1. A method for preparing non-lamellar bioresorbable microparticles to which protein substances, such as herein before described, are bonded, characterized in that it comprises the steps of: (i) preparing said microparticles from at least one bioresorbable polymer such as herein before described without stabilizer and without surfactant, and (ii) bonding said protein substances to the microparticles obtained in step (i) without surfactant.

2. The method as claimed in claim 1, wherein the polymer used in step (i) is a poly(a-hydroxylated acid) or a mixture of poly(a-hydroxylated acids).

3. The method as claimed in either of claims 1 and 2, wherein the bonding of the protein substances to the microparticles is carried out by adsorption.

4. The method as claimed in anyone of claims 1 to 3, wherein the protein substances is a antigen of viral origin.

5. The method as claimed in claim 4, wherein the antigen is an HIV virus antigen, preferably the p24 protein or a regulatory protein.

6. The method as claimed in claim 5, wherein the HIV virus antigen is a regulatory protein chosen from the Tat, Rev and Nef proteins.

7. The method as claimed in claim 4, wherein the antigen is an HCV virus antigen preferably a non-structural protein of the HCV virus.

8. The method as claimed in claim 7, wherein the antigen is the NS3 protein, preferably the NS3 helicase protein.

9. Bioresorbable non-lamellar microparticles to which protein substances are bonded, obtainable by the method as claimed in any one of the claims 1 to 8.

10. Method for preparing a medicinal product using the bioresorbable microparticles as claimed in claim 9.

11. A pharmaceutical composition in particular a vaccine, characterized in that it contains at least bioresorbable microparticle as claimed in claim 9 and where appropriate, a pharmaceutically acceptable excipient.

12. Antibodies directed against the bioresorbable microparticles as claimed in claim 9.

13. A diagnostic composition consisting of the bioresorbable microparticles as claimed in claim 9 or of the antibodies as claimed in claim 12.

The present invention relates to novel bioresorbable particles to which protein substances are bonded, that are useful in particular in the field of vaccination.
Protein substances, such as proteins and peptides, also called antigens, are widely used in the treatment of many diseases, such as diseases of viral origin, generally in the form of a vaccine formulation.
In order to increase the activity and the strength of the antigen and to improve the stability of the pharmaceutical compositions containing the antigens, these compositions contain adjuvants. In this regard, vaccine formulations often comprise immunological adjuvants for enhancing cell-mediated responses and humoral responses.
Despite the presence of such adjuvants, conventional vaccines often have the drawback that they provide unappropriate protection against the pathogenic agents targeted. The term "appropriate protection" is intended to mean protection involving both the cell-mediated response and the humoral response of the immune system.
In order to obtain an appropriate immune response, particulate carriers associated with antigens have been used, said antigens being either adsorbed onto the carrier, or trapped in the carrier. Such carriers possess multiple copies of the antigen of interest presented to the immune system and promote the trapping and retention of the antigens in the local lymph nodes. The particles can be phagocytozed by antigen-presenting cells and can increase antigen-presentation to the immune system. Examples of such carriers include poly{methyl methacrylate) polymers and also microparticles made of polylactides, such as poly(D- or
L-lactic acid) known as D-PLA or L-PLA, respectively, and of poly(lactide-co-glycolide)s known as PLG.
PLG-based microparticles in which antigens are trapped are capable of giving an immune response. By way of example, Moore et al. (1995, Vaccine, 13:1741-1749) have shown that the microencapsulated HIV virus gp!20 antigen induces an HIV-specific CD4+ and CD8+ T-cell response. Similarly, Vordemeier et al. (1995, Vaccine, 13-1576-1582) have shown that the PLG-trapped Mycobacterium tuberculosis antigen induces, in mice immunized with such an antigen, both a humoral response and a T-cell response.
Although these types of adjuvant offer some advantages compared with the other more toxic systems, they have the drawback that the production of microparticles is difficult and involves the use of corrosive chemical products, such as solvents, which can denature the antigen and destroy the immunogenicity thereof. Furthermore, the antigen may also be degraded due to the vigorous agitation required during the trapping process such as encapsulation.
It has thus been proposed to use microparticles which have the antigens adsorbed or grafted onto their surface (Rock K.L., Efficient MHC I presentation of exogenous Ag, PNAS 1993). Some authors, such as Eldrige et al., in Infect. Immun., 59:2978-2986 (1991), have, however, indicated that, in order to achieve an appropriate adjuvant effect, the antigens must be trapped in microparticles.
PCT patent application WO 97/02810 describes particles consisting of a biodegradable polymer, onto which antigens can be adsorbed. These particles are useful
for the delivery of these antigens. The drawback of these particles is their lamellar nature, such that it is not possible to control their size. In fact, particles intended for vaccination must have a submicronic size in order to be effective in transfection and in immunization.
Patent applications WO 98/33487 and WO 00/06123 describe, for their part, polylactide or PLG-based microparticles onto which antigens are adsorbed and the use thereof for stimulating immune responses. All the microparticles of these patent applications, onto which antigens are adsorbed, have been obtained using a surfactant in order to maintain the colloidal stability of said microparticles, and also a stabilizer, such as poly(vinyl alcohol), for the preparation of the polymer particles. The drawback of the microparticles thus obtained is their toxicity due to the presence in the microparticles of said surfactant and of said stabilizer.
The applicant has now discovered, against all expectations, that it is possible to obtain nonlamellar bioresorbable microparticles of at most micronic size, to which are bonded antigenic protein substances against which it is sought to trigger an immune response, and which are devoid of the above drawbacks, i.e. they are not toxic since they are prepared without stabilizer and without surfactant for the preparation of the polymeric microparticles, and without surfactant for the bonding of the antigens to the surface of the microparticles, without loss of colloidal stability.
Thus, a first subject of the invention consists of a method for preparing bioresorbable microparticles to which protein substances are bonded, characterized in
that it comprises the steps of:
(i) preparing said mi cropar tides from at least one
bioresorbable polymer without stabilizer and
without surfactant, and (ii) bonding said protein substances to the
microparticles obtained in step (i) without
surfactant.
The microparticles obtained by means of the method of the invention are devoid of stabilizer and of surfactant such that they are novel.
Thus, another subject of the invention consists of the bioresorbable microparticles to which protein substances are bonded, which are obtainable by the method of the invention.
Against all expectations, the particles of the invention conserve their colloidal stability.
The microparticles of the invention are useful for stimulating both a cell-mediated response and a humoral response, such that they are useful both in therapy and in diagnosis.
Thus, another subject of the invention consists of the use of the microparticles of the invention for preparing a medicinal product, and also the pharmaceutical compositions, in particular vaccines, comprising the microparticles of the invention.
Finally, another subject of the invention consists of the use of the microparticles of the invention for the in vitro diagnosis of pathological states related to the protein substance bonded to said microparticles.
The microparticles intended for vaccination must not be toxic for the organism that receives them, while at the same time conserving their colloidal stability.
The method of the invention, that uses neither surfactant nor stabilizer, makes it possible, against all expectations, to obtain such particles.
The term "microparticle" is intended to mean particles of at most micronic size so as to allow them to enter antigen-presenting cells.
The term "at most micronic size" is intended to mean a size of less than or equal to 999 /mi.
Preferably, the microparticles have a particle diameter of less than or equal to 3 /im. More preferably, the particles of the invention are of submicronic size, with preferably a diameter of between 150 and 900 nm, more preferably between 250 and 700 nm.
The size of the particles is readily determined by techniques known to those skilled in the art, such as, for example, using scanning electron microscopy, quasi-elastic light scattering or transmission electron microscopy.
The term "toxic microparticle" is intended to mean a microparticle comprising at least one compound capable of causing biological disorders, such as metabolic disturbances, in the organism having received the microparticle.
The first step of the method of the invention consists of the preparation of said microparticles from at least
one bioresorbable polymer without stabilizer and without surfactant.
The term "bioresorbable polymer" is intended to mean a polymer capable of degrading, in the organism into which it has been introduced, into compounds that can be eliminated via the natural pathways.
Examples of such bioresorbable polymer include, without limitation, poly(a-hydroxylated acids), poly(hydroxybutyric acids), polycaprolactones, poly-orthoesters and polyanhydrides. Preferably, the bioresorbable polymer used in the method of the invention is a poly (a-hydroxylated acid) such as poly(D-lactic acid), poly(L-lactic acid) (called PLA) , poly(glycolic acid) (called PLG), or else a mixture of poly (a-hydroxylated acids) , such as a mixture of poly(D- and L-lactic acids), a mixture of poly(L-lactic acid) and of poly(glycolic acid), a mixture of poly(D-lactic acid) and of poly(glycolic acid), or a mixture of poly(D-lactic and L-lactic acids) and of poly(glycolic acid), which constitutes an embodiment of the invention.
When the polymer used in the method of the invention is a mixture of poly(a-hydroxylated acids), the proportion of each constituent can be readily determined by those skilled in the art. Thus, for example, it is possible to use a racemic mixture of poly(D- and L-lactic acids) or a PLA/PLG mixture at various percentages known to those skilled in the art.
The preparation of the microparticles of the invention from at least one bioresorbable polymer can be carried out by any methods for preparing microparticles known to those skilled in the art, for which no stabilizer
and no surfactant are used. In fact, step (i) of the method of the invention is characterized in that such agents are not used.
The stabilizers normally used in methods for preparing microparticles include, for example, poly(vinyl alcohol), pluronics (copolymer of poly(ethylene oxide) and of poly(propylene oxide)), and cationic or anionic surfactants such as cetyltrimethylammonium bromide or sodium dodecyl sulfate.
Of course, when the stabilizer used in the methods of the prior art is a surfactant, said methods do not use an additional surfactant.
The surfactants normally used in the methods of the prior art are largely known to those skilled in the art and are described, for example, in patent application WO 98/33487.
By way of example of a method for preparing microparticles without stabilizer and without surfactant, mention may be made of dialysis, solvent displacement, emulsification-solvent evaporation and emulsification-diffusion, these said methods being largely known to those skilled in the art.
For example, the dialysis for preparing the microparticles of the invention can be carried out with a solution of bioresorbable polymer in a water-miscible solvent, such as acetone, DMSO or DMF, at a concentration by mass of 0.1% to 10%, dialyzed against 1000 times its volume of water for 12 hours.
The second step of the method of the invention consists in bonding protein substances to the microparticles
obtained in the first step of the method, without using surfactant.
This step of bonding the protein substance to the surface of the microparticles has the characteristic that it is carried out without surfactant. In fact, against all expectations, even in the absence of surfactant, the microparticles of the invention exhibit a colloidal stability that provides a range of particle size suitable for use in immunization.
By way of examples of a surfactant normally used for the bonding of protein substances to the surface of microparticles, reference may be made to the surfactants mentioned above.
The protein substances to be bonded to the surface of the microparticles obtained in step (i) of the method of the invention may be any protein substance against which it is sought to trigger an immune response.
The term "immune response" is intended to mean a cell-mediated response, a humoral response or both.
The term "cell-mediated response" is intended to mean a response mediated by T lymphocytes and/or other leukocytes. This response is reflected by the induction of a lytic activity by cytotoxic T lymphocytes and/or by cytokine production by suppressor CD8+ T lymphocytes or by helper T cells.
The term "humoral response" is intended to mean a response mediated by the antibody molecules secreted by B lymphocytes.
The protein substances that are suitable for the purposes of the invention may be of several origins, such as of viral or bacterial origin.
By way of example of such protein substances, mention may, for example, be made of antigens and epitopes or any protein substance having the role of an antigen after bonding to the microparticles.
The antigens are molecules capable of being recognized by an antibody, the synthesis of which they have induced via an immune response, and containing at least one epitope. This may be whole proteins or protein fragments having conserved the structure of interest.
The epitopes are peptides comprising between 3 and 15 and generally between 5 and 15 amino acids, having also conserved the structure of interest.
According to a particular embodiment of the invention, the protein substance is an antigen of viral origin.
When the protein substance is of viral origin, the suitable viruses are any viruses for which substances capable of an immune response are known.
By way of example, mention may be made, without any limitation, of herpesviruses, hepatitis viruses, such as hepatitis B virus (HBV) and hepatitis C virus (HCV), papilloma viruses (HPV) and human immunodeficiency viruses (HIV), such as HIV-1 and HIV-2.
The nucleic acid sequences of the viruses suitable for the purposes of the invention, and also the proteins encoded by said sequences, are largely known to those
skilled in the art and are available, for example, in databases such as GenBank.
Thus, for example, the HIV virus has genes which encode structural proteins of the virus. The gag gene encodes the protein that forms the core of the virion, including the p24 antigen. The pol gene encodes the enzymes responsible for reverse transcription (reverse transcriptase), for cleavage (protease) and for integration (integrase). The env gene encodes the envelope glycoproteins. It contains six other genes (tat, rev, nef, vif, vpr and vpu (HIV-1) or vpx (HIV-2)) which encode proteins involved in regulating the expression of the genes of the virus (regulatory proteins) . The HIV genome also comprises the 5' and 3' LTRs (Long Terminal Repeats) which comprise regulatory elements involved in the expression of the genes of the virus.
According to one embodiment of the invention, the protein substance used in the method of the invention is an HIV virus antigen. Preferably, the HIV virus antigen is a regulatory protein or the p24 protein, the preferred regulatory proteins being the Tat, Rev or Nef protein.
As regards HCV, the 5' end of its genome corresponds to an untranslated region adjacent to the genes which encode the structural proteins, the nucleocapsid core protein, the two envelope glycoproteins, El and E2, and a small protein called p7. The 5' untranslated region and the core gene are relatively well conserved in the various genotypes. The El and E2 envelope proteins are encoded by regions that are more variable from one isolate to another. The p7 protein is an extremely hydrophobic protein which is thought to constitute an
ion channel. The 3' end of the HCV genome contains the genes which encode the nonstructural proteins (NS2, NS3, NS4, NS5) and a 3' noncoding region that has a well-conserved domain (Major ME, Feinstone SM, Hepatology, June 1997, 25(6):1527-1538).
The NS3 nonstructural protein of HCV is a 630 amino acid protein which comprises two distinct structural domains: an N-terminal domain, of 81 amino acids, which has an active serine protease activity involved in the maturation of the viral protein (domain called NS3 protease), and a C-terminal domain, of 549 amino acids, comprising a helicase activity associated with an NTPase activity which plays a role in the replication of the viral genome (domain called NS3 helicase). This NS3 protein is relatively well-conserved among the various genotypes of the virus, such that this protein constitutes a "vaccine candidate" antigen of choice.
According to one embodiment of the invention, the protein substance of interest is an antigenic protein of HCV, preferably a nonstructural protein, more preferably the NS3 protein, and in particular the NS3 helicase protein being more preferred.
The protein substances suitable for the purposes of the invention can be obtained by the genetic engineering technique which comprises the steps of:
- culturing a microorganism or eukaryotic cells
transformed with a nucleotide sequence encoding the
protein substance of interest, and
- recovering said protein substance produced by said
microorganism or said eukaryotic cells.
This technique is well known to those skilled in the art. For further detail with regard thereto, reference

may be made to the manual hereinafter: Recombinant DNA Technology I, Editors Ales Prokop, Raskesh K Bajpai; Annals of the New-York Academy of Sciences, Volume 646, 1991.
The protein substances of interest, when they are small in size, can also be prepared by conventional peptide syntheses well known to those skilled in the art.
The bonding of the protein substances to the bioresorbable microparticles can be carried out by any method known to those skilled in the art.
Examples of such bonding include adsorption, and covalent bonding.
The adsorption can be carried out, for example, by mixing the microparticles with the protein substances and incubating with agitation, for example at ambient temperature or at 37°C.
The covalent bonding of the protein substances to the surface of the microparticles can be carried out using the techniques and reagents known in the literature, as described, for example, in Bioconjugate Techniques, G.T. Hermanson, Academic Press, London, 1996 and Chemical Reagents for Protein Modification, R.L. Lundblad, Ed. CRC Press, 1991.
According to a particular embodiment, the bonding of the protein substances to the microparticles is carried by adsorption.
The bioresorbable microparticles to which protein substances are bonded, prepared according to the method of the invention, are devoid of stabilizer and of
surfactant, such that they are novel and constitute another subject of the invention.
The microparticles of the invention, because of their ability to induce an immune response by virtue of the protein substance, and because of their lack of toxicity, are particularly suitable for the preparation of pharmaceutical compositions, in particular vaccines, that are useful in the treatment of pathologies associated with the protein substance bonded to the microparticles.
Thus, another subject of the invention consists of the use of the bioresorbable microparticles of the invention, for preparing a medicinal product.
In particular, the medicinal product prepared with the microparticles of the invention is particularly useful for the inhibition, prevention or treatment of an infection caused by a virus, such as, for example, the HIV or HCV virus or any other known virus, which constitutes another embodiment of the invention.
The invention also relates to a pharmaceutical composition, in particular a vaccine, containing at least one microparticle of the invention and, where appropriate, a pharmaceutically acceptable excipient.
The microparticles of the invention also make it possible to obtain antibodies, which constitutes another subject of the invention.
The antibodies according to the invention are polyclonal or monoclonal antibodies, monoclonal antibodies being preferred.
The abovementioned polyclonal antibodies can be obtained by immunization of an animal with at least one microparticle of the invention, followed by recovery of the desired antibodies in purified form, by taking a sample of the serum of said animal and separating said antibodies from the other constituents of the serum, in particular by affinity chromatography on a column to which is attached an antigen specifically recognized by the antibodies, in particular that of the microparticle of the invention.
The monoclonal antibodies can be obtained by means of the hybridoma technique, the general principle of which is recalled hereinafter.
Firstly, an animal, generally a mouse (or cells in culture in the case of in vitro immunizations) , is immunized with at least one microparticle of the invention, for which the B lymphocytes are then capable of producing antibodies against said microparticle. These antibody-producing lymphocytes are then fused with "immortal" (murine in the example) myeloma cells so as to give hybridomas. Using the heterogeneous mixture of the cells thus obtained, a selection of the cells capable of producing a specific antibody and of indefinitely multiplying is then carried out. Each hybridoma is multiplied in the form of a clone, each resulting in the production of a monoclonal antibody whose recognition properties with respect to the microparticle of the invention may be tested, for example, by ELISA, by one- or two-dimensional immunoblotting, by immunofluorescence, or using a biosensor. The monoclonal antibodies thus selected are subsequently purified, in particular according to the affinity chromatography technique described above.
The micropartides and the antibodies of the invention are also useful in the diagnosis of the pathological state associated with the protein substance bonded to the surface of said microparticles.
Specifically, the microparticles or antibodies of the invention can be used as a partner for the capture or detection of an analyte in any diagnostic technique using such partners, such as the ELISA method. For example, when the intention is to search for an antigen as an analyte, an antibody of the invention obtained from microparticles to which said antigen is bonded is used, whereas, if the intention is to search for antibodies, the microparticles of the invention are used. In the latter case, if the diagnostic test requires the use of a solid support, the microparticles may or may not play this role.
Thus, another subject of the invention consists of a diagnostic composition consisting of the bioresorbable microparticles or of the antibodies of the invention.
It also relates to the use of this diagnostic composition for the in vitro diagnosis of the pathological state related to the protein substance bonded to the bioresorbable microparticle, it being possible for the pathological state to be, according to one embodiment, a viral infection, as caused by the HIV virus or the HCV virus.
Here again, those skilled in the art will readily determine the amount of microparticles or antibodies to be used according to the diagnostic technique used.
The present invention will be understood more fully from the following examples given only by way of nonlimiting illustration, and also from figure 1, which
gives the result of CTL assays by immunizing mice with the DNA sequence corresponding to the HCV NS3NS4 polyprotein as a control (figure 1A) , with the NS3 helicase protein substance and Freund's adjuvant, without microparticle (figure IB) , with the NS3 helicase protein substance without microparticle (figure 1C) , with PLA microparticles without protein substance (figure ID) , and with the microparticles of the invention in which the polymer is PLA and the protein substance is the NS3 helicase peptide (figure IE) .
Example It Preparation of microparticles of the invention
1. Preparation of PLA particles
PLA 50 (50% of poly(L-lactic acid) and 50% of poly(D-lactic acid)) of molar mass 52 000 Da (Phusis ) was used.
This PLA was dissolved at 2% by total weight of solution in DMSO (Prolabo8) . 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 1, 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.
The following day, the solution of PLA particles was recovered and stored at 4°C.
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)xlOO.
2. Preparation of PLA/p24 particles
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.
The PLA microparticles were prepared as described in point 1 above and have a particle diameter of 515.7 +/-6.7 run, a solids content of 1.1% and a polydispersity index of 0.242 +/- 0.013.
A 10 mM phosphate buffer, pH 5.7, was prepared by mixing 10 ml of 0.1M phosphate buffer, pH 4.7 (NaH2P04.2 H20, M=15.60 g/1) and 1.1 ml of 0. 1M phosphate buffer, pH 9.2 (NaH2PO4.2 H2O, M=17.79 g/1), and diluting to l/10th with water.
200 /xl of the p24 protein diluted to 0.6 g/1 in the 10 mM phosphate buffer, pH 5.7 were mixed with 200 /zl 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/1.
3. Preparation of the PLA/Tat microparticles
The HIV-1 Tat protein, of sequence SEQ ID No. 1,
synthesized according to the procedure described in
Peloponese J.P., et al., 1999, The Journal of
Biological Chemistry, 274(17):11473-11478, was used.
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.
200 /il of the Tat protein diluted to 0.4 g/1 in a degassed 10 mM phosphate buffer, pH 6.8, prepared as indicated in point 1 above, except that 13.8 ml of 0.1M phosphate buffer, pH 9.2, were used, were mixed with 200 /zl 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/1.
4. Preparation of the PLA/NS3 helicase microparticles
The HCV NS3 helicase peptide of sequence SEQ ID No. 2 obtained in recombinant form as follows, was used.
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 pMHSO 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 MgCl2, 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.
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.
200 pil of the NS3 helicase peptide diluted to 0.327 g/1 in a 10 mM phosphate buffer, pH 6.5, prepared as described in point 1 above, except that 6.8 ml of 0. 1M phosphate buffer, pH 9.2, were used, were mixed with 200 /zl 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/1.
Example 2; Immunization of mice with the PLA/p24 microparticles of the invention
1. Animal model
The immunization experiments were carried out on female BALB/c (H-2d) mice 6 to 8 weeks old at the time of the first immunization.
2. Immunogens administered
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.
3. Immunizations
The mice received three successive doses (40 /ig or 10 fig each) of the immunogens described in point 2 above at 0, 2 and 4 weeks. All the injections were given subcutaneously.
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.
4. Immunological analyses
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 (IgGl, 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 IgGl antibody
(Southern Biotechnology Associates Inc., Cat no. 1070-05, Birmingham, Alabama, 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:IgGl isotypes, which makes it possible to judge the IFN-y - IL-4 tendency (respectively, Thl-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.
(i) CTL Assay: 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-2JCd-restricted CTL epitope, and of IL-2. Five days later, the effector population was restimulated with irradiated naive cells loaded with the peptide. The effector cytotoxic population was harvested after the
7th day and the CTL activity was measured using
51Cr-labeled P815 cells as targets.
(ii) ELISPOT: 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-y (Thl). The cell suspensions obtained from the spleens were restimulated in vitro with the peptide AMQMLKETI for 20 h in order to analyze the CDS-type responses.
96-well ELISPOT plates with PVDF membranes (Multiscreen IP, Millipore) were coated with an anti-IFN-y 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.
(Hi) 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).
5. Results
A first series of experiments was carried out with 15 mice (5 mice per branch) , three doses of 10 /zg of

immunogen and sacrifice of the mice at D38, and investigation of the humoral response and the CTL assay and proliferation as cellular response.
The results are given in table 1 below:
(Table Removed)

a Mean of the cpm values (specific stimulation stimulation with medium), cpm = counts per minute (Student's test, P=0.002)
b Number of mice having specific CTL activity out of the total number of mice of the branch
c Geometric mean of the anti-p24 IgGl titers of the mice of the branch
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.
The experiment was repeated with 13 mice (3 or 4 mice per branch), except that the immunogens were used at a rate of 40 jug and that the mice were sacrificed either at D42 (3 mice) or at D70 (4 mice).
The results are given in table 2 below.
Table 2

(Table Removed)
a Mean of the number of cells secreting IFN-y/106 total
cells, in response to a specific stimulus (peptide
AMQMLKETI) for 20 h
b Number of mice having specific CTL activity out of the
total number of mice of the branch
c Geometric mean of the anti-p24 IgGl titers of the mice
of the branch
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 /zg to 40 /zg 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 3; Immunization of mice with the PLA/Tat microparticles of the invention
The procedure indicated in example 2 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 /zg, that, for the humoral response, the Tat protein was used as capture partner and the mouse polyclonal antibodies as indicated in example 2, 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. Peptides used in the ELISPOT assay (Sygma Genosys)
CFHCQVCFTKKGLGI (SEQ ID No.4) SYGRKKRRQRRRSPQ (SEQ ID No.7) VCFTKKGLGISYGRK (SEQ ID No.5) KRRQRRRSPQDSETH (SEQ ID No.8) KGLGISYGRKKRRQR (SEQ ID No.6) RRSPQDSETHQVSLS (SEQ ID No.9)
The results are indicated in table 3 below.
Table 3

(Table Removed)
a Mean of the number of cells secreting IFN-y/106 total
cells, in response to a specific stimulus (pool of
peptides) for 20 h
b Mean of the number of cells secreting IFN-y/106 total
cells, in response to a specific stimulus (Tat protein)
for 42 h
c Geometric mean of the anti-Tat total IgG titers of the
mice of the branch
d Number of mice having a specific IgG2a response out of
the total number of mice of the branch
e Geometric mean of the anti-Tat IgG2a titers of the
mice which have responded
The results in table 3 above demonstrate that:
injection of the mi cropar tides of the invention makes it possible to induce IFN-y-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 104. The use of the Tat/PLA microparticles makes it possible to enhance both the frequency and the titer of the anti-Tat IgG2as.
Example 4; Immunization of mice with the PLA/NS3 helicase microparticles of the invention
1. Animal model
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).
2. Immunogens administered
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.
3. Immunizations
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 /xg each in the case of the proteins or of 100 /zg 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.
The animals were sacrificed approximately 70 days (D70) after the first injection and the blood and the spleen were taken for the immunological analyses.
4. Immunological analyses
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 naive cells loaded with the peptide. The effector cytotoxic population was harvested after the 7th day and the CTL activity was measured using 51Cr-labeled P815 cells as targets.
5. Results
The results are given in figure 1, representing graphs giving the percentage specific lysis as a function of the effector/target ratio, and where figure 1A gives the cellular response induced after injection of the DNA sequence corresponding to the HCV NS3NS4 polyprotein as a control, figure IB gives the cellular response induced after injection of the NS3 helicase/Freund's adjuvant combination without microparticle, figure 1C gives the cellular response induced after injection of the NS3 helicase peptide without microparticle, figure ID gives the cellular response induced after injection of the PLA microparticles without protein substance, and figure IE gives the cellular response induced after injection of the PLA/NS3 helicase microparticles of the invention.
These graphs show a CTL response specific for the NS3 helicase peptide, demonstrated when the PLA/NS3 microparticles of the invention are injected.

Example 5; Obtaining monoclonal antibodies with the PLA/p24 microparticles of the invention
1. Animal model
The immunization experiments were carried out on female BALB/c (H-2d) mice 6 to 8 weeks old at the time of the first immunization.
2. Immunogens administered
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.
3. Immunizations
The mice received 3 successive doses of 10 /zg 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 /xg of p24.
4. Monitoring of the appearance of the anti-p24 humoral
response
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).
5. Obtaining monoclonal antibodies
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-Agl4 myeloma cells so that they would fuse and become immortalized, according to the protocol described by Kohler and Milstein (Kohler, G. and Milstein, C., 1975, Nature, 256:495-497; Kohler, 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.
6. Results
The anti-p24 antibody titer in the serum of the mice was determined just before sacrifice, individually for each mouse.
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
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.
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.

WE CLAIM:
1. A method for preparing non-lamellar bioresorbable microparticles to which protein
substances, such as herein before described, are bonded, characterized in that it comprises the
steps of:
(i) preparing said microparticles from at least one bioresorbable polymer such as herein before described without stabilizer and without surfactant, and
(ii) bonding said protein substances to the microparticles obtained in step (i) without surfactant.
2. The method as claimed in claim 1, wherein the polymer used in step (i) is a poly(a-hydroxylated acid) or a mixture of poly(a-hydroxylated acids).
3. The method as claimed in either of claims 1 and 2, wherein the bonding of the protein substances to the microparticles is carried out by adsorption.
4. The method as claimed in anyone of claims 1 to 3, wherein the protein substances is a antigen of viral origin.
5. The method as claimed in claim 4, wherein the antigen is an HIV virus antigen, preferably the p24 protein or a regulatory protein.
6. The method as claimed in claim 5, wherein the HIV virus antigen is a regulatory protein chosen from the Tat, Rev and Nef proteins.
7. The method as claimed in claim 4, wherein the antigen is an HCV virus antigen preferably a non-structural protein of the HCV virus.
8. The method as claimed in claim 7, wherein the antigen is the NS3 protein, preferably the NS3 helicase protein.

9. Bioresorbable non-lamellar microparticles to which protein substances are bonded, obtainable by the method as claimed in any one of the claims 1 to 8.
10. Method for preparing a medicinal product using the bioresorbable microparticles as claimed in claim 9.
11. A pharmaceutical composition in particular a vaccine, characterized in that it contains at least bioresorbable microparticle as claimed in claim 9 and where appropriate, a pharmaceutically acceptable excipient.
12. Antibodies directed against the bioresorbable microparticles as claimed in claim 9.
13. A diagnostic composition consisting of the bioresorbable microparticles as claimed in claim 9 or of the antibodies as claimed in claim 12.