LENTIVIRAL VECTORS TARGETING ANTIGENS TO MHC-II PATHWAY AND INDUCING PROTECTIVE CD8+ AND CD4+ T-CELL IMMUNITY IN A HOST

Abstract

A recombinant lentiviral vector genome comprising a polynucleotide encoding a fusion polypeptide, wherein said fusion polypeptide comprises, arranged from N-terminal to C-terminal ends: a first polypeptide comprising (i) an MHC-ll-associated light invariant chain (li), or (ii)) the transmembrane domain of the transferrin receptor (TfR) and at least one antigenic polypeptide of a pathogen. The invention also relates to a lentiviral vector and pharmaceutical compositions comprising it.

Claims

1. A recombinant lentiviral vector genome comprising a polynucleotide encoding a fusion polypeptide, wherein said fusion polypeptide comprises, arranged from N-terminal to C-terminal ends: a first polypeptide comprising (i) an MHC-II-associated light invariant chain (li), preferably of SEQ ID No. 11, or (ii) the transmembrane domain of the transferrin receptor (TfR), preferably of SEQ ID No. 13, and at least one antigenic polypeptide of a pathogen.

2. The recombinant lentiviral vector genome according to claim 1, wherein said antigenic polypeptide is a mono-antigenic polypeptide comprising one antigen of a pathogen or immunogenic fragment thereof, or is a poly-antigenic polypeptide comprising at least two antigens of one or more pathogens or immunogenic fragments thereof.

3. The recombinant lentiviral vector genome according to claim 1, wherein the pathogen is a bacterial, parasite or viral pathogen, in particular a pathogen associated with an acute or chronic respiratory infectious disease in a mammal, more particularly is Mycobacterium tuberculosis, an influenza virus or a coronavirus such as SARS-CoV-2.

4. The recombinant lentiviral vector genome according to claim 3, wherein said antigenic polypeptide comprises one or more Mycobacterium tuberculosis (Mtb) antigens selected from EsxA, EspC, EsxH, PE19 or Ag85A, or an immunogenic fragment thereof, in particular one of the following Mtb antigenic combinations: (a) EsxH; (b) EsxH and EsxA; (c) EsxH, EsxA and PE19; (d) EsxH, EsxA, EspC and PE19; (e) EsxH, EsxA, EspC, PE19 and Ag85A; or immunogenic fragments thereof.

5. The recombinant lentiviral vector genome according to claim 1, wherein said genome is obtained from a pFLAP vector plasmid, in particular the vector plasmid of nucleotide sequence SEQ ID No. 20, wherein the polynucleotide encoding the fusion polypeptide has been cloned under control of a promoter functional in mammalian cells, in particular the CMV promoter, the human beta-2 microglobulin promoter, the SP1-human beta-2 microglobulin promoter of SEQ ID No. 21 or the composite BCUAG promoter of SEQ ID No. 22 and wherein the vector optionally comprises post-transcriptional regulatory element of the woodchuck hepatitis virus (WPRE), in particular a mutant WPRE as set forth in SEQ ID No. 23.

6. A DNA plasmid comprising the recombinant lentiviral vector genome according to claim 1, in particular wherein said genome is inserted within a pFLAP vector plasmid, preferably the vector plasmid of nucleotide sequence SEQ ID No. 20, wherein the fusion polypeptide encoded by the polynucleotide comprised within the recombinant lentiviral vector genome is inserted between restriction sites BamHI and Xhol in replacement of the GFP sequence.

7. A recombinant lentiviral vector particle which comprises the recombinant lentiviral vector genome according to claim 1.

8. The recombinant lentiviral vector particle according to claim 7, which is a recombinant integration-deficient lentiviral vector particle, in particular wherein the recombinant integration-deficient lentiviral vector particle is a HIV-1 based vector particle and is integrase deficient as a result of a mutation of the integrase gene encoded in the genome of the lentivirus in such a way that the integrase is not expressed or not functionally expressed, in particular the mutation in the integrase gene leads to the expression of an integrase substituted on its amino acid residue 64, in particular the substitution is D64V in the catalytic domain of the HIV-1 integrase encoded by Pol.

9. The recombinant lentiviral vector particle according to claim 7, wherein said recombinant lentiviral vector particle is a recombinant replication-incompetent pseudotyped lentiviral vector particle, in particular a replication-incompetent pseudotyped HIV-1 lentiviral vector particle, in particular wherein the lentiviral vector particle is pseudotyped with the glycoprotein G from a Vesicular Stomatitis Virus (V-SVG) of Indiana or of New-Jersey serotype.

10. A host cell, preferably a mammalian host cell, transfected with a DNA plasmid according to claim 6, in particular wherein said host cell is a HEK-293T cell line or a K562 cell line.

11. A pharmaceutical composition, in particular a vaccine composition, suitable for administration to a mammalian host, comprising a recombinant lentiviral vector particle of claim 7 together with one or more pharmaceutically acceptable excipient(s) suitable for administration to a host in need thereof, in particular a human host.

12. The pharmaceutical composition of claim 11, further comprising an adjuvant, in particular a pro-Th1 and/or pro-Th17 adjuvant such as polyinosinic-polycytidylic acid (polyI:C) or a derivative thereof, or a cyclic dinucleotide adjuvant, in particular cyclic Guanine-Adenine dinucleotide (cGAMP).

13. The pharmaceutical composition of claim 11, for use in the elicitation of a protective, preferentially prophylactic, immune response by the elicitation of antibodies directed against the antigenic polypeptide or immunogenic fragments thereof in a host in need thereof, in particular a human host.

14. The pharmaceutical composition of claim 13, wherein the immune response involves the induction of MHC-I restricted presentation and MHC-II restricted presentation of the antigenic polypeptide or immunogenic fragments thereof, by an antigen-presenting cell, in particular a dendritic cell, and the induction of a CD4- and CD8-mediated cellular immune response.

15. The pharmaceutical composition of claim 11, for use in preventing and/or treating an infection by a pathogen in a mammalian host in need thereof, in particular a human host in particular an infection by a pathogen associated with an acute or chronic respiratory infectious disease in a mammal.

16. A method for the preparation of recombinant lentiviral vector particles suitable for the preparation of a pharmaceutical composition, in particular a vaccine composition, comprising the following steps: a) transfecting the recombinant lentiviral transfer vector carrying the lentiviral vector genome comprising a polynucleotide encoding a fusion polypeptide, according to claim 1 in a host cell, for example a HEK-293T cell line or a K562 cell line; b) co-transfecting the cell of step a) with: (i) a plasmid vector encoding envelope proteins and with a plasmid vector encoding the lentiviral GAG and POL or mutated POL protein as packaging construct; and (ii) a plasmid encoding VSV-G Indiana or New Jersey envelope, c) culturing the host cell under conditions suitable for the production of recombinant lentiviral vector particles expressing the fusion polypeptide; d) recovering the recombinant lentiviral particles expressing the fusion polypeptide.

Description

LEGENDS OF THE FIGURES

[0232] FIG. 1. Intra-phagocyte quantitation of Ag85A/B and EsxA secretion by Beijing or non-Beijing Mtb clinical isolates. (A-B) Bone-marrow-derived DC (H-2.sup.b) were infected with various CFU/ml of each Mtb strain from a set of non-Beijing or Beijing clinical isolates, numbered as indicated in Table S1. After overnight incubation, MHC-II-restricted T-cell hybridomas specific to Ag85A/B (DE10) (A) or EsxA (NB11) () were added and the concentration of IL-2 produced by T-cell hybridomas, proportional to the amounts of Mtb antigens secreted inside the DC phagosomes, was determined by ELISA, after 24h incubation. (C) Intra-phagocyte amounts of Ag85A/B or EsxA secretion, as determined in DC infected with 410.sup.3 CFU/ml.

[0233] FIG. 2. Tailoring LV to direct antigens towards MHC-II processing pathway. (A) Failure of CD4.sup.+ T-cell induction by conventional LV. Cytometric analysis of splenocytes from C57BL/6 mice immunized with a conventional LV encoding for the fusion of EsxA-Ag85A-EspC-EsxH-PE19 Mtb immunogens. Shown are CD4.sup.+ and CD8.sup.+ T-splenocyte IFN- responses subsequent to in vitro stimulation with EspC: 45-54 peptide, which contains both MHC-I and -II-restricted epitopes in H-2.sup.b, or a negative control peptide. () Scheme of full length EsxH protein, with sequences potentially facilitating its routing through the MHC-II pathway added at its N- or C-ter extremity. (C) Presentation of MHC-I- or -II-restricted EsxH epitopes by DC (H-2.sup.d), transduced with 110.sup.6 TU/ml of LV encoding EsxH alone, li-EsxH, TfR-EsxH or SP-EsxH-MITD, and co-cultured at day 3 post-transduction with T-cell hybridomas specific to EsxH: 20-28 and restricted by K.sup.d (YB8) (top) or specific to EsxH: 74-88 and restricted by I-Ad (1H2) (bottom). Results are MeanSD of concentrations of IL-2, produced by T-cell hybridomas after overnight co-culture.

[0234] FIG. 3. Induction of systemic or mucosal CD4.sup.+ and CD8.sup.+ T-cell responses by the optimized LV. BALB/c (H-2.sup.d) mice (n=3/group) were immunized s.c. with 510.sup.7 TU of LV::li-EsxH alone (1) or adjuvanted with polyI:C (2) or cGAMP (3). At 11 dpi, EsxH-specific Th1 cytokine responses of splenocytes were analyzed by ICS in individual mice. (A) Gating strategy carried out on cytokine producing CD4.sup.+ or CD8.sup.+ T cells. (-C) Recapitulative frequencies of each (multi) functional population within the CD4.sup.+ () or CD8.sup.+ (C) T subset. (D-E) BALB/c mice (n=3/group) were immunized i.n. with 510.sup.7 TU of LV::li-EsxH alone or adjuvanted with polyI:C or cGAMP. At 13 dpi, EsxH-specific lung CD4.sup.+ or CD8.sup.+ T-cell responses were analyzed by co-culture of lymphocytes enriched from the lungs with homologous DC loaded with EsxH: 74-88 (MHC-II) (D) or with EsxH: 20-28 (MHC-I) (E). IL-2, IL-17A or IFN- contents in the co-culture supernatants were quantitated by ELISA.

[0235] FIG. 4. Characterization of mucosal CD4.sup.+ or CD8.sup.+ T-cell responses induced by the optimized LV. BALB/c (H-2.sup.d) mice (n=3/group) were immunized i.n. with 510.sup.7 TU of LV::li-EsxH alone or adjuvanted with polyI:C or cGAMP. At 13 dpi, lung CD4.sup.+ (A) or CD8.sup.+ (E) T cells were discriminated for their location inside the interstitium (CD45i.v) or in the vasculature (CD45.sub.i.v.sup.+) by an i.v. injection of PE-anti-CD45 mAb, 3 min before sacrifice. (, F) Profile of CD27 versus CD62L expression or (C, D, G, H) cytokine production, as detected by ICS in lung CD4.sup.+ or CD8.sup.+ T cells of the lung interstitium or vasculature. Results, representative of 2-3 independent experiments, were from lungs pooled per experimental groups to reach cell numbers high enough for accurate cytometric analyses.

[0236] FIG. 5. Characterization of mucosal innate immunity induced by LV i.n. administration. (A) Cytometric gating strategy used on total lung cells to analyze various mucosal innate immune cell populations. Shown are cells from PBS-injected negative controls. () Percentages of each innate immune subset versus total lung CD45.sup.+ cells in C57BL/6 mice injected i.n. with PBS, LV alone, or cGAMP-adjuvanted LV, as determined at 2 dpi. Results are from 3 individual mice/group. ns=not significant, as determined by One-tailed Mann Whitney test.

[0237] FIG. 6. Potential of the poly-antigenic LV::li-HAEP at inducing CD4.sup.+ and CD8.sup.+ T cells. (A) Presentation of MHC-I- or -II-restricted epitopes by H-2.sup.d or H-2.sup.b DC transduced with LV::li-HAEP or LV::TB as a negative control and co-cultured at day 3 post-transduction with T-cell hybridomas specific to EsxH: 20-28, restricted by K.sup.d (YB8), EsxH: 74-88, restricted by I-Ad (1G1), EsxA: 1-20 (NB11) or to PE19: 1-18 (IF6), restricted by I-Ab. (-D) C57BL/6 (H-2.sup.b) mice were immunized s.c. with 510.sup.8 TU of LV::li-HAEP or injected with PBS. At 11 dpi, the antigen-specific cytokine responses of splenocytes, determined as SFU (Spot Forming Unit) by ELISPOT () or ICS (C-D) in individual mice. (C-D) Shown are recapitulative frequencies of each (multi) functional population within the CD4.sup.+ (C) or CD8.sup.+ (D) T subsets, after removing the background signal observed with an irrelevant negative control peptide for each mouse.

[0238] FIG. 7. Features of mucosal T-cell responses induced by LV::li-HAEP. C57BL/6 (H-2.sup.b) mice were immunized i.n. with 510.sup.8 TU of LV::li-HAEP, adjuvanted with cGAMP (n=7) or instilled with PBS (n=3). At 13 dpi, following an i.v. injection of PE-anti-CD45 mAb, 3 min before sacrifice, CD4.sup.+ (A) or CD8.sup.+ () lung T-cell responses were analyzed by ICS after co-culture with homologous DC loaded with EsxA: 1-20 (MHC-II), PE19: 1-18 (MHC-II), EspC: 40-54 (MHC-I, and -II), EsxH: 20-28 (MHC-I) or an irrelevant negative control peptide. Shown are recapitulative absolute numbers of each (multi) functional population within the CD4.sup.+ (A) or CD8.sup.+ () T subsets located inside the interstitium (CD45i.v.sup.) or in the vasculature (CD45i.v.sup.+). (C, D) Phenotyping of interstitial (CD45.sub.i.v.sup.) CD4.sup.+ (C) or CD8.sup.+ (D). Results were generated with cells pooled from the lungs per group to reach enough number for cytometric analyses.

[0239] FIG. 8. Protective potential of an optimized poly-antigenic LV as a booster in TB vaccination. (A) MHC-II-restricted presentation of Ag85A, in parallel to EsxA, as detected on DC (H-2.sup.b) transduced with LV::li-HAEPA or LV::TB as a negative control and co-cultured at day 3 post-transduction with Ag85A- or EsxA-specific T-cell hybridomas harboring the gene encoding ZsGreen reporter under the control of IL-2 promoter. () Timeline of prime with BCG::ESX-1.sup.Mmar, boost with cGAMP-adjuvanted LV::li-HAEPA and challenge with Mtb H37Rv strain, performed in C57BL/6 mice (n=5-9/group). (C) Mycobacterial loads determined by CFU counting in the lungs and spleen of individual mice at week 5 post challenge. ns=not significant, * (p=0.0415), ** (p=0.0040) *** (p=0.00105), statistically significant, as determined by One-tailed Mann Whitney test. (D) Representative lung hematoxylin and eosin histopathological results. Analysis was performed on the left lung lobes of unvaccinated (left), BCG: ESX-1.sup.Mmar-vaccinated (middle) or BCG::ESX-1.sup.Mmar-primed and cGAMP-adjuvanted LV::li-HAEPA-boosted (right) C57BL/6 mice, at week 5 post Mtb challenge.

[0240] FIG. 9. Weak capacity of LV at inducing DC maturation. Bone-marrow-derived DC from C57BL/6 mice were left untreated (negative control), treated with Mtb at MOI=3 (positive control), or LV at the high MOI of 50. (A-C) Maturation of CD11c.sup.+CD11b.sup.+ cells (A), as monitored by flow cytometry after overnight incubation for the expression of CD40, CD80, CD86, MHC-I and MHC-II surface molecules (). (C) MFI or percentage of bright (hi) cells. Results are representative of two independent experiments.

[0241] FIG. 10. Non-dependence of LV-mediated CD8.sup.+ T-cell induction on IFNAR signaling in DC. (A) Verification of the IFNAR1 deficiency in DC of the KO mice by assessing the IFNAR1 surface expression by bone-marrow DC derived from hematopoietic stem cells of ifnar.sup.flox/flox pCD11c-Cre.sup. (WT) or ifnar.sup.flox/flox pCD11c-Cre.sup.+ (KO) mice. (-G) Mice, either ifnar.sup.flox/flox pCD11c-Cre.sup. (WT) or ifnar.sup.flox/flox pCD11c-Cre.sup.+ (KO), were immunized i.m. with 510.sup.7 TU of LV::OVA (-C) or LV::li-EsxH (D-G). At 11 dpi, antigen-specific CD8.sup.+ T splenocytes were assessed through tetramer staining, ELISPOT or ICS analysis. () Percentages of cells stained positively with OVA tetramer, as compared to total CD3+CD8.sup.+ T splenocytes. (C) Numbers of splenocytes secreting IFN- after ex vivo stimulation with OVA: 257-264, as detected by ELISPOT. (D) Numbers of splenocytes secreting IFN- or TNF-, after ex vivo stimulation with EsxH: 3-11 or a negative ctrl peptide or, as detected by ELISPOT (SFU=Spot Forming Unit). (E) Degranulation activity of the IFN--producing CD8.sup.+ T cells, as evaluated by the surface CD107a staining. (F) Gating strategy used in ICS analysis performed on CD8.sup.+ T splenocytes. (G) Recapitulative frequencies of various (poly) functional EsxH:3-11-specific CD8.sup.+ T-cell effectors.

[0242] FIG. 11. Non-dependence of LV-mediated CD4.sup.+ T-cell induction on IFNAR Signaling in DC. Ifnar.sup.flox/flox pCD11c-Cre.sup. (WT) and ifnar.sup.flox/flox pCD11c-Cre.sup.+ (KO) mice were immunized s.c. with 510.sup.8 TU of LV::li-HAEP. At 11 dpi, antigen-specific CD4.sup.+ T-cell responses were assessed through ICS. Shown are recapitulative frequencies of (poly) functional CD4.sup.+ T splenocytes, specific to EsxA or PE19, as detected after stimulation with EsxA: 1-20 or PE19: 1-18 peptides.

[0243] FIG. 12. Comparison of the immunogenicity of LV::li-HAEP injected via i.m. or s.c. systemic routes. C57BL/6 mice were immunized i.m. or s.c. with 510.sup.8 TU of LV::li-HAEP. At 14 dpi, antigen-specific, IFN- or TNF- T cell responses were assessed by ELISPOT.

[0244] FIG. 13. Maps of plasmids encoding EsxH variants or poly-antigenic fusion proteins. The codon-optimized cDNA sequences, encoding EsxH variants or poly-antigenic fusion proteins of vaccine interest (Table S3), were inserted under the SP1-2m promoter in a pFLAP backbone plasmid.

[0245] FIG. 14. Map of the pFLAP backbone plasmid containing GFP. The sequence of a GFP transgene was inserted under the SP1-2m promoter, with a WPREm sequence

[0246] FIG. 15. Maps of plasmids for human immunization. The codon-optimized cDNA sequences, encoding EsxH variants, were inserted under the SP1-B2m promoter in a pFLAP backbone plasmid. Panel A: the EsxH antigen is fused with human li. Panel B: the EsxH antigen is fused with hTfR.

EXAMPLES

Introduction

[0247] According to the World Health Organization, Mycobacterium tuberculosis (Mtb) causes more than 8 million new cases of pulmonary tuberculosis (TB) each year, and remains one of the top ten causes of death and the first cause of mortality due to an infectious pathogen worldwide. Of the estimated 1.7 billion asymptomatic people latently infected with Mtb, 5 to 15% will evolve toward active TB. The risk of developing the disease is higher in individuals co-infected with HIV or affected by under-nutrition, diabetes, smoking or alcoholism (1). The only current TB vaccine is the widely administrated Mycobacterium bovis Bacillus Calmette-Gurin (BCG), given early after birth is particularly effective at inducing Th1-biased responses in infants. Although BCG is effective in protecting children against pulmonary and disseminated forms, it has a limited impact on adolescent and adult pulmonary TB and reactivation of latent TB and thus cannot prevent global bacillary spread (2). Therefore, there is an urgent need for new immunization strategies: (i) effective as pre-exposure vaccines, (ii) able to decrease the risk of primary Mtb infection, (iii) preventive against latent TB progression to active disease, or (iv) usable in TB immune-therapy.

[0248] Even though the immune correlates of TB protection are poorly understood, it is well established that protective immunity against the intracellular pathogen Mtb is mainly dependent on cell-mediated immunity. The contribution of appropriate innate and T cell-mediated immunity, notably IFN-/TNF--producing CD4.sup.+ Th1 cells, and at a lesser extent CD8.sup.+ T cells, is instrumental in anti-mycobacterial host defense, even not sufficient to reach full protection (3, 4). Since 1921, four billion people have been vaccinated by BCG and numerous improved live-attenuated vaccine candidates are in development (5). The immunity conferred by BCG is of variable duration, but admittedly limited to 10 years. Homologous boosting with live-attenuated vaccines and repeated administration of mycobacteria may cause adverse necrotic inflammation, namely the Koch phenomenon, characterized by strong expression of IL-6, IL-17, TNF- and CXCL2, and massive recruitment of neutrophils (6). In this context, heterogeneous prime-boost regimen, relying on priming with improved live-attenuated vaccines followed by boosting with subunit vaccines, is an attractive approach to synergistically enhance the Mtb-specific protective immunity (7). We have previously elaborated a promising live-attenuated TB vaccine candidate based on BCG Pasteur strain stably complemented with the esx-1 genomic region of a fish pathogen phylogenetically related to Mtb, i.e., Mycobacterium marinum (BCG::ESX-1.sup.Mmar) (8). Compared to BCG, this strain shows largely improved protective potential against TB in animal models, which is consistent with its known properties. In fact, this strain displays an enlarged antigenic repertoire, capacity to trigger the cGAS (cyclic GMP-AMP Synthase)/STING (STimulator of INterferon Genes)/IRF3 (Interferon Regulatory Factor 3)/IFN-I (type-I IFN) axis, and to reinforce the NLRP3 (NOD-Like Receptor family Protein 3) and the cytosolic DNA sensor, AIM-2 (Absent In Melanoma-2) inflammasome pathways, while displaying attenuated virulence (8, 9). In the murine TB model, BCG::ESX-1.sup.Mmar vaccination reduces mycobacterial loads better than the parental BCG, but does not yet lead to sterilizing immunity, leaving the possibility to evaluate the protective potential of booster vaccines.

[0249] The use of recombinant viral vaccine vectors, expressing potent Mtb antigens, may result in a synergistic enhancement of anti-mycobacterial immunity in individuals primed with live-attenuated vaccine candidates. This can be achieved by increasing the frequencies of antigen-specific T cells, improving T-cell avidity, as well as enhancing the CD8.sup.+ T-cell responses that are not efficaciously induced by mycobacteria in general. Replication-defective, Lentiviral Vectors (LV) are powerful delivery systems and attractive immunization tools, based on their: (i) low genotoxic potential, (ii) capacity to accept up to 8 kb inserts, (iii) strong ability to transduce in vivo both replicating and non-dividing cells, (iv) persistent antigen expression, and (v) advantage of not being the target of preexisting anti-vector immunity in the human populations (10-15). However, one limitation of LV is their inability to target antigens to the Major Histocompatibility Complex class II (MHC-II) pathway to trigger CD4.sup.+ T cells, which are considered so far as the best correlates of protection against TB and multiple other diseases.

[0250] Here, we describe a new generation of LV, with the capacity to route immunogens toward the MHC-II machinery, created by adding of the MHC-II light invariant chain (li) at the N-ter part of the antigen(s) encoded by the vector. This approach and immunization strategies via the systemic or mucosal route, allowed proper implementation of antigen trafficking to the MHC-II machinery, thereby inducing in addition to CD8.sup.+ T cells, appropriate CD4.sup.+ T cells, that we thoroughly characterized for their phenotype, functions and pulmonary localization. We further report the significant protective potential of a selected optimized LV, encoding five potent Mtb antigens, used as a booster, administered via systemic and intranasal (i.n.) routes into BCG::ESX-1.sup.Mmar-primed mice.

Results

Rational Selection of Mtb Immunogens

[0251] Since the T-cell targets of Mtb are predominantly secreted proteins (16, 17), to develop a poly-antigenic LV-based vaccine, we selected the following virulence-related factors: (i) EsxA (Rv3875), (ii) ESX-1 secretion-associated proteins (Esp) C (Rv3615c), both secreted via ESX-1 Type VII Secretion System (T7SS), (iii) EsxH (Rv0288, TB10.4), secreted via ESX-3 T7SS, (iv) PE19 (Rv1791), secreted via ESX-5 T7SS (18-21), and (v) Ag85A (Rv3804c) from the mycolyl transferase Ag85 complex, secreted via the Tat system (17, 22). Concerning the latter, we had previously observed that some Beijing clinical isolates expressed only minute amounts of Ag85A/B, which called into question the pertinence of the inclusion of these antigens in vaccine candidates (23). Here, to re-evaluate this assumption, we comparatively quantitated the intra-phagocyte secretion of Ag85A/B (FIG. 1A) versus EsxA (FIG. 1B) inside Dendritic Cells (DC) infected with each of the 15 non-Beijing or 31 Beijing clinical Mtb isolates listed in Table S1. This was performed using T-cell hybridomas specific to Ag85A/B or EsxA (Table S2), to measure the MHC-II-mediated presentation of T-cell epitopes derived from these antigens, which is proportional to their intra-phagocyte secretion (23). Although the average of Ag85A/B expression of the Beijing clinical isolates was statistically lower than that of the non-Beijing strains, the expression of these antigens was quite variable and many Beijing isolates were found to produce large amounts of Ag85A/B (FIG. 1C). This observation determined that Ag85A/B is in fact a relevant vaccine target.

LV Optimization to Route Mtb Immunogens to MHC-II Pathway

[0252] Highly efficient at routing endogenously produced antigens to MHC-I, viral vectors are however poorly effective, if not inoperative, in antigen delivery to MHC-II machinery. This was confirmed by the fact that our initial conventional LV, encoding individual EsxA, EspC, EsxH, PE19 or Ag85A, or various fusions of them, did not trigger CD4.sup.+ T-cell responses in mice. This was exemplified by the absence of EspC-specific CD4.sup.+ T cells, despite the presence of EspC-specific CD8.sup.+ T cells in C57BL/6 mice immunized with a conventional LV encoding a fusion of Mtb antigens including EsxA, Ag85A, EspC, EsxH, and PE19 (LV::TB) (FIG. 2A). It is noteworthy that, in addition to the MHC-II-restricted epitope within EspC:45-54 (23), this segment contains an MHC-I-restricted epitope, so far uncovered in H-2.sup.b mice, and evidenced here by use of LV. To overcome LV inability to induce inducing CD4.sup.+ T cells, we sought to optimize this vector, as detailed below and using EsxH as reporter antigen, considering the availability of EsxH-specific, MHC-I- or -II-restricted T-cell hybridomas (Table S2) (24, 25).

[0253] We generated a series of LV encoding: (i) EsxH alone (LV::EsxH), (ii) EsxH added at its N-ter part with the murine MHC-II li light invariant chain (LV::li-EsxH), to target the translated antigen to the MHC-II compartment (26, 27), (iii) EsxH added at its N-ter part with the 1-118 transmembrane domain of the human Transferrin Receptor (LV::TfR.sub.1-118-EsxH), to generate a membrane-bound protein which should traffic through endosomes, to potentially gain access to the MHC-II pathway (26, 27), or (iv) EsxH added at its N-ter and C-ter ends, respectively with HLA-B-derived leader SP peptide and MHC-I Trafficking signal (LV::SP-EsxH-MITD), since the MHC-I molecules also traffic via endosomes (28) (FIG. 2B, FIG. 13). DC transduced with LV encoding either EsxH variants, were able to present efficiently the MHC-I-restricted EsxH: 20-28 epitope to specific T-cell hybridoma (FIG. 2C). In net contrast to the conventional LV::EsxH, only the optimized LV::li-EsxH, and to a lesser extent, LV::TfR.sub.1-118-EsxH, were able to induce efficient MHC-II-restricted EsxH: 74-88 epitope presentation to specific T-cell hybridoma

[0254] (FIG. 2C). LV::SP-EsxH-MITD did not enable antigen presentation by MHC-II. For further experiments described below, we thus selected the li flanking strategy, which resulted in the highest presentation level via MHC-II, without impacting the presentation via MHC-I. In sum, we elaborated a new generation of LV which gains the instrumental property to provide appropriate antigen presentation, i.e., signal 1 (29), not only via MHC-I, but also through MHC-II pathway.

Very Minor Impact of LV on Innate Immunity Versus their Noticeable T-Cell Immunogenicity

[0255] Before exploring the potential of the optimized LV at inducing CD4.sup.+ T cells in vivo, we assessed some properties of LV regarding induction of DC maturation or inflammatory cytokine signaling, i.e., signal 2-3 (29). A preparation of LV, not characterized for endotoxin content, has been described to induce a few inflammatory responses in vivo (30). Another study reported some degrees of LV-induced DC maturation in vitro, attributed to the Vesicular Stomatitis Virus (VSV) G envelop glycoprotein, with which the LV are pseudo-typed (31). Murine DC, even when confronted to high amounts (MOI of 50) of our pre-GMP quality, VSV-G-pseudo-typed LV, displayed very slight phenotypic maturation, as judged by only a very minor CD86 upregulation and minute increases in the percentages of MHC-I.sup.hi or -II.sup.hi cells (FIG. 9A-B, C In terms of functional maturation, DC transduced with LV secreted readily detectable amounts of IFN-, CCL5 and IL-10 and very mild amounts of IFN-. Importantly, no IL-1, IL-1, IL-6 or TNF- were detected, indicating a poor inflammatory and even anti-inflammatory properties of LV (FIG. 9 D).

[0256] Based on the potency of LV at inducing IFN-I (30, 32) and the unique capacity of DC to activate nave T cells, we then evaluated the dependence of LV-mediated CD8.sup.+ T-cell induction (12) on IFN-I signaling in DC. To this end, we used conditional C57BL/6 mutants, ifnar1.sup.flox/flox pCD11c-Cre.sup.+ (with IFNAR-deficient DC) versus ifnar1.sup.flox/flox pCD11c-Cre.sup. (with IFNAR-proficient DC), after preliminary confirmation that DC derived from the former displayed a large reduction of surface IFNAR expression (FIG. 10A). Mice, ifnar1.sup.flox/flox pCD11c-Cre.sup. or Cre.sup.+, originated from the same litters, were immunized s.c. with 510.sup.7 Transduction Unit (TU) of LV::OVA or LV::li-EsxH. Eleven days post-immunization (dpi), tetramer staining, ELISPOT or Intracellular Cytokine Staining (ICS) assays detected in both mouse types strong and comparable CD8.sup.+ T splenocyte responses, specific to OVA (FIG. 10B-C) or EsxH (FIG. 10D-G), including similar proportions of IFN-.sup.+CD107a.sup.+ degranulating or polyfunctional CD8.sup.+ T cells. Therefore, the capacity of LV to induce CD8.sup.+ T-cell responses is not governed by IFNAR signaling in conventional DC.

[0257] The very slight DC stimulatory capacity of LV highlights the intrinsically minor inflammatory properties of these efficient vectors. In addition, the non-dependence of LV-mediated T-cell induction on DC signaling through the rare inflammatory mediators that they can induce (IFN-I) suggests that the underlying innate immune mechanisms are reduced to strict minimum.

Substantial Capacity of the Optimized LV at Inducing Systemic and Mucosal CD4.SUP.+ T-Cell Immunity

[0258] We then evaluated the potential of the optimized LV::li-EsxH at inducing CD4.sup.+ T-cell responses at the systemic or mucosal levels. Taking into the account the mild impact of LV on innate immune system and the so far uncharacterized CD4.sup.+ T-cell immunogenicity of this vector, we investigated the optimized LV::li-EsxH, either alone or adjuvanted with the pro-Th1 polyinosinic-polycytidylic acid (polyI:C) or the pro-Th1/Th17 cyclic Guanine-Adenine dinucleotide (cGAMP) (33). First, BALB/c mice were immunized s.c. with 510.sup.7 TU of LV::li-EsxH, alone or adjuvanted. At 11 dpi, ICS analysis detected remarkable amounts of EsxH-specific, Th1 cytokine-producing CD4.sup.+ (FIG. 3A, B), as well as CD8.sup.+ (FIG. 3A, C) T splenocytes. No significant impact of adjuvantation was observed in such responses induced by systemic immunization (FIG. 3B, C). Then, mucosal immunization of BALB/c mice was performed via intranasal (i.n.) route with 510.sup.7 TU of LV::li-EsxH, alone or adjuvanted. At 13 dpi, lung T cells were co-cultured with syngeneic DC loaded with EsxH: 74-88 or EsxH: 20-28 peptides, bearing respectively MHC-II or -I H-2.sup.d T-cell epitopes (24, 25). Mucosal antigen-specific IL-2- or IL-17A-producing CD4.sup.+ T cells were only detected in the lungs of mice immunized with cGAMP-adjuvanted LV::li-EsxH (FIG. 3D). In parallel, mucosal antigen-specific IL-2- or IL-17A-producing CD8.sup.+ T cells were detected in the lungs of mice immunized with LV::li-EsxH combined with either adjuvant (FIG. 3E). Antigen-specific IFN--producing lung CD4.sup.+ or CD8.sup.+ T cells were detected in all immunized groups (FIG. 3D, E).

[0259] Intravenous (i.v.) injection of mice with PE-anti-CD45 mAb, 3 min before sacrifice, allows distinction of hematopoietic cells located inside the lung interstitium from those in the lung vasculature (34). Compared to the PBS-injected controls, mice immunized with LV::li-EsxH alone possessed notable percentages of CD45i.v CD4.sup.+ (FIG. 4A) or CD45.sub.i.v.sup. CD8.sup.+ (FIG. 4E) T cells in the interstitium. This T-cell recruitment/expansion increased in mice immunized with adjuvanted LV::li-EsxH. Percentages of CD27.sup. CD62L.sup. recent migrant effectors, among interstitial CD4.sup.+ (FIG. 4B) or CD8.sup.+ (FIG. 4F) CD45.sub.i.v.sup. T cells, were also higher in mice immunized with adjuvanted LV::li-EsxH. Notable amounts of antigen-specific IFN-/TNF--producing CD4.sup.+ (FIG. 4C) or CD8.sup.+ (FIG. 4G) T-cell effectors were detected in the interstitium of mice immunized with LV::li-EsxH alone and even more amounts of these T cells were detected in their counterparts immunized with adjuvanted LV::li-EsxH. Immunization with cGAMP-adjuvanted LV::li-EsxH generated Th17 (FIG. 4D) and Tc17 (FIG. 4H) cells, as detected in the interstitium and consistent with IL-17A released in the supernatants of lung T cells stimulated in vitro with EsxH: 74-88 or EsxH: 20-28 peptides (FIG. 3D, E). CD45i.v.sup.+ Th1 cytokine-producing CD4.sup.+ or CD8.sup.+ T cells were also detected in the vasculature (FIG. 4C, G), showing that the i.n. immunization also generated antigen-specific T cells that gain access to the blood circulation and can thus contribute to systemic immunity.

[0260] As determined by cytometry at 1 dpi, i.n. administration of LV alone did not have a significant impact on the proportions of various lung innate immune cell subsets, compared to PBS treatment alone (FIG. 5A, B). Following i.n. instillation of PolyI:C- or cGAMP-adjuvanted LV, minor and statistically unsignificant increases in the percentages of DC and interstitial macrophages were detected. Notably, the proportions of pro-allergenic mast cells or basophils, and inflammatory Ly6C.sup.+ macrophages/monocytes or neutrophilspotentially harmful in the context of mycobacterial infection (38)remained unchanged in LV-treated mice.

Generation of an Optimized Poly-Antigenic LV

[0261] We then generated an optimized LV encoding for a fusion of li and juxtaposed sequences of EsxH, EsxA, EspC and PE19 (LV::li-HAEP) (Table S3, FIG. 5S). LV::li-HAEP-transduced DC were able to present the MHC-I- or -II-restricted epitopes of these immunogens to specific T-cell hybridomas (FIG. 6A). As determined by ELISPOT, in C57BL/6 mice, systemic s.c. immunization with 510.sup.8 TU of LV::li-HAEP alone induced IFN-/TNF--producing CD4.sup.+ or CD8.sup.+ T splenocytes specific to all included immunogens (FIG. 6B), with notable bi- or polyfunctionality in the both subsets (FIG. 6C, D). Like for LV-mediated CD8.sup.+ T-cell induction, with this optimized LV, we did not detect any dependence of CD4.sup.+ T-cell induction on DC IFNAR signaling (FIG. 11). We also established that immunization via s.c. or intramuscular (i.m.) systemic route with LV::li-HAEP alone resulted in comparable IFN- or TNF- CD4.sup.+ and CD8.sup.+ T splenocyte responses (FIG. 12).

[0262] Mucosal i.n. immunization of C57BL/6 mice with cGAMP-adjuvanted LV::li-HAEP elicited (poly) functional CD4.sup.+ (FIG. 7A) or CD8.sup.+ (FIG. 7B) T cells specific to each of the 4 Mtb antigens in lung interstitium, and also at a lesser extent in the vasculature. The CD45.sub.i.v..sup. interstitial CD4.sup.+ or CD8.sup.+ T subset in the vaccinated mice contained increased proportions of CD27.sup. CD62L.sup. migrant effectors and CD69.sup.+ CD103.sup.+ lung-tissue resident cells (FIG. 7C). The majority of CD69.sup.+ CD103.sup.+ CD4.sup.+ T cells displayed a CD44.sup.+ CXCR3.sup.+ phenotype (FIG. 7C bottom), reminiscent of CD8.sup.+ T-cell resident-memory phenotype (39, 40).

Evaluation of the Protective Booster Potential of the Optimized Poly-Antigenic LV

[0263] Considering the interest of Ag85A/B antigens as vaccine targets (FIG. 1), to maximize the boosting potential of the elaborated vector, we added the Ag85A: 241-260 immunogenic region (41, 42) to the C-ter end of HAEP in LV::li (LV::li-HAEPA) (Table S3, FIG. 13). LV::li-HAEPA-transduced DC were able to induce MHC-II-restricted presentation of Ag85A: 241-260 to specific T-cell hybridoma (FIG. 8A), in addition to the presentation of the other Mtb antigens, as exemplified by EsxA and as detected by specific T-cell hybridoma (ref 23) To evaluate the booster efficacy of LV::li-HAEPA, C57BL/6 mice were either left unvaccinated or primed s.c. at week 0 with 110.sup.6 CFU of BCG::ESX-1.sup.Mmar vaccine candidate with increased protective capacity compared to the parental BCG (8) (FIG. 8B). The advantage of this live-attenuated vaccine candidate over BCG in prime immunization is linked to its capacity to secrete EsxA and EspC via the orthologous ESX-1 T7SS, which allows here to boost the T-cell responses against all the Mtb antigens included in the optimized poly-antigenic LV. A group of BCG::ESX-1.sup.Mmar primed mice was boosted s.c. with 510.sup.8 TU of cGAMP-adjuvanted LV::li-HAEPA at week 5, and then again boosted i.n. at week 10 with the same amount of cGAMP-adjuvanted LV::li-HAEPA in order to attract the induced immune effectors to the lung mucosa. At week 12, mice were challenged with 200 CFU of virulent Mtb H37Rv strain via aerosol and mycobacterial burdens were determined in the lungs and spleen at week 17. The lung mycobacterial load average in the primed-boosted mice was decreased by 2.5 log 10 compared to unvaccinated controls (Mann-Whitney test, p value=0,0005), and by 1 log.sub.10 compared to their BCG::ESX-1.sup.Mmar-vaccinated counterparts (Mann-Whitney test, p value=0,0415) (FIG. 8C). This significant decrease in the primed and boosted mice compared to the mice vaccinated only with BCG::ESX-1.sup.Mmar seems nevertheless without major impact on lung histopathology (FIG. 8D). In the spleen, the boost with cGAMP-adjuvanted LV::li-HAEPA led to a net trend to reduced mycobacterial loads, which however did not reach statistical significance. This could be explained by the particularly strong and hardly improvable protective effect of ESX-1-complemented BCG strains against dissemination to the spleen in the mouse and guinea pig models (22, 43, 44).

Discussion

[0264] Extensive investigations of human immune cells by omics approaches in healthy donors or individuals with latent versus active TB allowed identification of biomarkers for developing host response-based diagnosis of active TB (45). However, these studies did not provide a thorough view of multifactorial processes which result in immune failure of granulomas and progression to active TB. Therefore, reliable correlates of optimal protection, and causative biomarkers of non-progression of latent to active TB remain largely elusive. In this context, rational design of new generation of TB vaccines is challenging (46). One consensus in the domain consists of prime-boost immunization approaches. BCG displayed an excellent safety record over the last 80 years and shows a high rate of protector effect against disseminated forms of TB in children. Therefore, the use of: (i) BCG or an improved live-attenuated vaccine for priming, and (ii) subunit vaccine candidates for boosting, represent a promising strategy.

[0265] Viral vectors, notably Modified Vaccinia Ankara (MVA) or adenoviral vectors have been used in immunization against Mtb (7). Despite its remarkable success in pre-clinical animal models, an MVA encoding Ag85A, was poorly immunogenic in clinical trials and was unable to induce protection (47). Another LV encoding Ag85A, together with an NF-kB activator, induced systemic and mucosal T-cell immunity, but did not afford protection against a BCG challenge in the mouse model (48). In BCG-primed mice, a boost with an LV encoding an Ag85B-PPE57 fusion, increased the amplitude of T-cell responses and protection against a high-dose i.v. Mtb challenge (49). In these studies, the LV encoded only for one or two Mtb antigens and were not optimized to target the antigens to the MHC-II presentation pathway, which can explain their poor capacity to induce protection against Mtb. In fact, despite their remarkable ability to target endogenously produced antigens into the MHC-I pathway of the transduced antigen presenting cells, viral vectors, including LV, mostly fail to deliver antigens to the MHC-II machinery for CD4.sup.+ T-cell induction. Here, we generated a new generation of LV in which the genes encoding multiple potent Mtb antigens were engineered to allow trafficking of the resulted fusion proteins through the MHC-II pathway. Addition of the li or TfR at the N-ter of a single or a poly-antigenic protein, achieved proper antigen routing to the MHC-II machinery and robust triggering of CD4.sup.+ T cells, without any propensity to reduce MHC-I presentation or CD8.sup.+ T-cell induction. However, li fusion to the N-ter of protein sequences might not be always sufficient and preservation of the native tertiary structure of the resulting proteins seems to matter as well. For instance, LV encoding for a fusion of li and a cluster of predicted T-cell epitopes, derived from EsxH, EsxA, EspC and PE19, failing to preserve protein folding and enriching the sequences in hydrophobic residues, did not induce efficient antigen routing to MHC-II machinery.

[0266] The choice of the Mtb immunogens included in the poly-antigen inserted in the optimized LV was based on their direct relationship with the mycobacterial virulence in vivo and active secretion by the ESX-1, -3, -5 T7SS or Tat systems, throughout various TB phases (16, 17). Among these proteins, PE19 is of particular interest. As a single antigen, PE19 harbors T-cell epitopes which are shared with its several homologs. The Mtb genome contains up to one hundred of pe (and ppe) genes. The resulted PE/PPE proteins, named after their N-ter PE or PPE motifs (18, 50, 51), form large multigenic families of proteins, which are secreted or cell wall-attached and many are related to pathogenic potential (18-21). Resulting from ancestral gene duplication, PE/PPE proteins display substantial sequence homologies and thus share plethora of T-cell epitopes (42). The arbitrary insertion of the pe/ppe genes all over the Mtb genome led to their expression by an array of independent promoters, which generates unprecedented degrees of variability in their expression profiles at distinct infection phases (52). This situation can readily generate consecutive display of groups of shared PE (/PPE) epitopes, during various TB phases (42, 53-55).

[0267] As we recently demonstrated with LV-based vaccination against SARS-COV-2 (56), systemic immune responses, even of high quality, may not always reach the site of the infection in the lung mucosa to prevent replication of pulmonary pathogens. Mucosal immunity, including antibodies and tissue-resident lymphocytes, have been shown to be instrumental in pathogen clearance from the respiratory tracts (56-60). In TB vaccination, our previous results demonstrated the advantages of i.n. immunization with Esx or PE/PPE antigens in various formulations (9, 61). Moreover, the protection against pulmonary TB has been correlated with the presence of antigen-specific resident-memory CD4.sup.+ T cells (62-65). Here, we thoroughly characterized the functions and phenotype of CD4.sup.+ and CD8.sup.+ T cells, induced through systemic or i.n. administration of the optimized LV encoding EsxH or HAEP(A) poly-antigen. Most notably, mucosal immunization induced lung CD4.sup.+ and CD8.sup.+ T cells with polyfunctional effector features, accompanied by activated, tissue-resident and memory phenotypes. When formulated with cGAMP adjuvant and administered via i.n., the optimized LV also triggered lung Th17 and Tc17 responses with prospective implications in the protection against Mtb (66, 67).

[0268] The very mild impact of LV on DC maturation in vitro, and the very slight modification of the lung innate immune cell composition subsequent to i.n. administration of LV alone, indicate the intrinsically low inflammatory properties of these vectors. Interestingly, DC signaling through IFN-I, i.e., the rare inflammatory factors induced by LV, is not involved in CD4.sup.+ or CD8.sup.+ T-cell induction by these vectors. This suggests a minimalist involvement of innate immune pathways engaged by LV to induce robust T-cell immunity. These characteristics, together with the non-replicative property of LV, reflect favorably on their safety for veterinary or human vaccination, notably via the mucosal pathways. In addition, due to the mucosal barrier, i.n. immunization could even generate minimized systemic adverse effects (68).

[0269] Finally, we investigated the protective potential of a vaccination approach based on priming with the improved live-attenuated vaccine, BCG::ESX-1.sup.Mmar (8), and boosting with the optimized LV::li-HAEPA formulated in cGAMP in the prophylactic C57BL/6 mouse TB model. BCG::ESX-1.sup.Mmar per se triggered a substantial reduction in the Mtb loads in the lungs and spleen, while LV boosting via systemic and nasal routes achieved significant additional decrease of bacterial loads in the lungs, accompanied by a net trend to weakened dissemination to the spleen. These data provide the proof-of-concept evidences that, in the context of LV, not only single small antigens like EsxH, but also fusion of multiple antigens, fused to li are able to gain access to the MHC-II presentation pathway to induce CD4.sup.+ T cells, without reduction of CD8.sup.+ T-cell triggering. In addition, i.n. immunization with the optimized LV induces recruitment and establishment of poly-specific lung CD4.sup.+ and CD8.sup.+ T-cell immunity with resident-memory phenotype. This approach can optionally be improved by the addition of appropriate adjuvants.

[0270] Whether the i.n. immunization involves the mediastinal lymph nodes, immune cells directly recruited and located into the lung parenchyma or the highly organized ectopic lymphoid-like structures of tertiary lymphoid organs remains to be uncovered. The latter mimic the immune germinal centers in the mucosal tissues, providing local and controlled inflammation and an optimal environment for innate and adaptive immune cell cross-talk to reinforce anti-microbial host immunity at the site of the potential 5 infection (74).

[0271] The non-replicative and very weakly inflammatory properties of LV, now optimized to induce CD4.sup.+ T-cell responses, predict these vectors as tools of choice for mucosal vaccination, especially via the i.n. route. The prospects for development of these LV-based strategies go far beyond mycobacterial infections, extending the approach to acute or chronic respiratory infectious diseases.

Materials and Methods

Construction of Transfer Plasmids Encoding Mtb (Poly) Antigen and LV Generation

[0272] Codon-optimized genes encoding EsxH alone or in fusion with the II, TfR, and MITD or encoding II-HAEP or II-HAEPA were synthetized by Eurofins were then cloned downstream of the SP1 promoter: (i) based on human 32 microglobulin (2m) promoter which derives antigen expression predominantly in immune cells and notably activated APCs (70), and (ii) containing inserted/substituted regions originated from the CMV promoter albeit with minimal proximal enhancers and thus improved vector safety (our unpublished results). The promoter is located between BamHI and Xhol sites of the pFLAPAU3 transfer plasmid (14) (FIG. 13) containing a mutated WPRE (Woodchuck Posttranscriptional Regulatory Element) sequence to increase gene transcription. Production and titration of LV were performed as described elsewhere (56)

Mycobacteria

[0273] Mtb (H37Rv strain) or BCG: ESX-1.sup.Mmar (8), were cultured to exponential phase in Dubos broth, complemented with Albumine, Dextrose and Catalase (ADC, Difco, Becton Dickinson, Le Pont-de-claix, France). Non-Beijing and Beijing clinical Mtb isolates, representative of the most prevalent genotypes in France, have been submitted to the National Reference Centre for TB for drug-resistance characterization and Mycobacterial Interspersed Repetitive-Unit-Variable-Number Tandem-Repeat (MIRU-VNTR) genotyping (75). Mtb clinical isolates were grown in Dubos broth, complemented with oleic ADC (OADC, Difco). Titers of the mycobacterial cultures were determined by OD.sub.600 measuring. Experiments with pathogenic mycobacteria were performed in BSL3, following the hygiene and security recommendations of Institut Pasteur.

Detection of MHC-I or -II Restricted Antigenic Presentation In Vitro

[0274] Histocompatible bone-marrow derived DC were plated at 510.sup.5 cells/well in 24-well plates in RPMI 1640 containing 5% FBS. When adherent, cells were transduced with LV vectors, or were loaded with 1 g/ml of homologous or control synthetic peptides. At 24 h post infection 510.sup.5 appropriate T-cell hybridomas were added and the co-culture supernatants were assessed for IL-2 production at 24h by ELISA. In this assay, the amounts of released IL-2 is proportional to the efficacy of antigenic presentation by MHC molecules. The peptides harboring MHC-I or -II-restricted epitopes were synthesized by Proteogenix (Schiltigheim, France) and were reconstituted in H.sub.2O containing 5% Di-Methyl Sulfoxyd (DMSO) (Sigma-Aldrich). When indicated antigenic presentation was assessed by use of reporter T-cell hybridomas, transduced to emit fluorescent signals subsequent to TCR triggering, as recently described (23).

Mice, Immunization

[0275] Female BALB/c (H-2.sup.d) and C57BL/6 (H-2.sup.b) (Janvier Labs, Le Genest-Saint-Isle, France) were immunized after at least one week of acclimatation, with the indicated dose of LV contained in 50 l/mouse for i.m. injection, in 200 l/mouse for s.c. at the basis of the tail, or in 20 l/mouse for i.n. instillation. The i.n. administration was performed under general anesthesia, obtained by i.p. injection of 100 l of PBS containing weight-adapted quantities of Imalgne1000 (Ktamine, i.e., 100 mg/kg, Merial, France) and Rompun 2% (Xylazine solution, 10 mg/kg, Bayer, Germany). When indicated LV was adjuvanted with 10 ug/mouse of polyI:C or cGAMP (Invivogen).

[0276] The hemizygous C57BL/6 (H-2.sup.b) mice, carrying the gene encoding Cre DNA recombinase, under the regulation of murine CD11c promoter (76), were crossed with C57BL/6 mice homozygous for the floxed ifnar1 allele (77) to obtain litters of homozygous ifnar1.sup.flox/flox mice that carry or not the Cre transgene. In ifnar1.sup.flox/flox pCD11c-Cre.sup.+ mice, with the exception of CD11c-expressing plasmacytoid DC, all other DC populations lacked IFNAR1 (77). The breeding was performed at the central animal facilities of Institut Pasteur, under SPF conditions.

[0277] All the mice were used between the age of 8 and 16 weeks, in accordance with the European and French directives (Directive 86/609/CEE and Decree 87-848 of 19 Oct. 1987), after approval by the Institut Pasteur Safety, Animal Care and Use Committee, under local ethical committee protocol agreement #CETEA 2013-0036 and CETEA 2012-0005 (APAFIS #14638-2018041214002048).

Intracellular Cytokine Staining

[0278] Splenocytes from immunized mice were obtained by tissue homogenization and passage through 100-m nylon filters (Cell Strainer, BD Biosciences) and were plated at 410.sup.6 cells/well in 24-well plates. Lungs were treated with 400 U/ml type IV collagenase and DNase I (Roche) for 30 min at 37 C. and homogenized by use of GentleMacs (Miltenyi). Cells were then filtered through 70-m nylon filters (Cell Strainer, BD Biosciences), and centrifuged for 20 min at 3000 rpm at RT without brake on Ficoll gradient medium (Lympholyte M, Cedarlane Laboratories). Lung T-cell-enriched fractions were co-cultured at 410.sup.6 cells/well with histocompatible bone-marrow-derived DC (810.sup.5 cells/well) in 24-well plates. Splenocytes or lung T cells were co-cultured during 6h in the presence of 10 g/ml of homologous or control peptide, 1 g/ml of anti-CD28 (clone 37.51) and 1 g/ml of anti-CD49d (clone 9C10-MFR4.B) mAbs (BD Biosciences). During the last 3h of incubation, cells were treated with a mixture of Golgi Plug and Golgi Stop, both from BD Biosciences. When indicated, PE-Cy7-anti-CD107a (clone 1D4B, BioLegend) mAb was also added to the cultures at this step. Cells were then collected, washed with PBS containing 3% FBS and 0.1% NaN.sub.3 (FACS buffer) and incubated for 25 min at 4 C. with a mixture of FcII/III receptor blocking anti-CD16/CD32 (clone 2.4G2) and APC-eFluor780-anti-CD3E (clone17A2), eF450-anti-CD4 (clone RM4-5), BV711-anti-CD8 (clone 53-6.7) mAbs (BD Biosciences or eBioscience). Cells were washed twice in FACS buffer, then permeabilized by use of Cytofix/Cytoperm kit (BD Bioscience). Cells were then washed twice with PermWash 1 buffer from the Cytofix/Cytoperm kit and incubated with a mixture of AF488-anti-IL-2 (clone JES6-5H4, BD Biosciences), PE/Dazzle 594-anti-TNF- (MP6-XT22, BioLegend), and APC-anti-IFN- (clone XMG1.2, BD Biosciences) mAbs or a mixture of appropriate control Ig isotypes, during 30 min at 4 C. Cells were then washed twice in PermWash and once in FACS buffer, then fixed with Cytofix (BD Biosciences) overnight at 4 C. Cells were acquired in an Attune NxT cytometer system (Invitrogen) and data analysis was performed using FlowJo software (Treestar, OR, USA).

Lung Cell Phenotyping

[0279] Lymphocyte-enriched lung cells from mice, injected i.v. with PE-anti-CD45 (clone 30-F11, BioLegend) 3 min before sacrifice, were prepared as described above and stained with a mixture of APC-eFluor780-anti-CD3 (clone17A2, eBioscience), eF450-anti-CD4 (clone RM4-5, eBioscience), BV711-anti-CD8 (clone 53-6.7, BD Biosciences) mAbs, with either: (i) PE-Cy7-anti-CD27 (clone LG.7F9, eBioscience) and AF700-anti-CD62L (clone MEL-14, BD Biosciences) mAbs, or (ii) BV605-anti-CD69 (clone H1.2F3, BioLegend), FITC-anti-CD103 (clone 2E7, BioLegend), PE-Cy7-anti-CD49a (clone HM1, BioLegend), AF700-anti-CD44 (clone IM7, BioLegend) and APC-Fire750-anti-CXCR3 (clone CXCR3-173, BioLegend) mAbs, all in the presence of FcII/III receptor blocking anti-CD16/CD32 (BD Biosciences). After 25 min incubation at 4 C., the cells were washed twice in FACS buffer and fixed by incubation with Cytofix (BD Bioscience) overnight at 4 C. Cytometric analysis of lung innate immune cells was recently detailed elsewhere (56).

Elispot Assay

[0280] Splenocytes from individual mice were homogenized and filtered through 100 m-pore filters and centrifuged at 1500 rpm during 5 min. Cells were then treated with Red Blood Cell Lysing Buffer (Sigma), washed twice in PBS and counted in a MACSQuant-10 cytometer (Miltenyi Biotec). Splenocytes were then plated at 110.sup.5 cells/well in 200 l of RPMI-GlutaMAX, containing 10% heat-inactivated FBS, 100 U/ml penicillin and 100 g/ml streptomycin, 110.sup.4 M non-essential amino-acids, 1% vol/vol HEPES, 110.sup.3 M sodium pyruvate and 510.sup.5 M of -mercaptoethanol in ELISPOT plates (Mouse IFN- or TNF- ELISPOTPLUS, Mabtech). Cells were left unstimulated or were stimulated with 2 g/ml of appropriate synthetic peptides (Proteogenix) or 2.5 g/ml of

[0281] Concanavalin A (Sigma), as a functionality control. For each mouse, the assay was performed in triplicates, according to the manufacturer's recommendations. Plates were analyzed in an ELR04 ELISPOT reader (AID, Strassberg, Germany).

Protection Assay

[0282] C57BL/6 mice were primed s.c. with 110.sup.6 CFU/mouse of BCG: ESX-1.sup.Mmar (8) at day 0, boosted s.c. with 510.sup.8 TU/mouse of adjuvanted LV at week 5, and boosted again i.n. with 510.sup.8 TU/mouse of adjuvanted LV at week 10. The immunized mice, as well as age-matched, unvaccinated controls, were challenged 2 weeks after the i.n. boost by use of a homemade nebulizer via aerosol, as previously described (9). Briefly, 5 ml of a suspension of 1.710.sup.6 CFU/ml of H37Rv Mtb strain were aerosolized to deliver an inhaled dose of 200 CFU/mouse. The infected mice were placed in isolator in BSL3 facilities at Institut Pasteur. Five weeks later, lungs or spleen of the infected mice were homogenized by using a MillMixer homogenizer (Qiagen, Courtaboeuf, France) and serial 5-fold dilutions prepared in PBS were plated on 7H11 Agar complemented with ADC (Difco, Becton Dickinson). CFU were counted after 3 weeks of incubation at 37 C. Significance of inter-group CFU differences was determined by Mann-Whitney t-test by use of Prism v8.01 (GraphPad Software, Inc.).

TABLE-US-00001 TABLE S1 Non-Beijing or Beijing Mtb clinical isolates from MIRU-VNTR, tested for the intra-phagocyte Ag85A/B and EsxA expression. Corresponding Number Clinical Isolate on Heat Maps No. lineage Non Beijing 1 661603069400 2 661610071089 3 661702081724 4 661612077758 5 661708094529 6 661801095406 7 661805075095 8 661412076132 9 661404013957 10 661407086878 11 661405051259 12 661404032252 13 661408059503 14 661409034117 15 661410060829 Beijing 1 661701115366 2 661711076067 3 661501008044 4 661508077689 5 661611080910 6 661602107040 7 661604058591 8 661609072250 9 661612100794 10 661701037389 11 661706099221 12 661707013366 13 661707023069 14 661709001208 15 661709065148 16 661805075112 17 661711117831 18 661406088111 19 661406088102 20 CR 1201 6122 21 CR 1104 7352 22 CR 1005 5423 23 CR 1010 5929 24 CR 1008 5736 25 CR 1010 5930 26 661305027157 27 CR 0910 6843 28 CR 0910 6844 29 CR 0905 6418 30 CR 0905 6417 31 CR 1211 7109

TABLE-US-00002 TABLES2 MHC-Ior-IIrestrictedT-cellhybridomasspecifictothe selectedMtbimmunogens MHC T-cell Restricting Antigen epitope Epitopesequence hybridoma element EsxH 20-28 GYAGTLQSL YB8 K.sup.d (SEQIDNo.15) EsxH 74-88 STHEANTMAMMARDT 1H2 I-A.sup.d (SEQIDNo.16) EsxA 1-20 MTEQQWNFAGIEAAASAIQG NB11 I-A.sup.b (SEQIDNo.17) PE19 1-18 MSFVTTQPEALAAAAANL IF6 I-A.sup.b (SEQIDNo.18) Ag85A 241-260 QDAYNAGGGHNGVFDFPDSG DE10 I-A.sup.b (SEQIDNo.19)

TABLE-US-00003 TABLES3 SequencesoffusedliandselectedMtbantigens,insertedinLV Insert Poly-antigenicLV length(a.a.) Sequence LV::li-EsxH-EsxA-EspC- 617 MDDQRDLISNHEQLPILGNRPREPERCSRGALYTGVSVLVA PE19 LLLAGQATTAYFLYQQQGRLDKLTITSQNLQLESLRMKLPK (LV::li-HAEP) SAKPVSQMRMATPLLMRPMSMDNMLLGPVKNVTKYGNM (SEQIDNo.1) TQDHVMHLLTRSGPLEYPQLKGTFPENLKHLKNSMDGVN WKIFESWMKQWLLFEMSKNSLEEKKPTEAPPKEPLDME DLSSGLGVTRQELGQVTLGGGDSQIMYNYPAMLGHAGDMA GYAGTLQSLGAEIAVEQAALQSAWQGDTGITYQAWQAQWN QAMEDLVRAYHAMSSTHEANTMAMMARDTAEAAKWGGN TEQQWNFAGIEAAASAIQGNVTSIHSLLDEGKQSLTKLA AAWGGSGSEAYQGVQQKWDATATELNNALQNLARTIS EAGQAMASTEGNVTGMFANNGGTENLTVQPERLGVLAS HHDNAAVDASSGVEAAAGLGESVAITHGPYCSQFNDTLNV YLTAHNALGSSLHTAGVDLAKSLRIAAKIYSEADEAWRKAI DGLFTNNDDSFVTTQPEALAAAAANLQGIGTTMNAQNAAA AAPTTGVVPAAADEVSALTAAQFAAHAQMYQTVSAQAAAIH EMFVNTLVASSGSYAATEAANAAAAG LV::li-EsxH-EsxA-EspC- 636 MDDQRDLISNHEQLPILGNRPREPERCSRGALYTGVSVLVA PE19-Ag85A:241-260 LLLAGQATTAYFLYQQQGRLDKLTITSQNLQLESLRMKLPK (LV::li-HAEPA) SAKPVSQMRMATPLLMRPMSMDNMLLGPVKNVTKYGNM (SEQIDNo.3) TQDHVMHLLTRSGPLEYPQLKGTFPENLKHLKNSMDGVN WKIFESWMKQWLLFEMSKNSLEEKKPTEAPPKEPLDME DLSSGLGVTRQELGQVTLGGGDSQIMYNYPAMLGHAGDMA GYAGTLQSLGAEIAVEQAALQSAWQGDTGITYQAWQAQWN QAMEDLVRAYHAMSSTHEANTMAMMARDTAEAAKWGGN TEQQWNFAGIEAAASAIQGNVTSIHSLLDEGKQSLTKLA AAWGGSGSEAYQGVQQKWDATATELNNALQNLARTIS EAGQAMASTEGNVTGMFANNGGTENLTVQPERLGVLAS HHDNAAVDASSGVEAAAGLGESVAITHGPYCSQFNDTLNV YLTAHNALGSSLHTAGVDLAKSLRIAAKIYSEADEAWRKAI DGLFTNNDDSFVTTQPEALAAAAANLQGIGTTMNAQNAAA AAPTTGVVPAAADEVSALTAAQFAAHAQMYQTVSAQAAAIH EMFVNTLVASSGSYAATEAANAAAAQDAYNAGGGHNGVFD FPDSG LV::li-EsxH 315 MDDQRDLISNHEQLPILGNRPREPERCSRGALYTGVSVLVA (SEQIDNo.5) LLLAGQATTAYFLYQQQGRLDKLTITSQNLQLESLRMKLPK SAKPVSQMRMATPLLMRPMSMDNMLLGPVKNVTKYGNM TQDHVMHLLTRSGPLEYPQLKGTFPENLKHLKNSMDGVN WKIFESWMKQWLLFEMSKNSLEEKKPTEAPPKEPLDME DLSSGLGVTRQELGQVTLGAGAMSQIMYNYPAMLGHAGDM AGYAGTLQSLGAEIAVEQAALQSAWQGDTGITYQAWQAQW NQAMEDLVRAYHAMSSTHEANTMAMMARDTAEAAKWGG TfR.sub.1-118-EsxH 218 MMDQARSAFSNLFGGEPLSYTRFSLARQVDGDNSHVEMK (SEQIDNo.7) LAVDEEENADNNTKANVTKPKRCSGSICYGTIAVIVFFLIGF MIGYLGYCKGVEPKTECERLAGTESPVREEPGEDFPAGAGA MSQIMYNYPAMLGHAGDMAGYAGTLQSLGAEIAVEQAALQS AWQGDTGITYQAWQAQWNQAMEDLVRAYHAMSSTHEANT MAMMARDTAEAAKWGG Double-underlined sequences are junctions inserted to avoid neo-epitope generation.

REFERENCES

[0283] 1. WHO. 2018. http://www.who.int/tb/publications/global_report/en/. [0284] 2. Dockrell H M, Smith S G. 2017. What Have We Learnt about BCG Vaccination in the Last 20 Years? Front Immunol 8:1134. [0285] 3. Ernst J D. 2018. Mechanisms of M. tuberculosis Immune Evasion as Challenges to T B Vaccine Design. Cell Host Microbe 24:34-42. [0286] 4. Lyadova I, Nikitina I. 2019. Cell DifferentiationDegree as a Factor Determining the Role for Different T-Helper Populations in Tuberculosis Protection. Front Immunol 10:972. [0287] 5. Scriba T J, Kaufmann S H, Henri Lambert P, Sanicas M, Martin C, Neyrolles O. 2016. Vaccination Against Tuberculosis With Whole-Cell Mycobacterial Vaccines. J Infect Dis 214:659-64. [0288] 6. Cruz A, Fraga A G, Fountain J J, Rangel-Moreno J, Torrado E, Saraiva M, Pereira D R, Randall T D, Pedrosa J, Cooper A M, Castro A G. 2010. Pathological role of interleukin 17 in mice subjected to repeated BCG vaccination after infection with Mycobacterium tuberculosis. J Exp Med 207:1609-16. [0289] 7. Dalmia N, Ramsay A J. 2012. Prime-boost approaches to tuberculosis vaccine development. Expert Rev Vaccines 11:1221-33. [0290] 8. Groschel M I, Sayes F, Shin S J, Frigui W, Pawlik A, Orgeur M, Canetti R, Honore N, Simeone R, van der Werf T S, Bitter W, Cho S N, Majlessi L, Brosch R. 2017. Recombinant BCG Expressing ESX-1 of Mycobacterium marinum Combines Low Virulence with Cytosolic Immune Signaling and Improved T B Protection. Cell Rep 18:2752-2765. [0291] 9. Sayes F, Pawlik A, Frigui W, Groschel M I, Crommelynck S, Fayolle C, Cia F, Bancroft G J, Bottai D, Leclerc C, Brosch R, Majlessi L. 2016. CD4.sup.+ T Cells Recognizing P E/PPE Antigens Directly or via Cross Reactivity Are Protective against Pulmonary Mycobacterium tuberculosis Infection. PLOS Pathog 12: e1005770. [0292] 10. Arhel N J, Souquere-Besse S, Munier S, Souque P, Guadagnini S, Rutherford S, Prevost M C, Allen T D, Charneau P. 2007. HIV-1 DNA Flap formation promotes uncoating of the pre-integration complex at the nuclear pore. EMBO J 26:3025-37. [0293] 11. Di Nunzio F, Felix T, Arhel N J, Nisole S, Charneau P, Beignon A S. 2012. HIV-derived vectors for therapy and vaccination against HIV. Vaccine 30:2499-509. [0294] 12. Hu B, Tai A, Wang P. 2011. Immunization delivered by lentiviral vectors for cancer and infectious diseases. Immunol Rev 239:45-61. [0295] 13. Sirven A, Pflumio F, Zennou V, Titeux M, Vainchenker W, Coulombel L, Dubart-Kupperschmitt A, Charneau P. 2000. The human immunodeficiency virus type-1 central DNA flap is a crucial determinant for lentiviral vector nuclear import and gene transduction of human hematopoietic stem cells. Blood 96:4103-10. [0296] 14. Zennou V, Petit C, Guetard D, Nerhbass U, Montagnier L, Charneau P. 2000. HIV-1 genome nuclear import is mediated by a central DNA flap. Cell 101:173-85. [0297] 15. Zennou V, Serguera C, Sarkis C, Colin P, Perret E, Mallet J, Charneau P. 2001. The HIV-1 DNA flap stimulates HIV vector-mediated cell transduction in the brain. Nat Biotechnol 19:446-50. [0298] 16. Lindestam Arlehamn C S, Gerasimova A, Mele F, Henderson R, Swann J, Greenbaum J A, Kim Y, Sidney J, James E A, Taplitz R, Mckinney D M, Kwok W W, Grey H, Sallusto F, Peters B, Sette A. 2013. Memory T cells in latent Mycobacterium tuberculosis infection are directed against three antigenic islands and largely contained in a CXCR3+CCR6+ Th1 subset. PLOS Pathog 9: e1003130. [0299] 17. Majlessi L, Prados-Rosales R, Casadevall A, Brosch R. 2015. Release of mycobacterial antigens. Immunol Rev 264:25-45. [0300] 18. Cole S T, Brosch R, Parkhill J, Garnier T, Churcher C, Harris D, Gordon S V, Eiglmeier K, Gas S, Barry C E, 3rd, Tekaia F, Badcock K, Basham D, Brown D, Chillingworth T, Connor R, Davies R, Devlin K, Feltwell T, Gentles S, Hamlin N, Holroyd S, Hornsby T, Jagels K, Krogh A, McLean J, Moule S, Murphy L, Oliver K, Osborne J, Quail M A, Rajandream M A, Rogers J, Rutter S, Seeger K, Skelton J, Squares R, Squares S, Sulston J E, Taylor K, Whitehead S, Barrell B G. 1998. Deciphering the biology of Mycobacterium tuberculosis from the complete genome sequence. Nature 393:537-44. [0301] 19. Banu S, Honore N, Saint-Joanis B, Philpott D, Prevost M C, Cole S T. 2002. Are the PE-PGRS proteins of Mycobacterium tuberculosis variable surface antigens? Mol Microbiol 44:9-19. [0302] 20. Iantomasi R, Sali M, Cascioferro A, Palucci I, Zumbo A, Soldini S, Rocca S, Greco E, Maulucci G, De Spirito M, Fraziano M, Fadda G, Manganelli R, Delogu G. 2011. PE_PGRS30 is required for the full virulence of Mycobacterium tuberculosis. Cell Microbiol doi: 10.1111/j. 1462-5822.2011.01721.x: doi: 10.1111/j. 1462-5822. [0303] 21. Abdallah A M, Verboom T, Weerdenburg E M, Gey van Pittius N C, Mahasha P W, Jimenez C, Parra M, Cadieux N, Brennan M J, Appelmelk B, Bitter W. 2009. PPE and PE_PGRS proteins of Mycobacterium marinum are transported via the type VII secretion system ESX-5. Molecular Microbiology 73:329-340. [0304] 22. Groschel M I, Sayes F, Simeone R, Majlessi L, Brosch R. 2016. ESX secretion systems: mycobacterial evolution to counter host immunity. Nat Rev Microbiol 14:677-691. [0305] 23. Sayes F, Blanc C, Ates L S, Deboosere N, Orgeur M, Le Chevalier F, Groschel M I, Frigui W, Song O R, Lo-Man R, Brossier F, Sougakoff W, Bottai D, Brodin P, Charneau P, Brosch R, Majlessi L. 2018. Multiplexed Quantitation of Intraphagocyte Mycobacterium tuberculosis Secreted Protein Effectors. Cell Rep 23:1072-1084. [0306] 24. Hervas-Stubbs S, Majlessi L, Simsova M, Morova J, Rojas M J, Nouze C, Brodin P, Sebo P, Leclerc C. 2006. High frequency of CD4.sup.+ T cells specific for the TB10.4 protein correlates with protection against Mycobacterium tuberculosis infection. Infect Immun 74:3396-407. [0307] 25. Majlessi L, Rojas M J, Brodin P, Leclerc C. 2003. CD8.sup.+-T-cell responses of Mycobacterium-infected mice to a newly identified major histocompatibility complex class I-restricted epitope shared by proteins of the ESAT-6 family. Infect Immun 71:7173-7. [0308] 26. Diebold S S, Cotten M, Koch N, Zenke M. 2001. MHC class II presentation of endogenously expressed antigens by transfected dendritic cells. Gene Ther 8:487-93. Rowe H M, Lopes L, Ikeda Y, Bailey R, Barde I, Zenke M, Chain B M, Collins M K. 2006. 27. Immunization with a lentiviral vector stimulates both CD4 and CD8 T cell responses to an ovalbumin transgene. Mol Ther 13:310-9. [0309] 28. Kreiter S, Selmi A, Diken M, Sebastian M, Osterloh P, Schild H, Huber C, Tureci O, Sahin U. 2008. Increased antigen presentation efficiency by coupling antigens to MHC class | trafficking signals. J Immunol 180:309-18. [0310] 29. Liechtenstein T, Dufait I, Bricogne C, Lanna A, Pen J, Breckpot K, Escors D. 2012. P D-L1/PD-1 Co-Stimulation, a Brake for T cell Activation and a T cell Differentiation Signal. J Clin Cell Immunol S12. [0311] 30. Cousin C, Oberkampf M, Felix T, Rosenbaum P, Weil R, Fabrega S, Morante V, Negri D, Cara A, Dadaglio G, Leclerc C. 2019. Persistence of Integrase-Deficient Lentiviral Vectors Correlates with the Induction of STING-Independent CD8 (+) T Cell Responses. Cell Rep 26:1242-1257 e7. [0312] 31. Pichlmair A, Diebold S S, Gschmeissner S, Takeuchi Y, Ikeda Y, Collins M K, Reis e Sousa C. 2007. Tubulovesicular structures within vesicular stomatitis virus G protein-pseudotyped lentiviral vector preparations carry DNA and stimulate antiviral responses via Toll-like receptor 9. J Virol 81:539-47. [0313] 32. Brown B D, Sitia G, Annoni A, Hauben E, Sergi L S, Zingale A, Roncarolo M G, Guidotti L G, Naldini L. 2007. In vivo administration of lentiviral vectors triggers a type I interferon response that restricts hepatocyte gene transfer and promotes vector clearance. Blood 109:2797-805. [0314] 33. Van Dis E, Sogi K M, Rae C S, Sivick K E, Surh N H, Leong M L, Kanne D B, Metchette K, Leong J J, Bruml J R, Chen V, Heydari K, Cadieux N, Evans T, McWhirter S M, Dubensky T W, Jr., Portnoy D A, Stanley S A. 2018. STING-Activating Adjuvants Elicit a Th17 Immune Response and Protect against Mycobacterium tuberculosis Infection. Cell Rep 23:1435-1447. [0315] 34. Anderson K G, Mayer-Barber K, Sung H, Beura L, James B R, Taylor J J, Qunaj L, Griffith T S, Vezys V, Barber D L, Masopust D. 2014. Intravascular staining for discrimination of vascular and tissue leukocytes. Nat Protoc 9:209-22. [0316] 35. Vroman H, Hendriks R W, Kool M. 2017. Dendritic Cell Subsets in Asthma: Impaired Tolerance or Exaggerated Inflammation? Front Immunol 8:941. [0317] 36. Gibbings S L, Thomas S M, Atif S M, McCubbrey A L, Desch A N, Danhorn T, Leach S M, Bratton D L, Henson P M, Janssen W J, Jakubzick C V. 2017. Three Unique Interstitial Macrophages in the Murine Lung at Steady State. Am J Respir Cell Mol Biol 57:66-76. [0318] 37. Kubo M. 2018. Mast cells and basophils in allergic inflammation. Curr Opin Immunol 54:74-79. [0319] 38. Lyadova I V. 2017. Neutrophils in Tuberculosis: Heterogeneity Shapes the Way? Mediators Inflamm 2017:8619307. [0320] 39. Schenkel J M, Masopust D. 2014. Tissue-resident memory T cells. Immunity 41:886-97. [0321] 40. Turner D L, Bickham K L, Thome J J, Kim C Y, D'Ovidio F, Wherry E J, Farber D L. 2014. Lung niches for the generation and maintenance of tissue-resident memory T cells. Mucosal Immunol 7:501-10. [0322] 41. D'Souza S, Rosseels V, Romano M, Tanghe A, Denis O, Jurion F, Castiglione N, Vanonckelen A, Palfliet K, Huygen K. 2003. Mapping of murine Th1 helper T-Cell epitopes of mycolyl transferases Ag85A, Ag85B, and Ag85C from Mycobacterium tuberculosis. Infect Immun 71:483-93. [0323] 42. Sayes F, Sun L, Di Luca M, Simeone R, Degaiffier N, Fiette L, Esin S, Brosch R, Bottai D, Leclerc C, Majlessi L. 2012. Strong immunogenicity and cross-reactivity of Mycobacterium tuberculosis ESX-5 type VII secretion: encoded PE-PPE proteins predicts vaccine potential. Cell Host Microbe 11:352-63. [0324] 43. Majlessi L, Brodin P, Brosch R, Rojas M J, Khun H, Huerre M, Cole S T, Leclerc C. 2005. Influence of ESAT-6 secretion system 1 (RD1) of Mycobacterium tuberculosis on the interaction between mycobacteria and the host immune system. J Immunol 174:3570-9. [0325] 44. Pym A S, Brodin P, Majlessi L, Brosch R, Demangel C, Williams A, Griffiths K E, Marchal 44. G, Leclerc C, Cole S T. 2003. Recombinant BCG exporting ESAT-6 confers enhanced protection against tuberculosis. Nat Med 9:533-9. [0326] 45. Haas C T, Roe J K, Pollara G, Mehta M, Noursadeghi M. 2016. Diagnostic omics for active tuberculosis. BMC Med 14:37. [0327] 46. Bhatt K, Verma S, Ellner J J, Salgame P. 2015. Quest for correlates of protection 46. against tuberculosis. Clin Vaccine Immunol 22:258-66. [0328] 47. Tameris M D, Hatherill M, Landry B S, Scriba T J, Snowden M A, Lockhart S, Shea J E, McClain J B, Hussey G D, Hanekom W A, Mahomed H, McShane H, Team MATS. 2013. Safety and efficacy of MVA85A, a new tuberculosis vaccine, in infants previously vaccinated with BCG: a randomised, placebo-controlled phase 2b trial. Lancet 381:1021-8. [0329] 48. Britton G, MacDonald D C, Brown J S, Collins M K, Goodman A L. 2015. Using a prime and pull approach, lentivector vaccines expressing Ag85A induce immunogenicity but fail to induce protection against Mycobacterium bovis bacillus Calmette-Guerin challenge in mice. Immunology 146:264-70. [0330] 49. Xu Y, Yang E, Wang J, Li R, Li G, Liu G, Song N, Huang Q, Kong C, Wang H. 2014. Prime-boost bacillus Calmette-Guerin vaccination with lentivirus-vectored and DNA-based vaccines expressing antigens Ag85B and Rv3425 improves protective efficacy against Mycobacterium tuberculosis in mice. Immunology 143:277-86. [0331] 50. Bottai D, Brosch R. 2009. Mycobacterial P E, PPE and ESX clusters: novel insights into the secretion of these most unusual protein families. Mol Microbiol 73:325-8. [0332] 51. Brennan M J, Delogu G. 2002. The P E multigene family: a molecular mantra for mycobacteria. Trends Microbiol 10:246-9. [0333] 52. Voskuil M I, Schnappinger D, Rutherford R, Liu Y, Schoolnik G K. 2004. Regulation of the Mycobacterium tuberculosis P E/PPE genes. Tuberculosis (Edinb) 84:256-62. [0334] 53. Bottai D, Di Luca M, Majlessi L, Frigui W, Simeone R, Sayes F, Bitter W, Brennan M J, Leclerc C, Batoni G, Campa M, Brosch R, Esin S. 2012. Disruption of the ESX-5 system of Mycobacterium tuberculosis causes loss of PPE protein secretion, reduction of cell wall integrity and strong attenuation. Mol Microbiol 83:1195-209. [0335] 54. Fishbein S, van Wyk N, Warren R M, Sampson S L. 2015. Phylogeny to function: P E/PPE protein evolution and impact on Mycobacterium tuberculosis pathogenicity. Mol Microbiol 96:901-16. [0336] 55. Gey van Pittius N C, Sampson S L, Lee H, Kim Y, van Helden P D, Warren R M. 2006. Evolution and expansion of the Mycobacterium tuberculosis P E and PPE multigene families and their association with the duplication of the ESAT-6 (esx) gene cluster regions. BMC Evol Biol 6:95. [0337] 56. Ku M W, Bourgine M, Authie P, Lopez J, Nemirov N, Moncoq F, Noirat A, Vesin B, Nevo F, Blanc C, Souque P, Simon E, Tabbal H, Mouquet H, Anna F, Martin A, Escriou, Majlessi L, Charneau P. 2020. Intranasal Immunization with a Lentiviral Vector Coding for SARS-COV-2 Spike Protein Confers Vigorous Protection in Pre-Clinical Animal Models. BioRxiv [0338] 57. Chiu C, Openshaw P J. 2015. Antiviral B cell and T cell immunity in the lungs. Nat Immunol 16:18-26. [0339] 58. Holmgren J, Czerkinsky C. 2005. Mucosal immunity and vaccines. Nat Med 11: S45-53. [0340] 59. Mueller S N, Mackay L K. 2016. Tissue-resident memory T cells: local specialists in immune defence. Nat Rev Immunol 16:79-89. [0341] 60. Park C O, Kupper T S. 2015. The emerging role of resident memory T cells in protective immunity and inflammatory disease. Nat Med 21:688-97. [0342] 61. Dong H, Stanek O, Salvador F R, Langer U, Morillon E, Ung C, Sebo P, Leclerc C, Majlessi L. 2013. Induction of protective immunity against Mycobacterium tuberculosis by delivery of ESX antigens into airway dendritic cells. Mucosal Immunol 6:522-34. [0343] 62. Bull N C, Stylianou E, Kaveh D A, Pinpathomrat N, Pasricha J, Harrington-Kandt R, Garcia-Pelayo M C, Hogarth P J, McShane H. 2019. Enhanced protection conferred by mucosal BCG vaccination associates with presence of antigen-specific lung tissue-resident PD-1 (+) KLRG1 () CD4 (+) T cells. Mucosal Immunol 12:555-564. [0344] 63. Florido M, Muflihah H, Lin LCW, Xia Y, Sierro F, Palendira M, Feng C G, Bertolino P, Stambas J, Triccas J A, Britton W J. 2018. Pulmonary immunization with a recombinant influenza A virus vaccine induces lung-resident CD4 (+) memory T cells that are associated with protection against tuberculosis. Mucosal Immunol 11:1743-1752. [0345] 64. Perdomo C, Zedler U, Kuhl A A, Lozza L, Saikali P, Sander L E, Vogelzang A, Kaufmann S H, Kupz A. 2016. Mucosal BCG Vaccination Induces Protective Lung-Resident Memory T Cell Populations against Tuberculosis. mBio 7. [0346] 65. Sakai S, Kauffman K D, Schenkel J M, McBerry C C, Mayer-Barber K D, Masopust D, Barber D L. 2014. Cutting edge: control of Mycobacterium tuberculosis infection by a subset of lung parenchyma-homing CD4 T cells. J Immunol 192:2965-9. [0347] 66. Desel C, Dorhoi A, Bandermann S, Grode L, Eisele B, Kaufmann S H. 2011. Recombinant BCG DeltaureC hly+ induces superior protection over parental BCG by stimulating a balanced combination of type 1 and type 17 cytokine responses. J Infect Dis 204:1573-84. [0348] 67. Shen H, Chen Z W. 2018. The crucial roles of Th17-related cytokines/signal pathways in M. tuberculosis infection. Cell Mol Immunol 15:216-225. [0349] 68. Raeven RHM, Rockx-Brouwer D, Kanojia G, van der Maas L, Bindels THE, Ten Have R, van Riet E, Metz B, Kersten GFA. 2020. Intranasal immunization with outer membrane vesicle pertussis vaccine confers broad protection through mucosal IgA and Th17 responses. Sci Rep 10:7396. [0350] 69. Kim T S, Gorski S A, Hahn S, Murphy K M, Braciale T J. 2014. Distinct dendritic cell subsets dictate the fate decision between effector and memory CD8 (+) T cell differentiation by a CD24-dependent mechanism. Immunity 40:400-13. [0351] 70. Ng S L, Teo Y J, Setiagani Y A, Karjalainen K, Ruedl C. 2018. Type 1 Conventional CD103 (+) Dendritic Cells Control Effector CD8 (+) T Cell Migration, Survival, and Memory Responses During Influenza Infection. Front Immunol 9:3043. [0352] 71. Zelante T, Wong A Y, Ping T J, Chen J, Sumatoh H R, Vigano E, Hong Bing Y, Lee B, Zolezzi F, Fric J, Newell E W, Mortellaro A, Poidinger M, Puccetti P, Ricciardi-Castagnoli P. 2015. CD103 (+) Dendritic Cells Control Th17 Cell Function in the Lung. Cell Rep 12:1789-801. [0353] 72. Kaufmann E, Sanz J, Dunn J L, Khan N, Mendonca L E, Pacis A, Tzelepis F, Pernet E, Dumaine A, Grenier J C, Mailhot-Leonard F, Ahmed E, Belle J, Besla R, Mazer B, King I L, Nijnik A, Robbins C S, Barreiro L B, Divangahi M. 2018. BCG Educates Hematopoietic Stem Cells to Generate Protective Innate Immunity against Tuberculosis. Cell 172:176-190 e19. [0354] 73. Kaufmann S H. 2010. Future vaccination strategies against tuberculosis: thinking outside the box. Immunity 33:567-77. [0355] 74. Jones G W, Hill D G, Jones S A. 2016. Understanding Immune Cells in Tertiary Lymphoid Organ Development: It Is All Starting to Come Together. Front Immunol 7:401. [0356] 75. Allix-Beguec C, Harmsen D, Weniger T, Supply P, Niemann S. 2008. Evaluation and strategy for use of MIRU-VNTRplus, a multifunctional database for online analysis of genotyping data and phylogenetic identification of Mycobacterium tuberculosis complex isolates. J Clin Microbiol 46:2692-9. [0357] 76. Caton M L, Smith-Raska M R, Reizis B. 2007. Notch-RBP-J signaling controls the homeostasis of CD8-dendritic cells in the spleen. J Exp Med 204:1653-64. [0358] 77. Le Bon A, Thompson C, Kamphuis E, Durand V, Rossmann C, Kalinke U, Tough D F. 2006. Cutting edge: enhancement of antibody responses through direct stimulation of B and T cells by type | IFN. J Immunol 176:2074-8.