Vaccine xxx (2016) xxxxxx

Contents lists available at ScienceDirect

Vaccine
journal homepage: www.elsevier.com/locate/vaccine

Protection of rhesus macaques against inhalational anthrax with a

Bacillus anthracis capsule conjugate vaccine
Donald J. Chabot a, Wilson J. Ribot a, Joseph Joyce b, James Cook b, Robert Hepler b, Debbie Nahas b,
Jennifer Chua a, Arthur M. Friedlander a,c,
a
United States Army Medical Research Institute of Infectious Diseases, 1425 Porter Street, Frederick, MD 21702, USA
b
Merck & Co., Inc., Kenilworth, NJ, USA
c
Department of Medicine, Uniformed Services University of the Health Sciences, Bethesda, MD 20814, USA

a r t i c l e i n f o a b s t r a c t

Article history: The efficacy of currently licensed anthrax vaccines is largely attributable to a single Bacillus anthracis
Received 2 March 2016 immunogen, protective antigen. To broaden protection against possible strains resistant to protective
Received in revised form 13 May 2016 antigen-based vaccines, we previously developed a vaccine in which the anthrax polyglutamic acid cap-
Accepted 9 June 2016
sule was covalently conjugated to the outer membrane protein complex of Neisseria meningitidis serotype
Available online xxxx
B and demonstrated that two doses of 2.5 lg of this vaccine conferred partial protection of rhesus maca-
ques against inhalational anthrax . Here, we demonstrate complete protection of rhesus macaques
Keywords:
against inhalational anthrax with a higher 50 lg dose of the same capsule conjugate vaccine. These
Anthrax
Vaccine
results indicate that B. anthracis capsule is a highly effective vaccine component that should be consid-
Capsule ered for incorporation in future generation anthrax vaccines.
Conjugate Published by Elsevier Ltd.
Polyglutamate
Rhesus macaque

1. Introduction strains [4] that may prove to be resistant to PA-based vaccines

The 2001 U.S. domestic terrorism incident involving mailing of
letters containing Bacillus anthracis spores heightened awareness
of the threat of using microbial agents to intentionally cause dis-
ease. Without treatment the mortality rate for inhalational anthrax
is about 90%, and even with antibiotics and supportive therapy,
mortality from inhalational anthrax in the 2001 anthrax letter
cases was 45% [1]. The efficacy of current and next generation
anthrax vaccines is attributable to a single secreted bacterial pro-
tein, protective antigen (PA) [2], the host cell-receptor binding
component common to anthrax lethal and edema toxins. However,
reliance on a single immunogen is of concern because of possible
naturally occurring [3] or genetically engineered B. anthracis
Abbreviations: PA, protective antigen; PGGA, poly-c-D-glutamic acid; OMPC,
Neisseria meningitidis serotype B outer membrane protein complex.
Corresponding author at: United States Army Medical Research Institute of
Infectious Diseases, 1425 Porter Street, Frederick, MD 21702, USA.
E-mail addresses: donald.j.chabot.ctr@mail.mil (D.J. Chabot), wilson.j.ribot.civ@
mail.mil (W.J. Ribot), Joseph_Joyce@Merck.com (J. Joyce), James_Cook@merck.com
(J. Cook), rhepler2@its.jnj.com (R. Hepler), debbie_nahas@merck.com (D. Nahas),
Jennifer.chua.ctr@mail.mil (J. Chua), arthur.m.friedlander.civ@mail.mil (A.M.
Friedlander).
and because some individuals may not respond optimally to a
PA-based vaccine [57]. It is postulated that anthrax vaccine cover-
age and efficacy might be improved by inclusion of a second major
anthrax virulence factor, the poly-c-D-glutamic acid (PGGA) cap-
sule [8,9].
Like many bacterial capsules, the PGGA capsule is a poorly
immunogenic homopolymer and a T cell-independent antigen
[10]. When used alone as a vaccine it tends to elicit a predomi-
nantly IgM antibody response which is poorly protective [11,12].
Covalent conjugation or coupling native capsule or synthetic PGGA
peptides to various carrier proteins converts it to a highly immuno-
genic T cell-dependent antigen with concomitant IgG class-
switching resulting in high levels of anti-capsule IgG [1017]. In
a previous study we reported for the first time that a capsule-
based vaccine can partially protect mice against challenge with a
virulent encapsulated non-toxigenic B. anthracis strain, and, impor-
tantly, that capsule combined with PA was significantly more pro-
tective than PA alone against challenge with the wild-type
encapsulated toxigenic B. anthracis Ames strain [11]. Subsequent
studies showed monoclonal and polyclonal anti-capsule antibodies
provided some level of passive protection in mice against B. anthra-
cis challenge [12,18]. Other reports have demonstrated that

http://dx.doi.org/10.1016/j.vaccine.2016.06.031
0264-410X/Published by Elsevier Ltd.

Please cite this article in press as: Chabot DJ et al. Protection of rhesus macaques against inhalational anthrax with a Bacillus anthracis capsule conjugate
vaccine. Vaccine (2016), http://dx.doi.org/10.1016/j.vaccine.2016.06.031
2 D.J. Chabot et al. / Vaccine xxx (2016) xxxxxx
synthetic PGGA peptides conjugated to PA protected guinea pigs
against virulent anthrax challenge [16,17], although it was not pos-
sible to know conclusively whether any of the observed efficacy
was attributable to the PGGA peptide. However, one of these stud-
ies showed that the PGGA peptide component of the PGGA
peptide-PA conjugate protected guinea pigs against challenge with
an attenuated PA null strain [17]. In another report, a multi-
component vaccine consisting of extracted capsule-peptidoglycan
complex combined with PA provided superior protection in mice
against a virulent challenge strain compared to PA alone. Although
it was not definitively established whether the enhanced efficacy
was the result of inclusion of capsule, peptidoglycan or both, an
additional experiment suggested it was due to the peptidoglycan
component [19].
Previous experiments from our laboratory showed that vaccina-
tion with the capsule conjugated to Neisseria meningitidis serotype
B outer membrane protein complex (OMPC) in the absence of a PA
component completely protected mice against challenge with the
virulent B. anthracis Ames strain [12], though only partial protec-
tion was observed in rhesus macaques, and rabbits were not pro-
tected [20]. In the present report we demonstrate that higher
vaccine doses of the capsule-OMPC vaccine, while not protecting
rabbits, provide complete protection in the rhesus macaque non-
human primate model of inhalational anthrax.
2. Materials and methods
2.1. Bacteria
B. anthracis Ames was obtained from the USAMRIID strain
collection.
2.2. Vaccinations and challenge
Purified PGGA capsule extracted from B. anthracis Ames and
capsule-OMPC conjugate were prepared as described [12]. Adult
male rhesus macaques (Macaca mulatta, Walter Reed Army Insti-
tute of Research, Washington, DC), 5 per group, weighing 8.4
13.9 kg, were vaccinated intramuscularly (IM) with two doses of
10 or 50 lg capsule conjugated to 125 or 625 lg OMPC, respec-
tively, on days 0 and 28. Each dose consisted of two 0.5 ml injec-
tions, one in each thigh. Control groups were vaccinated with
50 lg capsule or 625 lg OMPC alone by the same schedule. All vac-
cines were adsorbed to 0.2% Alhydrogel adjuvant (Invivogen, San
Diego, CA). The ratio of protein (capsule plus OMPC) to Alhydrogel
was 0.0675 for the 10 lg capsule-OMPC vaccine and 0.337 for the
50 lg capsule-OMPC vaccine. Blood was collected for serological
analysis prior to vaccination, just before the second vaccination
(day 28) and just before challenge (day 56). Rhesus macaques were
challenged with a mean inhaled dose of 77 LD50 (range 10284) of
B. anthracis Ames spores in a head-only chamber, as described [20].
Female New Zealand white rabbits (NZWR, Charles River, Freder-
ick, MD), 10 per group, weighing 2.43.35 kg, were vaccinated with
the same doses and schedule and challenged similarly with a mean
inhaled dose of 50 LD50 (range 2.1174). Research was conducted
under an IACUC approved protocol in compliance with the Animal
Welfare Act, PHS Policy, and other federal statutes and regulations
relating to animals and experiments involving animals. The facility
where this research was conducted is accredited by the Association
for Assessment and Accreditation of Laboratory Animal Care, Inter-
national and adheres to principles in Guide for the Care and Use of
Laboratory Animals, National Research Council, 2011.
Please cite this article in press as: Chabot DJ et al. Protection of rhesus macaques against inhalational anthrax with a Bacillus anthracis capsule conjugate
vaccine. Vaccine (2016), http://dx.doi.org/10.1016/j.vaccine.2016.06.031
2.3. ELISAs
Anti-capsule IgG and IgM concentrations were measured by
ELISA as follows. Immulon 4XHB 96-well plates (Fisher Scientific,
Pittsburgh, PA) were coated with 0.5 lg capsule in 60 ll 0.1 M
sodium borate, pH 9.3, and dried overnight at room temperature.
Rhesus macaque normal IgG and IgM standards (Southern Biotech,
Birmingham, AL) diluted in PBS were also applied to plates over-
night. Plates were washed three times with 0.1% Tween-20 in
PBS (Teknova, Hollister, CA), and serum samples tested at 3-fold
serial dilutions, starting with 1:50. Serum diluent was wash buffer
containing 5% non-fat dry milk (Bio-Rad, Hercules, CA) and 0.5%
bovine gelatin (Sigma-Aldrich, St. Louis, MO). After washing five
times, horseradish peroxidase (HRP)-coupled goat anti-monkey
IgG (KPL, Gaithersburg, MD) or HRP-coupled goat anti-monkey
IgM (Fitzgerald Industries, Acton, MA) was added. After washing
five times, plates were developed using ABTS (KPL, Inc., Gaithers-
burg, MD) for 30 min at 37 C. Absorbance was measured at
405 nm. The same methods were used to quantitate rabbit anti-
body. Rabbit IgG and IgM standards and HRP-labeled goat anti-
rabbit IgG were also obtained from Southern Biotech. HRP-
labeled goat anti-rabbit IgM was obtained from KPL.
2.4. Opsono-adherence assay
B. anthracis Ames was grown shaking in 50% BHI broth, 40%
Fetal Bovine Serum (FBS) and 0.8% sodium bicarbonate overnight
in 20% CO2 at 37 C. Bacilli were fixed with 4% paraformaldehyde
and labeled overnight at 4 C with fluorescein isothiocyanate (FITC)
[20]. RAW 264.7 mouse macrophages (ATCC, Manassas, VA) seeded
at 1.4  104/well in Dulbeccos Modified Eagle Medium with 10%
heat inactivated FBS (DF10) in 96-well 0.15 lm-thick glass bottom
plates (In Vitro Scientific, Sunnyvale, CA) were used at about 90%
confluency, three days following seeding. Two-fold dilutions of
heat-inactivated (56 C, 30 min) immune sera were incubated with
FITC-bacilli, 10 ll baby rabbit complement (Cedarlane Labs, Ontar-
io, Canada) in a volume of 105 ll at 37 C for 30 min, using DF10 as
diluent. The serum-treated bacterial mixtures were added to
macrophages (multiplicity of infection = 20) in duplicate wells
and incubated at 37 C for 30 min. Wells were washed five times
with PBS, cells fixed with 4% paraformaldehyde for 1 h and macro-
phages stained for 30 min with HCS Orange Cell Stain (diluted
1:10,000 in PBS; Life Technologies, Grand Island, NY).
Ten randomly pre-selected sites were imaged per well, using
the Zeiss LSM 700 microscopy system with 40 NA 0.6 dry objec-
tive, AxioCam HRc camera, Definite Focus module, and Zen 2011
(Blue Edition) software (Carl Zeiss, Thornwood, NY). The average
number of adherent bacilli per macrophage from duplicate wells
was determined by counting >300 cells/well.
2.5. Statistical analysis
All statistics for these studies were calculated using GraphPad
Prism V.6.04 software (GraphPad Software, La Jolla, CA). Differ-
ences in survival among the vaccine groups were determined by
Fishers exact test and in time to death by log-rank analysis of
Kaplan-Meier survival curves.
Antibody concentrations from individual serum samples were
calculated from ELISA data by interpolation from standard curves
using non-linear regression analysis. Geometric mean concentra-
tions (GMC) for each of the vaccine groups were then calculated.
ANOVA with least significant difference (LSD) posthoc analysis of
log transformed data was used to calculate P values comparing
vaccine groups.
Opsono-adherence titers were determined by nonlinear regres-
sion analysis as described [20]. ANOVA with LSD posthoc analysis
 D.J. Chabot et al. / Vaccine xxx (2016) xxxxxx
of log transformed data were used to calculate P values comparing
the geometric mean (GM) titer of vaccine groups.
3. Results
Serum anti-capsule responses in rhesus macaques were evalu-
ated using an ELISA to detect anti-capsule IgG and IgM and a
macrophage opsono-adherence assay. Two doses of 10 or 50 lg
capsule-OMPC vaccine (Fig. 1A), resulted in higher anti-capsule
IgG levels than did a single dose (P = 0.003 for 10 lg dose;
P = 0.006 for 50 lg dose). After 2 doses of vaccine, there were no
significant differences in anti-capsule IgG GMC (Fig. 1A) or
opsono-adherence titers (Fig. 2A) between the 10 and 50 lg
groups, suggesting that 10 lg results in a maximum antibody
response. Vaccination with unconjugated capsule failed to elicit a
measurable anti-capsule IgG or opsono-adherence antibody
response relative to the OMPC control group (Fig. 1A and B). As
expected, animals immunized with either 10 or 50 lg doses of
the capsule-OMPC vaccine developed anti-capsule IgM levels
which were significantly lower than corresponding IgG levels and
which showed no booster effect (data not shown).
Four of five rhesus macaques receiving 10 lg and all of five
receiving 50 lg of the capsule-OMPC vaccine were protected from
challenge, while only one of five receiving PGGA capsule alone and
none of five receiving OMPC alone survived (Fig. 3). Protection in
both capsule-OMPC groups was significantly better relative to
the OMPC control group determined by overall survival (10 lg
capsule-OMPC, P = 0.048 and 50 lg capsule-OMPC, P = 0.008, by
Fishers exact test) or mean survival times (10 lg capsule-OMPC,
P = 0.023 and 50 lg capsule-OMPC, P = 0.002, by log-rank analysis
of Kaplan Meier survival curves). No significant difference in sur-
vival was observed between the PGGA group and OMPC control
(P = 1.000 by Fishers exact test, P = 0.202 by log-rank analysis).
The one animal in the 10 lg capsule-OMPC vaccine group that died
had the lowest anti-capsule IgG level in the group (124 lg/ml vs. a
GMC of 676 lg/ml for the 4 survivors) and the lowest opsono-
adherence titer (1490 vs. a GM of 5425 for the 4 survivors).
A corresponding immunization study was performed in rabbits
using 10 or 50 lg capsule-OMPC vaccine and the same vaccination
schedule. Rabbits immunized with 2 doses of 10 lg developed
lower levels of anti-capsule IgG than rhesus macaques (Fig. 1,
Rhesus macaques
P = 0.812
P = 0.987
P = 0.003 P = 0.006
A B
Fig. 1. IgG anti-capsule antibody responses of rhesus macaques (A) and rabbits (B) to one and two doses capsule-OMPC vaccine. Geometric means of 5 animals/group are
graphed. Error bars indicate 1 SEM. ANOVA with LSD posthoc test was used to calculate P values comparing vaccine groups.
Please cite this article in press as: Chabot DJ et al. Protection of rhesus macaques against inhalational anthrax with a Bacillus anthracis capsule conjugate
vaccine. Vaccine (2016), http://dx.doi.org/10.1016/j.vaccine.2016.06.031
3
GMC = 73 lg/ml in rabbits and 481 lg/ml in rhesus macaques,
P = 0.002). Those given 2 doses of 50 lg capsule-OMPC vaccine
developed comparable anti-capsule serum IgG levels as rhesus
macaques (Fig. 1, GMC = 518 lg/ml in rabbits and 412 lg/ml in
rhesus macaques, P = 0.688). Opsono-adherence titers were gener-
ally lower in rabbits (Fig. 2). After 2 doses of 10 lg of the capsule-
OMPC vaccine rabbits had an opsono-adherence GM titer of 482 vs.
4189 in rhesus macaques (P = 0.006), and rabbits given 50 lg
capsule-OMPC vaccine had an opsono-adherence GM titer of 684
vs. 2315 in rhesus macaques (P = 0.091). Correlated with this lower
functional antibody response, none of the vaccinated rabbits sur-
vived and there were only minimal increases in mean survival
times in groups receiving the capsule-OMPC vaccine relative to
the OMPC control (Fig. 4, 10 lg capsule-OMPC, P = 0.048 and
50 lg capsule-OMPC, P = 0.053, by log-rank analysis).
4. Discussion
We previously showed that active immunization with capsule
alone confers partial protection in mice against a nontoxigenic
encapsulated strain [11] and that complete protection against chal-
lenge with a fully virulent strain is provided by vaccination with a
capsule-OMPC conjugate vaccine [12]. Additional support for the
inclusion of capsule as a vaccine component is provided by studies
in which mice passively treated with anti-capsular monoclonal or
polyclonal antibodies were protected from challenge with virulent
strains [12,18]. We subsequently demonstrated that the capsule-
OMPC vaccine conferred significant protection against inhalational
anthrax in a nonhuman primate model, the only non-toxin based
vaccine shown to do so [20]. Two doses of 2.5 lg capsule-OMPC
protected three of five rhesus macaques against aerosol B. anthracis
Ames challenge, while two of five animals were protected by a sin-
gle vaccine dose.
The current study was designed to investigate whether
increased dosage of the capsule-OMPC vaccine could increase pro-
tection of rhesus macaques against inhalational anthrax. Our
results showed that eighty percent (four of five) of animals were
protected with two 10 lg doses while 100% (five of five) survived
after two 50 lg doses. The difference in protection between the
10 lg and 50 lg groups was not significant (P = 0.317, log-rank
analysis). However, taken together with the earlier study, the
Rabbits
P < 0.001
P = 0.048
P < 0.001 P < 0.001
4 D.J. Chabot et al. / Vaccine xxx (2016) xxxxxx
Rhesus macaques
P < 0.001
A P < 0.001
P = 0.364
P = 0.870
Fig. 2. Opsono-adherence antibody responses of rhesus macaques (A) and rabbits (B) to one and two doses capsule-OMPC vaccine. Geometric means of 5 animals/group are
graphed. Error bars indicate 1 SEM. ANOVA with LSD posthoc test was used to calculate P values comparing vaccine groups.
Fig. 3. Efficacy of capsule-OMPC conjugate vaccine against B. anthracis aerosol
challenge in rhesus macaques. Rhesus macaques (5/group) were vaccinated and
challenged as described in Methods with a mean inhaled mean dose of 77 LD50.
Survival was significantly higher than controls in the 10 and 50 lg capsule-OMPC
vaccine groups (10 lg capsule-OMPC vs. OMPC, P = 0.023 and 50 lg capsule-OMPC
vs. OMPC, P = 0.002 by log-rank analysis).
Fig. 4. Efficacy of capsule-OMPC conjugate vaccine against B. anthracis aerosol
challenge in rabbits. Rabbits (10/group) were vaccinated and challenged as
described in Methods with a mean inhaled mean dose of 50 LD50. Though all
vaccinated rabbits succumbed to infection, survival time was significantly greater
compared to the OMPC control group in the 10 lg capsule-OMPC group (P = 0.048
by log-rank analysis), and approached statistical significance in the 50 lg capsule-
OMPC group (P = 0.053 by log-rank analysis).
Please cite this article in press as: Chabot DJ et al. Protection of rhesus macaques against inhalational anthrax with a Bacillus anthracis capsule conjugate
vaccine. Vaccine (2016), http://dx.doi.org/10.1016/j.vaccine.2016.06.031
Rabbits
P = 0.006
B P = 0.002
P = 0.596
P = 0.630
results suggest an apparent dose-ranging effect. The rhesus maca-
que serological responses to 10 lg and 50 lg doses were also not
significantly different, although the one animal in the 10 lg group
that did not survive had the lowest IgG level and opsono-
adherence antibody titer in that group, suggesting a correlation
between antibody levels and protection. Further studies will be
required to confirm this observation, but it is anticipated to be
highly likely given the established mechanism of action for similar
bacterial capsule vaccines for prevention of Haemophilus influenzae
and Streptococcus pneumoniae infections [21,22] and the fact that
antibody to anthrax capsule can confer passive protection. While
OMPC, the carrier used in the capsule conjugate, in contrast to
other protein carriers, acts as an adjuvant as well as a carrier
[23] an additional component to consider for enhancing the overall
immune response to a combined PA and capsule conjugate vaccine
would be adjuvant choice. A variety of new adjuvants have been
introduced in recent years which can act alone or in combination
with aluminium salts, including oil-in-water and TLR4 agonists
that have been licensed for human use [24]. The current work also
strongly supports future studies evaluating the capsule-OMPC vac-
cine co-administered with PA to determine if such a combination
vaccine provides a synergistic benefit as suggested by prior mouse
studies [11]. In our previous report, we were unable to protect rab-
bits using 1 lg of capsule-OMPC vaccine [20]. In agreement with
that finding, in the current study using doses of 10 or 50 lg we
observed no effect on overall survival and only a minimal delay
in time to death (Fig. 4). There was a suggestion that rabbits pro-
duced anti-capsule antibody with lower opsono-adherence activity
than rhesus macaques (Fig. 2, P = 0.006 for 10 lg capsule-OMPC;
P = 0.091 for 50 lg capsule-OMPC), but further study is required
to evaluate this finding. The lack of protection in rabbits is consis-
tent with older reports that vaccination with killed encapsulated
bacilli does not protect rabbits whereas serum from those same
vaccinated rabbits passively protects mice [25]. Our results suggest
rabbits are not predictive of the efficacy of capsule-based vaccines
in nonhuman primates.
Protection against inhalational anthrax in the nonhuman pri-
mate model using a capsule-OMPC conjugate vaccine strongly sug-
gests that an immunogenic capsule component be combined with
PA in future anthrax vaccines to protect against a broader range of
anthrax strains, protect individuals who might not respond opti-
mally to PA, and possibly to increase the efficacy of PA-based
vaccines.
 D.J. Chabot et al. / Vaccine xxx (2016) xxxxxx
Funding
This work was supported by the Medical Biological Defense
Research Program, U.S. Army Medical Research and Materiel Com-
mand. Project No. 921175.
Role of the funding source
The sponsor had no role in the conduct of the research or in the
preparation of the report.
Contributors
AMF designed the experiments. DJC, WJR, JJ, JC, RH, DN and JC
performed the experiments. DJC, JC and AMF analyzed the data.
DJC and AMF wrote the manuscript with critical review by JJ. All
authors approved the final version.
Conflict of interest statement
The authors from USAMRIID do not have a commercial or other
association that might pose a conflict of interest. JJ, JC, RH, and DN
were employed by Merck and Company during this study.
Disclaimer
Opinions, interpretations, conclusions, and recommendations
are those of the authors and are not necessarily endorsed by the
U.S. Army.
Acknowledgements
The authors thank the USAMRIID Veterinary Medicine Division,
Tammy Putmon-Taylor and Kyle J. Fitts for excellent technical
assistance and Nathan Hoyt for help with rhesus macaques.
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