NAOSITE: Nagasaki University's Academic Output SITENAOSITE: Nagasaki University's Academic Output SITE
TitleA parenteral DNA vaccine protects against pneumonic plague.
Yamanaka, Hitoki; Hoyt, Teri; Yang, Xinghong; Bowen,
Richard; Golden, Sarah; Crist, Kathryn; Becker, Todd;
Maddaloni, Massimo; Pascual, David W
Citation Vaccine, 28(18), pp.3219-3230; 2010
Copyright © 2010 Elsevier Ltd All rights reserved.
A Parenteral DNA vaccine protects against pneumonic plague
Hitoki Yamanaka1,3, Teri Hoyt1, Xinghong Yang1, Richard Bowen2, Sarah Golden1, Kathryn
Crist1, Todd Becker1, Massimo Maddaloni1, and David W. Pascual1 *
1 Veterinary Molecular Biology, Montana State University, Bozeman, MT 59717
2 Department of Microbiology, Immunology, and Pathology, Colorado State University,
Fort Collins, CO 80521
3 Division of Comparative Medicine, Center for Frontier Life Sciences, Nagasaki University,
1-12-4 Sakamoto, Nagasaki 852-8523, Japan
*Corresponding Author. Mailing address: Veterinary Molecular Biology, Montana State
University, P.O. Box 173610, Bozeman, Montana 59717-3610. Telephone: (406) 994-6244.
Fax: (406) 994-4303. E-mail: firstname.lastname@example.org
The chemokine, lymphotactin (LTN), was tested as a molecular adjuvant using bicistronic
DNA vaccines encoding the protective Yersinia capsular (F1) antigen and virulence antigen
(V-Ag) as a F1-V fusion protein. The LTN-encoding F1-V or V-Ag vaccines were given by
the intranasal (i.n.) or intramuscular (i.m.) routes, and although serum IgG and mucosal IgA
antibodies (Abs) were induced, F1-Ag boosts were required for robust anti-F1-Ag Abs.
Optimal efficacy against pneumonic plague was obtained in mice i.m.-, not i.n.-immunized
with these DNA vaccines. These vaccines stimulated elevated Ag-specific Ab-forming cells
and mixed Th cell responses, with Th17 cells markedly enhanced by i.m. immunization.
These results show that LTN can be used as a molecular adjuvant to enhance protective
immunity against plague.
Running title: DNA vaccines for plague
Keywords: mucosal, IgA, vaccines, pneumonic plague, lymphotactin
Plague is a zoonotic disease caused by Yersinia pestis and assumes three forms of disease
in humans: bubonic, septicemic, and pneumonic. Bubonic and septicemic plague arise from
flea bites in which this vector has previously fed on infected animals [1,2]. Without treatment,
even bubonic plague results in high mortality, as does septicemic plague, and also causes
secondary pneumonic plague . Pneumonic plague is considered the most infectious form
because this disease can be readily transmitted from person to person via inhalation of
contaminated airborne droplets, and because of its rapid disease progression, there is a high
mortality rate . Throughout history, three major pandemics of plague disease have resulted
in an estimated 200 million deaths, and plague still remains endemic in regions of Africa,
Asia, and North and South America [1,2]. Therefore, development of efficacious vaccines
for plague is warranted.
At present, there are no licensed plague vaccines in the United States. For development of
a subunit vaccine to plague, efforts have focused on two primary Y. pestis antigens (Ags), the
outer capsule protein (F1-Ag), which is believed to help avoid phagocytosis [4,5], and the
low calcium response (LcrV) protein, V-Ag, which has been implicated in mediating a
suppressive effect upon Th1 cells via the stimulation of IL-10 . These individual vaccine
candidates are protective against bubonic and pneumonic plague [7,8]; however, these
vaccines, when applied in combination or in a fusion form, act synergistically in conferring
protection [9-12]. Although the observed protective immunity is largely Ab-dependent, Y.
pestis is an intracellular pathogen, and new data have shown that during early infection
events cellular immunity can contribute to effective protective immunity against plague
Lymphotactin (LTN; XCL1) is a member of the chemokine superfamily and classified into
the C chemokine family as a single C motif-1 chemokine in both mice and humans [16,17].
LTN is produced mainly by CD8+ T cells and NK cells and has chemotactic activity for
lymphocytes, CD4+ and CD8+ T cells, and NK cells upon binding to its specific receptor, XC
chemokine receptor-1 (XCR1) [18-22]. In addition, Boismenu et al. reported that TCRγδ
TCR+ intraepitheral lymphocytes (IELs) also produce LTN and induce innate and adaptive
immunity via chemotaxis for T cells and NK cells [19,23]. Thus, we hypothesize that LTN
can enhance recruitment of lymphocytes to react to the encoded plague DNA vaccines,
which should result in improved vaccine efficacy when given either by the mucosal or
parenteral routes similar to that previously shown .
To develop an effective vaccine against pneumonic plague, we constructed LTN-based
DNA vaccines that co-express V-Ag or F1-V fusion protein, using a bicistronic eukaryotic
expression vector, and assessed their vaccine efficacy against pneumonic plague challenge.
This is the first example of using an immunization approach with LTN DNA vaccines for
plague. These DNA vaccines did effectively prime and, with subsequent nasal F1-Ag protein
boosts, were able to confer variable protection against pneumonic plague. Thus, the LTN
DNA vaccine can be used to prime for protection against plague.
2. Materials and Methods
To develop the lymphotactin (LTN) DNA vaccines, the LTN cDNA was PCR-amplified
from pGT146-mLTN (Invivogen, San Diego, CA) as a template similar to that previously
described . Primers contained restriction sites for HindIII at the 5'-teminus and BamHI
at the 3'-terminus. After TA cloning (TOPO cloning kit, Invitrogen Corp., Carlsbad, CA) and
verification of the PCR products’ sequence, the LTN fragment was excised from the TA
vector and inserted into the pBudCE4.1 vector (Invitrogen Corp.) cut with HindIII and
BamHI, resulting in the plasmid pBud-LTN. The V and F1-V fusion Ags were then
amplified by PCR from a synthetic gene (GenScript, Piscataway, NJ), which was optimized
for mouse codon usage similar to that previously described , using primers which
contained sequences for NotI at the 5'-terminus and for KpnI at the 3'-terminus. The F1-V
fusion protein contained a linker sequence, Pro-Gly-Gly, between the F1 and V-Ag.
Following sequence confirmation of the TA cloned (TOPO cloning kit) PCR products, each
fragment was excised and inserted into the vectors, resulting in pBud-LTN/V and
pBud-LTN/F1-V. These DNA plasmids were purified with a commercially available plasmid
purification kit (Qiagen, Inc., Valencia, CA) and resuspended with DNase-free water.
To evaluate the expression of LTN, V-Ag, and F1-V fusion protein, we used supernatants
and lysates of 293A cells (ATCC, Manassas, VA) that were transfected with each DNA
plasmid using Lipofectamine LTX (Invitrogen). The 293A cells were cultured in a complete
medium (CM): RPMI-1640 (Invitrogen) containing 10% FBS (Atlanta Biologicals, GA), 10
mM HEPES buffer, 10 mM nonessential amino acids, 10 mM sodium pyruvate, 100 U/ml
penicillin, and 100 µg/ml streptomycin. The cell culture supernatants and lysates were
subjected to ELISA and immunoblotting 2 days after transfection, respectively, as described
2.3 Plasmid LTN production
To measure LTN expression in collected cell supernatants from transfected 293A cells, a
sandwich ELISA was used. Briefly, the anti-mouse XCL/lymphotactin mAb (8 µg/ml; R&D
Systems, MN) in sterile PBS was coated onto Maxisorp Immunoplate II microtiter plates
(Nunc, Roskilde, Denmark) at 50 µl/well. After overnight incubation at room temperature,
wells were blocked with PBS containing 1% BSA for 2 hour at 37 °C. Cell supernatants
from DNA vaccine-transfected 293A cells were loaded to individual wells, and to determine
the amount of LTN present in these supernatants, serially diluted recombinant mouse LTN
(R&D Systems, MN) was used to generate a standard curve. After overnight incubation at 4
°C, captured LTN was reacted with 0.4 µg/ml of biotinylated goat anti-mouse lymphotactin
Ab (R&D Systems, MN) for 1 hr at 37 °C. The specific reactions were detected by
anti-biotin HRP conjugated Ab (Vector Laboratories, CA) with incubation for 90 min at
room temperature. To visualize the specific reactions, ABTS substrate (Moss, Inc., Pasadena,
CA) was used, and absorbance was measured at 415 nm after 1 hour incubation at room
temperature using Bio-Tek Instruments ELx808 microtiter plate reader (Winooski, VT).
Transfected 293A cells were lysed in Milli-Q water; 30 µg of total protein were
electrophoresed on a 12% SDS-polyacrylamide gel, and then transferred onto a
nitrocellulose membrane (Bio-Rad Lab., Hercules, CA). The membrane was incubated with
anti-V-Ag rabbit serum  overnight at 4 °C and then with HRP-conjugated goat anti-rabbit
IgG (Southern Biotechnology Associates, Birmingham, AL) for 90 min at room temperature.
The reaction was visualized using the substrate 4-chloro-1-naphtol chromogen and H2O2
(Sigma-Aldrich, St. Louis, MO).
2.5 Immunizations and challenge
Female BALB/c mice were obtained from the National Cancer Institute (Frederick
Cancer Research Facility, Frederick, MD). Mice were maintained at Montana State
University Animal Resources Center under pathogen-free conditions in individual ventilated
cages under HEPA-filtered barrier conditions and were fed sterilized food and water ad
For intranasal (i.n.) immunization study, mice at 8-10 wks of age were immunized with
each DNA vaccine (80 µg/dose) on wks 0, 1, and 2 with each dose administered over a
two-day period. On wks 8 and 9, mice were nasally boosted with 25 µg of recombinant
F1-Ag protein  plus 2.5 µg of cholera toxin (CT; List Biological Laboratories, Campbell,
CA) adjuvant. Before challenge, a final boost of DNA vaccine (100 µg) and F1-Ag protein
(25 µg) plus CT adjuvant was given i.n. on week 12. One group of mice was immunized only
with Fl-Ag, as described.
For intramuscular (i.m.) immunization study, mice were immunized i.m. with each DNA
vaccine on wks 0, 1, and 2. For i.m. immunizations, 100 µg DNA were administered with a
needle into the tibialis anterior muscles of the two hind legs, as previously described .
On wks 8 and 9, mice were nasally boosted with 25 µg of F1-Ag protein plus 2.5 µg of CT
(List Biological Laboratories) adjuvant. Before challenge, a final boost of DNA vaccine
(100 µg) i.m. and F1-Ag protein (25 µg) plus CT adjuvant was given i.n. on week 12.
To test the efficacy of the LTN DNA vaccines against pneumonic challenge, immunized
mice were transported to Colorado State University, acclimated for at least 7 days, and
subjected to nasal challenge with 100 LD50 of Y. pestis Madagascar strain (MG05) >2 weeks
after the last immunization, as previously described [25,27]. All mice care and procedures
were in accordance with institutional policies for animal health and well-being.
2.6 Collection of serum and mucosal samples
Blood was collected from the saphenous vein. Fresh fecal pellets from individual mice
were solubilized in sterile PBS containing 50 µg/ml of soybean trypsin inhibitor
(Sigma-Aldrich) by vortexing for 10 minutes at 4 °C. After microcentrifugation,
supernatants were collected and frozen at -30 °C until assay.
2.7 Measurement of anti-F1- and V-Ag Abs titers by ELISA
Serum and fecal Ab titers were determined by ELISA. Briefly, recombinant F1- or V-Ag
 in sterile PBS was coated onto Maxisorp Immunoplate II microtiter plates (Nunc) at 50
µl/well. After overnight incubation at room temperature, wells were blocked with PBS
containing 1% BSA for 1 hour at 37 °C; individual wells were loaded with serially diluted
mouse serum or fecal samples in ELISA buffer (PBS containing 0.5 % BSA and 0.5 % Tween
20) overnight at 4 °C. Ag-specific Abs were reacted with HRP-conjugated goat anti-mouse
IgG, IgA, IgG1, IgG2a, or IgG2b Abs (Southern Biotechnology Associates) for 90 minutes at
37 °C. The specific reactions were detected with soluble enzyme substrate, 50 µl of ABTS
(Moss), and absorbance was measured at 415 nm after 1 hour incubation at room temperature
using Bio-Tek Instruments ELx808 microtiter plate reader. Endpoint titers were determined
to be an absorbance of 0.1 OD above negative controls after 1 hour at room temperature.
2.8 Lymphocyte isolation
Lymphocytes were isolated from nasal-associated lymphoid tissues (NALT), nasal
passages (NPs), head and neck lymph nodes (HNLNs), submaxillary glands (SMGs), spleens,
small intestinal lamina propria (iLP), Peyer’s patches (PPs), lumbar lymph nodes (LLNs),
sciatic lymph nodes (SLNs), and popliteal lymph nodes (PopLNs). HNLN, splenic, PP, LLN,
SLN, and PopLN mononuclear cells were isolated by conventional methods using Dounce
homogenization [26,27]. To isolate the mononuclear cells from NALT, NPs, SMGs, and iLP,
the tissues were minced and digested using 300 units/ml of Clostridium histolyticum Type IV
collagenase (Worthington, Freehold, NJ) for 30 min at 37 °C in spinner flasks . After
incubation, the digestion mixtures were passed through Nitex mesh (FairviewFabrics,
Hercules, CA) to remove undigested tissues. Mononuclear cells were separated by Percoll
(Pharmacia, Uppsala, Sweden) density gradient centrifugation with cells interfacing between
40 % and 60 % Percoll. Greater than 95 % viability was obtained for all lymphocytes
isolated from each tissue, as determined by trypan blue exclusion.
2.9 Ab ELISPOT
On wk 14, sets of studies were terminated to collect NALT, NP, HNLN, SMG, splenic, iLP,
PP, LLN, and PopLN mononuclear cells from the immunized mice. NALT, NP, HNLN, SMG,
splenic, iLP, and PP mononuclear cells were used from i.n.-immunized mice, and NP, HNLN,
splenic, iLP, LLN, and PopLN mononuclear cells were used from i.m.-immunized mice.
Ag-specific Ab-forming cell (AFC) responses by the ELISPOT method were detected, using
mixed cellulose ester membrane-bottom microtiter plates (MultiScreen-HA; Millipore,
Bedford, MA) by coating with 5 µg/ml F1- or V-Ag in sterile PBS, as previously described
. For total IgA or IgG AFC responses, wells were coated with 5 µg/ml goat anti-mouse
IgA or IgG Abs (Southern Biotechnology Associates) in sterile PBS.
2.10 Cytokine analysis
On wk 7 or 14, groups of i.n.- or i.m.-immunized mice, respectively, were evaluated for
cytokine responses to F1- and V-Ags. I.m.-immunized mice were boosted nasally with
F1-Ag protein at 8 and 9 wks and with both DNA and nasally dosed with F1-Ag protein at 12
wks. From i.n.-immunized mice, HNLN, splenic, and PP mononuclear cells were obtained,
and HNLN, splenic, and peripheral lymph nodes (PLNs), containing LLN, SLN, and PopLN
mononuclear cells, were obtained from i.m.-immunized mice. Total mononuclear cells from
each lymph tissue were resuspended in CM. Mononuclear cells were restimulated with 10
µg of recombinant F1-Ag, V-Ag, or with media as control in the presence of 10 U/ml human
IL-2 (PeproTech) for 2 days at 37 °C in a humidified 5 % CO2 incubator. Cells were washed
and resuspended in CM, and then these stimulated lymphocytes were evaluated by IFN-γ-,
IL-4-, IL-5-, IL-10-, and IL-13-specific ELISPOT assays, as described previously
To determine cytokine responses to F1- and V-Ags, on wk 7 or 14, groups of immunized
i.n. or i.m. mice were used, respectively. Cell cultures supernatants were collected from the
restimulated lymphocytes used in cytokine ELISPOT assay. These supernatants were used
to quantify the presence of IFN-γ, IL-6, IL-10, IL-17, and TGF-β by sandwich ELISA, as
previously described [25,29,30].
2.11 Statistical analysis
An ANOVA followed by Tukey’s method was used to evaluate differences in expression of
LTN, Ab titers, and IgG subclass responses; the Mann-Whitney U-test was used to evaluate
differences in AFC and CFC responses. The Kaplan-Meier method (GraphPad Prism,
GraphPad Software, Inc., San Diego, CA) was applied to obtain the survival fractions
following pneumonic Y. pestis challenges in LTN DNA vaccine immunized mice. Using the
Mantel-Haenszel log rank test, the P-value for statistical differences between surviving
plague challenges among the vaccinated groups versus those dosed with PBS was discerned
at the 95% confidence interval.
3.1 Expression of LTN, V-Ag, and F1-V fusion proteins
DNA vaccines for plague were generated using a bicistronic expression plasmid carrying 8
the molecular adjuvant, LTN, and under a separate promoter, V-Ag or F1-V fusion protein
sequences (Fig. 1A). These are called LTN/V-Ag and LTN/F1-V, respectively. A LTN-based
DNA vaccine encoding solely F1-Ag was found to be as immunogenic as the LTN/F1-V
vaccine and, thus, was not used for these studies. To verify the expression of LTN, V-Ag, and
F1-V fusion proteins, replicate cultures of 293A cells were transfected with each LTN DNA
vaccine, and cell culture supernatants and lysates were collected (Fig. 1B and C). LTN could
readily be detected in each of the cell supernatants from the transfected 293A cells when
compared to supernatants from DNA plasmids lacking LTN using a LTN-specific ELISA
(Fig. 1B). To detect the expression of V-Ag and F1-V fusion proteins, cell lysates were used
for immunoblotting. The V-Ag and the F1-V could be detected using the anti-V-Ag serum
(Fig. 1C). The F1-V protein migrated with an apparent MW of 54 kDa, which represents the
expected molecular mass for F1-Ag (17 kDa) plus V-Ag (37 kDa).
3.2 Antibody responses to F1- and V-Ag 1
To evaluate the relative immunogenicity of the LTN DNA vaccines, samples were
collected at 6 wks post-primary immunization and subsequently at two wk intervals. Past
studies with other DNA vaccines show that Ab responses are delayed and peak between 8
and 10 wks post-primary immunization . Ag-specific Ab titers in sera and fecal extracts
were measured by ELISA using F1- or V-Ag coated wells (Fig. 2). By 6 wks post-primary
immunization to F1- and V-Ag, significant Ab titers were detected in the i.n.- (Fig. 2A and B)
and i.m.-immunized groups (Fig. 2C and D), and Ab titers in the i.m.-immunized mice were
slightly greater than those in nasally immunized mice on wk 6. While Ab responses to
F1-Ag in i.n.-immunized mice steadily increased with time, the anti-F1- or -V-Ag Ab
responses in i.m.-immunized mice did not (Fig. 2C and D), nor did the anti-V-Ag Ab
responses in nasally immunized mice (Fig. 2B). Thus, to enhance Ab responses, mice were
boosted nasally with 25 µg of recombinant F1-Ag plus CT on wks 8 and 9, resulting in robust
mucosal IgA and serum IgG titers against both F1- and V-Ags by wk 12 in both i.n.- (Fig. 2A
and B) and i.m.-immunized mice (Fig. 2C and D). Before challenge study, a final boost with
DNA vaccine, as well as with recombinant F1-Ag plus CT, was given on wk 12.
3.3 IgG subclasses responses with i.n. or i.m. LTN DNA vaccinations
IgG subclass responses were determined using serum samples from i.n. or i.m. LTN DNA
vaccine immunized mice on wk 12 (Fig. 3). Nasal LTN DNA vaccinations induced
equivalent IgG1, IgG2a, and IgG2b anti-F1-Ag and -V-Ag Ab responses (Fig. 3A and B). In
the i.m. LTN DNA-immunized mice, significant differences were shown in responses
between each IgG subclass (Fig. 3C and D). LTN/V-Ag DNA vaccination induced greater
IgG1 anti-F1-Ag responses than IgG2a or IgG2b responses. The LTN/F1-V DNA vaccine
stimulated greater IgG2a endpoint titers than IgG1 or IgG2b anti-F1-Ag endpoint titers (Fig.
3C). These results show that LTN DNA vaccinations could induce mixed IgG subclass
responses, but these differences were influenced by the route and composition of the LTN
3.4 Vaccinations with LTN DNA vaccine protects against pneumonic plague
To test the efficacy of these nasal or i.m. DNA vaccines against pneumonic plague, LTN
DNA plus F1-Ag-immunized mice were challenged nasally with 100 LD50 Y. pestis
Madagascar strain >2 wks after the final boost, and the mean survival rates were determined
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(Fig. 4A and B). All mice dosed with PBS succumbed to challenge within 3 days (Fig. 4A and
B). Mice nasally vaccinated with LTN/βgal, LTN/V-Ag, or LTN/F1-V DNA showed partial
protection, 60% (P < 0.001), 20% (P < 0.001) and 40% (P < 0.005) survival, respectively (Fig.
4A). Mice vaccinated i.m. with LTN/V-Ag or LTN/F1-V showed better efficacy, 75% (P <
0.001) and 62.5 % (P < 0.001) survival, respectively (Fig. 4B). Mice i.m.-vaccinated with
LTN/βgal showed only partial protection, 36.5% (P < 0.001). The efficacy conferred by the
nasal LTN/V DNA vaccine plus F1-Ag protein-dosed mice was similar to the efficacy obtained
with mice nasally dosed with F1-Ag protein only (20% survival; P < 0.005) (Fig. 4A), and this
level of protection was significantly less than that conferred in i.m.-immunized mice (P < 0.05)
(Fig. 4B). Thus, the nasal LTN/V-Ag DNA vaccine was minimally protective. These results
show that the LTN DNA vaccines contribute to optimal protection against pneumonic plague
when given by the parenteral route rather than the mucosal route.
3.5 Nasal immunization with LTN vaccine stimulates weak mucosal IgA anti-V-Ag Abs
To assess the differences between parenteral and nasal immunizations with LTN vaccines,
nasal washes from mice immunized with the vaccine regimen were used for the challenge
studies (Fig. 5). As evident from the challenge studies, i.m. immunization showed the
protective responses, and both LTN/F1-V and LTN/V-Ag vaccines elicited similar nasal IgA
and IgG Ab titers to V-Ag and F1-Ag, except the LTN/V-Ag mice induced significantly
enhanced nasal IgG anti-V-Ag Ab titers (Fig. 5A). Since the nasal LTN/F1-V vaccine
showed the best protection against pneumonic plague challenge, analysis of nasal washes
from mice immunized with this vaccine regimen still showed weak nasal IgA or IgG
anti-V-Ag Abs, although IgA and IgG anti-F1-Ag Abs were induced (Fig. 5B), but with
diminished magnitude when compared to i.m. vaccinated mice. Thus, i.m. DNA priming