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  • C12N2760/18011—Paramyxoviridae
  • C12N2760/18411—Morbillivirus, e.g. Measles virus, canine distemper
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  • C12N2760/18511—Pneumovirus, e.g. human respiratory syncytial virus
  • C12N2760/18522—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes

Definitions

• This invention relates to isolated, recombinantly-generated, attenuated measles virus or human respiratory syncytial virus subgroup B having specified attenuating mutations.
• This invention was made with Government support under a grant awarded by the Public Health Service. The Government has certain rights in the invention.
• Enveloped, negative-sense, single stranded RNA viruses are uniquely organized and expressed.
• the genomic RNA of negative-sense, single stranded viruses serves two template functions in the context of a nucleocapsid: as a template for the synthesis of messenger RNAs (mRNAs) and as a template for the synthesis of the antigenome (+) strand.
• Negative- sense, single stranded RNA viruses encode and package their own RNA dependent RNA Polymerase.
• Messenger RNAs are only synthesized once the nucleocapsid has entered the cytoplasm of the infected cell. Viral replication occurs after synthesis of the mRNAs and requires the continuous synthesis of viral proteins.
• the newly synthesized antigenome (+) strand serves as the template for generating further copies of the (-) strand genomic RNA.
• the polymerase complex actuates and achieves transcription and replication by engaging the cis- acting signals at the 3 1 end of the genome, in particular, the promoter region.
• Viral genes are then transcribed from the genome template unidirectionally from its 3' to its 5' end. There is always less mRNA made from the downstream genes (e.g., the polymerase gene (L) ) relative to their upstream neighbors (i.e., the nucleoprotein gene (N) ) . Therefore, there is always a gradient of mRNA abundance according to the position of the genes relative to the 3" -end of the genome.
• This Order contains three families of enveloped viruses with single stranded, nonsegmented RNA genomes of minus polarity (negative-sense) . These families are the Paramyxoviridae, Rhabdoviridae and Filoviridae. The family Paramyxoviridae has been further divided into two subfamilies, Paramyxovirinae and Pneumovirinae . The subfamily Paramyxovirinae contains three genera, Para yxovirus , Rubulavirus and Morbillivirus . The subfamily Pneumovirinae contains the genus Pneumovirus .
• the new classification is based upon morphological criteria, the organization of the viral genome, biological activities and the sequence relatedness of the genes and gene products.
• the morphological distinguishing feature among enveloped viruses for the subfamily Paramyxovirinae is the size and shape of the nucleocapsids (diameter 18mm, 1mm in length, pitch of 5.5 nm) , which have a left-handed helical symmetry.
• the biological criteria are: 1) antigenic cross-reactivity between members of a genus, and 2) the presence of neuraminidase activity in the genera Paramyxovirus , Rubulavirus and its absence in genus Morbillivirus .
• Pneumoviruses can be distinguished from Paramyxovirinae morphologically because they contain narrow nucleocapsids .
• pneumoviruses have major differences in the number of protein-encoding cistrons (10 in pneumoviruses versus 6 in
• Paramyxovirinae and an attachment protein (G) that is very different from that of Paramyxovirinae.
• the paramyxoviruses and pneumoviruses have six proteins that appear to correspond in function (N, P, M, G/H/HN, F and L) , only the latter two proteins exhibit significant sequence relatedness between the two subfamilies .
• Several pneumoviral proteins lack counterparts in most of the paramyxoviruses, namely the nonstructural proteins NS1 and NS2, the small hydrophobic protein SH, and a second protein M2.
• Some paramyxoviral proteins, namely C and V lack counterparts in pneumoviruses.
• Subfamily Paramyxovirinae Genus Paramyxovirus Sendai virus (mouse parainfluenza virus type 1)
• Bovine parainfluenza virus type 3 Genus Rubulavirus Simian virus 5 (SV) (Canine parainfluenza virus type 2) Mumps virus
• Newcastle disease virus (avian Paramyxovirus 1)
• Canine distemper virus Peste-des-petits-ruminants virus Phocine distemper virus Rinderpest virus Subfamily Pneumovirinae
• a variety of approaches can be considered in seeking to develop such vaccines, including the use of: (1) purified individual viral protein vaccines (subunit vaccines) ; (2) inactivated whole virus preparations; and (3) live, attenuated viruses.
• Subunit vaccines have the desirable feature of being pure, definable and relatively easily produced in abundance by various means, including recombinant DNA expression methods. To date, with the notable exception of hepatitis B surface antigen, viral subunit vaccines have generally only elicited short-lived and/or inadequate immunity, particularly in naive recipients . Formalin inactivated whole virus preparations of polio (IPV) and hepatitis A have proven safe and efficacious.
• IPV polio
• hepatitis A have proven safe and efficacious.
• RSV vaccine candidates were generated by cold passage or chemical mutagenesis. These RSV strains were found to have reduced virulence in seropositive adults. Unfortunately, they proved either over or under- attenuated when given to seronegative infants; in some cases, they also were found to lack genetic stability (5,6). Another vaccination approach using parenteral administration of live virus was ineffective and efforts along this line were discontinued (7) . Notably, these live RSV vaccines were never associated with disease enhancement as observed with the formalin- inactivated RSV vaccine described above. Currently, there are no RSV vaccines approved for administration to humans, although clinical trials are now in progress with cold-passaged, chemically mutagenized strains of RSV designated A2 and B-l.
• Appropriately attenuated live derivatives of wild-type viruses offer a distinct advantage as vaccine candidates.
• live, replicating agents they initiate infection in recipients during which viral gene products are expressed, processed and presented in the context of the vaccinee 's specific MHC class I and II molecules, eliciting humoral and cell-mediated immune responses, as well as the coordinate cytokine and chemokine patterns, which parallel the protective immune profile of survivors of natural infection.
• This propagation/passage scheme typically leads to the emergence of virus derivatives which are temperature sensitive, cold-adapted and/or altered in their host range -- one or all of which are changes from the wild-type, disease-causing viruses -- i.e., changes that may be associated with attenuation.
• live virus vaccines including those for the prevention of measles and mumps (which are paramyxoviruses) , and for protection against polio and rubella (which are positive strand RNA viruses) , have been generated by this approach and provide the mainstay of current childhood immunization regimens throughout the world.
• Rational vaccine design would be assisted by a better understanding of these viruses, in particular, by the identification of the virally encoded determinants of virulence as well as those genomic changes which are responsible for attenuation.
• one or more attenuating mutations are selected from the group consisting of: (1) for the N gene, nucleotide changes which produce changes in an amino acid selected from the group consisting of residues 129 (glutamine — lysine) , 148 (glutamic acid — > glycine) and 479 (serine — > threonine) ; (2) for the P gene, nucleotide changes which produce changes in an amino acid selected from the group consisting of residues 225 (glutamic acid — > glycine) , 275 (cysteine —» tyrosine) and 439 (leucine - proline) ; (3) for the C gene, nucleotide changes which produce changes in an amino acid selected from the group consisting of residues 73 (alanine —» valine) , 104 (methionine —» threonine) and 134 (serine - tyrosine) ; and
• these attenuated measles or human respiratory syncytial subgroup B viruses are used to prepare vaccines which elicit a protective immune response against the wild- type form of each virus .
• an isolated, positive strand, antigenomic message sense nucleic acid molecule (or an isolated, negative strand genomic sense nucleic acid molecule) having the complete viral nucleotide sequence (whether of wild- type virus or virus attenuated by non-recombinant means) is manipulated by introducing one or more of the attenuating mutations described in this application to generate an isolated, recombinantly-generated attenuated measles or human respiratory syncytial subgroup B virus. Each attenuated virus is then used to prepare vaccines which elicit a protective immune response against the wild- type form of each virus.
• Figure 1 depicts the passage history of the
• Edmonston measles virus (15) .
• the abbreviations have the following meanings: HK - human kidney; HA - human amnion; CE(am) - chick embryo; CEF - chick embryo fibroblast; DK - dog kidney; WI-38 - human diploid cells; SK - sheep kidney; * - plaque cloning.
• the number following each abbreviation represents the number of passages.
• Figure 2 depicts a map of the measles virus genome showing putative cis-acting regulatory elements at and near the genome and antigenome termini .
• Top a schematic map of the measles virus genome, beginning at the 3' end with 52 nucleotides of leader sequence (1) and ending at the 5' terminus with 37 nucleotides of trailer sequence (t) . Gene boundaries are denoted by vertical bars; below each gene is the number of cistronic nucleotides.
• Bottom an expanded schematic view of the 3 ' extended genomic promoter regions of genome and antigenome, showing the position and sequence of the two highly conserved domains, A and B. The intervening intergenic trinucleotide is denoted as well. Nascent 5' RNAs encompassing the A' to B ' regions are presumed to contain the regulatory sequence at which the N protein encapsidation initiates.
• Figure 3 depicts a genetic map of the RSV subgroup B wild-type strains designated 2B and 18537 (top portion) , the intergenic sequences of those strains (middle portion) and the 68 nucleotide overlap between the M2 and L genes (bottom portion) .
• the RSV 2B stain has six fewer nucleotides in the G gene, encoding two fewer amino acid residues in the G protein, as compared to the 18537 strain.
• the 2B strain has 145 nucleotides in the 5' trailer region, as compared to 149 nucleotides in the 18537 strain.
• the 2B strain has one more nucleotide in each of the NS-1, NS-2 and N genes, and one fewer nucleotide in each of the M and F genes, as compared to the 18537 strain.
• Figure 4 depicts a Northern blot analysis of poly (A) + RNA isolated from Vero cells infected with RSV subgroup A and B viruses .
• a 1.5 hour autoradiograph of the blot is shown.
• Lanes 1-2 contain RNA isolated from cells infected with RSV subgroup A strains (lane 1, 3A; lane 2, A2) .
• Lanes 3-7 contain RNA isolated from cells infected with RSV subgroup B strains: lane 3, Bl; lane 4, 2B; lane 5, 2B33F; lane 6, 2B33F TS(+); lane 7, 18537.
• Monocistronic M gene and bicistronic M:SH gene transcripts are indicated by arrows .
• Figure 5 depicts a ribonuclease protection assay of poly (A) + RNA extracted from Vero cells infected with RSV subgroup B wild- type 2B, mutant
• RNA markers were included as size standards: Lane 1, yeast RNA minus RNase; lane 2, yeast RNA plus RNase; lane 3, 2B RNA plus RNase; lane 4, 2B33F RNA plus RNase; lane 5, 2B33F TS+ RNA plus RNase; lane 6, yeast RNA plus RNase; lane 7, yeast RNA minus RNase.
• RSV 2B- specific probe was used in lanes 1-3 and 2B33F-specific probe was used in lanes 4-7.
• Figure 6 depicts a Northern blot analysis of poly (A) + RNA isolated from Vero cells infected with RSV subgroup A and B viruses .
• a 1.5 hour autoradiograph of the blot is shown.
• Lanes 1-2 contain RNA isolated from cells infected with RSV subgroup A strains (lane 1, 3A; lane 2, A2) .
• Lanes 3-7 contain RNA isolated from cells infected with RSV subgroup B strains (lane 3, Bl; lane 4, 2B; lane 5, 2B33F; lane 6, 2B33F TS(+); lane 7, 18537).
• Monocistronic SH gene and bicistronic M:SH gene transcripts are indicated by arrows .
• RNA viral genomes Transcription and replication of negative- sense, single stranded RNA viral genomes are achieved through the enzymatic activity of a multimeric protein acting on the ribonucleoprotein core (nucleocapsid) . Naked genomic RNA cannot serve as a template. Instead, these genomic sequences are recognized only when they are entirely encapsidated by the N protein into the nucleocapsid structure. It is only in that context that the genomic and antigenomic terminal promoter sequences are recognized to initiate the transcription or replication pathways.
• All paramyxoviruses require the two viral proteins, L and P, for these polymerase pathways to proceed.
• the pneumoviruses, including RSV also require the transcription elongation factor, M2, for the transcriptional pathway to proceed efficiently.
• Additional cofactors may also play a role, including perhaps the virus-encoded NSl and NS2 proteins, as well as perhaps host-cell encoded proteins.
• L protein which performs most, if not all, the enzymatic processes associated with transcription and replication, including initiation, and termination of ribonucleotide polymerization, capping and polyadenylation of mRNA transcripts, methylation and perhaps specific phosphorylation of P proteins.
• the L protein's central role in genomic transcription and replication is supported by its large size, sensitivity to mutations, and its catalytic level of abundance in the transcriptionally active viral complex (16) .
• the invention is believed to encompass a set of changes in several measles virus genes and one cis-acting regulatory domain, and one change in an RSV subgroup B cis-acting regulatory domain, which result in attenuation of each virus while retaining sufficient ability of the virus to replicate. Therefore, as part of a rational vaccine design, such mutations are introduced to provide the desired balance of replication efficiency and immunogenicity: so that the virus vaccine is no longer able to produce disease, yet retains its capacity to infect the vaccinee 's cells, to express sufficiently abundant gene products to elicit the full spectrum and profile of desirable immune responses, and to reproduce and disseminate sufficiently to maximize the abundance of the immune response elicited.
• the attenuating mutations described herein may be introduced into viral strains by two methods:
• a preferred means of introducing attenuating mutations comprises making predetermined mutations using site-directed mutagenesis. These mutations are identified either by method (1) or by reference to closely-related viruses whose attenuating mutations are already known. One or more mutations as defined herein are introduced into measles virus or RSV subgroup B strains. Cumulative effects of different combinations of coding and non-coding changes can also be assessed.
• the mutations to the N, P and/or C genes and/or the F gene-end signal of measles virus, or to the M gene-end signal of RSV subgroup B are introduced by standard recombinant DNA methods into a DNA copy of the viral genome.
• This may be a wild-type or a modified viral genome background (such as viruses modified by method (1) ) , thereby generating a new virus.
• Infectious clones or particles containing these attenuating mutations are generated using the cDNA
• rescue system which has been applied to a variety of viruses, including Sendai virus (18) ; measles virus (19); respiratory syncytial virus (20); PIV-3 (21); rabies (22) ; vesicular stomatitis virus (VSV) (15) ; and rinderpest virus (23); these references are hereby incorporated by reference. See, for measles virus rescue, published International patent application WO 97/06270, designating the United States (24); for RSV rescue, published International patent application WO 97/12032, designating the United States (25); these applications are hereby incorporated by reference.
• RNA polymerase promoter e.g., the T7 RNA polymerase promoter
• ribozyme sequence e.g., the hepatitis delta ribozyme
• This transcription vector provides the readily manipulable DNA template from which the RNA polymerase (e.g., T7 RNA polymerase) can faithfully transcribe a single-stranded RNA copy of the viral antigenome (or genome) with the precise, or nearly precise, 5' and 3' termini.
• the orientation of the viral genomic DNA copy and the flanking promoter and ribozyme sequences determine whether antigenome or genome RNA equivalents are transcribed.
• virus-specific trans-acting proteins needed to encapsidate the naked, single-stranded viral antigenome or genome RNA transcripts into functional nucleocapsid templates: the viral nucleocapsid (N or NP) protein, the polymerase-associated phosphoprotein (P) and the polymerase (L) protein. These proteins comprise the active viral RNA-dependent RNA polymerase which must engage this nucleocapsid template to achieve transcription and replication.
• the trans-acting proteins required for measles virus rescue are the encapsidating protein N, and the polymerase complex proteins, P and L.
• the virus-specific trans-acting proteins include N, P and L, plus an additional protein, M2, the RSV-encoded transcription elongation factor.
• these viral trans-acting proteins are generated from one or more plasmid expression vectors encoding the required proteins, although some or all of the required trans-acting proteins may be produced within mammalian cells engineered to contain and express these virus-specific genes and gene products as stable transformants.
• the typical (although not necessarily exclusive) circumstances for rescue include an appropriate mammalian cell milieu in which T7 polymerase is present to drive transcription of the antigenomic (or genomic) single-stranded RNA from the viral genomic cDNA-containing transcription vector. Either cotranscriptionally or shortly thereafter, this viral antigenome (or genome) RNA transcript is encapsidated into functional templates by the nucleocapsid protein and engaged by the required polymerase components produced concurrently from co- transfected expression plasmids encoding the required virus-specific trans-acting proteins. These events and processes lead to the prerequisite transcription of viral mRNAs, the replication and amplification of new genomes and, thereby, the production of novel viral progeny, i.e., rescue.
• T7 polymerase is provided by recombinant vaccinia virus VTF7-3.
• This system requires that the rescued virus be separated from the vaccinia virus by physical or biochemical means or by repeated passaging in cells or tissues that are not a good host for poxvirus.
• MV cDNA rescue this requirement is avoided by creating a cell line that expresses T7 polymerase, as well as viral N and P proteins. Rescue is achieved by transfecting the genome expression vector and the L gene expression vector into the helper cell line.
• MVA-T7 which expresses the T7 RNA polymerase, but produces little or no infectious progeny in mammalian cells, are exploited to rescue RSV, Rinderpest virus and MV.
• synthetic full length antigenomic viral RNA are encapsidated, replicated and transcribed by viral polymerase proteins and replicated genomes are packaged into infectious virions.
• genome analogs have now been successfully rescued for Sendai and PIV-3 (21,26) .
• the rescue system thus provides a composition which comprises a transcription vector comprising an isolated nucleic acid molecule encoding a genome or antigenome of a nonsegmented, negative-sense, single stranded RNA virus of the Order Mononegavirales having (a) for measles virus, at least one attenuating mutation in the N, P or C genes or the F gene-end signal; and (b) for RSV subgroup B, at least one attenuating mutation in the M gene-end signal, together with at least one expression vector which comprises at least one isolated nucleic acid molecule encoding the trans-acting proteins necessary for encapsidation, transcription and replication (e.g., N, P and L for measles virus; N, P, L and M2 for RSV) .
• Host cells are then transformed or transfected with the at least two expression vectors just described.
• the host cells are cultured under conditions which permit the co- expression of these vectors so as to produce the infectious attenuated virus.
• the rescued infectious virus is then tested for its desired phenotype (temperature sensitivity, cold adaptation, plaque morphology, and transcription and replication attenuation), first by in vi tro means.
• the mutations in the N, P or C genes or the F gene-end signal of measles virus and in the M gene-end signal of RSV subgroup B are also tested using the minireplicon system where the required trans-acting encapsidation and polymerase activities are provided by wild-type or vaccine helper viruses, or by plasmids expressing the N, P and different L genes harboring gene-specific attenuating mutations (19,27,83).
• Non-human primates provide the preferred animal model for the pathogenesis of human disease. These primates are first immunized with the attenuated, recombinantly-generated virus, then challenged with the wild- type form of the virus. Monkeys are infected by various routes, including but not limited to intranasal, intratracheal or subcutaneous routes of inoculation (28) . Experimentally infected rhesus and cynomolgus macaques have also served as animal models for studies of vaccine-induced protection against measles (29) .
• the cotton rat or mouse models may also be used (30,31) . Protection is measured by such criteria as disease signs and symptoms, survival, virus shedding and antibody titers. If the desired criteria are met, the attenuated, recombinantly-generated virus is considered a viable vaccine candidate for testing in humans.
• the "rescued" virus is considered to be "recombinantly- generated” , as are the progeny and later generations of the virus, which also incorporate the attenuating mutations.
• a codon containing an attenuating point mutation may be stabilized by introducing a second or a second plus a third mutation in the codon without changing the amino acid encoded by the codon bearing only the attenuating point mutation.
• Infectious virus clones containing the attenuating and stabilizing mutations are also generated using the cDNA "rescue" system described above.
• measles virus mutations are assessed, because sequence data are now available as described herein for the disease- causing wild-type virus and for the disease-preventing vaccines which have a demonstrated history of efficacy.
• Measles virus was first isolated in tissue culture in 1954 (32) from an infected patient named David Edmonston.
• This Edmonston strain of measles became the progenitor for many live-attenuated measles vaccines including Moraten, which is the current vaccine in the United States (AttenuvaxTM; Merck Sharp & Dohme, West Point, PA) and was licensed in 1968 and has proven to be efficacious.
• Aggressive immunization programs instituted in the mid to late 1960s resulted in the precipitous drop in reported measles cases from near 700,000 in 1965 to 1500 in 1983.
• other vaccine strains were also developed from the Edmonston strain
• MV measles virus
• H hemagglutinin
• F 12 fusion glycoproteins
• Antibodies to H and/or F are considered protective since they neutralize the virus' ability to initiate infection (35,36,37).
• the matrix (M; approximately 37 kD) protein is the amphipathic protein lining the membrane's inner surface, which is thought to orchestrate virion morphogenesis and thus consummate virus reproduction (38) .
• the virion core contains the 15,894 nucleotide long genomic RNA upon which template activity is conferred by its intimate association with approximately 2600 molecules of the approximately 60 kD nucleocapsid (N) protein (39,40,41).
• Loosely associated with this approximately one micron long helical ribonucleoprotein particle are enzymatic levels of the viral RNA dependent RNA polymerase (L; approximately 240 kD) which in concert with the polymerase cofactor (P; approximately 70 kD) , and perhaps yet other virus-specified as well as host-encoded proteins, transcribes and replicates the MV genome sequences (42) .
• L viral RNA dependent RNA polymerase
• P polymerase cofactor
• C approximately 20 kD
• V approximately 45 kD
• C approximately 20 kD
• V approximately 45 kD
• C approximately 20 kD
• V approximately 45 kD
• MV cotranscriptionally edited P gene-derived mRNA which encodes a hybrid protein having the amino terminal sequences of P and a new zinc finger-like cysteine-rich carboxy terminal domain (16) .
• the MV genome contains distinctive non-protein coding domains resembling those directing the transcriptional and replicative pathways of related viruses (16,43) .
• regulatory signals lie at the 3 ' and 5 ' ends of the MV genome and in short internal regions spanning each intercistronic boundary.
• the former encode the putative promoter and/or regulatory sequence elements directing genomic transcription, genome and antigenome encapsidation, and replication.
• the latter signal transcription termination and polyadenylation of each monocistronic viral mRNA and then reinitiation of transcription of the next gene.
• the MV polymerase complex appears to respond to these signals much as the
• RNA-dependent RNA polymerases of other non-segmented negative strand RNA viruses (16,43,44,45).
• Transcription initiates at or near the 3 ' end of the MV genome and then proceeds in a 5 ' direction producing monocistronic mRNAs (41,43,46).
• stop/start signals which, in 3 ' to 5' order, are: a semi-conserved transcription termination/polyadenylation signal (A/G U/C UA A/U NN A 4 , where N may be any of the four bases) at which each monocistronic RNA is completed; a non-transcribed intergenic trinucleotide punctuation mark (CUU; except at the H:L boundary where it is CGU) ; and a semiconserved start signal for transcription initiation of the next gene (AGG A/G NN C/A A A/G G A/U, where N may be any of the four bases) (46,47) .
• A/G U/C UA A/U NN A 4 where N may be any of the four bases
• the 3 ' and 5 ' MV genomic termini contain non-protein coding sequences with distinct parallels to the leader and trailer RNA encoding regions of VSV (43) .
• Nucleotides 1-55 define the region between the genomic 3' terminus and the beginning of the N gene, while 37 additional nucleotides can be found between the end of the L gene and the 5 ' terminus of the genome.
• MV does not transcribe these terminal regions into short, unmodified (+) or (-) sense leader RNAs (48,49,50).
• leader readthrough transcripts including full-length polyadenylated leader :N, leader :N:P, leader:N:P:M, and of course full-length antigenome MV RNAs are transcribed (49,50).
• the short leader transcript the key operational element determining the switch from transcription to replication of the VSV single-stranded, negative polarity genome (51,52,53), seems absent in MV. This leads to consideration and exploration of alternative models for this crucial reproductive event (43) .
• nucleotides of both the genome and antigenome are two short regions of shared nucleotide sequence: 14 of 16 nucleotides at the absolute 3 ' ends of the genome and antigenome are identical. Internal to those termini, an additional region of 12 nucleotides of absolute sequence identity have been located. Their position at and near the sites at which the transcription of the MV genome must initiate and replication of the antigenome must begin, suggests that these short unique sequence domains encompass an extended promoter region.
• these discrete sequence elements may dictate alternative sites of transcription initiation -- the internal domain mandating transcription initiation at the N gene start site, and the 3' terminal domain directing antigenome production (43,49,54).
• these 3 ' extended genomic and antigenomic promoter regions encode the nascent 5 ' ends of antigenome and genome RNAs, respectively.
• Within these nascent RNAs reside as yet unidentified signals for N protein nucleation, another key regulatory element required for nucleocapsid template formation and consequently for amplification of transcription and replication.
• Figure 2 schematically shows the location and sequence of these highly conserved, putative cis-acting regulatory domains .
• Terminal non-protein coding regions similar in location, size and spacing are present in the genomes of other members of the genus Paramyxoviridae, though only 8-11 of their absolute terminal nucleotides are shared by MV (43,55) .
• the genomic terminii of the Morbillivirus canine distemper virus (CDV) displays a greater degree of homology with its MV relative: 73% of the nucleotides of the leader and trailer sequences of these two viruses are identical, including 16 of 18 at the absolute 3' termini and 17 of 18 at their 5' ends (56) . No accessory internal CDV genomic domain- sharing homology to that of the MV extended promoter has been found.
• CDV genomic nucleotides 85 and 104 and 15,587 and 15,606 in which 15 of the 20 nucleotides are complementary.
• CDV like MV contains an additional region within its non-coding 3 ' genomic and antigenomic ends that may provide important cis-acting promoter and/or regulatory signals (56) .
• the precise length of the 3 ' - leader region is identical among several members of the Family Paramyxoviridae (MV, CDV, PIV-3, BPV-3, SV and NDV) . Further evidence for the importance of these extended, non-protein coding regions comes from analyses of a large number of distinct copy-back Defective Interfering genomes (DIs) recently cloned from subacute sclerosing panencephalitis (SSPE) brain tissue. No DI with a stem shorter than the 95 5 ' terminal genomic nucleotides was found. This indicates that the minimal signals needed for MV DI RNA replication and encapsidation extend well beyond the 37 nucleotide long trailer sequence to encompass the additional internal putative regulatory domain (57) .
• DIs Defective Interfering genomes
• the cis-acting signals required for essential viral functions, including replication, transcription and encapsidation are contained in the non-coding genomic termini.
• the obligatory trans-acting elements for functionality are contained in the N, P and L genes . Mutations in any of these regions may result in alteration of vital functions, including attenuation of viral transcription/replication efficiency.
• the attenuation potential in the cis-acting sequence elements and in the trans-acting protein genes has been demonstrated in several viral systems by sequence analysis of their genomes (58,59).
• the measles embodiment of this invention involved an analysis of the nucleotide sequences of the progenitor Edmonston wild- type MV isolate, together with available measles vaccine strains derived from this isolate (see Figure 1) . Independent other wild- type isolates were examined for comparative purposes as well. In particular, the emphasis was on regions other than the 3' genomic promoter region and the L gene; those regions were the focus of International application PCT/US97/16718.
• nucleotide sequences in positive strand, antigenomic, message sense
• amino acids in positive strand, antigenomic, message sense
• Each measles virus genome listed above is 15,894 nucleotides in length.
• Translation of the N gene starts with the codon at nucleotides 108-110; the translation stop codon is at nucleotides 1683-1685.
• the translated N protein is 525 amino acids long.
• Translation of the P gene starts with the codon at nucleotides 1807-1809; the translation stop codon is at nucleotides 3328-3330.
• the translated P protein is 506 amino acids long.
• Translation of the C gene starts with the codon at nucleotides 1829-1831; the translation stop codon is at nucleotides 2387-2389.
• the translated C protein is 189 amino acids long.
• the F gene stop/polyadenylation signal includes an eleven nucleotide gene-end signal at nucleotides 7237-7247, followed by an intergenic region at nucleotides 7248-7250 and the H gene-start signal at nucleotides 7251-7260.
• nucleotide 2499 of 1983 wild-type measles virus is indicated as W G" in SEQ ID NO:3.
• the base is actually a mixture of W G" and W C" .
• nucleotide 2143 of RubeovaxTM vaccine virus is indicated as "T” in SEQ ID NO: 5.
• this base was W T" in seven and "C” in two; thus, this base can be W T" or "C” .
• the Schwarz vaccine virus genome is identical to that of the Moraten vaccine virus genome (SEQ ID NO: 6).
• nucleotide and amino acid differences distinguishing the N, P and C gene and protein sequences, and nucleotide differences distinguishing the F gene-end signal in the F/H intercistronic region of the Edmonston wild-type isolate, vaccine strains and other independently isolated wild-type viruses were compared and aligned (see Tables 3-5 in Example 1 below) .
• the attenuating phenotype of these three N gene mutations may be based upon the following discussion.
• the N protein must serve a number of functions in the measles virus life cycle. It must be able to interact with the viral genomic RNA and other copies of N to form the nucleocapsid complex in which the MV genome is always found. It interacts with the polymerase complex to allow transcription and replication. It has also been found to interact with the P protein separate from the replication complex. All of these functions could be points at which mutations might result in attentuation by affecting replicational efficiency of the virus.
• This unique mutation in the P protein is in one of the less conserved Morbillivirus amino acids: It is glutamic acid in both the wild- type and vaccine strains of the rinderpest virus, and cysteine in the canine distemper and phocine distemper virus strains (58) . However, because this unique mutation is common to all the MV vaccine strains examined, but was not present in the later wild-type isolates, it is viewed as a potentially attenuating mutation.
• the P and V mRNAs share the same start codon and the first 231 amino acids of the P and V proteins are identical .
• the V mRNA has an extra "G" between nucleotides 2498 and 2499 of the P mRNA. Editing takes place during transcription when an extra non-template- directed W G" residue is inserted between nucleotides 2498 and 2499, causing a shift in the reading frame, whereby the carboxy-terminal 276 amino acids of the P protein are replaced with a 68 amino acid cysteine-rich carboxy-terminus of the V protein.
• the mutation encoding amino acid 225 is located before the extra "G” , so that mutation is potentially attenuating for both the P and the V proteins .
• the function of the V protein is not known.
• the second change in the P gene from W G" to "A” at nucleotide 2630, resulted in a mutation from cysteine to tyrosine at amino acid 275.
• the third change in the P gene from W T" to "C” at nucleotide 3122, resulted in a mutation from leucine to proline at amino acid 439.
• the first change in the C gene from W C" to "T" at nucleotide 2046, resulted in a mutation from alanine to valine at amino acid 73 for all later wild- type isolates, as well as for all vaccine strains. Because of the presence of this mutation in the vaccine strains, but not in the progenitor wild-type strain, this mutation is viewed as potentially attenuating.
• A at nucleotide 2229, resulted in a mutation from serine to tyrosine at amino acid 134. This mutation was unique to the AIK-C vaccine strain -- it is not present in the other vaccine strains or in any of the other wild-type strains -- and, therefore, it is viewed as potentially attenuating.
• the regions comprising the cis-acting elements which control transcription initiation and termination for each MV gene were found to be highly conserved among all measles viruses examined. With one exception, the sequences of the gene-start and gene-end signals were identical for all the viruses analyzed.
• This F gene-end signal mutation is thought to affect the efficiency of transcription termination, which in turn could affect the levels of F gene expression, as well as downstream H gene expression. Decreases in F and H gene expression potentially are partially responsible for attenuation of the Moraten and Schwarz vaccine strains. This is consistent with the suggestion that a decrease in MV F gene expression may be a factor in attenuation (61) .
• the importance of mutations in gene-start and gene-end signals was exemplified by the observation that a change in the RSV M2 gene-start signal had a profound attenuating effect on the virus and was associated with the ts phenotype of the virus (62) .
• the key attenuating sites for the MV N protein are as follows: amino acid residues 129 (glutamine —» lysine) , 148 (glutamic acid — > glycine) and 479 (serine -» threonine) .
• the key attenuating sites for the P and C proteins are as follows: amino acid residues 225 (glutamic acid — glycine) , 275 (cysteine —» tyrosine) and 439 (leucine —>• proline) for the P protein and amino acid residues 73 (alanine — valine) , 104 (methionine — threonine) and 134 (serine —» tyrosine) for the C protein.
• nucleotide changes responsible for these amino acid changes are not limited to those set forth in Tables 3 and 4 of Example 1 below; all changes in nucleotides which result in codons which are translated into these amino acids are within the scope of this invention.
• the key attenuating mutation for the F gene-end signal is nucleotide 7243 (T —» C) (in antigenomic, message sense) .
• the measles virus phenotype is further attenuated by combining one or more of the above-referenced N, P or C gene or F gene-end signal mutations with one or more of each of the coordinate 3 ' genomic promoter region and L gene mutations described in International application PCT/US97/16718, which are as follows: for the MV 3' genomic promoter region, the mutations are nucleotide 26 (A ⁇ T) , nucleotide 42 (A ⁇ T or A ⁇ C) and nucleotide 96 (G -» A) (in antigenomic, message sense) , while for the L protein the mutations are amino acid residues 331 (isoleucine —» threonine) , 1409 (alanine —» threonine) , 1624 (threonine — > alanine) , 1649 (arginine ⁇ methionine) , 1717 (as
• RSV Human respiratory syncytial virus
• RSV Human respiratory syncytial virus
• RSV belongs to the Subfamily Pneumovirinae and the genus Pneumovirus (see Table 1) .
• a and B Two major subgroups of human RSV, designated A and B, have been identified based on reactivities of the F and G surface glycoproteins with monoclonal antibodies (63) . More recently, the A and B lineages of RSV strains have been confirmed by sequence analysis (64,65). Bovine, ovine, and caprine strains of this virus have also been isolated. The host specificity of the virus is most clearly associated with the G attachment protein, which is highly divergent between the human and the bovine/ovine strains (66,67), and may be influenced, at least in part, by receptor binding.
• RSV is the primary cause of serious viral pneumonia and bronchiolitis in infants and young children.
• Serious disease i.e., lower respiratory tract disease (LRD)
• LFD lower respiratory tract disease
• RSV additionally is associated with asthma and hyperreactive airways and it is a significant cause of mortality in "high risk" children with bronchopulmonary dysplasia and congenital heart disease (CHD) .
• CHD congenital heart disease
• RSV In adults, RSV generally presents as uncomplicated upper respiratory illness; however, in the elderly it rivals influenza as a predisposing factor in the development of serious LRD, particularly bacterial bronchitis and pneumonia. Disease is always confined to the respiratory tract, except in the severely immunocompromised, where dissemination to other organs can occur. Virus is spread to others by fomites contaminated with virus-containing respiratory secretions, and infection initiates through the nasal, oral, or conjunctival mucosa.
• RSV disease is seasonal and virus is usually isolated only in the winter months, e.g., from November to April in northern latitudes. The virus is ubiquitous, and over 90% of children have been infected at least once by 2 years of age. Multiple strains cocirculate. There is no direct evidence of antigenic drift (such as that seen with influenza A viruses) , but sequence studies demonstrating accumulation of amino acid changes in the hypervariable regions of the G protein and SH proteins suggest that immune pressure may drive virus evolution.
• the RSV virion consists of a ribonucleoprotein core contained within a lipoprotein envelope.
• the virions of pneumoviruses are similar in size and shape to those of all other paramyxoviruses. When visualized by negative staining and electron microscopy, virions are irregular in shape and range in diameter from 150-300 nm (75) .
• the nucleocapsid of this virus is a symmetrical helix similar to that of other paramyxoviruses, except that the helical diameter is 12-15 nm rather than 18nm.
• the envelope consists of a lipid bilayer that is derived from the host membrane and contains virally coded transmembrane surface glycoproteins .
• the viral glycoproteins mediate attachment and penetration and are organized separately into virion spikes .
• All members of the paramyxovirus subfamily have hemagglutinating activity, but this function is not a defining feature for pneumoviruses, being absent in RSV but present in Pneumovirus of mice (PVM) (76) .
• PVM Pneumovirus of mice
• Neuraminidase activity is present in members of the genera Paramyxovirus, Rubulavirus, and is absent in Morbillivirus and Pneumovirus (76) .
• RSV possesses two subgroups, designated A and
• the wild-type RSV (strain 2B) genome is a single strand of negative-sense RNA of 15,218 nucleotides (SEQ ID NO: 9) that are transcribed into ten major subgenomic mRNAs which encode eleven gene products.
• Each of the ten mRNAs encodes a major polypeptide chain: Three are transmembrane surface proteins (G, F and SH) ; three are the proteins associated with genomic RNA to form the viral nucleocapsid (N, P and L) ; two are nonstructural proteins (NSl and NS2) which accumulate in the infected cells but are also present in the virion in trace amounts and may play a role in regulating transcription and replication; one is the nonglycosylated virion matrix protein (M) ; and the last is M2, another nonglycosylated protein recently shown to be an RSV- specified transcription elongation factor (see Figure 3) (another gene product is also encoded by the M2 gene) . These ten viral proteins account for nearly all of the viral coding capacity.
• the viral genome is encapsidated with the major nucleocapsid protein (N) , and is associated with the phosphoprotein (P) , and the large (L) polymerase protein. These three proteins have been shown to be necessary and sufficient for directing RNA replication of cDNA encoded RSV minigenomes (77) . Further studies have shown that for transcription to proceed with full processing, the M2 protein (ORF 1) is required (75) . When the M2 protein is missing, truncated transcripts predominate, and rescue of the full length genome does not occur (75) . Both the M (matrix protein) and the M2 proteins are internal virion-associated proteins that are not present in the nucleocapsid structure.
• the M protein is thought to render the nucleocapsid transcriptionally inactive before packaging and to mediate its association with the viral envelope.
• the NSl and NS2 proteins have only been detected in very small amounts in purified virions, and at this time are considered non-structural . Their functions are uncertain, though they may be regulators of transcription and replication.
• Three transmembrane surface glycoproteins are present in virions: G, F, and SH.
• G and F (fusion) are envelope glycoproteins that are known to mediate attachment and penetration of the virus into the host cell. In addition, these glycoproteins represent major independent immunogens (78) .
• Genomic RNA is neither capped nor polyadenylated (80) . In both the virion and intracellularly, genomic RNA is tightly associated with the N protein.
• the 3 ' end of the genomic RNA consists of a 44-nucleotide extragenic leader region that is presumed to contain the major viral promoter (Fig. 3).
• the 3' genomic promoter region is followed by ten viral genes in the order 3 ' -NS1-NS2-N-P-M-SH-G-F-M2-L-5 ' (Fig. 3).
• the L gene is followed by a 145-149 nucleotide extragenic trailer region (see Figure 3) .
• Each gene begins with a conserved nine-nucleotide gene start signal 3 ' -GGGGCAAAU (except for the ten-nucleotide gene start signal of the L gene, which is 3 ' -GGGACAAAAU; and the gene start signal of the SH gene for 2B and 18537, which is 3' -GGGGUAAAU; differences underlined).
• transcription begins at the first nucleotide of the signal.
• Each gene terminates with a semi- conserved 12-14 nucleotide gene end (3 ' -A G U/G U/A ANNN U/A A 3 . 5 ) (where N can be any of the four bases) that directs transcription termination and polyadenylation (Fig. 3) .
• the first nine genes are non-overlapping and are separated by intergenic regions that range in size from 3 to 56 nucleotides for RSV B strains (Fig. 3) .
• the intergenic regions do not contain any conserved motifs or any obvious features of secondary structure and have been shown to have no influence on the preceding and succeeding gene expression in a minreplicon system (Fig. 3) .
• the last two RSV genes overlap by 68 nucleotides (Fig. 3) .
• the gene-start signal of the L gene is located inside of, rather than after, the M2 gene.
• This 68 nucleotide overlap sequence encodes the last 68 nucleotides of the M2 mRNA (exclusive of the Poly-A tail) , as well as the first 68 nucleotides of the L mRNA.
• Ten different species of subgenomic polyadenylated mRNAs and a number of polycistronic polyadenylated read-through transcripts are the products of genomic transcription (75) .
• Transcriptional mapping studies using UV light mediated genomic inactivation showed that RSV genes are transcribed in their 3 ' to 5' order from a single promoter near the 3' end (81). Thus, RSV synthesis appears to follow the single entry, sequential transcription model proposed for all Mononegavirales (16,82).
• the polymerase (L) contacts genomic RNA in the nucleocapsid form at the 3 ' genomic promoter region and begins transcription at the first nucleotide.
• RSV mRNAs are co-linear copies of the genes, with no evidence of mRNA editing or splicing.
• nucleotides 21-25 are the first ten nucleotides (presumably acting as a promoter) , nucleotides 21-25, and the gene start signal located at nucleotides 45-53 (84) .
• the remainder of the leader and non-coding region of NSl gene of RSV was found to be highly tolerant of insertions, deletions and substitutions (84) .
• gene-start and gene-end motifs were shown to be signals for mRNA synthesis and appear to be self- contained and largely independent of the nature of adjoining sequence (85) .
• the L gene start signal lies 68 nucleotides upstream of the M2 gene-end signal, resulting in gene overlap (Fig. 3) (75) .
• the presence of the M2 gene-end signal within the L gene results in a high frequency of premature termination of L gene transcripts .
• Full length L mRNA is much less abundant and is made when the polymerase fails to recognize the M2 gene-end motif. This results in much lower transcription of L mRNA.
• the gene overlap seems incompatible with a model of linear sequential transcription.
• RSV RNA replication is thought (75) to follow the model proposed from studies with vesicular stomatitis virus and Sendai virus (16,82). This involves a switch from the stop-start mode of mRNA synthesis to an antiterminator read- through, the latter resulting in synthesis of positive sense replication- intermediate (RI) RNA that is an exact complementary copy of genomic RNA. This serves in turn as the template for the synthesis of progeny genomes. The mechanism involved in the switch to the antiterminator mode is proposed to involve cotranscriptional encapsidation of the nascent RNA by N protein (16,82) . RNA replication in RSV like other nonsegmented negative-strand RNA viruses is dependent on ongoing protein synthesis (86) .
• RI RNA has been detected for the standard virus as well as RSV-CAT minigenome (75,86). RI RNA was 10-20 fold less abundant intracellularly than was the progeny genome both for the standard and the minigenome system.
• the RSV subgroup B embodiment of this invention involved an analysis of the nucleotide sequences of various wild- type, vaccine and revertant RSV strains. In particular, the emphasis was on regions other than the 3 ' genomic promoter region and the L gene; those regions were the focus of International application PCT/US97/16718.
• nucleotide sequences in positive strand, antigenomic, message sense
• nucleotide sequences in positive strand, antigenomic, message sense
• the M gene stop/polyadenylation signal includes a twelve nucleotide gene-end signal at nucleotides 4196-4207, followed by an intergenic region at nucleotides 4208- 4216 and the SH gene-start signal at nucleotides 4217- 4225.
• the third nucleotide of the M gene-end signal of the wild-type 2B, vaccine 2B33F and revertant 2B33F TS(+) strains was a G, while the third nucleotide of all other RSV subgroup B gene-end signals was a T.
• the sequence of the M gene-end signal impacts SH gene expression.
• the RSV subgroup B 2B33F mutant and its TS(+) revertant have this unique nucleotide at this fourth position of the M gene-end signal (at nucleotide 4200 in the mutant and revertant) that is not found in their 2B parental strain (at nucleotide 4199) or in any of the other RSV subgroup B or A viruses analyzed.
• the strains containing this mutation have down-regulated SH expression compared to the 2B parental strain, and have essentially stopped producing the SH protein.
• This shift to the predominance of bicistronic transcription products and the concomitant down-regulation of the downstream gene product, SH provides evidence that the mutation in the M gene-end signal contributes to the attenuation phenotype of these two strains .
• the key attenuating mutation for the M gene-end signal is nucleotide 4199 (T — > C) (in antigenomic, message sense) .
• T — > C in antigenomic, message sense
• the SH gene of the 2B33F mutant is blistered with nucleotide changes (biased hypermutation) compared to that of the 2B parent (see Table 6) , some of which would result in amino acid substitutions in the SH protein.
• One change at nucleotide 4498 of 2B33F converts the predicted SH translation stop codon to glutamine. This results in a predicted length for the 2B33F SH protein that is substantially longer than that of the 2B parent.
• the RSV subgroup B phenotype is further attenuated by combining the above-referenced M gene-end signal mutation with one or more of each of the coordinate 3' genomic promoter region and L gene mutations described in International application PCT/US97/16718, which are as follows : for the RSV subgroup B 3 ' genomic promoter region, the mutations are nucleotide 4 (C —» G) , and the insertion of an additional A in the stretch of A's at nucleotides 6-11 (in antigenomic message sense) , while for the L protein the mutations are amino acid residues 353 (arginine —* ⁇ lysine) , 451 (lysine -» arginine) , 1229 (aspartic acid — > asparagine) , 2029 (threonine —» isoleucine) and 2050 (asparagine — aspartic acid) .
• the mutations are nucleotide 4 (C —» G
• the attenuated viruses of this invention exhibit a substantial reduction of virulence compared to wild-type viruses which infect human and animal hosts .
• the extent of attenuation is such that symptoms of infection will not arise in most immunized individuals, but the virus will retain sufficient replication competence to be infectious in and elicit the desired immune response profile in the vaccinee.
• the attenuated viruses of this invention may be used to formulate a vaccine. To do so, the attenuated virus is adjusted to an appropriate concentration and formulated with any suitable vaccine adjuvant, diluent or carrier.
• Physiologically acceptable media may be used as carriers. These include, but are not limited to: an appropriate isotonic medium, phosphate buffered saline and the like.
• Suitable adjuvants include, but are not limited to MPLTM (3-O-deacylated monophosphoryl lipid A; RIBI ImmunoChem Research, Inc., Hamilton, MT) and IL-12 (Genetics Institute, Cambridge, MA) .
• the formulation including the attenuated virus is intended for use as a vaccine.
• the attenuated virus may be mixed with cryoprotective additives or stabilizers such as proteins (e.g., albumin, gelatin), sugars (e.g., sucrose, lactose, sorbitol), amino acids (e.g., sodium glutamate) , saline, or other protective agents.
• cryoprotective additives or stabilizers such as proteins (e.g., albumin, gelatin), sugars (e.g., sucrose, lactose, sorbitol), amino acids (e.g., sodium glutamate) , saline, or other protective agents.
• This mixture is maintained in a liquid state, or is then dessicated or lyophilized for transport and storage and mixed with water immediately prior to administration.
• Formulations comprising the attenuated viruses of this invention are useful to immunize a human or animal subject to induce protection against infection by the wild- type counterpart of the attenuated virus.
• this invention further provides a method of immunizing a subject to induce protection against infection by an RNA virus of the Order Mononegavirales by administering to the subject an effective immunizing amount of a vaccine formulation incorporating an attenuated version of that virus as described hereinabove.
• a sufficient amount of the vaccine in an appropriate number of doses must be administered to the subject to elicit an immune response.
• Persons skilled in the art will readily be able to determine such amounts and dosages.
• Administration may be by any conventional effective form, such as intranasally, parenterally, orally, or topically applied to any mucosal surface such as intranasal, oral, eye, lung, vaginal or rectal surface, such as by an aerosol spray.
• the preferred means of administration is by intranasal administration.
• Moraten MV vaccine virus was grown once, directly from the AttenuvaxTM vaccine vial (Lot #0716B) , the Schwarz vaccine virus was grown once (Lot 96G04/M179 G41D) , while the Zagreb and RubeovaxTM vaccine viruses were each grown twice in the Vero cells before RNAs were made for sequence analysis.
• MV wildtype isolate Montefiore (57) was passed 5-6 times in Vero cells before extraction of RNA materials and similarly, MV wildtype isolates 1977, 1983 (14) were grown 5-7 times before extracting materials for analysis.
• Edmonston wild- type isolate received from Dr. J. Beeler (CBER) (see Fig. 1) was the original Edmonston isolate already passaged seven times in human kidney cells and three times in Vero cells before receipt and further passaged once in Vero cells before using for sequence analysis.
• RNA was prepared by infecting Vero cells at a multiplicity of infection (m.o.i.) of 0.1 to 1.0 and allowed to reach maximum cytopathology before being harvested. Total RNA from measles virus-infected cells was extracted using TrizolTM reagent (Gibco-BRL) .
• RNA isolated from Vero cell passage material was amplified by the Reverse Transcriptase-PCR (Perkin-Elmer/Cetus) procedure using measles (Edmonston B strain (19)) specific primer pairs spanning the 3' and 5 ' promoter regions and the L gene of the viral genome. Table 2 presents these primer sequences.
• the primers of SEQ ID NOS:15-34, 54, 57 and 58 are in antigenomic message sense.
• the primers of SEQ ID NOS: 35-53, 55, 56 and 59 are in genomic negative-sense.
• GGGTTGGTACATAGCTCTGC 13401 (SEQ ID NO: 47) 13767 CACCCATCTGATATTTCCCTGATGG 13743 (SEQ ID NO: 48)
• GGGAAGCTT 15801 AACCCTAATCCTGCCCTAGGTGG 15823 (SEQ ID NO: 58)
• nucleotide sequences were determined for regions other than the genomic promoter region and the L gene of the progenitor Edmonston wild-type MV isolate, for the available vaccine strains derived from this isolate, as well as for other wild-type strains. Significant nucleotide (in antigenomic, message sense) and amino acid differences were then compared and aligned as set forth in Tables 3-5 (differences are in italics) :
• the temperature-sensitive (ts) phenotype is strongly associated with attenuation in vivo ⁇ in addition, some non-ts mutations may also be attenuating. Identification of ts and non-ts attenuating mutations was achieved by sequence analysis and evaluation of ts, cold-adapted (ca) , and in vivo growth phenotypes of RSV mutants and revertants.
• the genomes of the following three RSV 2B strains have been completely sequenced: The 2B wild- type parent, a ts and ca derivative thereof designated 2B33F and one ts(+) revertant designated 2B33F TS(+).
• the 2B33F strain is described in U.S. Serial No. 08/059,444 (88), which is hereby incorporated by reference.
• nine additional isolates of 2B33F "revertants" obtained following in vi tro passaging at 39°C and in vivo passaging in African green monkeys or chimpanzees have been sequenced in those previously- identified mutant regions.
• the ts, ca , and attenuation phenotypes of many of these revertants have now been characterized and assessed. Correlations between ts phenotype, virus attenuation and sequence changes have been identified.
• nucl. pos. numbers are one larger than for 2B for M, SH & L genes At pos. 9853, the Lys-Arg change has reverted back to Lys in the 2B33F TS(+) strain Table 7
• Northern blot analysis of poly (A) + RNA isolated from RSV-infected Vero cells was performed using a mixture of 32 P-labelled riboprobes specific for the M gene of RSV subgroup A and B viruses to determine the effect of the M gene-end signal sequences on M gene transcription.
• the negative sense hybridization probes were T7 RNA polymerase transcripts synthesized in vi tro from PCR products containing the T7 promoter sequence.
• the wild-type subgroup B strains 2B, Bl and 18537 produced a slight excess of bicistronic over monocistronic mRNAs (lanes 3, 4, 7) .
• the subgroup A 3A virus showed comparable levels of monocistronic and bicistronic products (lane 1) .
• the M gene transcription pattern of the A2 virus (lane 2) .
• monocistronic M transcripts were produced in abundance, while read- through transcription was minimal .
• Northern blot results were confirmed by a ribonuclease protection assay which was performed on the same poly (A) + RNA that was used for the Northern blot, in order to assess the ratio of monocistronic M and SH mRNAs to bicistronic M:SH for each RSV subgroup B virus .
• Riboprobes were used which were negative sense virus-specific 32 P-labelled transcripts of 284 nucleotides in length (B viruses) that spanned the M:SH gene junction. The full-length probe should be protected from RNase digestion predominantly by bicistronic M:SH transcripts.
• Monocistronic M and SH transcripts are expected to protect approximately 165 and 110 nucleotide probe fragments, respectively, from RNase digestion.
• viral RNA or control yeast RNA was hybridized with 5 X 10 4 cpm of probe; thereafter, the hybridized RNA was then digested with RNase Tl (diluted 1:100), precipitated with ethanol, and the protected probe fragments were separated on a denaturing 6% polyacrylamide gel.
• RSV 2B33F differs from parental RSV 2B by two changes at the 3 ' genomic promoter region, two changes at the non-coding 5 '-end of the M gene, including one in the M gene-end signal, and four coding changes plus one non-coding (poly (A) motif) change in the RNA dependent RNA polymerase coding L gene.
• An attenuating mutation can be identified in the M gene-end signal of the attenuated virus strain 2B33F: The shift from a slight excess of bicistronic over monocistronic mRNAs in the wild-type 2B strain to the predominance of bicistronic transcription products in the 2B33F vaccine strain and the 2B33F TS(+) revertant strain provides evidence that the mutation in the M gene-end signal contributes to the attenuation phenotype of these latter two viral strains.

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