Protective response to subunit vaccination against intranasal Burkholderia mallei and B. pseudomallei challenge
Burkholderia mallei and B. pseudomallei are Gram-negative pathogenic bacteria, responsible for the diseases glanders and melioidosis, respectively. Furthermore, there is currently no vaccine available against these Burkholderia species. In this study, we aimed to identify protective proteins against these pathogens. Immunization with recombinant B. mallei Hcp1 (type VI secreted/structural protein), BimA (autotransporter protein), BopA (type III secreted protein), and B. pseudomallei LolC (ABC transporter protein) generated significant protection against lethal inhaled B. mallei ATCC23344 and B. pseudomallei 1026b challenge. Immunization with BopA elicited the greatest protective activity, resulting in 100% and 60% survival against B. mallei and B. pseudomallei challenge, respectively. Moreover, sera from recovered mice demonstrated reactivity with the recombinant proteins. Dendritic cells stimulated with each of the different recombinant proteins showed distinct cytokine patterns. In addition, T cells from immunized mice produced IFN-γ following in vitro re-stimulation. These results indicated therefore that it was possible to elicit cross-protective immunity against both B. mallei and B. pseudomallei by vaccinating animals with one or more novel recombinant proteins identified in B. mallei.
Procedia in Vaccinology 2 (2010) 73–77
vailable online at www.sciencedirect.com
1877-282X/$–see front matter © 2010 Elsevier B.V. All rights reserved.
3rd Global Vaccine Congress, Singapore 2009
Protective response to subunit vaccination against intranasal
Burkholderia mallei and B. pseudomallei challenge
Gregory C. Whitlock1,4, Arpaporn Deeraksa2, Omar Qazi5, Barbara M. Judy2,
Katherine Taylor2, Katie L. Propst8, Angie J. Duffy8, Kate Johnson5,
G. Barrie Kitto5,6, Katherine A. Brown5,6,7, Steven W. Dow8, Alfredo G. Torres1,2,3
and D. Mark Estes1,2,3*
1Department of Microbiology and Immunology, 2Department of Pathology and
3The Sealy Center for Vaccine Development, 4Department of Clinical Laboratory Sciences University of Texas Medical Branch,
Galveston, Texas 77555-1070;
5Institute for Cellular and Molecular Biology, 6Department of Chemistry and Biochemistry
,University of Texas at Austin, Austin Texas 78712;
7Department of Life Sciences, Imperial College London, London, UK SW7 2AZ
8Department of Microbiology, Immunology and Pathology and 8Rocky Mountain Regional Center of Excellence
Colorado State University,College of Veterinary Medicine, Fort Collins, CO 80523 1
Burkholderia mallei and B. pseudomallei are Gram-negative pathogenic bacteria, responsible for the diseases glanders and melioidosis,
respectively. Furthermore, there is currently no vaccine available against these Burkholderia species. In this study, we aimed to identify
protective proteins against these pathogens. Immunization with recombinant B. mallei Hcp1 (type VI secreted/structural protein), BimA
(autotransporter protein), BopA (type III secreted protein), and B. pseudomallei LolC (ABC transporter protein) generated significant
protection against lethal inhaled B. mallei ATCC23344 and B. pseudomallei 1026b challenge. Immunization with BopA elicited the
greatest protective activity, resulting in 100% and 60% survival against B. mallei and B. pseudomallei challenge, respectively. Moreover,
sera from recovered mice demonstrated reactivity with the recombinant proteins. Dendritic cells stimulated with each of the different
recombinant proteins showed distinct cytokine patterns. In addition, T cells from immunized mice produced IFN-J following in vitro re-
stimulation. These results indicated therefore that it was possible to elicit cross-protective immunity against both B. mallei and B.
pseudomallei by vaccinating animals with one or more novel recombinant proteins identified in B. mallei.
© 2010 Published by Elsevier Ltd.
Key words: Burkholderia,B. mallei,B. pseudomallei, vaccine, subunit vaccination, intranasal infection
Vaccines are the most efficacious and cost-effective means of protecting human and animal populations from infection.
At present, there are no approved vaccines available for use in protecting animals or humans against the Gram-negative
1* Corresponding author
Phone: (409) 266-6523; Fax: (409) 266-6810
74 Gregory C. Whitlock et al. / Procedia in Vaccinology 2 (2010) 73–77
bacterial pathogens Burkholderia mallei and B. pseudomallei. Therefore, we sought to develop and evaluate vaccines
that might be used to generate cross-protective immunity against both pathogens.
B. mallei is a non-motile bacterium responsible for glanders. This disease mainly affects horses, which are
considered to be the natural reservoir for infection, although mules and donkeys are also susceptible (1). Humans are
accidental hosts of B. mallei following prolonged and close contact with infected animals. B. mallei infects humans by
entering through open wounds and surfaces of the eyes or nose. Symptoms of glanders are dependent on the route of
infection (2). B. pseudomallei are motile bacteria causing melioidosis (3). Melioidosis is a life-threatening disease that
is mainly acquired through skin inoculation or pulmonary contamination, although other routes have been documented.
This saprophyte inhabitant of soil environments is mainly encountered in Southeast Asia and Northern Australia, but is
sporadically isolated in subtropical and temperate countries (4).
Both Burkholderia species are highly pathogenic and are classified as such in list B by the Centers for Disease
Control and Prevention (5). Burkholderia infections are difficult to treat with antibiotics and there are several reports
that indicate it is feasible to protect against melioidosis, at least in animal models of disease, with non-living vaccines
(6). There has also been some progress in identifying partially protective subunits. Passively administered antisera
raised against flagellin, polysaccharide, or conjugates of polysaccharide and flagellin, protect diabetic rats against
challenge with B. pseudomallei (7-9). However, B. mallei are not motile and do not produce flagella. Moreover, the
ability of flagellin to induce protection against an aerosol, or intranasal challenge has not been reported. Therefore, we
assessed flagellin as a potential candidate for inclusion in a Burkholderia vaccine and found it unsuitable (our
unpublished data). In contrast, all of the current evidence indicates that other surface-expressed or secreted proteins are
immunogenic and structural similarity exists between the proteins in B. pseudomallei and B. mallei (10-11). In this
study, we aimed to identify Burkholderia protective proteins that could be administered in vaccines to generate cross-
protective immunity against both B. mallei and B. pseudomallei. We hypothesize that cross-protection is possible based
on the similarities in antigenic composition and mechanisms of protection between these organisms, and if this is true,
development of a single vaccine which stimulates T-cell and antibody responses against melioidosis and glanders-
producing bacterial agents is feasible. If cross-protective immunity is observed, then it may be possible to develop a
single vaccine capable of generating protection against both melioidosis and glanders.
2. Recombinant protein expression and purification
Bioinformatics analysis of target sequences was used to indicate the presence (or absence) of an N-terminal
secretion sequence, transmembrane domains and homology to published crystal structures. The programs used were
SignalP v.3.0, TMHMM v.2.0 and PHYRE v.0.2, respectively (12-14). DNA sequences coding for B. mallei proteins
BopA (BMA_A1521; AA 23 - 512), BimA (BMA_A0749; residues 19 - 265), Hcp1 (BMA_A0742; residues 1 - 169)
and the B. pseudomallei protein LolC (BPSL2277; residues 44 - 266) (15) were cloned into the pET28a (+) expression
vector (Novagen). Primers were designed to PCR-amplify and clone the selected sequences in frame with a C-terminal
6x Histidine tag, for all four targets. Expand high fidelity DNA polymerase (Roche) was used to amplify targets from
B. mallei ATCC 23344 or B. pseudomallei K96243 genomic DNA. Once ligated into pET28a (+), plasmid DNA was
electroporated into Escherichia coli DH5Į. Cloned sequences were verified by DNA sequencing, using T7 promoter /
terminator oligonucleotide primers.
Target protein expression in E. coli (
ȜDE3) Rosetta was induced by growth in Overnight Express instant TB
medium (Novagen) for 18 – 20 h. Bacterial pellets were lysed using 10x CelLytic B (Sigma), and 6x His-tagged
proteins were purified by Ni2+ affinity chromatography. Purified proteins were dialyzed against two changes of 10 mM
Hepes / 150 mM NaCl, pH 7.4, aliquoted and stored at -80 ºC. Protein concentrations were determined using the BCA
kit (Pierce) using bovine serum albumin (BSA) as a standard, and sample purity assessed by SDS-PAGE.
3. Vaccination and challenge with B. pseudomallei or B. mallei
To evaluate the potential of Burkholderia surface expressed or secreted proteins to generate protective immunity, the
purified recombinant proteins were used individually or in combination to vaccinate mice via the intranasal (i.n.) route.
For immunization of mice against B. mallei challenge, 6-8 week old female BALB/c mice (n = 8 per group) were
primed with 10 Pg of recombinant proteins mixed with adjuvant (12.5 Pg of CpG oligodeoxynucleotide (ODN) 2395
(Coley Pharmaceuticals) and mixed with 12.5 Pg immune-stimulating complex (ISCOM) AbISCO 100 (Isconova AB),
followed by a 2 week boost of 5 Pg recombinant proteins with adjuvant. Four weeks post-boost, animals were infected
by intranasal inoculation with 2 LD50 of B. mallei ATCC 23344 administered i.n. to anesthetized mice. Control animals
were vaccinated with non-specific protein (Bovine Serum Albumin, BSA) and adjuvant. Animals were observed
closely following challenge and euthanized immediately when pre-determined endpoints were reached and these time
points were used to calculate survival times. All animal studies were approved by the Institutional Animal Care and
Use Committee at UTMB.
Gregory C. Whitlock et al. / Procedia in Vaccinology 2 (2010) 73–77 75
Following challenge with B. mallei, 12.5% of control animals survived for > 21 days (Figure 1A). In contrast,
survival percentages were significantly increased to 100% up to 21 days post-infection in mice vaccinated with
recombinant BimA or BopA. The surviving animals were euthanized at day 21 post-challenge, and the lungs and
spleens were homogenized and bacterial counts determined. In all the surviving animals, B. mallei were not recovered
from the lungs. However, B. mallei were recovered from the spleens of all surviving animals (data not shown).
To determine whether antibodies from infected mice reacted with the recombinant Burkholderia proteins, serum was
obtained from surviving animals. These sera were then tested for recognition of the purified recombinant Burkholderia
antigens Hcp1, BimA, BopA and LolC by Western blot. Serum from infected mice recognized each of the recombinant
antigens tested except for the LolC protein (Figure 2A) and were recognized by both IgG1 and IgG2a isotypes (Figure
Experiments were also conducted to determine whether the Burkholderia recombinant antigens were also capable of
generating protective immunity against B. pseudomallei challenge (Figure 1B). For these experiments BALB/c mice,
(n = 5 per group) were primed by i.n. inoculation with two adjuvant systems and immunized with 2 Pg of the purified
recombinant BimA, BopA, or LolC proteins given with CLDC adjuvant (cationic liposome-DNA complex), and then
boosted 2 weeks later and again 2 weeks after that. The adjuvant used for these studies consisted of cationic liposome-
DNA complexes, as reported previously (16) for use in non-specific immunotherapy of B. pseudomallei. Controls
consisted of mice administered CLDC adjuvant alone, diluent only or BopA antigen alone. Two weeks after the last
immunization, mice were subjected to lethal i.n. challenge with 2 LD50 of B. pseudomallei strain 1026b, as described
previously (17). Survival times were determined as noted above, and all the B. pseudomallei animal challenge studies
were approved by the Institutional Animal Care and Use Committee at Colorado State University.
Our data indicate that the vaccine candidates protected the animals from an initial, acute infection, but failed to
confer sterilizing immunity. Our future studies are focusing on the identification of the immunogenic domains in the
proteins coupled with optimization in the vaccination strategies to develop a fully protective vaccine.
We observed that immunization separately or as a combination of each of the four recombinant Burkholderia
antigens conferred at least 75% protection against B. mallei infection at 21 days compared to control mice (Figures 1A
and1B). Notably, one of the vaccine antigens (BopA) also conferred significant 60% longer term (> 60 days) protection
against B. pseudomallei challenge. Furthermore, when surviving BopA-vaccinated mice were sacrificed and lung,
spleen, and liver tissues plated for detection of B. pseudomallei, 25% of the surviving animals were sterile, at least
within the limits of detection of the organ culture (typically 50 CFU/organ).
0 2 4 6 8 10 12 14 16 18 20 22
Days post infect ion
Percent sur vival
0 2 4 6 8 10 12 14 16 18 20 22
Days post infect ion
Percent sur vival
Fig. 1. Survival of BALB/c mice immunized with different recombinant proteins and challenged with B. mallei ATCC 23344 and
. pseudomallei 1026b.
BALB/c mice were challenged i.n. with 2 LD50 B. mallei 4 weeks following intranasal vaccination with BimA (n= 2), BopA (n=5),
Combo (n=5), LolC (n=6), Hcp1 (n=8) or Control (n= 8). BopA- and BimA-vaccinated animals resulted in 100% survival up to 21 days
post-challenge. (B) BALB/c mice (n = 15, pooled data from 3 separate experiments) were immunized 3 times with the indicated antigens, then
challenged i.n. with 2 LD50 B. pseudomallei strain 1026b and survival times determined,.
76 Gregory C. Whitlock et al. / Procedia in Vaccinology 2 (2010) 73–77
Fig. 2. (A). B. mallei antibody response post-vaccination. Western blots were performed on sera collected 2 weeks post-boost to determine IgG
reactivity. Mice vaccinated with recombinant BopA, BimA, LolC and Hcp1, individually or in combination (combo), demonstrated response to all
proteins except LolC. Individually vaccinated mice produced a robust humoral response, although LolC was lacking. (B) Isotype-specific responses
to the vaccine candidates (IgG1 and IgG2a) detected from vaccinated and challenged mice.
4. ELISA assay for humoral responses to vaccination
Blood was removed from the orbital veins of immunized mice 2 days post-boost. The blood was allowed to clot at
room temperature prior to centrifugation at 5,000 x g. Serum was collected and stored at -20 ºC. The recombinant
Hcp1-, LolC-, BimA- and BopA-specific protein responses were determined by ELISA (Table 1). Briefly, microtiter
plates were coated with 5 Pg/ml of the appropriate recombinant protein in PBS overnight. Non-specific binding was
blocked using 1% (w/v) ovalbumin in PBS (OVA-PBS) for 1 h at RT. The plates were washed three times using 0.05%
(v/v) Tween 20 in PBS, and appropriate dilutions of sera in OVA-PBS were added in triplicate and incubated for 2 h at
37 ºC. Following the washes, biotinylated-rat-anti-mouse IgG1or IgG2a (BD Biosciences) diluted in OVA-PBS was
added and incubated for 2 h at 37 ºC. Next, HRP-conjugated Streptavidin (BD Biosciences) was added and incubated
for 25 min at RT. The substrate ABTS was added and the absorbance at 405 nm was measured. Antibody
concentrations (in µg/ml) were calculated from standard curves generated with IgG1 or IgG2a specific antibodies.
Table 1 Immune response to Recombinant Burkholderia Proteins
Protein IgG1a(Pg/ml) IgG2aa (Pg/ml)
Hcp1 95 40
LolC 0.104 0.902
BimA 2,970 5,700
BopA 2,070 6,080
The IgG1 and IgG2a responses were analyzed by ELISA using sera removed from immunized and infected mice.
LolCHcp BimA BopALolCHcp BimA BopA
combo LolC BimA BopA unvaxHcP
LolCHcp BimA BopALolCHcp BimA BopALolCHcp BimA BopALolCHcp BimA BopA
combo LolC BimA BopA unvaxHcP
combo LolC BimA BopA unvaxHcP
Gregory C. Whitlock et al. / Procedia in Vaccinology 2 (2010) 73–77 77
Immunization with recombinant LolC, BimA, Hcp1HCP and BopA proteins provided significant protection against
B. mallei ATCC 23344 and B. pseudomallei 1026b (Figures 1A and B ), in which B. mallei BopA gave the best results.
The combination of all subunits for protection from B. mallei may have some utility as a combined vaccine, although
not remarkably better than BopA alone. The serological results suggest that optimal level of Th1 (IgG2a) and Th2
(IgG1) responses are important for protection in B. mallei infection. An interesting observation is that in control group
mice, which received only CpG2395 and ISCOM, we have observed over 78% survival, whereas in a previous study,
mice receiving only ISCOM had ~12% survival after infection with 2 LD
of B. mallei ATCC 23344, indicating that
CpG2395 itself also offers protection (data not shown). There are several reports showing immune-enhancing activity
of CpG (18-24). Consistent with this study, all future studies will be repeated using a more prolonged time to challenge
at 4 weeks to reduce background protection offered by CpG2395, and a control group without CpG treatment will be
included as a control as was done in these studies.
This work was supported by NIH National Institute of Allergy and Infectious Diseases, Western Regional Center for
Biodefense and Emerging Infections (D.M.E., A.G.T and K.A.B) and a fellowship award to G.C.W. from the UTMB
Sealy Center for Vaccine Development. The work was also supported by NIH-NIAID grant U54 AI065357 (KP, AD,
1. Neubauer H, Sprague LD, Zacharia R, Tomaso H, Al Dahouk S, Wernery R,et al. 2005. Serodiagnosis of Burkholderia mallei infections in
horses: state-of-the-art and perspectives. Journal of Veterinary Medicine Series B 52:201-5.
2. Srinivasan A, Kraus CN, DeShazer D, Becker PM, D. DJ, Spacek L, et al. 2001. Glanders in a military research microbiologist. N Engl J Med
3. Dance DAB. 1991. Melioidosis - the tip of the iceberg. Clin Microbiol Rev 4:52-60.
4. Stone R. 2007. Infectious disease. Racing to defuse a bacterial time bomb. Science 317:1022-4.
5. Horn JK. Bacterial agents used for bioterrorism. 2003. Surgical Infections. 4:281-7.
6. Nelson M, Prior JL, Lever MS, Jones HE, Atkins TP, Titball RW, et al. 2004. Evaluation of lipopolysaccharide and capsular polysaccharide
as subunit vaccines against experimental melioidosis. J Med Microbiol 53:1177-82.
7. Brett P, Mah D, Woods D. 1994. Isolation and characterization of Pseudomonas pseudomallei flagellin proteins. Infect Immun 62:1914-9.
8. Brett PJ, Woods DE. 1996. Structural and immunological characterization of Burkholderia pseudomallei O-polysaccharide-flagellin protein
conjugates. Infect Immun. 64:2824-8.
9. Bryan LE, Wong SE, Woods D, Dance D, Chaowagul W. 1994. Passive protection of diabetic rats with antisera specific for the polysaccharide
portion of the lipopolysaccharide isolated from Pseudomonas pseudomallei. Can J Infect Dis. 5:170-8.
10. Whitlock GC, Estes DM, Torres AG. 2007. Glanders: off to the races with Burkholderia. FEMS Microbiol. Lett. 277:115-22.
11. Whitlock GC, Estes DM, Young GM, Young B, Torres AG. 2008. Construction of a reporter system to study Burkholderia mallei type III
secretion and identification of the BopA effector protein function in intracellular survival. Transactions of the Royal Society of Tropical
Medicine & Hygiene 102 Suppl 1:S127-33.
12. Bendtsen JD, von Heijne G, Brunak S, Bendtsen JD, Nielsen H, et al. 2004. Improved prediction of signal peptides: SignalP 3.0. Journal of
Molecular Biology 340:783-95.
13. Krogh A, Larsson B, von Heijne G, Sonnhammer EL. 2001. Predicting transmembrane protein topology with a hidden Markov model:
application to complete genomes. Journal of Molecular Biology 305:567-80.
14. Kelley LA, Sternberg MJ, Kelley LA, Sternberg MJE. 2009. Protein structure prediction on the Web: a case study using the Phyre server.
Nature Protocols 4:363-71.
15. Harland DN, Chu K, Haque A, Nelson M, Walker NJ, Sarkar-Tyson M, et al. 2007. Identification of a LolC homologue in Burkholderia
pseudomallei, a novel protective antigen for melioidosis. Infection & Immunity 75:4173-80.
16. Zaks K, Jordan M, Guth A, Sellins K, Kedl R, Izzo A, et al. 2006. Efficient immunization and cross-priming by vaccine adjuvants containing
TLR3 or TLR9 agonists complexed to cationic liposomes. J Immunol. 176:7335-45.
17. Goodyear A, Kellihan L, Bielefeldt-Ohmann H, Troyer R, Propst K, Dow S, et al. 2009.Protection from pneumonic infection with
burkholderiaBurkholderia species by inhalational immunotherapy. Infection & Immunity 77:1579-88.
18. Chen Y-S, Hsiao Y-S, Lin H-H, Liu Y, Chen Y-L. 2006. CpG-modified plasmid DNA encoding flagellin improves immunogenicity and
provides protection against Burkholderia pseudomallei infection in BALB/c mice. Infection & Immunity 74:1699-705.
19. Elvin SJ, Healey GD, Westwood A, Knight SC, Eyles JE, Williamson ED. 2006. Protection against heterologous Burkholderia pseudomallei
strains by dendritic cell immunization. Infection & Immunity 74:1706-11.
20. Krieg AM. Therapeutic potential of Toll-like receptor 9 activation. 2006. Nature Reviews Drug Discovery 5:471-84.
21. Utaisincharoen P, Kespichayawattana W, Anuntagool N, Chaisuriya P, Pichyangkul S, Krieg AM, et al. 2003. CpG ODN enhances uptake
of bacteria by mouse macrophages. Clinical & Experimental Immunology 132:70-5.
22. Vollmer J, Krieg AM. 2009. Immunotherapeutic applications of CpG oligodeoxynucleotide TLR9 agonists. Advanced Drug Delivery Reviews
23. Waag DM, McCluskie MJ, Zhang N, Krieg AM. 2006. A CpG oligonucleotide can protect mice from a low aerosol challenge dose of
Burkholderia mallei. Infection & Immunity 74:1944-8.
24. Wongratanacheewin S, Kespichayawattana W, Intachote P, Pichyangkul S, Sermswan RW, Krieg AM, et al. 2004. Immunostimulatory
CpG oligodeoxynucleotide confers protection in a murine model of infection with Burkholderia pseudomallei. Infection & Immunity 72:4494-