Evaluation of different ways of presenting LipL32
to the immune system with the aim of developing
a recombinant vaccine against leptospirosis
Fabiana Ko ¨mmling Seixas, Claudia Hartleben Fernandes, Daiane Drawanz Hartwig,
Fabricio Rochedo Conceic ¸a ˜o, Jose ´ Anto ˆnio Guimara ˜es Aleixo, and Odir
Anto ˆnio Dellagostin
Abstract: Leptospirosis, caused by bacteria of the genus Leptospira, is a direct zoonosis with wide geographical distribu-
tion. The implications in terms of public health and the economical losses caused by leptospirosis justify the use of a vac-
cine against Leptospira in human or animal populations at risk. In this study, we used the external membrane protein
LipL32 as a model antigen, as it is highly immunogenic. The LipL32 coding sequence was cloned into several expression
vectors: (i) pTarget, to create a DNA vaccine; (ii) pUS973, pUS974, and pUS977 for expression in BCG (rBCG); and (iii)
pAE, to express the recombinant protein in Escherichia coli, for a subunit vaccine. Mice were immunized with the various
constructs, and the immune response was evaluated. The highest humoral immune response was elicited by the subunit
vaccine (rLipL32). However, with rBCG, the titer was still rising at the end of the experiment. The serum of vaccinated
animals was able to recognize LipL32 on the membrane of the Leptospira, detected by indirect immunofluorescence. A
monoclonal antibody anti-LipL32 was shown to inhibit the growth of Leptospira in vitro, indicating potential protection in-
duced by the LipL32 antigen.
Key words: Leptospira, LipL32, recombinant BCG, subunit vaccine, DNA vaccine.
Re ´sume ´ : La leptospirose, cause ´e par des bacte ´ries du genre Leptospira, est une zoonose directe dont la distribution ge ´o-
graphique est vaste. Les implications en termes de sante ´ des populations et de perte e ´conomique cause ´e par la leptospirose
justifient l’utilisation d’un vaccin dirige ´ contre Leptospira chez l’humain ou la population animale a ` haut risque. Dans
cette e ´tude, nous avons utilise ´ la prote ´ine LipL32 de la membrane externe de la bacte ´rie comme antige `ne mode `le car elle
est hautement immunoge `ne. La se ´quence codante de LipL32 a e ´te ´ clone ´e dans plusieurs vecteurs d’expression : (i) pTar-
get, pour cre ´er un vaccin a ` ADN; (ii) les vecteurs pUS973, pUS974 et pUS977 pour permettre l’expression dans la BCG
(rBCG); et (iii) pAE pour exprimer la prote ´ine recombinante chez Escherichia coli dans la perspective d’un vaccin a ` sous-
unite ´. Les souris ont e ´te ´ immunise ´es avec les diffe ´rentes constructions et la re ´ponse immune a e ´te ´ e ´value ´e. La re ´ponse im-
mune humorale la plus forte a e ´te ´ obtenue avec le vaccin a ` sous-unite ´ (rLipL32). Cependant le titre du rBCG continuait
d’augmenter a ` la fin de l’expe ´rience. Le se ´rum des animaux vaccine ´s e ´tait capable de reconnaı ˆtre LipL32 membranaire de
Leptospira, tel que de ´montre ´ par immunofluorescence. Un anticorps monoclonal anti-LipL32 a pu inhiber la croissance de
Leptospira in vitro, indiquant une protection potentielle induite par l’antige `ne LipL32.
Mots-cle ´s : Leptospira, LipL32, BCG recombinant, vaccin a ` sous-unite ´, vaccin a ` ADN.
[Traduit par la Re ´daction]
Leptospirosis, a worldwide zoonotic infection with a high
incidence rate in tropical regions, is classified as an emerg-
ing infectious disease (McBride et al. 2005). Transmission
to humans occurs either through direct contact with an in-
fected animal or through indirect contact via soil or water
contaminated with urine from an infected animal (Faine
1982). There are more than 230 serovars among pathogenic
leptospiras. The local variability in serovars of endemic lep-
Received 19 September 2006. Revision received 24 November 2006. Accepted 27 November 2006. Published on the NRC Research
Press Web site at cjm.nrc.ca on 25 May 2007.
F.K. Seixas and D.D. Hartwig. Centro de Biotecnologia, Universidade Federal de Pelotas, C.P. 354, 96010-900, Pelotas, Brazil.
C.H. Fernandes and F.R. Conceic ¸a ˜o. Centro de Biotecnologia, Universidade Federal de Pelotas, C.P. 354, 96010-900, Pelotas, Brazil;
Faculdade de Veterina ´ria, Universidade Federal de Pelotas, C.P. 354, 96010-900, Pelotas, Brazil.
J.A.G. Aleixo. Centro de Biotecnologia, Universidade Federal de Pelotas, C.P. 354, 96010-900, Pelotas, Brazil; Faculdade de Nutric ¸a ˜o,
Universidade Federal de Pelotas, C.P. 354, 96010-900, Pelotas, Brazil.
O.A. Dellagostin.1Centro de Biotecnologia, Universidade Federal de Pelotas, C.P. 354, 96010-900, Pelotas, Brazil; Instituto de Biologia.
Universidade Federal de Pelotas, C.P. 354, 96010-900, Pelotas, Brazil.
1Corresponding author (e-mail: firstname.lastname@example.org).
Can. J. Microbiol. 53: 472–479 (2007)doi:10.1139/W06-138
##2007 NRC Canada
tospiral strains complicates the development of a vaccine
that could be used worldwide (Levett 2001). Therefore, the
development of new strategies for the prevention of lepto-
spirosis is necessary. Immunogenic proteins, especially the
outer membrane surface proteins, of pathogenic Leptospira,
may be effective vaccinogens. LipL32, also called haemoly-
sis associated protein-1 (Hap-1), is a promising vaccine can-
didate. It is the major outer membrane protein and it is
surface exposed (Cullen et al. 2005). Antigenicity of
LipL32 was first identified in naturally infected dogs (Gitton
et al. 1994), and it was demonstrated to be expressed during
infection in hamsters (Haake et al. 2000). Subsequently, it
was demonstrated that over 95% of human patients with lep-
tospirosis produce antibodies to LipL32 during infection
(Flannery et al. 2001). The nucleotide sequence coding for
LipL32 is conserved among pathogenic Leptospira, whereas
it is absent in nonpathogenic Leptospira (Haake et al. 2000).
Vaccination with an adenovirus vector encoding the lipL32/
hap-1 gene induced cross-protection in the gerbil model of
leptospirosis (Branger et al. 2001). The lipL32/hap-1 gene
derived from Leptospira interrogans serovar Autumnalis
provided protective immunity against a challenge with a het-
erologous strain of L. interrogans serovar Canicola (Branger
et al. 2005).
Possible strategies for developing LipL32 into a recombi-
nant vaccine against leptospirosis include its use as a subunit
vaccine or as a DNA vaccine, or the use of a vaccine vector,
such as Mycobacterium bovis BCG as a carrier for the anti-
gen. BCG offers unique advantages as a vaccine: it is unaf-
fected by maternal antibodies and therefore it can be given
at any time after birth, it is usually given as a single dose
eliciting a long-lasting immunity, it is stable and safe, it can
be administrated orally, and it is inexpensive to produce
when compared with other live vaccines (Ohara and Ya-
mada 2001). Subunit vaccines have the potential of inducing
protection without the risk of causing the illness, whereas
DNA vaccines are simple to make and deliver and can elicit
both humoral and cellular immunity (Shams 2005).
The purpose of the present study was to investigate the
immunogenicity of the LipL32 antigen presented to the im-
mune system under different forms. We evaluated LipL32 as
a subunit vaccine, as a DNA vaccine, and as a live recombi-
nant BCG vaccine expressing the LipL32 antigen.
Materials and methods
Bacterial strains and culture conditions
The bacterial strains and plasmids used in this study are
listed in Table 1. Leptospiras were grown at 30 8C in Elling-
hausen–McCullough–Johnson–Harris (EMJH) medium sup-
plemented with Leptospira enrichment EMJH (Difco, USA).
Escherichia coli strains DH5a and BL21(DE3) pLysS were
grown in Luria–Bertani medium at 37 8C with the addition
of the appropriate antibiotic (50 mg/mL kanamycin or
100 mg/mL ampicillin ). Mycobacterium bovis BCG Pasteur
strain 1173P2 was cultivated in stationary 25 cm2tissue cul-
ture flasks or in 50 mL tubes agitated at 250 r/min at 37 8C
containing Middlebrook 7H9 (Difco) liquid medium supple-
mented with oleic acid – albumin–dextrose–catalase (Difco),
0.05% Tween 80, and 0.2% glycerol.
DNA extraction, PCR amplification, and cloning
The genomic DNA from L. interrogans serovar Copenha-
geni strain Fiocruz L1-130 was prepared according to Sam-
Table 1. Bacterial strains, plasmids, and primers used in this study.
Strain, plasmid, or primerRelevant information Source or reference
Escherichia coli DH5a
E. coli BL21(DE3) pLysS
F–, lacZ DM15, endA1, recA1, supE44, relA1
F–ompT hsdSB (rB–mB–) gal dcm D(srl-recA)306::Tn10(TcR) (DE3)
Strain Fiocruz L1-130 was isolated from a patient during an outbreak
of leptospirosis in Salvador, Brazil
Mycobacterium bovis BCG Pasteur
Ko et al. 1999
Mammalian expression vector, Ampr, CMV promoter
Cloning and expression vector, Ampr, T7 promoter
E. coli – mycobacteria shuttle vector, Kanr, oriM, promoter hsp60
from Mycobacterium tuberculosis
E. coli – mycobacteria shuttle vector, Kanr, oriM, hsp60 promoter
and signal sequence of M. tuberculosis antigen 19 (MT19)
E. coli – mycobacteria shuttle vector, Kanr, oriM, promoter PANfrom
Ramos et al. 2004
Medeiros et al. 2002
pUS974Medeiros et al. 2002
Medeiros et al. 2002
Note: In primer sequences, lowercase letters denote nucleotides added or modified to facilitate incorporation of restriction sites, marked in bold.
Seixas et al. 473
##2007 NRC Canada
brook and Russel (2001). The lipL32 gene, excluding the
signal peptide coding sequence, was amplified by polymer-
ase chain reaction (PCR) using the oligonucleotide primers
listed in Table 1. PCR was carried out with the following
program: 95 8C for 5 min; followed by 35 cycles of 95 8C
for 30 s, 50 8C for 30 s, and 72 8C for 30 s; with a final
extension of 72 8C for 7 min. The reactions were performed
in a final volume of 25 mL containing 2.5 mL of 10? buffer,
0.5 mL of 10 mmol/L dNTP, 150 ng of each primer, 0.5 mL
(1 unit) of Platinum1Taq DNA Polymerase High Fidelity
(Invitrogen, USA), 0.5 mL of 50 mmol/L MgCl2, 18 mL of
Milli-Q water, and 2 mL of template DNA containing
25 ng/mL, in an Eppendorf Mastercycler thermocycler. The
PCR products were subsequently cloned into vectors listed
in Table 1. All cloning procedures were carried out accord-
ing to standard procedures (Sambrook and Russel 2001).
The plasmid used for nucleic acid immunization was the
pTARGETTMexpression vector (Promega, USA). The PCR-
amplified lipL32 DNA fragment was cloned into pTAR-
GETTM, resulting in the plasmid named pTARGET/lipL32.
A colony of E. coli DH5a containing the recombinant plas-
mid was cultured in Luria–Bertani broth containing ampicil-
lin. Large-scale plasmid DNA isolation was performed using
the Perfectprep1Plasmid Maxi kit, according to the manu-
facturer’s directions (Eppendorf, Germany). The DNA was
finally resuspended in phosphate-buffered saline (PBS) at a
concentration of 1 mg/mL. DNA concentration and purity
were determined by optical density, and the A260/A280ratio
was typically greater than 1.9. The pTARGET/lipL32 plas-
mid was verified by restriction digestion and by sequencing
of the entire insert with an automated DNA sequencer (Meg-
aBACE – Amersham Biosciences, USA).
Expression and purification of recombinant His6-LipL32
The E. coli expression vector named pAE (Ramos et al.
2004) was used for cloning the LipL32 coding sequence, re-
sulting in the plasmid named pAE-lipL32. This plasmid was
used to transform E. coli BL21(DE3) pLysS, which was cul-
tivated in liquid medium containing ampicillin. When the
absorbance at 600 nm reached 0.8, isopropyl-1-thio-b-D-gal-
actoside was added to a final concentration of 1 mmol/L,
and the cells were harvested by centrifugation 3 h later.
After centrifugation at 10 000g for 5 min at 4 8C, the cells
were lysed by sonication, centrifuged once more (10 000g
for 5 min), and the supernatant submitted to protein purifica-
tion by affinity chromatography in a nickel-charged Sephar-
ose column using the A¨KTAPrime chromatography system
(Amersham Biosciences, USA). Fractions of the purified
proteins were analyzed by 15% sodium dodecyl sulfate –
polyacrylamide gel electrophoresis (SDS–PAGE) and West-
ern blot (WB) using the 1D9 monoclonal antibody (MAb)
(Lu ¨dtke et al. 2003). Fractions containing rLipL32 were dia-
lyzed against PBS and glycine 0.1%, pH 8.0, for approxi-
mately 16 h at 4 8C. Protein in the final preparation was
quantified by the Bradford method (Bradford 1976).
Preparation of rBCG and WB analysis
The pUS973/lipL32, pUS974/lipL32, and pUS977/lipL32
vectors were introduced into M. bovis BCG Pasteur strain
by electroporation, as previously described (Bastos et al.
2002). BCG transformants were grown for 15 days in Mid-
dlebrook 7H9 medium (Difco) containing 15 mg/mL of ka-
namycin. A volume of 3 mL was centrifuged at 14 000g for
5 min, the pellet resuspended in 0.5 mL of 50 mmol/L Tris
(pH 7.5), and the cells lysed three times (40 s) using a Ribo-
lyser (Hybaid) at speed 4. The lysate was centrifuged at
14 000g for 5 min and the supernatant recovered. A volume
of 50 mL of 2? loading buffer (50 mmol/L Tris–HCl
(pH 6.8), 100 mmol/L DTT, 2% SDS, 10% glycerol, and
0.1% bromophenol blue) was added to 50 mL of the super-
natant, the suspension was heated to 100 8C for 10 min,
and a volume of 10 mL was submitted to SDS–PAGE.
For WB analysis, a total of 5 mg of rLipL32 or 50 mg of
rBCG cell lysates and negative controls was separated in
15% SDS–PAGE resolving gels and electrotransferred to ni-
trocellulose Hybond-C Super membrane (Amersham Bio-
sciences). After blocking with 5% nonfat dry milk, the
membranes were incubated for 1 h at 37 8C with 1D9 MAb
or pooled human sera at 1:100 dilution in PBS from conva-
lescent patients diagnosed with leptospirosis (microaggluti-
nation test titer of 25 000). After three washes of 20 min
each with PBS containing 0.1% Tween 20 (PBS-T), the
membranes were incubated for 1 h at room temperature
with a secondary antibody conjugated with goat anti-mouse
immunoglobulin G (IgG) peroxidase conjugate (Sigma) or
goat anti-human IgG peroxidase conjugate, diluted in PBS-
T and detected with ECLTMWB detection reagents (Amer-
sham Biosciences, USA).
BALB/c female mice were obtained from the University
of Sa ˜o Paulo, Brazil, (ICB-USP) animal house. Experimen-
tal animals were housed at the animal facility of the Bio-
technology Center of the Federal University of Pelotas
(UFPel). The animals were maintained in accordance with
the guidelines of the Ethics Committee in Animal Experi-
mentation of the UFPel throughout the experimental period.
Inoculation of mice and humoral immune response
For evaluating the humoral immune response induced by
the different vaccine preparations, mice aging from 5 to
6 weeks were allocated into eight groups each containing
five animals and inoculated twice, at days 0 and 21, as de-
scribed in Table 2. Sera from each group was collected from
the retro-orbital plexus at days 0, 21, 42, 63, 84, 105, 126,
147, and 168 to monitor antibody responses, determined by
ELISA against rLipL32. ELISA plates (Nunc Polysorp,
Nalge Nunc International, USA) were coated overnight with
500 ng of rLipL32 per well, diluted in carbonate–bicarbon-
ate buffer (pH 9.6), washed three times, and sera diluted
1:50 in PBS-T were added. After incubation for 1 h at
37 8C, followed by three washes with PBS-T, peroxidase-
conjugated rabbit anti-mouse immunoglobulins (Sigma,
USA) were added and the reaction visualized with o-phenyl-
enediamine dihydrochloride (Sigma) and hydrogen peroxide.
Optical density at 450 nm was determined in a Multiskan
MCC/340 (Titertek Instruments,
15 min later. To avoid plate differences, the actual absor-
bances were transformed into seroconversions by dividing
474Can. J. Microbiol. Vol. 53, 2007
##2007 NRC Canada
the actual absorbances by the absorbance of day 0 of the se-
rum from the same animal.
For WB analysis, a pool of sera of each experimental
group from day 0 and day 168 post inoculation was used.
Cultures of L. interrogans L1-130 in log phase were har-
vested, washed in PBS, resuspended in SDS–PAGE sample
buffer, and boiled for 10 min prior to separation by SDS–
PAGE and electrotransference onto a PVDF membrane
(Amersham Biosciences, USA). Following blocking in 5%
nonfat dry milk, the pool of sera diluted 1:25 was incubated
at room temperature for 1 h. After five PBS-T washes, the
membrane was incubated at room temperature for 1 h with
1:2000 goat anti-mouse IgG peroxidase conjugate. The reac-
tion was revealed with 3-4-chloronaphthol after five PBS-T
washes. A BenchmarkerTMpre-stained protein ladder (Invi-
trogen, USA) was used as the molecular mass standard.
Microscope slides (ICN Biomedicals, Inc.) were coated
with a 0.01% poly L-lysine solution (Sigma, USA) and dried
for 1 h at room temperature. A 7 day culture of
L. interrogans L1-130 was washed once in PBS and sus-
pended in PBS to a density of 108cells/mL. A volume of
approximately 1 mL was placed on the slide and incubated
for 2 h at 30 8C. The slides were washed twice with lepto-
spiral culture medium (LCM) and coated with a pool of sera
diluted 1:10 in LCM. The following groups were used in
this experiment: (i) pTARGET/lipL32, (ii) recombinant
LipL32, (iii) rBCG (pUS973/lipL32), (iv) rBCG (pUS974/
lipL32), (v) rBCG (pUS977/lipL32), (vi) MAb against
LipL32 (1D9), (vii) MAb against Salmonella outer mem-
brane protein, (viii) rabbit anti-mouse FITC (fluorescein iso-
thiocyanate) conjugate applied to slides without primary
antibody, (ix) GroEL antiserum, and (x) control mouse se-
rum used as primary antibody. After incubating for 1 h at
30 8C, the slides were washed twice with LCM, and a
1:100 dilution of rabbit anti-mouse FITC conjugate was
added and incubated for 1 h in a dark, humid chamber at
30 8C. After washing with LCM, a drop of mounting me-
dium was added and a coverslip was sealed with acrylic.
Stainingwas visualized by
(Olympus) with an excitation wavelength of 450 nm.
In vitro growth inhibition
Leptospiral in vitro growth inhibition was performed as
previously described by Tabata et al. (2002), with modifica-
tions. Briefly, a 7 day culture of L. interrogans L1-130 and
Leptospira biflexa Patoc were grown to 2 ? 108cells/mL
and diluted in tubes with fresh leptospiral culture medium
at final concentration of approximately 2 ? 107cells/mL. A
volume of 0.5 mL was incubated with twofold dilutions of
heat-inactivated ascite fluid of anti-LipL32 MAb, with the
dilutions ranging from 1:5 to 1:320. Experiments were per-
formed in triplicate for all dilutions tested. For the test con-
trols, three tubes were added with heat-inactivated ascite
fluid of an unrelated MAb, three tubes were added with
heat-inactivated normal mouse serum (NMS) with 1:5, 1:10,
and 1:20 final dilutions, and three tubes without any addi-
tion. All experiments were performed under sterile condi-
tions; ascite MAbs and NMS were passed through a
0.22 mm membrane (Millipore). The cell growth was ob-
served every other day by dark-field microscopy to verify
cell viability, including movement, morphology, and aggluti-
nation. After 7 days, the cultures were counted in a Petroff–
Hausser chamber to determinethe bacterial population.
Growth inhibition was estimated by comparing the number
of live cells in the presence of MAbs with the number of
live cells in the presence of NMS.
The results of mice serological assays were compared us-
ing analysis of variance and t test. Any differences were
considered significant at P £ 0.05.
The lipL32 gene was amplified and cloned into the
pTARGET plasmid vector. A recombinant clone was se-
lected, and the presence and integrity of the insert was con-
firmed by BamHI restriction digestion and DNA sequencing.
A large-scale plasmid DNA preparation was carried out, and
the DNA concentration was determined to be at 1 mg/mL.
This plasmid preparation was subsequently used for mice
Expression of the rLipL32
Escherichia coli BL21 (DE3) pLysS transformed with the
expression plasmid pAE/lipL32 expressed a soluble re-
combinant protein of the expected size (32 kDa). Purifica-
tion of rLipL32 from E. coli by affinity chromatography
was highly efficient, resulting in approximately 40 mg/L of
medium. A single band was observed when the protein was
submitted to SDS–PAGE (Fig. 1A).
The expression of LipL32 in BCG was evaluated by WB
using the 1D9 MAb (Fig. 1B). This MAb recognized a pro-
Table 2. Groups of mice and vaccine preparations used in the experiment.
100 mg of DNA
100 mg of DNA
15% Aluminum hydroxide
100 mg of rLipL32 + 15% aluminum hydroxide
106CFU of BCG
106CFU of BCG
106CFU of BCG
106CFU of BCG
Note: CFU, colony-forming units; IP, intraperitoneal injection; IM, intramuscular.
Seixas et al.475
##2007 NRC Canada
tein of approximately 30 kDa in rBCG cell lysates and
L. interrogans extracts. In contrast, no band was detected in
wild-type BCG extracts, demonstrating the specificity of this
antibody. Based on band intensities, the level of expression
of rLipL32 shown by recombinant BCG grown in vitro was
considered to be similar for vectors pUS974/lipL32 and
pUS977/lipL32 but lower for pUS973/lipL32 (Fig. 1B). WB
with pooled sera from patients diagnosed with leptospirosis
and with crude extracts of rBCG strains expressing LipL32
or the purified rLipL32 revealed a positive reaction (data
Humoral immune response
Seroconversions of BALB/c mice inoculated with the dif-
ferent vaccine preparations varied. The groups vaccinated
with DNA vaccine (pTARGET/lipL32), rBCG (pUS974/
lipL32 and pUS977/lipL32), and rLipL32 showed a serocon-
version statistically different (P < 0.05) from the other
groups (Fig. 2). The seroconversion of mice inoculated with
rBCG/lipL32 and pUS974/lipL32 or with rBCG/lipL32 and
pUS977/lipL32 was still rising at the end of the experiment.
As expected, the control groups inoculated with saline,
pTarget, and wild-type BCG did not show any seroconver-
sion; however, rBCG (pUS973/lipL32) also failed to stimu-
late a humoral immune response in mice.
WB analysis showed that sera from mice inoculated
with DNA vaccine (pTARGET/lipL32), rBCG (pUS974/
lipL32 and pUS977/lipL32), and rLipL32 recognized na-
tive LipL32 present in L. interrogans extract (Fig. 3). No
immuno-reactive bands were detected by WB using sera
from the control animals and rBCG transformed with
Fig. 2. Mean seroconversion, determined by ELISA, of anti-
LipL32 systemic antibodies from mice inoculated with different
vaccine preparations. Mice were inoculated at days 0 and 21 of the
experiment. (A) Evaluation of the immune response elicited by the
DNA vaccine pTARGET/lipL32 and the rBCG constructs. (B) Eva-
luation of the immune response elicited by the subunit vaccine
Fig. 3. Western blot analysis of pooled sera from mice inoculated
with different vaccines against crude extract of Leptospira interro-
gans. Lane 1, BenchmarkerTMprestained protein ladder (Invitrogen);
lane 2, pTARGET/lipL32 (day 0); lane 3, pTARGET/lipL32 (day
168); lane 4, rBCG (pUS974/lipL32) (day 0); lane 5, rBCG
(pUS974/lipL32) (day 168); lane 6, rBCG (pUS977/lipL32) (day 0);
lane 7, rBCG (pUS977/lipL32) (day 168); lane 8, rLipL32 (day 0);
lane 9, rLipL32 (day 168); lane 10, monoclonal antibody 1D9.
Fig. 1. (A) Sodium dodecyl sulfate – polyacrylamide gel electro-
phoresis of purified rLipL32. Lane 1, protein ladder (Invitrogen);
lanes 2–4, purified rLipL32 fractions. (B) Western blot with mono-
clonal antibody 1D9 demonstrating LipL32 expression in BCG.
Lane 1, rBCG transformed with pUS973/lipL32; lane 2, rBCG
transformed with pUS974/lipL32; lane 3, rBCG transformed with
pUS977/lipL32; lane 4, BCG (control).
476Can. J. Microbiol. Vol. 53, 2007
##2007 NRC Canada
pUS973/lipL32, demonstrating that the humoral immune
response induced by the different vaccine preparations
was specific (data not shown).
An indirect immunofluorescence assay revealed that anti-
bodies induced by rBCG, DNA vaccine, and rLipL32 were
able to bind to LipL32 on the surface of the bacterium, sug-
gesting that the antigen was properly presented to the im-
mune system. Sera from groups of mice that showed
seroconversion by ELISA, also recognized the LipL32 with
intense fluorescence on intact bacterial cells (Fig. 4). When
pooled sera from control groups was used, no fluorescence
In vitro growth inhibition by anti-rLipL32 MAb
This approach was used to investigate the ability of an
anti-rLipL32 MAb to inhibit the growth of Leptospira in vi-
tro. The 1D9 anti-LipL32 MAb was bacteriostatic, since cell
population remained constant over the incubation period in
all dilutions tested. A slight morphology change and reduc-
tion in cell movement was observed after 7 days of incuba-
tion with the 1D9 MAb. Bacterial growth was not affected
by either a MAb against an unrelated antigen or NMS. No
agglutination of bacterial cells was observed in any of the
Leptospirosis is an important zoonotic disease distributed
worldwide (McBride et al. 2005). Immunization of livestock
animals with bacterins is widely used, but the immune re-
sponse is short-lived, and animals require periodic boosters.
against the different serogroups of pathogenic leptospires
(Faine 1982; Yan et al. 2003; Martinez et al. 2004). There-
fore, the development of new strategies for the prevention of
leptospirosis is necessary. Several studies indicate that the
lipoprotein LipL32 is a promising vaccine antigen candidate.
It is the major outer membrane protein and is surface ex-
posed (Cullen et al. 2005). Over 95% of patients with lepto-
spirosis produce antibodies to LipL32 during infection
(Flannery et al. 2001). In the present study, three different
experimental immunization strategies against leptospirosis
using LipL32 were evaluated: a subunit vaccine using puri-
fied recombinant LipL32, a live recombinant M. bovis BCG
expressing LipL32, and a DNA vaccine. All approaches
were able to induce high levels of anti-LipL32 antibodies in
BALB/c mice, which recognized the native LipL32 from
L. interrogans by WB and indirect immunofluorescence. In
addition, an anti-LipL32 MAb was shown to be able to in-
hibit growth of Leptospira in vitro, indicating potential pro-
tection by the LipL32 antigen.
The expression of LipL32 in BCG was achieved with the
use of three different expression vectors. The plasmids
pUS973 and pUS974 contain the mycobacterial hsp60 gene
promoter, which has been widely used to express heterolo-
gous antigens in BCG (Stover et al. 1991; Ohara and Ya-
mada 2001; Dennehy and Williamson 2005). In addition,
the plasmid pUS974 carries a signal sequence from the My-
cobacterium tuberculosis 19 kDa antigen (MT19). The
MT19 signal sequence has been used to express heterolo-
gous proteins as lipoproteins on the mycobacterial surface
(Stover et al. 1993; Ohara and Yamada 2001; Bastos et al.
2002). The pUS977 vector carries the PANpromoter of My-
cobacterium paratuberculosis, first characterized and iso-
lated by Murray et al. (1992). Since then, it has been used
to express heterologous antigens from different pathogens
(Ohara and Yamada 2001; Dennehy and Williamson 2005).
Recombinant BCG transformed with pUS973/lipL32 did not
stimulated seroconversion against the recombinant antigen.
The reason for this might be the in vivo instability of the
plasmid construction. The strong promoter, present in
pUS973 vector, has been shown to cause instability of the
vector, both in vitro and in vivo (Medeiros et al. 2002).
However, the pUS974/lipL32 construct, which carries the
same promoter, did not show the same instability, possibly
because of the presence of the MT19 signal sequence.
To evaluate LipL32 as a naked DNA vaccine, we used the
pTargetTMvector. The cytomegalovirus (CMV) promoter
present in this vector can drive high levels of expression of
recombinant antigens in eukaryotic cells, stimulating an ef-
fective immune response for over 1 year (Wolff et al.
1992). Mice inoculated with pTarget/lipL32 presented a sig-
nificant humoral immune response, which remained high for
the duration of the experiment. A DNA vaccine containing
the lipL32 (hap-1) coding sequence cloned into the
pCDNA3.1 eukaryotic expression vector can induce protec-
tion against a lethal challenge with L. interrogans serovar
Canicola in the gerbil model (Branger et al. 2005). It re-
mains to be evaluated whether the pTarget/lipL32 construct
is able to afford a similar level of protection.
Mice that received two doses of rLipL32 expressed in
E. coli presented a high seroconversion 42 days after the
first inoculation. The seroconversion remained high until
the end of the experiment, 160 days after the first inocula-
tion. Previous attempts to induce protection in animal mod-
els with recombinant LipL32 failed (Branger et al. 2001,
Fig. 4. Indirect immunofluorescence with intact Leptospira interrogans. (A) Pooled sera from animals vaccinated with the DNA vaccine
(pTARGET/lipL32), (B) rBCG (pUS974/lipL32), (C) rBCG (pUS977/lipL32), (D) rLipL32, (E) pooled sera from the saline group.
Seixas et al.477
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2005). It is conceivable that the modes of production and (or)
extraction of this recombinant protein or its presentation to
the immune system are unsuitable to induce a protective im-
mune response, even though antibody production is obtained.
Comparing the immunization methods, all of them were
able to develop a specific humoral immune response against
LipL32. Although the highest seroconversions were obtained
with the purified recombinant protein, this might not be the
most adequate form of presenting the antigen to the immune
system. DNA immunization using the lipL32 coding se-
quence can be protective against a lethal challenge (Branger
et al. 2005). The use of BCG as a vaccine vector for the
LipL32 antigen may constitute an efficient form of present-
ing this antigen to the immune system. As a live replicating
bacterium, it keeps stimulating the immune response for a
long period of time. Indeed, the groups of animals vacci-
nated with rBCG showed antibody titers that were still in-
creasing at the end of the experiment. In addition,
recombinant BCG can induce a potent cellular immune re-
sponse against foreign antigens (Bastos et al. 2002; Mi-
chelon et al. 2006). The protective immune response against
leptospirosis is not fully characterized; however, there is in-
creasing evidence that the cellular immune response plays a
major role (Naiman et al. 2001; Vernel-Pauillac and Merien
2006). Similar to the DNA vaccine using lipL32, which is
protective possibly because of its capacity to induce primar-
ily a cellular immune response, the rBCG vaccine express-
ing LipL32 may also be protective, as it is known to induce
a strong cellular response. The main advantages of using
rBCG are the low cost of production and the long-lived im-
One particularly important finding is that mice inoculated
with DNA vaccine, rLipL32, and rBCG developed antibod-
ies that recognized the native protein in the intact membrane
of L. interrogans by indirect immunofluorescence. This find-
ing provides further evidence of LipL32 exposure on the
surface of the bacterial cell.
The 1D9 MAb anti-LipL32, when incubated with live lep-
tospiral cells, showed a bacteriostatic effect. This finding
showed that the MAb was able to attach to a cell-surface
epitope and hamper the protein biological function in the
outer membrane, indicating that an antibody immune re-
sponse against LipL32 may contribute to protecting an indi-
vidual against infection by pathogenic leptospiras.
The present study described different strategies for induc-
ing immune response against leptospirosis, including the use
of LipL32 as a recombinant subunit vaccine, a DNA vac-
cine, and a live recombinant BCG vaccine. To our knowl-
edge, this is the first report of expression of LipL32 in
BCG. Humoral immune response, specific to the recombi-
nant antigen, was demonstrated also in rBCG. Since the
amino acid sequence of LipL32 is highly conserved among
LipL32 may be broadly protective. We are currently investi-
gating the efficacy of the different strategies of presenting
the LipL32 to the immune system in inducing protection
against challenge with pathogenic L. interrogans, using a
This work was supported by the Brazilian Government
through CNPq and CAPES. We are grateful to Dr. A. Ko
from FIOCRUZ, Salvador, Brazil, for providing the L1-130
strain and for fruitful discussions.
Bastos, R.G., Dellagostin, O.A., Barletta, R.G., Doster, A.R., Nel-
son, E., and Osorio, F.A. 2002. Construction and immunogeni-
city of recombinant Mycobacterium bovis BCG expressing GP5
and M protein of porcine reproductive respiratory syndrome
virus. Vaccine, 21: 21–29. doi:10.1016/S0264-410X(02)00443-
Bradford, M.M. 1976. A rapid and sensitive for the quantification
of microgram quantities of protein utilizing the principle of pro-
tein-dye binding. Anal. Biochem. 72: 248–254. doi:10.1016/
Branger, C., Sonrier, C., Chatrenet, B., Klonjkowski, B., Ruvoen-
Clouet, N., Aubert, A., Andre-Fontaine, G., and Eloit, M. 2001.
Identification of the hemolysis-associated protein 1 as a cross-
protective immunogen of Leptospira interrogans by adenovirus-
mediated vaccination. Infect. Immun. 69: 6831–6838. doi:10.
Branger, C., Chatrenet, B., Gauvrit, A., Aviat, F., Aubert, A., Bach,
J.M., and Andre-Fontaine, G. 2005. Protection against Leptos-
pira interrogans sensu lato challenge by DNA immunization
with the gene encoding hemolysin-associated protein 1. Infect.
Immun. 73: 4062–4069. doi:10.1128/IAI.73.7.4062-4069.2005.
Cullen, P.A., Xu, X., Matsunaga, J., Sanchez, Y., Ko, A.I., Haake,
D.A., and Adler, B. 2005. Surfaceome of Leptospira spp. Infect.
Immun. 73: 4853–4863. doi:10.1128/IAI.73.8.4853-4863.2005.
Dennehy, M., and Williamson, A.L. 2005. Factors influencing the
immune response to foreign antigen expressed in recombinant
BCG vaccines. Vaccine, 23: 1209–1224. doi:10.1016/j.vaccine.
Faine, S. 1982. Guidelines for the control of leptospirosis. World
Health Organization, Geneva, Switzerland.
Flannery, B., Costa, D., Carvalho, F.P., Guerreiro, H., Matsunaga,
J., Da Silva, E.D., et al. 2001. Evaluation of recombinant Lep-
tospira antigen-based enzyme-linked immunosorbent assays for
the serodiagnosis of leptospirosis. J. Clin. Microbiol. 39: 3303–
3310. doi:10.1128/JCM.39.9.3303-3310.2001. PMID:11526167.
Gitton, X., Daubie, M.B., Andre, F., Ganiere, J.P., and Andre-Fon-
taine, G. 1994. Recognition of Leptospira interrogans antigens
by vaccinated or infected dogs. Vet. Microbiol. 41: 87–97.
Haake, D.A., Chao, G., Zuerner, R.L., Barnett, J.K., Barnett, D.,
Mazel, M., et al. 2000. The leptospiral major outer membrane
protein LipL32 is a lipoprotein expressed during mammalian in-
fection. Infect. Immun. 68: 2276–2285. doi:10.1128/IAI.68.4.
Ko, A.I., Galvao, R.M., Ribeiro Dourado, C.M., Johnson, W.D., Jr.,
and Riley, L.W. 1999. Urban epidemic of severe leptospirosis in
Brazil. Salvador Leptospirosis Study Group. Lancet, 354: 820–
Levett, P.N. 2001. Leptospirosis. Clin. Microbiol. Rev. 14: 296–
326. doi:10.1128/CMR.14.2.296-326.2001. PMID:11292640.
Lu ¨dtke, C.B., Coutinho, M.L., Jouglard, S.D.D., Moreira, C.N.,
Fernandes, C.H.P., Brod, C.S., et al. 2003. Monoclonal antibo-
dies against an outer membrane protein from pathogenic Leptos-
pira. Braz. J. Microbiol. 34: 1–4.
Martinez, R., Perez, A., Quinones, M.C., Cruz, R., Alvarez, A., Ar-
mesto, M., et al. 2004. Efficacy and safety of a vaccine against
478Can. J. Microbiol. Vol. 53, 2007
##2007 NRC Canada
human leptospirosis in Cuba. Rev. Panam. Salud Publica, 15: Download full-text
McBride, A.J., Athanazio, D.A., Reis, M.G., and Ko, A.I. 2005.
Medeiros, M.A., Dellagostin, O.A., Armoa, G.R., Degrave, W.M.,
Mendonca-Lima, L., Lopes, M.Q., et al. 2002. Comparative eva-
luation of Mycobacterium vaccae as a surrogate cloning host for
use in the study of mycobacterial genetics. Microbiology
(Reading, U.K.), 148: 1999–2009. PMID:12101288.
Michelon, A., Conceicao, F.R., Binsfeld, P.C., da Cunha, C.W.,
Moreira, A.N., Argondizzo, A.P., et al. 2006. Immunogenicity
of Mycobacterium bovis BCG expressing Anaplasma marginale
MSP1a antigen. Vaccine, 24: 6332–6339. doi:10.1016/j.vaccine.
Murray, A., Winter, N., Lagranderie, M., Hill, D.F., Rauzier, J.,
Timm, J., et al. 1992. Expression of Escherichia coli b-galacto-
sidase in Mycobacterium bovis BCG using an expression system
isolated from Mycobacterium paratuberculosis which induced
humoral and cellular immune responses. Mol. Microbiol. 6:
Naiman, B.M., Alt, D., Bolin, C.A., Zuerner, R., and Baldwin, C.L.
2001. Protective killed Leptospira borgpetersenii vaccine in-
duces potent Th1 immunity comprising responses by CD4 and
gd T lymphocytes. Infect. Immun. 69: 7550–7558. doi:10.1128/
Ohara, N., and Yamada, T. 2001. Recombinant BCG vaccines.
Vaccine, 19: 4089–4098. doi:10.1016/S0264-410X(01)00155-4.
Ramos, C.R., Abreu, P.A., Nascimento, A.L., and Ho, P.L. 2004. A
high-copy T7 Escherichia coli expression vector for the produc-
tion of recombinant proteins with a minimal N-terminal His-
tagged fusion peptide. Braz. J. Med. Biol. Res. 37: 1103–1109.
Sambrook, J., and Russel, D.W. 2001. Molecular cloning. Cold
Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.
Shams, H. 2005. Recent developments in veterinary vaccinology.
Vet. J. 170: 289–299. doi:10.1016/j.tvjl.2004.07.004. PMID:
Stover, C.K., de la Cruz, V.F., Fuerst, T.R., Burlein, J.E., Benson,
L.A., Bennett, L.T., et al. 1991. New use of BCG for recombi-
nant vaccines. Nature, 351: 456–460. doi:10.1038/351456a0.
Stover, C.K., Bansal, G.P., Hanson, M.S., Burlein, J.E., Palas-
zynski, S.R., Young, J.F., et al. 1993. Protective immunity eli-
cited by recombinant bacille Calmette-Guerin (BCG) expressing
outer surface protein A (OspA) lipoprotein: a candidate Lyme
disease vaccine. J. Exp. Med. 178: 197–209. doi:10.1084/jem.
Tabata, R., Neto, H.S., Zuanaze, M.A.F., Oliveira, E.M., Dias,
R.A., Morais, Z.M., ItoFumio, H., and Vasconcellos, S.A. 2002.
Cross neutralizing antibodies in hamsters vaccinated with leptos-
piral bacterins produced with three serovars of serogroup sejroe.
Braz. J. Microbiol. 33: 265–268.
Vernel-Pauillac, F., and Merien, F. 2006. Proinflammatory and im-
munomodulatory cytokine mRNA time course profiles in ham-
sters infected with a virulent variant of Leptospira interrogans.
Wolff, J.A., Ludtke, J.J., Acsadi, G., Williams, P., and Jani, A.
1992. Long-term persistence of plasmid DNA and foreign gene
expression in mouse muscle. Hum. Mol. Genet. 1: 363–369.
Yan, Y., Chen, Y., Liou, W., Ding, J., Chen, J., Zhang, J., et al.
2003. An evaluation of the serological and epidemiological ef-
fects of the outer envelope vaccine to Leptospira. J. Chin. Med.
Assoc. 66: 224–230. PMID:12854874.
Seixas et al.479
##2007 NRC Canada