VACCINE COMPOSITION FOR PREVENTING OR TREATING
LEISHMANIASES
The present invention relates to a vaccine composition,
5 based on excretory/secretory proteins from
promastigotes and/or amastigotes of Leishmania sp., for
preventing or treating leishmaniases in mammals,
preferably in the Canidae.
10 Leishmaniases are endemic parasitic infections caused
by a flagellate protozoan of the family Trypanosomidae
and of the genus Leishmania, transmitted by a sandfly
(for review, see : Chang, Leishmaniases, Encyclopedia of
Life Sciences, 2005, John Wiley & Sons, Ltd.; Bates,
15 Leishmania, Encyclopedia of Life Sciences, 2006, John
Wiley & Sons, Ltd.). A very large number of mammalian
species, including humans and dogs, are infected with
this parasite. The female sandflies, when they bite a
mammalian host, transmit to said mammalian host the
20 parasite in its infecting flagellate form, the
metacyclic promastigote. The metacyclic promastigotes
are then phagocytosed by the macrophages present in the
dermis and transform into a nonflagellate form called
an amastigote.
25
Eleven species of Leishmania, divided up into two
subgenera, Leishmania leishmania and Leishmania
viannia, are of medical importance in humans. Each
Leishmania species is responsible for a well-defined
30 clinical manifestation: visceral, cutaneous, diffuse
cutaneous and cutaneous-mucosal, depending on whether
the parasite infects the mononuclear phagocytes of the
viscera, of the dermis or of the mucous membranes.
35 Leishmaniasis is endemic in 88 countries (including
India, Bangladesh, Nepal, Sudan and Brazil), posing a
threat to approximately 350 million people. Visceral
leishmaniasis, caused by Leishmania (L) donovani and
Leishmania (L) infantum (or L. chagasi) , affects
500 000 people each year throughout the world. It
represents the most severe form of leishmaniases.
5
In the areas affected by visceral leishmaniasis caused
by L. infantum, in particular in the Mediterranean
region, dogs, which are massively infected, represent a
reservoir of parasites for humans.
10
In humans, leishmaniases are treated mainly with
antimony derivatives : meglumine antimoniate
( GlucantimeB) and sodium stibogluconate (Pen tos tam) .
However, in certain regions of the world, the parasite
15 has become resistant to antimony. Arnphotericin B
(Ambisome8) is therefore now used in said regions for
treating visceral or cutaneous-mucosal leishmaniasis.
However, there is for the moment no vaccine nor any
20 prophylactic medicament for preventing leishmaniases in
humans. Indeed, the development of a vaccine
composition continues to be a problem today.
Several vaccine compositions have been proposed for
25 protecting humans and/or dogs against leishmaniasis:
- a vaccine composition based on an antigenic membrane
fraction of Leishmania donovani (known as FML,
"fucose mannose ligand", antigen) and on an adjuvant
such as alum (aluminum-based adjuvant) , FIA (Freund' s
30 Incomplete Adjuvant) , BCG (Bacillus Calmette Guerin) ,
interleukin 12, mixtures of saponins such as Riedelde
Haen saponin R (Sigma-Aldrich) or Quil A
(Brenntag) and the purified saponin QS21 (for review,
see Palatnik-de-Sousa et al., Expert. Rev. Vaccines,
3 5 2008, 7:833-851). The FML antigen membrane fragment
comprises a glycosylated glycoprotein GP36 and also
several proteins which have not all been
characterized;
- a vaccine composition based on excretory/secretory
proteins (ESPs) from promastigotes and/or from
amastigotes of Leishmania sp. and on MDP (muramyl
5 dipeptide) as adjuvant inducing a Thl-type immune
response (Bourdoiseau et al., Vet. Immunol.
Immunopathol., 2009, 128:71-78; Lemesre et al.,
Vaccine, 2007, 25:4223-4234; Lemesre et al., Vaccine,
2005, 23:2825-2840; international applications Nos
10 WO 94/26899 and WO 2002/100430);
- a vaccine composition comprising two peptides (A16E
and A16G) from Leishmania and/or their derivatives
and an adjuvant such as MDP (international
application WO 03/025012) or QS21. However, this
15 composition has the disadvantage of inducing an
immunity directed against a limited number of
antigens ;
- a vaccine composition based on ultrasound-inactivated
Leishmania braziliensis promastigotes and on the
20 saponin mixture Riedel-de Haen saponin R as adjuvant
(Giunchetti et al., Vaccine, 2007, 25:7674-7686 and
Giunchetti et al., Vaccine, 2008, 26: 623-638;
Brazilian patent application PI 0601225-6). However,
this composition has the drawback of containing a
25 mixture of uncharacterized antigens that may pose
problems in terms of innocuousness and some of which
may induce immune responses of Thl type and others of
Th2 type;
- a vaccine composition comprising the recombinant A2
3 0 protein originating from the amastigote form of
Leishmania donovani and an adjuvant such as the
saponin mixture Riedel-de Haen saponin R (Fernandes
et al., Vaccine, 2008, 26:5888-5895, international
application WO 2008/009088).
35
For dogs, there are currently two vaccines marketed in
Brazil: a vaccine composition called LeishmuneB,
c o n s i s t i n g of the FML antigen, of the saponin mixture
Riedel-de Haen saponin R and of NaCl ( i n t e r n a t i o n a l
a p p l i c a t i o n No. WO 2006/122382; Palatnik-de-Sousa et
a l . , 2008, mentioned above), and a vaccine composition
5 c a l l e d LeishTecB, c o n s i s t i n g of the A2 antigen and of a
saponin mixture.
It emerges from the numerous s t u d i e s r e l a t i n g t o the
immune response of hosts ( i n p a r t i c u l a r i n dogs),
10 during i n f e c t i o n with Leishmania, t h a t the e f f i c a c y of
a p o t e n t i a l vaccine candidate must be r e f l e c t e d by the
a c t i v a t i o n i n the host of a Thl-type immune response
with production of i n t e r f e r o n gamma ( B a r b e r , P a r a s i t e
Immunol., 2006, 28:329-337, Review; Alvar et a l . , Adv.
15 P a r a s i t o l . , 2004, 57:l-88; Ravindran and A l i , Curr.
Mol. Med., 2004, 4:697-709 and Gradoni, Vet.
P a r a s i t o l . , 2001, 100:87-103). Numerous adjuvants, such
as t h e p u r i f i e d saponin QS21, are capable of
s t i m u l a t i n g a Thl-type immune response.
20
Saponins of Q u i l l a j a type are a mixture of t r i t e r p e n e
glycosides extracted from the bark of the Q u i l l a j a
saponaria t r e e . They are n a t u r a l products which are
characterized by a c e r t a i n number of common p r o p e r t i e s ,
25 i n p a r t i c u l a r the a b i l i t y t o produce foam i n an aqueous
s o l u t i o n . Additional c h a r a c t e r i s t i c s are hemolytic
a c t i v i t y , t o x i c i t y with respect t o f i s h , t h e formation
of complexes with c h o l e s t e r o l and, i n c e r t a i n cases,
a n t i b i o t i c a c t i v i t y (Kofier, Die Saponine (Springer
30 Verlag), Berlin, 1927; Tschesche et a l . , Chemie und
Biologie der Saponine. Fortscher. 30 Chem. Org.
Naturst. XXXf Abl (1972) ) .
The Q u i l l a j a saponin mixtures a v a i l a b l e on the market
35 are crude mixtures which, because of t h e i r v a r i a b i l i t y
and t h e i r lack of complete c h a r a c t e r i z a t i o n , are not
d e s i r a b l e f o r use i n v e t e r i n a r y p r a c t i c e or i n
pharmaceutical compositions for humans. The impurities
present in the products available on the market can
lead to adverse reactions. Furthermore, the content of
active substance from one batch of saponin mixtures to
5 another can vary, thereby decreasing reproducibility.
Saponin adjuvants have been identified and purified by
high performance liquid chromatography (HPLC) . The
method for producing these purified fractions is
10 described in American patent US 5 057 540.
Among them, the substantially pure QA21 saponin (or QA-
21) is characterized by the following properties:
- immune adjuvant activity,
15 - contains approximately 22% of carbohydrates (analyzed
using anthrone) by dry weight,
- has a maximum UV absorption at 205-210 nm,
- has a retention time of approximately 51 minutes by
RP-HPLC on a Vydac C4 column having particles of 5 pm,
20 pores of 330 A, an internal diameter of 4.6 mm and a
length of 25 cm, in a solvent of acetic acid at 40 mM
in methano1:water (58 :42; v:v) , at a flow rate of
1 ml per minute,
- is eluted with 69 to 70% methanol from a Vydac C4
25 column having particles of 5 pm, pores of 330 A, an
internal diameter of 10 mm and a length of 25 cm, in
a solvent of acetic acid at 40 mM with an elution
gradient of from 50 to 80% of methanol,
- has a critical micellar concentration of 0.03% (w:v)
30 in water and of 0.02% (w:v) in phosphate buffered
saline,
- causes sheep red blood cell hemolysis at
concentrations of 25 pg per ml or more, and
- contains the terminal rhamnose, terminal arabinose,
3 5 terminal apiose, terminal xylose, 4-rhamnose,
terminal glucose, terminal galactose, 2-fucose, 3-
xylose, 3,4-rhamnose and 2,3-glucuronic acid
monosaccharides.
The term "substantially pure" means substantially free
of compounds normally associated with saponin in its
5 natural state and giving rise to a chromatographic
response, elution profiles and a biological activity
that are constant and reproducible.
The substantially pure QA21 saponin previously
10 described is also called QS21 (or QS-21) (for review,
see Kensil et al., Pharm. Biotechnol., 1995, 6: 525-541;
Kensil and Kammer, Expert Opin. Investig. Drugs., 1998,
7:1475-1482 and Soltysik et al., Vaccine, 1995,
13: 1403-10) . QS21 is an amphipathic (water-soluble)
15 molecule which has a molar mass of 1988 g/mol. Its
structural formula is represented in figure 1. A method
for obtaining the QS21 is described in American patent
US 5 057 540. Nevertheless, QS21 can also be purified
from saponin mixtures such as Riedel-de Haen saponin R
20 or Quil A. The efficacy of QS21 has been evaluated in
human and veterinary immunology, in its pure form, for
the treatment of cancers, AIDS or malaria, or feline
leukosis (LeucogenB vaccine - Virbac) . In the saponin
mixtures Riedel-de Haen saponin R and Quil A, QS21
25 appears to represent the major saponin fraction,
respectively in the region of 18% and 40-50% (Palatnikde-
Sousa et al., 2008, mentioned above, and Parra et
al., Vaccine, 2007, 25:2180-2186). The proportion of
QS21 in the saponin mixtures Riedel-de Haen saponin R
30 and Quil A vary, however, depending on the publication:
for example, in the Quil A mixture, according to Kensil
et al. (J. Immunol., 1991, 146:431-437), QS21 does not
represent the major saponin fraction, whereas,
according to other publications by the same authors, it
35 supposedly represents 40% (Parra et al., 2007,
mentioned above) or 49% (Santos et al., 2002, Vaccine,
21, 30-43) of the mixture. This variability in amount
of QS21 may point to a low reproducibility in the
production of these saponin mixtures. QS21 induces a
good Thl-type immune response, but has, however,
several side effects, among which mention may be made
5 of: edema at the point of injection owing to a strong
local inflammatory reaction, local pain, anorexia,
apathy, vomiting and diarrhea. These side effects
therefore limit its use. One solution proposed for
remedying these disadvantages consists in combining
10 QS21 with compounds which reduce its side effects, such
as, for example, a sterol (international application
WO 96/33739).
The inventors set themselves the objective of providing
15 a novel vaccine composition for preventing or treating
leishmaniases which would provide long-lasting
protection of mammals against these diseases, which
more successfully meets the practical needs than the
vaccine compositions previously proposed and which
20 causes few or no side effects.
Consequently, the subject of the present invention is a
vaccine composition for preventing or treating
leishmaniasis in a mammal, consisting:
25 - of excretory/secretory proteins (ESPs) from
promastigotes and/or amastigotes, preferably
promastigotes, of Leishmania sp., produced in an
axenic, serum-free medium;
- of substantially pure QA21 saponin; and
30 - optionally, of a pharmaceutically acceptable
excipient.
The expression "preventing leishmaniasis" is intended
to mean inducing a protection or an immune response
35 against Leishmania infections.
The expression "treating leishmaniasis" is intended to
mean reducing the symptoms of leishmaniasis, or
reducing or eliminating Leishmania in the infected
mammal.
5 Said excretory/secretory proteins (ESPs) can be
obtained by implementing the methods described in
international applications WO 94/26899 and
WO 2002/100430.
10 According to one preferred embodiment of the present
invention, said excretory/secretory proteins are from
promastigotes and/or amastigotes, preferably
promastigotes only, of Leishmania (L) infantum.
15 According to one advantageous embodiment of the
invention, the amount of substantially pure QA21
saponin in said vaccine composition is from 30 to
120 pg per vaccine dose, preferably from 50 to
90 pg/vaccine dose and more preferentially
20 60 pg/vaccine dose.
The term "vaccine dose" is intended to mean the volume
of the vaccine composition according to the present
invention administered to a mammal. The vaccine dose
25 varies according to the size of the mammal to which the
vaccine composition is administered. This dose is
generally from 0.1 to 5 ml. Preferably, the vaccine
dose is 1 ml.
30 The inventors have shown that, surprisingly, a dose of
QA21 of 60 pg per vaccine dose gave the best
efficacy/innocuousness ratio for the dogs treated.
Indeed, the systemic reactions (side effects) of the
animals vaccinated with this dose were nonexistent and
35 the local reactions were weak. Furthermore, at this
dose, the humoral response (kinetics and titer) was
preserved with respect to the higher doses. In
addition, it is not necessary to combine QA21 with
compounds such as sterols, as recommended in the prior
art, in order to reduce its side effects, or to use it
in saponin mixtures, such as Riedel-de Haen saponin R
or Quil A, thus making the manufacture of a vaccine
composition simpler, said composition containing an
adjuvant which is characterized, defined and
reproducible, and therefore controlled more easily, and
inducing an activity/innocuousness ratio that is
constant over time.
According to one advantageous embodiment of the
invention, the amount of ESPs in said vaccine
composition is from 10 to 140 1-19 per vaccine dose,
preferably from 80 to 140 1-19 per vaccine dose,
particularly from 90 to 130 1-19 per vaccine dose and
more preferentially 110 1-19 per vaccine dose.
According to another embodiment of the invention, the
substantially pure QA21 saponin is the only saponin
adjuvant of the composition.
According to an even more advantageous embodiment, the
substantially pure QA21 saponin is the only adjuvant of
the composition (no other element of the composition
can adjuvant the antigens).
The pharmaceutically acceptable excipient may be any
excipient known to those skilled in the art.
Preferably, it comprises or consists of one or more of
the compounds chosen from the group consisting of
sodium chloride (NaCl), lyophilization agents such as
mannitol and sucrose, buffers such as Tris
(trishydroxymethylaminomethane), preservatives or
stabilizers.
According to another advantageous embodiment of the
invention, the vaccine composition is for preventing or
treating leishmaniasis in a mammal chosen from the
group comprising humans and the Canidae, preferably the
species Canis lupus (dog) .
5
According to another advantageous embodiment of the
invention, said vaccine composition is for preventing
or treating visceral leishmaniasis, more particularly
that caused by Leishmania (L) infantum, Leishmania (L)
10 chagasi and Leishmania (L) donovani.
According to this embodiment, said vaccine composition
can be in a lyophilized form.
15 The subject of the present invention is also a method
for manufacturing a vaccine composition for preventing
or treating leishmaniasis in a mammal, comprising the
mixing :
- of excretory/secretory proteins (ESPs) from
20 promastigotes and/or amastigotes of Leishmania sp.,
preferably from promastigotes of Leishmania (L)
infantum, produced in an axenic, serum-free medium,
as defined above,
- of substantially pure QA21 saponin, and
25 - optionally, of a pharmaceutically acceptable
excipient, as defined above.
According to one advantageous embodiment of the method
of the invention, the amount of substantially pure QA21
30 saponin in said composition is from 30 to
120 pg/vaccine dose, preferably from 50 to
90 pg/vaccine dose and more preferentially
60 pg/vaccine dose, and the amount of ESPs is from 10
to 140 pg/vaccine dose, preferably from 80 to
35 140 pg/vaccine dose, more preferably from 90 to
130 pg/vaccine dose and more preferentially
110 pg/vaccine dose.
The subject of the present invention is also a kit
comprising at least one injectable dose of a vaccine
composition according to the present invention.
5
The subject of the present invention is also a method
for treating or preventing leishmaniasis in a mammal,
comprising the administration to said mammal of a
vaccine composition as defined above. Preferably, the
10 administration of the vaccine composition is carried
out by subcutaneous injection.
In addition to the above arrangements, the invention
also comprises other arrangements that will become
15 apparent from the following description, which refers
to exemplary embodiments of the vaccine composition
which is the subject of the present invention and also
to the appended drawings:
- figure 1 illustrates the structural formula of QS21;
20 - figure 2 is a graph representing the mean of the
titers (in loglo) of anti-ESP IgGl as a function of
time for each group of dogs vaccinated with
110 pg/dose of excretory/secretory proteins (ESPs)
from promastigotes of L. Infantum and, as adjuvant,
25 an amount of QA21 respectively of 500 pg/dose
(vaccine composition LSH303), 250 pg/dose (vaccine
composition LSH304), 125 pg/dose (vaccine composition
LSH305) and 60 pg/dose (vaccine composition LSH306) ;
- figure 3 is a graph representing the mean of the
30 titers (in loglo) of anti-ESP IgG2 as a function of
time for each group of dogs vaccinated with the
vaccine composition LSH303, LSH304, LSH305 or LSH306;
- figure 4 is a graph representing the titers (in
loglo) of anti-ESP IgGl as a function of time in the
35 two dogs vaccinated with the vaccine composition
LSH304 (figure 4A) and in the two dogs vaccinated
with the vaccine composition LSH206 (figure 4B);
- figure 5 is a graph representing the titers (in
loglo) of anti-ESP IgG2 as a function of time in the
two dogs vaccinated with the vaccine composition
LSH304 (figure 5A) and in the two dogs vaccinated
5 with the vaccine composition LSH306 (figure 5B);
- figure 6 is a graph representing, as a function of
time, the leismanicidal activity (percentage of L.
i n f a n t u m amastigotes killed) of the macrophages of
the two dogs vaccinated with the vaccine composition
10 LSH304 (figure 6A) and of the two dogs vaccinated
with the vaccine composition LSH306 (figure 6B);
- figure 7 is a graph representing the production of NO
(nmol/10~ cells/72 h) as a function of time in the two
dogs vaccinated with the vaccine composition LSH304
15 (figure 7A) and in the two dogs vaccinated with the
vaccine composition LSH306 (figure 7B);
- figure 8 is a graph representing the amount (as
percentage) of the NO-synthase enzyme inducible in
the supernatant of the cocultures of autologous
20 macrophages and lymphocytes originating from the two
dogs vaccinated with the vaccine composition LSH304
(figure 8A) or from the two dogs vaccinated with the
vaccine composition LSH306 (figure 8B).
25 EXAMPLE 1: Study of innocuousness and of efficacy, in
dogs, of three injections of a vaccine composition
comprising ESPs of L. infantum and QA21
The purpose of this study was to test various
30 concentrations of QA21 in a vaccine composition
comprising ESPs from promastigotes of L. i n f a n t u m and
QA21 as the only adjuvant, in order to determine the
best efficacy/innocuousness ratio.
35 1.1. Materials and methods
Obtaining the ESPs
The excretory/secretory proteins (ESPs) from
promastigotes of Leishmania infantum, produced in an
axenic, serum-f ree medium, were obtained in accordance
5 with the method described in international applications
WO 2002/100430 and WO 94/26889 using the culture media
and parameters adapted to industrial production.
Vaccine compositions
10
Vaccine compositions consisting of 110 pg/dose of
excretory/secretory proteins (ESPs) from promastigotes
of Leishmania infantum, produced in an axenic, serumfree
medium, of decreasing amounts of QA21: 500 pg/dose
15 (vaccine composition LSH303), 250 pg/dose (vaccine
composition LSH304), 125 pg/dose (vaccine composition
LSH305) and 60 pg/dose (vaccine composition LSH306),
and of a pharmaceutically acceptable excipient
(excipient TRIS mannitol , sucrose and NaC1) were
20 prepared.
Dogs: Nine dogs of the Beagle breed, at least 6 months
old, conventionally vaccinated and wormed one week
before the first vaccination with the vaccine
25 composition based on ESPs and QA21.
Vaccination protocol
Two dogs each received 3 injections of the vaccine
30 composition LSH303, 4 weeks apart, on DO, 27 and 55;
Two dogs each received 3 injections of the vaccine
composition LSH304, 4 weeks apart, on DO, 27 and 55;
35 Two dogs each received 3 injections of the vaccine
composition LSH305, 4 weeks apart, on DO, 27 and 55;
Three dogs each received 3 injections of the vaccine
composition LSH306, 4 weeks apart, on DO, 27 and 55.
The vaccinations were carried out subcutaneously.
5
Immune response evaluation criteria
(i) Local and general clinical follow-up: a local and
general clinical follow-up (including measurement of
10 rectal temperature and of weight) was carried out on
each day of vaccination and 1 and 3 days after
vaccination. In the event of persistence of the
clinical signs 3 days after vaccination, this follow-up
was carried out twice a week until the symptoms had
15 completely disappeared. A complete clinical
examination, consisting in verifying the general
condition of the dogs and including, inter alia,
measurement of rectal temperature and of weight, was
carried out once a week.
20
(ii) Serological analysis:
Blood samples were taken regularly in order to measure
the serological response of the dogs. The anti-ESP IgGl
25 and IgG2 antibodies in the serum of the vaccinated dogs
was assayed using the ELISA (Enzyme Link Immunosorbent
Assay) method, with peroxidase-labeled goat anti-dog
IgGl antibodies (ref. Bethyl Laboratories A40-120P) and
peroxydase-labeled sheep anti-dog IgG2 antibodies (ref.
30 Bethyl Laboratories A40-121P). The evaluation of the
IgGl and IgG2 response against the ESPs was carried out
by means of a Western blotting test.
1 . 2 R e s u l t s
General clinical examination
- for the group of dogs which received the vaccine
composition LSH303: a hypothermia peak was observed
after the 2nd vaccination (39.7"~a nd 4 0 . 8 " ~ )a~nd
for one of them also after the 3rd vaccination
5 (39.7"~). These dogs showed no symptom of general
type. The weight losses observed in this group never
exceeded 3% and are compatible with values observed
in the other three groups;
10 - for the groups of dogs which received the vaccine
composition LSH304, LSH305 or LSH306: no general sign
was observed after the vaccination. The transient and
isolated variations in weight gain observed in these
groups are compatible with the growth curves for dogs
15 of this age and are not related to the vaccinations.
Slight increases in rectal temperature were sometimes
observed 24 hours after the injection of the vaccine
in the groups having received the composition LSH304
or LSH306, but are not significant.
20
Local clinical examination
- for the group of dogs which received the vaccine
composition LSH303: severe local reactions were
25 observed 24 hours after the vaccination, including
tumefactions of more than 2 cm in diameter, regular
pain at the point of injection (simple sensitivity
during palpation or persistent pain) and cases of
edema of a large surface area. These signs had
30 disappeared within a maximum of 7 days after
injection of the vaccine composition as regards the
pain, within a maximum of 6 days as regards the edema
and within a maximum of 17 days as regards the
swellings;
35
- for the group of dogs which received the vaccine
composition LSH304: the local reactions observed
24 hours after the vaccinations were tumefactions
between 1 and 2 cm in diameter, and sensitivity at
the point of injection of the 2nd vaccination
(2 dogs) and of the 3rd vaccination (1 dog). Slight
5 pruritus (less than 1 minute) was observed
immediately after the 3rd vaccination (1 dog).
Moderate (localized) to severe (large surface area)
edema was observed, respectively, at the 2nd and 3rd
point of injection. These signs had disappeared
10 within a maximum of 3 days after the injections as
regards the pain, within a maximum of 6 days as
regards the edema and withing a maximum of 17 days as
regards the swellings;
15 - for the group of dogs which received the vaccine
composition MSH305: the local reactions observed
24 hours after the vaccination were similar in
intensity to those observed for the group of dogs
having received the vaccine composition LSH304, with
20 tumefactions between 1 and 2 cm in diameter, and
sensitivity at the site of injection of the 2nd
vaccination (1 dog). Moderate (localized) to severe
(large surface area) edema was also observed at the
3rd point of injection. These signs had disappeared
25 within a maximum of 3 days after the injections as
regards the pain, within a maximum of 6 days as
regards the edema and within a maximum of 17 days as
regards the swellings;
30 - for the group of dogs which received the vaccine
composition LSH306: the clinical signs remained
benign and amounted to 2 cases of slight pruritus
(less than 1 minute) observed immediately after the
3rd injection. A tumefaction up to 1-2 cm in diameter
35 was observed 24hours after the 1st and 2nd
vaccinations in all the dogs. This tumefaction had
disappeared within a maximum of 13 days after the
injection. Only one dog also showed a tumefaction for
24 hours after the 3rd vaccination, but this
disappeared 6 days after the injection.
Serological follow-up
The result of the assaying of the anti-ESP IgGl and
IgG2 antibodies is given in figures 2 and 3,
respectively.
10
The change in the mean of the anti-ESP IgGl and IgG2
titers was similar for the groups of dogs having
received the vaccine composition LSH303 or LSH304:
- the IgGls reached a mean titer of 103.13 and l ~ ~ . ~ 15 (titer in loglo) two weeks after the final injection,
respectively in the group having received the vaccine
composition LSH303 and in the group having received
the vaccine composition LSH304;
- the 1gG2s reached a mean titer of 103.13 (titer in
20 loglo) two weeks after the final injection in the two
groups.
The change in the mean of the anti-ESP IgGl and IgG2
titers was smaller for the groups of dogs having
25 received the vaccine composition LSH305 or LSH306:
- the IgGls reached a mean titer of 1 0 ~ .and~ ~
(titer in loglo) two weeks after the final injecton,
respectively in the groups having received the
vaccine composition LSH305 and in the group having
30 received the vaccine composition LSH306;
- the IgG2s reached a mean titer of 103.13 and 102.65
(titer in loglo) two weeks after the final injection,
respectively in the group having received the vaccine
composition LSH305 and in the group having received
35 the vaccine composition LSH306.
The decrease in the serological response, observed with
the lowest concentrations of adjuvant, is greater with
the IgGl isotype.
Finally, no significant increase in the serological
5 response was observed after the second injection,
regardless of the vaccine composition used.
The specific IgGl and IgG2 response against the ESPs,
analyzed by Western blotting, revealed 100%
10 seroconversion in the 4 groups of dogs.
Conclusion
The general innocuousness of a vaccine composition
15 according to the present invention formulated with
60 pg/dose of QA21 is good. On the other hand, edema
and pain were observed with the vaccine compositions
formulated with 500, 250 and 125 pg/dose of QA21.
20 The data on the local innocuousness are in favor of an
amount of 60 pg/dose of QA21 - an amount for which only
slight tumefactions are observed, for less than
13 days.
25 The serological follow-up shows maintenance of anti-ESP
IgG2 antibodies and a decrease in anti-ESP IgGl
antibodies for the lowest QA21 concentrations: 60 and
125 pg/dose.
30 A dose of 60 pg of QA21 therefore induces very moderate
local reactions while at the same time maintaining a
high humoral response and consequently exhibits the
best efficacy/innocuousness ratio for the dogs treated.
35 EXAMPLE 2: Study of innocuousness and efficacy, in
dogs, of two injections of a vaccine composed of ESPs
of L. infantum and of QA21 as adjuvant; analysis of the
immune response
The purpose of this study was to compare the efficacy
and the innocuousness of two formulations of vaccine
5 based on ESPs from promastigotes of L. infantum and
containing different amounts of QA21 (60 pg/dose or
250 pg/dose). The efficacy was studied by analyzing the
humoral and cellular immune response. The innocuousness
was studied by evaluating the local and general
10 reactions.
2.1. Materials and methods
Vaccine compositions: LSH304 and LSH306 (see example
15 4.1).
Dogs: Six SPF dogs aged 21 weeks.
Vaccination protocol:
20
Two dogs each received 2 injections of the vaccine
composition LSH304, 4 weeks apart, on DO and D28;
Two dogs each received 2 injections of the vaccine
25 composition LSH306, 4 weeks apart, on DO and D28;
Two dogs were not vaccinated.
The vaccinations were carried out subcutaneously.
30
Preparation of leishmanin
A strain of Leishmania infantum was thawed on DO and
placed in culture. After 5 to 6 days of culture, an
35 amplification was carried out by increasing the culture
volume. The parasites were maintained in culture until
they reached the stationary growth phase. The parasites
were then harvested, and the suspension was washed
three times with PBS. The parasites were lysed by means
of 3 freezing/thawing cycles. The leishmanin thus
obtained was stored at I -70°C.
5
DTH test
The DTH (Delayed Type Hypersensitivity) skin test, well
known to those skilled in the art, makes it possible to
10 determine whether a cell-mediated response is of Thl or
Th2 type (Black C.A., Dermatol. Online J., 1999, 5:7
http://dermatology.cdlib.org/D0Jvol5numl/reviews/black.
html; Kid P., Alternative Medicine Review, 2003, 8 : 233-
246).
15
Four weeks after the final injection (D56), each dog
received, intradermally, 0.1 ml of leishmanin in the
right side of the muzzle and 0.1 ml of control solution
(0.9% of NaCl and 0.4% of phenol) in the left side of
20 the muzzle.
Change in leishmanicidal activity:
Peripheral blood mononuclear cells or PBMCs are
25 obtained by centrifugation on a Ficoll gradient, and
then the monocytes are separated from the lymphocyte
population by adhesion on plastic, and each population
is incubated at 37°C in order to allow the monocytes to
become mature macrophages. The resulting macrophages
30 are then infected with Leishmania parasites. After
incubation, the excess parasites are removed and then
the macrophages are incubated for 24 h in complete
medium. The autologous lymphocytes are then added to
half the wells in order to evaluate the ability of the
35 macrophages to kill the parasites after activation by
the lymphocytes. The wells without lymphocytes act as
controls for multiplication of the parasite. After 72
h, the culture supernatants are recovered and stored,
the lymphocytes are removed and the macrophages are
fixed in the wells with methanol. A part of the
macrophages is stained with Giemsa and the percentage
5 inhibition of the parasitic index is evaluated. The
other part of the macrophages is used for the detection
of inducible NO synthase (iNOS) using specific
antibodies and/or by immunolabling with the avidinbiotin/
peroxidase-complex technique. The production of
10 NO2 (involved in the NO cascade) is evaluated in the
culture supernatants using the modified Griess
reference technique.
Immune response evaluation criteria:
15
During the "vaccination phase": the general and local
reactions were evaluated from DO to D4 and from D28 to
D32, as described in example 4.1; the serological
analysis was carried out as described in example 4.1;
20 the leishmanicidal activity of the macrophages was
measured; the CD~+CD~an+d CD~+CD~T +c ells, and the B
cells, were counted using a flow cytometry method.
During the "DTH phase" (subsequent to the leishmanin
25 injection): the general reaction was evaluated every
day from D56 to D63; the local reactions were measured
every day with a caliper rule; the serological analysis
was carried out using the ELISA method and by Western
blotting, as described in example 4. 1; the
30 leishmanicidal activity of the macrophages was
measured; the CD~+CD~an+d CD~+CD~T +c ells, and the B
cells, were counted.
2 . 2 . R e s u l t s
35
2.2.1 Results during the "vaccination phase"
General clinical examination
One of the two dogs vaccinated with the vaccine
composition LSH304 (containing a dose of 250 pg of
5 QA21) suffered hyperthermia for one day, associated
with a dip for 2 days, after the 1st vaccination. The
two dogs showed hypertrophy of the popliteal and
subcapsular lymph nodes after the 2nd vaccination; this
hypertrophy disappeared in 4 days. The two dogs
10 vaccinated with the vaccine composition LSH306
(containing a dose of 60 pg of QA21) showed only a
slight hypertrophy of the popliteal and subcapsular
lymph nodes after the 2nd vaccination, which
disappeared in 4 days. All the animals showed a weight
15 gain of 15% and 37% between DO and D56.
Local clinical examination
Local reactions of various intensities were observed in
20 the two groups of dogs vaccinated:
- in the group of dogs vaccinated with the vaccine
composition LSH304: observation of edema from 3 to
4 cm in diameter associated with pain after the 1st
vaccination. For one of the two dogs, the edema
25 progressed to a nodule (1 cm), which disappeared in
16 days. Observation of severe dependent edema 5 to
10 cm in diameter after the 2nd vaccination, which
disappeared in 4 days;
- in the group of dogs vaccinated with the vaccine
30 composition LSH306: observation of a tumefaction less
than 1.5 cm in diameter after the 1st vaccination,
which disappeared 1 day later. For one of the two
dogs, observation of dependent edema 3 to 5 cm in
diameter after the 2nd vaccination, which disappeared
35 in 3 days.
Serological analysis
All the dogs were seronegative with respect to anti-ESP
antibodies before the 1st vaccination.
5 The dogs vaccinated with the vaccine composition LSH304
exhibited high titers of anti-ESP IgGl and IgG2
antibodies (see figures 4A and 4B). The IgGl and IgG2
seroconversion took place starting from the 1st
vaccination.
10
The dogs vaccinated with the vaccine composition LSH306
exhibited very low titers of anti-ESP IgGl and IgG2
antibodies (see figures 5A and 5B). In the case of one
of the two dogs, the IgG2 seroconversion took place
15 starting from the 1st vaccination, whereas the IgGl
seroconversion occurred after the 2nd vaccination. In
the case of the other dog, the seroconversion for the
two serotypes took place after the 1st vaccination.
20 Figures 4 and 5 also show that the IgGl and IgG2
antibody titers correlate with the dose of the adjuvant
in the vaccine composition.
For the two group of dogs, the IgG2 antibodies directed
25 against the ESPs, revealed by the Western blotting
method, appeared starting from the 1st vaccination,
whereas the IgGl antibodies appeared starting from the
2nd vaccination.
30 Leishmanicidal activity of the macrophaqes
All the dogs were negative for leishmanicidal activity
of the macrophages before the 1st vaccination. The
macrophages of the dogs of the two vaccinated groups
35 were capable of killing the L. infantum amastigotes
after the 2nd vaccination when they were cocultured
with autologous lymphocytes. The level of the
leishmanicidal activity of the macrophages was similar
in the two vaccinated groups (figures 6A and 6B). On
the other hand, no leishmanicidal activity of the
macrophages of the dogs in the control group was
5 measured during the study.
In order to determine whether nitric oxide (NO) played
a role in the death of the amastigote form of L.
infantum after it had been brought into contact with
10 the macrophages of the vaccinated dogs, the production
of NO and the presence of the inducible NO synthase
(iNOS) enzyme were measured in the supernatant of the
cocultured cells (macrophages and lymphocytes).
15 The results are represented in figures 7 and 8. Only
the cocultures of the cells of the vaccinated dogs
exhibited an enhancement of the production of NO. This
increase correlates with an increase in the
concentration of the inducible NO synthase (iNOS)
20 enzyme.
Counting of CD~+CD~a'n d CD~+CD~T' cells, and of B cells
The number of helper T cells (cD~+cD~+o)f, cytotoxic T
25 cells (cD~+cD~a+n)d of B lymphocytes (CD21 cells) was
evaluated by cytometry before and after vaccination.
The number of blood cells, of cytotoxic cells and of B
lymphocytes remained stable for all the animals
(vaccinated and nonvaccinated) during the study. One of
30 the two dogs vaccinated with the vaccine composition
LSH304 and the two dogs vaccinated with the vaccine
composition LSH306 showed a slight but nonsignificant
increase in the number of helper T cells, after the 2nd
vaccination.
35
2.2.2 Results during the "DTH phase"
General clinical examination
No general reaction was observed after the injection of
the leishmanin in the dogs vaccinated with the vaccine
5 composition LSH304 or LSH306. All the animals continued
to gain weight (between +4% and +7% of body weight)
between D56 and D63.
Local clinical examination
10
The two dogs vaccinated with the vaccine composition
LSH304 exhibited, at the point of injection of the
leishmanin, a hardening associated with pain and
pruritus, which disappeared in 4 days.
The two dogs vaccinated with the vaccine composition
LSH306 exhibited, at the point of injection of the
leishmanin, a hardening associated with pain and
pruritus, which disappeared in 4 days.
On the other hand, none of the vaccinated dogs showed
any local reaction at the point of injection of the
control solution. Moreover, the two dogs of the control
group exhibited only an erythema at the point of
25 injection of the leishmanin.
The slight reactions observed in the control group are
the normal consequence of an intradermal injection of
100 p1 of a solution of antigens, whereas the severe
30 reactions in the vaccinated group are the sign of an
antigen-specific cell-mediated immune response which is
directed against the leishmanin injected.
Serological analysis
35
The results are represented in figures 4 and 5. The
injection of leishmanin does not modify the titer of
the anti-ESP IgGl or IgG2 antibodies in the dogs
vaccinated with the vaccine composition LSH304. On the
other hand, the injection of leishmanin caused a slight
increase in the level of anti-ESP IgGl or IgG2
5 antibodies in the dogs vaccinated with the vaccine
composition LSH306.
Leishmanicidal activity of the macrophages
10 The ability of the macrophages to kill the amastigote
forms of L. infanturn was increased after injection of
leishmanin (see figure 6). This result is in contrast
to the absence of leishmanicidal activity of the
macrophages of the control group throughout the study.
15 This increase in the leishmanicidal activity of the
macrophages correlates with an increase in the
production of NO and also the presence of inducible NO
synthase (iNOS) (see figures 7 and 8).
20 2.2.3 Conclusion
It is apparent from this study that the best ratio
between efficacy and innocuousness is observed with the
vaccine composition LSH306 (containing 60 pg of QA21) .
25 Indeed, a local reaction was observed only for one dog
after the 2nd vaccination. In addition, no hyperthermia
or dip was observed. The anti-ESP humoral response was
well developed. The macrophages of the dogs vaccinated
with this vaccine composition were capable of killing
30 the L. infanturn amastigotes through the activation of
the iNOS enzyme (which produces NO). In addition, the
two vaccinated dogs developed an intradermal reaction
48 hours after the injection of leishmanin, which is
the sign of a cell-mediated immune response.
CLAIMS
1. A vaccine composition for preventing or treating
leishmaniasis in a mammal, characterized in that
5 it consists:
- of excretory-secretory proteins (ESPs) from
promastigotes and/or amastigotes of Leishmania
sp., produced in an axenic, serum-free medium;
- of substantially pure QA21 saponin; and
10 - optionally, of a pharmaceutically acceptable
excipient.
2. The composition as claimed in claim 1,
characterized in that the amount of substantially
15 pure QA21 saponin is between 30 and 120 pg per
vaccine dose.
3. The composition as claimed in either one of claims
1 and 2, characterized in that the amount of
20 substantially pure QA21 saponin is between 50 and
90 pg per vaccine dose.
4. The composition as claimed in any one of claims 1
to 3, characterized in that the amount of
25 substantially pure QA21 saponin is 60 pg per
vaccine dose.
5. The composition as claimed in any one of the
preceding claims, characterized in that the
30 substantially pure QA21 saponin is the only
saponin adjuvant.
6. The composition as claimed in any one of the
preceding claims, characterized in that the
35 substantially pure QA21 saponin is the only
adjuvant.
7. The composition according to any one of claims 1
to 6, characterized in that the amount of ESPs is
between 10 and 140 pg per vaccine dose, preferably
between 80 and 140 pg per vaccine dose.
5
8. The composition as claimed in claim 7,
characterized in that the amount of ESPs is
110 pg/vaccine dose.
10 9. The composition as claimed in any one of claims 1
to 8, characterized in that said excipient
comprises or consists of one or more of the
compounds chosen from the group consisting of
sodium chloride, lyophilization agents, buffers,
15 preservatives and stabilizers.
10. The composition as claimed in claim 9,
characterized in that said excipient comprises
Tris (trishydroxymethylaminomethane), mannitol and
20 sucrose.
11. The composition as claimed in any one of claims 1
to 10 I characterized in that said
excretory/secretory proteins a re from
25 promastigotes or Leishmania sp.
12. A method for manufacturing a vaccine composition
for preventing or treating leishmaniasis in a
mammal, characterized in that it comprises the
30 mixing:
- of excretory/secretory proteins (ESPs) from
promastigotes and/or amastigotes of Leishmania
sp., produced in an axenic, serum-free medium,
- of substantially pure QA21 saponin, and
- optionally, of a pharmaceutically acceptable
excipient.