Lentiviral vectors for expression of hepatitis B virus (HBV) antigens

Abstract

The invention relates to nucleic acids, including lentiviral vectors and lentiviral vector particles, encoding at least one Hepatitis B virus (HBV) envelop surface of genotypes A and/or C antigen, at least one polymerase of genotypes A and/or C antigen, at least one HBX protein of genotypes A and/or C antigen, at least one HBV consensus core of genotypes A and/or C antigen, and at least one HBV consensus core MHCI and MHCII epitopes of genotypes A and/or C antigen. The invention encompasses these lentiviral vectors and lentiviral vector particles, methods of making the vectors, and their use, including medicinal uses. The lentiviral vectors and lentiviral vector particles are for use in administering to humans to induce immune responses against the HBV antigens.

Claims

1. A lentiviral vector encoding: at least one Hepatitis B virus (HBV) envelop surface of genotype A antigen, genotype C antigen or genotypes A and C antigen, at least one polymerase of genotype A antigen, genotype C antigen or genotypes A and C antigen, at least one HBX protein of genotype A antigen, genotype C antigen or genotypes A and C antigen, at least one HBV consensus core of genotype A antigen, genotype C antigen or genotypes A and C antigen, and at least one HBV consensus core MHCI and/or MHCII epitope of genotype A antigen, genotype C antigen or genotypes A and C antigen.

2. The lentiviral vector of claim 1, wherein the lentiviral vector encodes at least the amino acid sequence of SEQ ID NO:24.

3. The lentiviral vector of claim 1, wherein the vector encodes at least the amino acid sequence of SEQ ID NO:25.

4. The lentiviral vector according to claim 1, wherein the lentiviral vector encodes at least the amino acid sequence of SEQ ID NO:24 and at least the amino acid sequence of SEQ ID NO:25.

5. The lentiviral vector according to claim 1, wherein the lentiviral vector comprises a 2-microglobulin promoter.

6. The lentiviral vector according to claim 1, wherein the lentiviral vector is a DNA.

7. A lentiviral vector particle encoding: at least one Hepatitis B virus (HBV) envelop surface of genotype A antigen, genotype C antigen or genotypes A and C antigen, at least one polymerase of genotype A antigen, genotype C antigen or genotypes A and C antigen, at least one HBX protein of genotype A antigen, genotype C antigen or genotypes A and C antigen, at least one HBV consensus core of genotype A antigen, genotype C antigen or genotypes A and C antigen, and at least one HBV consensus core MHCI and/or MHCII epitope of genotype A antigen, genotype C antigen or genotypes A and C antigen.

8. The lentiviral vector particle according to claim 7, wherein the vector encodes the amino acid sequence of SEQ ID NO:24.

9. The lentiviral vector particle according to claim 7, wherein the vector encodes the amino acid sequence of SEQ ID NO:25.

10. The lentiviral vector particle according to claim 7, wherein the vector encodes at least the amino acid sequence of SEQ ID NO:24 and at least the amino acid sequence of SEQ ID NO:25.

11. The lentiviral vector particle according to claim 7, wherein the lentiviral vector particle comprises a functional lentiviral integrase protein.

12. The lentiviral vector particle according to claim 7, wherein the lentiviral vector particle comprises a vesicular stomatitis virus glycoprotein.

13. The lentiviral vector particle according to claim 7, wherein the lentiviral vector particle comprises HIV-1 subtype D Gag and Pol proteins.

14. An isolated cell comprising the lentiviral vector according to claim 1.

15. A composition comprising a lentiviral vector according to claim 1.

16. An isolated cell comprising the lentiviral vector particle according to claim 7.

17. A composition comprising a lentiviral vector according to claim 7.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIG. 1 depicts a lentiviral vectored HBV vaccine construct HBV-3 according to the invention. This construct comprises the amino acid sequence of SEQ ID NO:24 (HBV Genotype A & C Surface, Pol, HBX and consensus Core MHCI and MHCII epitopes sequence) and the amino acid sequence of SEQ ID NO:25 (Chimeric VLP with Surface B cell epitopes of genotype A&C sequence).

(2) FIG. 2 depicts cumulated immune response and diversity of the T-cell response (IFN- secretion) in C57Bl/6j mice with the HBV-3 lentiviral vector.

(3) Different doses of injected lentiviral vectors are compared (1.Math.10.sup.7 TU and 1.Math.10.sup.6 TU) as well as different administration strategies (prime/boost (P/B) or prime (P)). From the bottom to the top of each bar: VLP, PolA, EnvA, XA, PoIC, EnvC, XC and Core. A and C are HBV genotypes.

DETAILED DESCRIPTION OF THE INVENTION

(4) The inventors have discovered that the intramuscular administration to an animal of a lentiviral vector encoding an antigen results in a high immune response against the protein and can lead to elimination of the integrated vector from the animal. Thus, the invention provides for new lentivectors having high immune responses and increased safety for human administration.

(5) The present invention encompasses lentiviral vectors encoding: at least one Hepatitis B virus (HBV) envelop surface of genotypes A and/or C antigen, at least one polymerase of genotypes A and/or C antigen, at least one HBX protein of genotypes A and/or C antigen, at least one HBV consensus core of genotypes A and/or C antigen, and at least one HBV consensus core MHCI and MHCII epitopes of genotypes A and/or C antigen,
and their use for the induction of immune responses in a host, preferably in a human being, in particular by intramuscular administration.

(6) Lentiviral vectors can enable the induction of a strong, lasting and broad cell-mediated response and humoral response. LVs can incorporate multi-antigenic cassettes allowing the development of multivalent vaccines in a single dose. Filovirus outbreaks occur unexpectedly and spread rapidly. Thus, under these settings, short vaccine regimens may be required. Due to high expression levels of the inserts and the tropism for dendritic cells, THVs Lentiviral vectored vaccines can elicit strong immune responses in short period. Conversely, long-term protective immunity will likely involve a prime-boost regimen consisting of two or more injections that can induce durable T-cell memory.

(7) Proteins

(8) The invention encompasses lentiviral vector constructions encoding proteins comprising at least one: at least one Hepatitis B virus (HBV) envelop surface of genotypes A and/or C antigen, at least one polymerase of genotypes A and/or C antigen, at least one HBX protein of genotypes A and/or C antigen, at least one HBV consensus core of genotypes A and/or C antigen, and at least one HBV consensus core MHCI and MHCII epitopes of genotypes A and/or C antigen.

(9) Preferably, the at least one hepatitis B virus (HBV) envelop surface of genotypes A and/or C antigen is inserted in the at least one HBV consensus core of genotypes A and/or C antigen, thus forming a chimeric protein. More preferably, said chimeric protein comprises or consists in the amino acid sequence SEQ ID NO: 25.

(10) The HBV consensus core MHCI and MHCII epitopes of genotypes A and/or C antigen consists of different MHCI and MHCII epitopes of core HBV fused and/or linked to one another by short linking sequences, preferably via a short sequence of two amino acids GS, and preferably comprises or consists in the amino acid sequence SEQ ID NO: 26:

(11) TABLE-US-00001 (SEQIDNO:26) MDIDPYKEFGATVELLSFLPSDFFPSVRDLGSALESPEHCSPHHTALRQA ILGSELMTLATWVGSSRDLVVNYVNTNMGLKIRQLLWFHISCLTFGRETV LEYLVSFGVWIRTPPGSILSTLPETTVVRRRDRGRSPRRRT.

(12) Preferably, the at least one polymerase of genotypes A and/or C antigen, the at least one HBX protein of genotypes A and/or C antigen, and the at least one HBV consensus core MHCI and MHCII epitopes of genotypes A and/or C antigen are comprised in or form the amino acid sequence SEQ ID NO: 24.

(13) Preferably, the at least one polymerase of genotypes A and/or C antigen according to the invention is at least one polymerase of genotype A and at least one polymerase of genotype C.

(14) Preferably, the at least one HBX protein of genotypes A and/or C antigen according to the invention is at least one HBX protein of genotype A and at least one HBX protein of genotype C.

(15) Preferably, the at least one Hepatitis B virus (HBV) envelop surface of genotypes A and/or C antigen according to the invention is at least one Hepatitis B virus (HBV) envelop surface of genotype A and at least one Hepatitis B virus (HBV) envelop surface of genotype C.

(16) In a preferred embodiment: the at least one polymerase of genotypes A and/or C antigen according to the invention is at least one polymerase of genotype A and at least one polymerase of genotype C; the at least one HBX protein of genotypes A and/or C antigen according to the invention is at least one HBX protein of genotype A and at least one HBX protein of genotype C; and the at least one Hepatitis B virus (HBV) envelop surface of genotypes A and/or C antigen according to the invention is at least one Hepatitis B virus (HBV) envelop surface of genotype A and at least one Hepatitis B virus (HBV) envelop surface of genotype C.

(17) HBV Genotype A & C Surface, Pol, HBX and core MHC I and MHCII epitopes sequence:

(18) TABLE-US-00002 (SEQIDNO:24) MWPAANQVGVGAFGPGLGSMQWNSTAFHQALQDPRVRGLGSSISARTGDP VGSVLQAGFFLLTRILTIPQSLDSWWTSLNFLGGSPVCLGQNSQSPTSNH SPTSCPPICPGYRWMCLRRFGSIIFLFILLLCLIFLLVLLDYQGMLGSCT TPAQGNSMFPSCCCTKPTDGNGSIPSSWAFAGSRFSWLSLLVPFVQWFVG LSPTVWLSAIWMMWYWGPSLYSIVSPFIPLLPIFFCLWVYIGSNVSIPWT HKVGNFTGLYSSTVPIGSVGPLTVNEKRRLKLIGSRHYLHTLWKAGILYK GSTPARVTGGVFGSESRLVVDFSQFSRGITRVSWPKFAVPNLQSLTNLLS SNLSWLSLDVSAAFYHIPLHPAAMPHLLIGSSGLSRYVARLGSLHLYSHP IVLGFRKIGSSAICSVVRRAFPHCLAFSYMDDVVLGAGSFLLSLGIHLGS KHCFRKLPVNRPIDWGSYPALMPLYACIQAKQAFTFSPTYKAFLSGSLCQ VFADATPTGWGLGSKYTSFPWLLGCTANWILRGTSFVYVPGSSLYAVSPS VHLSLRGLPVGSVLHKRTLGLPAMSTTDLGSCVFKDWEELGEEIRLKVLF VLGGCRHKLGSWPEANQVGAGAFGPGFGSMQWNSTTFHQALLDPRVRGLY FPAGGSSSGGSTSLNFLGGAPTCPGQGSCTIPAQGTSMFPSCCCTKPSDG NCTCGSGLSPTVWLSVIWMMWYWGPSLYNILSPFLPLLPIFFCLWVYIGS NVSIPWTHKVGNFTGLYSSTVPVGSLHLYSHPIILGFRKIGSKQCFRKLP VNRPIDWGSKQAFTFSPTYKAFLCGSLCQVFADATPTGWGLGSAANWILR GTSFVYVPGSVLHKRTLGLSAMSTTDLGSCLFKDWEELGEEIRLMIFVLG GCRHKLGSMDIDPYKEFGATVELLSFLPSDFFPSVRDLGSALESPEHCSP HHTALRQAILGSELMTLATWVGSSRDLVVNYVNTNMGLKIRQLLWFHISC LTFGRETVLEYLVSFGVWIRTPPGSILSTLPETTVVRRRDRGRSPRRRT.

(19) Chimeric VLP with Surface B cell epitopes sequence of genotype A&C:

(20) TABLE-US-00003 (SEQIDNO:25) MDIDPYKEFGATVELLSFLPSDFFPSVRDLLDTASALYREALESPEHCSP HHTALRQAILCWGELMTLATWVGNNLEDGGGGSGGGGTPLGFFPDHQLDP AFGANSNNPDWDFNGSASTNRQSGRQPTPISPPLRDSHPQMQWNSTAFHQ ALQDPRVRGLYFPAGGSSSGTVNPAPNIASHISSISARTGDGSCKTCTTP AQGNSMFPSCCCTKPTDGNCTCIPIPSSWAFAKYLWEWASGSPFVQWFVG LAMQWNSTTFHQALLDPRVRGLYFPAGGSSSGTVNPVPTTASPISSIFSR TGDGSCKTCTIPAQGTSMFPSCCCTKPSDGNCTCIPIPSSWAFARFLWEW ASGSGGGGSGGGGSRDLVVNYVNTNMGLKIRQLLWFHISCLTFGRETVLE YLVSFGVWIRTPPAYRPPNAPILSTLPETTVVRRRDRGRSPRRRTPSPRR RRSQSPRRRRSQSRESQC.

(21) The proteins can be purified.

(22) Preferably, the purified protein is more than 50%, 75%, 85%, 90%, 95%, 97%, 98%, or 99% pure. Within the context of this invention, a purified protein that is more than 50% (etc.) pure means a purified protein sample containing less than 50% (etc.) other proteins. For example, a sample of a recombinant protein purified from a host cell can be 99% pure if it contains less than 1% contaminating host cell proteins.

(23) Nucleic Acids

(24) The invention encompasses nucleic acids encoding: at least one Hepatitis B virus (HBV) envelop surface of genotypes A and/or C antigen, at least one polymerase of genotypes A and/or C antigen, at least one HBX protein of genotypes A and/or C antigen, at least one HBV consensus core of genotypes A and/or C antigen, and at least one HBV consensus core MHCI and MHCII epitopes of genotypes A and/or C antigen.

(25) The nucleic acid can be single-stranded or double-stranded. The nucleic acid can be an RNA or DNA molecule.

(26) Preferred nucleic acids encode an amino acid sequence of any of the SEQ ID NOs detailed herein, either alone or combined, such as SEQ ID NO:24 and SEQ ID NO:25. The invention encompasses an isolated nucleic acid of the invention inserted into a vector.

(27) The nucleic acid can be purified. Preferably, the purified nucleic acid is more than 50%, 75%, 85%, 90%, 95%, 97%, 98%, or 99% pure. Within the context of this invention, a purified nucleic acid that is more than 50% pure means a purified nucleic acid sample containing less than 50% other nucleic acids. For example, a sample of a plasmid purified from a host bacteria can be 99% pure if it contains less than 1% contaminating bacterial DNA.

(28) Preferably, the at least one polymerase of genotypes A and/or C antigen encoded by a nucleic acid according to the invention is at least one polymerase of genotype A and at least one polymerase of genotype C.

(29) Preferably, the at least one HBX protein of genotypes A and/or C antigen encoded by a nucleic acid according to the invention is at least one HBX protein of genotype A and at least one HBX protein of genotype C.

(30) Preferably, the at least one Hepatitis B virus (HBV) envelop surface of genotypes A and/or C antigen encoded by a nucleic acid according to the invention is at least one Hepatitis B virus (HBV) envelop surface of genotype A and at least one Hepatitis B virus (HBV) envelop surface of genotype C.

(31) In a preferred embodiment: the at least one polymerase of genotypes A and/or C antigen encoded by a nucleic acid according to the invention is at least one polymerase of genotype A and at least one polymerase of genotype C; the at least one HBX protein of genotypes A and/or C antigen encoded by a nucleic acid according to the invention is at least one HBX protein of genotype A and at least one HBX protein of genotype C; and the at least one Hepatitis B virus (HBV) envelop surface of genotypes A and/or C antigen encoded by a nucleic acid according to the invention is at least one Hepatitis B virus (HBV) envelop surface of genotype A and at least one Hepatitis B virus (HBV) envelop surface of genotype C.

(32) Vectors

(33) The invention encompasses vectors encoding: at least one Hepatitis B virus (HBV) envelop surface of genotypes A and/or C antigen, at least one polymerase of genotypes A and/or C antigen, at least one HBX protein of genotypes A and/or C antigen, at least one HBV consensus core of genotypes A and/or C antigen, and at least one HBV consensus core MHCI and MHCII epitopes of genotypes A and/or C antigen, and preferably comprising a SEQ ID of the invention.

(34) Preferably, the at least one polymerase of genotypes A and/or C antigen encoded by a vector, in particular a lentiviral vector, according to the invention is at least one polymerase of genotype A and at least one polymerase of genotype C.

(35) Preferably, the at least one HBX protein of genotypes A and/or C antigen encoded by a vector, in particular a lentiviral vector, according to the invention is at least one HBX protein of genotype A and at least one HBX protein of genotype C.

(36) Preferably, the at least one Hepatitis B virus (HBV) envelop surface of genotypes A and/or C antigen encoded by a vector, in particular a lentiviral vector, according to the invention is at least one Hepatitis B virus (HBV) envelop surface of genotype A and at least one Hepatitis B virus (HBV) envelop surface of genotype C.

(37) In a preferred embodiment: the at least one polymerase of genotypes A and/or C antigen encoded by a vector, in particular a lentiviral vector, according to the invention is at least one polymerase of genotype A and at least one polymerase of genotype C; the at least one HBX protein of genotypes A and/or C antigen encoded by a vector, in particular a lentiviral vector, according to the invention is at least one HBX protein of genotype A and at least one HBX protein of genotype C; and the at least one Hepatitis B virus (HBV) envelop surface of genotypes A and/or C antigen encoded by a vector, in particular a lentiviral vector, according to the invention is at least one Hepatitis B virus (HBV) envelop surface of genotype A and at least one Hepatitis B virus (HBV) envelop surface of genotype C.

(38) Preferred vectors comprise a nucleic acid sequence encoding an amino acid sequence of any of the SEQ ID NOs detailed herein, either alone or combined, and in particular SEQ ID NO:24 and/or SEQ ID NO:25, preferably SEQ ID NO:24 and SEQ ID NO:25.

(39) The vector can be an expression vector. The vector can be a plasmid vector. Preferably, the vector is a lentiviral vector.

(40) Within the context of this invention, a lentiviral vector means a non-replicating vector for the transduction of a host cell with a transgene comprising cis-acting lentiviral RNA or DNA sequences, and requiring lentiviral proteins (e.g., Gag, Pol, and/or Env) that are provided in trans. The lentiviral vector lacks expression of functional Gag, Pol, and Env proteins. The lentiviral vector may be present in the form of an RNA or DNA molecule, depending on the stage of production or development of said retroviral vectors.

(41) The lentiviral vector can be in the form of a recombinant DNA molecule, such as a plasmid. The lentiviral vector can be in the form of a lentiviral vector particle, such as an RNA molecule(s) within a complex of lentiviral and other proteins. Typically, lentiviral particle vectors, which correspond to modified or recombinant lentivirus particles, comprise a genome which is composed of two copies of single-stranded RNA. These RNA sequences can be obtained by transcription from a double-stranded DNA sequence inserted into a host cell genome (proviral vector DNA) or can be obtained from the transient expression of plasmid DNA (plasmid vector DNA) in a transformed host cell.

(42) Preferably the lentiviral vector particles have the capacity for integration. As such, they contain a functional integrase protein.

(43) Non-integrating vector particles have one or more mutations that eliminate most or all of the integrating capacity of the lentiviral vector particles. For, example, a non-integrating vector particle can contain mutation(s) in the integrase encoded by the lentiviral pol gene that cause a reduction in integrating capacity. In contrast, an integrating vector particle comprises a functional integrase protein that does not contain any mutations that eliminate most or all of the integrating capacity of the lentiviral vector particles.

(44) Lentiviral vectors derive from lentiviruses, in particular human immunodeficiency virus (HIV-1 or HIV-2), simian immunodeficiency virus (SIV), equine infectious encephalitis virus (EIAV), caprine arthritis encephalitis virus (CAEV), bovine immunodeficiency virus (BIV) and feline immunodeficiency virus (FIV), which are modified to remove genetic determinants involved in pathogenicity and introduce new determinants useful for obtaining therapeutic effects.

(45) Such vectors are based on the separation of the cis- and trans-acting sequences. In order to generate replication-defective vectors, the trans-acting sequences (e.g., gag, pol, tat, rev, and env genes) can be deleted and replaced by an expression cassette encoding a transgene.

(46) Efficient integration and replication in non-dividing cells generally requires the presence of two cis-acting sequences at the center of the lentiviral genome, the central polypurine tract (cPPT) and the central termination sequence (CTS). These lead to the formation of a triple-stranded DNA structure called the central DNA flap, which acts as a signal for uncoating of the pre-integration complex at the nuclear pore and efficient importation of the expression cassette into the nucleus of non-dividing cells, such as dendritic cells.

(47) In one embodiment, the invention encompasses a lentiviral vector comprising a central polypurine tract and central termination sequence referred to as cPPT/CTS sequence as described, in particular, in the European patent application EP 2 169 073.

(48) Further sequences are usually present in cis, such as the long terminal repeats (LTRs) that are involved in integration of the vector proviral DNA sequence into a host cell genome. Vectors may be obtained by mutating the LTR sequences, for instance, in domain U3 of said LTR (U3) (Miyoshi H et al, 1998, J Virol. 72(10):8150-7; Zufferey et al., 1998, J Virol 72(12):9873-80) as shown in FIG. 1.

(49) Preferably, the vector does not contain an enhancer. In one embodiment, the invention encompasses a lentiviral vector comprising LTR sequences, preferably with a mutated U3 region (U3) removing promoter and enhancer sequences in the 3 LTR.

(50) The packaging sequence (psi) can also be incorporated to help the encapsidation of the polynucleotide sequence into the vector particles (Kessler et al., 2007, Leukemia, 21(9):1859-74; Paschen et al., 2004, Cancer Immunol Immunother 12(6):196-203).

(51) In one embodiment, the invention encompasses a lentiviral vector comprising a lentiviral packaging sequence (psi).

(52) Further additional functional sequences, such as a transport RNA-binding site or primer binding site (PBS) or a Woodchuck Post Transcriptional Regulatory Element (WPRE), can also be advantageously included in the lentiviral vector polynucleotide sequence of the present invention, to obtain a more stable expression of the transgene in vivo.

(53) In one embodiment, the invention encompasses a lentiviral vector comprising a PBS. In one embodiment, the invention encompasses a lentiviral vector comprising a WPRE and/or an IRES.

(54) Thus, in a preferred embodiment, the lentiviral vector comprises at least one cPPT/CTS sequence, one sequence, one (preferably 2) LTR sequence, and an expression cassette including a transgene under the transcriptional control of a 2m or class I MHC promoter.

(55) Promoter

(56) In various embodiments, the promoter drives high expression in antigen presenting cells, including dendritic cells, to induce maximal immune responses. Preferably, the promoter drives expression in other transduced cell types sufficient for elimination by the induced immune response. Most preferably, the promoter lacks an enhancer element to avoid insertional effects.

(57) Most preferably, the promoter is not a CMV promoter/enhancer. Preferably, the promoter is not a dectin-2 or MHCII promoter.

(58) The sequences of various mammalian (human) MHC class I promoters are shown below:

(59) TABLE-US-00004 HLA-A2(MHCI): (SEQIDNO:1) attggggagtcccagccttggggattccccaactccgcagtttcttttct ccctctcccaacctatgtagggtccttcttcctggatactcacgacgcgg acccagttctcactcccattgggtgtcgggtttccagagaagccaatcag tgtcgtcgcggtcgcggttctaaagtccgcacgcacccaccgggactcag attctccccagacgccgagg HLA-B7(MHCI): (SEQIDNO:2) ggggaggcgcagcgttggggattccccactcccctgagtttcacttcttc tcccaacttgtgtcgggtccttcttccaggatactcgtgacgcgtcccca cttcccactcccattgggtattggatatctagagaagccaatcagcgtcg ccgcggtcccagttctaaagtccccacgcacccacccggactcagag HLA-Cw5(MHCI): (SEQIDNO:3) cactggggaggcgccgcgttgaggattctccactcccctcagtttcactt cttctcccaacctgcgtcgggtccttcttcctgaatactcatgacgcgtc cccaattcccactcccattgggtgtcgggttctagagaagccaatcagcg tctccgcagtcccggtctaaagtccccagtcacccacccggactcagatt ctccccagacgccgag HLA-E(MHCI): (SEQIDNO:4) taagaactgctgattgctgggaaactctgcagtttcccgttcctctcgta acctggtcatgtgtccttcttcctggatactcatgacgcagactcagttc tcattcccaatgggtgtcgggtttctagagaagccaatcagcgtcgccac gactcccgactataaagtccccatccggactcaagaagttctcaggactc agagg HLA-F(MHCI): (SEQIDNO:5) aggccccgaggcggtgtctggggttggaaggctcagtattgagaattccc catctccccagagtttctctttctctcccaacccgtgtcaggtccttcat cctggatactcataacgcggccccatttctcactcccattgggcgtcgcg tttctagagaagccaatcagtgtcgccgcagttcccaggttctaaagtcc cacgcaccccgcgggactcatatttttcccagacgcggaggttggggtca tg

(60) A sequence of the human 2-microglobulin promoter is shown below:

(61) TABLE-US-00005 (SEQIDNO:6) Aacatcacgagactctaagaaaaggaaactgaaaacgggaaagtccctct ctctaacctggcactgcgtcgctggcttggagacaggtgacggtccctgc gggccttgtcctgattggctgggcacgcgtttaatataagtggaggcgtc gcgctggcgggcattcctgaagctgacagcattcgggccgag.

(62) The MHCI and 2m promoters do not contain an enhancer. Moreover, these promoters are dendritic-specific (APCs) in that expression of the promoter in BDCA+ dendritic cells is higher than the expression in kidney, smooth muscle, liver, and heart cells. They also have relatively high expression in other transduced cell types, for example, expression of the promoter in BDCA+ dendritic cells is only 12-100 times the expression of that promoter in skeletal muscle cells, in contrast to 900 times with the MHCII HLA-DR promoter. Id.

(63) In various embodiments, the lentiviral vector comprises a 2m or MHC class I promoter. Preferably, the MHC class I promoter is an HLA-A2 promoter, an HLA-B7 promoter, an HLA-Cw5 promoter, an HLA-F, or an HLA-E promoter. In various embodiments, the promoter sequence comprises a polynucleotide sequence that shares more than 90%, preferably more than 95%, more preferably more than 99% identity with the promoter sequence of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, or SEQ ID NO:6.

(64) The invention encompasses dendritic cell-specific promoters. A dendritic cell-specific promoter is one in which expression of the promoter in BDCA+ dendritic cells is higher than the expression in kidney, smooth muscle, liver, and heart cells. Preferably, expression of the promoter in BDCA+ dendritic cells is at least 2, 3, or 4 higher than the expression in kidney, smooth muscle, liver, and/or heart cells. Whether a promoter is (2, 3, or 4) higher than the expression in kidney, smooth muscle, liver, and/or heart cells can be determined by reference to the data sets at http://biogps.org, which are hereby incorporated by reference or by the use of the Affimetrix probes, chips, and methods used to generate these data sets (particularly HG-U133 set). The 2m, HLA-A2, HLA-B7, HLA-Cw5, HLA-F, and HLA-E promoters are dendritic cell-specific promoters. The EF1 promoter is not. Thus, preferably the promoter is not an EF1 promoter.

(65) Preferably, the promoter drives expression in other transduced cell types, such as kidney, smooth muscle, liver, and/or heart cells, and skeletal muscle. Preferably, the expression of the promoter in BDCA+ dendritic cells is 12-100 times the expression of that promoter in skeletal muscle cells. Whether the expression of the promoter in BDCA+ dendritic cells is 12-100 times the expression of that promoter in skeletal muscle cells can be determine by reference to the data sets at http://biogps.org, which are hereby incorporated by reference or by the use of the Affimetrix probes, chips, and methods used to generate these data sets (particularly HG-U133 set). Most preferably, the expression of the promoter in BDCA+ dendritic cells is 12-100 times the expression of that promoter in skeletal muscle cells and expression of the promoter in BDCA+ dendritic cells is higher than the expression in kidney, smooth muscle, liver, and heart cells. The 2m, HLA-A2, HLA-B7, HLA-Cw5, HLA-F, and HLA-E promoters are promoters where expression of the promoter in BDCA+ dendritic cells is 12-100 times the expression of that promoter in skeletal muscle cells. The HLA-A2 promoter and UBC promoter are not. Thus, preferably the promoter is not an HLA-A2 (MHCII) promoter or UBC (Ubiquitin) promoter.

(66) In some embodiments, the expression of the promoter in BDCA+ dendritic cells is at least 10, 12, 15, 20, 25, 30, 35, 40, 50, or 60 times the expression of that promoter in skeletal muscle cells.

(67) In one embodiment, the invention encompasses lentiviral vector particles comprising a lentiviral vector that comprises a dendritic cell-specific promoter directing expression of at least one Hepatitis B virus (HBV) envelop surface antigen, polymerase, HBX protein and HBV consensus core of genotypes A and C antigen, wherein the lentiviral vector particles exhibit higher expression of the antigen in BDCM cells than in HEK 293 T cells.

(68) In various embodiments, the lentiviral vector comprising the promoter induces a greater CTL response in vivo against the encoded immunogenic polypeptide than a vector in which the transgene sequence is under the transcriptional control of a CMV promoter. Within the context of this invention, whether a vector induces a greater CTL response in vivo against the encoded immunogenic polypeptide than a vector in which the transgene sequence is under the transcriptional control of a CMV promoter can be determined using the assay set forth in the examples. Other assays that provide similar results can also be used.

(69) In various embodiments, the lentiviral vector comprising the promoter induces a greater CTL response in vivo against the encoded immunogenic polypeptide than a vector in which the transgene sequence is under the transcriptional control of an EF1 promoter. Preferably, the CTL response with the promoter is at least 2-fold or 3-fold higher than with the EF1 promoter. Within the context of this invention, whether a vector induces a greater CTL response in vivo against the encoded immunogenic polypeptide than a vector in which the transgene sequence is under the transcriptional control of an EF1 promoter can be determined using the assay set forth in the examples. Other assays that provide similar results can also be used.

(70) In various embodiments, the lentiviral vector comprising the promoter induces a greater CTL response in vivo against the encoded immunogenic polypeptide than a vector in which the transgene sequence is under the transcriptional control of an Ubiquitin promoter. Within the context of this invention, whether a vector induces a greater CTL response in vivo against the encoded immunogenic polypeptide than a vector in which the transgene sequence is under the transcriptional control of an Ubiquitin promoter can be determined using the assay set forth in the examples. Other assays that provide similar results can also be used.

(71) The invention encompasses lentiviral vectors containing a promoter that does not contain an enhancer.

(72) The invention encompasses the insertion of an MHC Class I (MHCI) or 2 microglobulin promoter (2m) promoter into a lentiviral vector. As used herein, an MHC Class I (MHCI) promoter or 2 microglobulin promoter includes a naturally occurring or synthetic MHC Class I promoter or 2 microglobulin promoter. The term MHC Class I promoter does not include a 2m promoter.

(73) In one embodiment, the lentiviral vector particles comprising the promoter exhibit higher expression of the antigen in BDCM cells than in HEK 293 T cells.

(74) The promoter can be a naturally occurring promoter. Examples of naturally occurring promoters are the human 2m, HLA-A2, HLA-B7, HLA-Cw5, HLA-E, HLA-F gene promoters. These naturally occurring MHCI promoters are generally cloned or reproduced from the promoter region of a gene encoding the MHC class I protein, or referred to as putatively encoding such proteins in genome databases (ex: NCBI polynucleotide database http://www.ncbi.nlm.nih.gov/guide/dna-rna). Both 2m and class I MHC proteins enter the Major Histocompatibility Complex (MHC).

(75) The proteins encoded by these genes are found in almost all cell types. MHCI proteins are generally present at the surface of the membrane of leucocytes, where they are associated with the 2-microglobulin (2m). The role of these associated proteins is to present peptides from endogenous sources to CD8+ T cells. They thus play a central role to the generation of the antigen-specific immune response. Because MHC class I proteins have been widely studied and described for many years, their genes are well characterized and detectable using sequence comparison tools, such as the BLAST method (Altschul, S. F. et al. (1990). Basic local alignment search tool. J. Mol. Biol. 215(3):403-410).

(76) MHC class I promoters share the ability to be strongly activated in antigen presenting cells, including dendritic cells, as well as, to lower intensity, in the majority of the other human body tissues.

(77) The promoters of the invention can contain further regulatory elements, such as one or more Sp1 and ETs binding sites. In a preferred embodiment, the MHC class I promoter contains 2 Sp1 binding sites and 1 Ets binding site. In other embodiments, Ap1 and/or Ap2 sites are further contained in the promoter.

(78) Preferred promoters are naturally occurring human 2m, HLA-A2, HLA-B7, HLA-Cw5, HLA-E and HLA-F promoters.

(79) Promoters can also be synthetic. Synthetic promoters include promoters that are synthesized using molecular biological techniques to assemble the individual components of a promoter or that are derived from naturally occurring promoters using molecular biological techniques.

(80) In various embodiments, the synthetic promoter comprises a polynucleotide sequence that shares more than 90%, preferably more than 95%, more preferably more than 99% identity, or 100% with the promoter sequence of a 2m or MHC class I gene promoter (e.g., SEQ ID NOs: 1-6 and 19).

(81) The transcription of MHC class genes are usually mediated by two major regulatory elements: Interferon stimulated response element (ISRE) and the SXY module (encompassing the W/S, X1X2/Site and Y/enhancer B regulatory elements) (see FIG. 1). See also Van den Elsen, Immunogenetics (1998) 48:208-211.

(82) These regulatory promoter elements are localized in a region extending approximately from nucleotides 220 to 95 upstream of the transcription initiation site. They mediate tissue-specific and cytokine-induced transcription of MHC class I genes.

(83) The ISRE of MHC class I gene promoters generally contains binding sites for interferon regulatory factor (IRF) family members. It is thus a property of MHC class I promoters to bind to interferon regulatory factor (IRF) family members. This may be verified, for example, by gel shift assays.

(84) Another regulatory element, the enhancer A (containing binding sites for nuclear transcription factor B (NF-B)) is present in most cases. It is thus a property of MHC class I promoters to bind to nuclear transcription factor B (NF-B). This may be verified, for example, by gel shift assays.

(85) In addition to ISRE, MHC class I promoters generally share another set of conserved upstream sequence motifs, consisting of three regulatory elements: the S or W box, the X1/CREX2 boxes or site , and the Y box or enhancer B, which together are termed the SXY module. This SXY module is generally cooperatively bound by a multiprotein complex containing regulatory factor X (RFX; consisting of RFX5, RFXB/ANK and RFXAP), cAMP response element binding protein (CREB)/activating transcription factor (ATF), and nuclear factor Y (NFY), which acts as an enhanceosome driving transactivation of these genes. It is thus a property of MHC class I promoters to bind to these factors. This may be verified, for example, by gel shift assays.

(86) In contrast, MHC class II promoters do not display enhancer A nor ISRE elements (Van den Elsen, P. J. et al, 1998, Immunogenetics. 48:208-221). Furthermore, RFX and CIITA in MHC class II gene regulation have been found of crucial importance as illustrated by studies with cell lines established from patients with the bare lymphocyte syndrome (BLS), a severe combined immunodeficiency due to mutations in one of the RFX subunits or CIITA (DeSandro, A. et al., 1999, Am J Hum Genet, 65:279-286). Also, lack of either CIITA or one of the RFX subunits affects the functioning and assembly of the MHC enhanceosome, respectively, leading to a lack of MHC class II and reduced levels of MHC class I transcription (Van den Elsen, P. J. et al. 2004, Current Opinion in Immunology, 16:67-75).

(87) In one embodiment, the invention encompasses a method comprising inserting a promoter of the invention, particularly a 2m or MHC class I promoter, into a lentiviral vector to direct expression of a transgene, which preferably encodes an immunogenic polypeptide, most preferably encoding at least one Hepatitis B virus (HBV) envelop surface antigen, polymerase, HBX protein and HBV consensus core of genotypes A and C antigen. The method can further comprise inserting any of the other nucleic acid elements mentioned herein, such as a DNA flap sequence.

(88) Isolated Cells

(89) The invention encompasses cells comprising vectors and lentiviral vector particles encoding: at least one Hepatitis B virus (HBV) envelop surface of genotypes A and/or C antigen, at least one polymerase of genotypes A and/or C antigen, at least one HBX protein of genotypes A and/or C antigen, at least one HBV consensus core of genotypes A and/or C antigen, and at least one HBV consensus core MHCI and MHCII epitopes of genotypes A and/or C antigen.

(90) In one embodiment, the cell contains the vector integrated into the cellular genome.

(91) In one embodiment, the cell contains the vector transiently expressing the at least one Hepatitis B virus (HBV) envelop surface of genotypes A and/or C antigen, at least one polymerase of genotypes A and/or C antigen, at least one HBX protein of genotypes A and/or C antigen, at least one HBV consensus core of genotypes A and/or C antigen, and at least one HBV consensus core MHCI and MHCII epitopes of genotypes A and/or C antigen.

(92) In one embodiment, the cell produces lentiviral vector particles encoding the at least one Hepatitis B virus (HBV) envelop surface of genotypes A and/or C antigen, at least one polymerase of genotypes A and/or C antigen, at least one HBX protein of genotypes A and/or C antigen, at least one HBV consensus core of genotypes A and/or C antigen, and at least one HBV consensus core MHCI and MHCII epitopes of genotypes A and/or C antigen.

(93) In various embodiments, the invention encompasses a cell line, a population of cells, or a cell culture comprising vectors and lentiviral vector particles according to the invention.

(94) Preferably, the at least one polymerase of genotypes A and/or C antigen encoded by a vector, in particular a lentiviral vector, or lentiviral vector particle according to the invention is at least one polymerase of genotype A and at least one polymerase of genotype C.

(95) Preferably, the at least one HBX protein of genotypes A and/or C antigen encoded by a vector, in particular a lentiviral vector, or lentiviral vector particle according to the invention is at least one HBX protein of genotype A and at least one HBX protein of genotype C.

(96) Preferably, the at least one Hepatitis B virus (HBV) envelop surface of genotypes A and/or C antigen encoded by a vector, in particular a lentiviral vector, or lentiviral vector particle according to the invention is at least one Hepatitis B virus (HBV) envelop surface of genotype A and at least one Hepatitis B virus (HBV) envelop surface of genotype C.

(97) In a preferred embodiment: the at least one polymerase of genotypes A and/or C antigen encoded by a vector, in particular a lentiviral vector, or lentiviral vector particle according to the invention is at least one polymerase of genotype A and at least one polymerase of genotype C; the at least one HBX protein of genotypes A and/or C antigen encoded by a vector, in particular a lentiviral vector, or lentiviral vector particle according to the invention is at least one HBX protein of genotype A and at least one HBX protein of genotype C; and the at least one Hepatitis B virus (HBV) envelop surface of genotypes A and/or C antigen encoded by a vector, in particular a lentiviral vector, or lentiviral vector particle according to the invention is at least one Hepatitis B virus (HBV) envelop surface of genotype A and at least one Hepatitis B virus (HBV) envelop surface of genotype C.
Lentiviral Vector Particles

(98) The present invention provides a method for producing a lentiviral vector particle. A lentiviral vector particle (or lentiviral particle vector) comprises a lentiviral vector in association with viral proteins. The vector is preferably an integrating vector.

(99) In one embodiment, the lentiviral vector particles encode: at least one Hepatitis B virus (HBV) envelop surface of genotypes A and/or C antigen, at least one polymerase of genotypes A and/or C antigen, at least one HBX protein of genotypes A and/or C antigen, at least one HBV consensus core of genotypes A and/or C antigen, and at least one HBV consensus core MHCI and MHCII epitopes of genotypes A and/or C antigen.

(100) Preferably, the at least one polymerase of genotypes A and/or C antigen encoded by a lentiviral vector particle according to the invention is at least one polymerase of genotype A and at least one polymerase of genotype C.

(101) Preferably, the at least one HBX protein of genotypes A and/or C antigen encoded by a lentiviral vector particle according to the invention is at least one HBX protein of genotype A and at least one HBX protein of genotype C.

(102) Preferably, the at least one Hepatitis B virus (HBV) envelop surface of genotypes A and/or C antigen encoded by a lentiviral vector particle according to the invention is at least one Hepatitis B virus (HBV) envelop surface of genotype A and at least one Hepatitis B virus (HBV) envelop surface of genotype C.

(103) In a preferred embodiment: the at least one polymerase of genotypes A and/or C antigen encoded by a lentiviral vector particle according to the invention is at least one polymerase of genotype A and at least one polymerase of genotype C; the at least one HBX protein of genotypes A and/or C antigen encoded by a lentiviral vector particle according to the invention is at least one HBX protein of genotype A and at least one HBX protein of genotype C; and the at least one Hepatitis B virus (HBV) envelop surface of genotypes A and/or C antigen encoded by a lentiviral vector particle according to the invention is at least one Hepatitis B virus (HBV) envelop surface of genotype A and at least one Hepatitis B virus (HBV) envelop surface of genotype C.

(104) Preferably, the lentiviral vector particles comprise a nucleic acid sequence encoding an amino acid sequence of any of the SEQ ID NOs detailed herein, either alone or combined, such as SEQ ID NO:24 and/or SEQ ID NO:25, and preferably encoding at least SEQ ID NO: 24 and SEQ ID NO: 25.

(105) In one embodiment, the lentiviral vector particle comprises HIV-1 Gag and Pol proteins. Preferably, the lentiviral vector particle comprises subtype D, especially HIV-1.sub.NDK, Gag and Pol proteins.

(106) According to one embodiment of this method, the lentivector particles are obtained in a host cell transformed with a DNA plasmid.

(107) Such a DNA plasmid can comprise: bacterial origin of replication (ex: pUC ori); antibiotic resistance gene (ex: KanR) for selection; and more particularly: a lentiviral vector comprising at least one transgene transcriptionally linked to a MHC class I promoter.

(108) Such a method allows producing a recombinant vector particle according to the invention, comprising the following steps of: i) transfecting a suitable host cell with a lentiviral vector; ii) transfecting said host cell with a packaging plasmid vector, containing viral DNA sequences encoding at least structural and polymerase (+ integrase) activities of a retrovirus (preferably lentivirus); Such packaging plasmids are described in the art (Dull et al., 1998, J Virol, 72(11):8463-71; Zufferey et al., 1998, J Virol 72(12):9873-80). iii) culturing said transfected host cell in order to obtain expression and packaging of said lentiviral vector into lentiviral vector particles; and iv) harvesting the lentiviral vector particles resulting from the expression and packaging of step iii) in said cultured host cells.

(109) For different reasons, it may be helpful to pseudotype the obtained retroviral particles, i.e. to add or replace specific particle envelope proteins. For instance, this may be advantageous to have different envelope proteins in order to distinguish the recombinant particle from natural particles or from other recombinant particles. In matter of vaccination strategy, pseudotyped particle vectors are more likely to escape the immune system, when this latter already developed immunity against lentiviruses. This is particularly helpful when successive injections of similar particle vectors are required for immunizing a patient against a disease.

(110) In order to pseudotype the retroviral particles of the invention, the host cell can be further transfected with one or several envelope DNA plasmid(s) encoding viral envelope protein(s), preferably a VSV-G envelope protein.

(111) An appropriate host cell is preferably a human cultured cell line as, for example, a HEK cell line.

(112) Alternatively, the method for producing the vector particle is carried out in a host cell, which genome has been stably transformed with one or more of the following components: a lentiviral vector DNA sequence, the packaging genes, and the envelope gene. Such a DNA sequence may be regarded as being similar to a proviral vector according to the invention, comprising an additional promoter to allow the transcription of the vector sequence and improve the particle production rate.

(113) In a preferred embodiment, the host cell is further modified to be able to produce viral particle in a culture medium in a continuous manner, without the entire cells swelling or dying. One may refer to Strang et al., 2005, J Virol 79(3):1165-71; Relander et al., 2005, Mol Ther 11(3):452-9; Stewart et al., 2009, Gene Ther, 16(6):805-14; and Stuart et al., 2011, Hum gene Ther., with respect to such techniques for producing viral particles.

(114) An object of the present invention consists of a host cell transformed with a lentiviral particle vector.

(115) The lentiviral particle vectors can comprise the following elements, as previously defined: cPPT/CTS polynucleotide sequence; and a transgene sequence under control of a promoter of the invention, and optionally one of the additional elements described above.

(116) Preferably, the lentivector particles are in a dose of 10.sup.6, 210.sup.6, 510.sup.6, 10.sup.7, 210.sup.7, 510.sup.7, 10.sup.8, 210.sup.8, 510.sup.8, or 10.sup.9 TU.

(117) Methods for Expressing a Transgene in a Cell

(118) The present invention encompasses methods for expressing a transgene in a cell, preferably a non-dividing cell. The method comprises transducing a cell with a lentiviral vector or lentiviral particle vector of the invention under conditions that allow the expression of the transgene.

(119) The cells are preferably mammalian cells, particularly human cells. Particularly preferred are human non-dividing cells.

(120) The transgene preferably encodes at least one Hepatitis B virus (HBV) envelop surface antigen, polymerase, HBX protein and HBV consensus core of genotypes A and C antigen. The method can further comprise harvesting or isolating the polypeptide.

(121) The lentiviral vector or lentiviral particle vector preferably comprises a promoter of the invention.

(122) In one embodiment, the invention encompasses a method for expressing a transgene comprising inserting promoter of the invention into a lentiviral vector such that it direct the expression of a transgene and transducing a cell with the vector containing the promoter.

(123) Therapeutic Use of Lentiviral Vectors

(124) The present invention further relates to the use of the lentiviral vectors according to the invention, especially in the form of lentiviral vector particles, for the preparation of therapeutic compositions or vaccines which are capable of inducing or contributing to the occurrence or improvement of an immunogical reaction against epitopes, more particularly those encoded by the transgene present in the vectors under the transcriptional control of any of the promoters of the invention.

(125) The invention encompasses methods of administration of a lentiviral vector (or lentivector) to a human. Preferably, the lentivector contains a promoter that drives high expression of an antigen in antigen presenting cells, including dendritic cells, and drives expression in other transduced cell types sufficient for elimination by the induced immune response. Most preferably, the promoter lacks an enhancer element to avoid insertional effects.

(126) Preferably, the administration is intramuscular. In one embodiment, the lentivector is injected into the muscle using a needle.

(127) The antigens according to the invention are: at least one Hepatitis B virus (HBV) envelop surface of genotypes A and/or C antigen, at least one polymerase of genotypes A and/or C antigen, at least one HBX protein of genotypes A and/or C antigen, at least one HBV consensus core of genotypes A and/or C antigen, and at least one HBV consensus core MHCI and MHCII epitopes of genotypes A and/or C antigen.

(128) Preferably, the lentivector particle is an integrating lentivector particle, comprising a functional integrase protein.

(129) Most preferably, the administration eliminates at least 95%, 99%, 99.9%, or 99.99% of the lentiviral DNA integrated in the muscle cells of an animal model at day 4 after administration is eliminated by day 21 after administration.

(130) In one embodiment, the invention comprises a method for inducing an immune response in a human comprising intramuscularly administering lentiviral vector particles comprising a functional integrase protein and a lentiviral vector to a human; wherein the integrating lentiviral vector comprises a promoter directing expression of at least one Hepatitis B virus (HBV) envelop surface of genotypes A and/or C antigen, at least one polymerase of genotypes A and/or C antigen, at least one HBX protein of genotypes A and/or C antigen, at least one HBV consensus core of genotypes A and/or C antigen, and at least one HBV consensus core MHCI and MHCII epitopes of genotypes A and/or C antigen, wherein the promoter does not contain an enhancer, and wherein the lentiviral vector particles exhibit higher expression of the antigens in BDCM cells than in HEK 293 T cells; integrating the DNA of the lentiviral vector into cells of the human; expressing the antigens in the cells of the human; and generating an immune response against the antigens.

(131) In one embodiment, the invention comprises a method for inducing an immune response in a human comprising intramuscularly administering lentiviral vector particles comprising a functional integrase protein and a lentiviral vector to a human; wherein the integrating lentiviral vector comprises a promoter directing expression of at least one Hepatitis B virus (HBV) envelop surface of genotypes A and/or C antigen, at least one polymerase of genotypes A and/or C antigen, at least one HBX protein of genotypes A and/or C antigen, at least one HBV consensus core of genotypes A and/or C antigen, and at least one HBV consensus core MHCI and MHCII epitopes of genotypes A and/or C antigen, wherein the promoter does not contain an enhancer, wherein expression of the promoter in BDCA+ dendritic cells is higher than the expression in kidney, smooth muscle, liver, and heart cells; integrating the DNA of the lentiviral vector into cells of the human; expressing the antigens in the cells of the human; and generating an immune response against the antigens.

(132) In one embodiment, the invention comprises a method for inducing an immune response in a human comprising intramuscularly administering lentiviral vector particles comprising a functional integrase protein and a lentiviral vector to a human; wherein the integrating lentiviral vector comprises a promoter directing expression of at least one Hepatitis B virus (HBV) envelop surface of genotypes A and/or C antigen, at least one polymerase of genotypes A and/or C antigen, at least one HBX protein of genotypes A and/or C antigen, at least one HBV consensus core of genotypes A and/or C antigen, and at least one HBV consensus core MHCI and MHCII epitopes of genotypes A and/or C antigen, wherein the promoter does not contain an enhancer, wherein the expression of the promoter in BDCA+ dendritic cells is 12-100 times the expression of that promoter in skeletal muscle cells; integrating the DNA of the lentiviral vector into cells of the human; expressing the antigens in the cells of the human; and generating an immune response against the antigens.

(133) In one embodiment, the invention comprises a method for inducing an immune response in a human comprising intramuscularly administering lentiviral vector particles comprising a functional integrase protein and a lentiviral vector to a human; wherein the integrating lentiviral vector comprises a promoter directing expression of at least one Hepatitis B virus (HBV) envelop surface of genotypes A and/or C antigen, at least one polymerase of genotypes A and/or C antigen, at least one HBX protein of genotypes A and/or C antigen, at least one HBV consensus core of genotypes A and/or C antigen, and at least one HBV consensus core MHCI and MHCII epitopes of genotypes A and/or C antigen, wherein the promoter does not contain an enhancer, wherein expression of the promoter in BDCA+ dendritic cells is higher than the expression in kidney, smooth muscle, liver, and heart cells, and wherein the expression of the promoter in BDCA+ dendritic cells is 12-100 times the expression of that promoter in skeletal muscle cells; integrating the DNA of the lentiviral vector into cells of the human; expressing the antigens in the cells of the human; and generating an immune response against the antigens.

(134) In some embodiments, the method comprises eliminating at least 95%, 99%, 99.9% or 99.99% of the lentiviral DNA integrated in the muscle cells of an animal model at day 4 after administration by day 21, day 25, day 33, or day 66 after administration. Elimination of integrated lentiviral in muscle cells can be measured as described in the examples and by other similar techniques. For example, a biopsy can be taken at the site of the injection of a mouse or rat at 4 days and at 21 days and the amount of integrated DNA in the cells determined at these time points by PCR.

(135) In one embodiment, the invention comprises a method for inducing an immune response in a human comprising intramuscularly administering lentiviral vector particles comprising a functional integrase protein and a lentiviral vector to a human; wherein the integrating lentiviral vector comprises a promoter directing expression of at least one Hepatitis B virus (HBV) envelop surface of genotypes A and/or C antigen, at least one polymerase of genotypes A and/or C antigen, at least one HBX protein of genotypes A and/or C antigen, at least one HBV consensus core of genotypes A and/or C antigen, and at least one HBV consensus core MHCI and MHCII epitopes of genotypes A and/or C antigen, wherein the promoter does not contain an enhancer; integrating the DNA of the lentiviral vector into cells of the human; expressing the antigens in the cells of the human; and generating an immune response.

(136) Preferably, the lentivector particles are in a dose of 10.sup.6, 210.sup.6, 510.sup.6, 10.sup.7, 210.sup.7, 510.sup.7, 10.sup.8, 210.sup.8, 510.sup.8, or 10.sup.9 TU. More preferably, the lentivector particles are in a dose of at least 110.sup.7 TU, in particular at least 710.sup.7 TU.

(137) The immune response induced by the lentiviral vector can be a B cell response, a CD4+ T cell response, and/or a CD8+ T cell response.

(138) The present invention thus provides vectors that are useful as a medicament or vaccine, particularly for intramuscular administration.

(139) These vectors are preferentially for use in the treatment or prophylaxis of infectious diseases, especially diseases associated with virus infection and more particularly, with HBV infection.

(140) As the vectors of the invention more specifically target dendritic cells to obtain a cell-mediated immune response and especially the CTL response associated with the antigen expressed by the transgene in these cells, they are particularly useful as vaccines targeting HBV.

(141) Accordingly, the invention relates to an immunogenic composition comprising a lentiviral vector as previously defined.

(142) The immunogenic compositions of the invention preferably contain cPPT and CTS sequences in the vector and vector particles to induce or to stimulate the nuclear import of the vector genome in the target cells.

(143) During reverse transcription, cPPT and CTS sequences induce the formation of a three stranded DNA structure referred as DNA triplex, which stimulates the nuclear import of DNA vector sequence. Preferably, the vector comprises a transgene and regulatory signals of retrotranscription, expression and encapsidation of retroviral or retroviral-like origin, wherein the composition is capable of inducing or of stimulating a CTL (Cytotoxic T Lymphocytes) and/or a CD4 response against one or several epitopes encoded by the transgene sequence present in the vector.

(144) The expression of the transgene is greatly improved by inclusion of a promoter of the invention in the vector.

(145) Thus, the lentiviral vectors according to the invention have the ability to induce, improve, or in general be associated with the occurrence of a B cell response, a CD4+ T cell response, and/or a CD8+ T cell response, especially a memory CTL response, in particular against HBV. In other words, they can be used for the preparation of therapeutic composition for the treatment of allergies, autoimmune diseases, tumor diseases, or infectious diseases, in particular of HBV, by induction of, stimulation of, or participation in the occurrence of a cell-mediated immune response, especially a CTL response or a memory response.

(146) The lentiviral vectors of the invention can be used in methods of treatment and methods of inducing an immune response, in particular against HBV, comprising administering the lentiviral vector to a host and generating a specific immune response against the transgene in the host. The cells and antibodies generated in these hosts can be used as diagnostic reagents.

(147) The lentiviral vectors according to the invention are preferably for intramuscular administration, most preferably by injection with a needle.

(148) In a particular embodiment, the immunogenic composition according to the invention can be directly administered to the patient, in such a way that it will induce, improve, or participate in vivo in the occurrence of a B cell response, a CD4+ T cell response, and/or a CD8+ T cell response, especially a CTL-mediated immune response, in particular against HBV.

(149) In another embodiment, the immunogenic compositions are used once or upon repeated administration, preferably at least upon two administrations, so that they can enable the occurrence of a long-term memory cell mediated response.

(150) A particular advantage of the immunogenic compositions of the invention is that they can be used to elicit or stimulate a cell-mediated immune response against multiple epitopes encoded by the nucleotides sequence of interest or transgene present in the vector or vector particles, in particular against HBV, and they can also be used to elicit or stimulate a cell-mediated immune response against the product of the entire sequence of a gene.

(151) The invention also encompasses a lentiviral vector comprising a nucleotide sequence encoding a multiple repeat (at least 2 identical sequences) of said amino acid sequence inducing a cellular response and/or an amino acid sequence containing at least 2 different sequences.

(152) As a result, the invention encompasses a composition that could be used in prophylactic and/or therapeutic vaccination protocols, in particular against HBV.

(153) In particular, it can be used in combination with adjuvants, other immunogenic compositions, chemotherapy, or any other therapeutic treatment, in particular against HBV.

(154) The invention encompasses a composition for intramuscular administration to a human comprising lentiviral vector particles comprising a functional integrase protein and a lentiviral vector; wherein the DNA of the lentiviral vector comprises a promoter directing expression of an amino acid comprising or consisting of at least one Hepatitis B virus (HBV) envelop surface of genotypes A and/or C antigen, at least one polymerase of genotypes A and/or C antigen, at least one HBX protein of genotypes A and/or C antigen, at least one HBV consensus core of genotypes A and/or C antigen, and at least one HBV consensus core MHCI and MHCII epitopes of genotypes A and/or C antigen, particularly encoded by an amino acid sequence of any of the SEQ ID NOs detailed herein.

(155) In one embodiment, the invention encompasses a composition for intramuscular administration to a human comprising lentiviral vector particles comprising a functional integrase protein and a lentiviral vector; wherein the DNA of the lentiviral vector comprises a promoter directing expression of an amino acid comprising or consisting of at least one Hepatitis B virus (HBV) envelop surface of genotypes A and/or C antigen, at least one polymerase of genotypes A and/or C antigen, at least one HBX protein of genotypes A and/or C antigen, at least one HBV consensus core of genotypes A and/or C antigen, and at least one HBV consensus core MHCI and MHCII epitopes of genotypes A and/or C antigen, particularly encoded by an amino acid sequence of any of the SEQ ID NOs detailed herein, wherein the promoter does not contain an enhancer, and wherein the expression of the promoter in BDCA+ dendritic cells is 12-100 times the expression of that promoter in skeletal muscle cells and expression of the promoter in BDCA+ dendritic cells is higher than the expression in kidney, smooth muscle, liver, and heart cells.

(156) The invention encompasses use of a composition comprising lentiviral vector particles for intramuscular administration to a human, wherein the lentiviral vector particles comprise a functional integrase protein and a lentiviral vector; wherein the DNA of the lentiviral vector comprises a promoter directing expression of an amino acid comprising or consisting of at least one Hepatitis B virus (HBV) envelop surface of genotypes A and/or C antigen, at least one polymerase of genotypes A and/or C antigen, at least one HBX protein of genotypes A and/or C antigen, at least one HBV consensus core of genotypes A and/or C antigen, and at least one HBV consensus core MHCI and MHCII epitopes of genotypes A and/or C antigen, particularly encoded by an amino acid sequence of any of the SEQ ID NOs detailed herein.

(157) In one embodiment, the invention encompasses use of a composition comprising lentiviral vector particles for intramuscular administration to a human, wherein the lentiviral vector particles comprise a functional integrase protein and a lentiviral vector; wherein the DNA of the lentiviral vector comprises a promoter directing expression of an amino acid comprising or consisting of at least one Hepatitis B virus (HBV) envelop surface of genotypes A and/or C antigen, at least one polymerase of genotypes A and/or C antigen, at least one HBX protein of genotypes A and/or C antigen, at least one HBV consensus core of genotypes A and/or C antigen, and at least one HBV consensus core MHCI and MHCII epitopes of genotypes A and/or C antigen, particularly encoded by an amino acid sequence of any of the SEQ ID NOs detailed herein, wherein the promoter does not contain an enhancer, and wherein the expression of the promoter in BDCA+ dendritic cells is 12-100 times the expression of that promoter in skeletal muscle cells and expression of the promoter in BDCA+ dendritic cells is higher than the expression in kidney, smooth muscle, liver, and heart cells.

(158) Having thus described different embodiments of the present invention, it should be noted by those skilled in the art that the disclosures herein are exemplary only and that various other alternatives, adaptations, and modifications may be made within the scope of the present invention. Accordingly, the present invention is not limited to the specific embodiments as illustrated herein.

EXAMPLES

Example 1. Molecular Constructions

(159) PCR amplification of the proviral region of the pTRIPU3-CMV-GFP(15) was performed using direct (5-CTTACTAGTTGGAAGGGCTAATTCACT CCCAAC-3; SEQ ID NO: 7) and reverse (5-CATTCTAGAACTGCTAGAG ATTTTCCACACTG-3; SEQ ID NO: 8) oligonucleotides encompassing respectively the SpeI and XbaI restriction sites. The resulting fragment was digested and cloned between the SpeI and XbaI sites of the pVAX-1 plasmid (Invitrogen, Lifetech) from which the MluI site have been deleted. The resulting plasmid was named pFLAP-CMV-GFP. The SV40 sequence was amplified by PCR from the pTRIPU3-CMV-GFP plasmid (using the 5-TACCCCGGGCCA TGGCCTCCAAAAAAGCCTCCTCACTACTTC-3 (SEQ ID NO: 9) and 5-ACTCCCGGGTAATTTTTTTTATTTATGCAGAGGCCGAG GCCGCC-3 (SEQ ID NO: 10) oligonucleotides), and cloned into the Pml1 site of the pFLAP-CMV-GFP, the resulting plasmid being then named pFLAP-CMV-GFP-SV. The CMV promoter was amplified with direct (5-TACACGCGTGGAGTTCCGCGTTACATA ACTTACGG-3; SEQ ID NO: 11) and reverse (5-CGTGGATCCGATCGCGGT GTCTTCTATGGAGGTCAAAAC-3; SEQ ID NO: 12) oligonucleotides encompassing the MluI and BamHI sites, respectively. The resulting fragment was cloned back between the MluI and BamHI sites of the pFlap-CMV-GFP-SV allowing the easy replacement of the promoters inside the lentiviral vectors. The promoter was then amplified by PCR from HEK 293T cells DNA with 5-GCCGGCGCGCCGAGAAACCCTGCAGGGAATTCCC-3 (SEQ ID NO: 13) and 5-CGTGGATCCGATCGCTCGGCCCGAATGCTGTCAGCTTCAGG-3 (SEQ ID NO: 14) for the 2m promoter and cloned between the MluI and BamH1 sites of pFLAP-CMV-GFP-SV to create pFlap-2m-SV. The amplified 2m promoter sequence is the following:

(160) GAGAAACCCTGCAGGGAATTCCCCAGCTGTAGTTATAAACAGAAGTTC TCCTTCTGCTAGGTAGCATTCAAAGATCTTAATCTTCTGGGTTTCCGTTTTCTC GAATGAAAAATGCAGGTCCGAGCAGTTAACTGGCGGGGGCACCATTAGCAA GTCACTTAGCATCTCTGGGGCCAGTCTGCAAAGCGAGGGGGCAGCCTTAAT GTGCCTCCAGCCTGAAGTCCTAGAATGAGCGCCCGGTGTCCCAAGCTGGGG CGCGCACCCCAGATCGGAGGGCGCCGATGTACAGACAGCAAACTCACCCAG TCTAGTGCATGCCTTCTTAAACATCACGAGACTCTAAGAAAAGGAAACTGAAA ACGGGAAAGTCCCTCTCTCTAACCTGGCACTGCGTCGCTGGCTTGGAGACAG GTGACGGTCCCTGCGGGCCTTGTCCTGATTGGCTGGGCACGCGTTTAATATA AGTGGAGGCGTCGCGCTGGCGGGCATTCCTGAAGCTGACAGCATTCGGGCC GAG (SEQ ID NO: 15). The HBV antigen can be synthetized and cloned between the BamHI and XhoI sites of the pFlap-2m-SV, in place of the GFP gene.

(161) For example, pFlap-2m-GFP-SV can be digested by BamHI and XhoI, and a DNA linker containing a Multiple Cloning Site (MCS, carrying SalI, SadI, NdeI, AscI and NheI restriction sites) can be cloned between those sites, in place of the GFP gene to allow insertion of a nucleic acid sequence encoding the HBV antigen.

(162) The packaging plasmid pTHV-GP-N was constructed by amplifying the HIV-NDK genome by PCR (using the following oligonucleotides with 5-atgcatgcgtcgacctcgagttaatcctcatcctgtctacttgccac-3 (SEQ ID NO: 16) and 5-gcatgcatcggccggggcggcgactgGTgagagGCCACCatgggtgcgagagcgtcagtattaag-3 (SEQ ID NO: 17). The resulting fragment has been digested by EagI and SalI restriction enzymes and inserted in the p8.74 packaging plasmid (15) from which the Eag1-SalI fragment had been previously removed.

(163) Pseudotyping plasmids were generated by synthesizing the codon optimized genes corresponding to the vesicular stomatitis virus Indiana (GenBank # CAX62728.1), New Jersey GenBank # CAX62729.1) and Cocal (GenBank # CAX62731.1) strains. Those genes were then digested with EcoR1 and BamH1 and cloned between the corresponding restriction sites of the pVAX1 plasmid (Invitrogen, Lifetech).

(164) The plasmids can produced using Nucleobond Xtra Maxi EF column according to manufacturer's instructions (Macherey Nagel).

Example 2. Lentiviral Production

(165) R&D productions: Vectors can be produced by transient calcium-phosphate transfection of HEK 293T as previously describe (25).

(166) Preclinical and GMP productions: The day before transfection, HEK 293T cells are seeded in culture medium on 24 units of Cell Factory 10 (CF-10, Nunc). Cells are transfected by a calcium-phosphate method as reported previously (25). 18 to 24 hours post-transfection, culture medium is changed with production medium corresponding to Dubelcco's modified Eagle's medium (DMEM/High modified, Hyclone) supplemented with 2% heat-inactivated fetal calf serum (FCS, PAA), 1% L-Glutamine (Gibco by Life technologies), 1% Penicillin-Streptomycin (Gibco by Life technologies), 1% Sodium Pyruvate (GIBCO Gibco by Life technologies), BENZONASE (pharma grade 1, 100 000U, Merck Millipore) and MgCL.sub.2 1M. Minimum 24 hours after medium renewal, supernatant of the 24 CF-10 is harvested and pooled. After a second BENZONASE treatment, supernatant is clarified by filtration on KLEENPAK NOVA PROFILE II cartridge (Pall). After clarification, a third BENZONASE treatment is applied overnight at +2/+8 C. Viral vector were purified using Anion exchange chromatography on Mustang-Q XT cassette (Pall). Lentiviral particles are eluted in two steps with 0.5M and 1.2M NaCl. Both fractions are diluted to decrease NaCl concentration up to +150 mM before pooling. 1EX eluate is further concentrated approximately 40 fold by ultrafiltration using a 100 KDa Omega T series filter, 0.1 m.sup.2 (Pall) and diafiltrated with PBS-Lactose 40 mg.Math.L1. Purified bulk (Drug substance) is finally filtered through a 0.2 M Sartobran H5 filter, 300 cm.sup.2 (Sartorius Stedim) and aseptically distributed on 2R 3 mL-glass vials with a target filling volume of 6504 (12004 for pilot batches). After visual inspection of all the vials (about 350 vials by clinical batch), drug product is stored at 70 C.10 C.

(167) For product characterization and pharmaceutical release, quality tests can be performed according to regulatory texts on vaccines: the quality control required for vaccines as per the European Pharmacopeia (section 6.16), the guideline on quality, non-clinical and clinical aspects of live recombinant viral vectored vaccines (EMA/CHMP/141697/2009), the guideline on development and manufacture of lentiviral vectors (CHMP/BWP/2458/03); regulatory text on gene therapy medicinal products: the quality controls required for gene transfer medicinal products for human use as per the European Pharmacopeia (section 5.14), the quality controls specific to gene therapy products as defined in the note for guidance on the quality, preclinical and clinical aspects of gene transfer medicinal products (CHMP/BWP/3088/99); regulatory texts on biotechnological products (ICH Q5A to ICH Q5E); regulatory texts on specifications (ICH Q6A and ICH Q6B) and the quality controls required for parenteral preparations as per the European Pharmacopeia (section 7.0).

Example 3. Lentiviral Vector Titration

(168) qPCR reactions: HEK 293T cells are seeded in 6-well plates (BD Falcon) in culture medium and incubated for 4 h at 37 C., 5% CO2 in moist atmosphere. Cells are transduced with 3 successive dilutions of lentiviral vector. 72 h post-incubation, cells are harvested and transduced HEK 293T cell pellets are produced. Total genomic DNA from transduced cell-pellets is extracted using a method based on QIAGEN QIAamp DNA mini kit handbook. Proviral quantification is performed using Taqman qPCR. The amplification is performed with the Master Mix (Fermentas Thermo Scientific), the Flap A (CCCAAGAACCCAAGGAACA; SEQ ID NO: 18) and Flap S (AGACAA GATAGAGGAAGAGCAAAAC; SEQ ID NO: 19) primers and LENTI TM probe (6FAM-AACCATTAGGAGTAGCACCCACCAAGG-BBQ; SEQ ID NO: 20). Normalization is performed with the quantification of the actin gene (same Mix, Actine A-CGGTGAGGATCTTCATGAGGTAGT- (SEQ ID NO: 21), Actine S-AACACCCCAGCCATGTACGT- (SEQ ID NO: 22) primers and HUMURA ACT TM probe-6FAM-CCAGCCAGGTCCAGACGCAGGA-BBQ- (SEQ ID NO: 23). Both reactions are achieved on MasterCycler Ep Realplex S (Eppendorf, 2 min at 50 C., 10 min at 95 C. and 40 cycles of 15 seconds at 95 C. and 1 min at 63 C.). The analysis is performed on MasterCycler Ep Realplex Software.

(169) FACS analyses are then performed.

Example 4. T-Specific Response (Median) in C57Bl/6j Mice with or without Prime-Boost Immunization

(170) Animals: Female mice (C57Bl/6J) of four weeks or Sprague Dawley RjHan:SD (Sprague Dawley) female mice of eight weeks were purchased from Janvier Laboratories (France).

(171) The animals were housed in Institute Pasteur animal facility in accordance with Institute regulations on the respect of animal experimentation ethical procedures.

(172) Two groups of 5 mice were intramuscularly immunized according to a prim-boost strategy (P/B):

(173) (i) the first one with 710.sup.7 TU of lentiviral vectored HBV construct HBV-3 of the invention in prime and 710.sup.7 TU of lentiviral vectored HBV construct HBV-3 of the invention in boost;

(174) (ii) the second group with 710.sup.6 TU of lentiviral vectored HBV construct HBV-3 in prime and 710.sup.6 TU of lentiviral vectored HBV construct HBV-3 in boost. In these two groups, 7 weeks separate the prime and the boost injections.

(175) Two other mice groups were intramuscularly immunized according to a prime injection strategy (P):

(176) (i) the first one (2 mice) with 710.sup.7 TU of lentiviral vectored HBV construct HBV-3;

(177) (ii) the second group (5 mice) with 710.sup.6 TU of lentiviral vectored HBV construct HBV-3.

(178) Control mice were intramuscularly injected with PBS-lactose.

(179) The lentivectors in which the HBV-3 construct expression of the invention is driven by a promoter (e.g., 2m or MHCI). 15 days after prime immunization, or after boost immunization for the two groups concerned, the specific T-cell responses can be monitored in mice splenocytes by IFN- ELISPOT.

(180) Ninety-six-well tissue culture plates (Millipore) are coated overnight at 4 C. with 50 l/well of 5 g/ml anti-mouse IFN- mAb (Mouse IFN- Elispot pair; BD Biosciences Pharmingen). The plates are washed three times with 200 l DPBS/well and blocked with 200 l/well of DPBS/10% fetal bovine serum for 2 h at 37 C. The plates are washed three times with 200 l DPBS/well. Splenocytes are added to the plates in triplicate at 2.5, 4.1, or 5.110.sup.5 cells/well and stimulated with 2 g/ml of stimulatory peptides (specific to the antigen), concanavalin A (5 g/ml; source), or culture medium alone. The plates are incubated for 18 h at 37 C. and then rinsed three times with 200 l/well of DPBS/0.05% Tween 20 and three times with 200 l/well of DPBS. For detection, 50 l/well of 2 g/ml anti-mouse IFN--biotinylated monoclonal antibody (BD Pharmingen) are added for 2 h at room temperature. Plates are washed and 100 l/well of streptavidin-alkaline phosphatase (Roche) diluted 1:2000 in Dulbecco's PBS for 90 min at room temperature. After washing the plates, spots (IFN--secreting cells) are visualized by adding 60 l/well of BCIP/NBT solution (Sigma). Plates are incubated for 15-30 min at room temperature until blue spots develop and are thoroughly washed with running tap water and air-dried for 24 h. Finally, the spots were counted using a Bioreader 2000 (Biosys).

(181) The cumulated immune response and diversity of the T immune response measured in this experiment is represented in FIG. 2. FIG. 2 moreover illustrates the proportion of immune response for each antigen in the cumulated results.

(182) The results obtained demonstrate that a significant immune response is obtained compared to the control group when injected (in both prime (P) and prime/boost (P/B) groups) with the vectors at the dose of 710.sup.7 TU.

(183) An improved immune response is obtained compared to the control group when injected with vectors comprising the construction of the invention, at the dose of 710.sup.6 TU according to a prime/boost strategy (P/B).

(184) Better immune response results are obtained when injected at a dose of 710.sup.7 TU compared to the dose of 710.sup.6 TU, both with the (P/B) and (P) strategies.

(185) At similar doses (i.e. 710.sup.7 TU or 710.sup.6 TU), the immune response induced by vectors administrated according to a prime-boost strategy (P/B) is significantly higher than the immune response induced by the vectors administrated according to a prime strategy (P).

(186) Said induced immune response is more particularly strong against Core, PolA, XC, EnvC and VLP in the group of mice immunized according to a prim-boost strategy (P/B) with 710.sup.7 TU.

(187) The induced immune response is more particularly strong against XC and EnvC in the group of mice immunized according to a prim-boost strategy (P/B) with 710.sup.6 TU.

(188) The induced immune response is more particularly strong against Core, PolA, PoIC and XA, in particular against Core in the group of mice immunized according to a prim strategy (P) with 710.sup.7 TU.

Example 5. HBV Antigens

(189) HBV therapeutic vaccine, designed for use in the general population, needs to be based on numerous epitopes restricted by multiple HLA types in order to provide acceptable levels of population coverage.

(190) To induce multispecific CTL responses, which are key to resolution of HBV infection, a lentiviral vector carrying envelop surface antigen, polymerase, HBX protein and HBV consensus core of genotypes A and C antigens was constructed.

(191) HBV core protein based chimeric VLP can be constructed to stimulate multivalent immunity against surface antigen and HBV core, since, immune responses specific for both HBV core Ag and HBV surface Ag play an important role in controlling HBV infection. HBV surface Ag-specific antibodies mediate elimination of virions at an early stage of infection and prevent the spread of virus. The chimeric VLP can be constructed by inserting the immunodominant, antibody-binding determinant of HBV surface Ag into the HBV core Ag.

(192) More than a thousand Hepatitis genotype A and genotype C antigen sequences were collected from HBV database, HBVdb, (see online HBVdb/HBVdbIndex) and the genotype-specific antigen consensus sequences were obtained by performing multiple sequence alignments.

(193) CLUSTALW, an alignment software product, (see HBVdb/HBVdbDataset?seqtype=2) was used to create multiple alignments needed to generate a consensus sequence.

(194) Highly conserved HBV-derived peptides that bind multiple HLA class I and class II alleles with high affinity are recognized as CTL epitopes in acutely infected patients are selected (Reignat et al., J. Exp. Med. 195:1089-1101 (2002); Mizukoshi et al., J. Immunol. 173:5863-5871 (2004); Su et al., Proc Natl Acad Sci USA 1997; 94:8744-8749; Desmond et al., Antiviral Therapy 2007; 13:161-175; Bertoni et al., J Clin Invest. 1997 Aug. 1; 100(3):503-13; Nayersina et al., J Immunol. 1993 May 15; 150(10):4659-71; Rehermann et al., J Clin Invest. 1996 April 1; 97(7):1655-65; Kakimi et al., J Virol. 2002 September; 76(17):8609-20; Mizukoshi et al., J Immunol. 2004 Nov. 1; 173(9):5863-71; Depla et al., J Virol. 2008 January; 82(1):435-50; Ding et al., Hepatology. 2009 May; 49(5):1492-502).

(195) The order of the HLA epitopes was selected to distribute epitopes throughout the construct. The construct is shown in FIG. 1.

(196) To avoid toxic long-term drug treatment or to support only partially effective antiviral therapy, LV based HBV therapeutic vaccine can augment the patient's immune system effectually to combat and control the virus.

(197) Inclusion of key viral antigens ensures effective stimulation of T-cell responsesin addition activation of a humoral immune response.

(198) The HBV therapeutic vaccine can break tolerance and induce T-cells infiltration into the liver. The multi-antigen vaccine construct designed to elicit multi-specific, multifunctional cellular and humoral responses, mimicking clearance of natural HBV infection. HBV core based chimeric VLP can act as an adjuvant. Since it can be used without adjuvant, it can eliminate the majority of the adverse reactions.

Example 6. Preclinical Evaluation

(199) Animal Efficacy Studies:

(200) HBV transgenic mice (HBV-Tg) of both genders will be used for the proposed work. They will first be subjected to a partial hepatectomy to assess virus replication levels in the liver prior to vaccination.

(201) After the HBV-Tg mice recover from hepatectomy, they will be vaccinated once, and three weeks later, vaccinated again (boosted).

(202) The vaccine is composed of a lentivirus vector expressing a combination of HBV antigens.

(203) Three weeks after the boost, the mice will be retro-orbitally bled and tested for the presence of HBV DNA in blood by real-time qPCR.

(204) Serum collected prior to immunization, and three weeks after the boost will be tested for levels of HBV associated biomarkers, preferably HBsAg, anti-HBs and ALT.

(205) At the end of the observation period, mice will be euthanized and intrahepatic templates supporting HBV replication will be analyzed and quantified by Southern blot hybridization.

(206) The presence of integrated HBV DNA will also be assessed. Livers will be stained for HBV associated biomarkers, preferably HBsAg, anti-HBs and ALT.

(207) The results will be compared between prior and after vaccination in test and control groups.

(208) As a control for these studies, nontransgenic littermates will also be used. Another two groups of mice will be vaccinated as described above, but will also be injected i,p. with anti-PD-1 at the time of initial immunization and again at the time of boost.

(209) Toxicity studies will be performed using the maximal injectable volume in rat, 200 l in the left plus 200 l in the right hind limb muscle, via intramuscular injection. Hence, toxicity of the maximal feasible dose will be assessed using the clinical chosen route. Intravenous injection (slow infusion in the caudal vein) using the same total volume (400 l) will be performed in other animals to assess systemic exposure. The dose corresponding to this volume is not yet known as the titer of the batches (expressed in TU mL-1) varies from one batch to another. In case of an adverse event considered as significant occurs, a second, lower, dose will be assessed. This lower dose will be of the MAD to be consistent with i. The preclinical data gathered on the therapeutic HIV vaccines and other preclinical development plans; ii. the sensitivity of the methods used for vector titration and during injection. Following guidelines recommendations (The WHO guidelines on nonclinical evaluation of vaccines), groups will be constituted by 10 males and 10 females plus control groups (control to be determined as the final composition of the vectors is to be defined) except for the local tolerance study during which 5 animals/sex/group are planned. Toxicity following IV injection will be assessed without assessment of local tolerance

(210) Intramuscularly (the clinical route), two sacrifice dates are planned: at day 4 for interim sacrifice to assess local tolerance (at the injection site) and at day 15 and day 47 for the terminal sacrifice with an ELISPOT to assess immunogenicity after HBV-3 injection. Animals injected intravenously will be sacrificed only at D15 and no ELISPOT will be performed.

(211) Assessment of toxicity will be performed by monitoring the mortality, clinical signs and body weight (once a day), food consumption (once a week), body temperature (before injection, 6 h post-injection then once a day until day 5 then once every 2 days), hepatic enzymes (ASAT, ALAT, on day 2). Microscopic examination of the liver will be performed after terminal sacrifice.

(212) Studies have been designed to assess the biodistribution, shedding and persistence of the integrated vectors sequences of the injected lentiviral vectors.

(213) Each of the lentiviral vaccines will be injected at the maximal achievable dose in Sprague-Dawley rats, using the clinical chosen route, i.e. intramuscular injection. Sacrifices of 5 males+5 females will be performed at days 3, 21, 33 and 56 days; to be consistent with data previously gathered on the THV01 therapeutic HIV vaccines and other lentiviral vectors developed by the Applicant. Indeed, similar studies were performed on the THV01 vaccines, therapeutic HIV vaccines with similar vector's design but encoding a different antigen. Significant positive signals were detected only at the injection site, the draining lymph nodes and the spleen at days 4 and 21. Residual test items target sequences were found at Day 33 and 56 but results not significantly different from controls.

(214) Blood, urine and faeces will be collected one day before injection, and during 3 days post injection to assess vector' shedding at several late time points. This will be performed following RNA extraction on these samples.

(215) The biodistribution studies described above will enable to gain data on the biodistribution of integrated vector.

(216) The biological response induced by the HBV-3 lentiviral vector will be measured by quantifying the generated T-cell mediated immune response by ELISPOT IFN- assay. Indeed, the expected efficacy of these vaccines relies on the induction of a strong, diverse and long lasting T-cell immune response. Hence, rather than generating non-clinical evidence supporting the potential clinical effect, the related biological effect will be assessed.

(217) The humoral response against HBV-3 vector will consist mostly in antibodies generated against the VSV envelope proteins used for vectors' pseudotyping. This humoral response is therefore considered as an unwanted immunogenicity.

(218) The cellular immune response will be evaluated by performing ELISPOT that will measure the number of specific effector T-lymphocytes via IFN- secretion. Lentiviral vectors will be injected into animals, then their splenocytes isolated and the immune response assessed against a panel of HBV peptides representatives of the epitopes as indicated here-above. These studies will enable evaluation of the efficient dose.

(219) Finally, characterisation of the immune response will be performed by quantifying the CD4+ T cell and the CD8+ T cell response.

(220) Pre-existing immunity and induction of an immune response to the VSV-G protein may result in a decreased efficacy of the vaccination. Indeed, if host's antibodies bind to the HBV-3 vector particles, less DCs will be transduced by these particles, which might lead to the induction of immune responses of lower magnitude.

(221) To assess this risk, both the presence of pre-existing anti-VSV-G antibodies and the induction of these antibodies after each vaccination was or will be assessed:

(222) The prevalence of anti-VSV-G antibodies has been assessed using human sera and an ELISA developed in-house. Briefly, vectors pseudotyped by the VSV-G serotypes but encoding the luciferase were incubated at several dilutions with approximately 100 human sera. The percentage of transduced cells was quantified by FACS after addition of luciferine: the higher the percentage, the less neutralizing activity. This neutralizing activity is considered to be due to antibodies against the specific envelope serotype; hence indirect assessment of the prevalence is performed. Results showed that a negligible percentage of the patients display pre-existing anti-VSV-G antibodies. Such an evaluation might be relevant to be performed in clinic if trials are held in regions where the prevalence of VSV infection is higher than Europe.

(223) ELISA will be performed on blood samples taken from rats injected with the preclinical batches of the vectors and will assess the humoral response specific to each of the serotypes.

(224) Local tolerance will be assessed during the acute toxicity study by macroscopic and post-mortem microscopic observation of the injection site.

(225) The expected effect of the HBV-3 treatment is the induction of a cellular immune response. This will lead to elimination of the infected cells as well as those transduced by the HBV-3 vector from the host.

(226) As the vectors will be injected intramuscularly and not systemically, the risk of germline transmission is reduced.

(227) A phase I/II study will be carried out in chronically infected patients.

(228) In addition to the primary endpoints, the cellular immune response elicited by the vaccine candidate will be studied by monitoring the cellular immune response by cytokines and integrins quantification.