LENTIVIRAL VECTORS ENABLING ROUTING ANTIGENS TO MHC-II PATHWAY AND INDUCING CD4+ AND CD8+ T-CELL RESPONSES IN A HOST

Abstract

The invention relates to a recombinant lentiviral vector genome comprising a polynucleotide encoding a fusion polypeptide, wherein said fusion protein comprises arranged from N-terminal to C-terminal ends: (i) a first polypeptide comprising a multimerization scaffold which comprises at least one collectin or a fragment thereof suitable to enable self-assembly of multimers of the first polypeptide, fused with at least one antigenic polypeptide; (ii) a second polypeptide comprising a CD40L ectodomain or a receptor binding fragment thereof, in particular the CD40L ectodomain of the human CD40L. 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 recombinant polypeptide and a second polypeptide, wherein: (i) said first recombinant polypeptide comprises a multimerization scaffold which comprises at least one collectin or a fragment thereof suitable to enable self-assembly of multimers of the first polypeptide, fused with at least one antigenic polypeptide; (ii) said second polypeptide comprises a CD40L ectodomain or a receptor binding fragment thereof, in particular the CD40L ectodomain of the human CD40L, preferably the CD40L ectodomain of SEQ ID No. 19.

2. The recombinant lentiviral vector genome according to claim 1, wherein said collectin or fragment thereof comprises arranged from N-terminal to C-terminal ends: at least one crosslinking region of a collectin; at least one collagen-like region of a collectin; and at least one neck region of a collectin.

3. The recombinant lentiviral vector genome according to claim 2, wherein the polynucleotide encoding the said at least one antigenic polypeptide is fused in-frame within the nucleotide sequence of the collagen-like region of said collectin.

4. The recombinant lentiviral vector genome according to any one of claims 1 to 3, wherein said collectin or fragment thereof is a carbohydrate recognition domain (CRD)-truncated form of collectin, wherein said at least one antigenic polypeptide is inserted within the collagen-like region of the collectin.

5. The recombinant lentiviral vector genome according to any one of claims 1 to 4, wherein said fusion polypeptide further comprises a polypeptide comprising a chemokine, in particular selected from CCL20, a pro-inflammatory Th1-related chemokine such as CXCL9, CXCL10, CCL3, CCL4 and/or CCL5, and/or a Th17-promoting chemokine such as CXCL20, or a receptor binding domain thereof, preferably wherein said chemokine is the CCL20 domain of SEQ ID No. 20, in particular wherein said chemokine or fragment thereof is inserted within the collagen-like domain of said collectin.

6. The recombinant lentiviral vector genome according to any one of claims 1 to 5, wherein said collectin is selected from mannan-binding lectin (MBL), surfactant protein D (SP-D), surfactant protein A (SP-A), collectin liver 1 (CL-L1), collectin placenta 1 (CL-P1), conglutinin collectin of 43 kDa (CL-43), collectin of 46 kDa (CL-46),and collectin kidney 1 (CL-K1), preferably wherein said collectin is a human collectin selected from MBL (SEQ ID No. 17), SP-D (SEQ ID No. 18), CL-L1 (SEQ ID No. 34), SP-A1 (SEQ ID No. 35), SP-A2 (SEQ ID No. 36), CL-P1 (SEQ ID No. 37) and CL-K1 (SEQ ID no. 38).

7. The recombinant lentiviral vector genome according to any one of claims 1 to 6, wherein said antigenic polypeptide is a mono-antigenic polypeptide comprising one antigen or immunogenic fragment thereof, or is a poly-antigenic polypeptide comprising at least two antigens or immunogenic fragments thereof.

8. The recombinant lentiviral vector genome according to any one of claims 1 to 7, wherein the at least one antigen or immunogenic fragment thereof is selected from a bacterial, parasite or viral pathogen, in particular from Mycobacterium tuberculosis, an influenza virus or a coronavirus such as SARS-CoV-2 or is a tumoral antigen or immunogenic fragment thereof.

9. The recombinant lentiviral vector genome according to any one of claims 1 to 8, wherein said antigenic polypeptide comprises one or more Mycobacterium tuberculosis (Mtb) antigens selected from EsxA, EspC, EsxH, PE19, Hypoxic response protein 1 (Hrp1) and Resuscitation promoting factor D (RpfD), 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, PE19 and EspC; (e) EsxA, PE19, EspC, HRp1 and RpfD; or an immunogenic fragment thereof.

10. The recombinant lentiviral vector genome, wherein said genome is obtained from the pTRIP vector plasmid of nucleotide sequence SEQ ID No. 21, 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 composite BCUAG promoter of SEQ ID No. 22 and wherein the vector optionally comprises post-transcriptional regulatory element of the woodchuck hepatitis virus (WPRE).

11. A DNA plasmid comprising the recombinant vector genome according to any one of claims 1 to 10, in particular wherein said genome is inserted within the pTRIP vector plasmid of nucleotide sequence SEQ ID No.21 or within the pFlap-SP1beta2m-GFP-WPREm deposited at the CNCM (Paris, France) on Feb. 16, 2021 under number CNCM I-5657 or variants thereof.

12. A recombinant lentiviral vector particle which comprises the recombinant lentiviral vector genome according to any one of claims 1 to 10.

13. The recombinant lentiviral vector particle according to claim 12 which is a recombinant integration-deficient lentiviral vector particle, in particular the recombinant integration-deficient lentiviral vector is a HIV-1 based vector 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.

14. The recombinant lentiviral vector particle according to any one of claim 12 or 13, wherein said recombinant lentiviral vector genome is the genome of a replication-incompetent pseudotyped lentiviral vector, in particular a replication-incompetent pseudotyped HIV-1 lentiviral vector, in particular wherein the vector is pseudotyped with the glycoprotein G from a Vesicular Stomatitis Virus (V-SVG) of Indiana or of New-Jersey serotype.

15. A host cell, preferably a mammalian host cell, transfected with a DNA plasmid according to claim 11, in particular wherein said host cell is a HEK-293T cell line or a K562 cell line.

16. A pharmaceutical composition, in particular a vaccine composition, suitable for administration to a mammalian host, comprising a recombinant lentiviral vector particle of any one of claims 12 to 14 together with one or more pharmaceutically acceptable excipient(s) suitable for administration to a host in need thereof, in particular a human host.

17. The pharmaceutical composition of claim 16, 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, and/or cellular and/or humoral response in a host in need thereof, in particular a human host.

18. The pharmaceutical composition of claim 16 or 17, wherein the immune response involves the induction of 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-mediated cellular immune response.

19. The pharmaceutical composition of any one of claims 16 to 18, for preventing and/or treating an infection by a pathogen in a mammalian host in need thereof, in particular a human host.

20. A method for the preparation of recombinant lentiviral vector particles suitable for the preparation of a pharmaceutical composition, in particular a vaccine, comprising the following steps: a) transfecting the recombinant lentiviral transfer vector carrying the lentiviral vector genome according to any one of claims 1 to 10, or the DNA plasmid according to claim 11 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 antigenic polypeptide, or an immunogenic fragment thereof; d) recovering the recombinant lentiviral particles expressing the antigenic polypeptide, or an immunogenic fragment thereof.

Description

LEGENDS OF THE FIGURES

[0233] FIG. 1. Schematic structure of MBL or SPD collectin polymers. (A) Structural domains of MBL or SPD. CRD=Carbohydrate-Recognition Domain. (B) MBL or SPD self-assembled, collagen-like triple helixes, formed by interchain cysteine bonds. (C) SPD cross-shaped dodecamer. (D) SPD or MBL “tulip-bouquet” octodecamer. Adapted from.sup.34. (E-F) Schematic primary structure of the designed M40 (E) or S40 (F) monomers, harboring selected Mtb antigens. Crosslinking region (S), Collagen-like region (Coll), Neck region (N).

[0234] FIG. 2. Properties of LV::M40 at inducing antigenic presentation by both MHC-I and -II pathways. (A) BM-DC from BALB/c (H-2.sup.d) or C57BL/6 (H-2.sup.b) mice were transduced (MOI=20) with LV::M40-H, -HA, -HAP or -HAPE, under the transcriptional control of BCUAG promoter. Control cells were transduced with LV::EsxH alone. (B) BM-DC from BALB/c or C57BL/6 mice were incubated with successive dilutions of supernatants of HEK-293T cells, transduced (MOI=20) for 48 h by each of the indicated LV. (C) At day 3 after the addition of LV, or at day 1 after incubation with the HEK-293T cell supernatants or peptides, presentation of MHC-I- or -II-restricted epitopes of the EsxH, EsxA, PE19 or EspC mycobacterial antigens by DC were assessed by their co-culture with T-cell hybridomas specific to EsxH:20-28 (YB8 cell line, restricted by K.sup.d), EsxH:74-88 (1G1 cell line, restricted by I-A.sup.d), EsxA:1-20 (NB11 cell line, restricted by I-A.sup.b), PE:19:1-18 (IF6 cell line, restricted by I-A.sup.b), or EspC:45:54 (IF1 cell line, restricted by I-A.sup.b). (D) BM-DC from BALB/c or C57BL/6 mice were incubated with successive dilutions of supernatants of HEK-293T cells, transduced (MOI=20) for 48 h by each of the indicated LV. Results are concentrations of IL-2 produced by T-cell hybridomas 24 h after the beginning of the co-cultures. The amounts of IL-2 produced in the co-culture supernatants are proportional to the efficacy of antigenic presentation by DC and TCR triggering.

[0235] FIG. 3. Phenotypic Maturation of DC Induced by M40 or S40. (A-B) Phenotypic maturation of BM-DC from C57BL/6 mice infected at MOI of 5 with Mtb, as positive control, or incubated with supernatants from HEK-293T cells transduced (MOI=20) with LV::EsxH alone (H=Ctrl), LV::M40-H or LV::S40-H. Expression of co-stimulatory or MHC molecules were assessed by cytometry on CD11b.sup.+ CD11c.sup.+ cells at 24 h p.i. (B) Heatmaps recapitulating the Mean Fluorescence Intensity (MFI) of CD40 or CD80 surface expression or percentages of CD86.sup.hi, MHC-I.sup.hi, or MHC-II.sup.hi DCs.

[0236] FIG. 4. T-cell immunogenicity of LV encoding for M40-H. (A) IFN-γ- or TNF-α-producing CD8.sup.+ (top) or CD4.sup.+ (bottom) T-cell responses, as assessed by ELISPOT at day 13 post-immunization, in the spleen of individual BALB/c mice (n=3), immunized s.c. with 1×10.sup.8 TU/mouse of LV::M40-H, harboring β2m, CMV or BCUAG promoters. Frequencies of Spot Forming Cells (SFC) were determined subsequent to in vitro stimulation of splenocytes with EsxH:20-28 (top) or EsxH:74-88 (bottom) synthetic peptide. Shown are Median with two tailed values and 95% confidence. Quantitative differences between various groups were not statistically significant (non-parametric Mann & Whitney test, p<0.05). (B) Cytometric gating strategy of CD4.sup.+ or CD8.sup.+ T splenocytes and Representative IFN-γ.sup.+ or IFN-γ.sup.− CD8.sup.+ or CD4.sup.+ T cells, expressing TNF-α and/or IL-2. (C) Recapitulative percentages of each functional subsets within the CD8.sup.+ or CD4.sup.+ T-cell population from the mice immunized with LV::M40-H harboring β2m, CMV or BCUAG promoters. Shown are Mean +/−SD Quantitative differences between various groups were not statistically significant (non-parametric Mann & Whitney test, p<0.05).

[0237] FIG. 5. Immunogenicity of the poly-antigenic LV::M40-HAPE. (A) IFN-γ T-cell responses, as assessed by ELISPOT at day 14 post-immunization, in the spleen of individual C57BL/6 mice (n=3), immunized s.c. with 1×10.sup.8 TU/mouse of LV::M40-HAPE harboring β2m, CMV or BCUAG promoters. The frequencies of responding T cells were determined subsequent to in vitro stimulation with EsxH:3-11 (containing MHC-I-restricted epitope) or EsxA:1-20 (containing MHC-II-restricted epitope), PE10:-1-18 (containing MHC-II-restricted epitope), or EspC:45-54 (containing MHC-I and II-restricted epitopes) synthetic peptide. Shown are Median with two tailed values and 95% confidence. Quantitative differences among the groups of mice immunized with LV::M40-HAPE, harboring β2m, CMV or BCUAG promoter, were not statistically significant (non-parametric Mann & Whitney, p<0.05). (B) Representative gating strategy (C) and dot plots of TNF-α.sup.+ vs IFN-γ.sup.+ or IL-2.sup.+ vs IFN-γ.sup.+ inside the CD8.sup.+ T splenocyte subset, subsequent to stimulation with a negative control, EsxH:3-11, or EspC:45-54 peptide. (D-E) Heatmap CD8.sup.+ (D) recapitulating percentages of each CD4.sup.+ (E) or functional subsets specific to EsxH or EspC antigens in mice immunized with LV::M40-HAPE, harboring β2m, CMV or BCUAG promoters or injected with PBS. Quantitative differences among the groups of mice immunized with LV::M40-HAPE, harboring distinct promoter were not statistically significant (non-parametric Mann & Whitney, p<0.05). The immunized C57BL/6 mice were those detailed in the FIG. 7.

[0238] FIG. 6. Immunogenicity of the Multi-Antigenic LV::S40-HAPEHR or LV::S40-HAPEHR-20. (A) IFN-γ T-cell responses, as assessed by ELISPOT at day 13 post-immunization, in the spleen of individual C57BL/6 mice (n=3), immunized s.c. with 1×10.sup.8 TU/mouse of LV::S40-HAPEHR or LV::S40-HAPEHR-20. The frequencies of responding T cells were determined following in vitro stimulation with the indicated synthetic peptides. Shown are Median with two tailed values and 95% confidence. Quantitative differences among the groups of mice immunized with LV::S40-HAPEHR or LV::S40-HAPEHR-20, were not statistically significant (non-parametric Mann & Whitney, p<0.05). (B) Epitope mapping of Hrp-1 and RfpD as determined by the pooled splenocytes from C57BL/6 mice, injected with PBS or immunized s.c. with 1×10.sup.8 TU/mouse of LV::S40-HAPEHR, subsequent to stimulation with each of the individual peptides from the Hrp-1- or RfpD-derived overlapping 15-mers offset by 5 a.a. (C) Cytometric analysis of intracellular IFN-γ versus IL-2 staining of CD4+ T splenocytes after stimulation with 10 μg/ml of the indicated peptides encompassing the immunodominant epitopes, identified in (B).

[0239] FIG. 7. Features of mucosal CD4.sup.+ T cells triggered by i.n. immunization with LV::S40-HAPEHR or LV::S40-HAPEHR-20. C57BL/6 mice were immunized i.n. with 1×10.sup.8 TU of LV::S40-HAPEHR or LV::S40-HAPEHR-20. At 14 dpi, lung CD4.sup.+ T cells were distinguished for their location within the interstitium (CD45.sub.i.v-) or in the vasculature (CD45.sub.i.v.sup.+) by an i.v. PE-anti-CD45 mAb injection. (A) Profile of CD27 versus CD62L or CD45RB, and (B) CD103 vs CD69, or CD44 vs CXCR3 of the lung CD4.sup.+ T cells of the interstitium or vasculature. (C) Heatmap recapitulating percentages of (poly)functional CD4.sup.+ T cells specific to EsxA, PE19 or EspC in the lung interstitium or vasculature, as determined by ICS. Results, representative of 2 independent experiments, are from pooled lungs per group to reach enough cells for accurate cytometric analyses.

[0240] FIG. 8. Features of mucosal CD8.sup.+ T cells induced by i.n. immunization with LV::S40-HAPEHR or LV::S40-HAPEHR-20. The immunized C57BL/6 mice are those studied in the FIG. 7. (A) Shown are lung CD8.sup.+ T cells, distinguished for their location within the interstitium (CD45.sub.i.v.sup.−) or in the vasculature (CD45.sub.i.v.sup.+). Profile of CD27 versus CD62L or CD45RB, and (B) CD103 vs CD69, or CD44 vs CXCR3 of the lung CD8.sup.+ T cells from the interstitium or vasculature. (C) Heatmap recapitulating percentages of (poly)functional CD8.sup.+ T cells specific to EsxH or EspC in the lung interstitium or vasculature, as determined by ICS. Results, representative of 2 independent experiments, are from pooled lungs per group to reach enough cells for accurate cytometric analyses.

[0241] FIG. 9. Protective potential of an optimized poly-antigenic LV as a booster against Mtb. (A) Time line of prime-boost-challenge performed in C57BL/6 mice (n=5-9/group). (B) Mtb burdens as quantitated by CFU counting in the lungs and spleen of mice at week 5 post challenge. NS=not significant, *, **, ***=statistically significant, as determined by One Tail Mann Whitney test, p=0.0415, p=0.0040, p=0.00105, respectively.

[0242] FIG. 10. Sensitivity of the T-cell hybridomas used in the Mtb antigen presentation assays. BM-DC from BALB/c (H-2.sup.d) or C57BL/6 (H-2.sup.b) mice were incubated with various concentrations of homologous or negative control peptides. At day 1, presentation of MHC-I- or -II-restricted epitopes were assessed by use of T-cell hybridomas specific to EsxH:20-28 (YB8, restricted by K.sup.d), EsxH:74-88 (1G1, restricted by I-A.sup.d), EsxA:1-20 (NB11, restricted by I-A.sup.b), PE:19:1-18 (IF6, restricted by I-A.sup.b), or EspC:45:54 (IF1, restricted by I-A.sup.b). Results are concentrations of IL-2 produced by T-cell hybridomas 24 h after the T-cell addition.

[0243] FIG. 11 (A) Schematic description of the three used promoters. β2m promoter (RefSeq: LRG-1215; from 4556 to 5070), hCMV promoter (RefSeq: MN920393.1; from 174188 to 174714), BCUAG is a combination of β2m and hCMV promoters with the addition of “Inf” (Inflammation-related) cluster, a set of cis-regulating motifs associated with inflammation. (B) Sequence of Inf Cluster. Cis regulating motifs and associated transcriptional factor are indicated under the sequence.

[0244] FIG. 12. Map of the segment of the pFLAP plasmid containing M40 or S40 harboring the selected mycobacterial antigens, under β2m, CMV or BCUAG promoter. The codon-optimized cDNA sequences, encoding for EsxH variants or poly-antigenic fusion proteins of vaccine interest, were inserted under the SP1-β2m promoter.sup.36 in a pFLAP backbone plasmid.

[0245] FIG. 13. Structure of a human SPD-40 polypeptide harboring the antigen EsxH. P1-106: fragment of Pulmonary surfactant-associated protein D (UniProtKB—P35247 p1-106), ensures secretion via signal peptide (p1-20), enables antigen access to APC surrounding transduced cells and 2 levels of multimerization: collagen-like domain (p46-106), generates a trimerization with hydrogen bounds and cystein-rich region (p21-45), generates a covalent multimerization (n>3) via disulfide bounds. The scaffold is expected to increased bioavailability and CD40L functionality mimicking its natural trimerization. P208-273: fragment from Mannose-binding protein C (UniProtKB—P11226 p65-130). Multimerization via collagen-like domain (p208-243) and coiled-coil region (p244-273). Coiled-coil region is also a rigid spacer between transgene and CD40L ectodomain preventing deleterious interactions. P277-422: fragment of CD40L ectodomain (UniProtKB—P29965 p116-261). Ensures APC targeting and maturation through interaction with its native receptor CD40. Peptide sequence starts after EMQK motif preventing the natural methionine cleavage that occurs in wild type CD40L full-length.

EXAMPLES

Introduction

[0246] Lentiviral Vectors (LV) provide one of the most efficient vaccine platforms, relied on their outstanding potential of gene transfer to the nuclei of the host cells, including notably Antigen Presenting Cells (APC). Such nuclear transfer of genes initiates expression of antigens which readily access the Major Histocompatibility Complex Class-I (MHC-I) presentation machinery, i.e., proteasome, for further triggering of CD8.sup.+ T cells.sup.1-3. In net contrast with their substantial ability at routing the endogenously produced antigens into the MHC-I pathway, viral vectors, including LV, are barely effective or inoperative in delivery of non-secreted antigens to the endosomal MHC-II compartment (MIIC) and unable to trigger CD4.sup.+ T cells. Although CD8.sup.+ T cells contribute largely to the immune control of infectious diseases or tumor growth, CD4.sup.+ T cells are the major immune players. In addition to their long lifespan and their own direct effector functions, CD4.sup.+ T cells orchestrate the immune system by regulating innate immunity, tailoring B-cell responses and supporting CD8.sup.+ T-cell effector functions.sup.4. Therefore, leveraging the potential of LV to induce CD4.sup.+ T cells will maximize their success rate in vaccine strategies.

[0247] Implication of CD4.sup.+ T cells is notably of utmost importance in the immune protection against the leading cause of death from a single infectious agent, Mycobacterium tuberculosis (Mtb), the etiologic agent of human pulmonary tuberculosis (TB).sup.5. During the chronic infection, this intracellular bacillus is lodged inside the phagosomes of infected phagocytes, which results in the presentation of its antigens essentially by MHC-II molecules. Consequently, it is via MHC-II that the adaptive immune effector cells can recognize the infected cells to eradicate them or to strengthen their intracellular microbicidal arsenal.sup.6,7. In order to develop a poly-antigenic and multistage anti-TB subunit vaccine, we engineered a new generation of LV able to induce MHC-II antigen presentation, resulting in CD4.sup.+ T-cell initiation. In our rational design, we also took into the account the fact that direct delivery of antigens to APC, by their addressing to appropriate co-stimulatory cell surface receptors, substantially increases the immunogenicity by lowering the threshold of required antigen amounts and by providing a slight and local adjuvant effect.sup.8,9.

[0248] We generated a platform of LV encoding for secreted monomers of collagen-containing C-type lectins (collectins), i.e., Mannan-Binding Lectin (MBL) or Surfactant-associated Protein D (SPD).sup.10, as scaffold protein carriers. The latter are engineered to harbor multiple Mtb immunogens and the ectodomain of the Tumor Necrosis Factor (TNF) family member CD40 ligand (CD40L, CD154), with the perspective of targeting and activating APC via the co-stimulatory receptor CD40.sup.11. Transduction of host cells with such LV results in secretion of antigen-bearing MBL-CD40L (“M40”) or SPD-CD40L (“S40”) monomers. Such monomers can spontaneously self-assemble into helicoidal trimers, as the first structural units. This leads potentially to a CD40L homo-trimeric configuration, required to cluster CD40. In their turn, the trimers can further tetra- or hexamerize to form soluble macromolecule carriers, able to circulate in biological fluids or be locally taken up by bystander APC. Therefore, such macromolecule carriers can be more efficiently delivered to CD40.sup.+ APC and notably dendritic cells (DC), in which they gain access to MIIC. Moreover, the C-ter CD40L trimeric motifs will mimic the trivalent membrane CD40L on CD4.sup.+ T cells to activate DC, similar to an adjuvant. Our results provided proof-of-concept evidences that, in contrast to conventional LV, this new generation of LV encoding for M40 or S40 carriers which bear multistage Mtb immunogens: (i) induces MHC-II-restricted antigen presentation, (ii) triggers CD4.sup.+—and CD8.sup.+—T cells when used in systemic or intranasal (i.n.) immunization, and (iii) displays a significant booster protective effect in the TB mouse model. This innovating approach can be largely extended to LV-based vaccine candidates against numerous other bacterial or viral infectious diseases or cancers, with the critical advantage of inducing robust CD4.sup.+ T-cell responses, a rare property for a viral vector vaccine.

Results

Rational Selection of Antigens

[0249] To generate an LV-based vaccine vector encoding for M40 or S40 antigen carriers and potentially usable in an infectious disease controllable by CD4.sup.+ T cells, we selected EsxA, EspC (ESX-1 secretion-associated protein C), EsxH, PE19, Hypoxic response protein 1 (Hrp1), and Resuscitation promoting factor D (RpfD) immunogens from Mtb (Table 1). EsxA and EspC virulence factors are strongly immunogenic, due to their small size and their active secretion through the ESX-1 Type VII Secretion System (T7SS), which favor their access to MHC presentation machineries of the host phagocytes.sup.12,13. The highly immunogenic EsxH.sup.14-16, secreted through the ESX-3 T7SS, has shown protective potential in several subunit vaccine candidates. EsxH, and its close relative EsxR, are present in all the Mtb clinical isolates so far studied.sup.17. Inclusion of PE19 from the large family of PE/PPE Mtb proteins, secreted through ESX-5 T7SS.sup.13, 18-23, was based on its contents in T-cell epitopes, shared by numerous homologous members of the large PE multigenic family, with various expression profiles at the distinct stages of infection, which may generate a constant display of such shared T-cell epitopes during the course of infection.sup.20, 23-25.

[0250] As Mtb evolves from acute to persistence phase, its adaptation to starvation, hypoxia, nitrogen stress or host immune pressure, is regulated by the dosRS two-component regulatory system.sup.26, which initiates transcription of 48 genes, including rv2626c, among the most strongly induced.sup.27. The resulted Hrp1 can be target of the host adaptive immunity when the bacilli are quiescent within the granuloma.sup.28. Immunity to Hrp1 may dampen latent TB reactivation. In addition, Mtb possesses five Resuscitation promoting factors (RpfA-E). Rpf are cell-wall associated or secreted enzymes with peptidoglycan hydrolase, trans-glycosylase and lytic activities. These enzymes contribute to biosynthesis of muropeptides, involved in cell wall remodeling during the bacterial division, at both acute or reactivation phases.sup.29-31. We selected RpfD because of its demonstrated immunogenicity in both mice.sup.32 and latent TB humans.sup.33. Both Hrp-1 and Rpf lack human homologs.

Design of an LV Encoding for Collectin Scaffolds Harboring Mtb Antigens and a DC Targeting Segment

[0251] Collectins are soluble Pattern Recognition Receptors (PRRs), able to bind oligosaccharides or lipids at the surface of microorganisms and thereby contributing to their elimination by opsonization or complement activation.sup.34. Among collectins, MBL and SPD are composed of four distinct segments: (i) N-terminal cysteine-rich crosslinking domain, (ii) collagen-like domain, (iii) α-helical neck domain, and (iv) Carbohydrate-Recognition Domain (CRD) (FIG. 1A). A self-assembled collagen-like triple helix forms the first structural MBL or SPD unit (FIG. 1 B). SPD triple-subunits themselves can tetramerize to form cross-shaped dodecamer (FIG. 1C). SPD or MBL triple-subunit can also hexamerize to form “tulip-like nano-bouquet” octodecamer (FIG. 1D). The resulted secreted polymers are soluble.sup.34. We first engineered the murine MBL to harbor complete sequences of: (i) EsxH alone, (ii) EsxH and EsxA, (iii) EsxH, EsxA, and PE19, or (iv) EsxH, EsxA, PE19 and EspC within the collagen-like region, while replacing its CRD by the murine CD40L.sub.115-260 ectodomain. The prospective MBL polymers will be referred to as “M40-H”, “M40-HA”, “M40-HAP”, or “M40-HAPE”, respectively (FIG. 1E, Table S1).

[0252] In parallel, we engineered SPD to harbor: (i) EsxH alone, (ii) EsxH, EsxA, PE19, EspC, or (iii) EsxH, EsxA, PE19, EspC, Hrp1 and RpfD.sub.42-154 within the collagen-like region and we substituted its CRD by CD40L.sub.115-260 (FIG. 1F, Table S1). The expected SPD polymers are referred to as “S40-H”, “S40-HAPE”, or “S40-HAPEHR”, respectively. To confer some chemo-attracting properties to the resultant fusion protein, we also designed an S40-HAPEHR, which harbors the murine CCL20.sub.28-97 segment within the collagen-like domain (“S40-HAPEHR-20”). CCL20 is the CCR6 ligand, largely involved in the migration and recruitment of DC and lymphocytes.sup.35.

Induction of MHC-II-Restricted Antigen Presentation by LV::M40

[0253] DCs (H-2.sup.d or H-2.sup.b) were directly transduced with LV::M40-H, -HA, -HAP or -HAPE, using the BCUAG promoter. Control DC were transduced with a conventional LV encoding for EsxH without being inserted into an engineered scaffold. Three days post-transduction, the DCs were co-cultured with T-cell hybridomas, specific to the immunodominant epitopes of each of the Mtb antigens. The DCs transduced with either LV were largely able to induce presentation of EsxH via MHC-I (FIG. 2A). In contrast to the conventional LV::EsxH, the LV encoding for M40-H, -HA, -HAP or -HAPE induced the presentation of EsxH, EsxA and PE19, when these antigens were included. No MHC-II presentation of EspC was detected in this context, which can be explained by the relatively weak sensitivity of the anti-EspC T-cell hybridoma (FIG. 10). To evaluate whether M40 or S40 carriers secreted by transduced cells induce presentation through MHC-II, DC were incubated with successive dilutions of M40-H-, -HA-, -HAP-, or -HAPE-containing supernatants from transduced HEK-293T cells (FIG. 2B). At day 1 after incubation, co-culture of the DCs with T-cell hybridomas, showed that these DCs were unable to present EsxH via MHC-I, strongly suggesting that endocytosis/micropinocytosis or CD40-mediated cell entry of the M40 carrier does not allow their access to MHC-I machinery, in contrast to observations made by others.sup.38. In net contrast, DCs incubated with M40-H-, -HA-, -HAP-, or -HAPE-containing supernatants were very efficient at inducing presentation of the respective antigens via MHC-II, including EspC. It was noticed that the intensity of antigen presentation had a propensity to decrease with growing number of antigens carried by the M40 scaffold (FIG. 2A, B). In a mutually non-exclusive manner, this may result from: (i) a slight structural instability of the carriers with the insertion of increasing number of antigens, (ii) a competition among the multiple T-cell epitopes for the available MHC presentation sites.

[0254] Direct transduction of DCs with LV::S40-HAPE, -HAPEHR or -HAPEHR-20 induced also efficacious MHC-I- or -II-restricted presentation of the selected Mtb antigens (FIG. 2C). Incubation of DC with successive dilutions of supernatants from HEK-293T cells transduced with LV::S40-HAPE, -HAPEHR or -HAPEHR-20 induced MHC-II-restricted presentation of the Mtb antigens (FIG. 2D). In the absence of identified T-cell epitope or T-cell hybridoma specific to Hrp1 or RpfD, the immunogenicity of these antigens in the context of the developed vectors was studied in vivo, as detailed below. Notably, the addition of Hrp1 and RpfD and CCL20 to the S40 scaffold did not impact the efficacy of the presentation of the other antigens. The assay performed with DC incubated with synthetic peptides harboring the homologous T-cell epitopes showed the sensitivity of the T-cell hybridoma-based presentation assay (FIG. 10).

[0255] These results showed that, in net opposition to conventional LV, this new generation of LV encoding for secreted scaffolds which can incorporate numerous antigens and immune mediators, possess a strong capacity at inducing MHC-II-restricted antigen presentation and thus provide a valuable platform for both CD4.sup.+ and CD8.sup.+ T cell induction.

M40 and S40 Potential at Inducing DC Maturation

[0256] To evaluate the potential of M40 and S40 carriers to induce DC maturation, BM-DCs were incubated with supernatants from HEK-293T cells transduced with LV::M40-H or LV::S40-H. In parallel, DCs were incubated with supernatants from HEK-293T cells transduced with the conventional LV::H, as a negative control or were infected with Mtb, as a positive control. The expression of surface co-stimulatory and MHC molecules was assessed on CD11b.sup.+ CD11c.sup.+ cells at day 1 post incubation (FIG. 3A, B). On DCs incubated with M40-H or S40-H, no increase of CD40 surface expression was detected, probably as a consequence of direct interaction of CD40 with M40-H or S40-H (FIG. 3A, B). CD80 upregulation was only detected on DCs incubated with S40-H, while CD86 upregulation and increase in the percentages of MHC-I.sup.hi or MHC-II.sup.hi cells was detected on DCs incubated with M40-H or S40-H. Therefore, through induction of M40 or S40 secretion, this new generation of LV is able to induce DC maturation, instrumental for appropriate T-cell activation.

T-Cell Immunogenicity of LV Encoding for M40 or S40 Carrying a Single or Multiple Mtb Immunogens

[0257] To assess the immunogenicity of this new generation of LV, BALB/c mice (n=3/group) were immunized s.c. with LV::M40-H harboring human β2-microglobulin (β2m) promoter.sup.36, human CytoMegaloVirus (CMV) immediate early enhancer and promoter (CMV).sup.37, or a composite β2m-CMV promoter (“BCUAG”) to get insights on possible consequences of distinct antigen carrier transcription profile on the induction of immune responses (FIG. 11). At day 13 post injection (dpi), stimulation of the splenocytes with EsxH:20-28 (MHC-I) or EsxH:74-88 (MHC-II) peptides.sup.15,16 detected both CD8.sup.+ T and CD4.sup.+ T cells by ELISPOT (FIG. 4A). Intracellular Cytokine Staining (ICS) showed the multifunctional properties of these CD8.sup.+ or CD4.sup.+ (FIG. 4B, C) T cells. Functional CD8.sup.+ T cells effectors were mainly distributed among IFN-γ.sup.+ (single positive), IFN-γ.sup.+ TNF-α.sup.+ (double positive), or IFN-γ.sup.+ TNF-α.sup.+ IL-2.sup.+ (triple positive) subsets, while CD4.sup.+ T cells were essentially IFN-γ.sup.+ (single positive), or IFN-γ.sup.+ TNF-α.sup.+ IL-2.sup.+ (triple positive) (FIG. 4C). It is noteworthy that conventional LV encoding EsxH as single do not induce such CD4.sup.+ T-cell responses.sup.39.

[0258] To evaluate the immunogenic potential of the developed poly-antigenic LV::M40-HAPE, C57BL/6 mice (n=3/group) were immunized s.c. with LV::M40-HAPE harboring β2m, CMV or BCUAG promoter. At 14 dpi, CD8.sup.+ and CD4.sup.+ T splenocyte responses, specific to EsxH:3-11 (MHC-I), EsxA:1-20 (MHC-II), PE19:1-18 (MHC-II), or EspC:45-54 (MHC-I and -II).sup.39, were detected in all mice, as assessed by ELISPOT (FIG. 5A). ICS analysis of the splenocytes from the same mice showed the multifunctional properties of the induced CD8.sup.+ (FIG. 5B) or CD4.sup.+ (FIG. 5C) T cells. Functional CD8.sup.+ T cell effectors were again mainly distributed among IFN-γ.sup.+ single positive, IFN-γ.sup.+ TNF-α.sup.+ double positive, or IFN-γ.sup.+ TNF-α.sup.+ IL-2.sup.+ triple positive subsets. CD4.sup.+ T cells specific to EsxA, PE10 or EspC antigen were preferentially distributed among IFN-γ.sup.+ single positive, IFN-γ.sup.+ TNF-α.sup.+ double positive or IFN-γ.sup.+ TNF-α.sup.+ IL-2.sup.+ triple positive subsets (FIG. 5D, E). No consistent quantitative or qualitative differences were detected in the T-cell responses in the mice immunized with LV::M40 harboring each of the distinct promoters. Again, conventional LV encoding for these Mtb proteins as a poly-antigen, is unable to induce CD4.sup.+ T-cell responses.sup.39.

[0259] We further established the induction of both CD8.sup.+ and CD4.sup.+ T cells specific to EsxH, EsxA, PE19 and EspC in C57BL/6 mice (n=3/group) immunized s.c. with LV::S40-HAPEHR or LV::S40-HAPEHR-20 (FIG. 6A). The immunogenicity of Hrp-1 and RpfD was assessed by their epitope mapping by use of splenocytes from LV::S40-HAPEHR-immunized mice in ELISPOT assay (FIG. 6B). Hrp-1:77-91 (SIYYVDANASIQEML), RpfD:57-71 (IAQCESGGNWAANT) and RpfD:87-101 (SNGGVGSPAAASPQQ) immunogenic regions were identified.

[0260] Altogether, these results provide evidence of the induction of robust, polyfunctional CD4.sup.+ T-cell responses by immunization with the developed new generation of LV.

Immunogenicity of the Poly-Antigenic Multistage LV::S40 at the Mucosal Level

[0261] We then evaluated the immunogenicity of LV::S40-HAPEHR or LV::S40-HAPEHR20 in C57BL/6 mice immunized (i.n.) with 1×10.sup.8 TU. At 14 dpi, intravenous (i.v.) injection of the immunized mice with PE-anti-CD45 mAb, 3 min before sacrifice, allowed detection of massive T-cell recruitment to the lung interstitium distinct from those in the vasculature.sup.40. The lung interstitial (CD45.sub.i.v.−) CD4.sup.+ (FIG. 7A) or CD8.sup.+ (FIG. 8A) T cells of these LV::S40-HAPEHR- or LV::S40-HAPEHR20-vaccinated mice contained increased frequencies of CD27.sup.− CD45RB.sup.− CD62L.sup.− migrant effectors and CD69.sup.+ CD103.sup.+ resident cells (FIG. 7B, FIG. 8B) compared to their PBS-injected counterparts. Most of the CD69.sup.+ CD103.sup.+ CD4.sup.+ or CD8.sup.+ T cells were CD44.sup.+ CXCR3.sup.+. ICS analysis of these cells indicated the presence of (poly)functional CD4.sup.+ (FIG. 7C) or CD8.sup.+ (FIG. 8C) T cells specific to EsxA, EspC, EsxH or PE19, and essentially located in the lung interstitium.

Booster Protective Effect of LV::S40-HAPEHR-20 Against Mtb Infection

[0262] Prime-boost strategies using BCG or an improved live-attenuated vaccine for priming, and subunit vaccine candidates for boosting, is a promising approach to improve the incomplete efficacy of BCG. To assess the booster potential of LV::S40-HAPEHR-20, C57BL/6 mice were left unvaccinated or were immunized s.c. at week 0 with 1×10.sup.6 CFU of a genetically improved BCG, i.e., BCG::ESX-1.sup.Mmar vaccine candidate.sup.41 (FIG. 9A). The latter provide the opportunity to perform a prime-boost with the developed LV vaccine as this live-attenuated vaccine actively secretes EsxA and EspC. A group of BCG::ESX-1.sup.Mmar-primed mice was boosted s.c. with 1×10.sup.8 TU of LV::S40-HAPEHR-20 at week 5, and then again boosted i.n. at week 10 with the same LV to recruit the induced immune effectors to the lung mucosa. At week 12, mice were challenged with ≈200 CFU of Mtb H37Rv via aerosol and lung and spleen mycobacterial burdens were determined at week 17 (FIG. 9B). The average lung Mtb load in the primed-boosted mice was decreased by ≈2.5 log.sub.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). The LV::S40-HAPEHR-20 boost resulted in a tendency to reduce in the spleen Mtb loads, which was however not statistical significance. An explanation for this is the particularly strong protective effect of ESX-1-complemented BCG strains against dissemination to the spleen.sup.12,42,43.

Discussion

[0263] We developed a new generation of multifunctional LV which, in comparison to the conventional vector, has been leveraged to: (i) facilitate poly-antigen delivery, (ii) target antigens to APC that it activates, (iii) route antigens through MHC-II pathway, and (iv) induce, in addition to CD8.sup.+ T cells, robust and polyfunctional CD4.sup.+ T-cell response. Such LV are tailored to induce secretion of multimeric protein carriers formed by truncated collectin-based scaffolds, able to harbor several antigens, as well as protein components with adjuvant or chemo-attracting properties. This is achieved by insertion of potent immunogens within the collagen-rich regions of MBL or SPD, substituted with CD40L ectodomain at their CRD region. Self-assemblage and polymerization of the monomers produced in the LV-transduced cells in vitro or in vivo, results in secreted multimeric carriers able to interact with CD40.sup.+ cells, including APC and notably DC. It is known that antigen delivery to appropriate surface receptors of DC improves the efficacy of antigenic presentation by several orders of magnitude.sup.8,44,45. The conventional LV per se, as prepared in our conditions, is barely inflammatory and induces almost no DC phenotypic or functional maturation, even used at very high doses. The capacity of LV to induce transitorily minute levels of IFN-I in vivo and IFN-I signaling in DC in vivo is not linked either to its outstanding T-cell immunogenicity.sup.39. Unlike the conventional LV, the new generation of LV described here, induces some degrees of DC maturation, via CD40 clustering by the trimeric extremities of M40 or S40 carriers. Therefore, these vectors assemble: (i) the intrinsic and outstanding CD8.sup.+ T-cell immunogenicity of the conventional LV and (ii) the properties of slight adjuvantation, antigen delivery to DC surface receptors, antigen routing to MHC-II and CD4.sup.+ T-cell immunogenicity of the secreted multimeric scaffolds that they encode.

[0264] Since TB is a disease primarily controlled by CD4.sup.+ T responses, as a first application, we investigated these optimized LV for their potential at inducing T-cell responses against selected Mtb antigens with preferential expression at distinct infection phases. We demonstrated in vitro a slight DC activating property of the secreted M40 or S40 carriers and their large efficiency at MHC-II- (and -I)-restricted presentation of the Mtb antigens inserted within their collagen-like domains. We then evidenced in vivo efficient induction of both (poly)functional CD8.sup.+ and CD4.sup.+ T-cell effectors, at the both systemic or mucosal levels, following s.c. or i.n. immunization. Notably only one shot of i.n. immunization generates high quality CD8.sup.+ and CD4.sup.+ T-cell effectors, with activated/effector/resident memory phenotype and located at the pulmonary interstitium. In future experiments, it will be informative to determine whether such T cells are located in the lung tertiary lymphoid organs.sup.46.

[0265] One of the multimeric carriers, i.e., S40-HAPEHR also harbors a segment of CCL20, a strong chemo-attracting chemokine. The receptor for CCL20, CCR6 is expressed on lymphocytes and DC, thus S40-HAPEHR-20 should reinforce the recruitment of immune cells. It is admitted that prime immunization with improved live-attenuated vaccine candidates and boosting with subunit vaccines is a promising approach. We used LV::S40-HAPEHR-20 as subunit booster in the mouse TB model after prime with BCG::ESX-1.sup.Mmar vaccine candidate, with largely improved protective potential compared to the parental BCG.sup.41. We observed that the lung Mtb burdens were statistically reduced by ˜1 log.sub.10 after LV::S40-HAPEHR-20 boosting.

[0266] A plasmid DNA encoding SPD-CD40L has been already used as an adjuvant when mixed with another plasmid DNA encoding for the HIV-1 Gag protein and led to a significant enhancement of CD8.sup.+ T cell responses (43). In net contrast to the LV platform developed here, this plasmid adjuvant was unable to induce CD4.sup.+ T-cell proliferative or cytokine production. Insertion of SPD-Gag-CD40L into an adenoviral vector serotype 5 (Ad5) has been more recently demonstrated to elicit much stronger Gag-specific CD8.sup.+ T-cell responses and a protection from a Gag-expressing vaccinia virus in the mouse model.sup.47. Co-immunization with a plasmid DNA encoding for SPD-gp100-CD40L, bearing the tumor gp100 antigen, and plasmids encoding for IL-12p70 and Granulocyte-Macrophage Colony Stimulating Factor (GM-CSF), increased immune control of the melanoma cells in mice.sup.48. However, induction of MHC-II-restricted antigenic presentation or CD4.sup.+ T-cell initiation have not been addressed in these studies. Compared to these previous studies, our project used polymers of M40 or S40 to generate, not only CD8.sup.+ T cells, but also notably (poly)functional CD4.sup.+ T-cell responses and some constructs harbor the CCL20 chemoattractant component, without need for additional other adjuvant or immune-stimulatory molecules. This new property of LV at inducing CD4.sup.+ T cells is of utmost importance, as CD4.sup.+ T cells are major immune players, based on their: (i) long lifespan, (ii) direct effector functions, (iii) capacity at orchestrating the immune system by regulating innate immunity, (iv) helper functions at tailoring B-cell responses, and (v) helper functions at supporting CD8.sup.+ T-cell effector pathways 4.

[0267] Altogether, we set up a new generation of LV vector leveraged to target and activate DCs, to route immunogens to MHC-II pathway and to induce both CD4.sup.+ and CD8.sup.+ T-cell responses. The applications of this innovating strategy are much larger and can be extended to vaccine LV against multiple other bacterial, viral, parasitic infectious diseases or cancers.

Material and Methods

Construction of Transfer pFLAP Plasmids Encoding for MBL or SPD Collectin Scaffolds, Harboring Selected Mycobacterial Antigens, CD40L and/or CCL20

[0268] Genes encoding for Mus musculus Mannan-Binding Lectin (MBL) or Surfactant-associated Protein D (SPD), engineered to harbor selected mycobacterial antigens and/or CCL20 within their collagen-like domains, and murine CD40L ectodomain instead of their CRD, were synthetized by GenScript after codon optimisation. Each of these genes were inserted into the sites BamHI and Xhol of the transfer pFLAPΔU3 plasmid.sup.49. Transcription is under control of the native human CMV, human β2-microglobulin β2m or BCUAG promoters, the two latter replacing CMV promoter after insertion between Mlul and BamHI sites. The human β2-microglobulin promoter has been previously described.sup.50. The BCUAG promoter is a hybrid promoter comprising CMV enhancer, inflammation-related cis-regulating region and β2m core promoter (FIG. 11). The pFLAPΔU3 plasmid contains also a mutated WPRE (Woodchuck Posttranscriptional Regulatory Element) sequence to improve protein expression.

Plasmid Amplification and Purification

[0269] Plasmid DNA were amplified in DH5α Escherichia coli in Lysogeny Broth (LB) completed with 50 μg/ml of kanamycin. The plasmid DNA was then purified by use of the NucleoBond Xtra Maxi EF Kit (Macherey Nagel). After drying, the DNA pellets were resuspended in Tris-EDTA Endotoxin-Free (TE-EF) buffer overnight, quantitated in a NanoDrop 2000c spectrophotometer (Thermo Scientific), adjusted to 1 μg/pl in TE-EF buffer, aliquoted and stored at −20° C. The quality of the plasmid DNA was controlled: (i) either undigested or subsequent to digestion with a mixture of 2 plasmid-specific appropriate restriction enzymes prior to gel electrophoresis, and (ii) by sequencing the inserts in each pFLAP plasmid.

Production and Titration of LV

[0270] Non-replicative integrative LV were produced in Human Embryonic Kidney (HEK)-293T cells, as previously detailed (Zennou et al., 2000). Briefly, 1×10.sup.7 cells/Petri dish were cultured in DMEM and were co-transfected in a tripartite manner with 1 ml of a mixture of: (i) 2.5 μg/ml of the pSD-GP-NDK packaging plasmid, encoding for codon optimized gag-pol-tat-rre-rev, (ii) 10 μg/ml of VSV-G Indiana envelop plasmid, and (iii) 10 μg/ml of “transfer” pFLAP plasmid in Hepes 1× containing 125 mM of Ca(ClO.sub.3).sub.2. Supernatants were harvested at 48 h post-transfection, clarified by 6-minute centrifugation at 2500 rpm and concentrated by 1-hour ultracentrifugation at 22,000 rpm at 4° C. LV were then aliquoted in PBS 1×, PIPES 20 mM, sucrose 2.5%, NaCl 75 mM and conserved at −80° C.

[0271] To determine the titers of the produced LV, HEK-293T were distributed at 4×10.sup.4 cell/well in flat-bottom 96-well-plates in complete DMEM in the presence of 8 μM aphidicolin (Sigma) to blocks the cell growth. The cells were then transduced with serial dilutions of concentrated LV. The titers, proportional to efficacy of the nuclear gene transfer, were determined as “Transduction Unit” (TU)/ml by quantitative real-time PCR on total lysates at day 3 post-transduction, by use of forward 5′-TGG AGG AGG AGA TAT GAG GG-3′ and reverse 5′-CTG CTG CAC TAT ACC AGA CA-3′ primers, specific to pFLAP plasmid and forward 5′-TCT CCT CTG ACT TCA ACA GC-3′ and reverse 5′-CCC TGC ACT TTT TAA GAG CC-3′ primers specific to the host housekeeping gene gadph, as described elsewhere.sup.51.

Mice, Immunization

[0272] C57BL/6JRj or BALB/cJ mice (Janvier, Le Genest Saint Isle, France) were used between the age of 7 and 10 weeks. Experimentation on mice was performed in accordance with the European and French guidelines (Directive 86/609/CEE and Decree 87-848 of 19 Oct. 1987) subsequent to approval by the Institut Pasteur Safety, Animal Care and Use Committee, under local ethical committee protocol agreement #CETEA 2013-0036, #CETEA DAP180030, and CETEA 2012-0005 (APAFIS #14638-2018041214002048). Mice were immunized subcutaneously (s.c.) at the basis of the tail with the indicated amounts of LV contained in 200 μl. When indicated, mice were immunized intranasally (i.n.) with the indicated amounts of LV contained in 20 μl, as previously detailed.sup.52. The i.n. administration was realized under anesthesia, obtained by peritoneal injection of a mixture of Xylazine (Rompun, 10 mg/kg) and Ketamine (Imalgene, 100 mg/kg).

T-Cell Assay by ELISPOT

[0273] At day 11-14 post-immunization, splenocytes from individual mice (n=3/group) were homogenized and filtered through 100 μm-pore filters and centrifuged at 1300 rpm during 5 minutes. Cells were then treated with Red Blood Cell Lysing Buffer (Sigma), washed twice in PBS and counted in a MACSQuant10 cytometric system (Miltenyi Biotec). Splenocytes were then seeded at 0.5-1×10.sup.5 cells /well in 200 μl of RPMI-GlutaMAX, containing 10% heat-inactivated FBS, 100 U/ml penicillin and 100 μg/ml streptomycin, 1×10.sup.−4 M non-essential amino-acids, 1% vol/vol HEPES, 1×10.sup.−3 M sodium pyruvate and 5×10.sup.−5M of β-mercapto-ethanol in the wells of IFN-γ or TNF-α ELISPOT plates (Mouse ELISPOT.sup.PLus, Mabtech). Cells were left unstimulated or were stimulated with 2 μg/ml of synthetic peptide (Proteogenix, Strasbourg, France), harboring the well-defined MHC-I-, or -II-restricted T-cell epitopes of each mycobacterial antigen. In parallel, splenocytes were stimulated with 2.5 μg/ml of Concanavalin A (Sigma), as a functionality control. For each individual, the assays were run in technical triplicates, following Mabtech's recommendations. Spots were quantified in an ELR04 ELISPOT reader (AID, Strassberg, Germany).

T-Cell Assay by Intracellular Cytokine Staining, Lung T-Cell Phenotyping

[0274] Splenocytes from immunized mice were obtained by tissue homogenization and passage through 100 μm-pore filter and were cultured during 6 h at 8×10.sup.6 cells/well in 24-well plates in the presence of 10 μg/ml of homologous or control peptide, 1 μg/ml of anti-CD28 (clone 37.51) and 1 μg/ml of anti-CD49d (clone 9C10-MFR4.B) mAbs (BD Pharmingen). During the last 3 h of incubation, cells were added with a mixture of Golgi Plug and Golgi Stop (BD Pharmingen). Cells were then collected, washed with PBS containing 3% heat-inactivated FBS and 0.1% NaN.sub.3 (FACS buffer) and incubated for 25 minutes at 4° C. with a mixture of FcγII/III receptor blocking anti-CD16/CD32 (clone 2.4G2), APC eF780-anti-CD3ε (clone 17A2), eFluor450-anti-CD4 (RM4-5) and BV711-anti-CD8α (53-6.7), mAbs (BD Pharmingen and eBioscience). Cells were then washed twice in FACS buffer, permeabilized by use of Cytofix/Cytoperm kit (BD Pharmingen). Cells were then washed twice with PermWash 1× buffer from the Cytofix/Cytoperm kit and incubated with a mixture of FITC-anti-IL-2 (clone JES6-5H4, eBioscience), PE-Dazzle-anti-TNF-α (MP6-XT22, Biolegend) and APC-anti-IFN-γ (clone XMG1.2, BD Pharmingen) mAbs or a mixture of appropriate control Ig isotypes, during 30 minutes at 4° C. Cells were then washed twice in PermWash and once in FACS buffer and then fixed with Cytofix (BD Pharmingen) overnight at 4° C. The cells were acquired in an Attune NxT cytometer system (Invitrogen). Data were analyzed by FlowJo software (Treestar, OR, USA). Lung T-cell phenotyping was performed as recently described.sup.39.

Antigenic Presentation Assay

[0275] Bone-marrow derived DC were plated at 5×10.sup.5 cells/well in 24-well plates in RPMI 1640 containing 5% FBS. Cells were transduced with LV or were loaded homologous or control synthetic peptides. At 24 h post infection 5×10.sup.5 appropriate T-cell hybridomas.sup.53 were added and the culture supernatants were quantitated for IL-2 production at 24 h by ELISA. Synthetic peptides were synthesized by Proteogenix (Schiltigheim, France).

Protection Assay

[0276] Mtb H37Rv strain or BCG::ESX-1.sup.Mmar 41, were cultured in Dubos broth, complemented with Albumine, Dextrose and Catalase (ADC, Difco, Becton Dickinson, Le Pont-de-Claix, France). Experiments with pathogenic mycobacteria were performed in BSL3, following the hygiene and security recommendations of Institut Pasteur. C57BL/6 mice were primed s.c. with 1×10.sup.6 CFU/mouse of BCG::ESX-1.sup.Mmar 41 at day 0, boosted s.c. with 5×10.sup.8 TU of SPD40-HAPEHR-20 at week 5, and boosted i.n. with 5×10.sup.8 TU of SPD40-HAPEHR-20 at week 10. The mice were challenged 2 weeks after the mucosal boost by use of a homemade nebulizer via aerosol, as previously described.sup.52. Briefly, 5 ml of a suspension of 1.7×10.sup.6 CFU/ml of Mtb H37Rv strain were aerosolized in order to deliver an inhaled dose of ≈200 CFU/mouse. The mice were then placed in isolator. 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. Statistical significance of inter-group Mtb load differences was determined by Mann-Whitney t-test by use of Prism v8.01 (GraphPad Software, Inc.).

Tables

[0277]

TABLE-US-00001 TABLE 1 Mtb proteins rationally selected as target antigens to be incorporated in the prospective multistage anti-TB LV. Mtb Locus Size immunogen in H37Rv a.a Major characteristics EsxA rv3875 95 Early Secreted Antigenic Target 6 kDa (ESAT-6) secreted by ESX-1 T7SS EspC rv3615c 103 ESX-1 secretion-associated proteins C secreted by ESX-1 T7SS EsxH rv0288 96 Virulence-related factor (TB10.4) secreted by ESX-3 T7SS PE19 rv1791 99 Virulence-related factor, with numerous homologous secreted by ESX-5 T7SS Hrp1 rv2626c 143 Dormancy-related Hypoxic response protein 1 RpfD rv2389c 154 Reactivation-related (42- Resuscitation promoting factor D (mb) 154)* *Only the RpfD.sub.42-154 ectodomain was included to minimize the hydrophobicity of the resulted protein.

TABLE-US-00002 SUPPLEMENTAL TABLE 1 Various M40 and S40 scaffolds designed to harbor the selected Mtb antigens and/or CCL20. Monomer Chemo- Lenght Carrier Antigens attractant Nomenclature (a.a) MBL40 EsxH — M40-H 414 EsxH-EsxA — M40-HA 556 EsxH-EsxA-PE19 — M40-HAP 721 EsxH-EsxA-PE19-EspC — M40-HAPE 885 SPD40 EsxH — S40-H XX EsxH-EsxA-PE19-EspC — S40-HAPE 736 EsxH-EsxA-PE19-EspC- — S40-HAPEHR 1004 Hrp1-RpfD — EsxH-EsxA-PE19-EspC- CCL20 S40-HAPEHR- 1128 Hrp1-RpfD 20

TABLE-US-00003 TABLE S2 Sequences of MBL fused with selected Mtb antigens and CCL20 as coded by LV Insert Poly-antigenic length LV (a.a.) Sequence LV::M40-EsxH 414 MSIFTSFLLLCVVTVVYAETLTEGVQNSCPVVTCSSPGLNGFPGKDGRDGAK (LV::M40-H) GEKGEPGQGLRGLQGPPGAVGPTGPPGNPGLKGAVGPKGDRGDRGGGSQIM YNYPAMLGHAGDMAGYAGTLQSLGAEIAVEQAALQSAWQGDTGITYQA WQAQWNQAMEDLVRAYHAMSSTHEANTMAMMARDTAEAAKWGGGS GLRGLQGPPGALGPPGSVGSPGSPGPKGQKGDHGDNRAIEEKLANMEAEIRI LKSKLQLTNKLHAFSMGGGSGDEDPQIAAHVVSEANSNAASVLQWAKKGYY TMKSNLVMLENGKQLTVKREGLYYVYTQVTFCSNREPSSQRPFIVGLWLKP SSGSERILLKAANTHSSSQLCEQQSVHLGGVFELQAGASVFVNVTEASQVIHR VGFSSFGLLKL LV::M40-EsxH- 556 MSIFTSFLLLCVVTVVYAETLTEGVQNSCPVVTCSSPGLNGFPGKDGRDGAK EsxA GEKGEPGQGLRGLQGPPGAVGPTGPPGNPGLKGAVGPKGDRGDRGGGSQIM (LV::M40-HA) YNYPAMLGHAGDMAGYAGTLQSLGAEIAVEQAALQSAWQGDTGITYQA WQAQWNQAMEDLVRAYHAMSSTHEANTMAMMARDTAEAAKWGGGS GFPGPPGPKGEPGSPAGRGERGFQGSPGKMGPAGSKGEPGGGSG GGGSGLRGLQGPPGA LGPPGSVGSPGSPGPKGQKGDHGDNRAIEEKLANMEAEIRILKSKLQLTNKL HAFSMGGGSGDEDPQIAAHVVSEANSNAASVLQWAKKGYYTMKSNLVMLE NGKQLTVKREGLYYVYTQVTFCSNREPSSQRPFIVGLWLKPSSGSERILLKAA NTHSSSQLCEQQSVHLGGVFELQAGASVFVNVTEASQVIHRVGFSSFGLLKL LV::M40-EsxH- 721 MSIFTSFLLLCVVTVVYAETLTEGVQNSCPVVTCSSPGLNGFPGKDGRDGAK EsxA-PE19 GEKGEPGQGLRGLQGPPGAVGPTGPPGNPGLKGAVGPKGDRGDRGGGSQIM (LV::M40-HAP) YNYPAMLGHAGDMAGYAGTLQSLGAEIAVEQAALQSAWQGDTGITYQA WQAQWNQAMEDLVRAYHAMSSTHEANTMAMMARDTAEAAKWGGGS GFPGPPGPKGEPGSPAGRGERGFQGSPGKMGPAGSKGEPGGGSG GGGSGLPGRDGRDG REGPRGEKGDPGLPGPMGLSGLQGPTGPVGPKGENGSAGEPGPKGERGLSG GGGSFVTTQPEALAAAAANLQGIGTTMNAQNAAAAAPTTGVVPAAADEVSALT AAQFAAHAQMYQTVSAQAAAIHEMFVNTLVASSGSYAATEAANAAAAGGGSG LRGLQGPPGALGPPGSVGSPGSPGPKGQKGDHGDNRAIEEKLANMEAEIRIL KSKLQLTNKLHAFSMGGGSGDEDPQIAAHVVSEANSNAASVLQWAKKGYYT MKSNLVMLENGKQLTVKREGLYYVYTQVTFCSNREPSSQRPFIVGLWLKPS SGSERILLKAANTHSSSQLCEQQSVHLGGVFELQAGASVFVNVTEASQVIHRV GFSSFGLLKL LV::M40-EsxH- 885 MSIFTSFLLLCVVTVVYAETLTEGVQNSCPVVTCSSPGLNGFPGKDGRDGAK EsxA-PE19-EspC GEKGEPGQGLRGLQGPPGAVGPTGPPGNPGLKGAVGPKGDRGDRGGGSQIM (LV::M40-HAPE) YNYPAMLGHAGDMAGYAGTLQSLGAEIAVEQAALQSAWQGDTGITYQA WQAQWNQAMEDLVRAYHAMSSTHEANTMAMMARDTAEAAKWGGGS GFPGPPGPKGEPGSPAGRGERGFQGSPGKMGPAGSKGEPGGGSG GGGSGLPGRDGRDG REGPRGEKGDPGLPGPMGLSGLQGPTGPVGPKGENGSAGEPGPKGERGLSG GGGSFVTTQPEALAAAAANLQGIGTTMNAQNAAAAAPTTGVVPAAADEVSALT AAQFAAHAQMYQTVSAQAAAIHEMFVNTLVASSGSYAATEAANAAAAGGCPG LPGAAGPKGEAGAKGDRGESGLPGIPGKEGPTGPKGNQGEKGIRGEKGDSGP SGGGSTENLTVQPERLGVLASHHDNAAVDASSGVEAAAGLGESVAITHGP YCSQFNDTLNVYLTAHNALGSSLHTAGVDLAKSLRIAAKIYSEADEAWR KAIDGLFTGGGSGLRGLQGPPGALGPPGSVGSPGSPGPKGQKGDHGDNRAIE EKLANMEAEIRILKSKLQLTNKLHAFSMGGGSGDEDPQIAAHVVSEANSNAA SVLQWAKKGYYTMKSNLVMLENGKQLTVKREGLYYVYTQVTFCSNREPSS QRPFIVGLWLKPSSGSERILLKAANTHSSSQLCEQQSVHLGGVFELQAGASVF VNVTEASQVIHRVGFSSFGLLKL

TABLE-US-00004 TABLE S3 Sequences of SPD fused with selected Mtb antigens and CCL20 as coded by LV Insert Polyantigenic length LV (a.a.) Sequence LV::S40-EsxH 506 MLPFLSMLVLLVQPLGNLGAEMKSLSQRSVPNTCTLVMCSPTENGLPGRDG (LV::S40-H) RDGREGPRGEKGDPGLPGPMGLSGLQGPTGPVGPKGENGSAGEPGPKGERG LSGGSGSQIMYNYPAMLGHAGDMAGYAGTLQSLGAEIAVEQAALQSAW QGDTGITYQAWQAQWNQAMEDLVRAYHAMSSTHEANTMAMMARDT AEAAKWGGGSGPPGLPGIPGPAGKEGPSGKQGNIGPQGKPGPKGEAGPKGE VGAPGMQGSTGAKGSTGPKGERGAPGVQGAPGNAGAAGPAGPAGPQGAPG SRGPPGLKGDRGVPGDRGIKGESGLPDSAALRQQMEALKGKLQRLEVAFSH YQKAALFPDGGGSGDEDPQIAAHVVSEANSNAASVLQWAKKGYYTMKSNL VMLENGKQLTVKREGLYYVYTQVTFCSNREPSSQRPFIVGLWLKPSSGSERI LLKAANTHSSSQLCEQQSVHLGGVFELQAGASVFVNVTEASQVIHRVGFSSF GLLKL LV::S40-EsxH- 736 MLPFLSMLVLLVQPLGNLGAEMKSLSQRSVPNTCTLVMCSPTENGLPGRDG EsxA-PE19-EspC RDGREGPRGEKGDPGLPGPMGLSGLQGPTGPVGPKGENGSAGEPGPKGERG (LV::S40-HAPE) LSGGSGSQIMYNYPAMLGHAGDMAGYAGTLQSLGAEIAVEQAALQSAW QGDTGITYQAWQAQWNQAMEDLVRAYHAMSSTHEANTMAMMARDT AEAAKWGGGSGG GGGSGGSFVTTQPEALAAAAANLQGIGTTMNAQNAAAAAPTTGVVP AAADEVSALTAAQFAAHAQMYQTVSAQAAAIHEMFVNTLVASSGSYAATEAAN AAAAGGGSGGTENLTVQPERLGVLASHHDNAAVDASSGVEAAAGLGESV AITHGPYCSQFNDTLNVYLTAHNALGSSLHTAGVDLAKSLRIAAKIYSEA DEAWRKAIDGLFTGSGGSGGLRGLQGPPGALGPPGSVGSPGSPGPKGQKGD HGDNRAIEEKLANMEAEIRILKSKLQLTNKLHAFSMGGGSGDEDPQIAAHVV SEANSNAASVLQWAKKGYYTMKSNLVMLENGKQLTVKREGLYYVYTQVTF CSNREPSSQRPFIVGLWLKPSSGSERILLKAANTHSSSQLCEQQSVHLGGVFE LQAGASVFVNVTEASQVIHRVGFSSFGLLKL LV::S40-EsxH- 1004 MLPFLSMLVLLVQPLGNLGAEMKSLSQRSVPNTCTLVMCSPTENGLPGRDG EsxA-PE19-EspC- RDGREGPRGEKGDPGLPGPMGLSGLQGPTGPVGPKGENGSAGEPGPKGERG Hrp1-RpfD LSGGSGSQIMYNYPAMLGHAGDMAGYAGTLQSLGAEIAVEQAALQSAW LV::S40-HAPEHR QGDTGITYQAWQAQWNQAMEDLVRAYHAMSSTHEANTMAMMARDT AEAAKWGGGSGG GGGSGGSFVTTQPEALAAAAANLQGIGTTMNAQNAAAAAPTTGVVP AAADEVSALTAAQFAAHAQMYQTVSAQAAAIHEMFVNTLVASSGSYAATEAAN AAAAGGGSGGTENLTVQPERLGVLASHHDNAAVDASSGVEAAAGLGESV AITHGPYCSQFNDTLNVYLTAHNALGSSLHTAGVDLAKSLRIAAKIYSEA DEAWRKAIDGLFTGSGGSGGTTARDIMNAGVTCVGEHETLTAAAQYMREHD IGALPICGDDDRLHGMLTDRDIVIKGLAAGLDPNTATAGELARDSIYYVDANASI QEMLNVMEEHQVRRVPVISEHRLVGIVTEADIARHLPEHAIVQFVKAICSPMAL ASGGGSGSGG GGSGGLRGLQGPPGALGPPGSVGSPGSPGPKG QKGDHGDNRAIEEKLANMEAEIRILKSKLQLTNKLHAFSMGGGSGDEDPQI AAHVVSEANSNAASVLQWAKKGYYTMKSNLVMLENGKQLTVKREGLYYVY TQVTFCSNREPSSQRPFIVGLWLKPSSGSERILLKAANTHSSSQLCEQQSVHL GGVFELQAGASVFVNVTEASQVIHRVGFSSFGLLKL LV::S40-EsxH- 1128 MLPFLSMLVLLVQPLGNLGAEMKSLSQRSVPNTCTLVMCSPTENGLPGRDG EsxA-PE19-EspC RDGREGPRGEKGDPGLPGPMGLSGLQGPTGPVGPKGENGSAGEPGPKGERG Hrp1-RpfD-CCL20 LSGGSGSQIMYNYPAMLGHAGDMAGYAGTLQSLGAEIAVEQAALQSAW LV::S40 HAPEHR- QGDTGITYQAWQAQWNQAMEDLVRAYHAMSSTHEANTMAMMARDT 20 AEAAKWGGGSGG GGGSGGSFVTTQPEALAAAAANLQGIGTTMNAQNAAAAAPTTGVVP AAADEVSALTAAQFAAHAQMYQTVSAQAAAIHEMFVNTLVASSGSYAATEAAN AAAAGGGSGGTENLTVQPERLGVLASHHDNAAVDASSGVEAAAGLGESV AITHGPYCSQFNDTLNVYLTAHNALGSSLHTAGVDLAKSLRIAAKIYSEA DEAWRKAIDGLFTGSGGSGGTTARDIMNAGVTCVGEHETLTAAAQYMREHD IGALPICGDDDRLHGMLTDRDIVIKGLAAGLDPNTATAGELARDSIYYVDANASI QEMLNVMEEHQVRRVPVISEHRLVGIVTEADIARHLPEHAIVQFVKAICSPMAL ASGGGSGSGG GGSGGGFPGPPGPKGEPGSPAGRGERGFQG SPGKMGPAGSKGEPGGSGSGGASNYDCCLSYIQTPLPSRAIVGFTRQMAD EACDINAIIFHTKKRKSVCADPKQNWVKRAVNLLSLRVKKMGSGSGSGG LRGLQGPPGALGPPGSVGSPGSPGPKGQKGDHGDNRAIEEKLANMEAEIRIL KSKLQLTNKLHAFSMGGGSGDEDPQIAAHVVSEANSNAASVLQWAKKGYYT MKSNLVMLENGKQLTVKREGLYYVYTQVTFCSNREPSSQRPFIVGLWLKPS SGSERILLKAANTHSSSQLCEQQSVHLGGVFELQAGASVFVNVTEASQVIHRV GFSSFGLLKL

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