of subunit vaccines for immunization against
45
melioidosis.
46 47
© Crown Copyright 2014. Published with the permission of the Defence Science and
48
Technology Laboratory on behalf of the controller of HMSO.
49 50
51
Introduction
52
Burkholderia pseudomallei is a Gram-negative, facultative intracellular pathogen that causes
53
melioidosis, a serious and often fatal disease in humans and animals (1). While the incidence
54
of melioidosis is particularly high in Southeast Asia and northern Australia, recent reports
55
have expanded the endemic region to include India and Brazil. Sporadic cases of melioidosis
56
have also been reported in various parts of the Caribbean, Central and South America, the
57
Middle East and Africa (2, 3). B. pseudomallei can be isolated from environmental niches
58
such as rice paddies, still or stagnant waters and moist soils which predominate in the tropics
59
(4), and it is believed that these habitats are the primary reservoirs from which susceptible
60
hosts acquire infections. Routes of infection include inhalation, ingestion and percutaneous
61
inoculation (5).
62
Melioidosis has a broad clinical spectrum and may manifest as acute localized
63
infections, acute pulmonary infections and fulminating septicemias or as chronic disease (6,
64
7). Remarkably, 20% of all community-acquired septicemias in northeast Thailand and 32%
65
of community-acquired bacteremic pneumonias in northern Australia are due to B.
66
pseudomallei infections (8, 9). Because of the non-specific presentation, the lack of rapid
67
diagnostic tests and the intrinsic resistance of B. pseudomallei to commonly used antibiotics,
68
diagnosis and treatment of melioidosis can be challenging. In the absence of optimal
69
chemotherapeutic intervention, mortality rates associated with acute human melioidosis
70
remain unacceptably high (40 - 50% in northeast Thailand and 19% in Australia (9, 10)). At
71
present, there are no human vaccines available for immunization against this emerging
72
infectious disease (for recent reviews see (11-13)). Due to the high risk of aerosol infection,
73
the severe course of disease and the potential for malicious use, B. pseudomallei is currently
74
classified as a CDC Tier 1 Select Agent.
75
Several studies have demonstrated that B. pseudomallei expresses a number of factors
76
that are required for virulence in animal models of infection. Included amongst these are the
77
Bsa type III secretion system, the VirAG two-component regulatory system and the cluster 1
78
type VI secretion system (for recent reviews see (5, 14)). Additionally, studies in our
79
laboratories and others have shown that the O-polysaccharide (OPS) component of B.
80
pseudomallei lipopolysaccharides (LPS) and the manno-heptose capsular polysaccharide
81
(CPS) are both virulence determinants (15-17) and protective antigens (18). Consequently,
82
these carbohydrate moieties have become important components of the various subunit
83
vaccines that are currently being developed for immunization against melioidosis (19, 20).
84
Unlike other Gram-negative pathogens, B. pseudomallei isolates appear to express
85
only a limited repertoire of OPS (21) and CPS antigens (16, 22). At present, the significance
86
of these observations with regards to virulence and evasion of host immune responses
87
remains to be fully determined. Nevertheless, these attributes bode well from a vaccine
88
development standpoint. Relevant to the current study, a number of studies have shown that
89
monoclonal and polyclonal antibodies specific for OPS and CPS can be used to passively
90
immunize mice against lethal challenges of B. pseudomallei (18, 20, 23-26). Such findings
91
confirm the protective capacity of these surface exposed antigens and support the rationale
92
for exploring the use of OPS and CPS antigens to actively immunize against melioidosis.
93
Immunologically, antigens can be classified as either T cell-dependent (TD) or T cell-
94
independent Type 1 or Type 2 (TI-1 or TI-2). Carbohydrates such as capsular antigens and O-
95
polysaccharides are generally considered to be TI-2 antigens (27). Typically, high molecular
96
weight TI-2 antigens such as capsular polysaccharides are immunogenic due to their ability to
97
crosslink multiple surface immunoglobulin molecules present on antigen-specific B cells
98
(28), but without the involvement of T helper (Th) cells, TI-2 antigens induce poor
99
immunological memory, and only limited affinity maturation and isotype switching (29).
100
Additionally, without dosing at frequent intervals antibody levels often decline. Efforts to
101
overcome the poor immunogenicity of many clinically relevant polysaccharides have led to
102
the development of glycoconjugate vaccines (30, 31), a number of which are currently
103
licensed for human use (32). Covalent linkage of polysaccharides to carrier proteins promotes
104
Th cell involvement which improves immunological memory (33) and increases isotype
105
switching. The affinities of the antibodies elicited by glycoconjugates may also be higher
106
than those produced by polysaccharides alone (29).
107
In the present study, B. pseudomallei OPS- and CPS-based glycoconjugates were
108
constructed and evaluated their immunogenic potential and protective capacities using a
109
murine model of melioidosis. Collectively, the results suggest that CPS-based
110
glycoconjugates are promising candidates for the development of subunit vaccines for
111
immunization against melioidosis.
112 113
Materials and methods
114
Strain and growth conditions
115
The bacteria used in this study were B. pseudomallei strains K96243 (34), RR2808 (ΔpurM,
116
ΔwcbB) (35) and RR2683 (ΔpurM, ΔrmlD) (19) and E. coli BL21 Star(DE3)(pLysS)
117
(pCRT7/NT-TOPO-LolC) (36). B. pseudomallei strains RR2808 and RR2683 are derivatives
118
of the adenine auxotroph Bp82 (37), a Select Agent excluded ΔpurM derivative of strain
119
1026b. Strain RR2808 (CPS mutant; source of OPS) and RR2683 (OPS mutant; source of
120
CPS) enable the production of highly purified polysaccharides without the requirement for
121
BSL3 facilities. B. pseudomallei K96243 and E. coli BL21 were cultured in L-broth and on
122
L-agar at 37°C. B. pseudomallei RR2808 and RR2683 were cultured in L-broth and on L-
123
agar supplemented with thiamine (5μg/mL) and adenine (100μg/mL) at 37°C. Bacterial
124
stocks were maintained at –80°C as 20% glycerol suspensions. Wild type B. pseudomallei
125
K96243 was handled at Advisory Committee for Dangerous Pathogens (ACDP) containment
126
level 3.
127 128
For animal challenges, B. pseudomallei K96243 was inoculated from a glycerol stock into
129
100ml L-broth and incubated for 24 hours at 37°C with shaking (180 rpm). The OD590 was
130
adjusted to 0.4 (corresponding to approximately 4 x 108 CFU / ml) and diluted in L-broth to
131
the correct concentration for challenge.
132 133
OPS and CPS purification
134
Broth in 2 L baffled Erlenmeyer flasks was inoculated with B. pseudomallei RR2808 or
135
RR2683 and incubated overnight at 37°C with vigorous shaking. Cell pellets were obtained
136
by centrifugation and extracted using a modified hot aqueous-phenol procedure (38). Purified
137
OPS and CPS antigens were then obtained essentially as previously described (19, 39).
138 139
Glycoconjugate synthesis
140
The OPS2B1 and CPS2B1 glycoconjugates used in this study were synthesized using
141
reductive amination chemistry as previously described (19, 39). Briefly, purified OPS or CPS
142
samples were solubilized at 5 mg/ml in PBS and added to a small amber vials. To each ml of
143
the solutions was added 6 mg (~30 mM) of sodium meta-periodate (NaIO4; Pierce). Once the
144
crystals had dissolved by gentle agitation, the reaction mixtures were gently stirred at room
145
temperature for 40 minutes. To remove any excess oxidizing agent, the reaction mixtures
146
were applied to a Zeba Desalt Spin Columns (Pierce) equilibrated with PBS and the eluates
147
collected. To facilitate conjugation of the OPS or CPS antigens to cationized bovine serum
148
albumin (cBSA; Pierce), the activated polysaccharides were added to small amber vials. To
149
each ml of the OPS or CPS solutions was added 0.5 ml of the carrier proteins (5 mg/ml in
150
PBS). After mixing by gentle agitation, 10μl aliquots of a sodium cyanoborohydride stock (1
151
M NaBH3CN in 10 mM NaOH) were added to each ml of the conjugation mixtures and the
152
reactions were gently stirred at room temperature for 4 days. Following this, 10μl aliquots of
153
a sodium borohydride stock (1M NaBH4 in 10mM NaOH) were added to each ml of the
154
conjugation mixtures and the reactions were stirred for 40 minutes. The conjugate reactions
155
were then brought to 5ml with ultrapure water, dialyzed against distilled water and then
156
lyophilized. The resulting preparations were re-suspended in ultrapure water as 1mg/ml
157
stocks and stored at -20°C until required for use. BCA assays were used to quantitate the
158
protein concentrations of the glycoconjugate stocks (the remaining masses of which were
159
assumed to be polysaccharide).
160 161
LolC protein preparation
162
LolC protein (encoded by BPSL2277 in B. pseudomallei K96243) was purified from E. coli
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BL21 Star(DE3)(pLysS) (pCRT7/NT-TOPO-LolC) as previously described (36). Briefly,
164
cells were grown to mid-log phase at 37°C, induced by the addition of 1mM IPTG and cooled
165
to 20°C for overnight incubation. Following cell disruption by sonication, LolC was loaded
166
onto HisTrap FF columns (GE Healthcare) and eluted in buffer (40mM Tris-Cl, 750 mM
167
NaCl, 1% glucose, 5% glycerol, pH 7.5) using imidazole in steps to 500mM. The recovered
168
material was dialysed against PBS and protein purity assessed using SDS-PAGE and
169
concentration determined using a BCA assay.
170 171
SDS-PAGE and Western immunoblotting
172
Glycoconjugate samples were analysed using SDS-PAGE and Western immunoblotting as
173
previously described (19, 39). Briefly, samples solubilized in 1X SDS-PAGE sample buffer
174
and heated to 100°C for 5 minutes prior to electrophoresis on 12% Precise gels (Pierce).
175
Proteins were visualized via staining with Coomassie Blue R-250. For Western immunoblot
176
analyses, the glycoconjugate samples and controls were separated on the same 12% gels and
177
electrophoretically transferred to nitrocellulose membranes, before hybridization to either B.
178
pseudomallei OPS-specific mAb (Pp-PS-W) or a CPS-specific mAb (3C5).
179 180
Animal studies
181
All investigations involving animals were carried out according to the requirements of the
182
UK Animal (Scientific Procedures) Act 1986. Studies were performed using 6 – 8 week old
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female BALB/c mice (Charles River) randomly allocated into cages of five upon arrival.
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Mice were held under a 12 hour light/dark cycle with free access to food and water and were
185
implanted with a microchip to allow tracking of individual mice. After challenge with B.
186
pseudomallei, animals were handled under ACDP containment level 3 conditions within a
187
rigid-wall half-suit isolator. Mice were checked at least twice daily following challenge and
188
clinical signs for each mouse recorded. Upon reaching pre-determined humane end-points,
189
mice were culled via cervical dislocation.
190
Mice were immunized subcutaneously (s.c.) on days 0, 21 and 35 with the various
191
immunogens. All immunogens were formulated in Alhydrogel 2% (500 μg/mouse; Brenntag)
192
plus ODN 2006 (CpG; 20 μg/mouse; InvivoGen). The mice received 5µg per dose of OPS or
193
CPS as a conjugate, 5µg per dose of unconjugated OPS or CPS and 10µg per dose of cBSA
194
or LolC. Challenges with B. pseudomallei K96243 were delivered at day 70 via the intra-
195
peritoneal route (i.p.; the MLD at day 35 for B. pseudomallei K96243 via this route was
196
calculated to be 744 CFU (unpublished)). For organ bacterial enumeration, animals were
197
culled and organs removed. These were then mashed through 40μm sieves into PBS, serially
198
diluted and plated onto L-agar.
199
200
Analysis of antibody responses
201
Approximately 0.1ml of blood was collected from the tail veins of mice two weeks after the
202
final boost was administered. After clotting at 4°C, the serum was removed and stored at -
203
20°C until required for use. Responses directed against the OPS, CPS or LolC antigens were
204
assessed by ELISA essentially as previously described (40). A reading of twice background
205
or above was considered positive and the titer was determined to be the reciprocal of the final
206
positive dilution.
207 208
Statistical analysis
209
All graphs were produced using the program GraphPad PRISM V5.0. Survival data was
210
analysed using a log rank (Mantel-Cox) test. Bacterial burden data was transformed to the
211
logarithm of 10 and compared using a Mann-Whitney U-test. Antibody data was transformed
212
to the logarithm of 10 and compared using a Mann-Whitney U-test.
213 214
Results
215
Glycoconjugate synthesis
216
Polysaccharide-based glycoconjugates represent a rational approach for immunizing against
217
melioidosis. However, until recently isolating OPS and CPS antigens to develop these
218
vaccine candidates has been hampered by the fact that B. pseudomallei is a Select Agent
219
requiring specialized handling and containment practices. To address this issue, B.
220
pseudomallei RR2808 and RR2683 (derivatives of the Select Agent-excluded strain Bp82, a
221
ΔpurM derivative of 1026b) were created to enable us to produce these polysaccharides in a
222
safer and more cost-effective manner without the requirement for BSL-3 containment (19,
223
35).
224
To facilitate the construction of the glycoconjugates described in this study, the OPS
225
and CPS antigens purified from RR2808 and RR2683 were chemically activated with sodium
226
periodate and covalently linked to cBSA via reductive amination to produce OPS2B1 and
227
CPS2B1 (Figure 1A). Following conjugation, the samples were examined by SDS-PAGE.
228
Results of these analyses demonstrated that in both instances the polysaccharides had
229
covalently linked to the protein carriers as indicated by the shifts in molecular weights of the
230
glycoconjugates relative to the unconjugated cBSA controls (Figure 1B). Additionally,
231
Western immunoblotting confirmed that the structural integrity/antigenicity of the OPS and
232
CPS moieties remained intact following chemical activation and linkage to the protein
233
carriers based upon their reactivity with the Pp-PS-W or 3C5 mAbs (data not shown). Further
234
analysis of the constructs revealed that OPS2B1 and CPS2B1 contained 60% OPS or 53%
235
CPS (w/w) respectively.
236 237
Immunogenicity of the glycoconjugates
238
To compare the immunogenicity of the glycoconjugates, groups of BALB/c mice were
239
immunized with OPS2B1 or CPS2B1. For control purposes, groups of mice were also
240
immunized with a matching amount of antigen composed of mixed but unconjugated
241
polysaccharide and cBSA. Two weeks after the final boost, serum was obtained from the
242
mice and the LPS and CPS specific IgG titers were determined by ELISA (Figure 2). In both
243
cases, the glycoconjugates induced significantly higher antigen-specific IgG titers than the
244
unconjugated controls (p
