JP2002541798A - Production of attenuated (-) strand RNA virus vaccines from cloned nucleotide sequences - Google Patents

Abstract

  (57) [Summary] An attenuated recombinant (-) strand RNA virus suitable for vaccine use is produced from one or more isolated polynucleotide molecules encoding the virus. The recombinant genome or antigenome of this subject virus is abrupt inside the recombinant protein of the virus at one or more amino acid positions corresponding to the site of the attenuating mutation in the heterologous mutant (-) strand RNA virus. It is modified to encode a mutation. Thus, an attenuating mutation similar to that identified in the heterologous (-) strand RNA virus is incorporated into the corresponding site inside the recombinant virus, giving the recombinant virus an attenuated phenotype. The attenuating mutation incorporated into the recombinant virus can be identical or conservative with respect to the attenuating mutation identified in the heterologous mutant virus. By introducing the mutation into the recombinant (-) strand RNA virus in this manner, the candidate vaccine virus is designed to elicit the desired immune response against the subject virus in a host susceptible to infection thereby.

Description

DETAILED DESCRIPTION OF THE INVENTION

BACKGROUND OF THE INVENTION [0001] (-) RNA RNA viruses comprise a diverse and important and highly disruptive pathogen, the mononegavirus. Human pathogens in this group include several genotypes of rabies virus (RaV), measles virus (MeV), mumps virus (MuV), respiratory syncytial virus (RSV) and parainfluenza virus (PIV). It is. In the case of RSV, this pathogen is more important than other microbial pathogens as a cause of pneumonia and bronchitis in infants under one year of age. RSV is one of five children hospitalized for respiratory disease
It is responsible for more than one case, with an estimated 91,000 hospitalizations and 4,500 deaths annually in the United States alone. The human PIV virus (eg, HPIV1, HPIV2 and HPIV3) also causes bronchitis, pharyngitis and pneumonia, mainly in infants and children, causing devastating damage to the human population. Karron et al., J. Infect. Dis. 172: 1445-5.
0 (1995); Collins et al. "Parainfluenza Viruses", p. 1205-1243. BN Fi
elds et al., eds., Fields Virology, 3rd ed., vol. 1.Lippincott-Raven Pu
bl., Philadelphia (1996); Murphy et al., Virus Res. 11: 1-15 (1988). Infections with the human PIV virus cause about 20% of infants and children hospitalized for respiratory tract infections.

[0002] Despite decades of research to develop effective vaccines against RSV and PIV, no safe and effective vaccine has been approved to prevent severe morbidity and significant mortality associated with the virus . Efficient vaccine development is awaited for other important members of the mononegavirus, or improved vaccines would benefit. One obstacle to the development of live vaccines against (-) strand RNA viruses is that it is difficult to strike a proper balance between attenuation and immunogenicity. Gene stability of the attenuated virus can also be a problem. Vaccine development in the case of RSV has also been hampered by the relatively poor growth of RSV in cell culture and the instability of virus particles. Another feature of RSV infection is that the immunity elicited does not completely protect against subsequent infections. This includes the relative inefficiencies of the immune system in limiting viral infection to the airway lumen surface,
Short-term local mucosal immunity, rapid and widespread virus replication, low immunoreactivity due to immunological immaturity of young people, immunosuppression by maternal serum antibodies derived from placenta, and A number of factors contribute, such as the viral properties. Also, as described below, RSV exists as two antigenic subgroups A and B, and immunization against one subgroup does not work against the other.

In the mid-1960s, formalin-inactivated RSV was tested as a vaccine against RSV, but failed to protect against RSV infection or disease,
In fact, it exacerbated the symptoms of subsequent infection by the virus (Kim et al., Am. J. Epide
miol., 89: 422-434 (1969), Chin et al., Am. J. Epidemiol., 89: 449-463 (
1969); Kapikian et al., Am. J. Epidemiol., 89: 405-421 (1969)). More recently, vaccine development for RSV has focused on attenuated RSV mutants. Friede
wald et al., J. Amer. Med. Assoc. 204: 690-694 (1968) reported a cold-passage mutant (cpRSV) of RSV that appeared to be sufficiently attenuated to be a candidate vaccine. . This mutant showed slightly increased growth efficiency at 26 ° C. compared to its wild-type (wt) parent virus, but its replication was neither temperature-sensitive nor significantly cold-adapted. However, this cold passage mutant was attenuated for adults. Although satisfactorily attenuated and immunogenic in infants and children (seropositive) previously infected with RSV, this cpRSV mutant
The seronegative infant maintained low levels of virulence in the upper respiratory tract.

[0004] Similarly, Gharpure et al., J. Virol. 3: 414-421 reported the isolation of a temperature-sensitive RSV (tsRSV) mutant, which is also a promising vaccine candidate. One mutant, ts-1, was evaluated in the lab and in volunteers. This mutant produced an asymptomatic infection in adult volunteers and rendered resistant to challenge with the wild-type virus 45 days after immunization. Again, seropositive infants and children received asymptomatic infections, while seronegative infants developed signs of rhinitis and other minor symptoms. In addition, instability of the ts phenotype was detected. However, the virus that partially or completely lost temperature sensitivity was only a small percentage of the virus recovered from the vaccine and was not associated with any signs of disease other than minor rhinitis.

[0005] These studies show that certain low-passage and temperature-sensitive RSV strains are not sufficiently attenuated by certain vaccines, and cause minor disease symptoms, especially in seronegative infants. Others have been over-attenuated, indicating that they cannot replicate sufficiently to elicit a protective immune response (Wright et al., Infect. Immun., 37:
397-400 (1982)). In addition, the genetic instability of the candidate vaccine mutant results in the loss of its temperature-sensitive phenotype, further hindering the development of an effective RSV vaccine. For an overview, Hodes et al., Proc. Soc. Exp. Biol. Med. 145: 1158-11.
64 (1974), McIntosh et al., Pediatr. Res. 8: 689-696 (1974), and Belshe
See et al., J. Med. Virol., 3: 101-110 (1978).

Properly attenuated via uncontrollable biological methods such as cold passage
Investigators who abandoned the method of creating RSV strains have tested subunit vaccine candidates using purified RSV coat glycoprotein. This glycoprotein induced resistance to RSV infection in the lungs of cotton rats. Walsh et al., J. Infect.
Dis. 155: 1198-1204 (1987). However, this antibody has very weak neutralizing activity, and when immunizing rodents with the purified subunit vaccine, the previously treated RS
V vaccine resulted in an enhanced disease (Murphy et al., Vaccine 8: 4
97-502 (1990)).

[0007] Vaccinia virus recombinant-based vaccines expressing F or G coat glycoprotein have also been studied. These recombinants express a glycoprotein of RSV that is indistinguishable from the original virus control, and rodents infected intradermally with vaccinia-RSV F and G recombinants neutralize viral infection Expressed high levels of specific antibodies. Indeed, infection of cotton rats with the vaccine-F recombinant promoted almost complete resistance to RSV replication in the lower respiratory tract and significant resistance in the upper respiratory tract (Olmsted
et al., Proc. Natl. Acad. Sci. USA 83: 7462-7466 (1986)). However, immunization of chimpanzees with vaccinia-F and -G recombinants resulted in RS in upper respiratory tract.
Provides little protection against the V challenge (Collins et al., Vaccine 8: 164-
168 (1990)), and protection was inconsistent in the lower respiratory tract (Crowe et al., Vaccine 11).
: 1395-1404 (1993)).

[0008] Biologically attenuated virus strains, subunit vaccines, and RSV and other (-
) Because other vaccine development strategies for treating strand RNA viruses are not promising,
There is a need for new ways to develop new vaccines, especially those that manipulate recombinant vaccine candidates to incorporate genetic alterations to produce new phenotypic characteristics in viable, attenuated recombinants. Be emphasized. However, RSV and other (-) chains
Manipulating the genomic RNA of RNA viruses has traditionally been difficult. The major obstacles in this are the lack of infectivity of the naked genomic RNAs of these viruses, inadequate viral growth in tissue culture, replication cycle redundancy, virion instability, genomic complexity, and gene products. The difficulty of organizing is included.

In the case of PIV3, two live attenuated vaccine candidates are of particular interest. One such candidate is the bovine PIV3 (BPIV3) strain, which has been shown to be antigenically related to HPIV3 and protect animals against HPIV3. BPIV3 is attenuated, genetically stable and immunogenic in human infants and children (Karron et al., J. Inf. Dis. 171:
1107-14 (1995a); Karron et al., J. Inf. Dis. 172: 1445-1450, (1995b))
. A second PIV vaccine candidate, JS cp45, is a cold-adapted mutant of the JS wild (wt) strain of HPIV3 (Karron et al., (1995b), supra; Belshe et al., J. Med.
Virol. 10: 235-42 (1982)). This live attenuated, cold-passaged (cp) PIV3 vaccine candidate is stable after virus replication in vivo, has expression of temperature sensitivity (ts), cold adaptation (ca), and attenuation (att) Indicates the type. The cp45 virus protects against the human PIV3 challenge in laboratory animals, is attenuated, is genetically stable and immunogenic in seronegative human infants and children (Hall et al., Virus R
es. 22: 173-184 (1992); Karron et al., J. Infect. Dis. 172 (6): 1445-145.
0 (1995b), ibid).

[0010] Recombinant DNA technology involves recovering infectious (-) stranded RNA virus from cDNA, genetically manipulating the virus clone to construct a novel vaccine candidate, stabilizing its attenuation level and phenotype (See for an overview, Conz
elmann, J. Gen. Virol. 77: 381-89 (1996); Palese et al., Proc. Natl. Aca
d. Sci. USA 93: 11354-58, (1996)). In this context, recombinant rescue from cDNA-encoded antigenomic RNA in the presence of essential viral proteins has been reported to include infectious respiratory syncytial virus (RSV),
Human parainfluenza virus 3 (HPIV3), rabies virus (RaV), vesicular stomatitis virus (VSV), measles virus (MeV), Sendai virus (SeV), Linderpest virus, Simian virus type S, and bovine RSV (Eg, Garcin et al., EMBO J. 14: 6087-6094 (1995); Lawson et al., Proc. Natl.
Acad. Sci. USA 92: 4477-81 (1995); Radecke et al., EMBO J. 14: 57.
73-5784 (1995); Schnell et al., EMBO J. 13: 4195-203 (1994); Whelan et a
Natl. Acad. Sci. USA 92: 8388-92 (1995); Hoffman et al.,
J. Virol. 71: 4272-4277 (1997); Kao et al., Genes to Cells 1: 569-579 (1
996), Roberts et al., Virology 247 (1), 1-6 (1998); Baron et al., J. Vir
ol. 71: 1265-1271 (1997); International Patent Application No. WO 97/06270; Collins et al., P.
roc. Natl. Acad. Sci. USA 92: 11563-11567 (1995); Durbin et al., Virolog.
y 235: 323-332 (1997); U.S. patent application Ser. No. 08 / 892,40, filed Jul. 15, 1997.
No. 3 [Published International Patent Application No. WO98 / 02530 and Priority US Patent Application No. 60 / 047,634]
No. {filed May 23, 1997}, No. 60 / 046,141 {filed May 9, 1997}, and No. 60 / 046,141
No. 021,773 {filed on July 15, 1996}]; Juhasz et al., J. Virol. 71
(8): 5814-5819 (1997); He et al. Virology 237: 249-260 (1997); Whitehead
et al., Virology 247 (2): 232-9 (1998a); Whitehead et al., J. Virol. 72
(5): 4467-4471 (1998b); Jin et al. Virology 251: 206-214 (1998); Buchol
z et al. J. Virol. 73: 251-259 (1999); and Whitehead et al., J. Virol.
73: 3438-3442 (1999), both of which are incorporated herein by reference).

[0011] Reverse genetic engineering methods for recovering and modifying recombinant (-) strand RNA viruses are available, but have severe health problems due to RSV, PIV and other pathogens in mononegavirus. There remains a critical need in the art for additional tools and methods for designing safe and effective vaccines to mitigate. The challenge remaining in this context is the difficulty in achieving appropriately attenuated, immunogenic, genetically stable vaccine candidates. To achieve this goal, existing methods for identifying attenuating mutations and incorporating them into recombinant virus strains must be further expanded. Surprisingly, the present invention achieves this goal and offers additional advantages as described below.

SUMMARY OF THE INVENTION [0012] The present invention provides novel methods and compositions for designing and producing attenuated recombinant (-) stranded RNA virus suitable for vaccine use. According to the method of the present invention, a recombinant (-) strand RNA virus is produced from one or more isolated polynucleotide molecules encoding the virus. This means that one or more polynucleotide molecules encoding the recombinant genome or antigenome of this virus, together with the essential viral proteins necessary to produce an infectious virus or virus particle, may be used in a cell or cell-free system. At the same time.

[0013] The recombinant genome or antigenome of the virus is a heterologous mutant (-) strand RNA.
It is modified to encode a mutation within the recombinant protein of the virus at one or more amino acid positions corresponding to the site of the attenuating mutation in the virus. Mutations thus "introduced" in the form of integration or repetition,
Surprisingly, it gives the recombinant virus an attenuated phenotype. In this way,
Candidate vaccine virus is a host susceptible to infection by the subject virus,
It is recombinantly designed to elicit an immune response against the selected (-) strand RNA virus.

In a related aspect of the invention, there are provided isolated polynucleotide molecules and vectors encoding an attenuated recombinant (−) strand viral genome or antigenome. In accordance with the above aspects, the genome or antigenome of the present invention may comprise a heterologous mutation (-)
The mutation encodes a mutation within a selected recombinant protein of the virus at an amino acid position corresponding to the site of the attenuating mutation in the strand RNA virus.

[0015] Also provided in the present invention are methods and compositions for incorporating an attenuated recombinant (-) stranded RNA virus mutated as described above for the prevention or treatment of infection by a subject virus. .

In a preferred embodiment of the invention, the recombinant (-) strand RNA virus is any of respiratory syncytial virus (RSV), parainfluenza virus (PIV) or measles virus. In each of the above embodiments, the heterologous mutant (−) strand RNA virus is a heterologous RSV, a human parainfluenza virus (HPIV1, HPIV2, HPIV3), bovine PI
V (BPIV), Sendai virus (SeV), Newcastle disease virus (NDV), Simian virus 5 (SV5), mumps virus (MuV), measles virus (MeV),
Canine distemper virus (CDV), rabies virus (RaV), or vesicular stomatitis virus (VSV).

According to the method of the present invention, a variety of target proteins are applied to introduce an attenuating mutation from one (-) stranded RNA virus at a corresponding site within a heterologous protein. Strictly speaking, five target proteins are conserved throughout the Mononegavirus order, exhibiting moderate to high sequence identity for a particular region or domain. In particular, all known members of this order share the sequence of five homologous proteins: nucleocapsid protein (N), nucleocapsidolin protein (P), non-glycosylated matrix (M) protein, at least one surface Glycoproteins (HN, H or G), and large polymerase (L) proteins. All of these proteins have 1 site in the recombinant virus
Altering species or more conserved residues makes it a useful target for incorporating attenuating mutations.

In a more detailed aspect of the invention, additional proteins that are shared only between certain families, subfamilies, genera or species within the mononegavirus may be targeted. For example, all members of the Paramyxoviridae family have at least two surface glycoproteins, H
It has N (or H or G) and F. Almost all members of the genus Respirovirus, Rubravirus and Morbillivirus have cysteine-rich V proteins. Respiroviruses and morbilliviruses also encode homologous C proteins. Subset of Pneumovirus and Rubravirus (Simian virus)
5 (SV5) and mumps virus (MuV) share a homologous surface glycoprotein SH. Within the genus Pneumovirus (including bovine, ovine and goat RSV and mouse pneumovirus [herein referred to as mouse RSV]), several additional proteins, NS1, NS2, M2 (ORF1) and M2 (ORF1) ORF2) is stored. Avian pneumovirus lacks NS1 and NS2 but has M2 (ORF1), M2 (ORF2) and SH proteins. Any of the above proteins provides a useful target for heterologous introduction of attenuating mutations between taxa sharing the target protein of interest.

In this context, the method of the invention is based on the identification of an attenuating mutation in the first (−)-strand RNA virus. Mutations are identified for mutants against the wild-type sequence at the amino acid position that characterizes the mutation site, but provide an indication of sequence comparison to homologous proteins in different viruses that are target viruses for recombinant attenuation. Attenuating mutations can be identified by mutagenesis and reverse genetics techniques that are already known or that are applied to produce and characterize biologically derived mutant viruses. In another approach, the attenuating mutation of interest may be produced and characterized de novo, for example, by site-directed mutagenesis or conventional screening methods.

Each attenuating mutation identified in the (-) strand RNA virus provides an indication of sequence comparison to homologous proteins in one or more heterologous (-) strand viruses. In this context, existing sequence alignments can be analyzed or, using conventional sequence alignment techniques, sequence comparisons can be produced for analysis using the protein responsible for the attenuating mutation and the attenuated target recombinant virus. The corresponding protein regions and amino acid positions are identified between homologous proteins of a different virus. A genome or antigenome of a target virus is altered if one or more of the residues characterizing the attenuating mutation has changed from a `` wild-type '' identity conserved at the corresponding amino acid position in the target virus protein. , Which are recombinantly modified to encode amino acid deletions, substitutions or insertions, changing conserved residues in the target viral protein, thereby conferring an attenuated phenotype similar to the recombinant virus.

In this rational design method for constructing an attenuated recombinant (−) chain virus, one (−)
3.) The identity of the wild-type residue at the amino acid position that characterizes the attenuating mutation in the strand RNA virus can be conserved at the corresponding amino acid position in the target viral protein by strict or conservative substitutions. Thus, the corresponding residue in the target viral protein is identical or amino acid side to the wild-type residue found to be altered by the attenuating mutation in the heterologous mutant virus. It may be conservatively related to chain structure and function. In each case, a similar attenuation in the recombinant virus is achieved by the method of the present invention, wherein the recombinant genome or antigen of the target virus is encoded to encode an amino acid deletion, substitution or insertion that alters a conserved residue. This can be achieved by modifying the genome. In this context, it is preferred to modify the genome or antigenome to encode conservative residue changes that conservatively correspond to the changes characterizing the attenuating mutation in the heterologous mutant virus. For example, if a substitution of an amino acid characterizes a mutation site in a mutant virus relative to the corresponding wild-type sequence, a substitution should be designed at the corresponding residue in the recombinant virus. Preferably, the substitution will be identical or conservative to the replacement residue present in the mutant viral protein. However, it is also possible to non-conservatively change the native amino acid residue at the mutation site with respect to the replacement residue in the mutein (eg, using other amino acids to identify the identity of the wild-type residue). And disrupting or disrupting functioning). In the case of mutations characterized by deletions and insertions, these can be introduced as corresponding deletions or insertions into the recombinant virus, but the specific size and amino acid sequence of the deleted or inserted protein fragment varies. Can change.

In another aspect of the invention, the mutations incorporated into the recombinant (-) chain virus may confer various phenotypes in addition to or in association with the desired attenuation phenotype. Thus, representative mutations incorporated into the recombinant protein of this virus may be associated with attenuated phenotypes, such as temperature sensitivity (ts), cold adaptation (ca), small plaques (sp) , Or a host range restriction (hr) phenotype, or a change in growth or immunogenicity.

In an exemplary embodiment of the invention, the ts or non-ts mutation is a (−) chain virus, eg, H
It is incorporated into the large polymerase L protein of PIV3. Attenuating mutations have been identified in heterologous mutant (-) strand RNA viruses (eg, RSV),
It can be any virus that shares a conservative protein structure relationship with PIV3, including the mutation of interest. In a more particular aspect, the attenuating mutation in the heterologous mutant virus comprises an amino acid substitution of phenylalanine at position 521 of the L protein of human RSV cpts530 (ATCC VR 2452). The non-ts attenuating mutation is a parainfluenza virus (PIV) such as human PIV1 (HPIV1), human PIV2 (HPIV2), human PIV3 (HPI
V3), incorporated into recombinant proteins of bovine PIV (BPIV) or mouse PIV (MPIV or Sendai virus). The recombinant genome or antigenome is modified to encode amino acid substitutions, deletions or insertions within the recombinant virus N, P, C, D, V, M, F, HN or L proteins. This mutation causes ts
Alternatively, it may confer a non-ts attenuated phenotype. In a preferred aspect, the heterologous mutation (-)
Mutant HPIV3 in which an attenuating mutation in a strand RNA virus is biologically induced
It is an amino acid substitution corresponding to a mutation in strain JS cp45, preferably within the L protein of HPIV3 JS cp45. Representative mutations in this context include HPIV3
Amino acid substitution of tyrosine at position 942 of L protein of JS cp45, HPIV3 JS cp45
Amino acid substitution of leucine at position 992 of the L protein of and / or HPIV3 JS c
Includes an amino acid substitution of threonine at position 1558 of the L protein of p45.

Still additional mutations identified in the heterologous (-) stranded RNA virus used to construct the recombinant PIV of the present invention include amino acid substitutions within the F protein of HPIV3 JS cp45. In one example, the attenuating mutation in the heterologous virus comprises an amino acid substitution of isoleucine at position 420 of the F protein of HPIV3 JS cp45. In another approach, the attenuating mutation is the HPIV3 JS cp45 F protein 45
It may include an amino acid substitution of alanine at position 0. Similarly, attenuation of a recombinant PIV can be achieved by modifying the recombinant PIV genome or antigenome to encode a mutation similar to the attenuating mutation identified in RSV. In such examples described below, the attenuating mutation identified in RSV is RSV
Includes an amino acid substitution of phenylalanine at position 521 of the L protein. The PIV genome or antigenome is modified to encode conservative residue changes that conservatively correspond to changes characterizing an attenuating mutation in a heterologous RSV mutant. In one embodiment, the mutation is incorporated inside a recombinant HPIV3 protein and the HPIV3
The amino acid substitution of phenylalanine at the corresponding position 456 of the L protein.

[0025] Still additional PIV candidate vaccine candidates within the present invention are Sendai virus (SeV)
Recombines to encode a mutation similar to the attenuating mutation identified in
This can be achieved by modifying the PIV genome or antigenome. In such an embodiment described below, the attenuating mutation involves an amino acid substitution of phenylalanine at position 170 of the C protein of SeV. The PIV genome or antigenome is a heterologous S
It is modified to encode a conservative residue change that conservatively corresponds to the change characterizing the attenuating mutation in the eV mutant. In one embodiment, the mutation is recombinant H
It is incorporated inside the PIV3 protein and contains an amino acid substitution of phenylalanine at position 164 of the HPIV3 C protein.

In a further exemplary embodiment, the ts or non-ts attenuating mutation is a measles virus (MeV)
Is incorporated into the recombinant protein. Heterologous mutant (-) stranded RNA viruses identified as attenuating mutations include respiratory syncytial virus (RSV), human parainfluenza virus (HPIV) 1, HPIV2, HPIV3, bovine PIV (BPIV), Sendai virus (SeV), Newcastle disease virus (NDV), Simian virus 5 (SV5)
, Mumps virus (MuV), measles virus (MeV), canine distemper virus (CDV), rabies virus (RaV), or vesicular stomatitis virus (VSV). Preferably, the heterologous mutant (-) chain virus is HPIV3 JS cp45. In a more detailed embodiment, the attenuating mutation identified in HPIV3 JS cp45 comprises an amino acid substitution of tyrosine at position 942 or leucine at position 992 of the L protein.

[0027] Still further aspects of the invention are provided wherein the recombinant (-) stranded RNA virus is a chimeric virus. In particular, the virus replaces the genome or antigenome of some or all of the (-) strand RNA virus of one species, subgroup or strain with a different species, subgroup or strain (
-) Having a recombinant genome or antigenome comprising in combination with a heterologous gene or gene segment of a strand RNA virus. Attenuating mutations can be incorporated, if desired, within a protein or protein region encoded by a heterologous gene or gene segment. In a preferred aspect, the chimeric virus has one PIV
The genome or antigenome of some or all of the species, subgroups or strains of
PIV having in combination with at least one gene or gene segment of the HN and F glycoprotein genes of a subgroup or strain. In other embodiments, the chimeric virus is RSV
Wherein the recombinant genome or antigenome incorporates one gene or gene segment of the F, G and SH glycoprotein genes from a heterologous RSV species, subgroup or strain. Preferably, the human RSV subgroup A F and G glycoprotein genes have been replaced by the human RSV subgroup B F and G glycoprotein genes in the RSV A background.

In another aspect of the invention, the recombinant (-) stranded RNA virus is further modified or attenuated by additional changes to the recombinant genome or antigenome. In one embodiment, the recombinant genome or antigenome is a biologically-derived mutation (
-) Is further modified to encode one or more additional attenuating mutations taken from the strand RNA virus. For example, in a recombinant RSV, the genome or antigenome is modified to encode at least one and up to a total number of attenuating mutations present in a panel of biologically derived mutant RSV strains, wherein said panel Is ATCC
VR 2450, cpts RSV 248/404 (ATCC VR 2454), cpts RSV 248/955 (ATCC VR
2453), cpts RSV 530 (ATCC VR 2452), cpts RSV 530/1009 (ATCC VR 2451)
, Cpts RSV 530/1030 (ATCC VR 2455), RSV B-1 cp52 / 2B5 (ATCC VR 2542)
, And RSV B-1 cp-23 (ATCC VR 2579). Alternatively, recombinant P
In IV, the recombinant genome or antigenome encodes at least one and up to attenuating mutations present within HPIV3 JS cp45. Preferably, at least one of the attenuating mutations contemplated herein is stabilized by multiple nucleotide changes in the codon identifying the mutation.

[0029] In another aspect of the invention, the recombinant (-) stranded RNA virus is characterized by altered growth characteristics, attenuation, temperature sensitivity, cold adaptation, small plaque size, restricted host range, or altered immunogenicity. It is further modified or attenuated by nucleotide modifications that specify a phenotypic change of choice. For example, in RSV, the recombinant genome or antigenome may incorporate modifications of the SH, NS1, NS2, or G genes, such as deletion of the gene or exclusion of gene expression. Other nucleotide modifications in RSV and other (-) strand RNA viruses include deletion, insertion, addition or rearrangement of cis-acting regulatory sequences or nucleotides within the recombinant genome or antigenome.

In another related aspect, the invention relates to an isolated recombinant (−) strand RNA that is attenuated to elicit an immune response in a host susceptible to the virus.
Provide the virus. The virus has a recombinant genome or antigenome,
Contains viral proteins necessary to produce infectious viral particles of RNA viruses. The recombinant genome or antigenome encodes a mutation within the recombinant protein of the virus at an amino acid position corresponding to that of the attenuating mutation identified in the heterologous mutant (-) strand RNA virus. To be modified. Mutations incorporated within this recombinant protein confer a phenotype of attenuation on the recombinant virus.

[0031] Further provided is an isolated polynucleotide molecule encoding a recombinant genome or antigenome of a recombinant (-) strand RNA virus. The recombinant genome or antigenome is also abrupt within the recombinant protein of the virus at an amino acid position corresponding to the amino acid position of the attenuating mutation identified in the heterologous mutant (-) strand RNA virus. It is modified to encode a mutation. Mutations incorporated within this recombinant protein confer a phenotype of attenuation on the recombinant virus. In a related aspect, an expression vector comprising a operably linked transcription promoter, a polynucleotide molecule encoding a recombinant genome or antigenome of a recombinant (-) strand RNA virus as described above, and a transcription terminator is provided. Provided.

The present invention also provides a method of stimulating an individual's immune system to elicit protection against (-) strand RNA viruses. This method includes attenuated recombination (-) as described above.
Administering an immunologically sufficient amount of the chain virus together with a physiologically acceptable carrier to the individual is included. In a related aspect, an immunogenic composition is provided that elicits an immune response against a (-) strand RNA virus. The composition comprises an immunologically sufficient amount of the attenuated recombinant (-) chain virus of the invention together with a physiologically acceptable carrier. Preferably, the attenuated recombinant (-) chain virus is RSV, PIV or measles virus.

DESCRIPTION OF SPECIFIC EMBODIMENTS The methods of the present invention provide an attenuated recombinant (−) strand RNA virus suitable for vaccine use. Recombinant (-) strand RNA virus is produced from one or more isolated polynucleotide molecules encoding the virus. Production of recombinant virus
i) encodes the recombinant genome or antigenome of this virus and (ii) the essential viral proteins necessary to produce infectious virus or subviral particles
This is achieved by co-expressing one or more polynucleotide molecules in a cell or cell-free system.

The recombinant genome or antigenome of the recombinant virus has a heterologous mutation (−
A) at one or more amino acid positions corresponding to the site of the attenuating mutation in the strand RNA virus, modified to encode the mutation within a recombinant protein of the virus; Attenuating mutations identified within the heterologous (-) strand RNA virus are thus "introduced" to the corresponding site inside the recombinant virus, giving the recombinant virus an attenuated phenotype. The introduced mutation is typically identical or conservative to the attenuating mutation identified in the heterologous mutant virus, but may also make non-conservative substitutions. In this way, by incorporating the mutation introduced into the recombinant (-) strand RNA virus, the candidate vaccine virus will elicit the desired immune response against that virus in a host susceptible to the virus. Designed to.

The present invention embodies a novel paradigm for rationally designing attenuated vaccine viruses based on the identification of attenuating mutations in heterologous (-) strand RNA viruses.
Attenuating mutations in a heterologous virus can be, for example, by comparing one or more amino acids in the protein of interest in the heterologous mutant virus by conventional nucleotide or amino acid sequence comparisons between the mutant virus and its non-attenuated parent virus. Maps to a deletion, substitution or insertion. In this context, the parent virus is typically a biologically derived strain that is wild type for at least the attenuated phenotype. However, partially attenuated mutant strains may also be used. Here, additional attenuating mutations may result from artificial mutagenesis or natural polymorphisms. In addition, parental viruses into which attenuating mutations have been introduced and which can be mapped later include artificially produced viruses such as those provided from cDNA virus clones by known reverse genetics methods.

Thus, the attenuating mutation identified in the heterologous (-) strand RNA virus is already known, or is produced and / or identified by conventional mutagenesis and / or reverse genetics techniques. Can be done. These techniques include, for example, site-directed mutagenesis of wild-type or non-attenuated mutant virus cDNA clones in combination with conventional screening methods to identify attenuated derivatives. To produce and characterize, or to generate and characterize the attenuating mutations of interest de novo. Reverse genetic methods for rescue and genetic manipulation of infectious virus clones are known for representative groups of viruses throughout the mononegavirus (for review, see Conzelmann, J. Gen. Virol. 77: 3
81-89 (1996); Palese et al., Proc. Natl. Acad. Sci. USA 93: 11354-5.
8, (1996)).

For example, viral proteins essential for infection, ie, nucleocapsid N, phosphoprotein P, and large polymerase subunit L (eg, Garcin et al., EMB
O J. 14: 6087-6094 (1995); Lawson et al., Proc. Natl. Acad. Sci. US A
92: 4477-81 (1995); Radecke et al., EMBO J. 14: 5773-5784 (1995); Schn.
ell et al., EMBO J. 13: 4195-203 (1994); Whelan et al., Proc. Natl. Acad.
Sci. USA 92: 8388-92 (1995); and WO 97/06270, both of which are incorporated herein by reference). Rescue of infectious virus clones from antigenomic RNA has been demonstrated in rabies virus (RaV), vesicular stomatitis virus (VSV), measles virus (MeV), and Sendai virus (SeV) Linderpest (Baron et al., J . Vir
ol. 71: 1265-1271 (1997)) and Simian virus S (He et al. Virology 23).
7: 249-260 (1997), p. 5).

[0038] Rescue of infectious respiratory syncytial virus (RSV) has also been demonstrated with nucleocapsid N,
It has been achieved by the development of a novel system using cDNA-encoding antigenomic RNA that is co-expressed with phosphoprotein P, large polymerase subunit L, and the product of the previously uncharacterized M2 ORF1 gene (1996). US patent application Ser. No. 08 / 720,132 filed Sep. 27, 1995 [this is US Provisional Application Ser.
007,083] and US patent application Ser. No. 08 / 892,40, filed Jul. 15, 1997.
No. 3 [corresponding to published International Patent Application No. WO 98/02530;
No. 60 / 047,634, filed on May 9, 1997, and 60 / 021,773, filed on July 15, 1997, which is a continuation-in-part application of thing. All of which are incorporated herein by reference). The disclosure above includes representative constructs for use in producing infectious recombinant RSV, including recombinant RSV clones that incorporate attenuating mutations taken from biologically-derived RSV mutants. Description is included. One such construct is the R53, designated D53-530-site ().
It is a recombinant virus clone that has incorporated an attenuating mutation of the cpsts530 SV mutant. Further description regarding methods and compositions for rescue and genetic manipulation of RSV clones can be found in Collins et al., Proc. Natl. Acad. Sci. USA 92: 11563-115.
67 (1995); Juhasz et al., Vaccine 17: 1416-1424 (1999); Juhasz et al., J
Virol. 71 (8): 5814-5819 (1997); Whitehead et al., Virology 247 (2): 2
32-9 (1998a); Whitehead et al., J. Virol. 72 (5): 4467-4471 (1998b); and Whitehead et al., J. Virol. 73 (4): 3438-3442 (1999). Provided. These are incorporated herein by reference.

In another important example, rescue of infectious parainfluenza virus (PIV) has also been achieved by using cDNA-encoding antigenomic RNA that is co-expressed with N, P and L proteins ( For example, U.S. patent application Ser. Nos. 60 / 047,575 and 60 / 059,385
This is a continuation-in-part application of this issue. All of which are incorporated herein by reference). The above references include a description of the following plasmids used to generate infectious PIV clones: p3 / 7 (131) (ATCC 9
7990); p3 / 7 (131) 2G (ATCC 97989); and p218 (131) (ATCC 97991); both according to the terms of the Budapest Treaty, Manassas, Virginia, Boulevard University, 10801, American Type Culture Collection (ATCC), 20110-2
Stored in 2092.

The development of a reverse genetics system for the rescue and manipulation of (−) strand RNA viruses has allowed detailed analysis and mapping of attenuating mutations to develop useful recombinant vaccines specific for the subject virus species. Will be possible. To date, in the order of the mononegavirus, the introduction of attenuating mutations by recombinant methods has not been tested or achieved. Nevertheless, a wide range of attenuated mutant strains identified for representative taxa, such as RSV, PIV, measles and other mononegavirus members, have been identified in combination with the powerful tools provided by reverse genetics. It serves as a rich source for determining useful mutations for heterologous transfer in the methods of the invention.

Attenuating mutations in a heterologous (-) strand RNA virus can be identified in a biologically derived mutant strain for incorporation into a heterologous recombinant virus of the invention. The subject mutations can occur naturally or can be introduced into the wild-type or partially attenuated parent strain by known mutagenesis techniques. For example, an attenuated mutant virus strain may be a suboptimal temperature of a virus that introduces a growth-limiting mutation by chemical mutagenesis during growth of the virus in cell culture to which a chemical mutagen is added. It can be produced by selection or by selection of mutated viruses that produce small plaque (sp) or temperature sensitive (ts) viruses in cell culture (see, eg, US Patent Application No. 08 / 327,263, which is hereby incorporated by reference in its entirety). Which are incorporated herein by reference).

By “biologically derived” mutant is meant a mutant virus that is not produced by recombinant means. Thus, biologically derived viruses include naturally occurring mutants having genomic mutations from a reference wild-type sequence, such as a partially attenuated mutant PIV strain. Similarly, biologically derived mutants include those derived from the parental virus strain without the use of recombinant methods, particularly artificial mutagenesis and selection methods.

One well-known method of producing biologically-derived (−)-strand RNA viruses is to monetize wild-type or partially attenuated viruses into cell cultures at progressively lower attenuating temperatures. including. For example, in the case of RSV, the wild-type virus is typically about 34-37
Incubate at ℃. Partially attenuated mutants may be used in cell culture (eg, primary bovine kidney cells)
At a suboptimal temperature, for example, 20-26 ° C. Thus, cp mutants or other partially attenuated strains, such as ts-1 or spRSV, may have MRC-5 or
Due to passage in Vero cells, lower temperatures, about 20-24 ° C, preferably 20-22 ° C
Adapted to grow efficiently. Selection of the mutant RSV during this cold passage substantially eliminates residual virulence in the derived strain as compared to the partially attenuated parent strain.

In another approach, certain mutations can be introduced into biologically derived viruses by chemical mutagenesis of the wild-type or partially attenuated parent virus. For example, enough additional ts mutations to introduce a ts mutation or, in the case of a virus that is already ts, enough to confer attenuation and / or enhance the stability of the ts phenotype to the attenuated derivative. Is introduced. Means for introducing a ts mutation into a (−) strand RNA virus include, for example, Gharpure et al., J. Virol. 3: 414-421 (1969) and Rich
3: 91-100 (1978), at a concentration of about 10 ⁻³ to 10 ⁻⁵ M, preferably 10 ⁻⁴ M, by the general method described in ardson et al., J. Med. Replicating the virus in the presence of a mutagen such as fluorouridine or 5-fluorouracil, about 100 μg /
Includes exposing the virus to ml of nitrosoguanidine. Other chemical mutagens may also be used. Although attenuation can occur from ts mutations in most viral genes, a particularly suitable target for this purpose has been found to be the highly conserved polymerase (L) gene.

The temperature sensitivity level of replication in a representative attenuated virus for use in the present invention is determined by comparing replication at several limiting temperatures to that at permissive temperatures. The lowest temperature at which viral replication falls 100 or more times compared to replication at permissive temperatures is called the shutoff temperature. In laboratory animals and humans, the replication and virulence of a representative mutant RSV strain are both approximately correlated with the shut-off temperature of this mutant. Mutants with a shutoff temperature of 39 ° C are moderately restricted in replication, whereas mutants with a shutoff temperature of 38 ° C do not replicate well and disease symptoms are mainly restricted to the upper respiratory tract . Viruses with a shutoff temperature of 35-37 ° C are typically completely attenuated in humans. Thus, ts
The attenuated biologically induced mutant RSV used in the present invention is
It has a shut-off temperature in the range of about 35-39 ° C, and preferably 35-38 ° C.
Addition of the ts mutation to a partially attenuated strain produces a multi-attenuated virus useful in the vaccine compositions of the present invention.

A number of attenuated RSV strains as candidate vaccines have been developed by using multiple rounds of chemical mutagenesis to introduce multiple mutations into an already attenuated virus during cold passage. (Eg, Connors et al., Virology 208: 478-48
4 (1995); Crowe et al., Vaccine 12: 691-699 (1994a); and Crowe et al.,
Vaccine 12: 783-790 (1994b), which is incorporated herein by reference). The evaluation of accepted rodent and chimpanzee models, and of these biologically induced mutants in human adults and infants, indicates that some of these candidate vaccine strains are genetically stable and highly Indicates immunogenic and attenuated. Similar descriptions for attenuated mutant viruses are provided for PIV and other (-) strand RNA viruses of the invention (eg, US patent application Ser. No. 08 / 892,403, and corresponding international patent application WO98). / 02530; see U.S. Patent Application No. 09 / 083,793 and corresponding International Patent Application No. WO 98/53078, both of which are incorporated herein by reference).

According to the method of the present invention, utilizing the nucleotide sequence analysis of the attenuated mutant virus, including comparison with that of the mutant DNA or amino acid parent strain, eg, wild-type or partially attenuated strain, Attenuating mutations can be mapped to specific nucleotide and amino changes. Often, these changes involve individual amino acid substitutions, but other mutations conservatively introduced by the methods of the invention include multiple amino acid substitutions, amino acid insertions or deletions, and conserved regions in the target protein. Or more extensive changes in domains.

By utilizing the reverse genetics method described above, it is possible to introduce nucleotide and amino acid changes identified in an attenuated mutant virus into a previously characterized cDNA-encoding virus. This allows one skilled in the art to distinguish between silent accidental mutations and those responsible for the desired phenotypic change. In this regard, the subject mutations are separately and in various combinations,
Introduced into the genome or antigenome of an infectious RSV clone. In this way,
The phenotypic characteristics of the parent and derived viruses are evaluated, and mutations responsible for the desired characteristics such as attenuation, temperature sensitivity, bass adaptation, small plaque size, host restriction, etc. are further identified.

[0049] The mutations thus identified and mapped provide a rich source of candidate mutations for designing recombinant (-) stranded RNA viruses using the heterologous transfer methods described herein. provide. Representative disclosures that identify and characterize such attenuating mutations in (-) strand RNA viruses are described in US patent application Ser. No. 08 / 892,403, and corresponding International Patent Application No. WO 98/02530; Application No. 08 / 083,793 and corresponding International Patent Application No. WO 98/53078. These and other references, incorporated herein by reference, provide a "menu" of attenuating mutations that are candidates for introduction into a heterologous virus clone by the methods of the present invention.

For example, US patent application Ser. No. 08 / 892,403 and corresponding International Patent Application No. WO 98/025
No. 30 is cpts RSV 248 (ATCC VR 2450), cpts RSV 248/404 (ATCC VR 2454)
, Cpts RSV 248/955 (ATCC VR 2453), cpts RSV 530 (ATCC VR 2452), cpts
RSV 530/1009 (ATCC VR 2451), cpts RSV 530/1030 (ATCC VR 2455), RSV B
Low temperature of RSV (Pneumovirus subfamily; Pneumovirus), including representative mutants designated -1 cp52 / 2B5 (ATCC VR 2542) and RSV B-1 cp-23 (ATCC VR 2579). Passage (cp) and temperature sensitive (ts) mutants are described. From the above biologically derived mutants, a representative panel of attenuating mutations has been mapped and characterized, including Leu for Phe ₅₂₁ and Leu for Gln ₈₃₁ .
Results in an amino acid substitution at the parent residue / sequence position of Phe ₅₂₁ , Gln ₈₃₁ , Met ₁₁₆₉ , Tyr ₁₃₂₁ , as exemplified by attenuating substitutions such as Val for Met ₁₁₆₉ and Asn for Tyr ₁₃₂₁ . , Specific nucleotide changes in the large polymerase gene L. Each of these mutations occurs in the highly conserved L protein, conferring a ts phenotype on the mutant virus. However, additional mutations have been identified in RSV and other (-) strand RNA viruses, as well as other conserved proteins, which are associated with ts, such as cold passage (cp), small plaque (sp), cold adaptation (ca) Or a wide range of attenuated phenotypes, including non-ts attenuated phenotypes such as those present in host range restriction (hr) mutants.

Thus, an additional menu of representative mutations incorporated into the recombinant (−) chain virus according to the method of the present invention includes distantly related paramyxoviruses, human PIV3 (paramyxovirinae; respiroviruses) Genus) has also been identified. One such panel of mutations has been identified and characterized in the biologically-derived (cold-passaged) HPIV3 mutant virus strain JS cp45 (incorporated herein by reference). No. 08 / 083,793 and corresponding International Patent Application No. WO 98/53078). Mutations mapped and characterized within this strain include nucleotide changes encoding a ts attenuated amino acid substitution in the polymerase L gene at the parent residue / sequence position of Tyr ₉₄₂ , Leu ₉₉₂ and / or Thr _1558. There is. In a representative JS cp45 mutant L protein, Tyr ₉₄₂ is replaced by His, Leu ₉₉₂ is replaced by Phe, and Thr ₁₅₅₈ is replaced by Ile. These mutations incorporate the numbered mutations alone or in combination, r942
, R992, r1558, r942 / 992, r992 / 1558, r942 / 1558 and r942 / 992/1558, have been successfully incorporated into various PIV recombinants.

Another exemplary mutation identified in HPIV3 JS cp45 has been mapped and characterized to encode an attenuated amino acid substitution in the HPIV3 F and C proteins. This includes JS HPIV3 as exemplified by the substitution of Ile ₉₆ for Thr.
A mutation encoding a non-ts attenuating amino acid substitution in the C protein of the P gene at Ile ₉₆ at the parent residue / position of is included. Additional representative mutations identified in the F protein of HPIV3 are the parent residues / sequences of Ile ₄₂₀ and Ala ₄₅₀ , as exemplified by the substitution of Ile ₄₂₀ for Val and Ala ₄₅₀ for Thr. Encodes an amino acid substitution at a position.

[0053] Non-coding regions of the (-) chain viral gene are also identified in this manner. For example, an attenuating mutation can include a single or multiple base changes in the gene start sequence, as exemplified by an attenuating base substitution at nucleotide 7605 in the RSV M2 gene start sequence. Where such mutations are mapped to conserved nucleotide positions within a heterologous (-) strand RNA virus, they also apply to the introduction between heterologous taxa by the methods of the invention.

Each attenuating mutation thus identified in a (-) strand RNA virus provides an indication of sequence comparison to homologous proteins in one or more heterologous (-) strand viruses. To practice this aspect of the invention, existing sequence alignments can be analyzed, or sequence comparisons performed using conventional sequence alignment methods to identify the attenuated species identified in one (-) strand RNA virus. Corresponding protein regions and amino acid positions are identified between the protein responsible for the mutation and the homologous protein of a different virus that is the target virus for recombinant attenuation. The focus of this practice is
By identifying one or more residues that are associated with an attenuating mutation in the first (heterologous) virus, ie, the parent strain has changed from the parental sequence lacking the mutant phenotype is there. Sequence alignment determines whether the parent sequence of the mutant is conserved by the presence of identical or conserved amino acid residues at corresponding amino acid positions in the target (recombinant) viral protein. . Typically, such conserved "wild-type" sequence elements occur in conserved regions or domains of the protein, but the isolated residues and blocks of amino acid residues also differ in the classification of mononegavirus. It is widely conserved between groups and may provide a target that is equally useful for heterologous transfer of attenuating mutations between heterologous RNA viruses (here, all or some of the conserved sequence elements responsible for the mutational change). Is copied or transferred to a recombinant virus to produce a novel attenuated derivative).

The various conserved proteins between mononegaviruses are useful for introducing attenuating mutations found or produced in one heterologous (-) stranded RNA virus into different recombinant viruses of the invention. Provide a target. This is due to the significant conservation of structure and function among the various taxa within the mononegavirus.
Throughout this eye, five target proteins are universally conserved as accepted homologous proteins derived from past common ancestral viruses. The proteins typically exhibit moderate to high sequence identity, particularly in certain regions or domains that are alleged to share common functional attributes. If you specify
Known (-) strand RNA viruses include nucleocapsid protein (N), nucleocapsidolin protein (P), non-glycosylated matrix (M) protein, at least one surface-attached glycoprotein (HN, H or G), And large polymerase (L)
Share the composition of homologous proteins comprising the proteins. These proteins are shared between the target virus and the heterologous parent virus, whereby one or more of the mutant derivatives or constructs are identified that exhibit amino acid changes that specify the attenuating phenotype. The conserved sequence elements that are representative of targets useful for introducing attenuating mutations due to the above conserved residue changes are shown below. Additional proteins that are shared only between certain families, subfamilies, genera or species of mononegavirus are also targeted in this way.

The order of the Mononegaviruses includes the families of filoviruses, paramyxoviruses, bornaviruses and rhabdoviruses, each of which consists of viruses having a single (−)-strand RNA genome. Pringle, Arch Virol. 117: 1377-140 (1991). A summary of the nomenclature of this group, including identification of family / subfamily / general type-specific groupings, is provided in Pringle, Arch Virol. 142 (11): 2321-2326 (1997).
Representative mononegavirus classifications and expanded paramyxovirus classifications are shown in Table 1 herein. Due to the common features evident in the gene organization of these three virions with linear (-) strand RNA genomes, genetic recombination, if any, occurs only rarely, and therefore phenotypically, in these viruses. The fact that relationships reflect genetic continuity justifies grouping them together by eye.

[0057]

[Table 1]

The Bornavirus and Filoviridae are represented by a single genus. The Bornavirus genus contains only a single species (Borna disease virus). Four species have been identified in the genus Filovirus (representative species: Marburg virus) due to the diversity and expression of the nucleotide sequence and antigenicity of the attachment (G) protein. The Rhabdoviridae family includes five genera, vesiculovirus, lyssavirus, ephemerovirus, cytorhabdovirus, and nucleorhabdovirus, which differ in sequence and antigenicity as well as host range and the presence of accessory genes. , And the site of proliferation within the cell. The Paramyxoviridae is represented by two subfamilies: three genera, respirovirus (typical species: HPIV1), morbillivirus (typical species: MeV).
And a paramyxovirinae subfamily having the same and Rubravirus (representative species: MuV) and a pneumovirus subfamily (representative species: human RSV). It is contemplated to add a genus that includes avian pneumovirus.

There are 5 to 10 genes in all members of this genus and transcription is initiated by a viral RNA-dependent RNA polymerase from a single putative 3′-end promoter. With the exception of certain pneumoviruses, the order of the genes is strictly conserved. Prin
gle, Arch Virol. 142 (11): 2321-2326 (1997) .Figure 1 compares the genes of 16 viruses representing different families, subfamilies and genera within a mononegavirus. The homologues among the various taxa described are classified and grouped. Thus, VSV has been shown to have a minimum of 5 genes:
Nucleoprotein (N), Phosphoprotein (P), Matrix protein (M
), Adhesion protein (G), and polymerase protein (L). Eight members of the Bornavirus family, Borna disease virus, Filoviridae, Ebola virus, and Rhabdovirus show five basic gene patterns (NPMGL), and G in Ebola virus and four of the eight Rhabdoviruses. One or more genes are inserted between L or between P and M in the remaining four rhabdoviruses.
Paramyxoviruses of the subfamily Paramyxoviridae show increased complexity; the five basic gene patterns are the multiple coding of the P gene's genetic information and the additional coat protein gene between M and the adhesion protein (H or HN). It is enhanced by the insertion of (F) and is further enhanced by SH in certain rubraviruses. All paramyxoviruses have at least two surface glycoproteins, HN (or H or G) and F. Almost all members of the genus Respirovirus, Rubravirus and Morbillivirus have cysteine-rich V proteins. Respirovirus and morbillivirus also encode homologous C proteins. Subsets of pneumovirus and rubravirus (Simian virus 5 (SV5) and mumps virus (MuV)) share a homologous surface glycoprotein, SH. Within the genus Pneumovirus (including bovine, ovine and goat RSV and murine pneumovirus [herein referred to as mouse RSV]), several additional proteins, NS1,
NS2, M2 (ORF1) and M2 (ORF2) are stored. Paramyxoviruses of the subfamily Pneumovirus exhibit unique mutations from a basic pattern, most importantly having additional genes. In avian, human and mouse pneumoviruses, the M2 gene encodes two proteins in different reading frames, both of which have significant effects on RNA synthesis in vitro. In human and mouse pneumoviruses, two genes encoding NS1 and NS2, non-structural proteins of unknown function, are located between the 3'-leader region and N. Avian pneumovirus is closest to the basic pattern, lacks two unique 3'-terminal NS genes,
Except for the insertion of the gene, it retains the standard paramyxovirus gene order. This conserved pattern of genomic composition suggests expansion of the gene region and evolution through gene duplication rather than the introduction of genetic information from outside. Pringle, Semin. Virol. 8:
49-57, 1997.

As mentioned above, the method of the present invention is based on the identification of an attenuating mutation in the first (−)-strand RNA virus, wherein the mutation compares the mutant with the wild-type sequence. To identify the subject amino acid positions that characterize the mutation site. Preferably, these mutations are incorporated into a non-attenuated or partially attenuated recombinant virus clone to prove that the subject change does indeed specify an attenuated phenotype. A group of representative attenuating mutations in this context are provided in the disclosure herein, and other attenuating mutations can also be readily identified by known mutagen and reverse gene techniques described herein. . Each attenuating mutation identified in one (-) strand RNA virus provides an indicator for comparing sequences to homologous proteins in one or more heterologous (-) strand viruses.

In practicing the above aspects of the invention, existing sequence alignments can be analyzed, or conventional sequence alignment methods can be used to produce sequence comparisons for analysis and carry attenuating mutations. Corresponding protein regions and amino acid positions are identified between the protein and homologous proteins of different viruses that are target recombinant viruses for attenuation. One or more residues that characterize an attenuating mutation are conserved at corresponding amino acid positions in the target viral protein (ie, represented by identical or conservatively related amino acid residues). The target virus genome or antigenome is modified recombinantly to encode amino acid deletions, substitutions or insertions, if the parental, eg, wild-type, identity is altered. Altering conserved residues in the viral proteins, thereby conferring an attenuated phenotype similar to the recombinant virus.

Sequence alignments and analyzes embodying this aspect of the invention focus on homologous “pair” genes, gene segments, proteins and / or protein domains, and here provide a basis for statistical sequence comparisons. The polynucleotide or amino acid reference sequence is used as the defined sequence. For example, a reference sequence can be a defined segment of a cDNA or gene, a complete cDNA or gene sequence, or a sequence of a protein or a portion thereof.

Generally, a reference sequence for use in defining a paired gene or gene segment is at least 20 nucleotides in length, frequently at least 25 nucleotides in length, and often at least 50 nucleotides in length. , Proteins and protein segments are at least 20 amino acids in length. The two polynucleotides or amino acid sequences each include (1) a sequence that is similar between the two polynucleotides or proteins (ie, a portion of the complete polynucleotide or protein sequence); and (2) Sequence further between two (or more) polynucleotides or proteins, typically includes two or more sequences that vary between the two polynucleotides or proteins. It is performed by comparing the sequences of the subject sequences to identify and compare the sequence identity or similarity of the local regions. A `` comparison window, '' as used herein, refers to a conceptual segment consisting of at least 20 contiguous nucleotide or amino acid positions, where a reference to at least 20 contiguous nucleotides or amino acids is provided. A sequence is compared to a sequence, wherein the portion of the polynucleotide or amino acid in the comparison window matches the reference sequence (for optimal alignment of the two sequences)
20% or less additions or deletions (ie, not including additions or deletions) (ie,
Gap).

An optimal juxtaposition method for juxtaposing comparison windows is described in Smith & Waterman, Adv. App.
l. Math. 2: 482 (1981) local homology algorithm, Needleman & Wunsch, J. Mo.
l. Biol. 48: 443 (1970) homology alignment algorithm, Pearson & Lipman, Proc.
Natl. Acad. Sci. USA 85: 2444 (1988), similarity search method (both of which are incorporated herein by reference), computerized methods of the above algorithm (Geneti
cs Computer Group, 575 Science Dr., Wisconsin Genetics Sof at Madison, WI
GAP, BESTFIT, FASTA and TFASTA of tware Package Release 7.0; all of which are incorporated herein by reference), or optimal juxtaposition (ie, comparison window) that can be performed by inspection and produced by various methods Which result in the highest overall sequence similarity).

The term “sequence identity”, as used herein, is that the sequences of two polynucleotides or proteins are identical throughout the comparison window (ie, nucleotide-nucleotide or residue-residue). On a group basis). The term "sequence identity ratio" refers to comparing two optimally aligned sequences over a comparison window, the same nucleobase (eg, A, T, C, G, U or I) or amino acid residue Determining the number of positions that occur between the two sequences yielding the number of matched positions, dividing the number of matched positions by the total number of positions in the comparison window (ie, the size of the window),
And the result is multiplied by 100 to obtain the percent sequence identity ratio.

The term “substantial identity” as used herein refers to a comparison window of at least 20 nucleotide or amino acid positions, often at least 25-50
Two polynucleotides that share at least 85% sequence identity, preferably at least 90-95% sequence identity, and sometimes 99% or more sequence identity over the entire nucleotide or amino acid window A sequence property of amino acids, where the sequence identity ratio is determined by comparing a reference sequence to a reference sequence which may include a deletion or addition of 20% or less of the reference sequence over the entire comparison window. Is calculated. The reference sequence can be a subset of a larger sequence.

As applied to proteins and structural elements within proteins, the term “sequence identity” means that the sequences share one or more identical amino acids at corresponding positions. . The term “sequence similarity” means that two sequences share one or more conservatively related amino acids at corresponding positions, eg, by conservative substitutions. The term "substantial sequence identity" refers to 2
At least 85% sequence identity, preferably at least 90% sequence identity, more preferably at least 95% when the species peptide sequences are optimally aligned, such as by the GAP or BESTFIT programs using default gap weights Or more (eg, 99%) sequence identity. The term "substantial similarity" means that two peptide sequences share a ratio corresponding to sequence similarity.

[0068] Conservative sequence relatedness also exists when the amino acid residues at corresponding positions between the two sequences are not identical, but differ in conservative amino structure relatedness. In this context, conservative amino acid substitutions refer to the general interchangeability of amino acid residues having similar side chains. For example, the group of amino acids having an aliphatic side chain is glycine, alanine, valine, leucine and isoleucine; the group of amino acids having an aliphatic-hydroxyl side chain is serine and threonine; The group of amino acids is asparagine and glutamine; the group of amino acids having aromatic side chains is phenylalanine, tyrosine and tryptophan; the group of amino acids having basic side groups is lysine, arginine and histidine; The group of amino acids having side chains is cysteine and methionine. Preferred groups of conservative amino acid substitutions are: valine-leucine-isoleucine, phenylalanine-
Tyrosine, lysine-arginine, alanine-valine, and asparagine-glutamine. Twenty stereoisomers of conventional amino acids (eg, D-amino acids), unnatural amino acids such as α, α-substituted amino acids, N-alkyl amino acids, lactic acid, and other non-conventional amino acids are also included in the present invention. It can be a suitable component for a polypeptide. Examples of non-conventional amino acids include: 4-hydroxyproline, γ-carboxyglutamate, ε-N, N, N-
Trimethyllysine, ε-N-acetyllysine, O-phosphoserine, N-acetylserine, N-formylmethionine, 3-methylhistidine, 5-hydroxylysine, ω-N-methylarginine, and other similar amino acids and imino acids ( For example, 4-hydroxyproline). In addition, amino acids can be modified by glycosylation, phosphorylation, and the like.

To facilitate the practice of the above aspects of the invention, reference is made to the known molecular phylogeny of viral proteins conserved in the order of the mononegavirus. In this regard, extensive research has provided an accurate assessment of protein relevance at the molecular level. These studies include detailed sequence comparisons of conserved proteins between mononegaviruses, and broad acceptance of conserved structural domains, sequence elements, and constrained, isolated residues within these proteins Map has been brought. Each of these conserved protein domains, sequence elements, and tethered residues are useful for conservatively introducing an attenuating mutation from a heterologous attenuated mutant virus into a different recombinant virus by the methods of the present invention. Providing useful targets facilitates the practice of the present invention.

For example, Poch et al., J. Gen. Virol. 5: 1153-62, (1990) (incorporated herein by reference) include rhabdoviruses (VSV and RaV) and paramyxoviruses. Detailed comparisons of deduced amino acid sequences are provided for the five L proteins in the mononegavirus, including the L proteins of (SeV, NDV and MeV). Conventional juxtaposition methods (incorporated herein by reference) indicate that these L proteins from various virions have a high degree of homology along most of their lengths--strongly invariant amino acids Is embedded in a storage block separated by a relatively well-preserved area. Thus, these different L proteins from heterologous (-) strand RNA viruses have a conserved structure of linked functional domains. For example, the L protein contains a highly conserved central region that is thought to contain the active site for RNA synthesis. Other conserved structural domains, motifs and sequence elements, including stringent conserved isolated residues, have also been identified, some of which are distributed around the conserved central core and are considered important for polymerase activity Have been. These conserved sequence elements identified in the L protein (Poch et al., J. Gen. Virol. 5: 1153-62, (1990) and Po
ch et al., EMBO J. 8 (12) 3867-3874, (1989), especially see FIG. 1) shows that an attenuating mutation was produced and compared to the mapped heterologous parental L protein sequence. Provide detailed information for Thus, using the above and other sequencing methods and data, including those provided in the publications incorporated herein, the residues characterizing the attenuating mutation were identified. The target virus L
Whether it has changed from the identity of the parent, eg, wild-type, conserved at the corresponding amino acid position in the protein (ie, identical or represented by conservatively related amino acid residues) Can be easily determined. This determination indicates a high probability of successful integration of the attenuating mutation identified in the heterologous virus into the antigenome or genome of the recombinant target virus.

Further details regarding structural conservation between the various L protein homologs throughout the mononegavirus can be found in Stec et al., Virology 183, which is incorporated herein by reference.
: 273-287 (1991). This sequence analysis shows three types of paramyxovirus,
Two species from the genus Respirovirus (PIV3 and SeV) and one from the genus Rubravirus (N
DV), one paramyxovirus from the genus Morbillivirus (MeV), and two viruses from the Rhabdoviridae family (RaV and VSV) based on published sequences of L genes and proteins ( For example, Schubert et al., 1985; Shioda
et al., 1986; Yusoff et al., 1987; Blumberg et al., 1988; Galinski et al
, 1988; and Teart et al., 1988. All of which are incorporated herein by reference), provide additional sequence information and alignment results for RSV. The above results confirm the teachings of Poch et al., Supra, for significant sequence conservation within the Paramyxovirus and Rhabdoviridae families, and identify additional conserved structural domains, motifs and sequence elements.

Briefly, the disclosure of Stec et al. Relies on conventional juxtaposition, which is useful in the present invention, among other similar methods. In particular, Stec et al. Reported that the penalties for 12 and 6 deletions and gaps, respectively, and Dayhoff et al., "Atlas of P
rotein Sequence and Structure "(MO Dayhoff, Ed.), Vol. 5, Suppl. 3, p
Accepted Wilbur and Lipman, Proc using the similarity scoring matrix of p. 345-352, Natl. Biomed. Res. Found., Silver Spring, MD (1978).
Natl. Acad. Sci. USA 80: 726-730 (1983).
The strand RNA viral sequences were aligned (both of which are incorporated herein by reference). The similarity scoring system uses the RSVs listed in Table 1 and other (-)
It was used to construct a paired global dot matrix alignment between the L proteins of the strand RNA virus. By the above method, a distinct sequence-related region was detected between the RSV L protein and the distantly related L proteins of paramyxovirus and rhabdovirus. As shown in FIG. 3, the sequence-related regions of the RSV L protein and each of the others are linearly aligned in the same order, concentrated in the amino-proximal region, and representing about 1/5 of each molecule. Identical sequence similarity patterns were obtained from other paramyxovirus and rhabdovirus L, including five group juxtapositions of the SeV, MeV, NDV, RaV and VSV L proteins performed by Poch et al. (1989), supra. Was found in the former juxtaposition of proteins (B
lumberg et al., 1988, Galinski et al., 1988; Teart et al., 1988, both of which are incorporated herein by reference).

In the 7 group juxtaposition of Stec et al., Supra, the RSV L protein had an amino-terminal extension of about 70 amino acids and an amino-terminal about 100 aminocarboxy termini compared to other (-) strand RNA viruses. It was determined to contain a piece. However, this change does not affect the reliability of the juxtaposition method for identifying conservative structural elements, and the RSV L protein overlaps the upstream neighboring M2 gene (formerly called 22K). It is believed to be due to the fact that it is partially encoded by a gene (Collins et al., 1987).
, Incorporated herein by reference).

The disclosure of Stec et al., Supra, confirms the presence of a large number of short segments in the L protein of a heterologous (-) stranded RNA virus that are almost exactly conserved among the various taxa. These nearly identical segments are conserved in the RSV L protein and are highly conserved regions shown in FIG. 4 of Stec et al. (See boxed sequences incorporated herein by reference). It is also shown that it exists in. The identity of the amino acids in these six segments ranges from 30 to
80% different. Numerous residues within the identified segments are constant among the seven different (-) strand RNA viruses subjected to the alignment. Further, it is noted that when substitutions occur within these conserved segments, many are characterized by conservative substitutions. As noted previously, the high degree of identity of these segments between each member of the two viridae suggests that they are important for L protein function.

In more specific details, the most highly conserved region of the L protein juxtaposed among the various taxa within the mononegavirus in FIG. 4 of Stec et al.
al., (1989), also contains four characteristic polymerase motifs (underlined and designated AD) that correspond to the motifs identified by Id. These regions homologous to the four polymerase motifs were identified in the RSV L protein between amino acids 696-887. Three of the four motifs (AC) correspond to the highly conserved segments of the L proteins of the paramyxovirus and rhabdovirus. Although the D segment is poorly conserved among these viruses, the single invariant lysine and conserved glycine typical of this element (RSV K-886 and G-87, respectively)
Contains 7).

Similarly detailed sequence alignments and analyzes of other proteins that are shared between (−) strand RNA viruses and that are useful as target proteins of the present invention have been published. In this regard, Barr et al., J. et al., Incorporated herein by reference.
Gen. Virol. 72: 677-685 (1991) describes N among heterogeneous members of the mononegavirus.
The sequence conservation of the protein was analyzed. In particular, this study showed that there was a high level of amino acid identity (60%) between the predicted amino acid sequences of the N proteins of RSV and PVM (see FIG. 7, incorporated herein by reference). Thing). Amino acid residue 1
150150 and 150-393 contain 38% and 74% identity, respectively, whereas residues 245-
315 contains 68 of 71 identical amino acids (96% identity). This high conservation of these proteins is consistent with the observation that the N proteins of RSV and PVM are serologically related (Gimenez et al., 1984; Ling & Pringle, 1989).
).

A computer matrix comparison of the amino acid sequences of the N proteins of PVM and the more diverse members of the mononegavirus also reveals conserved regions (Barr et al.,
Id., Fig. 5). Sequence conservation in this context extends between the PVM and the N protein of the Ebola virus. Furthermore, the hydropathy profile of N proteins from members of a broad group of non-segmented (-) chain viruses
Are similar to each other in the region of greatest sequence similarity between paramyxoviruses and mononegaviruses (Galinski et al., 1985, incorporated herein by reference). Approximately 180 amino acids, starting at 130-170 residues from the amino terminus of each protein, form alternating hydrophobic and hydrophilic regions. Secondary structure predictions for these amino acid sequences (Garnier et al., 1978, incorporated herein by reference) indicate that this conserved hydropathic region begins with a high proportion of α-helices and a high proportion of β-helix. -Indicates that you may end up with a sheet and a reverse turn. The above data suggest that these conserved proteins may have similar folds across similar hydropathic regions.

Further juxtaposition studies on representative N proteins concentrated in the conserved hydropathic region using the famous CLUSTAL program (Higging & Sharp, Gene 33: 237-2
44 (1988), which is incorporated herein by reference), further points out additional conserved protein domains, structural motifs, and isolated conserved amino acid residues (
See Barr et al., FIG. 7, boxes A, B and C). From this research, SeV, VS
Conservation between the V and N proteins of PVM was found to be particularly high in the defined region of the carboxy terminus of the PVM sequence (Figure 7, box C). This region was also identified in a DIAGON comparison of Ebola virus and PVM using a window less than 99. Within these sequences, two short similar regions were also detected. Both contain hydrophobic regions interrupted by a single conserved basic amino acid (K or R; boxes A and B in FIG. 7).
. In the corresponding region there are similar gaps with the N proteins of other paramyxoviruses and rhabdoviruses. The level of identity in these regions was determined by Barr et al.
Table 1 of. Although the highest identity ratios are found in certain virions, these regions between virions also show a high level of similarity, especially when conservative substitutions are considered.

Facilitating the practice of the present invention is a separate published juxtaposition that confirms a high degree of molecular conservation between heterologous members of the mononegavirus. For example, Parks et al., Virus Res. 22: 259-279, (1992), incorporated herein by reference, discloses a computer-assisted alignment of the amino acid sequences of N proteins from ten different paramyxoviruses. Will be described. As shown in Figure 3 of reference, (residues 323-340 of SV5) characteristic region in the vicinity of the center of the N protein is between the N protein of different taxa, invariant hydrophobic motif FX ₄ - Contains a wide range of sequence conservation including YX ₄ -SYAMG, where X is any residue. The second highly conserved domain, rich in the negatively charged amino acids glutamate and aspartate, is the C protein of the N protein.
The terminal region was identified (SV5 residues 455-469).

Another study on the molecular conservation of the N protein, Miyahara et al., Arch. Virol. 124: 255-268 (1992), which is incorporated herein by reference, describes HPIV-1
The amino acid sequence of the N protein of HPIV-2 (Yua
sa et al., Virology 179: 777-784 (1990)); HPIV3 (Galinski et al., Virol
ogy 149: 139-151 (1986)), HPIV-4A and 4B (Kondo et al., Virology 174: 1-
8 (1990)), SV5 and SV41 (Tsurudome et al., J. Gen. Virol. 72: 2282-2292).
(1991)), MuV (Elango, Virus Res. 12: 77-86 (1989)), SeV (Morgan et al.
, Virology 135: 279-287 (1984)), PIV-3 (Sakai et al., Nucleic Acids Res.
&& 2927-2944 (1987)), NDV (Ishida et al., Nucleic Acids Res. 14: 6551
-6564 (1986)), MeV, and CDV (Rozenblatt et al., J. Virol. 53: 684-690 (
1985)) (the above references are each incorporated herein by reference).

As shown in FIG. 3 of Miyahara et al., The N gene of HPIV1 shows extensive homology to SeV; nucleotide and amino acid identities were 70.8% and 87.8%, respectively.
%Met. The N protein of HPIV-1 also showed high amino acid identity with HPIV-3 (63.1%) and BPIV-3 (63.3%), but NDV (20.9%), MeV (20.5%), CDV (19.6%
), SV41 (18.6%), SV5 (17.9%), HPIV-4A (17.9%), HPIV-4B (17.5%)
, HPIV-2 (11.5%) and MuV (17.1%) showed low identity. A protease-sensitive "hinge" was found at the two defined domain junctions of the SeV K protein; (1) an amino-terminal domain that interacts directly with RNA; and (2) located on the surface of the assembled nucleocapsid Carboxyl-terminal domain (Heggenes
s et al., Virology 114: 555-562 (1981), which is incorporated herein by reference).

As described in detail in Miyahara et al., 26 amino acids are conserved in the N protein of any paramyxovirus and a conserved N domain is identified for the N protein between amino acids 260-360. Was done. In addition, HPIV-1 and SV N proteins conserve 37 of 40 glycines and 13 of 13 prolines, and these SeV proteins maintain a common tertiary structure. It suggests.

Miyahara also compared the M protein sequence between the HPIV-1 M protein and 13 other paramyxoviruses (see, eg, Miyahara et al., FIG. 4). These heterologous viruses also showed a high degree of structural conservation of the M protein sequence element. For example, HPIV-1 and SeV showed 72.6% and 88.4% nucleotide and amino acid identity levels for M, respectively. HPIV-1 also has high levels of storage with HPIV-3 (65.7%) and BPIV-3 (65.4%), MeV (36.4%), CDV (34.6%) and RP
V (36.7%) showed moderate storage stability. All of the five cysteines of HPIV-1, 20 of the 22 prolines, and 24 of the 25 glycines were conserved in SeV, and almost all of them were also conserved in PIV-3. The above findings indicate that the tertiary structure of the M protein is H
Indicates that it may be stored in PIV-1, SeV, HPIV-3 and BPIV-1. In addition, 14 amino acids were conserved in the M proteins of all paramyxoviruses compared.

Additional sequence alignments and analyzes have been published for the P protein, which is also a universally conserved target in mononegaviruses and is therefore a particularly useful target for heterologous introduction of mutations in the present invention. See, for example, Kondo et al., Virology 178: 321-326 (1990), which is incorporated herein by reference. In this context, SeV (Giorgi et al., Cell 35: 82-836 (1983); Neubert, Nucleic A
cids Res. 17: 10-101 (1989), which is incorporated herein by reference) and PIV
3 (Luk et al., Virology 153: 318-325 (1986), incorporated herein by reference) are similar in size, 568 and 603, respectively.
Consists of amino acids. P in various taxa, such as MuV, SV5, PIV-2 and NDV
The gene encodes a protein containing 391, 392, 395 and 395 amino acids, respectively (Takeuchi et al., J. Gen. Virol. 69: 2043-2049 (1988); Thom
as et al., Cell 54: 891-902 (1988); and Sato et al., Virus Res. 7: 241-
255 (1987)). The MeV and CDV P proteins are intermediate in size (Bar
ret et al., Virus Res. 3: 367-372 (1985); Bellini et al., J. Virol. 53:
908-919 (1985)).

The P-specific regions of all heterologous viruses compared by Kondo et al., Virology 178: 321-326 (1990) are juxtaposed by the conventional method employed, which is incorporated herein by reference. Can be done. PIV-4A, PIV-4B, SV5, PIV-2, MuV, NDV, MeV, C
Characteristic conservative structural elements were identified among DV, PIV-3 and SeV (see FIG. 2 of the Kondo paper, which is incorporated herein by reference). Thus, this and other published studies on the molecular phylogeny of the P protein suggest that mutation sites may be used to assess the target of incorporation of an attenuating mutation from a heterologous mutant virus into a recombinant virus strain. Identify additional conserved protein domains, structural motifs, amino acid segments and isolated residues for juxtaposition.

Additional sequence alignments and analyzes that facilitate the practice of the present invention as applied to the full range of proteins represented within the mononegavirus are also provided. Briefly, Yuasa et al., Virology 179: 777-784 (1990), incorporated herein by reference, describes human and non-human parainfluenza viruses, PIV-4A, PIV-4B, MuV, NDV. , Me
Identify structural elements conserved in the 3 'end of the V, PIV-3, BPIV-3, SeV and RSV genes and the N gene (see, eg, FIGS. 2 and 4). Kawano et al., Virology 174: 308-3
13 (1990) provide exemplary alignment and molecular analysis of HN proteins for eight heterologous paramyxoviruses (see, eg, FIG. 2). Tsurudome et al.,
J. Gen. Virol. 72: 2282-2292 (1991) provides an exemplary alignment and analysis for HPIV-2, SV5 and SV41 N proteins (see, eg, FIG. 3). Spr
iggs et al., 1986, Virology 152: 241-251 (1986) identify elements of the F protein that are structurally conserved in heterologous paramyxoviruses, including RSV. Higuchi et
al., J. Gen. Virol. 73: 1005-1010 (1992) (see, for example, FIG. 4), Kawano et a
l., Nuc. Acids Res. 19 (10): 2739-2746 (1991) (see, for example, FIGS. 2 and 6), M
uhlberger et al, Virology 187: 534-547 (1992) (see, eg, FIGS. 4 and 6) and Ogawa et al., J. Gen. Virol. 73: 2743-2750 (1992) (see, eg, FIG. 3).
Are for the L protein and the 3 'and 5' non-coding genomic ends between the large group of mononegaviruses. N, P, C, L, M, H in mononegavirus
A more comprehensive review, including N, F, SH, V, D, and additional references citing molecular conservation between additional ORFs and gene products, can be found in Collins et al., Fields Virology, Fi
elds et al. eds., 3rd edition, Chapter 41: 1205-1241, Lippincott-Raven,
Provided by Philadelphia (1996). All of the above studies are hereby incorporated by reference and describe the construction of a conserved structural element at a defined position representing a target site for incorporating an attenuating mutation in a recombinant vaccine virus according to the teachings of the present invention. Includes identifying juxtapositions and charts.

In a more detailed aspect of the invention, the recombinant (−) strand RNA virus attenuated by the introduction of a mutation identified in a heterologous virus is designed as a chimeric virus, eg, a chimeric RSV or PIV virus. . The chimeric (-) chain virus of the present invention is recombinantly designed to incorporate nucleotide sequences from one or more virus strains or subgroups producing infectious chimeric viruses or subviral particles. In this way, candidate vaccine viruses are recombinantly designed to elicit an immune response in a mammalian host, including humans and non-human primates. A chimeric virus according to the present invention may elicit an immune response against a particular virus subgroup or strain, or elicit a multispecific response against multiple virus subgroups or strains.

In an exemplary embodiment, the chimeric virus that incorporates an attenuating mutation as described above is also a subject virus, eg, by replacing a paired sequence from a heterologous RSV to produce a chimeric RSV genome or antigenome. May have heterologous genes or gene segments of a heterologous virus that is added to or incorporated into a recombinant genome or antigenome. In this way, the chimeric viruses of the present invention may include a portion or part of a virus strain or subgroup virus from one virus strain or subgroup virus in combination with an additional or replacement "donor" gene or gene segment of a different virus strain or subgroup. The entire "recipient" viral genome or antigenome is included.

In a preferred aspect of the invention, the chimeric attenuated virus is a different human RSV A or B
RSV consisting of the genome or antigenome of part or all of the human RSV A or B subgroup in complex with a heterologous gene or gene segment from a subgroup virus. To produce this recombinant virus, a heterologous donor gene or gene segment from one RSV strain or subgroup is combined with a recipient genome or antigenome that serves as an insertion or additional backbone for the donor gene or gene segment, Or replace with it. Thus, the recipient genome or antigenome acts as a vector to transfer and express heterologous genes or gene segments, producing chimeric RSV that exhibits novel structural and / or phenotypic characteristics. Preferably, the addition or replacement of the heterologous gene or gene segment in the selected recipient RSV strain comprises:
It produces a novel phenotypic effect compared to the corresponding phenotype of the unmodified recipient and / or donor, such as attenuation, growth changes, immunogenicity changes, or other desired phenotypic changes.

A representative, attenuated chimeric RSV that incorporates both human RSV B subgroup glycoprotein genes F and G, replacing the paired F and G glycoprotein genes in the RSV A genome, has been developed and characterized It has been attached. This representative chimera has (i) Gln ₈₃₁ to Leu and
Panel of mutations specifying a temperature-sensitive amino acid substitution of Tyr ₁₃₂₁ to Asn;
(Ii) temperature-sensitive nucleotide substitutions in the gene initiation sequence of the M2 gene; and (iii) substitution of Val ₂₆₇ for Ile in the RSV N gene, and substitution of Cys ₃₁₉ for Tyr and His _{1690 for} the RSV polymerase gene L. A panel of attenuating mutations taken from cold-passage RSV identifying amino acid substitutions for substitutions to Tyr; or (iv) further modified to incorporate an attenuating point mutation selected from deletion of the SH gene (See, eg, US patent application Ser. No. 09 / 291,894). Preferably, the above and other examples of chimeric viruses incorporate at least two attenuating mutations that can be derived from the same or different mutant viruses.

US Patent Application No. 09 / 083,793, filed May 22, 1998 (WO 98/53)
HPIV-1 and HPIV-3, as described in priority provisional patent application No. 06 / 047,575, filed May 23, 1997, both of which are incorporated herein by reference.
Representative, attenuated chimeric PIVs have also been developed and characterized that incorporate the heterologous sequence from, as well as the attenuating mutation taken from the PIV3 mutant, cp45. More recently, all of the attenuating mutations identified in cp45, with the exception of those in the F protein, have been successfully incorporated in attenuated recombinant HPIV-3 chimeras.

Chimeric (−) by introducing heterologous immunogenic proteins, domains and epitopes
Producing a strand RNA virus is particularly useful for producing a novel immune response in an immunized host. By adding or replacing an immunogenic gene or gene segment from one donor virus subgroup or strain within the recipient genome or antigenome of a different virus subgroup or strain, the donor subgroup or strain, recipe An immune response directed against a subgroup or strain of the ent, or both the donor and the recipient, can be generated. To this end, chimeric proteins, e.g., those expressing an immunogenic protein having a cytoplasmic tail and / or transmembrane domain specific for one virus strain or subgroup fused to the ectodomain of a different virus Viruses can also be constructed. Other exemplary recombinants of this type may express the same protein region, such as the same immunogenic region.

It is often useful to add or replace entire genes (including cis-acting elements and coding regions) in the chimeric or antigenome, but to introduce only a portion of the donor gene of interest. It is also useful. Most commonly, non-coding nucleotides such as cis-acting regulatory elements and intragenic sequences need not be introduced with the donor gene coding region. In addition, a wide variety of gene segments provide useful donor polynucleotides contained within a chimeric genome or antigenome that express chimeric viruses with novel and useful properties. Thus, a heterologous gene segment advantageously contains the cytoplasmic tail, transmembrane domain or ectodomain, epitope site or region, binding site or region, active site or active site of a selected protein from one virus. Regions, etc. may be coded. The above and other gene segments may be added or substituted for another viral countergene segment, resulting in new chimeric recombinants, such as the cytoplasmic tail of one viral glycoprotein fused to the ectodomain of another viral glycoprotein and And / or producing a recombinant that expresses a chimeric protein having a transmembrane domain. Genomic segments useful in this regard include small functional domains of proteins, such as from about 15-35 nucleotides for gene segments encoding epitope sites, to about 50, for gene segments encoding larger domains or protein regions. It ranges from 75, 100, 200-500 and 500-1500 or more nucleotides.

To construct a chimeric virus carrying the introduced attenuating mutation, all or part of the heterologous gene is added or substituted to the background genome or antigenome to form the chimeric genome or antigenome. I can do it. The mutation may be present with one or more additional mutations in the heterologous gene (ie, the donor gene) or gene segment, or may be partially or completely in the recipient antigenome or genomic "background". ". In the case of chimeras produced by substitution, a selected protein or protein region from a virus (eg, cytoplasmic tail, transmembrane domain or ectodomain, epitope site or region, binding site or region, active site or active site containing region) , Etc.) are replaced for paired genes or gene segments in different viral genomes or antigenomes, producing new recombinants with the desired phenotypic change compared to wild-type or parental strains. As used herein, a "pair" gene,
A gene segment, protein or protein region refers to two paired polynucleotides from different sources, including different genes of a single species or strain, including species and allelic variants between different virus subgroups or strains, or the same. Different variants of the gene are included.

[0095] Paired genes and gene segments typically share at least moderate structural similarity. For example, a pair of gene segments can encode a common structural domain of the protein of interest, such as a cytoplasmic domain, transmembrane domain, ectodomain, binding site or region, epitope site or region, and the like. Typically, they also share a common biological function. For example, protein domains encoded by paired gene segments can provide common membrane spanning functions, specific binding activities, immunological recognition sites, and the like. The paired genes and gene segments used to construct the attenuated chimeric virus of the present invention encompass a large number of exchangeable species having a wide range of size and sequence variations. However, the choice of paired genes and gene segments depends on the substantial sequence identity between the subject controls, as defined above. In this context, the reference sequence of the selected polynucleotide is
A sequence or portion of a sequence present in either the genome or antigenome of the donor or recipient. This reference sequence is used as the reference sequence and provides the basis for sequence comparison. For example, a reference sequence can be a defined segment of a cDNA or gene, or a complete cDNA or gene sequence.

[0096] Well-known cDNA-based methods are useful for constructing large panels of recombinant chimeric viruses and subviral particles that incorporate attenuating mutations identified in heterologous viruses. These recombinant constructs offer improved attenuation and immunogenic characteristics for use as vaccines. Among the phenotypic changes desired in this context are resistance to reversion from the attenuated phenotype, improved attenuation in culture or in a selected host environment, (eg, an enhanced immune response or There are immunogenic characteristics (as determined by elimination), up-regulation or down-regulation of transcription and / or translation of the selected viral product, and the like.

In an additional aspect of the invention, an attenuated recombinant virus that incorporates an attenuating mutation identified in a heterologous virus comprises one or more additional attenuators that specify an altered attenuation phenotype. It is further modified by introducing mutations.
The above mutations are produced de novo and can be tested for attenuating effects by rational design of mutagenesis strategies. In another approach, an attenuating point mutation is identified in a biologically induced mutation, such as a RSV or PIV ts or cp mutant, which is then incorporated into an attenuated recombinant virus of the invention. Recombinants that are further attenuated in this way can be chimeric viruses.

Further attenuating mutations in biologically-derived RSV for incorporation into vaccine strains can occur naturally or can be obtained by known mutagenesis methods described above, and It can be introduced into a wild strain in USSN 08 / 327,263, incorporated herein.

“Biologically derived” mutant refers to a mutant virus that is not produced by recombinant means. Thus, the biologically derived virus can be any (
-) Strand RNA virus species, subgroup or strain, eg, a naturally occurring RSV or PIV with a genomic sequence of the mutation, or a genomic mutation from a reference wild-type sequence (eg, identifying an attenuation phenotype) RSV or PIV). Similarly, biologically derived mutants include mutants derived from the parent strain, particularly by artificial mutagenesis and selection methods.

[0100] Additional attenuating mutations identified as above are compiled into the "Menu"
The candidate vaccine virus, introduced alone or in combination, as desired, is adjusted to the desired suitable level of attenuation, immunogenicity, genetic resistance to reversion from the attenuated phenotype, and the like. Preferably, the recombinant mutant virus of the present invention is attenuated by incorporation of at least one, and more preferably, two or more attenuating point mutations identified from a menu such as: : For example, cpts
RSV 248 (ATCC VR 2450), cpts RSV 248/404 (ATCC VR 2454), cpts RSV 24
8/955 (ATCC VR 2453), cpts RSV 530 (ATCC VR 2452), cpts RSV 530/1009
(ATCC VR 2451), cpts RSV 530/1030 (ATCC VR 2455), RSV B-1 cp52 / 2B5
(ATCC VR 2542) and a panel of RSV mutants such as RSV B-1 cp-23 (ATCC VR 2579). Additional mutations include, for example, small plaque (sp), cold adapted (ca
) Or non-specific or heterologous viruses, including those with ts, cp, or non-ts or non-cp attenuating mutations, as identified in host range restriction (hr) mutants. The attenuating mutation is the coding portion of the gene, or
It can be selected in non-coding regions such as cis regulatory sequences. For example, an attenuating mutation can include a single or multiple base change in the gene start sequence, as exemplified by a single base substitution at nucleotide 7605 in the RSV M2 gene start sequence. Thus, attenuation of a recombinant vaccine candidate may be fine-tuned for use in one or more patient classes, including seronegative infants. c
The ability to produce virus from DNA allows the above mutations to be routinely introduced into full-length cDNA clones, either individually or in variously selected combinations, after which the introduced suddenly The phenotype of the recovered recombinant virus containing the mutation can be easily determined.

By identifying a specific attenuating mutation associated with a desired phenotype, for example, the cp or ts phenotype, and incorporating it into an infectious recombinant clone, the present invention provides a method for identifying where a mutation is identified. , Or in the immediate vicinity thereof, provide other site-specific modifications. Whereas most attenuating mutations produced in biologically derived viruses are single amino acid substitutions, other "site-specific" mutations also It can be incorporated into a recombinant virus. As used herein, site-specific mutations include 1-3, about 5-15 or more changes (
For example, insertion, substitution, deletion or rearrangement of nucleotides (changed from the wild-type sequence, from the sequence of the selected mutant strain, or from the parent recombinant clone, by mutagenesis). Such site-specific mutations can be incorporated either at the selected attenuating mutation or within the region. In another approach, the mutation may be in various other contexts within the viral clone, such as a cis-acting regulatory sequence, or a nucleotide sequence encoding the active site, binding site, immunogenic epitope, etc. Can be introduced nearby. Site-specific virus mutants typically retain the desired attenuated phenotype, but have a substantially altered phenotypic trait that is independent of attenuation, e.g., enhanced or expanded. Immunogenicity,
Or may show improved growth. Further examples of desired site-specific mutants include recombinant viruses that incorporate additional, stabilizing nucleotide mutations at codons that specify attenuating mutations. Where possible, introduce two or more nucleotide substitutions at the codon that identifies the attenuating amino acid change in the parent mutant or recombinant clone and genetically resist reversion from the attenuated phenotype Obtain the recombinant. In other embodiments, site-specific nucleotide substitutions, additions, deletions, or rearrangements are made up to, for example, 1-3, 5-10, and 15 nucleotides relative to the targeted nucleotide position (encoded viral protein). N-terminal direction) or downstream (C-terminal direction), or even 5 ′ or 3 ′, eg, to construct or remove existing cis-acting regulatory elements.

In addition to single and multiple point and site-directed mutations, changes to the attenuated recombinant virus of the present invention include insertion, replacement or rearrangement of an entire gene or gene segment. The mutations described above may, depending on the type of change, be made from a small number of bases (eg, 15-30 bases, 35-
Up to 50 bases or more) or large nucleotide blocks (e.g., 50-
100, 100-300, 300-500, 500-1000 bases) (i.e., a small number of bases are altered to insert or remove immunogenic epitopes, or to alter small gene segments). Whereas large blocks of bases are involved when genes or large gene segments are added, substituted or rearranged).

In an additional aspect, the invention relates to an additional type of mutation involving the same or a different gene of a recombinant virus to be further modified, wherein the mutation is introduced into a recombinant (−) strand viral cDNA from a heterologous virus. Mutations (eg, cp and ts mutations) are supplemented. In this regard, the viral proteins can be selectively altered with respect to expression levels, or alone or with other desired modifications, in whole or in part, additions,
It can be deleted, replaced, producing a recombinant attenuated virus with additional novel vaccine characteristics.

Thus, in addition to, or in combination with, attenuating mutations taken from heterologous virus mutants, the present invention also provides for attenuating recombinant vaccine candidates based on recombinant engineering, Or a range of additional methods for modifying in other ways. According to this aspect of the invention, a wide variety of alterations can be produced in the isolated polynucleotide sequence encoding the recombinant genome or antigenome. More specifically, in order to achieve the desired structural and phenotypic changes in the recombinant PIV, the present invention provides for deletion, substitution, introduction, or deletion of selected nucleotides or nucleotides from the parent genome or antigenome. Or the modification to be rearranged, as well as the introduction of mutations that delete, replace, introduce or rearrange an entire gene or gene segment in the parent genome or antigenome.

The desired modification of the attenuated recombinant virus typically results in a desired phenotypic change, eg, a change in virus growth, temperature sensitivity, the ability to elicit a host immune response, attenuation, etc. Is selected to identify Such alterations may be, for example, gene segments encoding specific genes or gene regions (eg, structural domains of proteins such as cytoplasmic, transmembrane or extracellular domains, immunogenic epitopes, binding regions, active sites, etc.). Mutagenesis of the parent clone, which removes, introduces or rearranges, can occur in either the donor or recipient genome or antigenome. Genes of interest in this regard include any viral gene, for example, 3'-NS1-NS2-NPM-SH-GF-M2-L-5 'for RSV, as well as heterologous genes from other viruses. It is.

Also provided are, for example, introducing a stop codon into the selected RSV coding sequence, changing the position of the gene relative to an operably linked promoter, starting codons upstream To change the expression rate by introducing
Modification of the transcription signal (eg, by repositioning, altering an existing sequence, or replacing an existing sequence with a heterologous sequence) results in a phenotype (eg, growth, temperature restriction to transcription, etc.). Selected by alteration and other deletions, substitutions, additions and rearrangements that identify quantitative or qualitative changes in viral replication, transcription of selected genes, or translation of selected proteins. A modification in a recombinant vaccine candidate that alters or limits the expression of a gene.

The ability to analyze other types of attenuating mutations and incorporate them into recombinant vaccine candidates extends to a wide range of targeted changes in recombinant clones. For example, RS
Deletion of the SH gene in V results in a recombinant RSV with novel phenotypic characteristics, including enhanced proliferation. Thus, in the recombinant RSV of the present invention, the deletion of the SH gene (or the deletion of other non-essential genes or gene segments) can be used to identify one or more attenuating phenotypes in the recombinant virus. One or more additional mutations (eg, one derived from a heterologous virus, optionally supplemented by one or more additional attenuating mutations derived from a biologically-derived attenuating mutant) Or more point mutations). In some embodiments, the RSV SH or NS2 gene is one or more cp and / or ts aborted from cpts248 / 404, cpts530 / 1009, cpts530 / 1030, or other selected mutant RSV strain. Deleted with the mutation, the combined effect of the various mutations produces a recombinant RSV with increased virus yield, enhanced attenuation, and resistance to phenotypic reversion.

For one representative SH (-) RSV clone, the modified viral genome was 14,82
5 nt long, 398 nucleotides less than wild type. By designing similar mutations that reduce genome size in other coding or non-coding regions everywhere in the RSV genome, such as the P, M, F and M2 genes,
The present invention provides several readily available methods and materials for improving the growth of chimeric RSV.

In addition, a wide variety of other genetic alterations can be produced in the recombinant or antigenome for incorporation into infectious viruses attenuated by the methods of the present invention.
Additional heterologous genes or gene segments (e.g., from different viral genes, or from different viral strains or types) may be inserted in whole or in part, altering the order of the genes, removing duplication of genes, and removing genomic promoters. It may be replaced with the antigenome pair, a part of the gene removed or replaced, and even the entire gene deleted. Various or additional modifications in the sequence can be made to facilitate various manipulations, such as inserting unique restriction sites in various intragenic regions or anywhere. Untranslated gene sequences can be removed to increase the ability to insert foreign sequences.

Further provided in the present invention are attenuated recombinant vaccine candidates that alter or suppress the expression of a selected gene or gene segment without removing the gene or gene segment from the recombinant clone. Genetic modification in the virus. For example, this may involve introducing a stop codon within the selected coding sequence, changing the location of the gene, or introducing an upstream start codon to alter its rate of expression, or altering the transcription signal. Phenotype (e.g.,
Growth, temperature limits on transcription, etc.).

Preferred mutations in this context include mutations directed against cis-acting signals, which can be identified, for example, by mutational analysis of the viral minigenome. For example, insertion and deletion analysis of leader and trailer and flanking sequences identified viral promoters and transcriptional signals, and provided a line of mutations associated with varying degrees of repression of RNA replication or transcription. Saturation mutagenesis of these cis-acting signals, which in turn modifies each position for each of the nucleotide choices, also inhibits (or increases, in two cases, increases) RNA replication or transcription. Mutations were identified. Any of these mutations can be inserted into a recombinant antigenome or genome, as described herein.

Trans-acting proteins and cis-acting RNs using complete antigenomic cDNA
Evaluating and manipulating A is assisted by the use of the viral minigenome (see, for example, Grosfeld et al., J. Virol. 69: which is incorporated herein by reference:
5677-5686 (1995)), the helper-dependent state of which is so inhibitory that it is useful in characterizing mutants that are not recovered in replication-independent infectious viruses.

Other mutations in the recombinant viruses of the invention include replacing the 3 ′ end of the genome with its pair from the antigenome, which is associated with changes in RNA replication and transcription. I have. In addition, regions within the gene (Collins et al., Proc. Natl.
Acad. Sci. USA 83: 4594-4598 (1986), incorporated herein by reference) can be shortened or lengthened, or altered in sequence content,
Naturally occurring gene duplications (Collins et al., Proc. Natl. Acad. Sci. USA 84:
5134-5138 (1987), incorporated herein by reference) can be removed by the methods described herein or can be changed to a different intragenic region.

In one exemplary embodiment, the RSV protective F and G antigens are substituted by replacing the natural codon usage with one designed to match efficient translation and assembled into a synthetic cDNA. The expression level of a particular protein such as can be increased. In this context, codon usage has been shown to be a major factor in the translational level of mammalian viral proteins (Haas et al., Current Biol. 6: 315-324 (1996).
). Examination of the codon usage of mRNA encoding the primary protective antigen, the RSV F and G proteins, indicates that this usage is consistent with weak expression. Thus, the codon usage can be improved by the recombinant methods of the present invention to achieve improved expression for the selected gene.

In another exemplary embodiment, the sequence surrounding the translation start site (preferably including the nucleotide at position 3) of the selected viral gene is alone or in combination with the introduction of an upstream start codon. Modified to modulate gene expression of the attenuated recombinant virus by specifying up or down regulation of translation.

In a more particular embodiment, expression of the attenuated RSV gene may be modulated by altering the transcribed GS signal of a selected gene of the virus. In one exemplary embodiment, the NS2 GS signal is a defined mutation (eg, 404 as described below).
(M2) mutation) to add to the ts restriction on viral replication. Still additional attenuated RSV clones can incorporate various modifications to the transcribed GE signal. For example, producing RSV clones with GE signals replaced or mutated for the N gene of the NS1 and NS2 genes,
This results in decreased A levels and increased protein expression from downstream genes. The resulting recombinant virus shows increased growth kinetics and increased plaque size, and demonstrates an example in which the modification of cis-acting regulatory elements in the RSV genome alters the growth characteristics of RSV.
Only examples are provided.

In another example, the specific expression of the G protein of the attenuated recombinant RSV is Gm
Increased by RNA modification. The G protein is expressed as both membrane-bound and secreted, the latter form being expressed by translation initiation at the start site within the G translation open reading frame. This secreted form may account for as much as half of the G protein expressed. Elimination of the internal start site (due to sequence changes, deletions, etc.), alone or in altering the sequence context of the upstream start site, results in a desired change in G protein expression. Elimination of the secreted form of G improves the quality of the host immune response to the exemplary chimeric RSV, since the soluble form of G may act as a `` bait '' to capture neutralizing antibodies. is there. Soluble G proteins have also been linked to enhancing immunopathology by preferentially stimulating Th2-biased responses.

In another aspect, the expression level of the attenuated recombinant virus gene is modified at the transcript level. In one aspect, the location of the gene selected in the RSV gene map is
The gene will be changed more proximal to the promoter or more distal to the promoter, so that the gene will be expressed more efficiently or less efficiently, respectively. According to this aspect, modulation of expression for a particular gene is achieved, resulting in a reduction or increase in gene expression by a factor of 2, typically 4 fold, 10 fold or more, relative to wild-type levels . In one example, the NS2 gene of RSV (second order in the RSV gene map) is replaced in position with the SH gene (sixth order), resulting in a predicted decrease in NS2 expression. The change in the selected RSV gene expression due to the change in position is 10
Up to two-fold or more can be achieved, but often with a concomitant decrease in expression levels for repositioned genes.

In another exemplary embodiment, the viral gene, alone or together, is translocated to a site more proximal or more distal to the promoter within the recombinant viral gene map, and Each achieves a level of gene expression. These and other transposition changes produce new clones with an attenuated phenotype, for example, by reducing expression of selected viral proteins involved in RNA replication.

In a more detailed aspect of the invention, the attenuated recombinant virus incorporates expression of a viral gene, eg, the NS2 gene of RSV, eg, with two tandem translation stop codons into the open reading frame of translation (ORF). Thereby further modifying the gene or a segment thereof by suppressing it at the translational level without deleting it. This produces a viable virus in which the selected gene becomes quiescent at the level of translation without deleting the gene. these"
The form of the "knockout" virus shows a reduced growth rate and small plaque size in tissue culture. Thus, the methods and compositions of the present invention provide an additional new type of attenuating mutation that suppresses the expression of viral genes that are neither one of the major viral protective antigens nor essential for viral growth. I will provide a. In this context, "knock-out" produced without deletion of a gene or gene segment
The viral phenotype is selectively produced by deletion mutagenesis, as described herein, to effectively eliminate correctional mutations that may restore target protein synthesis. obtain. Methods for producing these and other knockout viruses are well known in the art (see, for example, Kretzschmar et al., Virolo).
gy 216: 309-316 (1996); Radecke et al., Virology 217: 418-422 (1996); and Kato et al., EMBO J. 16: 178-187 (1987); and Schneider et al., Virol
ogy 277: 314-322 (1996), both of which are incorporated herein by reference).

The infectious recombinant virus clones of the present invention also have enhanced immunogenicity and are provided by infection with a wild-type or parental recombinant virus by the methods and compositions disclosed herein. It can be designed to induce the above protection levels. For example, an immunogenic epitope from a heterologous virus strain or type, eg, PIV, can be added to a recombinant clone, eg, RSV, by appropriate nucleotide changes in the polynucleotide sequence encoding the chimeric genome or antigenome. Alternatively, the virus may be designed to add or remove (eg, by amino acid insertion, substitution or deletion) an immunogenic epitope associated with a desired or undesired immune response.

In the methods of the present invention, additional genes or gene segments can be inserted into or near the recipient's genome or antigenome. These genes can be controlled with the recipient gene or can be under the control of an independent set of transcriptional signals. Genes of interest include cytokines (eg, IL-2 to IL-15, especially I
L-2, IL-6 and IL-12), γ-interferon, and those encoding proteins abundant in helper T cell epitopes. These additional proteins can be expressed either as separate proteins or as chimeras designed from one second copy of a viral protein, such as SH. This provides the ability to quantitatively and qualitatively modify and improve the immune response to the virus.

In an exemplary embodiment of the invention, the insertion of a foreign gene or gene segment, and possibly non-coding nucleotide sequences, into the recombinant viral genome is
This results in the desired increase in genome length, causing additional desired phenotypic effects. Increased genome length results in attenuation of the generated virus, depending in part on the length of the insert. In addition, the expression of certain proteins, eg, cytokines, from heterologous sources in the recombinant attenuated virus of the invention results in further attenuation of the virus by the action of the protein. This has been described for IL-2 expressed in vaccinia virus (eg, Flexner et al.
al., Nature 33: 259-62 (1987)), and γ-interferon is also expected.

Deletions, insertions, substitutions and other mutations involving alterations of whole viral genes or gene segments within the recombinant viruses of the invention produce highly stable vaccine candidates, which are immunosuppressive. This is particularly important in the case of isolated individuals. While many of these changes result in attenuation of the resulting vaccine strain, others identify various types of desired phenotypic changes. For example, certain viral genes are known that encode proteins that specifically interfere with host immunity (see, for example, Kato et al., EMBO J. 16: 578-87 (1997); Which are incorporated herein by reference). Deletion of such genes in the recombinant vaccine virus is expected to suppress virulence and pathogenesis and / or improve immunogenicity.

Additional mutations to further attenuate the recombinant viruses of the present invention include heterologous genes or cis-acting elements that confer preferred host range restrictions and other desired phenotypes for vaccine use. Includes introduction. In representative embodiments, for example, Pastey
et al., J. Gen. Virol. 76: 193-197 (1993); Pastey et al., Virus Res. 29:
195-202 (1993); Zamora et al., J. Gen. Virol. 73: 737-741 (1992); Malli
peddi et al., J. Gen. Virol. 74: 2001-2004 (1993); Mallipeddi et al., J.
Gen. Virol. 73: 2441-2444 (1992); and Zamora et al., Virus Res. 24: 115.
-121 (1992), already known aspects of the structure and function of bovine RSV, as provided in and by the teachings disclosed herein, both of which are incorporated herein by reference. , A bovine RSV sequence for introduction into human RSV is selected. In another aspect of the invention, the mutation of interest for introduction into the chimeric RSV is a non-pathogenic mouse murine pneumovirus (the mouse counterpart of human RSV) that lacks the cytoplasmic tail of the G protein and is compatible with tissue culture. Is modeled according to sex strains (Randh
awa et al., Virology 207: 240-245 (1995)). Thus, in one aspect of the invention, one or more human RSV glycoproteins are provided to achieve the desired attenuation.
The cytoplasmic and / or transmembrane domains of F, G and SH are attenuated using heterologous paired sequences (eg, sequences from the cytoplasmic or transmembrane domains of the F, G or SH proteins of murine pneumovirus). Added, deleted, modified or substituted in the recombinant RSV. As another example, to achieve novel effects on virus growth and / or infectivity and pathogenesis in tissue culture, putative attachment of nucleotide sequences at or near the cleavage site of the F protein, or G protein Domains can be modified by point mutations, site-specific changes, or changes involving the entire gene or gene segment.

In addition to the above modifications to the recombinant vaccine virus, various or additional modifications in the viral clones to facilitate manipulations such as inserting unique restriction sites into various intergenic regions or anywhere. Can be done. Untranslated gene sequences can be removed to increase capacity to insert foreign sequences.

In another aspect of the invention, a composition that produces an isolated attenuated infectious virus, such as a recombinant that incorporates a mutation identified in a heterologous virus (eg,
-) Isolated polynucleotides and vectors comprising the strand RNA viral genome). Using the compositions and methods described above, viral proteins selected as recombinant genomes or antigenomes, such as (for RSV) nucleocapsid (N) protein, nucleocapsidolin protein (P), large (L) Infectious virus and subviral particles are produced from the polymerase protein and the RNA polymerase elongation factor. In a related aspect of the invention, methods and compositions are provided for introducing the above structural and phenotypic changes into a recombinant virus to produce an infectious, attenuated vaccine virus.

The introduction of a mutation as defined above into an infectious recombinant virus is achieved by various known methods as referred to above. By "infectious clone" is meant a cDNA or the product of its synthesis or other methods that can be transcribed into genomic or antigenomic RNA that can serve as a template for producing infectious virus or subviral particles. Thus, defined mutations can be introduced into genomic or antigenomic cDNA copies by conventional techniques (eg, site-directed mutagenesis). As described herein, assembling a complete antigenome or genomic cDNA using antigenome or genomic cDNA subfragments requires that each region be individually manipulated (smaller
The advantage is that cDNAs are easier to manipulate than larger ones) and then are easily assembled into complete cDNAs. Thus, the complete antigenome or genomic cDNA, or a subfragment thereof, can be used as a template for mutagenesis directed to the oligonucleotide. This is because Bio-Rad Laboratories (Richmond, CA)
Intermediates in the form of single-chain phagemids, such as using the Muta-gene® kit from K.K. Alternatively, it can be accomplished by the polymerase chain reaction utilizing either an oligonucleotide primer or a template containing the mutation of interest. The mutated subfragment can then be assembled into a complete antigenomic or genomic cDNA. A wide variety of other mutagenesis techniques are known and available for use in producing the mutation of interest in antigenome or genomic cDNA. Mutation from a single nucleotide change
It can vary up to replacement of large cDNA sections containing one or more genes or genomic regions.

Thus, in one exemplary embodiment, the mutation is introduced by using in vitro mutagenesis of the Muta-gene phagemid available from Bio-Rad. CDNA encoding part of the RSV genome or antigenome is cloned into plasmid pTZ18U and used to transform CJ236 cells (Life Technologies).
Phagemid preparations are prepared as recommended by the manufacturer. Oligonucleotides are designed for mutagenesis by introducing altered nucleotides at desired locations in the genome or antigenome. The plasmid containing the genetically altered genomic or antigenomic fragment is then amplified and the mutated section reintroduced into a full-length genomic or antigenomic clone.

The invention also provides for the production of an attenuated, infectious, recombinant virus that incorporates a mutation identified in a heterologous virus from one or more isolated polynucleotides, such as one or more cDNAs. Provide a way to The cDNA encoding the subject viral genome or antigenome is constructed, for example, to co-express intracellularly or in vitro with the viral proteins necessary to form infectious RSV. "
By "antigenome" is meant an isolated (+) sense polynucleotide molecule that serves as a template for the synthesis of the progeny viral genome. Preferably, the RN of the replication intermediate
A, or a complementary sequence encoding a protein that promotes the production of a nucleocapsid that constructs a cDNA that is a template for synthesizing a (+) sense version of the genome corresponding to the antigenome, and that transcribes and replicates it. Minimize the possibility of hybridizing with the sense transcript. For the purposes of the present invention, the genome or antigenome of the recombinant virus may contain the gene or part thereof necessary for the particles of the virus or subvirus encoded thereby to be infectious. Further, the gene or portion thereof may be provided by one or more polynucleotide molecules. That is, the gene may be provided by complementation from a separate nucleotide molecule or the like.

Recombinant virus means an intact virus or virus-like subviral particle derived directly or indirectly from a recombinant expression system or propagated from a virus or subviral particle produced therefrom. I do. A recombinant expression system comprises at least one genetic element or elements that have a regulatory role in viral gene expression, such as a promoter, a structural or coding sequence transcribed into viral RNA, and appropriate transcriptional start and stop sequences. Utilizing a recombinant expression vector comprising an operably linked transcription unit comprising the assembly of

To produce an infectious virus from a cDNA-expressing genome or antigenome, by known methods, (i) producing a nucleocapsid capable of RNA replication, and (ii)
The genome or antigenome is expressed so that the progeny nucleocapsid RNA is competent for both RNA replication and transcription. Transcription by the nucleocapsid of the genome provides other proteins and initiates productive infection. Alternatively, additional viral proteins essential for productive infection can be supplied by co-expression.

The antigenome of the virus may be cloned for use in the present invention, for example, by cloning.
Assembling cDNA segments, assembling complete antigenomes, polymerase chain reaction (PCR; eg, US Pat. No. 4,683,195, incorporated herein by reference) of reverse transcribed copies of viral or genomic RNA. No. and 4,683,202
And PCR Protocols: A Guide to Methods and Applications, Innis et al
, eds., Academic Press, San Diego (1990)). For example, a cDNA containing the antigenome or a portion thereof is assembled in a plasmid or a suitable expression vector such as a cosmid, phage, or DNA viral vector, which is variously utilized. This vector is mutagenized and / or
Alternatively, they can be modified by insertion of a synthetic polylinker containing unique restriction sites designed to facilitate assembly. In some cases, such as RSV, the use of a particular vector (pBR322) stabilizes viral sequences that might otherwise support nucleotide deletion or insertion. Similarly, propagation of the plasmid may be facilitated by selection of a particular strain (eg, DH10B) that avoids artificial duplication and insertion that would otherwise occur (eg, near nt4499 of RSV).

In one aspect of the invention, the complementary sequence encoding a protein necessary to produce the nucleocapsid of the transcribing and replicating virus is provided by one or more helper viruses. Such helper virus can be wild-type or mutant. Preferably, the helper virus can be phenotypically distinguished from the virus encoded by the cDNA. For example, it would be desirable to provide a monoclonal antibody that reacts immunologically with a helper virus but does not react with a virus encoded by cDNA. Such an antibody can be a neutralizing antibody.
In one embodiment, the antibody is used in affinity chromatography to allow the helper virus to be separated from the recombinant virus. To assist in obtaining such antibodies, mutations can be introduced into the cDNA to provide antigenic diversity from the helper virus, as in the RSV HN or F glycoprotein gene.

[0135] A wide variety of nucleotide insertions and deletions can be made in the genome or antigenome of the virus to produce modified, attenuated virus clones. Paramyxoviruses and members of the morbillivirus genus typically follow the "rule of six". That is, the genome (or minigenome) is a multiple of six nucleotides in length (considered a necessary condition for accurate spacing of nucleotide residues relative to the N protein encapsulated in the capsid). Duplicate only when.

Another means of constructing a cDNA encoding a recombinant (−) strand RNA virus that incorporates a mutation identified in a heterologous virus is one in which the number of subunit cDNA components is one.
To as few as two fragments (eg, Cheng et al., Proc. Natl. Acad. Sci. USA 91: 5695-5699, which is incorporated herein by reference).
(As described in (1994)) by reverse transcription PCR using improved PCR conditions. In other embodiments, various promoters (eg, T3, SP6) or ribozymes (eg, that of hepatitis delta virus) can be used. Various DNA vectors (eg, cosmids) can be used for propagation to better accommodate large size genomes or antigenomes.

An isolated polynucleotide encoding the genome or antigenome of a virus (eg,
cDNA) and the required viral proteins, either isolated or in cis, are capable of supporting productive viral infection by transfection, electroporation, mechanical insertion, transduction, etc., eg, HEp-2 , FRhL-DBS2, MRC, and Vero cells. Transfection of the isolated polynucleotide sequence can be performed, for example, by calcium phosphate mediated transfection (Wigler et al.
., Cell 14: 725 (1978); Corsaro and Pearson, Somatic Cell Genetics 7: 60
3 (1981); Graham and Van der Eb, Virology 52: 456 (1973)), electroporation (Neumann et al., EMBO J. 1: 841-845 (1982)), DEAE-dextran mediated transfection ( Ausubel et al., (Ed.) Current Protocols in
Molecular Biology, John Wiley and Sons, Inc., NY (1987)), cationic lipid-mediated transfection (Hawley-Nelson et al., Focus 15: 73-79 (1993)).
) Or a commercially available transfection reagent such as LipofectACE® (Life
Technologies) into cultured cells (all of the above references are incorporated herein by reference).

[0138] The viral proteins are encoded by one or more expression vectors, which may be the same or different from those encoding the genome or antigenome, and various combinations thereof. The additional protein may be encoded by its own vector or a vector encoding an essential viral protein, and may be included as desired. Expression of proteins from genomic or antigenome, and transfected plasmids can be performed, for example, using (T7 RNA polymerase expression system, eg, infection, transfection using a recombinant vaccinia virus MVA strain expressing T7 RNA polymerase). Or provided by transduction) T7 RNA
Achieved by each cDNA under the control of the polymerase promoter (
Wyatt et al., Virology, 210: 202-205 (1995), which is incorporated herein by reference). Viral proteins, and / or T7 RNA polymerase, can also be provided from transformed mammalian cells or by transfection of mRNA-formed mRNA or protein.

In another approach, cells are transfected after antigenome or genomic synthesis has been performed in vitro (cell-free) in a combined transcription-translation reaction. Alternatively, antigenomic or genomic RNA can be synthesized in vitro and transfected into cells expressing viral proteins.

To select candidate vaccine viruses according to the present invention, criteria for viability, attenuation and immunogenicity are determined by known methods. The viruses that would be most desirable in the vaccines of the present invention maintain viability, have a stable attenuated phenotype, and exhibit replication (even at lower levels) in the immunized host. The production of an immune response sufficient to confer protection against serious illness caused by subsequent infection from the wild-type virus must be effectively elicited in the vaccinated person. In this context, the virus of the present invention is not only viable and attenuated than conventional mutations, but also more in vitro than previously studied.
It is genetically more stable in vivo, stimulates a protective immune response, and in some cases extends the protection provided by multiple modifications (eg, induces protection against various virus strains or subgroups, Or protected by various immunological bases, eg, secretion versus serum immunoglobulin, cell-mediated immunity, etc.).

[0141] To propagate a recombinant (-) stranded RNA virus that incorporates a mutation identified in a heterologous virus, a number of cell lines that allow viral growth may be used. The preferred cell line for growing attenuated virus for vaccines is DBS-FRhL
-2, MRC-5 and Vero cells. The highest RSV yields are usually achieved with Vero
This is the case when an epithelial cell line such as a cell is used. Cells are typically about 0.01-1.
The virus is inoculated at a multiplicity of infection in the range of 0 or more and under conditions acceptable for virus replication, for example, at about 30-37 ° C. for about 3-5 days, or when the virus has a sufficient titer. Cultivated as long as necessary to reach The virus is removed from the cell culture, typically separated from the cellular components by known purification methods (eg, centrifugation), and may be further purified as desired using methods well known to those skilled in the art.

[0142] Recombinant viruses that have been attenuated as described herein can be subjected to various attenuations, resistance to phenotypic reversion, and immunogenicity to ensure sufficient attenuation for use in vaccines. It can be tested in known, generally accepted in vitro and in vivo models. In vitro assays use modified viruses (eg, variously attenuated,
Biologically derived or recombinant viruses) are tested for temperature sensitivity of virus replication (ie, ts phenotype), and small plaque phenotype. The modified virus is further tested in animal models of viral infection. A wide variety of animal models for RSV are described in Meignier et al., Eds., Animal Models of Respirato
ry Syncytial Virus Infection, Merieux Foundation Publication, (1991), which is hereby incorporated by reference. A cotton rat model of RSV infection is described in U.S. Patent No. 4,800,078 and Prince et al., Virus Res.
3: 193-196 (1985), which is incorporated herein by reference, and is believed to reasonably predict attenuation and efficacy in humans and non-human primates. In addition, a primate model of RSV infection using chimpanzees has been described by Richardson et al.
al., J. Med.Virol. 3: 91-100 (1978); Wright et al., Infect. Immun. 37:
Attenuation and potency in humans, as described in detail in 397-400 (1982); Crowe et al., Vaccine 11: 1396-1404 (1993), both incorporated herein by reference. Is reasonably predicted. Other models are known for a wide range of (-) strand RNA viruses. The correlation of the data derived from the above animal models with respect to the level of attenuation and immunogenicity of the recombinant (-) strand RNA virus is generally accepted. For example, the therapeutic effect of RSV neutralizing antibodies in infected cotton rats has been shown to be highly correlated with subsequent experience with immunotherapy in monkeys and humans infected with RSV. Indeed, cotton rats appear to be a well-reliable experimental surrogate for the responsiveness of infected monkeys, chimpanzees and humans to immunotherapy with RSV neutralizing antibodies. For example, RSV associated with therapeutic effect in cotton rats, as measured by antibody levels in the serum of the treated animal
The amount of neutralizing antibody (ie, serum RSV neutralizing titer between 1: 302 and 1: 518) is indicated for monkeys (titers of 1: 539) or human infants or children (ie, 1: 877) In the same range as the one. The therapeutic effect in cotton rats is 100% of the virus titer in the lung.
It was evident by a two-fold or more reduction (Prince et al., J. Virol. 61: 1
851-1854), in monkeys, the therapeutic effect was observed to be a 50-fold reduction in lung viral titer (Hemming et al., J. Infect. Dis. 152: 1083-1087 (1985)). Finally,
The therapeutic effect in infants and children hospitalized with severe RSV bronchitis or pneumonia was a significant increase in oxygen evolution in the treatment group and RSV recovered from the upper respiratory tract of treated patients
(Hemming et al., Antimicrob. Agents C
hemother. 31: 1882-1886 (1987)). Thus, based on the above test results, cotton rats constitute a model that can predict the success of chimeric and non-chimeric RSV vaccines in infants and children. Other rodents, including mice, will be useful as well.
Because these animals moderately tolerate RSV replication and have a core temperature similar to humans (Wright et al., J. Infect. Dis. 122: 501-512 (1970) and
Anderson et al., J. Gen. Virol. 71: (1990)). Similar models are available for other (−) strand RNA viruses and are widely known.

According to the above description and based on the following examples, the present invention also provides isolated infectious attenuated virus compositions for vaccine use. The attenuated virus, a component of the vaccine, is in isolated, typically purified form. By isolated is meant RSV present outside the native environment of the wild-type virus, such as the nasopharynx of an infected individual. More generally, isolated means containing the attenuated virus as a component of a cell culture or other artificial medium. For example,
The attenuated RSV of the present invention was separated from the cell culture and added to the stabilizer,
It can be produced by culturing infected cells.

The vaccines of the present invention contain, as an active ingredient, an immunologically effective amount of a recombinant (-) strand RNA virus carrying a mutation identified in a heterologous virus as described herein. The recombinant virus can be used directly in a vaccine formulation or can be lyophilized. Lyophilized virus is typically maintained at about 4 ° C. When ready for use, the lyophilized virus may be combined with a stabilizing solution, such as saline, or a solution comprising SPG, Mg ⁺⁺ and HEPES, with an adjuvant as further described below. Or not together, restored. The biologically derived or recombinantly modified virus can be introduced into a host with a physiologically acceptable carrier or adjuvant. Useful carriers are well known in the art,
Examples include water, buffered water, 0.4% saline, 0.3% glycine, hyaluronic acid, and the like. The resulting aqueous solution can be packaged for use as is,
Alternatively, the lyophilized preparation may be combined with a sterile solution prior to administration, as described above. The composition may contain pharmaceutically acceptable auxiliary substances as required to approximate physiological conditions, such as pH and buffering agents, tonicity adjusting agents, wetting agents, etc. Sodium, sodium lactate, sodium chloride,
It may contain potassium chloride, calcium chloride, sorbitan monolaurate, triethanolamine oleate, and the like. Acceptable adjuvants include incomplete Freund's adjuvant, aluminum phosphate, aluminum hydroxide, or alum, which are materials well known in the art. Preferred adjuvants also include Stimulon ^™ QS-21 (Aquila Biopharmaceuticals, Inc., Uchester, MA), MPL ™ (3-0-deacylated monophosphoryl lipid A; RIBI ImmunoChem Re
search, Inc., Hamilton, MT), and Interleukin-12 (Genetics Institute, Cambridge, MA).

When immunized with an attenuated viral vaccine composition as described herein by aerosol, droplet, oral, topical, or other routes, the host immune system will have one or more viral proteins, Respond to the vaccine by, for example, producing antibodies specific for RSV F and / or G glycoprotein. As a result of the vaccination, the host becomes at least partially or fully immunized against infection by the subject virus or becomes resistant to developing moderate to severe illnesses associated therewith. .

The vaccine of the present invention may comprise a single virus strain or an antigenic subgroup, such as RSV A or B
Or an attenuated virus that elicits an immune response against or against multiple strains or subgroups. In this context, the chimeric vaccine virus can elicit a monospecific immune response, or a polyspecific immune response against multiple strains or subgroups. In another approach, chimeric viruses with various immunogenic characteristics can be conjugated in a vaccine mixture to elicit effective protection against one RSV strain, or multiple RSV strains or subgroups, Or they may be administered separately in a tailored treatment protocol.

The host to which the vaccine is administered can be a mammal susceptible to infection by the subject or related viruses and capable of producing a protective immune response against the antigen of the vaccinated virus. Thus, suitable hosts include humans, non-human primates, cows, horses, pigs, sheep, goats, rabbits, rodents, and the like. Thus, the present invention provides a method of creating a vaccine for use on a wide variety of humans and livestock.

A vaccine composition containing an attenuated recombinant virus of the invention may be used to immunize each individual against a subject virus at risk of being susceptible or otherwise infected with the subject virus. "Is sufficient to induce or enhance responsiveness"
An "immunologically effective amount" is administered. In humans, the attenuated virus of the present invention may be, for example, Wright et al., Infect Immun. 37: 397-400 (1982), Kim et al., Pediatri.
cs 52: 56-63 (1973), and Wright et al., J. Pediatr. 88: 931-936 (1976)
It is administered by a well-established human vaccine protocol as described for RSV (both incorporated herein by reference). Briefly, adults or children are nasally inoculated with a droplet of an immunologically effective amount of a RSV vaccine, typically in a 0.5 ml volume of a physiologically acceptable diluent or carrier. This has the advantage of simplicity and safety compared to parenteral immunization with non-replicating vaccines. It also provides a direct stimulation of local airway immunity, which plays a major role in resistance to RSV. Furthermore, this form of vaccination effectively avoids the immunosuppressive effects of RSV-specific maternal serum antibodies normally found in very young people. Also, while parenteral administration of RSV antigen has sometimes been associated with immunopathological complications, this has never been observed with live viruses.

In any subject, the precise amount of attenuated virus vaccine to be administered and the timing and frequency of administration will be determined based on the patient's health and weight, mode of administration, nature of the formulation, etc. . Dosages generally range from about 10 ³ to about 10 ⁶ plaque forming units (RFU) or more virus per patient, more usually about 10 ⁴ to 10 ⁵ PFU virus per patient. In each case, the vaccine formulation should provide an amount of the attenuated virus of the invention sufficient to effectively stimulate or induce an antiviral immune response, which, among other methods, For example, complement fixation, plaque neutralization, and / or
Alternatively, it can be determined by an enzyme-linked immunosorbent assay. In this regard, each individual is monitored for signs and symptoms of upper respiratory illness. For administration to chimpanzees,
The attenuated virus of the vaccine of the present invention may be about 10-fold or more lower than the wild-type virus in the nasopharynx of the vaccinated or compared to the level of the partially attenuated virus. Proliferate at a level two times or more lower.

In neonates and infants, frequent administration may be required to elicit sufficient levels of immunity. For RSV infection, treatment should be started within one month of birth, and in childhood, two months, as needed to maintain sufficient levels of protection against native RSV infection. It should be administered at intervals such as 6 months, 1 year and 2 years. Similarly, adults particularly susceptible to recurrent or severe RSV infection, such as, for example, medical workers, day care workers, infant families, the elderly, individuals with compromised cardiorespiratory function, may have a protective immune response. Frequent immunizations may be required to establish and / or maintain. The level of immunity induced can be monitored by measuring the amount of neutralizing secreted and serum antibodies and adjusting the dose as necessary to maintain the desired level of protection Alternatively, it is possible to repeat the vaccination. In addition, different vaccine viruses may be applied for administration to different groups of recipients. For example, engineered recombinants that express additional proteins that are rich in cytokines or T cell epitopes may be particularly advantageous for adults over infants. A vaccine produced according to the present invention can be combined with a virus that expresses an antigen of a virus of another subgroup or strain to achieve protection against multiple subgroups or strains. In another approach, a vaccine virus may incorporate the designed epitopes of multiple virus strains or subgroups into a single clone as described herein.

Typically, when different vaccine viruses are used, they are administered simultaneously in a mixture, but they can also be administered separately. For example, since the F glycoproteins of the two RSV subgroups differ by only about 11% in amino acid sequence, this similarity is observed in animals immunized with RSV or F antigen and challenged with heterologous strains. Underlies the cross-protective immune response. Thus, immunization with one strain may result in protection against the same or different strains of different subgroups. However, it may be preferable to have the G and F glycoproteins of the antigenic subgroup A or B of RSV present in the vaccine.

The attenuated recombinant vaccines of the present invention induce the production of an immune response that protects against serious diseases such as pneumonia and bronchitis when an individual is later infected with the wild-type virus. Although naturally circulating viruses may still be able to cause infection, the chances of producing disease symptoms as a result of vaccination and possible additional resistance due to subsequent infection with wild-type virus are greatly reduced. ing. Following vaccination, there are detectable levels of serum and secreted antibodies produced by the host that can neutralize homologous (of the same subgroup) wild-type virus in vitro and in vivo. In many cases, this host antibody will also neutralize different non-vaccine subgroups of wild-type virus.

Preferred attenuated viruses of the present invention show a very substantial reduction in virulence as compared to wild-type viruses circulating naturally in humans. Chimeric viruses are sufficiently attenuated and symptoms of infection do not occur in most immunized individuals. In some cases, the attenuated virus may still be able to spread to unvaccinated individuals. However, this virulence is sufficiently weakened that no severe lower respiratory tract infection occurs in vaccinated or accidental hosts.

The level of attenuation of the vaccine virus of the invention can be determined, for example, by quantifying the amount of virus present in the immunized host, eg, in the respiratory tract, and by wild-type or other attenuated virus evaluated as a candidate vaccine strain. It can be determined by comparing the amount produced to that amount. For example, the attenuated RSV of the present invention can be used in the upper respiratory tract of highly susceptible hosts, such as chimpanzees, compared to the level of replication of the wild-type virus.
Replication is more highly restricted, for example to 10-1000 times or less. Furthermore, the level of replication of the attenuated RSV vaccine strain in the upper respiratory tract of chimpanzees should be lower than that of the RSV A2 ts-1 mutation, which was previously shown to be incompletely attenuated in seronegative human infants. is there. To further reduce the development of rhinorrhea associated with viral replication in the upper respiratory tract, an ideal vaccine candidate virus should exhibit limited replication levels in both the upper and lower respiratory tract. However, the attenuated virus of the present invention is sufficiently infectious and immunogenic to provide protection to vaccinated individuals. Methods for determining the level of RSV in the nasopharynx of an infected host are well known in the literature. The specimen is obtained by aspirating or washing out the secretions of the nasopharynx, and the virus is quantified in a tissue culture or the like by a laboratory method. For example, Belshe et al., J. Med. Virology 1: 157-162 (1997), Friedew
ald et al., J. Amer. Med.Assoc. 204: 690-694 (1968); Gharpure et al., J
Virol. 3: 414-421 (1969); and Wright et al., Arch. Ges. Virusforsch.
41: 238-247 (1973). All of which are incorporated herein by reference. The virus can be conveniently measured in the nasopharynx of a host animal such as a chimpanzee. Other methods for determining the level and virulence of additional (-) strand RNA are widely known and readily implemented.

The present invention disclosed herein will be further clarified by the following examples, which are provided by way of illustration and not limitation. All publications and patent documents cited herein for the description of the present invention are hereby incorporated by reference for all purposes.

[0156]

【Example】

Example I: Mapping of attenuating mutations identified in HPIV3 JS cp45 and RSV cp530 and Sendai virus to sequence elements conserved in heterologous (-) stranded RNA virus. -) Shows that the mutations identified in the strand RNA virus can be easily mapped to the corresponding conserved amino acid sequence element in the heterologous virus within the order of the Mononegavirus. These mutations, which are characterized by structural changes relative to the parental sequence that are conserved (identical or conservative) in one or more heterologous (-) stranded RNA viruses, are the sequence of the parent protein Provide candidate mutations that may be incorporated into recombinant viruses that share the element.

To embody this aspect of the invention, a panel of known mutations within JS cp45 of the HPIV3 mutant was analyzed. This mutation panel includes ts-attenuating amino acid substitutions in the polymerase L gene at parental residues / sequence positions of Tyr ₉₄₂ , Leu ₉₉₂ and / or Thr ₁₅₅₈ . More specifically, the JS cp45 mutant L protein exhibits an attenuating mutation in which the parent Tyr ₉₄₂ has been replaced by His, Leu ₉₉₂ has been replaced by Phe, and / or Thr ₁₅₅₈ has been replaced by Ile. (See US patent application Ser. No. 08 / 083,793 and corresponding International Patent Application No. WO 98/53078, which are incorporated herein by reference). According to a preferred aspect of the present invention, these mutations were not only mapped to the parent sequence to identify this change, but also their attenuating effect and potential for recovery to the cloned infectious virus. In order to confirm that, PIV recombinant alone (r942, r992, r1558, r942 / 992, r992 / 1558, r942 / 1
558 and r942 / 992/1558).

[0158] Additional representative mutations were evaluated in the HPIV3 JS cp45 mutation and the HPIV3 F
And were mapped and characterized to attenuate amino acid substitutions in the C protein. This mutation includes JS as exemplified by the substitution of Ile ₉₆ for Thr.
Includes a non-ts attenuated amino acid substitution in the C protein of the P gene at Ile ₉₆ at the parent residue / position of HPIV3. Additional representative mutations identified in the F protein of HPIV3 are the parent residues / sequences of Ile ₄₂₀ and Ala ₄₅₀ , as exemplified by the substitution of Ile ₄₂₀ for Val and Ala ₄₅₀ for Thr. Encodes an amino acid substitution at a position.

Each of the attenuating mutations thus identified in HPIV3 has a different (-)
An index of sequence comparison to homologous proteins in the chain virus was provided. To demonstrate this aspect of the invention, conventional sequence alignments were performed by the methods outlined above to identify the mutations identified in the HPIV3 L and F proteins as heterologous (-) stranded RNA.
Virus panel (HPIV1, Sendai virus, HPIV2, BPIV3, MeV and RSV)
It mapped to the corresponding part in. As shown in Table 2, this implementation demonstrated very high degrees of conservation between the parental sequence elements of the selected mutants and the wild-type sequence of the heterologous virus compared thereto. What surprised these results was that
Evolutionarily conserved sequence elements are of significant functional significance and, when mutated, have a more severe phenotypic effect (eg, loss of viability or infectivity) than mere attenuation. It is expected to occur.

[0160]

[Table 2]

[0161]

[Table 3]

Looking at the exemplary alignments shown in Table 2, each of the L and F mutations analyzed is substantially conserved and is characterized by the presence of identical or conservative amino acid residues. It is noted that the parent sequence elements were changed at corresponding positions between the indicated virus taxa. For example, a mutation in the cp45 L protein that changes the parent residue Tyr ₉₄₂ to His is due to the retention of the same tyrosine residue at the corresponding wild-type position in Sendai, HPIV2, HPIV3, BPIV3, and MeV L proteins, respectively. As shown, they were very conservatively mapped (Table 2). Similarly, the cp45 L mutation featuring Leu ₉₉₂ replaced by Phe maps to a parental residue / position that is identically conserved among Sendai, HPIV3, BPIV3 and MeV L proteins. Ile ₄₂₀ to Val, and Ala ₄₅₀
There has been substituted to Thr, also two identified mutations in cp45 F protein, shows the conserved residues / positions in the same between HPIV3 and BPIV3, from Ile ₄₂₀
The parent residue of the mutant to Val is also identically conserved in HPIV1. HPIV3
Additional conserved sequence elements corresponding to the site of the attenuating mutation identified in JS cp45 are also shown in Table 2.

Another heterologous sequence alignment was performed to evaluate the conservation of the exemplary RSV attenuating mutation identified in mutant strain RSV cp530. This mutation is characterized by a substitution of leucine for phenylalanine at position 521 in the L polymerase of cp530, which occurs within the larger conserved structural domain of the protein. As shown in Table 3, the mutation at Phe ₅₂₁ was also highly conservative, as indicated by the retention of the same phenylalanine residue at the corresponding wild-type position in the 13 subject taxa. Mapped (Table 3; Figure 1, Panel A)
.

[0164]

[Table 4]

[0165] Yet another heterologous sequence alignment was performed to demonstrate the ability to identify conserved structural elements corresponding to known attenuating mutation sites. In this example, the attenuating mutation identified at position 170 of the C protein of the Sendai virus was modified with the heterologous virus, HPIV-1,
Maps to the corresponding sequences in the HPIV-3 and BPIV-3 C proteins (see Table 4;
Number the position of the first residue in the corresponding sequence). Again, this mutation was characterized by an amino acid change at the parent residue / sequence position that was identically conserved among the various taxons.

[0166]

[Table 5]

Summarizing the above findings, we show the surprising conservation of sequence elements corresponding to the site of the attenuating mutation. On this basis, we start the rational design method of the present invention and convert the attenuated sequence changes identified in one heterologous virus to a different recombinant virus, e.g., the same or conservative changes in the recombinant genome or antigenome. And produced an attenuated recombinant clone. The practical development of the above method is demonstrated by the following example.

Example II: Heterologous introduction of attenuating mutations from RSV cpts530L into recombinant HPIV3 vaccine candidates Thus, corresponding to structural elements conserved between heterologous taxa within the order of the Mononegavirus Attenuating mutations were identified at the site and the concept of cross-introducing attenuating mutations across phylogenetic boundaries was validated using the key disease virus RSV as a model. In this context, this example can introduce an attenuating (att) mutation identified in RSV, a virus of the Pneumovirus genus of the Paramyxoviridae family, into PIV3, a member of the closely related respirovirus genus. It indicates that. The viruses represent two different subfamilies within the Paramyxoviridae family, respectively, Pneumovirus and Paramyxovirus.

More specifically, a first heterologous virus (RSV cpts5) in which the parental RSV sequence was altered at the defined site of the mutation (Phe _{521 of the} RSV L protein) that specified the attenuation phenotype.
The attenuating mutation in 30) was mapped to an identically conserved residue at the corresponding position (F456L) in the L protein of the selected target virus, HPIV3 (
Table 3). Therefore, the sequence elements of wild-type HPIV3 thus conserved were tested as potential targets for heterologous introduction of attenuating mutations between RSV and HPIV3.

To accomplish this introduction, all or part of a conserved sequence element carrying the attenuating mutation is preferably copied or transferred into the recombinant virus, and the new attenuated derivative is Produce. Preferably, the mutation changes are copied identically to the recombinant virus, but this level of fidelity is not required. Conversely, the parent or wild-type residue thus identified as a target, at the site of the mutation comprising one or more residues that may be independent of the residue characterizing the mutation , Can be deleted or replaced by amino acid insertions. Preferably, if the subject mutation is characterized by one or more amino acid substitutions, the residues of the parental clone of the recombinant virus corresponding in position to the site of the mutation are identified in the sequence of the mutant. Is replaced by one or more residues conservatively related to the replacement residue made. More preferably, the residue in the parent clone of the recombinant virus corresponding to the site of the mutation is replaced by one or more residues that are identical to those present in the sequence of the mutant.

Thus, in this example, it was characterized that the mutation of phenylalanine to leucine at aa at position 521 in the L polymerase of cpts530 specifies the ts and attenuated (att) phenotypes. . Sequence alignment of this region in the L proteins of some distantly related paramyxoviruses (Table 3) revealed that this phenylalanine is widely conserved. Using reverse genetics, a similar phenylalanine at position 456 in the L protein of wild-type PIV3 was mutated to leucine (F
456L). This produced virus, designated r456 _L , is ts (40 ° C. plaque formation shutoff temperature) and replicates in the upper but not the lower respiratory tract of hamsters by 10% compared to that of recombinant wild-type (rwt) PIV3. Had dropped twice. The above results indicate that the introduced phenylalanine to leucine mutations have identified equivalent levels of temperature sensitivity and attenuation in the two closely related paramyxoviruses.

Also, in this example, F456 was transferred to two rPIV3 candidate vaccine viruses (one rcp45 _L carrying three cp45 ts missense mutations in the L protein and the other rcp45 carrying all 15 cp45 mutations). The introduction of the mutation has been shown to further attenuate the virus in vivo. More specifically, two recently developed recombinant PIV3
The F456L mutation was introduced into a vaccine candidate. One is rcp45, a recombinant version of cp45, a biologically derived PIV3 vaccine candidate, and the other is rcp4
5 _L is a version in which only three amino acid substitutions in the L protein exist as cp45 mutations (published International Patent Application No. 98/53078; Skiadopoulos et
al., J. Virol. 72 (3): 1762-8 (1998); Skiadopoulos et al., J. Virol. 73
(2): 1374-1381 (1999), both of which are incorporated herein by reference). F4
Adding the 56L mutation to rcp45 or rcp45 _L increased the temperature sensitivity and the level of attenuation in vivo, rcp45-456 was immunogenic and phenotypically stable. The rcp45 _L- 456 and rcp45-456 viruses were 100-1000 fold more restricted in replication in hamsters than their parental rcp45 _L and rcp45. Immunization with rcp45-456 elicited moderate levels of resistance to PIV3 challenge virus replication. In chimpanzees, rcp45-456 was more than 5-fold more restricted in the respiratory tract than rcp45 and elicited comparable medium to high levels of PIV3-specific serum antibodies. Rc isolated from chimpanzee
The p45 and rcp45-456 viruses maintained the same level of temperature sensitivity as the respective starting viruses during the course of replication for two weeks, demonstrating their phenotypic stability.

Thus, introduction of the F456L mutation corresponding to the RSVF521L mutation into the cp45 recombinant virus resulted in a gradual increase in attenuation of the recombinant virus, and this Demonstrates the utility of mutagenesis. The ability to introduce mutations identified in a heterologous paramyxovirus, which in this example represents various subfamilies, greatly enhances the ability to develop new parainfluenza virus vaccines.

Viruses and Cells rPIV3 JS wt (referred to herein as rwt), rcp4 used as controls in this example
The 5 _L and rcp45 viruses have been produced previously (Published International Patent Application No. 98/53078; Durbin et al., Virology 235: 323-332 (1997); Skiadopoul
os et al., J. Virol. 72 (3): 1762-8 (1998); Skiadopoulos et al., J. Viro.
l. 73 (2): 1374-1381 (1999), all of which are incorporated herein by reference)
. rPIV3, as previously reported (Durbin et al., Virology 235: 323-332 (1997);
ll et al., Virus Res. 22 (3): 173-184, 1992; Skiadopoulos et al., J. Vir.
ol. 72 (3): 1762-8 (1998), which is incorporated herein by reference), grew in Simian LLC-MK2 cells (ATCC CCL 7.1). Ankara (MVA-T7), a modified vaccinia virus expressing T7 polymerase (Wyatt et al., Virology
210 (1): 202-205 (1995)) was kindly provided by Linda Wyatt and Bernard Moss. HEp-2 (ATCC CCL 23) and LLC-MK2 cells were from OptiMEM I (Life Technologies, Gaithersburg, MD) supplemented with 2% FBS and gentamicin sulfate (50 μg / mL) or 10% FBS, gentamicin sulfate. (50 μg / mL) and maintained in EMEM supplemented with 4 mM glutamine.

Construction of Point Mutations in rwt Antigenomic cD of PIV3 JS wt Used Once to Recover Infectious Virus
The NA clone p3 / 7 (131) 2G + (Durbin et al., Virology 235: 323-332 (
1997); Skiadopoulos et al., J. Virol. 72 (3): 1762-8 (1998); Skiadopoulo
s et al., J. Virol. 73 (2): 1374-1381 (1999)), PIV3 nt7437-11312 (Xh
The subgenomic fragment containing oI-SphI) was cloned into a pUC19 vector modified to accept this fragment using standard molecular cloning techniques. Previously reported (Skiadopoulos et al., J. Virol. 72 (3): 1762-8 (1998);
Transfo as in Skiadopoulos et al., J. Virol. 73 (2): 1374-1381 (1999)).
This cDNA was modified by introducing two point mutations using the rmer Mutagenesis kit (Clonetech, CA). The above two changes are due to the rPIV3 antigenome.
Counted by the complete (+) sense sequence of the RNA, positions 10011 (T to C) and 10013 (C
To G). This introduced a codon change in F456L as well as overlapping silent Xmn sites as markers (FIG. 1). After mutagenesis, the restriction endonuclease fragment is fully sequenced and cloned directly into the full-length clone, p3 / 7 (131) 2G +, using standard molecular cloning techniques, or cp45. It was cloned into the derivative responsible for the mutation.

Recovery of Recombinant PIV3 Carrying F456L Mutation The F456L mutation and three support plasmids, pTM (N), pTM (P no C) and pTM
The full-length antigenomic cDNA derivative responsible for (L) was purchased from LipofectACE (Life Technolog
ies, MD) and transfected into a HEp-2 monolayer of a 6-well plate (Costar, MA) and this monolayer was previously reported (Durbin et al., Virology 235: 323-332 (1997); Ski
adopoulos et al., J. Virol. 72 (3): 1762-8 (1998)). Plasmid pTM (P no C) is a previously reported pTM (P) (Durbin et al., Virolo
gy 235: 323-332 (1997)) a plasmid whose translation initiation codon changes from AUG to ACG (
This is a modified version of the CORF to change (from methionine to threonine). After 4 days incubation at 32 ° C, the transfection harvest was
Passaged on LLC-MK2 cells in 5 flasks, which were incubated at 32 ° C. for 4-8 days. The clarified medium supernatant was used previously (Durbin et al., Virology 235: 323-332
(1997); Hall et al., Virus Res. 22 (3): 173-184, 1992; Skiadopoulos et.
Plaque purification was performed three times on LLC-MK2 cells as in al., J. Virol. 72 (3): 1762-8 (1998),). LLC-MK was used for each biologically cloned recombinant virus.
Amplified twice at 32 ° C. in 2 cells and produced virus for further characterization. Virus was concentrated from the clarified medium by polyethylene glycol precipitation (
Mbiguino and Menezes, J. Virol. Methods 31: 161-170 (1991)), virus RN
A (vRNA) was extracted with TRIzol Reagent (Life Technologies). Superscript II Preamplification System using random hexamer primers (Life Techn
reverse transcription was performed on vRNA using The fragments for restriction endonuclease digestion and / or sequence analysis were amplified using the Advantage cDNA PCR kit (Clonetech, CA) and sense and antisense primers specific for various parts of the PIV3 genome. This PCR fragment was analyzed by sequencing and / or restriction analysis with each of the restriction enzymes for which a recognition site was created or deleted during the construction of the mutation.

Plaque Formation Efficiency at Permissive and Restricted Temperatures of rPIV3 Carrying the F456L Mutation (EOP) The temperature-sensitive levels of in vitro plaque formation of control and recombinant viruses were determined by previously reported methods (Hall et al., Virus Res. 22 ( 3): In a monolayer culture of LLC-MK2 cells incubated for 6 days as in 173-184, 1992), the temperature was determined at 32 ° C and at a temperature ranging from 35 ° C to 41 ° C. Plaques were counted by removing the layer of methylcellulose after hemocyte adsorption with guinea pig erythrocytes, or in other ways, the viral plaque present in the monolayer was replaced by two PIV3-specific anti-HN mouse mAbs, 101/1 And immunoperoxidase staining with a mixture of 250-fold dilutions of 454 and 11 (Murphy et al., Vaccine 8 (5): 497-
502 (1990); Murphy et al. (1990); van Wyke Coelingh, Winter, and Murphy
Virology 143 (2): 569-582 (1985), both incorporated herein by reference).

Evaluation in hamsters for the att phenotype of rPIV3 mutant virus Four week old Golden Syrian hamsters (Charles) that are seronegative for PIV3
River Laboratories, the NY), the L15 medium 0.1ml containing one 10 ^6.0 TCID ₅₀ of rwt or mutated rPIV3 were inoculated intranasally. Hamsters were sacrificed on day 4 of infection, lungs and nasal turbinates were collected and reported previously (Durbin et al., Virology 235: 323-332, 1997; Skia
Virus was quantified as in dopoulos et al., J. Virol. 72 (3): 1762-8 (1998). The average log ₁₀ TCID ₅₀ / gram at 32 ° C. was calculated for each group of hamsters.

[0179] The immunogenicity and efficacy 5 hamsters 3 group in hamsters rcp45-456, (i) L15 medium (placebo), (ii) rcp45 of 10 ^6.0 TC
ID _50, or 0.1ml of (iii) rcp45-456 of 10 ^6.0 TCID ₅₀ were inoculated intranasally. Blood was collected from hamsters before and on day 42 post-infection, and serum hemagglutination inhibition (HA
I) Antibody titers were determined as previously reported (van Wyke Coelingh, Winter, and Murphy, 1985). Day 44, were challenged hamster administration 10 ^6.0 TCID ₅₀ rwt intranasally. Four days later, the nasal turbinates and lungs were collected and the rwt titers in the tissue homogenates were determined as described above.

Evaluation in chimpanzees for the att phenotype of the rPIV3 mutant virus Young male and female chimpanzees, both seronegative for RSV and PIV3 and weighing 6.6 to 10.0 kg, were housed in large glass isolation rooms in pairs and reported in (Crowe et
al., 1994a; Hall et al., J. Infect. Dis. 167: 958-962 (1993)). Groups of chimpanzees were infected with each part by inoculum 1ml intranasally (IN) and intratracheally (IT) route containing 10 ^6.0 TCID ₅₀ of rcp45 or Rcp45-456. After inoculation of the virus, nasopharyngeal swabs and tracheal lavage samples were previously reported for quantification of the escaping virus (
Hall et al., 1993; Karron et al., 1997). Nasopharyngeal swab samples were collected on days 1-10 and 13; tracheal lavage samples were 2, 4, 6, 8 and 10
Collected on day. The extent of rhinorrhea was evaluated daily and scored from 0 to 4 (0 = no rhinorrhea; 1 = trace; 2 = mild; 3 = moderate; 4 = severe). The rcp45 and rcp45-456 viruses were evaluated in two separate experiments following the same protocol. In experiment 1, two chimpanzees were inoculated with rcp45 and four were inoculated with rcp45-456, and in experiment 2 each virus was given to two animals. The second experiment was performed to confirm the differences in growth between the two viruses observed in the first experiment. The two sets of data could not be distinguished with respect to virus growth and immunogenicity, and were therefore averaged together. IN and data previously reported from 4 animals that received 10 ⁴ TCID ₅₀ of wt JS strain of PIV3 by IT route (Hall
et al., 1993) and are presented here for comparison.

Characterization of rPIV Replication in Chimpanzees The amount of virus in nasopharyngeal swabs and tracheal lavage samples was determined previously (Crowe et al.
al., 1994a; Hall et al., 1993), determined at 32 ° C. in LLC-MK2 monolayers and expressed as log ₁₀ TCID ₅₀ / ml. Virus present in chimpanzee nasopharyngeal and tracheal lavage samples was grown at 32 ° C. in LLC-MK2 monolayers in 24-well plates until extensive cytopathic effects were detected. The temperature sensitivity level of the rPIV3 isolate was evaluated by determining the plaque formation efficiency on LLC-MK2 monolayer as described above. All tests with chimpanzee specimens or isolates included clindamycin (100 μg / ml), ciprofloxacin (100 μg / ml), gentamicin (100 μg / ml), and amphotericin B (2.5 μg / ml). ) Was included.

Results Introducing the F456L Mutation into rwt Gives a ts Phenotype As described above, RSV cpts530 is a ts and is attenuated for replication in the respiratory tract of mice (Crowe et al., 1994b). Single amino acid substitution with phenylalanine-521 in the L protein of RSV cpts530 causes temperature sensitivity (39 ° C shutoff temperature for plaque formation) and attenuation (10-fold reduction in replication in the upper respiratory tract of mice) ( Crowe et al., 1994b; Juhasz et al., 1997). The sequence alignment of the L protein of 13 different paramyxoviruses, including RSV and PIV3,
Position phenylalanine was found to be highly conserved (Table 3; FIG. 1,
Panel A). In the case of HPIV3, the corresponding amino acid appears at position 456 of PIV3 L polymerase. The difference in the position number of this residue between RSV (521) and PIV3 (456) indicates that RSV L
Consistent with previous observations that the protein has an amino-terminal extension of about 70 amino acids compared to paramyxoviruses from other genera (Stec et al.
al., Virology 183: 273-287 (1991)). To determine if the mutation at position 456 of the L protein of PIV3 gives a ts and att phenotype similar to that of the heterologous RSV cpts mutant, Was mutagenized to leucine (F456L). This coding change was designed to include two nucleotide substitutions to reduce the probability of reverting to wt. This mutation was also characterized by the introduction of a silent XmnI restriction endonuclease recognition site (FIG. 1, panel C). This mutation was introduced into a full-length infectious rwt cDNA plasmid and reported previously (Durbin et al., Virology 235: 323-332 (1997); Ski
adopoulos et al., J. Virol. 72 (3): 1762-8 (1998); Skiadopoulos et al.,
J. Virol. 73 (2): 1374-1381 (1999)). Recovered R456 _L mutants (Figure 1, panel C), based on the analysis by RT-PCR and restriction enzyme digestion and sequencing of purified vRNA, confirmed to possess XmnI marker and F456L mutations Was done. Introduction of the F456L mutation into rwt provided a shutoff temperature of 40 ° C (Table 5), which was 1 ° C higher than that of RSV cpts530, and the ts mutation identified in pneumovirus was a closely related respirovirus. To indicate that they can give equivalent, but not identical, ts phenotypes.

[0183]

[Table 6]

Introduction of F456L Mutation into Recombinant rcp45-Based Attenuated Virus Increases Temperature Sensitivity cp45 has three specific amino acid substitutions in the L protein, which are due to ts and att
It has already been shown to be a major determinant of phenotype (Skiadopoulos et al.
., J. Virol. 72 (3): 1762-8 (1998)). cp45 also has 12 other mutations outside of L, including the ts and att mutations (Skiadopoulos et al.
, J. Virol. 73 (2): 1374-1381 (1999)). F456L was introduced into rcp45 and a virus carrying only three mutations in the rcp45 L gene (rcp45 _L ). What did this
The temperature sensitivity identified by the F456L mutation was 3 cp45 L mutations or 15
This is to determine if it is additive to that specified by the full set of cp45 mutations. Remarkably, rcp45 _L -456 was compared to 39 ° C with rcp45 _L
It had a shutoff temperature of 35 ° C. and revealed a significant increase in the temperature sensitivity level (Table 5). The rcp45-456 mutant had an intermediate shutoff temperature of 37 ° C, as compared to 38 ° C with rcp45. The temperature sensitivity identified by the F456L mutation is r
Although additive to that specified by mutations in cp45 _L and rcp45, the magnitude of this effect was clearly different for the two viruses. in this way,
Rcp45-456, which contains a higher number of mutations, is unexpectedly more tcp than rcp45 _L- 456
Not s. Evidence for complex interactions between cp45 ts mutations has been previously observed for other rPIV3 viruses containing various combinations of cp45 mutations, but the underlying underlying effects are not understood (Skiadopoulos et al.,
J. Virol. 72 (3): 1762-8 (1998) and Skiadopoulos et al., J. Virol. 73
(2): 1374-1381 (1999)).

[0185] the replication and immunogenicity hamsters in hamsters mutants RPIV3, including viruses responsible 10 ^6.0 TCID ₅₀ of rwt or F456L mutation alone or together with a cp45-specific mutations, several mutations IN one of rPIV3
Inoculated (Table 6). Four days later, lungs and turbinates were collected to determine the level of replication of each virus. F456L alone has a modest effect on r456 _L replication, with about 10
And resulted in a two-fold reduction, with no apparent restriction on replication in the lung. This level of attenuation is comparable to that of the RSV cpts530 mutant in the upper respiratory tract of mice (Crowe et al., Vaccine 12 (8), 691-699 (1994a)). Surprisingly, the addition of the F456L mutation to either rcp45 _L or rcp45 resulted in a recombinant whose replication was significantly more restricted in the upper respiratory tract. This indicates that the F456L mutation can greatly enhance the attenuation of replication in both hamster upper respiratory tracts of the mutant virus.

[0186]

[Table 7]

To determine whether the rcp45-456 transduction mutant elicits an immune response,
Rcp45-456 to seronegative hamster, rcp45, or L15 of medium 10 ^6.0 TCID ₅₀ were IN administration. 42 days later, serum samples were collected and on day 44 the animals were challenged with rwt. rcp
Immunization with 45-456 or rcp45 elicited moderate or high titers of serum HAI antibodies, respectively, and induced resistance to rwt challenge virus replication (Table 7). rcp4
The level of antibody and resistance induced by infection with 5-456 was less than that induced by rcp45. The lower level of immune response and protection conferred by rcp45-456 may reflect its significantly lower level of replication in the hamster airways.

[0188]

[Table 8]

The rcp45-456 mutant is more attenuated than rcp45 in chimpanzees Since the rcp45-456 mutation appeared to be over attenuated in hamsters,
We sought to determine its level of replication and immunogenicity in chimpanzee, a non-human primate most closely related to humans. In two separate confirmation experiments, groups of chimpanzees received either rcp45-456 or rcp45 at 10 ^6.0 TCID ₅₀ per site.
Was inoculated with IN and IT. On day 10 and day 13 after infection, tracheal lavage samples and nasopharyngeal swabs were collected, respectively, and the titer of virus in each specimen was determined. rcp45-456
And rcp45 are much more restricted in replication in the upper and lower respiratory tract compared to PIV3 wt (Table
8), each mutant virus was shown to be attenuated for replication in chimpanzees. As described above (Table 6), the effect of adding the F456L mutation to cp45 reduced replication by more than 2500-fold in the upper respiratory tract of hamsters. In contrast, when evaluated in chimpanzees, this effect was only a five-fold decrease in both the upper and lower airways (Table 8 and FIG. 2). This difference was observed in two independent experiments. In the first experiment two animals received rcp45, four animals received rcp45-456, and in the second experiment each virus was administered to two animals.

[0190]

[Table 9]

A comparison of the daily variability pattern of virus efflux in the upper respiratory tract showed that rcp45-456 virus reached lower peak titers compared to rcp452, but its efflux decayed more slowly (Figure 2). This pattern was previously shown to be characterized by increased levels of attenuation of RSV mutants in non-human primates (Prince et al., Infect. Immun. 26 (3): 1009-13, 1979). This increased level of attenuation was also reflected in significantly lower mean peak rhinorrhea scores (Table 8).
). Thus, introduction of the F456L mutation into rcp45 resulted in a gradual increase in attenuation of the chimpanzee airways.

The ts phenotype of rcp45 and rcp45-456 is maintained after replication in chimpanzees The temperature-sensitive levels of isolates from chimpanzees infected with rcp45-456 and rcp45 were tested and the two sets were tested. After replication in a highly permissive host the t
It was determined whether to retain the s phenotype. Due to the large number of isolates obtained, the isolates were evaluated in two trials. The analysis of isolates from each two animals from Experiment 1 is shown in Table 9 and a summary of all animals is shown in Table 10. The temperature sensitivity levels of the same control virus suspension differed by 1 ° C. in the two experiments (Table 10), indicating experimental variability in this assay. For example, the temperatures at which no plaque was observed with rcp45-456 and rcp45 were 38 ° C. and 39 ° C. in Experiment 1, respectively, and 39 ° C. and 40 ° C. in Experiment 2, respectively (Table 10). This degree of experimental variability is sometimes observed. Importantly, the difference in plaque formation shutoff temperature between rcp45-456 and rcp45 was consistently 1 ° C. in both experiments. Due to the experimental variability between the two tests, they are summarized separately in Table 10. Each isolate retained the ts phenotype and the temperature-sensitive levels of the isolates were not different from those of the control input virus throughout the course of replication in chimpanzee airways. Importantly, the proportion of rcp45-456 isolates plaque forming at 38 ° C. and 39 ° C. is lower than that of rcp45 isolates, in v
The higher level of temperature sensitivity of rcp45-456 observed in itro was shown to be maintained after replication in vivo.

[0193]

[Table 10]

[0194]

[Table 11]

The above results indicate that the mutation in RSV cpts530, ie, the replacement of the parent phenylalanine at position 521 of the L protein, is as temperature sensitive and attenuated as when introduced into the corresponding position (456) of the L protein of PIV3. Indicates that a level is given. RSV and PIV
3 represents individual subfamilies of the Paramyxoviridae family, namely Pneumovirus and Paramyxovirus, respectively. Both viruses have, in particular, the amino terminus (aa422-938 of RSV).
And PIV3 357-896) and Poch et al. (J. Gen. Virol. 5: 1153-62)
(1990); and Stec et al. Virology 183: 273-287 (1991)), a region of about 540-570 amino acids encompassing the four highly conserved polymerase motifs. Share a statistically significant sequence relatedness (Stec et al. Virology 183: 273-287 (1991)). The mutation described above is RSV 521
Although present in this general region at position 456 of PIV3, it is approximately 175 residues upstream of the first conserved motif identified by Poch et al. Notable is that
This residue is strictly conserved among the 13 heterologous paramyxoviruses tested in this study, with the leucine residue located 12 residues from the C-terminus. (Figure 1, Panel A). Despite this stringent conservation, if the 521 L mutation was previously identified in the RSV cpts mutant and the mutation was not successfully introduced into the conserved position of the HPIV3 L protein, PIV3 L Among the 2233 positions in the amino acid sequence of the protein, it would not have been possible to predict that this residue would be a suitable target site for mutagenesis to produce a conditionally lethal attenuated recombinant . In light of the above results, it is possible to extend the method of the invention to include a number of attenuating mutations recently identified among diverse members of mononegaviruses, especially paramyxoviruses (Bukreyev et al. ., J. Virol. 71 (1
2), 8973-8982; Garcin et al., Virology 238 (2): 424-431 (1997); Juhasze
t al., Vaccine 17: 1416-1424 (1999); Juhasz et al., J. Virol. 71 (8): 58
14-5819 (1997); Kato et al., EMBO J. 16 (3): 578-587 (1997a); Kato et al.
., J. Virol. 71 (10): 7266-7272 (1979b); Kondo et al., J. Biol. Chem. 26
8 (29): 21924-21930 (1993); Kurotani et al., Genes Cells 3 (2): 111-124
(1998); Skiadopoulos et al., J. Virol. 72 (3): 1762-8 (1998); Whitehead
et al., J. Virol. 73: 871-877 and 73: 3438-3442 (1999); Whitehead et al.
., Virology 247 (2): 232-239 (1998a); Whitehead et al., J. Virol. 73 (2).
: 871-877 (1999b); Whitehead et al., J. Virol. 72 (5): 4467-4471 (1998b)
, Both of which are incorporated herein by reference).

The above findings provide new PIV vaccine candidates and also allow the introduction of attenuating mutations from RSV into other paramyxoviruses. Here the mutations are mapped to conserved parent residues / positions. Further in this context, the 521L mutation in RSV
Following hemagglutination of wt PIV3-transfer of the neuraminidase (HN) and F genes to a recently developed chimeric PIV3 recombinant in which the PIV1 was replaced by that of PIV1 (Tao et al., J. V.
irol. 72 (4), 2955-2961 (1998), which is incorporated herein by reference). This chimeric recombinant can be further attenuated by the incorporation of one or more additional mutations identified in the cp45 mutant, but this mutation is now replaced within the JS wt PIV3 background. (Except for the three mutations that occur in the F and HN genes) as such in the infectious, attenuated chimeric PIV3-1 clone that carries the HN and F genes of PIVI.

Example III Heterologous introduction of an attenuating mutation in the C protein of Sendai virus into a recombinant HPIV3 vaccine candidate This example demonstrates the substitution of phenylalanine (F) at position 170 with serine (S). A known attenuating mutation in the C protein of Sendai virus (SeV), characterized by (Itoh et al., J. Gen. Virol. 78: 3207-3215 (1997), incorporated herein by reference. Heterologous introduction into the corresponding position in the recombinant HPIV3 clone. As shown above and in Table 4, the parental sequence element of F170 maps to F164, an identically conserved sequence position / residue in the C protein of HPIV3, but this element is also conserved in SeV and BPIV-3. Have been.

By introducing a point mutation into the HPIV3 CORF, the SeV F170S mutation was introduced into the recombinant HPIV3 virus rF164S, and the amino acid at position 164 was changed from phenylalanine (F) to serine (S). Changed. The resulting rF164S recombinant was surprisingly more attenuated in the upper respiratory tract than in the lower respiratory tract. This pattern is opposite to that seen with a temperature-sensitive attenuating mutation, whereby inclusion of this novel mutation in a recombinant live attenuated vaccine virus reduces residual virulence in the upper respiratory tract Would prove useful. This rF164S recombinant also conferred protection against challenge with wild-type HPIV3.

The P and C proteins of HPIV3 are translated from separately overlapping ORFs of the mRNA (FIG.
3). All paramyxoviruses encode the P protein, whereas only members of the genus Respirovirus and Morbillivirus encode the C protein. Each virus has the number of proteins expressed from the CORF and the number of proteins in the virus.
They vary widely in the importance of replication in vitro and in vivo. HPIV3 and measles virus (MeV) express only a single C protein (Bellini et al., J. Vir
ol. 53: 908-19 (1985); Sanchez et al., Virology 147: 177-86 (1985); Spri.
ggs et al., J. Gen. Virol. 67: 2705-2719 (1986)), whereas Sendai virus (SeV) contains four proteins, C 'and C, which begin to be read independently of the CORF. , Y
Expresses 1 and Y2 (these translation initiation sites appear in the mRNA in this order)
(Curran, et al., Enzyme 44: 244-9 (1990); Lamb et al., P. 181-214, D. K.
ingsbury (ed.), The Paramyxoviruses, Plenum Press, New York (1991)).

A viable recombinant SeV in which all four C-derived proteins have been eliminated is
Was found to replicate very inefficiently (Kurotani et al., Genes Cel
ls 3: 111-124 (1998)), and individual elimination of the C protein had a complex effect (Cadd et al., J. Virol. 70: 5067-74 (1996); Curran et al., Virology 189).
: 647-56 (1992); Latorre et al., J. Virol. 72: 5984-93 (1998)).

Recombinant SeV, which carries a single point mutation resulting in a phenylalanine (F) to serine (S) substitution at position 170 of the C protein was attenuated in mice, but in cell culture, its replication was Not inhibited (Garcin et al., Virology 238: 424-431 (19
97); Itoh et al., J. Gen. Virol. 78: 3207-15 (1997)). In sharp contrast to SeV, C (-) measles virus (MeV) replicated efficiently in Vero cells (Rad
ecke et al., Virology 217: 418-21 (1996)), which showed limited replication in human peripheral blood and only a modest attenuation in vivo (Escoffier et al., J
Virol. 73: 1695-8 (1999); Valsamakis et al., J. Virol. 72: 7754-61 (19
98)).

The altered replication of various MeV and SeV C mutants in animals indicates that this protein is a potential target for the introduction of attenuating mutations useful in the development of live attenuated HPIV3 vaccines Suggests. In this example, using the method of reverse genetics, a mutation was introduced into the HPIV3 CORF to create an F → S change at 162 amino acids, which is a heterologous attenuated SeV mutant. Then F at the amino acid at position 170
→ It corresponds to S change.

Cells and Viruses HEp-2 and Simian LLC-MK2 monolayer cell cultures were prepared using OptiMEM I (Life Technologies) supplemented with 2% fetal serum, gentamicin sulfate (50 μg / mL) and 4 mM glutamine.
, Gaithersburg, MD). The modified vaccinia strain Ankara (MVA) recombinant virus expressing the bacteriophage T7 polymerase is
Courtesy of Drs. Wyatt and B. Moss (Wyatt et al., Virology, 21
0: 202-205 (1995)). JS wild-type (wt) PIV3 strain and its attenuated ts derivative, JS
LLC-MK2 was used as previously reported (Hall et al., Virus Res. 22: 173-184 (1992)).
Proliferated in cells.

A full-length cDNA clone encoding the complete 15462 nt antigenome {p3 / 7 (131) 2G} of the cDNA JS wt virus was previously described (Genbank accession number: # Z11575) (D
urbin et al., Virology 235: 323-332 (1997)). This clone was used as a template to construct a mutated cDNA encoding a phenylalanine to serine change at amino acid position 164 of the CORF (Table 11, FIG. 3). Pml1-Ba of p3 / 7 (131) 2G
Plasmid pUC119 ｛pUC119 (Pml1-BamHI) in which the mHI fragment (nt1215-3903 of the PIV3 antigenome) has been modified to include a PmlI site in the multiple cloning region.
) Subcloned into ①. Then, the method of Kunkel (Kunkel et al., Methods E
Site-directed mutagenesis was performed on pUC119 (Pml1-BamHI) using nzymol. 154: 367-382, 1987) to introduce various mutations into the CORF.

[0205]

[Table 12]

Antigenome encoding a virus with the F164S mutation in the C ORF (rF164S)
cDNA Construction Using a mutagenic primer to introduce an A to G change at position nt2284 of the full-length antigenome, the phenylalanine (F) to serine (S) change at amino acid position 164 of the CORF was detected. Created. This mutation was also silent in the PORF. The PmlI-BamHI fragment of this full-length clone was sequenced as-is to confirm the mutations introduced and to confirm that no other mutations were accidentally introduced. This fragment was then previously reported (Durbin et al., Virology 235: 323-332 (1997)).
) And cloned back to the full-length clone p3 / 7 (131) 2G to create an antigenomic cDNA clone.

Recovery of Recombinant CF164S Mutant from cDNA The full-length antigenomic cDNA carrying the CF164S mutation was ligated to LipofectACE (Life Tec
hnologies) using the support plasmids ｛pTM (N), pTM (P no C) and pT
Transfect HEp-2 cells in 6-well plates (Costar, Cambridge, MA) with M (L)｝ and use MVA-T7 as described previously (Durbin et al., Virology 235: 323-332, 1997). Infected. pTM (P no C) was previously reported (Durbin et al., Virology
234: 74-83 (1997)), except that the C transcription start site is mutated from AUG to ACG and the first amino acid of the CORF is changed from methionine to threonine. ing. After incubation at 32 ° C. for 3 days, the transfection harvest was passaged on fresh LLC-MK2 cell monolayers in T-25 flasks and incubated at 32 ° C. for 5 days (referred to as passage 1). The amount of virus present in the F164S harvest is visualized by immunoperoxidase staining using a PIV3 HN-specific monoclonal antibody, as previously reported (Durbin et al., Virology 235: 323-332 (1997)). Determined by plaque titer assay on LLC-MK2 monolayer cultures with plaques.

The F164S recombinant virus present in the supernatant of the passage 1 harvest was previously reported (Hall et al.,
Plaque purification was performed three times on LLC-MK2 cells as in Virus Res. 22: 173-184 (1992). The biologically cloned recombinant virus from the third plaque purification was amplified twice at 32 ° C. in LLC-MK2 cells to produce the virus for further characterization.

Sequence Analysis of Recovered Recombinant Virus As described previously (Durbin et al., Virology 235: 323-332 (1997)), virus was purified by polyethylene glycol precipitation from a medium clarified from a monolayer of infected cells. Was concentrated. Following the manufacturer's recommendations, use this RNA with TRIzol reagent (Life Technologi
es). Advantage RT for PCR kit (Clontech, Palo Alto, CA
), RT-PCR was performed by the recommended method. The control reaction was identical except that the reverse transcriptase was omitted from the reaction, confirming that the PCR product was derived only from the viral RNA and not from any possible mixed cDNA plasmid. A variety of primers were used to generate PCR fragments spanning nucleotides 1595-3104 of the full-length antigenome. This fragment contains the entire C, D and V ORF of the recombinant virus. Then, cycle dideoxynucleotide sequence analysis (New Englan
d Biolabs, Beverly, MA) was used to determine the sequence.

Analysis of Proteins by Immunoprecipitation Two different C-peptides (Research Gene
tics, Huntsville, AL) were raised in rabbits. The two peptides spanned the amino acid regions 30-44 and 60-74 of the C protein. Eight copies of each C-peptide were placed on a branched carrier core and injected separately into rabbits (2 rabbits per peptide) with Freund's adjuvant. The rabbits were boosted at weeks 2 and 4 with the indicated MAPs and bled at weeks 4, 8, and 10. Both antisera recognized the C peptide used as an immunogen with high titer and precipitated the C protein from HPIV3-infected cells in a radioimmunoprecipitation assay (RIPA).

[0211] T25 cell monolayers of LLC-MK2 cells were infected with either rF164S or recombinant JS wt virus (rJS) at a multiplicity of infection of 5 or mock infected and incubated at 32 ° C. Twenty-four hours after infection, the monolayer was washed with methionine (-) DMEM (Life Technologies) and incubated for an additional 6 hours in methionine (-) DMEM in the presence of 10 μCi / μl ³⁵ S-methionine. The cells were then harvested, washed three times, and 1 ml of RIPA buffer ｛1% (w / v) sodium deoxycholate, 1% (v / v) Triton X-100, 0.1%.
Resuspended in 2% (w / v) SDS, 150 mM NaCl, 50 mM Tris-HCl, pH 7.4｝, freeze-thaw, and pelleted at 65000 × G. The cell extract was transferred to a fresh Eppendorf tube, and a mixture of both C antisera (5 μl each) was added to each sample and incubated at 4 ° C. for 2 hours with constant mixing. Recognizes HIV glycoprotein of HPIV3 (van
Wyke Coelingh et al., J. Virol. 61: 1473-1477 (1987)), mAb 454/11 and 1
10 μl of the 01/1 mixture was added to each sample to confirm that the recovered virus was indeed HPIV3. Add Protein A Sepharose beads (Sigma,
The immune complexes were precipitated by adding 200 μl of a 10% suspension (St. Louis, MO) and mixing constantly at 4 ° C. overnight. Each sample was denatured, reduced, and 4-12% polyacrylamide gel (NuPAGE, Novex, San Diego, CA, recommended by the manufacturer).
). The gel was dried and analyzed by autoradiography.

Multi-Cycle Replication of rPIV3 A monolayer of LLC-MK2 cells in a T25 flask was infected with rF164S or rJS at the MOI of 0.1 with the same two specimens and incubated at 32 ° C. in 5% CO ₂ . A 250 μl sample was taken from each flask every 24 hours for 5 consecutive days and replaced with the same amount of fresh medium at each time point. Each sample was incubated at 32 ° C for 7 days in a LLC-MK2 cell monolayer in a 96-well plate and assayed for titer. Virus is detected by blood cell adsorption and log ₁₀ TCID ₅₀ / ml
Recorded as.

Animal Testing Groups of 21 Golden Syrian hamsters, 4-6 weeks of age, received rF164S, rJS, cp45 (
Live attenuated derivative of JS wt virus induced biological), or EMEM containing 10 ⁵ PFU of respiratory syncytial virus (RSV) and (Life Technologies) for each animal
0.1 ml was inoculated intranasally. On days 3, 4 and 5 after the inoculation, except for the hamsters receiving RSV, 5 hamsters in each group were sacrificed, and the lungs and turbinates were collected. Homogenize the nasal turbinate and lung, and 10% and 20% (w / v) of L-15 (Quality Biologicals)
, Gaithersburg, MD) suspension was prepared and the sample was frozen immediately.
For viruses present in the sample, 3 in a 96-well plate LLC-MK2 cell monolayer
Incubated at 2 ° C for 7 days and assayed for titer. The virus was detected by blood cell adsorption, and the average log ₁₀ TCID ₅₀ / g was calculated for each day of each group of 5 hamsters. Serum was collected from the remaining six animals in each group on days 0 and 28 after inoculation. Serum antibody responses to each virus were reported previously (van Wyke Coelingh et al., Virology 143: 569-582 (1985)).
) Was evaluated by the hemagglutination inhibition (HAI) assay. On day 28, the remaining hamsters in each group, including those immunized with RSV, were tested for biologically-derived PIV3 JS wt virus.
Intranasal challenge with 10 ⁶ PFU. Animals were sacrificed 4 days post-challenge and lungs and turbinates were harvested and processed as described above. The amount of virus present in the challenge sample was determined as described above.

[0214] Groups of four African Green Monkeys (AGM) each received 10 rF164S, JSwt, or cp45. ⁶ PFU was used in previous studies in rhesus monkeys (Durbin et al., Vaccine 16: 1324-30 (
1998)). Inoculated intranasally and intratracheally. 12 days after inoculation
Consecutive nasopharyngeal swab samples were collected and tracheal washed on days 2, 4, 6, 8, and 10 after ingestion
A clean sample was taken. This sample is flash frozen until all samples have been collected.
Stored at -70 ° C. Incubate the virus present in the sample at 32 ° C for 7 days.
The titer was assayed on LLC-MK2 cell monolayers from 96-well plated plates. Blood cell adsorption
Virus, and average log per day_TenTCID₅₀/ Ml was calculated. Inoculation
On days 0 and 28, sera were collected from each monkey and various mutants and PIV3 wild-type (JS)
The response of the PIV3 HAI antibody to the experimental infection used was determined. 28 days after inoculation,
10 biologically derived PIV3 wild-type viruses⁶PFU was intranasally and intravenously with 1 ml of inoculum.
AGM was challenged by intravenous administration. 0, 1, 2, 4, 6, 8, 10 and after challenge
Nasopharyngeal swab samples were collected on days 12 and 12, and on days 2, 4, 6, 8, and 10 after challenge
A tracheal lavage sample was taken on day 1. Sample is flash frozen, stored, and virus present
Was titered as described above.

Results Recovery of Recombinant Mutant rF164S An antigenomic cDNA of HPIV3 encoding the mutant virus rF164S, in which aa residue 164 of the C protein was changed from F to S, was prepared. This mutation causes the overlapping P
The ORF was translationally silent (Table 11).

The antigenome cDNA was transformed into three PIV3 support plasmids ｛pTM (P no C), pTM (N
), PTM (L)｝, and transfected into HEp-2 cells.
Co-infection was performed with MVA expressing A polymerase. R containing mutations in the C ORF
The recovery of PIV3 was similar to that of similarly transfected-infected HEp-2 cell culture,
p3 / 7 (131) 2G [plasmid expressing the full-length PIV3 antigenome from which recombinant JSwt PIV3 (rJS) was recovered (Durbin et al., Virology 235: 323-332 (1997
))]. After incubation at 32 ° C. for 3 days, the transfected cells were harvested, and the supernatant was subcultured on a fresh LLC-MK2 cell monolayer in a T-25 flask and incubated at 32 ° C. for 5 days ( Passage 1). After 5 days at 32 ° C., rJS and rF164S LLC-MK2 cell monolayers showed 3-4 + cytopathic effects (CPE). After three biological clonings by plaque isolation, the recombinant mutant was
Amplified twice in LC-MK2 cells to produce a suspension of the virus for further characterization.

To confirm that the recovered virus was indeed the predicted rF164s mutant, the HPIV3 antigenome containing the P gene portion containing the C, D and V ORFs
RT-PCR using primer pairs to amplify fragments of DNA spanning nt1595-3104
Analyzed the cloned virus. Production of the PCR product was dependent on the inclusion of RT, indicating that it was derived from RNA and not from mixed cDNA. Nucleotide sequencing was performed on this RT-PCR product to confirm the presence of the introduced mutation. The introduced mutation was confirmed to be present in the RT-PCR fragment spanning nt1595-3104 amplified from the cloned recombinant, and no other accidental mutations were found.

A radioimmunoprecipitation assay (RIPA) was performed to convert the rF164S mutant virus to rJS wt
Compared to virus. Cells were infected and incubated in the presence of ³⁵ S-methionine for 24-30 hours post infection to prepare cell lysates. Equal amounts of all proteins were incubated with anti-C and anti-HN antibodies, and then the antibodies were bound to protein A sepharose beads. Both rJS and rF164S encoded HN and C proteins (FIG. 4). The C protein expressed by rF164S was the same size as that expressed by the parent rJS, and was expressed in the same amount (FIG. 4).

Replication of rF164S in Cell Culture The same two LLC-MK2 cell monolayer cultures were infected with rF164S or rJS at a MOI of 0.1 and
Incubated at 2 ° C. for 5 days. Medium was sampled from each culture every 24 hours, and the titer of this material was later assayed to evaluate the replication of each virus in the cell culture. Replication of rF164S was virtually indistinguishable from that of the parental rJS wt with respect to both the rate of production and final titer of the virus, as well as its ability to replicate at elevated temperatures.

Replication of rF164S in Hamsters Next, the rF164s mutant virus was compared to the parental rJS wt for replication ability in the upper and lower respiratory tracts of hamsters. Groups of hamsters, rF164S or cp45 is a vaccine candidates derived biologically inoculated in 10 ⁵ pfu intranasally per animal. rJS
RF164S replication was reduced 100-500-fold in the upper respiratory tract and more than 10-fold in the lower respiratory tract of hamsters (Table 12). Hamsters infected with rF164S and rJS are H
It had a significant antibody response to PIV3, indicating a high level restriction of PIV3 challenge virus replication.

[0221]

[Table 13]

[0222]

[Table 14]

Replication of rF164S in primates To further evaluate the attenuated phenotype and protective efficacy of rF164S, the recombinant mutant virus was administered to groups of four AGM and their replication in upper and lower respiratory tract To
It was compared with that of cp45 and JSwt. rF164S replication was reduced 100-fold or more in the upper respiratory tract of AGM. The attenuation observed in the upper respiratory tract for this virus was cp45
(Table 14). rF164S was moderately (<10-fold) restricted in the lower respiratory tract of AGM. This recombinant virus elicited a HAI antibody response to HPIV3 (Table 14). On day 28, the AGM immunized, IN and IT to 10 ⁶ PFU of JS wt virus derived biologically
And challenged. Animals receiving the rF164S or cp45 vaccine candidate virus fully protected against replication of the challenge virus in both the upper and lower respiratory tracts of AGM.

[0224]

[Table 15]

Summarizing the above examples, the F170S mutation in SeV was biologically produced by adapting the virus to growth in LLC-MK2 simian cells (
Garcin et al., Virology 238: 424-431 (1997), and Itoh et al., J. Gen. V
irol. 78: 3207-15 (1997)). Recombinant SeV with the F170S mutation shows enhanced replication in vitro, but this mutant is very restricted in replication in mice as replication of the SeV C '/ C mutant is inhibited (Garcin et al., Virology 238: 424-431 (1997) and Latorre et al., J. Vi.
rol. 72: 5984-93 (1998)). Since the C proteins of SeV and HPIV3 are only 38% homologous, it was surprising to find that transferring the F170S mutation of SeV into HPIV3 caused attenuation in vivo.

The recombinant HPIV3, rF164S, is responsible for mutating the amino acid residue of HPIV3 corresponding to position 170 of SeV and was not restricted in replication in vitro, but was associated with the upper respiratory tract of hamsters. In both lower orbits, AGM was significantly attenuated in the upper and lower airways. rF164S elicited a serum antibody response comparable to wtPIV infection and completely protected both hamsters and AGM against challenge with wt HPIV3. The above results indicate that the rF164S mutation would be useful in developing a live attenuated vaccine against HPIV3.

Storage of Biological Materials The following materials are stored in VA, Manassas, Boulevard University, 10801, American Type Culture Collection, 20110-22092, under the terms of the Budapest Treaty. Virus Accession number Storage date p3 / 7 (131) 2G (ATCC 97989) April 18, 1997 p3 / 7 (131) (ATCC 97990) April 18, 1997 p218 (131) (ATCC 97991) April 1997 18 cpts RSV 248 (ATCC VR 2450) March 22, 1994 cpts RSV 530/1009 (ATCC VR 2451) March 22, 1994 RSV cpts530 (ATCC VR 2452) March 22, 1994 cpts RSV 248/955 (ATCC VR 2453) March 22, 1994 cpts RSV 248/404 (ATCC VR 2454) March 22, 1994 cpts RSV 530/1030 (ATCC VR 2455) March 22, 1994 RSV B-1 cp52 / 2B5 (ATCC VR 2542) September 26, 1996 A2 ts530-s c11cp or ts530-sites (ATCC VR 2545) October 17, 1996 RSV B-1 cp-23 (ATCC VR 2579) July 15, 1997.

Although the foregoing invention has been described in some detail by way of figures and examples for clarity of understanding, it will be apparent that certain changes and modifications may be practiced within the scope of the appended claims. .

[Sequence list]

[Brief description of the drawings]

BRIEF DESCRIPTION OF THE FIGURES FIG. 1, Panel A shows RSV (SEQ ID NO: 1) containing RSV Phe-521 (indicated by the arrow).
NO. 44), PIV3 (SEQ ID NO. 45), measles (SEQ ID NO. 46) and Sendai (SEQ.
ID NO. 47) Provides alignment of the amino acid sequence of the viral L polymerase protein. A consensus sequence (SEQ ID NO. 48) was generated from an exact match of at least three residues of each protein. Shows the numerical position of the first amino acid in the sequence shown. Residues conserved in all four viruses are shown in bold. Underlined residues are PIV2, canine distemper virus, simian parainfluenza virus
41, Simian parainfluenza virus 5, avian pneumovirus, Newcastle disease virus, Hendra virus, and Linder pest virus (accession numbers: P26676, P24658, P35341, Q88434, Y09630, U665312, X05399, respectively)
, AF017149, P41357) are also conserved. RSV cpt
Mutation of phenylalanine at position 521 to leucine in s530 corresponds to aa456 in LIV polymerase of PIV3. FIG. 1, Panel B provides a schematic of the F456L and cp45 mutations introduced into PIV3 rwt. The relative position of the introduced cp45 mutation is ( ^* ) and the F456L mutation is (◆). FIG. 1, panel C shows the F456L mutation (SEQ ID NO. 49) compared to the wt sequence (SEQ ID NO. 49).
No. 50) (nucleotide sequence ((+) strand)). The PIV3 nucleotide sequence numbered by the complete rwt antigenome is shown for the wt virus and the sequence of the mutant is shown below. Nucleotide changes are underlined and the codon responsible for the F456L mutation is shown in bold. The Xmn1 restriction endonuclease recognition site introduced into the mutant sequence is shown in italics.

FIG. 2 shows replication of rcp45 and rcp45-456 in chimpanzee upper respiratory tract. Average virus titers in nasopharyngeal swab specimens collected at the indicated days after infection from animals infected with rcp45-456 (□; n = 6) or rcp45 (■ (closed square); n = 4) Indicates a value. The statistically significant differences between the indicated values are: (a) p <0.005; (b)
p <0.05; (c) p <0.025 (Student's t-test). (D) The detection limit is
0.5 log ₁₀ TCID ₅₀ / ml or less.

FIG. 3 illustrates the organization of the HPIV3 P / C / D / V ORF (scale not correct). Shows three reading frames of P mRNA (+1, +2 and +3), P, C,
The ORF of D and V is represented by a rectangle. Indicates the length of the amino acid. The location of the RNA editing site is shown as a vertical line, its sequence motif is shown, and is numbered by nucleotide position in the complete HPIV3 antigenome sequence. FIG. 3, Panel A, illustrates the organization of unedited P mRNA. The sequences containing the translation start sites of the P and C ORFs are shown and are numbered by the complete antigenome sequence. The nucleotide positions of the P, C, D and V ORFs in the complete antigenome sequence are: P, 1784-359
5; C, 1794-2392; D, 2442-2903; V, 2792-3066. The AUG that opens the P ORF for P mRNA is at positions 80-82. FIG. 3, Panel B, illustrates the organization of an edited version of a P mRNA containing an insertion of two non-template G residues (GG) (SEQ ID NO. 53) at the editing site (SEQ ID NO. 51). This changes the reading register so that the upstream end of the P ORF at frame +2 fuses with the D ORF at frame +3. The resulting chimeric protein contains the N-terminal 241 amino acids encoded by the P ORF fused to the C-terminal 131 amino acids encoded by the D ORF.

FIG. 4 shows the results of a radioimmunoprecipitation assay showing expression of the C protein in rF164S. In lane (a), ³⁵ S-labeled cell lysates were immunoprecipitated with a polyclonal C-specific rabbit antiserum. A 22 kD band corresponding to the C protein (open arrow) is clearly present in the rJS and rF164 lysates. Lane (b)
In, cell lysates were immunoprecipitated with a mixture of two monoclonal antibodies specific for the HPIV3 HN protein. The presence of a 64 kD band (closed arrow) corresponding to the HN protein in each virus lysate confirmed that they were actually HPIV3 and expressed the same level of protein. Sham lanes show lysates of tissue culture taken from uninfected cells.

FIG. 5 shows the recombinant mutant virus rF164S compared to the parent virus rJS.
2 shows the results of multi-cycle replication. Virus titers are given as TCID ₅₀ / ml,
Average of two identical samples.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) C12R 1:93) (81) Designated country EP (AT, BE, CH, CY, DE, DK, ES, FI) , FR, GB, GR, IE, IT, LU, MC, NL, PT, SE), OA (BF, BJ, CF, CG, CI, CM, GA, GN, GW, ML, MR, NE, SN , TD, TG), AP (GH, GM, KE, LS, MW, SD, SL, SZ, TZ, UG, ZW), EA (AM, AZ, BY, KG, KZ, MD, RU, TJ, TM), AE, AG, AL, AM, AT, AU, AZ, BA, BB, BG, BR, BY, CA, CH, CN, CR, CU, CZ, DE, DK, DM, Z, EE, ES, FI, GB, GD, GE, GH, GM, HR, HU, ID, IL, IN, IS, JP, KE, KG, KP, KR, KZ, LC, LK, LR, LS , LT, LU, LV, MA, MD, MG, MK, MN, MW, MX, NO, NZ, PL, PT, RO, RU, SD, SE, SG, SI, SK, SL, TJ, TM, TR, TT, TZ, UA, UG, US, UZ, VN, YU, ZA, ZW (72) Inventor Durbin, Ana Pi 20912, Maryland, USA, Tacoma Park, Hudson Avenue 806 (72) Invention Sukiadopoulos, Mario H. 20854, Maryland, USA, Accord Road, Potomac, 8303 F-term (reference) 4B024 AA01 BA32 CA04 CA06 DA02 EA02 FA02 GA11 HA01 HA17 4B065 AA97X AA97Y AB01 AC20 BA01 CA44 CA45 4C085 AA03 BA57 CC08 DD01 DD62 EE01 EE05

Claims (126)

[Claims] 1. An isolated and attenuated recombinant (-) strand (negative stranded)
A) A method for producing said (-) strand RNA virus from one or more isolated polynucleotide molecules encoding an RNA virus, comprising: encoding a recombinant genome or antigenome in a cell or cell-free system. Co-expressing one or more expression vectors comprising one or more polynucleotide molecules and essential viral proteins required to produce infectious viral particles of said recombinant (-) strand RNA virus. Wherein the recombinant genome or antigenome comprises, within the recombinant protein of the recombinant virus, an amino acid position corresponding to the amino acid position of the attenuating mutation identified in the heterologous mutant (-) strand RNA virus. Modified to encode the mutation at Such a method, wherein said recombinant virus is given an attenuated phenotype. 2. The method according to claim 1, wherein the recombinant (−) strand RNA virus is a respiratory syncytial virus (RS).
2. The method of claim 1, wherein V). 3. The RSV subgroup A, human RSV subgroup B, bovine RSV, mouse RS
3. The method of claim 2, wherein the method is V or avian pneumovirus. 4. The heterologous mutant (−) strand RNA virus is a heterologous RSV, human parainfluenza virus (HPIV) 1, HPIV2, HPIV3, bovine PIV (BPIV), Sendai virus (SeV), Newcastle disease virus (NDV) ), Simian virus 5
(SV5), mumps virus (MuV), measles virus (MeV), canine distemper virus (CDV), rabies virus (RaV), or vesicular stomatitis virus (VSV)
3. The method of claim 2, wherein the method is: 5. The method according to claim 5, wherein the heterologous mutant (−) strand RNA virus is RSV cpts248 (ATC
CVR 2450), RSV cpts248 / 404 (ATCC VR 2454), RSV cpts248 / 955 (ATCC VR
2453), RSV cpts530 (ATCC VR 2452), RSV cpts530 / 1009 (ATCC VR 2451)
, RSV cpts530 / 1030 (ATCC VR 2455), RSV B-1 cp52 / 2B5 (ATCC VR 2542),
5. The method according to claim 4, which is RSV B-1 cp-23 (ATCC VR 2579). 6. The recombinant genome or antigenome is RSV NS1, NS2, N, P,
3. The method of claim 2, wherein the method is modified to encode an amino acid substitution, deletion or insertion within an M, SH, M2 (ORF1), M2 (ORF2), L, F or G protein. 7. The mutation incorporated within the recombinant protein confers a temperature sensitive (ts) phenotype on the recombinant (−) strand RNA virus.
The method described in. 8. The method according to claim 8, wherein the mutation incorporated inside the recombinant protein is
2. The method of claim 1, wherein the method comprises an amino acid substitution within a RSV L protein. 9. The attenuating mutation identified in the heterologous mutant (−) strand RNA virus comprises an amino acid substitution of phenylalanine at position 521 of the L protein of human RSV cpts530 (ATCC VR 2452). 8. The method according to 8. 10. The method of claim 1, wherein said recombinant (−) strand RNA virus is parainfluenza virus (PIV). 11. The method according to claim 11, wherein the PIV is human PIV1 (HPIV1), human PIV2 (HPIV2), human PIV.
11. The method according to claim 10, wherein the method is IV3 (HPIV3), bovine PIV (BPIV), or mouse PIV (MPIV). 12. The method according to claim 12, wherein the heterologous mutant virus is a respiratory syncytial virus (RS).
V), human parainfluenza virus (HPIV) 1, HPIV2, HPIV3, bovine PIV (BPI
V), Sendai virus (SeV), Newcastle disease virus (NDV), Simian virus 5 (SV5), mumps virus (MuV), measles virus (MeV), dog
11. The method according to claim 10, wherein the method is distemper virus (CDV), rabies virus (RaV), or vesicular stomatitis virus (VSV). 13. The method according to claim 13, wherein said recombinant genome or antigenome is PIV N, P, C, D, V
11. The method of claim 10, wherein the method is modified to encode amino acid substitutions, deletions or insertions within the M, F, HN or L protein. 14. The method of claim 10, wherein the mutation incorporated within the recombinant protein confers a temperature sensitive (ts) phenotype on the recombinant (−) strand RNA virus. 15. The heterologous mutant (−) strand RNA virus is HPIV3 JS cp45.
11. The method of claim 10, wherein 16. The attenuating mutation identified in the heterologous mutant (-) strand RNA virus comprises an amino acid substitution within a HPIV3 JS cp45 L protein.
The method according to claim 15. 17. The method of claim 16, wherein the attenuating mutation identified in the heterologous mutant (-) strand RNA virus comprises a tyrosine amino acid substitution at position 942 of the L protein of HPIV3 JS cp45. . 18. The method of claim 16, wherein the attenuating mutation identified in the heterologous mutant (-) strand RNA virus comprises a leucine amino acid substitution at position 992 of the L protein of HPIV3 JS cp45. . 19. The method of claim 16, wherein the attenuating mutation identified in said heterologous mutant (-) strand RNA virus comprises an amino acid substitution of threonine at position 1558 of the L protein of HPIV3 JS cp45. . 20. The attenuating mutation identified in the heterologous mutant (-) strand RNA virus comprises an amino acid substitution within the HPIV3 JS cp45 F protein.
The method according to claim 15. 21. The method of claim 20, wherein the attenuating mutation identified in the heterologous mutant (-) strand RNA virus comprises an amino acid substitution of isoleucine at position 420 of the F protein of HPIV3 JS cp45. . 22. The method of claim 20, wherein the attenuating mutation identified in the heterologous mutant (-) strand RNA virus comprises an amino acid substitution of alanine at position 450 of the F protein of HPIV3 JS cp45. . 23. The attenuating mutation identified in the heterologous mutant (−) strand RNA virus comprises an amino acid substitution of phenylalanine at position 521 of the L protein of human RSV cpts530 (ATCC VR 2452). The method according to 10. 24. The recombinant (−) strand RNA virus is human PIV (HPIV3), and the mutation incorporated into the recombinant protein is an amino acid substitution of phenylalanine at position 456 of the HPIV3 L protein. 24. The method of claim 23, comprising: 25. The method of claim 10, wherein the attenuating mutation identified in the heterologous mutant (-) strand RNA virus comprises an amino acid substitution of phenylalanine at position 170 of the SeV C protein. 26. The recombinant (-) strand RNA virus is human PIV (HPIV3), and the mutation incorporated into the recombinant protein is an amino acid substitution of phenylalanine at position 164 of the HPIV3 C protein. 26. The method of claim 25, comprising: 27. The method of claim 1, wherein said recombinant (−) strand RNA virus is measles virus (MeV). 28. The reproductive syncytial virus (RS)
V), human parainfluenza virus (HPIV) 1, HPIV2, HPIV3, bovine PIV (BPI
V), Sendai virus (SeV), Newcastle disease virus (NDV), Simian virus 5 (SV5), mumps virus (MuV), measles virus (MeV), dog
28. The method of claim 27, wherein the method is distemper virus (CDV), rabies virus (RaV), or vesicular stomatitis virus (VSV). 29. The heterologous mutant (−) strand RNA virus is HPIV3 JS cp45.
28. The method of claim 27, wherein 30. The attenuating mutation identified in the heterologous mutant (−) strand RNA virus is a tyrosine or 9 at position 942 of the L protein of HPIV3 JS cp45.
30. The method of claim 29 comprising a leucine amino acid substitution at position 92. 31. The recombinant (−) strand RNA virus is of one species, subgroup or strain of (−)
) Having a recombinant genome or antigenome comprising part or all of the genome or antigenome of a strand RNA virus in combination with a heterologous gene or gene segment of a (-) strand RNA virus of a different species, subgroup or strain 2. The method according to claim 1, which is a chimeric virus. 32. The method of claim 31, wherein said attenuating mutation is incorporated within a protein or protein region encoded by said heterologous gene or gene segment. 33. A chimeric virus comprising the genome or antigenome of part or all of one PIV species, subgroup or strain at least one of the HN and F glycoprotein genes of a heterologous PIV species, subgroup or strain. Parainfluenza virus (PI) having a recombinant genome or antigenome comprising a combination of two genes or gene segments
32. The method of claim 31, wherein V). 34. The method of claim 33, wherein the human PIV3 HN and F genes are replaced by human PIV1 HN and F genes. 35. A chimeric virus comprising the genome or antigenome of part or all of one RSV species, subgroup or strain, for the F, G and SH glycoprotein genes of a heterologous RSV species, subgroup or strain. Respiratory syncytial virus (RSV) having a recombinant genome or antigenome comprising in combination with at least one gene or gene segment
32. The method of claim 31, wherein 36. The method of claim 35, wherein the human RSV subgroup A F and G glycoproteins are replaced by human RSV subgroup B F and G glycoprotein genes. 37. The recombinant genome or antigenome is further modified to encode one or more additional attenuating mutations derived from a biologically-derived mutant (-) strand RNA virus. 2. The method of claim 1, wherein the method is performed. 38. The recombinant (−) strand RNA virus is a respiratory syncytial virus (RSV).
) Wherein the recombinant genome or antigenome is biologically derived
38. The method of claim 37, wherein the panel encodes at least one and up to a total number of attenuating mutations present in the panel of strains, wherein the panel comprises cpts RSV248 (ATCC VR24).
50), cpts RSV 248/404 (ATCC VR 2454), cpts RSV 248/955 (ATCC VR 2453)
), Cpts RSV 530 (ATCC VR 2452), cpts RSV 530/1009 (ATCC VR 2451), cp
The method comprising ts RSV 530/1030 (ATCC VR 2455), RSV B-1 cp52 / 2B5 (ATCC VR 2542), and RSV B-1 cp-23 (ATCC VR 2579). 39. The recombinant (-) strand RNA virus is a parainfluenza virus (PIV) and the recombinant genome or antigenome is capable of harboring at least one and up to a total number of attenuating mutations present within HPIV3 JS cp45. 38. The method of claim 37, wherein the method encodes. 40. The method of claim 37, wherein the recombinant genome or antigenome comprises at least one attenuating mutation stabilized by multiple nucleotide changes in the codon specifying the mutation. 41. The recombinant genome or antigenome has a phenotypic change selected from altered growth characteristics, attenuation, temperature sensitivity, cold adaptation, small plaque size, restricted host range, or altered immunogenicity. 2. The method of claim 1, further modified by the specified nucleotide modification. 42. The recombinant (−) strand RNA virus is a respiratory syncytial virus (RSV).
42. The method of claim 41, wherein the recombinant genome or antigenome incorporates a modification of the SH, NS1, NS2 or G gene. 43. The method of claim 42, wherein the SH, NS1 or NS2 gene has been deleted or the expression of that gene has been excluded. 44. A nucleotide modification comprising the deletion, insertion, or deletion of a nucleotide in a cis-acting regulatory sequence of a selected RSV gene within a recombinant genome or antigenome.
43. The method of claim 42, comprising adding or rearranging. 45. The method of claim 1, wherein the recombinant (−) strand RNA virus is a subviral particle. 46. An isolated, attenuated, recombinant (-) strand RNA virus comprising: a recombinant genome or antigenome and an infectious viral particle of said recombinant (-) strand RNA virus. Comprising the necessary essential viral proteins, wherein said recombinant genome or antigenome contains an attenuating mutation identified in a heterologous mutant (-) strand RNA virus within a recombinant protein of said recombinant virus. The virus wherein the virus has been modified to encode a mutation at an amino acid position corresponding to an amino acid position, and wherein the mutation incorporated within the recombinant protein confers an attenuated phenotype on the recombinant virus. 47. The recombinant (−) strand RNA virus is a respiratory syncytial virus (
47. The attenuated recombinant (-) strand RNA virus of claim 46, which is RSV). 48. The said RSV is human RSV subgroup A, human RSV subgroup B, bovine RSV, mouse
48. The attenuated recombinant (-) strand RNA virus of claim 47, which is RSV or avian RSV. 49. The heterologous mutant (−) strand RNA virus is selected from respiratory syncytial virus (RSV), human parainfluenza virus (HPIV) 1, HPIV2, HPIV3, bovine PIV (BPIV), and Sendai virus (SeV). , Newcastle disease virus (NDV)
, Simian virus 5 (SV5), mumps virus (MuV), measles virus (MeV)
48. The attenuated recombinant (-) strand RNA virus of claim 47, which is a canine distemper virus (CDV), rabies virus (RaV), or vesicular stomatitis virus (VSV). 50. The heterologous mutant (−) strand RNA virus is RSV cpts248 (A
TCC VR 2450), RSV cpts248 / 404 (ATCC VR 2454), RSV cpts248 / 955 (ATCC
VR 2453), RSV cpts530 (ATCC VR 2452), RSV cpts530 / 1009 (ATCC VR 2451)
), RSV cpts530 / 1030 (ATCC VR 2455), RSV B-1 cp52 / 2B5 (ATCC VR 2542)
50. The attenuated recombinant (-) strand RNA virus of claim 49, which is RSV B-1 cp-23 (ATCC VR 2579). 51. The recombinant genome or antigenome is RSV NS1, NS2, N, P
, M, SH, M2 (ORF1), M2 (ORF2), substitution of amino acids within the L, F or G gene;
48. The attenuated recombination of claim 47, which is modified to encode a deletion or insertion.
-) Strand RNA virus. 52. The attenuated recombination (-) according to claim 47, wherein the mutation incorporated within the recombinant protein confers a temperature sensitive (ts) phenotype on the recombinant (-) strand RNA virus. ) Strand RNA virus. 53. The mutation incorporated into the recombinant protein is RS
The attenuated recombination according to claim 47, comprising an amino acid substitution within the VL protein (-
) Strand RNA virus. 54. The attenuating mutation identified in said heterologous mutant (-) strand RNA virus comprises an amino acid substitution of phenylalanine at position 521 of the L protein of human RSV cpts530 (ATCC VR 2452). Attenuated recombination according to 47 (
-) Strand RNA virus. 55. The attenuated recombinant (−) strand RNA virus of claim 46, wherein said recombinant (−) strand RNA virus is parainfluenza virus (PIV). 56. The method according to claim 56, wherein the PIV is human PIV1 (HPIV1), human PIV2 (HPIV2),
56. The attenuated recombinant (-) strand RNA virus of claim 55, which is an IV3 (HPIV3), a bovine PIV (BPIV), or a mouse PIV (MPIV). 57. The heterologous mutant virus is respiratory syncytial virus (RS)
V), human parainfluenza virus (HPIV) 1, HPIV2, HPIV3, bovine PIV (BPI
V), Sendai virus (SeV), Newcastle disease virus (NDV), Simian virus 5 (SV5), mumps virus (MuV), measles virus (MeV), dog
56. The attenuated recombinant (-) strand RNA virus of claim 55, which is a distemper virus (CDV), a rabies virus (RaV), or a vesicular stomatitis virus (VSV). 58. The method according to claim 58, wherein the recombinant genome or antigenome is PIV N, P, C, D, V
56. The attenuated recombinant (-) strand RNA virus of claim 55, wherein the virus is modified to encode an amino acid substitution, deletion or insertion within a, M, F, HN or L protein. 59. The attenuated recombinant (−) strand RNA virus of claim 55, wherein the mutation incorporated within the recombinant protein confers a ts phenotype on the recombinant (−) strand RNA virus. 60. The heterologous mutant (−) strand RNA virus is HPIV3 JS cp45.
56. The attenuated recombinant (-) strand RNA virus of claim 55, wherein 61. The attenuating mutation identified in the heterologous mutant (-) strand RNA virus comprises an amino acid substitution within a HPIV3 JS cp45 L protein.
61. The attenuated recombinant (-) strand RNA virus of claim 60. 62. The attenuator of claim 60, wherein the attenuating mutation identified in the heterologous mutant (-) strand RNA virus comprises a tyrosine amino acid substitution at position 942 of the L protein of HPIV3 JS cp45. Recombinant (-) strand RNA virus. 63. The attenuator of claim 60, wherein the attenuating mutation identified in the heterologous mutant (-) strand RNA virus comprises a leucine amino acid substitution at position 992 of the HPIV3 JS cp45 L protein. Recombinant (-) strand RNA virus. 64. The attenuator of claim 60, wherein the attenuating mutation identified in the heterologous mutant (-) strand RNA virus comprises a threonine amino acid substitution at position 1558 of the L protein of HPIV3 JS cp45. Recombinant (-) strand RNA virus. 65. The attenuating mutation identified in the heterologous mutant (-) strand RNA virus comprises an amino acid substitution within the HPIV3 JS cp45 F protein.
61. The attenuated recombinant (-) strand RNA virus of claim 60. 66. The attenuator of claim 60, wherein the attenuating mutation identified in the heterologous mutant (−) strand RNA virus comprises an amino acid substitution of isoleucine at position 420 of the F protein of HPIV3 JS cp45. Recombinant (-) strand RNA virus. 67. The attenuator of claim 60, wherein the attenuating mutation identified in the heterologous mutant (−) strand RNA virus comprises an amino acid substitution of alanine at position 450 of the HPIV3 JS cp45 F protein. Recombinant (-) strand RNA virus. 68. The attenuating mutation identified in the heterologous mutant (−) strand RNA virus comprises an amino acid substitution of phenylalanine at position 521 of the L protein of human RSV cpts530 (ATCC VR 2452). Attenuated recombination according to 55 (
-) Strand RNA virus. 69. The recombinant (−) strand RNA virus is human PIV (HPIV3), and the mutation incorporated into the recombinant protein is an amino acid substitution of phenylalanine at position 456 of the HPIV3 L protein. 70. The attenuated recombinant (-) stranded RNA virus of claim 68, comprising: 70. The attenuated set of claim 55, wherein the attenuating mutation identified in said heterologous mutant (-) strand RNA virus comprises an amino acid substitution of phenylalanine at position 170 of the SeV C protein. Recombinant (-) strand RNA virus. 71. The recombinant (−) strand RNA virus is human PIV (HPIV3), and the mutation incorporated into the recombinant protein is an amino acid substitution of phenylalanine at position 164 of the HPIV3 C protein. 71. The attenuated recombinant (-) strand RNA virus of claim 70, wherein the virus comprises: 72. The attenuated recombinant (−) strand RNA virus of claim 46, wherein said recombinant (−) strand RNA virus is measles virus (MeV). 73. The heterologous mutant virus is respiratory syncytial virus (RS)
V), human parainfluenza virus (HPIV) 1, HPIV2, HPIV3, bovine PIV (BPI
V), Sendai virus (SeV), Newcastle disease virus (NDV), Simian virus 5 (SV5), mumps virus (MuV), measles virus (MeV), dog
73. The attenuated recombinant (-) strand RNA virus of claim 72, which is a distemper virus (CDV), a rabies virus (RaV), or a vesicular stomatitis virus (VSV). 74. The heterologous mutant (−) strand RNA virus is HPIV3 JS cp45.
73. The attenuated recombinant (-) strand RNA virus of claim 72, wherein 75. The attenuator of claim 74, wherein the attenuating mutation identified in the heterologous mutant (-) strand RNA virus comprises a tyrosine amino acid substitution at position 942 of the L protein of HPIV3 JS cp45. Recombinant (-) strand RNA virus. 76. The attenuator of claim 74, wherein the attenuating mutation identified in the heterologous mutant (-) strand RNA virus comprises a leucine amino acid substitution at position 992 of the HPIV3 JS cp45 L protein. Recombinant (-) strand RNA virus. 77. An isolated polynucleotide molecule encoding a recombinant genome or antigenome of a recombinant (-) strand RNA virus, wherein said recombinant genome or antigenome is a recombinant protein of said virus. Internally, it has been modified to encode a mutation at an amino acid position corresponding to the amino acid position of the attenuating mutation identified in the heterologous mutated (-) strand RNA virus, and is suddenly incorporated into the recombinant protein. The polynucleotide, wherein the mutation confers an attenuated phenotype on the recombinant virus. 78. The recombinant (−) strand RNA virus is a respiratory syncytial virus (
80. The isolated polynucleotide of claim 77, which is RSV). 79. The RSV subgroup A, human RSV subgroup B, bovine RSV, mouse
79. The isolated polynucleotide of claim 78 which is RSV or avian pneumovirus RSV. 80. The heterologous mutant (−) strand RNA virus is a heterologous RSV, human parainfluenza virus (HPIV) 1, HPIV2, HPIV3, bovine PIV (BPIV), Sendai virus (SeV), Newcastle disease virus (NDV) ), Simian virus
5 (SV5), mumps virus (MuV), measles virus (MeV), canine distemper virus (CDV), rabies virus (RaV), or vesicular stomatitis virus (VSV)
81. The isolated polynucleotide of claim 78, which is 81. The recombinant genome or antigenome is RSV NS1, NS2, N, P
79. The isolated poly- according to claim 78, which is modified to encode amino acid substitutions, deletions or insertions within the, M, SH, M2 (ORF1), M2 (ORF2), L, F or G proteins. nucleotide. 82. The isolated polynucleotide of claim 78, wherein the mutation incorporated within the recombinant protein confers a temperature sensitive (ts) phenotype on the recombinant (−) strand RNA virus. 83. The isolated polynucleotide of claim 77, wherein said mutation incorporated into said recombinant protein comprises an amino acid substitution within RSV L protein. 84. The attenuating mutation identified in said heterologous mutant (-) strand RNA virus comprises an amino acid substitution of phenylalanine at position 521 of the L protein of human RSV cpts530 (ATCC VR 2452). 83. The isolated polynucleotide according to 83. 85. The isolated polynucleotide of claim 77, wherein said recombinant (−) strand RNA virus is a parainfluenza virus (PIV). 86. The PIV is human PIV1 (HPIV1), human PIV2 (HPIV2), human PIV
86. The isolated polynucleotide of claim 85, which is an IV3 (HPIV3), a bovine PIV (BPIV), or a mouse PIV (MPIV). 87. The heterologous mutant virus is respiratory syncytial virus (RS)
V), human parainfluenza virus (HPIV) 1, HPIV2, HPIV3, bovine PIV (BPI
V), Sendai virus (SeV), Newcastle disease virus (NDV), Simian virus 5 (SV5), mumps virus (MuV), measles virus (MeV), dog
86. The isolated polynucleotide of claim 85, which is a distemper virus (CDV), rabies virus (RaV), or vesicular stomatitis virus (VSV). 88. The method wherein the recombinant genome or antigenome is PIV N, P, C, D, V
86. The isolated polynucleotide of claim 85, wherein the polynucleotide is modified to encode an amino acid substitution, deletion or insertion within a, M, F, HN or L protein. 89. The isolated polynucleotide of claim 85, wherein the mutation incorporated within the recombinant protein confers a temperature sensitive (ts) phenotype on the recombinant (-) strand RNA virus. 90. The heterologous mutant (−) strand RNA virus is HPIV3 JS cp45
86. The isolated polynucleotide of claim 85, wherein 91. The attenuating mutation identified in the heterologous mutant (−) strand RNA virus comprises an amino acid substitution within an HPIV3 JS cp45 L protein.
90. The isolated polynucleotide of claim 90. 92. The method of claim 91, wherein the attenuating mutation identified in the heterologous mutant (−) strand RNA virus comprises a tyrosine amino acid substitution at position 942 of the HPIV3 JS cp45 L protein. Isolated polynucleotide. 93. The method of claim 91, wherein the attenuating mutation identified in the heterologous mutant (-) strand RNA virus comprises a leucine amino acid substitution at position 992 of the HPIV3 JS cp45 L protein. Isolated polynucleotide. 94. The method of claim 91, wherein the attenuating mutation identified in the heterologous mutant (−) strand RNA virus comprises an amino acid substitution of threonine at position 1558 of the L protein of HPIV3 JS cp45. Isolated polynucleotide. 95. The attenuating mutation identified in the heterologous mutant (-) strand RNA virus comprises an amino acid substitution within the F protein of HPIV3 JS cp45.
90. The isolated polynucleotide of claim 90. 96. The method of claim 95, wherein the attenuating mutation identified in the heterologous mutant (−) strand RNA virus comprises an amino acid substitution of isoleucine at position 420 of the F protein of HPIV3 JS cp45. Isolated polynucleotide. 97. The method of claim 95, wherein the attenuating mutation identified in the heterologous mutant (−) strand RNA virus comprises an alanine amino acid substitution at position 450 of the F protein of HPIV3 JS cp45. Isolated polynucleotide. 98. The attenuating mutation identified in the heterologous mutant (−) strand RNA virus comprises a phenylalanine amino acid substitution at position 521 of the L protein of human RSV cpts530 (ATCC VR 2452). 85. The isolated polynucleotide according to 85. 99. The recombinant (−) strand RNA virus is human PIV (HPIV3), and the mutation incorporated into the recombinant protein is an amino acid substitution of phenylalanine at position 456 of the HPIV3 L protein. 100. The isolated polynucleotide of claim 98, comprising: 100. The isolated polymorph of claim 85, wherein the attenuating mutation identified in said heterologous mutant (-) strand RNA virus comprises an amino acid substitution of phenylalanine at position 170 of the SeV C protein. nucleotide. 101. The recombinant (−) strand RNA virus is human PIV (HPIV3),
1 1. The isolated polynucleotide of claim 100, wherein the mutation incorporated into the recombinant protein comprises an amino acid substitution of phenylalanine at position 164 of the HPIV3 C protein. 102. The recombinant (−) strand RNA virus is a measles virus (MeV)
78. The isolated polynucleotide of claim 77, which is 103. The heterologous mutant virus is a respiratory syncytial virus (
RSV), human parainfluenza virus (HPIV) 1, HPIV2, HPIV3, bovine PIV (B
PIV), Sendai virus (SeV), Newcastle disease virus (NDV), Simian virus 5 (SV5), mumps virus (MuV), measles virus (MeV), canine distemper virus (CDV), rabies virus (RaV), 103. The isolated polynucleotide of claim 102, which is or vesicular stomatitis virus (VSV). 104. The heterologous mutant (−) strand RNA virus is HPIV3 JS cp4
103. The isolated polynucleotide of claim 102, which is 5. 105. The method of claim 103, wherein the attenuating mutation identified in the heterologous mutant (-) strand RNA virus comprises a tyrosine amino acid substitution at position 942 of the L protein of HPIV3 JS cp45. Isolated polynucleotide. 106. The method of claim 103, wherein the attenuating mutation identified in the heterologous mutant (−) strand RNA virus comprises a leucine amino acid substitution at position 992 of the HPIV3 JS cp45 L protein. Isolated polynucleotide. 107. A recombinant (-) strand RNA virus in which the genome or antigenome of part or all of a (-) strand RNA virus of one species, subgroup or strain is replaced by a different species, subgroup or strain 81. The isolated polynucleotide of claim 77, which is a chimeric virus having a recombinant genome or antigenome comprising in combination with a heterologous gene or gene segment of a (-) strand RNA virus. 108. The attenuating mutation is incorporated within a protein or protein region encoded by the heterologous gene or gene segment.
108. The isolated polynucleotide of claim 107. 109. A chimeric virus wherein the genome or antigenome of part or all of one PIV species, subgroup or strain is replaced by at least one of the HN and F glycoprotein genes of the heterologous PIV species, subgroup or strain. Parainfluenza virus having a recombinant genome or antigenome comprising in combination with two genes or gene segments (
108. The isolated polynucleotide of claim 107, which is PIV). 110. The isolated polynucleotide of claim 109, wherein the human PIV3 HN and F genes are replaced by human PIV1 HN and F genes. 111. A chimeric virus that replaces the genome or antigenome of part or all of one RSV species, subgroup or strain with the F, G and SH glycoprotein genes of a heterologous RSV species, subgroup or strain. Respiratory syncytial virus (RS) having a recombinant genome or antigenome comprising in combination with at least one gene or gene segment (RS
108. The isolated polynucleotide of claim 107, which is V). 112. The F and G glycoproteins of human RSV subgroup A are human RSV subgroup B F
111. The isolated polynucleotide of claim 111, wherein said polynucleotide is replaced by a G glycoprotein gene. 113. The recombinant genome or antigenome is further modified to encode one or more additional attenuating mutations taken from the biologically-derived mutant (-) strand RNA virus. 81. The isolated polynucleotide of claim 77, wherein 114. The recombinant (−) strand RNA virus is a respiratory syncytial virus (RS)
V), wherein the recombinant genome or antigenome is biologically derived
114. The isolated polynucleotide of claim 113, wherein the panel encodes at least one and up to a total number of attenuating mutations present in the panel of strains, wherein the panel comprises cpts RS.
V 248 (ATCC VR 2450), cpts RSV 248/404 (ATCC VR 2454), cpts RSV 248 /
955 (ATCC VR 2453), cpts RSV 530 (ATCC VR 2452), cpts RSV 530/1009 (A
TCC VR 2451), cpts RSV 530/1030 (ATCC VR 2455), RSV B-1 cp52 / 2B5 (AT
CCVR 2542), and said isolated polynucleotide comprising RSV B-1 cp-23 (ATCC VR 2579). 115. The recombinant (-) strand RNA virus is a parainfluenza virus (PIV), wherein the recombinant genome or antigenome is capable of harboring at least one and up to a total number of attenuating mutations present within PIV3 JS cp45. 114. The isolated polynucleotide of claim 113, which encodes. 116. The isolated polynucleotide of claim 77, wherein the recombinant genome or antigenome comprises at least one attenuating mutation stabilized by multiple nucleotide changes in the codon specifying the mutation. . 117. The recombinant genome or antigenome has a phenotypic change selected from altered growth characteristics, attenuated, temperature sensitive, cold adapted, small plaque size, restricted host range, or altered immunogenicity. 78. The isolated polynucleotide of claim 77, further modified by the specified nucleotide modification. 118. The recombinant (-) stranded RNA virus is a respiratory syncytial virus (RS).
118. The isolated polynucleotide of claim 117, wherein the recombinant genome or antigenome incorporates a modification of the SH, NS1, NS2 or G gene. 119. The isolated polynucleotide of claim 117, wherein the SH, NS1 or NS2 gene has been deleted or expression of that gene has been excluded. 120. The method of claim 117, wherein the nucleotide modification comprises a deletion, insertion, addition or rearrangement of a nucleotide in a cis-acting regulatory sequence of a selected RSV gene internal to the recombinant genome or antigenome. An isolated polynucleotide. 121. An operably linked transcription promoter, recombinant (-)
An expression vector comprising a polynucleotide molecule encoding a recombinant genome or antigenome of a strand RNA virus, and a transcription terminator, wherein the recombinant genome or antigenome is contained within a recombinant protein of the virus.
The mutation modified to encode a mutation at an amino acid position corresponding to the amino acid position of the attenuating mutation identified in the heterologous mutant (-) strand RNA virus, wherein the mutation incorporated into the recombinant protein is Such a vector, which confers an attenuated phenotype on the recombinant virus. 122. A method of stimulating the immune system of an individual to induce protection against (-) strand RNA virus, comprising immunologically the attenuated recombinant (-) strand virus of claim 46. Such a method, comprising administering to the individual a sufficient amount together with a physiologically acceptable carrier. Wherein 123] attenuated recombinant (-) strand viruses are administered by 10 ³ to 10 ⁶ PFU doses, The method of claim 122. 124. The method of claim 122, wherein the attenuated recombinant (−) strand virus is a respiratory syncytial virus (RSV) or a parainfluenza virus (PIV). 125. Inducing an immune response to a (-) strand RNA virus comprising an immunologically sufficient amount of the attenuated recombinant (-) strand virus of claim 46 together with a physiologically acceptable carrier. An immunogenic composition. 126. The immunogenic composition of claim 125, wherein said attenuated recombinant (-) chain virus is a respiratory syncytial virus (RSV) or a parainfluenza virus (PIV).