Chimeric non-integrating lentiviral genomes as vaccines against HIV-1

Abstract

Novel nucleic acids include non-integrative chimeric retroviral genomes including the 5 and 3 long terminal repeat sequences (LTRs) of the caprine lentivirus: the Caprine Arthritis Encephalitis Virus (CAEV) or of another retrovirus not integrating human cells and at least one viral gene of another retrovirus. A vector including such a nucleic acid, an immunogenic or vaccinal composition including the vector or the nucleic acid, as well as their use for treating and/or preventing an infection by a retrovirus or a disease induced by a pathogenic agent are also described.

Claims

1. A nucleic acid comprising a chimeric retroviral genome, in which said chimeric retroviral genome comprises: Long terminal repeat sequences (LTR) positioned 5 and 3 of a first retrovirus, said first retrovirus being the Caprine Arthritis Encephalitis Virus (CAEV), and at least one viral gene of a second retrovirus, said second retrovirus not being the first retrovirus, wherein the chimeric retroviral genome comprises the gag, pol, vif, vpx, vpr, nef, tat, rev, vpu and env genes of said second retrovirus, and wherein said pol gene is mutated to delete nucleic acid sequences encoding an integrase protein to an extent sufficient to abolish any integrating activity of said integrase protein.

2. The nucleic acid according to claim 1, wherein said second retrovirus is a lentivirus.

3. The nucleic acid according to claim 2, wherein said lentivirus is selected from the human immunodeficiency virus of type I (HIV-1), the HIV-2, the simian immunodeficiency virus (SIV), the feline immunodeficiency virus (FIV), or the equine infectious anaemia virus (EIAV).

4. A recombinant vector comprising the nucleic acid according to claim 1.

5. An immunogenic or vaccinal composition comprising the nucleic acid according to claim 1.

6. A nucleic acid comprising a chimeric retroviral genome, in which said chimeric retroviral genome comprises: Long terminal repeat sequences (LTR) positioned 5 and 3 of a first retrovirus, said first retrovirus being the Caprine Arthritis Encephalitis Virus (CAEV), and at least one viral gene of a second retrovirus, said second retrovirus not being the first retrovirus; wherein the chimeric retroviral genome further comprises at least one viral gene of a third retrovirus, said third retrovirus being different from said first and second retroviruses; and wherein the chimeric retroviral genome comprises the gag, pol, vif, vpx, vpr, nef, tat, rev, vpu, and env genes each of said genes being of either but not both of said second or third retrovirus, and wherein if said at least one viral gene of said second retrovirus or said at least one viral gene of said third retrovirus is a pol gene then said pol gene is mutated to delete nucleic acid sequences encoding an integrase protein to an extent sufficient to abolish any integrating activity of said integrase protein.

7. The nucleic acid according to claim 6, wherein said at least one gene of said second retrovirus, or said at least one gene of said third retrovirus, is selected from the gag, pol, vif, vpx, vpr, nef, tat, rev, vpu and env genes.

8. The nucleic acid according to claim 6, wherein the chimeric retroviral genome comprises the gag, pol, vif, vpx, vpr, nef genes of said second retrovirus and the tat, rev, vpu and env genes of said third retrovirus.

9. The nucleic acid according to claim 6, wherein said second and third retroviruses are each a lentivirus.

10. The nucleic acid according to claim 9, wherein said lentivirus is selected from the group consisting of human immunodeficiency virus type I (HIV-1), human immunodeficiency virus type II (HIV-2), simian immunodeficiency virus (SIV), feline immunodeficiency virus (FIV), or equine infectious anaemia virus (EIAV).

11. The nucleic acid according to claim 6, wherein said retroviral chimeric genome comprises the gag, pol, vif, vpx, and vpr genes of SIV and the nef, tat, rev, vpu and env genes of HIV-1.

12. The nucleic acid according to claim 6, wherein said retroviral chimeric genome comprises at least one of the gag, pol, vif, vpx, vpr, nef, tat, rev, vpu and env genes of HIV-1 and comprises at least one of the gag, pol, vif, vpx, vpr, nef, tat, rev, vpu and env genes of HIV-2.

13. The nucleic acid according to claim 6, wherein said second retrovirus is a lentivirus.

14. The nucleic acid according to claim 6, wherein said chimeric retroviral genome comprises one of the nucleic acid sequences according to SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 6, and wherein said pol gene, when it is present, is mutated to delete nucleic acid sequences encoding an integrase protein to an extent sufficient to abolish any integrating activity of said integrase protein.

15. A recombinant vector comprising the nucleic acid according to claim 6.

16. An immunogenic or vaccinal composition comprising the nucleic acid according to claim 6.

17. A nucleic acid comprising a chimeric retroviral genome, wherein said chimeric retroviral genome comprises one of the nucleic acid sequences according to SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 6.

Description

DESCRIPTION OF THE FIGURES

(1) FIG. 1: Schematic illustration of the nucleic acid coded by the sequence SEQ ID NO.: 2.

(2) FIG. 2: Schematic illustration of the nucleic acid coded by the sequence SEQ ID NO.: 4.

(3) FIG. 3: Schematic illustration of the nucleic acid coded by the sequence SEQ ID NO.: 5.

(4) FIG. 4: Schematic illustration of the nucleic acid coded by the sequence SEQ ID NO.: 6.

(5) FIG. 5: Schematic illustration of the nucleic acid coded by the sequence SEQ ID NO.: 7.

(6) FIG. 6: Schematic illustration of the nucleic acid coded by the sequence SEQ ID NO.: 1.

(7) FIG. 7: Schematic illustration of the nucleic acid coded by the sequence SEQ ID NO.: 12.

(8) FIG. 8: Schematic illustration of the nucleic acid coded by the sequence SEQ ID NO.: 13.

(9) FIG. 9: Schematic illustration of the nucleic acid coded by the sequence SEQ ID NO.: 14.

(10) FIG. 10: Schematic illustration of the nucleic acid coded by the sequence SEQ ID NO.: 15.

(11) FIG. 11: Schematic illustration of the construction of the genome of CAEV, of the plasmid pSHIV.sub.KU2, pCA-LTR-SHIV.sub.KU2IN- and of p4SHIV.sub.KU2.

(12) FIG. 12: Evaluation of the number of T lymphocytes secreting IFN- of Balb/c mice. Spleen cells of control BALB/c immunocompetent mice and immunized with p4SHIV.sub.KU2 and pCA-LTR-SHIV.sub.KU2IN- and stimulated by the Gag, Env peptides and the peptide Tat+Rev+Nef pool. The number of spots was calculated for 1 million PBMCs.

(13) FIG. 13: Evaluation of the number of human T Lymphocytes secreting IFN- in immunized NOD/SCID-hu mice. Spleen cells of immunodeficient mice reconstructed by mononuclear cells of human blood and immunized with p4SHIV.sub.KU2 or pCA-LTR-SHIV.sub.KU2IN- or pSHIV.sub.KU2 are stimulated with Gag, Env peptides and with the peptide pool Tat+Rev+Nef. The number of spots was calculated for 1 million PBMCs and normalized to 20%.

(14) FIG. 14: Illustration of the preparation of the vector CA-LTR-SHIV.sub.KU2-IN-.

(15) FIG. 15: Schematic illustration of the vector CA-LTR-SHIV.sub.KU2.

(16) FIG. 16: Schematic illustration of the vector CA-LTR-SHIV.sub.KU2-IN-.

(17) FIG. 17: Illustration of the preparation of the vector CAL-HIV-IN-.

(18) FIG. 18: Schematic illustration of the vector CAL-HIV-IN-.

DESCRIPTION OF THE SEQUENCES

(19) SEQ ID NO: 1 Represents the sequence of a chimeric retroviral genome comprising the LTRs of the SAEV and the genome of SHIV deleted from the sequences coding for the integrase. SEQ ID NO: 2 Represents the sequence of a chimeric retroviral genome comprising the LTRs of the CAEV and the genome of the HIV-1. SEQ ID NO: 3 Represents the sequence of the LTR of the CAEV. SEQ ID NO: 4 Represents the sequence of a chimeric retroviral genome comprising the LTRs of the CAEV and the genome of SIV. SEQ ID NO: 5 Represents the sequence of a chimeric retroviral genome comprising the LTRs of the CAEV and the genome of HIV-2. SEQ ID NO: 6 Represents the sequence of a chimeric retroviral genome comprising the LTRs of the CAEV and the genome of the FIV. SEQ ID NO: 7 Represents the sequence of a chimeric retroviral genome comprising the LTRs of the CAEV and the genome of SHIV. SEQ ID NO: 8 Represents the sequence of the integrase of SIV. SEQ ID NO: 9 Represents the sequence of the integrase of HIV-1. SEQ ID NO: 10 Represents the sequence of the integrase of HIV-2. SEQ ID NO: 11 Represents the sequence of the integrase of FIV. SEQ ID NO: 12 Represents the sequence of a chimeric retroviral genome comprising the LTRs of the CAEV and the genome of HIV-1 deleted from the sequences coding for the integrase. SEQ ID NO: 13 Represents the sequence of a chimeric retroviral genome comprising the LTRs of the CAEV and the genome of HIV-2 deleted from the sequences coding for the integrase. SEQ ID NO: 14 Represents the sequence of a chimeric retroviral genome comprising the LTRs of the CAEV and genome of FIV deleted from the sequences coding for the integrase. SEQ ID NO: 15 Represents the sequence of a chimeric retroviral genome comprising the LTRs of the CAEV and the genome of SIV deleted from the sequences coding for the integrase. SEQ ID NO: 16 Represents the sequence of the vector pCA-LTR-SHIV.sub.KU2. SEQ ID NO: 17 Represents the sequence of the vector pCA-LTR-SHIV.sub.KU2-IN-. SEQ ID NO: 18 Represents the sequence of the vector CAL-HIV-IN-.

EXAMPLES

(20) 1. Material and Methods

(21) 1.1. The Vaccinating Vectors (FIG. 1)

(22) 1.1.1. The Vectors pCA-LTR-SHIV.sub.KU2 and pCA-LTR-SHIV.sub.KU2-IN-

(23) The vector CA-LTR-SHIV.sub.KU2 contains the genome of the Simian and Human Immunodeficiency Virus (SHIV) deleted from the LTRs of SIV and replaced with the LTRs of CAEV. The SHIV contains a chimeric genome consisting of the one of SIV-mac239 in which the tat, env and rev genes of the SIV were deleted and replaced with the vpu, tat, env and rev genes of the HIV-1. The vector therefore bears the vpr, vpx, gag, pol, vif and nef genes of SIV and the tat, rev, vpu and env genes of HIV-1 under the transcriptional control of the LTRs in 5 and 3 of the CAEV. The pol gene was deleted from the sequences coding for the integrase (in). The vaccinating vector pCA-LTR-SHIV.sub.KU2 non-deleted from the sequences coding for the integrase consists in the sequence SEQ ID NO: 16 (FIG. 15). The vector pCA-LTR-SHIV.sub.KU2-IN-, deleted from the sequences coding for the integrase consists in the SEQ ID NO: 17 (FIG. 16).

(24) The construction of a vector CA-LTR-SHIV.sub.KU2-IN- was achieved in the following way (FIG. 14). The vector SHIV-.sub.KU2 was digested with EcoR1 and Nar1, and then the LTR fragment of 0.8 kb was removed. The CAEV-pBSCA vector was then digested with EcoR1 and Nar1 and the fragment LTR of 0.5 kb was purified. Both fragments were then subject to ligation. The vector SHIV-1 LTRCA was then digested with Stu1 and Ava1 and the LTR fragment of 0.8 kb was removed. The LTR in 3 of the CAEV was amplified with timers Stu1 and Ava1, the PCR products were digested by Stu1 and Ava1 and the LTR fragment of 0.5 kb was purified. Both fragments were subject to ligation for generating the CAL-SHIV.sub.KU2. Finally a digestion with Kpn1 and Acc1 in the pol gene was achieved in order to remove 314 bp of the gene of the integrase of SHIV for generating the CAL-SHIV.sub.KU2-IN-.

(25) 1.1.2. Vectors pSHIV.sub.KU2 and 4SHIV.sub.KU2

(26) The plasmids pSHIV.sub.KU2 and p4SHIV.sub.KU2 are plasmids used as controls. Their constructions have been described in many publications (Liu Z Q et al., 2006, Ramakrisna Hegde et al., 2005).

(27) 1.2 Production of Vaccinal DNA

(28) 1.2.1. Bacterial Culture

(29) E. coli K12 (JM109) bacteria containing the plasmid are put in a pre-culture in 5 ml of BL medium containing 0.05 mg/ml of kanamycin and then incubated for one night at 30 C. with stirring at 150 rpm. From the pre-culture, the bacterial suspension was diluted to 1:100,000 in the BL medium, and then 50 l of the dilution are spread over the surface of the agar/BL/Kanamycin contained in a Petri dish which is then incubated at 32 C. for one night. The isolated colonies developed on the agar of the Petri dish are sown into 5 ml of liquid medium BL containing 0.05 mg/ml of kanamycin and cultivated with stirring at 150 rpm at 30 C. for one night. A fraction (1 ml) of the culture is used for rapid extraction of the DNA by means of the Mini-prep kit of Macherey-Nagel or Qiagen, according to the recommended procedure, and the extracted DNA is then separated on a 1% agarose gel for checking its quality. The bacteria corresponding to the DNA estimated to be satisfactory are used for sowing the 1 L cultures which are cultivated under the same conditions as earlier, for the isolation of DNA in a maxi-preparation.

(30) 1.2.2. Maxi-Preparation: Plasmid Extraction

(31) The bacteria cultivated with stirring (150 rpm) at 30 C. for one night are harvested as a sediment by centrifugation (4000 g, 4 C., 15 min) and the sediment is re-suspended in 8 ml of re-suspension buffer (Tris-HCl 50 mM pH8, EDTA 10 mM). The cells are then lyzed by adding 8 ml of alkaline lysis buffer (NaOH 200 mM, 1% SDS) in order to release the plasmid DNA. The lyzate is neutralized by adding 8 ml of neutralization buffer (potassium acetate 3M pH 5.5). The mixture is then incubated for 5 min in ice and then centrifuged for 15 minutes at 15,000 g at 4 C. The solution containing the DNA is transferred into a column equilibrated beforehand, allowing retention of the plasmid DNA. The column is washed three times with the washing buffer and then the DNA is eluted and precipitated with isopropanol. The precipitated DNA sediment is obtained by centrifugation (30 min, 15,000 g at 4 C.). The DNA is then washed with 2 ml of 70% ethanol and centrifuged for 10 min at 4 C. at 15,000 g in order to remove the excess impurities and salts and then the sediment is dried and re-suspended in a suitable volume of ultra-pure water.

(32) The concentration of the DNA solution was then determined by spectrophotometry at a wavelength equal to 260 nm and the quality of the plasmid is then checked by electrophoretic migration on a 1% agarose gel. The size of the plasmid and the integrity of the plasmid are checked on an agarose gel after digestion by restriction enzymes Bam H1 and Eco R1 for example.

(33) 1.2.3. Checking the Plasmid pCA-LTR-SHIV.sub.KU2-IN- by Enzymatic Digestion

(34) An aliquot fraction of 0.5 g of the plasmid is subjected to digestion with 2 units of enzymes EcoR1, BamH1 or Sph1 for 60 minutes at 37 C. in a suitable 1 buffer for each enzyme, and in a final volume of 20 l. The profile of the digestion is checked by electrophoretic migration in a 1% agarose gel with a TAE 1 buffer and revealed with ethidium bromide (ETB) and observation of the gel under UVs.

(35) 1.3 Cell Cultures and Treatments

(36) 1.3.1. Cell Models and Cultivation Conditions

(37) The cell lines were obtained from the National Institute of Health AIDS Research and Reference Reagent Program in the United States. The cells are cryo-preserved in 10% of dimethylsulfoxide (DMSO), at 170 C. in liquid nitrogen. They are defrosted and then cultivated in cultivation flasks.

(38) HEK293T (immortalized Human Embryonic Kidney 293) cells are a permanent line of human embryonic kidney cells. They are used because they are very easy to transfect, with very high transfection efficiencies which may attain 100%. The lentiviral genome is strongly expressed in these cells and the proteins assemble into infectious particles and their co-cultivation with the indicating cells (CEM or M8166) allows the formation of typical syncytia. The HEK293T cells are adherent, cultivated in a monolayer at the surface of the flasks in a MEM medium supplemented with 10% of fetal calf serum (FCS), 1% of penicillin 5,000 Units/ml streptomycin 5,000 g/ml and 1% of gentamycin 10 mg/ml. The cells are maintained at 37 C. under a humid atmosphere with 5% CO.sub.2. The culture medium is changed every three days. In order to carry out sub-cultivations, the nutritional medium is removed, the cells are washed with d PBS/EDTA and incubated for 1 minute at 37 C. in the presence of 0.5% trypsin-0.01% EDTA. After detachment of the cells, a volume of MEM medium is immediately added to the cells and the cells are then homogenized and transferred into new flasks.

(39) The cells CEMx174 and M8166 are human CD4+ T lymphocytes which are permissive to infection by human and simian lentiviruses and form typical cytopathogenic effects (CPE). They are non-adherent and are cultivated in RPMI medium supplemented with 10% of SCS, 1% of penicillin-streptomycin and 1% of gentamycin. These cells are maintained at 37 C. under a humid atmosphere with 5% CO.sub.2. The medium is changed every three days by carrying out a centrifugation step at 1,500 G for 5 minutes. The sediment is then re-suspended by successive suctions and discharges into a suitable volume of culture medium.

(40) 1.3.2. Functional Evaluation with an In Vitro Biological Test

(41) Transfection of the HEK293T with the Plasmids pCA-LTR-SHIV.sub.KU2-IN- or pSHIV.sub.KU2

(42) The transfection method used is the method with ExGen500. ExGen500 (Euromedex, France) consists of a cationic polymer based on linear polyethylenimine. This polymer has a very large cationic charge density allowing it to form complexes with DNA through ionic bonds. These ExGen500/DNA complexes are then capable of interacting with the plasma membranes of the generally anionic cells (interaction via the sulfated proteoglycans). Endocytosis of the complex by the cells ensues as well as their transport towards endosomes/lysosomes. By its protonation capacity at acid pH, ExGen500 gives the possibility of buffering the medium of acid vesicles thereby preventing degradation of the transfected DNA. This property also causes an osmotic shock which allows the DNA to be released in the cytoplasm of the cell. ExGen500 then promotes the transport of the DNA towards the nucleus and avoid its degradation by cytoplasm nucleases.

(43) Five g of the DNA of the plasmid are added to 350 l of 150 mM NaCl solution and to 15 l of ExGen 500. The mixture is incubated for 40 minutes at room temperature. Next, the mixture is added into the flasks containing the HEK-293T covered with freshly renewed medium.

(44) Infection of the CEMx174 and Amplification of the Viral Stock

(45) The HEK-293T transfected with the DNA of pCA-LTR-SHIV.sub.KU2-IN- or of pSHIV.sub.KU2 are co-cultivated with the CEMx174, and from 48 hours onwards, the CEMx174 develop signs of infection expressed by the formation of ECP which results from the merging of the CEMx174 for forming syncytia. The infected CEMx174 are transferred into a new flask in the presence of fresh CEMx174 for amplifying the virus which is harvested in the supernatant.

(46) Viral Production

(47) The harvesting of the viral stock is carried out from 48 hours onwards by means of a syringe, and the cell debris are then removed by having them pass through a filter with a diameter of 0.22 m before being put into tubes which are stored at 80 C.

(48) Inoculation of the Cells with the Virus

(49) An aliquot fraction (10-100 l) of the virus supernatant is used for inoculating the target cells in order to evaluate its infectiousity by cytopathic development or by detection of expression of marker genes.

(50) Titration of the Virus on Non-Adherent Cells

(51) The supernatant containing the virus is diluted in tenfold steps (successive dilutions) in a medium for obtaining dilutions from 10.sup.1 to 10.sup.6 which are used for inoculating in quadruplicate wells containing 1.Math.10.sup.5 cells per well in 0.5 to 1 ml of RPMI medium in a 24-well plate. The thereby inoculated cells are incubated at 37 C. and with 5% CO.sub.2 and sustained by changing medium every 3 days. They are regularly observed for development of ECP.

(52) 1.4. Electron Microscopy of HEK293T Cells Transfected with pCA-LTR-SHIV.sub.KU2-IN-

(53) With the goal of examining whether the proteins produced by the vaccinal genome pCA-LTR-SHIV.sub.KU2-IN- assembled into viral particles, the inventors conducted morphological studies by electron microscopy (EM).

(54) The samples of HEK293T cells transfected with the pCA-LTR-SHIV.sub.KU2-IN-, pSHIV.sub.KU2 or 4SHIV.sub.KU2 are fixed in a 2.5% glutaraldehyde solution diluted in a cacodylate buffer (0.1M sodium cacodylate). They are then post-fixed, at 4 C., in cacodylate buffer containing 1% osmium tetraoxide (OsO4), for 60 minutes. The samples are then incubated overnight at 4 C. in darkness in uranyl acetate at pH 4. The samples are then immersed in successive baths for 10 minutes of ethanol respectively diluted to 30%, 60%, 90% and 100%. Next, the samples are immersed for two hours in a 50/50 mixture of pure ethanol and of epoxy resin (8 ml of DDSA, 7 ml of MNA, or 13 ml of epoxy). The samples are then placed for two hours in pure epoxy resin before being included in Beem capsules and set to polymerize for 48 hours at 60 C.

(55) Ultrathin cuts of these regions are carried out by means of a diamond cutter with an ultra-microtome. These cuts with a thickness of 70 nm are deposited on copper grids so as to be observed under a voltage of 80 kV by means of a Jeol 1200 EX transmission electron microscope.

(56) 1.5. Immunization of Mice with pCA-LTR-SHIV.sub.KU2-IN- Vaccinal DNA

(57) 1.5.1. Humanization and Vaccination of NOD/SCID Mice

(58) The 6 week old mice are irradiated with a dose of 120 Centigrays of gamma radiation for 50 seconds.

(59) Humanization of the Mice with PBMC of Human Blood

(60) The total sample blood on sodium citrate is centrifuged (2000 g, 10 min, 20 C.) in order to recover the layer of white cells between the plasma and the red cells. The cells are diluted 3 times in PBS/EDTA, delicately deposited above a Ficoll cushion (a medium for separating lymphocytes) and then centrifuged for 45 minutes at 2,000 g at 20 C. The PBMCs are recovered, washed several times in PBS/EDTA and re-suspended in PBSx1 and then 50.Math.10.sup.6 PBMCs in 0.1 ml are injected for each mouse via an intra-peritoneal route.

(61) Immunization of the Mice

(62) After 48-72 h of post-humanization, the SCID-hu cells are injected via an intra-muscular route (IM) with 50 g of DNA of the pCA-LTR-SHIV.sub.KU2-IN-, pSHIV.sub.KU2 or p4SHIV.sub.KU2. The BALB/c mice, 6-8 weeks old, are directly immunized by IM injection with 100 g of each of the DNAs.

(63) 1.5.2. Method for Evaluating the Humoral Response

(64) Abnova Sandwich ELISA Test:

(65) This test is based on the detection of the antibodies directed against the viral antigens and which are bound to the bottom of the wells of the 96-well plate. The sera to be tested (recovered at different times post-immunization), the positive and negative controls are deposited in the wells. The anti-HIV Ac (antibodies) optionally present, bind onto the viral antigens. After several washings of the wells for removing the excess and the non-specific bindings, a secondary detection Ac is added which bears a biotin molecule which interacts with a streptavidin coupled to the HPRO enzyme. The formed antigens/Ac complexes will then be detected by adding the substrate of the enzyme, TMB, which will give rise to a colored reaction. The coloration is expressed in optical densities by readout with an ELISA reader photometer.

(66) The reagents and products are brought to room temperature. The negative and positive controls (100 l) provided in the kit, the blanks (100 l) and the 10 l and 50 l serum samples are deposited in the wells in a final volume of 100 l. The plate is incubated for 30 minutes at 37 C., and the rinsed with the washing solution. The secondary Ac solution diluted to 1:100 is added (100 l) in all the wells except in the blanks. The plate is then covered with parafilm paper and incubated for 20 minutes at 37 C. A TMB solution A and B is added after a washing step and the plate is incubated 15 mins at the temperature of the laboratory. For stopping the coloration reaction, 100 l of 2N H.sub.2SO.sub.4 are added per well. Readout of the plate is performed at 450 nm.

(67) The samples having an absorbance value equal to or greater than the threshold value are considered to be positive, the threshold value being determined according to the formula: Cutoff Value=NCx+0.100 with NCx=the average of the absorbance values of the two negative controls.

(68) Sero-Neutralization Test

(69) This technique is based on the capability of sero-neutralizing Acs (sero-N) of inhibiting infection of cells sensitive to the virus. For the detection and evaluation of the seric sero-N Acs, a constant amount of infectious viruses is put into contact with serial dilutions of the serum to be tested, and the mixture is then inoculated to a permissive cell culture on microplates and incubated for 3 to 5 days. The virus is most often a cytopathogenic strain, and therefore the absence or the reduction in number of ECPs expresses the presence of Acs in the tested serum.

(70) The viral stock SHIV.sub.KU2 is diluted to 1:1000 in RPMI (the supernatant volume of the virus is determined for 100 TCID.sub.50, which corresponds to the dilution of a virus for which 50% of the wells have syncytia) and the serums recovered from the NOD/SCID control mice, humanized and vaccinated with pCA-LTR-SHIV.sub.KU2-IN- or pSHIV.sub.KU2 are diluted in the same medium (at 10, 20, 40, 80, 160 and 320 dilutions). The diluted virus and the serum dilutions are mixed in a 96-well plate (100 l/well) and the mixture is incubated for 1 h at 4 C. The mixture is then deposited on M8166 cells (1.Math.10.sup.5 cells/well) cultivated beforehand in a 24-well plate. In this experiment, the positive control is represented by the virus stemming from pSHIV.sub.KU2 without any serum and the negative control corresponds to the cells alone.

(71) Method for Evaluating the Cell Response: Elispot Test

(72) This test has the purpose of detecting and evaluating the proportion of T lymphocytes (TL) which secretes IFN- as a response specific to antigenic stimulation.

(73) The mice are first deeply anesthetized and then the total blood and the spleen are taken. The spleens of the mice are put in RPMI medium in ice. The blood in dry tubes is used for isolating the serum. The splenocytes are isolated as a result of milling the spleen in a Petri dish between a pair of blades in the presence of PBS and 1% EDTA. The cells are washed twice in PBS/EDTA (centrifugation 2,000G, 5 min at 20 C.) in order to purify and to enrich them with splenocytes. After sowing the wells with the cells, the plate is washed with PBS and then incubated for 30 min with PBS+10% SCS at the temperature of the laboratory. The cells (5.Math.10.sup.5 splenocytes) are sown in each well, and are inoculated with pools of peptides of the Gag, Env, Tat, Rev and Nef proteins at a final concentration of 2 g/ml. Positive controls (CD3-2 included in the kit) and negative controls, are added to the test. The plate is covered with an aluminium foil pouch and incubated at 37 C. for 19 hours. The cells and peptides are washed, and then the biotinylated (7-b6-biotin) anti-IFN- monoclonal antibody is added, and the plate is covered and incubated for 2 h at room temperature. The streptavidin diluted in PBS containing 0.5% of FCS is added (100 l/well) and the plate is incubated for 1 h at room temperature. The TMB, a developer substrate, is added subsequently after another washing step, and the plate is then washed and dried after the emergence of blue spots. Readout of the plate is accomplished with the binocular magnifying glass at a magnification of 40.

(74) The positivity criteria of a well are determined for each condition by calculating the average of the number of spots of the duplicates, as well as the standard deviations. The number of spots is calculated for 1 million PBMCs and is normalized to 20% for the data obtained in NOD/SCID mice. The test is considered as being positive if the value of the average of the spots is greater than 10 spots per million of PBMCs which corresponds to the average of spots obtained with the control cultures.

(75) 1.6. Phenotype and Functional Examinations of the Specific T Cells of the Antigen by Flow Cytometry

(76) 1.6.1. Isolation of the Peripheral Mononuclear Cells

(77) The mononuclear cells of human peripheral blood are prepared as indicated above. The splenocytes of mouse spleens isolated according to the procedure described above are also re-suspended in the AIM V medium without any serum for cultivation and cytometry tests.

(78) 1.6.2. Antigenic Stimulation and Cultivation of the Cells

(79) In order to examine whether the specific cells of the antigen are capable of proliferating and of producing cytokines and lytic molecules, the splenocytes and the PBMCs are marked with CFSE (1 g/ml) for 10 min at 37 C., and the cells are then washed with PBS 1 for removing the excess. The marked cells are sown in deep wells of 96-well plates in an amount of 2.Math.10.sup.6/well in 1 ml of AIM V medium, and then stimulated with different pools of peptides (Gag, Env and Tat+Rev+Nef) in an amount of 2 g/ml in the presence of anti-CD49 and CD28 co-stimulation Acs. Cells without any peptides are used as a negative control and cells added with phytohemagglutinin (PHA) at 2 g/ml are added as a positive control. The cells are cultivated for 5 days (37 C. with humidity) and then re-stimulated with the same pools of peptides for 6 hours before marking them. The cells are harvested by centrifugation (2,000G, 5 min, 4 C.), re-suspended in 100 l of PBS and first marked with surface Acs (CD3, CD4 and CD8) [Pacific Blue anti-human CD3 (5 l), PE anti-human CD4 (10 l) and APC/Cy7 anti-human CD8 (10 l)] for 30 mins at room temperature. The cells are then centrifuged and washed with PBS 1 and then fixed and made permeable in 100 l of Cytofix Cytoperm of BD. The cells are then incubated for 20 minutes at 4 C. with anti-human IFN- PE-Cy7 and Alexa Fluor 647 anti-human Granzyme A Acs (5 l) for cytoplasm markings. The cells are finally washed with PBS and fixed with 4% PFA before acquisition and analysis with the flow cytometer.

(80) 1.6.3. Instrumentation

(81) An LSRII flow cytometer from BD connected to the BD FACSDiva6 software package was used. This instrument allows measurement of up to 13 fluorescence parameters and two physical parameters which are the FSC size (Forward Scatter) and the complexity or granulosity SSC (Side Scatter). The instrument is equipped with three lasers. The blue laser emitting at 488 nm may independently excite several fluorochromes (FITC, PE, PE-Cy7). The red laser which emits at 633 nm may excite the APC and APC-Cy7 fluorochromes and finally the violet laser which emits at 405 nm may excite the Pacific Blue fluorochrome.

(82) 1.7. Immunization of the Macaques

(83) A total of 12 cynomolgus macaques is used in the study. Six macaques form the control group and six other ones form the vaccinated group. The animals were immunized with a single double injection of DNA via the intramuscular route (4 mg/animal) and 1 mg/animal by electroporation (EP).

(84) The reasons for this immunization strategy with a single dose of the vaccine are multiple. One of the main reasons is not to perturb the generation, maturation and amplification of the memory T cells by the primary effector T cells associated with each re-immunization step.

(85) The immunized animals were subject to longitudinal follow up (once a week for 4 weeks and then 1 week out of 2 up to week 32, and then 1 week out of 4 for 10 months) for examining the immune responses induced by the vaccine. The blood samples taken on weeks 2 and 1 before immunization were used for examining possible basal responses. The mononuclear cells of peripheral blood (PBMCs) are isolated and used for evaluating the responses of the T cells with the ELISPOT IFN- test, by surface and intracytoplasm markings, and analysis by flow cytometry.

(86) 2. Results

(87) 2.1. Qualitative and Quantitative Checking of the Construction of the pCA-LTR-SHIV.sub.KU2-IN- Plasmid

(88) 2.1.1. Presence of the pCA-LTR-SHIV.sub.KU2-IN- Plasmid

(89) When the plasmid DNA was isolated from a polyclonal bacterial culture obtained from a preculture in a BL medium used for sowing a large volume of BL liquid medium, a significant proportion of episome is observed around 2000 bp. The electrophoretic profile also shows the presence of a single DNA band around 14,000 bp (which theoretically is 13,739 bp) corresponding to the plasmid.

(90) When the DNA is isolated from a bacterial culture first produced on a Petri dish in order to obtain isolated colonies, these colonies having been used for preculture and bulk culture used for isolating and purifying the plasmid DNA, the electrophoretic profile obtained after separation of 0.5 g of DNA shows the absence of any episome and shows three bands of high molecular weight DNA corresponding to the circular, wound and superwound forms of the pCA-LTR-SHIV.sub.KU2-IN- plasmid. The purity and the quantitative evaluation of the plasmid DNAs of our two preparations were checked by spectrophotometry. The values of the measurements of absorbents at wavelengths 230, 260 and 280 nm were used for determining the 260/280 ratios which were 1.75 and 1.82 and at 260/230 of 2.04 and 1.92 respectively, show satisfactory quality of our DNA. The DNA concentrations are 545 g/ml and 765 g/ml, respectively.

(91) 2.1.2. Enzymatic Digestion of pCA-LTR-SHIV.sub.KU2-IN-

(92) The profile of the digestion of the plasmid with EcoR1 reveals the presence of two bands of about 5,000 and 7,500 bp from cuts at both EcoRI sites, three bands with Bam H1 of 2400 bp, 4,900 bp and 7,400 bp resulting from cuts at both Bam H1 sites, and a band with Sph1 located at 10,000 bp resulting from the cut at the single Sph1 site. An additional band of 2,400 bp is also observed with the BamH1 digestion and it would result from the episome.

(93) 2.2. Evaluation of the Functionality: Effect of the Plasmid DNA on the Cells

(94) The HEK293T cells were transfected with a control plasmid pCG-GFP expressing GFP, the plasmid pSHIV.sub.KU2, and then with the plasmid pCA-LTR-SHIV.sub.KU2-IN-. The cells transfected with the plasmid pCG-GFP gave the possibility of estimating the efficiency of transfection by evaluating the number of GFP+ cells.

(95) In order to check that the HEK293T cells transfected with pCA-LTR-SHIV.sub.KU2-IN- or pSHIV.sub.KU2 produce virions which induce typical syncytia, these cells were co-cultivated with CEMx174, and the occurrence of the ECPs was then followed by observation under the microscope. Both co-cultures produced characteristic ECPs.

(96) In order to check whether the SHIV.sub.KU2 virus produced by the transfected cells replicates several times, the supernatant of the transfected cells was used for infecting the M8166 human CD4+ T lymphocyte line. The results obtained with the SHIV.sub.KU2, clearly show that it infects and induces ECPs especially with the highly permissive M8166 cell line. These ECPs appear as soon as after 48 hours but also at belated stages.

(97) Next, in order to check that the DNA of the vaccinal vector pCA-LTR-SHIV.sub.KU2-IN- only allows a single replication cycle (as it is deleted from the in gene), the recovered supernatant containing the CA-LTR-SHIV.sub.KU2-IN- virus was inoculated a first time, and then a second time to M8166 cells being cultivated. The first inoculation produced ECPs from 48 hours onwards and at the end of 76 hours they were more numerous. The supernatant of these infected cells was recovered and inoculated once again to M8166. Unlike the previous inoculation, no ECP is visible either 48 or at 76 hours after inoculation. These results allow the conclusion to be drawn that pCA-LTR-SHIV.sub.KU2-IN- gives the possibility of transducting only once the target cells being cultivated.

(98) The presence of typical ECPs, suggest that the plasmid DNA is replicated in the HEK293T cells and has produced CA-LTR-SHIV.sub.KU2-IN- virions. The ECPs of the first infection show the infectivity of the M8166 human CD4+ TL line by the produced particles, and their absence during the second infection shows the productive replication deficit in the cells.

(99) 2.3. Electron Microscopy Analysis of the Morphogenesis of Viral Particles in HEK293T Cells Transfected with pCA-LTR-SHIV.sub.KU2-IN-

(100) It was next determined whether the proteins produced by the vaccinal pCA-LTR-SHIV.sub.KU2-IN- genome properly assembled into viral particles in the HEK293T cells. The morphology of the HEK293T cells transfected with the plasmid pCA-LTR-SHIV.sub.KU2-IN- was examined by EM.

(101) The results showed that the viral particles are present at the surface of the cells in the form of buds and mature viral particles which have detached from the cells. Thus, the viral proteins of the pCA-LTR-SHIV.sub.KU2-IN- vaccine assemble in order to give viral particles which bud on the outside of the cells.

(102) 2.4. In Vivo Test of the Vaccine on Humanized SCID Mice and on BALB/c Mice

(103) 2.4.1. Evaluation of the Humoral Response in SCID-Hu Mice Vaccinated with pCA-LTR-SHIV.sub.KU2-IN-

(104) The serum samples of the immunized mice, taken at about 1 month after immunization are examined for the presence of Acs which specifically bind to the antigens of the virus by means of a commercial ELISA test. The results of this analysis are summarized in Table 1 (below). These results show that about half of the samples from the mice immunized with vaccinal pCA-LTR-SHIV.sub.KU2-IN- DNA, like those immunized with DNA of SHIV.sub.KU2, have lesser proportions of positives (44% and 37%, respectively) unlike those from the mice immunized with p4SHIV.sub.KU2 (50%). These results demonstrate the capability of the vaccinal pCA-LTR-SHIV.sub.KU2-IN- DNA of inducing humoral responses in NOD/SCID-hu mice. One sample out of three of serum of the immunized mice with p4SHIV.sub.KU2, has its OD value greater than those obtained with the samples of serums of immunized mice with pCA-LTR-SHIV.sub.KU2-IN- and pSHIV.sub.KU2. This plasmid is non replicative, the proteins of the virus remain associated with the membrane of the transfected cells and therefore should induce less Acs.

(105) TABLE-US-00001 TABLE 1 Detection of antibodies in samples of sera of control SCID, SCID-hu and SCID- hu mice, immunized with p4SHIV.sub.KU2, pCA-LTR-SHIV.sub.KU2-IN- and pSHIV.sub.KU2 and of the controls. Samples CA-LTR SHIV.sub.KU2- Positive Negative 4SHIV IN- SHIV controls controls controls Blanks Number of 6 16 19 2 2 2 2 samples Positivity 3 7 7 2 2 2 2 number Absorbance 0.3-1.823 0.26-0.9 0.34-1.64 0.01-0.19 1.9-2.2 0.15-0.16 0.01-0.03 positivity Interval
2.4.2. Analysis of the Neutralizing Activity by Sero-Neutralization

(106) The serums of SCID-hu mice vaccinated with the selected plasmids pCA-LTR-SHIV.sub.KU2-IN- or SHIV.sub.KU2 are those for which the OD was found positive by ELISA. Because of the small amount of serum, certain samples with a strong OD value for the ELISA test, were not able to be examined by sero-neutralization. A serum sample of SCID-hu mice and vaccinated with pCA-LTR-SHIV.sub.KU2-IN- found to be negative was also used for the ELISA test for ensuring the reliability of the test.

(107) TABLE-US-00002 TABLE 2 Analysis of the neutralizing activity of the sera of immunized mice. Dilutions ( 1/10, 1/20, . . . 1/320) were mixed with the virus SHIV.sub.KU2 (100 TCID.sub.50), incubated and then used for inoculating the M8166 cells. After 5 days after inoculation, the induced EPCs are listed and used for evaluating the sero-neutralizing activity. Caption: +++ correspond to strong neutralization, ++ quite high neutralization, + less neutralization, no neutralization action. For the CA-LTR-SHIV.sub.KU2 etc, two types of samples are represented, a type of sample having a less neutralizing profile as compared with the two other samples. Samples CA-LTR-SHIV.sub.KU2-IN- (3 samples) SHIV (1 sample) Dilutions Neutralization ECP interval Neutralization ECP interval 10 +++/+++ 5-9 +++ 4 20 ++/++ 14-19 ++ 12 40 +/ 25-44 ++ 20 80 +/ 27-49 ++ 15 160 +/ 31-57 + 26 320 +/ 28-69 44

(108) The number of ECPs obtained is higher in the M8166 cells infected by the SHIV.sub.KU2 virus incubated without any serum (76 ECPs) or with the serum of the non-immunized mice (42 ECPs) (negative control), which allowed us to set the negativity threshold of viral neutralization to 42 ECPs (data not shown in the table). On the other hand, the number of ECPs becomes quasi zero when the virus is incubated with 1/10 diluted serum of mice immunized with the DNA of CA-LTR-SHIV.sub.KU2-IN- or SHIV.sub.KU2. This number increases gradually as increases the dilution of the serums indicating a dose effect. For example, with the serum of a mouse immunized with pCA-LTR-SHIV.sub.KU2-IN- at 1/10 dilution, 5 ECPs are obtained, whereas at 1/320 dilution, a value of 69 ECPs is obtained, a value similar to the one of the control without any serum.

(109) 2.4.3. Evaluation of the Immunogenicity of pCA-LTR-SHIV.sub.KU2-IN- in Vaccinated BALB/c Mice

(110) In order to study the immunogenicity of the pCA-LTR-SHIV.sub.KU2-IN- vaccine in BALB/c mice, the animals were injected with a single dose of 100 g of DNA via an intramuscular route. The proportion of specific spleen cells (splenocytes) of the antigens was examined by ELISPOT. The results of this study actually show the capability of the DNAs used of inducing specific immune responses directed against all the studied antigens (FIG. 12). The T cell responses with the plasmid p4SHIV.sub.KU2 are twice higher than with the plasmid pCA-LTR-SHIV.sub.KU2-IN-. This small difference in efficiency between both DNAs may be related to better quality of the DNA of p4SHIV.sub.KU2 than that of the pCA-LTRSHIV.sub.KU2-IN-. These results nevertheless give the possibility of determining the base level of the immune responses induced by these vaccines in normal mice and give the possibility of performing a comparison with the immune responses obtained in the SCID-hu mouse.

(111) 2.4.4. Evaluation of the Cell Response in NOD/SCID-Hu Mice Immunized with the Different DNAs

(112) The NOD/SCID-hu mice were immunized by intramuscular injection with a single dose of 50 g of DNA of CA-LTR-SHIV.sub.KU2-IN-, 4SHIV.sub.KU2 or SHIV.sub.KU2, and the splenocytes were then used for examining the immune response by ELISPOT for evaluating the proportion of specific cells of the antigen and producing IFN-. As shown in FIG. 13, there is a significant number of T cells producing human INF- in response to stimulation by the Gag, Env or Tat+Rev+Nef antigens in the form of SIV or HIV peptides. It is interesting to note that the responses obtained after immunization with pCA-LTR-SHIV.sub.KU2-IN- are quasi-similar to those obtained with pSHIV.sub.KU2 and which are both clearly greater than those obtained after immunization with p4SHIV.sub.KU2. The predominancy of the responses induced by pCA-LTR-SHIV.sub.KU2 is against the Gag and Tat+Rev+Nef antigens.

(113) The whole of these results demonstrates that the DNA of pCA-LTR-SHIV.sub.KU2 is highly immunogenic in NOD/SCID-hu mice and that it preferentially induces responses against the antigens known for being associated with the protection against pathogenic viruses.

(114) 2.5. Phenotype and Functional Examinations of Specific T Cells of the Antigen by Flow Cytometry

(115) 2.5.1. Monomarking Carried Out on Day Zero (Performed on the Day when the Mouse Spleens were Recovered)

(116) These mono-markings are used in order to examine whether the selected antibodies actually detect those targets on the one hand, and for evaluating the presence and the proportion of human cells in spleens of SCID-hu mice on the other hand.

(117) The non-marked lymphocytes of humanized and non-humanized mice were analyzed in flow cytometry for measuring the fluorescence in the basal state of the cells (negative control).

(118) The detection of CD3+ TLs is carried out with human anti-CD3 Acs coupled with the fluorochrome Pacific Blue. These cells form a peak at 10.sup.2 in the profile of cells isolated in SCID-hu cells vaccinated with the plasmid pCA-LTR-SHIV.sub.KU2-IN-. This peak is not present in the cells recovered in SCID-non-hu mice.

(119) No significant peak was observed for cells mono-marked with anti-CD4+ Ac (CD4 anti-human PE), whether this is for the control or for our tested sample. This would be due to the anti-CD4 Ac used which would be non-functional.

(120) For detecting CD8+ TLs, a human anti-CD8 monoclonal Ac coupled with the fluorochrome APC/Cy7 was used. It allows detection of a peak located between 10.sup.2 and 10.sup.3 in the profile of the cells isolated in SCID-hu mice vaccinated with pCA-LTR-SHIV.sub.KU2-IN- and not in SCID-non-hu mice.

(121) The proportion of lymphocytes producing GRA molecules was not able to be evaluated because no peak difference was observed on the cells from immunized and non-immunized mice marked with anti-GRA Ac coupled with Alexa Fluor 647.

(122) On the other hand, the cells marked with human anti-IFN- Ac coupled with the fluorochrome PE-Cy7 showed a clear peak located at 10.sup.2 with cells of SCID-hu mice vaccinated with pCA-LTR-SHIV.sub.KU2-IN- which is absent with cells of non-immunized SCID-non-hu mice.

(123) 2.5.2. Phenotyping and Functions of the T Cells in Immunized Animals

(124) In order to examine the phenotype and the functions of the specific T cells of the antigens of the virus, the cells marked with CFSE are incubated for five days with the (Gag, Env and Tat+Rev+Nef) peptides and then re-stimulated for 6 hours with the same peptides. The cells are then marked with the Acs and examined as indicated above.

(125) The results of this analysis show the presence of CD3+ and CD8+ cells which produce IFN- and which have GRA molecules especially with the cells from vaccinated NOD/SCID-hu mice. Indeed, at least 6% of the CD8+ TLs produce IFN- and 1.7% produce GRA A in immunized NOD/SCID-hu mice, versus only 2.5% and 0.6% respectively in control mice. These results demonstrate the presence of CD3+, CD8+ activated cells which produce GRA and IFN-, which corresponds to an effector cellular immune response induced by our vaccine pCA-LTR-SHIV.sub.KU2-IN-.

(126) 2.6. Analysis of the Immune and Humoral Responses in Macaques

(127) 2.6.1. Analysis of the Immune Responses of T Cells by the ELISPOT IFN- Test

(128) A fraction of the mononuclear cells isolated from blood samples taken was used for evaluating the proportion of cells secreting IFN- in response to stimulation by the viral Gag, Pol, Env and Tat+Rev+Nef antigens by means of a commercial kit. The results of the first 20 weeks of the analysis are shown in table 3. They clearly show that following a single administration of the vaccinal vector CAL-SHIV-IN-, all the animals developed cellular immune responses characterized by specific cells of the antigens, which secrete IFN-. These responses are heterogenous according to the animals which have CMH-1 haplotypes different from each other. The immune responses are characterized by the presence of a first primary response peak at 2-4 weeks post-immunization (PI), and then of more belated responses from 8-10 weeks PI, and this in the absence of a second immunization. It is of great interest to note that the intensity of the second peak is often much greater than that of the first peak especially for the BX80 animal where the number of cells secreting IFN- is multiplied by 3.

(129) TABLE-US-00003 TABLE 3 Summary of the proportions of T cells secreting gamma interferon cytokine (IFN-) in response to the stimulation by the viral antigens (Gag, Pol, Env and Tat + Rev + Nef) expressed by the vaccine in vaccinated monkeys. The figures correspond to the numbers of secreting cells forming one spot per million (10.sup.6) of mononuclear cells of peripheral blood (PBMC). The weeks of analysis are indicated at the top of the table. Post-Immunization week 1 2 3 4 6 8 10 12 14 16 18 20 Animal Ag Number of spots/million PBMCs in response to the antigens (Ag) BX80 Gag 208 821 666 472 317 504 538 474 226 574 621 862 Pol 0 105 17 350 15 32 0 97 27 11 11 168 Env 0 84 1 7 124 625 753 67 36 41 48 132 TRN 0 64 0 107 137 1238 1663 1084 728 1215 800 1752 BX83 Gag 0 565 1033 443 147 0 488 377 69 152 96 298 Pol 0 33 75 193 0 0 340 145 19 0 0 12 Env 0 87 49 72 0 0 1035 60 40 25 9 20 TRN 0 51 204 116 0 0 731 393 195 224 143 432 BX84 Gag 0 1279 703 752 300 380 161 427 155 461 91 1269 Pol 1 88 36 24 15 5 8 80 0 19 9 44 Env 1 340 85 105 48 57 21 72 13 96 57 217 TRN 0 205 73 128 27 416 417 761 59 272 79 228 BX72 Gag 16 388 1989 1220 813 885 840 1795 0 85 472 1344 Pol 0 205 17 45 8 0 13 0 0 0 7 120 Env 7 79 57 56 12 168 56 5 0 0 12 132 TRN 5 35 161 192 92 692 445 736 0 25 119 587 BX78 Gag 8 668 163 213 45 52 76 555 24 83 80 227 Pol 0 548 60 20 0 31 0 104 132 1 0 59 Env 0 491 68 97 437 181 0 259 24 15 19 73 TRN 3 296 279 39 0 84 824 685 17 69 39 275
2.6.2. Analysis of the Immune Responses of T Cells by Multiparameter Flow Cytometry in Vaccinated Monkeys

(130) The results of the analysis by multiparameter flow cytometry will confirm those obtained by ELISPOT by revealing that all the animals developed a response consisting of T cells which proliferate and which are specific of all the antigens expressed by the vaccinal vector (Table 4). These responses are heterogenous among the animals and also reveal a first primary response phase which extends up to about 8 weeks PI, followed by a contraction phase (2-4 weeks) and then by a re-emergence phase. This longitudinal tracking of the immune response by multiparameter flow cytometry is continued until the virulent test with the test virus SIVmac251 which is conducted in week 52.

(131) TABLE-US-00004 TABLE 4 Summary of the values of the CD4+ and CD8+ proliferating T cells in response to the antigenic stimulations at the moment of the primary expansion phases, of the contraction and finally re- emergence or secondary expansion phases in vaccinated monkeys. The weeks corresponding to each of the phases are indicated. The numbers correspond to the percentages of specific T cells of each of the antigens which proliferate in response to the stimulation as compared with the total number of T cells. Proportion of T cells which proliferate in Animals Ag response to the antigens (Ag) Week Week Week Week Type (1) (3-6) (8-20) (22-26) BX80 Gag CD8+ 0.5 3.6 0.4 1.5 CD4+ 0.1 0.1 0.1 0.4 Pol CD8+ ND 0.6 0.2 0.7 CD4+ ND 0.7 0.1 0.6 Env CD8+ 0.3 0.5 0.1 1.4 CD4+ 0.5 1.3 0.2 0.5 TRN CD8+ 0 4.8 0.8 1.6 CD4+ 0.3 1.2 0.2 0.5 Week Week Week Week Type (1) (2) (8-20) (22-26) BX83 Gag CD8+ 0.2 1.7 0.5 1.6 CD4+ 0.1 0.6 0.4 0.9 Pol CD8+ ND ND 0.3 0.7 CD4+ ND 0.3 0.2 0.5 Env CD8+ 0.1 ND 0.5 1.2 CD4+ 0.3 ND 0.3 0.7 TRN CD8+ 0.4 1.5 0.6 2.3 CD4+ 0.2 0.8 0.3 0.5 Week Week Week Week Type (1) (3-6) (8-22) (24-26) BX84 Gag CD8+ 0.1 1.1 0.3 2.6 CD4+ 0.1 0.3 0.1 0.8 Pol CD8+ ND 0.3 0.2 0.7 CD4+ ND 0.3 0.2 0.5 Env CD8+ 0.1 1.1 0.3 0.9 CD4+ 0.2 0.8 0.3 0.7 TRN CD8+ 0 1.3 0.7 1.1 CD4+ 0.2 0.8 0.3 0.5 Week Week Week Week Type (1) (3-6) (8-20) (22-26) BX72 Gag CD8+ 0 4.2 0.7 3.0 CD4+ 0 0.6 0.2 0.4 Pol CD8+ ND 0.7 0.2 0.7 CD4+ ND 1.3 0.1 0.1 Env CD8+ 0.2 1.0 0.5 0.6 CD4+ 0.3 1.4 0.5 2.3 TRN CD8+ 0.1 0.9 0.3 1.5 CD4+ 0.1 1.7 0.4 0.9 Week Week Week Week Type (1) (3-6) (8-18) (20-32) BX78 Gag CD8+ 0 3.4 1.1 3.7 CD4+ 0.1 1.3 0.6 2.9 Pol CD8+ ND 1.1 0.3 1.1 CD4+ ND 0.6 0.2 1.3 Env CD8+ 0.2 1.4 0.4 1.2 CD4+ 0.3 0.6 0.6 2.5 TRN CD8+ 0.3 1.9 0.8 2.1 CD4+ 0.3 1.2 0.5 2.1
2.6.3. Analysis of the Humoral Response in Macaques

(132) Detection of the anti-antigen antibodies of SHIV was carried out with a commercial ELISA kit which allows detection of the anti-Env antibodies of the HIV-1. The longitudinal examination of the serums harvested at each blood sampling point showed the presence of anti-Env antibodies from week 20 PI in the BX80 animal and from week 8 for the BX73 animal. The presence of antibodies in positive sera was confirmed by Western blot against proteins of the SHIV showing a strong signal against the Gag-p27 protein as well as a signal against the gp160/gp120 glycoproteins.

(133) 2.6.4. Conclusion

(134) These results clearly demonstrate that a single injection of vaccinal DNA (CAL-SHIV-IN-) gives the possibility of inducing T cell and humoral (antibody) immune responses. The T cell responses are directed against all the viral antigens expressed by the vaccinal vector. They are persistent and follow a conventional expansion, contraction and storage-in-memory scheme. The presence of memory T cells of the central type and of memory effector cells was confirmed by the phenotyping.

(135) 2.7. Construction and Functional Analysis of a Novel Vector Expressing the Whole of the Antigens of the HIV-1: The Vector CAL-HIV-IN-

(136) From the genome of the vector CAL-SHIV.sub.KU2-IN-, the gag, pol, vif, vpx and vpr genes of the SIV were deleted following double digestion with the Nar1 and Sph1 enzymes, and the nef gene of the SIV was deleted following the partial digestion with the Nru1 enzyme and the total digestion with the Not 1 enzyme. The remaining fragment was then purified. This fragment, bearing the tat, rev and env genes of the HIV framed with the RTLs of the CAEV and transported by the plasmid pET, was used for introducing the 5 kb fragment bearing the gag, pol, vif, vpx and vpr genes of the HIV-1 and the 620 bp fragment bearing the nef gene of the HIV-1, and generating the vector CAL-HIV-IN- (FIG. 17). The CAL-HIV-IN- vector, deleted from the sequences coding for the integrase, consists in the sequence SEQ ID NO: 18.

(137) 2.7.1. Evaluation of the Functionality: Effect of Plasmid DNA on the Cells

(138) The DNA of this vector was introduced into the GHOST-CXCR4 and HEK-293 T cells by transfection using the ExGen and the procedure recommended by the manufacturer. The transfected GHOST-CXR4 cells had then become fluorescent confirming expression of the viral proteins of the HIV-1 by the vaccinal vector and more particularly the Tat protein which transactivates the expression of the GFP (Green Fluorescent Protein) gene under the control of the RTL of the HIV.

(139) The supernatant of the HEK-293T cells transfected with the vaccinal vector CAL-HIV-IN- was used for inoculating the M8166, indicating CD4+ T cells which developed cytopathic effects, characteristics of HIV infection. These results provide the proof that the proteins expressed by the vaccinal vector assembled into viral particles allowing infection of the M8166 indicator cells. These cells with the cytopathic effects did not produce any viruses capable of again infecting the M8166 indicator cells and inducing cytopathic effects. This result indicates that CAL-HIV-IN- is associated with a single replication cycle in the absence of integration.

(140) In order to evaluate the viral proteins produced after transfection, the supernatants of the transfected HEK-293T cells with the vaccinel vector, were harvested 24 h, 48 h and 72 h post-transfection and then examinated for the presence of Gag p24 antigens with ELISA. The measurements of the amounts of this protein are indicated in Table 5. They show increasing accumulation of this protein ranging from 100 ng/ml at 24 h up to 135 ng/ml at 72 h post-transfection.

(141) TABLE-US-00005 TABLE 5 Evaluation of the Gag p24 protein of the HIV-1 secreted in the supernatant of HEK 293T cells transfected with the vaccinal vector CAL-HIV-IN-. Quantification of the protein p24 accumulated in the supernatant of the HEK 293T cells after 24, 48 and 72 hours post-transfection with the DNA of the vector CAL-HIV-IN-. 24 h 48 h 72 h Gag p24 concentration 100 ng/ml 110 ng/ml 135 ng/ml
2.7.2. Immunization of BALB/C Mice and Characterization of the Induced Immune Responses

(142) Three groups of BALB/c mice (6 per group) of 6 weeks old were used: two groups were used for immunization and the third group is a control group. The two groups of immunized mice were injected with 100 g/mouse DNA of the vector CAL-HIV-IN- via the intramuscular route. The animals of a group were sacrificed after two weeks and the other ones at three weeks post-immunization (PI). The control mice were sacrificed at two weeks PI. The spleens of each of the mice were taken, the splenocytes were isolated and then used either for the ELISPOT test or for the analysis in multiparameter flow cytometry as described above. The results of the analysis by ELISPOT are indicated in Table 6. They show the presence of specific cells of all the antigens which secrete IFN-. The majority of these cells are specific of Gag and Tat+Rev+Nef antigens. The responses at 2 and 3 weeks post-immunization are substantially similar.

(143) TABLE-US-00006 TABLE 6 Summary of the results of the analysis by ELISPOT on the splenocytes of BABL/c mice immunized with the vaccinal vector CAL-HIV-IN- at 2 and 3 weeks post-immunization. The isolated splenocytes of the mouse spleens immunized with 100 g per mouse via an intramuscular route were examined with the ELISPOT IFN- test for evaluating the number of T cells secreting the cytokine in response to the stimulations with the pools of peptides (Gag, Env and Tat + Rev + Nef, TRN). The averages of the number of spots for each antigen and for the 6 examined mice after 2 and 3 weeks post-immunization are indicated. Medium without any peptide Gag Tat + Rev + Nef Env 2 weeks 10 45 50 17 3 weeks 5 35 55 15

(144) The results of the analysis by flow cytometry (Table 7) demonstrate specific CD4+ and CD8+ T cell immune responses of all the antigens expressed by the vaccinal vector CAL-HIV-IN-.

(145) TABLE-US-00007 TABLE 7 Summary of the results of the analysis by multiparameter flow cytometry. The figures correspond to the percentages of specific T cells of each of the antigens which proliferate in response to the antigenic stimulation, based on the total number of T cells. Medium without any peptide Gag Tat + Rev + Nef Env CD3 + CD4+ 0.16 0.26 0.40 0.18 CD3 + CD8+ 0.20 0.27 0.50 0.25