Cysteine proteinase type I, encapsulated in solid lipid nanoparticles
induces substantial protection against Leishmania major infection
in C57BL⁄6 mice
D. DOROUD,1,2*F. ZAHEDIFARD,1*A. VATANARA,2A. R. NAJAFABADI2?& S. RAFATI1?
1Molecular Immunology and Vaccine Research Laboratory, Pasteur Institute of Iran, Tehran, Iran,2Department of Pharmaceutics, School
of Pharmacy, Tehran University of Medical Sciences, Tehran, Iran
Appropriate adjuvant, proper antigen(s) and a suitable for-
mulation are required to develop stable, safe and immuno-
genic vaccines. Leishmanial cysteine proteinase type I
(CPB) is a promising vaccine candidate; nevertheless, it
requires a delivery system to induce a potent immune
response. Herein, solid lipid nanoparticles (SLN) have been
applied for CPB [with and without C-terminal extension
(CTE)] formulation to utilize as a vaccine against Leish-
mania major infection in C57BL⁄6 mice. Therefore, SLN-
CPB and SLN-CPB)CTEformulations were prepared from
cetyl palmitate and cholesterol, using melt emulsification
method. After intraperitoneal vaccination and subsequent
L. major challenge, a strong antigen-specific T-helper type 1
(Th1) immune response was induced compared to control
groups. Lymph node cells from immunized mice displayed
lower parasite burden, higher IFN-c, IgG2a and lower IL-4
production, indicating that robust Th1 immune response had
been induced. Our results revealed that CTE is not neces-
sary for inducing protective responses against L. major
infection as the IFN-c⁄IL-4 ratio was significantly higher,
whereas IgG1 responses were lower in the SLN-CPB)CTE
vaccinated group, post-challenge. Thus, SLN-CPB)CTEwas
shown to induce specific Th1 immune responses to control
L. major infection, through effective antigen delivery to the
peritoneal antigen presenting cells.
Keywords cysteine proteinase type I, delivery systems,
Leishmania major, solid lipid nanoparticle, vaccination
Leishmaniasis is an important vector-borne parasitic infec-
tion which can cause a spectrum of diseases, ranging from
a clinically silent to a fatal progressive disease in human
and is a major public health problem in many Mediterra-
nean, Asian, African, Central and South American, Carib-
bean, Near and Middle East countries including Iran
(World Health Organization website, http://www.who.int/
In the recent years, much interest has been directed
towards finding a vaccination as effective prevention is not
available, and current curative therapies are costly, often
poorly tolerated and not always effective. Furthermore,
drug resistance exists in endemic areas. The existence of
14 million clinical cases of leishmaniasis worldwide and
350 million people living at risk of infection directs essential
research and trials towards vaccination to stop the incidence
of more than 2 million newly infected cases annually (World
Health Organization website, http://www.who.int/vaccine_
research/diseases/soa_parasitic/en/index3.html). However, over
the years, even though there has been research on different
vaccine generations ranging from killed, live-attenuated to
recombinant, synthetic and even naked DNAvaccines, there
is no routine vaccine available against leishmaniasis world-
Immunity against Leishmania is dependent on the devel-
opment of a strong T-cell response (mainly Th1) involving
the production of cytokines such as IFN-c and IL-12 that
Correspondence: Sima Rafati, Molecular Immunology and Vaccine
Research Lab, Pasteur Institute of Iran, Tehran, Iran
(e-mail: email@example.com or firstname.lastname@example.org) and
Abdolhossein R. Najafabadi, Department of Pharmaceutics,
School of Pharmacy, Tehran University of Medical Sciences,
Tehran, Iran (e-mail: email@example.com or Roholami@
Received: 16 January 2011
Accepted for publication: 9 March 2011
*Equally participated as first author.
?Equally participated as corresponding author.
Parasite Immunology, 2011, 33, 335–348DOI: 10.1111/j.1365-3024.2011.01289.x
? 2011 Blackwell Publishing Ltd
Although live vaccines can induce these protective long-
lasting immunity responses, vaccination using this strategy
is being suspended because of undesirable side effects and
clinical complications. On the other hand, Leishmania vac-
cines based on recombinant proteins and pDNAs encod-
immunogenic, and it is critical to co-administer an effec-
tive safe T-cell adjuvant and⁄or delivery system (2).
One of the vaccine candidates studied by our research
group is cysteine proteinase (CP) type I or CPB. This
enzyme is produced by a variety of organisms, including
Leishmania, and belongs to the papain superfamily and
has been reported to be an attractive target for drug devel-
opment (3). Another important point is the presence of an
unusual C-terminal extension (CTE) that differentiates
CPB from the other CPs in the papain superfamily (3).
Comparison of Leishmanial CPBs showed that the CTE is
highly variable (4). In some cases, the CTE is glycosylated
and may be partially removed by proteolytic cleavage dur-
ing processing of the enzyme to its mature form (5).
Hence, the CTE fragment is not essential for enzyme
activity and intracellular trafficking, but it has been postu-
lated that it is highly immunogenic and responsible for
immune evasion and plays a role in the diversion of the
host immune responses (6).
L. major rCPB to elicit a protective immune response
against infectious challenge in BALB⁄c mice. We demon-
strated that immunization with rCPB in combination with
Poloxamer 407 as an adjuvant could partially protect the
mice against infectious challenge (7). Subsequent to these
observations, we have had two major concerns. The first
was utilizing Poloxamer 407 as an adjuvant, which has no
relevant advantage for our forthcoming practical vaccina-
tion studies for the reason that it has been reported that a
single intraperitoneal (i.p.) injection of this adjuvant pro-
duces marked physio-pathological changes in rodents such
as mice (8,9).
The second concern was about the production of a pre-
dominant IgG1 response that antigenic rCTE of L. infan-
tum elicited in our previous studies which confirmed that
rCTE is not protective as a vaccine candidate (10). There-
fore, there was still a need to further explore and examine
the protective potential of rCPB without the CTE frag-
On the other hand, rCPB, with or without CTE, in
common with other subunit antigen requires a suitable
adjuvant and⁄or delivery system to enhance and direct the
induced immunity. The delivery system must not only pro-
tect the antigen from extracellular enzymatic degradation
but also target it to the relevant immune cells. Polymeric
particulate drug delivery systems as well as lipidic delivery
systems, such as fat emulsions, liposomes and solid lipid
nanoparticles (SLN), are particulate carriers that can deli-
ver cargos such as proteins efficiently.
It has been shown that SLN are highly biocompatible
and have the ability to encapsulate antigens and release
them in a controlled manner (11). In the present study, we
investigated the preparation of L. major rCPB with and
without CTE (rCPB)CTE) and their entrapment in SLN.
The ability of formulations to induce the required immune
responses for protective immunity against L. major infec-
tion in the resistant C57BL⁄6 mice was evaluated via
administration of vaccine through an i.p. route. This route
of vaccine administration might be helpful because of high
accessibility to antigen-presenting cells (APCs) in the peri-
toneal cavity and lymph nodes (LNs) (12) and has been
reported in several studies for evaluation of protein deliv-
ery when using lipidic colloidal carriers (13,14).
MATERIALS AND METHODS
All solutions were prepared using MilliQ?
(Milli-Q-System, Millipore, Molsheim, France) and apyro-
genic water. Cetyl palmitate, Tween-80 and cholesterol
were purchased from Merck (Darmstadt, Germany).
Sodium dodecyl sulphate (SDS) was purchased from
Sigma-Aldrich (Deisenhofen, Germany). The materials
acquired from Sigma (Darmstadt, Germany) and those
applied for PCR and enzymatic digestion were provided
by Roche Applied Sciences (Mannheim, Germany). Cell
culture reagents including fetal calf serum (FCS) and
RPMI were sourced from Gibco (Gibco, Life Technologies
GmbH, Karlsruhe, Germany) and Sigma, respectively.
Mice and parasite
20 € 5 g) were obtained from the Pasteur Institute of Iran
and were housed in plastic cages with free access to tap
water and standard rodent pellets in an air-conditioned
room under a constant 12:12-h light–dark cycle and kept
at room temperature of 25?C with relative humidity (50–
60%). The appropriate review board ethics committee of
the Pasteur Institute of Iran has reviewed the use of exper-
iments involving animals.
The L. major strain (MRHO⁄IR⁄75⁄ER) was kept in a
virulent state by continuous passage in BALB⁄c mice. A
homogenized lymph node from an infected BALB⁄c
mouse was cultured in RPMI-1640 media supplemented
with 10% FCS and 100 lg of gentamycin⁄mL. Five-day
D. Doroud et al.
? 2011 Blackwell Publishing Ltd, Parasite Immunology, 33, 335–348
ratio was significantly higher and IgG1 was significantly
lower after the challenge in the SLN-CPB)CTEvaccinated
group (Figures 5, 6 and 7c). Protection also correlated
with a strong immune response as evidenced by enhanced
IgG2a isotype antibodies, which is possibly because of
stimulation of a Th1 type of immune response. Also, the
high induction of this antibody proves the ability of SLN
to preserve antigen integrity and to result in long-lasting
protection, as the induction of IgG2a might be more sensi-
tive to antigen integrity than that of IgG1 (29). The pro-
longed antibody induction is consistent with the antigen
release profile that also revealed the depot effect (antigen
localization). This aptitude of SLNs is primarily attributed
to an increased antigen uptake by APC endocytosis that
consequently enhances antigen presentation to T cells.
Hence, SLN seems to be a suitable adjuvant and antigen
delivery systems for designing subunit vaccines based on
CPs. It would have been interesting to assess whether the
vaccinated mice were immune to reinfection, but we used
C57Bl⁄6 mice that are believed to maintain a life-long
immunity after infection (30,31); this idea will be consid-
ered in our next study using the BALB⁄c mice.
Our future studies are being designed to assess SLN
ability to expand the sustained release profile to an active
targeting. Further vaccination studies via the subcutane-
ous route using other mouse strains are also underway in
D. Doroud thanks Tehran University of Medical sciences
and Pasteur Institute of Iran for the grants supporting her
PhD studentship. The authors thank Dr Crispin Williams
for critical English revision of the manuscript. The authors
also thank Mr A. Javadi (Pasteur Institute of Iran, Depart-
ment of Immunology) and also Mr Sh. Alizadeh (Pasteur
institute of Iran, Molecular Immunology and Vaccine
Research Laboratory) for their technical assistance.
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