Paramyxovirus immunogens and related materials and methods

Abstract

The present invention includes methyltransferase (MTase)-defective recombinant viruses as live vaccine candidates for human metapneumovirus (hMPV), human respiratory syncytial virus (hRSV), and human parainfluenza virus type 3 (PIV3). Here the inventors provide the technical description for generating MTase-defective paramyxoviruses useful as immunogens, as well as related materials and methods.

Claims

1. A recombinant paramyxovirus composition comprising a nucleic acid molecule which encodes large RNA polymerase (L) protein having a defective mRNA cap MTase domain in a paramyxovirus viral genome, wherein the paramyxovirus viral genome is human respiratory syncytial virus (hRSV); and, wherein the nucleic acid molecule which encodes large RNA polymerase (L) protein having a defective mRNA cap MTase domain carries at least one mutation in a S-adenosyl methionine (SAM) binding site; wherein the at least one mutation in the SAM binding site is in a GGG . . . D/E/W site in an amino acid sequence corresponding to amino acid positions 31-90 of SEQ ID NO: 3, wherein the mutations comprise: at position 31, AGG . . . D; at position 33, GAG . . . D; at position 35, GGA . . . D; and, at position 90, GGG . . . A.

2. A method of eliciting an immune response in a human comprising: administering a recombinant paramyxovirus composition of claim 1.

3. The method of claim 2 wherein the composition is administered orally.

4. The method of claim 2 wherein the composition is administered intranasally.

5. A method of preparing an immunogenic composition comprising: mixing a paramyxovirus composition of claim 1 with a suitable excipient or carrier, thereby forming an immunogenic composition.

6. The method of claim 5 wherein the pharmaceutical composition is formulated for oral administration.

7. The method of claim 5 wherein the pharmaceutical composition is formulated for intranasal administration.

8. An isolated live human respiratory syncytial virus (hRSV) rRSV-G1853A, as deposited with American Type Culture Collection (ATCC) under the Accession Number PTA-122916.

9. An isolated live human respiratory syncytial virus (hRSV) rRSV-G1857A, as deposited with American Type Culture Collection (ATCC) under the Accession Number PTA-122915.

Description

BRIEF DESCRIPTIONS OF THE FIGURES

(1) The patent or application file contains at least one drawing executed in color and/or one or more photographs. Copies of this patent or patent application publication with color drawing(s) and/or photograph(s) will be provided by the Patent Office upon request and payment of the necessary fee. FIG. 1. Sequence alignment of conserved domain VI of L proteins of human paramyxoviruses and modeling with two known 2-O MTase structures, VP39 (SEQ ID NO: 11) and RRMJ (SEQ ID NO: 10).

(2) STR: structure of RRMJ and VP39. Predicted or known alpha-helical regions are shown as cylinders and the -sheet regions as arrows. The conserved motifs (X and I to VIII) correspond to the SAM-dependent MTase superfamily are indicated. The predicted MTase active site (K-D-K-E tetrad) is shown by yellow boxes. The predicted SAM binding site (GGG - - - D/E/W) is shown by grey boxes. The conserved aromatic amino acid resides are shown by red boxes. Representative members of Paramyxoviridae (HMPV, human metapneumovirus (SEQ ID NO: 1); AMPVC, avian metapneumovirus type C (SEQ ID NO: 2); HRSV, human respiratory syncytial virus (SEQ ID NO: 3); BRSV, bovine respiratory syncytial virus (SEQ ID NO: 4); PVM, pneumonia virus of hamsters (SEQ ID NO: 5); PIV3, human parainfluenza virus type 3 (SEQ ID NO: 6); NDV, Newcastle disease virus (SEQ ID NO: 7)), Filoviridae (EBOM, Ebola virus (SEQ ID NO: 8)), Rhabdoviridae (VSIV, vesicular stomatitis virus Indiana serotype (SEQ ID NO: 9)) are shown.

(3) FIG. 2. Examination of the function of hMPV L protein by a minigenome assay.

(4) A minigenome system was established to analyze the function of L protein carrying mutations in catalytic site or SAM binding site. To establish this system, the full-length genomic cDNA of hMPV in the plasmid (phMPV) was replaced by green fluorescent protein (GFP) flanked hMPV trailer and gene end sequences on one side and hMPV gene start and leader sequences on the other side, followed by the HDV ribozyme, and a T7 terminator, to yield phMPV-GFP. To achieve maximum level of minigenome replication, vaccinia vTF-7 was used as the source of T7 polymerase. Briefly, BHK cells were infected by vaccinia vTF-7 at a MOI of 10, followed by transfection of phMPV-GFP together with support plasmids (pN, pP, pL and pM2-1) using a standard protocol recommended by Invitrogen. Two days later, GFP expression was observed by fluorescence microscopy. Each amino acid residue in the MTase catalytic site and SAM binding site was substituted into alanine in the L gene of hMPV (pL) by site-directed mutagenesis. All plasmids were sequenced to confirm the presence of the designed mutation. The effect of each L gene mutation on GFP expression was analyzed by the minigenome assay as described above.

(5) FIG. 3. Recovery of recombinant hMPV from full-length genomic cDNA clones.

(6) A schematic hMPV genome comprising a leader region (Le); eight genes that encode the viral N, P, M, F, M2, SH, G and L proteins, and a trailer region (Tr), is shown. Recombinant hMPV was recovered by transfection of plasmids encoding the full-length hMPV genome (phMPV), pM2, pL, pP, and pN into BSRT7 cells which stably expressing T7 RNA polymerase. Six days post transfection, the cells were subjected to three freeze-thaw steps and the supernatant was used to infect Vero-E6 cells (ATCC). TPCK-typsin (0.5 g/ml) was added to cells at day 2 post-infection since hMPV requires typsin to grow. Cytopathic effects (CPE) were observed after 5 day post-infection. Viruses were further amplified in Vero-E6 cells. Each amino acid residue in the MTase catalytic site and SAM binding site was substituted into alanine in the L gene of hMPV genome by site-directed mutagenesis. All plasmids were sequenced to confirm the presence of the designed mutation. Recombinant hMPVs carrying mutations in MTase catalytic and SAM binding site were recovered by an identical procedure as described above. Vero E6 cells were infected by the recovered hMPV and viral replication and protein synthesis was determined.

(7) FIG. 4: Summary of recombinant viruses carrying mutations in either MTase catalytic site or SAM binding site.

(8) Recombinant hMPV, RSV and PIV3 carrying mutations in either MTase catalytic site or SAM binding site were recovered by the procedure described in FIG. 2. Viral RNA was extracted from each recombinant virus. The entire L gene of each recombinant virus was amplified by reverse transcription-PCR (RT-PCR), and sequence analysis confirmed the presence of the desired mutation.

(9) FIG. 5: Characterization of recombinant MTase-defective hMPV by immunostaining.

(10) Recombinant hMPV forms plaques visualized by immunostaining. Vero E6 cells were infected with the indicated virus and overlayed with 2% methyl cellulose. After 5 days, the methyl cellulose was removed, and cells were incubated with a monoclonal antibody against hMPV N protein (Santa Cruz Biotechnology, Inc.), followed by incubation with horseradish peroxidase-labeled rabbit anti-guinea pig antibodies (Invitrogen). After incubation with AEC substrate chromogen (Invitrogen), viral plaques were visualized under the microscope.

(11) FIG. 6: Characterization of recombinant MTase-defective hMPV by agarose overlay plaque assay.

(12) Recombinant hMPV forms small rounded plaques visualized by crystal violet staining. Vero-E6 cells were infected with indicated virus. After 1 h infection, the cells were overlaid with 2 ml of cell culture medium containing 0.5% agarose and 5% FBS, and incubated for 6 days. The plates were fixed by 10% formaldehyde, followed by staining the crystal violet.

(13) FIG. 7: MTase-defective recombinant hMPV has a delayed cytopathic effect (CPE) in Vero cells.

(14) Recombinant MTase-defective rhMPV-G1696A was shown as an example. Vero E6 cells were infected with wild type rhMPV or rhMPV-G1696A at a MOI of 1, and the cytopathic effect (CPE) was observed at day 0, 3 and 5 post-infection by light microscopy.

(15) FIG. 8: Single step growth curves of MTase-defective rhMPV.

(16) Confluent Vero E6 cells were infected with individual viruses at an MOI of 1. After 1 h of incubation, the inoculum was removed, the cells were washed with DMEM, and fresh medium (containing 2% fetal bovine serum) was added, followed by incubation at 37 C. Samples of supernatant were harvested at the indicated intervals over a 48-h time period, and the virus titer was determined by plaque assay using immunostaining. Titers are the average of three independent experiments.

(17) FIG. 9. Analysis of viral protein synthesis in virus-infected cells.

(18) Confluent Vero E6 cells were infected with either rhMPV or MTase-defective rhMPV at a MOI of 1. After 48 h postinfection, cells were washed with methionine- and cysteine-free (M.sup.C.sup.) medium and incubated with fresh M.sup.C.sup. medium supplemented with actinomycin D (15 g/ml). After 1 h of incubation, the medium was replaced with M.sup.C.sup. medium supplemented with EasyTag .sup.35S-Express (4 Ci/ml; Perkin-Elmer, Wellesley, Mass.). After 24 h of incubation, cytoplasmic extracts were prepared and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) as described previously. Labeled proteins were detected either by autoradiography or by using a phosphorimager.

(19) FIG. 10A-FIG. 10B. Pathogenicity of MTase-defective hMPV in a hamster model

(20) (A) Diagram of proposed animal experiments. (B) Two-week-old female hamsters (Charles River laboratories, Wilmington, Mass.) were inoculated intranasally with three different doses (6.4810.sup.6, 1.010.sup.5, 1.010.sup.4 PFU) of the wild type hMPV or MTase-defective hMPV. In one group, hamsters were inoculated with cell culture medium (DMEM) and served as uninfected controls. After inoculation, the animals were evaluated on a daily basis for mortality, weight loss, and the presence of any respiratory symptoms of hMPV. At day 4 post-infection, five hamsters from each group were sacrificed, and their lungs and nasal turbinates were removed for pathogenicity studies as follows. (i) Virus titer in lung. One lung from each animal was weighed and homologized in 1 ml of phosphate-buffered saline (PBS). Viral titer was determined by plaque assay and viral RNA was quantified by real-time reverse-transcriptase polymerase chain reaction (RT-PCR). (ii) Virus titer in nasal turbinate. Nasal turbinate from each hamster was removed, weighed, and virus titer was determined by plaque assay. (iii) Pulmonary histopathology. One lung from each hamster was inflated with 10% buffered formalin, embedded in paraffin, sectioned, and stained with hematoxylin-eosin. Histopathological changes were scored include the extent of inflammation (focal or diffuse), the pattern of inflammation (peribronchilolar, perivascular, interstitial, alveolar), and the nature of the cells making up the infiltrate (neutrophils, eosinophils, lymphocytes, macrophages). Deparaffinized sections were also stained with polyclonal antiserum to determine the distribution of viral antigen. Five animals per cohort was used in these experiments.

(21) FIG. 11. Immunogenicity of MTase-defective hMPV in a hamster model

(22) Two-week-old female hamsters were inoculated intranasally with two different doses (10.sup.5 and 10.sup.4 PFU) of the MTase-defective hMPV strains. As the controls, hamsters were inoculated with PBS. Serum samples were collected at days 7, 14, 21, and 28 post-inoculation for the detection of humoral immune response. At day 28 post-inoculation, hamsters were sacrificed, and spleen samples were isolated for the detection of cellular immune response. The hamsters were also challenged with 10.sup.6 PFU of the wild-type hMPV. After the challenge, each animal was evaluated on a daily basis for weight loss and the presence of any respiratory symptoms. At day 4 post-challenge, all the animals were sacrificed and lung samples were collected for virus detection and pathological examination. The immunogenicity of the MTase-defective hMPV was evaluated as the following: (i) humoral immunity was determined by virus-serum neutralization assay using an end-point dilution plaque reduction assay. (ii) Cellular immunity was determined by a T cell proliferation assay. (iii) Viral clearance in the lungs. Lung samples were homogenized in PBS. Viral titer was determined by plaque assay and viral RNA was quantified by real-time RT-PCR. (iv) Evaluation of the protection efficacy after challenge. The protection was evaluated with respect to weight loss, respiratory symptoms, and pulmonary histopathology as described above.

DETAILED DESCRIPTION

(23) The present invention provides methyltransferase (MTase)-defective recombinant viruses as live vaccine candidates for hMPV, RSV and PIV3. Messenger RNA (mRNA) modification is the essential issue in paramyxovirus gene expression and replication. During viral RNA synthesis, paramyxoviruses produce capped, methylated, and polyadenylated mRNAs. Methylation of the mRNA directly impacts the subsequent translation of viral proteins, which in turn affects viral genome replication, virus assembly, and budding. Viruses lacking MTase would likely be attenuated without affecting immunogenicity, since the MTase is located in L protein, which is not a neutralizing antibody target. Therefore, MTase is a novel and new target for the development of a stable and efficacious live vaccine. It is known that viral large RNA polymerase (L) protein of paramyxovirus contains mRNA cap MTase activity. L protein is a 230-250-KDa multifunctional protein consisting of 2005-2200 amino acids. Amino acid sequence alignments of the L protein of paramyxoviruses identified six conserved domains numbered I to VI. The MTase activity is located in the conserved domain VI of the L protein. Using a reverse genetics system, the inventors have successfully generated a panel of recombinant hMPV, RSV and PIV3 that are defective in MTase. These recombinant viruses were attenuated in cell culture as well as in hamster models. More importantly, these attenuated viruses elicited high level of neutralizing antibody and cellular immune response in hamsters, and protected hamsters from challenge of virulent viruses. Taken together, these MTase-defective viruses are excellent candidates for live attenuated vaccine for RSV, hMPV and PIV3.

(24) MTase-Defective Viruses as Live Vaccine Candidates for hMPV.

(25) Specifically, provided are MTase-defective hMPV carrying mutations in MTase catalytic site (rhMPV-K1673A, rhMPV-D1779A, rhMPV-K1817A, rhMPV-E1848A,) and SAM binding site (rhMPV-G1696A, rhMPV-G1698A, rhMPV-G1700A, and rhMPV-D1755A). All MTase-defective hMPV were attenuated in cell culture as well as in animal, and remained excellent immunogenicity. Therefore, MTase-defective hMPVs are excellent live vaccine candidates.

(26) MTase-Defective Viruses as Live Vaccine Candidates for RSV.

(27) Specifically, provided are MTase-defective RSV carrying mutations in MTase catalytic site (rRSV-K1831A, rRSV-D1936A, rRSV-K1973A, rRSV-E2004A,) and SAM binding site (rRSV-G1853A, rRSV-G1855A, rRSV-G1857A, and rRSV-D1912A). All MTase-defective RSV were attenuated in cell culture as well as in animal, and remained excellent immunogenicity. Therefore, MTase-defective RSVs are excellent live vaccine candidates. The virus strains that have been deposited with the American Type Culture Collection (ATCC) located at 10801 University Boulevard, Manassas, Va. 20110-2209 under the Accession Numbers: rRSV-G1853A having ATCC Accession NO: PTA-122916, deposited on Mar. 10, 2016; and, rRSV-G1857A having ATCC Accession NO: PTA-122915, deposited on Mar. 10, 2016; in accordance with the provisions of the Budapest Treaty, or any descendant or progeny of one of the aforementioned strains.

(28) MTase-Defective Viruses as Live Vaccine Candidates for PIV3.

(29) Specifically, provided are MTase-defective PIV3 carrying mutations in MTase catalytic site (rPIV3-K1786A, rPIV3-D1905A, rPIV3-K1941A, rPIV3-E1978A,) and SAM binding site (rPIV3-G1808A, rPIV3-G1810A, rPIV3-G1812A, and rPIV3-W1880A). All MTase-defective PIV3 were attenuated in cell culture as well as in animal, and remained excellent immunogenicity. Therefore, all MTase-defective PIV3 are excellent live vaccine candidates.

EXAMPLES

Example 1

Identification of Critical Amino Acid Residues that are Essential for mRNA Cap MTase

(30) To develop MTase-defective hMPV as live vaccine candidates, the inventors characterized critical amino acid residues that are essential for mRNA cap MTase. The SAM-dependent MTase superfamily contains six motifs involved in either SAM binding (motifs I, III, IV) or in the catalytic reaction (motifs IV, VI, VIII, X) (FIG. 1). Sequence alignment and structural modeling with crystal structure-solved ribose 2-O MTases, vaccinia virus VP39 and E. coli RRMJ, identified these motifs in the conserved domain VI of paramyxovirus L proteins.

(31) 1.1. Putative catalytic site of the hMPV MTases. The SAM-dependent MTase superfamily contains a K-D-K-E tetrad that functions as the catalytic residues of the MTase. Structural modeling and amino acid sequence alignments indicate that residues K1673, D1779, K1817, and E1848 of the hMPV L protein correspond to the catalytic K-D-K-E tetrad (FIG. 1). In RSV, these amino acids are K1831, D1936, K1973, and E2004 (FIG. 1). In PIV3, these amino acids are K1786, D1905, K1941, and E1978 (FIG. 1).

(32) 1.2. Putative SAM binding site of the hMPV MTases. In methylation reactions, a G-rich motif and an acidic residue (D/E/W) are involved in binding the methyl donor, SAM. Indeed, this GGG . . . D/E/W motif is conserved in all paramyxoviruses. Sequence alignments indicate that the SAM binding site residues of hMPV L protein include G1696, G1698, G1700 and D1755 (FIG. 1). In RSV these amino acids are G1853, G1855, G1857, and D1912 (FIG. 1). In PIV3, these amino acids are G1808, G1810, G1812, and W1880 (FIG. 1).

Example 2

Examination of the Effect of Mutations to Catalytic Site and SAM Binding Site on Gene Expression by a Minigenome System

(33) The inventors used the minigenome assay to determine whether L protein is functional in replication and RNA synthesis in cells. If a mutant L is functional in the minigenome assay, the inventors may be able to recover recombinant hMPV carrying this mutation by reverse genetics. Briefly, a minigenome plasmid phMPV-GFP containing green fluorescent protein (GFP) flanked hMPV trailer and gene end sequences on one side and hMPV gene start and leader sequences on the other side was constructed. BHK cells were infected by vaccinia vTF-7 at a MOI of 10, followed by transfection of phMPV-GFP together with support plasmids expressing hMPV proteins (pN, pP, pL and pM2-1). Two days later, GFP expression was observed by fluorescence microscopy. As shown in FIG. 2, a strong GFP signal was observed when minigenome was transfected with wild type hMPV L (pL). As a negative control, no GFP expression was observed when pL was omitted from transfection. However, mutations in MTase catalytic site (pL-K1673A, pL-D1779A, pL-K1817A, and pL-E1848A) and SAM binding site (pL-G1696A, pL-G1698A, pL-G1700A, and pL-D1755A) had a diminished GFP expression level. This result demonstrated that mutations in catalytic site and SAM binding site of hMPV L protein were functional but had a diminished replication and/or gene expression. This result also suggests that we may be able to recover viable recombinant hMPV carrying these mutations since they are not lethal in replication and gene expression.

Example 3

Recovery of MTase-Defective Paramyxoviruses from Full-Length cDNA Clones

(34) The inventors have successfully generated a panel of recombinant hMPV that are defective in mRNA cap MTase. The putative MTase catalytic K-D-K-E tetrad and potential SAM binding site GGG . . . D/E/W motif was individually mutated to alanine in the hMPV infectious clone. The mutations in MTase catalytic site were named K1673A, D1779A, K1817A, and E1848A. The mutations in SAM binding site were named G1696A, G1698A, G1700A and D1755A. Using the reverse genetics technique (FIG. 3), recombinant hMPV viruses carrying these mutations were recovered from an infectious cDNA clones. Briefly, recombinant hMPV was recovered by co-transfection of plasmid encoding full-length genomic cDNA of hMPV (phMPV) and support plasmids encoding viral N (pN), P (pP), L (pL) and M2-1 (pM2-1) proteins into BHK.SR19T7pac cells stably expressing T7 RNA polymerase. Six days post transfection, the cells were subjected to three freeze-thaw steps and the supernatant was used to infect Vero-E6 cells (ATCC). TPCK-typsin (0.5 g/ml) was added to cells at day 2 post-infection since hMPV requires typsin to grow. Cytopathic effects (CPE) were observed after 5 day post-infection. Viruses were further amplified in Vero-E6 cells. Recombinant hMPV (rhMPV) carrying mutations in MTase catalytic site were named rhMPV-K1673A, rhMPV-D1779A, rhMPV-K1817A, and rhMPV-E1848A. Recombinant hMPV carrying mutations in SAM binding site were named rhMPV-G1696A, rhMPV-G1698A, rhMPV-G1700A and rhMPV-D1755A. All recombinant viruses were sequenced to confirm the presence of the designated amino acid changes in the L gene. These recombinant MTase-defective hMPVs were summarized in FIG. 4.

(35) Using similar approaches, the inventors recovered a panel of MTase-defective RSV. Specifically, these MTase-defective RSVs carrying mutations in MTase catalytic site (rRSV-K1831A, rRSV-D1936A, rRSV-K1973A, rRSV-E2004A,) and SAM binding site (rRSV-G1853A, rRSV-G1855A, rRSV-G1857A, and rRSV-D1912A). All recombinant viruses were sequenced to confirm the presence of the designated amino acid changes in the L gene. These recombinant MTase-defective RSVs were summarized in FIG. 4.

(36) Using similar approaches, the inventors recovered a panel of MTase-defective PIV3. Specifically, these MTase-defective PIV3 viruses carrying mutations in MTase catalytic site (rPIV3-K1786A, rPIV3-D1905A, rPIV3-K1941A, rPIV3-E1978A,) and SAM binding site (rPIV3-G1808A, rPIV3-G1810A, rPIV3-G1812A, and rPIV3-W1880A). All recombinant viruses were sequenced to confirm the presence of the designated amino acid changes in the L gene. These recombinant MTase-defective PIV3 were summarized in FIG. 4.

Example 4

MTase-Defective hMPVs were Attenuated in Cell Culture

(37) The attenuation of MTase-defective hMPV in cell culture was determined by evaluation of the size of viral plaque, single-step virus growth curve, viral RNA synthesis, and viral protein synthesis. The inventors found that all the MTase-defective viruses carrying mutations either in MTase catalytic site (rhMPV-K1673A, rhMPV-D1779A, rhMPV-K1817A, and rhMPV-E1848A) or SAM binding site (rhMPV-G1696A, rhMPV-G1698A, rhMPV-G1700A and rhMPV-D1755A) were attenuated in cell culture. Specifically, all MTase-defective hMPV formed significantly smaller plaque size, had a delayed viral replication and single step growth curve, and had significantly less protein synthesis as compared to wild type hMPV.

(38) FIG. 5 showed the viral plaque size by immunostaining assay. Briefly, Vero E6 cells were infected with the indicated virus and overlayed with 2% methyl cellulose. After 5 days, the methyl cellulose was removed, and cells were incubated with a monoclonal antibody against hMPV N protein (Santa Cruz Biotechnology, Inc.), followed by incubation with horseradish peroxidase-labeled rabbit anti-guinea pig antibodies (Invitrogen). After incubation with AEC substrate chromogen (Invitrogen), viral plaques were visualized under the microscope. As shown in FIG. 5, wild type hMPV formed big plaques after 5 days post-infection. However, recombinant MTase-defective hMPV formed significantly smaller plaques as compared to wild type hMPV. This result suggested that MTase-defective hMPV had a defect in cell-to-cell spread, replication or gene expression.

(39) FIG. 6 showed viral plaque size by agarose overlay plaque assay. Briefly, Vero-E6 cells were infected with indicated virus. After 1 h infection, the cells were overlaid with 2 ml of cell culture medium containing 0.5% agarose and 5% FBS, and incubated for 6 days. The plates were fixed by 10% formaldehyde, followed by staining the crystal violet. As shown in FIG. 6, wild type hMPV forms rounded plaques visualized by crystal violet staining. However, MTase-defective hMPV formed smaller plaques as compared to wild type hMPV. Again, this data suggest that MTase-defective hMPV had a defect in cell-to-cell spread, replication or gene expression.

(40) FIG. 7 showed cytopathic effect (CPE) of MTase-defective hMPV in Vero-E6 cells. Briefly, confluent Vero E6 cells were infected with individual viruses at an MOI of 1. CPE was monitored every day after infection. As shown in FIG. 7, wild type hMPV exhibited extensive CPE after 3 days post-infection and cells were killed at day 5 post-infection. However, MTase-defective hMPV had a significant delayed CPE. CPE was observed until day 8-10 days post-infection. Cells were not killed until day 14 post-infection. This results demonstrated MTase-defective hMPV had a defect in viral growth.

(41) FIG. 8 showed a single-step growth curve of MTase-defective hMPV in cell culture. Briefly, confluent Vero E6 cells were infected with individual viruses at an MOI of 1. After 1 h of incubation, the inoculum was removed, the cells were washed with DMEM, and fresh medium (containing 2% fetal bovine serum) was added, followed by incubation at 37 C. Samples of supernatant were harvested at the indicated intervals over a 48-h time period, and the virus titer was determined by plaque assay using immunostaining. As shown in FIG. 8, wild type hMPV grew to high titer at 5 days post-infection and remained high titer after 5-10 days post-infection. After 10 days, virus titer gradually decreased. However, MTase-defective hMPV (rhMPV-G1696A and rhMPV-D1755A) had significant defects in viral growth curve. MTase-defective hMPV replicated to peak titer around 8-10 days post-infection and viral titer gradually decreased after 12 days post-infection. These results demonstrated that MTase-defective hMPV had significant defects in viral replication.

(42) FIG. 9 showed viral protein synthesis of MTase-defective hMPV in Vero cells. Briefly, confluent Vero E6 cells were infected with either wild type rhMPV or MTase-defective rhMPV at a MOI of 1. After 48 h postinfection, cells were metabolically labeled with [35S] methionine. After 24 h of incubation, cytoplasmic extracts were prepared and analyzed by SDS-PAGE. Labeled proteins were detected by using a phosphorimager. As shown in FIG. 9, viral N, P and M proteins were detected in wild type hMPV. However, the abundance of viral proteins significantly diminished for MTase-defective hMPV. Only 20-30% and 60-70% of viral proteins were detected for recombinant rhMPV-G1696A and rhMPV-D1755A respectively. This result demonstrated that MTase-defective hMPV had defects in viral protein synthesis.

(43) Taken together, MTase-defective hMPV was attenuated in cell culture as judged by viral plaque size, replication, growth curve and gene expression.

Example 5

MTase-Defective RSVs were Attenuated in Cell Culture

(44) Using the techniques of the previous examples, the inventors found that certain MTase-defective RSVs carrying mutations in MTase catalytic site (rRSV-K1831A, rRSV-D1936A, rRSV-K1973A, rRSV-E2004A,) and SAM binding site (rRSV-G1853A, rRSV-G1855A, rRSV-G1857A, and rRSV-D1912A) were attenuated in cell culture.

Example 6

MTase-Defective PIV3s were Attenuated in Cell Culture

(45) Using similar approaches, the inventors found that MTase-defective PIV3 viruses carrying mutations in MTase catalytic site (rPIV3-K1786A, rPIV3-D1905A, rPIV3-K1941A, rPIV3-E1978A,) and SAM binding site (rPIV3-G1808A, rPIV3-G1810A, rPIV3-G1812A, and rPIV3-W1880A) were attenuated in cell culture.

Example 7

Genetic Stability of MTase-Defective hMPV in Cell Culture

(46) All MTase-defective were passed 10 times in Vero-E6 cells. At each passage, the L gene for each virus was sequenced to confirm the presence of the designed mutation. No additional mutation was found. These data indicated that MTase-defective hMPV is genetically stable in cell culture.

Example 8

MTase-Defective hMPV were Attenuated in Animal Models

(47) To determine whether MTase-defective hMPVs are attenuated in animal, all recombinant viruses were inoculated into two-week-old specific-pathogen-free female hamsters (Charles River laboratories, Wilmington, Mass.). After inoculation, the animals were evaluated on a daily basis for mortality, weight loss, and the presence of any respiratory symptoms of hMPV. At day 4 post-infection, five hamsters from each group were sacrificed, and their lungs were removed for pathogenicity studies as follows. (i) Lung virus titer. One lung from each animal were weighed and homologized in 1 ml of phosphate-buffered saline (PBS). Viral titer was determined by plaque assay. (ii) Pulmonary histopathology. One lung from each hamster was inflated with 10% buffered formalin, embedded in paraffin, sectioned, and stained with hematoxylin-eosin. Histopathological changes were scored include the extent of inflammation (focal or diffuse), the pattern of inflammation (peribronchilolar, perivascular, interstitial, alveolar), and the nature of the cells making up the infiltrate (neutrophils, eosinophils, lymphocytes, macrophages). Deparaffinized sections were also stained with polyclonal antiserum to determine the distribution of viral antigen. Five animals per cohort were used in these experiments. The present results demonstrated that wild type hMPV was virulent to hamster (FIG. 10B). Specifically, hamsters inoculated with wild type hMPV exhibited mild clinical signs of respiratory tract infection, including ruffled fur, tendency to huddle, heavy breathing, and body weight losses. Wild type hMPV caused moderate to severe damage lung as confirmed by pulmonary histopathology. In contrast, all MTase-defective hMPV were highly attenuated in hamsters. Specifically, hamsters inoculated with MTase-defective hMPV did not exhibit any clinical sign for respiratory tract infection or weight loss. In addition, MTase-defective hMPV did not cause or only caused mild pulmonary histopathological changes (FIG. 10B). Taken together, these results demonstrated that MTase-defective hMPV were highly attenuated in hamsters and may be good live vaccine candidates for hMPV.

Example 9

MTase-Defective RSVs were Attenuated in Animal Models

(48) Using similar approaches, the inventors found that MTase-defective RSVs carrying mutations in MTase catalytic site (rRSV-K1831A, rRSV-D1936A, rRSV-K1973A, rRSV-E2004A,) and SAM binding site (rRSV-G1853A, rRSV-G1855A, rRSV-G1857A, and rRSV-D1912A) were attenuated in animals.

Example 10

MTase-Defective PIV3s were Attenuated in Animal Models

(49) Using similar approaches, the inventors found that MTase-defective PIV3 viruses carrying mutations in MTase catalytic site (rPIV3-K1786A, rPIV3-D1905A, rPIV3-K1941A, rPIV3-E1978A,) and SAM binding site (rPIV3-G1808A, rPIV3-G1810A, rPIV3-G1812A, and rPIV3-W1880A) were attenuated in animals.

Example 11

MTase-Defective hMPVs as Immunogens

(50) To determine whether MTase-defective hMPV can be used as live vaccine candidates, the inventors examined the immunogenicity of these recombinant viruses. All MTase-defective hMPV were inoculated intranasally into two-week-old female hamsters (10 hamsters per group). Serum samples were collected at days 7, 14, 21, and 28 post-inoculation for the detection of humoral immune response. At day 28 post-inoculation, 5 hamsters from each group were sacrificed, and spleen samples were isolated for the detection of cellular immune response. The remaining 5 animals were challenged with 10.sup.6 PFU of the wild-type hMPV. After the challenge, each animal was evaluated on a daily basis for weight loss and the presence of any respiratory symptoms. At day 4 post-challenge, all the animals were sacrificed and lung samples were collected for virus detection and pathological examination. The immunogenicity of the MTase-defective hMPV was evaluated as the following: (i) humoral immunity was determined by virus-serum neutralization assay using an end-point dilution plaque reduction assay. (ii) Cellular immunity was determined by a T cell proliferation assay. (iii) Viral clearance in the lungs. Lung samples were homogenized in PBS. Viral titer was determined by plaque assay. (iv) Evaluation of the protection efficacy after challenge. The protection was evaluated with respect to weight loss, respiratory symptoms, and pulmonary histopathology as described above. The present results demonstrated that all MTase-defective hMPV elicited high level of neutralizing antibody and T cell immune response in hamsters, and protected hamsters from virulent challenge (FIG. 11). Specifically, severe lung damage was observed in PBS challenge control. However, no or only mild lung damage was observed in hamsters that were immunized by MTase-defective hMPV. Protection rate for each MTase-defective hMPV was 100%. These results demonstrated that MTase-defective hMPVs have excellent immunogenicity. Thus, MTase-defective hMPVs are excellent live vaccine candidates.

Example 12

MTase-Defective RSVs as Immunogens

(51) Using similar approaches, the inventors found that MTase-defective RSVs carrying mutations in MTase catalytic site (rRSV-K1831A, rRSV-D1936A, rRSV-K1973A, rRSV-E2004A,) and SAM binding site (rRSV-G1853A, rRSV-G1855A, rRSV-G1857A, and rRSV-D1912A) retained excellent immunogenicity.

Example 13

MTase-Defective PIV3s as Immunogens

(52) Using similar approaches, the inventors found that MTase-defective PIV3 viruses carrying mutations in MTase catalytic site (rPIV3-K1786A, rPIV3-D1905A, rPIV3-K1941A, rPIV3-E1978A,) and SAM binding site (rPIV3-G1808A, rPIV3-G1810A, rPIV3-G1812A, and rPIV3-W1880A) retained excellent immunogenicity.