Bioresorbable nonlamellar microparticles and uses thereof
10465012 ยท 2019-11-05
Assignee
Inventors
- Yasemin Ataman-Onal (Lyons, FR)
- Thierry Delair (Echalas, FR)
- Genevieve Inchauspe (Lyons, FR)
- PASCALE JEANNIN (BOUCHEMAINE, FR)
- Glaucia Paranhos-Baccala (Lyons, FR)
- Bernard Verrier (Mornant, FR)
Cpc classification
A61K47/34
HUMAN NECESSITIES
A61K2039/6093
HUMAN NECESSITIES
C12N2740/16322
CHEMISTRY; METALLURGY
A61K39/00
HUMAN NECESSITIES
A61K9/167
HUMAN NECESSITIES
A61P43/00
HUMAN NECESSITIES
C12N2770/24222
CHEMISTRY; METALLURGY
C12N2740/16222
CHEMISTRY; METALLURGY
International classification
A61K47/69
HUMAN NECESSITIES
A61K9/16
HUMAN NECESSITIES
A61K47/34
HUMAN NECESSITIES
G01N33/543
PHYSICS
Abstract
Nonlamellar bioresorbable microparticles to which protein substances are bonded, and a method for preparing the microparticles, comprising: (i) preparing the microparticles from at least one bioresorbable polymer without stabilizer and without surfactant; and (ii) bonding the protein substances to the microparticles obtained in step (i) without surfactant.
Claims
1. Bioresorbable nonlamellar microparticles to which protein substances are bonded, consisting of poly(lactic acid), the microparticles being obtained by: (i) preparing, without stabilizer and without surfactant, said microparticles by a solvent displacement method; and (ii) bonding said protein substances to the microparticles obtained in step (i) without surfactant; wherein the microparticles to which are bonded the protein substances exhibit colloidal stability, and have a diameter in a range of 150 to 250 nm.
2. The bioresorbable nonlamellar microparticles according to claim 1, wherein the protein substance is an antigen of viral origin.
3. The bioresorbable nonlamellar microparticles according to claim 1, wherein the bonding of the protein substances to the microparticles is carried out by adsorption.
4. A pharmaceutical composition, comprising at least one bioresorbable microparticle according to claim 1.
5. The pharmaceutical composition according to claim 4, wherein the pharmaceutical composition is a vaccine.
6. The pharmaceutical composition according to claim 4, further comprising a pharmaceutically acceptable excipient.
7. A diagnostic composition comprising the bioresorbable microparticles according to claim 1.
8. Bioresorbable nonlamellar microparticles to which protein substances are bonded, comprising poly(lactic acid), wherein the microparticles exhibit colloidal stability, are devoid of stabilizer and surfactant, and have a diameter in a range of 150 to 250 nm.
Description
(1) The present invention will be understood more fully from the following examples given only by way of nonlimiting illustration, and also from
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EXAMPLE 1: PREPARATION OF MICROPARTICLES OF THE INVENTION BY Dialysis
(11) 1. Preparation of PLA Particles
(12) PLA 50 (50% of poly(L-lactic acid) and 50% of poly(D-lactic acid)) of molar mass 52 000 Da (PHUSIS) was used.
(13) This PLA was dissolved at 2% by total weight of solution in DMSO (PROLABO). The organic solution of PLA was subsequently introduced into a dialysis membrane with a cut off of 15 000 Da (SPECTRUM) and the assembly was placed in a water bath of double-distilled water (4 l, MILLIQ), stirred and changed regularly every hour, for 6 h. The final dialysis bath was continued overnight in order to obtain particles as a precipitate.
(14) The following day, the solution of PLA particles was recovered and stored at 4 C.
(15) The particles thus obtained were characterized in terms of their size, their polydispersity index and their charge using the Zetasizer 3000 HS device (MALVERN INSTRUMENTS). Their solids content was also evaluated after weighing, by means of the calculation: (mass of dry extract/mass of wet extract)100.
(16) 2. Preparation of PLA/p24 Particles
(17) The HIV-1 p24 protein was prepared in recombinant form in E. coli and was purified by metal-chelate affinity chromatography according to the technique of Cheynet V., et al., 1993, Protein Expr. Purif., 4:367-372.
(18) The PLA microparticles were prepared as described in point 1 above and have a particle diameter of 515.7+/6.7 nm, a solids content of 1.1% and a polydispersity index of 0.242+/0.013.
(19) A 10 mM phosphate buffer, pH 5.7, was prepared by mixing 10 ml of 0.1M phosphate buffer, pH 4.7 (NaH.sub.2PO.sub.4.2H.sub.2O, M=15.60 g/l) and 1.1 ml of 0.1M phosphate buffer, pH 9.2 (NaH.sub.2PO.sub.4.2H.sub.2O, M=17.79 g/1), and diluting to 1/10th with water.
(20) 200 l of the p24 protein diluted to 0.6 g/l in the 10 mM phosphate buffer, pH 5.7 were mixed with 200 l of the microparticles, and stirring was carried out overnight on a wheel at ambient temperature. Centrifugation was then carried out for 5 min at 5000 rpm and the supernatant was drained, which made it possible to assay the amount of nonadsorbed p24 (BCA Protein Assay kit from Pierce) and to deduce therefrom the concentration of p24 adsorbed onto the microparticles, which comes to 0.2 g/l.
(21) 3. Preparation of the PLA/Tat Microparticles
(22) The HIV-1 Tat protein, of SEQ ID NO: 1, synthesized according to the procedure described in Ploponse J. P., et al., 1999, The Journal of Biological Chemistry, 274(17):11473-11478, was used.
(23) The PLA microparticles were prepared as described in point 1 above and have a particle diameter of 420.1+/10.7 nm, a solids content of 1.02% and a polydispersity index of 0.241+/0.040.
(24) 200 l of the Tat protein diluted to 0.4 g/l in a degassed 10 mM phosphate buffer, pH 6.8, prepared as indicated in point 2 above, except that 13.8 ml of 0.1M phosphate buffer, pH 9.2, were used, were mixed with 200 l of the microparticles, and stirring was carried out overnight on a wheel at ambient temperature. Centrifugation was then carried out for 5 min at 5000 rpm and the supernatant was drained, which made it possible to assay the amount of nonadsorbed Tat (BCA Protein Assay kit from Pierce) and to deduce therefrom the concentration of Tat adsorbed onto the microparticles, which comes to 0.1 g/l.
(25) 4. Preparation of the PLA/NS3 Helicase Microparticles
(26) The HCV NS3 helicase peptide of SEQ ID NO: 2 obtained in recombinant form as follows, was used.
(27) The gene encoding amino acids 1192-1458 corresponding to the helicase domain of the HCV NS3 protein as a fusion with hexahistidine was cloned into the prokaryotic expression vector pMH80 and expressed in E. coli JM109 bacteria (Promega). The expression of the recombinant protein was carried out at 30 C. after 3 hours of induction with 1 mM IPTG (isopropyl-beta-D-thiogalactopyranoside, Promega). After centrifugation, the bacteria were lysed by sonication in the buffer solution: 10 mM Tris-HCl, pH 8, 5 mM MgCl.sub.2, 1% Triton X100, 1 tablet of anti-protease (Boehringer), 250U benzonase (Merck). After lysis and centrifugation, the soluble fraction was purified on a Ni-agarose column and eluted in 10 mM sodium phosphate buffer solution, pH 7.2, containing 300 mM NaCl and 300 mM imidazole. The pure protein was thus dialyzed against PBS, pH 7.2. After purification, the protein was analyzed by acrylamide gel electrophoresis in the presence of sodium dodecyl sulfate (SDS), and mass spectrometry. The degree of purity of the NS3 protein, helicase domain, is estimated at greater than 95%. The absence of endotoxin was verified by measuring the degree of endotoxins (LPS) with an in vitro LAL and functional assay.
(28) The PLA microparticles were prepared as described in point 1 above and have a particle diameter of the order of 600 nm, a solids content of 1% and a polydispersity index of 0.2.
(29) 200 l of the NS3 helicase peptide diluted to 0.327 g/l in a 10 mM phosphate buffer, pH 6.5, prepared as described in point 2 above, except that 6.8 ml of 0.1M phosphate buffer, pH 9.2, were used, were mixed with 200 l of the microparticles, and stirring was carried out overnight on a wheel at ambient temperature. Centrifugation was then carried out for 5 min at 5000 rpm and the supernatant was drained, which made it possible to assay the amount of nonadsorbed NS3 helicase (BCA Protein Assay kit from Pierce) and to deduce therefrom the concentration of NS3 helicase adsorbed onto the microparticles, which comes to 0.28 g/l.
EXAMPLE 2: PREPARATION OF MICROPARTICLES OF THE INVENTION BY SOLVENT DISPLACEMENT
(30) 1. Preparation of PLA Particles
(31) PLA 50 (50% of poly(L-lactic acid) and 50% of poly(D-lactic acid)) of molar mass 52 000 Da (PHUSIS) was used.
(32) This PLA was dissolved at 2% by a total weight of solution in acetone. The solution of PLA in acetone was then added, dropwise, to 35 ml of water and the solvent was evaporated off under reduced pressure for 35 min.
(33) The particles thus obtained were characterized in terms of their size, their polydispersity index and their charge using the Zetasizer 3000 HS device (MALVERN INSTRUMENTS). Their solids content was also evaluated after weighing, by means of the calculation: (mass of dry extract/mass of wet extract)100.
(34) 2. Preparation of the PLA/NS3 Helicase Microparticles
(35) The HCV NS3 helicase peptide of SEQ ID NO: 2 obtained in recombinant form as indicated in example 1, point 4 above was used.
(36) The PLA microparticles were prepared as described in point 1 above and have a particle diameter of the order of 250 nm, a solids content of 1% and a polydispersity index of 0.3.
(37) 200 l of the NS3 helicase peptide diluted to 0.327 g/l in a PBS buffer (Phosphate Buffered Saline buffer; 150 mM NaCl, pH 7.1), prepared as described in point 1 above, except that 6.8 ml of 0.1M PBS buffer, pH 9.2, were used, were mixed with 200 l of the microparticles, and stirring was carried out overnight on a wheel at ambient temperature. Centrifugation was then carried out for 5 min at 5000 rpm and the supernatant was drained, which made it possible to assay the amount of nonadsorbed NS3 helicase (BCA Protein Assay kit from Pierce) and to deduce therefrom the concentration of NS3 helicase adsorbed onto the microparticles, which comes to 0.34 g/l.
EXAMPLE 3: IMMUNIZATION OF MICE WITH THE PLA/P24 MICROPARTICLES OF THE INVENTION
(38) 1. Animal Model
(39) The immunization experiments were carried out on female BALB/c (H-2.sup.d) mice 6 to 8 weeks old at the time of the first immunization.
(40) 2. Immunogens Administered
(41) In this experiment, the p24 protein alone, the PLA/p24 microparticles of the invention prepared as indicated in example 1, point 2 above, and also the p24-Freund's adjuvant (Sigma) composition prepared in the form of a water-in-oil emulsion, and which is known to exhibit a good immunogenic capacity (positive control), were used.
(42) 3. Immunizations
(43) The mice received three successive doses (40 g or 10 g each) of the immunogens described in point 2 above at 0, 2 and 4 weeks. All the injections were given subcutaneously.
(44) The animals were sacrificed 10 days (D38), 14 days (D42) or else 42 days (D70) after the third injection and the blood and the spleen were taken for the immunological analyses.
(45) 4. Immunological Analyses
(46) The humoral response and the cellular response were investigated as follows: Humoral response: a blood sample was taken from the mice before they were sacrificed. The presence of anti-p24 antibodies (IgG1, IgG2a and IgG) was determined by ELISA. The p24 protein was used for capture and the specific antibodies present in the serum were revealed with anti-mouse polyclonal antibodies as detection antibodies, which antibodies bind to the antibodies being sought and are, respectively, a horseradish peroxidase-labeled goat anti-mouse IgG1 antibody (Southern Biotechnology Associates Inc., Cat no. 1070-05, Birmingham, Ala., USA), a horseradish peroxidase-labeled goat anti-mouse IgG2a antibody (Southern Biotechnology Associates Inc., Cat no. 1080-05), and a horseradish peroxidase-conjugated AffiniPure goat anti-mouse IgG antibody (H+L, Jackson Immunoresearch, Cat no. 115-035-062). The titer is the inverse of the dilution for which an absorbance of 0.3 OD unit is obtained with the ELISA protocol used. The ratio of the IgG2a:IgG1 isotypes, which makes it possible to judge the IFN--IL-4 tendency (respectively, Th1-Th2) of the immune response, was also determined by indirect ELISA. Cellular response: after sacrifice of the mice, the spleens were removed sterilely so as to prepare a cell suspension. The following analyses were carried out on the cell suspensions obtained, each mouse having been analyzed individually.
(47) (i) CTL Assay:
(48) The cell suspension was placed in culture in the presence of a 9-mer peptide (AMQMLKETI, SEQ ID NO: 3) which corresponds to an immunodominant H-2K.sup.d-restricted CTL epitope, and of IL-2. Five days later, the effector population was restimulated with irradiated nave cells loaded with the peptide. The effector cytotoxic population was harvested after the 7th day and the CTL activity was measured using .sup.51Cr-labeled P815 cells as targets.
(49) (ii) ELISPOT:
(50) The ELISPOT makes it possible to determine the number of cells secreting a given cytokine in response to a specific stimulus. We were interested in the cytokine IFN- (Th1). The cell suspensions obtained from the spleens were restimulated in vitro with the peptide (AMQMLKETI, SEQ ID NO: 3) for 20 h in order to analyze the CD8-type responses.
(51) 96-well ELISPOT plates with PVDF membranes (Multiscreen IP, Millipore) were coated with an anti-IFN- antibody. During the restimulation, the splenocyte suspensions were incubated in these plates so as to capture the cytokines secreted by each cell. The spots corresponding to each cell secreting the cytokine of interest were visualized with a biotinylated detection antibody specific for the cytokine of interest.
(52) (iii) Proliferation: The splenocytes were stimulated in the presence of the p24 protein for 5 days. The cells were pulsed for 18 h with tritiated thymidine, which incorporates into the DNA of the cells undergoing proliferation. Following the pulse, the cells were harvested on a membrane which retains the DNA and makes it possible to eliminate the nonincorporated labeled thymidine by washing. The more the cells proliferate in response to the specific stimulus, the more the DNA is labeled; in other words, the greater the cellular response against the immunogen (p24).
(53) 5. Results
(54) A first series of experiments was carried out with 15 mice (5 mice per branch), three doses of 10 g of immunogen and sacrifice of the mice at D38, and investigation of the humoral response and the CTL assay and proliferation as cellular response.
(55) The results are given in table 1 below:
(56) TABLE-US-00001 TABLE 1 p24 alone p24/Freund's p24/PLA Proliferation (cpm).sup.a 3000 3900 8000 CTL.sup.b 0/5 0/5 0/5 IgG1 antibodies (titer).sup.c 0.1 10.sup.5 10 10.sup.5 7 10.sup.5 .sup.aMean of the cpm values (specific stimulation stimulation with medium), cpm = counts per minute (Student's test, P = 0.002) .sup.bNumber of mice having specific CTL activity out of the total number of mice of the branch .sup.cGeometric mean of the anti-p24 IgG1 titers of the mice of the branch
(57) This table demonstrates that: enhanced proliferative responses are obtained with the p24/PLA microparticles of the invention, compared with the p24 protein alone or adjuvanted with Freund's, no CTL activity is detected with any one of the immunogens, and the p24/PLA microparticles of the invention make it possible to obtain a specific antibody titer that is largely superior to that obtained when p24 alone is administered, the responses obtained being within the order of magnitude of the antibody titers obtained with the p24/Freund's adjuvant combination.
(58) The experiment was repeated with 13 mice (3 or 4 mice per branch), except that the immunogens were used at a rate of 40 g and that the mice were sacrificed either at D42 (3 mice) or at D70 (4 mice).
(59) The results are given in table 2 below.
(60) TABLE-US-00002 TABLE 2 p24/PLA p24/PLA p24 alone p24/Freund's D 42 D 70 ELISPOT IFN- CD8.sup.a 6 100 50 330 CTL.sup.b 0/3 0/3 0/3 4/4 IgG1 antibodies (titer).sup.c 0.7 10.sup.5 10 10.sup.5 8 10.sup.5 30 10.sup.5 .sup.aMean of the number of cells secreting IFN-/10.sup.6 total cells, in response to a specific stimulus (peptide AMQMLKETI, SEQ ID NO: 3) for 20 h .sup.bNumber of mice having specific CTL activity out of the total number of mice of the branch .sup.cGeometric mean of the anti-p24 IgG1 titers of the mice of the branch
(61) The results in the table demonstrate that: if the antibody titers reported in table 1 are compared with those reported here in table 2, increasing the dose from 10 g to 40 g makes it possible to give comparable results, and a more long-term response after the final injection, reflected by a later sacrifice of the mice, makes it possible to demonstrate a CTL response in all the mice of the p24/PLA group, and also a response by ELISPOT and in terms of enhanced antibody titers.
EXAMPLE 4: IMMUNIZATION OF MICE WITH THE PLA/TAT MICROPARTICLES OF THE INVENTION
(62) The procedure indicated in example 3 was repeated, except that the PLA/Tat microparticles as prepared in example 1, point 3 above, the Tat protein alone and the Tat protein/Freund's adjuvant (Sigma) combination prepared in the form of a water-in-oil emulsion were used as immunogen, that the injection doses were each 20 g, that, for the humoral response, the Tat protein was used as capture partner and the mouse polyclonal antibodies as indicated in example 3, point 4 above were used as detection partner, and that, for the humoral response, only an ELISPOT assay was carried out, using as stimulus either the six peptides as indicated hereinafter, for 20 h, for analyzing the CD8-type responses, or the Tat protein, for 42 h, for analyzing the CD4-type responses.
(63) Peptides Used in the ELISPOT Assay (Sygma Genosys)
(64) TABLE-US-00003 (SEQIDNo.4) CFHCQVCFTKKGLGI (SEQIDNo.5) VCFTKKGLGISYGRK (SEQIDNo.6) KGLGISYGRKKRRQR (SEQIDNo.7) SYGRKKRRQRRRSPQ (SEQIDNo.8) KRRQRRRSPQDSETH (SEQIDNo.9) RRSPQDSETHQVSLS
(65) The results are indicated in table 3 below.
(66) TABLE-US-00004 TABLE 3 Tat alone Tat/Freund's Tat/PLA ELISPOT IFN- CD8.sup.a 3 3 24 ELISPOT IFN- CD4.sup.b 4 and 6 4 and 8 28 and 50 IgG antibodies (titer).sup.c 0.1 10.sup.5 0.2 10.sup.5 1.7 10.sup.5 IgG2a antibodies (frequency).sup.d 2/4 1/4 3/4 IgG2a antibodies (titer).sup.e 730 560 3900 .sup.aMean of the number of cells secreting IFN-/10.sup.6 total cells, in response to a specific stimulus (pool of peptides) for 20 h .sup.bMean of the number of cells secreting IFN-/10.sup.6 total cells, in response to a specific stimulus (Tat protein) for 42 h .sup.cGeometric mean of the anti-Tat total IgG titers of the mice of the branch .sup.dNumber of mice having a specific IgG2a response out of the total number of mice of the branch .sup.eGeometric mean of the anti-Tat IgG2a titers of the mice which have responded
(67) The results in table 3 above demonstrate that: injection of the microparticles of the invention makes it possible to induce IFN--secreting cells, whereas injection of the Tat protein alone or of the Tat/Freund's composition, and bonding of the Tat protein to the PLAs makes it possible to enhance the antibody titers (total IgGs) by approximately 1 log compared with the titers obtained with the Tat protein alone or adjuvanted with Freund's, which are of the order of 10.sup.4. The use of the Tat/PLA microparticles makes it possible to enhance both the frequency and the titer of the anti-Tat IgG2as.
EXAMPLE 5: IMMUNIZATION OF MICE WITH THE PLA/NS3 HELICASE MICROPARTICLES OF THE INVENTION
(68) 1. Animal Model
(69) The immunization experiments were carried out on 14 C57BL/6 mice transgenic for the HLA-A2 molecule (Pascolo S., et al. (1997), J. Exp Med., 185(12), 2043-2051).
(70) 2. Immunogens Administered
(71) In this experiment, naked DNA corresponding to the NS3NS4 nucleic acid sequence (SEQ ID NO: 10) as a positive control, the NS3 helicase peptide alone, the PLA particles alone, as prepared in example 1, points 1 and 4 above, the NS3 helicase/Freund's adjuvant (Sigma) composition prepared in the form of a water-in-oil emulsion and also the PLA/NS3 helicase microparticles of the invention as prepared in example 1, point 4 above, were used.
(72) 3. Immunizations
(73) The mice received three successive doses of the immunogens described in point 2 above, at 0, 2 and 4 weeks, at a rate of 50 g each in the case of the proteins or of 100 g each in the case of the naked DNA. All the injections were given subcutaneously, with the exception of the naked DNA, which was administered intramuscularly.
(74) The animals were sacrificed approximately 70 days (D70) after the first injection and the blood and the spleen were taken for the immunological analyses.
(75) 4. Immunological Analyses
(76) The CTL cellular response was investigated as follows: after sacrifice of the mice, the spleens were removed sterilely in order to prepare a cell suspension. The cell suspension was placed in culture in the presence of the KLV peptide (KLVALGVNAV, SEQ ID NO: 11), which corresponds to a CTL epitope contained in the NS3 protein, and of IL-2. Five days later, the effector population was restimulated with irradiated nave cells loaded with the peptide. The effector cytotoxic population was harvested after the 7th day and the CTL activity was measured using .sup.51Cr-labeled P815 cells as targets.
(77) 5. Results
(78) The results are given in
(79) These graphs show a CTL response specific for the NS3 helicase peptide, demonstrated when the PLA/NS3 microparticles of the invention are injected.
EXAMPLE 6: IMMUNIZATION OF RABBITS WITH THE PLA/P24 MICROPARTICLES OF THE INVENTION
(80) 1. Animal Model
(81) The immunization experiments were carried out on New Zealand White strain rabbits weighing approximately 2.5 kg at the time of the first immunization.
(82) 2. Immunogens Administered
(83) In this experiment, the PLA/p24 microparticles of the invention prepared as indicated in example 1, point 2 above, and also the p24-Freund's adjuvant (Sigma) composition prepared in the form of a water-in-oil emulsion, and which is known to exhibit a good immunogenic capacity (positive control), were used.
(84) 3. Immunizations
(85) The rabbits received five successive doses of 200 g of the immunogens described in point 2 above at 0, 1, 2, 3 and 4 months. All the injections were given subcutaneously or intradermally.
(86) 4. Monitoring of the Appearance of the Anti-p24 Humoral Response
(87) In order to follow the appearance of the anti-p24 antibodies, blood samples were taken regularly from the animals. The presence of the anti-p24 antibodies was then tested using the ELISA assay similar to that described in example 3, point 4, except that the visualizing conjugate was replaced with a horseradish peroxidase-conjugated AFFINIPURE goat anti-rabbit IgG antibody (H+L, Jackson Immunoresearch, Cat no. 111-035-003).
(88) 5. Results
(89) The results are given in
(90) The results obtained show that the immunization with the PLA/p24 particles of the invention gives good titers in all the animals, irrespective of whether the antigen was administered subcutaneously or intradermally. However, in the model chosen, intradermal administration appears to be slightly better than subcutaneous administration. It may be noted that the titers obtained with the immunization of Freund's/p24 are substantially comparable to those obtained with a PLA/p24 immunization (210.sup.7 versus 510.sup.6). The PLA/p24 microparticles can therefore be used to induce a polyclonal serum in rabbits.
EXAMPLE 7: IMMUNIZATION OF MACAQUES WITH THE PLA/P24 MICROPARTICLES OF THE INVENTION
(91) 1. Animal Model
(92) The immunization experiments were carried out on cynomolgus macaques housed at the CEA.
(93) 2. Immunogens Administered
(94) In this experiment, the PLA/p24 microparticles of the invention prepared as indicated in example 1, point 2 above, were used.
(95) 3. Immunizations
(96) Two cynomolgus macaques were immunized with an injection of PLA/p24 (500 g per animal administered IM), followed by an identical booster 6 weeks later. A third injection is given under the same conditions 6 months later.
(97) 4. Monitoring of the Appearance of the Anti-p24 Humoral Response
(98) In order to follow the appearance of the anti-p24 antibodies, blood samples were taken from the macaques at weeks 0 (the day of the primer immunization), 2, 4, 6 (the day of the booster injection), 8, 10 and 12. The presence of the anti-p24 antibodies was then tested using the ELISA assay similar to that described in example 3, point 4 above, with the exception that the visualizing conjugate was replaced with a horseradish peroxidase-conjugated AFFINIPURE mouse anti-human IgG Fc gamma fragment antibody (H+L, Jackson Immunoresearch, Cat no. 209-035-098). The same ELISA format was also used to analyze the IgG subclasses present, using anti-human IgG1 (Cat no. 05-3320, Zymed), anti-human IgG2 (Cat no. 05-0520, Zymed), anti-human IgG3 (Cat no. 05-3620, Zymed) and anti-human IgG4 (Cat no. 05-3820, Zymed) horseradish peroxidase-conjugated antibodies.
(99) 5. Monitoring of the Appearance of the Anti-p24 Cellular Response by ELISPOT
(100) This procedure makes it possible to determine the number of cells secreting interferon gamma (IFN-gamma) in response to an antigenic stimulation at a final concentration of 5 g/ml for 48 h. This procedure was used successfully with freshly isolated peripheral blood mononuclear cells (PBMCs), PBMCs cryoconserved beforehand, T lymphocytes lines derived from PBMCs stimulated in vitro and from PBMCs pre-depleted of CD4.sup.+ cells (using an anti-CD4 antibody) or of CD8.sup.+ cells (using an anti-CD8 antibody). The CD4.sup.+ or CD8.sup.+ cell sorting was carried out using the MACS reagents, CD4 microbeads (Cat no. 130-091-102) and CD8 microbeads kit (Cat no. 130-091-112) from Miltenyi Biotec, according to the manufacturer's instructions.
(101) The PMA-ionomycin couple (PMA for Phorbol Myristate Acetate), which mimics the effect of an antigenic activation of T lymphocytes, was used as a positive control.
(102) 96-well ELISPOT plates with PVDF membranes (Multiscreen, Millipore) were coated with the anti-macaque IFN gamma monoclonal antibody, clone GZ-4 (Mabtech, ref: 3420M-3) at 1 g/ml in sterile PBS, overnight at +4 C. The plates were then washed and saturated. In parallel, PBMCs were isolated from the blood samples on a Ficoll gradient, according to the usual techniques. 10.sup.5 cells in 100 p1 of culture medium/well and the antigen source in 100 l of culture medium/well were deposited. According to the experiments, the antigen source is either the p24 protein, or a pool of gag peptides as defined hereinafter, for which it was verified beforehand that they make it possible to obtain a positive response in ELISPOT. In order to produce positive controls for stimulation, 410.sup.3 cells in 200 p1 of culture medium were deposited/well containing 50 ng/ml of PMA and 500 ng/ml of ionomycin. The plates were then incubated for 24 h at 37 C. in a humid atmosphere at 5% CO.sub.2, and then washed with PBS. The remaining cells were then lysed using a treatment with ice-cold water for 10 minutes, and the plates were again washed. The visualizing antibody, the biotinylated monoclonal directed against human IFN-gamma, clone 7-B6-1 (Mabtech, ref: 3420-6) was then added at 0.1 g/well (incubation for 2 h at 37 C. or overnight at 4 C.). The spots were visualized by adding extravidin-alkaline phosphatase and the 5-bromo-4-chloro-3-indolyl phosphate/nitro blue tetrazolium (BCIP/NBT) substrate. The sequences of the gag peptides used are as follows:
(103) TABLE-US-00005 (SEQIDNo.12) EQIGWMTNNPPIPVG (SEQIDNo.13) WMTNNPPIPVGEIYK (SEQIDNo.14) NPPIPVGEIYKRWII (SEQIDNo.15) PVGEIYKRWIILGLN (SEQIDNo.16) IYKRWIILGLNKIVR (SEQIDNo.17) WIILGLNKIVRMYSP (SEQIDNo.18) GLNKIVRMYSPISIL (SEQIDNo.19) IVRMYSPTSILDIRQ (SEQIDNo.20) YSPTSILDIRQGPKE (SEQIDNo.21) SILDIRQGPKEPFRD (SEQIDNo.22) IRQGPKEPFRDYVDR (SEQIDNo.23) PKEPFRDYVDRFYKT (SEQIDNo.24) FRDYVDRFYKTLRAE (SEQIDNo.25) VDRFYKTLRAEQASQ (SEQIDNo.26) YKTLRAEQASQEVKN (SEQIDNo.27) RAEQASQEVKNWMTE (SEQIDNo.28) ASQEVKNWMTETLLV (SEQIDNo.29) VKNWMTETLLVQNAN (SEQIDNo.30) MTETLLVQNANPDCK (SEQIDNo.31) LLVQNANPDCKTILK (SEQIDNo.32) NANPDCKTILKALGP
(104) 6. Results
(105) 6.1 Demonstration of the ELISPOT Responses
(106) The results of the IFN-gamma ELISPOT assays for the two macaques (M1 and M2) having been given the PLA/p24 preparation, and the PBMCs of which were stimulated, are reproduced in
(107) These results demonstrate that the PLA/p24 microparticles of the invention make it possible to induce an IFN-gamma ELISPOT response in the two animals tested. The first monkey (M1) only develops a specific response after the second injection, whereas the second animal (M2) has already developed a response at the first sample tested, after the first injection. In both cases, the effect of the booster injection is very significant (boost effect) since the ELISPOT responses obtained are high. Furthermore, the responses obtained are relatively long-lasting, given the fact that a non-replicative immunogen is involved; the responses remain significant approximately 40 days after the second injection.
(108) 6.2 Nature of the ELISPOT Responses
(109) The nature of the ELISPOT responses makes it possible to demonstrate what types of effector immune cells, CD4+ or CD8+, are responsible for the IFN-gamma secretion measured.
(110) The results are given in
(111) The results obtained in
(112) 6.3 Analysis of the Humoral Response
(113) At the same time as the analysis of the cellular responses on the PBMCs, we also analyze the humoral response on the sequential sera obtained from the monkeys. Among the two animals, only the M2 monkey developed an antibody response, which reached an anti-p24 IgG titer of approximately 10.sup.5 following the second injection. The M1 monkey did not develop a significant antibody response after two injections of PLA/p24, the titer remaining less than 10.sup.3. This result is not very surprising because, during the analysis of the cellular responses, we were able to demonstrate that the M1 monkey was a poor responder, requiring an additional injection compared with the M2 monkey in order to develop an immune response.
(114) The results of the ELISA assay with the M2 monkey are given in
(115) 6.4 Conclusion
(116) All these experiments carried out in the cynomolgus macaque model have made it possible to show that the microparticles of the invention make it possible to induce good immune responses in a nonhuman primate, with CD4+, CD8+ cellular responses and antibody responses.
EXAMPLE 8: IMMUNIZATION OF MICE WITH DENDRITIC CELLS SENSITIZED WITH THE PLA/P24 MICROPARTICLES OF THE INVENTION
(117) 1. Animal Model
(118) The immunization experiments were carried out on female CBA/J (H-2.sup.k) mice 6 to 8 weeks old at the time of the first immunization.
(119) 2. Immunogens Administered
(120) In this experiment, the SRDC (H-2.sup.k) murine spleen cell line was used in order to transport the various immunogens to be tested. This dendritic cell line was sensitized for 12 h with one of the following immunogens: PLA/p24 microparticles of the invention prepared as indicated in example 1, point 2 (at 10 g/ml of p24/PLA in terms of p24 equivalent), PLA-OVA microparticles, prepared in the same way, which will serve as a negative control, or p24 protein.
(121) 3. Immunizations
(122) The mice were divided up into 4 batches of 6 animals. Each mouse received 510.sup.5 sensitized SRDC cells at 0, 2 and 4 weeks, subcutaneously. The 4 batches of mice received either nonsensitized cells (negative control), or cells sensitized with the PLA/p24 microparticles or PLA/OVA microparticles (microparticle negative control) or the p24 protein.
(123) 4. Monitoring of the Appearance of the Anti-p24 Humoral Response
(124) In order to monitor the appearance of the anti-p24 antibodies, blood samples were taken from the mice every 10 days after immunization. The presence of the anti-p24 antibodies was then tested using the ELISA assay similar to that described in the preceding examples.
(125) 5. Results
(126) The analysis of the humoral response is presented in
(127) The results show that the injection of SRDC cells induces antibodies only when the SRDCs are sensitized with the PLA-p24 microparticles. The sensitization with the soluble p24 protein does not make it possible to obtain an immune response.
(128) 6. Study of the Cellular Response
(129) The study was carried out using both splenic and mucosal lymphocytes isolated from the mice. The analysis of the stimulated T lymphocyte subpopulations was carried out using lymphoproliferation and cytokine secretion assays so as to evaluate their ability to respond to the antigen, two weeks after the final immunization.
(130) For the lymphoproliferation assays, the T lymphocytes isolated from the spleen and from the mucosal lymph nodes were placed in culture for 5 days in complete culture medium in the presence of various concentrations of the antigen of interest, and then radiolabeled with .sup.3H thymidine for 18 h. The level of thymidine incorporation corresponds to the degree of the lymphoproliferative response.
(131) For the cytokine secretion assays, the T lymphocytes isolated from the spleen and from the mucosal lymph nodes were placed in culture for 3 days in complete culture medium in the presence of various concentrations of the antigen of interest, and then the cytokine secreted into the culture supernatant were assayed using commercial ELISA kits.
(132) 7. Results
(133) Table 4 below gives a summary of the results obtained.
(134) TABLE-US-00006 TABLE 4 Number of mice Lymphopro- having developed an liferation antibody (IgG) Stimulation Groups of response after index Spleen- IFN-gamma mice the 3.sup.rd injection lymph node secretion Control 0/2 1-1 No SRDC-p24 0/2 1-1 No SRDC-PLA-Ova 0/2 1.4-1.4 No SRDC-PLA-p24 2/2 2.8-2.2 +spleen, ++lymph node
(135) As above, the results show that the SRDC-PLA-p24 group is the only group for which it is possible to demonstrate a specific immune response. The stimulation index is 2.8 for the cells isolated from the spleen and 2.2 for those isolated from the mesenteric lymph nodes. For the other groups, there is no increase in the stimulation index or it is not significant. Similarly, the SRDC-PLA-p24 group is the only group which makes it possible to induce secretion of IFN-gamma, which is a Th1-type cytokine. This secretion in the presence of the antigen can be demonstrated using both the spleen cells and those of the mesenteric lymph nodes.
(136) 8. Conclusion
(137) All these experiments have enabled us to show that it is also possible to use the PLA-p24 microparticles in order to sensitize dendritic cells with an antigen of interest, in this case p24. Although administration of the SRDCs, when they are loaded with the PLA-p24s, makes it possible to induce both cellular and humoral specific responses, the SRDCs sensitized with the soluble p24 do not make it possible to induce anti-p24 responses. Thus, the PLA microparticles can also be used successfully in immunotherapy applications based on the transfer of dendritic cells sensitized in vitro.
(138) When the antigen of interest cannot be loaded into the dendritic cells in its soluble form, the PLA microparticles carrying the antigen can be used to facilitate its uptake by the dendritic cells. The use of the microparticles of the invention makes it possible to considerably enhance the specific immune responses obtained.
EXAMPLE 9: IMMUNIZATION OF MICE WITH THE PLA/NS3 HELICASE MICROPARTICLES OF THE INVENTION: EARLY AND LOCALIZED CELLULAR RESPONSE
(139) 1. Animal Model
(140) The immunization experiments were carried out on female BALB/c (H-2.sup.d) mice 6 to 8 weeks old at the time of the first immunization.
(141) 2. Immunogens Administered
(142) In this experiment, 5 mice per immunization group were used: the NS3 helicase genotype 1b protein (NS3h) alone, the PLA/NS3h microparticles of the invention prepared by dialysis (PLADYS) as indicated in example 1, point 4 above, the PLA/NS3h microparticles of the invention prepared by solvent displacement (PLADDS) as indicated in example 2, point 2 above, and also the NS3h-Alum (Pierce) composition prepared in the form of an emulsion and known to be an adjuvant in commercial vaccines (positive control).
(143) 3. Immunizations
(144) The mice received one dose (100 g) of the immunogens described in point 2 above, subcutaneously into the plantar footpad.
(145) The animals were sacrificed 10 days after the first injection and the popliteal lymph nodes were removed for immunological analysis.
(146) 4. Immunological Analyses
(147) The early and localized NS3h protein-specific dose-response cellular response was investigated as follows:
(148) Proliferation of the Popliteal Lymph Node Cells after Stimulation with Various Concentrations of the NS3h Protein
(149) the popliteal lymph node cells were stimulated in the presence of 0, 0.1, 0.3 and 1 l of the NS3h protein for 3 days. The cells were pulsed for 18 h with tritiated thymidine, which incorporates into the DNA of the cells undergoing proliferation.
(150) Following the pulse, the cells were harvested on a membrane which retains the DNA and makes it possible to eliminate the nonincorporated labeled thymidine by washing. The more the cells proliferate in response to the specific stimulus, the more the DNA is labeled; in other words, the greater the cellular response against the NS3h immunogen.
(151) 5. Results
(152) The results of the proliferation of the popliteal lymph node cells after stimulation with the NS3h protein are indicated in
(153) This graph shows a cellular response specific for the NS3h protein when the PLA/NS3h microparticles prepared by solvent displacement (DDS) and the PLA/NS3h microparticles prepared by dialysis (DYS) are injected. The specific cellular responses of the mice injected with the PLADDS/NS3hs of the invention are greater than those obtained with the PLADYS/NS3hs of the invention, the positive control (Alum/NS3h) and the PBS/NS3h control for all the concentrations of NS3h antigens tested (PLADDS/NS3h>PLADYS/NS3h>Alum/NS3h=PBS/NS3h).
(154) The results show that enhanced cellular proliferative responses are obtained with the PLADDS/NS3h and PLADYS/NS3h microparticles of the invention, compared with the NS3h protein alone or adjuvanted with Alum.
EXAMPLE 10: IMMUNIZATION OF MICE WITH THE PLA/NS3 HELICASE MICROPARTICLES OF THE INVENTION: LOCALIZED AND SYSTEMIC CELLULAR RESPONSE AND INHIBITION OF THE SYSTEMIC CELLULAR RESPONSE WITH ANTI-CD4+ ANTIBODIES
(155) 1. Animal Model
(156) The immunization experiments were carried out on female BALB/c (H-2.sup.d) mice 6 to 8 weeks old at the time of the first immunization.
(157) 2. Immunogens Administered
(158) In this experiment, 5 mice per immunization group were used: the NS3 helicase genotype 1b protein (NS3h) alone, the PLA/NS3h microparticles of the invention prepared by dialysis (PLADYS) as indicated in example 1, point 4 above, the PLA/NS3h microparticles of the invention prepared by solvent displacement (PLADDS) as indicated in example 2, point 2 above, and also the NS3h-Alum (Pierce) composition prepared in the form of an emulsion and known to be an adjuvant in commercial vaccines (positive control).
(159) 3. Immunizations
(160) The mice received 2 doses (100 pig) of the immunogens described in point 2 above, the first dose having been given subcutaneously into the plantar footpad at day 0 and the second subcutaneously at the base of the tail at day 7.
(161) The animals were sacrificed 7 days after the second injection and the popliteal lymph nodes and the spleen were removed for immunological analysis.
(162) 4. Immunological Analyses
(163) The NS3h protein-specific dose-response cellular response localized in the popliteal lymph nodes and systemic in the spleen was investigated as follows:
(164) Proliferation of the Popliteal Lymph Node Cells after Stimulation with Various Concentrations of the NS3h Protein
(165) the cells of the popliteal lymph nodes and of the spleen were stimulated in the presence of 0, 0.1, 0.3 and 1 M of the NS3h protein for 3 days. After 3 days of culture, 50 l of supernatant were removed. The cells were pulsed for 18 h with tritiated thymidine, which incorporates into the DNA of the cells undergoing proliferation. Following the pulse, the cells were harvested on the membrane that retains the DNA and that makes it possible to eliminate the nonincorporated labeled thymidine by washing. The more the cells proliferate in response to the specific stimulus, the more the DNA is labeled; in other words, the greater the cellular response against the NS3h immunogen.
(166) It was sought to inhibit the cellular response with anti-CD4.sup.+ antibodies (GK1.5; American Type Culture Collection (ATCC)) in the cells originating from the spleen as follows:
(167) (i) Inhibition, with the Anti-CD4.sup.+ Antibody, of the Proliferation of the Spleen Cells after Stimulation with 1.sub.4M of the NS3h Protein
(168) the spleen cells were stimulated in the presence of 1 M of the NS3h protein and incubated with 10 g of anti-CD4.sup.+ antibody for 3 days. The cells were pulsed for 18 h with tritiated thymidine, which incorporates into the DNA of the cells undergoing proliferation. Following the pulse, the cells were harvested on a membrane that retains the DNA that makes it possible to eliminate the nonincorporated labeled thymidine by washing. The more the cells proliferate in response to the specific stimulus, the more the DNA is labeled; in other words, the greater the cellular response against the NS3h immunogen.
(169) (ii) Assaying of Interferon
(170) the interferon was assayed using the BD Cytometric Bead Array kit, Mouse Th1/Th2 Cytokine CBA (BD Biosciences, Cat. No. 551287). Five populations of beads with distinct fluorescence intensities were coated with capture antibodies specific for IL-2, IL-4, IL-5, IFN-gamma, and TNF-alpha proteins. The five populations of beads were mixed together to form the CBA, which is resolved in the FL3 channel of a flow cytometer such as the BD FACScan Coule Cytometer. The cytokine capture beads were mixed with phycoerythrin-conjugated detection antibodies, and then incubated according to the supplier's recommendations. The sample data acquisition using the flow cytometer to produce the results was carried out using the BD CBA analytical software.
(171) 5. Results
(172) 5.1 Localized and Systemic Cellular Response
(173) The results of the proliferation of the spleen cells after stimulation with the NS3h protein are indicated in
(174) These results demonstrate a cellular response that is systemic (spleen cells
(175) 5.2 Inhibition of the Systemic Cellular Response with the Anti-CD4 Antibody.
(176) The results are indicated in
(177) The results show that the systemic cellular response is inhibited by at least a factor of 5 in the presence of the anti-CD4+ antibody, suggesting that the NS3h protein-specific response is of the Th2 type.
(178) 5.3 Supplementary Results
(179) At the third day of proliferation after stimulation with the NS3h protein, interferon gamma (IFN-gamma) secretion was observed under all the conditions used, which secretion is decreased with the anti-CD4.sup.+ antibody.
(180) The results of the IFN-gamma assay at the third day after stimulation with the NS3h protein are given in table 5 below:
(181) TABLE-US-00007 TABLE 5 Interferon gamma Type of stimulation assay pg/ml PBS/NS3h 1 M of NS3h 316 1 M of NS3h + anti-CD4+ 60 PLADDS/NS3h 1 M of NS3h 556 1 M of NS3h + anti-CD4+ 196 PLADYS/NS3h 1 M of NS3h 606 1 M of NS3h + anti-CD4+ 249 Alum/NS3h 1 M of NS3h 583 1 M of NS3h + anti-CD4+ 144
EXAMPLE 11: IMMUNIZATION OF MICE WITH THE PLA/NS3 HELICASE MICROPARTICLES OF THE INVENTION: HUMORAL RESPONSE
(182) 1. Animal Model
(183) The immunization experiments were carried out on female BALB/c (H-2.sup.d) mice 6 to 8 weeks old at the time of the first immunization.
(184) 2. Immunogens Administered
(185) In this experiment, 5 mice per immunization group were used, the NS3 helicase genotype 1b protein (NS3h) alone, the PLA/NS3h microparticles of the invention prepared by dialysis (PLADYS) as indicated in example 1, point 4 above, the PLA/NS3h microparticles of the invention prepared by solvent displacement (PLADDS) as indicated in example 2, point 2 above, and also the NS3h-Alum (Pierce) composition prepared in the form of an emulsion and known to be an adjuvant in commercial vaccines (positive control).
(186) 3. Immunizations
(187) The mice received 3 doses (50 g) of the immunogens described in point 2 above, subcutaneously at the base of the tail at 0, 2 and 4 weeks. The sera were taken at day 13, day 27 and day for the analyses of the specific humoral response against the NS3h protein using an ELISA assay as indicated hereinafter.
(188) The animals were sacrificed at 10 days after the first injection and the popliteal lymph nodes were removed for immunological analysis.
(189) 4. Immunological Analyses
(190) The early and localized NS3h protein-specific humoral response was investigated as follows:
(191) Qualitative and Quantitative Humoral Response Against the NS3h Protein
(192) a blood sample was taken from the mice before the first injection (DO), and at D13, D27 and D45. The presence of the specific anti-NS3h antibodies, the antibody titer and the immunoglobulin isotypes (IgG, IgG1, IgG2a) were determined by ELISA. The microtitration plates were sensitized with the NS3h protein and the specific antibodies against the NS3h protein that were present in the serum of immunized mice were visualized using peroxidase-labeled goat anti-mouse IgG serum (H+L, Jackson Immunoresearch, Cat no. 115-035-062). For the determination of the antibody titer, the immunized mice sera were serially diluted. For the determination of the isotyping, the reaction was visualized using peroxidase-labeled goat anti-mouse IgG1 serum (Southern Biotechnology Associates Inc., Cat no. 1070-05, Birmingham, Ala., USA), and a peroxidase-labeled goat anti-mouse IgG2a antibody (Interchim, UPB 90520). The IgG2a/IgG1 isotype ratio, which makes it possible to interpret the IFN-gamma/IL-4 (respectively, Th1-Th2) tendency of the immune response was also determined.
(193) 5. Results
(194) The results of the specific anti-NS3h total IgG antibody titer at day 30 and day 45 are given in table 6 below:
(195) TABLE-US-00008 TABLE 6 Specific anti-NS3h antibody titer Day 30 Day 45 NS3h 3.3 10.sup.3 4.5 10.sup.4 PLADYS/NS3h 3.5 10.sup.4 2.6 10.sup.5 Freund's adjuvant/NS3h 7.1 10.sup.4 1.9 10.sup.5
(196) The results demonstrate that the bonding of the NS3h protein to the PLAs makes it possible to enhance the antibody (total IgG) titers by approximately 1 log relative to the titer obtained with the NS3h protein alone. Moreover, the titers obtained are also comparable to those obtained with the Freund's adjuvant/NS3h formulation.
(197) The use of the PLA/NS3h microparticles makes it possible to obtain an essentially IgG1 antibody response specific for the NS3h protein, suggesting that the response is of the Th2 type.
EXAMPLE 12: ACTION OF THE PLA/NS3HS DURING THE DIFFERENTIATION OF MONOCYTES TO DENDRITIC CELLS
(198) This study consists in studying the effect on antigen-presenting cells of the NS3h protein adsorbed onto PLA nanoparticles. To do this, the procedure is carried out in the presence of dendritic cells generated using monocytes isolated from human peripheral blood and differentiated.
(199) Analysis of the expansion of costimulatory molecules makes it possible to determine whether dendritic cells that are immature at the start (DCi) enter into a process of maturation.
(200) The ability of the potential adjuvant and of the PLA-NS3h formulation to promote the differentiation and maturation of monocytes into dendritic cells is also tested. This study enables us to understand more clearly their role in cell mediation, which is essential at the interface between innate and adaptive immunity. The screening is carried out by means of successive steps of analysis of phenotypic markers of differentiation and of maturation and by analysis of the profile of cytokines produced. The analysis of the cytokine production makes it possible to identify whether the PLA-NS3 formulation induces a Th1 and/or Th2 profile.
(201) 1. Purification of Monocytes from Human Peripheral Blood
(202) The monocytes were isolated from normal human peripheral blood (recovered at the Etablissement Francais du Sang [French bloodbank] in Lyon) by centrifugation on a Ficoll and Percoll (Amersham Biosciences) gradient. The Ficoll makes it possible to create a density gradient while at the same time conserving the integrity of the cells and their function. After centrifugation, the red blood cells and the polymorphonuclear cells that are more dense than the Ficoll are at the bottom of the tube. The PBMCs (peripheral blood mononuclear cells) comprising lymphocytes and monocytes remain at the interface between the plasma and the Ficoll. They are then purified on a Percoll gradient. After centrifugation, the lymphocytes, that are more dense than the Percoll, are in the pellet, whereas the monocytes remain at the interface between the medium and the Percoll.
(203) The monocytes were incubated with the mixture of antibodies (Ab) indicated hereinafter, in order to eliminate the remaining contaminants by depletion of T lymphocytes (mouse anti-CD3 Ab OKT3, ATCC, Rockville, Md., USA, directed against T lymphocytes), of B lymphocytes (mouse anti-CD19 Ab hybridoma 4G7, directed against B lymphocytes), of red blood cells (mouse anti-glycophorin A Ab, Immunotech) and of NK cells (mouse anti-CD56 Ab NKH1, Immunotech, directed against Natural Killer cells) and using magnetic beads (DYNAL). The Dynal beads are small magnetic beads coated with sheep antibodies directed against mouse antibodies. These antibodies will bind the mouse Ab/cell complexes and then, after the cell suspension has been passed over a magnetized carrier, the remaining cells will only be monocytes.
(204) The depletion was verified by FACScan (Becton Dickinson) flow cytometry analysis.
(205) The cell suspension was analyzed by isolated passage of the cells through a liquid matrix. The passage of these cells through a light beam results in two types of scattering referred to as small angle (Forward scatter-FS) and large angle (Side scatterSS), which represent the two cellular parameters taken into account, the size (scattering) and the granulometry (refraction) of the cells. The use of antibodies coupled to fluorochromes (FITC: Fluorescein IsoThioCyanate read on the X-axis FL1 and PE: PhycoErythrin read on the Y-axis in FL2) that are laser-excitable allows detectable fluorescence emission. An electrical signal amplification and analog-digital converter system allows the data to be computer-formatted.
(206) The cells were labeled with various types of antibodies: Anti-CD14 Ab labeled with FITC (monocyte specific) Anti-CD3 Ab labeled with PE (LT specific) Anti-CD56 Ab PE (NK cells) Anti-CD20 Ab PE (LB specific).
(207) The contaminant level is less than 10%.
(208) The monocytes were subsequently placed in culture (day 0) in 24-well plates at a rate of 110.sup.6 cells/ml in RPMI 1640 medium (Gibco), 2 mM of L-glutamine (Life Technologies), 10 mM Hepes (Life Technologies), 40 ng/ml of gentamycin (Life Technologies)+10% decomplemented fetal calf serum, and in the presence of GM-CSF (40 ng/ml) (Granulocyte Macrophage Colony Stimulating Factor) and recombinant human IL-4 (250U/ml). The GM-CSF and the IL-4 allow differentiation of the monocytes into immature dendritic cells.
(209) 2. Phenotypic Analysis of the Cells
(210) At the 5.sup.th day of differentiation, various tests were applied to the cells: LPS from 250 to 2000 pg/ml.fwdarw.LPS (lipopolysaccharide) is a component of the bacterial membrane and is recognized as a danger signal by DCis, systematically allowing their maturation. A range of LPS concentrations was realized in each of the assays in order to compare the degrees of maturation.
(211) A dose-response was carried out for the NS3h alone, the particles alone and the PL/NS3h particles. NS3h from 1 to 50 g/ml.fwdarw.control for ensuring that the protein alone does not induce any maturation. PLA from 0.01 to 1 mg/ml.fwdarw.test to determine whether the PLAs have an adjuvant effect. PLA/NS3h from 10 to 100 l.fwdarw.test to determine whether the PLAs have an adjuvant effect in the presence of the NS3h protein.
(212) At the 6.sup.th day, 200 l of the supernatant were conserved at 80 C. for assaying the cytokines. The cells were subsequently collected and then washed.
(213) Various control labelings were carried out: a control isotype labeling IgG1-FITC/IgG2a-PE: IgGs are capable of binding nonspecifically to cells possessing an Fc fragment receptor. This labeling therefore makes it possible to be sure of the specificity of the reaction and to eliminate the nonspecific fluorescence from the various assays. The use of IgG1 and of IgG2a is related to the fact that the anti-CD antibodies used are also of this type; a CD14-FITC (specific for monocytes)/CD1a-PE (specific for immature dendritic cells) labeling makes it possible to be sure that the differentiation has correctly functioned.
(214) The phenotype was subsequently determined by specific labeling of the mature dendritic cells with: anti-CD80 FITC Ab/anti-CD86 PE Ab anti-HLA-DR FITC Ab/anti-CD83 PE Ab anti-CD40 PE Ab.
(215) 3. Implementation of the Dendritic Cell Maturation Protocol
(216) The formulations were brought into contact with the immature dendritic cells in order to judge the ability of the PLA/NS3h particles to generate mature dendritic cells (adjuvant effect).
(217) The assays carried out were as follows: LPS range: 250-300-400-500-750-1000-2000 pg NS3h: 1-10-25-50 g PLA particles: 0.05-0.1-0.5-1 mg PLA/NS3h particles: 0.24 mg DYS/10 g NS3h (10 l)-0.6 mg/25 g (25 l)-1.2 mg/50 g (50 l)-2.4 mg/100 g (100 l).
(218) 4. Results
(219) The NS3h protein gives no positive labeling.
(220) The PLA DYS particles alone, added at D5 to DCis, make it possible to obtain a maturation phenotype (DCm CD83+, CD86+, CD40+). A dose-response effect on cell maturation was observed.
(221) The PLA/NS3h particles give very advantageous results. Specifically, it can be noted that the degree of activation of maturation is substantial. Right from the assay at 10 l (0.24 mg PLA/10 g helicase), maturation can be observed. The PLA/NS3hs added to DCis induce the expression of all the activation markers (CD83+, CD86+, CD80+, HLA-DR+, CD40+).
(222) The assay at 100 p1 was carried out in the knowledge that it is in large excess in the conditions used in vitro.
(223) In conclusion, the PLA formulations have an adjuvant effect on the HCV NS3h protein since they make it possible to obtain mature dendritic cells.
EXAMPLE 13: OBTAINING MONOCLONAL ANTIBODIES WITH THE PLA/P24 MICROPARTICLES OF THE INVENTION
(224) 1. Animal Model
(225) The immunization experiments were carried out on female BALB/c (H-2.sup.d) mice 6 to 8 weeks old at the time of the first immunization.
(226) 2. Immunogens Administered
(227) In this experiment, the PLA/p24 microparticles of the invention prepared as indicated in example 1, point 2, and also the p24-Freund's adjuvant (Sigma) composition prepared in the form of a water-in-oil emulsion, and which is known to have a good immunogenic capacity (positive control), were used.
(228) 3. Immunizations
(229) The mice received 3 successive doses of 10 g of the immunogens described in point 2 above, at 0, 2 and 4 weeks. All the injections were given subcutaneously. At D68 after the first injection, the humoral responses were restimulated with an intravenous injection of 50 g of p24.
(230) 4. Monitoring of the Appearance of the Anti-p24 Humoral Response
(231) In order to monitor the appearance of the anti-p24 antibodies, blood samples were taken regularly from the mice. The presence of the anti-p24 antibodies is then tested using the ELISA assay similar to that described in example 2, point 4. However, the visualizing conjugate is replaced with an alkaline phosphatase-conjugated AffiniPure goat anti-mouse IgG antibody (H+L, Jackson Immunoresearch, Cat no. 115-055-146).
(232) 5. Obtaining Monoclonal Antibodies
(233) Three days after the final injection, a mouse of the PLA-p24 group was sacrificed; the blood and the spleen were taken. The splenocytes obtained from the spleen were placed in culture with Sp2/0-Ag14 myeloma cells so that they would fuse and become immortalized, according to the protocol described by Khler and Milstein (Khler, G. and Milstein, C., 1975, Nature, 256:495-497; Khler, G. and Milstein, C., 1976, Eur. J. Immunol., 6:511-519). After an incubation period of 12-14 days, the supernatants of the hybridomas obtained were screened in order to determine the presence of anti-p24 antibodies using the ELISA assay described in point 4 of this example. The positive hybridoma colonies were subcloned twice according to the limiting dilution technique.
(234) 6. Results
(235) The anti-p24 antibody titer in the serum of the mice was determined just before sacrifice, individually for each mouse.
(236) The results are given in table 7 below.
(237) TABLE-US-00009 TABLE 7 PLA/p24 group Mouse 1 dilution 1/8000 >3.0 (saturating) dilution 1/64000 0.5 Mouse 2 dilution 1/8000 >3.0 (saturating) dilution 1/64000 0.5 Mouse 3 dilution 1/2000 >3.0 (saturating) dilution 1/8000 1.3 Freund's/p24 group Mouse 1 dilution 1/8000 >3.0 (saturating) dilution 1/64000 0.5 Mouse 2 dilution 1/8000 >3.0 (saturating) dilution 1/64000 0.5 Mouse 3 dilution 1/2000 >3.0 (saturating) dilution 1/8000 1.4
(238) The titers obtained are comparable in the two groups. As monoclonal antibodies had already been obtained by Freund's/p24 immunization, we sought to determine whether the PLA/p24 immunogen, which makes it possible to induce comparable titers, will also make it possible to obtain monoclonal antibodies.
(239) For this, a mouse of the PLA/p24 group (mouse 1) was sacrificed and the cells from its spleen were fused with myeloma cells. The hybridomas derived from the fusion were cloned by limiting dilution in 18 96-well plates. Screening of the hybridoma culture supernatants using an anti-p24 ELISA assay made it possible to identify 12 hybridoma clones which secrete a p24-specific antibody. The PLA/p24 microparticles can therefore also be used to obtain monoclonal antibodies.