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Single-dose combination nanovaccine induces both rapid and
long-lived protection against pneumonic plague
Danielle A Wagnera,1, Sean M Kellyb,1, Andrew C Petersena, Nathan Peroutka-Bigusa,c,
Ross J Darlinga, Bryan H Bellairea,c,d, Michael J Wannemuehlera,d,*, Balaji Narasimhanb,d,*
aDepartment of Veterinary Microbiology and Preventive Medicine, Iowa State University, Ames,
IA, United States
bDepartment of Chemical and Biological Engineering, Iowa State University, Ames, IA, United
States
cInterdepartmental Microbiology Program, Iowa State University, Ames, IA, United States
dNanovaccine Institute, Iowa State University, Ames, IA, United States
Abstract
Yersinia pestis
, the causative agent of pneumonic plague, induces a highly lethal infection if left
untreated. Currently, there is no FDA-approved vaccine against this pathogen; however,
USAMRIID has developed a recombinant fusion protein, F1-V, that has been shown to induce
protection against pneumonic plague. Many F1-V-based vaccine formulations require prime-boost
immunization to achieve protective immunity, and there are limited reports of rapid induction of
protective immunity (≤ 14 days post-immunization (DPI)). The STimulator of INterferon Genes
agonists cyclic dinucleotides (CDNs) have been shown to be promising vaccine adjuvants.
Polyanhydride nanoparticle-based vaccines (i.e., nanovaccines) have also shown to enhance
immune responses due to their dual functionality as adjuvants and delivery vehicles. In this work,
a combination nanovaccine was designed that comprised F1-V-loaded nanoparticles combined
with the CDN, dithio-RP,RP-cyclic di-guanosine monophosphate, to induce rapid and long-lived
protective immunity against pneumonic plague. All mice immunized with a single dose
combination nanovaccine were protected from
Y. pestis
lethal challenge within 14 DPI and
demonstrated enhanced protection over F1-V adjuvanted with CDNs alone at challenge doses
≥7000 CFU
Y. pestis
CO92. In addition, 75% of mice receiving the single dose of the combination
*Corresponding authors at: Nanovaccine Institute, Iowa State University, Ames, IA, United States. mjwannem@iastate.edu (M.J.
Wannemuehler), nbalaji@iastate.edu (B. Narasimhan).
1Indicates these authors contributed equally to this manuscript
CRediT authorship contribution statement
Danielle A Wagner: Conceptualization, Formal analysis, Writing - original draft. Sean M Kelly: Conceptualization, Formal analysis,
Writing - original draft. Andrew C Petersen: Data curation, Formal analysis, Writing - original draft. Nathan Peroutka-Bigus:
Formal analysis, Writing - original draft. Ross J Darling: Data curation, Formal analysis, Writing - original draft. Bryan H Bellaire:
Formal analysis, Writing - original draft. Michael J Wannemuehler: Writing - original draft. Balaji Narasimhan: Writing - original
draft.
Declaration of Competing Interest
The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be
construed as a potential conflict of interest.
Supplementary materials
Supplementary material associated with this article can be found, in the online version, at doi: 10.1016/j.actbio.2019.10.016.
HHS Public Access
Author manuscript
Acta Biomater
. Author manuscript; available in PMC 2020 February 11.
Published in final edited form as:
Acta Biomater
. 2019 December ; 100: 326–337. doi:10.1016/j.actbio.2019.10.016.
Author Manuscript Author Manuscript Author Manuscript Author Manuscript
nanovaccine were protected from challenge at 182 DPI, while maintaining high levels of antigen-
specific serum IgG. ELISPOT analysis of vaccinated animals at 218 DPI revealed F1-V-specific
long-lived plasma cells in bone marrow in mice vaccinated with CDN adjuvanted F1-V or the
combination nanovaccine. Microarray analysis of serum from these vaccinated mice revealed the
presence of serum antibody that bound to a broad range of F1 and V linear epitopes. These results
demonstrate that combining the adjuvanticity of CDNs with a nanovaccine delivery system
enables induction of both rapid and long-lived protective immunity against
Y. pestis
.
Keywords
Pneumonic plague; Polyanhydride; Nanovaccine; Cyclic dinucleotide; Combination
1. Introduction
Plague has relentlessly affected humans throughout history, accounting for an estimated 200
million deaths [1, 2] and continues to persist worldwide, with the latest outbreak occurring
in Madagascar in 2017. Plague is caused by the non-motile, facultative intracellular, Gram-
negative bacterium
Yersinia pestis
[3]. Pneumonic plague, the respiratory manifestation of
Y.
pestis
infection, is transmitted through aerosolized droplets [4]. In the case of pneumonic
plague, treatment with intravenous or oral ciprofloxacin and doxycycline over 48 h is
successful, though mortality rates quickly approach 90–100% if left untreated for 24–36 h
post-infection [1].
The threat of weaponization of
Y. pestis
is high, largely evidenced by its history of such use
[2]. Though the United States and 103 other countries co-signed an agreement to terminate
biological weapons programs in 1972,
Y. pestis
remains listed as a Tier 1 Select Agent.
Currently, there is no FDA-approved vaccine against
Y. pestis
, making it critical to develop a
protective vaccine with the capability to provide both rapid and long-lived immunity in the
event of mass exposure of aerosolized
Y. pestis
to civilian or military populations. As
Y.
pestis
is predominantly an extracellular pathogen, many vaccination strategies have focused
on developing strong humoral immunity, characterized by neutralizing antibodies against the
V antigen and opsonizing antibodies against the F1 capsule [5–8]. In mice, anti-F1 IgG
antibody titers ≥ 100,000 have been shown to correlate with protection [5]. Additionally,
monoclonal antibody (mAb) 7.3, which binds to the V antigen, has been shown to neutralize
Y. pestis
in vitro and provides passive protection against lethal challenge [6, 9].
The United States Army Medical Research Institute of Infectious Diseases (USAMRIID)
developed a recombinant fusion protein, F1-V, comprised of full length F1 and V antigens,
and shows promise as a target antigen for
Y. pestis
[10–13]. This vaccine has long shown
promise as a vaccine candidate against both pneumonic and bubonic plague in rodents [10–
12]. In fact, it was recently shown that F1-V adjuvanted with aluminum hydroxide (alum)
using an IM/SC prime-boost regimen provided complete protection against intranasal
challenge with virulent
Y. pestis
CO92 in mice, guinea pigs, and macaques [13].
Additionally, serum collected from immunized macaques conferred passive protection to
mice. Despite such success, this vaccine formulation is most often administered as a prime-
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boost regimen; in the event of mass exposure, it would be ideal to use single dose
formulations that can provide rapid immunity in as short a time frame as possible and that
can maintain long-lived protective immunity.
Cyclic dinucleotides (CDNs), a class of small molecule adjuvants, are recognized as
microbial-associated molecular patterns (MAMPs) by the pattern recognition receptor (PRR)
STING (STimulator of INterferon Genes) [14], resulting in the phosphorylation of
transcription factors NF
κ
B and IRF3 and the induction of type I interferon (IFN), which is
associated with anti-pathogenic activity [15, 16]. Cyclic di-guanosine monophosphate (3),
the most well-studied CDN to date, has been recognized as a universal secondary messenger
molecule in Gram-negative bacteria, playing a role in bacterial development, motility, and
virulence [17]. Vaccines adjuvanted with CDG induce a balanced immune response,
characterized by equal presence of IgG subclasses in serum [18], and have been shown to
elicit significantly higher antibody titers than alum-adjuvanted formulations [19].
Biodegradable polyanhydride particles, a novel class of adjuvants and delivery vehicles,
possess a multitude of beneficial characteristics to address challenges faced by many current
vaccines [20]. Comprised of 1,6-bis(
p
-carboxyphenoxy)hexane (CPH) and 1,8-bis(
p
-
carboxyphenoxy) −3,6-dioxaoctane (CPTEG), these materials are highly biocompatible
demonstrating minimal injection-site reactivity when administered as particles [21, 22] and
whose non-toxic dicarboxylic acid degradation products are safely excreted from the body
[23]. Their amphiphilic properties allow for stabilization of labile proteins [24–26], provide
sustained release of encapsulated proteins via surface erosion kinetics [25, 27–29], and allow
for enhanced shelf stability of encapsulated proteins, even at elevated temperatures (e.g.,
40 °C) [25]. Additionally, polyanhydride particles display inherent adjuvanticity
demonstrating chemistry-dependent internalization, persistence, and activation of antigen
presenting cells in vitro, as well as the ability to prime humoral and cell-mediated immune
responses in vivo [30, 31, 40–42, 32–39]. These properties have enabled the study of
polyanhydride nanoparticle-based vaccines (i.e., nanovaccines) against multiple bacterial
and viral pathogens [25, 29, 37, 43, 44].
Previous work from our laboratories demonstrated that encapsulation of F1-V into
polyanhydride particles maintained F1-V structure and prolonged antigen bioavailability
[24], and a single, intranasal dose of F1-V nanovaccine provided complete protection against
lethal challenge of
Y. pestis
for at least 280 days post-immunization (DPI) [37]. In addition
to long-lived protective immunity, it is also important that vaccines induce rapid (i.e., ≤ 14
days) protective immunity to counter acute outbreaks of disease. Designing next-generation
vaccine platforms that provide
both
rapid and long-lived immunity against highly lethal
pathogens will likely require novel approaches including developing new adjuvants or
vaccine regimen with combination adjuvants to enhance both rapid and long-lived protective
immunity. Herein, the design and evaluation of a combination nanovaccine, comprising of
F1-V-containing polyanhydride nanoparticles and a non-canonical CDG CDN adjuvant
(containing 2′,5′–3′ –5′ phosphate linkages) is described. This nanovaccine formulation
synergistically combines the adjuvant properties of polyanhydride nanoparticles and CDNs
to induce rapid immune responses and facilitate the induction of long-lived protective
immunity against pneumonic plague.
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2. Material and methods
2.1. Materials
Chemicals used for CPTEG and CPH diacid and polymer synthesis included 1,6-
dibromohexane, triethylene 4-
p
-hydroxybenzoic acid, and 1-methyl-2-pyrrolidinone,
purchased from Sigma-Aldrich (St. Louis, MO). Chloroform, petroleum ether, ethyl ether,
hexanes, sodium hydroxide, toluene, sulfuric acid, acetonitrile, dimethyl formamide, acetic
anhydride, methylene chloride, pentane, and potassium carbonate were purchased from
Fisher Scientific (Fairlawn, NJ). 4-
p
-fluorobenzonitrile was purchased from Apollo
Scientific (Cheshire, UK). Deuterated chloroform used for 1H NMR analysis was purchased
from Cambridge Isotope Laboratories (Andover, MA). Dithio-RP,RP-cyclic di-guanosine
monophosphate (R,R-Cyclic di-GMP (CDG)) was provided by Aduro Biotech (Berkeley,
CA). Complete cell culture medium reagents RPMI 1640 and penicillin-streptomycin were
purchased from Mediatech (Herndon, VA); heat inactivated fetal calf serum was purchased
from Atlanta Biologicals (Atlanta, GA).
Y. pestis
, strain CO92 (NR-641) and the
Y. pestis
fusion protein F1-V (NR-4526) were obtained from the Biodefense and Emerging Infections
Repository (Manassas, VA).
2.2. Polyanhydride synthesis
CPTEG and CPH diacids were synthesized as previously described [28, 35]. 20:80
CPTEG:CPH copolymer synthesis was performed using melt polycondensation [28].
Copolymer composition and molecular weight were estimated using end group analysis of
1H NMR (DXR 500) spectra.
2.3. Nanoparticle synthesis
10% (w/w) F1-V-loaded 20:80 CPTEG:CPH nanoparticles were synthesized using flash
nanoprecipitation, as described previously [30]. Briefly, F1-V and 20:80 CPTEG:CPH
copolymer was dissolved in methylene chloride at 2 mg/mL and 20 mg/mL, respectively,
sonicated at 30 Hz for approximately 30 s, and poured into pentane chilled to −20 °C at a
methylene chloride:pentane ratio of 1:250. Nanoparticles were imaged using scanning
electron microscopy (SEM; JEOL 840 A, JEOL Ltd., Tokyo, Japan), and nanoparticle mean
size and size distribution were determined using ImageJ (National Institutes of Health,
Bethesda, MD). Nanoparticle zeta potential was measured using Zetasizer Nano (Malvern
Instruments, Worcestershire, UK).
2.4. Protein release and encapsulation efficiency
To quantify released protein, approximately 2 mg of nanoparticles were suspended in 500 μL
of PBS, sonicated for 15 s, and placed on a shaker plate at 37 °C. Periodically, 400 μL
aliquots of sample supernatant were withdrawn and sample volumes were reconstituted with
400 μL of fresh PBS. After 32 days, 40 mM sodium hydroxide was used in place of PBS to
catalyze the release of any remaining protein. Protein released from F1-V-loaded 20:80
CPTEG:CPH nanoparticles was quantified using a microbichoninic acid (microBCA) assay
(Thermo Fisher Scientific, Waltham, MA), and percent by mass of protein released over time
was calculated as the cumulative protein released from nanoparticles at each time point
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divided by total mass of protein released. To quantify protein encapsulation efficiency,
approximately 1 mg of nanoparticles was suspended in 500 μL of 40 mM sodium hydroxide
solution, sonicated for 15 s, and placed on a shaker plate at 37 °C. Frequently, 400 μL
samples of supernatant were withdrawn and sample volumes were reconstituted with 400 μL
of 40 mM sodium hydroxide. Protein released from F1-V-loaded 20:80 CPTEG:CPH
nanoparticles was quantified using a microBCA assay, and the encapsulation efficiency was
calculated as the sum of the protein released from the nanoparticles divided by the initial
mass of protein.
2.5. Animals
Seven to eight-week old female C57BL/6NCrl mice were purchased from Charles River
(Wilmington, MA). Mice were housed under specific pathogen-free conditions where all
bedding, caging, water, and feed were sterilized prior to use. All studies were conducted
with the approval of the Iowa State University Institutional Animal Care and Use Committee
(IACUC).
2.6. Immunization and serum collection
Groups comprised of C57BL/6NCrl mice (
n
= 8–12/group) were immunized once
subcutaneously at the nape of the neck with either of the following formulations: 50 μg F1-V
+ 35 μg CDNs (R,R-CDG, Aduro Biotech), 50 μg F1-V encapsulated into 500 μg of 20:80
CPTEG:CPH nanoparticles, 50 μg F1-V encapsulated into 500 μg of 20:80 CPTEG:CPH
nanoparticles + 35 μg CDNs, or saline (
n
= 16/group) in a total volume of 200 μL. Blood
was collected from mice via saphenous vein and serum was separated following
centrifugation (10,000 rcf for 10 min) at 13, 14, 36, 49, 79, 101, 121, 150, and 178 DPI. An
additional serum sample was obtained from mice sacrificed for ELISPOT analysis at 218
DPI via cardiac exsanguination. Serum was stored at −20 °C until analysis. Bronchoalveolar
lavage (BAL) fluid was collected post-euthanization by introducing a catheter needle
through a small incision made in the trachea and injecting/aspirating 700 μL of PBS thrice.
2.7. ELISA
Anti-F1-V antibody titers were determined via ELISA, as previously described [29]. Briefly,
high-binding Costar 590 EIA/RIA microtiter plates (Corning) were coated overnight with
100 μL of a 0.5 μg/mL solution of F1-V at 4 °C. After washing the wells, microtiter plates
were blocked for two hours with a solution of 2.5% (w/v) powdered skim milk dissolved in
PBS-Tween with 0.05% Tween 20, pH 7.4, that had been incubated for two hours at 56 °C to
inactivate any endogenous phosphatase activity. Following block, microtiter plates were
washed thrice with PBS-T. Serum obtained from immunized mice was added at a dilution of
1:200 and serially diluted in PBS-T containing 1% (v/v) goat serum. Each sample was tested
in duplicate. Following incubation overnight at 4 °C, plates were washed thrice with PBS-T,
after which secondary antibody was added at a dilution of 1 μg/mL. Secondary antibodies
used in these studies were: alkaline phosphatase-conjugated goat anti-mouse IgG heavy and
light chain, IgG1 and IgG2c (Jackson ImmunoResearch). Plates were incubated for two
hours at room temperature and then washed three times with PBS-T. To each well, 100 μL of
alkaline phosphatase substrate (Fisher Scientific, Pittsburgh, PA) was added at a
concentration of 1 mg/mL dissolved in 50 mM sodium carbonate, 2 mM magnesium
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chloride buffer at pH 9.3 for colorimetric development. Plates were analyzed after 30 min
using a SpectraMax M3 microplate reader at a wave-length of 405 nm. Titer is reported as
the reciprocal of serum dilution at which the optical density (OD) value was at most 0.2, a
conservative endpoint greater than the average OD of saline-mouse serum, at a 1:200
dilution, plus two standard deviations. Avidity assays were performed similarly as described
for ELISA. Following overnight incubation with a 1:200 dilution of serum, plates were
washed thrice with PBS-T, then 150 μL sodium thiocyanate was added to the first well at a
concentration of 5 M in 0.1 M sodium phosphate buffer and serially diluted two-fold down
the plate five times to a final concentration of 0.15625 M. After 15 min, the plates were
washed five times with PBS-T, then the remaining steps were followed as described for
ELISA. An average of six non-treated wells was used as a control for each serum sample.
The relative avidity was calculated as the molar concentration of sodium thiocyanate which
yielded an OD that was 50% that of non-treated wells containing 0.1 M sodium phosphate
buffer.
2.8. ELISPOT
MultiScreen 96-well plates (Millipore Sigma, Billerica, MA) were pretreated with 35%
ethanol for one minute, washed three times with PBS, and coated overnight at 4 °C with 0.5
μg/mL F1-V in PBS. The following day, plates were dumped and blocked with complete
tissue culture medium consisting of RPMI 1640 (Gibco, Grand Island, NY), 10% (v/v) fetal
calf serum, 1% (v/v) penicillin–streptomycin, and 1% (v/v) l -glutamine for at least two
hours at 37 °C. Plates were dumped and single cell suspensions of bone marrow or splenic
lymphocytes harvested from mice between 214–218 days post-immunization were added to
the plates at 500,000 cells per well and placed in a 37 °C incubator for two hours. Plates
were then washed three times with PBS, another three times with PBS-T, and alkaline
phosphatase-conjugated goat anti-mouse IgG (
H
+
L
) secondary antibody (Jackson
ImmunoResearch) was added to the wells at a 1:500 dilution in 1% (v/v) goat serum PBS-T
for two hours at room temperature. Plates were washed six times with PBS-T and 25 μL per
well of BCIP/NBT liquid substrate (Millipore Sigma) was added to the wells and developed
for 15 min at room temperature. Plates were emptied, the bottoms removed, and gently
washed with nanopure water, after which they were left to dry completely. Spots were
counted using an AID Multispot Reader (AID, Strassberg, Germany). AID EliSpot software
(Version 6.0) was used for data analysis.
2.9. Peptide microarray printing and analysis
Twenty-seven 14- to 17-mer linear peptides (11 amino acid overlaps) spanning the full
length of F1 antigen and fifty-three 15-to 17-mer linear peptides (11 or 12 amino acid
overlaps) spanning the full length of V antigen, as well as full length proteins F1-V,
Bacillus
anthracis
protective antigen (PA), and chicken egg ovalbumin (OVA) were printed onto
Nexterion Slide AL (Schott, Louisville, KY) using a BioRobotics MicroGRID II microarray
printer (Genomic Solutions, Inc. Ann Arbor, MI). Peptides and proteins were dissolved into
DMSO at 10 mg/mL to ensure dissolution, then diluted 10-fold in water to 1 mg/mL. The
solution was diluted to bring the final concentration to 0.5 mg/mL in 1x print buffer (5%
(v/v) DMSO, 137 mM NaCl, 9 mM KOH, 11.3 mM NaH2PO4). The full length F1-V fusion
protein was used as a positive control, while PA and OVA were used as negative controls.
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Slides were printed with 16 arrays per slide, each array containing 16 × 16 spots per array,
with peptides printed in a serpentine pattern in triplicate. Following printing, slides were
vacuum sealed and stored at −80 °C until further use.
Microarray slides were warmed to room temperature and then placed on a hot plate at 37 °C
for 30 min to dry; the slides were then placed immediately in a blocking solution consisting
of 1% BSA (w/v) in PBS-T for one hour. Slides were subsequently blocked for one hour in
1% (v/v) goat serum PBS-T, then thrice washed with PBS-T and placed into a 16-well
incubation chamber (Nexterion IC-16) to separate individual arrays. Serum was added at a
1:20 dilution for one hour at room temperature with gentle agitation. Slides were washed
thrice with PBS-T and biotinylated goat anti-mouse IgG (
H
+
L
) secondary antibody
(Jackson ImmunoResearch) was added to wells at 1:1000 dilution for one hour at room
temperature with gentle agitation. Slides were washed thrice with PBS-T. Streptavidin Alexa
Fluor 555 conjugate was added to wells at a 1:10 0 0 dilution for 30 min at room
temperature with gentle agitation. Slides were removed from incubation chambers, washed
thrice with PBS-T, followed by another three washes with PBS. Slides were spun dry by
centrifugation and read on the Scanarray 50 0 0 laser scanner (GSI Lumonics, Bedford,
MA).
The scanned images of the microarray slides were analyzed using SoftWorRx Tracker v2.8
software (Applied Precision, Inc., Issaquah, WA) to detect and quantify the fluorescence of
each spot. R (v3.4.1) software was used to calculate the background-corrected fluorescence
of each spot as the mean fluorescence intensity of each spot minus the median background
fluorescence intensity surrounding the spot. The mean fluorescence intensity for each
peptide or protein was calculated as the average of the corresponding triplicate background-
corrected fluorescence spot values, and a heat map was generated based upon the magnitude
of individual mouse serum responses to each peptide. Saline-treated mouse serum was used
as a negative control.
2.10. Lethal challenge
Y. pestis
CO92 (NR-641) was obtained from BEI Resources. Frozen stocks were prepared in
advance of challenges.
Y. pestis
was initially grown on brain heart infusion (BHI) agar at
28 °C for 72 h; a single colony was isolated and inoculated in BHI broth and cultivated for
24 h at 28 °C while shaking at 120 rpm. Glycerol solution was added to the broth to bring
final glycerol concentration to 5% (v/v). Aliquots were snap frozen and stored at −72 °C.
Prior to challenge experiments, aliquots of the frozen stock were thawed and cultured on
tryptic soy agar (TSA) with 1% bovine hemoglobin at 37 °C and 5% CO 2 to check viability
and CFU enumeration. For challenge experiments, frozen
Y. pestis
stocks were thawed and
diluted in room temperature PBS. Groups (
n
= 8/group per vaccination group;
n
= 16/group
for naïve controls) of C57BL/6NCrl mice immunized as described previously were
anesthetized with an intraperitoneal injection of ketamine/xylazine cocktail and infected
intranasally with 5700, 7000, or 10,700 CFU (14 DPI) or 6500 CFU (218 DPI) of
Y. pestis
CO92 in a 50 μL volume.
Y. pestis
bacterial suspension was administered intranasally to
nares of mice by a micropipette. CFU enumeration was performed on infectious inoculum
preceding to and following infection of the animals to determine infectious dose
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administered. Animals were checked daily for morbidity and mortality over the course of
two weeks. All activities were performed in an animal biosafety level 3 (ABSL-3) laboratory
at Iowa State University with protocols approved by the Iowa State University Institutional
Animal Care and Use Committee and Institutional Biosafety Committee.
2.11. IFN γ and TNF α neutralization
Female C57BL/6NCrl mice (
n
= 12 per group) were immunized subcutaneously with the
Combination Nanovaccine and anti-F1-V total IgG antibody titers were evaluated at 13 DPI.
A cohort of mice (
n
= 4 per group) then received 1 mg each of anti-TNF
α
(rat IgG1,
XT3.11) mAb and anti-IFN
γ
(rat IgG1, XMG1.2) mAb delivered intraperitoneally in 500
μL PBS. The mice were challenged intranasally the following day (14 DPI) with 70 0 0 CFU
of
Y. pestis
CO92, as described above.
2.12. Statistical analyses
Statistical analyses on ELISA and survival were performed using the Mantel-Cox log rank
test and the one-way analysis of variance (ANOVA) using Tukey’s multiple comparisons
test. Statistical significance in microarray serum responses to each peptide between
vaccinated groups of animals was evaluated using Student’s
t
-test. A p-value ≤ 0.05 was
considered to be statistically significant. All analyses were performed using GraphPad Prism
v. 7.0 (GraphPad, La Jolla, CA).
3. Results
3.1. F1-V nanovaccine characterization
Following synthesis, the F1-V nanovaccine was characterized for size distribution, surface
charge, release kinetics, and encapsulation efficiency of encapsulated F1-V. The size of the
F1-V nanovaccine was consistent with previous work [37], with a mean diameter of 228
± 78 nm and a polydispersity index of 0.12, indicating a narrow size distribution (Fig. 1 A
and 1 B). Zeta potential measurements of the F1-V nanovaccine demonstrated a negative
surface charge, −34.8 ± 1.4 mV, consistent with that of empty nanoparticles, as reported
previously [41]. In order to provide sufficient antigen to induce a robust immune response
for rapid protection, this nanoparticle formulation was designed with a high (10% w/w)
loading of F1-V providing an initial burst of approximately 75% of the protein released
within the first 24 h (Fig. 1 C). The encapsulation efficiency of the F1-V within the various
nanoparticle batches was 51–72%.
3.2. Combination nanovaccine induces rapid protective immunity
In order to assess the potential of CDNs and nanovaccines to rapidly stimulate protective
immunity, C57BL/6NCrl mice (
n
= 8–12/group;
n
= 16/group for naïve controls) were
immunized subcutaneously with a single dose of one of the following vaccine formulations:
i) soluble F1-V adjuvanted with CDNs (CDN Vaccine), ii) F1-V encapsulated into 20:80
CPTEG:CPH nanoparticles (Nanovaccine), iii) F1-V encapsulated into 20:80 CPTEG:CPH
nanoparticles co-adjuvanted with CDNs (Combination Nanovaccine), or iv) saline alone.
Blood samples were collected from immunized animals at 13 DPI (Fig. 2 A, C, E). Anti-F1-
V total IgG antibody titers from CDN Vaccine- and Combination Nanovaccine-immunized
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animals evaluated at 13 DPI were similar in magnitude and showed significantly (
p
≤
0.0001) higher titers than sera from animals immunized with the Nanovaccine alone.
Consistent with these results, the avidity of the anti-F1-V serum IgG antibodies was similar
between CDN Vaccine and Combination Nanovaccine-immunized mice (Supplementary
Fig.1).
Mice were challenged (
n
= 8–12/group;
n
= 16/group for naïve controls) intranasally 14 DPI
with escalating lethal doses of
Y. pestis
CO92 in separate studies and survival was assessed
over a period of two weeks (Fig. 2 B, D, F). All naïve (i.e., saline-treated) mice succumbed
to infection within four days of challenge, while all Nanovaccine-immunized mice
succumbed over a period of eight days following challenge (Fig. 2 B). In contrast,
immunization with Combination Nanovaccine yielded 100% protection against all challenge
doses. Mice immunized with the CDN Vaccine showed similar protection against lethal
challenge with 5700 CFU
Y. pestis
, however lower levels of efficacy were observed with
increasing challenge doses. Immunization with the Combination Nanovaccine provided
significantly higher protection (
p
≤ 0.05) compared to the CDN Vaccine when challenged at
70 0 0 and 10,70 0 CFU.
The results indicate that while the CDN vaccine induced a similar anti-F1-V antibody titer as
did the Combination Nanovaccine formulation, only the latter formulation induced
protection at 14 DPI against all three challenge doses of
Y. pestis
CO92, suggesting a
qualitative difference in the immune response induced by the Combination Nanovaccine. In
addition, to investigate the contribution of cell-mediated immunity, a cohort of female
C57BL/6 mice (
n
= 4 per group) were immunized subcutaneously with the Combination
Nanovaccine as described above and were treated intraperitoneally with 1 mg each of anti-
IFN
γ
and anti-TNF
α
monoclonal antibodies at 13 DPI to neutralize these cytokines. Serum
responses analyzed at 13 DPI showed similar high titer anti-F1-V IgG titers between control
mice and those injected with the neutralizing antibodies (Supplementary Figure 2A). In
agreement with previous studies [45, 46], it was observed that the protection afforded to
mice injected with IFN
γ
/TNF
α
neutralizing antibodies was significantly (
p
≤ 0.05)
reduced compared to vaccinated mice not receiving the antibody cocktail indicating that
cell-mediated immunity contributed to the protection induced by the Combination
Nanovaccine (Supplementary Fig. 2B).
As current plague vaccine candidates undergoing clinical trials consist of F1-V adsorbed to
aluminum-based adjuvants, mice (
n
= 8 per group) were immunized with either the
Combination Nanovaccine or 50 μg F1-V adjuvanted with alhydrogel in order to compare
the two vaccine formulations at inducing anti-F1-V humoral immune responses. Anti-F1-V
serum antibody responses evaluated at 9 DPI revealed that the Combination Nanovaccine
induced significantly higher anti-F1-V IgG titers compared to alhydrogel-immunized (
p
≤
0.05) and saline-treated (
p
≤ 0.0 0 01) mice, whereas mice immunized with F1-V adjuvanted
with alhydrogel failed to induce significantly higher anti-F1-V IgG titers compared to saline-
treated mice (Supplementary Figure 3). Therefore, the Combination Nanovaccine elicited
more rapid and higher anti-F1-V IgG titer than the alhydrogel-adjuvanted F1-V vaccine.
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3.3. Combination nanovaccine enhances long-lived protective immunity
Previous work from our laboratories has shown the potential for a single dose nanovaccine,
consisting of a soluble bolus of F1-V and F1-V containing nanoparticles, to enhance long-
lived antibody responses for at least 280 DPI and provide protective immunity [29, 37]. To
evaluate the potential for a single dose of the CDN vaccine and the two nanovaccines (with
no soluble F1-V bolus) to induce long-lived protective immunity, separate groups of C57BL/
6NCrl mice (
n
= 8–12 per group) were immunized subcutaneously with the same
formulations as above and anti-F1-V total IgG titers were evaluated between 14 and 218 DPI
(Fig. 3 A and Supplementary Figure 4). At all time points, sera from both Combination
Nanovaccine-immunized and CDN Vaccine-immunized mice showed comparable and
significantly (
p
≤ 0.0 0 01) higher titers than sera from Nanovaccine-immunized mice.
Serum was analyzed at 14, 79 and 178 DPI for presence of class-switched anti-F1-V IgG1,
IgG2c and IgG3 antibodies (Supplementary Fig. 5). All vaccination regimens produced
detectable IgG1 at 14 and 79 DPI significant (
p
≤ 0.0 0 01) over background, with
Combination Nanovaccine and CDN Vaccine groups also showing significant (
p
≤ 0.04)
responses at 178 DPI (Supplementary Fig. 5A). Additionally, Combination Nanovaccine-
and CDN Vaccine-immunized animals showed significantly (
p
≤ 0.05) higher IgG1 titers
compared to Nanovaccine-immunized mice at 79 DPI. Sera from animals immunized with
both Combination Nanovaccine and CDN Vaccine exhibited significantly (
p
≤ 0.0 0 01)
higher IgG2c antibody titers at all time points, and IgG3 at 14 DPI, compared to sera from
Nanovaccine-immunized and Saline-treated mice (Supplementary Fig. 5B and C). Lastly, a
subset of mice (
n
= 2–4) were euthanized 214–218 DPI and BAL fluid was collected for
evaluation of anti-F1-V lung IgG (Supplementary Fig. 6) and IgA. The BAL fluid recovered
from the animals immunized with Combination Nanovaccine and CDN Vaccine was found
to have significantly (
p
≤ 0.05) higher total anti-F1-V IgG compared to that from animals
immunized with Nanovaccine or treated with Saline. No IgA was detected in the BAL fluid
(data not shown).
After the single s.c. immunization, mice were challenged intranasally at 182 DPI with a
lethal dose of
Y. pestis
CO92 (6500 CFU) and survival assessed over a two-week period
(Fig. 3 B). All naïve mice succumbed to infection within three days of challenge. Both the
Combination Nanovaccine and CDN Vaccine immunization regimens induced superior
protection (
p
≤ 0.01) when compared to the Nanovaccine- or saline-immunized mice.
Specifically, immunization with the Combination Nanovaccine and CDN Vaccine provided
similar levels of protection, with the Combination Nanovaccine providing 75% protection
from lethal challenge and the CDN Vaccine providing 62% protection.
3.4. Combination nanovaccine provides broad antibody IgG recognition to F1-V linear
epitopes
A linearly overlapping peptide array was used to identify F1-V- specific IgG (
H
+
L
)
antibodies in serum collected from mice immunized with either CDN Vaccine, Nanovaccine,
or Combination Serum from Nanovaccine and naïve control mice collected at 14, 79, and
178 DPI was reactive to 14- to 17-mer linear peptides spanning the full length of F1 antigen,
15- to 17-mer linear peptides spanning the full length of V antigen, and F1-V fusion protein
(Fig. 4). The antibody response to F1-V and all linear epitopes in mouse serum collected at
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14 DPI was noticeably lower than at 79 and 178 DPI. In addition, naïve mouse serum did not
bind appreciably to either F1-V, or any F1 or V peptides, at any time point evaluated.
Serum from CDN Vaccine- and Combination Nanovaccine-immunized animals elicited
antibodies that bound multiple linear peptides from F1 and V antigens across all time points,
with responses observed to peptides F1–3, F11, and V45–46 at all time points evaluated (
p
≤
0.05 compared to non-vaccinated mice). At 178 DPI, serum antibody from both CDN
Vaccine- and Combination Nanovaccine-immunized animals continued to react with
peptides F9, V18, V32, and V44 (Fig. 4, blue arrows). Serum from mice immunized with
Nanovaccine possessed fewer F1- and V-peptide specific antibodies compared to CDN
Vaccine- and Combination Nanovaccine-immunized animals, however, responses to peptides
F1–3 and V44–45 were observed at all time points evaluated.
3.5. Combination nanovaccine induces long-lived plasma cells
As anti-F1-V-specific serum antibody was found to persist for at least 218 days, ELISPOT
analysis was performed on cells harvested from spleen and bone marrow at 214–218 DPI to
demonstrate the presence of the long-lived antibody secreting cells (ASCs). Analysis of
bone marrow revealed a significantly (
p
≤ 0.05) higher number of F1-V-specific ASCs in
CDN Vaccine- and Combination Nanovaccine-immunized mice compared to non-vaccinated
(dashed line) mice (Fig. 5 A). There was no difference in the number of ASCs in the bone
marrow of Nanovaccine-immunized mice compared to non-vaccinated mice. Additionally,
there was no difference in the number of ASCs detected in the spleens harvested from
immunized mice versus naïve mice at 218 DPI (Fig. 5 B). Analysis of ASC-derived antibody
IgG subclass expression revealed a trend of a greater number of F1-V-specific IgG2c ASCs
present in the bone marrow of CDN Vaccine- and Combination Nanovaccine-immunized
mice compared to non-vaccinated mice 218 DPI; however, this difference was not
statistically significant (Supplementary Fig. 7). There was no difference in the number of
F1-V-specific IgG2c ASCs in the spleen 218 DPI, nor was there a difference in the number
of F1-V-specific IgG1 ASCs between all vaccine groups in either spleen or bone marrow.
4. Discussion
With increasing concern of biological weaponization, it is of paramount importance to
enhance preparedness with biodefense vaccines that can induce both rapid and long-lived
protective immunity. To date, there is no FDA-approved vaccine against the weaponizable
Y.
pestis
. An efficacious, single-dose vaccine would be vitally important to induce rapid and
long-lived protective immunity both in the critical window of time immediately following an
exposure event as well as for military personnel against potential exposure after deployment.
Previous work on pneumonic plague vaccines from other laboratories has shown some
immunization success using aluminum hydroxide-based and TLR-targeting adjuvants;
however, many of these studies required prime-boost vaccination [10–13, 47, 48]. A few
studies have demonstrated the ability to induce protective immunity within 14 DPI against
pneumonic plague challenge with strains other than CO92 [49–51].
The goal of this work was to incorporate the benefits of two novel adjuvants (i.e., CDNs and
polyanhydride nanoparticles) in order to design a single-dose, combination vaccine that
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would provide both rapid and long-lived immunity. Previously, it was shown that a single
dose of intranasally administered nanovaccine containing 40 μg soluble F1-V antigen and 10
μg F1-V encapsulated into polyanhydride nanoparticles provided 100% protection for at
least 280 DPI [37]. Additionally, it was previously demonstrated that intranasally
administered 20:80 CPTEG:CPH nanoparticles elicited demonstrable F1-V-specific antibody
titers as early as 14 DPI [30]. Motivated by these studies, a combination nanovaccine
including R,R-CDG CDNs and F1-V-containing 20:80 CPTEG:CPH nanoparticles was
designed with rapid release of ~ 70% of the antigen (Fig. 1) in order to initiate the adaptive
immune response. This current work highlights the potent immunostimulatory properties of
polyanhydride nanoparticle- and CDN-based combination adjuvants, which individually
could not provide rapid protection (i.e., at 14 DPI) when the challenge inoculum contained ≥
70 0 0 CFU
Y. pestis
; however, when administered together, rapid and complete protection
against all three lethal doses of
Y. pestis
was achieved (Fig. 2). Additionally, when compared
to F1-V adjuvanted with alhydrogel, the Combination Nanovaccine was able to induce
significantly higher anti-F1-V IgG serum responses, highlighting its potential as a vaccine
candidate in the case of an acute exposure event in the event to
Y. pestis
(Supplementary
Figure 3).
The focus on the importance of serum antibody to the immunity induced by plague vaccines
is based largely on the following: 1) human patients infected with
Y. pestis
demonstrate
long-lived antibody responses to F1 and V antigens with little to no demonstrable T cell
responses [52]; 2) monoclonal antibodies passively transferred to mice have been shown to
be protective against lethal challenge [7–9]; 3) direct neutralization of LcrV has been shown
to be necessary to prevent Yop translocation into macrophages in vitro [6]; 4) murine
challenge models have suggested that anti-F1 IgG1 antibody titer correlates with survival
[5]; and 5) mucosal and serum anti-V antigen antibodies are the best correlates for survival
against pneumonic plague in mice [53]. Regardless of the route of immunization, the
induction or presence of anti-V neutralizing antibodies in the lungs is likely critical for
protection against pneumonic plague; and, it has been demonstrated that intratracheally
administered mAb 7.3, which binds a conformational epitope on LcrV located between
amino acids (a.a.) 135 to 275 [7], protected mice against lethal intranasal challenge [54–56].
The results herein support this notion, as high IgG levels in peripheral blood and BAL fluid
were observed 218 DPI (Supplementary Figs. 4 and 6) following a single, subcutaneous
administration of the Combination Nanovaccine.
Elevated antibody responses observed in this work are in agreement with previously reported
studies that demonstrated that the use of CDG as an adjuvant enhances antibody responses
compared to traditional adjuvants [18, 19, 57, 58]. In this work, the resultant antibody
responses induced by both the Combination Nanovaccine and CDN Vaccine were
characterized by high affinity, high avidity class-switched F1-V-specific IgG1 and IgG2c by
14 DPI (Fig. 2 and Supplementary Figs. 1 and 5). Following a single SC immunization, the
antibody response was also characterized by a stable F1-V-specific antibody response over
218 DPI (Fig. 3 and Supplementary Fig. 4) that was accompanied by evidence of F1-V-
specific long-lived plasma cells in the bone marrow, however these titers were not
necessarily correlated with protection at 182 DPI (Supplementary Fig. 7 & Fig. 5).
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Using microarray analysis, potential protective immunodominant peptides were identified
(Fig. 4) that were consistent with previously published reports. Xiao et al. identified mAbs
that bound to peptides F1–2, V19–20, and V28 and together conferred protection against
lethal challenge [8]. In the current study, responses to F1–3 were observed in sera from
immunized mice at all time points. It has also been reported that a neutralizing mAb BA5
binds a linear epitope containing amino acids 196–225, corresponding to peptides V31–39,
and protects mice against systemic challenge [59]. Consistent with this study, serum
antibody recognized the V32 peptide suggesting that the CDN Vaccine and Combination
Nanovaccine elicited antibodies with potentially similar neutralizing capacity as mAb BA5.
Khan et al. previously characterized peptide ‘g’ (a.a. 256–270) as a B cell epitope [60].
Similar antibody responses were observed in this study to peptides V44–46 (a.a. 258–286).
Interestingly, there are no previous reports of antibody recognition of peptide F9 (a.a. 49–
65), F11 (a.a. 61–77) or V18 (a.a. 102–118), and, therefore, these regions may be of interest
for further research. Previous studies from our laboratories have shown that serum IgG from
mice immunized subcutaneously with an
α
-galactosidase-modified F1-V-based nanovaccine
bound to F1 peptide and peptides in the V32–37 regions, similar to what was observed in
this work [61]. However, other work from our laboratories showed that intranasal delivery of
an F1-V-based nanovaccine induced serum IgG responses to F18 and V1–2, which were not
observed in this work suggesting that the route of administration plays a role in inducing
serum IgG responses to epitopes on F1-V [30]. Together, these results suggest that both
CDN Vaccine and Combination Nanovaccine elicited polyclonal antibody responses that
recognized multiple linear epitopes.
Previous studies have also demonstrated a role of T cells in protecting against fully virulent
strains of
Y. pestis
[62, 63]. Further studies have highlighted a role of IFN Ɣ/TNF
α
in
providing protection against pneumonic plague suggesting the need for cell-mediated
immunity in protection against pneumonic plague [45, 46, 63, 64]. This appears to be in
agreement with observations that, despite generating high titer antibody against F1-V,
African Green Monkeys are highly susceptible to pneumonic plague infection [13, 65, 66].
As previously mentioned, polyanhydride particles possess inherent adjuvanticity
demonstrating the ability to prime humoral and cell-mediated immune responses in vivo [30,
31, 40–42, 67, 32–39]; therefore, we investigated the ability of the Combination
Nanovaccine to induce cell-mediated immunity to F1-V by neutralizing IFN
γ
and TNF
α
prior to challenge. We observed that neutralizing these cytokines reduced the survival rates
of mice vaccinated with the Combination Nanovaccine indicating that the nanovaccine
regimen may be activating cellular immune responses that contribute to the protection
against
Y. pestis
CO92 (Supplementary Fig. 2).
Encapsulation of antigen into polyanhydride nanoparticles can provide many benefits for
labile proteins including maintenance of protein structure and adjuvant activity for multiple
vaccine antigens [25, 68–70], including F1-V [24]. The surface eroding characteristics of
these particles enables chemistry-dependent release kinetics of antigen providing for single-
dose (i.e., no booster) vaccination [29, 37, 43]. Previously, polyanhydride nanoparticles have
shown the ability to adjuvant pneumococcal surface protein A (PspA) resulting in the
induction of complete protection of mice from a lethal challenge of
S. pneumoniae
that was
comparable to that induced by alum-adjuvanted PspA with markedly less injection site
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reactogenicity [21, 43]. In addition to the adjuvanticity of the polyanhydrides, previous
reports have demonstrated that protein structure and functional activity of the labile
recombinant protective antigen from
B. anthracis
was maintained upon encapsulation and
release from polyanhydride nanoparticles after storage for at least four months at
temperatures up to 40 °C [25]. Consequently, encapsulation of proteins into polyanhydride
nanoparticles has the capability to both enhance protective immunity and provide extended
shelf storage, both of which may be beneficial with respect to stockpiling biodefense
vaccines such as plague vaccines.
In summary, these results indicate that the Combination Nanovaccine is a promising vaccine
candidate against
Y. pestis
based on its ability to induce both rapid and long-lived protective
immunity. Immune responses to F1-V were characterized by long-lived high antibody titers,
increased breadth of antibody responses to a broader array of epitopes, and induction of
long-lived plasma cells in the bone marrow for at least 218 DPI. With the ability to enhance
immune responses to F1-V and potential for enhanced shelf stability of labile proteins, the
Combination Nanovaccine shows promise as a next-generation vaccine platform against
weaponized
Y. pestis
.
Supplementary Material
Refer to Web version on PubMed Central for supplementary material.
Acknowledgments
The authors acknowledge financial support from NIH -NIAID (R01 AI111466) and the Iowa State University
Nanovaccine Institute. The authors thank Drs. David Kanne, Chudi Ndubaku, and Thomas Dubensky Jr at Aduro
Biotech for providing the CDG used in these studies, as well as Dr. Chris Minion at Iowa State University for
providing the BioRobotics MicroGRID II printer used for printing microarray slides. The authors acknowledge Dr.
Thomas Waldschmidt at the University of Iowa for his guidance in ELISPOT assay development. The authors
would also like to thank Min Zhang at Iowa State University for assistance with the statistical analyses. B.N.
acknowledges the support of the Vlasta Klima Balloun Faculty Chair.
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Statement of significance
•
Yersinia pestis
, the causative agent of pneumonic plague, induces a highly
lethal infection if left untreated. Currently, there is no FDA-approved vaccine
against this biodefense pathogen.
•We designed a combination nanovaccine comprising of F1-V antigen-loaded
polyanhydride nanoparticles and a cyclic dinucleotide adjuvant to induce both
rapid and long-lived protective immunity against pneumonic plague.
•Animals immunized with the combination nanovaccine maintained high
levels of antigen-specific serum IgG and long-lived plasma cells in bone
marrow and the serum antibody showed a high affinity for a broad range of F1
and V linear epitopes.
•The combination nanovaccine is a promising next-generation vaccine
platform against weaponized
Y. pestis
based on its ability to induce both rapid
and long-lived protective immunity.
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Fig. 1. Characterization of F1-V Nanovaccine.
A) Representative scanning electron photomicrograph of F1-V nanovaccine (scale bar = 3
μm). B) Nanoparticle size distribution of the F1-V nanovaccine (228 ± 78 nm), determined
via ImageJ analysis. C) Cumulative in vitro F1-V release profile from the nanovaccine.
Nanoparticles were suspended in PBS (pH 7.4) and released protein was quantified via a
microBCA assay.
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Fig. 2. Combination Nanovaccine provides rapid protective immunity against lethal Y. pestis
challenge.
Groups of C57BL/6NCrl mice (
n
= 12–16 per group) were immunized subcutaneously with
the following groups: F1-V + CDNs (CDN Vaccine), F1-V encapsulated into nanoparticles
(Nanovaccine), F1-V encapsulated into nanoparticles + CDNs (Combination Nanovaccine)
or saline. (A,C,E) Serum was collected at 13 DPI and evaluated via ELISA for total anti-F1-
V IgG antibody titers. The dashed line represents anti-F1-V IgG (
H
+
L
) antibody titers from
saline-treated mice as a negative control. *
p
≤ 0.0 0 01 compared to Nanovaccine and
saline. There was no significant difference in anti-F1-V IgG serum responses observed
within each vaccination group between experiments. Mice were challenged at 14 DPI with
(B) 5700 CFU, (D) 70 0 0 CFU, or (F) 10,700 CFU
Y. pestis
CO92 and survival was
monitored for two weeks post-challenge. *
p
≤ 0.0 0 01 compared to saline-treated mice. +
p
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≤ 0.0 0 01 compared to Nanovaccine-immunized mice. #
p
≤ 0.05 compared to CDN
Vaccine-immunized mice.
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Fig. 3. Combination Nanovaccine provides long-lived protective immunity against lethal Y. pestis
challenge.
Groups of C57BL/6NCrl mice (
n
= 12–16 per group) were immunized subcutaneously with
the following treatments: F1-V + CDNs (CDN Vaccine), F1-V encapsulated into
nanoparticles (Nanovaccine), F1-V encapsulated into nanoparticles + CDNs (Combination
Nanovaccine) or saline. (A) Serum samples were collected at 14, 36, 79, 121, and 178 DPI
and analyzed for total anti-F1-V IgG (
H
+
L
) antibodies via ELISA. The dashed line
represents the background anti-F1-V IgG (
H
+
L
) antibody response from naive mice. #
p
≤
0.05 compared to CDN Vaccine. *
p
≤ 0.0 0 01 compared to Nanovaccine-immunized and
saline treated mice. (B) Mice were challenged at 182 DPI with 6500 CFU
Y. pestis
CO92
and survival was monitored for two weeks post-challenge. #
p
≤ 0.01 compared to
Nanovaccine-immunized mice. *
p
≤ 0.05 compared to Saline-treated mice.
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Fig. 4. Combination Nanovaccine and CDN Vaccine provide broad antibody responses to
potentially protective linear epitopes.
Serum samples collected from C57BL/6NCrl mice (
n
= 12–16 per group) immunized with
either saline, CDN Nanovaccine, Nanovaccine, or Combination Nanovaccine at 14, 79, and
178 DPI was analyzed for total anti-F1-V IgG antibodies against twenty-seven 14- to 17-mer
linear peptides (11 amino acid overlaps) from the F1 antigen and fifty-three 15- to 17-mer
linear peptides (11 or 12 amino acid overlaps) from the V antigen. The peptides were
covalently bound to microarray slides as described in Materials and Methods. Each row
corresponds to a specific peptide, the top row representing peptide F1 and the proceeding
downward rows corresponding to each following linear peptide incrementally through
peptide V53. Each column represents responses from a single mouse. The mean
fluorescence intensity of serum responses to each peptide is represented by a range of color
from white (no response) to purple (maximum response). The full-length F1-V fusion
protein was used as a positive control, and Bacillus anthracis protective antigen (PA) and
chicken egg ovalbumin (OVA) were used as negative controls. Black arrows indicate
significance (
p
≤ 0.05) of Combination Nanovaccine and CDN Vaccine serum compared to
naïve serum at all time points evaluated. Blue arrows indicate significance (
p
< –0.05) of
Combination Nanovaccine and CDN Vaccine serum compared to naïve serum at 179 DPI.
(For interpretation of the references to color in this figure legend, the reader is referred to the
web version of this article.)
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Fig. 5. Long-lived antibody responses to F1-V originate from long-lived plasma cells in the bone
marrow.
Bone marrow and splenic lymphocytes were harvested at 213–218 DPI from groups (
n
= 3–
4/group) of C57BL/6NCrl mice immunized with CDN Vaccine, Nanovaccine, Combination
Nanovaccine regimens, or treated with saline, and were analyzed via ELISPOT for the
number of F1-V-specific antibody secreting cells. Data represent the number of F1-V-
specific antibody secreting cells per 50 0,0 0 0 cells in either A) bone marrow or B) spleen
from individual mice from one experiment. The dashed line represents the number of F1-V-
specific ASCs per 50 0,0 0 0 cells determined from saline-treated mice as a negative control.
*
p
≤ 0.02 compared to saline-treated mice. #
p
≤ 0.03 compared to Nanovaccine-immunized
mice.
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