Intracellular Replication-Deficient

Leishmania donovani Induces Long Lasting

This information is current as
of June 20, 2017.
Protective Immunity against Visceral
Leishmaniasis
Angamuthu Selvapandiyan, Ranadhir Dey, Susanne Nylen,
Robert Duncan, David Sacks and Hira L. Nakhasi
J Immunol 2009; 183:1813-1820; Prepublished online 10
July 2009;
doi: 10.4049/jimmunol.0900276
http://www.jimmunol.org/content/183/3/1813

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Supplementary http://www.jimmunol.org/content/suppl/2009/07/13/jimmunol.090027
Material 6.DC1
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 The Journal of Immunology

Intracellular Replication-Deficient Leishmania donovani

Induces Long Lasting Protective Immunity against Visceral
Leishmaniasis

Angamuthu Selvapandiyan,* Ranadhir Dey,* Susanne Nylen,† Robert Duncan,* David Sacks,†
and Hira L. Nakhasi1*
No vaccine is currently available for visceral leishmaniasis (VL) caused by Leishmania donovani. This study addresses whether a
live attenuated centrin gene-deleted L. donovani (LdCen1ⴚ/ⴚ) parasite can persist and be both safe and protective in animals.
LdCen1ⴚ/ⴚ has a defect in amastigote replication both in vitro and ex vivo in human macrophages. Safety was shown by the lack
of parasites in spleen and liver in susceptible BALB/c mice, immune compromised SCID mice, and human VL model hamsters 10
wk after infection. Mice immunized with LdCen1ⴚ/ⴚ showed early clearance of virulent parasite challenge not seen in mice
immunized with heat killed parasites. Upon virulent challenge, the immunized mice displayed in the CD4ⴙ T cell population a

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significant increase of single and multiple cytokine (IFN-␥, IL-2, and TNF) producing cells and IFN-␥/IL10 ratio. Immunized mice
also showed increased IgG2a immunoglobulins and NO production in macrophages. These features indicated a protective Th1-
type immune response. The Th1 response correlated with a significantly reduced parasite burden in the spleen and no parasites
in the liver compared with naive mice 10 wk post challenge. Protection was observed, when challenged even after 16 wk post
immunization, signifying a sustained immunity. Protection by immunization with attenuated parasites was also seen in hamsters.
Immunization with LdCen1ⴚ/ⴚ also cross-protected mice against infection with L. braziliensis that causes mucocutaneous leish-
maniasis. Results indicate that LdCen1ⴚ/ⴚ can be a safe and effective vaccine candidate against VL as well as mucocutaneous
leishmaniasis causing parasites. The Journal of Immunology, 2009, 183: 1813–1820.

T he obligate kinetoplastid protozoan parasites of the genus
Leishmania are spread by sand fly vectors and cause a
spectrum of diseases collectively known as leishmaniasis
that ranges from self-healing cutaneous leishmaniasis (CL),2 mu-
cocutaneous leishmaniasis (MCL), and to fatal visceral leishman-
iasis (VL). Leishmaniasis is endemic to 88 countries in the tropical
and subtropical world, affecting 12 million people and threatening
350 million more. Drug treatment requires long-term medication,
which is expensive and highly toxic. A vaccine for leishmaniasis
has been a goal for a century, but there are still no effective vac-
cines (1). The knowledge that a cured disease either due to a nat-
ural infection or cutaneous leishmanization (1) protects the indi-
vidual from reinfection and the persistence of a few parasites in the
body can impart life-long protection against leishmaniasis, has en-
couraged researchers to develop live attenuated Leishmania vac-
cine candidates.
*Division of Emerging and Transfusion Transmitted Diseases, Center for Biologics
Evaluation and Research, Food and Drug Administration, Bethesda, Maryland,
20892; and †Laboratory of Parasitic Diseases, National Institute of Allergy and In-
fectious Diseases, National Institutes of Health, Bethesda, MD 20892
Received for publication January 27, 2009. Accepted for publication June 2, 2009.
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance
with 18 U.S.C. Section 1734 solely to indicate this fact.
1
Address correspondence and reprint requests to Dr. Hira Nakhasi, Division of
Emerging and Transfusion Transmitted Diseases, Center for Biologics Evaluation and
Research, Food and Drug Administration, Bethesda, MD 20892. E-mail address:
hira.nakhasi@fda.hhs.gov
2
Abbreviations used in this paper: CL, cutaneous leishmaniasis; MCL, mucocutane-
ous leishmaniasis; VL, visceral leishmaniasis; Wt, wild type; FTAg, freeze-thaw Ag;
WI, week immunized; WPC, week post challenge.
Copyright © 2009 by The American Association of Immunologists, Inc. 0022-1767/09/$2.00
So far, several procedures have been used to develop live at-
tenuated Leishmania parasites including long-term in vitro cul-
tures, selection for temperature sensitivity, chemical mutagenesis,
and irradiation (1, 2). Although such live attenuated lines have
shown substantial protection against challenge in animal models,
undefined random genetic mutations and concerns arising from
potential reversion to virulence make such vaccines unsuitable for
human vaccination. Indeed, the persistence of asymptomatic in-
fection especially in immunocompromised individuals raises the
risk of reversion to clinical disease. Moreover, attenuations due to
undefined genome alterations can reduce effective protective im-
munity, either they fail to persist long enough to elicit an immune
response or lack critical epitopes to evoke the protective response
(3). Alternatively, attenuation obtained through targeted genetic
disruptions of essential growth regulating or virulence genes by
homologous recombination is nonrevertible, hence can be safe (1).
Moreover, sustained exposure of parasite Ag to the host eliminates
the need of any adjuvants that are required in other nonliving vac-
cines (e.g., subunit vaccines).
There are several examples of targeted gene deletions that
have been conducted for developing Leishmania-attenuated
vaccine strains. Among the vaccine candidates studied for CL,
L. major dihydrofolate reductase thymidylate synthase (dhfr-
ts⫺) knockout parasites protect mice (4) but not Rhesus mon-
keys (5). L. major deficient in surface and secreted phospho-
glycans (lpg2⫺), although unable to survive in sand flies and
macrophages, retained the ability to persist indefinitely in mice
and conferred protection against virulent challenge, even in the
absence of a strong Th1 response (6, 7). Over time, however,
2 ⫺
lpg (a complete gene knockout) parasites unexpectedly re-
gained virulence (8). L. major deleted for phosphomanno-
mutase-protected mice, despite no increase in either effector or

www.jimmunol.org/cgi/doi/10.4049/jimmunol.0900276
1814 REPLICATION DEFICIENT Leishmania INDUCES PROTECTIVE IMMUNITY
memory response (9). In contrast, L. mexicana that also causes
CL, deficient in cysteine proteinase genes (⌬cpa and ⌬cpb),
conferred protection in mice and hamsters against homologous
challenge (10, 11). Among the vaccination studies in VL, mice
immunized with a L. donovani strain deleted for biopterin trans-
porter (BT1) were similarly protected (12). Recent attempts us-
ing partial knockout parasites A2-A2rel gene cluster in L. do-
novani (13) and SIR2 gene in L. infantum (14) as immunogens
induced protection against virulent challenge in BALB/c mice.
However, such mutants cannot be used as vaccine candidates,
because they still carry wild-type (Wt) allele/s and could cause
disease.
Despite limitations with most of the mutant strains, these ex-
periments clearly demonstrate the potential as well as the pitfalls
of generating live attenuated Leishmania vaccines by targeted gene
deletions. Hence it is critical to develop attenuated lines through
complete gene knockouts that generate avirulent organisms that
persist for a brief duration but eventually are completely elimi-
nated, thereby inducing effective immunity without clinical disease
or the risk of reactivation. Recently, we have developed a L. do-
novani strain completely deleted for the centrin1 gene (15). Cen-
trin1 is a calcium-binding basal body-associated protein involved
in cell division in protozoan parasites like Leishmania, Trypano-
soma, and Plasmodium (16 –18) and centrosome associated in
higher eukaryotes (19). We demonstrated that deletion of LdCen1
(LdCen1⫺/⫺) did not affect the growth of the promastigote in vitro,
however, the growth of the mammalian-infecting, amastigote form
of the parasite, was blocked. These amastigotes showed failure of
basal body duplication and cytokinesis, resulting in large multinu-
cleated cells in culture and ex vivo in human macrophages (15).
We tested this unique parasite line for its safety and protective
efficacy against homologous and heterologous virulent Leishmania
challenge and the immune response correlating with protection in
rodent animal models.
Materials and Methods
Animals and parasites
Five- to six-wk-old female BALB/c mice from National Cancer Institute,
SCID mice (BALB/c background) from The Jackson Laboratory, and
40 –50 gm male Syrian golden hamsters from Charles River Laboratories
were used in the experiments. Procedures used were reviewed and ap-
proved by the Animal Care and Use Committee, Center for Biologics Eval-
uation and Research, Food and Drug Administration. Among parasites, the
Wt, centrin1-deleted (LdCen1⫺/⫺) and centrin1 deleted but episomally
centrin1 protein expressing line (LdCen1⫺/⫺ AB) of L. donovani (15) were
used. The parasite culture procedure and the routine molecular biology
practices were as previously described (16).
Vaccinations and challenge studies
In independent experiments, the mice were inoculated/vaccinated via tail
vein with 3 million metacyclic cells of either L. donovani Wt, or Ld-
Cen1⫺/⫺ parasites. Infective-stage metacyclic promastigotes of L. dono-
vani were isolated from stationary cultures by density gradient centrifuga-
tion as described (20). Control groups (naive) corresponded to mice that
received a saline solution (PBS). Mice vaccinated with LdCen1⫺/⫺ para-
sites after different time periods were challenged via tail vein with 3 million
virulent Wt L. donovani metacyclic parasites. Age-matched naive mice as
control groups were also similarly challenged with 3 million virulent me-
tacyclic L. donovani parasites. In separate experiments, mice vaccinated
(with LdCen1⫺/⫺) or not (naive with saline) were also challenged by in-
jecting i.m. on left hind footpad with 3 million metacyclic L. braziliensis
parasites. Hamsters were inoculated intracardially with 10 million meta-
cyclics of Wt or LdCen1⫺/⫺ parasites. The hamsters vaccinated with Ld-
Cen1⫺/⫺ or not (naive with saline) were challenged after 5 wk intracardi-
ally with 10 million metacyclic virulent L. donovani parasites. After post
challenge periods, parasite load was recorded from spleens and livers from
the L. donovani-challenged mice and hamsters and footpads and lymph
nodes from the L. braziliensis-challenged mice by culturing the separated
host cell preparations by limiting dilutions as previously described (21). As
an additional confirmation of the presence of parasites in tissues, total DNA
samples obtained from infected mouse and hamster spleens were used as
templates in a Taqman-based real-time PCR. The amplification target was
on the kinetoplast minicircle DNA of the parasite. The primers and meth-
ods were as previously described (22), with the addition of a fluorescent
probe for detection. The probe had the sequence 5⬘-RAAARKKVRTRCA
GAAAYCCCGT-3⬘. A Black Hole Quencher moiety is coupled to the 3⬘
end and Calfluor Red is coupled to a C6 linker at the 5⬘ end. The degenerate
letter code is according to the Nomenclature Committee of the Interna-
tional Union of Biochemistry (http://www.chem.qmul.ac.uk/iubmb/misc/
naseq.html). The degenerate probe allows detection of sequence variants of
the minicircle found in Leishmania and is added to the reaction mixture at
a final concentration of 1.5 pmols/␮l. To evaluate the number of Leish-
mania cells that were represented by a given Ct value, a standard curve was
constructed by infecting macrophages ex vivo (macrophage CSF cytokine
differentiated primary human monocytes) with stationary phase Leishma-
nia. After 24 h, macrophages were trypsinized, scraped into PBS, the mac-
rophage cells/␮l were determined by hemocytometer, and the Leishmania
amastigotes per macrophage were determined microscopically from a
smear of Diff-Quick (Baxter Healthcare Corporation) stained cells. A cal-
culated volume of the cell suspension was added to DirectPCR Lysis Re-
agent (Viagen Biotech) to produce lysates of 10 parasites/␮l, 1 parasite/␮l,
and 0.1 parasite/␮l. One microliter of each concentration was used as tem-
plate in real-time minicircle PCR with four replicates to determine the
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mean Ct value.
Intracellular staining and flow cytometry
Splenocytes were plated in 24-well plates and stimulated with freeze-thaw
Ag (FTAg) (23) or no Ag (control) in complete RPMI 1640 medium. After
36 h at 37°C brefeldin A was added to the wells. After 7 h at 37°C, cells
were blocked with anti-CD 16/32 (5 ␮g/ml) for 20 min (4°C), surface
stained with PerCP anti-CD3, allophycocyanin-Cy7 anti-CD4, and PE-Cy7
anti CD8 Abs for 30 min (each with 1/300 dilution; 4°C), fixed with Cyto-
fix/Cytoperm kit for 20 min (room temperature), intracellular staining with
Alex Flour 700 anti-IFN-␥, FITC anti-TNF, Pacific blue anti-IL-2, and
APC anti-IL-10 or isotype-specific control for each fluorescence tagged Ab
was for 30 min (each with 1/300 dilution; 4°C). Isotype controls for each
Ab used under similar conditions indicated specific binding of the test Ab.
All Abs and reagents were purchased from BD Biosciences except Pacific
blue anti-IL-2, which was from eBioscience. Electronic compensation was
performed with single-stained cells with individual mAbs used in the test
samples. Cells were acquired on LSRII with DIVA software (BD Bio-
sciences) and analyzed by FlowJo software (Tree Star).
Ab responses
Specific Ab responses were measured by conventional ELISA. In brief,
ELISA plates were coated overnight at room temperature with FTAg. A
serial dilution of the sera was conducted to determine the titer, which is
defined as the inverse of the highest serum dilution factor giving an ab-
sorbance of ⬎0.2. The titers for the Abs were determined using the fol-
lowing HRP-conjugated secondary Abs: Rabbit anti-mouse IgG (H⫹L)-
HRP; Rabbit anti-mouse IgG1-HRP, Human Adsorbed, Rabbit anti-mouse
IgG2a-HRP; Human Adsorbed (Southern Biotechnology Associates; all
with 1/1000 dilutions). SureBlue (KPL) was used as a peroxidase substrate.
After 15 min, the reaction was stopped by the addition of 100 ␮l of 1M
H2SO4, and the absorbance was read at 450 nm.
NO quantifications
Splenocytes or macrophages obtained from peritoneal fluid (24) were cul-
tured in complete, endotoxin-free RPMI 1640 medium (certified LPS free
by the manufacturer). NO (nitrite/nitrate) production was determined for
the supernatants from unstimulated cultures and cultures stimulated with
FTAg for 48 h at 37°C by the Griess reaction kit (Sigma-Aldrich).
Statistical analysis
Statistical analysis of differences between means of groups was determined
by two-sample t test assuming unequal variance. A p value ⬍ 0.05 was
considered as highly significant.
Results
Growth attenuation and limited persistence of LdCen1⫺/⫺
parasites in mice
Centrin1-deleted L. donovani metacyclics when used to infect hu-
man macrophages in vitro or injected in susceptible BALB/c mice,
displayed an attenuated growth (i.e., reduction in the number of
The Journal of Immunology
FIGURE 1. Avirulence and immunoprotective properties of centrin
(LdCen1) knockout L. donovani (LdCen1⫺/⫺) parasites in mice. a, Sur-
vival of parasites of LdCen1⫺/⫺, LdCen1⫺/⫺ episomally expressing
LdCen1 protein (add back; LdCen1⫺/⫺ AB), and Wt in BALB/c mice.
Parasite burdens from the organs of mice infected with metacyclic parasites
at 5 wk post infection are shown. b, Centrin knockout L. donovani does not
survive for long in BALB/c and SCID (BALB/c) mice. Parasite load in
organs of mice infected with metacyclics of either Wt or LdCen1⫺/⫺ par-
asites were measured at 5 or 12 wk after infection. c, Protection of Ld-
Cen1⫺/⫺ vaccinated BALB/c mice against virulent challenge. Mice vac-
cinated with LdCen1⫺/⫺ parasites were challenged after different time
periods with virulent Wt L. donovani and the challenge parasite burdens
from the organs were measured after different time points post challenge.
The data presented are representative of two or more experiments with
similar results. Mean and SEM of four or more mice in each group are
shown. Cha, challenged; Imm, immunized; ND, not detected; WI, wk after
infection; WC, wk after challenge; ⴱ, p ⬍ 0.01.
infected macrophages over time; supplementary Fig. 1a),3 and the
number of parasites per spleen or liver 5 wk postinfection com-
pared with control (Fig. 1a). This is the direct consequence of
centrin1 deficiency, because LdCen1⫺/⫺ cells expressing centrin1
protein from a transfected plasmid were rescued for growth both in
macrophages and in mice (supplementary Fig. 1a and Fig. 1a). To
analyze the persistence of LdCen1⫺/⫺ in mice, BALB/c mice were
injected with LdCen1⫺/⫺ or Wt parasites and were monitored up to
12 wk post infection. Few LdCen1⫺/⫺ parasites were seen at 5 wk
post infection in spleen and liver, measured by limiting dilution,
and by 12 wk, the parasites were completely cleared from both
organs (Fig. 1b). The Wt-infected mice had significant parasite
burden in these organs at both the time points (Fig. 1b). Similar
results were obtained in a confirmatory real-time PCR study using
3
The online version of this article contains supplemental material.
1815
DNA from spleens of mice infected either with Wt or LdCen1⫺/⫺
parasites (supplementary Table 1). Detection of the parasite mi-
nicircle DNA target indicated the presence of a substantial number
of parasites in both Wt-infected (6 wk) and LdCen1⫺/⫺-infected (5
wk) mice. However, the mice infected with LdCen1⫺/⫺ for 12 wk
showed a level equivalent to naive mice, which can be considered
as a background, signifying either no LdCen1⫺/⫺ parasites or sig-
nificantly reduced parasite burden in mice at this time point.
Hence, it appears that the parasites are present for at least 5 wk in
the viscera as opposed to disappearing within just a few days.
Similarly, we did not observe any LdCen1⫺/⫺ parasites at 12 wk
postinfection in SCID mice, indicating that their clearance is not
dependent on functional T and B cells (Fig. 1b).
Protective efficacy of LdCen1⫺/⫺ parasites against virulent
infection
The ability of LdCen1⫺/⫺ parasite to protect against Leishmania
infection was determined in 5-wk immunized mice (WI) followed
by challenge with virulent Wt L. donovani. Spleen and liver were
analyzed for parasite burden 4, 8, and 10 wk post challenge
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(WPC), measured by limiting dilution. The results showed that
the immunized mice had ⬃2-log-fold reduced ( p ⬍ 0.01) parasite
burden in spleen and undetectable parasite in liver compared with
naive-challenged mice at 10 WPC (Fig. 1c). However, there was
no significant change in the weight of liver or spleen of immunized
or unimmunized mice. To evaluate the ability of LdCen1⫺/⫺ im-
munization to confer lasting protection, mice were challenged with
virulent Wt L. donovani 12 or 16 wk following LdCen1⫺/⫺ im-
munization, and evaluated after 10 wk. The parasite burden in the
spleen and liver indicated that the level of protection in both im-
munized groups (12 WI and 16 WI) was similar to the level ob-
served for the 5 WI group, indicating a sustained protective re-
sponse after immunization (Fig. 1c). A similar result was observed
in a separate experiment where mice were challenged after 24 wk
of immunization (data not shown). No protection was observed 8
WPC in mice immunized with heat killed 3 million LdCen1⫺/⫺
metacyclics challenged 5 wk after immunization (supplementary
Fig. 1b), suggesting that live attenuated parasites were required to
induce protective immunity. Overall, the results suggest that mice
immunized with LdCen1⫺/⫺ elicit a strong and sustained protec-
tion against the virulent challenge.
Induction of multifunctional Th1 effector cells correlates with
LdCen1⫺/⫺-induced protection
The involvement of IFN-␥-producing Th1 cells in immunity
against Leishmania is described (25). We also observed an ab-
solute requirement for IFN-␥ in LdCen1⫺/⫺-induced immunity.
IFN-␥ knockout mice immunized with LdCen1⫺/⫺ for 5 wk
followed by challenge were not protected. Parasite burden from
these mice was similar to naive-challenged control observed at
10 wk post challenge (Fig. 2). To further investigate the cell-
mediated responses induced by LdCen1⫺/⫺ parasites and find a
correlate of protective immunity, we measured the Th1 cyto-
kines IFN-␥, TNF, and IL-2 produced by splenic CD4⫹ and
CD8⫹ T cells from the BALB/c mice using multiparameter flow
cytometry. Spleen cells grown in vitro with or without freeze
thaw Ags followed by multicolor staining were gated based on
forward and side scatter in FlowJo to select only the lympho-
cyte population devoid of dead cells and other larger leukocytes
(Fig. 3a, left) and further gating of the lymphocytes for the
CD3⫹ (Fig. 3a, middle) followed by gating of CD3⫹ in to
CD4⫹ and CD8⫹ T cells (Fig. 3a, right). Seven distinct popu-
lations of cytokine-producing cells were defined from the CD4⫹
and CD8⫹ T cells based on different combinations of IFN- ␥,
1816 REPLICATION DEFICIENT Leishmania INDUCES PROTECTIVE IMMUNITY
FIGURE 2. Lack of protection of LdCen1⫺/⫺ vaccinated IFN-␥ knock-
out mice against virulent challenge. IFN-␥ knockout mice vaccinated with
LdCen1⫺/⫺ parasites were challenged after 5 wk with virulent Wt L. do-
novani and the challenge parasite burdens from organs were measured after
10 wk post challenge. Mean and SEM of four mice in each group are
shown.
TNF, and IL-2 (a representative analysis is shown in Fig. 3b).
The quality of the Th1 response is based on the relative fre-
quency of these distinct populations (26). Five weeks after im-
munization with LdCen1⫺/⫺, single cytokine-producing CD4⫹
and CD8⫹ T cells, making either IFN-␥ or IL-2, were more
predominant than multicytokine-producing subpopulations post
immunization (Fig. 3, c and d). Similar analysis conducted with
spleens of immunized mice analyzed 10 WPC showed a signif-
icantly higher percent (2- to 10-fold; p ⬍ 0.01) of the cytokine-
producing subpopulations than in the naive challenged mice
(Fig. 3, e and f). Importantly, the expansion of multifunctional
cells was clearly evident in the immunized mice 10 WPC. In-
terestingly, the percent of both CD4⫹ and CD8⫹ T cells that
make TNF alone increased significantly (up to 2-fold; p ⬍ 0.01)
after challenge in LdCen1⫺/⫺ immunized mice. The percentage
of T cells that produce Th1 cytokines increased after immuni-
zation and the increased frequency of multifunctional cells after
challenge strongly correlated with protection.
We also quantitated T cells that produce IL-10, a cytokine in-
volved in the pathogenesis of VL (27). The ratio of IFN-␥ to IL-10
serves as an additional correlate of immune protection (14). In the
restimulated CD4⫹ T cells from the spleen, the IFN-␥/IL-10 ratio
was significantly higher in the immunized mice both at the time of
challenge (5 WI) and after challenge (5 WI plus 10 WC) compared
with either naive or naive-challenged controls (Fig. 3g). The re-
sults thus indicate an increased IFN-␥ secretion coinciding with
reduced IL-10 production among the immunized mice, indicating
a strong Th1 response that could be accountable for protective
immunity.
Recent studies in CL infection in mice indicate a role for
nonregulatory T cells, which simultaneously produce IFN-␥ and
IL-10, for immune suppression during pathogenesis of a non-
healing lesion (28). Hence, we also looked at IFN-␥⫹ T cells
that coproduce IL-10 cytokine after immunization with Ld-
Cen1⫺/⫺. The results indicated a significantly higher percent-
age of CD4⫹ T cells positive for both IFN-␥ and IL-10 in the
5-wk-immunized mice than in controls (Fig. 3h). No increase of
such cells in the CD4⫹ population was observed either 12 or 16
wk after immunization (data not shown). The presence of IFN-␥
and IL-10 coproducing CD4⫹ cells early after immunization
(5W, a time point when LdCen1⫺/⫺ parasites are still detect-
able), might reflect immune modulation to reduce inflamma-
tion-mediated host damage.
Induction of humoral response in the immunized mice
We evaluated the humoral response in the immune-challenged
mice. Sera from BALB/c mice taken 10 wk post challenge after
5, 12, or 16 immunization weeks were measured for Leishma-
nia-specific IgG, IgG1, and IgG2a responses. Results indicated
a higher level of all three Ab populations in the immune-chal-
lenged groups compared with the naive challenged groups (Fig.
4a). Importantly, the immunization led to a selective increase in
Th1-driven IgG2a Ab levels during infection ( p ⬍ 0.01). The
results indicate a higher Leishmania specific humoral immune
response generated by immunization with LdCen1⫺/⫺ that cor-
relates with the increase in the Th1 response.
Increased NO production in spleen cells in the
immune-challenged mice
Because the production of NO by macrophages is a key factor in
killing Leishmania, we determined the level of NO produced by
in vitro Ag restimulated splenocytes that included macrophages
derived from the immune-challenged mice by ELISA. Interest-
ingly, significantly higher ( p ⬍ 0.01) levels of the reactive NO
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radical (nitrite) were demonstrated in the supernatants of
splenocytes of the three immune-challenged groups (Fig. 4b).
We also analyzed Ag-specific release of nitrite from the cul-
tured macrophages derived from the peritoneal fluids of mice.
There is a small increase in NO production in macrophages of
the naive mice, suggesting a background level in this assay, as
well as in the naive challenged mice with the addition of FTAg.
However, in the immunized, challenged mice the production of
NO in response to FTAg is significantly higher ( p ⬍ 0.01) than
the naive challenged mice, which clearly correlates with pro-
tection (supplemental Fig. 2).
Safety and efficacy of LdCen1⫺/⫺ in the hamster model
Because golden Syrian hamsters are considered a more appro-
priate model for VL (29), we evaluated the safety and protec-
tion of LdCen1⫺/⫺ against L. donovani challenge in this spe-
cies. The LdCen1⫺/⫺ parasite burden in spleens, measured by
limiting dilution, was 3-log-fold less than Wt L. donovani 5 wk
after inoculation in hamsters and no LdCen1⫺/⫺ parasites were
found in the liver, whereas at 10 wk post immunization, no
LdCen1⫺/⫺ parasites were observed in any of the organs in
contrast to hamsters inoculated with virulent parasites, which
had significantly higher number of parasites in both spleen and
liver (Fig. 5a). Further persistence of the parasites in the ham-
ster model was evaluated by performing real-time minicircle
PCR on spleen DNA samples collected more than 3 mo postin-
fection. Samples from Wt-infected hamsters gave a Ct value
indicating a substantial number of parasites, while at the same
time point, LdCen1⫺/⫺-infected samples gave a Ct value equiv-
alent to the uninfected hamsters. After 5 wk of immunization
with LdCen1⫺/⫺ and challenge with virulent L. donovani, the
immunized hamsters had significantly lower parasite burden in
both the organs, measured by limiting dilution, compared with
the naive-challenged animals, with a reduction of 99.9% in
spleen and 99.7% in liver ( p ⬍ 0.01) (Fig. 5b).
Centrin1 KO L. donovani cross protects mice against challenge
with L. braziliensis
We wanted to determine whether immunization with Ld-
Cen1⫺/⫺ could provide heterologous protection against infec-
tion involving other Leishmania species. For this purpose, 5 wk
LdCen1⫺/⫺ immunized as well as naive BALB/c mice were
challenged by s.c. inoculation in the footpad with 2 million
metacyclics purified from L. braziliensis, the causative agent of
The Journal of Immunology 1817

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FIGURE 3. Multiparameter flow cytometry based analysis for distinct Th1/Th2 cytokine populations. A seven color cytometry panel was used to
measure the four cytokines, IFN-␥, TNF, IL2, and IL10, with the help of three T cell membrane markers CD3, CD4, and CD8. a, The common gating
steps shown in this study through FlowJo analysis are as explained in the Results section. b, Representative fluorescence intensity distributions of
single, double, or triple positive cytokines (IFN-␥, TNF, and IL-2) expressing T cells in spleens of vaccinated (5 wk post vaccination) and naive
mice. CD3⫹CD4⫹ or CD3⫹CD8⫹ lymphocytes from spleens showing either IFN-␥⫹ or IFN-␥⫺ are each further separated into cells that also make
either TNF, IL-2, both TNF and IL-2, or none. c–f, Flow cytometric analysis of Leishmania Ag-specific multifunctional T cells. Leishmania
Ag-specific multifunctional effector cells that were measured from splenocytes of LdCen1⫺/⫺ vaccinated mice at the time of challenge (5WI, 5 wk
post immunization) (c and d) and after challenge (5WI plus 10WPC: 10 wk post challenge) (e and f). The percentages of CD4⫹ (c and e) and CD8⫹
(d and f) T cells producing the cytokines IFN-␥, TNF, and IL-2 and combinations of these. The analysis was conducted using multiparameter flow
cytometry (as in Methods). g, Ratio of IFN-␥ to IL-10 producing CD4⫹ T cells from spleens at the time of challenge (naive and immunized (5W))
and after challenge (naive-challenged and immune-challenged (5WI plus 10WPC)). h, CD4⫹ T cells from spleens that simultaneously produce IFN-␥
and IL-10 at 5W post immunization with LdCen1⫺/⫺. The data presented are representative of two or more experiments with similar results.
Mean and SEM of four or more mice in each group with error bar indicating SEM are shown. Cha, challenged; Imm, immunized; W, wk infected;
WI, wk after immunization; WPC, wk after challenge; ⴱ, p ⬍ 0.01.

MCL. The naive, challenged mice developed progressive le- lenged mice showed significantly lower parasite burdens both in
sions during the 7 wk of observation, whereas the immunized the footpads and lymph nodes compared with the controls (Fig.
mice developed significantly smaller lesions (⬍3-fold; p ⬍ 5d), with a 99.9% reduction in both the tissues ( p ⬍ 0.02; p ⬍
0.01) throughout the 7 wk (Fig. 5c). The immune-chal- 0.01). A similar type of experiment conducted to examine cross
1818 REPLICATION DEFICIENT Leishmania INDUCES PROTECTIVE IMMUNITY
FIGURE 4. a, Humoral response in the virulent challenged mice vac-
cinated with LdCen1⫺/⫺ parasites. ELISA measurement of IgG, IgG1, and
IgG2a Abs in sera from the mice immunized with LdCen1⫺/⫺ parasites for
5, 12, or 16 wk or naive before challenge with virulent L. donovani. Sera
were collected 10 wk post challenge. b, Leishmania Ag specific stimulation
of NO synthase (NOS2) in the splenocytes of naive, naive challenged and
LdCen1⫺/⫺ immunized and Wt challenged mice. The activity of NOS2,
indicated by the amount of released nitrite (NO) in the splenocyte super-
natants was measured by the Griess reaction. The data presented are rep-
resentative of two experiments with similar results. Mean and SEM of three
or more mice in each group are shown. Cha, challenged; Imm, immunized;
FTAg, Freeze-thaw Ag; WI, wk after immunization; WPC, wk after chal-
lenge; ⴱ, p ⬍ 0.01.
protection of LdCen1⫺/⫺ against L. major showed a moderate
but delayed protection (data not shown).
Discussion
In pursuit of a candidate live attenuated vaccine, we generated a L.
donovani parasite that had a complete deletion for a cell division
gene, centrin1 (LdCen1⫺/⫺) (15). We have demonstrated that
LdCen1⫺/⫺ attenuation is due to the failure of cytokinesis, result-
ing in multiplication of cellular organelles, cell enlargement, and
eventual programmed cell death. We think that the genetically tar-
geted and defined attenuation reduces the risk of reversal to viru-
lence, a concern generally raised for attenuated organisms that are
created by random genomic mutations. LdCen1 deletion specifi-
cally attenuated the amastigote stage of the parasite that replicates
inside macrophages, and has no effect on the growth of the pro-
mastigote form. This could be advantageous to grow large quan-
tities of parasites for vaccine trials.
FIGURE 5. Avirulence and immuno-protective properties of
LdCen1⫺/⫺ parasites in hamsters. a, Parasite load in organs of hamsters
infected with either Wt or LdCen1⫺/⫺ metacyclic parasites was measured
at 5 and 10 wk after infection. b, Protection of the LdCen1⫺/⫺ vaccinated
hamsters against virulent challenge. Hamsters vaccinated with LdCen1⫺/⫺
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parasites were challenged after 5 wk of immunization with virulent Wt L.
donovani and the challenge parasite burdens from the organs were mea-
sured after 4 wk. c, Cross-protection of BALB/c mice vaccinated with
LdCen1⫺/⫺ against heterologous challenge with L. braziliensis. Five wk
post vaccinated mice were infected with metacyclics of L. braziliensis in-
jecting i.m. in the left-hind footpad of each mouse. Graph shows footpad
swelling due to infection in the injected footpad calculated by measuring
the difference in the footpad size between the two hind footpads. d, Parasite
numbers per organ of infected footpad or draining lymph nodes from 7 wk
postchallenge with L. braziliensis are shown. The data presented are rep-
resentative of two experiments with similar results. Mean and SEM of four
animals in each group are shown. Cha, challenged; Imm, immunized; ND,
not detected; W, wk after infection; ⴱ, p ⬍ 0.02 and ⴱⴱ, p ⬍ 0.01.
To evaluate it as a vaccine candidate, the LdCen1⫺/⫺ parasite
was tested both in susceptible BALB/c mice and Syrian hamsters.
LdCen1⫺/⫺ parasites showed persistence of low numbers of par-
asites both in mice and hamsters for at least 5 wk. Ten (both in
mice and hamsters) or 16 wk postinfection and in some cases even
longer periods (24 wk; data not shown), LdCen1⫺/⫺ parasites were
not detected in liver or spleen of mice, suggesting the complete
clearance demanded of a safe vaccine candidate. Similar observa-
tions in SCID mice lacking both T and B cells reinforce its safety
even in the immune compromised host.
While searching for correlates of protection against homologous
challenge, we found that LdCen1⫺/⫺ immunized mice developed
an increased percent of Leishmania specific CD4⫹ and/or CD8⫹ T
cells expressing Th1 cytokines (IFN-␥, TNF, and IL-2) either sin-
gly or in multiple combinations. Multifunctional effector cells as-
sociated with protection have been described by others in mice
vaccinated with Leish-111f recombinant polyproteins plus adju-
vant (30) and with Leishmania MML protein expressed in a rep-
lication-defective adenovirus (26). We found an especially strong
increase of TNF-producing T cells, reinforcing prior data suggest-
ing that TNF along with IFN-␥ secretion are important cytokines
for protection in vivo (26). We also observed an increased IFN-
␥/IL-10 ratio among the CD4⫹ T cells in the LdCen1⫺/⫺ vacci-
nated mice both at the time of and after challenge, revealing an-
other correlate of protection. A similar polarization to an increased
IFN-␥ to IL-10 ratio in the splenocyte supernatant measured by
ELISA after L. infantum challenge in mice immunized with
SIR2⫹/⫺ L. infantum was observed (14). We also observed an
increase of CD4⫹ T cells that coproduce IFN-␥ and IL10 during
the initial period (5W) of infection with LdCen1⫺/⫺, suggesting
The Journal of Immunology
possible immune modulation at this time as also observed previ-
ously, during L. major infection in mice (28). In the L. major case,
the nonregulatory T cells (CD4⫹CD25⫺Foxp3⫺) that were the
source of IL-10 (most of them also produced IFN-␥) were immu-
nosuppressive during protozoan infection for the reduction of in-
fection mediated damage to host cells. Hence, the LdCen1⫺/⫺ im-
munization may be facilitating a desirable balance in the immune
response allowing brief parasite persistence that facilitates protec-
tion without causing host damage.
In the present study, we observed a robust Th1-specific serum
Ab (IgG2a) response in the immune-challenged mice, further sup-
porting the observation that LdCen1⫺/⫺ induces a generalized Th1
type response. In addition, the increased release of NO observed in
the splenocyte culture derived from the immune-challenged mice
coincided with the increase of Leishmania specific T cells produc-
ing IFN-␥, TNF, and IL-2 cytokines. NO production induced by
Th1 cytokines is a main leishmanicidal mechanism of murine mac-
rophages (31, 32). Thus, in the LdCen1⫺/⫺-immunized mice, NO
production by macrophages that are stimulated by Th1 cytokine
producing T cells correlates with the control of infection.
Importantly, immunization with LdCen1⫺/⫺ protected both
mice and hamsters against virulent homologous challenge, as in-
dicated by significantly reduced parasite burdens in the organs of
the immune-challenged mice and hamsters compared with naive
controls. The protective response was sustained for up to 24 wk
after immunization. Complete elimination of wild-type parasites
from the livers of LdCen1⫺/⫺ immunized mice after virulent chal-
lenge, is superior to the protection induced by L. donovani
BT1⫺/⫺, the only other complete gene knockout strain reported to
target VL, which reduced parasite level in liver by 75% of the level
in naive challenged mice (12). The dhfr-ts⫺ auxotropic L. major
line was safe in both mice and rhesus monkeys but protected only
the mice (4, 5). Because the immune responses due to dhfr-ts⫺
were not studied in those animals, it would be difficult to know if
there were weakness in the mouse immune response that could
have predicted failure in the monkey. However, the quality of
LdCen1⫺/⫺-induced immune response in mice and protection in
mice and hamsters encourage us to proceed with immune studies
in higher animal models. The level of protection achieved by
LdCen1⫺/⫺ was similar to the protection due to L. infantum
SIR2⫹/⫺ in mice (14). Because L. infantum SIR2⫹/⫺ is a partial
knockout, it may be at a greater risk of reversion and therefore is
unlikely to be pursued as a vaccine candidate. Lpg2⫺/⫺, the only
complete gene knockout that has reverted to virulence in mice, is
also known to establish prolonged persistence in the host (7),
which may have permitted the acquisition of compensating muta-
tions. Because LdCen1⫺/⫺ does not persists in mice and hamsters
beyond 2 mo, such a reversal to virulence is less likely.
There are several possible explanations for the protection by
LdCen1⫺/⫺ parasite vaccination. First, there could be residual par-
asite burden (beyond the detection sensitivity of our methods,
which would allow continued presence of Leishmania-specific ef-
fector cells and maintain anti-Leishmania immunity (21, 33). Sec-
ond, vaccination with LdCen1⫺/⫺ parasites could lead to genera-
tion of a central memory T cell response after the KO parasites are
cleared that could develop into an effector memory T cell response
upon challenge, and provide protection (34). Third, there could
be Ag persistence in peripheral tissues long after the parasites are
cleared. These persistent Ag “depots” may contribute to specific
memory T cells through the activation of naive T cells as has been
reported for some viral infections (35, 36). However, these hy-
potheses need to be tested and are the subject of future studies.
Finally, heterologous protection induced by infection with one
virulent species of Leishmania against infection with another spe-
1819
cies of Leishmania has been documented in animal model studies
(37–39). In mice, experimental vaccination using dp72 protein iso-
lated from L. donovani cross-protected against L. major infection
(40) and immunization with exogenous Ags (LmSEAgs) of L. ma-
jor cross-protected against L. donovani (41). Our results show that
LdCen1⫺/⫺-immunized mice were cross-protected to a high de-
gree against a heterologous challenge with L. braziliensis, that
causes MCL, and to some degree against L. major that causes CL.
Therefore, our results confirm that the live attenuated parasites also
confer resistance to infection with other species.
In the literature, several routes of administration of Leishmania
vaccine candidates have been studied with varied degree of pro-
tection (reviewed in Ref. 42). In the present study, we have used
the i.v. route in mice and intracardial route in hamster, however we
recognize that in general a recommended route of administration
of a vaccine in humans is either intradermal or i.m. Future studies
in our laboratory will focus on defining the ideal route of admin-
istration for optimal protection for LdCen1⫺/⫺ parasites.
In summary, this study demonstrates that in mice and hamsters,
the live attenuated LdCen1⫺/⫺ is highly immunogenic and confers
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a significant degree of protection against L. donovani and also is
capable of inducing cross protection against L. braziliensis. The
vaccine-elicited parasite Ag-specific Th1 responses coinciding
with a robust Ab response and NO production, all strongly corre-
lated to a sustained protection. This report thus presents a well
characterized amastigote specific, live attenuated L. donovani can-
didate vaccine that has been evaluated for its in vivo persistence,
immunological response, and protective (against homologous and
heterologous challenges) efficacy.
Acknowledgments
We acknowledge Kimberly Beacht for technical assistance and Sanjai Ku-
mar and Alain Debrabant for valuable suggestions.
Disclosures
The authors have no financial conflict of interest.
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