Recombinant measles viruses expressing epitopes of antigens of RNA viruses—use for the preparation of vaccine compositions

Abstract

The invention relates to a recombinant measles virus expressing a heterologous amino acid sequence derived from an antigen of a determined RNA virus, said recombinant measles virus being capable of eliciting a humoral and/or cellular immune response against measles virus or against said RNA virus or against both measles virus and against said RNA virus. It also relates to the use of said recombinant measles virus for the preparation of immunogenic composition.

Claims

1. An immunogenic composition comprising (i) a recombinant measles virus expressing a heterologous amino acid sequence, or a recombinant measles virus expression vector, and (ii) an acceptable vehicle, wherein said virus or vector comprises a sequence comprising: A) a nucleotide sequence encoding the full length antigenomic (+)RNA strand of a measles virus vaccine strain; B) a T7 promoter sequence comprising a GGG motif at its 3 end, operably linked to the nucleotide sequence of A; C) a hammerhead ribozyme sequence located adjacent to the GGG motif at one end and adjacent to the first nucleotide of the nucleotide sequence encoding the full length anti-genomic (+)RNA strand of the measles virus strain at the other end; D) a T7 terminator sequence operably linked to the nucleotide sequence of A; E) the sequence of a hepatitis delta virus ribozyme located adjacent to the last nucleotide of the nucleotide sequence encoding the full length anti-genomic (+)RNA strand of the measles virus; and F) a heterologous coding sequence encoding a heterologous amino acid sequence comprising an antigen of a heterologous RNA virus selected from a retrovirus and a flavivirus.

2. The composition according to claim 1, wherein the infectious recombinant measles virus is capable of eliciting a humoral immune response, a cellular immune response, or a humoral and cellular immune response against measles virus or against said heterologous RNA virus or against both measles virus and against said heterologous RNA virus.

3. The composition according to claim 1, wherein the infectious recombinant measles virus is a recombinant Schwarz strain and the full length anti-genomic (+)RNA strand has the sequence extending from position 83 to position 15976 of SEQ ID NO: 16.

4. The composition according to claim 1, wherein the expression vector comprises the nucleotide sequence of SEQ ID NO:16.

5. The composition according to claim 1, wherein the expression vector comprises the nucleotide sequence extending from nucleotide 29 to nucleotide 16202 of the sequence of SEQ ID NO:16.

6. The composition according to claim 1, wherein the expression vector comprises the nucleotide sequence extending from nucleotide 26 to nucleotide 16202 of the sequence of SEQ ID NO:16.

7. The composition according to claim 1, wherein the expression vector comprises the nucleotide sequence extending from nucleotide 9 to nucleotide 16202 of the sequence of SEQ ID NO:16.

8. The composition according to claim 1, wherein the heterologous coding sequence is cloned within the nucleotide sequence encoding the full length antigenomic (+)RNA strand of the measles virus Schwarz strain (from position 83 to position 15976 of SEQ ID NO: 16) at a position upstream of the N gene of the measles virus.

9. The composition according to claim 1, wherein the heterologous coding sequence is cloned between the P and M genes of the measles virus.

10. The composition according to claim 1, wherein the heterologous coding sequence is cloned between the H and L genes of the measles virus.

11. The composition according to claim 1, wherein the antigen of a heterologous RNA virus is a West Nile Virus antigen.

12. The composition according to claim 1, wherein the antigen of a heterologous RNA virus is a Yellow Fever Virus antigen.

13. The composition according to claim 12, wherein the Yellow Fever Virus antigen is selected from the Env antigen and the NS1 antigen.

14. The composition according to claim 1, wherein the antigen of a heterologous RNA virus is an HIV antigen.

15. The composition according to claim 1, wherein the HIV antigen is an envelope antigen of HIV-1 selected from gp120, gp140, and gp160.

16. The composition according to claim 1, wherein the HIV antigen is an HIV gag antigen (p17p24).

17. The composition according to claim 1, wherein the HIV antigen is HIV Tat antigen.

18. The composition according to claim 1, wherein the HIV antigen is selected from the group consisting of: A) gp160V3 (SEQ ID NO: 31), in which the V3 loop consisting of amino acids 299 to 334 of SEQ ID NO: 27 is replaced by the sequence AAELDKWASAA (SEQ ID NO: 8); B) gp160V1V2 (SEQ ID NO: 35), in which the V1 and V2 loops consisting of amino acids 131 to 159 and 164 to 198 of SEQ ID NO: 27, respectively, are deleted; C) gp160V1V2V3 (SEQ ID NO: 39) in which the V1 and V2 loops consisting of amino acids 131 to 159 and 164 to 198 of SEQ ID NO: 27, respectively, are deleted and the V3 loop consisting of amino acids 299 to 334 (SEQ ID NO: 27) is replaced by the sequence AAELDKWASAA (SEQ ID NO: 8); D) gp140V3 (SEQ ID NO: 29) in which the V3 loop consisting of amino acids 299 to 334 of SEQ ID NO: 25 is replaced by the sequence AAELDKWASAA (SEQ ID NO: 8); E) gp140V1V2 (SEQ ID NO: 33) in which the V1 and V2 loops consisting of amino acids 131 to 159 and 164 to 198 of SEQ ID NO: 25, respectively, are deleted; and F) gp140V1V2V3 (SEQ ID NO: 37) in which the V1 and V2 loops consisting of amino acids 131 to 159 and 164 to 198 of SEQ ID NO: 25, respectively, are deleted and the V3 loop consisting of amino acids 299 to 334 of SEQ ID NO: 25 is replaced by the sequence AAELDKWASAA (SEQ ID NO: 8).

19. The composition according to claim 1, wherein the heterologous amino acid sequence expressed from the recombinant measles virus expression vector is selected from the group consisting of: A) gp160V3 (SEQ ID NO: 31), in which the V3 loop consisting of amino acids 299 to 334 of SEQ ID NO: 27 is replaced by the sequence AAELDKWASAA (SEQ ID NO: 8); B) gp160V1V2 (SEQ ID NO: 35), in which the V1 and V2 loops consisting of amino acids 131 to 159 and 164 to 198 of SEQ ID NO: 27, respectively, are deleted; C) gp160V1V2V3 (SEQ ID NO: 39) in which the V1 and V2 loops consisting of amino acids 131 to 159 and 164 to 198 of SEQ ID NO: 27, respectively, are deleted and the V3 loop consisting of amino acids 299 to 334 (SEQ ID NO: 27) is replaced by the sequence AAELDKWASAA (SEQ ID NO: 8); D) gp140V3 (SEQ ID NO: 29) in which the V3 loop consisting of amino acids 299 to 334 of SEQ ID NO: 25 is replaced by the sequence AAELDKWASAA (SEQ ID NO: 8); E) gp140V1V2 (SEQ ID NO: 33) in which the V1 and V2 loops consisting of amino acids 131 to 159 and 164 to 198 of SEQ ID NO: 25, respectively, are deleted; and F) gp140V1V2V3 (SEQ ID NO: 37) in which the V1 and V2 loops consisting of amino acids 131 to 159 and 164 to 198 of SEQ ID NO: 25, respectively, are deleted and the V3 loop consisting of amino acids 299 to 334 of SEQ ID NO: 25 is replaced by the sequence AAELDKWASAA (SEQ ID NO: 8).

20. The composition according to claim 1, wherein the recombinant measles virus expression vector is selected from the following vectors deposited with the COLLECTION NATIONALE DE CULTURES DE MICROORGANISMES (CNCM): TABLE-US-00023 pTM-MVSchw2-GFPbis CNCM I-3034, pTM-MVSchw2-p17p24[delta]myr(HIVB) CNCM I-3035, pTM-MVSchw3-Tat(HIV89-6p) CNCM I-3036, pTM-MVschw3-GFP CNCM I-3037, pTM-MVSchw2-Es (YFV) CNCM I-3038, pTM-MVSchw2-gp140 [delta] V1 V2 V3(HIV89-6) CNCM I-3054, pTM-MVSchw2-gp140 [delta] V3(HIV89-6) CNCM I-3055, pTM-MVSchw2-gp160 [delta] V1 V2 V3(HIV89-6) CNCM I-3056, and pTM-MVSchw2-gpl 60 [delta] V1 V2(HIV89-6) CNCM I-3057.

21. The composition according to claim 1, wherein said composition is administered in one or multiple administration dose(s), in particular in a prime-boost administration regime.

22. The composition according to claim 1, wherein said composition is formulated for an administration through subcutaneous (sc) or intraperitoneal (ip) injection.

23. A method of inducing a protective immune response against a retrovirus or a flavivirus in a host, comprising administering the composition according to claim 1 to the host.

24. The method according to claim 23, wherein the flavivirus is West Nile Virus.

25. The method according to claim 23, wherein the flavivirus is Yellow Fever Virus.

26. The method according to claim 23, wherein the retrovirus is HIV.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIGS. 1A and 1B. HIV1 Env glycoprotein constructions. (A) gp160 constructions full-length and V3-AAELDKWASAA (SEQ ID NO: 8), V1V2 and V1V2V3 (SEQ ID NO: 8) mutants (from top to bottom). The BbsI and MfeI restriction sites used to introduce the V3 deletion in the other constructions are indicated. (B) gp140 constructions are the same as gp160 except that the intracytoplasmic and transmembrane regions of the gp41 have been deleted (AAELDKWASAA disclosed as SEQ ID NO: 8).

(2) FIG. 2A. Schematic map of the pTM-MV Schw plasmid. To construct the complete sequence, the six fragments represented in the upper part were generated and recombined step by step using the unique restriction sites indicated. T7=T7 promoter; hh=hammerhead ribozyme; hAv=hepatitis delta ribozyme (=); T7t=T7 RNA polymerase terminator.

(3) FIG. 2B. The pMV(+) vectors with ATU containing a green fluorescent protein (GFP) gene in position 2 and position 3. The MV genes are indicated: N (nucleoprotein), PVC (phosphoprotein and V C proteins), M (matrix), F (fusion), H (hemaglutinin), L (polymerase). T7: T7 RNA polymerase promoter; T7t: T7 RNA polymerase terminator; : hepatitis delta virus (HDV) ribozyme; ATU: additional transcription unit.

(4) ATU sequence: small letters represent additional sequences (copy of the N-P intergenic region of measles virus) plus cloning sites. Capital letters correspond to the inserted enhanced GFP sequence. This sequence is inserted at the SpeI site (position 3373) of the cDNA sequence of the Schwarz strain of the measles virus for ATU2 and at the SpeI site (position 9174) for the ATU3. The mutation which distinguishes normal ATU from bis (in pTM-MVSchw2-gfp and pTM-MVSchw2-GFPbis) is a substituted C (Capital letter) at the end of ATU.

(5) FIG. 3A: shows that ENV.sub.HIV89.6 expression was similar for passages 2 and 5, confirming the stability of expression of transgenes in this system.

(6) FIG. 3B: Construct of double recombinant pTM-MVSchw2-Gag-3gp140

(7) Some recombinant vectors expressing two different heterologous antigens have been constructed. They were obtained by ligation of two different recombinant pTM-MVSchw plasmids containing different inserts in position 2 and position 3. Plasmid pTM-MVSchw2-Gag-3-gp140 is shown. From this plasmid a recombinant virus was rescued that expressed both Gag and gp140 proteins (FIG. 3B(2) Western blot). Using appropriate constructions of the different inserted heterologous genes, such recombinant MV expressing two heterologous viral proteins may produce <<virus like particles>> (VLP) assembled in infected cells and secreted: Gag-Env from retroviruses or prM/E from flaviviruses. Such VLP are good immunogens. Produced in vivo by a live attenuated vaccine like MV, they should be even more immunogenic.

(8) FIG. 3C: Expression of HIV-1 envelope glycoproteins in recombinant pTM-MVSchw. Vero cells were infected with the different recombinant viruses for 48H and expression of HIV Env was determined by western blot. 30 g of each cell lysate were resolved on 4-12% SDS-PAGE, blotted onto nitrocellulose membranes and probed with a mouse monoclonal anti-HIV gp120 (Chessie, NIH) antibody. Anti-mouse IgG RPO conjugate was used as second antibody and proteins were detected using an ECL detection kit.

(9) FIG. 3D: Expression of HIV-1 gp140 and SIV239 Gag in recombinant pTM-MVSchw2-Gagsv (p17-p24 [delta] myr)-3-gp140.sub.HIV. HIV gp140 and SIV Gag were detected in lysates of infected Vero cells. (A) a mouse monoclonal anti-HIV gp120 and (B) serum from macaque infected with SlVmac251.

(10) FIG. 3E: Expression of HIV-1 Gag (p17-p24 myr) in recombinant pTM-MVSchw2-Gag.sub.HIV (p17-p24 [delta] myr). HIV Gag were detected in lysates of infected Vero cells with a mouse monoclonal anti-HIV Gag antibody.

(11) FIG. 3F: Expression of HIV-1 Tat protein in recombinant pTM-MVSchw. Vero cells were infected with MVSchw-Tat HIV recombinant or control MVSchw viruses for 48H and expression of HIV Tat was determined by western blot. 30 g of each cell lysate were resolved on 4-12% SDS-PAGE, blotted onto nitrocellulose membranes and probed with a mouse monoclonal anti-HIV Tat (BH10, NIH) antibody. Anti-mouse IgG RPO conjugate was used as second antibody and proteins were detected using an ECL detection kit.

(12) FIGS. 4A and 4B. Growth kinetics of recombinant MV.sub.EdB-En.sub.HIV viruses on Vero cells. Cells on 35 mm dishes were infected with recombinant viruses at different MOI (as indicated). At each time point, cells were collected and cell-associated virus titers were determined using the TCID.sub.50 method on Vero cells. (A) Infections with MV EdB-tag and different MV-HIV recombinant viruses at MOI=0.0001. (B) Infections with MV2-gp160.sub.HIV at two different MOI (0.0001 and 0.01).

(13) FIGS. 5A through 5F. Anti-HIV and anti-MV humoral immune responses in mice inoculated with recombinant MV.sub.EdB-Env.sub.HIV viruses. A-B Four groups of 3 mice were immunized with 10.sup.7 TCID.sub.50 of each MV-HIV recombinant virus. Antibody titers against MV (A) and HIV Env (B) were determined by ELISA in sera collected 28 days post inoculation. C-F: Anti-HIV and anti-MV antibody titers in IFNAR.sup.//CD46.sup.+/ mice immunized with MV-Env.sub.HIV viruses. (C) Anti-MV and anti-HIV titers detected 28 days after injection of increasing doses of MV.sub.EdB-gp160 (3 mice per group). (D) Anti-MV (black bars), anti-HIV (gray bars) and anti-ELDKWAS (Residues 3-9 of SEQ ID NO: 8; white bars) titers detected 28 days after injection of 5 106 TCID.sub.50 of MV-Env.sub.HIV viruses (6 mice per group). Results are expressed as the mean valuesSD.

(14) FIG. 6. Neutralizing activities against Bx08 of sera from mice immunized with MV2-gp140.sub.HIV89.6 and MV2-gp160.sub.HIV89.6 viruses. Primary isolate Bx08 was provided by C.Moog (Strasbourg, France) and propagated once on PHA-stimulated PBMC to obtain viral stocks. 2 ng of virus was incubated for 30 min at 37 C. with 25 l of each mouse serum (collected one month post-infection) before infection of P4R5 cells in a 96-well plate. Cells were then cultured in DMEM containing 10% of fetal calf serum until 2 days post-infection, at which time Galactosidase activity was measured with a chemiluminescence test (Roche, Germany). Lane 1: serum of a MV.sub.EdB-Tag immunized mouse; Lane 2: serum of a MV2-gp140.sub.HIV-1 immunized mouse; Lane 3: serum of a MV2-gp160.sub.HIV-1 immunized mouse; Lane 4: non-infected cells. All assays were performed in triplicate.

(15) FIGS. 7A through 7D. Edm-HIV Env vaccine candidate stimulates env-specific lymphocytes in vivo. Two groups of 3 mice were inoculated with 10.sup.7 TCID.sub.50 of MV2-gp160.sub.HIV virus, and euthanized 7 day and one 1 month post inoculation. (A) ELISpot assays performed with splenocytes from immunized mice. Stimulation with HIV-gp120 purified protein (black) or irrelevant BSA (white). (B) Splenocytes collected 7 days after immunization were stimulated either with medium alone (left panel), HIV gp120 (middle panel) or EdB-tag virus (right panel). Three-color cytofluorometry detected both CD8+(upper panel) and CD4+(lower panel) lymphocytes producing -IFN after HIV gp120 and measles virus stimulations. Percentages are given according to the total CD8+(upper panel) and CD4+(lower panel) lymphocyte gates respectively. (C and D). Anti-MV and anti-HIV antibody titers in mice and macaques immunized with MV2-gp140HIV89.6 virus months after MV priming. (C) Mice (3 per group) were vaccinated with 10.sup.5 TCID.sub.50 of EdB-tag MV then inoculated twice with 5 10.sup.6 TCID.sub.50 of MV2-gp140.sub.HIV89.6 virus as indicated (arrows). (D) Cynomolgus macaques (#432 and 404) were vaccinated with ROUVAX then inoculated twice with 5 10.sup.6 TCID.sub.50 of MV2(gp140.sub.HIV89.6 virus as indicated (arrows).

(16) FIGS. 8A and 8B. Schematic representation the additional transcription unit (ATU) (Residues 1817-1843 of SEQ ID NO: 16 and 3475-3498 of SEQ ID NO: 16, respectively, in order of appearance) and Schwarz MV vector plasmid. (A) Cis-acting elements of the ATU inserted in position 2 between phosphoprotein (P) and matrix (M) MV open reading frames. (B). Representation of the three positions of ATU insertion in the Schwarz MV vector plasmid.

(17) FIG. 9. Expression of YFV proteins by recombinant MV. Vero cells were infected by recombinant EdB-Env.sub.YFV and EdB-NS1.sub.YFV MV at an MOI of 0.01. Immunofluorescence was performed using a mouse polyclonal anti-YFV serum and a Cy3 secondary anti-mouse IgG antibody. All the syncytia observed in infected Vero cells were positive.

(18) FIG. 10. YFV challenge. Six 4-weeks old mice were inoculated with a mixture of EdB-Env.sub.YFV and EdB-NS1.sub.YFV viruses (10.sup.7 TCID.sub.50) and 6 control mice were inoculated with the same dose of standard EdB-tag virus. After 1 month, anti-MV serologies were determined and a similar level of antibodies was observed in the two groups. Mice were challenged and mortality was observed.

(19) FIGS. 11A through 11E. Complete nucleotide sequence of the pTM-MVSchw plasmid (CNCM I-2889; SEQ ID NO: 16). The sequence can be described as follows with reference to the position of the nucleotides:

(20) TABLE-US-00004 1-8 NotI restriction site 9-28 T7 promoter 29-82 Hammer head ribozyme 83-15976 MV Schwarz antigenome 15977-16202 HDV ribozyme and T7 terminator 16203-16210 NotI restriction site 16211-16216 ApaI restriction site 16220-16226 KpnI restriction site 16226-18967 pBluescript KS(+) plasmid (Stratagene)

(21) FIGS. 12A and 12B. (SEQ ID NO: 17):

(22) The flaviral sequences which have been expressed in MV are the following:

(23) YFV Env seq: This is the Env YFV 17D204 sequence cloned by the inventors.

(24) TABLE-US-00005 pos 1 3 START codon pos 4 51 Env signal peptide pos 52 1455 Env sequence pos 1456 1458 STOP codon

(25) The stop and start codons have been added.

(26) YFV NS1 seq: This is the NS1 YFV 17D204 sequence cloned by the inventors.

(27) TABLE-US-00006 pos 1 3 START codon pos 4 78 NS1 signal peptide pos 79 1110 NS1 sequence pos 1111 1113 STOP codon

(28) The stop and start codons have been added.

(29) FIG. 12C: WNV Env seq (SEQ ID NO: 18): this is the Env WNV sequence cloned by the inventors.

(30) TABLE-US-00007 pos 1 3 START codon pos 4 51 env signal peptide pos 52 1485 Env sequence pos 1486 1488 STOP codon

(31) The stop and start codons have been added.

(32) FIG. 12D: WNV NS1 seq (SEQ ID NO: 19): This is the NS1 WNV sequence cloned by the inventors.

(33) TABLE-US-00008 pos 1 3 START codon pos 4 78 NS1 signal peptide pos 79 1104 NS1 sequence pos 1105 1107 STOP codon pos 1108 1110 STOP codon (a second is added in order to respect the rule six.)

(34) The stop and start codons have been added.

(35) FIG. 13: Schematic representation of recombinant pTM-MVSchw-sE.sub.WNV. The MV genes are indicated: N (nucleoprotein), PVC (phosphoprotein and V, C proteins), M (matrix), F (fusion), H (hemmaglutinin), L (polymerase). T7: T7 RNA polymerase promoter; T7t: T7 RNA polymerase terminator; hepatitis delta virus (HDV) ribozyme; ATU: additional transcription unit.

(36) After rescue, the recombinant virus was grown on Vero cell monolayers. The procedure used to prepare the recombinant virus was similar to the standard procedures used to prepare the live attenuated measles vaccines, except for the lyophilization that was not used.

(37) The WNV sE expression in Vero cells infected by the MV-WN sE virus was verified by using indirect immunofluorescence assay as shown in FIG. 14.

(38) FIGS. 14A through 14D: Expression of sE protein from WNV in MV induced syncytia. Immunofluorescence detection of secreted WNV Env (sE) protein in syncytia induced by recombinant MV-WN sE in Vero cells. (A, B) sE protein detected at the external surface all around recombinant MV-induced syncytia. (C, D) intracellular sE protein in recombinant MV-induced syncytia.

(39) FIG. 15: Anti-MV serology 1 month after the first injection.

(40) FIGS. 16A through 16T: HIV-1 immunogenic sequences prepared for insertion in plasmid pTM-MVSchw2 illustrated in Example II (SEQ ID NOS: 24-43, respectively, in order of appearance).

EXAMPLE I: RECOMBINANT MEASLES VIRUSES EXPRESSING THE NATIVE ENVELOPE GLYCOPROTEIN OF HIV1 CLADE B, OR ENVELOPES WITH DELETED VARIABLE LOOPS, INDUCE HUMORAL AND CELLULAR IMMUNE RESPONSES

(41) Preparing a vaccine against HIV with its formidable ability at evading the host immune responses is certainly a daunting task. However, what we have learned about the immunopathogenesis of the infection and results already obtained with animal models indicate that it may be possible (Mascola, J. R., and G. J. Nabel. 2001. Vaccines for prevention of HIV-1 disease. Immunology. 13:489-495). Ideally, a preventive immunization should induce 1) antibodies that neutralize primary isolates, thereby preventing entry into host cells, and 2) CTL that eliminate the cells that were nevertheless infected. Antibodies and CTL should be directed at conserved epitopes that are critical for viral entry and replication into host cells.

(42) Several studies, in particular with various candidate vaccines, show that a good cellular immune response might be able to control viral load, although not to eliminate the agent (Mascola, J. R., and G. J. Nabel. 2001. Vaccines for prevention of HIV-1 disease. Immunology. 13:489-495). On the other hand humoral immune responses induced so far by subunit vaccines have been disappointing, mainly because the antibodies induced did not neutralize primary isolates of HIV. For example, recombinant vaccines expressing the SIV Env were able to protect macaques against an homologous, but not an heterologous, challenge (Hu, S., et al 1996. Recombinant subunit vaccines as an approach to study correlates of protection against primate lentivirus infection. Immunology Letters. 51:115-119). DNA immunization combined with boosting with soluble recombinant gp could protect macaques against an heterologous challenge but only against a strain of SIV genetically related to the vaccine (Boyer, J. et al 1997. Protection of chimpanzees from high-dose heterologous HIV-1 challenge by DNA vaccination. Nature Medicine. 3:526-532). More recently, various <<prime-boost>> regimen, using combinations of naked DNA and viral vectors such as MVA (Amara, R. et al. 2001. Control of a mucosal challenge and prevention of AIDS by a multiprotein DNA/MVA vaccine. Science. 292:69-74) or Adenovirus (Shiver, J. W., et al 2002. Replication-incompetent adenoviral vaccine vector elicits effective anti-immunodeficiency-virus immunity. Nature. 415:331-335), gave reasonable protection against a challenge with pathogenic SHIV89.6P. <<Prime-boost>> might not be an absolute requirement since using recombinant live attenuated polio virus vaccine protected macaques against an SIV251 challenge (Crotty, S., et al 2001. Protection against simian immunodeficiency virus vaginal challenge by using Sabin poliovirus vectors. J Virol. 75:7435-7452). It is interesting to note that in all these experiments, even when the animals were not protected against the infection, immunization caused a delay in, or even abrogated, clinical disease.

(43) As shown by crystallography, the V1 and V2 loops of gp120 mask the CD4 binding site and the V3 loop masks the binding sites for the CXCR4 and CCR5 co-receptors (Kwong, P. D., et al 2000. Structures of HIV-1 gp120 envelope glycoproteins from laboratory-adapted and primary isolates. Structure Fold Des. 8:1329-1339; Kwong, P. D. et al 1998. Structure of an HIV gp120 envelope glycoprotein in complex with the CD4 receptor and a neutralizing human antibody. Nature. 393:648-659; Kwong, P. D., et al 2000. Oligomeric modeling and electrostatic analysis of the gp120 envelope glycoprotein of human immunodeficiency virus. J Virol. 74:1961-1972). In spite of this, antibodies against the gp120 CD4 binding site are present in the sera of HIV seropositive individuals and are able to neutralize several HIV-1 isolates in in vitro tests (Burton, D. 1997. A vaccine for HIV type 1: the antibody perspective. Proceedings of the National Academy of Sciences of the United States of America. 94:10018-10023; Hoffman, T. L et al., 1999. Stable exposure of the coreceptor-binding site in a CD4-independent HIV-1 envelope protein. Proc Nat/Acad Sci USA. 96:6359-6364). Also, some epitopes which are buried in the 3-D structure of the glycoprotein but become exposed after binding to the co-receptor, can induce highly neutralizing antibodies (Muster, T., et al 1993. A conserved neutralizing epitope on gp41 of human immunodeficiency virus type 1. J Virol. 67:6642-6647). Furthermore, neutralizing monoclonal antibodies have been obtained from patient's B cells (Parren, P. W., et al 1997. Relevance of the antibody response against human immunodeficiency virus type 1 envelope to vaccine design. Immunol Lett. 57:105-112). They are directed at gp41 linear epitopes (2F5) (Muster, T., F. et al 1993. A conserved neutralizing epitope on gp41 of human immunodeficiency virus type 1. J Virol. 67:6642-6647), or at gp120 conformational epitopes (2G12, 17b, 48db12) (Thali, M., et al 1993. Characterization of conserved human immunodeficiency virus type 1 gp120 neutralization epitopes exposed upon gp120-CD4 binding. J Virol. 67:3978-3988; Trkola, A., et al. 1996. Human monoclonal antibody 2G12 defines a distinctive neutralization epitope on the gp120 glycoprotein of human immunodeficiency virus type 1. J Virol. 70:1100-1108). Used in synergy they can neutralize in vitro several primary isolates (Mascola, J. R. et al 1997. Potent and synergistic neutralization of human immunodeficiency virus (HIV) type 1 primary isolates by hyperimmune anti-HIV immunoglobulin combined with monoclonal antibodies 2F5 and 2G12. J Virol. 71:7198-7206) and protect macaques against a mucosal challenge with SHIV (Baba, T. W. et al, 2000. Human neutralizing monoclonal antibodies of the IgG I1 subtype protect against mucosal simian-human immunodeficiency virus infection. Nat Med. 6:200-206; Mascola, J. R., et al 1999. Protection of Macaques against pathogenic simian/human immunodeficiency virus 89.6PD by passive transfer of neutralizing antibodies. J Virol. 73:4009-4018; Mascola, J. R., et al 2000. Protection of macaques against vaginal transmission of a pathogenic HIV-1/SIV chimeric virus by passive infusion of neutralizing antibodies. Nat Med. 6:207-210). However in infected people, all these antibodies are present in very low amounts, diluted in large quantities of non-neutralizing antibodies directed mainly at the antigenically variable V1, V2 and V3 gp120 loops. Therefore, there is hope that if one could induce high levels of such cross-neutralizing antibodies one may achieve at least some degree of protection. A major goal is to design a vector that will favor the production of such neutralizing antibodies.

(44) For this reason, we engineered mutant gp160 (anchored) and gp140 (soluble) by deleting the hypervariable V1, V2 and V3 loops individually or in combination to expose conserved epitopes and induce antibodies able to neutralize primary isolates. In some of the constructions, we also replaced the V3 loop by the AAELDKWASAA (SEQ ID NO: 8) sequence, especially ELDKWAS (SEQ ID NO: 8) sequence flanked on both sides by two Alanine to maintain the conformation of this gp41 conserved epitope normally buried in the native protein but able to induce large spectrum neutralizing antibodies (Muster, T., F. at al 1993. A conserved neutralizing epitope on gp41 of human immunodeficiency virus type 1. J Virol. 67:6642-6647; Binley, J. M., et al 2000. A recombinant human immunodeficiency virus type 1 envelope glycoprotein complex stabilized by an intermolecular disulfide bond between the gp120 and gp41 subunits is an antigenic mimic of the trimeric virion-associated structure. J Virol. 74:627-643; Sanders, R. W., et al 2000. Variable-loop-deleted variants of the human immunodeficiency virus type 1 envelope glycoprotein can be stabilized by an intermolecular disulfide bond between the gp120 and gp41 subunits. J Virol. 74:5091-5100). The normal alpha helical structure of this peptide should be conserved when exposed in our constructions at the tip of a deleted V3 loop. These constructions, in which the immunological decoys have been eliminated and the neutralizing epitopes have been exposed, should be good candidates for the induction of robust neutralizing antibody responses.

(45) The HIV gp constructions were introduced into a measles vaccine vector because it induces very high titers (1/80,000) of neutralizing anti-measles antibodies. (This is probably because it replicates in a large number of cells of different types.) One may hope, therefore, that the antibody response against the engineered HIV gps will also be strong. Furthermore, measles vaccine is also a potent inducer of long lasting cellular responses. The recombinant vaccines induced cross-neutralizing antibodies as well as cellular immune responses after a single injection in CD46.sup.+/ IFN-/_R.sup./ mice. Furthermore, they induced immune responses against HIV in mice and macaques with a pre-existing anti-MV immunity.

(46) Construction of Mutant HIV-1 Envelope Glycoproteins.

(47) The envelope glycoproteins used in this study (FIG. 1) were derived from SHIV89.6P, a chimeric simian/human immunodeficiency virus which contains tat, rev, vpu and env genes of HIV1 in an SIVmac239 background (Reimann, K. A., et al 1996. A chimeric simian/human immunodeficiency virus expressing a primary patient human immunodeficiency virus type 1 isolate env causes an AIDS-like disease after in vivo passage in rhesus monkeys. J Virol. 70:6922-6928). The env gene is derived from a cytopathic primary HIV1 isolate, 89.6, which is tropic for both macrophages and T cells (Collman, R., et al 1992. An infectious molecular clone of an unusual macrophage-tropic and highly cytopathic strain of human immunodeficiency virus type 1. J Virol. 66:7517-7521). The env sequence was amplified from the plasmid pSHIV-KB9 (NIH) that was previously cloned after in vivo passages of the original virus (Karlsson, G. B., et al 1997. Characterization of molecularly cloned simian-human immunodeficiency viruses causing rapid CD4+ lymphocyte depletion in rhesus monkeys. J Virol. 71:4218-4225). The full-length env (gp160) was amplified by PCR (Pfu polymerase) using primers that contain unique BsiWI and BssHII sites for subsequent cloning in measles vector: 160E5 (5-TATCGTACGATGAGAGTGAAGGAGAAATAT-3; SEQ ID NO: 1) and 160E3 (5ATAGCGCGCATCACAAGAGAGTGAGCTCAA-3; SEQ ID NO: 2). The env sequence corresponding to the secreted form (gp140) was amplified using primers 160E5 and 140E3 (5-TATGCGCGCTTATCTTATATACCACAGCCAGT-3; SEQ ID NO: 3). A start and a stop codon were added at both ends of the genes as well as several nucleotides after the stop codon in order to respect the rule of six, stipulating that the number of nucleotides of MV genome must be a multiple of 6 (Calain, P., and L. Roux. 1993. The rule of six, a basic feature for efficient replication of Sendai virus defective interfering RNA. J Virol. 67:4822-4830; Schneider, H., et al 1997. Recombinant measles viruses defective for RNA editing and V protein synthesis are viable in cultured cells. Virology. 227:314-322). Both gp160 and gp140 env fragments were cloned in PCR2.1-TOPO plasmid (Invitrogen) and sequenced to check that no mutations were introduced.

(48) Mutants with loop-deletions were generated by PCR amplification of two overlapping fragments flanking the sequence to be deleted and annealing of these fragments by PCR. To replace the V3 sequence by the AAELDKWASAA (SEQ ID NO: 8) sequence containing the gp41 epitope (Muster, T., F. et al 1993. A conserved neutralizing epitope on gp41 of human immunodeficiency virus type 1. J Virol. 67:6642-6647), four primers were designed on both sides of BbsI and MfeI sites encompassing the V3 sequence:

(49) TABLE-US-00009 V3A1 (SEQIDNO:4) (5-ATAAGACATTCAATGGATCAGGAC-3), V3A2 (SEQIDNO:5) (5TGCCCATTTATCCAATTCTGCAGCATTGTTGTTGGGTCTTGTA CAATT-3,? V3B1 (SEQIDNO:6) (5GATAAATGGGCAAGTGCTGCAAGACAAGCACATTGTAACATTG T-3,? V3B2 (SEQIDNO:7) (5-CTACTCCTATTGGTTCAATTCTTA-3).
The underlined sequences in V3A2 and V3B1 correspond to the AAELDKWASAA (SEQ ID NO: 8) epitope with a 12 nucleotides overlap. PCR amplifications with primer pairs V3A1/V3A2 and V3B1/V3B2 produced two fragments of 218 and 499 bp respectively. After gel purification, these fragments were annealed together by 15 PCR cycles without primers and amplified with V3A1/V3B2 primers. The resulting 705 bp fragment was cloned in PCR2.1-TOPO plasmid and sequenced. After digestion by BbsI and MfeI, the fragment lacking the sequence encoding the V3 loop (V3-AAELDKWASAA; SEQ ID NO: 8)) was purified and introduced in place of the corresponding fragment in the gp160 and gp140 in PCR2.1-TOPO plasmids.

(50) The resulting plasmids were designated pMV2-gp160V3 and pMV2-gp140V3.

(51) The V1V2 mutants were produced using the same procedure. Two fragments were amplified on both sides of V1V2 loop using the following primers:

(52) TABLE-US-00010 160E5 (5-TATCGTACGATGAGAGTGAAGGAGAAATAT-3; SEQIDNO:1), V1V2A1 (5-ATTTAAAGTAACACAGAGTGGGGTTAATTT-3; SEQIDNO:9), V1V2B1 (5-GTTACTTTAAATTGTAACACCTCAGTCATTACACAGGCCTG T-3;SEQIDNO:10), V1V2B2 (5-TTGCATAAAATGCTCTCCCTGGTCCTATAG-3; SEQIDNO:11).
The italicized sequences in V1V2A1 and V1V2B1 correspond to a 12 nucleotide overlap generated between the two fragments. PCR amplifications with primer pairs 160E5/V1V2A1 and V1V2B1/V1V2B2 produced two fragments of 400 and 366 bp respectively. After gel purification, these fragments were annealed together by 15 PCR cycles without primers and amplified with 160E5/V1V2B2 primers. The resulting 766 bp fragment was cloned in PCR2.1-TOPO plasmid and sequenced. After digestion with BsiWI (in 160E5 primer) and BbsI, the fragment lacking the sequence encoding the V1V2 loop was purified and introduced in place of the corresponding fragment in the gp160 and gp140 in PCR2.1-TOPO plasmids.

(53) To obtain the V1V2V3 mutants, the BsiWI/BbsI fragment lacking the sequence encoding the V1V2 loop was introduced in place of the corresponding fragment in the PCR2.1-TOPO-gp140V3 and PCR2.1-TOPO-gp160V3 plasmids.

(54) After BsiWI/BssHII digestion of the different PCR2.1-TOPO plasmids, the native and mutant gp160 and gp140 sequences were cloned in the EdB-tag vector in ATU position 2 and ATU position 3 (FIG. 2B). The resulting plasmids were designated pMV2-gp160.sub.HIV, pMV2-gp140.sub.HIV.

(55) Cells were maintained in Dubelbecco's modified Eagle's medium (DMEM) supplemented with 5% fetal calf serum (FCS) for Vero cells (African green monkey kidney), or with 10% FCS, 1 mg/ml G418 for helper 293-3-46 cells (35) and for P4-CCR5 cells (Hela-CD4-CXCR4-CCR5-HIVLTR-LacZ) (12).

(56) Recovery of Recombinant MV.sub.EdB-Env.sub.HIV89.6 Virus.

(57) To recover the recombinant MV.sub.EdB-HIV viruses from the plasmids, the different EdB-HIV Env plasmids were used to transfect 293-3-46 helper cells.

(58) To recover the measles virus from the EdB-HIV-Envplasmids cDNA, we used the helper-cell-based rescue system described by Radecke et al. (Radecke, F., et al 1995. Rescue of measles viruses from cloned DNA. EMBO Journal. 14:5773-5784) and modified by Parks et al. (Parks, C. L., et al 1999. Enhanced measles virus cDNA rescue and gene expression after heat shock. J Virol. 73:3560-3566). Human helper cells stably expressing T7 RNA polymerase and measles N and P proteins (293-3-46 cells, disclosed by Radecke et al) were co-transfected using the calcium phosphate procedure with the EdB-HIV-Env plasmids (5 g) and a plasmid expressing the MV polymerase L gene (pEMC-La, 20 ng, disclosed by Radecke et al). The virus was rescued after cocultivation of transfected 293-3-46 helper cells at 37 C. with primate Vero cells (african green monkey kidney). In this case, syncytia appeared systematically in all transfections after 2 days of coculture.

(59) In a further experiment (FIGS. 3C-D), after overnight incubation at 37 C., the cells were heat shocked at 43 C. for 3 hours in fresh medium (40). Heat-shocked cells were incubated at 37 C. for 2 days, then transferred onto a 70% confluent Vero cells layer (10 cm Petri dishes). Syncytia appeared in Vero cells after 2-5 days of coculture. Single syncytia were harvested and transferred to Vero cells grown in 35 mm wells. The infected cells were expanded in 75 and 150 cm3 flasks. When syncytia reached 80-90% confluence, the cells were scraped in a small volume of OptiMEM (Gibco BRL) and frozen and thawed once. After centrifugation, the supernatant, which contained virus, was stored at 80 C.

(60) Expression of HIV1 Glycoproteins by Recombinant MV.

(61) The rescued recombinant viruses MV2-gp140, MV2-gp160, MV3-gp140V3 and MV2-gp160V3 were propagated on Vero cells and the expression of HIV Env glycoproteins was analyzed by western blotting and immunofluorescence. Infection of Vero cells by recombinant MV2 viruses (with transgene insertion in position 2) showed a high expression of the HIV Env gp160 and gp140. The cleaved recombinant Env protein (gp120) was also detected. The MV3 virus (with transgene insertion in position 3) expressed lower levels of transgene, as expected due to the transcription gradient observed in MV expression. Taken together, these results indicate that HIV1 Env glycoprotein and V3 mutant are efficiently expressed by the recombinant MVs.

(62) Virus titration. The titers of recombinant MV were determined by an endpoint limit dilution assay on Vero cells. 50% tissue culture infectious dose (TCID.sub.50) were calculated using the Karber method.

(63) Growth Capacity of the MV.sub.EdB-Env.sub.HIV89.6 Recombinant Viruses.

(64) To analyze the growth capacity of MV.sub.EdB-Env.sub.HIV89.6 viruses, Vero cells were infected at different MOI (0.01 and 0.0001), incubated at 37 C., and collected at different time points. Titers of cell-associated viruses were determined for each sample using the TCID.sub.50 method on Vero cells. FIG. 4 shows that using MOI of 0.0001, the growth kinetics of MV.sub.EdB-Env.sub.HIV89.6 viruses was delayed, as compared to standard MV.sub.EdB-tag. However, using an MOI of 0.01 the production of recombinant viruses was comparable to that of standard virus, and peak titers of 10.sup.7 TCID.sub.50/ml or even more were easily obtained.

(65) In particular, monolayers of Vero cells (T-25 flasks) were infected at an MOI of 0.05 with the recombinant viruses. When syncytia reached 80-90% confluence, cells were lysed in 150 mM NaCl, 50 mM Tris pH=8, 1% NP40, 0.5 mM PMSF and 0.2 mg/ml Pefabloc (Interbiotech, France). Chromatin was removed by centrifugation and the concentration of protein in the supernatant was determined with a Bradford assay. Proteins (50 g) were fractionated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to cellulose membranes (Amersham Pharmacia Biotech). The blots were probed with a mouse monoclonal anti-HIV gp120 antibody (Chessie 13-39.1, NIH-AIDS Research & Reference Reagent Program) or with a monoclonal anti-MV N antibody (Chemicon, Temecula, USA). A goat anti-mouse IgG antibody-horseradish peroxidase (HRP) conjugate (Amersham) was used as second antibody. Peroxidase activity was visualized with an enhanced chemiluminescence detection Kit (Pierce).

(66) Mice Immunizations

(67) Mice susceptible for MV infection were obtained as described previously (21). Transgenic FVB mice heterozygous for CD46 (32), the receptor for MV vaccine strains (24) were crossed with 129sv IFN-/R.sup./ mice lacking the type I interferon receptor (22). The F1 progeny was screened by PCR and the CD46.sup.+/ animals were crossed again with 129sv IFN-/R.sup./ mice. IFN-/R.sup./ CD46.sup.+/ animals were selected and used for immunization experiments. The same type of mice have already been shown to be susceptible to MV infection (20, 21).

(68) Six-weeks-old female CD46.sup.+/ IFN-/R.sup./ mice were inoculated intraperitoneally with 10.sup.7 TCID.sub.50 of MV2-gp140, MV2-gp160, MV3-gp140V3 or MV2-gp160V3 recombinant viruses prepared and titrated as described above. Mice were euthanized 7 days and 1 month post-infection. Spleens and whole blood were collected. Splenocytes were extracted from spleens and kept frozen in liquid nitrogen until use. Serums were decanted and serology was analyzed by ELISA for MV (Trinity Biotech, USA) and HIV (Sanofi Diagnostics, France).

(69) Monkey Immunization

(70) Two colony-bred rhesus macaques (Macaca mulatto) (seronegative for simian type D retrovirus, simian T-cell lymphotropic virus, simian immunodeficiency virus and MV) were vaccinated subcutaneously with 104 TCID.sub.50 of MV vaccine (Rouvax, Aventis Pasteur, France). They were boosted one year later by two injections of 5 106 TCID.sub.50 of MV2-gp140 recombinant virus done at 1 month interval. Blood samples were collected at different time points and anti-MV and anti-HIV antibodies were looked for.

(71) Humoral Immune Response to Rescued Recombinant Viruses.

(72) 1.sup.st Experiment

(73) Humoral immune responses against MV and HIV Env were analyzed by ELISA in serums collected 1 month after immunization of mice. Titers were determined by limiting dilutions. The results presented in FIG. 5 show that all the vaccinated mice responded to measles with high titers of antibodies (1/50000 to 1/80000) and to HIV Env with titers between 1/1000 and 1/5000 depending on the inserted sequence. The antibody titers between MV and HIV cannot be compared because the ELISA used have not the same sensitivity. The MV ELISA (Trinity Biotech, USA) detected the whole response against all MV proteins, while the HIV ELISA (Sanofi Diagnostics) detected only the anti-HIV Env antibodies. The capacity of these sera to neutralize a primary HIV clade B isolate was tested using indicator cells, P4R5, that express beta-galactosidase when infected with HIV (HeLa-CD4-CXCR4-CCR5-HIV LTR-LacZ cells). In preliminary experiments, we tested sera of mice immunized with recombinant MV-HIV viruses expressing native envelope glycoproteins (MV-gp160.sub.HIV-1 or MV.sub.EdB-gp140.sub.HIV89.6). The results showed that these sera had a 70-50% neutralizing activity against a primary isolate, Bx08, when used at a 1/20 dilution (FIG. 6). The neutralizing activity of sera raised against the genetically engineered Env molecules is currently under study.

(74) 2.sup.nd Experiment

(75) In another experiment (FIG. 5C-F), sera were collected one month after immunization and heat inactivated. Anti-MV (Trinity Biotech, USA) and anti-HIV Env (Sanofi Diagnostic Pasteur, Biorad, France) antibodies were detected using commercial ELISA kits. An anti-mouse antibody-HRP conjugate (Amersham) was used as the secondary antibody. Titers were determined by limiting dilutions and calculated as the highest dilution of serum giving twice the absorbence of a 1/100 dilution of a mixture of control sera. The same ELISA kits were used for sera from macaque monkeys. An anti-monkey IgG secondary antibody was used to detect anti-HIV antibodies. Anti-MV antibodies were detected with an anti-human IgG in order to be able to calibrate the assay with standards supplied in the MV ELISA kit. They were expressed in mIU/ml. A mixture of 5 samples from negative monkeys was used as the negative control. The titer of anti-ELDKWAS (Residues 3-9 of SEQ ID NO: 8) antibodies was determined by ELISA using 96-well NeutrAvidin plates (Pierce) coated with the ELDKWAS (Residues 3-9 of SEQ ID NO: 8) biotynilated peptide (Neosystem, 5 g/ml in NaHCO.sub.3 2M, Na.sub.2CO.sub.3.H.sub.2O 2M, pH 9.6). Sera from mice immunized with standard MV were used as negative controls. Peptide-bound antibodies were detected with anti-mouse antibody-HRP conjugate.

(76) HIV-1 neutralization assays. Sero-neutralization was tested against SHIV89.6p (A. M. Aubertin, Universite Louis Pasteur, Strasbourg, H. Fleury, Bordeaux, France), 92US660, 92US714, 92HT593 (NIH-AIDS Research & Reference Reagent Program), and a clade A primary isolate: 3253 (G. Pancino, Institut Pasteur, Paris). These viruses were propagated on PHA-stimulated human PBMC as already described (42). HIV-1 neutralization assays were performed using the P4-CCR5 indicator cell line (43). P4-CCR5 cells were seeded in 96-well plates (20 000 cells per well) and incubated at 37 C. in DMEM, 10% FCS for 24 h. The medium was replaced by 100 l DMEM, 10% FCS, DEAE dextran (100 g/ml) and the cells were incubated at 37 C. for 30 minutes. Virus (0.5 ir 1 ng p24) was incubated with serum dilutions in 50 l PBS at 37 C. for 20 minutes and the virus-serum mixtures were added to the cells in triplicate. After 48 hours of incubation, the (3-galactosidase activity was measured using a Chemiluminescence Reporter Gene Assay (Roche, USA).

(77) Cellular Immune Responses to Rescued Recombinant Viruses.

(78) The capacity of splenocytes from vaccinated mice to secrete -IFN upon in vitro stimulation was tested by flow-cytometry and ELISpot assays. Frozen cells from immunized mice were thawed 18 h before functional assays and incubated in RPMI medium supplemented with 10% 56 C.-heated FCS (Gibco) and 10 U rh-IL2 (Boehringer Mannheim). Cell viability was evaluated by trypan-blue exclusion.

(79) To perform -IFN ELISpot assay, multiscreen-HA 96-wells plates were coated with capture anti-mouse -IFN (R4-6A2, Pharmingen) in PBS solution (6 g/ml). After overnight incubation at 4 C., wells were washed 4 times with PBS. The remaining protein binding sites were blocked by incubating wells with 100 l RPMI/FCS 10% for 1 h at 37 C. Medium was withdrawn just before addition of cell suspensions (100 l) and stimulating agents (100 l). Splenocytes from immunized mice were plated at 5.10.sup.5 cell per well in duplicate in RPMI. Concanavalin A (5 g/ml, Sigma) was used as a positive control, and RPMI/IL2 (10 U/ml) as a negative control. Cells were stimulated either with 1 g/ml HIV1 gp120, 1 g/ml Bovine Serum Albumin (Sigma), or Edm-Tag virus (MOI=1). After incubation for 2 h at 37 C. for viral adsorption, heated-FCS (10 l) was added in each well (10% final concentration) and plates were incubated for 24-36 h at 37 C. To remove cells, the plates were washed twice with PBS, 4 times with PBS containing 0.05% TWEEN 20 (Sigma), and 2 times again with PBS. For detection, a biotinylated anti-mouse -IFN antibody (XMG1.2, Pharmingen) was added to each well (100 l, 4 g/ml in PBS-0.1% FCS). After incubation for 2 h at room temperature, plates were washed 4 times with PBS-0.1% TWEEN 20 and twice with PBS. Streptravidin-Alkaline Phosphatase (AP) conjugate (Roche) (100 l, 1/2000 dilution in PBS) was added and incubated for 1-2 hours at room temperature. The enzyme was removed by 4 washes with PBS-0.1% TWEEN 20 and 2 washes with PBS. Spots were then developed with BCIP/NBT color substrate (Promega) prepared in AP buffer pH 9.5 (1 M Tris, 1.5 M NaCl, 0.05 M MgCl2). Wells were monitored for spot formation by eye: after a 15-30 minutes incubation the reaction was stopped by washing under running tap water. After drying at least overnight at room temperature, colored spots were counted using an automated image analysis system ELISpot Reader (Bio-Sys).

(80) For Flow-cytometry assays, 5 10.sup.5 splenocytes (diluted in 100 l RPMI) were stimulated in V-bottomed 96-wells plates with either 1 g/ml HIV1 gp120 protein (AbCys) in RPMI/IL2 (10 U/ml), or EdB-tag virus (MOI=1) diluted in 100 l RPMI/IL2. Non stimulated control cells were incubated with RPMI/IL2 (10 U/ml). After incubation for 2 h at 37 C. for viral adsorption, 10 l FCS were added in each well (10% final concentration) and plates were incubated overnight at 37 C. The medium was then replaced by 150 l RPMI-10% FCS containing 10 U rh-IL2 and 10 g/ml Brefeldin A (Sigma). Cells were incubated for 4 hours at 37 C., harvested, stained with anti-mouse CD8-APC (Pharmingen) and anti-mouse CD4-CyCr (Pharmingen) for 20 minutes at room temperature, washed with PBS-BSA (0.5%), then fixed for 5 minutes at 37 C. in CytoFix (Pharmingen). After washing cells were resuspended in 100 l PBS-BSA (0.5%) containing 0.1% Saponin (Sigma) and incubated for 30 minutes at room temperature with anti-mouse -IFN-PE (Pharmingen). Cells were washed again and samples were analyzed using a FACSCalibur cytometer (Becton Dickinson). The data were analyzed using Cell Quest software.

(81) Recombinant MV Express HIV89.6 Env Glycoproteins and Replicate Efficiently.

(82) The anchored (gp160) and soluble (gp140) forms of the HIV Env glycoprotein (strain SHIV89.6p), with or without deletion of the V3 loop and insertion of an additional ELDKWAS (Residues 3-9 of SEQ ID NO: 8) epitope, were inserted into one of the ATU of the p(+)MV vector (FIG. 2). Recombinant viruses MV2-gp140, MV2-gp160, MV3-gp140V3 and MV2-gp160V3 were obtained after transfection of the plasmids into the 293-3-46 helper cell line and propagation in Vero cells. MV2- and MV3-refers to the site of the insertion, position 2 or 3 respectively, of the EnvHIV89.6 construction. Expression of the EnvHIV89.6 protein was analyzed by western blotting of infected-cells lysates (FIG. 3) and immunofluorescence (not shown). The MV2-gp140 and MV2-gp160 viruses showed a high level of expression of the EnvHIV89.6 protein (FIG. 3C, lanes 1, 2, 4). As expected, the MV2-gp160A viruses expressed the env gp160 precursor as well as the cleaved gp120 protein (FIG. 3C, lanes 2, 4). In contrast, the MV2-gp140 and MV3-gp140V3 viruses expressed only the secreted, uncleaved gp140 form. The MV3-gp140V3 virus expressed slightly lower levels of transgene than viruses of the MV2-series, as expected, due to the transcription gradient observed in MV expression (FIG. 3C, lane 3). Taken together, these results indicate that Env.sub.HIV89.6 and the V3 mutants were efficiently expressed and correctly matured. The recombinant MV were passaged 5 times on Vero cells and the expression of the transgene was compared to that of the MV nucleoprotein. FIG. 3 shows that Env.sub.HIV89.6 expression was similar for passages 2 and 5, confirming the stability of expression of transgenes in this system.

(83) The growth of MV-Env.sub.HIV89.6 recombinant viruses was analyzed on Vero cells using an MOI of 0.0001 or 0.01. The growth of recombinant viruses was only slightly delayed compared to that of standard EdB-tag MV rescued from p+(MV). Viruses expressing the secreted gp140 were less affected than viruses expressing the anchored gp160. The gp140V3 recombinant grew at the same rate as control MV. The delay observed with viruses expressing the anchored gp160 may be due either to lower replication rate, because of the larger size of the transgene, or to reduced MV budding because of the insertion of gp160 at the surface of the infected cells. Nevertheless, the final yield of recombinant viruses was comparable to that of control MV and peak titers of about 10.sup.6 to 10.sup.7TCID.sub.50/ml were obtained routinely.

(84) Induction of Humoral Immune Response to Recombinant MV in Susceptible Mice.

(85) The immunogenicity of MV-Env.sub.HIV89.6 viruses was tested in genetically modified mice expressing the human CD46 MV receptor and lacking the Type I IFN receptor. Increasing doses of MV2-gp160 virus (103-107 TCID.sub.50) were tested in 5 groups of 3 mice. Antibodies to MV and HIV Env were looked for by ELIA in sera collected 1 month after immunization (FIG. 5C). Both anti-MV and anti-HIV antibody titers increased when the dose of recombinant MV increased. Since high anti-MV titers were obtained when animals were inoculated with 10.sup.6 to 10.sup.7 TCID.sub.50, mice were immunized with 5.10.sup.6 TCID.sub.50 in all further experiments. At this dose, anti-MV antibody titers were six fold higher than anti-HIV titers. One should keep in mind that immunization was against HIV Env only, whereas all MV proteins were expressed during infection. To compare the immunogenicity of the different Env.sub.HIV constructs, four groups of 6 mice were inoculated intraperitoneally with various MV-Env.sub.HIV89.6 viruses (FIG. 5B, 5E). All mice responded to MV (mean anti-MV titer: 5 10.sup.4) and to HIV Env (mean anti-HIV titer: 8 10.sup.3). No difference in anti-MV or anti-HIV or antiHIV titers was observed between the four constructs tested. Interestingly, expression from the ATU 2 or the ATU 3 position of the MV vector did not affect the antibody response. Because the V3 constructions expressed an additional ELDKWAS (Residues 3-9 of SEQ ID NO: 8) epitope, the antibody response against this gp41 epitope was examined separately using a specific ELISA assay (FIG. 5F). The results showed that the V3-ELDKWAS (Residues 3-9 of SEQ ID NO: 8) constructions induced higher titers of anti-ELDKWAS (Residues 3-9 of SEQ ID NO: 8) antibodies. The titer of 1/50 000 corresponds to the dilution of an immune serum capable of recognizing the antigen administered for the immunization, in ELISA assay.

(86) MV-Env.sub.HIV89.6 Viruses Induce Neutralizing Anti-HIV Antibodies.

(87) The capacity of these sera to neutralize either homologous SHIV89.6p virus or various heterologous primary HIV-1 isolates was tested using a single cycle virus infectivity assay on P4-CCR5 indicator cells (43). P4-CCR5 cells express the CD4, CXCR4 and CCR5 HIV-1 receptors and have been stably transfected with an HIV LTR LacZ. Therefore, they are susceptible to HIV-1 isolates and express (3-galactosidase upon infection. The sero-neutralization assay was validated using a combination of anti-HIV immunoglobulin (HIVIG) and monoclonal antibodies (2F5 and 2G12) previously shown to synergistically neutralize primary HIV isolates (17). We also used sera from infected patients that neutralize primary HIV isolates (17). We also used sera from infected patients that neutralize primary HIV primary isolates using a standard neutralization assay on human PBMCs (42). The neutralizing activity of a serum (Table 1) is expressed as the ratio of the reduction of infection obtained with this serum over the reduction obtained with negative control sera used at the same dilution (sera from HIV negative individuals and from infected patients neutralized clade B and A viruses equally well in this assay.

(88) As shown in Table 1, antibodies induced in mice by the four MV-Env.sub.HIV89.6 viruses neutralized the homologous SHIV89.6p at both dilutions tested (1/30 and 1/60). No significant difference was observed between the sera obtained with the different Env constructs, indicating that the secreted and anchored from of HIV glycoprotein induced neutralizing antibodies against homologous virus equally well when expressed by MV. Deleting the V3 loop, known to contain type-specific neutralizing epitopes, had no significant effect on the induction of antibodies that neutralized the homologous virus. This suggests that the deletion might have been compensated either by the addition of a second ELDKWAS (Residues 3-9 of SEQ ID NO: 8) gp41 neutralizing epitope, or by the uncovering of other neutralizing epitopes.

(89) The antibodies induced by the recombinant viruses neutralized heterologous primary clade B isolates, except the 92HT593 isolate, as well as a clade A virus. In each case, antibodies induced by the anchored gp160 were slightly more neutralizing than antibodies induced by the secreted gp140, especially against the clade A 3253 virus. The antibodies induced by the V3-ELDKWAS (Residues 3-9 of SEQ ID NO: 8) Env.sub.HIV89.6 neutralized heterologous viruses more efficiently than those induced by the native envelope. This was particularly striking for the Bx08 virus which could be neutralized up to 90% by sera from mice immunized with MV2-gp160V3 (1/30 dilution) but not by sera from mice immunized with MV expressing the native Env.sub.HIV89.6. This neutralization was just as efficient as neutralization by positive control sera. These results show that replacing the V3 loop of Env.sub.HIV89.6 by an additional ELDKWAS (Residues 3-9 of SEQ ID NO: 8) gp41 epitope and expressing the construct with a MV vector allowed the induction of antibodies with cross-neutralizing activity against clade A and B HIV-1 primary isolates, at least in the context of recombinant MV infection of mice.

(90) TABLE-US-00011 TABLE 1 Neutralization of HIV-1 primary heterologous isolates by sera from MV- Env.sub.HIV89.6 immunized mice.sup.a. Positive controls Mab Human HIV Mice Sera (1/60) Mice Sera (1/30) sera.sup.c MV2 MV2 MV2 MV2 (2F5/ Virus isolate MV2 Gp140 MV2 Gp160 MV2 Gp140 MV2 Gp160 2G12/ 4 33 (subtype) Gp140 V3 Gp160 V3 Gp140 V3 Gp160 V3 HIV-IG 61/40) 1/30) SHIV 89.6 40 50 52 45 76 57 72 68 ND ND ND Bx08 (B) 0 31 0 40 0 76 18 90 94 94 90 92 US 660 (B) 2.5 15 13 17 ND ND ND ND ND ND ND 92 US 714 (B) 45 49 45 68 ND ND ND ND ND ND ND 92 HT 593 (B) 0 0 0 0 0 0 0 0 ND ND ND 3253 (A) 0 0 18 30 0 10 43 49 73 54 45 .sup.aSerum was evaluated for neutralizing antibodies at two dilutions. Values are % reduction in infection of primary HIV isolates on P4-CCR5 cells in presence of mice sera (three mice per point). Determinations were made in triplicate and the standard deviations were <10%. .sup.bMix of HIVIG (2.5 mg/ml) and Mabs 2F5 and 2G12 (25 g/ml). .sup.cNumbers correspond to the nomemclature used in Burrer et al.

(91) Induction of Cellular Immune Response Against Recombinant MV

(92) The results of these experiments performed with splenocytes from mice immunized with MV2-gp160.sub.HIV virus (FIG. 7) demonstrated that a single immunization with MV2-gp160.sub.HIV virus was able to prime HIV Env-specific lymphocytes in vivo. The -IFN-ELISpot assay is a sensitive method for antigen-specific cell numeration in fresh cells after in vivo immunization. This assay was used to determine whether HIV-Env-specific -IFN-secreting cells could be detected after a single immunization with the MV2-gp160.sub.HIV virus. FIG. 7A shows that a significant number of Env-specific cells were present in 2/3 mice tested, 7 days as well as 1 month after immunization. (For one mouse in each group the number of spots was the same after BSA or gp120 stimulation). The number of HIV-specific spots detected (up to 600/10.sup.6 cells) represents 15-20% of MV-specific spots detected in the same mice (not shown), indicating that recombinant MV is able to efficiently immunize against the foreign gene expressed.

(93) To assess the phenotype of these Env-specific cells, 3-color cytofluorometry experiments were performed on mice euthanized 7 days after immunization, at the theoretical peak of effector cells proliferation. A representative result is shown on FIG. 7B. The background -IFN production level for both CD4+ and CD8+ lymphocytes is shown on the left panel. For this animal, 0.09% of CD8+ lymphocytes (mean calculated for 3 mice: 0.31%) and 0.25% of CD4+ lymphocytes (mean: 0.41%) were spontaneously producing -IFN. The frequencies of HIV-gp120 T-cells (middle panel) in the CD8+ and CD4+ subsets were 1.76% (mean: 1.69%) and 0.92% (mean: 0.76%) respectively. It's interesting to take into account that in the same immunized mouse the frequencies of Measles specific cells in CD8+ and CD4+ subsets were 7.63% (mean: 7.03%) and 4.11% (mean: 3.50%) respectively. Indeed the recombinant MV2-gp160.sub.HIV virus expresses 6 measles proteins plus one gp160 foreign protein. Thus, the frequencies of antigen-specific lymphocytes followed the recombinant gene proportions. As a conclusion, 3-color cytofluorometry performed 7 days after MV2-gp160.sub.HIV virus vaccination showed that both CD8+(FIG. 7B, upper panel) and CD4+(FIG. 7B, lower panel) lymphocytes specific for HIV gp120 and measles virus were primed in vivo

(94) Inducing an Anti-HIV Response in Animals with Pre-Existing Anti-MV Immunity.

(95) We first tested the possibility of boosting the anti-HIV response by a second injection of recombinant MV. Mice immunized with 5.10.sup.6 TCID.sub.50 of MV2-gp140 recombinant virus (3 mice per group) were boosted with a second injection of the same recombinant MV one month after the first injection. The mean anti-MV and anti-HIV antibody titers at the time of boosting were 5 10.sup.4 and 8 10.sup.3 respectively. These titers increased to, respectively 5 10.sup.5 and 5 10.sup.4 one month after boosting. Thus, anti-MV and HIV responses can be boosted 10 times by injecting the same dose of recombinant MV one month after the first immunization.

(96) We then tested the ability of recombinant MV to induce anti-HIV antibodies in mice and monkeys in the presence of pre-existing anti-MV immunity. Mice (3 mice per point) were first immunized with 10.sup.5 TCID.sub.50 of EdB-tag MV (without an HIV insert). High levels of anti-MV antibodies were induced (FIG. 7C). The titer decreased slightly after 2 months and remained stable for the following 9 months. Mice were then inoculated with 5 10.sup.6 TCID.sub.50 of MV2-gp140.sub.HIV89.6, and boosted with the same dose one month later. The titer of anti-MV antibodies was increased 100 times and high titers of anti-HIV antibodies (5 10.sup.4) were induced. These titers were similar to those obtained after immunization of nave animals with two injections.

(97) The same experiment was performed with rhesus macaques (FIG. 7D). Two macaques were immunized with a standard dose (10.sup.4 TCID.sub.50) of MV vaccine (Rouvax, Aventis Pasteur). As for mice, high anti-MV antibody levels were induced and remained stable during one year. Macaques were then inoculated with 5 10.sup.6 TCID.sub.50 of MV2-gp140.sub.HIV89.6 twice at one month interval. Anti-MV titers increased 150 times after the first injection of MV-HIV, while the second injection had no or little effect. Anti-HIV antibodies were induced by the first MV2-gp140.sub.HIV89.6 injection despite the presence of pre-existing anti-MV immunity. One month after the second MV2-gp140.sub.HIV89.6 injection, the anti-HIV antibody level had increased about 10 times and had reached titers similar to those obtained in mice. This level remained stable for the following 5 months.

(98) The main goal of the present work was to test the immunogenicity of attenuated MV-Env.sub.HIV recombinant viruses. We showed that such recombinants were genetically stable, expressed the HIV Env protein at high levels, and induced high titers of antibodies against both MV and the HIV Env constructs in transgenic mice. The anti-HIV antibodies titers were approximately 15-20% of those of the anti-MV antibodies. This corresponds roughly to the ratio of HIV/MV proteins expressed by the recombinant viruses. HIV Env constructions with a deleted V3 loop and an additional ELDKWAS gp41 epitope induced twice as much anti-ELDKWAS (Residues 3-9 of SEQ ID NO: 8) antibodies as native constructions, suggesting that the native conformation of the additional peptide was conserved in spite of its ectopic position. A high level of HIV-specific CD8+ and CD4+ cells was also induced. As much as 1.5-2% of the total CD8+ T-cells and 0.9% of the total CD4+ T-cells were HIV-specific.

(99) However, the most important aspect of our results is that these anti-HIV antibodies were neutralizing for the homologous SHIV89.6p virus as well as for several heterologous clade A and clade B HIV-1 primary isolates. Interestingly, the anchored gp160 V3-ELDKWAS (Residues 3-9 of SEQ ID NO: 8) construction induced antibodies that neutralized heterologous viruses more efficiently than those induced by the native envelope. Their neutralizing titers were similar to those of reference human HIV-neutralizing sera. The broader neutralizing capacity of these antibodies could be due either to the addition of a second ELDKWAS (Residues 3-9 of SEQ ID NO: 8) gp41 neutralizing epitope, or to the exposure of previously masked conserved neutralizing epitopes. Several groups have inserted the ELDKWAS (Residues 3-9 of SEQ ID NO: 8) epitope into various immunogenic molecules (44, 45, 46, 47). These studies showed that the conformational context in which the epitope is displayed is essential for the induction of neutralizing antibodies. A j-turn-like constraint was shown to be the most likely conformation structure of the ELDKWAS (Residues 3-9 of SEQ ID NO: 8) epitope recognized by the 2F5 neutralizing antibody (46). In our constructions, the insertion of the short AAELDKWASAA (SEQ ID NO: 8) epitope in place of the V3 loop, which is flanked by j-strands (28, 29), may have such a j-turn-like conformation.

(100) It has been shown, already, that deleting the hyper-variable loops of HIV Env can enhance its immunogenicity (3, 48, 39). However, in previous studies neutralizing antibodies were obtained only after multiple injections of high amounts of soluble protein (23), or with a prime boost regimen using very large amounts of DNA and pure protein (3, 39). In contrast, we observed the same levels of neutralizing antibodies in mice injected with a single dose of MV-gp160V3-ELDKWAS (Residues 3-9 of SEQ ID NO: 8). Good immunogenicity in our system results probably from the fact that the HIV Env is expressed and processed by the immune system n the same way as proteins from the live MV vaccine, a highly potent immunogen. One may hope that such levels of neutralizing antibodies could at least induce partial protection in vaccinated individuals. According to the data of others (3, 39), it might be possible to increase the immunogenicity of M-HIV Env recombinants even further by deleting the V1 and V2 loops of HIV gp120, notably to induce antibodies directed against the CD4-binding site. However, it has been recently reported that this receptor-binding site can escape from the immune response by conformational and entropic masking (49).

(101) The presence of anti-MV immunity in nearly the entire adult human population would seem to restrict the use of MV recombinants to infants, an already worthy goal in any event. However, several studies showed that revaccinating already immunized individuals results in a boost of anti-MV antibodies, suggesting that the attenuated live vaccine replicated and expressed its proteins in spite of preexisting immunity (50). Under such circumstances, one might hope to be able to vaccinate adults against a foreign antigen with a MV recombinant. Indeed, our results demonstrate, both with mice and macaques, that high levels of anti-HIV neutralizing antibodies can be obtained in the presence of pre-existing anti-MV immunity.

(102) Various prime-boost regimen, using combinations of naked DNA and viral vectors such a sMVA (1) or Adenovirus (29), gave reasonable protection against a challenge with pathogenic SHIV89.6p. In the present study, we show that a single injection of MV is able to combine humoral and cellular responses at levels similar to those induced by these complex combinations.

(103) The same recombinants have been prepared using the cloned Schwarz strain as a vector. This should raise their immunogenicity even further.

EXAMPLE II: CONSTRUCTION OF SCHWARZ MEASLES VIRUSES (MVSCHW) EXPRESSING HIV-1 ANTIGENS

(104) In order to test their capacity as vaccine candidates against HIV infection, we constructed several recombinant Schwarz measles viruses (MV) expressing HIV-1 antigens. Different HIV-1 genes from different open reading frames were constructed and introduced in additional transcription units in the Schwarz MV cDNA that we previously cloned (pTM-MVSchw). After rescue of the different recombinant Schwarz measles viruses, the expression of the different HIV-1 proteins was analyzed by western blotting of infected-cells lysates (FIGS. 3A-D).

(105) Different immunogens were constructed from HIV-1 Env glycoprotein (hereafter 1-8), Gag protein (hereafter 9), and Tat protein (hereafter 10): 1. Secreted glycoprotein gp140 from HIV-1 89.6p 2. Anchored glycoprotein gp160 from HIV-1 89.6p 3. Secreted glycoprotein gp140 from HIV-1 89.6p deleted from hypervariable region V3 and additional AAELDKWASAA (SEQ ID NO: 8) epitope (gp140HIV.sub.89.6 V3-ELDKWAS (Residues 3-9 of SEQ ID NO: 8)) 4. Anchored glycoprotein gp160 from HIV-1 89.6p deleted from hypervariable region V3 with an additional AAELDKWASAA (SEQ ID NO: 8) epitope (gp160HIV.sub.89.6 V3-ELDKWAS(Residues 3-9 of SEQ ID NO: 8)) 5. Secreted glycoprotein gp140 from HIV-1 89.6p deleted from hypervariable regions V1-V2 (gp140HIV.sub.89.6 V1 V2) 6. Anchored glycoprotein gp160 from HIV-1 89.6p deleted from hypervariable regions V1-V2 (gp160HIV.sub.89.6 V1 V2) 7. Secreted glycoprotein gp140 from HIV-1 89.6p deleted from hypervariable regions V1-V2-V3 (gp140HIV.sub.89.6 V1 V2V3) 8. Anchored glycoprotein gp160 from HIV-1 89.6p deleted from hypervariable regions V1-V2-V3 (gp160HIV.sub.89.6 V1 V2V3) 9. Gag polyprotein (p17p24, delta myr) from HIV-1 (clade B consensus) truncated from the nucleoprotein ORF in C-terminal (p17p24myrHIV-1B) 10. Tat protein from HIV-1 89.6p (TatHIV.sub.89.6)

(106) The HIV env genes encoding the different forms of the Env protein were generated by PCR amplification from plasmid pSHIV-KB9 (NIH-AIDS Research & Reference Reagent Program). The specific sequences were amplified using PfuTurbo DNA polymerase (Stratagene) and specific primers. To generate the different deletions, overlapping fragments flanking the sequences to be deleted were generated and annealed together by PCR. They were then introduced by enzyme restriction cloning in place of the corresponding fragment in the gp160 and gp140 sequences already cloned in PCR2.1-TOPO plasmids (FIG. 1A). The different sequences generated include a start and a stop codon at both ends and respect the rule of six, stipulating that the nucleotides number of MV genome must be divisible by 6 (7, 8). After BsiWI/BssHII digestion, the different HIV sequences were introduced in the pTM-MVSchw vector in ATU position 2 or 3 (FIG. 1B). The resulting plasmids were designated: 1. pTM-MVSchw2-gp140.sub.HIV 2. pTM-MVSchw2-gp160.sub.HIV 3. pTM-MVSchw2-gp140V3.sub.HIV 4. pTM-MVSchw2-gp160V3.sub.HIV 5. pTM-MVSchw2-gp140.sub.HIV V1V2 6. pTM-MVSchw2-gp160.sub.HIV V1V2 7. pTM-MVSchw2-gp140.sub.HIV V1V2V3 8. pTM-MVSchw2-gp160.sub.HIV V1V2V3 9. pTM-MVSchw2-Gag.sub.HIV (p17-p24 myr) 10. pTM-MVSchw3-Tat.sub.HIV

(107) A recombinant virus expressing both Gag and gp140 in both positions 1 and 2 of the measles Schwarz vector was produced. 11. pTM-MVSchw2-Gag.sub.SIV239 (p17-p24 myr)-3-gp140.sub.HIV

(108) This virus expressed both proteins (Fig z). Such constructs allow the production of HIV, SHIV or SIV assembled Gag-Env virus like particles in cells infected by recombinant measles virus.

(109) The HIV-1 immunogenic sequences represented in FIG. 16 have been generated:

EXAMPLE III: RECOMBINANT MEASLES VIRUSES EXPRESSING DIFFERENT VIRAL TRANSGENES

(110) In order to demonstrate the immunizing and protective capacities of MV as a pediatric vaccination vector, a series of recombinant measles viruses expressing different viral transgenes (listed below) from other viruses were constructed and studied. The results presented here were obtained with the old EdB-tag vector. However, we have shown that the EdB-tag was 100 times less immunogenic than the Schwarz vaccine. Thus MV.sub.EdB recombinant viruses were inoculated at higher doses. All the inserted sequences with good immunological records can be obviously inserted in the Schwarz vector.

(111) Viral genes which have been already inserted in the recombinant measles viruses:

(112) TABLE-US-00012 HIV clade B 89.6P gp160 gp140 gp160 V3 gp140V3 gp160 V1V2 gp140V1V2 gp160V1V2V3 gp140V1V2V3 tat

(113) HIV clade B consensus codon optimized Gag (p17-p24)

(114) TABLE-US-00013 SIV Mac 239 Nef NefMyr Nef29-236 Tat HTLV-I Env Gag (p19-p24) Tax

EXAMPLE IV: RECOMBINANT MEASLES VIRUSES EXPRESSING ENV AND NS1 FROM YELLOW FEVER VIRUS HAVE IMMUNE CAPACITY

(115) Because a pediatric bivalent vaccine against measles and yellow fever should be useful, we constructed recombinant MV expressing the Env and NS1 proteins from Yellow Fever Virus (YFV 17D204, Pasteur vaccine strain) and tested their capacity to protect mice from a lethal YFV challenge.

(116) Construction of MV-YFV Recombinant Plasmids.

(117) The env gene was PCR amplified with Pfu polymerase using primers that contain unique BsiW and BssHII sites for subsequent cloning in MV vector: MV-YFVEnv5 (5-TATCGTACGATGCGAGTCGTGATTGCCCTACTG-3; SEQ ID NO: 12) and MV-YFVEnv3 (5-ATAGCGCGCTTATGTGTTGATGCCAACCCA-3; SEQ ID NO: 13). The Env protein thus generated (amino acids 270-753 in YFV polyprotein) contained the signal peptide in N-terminal and a part of the tramsmenbrane region in C-terminal. The NS1 sequence was PCR amplified in the same way with Pfu polymerase using primers: MVYFVNS5 (5-TATCGTACGATGAGAAACA TGACAATGTCC-3; SEQ ID NO: 14) and MVYFVNS3 (5-ATAGCGCGCTTAATGGCTTTCATGCGTTT TCC-3; SEQ ID NO: 15). The NS1 protein (amino acids 754-1122 in YFV polyprotein) contained its signal peptide sequence. A start and a stop codon were added at both ends of the genes as well as several nucleotides after the stop codon in order to respect the rule of six, stipulating that the nucleotides number of MV genome must be a multiple of 6 (7). Both env and NS1 fragments were cloned in PCR2.1-TOPO plasmid (Invitrogen) and sequenced to check that no mutations had been introduced. After BsiWI/BssHII digestion of the PCR2.1-TOPO plasmids, the env and NS1 sequences were cloned in the EdB-tag vector in ATU position 2 giving plasmids: EdB-Env.sub.YFV and EdB-NS1.sub.YFV.

(118) Recovery of Recombinant EdB-Env.sub.YFV and EdB-NS1.sub.YFV Viruses.

(119) EdB-Env.sub.YFV and EdB-NS1.sub.YFV plasmids were used to transfect 293-3-46 helper cells as described above, and recombinant viruses were rescued from transfected cells cocultivated with Vero cells. Recombinant viruses were passaged two times on Vero cells and tested for transgene expression.

(120) Expression of YFV Proteins by Recombinant MV.

(121) The rescued recombinant viruses MV2-Env.sub.YFV and MV2-NS1.sub.YFV were propagated on Vero cells and the expression of YFV proteins was analyzed by immunofluorescence. FIG. 9 shows that syncytia of Vero cells infected by recombinant MV2-YFV viruses showed a high expression of the YFV Env and NS1 proteins as detected with a mouse anti-YFV polyclonal serum. In order to determine whether the expression of YFV genes was stable, the rescued recombinant viruses were serially passaged on Vero cells. After 10 passages all the syncytia observed in infected cells were positive for YFV (not shown). Taken together, these results indicate that Env and NS1 proteins from YFV are efficiently and stably expressed over several passages by the recombinant MVs.

(122) Mice Immunization with MV-YFV Recombinant Viruses.

(123) A mixture of both MV2-Env.sub.YFV and MV2-NS1.sub.YFV viruses (10.sup.7 TCID.sub.50) was inoculated intraperitoneally to six CD46.sup.+/ IFN-/bR.sup./ mice as described above (see MV-HIV gp experiments). As a control, six other mice received the same dose of standard measles vaccine. After one month, mice were intracranially challenged with YFV 17D204 (10 LD.sub.50 determined on FVB mice). FIG. 10 shows that 65% of MV-YFV immunized animals were fully protected against the challenge, while all animals vaccinated with standard MV died between 6 and 7 days post-challenge. Moreover, a 4-days delay in mortality was observed in mice immunized with MV-YFV, and these mice did not die with the same encephalitic clinical symptoms than mice vaccinated with standard MV vaccine. The disease was attenuated and consisted of limb paralysis. It has to be noticed that IFN-/bR.sup./ mice are much more sensitive to viral infections than immunocompetent mice (10.sup.2-10.sup.4 times). For this reason, the lethal dose determined on immunocompetent mice was probably too high for IFN-/bR.sup./ mice. The same experiment is underway using several decreasing doses of YFV challenge viruses.

(124) In conclusion, this preliminary experiment shows that the immune responses induced by recombinant MV against YFV proteins are able to protect mice against a lethal challenge.

(125) The above constructs were made by using the sequences disclosed on FIGS. 12A and 12B.

(126) The same principles for the preparation of constructs would apply with sequences disclosed on FIGS. 12C and 12D.

EXAMPLE V: VACCINATION AGAINST WNV WITH A LIVE ATTENUATED MEASLES VIRUS (SCHWARZ STRAIN) EXPRESSING THE SECRETED FORM OF THE E GLYCOPROTEIN OF THE WNV (WEST NILE VIRUS)

(127) We constructed a recombinant Schwarz measles attenuated virus expressing the WNV E soluble form and tested its capacity as vaccine candidate against WN encephalitis. The WN cDNA corresponding to the sE protein of IS-98-ST1 strain of WNV was introduced in an additional transcription unit in the Schwarz MV cDNA (pTM-MVSchw CNCM I-2889). After rescue of the recombinant Schwarz measles virus, its capacity to protect mice from a lethal WNV encephalitis following intraperitoneal challenge was tested.

A) MATERIALS AND METHODS

(128) A.1 Cells and WN Virus

(129) The IS-98-ST1 strain of WN virus was produced on Aedes AP61 mosquito cells according to the protocol described in Despres et al (51), Mashimo et al (52) and Lucas et al (53). The Vero-NK cell clone used in this study was selected for its capacity to fuse after infection with measles virus and to amplify the WN virus.

(130) A.2 Titration of WN Virus on AP61 Mosquito Cells by Immunodetection of Focuses Viral Replication (Focus Immuno Assay, FIA).

(131) The titration was performed according to the protocol described in Despres et al (51), Mashimo et al (52) and Lucas et al (53).

(132) The infectious titer of WN virus on AP61 cells was determined as focus forming units on AP61 cells (AP61 UFF/ml).

(133) A.3 Purification of WN Virus Produced on AP 61 Cells.

(134) The purification was carried out according to the protocol described in Despres et al (51), Mashimo et al (52) and Lucas et al (53).

(135) Briefly, the viral particles present in supernatants of AP61 cells infected during 3 days with WN virus strain IS-98-ST1 (MOI 0.4) were concentrated in 7% PEG 6000 and then purified in 30-60% discontinuous saccharose gradient and in 10-50% linear saccharose gradient. WN virious in 30% saccharose were stored at 80 C. The obtained infectious titers were about 10.sup.10 AP61 FFU/ml.

(136) A.4 Anti-WN Antibody Detection in ELISA

(137) The anti-WN antibody titers of diluted sera (1:100) were determined by ELISA on a given quantity of 10.sup.6 AP61 FFU of WN IS-98-ST1 virions purified in saccharose gradient. The protocol is described in Despres et al (1993) and Mashimo et al (2002).

(138) A.5 Anti-WN Immune Sera

(139) Anti-WN immune sera were collected in adult mice genetically resistant to viral encephalitis (Mashimo et al2002) which were tested during at least one month with intraperitoneal inoculation of 10.sup.3 AP61 FFU of WN virus strain IS-98-ST1.

(140) The anti-WN antibody titer of 1:100 diluted immunsera were measured in ELISA and were about 1.4 DO units. The neutralizing titers (TNRF90) of anti-WN sera were about 1600.

(141) Ascites of mice (HMAF) against WN strain IS-98-ST1 were obtained from animals which had been hyperimmunized with brain homogenates of baby mice inoculated with WN. The ELISA titers of anti-WN HMAF, diluted to 1:1000 were about 1 DO unit.

(142) The anti-WN immune sera were used for indirect immunofluorescence and for passive seroprotection assays against the disease. Anti-WN HMAF were used for membrane immunodetection of viral proteins.

(143) A6. Construction of Recombinant Schwarz Measles Virus Expressing WN sE

(144) The WNV env gene encoding the secreted form of the protein was generated by RT-PCR amplification of viral RNA purified from viral particles (WNV IS-98-ST1 strain). The specific sequence was amplified using PfuTurbo DNA polymerase (Stratagene) and specific primers that contain unique sites for subsequent cloning in pTM-MVSchw vector: MV-WNEnv5 5-TATCGTACGATGAGAGTTGTGTTTGTCGTGCTA-3 (SEQ ID NO: 20; BsiWI site italicized) and MV-WNEnv3 5-ATAGCGCGCTTAGACAGCCTTCCCAACTGA-3 (SEQ ID NO: 21; BssHII site italicized). A start and a stop codon were added at both ends of the gene. The whole sequence generated is 1380 nucleotides long, including the start and the stop codons and respects the rule of six, stipulating that the nucleotides number of MV genome must be divisible by 6 [Calain, 1993 (7); Schneider, 1997 (28)]. The Env protein thus generated contains its signal peptide in N-term (18 aa) and no transmembrane region. Thus, It represents amino acids 275-732 in WNV polyprotein and has the following sequence:

(145) TABLE-US-00014 (SEQIDNO:22) atgagagttgtgtttgtcgtgctattgcttttggtggccccagcttaca gcttcaactgccttggaatgagcaacagagacttcttggaaggagtgtc tggagcaacatgggtggatttggttctcgaaggcgacagctgcgtgact atcatgtctaaggacaagcctaccatcgatgtgaagatgatgaatatgg aggcggtcaacctggcagaggtccgcagttattgctatttggctaccgt cagcgatctctccaccaaagctgcgtgcccgaccatgggagaagctcac aatgacaaacgtgctgacccagcttttgtgtgcagacaaggagtggtgg acaggggctggggcaacggctgcggattatttggcaaaggaagcattga cacatgcgccaaatttgcctgctctaccaaggcaataggaagaaccatc ttgaaagagaatatcaagtacgaagtggccatttttgtccatggaccaa ctactgtggagtcgcacggaaactactccacacaggttggagccactca ggcagggagattcagcatcactcctgcggcgccttcatacacactaaag cttggagaatatggagaggtgacagtggactgtgaaccacggtcaggga ttgacaccaatgcatactacgtgatgactgttggaacaaagacgttctt ggtccatcgtgagtggttcatggacctcaacctcccttggagcagtgct ggaagtactgtgtggaggaacagagagacgttaatggagtttgaggaac cacacgccacgaagcagtctgtgatagcattgggctcacaagagggagc tctgcatcaagctttggctggagccattcctgtggaattttcaagcaac actgtcaagttgacgtcgggtcatttgaagtgtagagtgaagatggaaa aattgcagttgaagggaacaacctatggcgtctgttcaaaggctttcaa gtttcttgggactcccgcagacacaggtcacggcactgtggtgttggaa ttgcagtacactggcacggatggaccttgcaaagttcctatctcgtcag tggcttcattgaacgacctaacgccagtgggcagattggtcactgtcaa cccttttgtttcagtggccacggccaacgctaaggtcctgattgaattg gaaccaccctttggagactcatacatagtggtgggcagaggagaacaac agatcaatcaccattggcacaagtctggaagcagcattggcaaagcctt tacaaccaccctcaaaggagcgcagagactagccgctctaggagacaca gcttgggactttggatcagttggaggggtgttcacctcagttgggaagg ctgtctaa (SEQIDNO:23) MRVVFVVLLLLVAPAYSFNCLGMSNRDFLEGVSGATWVDLVLEGDSCVT IMSKDKPTIDVKMMNMEAVNLAEVRSYCYLATVSDLSTKAACPTMGEAH NDKRADPAFVCRQGVVDRGWGNGCGLFGKGSIDTCAKFACSTKAIGRTI LKENIKYEVAIFVHGPTTVESHGNYSTQVGATQAGRFSITPAAPSYTLK LGEYGEVTVDCEPRSGIDTNAYYVMTVGTKTFLVHREWFMDLNLPWSSA GSTVWRNRETLMEFEEPHATKQSVIALGSQEGALHQALAGAIPVEFSSN TVKLTSGHLKCRVKMEKLQLKGTTYGVCSKAFKFLGTPADTGHGTVVLE LQYTGTDGPCKVPISSVASLNDLTPVGRLVTVNPFVSVATANAKVLIEL EPPFGDSYIVVGRGEQQINHHWHKSGSSIGKAFTTTLKGAQRLAALGDT AWDFGSVGGVFTSVGKAV*

(146) After agarose gel purification, the PCR fragment was cloned in PCR2.1-TOPO plasmid (Invitrogen) and sequenced to check that no mutations were introduced. After BsiWI/BssHII digestion of the PCR2.1-TOPO plasmid, the DNA fragment was cloned in the pTM-MVSchw vector in ATU position 2 giving plasmid: pTM-MVSchw-sE.sub.WNV according to FIG. 13.

(147) A7. Production of Recombinant Measles Virus Expressing WN sE

(148) To recover recombinant MV from plasmid, we used the helper-cell-based rescue system described by Radecke et al. [Radecke, 1995 (35)] and modified by Parks et al. [Parks, 1999 (40)]. Human helper cells stably expressing T7 RNA polymerase and measles N and P proteins (293-3-46 cells, a kind gift from MA Billeter, University of Zurich) were transfected using the calcium phosphate procedure with pTM-MVSchw-sE.sub.WNV plasmid (5 g) and a plasmid expressing the MV polymerase L gene (pEMC-La, 20 ng). After overnight incubation at 37 C., the transfection medium was replaced by fresh medium and a heat shock was applied (43 C. for two hours) [Parks, 1999 (40)]. After two days of incubation at 37 C., transfected cells were transferred on a CEF cells layer and incubated at 32 C. in order to avoid adaptation of the Schwarz vaccine that was originally selected on CEF cells and is currently grown on these cells. Infectious virus was recovered between 3 and 7 days following cocultivation. The recombinant virus was also rescued by the same technique after cocultivation of transfected 293-3-46 helper cells at 37 C. with Vero cells (african green monkey kidney, clone Vero-NK). In order to increase the yield of rescue and because these recombinant viruses were prepared to be used be used in mice experiments, we used Vero cells as producing cells in place of the usual chick embryo fibroblasts (CEF). Single syncytia were harvested and transferred to Vero cells grown in 35 mm wells in Dulbebecco's modified Eagle's medium (DMEM) supplemented with 5% fetal calf serum (FCS). The infected cells were expanded in 75 and 150 cm3 flasks. When syncytia reached 80-90% confluence (usually 36-48 hours post infection), the cells were scraped in a small volume of OptiMEM (Gibco BRL) and frozen and thawed once. After low-speed centrifugation to pellet cellular debris, the supernatant, which contained virus, was stored at 80 C. We have shown that two passages of the Schwarz virus on Vero cells did not change its immunogenic capacities in macaques.

(149) A8. Titration of Recombinant MV-WN Virus

(150) The titers of recombinant MV were determined by an endpoint limit dilution assay on Vero cells. 50% tissue culture infectious dose (TCID.sub.50) were calculated using the Karber method [Karber, 1931 (41)].

(151) A9. Immunofluorescence Detection of WNV sE Expressed in Vero Cells Infected by MV-WN sE Recombinant Virus.

(152) The expression of the WN sE protein in cells infected by recombinant MV-WN sE was detected by immunofluorescence. Vero cells were grown on polyornithine-coated coverslips and infected by MV-WN sE at an MOI of 0.05. After two days of infection, coverslips were washed twice in PBS and fixed for 15 minutes in paraformaldehyde (4% in PBS). In some cases, cells were permeabilized by Triton 100 (0.1%, 5 min). After two PBS washes, coverslips were incubated for 15 minutes at room temperature in PBS with 2% goat serum, then incubated for 1 hour at room temperature with mouse anti-WNV immune sera or mouse anti-WNV HMAF (see A5) diluted in PBS with 2% goat serum. After washing in PBS, cells were incubated for 45 minutes at room temperature with R-phycoerythrin-conjugated goat anti-mouse IgG (SBA, Birmingham). Following washing in PBS, coverslips were mounted on slides with fluoromount (Southern Biotech Associates inc., Birmingham, Ala.).

(153) A10. Anti-MV Antibody Detection by ELISA

(154) Anti-MV antibodies were detected using a standard ELISA kit (Trinity Biotech, USA). An anti-mouse antibody-HRP conjugate (Amersham) was used as the secondary antibody. Titers were determined by limiting dilutions and calculated as the highest dilution of serum giving twice the absorbence of a 1/100 dilution of a mixture of control sera.

(155) A.11 Neutralization Test by Reduction of Viral Replication Focuses (TNRF90) on VERO Cells.

(156) Successive dilutions of sera were prepared for testing in DMEM Glutamax with 2% decomplemented FCS (Fetal Calf Serum) in tubes of 0.5 ml.

(157) For 0.1 ml of diluted serum in DMEM Glutamax with 2% FCS, 0.1 ml of DMEM Glutamax/2% FCS containing 100 AP61 UFF of WN virus strain IS-98-ST1 was added.

(158) Control cell: 0.2 ml of DMEM 0.2% FCS

(159) Control virus: 0.2 ml of DMEM Glutamax/2% FCS containing 100 AP61UFF of WN virus strain IS-98-ST1.

(160) 2 hours with mild rotation at 37 C.

(161) Plates with 12 cups with 150 000 VERO HK cells per cup which are grown in monolayers for 24 hours in DMEM Glutamax 5% FCS

(162) 1 washing in DMEM of cell layers.

(163) Add 0.2 ml of DMEM Glutamax/2% SVF

(164) Add 0.2 ml of a mixture serum/WN virus on cell layers.

(165) Incubate 2 hours at 37 C. in CO.sub.2.

(166) Withdraw the serum/WN virus mixture of infected cell layers.

(167) 1 washing in DMEM of infected cell layers.

(168) Add 1 ml of DMEM 2% SVF per cup.

(169) Add 1 ml of CMC 1.6% diluted in DMEM Glutamax/2% SVF

(170) Incubate 2 days at 37 C. in CO.sub.2.

(171) The plaques were revealed through FIA technique. The last dilution of immunsera which neutralize at least 90 of 100 UFF of WN virus tested on VERO cells were determined (TNRF90: Test de Neutralisation par Reduction de Foyers de replication virale 90%). The titer of neutralizing antibodies of the sera was determined by TNRF90.

(172) A.12 Production of WN Virus Pseudo-Particles by Cell Line MEF/3T3.Tet-Off/Pr ME.WN # h2.

(173) Pseudo-particles of WN virus strain IS-98-ST1 composed of prME complexed glycoproteins were secreted by MEF/3T3.Tet-Off/pr ME.WN # h2 line induced for the expression of viral proteins (CNCM I-3018). They were purified for supernatants of 3-day cell culture according to the protocol used for WN virus purification.

(174) Passive seroprotection assay against WN virus in adult BALB/c mice.

(175) 6-week-old BALB/c mice were provided by the Janvier breeding Center. The dose for viral test is 100 ap61 UFF, i.e., 10 DL 50 (Tomoshi et al 2002) diluted in 100 l of DPBS supplemented with 0.2% BSA (Bovine Serum Albumine) pH7.5 (Sigma) which are inoculated intraperitoneally. The average time for lethal effect was 10 days. Animals were observed for 2 to 3 weeks.

(176) The sera to be tested for passive seroprotection in mice are diluted in 0.1% DPBS/0.2% BSA and inoculated 24 hours prior to viral test.

B) RESULTS AND CONCLUSIONS

(177) B1. Production of Recombinant Measles Virus Expressing WN sE

(178) cDNA encoding E protein of WNV strain IS-98-ST1 deleted for its transmembrane anchoring region was inserted in the genome of measles virus (Schwarz strain) according to FIG. 13.

(179) B.2. Preliminary Assays of Passive Seroprotection Against WN Virus in Mice

(180) Anti-WN immune sera to be tested were obtained from mice genetically resistant to the disease (52). The anti-WN sera, late taken, were injected at dilutions 1:10 (16 TNRF.sub.90) et 1:40 (4 TNRF.sub.90) in a final volume of 0.1 ml DPBS/0.2% SAB intraperitoneally in adult BALB/c mice genetically sensitive. The antibodies were administered only 24 hours prior to the viral test or 24 hours before and 24 hours after the test with 10 DL.sub.50 of strain IS-98-ST1 of WN virus. The negative control was the injection of normal serum of mice at 1:10. The neurovirulence of WN virus was evaluated in mice tested with DPBS/0.2% SAB. The results of passive protection after two weeks of viral tests were as follows:

(181) TABLE-US-00015 TABLE 1 Passive seroprotection against WNV encephalitis in adult BALB/c mice. Passive transfer Mortality MDOD* PBS/BSA (0.2%) 6\6 10.5 (1.5) normal serum (1:10) 6\6 12.5 (1.5) anti-WNV serum (1:10), 2 doses** 0\6 NA anti-WNV serum (1:40), 2 doses 0\6 NA anti-WNV serum (1:10), 1 dose*** 1\6 12 anti-WNV serum (1:40), 1 dose 0\6 NA (*Mean Day Of Death SD) (**Day 1 and Day +1 of virus challenge) (***Day 1 of virus challenge)

(182) To conclude, a unique injection of anti-WN antibodies (2.5 10 l of serum) obtained from mice genetically resistant to WN virus, said injection being carried out intraperitoneally in adult mice sensitive to viral encephalitis provides passive protection against a test dose.

(183) It is noted that the sera of BALB/c mice having received anti-WN protective antibodies and resisting to viral infection have anti-WN antibody titers by ELISA which are of about 1 DO unit (for a dilution of serum of 1:100) after one month of test. This indicates that the WN virus inoculated for the test has achieved replication in protected mice, inducing a humoral response. If passive seroprotection protects against lethal encephalitis due to WN virus, it does not seem to be appropriate in order to prevent viral propagration in infected individual.

(184) B.3. Vaccination of CD46.sup.+/ IFN-/R.sup./ Mice with MV/WN sE Virus

(185) Mice susceptible for MV infection were obtained as described previously [Mrkic, 1998 (21)]. FVB mice heterozygous for the CD46 MV receptor transgene [Yannoutsos, 1996 (32)] were crossed with 129Sv IFN-I3R.sup./ mice [Muller, 1994 (22)]. The F1 progeny was screened by PCR and the CD46.sup.+/ animals were crossed again with 129Sv IFN-/R.sup./ mice. IFN-/R.sup./ CD46.sup.+/ animals were selected and used for immunization experiments. Six-week-old CD46.sup.+/ IFN-/R.sup./ mice were inoculated intraperitoneally with a single dose of standard MV vaccine (10.sup.6 TCID.sub.50, 3 mice) or MV-WN sE recombinant virus (10.sup.4 or 10.sup.6 TCID.sub.50, 6 mice per dose) in 300 l phosphate buffer saline (PBS).

(186) A serum has been taken from eye after one month of vaccination with a unique dose in order to determine the production of anti-MV, anti-WN E and neutralizing antibodies against the test virus.

(187) b) Sera Diluted to 1:100 and Tested for Antibodies by ELISA on Purified NV Virion, for:

(188) TABLE-US-00016 DO unit Ascite of anti-WN mice: 1 (control+) Serum of anti-WN mice: 0.8 (control+) Serum of MV vaccinated mice: 0.110 0.005 Serum of MV/WN sE vaccinated mice, 10.sup.4 0.635 0.040 (males) DCIP.sub.50: Serum of MV/WN sE vaccinated mice, 10.sup.4 0.815 0.005 (females) DCIP.sub.50: Serum of MV/WN sE vaccinated mice, 10.sup.6 0.800 0.200 (males) DCIP.sub.50: Serum of MV/WN sE vaccinated mice, 10.sup.6 0.900 0.195 (females) DCIP.sub.50:
c) In Vitro Seroneutralization Test for WNV on VERO Cells.

(189) TNRF.sub.90 of pools of sera on 100 AP61UFF of strain IS-98-ST1 of WN virus in VERO cells:

(190) TABLE-US-00017 TNRF.sub.90 Serum of MV vaccinated mice: <10 Serum of MV vaccinated mice MV-WN sE, 10.sup.4 DCIP.sub.50: 400 Serum of MV vaccinated mice MV-WN sE, 10.sup.6 DCIP.sub.50: 800

(191) To conclude, antibodies directed against soluble E glycoprotein WN virus have the capacity to neutralize strain IS-98-ST1 used for the test by WN virus in mice in vitro.

(192) A vaccine boost in immunized CD46.sup.+/ IFN-/R.sup./ mice has been carried out 1 month after the beginning of vaccination with a unique dose, identical to the dose of the first injection.

(193) After 2 weeks of boosting, sera were tested by ELISA and in TNRF.sub.90 as above:

(194) a) Sera Diluted to 1:100 and Tested for Antibodies by ELISA on Purified WN Virion:

(195) TABLE-US-00018 DO Unit Ascite of anti-WN mice: 1.4 (control+) Serum of anti-WN mice: 1 (control+) Serum of MV vaccinated mice: 0.110 0.005 Serum of MV-WN sE vaccinated mice, 10.sup.4 0.810 0.100 (males) DCIP.sub.50: Serum of MV-WN sE vaccinated mice, 10.sup.4 1.150 0.015 (females) DCIP.sub.50: Serum of MV-WN sE vaccinated mice, 10.sup.6 0.965 0.230 (males) DCIP.sub.50: Serum of MV-WN sE vaccinated mice, 10.sup.6 1.075 0.240 (females) DCIP.sub.50:

(196) b) Seroneutralization Test in Vitro on VERO Cells

(197) TNRF.sub.90 of pools of sera on 100 AP61UFF of strain IS-98-ST1 of WN virus in VERO cells:

(198) TABLE-US-00019 TNRF.sub.90 Serum of boosted MV mice: <10 Serum of boosted MV-WN sE, 10.sup.4 DCIP.sub.50 mice: >1600 Serum of boosted MV-WN sE, 10.sup.6 DCIP.sub.50 mice: >1600

(199) After 4 weeks of boosting, the sera were tested by ELISA and in TNRF.sub.90 as above:

(200) a) Sera Diluted at 1:100 and Tested for Antibodies by ELISA on Purified WN Virion:

(201) TABLE-US-00020 DO unit Ascite of anti-WN mice: 1.7 (control+) Serum of anti-WN mice: 1.2 (control+) Serum of MV vaccinated mice: 0.2 Serum of MV-WN sE vaccinated mice, 10.sup.4 1.52 (0.15) DCIP.sub.50: Serum of MV-WN sE vaccinated mice, 10.sup.6 1.76 (0.10) DCIP.sub.50:

(202) b) Seroneutralization in Vitro on VERO Cells

(203) TNRF.sub.90 of pools of sera on 100 AP61UFF of strain IS-98-ST1 of WN virus on VERO cells:

(204) TABLE-US-00021 TNRF.sub.90 Serum of MV-WN sE vaccinated mice, 10.sup.4 DCIP.sub.50: 4000 (males) Serum of MV-WN sE vaccinated mice, 10.sup.4 DCIP.sub.50: 8000 (females) Serum of MV-WN sE vaccinated mice, 10.sup.6 DCIP.sub.50: 10 000-12 000

(205) To conclude, after a boost with a unique dose, the anti-WNV antibody titers and the anti-WNV neutralizing antibody titers were significantly increased by a 10-fold factor or more.

(206) Splenocytes of CD46.sup.+/ IFN-/R.sup./ mice immunized with two injections separated by 4 weeks with the MV-WN sE virus with doses of 10.sup.4 or 10.sup.6 DCIP.sub.50 are tested in ELISpot and flux/cytometry for the T CD4 and CD8 response after in vitro stimulation with purified viral pseudo-particules in saccharose gradients starting from supernatants of induced MEF/3T3.Tet-Off/prME.WN # h-2 (CNCM I-3018) cell line.

(207) B.4. Passive Anti-WN Seroprotection Test in BALB/c with Anti-E Antibodies

(208) Immune sera of CD46.sup.+/ IFN-/R.sup./ mice vaccinated with a unique dose of recombinant measles virus has been collected after one month. Various dilutions of these sera have been injected in a final volume of 0.1 ml in 6-week-old BALB/c mice and only 24 hours before inoculation of 100 AP61UFF of strain IS-98-ST1 of WN virus (10 DL.sub.50) intraperitoneally (see protocol in B2).

(209) The results of passive protection after two weeks of viral test are as follows:

(210) TABLE-US-00022 TABLE 2 Recombinant MV-WN sE induce antibodies that provide full protection against WNV encephalitis in BALB/c mice Passive transfer Mortality Day PBS/BSA (0.2%) 6\6 10 to 11 anti-WNV serum (1:10), 1 dose* 0\6 NA anti-WNV serum (1:40), 1 dose 1\6 20 anti-MV (1:10), 1 dose 4\6 10 to 11 anti-MV-WN sE 10e4 (1:10), 1 dose 3\6 8 to 10 anti-MV-WN sE 10e6 (1:10), 1 dose 0\6 NA anti-MV-WN sE 10e6 (1:40), 1 dose 0\6 NA anti-MV-WN sE 10e6 (1:100), 1 dose 3\6 10 to 11 (*Day 1 of virus challenge)

(211) To conclude, antibodies directed against WN-virus soluble glycoprotein E have the capacity to protect in vivo against WN-virus encephalitis. The vaccination of CD46.sup.+/ IFN-I3R.sup./ mice with a dose of 10.sup.6 DCIP.sub.50 of MV-WN sE virus as a unique injection is required to induce an anti-WN E humoral response on a four-week period of time which is capable of protecting against the disease by passive seroprotection. A minimal volume of 2.5 l of immune serum of mice vaccinated with MV-WN sE virus, is sufficient to provide a complete protection in adult BALB/c mice tested with a lethal dose of WN-virus (i.e., a ratio of about 0.1 ml of immune serum/kg). It is noted that anti-lethal sera diluted to 1:10 induce a partial protection (about 30%) against West Nile virus encephalitis.

(212) Sera obtained in vaccinated CD46.sup.+/ IFN-/R.sup./ mice which have then been boosted with a weak dose (10.sup.4 TCID.sub.50) will be tested for their capacity to provide passive protection in BALB/c mice.

(213) B.5. Viral Test on CD46.sup.+/ IFN-/R.sup./ Mice Vaccinated with MV-WN sE

(214) CD46.sup.+/ IFN- R.sup./ mice vaccinated 2 months after the 2 injections of 10.sup.6 DCIP.sub.50 of MV-WN sE virus, these injections being done at 4 weeks internal have been tested with 100 AP61UFF of strain IS-98-ST1 of WN virus administered intraperitoneally.

(215) The 2 mice vaccinated with standard measles virus died the 3rd day of the test. No morbidity or lethality was observed for mice vaccinated with MV-WN sE on the 7.sup.th day of the test. To conclude, CD46.sup.+/ IFN-/R.sup./ mice immunized against soluble gpE of WN virus are protected against a lethal test dose of WN virus in the absence of anti-viral activity of alpha-interferon.

(216) B6. New Test of Anti-WN Vaccination with an Antigen Boost

(217) Adult CD46.sup.+/ IFN-/R.sup./ mice are vaccinated on a 4 week period of time with MV-WN sE virus at a dose of 10.sup.4 DCIP.sub.50 which is proposed for human and a boost with an antigen is carried out with purified pseudo-particles of WN-virus which are secreted by the cell line MEF/3T3.Tet-Off/WN prME # h2.

BIBLIOGRAPHY

(218) 1. Amara, R. R., F. Villinger, J. D. Altman, S. L. Lydy, S. P. O'Neil, S. I. Staprans, D. C. Montefiori, Y. Xu, J. G. Herndon, L. S. Wyatt, M. A. Candido, N. L. Kozyr, P. L. Earl, J. M. Smith, H. L. Ma, B. D. Grimm, M. L. Hulsey, J. Miller, H. M. McClure, J. M. McNicholl, B. Moss, and H. L. Robinson. 2001. Control of a mucosal challenge and prevention of AIDS by a multiprotein DNA/MVA vaccine. Science. 292:69-74. 2. Baba, T. W., V. Liska, R. Hofmann-Lehmann, J. Vlasak, W. Xu, S. Ayehunie, L. A. Cavacini, M. R. Posner, H. Katinger, G. Stiegler, B. J. Bernacky, T. A. Rizvi, R. Schmidt, L. R. Hill, M. E. Keeling, Y. Lu, J. E. Wright, T. C. Chou, and R. M. Ruprecht. 2000. Human neutralizing monoclonal antibodies of the IgG1 subtype protect against mucosal simian-human immunodeficiency virus infection. Nat Med. 6:200-206. 3. Barnett, S. W., S. Lu, I. Srivastava, S. Cherpelis, A. Gettie, J. Blanchard, S. Wang, I. Mboudjeka, L. Leung, Y. Lian, A. Fong, C. Buckner, A. Ly, S. Hilt, J. Ulmer, C. T. Wild, J. R. Mascola, and L. Stamatatos. 2001. The ability of an oligomeric human immunodeficiency virus type 1 (HIV-1) envelope antigen to elicit neutralizing antibodies against primary HIV-1 isolates is improved following partial deletion of the second hypervariable region. J Virol. 75:5526-5540. 4. Binley, J. M., R. W. Sanders, B. Clas, N. Schuelke, A. Master, Y. Guo, F. Kajumo, D. J. Anselma, P. J. Maddon, W. C. Olson, and J. P. Moore. 2000. A recombinant human immunodeficiency virus type 1 envelope glycoprotein complex stabilized by an intermolecular disulfide bond between the gp120 and gp41 subunits is an antigenic mimic of the trimeric virion-associated structure. J Virol. 74:627-643. 5. Boyer, J., K. Ugen, B. Wang, M. Agadjanyan, L. Gilbert, M. Bagarazzi, M. Chattergoon, P. Frost, A. Javadian, W. Williams, Y. Refaeli, R. Ciccarelli, D. McCallus, L. Coney, and D. Weiner. 1997. Protection of chimpanzees from high-dose heterologous HIV-1 challenge by DNA vaccination. Nature Medicine. 3:526-532. 6. Burton, D. 1997. A vaccine for HIV type 1: the antibody perspective. Proceedings of the National Academy of Sciences of the United States of America. 94:10018-10023. 7. Calain, P., and L. Roux. 1993. The rule of six, a basic feature for efficient replication of Sendai virus defective interfering RNA. J Virol. 67:4822-4830. 8. Collman, R., J. W. Balliet, S. A. Gregory, H. Friedman, D. L. Kolson, N. Nathanson, and A. Srinivasan. 1992. An infectious molecular clone of an unusual macrophage-tropic and highly cytopathic strain of human immunodeficiency virus type 1. J Virol. 66:7517-7521. 9. Crotty, S., C. J. Miller, B. L. Lohman, M. R. Neagu, L. Compton, D. Lu, F. X. Lu, L. Fritts, J. D. Lifson, and R. Andino. 2001. Protection against simian immunodeficiency virus vaginal challenge by using Sabin poliovirus vectors. J Virol. 75:7435-7452. 10. Hoffman, T. L., C. C. LaBranche, W. Zhang, G. Canziani, J. Robinson, I. Chaiken, J. A. Hoxie, and R. W. Doms. 1999. Stable exposure of the coreceptor-binding site in a CD4-independent HIV-1 envelope protein. Proc Natl Acad Sci USA. 96:6359-6364. 11. Hu, S., P. Polacino, V. Stallard, J. Klaniecki, S. Pennathur, B. Travis, L. Misher, H. Kornas, A. Langlois, W. Morton, and R. Benveniste. 1996. Recombinant subunit vaccines as an approach to study correlates of protection against primate lentivirus infection. Immunology Letters. 51:115-119. 12. Karlsson, G. B., M. Halloran, J. Li, I. W. Park, R. Gomila, K. A. Reimann, M. K. Axthelm, S. A. Iliff, N. L. Letvin, and J. Sodroski. 1997. Characterization of molecularly cloned simian-human immunodeficiency viruses causing rapid CD4+ lymphocyte depletion in rhesus monkeys. J Virol. 71:4218-4225. 13. Kwong, P. D., R. Wyatt, S. Majeed, J. Robinson, R. W. Sweet, J. Sodroski, and W. A. Hendrickson. 2000. Structures of HIV-1 gp120 envelope glycoproteins from laboratory-adapted and primary isolates. Structure Fold Des. 8:1329-1339. 14. Kwong, P. D., R. Wyatt, J. Robinson, R. W. Sweet, J. Sodroski, and W. A. Hendrickson. 1998. Structure of an HIV gp120 envelope glycoprotein in complex with the CD4 receptor and a neutralizing human antibody. Nature. 393:648-659. 15. Kwong, P. D., R. Wyatt, Q. J. Sattentau, J. Sodroski, and W. A. Hendrickson. 2000. Oligomeric modeling and electrostatic analysis of the gp120 envelope glycoprotein of human immunodeficiency virus. J Virol. 74:1961-1972. 16. Mascola, J. R., M. G. Lewis, G. Stiegler, D. Harris, T. C. VanCott, D. Hayes, M. K. Louder, C. R. Brown, C. V. Sapan, S. S. Frankel, Y. Lu, M. L. Robb, H. Katinger, and D. L. Birx. 1999. Protection of Macaques against pathogenic simian/human immunodeficiency virus 89.6PD by passive transfer of neutralizing antibodies. J Virol. 73:4009-4018. 17. Mascola, J. R., M. K. Louder, T. C. VanCott, C. V. Sapan, J. S. Lambert, L. R. Muenz, B. Bunow, D. L. Birx, and M. L. Robb. 1997. Potent and synergistic neutralization of human immunodeficiency virus (HIV) type 1 primary isolates by hyperimmune anti-HIV immunoglobulin combined with monoclonal antibodies 2F5 and 2G12. J Virol. 71:7198-7206. 18. Mascola, J. R., and G. J. Nabel. 2001. Vaccines for prevention of HIV-1 disease. Immunology. 13:489-495. 19. Mascola, J. R., G. Stiegler, T. C. VanCott, H. Katinger, C. B. Carpenter, C. E. Hanson, H. Beary, D. Hayes, S. S. Frankel, D. L. Birx, and M. G. Lewis. 2000. Protection of macaques against vaginal transmission of a pathogenic HIV-1/SIV chimeric virus by passive infusion of neutralizing antibodies. Nat Med. 6:207-210. 20. Mrkic, B., B. Odermatt, M. Klein, M. Billeter, J. Pavlovic, and R. Cattaneo. 1999. Lymphatic dissemination and comparative pathology of recombinant measles viruses in genetically modified mice. Journal of Virology. 74:1364-1372. 21. Mrkic, B., J. Pavlovic, T. Rulicke, P. Volpe, C. J. Buchholz, D. Hourcade, J. P. Atkinson, A. Aguzzi, and R. Cattaneo. 1998. Measles virus spread and pathogenesis in genetically modified mice. J Virol. 72:7420-7427. 22. Mliller, U., U. Steinhoff, L. F. L. Reis, S. Hemmi, J. Pavlovic, R. M. Zinkernagel, and M. Aguet. 1994. Functional role of type I and type II interferons in antiviral defense. Science. 264:1918-1921. 23. Muster, T., F. Steindl, M. Purtscher, A. Trkola, A. Klima, G. Himmler, F. Ruker, and H. Katinger. 1993. A conserved neutralizing epitope on gp41 of human immunodeficiency virus type 1. J Virol. 67:6642-6647. 24. Naniche, D., G. Varior-Krishnan, F. Cervoni, T. F. Wild, B. Rossi, C. Rabourdin-Combe, and D. Gerlier. 1993. Human membrane cofactor protein (CD46) acts as a cellular receptor for measles virus. J Virol. 67:6025-6032. 25. Parren, P. W., M. C. Gauduin, R. A. Koup, P. Poignard, P. Fisicaro, D. R. Burton, and Q. J. Sattentau. 1997. Relevance of the antibody response against human immunodeficiency virus type 1 envelope to vaccine design. Immunol Lett. 57:105-112. 26. Reimann, K. A., J. T. Li, R. Veazey, M. Halloran, I. W. Park, G. B. Karlsson, J. Sodroski, and N. L. Letvin. 1996. A chimeric simian/human immunodeficiency virus expressing a primary patient human immunodeficiency virus type 1 isolate env causes an AIDS-like disease after in vivo passage in rhesus monkeys. J Virol. 70:6922-6928. 27. Sanders, R. W., L. Schiffner, A. Master, F. Kajumo, Y. Guo, T. Dragic, J. P. Moore, and J. M. Binley. 2000. Variable-loop-deleted variants of the human immunodeficiency virus type 1 envelope glycoprotein can be stabilized by an intermolecular disulfide bond between the gp120 and gp41 subunits. J Virol. 74:5091-5100. 28. Schneider, H., K. Kaelin, and M. A. Billeter. 1997. Recombinant measles viruses defective for RNA editing and V protein synthesis are viable in cultured cells. Virology. 227:314-322. 29. Shiver, J. W., T. M. Fu, L. Chen, D. R. Casimiro, M. E. Davies, R. K. Evans, Z. Q. Zhang, A. J. Simon, W. L. Trigona, S. A. Dubey, L. Huang, V. A. Harris, R. S. Long, X. Liang, L. Handt, W. A. Schleif, L. Zhu, D. C. Freed, N. V. Persaud, L. Guan, K. S. Punt, A. Tang, M. Chen, K. A. Wilson, K. B. Collins, G. J. Heidecker, V. R. Fernandez, H. C. Perry, J. G. Joyce, K. M. Grimm, J. C. Cook, P. M. Keller, D. S. Kresock, H. Mach, R. D. Troutman, L. A. Isopi, D. M. Williams, Z. Xu, K. E. Bohannon, D. B. Volkin, D. C. Montefiori, A. Miura, G. R. Krivulka, M. A. Lifton, M. J. Kuroda, J. E. Schmitz, N. L. Letvin, M. J. Caulfield, A. J. Bett, R. Youil, D. C. Kaslow, and E. A. Emini. 2002. Replication-incompetent adenoviral vaccine vector elicits effective anti-immunodeficiency-virus immunity. Nature. 415:331-335. 30. Thali, M., J. P. Moore, C. Furman, M. Charles, D. D. Ho, J. Robinson, and J. Sodroski. 1993. Characterization of conserved human immunodeficiency virus type 1 gp120 neutralization epitopes exposed upon gp120-CD4 binding. J Virol. 67:3978-3988. 31. Trkola, A., M. Purtscher, T. Muster, C. Ballaun, A. Buchacher, N. Sullivan, K. Srinivasan, J. Sodroski, J. P. Moore, and H. Katinger. 1996. Human monoclonal antibody 2G12 defines a distinctive neutralization epitope on the gp120 glycoprotein of human immunodeficiency virus type 1. J Virol. 70:1100-1108. 32. Yannoutsos, N., J. N. Ijzermans, C. Harkes, F. Bonthuis, C. Y. Zhou, D. White, R. L. Marquet, and F. Grosveld. 1996. A membrane cofactor protein transgenic mouse model for the study of discordant xenograft rejection [published erratum appears in Genes Cells 1996 August; 1(8):785]. Genes Cells. 1:409-419. 33. Griffin, D. 2001. Measles virus, P. 1401-1441. In D. Knipe and P. Howley (ed.), Field's Virology, 4.sup.th Edition, vol. 2. LippincottRaven Publishers, Philadelphia. 34. Hilleman, M. 2002. Current overview of the pathogenesis and prophylaxis of measles with focus on practical implications. Vaccine. 20:651-665. 35. Radecke, F., P. Spielhofer, H. Schneider, K. Kaelin, M. Huber, C. Dotsch, G. Christiansen, and M. A. Billeter. 1995. Rescue of measles viruses from cloned DANN. Embdo J. 14: 5773-5784. 36. Radecke, F., and Billeter. 1997. Reverse genetics meets the nonsegmented negative-strand RNA viruses. Reviews in Medical Virology. 7:49-63. Singh, M., R. Cattaneo, and M. A. Billeter. 1999. A recombinant measles virus expressing hepatitis B virus surface antigen induces humoral immune responses in genetically modified mice. J. Virol. 73: 4823-4828. Spielhofer, P., T. Bachi, T. Fehr, G. Christiansen, R. Cattaneo, K. Kaelin, M. Billeter, and H. Naim. 1998. Chimeric measles viruses with a foreign envelope. J. Virol. 72: 2150-2159. 39. Srivastava, I., K. Vandorsten, L. Vojtech, S. Barnett, and L. Stamatos. 2003. Changes in the immunogenic properties of soluble gp140 human immunodeficiency virus envelope constructs upon partial deletion of the second hypervariable region. J. Virol. 77:2310-2320. 40. Parks, C. L., R. A. Lerch, P. Walpita, M. S. Sidhu, and S. A. Udem. 1999. Enhanced measles virus cDNA rescue and gene expression after heat shock. J. Virol. 73: 3560-3566. 41. Karber, G. 1931. Breitag zur kollektiven Behandlung pharmakologischer Reihenversuche. Arch Exp Path Pharmak. 162: 480-483. 42. Burrer, R., D. Salmon-Ceron, S. Richert, G. Pancino, G. Spiridon, S. Haessig, V. Roques, F. Barre-Sinoussi, A. M. Aubertin, and C. Moog. 2001. Immunoglobulin G (IgG) and IgA, but also nonantibody factors, account for in vitro neutralization of human immunodeficiency virus (HIV) type 1 primary isolates by serum and plasma of HIV-infected patients. J Virol. 75: 5421-5424. 43. Charneau, P., G. Mirambeau, P. Roux, S. Paulous, H. Buc, and F. Clavel 1994. HIV-1 reverse transcription. A termination step at the center of the genome. J Mol Biol. 241:651-662. 44. Coeffier, E., J. Clement, V. Cussac, N. Khodaei-Boorane, M. Jehanno, M. Rojas, A. Dridi, M. Latour, R. El Habib, F. Barre-Sinoussi, M. Hofnung, and C. Leclerc. 2001. Antigenicity and immunogenicity of the HIV-1 gp41 epitope ELDKWA inserted into permissive sites of the MalE protein. Vaccine. 19:684-693. 45. Mascola, J. R., M. K. Louder, T. C. VanCott, C. V. Sapan, J. S. Lambert, L. R. Muenz, B. Bunow, D. L. Birx, and M. L. Robb. 1997. Potent and synergistic neutralization of human immunodeficiency virus (HIV)) type 1 primary isolates by hyperimmune anti-HIV immunoglobulin combined with monoclonal antibodies 2F5 and 2G12. J Virol. 71: 7198-7206. 45. Eckhart, L., W. Raffelberger, B. Ferko, A. Klima, M. Purtscher, H. Katinger, and F. Riker. 1996. Immunogenic presentation of a conserved gp41 epitope of human immunodeficiency virus type I on recombinant surface antigen of hepatitis B. virus. J. Gene. Virol. 77: 2001-2008. 46. Ho, J., K. MacDonald, and B. Barber. 2002. Construction of recombinant targeting immunogens incorporating an HIV-1 neutralizing epitope into sites of differing conformational constrain. Vaccine. 20: 1169-1180. 47. Liang, X., S. Munshi, J. Shendure, Mark, M. Davies, D. Freed, D. Montefiori, and J. Shiver. 1999. Epitope insertion into variable loops of HIV-1 gp120 as a potential means to improve immunogenicity of viral envelope protein. Vaccine. 17: 2862-2872. 48. Jeffs, S., C. Shotton, P. Balfe, and J. McKeating. 2002. Truncated gp120 envelope glycoprotein of human immunodeficiency virus 1 elicits broadly reactive neutralizing immune response. J. Gen. Virol. 83: 2723-2732. 49. Kwong, P., and e. al. 2002. HIV evades antibody-mediated neutralization through conformational masking of receptor-binding sites. Nature. 420-678-682. 50. Dilraj. A., F. T. Cutts, J. F. de Castro, J. G. Wheeler, D. Brown, C. Roth, H. M. Coovadia, and J. V. Benett. 2000, Lancet. 355:798-803. 51. Despres et al, 1993. Virology 196: 209-219 52. Mashimo et al. 2002. PNAS. USA 99: 11311-11316 53. Lucas et al. 2003. Immunol. Cell. Biol. 81(3): 230-6.