COMPOSITIONS AND METHODS COMPRISING MEASLES VIRUS DEFECTIVE INTERFERING PARTICLES FOR THE PREVENTION OF INFECTIOUS DISEASES

Abstract

The invention is in the field of prevention or treatment of diseases, in particular infectious diseases, and more particularly in the field of multivalent vaccines. The inventors characterized 5′ copy-back DI-RNAs produced by recombinant MV strains, including rMV-based vaccines and wild-type MV (wt-MV). The efficiency of these DI-RNAs productions in different cell types was compared. For the first time 5′ copy-back DI-RNAs specific binding to RIG-I, MDA5 and LGP2 was assessed and linked to functional outcome in type-I IFN signalling. The inventors provide a composition of products comprising at least (i) a mixture of particles of a rescued recombinant MV-derived virus encoding at least one antigen (ii) a recombinant and/or purified protein, comprising at least one antigen. Regardless of the presentation of the products, and in particular regardless of whether the products are separated or readily separable or presented as a mixture.

Claims

1. A composition of products comprising at least: a mixture of particles of a rescued recombinant MV-derived virus encoding at least one antigen (the “vectored antigen”), said mixture comprising infectious replicating viral particles and defective interfering (DI) particles and optionally DI-RNA; a recombinant and/or purified protein, comprising at least one antigen (the “protein antigen”).

2. A composition of products comprising at least: a mixture of particles of a rescued recombinant MV-derived virus encoding at least one antigen (the “vectored antigen”), said mixture comprising infectious replicating viral particles and defective interfering (DI) genome as DI-RNA; a recombinant and/or purified protein, comprising at least one antigen (the “protein antigen”).

3. The composition of claim 1 or 2, wherein the MV-derived viral particles encode at least one antigen which is not from MV.

4. The composition of any one of claim 1, 2 or 3, wherein the vectored antigen and the protein antigen are from at least two distinct infectious species, in particular from two distinct viruses.

5. The composition of any one of claims 1 to 4, wherein the vectored antigen and the protein antigen are the same, or wherein at least one vectored antigen is the same as at least one protein antigen.

6. The composition of any one of claims 1 to 5, wherein the DI genomes (i.e. the genomes of the DI particles) represent at least 0.00001% of the full-length MV genomic RNA in the composition.

7. The composition of any one of claims 1 to 6 wherein at least one antigen is from an arbovirus.

8. The composition of claim 7, wherein the arbovirus is selected from the group consisting of CHIKV, DV, WNV, YFV, ZV.

9. The composition of any one of claims 1 to 8, wherein at least one antigen is from a parasite such as a plasmodium, in particular Plasmodium falciparum.

10. The composition of any one of claims 7 to 9, wherein the antigens are from at least two distinct arboviruses and/or are from at least Plasmodium falciparum and an arbovirus or from Plasmodium falciparum.

11. The composition of claim 10, wherein at least one vectored antigen is from CHIKV and at least one protein antigen is from Plasmodium falciparum, or wherein at least one vectored antigen is from Plasmodium falciparum and at least one protein antigen is from CHIKV.

12. The composition from any one of claims 9 to 11 wherein the antigen from Plasmodium falciparum is a CS protein, in particular a recombinant CS protein.

13. The composition of any one of claims 8 to 12, wherein the antigen from CHIKV comprises structural proteins of the Chikungunya virus, in particular one or several CHIKV structural proteins E1, E2, E3, C and 6K.

14. The composition of any one of claims 1 to 13 wherein the MV-derived viral particles encode at least two distinct antigens.

15. The composition of any one of claims 1 to 14 wherein the MV-derived viral particles have at least one of the following modifications relatively to the wild-type MV: deletion of the C protein or of a portion thereof; deletion of the V protein or of a portion thereof; duplication of the N gene or of a portion thereof.

16. The composition of any one of claims 1 to 15, wherein the MV-derived viral particles are derived from a strain selected in the group consisting of Schwarz, Moraten, Edmonston-Zagreb or from the group consisting of CAM-70, TD 97, Leningrad-16, and Shanghai 191 (Ji-191), in particular a Schwarz strain.

17. The composition of any one of claims 1 to 16, for use in the prevention of infectious diseases in a patient.

18. The composition according to claim 17, for use in the prevention of infection by an arbovirus.

19. The composition according to any one of claim 17 or 18, for use in the prevention of infection by a parasite.

20. The composition according to any one of claims 17 to 19, for use in the prevention of an infectious disease in a patient, in an administration scheme wherein the MV-derived viral particles are administered simultaneously in time with the recombinant protein.

21. The composition according to any one of claims 17 to 20, for use in the prevention of an infectious disease in a patient, in an administration scheme wherein a composition, in particular a composition according to claim 4, comprising the MV-derived viral particles expressing an antigen as the vectored antigen and the same antigen as a protein antigen is administered to prime the immune response to said antigen and wherein said antigen is administered as a protein antigen separately in time as a protein to boost the immune response to said antigen.

22. The composition according to any one of claims 17 to 21, for use in the prevention of an infectious disease in a patient, in an administration scheme wherein the MV-derived viral particles encode a first vector antigen and a second antigen as a protein antigen are administered to prime the immune response to at least one of said antigens and either the MV-derived viral particles encoding the first antigen and the second antigen as a protein antigen or the first antigen as a protein antigen or the second antigen as a protein antigen or a mixture of the first antigen and the second antigen as protein antigens is or are administered to boost the immune response to the first, the second or both antigens.

23. The composition for use according to claim 22, wherein the first and second antigens are identical, or comprise overlapping portions of the same protein.

24. The composition according to any one of claims 17 to 23, for use in the prevention of an infectious disease in a patient, in an administration scheme wherein the MV-derived viral particles are administered to stimulate the innate immune response, and to prime the adaptive immune response to an antigen, and the recombinant protein is administered to prime an adaptive immune response to a second antigen, preferably in conditions of activated innate immunity.

25. The composition for use according to any one of claims 21 to 24, wherein the priming and boosting administrations are separated by at least 4, in particular at least 6, more particularly at least 12 weeks.

26. A method of producing an MV-derived viral particles comprising DI particles, comprising the steps of: a) rescuing recombinant MV-derived viral particles from helper cells transfected with cDNA comprising the antigenome of an MV-derived viral particle, wherein in particular said cells express the N, P and L protein of an MV and an RNA polymerase, in particular T7 polymerase; b) recovering the viral particles from the culture of helper cells, or from a co-culture of helper cells with passage cells, i.e. cells suitable for the passage of the MV-derived virus; c) infecting passage cells with the MV-derived virus, with an MOI of at least 0.09 and in particular at least 0.1 such as a MOI of 0.09 to 1 to; d) recovering MV-derived particles from the culture of passage cells of c).

27. A method of producing an MV-derived viral particle comprising DI particles, in particular a method according to claim 26, comprising the steps of: a) rescuing recombinant MV-derived viral particles from helper cells transfected with cDNA comprising the antigenome of an MV-derived viral particle, wherein said cells in particular express the N, P and L protein of an MV and an RNA polymerase, in particular T7 polymerase; e) infecting passage cells with the MV-derived viral particles, recovered from the helper cells of a), or from cells infected with the viral particles produced from said helper cells, in particular from passage cells co-cultivated with said helper cells; f) maintaining the passage cells in culture for at least 3 passages or at least 8 passages and/or for a time sufficient for at least 6 cell divisions, preferably at least 9 cell divisions, and more preferably for at least 15 cell divisions.

28. A method of producing a MV viral stock containing MV-derived viral particles comprising performing the method of claim 26 or 27 wherein the helper cells are human helper cells such as HEK293T cells or cells derived therefrom and the passage cells are CEF cells, VERO cells, or VERO-hSLAM cells or A549 cells.

29. Use of DI-RNA of a measles virus genome as an adjuvant in an immunogenic composition, in particular in a composition according to any one of claims 1 to 25, to enhance the immune response.

30. A viral stock which contains rescued MV-derived particles as defined in any one of claims 1 to 25, in particular MV-derived particles rescued from a vaccine Schwarz strain wherein said particles contain infectious MV-derived particles and DI-RNA and/or DI-RNA particles when said rescued MV-derived particles have been passaged on cells such as CEF or Vero cells infected at a MOI of 0.09 to 1 and when the number of passages is at least 2 and in particular from 2 to 10, in particular 8,

Description

LEGENDS OF FIGURES

[0110] FIG. 1. Detection of DI-RNAs produced by rMV.

[0111] (A) Schematic representation of 5′ copy-back DI-RNA generation by the viral polymerase (RdRp). Primers used for PCR are represented. (B) Total RNA was extracted 24 hours post-infection with MV-Schwarz, rMV-N, rMV-ΔV and rMV-AC at a MOI of 1. DI-RNAs and genomes were detected by RT-PCR in four different cell types (A549, HeLa, 293T, Vero).

[0112] FIG. 2. Absolute quantification of genomes and DI-RNAs for MV-Schwarz, rMV-N, rMV-ΔV, rMV-ΔC, rMV-CHIKV and rMV-p55Gag/Env in four different cell lines.

[0113] Total RNA (400 ng) purified from 24 hours post-infection cells was analyzed by RT-qPCR. Absolute quantification was performed using serial dilutions of in vitro transcribed MV DI-RNAs or MV genome RNA fragment. Results are expressed in copy number of RNA molecules. Samples were tested in triplicates.

[0114] FIG. 3. IFN response to rMVs on A549 and STING-37 cells.

[0115] (A) Growth curves of MV-Schwarz, rMV-N, rMV-ΔV, rMV-ΔC, rMV-CHIKV and rMV-p55Gag/Env during 60 hours after A549 cells infection at a MOI of 1. Titres are expressed in TCID.sub.50/ml. (B) IFN-β mRNA and (C) Mx1 mRNA fold induction 12, 24 and 36 hours post-infection of A549 cells at a MOI of 1. Quantification of mRNA was done by RT-qPCR gene expression assay. (D) IFN-β mRNA fold induction 12, 24 and 36 hours post-infection of A549 cells infected by UV-inactivated (UV-in.) rMV. (E) Luciferase reporter activity of STING 37 cells infected by rMVs at a MOI of 1 during 36 hours. IFN-α at 500UI/ml was used as positive control, non-infected cells (mock) as negative control. Experiments were performed two times and data represent means±SD of the technical triplicates of the most representative experiment.

[0116] FIG. 4. IFN response to DI-RNAs transfection on STING-37 and A549 cells.

[0117] STING-37 and A549 cells were transfected with 10 ng of eight in vitro transcribed DI-RNAs of different lengths, actin RNA, Poly I:C of high molecular weight (HMW) or low molecular weight (LMW) and 5′ triphosphate RNA (5′3P). (A) in vitro transcribed DI-RNAs analysis on Agilent bioanalyzer. (B) Luciferase reporter activity of STING-37 cells and (C) IFN-β mRNA expression profile in A549 cells were measured 24 hours post-transfection and normalized to mock transfected cells. Experiments were performed three times and data represent means±SD of the technical triplicates of the most representative experiment.

[0118] FIG. 5. 5′ copy-back DI-RNAs specific binding to Rig-I like receptors.

[0119] Ratio of genomes and DI-RNA molecules after to before One-STrEP tag affinity purification of ST-RLR cell lines. 10 ng of either total RNA and RNA obtained after affinity chromatography of STrEP tagged Cherry (negative control), LGP2, RIG-I and MDA5 was analyzed by RT-qPCR. Absolute quantification was performed as described for FIG. 2. Samples were analyzed in triplicates and two biological replicates were performed.

[0120] FIG. 6. 504 nucleotide-long 5′ copy-back DI-RNA of rMV-CHIKV.

[0121] (A) Total RNA was extracted 24 hours post-infection by rMV-CHIKV and rMVp55Gag/Env at a MOI of 1. DI-RNAs were detected by RT-PCR in Vero cells. (B) Schematic representation and exact nucleotide sequence of 504 nt-long DI-RNA of rMV-CHIKV.

[0122] FIG. 7. Expression profile of DHX58 mRNA in A549 cells transfected with in vitro transcribed DI-RNAs.

[0123] A549 cells were transfected with 10 ng of eight in vitro transcribed DI-RNAs of different lengths, actin RNA, Poly I:C of high molecular weigh (HMW) or low molecular weigh (LMW) and 5′ triphosphate RNA (5′3P). DHX58 mRNA were measured by qRT-PCR 24 hours post-transfection and results were normalized to mock-transfected cells. Experiments were performed three times and data represent means±SD of the technical triplicates of the most representative experiment.

[0124] FIG. 8. Western Blot of infected ST-RLR before (input) and after (output) One-STrEP tag affinity purification.

[0125] β-actin served as negative control.

[0126] FIG. 9: Analysis of type-I IFN activation by MV DI-RNAs in the presence of MV-N and P proteins. (A) Western Blot analysis of T7 polymerase, MV-N, MV-P and β-actin in HEK293-T7 and HEK293-T7-NP cell lysates. (B) Absolute quantification of 1212, 450 and 1260 nt-long MV DI-RNA produced by HEK293-T7 and HEK293-T7-NP cells transfected with p2RZ-DI 1212, 450 and 1260 vectors. Total RNA (400 ng) purified from cells 24 hours after transfection was treated with RQ1 DNase RNase-free and 100 ng was analysed by RT-qPCR. Absolute quantification was performed using serial dilutions of in vitro transcribed MV DI-RNAs and normalized with actin. Results are expressed in copy number of RNA molecules. Experiments were performed two times and samples were tested in triplicates. (C-D) HEK293-T7 and HEK293-T7-NP cells were transfected with either (C) vector p2RZ encompassing DI-RNAs or actin sequence or (D) in vitro transcribed RNAs (Actin, 5′3P or 3 different IVT DI-RNAs). Relative IFN-β mRNA expression was measured 24 hours post-transfection and normalized to GAPDH and compared to mock transfected cells. Experiments were performed two times and data represent means±SD of the technical triplicates of the most representative experiment.

[0127] FIG. 10: CS antibody response elicited in C57BL/6 mice. (A) IgG anti-CS titre in mice immunized with CS alone (blue) or with rMV-CHIKV (purple) or with alum (red, positive control) at day 28 (before the second immunization) and day 55 (4 weeks after the second immunization). Antibody titres are expressed in Log 2. (Below) IgG antibody titre at day 55

TABLE-US-00005 CS 5 μg CS5 μg + rMV-CHIKV CS 5 μg + Alum Day 55 1.70E+04 1.38E+05 3.29E+05

[0128] FIG. 11: Detection of Genome and DI-RNAs in Vero cells infected at a MOI of 1 with viral stocks resulting from a transfection of IVT-DI-RNA 450 followed by MV-Schwarz or rMV-N infection in HEK293T cells. Total RNA was extracted 48 hours post-infection. DI-RNAs and genomes were detected by RT-PCR

EXAMPLES

Example 1. Cells and Viruses

[0129] HEK-293T (human embryonic kidney cells), Vero (African green monkey kidney cells), A549 (adenocarcinomic human alveolar basal epithelial cells) and HeLa cells were maintained in Dulbecco's modified Eagle medium (#10566-016, Gibco™ DMEM) supplemented with 10% heat-inactivated fetal calf serum (#A15-101, GE Healthcare) and 10,000 U/ml of Penicillin-Streptomycin (#15140122, Life Technologies). Vero-SLAM (and HEK-293 cell lines expressing One-STrEP-tagged RIG-I (ST-RIG-I), MDA5 (ST-MDA5), LGP2 (ST-LGP2) and Cherry (ST-CH) (13) were maintained with the same medium supplemented with G418 (#G8168, SIGMA) at 500 μg/ml. The STING-37 cell line corresponded to HEK-293 cells stably transfected with an ISRE-luciferase reporter-gene (25).

[0130] The MV-Schwarz vaccine strain (26) and seven recombinant MVs, rMV-N expressing an additional nucleoprotein in ATU 2 (10), rMV-ΔC lacking MV protein C (27) and rMV-ΔV lacking MV protein V (13), rMV-CHIKV expressing five chikungunya structural proteins (E1, E2, E3, C and 6K) in ATU 2 (28), rMV-p55Gag/Env expressing the p55Gag polyprotein and the EnvΔV1V2 envelope glycoprotein in ATU2 (29), rMV-GFP expressing green fluorescent protein in ATU2 and rMV-CH expressing Cherry red fluorescent protein in ATU3 were used. Wild-type MV (wt-MV) was obtained from a urine patient sample. Stock of wt-MV was obtained by virus multiplication on Vero-SLAM cells at a MOI of 0.1.

[0131] Virus titres were determined by 50% TCID.sub.50 titration on Vero cells (or Vero-SLAM cells for wt-MV). Two different viral stocks from two technical replicates of rescues were obtained for rMV-ΔC, rMV-N and rMV-CHIKV using the protocol described by Radecke et al. (30) and modified by Parks et al. (31).

Example 2. Virus Growth Curves

[0132] Monolayers of A549 cells of 24-mm-diameter-dishes (6-well-plates) were infected with MV-Schwarz or rMVs at a MOI of 1. At various times post-infection, cells were scraped into culture medium. After freeze thawing of cells and medium, and clarification of cell debris, virus titres were determined. For this purpose, Vero cells were seeded into 96-well plates (7500 cells/well) and infected by serial 1:10 dilutions of virus sample in DMEM-5% FCS. After incubation for 7 days, cells were stained with crystal violet and the TCID.sub.50 values were calculated by use of the Karber (1931) method.

Example 3. Virus Infection for RT-PCR Detection of 5′ Copy-Back DI-RNAs

[0133] Cells were seeded into T25 flasks one day before infection. Virus infections were carried out with a MOI of 1. Viruses were diluted with Opti-MEM to obtain a final inoculum volume of 2 ml. Cells were incubated with virus for 2 hours at 37° C. Then, 4 ml of DMEM containing 10% FBS were added in each T25 flask, and cells were incubated at 37° C. until infections were stopped by cell lysis 24 hours later.

[0134] Total RNA was extracted with RNeasy mini kit (#74104, Qiagen). cDNA was generated from 300 ng of total RNA using Superscript III (#18080-093, Thermo Fisher Scientific) and primer A2 (Table 1) in a total volume of 20 μl. A total of 2 μl of the resultant cDNA was then amplified with primers A2 and JM402 (Table 1) for genome amplification, A2 and JM403 (Table 1) for DI-RNA amplification, using Phusion® High-Fidelity DNA Polymerase (#F-5305/L, Thermo Fisher Scientific) in a total volume of 50 μl (95° C. for 2 min; 40 cycles of 95° C. for 30 sec, 55° C. for 30 sec, 72° C. for 1 min; 72° C. for 10 min). At the end of the PCR, Taq DNA polymerase (#10342-053, Thermo Fisher Scientific) was added and incubated at 72° C. for 10 min. The products were analyzed on a 1% agarose gel with Smartladder (#MW-1700-10, Eurogentec) as the size standard, and gel purified with QIAquick® Gel Extraction Kit (#28704, Qiagen). The PCR-amplified products were cloned into pTOPO vector (#45-0640, Thermo Fisher Scientific) and sequenced.

TABLE-US-00006 TABLE 1  RT-PCR Primers and probes used in the study PCR primer 5′-3′ sequence A2 (Pfaller, 2014) AAAGCTGGGAATAGAAACTTCG JM 403 (Shingai, 2007) CGAAGATATTCTGGTGTAAGTCTAGTA JM 402 (Shingai, 2007) TTTATCCAGAATCTCAATCCGG

TABLE-US-00007 TABLE 2 Characterisation of DI-RNAs produced by different MV. DI-RNA Re- ssRNA dsRNA Length Breakpoint initiation loop stem Indels Rule virus Rescue (nt) site.sup.1 site.sup.1 (nt) (nt) (nt) of six MV-Schwarz a — — — — — — — rMV-N a 1212 14781 15797 1016 98 — 202 × 6 b 1140 14895 15755 860 140 — 190 × 6 rMV-ΔV a 1662 14428 15696 1264 199 — 277 × 6 1260 14793 15737 938 161 — 210 × 6 rMV-ΔC a 1440 14648 15702 1054 193 — 240 × 6 450 15557 15783 226 112 —  75 × 6 b 2094 13946 15750 1804 145 — 349 × 6 1032 15046 15712 666 183 — 172 × 6 696 15302 15792 490 103 — 116 × 6 c 1218 14839 15733 894 162 — 203 × 6 636 15351 15803 452 92 — 106 × 6 rMV-GFP a 402 15591 15797 206 98 —  67 × 6 rMV-CH a 978 15062 15750 688 145 — 163 × 6 rMV-CHIKV a 504 15521 15765 244 130 —  84 × 6 1260 14793 15737 938 161 — 210 × 6 b 462 15530 15798 268 97 —  77 × 6 rMV- a 504 15542 15744 202 151 —  84 × 6 p55Gag/Env 1212 14781 15797 1016 98 — 202 × 6 Wildtype — 1212 14781 15797 1016 98 — 202 × 6 .sup.1 nucleotide positions of breakpoints and reinitiation sites as aligned to the reference sequence for Schwarz vaccine virus (GenBank: AF266291.1)

Example 4. TaqMan RT-qPCR Analysis of DI-RNAs and Full-Length MV Genomes

[0135] The analysis was performed using Applied Biosystem StepOnePlus™ technology. Various MV DI-RNAs primers and probes were designed using Primer Express Software (Applied Biosystem) (Table 3). Reactions were performed on 400 ng of total RNA using TaqMan RNA-to-Ct 1-Step Kit (#4392938, Thermo Fisher Scientific) for one-step RT-qPCR analyses. Reactions were performed in a final volume of 20 μl in the presence of 100 nM of each TaqMan DI-RNA specific probe and 100 nM of each DI-RNA specific forward and reverse primers. For absolute quantification of DI-RNAs, standard curves were established using serially diluted RNAs obtained by in vitro transcription of plasmids encompassing DI-RNAs specific for each rMVs. The DI-RNAs sequences were cloned into the in vitro transcription vector P2RZ (#27664, Addgene) with HindIII and NheI restriction sites. In vitro transcription was carried out at 37° C. using T7 RiboMAX™ (#P1320, Promega), and RNA products were purified using RNeasy clean up (#74104, Qiagen). Protocol for absolute quantification of β-actin or MV genome was described elsewhere (10). The standard curves were generated by the StepOnePlus™ software system by plotting the Cts against the logarithm of the calculated initial copy numbers. The unknown initial sample copy numbers were then automatically calculated from their Cts, as compared with the RNA standard curves.

TABLE-US-00008 TABLE 3   RT-qPCR primers and probes used in the study 5′-3′ sequence: Forward primer Reverse primer Targeted RNA TaqMan probe (reporter dye) Measles genome TCAGGCATACCCACTAGTGTGAA TGACAGATAGCGAGTCCATAACG CATCAGAATTAAGAAAAACGTAG (VIC) β-actin ACCGAGCGCGGCTACAG CTTAATGTCACGCACGATTTCC CACCACCACGGCCGA (FAM) 504 nt-long  CGGAGTTCAACCAATTAGTCCTTAA DI-RNA TGTGCCCCCAGAATTTGC CAGGGCACTATCTAGG (FAM) 1212 nt-long  TTGCAAATAATGCCTAACCACCTA DI-RNA ACACTGCCTACCCACGTGACT CAGGATTAGGGTTCCGGG (FAM) 1662 nt-long  TTACCTTAAAAACCCACTCACGTTT DI-RNA AGATTGCCCCCTGAAATGG AACACAAGCAAGCACA (FAM) 450 nt-long  TTATCAACTTTTTGTTCCCGGAGTA DI-RNA ACCACCTAGGGCAGGATTAGG AGATAATTGGTTGAACTCCGGAA (FAM)

Example 5. Affinity Chromatography of RLR RNP Complexes and Subsequent RNA Purification

[0136] ST-RLR cells (4×10.sup.7) were infected at a MOI of 1 for 24 h by MV-Schwarz, rMV-ΔV, rMV-ΔC and rMV-CHIKV. Cells were washed twice with cold PBS and lysed in 4 ml of lysis buffer (20 mM MOPS-KOH pH7.4, 120 mM of KCl, 0.5% Igepal®, 2 mM (3-Mercaptoethanol), supplemented with RNasin® at 1/200 (#N2515, Promega) and Complete Protease Inhibitor Cocktail (#11873580001, Roche). Cell lysates were incubated on ice for 20 min with gentle mixing every 5 min, and then clarified by centrifugation at 16,000 g for 15 min at 4° C. A 100-μ1 aliquot of each cell lysate was used to perform total RNA purification using TRI Reagent LS (#T3934, Sigma). The rest of cell lysate was incubated for 2 hours on a spinning wheel at 4° C. with 200 μl of Strep-Tactin® Sepharose High Performance (#28-9355-99, GE Healthcare). Beads were collected by centrifugation (1,600 g for 5 min at 4° C.) and washed three times for 5 min on a spinning wheel with 5 ml of washing buffer (20 mM MOPS-KOH pH7.4, 120 mM of KCl, 2 mM (3-Mercaptoethanol) supplemented with RNasin® at 1/400 and Complete Protease Inhibitor Cocktail. RNA purification was performed using TRI Reagent LS. RNA was dissolved in 50 μl of DNase-free and RNase-free ultrapure water. Extracted RNAs were analyzed using Nanovue (GE Healthcare) and Bioanalyser RNA nano kit (#5067-1511, Agilent).

[0137] To validate the efficacy of RLR ribonucleoprotein complex purification protein extracts were resolved by SDS-polyacrylamide gel electrophoresis on 4-12% Criterion™ gels BioRad) with MOPS running buffer and transferred to cellulose membranes (GE Healthcare) with the Criterion™ Blotter system (BioRad). The following antibodies were used: an anti-STrEP-Tag (#34850, Qiagen), anti-LGP2 (#NBP1-85348, Novus), anti-MDA5 (#5321, Cell Signaling), anti-RIG-I (#D14G6, Cell Signaling) or monoclonal anti-β-actin antibody (#A5441, Sigma).

[0138] Absolute quantification of DI-RNAs, genomes and β-actin mRNA was done by RT-qPCR on total RNA and on RNA obtained after One-STrEP tag affinity purification. β-actin mRNA was used to normalise the quantity of RNA before to after affinity purification.

Example 6. Analysis of rMVs Immunostimulatory Activity

[0139] STING-37 cells (25), stably transfected with an ISRE-luciferase reporter-gene, were infected by MV-Schwarz and different rMVs at a MOI of 1, were non-infected (Mock), or were transfected with 10 ng of in vitro transcribed DI-RNAs, using TransIT®-mRNA Transfection Kit (#MIR2250, Mirus). Cells were lysed at different time points post-infection. The Firefly luciferase activity was measured using the Bright-Glo™ Luciferase Assay System (#E2650, Promega) following the manufacturer's recommendation.

Example 7. TaqMan RT-qPCR of β-IFN and ISG Expression

[0140] Gene expression qPCR analysis was performed using the Applied Biosystem StepOnePlus™ technology. Total RNA was extracted from A549 cells infected by different rMVs at different time points using the RNeasy Mini Kit (#74104, Qiagen). Expression levels of IFNβ or ISG (Mx1 and DHX58) were quantified by one-step real-time PCR using GAPDH mRNA expression as internal control. Hundred ng of total RNA was amplified with 20× custom TaqMan Gene Expression Assays (#Hs01077958_s1; #Hs00895608_m1; #Hs00225561_m1; #Hs99999905_m1) using TaqMan RNA-to-Ct 1-Step Kit in accordance with the manufacturers' instructions. All the measurements were performed in triplicate and analysed as generated by StepOnePlus™ software system.

Example 8. Various 5′Copy-Back DI-RNAs are Produced by Modified Recombinant MVs

[0141] Production of 5′ copy-back DI genomes has previously been shown for three modified rMVs, separately in independent studies: rMV-N (10), rMV-ΔV (13) and rMV-ΔC (23). By applying a unique RT-PCR protocol, we performed molecular characterization and studied profiles of 5′copy-back DI-RNAs produced by rMV-N, rMV-ΔV and rMV-ΔC in parallel and in four different cell types. MV-Schwarz, from which rMVs are derived, was used as a negative control for the DI genome production. DI genomes were detected by RT-PCR using two primers both of negative polarity in accordance to the full-length MV genome, so that only 5′ copy-back DI-RNAs could be amplified (FIG. 1A and Table 1). DI genomes presence was assessed 24 hours post-infection of Vero, A549, 293T and HeLa cells at a MOI of 1 on total RNA purified from infected cells. As expected, we observed that various DI-RNAs were produced upon infection with modified rMVs but not the control MV-Schwarz strain (FIG. 1B).

[0142] Further, we performed molecular characterization of various 5′copy-back DI-RNAs produced upon infection with rMV-N, rMV-ΔV and rMV-ΔC. Thus, cDNA molecules encompassing the corresponding DI genome fragments were extracted from agarose gel, cloned, amplified, sequenced and aligned on MV-Schwarz sequence. Then, the known structure of 5′copy-back DI genome allowed deduction of the entire sequence of the DI-RNA from the amplified cDNA. Table 2 summarizes important points that define each 5′ copy-back DI-RNAs: the breakpoint site corresponds to the last nucleotide incorporated by the RdRp before leaving the template antigenome strand; the re-initiation site corresponds to the first nucleotide where the polymerase begin the synthesis of a complementary RNA strand of opposite polarity. We validated that rMV-N produced a single population of 1212 nt-long DI-RNA (10). rMV-ΔV produced two DI-RNAs of 1662 and 1260 nt-long, and rMV-ΔC one major DI-RNA of 2094 nt-long, but also minor DI-RNAs of different sizes (1032 nts, 696 nts). We observed that a major DI-RNA was detected for each rMV in the four different cell types (FIG. 1B).

[0143] Thus, we identified 5′ copy-back DI-RNAs that were produced by rMVs. They differed in size for both loop and stem, but they all respected the so-called “rule of six” postulating that for the paramyxovirus subfamily Paramyxovirinae only hexameric-length genomes are replicated efficiently (32).

Example 9. Recombinant MVs that Express Heterologous Antigens are Efficient Producers of 5′Copy-Back DI Genomes

[0144] First, we assessed if any perturbation of MV genome would outcome in production of DI genomes. Previous studies have demonstrated that rMVs expressing green fluorescent protein (GFP) or the Cherry red fluorescent protein (CH) gene from additional transcription unit on the viral genome were poor producers of DI genomes and behaved similar to the parental virus (15). We found a 402 nt-long DI-RNA in our viral stock of rMV-GFP and a 978 nt-long DI-RNA in our viral stock of rMV-CH (Table 2).

[0145] Further we examined whether or not recombinant MV-based vaccines expressing heterologous antigens could produce DI genomes. Vero, A549, 293T and HeLa cells were infected with either rMV-CHIKV expressing the structural proteins of chikungunya virus (E1, E2, E3, C and 6K) (28) and rMV-p55Gag/Env expressing HIV-1 p55Gag polyprotein and EnvΔV1V2 envelope glycoprotein (29). As previously, total RNA was extracted and DI-RNAs were detected by RT-PCR using two DI genome specific primers (Table 1). We observed that upon infection both recombinant viruses produced 5′ copy-back DI genomes (FIG. 6A). As above we performed their molecular characterization. rMV-CHIKV produced two different 5′ copy-back DI genomes of 1260 nt- and 504 nt-long (Table 2, FIG. 6B). Interestingly rMV-dbp55Gag/Env produced two different DI RNAs of 1212 and 504 nt-long with the longest DI genome perfectly corresponding to the unique DI-RNA detected in cells infected with rMV-N. No insertions/deletions were found in any of the DI-RNAs in Table 2.

[0146] Therefore, we identify various 5′ copy-back DI RNAs produced by rMVs that express heterologous genes. Again, these DI genomes all respected the “rule of six”.

Example 10. Production of Various DI Genomes is an Intrinsic Quality of MV Infection

[0147] To see whether DI genomes were dependent only on the type of genetic modification of the viral genome or on the rescue itself, we characterized DI genomes produced by two different stocks of either rMV-ΔC, or rMV-N, or rMV-CHIKV viruses obtained from two different rescues. We observed that the pattern of DI genomes was different for each rescue (Table 2, see rescues a and b). Indeed, previous studies have shown that DI-RNAs were produced very early after the rescue and this initial pattern defined the DI genome pattern of the viral stock (23). To be sure that these DI-RNAs were not due to the rescue system, we amplified a wt-MV from a patient sample in Vero/hSLAM cells permissive to the wt virus. We next infected Vero/hSLAM cells with the wt-MV at a MOI of 1 and assessed RT-PCR analysis for the presence of 5′ copy-back DI genomes. Again, we identified the 1212 nt-long DI-RNA with identical breakpoint and re-initiation sites of the one produced by rMV-N and rMV-dbp55Gag/Env, but with differences in nucleotide sequence corresponding to the wt-MV (Measles virus genotype D4, Genbank accession number: KT732229).

[0148] These results validate that DI genomes production is an intrinsic quality of MV infection and that the same virus obtained from a different rescue can produce distinct 5′ copy-back DI genomes.

Example 11. Some DI Genomes are Produced as Efficient as the Full-Length Genome

[0149] DI genomes are designated as defective interfering, as they are able to interfere with standard virus replication (33). In order to assess the question of interference, we quantified by RT-qPCR the full length and DI genomes produced by five different recombinant viruses: rMV-N, rMV-ΔV, rMV-ΔC, rMV-CHIKV, rMV-dbp55Gag/Env. Vero, A549, 293T and HeLa cells were infected at a MOI of 1 and total RNA purified from infected cells 24 hours post-infection. Absolute quantification was performed using serial dilutions of in vitro transcribed specific DI-RNAs or a MV full-length genome RNA fragment. MV-Schwarz, corresponding to an “empty of DI-RNA rMV”, was used as a control. The absolute quantification of MV-Schwarz full-length genomes in the four different cell lines reached the same level of about 1.Math.10.sup.8 copy number of RNA molecules thus providing a “gold standard” for MV replication efficiency. We observed three different scenarii (FIG. 2): i) lack of the DI genome production (MV-Schwarz); ii) for some infection DI genomes production was as efficient as for the full-length genome (rMV-N and 2094 nt-long DI RNA of rMV-ΔC); iii) finally, some DI genomes were present in lower copy number compare to the higher level of the full-length genomes (rMV-ΔV, rMV-CHIKV and rMV-dbp55Gag/Env).

[0150] Further, the level of various DI genomes in each cell types was different. In most of the cases, Vero cells, that are interferon deficient cells (34), were more permissive to DI-RNAs production (FIG. 2). We failed to quantify some minor DI genomes by RT-qPCR (1032 nt-long DI-RNA of rMV-ΔC, 1260 nt-long DI-RNA of rMV-ΔV and rMV-CHIKV), probably due to their low level of production.

[0151] Thus, we observed that the ratio DI genome to the full-length MV genome varied greatly for the different rMVs.

Example 12. rMVs are Strong Inducers of the IFN Signalling Pathway in Cells

[0152] In the same experimental conditions we studied rMVs potential to induce type-I IFN signalling and compared it to the parental MV-Schwarz. First, we assessed the growth kinetics of MV-Schwarz, rMV-N, rMV-ΔV, rMV-ΔC, rMV-CHIKV and rMV-p55Gag/Env. A549 cells were infected with these five rMVs and the MV-Schwarz strain at a MOI of 1 and tittering was performed at six different time points. We observed that all rMVs grew slower than their parental MV-Schwarz, with a final titre varying from 1.Math.10.sup.4 to 1.Math.10.sup.6 TCID.sub.50/ml (FIG. 3A). Then, we applied two different approaches to study immunostimulatory activity of these rMVs: (i) we measured rMV potential to induce IFN-β transcription and the transcription of Interferon Stimulated Gene (ISG) on A549 cells; (ii) we measured the type-I IFN stimulatory activity of rMVs at the protein level by using STING-37 cells that express an Interferon-Stimulated Response Element (ISRE)-luciferase reporter gene.

[0153] A549 cells were infected with corresponding rMVs and lysed at different time points (12 h, 24 h, 36 h) in order to quantify the IFN response (IFN-β and Mx1) by qRT-PCR gene expression assays (FIG. 3B-C). Level of IFN-β mRNA was higher for all of them except rMV-N (FIG. 3B). It is important to note that 36 hours post-infection, MV-Schwarz and rMV-CHIKV have almost reached the same titre (FIG. 3A), but rMV-CHIKV infected cells produced two fold more IFN-β mRNA than MV-Schwarz (FIG. 3B). Additionally, at the same time point, rMV-p55Gag/Env infected cells produced as many IFN-β mRNA as MV-Schwarz infected cells, whereas the titre of this virus was at least one log less high than the parental strain. Furthermore, we observed temporal differences in the IFN-β response: (i) rMV-N (t-test, p<0.05) and rMV-ΔC (t-test, p<0.001) that produced a high quantity of DI-RNAs, showed an early increase of IFN-β mRNA production at 12 hours post-infection (FIG. 7); (ii) rMV-AV and rMV-ΔC that lack the expression of virulence proteins presented higher level of IFN-β mRNA at 24 hours post-infection. Finally, the activation of ISG was achieved by all viruses as attested by the same level of induction of Mx1 mRNA production at 24 hours post-induction (FIG. 3C). Nevertheless, the level of Mx1 mRNA produced by infected cells early at 12 hours post-infection was higher for rMV-N (t-test, p<0.001) and rMV-ΔC (t-test, p<0.01).

[0154] To confirm the type-I IFN stimulatory activity of rMVs at the protein level, we used STING-37 cell line, corresponding to HEK-293 cells stably transfected with an ISRE-luciferase reporter gene (25). We validated that all rMVs including the backbone MV-Schwarz were strong IFN inducers in comparison to non-infected cells (FIG. 3D). Interestingly, for rMV-CHIKV and rMV-p55Gag/Env that are vaccine candidates, the ISRE activation by IFN-β was observed later, at 36 hours post-infection, but at a high level as for rMV-N and rMV-ΔV strains.

[0155] Thus, we linked presence of DI-RNAs with high immunostimulatory effects on type-I IFN signalling pathway, despite their negative impact on viral growth.

Example 13. Efficiency of Type-I IFN Signalling is Independent of DI-RNA Size or Sequence

[0156] To compare type-I IFN activation potential of different DI-RNAs identified in this study (Table 2) we produced in vitro transcribed (IVT) DI-RNAs and transfected them in either STING-37 or A549 cells. We studied a total number of eight IVT DI-RNAs of different sizes and sequences: 1662, 1260, 1212, 1032, 696, 636, 504 and 450 nt-long DI-RNAs (FIG. 4A and Table 2). β-actin RNA was used as negative control, and three classical RLRs PAMPs were used as positive control: poly I:C of high molecular weight (HMW) or low molecular weight (LMW) and 5′ triphosphate RNA (5′3P). IFN transcription efficiency was assessed in A549 cells by qRT-PCR and type-I IFN protein level potential was observed using the ISRE-luciferase reporter assay (FIG. 4B,C and FIG. 8). We observed that our eight DI-RNAs efficiently activated the type-I interferon pathway with little difference between DI-RNAs for two approaches applied (FIG. 4B-C).

[0157] Thus, we showed that type-I IFN activation was achieved by all IVT DI-RNA, independently of their size or sequence.

Example 14. MV 5′ Copy-Back DI-RNAs are Specific Agonists of RIG-I and LGP2, but not MDA5

[0158] To test if each of the three RLRs are involved in DI genome sensing and to compare LGP2, MDA-5 and RIG-I efficiency in binding various 5′ copy-back DI genomes we used human HEK293 cell lines stably expressing tagged LGP2, or MDA5 or RIG-I proteins (assigned ST-RLRs (13)). These cells have been shown to be efficient and specific tool to purify viral agonists of RLR. ST-RLRs cells were infected with MV-Schwarz, rMV-ΔV, rMV-ΔC and rMV-CHIKV for 24 hours at a MOI of 1. RLR-specific RNA ligands were purified using previously published protocol (13). In addition, a stable cell line (assigned ST-CH) expressing the Cherry protein instead of tagged RLRs was used as a negative control to allow subtraction of non-specific RNA binding (13). The efficacy of the purification was assessed by western blot to confirm the enrichment of RLRs receptors and the depletion of β-actin (FIG. 8). Absolute quantification of DI-RNA, genomes and β-actin mRNA was done by qRT-PCR. Ratios of the full-length genomes to actin and DI-RNAs to actin showed that DI-RNAs were always enriched on RIG-I, to a lesser extent on LGP2 and never on MDA5 (FIG. 5). Full-length genome was equally depleted on the three RLRs for MV-Schwarz and any of the rMV infections.

[0159] Therefore, we validated that various DI-RNAs specifically interact with RIG-I and for the first time observed their binding to LGP2.

Example 15. The Capacity of MV DI-RNA to Activate Type-I IFN Response is Lost when DI-RNA is Encapsidated into MV Nucleocapsid

Material and Method:

[0160] HEK293-T7 and HEK293-T7-NP cells express the T7 RNA polymerase in the cytosol. HEK-293-T7-NP cells also stably express MV-N and MV-P proteins necessary for MV genome encapsidation. Both cells were transfected by p2RZ plasmids expressing different MV DI-RNAs. After 24 hours total RNA was extracted using the RNeasy Mini Kit and treated by RQ1 RNase-Free DNase (#M6101, Promega). Expression levels of IFN-β or Mx1 and DHX58 interferon stimulated genes (ISG) were quantified by one-step real-time PCR using GAPDH mRNA expression as internal control. RNA (100 ng) was amplified with 20× custom TaqMan Gene Expression Assays (IFN-β #Hs01077958_s1; Mx1 #Hs00895608_m1; DHX58 #Hs00225561_m1; GAPDH #Hs99999905_m1, Life Technologies) using TaqMan RNA-to-Ct1-Step Kit in accordance with the manufacturer's instructions. All measurements were performed in triplicate and analysed as generated by StepOnePlus™ software system.

Results:

[0161] To investigate whether MV DI-RNAs encapsidated within MV nucleocapsids were as efficient as non-encapsidated ones to stimulate type-I IFN, we used HEK293-T7 and HEK293-T7-NP cell lines that both stably express T7 RNA Polymerase but only HEK293-T7-NP cells also produce MV-N and P proteins (A). HEK293-T7 and HEK293-T7-NP cells were transfected with p2RZ vectors expressing 1212 nt-long DI-RNA (p2RZ-DI1212), 450 nt-long DI-RNA (p2RZ-DI450), 1260 nt-long DI-RNA (p2RZ-DI1260) or a 1212 nt-long RNA fragment of actin mRNA (p2RZ-actin). The p2RZ plasmid allows producing RNA transcripts under the control of T7 Polymerase promoter and possesses 3′-end cis-acting ribozyme of hepatitis delta virus (HDV) for direct transcriptional processing to precisely trim the RNA transcript 3′-end.

[0162] Upon transfection of p2RZ-DI1212, p2RZ-DI450 and p2RZ-DI1260 vectors both cell lines produced a similar amount of DI-RNA (B). However, only HEK293-T7 showed a significant increased level of IFN-β while HEK293-T7-NP cells did not. As expected, no type-I IFN activation was observed in both cell lines upon transfection with the negative control p2RZ-actin vector (C). Furthermore, we analysed the potential of DI-RNAs to stimulate type-I IFN by transfecting in vitro transcribed (IVT) DI-RNAs in HEK293-T7 and HEK293-T7-NP cells (D). As above, HEK293-T7-NP cells presented a lower type-I IFN activation than HEK293-T7 cells upon transfection of the IVT DI-RNAs, whereas control stimulation by 5′3P RNA was identical in the two cell lines. In that case, the presence of MV N and P in HEK293-T7-NP cells decreased type-I IFN activation by DI genomes but did not abrogate it. Importantly, we observed similar type-I IFN activation by plasmid-derived or in vitro transcribed DI-RNAs in HEK293-T7 cells, whereas in HEK293-T7-NP cells the plasmid-derived DI genomes were less potent. This indicates that the rapid encapsidation immediately after RNA synthesis from p2RZ-DI-RNA vectors in HEK293-T7-NP cells masked viral RNA and abolished its recognition and downstream activation of type-I IFN signalling.

Conclusion:

[0163] These results show that encapsidation of DI-RNA by MV-N and MV-P proteins abolished its capacity to stimulate type-I IFN. This demonstrates that naked DI-RNA is the molecular form that is recognized by RLRs to activate type-I IFN

Example 16. Administration of an MV-Derived Virus as an Adjuvant

[0164] Viruses and Antigens

[0165] MV-Schwarz and rMV-CHIKV design and production have been described previously (Combredet, J Virol, 2003; Brandler, Vaccine, 2013). Recombinant circumsporozoite proteins from Plasmodium falciparum (CSPf) were produced in E. coli (Malaria Parasite Biology and Vaccines Unit, Institut Pasteur, Paris). Alum was used as positive control to enhance immunogenicity of the CSPf.

[0166] Study Design

[0167] C57Bl/6 mice were obtained from Charles River Laboratory, with an average age of 5 weeks. Four Groups of 6 mice were inoculated intraperitoneally (i.p.) with 5 μg of CSPf alone, 5 μg of CSPf+10.sup.5 TCID.sub.50 of rMV-CHIKV, or 5 μg of CSPf+Alum. A second immunization was performed 4 weeks later, with the same dose and route of delivering. For antibody determination, blood samples were collected at day 0 (negative control), day 28 (before the boost) and at day 55 (4 weeks after the boost). All experiments were approved and conducted in accordance with the guidelines of the Office of Laboratory Animal Care at Pasteur Institute.

[0168] Humoral Response

[0169] The presence of anti-CS antibodies was assessed using enzyme-linked immunosorbent assay (ELISA). 96-well plates were coated with recombinant CS protein diluted in carbonate buffer at a concentration of 1 μg/ml. The plates were incubated overnight at +4° C. and washed with PBS-0.05% Tween 20. Unspecific interactions were blocked with 3% BSA in PBS for 1 hour at 37° C. After washing, plates were incubated with serial dilutions of mouse sera for 1 hour at 37° C. Horse-radish peroxidase-conjugated goat anti-mouse IgG was used as secondary antibody and plates were revealed using TMB substrate. The endpoint titers for each individual serum were calculated as the reciprocal of the last dilution giving twice the absorbance of negative control sera.

[0170] Results:

[0171] The recombinant CS protein was used as a model antigen. The CS alone is immunogenic and induced an antibody response, which was weakly boosted after the second immunization. The addition of rMV-CHIKV or Alum (positive control group) enhanced the antibody response. After the second immunization, we observed ten times higher level of antibodies in the group immunized with CS together with rMV-CHIKV compared to the group immunized with CS alone, highlighting the adjuvant effect of the virus to the recombinant protein.

[0172] Conclusion:

[0173] rMV-CHIKV has an adjuvant effect in mice in vivo when added to a recombinant protein. This observation allows adding recombinant antigens to the viral vector to benefit of a natural adjuvant effect.

Example 17. Capacity of MV-Schwarz to Encapsidate a Transfected RNA and to Maintain it in the New Viral Stock

[0174] Material and Method:

[0175] T25 flasks of HEK293T cells were transfected with 1 μg of IVT-DI-RNA-450 or were mock-transfected using Jet-Prime® polyplus transfection reagent. Seven hours later, cells were infected with either MV-Schwarz or rMV-N at a MOI of 1. Infections were stopped after 24 hours in 500 μl of Optimem® and frozen at −80° C. After thawing, 250 μl of the new virus stocks were used to infect T25 flasks of Vero cells. Two hours after infection, supernatants were removed and DMEM was added in each flask. Infections were stopped by cell lysis after removing the supernatant 48 hours later, and total RNA was extracted with RNeasy mini kit. Then, genome and DI-RNA were detected by RT-PCR (see above).

[0176] Results:

[0177] Measles genome was detected in each case, confirming that all new viral stocks were able to infect and disseminate in Vero cells (the presence of syncytia was also assessed by microscopy). 450 nt-long DI-RNA was detected in Vero cells infected with the new MV-Schwarz viral stock, confirming that this RNA was delivered into the cells via the viral particles. The same was observed with rMV-N that still delivered its 1212 nt-long DI-RNA but that also delivered the new 450 nt-long DI-RNA to the cells via the viral particles.

[0178] Conclusion:

[0179] MV-Schwarz or rMV are able to encapsidate a transfected RNA and to maintain it in the new viral stock so that we can detect it in new infected cells. These results highlighted two important points: (i) it is possible to control the presence of a defined DI-RNA in a MV-Schwarz viral stock; (ii) MV-Schwarz could potentially encapsidate any RNA that would respect the rule of six necessary for its encapsidation and deliver said RNA to cells.

Example 18. Testing the Adjuvant Effect of MV Expressing DI-RNA in Human Dendritic Cells

[0180] Material and Method:

[0181] Human myeloid dendritic cells (MoDCs) were used to analyse the presentation of HIV-1 Gag epitopes to HIV-1 Gag-specific CD4+ T cells. Obtained from HLA-DRb1*01+CD14+ monocytes incubated with IL-4 and GM-CSF during 4 days, DCs were exposed to recombinant Gagp24 protein at different concentrations (2 μg and 10 μg) alone, or with MV-Schwarz, or MV-Schwarz p8 at a MOI of 0.5 or 5. After 2 hours of incubation in RPMI at 37° C., 5% CO2, cells were washed and incubated in 24-well plates at 37° C., 5% CO2 for 24 hours. HIV-1-Gag specific CD4+ T cells (clone F12) were added at a ratio of 1:40. As a positive control, DCs previously incubated with HIV-1 peptides (SL9 and gag2) or with rMV-p55Gag/Env were also incubated with T cell clones at the same ratio. After 6 hours of coculture, the activation of CD4+ T cells was measured by intracellular cytokine staining (anti-MIP-1B and a pool of anti-IL-2, -IFNg and -TNFa conjugated to the same fluorochrome) using flow cytometry. Schwarz p8 was obtained after 8 passages of MV-Schwarz on Vero cells at a MOI of 0.1. The genome sequences of both viruses are identical but Schwarz p8 viral stock contains DI-RNA whereas MV-Schwarz does not.

[0182] Results:

[0183] DCs that are exposed simultaneously to recombinant Gagp24 antigen and Schwarz p8 activated a higher percentage of anti-Gagp24 CD4+ T cells than DCs exposed to recombinant protein alone or with MV-Schwarz. Moreover, these T cells clones presented higher polyfunctionality by the co-expression of MIP-1B, IL-2, IFNg and TNFa. Nevertheless, the effect was weak and lost at a higher MOI. Indeed, the clonal system is highly sensitive and rapidly reached a plateau of activation

[0184] Conclusion:

[0185] Schwarz p8 that is similar to MV-Schwarz in the sequence but only differs by the presence of DI-RNA is able to enhance DCs and T cell activation exposed to recombinant Gagp24 antigen. These results are consistent with the already known capacity of DI-RNA to induce a greater maturation of moDCs and higher levels of IFN and ISGs (Shivakoti R, 2013, J Virol), and emphasize the adjuvant role of DI-RNA in a viral stock.

Example 19. Discussion

[0186] DI-RNAs were known to be produced by some modified MV (rMV-N (10), rMV-ΔV (13), rMV-ΔC (23)), or by some measles vaccine strains after cell passages at a high MOI (8,20,22). By comparing different stocks of rMVs produced by the same manner, we observed that any modification of the MV genome increased its capacity to produce DI-RNAs. Indeed, no DI-RNA was found in the parental MV-Schwarz stock, whereas all rMVs tested produced one or more DI-RNAs. This includes addition of genetic material (rMV-GFP, rMV-CH, rMV-N, rMV-CHIKV, rMVp55Gag/Env) or lacking of the expression of virulence factors, C and V protein (rMV-ΔC or rMV-ΔV). Both proteins have at least two functions: modulation of the innate immune response (35-37) and regulation of viral RNA synthesis. C protein is described as an important factor that stabilizes the RNP-polymerase complex, as a lack of C protein results in more frequent chain termination occurring both during transcription and replication (15). And V protein helps maintaining the ratio of genome to antigenome synthesis during replication (38). But the exact mechanism used by the polymerase to stop the replication and start copying back remains unknown. Moreover, the pattern of DI-RNA is different for each viral stock and multiple breakpoint sites and initiation sites were identified, especially for rMV-ΔC. This pattern is defined early after the rescue, as showed by Pfaller et al. (23). Nevertheless, we found 1212 nt-long DI-RNA in three different viral stocks (rMV-N, rMV-P55Gag/Env, and wt-MV), suggesting that the polymerase may recognize some specific nucleotide sequences.

[0187] DI-RNAs length ranged from 402 nt to 2094 bp. The loop and stem sizes varied from one DI-RNA to another, but interestingly all identified sequences respected the “rule of six” that is necessary for encapsidation efficiency (21,23,32). The minimal size of the double stranded stem region, 93 nt, included the B′ box of the trailer sequence (39). This is also the minimal size observed by Pfaller et al (23) and is comparable to the minimal length of 96 nt observed in Sendai Virus copy-back DI-RNAs, an other paramyxovirus (40). This minimal sequence is necessary for genome encapsidation by N and P proteins (41,42). MV genome is encapsidated immediately after the beginning of the replication (4) and the rule of six necessary for encapsidation (32) may have advantaged the replication and transmission from cell to cell of DI-RNAs respecting this rule. However, Pfaller et al. found naked dsRNA in cells infected by rMV-ΔC (15), as well as DI-RNA with a length that does not respect this rule (23). As C protein stabilized the polymerase complex, its absence might enhance a higher production of DI-RNAs that overpasses the fitness of encapsidation.

[0188] Defective viral genomes are named as defective “interfering” RNA because DI-RNA particles were postulated to interfere with the replication of the standard virus by competing for viral proteins in the infected cell (43). In our study, all rMVs have a decreased growth compared to MV-Schwarz, meaning that the genetic modifications have a cost for viral fitness. The slowest growths were observed for rMV-N and rMV-ΔC that both produced as many DI-RNAs as genomes. These DI-RNAs may indeed decrease viral growth by interfering directly with the viral replication but also by enhancing the host response due to their presence in high quantity as PAMPs recognized by the innate immune system.

[0189] 5′ copy back DI-RNAs are known to be PAMPs to RLR-intracellular receptors, specifically RIG-I (12,44) but also MDA5 (45). We showed a direct interaction between DI-RNAs and RIG-I and LGP2 by infecting ST-RLR cell lines (13). These cells enabled isolation of RLR interactors by using chromatographic affinity purification. DI-RNAs were amplified in RNAs associated with RIG-I and LGP2 receptors and not MDA5. We described for the first time an interaction between LGP2 and DI-RNA. LGP2 differed from RIG-I and MDA5 by the absence of CARD domain that is necessary to activate the final common IFN signalling cascade via MAVS activation (46). The role of LGP2 as a regulator of MDA5 and RIG-I response is not well understood and controversial. It acts mainly as a negative regulator of RIG-I (47) and a positive regulator of MDA5 (48-50). Common ligands between MDA5 and LGP2 are suggested (13,51) and MDA5 may play a role in the induction of an antiviral state in cells infected by measles vaccine by recognizing others PAMPs of MV than DI-RNA (13,52). Here, we described DI-RNA as a common ligand between RIG-I and LGP2. As all rMVs shared high immunostimulatory properties on IFN and ISG expression, both regulatory functions of LGP2 could be involved: enhancement of RIG-I response, or down-regulation to avoid excessive response, but that still permit a high activation.

[0190] DI-RNAs sequences were highly preserved with no mutation observed for all DI-RNAs, except 1032 nt-long DI-RNA. This DI-RNA produced by rMV-ΔC virus accumulated clusters of A-to-G transitions. This pattern was already observed by Pfaller et al (23) for DI-RNAs produced by Moraten vaccine strain and wild-type IC-B strain lacking expression of C-protein. ADAR1-editing was hypothesized to destabilize unencapsidated DI-RNAs that have dsRNA structures (23). But in our case, A-to-G transitions, observed in 36 upon 286 A nucleotides (12.7%), were located in the loop sequence only. These mutations did not affect its immunostimulatory properties, as the general structure is conserved. Indeed, cells transfected with eight in vitro transcribed DI-RNAs different in sizes and sequences showed similar high level of IFN β and ISG expression on STING-37 and A549 cells. These experiments confirmed that the PAMP recognized by RIG-I and LGP2 is structure dependent and do not depend on the sequence or the size of ssRNA loop and dsRNA stem. Moreover, Ho et al (14) also confirmed that the 5′3P extremity play a major role for the recognition by RIG-I.

[0191] MV vaccine strains are stronger inducers of IFN pathway compared to wt-MV (20), even in the absence of DI-RNA. Here, we showed that all rMVs tested possessed higher immunostimulatory properties than the parental strain MV-Schwarz due to the production of DI-RNAs. These specific viral PAMPs are bound to RIG-I and LGP2, both cytosolic receptors of the innate immunity Measles-virus-based vaccine platform is of great interest since rMV-CHIKV vaccine have shown in a phase I clinical trial that pre-immunity against measles did not interfere with the induction of an immunogenicity against heterologous antigens expressed by the vector (24). Efficiency of vaccines depends on their immunostimulatory properties. 5′ copy-back DI-RNAs act as intrinsic adjuvants naturally produced by measles vector, as they enhance recognition and activation of the innate immune system. They certainly play a major role in MV efficiency as a vector against heterologous antigens and their presence should be considered of great importance.

REFERENCES

[0192] 1. Lazzarini, R. A., Keene, J. D. and Schubert, M. (1981) The origins of defective interfering particles of the negative-strand RNA viruses. Cell, 26, 145-154. [0193] 2. Dimmock, N. J. and Easton, A. J. (2014) Defective interfering influenza virus RNAs: time to reevaluate their clinical potential as broad-spectrum antivirals? Journal of virology, 88, 5217-5227. [0194] 3. Lopez, C. B. (2014) Defective viral genomes: critical danger signals of viral infections. Journal of virology, 88, 8720-8723. [0195] 4. Gubbay, O., Curran, J. and Kolakofsky, D. (2001) Sendai virus genome synthesis and assembly are coupled: a possible mechanism to promote viral RNA polymerase processivity. The Journal of general virology, 82, 2895-2903. [0196] 5. Bellini, W. J., Englund, G., Rozenblatt, S., Arnheiter, H. and Richardson, C. D. (1985) Measles virus P gene codes for two proteins. Journal of virology, 53, 908-919. [0197] 6. Cattaneo, R., Kaelin, K., Baczko, K. and Billeter, M. A. (1989) Measles virus editing provides an additional cysteine-rich protein. Cell, 56, 759-764. [0198] 7. Strahle, L., Garcin, D. and Kolakofsky, D. (2006) Sendai virus defective-interfering genomes and the activation of interferon-beta. Virology, 351, 101-111. [0199] 8. Shingai, M., Ebihara, T., Begum, N. A., Kato, A., Honma, T., Matsumoto, K., Saito, H., Ogura, H., Matsumoto, M. and Seya, T. (2007) Differential type I IFN-inducing abilities of wild-type versus vaccine strains of measles virus. Journal of immunology, 179, 6123-6133. [0200] 9. Mercado-Lopez, X., Cotter, C. R., Kim, W. K., Sun, Y., Munoz, L., Tapia, K. and Lopez, C. B. (2013) Highly immunostimulatory RNA derived from a Sendai virus defective viral genome. Vaccine, 31, 5713-5721. [0201] 10. Komarova, A. V., Combredet, C., Sismeiro, O., Dillies, M. A., Jagla, B., Sanchez David, R. Y., Vabret, N., Coppee, J. Y., Vidalain, P. O. and Tangy, F. (2013) Identification of RNA partners of viral proteins in infected cells. RNA biology, 10, 944-956. [0202] 11. Baum, A., Sachidanandam, R. and Garcia-Sastre, A. (2010) Preference of RIG-I for short viral RNA molecules in infected cells revealed by next-generation sequencing. Proceedings of the National Academy of Sciences of the United States of America, 107, 16303-16308. [0203] 12. Runge, S., Sparrer, K. M., Lassig, C., Hembach, K., Baum, A., Garcia-Sastre, A., Soding, J., Conzelmann, K. K. and Hopfner, K. P. (2014) In vivo ligands of MDA5 and RIG-I in measles virus-infected cells. PLoS pathogens, 10, e1004081. [0204] 13. Sanchez David, R. Y., Combredet, C., Sismeiro, O., Dillies, M. A., Jagla, B., Coppee, J. Y., Mura, M., Guerbois Galla, M., Despres, P., Tangy, F. et al. (2016) Comparative analysis of viral RNA signatures on different RIG-I-like receptors. eLife, 5. [0205] 14. Ho, T. H., Kew, C., Lui, P. Y., Chan, C. P., Satoh, T., Akira, S., Jin, D. Y. and Kok, K. H. (2015) PACT- and RIG-I-Dependent Activation of Type I Interferon Production by a Defective Interfering RNA Derived from Measles Virus Vaccine. Journal of virology, 90, 1557-1568. [0206] 15. Pfaller, C. K., Radeke, M. J., Cattaneo, R. and Samuel, C. E. (2014) Measles virus C protein impairs production of defective copyback double-stranded viral RNA and activation of protein kinase R. Journal of virology, 88, 456-468. [0207] 16. Saira, K., Lin, X., DePasse, J. V., Halpin, R., Twaddle, A., Stockwell, T., Angus, B., Cozzi-Lepri, A., Delfino, M., Dugan, V. et al. (2013) Sequence analysis of in vivo defective interfering-like RNA of influenza A H1N1 pandemic virus. Journal of virology, 87, 8064-8074. [0208] 17. Li, D., Lott, W. B., Lowry, K., Jones, A., Thu, H. M. and Aaskov, J. (2011) Defective interfering viral particles in acute dengue infections. PloS one, 6, e19447. [0209] 18. Prince, A. M., Huima-Byron, T., Parker, T. S. and Levine, D. M. (1996) Visualization of hepatitis C virions and putative defective interfering particles isolated from low-density lipoproteins. Journal of viral hepatitis, 3, 11-17. [0210] 19. Sun, Y., Jain, D., Koziol-White, C. J., Genoyer, E., Gilbert, M., Tapia, K., Panettieri, R. A., Jr., Hodinka, R. L. and Lopez, C. B. (2015) Immunostimulatory Defective Viral Genomes from Respiratory Syncytial Virus Promote a Strong Innate Antiviral Response during Infection in Mice and Humans. PLoS pathogens, 11, e1005122. [0211] 20. Kessler, J. R., Kremer, J. R. and Muller, C. P. (2011) Interplay of measles virus with early induced cytokines reveals different wild type phenotypes. Virus research, 155, 195-202. [0212] 21. Calain, P. and Roux, L. (1988) Generation of measles virus defective interfering particles and their presence in a preparation of attenuated live-virus vaccine. Journal of virology, 62, 2859-2866. [0213] 22. Whistler, T., Bellini, W. J. and Rota, P. A. (1996) Generation of defective interfering particles by two vaccine strains of measles virus. Virology, 220, 480-484. [0214] 23. Pfaller, C. K., Mastorakos, G. M., Matchett, W. E., Ma, X., Samuel, C. E. and Cattaneo, R. (2015) Measles Virus Defective Interfering RNAs Are Generated Frequently and Early in the Absence of C Protein and Can Be Destabilized by Adenosine Deaminase Acting on RNA-1-Like Hypermutations. Journal of virology, 89, 7735-7747. [0215] 24. Ramsauer, K., Schwameis, M., Firbas, C., Mullner, M., Putnak, R. J., Thomas, S. J., Despres, P., Tauber, E., Jilma, B. and Tangy, F. (2015) Immunogenicity, safety, and tolerability of a recombinant measles-virus-based chikungunya vaccine: a randomised, double-blind, placebo-controlled, active-comparator, first-in-man trial. The Lancet. Infectious diseases, 15, 519-527. [0216] 25. Lucas-Hourani, M., Munier-Lehmann, H., Helynck, O., Komarova, A., Despres, P., Tangy, F. and Vidalain, P. O. (2014) High-throughput screening for broad-spectrum chemical inhibitors of RNA viruses. Journal of visualized experiments: JoVE. [0217] 26. Combredet, C., Labrousse, V., Mollet, L., Lorin, C., Delebecque, F., Hurtrel, B., McClure, H., Feinberg, M. B., Brahic, M. and Tangy, F. (2003) A molecularly cloned Schwarz strain of measles virus vaccine induces strong immune responses in macaques and transgenic mice. Journal of virology, 77, 11546-11554. [0218] 27. Richetta, C., Gregoire, I. P., Verlhac, P., Azocar, O., Baguet, J., Flacher, M., Tangy, F., Rabourdin-Combe, C. and Faure, M. (2013) Sustained autophagy contributes to measles virus infectivity. PLoS pathogens, 9, e1003599. [0219] 28. Brandler, S., Ruffle, C., Combredet, C., Brault, J. B., Najburg, V., Prevost, M. C., Habel, A., Tauber, E., Despres, P. and Tangy, F. (2013) A recombinant measles vaccine expressing chikungunya virus-like particles is strongly immunogenic and protects mice from lethal challenge with chikungunya virus. Vaccine, 31, 3718-3725. [0220] 29. Guerbois, M., Moris, A., Combredet, C., Najburg, V., Ruffle, C., Fevrier, M., Cayet, N., Brandler, S., Schwartz, O. and Tangy, F. (2009) Live attenuated measles vaccine expressing HW-1 Gag virus like particles covered with gp160DeltaV1V2 is strongly immunogenic. Virology, 388, 191-203. [0221] 30. Radecke, F., Spielhofer, P., Schneider, H., Kaelin, K., Huber, M., Dotsch, C., Christiansen, G. and Billeter, M. A. (1995) Rescue of measles viruses from cloned DNA. The EMBO journal, 14, 5773-5784. [0222] 31. Parks, C. L., Lerch, R. A., Walpita, P., Sidhu, M. S. and Udem, S. A. (1999) Enhanced measles virus cDNA rescue and gene expression after heat shock. Journal of virology, 73, 3560-3566. [0223] 32. Kolakofsky, D., Roux, L., Garcin, D. and Ruigrok, R. W. (2005) Paramyxovirus mRNA editing, the “rule of six” and error catastrophe: a hypothesis. The Journal of general virology, 86, 1869-1877. [0224] 33. Pathak, K. B. and Nagy, P. D. (2009) Defective Interfering RNAs: Foes of Viruses and Friends of Virologists. Viruses, 1, 895-919. [0225] 34. Desmyter, J., Melnick, J. L. and Rawls, W. E. (1968) Defectiveness of interferon production and of rubella virus interference in a line of African green monkey kidney cells (Vero). Journal of virology, 2, 955-961. [0226] 35. Caignard, G., Guerbois, M., Labernardiere, J. L., Jacob, Y., Jones, L. M., Infectious Mapping Project, I. M., Wild, F., Tangy, F. and Vidalain, P. O. (2007) Measles virus V protein blocks Jak1-mediated phosphorylation of STAT1 to escape IFN-alpha/beta signaling. Virology, 368, 351-362. [0227] 36. Irie, T., Kiyotani, K., Igarashi, T., Yoshida, A. and Sakaguchi, T. (2012) Inhibition of interferon regulatory factor 3 activation by paramyxovirus V protein. Journal of virology, 86, 7136-7145. [0228] 37. Sparrer, K. M., Pfaller, C. K. and Conzelmann, K. K. (2012) Measles virus C protein interferes with Beta interferon transcription in the nucleus. Journal of virology, 86, 796-805. [0229] 38. Parks, C. L., Witko, S. E., Kotash, C., Lin, S. L., Sidhu, M. S. and Udem, S. A. (2006) Role of V protein RNA binding in inhibition of measles virus minigenome replication. Virology, 348, 96-106. [0230] 39. Parks, C. L., Lerch, R. A., Walpita, P., Wang, H. P., Sidhu, M. S. and Udem, S. A. (2001) Analysis of the noncoding regions of measles virus strains in the Edmonston vaccine lineage. Journal of virology, 75, 921-933. [0231] 40. Salinas, Y. and Roux, L. (2005) Replication and packaging properties of short Paramyxovirus defective RNAs. Virus research, 109, 125-132. [0232] 41. Bhella, D., Ralph, A. and Yeo, R. P. (2004) Conformational flexibility in recombinant measles virus nucleocapsids visualised by cryo-negative stain electron microscopy and real-space helical reconstruction. J Mol Biol, 340, 319-331. [0233] 42. Tapparel, C., Maurice, D. and Roux, L. (1998) The activity of Sendai virus genomic and antigenomic promoters requires a second element past the leader template regions: a motif (G)3 is essential for replication. Journal of virology, 72, 3117-3128. [0234] 43. Perrault, J. (1981) Origin and replication of defective interfering particles. Current topics in microbiology and immunology, 93, 151-207. [0235] 44. Martinez-Gil, L., Goff, P. H., Hai, R., Garcia-Sastre, A., Shaw, M. L. and Palese, P. (2013) A Sendai virus-derived RNA agonist of RIG-I as a virus vaccine adjuvant. Journal of virology, 87, 1290-1300. [0236] 45. Yount, J. S., Gitlin, L., Moran, T. M. and Lopez, C. B. (2008) MDA5 participates in the detection of paramyxovirus infection and is essential for the early activation of dendritic cells in response to Sendai Virus defective interfering particles. Journal of immunology, 180, 4910-4918. [0237] 46. Yoneyama, M., Kikuchi, M., Matsumoto, K., Imaizumi, T., Miyagishi, M., Taira, K., Foy, E., Loo, Y. M., Gale, M., Jr., Akira, S. et al. (2005) Shared and unique functions of the DExD/H-box helicases RIG-I, MDA5, and LGP2 in antiviral innate immunity. Journal of immunology, 175, 2851-2858. [0238] 47. Rothenfusser, S., Goutagny, N., DiPerna, G., Gong, M., Monks, B. G., Schoenemeyer, A., Yamamoto, M., Akira, S. and Fitzgerald, K. A. (2005) The RNA helicase Lgp2 inhibits TLR-independent sensing of viral replication by retinoic acid-inducible gene-I. Journal of immunology, 175, 5260-5268. [0239] 48. Rodriguez, K. R., Bruns, A. M. and Horvath, C. M. (2014) MDA5 and LGP2: accomplices and antagonists of antiviral signal transduction. Journal of virology, 88, 8194-8200. [0240] 49. Parisien, J. P., Bamming, D., Komuro, A., Ramachandran, A., Rodriguez, J. J., Barber, G., Wojahn, R. D. and Horvath, C. M. (2009) A shared interface mediates paramyxovirus interference with antiviral RNA helicases MDA5 and LGP2. Journal of virology, 83, 7252-7260. [0241] 50. Childs, K. S., Randall, R. E. and Goodbourn, S. (2013) LGP2 plays a critical role in sensitizing mda-5 to activation by double-stranded RNA. PloS one, 8, e64202. [0242] 51. Bruns, A. M., Leser, G. P., Lamb, R. A. and Horvath, C. M. (2014) The innate immune sensor LGP2 activates antiviral signaling by regulating MDA5-RNA interaction and filament assembly. Mol Cell, 55, 771-781. [0243] 52. Berghall, H., Siren, J., Sarkar, D., Julkunen, I., Fisher, P. B., Vainionpaa, R. and Matikainen, S. (2006) The interferon-inducible RNA helicase, mda-5, is involved in measles virus-induced expression of antiviral cytokines. Microbes Infect, 8, 2138-2144.