Live recombinant measles-M2 virus—its use in eliciting immunity against influenza viruses
11160858 · 2021-11-02
Assignee
Inventors
- Nicolas Robert Xavier Escriou (Paris, FR)
- Frederic Tangy (Les Lilas, FR)
- Ho Hong Hai Vo (Ivry sur Seine, FR)
Cpc classification
C12N2760/16134
CHEMISTRY; METALLURGY
A61K2039/55
HUMAN NECESSITIES
C12N2760/16122
CHEMISTRY; METALLURGY
C12N2760/18441
CHEMISTRY; METALLURGY
International classification
Abstract
The invention relates to an active ingredient which is a live attenuated recombinant measles virus expressing influenza A virus antigen(s) and to its use in the elicitation of immunity, in particular protective immunity and advantageously broad-spectrum protective immunity against influenza A virus. In particular, the influenza A virus is selected among epidemic seasonal viruses and/or endemic viruses circulating in the human population and advantageously encompasses a pandemic virus such as H1N1v.
Claims
1. A recombinant measles virus (MV) comprising a recombinant genome comprising a cDNA comprising (i) a nucleotide sequence that encodes the sequence of a full-length antigenomic RNA of MV, and wherein one additional transcription unit (ATU) has been inserted upstream of the N gene (ATU1) or in the intergenic region(s) between the P and M genes (ATU2) or between the H and L genes (ATU3) of MV and, (ii) operably linked in frame into said ATU or ATUs, a heterologous polynucleotide that encodes at least a M2 or M2e antigen wherein the heterologous polynucleotide is provided as the insert cloned into the cDNA encoding the full-length antigenomic RNA of MV in a transfer vector wherein the transfer vector is selected from the group consisting of: pTM-MV-ATU1-M2raw having the sequence of SEQ ID No.1, pTM-MV-ATU2-M2raw having the sequence of SEQ ID No.3, pTM-MV-ATU1-M2opt having the sequence of SEQ ID No.2, pTM-MV-ATU2-M2opt having the sequence of SEQ ID No.4, pTM-MV-ATU3-M2opt having the sequence of SEQ ID No.8; pTM-MV-ATU3-N-1xM2e having the sequence of SEQ ID No.5, pTM-MV-ATU3-N-3xM2e having the sequence of SEQ ID No.6 (N is the measles virus N protein); pTM-MV-M1&M2 having the sequence of SEQ ID No.9; pTM-MV-NPflu&M2 having the sequence of SEQ ID No.10 and pTM-MV-ATU2-NPflu having the sequence of SEQ ID No.11 which is the parental construct for pTM-MV-NPflu&M2; pTM-MV-ATU2-NPflu-3xM2e having the sequence of SEQ ID No.13; pTM-MV-ATU2-M1opt having the sequence of SEQ ID No.7; and pTM-MV-ATU2-N-3xM2e having the sequence of SEQ ID No.12, where N is the measles virus Nucleoprotein.
2. The recombinant measles virus according to claim 1, wherein the measles virus is a live attenuated strain selected in the group of the Schwarz strain, the Moraten strain, the Zagreb and the AIK-C strain.
3. An immunogenic composition comprising a recombinant measles virus according claim 1, optionally comprising influenza VLPs and further comprising a pharmaceutical vehicle suitable for administration to a host and optionally an adjuvant of the immune response wherein said composition is optionally formulated for the administration in children.
4. An immunogenic composition according to claim 3, which further comprises Influenza Virus-Like Particles and is obtained from a supernatant or a lysate of cells producing the recombinant measles virus.
5. A method of inducing an immune response for prophylactic protection against flu in a subject, comprising administering the recombinant measles virus according to claim 3 to a subject.
6. A method of inducing formation of antibodies against influenza A virus in a subject and/or inducing a cellular immune response against influenza A virus in a subject, comprising administering the recombinant measles virus according to claim 3 to a subject.
7. A method of protecting a subject against a condition or a disease resulting from the infection by an influenza virus A in a human host, comprising administering the recombinant measles virus according to claim 3 to the subject.
8. The method of claim 7, further comprising protecting the subject from a measles virus infection.
9. A method of protecting a subject against a condition or a disease resulting from the infection by an influenza virus A in a subject, comprising administering a multivalent vaccine comprising the recombinant measles virus according to claim 3 to the subject, such as a combined measles, mumps, rubella and influenza multivalent vaccine or a measles, mumps, rubella, varicella and influenza multivalent vaccine.
10. A transfer vector selected from the group consisting of: pTM-MV-ATU1-M2raw having the sequence of SEQ ID No.1, pTM-MV-ATU2-M2raw having the sequence of SEQ ID No.3, pTM-MV-ATU1-M2opt having the sequence of SEQ ID No.2, pTM-MV-ATU2-M2opt having the sequence of SEQ ID No.4, pTM-MV-ATU3-M2opt having the sequence of SEQ ID No.8; pTM-MV-ATU3-N-1xM2e having the sequence of SEQ ID No.5, pTM-MV-ATU3-N-3xM2e having the sequence of SEQ ID No.6 (N is the measles virus N protein); pTM-MV-M1&M2 having the sequence of SEQ ID No.9; pTM-MV-NPflu&M2 having the sequence of SEQ ID No.10 and pTM-MV-ATU2-NPflu having the sequence of SEQ ID No.11 which is the parental construct for pTM-MV-NPflu&M2; pTM-MV-ATU2-NPflu-3xM2e having the sequence of SEQ ID No.13; pTM-MV-ATU2-M1opt having the sequence of SEQ ID No.7; and pTM-MV-ATU2-N-3xM2e having the sequence of SEQ ID No.12, where N is the measles virus Nucleoprotein.
11. A rescue system for the assembly of infectious recombinant measles virus particles and optionally influenza A VLP, comprising a mammalian cell or cell line, transformed with plasmid vectors suitable for the expression of a polymerase, and for the expression of the N, P and L proteins of a measles virus, wherein said cell is further transfected with a vector according to claim 10.
12. A rescue system according to claim 11, wherein the cell is the 293-T7-NP cell line deposited on Jun. 14, 2006 with the CNCM (Paris, France) under number I-3618 or the 293-Tnls7-NP cell line deposited on Aug. 4, 2006 with the CNCM (Paris, France) under number I-3662.
13. A cell transformed with nucleotide sequences expressing a polymerase, and nucleotide sequences expressing the N, P and L proteins of a measles virus, wherein said cell is further transfected with a vector according to claim 10 in conditions enabling production of recombinant measles virus.
14. A cell culture supernatant or lysate recovered from cells according to claim 13.
15. An immunogenic composition prepared from the cell culture supernatant or lysate according to claim 14.
16. A polynucleotide which is selected from the group consisting of: pTM-MV-ATU1-M2raw having the sequence of SEQ ID No.1, pTM-MV-ATU2-M2raw having the sequence of SEQ ID No.3, pTM-MV-ATU1-M2opt having the sequence of SEQ ID No.2, pTM-MV-ATU2-M2opt having the sequence of SEQ ID No.4, pTM-MV-ATU3-M2opt having the sequence of SEQ ID No.8, pTM-MV-ATU3-N-1xM2e having the sequence of SEQ ID No.5, pTM-MV-ATU3-N-3xM2e having the sequence of SEQ ID No.6, pTM-MV-M1&M2 having the sequence of SEQ ID No.9, pTM-MV-NPflu&M2 having the sequence of SEQ ID No.10 and pTM-MV-ATU2-NPflu having the sequence of SEQ ID No.11, pTM-MV-ATU2-NPflu-3xM2e having the sequence of SEQ ID No.13, pTM-MV-ATU2-M1opt having the sequence of SEQ ID No.7, pTM-MV-ATU2-N-3xM2e having the sequence of SEQ ID No.12, a polynucleotide having the sequence of SEQ ID No.17or a polynucleotide having the sequence of SEQ ID No.18, a polynucleotide having the sequence of SEQ ID No.20, a polynucleotide having the sequence of SEQ ID No.22, a polynucleotide having the sequence of SEQ ID No.24, a polynucleotide having the sequence of SEQ ID No.26, a polynucleotide having the sequence of SEQ ID No.27, a polynucleotide having the sequence of SEQ ID No. 30, and a polynucleotide encoding any of the polypeptide the sequence of which consists of a sequence selected the group consisting of: SEQ ID No.19, SEQ ID No.23, SEQ ID No.25, SEQ ID No.28, SEQ ID No.29, SEQ ID No.31, and SEQ ID No.32.
Description
FIGURE LEGENDS
(1)
(2) A. Schematic representation of the pTM-MVSchw-ATU2 vector containing the Schwarz MV cDNA with a green fluorescent protein (eGFP) gene as an additional transcription unit (ATU) between the P and the M genes (ATU2). Wild-type (M2raw) and codon-optimized (M2opt) synthetic genes coding for the full-length M2 consensus protein were inserted into pTM-MVSchw-ATU2 between the BsiWI and BssHII sites of the ATU, in place of the eGFP gene. Both M2raw and M2opt genes were also inserted into pTM-MVSchw-ATU1 as an additional ATU upstream of the N gene (not shown).
(3) MV genes are indicated: N (nucleoprotein), P (phosphoprotein) and V/C (accessory proteins), M (matrix), F (fusion), H (hemaglutinin), L (polymerase). T7: T7 RNA polymerase promoter. hhR: hammerhead ribozyme. T7t: T7 RNA polymerase terminator. h∂vR: hepatitis delta virus (HDV) ribozyme.
(4) B. Growth kinetics of recombinant MV-M2 viruses. Vero NK cells were infected with parental MVSchw or recombinant MV-ATU2-M2raw or MV-ATU2-M2opt viruses at an MOI of 0.1. The cells were collected at the indicated time points, and the cell-associated virus titers were determined as described in Materials and Methods.
(5) C. Immunofluorescence staining of M2 polypeptides in syncytia of MV-M2-infected Vero NK cells. Cells were fixed 30-36 hours after infection with the indicated viruses and stained with mouse monoclonal anti-M2 antibody (14C2) and AF555-conjugated anti-mouse IgG antibodies. Magnification: ×40.
(6) D. and E. Western blot analysis of M2 polypeptides expression. M2 was detected in lysates of Vero-NK cells infected with the indicated MV-M2 or parental MVSchw viruses using mouse monoclonal anti-M2 antibody (14C2). Lysates of MV-infected cells were diluted 1 in 4 (D) or 1 in 10 (E) before being assayed. Lysates prepared from MDCK cells infected with A/Scotland/20/74 (SCOT) or A/PuertoRico/8/34 (PR8) influenza viruses at a MOI of 5 were used as positive controls. The positions of molecular weight markers (size in kDa) are indicated. D. Two viral clones (1, 2) of MV-M2raw and MV-M2opt were assayed as indicated. E. Lysates were harvested 24 or 36 hours post-infection as indicated.
(7)
(8) A. Schematic representation of the pTM-MVSchw-ATU3 vector containing the Schwarz MV cDNA with a green fluorescent protein (eGFP) gene as an ATU between the H and the L genes (ATU3). Genes encoding the MV N protein fused to a single copy of M2 ectodomain consensus sequence (N-1xM2e) or to three tandem copies of M2e (N-3xM2e) were inserted into pTM-MVSchw-ATU3 between the BsiWI and BssHII sites of the ATU, in place of the eGFP gene.
(9) MV genes are indicated as in
(10) B. Immunofluorescence staining in syncytia of MV-NM2e-infected Vero NK cells. Cells were fixed 30-36 hours after infection with the indicated viruses and stained with mouse monoclonal anti-M2 antibody (14C2) and AF555-conjugated anti-mouse IgG antibodies, and with rabbit polyclonal anti-MV-N and AF488-conjugated anti-rabbit IgG antibodies. Single-color and merged images are shown. Magnification: ×20.
(11) C. and D. Western blot analysis of N-M2e fusion polypeptides expression. N-M2e fusion proteins were detected in lysates of Vero-NK cells infected with the indicated MV-NM2e or parental MVSchw viruses using mouse monoclonal anti-M2 antibody (14C2, panel C) or rabbit polyclonal anti-MV-N(panel D). Two viral clones (1, 2) of MV-N-1xM2e and MV-N-3xM2e were assayed as indicated. Lysates prepared from Vero NK cells infected with MV-ATU1-M2opt or MV-ATU2-M2opt were used as positive controls. The positions of MV N protein and N-M2e fusion proteins as well as molecular weight markers (size in kDa) are indicated.
(12)
(13) Groups of 6 or 12 CD46-IFNAR mice were injected twice intraperitoneally at four-week interval with 10.sup.5 TCID.sub.50 of the indicated recombinant MV-M2 measles viruses or with parental MVSchw, as control. Another group of mice was immunized with two intranasal administration of 0.1 LD50 of mouse-adapted A/Scotland/20/74 (SCOT) influenza virus. Sera were collected 3 weeks after each injection (IS1 and IS2, respectively). M2e-specific (A, C) or MV-specific (B, D) IgG antibody titers were determined by indirect ELISA, as described in Material and Methods. A-D. Each symbol represents an individual mouse, and the short horizontal line indicates the mean value of the group. Detection limits of the assays are indicated by dotted lines.
(14) D-E. Survival curves for 5-6 mice per group were recorded for 22 days after intranasal challenge with 10 LD50 of mouse-adapted A/Scotland/20/74 (H3N2) or A/Paris/2590/09 (H1N1v) influenza viruses.
(15)
(16) Groups of CD46-IFNAR mice were injected twice intraperitoneally at four-week interval with 10.sup.5 TCID.sub.50 of the indicated recombinant MV-NM2e measles viruses or with parental MVSchw, as control. Another group of mice was immunized with MV-ATU2-M2opt. Sera were collected 3 weeks after each injection (IS1 and IS2, respectively). M2e-specific (A) or MV-specific (B) IgG antibody titers were determined by indirect ELISA.
(17) C. M2e-specific, IgG1 (filled circles) and IgG2a (open circles) isotype titers were determined for IS2 sera by indirect ELISA.
(18) D. Specific antibodies against the native form of the M2 protein were measured using a cell-based ELISA, as described in Material and Methods.
(19) A-D. Each symbol represents an individual mouse, and the short horizontal line indicates the mean value of the group. Detection limits of the assays are indicated by dotted lines.
(20) E. Survival curves were recorded for 21 days after intranasal challenge with 10 LD50 of mouse-adapted A/Scotland/20/74 (H3N2) influenza virus.
(21)
(22) Survival of C57BL/6 mice (6 mice/group) lethally challenged with 10 LD50 of mouse-adapted A/Scotland/20/74 (H3N2) influenza virus after passive transfer of pooled sera from CD46-IFNAR mice immunized with MV-ATU1-M2opt or MV-ATU2-M2opt recombinant virus. Control mice received sera from CD46-IFNAR mice immunized with empty parental MVSchw.
(23)
(24) A. Schematic representation of the pTM-MV-ATU2-M1 vector containing the Schwarz MV cDNA with the M1 codon-optimized consensus gene as an ATU between the P and the M genes (ATU2), of the pTM-MV-ATU3-M2 vector with the M2 codon-optimized consensus gene as an ATU between the H and the L genes (ATU3), and of the double recombinant pTM-MV-M1&M2 vector.
(25) MV genes are indicated as in
(26) B. and C. Western blot analysis of M1 and M2 polypeptide expression. M1 and M2 were detected in lysates of Vero-NK cells infected with the indicated recombinant or parental measles viruses using mouse monoclonal anti-M1 (GA2B, panel B) or anti-M2 antibody (14C2, panel C). Three viral clones (1, 2, 3) of each viral construct were assayed as indicated. Lysates prepared from MDCK cells infected with A/Scotland/20/74 (SCOT) influenza virus were used as positive controls. The positions of M1 and M2 proteins as well as molecular weight markers (size in kDa) are indicated.
(27)
(28)
(29) Groups of 12 CD46-IFNAR mice were injected twice intraperitoneally at four-week interval with 10.sup.5 TCID50 of the indicated single recombinant MV-ATU3-M2opt and MV-ATU2-M1opt viruses, with the dual recombinant MV-M1&M2 virus or with parental MVSchw, as control. Another group of mice was immunized with the former MV-ATU2-M2opt. Sera were collected 3 weeks after each injection (IS1 and IS2, respectively). M2e-specific (A) or MV-specific (B) IgG antibody titers were determined by indirect ELISA, as described in Materials and Methods. A-B. Each symbol represents an individual mouse, and the short horizontal line indicates the mean value of the group. Detection limits of the assays are indicated by dotted lines.
(30) C. Survival curves were recorded for 21 days after intranasal challenge with 10 LD50 of mouse-adapted A/Scotland/20/74 (H3N2) influenza virus.
(31) D. Weight curves of infected mice were recorded. Values represent the mean weight of surviving mice, expressed as the percentage of initial weight at day of infection. SD are represented by one-sided vertical bars. For clarity, filled symbols indicate that all mice of the group were alive on the day of monitoring, half-filled symbols that at least one mouse died previously and open symbols that a single mouse remained alive in the group.
(32)
(33) A. Schematic representation of the pTM-MV-ATU2-NPflu vector containing the Schwarz MV cDNA with the NP codon-optimized consensus gene as an ATU between the P and the M genes (ATU2), of the pTM-MV-ATU3-M2 vector with the M2 codon-optimized consensus gene as an ATU between the H and the L genes (ATU3), and of the dual recombinant pTM-MV-NPflu&M2 vector. MV genes are indicated as in
(34) B. Schematic representation of the pTM-MVSchw-ATU2 vector as described in
(35) C. and D. Western blot analysis of NP and M2 polypeptide expression. NP and M2 (or M2e) were detected in lysates of Vero-NK cells infected with the indicated recombinant or parental measles viruses using rabbit polyclonal anti-influenza virion antibodies (panel C) or mouse monoclonal anti-M2 antibody (14C2, panel D). Three or four viral clones (1, 2, 3, 4) of each viral construct were assayed as indicated. Lysates prepared from MDCK cells infected with A/Scotland/20/74 (SCOT) influenza virus were used as positive controls. The positions of NPflu, M2 and NPflu-3xM2e proteins as well as molecular weight markers (size in kDa) are indicated.
(36)
(37) Groups of CD46-IFNAR mice were injected twice intraperitoneally at four-week interval with 10.sup.5 TCID50 of the indicated single recombinant MV-ATU2-NPflu and MV-ATU3-M2opt viruses, with the dual recombinant MV-NPflu&M2 virus or with parental MVSchw, as control. Sera were collected 3 weeks after each injection (IS1 and IS2, respectively). M2e-specific (A) or MV-specific (B) IgG antibody titers were determined by indirect ELISA.
(38) C. Frequency of influenza-specific IFN-γ-producing T cells in the spleens of immunized mice was quantified by ELISPOT 7 to 10 days after a single injection of the indicated recombinant measles viruses. ELISPOT was performed as described in Materials and Methods, in response to the NP366 (H3N2) consensus peptide (class I), the NP366 (SCOT) peptide of the A/Scotland/20/74 challenge virus (class I) and the NP260-273 conserved peptide (class II).
(39) A-C. Each symbol represents an individual mouse, and the short horizontal line indicates the mean value of the group. Detection limits of the assays are indicated by dotted lines.
(40) D. Survival curves were recorded for 21 days after intranasal challenge with 10 LD50 of mouse-adapted A/Scotland/20/74 (H3N2) influenza virus.
(41) E. Weight curves of infected mice were recorded. Values represent the mean weight of surviving mice, expressed as the percentage of initial weight at day of infection. SD are represented by one-sided vertical bars. For clarity, filled symbols indicate that all mice of the group were alive on the day of monitoring and half-filled symbols that at least one mouse died previously.
(42)
(43) A. Schematic representation of the pTM-MVSchw-ATU2 vector containing the Schwarz MV cDNA with a green fluorescent protein (eGFP) gene as an ATU between the P and the M genes (ATU2). The gene encoding the MV N protein fused to three tandem copies of M2e (N-3xM2e) was inserted into pTM-MVSchw-ATU2 between the BsiWI and BssHII sites of the ATU, in place of the eGFP gene.
(44) MV genes are indicated as in
(45) B. and C. Western blot analysis of N-M2e fusion polypeptide expression. N-3xM2e fusion protein was detected in lysates of Vero-NK cells infected with the indicated virus using mouse monoclonal anti-M2 antibody (14C2, panel B) or rabbit polyclonal anti-MV-N (panel C). Two viral clones (1, 2) of MV-ATU2-N-3xM2e were assayed as indicated. Lysates prepared from Vero NK cells infected with MV-ATU3-N-3xM2e or MVSchw were used as positive controls. The positions of MV N protein and N-3xM2e fusion protein as well as molecular weight markers (size in kDa) are indicated.
EXAMPLES
(46) Materials and Methods
(47) Cell Lines and Viruses
(48) Vero-NK (African Green Monkey Kidney) cells were grown at 37° C. under 5% CO.sub.2 in complete DMEM (Dulbecco's modified Eagle medium with 4.5 mg/ml L-glucose, 100 U/ml penicillin and 100 μg/ml streptomycin), supplemented with 5% heat-inactivated fetal bovine serum (FBS). Helper 293-T7-MV cells stably expressing T7 RNA polymerase and N and P genes from Schwarz MV were grown in complete DMEM supplemented with 10% FBS.
(49) MDCK (Madin-Darby canine kidney) cells were grown at 37° C. under 5% CO.sub.2 in MEM (Minimum Essential Media), supplemented with penicillin, streptomycin and 10% FBS (MEM-10). MDCK-M2 cells that constitutively express the full-length M2 consensus sequence were generated by repeated transduction with a lentiviral TRIP-M2opt vector. Cells expressing high-levels of M2 on the cell surface were selected by fluorescence-activated cell sorting and stably maintained in culture in MEM-10 supplemented with 20 μg/ml of rimantadine, an M2 proton channel inhibitor (Sigma-Aldrich).
(50) The mouse-adapted influenza A/Scotland/20/74 (H3N2) virus was previously described (Ramisse et al., 1998). Working stocks were prepared from lung homogenates by two successive amplifications in MDCK cells at a multiplicity of infection (MOI) of 10.sup.−3 for 3 days at 35° C. in completed MEM supplemented with 1 μg/ml TPCK-treated trypsin. Virus titers were determined on MDCK cells by a standard plaque assay under Avicel® overlays and are expressed as plaque forming unit per ml (pfu/ml).
(51) The A/Paris/2590/09 virus (Jonges M. et al 2010) was isolated by the National Influenza Center (Northern-France) at the Institut Pasteur in Paris (France) from nasal swab collected in Paris during the 2009 pandemic, and passaged twice in MDCK cells. The eight genomic segments were cloned from viral RNA into a bidirectional transcription plasmid derived from pHW2000 (Hoffmann et al., 2002) to generate recombinant viruses. The rescued virus was adapted to mice by serial passage of pulmonary homogenates of infected to naive mice, and further amplified in MDCK cells as described above to constitute working stocks.
(52) Plasmid Constructs
(53) The MVSchw recombinant plasmid constructs were derived from the previously described pTM-MVSchw-ATU1, -ATU2 and -ATU3 plasmid vectors (Combredet et al., 2003). These vectors were cloned from a commercial batch of the licensed vaccine Rouvax (kindly provided by Sanofi Pasteur MSD, Marcy l'Etoile, France). They carry an infectious cDNA corresponding to the anti-genome of the Schwarz MV vaccine strain and an additional transcription unit containing unique BsiWI and BssHII restriction sites for the insertion of foreign open reading frames upstream from the N gene (ATU1), between the P and M genes (ATU2) and between the H and L genes (ATU3).
(54) A full-length M2 consensus sequence, reflecting circulating human influenza lineages (seasonal A/H3N2 and A/H1N1 strains, as well as the 2009 pandemic A/H1N1 variants i.e., H1N1v), was generated from complete M2 coding sequences available at the NCBI Influenza Virus Sequence Database, accessed in October 2011 (Bao et al., 2008). Briefly, a total of 1148 complete M2 coding sequences, representing the spectrum of H3N2 (540 sequences), H1N1 (386 sequences) and H1N1v (222 sequences) influenza A diversity in humans from 1918 to 2011, were selected and downloaded. From these data, sequence alignments and consensus sequences were computed for each of the three subtypes on the CLC Main Workbench platform (version 6.1.1) using the default settings. Next, a global consensus was generated by giving the same weight to each of H3N2, H1N1 and H1N1v M2 consensus and following the majority rule whenever applicable. When the three consensus differed at a given position, the H3N2 value was chosen to reflect the predominance of this subtype during the 2011-2012 flu season in the northern hemisphere at the time of design. The global consensus sequence was further edited to remove MV editing (A.sub.5G.sub.3)- and core gene end (A.sub.4CKT)-like sequences on both strands. This consensus sequence was named M2raw and has the sequence of SEQ ID No. 17.
(55) The full-length M2raw consensus sequence was chemically synthesized by Geneart (Life Technologies) with additional BsiWI and BssHII restriction sites at the 5′ and 3′ ends, respectively. The sequence respects the “rule of six”, which stipulates that the number of nucleotides of the MV genome must be a multiple of 6 (Calain and Roux, 1993; Schneider et al., 1997). A human codon-optimized version of the consensus (M2opt) was also synthesized and has the sequence of SEQ ID No. 18. In addition to codon bias optimization for high expression in mammalian cells, MV editing (A.sub.5G.sub.3)- and core gene end (A.sub.4CKT)-like sequences, and regions of very high (>80%) or low (<30%) GC content were avoided whenever possible. Furthermore, cis-acting sequence motifs such as internal TATA-boxes, chi-sites, ribosomal entry sites, ARE, INS, and CRS sequence elements, as well as repetitive sequences, RNA secondary structures and splice donor and acceptor sites, were avoided. Both M2raw and M2opt cDNAs were inserted into BsiWI/BssHII-digested pTM-MVSchw-ATU1(eGFP) vector, resulting in pTM-MV-ATU1-M2raw and pTM-MV-ATU1-M2opt plasmids. Similarly, pTM-MV-ATU2-M2raw and pTM-MV-ATU2-M2opt were generated by inserting both cDNAs into pTM-MVSchw-ATU2(eGFP).
(56) A construct encoding the MV N protein fused to a single copy of M2 ectodomain (M2e) derived from the consensus sequence was chemically synthesized by Geneart. This construct encompassed the 5′ extremity of the MV rescue plasmid from the T7 and hammerhead ribozyme sequence up to nt 2042 of MV antigenome. It contains unique BspE1 and BstB1 restriction enzyme sites in order to permit subsequent exchange of the M2e ectodomain sequence. In this synthetic gene, the M2e peptide (SLLTEVETPI RNEWGCRCND SSD SEQ ID No.21) is connected to the C-terminus of MV nucleoprotein through a flexible SGGSGG linker (N-1xM2e fusion protein). A second construct was obtained by exchange of the BspE1-BstB1 fragment with a synthetic and codon-optimized sequence encoding three tandem copies of M2e consensus connected by GGG spacers. A restriction enzyme site was also included after the third copy of M2e sequence for further subcloning. Next, the construct encoding the N-3xM2e fusion protein was used as a template for PCR amplification using the forward primer 5′-AGTCGTACGGAGATGGCCACACTTTTAAGG-3′ (SEQ ID No.33) containing BsiWI restriction site (underlined) and the reverse primer 5′-GGCCTTGAGAGCCCGGATG-3′ (SEQ ID No.34). The N-1xM2e coding sequence was amplified by PCR using the same forward primer and the reverse primer 5′-GTTGCGCGCTCGTTATCAATCAGAGCTGTCGTTGCAC-3′ (SEQ ID No.35) containing BssHII restriction site. After digestion with BsiWI and BssHII restriction enzymes, the resulting DNA fragments were inserted into the corresponding sites of pTM-MVSchw-ATU3(eGFP) plasmid and both pTM-MV-ATU3-N-1xM2e and pTM-MV-ATU3-N-3xM2e constructs were checked by sequencing of the insert.
(57) Rescue of Recombinant MV-M2 and MV-NM2e Viruses
(58) The pTM recombinant plasmids were used to rescue recombinant viruses using a helper-cell-based system as previously described (Combredet et al., 2003). Single viral clones were amplified on Vero-NK cells. All viral stocks were produced after infection at a MOI of 0.1, stored at −80° C. and titrated by an endpoint limiting dilution assay on Vero-NK cell monolayers. Infectious titers were determined as 50% tissue culture infectious doses (TCID.sub.50) according to the Reed and Muench method (Reed and Muench, 1938). Growth curves of recombinant and parental viruses were determined on Vero-NK cells infected at a MOI of 0.1, as described (Combredet et al., 2003).
(59) Immunofluorescence Assays
(60) Monolayers of Vero-NK cells plated on 20 mm glass coverslips in a 12-well plate were infected with the recombinant or parental MVSchw viruses at a MOI of 0.01. When syncytia were clearly visible but not yet confluent (30-36 hours post-infection), cells were washed in Dulbecco's PBS and fixed with PBS-4% paraformaldehyde for 20 minutes. In order to analyze the expression of N-M2e fusion proteins, cells were further permeabilized with PBS-0.2% triton X-100 for 10 minutes at 4° C. Coverslips were then incubated with 0.5 μg/ml of 14C2 mouse anti-M2e monoclonal antibody (Santa Cruz Biotechnology) or with rabbit polyclonal anti-MV-N(Covalab) diluted 1/1500 in PBS-1% donkey serum (DKS). After subsequent incubation with Alexa Fluor-labeled donkey anti-mouse or anti-rabbit IgG conjugates (Life Technologies, 1/500 dilution), the coverslips were mounted on slides with DAPI-containing Prolong Gold Antifade Reagent (Life Technologies) and analyzed under a DM IRB fluorescence microscope (Leica) using a 20× objective or 40× oil immersion objective. Pictures were acquired with a QICAM Fast 1394 camera (QImaging) and processed with the Qcapture Pro software (version 6.0.0.412, QImaging).
(61) Western Blots
(62) Monolayers of Vero-NK cells were infected at a MOI of 0.05 with the recombinant MV-M2, MV-NM2e or parental MVSchw viruses. 24 or 36 h post-infection, cell extracts were harvested in Laemmli sample buffer and denatured by heating at 95° C. for 10 min. Proteins were separated by 4-12% SDS Bis-Tris polyacrylamide gels (Life Technologies) and transferred onto a PVDF membrane prior to immunoblotting with 14C2 anti-M2e antibody (Santa Cruz Biotechnology, 0.2 μg/ml) or with anti-MV-N antibody (Covalab, 1/10000 dilution). Following incubation with Alexa Fluor 680-labeled donkey anti-mouse or anti-rabbit IgG conjugates (Life Technologies, 1/40000 dilution), fluorescence was captured with an Odyssey Infrared Imaging system (Li-Cor Biosciences).
(63) Mice Experiments and Characterization of Humoral Immune Responses
(64) All experiments were approved and conducted in accordance to the Pasteur Institute guidelines in compliance with European animal welfare regulations (http://ec.europa.eu/environment/chemicals/lab _animals/home_en.htm). The protocol was approved by the Institut Pasteur animal care and use committee. All experiments were conducted under enhanced biosafety level 2 conditions. To obtain CD46.sup.+/− IFNα/βR.sup.−/− mice permissive for measles vaccine (Mrkic et al., 1998), FVB mice heterozygous for the measles vaccine CD46 receptor transgene were backcrossed to 129/Sv mice lacking the type IFN (Combredet et al., 2003). After more than 10 generations of backcrossing in our breeding colony, the resulting CD46-IFNAR line acquired a uniform 129/Sv background.
(65) Six- to nine-week-old CD46-IFNAR mice were used to assess the immune response induced by recombinant MV-M2 and MV-NM2e viruses. Unless otherwise stated, groups of 6 mice were injected intraperitoneally (i.p.) with 10.sup.5 TCID.sub.50 of recombinant or parental MVSchw or with PBS as a control. Booster injections were administered four weeks thereafter. Serum samples were collected three weeks after each injection (IS1 and IS2 sera, respectively).
(66) Antibody response to the M2 protein in immunized mice was measured by indirect ELISA. Briefly, biotinylated M2e peptide corresponding to the consensus M2e sequence (SLLTEVETPIRNEWGCRCNDSSDK-biotin—SEQ ID No. 39, Eurogentec) was immobilized on streptavidine-coupled microtiter plates (Nunc) at a concentration of 1 μg/ml in 50 μl PBS. The M2e-coupled plates were subsequently incubated with serial dilutions of the test sera. Bound antibodies were revealed with mouse-specific anti-IgG secondary antibody conjugated to horseradish peroxidase (Southern Biotech, 1/8000) and TMB (3,3′-5,5′-tetramethylbenzidine, KPL). The isotype determination of the antibody responses was performed using isotype-specific (IgG1 and IgG2a) secondary antibodies coupled to horseradish peroxidase (Southern Biotech). The reaction was stopped by addition of an equal volume of H.sub.3PO.sub.4 (1 M) and absorbance of each well was read at 450 nm/620 nm. The M2e-specific antibody titers were calculated as the reciprocal of the highest dilution of individual serum, giving an absorbance of 0.5 over blank value. MV-specific antibodies were similarly measured using ELISA plates (Maxisorp, Nunc) coated with 50 ng/well of purified measles antigens (Jena Bioscience, Germany).
(67) Specific antibodies against the native form of the M2 protein were measured using a cell-based ELISA. Briefly, monolayers of MDCK and MDCK-M2 cells in 96-well microtiter plates were incubated with serial dilutions of the test sera. Bound antibodies were revealed with anti-mouse IgG secondary antibody and TMB substrate as described above. Readings from wells seeded with MDCK cells were subtracted from wells with MDCK-M2 cells and the M2-specific IgG titers were calculated as the reciprocal of the highest dilution of individual serum, giving an absorbance of 0.2.
(68) Challenge Infection of Animals with Influenza Virus
(69) Four weeks after the second immunization, animals were lightly anesthetized with ketamine/xylazine solution (50 mg/kg and 10 mg/kg respectively) and, unless otherwise stated, inoculated intranasally with 10 Lethal Dose 50 (LD50) of virus in 30 μl PBS. Mice were weighed every other day and monitored daily for signs of morbidity and mortality over 21 days. Animals that lost more than 30% of their initial weight were euthanized by cervical dislocation.
(70) Passive Immunization and Virus Challenge
(71) Immune sera were prepared from CD46-IFNAR mice previously immunized with MV-M2 recombinant viruses and control MVSchw virus. Blood was collected 3 and 4 weeks after the second administration of virus, and serum was prepared by clotting the blood at room temperature for 2 to 4 hours, followed by 2 hours incubation at 4° C. and centrifugation Sera were pooled per immunization group, filter-sterilized and kept at −20° C.
(72) Eight-week-old C57BL/6 mice (Charles River) were injected by the i.p. route with 400 μl of pooled immune serum, diluted in PBS up to 500 μl, or with 500 μl PBS alone as a control. The day after, the passively immunized mice were challenged with 10 LD50 of the mouse-adapted A/Scotland/20/74 (H3N2) strain and monitored, as described above.
(73) Supplementary Materials and Methods for the Construction and Characterization of Recombinant MV-M1 and MV-M1&M2 Viruses
(74) A full-length M1 consensus sequence, reflecting circulating human influenza lineages (seasonal A/H3N2 and A/H1N1 strains, as well as the 2009 pandemic A/H1N1 variants), was generated from complete M1 protein sequences available at the NCBI Influenza Virus Sequence Database, accessed in January 2013. Briefly, a total of 4686 complete M1 protein sequences, representing the spectrum of H3N2 (1480 sequences from the period of 2007-2012), H1N1 (1740 sequences from the period of 1977-2012) and H1N1v (1466 sequences) influenza A diversity in humans from 1977 or 2007 to 2012, were selected and downloaded. From these data, sequence alignments and consensus sequences were computed for each of the three subtypes on the CLC Main Workbench platform (version 6.7.1) using the default settings. Next, a global consensus was generated by giving the same weight to each of H3N2, H1N1 and H1N1v M1 consensus and following the majority rule whenever applicable. When the three consensus differed on a given position, the H3N2 value was chosen to reflect the overall predominance of this subtype during the 2011-2012 and 2012-2013 flu seasons in the northern hemisphere preceeding the time of design. The amino acid sequence of the M1 global consensus was then processed to generate a codon-optimized nucleotide sequence for high expression in mammalian cells. This coding sequence was further edited to inhibit alternative splicing and prevent synthesis of truncated M2-like polypeptide, and to avoid MV editing (A.sub.5G.sub.3)- and core gene end (A.sub.4CKT)-like sequences, and regions of very high (>80%) or low (<30%) GC content whenever possible. Furthermore, cis-acting sequence motifs such as internal TATA-boxes, chi-sites, ribosomal entry sites, ARE, INS, and CRS sequence elements, as well as repetitive sequences, RNA secondary structures and other cryptic splice donor and acceptor sites, were avoided. The optimized nucleotide sequence of the M1 global consensus (M1opt) was chemically synthesized (Geneart, Life Technologies) with additional BsiWI and BssHII restriction sites at the 5′ and 3′ ends, respectively. The sequence respects the “rule of six”, which stipulates that the number of nucleotides of the MV genome must be a multiple of 6.
(75) Insertion of M1opt and M2opt cDNAs in BsiWI/BssHII-digested pTM-MVSchw-ATU2(eGFP) and pTM-MVSchw-ATU3(eGFP) vectors resulted in pTM-MV-ATU2-M1 and pTM-MV-ATU3-M2 plasmids, respectively. These two plasmids were then digested with SaII restriction enzyme and ligated to produce the double recombinant pTM-MV-M1&M2 plasmid.
(76) Rescue and characterization of MV-ATU2-M1, MV-ATU3-M2 and MV-M1&M2 recombinant viruses were performed as described above. The GA2B anti-M1 mAb (Thermo Scientific, 0.2 μg/ml) was used for immunofluorescence and western blot assays of M1 expression.
(77) Supplementary Materials and Methods for the Design of a Consensus Nucleoprotein (NP) Gene and of a Construct Encoding the NP Consensus Protein Fused to 3 Copies of M2e (NPflu-3xM2e)
(78) A full-length nucleoprotein (NP) consensus sequence, reflecting circulating human influenza lineages (seasonal A/H3N2 and A/H1N1 strains, as well as the 2009 pandemic A/H1N1 variants), was generated from complete NP protein sequences available at the NCBI Influenza Virus Sequence Database, accessed in May 2015. Briefly, a total of 1494 complete NP protein sequences, representing the spectrum of H3N2 (746 sequences from the period of 1968-2015), H1N1 (249 sequences from the period of 1977-2015) and H1N1v (499 sequences) influenza A diversity in humans from 1968 to 2015, were selected (after collapsing identical sequences) and downloaded. From these data, sequence alignments and consensus protein sequences were computed for each of the three subtypes on the CLC Main Workbench platform (version 6.8.4) using the default settings. Next, a global consensus was generated by giving the same weight to each of H3N2, H1N1 and H1N1v NP consensus and following the majority rule whenever applicable. When the three protein consensus sequence differed on a given position, the H3N2 value was chosen to reflect the overall predominance of this subtype during the 2011-2012, 2012-2013, and 2014-2015 flu seasons in the northern hemisphere preceding the time of design. The amino acid sequence of the NP global consensus was then processed to generate a codon-optimized nucleotide sequence for high expression in mammalian cells. Regions of very high (>80%) or low (<30%) GC content were avoided whenever possible, and cis-acting sequence motifs like internal TATA-boxes, chi-sites, ribosomal entry sites, ARE, INS, and CRS sequence elements, as well as repetitive sequences, RNA secondary structures and splice donor and acceptor sites, were avoided. The sequence was further edited to remove MV editing (A.sub.5G.sub.3)- and core gene end (A.sub.4CKT)-like sequences on both strands. BsiWI and BssHII restriction sites were then added at the 5′ and 3′ ends, respectively, of the nucleotide sequence (NPflu) of the NP global consensus. The sequence respects the “rule of six”, which stipulates that the number of nucleotides of the MV genome must be a multiple of 6 and has the sequence of SEQ ID No. 30.
(79) A further construct encoding the NP consensus protein fused to three copies of M2e (NPflu-3xM2e) was generated by PCR amplification using the NPflu consensus gene as a template and the forward primer 5′-AGTCGTACGG CCACCATGGC CTCTC-3′ 5seq id No; 36) containing BsiWI restriction site (underlined) and the reverse primer 5′—TCGGCGCGCG ATCCTCCGGA GTTGTCGTAC TCTTCGGCGT TG-3′ (SEQ ID No.37) containing BspE1 and BssHII restriction enzyme sites (underlined). The resulting cDNA was cloned into the PCR4Blunt-TOPO plasmid. It contained unique BspE1 and BssHII restriction enzyme sites at the 3′ extremity of the NPflu coding sequence which permitted subsequent insertion of the synthetic and codon-optimized 3xM2e sequence described above.
(80) Supplementary Materials and Methods for the Construction and Characterization of Recombinant MV-NPflu&M2 and MV-NPflu-3xM2e Viruses
(81) Both NPflu and NPflu-3xM2e cDNAs were inserted into BsiWI/BssHII-digested pTM-MVSchw-ATU2(eGFP) vector, resulting in pTM-MV-ATU2-NPflu (SEQ ID No.11) and pTM-MV-ATU2-NPflu-3xM2e plasmids. (SEQ ID No.13) The plasmids pTM-MV-ATU2-NPflu and pTM-MV-ATU3-M2 were then digested with SaII restriction enzyme and ligated to produce the double recombinant pTM-MV-NPflu&M2 (SEQ ID No.10) plasmid.
(82) Rescue of MV-ATU2-NPflu, MV-NPflu&M2 and MV-ATU2-NPflu-3xM2e recombinant viruses was performed in 293T-T7-MV helper cells as described above. The viruses were characterized by sequencing of their genome and by western blot assay of influenza NP and M2 (or M2e) expression (
(83) Supplementary Materials and Methods for the Construction and Characterization of Recombinant MV-ATU2-N-3xM2e Virus
(84) The N-3xM2e coding sequence was obtained by digestion with BsiWI and BssHII restriction enzymes of the pTM-MV-ATU3-N-3xM2e plasmid. The resulting DNA fragment was then inserted into the corresponding sites of pTM-MVSchw-ATU2(eGFP) vector and the resulting pTM-MV-ATU2-N-3xM2e construct (SEQ ID No.12) was checked by sequencing of the insert.
(85) Rescue of MV-ATU2-N-3xM2e recombinant virus was performed in 293T-T7-MV helper cells as described above. The virus was characterized by sequencing of its genome and by western blot analysis of infected cell lysates. High level of expression of the N-3xM2e fusion protein was evidenced with anti-MV N antibodies and with the 14C2 anti-M2e monoclonal antibody, demonstrating that such fusion protein can be expressed either from ATU2 (
(86) Supplementary Materials and Methods for the Characterization of Cellular Immune Responses
(87) Spleen cells were collected 7 to 10 days after a single administration of recombinant or parental measles virus and the frequency of influenza virus-specific IFN-γ-producing T cells was quantified in a standard ELISPOT assay. Briefly, 96-wells Multi-screen PVDF plates (Millipore) were coated with 10 μg/ml rat anti-mouse IFN-γ antibodies (R4-6A2, Becton-Dickinson) in PBS. Plates were washed and blocked with complete RPMI medium (RPMI 1640 supplemented with 10% FCS, 10 mM Hepes, 5×10.sup.−5 M R-mercaptoethanol, non-essential amino acids, Sodium Pyruvate, 100 U/ml penicillin and 100 μg/ml styreptomycin) for 2 h. Various numbers of splenocytes (typically 4×10.sup.5, 2×10.sup.5 and 1×10.sup.5) from immunized and control mice were then plated in triplicate in the presence or absence of the appropriate peptide (10 μM) and IL2 (10 U/ml). The cells were incubated for 20 h at 37° C., and after extensive washes, the spots were revealed by successive incubations with biotinylated rat anti-mouse IFNγ antibodies (XMG1.2, Becton-Dickinson), alkaline phosphatase-conjugated streptavidin (Becton-Dickinson) and 5-bromo-4-chloro-3-indolylphosphate/nitroblue tetrazolium (BCIP/NBT, Sigma) as the substrate. The spots were counted using the automated S6 Ultimate-V analyzer and associated Immunosoft software (CTL Analyzer). For each mouse, the number of peptide-specific IFNγ-producing cells was determined by calculating the difference between the number of spots generated in the absence and in the presence of the peptide. Results were expressed as the number of spot-forming cells (SFCs) per 10.sup.6 splenocytes.
(88) The NP366 (H3N2) consensus peptide (ASNENMDNM—SEQ ID No.41), the NP366 (Scotland) peptide (ASNENMDTM—SEQ ID No.42) and the NP263-276 conserved peptide (ALILRGSVAHKSCL—SEQ ID No.43) were synthesized by Eurogentec and used to measure the frequency of influenza-specific T cells. The measles H22-30 (RIVINREHL—SEQ ID No.44) and H446-454 (SNHNNVYWL—SEQ ID No.45) peptides and the LCMV NP396-404 (FQPQNGQFI—SEQ ID No.46) peptide were used as positive and negative control peptides, respectively.
(89) Results
(90) Recombinant MVSchw Express the Full-Length M2 Consensus Protein and Replicate Efficiently
(91) A full-length M2 consensus sequence was designed by the inventors, reflecting circulating human influenza lineages, seasonal A/H3N2 and A/H1N1 strains, as well as the 2009 pandemic A/H1N1 variant strain, from complete coding sequences available at the NCBI in November 2011. This global consensus (M2raw) was generated from separate H3N2, H1N1 and H1N1v consensus, as described in materials and methods and was edited to remove potential MV editing- and polyadenylation sites. The global consensus amino acid sequence is identical to the H3N2 consensus with the exception of a single conservative substitution (Val51Ile) in the cytoplasmic domain, but different from the H1N1 (7 substitutions) and the H1N1v consensus (15 substitutions). When the M2 ectodomain (M2e) region only is considered, the global M2e consensus is identical to both H1N1 and H3N2 consensus, and differs at 4 positions from the H1N1v consensus (Table 1).
(92) Both M2raw and a human codon-optimized version (M2opt) of the consensus gene were inserted as an additional transcription unit (ATU) into MV vector, either in position 3 (ATU2,
(93) The growth of recombinant MV-ATU2 viruses was next analyzed in Vero-NK cells. Growth kinetics of both recombinant MV-ATU2-M2raw and MV-ATU2-M2opt were slightly delayed compared to that of parental MVSchw (
(94) Indeed, expression of M2 consensus protein at the surface of infected cells was evidenced by immunofluorescence analysis of non-permeabilized cells with the 1402 mouse monoclonal antibody directed against the extracellular M2 ectodomain (
(95) Recombinant MVSchw Express an Additional N Protein Fused to M2e Ectodomain and Replicate Efficiently
(96) To enhance M2e immunogenicity, we sought to express M2e as a fusion protein with measles nucleoprotein (N), thereby achieving multimerization and display of M2e on the viral nucleoprotein. To that end, one or three tandem copies of the 23 aa-consensus M2e sequence was genetically linked to the measles N C-terminus through a flexible SGGSGG linker (SEQ ID No.38), and the resulting protein was expressed from the ATU located in position 6 of the pTM-MVSchw-ATU3 vector (
(97) Both recombinant MV-N-1xM2e and MV-N-3xM2e viruses were successfully rescued in helper 293-T7-MV, as indicated by the formation of syncytia in MV-infected Vero-NK cells (
(98) MV-M2 and MV-N-M2e Induce Th1-Type Immune Response
(99) The immunogenicity of the recombinant MV-M2 viruses was investigated in genetically modified CD46-IFNAR mice susceptible to MV infection (Mrkic et al., 1998) and compared to the immunogenicity of M2 expressed during experimental influenza infection with the mouse-adapted A/Scotland/20/74 strain (
(100) M2e- and measles-specific antibody responses were evaluated for each individual mouse by indirect ELISA against M2e peptide and MV antigens, respectively. M2e peptide was biotinylated at its C-terminus and immobilized on streptavidine-coupled microtiter plates in order to ensure that the peptide was displayed onto the polystyrene wells in a conformation as close as possible to its natural conformation, where it is C-terminally linked to M2 transmembrane region and displayed on the cell surface. Indeed, preliminary experiments showed that recognition of the M2e peptide by the 14C2 monoclonal antibody as well as by polyclonal antibodies induced in mice by experimental influenza infection was much more efficient when the M2e peptide was biotinylated and immobilized, rather than adsorbed on the plastic surface of ELISA plates (not shown).
(101) High titers of anti-M2e IgG were raised in all mice to similar levels after the first injection of recombinant MV-ATU2-M2 viruses (
(102) Next, the immunogenicity of the recombinant MV-N-M2e viruses was compared to that of the MV-ATU2-M2opt virus. Significant titers of anti-M2e IgG were raised in all mice after the first injection of recombinant MV-N-3xM2e virus (2.7±0.2 log 10 titer), whereas sera from control animals and sera from animals that received the MV-N-1xM2e remained negative (log 10 titers <1.7,
(103) To analyze the polarization of the immune response induced by the recombinant MV vaccines, we next measured the level of IgG1 and IgG2a isotypes three weeks after the second injection (
(104) Interestingly, antibodies to MV were raised at similar levels in all mice that received either MVSchw, MV-M2 viruses (
(105) Altogether, these results demonstrated that the measles vector is capable of inducing very high levels of anti-M2e antibodies in CD46-IFNAR mice (H-2b 129/Sv background) whether the full-length M2 gene is expressed from an ATU or 3 tandem copies of the short 23-aa M2e sequence are fused to an extra copy of the N gene. Most interestingly, both vectorization strategy allowed to bypass the H-2 restriction of anti-M2e responses, which was described recently for DNA immunization, adenovirus vectorization and M2e-multiple antigenic peptides (MAP) immunization, and which evidenced very poor responsiveness in mice of the H2.sup.b haplotype (Misplon et al., 2010; Wolf et al., 2011). In addition, anti-M2e responses are likely Th1-skewed, a hallmark of live attenuated viruses and a highly desirable feature for an antiviral vaccine.
(106) Protection of Mice after Homologous and Heterologous Challenge
(107) To determine the protective efficacy of MV-ATU1-M2opt and MV-ATU2-M2opt, we examined the survival of CD46-IFNAR mice after intranasal lethal challenge with 10 LD50 of the homologous A/Scotland/20/74 (H3N2) strain or the heterologous A/Paris/2590/09 (H1N1v) strain. Mice were also monitored for weight change as a measure of illness. All mice immunized as a control with the parental MVSchw died within 10 days after challenge with A/Paris/2590/09 (H1N1v) (
(108) To confirm that the antibodies induced by vaccination with MV-M2 recombinant viruses were responsible for the observed protection, serum was prepared from CD46-IFNAR mice immunized with MV-ATU1-M2 or MV-ATU2-M2 and transferred to naïve C57BL/6 recipient mice. Each mouse received 400 μl of pooled immune serum by intraperitoneal injection, a dose sufficient to obtain anti-M2e circulating antibody titers in the recipient mice at approximately 1/10.sup.th the levels present in the donor mice (preliminary experiments, not shown). The day after transfer, mice were challenged with homologous A/Scotland/20/74 (H3N2) and survival curves were recorded (
(109) Together, these results indicate that recombinant MV-M2 viruses induced protective immunity against homologous H3N2 and heterologous H1N1v influenza in CD46-IFNAR mice, and that circulating antibodies against M2 present in the immunized animals contribute to the observed protection.
(110) Antibodies Induced by N-M2e Fusion Proteins Recognize Native Tetrameric M2 and Protect Mice Against Homologous Challenge
(111) Although the mechanism of protection by anti-M2e antibodies remains poorly understood, it relies largely on Fc receptor-dependent elimination of influenza virus-infected cells, ADCC and Ab-dependant cell-mediated phagocytosis (El Bakkouri et al., 2011; Jegerlehner et al., 2004). Therefore, it is critical that anti-M2e antibodies raised by immunization are capable of recognizing native M2e, which is presented as a tetrameric complex at the surface of influenza-infected cells as on influenza virions. To investigate this point, we produced MDCK cells constitutively expressing the full-length consensus M2 protein at the cell surface, as described in materials and methods. We used these MDCK-M2 cells in a cell-based indirect ELISA to analyze binding of sera from MV-N-1xMe- and MV-N-3xM2e-immunized mice (
(112) MV-N-3xM2e recombinant virus induced high levels of antibodies able to bind to MDCK-M2 cells (2.8±0.4 log 10 titer). The titers of anti-MDCK-M2 antibodies induced by N-displayed M2e in these MV-N-3xM2e-immunized mice were 20 times lower than those of antibodies induced by the native M2 consensus protein in MV-ATU2-M2-immunized mice (4.2±0.2 log 10 titer, p<10.sup.−3). Sera from mice immunized with MV-N-1xM2e remained negative (log 10 titer <1.7). The hierarchy of binding titers was indeed comparable to that observed with the peptide ELISA (
(113) Altogether, these results demonstrated that the M2e peptide, displayed and multimerized on measles RNP complexes is able to induce antibodies recognizing the native tetrameric M2 protein expressed at the cell surface of MDCK-M2 cells.
(114) To investigate the protective efficacy of MV-N-1xM2e and MV-N-3xM2e, we examined the survival of CD46-IFNAR mice after intranasal lethal challenge with the homologous A/Scotland/20/74 (H3N2) virus. All mice immunized with the parental MVSchw died within 9 days (
(115) Supplementary Results for the Construction and Characterization of Recombinant MV-M1 and MV-M1&M2 Viruses
(116) Recombinant MVSchw can be Engineered to Express Full-Length M1 and M2 Consensus Proteins from Two ATUs
(117) We designed a full-length M1 consensus sequence, reflecting circulating human influenza lineages, seasonal A/H3N2 and A/H1N1 strains, as well as the 2009 pandemic A/H1N1v strain, from complete protein sequences available at the NCBI in January 2013. This global consensus was generated from separate H3N2, H1N1 and H1N1v consensus, as described in materials and methods. The corresponding nucleotide coding sequence was then generated, codon-optimized for high expression in mammalian cells, and further edited to remove influenza splice sites and prevent synthesis of truncated M2-like polypeptides. It was also edited to remove potential MV editing- and polyadenylation sites. Taking advantage of the gradient of gene expression generated by MV replication (Plumet et al., 2005), the resulting M1opt consensus gene was inserted as an ATU in position 3 (ATU2,
(118) Then, a measles vector with M1opt and M2opt consensus genes inserted in two distinct ATUs was produced by SaII restriction and ligation. The single and double recombinant viruses were successfully rescued in helper 293-T7-MV, as indicated by the formation of syncytia in MV-infected Vero-NK cells and by the sequencing of the ATUs on the rescued virus genomes. All recombinant viruses grew to titers in the 10.sup.7-10.sup.8 TCID.sub.50/ml range, which are similar to those achieved by parental MVSchwarz. Interestingly, M2 toxicity for mammalian cells did not impair growth of MV-ATU3-M2opt and MV-M1&M2 viruses, as it did for MV-ATU2-M2opt, suggesting that M2 expression levels from the distal ATU3 were reduced below toxicity levels, as expected.
(119) Expression of M1 and M2 in Vero-NK cells infected by either the single and double recombinant virus was analyzed by fluorescent western blotting of cell lysates. M2 expression levels were shown to be higher than expression levels in MDCK cells infected with A.Scotland/20/74 influenza virus (
(120) Altogether, these data indicate that measles virus vector may be engineered in order to simultaneously express consensus genes coding for both M1 matrix and M2 ion channel from circulating influenza lineages. This double recombinant virus should drive the production of influenza virus-like particles (VLPs) covered with the M2 protein from infected cells and induce enhanced immune responses in immunized animals against both M1 and M2 influenza antigens.
(121) Dual Recombinant Measles Virus Expressing Both M1 and M2 Consensus Proteins Exhibit Higher Protection Efficiency in Mice than Single Recombinant Viruses.
(122) Immunogenicity of the dual recombinant MV-M1&M2 virus was investigated in CD46-IFNAR mice and compared to the immunogenicity of the single recombinant MV-ATU2-M2opt and MV-ATU3-M2opt viruses. High titers of anti-M2e IgG were raised in all mice after the first injection of either of the 3 recombinant viruses, whereas sera from control animals that received empty MVSchw or MV-ATU2-M1opt remained negative (
(123) Interestingly, antibodies to MV were raised at similar levels in all mice that received either MVSchw or MV-M1&M2 virus (
(124) To investigate the protective efficacy of the dual recombinant MV-M1&M2 virus, we examined the survival of CD46-IFNAR mice after intranasal lethal challenge with the homologous A/Scotland/20/74 (H3N2) virus. Mice immunized with MV-ATU2-M1opt or the parental MVSchw presented a massive and rapid weight loss from day 3 post-challenge and most died within 12 days (
(125) Recombinant MVSchw can be Engineered to Simultaneously Express Full-Length NP Consensus and M2 or M2e Consensus
(126) We designed a full-length NP consensus sequence, reflecting circulating human influenza lineages, seasonal A/H3N2 and A/H1N1 strains, as well as the 2009 pandemic A/H1N1v strain, from complete protein sequences available at the NCBI in May 2015. This global consensus was generated from separate H3N2, H1N1 and H1N1v consensus, as described in materials and methods. The corresponding nucleotide coding sequence was then generated, codon-optimized for high expression in mammalian cells, and further edited to remove potential MV editing- and polyadenylation sites.
(127) Two alternative strategies were then explored for simultaneous expression of NP and M2 consensus sequences.
(128) First, similarly to the strategy used to express M1 and M2 sequences (see above), the resulting NPflu consensus gene was inserted as an ATU in position 3 (ATU2,
(129) Second, NP and M2e were expressed as a chimeric NPflu-3xM2e antigen from the ATU located in position 3 of the pTM-MVSchw-ATU2 vector (
(130) Expression of NPflu and M2/M2e in Vero-NK cells infected by either single or double recombinant virus was analyzed by fluorescent western blotting of cell lysates. NPflu expressions levels were shown to be similar than expression levels in MDCK cells infected with A/Scotland/20/74 influenza virus (
(131) Expression of the NPflu-M2e fusion protein was demonstrated by the presence of a band of higher molecular weight and expected size (64.6 kDa) than that of authentic NP (56 kDa). This band reacted with both anti-influenza virion antibodies and 14C2 anti-M2e monoclonal antibody (
(132) Altogether, these data indicate that measles virus vector may also be engineered in order to simultaneously express consensus genes coding for both NP nucleoprotein and M2 ion channel from circulating influenza lineages. M2 can be expressed either as a full length integral protein from a dedicated ATU, such as in the dual MV-NPflu&M2 recombinant virus, or as a fusion protein between its 23 aa-core M2e ectodomain and NPflu, such as in the single MV-NPflu-3xM2e recombinant virus.
(133) Dual Recombinant Measles Virus Expressing Both NPflu and M2 Consensus Proteins Exhibit Higher Protection Efficiency in Mice than Single Recombinant Viruses.
(134) Immunogenicity of the dual recombinant MV-NPflu&M2 virus was investigated in CD46-IFNAR mice and compared to the immunogenicity of the single recombinant MV-ATU3-M2opt viruses.
(135) High titers of anti-M2e IgG were raised in all mice after the first injection of either of the recombinant viruses, whereas sera from control animals that received empty MVSchw or MV-ATU2-NPflu remained negative (
(136) Together, these results indicated that the dual recombinant MV-NPflu&M2 virus induces significantly better protective immunity against influenza in CD46-IFNAR mice than either of its parental MV-ATU2-NPflu or MV-ATU3-M2opt. Higher protective efficiency was correlated to the induction of both anti-M2e antibodies and anti-NP cellular responses.
(137) Tables
(138) TABLE-US-00001 TABLE 1 Alignment of the M2e consensus amino acid sequences global consensus.sup.† SLLTEVETPI RNEWGCRCND SSD (SEQ ID No. 21) H1N1 consensus ---------- ---------- --- H1N1v consensus ---------T -S--E---S- --- (SEQ ID No. 32) H3N2 consensus ---------- ---------- --- .sup.†used as immunogen in the reported experiments. -indicates matching residues.
CONCLUSION
(139) The objective of this preclinical study was to evaluate the proof-of-concept of a new universal influenza vaccine strategy based on a standard measles vaccine engineered to express a M2 consensus protein. The inventors found that the vaccines induced high titers of anti-M2e antibodies and protected mice from an intranasal lethal challenge with homologous or heterologous influenza viruses.
(140) After two successive immunizations with recombinant MV-M2 or MV-NM2e viruses, mice were partially protected from intranasal infectious challenge with mouse-adapted A/Scotland/20/74 (H3N2) and A/Paris/2590/09 (H1N1v) viruses and passive transfer experiments demonstrated that anti-M2 antibodies contributed to the protection. The anti-M2e responses are likely Th1-skewed, a hallmark of live attenuated viruses and a highly desirable feature for an antiviral vaccine.
(141) Interestingly, as already mentioned above, both vectorization strategy allowed to bypass the H-2 restriction of anti-M2e responses, which was evidenced recently and predicted very poor responsiveness of all mice of the H-2.sup.b MHC haplotype such as the CD46-IFNAR mice used in the study. This indicates that T cell responses induced against the measles vector likely supplied the T helper cellular response, which is needed to trigger B cells to produce anti-M2e antibodies. Remarkably, help was supplied whether M2 epitopes were linked to measles N within the N-M2e hybrid protein (like in a hapten-carrier conjugate) or co-expressed with MV proteins in the case of the MV-ATU1-M2 or MV-ATU2-M2 viruses.
(142) Most interestingly, protection was conferred against challenge with the A/Scotland/20/74 virus but also with the pandemic A/Paris/2590/09 H1N1v strain, whose M2e sequence has an avian origin and differs at 4 positions from the global consensus sequence used for immunization. This result may be predictive of broad protection against a variety of subtypes, since, as an example, avian H5N1 viruses differs only at 3 of the 4 above mentioned positions.
(143) Partial protection suggests that the recombinant vaccine might be improved. In that respect, expression of the hybrid N-M2e proteins as an additional nucleoprotein gene placed in position 6 of the genome (ATU3) did not affect replication in vitro and immunogenicity in mice. This gave the inventors the opportunity for further refinement of this strategy by placing the additional hybrid N-M2e genes upstream in the genome, such as in position 3 (ATU2) between the P and the M genes, thus increasing N-M2e expression and possibly immunogenicity and levels of protection. Levels of protection may also be improved by the co-expression of a second influenza consensus protein, such as an M1 or NP consensus, in a double recombinant measles vaccine. This approach would advantageously complete the induction of broad anti-M2 antibody responses by cellular responses targeting conserved influenza structural proteins. Indeed, the inventors showed that the measles vaccine can be further engineered to express either M1 and M2 consensus proteins, or NP and M2/M2e consensus proteins and that such double recombinant viruses provide enhanced protection against influenza in the IFNAR-CD46 mouse model than any single recombinant virus. Higher protection efficiency of the double MV-NP&M2 recombinant virus correlated to the induction of cross-reactive cellular responses against the NP protein. Interestingly, better protection efficiency of the double MV-M1&M2 recombinant virus correlated to the induction of higher anti-M2e antibody levels, strongly advocating in favor of the production of influenza VLP in cells infected by the double MV-M1&M2 recombinant virus.
(144) Alternatively, partial protection might be indicative of the stringency of the mouse model. The inventors used mouse-adapted viruses for challenge, and relied on stocks prepared after a limited number of passages (less than 2) in cell culture. At the challenge doses used (10 LD50 and less than 10.sup.3 PFU), most control mice died quickly, CD46-IFNAR mice within 8 days (
(145) Because recombinant MV-influenza vaccine can be easily and rapidly produced in large quantities and at low cost in most countries, recombinant MV-influenza vaccines might be used instead of the standard MV vaccine routinely used for infants worldwide, notably through the Expanded Program of Immunization of WHO. Interestingly, the induction of measles-specific immunity was not altered in the mouse preclinical model by the expression of any of the M1, M2 and NP transgenes, suggesting that MV-influenza vaccine might even replace the MV Schwarz strain in the combined measles, mumps, rubella vaccine or the measles, mumps, rubella, varicella vaccines.
(146) In conclusion, the inventors have produced new recombinant MV-influenza viruses able to induce high levels of anti-M2 antibodies and cellular responses to influenza and broad protection from intranasal challenge, thus making the proof-of-concept of this strategy for universal influenza vaccine development. It could also help achieve wide vaccine coverage in both children and adults against zoonotic influenza viruses, such as avian H5N1, H9N2 and H7N9 viruses, in the regions that are affected by cases of animal to human transmission. These characterized universal influenza vaccine candidates deserve to be evaluated in a more adapted, non-immuno-compromised and genetically diverse, non-human-primate model, in which the protective potential of the induced immune responses against field isolates of seasonal and zoonotic influenza strains could be addressed.
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