Paramyxovirus Immunogens and Related Materials and Methods

Abstract

Described herein are methyltransferase (MTase)-defective recombinant viruses as vaccines for human metapneumovirus (hMPV), human respiratory syncytial virus (hRSV), and human parainfluenza virus type 3 (PIV3); as well as related materials and methods.

Description

CROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application is a divisional application of U.S. Ser. No. 14/124,501 filed Feb. 14, 2014, now allowed, which is a national stage application filed under 35 USC .sctn.371 of international application PCT/US2012/041878 filed Jun. 11, 2012, which claims the priority claims the benefit of U.S. Provisional Application No. 61/495,119 filed Jun. 9, 2011, the disclosure of which is incorporated herein by reference.

REFERENCE TO SEQUENCE LISTING SUBMITTED VIA EFS-WEB

[0003] This application is being filed electronically via the USPTO EFS-WEB server, as authorized and set forth in MPEP.sctn.1730 II.B.2(a)(A), and this electronic filing includes an electronically submitted sequence (SEQ ID) listing. The entire content of this sequence listing is herein incorporated by reference for all purposes. The sequence listing is identified on the electronically filed .txt file as follows: 604_53068_SeqListing_OSU-2011-146.txt, created on Jun. 11, 2012 and is 19,469 bytes in size.

TECHNICAL FIELD OF THE INVENTION

[0004] The present invention relates to biotechnology, including modified viruses. The invention is in the field of medicine and immunology, including immunogen delivery to living cells and organisms.

BACKGROUND OF THE INVENTION

[0005] Paramyxoviruses are the leading causative agents of acute viral respiratory tract infections. Among the paramyxoviruses, human metapneumovirus (hMPV), human respiratory syncytial virus (RSV), and human parainfluenza virus type 3 (hPIV3) account for more than 70% of acute viral respiratory diseases. All of three viruses cause similar clinical signs and symptoms, ranging from mild respiratory problems to sever coughs, bronchiolitis, and pneumonia. All three viruses cause acute respiratory tract disease in individuals of all ages, especially in infants, children, the elderly, and immunocompromised individuals. In the United States, 60% of infants are infected during their first RSV season, and nearly all children will have been infected with the virus by 2-3 years of age. HMPV is a newly discovered human pathogen, first identified in 2001 in The Netherlands. Soon after its discovery, hMPV was recognized as a globally prevalent pathogen. Epidemiological studies suggest that 5 to 15% of all respiratory tract infections in infants and young children are caused by hMPV, a proportion second only to that of RSV. PIV3 is the third causative agent of viral respiratory infection in children and infants. All three pathogens are globally prevalent.

[0006] Despite the enormous economic losses and emotional burdens these viruses cause, vaccines and anti-viral drugs are currently not available. For decades, approaches to generate vaccines employing viral proteins or inactivated vaccines have failed either due to a lack of immunogenicity or the potential for causing enhanced pulmonary disease upon natural infection with the same virus.

[0007] Vaccination has been the most effective public health strategy to reduce morbidity and mortality associated with viral infections. The increasing clinical significance of RSV, hMPV and PIV3 infections suggest that there is an urgent need for a safe and efficacious vaccine against these viruses, particularly for the populations at high risk such as infants, children, elderly, and immunocompromised individuals. An effective vaccine would not only prevent acute respiratory tract infection caused by these viruses, but also block transmission routes and thus improve human and public health. In the current inventions, the inventors developed a panel of live attenuated vaccines against RSV, hMPV and PIV3.

[0008] However, development of vaccine against human paramyxoviruses has met serious challenge. With the exception of the influenza virus, there is no FDA approved vaccine for other viruses that cause acute upper and lower respiratory tract infections in human. Generally, inactivated and live attenuated vaccines are the two most common strategies used in vaccines against infectious diseases. For safety, an inactivated vaccine is preferred. However, development of an inactivated vaccine for the paramyxovirus RSV turned out to be a problem. A formalin-inactivated RSV vaccine developed and tested in the 1960s not only failed to induce a protective immune response in human, but led to an enhanced respiratory disease upon natural infection with RSV. Eighty percent of the vaccinated children were hospitalized following natural RSV infection, and two children died. Enhanced respiratory disease following vaccination with inactivated vaccine has been observed in other paramyxoviruses such as PIV-3, hMPV and measles virus. A recent study showed that cotton rats immunized with inactivated hMPV vaccine were protected against infection, but developed increased lung pathology. These observations suggest that inactivated vaccines are not the primary choices for three paramyxoviruses: hMPV, RSV, and PIV3, all of which cause extensive morbidity and mortality in the same population, infants and children.

[0009] In contrast to inactivated vaccines, enhanced lung diseases have not been observed for candidate live attenuated RSV vaccines. Therefore, live attenuated vaccines are the most promising vaccine candidates for hMPV, RSV, and PIV3. However, it has been technically challenging to isolate a virus with low virulence that retains high immunogenicity. In paramyxoviruses, spike proteins (F and G proteins for RSV and hMPV) are major determinants of virulence. Therefore, traditional attenuation strategies have been focused on engineering mutations in these two glycoproteins. However, F and G proteins are also viral immunogenic antigens that are responsible for immune response. As a consequence, mutations in glycoproteins may impair the immunogenicity of the attenuated live vaccine. Therefore, exploration of new attenuation approaches is urgently needed.

[0010] This invention develops new attenuated viruses as live vaccine candidates for major human paramyxoviruses including hMPV, RSV and PIV3 by targeting viral mRNA cap methyltransferase (MTase). Paramyxoviruses share a common strategy for replication and gene expression. During RNA synthesis, paramyxoviruses yield capped, methylated, and polyadenylated mRNAs. Methylation of the mRNA directly impacts the stability of mRNA and subsequent translation of viral proteins, which in turn affects viral genome replication, virus assembly, and budding. The large (L) polymerase protein catalyzes the mRNA cap MTases. Recombinant virus defective in MTase can be recovered from cloned full-length viral cDNA by a reverse genetics system. 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. Thus, ablating viral mRNA cap methylation provides a new avenue to rationally attenuate these viruses for development of live attenuated vaccines.

SUMMARY OF THE INVENTION

[0011] The present invention provides recombinant paramyxovirus compositions comprising a nucleic acid molecule which encodes a defective mRNA cap MTase in a paramyxovirus viral genome. Provided are those compositions wherein the composition is defective in mRNA cap MTase gene expression. Also provided are those compositions wherein paramyxovirus viral genome is selected from the group consisting of: a human metapneumovirus (hMPV); human respiratory syncytial virus (hRSV); and human parainfluenza virus type 3 (PIV3).

[0012] The present invention also provides compositions wherein the nucleic acid molecule which encodes a defective mRNA cap MTase carries at least one mutation in the MTase catalytic site. Also provided are those compositions wherein the at least one mutation in the MTase catalytic site is in at least one of the K-D-K-E tetrad sites. Also provided are those compositions wherein the paramyxovirus is human metapneumovirus (hMPV) and the at least one K-D-K-E tetrad site mutation is at least one of rhMPV-K1673A, rhMPV-D1779A, rhMPV-K1817A, rhMPV-E1848A, and rhMPV-E1848Q. Also provided are those compositions wherein the paramyxovirus is human respiratory syncytial virus (hRSV) and the at least one K-D-K-E tetrad site mutation is at least one of rRSV-K1831A, rRSV-D1936A, rRSV-K1973A, rRSV-E2004A. Also provided are those compositions wherein the paramyxovirus is human parainfluenza virus type 3 (PIV3) and the at least one K-D-K-E tetrad site mutation is at least one of rPIV3-K1786A, rPIV3-D1905A, rPIV3-K1941A, rPIV3-E1978A.

[0013] The present invention also provides compositions wherein the nucleic acid molecule which encodes a defective mRNA cap MTase carries at least one mutation in the SAM binding site. Also provided are those compositions according to claim 9, wherein the at least one mutation in the SAM binding site is in the G.times.G.times.G . . . D/E/W site. Also provided are those compositions wherein the paramyxovirus is human metapneumovirus (hMPV) and the at least one mutation in the G.times.G.times.G . . . D/E/W site is at least one of rhMPV-G1696A, rhMPV-G1698A, rhMPV-G1700A, and rhMPV-D1755A. Also provided are those compositions wherein the paramyxovirus is human respiratory syncytial virus (hRSV) and the at least one mutation in the G.times.G.times.G . . . D/E/W site is at least one of rRSV-G1853A, rRSV-G1855A, rRSV-G1857A, and rRSV-D1912A. Also provided are those compositions wherein the paramyxovirus is human parainfluenza virus type 3 (PIV3) and the at least one mutation in the G.times.G.times.G . . . D/E/W site is at least one of rPIV3-G1808A, rPIV3-G1810A, rPIV3-G1812A, and rPIV3-W1880A. Also provided are those compositions wherein the nucleic acid molecule which encodes a defective mRNA cap MTase carries at least one mutation in the MTase catalytic site and the SAM binding site.

[0014] The present invention also provides compositions wherein the at least one mutation in the MTase catalytic site is in at least one of the K-D-K-E tetrad sites and the at least one mutation in the SAM binding site is in the G.times.G.times.G . . . D/E/W site. Also provided are those compositions wherein the paramyxovirus is human metapneumovirus (hMPV) and the at least one K-D-K-E tetrad site mutation is at least one of rhMPV-K1673A, rhMPV-D1779A, rhMPV-K1817A, rhMPV-E1848A, and rhMPV-E1848Q and the at least one mutation in the G.times.G.times.G . . . D/E/W site is at least one of rhMPV-G1696A, rhMPV-G1698A, rhMPV-G1700A, and rhMPV-D1755A. Also provided are those compositions wherein the paramyxovirus is human respiratory syncytial virus (hRSV) and the at least one K-D-K-E tetrad site mutation is at least one of rRSV-K1831A, rRSV-D1936A, rRSV-K1973A, rRSV-E2004A and the at least one mutation in the G.times.G.times.G . . . D/E/W site is at least one of rRSV-G1853A, rRSV-G1855A, rRSV-G1857A, and rRSV-D1912A. Also provided are those compositions wherein the paramyxovirus is human parainfluenza virus type 3 (PIV3) and the at least one K-D-K-E tetrad site mutation is at least one of rPIV3-K1786A, rPIV3-D1905A, rPIV3-K1941A, rPIV3-E1978A and the at least one mutation in the G.times.G.times.G . . . D/E/W site is at least one of rPIV3-G1808A, rPIV3-G1810A, rPIV3-G1812A, and rPIV3-W1880A.

[0015] Also provided are those compositions which are mammalian immunogens.

[0016] Also provided are those compositions which are human immunogens.

[0017] Also provided are methods of eliciting an immune response in a mammal comprising administering to a mammal a recombinant paramyxovirus composition herein. Also provided are methods wherein the composition is administered orally. Also provided are those compositions wherein the composition is administered intranasally.

[0018] Also provided are methods of preparing a pharmaceutical composition for passive immunization of an individual in need of immunization comprising mixing a paramyxovirus composition herein with a suitable excipient or carrier, thereby forming a pharmaceutical composition. Also provided are pharmaceutical compositions formulated for oral administration. Also provided are compositions wherein the pharmaceutical composition is formulated for intranasal administration. Also provided are pharmaceutical compositions wherein the paramyxovirus virulence is attenuated or eliminated in any mammal susceptible to paramyxovirus.

BRIEF DESCRIPTIONS OF THE FIGURES

[0019] The patent or application file may contain one or more drawings 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 U.S. Patent and Trademark Office upon request and payment of the necessary fees.

[0020] 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).

[0021] STR: structure of RRMJ and VP39. Predicted or known alpha-helical regions are shown as cylinders and the .beta.-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 (G.times.G.times.G - - - 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.

[0022] FIG. 2. Examination of the function of hMPV L protein by a minigenome assay.

[0023] 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.

[0024] FIG. 3. Recovery of recombinant hMPV from full-length genomic cDNA clones.

[0025] 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 .mu.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.

[0026] FIG. 4: Summary of recombinant viruses carrying mutations in either MTase catalytic site or SAM binding site.

[0027] 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.

[0028] FIG. 5: Characterization of recombinant MTase-defective hMPV by immunostaining.

[0029] 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.

[0030] FIG. 6: Characterization of recombinant MTase-defective hMPV by agarose overlay plaque assay.

[0031] 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.

[0032] FIG. 7: MTase-defective recombinant hMPV has a delayed cytopathic effect (CPE) in Vero cells.

[0033] 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.

[0034] FIG. 8: Single step growth curves of MTase-defective rhMPV.

[0035] 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.degree. 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.

[0036] FIG. 9. Analysis of viral protein synthesis in virus-infected cells.

[0037] 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 .mu.g/ml). After 1 h of incubation, the medium was replaced with M.sup.-C.sup.- medium supplemented with EasyTag .sup.35S-Express (4 .mu.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.

[0038] FIG. 10A-FIG. 10B. Pathogenicity of MTase-defective hMPV in a hamster model.

[0039] (FIG. 10A) Diagram of proposed animal experiments.

[0040] (FIG. 10B) Two-week-old female hamsters (Charles River laboratories, Wilmington, Mass.) were inoculated intranasally with three different doses (6.48.times.10.sup.6, 1.0.times.10.sup.5, 1.0.times.10.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.

[0041] FIG. 11. Immunogenicity of MTase-defective hMPV in a hamster model 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

[0042] 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.

[0043] MTase-Defective Viruses as Live Vaccine Candidates for hMPV.

[0044] 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.

[0045] MTase-Defective Viruses as Live Vaccine Candidates for RSV.

[0046] 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.

[0047] MTase-Defective Viruses as Live Vaccine Candidates for PIV3.

[0048] 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

[0049] 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.

[0050] 1.1. Putative Catalytic Site of the hMPV MTases.

[0051] 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).

[0052] 1.2. Putative SAM Binding Site of the hMPV MTases.

[0053] In methylation reactions, a G-rich motif and an acidic residue (D/E/W) are involved in binding the methyl donor, SAM. Indeed, this G.times.G.times.G . . . . 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

[0054] 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

[0055] 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 G.times.G.times.G . . . 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 .mu.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.

[0056] Using Similar Approaches, the Inventors Recovered a Panel of MTase-Defective RSV.

[0057] 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.

[0058] Using Similar Approaches, the Inventors Recovered a Panel of MTase-Defective PIV3.

[0059] 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

[0060] 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.

[0061] 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.

[0062] 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.

[0063] 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.

[0064] 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.degree. 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.

[0065] 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.

[0066] 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

[0067] 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

[0068] 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

[0069] 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

[0070] 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

[0071] 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

[0072] 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

[0073] 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

[0074] 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

[0075] 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.

Sequence CWU 1

1

111194PRTHuman metapneumovirus 1Phe Val Phe Ser Ser Thr Gly Cys Lys Val Ser Leu Lys Thr Cys Ile 1 5 10 15 Gly Lys Leu Met Lys Asp Leu Asn Pro Lys Val Leu Tyr Phe Ile Gly 20 25 30 Glu Gly Ala Gly Asn Trp Met Ala Arg Thr Ala Cys Glu Tyr Pro Asp 35 40 45 Ile Lys Phe Val Tyr Arg Ser Leu Lys Asp Asp Leu Asp His His Tyr 50 55 60 Pro Leu Glu Tyr Gln Arg Val Ile Gly Glu Leu Ser Arg Ile Ile Asp 65 70 75 80 Ser Gly Glu Gly Leu Ser Met Glu Thr Thr Asp Ala Thr Gln Lys Thr 85 90 95 His Trp Asp Leu Ile His Arg Val Ser Lys Asp Ala Leu Leu Ile Thr 100 105 110 Leu Cys Asp Ala Glu Phe Lys Asp Arg Asp Asp Phe Phe Lys Met Val 115 120 125 Ile Leu Trp Arg Lys His Val Leu Ser Cys Arg Ile Cys Thr Thr Tyr 130 135 140 Gly Thr Asp Leu Tyr Leu Phe Ala Lys Tyr His Ala Lys Asp Cys Asn 145 150 155 160 Val Lys Leu Pro Phe Phe Val Arg Ser Val Ala Thr Phe Ile Met Gln 165 170 175 Gly Ser Lys Leu Ser Gly Ser Glu Cys Tyr Ile Leu Leu Thr Leu Gly 180 185 190 His His 2194PRTAvian metapneumovirus 2Phe Val Phe Ser Ser Thr Gly Cys Lys Ile Ser Val Lys Ala Cys Ile 1 5 10 15 Gly Lys Leu Ile Gln Asp Leu Asn Pro Thr Val Phe Tyr Phe Val Gly 20 25 30 Glu Gly Ala Gly Asn Trp Met Ala Arg Thr Ala Cys Glu Tyr Pro Asn 35 40 45 Ala Lys Phe Val Tyr Arg Ser Leu Lys Asp Asp Leu Asp His His Phe 50 55 60 Pro Leu Glu Phe Gln Arg Val Leu Gly Asn Met Asn Arg Val Ile Asp 65 70 75 80 Gly Gly Glu Gly Leu Ser Met Asp Thr Thr Asp Ala Thr Gln Lys Thr 85 90 95 His Trp Asp Leu Ile His Arg Ile Cys Lys Asp Ala Leu Leu Ile Thr 100 105 110 Leu Cys Asp Ala Glu Phe Lys Asp Arg Asp Asp Phe Phe Lys Met Val 115 120 125 Thr Leu Trp Arg Lys His Val Leu Ser Cys Arg Ile Cys Thr Thr Tyr 130 135 140 Gly Thr Asp Leu Tyr Leu Phe Ala Lys Tyr His Ala Lys Glu Gln Ser 145 150 155 160 Ile Lys Leu Pro Tyr Phe Val Arg Ser Ile Ala Thr Tyr Val Met Gln 165 170 175 Gly Ser Lys Leu Ser Gly Ser Glu Cys Tyr Val Leu Leu Thr Leu Ser 180 185 190 His His 3192PRTHuman respiratory syncytial virus 3Phe Val Phe Ser Ser Thr Gly Cys Lys Ile Ser Ile Glu Tyr Ile Leu 1 5 10 15 Lys Asp Leu Lys Ile Lys Asp Pro Asn Cys Ile Ala Phe Ile Gly Glu 20 25 30 Gly Ala Gly Asn Leu Leu Leu Arg Thr Val Val Glu Leu His Pro Asp 35 40 45 Ile Arg Tyr Ile Tyr Arg Ser Leu Lys Asp Cys Asn Asp His Ser Leu 50 55 60 Pro Ile Glu Phe Leu Arg Leu Tyr Asn Gly His Ile Asn Ile Asp Tyr 65 70 75 80 Gly Glu Asn Leu Thr Ile Pro Ala Thr Asp Ala Thr Asn Asn Ile His 85 90 95 Trp Ser Tyr Leu His Ile Lys Phe Ala Glu Pro Ile Ser Leu Phe Val 100 105 110 Cys Asp Ala Glu Leu Ser Val Thr Val Asn Trp Ser Lys Ile Ile Ile 115 120 125 Glu Trp Ser Lys His Val Arg Lys Cys Lys Tyr Cys Ser Ser Val Asn 130 135 140 Lys Cys Met Leu Ile Val Lys Tyr His Ala Gln Asp Asp Ile Asp Phe 145 150 155 160 Lys Leu Asp Asn Ile Thr Ile Leu Lys Thr Tyr Val Cys Leu Gly Ser 165 170 175 Lys Leu Lys Gly Ser Glu Val Tyr Leu Val Leu Thr Ile Gly Pro Ala 180 185 190 4192PRTBovine respiratory syncytial virus 4Phe Val Phe Ser Ser Thr Gly Cys Lys Ile Ser Thr Lys Leu Ile Leu 1 5 10 15 Lys Asp Leu Lys Ile Lys Asp Pro His Cys Ile Ala Phe Ile Gly Glu 20 25 30 Gly Ala Gly Asn Leu Leu Leu Arg Thr Val Val Glu Leu His Pro Asp 35 40 45 Ile Lys Tyr Ile Tyr Arg Ser Leu Lys Asp Cys Asn Asp His Ser Leu 50 55 60 Pro Ile Glu Phe Leu Arg Leu Tyr Asn Gly His Ile Ser Ile Asp Tyr 65 70 75 80 Gly Glu Asn Leu Thr Ile Pro Ala Thr Asp Ala Thr Asn Ala Ile His 85 90 95 Trp Ser Tyr Leu His Ile Arg Tyr Ala Glu Pro Ile Asn Leu Phe Val 100 105 110 Cys Asp Ala Glu Leu Pro Asp Leu Thr Asn Trp Ser Arg Ile Val Ser 115 120 125 Glu Trp Tyr Lys His Val Arg Cys Cys Lys Tyr Cys Ser Thr Ile Asp 130 135 140 Arg Ser Lys Leu Ile Val Lys Tyr His Ala Gln Asp Ile Thr Asp Phe 145 150 155 160 Lys Leu Asn Asn Ile Ser Ile Val Lys Thr Tyr Val Cys Leu Gly Ser 165 170 175 Lys Leu Lys Gly Ser Glu Val Tyr Leu Val Leu Thr Val Gly Pro Ser 180 185 190 5194PRTPneumonia virus of hamsters 5Phe Val Phe Ser Ser Thr Gly Cys Lys Val Ser Val Ile Asp Met Leu 1 5 10 15 Pro Lys His Phe Gln Arg Ser Asn Leu Lys Val Ile Cys Phe Ile Gly 20 25 30 Glu Gly Ala Gly Asn Leu Met Leu Arg Ala Val Leu Glu Val Gly Gly 35 40 45 Asn Ile Lys Leu Ile Tyr Arg Ser Leu Lys Asp Pro Asp Asp His His 50 55 60 Val Pro Val Glu Phe Leu Arg Leu Lys Pro Cys Tyr Pro Tyr Ile Asp 65 70 75 80 Thr Gly Gly Ser Leu Ser Leu Ala Ser Thr Asp Ala Thr Asn Lys Ala 85 90 95 His Trp Asp Tyr Leu His Leu His Trp Thr Asp Pro Leu Asn Leu Ile 100 105 110 Val Cys Asp Ala Glu Ile Ser Gly Val Lys His Trp Leu Lys Ile Leu 115 120 125 His Arg Trp Tyr Glu His Met Thr Ser Cys Lys His Cys Leu Lys Ser 130 135 140 Glu His Asp Lys Tyr Leu Ile Ile Lys Tyr His Ala Gln Asp Asp Leu 145 150 155 160 Ile Asp Leu Pro His Gly Val Arg Leu Leu Lys Cys Asn Ile Cys Leu 165 170 175 Gly Ser Lys Leu Ser Gly Ser Glu Ser Tyr Leu Leu Ile Gly Leu Gly 180 185 190 Leu Ser 6211PRTHuman parainfluenza virus 6Gly Ile Asn Ser Thr Ser Cys Leu Lys Ala Leu Glu Leu Ser Gln Ile 1 5 10 15 Leu Met Lys Glu Val Asn Lys Asp Gln Asp Arg Leu Phe Leu Gly Glu 20 25 30 Gly Ala Gly Ala Met Leu Ala Cys Tyr Asp Ala Thr Leu Gly Pro Ala 35 40 45 Val Asn Tyr Tyr Asn Ser Gly Leu Asn Ile Thr Asp Val Ile Gly Gln 50 55 60 Arg Glu Leu Lys Ile Phe Pro Ser Glu Val Ser Leu Val Gly Lys Lys 65 70 75 80 Leu Gly Asn Val Thr Gln Ile Leu Asn Arg Val Lys Val Leu Phe Asn 85 90 95 Gly Asn Pro Asn Ser Thr Trp Ile Gly Asn Met Glu Cys Glu Thr Leu 100 105 110 Ile Trp Ser Glu Leu Asn Asp Lys Ser Ile Gly Leu Val His Cys Asp 115 120 125 Met Glu Gly Ala Ile Gly Lys Ser Glu Glu Thr Val Leu His Glu His 130 135 140 Tyr Ser Val Ile Arg Ile Thr Tyr Leu Ile Gly Asp Asp Asp Val Val 145 150 155 160 Leu Ile Ser Lys Ile Ile Pro Thr Ile Thr Pro Asn Trp Ser Arg Ile 165 170 175 Leu Tyr Leu Tyr Lys Leu Tyr Trp Lys Asp Val Ser Ile Ile Ser Leu 180 185 190 Lys Thr Ser Asn Pro Ala Ser Thr Glu Leu Tyr Leu Ile Ser Lys Asp 195 200 205 Ala Tyr Cys 210 7217PRTNewcastle disease virus 7Gly Thr Ala Ser Ser Ser Trp Tyr Lys Ala Ser His Leu Leu Ser Val 1 5 10 15 Pro Glu Val Arg Cys Ala Arg His Gly Asn Ser Leu Tyr Leu Ala Glu 20 25 30 Gly Ser Gly Ala Ile Met Ser Leu Leu Glu Leu His Val Pro His Glu 35 40 45 Thr Ile Tyr Tyr Asn Thr Leu Phe Ser Asn Glu Met Asn Pro Pro Gln 50 55 60 Arg His Phe Gly Pro Thr Pro Thr Gln Phe Leu Asn Ser Val Val Tyr 65 70 75 80 Arg Asn Leu Gln Ala Glu Val Thr Cys Lys Asp Gly Phe Val Gln Glu 85 90 95 Phe Arg Pro Leu Trp Arg Glu Asn Thr Glu Glu Ser Asp Leu Thr Ser 100 105 110 Asp Lys Ala Val Gly Tyr Ile Thr Ser Ala Val Pro Tyr Arg Ser Val 115 120 125 Ser Leu Leu His Cys Asp Ile Glu Ile Pro Pro Gly Ser Asn Gln Ser 130 135 140 Leu Leu Asp Gln Leu Ala Ile Asn Leu Ser Leu Ile Ala Met His Ser 145 150 155 160 Val Arg Glu Gly Gly Val Val Ile Ile Lys Val Leu Tyr Ala Met Gly 165 170 175 Tyr Tyr Phe His Leu Leu Met Asn Leu Phe Ala Pro Cys Ser Thr Lys 180 185 190 Gly Tyr Ile Leu Ser Asn Gly Tyr Ala Cys Arg Gly Asp Met Glu Cys 195 200 205 Tyr Leu Val Phe Val Met Gly Tyr Leu 210 215 8202PRTEbola virus 8Gly Ile Val Ser Ser Met His Tyr Lys Leu Asp Glu Val Leu Trp Glu 1 5 10 15 Ile Glu Ser Phe Lys Ser Ala Val Thr Leu Ala Glu Gly Glu Gly Ala 20 25 30 Gly Ala Leu Leu Leu Ile Gln Lys Tyr Gln Val Lys Thr Leu Phe Phe 35 40 45 Asn Thr Leu Ala Thr Glu Ser Ser Ile Glu Ser Glu Ile Val Ser Gly 50 55 60 Met Thr Thr Pro Arg Met Leu Leu Pro Val Met Ser Lys Phe His Asn 65 70 75 80 Asp Gln Ile Glu Ile Ile Leu Asn Asn Ser Ala Ser Gln Ile Thr Asp 85 90 95 Ile Thr Asn Pro Thr Trp Phe Lys Asp Gln Arg Ala Arg Leu Pro Lys 100 105 110 Gln Val Glu Val Ile Thr Met Asp Ala Glu Thr Thr Glu Asn Ile Asn 115 120 125 Arg Ser Lys Leu Tyr Glu Ala Val Tyr Lys Leu Ile Leu His His Ile 130 135 140 Asp Pro Ser Val Leu Lys Ala Val Val Leu Lys Val Phe Leu Ser Asp 145 150 155 160 Thr Glu Gly Met Leu Trp Leu Asn Asp Asn Leu Ala Pro Phe Phe Ala 165 170 175 Thr Gly Tyr Leu Ile Lys Pro Ile Thr Ser Ser Ala Arg Ser Ser Glu 180 185 190 Trp Tyr Leu Cys Leu Thr Asn Phe Leu Ser 195 200 9201PRTVesicular stomatitis virus 9Gln Leu Pro Thr Gly Ala His Tyr Lys Ile Arg Ser Ile Leu His Gly 1 5 10 15 Met Gly Ile His Tyr Arg Asp Phe Leu Ser Cys Gly Asp Gly Ser Gly 20 25 30 Gly Met Thr Ala Ala Leu Leu Arg Glu Asn Val His Ser Arg Gly Ile 35 40 45 Phe Asn Ser Leu Leu Glu Leu Ser Gly Ser Val Met Arg Gly Ala Ser 50 55 60 Pro Glu Pro Pro Ser Ala Leu Glu Thr Leu Gly Gly Asp Lys Ser Arg 65 70 75 80 Cys Val Asn Gly Glu Thr Cys Trp Glu Tyr Pro Ser Asp Leu Cys Asp 85 90 95 Pro Arg Thr Trp Asp Tyr Phe Leu Arg Leu Lys Ala Gly Leu Gly Leu 100 105 110 Gln Ile Asp Leu Ile Val Met Asp Met Glu Val Arg Asp Ser Ser Thr 115 120 125 Ser Leu Lys Ile Glu Thr Asn Val Arg Asn Tyr Val His Arg Ile Leu 130 135 140 Asp Glu Gln Gly Val Leu Ile Tyr Lys Thr Tyr Gly Thr Tyr Ile Cys 145 150 155 160 Glu Ser Glu Lys Asn Ala Val Thr Ile Leu Gly Pro Met Phe Lys Thr 165 170 175 Val Asp Leu Val Gln Thr Glu Phe Ser Ser Ser Gln Thr Ser Glu Val 180 185 190 Tyr Met Val Cys Lys Gly Leu Lys Lys 195 200 10175PRTEscherichia coli 10Gly Leu Arg Ser Arg Ala Trp Phe Lys Leu Asp Glu Ile Gln Gln Ser 1 5 10 15 Asp Lys Leu Phe Lys Pro Gly Met Thr Val Val Asp Leu Gly Ala Ala 20 25 30 Pro Gly Gly Trp Ser Gln Tyr Val Gly Lys Gly Arg Ile Ile Ala Cys 35 40 45 Asp Leu Leu Pro Met Asp Pro Ile Val Gly Val Asp Phe Leu Gln Gly 50 55 60 Asp Phe Arg Asp Glu Leu Val Met Lys Ala Leu Leu Glu Arg Val Gly 65 70 75 80 Asp Ser Lys Val Gln Val Val Met Ser Asp Met Ala Pro Asn Met Ser 85 90 95 Gly Thr Pro Ala Val Asp Ile Pro Arg Ala Met Tyr Leu Val Glu Leu 100 105 110 Ala Leu Glu Met Cys Arg Asp Val Leu Ala Pro Gly Gly Ser Phe Val 115 120 125 Val Lys Val Phe Gln Gly Glu Gly Phe Asp Glu Tyr Leu Arg Glu Ile 130 135 140 Arg Ser Leu Phe Thr Lys Val Lys Val Arg Lys Pro Asp Ser Ser Arg 145 150 155 160 Ala Arg Ser Arg Glu Val Tyr Ile Val Ala Thr Gly Arg Lys Pro 165 170 175 11185PRTVaccinia virus 11Lys Leu Pro Tyr Gln Gly Gln Leu Lys Leu Leu Leu Gly Glu Leu Phe 1 5 10 15 Phe Leu Ser Lys Leu Gln Arg His Gly Ile Leu Asp Gly Ala Thr Val 20 25 30 Val Tyr Ile Gly Ser Ala Pro Gly Thr His Ile Arg Tyr Leu Arg Asp 35 40 45 His Phe Tyr Asn Leu Gly Met Ile Ile Lys Trp Met Leu Ile Asp Gly 50 55 60 Arg His His Asp Pro Ile Leu Asn Gly Leu Arg Asp Val Thr Leu Val 65 70 75 80 Thr Arg Phe Val Asp Glu Glu Tyr Leu Arg Ser Ile Lys Lys Gln Leu 85 90 95 His Pro Ser Lys Ile Ile Leu Ile Ser Asp Val Arg Ser Lys Arg Gly 100 105 110 Gly Asn Glu Pro Ser Thr Ala Asp Leu Leu Ser Asn Tyr Ala Leu Gln 115 120 125 Asn Val Met Ile Ser Ile Leu Asn Pro Val Ala Ser Ser Leu Lys Trp 130 135 140 Arg Cys Pro Phe Pro Asp Gln Trp Ile Lys Asp Phe Tyr Ile Pro His 145 150 155 160 Gly Asn Lys Met Leu Gln Pro Phe Ala Pro Ser Tyr Ser Ala Glu Met 165 170 175 Arg Leu Leu Ser Ile Tyr Thr Gly Glu 180 185

Claims

1. A recombinant paramyxovirus composition comprising a nucleic acid molecule which encodes a defective mRNA cap MTase in large (L) polymerase gene in a paramyxovirus viral genome; wherein the nucleic acid molecule which encodes a defective mRNA cap MTase carries at least one mutation in the MTase catalytic site; wherein the at least one mutation in the MTase catalytic site is in at least one of the K-D-K-E tetrad sites; and, wherein the paramyxovirus is human metapneumovirus (hMPV) and the at least one K-D-K-E tetrad site mutation is at least one of rhMPV-K1673A, rhMPV-D1779A, rhMPV-K1817A, rhMPV-E1848A, and rhMPV-E1848Q.

2. The recombinant paramyxovirus composition of claim 1, wherein the nucleic acid molecule which encodes a defective mRNA cap MTase carries at least one mutation in the SAM binding site.

3. The recombinant paramyxovirus composition according to claim 1, wherein the at least one mutation in the SAM binding site is in the G.times.G.times.G . . . D/E/W site; wherein the paramyxovirus is human metapneumovirus (hMPV) and the at least one mutation in the G.times.G.times.G . . . D/E/W site is at least one of rhMPV-G1696A, rhMPV-G1698A, rhMPV-G1700A, and rhMPV-D1755A.

4. The recombinant paramyxovirus composition of claim 1, wherein the nucleic acid molecule which encodes a defective mRNA cap MTase carries at least one mutation in the MTase catalytic site and the SAM binding site; wherein the at least one mutation in the MTase catalytic site is in at least one of the K-D-K-E tetrad sites and the at least one mutation in the SAM binding site is in the G.times.G.times.G . . . D/E/W site; and, wherein the paramyxovirus is human metapneumovirus (hMPV) and the at least one K-D-K-E tetrad site mutation is at least one of rhMPV-K1673A, rhMPV-D1779A, rhMPV-K1817A, rhMPV-E1848A, and rhMPV-E1848Q and the at least one mutation in the G.times.G.times.G . . . D/E/W site is at least one of rhMPV-G1696A, rhMPV-G1698A, rhMPV-G1700A, and rhMPV-D1755A.

5. The recombinant paramyxovirus composition of claim 1, which is a mammalian immunogen.

6. The recombinant paramyxovirus composition of claim 1, which is a human immunogen.

7. A method of eliciting an immune response in a mammal comprising: administering to the mammal a recombinant paramyxovirus composition of claim 1.

8. The method according to claim 7, wherein the composition is administered orally.

9. The method according to claim 7, wherein the composition is administered intranasally.

10. A method of preparing a pharmaceutical composition for passive immunization of an individual in need of immunization comprising: mixing a paramyxovirus composition of claim 1 with a suitable excipient or carrier, thereby forming a pharmaceutical composition.

11. The method according to claim 10, wherein the pharmaceutical composition is formulated for oral administration.

12. The method according to claim 10, wherein the pharmaceutical composition is formulated for intranasal administration.

13. A composition claim 1, wherein the paramyxovirus virulence is attenuated or eliminated in any mammal susceptible to paramyxovirus.

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