Recombinant modified vaccinia virus ankara (MVA) respiratory syncytial virus (RSV) vaccine

Abstract

Provided herein are recombinant modified vaccinia virus Ankara (MVA) strains as improved vaccines against infection with Respiratory Syncytial Virus (RSV virus) and to related products, methods and uses. Specifically, provided herein are genetically engineered recombinant MVA vectors comprising at least one nucleotide sequence encoding an antigenic determinant of an RSV membrane glycoprotein and at least one nucleotide sequence encoding an antigenic determinant of an RSV nucleocapsid protein. Also provided herein are products, methods and uses thereof, e.g., suitable to affect an immune response in a subject, or suitable to diagnose an RSV infection, as well as to determine whether a subject is at risk of recurrent RSV infection.

Claims

1. A recombinant modified vaccinia virus Ankara (MVA) comprising: (a) at least one nucleotide sequence encoding an antigenic determinant of a respiratory syncytial virus (RSV) membrane glycoprotein, wherein the nucleotide sequence encodes an RSV F antigenic determinant; (b) at least one nucleotide sequence comprising an open reading frame that encodes an RSV nucleocapsid antigenic determinant that is an RSV N nucleocapsid protein and an RSV nucleocapsid antigenic determinant that is an RSV M2 matrix protein; and further comprising: (c) at least one nucleotide sequence encoding an antigenic determinant of an RSV G membrane glycoprotein.

2. The recombinant MVA of claim 1, wherein the nucleotide sequence in (a) encodes a full-length RSV F membrane glycoprotein.

3. The recombinant MVA of claim 1, wherein the nucleotide in (a) is from RSV strain.

4. The recombinant MVA of claim 1, wherein the nucleotide sequence in (a) comprises a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 6 or comprises the nucleotide sequence of SEQ ID NO: 5.

5. The recombinant MVA of claim 1, wherein the nucleotide sequence in (b) encodes a full-length RSV N nucleocapsid protein and a full-length RSV M2 matrix protein.

6. The recombinant MVA of claim 1, wherein the nucleotide sequence in (b) is from strain.

7. The recombinant MVA of claim 1, wherein the nucleotide sequence in (b) comprises a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 35; or comprises the nucleotide sequence of SEQ ID NO: 34.

8. The recombinant MVA of claim 1, wherein the nucleotide sequence in (b) comprises a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 10; or comprises the nucleotide sequence of SEQ ID NO: 9.

9. The recombinant MVA of claim 1, wherein the nucleotide sequence in (b) comprises a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 33; or comprises the nucleotide sequence of SEQ ID NO: 32.

10. The recombinant MVA of claim 1, wherein the nucleotide sequence in (b) comprises a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 14; or comprises the nucleotide sequence of SEQ ID NO: 13.

11. The recombinant MVA of claim 1, wherein the nucleotide sequence in (b) further encodes a self-cleaving protease domain between the antigenic determinants of the RSV N nucleocapsid and the RSV M2 matrix proteins.

12. The recombinant MVA of claim 11, wherein the nucleotide sequence in (c) encodes a full-length RSV G membrane glycoprotein.

13. The recombinant MVA of claim 12, wherein the nucleotide sequence in (c) is from RSV strain A2.

14. The recombinant MVA of claim 13, wherein the nucleotide sequence in (c) comprises a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 2 or comprises the nucleotide sequence of SEQ ID NO: 1.

15. The recombinant MVA of claim 14, wherein the MVA used for generating the recombinant MVA is MVA-BN deposited at the European Collection of Cell Cultures (ECACC) under number V00083008.

16. A pharmaceutical composition comprising the recombinant MVA of claim 1.

17. A method for treating or preventing an RSV infection in a subject, comprising administering to the subject the recombinant modified vaccinia virus Ankara (MVA) of claim 1.

18. The recombinant MVA of claim 11, wherein the self-cleaving protease domain has the amino acid sequence set forth in SEQ ID NO:12.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIG. 1 shows the heterologous RSV genes used in the tested recombinant MVA-constructs, MVA-mBN199B, MVA-mBN201B, MVA-mBN201BM2, MVA-mBN294B, MVA-mBN295B and MVA-mBN330B.

(2) FIG. 2 shows serum RSV-specific IgG responses measured by IBL Hamburg-based ELISA. Mice were immunized (s.c. or i.n.) two or three times with TBS or 110.sup.8 TCID.sub.50 of either MVA-mBN199B, MVA-mBN201B or MVA-mBN201BM2. Control mice were immunized twice i.n. with 10.sup.6 pfu RSV. Sera were diluted 1/100 and analyzed using an RSV-specific IgG ELISA based on the IBL Hamburg kit using plates coated with RSV F and G proteins.

(3) FIG. 3 shows serum RSV-specific IgG responses measured by IBL Hamburg-based ELISA after serial dilution. Mice were immunized two or three times (s.c. or i.n.) with TBS or 110.sup.8 TCID.sub.50 of MVA-mBN199B, MVA-mBN201B or MVA-mBN201BM2. Control mice were immunized twice i.n. with 10.sup.6 pfu RSV. Sera were diluted (1/100, 1/200 and 1/400) and analyzed using an RSV-specific IgG ELISA based on the IBL Hamburg kit using plates coated with RSV F and G proteins.

(4) FIG. 4 shows serum RSV-specific IgG responses measured by IBL Hamburg-based ELISA using plates coated only with the F protein. Mice were immunized s.c. two or three times with TBS or 110.sup.8 TCID.sub.50 of MVA-mBN199B, MVA-mBN201B or MVA-mBN201BM2. Control mice were immunized twice i.n. with 10.sup.6 pfu RSV. Sera were diluted 1/100 and analyzed using an RSV-specific IgG ELISA based on the IBL Hamburg kit using plates coated with RSV F protein only.

(5) FIG. 5 shows serum RSV-specific IgG responses measured by Serion-based ELISA. Mice were immunized two or three times (s.c. or i.n.) with TBS or 110.sup.8 TCID.sub.50 of either MVA-mBN199B, MVA-mBN201B or MVA-mBN201BM2. Control mice were immunized twice i.n. with 10.sup.6 pfu RSV. Sera were diluted (1/100) and analyzed using an RSV-specific IgG ELISA based on the Serion kit using plates coated with an RSV lysate.

(6) FIG. 6 shows RSV-specific IgA versus IgG responses measured by IBL Hamburg-based ELISA in bronchoalveolar lavage (BAL) fluid and sera. Mice were immunized two or three times (s.c. or i.n.) with TBS or 110.sup.8 TCID.sub.50 of MVA-mBN199B, MVA-mBN201B or MVA-mBN201BM2. Control mice were immunized twice i.n. with 10.sup.6 pfu RSV. Sera and BAL fluid were diluted (1/100) and analyzed using an RSV-specific IgG or IgA ELISA based on the IBL Hamburg kit using plates coated with RSV F and G proteins.

(7) FIG. 7 shows RSV F-, RSV G- and RSV M2-specific T-cell responses measured by ELISPOT. Mice were immunized two or three times (s.c. or i.n.) with TBS or 110.sup.8 TCID.sub.50 of either MVA-mBN199B, MVA-mBN201B or MVA-mBN201BM2. Control mice were immunized twice i.n. with 10.sup.6 pfu RSV. Spleens were isolated on Day 48 and splenocytes were restimulated with three different RSV F-specific peptides (RSV-1 (SEQ ID NO:19), RSV-2 (SEQ ID NO:20) and RSV-3 (SEQ ID NO:21), one RSV G-specific peptide (RSV-4 (SEQ ID NO:22)), one RSV M2-specific peptide (RSV-9 (SEQ ID NO:27)) or MVA-BN. IFN-secreting cells were detected by ELISPOT. The stimulation index was calculated as explained in the Examples.

(8) FIG. 8 shows relative body weight loss after challenged with RSV(A2). Mice were immunized two or three times (s.c. or i.n.) with TBS or 110.sup.8 TCID.sub.50 of MVA-mBN199B, MVA-mBN201B or MVA-mBN201BM2. Control mice were immunized twice i.n. with 10.sup.6 pfu RSV. Mice were then challenged with 10.sup.6 pfu RSV(A2) on Day 49. Weight was measured daily from the day of challenge. The weight on the day of challenge was used as baseline to calculate percentage of relative body weight change.

(9) FIG. 9 shows RSV load in lungs measured by plaque assay. Mice were immunized two or three times (s.c. or i.n.) with TBS or 110.sup.8 TCID.sub.50 of MVA-mBN199B, MVA-mBN201B or MVA-mBN201BM2. Control mice were immunized twice i.n. with 10.sup.6 pfu RSV. Mice were then challenged with 10.sup.6 pfu RSV(A2) on Day 49. Lungs were isolated 4 days later and the RSV load (pfu per lung) was determined by plaque assay.

(10) FIG. 10 shows RSV load in lung measured by RT-qPCR. Mice were immunized two or three times (s.c. or i.n.) with TBS or 110.sup.8 TCID.sub.50 of MVA-mBN199B, MVA-mBN201B or MVA-mBN201BM2. Control mice were immunized twice i.n. with 10.sup.6 pfu RSV. Mice were then challenged with 10.sup.6 pfu RSV A2 on Day 49. Lungs were isolated 4 days later and the RSV load (estimated based on the number of L gene copies observed) was determined by RT-qPCR.

(11) FIG. 11 shows IL4 level in bronchoalveolar lavage (BAL) 4 days post RSV(A2) challenge measured by ELISA. Mice were immunized two times (s.c. or i.n.) with TBS or 110.sup.8 TCID.sub.50 of MVA-mBN199B or MVA-mBN201B. Control mice were immunized twice i.n. with 10.sup.6 pfu RSV. Mice were then challenged with 10.sup.6 pfu RSV A2. Lungs were washed with 1 ml PBS 4 days later and the IL4 level in BAL was determined by ELISA. (n.d.=not detectable)

(12) FIG. 12 shows IL5 level in bronchoalveolar lavage (BAL) 4 days post RSV(A2) challenge measured by ELISA. Mice were immunized two times (s.c. or i.n.) with TBS or 110.sup.8 TCID.sub.50 of MVA-mBN199B or MVA-mBN201B. Control mice were immunized twice i.n. with 10.sup.6 pfu RSV. Mice were then challenged with 10.sup.6 pfu RSV A2. Lungs were washed with 1 ml PBS 4 days later and the IL5 level in BAL was determined by ELISA. (n.d.=not detectable)

(13) FIG. 13 shows serum RSV-specific IgG responses measured by Serion-based ELISA. Mice were immunized s.c. twice 3 weeks apart with TBS or 110.sup.8 TCID.sub.50 of either MVA-mBN199B, MVA-mBN201B or MVA-mBN294A. Control mice were immunized twice i.n. with 10.sup.6 pfu RSV. Sera of 5 mice per group obtained 2 weeks after the last immunization were diluted and analyzed using an RSV-specific IgG ELISA based on the Serion kit using plates coated with an RSV lysate.

(14) FIG. 14 shows serum RSV-specific neutralizing antibody responses measured by PRNT. Mice were immunized s.c. twice 3 weeks apart with TBS or 110.sup.8 TCID.sub.50 of either MVA-mBN199B, MVA-mBN201B or MVA-mBN294A. Control mice were immunized twice i.n. with 10.sup.6 pfu RSV. Sera of 5 mice per group obtained 2 weeks after the last immunization analyzed by PRNT.

(15) FIG. 15 shows RSV F- and RSV M2-specific T-cell responses measured by ELISPOT. Mice were immunized s.c. twice 3 weeks apart with TBS or 110.sup.8 TCID.sub.50 of either MVA-mBN199B, MVA-mBN201B or MVA-mBN294A. Control mice were immunized twice i.n. with 10.sup.6 pfu RSV. Spleens were isolated on Day 34 and splenocytes were restimulated with one RSV F-specific peptides (RSV-2 (SEQ ID NO:20), one RSV M2-specific peptide (RSV-9 (SEQ ID NO:27)) or MVA-BN. IFN-secreting cells were detected by ELISPOT. The stimulation index was calculated as explained in the Examples.

(16) FIG. 16 shows RSV load in lungs measured by plaque assay. Mice were immunized s.c. twice 3 weeks apart with TBS or 110.sup.8 TCID.sub.50 of either MVA-mBN199B, MVA-mBN201B or MVA-mBN294A. Control mice were immunized twice i.n. with 10.sup.6 pfu RSV or i.m. with 50 l FI-RSV. Mice were then challenged with 10.sup.6 pfu RSV(A2) on Day 49. Lungs were isolated 4 days later and the RSV load (pfu per lung) was determined by plaque assay.

(17) FIG. 17 shows RSV load in lung measured by RT-qPCR. Mice were immunized s.c. twice 3 weeks apart with TBS or 110.sup.8 TCID.sub.50 of either MVA-mBN199B, MVA-mBN201B or MVA-mBN294A. Control mice were immunized twice i.n. with 10.sup.6 pfu RSV or i.m. with 50 l FI-RSV. Mice were then challenged with 10.sup.6 pfu RSV A2 on Day 49. Lungs were isolated 4 days later and the RSV load (estimated based on the number of L gene copies observed) was determined by RT-qPCR.

(18) FIG. 18 shows eosinophil and neutrophil infiltrations in bronchoalveolar lavage (BAL) fluids 4 days post RSV(A2) challenge. Mice were immunized s.c. twice 3 weeks apart with TBS or 1108 TCID.sub.50 of either MVA-mBN199B, MVA-mBN201B or MVA-mBN294A. Control mice were immunized twice i.n. with 10.sup.6 pfu RSV or i.m. with 50 l FI-RSV. Mice were then challenged with 10.sup.6 pfu RSV A2. Lungs were washed with 1 ml PBS 4 days later and the percentage of eosinophil and neutrophil in BAL fluid was determined.

BRIEF DESCRIPTION OF THE SEQUENCES

(19) SEQ ID NO:1 is a DNA sequence encoding full-length G protein from human RSV (hRSV) strain A2 (GenBank Accession No. M11486).

(20) SEQ ID NO:2 is the amino acid sequence of full-length G protein from hRSV strain A2 (GenBank Accession No. M11486).

(21) SEQ ID NO:3 is a DNA sequence encoding full-length F protein (BN variant) from hRSV strain A2.

(22) SEQ ID NO:4 is the amino acid sequence of full-length F protein (BN variant) from hRSV strain A2.

(23) SEQ ID NO:5 is a DNA sequence encoding full-length F protein (BN variant) from hRSV strain ALong.

(24) SEQ ID NO:6 is the amino acid sequence encoding full-length F protein (BN variant) from hRSV strain ALong.

(25) SEQ ID NO:7 is a DNA sequence encoding truncated G protein lacking the transmembrane and cytoplasmic domains from hRSV strain B (GenBank Accession No. P20896).

(26) SEQ ID NO:8 is the amino acid sequence of truncated G protein lacking the transmembrane and cytoplasmic domains from hRSV strain B (GenBank Accession No. P20896).

(27) SEQ ID NO:9 is a DNA sequence encoding N protein lacking a stop codon from hRSV strain A2 (Genbank Accession No. M11486).

(28) SEQ ID NO:10 is the amino acid sequence of N protein lacking a stop codon from hRSV strain A2 (Genbank Accession No. M11486).

(29) SEQ ID NO:11 is a DNA sequence encoding a fragment of protease 2A from Foot and Mouth Disease Virus lacking both start and stop codons.

(30) SEQ ID NO:12 is the amino acid sequence of a fragment of protease 2A from Foot and Mouth Disease Virus lacking both start and stop codons.

(31) SEQ ID NO:13 is a DNA sequence encoding full-length M2 protein lacking a start codon from hRSV strain A2 (GenBank Accession No. M11486).

(32) SEQ ID NO:14 is the amino acid sequence encoding full-length M2 protein lacking a start codon from hRSV strain A2 (GenBank Accession No. M11486).

(33) SEQ ID NO:15 is a DNA sequence encoding truncated F protein lacking the transmembrane and cytoplasmic domains (BN variant) from hRSV strain A2 (GenBank Accession No. M11486).

(34) SEQ ID NO:16 is the amino acid sequence of truncated F protein lacking the transmembrane and cytoplasmic domains (BN variant) from hRSV strain A2 (GenBank Accession No. M11486).

(35) SEQ ID NO:17 is a DNA sequence encoding N protein lacking a stop codon hRSV strain A2 (Genbank Accession No. M11486)+a DNA sequence encoding protease 2A fragment from Foot and Mouth Disease Virus, lacking both a start codon and a stop codon+a DNA sequence encoding full-length M2 protein lacking a start codon from hRSV strain A2 (GenBank Accession No. M11486).

(36) SEQ ID NO:18 is the amino acid sequence of N protein from hRSV strain A2 (Genbank Accession No. M11486)+the amino acid sequence of protease 2A fragment from Foot and Mouth Disease Virus, lacking a start codon+the amino acid sequence of full-length M2 protein lacking a start codon from hRSV strain A2 (GenBank Accession No. M11486).

(37) SEQ ID NO:19 is the amino acid sequence of RSV-1 peptide derived from RSV F protein.

(38) SEQ ID NO:20 is the amino acid sequence of RSV-2 peptide derived from RSV F protein.

(39) SEQ ID NO:21 is the amino acid sequence of RSV-3 peptide derived from RSV F protein.

(40) SEQ ID NO:22 is the amino acid sequence of RSV-4 peptide derived from RSV G protein.

(41) SEQ ID NO:23 is the amino acid sequence of RSV-5 peptide derived from RSV G protein.

(42) SEQ ID NO:24 is the amino acid sequence of RSV-6 peptide derived from RSV G protein.

(43) SEQ ID NO:25 is the amino acid sequence of RSV-7 peptide derived from RSV G protein.

(44) SEQ ID NO:26 is the amino acid sequence of RSV-8 peptide derived from RSV G protein.

(45) SEQ ID NO:27 is the amino acid sequence of RSV-9 peptide derived from RSV M2 protein.

(46) SEQ ID NO:28 is a DNA sequence encoding full-length F protein from hRSV strain A2.

(47) SEQ ID NO:29 is the amino acid sequence of full-length F protein from hRSV strain A2.

(48) SEQ ID NO:30 is a DNA sequence encoding full-length G protein from hRSV strain A2.

(49) SEQ ID NO:31 is the amino acid sequence of full-length G protein from hRSV strain A2.

(50) SEQ ID NO:32 is a DNA sequence encoding full-length M2 protein from hRSV strain A2.

(51) SEQ ID NO:33 is the amino acid sequence of full-length M2 protein from hRSV strain A2.

(52) SEQ ID NO:34 is a DNA sequence encoding full-length N protein from hRSV strain A2.

(53) SEQ ID NO:35 is the amino acid sequence of full-length N protein from hRSV strain A2.

(54) SEQ ID NO:36 is Primer 1 used in RT-qPCR.

(55) SEQ ID NO:37 is Primer 2 used in RT-qPCR.

(56) SEQ ID NO:38 is Probe 6 used in RT-qPCR.

(57) SEQ ID NO:39 is the nucleotide sequence of the PrS promoter.

(58) SEQ ID NO:40 is the nucleotide sequence of the Pr7.5 promoter.

(59) SEQ ID NO:41 is the nucleotide sequence of the PrSynllm promoter.

(60) SEQ ID NO:42 is the nucleotide sequence of the PrLE1 promoter.

(61) SEQ ID NO:43 is the nucleotide sequence of the PrH5m promoter.

EXAMPLES

Example 1: Construction of Recombinant MVAs

(62) Generation of recombinant MVA was done by insertion of the RSV coding sequences together with the indicated promoters (FIG. 1) into the MVA genome via homologous recombination in CEF cells using a selection marker to select for recombinant MVA. The use of intergenic regions (IGRs) as insertion sites is described in WO 03/097845. In order to delete the selection marker, a second step of homologous recombination was employed.

(63) MVA-BN virus was used as starting material for the generation of the recombinant MVA-mBN199B containing the genes for RSV-A2-G and RSV-F-A2_BN in IGR88/89. The PreMaster material of MVA-mBN199 was used as starting material for the generation of MVA-mBN201B described below.

(64) Insertions into IGR88/89 (MVA-mBN199B):

(65) The coding sequence for RSV-A2-G is based on the naturally occurring sequence of the RSV-A2-strain glycoprotein G. The coding sequence of the fusion protein RSV-F-A2 BN is also based on the RSV-A2 strain but was modified by Bavarian Nordic. Both inserted genes were synthesized by Geneart with human adapted codon usage and used for cloning of a recombination plasmid. The protein sequence of RSV-A2-G shows 100% identity to GenBank sequence P03423.1. The protein sequence of RSV-F-A2 BN shows only 99% identity to GenBank sequence P03420.1 due to one single amino acid exchange (P to A) on position 103.

(66) Insertions into IGR148/149 (MVA-mBN201B):

(67) The coding sequences for RSV-N-A2 and RSV-M2-A2 are based on the naturally occurring sequences of the respective RSV-A2-strain glycoproteins. Both genes are connected by a 2A self-cleaving peptide sequence [M. D. Ryan et al. (1991), J. Gen. Virol. 72(Pt 11):2727-2732] that allows the expression of two separate native proteins under the control of a single promoter. The coding sequences for RSV-G(B) and RSV-F A long BN were truncated to remove the transmembrane domains so that the expressed proteins can be secreted. All inserted genes were synthesized by Geneart with optimized codon usage and used for cloning of the recombination plasmid. The protein sequences of RSV-N-A2 and RSV-M2-A2 show 100% identity to GenBank sequence P03418.1 and P04545.1, respectively. The protein sequence of RSV-G(B) truncated shows 100% identity to GenBank sequence P20896.1. The coding sequence of RSV-F A long BN truncated was designed to contain the first 526 amino acids of the RSV-F protein as described by R. P. Du et al. (1994) Biotechnology (N Y) 12(8):813-818.

(68) Deletion Mutant in M2(A2) of MVA-mBN210BM2:

(69) MVA-mBN210BM2 includes a deletion mutation in the 12.sup.th codon of the M2(A2) gene not allowing a functional M2 to be expressed. This deletion causes the addition of the two amino acids threonine and alanine to the first 11 amino acids of M2 (A2) followed by a transcriptional stop (UGA stop codon).

Example 2: Immunogenicity and Efficacy of Recombinant MVA Vaccines Expressing RSV F Protein, RSV G Protein, RSV N Protein, and RSV M2 Protein

(70) Vaccine candidate MVA-mBN199B encodes the glycoprotein (G) and the fusion (F) protein of RSV, while MVA-mBN201BM2 and MVA-mBN201B express truncated versions of F and G in addition to full-length F and G proteins, the nucleocapsid protein (N) and in case of MVA-mBN201B also the matrix protein (M2) of RSV (see FIG. 1). The objective of these experiments was to analyze the immunogenicity and protective efficacy of MVA-mBN201BM2 and MVA-mBN201B compared to MVA-mBN199B after two or three immunizations via the subcutaneous (s.c.) or intranasal (i.n.) routes of administration.

(71) The efficacy of these constructs was tested using an RSV(A2) challenge model in BALB/c mice. Two immunizations with MVA-mBN199B or MVA-mBN201B offered partial protection as judged by real time quantitative polymerase chain reaction (RT-qPCR) when applied subcutaneously and an almost complete protection when applied by the intranasal route. The protection offered by MVA-mBN201B was better than that offered by MVA-mBN199B. There was no difference in the humoral immune responses induced by the two constructs, although major differences were observed in the T-cell responses. MVA-mBN199B induced a good RSV F-specific cellular response, whereas a strong M2-specific T-cell response with MVA-mBN201B was observed, as well as a more pronounced G-specific response compared to MVA-mBN199B. As the IgG and T-cell responses induced after subcutaneous and intranasal immunization were similar, the almost complete sterile immunity obtained by intranasal immunizations likely correlates with the induction and secretion of RSV-specific IgA at the mucosal infection site. For MVA-mBN201BM2, the lack of M2-specific T-cells responses correlated with a reduced protection compared to MVA-mBN201B and resulted in a similar protection than MVA-mBN199B.

(72) Study Design

(73) Mice were treated subcutaneously (s.c.) or intranasally (i.n.) with 110.sup.8 TCID.sub.50 MVA-mBN199B (Groups 3, 4 and 5), 110.sup.8 TCID.sub.50 MVA-mBN201B (Groups 6, 7 and 8), or 110.sup.8 TCID.sub.50 MVA-mBN201BM2 (Group 9). Mice were treated either two times (Groups 3, 4, 6, 7 and 9) or three times (Groups 5 and 8) according to Table 1. The two control groups were treated (s.c.) twice with TBS (Group 1) or i.n. with RSV (Group 2) according to Table 7. Blood was collected on the day before immunization or challenge as well as on the day of sacrifice. RSV-specific IgG titres were determined by Enzyme-Linked Immunosorbent Assay (ELISA). On Day 48, half of the mice were sacrificed. Their spleens were removed and prepared for the analysis of RSV-specific T-cell responses by Enzyme-Linked Immunosorbent Spot (ELISPOT). On Day 49, the remaining mice were challenged (i.n.) with 10.sup.6 pfu RSV A2. Appearance and body weight were monitored daily starting on the day of challenge. Four days post challenge, mice were sacrificed by injection of an elevated dose of Ketamine-Xylazine and end-bled. After lung lavage, the lungs were removed and RSV load was analyzed by plaque assay and by RT-qPCR.

(74) TABLE-US-00001 TABLE 1 Experimental Design Administration of Test or Reference Items Bleed Dose and Group Schedule per ELISPOT Challenge Group Size Cage Injections (Day)% Route Injection (Day)% (Day)%, # 1 5 A TBS 14 and s.c. n.a. 13, 34, 49 35 48 & 53 5 B 13, 34 and 48& 2 5 C RSV 14 and i.n. 10.sup.6 pfu 13, 34, 49 35 48 and 53 5 D 13, 34 and 48& 3 5 E MVA- 14 and s.c. 1 10.sup.8 13, 34, 49 mBN199B 35 TCID.sub.50 48 and 53 5 F 13, 34 and 48& 4 5 G MVA- 14 and i.n. 1 10.sup.8 13, 34, 49 mBN199B 35 TCID.sub.50 48 and 53 5 H 13, 34 and 48& 5 5 J MVA- 0, 21 and s.c. 1 10.sup.8 1, 20 34, 49 mBN199B 35 TCID.sub.50 48 and 53 5 K 1, 20 34 and 48& 6 5 L MVA- 14 and s. c. 1 10.sup.8 13, 34, 49 mBN201B 35 TCID.sub.50 48 and 53 5 M 13, 34 and 48& 7 5 N MVA- 14 and i.n. 1 10.sup.8 13, 34, 49 mBN201B 35 TCID.sub.50 48 and 53 5 P 13, 34 and 48& 8 5 Q MVA- 0, 21 and s.c. 1 10.sup.8 1, 20 34, 49 mBN201B 35 TCID.sub.50 48 and 53 5 R 1, 20 34 and 48& 9 5 W MVA- 14 and s.c. 1 10.sup.8 13, 34, 49 mBN201BM2 35 TCID.sub.50 48 and 53 %Relative to the first immunization. #Mice were challenged by the intranasal route with 10.sup.6 pfu of RSV A2. Four days after challenge, mice were bled and sacrificed under anesthesia. BAL fluid and lungs were sampled. &On Day 48, these mice were sacrificed and spleens were analyzed by ELISPOT.

(75) Study Schedule.

(76) The schedule of the in-life phase is summarized in Table 2.

(77) TABLE-US-00002 TABLE 2 Study schedule of the In-life Phase Day** Procedures 16 Arrival and import in animal facility of 85 BALB/c mice, cage card allocation and allocation of 5 mice per cage 1 Ear clipping, inclusion/exclusion examination of all mice 1 Pre-bleed of mice from cages J, K, Q and R (facial vein puncture right side) 0 1st administration of mice from cages J, K, Q and R 13 pre-bleed of all mice except mice from cages J, K, Q and R (facial vein puncture right side) 14 1st administration of all mice except mice from cages J, K, Q and R 20 Bleed of mice from cages J, K, Q and R (facial vein puncture left side) 21 2nd administration of mice from cages J, K, Q and R 34 Bleed of all mice (retro-bulbar vein puncture left eye) 35 2nd administration of all mice except mice from cages J, K, Q and R 3rd administration of mice from cages J, K, Q and R 48 Bleed of all mice (retro-bulbar vein puncture right eye) Final bleed for cages B, D, F, H, K, M, P and R 48 Spleens of mice from cages B, D, F, H, K, M, P and R will be removed for analysis by ELISPOT 49 Challenge of all remaining mice 49 to Appearance and body weight measurement daily 53 53 Final bleed, sacrifice & sampling of BAL & lung of remaining mice **Relative to the day of the 1.sup.st immunization.
Material and Methods

(78) Experimental Animals.

(79) Eighty-five female BALB/cJ Rj (H-2d) mice at the age of seven weeks were obtained from Janvier (Route des Chnes Secs, F-53940 Le Genest-Saint-Isle, France). All mice were specific pathogen free.

(80) Housing.

(81) The study was performed in room 117 of the animal facility at Bavarian Nordic-Martinsreid. This unit was provided with filtered air at a temperature of 20-24 C. and a relative humidity between 40% and 70%. The room was artificially illuminated on a cycle of 14 hours of light and 10 hours of darkness. The study acclimatization period was 15 days. The animals were housed in transparent SealSafe-cages (H Temp [polysulfon] cage Type II LEuro standard), with a floor area of 530 cm.sup.2. The cages were covered with an H-Temp SealSafe lid. The cages were placed in a TECNIPLAST-IVC SealSafe system with a SLIMLine circulation unit providing every single cage separately with HEPA-filtered air. Animal bedding was changed once a week.

(82) Diet and Water.

(83) Mice were provided with free access to irradiated maintenance diet (SSNIFF R/M-H, irradiated, V1534-727) and water (autoclaved at 121 C. for 20 minutes).

(84) Pre-Treatment Procedures: Identification of Animals.

(85) To individually mark animals within each cage, ear punching was done according to standard procedures.

(86) Inclusion/Exclusion Examination.

(87) Inclusion/exclusion examination was done according to standard procedures.

(88) Blood Sampling for Pre-Bleed.

(89) Blood samples of approximately 150 l were obtained by facial vein puncture according to standard procedures. Blood samples were transferred to the laboratory for further processing according to standard procedures.

(90) Treatment Procedures:

(91) Preparation and administration of Test Items 1 to 3 and Reference Item. Preparation and administration of test and reference items was performed in a class II microbiological safety cabinet (HERAsafe/class II type H, Kendro) according to standard procedures. Briefly, for s.c. administration, recombinant MVAs were diluted in TBS to obtain a working solution with a concentration of 210.sup.8 TCID.sub.50/ml. 110.sup.8 TCID.sub.50 in 500 l was injected s.c. according to standard procedures. For i.n. administration, recombinant MVAs were diluted in TBS to obtain a working solution with a concentration of 210.sup.9 TCID.sub.50/ml. 50 l of the diluted viruses was administered in one nostril of anesthetized (Xylazine/Ketamine) mice according to standard procedures. 500 l TBS was administered s.c. according to standard procedures.

(92) Preparation and Administration of Test Item 4/Challenge Virus.

(93) The RSV stock vial was thawed and used as quickly as possible due to virus instability (maximal 15 minutes on ice). Virus was kept on ice at all times and used immediately to challenge anaesthetized (Xylazine/Ketamine) mice with 100 l of the neat virus solution by the intranasal route according to standard procedures.

(94) Post-Treatment Procedures:

(95) Body Weight.

(96) Body weights were monitored on a daily basis from the day of challenge until sacrifice according to standard procedures.

(97) Blood Sampling.

(98) Blood samples (approximately 150 l) were obtained by retro-bulbar or facial venous puncture (for details see Table 1 and Table 2) according to standard procedures. Blood samples were transferred to the laboratory for further processing according to standard procedures.

(99) Euthanasia.

(100) Euthanasia of half of the mice was performed on Day 48 by cervical dislocation. On Day 53, the remaining mice received a double dose of Ketamine-Xylazine by intra-peritoneal injection and euthanasia was done by cutting the aorta within the peritoneal cavity.

(101) Spleen Removal.

(102) Spleens were removed aseptically. They were placed into tubes filled with medium according to standard procedures. These tubes had been imported into the animal facility and were then exported according to standard procedures.

(103) Lung Lavage and Lung Removal.

(104) Bronchoalveolar lavage (BAL) fluid was collected by flushing the lungs 4 times with 1 ml of PBS. The lungs were then removed and snap-frozen in two halves in liquid nitrogen for subsequent plaque assay and RNA extraction.

(105) Analysis: Blood Sample Processing and Storage of Sera.

(106) Following transfer to the laboratory, the blood samples were processed to serum according to standard procedures. After preparation the sera were stored at 20 C. (5 C.) until required for analysis.

(107) Analysis of RSV-Specific Antibody Titres from Serum Samples.

(108) The total RSV-specific IgG ELISA titres were determined from all serum samples using a modified ELISA kit (Serion ELISA classic, Catalog No. ESR113G): Instead of the Alkaline Phosphatase-conjugated anti-human IgG antibody supplied with the kit, an Alkaline Phosphatase-conjugated goat anti-mouse IgG (Serotec cat: 103004) was used as the secondary antibody.

(109) The RSV-F/G-specific IgG ELISA titers were determined from all serum samples and BAL fluid using a modified ELISA kit (IBL-Hamburg Ref. RE56881). Instead of the POD-conjugated anti-human IgG antibody supplied with the kit, an HRP-conjugated sheep anti-mouse IgG (ref. BN-687-95/96, Serotec cat: AAC10P) was used as the secondary antibody.

(110) Except for groups 4 and 7, The RSV-F-specific IgG ELISA titers were determined from serum samples of Day 48 using a modified ELISA kit (IBL-Hamburg Ref. RE56881 reagents and RSV (F-protein) IgG microtiter strips Ref. RE56692). Instead of the POD-conjugated anti-human IgG antibody supplied within the kit, an HRP-conjugated sheep anti-mouse IgG (ref. BN-687-95/96 Serotec cat: AAC10P) was used as the secondary antibody.

(111) The RSV-specific IgA ELISA titers in sera and BAL fluid were determined from Day 48 and Day 53 samples, respectively, using a modified ELISA kit (IBL-Hamburg Ref. RE56881): Instead of the POD-conjugated anti-human IgG antibody supplied within the kit, an HRP-conjugated sheep anti-mouse IgA (ref. BN-687-95/96 Serotec cat: STAR137P) was used as the secondary antibody.

(112) Analysis of RSV-Specific Cellular Immune Responses from Splenocytes.

(113) The RSV F-, RSV G- and RSV M2-specific cellular responses were determined two weeks after the last administration by re-stimulation of splenocytes with specific peptides as described elsewhere (see, e.g., S. M. Varga et al. (2000); S. Johnstone et al. (2004); S. Jiang et al., (2002); and A. B. Kulkarni et al., J. Virol. 67(7):4086-4092 (1993)) and detection of IFN release from the splenocytes by ELISPOT assay.

(114) ELISPOT Assay Method.

(115) The Mouse IFN-Gamma-Kit (BD Biosciences, Catalog No. 551083) was used for the ELISPOT assay. The assay was performed according to the manufacturer's instructions. Briefly, plates were coated with the capture antibody the day prior to splenocyte isolation. After isolation, cells were transferred to the ELISPOT plates and stimulated with different peptides (see Table 3) for 20 hours at 37 C. IFN production was detected using the detection antibody. Plates were developed using the BD ELISPOT AEC Substrate Set (BD Biosciences, Catalog No. 551951) according to the manufacturer's instructions.

(116) ELISPOT Stimulation Plan.

(117) All conditions were tested in duplicate. RSV-1, RSV-2, RSV-3, RSV-4, and RSV-5 peptides (see Table 3) were used at a final concentration of 5 g/ml (1 g/well) to stimulate 510.sup.5 and 2.510.sup.5 splenocytes per well. MVA (immunization control) was used at a Multiplicity of Infection (MOI) of 10 to stimulate 510.sup.5 and 2.510.sup.5 splenocytes per well and Concanavalin A (ConA [positive control]) was used at a final concentration of 0.5 g/ml to stimulate 2.510.sup.5 splenocytes. As a negative control, 510.sup.5 splenocytes were cultured in medium only (RPMI-1640 supplemented with Glutamax, penicillin, streptomycin, 10% Fetal Calf Serum and 10.sup.5M -mercaptoethanol.

(118) TABLE-US-00003 TABLE3 RSV-SpecificStimulation PeptideName Specificity PeptideSequence RSV-1 F TYMLTNSELL (SEQIDNO:19) RSV-2 F KYKNAVTEL (SEQIDNO:20) RSV-3 F ELQLLMQSTPAANNR (SEQIDNO:21) RSV-4 G WAICKRIPNKKPG (SEQIDNO:22) RSV-5 M2 SYIGSINNI (SEQIDNO:27)

(119) Analysis of BAL Fluid and Lungs.

(120) Cellular characterization of the BAL was not possible, due to staining issues. The RSV load in the lung samples was determined by RSV plaque assay and by RT-qPCR.

(121) RSV Plaque Assay.

(122) One half each of the snap-frozen lungs was homogenized in 1 ml cold medium using a French Press (Dulbecco's Modified Eagle Medium supplemented with 7% Fetal Calf Serum). After a brief centrifugation, two tubes of each supernatant were titrated in two-fold serial dilutions onto Vero cell monolayers grown in 48-well flat-bottomed plates. Six days later, the monolayers were washed and fixed with 1% Formaldehyde. After 24 hours, the monolayers were stained with 0.04% Neutral Red and plaques were counted.

(123) RSV RT-qPCR.

(124) 100 l of the homogenized lung tissue was removed immediately and RNA was isolated using the RNeasy Mini Kit from Qiagen (Catalog No. 74104). The reverse transcription reaction was performed using the High Capacity RNA-to-cDNA Kit from Applied Biosystems (Catalog No. 4387406). PCR specific for the RSV L gene was performed with the following parameters in a thermal cycler: (1) 50 C. for 2 minutes; (2) 95 C. for 10 minutes; (3) 45 cycles of (15 seconds at 95 C., 1 minute at 60 C.) using the Universal PCR Master Mix from Applied Biosystems (Catalog No. 4352042) and a mixture of three primers: (1) primer 1 (5-GAA CTC AGT GTA GGT AGA ATG TTT GCA-3; SEQ ID NO:36); (2) primer 2 (5-TTC AGC TAT CAT TTT CTC TGC CAA T-3; SEQ ID NO:37); and (3) probe 6 (5-TTT GAA CCT GTC TGA ACA TTC CCG GTT-3; (SEQ ID NO:38). Copy number was determined from a standard curve of pMISC202 plasmid vector containing a fragment of the RSV L gene. Similar reactions for murine beta-actin were used as internal controls for input cDNA using a VIC/MGB-labeled probe from Applied Biosystems (Catalog No. 4351315).

(125) Study Documentation.

(126) An in-life phase flow chart was prepared to collect all information during the individual steps of the in-life phase. In addition, mouse- or cage-specific information was recorded on the corresponding cage card. Cage cards are not considered as study raw data but a requirement from the Government of Upper Bavaria.

(127) An analysis phase flow chart was prepared to collect all information during the individual steps of the analysis phase. Assays were documented in assay-specific test records or Laboratory Note Books; cross-references were documented in the analysis phase flow chart. All assay documentation including raw data was reviewed according to standard procedures. In addition, sample tracking sheets for serum samples were prepared according to standard procedures.

(128) Data Processing.

(129) The raw data were transferred into the corresponding Excel files for further analysis according to standard procedures.

(130) ELISA.

(131) Mean values of the OD and standard errors of the mean were calculated using Excel.

(132) ELISPOT.

(133) ELISPOT plates were read with a CTL reader according to the manufacturer's instructions. The number of spot forming cells (SFC) was determined for each well and transferred into an Excel file for further evaluation. From the incubation with 510.sup.5 and 2.510.sup.5 cells per well, the number of spots per 110.sup.6 splenocytes was calculated for each well. The mean for the negative control was calculated and was subtracted from each individual value prior to the calculation of the mean value per mouse to obtain the Stimulation Index (SI) value (peptide-specific frequency of IFN- releasing splenocytes) per mouse.

(134) For the peptide stimulations, SI was obtained from the wells with 510.sup.5 and 2.510.sup.5 cells, except when the spots were too numerous to count or for the RSV immunized animals. In those cases only the concentration 2.510.sup.5 was used. For MVA-BN stimulation, SI was obtained from the wells with 510.sup.5, except when the spots were too numerous to count. In that case the concentration 2.510.sup.5 was used. Following determination of the SI for individual animals, the mean of SI (SFC per 110.sup.6 splenocytes) and standard error of the mean (SEM) was calculated per group.

(135) Body Weight Changes.

(136) Individual body weight values (in grams) prior to RSV challenge were taken as baseline values. With these baseline values, individual animal body weight changes (in %), as well as mean body weight changes of the groups were calculated for each monitored time point post challenge using Microsoft Excel.

(137) RSV Plaque Assay.

(138) The numbers of plaques were counted in the well with the three highest countable dilutions of virus. The average number of plaques adjusted by the dilution factor was then multiplied by 10 to obtain the titer of the solution in pfu/ml and finally multiplied by 2 to obtain the titer per lung.

(139) RSV RT-qPCR.

(140) PCR amplifications were measured in real time using the ABI 7500 from Applied Biosystems (Catalog No. 4351107) and analyzed using the System Software supplied by Applied Biosystems. All values were compared to the L gene standard and were normalized to the murine beta-actin determination for each sample.

(141) Results

(142) Analysis of the Humoral Immune Response: Analysis of RSV-Specific IgG Antibody Response from Serum Samples.

(143) Sera were first analyzed with an ELISA based on the IBL-Hamburg kit using plates coated only with recombinant RSV F and G proteins (FIG. 2 and FIG. 3). As shown in FIG. 2, similar RSV-specific IgG responses (ODs ranging between 0.870 and 1.347) were observed with all three constructs (MVA-mBN199B, MVA-mBN201BM2 and MVA-mBN201B) after a single immunization and independent of the route used for immunization (s.c. or i.n.). While the second immunization resulted in a 2.0- to nearly 3.5-fold increase in the antibody response (ODs ranging between 2.627 to 3.407), the third s.c. injection had only a minor effect on the B-cell response, producing an increase of approximately 0.500 OD units compared to ODs after the second immunization. Similar results were obtained with an ELISA based on the IBL Hamburg kit using plates coated only with recombinant RSV F protein (FIG. 4).

(144) After serial dilution of sera (1/100, 1/200 and 1/400) RSV F- and RSV G-specific ELISA results showed that MVA-mBN199B, MVA-mBN201BM2 and MVA-mBN201B induced similar RSV F- and RSV G-specific IgG responses despite the additional expression of a truncated RSV F protein and a truncated RSV G protein by MVA-mBN201BM2 and MVA-mBN201B (FIG. 3). After two s.c. immunizations with the constructs, the B-cell response was still lower compared to the immunization with RSV alone (positive control). To reach the level of antibody response induced by two i.n. applications of RSV required a third s.c. immunization with the MVA-mBN constructs. In contrast, 2 i.n. immunizations with MVA-mBN199B and MVA-mBN201B induced similar B-cell responses as two immunizations with RSV alone or 3 s.c. immunizations with MVA-mBN199B, MVA-mBN201BM2 and MVA-mBN201B, when analyzed with ELISA based on the IBL Hamburg kit (FIG. 3).

(145) When sera were analyzed again by ELISA based on the Serion kit using plates coated with an RSV lysate, again no differences between MVA-mBN199B, MVA-mBN201BM2 and MVA-mBN201B were found. No differences between 2 and 3 immunizations, or between the s.c. and i.n. routes of administration were observed either. In addition, the responses were all lower than the antibody response induced by 2 i.n. applications of RSV (FIG. 5).

(146) Analysis of RSV-Specific IgA Antibody Responses.

(147) RSV F- and RSV G-specific IgA (based on the IBL Hamburg kit) was measured in BAL fluid 4 days post-challenge (Day 53). In addition, also BAL and sera for RSV F- and RSV G-specific IgG by ELISA were analyzed. Results were compared to the results obtained in sera just before challenge (Day 48) and are shown in FIG. 6.

(148) As expected, IgA responses were detected only after i.n. application with RSV, MVA-mBN199B and MVA-mBN201B. Although IgG could also be detected in the BAL, IgA was detected at a higher level after i.n. application. Serum levels of IgA were much lower than IgG levels independent of the route of application.

(149) Analysis of RSV-Specific Cellular Immune Responses.

(150) T-cell responses were analyzed in the spleen by ELISPOT two weeks after the last immunization (FIG. 7). MVA-mBN199B administered by the i.n. or s.c. route induced a strong RSV-F specific T-cell response. This immune response was mainly directed against the RSV-F-specific peptide RSV-2, which is immunodominant in the absence of RSV-M2. The response was around 2000 spots per 10.sup.6 splenocytes after the 2.sup.nd s.c. immunization, and around 4000 after the 3.sup.rd s.c. injection or 2.sup.nd intranasal application. Similar to the response to RSV intranasal applications, a low G-specific response to peptide RSV-4 was detected after immunization with MVA-mBN199B (approximately 500 spots per 10.sup.6 splenocytes) and as expected, MVA-mBN199B did not induce M2-specific T-cells. The M2 peptide is the immuno-dominant peptide of RSV in mice. Consequently, RSV intranasal immunizations induced a good M2-specific T-cell response above 1000 spots per 10.sup.6 splenocytes and almost no F-specific T-cell response.

(151) Like MVA-mBN199B, MVA-mBN201B induced a strong T-cell response, but it was dominated by M2 (above 4000 spots per 10.sup.6 splenocytes independent of the number of doses administered or the route of administration). Even the G-specific response induced by MVA-mBN201B was at least 3-fold higher than the G-specific response induced by MVA-mBN199B or RSV. In contrast to MVA-mBN199B, the F-specific response induced by MVA-mBN201B was much lower, with less than 600 spots per 10.sup.6 splenocytes for the RSV-2 peptide.

(152) RSV Challenge with RSV A2 Strain.

(153) Mice were challenged intranasally with 10.sup.6 pfu of RSV(A2) two weeks after the last immunization. Body weight was monitored daily. Four days post challenge, mice were sacrificed. After lung lavage with 1 ml PBS, lungs were removed and the RSV load in lung was determined by plaque assay and RT-qPCR conducted as described above.

(154) Body Weight Changes.

(155) All mice lost weight one day post-challenge, most probably due to anesthesia or the i.n. challenge itself (FIG. 8). TBS-treated mice started to significantly lose weight 4 days post-RSV challenge. In contrast, mice that received RSV intranasally for the third time did not show body weight loss. All mice immunized s.c. with MVA-mBN199B, MVA-mBN201B or MVA-mBN201BM2 lost about 20% weight 4 days post challenge. Such weight loss was described earlier by Olszewska et al. (Vaccine 23:215 (2004)). However our RT-qPCR results (FIG. 10) show that it correlates to a better protection and earlier elimination of RSV from lung via the vaccine-primed CTL response compared to the normal clearance of primary RSV infection. When applied i.n., MVA-mBN201B immunized mice had a similar weight loss than s.c. immunized mice 2 days post-challenge, but had recovered 4 days post-challenge due to the low RSV load in lungs compared to the s.c. route (FIG. 10). Like the RSV-immunized group, mice immunized i.n. with MVA-mBN199B showed no weight loss.

(156) RSV Load Measured by Plaque Assay.

(157) Four days post challenge an average of 57671 pfu per lung for the non-immunized mice was detected (FIG. 9). As in the RSV-immunized control group, no RSV A2 plaques were detected in the lungs of animals immunized with MVA-mBN199B, MVA-mBN201B or MVA-mBN201BM2 after 2 s.c. or i.n. applications.

(158) RSV Load Measured by Quantitative Real-Time PCR.

(159) The RSV load in lung was also analyzed by RT-qPCR (FIG. 10). While RSV was not detected by plaque assay in any of the vaccinated mice, RSV genomes were still detectable in mice immunized three times with MVA-mBN199B. After 3 immunizations with MVA-mBN199B, the RSV load was 38 times lower compared to the TBS control group. RSV genomes were also detectable after three immunizations with MVA-mBN201B but the load was 158 times lower compared to the TBS control group. There was no major difference between mice immunized two or three times. Interestingly, the decrease in the RSV load observed with MVA-mBN201B was not observed after vaccination with MVA-mBN201BM2 which was in absence of M2 equivalent to MVA-mBN199B.

(160) Nearly complete protection comparable that obtained in the group treated with RSV was observed after i.n. immunization with MVA-mBN201B, although a few copies of the L gene were still detectable in one mouse out of five. Intranasal immunization with MVA-mBN199B also induced a strong decrease in the RSV load, but the L gene was still detected at a low level in three mice out of four.

(161) Discussion and Conclusions

(162) Although MVA-mBN201B expresses truncated versions of RSV F and G proteins in addition to the full-length RSV F and G proteins also included in the MVA-mBN199B construct, MVA-mBN201B induced a humoral immune response of similar magnitude. Both constructs induced an antibody response directed mostly against the RSV F protein as judged by similarly good responses measured in the RSV F-only ELISA compared to the RSV F and G ELISA. The antibody level following two i.n. applications was higher than after two s.c. applications. A third s.c. application was required to reach the antibody response level induced by two i.n. applications. In contrast, no major differences were observed in the T-cell responses induced using the s.c. versus i.n. routes, or using 2 versus 3 s.c. applications. However, MVA-mBN199B induced a good RSV F-specific cellular response, whereas a strong M2-specific T-cell response with MVA-mBN201B was observed. The RSV G-specific response induced by MVA-mBN201B was also more pronounced compared to MVA-mBN199B. The pattern of T-cell response induced by MVA-mBN201B was similar to the T-cell response induced by RSV immunization, albeit much higher.

(163) Independent of the routes or the number of applications, both constructs protected mice from challenge with RSV(A2), and no replicating virus could be recovered from the lungs. However, as previously observed, s.c. immunizations with MVA-mBN199B or MVA-mBN201B did not result in sterile immunity (i.e., immunity which persists even after the targeted infectious agent is cleared from the body). The genomic RSV load (measured by levels of the viral RNA polymerase (L) gene) in the lungs of mice immunized by s.c. application of MVA-mBN199B or MVA-mBN201B was significantly reduced but still detectable by quantitative RT-PCR, and a third s.c. immunization had no beneficial impact on viral load despite its increase in RSV-specific IgG levels. The reduction in RSV L protein expression was a little more pronounced after vaccination with MVA-mBN201B compared to MVA-mBN199B, which might be due to the increased M2-specific CD8+ T-cell response, as the RSV genomic load was higher in animals vaccinated with MVA-mBN201BM2 than for animals vaccinated with MVA-mBN201B, like MVA-mBN199B.

(164) Sterile immunity was almost obtained after two i.n. applications of MVA-mBN199B or MVA-mBN201B. This observation correlated with the induction and secretion of RSV-specific IgA at the mucosal infection site.

Example 3: Safety of Recombinant MVA Vaccines Expressing RSV F Protein, RSV G Protein, RSV N Protein, and RSV M2 Protein, Compared to FI-RSV

(165) Vaccine candidate MVA-mBN199B encodes the glycoprotein (G) and the fusion (F) protein of RSV, while MVA-mBN201B express truncated versions of F and G in addition to full-length proteins, the nucleocapsid protein (N) and the matrix protein (M2) of RSV (see FIG. 1). The objective of these experiments was to analyze the safety of MVA-mBN199B and MVA-mBN201B compared to FI-RSV after two immunizations via the subcutaneous (s.c.) or intranasal (i.n.) routes of administration.

(166) The safety of these constructs was tested using an RSV(A2) challenge model in BALB/c mice. Two immunizations with MVA-mBN199B or MVA-mBN201B did not induced increased IL4 and IL5 secretion in BAL following RSV(A2) challenge, compared to FI-RSV.

(167) Study Design

(168) Mice were treated twice three weeks apart subcutaneously (s.c.) or intranasally (i.n.) with 110.sup.8 TCID.sub.50 MVA-mBN199B (Groups 3 and 4), 110.sup.8 TCID.sub.50 MVA-mBN201B (Groups 5 and 6) according to Table x. The three control groups were treated (s.c.) twice with TBS (Group 1) or i.n. with RSV (Group 2) or i.m. with 30 g FI-RSV(Group 7), according to Table x. On Day 35, mice were challenged (i.n.) with 10.sup.6 pfu RSV A2. Four days post challenge, mice were sacrificed by injection of an elevated dose of Ketamine-Xylazine and end-bled. After lung lavage, IL4 and IL5 level were analyzed in BAL by ELISA.

(169) TABLE-US-00004 TABLE 4 Experimental Design Administration of Test or Reference Items Dose Group Schedule per Challenge Group Size Injections (Day)% Route Injection (Day)%, # 1 5 TBS 0 and 21 s.c. n.a. 35 2 5 RSV i.n. 10.sup.6 pfu 3 5 MVA- s.c. 1 10.sup.8 mBN199B TCID.sub.50 4 5 i.n. 1 10.sup.8 TCID.sub.50 5 5 MVA- s.c. 1 10.sup.8 mBN201B TCID.sub.50 6 5 i.n. 1 10.sup.8 TCID.sub.50 7 5 FI-RSV i.m. 30 g %Relative to the first immunization. #Mice were challenged by the intranasal route with 10.sup.6 pfu of RSV A2. Four days after challenge, mice were bled and sacrificed under anesthesia. BAL fluid were sampled.

(170) Study Schedule.

(171) The schedule of the in-life phase is summarized in Table 5.

(172) TABLE-US-00005 TABLE 5 Study schedule of the In-life Phase Day** Procedures 9 Arrival and import in animal facility of BALB/c mice, cage card allocation and allocation of 5 mice per cage 1 Ear clipping, inclusion/exclusion examination of all mice 0 1.sup.st administration of mice 21 2.sup.nd administration of mice 35 RSV(A2) Challenge 39 Final bleed, sacrifice & sampling of BAL **Relative to the day of the 1.sup.st immunization.
Material and Methods

(173) Experimental Animals.

(174) female BALB/cJ Rj (H-2d) mice at the age of seven weeks were obtained from Janvier (Route des Chnes Secs, F-53940 Le Genest-Saint-Isle, France). All mice were specific pathogen free.

(175) Housing.

(176) The study was performed in room 117 of the animal facility at Bavarian Nordic-Martinsreid. This unit was provided with filtered air at a temperature of 20-24 C. and a relative humidity between 40% and 70%. The room was artificially illuminated on a cycle of 14 hours of light and 10 hours of darkness. The study acclimatization period was 15 days. The animals were housed in transparent SealSafe-cages (H Temp [polysulfon] cage Type II LEuro standard), with a floor area of 530 cm.sup.2. The cages were covered with an H-Temp SealSafe lid. The cages were placed in a TECNIPLAST-IVC SealSafe system with a SLIMLine circulation unit providing every single cage separately with HEPA-filtered air. Animal bedding was changed once a week.

(177) Diet and Water.

(178) Mice were provided with free access to irradiated maintenance diet (SSNIFF R/M-H, irradiated, V1534-727) and water (autoclaved at 121 C. for 20 minutes).

(179) Pre-Treatment Procedures: Identification of Animals.

(180) To individually mark animals within each cage, ear punching was done according to standard procedures.

(181) Inclusion/Exclusion Examination.

(182) Inclusion/exclusion examination was done according to standard procedures.

(183) Blood Sampling for Pre-Bleed.

(184) Blood samples of approximately 150 l were obtained by facial vein puncture according to standard procedures. Blood samples were transferred to the laboratory for further processing according to standard procedures.

(185) Treatment Procedures:

(186) Preparation and administration of Test Items and Reference Item. Preparation and administration of test and reference items was performed in a class II microbiological safety cabinet (HERAsafe/class II type H, Kendro) according to standard procedures. Briefly, for s.c. administration, recombinant MVAs were diluted in TBS to obtain a working solution with a concentration of 210.sup.8 TCID.sub.50/ml. 110.sup.8 TCID.sub.50 in 500 l was injected s.c. according to standard procedures. For i.n. administration, recombinant MVAs were diluted in TBS to obtain a working solution with a concentration of 210.sup.9 TCID.sub.50/ml. 50 l of the diluted viruses was administered in one nostril of anesthetized (Xylazine/Ketamine) mice according to standard procedures. 500 l TBS was administered s.c. according to standard procedures.

(187) Preparation and Administration of RSV(A2) Virus.

(188) The RSV stock vial was thawed and used as quickly as possible due to virus instability (maximal 15 minutes on ice). Virus was kept on ice at all times and used immediately to challenge anaesthetized (Xylazine/Ketamine) mice with 100 l of the neat virus solution by the intranasal route according to standard procedures.

(189) Preparation and Administration of FI-RSV.

(190) 30 g FI-RSV in 40 l was injected intramuscularly.

(191) Euthanasia.

(192) On Day 35, the remaining mice received a double dose of Ketamine-Xylazine by intra-peritoneal injection and euthanasia was done by cutting the aorta within the peritoneal cavity.

(193) Lung Lavage.

(194) Bronchoalveolar lavage (BAL) fluid was collected by flushing the lungs 4 times with 1 ml of PBS.

(195) Analysis

(196) IL-4 and IL-5 levels were measured in bronchoalveolar lavage (BAL) supernatant using commercially available ELISA kits (mIL4 PLATINUM ELISA from eBIOSCIENCE Cat N BMS613 and READY-SET-GO MIL-5 ELISA from eBIOSCIENCE Cat N 88-7054-22).

(197) Study Documentation.

(198) An in-life phase flow chart was prepared to collect all information during the individual steps of the in-life phase. In addition, mouse- or cage-specific information was recorded on the corresponding cage card. Cage cards are not considered as study raw data but a requirement from the Government of Upper Bavaria.

(199) An analysis phase flow chart was prepared to collect all information during the individual steps of the analysis phase. Assays were documented in assay-specific test records or Laboratory Note Books; cross-references were documented in the analysis phase flow chart. All assay documentation including raw data was reviewed according to standard procedures. In addition, sample tracking sheets for serum samples were prepared according to standard procedures.

(200) Data Processing.

(201) The raw data were transferred into the corresponding Excel files for further analysis according to standard procedures.

(202) ELISA.

(203) Cytokine concentrations were determined from the standard curve of the respective ELISA kits.

(204) Results

(205) An increase of IL-4 (FIG. 11) and IL-5 (FIG. 12) production like that observed with FI-RSV was not observed for MVA-mBN199B or MVA-mBN201B. Both cytokines were below the detection level when mice were immunized i.n. with MVA-mBN199B or MVA-mBN201B.

(206) Discussion and Conclusions

(207) Both MVA-mBN199B and MVA-mBN201B do not induce enhanced disease compared to FI-RSV as assessed by TH2 response.

Example 4: Comparison of Immunogenicity Efficacy and Safety of Different Recombinant MVA Vaccines Expressing RSV F Protein, RSV G Protein, RSV N Protein, and RSV M2 Proteins

(208) Vaccine candidate MVA-mBN199B encodes the glycoprotein (G) and the fusion (F) protein of RSV, MVA-mBN201B expresses truncated versions of F and G in addition to full-length proteins, the nucleocapsid protein (N) and the matrix protein (M2) of RSV and MVA-mBN294B expresses one F and 2 G full-length proteins, the nucleocapsid protein (N) and the matrix protein (M2) of RSV (see FIG. 1). MVA-mBN294A is in an intermediate product in the cloning MVA-mBN294B which still has one cloning cassette in. This cloning cassette does not impact either transgene expression or the immunogenic properties of the transgenic proteins. The objective of this experiment was to analyze the Immunogenicity, efficacy and safety of MVA-mBN294A compared to MVA-mBN199B and MVA-mBN201B after two immunizations via the subcutaneous (s.c.) route of administration.

(209) The immunogenicity efficacy and safety of these constructs was tested using an RSV(A2) challenge model in BALB/c mice. We confirmed that despite the changes in MVA-mBN294A (equivalent to MVA-mBN294B) compared to MVA-mBN201B, it induced similar B- and T-cell responses and offered similar protection. This experiment showed that any constructs (MVA-mBN201B or MVA-mBN294A) expressing at least one antigenic determinant of an RSV membrane glycoprotein (F or G) and at least one antigenic determinant of an RSV nucleocapsid protein (N or M2) induces better protection than a construct expressing only antigenic determinants of RSV membrane glycoproteins (MVA-mBN199B)

(210) Study Design

(211) Mice were vaccinated (s.c.) with 110.sup.8 TCID.sub.50 MVA-mBN294A, MVA-mBN199B or MVA-mBN201B in a prime-boost schedule (Day 0 and 21) according to Table 6. The control groups were treated twice subcutaneously with TBS or with RSV-A2 according to Table 6. Formalin Inactivated (FI)-RSV was injected intramuscularly (i.m.) either once or twice according to Table 6.

(212) Blood was collected one day prior to each immunization and prior to challenge, as well as on the day of sacrifice. For 5 animals of groups 1 to 5 on Day 34, RSV-specific IgG titers and RSV-specific neutralizing antibody titers were determined by ELISA and PRNT respectively.

(213) On Day 34, some mice (Table 6) were sacrificed by injection of a lethal dose of ketamine-xylazine and final bleed. Spleens were removed and prepared for the analysis of RSV-specific T cell responses by ELISPOT.

(214) On Day 35, the remaining mice (Table 6) were challenged with 10.sup.6 pfu RSV-A2. Four days post-challenge, mice were sacrificed by injection of a lethal dose of ketamine-xylazine and final bleed. After lung lavage, the lungs were removed and RSV load was analyzed by plaque assay and RT-qPCR. Cellular infiltration and cytokines level in Bonchoalveolar lavage (BAL) fluids were analyzed.

(215) TABLE-US-00006 TABLE 6 Experimental Design Administration of Test or Reference Items Schedule for Group Injections Dose per Bleed Challenge Group Size Injections (Day) .sup.1 Route Injection (Day) .sup.1 (Day) .sup.1, 2 1 10 TBS 0 and 21 s.c. 1, 20, 34 and 35 39 5 1, 20 and 34.sup.& 2 10 RSV i.n. 10.sup.6 pfu 1, 20, 34 and 35 39 5 1, 20 and 34.sup.& 3 10 MVA-mBN199B s.c. 1 10.sup.8 1, 20, 34 and 35 TCID.sub.50 39 5 1, 20 and 34.sup.& 4 10 MVA-mBN201B 1, 20, 34 and 35 39 5 1, 20 and 34.sup.& 5 10 MVA-mBN294A 1, 20, 34 and 35 39 5 1, 20 and 34.sup.& 6 10 FI-RSV i.m. 50 l 1, 20, 34 and 35 39 5 1, 20 and 34.sup.& 7 5 FI-RSV 0 i.m. 50 l 1, 20, 34 and 35 39 .sup.1 relative to the first immunization .sup.2 Mice will be challenged by the intranasal route with 10.sup.6 pfu of RSV-A2. Four days after challenge, mice will be bled, sacrificed under anesthesia and BAL and lungs will be sampled .sup.&on Day 34, these mice will be sacrificed and spleens will be analyzed by ELISPOT

(216) Study Schedule.

(217) The schedule of the in-life phase is summarized in Table 7.

(218) TABLE-US-00007 TABLE 7 Study schedule of the Part 1 of the In-life Phase Day.sup.1 Procedures 9 Arrival and import in animal facility of BALB/c mice, cage card allocation and allocation of 5 mice per cage 1 Ear clipping, inclusion/exclusion examination of all mice 1 Pre-bleed of all mice (facial vein puncture right side) 0 1.sup.st administration 20 Bleed of all mice (facial vein puncture left side) 21 2.sup.nd administration 34 Final bleed, sacrifice and sampling of spleen for cages B, D, F, H, K and M 34 Bleed of all remaining mice (retro-bulbar vein puncture right eye) 35 Challenge of all remaining mice 35 to Appearance and body weight measurement daily 39 39 Final bleed, sacrifice and sampling of BAL and lung of mice .sup.1relative to the day of the 1.sup.st immunization
Material and Methods

(219) Experimental Animals.

(220) female BALB/cJ Rj (H-2d) mice at the age of seven weeks were obtained from Janvier (Route des Chnes Secs, F-53940 Le Genest-Saint-Isle, France). All mice were specific pathogen free.

(221) Housing.

(222) The study was performed in room 117 of the animal facility at Bavarian Nordic-Martinsreid. This unit was provided with filtered air at a temperature of 20-24 C. and a relative humidity between 40% and 70%. The room was artificially illuminated on a cycle of 14 hours of light and 10 hours of darkness. The study acclimatization period was 15 days. The animals were housed in transparent SealSafe-cages (H Temp [polysulfon] cage Type II LEuro standard), with a floor area of 530 cm.sup.2. The cages were covered with an H-Temp SealSafe lid. The cages were placed in a TECNIPLAST-IVC SealSafe system with a SLIMLine circulation unit providing every single cage separately with HEPA-filtered air. Animal bedding was changed once a week.

(223) Diet and Water.

(224) Mice were provided with free access to irradiated maintenance diet (SSNIFF R/M-H, irradiated, V1534-727) and water (autoclaved at 121 C. for 20 minutes).

(225) Pre-Treatment Procedures:

(226) Identification of Animals.

(227) To individually mark animals within each cage, ear punching was done according to standard procedures.

(228) Inclusion/Exclusion Examination.

(229) Inclusion/exclusion examination was done according to standard procedures.

(230) Blood Sampling for Pre-Bleed.

(231) Blood samples of approximately 150 l were obtained by facial vein puncture according to standard procedures. Blood samples were transferred to the laboratory for further processing according to standard procedures.

(232) Treatment Procedures:

(233) Preparation and administration of Test Items and Reference Item. Preparation and administration of test and reference items was performed in a class II microbiological safety cabinet (HERAsafe/class II type H, Kendro) according to standard procedures. Briefly, for s.c. administration, recombinant MVAs were diluted in TBS to obtain a working solution with a concentration of 210.sup.8 TCID.sub.50/ml. 110.sup.8 TCID.sub.50 in 500 l was injected s.c. according to standard procedures. 500 l TBS was administered s.c. according to standard procedures.

(234) Preparation and Administration of RSV(A2) Virus.

(235) The RSV stock vial was thawed and used as quickly as possible due to virus instability (maximal 15 minutes on ice). Virus was kept on ice at all times and used immediately to challenge anaesthetized (Xylazine/Ketamine) mice with 100 l of the neat virus solution by the intranasal route according to standard procedures.

(236) Preparation and Administration of FI-RSV:

(237) 50 l of FI-RSV was applied i.m.

(238) Post-Treatment Procedures:

(239) Blood Sampling.

(240) Blood samples (approximately 150 l) were obtained by retro-bulbar or facial venous puncture (for details see Table 7) according to standard procedures. Blood samples were transferred to the laboratory for further processing according to standard procedures.

(241) Euthanasia.

(242) Mice received a double dose of Ketamine-Xylazine by intra-peritoneal injection and euthanasia was done by cutting the aorta within the peritoneal cavity.

(243) Spleen Removal.

(244) Spleens were removed aseptically. They were placed into tubes filled with medium according to standard procedures. These tubes had been imported into the animal facility and were then exported according to standard procedures.

(245) Lung Lavage and Lung Removal.

(246) Bronchoalveolar lavage (BAL) fluid was collected by flushing the lungs 4 times with 1 ml of PBS. The lungs were then removed and snap-frozen in two halves in liquid nitrogen for subsequent plaque assay and RNA extraction.

(247) Analysis:

(248) Blood Sample Processing and Storage of Sera.

(249) Following transfer to the laboratory, the blood samples were processed to serum according to standard procedures. After preparation the sera were stored at 20 C. (5 C.) until required for analysis.

(250) Analysis of RSV-Specific Antibody Titres from Serum Samples.

(251) The total RSV-specific IgG ELISA titres were determined from all serum samples using a modified ELISA kit (Serion ELISA classic, Catalog No. ESR113G): Instead of the Alkaline Phosphatase-conjugated anti-human IgG antibody supplied with the kit, an Alkaline Phosphatase-conjugated goat anti-mouse IgG (Serotec cat: 103004) was used as the secondary antibody.

(252) Analysis of RSV-Specific Neutralizing Antibody Titres from Serum Samples.

(253) Briefly, 2-fold serial dilutions of the test sera were prepared and a defined number of RSV plaque forming units (pfu) were added to the serum dilution. After 185 min incubation at 36 C. (2 C.) and 5% CO.sub.2 (1%). it was added to pre-seeded plates containing Vero cells. Two days later plates were fixed, immuno-stained with a mixture of RSV-specific antibodies and plaques were counted.

(254) Analysis of RSV-Specific Cellular Immune Responses from Splenocytes.

(255) The RSV F- and RSV M2-specific cellular responses were determined two weeks after the last administration by re-stimulation of splenocytes with specific peptides as described elsewhere and detection of IFN release from the splenocytes by ELISPOT assay.

(256) ELISPOT Assay Method.

(257) The Mouse IFN-Gamma-Kit (BD Biosciences, Catalog No. 551083) was used for the ELISPOT assay. The assay was performed according to the manufacturer's instructions. Briefly, plates were coated with the capture antibody the day prior to splenocyte isolation. After isolation, cells were transferred to the ELISPOT plates and stimulated with different peptides (see Table 3) for 20 hours at 37 C. IFN production was detected using the detection antibody. Plates were developed using the BD ELISPOT AEC Substrate Set (BD Biosciences, Catalog No. 551951) according to the manufacturer's instructions.

(258) ELISPOT Stimulation Plan.

(259) All conditions were tested in duplicate. RSV-2 and RSV-5 peptides (see Table 8) were used at a final concentration of 5 g/ml (1 g/well) to stimulate 510.sup.5 and 2.510.sup.5 splenocytes per well. MVA (immunization control) was used at a Multiplicity of Infection (MOI) of 10 to stimulate 510.sup.5 and 2.510.sup.5 splenocytes per well and Concanavalin A (ConA [positive control]) was used at a final concentration of 0.5 g/ml to stimulate 2.510.sup.5 splenocytes. As a negative control, 510.sup.5 splenocytes were cultured in medium only (RPMI-1640 supplemented with Glutamax, penicillin, streptomycin, 10% Fetal Calf Serum and 10.sup.5 M -mercaptoethanol.

(260) TABLE-US-00008 TABLE8 RSV-SpecificStimulation PeptideName Specificity PeptideSequence RSV-2 F KYKNAVTEL (SEQIDNO:20) RSV-5 M2 SYIGSINNI (SEQIDNO:27)
Analysis of BAL Fluid:

(261) Two slides were prepared by cytospin centrifugation (800 rpm, 5 minutes) of 100 l of BAL fluid. Slides were dried overnight and then stained. Slides were analyzed by microscopy to determine the percentage of eosinophils and neutrophils. The rest of the BAL was then be centrifuged (12,000 rpm 5 minutes). After preparation, the BAL supernatants were stored at 20 C. (5 C.) until analysis. IL-4 and IL-5 levels were measured in bronchoalveolar lavage (BAL) supernatant using commercially available ELISA kits (mIL4 PLATINUM ELISA from eBIOSCIENCE Cat N BMS613 and READY-SET-GO MIL-5 ELISA from eBIOSCIENCE Cat N 88-7054-22).

(262) Analysis of RSV Load in the Lung

(263) The RSV load in the lung samples was determined by RSV plaque assay and by RT-qPCR.

(264) RSV Plaque Assay.

(265) One half each of the snap-frozen lungs was homogenized in 1 ml cold medium using a French Press (Dulbecco's Modified Eagle Medium supplemented with 7% Fetal Calf Serum). After a brief centrifugation, two tubes of each supernatant were titrated in two-fold serial dilutions onto Vero cell monolayers grown in 48-well flat-bottomed plates. Six days later, the monolayers were washed and fixed with 1% Formaldehyde. After 24 hours, the monolayers were stained with 0.04% Neutral Red and plaques were counted.

(266) RSV RT-qPCR.

(267) 100 l of the homogenized lung tissue was removed immediately and RNA was isolated using the RNeasy Mini Kit from Qiagen (Catalog No. 74104). The reverse transcription reaction was performed using the High Capacity RNA-to-cDNA Kit from Applied Biosystems (Catalog No. 4387406). PCR specific for the RSV L gene was performed with the following parameters in a thermal cycler: (1) 50 C. for 2 minutes; (2) 95 C. for 10 minutes; (3) 45 cycles of (15 seconds at 95 C., 1 minute at 60 C.) using the Universal PCR Master Mix from Applied Biosystems (Catalog No. 4352042) and a mixture of three primers: (1) primer 1 (5-GAA CTC AGT GTA GGT AGA ATG TTT GCA-3; SEQ ID NO:36); (2) primer 2 (5-TTC AGC TAT CAT TTT CTC TGC CAA T-3; SEQ ID NO:37); and (3) probe 6 (5-TTT GAA CCT GTC TGA ACA TTC CCG GTT-3; (SEQ ID NO:38). Copy number was determined from a standard curve of pMISC202 plasmid vector containing a fragment of the RSV L gene. Similar reactions for murine beta-actin were used as internal controls for input cDNA using a VIC/MGB-labeled probe from Applied Biosystems (Catalog No. 4351315).

(268) Study Documentation.

(269) An in-life phase flow chart was prepared to collect all information during the individual steps of the in-life phase. In addition, mouse- or cage-specific information was recorded on the corresponding cage card. Cage cards are not considered as study raw data but a requirement from the Government of Upper Bavaria.

(270) An analysis phase flow chart was prepared to collect all information during the individual steps of the analysis phase. Assays were documented in assay-specific test records or Laboratory Note Books; cross-references were documented in the analysis phase flow chart. All assay documentation including raw data was reviewed according to standard procedures. In addition, sample tracking sheets for serum samples were prepared according to standard procedures.

(271) Data Processing.

(272) The raw data were transferred into the corresponding Excel files for further analysis according to standard procedures.

(273) ELISA.

(274) Mean values of the OD and standard errors of the mean were calculated using Excel.

(275) PRNT.

(276) Plaques were transfer to a macro to calculate a PRNT titer according to standard procedures.

(277) ELISPOT.

(278) ELISPOT plates were read with a CTL reader according to the manufacturer's instructions. The number of spot forming cells (SFC) was determined for each well and transferred into an Excel file for further evaluation. From the incubation with 510.sup.5 and 2.510.sup.5 cells per well, the number of spots per 110.sup.6 splenocytes was calculated for each well. The mean for the negative control was calculated and was subtracted from each individual value prior to the calculation of the mean value per mouse to obtain the Stimulation Index (SI) value (peptide-specific frequency of IFN- releasing splenocytes) per mouse.

(279) For the peptide stimulations, SI was obtained from the wells with 510.sup.5 and 2.510.sup.5 cells, except when the spots were too numerous to count or for the RSV immunized animals. In those cases only the concentration 2.510.sup.5 was used. For MVA-BN stimulation, SI was obtained from the wells with 510.sup.5, except when the spots were too numerous to count. In that case the concentration 2.510.sup.5 was used. Following determination of the SI for individual animals, the mean of SI (SFC per 110.sup.6 splenocytes) and standard error of the mean (SEM) was calculated per group.

(280) RSV Plaque Assay.

(281) The numbers of plaques were counted in the well with the three highest countable dilutions of virus. The average number of plaques adjusted by the dilution factor was then multiplied by 10 to obtain the titer of the solution in pfu/ml and finally multiplied by 2 to obtain the titer per lung.

(282) RSV RT-qPCR.

(283) PCR amplifications were measured in real time using the ABI 7500 from Applied Biosystems (Catalog No. 4351107) and analyzed using the System Software supplied by Applied Biosystems. All values were compared to the L gene standard and were normalized to the murine beta-actin determination for each sample.

(284) Cytokines ELISA.

(285) Cytokine concentrations were determined from the standard curve of the respective ELISA kits.

(286) Results

(287) Analysis of the Humoral Immune Response:

(288) For both RSV-specific IgG (ELISA, FIG. 13) and RSV-specific neutralizing antibody responses (PRNT, FIG. 14) we did not observe any differences between the three constructs (MVA-mBN199B, MVA-mBN201B and MVA-mBN294A)

(289) Analysis of the Cellular Immune Response:

(290) As expected, MVA-mBN294A had a similar T-cell response pattern than MVA-mBN201B (FIG. 15), inducing both F and M2 specific responses dominated by the M2 T-cell response. In contrast, MVA-mBN199B only induced a F-specific response but at a higher level than MVA-mBN201B and MVA-mBN294A.

(291) Analysis of the RSV Load in the Lungs:

(292) RSV Challenge with RSV A2 Strain.

(293) Mice were challenged intranasally with 10.sup.6 pfu of RSV(A2) two weeks after the last immunization. Four days post-challenge, mice were sacrificed. After lung lavage with 1 ml PBS, lungs were removed and the RSV load in lung was determined by plaque assay and RT-qPCR conducted as described above.

(294) RSV Load Measured by Plaque Assay.

(295) Four days post challenge an average of 29842 pfu per lung for the non-immunized mice was detected (FIG. 16). As in the RSV-immunized control group, no RSV A2 plaques were detected in the lungs of animals immunized with MVA-mBN199B, MVA-mBN201B or MVA-mBN294A after 2 s.c. applications.

(296) RSV Load Measured by Quantitative Real-Time PCR.

(297) The RSV load in lung was also analyzed by RT-qPCR (FIG. 17). While RSV was not detected by plaque assay in any of the vaccinated mice, RSV genomes were still detectable in mice immunized s.c. twice with MVA-mBN199B, MVA-mBN201B or MVA-mBN294A. For MVA-mBN199B, the RSV load was 45 times lower compared to the TBS control group. RSV genomes were also detectable for MVA-mBN201B and MVA-mBN294A but the load was strongly reduced compared to MVA-mBN199B, 416 times and 281 times lower compared to the TBS control group, respectively.

(298) Analysis of the Enhanced Disease Signs

(299) In contrast to the batch of FI-RSV used in the experiments described in Example 3, the new batch used in this study did not show any increase of IL-4 or IL-5 production. However we were able with this batch to detect eosinophil and neutrophil infiltrations in the BAL fluid which is the main hallmark of enhanced diseases for FI-RSV. No signs of enhanced diseases were detectable for MVA-mBN199B, MVA-mBN201B, and MVA-mBN294A

(300) Discussion and Conclusions

(301) Despite the differences between MVA-mBN294A (equivalent to MVA-mBN294B) and MVA-mBN201B, both induced similar B- and T-cell responses and offer similar protection without inducing enhanced disease. Both constructs induced a better protection than MVA-mBN199B which expressed only antigenic determinants of membrane glycoproteins (F and G).

(302) Other embodiments of the invention will be apparent to those skilled in the art from consideration of the specification and practice of the invention disclosed herein. It is intended that the specification and examples be considered as exemplary only, with a true scope and spirit of the invention being indicated by the following claims.