DNA%20Vaccine%20Based%20on%20Leishmania%20major%20LACK%20Antigen%20Protects%20BALB/c%20Mice%20against%20Leishmania%20major%20Infection%20with%20Development%20of%20Th1%20Response

1
DNA Vaccine Based on Leishmania major LACK
Antigen Protects BALB/c Mice against Leishmania
major Infection with Development of Th1 Response
Yang LI1,2, Hajime HISAEDA1, Shinjiro HAMANO1,
Kazunari ISHII1, Yoichi MAEKAWA3, and Kunisuke
HIMENO1 1Department of Microbiology and
Immunology, Graduate School of Medical Sciences,
Kyushu University, Japan. 2Department of
Rheumatology,. 3Department of Parasitology and
Immunology, Tokushima University School of
Medicine, Japan.
Abstract
Susceptible BALB/c mice aberrantly develop Th2
cells in response to the infection with
Leishmania major. A large amount of IL-4 is
generated by oligoclonally expanded Vb4/Va8 CD4
T cells in response to a single epitope (aa
156-173) of parasite Leishmania homologue of
mammalian RACK1 (LACK) antigen, which is
responsible for the subsequent Th2 responses.
However, CD4 T cells secreting IFN-g still
exist. Here, we investigated different DNA
vaccines based on LACK, especially Th2
epitope-deleted LACK, and examined immune
responses induced by those vaccines. Five types
of plasmid expressing different regions of LACK
were constructed the full length of LACK (aa
1-312) Th2 epitope LACK156-173 dLACK, deleted
of aa 156-173 LACK1-155 and LACK174-312. BALB/c
mice (H-2d) were challenged to the footpad after
immunization with those plasmids by using a gene
gun. Vaccination with dLACK DNA protected mice
against the infection and resulted in
preferential induction of Th1 response.
LACK174-312 showed poor effect, while LACK1-155
appears to be more potent vaccine. After the
immunization, LACK-specific IgG was generated
only in the groups of full length LACK,
LACK?156-173 and LACK174-312, indicating that B
cell epitope(s) locates at LACK174-312. Both
IFN-g and IL-4 were secreted from splenocytes of
mice immunized with full length LACK under the
restimulation with recombinant LACK after the
immunization. In sharp contrast to the mice
immunized with LACK156-173, significant IFN-g but
not IL-4 was detected in the group of LACK1-155,
demonstrating the existence of epitope(s)
inducing Th1 responses. Further, immunization
with LACK1-155 developed Th1 response in DBA/2
mice (H-2d) but not in C57BL/6 mice (H-2b),
although LACK induced increased level of IFN-g in
both strain. Vaccination with DNA encoding aa
156-173-deleted LACK antigen can protect
susceptible BALB/c mice against infection with L.
major. The region of aa 1-155 is responsible for
the induction of Th1 response in a H-2d-restriced
manner.
Introduction
Next, to investigate the immune responses induced
after the DNA vaccination we immunized BALB/c
mice with those plasmids.
High levels of LACK-specific both IgG1 and IgG2a
were detected in mice immunized with full-length
LACK, dLACK and LACK-tail. However,
surprisingly, almost no Ig production was
detected in the mice vaccinated with LACK-head or
LACK-epitope. This indicated that the epitope(s)
recognized by B cells is located at the region of
a. a. 174-312 of LACK.
LACK-head induced Th1 responses in an H-2
restricted manner.
Mice from most inbred strains are resistant to
infection by Leishmania major, an obligate
intracellular parasite of macrophages in the
mammalian host. In contrast, mice from BALB/c
strains are unable to control infection and
develop progressive disease. Resistance and
susceptibility have been correlated with the
appearance of parasite-specific T helper 1 (Th1)
or T helper 2 (Th2) cells, respectively. LACK
(Leishmania homolog of mammalian RACKs, the
receptors for activated C kinase) is a highly
conserved protein among related leishmania
species and is expressed in both promastigote and
amastigote forms of the parasite. The early CD4
T cell response in BALB/c mice is dominated by
expansion of cells bearing Vß4/Va8 heterodimeric
TCR after recognition of a single I-Ad epitope
(amino acids 156-173) of the parasite LACK
antigen. Activation of these cells results in
the rapid production of IL-4 that drives the
subsequent Th2 response. Deletion of these
cells, either through thymic expression of LACK
as a transgene or superantigen-mediated deletion
of Vß4-expressing CD4 T cells, attenuated the
early production of IL-4 and promoted the
differentiation of Th1 effector cells that
controlled disease in infected BALB/c mice. In
the present study, we investigated different DNA
vaccines based on LACK, especially the DNA coding
the LACK antigen deleting of the Th2
response-inducing epitope (LACK?156-173), and
investigated the immune responses triggered. The
DNA vaccination of LACK?156-173 protected BALB/c
mice from subsequent L. major infection,
accompanying with a Th1-deviated immune response.
We found that the DNA sequence encoding a. a.
1-155 of LACK antigen is responsible for the
induction of IFN-?, thus plays an essential role
in the protective immunity. DNA sequence
encoding a. a. 174-312 of LACK antigen was proved
to be necessary for the generation of specific
antibody after the vaccination.
The N-terminal LACK is necessary for induction of
Th1 responses.
induced by LACK-head in other murine strains with
different MHC. DBA/2 mice, expressing the same
MHC class II molecules of I-Ad as BALB/c mice,
produced IFN-? after the vaccination with
indifferent of LACK or LACK-head. On the other
hand, a high amount of IFN-? was generated in
C57BL/6 mice (H-2b) that received LACK vaccine,
however, vaccination of B6 mice with
Experimental protocol
Also a typically Th1 immune response was detected
in mice with LACK-head vaccination but not in
mice received LACK-tail in response to the
secondary stimulation by rLACK in vitro. A high
level of IFN-? was also detected in mice received
LACK-head DNA when recombinant LACK-head instead
of full-length was used for stimulation. These
results suggested that the region of a. a. 1-155
in LACK antigen plays a critical role in the
induction of IFN-? by dLACK vaccination.
Conclusion
A Helios Gene Gun (Bio-Rad, Hercules, CA) was
used for gene transfer. The final tubing segment
resulted in delivery of 0.125 mg of gold
particles and 2 ?g of plasmid DNA. In some
experiments using IL-12-expressing plasmid, the
final tubing segment resulted in delivery of 1?g
of LACK plasmid or dLACK plasmid plus 1?g of
IL-12 plasmid DNA..
In conclusion, our results demonstrated that
epitope(s) inducing Th1 responses in an
H-2d-restriced manner were included in aa 1-155
of LACK antigen. Amino acid 174-312 is
responsible for LACK-specific antibodies
production. Both of the two regions composed the
potential DNA vaccine against L. major infection
in BALB/c mice. The forward region played a
major role, while the backward region was also
required to the vaccine efficacy.
Results
A DNA vaccine encoding LACK antigen deleted of
the Th2-inducing epitope protects BALB/c mice
against L. major.
Acknowledgements
It has been reported previously that a single
epitope of a. a. 156-173 from LACK antigen is
responsible for the induction of Th2 response in
susceptible BALB/c mice and that the vaccination
combined recombinant LACK antigen with
recombinant IL-12 can protect BALB/c mice against
infection with L. major, suggesting that immune
responses to LACK under Th1 promoting conditions
confer mice resistant to the infection. Thus, we
attempted to vaccinate those mice with plasmid
DNA encoding LACK depleted of aa 156 - 173
(dLACK). We immunized with the plasmid with or
without IL-12-expressing plasmid by using a gene
gun for 4 times at two-week intervals and footpad
swelling was monitored weekly after the challenge
infection. As shown here, DNA vaccination with
full-length LACK could not protect mice from the
following infection.
This work was supported by grants-in-aid from the
Ministry of Education, Culture, Sports, Science,
and Technology of Japan (15019075, 15025255,
15390136, 15659265).