Note: Descriptions are shown in the official language in which they were submitted.
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"Ii-ZMUNOGENIC COMPOSITIONS CONTAINING ANTIGENS, GENE
VECTORS AND ADJUVANTS IN BIODEGRADABLE MICROSPHERES"
The present invention is related to processes of
production of pharmaceutical compositions which are able to
stimulate a specific immune response, cellular and/or
humoral, in a host immunized with an antigen or a gene
vector that codifies the antigen of -interest. Such
compositions involve biodegradable microspheres based on a
copolymer derived from polyesters, capable of encapsulating
and controlling the release of antigens, gene vectors, and
adjuvants that stimulate mediators of the immune response.
It is known that a class of compounds, including 6-
mono and 6,6 diesters a,a-D-trehalose (among them the
ester groups that contain 30 to 90 atoms of carbon, known
as trehalose monomycolate and trehalose dimycolate,
respectively), are able to promote recruitment of antigen
presenting cells and cytokine induction, being therefore
potential candidates as adjuvants in vaccines and
immunotherapy. In the present invention, the term
"adjuvant" refers to a substance that does not share immune
epitopes with the antigen of interest itself, but, is able
to stimulate the immune response against the antigen of
interest.
Patent US 4,340,588 describes the use of trehalose
dimycolate as adjuvant in a vaccine composition containing
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antigens of Brucella abortus (BA, 45/20 strain). In this
composition, impure natural mixtures of trehalose
dimycolate were obtained from several sources and were
denominated "cord factor". These factors usually present
90o dimycolate and 10o monomycolate, as well as a small
amount of exogenous material, which contains from 95 to 990
trehalose dimycolate after appropriate purification by
chromatographic techniques.
The administration of vaccines containing antigen and
trehalose dimycolate in an oil-in-water emulsion induced a
significant reduction of the infection, when compared to a
composition based only on soluble antigens of BA 45/20
strain. In' this study the extract was mixed with oil as an
emulsion. The inclusion of an oil phase is justified by its
importance as a vehicle for extracts obtained from
mycobacteria cellular wall, with the objective of
stimulating an appropriate immune response, although it is
known that the presence of this oil pnase increas.~4 _.. v
occurrence of severe undesirable reactions in the
administration site and can cause granuloma.
Tn an attempt to overcome the problems attributed to
the presence ~of oil in preparations with immunogenic
activity, the use of aqueous suspensions containing an
insoluble fraction of mycobacteria cellular wall was
proposed (US 5,759,554). Aqueous suspensions are prepared
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by the following steps: (l) rupture of the bacteria and,
subsequent centrifugation; (ii) elimination of protein from
the fraction based on cellular wall (cellular debris
obtained in the. centrifugation step) employing proteolytic
enzymes; (iii) treatment with detergents and washing of
fraction resulting from step (ii), (iv) freeze-drying and
(v) suspension of the freeze-dried fraction in an aqueous
solution, such as saline solution. In that invention, the
stimulation of the immune system through the employment of
a suspension based on mycobacterial fractions stimulates
the human or animal organism to neutralize an infectious
agent or delay the growth of cancerous cells. However, this
procedure does not allow a controlled adjuvant release,
which could result in optimization of the immune response.
Processes that target the antigen, gene vectors
and/or adjuvant have also been described in the literature,
as in patent US 5,643,605. In this invention, the use of
adjuvant and/or microencapsulated antigens is described for
therapeutic or prophylactic purposes.
The composition presented under patent US 5,643,605
contains microspheres of PLGA (D-L-lactide-co-glycolide)
with an average diameter varying from 20 to 100 ~.m
encapsulating such immunoadjuvants..The adjuvant used was
saponin (QS21) or muramildipeptide (MDP), which are able to
stimulate an immune response where: (l) the volume of the
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aqueous adjuvant incorporated into the polymer is equal or
lower than 1 mL for 3 grams of polymer; (ii) the molar
ratio between monomers of lactic and glycolic acid can vary
from 100:0 to 0:100; (iii) the inherent viscosity of PLGA
S polymer varies from 0.1 to 1.2 dLlg; (iv) the diameters of
the microspheres vary from 20 to 100 ~tm, and (v) the
adjuvant is released from microspheres according to a
triphasic model in which, during the first phase, less than
30% of the adjuvant is released in a period of 1 day;
during the second phase, less than 10% of the adjuvant is
released after a period of 1 day, which varies between
approximately 30 and 200 days; and a third phase, where the
remaining adjuvant is released after the precedent phases.
However, it is important to point out that the diameter of
1S. the microspheres presented in this invention varies between
and 100 Vim, hindering the induction of the immune
response when used by administration routes such as oral or
pulmonary. The ideal diameter for these routes should be
comprised between 1 and 10 Vim, which, as described in the
20 literature, is also the ideal range for' subcutaneous
administration of immunogenic compositions. In this same
invention, saponin was dissolved in ethanol solution and
added to the polymeric phase and, in another formulation,
MDP was dissolved in an aqueous phase that was added to the
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polymeric phase. In both preparations, the phase
containing the adjuvant corresponds to the inner aqueous
phase of the preparation method. However, this procedure
does not allow the addition of adjuvants that are soluble
5' in non-polar solvents.
Several vaccine formulations use adjuvants with the
purpose of optimizing the immune response when administered
with the antigen, mainly in subunit vaccines. In the last
decade, the progresses in vaccine development allowed the
introduction of new strategies for obtaining and producing
antigens, as well as new ways to administer and present
those antigens to the immune system. These strategies
allowed innovations, particularly in the context of the
development of safer, effective and versatile vaccines. In
this context, the DNA vaccines appeared as an interesting
alternative to elicit protection, especially when cellular
immune response is required.
Patent US 6,048,551 describes a method of preparation
of microspheres containing gene vectors which, are employed
in the treatment of tumors. In this invention, the multiple
emulsion method was used. The gene vector was dissolved in
an aqueous phase and encapsulated, associated or not to
antiviral drugs that were also dissolved in the aqueous
phase. The diameter of the particles ranged between 1 and
200 Vim.
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Patent US 6,309,569 claims a method of encapsulating
DNA in a polymer microparticle where the excess of organic
solvent is extracted by adding the (water-in-oil)-in-water
emulsion to an aqueous phase to extract the solvent,
thereby forming polymer microparticles of a size up to 10
~m in diameter, containing DNA in aqueous solution.
So far, a method that allows the coencapsulation of an
antigen or a gene vector and an immunoadjuvant in the same
preparation of PLGA-derived biodegradable microspheres is
yet to be described. Therefore, in spite of the efforts
that have been made in the sense of offering controlled
delivery formulations, containing or not a co-adjuvant
molecule which is able to improve immune response, the
state of the art still lacks a passive phagocytic cell
targeting system based on microspheres, with an appropriate
particle diameter so that it could be administered through
different routes and still target specific cellular
populations.
An important reason for the development of such
system refers to the need of an effective vaccine against
tuberculosis.
This invention presents an immunogenic composition
involving, at least, adjuvant andlor protein; and/or
plasmids containing protein-expressing genes, entrapped
l5 into polymeric microspheres with an average alameter
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smaller than 20 ~.m, preferentially between 1 and 10 ~.m.
Therefore, the composition contains, besides the adjuvant,
an antigen or a gene vector that codifies the antigen
(preferentially from parasites and pathogenic agents),
which include antigens of extra and intracellular bacteria,
eucariotic organisms such as fungi and protozoa, among
others. The adjuvants are, preferentially, those which
stimulate citokyne production. Ideally, the plasmid
carrying the gene of Mycobacterium proteins as, for
instance, the hsp65 gene (heat shock protein of 65 kDa from
M. leprae), or still, the purified or recombinant protein
may be encapsulated in polymeric microspheres derived from
lactic acid and copolymers of lactic and glycolic acid
(monomer ratio varying between 100:0 and 0:100). Moreover,
the trehalose dimycolate may be encapsulated in
microspheres of PLGA. The system which includes trehalose
dimycolate and/or the plasmid containing the gene of hsp65
protein, or still the protein itself, can also be applied
in the prevention and therapy of tuberculosis, when the
disease is already installed in the human or animal host.
The microencapsulation of a gene vector, antigen
and/or.adjuvant according to the present invention can be
obtained by the following protocols:.
Protocol A:(i) dissolution of the polymer derived from
lactic and glycolic acids (ratio in o of weight between
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100:0 and 0:100) in an organic solvent, which is non
miscible or partially miscible in water in a concentration
between 0.2 and 10%, as for example, ethyl acetate,
methylene chloride, chloroform, among others;(ii)
dissolution of the lipophilic antigen and/or adjuvant in
the solution (i), that contains the polymer, (iii) addition
of the solution .obtained in step (ii) to a polyol solution,
for example, polyvinyl alcohol (1 to 100), aiming the
formation of an oil-in-water emulsion, (iv) stirring of
this emulsion under an appropriated speed (200 to 1000
rpm), in order to~ obtain microspheres with an average
diameter smaller than 10 ~.~.m, preferentially between 1 and 5
~.m, and (v) solvent elimination under agitation, which can
be done by using co-solvents, by vacuum evaporation or
other techniques that allow solvent removal, not
compromising the stability of the system. The suspension of
microspheres obtained may be, alternatively, collected by
centrifugation or filtration, washed with an aqueous
solvent, then freeze-dried. ,
Protocol B: (i) dissolution of the polymer derived
from lactic and glycolic acids (ratio in o of weight
between 100:0 and 0:100) in an organic solvent in a
concentration between 0.2 and 10s, -for example, methylene
chloride, ethyl acetate, chloroform, among others, with or
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without a lipophilic adjuvant such as trehalose
dimycolate;(ii) dissolution of the gene vector, antigen
and/or adjuvant of hydrophilic nature, in 0.1 to 1 mL of an
aqueous phase, (iii) addition of the solution obtained in
(ii) to the polymeric solution obtained in (i), followed by
agitation under a speed ranging between 500 to 15,.000 rpm,
leading to the formation of a water-in-oil emulsion (or
dispersion), (iv) addition of this primary emulsion (or
dis.persion), after adequate period of agitation (1 to 10
minutes), to an emulsifying aqueous phase, typically
composed by a polyol solution, for example, polyvinyl
alcohol (1 to 100), aiming the formation of a water-in-oil-
in-water emulsion; (v) agitation of the multiple emulsion
obtained in (iv) under appropriated speed in order to
remove the organic solvent (100 a 1000 rpm), resulting in
microspheres with an average diameter smaller than 10 (gym,
preferentially between 1 to 5 Vim, which may be collected by
filtration or centrifugation, washed with an aqueous
solvent and finally freeze-dried.
It is important to mention that other methods known by
the state of the art could be employed to obtain antigens
and/or adjuvants-loaded microspheres. This is the goal of
this invention, which is not limited by the techniques
presented herein.
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The present invention is described in details by the
following examples:
EXAMPLE 1 - Preparation of trehalose dimycolate
One hundred grams of Mycobacterium tuberculosis H37Rv
5 strain are homogenized several times in a chloroform-
methanol mixture (1:1, vol/vol). The extracted material (18
g) is fractionated as previously described for trehalose
dimycolate purification (Silva, C. L., S. M. Ekizlerian,
and R. A. Fazioli. 1985. Role of cord factor in the
10 modulation of infection caused by Mycobacteria. Am. J.
Pathol. 118:238-247). The purified glycolipid (0.530 g)
migrates as a unique band when submitted to thin-layer
chromatography, similarly to the commercial product (SIGMA,
St. Louis, USA).
EXAMPLE 2 - Preparation and characterization of
microspheres containing trehalose dimycolate
Mi.crospheres are prepared following the emulsion
and solvent evaporation technique (Lewis, D. H. 1990.
Controlled release of bioactive agents from
lactide/glicolide polymers. In Chasin M & Langer R.
Biodegradable polymers as drug delivery systems. Marcel
Dekker, New York, 1-43). Five mg of trehalose dimycolate
(SIGMA) and 125 mg.,of PLGA (50:50)biodegradable (Resomer RG
505, MW 78,000, Boehringer Ingelheim, Germany) were diluted
in 30 mL of methylene chloride. This organic phase was
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mixed with 100 mL of an aqueous phase containing 30 of
polyvinyl alcohol (Mowiol~ 40-88, SIGMA Aldrich Chemicals)
as surfactant, aiming the formation of an oil-in-water
stable emulsion. In order to allow organic solvent
evaporation, a mechanical agitation (800 rpm) was applied
for a 6-hour period at room temperature. Microspheres were
collected by centrifugation at 10.000 x g, washed 3 times
with sterile water or saline, freeze-dried and stored at
4°C. Presence of trehalose dimycolate in inicrospheres was
determined by thin-layer chromatography after dissolution
of the particles in methylene chloride. Diameter.of the
particles was determined by laser difractometry in a CILAS
1064 Liquid apparatus (Cilas, France) showing average
diameter between 1 and 5 ~.m. Results were expressed as
cumulative values of % under determined diameter. Unloaded
PLGA microspheres or microspheres containing trehalose
dibehenate (Sigma, St. Louis, USA) or control lipid as
unrelated control (a glycerides mixture, Sigma, St. Louis,
USA) are prepared by the same procedure and used as
controls for experimental protocols.
EXAMPLE 3 .- Preparation of plasmids
E. coli, DHSa strain, transformed with the gene
vectors pCDNA3 or pCDNA3modified (pCDNA3m), and with the
plasmids containing the hsp65 gene (pCDriA3-hwL-=-_ ..
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pCDNA3modified-hsp65) are cultured in LB BROTH BASE medium
(GIBCO BRL, Scotland) containing 100 ~.g/mL of ampicillin
(SIGMA). The construct pCDNA3modified-hsp65 is derived from
pCDNA3 vector (Invitrogen~, Carlsbad, CA, USA), which is
previously digested with Bam HI and Not I (Gibco BRL,
Gaithersburg, MD, USA) and then, a fragment of 3.3 kb
corresponding to the M. leprae hsp65 gene and the CMV
intron A (citomegalovirus) is inserted in the vector. The
vector without the hsp65 gene is used as control. Plasmids
are purified by anion exchange resin (Concert High Purity
Maxiprep System, GIBCO BRL). Evaluation of plasmid
concentration is carried out by spectrophotometry in 7~ -
260 and 280 nm using a Gene Quant II apparatus (Pha.rmacia
Biotech).
EXAMPLE 4 -Preparation of recombinant protein
E. coli, BL21 strain, transformed with pIL-161 plasmid
containing the M. leprae hsp65 gene, are cultured in 500 mL
of LB BROTH BASE liquid medium (GIBCO BRL, Scotland)
containing ampicillin (SIGMA) in the concentration of 100
2f ~g/mL and IPTG (Isopropylthio-(3-D-Galactoside, GIBCO BRL)
0.1 M, at 37°C, under agitation at 200 rpm in an incubator
shaker for 18 hours. After incubation, cells are collected
by centrifugation at 7,500 rpm for. 15 minutes in a J2HS
centrifuge (Beckman, rotor JS-7.5), ressuspended in 10 mL
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of CE buffer (30 mM Sodium citrate, 10 mM EDTA, pH 6.1)
and disrupted by means of 3 pulses of 1 minute and 100 W,
using a Vibra CellTM ultrasound probe (Sonics & Materials,
USA) in ice bath. After a 16,500 rpm centrifugation for 20
minutes, the supernatant is discharged and the pellet is
ressuspended in 30 mL of UPE buffer (6 M urea, 20 mM EDTA,
50 mM monobasic potassium phosphate, pH 7.0) and vortexed
for 3 minutes. Suspension is kept under gentle agitation at
room temperature for 15 minutes and then centrifuged at
1.0 16,500 rpm for 20 minutes. Ammonium sulfate is added to the
supernatant to a final concentration of 1 M and incubated
in ice bath for 30 minutes. After centrifugation at 16,500
rpm for 20 minutes, the pellet is ressuspended in 3 mL of
PBS and exhaustively dialyzed against PBS.
EXAMP?~E 5 - Preparation of Plasmi.d or recombinant ~rrotein-
loaded microspheres
Microspheres are obtained by multiple emulsion
followed by solvent evaporation method: the aqueous phase
(0.3 mL) containing only the plasmid vector that does not
codify the hsp65 protein (pCDNA3 or PcDNA3modified); or the
vector containing the gene that codifies the hsp65 protein
(pCDNA3-hsp65, pCDNA3modified-hsp65); or only the
recombinant hsp65 protein is emulsified in 30 mL of
methylene chloride containing 400 mg of PLGA 50:50 (Resomer
RG 505 from Boehringer Ingelheim), under strong agitation
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(8000 rpm) using an Ultraturrax T50 homogenizer (IKA~ -
Labortechnik, Germany), in order to obtain a water-in-oil
primary emulsion. For preparation of microspheres
containing plasmid, 4 mg of plasmid are dissolved into 0.3
mL of aqueous phase and, in ease of recombinant hsp65, 1 mg
of protein is dissolved into 0.3 mL of aqueous phase. This
emulsion is added to an external aqueous phase (100 mL),
containing 3~ of polyvinyl alcohol (Mowiol~ 40-88, Aldrich
Chemicals) as surfactant, and homogenized to form a
multiple water-in-oil-in-water emulsion. The organic
solvent is removed by evaporation at room temperature under
agitation (300 to 800 rpm) using a Eurostar homogenizer
(IKA~ - Labortechnik, Germany) for ~ to 10 hours.
Microspheres are collected by centrifugation at 10.000 x g
in a Himac CR21 centrifuge (Hitachi, rotor R20A2), washed 3
times with sterile water, freeze-dried, then stored at 4°C.
Particle diameter is evaluated by laser difractometry in a
CILAS 1064 Liquid apparatus (Cilas, France). The average
particle diameter should be comprised between 1 and 5 Vim.
EXAMPhE 6 - Preparation of dimycolate and plasmid or
dimycolate and protein-loaded mi.crospheres
Microspheres are obtained by multiple emulsion and
solvent evaporation method: the aqueous phase (0.3 mL)
containing pCDNA3, PcDNA3modified, pCDNA3-hsp65,
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PcDNA3modified-hsp65 or recombinant hsp65 (4 mg for
plasmid or 1 mg for protein), is emulsified in 30 mL of
methylene chloride containing 0.5 mg of trehalose
dimycolate and 400 mg of PLGA 50:50 (Resomer RG 505 da
5 Boehringer Ingelheim) under strong agitation (8,000 rpm),
using an Ultraturrax T50 homogenizer (IKA~ - Labortechnik,
Germany), in order to obtain a water-in-oil primary
emulsion. This emulsion is added to an external aqueous
phase (100 mL), containing 30 of polyvinyl alcohol
10 (Mowiol~ 40-88, Aldrich Chemicals) as surfactant, and
homogenized to form a multiple water-in-oil-in-water
emulsion. The organic solvent is removed by evaporation at
room temperature under agitation (300 to 800 rpm) , using a
Eurostar homogenizer (IKA~ - Labortechnik, Germany) for 6
15 hours. Microspheres are collected by centrifugation at
10.000 x g in a Himac CR21 centrifuge (Hitachi, rotor
RZOA2), washed 3 times with sterile water, freeze-dried and
stored at 4°C. Particle diameter is evaluated by laser
difractometry in a CILAS 1064 Liquid apparatus (Cilas,
France). The average particle diameter should be between 1
and 5 Vim.
EXAMPLE 7 - Evaluation of hsp65 expression in eucariotic
cells transfected with plasmid-loaded microspheres
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J774 cells (tumoral lineage cells originally from
BALB/c mice) and J774-hsp65 (J774 cells transfected with
retroviral vector [pZIPNeoSV(x)] carrying the M. Leprae
hsp65 gene) are cultured in 1640 RPMI medium (SIGMA)
supplemented with 2 % of heat inactivated fetal calf serum,
at 37 °C in a 5 % C02 atmosphere. One hundred ~L of cell
suspension (5x105 cells) are placed on sterile cover slips
in a 24 well plate and incubated for 3 hours to allow cell
adhesion. After incubation, 1 mL of RPMI medium, is added
to each sample and treated as follows:
A) J774 cells receive 1 mL of RPMI medium and are used as
negative control for hsp65 expression;
B) J774-hsp65 cells receive 1 mL of RPMI medium and are
used as positive control for hsp65 expression;
C) J774 cells receive 1 mL of RPMI medium containing
pCDNA3modified-hsp65-loaded microspheres (10
microspheres/cell). Particles are ressuspended in medium
and the concentration of this suspension is adjusted to
5x106 particles/mL, counting the particles in a Neubauer
chamber;
D) J774 cells receive 1 mL of RPMI medium containing
pCDNA3modified-hsp65 complexed with Lipofectin~ (GIBCO
BRL ) .
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Twenty-four hours after incubation, the culture
supernatants are removed and replaced with 1 mZ of RPMI
medium. Medium is substituted every 48 hours.
Immunocytochemistry analyses are performed 10 and 20 days
after the beginning of the test.
For the immunocytochemistry evaluation, the cells are
washed 3 times in PBS solution containing 1% of bovine
serum albumin (BSA) (SIGMA). They are fixed in 4%
paraformaldehyde solution for 30 minutes at room
temperature and then rewashed . All the washes are
performed 3 times. After that, endogenous peroxidase
blockage is carried out by incubation with PBS containing
3% of H202 for 30 minutes at room temperature. After
washing, the cells are incubated with blocking buffer (3 %
of rabbit serum, 1% of BSA and 0.01% of Triton X 100) for 1
hour at room temperature, in order to block nonspecific
linkages. Following that, anti-hsp65 antibody is added and
cells are incubated in humid chamber at 4°C overnight. The
monoclonal antibody is diluted (1:100) in blocking buffer
and, after incubation and washing, samples are incubated
with biotinilated anti-mouse IgG antibody (B-7022, Sigma),
diluted (1:200) in blocking buffer for 1 hour, at room
temperature. After washing, samples are further incubated
for 30 minutes at room temperature with streptavidin
biotin-peroxidase complex (Streptoavidin-Dako Corporation,
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CA-USA). They are then washed and revealed with 3-3'-
Diaminobenzidine substrate (SIGMA) (5 mg in 10 ml of PBS),
adding 180 ~L of H202 (20 volumes). The reaction is stopped
with distilled water and the cells are stained with Mayer
hematoxilin for 5 to 10 minutes at, room temperature. After
washing with distilled water, they are treated with 0.50
ammonium hydroxide solution. Following that, they are again
washed, dried at room temperature, and placed on slides
with Canadian balsam. Negative control is obtained by
replacing primary antibodies for PBS or irrelevant
antibodies with same isotypes. The cells are visualized and
pictures are taken using an Aristoplan microscope (Leitz),
equipped with the MC80DX photography system. It was
previously demonstrated that the encapsulating process does
not affect the DNA functionality, since macrophages
submitted to the procedure describe above are able to
express the hsp65 protein.
EXAMPLE 8 - Immunization and challenge infection
BALB/c mice, 6 to 8 weeks old, were obtained from the
animal facilities at the School of Medicine of Ribeirao
Preto, University of Sao Paulo, and maintained in standard
laboratory conditions. All procedures were carried out
according to the Ethic Committee recommendations.
Mice were immunized with different microsphere
formulations by intramuscular or intratracheal route.
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Microsphere doses varied from 100 to 500 mg/kg and the
schedules were of single dose for intratracheal route and 1
or 3 doses for intramuscular route. Mice were either
challenged or killed, 30 or 60 days after administration of
the microspheres, and the immune response was evaluated.
As challenge infection, mice received, by
intratracheal route, a dose of 105 colony-forming units
(CFU) of M. tuberculosis H37Rv strain, under anesthesia
with 200 ~1 of a 2.50 of tribromoethanol (Sigma) solution
in PBS. Infected animals were kept under suitable biosafety
conditions. Infected animals were killed in different time
intervals in order to characterize inflammatory reaction in
lungs and evaluate protection.
EXAMPLE 9 - Cytokine measurement
Cytokine levels produced by lung and spleen cells from
mice immunized with microspheres are measured by ELISA.
Total lung tissue is homogenized for 5 minutes in an
Ultraturrax T 25 IKA (Labortechnik; Germany) at 4°C.
Homogenized tissue is centrifuged at 10,000 x g for 15
minutes and the supernatant is filtered through a 0.22 ~.m
membrane, and used to measure cytokine production. Spleen
cells are cultured at 37°C in 5% COz atmosphere and
stimulated in vitro with concanavalin A, as nonspecific
stimulus, or with recombinant hsp65 as specific stimulus.
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Forty-eight hours later, the culture supernatant is
collected for cytokine analysis. Capture and biotinilated
monoclonal antibodies specific for TNF-a (MP6-XT22, MP6-
XT3), IL-10 (JESS-2A5, JESS-16E3), IL-6 (MP5-20F3, MP5-
5 32C11), IL-4 (BVD4-1D11, BVD6-24G2), IFN-y (R4-6A2, XMG1.2)
and IL-12 (C15.6, C17.8), as well as recombinant cytokines,
may be purchased from Pharmingen (San Diego, CA). Levels of
TNF-a, IL-10, IL-6, IL-4, IFN-y and IL-12 are determined in
supernatant by ELISA technique, as previously described
10 (Haagmans, B. L., A. J. van den Eertwegh, E. Claassen, M.
C. Horzinek, and V. E. Schijns. 1994. Tumor necrosis
factor-alpha production during cytomegalovirus infection in
immunosuppressed rats. J. Gen. Virol. 75:779-787),
according to manufacturer instructions (Pharmingen, San
15 Diego, CA). For each assay, a standard curve is built with
recombinant mouse rTNF-a, rIL-10, rIL-6, rIL-4, rIFN-y,. or
rIL-12. The limit of detection for all evaluated cytokines
is 15 pg/mL.
EXAMPLE 10 - Nitric oxide measurement
20 Cells from bronchoalveolar lavage recovered from mice
injected with microspheres containing trehalose dimycolate
are cultured in RPMI medium containing 10% of fetal calf
serum (GIBCO-BRL, Grand Island, NY), 10 mM Hepes, 20 mM
sodium bicarbonate, penicillin, and streptomycin at 100
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~g/ml (GIBCO-BRL). Production of nitric oxide is evaluated
by measuring nitrite (N02) production in cell culture
supernatant by Greiss method (Stuehr, D.J., and C. F.
Nathan. 1989. Nitric oxide. A macrophage product
responsible for cystostasis and respiratory inhibition in
tumor target cells. J. Exp. Med. 169:1543-1555). The
standard curve is constructed using a serial dilution of a
200 ~.M sodium nitrite solution. The limit of detection is 3
~M per 105 cells .
EXAMPLE 11 - Evaluation of protection against challenge
infection
In order to evaluate the protection conferred by the
different formulations against challenge infection with M.
Tuberculosis H37Rv strain, the animals previously immunized
and challenged (as described in EXAMPLE 8) are killed.
Lungs from these animals are removed, weighed, and
homogenized. Serial dilutions of the lung homogenate are
plated on 7H11 medium. After 21 days of incubation at 37°,
the CFU number in each plate is counted.
In all experiments, the formulations which are object
of the present invention presented suitable
characteristics, such as mean diameter smaller than 10 ~m
(more precisely between 1 and 5 Vim), therefore being easily
taken up by phagocytic cells, allowing passive targeting of
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the encapsulated material. The process of encapsulation
does not affect the DNA functionality, since macrophages
treated with pCDNA3modified-hsp65-loaded microspheres are
able to express the protein..
The present invention is further explained by the
annexed tables. Table I and II show that recombinant-hsp65-
loaded. microspheres are able to elicit specific immune
response in both local an systemic levels after
intratracheal administration. This response was evaluated
by measurement of interferon-y in spleen cells culture
supernatant (Table I) and by measurement of IZ-12 levels in
lung homogenate (Table II) from immunized BAZB/c mice.
Interferon-gamma (IFN-y) levels were significant after in
vitro stimulation with recombinant hsp65 in mice immunized
with recombinant hsp65-loaded microspheres. Mice immunized
with recombinant hsp65 (r-hsp65) in PBS or unloaded
microspheres did not produce significant levels of IFN-y.
IFN-y levels produced after in vitro stimulation with
concanavalin A (ConA) were used as positive control.
Interleukin 12 (IZ-12) levels were significant in mice
which received r-hsp65 in PBS or loaded into microspheres.
This result confirms the possibility of administering the
formulations resulting from the present invention by a
route other than the parenteral one.
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TABLE I: IFN-y production (pg/mL) in spleen cells culture
supernatant from immunized BALB/c mice, 30 days after
immunization
Formulations Stimuli
used in ~ Medium ConA Hsp65
immuni zation
r-hsp65 in PBS 140.4 (184.45) 7,264.9( 274.8) 470( 32.1)
r-hsp65 into
microspheres 64.7 (11.9) 5,274.8( 628.3) 1,036.6( 93.1)
Unloaded
Microspheres 157.3 (123.9) 8,276.1( 482.7) 336.7( 47.3)
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TABLE II: IL-12 production (pg/g) in lungs homogenate from
immunized BALB/c mice, 30 days after immunization
Formulations used in immunizationIL-12
(pg/g of lung)
r-hsp65 in PBS 17,360.62 ( 893.4)
r-hsp65 into microspheres 17,827.36 ( 978.6)
Unloaded microspheres 6,506.60 ( 843.4)
However, after parenteral administration, the
formulations object of the present invention were also able
to elicit specific immune response. BALB/c mice were
immunized with a single dose of 5 mg of microspheres
containing plasmid/mouse by intramuscular route. Sixty days
after immunization, there was evidence of specific immune
response, demonstrated by production of~IFN-y, IL-12, and
IL-6 in spleen cells culture supernatant As shown in Tables
III, IV and V, this response was achieved with a reduced
dose of plasmid compared to conventional vaccination
protocol, in which DNA is administered as a solution in
three separated doses , as described in invention patent
W095/31216 and in studies from Silva and co-workers
(Lowrie et al., Nature 400:269-271, 1999). A single dose of
5mg of microspheres (containing 30 ~g of plasmid) was able
to elicit an immune response similar to that elicited by
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intramuscular injection of three doses of 100 ~g of naked
plasmid. This result demonstrates the advance attained by
this invention, in regard to reduction of the amount of DNA
necessary to elicit a significant immune response, thus
5 reducing the number of injections required.
TABLE III: IFN-y production (ng/mL) in spleen cells
supernatant from BALB/c mice immunized by intramuscular
injection with plasmid or r-hsp65 encapsulated into
microspheres , 60 days after immunization
Formulations usedStimuli
in immunization Medium ConA hsp65
pCDNA3m into 2.94(t0.82) 125.07(7.78) 3.71(t1.14)
microspheres
pCDNA3m-hsp65 2,41(0.31) 239.92 012.79) 24.95( 1.24)
into
microspheres
r-hsp65 into 2,75(p.15) 138.47(13.15) 5.21(0.28)
microspheres
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Table IV: IL-12 production (pg/mL) in spleen cells culture
supernatant from BALB/c mice immunized by intramuscular
injection with plasmid and r-hsp65 loaded into
microspheres, 60 days after immunization
Formulations used Stimuli
in
immunization Medium hsp65
pCDNA3m into
microspheres 699.67 (83.15) 735.67 (217.68)
pCDNA3m-hsp65 into
microspheres 1,053.30 (234.42) 1,920.67 ( 320.20)
r-hsp65 into
microspheres 719.67 (129.31) 856.01 (44.23)
Table V: IL-6 production (pg/mL) in spleen cells culture
supernatant from BALB/c mice immunized by intramuscular
injection with plasmid and r-hsp65 encapsulated into
microspheres , 60 days after immunization
FormulationsStimuli
used in Medium ConA hsp65
immunization
pCDNA3m into
microspheres364.83 (25.7) 5,242.79 (705.4) 1,816.03 (1,490.5)
pCDNA3m-
hsp65 into 565.67 (83.8) 4,309.89 (359.1) 3,730.44 ( 703.5)
microspheres
r-hsp65 into
microspheres494.40 (148.4) 5,178.01 (815.7) 7,436.17 ( 281.0)
Concerning immunostimulatory properties of TDM-loaded
microspheres, Tables VI and VII show that, after
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intratracheal administration, the formulation obtained in
this invention was able to elicit the production of several
cytokines by lung cells, as well as the activation of
alveolar macrophages as determined by .nitric oxide
production. Table VI illustrates the results of cytokine
production by lung cells from mice injected with
microspheres containing TDM and controls (PBS, control
lipid CtLip and trehalose dibehenate DBT). The animals were
injected by intratracheal route. Sixty days later, their
lungs were removed and homogenized for measurement of
cytokine in supernatant by ELISA. Data are representative
of one experiment, repeated 3 times.
Table VI: Cytokine production in lung homogenate from
BALB/c mice treated by intratracheal route, 60 days after
inj ection
Treatment Cytokines
(pg/mL)
IFN-y IL-12 IL-10 IL-6 IL-4 TNF-a
PBS 9,720 13,125 22,835 7,783 49,684 2,804
(841) (2,203) (10,801) (1,412) (22,788)(418)
Control 8, 920 11, 880 23, 935 9, 624 43, 342 3, 548
lipid into (421) (346) (2,744) (707) (5,764) (724)
microspheres
DBT into 9, 680 15, 915 27, 591 11, 985 60, 688 3, 659
microspheres(g5p) (2,034) (4,138) (1,758) (13,261)(412)
TDM into 19,200 26,577 47,163 17,781 111,504 5,855
microspheres04,111) (f2,361) (3,707) (2,726) (1,786) (287)
Table VII illustrates the results of nitric oxide
production by cells from bronchoalveolar lavage from mice
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injected with microspheres containing TDM and controls
(PBS, control lipid, and trehalose dibehenate). Around 105
cells from bronchoalveolar. lavage were obtained in each
experiment and cultured for 24 hours at 37°C in 5% C02
atmosphere. Following that, culture supernatant was
collected and the nitrite concentration was measured by
Greiss method. Statistical analysis was carried out by
multiple comparison Dunnett~s test and showed significant
differences between treatment with TDM (* p<0.01) and
DBT(*p<0.01), compared to PBS injected mice.
Table VII: Evaluation of nitric oxide levels in
broncoalveolar lavage cells culture supernatant from BALB/c
mice treated by intratracheal route.
Concentration
(~M) of N02
Treatment
per 1 05 cells
PBS 0.455 (0.63)
Unloaded microspheres 0.045 (0.05)
Control lipid into microspheres 0.045 (0.05)
DBT into microspheres 6.5 (0.28)
TDM into microspheres 8.6 (1.41)
Table VIII illustrates the IFN-y levels produced in
spleen cell culture supernatant from BALB/c mice immunized
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with microspheres containing plasmid plus TDM and controls
Table VIIT: IFN-y production (pg/mL) in spleen cell s
culture supernatant from BALB/c mice immunized, 30 days
after immunization
Formulations used in Stimuli
immunization Medium ConA hsp65
Nonimmunized 856.2 26,761.1 959.8
(86.3) 03,840.3) (t50.0)
TDM into microspheres
1,204.1 14,043.2 1,153.4
(t264.6) 03,541.4) (428.8)
pCDNA3m and TDM into
microspheres 1,010.5 21,886.6 1,644.1
0141.2) (+11,395.6) (576.3)
pCDNA3m-hsp65 and TDM
into microspheres 1,077.4 14,711.3 18,806.4
(373.5) (+5,129.8) (1,057.4)
Table IX illustrates the levels of antibodies in serum
from BALB/c mice immunized with microspheres containing_
plasmid plus TDM and controls, 30 days after immunization.
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TABLE IX: Evaluation of antibody production in serum from
BALBIc mice immunized by intramuscular injection of
microspheres containing plasmid plus TDM, 30 days after
immunization
Formulations used in Title (hog
immuni nati 2)
n
o IgG IgGl. IgG2a
Nonimmunized 1.90( 0.13) 0.64( 0.04) 0.91( 0.22)
Unloaded microspheres 2.56( 0.17) 0.84( 0.04) 1.63( 0.19)
TDM into microspheres 2,25( 0.34) 1.11( 0.35) 1.21( 0.03)
PCDNA3m plus TDM into 3.23(t 0.39) 2.99( 0.47) 2.64( 0.14)
microspheres
pCDNA3m-hsp65 plus TDM g,95( 0.10) 2.61( 0.07) 10.15( 0.04),
into microspheres
5
Tables
X,
XI
and
XII
illustrate
cytokine
levels
produced in lung homogenate from BALB/c mice challenged
with M. tuberculosis H37Rv strain after injection of
microspheres containing plasmid plus TDM and controls.
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Table X: IL-10 production (pg/mL) in lung homogenate from
BALB/c mice immunized and challenged with M. tuberculosis
Formulations used in immunization IL-10 (pg/g of lung)
Nonimmunized 4,799.15 (t 1,503.7)
Unloaded microspheres 3,525.32 ( 2,594.4)
TDM into microspheres 2,428.69 ( 706.8)
pCDNA3m and TDM into microspheres 4.654.18 ( 300.9)
pCDNA3m-hsp65 a.nd TDM into 14,250.30 ( 6,580.9)
microspheres
ldlJl~ dil: 1L-1G proauction tpg~mL) in lung homogenate from
BALB/c mice immunized and challenged with M. tuberculosis
Formulations used in immunization IL-12 (pg/g of lung)
Nonimmunized 25, 817.28 ( 3, 286.2)
Unloaded microspheres 26,945.16 ( 2,346.0)
TDM into microspheres 15,852.67 ( 1,002.4)
pCDNA3m and TDM into microspheres 27,269.74 ( 1,610.9)
pCDNA3m-hsp65 and TDM into microspheres36,825.70 ( 662.0)
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Table XII: IFN-y production (pg/mL) in lung homogenate
from BALB/c mice immunized and challenged with M.
tuberculosis
Formulations used in immunization IFN-y (pg/g
of
lung)
Nonimmunized 2, 711. (-!-1,.225 .
34 8 )
Unloaded microspheres 2,422.64 (+_2,077.9)
TDM into microspheres 1,728.51 (t363.9)
pCDNA3m and TDM.into microspheres 1,411.67 ( 758.9)
pCDNA3m-hsp65 and TDM into microspheres7,775.13 ( 2,855.2)
Table XIII illustrates the ability of different microsphere
formulations containing DNA or protein plus TDM, to confer
protection in BALB/c mice against challenge with virulent
strain of M. Tuberculosis.
Formulations used in CFU
immunization number/g
of
lung(log
10)
Nonimmunized 6,32 ( 0.033)
Unloaded microspheres 5,27 ( 0.08)
TDM into microspheres 6, ( 0.380)
g4
PCDNA3m plus TDM into 3, ( 0.278)
microspheres g5
pCDNA3m-hsp65 plus TDM into 6,9g ( 0.023)
microspheres
As shown in Table XIII, only the formulation
containing pCDNA3m-hsp65 plus TDM loaded into microspheres
was able to protect mice from infection. It is important to
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point out that a single administration, containing a dose
10-fold lower of plasmid was required to induce protection
similar to that obtained by naked DNA protocol. The optimal
encapsulation rate of protein was up to 950; and varied
from 60 to 70o for plasmid. The optimal adjuvant/polymer
rate was 6 ~g of adjuvant to 2 mg of polymer.
