Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
  • A61P31/12—Antivirals
  • A61P31/14—Antivirals for RNA viruses
  • A61P31/18—Antivirals for RNA viruses for HIV
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
  • C07K16/08—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from viruses
  • C07K16/10—RNA viruses
  • C07K16/112—Retroviridae (F), e.g. leukemia viruses
  • C07K16/114—Lentivirus (G), e.g. human immunodeficiency virus [HIV], feline immunodeficiency virus [FIV] or simian immunodeficiency virus [SIV]
  • C07K16/1145—Env proteins, e.g. gp41, gp110/120, gp160, V3, principal neutralising domain [PND] or CD4-binding site
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N2740/00—Reverse transcribing RNA viruses
  • C12N2740/00011—Details
  • C12N2740/10011—Retroviridae
  • C12N2740/16011—Human Immunodeficiency Virus, HIV
  • C12N2740/16111—Human Immunodeficiency Virus, HIV concerning HIV env
  • C12N2740/16122—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes

Definitions

• the subject of the present invention is a new antigen and its use in an NIH vaccine and more particularly relates to a chemically modified NIH envelope glycoprotein capable of inducing antibodies neutralizing primary NIH isolates.
• the NIH envelope glycoprotein has been shown to be an oligomer composed of an extracellular domain, gpl20, and a transmembrane domain gp41 (Gallaher et al AIDS Research & Human Retro viruses 11 (2): 191- 202, 1995). Léonard et al showed that gpl60 included 20 cysteine residues forming 10 disulfide bridges.
• NIH binding and cell membrane fusion and / or exposing initially hidden epitopes have proposed to modify the structure of gpl60 in order to obtain a protein closer to that present on the surface of the virus during the stage.
• RAXaCasse et al (Science, 283: 357-362, 01/15/1999) described the preparation of a vaccine comprising whole cells fixed to formaldehyde which would reproduce the transient structure of envelope protein / CD4 / co-receptor present at during HIV infection. The use of such a preparation would lead in a transgenic mouse model to the neutralization of many primary HIV isolates. The reproduction of this experiment could not be carried out.
• Neutralizing antibody responses as described in the prior art mentioned above, have the disadvantage of either being specific for a given serotype, or of being incapable of leading to the neutralization of primary isolates of HTV. Due to the very large genetic variability of the AIDS virus, such immune responses are therefore of little or no interest from a vaccine point of view. There is therefore a need for a vaccine capable of inducing neutralizing immunity against the primary HIV isolates.
• the present invention therefore relates to an HIV envelope glycoprotein in purified form capable of being obtained by a process comprising the following steps:
• the glycoprotein (1) is in dimeric form and preferably corresponds to a gpl60MN / LAI.
• step (2) is implemented by adding a reducing agent in a molar ratio (moles of reducing agent) / (moles of sulfhydryl groups) from 1 to 500 times.
• step (3) is implemented by adding an alkylating agent in a molar ratio (moles of alkylating agent)
• EM is used as an alkylating agent in a molar ratio (moles of NEM) / (moles of sulfhydryl groups) from 1 to 100, preferably 10.
• the present invention relates to a composition
• a composition comprising a mixture of chemically modified proteins as defined above.
• the present invention relates to an antibody directed against a chemically modified envelope glycoprotein as defined above; this antibody preferably being monoclonal.
• the subject of the present invention is an HIV vaccine comprising:
• the vaccine according to the invention is used to induce antibodies neutralizing the VEH in a human subject as a therapeutic or prophylactic.
• the present invention relates to a diagnostic method comprising bringing a biological fluid into contact with an antibody as defined above and determining the immune complexes thus formed.
• envelope glycoprotein is meant in the context of the present invention a glycosylated protein gpl60, gp 120 or gp 140.
• the envelope protein is in monomeric, dimeric or multimeric form, it will preferably be in dimeric form.
• This envelope protein may or may not be a recombinant protein and may also consist of a hybrid protein; the hybrid term being used here in its conventional acceptance, namely a protein comprising sequences originating from envelope proteins of different strains of virus adapted in the laboratory or from primary isolates from NIH.
• Envelope proteins whose amino acid sequence differs from that of the native protein by mutation (s), deletion (s), insertion (s), or substitution (s) of amino acid (s) are also included in the definition above as long as these modifications do not eliminate the formation of antibodies capable of neutralizing primary isolates of NIH. This characteristic can easily be determined using the tests provided in the present application.
• the gpl60M ⁇ / LAI as described in Example 1 below;
• the envelope glycoprotein of step (1) is used in a substantially purified isolated form.
• isolated and substantially purified protein means a protein having a purity level of at least 75%, preferably at least 80%, as determined by the method of electrophoresis on acrylamide gel (SDS PAGE) (LAEMMLI UK 1970. Nature 27: 680-685.) And densitometric analysis. Reference is made in the present application to such a protein under the term of “protein in purified form”.
• Various methods for purifying the NEH envelope protein, natural or recombinant have been described in the literature. One can refer for example to the articles of Pialoux et al (Aids Res. Hum. Retr., 11, 373-381, 1995) and of Sakmon-Ceron et al (Aids Res. Hum. Retr., 12, 1479-1486 , 1995) or in the text WO91 / 13906.
• glycoproteins thus purified have inter-chain disulfide bridges, regardless of the nature of the host or of the vector used.
• the glycoproteins thus associate in part in covalent dimers visible on SDS PAGE gel.
• the envelope glycoprotein in purified form is first subjected to a step of partial or total reduction of the intra-chain and / or inter-chain disulfide bridges in which at least one disulfide bridge is reduced.
• the reduction step is carried out by reaction of the envelope glycoprotein of step (1) with a reducing agent, at ambient temperature and with gentle stirring.
• the reducing agent can be chosen from the molecules of dithiothreitol (DIT), beta-mercaptoethanol, reduced glutathione and sodium borohydride, for example.
• the quantity of reducing agent expressed by the molar ratio (moles of reducing agent) / (moles of sulfhydryl groups) varies between 1 and 0.5 ⁇ 10 4 and preferably corresponds to a molar ratio of 50.
• the reduction is carried out works at a basic pH of 7 to 10, preferably at a pH of 7.8. Control of the pH value is obtained by adding a buffer; any suitable buffer for this can be used.
• a sodium phosphate buffer is used.
• the reaction is carried out for approximately 15 minutes, the molar ratio moles of DTT / moles of SH used is from 1 to 0.510 4 and preferably 50 times.
• the duration of the reduction reaction is variable and depends on the molar ratio and the reducing agent chosen.
• the conditions of the reduction reaction allowing the reduction of at least one disulfide bridge are easily determinable by a person skilled in the art from the teaching provided here.
• the reduction can be checked by SDS PAGE analysis insofar as the reduction of the interchain disulphide bridges transforms the dimers into monomers. Finer controls of this reduction are possible by using N-ethyl-maleimide (NEM) labeled with 14 C or more simply by the use of a colorimetric assay based on dithio-nitro-benzoic acid (DTNB).
• NEM N-ethyl-maleimide
• DTNB dithio-nitro-benzoic acid
• alkylating agent in the context of the present invention means any reagent capable of reacting specifically with the -SH groups to give a covalent bond.
• N-ethyl-maleimide N-ethyl-maleimide
• iodo-acetamide N-ethyl-maleimide
• the amount of alkylating agent used, expressed by the molar ratio (moles of alkylating agent) / (moles of sulfydryl groups) is from 1 to 100 times, preferably from 10 to 100 times. It is necessary to ensure that there is an excess of alkylating agent relative to the reducing agent to neutralize the action of the latter.
• the alkylation reaction is carried out at a pH of 6 to 8, preferably at a pH of 7, at room temperature.
• Control of the pH value is obtained by adding a buffer; any suitable buffer for this can be used.
• a sodium phosphate buffer is used.
• the conditions of the alkylation reaction allowing the alkylation of at least two -SH groups are readily determinable by a person skilled in the art from the teaching provided here.
• the alkylation can be controlled by the use of 14 C-NEM as described below in the examples.
• the product from step (3) can be subjected to an oxidation step during which the remaining free sulfhydryl groups are oxidized in the presence of an oxidizing agent. If free sulfhydryl groups are still present at the end of step (3), an oxidation step is preferably carried out before the denaturation step.
• the term “oxidizing agent” means any molecule linked by disulfide bridges such as oxidized glutathione or cystine, but it may also be other molecules such as quinones, oxygen, etc.
• the reduced glutathione / oxidized glutathione mixture In this mixture, the reduced glutathione allows the disulfide bridges to dissociate to reassociate in a more stable thermodynamic state.
• the oxidation reaction is carried out at a pH of 7 to 9, preferably at pH 7.8, at a temperature of 4 to 25 ° C.
• the oxidizing agent is used in a molar ratio (moles of oxidizing agent) / (moles of sulfhydryl groups) of 50 to 5000, preferably 500.
• the reaction is carried out with an oxidized glutathione level from 1 to 1000 times higher than the reduced glutathione level.
• a ratio of 500 molecules of oxidized glutathione per mole of gpl60MN / LAI can be advantageously used.
• the duration of the oxidation step can vary between 5 minutes and 24 hours and preferably corresponds to 30 minutes.
• the conditions of the oxidation reaction allowing the oxidation of the free sulfhydryl groups are easily determinable by a person skilled in the art from the teaching provided here. Oxidation can be controlled by a process similar to that used for the control of the reduction step, paying close attention to the positive controls of the test.
• the product from step (3) or (4) is then denatured by the action of one or more denaturing agent (s). Used at a rate of 0.1 to 5% ( weight / vol) so as to modify the conformation of the glycoprotein.
• one or more detergent (s) preferably ionic (s) or one or more chaotropic agent (s) can be used for example.
• ionic detergents the salts of dodecyl sulfate, in particular sodium dodecyl sulfate (SDS) or of lithium, the dioctyl sulfosuccinate salts (of sodium, for example), the cetryltrimethylammonium salts (of bromine , for example), DTAB, cetylpyridinium (chlorine, for example) salts, N-dodecyl- or N-tetradecyl-sulfobetaine, zwittergents 3-14, and 3 - [(3-cholamidopropyl) -dimethylamino] -l-propane sulfonate (CHAPS), and the following neutral detergent (s): tween20®, tween80®, octylglucoside, lauryl-maltoside, hecameg®, lauryl-dimethylamine,
• SDS sodium dodecyl sulf
• SDS is preferably used in the context of the present invention, in particular at a concentration of 0.1% (weight / vol.).
• the denaturation reaction is carried out at neutral or alkaline pH at room temperature.
• Denaturation can be controlled by spectrophotometric measurement by measuring the absorbance of the tyrosine, phenylalanine and tryptophan residues of the molecule or by circular dichroism.
• the glycoprotein thus denatured is then subjected to a renaturation step which can be carried out by dialysis against 1000 volumes of a buffer free of detergent, preferably a phosphate buffer containing sodium chloride (PBS).
• a buffer free of detergent preferably a phosphate buffer containing sodium chloride (PBS).
• the efficiency of the dialysis step can be easily determined by colorimetric analysis of the residual oxidizing agents or by HPLC by showing the disappearance of certain reagents used in the manufacture of the antigen.
• the dialysis can be carried out overnight at room temperature, with gentle stirring, against a PB S buffer.
• the gpl60MN LAI in purified form (1) is chemically modified by a process comprising steps of: (2) reduction by incubation with DTT in a molar ratio (moles of DTT) / (moles of SH groups) of 50 at a pH of 7, for a period of approximately 15 minutes at room temperature, ( 3) alkylation by incubation with NEM in a molar ratio (moles of NEM) / (moles of SH groups) of 10 at a pH of 7, for a period of approximately 15 minutes, at room temperature, (4) oxidation by incubation of the product of step (3) with a reduced glutathione / oxidized glutathione mixture in a molar ratio (moles of oxidized glutathione) / (moles of SH groups) of 500 with a reduced glutathione / oxidized glutathione ratio of 10, to pH 7.8 for approximately 30 minutes, (5) denat uration of the product from step 4
• the present invention relates to a composition comprising a mixture of chemically modified glycoproteins as defined above.
• these chemically modified glycoproteins can be differentiated, for example, by the nature of the constitutive envelope glycoprotein (for example glycoproteins originating from different primary strains or isolates, some of which may also correspond to hybrid proteins) or by their preparation process; the parameters of the latter may vary, such as the concentration and the nature of the reagents. Any conceivable mixture comprising one or more chemically modified envelope glycoprotein (s) is included within the scope of the present invention.
• the subject of the present invention is also the antibodies directed against the chemically modified envelope glycoproteins as described above.
• the preparation of such antibodies is carried out by conventional techniques for obtaining polyclonal and monoclonal antibodies (Kohler G et al European Journal of Immunology. 6 (7): 511-9, 1976 Jul). These antibodies are particularly suitable for use in a passive immunization scheme.
• the present invention also relates to vaccines useful for therapeutic and prophylactic purposes.
• the vaccines according to the present invention comprise a chemically modified envelope glycoprotein as defined above or a mixture of such glycoproteins, a pharmaceutically acceptable carrier or diluent and optionally an adjuvant.
• the vaccine according to the present invention can therefore contain a single type of chemically modified envelope glycoprotein or a mixture of various types of chemically modified envelope glycoprotein as defined above.
• the vaccine according to the present invention comprises chemically modified envelope anti-glycoprotein antibodies.
• any mixture of antibodies, monoclonal or polyclonal, directed against different parts of the same chemically modified envelope glycoprotein or against different chemically modified envelope glycoproteins is part of the present invention.
• the amount of chemically modified envelope glycoprotein in the vaccine according to the present invention depends on many parameters as will be understood by those skilled in the art, such as the nature of the chemically modified glycoprotein, the route of administration and the state of the person to be treated (weight, age, clinical condition, etc.).
• An appropriate amount is an amount such that a humoral immune response capable of neutralizing primary HIV isolates is induced after administration of the latter.
• the vaccines according to the present invention may also contain an adjuvant. Any pharmaceutically acceptable adjuvant or mixture of adjuvants may be used for this purpose.
• Conventional auxiliary agents such as wetting agents, fillers, emulsifiers, buffers etc. can also be added to the vaccine according to the invention.
• the vaccines according to the present invention can be prepared by any conventional method known to those skilled in the art.
• the antigens are mixed with a pharmaceutically acceptable carrier or diluent, such as water or phosphate-buffered saline.
• a pharmaceutically acceptable carrier or diluent such as water or phosphate-buffered saline.
• the support or diluent will be selected according to the dosage form chosen, method and route of administration as well as pharmaceutical practice.
• the appropriate carriers or diluents as well as the pharmaceutical formulation requirements are described in detail in Remington's Pharmaceutical Sciences, which represents a reference work in this field.
• the vaccines mentioned above can be administered by any conventional route, usually used in the field of vaccines, such as the parenteral route (intravenous, intramuscular, subcutaneous, etc.).
• the administration can be carried out by the injection of a single dose or of repeated doses, for example on D0, at 1 month, at 3 months, at 6 months and at 12 months.
• the present invention also intends to cover a chemically modified envelope glycoprotein as defined above and the vaccine containing such a glycoprotein or mixture of such glycoproteins for their use for inducing antibodies neutralizing primary isolates of VEH.
• the Applicant has surprisingly demonstrated that the chemically modified envelope glycoproteins according to the invention are capable, after administration, of inducing antibodies capable of neutralizing primary isolates of HIV. These antigens therefore represent valuable candidates for the development of a vaccine usable for the protection and / or treatment of a large number, even all of those at risk or infected with HIV.
• Example 1 Preparation of the gpl60MN / LA1 glycoprotein
• the gpl60MN / LAI glycoprotein is a soluble hybrid glycoprotein in which the gpl20 subunit derives from VEH-1 MN and the gp41 subunit derives from the LAI isolate.
• the DNA sequences corresponding to these two components are fused using of a Smal restriction site which does not modify either the amino acid sequence of gpl20 or that of gp41. The preparation of this protein is described below.
• sequence coding for gpl20MN is amplified by PCR from the SupT1 cells infected with VEH-MN, using oligonucleotides introducing the restriction sites SphI and Smal respectively immediately downstream of the sequence coding for the leader peptide and upstream of the cleavage sites located between gpl20 and gp41.
• the sequence coding for the gp41 subunit is produced as follows: the complete sequence coding for the env protein of VEH 1 -LAI is placed under the control of the promoter pH5R of the vaccinia virus. Several modifications are introduced in this coding region.
• a SphI restriction site is created immediately downstream of the sequence coding for the leader peptide, without alteration of the amino acid sequence.
• a Smal restriction site is created immediately upstream of the sequence coding for the cleavage sites located between gpl20 and gp41, without alteration of the amino acid sequence.
• the two cleavage sites in position 507-516 were mutated (ie the original sequence KRR ... REKR has been transferred to QNH ... QEHN).
• the plasmid into which the LAI sequence is inserted between the homologous regions of the TK gene of the vaccinia virus is cut with SphI and SmaI then ligated with the sequence of gpl20MN.
• the WTG9150 virus is then constructed by conventional homologous recombination.
• the recombinant vaccinia virus vector WTG9150 thus produced is used for the production of gpl60MN / LAI.
• the vector is propagated on BHK21 cells.
• Gpl60 MN / LAI is produced on BHK21 cells infected for 72 hours with the recombinant vaccinia virus NVTG9150.
• the supernatant is collected, filtered and ultrafiltered to give the concentrated harvest.
• the purification takes place in three stages. Certain contaminants of gpl60 M ⁇ / LAI are fixed on an anion exchange column.
• the unfixed fraction is chromatographed on an immunoafinity column using a monoclonal antibody.
• the gpl60 MN / LAI is desalted by gel filtration chromatography in PBS buffer. To inactivate the residual vaccinia, the glycoprotein is heated at 60 ° C for 1 hour before being filtered to give the purified antigen.
• concentration of gpl60 MN / LAI used for the preparation of the chemically modified proteins is 1 mg / ml of proteins (determined by colorimetric assay kit BCA, Pierce TM) and its purity of 77% (determined by SDS PAGE electrophoresis and analysis by optical densitometry using the ScannerGS700 from Biorad TM).
• the glycoprotein is in a phosphate buffer of the following composition: 137 mM NaCl; 2.7 mM KC1; Na 2 HPO 4 6.5 mM; KH 2 PO 1.5 mM; pH 7.4 (PBS).
• the gpl ⁇ OMNZLAI thus obtained, has a molecular weight of 140kD in SDS-PAGE.
• Example 2 Preparation of chemically modified glycoproteins according to the invention From 172 ⁇ l of purified gp160 (1 mg / ml), 21 ⁇ l of 1M sodium phosphate buffer pH7.8 is added; 2 ⁇ l of distilled water and 19.5 ⁇ l of 50 mM dithiothreitol (DTT), the mixture is vortexed for 15 s, and incubated for 15 min at 25 ° C.
• DTT dithiothreitol
• 16 ⁇ l of 1M sodium phosphate buffer (NaH2PO4) are added to lower the pH to 7, then the sulfhydryl groups are blocked by the addition of 14 ⁇ l of 100 mM N-ethyl-maleimide (NEM) and incubated for 15 min at 25 ° vs.
• the sulfhydryl groups are reoxidized by addition of sodium phosphate buffer at pH 7.8 and a mixture of 4.8 ⁇ l of glutathione reduced to 150 mM and 71.6 ⁇ l of oxidized glutathione at 100 mM is added, the mixture is incubated for 30 min at 25 ° C.
• the gp160 dimers are then dissociated by the addition of 12 ⁇ l of sodium dodecyl sulfate (SDS) at 3%.
• SDS sodium dodecyl sulfate
• the sample is placed in a dialysis cassette with a capacity of 3 ml against 1000 volumes of PBS buffer (without detergent). Dialysis is carried out overnight at room temperature with gentle stirring.
• the gpl60 thus treated are found in the form of monomers and dimers.
• the protein thus obtained is called BA29.
• Example 3 Preparation of chemically modified glycoproteins with variation in the concentration of alkylating agent.
• the BA53 preparation is carried out according to the method described in Example 2 for BA29 in which the NEM has been omitted.
• the BA55 preparation is carried out according to the method described in Example 2 for the
• the BA56 preparation is carried out according to the method described in Example 2 for BA29 in which the NEM concentration used is 10 times greater than that indicated in Example 2.
• antigens were prepared in parallel to be injected into animals and for a biochemical measurement of the quantity of NEM fixed per molecule of gpl60. For this, the NEM marked with 14 C was used. About 4MBq of 14 C NEM was added per mM of
• Non-radioactive EOD The radioactivity measured is then directly proportional to the concentration of NEM. It was checked during the final dialysis step that the elimination of radioactive NEM was well done exhaustively and that only the radioactivity covalently attached to the protein remained in the sample. Aliquots of the antigens produced according to the different protocols were then placed in the scintillation vials and the ⁇ radiation emitted by the decay of the 1 C atoms was recorded for one minute. The radioactivity counts are directly proportional to the amount of NEM fixed. Knowing the amount of gpl60 present in each aliquot, the ratio of the number of NEM molecules per molecule of g l60 could be established.
• the results obtained show that unreduced gpl60 cannot fix NEM (control).
• the gpl60 treated according to the invention (BA29) fixes 8 molecules of NEM by gpl60 molecule. Therefore, there are at least 4 modified disulfide bridges inside this antigen. It has been shown that the use of a concentration of NEM ten times lower (BA55) made it possible to fix the NEM only on 2 (1.6 to 1.8) sulfydryls per molecule of gpl60. It is possible that only one disulfide bridge is modified within this antigen. The use of a concentration of NEM ten times higher (BA56) has been shown to abolish the immunological properties of the molecule.
• the chemically modified glycoproteins are diluted sterile in a stabilizing medium and then adsorbed on aluminum phosphate.
• the stabilizing mixture is composed of a mixture of amino acids and "Dulbecco's Modified Eagle" medium.
• DMEM-F12 Medium “DMEM-F12 (Gibco, France).
• the chemically modified glycoproteins prepared in Examples 2-4 (BA29, BA29 (7.8), BA52, BA53 and BA55 and BA56) are diluted in the stabilizer mixture before an equal volume 6.3 mg / ml aluminum phosphate in PBS is added to this mixture.
• the chemically modified glycoproteins named BA53 and BA52 are obtained by using the method described in Example 2 for BA29 in which the step of alkylation with NEM has been omitted (preparation BA53); or the denaturation step by SDS has been omitted (preparation BA52).
• a group of 5 female Dunkin-Hartley (Charles River) albino guinea pigs of 400 g is used. Each guinea pig receives 5 ⁇ g of antigen intramuscularly, in the right and left thighs (0.5 ml in each thigh) on Dl and D29. A volume of 3 ml of blood is taken by cardiac puncture under anesthesia on days -1, 28 and 56 (final bleeding about 30 ml).
• the guinea pig sera thus obtained were analyzed by ELISA assay against the native gpl60 MN / LAI.
• the gpl60 MN / LAI is immobilized on the solid phase at a rate of 130 ng per well for 1 hour at 37 ° C.
• the plate is emptied then saturated with a PBS buffer, 0.1% Tween 20 containing 5% of skimmed milk powder.
• Each serum is diluted on the plate according to serial dilutions of reason 3, between l / 100 e and 1/100000 6 as the case may be, in saturation buffer and incubated for 1 hour 30 minutes at 37 ° C.
• a rabbit anti-guinea pig antibody (Sigma, St Louis) coupled with peroxidase, diluted to 1/3000 e , makes it possible to reveal the presence of antibodies specific for gpl ⁇ OMNZLAI.
• the titles are calculated automatically by the reader from the optical density read and from the straight line obtained with a standard serum.
• the mean values of the immunoglobulin specific for gp160 MN / LAI for each group of guinea pigs are between 10. 5 and 10. 6 .
• the control group injected with untreated gpl60 MN / LAI is identified under the code BAI.
• Preparations BA55 and BA29 induce specific antibodies; BA29 leading to a titer higher than 5.10 5 . No specific antibody to gpl60 MN / LAI was detected in the pre-immune sera.
• the figures indicate the inverse of the dilution of the serum for which neutralization was observed.
• the antigens according to the present invention are produced from a gpl60MN / LAI, from an NIH-1 virus adapted in the laboratory.
• these antigens make it possible to induce in the immunized animal a humoral response capable of neutralizing primary isolates of the NEH-1 virus, which constitutes an improvement compared to current knowledge on this subject.

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