CVI Accepted Manuscript Posted Online 3 June 2015 Clin. Vaccine Immunol. doi:10.1128/CVI.00137-15 Copyright © 2015, American Society for Microbiology. All Rights Reserved.
2
Bacillus anthracis capsular conjugates elicit chimpanzee polyclonal antibodies that protect mice from pulmonary anthrax
3
Zhaochun Chena#, Rachel Schneersonb, Julie A. Lovchikc, Zhongdong Daib, Joanna Kubler-Kielbb, Liane
4
Agultoa, Stephen H. Lepplad, Robert H. Purcella
1
5
a
Laboratory of Infectious Diseases and dLaboratory of Parasitic Diseases, National Institute of Allergy and
6
Infectious Diseases, bProgram in Developmental and Molecular Immunity, National Institute of Child
7
Health and Human Development, National Institutes of Health, Bethesda, MD 20892; and cDepartment
8
of Internal Medicine, University of New Mexico Health Sciences Center, Albuquerque, NM 87131
9 10
Running title: Polyclonal antibodies against Bacillus anthracis capsule
11 12 13
Key words: Bacillus anthracis; Immunogenicity; Protective antigen; Poly-γ-D-glutamic acid; Antibodies; Protection
14 15
#Correspondence
16
50 south Drive, Bldg 50, Room 7527, NIH, Bethesda, MD 20892.
17
Phone: 301-594-2308; Fax: 301-402-0524.
18
E-mail: [email protected] (Z. Chen)
19 20 21 22 23 24 25 1
26 27
Abstract The immunogenicity of Bacillus anthracis capsule (poly-γ-D-glutamic acid, PGA) conjugated to
28
recombinant B. anthracis protective antigen (rPA) or to tetanus toxoid (TT) was evaluated in two
29
anthrax-naïve juvenile chimpanzees. In a previous study of these conjugates, highly protective
30
monoclonal antibodies (mAbs) against PGA were generated. This study examines the polyclonal
31
antibody response of the same animals. Preimmune antibodies to PGA with titers of >103 were detected
32
in the chimpanzees. The maximal titer of anti-PGA was induced within 1-2 weeks following the 1st
33
immunization, with no booster effects following 2nd and 3rd immunizations. Thus, the anti-PGA response
34
in the chimpanzees resembled a secondary immune response. Screening of sera from nine unimmunized
35
chimpanzees and six humans revealed antibodies to PGA in all samples, with an average titer of 103. An
36
anti-PA response was also observed following immunization with PGA-rPA conjugate, similar to that
37
seen following immunization with rPA alone. However, in contrast to anti-PGA, preimmune anti-PA
38
antibody titers and those following the 1st immunization were ≤300, with the antibodies peaking above
39
104 following the 2nd immunization. The polyclonal anti-PGA shared the mAb 11D epitope and, similar to
40
the mAbs, exerted opsonophagocytic killing of B. anthracis. Most importantly, the PGA-TT-induced
41
antibodies protected mice from a lethal challenge with virulent B. anthracis spores. Our data support
42
the use of PGA conjugates, especially PGA-rPA targeting both toxin and capsule, as second generation
43
anthrax vaccines.
44
2
45 46 47
Introduction Bacillus anthracis, the causative agent of anthrax, has two obligatory virulence factors: the
48
toxins and capsule. The toxins consist of protective antigen (PA), lethal factor (LF) and edema factor (EF).
49
PA is the cell-receptor-binding component common to the lethal and edema toxins (1).The capsule is
50
composed of poly-γ-D-glutamic acid (γ-D-PGA). Although licensed PA-based anthrax vaccines are safe
51
and effective, expanding protection by including additional antigens in the vaccine would be desirable as
52
defense against bioterrorism (2, 3). Given the capsule’s role in virulence, induction of anti-capsular
53
antibodies has been recommended (4-10).
54
The capsule, present in vegetative B. anthracis, is encoded by the capBCADE operon located on
55
plasmid pXO2 (11-14). Strains that lack pXO2, and capsule, are highly attenuated (15-17) and have been
56
used as vaccines to prevent anthrax in domesticated animals for more than 50 years and in some
57
countries for humans as well (18). The capsule of B. anthracis contributes to the organism’s virulence by
58
its antiphagocytic action (13, 19-21). The γ-D-PGA is poorly immunogenic and acts as a T-cell
59
independent antigen (21, 22), but γ-D-glutamic acid peptides conjugated to carrier proteins such as PA,
60
bovine serum albumin (BSA) or tetanus toxoid (TT) are highly immunogenic in mice, guinea pigs, rabbits
61
and monkeys (4-9).
62
To further evaluate PGA-based conjugates as vaccine candidates, we immunized chimpanzees
63
with PGA-TT or PGA-rPA and monitored both anti-PGA and anti-PA antibody responses. We also
64
determined the protection afforded by the PGA-TT-induced antibodies in a mouse inhalational model
65
following challenge with virulent B. anthracis spores. We found that IgG anti-PGA is protective, and
66
therefore suggest that PGA-rPA conjugates be developed as second generation anthrax vaccines.
67 3
68 69
Materials and Methods:
70
Antigens and sera.
71
B. anthracis γ-D-PGA purified from the culture supernatants, synthetic γ-D-PGA peptide
72
conjugates of recombinant protective antigen (rPA), and tetanus toxoid (TT) were described previously
73
(4). The γ-D,L-PGA from B. subtilis (23) was a gift from Vedan Enterprise Corporation, Taiwan. Naïve
74
human volunteer sera were purchased from Millennium Biotech, Inc.
75
Immunization.
76
Two anthrax-naïve juvenile chimpanzees (approximately 6 yrs of age) were immunized i.m. with
77
alum-adsorbed PGA peptide conjugates shown to induce high level antibody responses in mice (4).
78
Chimpanzee AOA006 received PGA bound to TT and chimpanzee AOA007 received PGA coupled to rPA.
79
The chimpanzees were injected with 25 µg PGA in the conjugate, three times at 6 week intervals.
80
Chimpanzees 1603 and 1609 (also approximately 6 yrs of age) were previously immunized with 50 µg of
81
alum-adsorbed rPA, three times at 2-week intervals (24). The immunized chimpanzees were bled
82
weekly. The housing and care of the chimpanzees were in compliance with all relevant guidelines and
83
requirements, in facilities fully accredited by the Association for Assessment and Accreditation of
84
Laboratory Animal Care International. All animal study protocols involving chimpanzees (LID 26, LID 64)
85
were approved by Animal Care and Use Committees of the National Institute of Allergy and Infectious
86
Diseases and the Animal Care and Use Committee of the facility housing the animals.
87
Preparation of polyclonal anti-PGA antibodies.
4
88
Remaining sera (after antibody assays) collected from weekly bleedings of chimpanzee AOA006
89
immunized with PGA-TT were pooled and concentrated approximately 10-fold using ammonium sulfate
90
precipitation (25-45%), caprylic acid precipitation and Amicon Ultra-15 centrifugal filters. The anti-PGA
91
concentration of this preparation was measured by ELISA, using mouse mAb anti-human IgG and rat
92
anti-mouse for detection, and a 1 mg/ml solution of mAb D11 as the standard (25).
93
Antibody assays by enzyme-linked immunosorbent assay (ELISA).
94
Serum antibody titers were measured by ELISA. Briefly, 96-well Nunc-Immuno™ plates (Thermo,
95
Milford, MA) were coated with 100 µL of purified antigen (rPA or PGA) at a concentration of 4.5-5 µg/ml
96
in phosphate-buffered saline (PBS), pH 7.4. Coated plates were washed with PBS containing 0.1%
97
Tween-20 (PBS-T) and blocked with 3% nonfat dry milk in PBS for 2 h at 370C. Serial 3-fold dilutions of
98
each serum were made beginning at 1:100 and incubated in the coated plates for 2 h at room
99
temperature (RT). After washing, the binding of antibodies to the antigen was detected by incubation
100
with goat anti-human IgG (Fab)2 conjugated with horseradish peroxidase (Jackson ImmunoResearch,
101
West Grove, PA)(1:5000 dilution) for 1 h at RT. Color was developed with tetramethylbenzidine solution
102
(TMB, KPL, Gaithersburg, MD). ELISA titers were calculated using the reciprocal of the highest serum
103
dilution that yielded an absorbance value that was 3-fold higher than the average of the background
104
absorbance. Antibody titers were assigned a value of
2
Bacillus anthracis capsular conjugates elicit chimpanzee polyclonal antibodies that protect mice from pulmonary anthrax
3
Zhaochun Chena#, Rachel Schneersonb, Julie A. Lovchikc, Zhongdong Daib, Joanna Kubler-Kielbb, Liane
4
Agultoa, Stephen H. Lepplad, Robert H. Purcella
1
5
a
Laboratory of Infectious Diseases and dLaboratory of Parasitic Diseases, National Institute of Allergy and
6
Infectious Diseases, bProgram in Developmental and Molecular Immunity, National Institute of Child
7
Health and Human Development, National Institutes of Health, Bethesda, MD 20892; and cDepartment
8
of Internal Medicine, University of New Mexico Health Sciences Center, Albuquerque, NM 87131
9 10
Running title: Polyclonal antibodies against Bacillus anthracis capsule
11 12 13
Key words: Bacillus anthracis; Immunogenicity; Protective antigen; Poly-γ-D-glutamic acid; Antibodies; Protection
14 15
#Correspondence
16
50 south Drive, Bldg 50, Room 7527, NIH, Bethesda, MD 20892.
17
Phone: 301-594-2308; Fax: 301-402-0524.
18
E-mail: [email protected] (Z. Chen)
19 20 21 22 23 24 25 1
26 27
Abstract The immunogenicity of Bacillus anthracis capsule (poly-γ-D-glutamic acid, PGA) conjugated to
28
recombinant B. anthracis protective antigen (rPA) or to tetanus toxoid (TT) was evaluated in two
29
anthrax-naïve juvenile chimpanzees. In a previous study of these conjugates, highly protective
30
monoclonal antibodies (mAbs) against PGA were generated. This study examines the polyclonal
31
antibody response of the same animals. Preimmune antibodies to PGA with titers of >103 were detected
32
in the chimpanzees. The maximal titer of anti-PGA was induced within 1-2 weeks following the 1st
33
immunization, with no booster effects following 2nd and 3rd immunizations. Thus, the anti-PGA response
34
in the chimpanzees resembled a secondary immune response. Screening of sera from nine unimmunized
35
chimpanzees and six humans revealed antibodies to PGA in all samples, with an average titer of 103. An
36
anti-PA response was also observed following immunization with PGA-rPA conjugate, similar to that
37
seen following immunization with rPA alone. However, in contrast to anti-PGA, preimmune anti-PA
38
antibody titers and those following the 1st immunization were ≤300, with the antibodies peaking above
39
104 following the 2nd immunization. The polyclonal anti-PGA shared the mAb 11D epitope and, similar to
40
the mAbs, exerted opsonophagocytic killing of B. anthracis. Most importantly, the PGA-TT-induced
41
antibodies protected mice from a lethal challenge with virulent B. anthracis spores. Our data support
42
the use of PGA conjugates, especially PGA-rPA targeting both toxin and capsule, as second generation
43
anthrax vaccines.
44
2
45 46 47
Introduction Bacillus anthracis, the causative agent of anthrax, has two obligatory virulence factors: the
48
toxins and capsule. The toxins consist of protective antigen (PA), lethal factor (LF) and edema factor (EF).
49
PA is the cell-receptor-binding component common to the lethal and edema toxins (1).The capsule is
50
composed of poly-γ-D-glutamic acid (γ-D-PGA). Although licensed PA-based anthrax vaccines are safe
51
and effective, expanding protection by including additional antigens in the vaccine would be desirable as
52
defense against bioterrorism (2, 3). Given the capsule’s role in virulence, induction of anti-capsular
53
antibodies has been recommended (4-10).
54
The capsule, present in vegetative B. anthracis, is encoded by the capBCADE operon located on
55
plasmid pXO2 (11-14). Strains that lack pXO2, and capsule, are highly attenuated (15-17) and have been
56
used as vaccines to prevent anthrax in domesticated animals for more than 50 years and in some
57
countries for humans as well (18). The capsule of B. anthracis contributes to the organism’s virulence by
58
its antiphagocytic action (13, 19-21). The γ-D-PGA is poorly immunogenic and acts as a T-cell
59
independent antigen (21, 22), but γ-D-glutamic acid peptides conjugated to carrier proteins such as PA,
60
bovine serum albumin (BSA) or tetanus toxoid (TT) are highly immunogenic in mice, guinea pigs, rabbits
61
and monkeys (4-9).
62
To further evaluate PGA-based conjugates as vaccine candidates, we immunized chimpanzees
63
with PGA-TT or PGA-rPA and monitored both anti-PGA and anti-PA antibody responses. We also
64
determined the protection afforded by the PGA-TT-induced antibodies in a mouse inhalational model
65
following challenge with virulent B. anthracis spores. We found that IgG anti-PGA is protective, and
66
therefore suggest that PGA-rPA conjugates be developed as second generation anthrax vaccines.
67 3
68 69
Materials and Methods:
70
Antigens and sera.
71
B. anthracis γ-D-PGA purified from the culture supernatants, synthetic γ-D-PGA peptide
72
conjugates of recombinant protective antigen (rPA), and tetanus toxoid (TT) were described previously
73
(4). The γ-D,L-PGA from B. subtilis (23) was a gift from Vedan Enterprise Corporation, Taiwan. Naïve
74
human volunteer sera were purchased from Millennium Biotech, Inc.
75
Immunization.
76
Two anthrax-naïve juvenile chimpanzees (approximately 6 yrs of age) were immunized i.m. with
77
alum-adsorbed PGA peptide conjugates shown to induce high level antibody responses in mice (4).
78
Chimpanzee AOA006 received PGA bound to TT and chimpanzee AOA007 received PGA coupled to rPA.
79
The chimpanzees were injected with 25 µg PGA in the conjugate, three times at 6 week intervals.
80
Chimpanzees 1603 and 1609 (also approximately 6 yrs of age) were previously immunized with 50 µg of
81
alum-adsorbed rPA, three times at 2-week intervals (24). The immunized chimpanzees were bled
82
weekly. The housing and care of the chimpanzees were in compliance with all relevant guidelines and
83
requirements, in facilities fully accredited by the Association for Assessment and Accreditation of
84
Laboratory Animal Care International. All animal study protocols involving chimpanzees (LID 26, LID 64)
85
were approved by Animal Care and Use Committees of the National Institute of Allergy and Infectious
86
Diseases and the Animal Care and Use Committee of the facility housing the animals.
87
Preparation of polyclonal anti-PGA antibodies.
4
88
Remaining sera (after antibody assays) collected from weekly bleedings of chimpanzee AOA006
89
immunized with PGA-TT were pooled and concentrated approximately 10-fold using ammonium sulfate
90
precipitation (25-45%), caprylic acid precipitation and Amicon Ultra-15 centrifugal filters. The anti-PGA
91
concentration of this preparation was measured by ELISA, using mouse mAb anti-human IgG and rat
92
anti-mouse for detection, and a 1 mg/ml solution of mAb D11 as the standard (25).
93
Antibody assays by enzyme-linked immunosorbent assay (ELISA).
94
Serum antibody titers were measured by ELISA. Briefly, 96-well Nunc-Immuno™ plates (Thermo,
95
Milford, MA) were coated with 100 µL of purified antigen (rPA or PGA) at a concentration of 4.5-5 µg/ml
96
in phosphate-buffered saline (PBS), pH 7.4. Coated plates were washed with PBS containing 0.1%
97
Tween-20 (PBS-T) and blocked with 3% nonfat dry milk in PBS for 2 h at 370C. Serial 3-fold dilutions of
98
each serum were made beginning at 1:100 and incubated in the coated plates for 2 h at room
99
temperature (RT). After washing, the binding of antibodies to the antigen was detected by incubation
100
with goat anti-human IgG (Fab)2 conjugated with horseradish peroxidase (Jackson ImmunoResearch,
101
West Grove, PA)(1:5000 dilution) for 1 h at RT. Color was developed with tetramethylbenzidine solution
102
(TMB, KPL, Gaithersburg, MD). ELISA titers were calculated using the reciprocal of the highest serum
103
dilution that yielded an absorbance value that was 3-fold higher than the average of the background
104
absorbance. Antibody titers were assigned a value of
