Vaccination with Heat-killed Leishmania Antigen or

Recombinant Leishmanial Protein and CpG

Oligodeoxynucleotides Induces Long-Term Memory CD4
and CD8 T Cell Responses and Protection Against
Leishmania major Infection
Elizabeth G. Rhee,1 Susana Mendez,2 Javeed A. Shah,1
Chang-you Wu,1 Joanna R. Kirman,1 Tara N. Turon,1 Dylan F. Davey,1
Heather Davis,3 Dennis M. Klinman,4 Rhea N. Coler,5 David L. Sacks,2
and Robert A. Seder1

1Cellular Immunology Section,Vaccine Research Center and 2Laboratory of Parasitic Diseases, National Institute of
Allergy and Infectious Diseases, National Institutes of Health (NIH), Bethesda, MD 20892
3Coley Pharmaceutical Group, Ottawa, Ontario K1Y 4S1, Canada
4Laboratory of Retrovirology and Immunology, Center for Biologics Evaluation and Research, Federal Drug

Administration, Bethesda, MD 20892

5Infectious Disease Research Institute, Seattle, WA 98104

Abstract
CpG oligodeoxynucleotides (ODN) have potent effects on innate and adaptive cellular immune
responses. In this report, the ability of CpG ODN to confer long-term immunity and protec-
tion when used as a vaccine adjuvant with a clinical grade of leishmanial antigen, autoclaved
Leishmania major (ALM), or a recombinant leishmanial protein was studied. In two different
mouse models of L. major infection, vaccination with ALM plus CpG ODN was able to con-
trol infection and markedly reduce lesion development in susceptible BALB/c and resistant
C57BL/6 (B6) mice, respectively, up to 12 wk after immunization. Moreover, B6 mice immu-
nized with ALM plus CpG ODNs were still protected against infectious challenge even 6 mo
after vaccination. In terms of immune correlates of protection, ALM plus CpG ODN-vacci-
nated mice displayed L. major–specific T helper cell 1 and CD8 responses. In addition, com-
plete protection was markedly abrogated in mice depleted of CD8 T cells at the time of vacci-
nation. Similarly, mice vaccinated with a recombinant leishmanial protein plus CpG ODN also
had long-term protection that was dependent on CD8 T cells in vivo. Together, these data
demonstrate that CpG ODN, when used as a vaccine adjuvant with either a recombinant pro-
tein or heat-killed leishmanial antigen, can induce long-term protection against an intracellular
infection in a CD8-dependent manner.
Key words: CD4 T cells • CD8 T cells • DNA vaccines • parasitic infection • Th cells

Introduction
The induction and maintenance of cellular immune re- leishmaniasis, is known to require IL-12–dependent pro-
sponses is the primary goal of vaccines for a variety of intra- duction of IFN- from CD4 T cells (1–3). Resistant
cellular infections including Mycobacterium tuberculosis, Plas- C57BL/6 (B6)* mice develop protective Th1 responses
modium species, and Leishmania major. Effective primary that control infection, whereas susceptible BALB/c mice
immunity against L. major, the causative agent of cutaneous are unable to control infection due to an aberrant Th2 re-
sponse produced by a restricted population of V4V8
E.G. Rhee and S. Mendez contributed equally to this work. CD4 T cells (4, 5). The treatment of BALB/c mice with
Address correspondence to Robert A. Seder, Cellular Immunology
Section, Vaccine Research Center, Building 40, Room 3512, 40 Con- *Abbreviations used in this paper: ALM, autoclaved Leishmania major; B6,
vent Drive, MSC 3025, Bethesda, MD 20892. Phone: 301-594-8483; C57BL/6 mice; DC, dendritic cells; ODN, oligodeoxynucleotides; SLA,
Fax: 301-480-2565; E-mail: [email protected] soluble leishmanial antigen.

1565 The Journal of Experimental Medicine • Volume 195, Number 12, June 17, 2002 1565–1573
http://www.jem.org/cgi/doi/10.1084/jem.20020147
IL-12 protein or neutralizing Ab to IL-4 at the time of in- In studying the role of CpG ODN as a prophylactic vac-
fection has been shown to shift the immune response to a cine adjuvant, leishmanial proteins plus CpG ODN have
Th1 profile and prevent susceptibility to infection (1, 6). been reported to confer some protection after a challenge
This ability to alter Th responses in BALB/c mice and to with L. major (24, 25). In addition, susceptible BALB/c
assess the functionality of these responses with a biologic mice treated only with CpG ODN up to 2 wk before or
correlate has made this a useful model for vaccine develop- 20 d after infection with L. major were able to control in-
ment against diseases requiring Th1 immunity. fection (26, 27). It is notable that in the latter study, a Th1
Previous vaccine studies in BALB/c mice have demon- response was induced in the course of an ongoing Th2 re-
strated that long-lasting control (up to 12 wk after vaccina- sponse in mice treated with CpG ODN after infection
tion) of L. major infection can result from vaccination with (27). In striking contrast, treatment of susceptible BALB/c
plasmid DNA encoding of a specific leishmanial Ag (7). In mice with IL-12 protein alone was only sufficient to con-
contrast, vaccination with either soluble leishmanial Ag trol infection if administered at the time of infection with
(SLA) or a recombinant leishmanial protein plus IL-12 pro- L. major and continued for up to 1 wk (1). Together, these
tein conferred short-term (4, 8) but not long-term protec- data suggest that CpG ODN is more potent and durable
tion (9). However, if leishmanial protein plus IL-12 pro- than IL-12 protein in terms of immune and biologic effects
tein–vaccinated mice were repeatedly boosted with IL-12 in susceptible BALB/c mice after infection with L. major.
protein, control of infection was better sustained (10). Fur- In view of the potent in vivo effects on the cellular im-
thermore, mice vaccinated with leishmanial protein plus mune response elicited by CpG ODN, the data presented
IL-12 DNA also had long-term Th1 immunity and protec- here determined whether mice vaccinated with CpG
tion (9). These data suggested that persistent IL-12 is neces- ODN and either a clinical grade of autoclaved (heat-killed)
sary to sustain immunity sufficient for protection against L. L. major (ALM) Ag or recombinant leishmanial protein had
major when using a protein vaccine. Similarly, in a low dose sustained immunity and protection in two different mouse
challenge model that more closely mimics the human dis- models of L. major infection.
ease in terms of route and dose of infection, vaccination
with DNA again has been shown superior to vaccination
with leishmanial protein plus IL-12 in protecting against le- Materials and Methods
sion development and parasite burden in resistant B6 mice Mice. Female BALB/c mice were purchased from Taconic
challenged 12 wk after immunization (11, 12). Farms, Inc., and female B6 mice were purchased from the Divi-
To better understand why protein and DNA vaccines sion of Cancer Treatment, National Cancer Institute. All mice
have varying efficacy in the Leishmania mouse model, the were maintained in the National Institute of Allergy and Infectious
Diseases Animal Care Facility or Vaccine Research Center Animal
immune correlates of protection were analyzed. As previ-
Care Facility (Bethesda, MD) under pathogen-free conditions.
ously noted, protein vaccines that primarily elicit CD4 T ODN. Phosphorothioate-modified ODN sequence 1826
cell responses require persistent IL-12 for control of infec- containing two CpG motifs (underlined: TCCATGACGTTC-
tion in this model (10). In contrast, DNA vaccination in- CTGACGTT) was provided by Coley Pharmaceutical Group
duces Ag-specific CD4 and CD8 T cells that are required and used in most experiments. ODN sequence 1982 was used as
for long-term immunity (7, 13). In addition, plasmid DNA a control in some experiments (TCCAGGACTTCTCTCAG
vaccination may offer an advantage over protein vaccines GTT). The ODN contained endotoxin levels 0.1 EU/mg us-
by inducing a qualitatively and/or quantitatively different ing the limulus amebocyte lysis assay (Associates of Cape Cod,
type of cellular immune response through specific immu- Inc.). For the 6-mo challenge study in B6 mice, CpG ODN were
nostimulatory CpG sequences contained within the vector synthesized at the Center for Biologics Evaluation and Research
(14, 15). In this regard, nucleotide sequences containing Core Facility (Bethesda, MD). Sequences were TCAACGTTGA
and GCTAGACGTTAGCGT.
these CpG motifs have been synthesized and studied as
Immunization. BALB/c and B6 mice were injected subcuta-
possible immune adjuvants for diseases requiring Th1 neously in their hind footpad with either 50 g ALM prepared
immune responses (16, 17). CpG oligodeoxynucleotides from whole cell, heat-killed L. major promastigotes or 25 g of a
(ODN) have been shown to stimulate macrophages and recombinant leishmanial protein containing three Ag: LmSTI1
dendritic cells (DC) to synthesize several cytokines includ- (28), TSA (29), and LeIF (30). This three-Ag leishmanial protein
ing IL-12, IL-18, TNF-, IFN-, IFN-, and IFN- and vaccine is referred to as Trifusion (Corixa Corporation). ALM is
to up-regulate costimulatory molecules such as CD40 and a clinical grade reagent and contained 10 EU/dose. Trifusion
MHC class II (18–20). The range and level of cytokine protein contained levels of endotoxin at the lower limits of de-
production vary according to each ODN sequence and its tection in the limulus lysate assay. ALM was given alone or with
particular modifications (21). Moreover, CpG ODN have 1 g recombinant IL-12 (Genetics Institute) or 50 g CpG
ODN. Trifusion protein was given alone or with 25 g CpG
been shown to activate DC, leading to the presentation of
ODN. Each injection was suspended in sterile PBS and given in a
soluble protein to class I–restricted T cells and the induc- volume of 50 l. In some experiments, as a positive control, na-
tion of CTL responses (20, 22, 23). This ability of CpG ive BALB/c mice were treated with 1 g neutralizing murine Ab
ODN to induce both innate and adaptive cellular immune against IL-4 (clone 11B11; provided by W. Paul, NIH, Bethesda,
responses has made it a potential treatment and/or prophy- MD) 2 d before, and at the time of, infection. In other experi-
lactic vaccine adjuvant, respectively, for diseases requiring ments, as an additional positive control for long-term protection,
cellular immunity. mice were vaccinated with a “cocktail” of DNA containing 33

1566 Leishmanial Ag plus CpG ODN Vaccination Confers Sustained Immunity

g each of plasmid DNA encoding LACK Ag, LmSTI1 Ag, or tained as previously described. For intracellular staining of IFN-,
TSA Ag, combined for a total of 99 g DNA/dose (11). In the cells were stimulated with DC or DC pulsed with L. major for 6 h
6-mo challenge study, B6 mice were vaccinated intradermally in and then 10 g/ml brefeldin A was added. The cells were cul-
a volume of 10 l into the ventral surface of the ear. In all exper- tured for an additional 18 h and then fixed in 4% paraformalde-
iments, mice were boosted 2 wk after the initial injection into the hyde. Staining of surface and cytoplasmic markers was performed
same site with their initial regimen. To determine the role of as previously described (11). For each sample, at least 100,000
CD8 T cells in mediating protection with ALM plus CpG cells were analyzed.
ODN or Trifusion protein plus CpG ODN, mice were treated
1 d before, and at the time of, each vaccination with 0.5 g of a
rat Ig control Ab or rat anti–mouse CD8 Ab (2.43) that has been Results
shown to deplete 95% of CD8 T cells and was sufficient to ab-
Vaccination with ALM plus CpG ODN Confers Durable
rogate protection to DNA vaccination in this model (7, 13). In
addition, treatment with anti-CD8 did not diminish the number Protection Against L. major in BALB/c Mice. In previous
or function (e.g., IL-12 production) of CD11c cells (13). reports, vaccination with SLA or recombinant leishmanial
Infectious Challenge. L. major clone V1 (MHOM/IL/80/ and IL-12 protein was sufficient for the protection of sus-
Friedlin) promastigotes were grown as previously described (11). ceptible BALB/c mice against challenge with L. major at 2
Infective stage promastigotes (metacyclics) of L. major were iso- but not 12 wk after vaccination (9). To compare CpG
lated from stationary cultures (4–5 d old) by negative selection ODN with IL-12 protein as a vaccine adjuvant with a
using peanut agglutinin (Vector Laboratories). B6 mice were heat-killed preparation of leishmanial Ag for long-term im-
challenged intradermally at 2 or 12 wk after vaccination in both munity, BALB/c mice were vaccinated and boosted with
ears using 500 metacyclic promastigotes. In the 6-mo challenge ALM alone, ALM plus IL-12 protein, ALM plus CpG
experiment, infection was done in the ear opposite to vaccina-
ODN, or normal saline and infected 2 or 12 wk later.
tion. The lesions were monitored by measuring the diameter of
the induration of the ear lesion with a metric caliper. BALB/c
Consistent with previous reports (9, 24, 25), mice vacci-
mice were infected either 2 or 12 wk after the boost with 105 nated with ALM plus CpG ODN or ALM plus IL-12 pro-
metacyclic promastigotes. Parasites were injected into the footpad tein were able to control the infection when challenged in
subcutaneously contralateral from the site of vaccination at a vol- the opposite footpad 2 wk after vaccination (Fig. 1 A). In
ume of 50 l. Weekly footpad swelling measurements were re- contrast, mice vaccinated with ALM alone or with normal
corded using a metric caliper. saline developed ulcerations and were killed 3–4 wk after
Parasite Quantitation. Parasite loads in the ears of B6 mice infection. Mice vaccinated with ALM plus CpG ODN but
were determined as previously described (11). The number of vi- not ALM plus IL-12 protein were able to control the infec-
able parasites in each ear was determined from the highest dilu- tion when challenged 12 wk after vaccination (Fig. 1 B).
tion that promastigotes could be grown out after 7 d of incuba- Moreover, in the same experiment, mice that received
tion at 26 C. The number of parasites was also determined in the
ALM plus CpG ODN were able to sustain control of in-
local draining LN (popliteal) of BALB/c mice. The LN from
each mouse was recovered and mechanically dissociated using a fection up to 3 mo after challenge. The control of infection
pellet pestle and then serially diluted as previously described. For after ALM plus CpG ODN vaccination was comparable to
most experiments, ears or draining LN from at least three indi- or better than that in mice treated with anti–IL-4 (an addi-
vidual mice were analyzed for parasite burden. Statistical analysis tional positive control) at the time of infection (Fig. 1 C).
was done as previously described (11). To compare the protection from CpG ODN to that
Measurement of Leishmania-specific Production of IFN- from elicited by a DNA vaccine, an additional group of mice
CD4 T Cells after Infection. 4 wk after infection, draining LN was given a cocktail of DNA plasmids encoding the leish-
were harvested from different groups of vaccinated BALB/c manial Ag LACK, LmSTI1, and TSA previously shown to
mice. The nodes from a minimum of three mice in each group elicit long-term protection (Fig. 1 D). Mice vaccinated
were pooled and then purified into CD4 T cell subpopulations
with ALM plus CpG ODN were equally able to control
using magnetic-activated cell sorting columns (Miltenyi Biotec).
Flow cytometry confirmed 95% purity of CD4 T lympho-
infection, as measured by footpad swelling, as mice vacci-
cytes. The cells were then plated in triplicate in a 96-well micro- nated with the DNA vaccine. To verify that CpG ODN
titer plate at 2 105 cells/200 l and cultured with media, mac- alone had no protective effect, mice vaccinated and
rophages (5 104), or macrophages plus L. major. Macrophages boosted only with CpG ODN were not protected when
were obtained by intraperitoneal washings of naive BALB/c challenged 12 wk after vaccination (Fig. 1 D). In addi-
mice and then pulsed with or without L. major before being tional experiments, mice vaccinated with CpG ODN
added to the CD4 T cell cultures. Supernatants were collected alone or ALM plus control ODN (without CpG) were not
48 h later and assessed for production of IFN- by specific ELISA able to control infection when challenged 2 wk after boost
(BD PharMingen). The lower limit of detection of IFN- was (unpublished data).
31.3 pg/ml. Vaccination with ALM plus CpG ODN Confers Durable
Assessment of the Frequency of Leishmania-specific CD4 and
Protection Against L. major in B6 Mice. In contrast to the
CD8 Cytokine-producing Cells after Vaccination. 6 mo after vac-
cination, B6 mice were injected in both ears with a combination BALB/c model, it has been demonstrated that vaccination
of living and killed Ag comprising 106 metacyclic L. major pro- with ALM plus IL-12 confers durable protection against le-
mastigotes and 12.5 g SLA prepared from 33 freeze-thawed sta- sion development in the innately resistant B6 mouse (11).
tionary phase L. major promastigotes. 48 h later, three mice per As previously noted, the route and dose of infection in the
group were killed and cells from the local draining LN were ob- B6 mouse differ from those in the BALB/c mouse, offering

1567 Rhee et al.

 Figure 1. Footpad swelling of BALB/c mice after in-
fection with L. major 2 or 12 wk after vaccination.
Mice (n 6–8 per group) were immunized in the
footpad and boosted 2 wk later with ALM, ALM plus
IL-12, ALM plus CpG ODN, or normal saline. Mice
were then challenged in the contralateral footpad with
105 L. major (A) 2 or (B) 12 wk after the boost and
footpad swelling measurements were recorded weekly.
These data are representative of three independent ex-
periments. (C) As a positive control in the same exper-
iment, a group of naive mice (n 6) was administered
anti–IL-4 3 d before, and at the time of, infection. (*)
Two mice in the anti–IL-4 treatment group were
killed 4 wk due to ulceration. (D) In a separate experi-
ment, mice were immunized with CpG ODN alone,
ALM, ALM plus CpG ODN, a cocktail of DNA plas-
mids encoding different leishmanial Ag, or normal sa-
line (n 6–8 per group). Challenge was then done 12
wk after the boost. Data represent the mean size of the
individual footpads SEM.

another model to assess the utility of CpG ODN as a vac- Mice Vaccinated with ALM plus CpG ODN Have a Reduc-
cine adjuvant. B6 mice were vaccinated with ALM alone, tion in Parasite Burden Compared with ALM plus IL-12 Protein
ALM plus IL-12, ALM plus CpG ODN, or normal saline after Infectious Challenge with L. major. To determine
and then challenged intradermally in both ears 2 or 12 wk whether the reduction in footpad swelling (BALB/c mice)
after the boost (Fig. 2). Mice vaccinated with ALM plus or dermal lesions (B6 mice) previously shown correlated
CpG ODN or IL-12 protein developed minimal lesions with a decrease in parasite burden, mice in each of the vac-
when challenged 2 or 12 wk after vaccination (Fig. 2, A cine groups were killed 4 wk after infection and parasite
and B). As a control in a separate experiment, mice given burden was assessed. First, BALB/c mice vaccinated with
CpG ODN alone at the time of vaccination and boost ALM plus CpG ODN had a 40-fold decrease in the para-
were not protected and developed lesions comparable to site burden from draining the LN of mice infected 12 wk
those of nonvaccinated mice when challenged 2 wk after after vaccination compared with other groups (1.6 103 in
the boost (Fig. 2 C). Finally, in an additional experiment the ALM plus CpG ODN group compared with 6.3 104
(Fig. 2 D), the durability of ALM plus CpG ODN vaccina- in the saline group and 1.3 105 in the ALM group). ALM
tion was comparable to that of DNA vaccination in elicit- plus CpG ODN–vaccinated mice were the only group that
ing protection when challenged 12 wk after vaccination. was significantly different (P  0.05) from control saline-

Figure 2. Ear lesion size of B6 mice after infection

with L. major 2 or 12 wk after vaccination. Mice (n
6–8 per group) were immunized and boosted 2 wk
later with ALM, ALM plus IL-12, ALM plus CpG
ODN, or normal saline in the footpad. Mice were
challenged (A) 2 or (B) 12 wk after the boost by intra-
dermal inoculation of 500 L. major metacyclic promas-
tigotes in both ears, and lesion diameter was moni-
tored. These data are representative of three
independent experiments. (C) In a separate experi-
ment, mice were vaccinated with CpG ODN, ALM,
ALM plus CpG ODN, or normal saline and then chal-
lenged 2 wk after the boost. (D) In another experi-
ment, mice were immunized with ALM plus CpG
ODN, a cocktail of DNA plasmids encoding different
leishmanial Ag, or normal saline and challenged 12 wk
after the boost. Data represent the mean lesion diame-
ter SEM of four to eight mice, 8–16 ears per group.

1568 Leishmanial Ag plus CpG ODN Vaccination Confers Sustained Immunity

vaccinated mice in terms of a decrease in parasite burden ALM plus CpG ODN Sustains Protection Against L. major
(Fig. 3 A). Of note, there was also an 20-fold decrease in up to 6 mo after Vaccination in B6 Mice. To further evaluate
the number of parasites in ALM plus CpG ODN compared the longevity of ALM plus CpG ODN immunization in
with ALM plus IL-12–vaccinated mice. Second, in the eliciting protection against L. major, B6 mice were vacci-
low-dose dermal challenge model in B6 mice, both the nated intradermally in the ear and then infected 6 mo later
ALM plus IL-12 and ALM plus CpG ODN groups dem- in the opposite ear. First, to assess the immune correlates of
onstrated fewer parasites compared with the ALM and sa- protection, we evaluated the immune responses of mice 6
line groups (2.0 105 for ALM plus IL-12 and 3.2 104 mo after the last vaccination by measuring the frequency of
for ALM plus CpG ODN vs. 1.5 106 for ALM and IFN-–producing T cells after a brief in vivo infection
6
2.5 10 for saline; Fig. 3 B). Most significantly, the mice (48 h) followed by in vitro stimulation. This method of in
that received ALM plus CpG ODN had a greater than vivo followed by in vitro Ag stimulation provides a sensi-
2-log decrease in parasite burden compared with mice that tive way to measure low level memory immune responses
only received saline (P  0.05). Moreover, such mice had after vaccination with ALM (11). As shown in Fig. 5 A, there
approximately a sevenfold decrease in parasite burden com- was a demonstrable number of both CD4 and CD8 T
pared with ALM plus IL-12 protein–vaccinated mice. To- cells producing IFN- after stimulation with DC pulsed
gether, these data establish the enhanced biologic potency with L. major. In contrast, nonvaccinated mice demon-
of ALM plus CpG ODN compared with ALM plus IL-12 strated very low frequencies of IFN-–producing CD4 or
vaccination in terms of efficient parasite killing in both CD8 T cells (0.1–0.2%). It was striking that the ALM plus
mouse models. CpG ODN–vaccinated group had similar or higher fre-
ALM plus CpG ODN Immunization Induces CD4 T Cell quencies of CD4 and CD8 IFN-–producing cells than
Production of IFN- after Infection with L. major. Protec- the definitive positive control (previously infected mice
tion against L. major is dependent on Ag-specific production that had healed; unpublished data). To correlate these find-
of IFN- from CD4 T cells. To test whether the adminis- ings with an infectious challenge, mice were injected with
tration of ALM plus CpG ODN elicits Th1 responses, pro- L. major intradermally in the ear 6 mo after vaccination.
duction of IFN- from CD4 T cells was assessed from Mice that received ALM plus CpG ODN demonstrated lit-
draining the LN 4 wk after infection from mice that were tle or no pathology after challenge as assessed by lesion size
challenged 12 wk after vaccination. As shown in Fig. 4, (Fig. 5 B). Furthermore, the degree of protection induced
CD4 T cells from BALB/c mice vaccinated with ALM by ALM plus CpG ODN was comparable to that in previ-
plus CpG ODN stimulated in vitro with macrophages ously infected mice that had healed or mice that had been
pulsed with L. major secreted substantially higher levels of vaccinated with a cocktail of plasmid DNA encoding re-
IFN- compared with CD4 T cells from the other groups combinant leishmanial Ag (unpublished data).
(Fig. 4). Although not shown in this experiment, in previ- CD8 T Cell Depletion at the Time of Vaccination Mitigates
ous studies (9, 10) as well as in the experiment shown in Protection Conferred by Vaccination with ALM or Recombinant
Fig. 1 A, vaccination with ALM or recombinant leishmanial Leishmanial Protein plus CpG ODN. We have previously
protein plus IL-12 protein does not confer long-term pro- shown that the depletion of CD8 T cells at the time of
tection or induce sustained Th1 responses in BALB/c mice. vaccination or infection abrogates protective immunity in
BALB/c mice vaccinated with plasmid DNA encoding
LACK Ag (7, 13) and B6 mice vaccinated with a cocktail
of DNA Ag (unpublished data). Because ALM plus CpG
Figure 3. Parasite burden in ODN immunization was able to induce CD8 T cell re-
the draining LN and ears of in- sponses 6 mo after vaccination (Fig. 5), the role of CD8 T
fected BALB/c and B6 mice af- cells in mediating protection was determined by depleting
ter vaccination and infection. (A)
BALB/c mice were vaccinated CD8 T cells in vivo at the time mice were vaccinated. In
and then challenged 12 wk after these experiments, the depletion of CD8 T cells at the
boost with L. major in the foot- time of vaccination rather than at the time of infection was
pad (groups shown in Fig. 1 B).
Parasite quantitation in the drain-
ing LN of three individual mice Figure 4. Leishmania-spe-
was done 4 wk after challenge. cific production of IFN- from
(B) B6 mice were vaccinated and CD4 T cells after infection
then challenged 12 wk after with L. major. BALB/c mice
boost with L. major in both ears were vaccinated and then chal-
(groups shown in Fig. 2 B). Par- lenged with L. major 12 wk after
asite loads in the ears were deter- boost. CD4 T cells were iso-
mined 4 wk after challenge from lated from the draining LN 4 wk
four individual mice in each after challenge and stimulated in
group (n 8 ears). Results are culture with media alone, mac-
expressed as geometric mean rophages, or macrophages plus
SEM. *, P  0.05 when ALM L. major. IFN- levels were
plus CpG ODN is compared measured 48 h later by ELISA of culture supernatants. Data are the mean
with saline-vaccinated mice. concentrations of triplicate assays SD.

1569 Rhee et al.

 Figure 6. Effect of CD8 T cell depletion on lesion development and
Figure 5. Assessment of cellular immune responses and protection in parasite load in mice vaccinated with ALM plus CpG ODN. (A) Mice
B6 mice 6 mo after vaccination. (A) IFN- responses by CD4 and (n 6–8 per group) were immunized and boosted 2 wk later with ALM,
CD8 T cells in B6 mice were assessed 6 mo after intradermal vaccination ALM plus CpG ODN, or normal saline in the footpad. In some
with ALM plus CpG ODN or saline. Draining LN cells were obtained groups, mice were treated 1 d before, and at the time of, vaccination
48 h after intradermal injection of metacyclic promastigotes and SLA into the with anti-CD8 (2.43) or rat Ig Ab. Mice were then challenged 2 wk later
nonvaccinated ear and 24 h after in vitro restimulation with uninfected in the ear and monitored as described in Fig. 2. (B) Parasite loads in the
DC or infected DC (three mice per group, six LN). Analyses are gated on ears and draining LN were determined 4 wk after challenge from four
CD3 cells. Numbers represent the percentage of CD4 or CD8 T cells mice in each group (n 8 ears) or pooled draining LN from four mice.
positive for IFN-. Nonvaccinated mice were included as negative con- Results for the parasite load in the ears are expressed as geometric
trols. (B) In the same experiment, mice vaccinated with ALM plus CpG mean SEM. *, P  0.05 in comparing parasites per ear harvested from
ODN were challenged 6 mo after boost with L. major in the ear opposite ALM plus CpG ODN–vaccinated mice depleted of CD8 T cells.
from which they were vaccinated and ear lesions were monitored. Data
represent the mean lesion diameter SEM of six to eight mice.

adjuvant. In this regard, mice were immunized with CpG

done to limit induction of Ag-specific CD8 T cells but ODN and a recombinant Trifusion protein comprising
not limit the role of the endogenous repertoire of CD8 T three different leishmanial proteins in the presence or ab-
cells during the natural course of infection, which we have sence of CD8 depletion at the time of immunization.
recently shown to be important in mediating primary pro- These recombinant leishmanial proteins have been previ-
tection in the B6 mouse model (31). As shown in Fig. 6 A, ously shown to confer protection in both mouse and pri-
depletion of CD8 T cells at the time of vaccination with mate models of L. major infection (11, 28–30, 32). As
ALM plus CpG ODN resulted in an increase in ear lesion shown in Fig. 7, B6 mice immunized with Trifusion pro-
swelling compared with that in mice vaccinated with ALM tein plus CpG ODN had minimal lesions when challenged
plus CpG ODN alone or treated with a control rat Ig Ab. with L. major 2 (Fig. 7 A) or 12 wk (Fig. 7 B) after vaccina-
Because the peak number of parasites in the site is found tion. Similar to the results shown above, the treatment of
just before the onset of lesion development in the natural Trifusion plus CpG ODN–immunized mice with anti-
course of the low-dose intradermal challenge model (12), CD8 at the time of vaccination resulted in an increase in
parasite loads were quantitated 4 wk after infection when the lesion size and parasite load (Fig. 7 C) after infection.
there was as yet little difference in the size of the dermal le- Finally, mice vaccinated with Trifusion DNA had minimal
sions between the anti-CD8–treated and control-treated ear lesions after infection 12 wk after immunization, which
mice. There was, however, a 1–2-log increase in parasite was comparably altered by treatment with anti-CD8 at the
load in the ears and draining LN (Fig. 6 B) in ALM plus time of vaccination (unpublished data). Thus, vaccination
CpG ODN–vaccinated mice treated with anti-CD8. with either leishmanial DNA (7, 13) or leishmanial protein
These findings suggest that CpG ODN induces CD8 T or killed Ag plus CpG ODN induces long-term protection
cell responses by enhancing the processing of proteins con- against L. major in a CD8 T cell–dependent manner.
tained within the ALM. Because ALM is an autoclaved
preparation of whole L. major, it was of interest to perform
similar studies using a recombinant leishmanial protein to Discussion
provide additional evidence that CD8 T cells were impor- This study demonstrates in two different mouse models
tant in mediating protection using CpG ODN as a vaccine of L. major infection that CpG ODN provides long-term
1570 Leishmanial Ag plus CpG ODN Vaccination Confers Sustained Immunity
 demonstrated that the effects of CpG ODN as an adjuvant
were partial and limited, as protection was demonstrated in
only 40% of BALB/c mice challenged 3 wk after vaccina-
tion. The greater efficacy of the leishmanial protein plus
CpG ODN in our study may be due to several factors.
First, the leishmanial Ag used in the other studies was a
preparation of SLA, which might be less immunogenic
than ALM, a clinical grade Ag that has been used in human
vaccine trials against L. major (33, 34). Second, the parasite
dose and strain differed in our studies compared with the
other studies. Third, the CpG ODN sequence we used was
different than that used in one of the previous studies
showing partial long-term immunity. Nevertheless, the re-
sults presented here showing that CpG ODN provide
long-term protection have been repeated multiple times in
both BALB/c and B6 mouse models of leishmania infec-
tion with consistent results and little variability.
With regard to the mechanism by which CpG ODN
mediate their function in vivo and why they are better
than IL-12 protein as a vaccine adjuvant, their role in en-
hancing APC function needs to be considered. At the
APC level, CpG ODN through toll-like receptor 9, aug-
ments both the activation and maturation of DC as well as
the induction of proinflammatory cytokines (35). Thus, in
comparison to the relatively short-lived effect of exoge-
nous IL-12 protein as an adjuvant, the endogenous pro-
duction of IL-12, IL-18, and other soluble mediators from
activated DC induced by CpG ODN are likely to result in
a more physiologic cognate interaction between the DC
Figure 7. Effect of CD8 depletion in mice vaccinated with recombi- and T cell, resulting in both a qualitatively and quantita-
nant leishmanial protein plus CpG ODN. Mice (n 6–8 per group) were
immunized and boosted 2 wk later with Trifusion protein, Trifusion pro- tively different type of CD4 and CD8 T cell response.
tein plus CpG ODN, or normal saline (nonvaccinated) in the footpad. In These data highlight the potential importance of targeting
some groups, mice were treated 1 d before, and at the time of, vaccina- DC, especially for vaccines against diseases requiring cellu-
tion with anti-CD8 (2.43) or rat Ig control Ab. Mice were challenged (A) lar immune responses.
2 or (B) 12 wk after the last immunization in the ear and lesion size was
monitored as described in Fig. 2. (C) Parasite loads in the ears from mice In terms of immune correlates of protection, it is well es-
challenged 2 wk after vaccination were determined as described in Fig. 6. tablished that CpG ODN are potent inducers of Th1 re-
Tri, Trifusion; *, P  0.05 in comparing parasites per ear harvested from sponses, which is consistent with our findings that mice
Trifusion plus CpG ODN–vaccinated mice depleted of CD8 T cells. vaccinated with ALM plus CpG ODN had striking en-
hancement in the frequency and production of IFN- from
CD4 T cells 6 mo after vaccination in B6 mice (Fig. 5)
protection when used as a vaccine adjuvant with either and after infection in BALB/c mice, respectively (Fig. 4).
ALM or recombinant leishmanial protein. In addition, sim- Moreover, an additional possibility relating to the effective-
ilar to our previous studies with DNA vaccines, CpG ness of CpG ODN as a vaccine adjuvant compared with
ODN conferred more potent and durable protection when IL-12 protein in the BALB/c and B6 models is the role of
compared with IL-12 protein as an adjuvant with ALM. CD8 T cells. As previously mentioned, CD8 T cell re-
These data strongly suggest that a heat-killed Ag and/or a sponses are not seen in vaccination with recombinant leish-
recombinant protein vaccine can be useful for inducing du- manial protein plus IL-12 but have an important role in
rable immunity for diseases requiring cellular immune re- maintaining the protection elicited by DNA vaccination (7,
sponses depending on the adjuvant used. 13). In this regard, CpG ODN have been shown to elicit
Our data demonstrating that ALM or Trifusion protein CD8 T cell responses in mice when given with a protein
plus CpG ODN provides long-term protection are in con- Ag such as OVA (20). Thus, our findings that there were
trast to previous studies using CpG ODN as a vaccine adju- significant frequencies of Ag-specific CD8 IFN- cells in
vant with other leishmanial Ag preparations. In the study B6 mice 6 mo after vaccination with ALM plus CpG
by Stacey and Blackwell (24), partial protection was dem- ODN and that the depletion of CD8 T cells at the time of
onstrated in BALB/c mice infected 6 wk or 6 mo after vac- vaccination enhances the size of the lesions and the parasite
cination, but the quality of protection at any time after vac- load in B6 mice, provide compelling evidence for the im-
cination was moderate because all of the mice developed portance of CD8 T cells in mediating long-term immu-
significant footpad swelling. Similarly, Walker et al. (25) nity. Furthermore, the data showing that lesion develop-
1571 Rhee et al.
ment is altered in B6 mice immunized with a recombinant Exp. Med. 177:1797–1802.
leishmanial protein plus CpG ODN depleted of CD8 T 3. Scharton-Kersten, T., L.C. Afonso, M. Wysocka, G. Trin-
cells at the time of vaccination provides additional support chieri, and P. Scott. 1995. IL-12 is required for natural killer
for CpG ODN inducing CD8 T cell responses with a de- cell activation and subsequent T helper 1 cell development in
experimental leishmaniasis. J. Immunol. 154:5320–5330.
fined parasitic protein Ag. Finally, although lesion size in
4. Julia, V., M. Rassoulzadegan, and N. Glaichenhaus. 1996.
leishmanial Ag plus CpG ODN–vaccinated mice depleted Resistance to Leishmania major induced by tolerance to a sin-
of CD8 T cells was increased, these mice had demonstra- gle antigen. Science. 274:421–423.
bly smaller lesions than control vaccinated mice. This is 5. Launois, P., I. Maillard, S. Pingel, K.G. Swihart, I. Xenarios,
consistent with the induction of both CD4 and CD8 T H. Acha-Orbea, H. Diggelmann, R.M. Locksley, H.R.
cells by such a vaccine. Together, these data suggest that MacDonald, and J.A. Louis. 1997. IL-4 rapidly produced by
vaccines that elicit both CD4 and CD8 T cell responses V4V8 CD4 T cells instructs Th2 development and
are sufficient to confer long-term protection against L. ma- susceptibility to Leishmania major in BALB/c mice. Immunity.
jor in mice. 6:541–549.
Although the vaccine approach described here poten- 6. Sadick, M.D., F.P. Heinzel, B.J. Holaday, R.T. Pu, R.S.
tially has immediate clinical use because ALM is already a Dawkins, and R.M. Locksley. 1990. Cure of murine leish-
maniasis with anti-interleukin 4 monoclonal antibody. Evi-
widely tested vaccine Ag in humans, it remains to be deter-
dence for a T cell–dependent, interferon-–independent
mined whether the effects of CpG ODN on the cellular mechanism. J. Exp. Med. 171:115–127.
immune response in humans will be comparable to those 7. Gurunathan, S., D.L. Sacks, D.R. Brown, S.L. Reiner, H.
observed in mice. One encouraging recent report related to Charest, N. Glaichenhaus, and R.A. Seder. 1997. Vaccina-
this issue showed that there was a decrease in lesion size af- tion with DNA encoding the immunodominant LACK para-
ter infectious challenge with L. major in primates vacci- site antigen confers protective immunity to mice infected
nated with a killed preparation of leishmanial Ag and a spe- with Leishmania major. J. Exp. Med. 186:1137–1147.
cific type of CpG ODN compared with vaccination with 8. Afonso, L.C., T.M. Scharton, L.Q. Vieira, M. Wysocka, G.
Ag alone (36). To conclude, it is worth noting that the his- Trinchieri, and P. Scott. 1994. The adjuvant effect of inter-
torical standard for effective vaccination against L. major in leukin-12 in a vaccine against Leishmania major. Science. 263:
235–237.
humans is leishmaniazation (attenuated live infection).
9. Gurunathan, S., C. Prussin, D.L. Sacks, and R.A. Seder.
Moreover, the fact that previous infection to L. major con- 1998. Vaccine requirements for sustained cellular immunity
fers life-long protection in humans and that there is evi- to an intracellular parasitic infection. Nat. Med. 4:1409–1415.
dence for the persistence of parasitic Ag raises the critical is- 10. Stobie, L., S. Gurunathan, C. Prussin, D.L. Sacks, N. Gla-
sue of whether the induction of potent CD4 and CD8 T ichenhaus, C.Y. Wu, and R.A. Seder. 2000. The role of an-
cell responses induced after DNA or a killed and/or re- tigen and IL-12 in sustaining Th1 memory cells in vivo: IL-12
combinant leishmanial Ag plus CpG ODN will be suffi- is required to maintain memory/effector Th1 cells sufficient
cient to confer life-long protection in humans. This dis- to mediate protection to an infectious parasite challenge. Proc.
tinction between the apparent lack of an Ag requirement to Natl. Acad. Sci. USA. 97:8427–8432.
maintain cellular immune memory for CD4 and CD8 T 11. Mendez, S., S. Gurunathan, S. Kamhawi, Y. Belkaid, M.A.
cell responses for nonliving vaccines versus the potential re- Moga, Y.A. Skeiky, A. Campos-Neto, S. Reed, R.A. Seder,
and D. Sacks. 2001. The potency and durability of DNA-
quirement of Ag for mediating biologic protection will be
and protein-based vaccines against Leishmania major evaluated
the critical determinant in whether specific vaccines will be using low-dose, intradermal challenge. J. Immunol. 166:
successful over the course of a lifetime against many infec- 5122–5128.
tions requiring cellular immunity in humans. 12. Belkaid, Y., S. Mendez, R. Lira, N. Kadambi, G. Milon, and
D. Sacks. 2000. A natural model of Leishmania major infection
We thank Brenda Marchal for editorial assistance. reveals a prolonged “silent” phase of parasite amplification in
E. Rhee and J. Shah are Howard Hughes Medical Institute-NIH the skin before the onset of lesion formation and immunity.
Research Scholars. J. Immunol. 165:969–977.
Submitted: 29 January 2002 13. Gurunathan, S., L. Stobie, C. Prussin, D.L. Sacks, N. Glaich-
Revised: 27 March 2002 enhaus, A. Iwasaki, D.J. Fowell, R.M. Locksley, J.T. Chang,
Accepted: 26 April 2002 C.Y. Wu, et al. 2000. Requirements for the maintenance of
Th1 immunity in vivo following DNA vaccination: a poten-
tial immunoregulatory role for CD8 T cells. J. Immunol.
165:915–924.
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1573 Rhee et al.