WO2016120489A2 - Lentiviral vectors for expression of mycobacterium tuberculosis antigens - Google Patents

Abstract

The invention relates to nucleic acids, including lentiviral vectors and lentiviral vector particles, encoding M. tuberculosis antigens. The invention encompasses these vectors, methods of making the vectors, and methods of using them, including medicinal uses. The vectors can be used for administration to humans via intramuscular injection to induce immune responses against the M. tuberculosis antigens.

Description

LENTIVIRAL VECTORS FOR EXPRESSION OF

MYCOBACTERIUM TUBERCULOSIS ANTIGENS

TECHNICAL FIELD

[1] The present invention is in the field of recombinant vaccine technology and relates to improvements of lentiviral vectors, which can be used as therapeutic and prophylactic vaccines. The vectors provide improved induction of immune responses over other vectors. BACKGROUND

[2] Recombinant vaccines have been developed with the progress of recombinant DNA technology, allowing the modification of viral genomes to produce modified viruses. In this manner, it has been possible to introduce genetic sequences into non-pathogenic viruses, so that they encode immunogenic proteins to be expressed in target cells upon infection or transduction, in order to develop a specific immune response in their host.

[3] Such vaccines constitute a major advance in vaccine technology (Kutzler et al., Nat Rev Genet, 9(10): 776-788, 2008). In particular, they have the advantage over traditional vaccines of avoiding live (attenuated) virus and eliminating risks associated with the manufacture of inactivated vaccines.

[4] Gene delivery using modified retroviruses (retroviral vectors) was introduced in the early 1980s by Mann et al. {Cell, 33(1 ): 153-9, 1983). The most commonly used oncogenic retroviral vectors are based on the Moloney murine leukemia virus (MLV). They have a simple genome from which the polyproteins Gag, Pol and Env are produced and are required in trans for viral replication (Breckpot et al., 2007, Gene Ther, 14(1 1 ):847-62; He et al. 2007, Expert Rev vaccines, 6(6):913-24). Sequences generally required in cis are the long terminal repeats (LTRs) and its vicinity: the inverted repeats (IR or att sites) required for integration, the packaging sequence Ψ, the transport RNA-binding site (primer binding site, PBS), and some additional sequences involved in reverse transcription (the repeat R within the LTRs, and the polypurine tracts, PPT, necessary for plus strand initiation). To generate replication-defective retroviral vectors, the gag, pol, and env genes are generally entirely deleted and replaced with an expression cassette.

[5] Retroviral vectors deriving from lentivirus genomes (i.e. lentiviral vectors) have emerged as promising tools for both gene therapy and immunotherapy purposes, because they exhibit several advantages over other viral systems. In particular, lentiviral vectors themselves are not toxic and, unlike other retroviruses, lentiviruses are capable of transducing non-dividing cells, in particular dendritic cells (He et al. 2007, Expert Rev vaccines, 6(6):913-24), allowing antigen presentation through the endogenous pathway.

[6] Lentiviruses are linked by similarities in genetic composition, molecular mechanisms of replication and biological interactions with their hosts. They are best known as agents of slow disease syndromes that begin insidiously after prolonged periods of subclinical infection and progress slowly; thus, they are referred to as the "slow" viruses (Narayan et al., 1989, J Gen Virol, 70(7): 1617-39). They have the same basic organization as all retroviruses but are more complex due to the presence of accessory genes (e.g., vif, vpr, vpu, nef, tat, and rev), which play key roles in lentiviral replication in vivo.

[7] Lentiviruses represent a genus of slow viruses of the Retroviridae family, which includes the human immunodeficiency viruses (HIV), the simian immunodeficiency virus (SIV), the equine infectious encephalitis virus (EIAV), the caprine arthritis encephalitis virus (CAEV), the bovine immunodeficiency virus (BIV) and the feline immunodeficiency virus (FIV). Lentiviruses can persist indefinitely in their hosts and replicate continuously at variable rates during the course of the lifelong infection. Persistent replication of the viruses in their hosts depends on their ability to circumvent host defenses.

[8] The design of recombinant integrating lentiviral vectors is based on the separation of the cis- and frans-acting sequences of the lentivirus. Efficient transduction in non-dividing cells requires the presence of two c/s-acting sequences in 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 maximizes the efficiency of gene import into the nuclei of non-dividing cells, including dendritic cells (DCs) (Zennou et ai, 2000, Cell, 101 (2) 173-85; Arhel et ai, 2007, EMBO J, 26(12):3025-37).

[9] Dendritic cells are of primary importance for antigen presentation because they constitute the main class of antigen presenting cells (APCs) whose primary function is to present antigens and initiate an immune response.

[10] To generate an immune response, antigenic proteins must be processed by cells into peptides that are displayed on the cell surface by major histocompatibility complex proteins (MHCs). Circulating APCs present the peptide-MHC complexes to T cells in the draining lymph nodes, where they interact with T cell receptors, and, in conjunction with co-stimulatory signals, activate the T cells.

[1 1 ] A variety of studies have shown that inoculation with lentiviral vectors leads to antigen presentation by DCs and strong activation of antigen specific cytotoxic T lymphocytes (CTLs; CD8⁺ T cells). Therefore, lentiviral vectors have been engineered for the last 10 years for gene transfer and immunotherapy applications.

[12] The vectors routinely contain strong constitutive promoters containing enhancers, such as the CMV promoter. Michelini et al., Vaccine 27(34):4622-29 (2009); Karwacz et al., J. Virol. 83(7):30943103 (2009); Negri et al., Molecular Therapy 15(9): 1716-23 (2007); and Buffa et al., J. General Virology 87:1625-1634 (2006).

[13] Lentiviral vectors have been improved in their safety by removal of the LTR U3 sequence, resulting in "self-inactivating" vectors that are entirely devoid of viral promoter and enhancer sequences originally present within the LTRs.

[14] The lentiviral particles, which contain lentiviral vectors, can be produced by recombinant technology upon transient transfection of cells, for example HEK 293T human cultured cells, by different DNA plasmids:

(i) a packaging plasmid, which expresses at least the Gag, Pol Rev, Tat and, in some cases, structural and enzymatic proteins necessary for the packaging of the transfer construct;

(ii) a proviral transfer plasmid, containing an expression cassette and HIV cis- acting factors necessary for packaging, reverse transcription, and integration; and

(iii) an envelope-encoding plasmid, in most cases the glycoprotein of vesicular stomatitis virus (VSV.G), a protein that allows the formation of mixed particles (pseudotypes) that can target a wide variety of cells, especially major histocompatibility (MHC) antigen-presenting cells (APCs), including DCs.

[15] This procedure allows obtaining transient production of lentiviral particle vectors by the transfected cells. However, the lentiviral particle vectors may also be continuously produced by cells by stably inserting the packaging genes, the proviral coding DNA, and the envelope gene into the cellular genome. This allows the continuous production of lentiviral particle vectors by the cells without the need for transient transfection. Of course, a combination of these procedures can be used, with some of the DNAs/plasmids integrated into the cellular genome and others provided by transient transfection.

[16] Non-integrating lentiviral vectors have been designed to mitigate the risks of potential oncogenesis linked to insertional mutagenesis events, particularly for vaccination purposes. Examples of non-integrating lentiviral vectors are provided in Coutant et al., PLOS ONE 7(1 1 ):e48644 (2102), Karwacz et al., J. Virol. 83(7):3094- 3103 (2009), Negri et al., Molecular Therapy 15(9): 1716-1723 (2007); Hu et al., Vaccine 28:6675-6683 (2010). Consequently, it has been reported that a non-integrating lentiviral vector system can mitigate the potential risk of insertional mutagenesis as compared to an integrating system. Hu et al., Vaccine 28:6675-6683 (2010). It has been further reported that functional analysis indicates that both the magnitude and quality of the immune responses elicited by DC-directed integration-defective lentiviral vectors (IDLVs) are comparable to that of its integrating counterpart. Id. Thus, integration-defective lentiviral vectors (IDLVs) have been considered safer vectors than integrating vectors for human administration, with comparable effectiveness.

[17] In addition, deletion in the U3 region of the 3' LTR of the viral promoter and enhancer sequences in self-inactivating lentiviral vectors limits the likelihood of endogenous promoter activation. These concerns with safety directly address the experiences gained from the SCID-X1 gene therapy trial carried out in 1998-1999, performed with Moloney virus-based retroviral vectors on children suffering from a rare form of X-linked (SCID-X1 gene) severe immunodeficiency disease (Cavazzana-Calvo et al., 2000, Science., 288(5466):669-72). During this trial, four of nine children developed leukemia as a result of the integration of the Moloney-derived retroviral vector at close proximity to the human LM02 proto-oncogene (Hacein-Bey-Abina et al., 2008, J.Clin. Invest, 1 18(9):3132-3142). It was demonstrated that malignancy was the consequence of the proximity of the viral U3 promoter/enhancer to the LM02 proto- oncogene. As a result, safety is a major concern for the administration of lentivectors to humans.

[18] Enhancers are cis-acting sequences, which can act as transcriptional activators at a distance. They have been widely employed in viral derived vectors because they appear to be the most efficient for obtaining transgene strong expression in a variety of cell types, in particular DCs (Chinnasamy et al., 2000, Hum Gene Ther 1 1 (13): 1901 -9; Rouas et al., 2008, Cancer Gene Ther 9(9):715-24; Kimura et al., 2007, Mol Ther 15(7): 1390-9; Gruh et al., 2008, J Gene Med 10(1 ) 21 -32). However, given the safety issue of insertional mutagenesis, such transcriptional enhancer sequences should be deleted from the lentiviral vector constructs to abolish the risk of insertional mutagenesis by enhancer proximity effect. This enhancer proximity effect is by far the most frequent mechanism of insertional mutagenesis and is the only effect described in human or animal cases of tumorigenic events after gene transfer.

[19] Thus, there is a need to develop retroviral, particularly lentiviral vectors, which do not include viral enhancers, but still allow sufficient expression of transgenes encoding immunogenic peptides, if possible, as much expression as that observed when using the CMV promoter.

[20] Recent studies has reported on the replacement of viral promoters by DC- specific promoters deriving from major histocompatibility complex class II genes (MHC class II) (Kimura et al., 2007, Mol Ther 15(7):1390-9) and dectin-2 genes (Lopes et al., 2008, J Virol 82(1 ):86-95). The dectin-2 gene promoter used in Lopes et al. contains a putative enhancer and an adenoviral conserved sequence (inverted terminal repeats in adenovirus promoter) (Bonkabara et al., 2001 , J. Immunology, 167:6893-6900). The MHC class II gene promoter used by Kimura et al. does not contain any known enhancer.

[21 ] Yet, without an enhancer, the MHC class II promoter was found not to provide sufficient transgene expression in DCs, when administered intravenously. In particular, lentiviral vectors including MHC class II promoters did not provoke an immune reaction in immunocompetent C57BL/6 mice, in contrast to the immune responses observed with CMV promoters/enhancers. Although integration and persistent transgene expression were observed after injection in mice, the lentiviral vectors transcribed through MHC class II promoters failed to stimulate an antigen- specific CD8+ cytotoxic T-lymphocyte response, even after vaccination boost. The authors of these studies therefore concluded that the use of MHC class II promoters was of interest only for applications where persistence of expression is sought as in gene replacement therapy, but not in the context of immunotherapy. Of note, MHC class II promoters are expressed poorly in most cell types.

[22] Thus, the MHC class II promoter is not an adequate promoter for lentiviral vectors for induction of an immune response against an antigen via IV injection. Moreover, the dectin-2 promoter is expressed poorly in most cell types and appears to contain an enhancer. Thus, the dectin-2 promoter is not a good promoter for lentiviral vectors for safety reasons.

[23] Preferably, in immunotherapy, lentiviral vectors provide effective expression of the transgene that elicits a desired specific immune response. This requires that the expression is at a high level in APCs, such as dendritic cells.

[24] It is also preferable that the cells transduced by the lentiviral vectors are eliminated by the immune response to provide a higher degree of safety. That is, the immune response generated against the transgene can elicit an immune response in the host sufficient to eliminate the cells that are transduced by the lentiviral vectors. The elimination of transduced cells eliminates the persistence of the lentiviral vector in the host, and possible secondary effects of the vector. In order for the transduced cells to be eliminated, expression is required in non-dendritic cells at a level that allows elimination by the immune response. Thus, appropriate expression of an antigen is desirable.

[25] At the same time, the promoter should maximize immune stimulation through the key cells (i.e., dendritic cells) involved in the activation of na^'ive and memory T cells, and should minimize the risk of insertional mutagenesis and genotoxicity in stem cells, leading to malignancies. Thus, the promoter should have sufficiently high activity in dendritic and other cells, but not contain an enhancer. Based on these criteria, viral promoters, such as the CMV promoter, are not ideal because of the presence of strong enhancers. These criteria are summarized as follows:

1 . high expression in antigen presenting cells, including dendritic cells, to induce maximal immune responses;

2. expression in other transduced cell types sufficient for elimination by the induced immune response; and

3. lack of an enhancer element to avoid insertional effects.

[26] Tuberculosis (TB) is the most common cause of infectious disease-related global deaths. TB is caused by Mycobacterium tuberculosis {M. tuberculosis), a slow- growing obligate aerobe and a facultative intracellular parasite. Humans are the only known reservoir for M. tuberculosis. The organism spreads primarily as an airborne aerosol from an individual who is in the infectious stage of TB. In immunocompetent individuals, exposure to M. tuberculosis usually results in a latent/dormant infection. The infection may be cleared by the host immune system or altered into an inactive form called latent tuberculosis infection (LTBI). Only about 5% of these individuals later show evidence of clinical disease. Weak host immune system can allow reactivation of M tuberculosis organisms.

[27] TB results from a combination of direct effects from the replicating M. tuberculosis and pathology associated with host immune responses to M. tuberculosis antigens. The lungs are the most common site for the development of TB. Extra pulmonary TB can occur as part of a primary or late, generalized infection and occurs mainly in patients with concurrent AIDS and tuberculosis. An extra pulmonary location may also serve as a reactivation site; extra pulmonary reactivation may coexists with pulmonary reactivation.

[28] The current control programs against TB include chemotherapy and

Bacillus Calmette-Guerin (BCG) vaccination. The present-day first-line treatment for TB is a multidrug regimen comprising of rifampin, isoniazid, pyrazinamide, and ethambutol (RHZE). It must be taken for a minimum of 6 months to be efficient. Adverse events in response to anti-TB drugs are very common and add to the problem of compliance. [29] BCG, the currently available prophylactic vaccine against TB, has been used in humans since 1921. BCG considerably protects new born babies and children from the infection of the meninges (brain and spinal cord) which can result in the development of a life-threatening condition known as meningeal TB and miliary TB- potentially lethal form of tuberculosis resulting from massive lymphohaematogeneous dissemination of bacilli. However, its efficacy in preventing most prevalent form of TB, namely pulmonary TB in adults, remains controversial. One potential explanation that has been suggested for the failure of BCG in adults is that the protective immune responses induced by BCG inoculation at infant waned as children grow up. Regardless of the relative efficacy of BCG in infants, the major unknown is why BCG fails to prevent pulmonary TB in adolescents.

[30] The increasing prevalence of multidrug-resistant (MDR; resistance to at least rifampin and isoniazid) and extensively drug-resistant (XDR; MDR resistance plus resistance to a fluoroquinolone and an aminoglycoside) TB is a grave concern. Resistant M. tuberculosis arises due to partial elimination of bacteria, generation of mutants, and selection of mutants over wild-type strains. Second-line drugs for drug- resistant TB are less effective, more toxic, and require longer use than first-line drugs.

[31] The high incidence of TB at 8-9 million cases per year reflects the partial failure of existing therapies. Even though tuberculosis remains a significant public health issue globally, progress has been made in the recent years in the development of new tools to limit the pandemic. These comprise fast cost-effective diagnostic tests, novel drug treatments, and new drug delivery methods. On the preventive measure side, there are presently more than a dozen tuberculosis vaccines assessed in human trials, based on diverse platforms, including viral-vectored, recombinant BCG, and protein/peptide. Unfortunately, eradication of tuberculosis is complicated by a number of issues, not the least of which are the interactions with HIV infection and the surge of MDR and XDR M. tuberculosis strains. Treatment of M-XDR TB can be as much as 1 ,000 times more expensive than drug-susceptible TB and requires two or more years of continuous therapy (WHO, 2007). The emergence of a type of TB resistant to all currently available first- and second-line drugs— totally drug-resistant (TDR)-TB or very extensively drug-resistant (XXDR)-TB— has been reported in the last few years. In parts of the globe where MDR and XDR strains are common, management of patients with tuberculosis is challenging because of the dearth of effective medications. Despite ongoing huge efforts (financial, scientific, clinical etc.) to generate new tuberculosis vaccines, most of them are still intended as prophylactic vaccines to thwart advancement to pulmonary disease in newly infected individuals. And no TB prophylactic vaccines have been licensed yet. In recently concluded MVA85A efficacy trials, though the primary outcome safety was met, the secondary outcome of efficacy either against TB or infection was not seen.

[32] The exact mechanism that mediates protective immunity and possibly sterile eradication of M. tuberculosis is unknown. Most of the subunit vaccines used in clinical trials are based on antigens expressed only in replicating stage, which may limit their efficacy. During M. tuberculosis infection, including initial infection stage, active TB and latent TB, tubercle bacilli population consists of growing, slow-growing and non- growing sub populations with various metabolic states in a continuum and the subpopulations can interconvert to each other. Therefore, ideal subunit vaccines, either prophylactic or therapeutic, should target all mycobacterial subpopulations. In order to develop effective multi-stage tuberculosis subunit vaccines, antigens expressed by the bacilli in various metabolic stages were selected to construct fusion proteins

[33] Antigens chosen are known to be involved in attachment, virulence, immune regulation, metabolism e.g. proteases, kinases, chaperones. Some of the selected antigens protect animals as vaccines- Mice, Guinea pigs, NHP. Antigens are also based on the human immune response to actively infected or non-progressive latent infection. Several of the antigens are from genome peptide libraries, and induced cytokine responses, T cell clones, and stimulated immune responses that recognized TB infected cells. Antigens incorporated into the THV03 lentiviral vectors have identified and potential human T cell epitopes. Selected antigens carried by lentiviral vector vaccine shows no homology with human proteins.

[34] It has been hypothesized that cellular immunity elicited by immunization could increase pathology or trigger reactivation of persistent lung bacteria. Indeed, semi-purified culture supernatants of M. tuberculosis, termed tuberculin, induced exacerbated immunopathology probably leading to tissue destruction and sometimes to systemic dissemination of M. tuberculosis, with fatal consequences. All attempts of therapeutic vaccination using killed or attenuated mycobacteria failed. More recently idea of therapeutic vaccination was revitalized. Modern immune therapeutic approaches aim to "realign" or improve the immune response either by promoting protective (Th1 ) immunity or by blocking harmful immune (Th2) responses. Immunotherapy agents that optimize the Th1 response by down-regulating the Th2 response are favored.

[35] Immunotherapeutic agents have been studied in mice (Hsp65 DNA vaccine, anti-TGF-β, anti-IL-4), humans (human immunoglobulin), or both (killed Mycobacterium vaccae, HE2000, rh-IFNy, rh-IL-2). As mention below number of immunotherapies have been evaluated in humans, and found to be safe although not all have been studied in patients with TB.

[36] Current candidate TB immunotherapies are at different stages of clinical development:

i. Heat-killed Mycobacterium vaccae, which is in phase III clinical trials, was shown to be safe in HIV patients who had previously received BCG.

ii. UTI is composed of detoxified fragments representing a whole range of inactivated latency-associated antigens. In phase I clinical trials, RUTTs safety and immunogenicity has been demonstrated. However, it has no therapeutic effect in late progressive TB disease in the absence of chemotherapy. It could be due to the fact that RUTI also induces a Th2 and large antibody response and may also induce TGF-β. iii. Mycobacterium smegmatis and Mycobacterium indicus pranii (MIP), two other saprophytic non-TB mycobacteria (NTM) that also share antigens with M. tuberculosis, similar Mycobacterium vaccae, were found to be safe in humans.

iv. V-5 immunitor (V5)— an oral therapeutic vaccine initially developed for management of chronic hepatitis— has been shown in several studies to be safe in TB patients.

[37] Several immunotherapy constructs are active in mouse TB models (with or without chemotherapy) but have not yet been tested on humans. DNA vaccines (which express Hsp65, IL-12; Ag85A, PST S3, IL-12; Ag85B; Hsp70/CD80; ESAT-6 in flu vector) show a one to three log increase in bacterial clearance in comparison to untreated mice. DNA vaccines harbouring a mycobacterial {Mycobacterium leprae) stress protein (Hsp65) or Hsp70 fused to CD80 are therapeutic when injected to tuberculosis mice. A DNA vaccine expressing ESAT-6 in a flu vector is efficient as adjunctive therapy when given with chemotherapy. Immunotherapy with Hsp65 as an adjunct to chemotherapy is concomitant with quick and effective response to treatment of MD -TB in mice. There is synergy between chemotherapy (moxifloxacin) and DNA vaccine in BCG-immunized, TB-challenged mice. Monkeys vaccinated with Hsp70/CD80 before being infected with TB survived; placebo or administered with BCG, died. The therapeutic effect of DNA vaccines is correlated with a switch from a predominantly type 2 to a predominantly type 1 response. DNA vaccines enhanced IFNy production and CD8+ CTL, down-regulated IL-4 production and eliminate persisting organisms.

[38] In summary, recombinant DNA vaccines may be therapeutic or work as adjuncts to chemotherapy to enhance bacterial killing, reduce pathology, eliminate organisms that persist, and protect against reinfection. In general, the immunotherapies tested over the last decades have been well tolerated. Despite initial concerns, there have not been side effects of the magnitude and frequency seen in Koch's time.

[39] While latency is characteristically induced by stress imposed by the host on M. tuberculosis, persistence signifies the survival of M. tuberculosis under harsh environment, be they caused by drug treatment or host immunity. Conventional antituberculosis drugs target biosynthetic processes involved in bacterial growth, including RNA transcription (RIF), protein translation (streptomycin), and cell wall. They preferentially target replicating organisms; therefore, a non-replicative subpopulation of the bacteria present in chronic infections will show resistance to drugs. New TB drugs are currently being tested for their activity against persistent bacteria, with the hope of shortening current regimens such as delamanid, bedaquiline, and the linezolid derivative sutezolid. Though animal models are developed to assess elimination of persisters by using sterilization as an endpoint pharmacokinetics/pharmacodynamics (PK/PD) in these animals are quite different from that of humans. Thus the strategy to optimize the use of current and novel drug therapy may however ultimately fail to eliminate persisters. [40] Robert Koch was the first to use mouse as an experimental model for TB research. Later, infections were successfully established in a range of animal models (mice, rabbits, guinea pigs, rats, monkeys etc.). Mouse and guinea pig, as animal models of pulmonary tuberculosis, are being widely used and provide information about the host response in the lungs, changes in immunopathology and the protective effect of vaccine candidates. Major advantage of these models is that these animals can be easily infected by pulmonary route; similarly to the way Human acquire infection as a result of few virulent tubercle bacilli get deposited in alveolar space. Further, it is easy to study various stages of TB progression like granuloma formation, liquefaction, cavity formation and haematogenous spread of the disease in animal models. The symptoms of the disease like fever, loss in weight, abnormal X-rays and respiratory distress can also be observed in these models. Moreover, the animals eventually die if untreated, like Human. Altogether, due to these resemblances between animal models and humans in the vulnerability as well as resistance to TB, disease progression and finally death, mice, guinea pigs and monkeys are considered good models for evaluating new anti-TB vaccines.

[41] Since the time of (1890s) Robert Koch, mouse is one of the most popular and economical TB animal model. They can be easily infected via aerosol with a low dose of M. tuberculosis, proliferating in lungs and then disseminating to liver and spleen. The infection is controlled but not abolished, by T-cell mediated immunity. Key immune correlates resulting from mouse infection have been shown to be similar to humans, including interleukin-12 (IL -12), CD4+T cells, and tumor necrosis factor-a (TNF-a). C57BL/6 as well as BALB/c mice strains are well characterized and their survival rate is twice that of DBA/2 and C3H/HeJ mice strains.

[42] In mice, vaccine candidates are assessed by their ability to reduce the bacterial load to a level which is statistically and significantly less than that of the saline control; BCG is taken as a positive control. In this model, one log reductions in bacillary counts were shown in experiments with DNA vaccines encoding ESAT6 and Ag85B. The main advantage of murine model for vaccine development lies in its capability to screen the vaccines at a limited cost, ease of use and study vaccine mediated protection. Availability of the reagents required for experiments are one of the reason mouse model is extensively used. Due to these reasons, several therapeutic vaccines against TB are being tested using mouse as an efficacy model. Coler et al described the use of mouse models of tuberculosis in which they tested the ability of a vaccine that incorporates 4 mycobacterial antigens. The inbred Swiss background (SWR) mouse strain, which is hyper susceptible, was selected for these experiments: it shows a fast progression to lethal disease resulting from pulmonary infection and failure of anti-TB drugs to completely eradicate bacilli from the tissues. These features provided the authors the opportunity to detect the consequence of immunizing the infected, drug- treated mice 3 times with their vaccine and to measure a substantial improvement in the response of the mice to the 2 drugs. It has recently been demonstrated that chemotherapy and immunotherapy with the combined DNA vaccine encoding M. tuberculosis antigens had showed efficient tuberculosis treatment in mice. In another tuberculosis mice model, immunotherapy with plasmid DNA encoding the Mycobacterium leprae 65 kDa heat-shock protein (hsp65 ) in association with chemotherapy demonstrated to be more rapid and effective treatment for TB. The antigen 85A (Ag85A), protein which is responsible for the high affinity of mycobacteria for fibronectin, coding DNA vaccines alone or in combination with chemotherapy reduced the pulmonary and splenic bacterial loads in mice. These studies support the relevance of mouse as animal model for the screening of vaccine candidates for immunological parameters as well as for efficacy of new candidate vaccines for TB.

[43] Multidrug-resistant TB (MDR-TB) is caused by M. tuberculosis strains that are resistant to at least the two most effective anti-TB drugs, isoniazid and rifampicin. Extensively drug-resistant TB (XDR-TB) is a M. tuberculosis strain that is resistant to isoniazid and rifampicin (i.e. MDR-TB) as well as any fluoroquinolone and any of the second-line anti-TB injectable drugs (amikacin, kanamycin or capreomycin).

[44] About 3.6% of new tuberculosis (TB) patients in the world have multidrug- resistant strains. Levels are much higher - about 20% - in those previously treated for TB. The frequency of MDR-TB varies substantially between countries. About 10% of MDR-TB cases are also resistant to the two most important second-line drug classes, or extensively drug-resistant TB. By September 2013, 92 countries had reported at least one XDR-TB case (WHO data). The WHO estimates that there were about 450,000 new MDR-TB cases in the world in 2012. More than one half of these cases occurred in China, India, and the Russian Federation.

[45] Although 48% of patients with MDR-TB enrolled on treatment (2010 WHO survey) were reported to have been successfully treated; the mortality rate remains quite high. The WHO estimates that 170,000 deaths related to MDR-TB infection occurred in 2012. Consequently, MDR-TB (and XDR-TB) is a severe disease for which alternative therapy and especially a therapeutic vaccine is needed.

[46] Thus, a need exists in the art for improved vectors and methods for immunizing humans. The present invention fulfills these needs in the art.

SUMMARY OF THE INVENTION

[47] The invention encompasses nucleic acid molecules and vectors encoding M. tuberculosis antigens and methods of using the nucleic acid molecules and vectors. Preferably, the M tuberculosis antigen is selected from Ag85A, ESAT6, Mpt64, EspC, RVBD, Rv1813c, HRP1 , HspX, Mtb39A, Rv2627c, Rv2628, PfkB, Mtb32A, EsxV, EsxW, HSP65, Rv2660, Archease, PE42, EsxJ, Rv1088, USP, PPE42, and PE35 antigens.

[48] Most preferably, the nucleic acid molecules and vectors encode M. tuberculosis antigens Ag85A, ESAT6, Mpt64(Rv1980c), RVBD(Rv0140), HRP1 (Rv2626c), RV2028c, HspX(Rv2031 c), and Mtb32A(Rv1 196), most preferably as a fusion protein in the order: Ag85A, ESAT6, Mpt64(Rv1980c), RVBD(Rv0140), HRP1 (Rv2626c), RV2028c, HspX(Rv2031 c), and Mtb32A(Rv1 196).

[49] Preferably, the vector is a lentiviral vector. In one embodiment, the vector comprises a 2-microglobulin promoter. In one embodiment, the vector comprises a Woodchuck PostTranscriptional Regulatory Element (WPRE).

[50] In one embodiment, the vector is a DNA. Preferably, the vector comprises a nucleic acid sequence encoding an M. tuberculosis antigen.

[51 ] The invention encompasses a lentiviral vector particle encoding an M. tuberculosis antigen.

[52] In one embodiment, the lentiviral vector particle comprises a functional lentiviral integrase protein. In one embodiment, the lentiviral vector particle comprises a vesicular stomatitis virus glycoprotein. In one embodiment, the lentiviral vector particle comprises HIV-1 subtype D Gag and Pol proteins.

[53] The invention encompasses an isolated cell comprising a vector of the invention.

[54] The invention encompasses the use of a vector of the invention for inducing an immune response in a human by intramuscular administration.

[55] The invention encompasses a method for inducing an immune response in a human comprising intramuscularly administering a lentiviral vector particle of the invention to a human.

BRIEF DESCRIPTION OF THE DRAWINGS

[56] Figure 1 depicts THV03-TB1 insert in the lentiviral vector. For the production transgene carrying lentiviral vector, M tuberculosis genes or part of the genes of Ag85A, ESAT6, Mpt64 (Rv1980c) only 9 amino acids, EspC (Rv3615c) , RVBD (Rv0140) , Rv1813c and HRP1 (Rv2626c) were synthesized as a fusion construct. The placement of the genes in the fusion construct is shown. THV03-TB1 construct contains genes expressed at active as well as latent stage of tuberculosis. In addition, this construct harbors a gene expressed at the reactivation stage of the tuberculosis. This fusion construct was subsequently cloned into a lentiviral vector to obtain THV03-TB1 and the construct was verified by sequencing. THV03-TB1 was pseudotyped by the Indiana serotype of the vesicular stomatitis virus G protein (VSV-G) and it was generated in HEK293 cell.

[57] Figure 2 depicts specific T-cell cumulative response (IFNy secretion) in C57BI/6j mice (median group value) with the THV03-TB1 lentiviral vector.

[58] Figure 3 depicts IFN-γ ELISPOT results of LV vectored TB antigens at the dose of 10e8 with the THV03-TB1 lentiviral vector.

[59] Figure 4 depicts THV03-TB2 insert in the lentiviral vector. For the production transgene carrying lentiviral vector, M tuberculosis genes or part of the genes of Hspx(Rv2626c), Mtb39A(Rv1 196), Mt2702(Rv2627c) 20amino acids, Mt2703(Rv2628) 20 aminoacids, PfkB (Rv2029c) 20 amino acids, Mtb32A (Rv0125) and EsxV (Rv3619c) were synthesized as a fusion construct. The order of the genes in the fusion construct is shown. THV03-TB2 construct contains genes expressed at active as well as latent stage of tuberculosis. The fusion construct was subsequently cloned into a lentiviral vector to obtain THV03-TB2 and the construct was verified by sequencing. THV03-TB2 was pseudotyped by the Indiana serotype of the vesicular stomatitis virus G protein (VSV-G) and it was generated in HEK293 cells.

[60] Figure 5 depicts depicts IFN-γ ELISPOT results of LV vectored TB antigens with the THV03-TB2 lentiviral vector.

[61 ] Figure 6 depicts depicts IFN-γ ELISPOT results of LV vectored TB antigens with the THV03-TB2 lentiviral vector.

[62] Figure 7 depicts THV03-TB3 insert in the lentiviral vector. For the production transgene carrying lentiviral vector, M tuberculosis genes or part of the genes of EsxW(Rv3620c), HSP65(Rv0440), Rv2660c, Mtb32A (Rv0125), EsxV (Rv3619c), Archease(Rv2630),PG42(Rv2608) 9 amino acids, EsxJ(Rv1038c)1 1 amino acids and Rv1088 15 amino acids were synthesized as a fusion construct. The order of the genes in the fusion construct is shown. THV03-TB3 construct contains genes expressed at active as well as latent stage of tuberculosis. The fusion construct was subsequently cloned into a lentiviral vector to obtain THV03-TB3 and the construct was verified by sequencing. THV03-TB3 is pseudotyped by the Indiana serotype of the vesicular stomatitis virus G protein (VSV-G) and it was generated in HEK293 cells.

[63] Figure 8 depicts specific T-cell cumulative response (IFNy secretion) in

C57BI/6j mice (median group value) with the THV03-TB3 lentiviral vector.

[64] Figure 9 depicts IFN-γ ELISPOT results of LV vectored TB antigens at the dose of 10e8 with the THV03-TB3 lentiviral vector.

[65] Figure 10 depicts depicts IFN-γ ELISPOT results of LV vectored TB antigens at the designated doses with the THV03-TB3 lentiviral vector

[66] Figure 1 1 depicts THV03-TB4 insert in the lentiviral vector. For the production transgene carrying lentiviral vector, M tuberculosis genes USP(Rv2028c), PPE42(Rv2608) and PE35 (Rv3872) were syntheised as a fusion construct. The order of the genes in the fusion construct is in the order shown. THV03-TB4 construct consists of genes expressed at active as well as latent stage of tuberculosis. The fusion construct was subsequently cloned in to Lentiviral vector to obtain THV03-TB4 and the construct was verified by sequencing. THV03-TB4 was pseudotyped by the Indiana serotype of the vesicular stomatitis virus G protein (VSV-G) and it was generated in HEK293 cells.

[67] Figure 12 depicts specific T-cell cumulative response (IFNy secretion) in C57BI/6j mice (median group value) with the THV03-TB4 lentiviral vector.

[68] Figure 13 depicts IFN-γ ELISPOT results of LV vectored TB antigens at the dose of 8,2910e7.

[69] Figure 14 depicts THV03-TB5 and THV03-TB6 inserts in the lentiviral vectors. For the production transgene carrying lentiviral vector, M tuberculosis genes 85A and ESAT6 were synthesized as a fusion construct. The fusion construct was subsequently cloned into a lentiviral vector in two orientations giving rise THV03-TB5 and THV03-TB6. The order of the genes in the fusion construct is in the order shown. THV03-TB5 and 6 constructs contain genes expressed only at active stage of tuberculosis. The construct was verified by sequencing. THV03-TB5 and THV03-TB6 were pseudotyped by the Indiana serotype of the vesicular stomatitis virus G protein (VSV-G) and generated in HEK293 cells.

[70] Figure 15 depicts T cell responses with THV03-TB5 and THV03-TB6 lentiviral vectors.

[71 ] Figure 16 depicts THV03-TB7 insert in the lentiviral vector. For the production transgene carrying lentiviral vector, M tuberculosis genes or part of the genes of Ag85A, ESAT6, Mpt64(Rv1980c) 9 amino acids, RVBD(Rv0140), HRP1 (Rv2626c), RV2028c, HspX(Rv2031 c), and Mtb32A(Rv1 196), were synthesized as a fusion construct. The order of the genes in the fusion construct is shown. THV03- TB7 construct contains genes expressed at active as well as latent stage of tuberculosis. In addition, this construct harbors a gene expressed at the reactivation stage of the tuberculosis. The fusion construct was subsequently cloned into a lentiviral vector to obtain THV03-TB7 and the construct was verified by sequencing. THV03-TB7 was pseudotyped by the Indiana serotype of the vesicular stomatitis virus G protein(VSV-G) and it was generated in HEK293 cells.

[72] Figure 17 depicts depicts IFN-γ ELISPOT results of LV vectored TB antigens with the THV03-TB7 lentiviral vector. [73] Figure 18 depicts depicts IFN-γ ELISPOT results of LV vectored TB antigens with the THV03-TB7 lentiviral vector.

[74] Figure 19 depicts the number of CD4+ cells, stained for Tuberculosis antigen 85B, producing IFN-γ and TNF-a in the inferior caval-lobe of C57/BI6 mice infected with Mycobacterium tuberculosis H37Rv and treated either with Isoniazid alone (Group 1 ) or with Isoniazid and THV03 (Group 2).

[75] Figure 20 depicts the number of CD4+ cells, stained for Tuberculosis antigen ESAT6, producing IFN-γ and TNF-a in the inferior caval-lobe of C57/BI6 mice infected with Mycobacterium tuberculosis H37Rv and treated either with Isoniazid alone (Group 1 ) or with Isoniazid and THV03 (Group 2).

DETAILED DESCRIPTION OF THE INVENTION

[76] 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.

[77] The present invention encompasses lentiviral vectors encoding M. tuberculosis antigens, and their use for the induction of immune responses in a host by intramuscular administration.

PROTEINS

[78] The invention encompasses proteins comprising M tuberculosis antigens, preferably Ag85A, ESAT6, Mpt64, EspC, RVBD, Rv1813c, HRP1 , HspX, Mtb39A, Rv2627c, Rv2628, PfkB, Mtb32A, EsxV, EsxW, HSP65, Rv2660, Archease, PE42, EsxJ, Rv1088, USP, PPE42, and PE35 antigens. Preferably, the M. tuberculosis antigen consists of the amino acid sequence of any of the SEQ ID NOs detailed herein.

[79] Preferred M. tuberculosis antigens are M. tuberculosis Ag85A, ESAT6, Mpt64, EspC, RVBD, Rv1813c, HRP1 , HspX, Mtb39A, Rv2627c, Rv2628, PfkB, Mtb32A, and EsxV antigens. [80] M. tuberculosis antigens comprising or consisting of the following the following amino acid sequences or fragments thereof, especially those in bold, are preferred.

[81] Ag85A (338aa)

MQLVDRVRGAVTGMSRRLVVGAVGAALVSGLVGAVGGTATAGAFSRPGLPVEYLQV PSPSMGRDIKVQFQSGGANSPALYLLDGLRAQDDFSGWDINTPAFEWYDQSGLSVV MPVGGQSSFYSDWYQPACGKAGCQTYKWETFLTSELPGWLQANRHVKPTGSAVVG LSMAASSALTLAIYHPQQFVYAGAMSGLLDPSQAMGPTLIGLAMGDAGGYKASDMWG PKEDPAWQRNDPLLNVGKLIANNTRVWVYCGNGKPSDLGGNNLPAKFLEGFVRTSNI KFQDAYNAGGGHNGVFDFPDSGTHSWEYWGAQLNAMKPDLQRALGATPNTGPAPQ GA (SEQ ID NO:24)

[82] ESAT6 (95aa)

MTEQQWNFAGIEAAASAIQGNVTSIHSLLDEGKQSLTKLAAAWGGSGSEAYQGVQQK WDATATELNNALQNLARTISEAGQAMASTEGNVTGMFA (SEQ ID NO:25)

[83] Mpt64(Rv1980c) (228aa)

MRIKIFMLVTAVVLLCCSGVATAAPKTYCEELKGTDTGQACQIQMSDPAYNINISLPSYY PDQKSLENYIAQTRDKFLSAATSSTPREAPYELNITSATYQSAIPPRGTQAVVLKVYQN AGGTHPTTTYKAFDWDQAYRKPITYDTLWQADTDPLPWFPIVQGELSKQTGQQVSIA PNAGLDPVNYQNFAVTNDGVIFFFNPGELLPEAAGPTQVLVPRSAIDSMLA (SEQ ID NO:26)

[84] EspC(Rv3615c) (103aa)

MTENLTVQPERLGVLASHHDNAAVDASSGVEAAAGLGESVAITHGPYCSQFNDTLNV YLTAHNALGSSLHTAGVDLAKSLRIAAKIYSEADEAWRKAIDGLFT (SEQ ID NO:27)

[85] RVBD(Rv0140) (126aa)

MSNRIVLEPSADHPITIEPTNRRVQVRVNGEWADTAAALCLQEASYPAVQYIPLADVV QDRLIRTETSTYCPFKGEASYYSVTTDAGDIVDDVMWTYENPYPAVAAIAGHVACYPD KAEISIFPG (SEQ ID NO:28)

[86] Rv1813c (MT1861 ) (143aa)

MITNLRRRTAMAAAGLGAALGLGILLVPTVDAHLANGSMSEVMMSEIAGLPIPPIIHYGAI AYAPSGASGKAWHQRTPARAEQVALEKCGDKTCKWSRFTRCGAVAYNGSKYQGGT GLTRRAAEDDAVNRLEGGRIVNWACN (SEQ ID NO:29) [87] HRP1 (Rv2626c)(143aa)

MTTARDIMNAGVTCVGEHETLTAAAQYMREHDIGALPICGDDDRLHGMLTDRDIVIKGL AAGLDPNTATAGELARDSIYYVDANASIQEMLNVMEEHQVRRVPVISEHRLVGIVTEAD IARHLPEHAIVQFVKAICSPMALAS (SEQ ID NO:30)

[88] HspX(Rv2031 c) (144aa)

MATTLPVQRHPRSLFPEFSELFAAFPSFAGLRPTFDTRLMRLEDEMKEGRYEVRAELP GVDPDKDVDIMVRDGQLTIKAERTEQKDFDGRSEFAYGSFVRTVSLPVGADEDDIKAT YDKGILTVSVAVSEGKPTEKHIQIRSTN (SEQ ID N0:31 )

[89] Mtb39A(Rv1 196)(391 aa)

MVDFGALPPEINSARMYAGPGSASLVAAAQMWDSVASDLFSAASAFQSWWGLTVG SWIGSSAGLMVAAASPYVAWMSVTAGQAELTAAQVRVAAAAYETAYGLTVPPPVIAE NRAELMILIATNLLGQNTPAIAVNEAEYGEMWAQDAAAMFGYAAATATATATLLPFEEA PEMTSAGGLLEQAAAVEEASDTAAANQLMNNVPQALQQLAQPTQGTTPSSKLGGLW KTVSPHRSPISNMVSMANNHMSMTNSGVSMTNTLSSMLKGFAPAAAAQAVQTAAQN GVRAMSSLGSSLGSSGLGGGVAANLGRAASVGSLSVPQAWAAANQAVTPAARALPL TSLTSAAERGPGQMLGGLPVGQMGARAGGGLSGVLRVPPRPYVMPHSPAAG

(SEQ ID NO:32)

[90] Rv2627c (Mt2702) (413aa)

MASSASDGTHERSAFRLSPPVLSGAMGPFMHTGLYVAQSWRDYLGQQPDKLPIARP TIALAAQAFRDEIVLLGLKARRPVSNHRVFERISQEVAAGLEFYGNRGWLEKPSGFFAQ PPPLTEVAVRKVKDRRRSFYRIFFDSGFTPHPGEPGSQRWLSYTANNREYALLLRHPE PRPWLVCVHGTEMGRAPLDLAVFRAWKLHDELGLNIVMPVLPMHGPRGQGLPKGAV FPGEDVLDDVHGTAQAVWDIRRLLSWIRSQEEESLIGLNGLSLGGYIASLVASLEEGLA CAILGVPVADLIELLGRHCGLRHKDPRRHTVKMAEPIGRMISPLSLTPLVPMPGRFIYAG IADRLVHPREQVTRLWEHWGKPEIVWYPGGHTGFFQSRPVRRFVQAALEQSGLLDAP RTQRDRSA (SEQ ID NO:33)

[91 ] Rv2628 (Mt2703) (120aa)

MSTQRPRHSGIRAVGPYAWAGRCGRIGRWGVHQEAMMNLAIWHPRKVQSATIYQVT DRSHDGRTARVPGDEITSTVSGWLSELGTQSPLADELARAVRIGDWPAAYAIGEHLSV EIAVAV (SEQ ID NO:34) [92] PfkB(Rv2029c)(339aa)

MTEPAAWDEGKPRIITLTMNPALDITTSVDWRPTEKMRCGAPRYDPGGGGINVARIV HVLGGCSTALFPAGGSTGSLLMALLGDAGVPFRVIPIAASTRESFTVNESRTAKQYRFV LPGPSLTVAEQEQCLDELRGAAASAAFWASGSLPPGVAADYYQRVADICRRSSTPLIL DTSGGGLQHISSGVFLLKASVRELRECVGSELLTEPEQLAAAHELIDRGRAEWWSLG SQGALLATRHASHRFSSIPMTAVSGVGAGDAMVAAITVGLSRGWSLIKSVRLGNAAG AAMLLTPGTAACNRDDVERFFELAAEPTEVGQDQYVWHPIVNPEASP

(SEQ ID NO:35)

[93] Mtb32A(Rv0125)(355aa)

MSNSRRRSLRWSWLLSVLAAVGLGLATAPAQAAPPALSQDRFADFPALPLDPSAMVA QVGPQWNINTKLGYNNAVGAGTGIVIDPNGVVLTNNHVIAGATDINAFSVGSGQTYGV DVVGYDRTQDVAVLQLRGAGGLPSAAIGGGVAVGEPWAMGNSGGQGGTPRAVPG RVVALGQTVQASDSLTGAEETLNGLIQFDAAIQPGDSGGPVVNGLGQVVGMNTAASD NFQLSQGGQGFAIPIGQAMAIAGQIRSGGGSPTVHIGPTAFLGLGVVDNNGNGARVQR WGSAPAASLGISTGDVITAVDGAPINSATAMADALNGHHPGDVISVTWQTKSGGTRT GNVTLAEGPPA (SEQ ID NO:36)

[94] EsxV(Rv3619c)(94aa)

MTINYQFGDVDAHGAMIRAQAGSLEAEHQAIISDVLTASDFWGGAGSAACQGFITQLG RN FQVI YEQAN AHGQKVQAAG N N MAQTDSAVGSS WA (SEQ ID NO:37)

[95] M tuberculosis antigens comprising or consisting of the following combined amino acid sequences or fragments thereof are particularly preferred:

[96] THV-TB1 (957aa X3= 2871 bp)

MQLVDRVRGAVTGMSRRLVVGAVGAALVSGLVGAVGGTATAGAFSRPGLPVEYLQV PSPSMGRDIKVQFQSGGANSPALYLLDGLRAQDDFSGWDINTPAFEWYDQSGLSVV MPVGGQSSFYSDWYQPACGKAGCQTYKWETFLTSELPGWLQANRHVKPTGSAWG LSMAASSALTLAIYHPQQFVYAGAMSGLLDPSQAMGPTLIGLAMGDAGGYKASDMWG PKEDPAWQRNDPLLNVGKLIANNTRVWVYCGNGKPSDLGGNNLPAKFLEGFVRTSNI KFQDAYNAGGGHNGVFDFPDSGTHSWEYWGAQLNAMKPDLQRALGATPNTGPAPQ GAMTEQQWNFAGIEAAASAIQGNVTSIHSLLDEGKQSLTKLAAAWGGSGSEAYQGVQ QKWDATATELNNALQNLARTISEAGQAMASTEGNVTGMFAFAVTNDGVIMTENLTVQ PERLGVLASHHDNAAVDASSGVEAAAGLGESVAITHGPYCSQFNDTLNVYLTAHNALG SSLHTAGVDLAKSLRI AAKI YSEADEAWRKAI DGLFTMSN RIVLEPSADH PITI EPTN RRV QVRVNGEWADTAAALCLQEASYPAVQYIPLADWQDRLIRTETSTYCPFKGEASYYS VTTDAGDIVDDVMWTYENPYPAVAAIAGHVACYPDKAEISIFPGMITNLRRRTAMAAAG LGAALGLGILLVPTVDAHLANGSMSEVMMSEIAGLPIPPIIHYGAIAYAPSGASGKAWHQ RTPARAEQVALEKCGDKTCKWSRFTRCGAVAYNGSKYQGGTGLTRRAAEDDAVNR LEGGRIVNWACNMTTARDIMNAGVTCVGEHETLTAAAQYMREHDIGALPICGDDDRLH GMLTDRDIVIKGLAAGLDPNTATAGELARDSIYYVDANASIQEMLNVMEEHQVRRVPVI SEHRLVGIVTEADIARHLPEHAIVQFVKAICSPMALAS (SEQ ID NO:38)

[97] THV-TB2 (1044aa X3= 3132bp)

MATTLPVQRHPRSLFPEFSELFAAFPSFAGLRPTFDTRLMRLEDEMKEGRYEVRAELP GVDPDKDVDIMVRDGQLTIKAERTEQKDFDGRSEFAYGSFVRTVSLPVGADEDDIKAT YDKGILTVSVAVSEGKPTEKHIQIRSTNMVDFGALPPEINSARMYAGPGSASLVAAAQM WDSVASDLFSAASAFQSVVWGLTVGSWIGSSAGLMVAAASPYVAWMSVTAGQAELT AAQVRVAAAAYETAYGLTVPPPVIAENRAELMILIATNLLGQNTPAIAVNEAEYGEMWA QDAAAMFGYAAATATATATLLPFEEAPEMTSAGGLLEQAAAVEEASDTAAANQLMNN VPQALQQLAQPTQGTTPSSKLGGLWKTVSPHRSPISNMVSMANNHMSMTNSGVSMT NTLSSMLKGFAPAAAAQAVQTAAQNGVRAMSSLGSSLGSSGLGGGVAANLGRAASV GSLSVPQAWAAANQAVTPAARALPLTSLTSAAERGPGQMLGGLPVGQMGARAGGGL SGVLRVPPRPYVMPHSPAAGVLSGAMGPFMHTGLYVAQSWIRAVGPYAWAGRCGRI GRWGITVGLSRGWSLIKSVRLGNAMSNSRRRSLRWSWLLSVLAAVGLGLATAPAQAA PPALSQDRFADFPALPLDPSAMVAQVGPQWNINTKLGYNNAVGAGTGIVIDPNGWL TNNHVIAGATDINAFSVGSGQTYGVDWGYDRTQDVAVLQLRGAGGLPSAAIGGGVA VGEPVVAMGNSGGQGGTPRAVPGRWALGQTVQASDSLTGAEETLNGLIQFDAAIQP GDSGGPWNGLGQVVGMNTAASDNFQLSQGGQGFAIPIGQAMAIAGQIRSGGGSPTV HIGPTAFLGLGWDNNGNGARVQRVVGSAPAASLGISTGDVITAVDGAPINSATAMAD ALNGHHPGDVISVTWQTKSGGTRTGNVTLAEGPPAMTINYQFGDVDAHGAMIRAQAG SLEAEHQAIISDVLTASDFWGGAGSAACQGFITQLGRNFQVIYEQANAHGQKVQAAGN NMAQTDSAVGSSWA (SEQ ID NO:39)

[98] THV-TB3 (927aa X3= 2781 bp)

MTSRFMTDPHAMRDMAGRFEVHAQTVEDEARRMWASAQNISGAGWSGMAEATSLD TMTQMNQAFRNIVNMLHGVRDGLVRDANNYEQQEQASQQILSSMAKTIAYDEEARRG LERGLNALADAVKVTLGPKGRNWLEKKWGAPTITNDGVSIAKEIELEDPYEKIGAELVK EVAKKTDDVAGDGTTTATVLAQALVREGLRNVAAGANPLGLKRGIEKAVEKVTETLLK GAKEVETKEQIAATAAISAGDQSIGDLIAEAMDKVGNEGVITVEESNTFGLQLELTEGM RFDKGYISGYFVTDPERQEAVLEDPYILLVSSKVSTVKDLLPLLEKVIGAGKPLLIIAEDV EGEALSTLWNKIRGTFKSVAVKAPGFGDRRKAMLQDMAILTGGQVISEEVGLTLENAD LSLLGKARKVWTKDETTIVEGAGDTDAIAGRVAQIRQEIENSDSDYDREKLQERLAKL AGGVAVIKAGAATEVELKERKHRIEDAVRNAKAAVEEGIVAGGGVTLLQAAPTLDELKL EGDEATGANIVKVALEAPLKQIAFNSGLEPGVVAEKVRNLPAGHGLNAQTGVYEDLLA AGVADPVKVTRSALQNAASIAGLFLTTEAWADKPEKEKASVPGGGDMGGMDFMIAG VDQALAATGQASQRAAGASGGVTVGVGVGTEQRNLSWAPSQFTFSSRSPDFVDET AGQSWCAILGLNQFHMLHRDDHINPPRPRGLDVPCARLRATNPLRALARCVQAGKPG TSSGHRSVPHTADLRIEAWAPTRDGCIRQAVLGTVESFLDLESAHAVHTRLRRLTADR DDDLLVAVLEEVIYLLDTVGETPVDLRLRDVDGGVDVTFATTDASTLVQVGAVPKAVSL NELRFSQGRHGWRCAVTLDVSAAIAGLFGQTVEDEARRMWRLFNANAEEYHALSA

(SEQ ID NO:40)

[99] THV-TB4 (958aa X3= 2874bp)

MNQSHKPPSIWGIDGSKPAVQAALWAVDEAASRDIPLRLLYAIEPDDPGYAAHGAAA RKLAAAENAVRYAFTAVEAADRPVKVEVEITQERPVTSLIRASAAAALVCVGAIGVHHF RPERVGSTAAALALSAQCPVAIVRPHRVPIGRDAAWIVVEADGSSDIGVLLGAVMAEA RLRDSPVRWTCRQSGVGDTGDDVRASLDRWLARWQPRYPDVRVQSAAVHGELLD YLAGLGRSVHMWLSASDQEHVEQLVGAPGNAVLQEAGCTLLWGQQYLMNFAVLPP EVNSARIFAGAGLGPMLAAASAWDGLAEELHAAAGSFASVTTGLAGDAWHGPASLAM TRAASPYVGWLNTAAGQAAQAAGQARLAASAFEATLAATVSPAMVAANRTRLASLVA ANLLGQNAPAIAAAEAEYEQIWAQDVAAMFGYHSAASAVATQLAPIQEGLQQQLQNVL AQLASGNLGSGNVGVGNIGNDNIGNANIGFGNRGDANIGIGNIGDRNLGIGNTGNWNI GIGITGNGQIGFGKPANPDVLVVGNGGPGVTALVMGGTDSLLPLPNIPLLEYAARFITPV HPGYTATFLETPSQFFPFTGLNSLTYDVSVAQGVTNLHTAIMAQLAAGNEVWFGTSQ SATIATFEMRYLQSLPAHLRPGLDELSFTLTGNPNRPDGGILTRFGFSIPQLGFTLSGAT PADAYPTVDYAFQYDGVNDFPKYPLNVFATANAIAGILFLHSGLIALPPDLASGVVQPV SSPDVLTTYILLPSQDLPLLVPLRAIPLLGNPLADLIQPDLRVLVELGYDRTAHQDVPSPF GLFPDVDWAEVAADLQQGAVQGVNDALSGLGLPPPWQPALPRLFMEKMSHDPIAADI GTQVSDNALHGVTAGSTALTSVTGLVPAGADEVSAQAATAFTSEGIQLLASNASAQDQ LHRAGEAVQDVARTYSQIDDGAAGVFAE (SEQ ID NO:41 )

[100] THV-TB5(433aa X3= 1299bp)

MQLVDRVRGAVTGMSRRLVVGAVGAALVSGLVGAVGGTATAGAFSRPGLPVEYLQV PSPSMGRDIKVQFQSGGANSPALYLLDGLRAQDDFSGWDINTPAFEWYDQSGLSVV MPVGGQSSFYSDWYQPACGKAGCQTYKWETFLTSELPGWLQANRHVKPTGSAWG LSMAASSALTLAIYHPQQFVYAGAMSGLLDPSQAMGPTLIGLAMGDAGGYKASDMWG PKEDPAWQRNDPLLNVGKLIANNTRVWVYCGNGKPSDLGGNNLPAKFLEGFVRTSNI KFQDAYNAGGGHNGVFDFPDSGTHSWEYWGAQLNAMKPDLQRALGATPNTGPAPQ GAMTEQQWNFAGIEAAASAIQGNVTSIHSLLDEGKQSLTKLAAAWGGSGSEAYQGVQ QKWDATATELNNALQNLARTISEAGQAMASTEGNVTGMFA (SEQ ID NO:42)

[101] THV-TB6 (433aa X3= 1299bp)

MTEQQWNFAGIEAAASAIQGNVTSIHSLLDEGKQSLTKLAAAWGGSGSEAYQGVQQK WDATATELNNALQNLARTISEAGQAMASTEGNVTGMFAMQLVDRVRGAVTGMSRRL WGAVGAALVSGLVGAVGGTATAGAFSRPGLPVEYLQVPSPSMGRDIKVQFQSGGAN SPALYLLDGLRAQDDFSGWDINTPAFEWYDQSGLSVVMPVGGQSSFYSDWYQPACG KAGCQTYKWETFLTSELPGWLQANRHVKPTGSAWGLSMAASSALTLAIYHPQQFVY AGAMSGLLDPSQAMGPTLIGLAMGDAGGYKASDMWGPKEDPAWQRNDPLLNVGKLI ANNTRVWVYCGNGKPSDLGGNNLPAKFLEGFVRTSNIKFQDAYNAGGGHNGVFDFP DSGTHSWEYWGAQLNAMKPDLQRALGATPNTGPAPQGA (SEQ ID NO:43)

[102] Preferred M. tuberculosis antigens include EsxW, HSP65, Rv2660, Archease, PE42, EsxJ, Rv1088, USP, PPE42, and PE35 antigens. M. tuberculosis antigens comprising or consisting of the following the following amino acid sequences or fragments thereof, especially those in bold, are also preferred.

[103] EsxW(Rv3620c) (98aa)

MTSRFMTDPHAMRDMAGRFEVHAQTVEDEARRMWASAQNISGAGWSGMAEATSLD TMTQMNQAFRNIVNMLHGVRDGLVRDANNYEQQEQASQQILSS (SEQ ID NO:44)

[104] HSP65(Rv0440)(540aa)

MAKTIAYDEEARRGLERGLNALADAVKVTLGPKGRNWLEKKWGAPTITNDGVSIAKEI ELEDPYEKIGAELVKEVAKKTDDVAGDGTTTATVLAQALVREGLRNVAAGANPLGLKR GIEKAVEKVTETLLKGAKEVETKEQIAATAAISAGDQSIGDLIAEAMDKVGNEGVITVEE SNTFGLQLELTEGMRFDKGYISGYFVTDPERQEAVLEDPYILLVSSKVSTVKDLLPLLEK VIGAGKPLLIIAEDVEGEALSTLVVNKIRGTFKSVAVKAPGFGDRRKAMLQDMAILTGGQ VISEEVGLTLENADLSLLGKARKWVTKDETTIVEGAGDTDAIAGRVAQIRQEIENSDSD YDREKLQERLAKLAGGVAVIKAGAATEVELKERKHR1EDAVRNAKAAVEEGIVAGGGV TLLQAAPTLDELKLEGDEATGANIVKVALEAPLKQIAFNSGLEPGVVAEKVRNLPAGHG LNAQTGVYEDLLAAGVADPVKVTRSALQNAASIAGLFLTTEAWADKPEKEKASVPGG GDMGGMDF (SEQ ID NO:45)

[105] Rv2660 (75aa)

MIAGVDQALAATGQASQRAAGASGGVTVGVGVGTEQRNLSVVAPSQFTFSSRSPDFV DETAGQSWCAILGLNQFH (SEQ ID NO:46)

[106] Archease (Rv2630) (179aa)

MLHRDDHINPPRPRGLDVPCARLRATNPLRALARCVQAGKPGTSSGHRSVPHTADLRI EAWAPTRDGCIRQAVLGTVESFLDLESAHAVHTRLRRLTADRDDDLLVAVLEEVIYLLD TVGETPVDLRLRDVDGGVDVTFATTDASTLVQVGAVPKAVSLNELRFSQGRHGWRCA VTLDV (SEQ ID NO:47)

[107] PG42(Rv2608)(694aa)

MSLVIATPQLLATAALDLASIGSQVSAANAAAAMPTTEWAAAADEVSAAIAGLFGAHA RQYQALSVQVAAFHEQFVQALTAAAGRYASTEAAVERSLLGAVNAPTEALLGRPLIGN GADGTAPGQPGAAGGLLFGNGGNGAAGGFGQTGGSGGAAGLIGNGGNGGAGGTG AAGGAGGNGGWLWGNGGNGGVGGTSVAAGIGGAGGNGGNAGLFGHGGAGGTGG AGLAGANGVNPTPGPAASTGDSPADVSGIGDQTGGDGGTGGHGTAGTPTGGTGGD GATATAGSGKATGGAGGDGGTAAAGGGGGNGGDGGVAQGDIASAFGGDGGNGSD GVAAGSGGGSGGAGGGAFVHIATATSTGGSGGFGGNGAASAASGADGGAGGAGGN GGAGGLLFGDGGNGGAGGAGGIGGDGATGGPGGSGGNAGIARFDSPDPEAEPDVV GGKGGDGGKGGSGLGVGGAGGTGGAGGNGGAGGLLFGNGGNGGNAGAGGDGGA GVAGGVGGNGGGGGTATFHEDPVAGVWAVGGVGGDGGSGGSSLGVGGVGGAGG VGGKGGASGMLIGNGGNGGSGGVGGAGGVGGAGGDGGNGGSGGNASTFGDENSI GGAGGTGGNGGNGANGGNGGAGGIAGGAGGSGGFLSGAAGVSGADGIGGAGGAG GAGGAGGSGGEAGAGGLTNGPGSPGVSGTEGMAGAPG (SEQ ID NO:48) [108] EsxJ(Rv1038c) (98aa)

MASRFMTDPHAMRDMAGRFEVHAQTVEDEARR WASAQNISGAGWSGMAEATSLD TMTQMNQAFRNIVNMLHGVRDGLVRDANNYEQQEQASQQILSS (SEQ ID NO:49)

[109] Rv1088(144aa)

MSYMIATPAALTAAATDIDGIGSAVSVANAAAVAATTGVLAAGGDEVLAAIARLFNANA EEYHALSAQVAAFQTLFVRTLTGGCGVFRRRRGRQCVTAAEHRAAGAGRRQRRRRS GDGQWRLRQQRHFGCGGQPEFRQHSEHRR (SEQ ID NO:50)

[1 10] USP (Rv2028c) (279aa)

MNQSHKPPSIWGIDGSKPAVQAALWAVDEAASRDIPLRLLYAIEPDDPGYAAHGAAA RKLAAAENAVRYAFTAVEAADRPVKVEVEITQERPVTSLIRASAAAALVCVGAIGVHHF RPERVGSTAAALALSAQCPVAIVRPHRVPIGRDAAWIVVEADGSSDIGVLLGAVMAEA RLRDSPVRWTCRQSGVGDTGDDVRASLDRWLARWQPRYPDVRVQSAAVHGELLD YLAGLGRSVHMWLSASDQEHVEQLVGAPGNAVLQEAGCTLLVVGQQYL

(SEQ ID N0:51 )

[1 1 1 ] PPE42(Rv2608)(580aa)

MNFAVLPPEVNSARIFAGAGLGPMLAAASAWDGLAEELHAAAGSFASVTTGLAGDAW HGPASLAMTRAASPYVGWLNTAAGQAAQAAGQARLAASAFEATLAATVSPAMVAAN RTRLASLVAANLLGQNAPAIAAAEAEYEQIWAQDVAAMFGYHSAASAVATQLAPIQEG LQQQLQNVLAQLASGNLGSGNVGVGNIGNDNIGNANSGFGNRGDANIGIGNIGDRNLGI GNTGNWNIGIGITGNGQIGFGKPANPDVLWGNGGPGVTALVMGGTDSLLPLPNIPLLE YAARFITPVHPGYTATFLETPSQFFPFTGLNSLTYDVSVAQGVTNLHTAIMAQLAAGNE WVFGTSQSATIATFEMRYLQSLPAHLRPGLDELSFTLTGNPNRPDGGILTRFGFSIPQ LGFTLSGATPADAYPTVDYAFQYDGVNDFPKYPLNVFATANAIAGILFLHSGLIALPPDL ASGWQPVSSPDVLTTYILLPSQDLPLLVPLRAIPLLGNPLADLIQPDLRVLVELGYDRTA HQDVPSPFGLFPDVDWAEVAADLQQGAVQGVNDALSGLGLPPPWQPALPRLF

(SEQ ID NO:52)

[1 12] PE35(Rv3872)(99aa)

MEKMSHDPIAADIGTQVSDNALHGVTAGSTALTSVTGLVPAGADEVSAQAATAFTSEG IQLLASNASAQDQLHRAGEAVQDVARTYSQIDDGAAGVFAE (SEQ ID NO:53) [1 13] Preferably, the protein is a fusion protein comprising amino acid sequences from at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , or 12 different M. tuberculosis proteins. Preferably, the amino acid sequences consist of at least 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 30, 40, 50, 100, 200, 300, 400, or 500 consecutive amino acids of one of the SEQ ID NOs detailed herein.

[1 14] The protein can be purified. 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.

NUCLEIC ACIDS

[1 15] The invention encompasses nucleic acids encoding an M. tuberculosis antigen. The nucleic acid can be single-stranded or double-stranded. The nucleic acid can be an RNA or DNA molecule. Preferred nucleic acids encode an amino acid sequence of any of the SEQ ID NOs detailed herein. Preferably, the nucleic acid encodes amino acid sequences from at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , or 12 different M. tuberculosis proteins. Preferably, the amino acid sequences consist of at least 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 30, 40, 50, 100, 200, 300, 400, or 500 consecutive amino acids of one of the SEQ ID NOs detailed herein. The invention encompasses an isolated nucleic acid of the invention inserted into a vector.

[1 16] 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. VECTORS

[1 17] The invention encompasses vectors encoding an M tuberculosis antigen. Preferred vectors comprise a nucleic acid sequence encoding an amino acid sequence of any of the SEQ ID NOs detailed herein.

[1 18] Preferably, the vector encodes an Ag85A, ESAT6, Mpt64, EspC, RVBD,

Rv1813c, HRP1 , HspX, Mtb39A, Rv2627c, Rv2628, PfkB, Mtb32A, EsxV, EsxW, HSP65, Rv2660, Archease, PE42, EsxJ, Rv1088, USP, PPE42, or PE35 antigen.

[1 19] A preferred vector encodes Ag85A, ESAT6, Mpt64, EspC, RVBD, Rv1813c, and HRP1 antigens. A particularly preferred vector encodes the amino acid sequence of SEQ ID NO:38.

[120] A preferred vector encodes HspX, Mtb39A, Rv2627c, Rv2628, PfkB, Mtb32A, and EsxV antigens. A particularly preferred vector encodes the amino acid sequence of SEQ ID NO:39.

[121] A preferred vector encodes EsxW, HSP65, Rv2660, Archease, PE42, EsxJ, and Rv1088 antigens. A particularly preferred vector encodes the amino acid sequence of SEQ ID NO:40.

[122] A preferred vector encodes USP, PPE42, and PE35 antigens. A particularly preferred vector encodes the amino acid sequence of SEQ ID NO:41.

[123] A preferred vector encodes Ag85A and ESAT6 antigens. A particularly preferred vector encodes the amino acid sequence of SEQ ID NO:42 or SEQ ID NO:43.

[124] Most preferably, the vector encodes M. tuberculosis antigens Ag85A, ESAT6, Mpt64(Rv1980c), RVBD(Rv0140), HRP1 (Rv2626c), RV2028c, HspX(Rv2031 c), and Mtb32A(Rv1 196), most preferably as a fusion protein in the order: Ag85A, ESAT6, Mpt64(Rv1980c), RVBD(Rv0140), HRP1 (Rv2626c), RV2028c, HspX(Rv2031 c), and Mtb32A(Rv1 196). A particularly preferred vector encodes the following amino acid sequence:

MQLVDRVRGAVTGMSRRLVVGAVGAALVSGLVGAVGGTATAGAFSRPGLPVEYLQV PSPSMGRDIKVQFQSGGANSPALYLLDGLRAQDDFSGWDINTPAFEWYDQSGLSVV MPVGGQSSFYSDWYQPACGKAGCQTYKWETFLTSELPGWLQANRHVKPTGSAWG LSMAASSALTLAIYHPQQFVYAGAMSGLLDPSQAMGPTLIGLAMGDAGGYKASDMWG

PKEDPAWQRNDPLLNVGKLIANNTRVWVYCGNGKPSDLGGNNLPAKFLEGFVRTSNI KFQDAYNAGGGHNGVFDFPDSGTHSWEYWGAQLNAMKPDLQRALGATPNTGPAPQ GAMTEQQWNFAGIEAAASA!QGNVTSIHSLLDEGKQSLTKLAAAWGGSGSEAYQGVQ QKWDATATELNNALQNLARTISEAGQAMASTEGNVTGMFAFAVTNDGVIMSNRIVLEP SADHPITIEPTNRRVQVRVNGEWADTAAALCLQEASYPAVQYIPLADVVQDRLIRTET STYCPFKGEASYYSVTTDAGDIVDDVMWTYENPYPAVAAIAGHVACYPDKAEISIFPG MTTARDIMNAGVTCVGEHETLTAAAQYMREHDIGALPICGDDDRLHGMLTDRDIVIKGL AAGLDPNTATAGELARDSIYYVDANASIQEMLNVMEEHQVRRVPVISEHRLVGIVTEAD IARHLPEHAIVQFVKAICSPMALASMNQSHKPPSIWGIDGSKPAVQAALWAVDEAASR DIPLRLLYAIEPDDPGYAAHGAAARKLAAAENAVRYAFTAVEAADRPVKVEVEITQERP VTSLIRASAAAALVCVGA1GVHHFRPERVGSTAAALALSAQCPVAIVRPHRVPIGRDAA WIWEADGSSDIGVLLGAVMAEARLRDSPVRVVTCRQSGVGDTGDDVRASLDRWLAR WQPRYPDVRVQSAAVHGELLDYLAGLGRSVHMVVLSASDQEHVEQLVGAPGNAVLQ EAGCTLLVVGQQYLMATTLPVQRHPRSLFPEFSELFAAFPSFAGLRPTFDTRLMRLED EMKEGRYEVRAELPGVDPDKDVDIMVRDGQLTIKAERTEQKDFDGRSEFAYGSFVRT VSLPVGADEDDIKATYDKGILTVSVAVSEGKPTEKHIQIRSTNMSNSRRRSLRWSWLLS VLAAVGLGLATAPAQAAPPALSQDRFADFPALPLDPSAMVAQVGPQVVNINTKLGYNN AVGAGTGIVIDPNGVVLTNNHVIAGATDINAFSVGSGQTYGVDWGYDRTQDVAVLQL RGAGGLPSAAIGGGVAVGEPWAMGNSGGQGGTPRAVPGRVVALGQTVQASDSLTG AEETLNGLIQFDAAIQPGDSGGPVVNGLGQWGMNTAASDNFQLSQGGQGFAIPIGQA MAIAGQIRSGGGSPTVHIGPTAFLGLGWDNNGNGARVQRVVGSAPAASLGISTGDVI TAVDGAPINSATAMADALNGHHPGDVISVTWQTKSGGTRTGNVTLAEGPPA

(SEQ ID NO:54).

[125] The vector can be an expression vector. The vector can be a plasmid vector. Preferably, the vector is a lentiviral vector.

[126] 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. [127] 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.

[128] Preferably, the lentiviral vector particles have the capacity for integration.

As such, they contain a functional integrase protein. 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.

[129] 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.

[130] Such vectors are based on the separation of the cis- and frans-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.

[131] Efficient integration and replication in non-dividing cells generally requires the presence of two c/s-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.

[132] 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.

[133] 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 the LTR (AU3) (Miyoshi H et al, 1998, J Virol. 72(10):8150-7; Zufferey et al., 1998, J Virol 72(12):9873-80).

[134] 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 (AU3) removing promoter and enhancer sequences in the 3' LTR.

[135] The packaging sequence Ψ (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).

[136] In one embodiment, the invention encompasses a lentiviral vector comprising a lentiviral packaging sequence Ψ (psi).

[137] Further additional functional sequences, such as a transport RNA-binding site or primer binding site (PBS) or a Woodchuck PostTranscriptional 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.

[138] 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. [139] Thus, in a preferred embodiment, the lentiviral vector comprises at least one cPPT/CTS sequence, one Ψ sequence, one (preferably 2) LTR sequence, and an expression cassette including a transgene under the transcriptional control of a β2ηη or class I MHC promoter.

[140] The invention encompasses methods for generating a vector comprising inserting a nucleic acid encoding the amino acid sequence of any of the SEQ ID NOs detailed herein into a vector, preferably a lentiviral vector.

[141] Preferably, the vector comprises Vpx protein. SIVmac251 protein Vpx enhances the transduction efficiency of the vectors in myeloid cells. The incorporation of Vpx into the lentiviral vector is based on results demonstrating that this accessory protein of HIV-2 and SIV, counteracts the SAMHD1 restriction factor in myeloid and resting T cells, thus rendering them permissive to transduction. Several groups have demonstrated that adding Vpx to lentiviral vectors increases indeed their transduction efficacy (Bobadilla, Sunseri et al., Gene Ther 20(5): 514-520, 2013; Tareen, Kelley- Clarke et al., Mol Ther 22(3): 575-587, 2014). The envelope plasmid used during vector production embeds the sequence of the Vpx which generates lentiviral particles packaged with Vpx.

PROMOTER

[142] 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.

[143] Most preferably, the promoter is not a CMV promoter/enhancer.

Preferably, the promoter is not a dectin-2 or MHCII promoter.

[144] The sequences of various mammalian (human) MHC class I promoters are shown below: HLA-A2 (MHC I):

attggggagtcccagccttggggattccccaactccgcagtttcttttctccctctcccaacct atgtagggtccttcttcctggatactcacgacgcggacccagttctcactcccattgggtgtcg ggtttccagagaagccaatcagtgtcgtcgcggtcgcggttctaaagtccgcacgcacccaccg ggactcagattctccccagacgccgagg

(SEQ ID NO: 1)

HLA-B7 (MHC I):

ggggaggcgcagcgttggggattccccactcccctgagtttcacttcttctcccaacttgtgtc gggtccttcttccaggatactcgtgacgcgtccccacttcccactcccattgggtattggatat ctagagaagccaatcagcgtcgccgcggtcccagttctaaagtccccacgcacccacccggact cagag (SEQ ID NO: 2)

HLA-Cw5 (MHC I):

cactggggaggcgccgcgttgaggattctccactcccctcagtttcacttcttctcccaacctg cgtcgggtccttcttcctgaatactcatgacgcgtccccaattcccactcccattgggtgtcgg gttctagagaagccaatcagcgtctccgcagtcccggtctaaagtccccagtcacccacccgga ctcagattctccccagacgccgag

(SEQ ID NO: 3)

HLA-E (MHC I):

taagaactgctgattgctgggaaactctgcagtttcccgttcctctcgtaacctggtcatgtgt ccttcttcctggatactcatgacgcagactcagttctcattcccaatgggtgtcgggtttctag agaagccaatcagcgtcgccacgactcccgactataaagtccccatccggactcaagaagttct caggactcagagg (SEQ ID NO: 4)

HLA-F (MHC I):

aggccccgaggcggtgtctggggttggaaggctcagtattgagaattccccatctccccagagt ttctctttctctcccaacccgtgtcaggtccttcatcctggatactcataacgcggccccattt ctcactcccattgggcgtcgcgtttctagagaagccaatcagtgtcgccgcagttcccaggttc taaagtcccacgcaccccgcgggactcatatttttcccagacgcggaggttggggtcatg (SEQ ID NO: 5)

[145] A sequence of the human p2-microglobulin promoter is shown below: aacatcacgagactctaagaaaaggaaactgaaaacgggaaagtccctctctctaacctggcac tgcgtcgctggcttggagacaggtgacggtccctgcgggccttgtcctgattggctgggcacgc gtttaatataagtggaggcgtcgcgctggcgggcattcctgaagctgacagcattcgggccgag (SEQ ID NO: 6) .

[146] The MHCI and β2ηη 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-DRa promoter. Id.

[147] In various embodiments, the lentiviral vector comprises a β2ηη 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.

[148] 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 2X, 3X, or 4X higher than the expression in kidney, smooth muscle, liver, and/or heart cells. Whether a promoter is "(2X, 3X, or 4X) 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 p2m, HLA-A2, HLA-B7, HLA-Cw5, HLA-F, and HLA-E promoters are "dendritic cell-specific promoters." The EF1 a promoter is not. Thus, preferably the promoter is not an EF1 a promoter.

[149] 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 p2m, 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.

[150] 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.

[151 ] In one embodiment, the invention encompasses lentiviral vector particles comprising a lentiviral vector that comprises a dendritic cell-specific promoter directing expression of a microbial or tumor antigen, wherein the lentiviral vector particles exhibit higher expression of the antigen in BDCM cells than in HEK 293 T cells.

[152] 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.

[153] 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 a promoter. Preferably, the CTL response with the promoter is at least 2-fold or 3- fold higher than with the EF1 a 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 a promoter" can be determined using the assay set forth in the examples. Other assays that provide similar results can also be used.

[154] 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.

[155] The invention encompasses lentiviral vectors containing a promoter that does not contain an enhancer.

[156] The invention encompasses the insertion of an MHC Class I (MHCI) or β2 microglobulin promoter (β2ηη) promoter into a lentiviral vector. As used herein, an "MHC Class I (MHCI) promoter" or "β2 microglobulin promoter" includes a naturally occurring or synthetic MHC Class I promoter or β2 microglobulin promoter. The term "MHC Class I promoter" does not include a β2ηη promoter.

[157] 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.

[158] The promoter can be a naturally occurring promoter. Examples of naturally occurring promoters are the human p2m, 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 β2ηη and class I MHC proteins enter the Major Histocompatibility Complex (MHC). [159] 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 p2-microglobulin (P2m). 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).

[160] 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.

[161] 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.

[162] Preferred promoters are naturally occurring human β2ηη, HLA-A2, HLA-B7, HLA-Cw5, HLA-E and HLA-F promoters.

[163] 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.

[164] 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 β2ηη or MHC class I gene promoter (e.g., SEQ ID NOs: 1 -6 and 19).

[165] 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 a and Y/enhancer B regulatory elements). See also Van den Elsen, Immunogenetics (1998) 48:208-21 1. [166] 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.

[167] 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.

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

[169] 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 a, 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.

[170] 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). [171] In one embodiment, the invention encompasses a method comprising inserting a promoter of the invention, particularly a β2ηη or MHC class I promoter, into a lentiviral vector to direct expression of a transgene, which preferably encodes an M tuberculosis, most preferably encoding a protein comprising an amino acid sequence of any of the SEQ ID NOs detailed herein. The method can further comprise inserting any of the other nucleic acid elements mentioned herein, such as a DNA flap sequence.

ISOLATED CELLS

[172] The invention encompasses cells comprising vectors and lentiviral vector particles encoding an M. tuberculosis antigen, particularly an amino acid sequence of any of the SEQ ID NOs detailed herein.

[173] In one embodiment, the cell contains the vector integrated into the cellular genome. In one embodiment, the cell contains the vector transiently expressing the M tuberculosis antigen. In one embodiment, the cell produces lentiviral vector particles encoding the M tuberculosis antigen.

[174] In various embodiments, the invention encompasses a cell line, a population of cells, or a cell culture comprising vectors and lentiviral vector particles encoding the M tuberculosis antigen. LENTIVIRAL VECTOR PARTICLES

[175] 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.

[176] In one embodiment, the lentiviral vector particles encode an M tuberculosis antigen. Preferably, the lentiviral vector particles comprise a nucleic acid sequence encoding an amino acid sequence of any of the SEQ ID NOs detailed herein.

[177] 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 NDK, Gag and Pol proteins.

[178] According to one embodiment of this method, the lentivector particles are obtained in a host cell transformed with a DNA plasmid. 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, preferably encoding a protein comprising an amino acid sequence of any of the SEQ ID NOs detailed herein, transcriptionally linked to a MHC class I promoter.

[179] 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(1 1 ):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.

[180] 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.

[181 ] 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.

[182] An appropriate host cell is preferably a human cultured cell line as, for example, a HEK cell line. [183] 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.

[184] 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 ai, 2005, J Virol 79(3): 1 165-71 ; Relander ef a/., 2005, Mol Ther 1 1 (3):452-9; Stewart et al., 2009, Gene Ther, 16(6):805- 14; and Stuart et ai, 201 1 , Hum gene Ther., with respect to such techniques for producing viral particles.

[185] An object of the present invention consists of a host cell transformed with a lentiviral particle vector.

[186] The lentiviral particle vectors can comprise the following elements, as previously defined:

- cPPT/CTS polynucleotide sequence; and

- a transgene sequence, preferably encoding a protein comprising an amino acid sequence of any of the SEQ ID NOs detailed herein, under control of a promoter of the invention, and optionally one of the additional elements described above.

[187] Preferably, the lentivector particles are in a dose of 10⁶, 2 x 10⁶, 5x 10⁶, 10⁷, 2 x 10⁷, 5 x 10⁷, 10⁸, 2 x 10⁸, 5 x 10⁸, or 10⁹ TU.

METHODS FOR EXPRESSING A TRANSGENE IN A CELL

[188] The present invention encompasses methods for expressing a transgene, preferably encoding a protein comprising an amino acid sequence of any of the SEQ ID NOs detailed herein, 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.

[189] The cells are preferably mammalian cells, particularly human cells.

Particularly preferred are human non-dividing cells. [190] The transgene preferably encodes an immunogenic polypeptide, preferably encoding a protein comprising an amino acid sequence of any of the SEQ ID NOs detailed herein. The method can further comprise harvesting or isolating the polypeptide.

[191] The lentiviral vector or lentiviral particle vector preferably comprises a promoter of the invention.

[192] 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, preferably encoding a protein comprising an amino acid sequence of any of the SEQ ID NOs detailed herein, and transducing a cell with the vector containing the promoter.

USE OF LENTIVIRAL VECTORS

[193] 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 immunogenic and/or therapeutic compositions and/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, preferably encoding a protein comprising an amino acid sequence of any of the SEQ ID NOs detailed herein, present in the vectors under the transcriptional control of any of the promoters of the invention.

[194] The invention encompasses methods for expressing a protein comprising any of the amino acid sequences of the invention. Preferably, the method comprises administering a nucleic acid encoding any of the amino acid sequences of the invention into a cell under conditions that allow expression of the protein. The nucleic acid can be any of the vectors of the invention, preferably a lentiviral vector. In a preferred embodiment, the method comprises contacting a cell with a lentiviral vector comprising a nucleic acid encoding any of the amino acid sequences of the invention under conditions that allow entry and reverse transcription of the lentiviral vector and expression of the protein [195] 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.

[196] Preferably, the administration is intramuscular. In one embodiment, the lentivector is injected into the muscle using a needle.

[197] Preferably, the antigen is an M. tuberculosis antigen, particularly encoded by an amino acid sequence of any of the SEQ ID NOs detailed herein.

[198] Preferably, the lentivector particle is an integrating lentivector particle, comprising a functional integrase protein.

[199] 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.

[200] 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 a M. tuberculosis antigen, wherein the promoter does not contain an enhancer, and wherein the lentiviral vector particles exhibit higher expression of the antigen in BDCM cells than in HEK 293 T cells; integrating the DNA of the lentiviral vector into cells of the human; expressing the M. tuberculosis antigen in the cells of the human; and generating an immune response against the M. tuberculosis antigen.

[201] 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 a M. tuberculosis 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 M. tuberculosis antigen in the cells of the human; and generating an immune response against the M. tuberculosis antigen.

[202] 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 a M. tuberculosis 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 M. tuberculosis antigen in the cells of the human; and generating an immune response against the M. tuberculosis antigen.

[203] 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 a M. tuberculosis 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 M. tuberculosis antigen in the cells of the human; and generating an immune response against the M. tuberculosis antigen.

[204] 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 timepoints by PCR.

[205] 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 a M. tuberculosis antigen, wherein the promoter does not contain an enhancer; integrating the DNA of the lentiviral vector into cells of the human; expressing the M. tuberculosis antigen in the cells of the human; and generating an immune response.

[206] Preferably, the lentivector particles are in a dose of 10⁶, 2 x 10⁶, 5x 10⁶, 10⁷, 2 x 10⁷, 5 x 10⁷, 10⁸, 2 x 10⁸, 5 x 10⁸, or 10⁹ TU.

[207] 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.

[208] The present invention thus provides vectors that are useful as a medicament or vaccine, particularly for intramuscular administration.

[209] These vectors are preferentially used for the treatment or prophylaxis of infectious diseases.

[210] 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 slow or endogenous pathogenic microorganisms such as Mycobacteria.

[21 1 ] Accordingly, the invention relates to an immunogenic composition comprising a lentiviral vector as previously defined.

[212] 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.

[213] 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. [214] The expression of the transgene is greatly improved by inclusion of a promoter of the invention in the vector.

[215] 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 other words, they can be used for the preparation of therapeutic composition for the treatment of diseases by induction of, stimulation of, or participation in the occurrence of a cell-mediated immune response, especially a CTL response or a memory response.

[216] The lentiviral vectors of the invention can be used in methods of treatment and methods of inducing an immune response 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.

[217] The lentiviral vectors according to the invention are preferably for intramuscular administration, most preferably by injection with a needle.

[218] 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.

[219] In another embodiment, the immunogenic compositions are used once or upon repeated administration so that they can enable the occurrence of a long-term memory cell mediated response.

[220] 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, 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.

[221] 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, 3, 4 ,5, 6, 7, 8, 9, 10, 1 1 , or 12 different sequences.

[222] As a result, the invention encompasses a composition that could be used in prophylactic and/or therapeutic vaccination protocols.

[223] In particular, it can be used in combination with adjuvants, other immunogenic compositions, chemotherapy, or any other therapeutic treatment.

[224] 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 an M. tuberculosis antigen, particularly encoded by an amino acid sequence of any of the SEQ ID NOs detailed herein.

[225] 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 an M. tuberculosis 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.

[226] 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 an M. tuberculosis antigen, particularly encoded by an amino acid sequence of any of the SEQ ID NOs detailed herein.

[227] 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 an M. tuberculosis 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.

[228] 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

[229] PCR amplification of the proviral region of the pTRIPAU3-CMV-GFP(15) was performed using direct (5'-CTTACTAGTTGGAAGGGCTAATTCACTCCCAAC-3'; SEQ ID NO:7) and reverse (5 '-CATTCTAG AACTGCTAG AG ATTTTCCACACTG-3 ' ; SEQ ID NO:8) oligonucleotides encompassing respectively the Spel and Xbal restriction sites. The resulting fragment was digested and cloned between the Spel and Xbal sites of the pVAX-1 plasmid (Invitrogen, Lifetech) from which the Mlul site have been deleted. The resulting plasmid was named pFLAP-CMV-GFP. The SV40 sequence was amplified by PCR from the pTRIPAU3-CMV-GFP plasmid (using the 5'- TACCCCGGGCCATGGCCTCCAAAAAAGCCTCCTCACTACTTC-3' (SEQ ID NO:9) and 5'-ACTCCCGGGTAATTTTTTTTATTTATGCAGAGGCCGAGGCCGCC-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'-TACACGCGTGGAGTTCCGCGTTACATAACTTACGG-3'; SEQ ID NO:1 1 ) and reverse (5'-

CGTGGATCCGATCGCGGTGTCTTCTATGGAGGTCAAAAC-3'; SEQ ID NO: 12) oligonucleotides encompassing the Mlul and BamHI sites, respectively. The resulting fragment was cloned back between the Mlul 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 β2ηη promoter and cloned between the Mlul and BamHI sites of pFLAP-CMV- GFP-SV to create pFlap-p2m-SV. The amplified β2ηη promoter sequence is the following

GAGAAACCCTGCAGGGAATTCCCCAGCTGTAGTTATAAACAGAAGTTCTCCTTCTG CTAGGTAGCATTCAAAGATCTTAATCTTCTGGGTTTCCGTTTTCTCGAATGAAAAAT GCAGGTCCGAGCAGTTAACTGGCGGGGGCACCATTAGCAAGTCACTTAGCATCTC TGGGGCCAGTCTGCAAAGCGAGGGGGCAGCCTTAATGTGCCTCCAGCCTGAAGT CCTAGAATGAGCGCCCGGTGTCCCAAGCTGGGGCGCGCACCCCAGATCGGAGGG CGCCGATGTACAGACAGCAAACTCACCCAGTCTAGTGCATGCCTTCTTAAACATCA CGAGACTCTAAGAAAAGGAAACTGAAAACGGGAAAGTCCCTCTCTCTAACCTGGCA CTGCGTCGCTGGCTTGGAGACAGGTGACGGTCCCTGCGGGCCTTGTCCTGATTG GCTGGGCACGCGTTTAATATAAGTGGAGGCGTCGCGCTGGCGGGCATTCCTGAA G CTG AC AG C ATTC GG G C CG AG (SEQ ID NO: 15). The M. tuberculosis antigen(s) can be synthetized and cloned between the BamHI and Xhol sites of the pFlap-p2m-SV, in place of the GFP gene.

[230] For example, pFlap-p2m-GFP-SV can be digested by BamHI and Xhol, and a DNA linker containing a Multiple Cloning Site (MCS, carrying Sail, Sacll, Ndel, AscI and Nhel restriction sites) can be cloned between those sites, in place of the GFP gene to allow insertion of a nucleic acid sequence encoding the M. tuberculosis antigen(s).

[231 ] The packaging plasmid pTHV-GP-N was constructed by amplifying the HIV-1 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 Eagl and Sail restriction enzymes and inserted in the p8.74 packaging plasmid (15) from which the Eag1 -Sall fragment had been previously removed.

[232] 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).

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

Example 2. Lentiviral production

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

[235] 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 by Life technologies), BENZONASE® (pharma grade I, 100 000U, Merck Millipore) and MgCL₂ 1 M. 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 NaCI. Both fractions are diluted to decrease NaCI concentration up to ± 150mM before pooling. I EX eluate is further concentrated approximately 40 fold by ultrafiltration using a 100KDa Omega T series filter, 0,1 m² (Pall) and diafiltrated with PBS-Lactose 40 mg.L-1. Purified bulk (Drug substance) is finally filtered through a 0.2 μΜ Sartobran H5 filter, 300cm² (Sartorius Stedim) and aseptically distributed on 2R 3mL-glass vials with a target filling volume of 650μΙ_ (1200μΙ_ 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.

[236] 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

[237] 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% C02 in moist atmosphere. Cells are transduced with 3 successive dilutions of lentiviral vector. 72h 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.

Example 4. T -specific response (median) in C57BI/6j mice

[238] Animals: For non-GLP studies, C57BL/6J Rj (C57BI/6J) female mice of four weeks or Sprague Dawley RjHan:SD (Sprague Dawley) female mice of eight weeks can be purchased from Janvier Laboratories (France). C57BI/6j mice can be immunized with 1 x 10⁶TU of lentivectors in which M. tuberculosis antigen expression is driven by a promoter (e.g., β2ηη or MHCI). 12 days after immunization, the specific T- cell responses can be monitored in mice splenocytes by IFN-γ ELISPOT.

[239] Ninety-six-well tissue culture plates (Millipore) are coated overnight at 4 °C with 50 μΙ/well of 5 μg/ml anti-mouse IFNy mAb (Mouse IFNy Elispot pair; BD Biosciences Pharmingen). The plates are washed three times with 200 μΙ DPBS/well and blocked with 200 μ l/well of DPBS/10% fetal bovine serum for 2 h at 37°C. The plates are washed three times with 200 μΙ DPBS/well. Splenocytes are added to the plates in triplicate at 2.5, 4.1 , or 5.1 *10⁵ cells/well and stimulated with 2 μg/ml of stimulatory peptides (specific to the antigen), concanavalin A (5 μg/ml; source), or culture medium alone. The plates are incubated for 18 h at 37°C and then rinsed three times with 200 μΙ/well of DPBS/0.05 % Tween 20 and three times with 200 μ l/well of DPBS. For detection, 50 μΙ/well of 2 μg/ml anti-mouse IFNy-biotinylated monoclonal antibody (BD Pharmingen) are added for 2 h at room temperature. Plates are washed and 100 μΙ/well of streptavidin-alkaline phosphatase (Roche) diluted 1 :2000 in Dulbecco's PBS for 90 min at room temperature. After washing the plates, spots (IFNy - secreting cells) are visualized by adding 60 μΙ/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). Example 5. Mycobacterium tuberculosis Antigens

[240] An effective subunit vaccine should comprise multiple epitopes to ensure a broad coverage of a genetically heterogeneous population infected with M. tuberculosis. It is important to have multi epitope vaccine not only to cover the genetic restriction imposed by MHC molecules but also to deal with complexity of the host immune response against tuberculosis. Recently, it has been demonstrated that vaccination with a fusion protein consisting of Ag85B and ESAT6 (Hybridl ) induced a strong immune response, which is highly protective against TB in the mouse, guinea pig, and non human primate models. This fusion Ag is also efficient if delivered in a viral vector or as a DNA vaccine. Significantly, Hybridl was more protective in both mouse and guinea pig animal models than either of the single proteins. The ability to establish a lifelong, persistent infection is a characteristic feature of M. tuberculosis. As the bacillus adapts to environments in the host, a substantial part of the bacterial population is believed to transform from metabolically active to a state of non-replicating persistence with low metabolic activity and essentially altered gene expression profile. Consequently, in latent TB, it is likely that some of the bacteria exist in a different state compared with active disease. Thus inhibiting reactivation may necessitate targeting several different bacterial antigens expressed at various metabolic states. To boost long-term efficacy, our approach is to develop a multistage therapeutic vaccine that combines classical preventive vaccine target antigens and key latency-associated antigens highly expressed as the bacteria adapt to survive in the host. The vaccines currently in clinical trials may improve or boost BCG efficacy in the short term, but they have not been designed to contain latent TB and prevent reactivation of disease. If THV03 in human clinical trials also promotes a response that controls the late stages of M. tuberculosis infection and contains latent TB (i.e., prevents reactivation), this vaccine could have a huge impact on TB transmission and the global TB epidemic.

[241] To eradicate persisters, therapeutic vaccines should express latency- associated antigens found in dormant M. tuberculosis. The THV03 vectors have been designed so that they encompass fragments of latency antigens. THV03 could be used to increase the immune response during the continuation phase of TB therapy, in which the remaining bacteria are poorly sensitive to anti mycobacterial agents, and potentiate chemotherapy. By reducing bacterial load by chemotherapy, the cytokine storm which causes the Th2-related exacerbated immune response can be prevented; this is essential for therapeutic vaccination. Since therapeutic vaccine in a synergy with chemotherapy has the potential to eliminate persisters, it is anticipated that shorter drug regimens resulting in enhanced treatment success. For the first time, MDR TB patients will be immunized with latent antigen which is part of THV03 therapeutic vaccine.

[242] Vectors have been generated encoding the following M. tuberculosis antigens:

• Ag85A: The antigen 85 protein is responsible for the high affinity of mycobacteria for fibronectin.

• ESAT6: Host cell surface binding. Secreted via the ESX-1 / type VII secretion system

• Mpt64: Cellular response to starvation. Showed protection in combination with other M. tuberculosis antigens in a DNA vaccine cocktail (Delogu et al., Infect Immun, 70 (2002), pp. 292-302) or in a multigene construct, as in the plasmid expressing the fusion protein (Sali et al., Microbes Infect, 10 (2008), pp. 605-612).

• EspC: Virulent factor. Suppress IL-12. Secreted via the ESX-1 / type VII secretion system. Suppress IL-12. Secreted via the ESX-1 / type VII secretion system

• RVBD(RV0140): Involved in reactivation of the latent TB. After stimulation of whole blood from TB patients, the antigen RVBD (Rv0140) induced the cytokines IL-2,

IL-6, and IL-17(Kassa et al., Clin Vaccine Immunol. 2012 Dec; 19(12):1907-15). RVBD (Rv0140) induced significantly higher T-cell responses from PBMCs of latently M tuberculosis- nfected (LTBI) donors compared to TB patients.

• Rv1813c: A member of the dormancy regulon. Induced in response to reduced hypoxia, low levels of NO and CO. Two-component regulatory signal systems MprAB and DosRS-DosT , have been associated with aspects of M tuberculosis persistence in vitro and in vivo.

• HRP1 : A member of the dormancy regulon. Induced in response to reduced oxygen tension (hypoxia), low levels of nitric oxide (NO) and carbon monoxide (CO). HRP1 consistently induced very strong T- and B-cell responses in mice (Roupie et al., Infect Immun. 2007 Feb;75(2):941 -9). • HspX: Acts as a chaperone and Induced in stationary phase. Induced by anoxia. Has a proposed role in maintenance of long-term viability during latent, asymptomatic infections, and a proposed role in replication during initial infection.

• Mtb39A: Induction by symbiont of host immune response, modification by symbiont of host protein by phosphorylation, modulation by symbiont of host transcription, negative regulation by symbiont of host inflammatory response.

• Rv2627c: A member of the dormancy regulon. Induced in response to reduced oxygen tension (hypoxia), low levels of nitric oxide (NO) and carbon monoxide (CO)

• Rv2628: A member of the dormancy regulon. Induced in response to reduced oxygen tension (hypoxia), low levels of nitric oxide (NO) and carbon monoxide (CO)

• PfkB: A member of the dormancy regulon. Belongs to the carbohydrate kinase PfkB family

• Mtb32A: Hydrolase, Protease 1 and serine-type endopeptidase activity. Predicted to be essential for in vivo survival and pathogenicity (Ribeiro-Guimaraes et al., Microb Pathog (2007) 43(5-6): 173-8).

• EsxV: Cell wall protein

• EsxW: Extracellular protein

• HSP65: Involved in adhesion to host, growth, protein refolding, response to heat, response to hypoxia. HSP65 regulates host cellular response by activating p38mapk and ERK1/2 signaling pathways and that these pathways cooperate to regulate pro-inflammatory cytokine production by human monocytes (Lewthwaite et al., Int Immunopharmacol. 2007 Feb;7(2):230-40).

• Rv2660: Immunogenic protein. Up-regulated at high temperatures, and highly up-regulated after 24h and 96h (very highly) of starvation.

· Archease: Chaperone or modulator of proteins involved in DNA or RNA. processing. A member of the dormancy regulon. Induced in response to reduced oxygen tension (hypoxia), low levels of nitric oxide (NO) and carbon monoxide (CO)

• PG42: Epitope expressed during infection of the host. It has been demonstrated that rBCG::Ag85BESAT6- Rv2608(PPE42) could elicit the stronger T cell response than the classical BCG in the mice model and could also induce significantly stronger B cell response. PPE42 was shown to induce partial protection against M. tuberculosis in mice.

• EsxJ: Epitope ESAT-6-like protein located in Cell wall, Extracellular region and Plasma membrane

· v1088: PE family protein

• USP(Rv2028c): Belongs to Universal stress protein A family. A member of the dormancy regulon. Induced in response to reduced oxygen tension (hypoxia), low levels of nitric oxi Rv2028c is identified by proteomics and is functionally involved in virulence, detoxification, adaptation. Rv2028c is dormancy regulon(DosR)-controlled protein and its expression is also up regulated in macrophages. Based on these facts it has been hypothesize that they may be playing a role in persistence and/or intracellular survival.de (NO) and carbon monoxide (CO). Cellular response to nitrosative stress.

• PPE42: Expressed during infection of the host

• PE35: Controls the expression of ESAT-6 and EsxB

[243] The vectors can be assessed for the induced immune responses in naive mice and in mice infected via aerosol with a low dose of M tuberculosis.

Example 6. Lentivectors

[244] THV03 treatment consists of therapeutic vaccination by 2 lentiviral vectors, encoding identical antigens that will elicit a cellular mediated immunity in M tuberculosis infected patients.

[245] The THV03 treatment comprises the two live recombinant lentiviral vectored vaccines THV03-1 , and THV03-2. The transgene encoded by these vectors is derived from the M tuberculosis. Two dose levels will be tested during a Phase l/ll trial.

[246] THV03-1 and THV03-2, are non-pathogenic and non-replicative vectors derived from the NL4-3 strain of HIV-1 . They are produced by tri-transfection with a plasmid encoding a provirus (encompassing the M tuberculosis antigens), a plasmid carrying structural and enzymatic proteins, and a third plasmid encoding the VSV.G protein.

[247] THV03-1 and THV03-2 code for an identical antigen selected based on their immunogenic potential from different metabolic stages of Mtb and their codons are optimized for expression in human cells. However, THV03-1 and THV03-2 are pseudotyped with different serotypes of the G protein of the vesicular stomatitis virus (VSV-G). This allows a broader tropism of the vectors, as they are hence able to transduce all type of cells. Experiments were performed to evaluate the absence of cross neutralization between the two VSV-G serotypes (as antibodies generated against one envelope after the prime injection might neutralize a vector pseudotyped by a different serotype of envelope). This enables iterative injections of THV03-1 then THV03-2 without a reduction in therapeutic immunogenicity.

[248] The production processes of the lentiviral vectors is as follows: Cells are transfected with plasmids, viral particles are harvested and clarified by filtration, and viral particles are purified and sterilized by filtration.

Example 7. Vaccine design evaluation

[249] The biological effect of the THV03 vectors will be evaluated during immunomonitoring studies. Studies will assess duration and breadth of the cellular immune response induced by vaccination with THV03-1 and THV03-2 alone or following their successive injection (to mimic the clinical trial design).

[250] Biodistribution will be performed to assess timepoints of integration and clearance of the integrated vectors once injected in rats (single injection, several doses). Quantification of the integrated DNA vector sequences will be performed by highly sensitive qPCR.

[251 ] Pharmacokinetic studies will evaluate the kinetic of the immune response, following single injection of the THV03 vectors or successive injections.

[252] Toxicity studies, designed to mimic the clinical trial successive injection of the two vectors, will be performed under GLP environment, on rats, and using the maximal achievable dose (maximal injectable volume in animal model).

[253] A multi-center, randomized, double-blind, placebo-controlled Phase l/l I dose-escalation study will be performed to evaluate the safety, tolerability, and efficacy of the multistage vaccine (THV03) given in patients with newly diagnosed or previously treated Sputum Smear-positive Pulmonary Multi-Drug Resistant Tuberculosis (MDR- TB). The Phase l/l I trial will be a randomized, placebo-controlled, 2-parallel groups trial to compare the safety, tolerability and immunogenicity of the THV03 vaccination versus placebo. Eligible patients must be i. Positive for Acid-Fast Bacilli (AFB) on direct smear examination of expectorated sputum specimen; ii. Patients with newly diagnosed for MDR-TB based on a Drug Sensitivity Tests (DST) showing resistance to both rifampicin and isoniazid; iii. Patients with pre-extensively Drug Resistant (pre-XDR-TB) defined as TB due to infection with an MDR strain of M. tuberculosis that is resistant either to at least one of the injectable second-line drugs (amikacin, kanamycin, or capreomycin) or to any fluoroquinolone, but not both. Example 8. Preclinical evaluation

[254] BALB/c mice will be infected through an intratracheal administration with 10⁵ colony forming units (CFU) of M. tuberculosis H37Rv. Treatment will be initiated four weeks post-infection. Groups of animals are described below.

• untreated mice

· THV03 (2 intramuscular injection of 4-week intervals)

• BCG (single intradermic injection of about 10⁵ live bacteria)

• Drug (treatment with a standard diet supplemented with isoniazid and pyrazinamide for 24 weeks)

• Drug +THV03

· Drug +BCG

[255] Mice will be killed at intervals (0, 1 , 3, and 6 months after the beginning of treatment) and bacteria in lungs will be counted as CFU.

[256] Evaluation of cytokine production (IFN-γ, IL-2 and IL-4) in vitro and IFN- γ ELISPOT assay will be carried out to study the immune potency of THV03 compare to BCG.

[257] Toxicity studies will be performed using the maximal injectable volume in rat, 200μΙ in the left plus 200 μ I 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 (400μΙ) 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 1 /5 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

[258] 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 each pseudo type, respectively THV03-1 and THV03-2injection. Animals injected intravenously will be sacrificed only at D15 and no ELISPOT will be performed.

[259] 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, 6h 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.

[260] Studies have been designed to assess the biodistribution, shedding and persistence of the integrated vectors sequences of the injected lentiviral vectors.

[261 ] 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.

[262] 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.

[263] The biodistribution studies described above will enable to gain data on the biodistribution of integrated vector.

[264] The biological response induced by the THV03 lentiviral vectors will be measured by quantifying the generated T-cell mediated immune response by ELISPOT IFN-g 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.

[265] The humoral response against THV03 vectors 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.

[266] The cellular immune response will be evaluated by performing ELISPOT that will measure the number of specific effector T-lymphocytes via IFN-g secretion. Lentiviral vectors will be injected into animals, then their splenocytes isolated and the immune response assessed against a panel of TB peptides representatives of the epitopes. These studies will enable evaluation of the efficient dose.

[267] Finally, characterisation of the immune response will be performed by quantifying the CD4+ T cell and the CD8+ T cell response.

[268] 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 THV03 vector particles, less DCs will be transduced by these particles, which might lead to the induction of immune responses of lower magnitude.

[269] 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: [270] 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.

[271] 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.

[272] Local tolerance will be assessed during the acute toxicity study by macroscopic and post-mortem microscopic observation of the injection site.

[273] As the THV03 vectors are integrative, activation of proto-oncogene or disruption of tumour suppressor gene leading to oncogenesis cannot be totally ruled out. However, lentiviral site selection for integration is not random: contrarily to gammaretroviruses, lentiviruses do not integrate preferentially in 5' flanking regions or CpG islands found near transcription start sites but rather into active transcription units. Moreover, no lentivirus induced tumour has been reported in seropositive patients and they do not induce tumorigenesis in cancer prone mice model. Finally, data collected on 65 patients who were enrolled in clinical trial using lentiviral vectors seem to confirm the safety of this approach (McGarrity, 2013).

[274] The expected effect of the THV03 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 THV03 vectors from the host. This mechanism adds to the safety of use of the THV02 vectors as they will be cleared from the host.

[275] Therefore, no expect adverse events linked to insertional mutagenesis as those that have been observed in past gene therapy clinical trials using viral vectors is expected. To gain however information on the preferential integration sites of the THV03 lentiviral vectors, LAM-PC (linear amplification mediated-PCR) experiments will be performed.

[276] Biodistribution studies conducted on the THV01 vectors demonstrated the absence of integrated vector sequence in gonads. This will be assessed again for the THV03 vectors during the GLP biodistribution studies. If absence of localisation in the gonads of the THV03 vectors is confirmed, no specific germline transmission study will be performed.

[277] In addition, as the vectors will be injected intramuscularly and not systemically, the risk of germline transmission is reduced.

[278] The THV03 lentiviral vectors are non-replicative. To assess whether replication competent vector has been generated during the manufacturing process, a specific test will be implemented during the manufacturing process and will be part of the release tests.

[279] A phase l/l I study will be carried out in drug-resistant patients, MDR- and XDR-TB.

[280] 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.

[281 ] Two different doses will be tested sequentially. The dose escalation will be based on the occurrence of Dose Limiting Toxicities experienced by the patients. The NOAEL or MAD (whichever is the lowest) will be used as the lowest dose tested in patients. This position is supported by the on-going clinical study (THV01 -1 1 -01 ) that demonstrates an excellent safety profile although some patients have been treated with a vaccine dose superior to the maximum dose injected in animal model. Indeed, in these GLP toxicity studies, the MAD was 1 .06 10e+8 for THV01 -1 and 1 .88 10e+8 for THV02-2 whereas patients enrolled in Cohort 3 of the THV01 -1 1 -01 trial received the vaccines at 5.0 10e+8. The harrowing nature of drug-resistant TB is a strong argument supporting the use of the NOAEL as the first dose tested in humans in the planned Phase l/l I trial. [282] Based on this information, studies were conducted to evaluate the protective efficacy of THV03 vaccine in combination with isoniazid in C57BI/6 mice compared to isoniazid alone.

[283] Groups of C57/BI6 mice were infected with Mycobacterium tuberculosis H37Rv via the low dose aerosol route and treated with either 8 weeks of isoniazid (Group 1 ) or with isoniazid for 8 weeks and immunized with THV03 at 1 and 9 weeks after starting chemotherapy with isoniazid (Group 2).

[284] Treatment with isoniazid is initiated 4 weeks after the infection.

[285] Figures 19 and 20 present the measured number of CD4+ cells producing IFN-γ and TNF-a in the inferior caval-lobe.

[286] Intracellular cytokine staining for Tuberculosis antigen 85B (Figure 19) and ESAT6 (Figure 20) specific T-cell responses were carried out at 7 weeks after 1 ^st immunization with THV03 or 8 weeks after isoniazid treatment or 12 weeks after infection. Cells were stimulated with respective TB antigen 85B (Figure 19) or ESAT6 (Figure 20) and stained with fluorochrome-conjugated antibodies against CD4, IFN-γ and TNF-a.

[287] These results clearly illustrate that post-infection immunization with THV03 vaccine administered with isoniazid (Group 2) induced a significantly higher number of CD4+ T cells specific to 85B or to ESAT6, as measured by IFN-γ and TNF-a producing T cells, compared to animals treated with isoniazid alone (Group 1 ).

[288] The THV03 vaccine administered adjunctively with a Tuberculosis drug is successful at stimulating a significantly more robust, high-quality (polyfunctionnal) CD4+ T cell response in the lung where the pathogen Mycobacterium tuberculosis resides.

Claims

CLAIMS We claim:

1. A lentiviral vector encoding an Ag85A, ESAT6, Mpt64, EspC, RVBD, Rv1813c, HRP1 , HspX, Mtb39A, Rv2627c, Rv2628, PfkB, Mtb32A, EsxV, EsxW,

HSP65, Rv2660, Archease, PE42, EsxJ, Rv1088, USP, PPE42, or PE35 antigen.

2. The vector of claim 1 , wherein the vector encodes Ag85A, ESAT6, Mpt64, RVBD, HRP1 , RV2028C, HspX, and Mtb32A antigens.

3. The vector of claim 1 , wherein the vector encodes SEQ ID NO:54.

4. The vector of claim 1 , wherein the vector encodes RVDB and/or Rv1813c

(MT1861 ) antigens.

5. The vector of any of claims 1 -4, wherein the vector comprises a β2- microglobulin promoter.

6. The vector of any of claims 1 -5, wherein the vector is a DNA.

7. A lentiviral vector particle encoding an Ag85A, ESAT6, Mpt64, EspC,

RVBD, Rv1813c, HRP1 , HspX, Mtb39A, Rv2627c, Rv2628, PfkB, Mtb32A, EsxV, EsxW, HSP65, Rv2660, Archease, PE42, EsxJ, Rv1088, USP, PPE42, or PE35 antigen.

8. The lentiviral particle of claim 7, wherein the lentiviral vector particle encodes Ag85A, ESAT6, Mpt64, RVBD, HRP1 , RV2028c, HspX, and Mtb32A antigens.

9. The lentiviral particle of claim 7, wherein the lentiviral vector particle encodes SEQ ID NO:54.

10. The lentiviral particle of claim 7, wherein the lentiviral vector particle encodes RVDB and/or Rv1813c (MT1861 ) antigens.

1 1 . The lentiviral vector particle of any of claims 7-10, wherein the lentiviral vector particle comprises a functional lentiviral integrase protein.

12. The lentiviral vector particle of any of claims 7-1 1 , wherein the lentiviral vector particle comprises a vesicular stomatitis virus glycoprotein.

13. The lentiviral vector particle of any of any of claims 7-12, wherein the lentiviral vector particle comprises HIV-1 subtype D Gag and Pol proteins.

14. An isolated cell comprising the vector or lentiviral vector particle of any of claims 1 -13.

15. Use of the vector of any of claims 1 -6 or the lentiviral vector particle of any of claims 7-13 for inducing an immune response in a human.

16. A method for inducing an immune response in a human comprising intramuscularly administering the lentiviral vector particle of any of claims 7-13 to a human.