EP0932684A2 - Mutationen in der genomischen 3'promoterregion und im polymerasegen, die für die attenuierung von viren der ordnung mononegavirales verantwortlich sind - Google Patents

Mutationen in der genomischen 3'promoterregion und im polymerasegen, die für die attenuierung von viren der ordnung mononegavirales verantwortlich sind

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EP0932684A2
EP0932684A2 EP97942613A EP97942613A EP0932684A2 EP 0932684 A2 EP0932684 A2 EP 0932684A2 EP 97942613 A EP97942613 A EP 97942613A EP 97942613 A EP97942613 A EP 97942613A EP 0932684 A2 EP0932684 A2 EP 0932684A2
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leu
ser
val
virus
arg
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French (fr)
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Stephen A. Udem
Mohinderjit S. Sidhu
Joanne M. Tatem
Brian R. Murphy
Valerie B. Randolph
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Wyeth Holdings LLC
US Government
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US Department of Health and Human Services
American Cyanamid Co
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
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    • C07—ORGANIC CHEMISTRY
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    • C12N7/00—Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • C12N2760/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses negative-sense
    • C12N2760/00011—Details
    • C12N2760/18011—Paramyxoviridae
    • C12N2760/18411—Morbillivirus, e.g. Measles virus, canine distemper
    • C12N2760/18422—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2760/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses negative-sense
    • C12N2760/00011—Details
    • C12N2760/18011—Paramyxoviridae
    • 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
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2760/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses negative-sense
    • C12N2760/00011—Details
    • C12N2760/18011—Paramyxoviridae
    • C12N2760/18611—Respirovirus, e.g. Bovine, human parainfluenza 1,3
    • C12N2760/18622—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, nonsegmented, negative-sense, single stranded RNA viruses of the Order designated Mononegavirales having at least one attenuating mutation in the 3 * genomic promoter region and having at least one attenuating mutation in the RNA polymerase gene.
  • This invention was made with Government support under a grant awarded by the Public Health Service. The Government has certain rights in the inventio .
  • 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 virus has been uncoated in 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' 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, Paramyxovirus , RuJulavirus and Morbillivirus .
  • the subfamily Pneumovirinae contains the genus Pneumov ⁇ ru ⁇ .
  • the new classification is based upon morphological criteria, the organization of the viral genome, biological activities and the sequence relationships of the proteins .
  • 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, Ru ulavirus and its absence in genus Mox ⁇ illiv ⁇ rus .
  • variations in the coding potential of the P gene are considered, as is the presence of an extra gene (SH) in Rubulaviruses.
  • 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.
  • G attachment protein
  • 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.
  • 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.
  • C and V lack counterparts in pneumoviruses.
  • the basic genomic organization of pneumoviruses and paramyxoviruses is the same. The same is true of rhabdoviruses and filoviruses. Table 1 presents the current taxonomical classification of these viruses, together with examples of each genus.
  • Sendai virus (mouse parainfluenza virus type 1) Human parainfluenza virus (PIV) types 1 and 3
  • Bovine parainfluenza virus type 3 Genus Rubulavirus Simian virus 5 (SV) (Canine parainfluenza virus type 2) Mumps virus
  • Newcastle disease virus (avian Paramyxovirus 1) Human parainfluenza virus types 2, 4a and 4b Genus Morbilliv ⁇ rus
  • MV Measles virus
  • CDV Painschivirus Canine distemper virus
  • Peste-des-petits-ruminants virus Phocine distemper virus Rinderpest virus Subfamily Pneumovirinae Genus Pneu ovirus
  • Marburg virus For many of these viruses, no vaccines of any kind are available. Thus, there is a need to develop vaccines against such human and animal pathogens. Such vaccines would have to elicit a protective immune response in the recipient. The qualitative and quantitative features of such a favorable response are extrapolated from those seen in survivors of natural virus infection, who, in general, are protected from reinfection by the same or highly related viruses for some significant duration thereafter.
  • 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 .
  • IPV polio
  • hepatitis A Formalin inactivated whole virus preparations of polio (IPV) and hepatitis A have proven safe and efficacious.
  • immunization with similarly inactivated whole viruses such as respiratory syncytial virus and measles virus vaccines elicited unfavorable immune responses and/or response profiles which predisposed vaccinees to exaggerated or aberrant disease when subsequently confronted with the natural or "wild-type M virus.
  • 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 patterns, which parallel the protective immune profile of survivors of natural infection.
  • This favorable immune response pattern is contrasted with the delimited responses elicited by inactivated or subunit vaccines, which typically are largely restricted to the humoral immune surveillance arm.
  • the immune response profile elicited by some formalin inactivated whole virus vaccines e.g., measles and respiratory syncytial virus vaccines developed in the 1960's, have not only failed to provide sustained protection, but in fact have led to a predisposition to aberrant, exaggerated, and even fatal illness, when the vaccine recipient later confronted the wild- type virus.
  • 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. Nevertheless, this means for generating attenuated live virus vaccine candidates is lengthy and, at best, unpredictable, relying largely on the selective outgrowth of those randomly occurring genomic mutants with desirable attenuation characteristics. The resulting viruses may have the desired phenotype in vi tro , and even appear to be attenuated in animal models. However, all too often they remain either under- or overattenuated in the human or animal host for whom they are intended as vaccine candidates.
  • At least one attenuating mutation in the 3 ' genomic promoter region is selected from the group consisting of nucleotide 26 (A ⁇ T) , nucleotide 42 (A ⁇ T or A ⁇ C) and nucleotide 96 (G - A) , where these nucleotides, as well as others delineated in this application (unless stated otherwise) , are presented in positive strand, antigenomic, that is, message (coding) sense, and at least one attenuating mutation in the RNA polymerase gene is selected from the group consisting of nucleotide changes which produce changes in an amino acid selected from the group consisting of residues 331 (isoleucine — > threonine) , 1409 (alanine -» threonine) , 1624 (threonine —> alanine) , 1649 (arginine — > methionine) , 1717 (aspartic acid — a
  • At least one attenuating mutation in the 3' genomic promoter region is selected from the group consisting of nucleotide 23 (T — > C) , nucleotide 24 (C -> T) , nucleotide 28 (G - T) and nucleotide 45 (T - A)
  • at least one attenuating mutation in the RNA polymerase gene is selected from the group consisting of nucleotide changes which produce changes in an amino acid selected from the group consisting of residues 942 (tyrosine —> histidine) , 992 (leucine —» phenylalanine) , 1292 (leucine —> phenylalanine) , and 1558 (threonine —» isoleucine) .
  • At least one attenuating mutation in the 3 ' genomic promoter region is selected from the group consisting of nucleotide 4 (C — G) and the insertion of an additional A in the stretch of A's at nucleotides 6-11, and at least one attenuating mutation in the RNA polymerase gene is selected from the group consisting of nucleotide changes which produce changes in an amino acid selected from the group consisting of residues 353 (arginine — > lysine) , 451 (lysine — arginine) , 1229 (aspartic acid — asparagine) , 2029 (threonine —> isoleucine) and 2050 (asparagine — ⁇ aspartic acid) .
  • Attenuated virus is used to prepare vaccines which elicit a protective immune response against the wild- type form of the 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 virus. This virus is then used to prepare vaccines which elicit a protective immune response against the wild-type form of the virus.
  • such a complete wild- type or vaccine viral nucleotide sequence is used: (1) to design PCR primers for use in a PCR assay to detect the presence of the corresponding virus in a sample; or (2) to design and select peptides for use in an ELISA to detect the presence of the corresponding virus in a sample.
  • Figure 1 depicts the passage history of the Editionston 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.
  • 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 transcriptional 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) .
  • L proteins consist of a linear array of domains whose concatenated structure integrates discrete functions (17) .
  • three such delimited, discrete elements within the negative-sense virus L protein have been identified based on their relatedness to defined functional domains of other well-characterized proteins. These include: (1) a putative RNA template recognition and/or phosphodiester bond formation domain; (2) an RNA binding element; and (3) an ATP binding domain. All prior studies of L proteins of nonsegmented negative-sense, single stranded RNA viruses have revealed these putative functional elements (17) .
  • the invention is believed to encompass a coordinate set of changes between the cis- acting regulatory signal (3' genomic promoter region) and the polymerase gene (L) which results in attenuation of the virus while retaining sufficient ability of the virus to replicate.
  • Attenuation is optimized by rational mutations of the 3 ' genomic promoter region and the polymerase gene, which provide the desired balance of replication efficiency: 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.
  • Attenuating mutations in the extended promoter (3 * genomic promoter region) and in the polymerase gene are believed to affect the display of cis-acting signals and the conformation of the polymerase complex engaging these signals.
  • the promoter RNA when encapsidated, the promoter RNA is coiled in a helical array. Changes in promoter sequence may affect the relative positions at which the conserved signals are displayed relative to one another.
  • the measles wild-type 3' genomic promoter region has a pyrimidine (uracil) at positions 26 and 42 (the antigenomic message sense sequences have the purine adenine) .
  • the vaccine strains have purines at those positions (the antigenomic message sense sequences have the corresponding pyrimidines; see Table 3 in Example 1 below) .
  • the larger purines may change the distance and/or angular display between the conserved domains of the promoter (e.g, in measles, positions 1-11 and 87- 98) , resulting in an altered spatial presentation of the cis-acting signals to the polymerase.
  • 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 are introduced into each of the 3 ' genomic promoter region and the polymerase gene. Cumulative effects of different combinations of coding and non-coding changes can also be assessed.
  • the mutations to the 3 ' genomic promoter region and polymerase gene 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) ; rabies (21) ; vesicular stomatitis virus (VSV) (15) ; and rinderpest virus (23); these references 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 encapsidating protein is designated NP
  • the polymerase complex proteins are also referred to as 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 mammallian 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.
  • 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.
  • 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 does not replicate 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 (25,27).
  • 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 at least one attenuating mutation in the 3 ' genomic promoter region and having at least one attenuating mutation in the RNA polymerase gene, together with at least one expression vector which comprises at least one isolated nucleic acid molecule encoding the transacting proteins necessary for encapsidation, transcription and replication (e.g., N, P and L for measles virus; NP, P and L for PIV-3; 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 at the cis-acting 3 ' genomic promoter region 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 (19,28).
  • 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 (29) . Experimentally infected rhesus and cynomolgus macaques have also served as animal models for studies of vaccine-induced protection against measles (30) . Protection is measured by such criteria as disease signs and symptoms, survival, virus shedding and antibody titers.
  • 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 serves as a useful model for this invention, 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 (31) from an infected patient named David Editionston.
  • 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 Editionston strain
  • Live measles virus vaccine provides a success story of the development of an efficacious vaccine and provides a model for understanding the molecular mechanisms of viral vaccine attenuation among nonsegmented, negative-sense, single stranded RNA viruses. Because of its significance as a major cause of human morbidity and mortality, measles virus (MV) has been quite extensively studied. MV is a large, relatively spherical, enveloped particle composed of two compartments, a lipoprotein membrane and a ribonucleoprotein particle core, each having distinct biological functions (33) .
  • the virion envelope is a host cell-derived plasma membrane modified by three virus-specified proteins: The hemagglutinin (H; approximately 80 kilodaltons (kD) ) and fusion (F 12 ; approximately 60 kD) glycoproteins project on the virion surface and confer host cell attachment and entry capacities to the viral particle (16) . Antibodies to H and/or F are considered protective since they neutralize the virus' ability to initiate infection (34,35,36).
  • 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 (37) .
  • 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 (38,39,40). 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 (41) .
  • L viral RNA dependent RNA polymerase
  • P polymerase cofactor
  • C approximately 20 kD
  • V approximately 45 kD
  • the MV genome contains distinctive non-protein coding domains resembling those directing the transcriptional and replicative pathways of related viruses (16,42). These regulatory signals lie at the 3 ' and 5 ' ends of the MV genome and in short internal regions spanning each intercistroni ⁇ 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 (16,42,43,44). Transcription initiates at or near the 3' end of the MV genome and then proceeds in a 5 ' direction producing monocistronic mRNAs (40,42,45).
  • 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) (45,46).
  • A/G U/C UA A/U NN A 4 where N may be any of the four bases
  • each MV mRNA diminishes in parallel with the distance of the encoding gene from the genomic 3 ' end. This mRNA gradient directly corresponds to the relative abundance of each virus-specified protein. This indicates that MV protein expression is ultimately controlled at the transcriptional level (44) .
  • 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 (42) .
  • 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 (47,48,49) .
  • 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 (48,49).
  • the short leader transcript the key operational element determining the switch from transcription to replication of the VSV single- stranded, negative polarity genome (50,51,52), seems absent in MV. This leads to consideration and exploration of alternative models for this crucial reproductive event (42) .
  • Measles virus as well as all other Mononegavirales except the rhabdoviruse ⁇ , appears to have extended its terminal regulatory domains beyond the confines of leader and trailer encoding sequences (42) .
  • these regions encompass the 107 3' genomic nucleotides (the "3' genomic promoter region”, also referred to as the “extended promoter”, which comprises 52 nucleotides encoding the leader region, followed by three intergenic nucleotides, and 52 nucleotides encoding the 5 ' untranslated region of N mRNA) and the 109 5' end nucleotides (69 encoding the 3 ' untranslated region of L mRNA, the intergenic trinucleotide and 37 nucleotides encoding the trailer) .
  • the 3' genomic promoter region also referred to as the "extended promoter” which comprises 52 nucleotides encoding the leader region, followed by three intergenic nucleotides, and 52 nucleotides encoding the
  • 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 (42,48,53).
  • 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 (42,54) .
  • the genomic terminii of the -brbillivirus 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 (55) . 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 (55) .
  • 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 Viruses (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 (56) .
  • DIs Defective Interfering Viruses
  • this invention is directed to the concept that important virulence/attenuation determinants reside in viral genomic non-protein coding regulatory regions and in the transacting transcription/replication enzyme complex with which these cis-acting elements must interact.
  • the cis-acting domains are found both at the 3' and 5' ends of the MV genome, flanking the six contiguous genes encoding viral structural proteins; and within the MV genome as short regions encompassing internal intergenic boundaries .
  • the former encode the putative promoter and/or regulatory sequence elements directing the vital processes of genomic transcription, genome and antigenome encapsidation, and replication.
  • RNA dependent RNA polymerase molecule can modulate transcription and/or replicative efficiency, thereby determining the abundance of cytopathic viral gene products and/or virion progeny.
  • Proof of the concept of this invention for measles virus is obtained by first determining the nucleotide sequences of the non-coding regulatory regions (3 1 genomic promoter region) and the coding regions of the L gene (with predicted amino acid sequences) of the progenitor Editionston wild-type MV isolate, together with available measles vaccine strains derived from this isolate (see Figure 1) .
  • Each measles virus genome listed above is 15,894 nucleotides in length.
  • Translation of the L gene starts with the codon at nucleotides 9234-9236; the translation stop codon is at nucleotides 15783- 15785.
  • the translated L protein is 2,183 amino acids long.
  • nucleotide 2499 of 1983 wild-type measles virus is indicated as "G” in SEQ ID NO: 5.
  • 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: 9. In nine clones sequenced, this base was "T” in seven and W C" in two; thus, this base can be M T" or "C” .
  • the Schwarz vaccine virus genome is identical to that of the Moraten vaccine virus genome (SEQ ID NO:ll), except that at nucleotides 4917 and 4924, Schwarz has a W C" instead of a "T” .
  • Nucleotide differences distinguishing the 3 ' genomic promoter region and nucleotide and amino acid differences distinguishing the L gene and L protein sequences of the Edmonston wild- type isolate, vaccine strains and other independently isolated wild- type viruses were then compared and aligned (see Tables 3-5 in Example 1 below) .
  • AIK-C vaccine strain nucleotide sequence differs from the published sequence (33) at 21 positions, including one insertion and one deletion.
  • Several of these differences result in coding changes including two in the L gene (at amino acids 1477 and 2008) .
  • the additional changes accrued within the L gene sequence as the measles progenitor strain is progressively attenuated to achieve a replicative capacity optimized for live vaccine purposes appears to be constrained and delimited.
  • this limited tolerance in the number and location of L gene changes is imposed not only by the need to preserve the multifunctional capacities of the polymerase, but also by the preexisting 3' promoter changes with which the evolving L protein must interact to achieve transcription and replication.
  • optimal virus attenuation requires coordinate (i.e., linked) changes in the polymerase protein and the cis-acting regulatory elements on which it acts.
  • the 3 '-leader displays the least tolerance for change, allowing highly selected changes during the attenuation process at nucleotide position 26 (always the change of from “A” to “T"), and at position 42 (the change of from “A” to “C” or from “A” to “T”) (in antigenomic, message sense) .
  • Zagreb only, there is a single further change, from "G” to "A” at position 96, which may be important when combined with Zagreb L gene-specific changes.
  • the 3 ' -leader region seems to have undergone only one instance of genetic drift since 1954, with a change of "G” to "A” at position 50 (see Table 3) .
  • the net change in the 3 ' genomic promoter region during the attenuation process is the replacement of two pyrimidines by two purines in genomic sense in all MV vaccine strains.
  • the co- evolution of the L gene during these attenuation processes is believed to reflect selection of subtle changes favoring reproduction of the viruses in different host cells.
  • All the vaccine strains were grown in chick embryo (CE) or chick embryo fibroblast (CEF) cells during their attenuation process ( Figure 1) .
  • some vaccine strains have been exposed to unique host cells; i.e., Zagreb vaccine was grown in dog kidney cells and human diploid cells, while the AIK-C vaccine was adapted to sheep kidney cells. Moraten and RubeovaxTM were exclusively developed in CE and CEF.
  • lineage-specific L gene changes position 1649 in RubeovaxTM, Moraten and Schwarz vaccines and the change at position 1717 in all vaccines
  • individual vaccine-specific changes may provide additional fine tune modulation of virus replication/transcription for each vaccine strain.
  • nucleotide 26 A — > T
  • nucleotide 42 A ⁇ T or A ⁇ C
  • nucleotide 96 G ⁇ A (in antigenomic, message sense)
  • the key attenuating sites for the L protein are as follows: amino acid residues 331 (isoleucine -» threonine) , 1409 (alanine - threonine) , 1624 (threonine — alanine) , 1649 (arginine —» methionine) , 1717 (aspartic acid —> alanine) , 1936 (histidine -» tyrosine), 2074 (glutamine -> arginine) and 2114 (arginine — > lysine) .
  • HPIV-3 Human parainfluenza virus type 3 (HPIV-3) is another nonsegmented, negative-sense, single stranded enveloped RNA virus.
  • HPIV-3 belongs to the Family Paramyxoviridae (see Table 1) .
  • the genome of HPIV-3 is 15,462 nucleotides long and encodes six non-overlapping protein-encoding genes (57) .
  • NP corresponding to the N protein of MV
  • M corresponding to the N protein of MV
  • F hemagglutinin-neura inidase
  • L hemagglutinin-neura inidase
  • HPIV-3 Like MV, HPIV-3 consists of a 3 ⁇ -nonprotein coding leader region of 55 nucleotides, but unlike measles (where it is 37 nucleotides) , it has a 44 nucleotide long 5' -trailer region.
  • the polymerase transcribes the genome in a linear, sequential, start- stop manner which is guided by transcription signals in the RNA template .
  • Attempts to develop a live attenuated HPIV-3 vaccine by passaging the wild-type virus JS strain through cell culture at sub-optimal temperature has produced promising results (7,57).
  • cp "cold passage" mutants were isolated for evaluation from different passage levels of the JS strain. One such mutant resulted from 45 serial passages and was designated cp45.
  • This virus exhibited three interesting properties: (1) cold adaptation (ca) : the ability to replicate efficiently at the suboptimal temperature of 20°C; (2) temperature sensitivity (ts) : inability to replicate in vi tro at temperatures greater than or equal to 39°C; and (3) small plaque morphology.
  • This mutant appeared to be a promising vaccine candidate because: (a) its ca , ts and small plaque phenotype is stable after passage in cell culture; (b) its replication is restricted in both the upper and lower respiratory tract of hamsters; and (c) it induced significant protection in hamsters against subsequent challenge with wild- type HPIV-3 (58,59).
  • the cp45 strain has been grown in both fetal rhesus lung (FRhL) and Vero cells as follows:
  • the PIV- 3 cp45 virus grown in FRhL cells was prepared by inoculating confluent FRhL cell monolayers in tissue culture flasks at an MOI 0.1-1.0.
  • the infected cell cultures were fed with EMEM medium and incubated at 32°C.
  • the virus was harvested by subjecting the cultures to one freeze-thaw cycle, pooling the fluids and then storing the virus at -70 °C.
  • the PIV-3 cp45 virus grown in Vero cells was prepared by inoculating with virus a bioreactor culture of confluent monolayers of Vero cells on microcarrier beads which was continuously stirred. The infected bioreactor culture was maintained at 30°C. The virus was harvested 4-5 days later when syncytial CPE was observed. The culture fluid containing the virus was stored at -70 °C.
  • nucleotide sequences (in positive strand, antigenomic, message sense) of the HPIV-3 JS wild- type strain (89) and the cp45 vaccine strain grown in FRhL and Vero cells, as well as the deduced amino acid sequences of the RNA polymerase (L protein) of these HPIV-3 viruses, are set forth as follows with reference to the appropriate SEQ ID NOS. contained herein:
  • Each PIV-3 virus genome listed above is 15,462 nucleotides in length. Translation of the L gene starts with the codon at nucleotides 8646-8648; the translation stop codon is at nucleotides 15345- 15347. The translated L protein is 2,233 amino acids long.
  • the key attenuating mutations for the HPIV-3 3' genomic promoter region are nucleotide 23 (T — > C) , nucleotide 24 (C — > T) , nucleotide 28 (G — > T) and nucleotide 45 (T — A) (in antigenomic, message sense).
  • key attenuating sites for the L protein of HPIV- 3 include the following: amino acid residues 942 (tyrosine —> histidine) , 992 (leucine — phenylalanine) and 1558 (threonine —> isoleucine) .
  • the Vero-grown cp45 mutant vaccine strain contains an additional mutation resulting from a coding change in the L gene at amino acid residue 1292 (leucine - phenylalanine) . It is understood that the nucleotide changes responsible for these amino acid changes are not limited to those set forth in Example 2 below; all changes in nucleotides which result in codons which are translated into these amino acids are within the scope of this invention.
  • 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 (62) . More recently, the A and B lineages of RSV strains have been confirmed by sequence analysis (63,64). 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 (65,66), 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 (74) .
  • 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 paramyxovirus subfamily have hemagglutinating activity, but this function is not a defining feature for pneumoviruses, being absent in RSV but present in PVM (75) . Neuraminidase activity is present in members of the genera Paramyxovirus, Rubulavirus, and is absent in Morbillivirus and Pneumovirus of mice (PVM) (75) .
  • RSV possesses two subgroups, designated A and B.
  • the wild- type RSV (strain 2B) genome is a single strand of negative-sense RNA of 15,218 nucleotides (SEQ ID NO: 23) that are transcribed into ten major subgenomic mRNAs.
  • 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) . 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 (76) . Further studies have shown that for transcription to proceed with full processing, the M2 protein (ORF 1) is required (74) . When the M2 protein is missing, truncated transcripts predominate, and rescue of the full length genome does not occur (74) . 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 (77) .
  • Genomic RNA is neither capped nor polyadenylated (79) . 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; 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.
  • the L gene start signal lies 68 nucleotides upstream of the M2 gene-end signal, resulting in gene overlap (Fig. 3) (74) .
  • 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. It is not known whether the polymerase that exits the M2 gene jumps backward to the L gene-start signal or whether there is a second, internal promoter for L gene transcription (74) .
  • the L gene is accessible by a small fraction of polymerases that fail to start transcription at the M2 gene-start signal and slide down the M2 gene to the L gene-start signal.
  • the relative abundance of each RSV mRNA decreases with the distance of its gene from the promoter, presumably due to polymerase fall-off during sequential transcription (80) .
  • Gene overlap is a second mechanism that reduces the synthesis of full length L mRNA.
  • certain mRNAs have features that might reduce the efficiency of translation.
  • the initiation codon for SH mRNA is in a suboptimal Kozak sequence context, while the G ORF begins at the second methionyl codon in the mRNA.
  • RSV RNA replication is thought (74) to follow the model proposed from studies with vesicular stomatitis virus and Sendai virus (16,81). This involves a switch from the stop-start mode of mRNA synthesis to an antiterminator read- through mode. This results in synthesis of positive sense replication- intermediate (Rl) 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 (16,81). RNA replication in RSV like other nonsegmented negative-strand RNA viruses is dependent on ongoing protein synthesis (85) .
  • Rl RNA has been detected for the standard virus as well as RSV-CAT minigenome (74,85).
  • Rl RNA was 10-20 fold less abundant intracellularly than was the progeny genome both for the standard and the minigenome system.
  • the nucleotide sequences (in positive strand, antigenomic, message sense) of various wild- type, vaccine and revertant RSV strains, as well as the deduced amino acid sequences of the RNA polymerase (L protein) of these RSV viruses, are set forth as follows with reference to the appropriate SEQ ID NOS. contained herein: L Protein Sequence
  • Each RSV virus genome encodes an L protein that is 2,166 amino acids long. Genome length and other nucleotide information is as follows:
  • the key attenuating mutations for the RSV subgroup B 3 ' genomic promoter region 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) .
  • the key potentially attenuating sites for the L protein of RSV are as follows: amino acid residues 353 (arginine —> lysine), 451 (lysine —> arginine), 1229 (aspartic acid -» asparagine) , 2029 (threonine — isoleucine) and 2050 (asparagine — > aspartic acid) . It is understood that the nucleotide changes responsible for these amino acid changes. are not limited to those set forth in Example 3 below; all changes in nucleotides which result in codons which are translated into these amino acids are within the scope of this invention.
  • 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, vaginal or rectal surface, such as by an aerosol spray.
  • the preferred means of administration is by intranasal administration.
  • an isolated nucleic acid molecule having the complete viral nucleotide sequence of either the wild- type viruses or vaccine viruses described herein is used to generate oligonucleotide probes (from either positive strand antigenomic message sense or negative strand complementary genomic sense) and to express peptides (from positive strand antigenomic message sense only) , which are used to detect the presence of those wild- type virus and/or vaccine strains in samples of body fluids and tissues.
  • the nucleotide sequences are used to design highly specific and sensitive diagnostic tests to detect the presence of the virus in a sample.
  • Polymerase chain reaction (PCR) primers are synthesized with sequences based on the viral wild- type or vaccine sequences described herein.
  • test sample is subjected to reverse transcription of RNA, followed by PCR amplification of selected cDNA regions corresponding to the nucleotide sequence described herein which have nucleotides which are distinct for a defined strain of virus. Amplified PCR products are identified on gels and their specificity confirmed by hybridization with specific nucleotide probes.
  • ELISA tests are used to detect the presence of antigens of the wild-type or vaccine viral strains.
  • Peptides are designed and selected to contain one or more distinct residues based on the wild- type or vaccine sequences described herein. These peptides are then coupled to a hapten (e.g., keyhole limpet hemocyanin (KLH) and used to immunize animals (e.g., rabbits) for the production of monospecific polyclonal antibody.
  • KLH keyhole limpet hemocyanin
  • a selection of these polyclonal antibodies, or a combination of polyclonal and monoclonal antibodies can then be used in a "capture ELISA" to detect antigens produced by those viruses.
  • 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 (56) 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
  • 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: 35-54, 74, 77 and 78 are in antigenomic message sense .
  • the primers of SEQ ID NOS: 55-73, 75, 76 and 79 are in genomic negative-sense. Table 2
  • Vero-grown cp45 mutant vaccine strain contains an additional mutation resulting from a coding change in the L gene (marked with an asterisk in Table 6) at amino acid residue 1292 (leucine -» phenylalanine) .
  • the first two amino acid changes in the L protein map to one of the highly conserved areas among all Paramyxovirus L genes.
  • the fourth amino acid change maps to the area joining two conserved blocks corresponding to the change at amino acid 1717 in the MV vaccine strains.
  • 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.
  • 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 8 Sequence comparison between RSV 2B and 2B20L strains
  • nucl. pos. numbers are one larger than for 2B for L gene
  • 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 gene, and four coding changes plus one non-coding (poly (A) motif) change in the RNA dependent RNA polymerase coding L gene.
  • RSV 2B20L differs from its RSV 2B parent only at seven nucleotide positions, of which three are common with 2B33F virus, including two changes at the 3' genomic promoter and one coding change in the L gene. Two additional unique changes of 2B20L virus mapped to the coding region of the L gene. Potentially attenuating mutations at the non-coding 3' genomic promoter region and the RNA dependent RNA polymerase gene have been identified.
  • This amino acid 451 mutation (Lys —> Arg) is amenable to stabilization in cDNA infectious clone constructs, by inserting a second mutation to stabilize the codon, thereby lessening the likelihood that it will revert back to Lys .
  • Another wild- type RSV designated 18537 was also sequenced and compared to the sequence of the wild-type RSV 2B strain. With one exception, at all the critical residues described above, the two wildtype strains were identical.
  • the codon ACA at nucleotides 14586-14588 encodes a Thr at amino acid 2029 of the L protein
  • the codon ATT at nucleotides 14593-14595 encodes an lie at amino acid 2029 (the L gene start codon is at nucleotides 8509- 8511 in 18537, compared to 8502-8504 in 2B) .
  • Bronchoalveolar lavage and transbronchial biopsies performed two days after admission to the hospital demonstrated reactive hyperplasia and alveolar lining cell desquamation with minimal chronic inflammation. No microorganisms were revealed by Gram, methenamine silver, or PAS stains. CT scans of the chest showed multiple, ill-defined, confluent nodules at the left lung base. Despite administration of empiric antimicrobials for opportunistic bacterial, mycobacterial, and fungal pathogens commonly responsible for pulmonary complications of advanced HIV disease, the patient became and remained febrile to
  • Rhesus monkey kidney (RMK) tissue culture cells inoculated with the patient's lung biopsy material revealed cytopathic changes characteristic of measles virus infection. Confirmation was obtained using an immunofluorescence assay with monoclonal antibodies directed to measles virus. Based upon this diagnosis, oral ribavirin lOOO g B.I.D. was given for 14 days. Unfortunately, the patient progressively deteriorated, eventually dying two months later.
  • the measles virus vaccine strain (Moraten) currently used in the United States as a component of the trivalent MMR vaccines, was obtained in its univalent form (AttenuvaxTM, Merck, Sharpe, & Dohme) . This virus was passaged once in Vero cells and total vaccine infected cellular RNA then was extracted as described above.
  • RNA preparations were reverse transcribed (RT) to cDNA using random hexameric primers and Maloney murine leukemia virus reverse transcriptase (Perkin-Elmer/Cetus RT-PCR kit reagents, Perkin-Elmer- Cetus, Branchburg, NJ) .
  • the cDNA then was amplified by PCR using measles virus-specific oligodeoxynucleotide primer pairs whose design was based on the Edmonston measles virus sequence described above.
  • These PCR products comprised a set of overlapping DNA fragments spanning the entire 15,894 nucleotide long measles genome.
  • a consensus genomic sequence was established by direct analysis of each PCR product, without cloning, using the dideoxy terminator cycle-sequencing method established by the manufacturer (ABI PRISM 377 sequencer and ABI PRISM DNA sequencing kit; Perkin- Elmer/Cetus, Foster City, CA) . Both strands of the PCR-amplified DNA products were analyzed to eliminate possible sequencing ambiguities.
  • An ELISA test is used to detect the presence of RSV.
  • Peptides are designed and selected based on homologies to the RSV sequences described herein to be specific for all subgroup B strains, or for individual wild- type, vaccine or revertant RSV subgroup B strains described herein. These peptides are then coupled to KLH and used to immunize rabbits for the production of monospecific polyclonal antibody. A selection of these polyclonal antibodies, or a combination of polyclonal and monoclonal antibodies is then used in a "capture ELISA" to detect the presence of an RSV antigen.
  • GAAACAAACC CAGGATTGCT GAAATGATAT GTGACATTGA TACATATATC GTAGAGGCAG 900
  • AAATGGGGGA AACTGCACCC TACATGGTAA TCCTGGAGAA CTCAATTCAG AACAAGTTCA 1080
  • CCCATCTTCC AACCGGCACA CCCCTAGACA TTGACACTGC ATCGGAGTCC AGCCAAGATC 1560
  • GGCAGAGATT CAGGCCGAGC ACTGGCCGAA GTTCTCAAGA AACCCGTTGC CAGCCGACAA 3060
  • AGTATAGCCT ATCCGACGCT GTCCGAGATT AAGGGGGTGA TTGTCCACCG GCTAGAGGGG 6360
  • GTGTGCAGCC AAAATGCCTT GTACCCGATG AGTCCTCTGC TCCAAGAATG CCTCCGGGGG 6540
  • GGTTAGTCCC AACCTCTTCA CTGTCCCAAT TAAGGAAGCA GGCGAAGACT GCCATGCCCC 8760
  • CAACTGTGTG CAACATGGTT TACACATGCT ATATGACCTA CCTCGACCTG TTGTTGAATG 13980
  • Asp Ser Pro lie Val Thr Asn Lys lie Val Ala He Leu Glu Tyr Ala 20 25 30
  • MOLECULE TYPE DNA (genomic)
  • GGAACAAACC CAGGATTGCT GAAATGATAT GTGACATTGA TACATATATC GTAGAGGCAG 900
  • AAATGGGGGA AACTGCACCA TACATGGTAA TCCTGGAGAA CTCAATTCAG AACAAGTTCA 1080
  • CCCATCCTCC AACCGACACA CCCTTAGACA TTGACACTGC ATCGGAGTCC AGCCAAGATC 1560
  • GGCAGAGATT CAGGCCGAGC ACTGGCTGAA GTTCTCAAGA AACCCGTTGC CAGCCGACAA 3060
  • AGTATAGCCT ACCCGACGCT GTCCGAGATC AAGGGGGTGA TTGTCCACCG GCTAGAGGGG 6360
  • GGTTAGTCCC AACCTCTTCA CTGTCCCAAT TAAGGAAGCA GGCGAAGACT GCCATGCCCC 8760
  • CAACTGTGTG CAACATGGTT TACACATGCT ATATGACCTA CCTCGACCTG TTGTTGAATG 13980
  • TTGTAGACCA TTACTCATGC TCTCTGACTT ATCTTCGGCG AGGATCGATC AAACAGATAA 14280 GATTGAGAGT TGATCCAGGA TTCATTTTCG ACGCCCTCGC TGAGGTAAAT GTCAGTCAGC 14340

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EP97942613A 1996-09-27 1997-09-19 Mutationen in der genomischen 3'promoterregion und im polymerasegen, die für die attenuierung von viren der ordnung mononegavirales verantwortlich sind Withdrawn EP0932684A2 (de)

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