Recombinant viral vectors and methods for inducing a heterosubtypic immune response to influenza A viruses

Abstract

The present invention relates to recombinant viral vectors and methods of using the recombinant viral vectors to induce an immune response to influenza A viruses. The invention provides recombinant viral vectors based, for example, on the non-replicating modified vaccinia virus Ankara. When administered according to methods of the invention, the recombinant viral vectors are designed to be cross-protective and induce heterosubtypic immunity to influenza A viruses.

Claims

1. A recombinant modified vaccinia virus Ankara (rMVA) comprising a gene cassette encoding a fusion protein comprising at least one influenza A M2 extracellular domain (M2e) polypeptide inserted in an influenza A headless hemagglutinin (h1HA) polypeptide set out in SEQ ID NO: 15.

2. The rMVA of claim 1 wherein the fusion protein comprises one selected from the group consisting of i) the h1HA/M2e fusion protein amino acid sequence set out in SEQ ID NO: 2, ii) the VN/1203 HA amino acid sequence set out in SEQ ID NO: 3, and iii) HA1 amino acids 17-58 of SEQ ID NO: 3, a peptide linker, at least one M2e polypeptide, a peptide linker, HA1 amino acids 290-343 of SEQ ID NO: 3 and HA2 amino acids 344-568 of SEQ ID NO: 3.

3. The rMVA of claim 2, wherein the peptide linkers linking the HA amino acids and M2e amino acids comprise the amino acids GGG set out in SEQ ID NO: 4.

4. The rMVA of claim 1, wherein the fusion protein comprises i) the H5N1 M2e amino acid sequence set out in SEQ ID NO: 5, ii) the H1N1 M2e amino acid sequence set out in SEQ ID NO: 6, iii) the H9N2 M2e amino acid sequence set out in SEQ ID NO: 7, and iv) the H7N2 M2e amino acid sequence set out in SEQ ID NO: 8.

5. The rMVA of claim 1, wherein the fusion protein comprises more than one M2e polypeptide and the M2e polypeptides are linked by a peptide linker.

6. The rMVA of claim 5, wherein the peptide linker linking the M2e polypeptides comprises the amino acids GSAGSA set out in SEQ ID NO: 9.

7. The rMVA of claim 1, wherein expression of the h1HA/M2e fusion protein from the gene cassette is under the control of an mH5 promoter or a selP promoter.

8. A recombinant vaccinia virus comprising the gene cassette set out in SEQ ID NO: 1.

9. The rMVA of claim 1, further comprising a gene cassette encoding an influenza A matrix protein 1 (M1) and a gene cassette encoding an influenza A nucleoprotein (NP).

10. A recombinant modified vaccinia virus Ankara (rMVA) comprising a first gene cassette encoding a fusion protein comprising at least one influenza A M2 extracellular domain (M2e) polypeptide inserted in an influenza A headless hemagglutinin (h1HA) polypeptide and a second gene cassette encoding influenza A nucleoprotein (NP) set out in SEQ ID NO: 14.

11. The rMVA of claim 10, wherein the fusion protein comprises one selected from the group consisting of i) the h1HA/M2e fusion protein amino acid sequence set out in SEQ ID NO: 2, ii) the VN/1203 HA amino acid sequence set out in SEQ ID NO: 3, and iii) HA1 amino acids 17-58 of SEQ ID NO: 3, a peptide linker, at least one M2e polypeptide, a peptide linker, HA1 amino acids 290-343 of SEQ ID NO: 3 and HA2 amino acids 344-568 of SEQ ID NO: 3.

12. The rMVA of claim 11, wherein the peptide linkers linking the HA amino acids and M2e amino acids comprise the amino acids GGG set out in SEQ ID NO: 4.

13. The rMVA of claim 10, wherein the fusion protein comprises i) the H5N1 M2e amino acid sequence set out in SEQ ID NO: 5, ii) the H1N1 M2e amino acid sequence set out in SEQ ID NO: 6, iii) the H9N2 M2e amino acid sequence set out in SEQ ID NO: 7, and iv) the H7N2 M2e amino acid sequence set out in SEQ ID NO: 8.

14. The rMVA of claim 10, wherein expression of the h1HA/M2e fusion protein from the first gene cassette is under the control of an mH5 promoter or a selP promoter.

15. The rMVA of claim 10, wherein expression of NP from the second gene cassette is under the control of an mH5 promoter or selP promoter.

16. A pharmaceutical composition comprising the rMVA of claim 1.

17. A method for inducing a heterosubtypic immune response to influenza A viruses in an individual, the method comprising the step of administering a pharmaceutical composition comprising the rMVA of claim 10 to the individual.

Description

FIGURES

(1) FIG. 1 shows the amino acid sequence (SEQ ID NO: 15) of the headless HA protein encoded by recombinant MVA (rMVA) of the invention. The protein contains a signal sequence (grey), HA1 residues (red), a linker peptide of four glycines (black), HA1 residues (red), and the HA2 stalk region (black). Cysteines 58 and 63 and the polybasic cleavage site (amino acids 112-119) are underlined. FIG. 2 shows the nucleotide sequence (SEQ ID NO: 14) of the headless HA protein encoded by rMVA of the invention.

(2) FIG. 3 shows the amino acid sequence (SEQ ID NO: 2) of headless HA/M2e fusion protein. The designed protein contains a signal sequence (grey), HA1 residues (red), a linker peptide of three glycines (black), the M2e of H5N1 (blue), the six amino acid linker GSAGSA (black), the M2e of H1N1 (equivalent to H2N2, H3N2; green), the six amino acid linker GSAGSA, the M2e of H9N2 (orange), the six amino acid linker GSAGSA, the M2e of H7N2 (pink), a linker peptide of three glycines (black), HA1 residues (red) and the HA2 stalk region (black). The polybasic cleavage site (amino acids 224-231) is underlined. FIG. 4 shows the nucleotide sequence (SEQ ID NO: 1) of the headless HA/M2e fusion protein encoded by rMVA of the invention.

(3) FIG. 5 shows single-insert rMVAs containing influenza genes. A) indicates the h1HA, h1HA/M2e, M2, PB1, or M1 gene cassettes that are located in the recombinant MVA D4R/D5R intergenic locus, at the position corresponding to nucleotide 87,281 of wild type MVA (Antoine et al, supra). B) indicates the NP gene cassette is located in the del 111 locus at the position corresponding to nucleotide 142,992 of wild type MVA.

(4) FIG. 6 shows a Western Blot of chicken cell lysates tested for influenza virus antigens. A) Expression of headless HA and the headless HA/M2e fusion protein using a detection antibody directed against HA. Lane 1, protein ladder, size in kDa; lane 2, MVA-h1HA; lane 3, MVA-h1HA/M2e; lane 4, MVA wt (negative control); and lane 5, MVA-HA-VN (positive control). B) Expression of the headless HA/M2e fusion protein using a detection antibody directed against M2. Lane 1, protein ladder, size in kDa; lane 2, MVA-M2-VN; lane 3, MVA-h1HA/M2e; and lane 4, MVA wt (negative control). The recombinant MVA-M2-VN expresses the M2 protein (weak band below 15 kDa). The anti-M2-antibody binds a peptide at the N-terminus of the M2 protein; thus the expression of the h1HA/M2e fusion protein is also detectable at around 70 kDa (lane 3).

(5) FIG. 7 shows double-insert rMVAs containing influenza genes. The h1HA or h1HA/M2e gene cassette is located in the D4R/D5R intergenic locus, at the position corresponding to nucleotide 87,281 of wild type MVA. The NP gene cassette is located in the del 111 locus at the position corresponding to nucleotide 142,992 of the wild type MVA.

(6) FIG. 8 shows a Western Blot of chicken cell lysates tested for influenza virus antigens. A) Expression of headless HA and the headless HA/M2e fusion protein using a detection antibody directed against HA. Lanes 1 and 7, protein ladder, size in kDa; lane 2, MVA-HA-VN (positive control); lane 3, MVA-h1HA; lane 4, MVA wt (negative control); lane 5, MVA-h1HA/M2e-NP; and lane 6, MVA-h1HA-NP. The h1HA/M2e fusion protein expressed by MVA-h1HA/M2e is visible at around 70 kDa (lane 5). The lower bands at around 40 kDa represent the h1HA expressed by MVA-h1HA-NP and MVA-h1HA. The control construct (MVA-HA-VN), expressing the full length HA protein express the HA0 (band around 80 kDa), the HA1 (band around 55 kDa, and the HA2 (band around 25 kDa). The expression of the HA2 protein is also visible in lanes 3, 5 and 6 as the h1HA and h1HA/M2e proteins also contain the polybasic cleavage site. The specific HA bands are absent in the negative control (lane 4). B) NP expression detected with an NP-specific antibody. Lane 1, protein ladder, size in kDa; lane 2, MVA-D3-NP-VN; lane 3, MVA-h1HA-NP; lane 4, MVA-h1HA/M2e-NP; and lane 5, MVA wt (negative control).

(7) FIG. 9 shows monitoring of weight (A, B), clinical symptoms (C, D) and survival (E, F) after vaccination with recombinant MVAs and challenge with H5N1. As controls, mice were vaccinated with MVA-HA-VN, expressing the full-length HA of A/Vietnam/1203/2004, wt MVA or were treated with PBS (panels A, C, E). Mice were vaccinated with the single recombinant MVA-h1HA, MVA-h1HA/M2e, MVA-NP-VN or the double recombinants MVA-h1HA-NP and MVA-h1HA/M2e-NP (panels B, D, F). After challenge with wild-type H5N1, mice were monitored for 14 days.

(8) FIG. 10 shows monitoring of weight (A, B), clinical symptoms (C, D) and survival (E, F) after vaccination with recombinant MVAs and challenge with H9N2 virus. As controls, mice were vaccinated with the whole virus preparation of H9N2, wt MVA or were treated with PBS (panels A, C, E). Mice were vaccinated with the single recombinant MVA-h1HA, MVA-h1HA/M2e, MVA-NP-VN or double recombinant MVA-h1HA-NP and MVA-h1HA/M2e-NP (panels B, D, F). After challenge with virulent mouse-adapted H9N2 influenza virus, mice were monitored for 14 days.

(9) FIG. 11 shows triple-insert rMVAs containing influenza genes. The h1HA or h1HA/M2e and M1gene cassettes will be located in the D4R/D5R intergenic locus, at the position 87,281 nt of the wt MVA sequence. The NP gene cassette will be located in the del 111 locus at the position 142,992 nt of the wt MVA sequence.

EXAMPLES

(10) The present invention is illustrated by the following examples wherein Example 1 describes the choice and design of influenza A antigens in exemplary recombinant MVA of the invention, Example 2 details the production of single-insert recombinant MVAs, Example 3 describes animal experiments with the single-insert MVAs, Example 4 details the production of double-insert recombinant MVAs, Example 5 describes animal experiments with the double-insert MVAs, Example 6 details the production of triple-insert recombinant MVAs and Example 7 describes animal experiments with the triple-insert MVAs.

Example 1

Choice and Design of Influenza a Antigens

(11) Influenza headless HA, a headless HA/M2e fusion protein, NP, M1, M2 and PB1 were the influenza A antigens chosen to be encoded by exemplary recombinant MVA of the invention.

(12) Monoclonal antibodies against the HA stalk domain, the HA2 region, are broadly cross-reactive and neutralize several subtypes of viruses (Ekiert et al. 2009; Kashyap et al. 2008; Okuno et al. 1993; Sanchez-Fauquier et al. 1987; Sui et al. 2009; Throsby et al. 2008). The antibodies target the HA2 region of the molecule and presumably act by preventing the conformational change of HA at low pH, thus presumably blocking fusion of viral and host membranes during influenza infection. However, the production of soluble, native (neutral pH-like) HA2 immunogen has proven to be difficult, owing to the metastable nature of HA (Chen et al. 1995). To induce an immune response against the neutral pH conformation, a headless HA was chosen as an antigen. The headless HA consists of two HA1 regions that interact with an HA2 subunit, stabilizing the neutral pH conformation (Bommakanti et al., supra; Steel et al., supra).

(13) The extracellular domain of the M2 protein (M2e, 23AS) is highly conserved across influenza A virus subtypes. In animals, M2e specific antibodies reduce the severity of infection with a wide range of influenza A virus strains (Fan et al. 2004; Neirynck et al. 1999). Many groups have reported M2e-based vaccine candidates in different forms (De Filette et al. 2008; Denis et al. 2008; Eliasson et al. 2008; Fan et al., supra; Neirynck et al., supra). Recently, Zhao et al. reported that a tetra-branched multiple antigenic peptide vaccine based on H5N1 M2e induced strong immune responses and cross protection against different H5N1 clades and even heterosubtypic protection from 2009 H1N1 (Zhao et al. 2010b; Zhao et al. 2010a).

(14) Vaccination using vectors expressing the conserved influenza NP, or a combination of NP and matrix protein has been studied in animal models and various degrees of protection against both homologous and heterologous viruses have been demonstrated (Price et al., supra; Ulmer et al. 1993). NP elicit a robust CD8.sup.+ T cell response in mice and in humans (McMichael et al., 1986; Yewdell et al., 1985) that, as epidemiological studies suggest, may contribute to resistance against severe disease following influenza A virus infection (Epstein 2006).

(15) The headless HA included in rMVA of the invention is a new headless HA (h1HA) based on the VN/1203 influenza strain. The h1HA contains a polybasic cleavage site which is cleaved during expression from the rMVA exposing the fusion peptide for the immune system. The amino acid sequence of the h1HA is set out in FIG. 1 and in SEQ ID NO: 15. The nucleotide sequence of the MVA insert is set out FIG. 2 and SEQ ID NO: 14.

(16) The amino acid sequence of the headless HA/M2e fusion protein included in rMVA of the invention is set out in FIG. 3 below and in SEQ ID NO: 2. The nucleotide sequence of the fusion protein is set out in FIG. 4 below and in SEQ ID NO: 1. In the fusion protein, the M2e domains of H5N1, H9N2, H7N2 and H1N1 (equivalent to H2N2, H3N2) form an M2e head on the h1HA. The four particular M2e domains were chosen to represent the M2e from seasonal and pandemic strains.

Example 2

Construction and Characterization of Single-insert MVA Vectors

(17) The following single-insert, recombinant MVA (rMVA) are utilized in the experiments described herein.

(18) TABLE-US-00002 TABLE 2 rMVA Inserted influenza gene NCBI gene acc no. 1. MVA-h1HA headless HA based on AY818135 2. MVA-h1HA/M2e headless HA/M2e fusion based on AY818135 3. MVA-M1-VN Matrix protein 1 AY818144 4. MVA-M2-VN Matrix protein 2 EF541453 5. MVA-PB1-VN Polymerase subunit PB1 AY818129 6. MVA-mNP Nucleoprotein AY818138 7. Control MVA-HA-VN Hemagglutinin AY818135 8. Control MVA-wt No insert 9. Control PBS No insert

(19) For construction of single-insert rMVA vectors expressing h1HA, the h1HA/M2e fusion protein or PB1, the h1HA, h1HA/M2e and PB1 genes were chemically synthesized (Geneart, Inc., Regensburg, Germany). The synthetic genes are driven by the strong vaccinia early/late promoter mH5 (Wyatt et al. 1996) and terminated with a vaccinia virus specific stop signal downstream of the coding region that is absent internally. The gene cassettes were cloned in the plasmid pDM-D4R (Ricci et al., 2011) resulting in plasmids pDM-h1HA, pDM-h1HA/M2e and pDM-PB1-VN, respectively. The introduction of the foreign genes into the D4R/D5R intergenic region of MVA was done as described elsewhere (Ricci et al. 2011) resulting in viruses MVA-h1HA, MVA-h1HA/M2e, MVA-PB1-VN.

(20) For the construction of the rMVA expressing M1, the M1 sequence (accession number AY818144) was placed downstream of the strong vaccinia early/late promoter selP (Chakrabarti et al. 1997) and cloned in pDM-D4R, resulting in pDM-M1-VN. The expression cassette of pDD4-M2-VNincluding the M2 sequence (accession number EF541453) under the control of the mH5 promoterwas cloned in pDM-D4R resulting in pDM-M2-VN. The plasmids were then used for recombination with MVA according to Holzer et al, supra resulting in the viruses MVA-M1-VN and MVA-M2-VN, respectively as shown in FIG. 5A.

(21) For the construction of single-insert MVAs expressing the NP protein, the NP expression cassette of pDD4-mH5-mNP-VN (Mayrhofer et al., supra) was cloned in plasmid pd3-lacZ-gpt, resulting in pd3-lacZ-mH5-NP-VN. Plasmid pd3-lacZ-gpt contains a lacZ/gpt selection marker cassette and a multiple cloning site (MCS) for insertion of genes of interest. The sequences are framed by genomic MVA sequences of the del III region. The marker cassette is destabilized by a tandem repeat of MVA del III flank, thus the final recombinant is free of any auxiliary sequences. The insertion plasmid directs the gene cassettes into the MVA deletion III (del III) region. After infection of primary chicken embryo cells with MOI 1, cells were transfected with pd3-lacZ-mH5-NP-VN according to the calcium phosphate technique (Graham and van der Eb 1973), resulting MVA-NP-VN shown in FIG. 5B. The MVA strain (MVA 1974/NIH clone 1) was kindly provided by B. Moss (National Institutes of Health). Recombinant virus is selected using the transient marker stabilization method (Scheiflinger et al, 1998).

(22) The single-insert MVA vectors expressing the NP, PB1, M1, M2, h1HA, and h1HA/M2e were characterized by PCR and Western blot as described in Hessel et al, supra. Recombinant viruses were grown in CEC or DF-1 cells and purified by centrifugation through a sucrose cushion. Primary CEC were produced in-house and cultivated in Med199 (Gibco) supplemented with 5% fetal calf serum (FCS). The DF-1 (CRL-12203) cell line was obtained from the ATCC (American Type Culture Collection) and cultivated in DMEM (Biochrom, Inc.) supplemented with 5% FCS.

(23) The correct expression of the influenza proteins by the rMVAs was confirmed by Western blotting. For this purpose CEC or the permanent chicken cell line DF-1 were infected with a MOI of 0.1 and cell lysates were prepared 48-72 hrs post infections. The recombinant MVAs that express the h1HA (MVA-h1HA and MVA-h1HA/M2e) were analyzed in a Western blot using an anti-influenza A/Vietnam/1194/04 (H5N1) polyclonal serum (NIBSC 04/214) for detection. Donkey-anti-sheep alkaline phosphatase-conjugated IgG (Sigma Inc.) was used as a secondary antibody. The recombinant MVAs that express the M2 and M2e (MVA-M2-VN and MVA-h1HA/M2e) were analyzed in Western Blots using an anti-avian influenza M2 antibody binding a peptide present at the amino terminus of the H5N1 M2 (ProSci, Cat#4333). Goat-anti-rabbit alkaline phosphatase-conjugated IgG (Sigma Inc.) antibody was used as a secondary antibody. As shown in FIG. 6A, the recombinant MVAs expressing the h1HA (MVA-h1HA and MVA-h1HA/M2e) gene inserts induced expression of the HA containing antigens in avian DF-1 cells. The bands around 40 kDa in lane 2 represent the h1HA. The larger band at around 70 kDa in lane 3 represents the h1HA/M2e. The large band at around 80 kDa in lane 5 represents the HA0 hemagglutinin-precursor, which is cleaved into the HA1 and HA2 subunits represent by the bands at approximately 55 and 25 kDa. The specific h1HA, h1HA/M2e or HA bands are absent in the wild-type MVA control (lane 4).

(24) FIG. 6B shows the M2 expression by MVA-M2-VN (lane 2) or MVA-h1HA/M2e (lane 3). The weak but specific band around 10 kDa in lane 2 represents the wild-type M2 protein whereas the larger band around 70 kDa represents the h1HA/M2e protein. Both bands are absent in the wild-type MVA control (lane 4).

(25) The expression of the M1, NP and PB1 protein is detected with polyclonal guinea-pig anti-influenza H5N1 serum produced in house, a polyclonal goat antibody detecting the PB1 of Influenza A virus (Santa Cruz, Cat#: vC-19), and a monoclonal mouse-anti-NP-antibody (BioXcell, Cat# BE0159), respectively. The MVA-M1-VN and MVA-NP-VN induce expression of the M1 protein (around 27 kDa) and the NP protein (around 60 kDa) (not shown).

Example 3

Animal Experiments with the Single-insert Vaccines

(26) Protection Experiment

(27) A standard protection experiment consists of two arms (primed with about 110.sup.3-110.sup.5 TCID.sub.50 H1N1v CA/07 and unprimed) of nine groups of mice each (respectively vaccinated i.m. with 110.sup.6 pfu of the nine vaccines and controls shown in Table 2), a group consisting of six animals resulting in 108 animals, defines one set. The animals of one set are challenged with one of the six challenge viruses shown in Table 3 below.

(28) TABLE-US-00003 TABLE 3 Pre-treatment Challenge strain Subtype Abbreviation H1N1v/unprimed A/California/07/2009 H1N1 CA/07 H1N1v/unprimed A/Vietnam/1203/2004 H5N1 VN/1203 H1N1v/unprimed A/HongKong/G9/ H9N2 HK/G9 H1N1v/unprimed A/Victoria/210/2009 H3N2 VF09 H1N1v/unprimed A/FPV/Rostock/34 H7N1 RO/34 H1N1v/unprimed A/PR8/1934 H1N1 PR8

(29) Female Balb/c mice are 8-10 weeks old at the pre-treatment time point and 14-16 weeks old at the time point of immunization with the vaccines and controls shown in Table 2. Mice were immunized intramuscularly twice (days 42 and 63) with 10.sup.6 pfu of the vaccines or wild type MVA, 3.75 g whole virus preparation H9N2 A/HongKong/G9/1997 or with buffer (PBS). At day 84, mice were challenged intranasally with 10.sup.3 TCID.sub.50 H5N1 A/Vietnam/1203/2004 (H5N1, CDC #2004706280), with 2.510.sup.4 TCID.sub.50 mouse adapted H9N2 A/HongKong/G9/1997 or with 1.6610.sup.4 TCID.sub.50 H7N1 A/FPV/Rostock/34. The challenge doses correspond to approx. 30 LD50 for the H5N1 challenge and 32 LD50 for the H9N2 challenge per animal. Sera are collected at days 41, 62 and 85 and analyzed for HA-specific IgG concentration by HI titer or microneutralization assay.

(30) The primary outcome of the animal experiments is protection as measured by lethal endpoint, weight loss, or lung titer. Further the ELISA titers of pooled pre-challenge sera measured against inactivated whole virus H5N1 strain A/Vietnam/1203/2004 are determined.

(31) T Cell Experiments

(32) Frequencies of influenza-specific CD4 and CD8 T cells are determined in immunized mice by flow cytometry. In a standard experiment, groups of 5 female BALB/c mice are immunized twice with the vaccines or controls listed in Table 2. Splenocytes are re-stimulated in-vitro using inactivated whole virus antigens of different influenza strains for CD4 T-cells and, when available, peptides representing the CD8 T-cell epitopes of the vaccine insert constructs and IFN- production are measured. All experiments are performed twice, using a total of 140 animals.

(33) Other Experiments

(34) An evaluation of the cell-mediated immunity after a single immunization, demonstration of functional activity of cytotoxic T-cells in a VITAL assay and assessment of recruitment of influenza-specific T-cells into the lungs of challenged animals are also carried out. The induction/expansion of vaccine-specific T-cells is also monitored in the primed mouse model by immunizing mice which resolved a influenza virus infection once with these vaccines.

Example 4

Construction and Characterization of Double-insert rMVA Vectors

(35) The following double-insert, rMVA and controls are utilized in the experiments described herein.

(36) TABLE-US-00004 TABLE 4 rMVA Inserted influenza gene Comment 1. MVA-h1HA-NP headless HA + NP Double insert construct 2. MVA-h1HA/M2e-NP headless HA/m2e fusion protein + NP Double insert construct 3. MVA-NP-VN nucleoprotein Control 4. MVA-HA-VN hemagglutinin Control 5. MVA-wt Empty vector Neg. control 6. PBS Neg. control

(37) For the construction of the double insert rMVA vector co-expressing either the h1HA or h1HA/M2e gene cassette in combination with the NP protein gene cassette, the single insert MVA recombinants of Example 2 containing the h1HA or h1HA/M2e gene cassette are used. CEC cells were infected with MVA-h1HA or MVA-h1HA/Me2 and afterwards transfected with pd3-lacZ-mH5-NP-VN (see Example 2). Homologous recombination and propagation of the recombinant MVA vectors are performed as described in Example 2. The resulting double insert MVA vectors, named MVA-h1HA-NP or MVA-h1HA/M2e-NP, contain the h1HA or h1HA/M2e expression cassette in the D4R/D5R locus and the NP expression cassette in the del III locus. See FIG. 7.

(38) The recombinant MVAs were characterized by Western Blot as described in Example 2. FIG. 8A shows the expression of the h1HA and h1HA/M2e after infection of CEC with MVA-h1HA-NP (lane 6) or MVA-h1HA/M2e-NP (lane 5). The bands around 40 kDa in lanes 3 and 6 represent the h1HA of the MVA-h1HA and MVA-h1HA-NP constructs. The band around 70 kDa in lane 5 represents the h1HA/M2e fusion protein. The HA bands are absent in the wild-type control in lane 4. The same samples were used for detection of NP protein expression in Western Blots (as described in Example 2). As shown in FIG. 8B, the recombinant MVAs MVA-h1HA-NP and MVA-h1HA/M2e-NP also induced expression of the NP protein in avian CEC cells. The bands around 60 kDa in lanes 2 to 4 represent the NP.

Example 5

Animal Experiments with the Double-insert Vaccines or Vector Combinations

(39) Protection Experiment

(40) A standard experiment included eight groups of mice (vaccinated with the six vaccines and controls shown in Table 5) each group consisting of six animals. The protection experiments were carried out as described in Example 3. After challenge mice were monitored over a time period of 14 days and weight loss or symptoms including ruffled fur (score of 1), curved posture (score of 2), apathy (score of 3), and death (score of 4) were recorded. For ethical reasons, mice were euthanized after weight loss of 25%. Protection results are compiled in Table 5 and displayed in FIGS. 9 and 10.

(41) TABLE-US-00005 TABLE 5 Protection of mice from death after double dose vaccinations with recombinant MVAs and homologous or heterologous challenge. VN1203.sup.(1) challenge HK/G9.sup.(2) After H5N1 Clinical Protection After H9N2 Clinical challenge n/nt Gr Vaccine score at day 14 n/nt.sup.(3) (%) score at day 14 (%) 1 MVA-h1HA-NP 2.83 2/6 (33) 0 6/6 (100) 2 MVA-h1HA/M2e-NP 1 5/6 (83) 0 6/6 (100) 3 MVA-h1HA 2.67 2/6 (33) 3.33 1/6 (17) 4 MVA-h1HA/M2e 4 0/6 (0) 2.67 2/6 (33) 5 MVA-NP-VN 3.33 2/6 (33) 0 6/6 (100) 6 Homologous 0 6/6 (100) 0 6/6 (100) control vaccine.sup.(4) 7 MVA-wt(5) 4 0/6 (0) 2.83 2/6 (33) 8 PBS 2.67 2/6 (33) 4 0/6 (0) .sup.(1)VN1203, challenge strain A/Vietnam/1203/2004; .sup.(2)HK/G9, challenge strain A/HongKong/G9/1997; .sup.(3)n/nt, survival per group, .sup.(4)Homologous control vaccine; (5)wild-type MVA (NIH74 LVD clone 6).

(42) As positive control mice were vaccinated with homologous control constructs. In case of H5N1 challenge mice were vaccinated with MYA-HA-VN (Hessel et al., 2011) and in case of H9N2 challenge mice were vaccinated with an inactivated whole virus preparation of the H9N2 A/HongKong/G9/1997 influenza virus. Both controls induced full protection (Table 5; FIGS. 9 and 10, panels E). In the wild-type MVA and buffer groups all mice showed marked weight loss compared to the positive control groups and nearly all mice died after challenge. Mice vaccinated with the single recombinant MVAs (MVA-h1HA, MVA-h1HA/M2e, MVA-D3-NP-VN) showed no significantly better protection after the strong H5N1 challenge compared to the negative control groups (FIG. 9 A-F). Also against heterosubtypic (H9N2) challenge no significant protection was seen in MVA-h1HA and MVA-h1HA/M2e vaccinated groups (FIG. 10).

(43) Surprisingly, however, vaccination with the double construct expressing the fusion protein h1HA/M2e and the NP protein resulted in nearly full protection (FIG. 9 B, D, F) after the H5N1 challenge with approx. 30 LD50 per animal. Also after heterosubtypic challenge (with approx. 32 LD50 H9N2 virus) mice were fully protected after vaccination with the double recombinant MVA-h1HA/M2e-NP. Furthermore, the double recombinant MVA-h1HA-NP and the single recombinant MVA-NP-VN induced full protection against the heterosubtypic challenge with H9N2 (FIG. 10, B, D, F). As can be seen in the weight monitoring (FIGS. 9 and 10, panels B) and in the clinical scores (FIGS. 9 and 10, panels D), the double construct MVA-h1HA/M2e-NP showed the best results presumably by combined beneficial effects contributed by the different influenza antigens.

(44) T Cell Experiments

(45) Frequencies of influenza-specific CD4 and CD8 T cells are determined in immunized mice by flow cytometry. In a standard protocol experiment, groups of 5 female BALB/c mice are immunized twice with the vaccines or controls listed in Table 4. Splenocytes are re-stimulated in-vitro using inactivated whole virus antigens of different influenza strains for CD4 T-cells and, when available, peptides representing the CD8 T-cell epitopes of the vaccine insert constructs and IFN- production are measured. All experiments are performed twice.

(46) Other Experiments

(47) An evaluation of the cell mediated immunity after a single immunization, demonstration of functional activity of cytotoxic T-cells in a VITAL assay and assessment of recruitment of influenza-specific T-cells into the lungs of challenged animals are also carried out. The induction/expansion of vaccine-specific T-cells is also monitored in the primed mouse model by immunizing mice which resolved a influenza virus infection once with these vaccines.

Example 6

Construction and Characterization of Triple-insert rMVA Vectors and Virus-like Particles

(48) Influenza virus-like particles (VLPs) induce humoral and cellular responses and can protect against lethal challenges (Bright et al. 2007; Pushko et al. 2005; Song et al. 2010). VLPs chosen for experiments herein comprise either h1HA or h1HA/M2e in combination with NP and M1. The VLPs are generated from triple-insert MVA vectors.

(49) For the construction of the triple-insert MVA vectors co-expressing either h1HA or h1HA/M2e in combination with the M1 (SEQ ID NO: 11) and the NP protein (SEQ ID NO: 13), the M1 gene (SEQ ID NO: 10) of pDD4-M1-VN is cloned downstream of the synthetic early/late promotor selP (Chakrabarti et al. 1997). The resulting gene cassette is cloned downstream of the h1HA or h1HA/M2e gene cassette in pDM-h1HA or pDM-h1HA/M2e. The resulting plasmids harboring a double gene cassette (pDM-h1HA-M1 and pDM-h1HA/M2e-M1) are used for recombination into defective MVA as described above. Afterwards, a recombination with an NP gene cassette (SEQ ID NO: 12)-containing plasmid (pD3-lacZ-gpt-NP-VN) is done resulting in a triple-insert MVA virus. This triple-insert MVA is plaque purified under transient marker selection.

(50) The triple-insert MVA vectors, named MVA-h1HA-M1-NP or MVA-h1HA/M2e-M1-NP contain the h1HA or h1HA/M2e expression cassette and M1 expression cassette in tandem order in the D4R/D5R locus and the NP expression cassette in the del III locus (FIG. 7).

(51) Detection of VLPs is as follows. HeLa or 293 cells are seeded into T 175 cm.sup.2 flasks and grown in DMEM+10% FCS+Pen/Strep. To generate VLPs, cells are infected with 1 MOI of single-insert MVA or triple-insert MVA recombinants, respectively. Empty MVA vectors or single-insert MVA recombinants without M1 are used as controls. At 1 h post infection (p.i.), the medium is replaced by DMEM+Pen/Strep and culture medium is harvested 48 h p.i. and cellular debris is pelleted by centrifugation at 2.000g for 10 min. The procedure for analyzing VLPs by sucrose gradient density flotation and sucrose cushion has been described previously (Chen et al. 2007; Chen et al. 2005; Gomez-Puertes et al. 2000). The samples are then analyzed by immunoblotting. Additionally, electron microscopy (EM) analysis with medium of infected cells is performed.

Example 7

Animal Experiments with the Triple-insert Vaccines or Vector Combinations

(52) A standard experiment includes 6 groups of primed and unprimed mice (vaccinated with the 6 vaccines and controls shown in Table 5), each group consisting of 6 animals, resulting in 36 animals (1 set). The animals are challenged with one of the 6 challenge viruses shown in Table 3. In sum, there are 6 sets of 72 animals each requiring 432 mice to assess cross-protection in the primed and naive models.

(53) TABLE-US-00006 TABLE 5 rMVA Inserted influenza gene(s) comment 1. MVA-h1HA-M1-NP headless HA + nucleoprotein + 3 inserts matrix 1 2. MVA-h1HA/M2e-M1-NP headless HA/m2e fusion 3 inserts protein + nucleoprotein + matrix 1 3. MVA-tbd best construct from previous control screening 4. MVA-HA-VN hemagglutinin control 5. MVA-wt empty vector neg. control 6. PBS neg. control

(54) The present invention is illustrated by the foregoing examples and variations thereof will be apparent to those skilled in the art. Therefore, no limitations other than those set out in the following claims should be placed on the invention.

(55) All documents cited in this application are hereby incorporated by reference in their entirety for their disclosure described.

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