U.S. patent number 6,713,066 [Application Number 09/611,829] was granted by the patent office on 2004-03-30 for production of attenuated respiratory syncytial virus vaccines involving modification of m2 orf2.
This patent grant is currently assigned to The United States of America as represented by the Department of Health and Human Services, The United States of America as represented by the Department of Health and Human Services. Invention is credited to Alison Bermingham, Peter L. Collins, Brian R. Murphy.
United States Patent |
6,713,066 |
Collins , et al. |
March 30, 2004 |
**Please see images for:
( Certificate of Correction ) ** |
Production of attenuated respiratory syncytial virus vaccines
involving modification of M2 ORF2
Abstract
Recombinant respiratory syncytial virus (RSV) are provided in
which expression of the second translational open reading frame
encoded by the M2 gene (M2ORF2) is reduced or ablated to yield
novel RSV vaccine candidates. Expression of M2 ORF2 is reduced or
ablated by modifying a recombinant RSV genome or antigenome to
incorporate a frame shift mutation, or one or more stop codons in
M2 ORF2. Alternatively, M2 ORF2 is deleted in whole or in part to
render the M2-2 protein partially or entirely non-functional or to
disrupt its expression altogether. M2 ORF2 deletion and knock out
mutants possess highly desirable phenotypic characteristics for
vaccine development. These changes specify one or more desired
phenotypic changes in the resulting virus or subviral particle.
Vaccine candidates are generated that show a change in mRNA
transcription, genomic or antigenomic RNA replication, viral growth
characteristics, viral antigen expression, viral plaque size,
and/or a change in cytopathogenicity. In addition, M2-2 knock out
or deletion virus exhibits increased levels of synthesis of viral
proteins in cell culture, providing an enriched source of viral
antigen or protein for purification and use as a noninfectious
subunit vaccine.
Inventors: |
Collins; Peter L. (Rockville,
MD), Murphy; Brian R. (Bethesda, MD), Bermingham;
Alison (Silver Spring, MD) |
Assignee: |
The United States of America as
represented by the Department of Health and Human Services
(Washington, DC)
|
Family
ID: |
31999821 |
Appl.
No.: |
09/611,829 |
Filed: |
July 7, 2000 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
Issue Date |
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291894 |
Apr 13, 1999 |
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892403 |
Jul 15, 1997 |
5993824 |
Nov 30, 1999 |
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Current U.S.
Class: |
424/199.1;
424/202.1; 424/205.1; 424/211.1; 435/69.1 |
Current CPC
Class: |
C07K
14/005 (20130101); C12N 7/00 (20130101); A61K
39/00 (20130101); A61K 2039/5254 (20130101); C12N
2760/18522 (20130101); C12N 2760/18543 (20130101); C12N
2760/18561 (20130101) |
Current International
Class: |
C07K
14/005 (20060101); C07K 14/135 (20060101); C12N
7/04 (20060101); A61K 39/00 (20060101); A61K
039/12 (); A61K 039/295 () |
Field of
Search: |
;424/199.1,202.1,205.1,211.1 ;435/69.1 |
References Cited
[Referenced By]
U.S. Patent Documents
Primary Examiner: Housel; James
Assistant Examiner: Chen; Stacy B.
Attorney, Agent or Firm: Graybeal Jackson Haley LLP
Parent Case Text
CROSS-REFERENCES TO RELATED APPLICATIONS
This application claims the priority benefit of U.S. Provisional
Application No. 60/143,097, filed by Peter L. Collins et al. on
Jul. 9, 1999. The present application also claims the benefit of
and is a continuation-in-part of U.S. patent application Ser. No.
09/291,894, filed Apr. 13, 1999, which is a continuation-in-part of
U.S. patent application Ser. No. 08/892,403, filed Jul. 15, 1997,
issued on Nov. 30, 1999 as U.S. Pat. No. 5,993,824, which is
entitled to priority from U.S. Provisional Application No.
60/047,634, filed May 23, 1997; No. 60/046,141, filed May 9, 1997;
and No. 60/021,773, filed Jul. 15, 1996.
Claims
What is claimed is:
1. An isolated infectious recombinant respiratory syncytial virus
(RSV) comprising a RSV genome or antigenome, a major nucleocapsid
(N) protein, a nucleocapsid phosphoprotein (P), a large polymerase
protein (L), and a M2(ORF1) RNA polymerase elongation factor,
wherein a modification is introduced in the genome or antigenome
comprising a partial or complete deletion of M2 ORF2 or one or more
nucleotide change(s) that reduce or ablate expression of M2
ORF2.
2. The recombinant RSV of claim 1, wherein expression of M2 ORF2 is
ablated by introduction of one or more stop codons.
3. The recombinant RSV of claim 2 which is rA2-K5.
4. The recombinant RSV of claim 1, wherein expression of M2 ORF2 is
ablated by introduction of a frame shift mutation.
5. The recombinant RSV of claim 4 which is rA2-NdeI.
6. The recombinant RSV of claim 1, wherein M2 ORF2 is deleted in
whole or in part.
7. The recombinant RSV of claim 1, wherein the modification in the
genome or antigenome specifies one or more desired phenotypic
changes in the recombinant RSV selected from (i) a change in mRNA
synthesis, (ii) a change in the level of viral protein expression;
(iii) a change in genomic or antigenomic RNA replication, (iv) a
change in viral growth characteristics, (v), a change in viral
plaque size, and/or vi) a change in cytopathogenicity.
8. The recombinant RSV of claim 7, wherein the phenotypic change
comprises attenuation of viral growth compared to growth of a
corresponding wild-type or mutant parental RSV strain.
9. The recombinant RSV of claim 1, wherein the RSV genome comprises
one or more shifted RSV gene(s) or genome segment(s) that is/are
positionally shited within genome or antigenome to a more
promoter-proximal or promoter-distal position relative to a
position of said RSV gene(s) or genome segment(s) within a wild
type RSV genome or antigenome.
10. The recombinant RSV of claim 9, wherein said one or more
shifted gene(s) or genome segment(s) is/are shifted to a more
promoter-proximal or promoter-distal position by deletion or
insertion of one or more displacement polynucleotide(s) within said
partial or complete genome or antigenome.
11. The recombinant RSV of claim 7, wherein the phenotypic change
comprises delayed kinetics of viral mRNA synthesis compared to
kinetics of mRNA synthesis of a corresponding wild-type or mutant
parental RSV strain.
12. The recombinant RSV of claim 7, wherein the phenotypic change
comprises a change in cumulative MRNA synthesis compared to
cumulative mRNA synthesis of a corresponding wild-type or mutant
parental RSV strain.
13. The recombinant RSV of claim 12, wherein the increase in
cumulative viral mRNA synthesis is approximately 1.3 to 2-fold or
greater at 24 hours post-infection compared to cumulative mRNA
synthesis of the corresponding wild-type or mutant parental RSV
strain.
14. The recombinant RSV of claim 7, wherein the phenotypic change
comprises increased viral protein accumulation in infected cells
compared to viral protein accumulation in cells infected with a
corresponding wild-type or mutant parental RSV strain.
15. The recombinant RSV of claim 7, wherein accumulation of one or
more viral proteins is increased approximately 2- to 3-fold or
greater compared to viral protein accumulation in cells infected
with the corresponding wild-type or mutant parental RSV strain.
16. The recombinant RSV of claim 7, wherein the phenotypic change
comprises increased expression of one or more viral antigens
compared to expression of said one or more viral antigens by the
corresponding wild-type or mutant parental RSV strain.
17. The recombinant RSV of claim 7, wherein the phenotypic change
comprises a change in viral RNA replication compared to viral RNA
replication of a corresponding wild-type or mutant parental RSV
strain.
18. The recombinant RSV of claim 17, wherein accumulation of
genomic and antigenomic RNA is decreased compared to accumulation
of genomic and antigenomic RNA of the corresponding wild-type or
mutant parental RSV strain.
19. The recombinant RSV of claim 7, wherein the phenotypic change
comprises an increase in cumulative mRNA synthesis and a reduction
in viral RNA replication compared to cumulative mRNA synthesis and
viral RNA replication of a corresponding wild-type or mutant
parental RSV strain.
20. The recombinant RSV of claim 19, wherein a cumulative molar
ratio of mRNA to genomic RNA is increased approximately 7- to
18-fold or greater compared to a cumulative molar ratio of mRNA to
genomic RNA observed for the corresponding wild-type or mutant
parental RSV strain.
21. The recombinant RSV of claim 7, wherein the phenotypic change
comprises a larger plaque phenotype compared to plaque phenotype of
a corresponding wild-type or mutant parental RSV strain.
22. The recombinant RSV of claim 7, wherein the phenotypic change
comprises a change in cytopathogenicity compared to
cytopathogenicity of a corresponding wild-type or mutant parental
RSV strain.
23. The recombinant RSV of claim 1, wherein the genome or
antigenome is further modified by introduction of one or more
attenuating mutations identified in a biologically derived mutant
human RSV.
24. The recombinant RSV of claim 23, wherein the genome or
antigenome incorporates at least one and up to a full complement of
attenuating mutations present within a panel of biologically
derived mutant human RSV strains, said panel comprising 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-1
cp52/2B5 (ATCC VR 2542), and RSV B-1 cp-23 (ATCC VR 2579).
25. The recombinant RSV of claim 23, wherein the genome or
antigenome incorporates at least one and up to a fall complement of
attenuating mutations specifying an amino acid substitution at
Val267 in the RSV N gene, Glu218 and/or Thr523 in the RSV F gene,
Asn43, Cys319 Phe521, Gln831, Met1169, Tyr1321 and/or His1690 in
the RSV polymerase gene L, and a nucleotide substitution in the
gene-start sequence of gene M2.
26. The recombinant RSV of claim 23, wherein the genome or
antigenome incorporates at least two attenuating mutations.
27. The recombinant RSV of claim 23, wherein the genome or
antigenome includes at least one attenuating mutation stabilized by
multiple nucleotide changes in a codon specifying the mutation.
28. The recombinant RSV of claim 1, wherein the genome or
antigenome comprises an additional nucleotide modification
specifying a phenotypic change selected from a change in growth
characteristics, attenuation, temperature-sensitivity,
cold-adaptation, plaque size, host-range restriction, antigen
expression, or a change in immunogenicity.
29. The recombinant RSV of claim 28, wherein the additional
nucleotide modification alters a SH, NS1, NS2, or G gene of the
recombinant RSV.
30. The recombinant RSV of claim 29, wherein a SH, NS1, NS2, or G
gene is deleted in whole or in part or expression of the gene is
reduced or ablated by a frame shift or introduction of one or more
stop codons in an open reading frame of the gene or a modification
of a tranlational start site.
31. The recombinant RSV of claim 28, wherein the nucleotide
modification comprises a nucleotide deletion, insertion,
substitution, addition or rearrangement of a cis-acting regulatory
sequence of a selected gene within the recombinant RSV genome or
antigenome.
32. The recombinant RSV of claim 31, wherein a gene end (GE) signal
of the NS1 or NS2 gene is modified.
33. The recombinant RSV of claim 28, wherein the nucleotide
modification comprises an insertion, deletion, substitution, or
rearrangement of a translational start site within the recombinant
RSV genome or antigenome.
34. The recombinant RSV of claim 33, wherein the translational
start site for a secreted form of the RSV G glycoprotein is
ablated.
35. The recombinant RSV of claim 28, wherein the genome or
antigenome is modified to encode a non-RSV molecule selected from a
cytokine, a T-helper epitope, a restriction site marker, or a
protein of a microbial pathogen capable of eliciting a protective
immune response in a mammalian host.
36. The recombinant RSV of claim 28, wherein the genome or
antigenome incorporates a gene or genome segment from parainfluenza
virus (PIV).
37. The recombinant RSV of claim 36, wherein the gene or genome
segment encodes a PIV HN or F glycoprotein or immunogenic domain or
epitope thereof.
38. The recombinant RSV of claim 37, wherein the genome segment
encodes an ectodomain or immunogenic epitope of HN or F of PIV1,
PIV2, or PIV3.
39. The recombinant RSV of claim 1, wherein the genome or
antigenome comprises a partial or complete RSV background genome or
antigenome of a human or bovine RSV combined with a heterologous
gene or genome segment of a different RSV to form a human-bovine
chimeric RSV genome or antigenome.
40. The recombinant RSV of claim 39, wherein the heterologous gene
or genome segment encodes a RSV F, G or SH glycoprotein or an
immunogenic domain or epitope thereof.
41. The recombinant RSV of claim 39, wherein the heterologous gene
or genome segment is substituted for a counterpart gene or genome
segment in a partial RSV background genome or antigenome.
42. The recombinant RSV of claim 39, wherein the heterologous gene
or genome segment is added adjacent to or within a noncoding region
of the partial or complete RSV background genome or antigenome.
43. The recombinant RSV of claim 39, wherein the chimeric genome or
antigenome comprises a partial or complete human RSV background
genome or antigenome combined with a heterologous gene or genome
segment from a bovine RSV.
44. The recombinant RSV of claim 39, wherein the chimeric genome or
antigenome comprises a partial or complete bovine RSV background
genome or antigenome combined with a heterologous gene or genome
segment from a human RSV.
45. The recombinant RSV of claim 44, wherein one or more human RSV
glycoprotein genes F, G and SH or a genome segment encoding a
cytoplasmic domain, transmembrane domain, ectodomain or immunogenic
epitope thereof is substituted for a counterpart gene or genome
segment within the bovine RSV background genome or antigenome.
46. The recombinant RSV of claim 45, wherein one or both human RSV
glycoprotein genes F and G is substituted to replace one or both
counterpart F and G glycoprotein genes in the bovine RSV background
genome or antigenome.
47. The recombinant RSV of claim 46, wherein both human RSV
glycoprotein genes F and G are substituted to replace counterpart F
and G glycoprotein genes in the bovine RSV background genome or
antigenome.
48. The recombinant RSV of claim 45, wherein the heterologous gene
or genome segment is from a subgroup A or subgroup B human RSV.
49. The recombinant RSV of claim 45, wherein the human-bovine
chimeric genome or antigenome incorporates antigenic determinants
from both subgroup A and subgroup B human RSV.
50. The recombinant RSV of claim 1 which is a complete virus.
51. The recombinant RSV of claim 1 which is a subviral
particle.
52. A method for stimulating the immune system of an individual to
elicit an immune response against RSV which comprises administering
to the individual an immunologically sufficient amount of the
recombinant RSV of claim 1 combined with a physiologically
acceptable carrier.
53. The method of claim 52, wherein the recombinant RSV is
administered in a dose of 10.sup.6 to 10.sup.7 PFU.
54. The method of claim 52, wherein the recombinant RSV is
administered to the upper respiratory tract.
55. The method of claim 52, wherein the recombinant RSV is
administered by spray, droplet or aerosol.
56. The method of claim 52, wherein the recombinant RSV is
administered to an individual seronegative for antibodies to RSV or
possessing transplacentally acquired maternal antibodies to
RSV.
57. The method of claim 52, wherein the recombinant RSV is
attenuated and exhibits increased antigen expression compared to
growth and antigen expression of a corresponding wild-type or
mutant parental RSV strain.
58. The method of claim 52, wherein the recombinant RSV elicits an
immune response against human RSV A, human RSV B, or both.
59. An immunogenic composition to elicit an immune response against
RSV comprising an immunologically sufficient amount of the
recombinant RSV of claim 1 in a physiologically acceptable
carrier.
60. The immunogenic composition of claim 59 formulated in a dose of
10.sup.3 to 10.sup.7 PFU.
61. The immunogenic composition of claim 59, formulated for
administration to the upper respiratory tract by spray, droplet or
aerosol.
62. The immunogenic composition of claim 59, wherein the
recombinant RSV exhibits attenuated growth and increased antigen
expression compared to growth and antigen expression of a
corresponding wild-type or mutant parental RSV strain.
63. The immunogenic composition of claim 62 which elicits an immune
response against human RSV A, human RSV B, or both.
Description
BACKGROUND OF THE INVENTION
Human respiratory syncytial virus (HRSV) is the leading viral agent
of serious pediatric respiratory tract disease worldwide (Collins,
et al., Fields Virology 2:1313-1352, 1996). RSV outranks all other
microbial pathogens as a cause of pneumonia and bronchiolitis in
infants under one year of age. Virtually all children are infected
by two years of age, and reinfection occurs with appreciable
frequency in older children and young adults (Chanock et al., in
Viral Infections of Humans, 3rd ed., A. S. Evans, ed., Plenum
Press, N.Y., 1989). RSV is responsible for more than one in five
pediatric hospital admissions due to respiratory tract disease, and
in the United States alone causes nearly 100,000 hospitalizations
and 4,500 deaths yearly. (Heilman, J. Infect. Dis. 161:402-6,
1990). In addition, there is evidence that serious respiratory
tract infection early in life can initiate or exacerbate asthma
(Sigurs, et al., Pediatrics 95:500-5, 1995).
While HRSV usually is thought of in the context of the pediatric
population, it also is recognized as an important agent of serious
disease in the elderly (Falsey, et al., J. Infect. Dis.
172:389-394, 1995). HRSV also causes life-threatening disease in
certain immunocompromised individuals, such as bone marrow
transplant recipients (Fouillard, et al., Bone Marrow Transplant
9:97-100, 1992).
For treatment of HRSV, one chemotherapeutic agent, ribavirin, is
available. However, its efficacy and use are controversial. There
are also licensed products for RSV intervention which are composed
of pooled donor IgG (Groothuis, et al. N. Engl. J. Med.
329:1524-30, 1993) or a humanized RSV-specific monoclonal antibody.
These are administered as passive immunoprophylaxis agents to high
risk individuals. While these products are useful, their high cost
and other factors, such as lack of long term effectiveness, make
them inappropriate for widespread use. Other disadvantages include
the possibility of transmitting blood-borne viruses and the
difficulty and expense in preparation and storage. Moreover, the
history of the control of infectious diseases, and especially
diseases of viral origin, indicates the primary importance of
vaccines.
Despite decades of investigation to develop effective vaccine
agents against RSV, no safe and effective vaccine has yet been
approved to prevent the severe morbidity and significant mortality
associated with RSV infection. Failure to develop successful
vaccines relates in part to the fact that small infants have
diminished serum and secretory antibody responses to RSV antigens.
Thus, these individuals suffer more severe infections from RSV,
whereas cumulative immunity appears to protect older children and
adults against more serious impacts of the virus.
The mechanisms of immunity in RSV infection have recently come into
focus. Secretory antibodies appear to be most important in
protecting the upper respiratory tract, whereas high levels of
serum antibodies are thought to have a major role in resistance to
RSV infection in the lower respiratory tract. RSV-specific
cytotoxic T cells, another effector arm of induced immunity, are
also important in resolving an RSV infection. However, while this
latter effector can be augmented by prior immunization to yield
increased resistance to virus challenge, the effect is short-lived.
The F and G surface glycoproteins are the two major protective
antigens of RSV, and are the only two RSV proteins which have been
shown to induce RSV neutralizing antibodies and long term
resistance to challenge (Collins et al., Fields Virology, Fields et
al. eds., 2:1313-1352, Lippincott-Raven, Philadelphia, 1996;
Connors et al., J. Virol. 65(3):1634-7, 1991). The third RSV
surface protein, SH, did not induce RSV-neutralizing antibodies or
significant resistance to RSV challenge.
An obstacle to developing live RSV vaccines is the difficulty in
achieving an appropriate balance between attenuation and
immunogenicity. Other obstacles include the genetic instability of
some attenuated viruses, the relatively poor growth of RSV in cell
culture, and the instability of the virus particle. In addition the
immunity which is induced by natural infection is not fully
protective against subsequent infection. A number of factors
probably contribute to this, including the relative inefficiency of
the immune system in restricting virus infection on the luminal
surface of the respiratory tract, the short-lived nature of local
mucosal immunity, rapid and extensive virus replication, reduced
immune responses in the young due to immunological immaturity,
immunosuppression by transplacentally derived maternal serum
antibodies, and certain features of the virus such as a high degree
of glycosylation of the G protein. Also, as will be described
below, HRSV exists as two antigenic subgroups A and B, and immunity
against one subgroup is of reduced effectiveness against the
other.
Although RSV can reinfect multiple times during life, reinfections
usually are reduced in severity due to protective immunity induced
by prior infection, and thus immunoprophylaxis is feasible. A
live-attenuated RSV vaccine would be administered intranasally to
initiate a mild immunizing infection. This has the advantage of
simplicity and safety compared to a parenteral route. It also
provides direct stimulation of local respiratory tract immunity,
which plays a major role in resistance to RSV. It also abrogates
the immunosuppressive effects of RSV-specific maternally-derived
serum antibodies, which typically are found in the very young.
Also, while the parenteral administration of RSV antigens can
sometimes be associated with immunopathologic complications (Murphy
et al., Vaccine 8(5):497-502, 1990), this has never been observed
with a live virus.
A formalin-inactivated virus vaccine was tested against RSV in the
mid-1960s, but failed to protect against RSV infection or disease,
and in fact exacerbated symptoms during subsequent infection by the
virus. (Kim et al., Am. J. Epidemiol., 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. Friedewald et al., (J. Amer. Med. Assoc.
204:690-694, 1968) reported a cold passaged mutant of RSV (cpRSV)
which appeared to be sufficiently attenuated to be a candidate
vaccine. This mutant exhibited a slightly increased efficiency of
growth at 26.degree. C. compared to its wild-type (wt) parental
virus, but its replication was neither temperature sensitive nor
significantly cold-adapted. The cold-passaged mutant, however, was
attenuated for adults. Although satisfactorily attenuated and
immunogenic for infants and children who had been previously
infected with RSV (i.e., seropositive individuals), the cpRSV
mutant retained a low level virulence for the upper respiratory
tract of seronegative infants.
Similarly, Gharpure et al., (J. Virol. 3:414-421, 1969) reported
the isolation of temperature sensitive RSV (tsRSV) mutants which
also were promising vaccine candidates. One mutant, ts-1, was
evaluated extensively in the laboratory and in volunteers. The
mutant produced asymptomatic infection in adult volunteers and
conferred resistance to challenge with wild-type virus 45 days
after immunization. Again, while seropositive infants and children
underwent asymptomatic infection, seronegative infants developed
signs of rhinitis and other mild symptoms. Furthermore, instability
of the ts phenotype was detected. Although virus exhibiting a
partial or complete loss of temperature sensitivity represented a
small proportion of virus recoverable from vaccinees, it was not
associated with signs of disease other than mild rhinitis.
These and other studies revealed that certain cold-passaged and
temperature sensitive RSV strains were underattenuated and caused
mild symptoms of disease in some vaccinees, particularly
seronegative infants, while others were overattenuated and failed
to replicate sufficiently to elicit a protective immune response,
(Wright et al., Infect. Immun., 37:397-400, 1982). Moreover,
genetic instability of candidate vaccine mutants has resulted in
loss of their temperature sensitive phenotype, further hindering
development of effective RSV vaccines. See generally, (Hodes et
al., Proc. Soc. Exp. Biol. Med. 145:1158-1164, 1974; McIntosh et
al., Pediatr. Res. 8:689-696, 1974; and Belshe et al., J. Med.
Virol., 3:101-110, 1978).
As an alternative to live-attenuated RSV vaccines, investigators
have also tested subunit vaccine candidates using purified RSV
envelope glycoproteins. The glycoproteins induced resistance to RS
virus infection in the lungs of cotton rats, (Walsh et al., J.
Infect. Dis. 155:1198-1204, 1987), but the antibodies had very weak
neutralizing activity and immunization of rodents with purified
subunit vaccine led to disease potentiation (Murphy et al., Vaccine
8:497-502, 1990).
Recombinant vaccinia virus vaccines which express the F or G
envelope glycoprotein have also been explored. These recombinants
express RSV glycoproteins which are indistinguishable from the
authentic viral counterpart, and rodents infected intradermally
with vaccinia-RSV F and G recombinants developed high levels of
specific antibodies that neutralized viral infectivity. Indeed,
infection of cotton rats with vaccinia-F recombinants stimulated
almost complete resistance to replication of RSV in the lower
respiratory tract and significant resistance in the upper tract.
(Olmsted et al., Proc. Natl. Acad. Sci. USA 83:7462-7466, 1986).
However, immunization of chimpanzees with vaccinia-F and -G
recombinant provided almost no protection against RSV challenge in
the upper respiratory tract (Collins et al., Vaccine 8:164-168,
1990) and inconsistent protection in the lower respiratory tract
(Crowe et al., Vaccine 11:1395-1404, 1993).
Despite these various efforts to develop an effective RSV vaccine,
no licensed vaccine has yet been approved for RSV. The unfulfilled
promises of prior approaches underscores a need for new strategies
to develop RSV vaccines, and in particular methods for manipulating
recombinant RSV to incorporate genetic changes that yield new
phenotypic properties in viable, attenuated RSV recombinants.
However, manipulation of the genomic RNA of RSV and other
non-segmented negative-sense RNA viruses has heretofore proven
difficult. Major obstacles in this regard include non-infectivity
of naked genomic RNA of these viruses and, in the case of RSV, poor
viral growth in tissue culture, lengthy replication cycles, virion
instability, a complex genome, and a refractory organization of
gene products.
Recombinant DNA technology has made it possible to recover
infectious non-segmented negative-stranded RNA viruses from cDNA,
to genetically manipulate viral clones to construct novel vaccine
candidates, and to rapidly evaluate their level of attenuation and
phenotypic stability (for reviews, see Conzelmann, J. Gen. Virol.
77:381-89, 1996; Palese et al., Proc. Natl. Acad. Sci. U.S.A.
93:11354-58, 1996). In this context, recombinant rescue has been
reported for infectious respiratory syncytial virus (RSV),
parainfluenza virus (PIV), rabies virus (RaV), vesicular stomatitis
virus (VSV), measles virus (MeV), rinderpest virus and Sendai virus
(SeV) from cDNA-encoded antigenomic RNA in the presence of
essential viral proteins (see, e.g., Garcin et al., EMBO J.
14:6087-6094, 1995; Lawson et al., Proc. Natl. Acad. Sci. U.S.A.
92:4477-81, 1995; Radecke et al., EMBO J. 14:5773-5784, 1995;
Schnell et al., EMBO J. 13:4195-203, 1994; Whelan et al., Proc.
Natl. Acad. Sci. U.S.A. 92:8388-92, 1995; Hoffman et al., J Virol.
71:4272-4277, 1997; Pecters et al., J. Virol. 73:5001-5009, 1999;
Kato et al., Genes to Cells 1:569-579, 1996; Roberts et al.,
Virology 247(1), 1-6, 1998; Baron et al., J Virol. 71:1265-1271,
1997; International Publication No. WO 97/06270; U.S. Provisional
Patent Application No. 60/007,083, filed Sep. 27, 1995; U.S. patent
application Ser. No. 08/720,132, filed Sep. 27, 1996; U.S.
Provisional Patent Application No. 60/021,773, filed Jul. 15, 1996;
U.S. Provisional Patent Application No. 60/046,141, filed May 9,
1997; U.S. Provisional Patent Application No. 60/047,634, filed May
23, 1997; U.S. Pat. No. 5,993,824, issued Nov. 30, 1999
(corresponding to International Publication No. WO 98/02530); U.S.
patent application Ser. No. 09/291,894, filed by Collins et al. on
Apr. 13, 1999; U.S. Provisional Patent Application No. 60/129,006,
filed by Murphy et al. on Apr. 13, 1999; Collins, et al., Proc Nat.
Acad. Sci. USA 92:11563-11567, 1995; Bukreyev, et al., J. Virol.
70:6634-41, 1996, Juhasz et al., J. Virol. 71(8):5814-5819, 1997;
Durbin et al., Virology 235:323-332, 1997; He et al. Virology
237:249-260, 1997; Baron et al. J. Virol. 71:1265-1271, 1997;
Whitehead et al., Virology 247(2):232-9, 1998a; Buchholz et al. J.
Virol. 73:251-9, 1999; Whitehead et al., J. Virol. 72(5):4467-4471,
1998b; Jin et al. Virology 251:206-214, 1998; and Whitehead et al.,
J. Virol. 73:(4)3438-3442, 1999, and Bukreyev, et al., Proc. Nat.
Acad. Sci. USA 96:2367-72, 1999; Collins et al., Virology
259:251-255, 1999, each incorporated herein by reference in its
entirety for all purposes).
Based on these developments in recombinant DNA technology, it is
now possible to recover infectious RSV from cDNA and to design and
implement various genetic manipulations to RSV clones to construct
novel vaccine candidates. Thereafter, the level of attenuation and
phenotypic stability, among other desired phenotypic
characteristics, can be evaluated. The challenge which thus
presents itself is to develop a broad and diverse menu of genetic
manipulations that can be employed, alone or in combination with
other types of genetic manipulations, to construct infectious,
attenuated RSV clones that are useful for broad vaccine use. In
this context, an urgent need remains in the art for additional
tools and methods that will allow engineering of safe and effective
vaccines to alleviate the serious health problems attributable to
RSV. Surprisingly, the present invention fulfills this need by
providing additional tools for constructing infectious, attenuated
RSV vaccine candidates.
SUMMARY OF THE INVENTION
The present invention provides recombinant RSV (rRSV) in which
expression of the second translational open reading frame encoded
by the M2 gene (M2ORF2) (Collins and Wertz, J. Virol. 54:65-71,
1985; Collins et al., J. Gen. Virol. 71:3015-3020, 1990, Collins et
al., Proc. Natl. Acad. Sci. USA 93:81-85, 1996, each incorporated
herein by reference) is reduced or ablated to yield novel RSV
vaccine candidates. In preferred aspects of the invention,
expression of M2 ORF2 is reduced or ablated by modifying a
recombinant RSV genome or antigenome to incorporate a frame shift
mutation or one or stop codons in M2 ORF2 yielding a "knock out"
viral clone. Alternatively, M2 ORF2 is deleted in whole or in part
to render the M2-2 protein partially or entirely non-functional or
to disrupt its expression altogether to yield a "deletion mutant"
RSV. Alternatively, the M2-2 ORF may be transpositioned in the
genome or antigenome to a more promoter-proximal or promoter-distal
position compared to the natural gene order position of M2-2 gene
to up-regulate or down-regulate expression of the M2-2 ORF. In
additional embodiments, the M2-2 ORF is incorporated in the genome
or antigenome as a separate gene having a gene start and gene end
gene end signal, which modification results in up-regulation of the
M2-2 ORF.
The recombinant RSV of the invention having mutations in M2 ORF2
possess highly desirable phenotypic characteristics for vaccine
development. The above identified modifications in the recombinant
genome or antigenome specify one or more desired phenotypic changes
in the resulting virus or subviral particle. Vaccine candidates are
thus generated that exhibit one or more characteristics identified
as (i) a change in mRNA transcription, (ii) a change in the level
of viral protein expression; (iii) a change in genomic or
antigenomic RNA replication, (iv) a change in viral growth
characteristics, (v), a change in viral plaque size, and/or (vi) a
change in cytopathogenicity.
In exemplary RSV recombinants described herein, desired phenotypic
changes include attenuation of viral growth compared to growth of a
corresponding wild-type or mutant parental RSV strain. In more
detailed aspects, viral growth in cell culture may be attenuated by
approximately 10-fold or more attributable to mutations in M2 ORF2.
Kinetics of viral growth are also shown to be modified in a manner
that is beneficial for vaccine development.
Also described herein are recombinant RSV that exhibit delayed
kinetics of viral mRNA synthesis compared to kinetics of mRNA
synthesis of corresponding wild-type or mutant parental RSV
strains. Despite these delayed transcription kinetics, these novel
vaccine candidates exhibit an increase in cumulative mRNA synthesis
compared to parental virus. These phenotypic changes typically are
associated with an increase in viral protein accumulation in
infected cells compared to protein accumulation in cells infected
with wild-type or other parental RSV strains. At the same time,
viral RNA replication is reduced in M2 ORF2 mutants compared to
that of a parental RSV strain, whereby accumulation of genomic or
antigenomic RNA is reduced.
Within preferred aspects of the invention, recombinant M2 ORF2
deletion and "knock out" RSV are engineered to express undiminished
or, more typically, increased levels of viral antigen(s) while also
exhibiting an attenuated phenotype. Immunogenic potential is thus
preserved due to the undiminished or increased mRNA transcription
and antigen expression, while attenuation is achieved through
concomitant reductions in RNA replication and virus growth. This
novel suite of phenotypic traits is highly desired for vaccine
development. Other useful phenotypic changes that are observed in
M2 ORF2 deletion and knock out mutants include a large plaque
phenotype and altered cytopathogenicity compared to corresponding
wild-type or mutant parental RSV strains.
In related aspects of the invention, a method for producing one or
more purified RSV protein(s) is provided which involves infecting a
host cell permissive of RSV infection with a recombinant, M2-ORF 2
deletion or knock out mutant RSV under conditions that allow for
RSV propagation in the infected cell. After a period of replication
in culture, the cells are lysed and recombinant RSV is isolated
therefrom: One or more desired RSV protein(s) is purified after
isolation of the virus, yielding one or more RSV protein(s) for
vaccine, diagnostic and other uses.
In combination with the phenotypic effects provided in recombinant
RSV bearing M2 ORF2 deletion or knock out mutations, it is often
desirable to adjust the attenuation phenotype by introducing
additional mutations that increase or decrease attenuation of the
recombinant virus. Thus, candidate vaccine strains can be further
attenuated by incorporation of at least one, and preferably two or
more different attenuating mutations, for example mutations
identified from a panel of known, biologically derived mutant RSV
strains. Preferred human mutant RSV strains are cold passaged (cp)
and/or temperature sensitive (ts) mutants, for example the mutants
designated "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-1 cp52/2B5 (ATCC VR 2542), and RSV B-1 cp-23 (ATCC VR
2579)" (each deposited under the terms of the Budapest Treaty with
the American Type Culture Collection (ATCC) of 10801 University
Boulevard, Manassas, Va. 20110-2209, U.S.A., and granted the above
identified accession numbers). From this exemplary panel of
biologically derived mutants, a large "menu" of attenuating
mutations is provided, each of which can be combined with any other
mutation(s) within the panel for calibrating the level of
attenuation and other desirable phenotypes in M2 ORF2 deletion and
knock out mutants for vaccine use. Additional mutations which can
be thus adopted or transferred to M2 ORF2 deletion and knock out
mutants within the invention may be identified in various
temperature sensitive (ts), cold passaged (cp), small plaque (sp),
cold-adapted (ca) or host-range restricted (hr) mutant RSV strains.
Additional attenuating mutations may be identified in non-RSV
negative stranded RNA viruses and incorporated in RSV mutants of
the invention by mapping the mutation to a corresponding,
homologous site in the recipient RSV genome or antigenome and
mutating the existing sequence in the recipient to the mutant
genotype (either by an identical or conservative mutation), as
described in U.S. Provisional Patent Application Serial No.
60/129,006, filed Apr. 13, 1999. Additional useful mutations can be
determined empirically by mutational analysis using recombinant
minigenome systems and infectious virus as described in the
references incorporated herein.
M2 ORF2 deletion and knock out mutants of the invention selected
for vaccine use often have at least two and sometimes three or more
attenuating mutations to achieve a satisfactory level of
attenuation for broad clinical use. In one embodiment, at least one
attenuating mutation occurs in the RSV polymerase gene L (either in
the donor or recipient gene) and involves one or more nucleotide
substitution(s) specifying an amino acid change in the polymerase
protein specifying an attenuation phenotype which may or may not
involve a temperature-sensitive (ts) phenotype. Recombinant RSV of
the invention may incorporate a ts mutation in any additional RSV
gene besides L, for example in the M2 gene. However, preferred
vaccine candidates in this context incorporate one or more
nucleotide substitutions in the large polymerase gene L resulting
in an amino acid change at amino acid Asn43, Cys319, Phe521,
Gln831, Met1169, Tyr1321 and/or His1690, as exemplified by the
changes, Ile for Asn43, Leu for Phe521, Leu for Gln831, Val for
Met1169, and Asn for Tyr1321. Other alternative amino acid changes,
particularly conservative changes with respect to identified mutant
residues, at these positions can of course be made to yield a
similar effect as the identified, mutant substitution. Additional
desired mutations for incorporation into recombinant RSV of the
invention include attenuating mutations specifying an amino acid
substitution at Val267 in the RSV N gene, Glu218 and/or Thr523 in
the RSV F gene, and a nucleotide substitution in the gene-start
sequence of gene M2. Any combination of one or more of the
attenuating mutations identified herein, up to and including a full
complement of these mutations, may be incorporated in M2 ORF2
deletion or knock out RSV to yield a suitably attenuated
recombinant virus for use in selected populations or broad
populations of vaccine recipients.
Attenuating mutations may be selected in coding portions of an M2
ORF2 deletion or knock out mutant genome or antigenome or in
non-coding regions such as a cis-regulatory sequence. Exemplary
non-coding mutations include single or multiple base changes in a
gene start sequence, as exemplified by a single or multiple base
substitution in the M2 gene start sequence at nucleotide 7605
(nucleotide 7606 in an exemplary recombinant sequence).
In addition to the above described mutations, infectious M2 ORF2
deletion and knock out mutants according to the invention can
incorporate heterologous, coding or non-coding nucleotide sequences
from any RSV or RSV-like virus, e.g., human, bovine, ovine, murine
(pneumonia virus of mice), or avian (turkey rhinotracheitis virus)
pneumovirus, or from another enveloped virus, e.g., parainfluenza
virus (PIV). Exemplary heterologous sequences include RSV sequences
from one human RSV strain combined with sequences from a different
human RSV strain in an M2 ORF2 deletion or knock out mutants. For
example, recombinant RSV of the invention may incorporate sequences
from two or more wild-type or mutant RSV strains, for example
mutant strains selected from cpts RSV 248, cpts 248/404, cpts
248/955, cpts RSV 530, cpts 530/1009, or cpts 530/1030.
Alternatively, M2 ORF2 deletion and knock out RSV mutants may
incorporate sequences from two or more, wild-type or mutant human
RSV subgroups, for example a combination of human RSV subgroup A
and subgroup B sequences (see, International Application No.
PCT/US/08802 and related U.S. patent application Nos. 60/021,773,
60/046,141, 60/047,634, Ser. Nos. 08/892,403, 09/291,894, each
incorporated herein by reference). In yet additional aspects, one
or more human RSV coding or non-coding polynucleotides are
substituted with a counterpart sequence from a heterologous RSV or
non-RSV virus, alone or in combination with one or more selected
attenuating mutations, e.g., cp and/or ts mutations, to yield novel
attenuated vaccine strains.
In related aspects of the invention, the disclosed modifications
relating to M2-2 are incorporated within chimeric human-bovine RSV,
which are recombinantly engineered to incorporate nucleotide
sequences from both human and bovine RSV strains to produce an
infectious, chimeric virus or subviral particle. Exemplary
human-bovine chimeric RSV of the invention incorporate a chimeric
RSV genome or antigenome comprising both human and bovine
polynucleotide sequences, as well as a major nucleocapsid (N)
protein, a nucleocapsid phosphoprotein (P), a large polymerase
protein (L), and a RNA polymerase elongation factor. Additional RSV
proteins may be included in various combinations to provide a range
of infectious subviral particles, up to a complete viral particle
or a viral particle containing supernumerary proteins, antigenic
determinants or other additional components.
Chimeric human-bovine RSV for use within the invention are
generally described in U.S. Patent Application entitled PRODUCTION
OF ATTENUATED, HUMAN-BOVINE CHIMERIC RESPIRATORY SYNCYTIAL VIRUS
VACCINES, filed by Bucholz et al. on Jun. 23, 2000, and in its
priority U.S. Provisional Patent Application Serial No. 60/143,132
(each incorporated herein by reference). These chimeric recombinant
RSV include a partial or complete "background" RSV genome or
antigenome derived from or patterned after a human or bovine RSV
strain or subgroup virus combined with one or more heterologous
gene(s) or genome segment(s) of a different RSV strain or subgroup
virus to form the human-bovine chimeric RSV genome or antigenome.
In certain aspects of the invention, chimeric RSV incorporate a
partial or complete bovine RSV background genome or antigenome
combined with one or more heterologous gene(s) or genome segment(s)
from a human RSV. In alternate aspects of the invention M2 ORF2
deletion and knock out RSV incorporate a partial or complete human
RSV background genome or antigenome combined with one or more
heterologous gene(s) or genome segment(s) from a bovine RSV.
Yet additional aspects of the invention involve changing the
position of a gene or altering gene order to create or modify a M2
ORF2 deletion or knock out mutant RSV. In this context, a number of
the foregoing incorporated references have focused on modification
of the naturally-occurring order in RSV and other viruses. For
example, in RSV the NS1, NS2, SH and G genes were deleted
individually, and the NS1 and NS2 gene were deleted together,
thereby shifting the position of each downstream gene relative to
the viral promoter. For example, when NS1 and NS2 are deleted
together, N is moved from position 3 to position 1, P from position
4 to position 2, and so on. Alternatively, deletion of any other
gene within the gene order will affect the position (relative to
the promoter) only of those genes which are located further
downstream. For example, SH occupies position 6 in wild type virus,
and its deletion does not affect M at position 5 (or any other
upstream gene) but moves G from position 7 to 6 relative to the
promoter. It should be noted that gene deletion also can occur
(rarely) in a biologically-derived mutant virus. For example, a
subgroup B RSV that had been passaged extensively in cell culture
spontaneously deleted the SH and G genes (Karron et al., Proc.
Natl. Acad. Sci. USA 94:13961-13966, 1997; incorporated herein by
reference). Note that "upstream" and "downstream" refer to the
promoter-proximal and promoter-distal directions, respectively (the
promoter is at the 3' leader end of negative-sense genomic
RNA).
Gene order shifting modifications (i.e., positional modifications
moving one or more genes to a more promoter-proximal or
promoter-distal location in the recombinant viral genome) to create
or modify M2 ORF2 deletion and knock out RSV of the invention
result in viruses with altered biological properties. For example,
RSV lacking NS1, NS2, SH, G, NS1 and NS2 together, or SH and G
together, have been shown to be attenuated in vitro, in vivo, or
both. It is likely that this phenotype was due primarily to the
loss of expression of the specific viral protein. However, the
altered gene map also likely contributed to the observed phenotype.
This effect is well-illustrated by the SH-deletion virus, which
grew more efficiently than wild type in some cell types, probably
due to an increase in the efficiency of transcription, replication
or both resulting from the gene deletion and resulting change in
gene order and possibly genome size. In other viruses, such as RSV
in which NS1 and/or NS2 were deleted, altered growth that might
have occurred due to the change in gene order likely was obscured
by the more dominant phenotype due to the loss of expression of the
RSV protein(s).
Yet additional changes will be introduced to change the gene order
of M2 ORF2 deletion and knock out RSV in an effort to improve its
properties as a live-attenuated vaccine (see, U.S. Provisional
Patent Application Ser. No. 60/213,708 entitled RESPIRATORY
SYNCYTIAL VIRUS VACCINES EXPRESSING PROTECTIVE ANTIGENS FROM
PROMOTOR-PROXIMAL GENES, filed by Krempl et al., Jun. 23, 2000,
incorporated herein by reference). In particular, the G and F genes
may be shifted, singly and in tandem, to a more promoter-proximal
position relative to their wild-type gene order. These two proteins
normally occupy positions 7 (G) and 8 (F) in the RSV gene order
(NS1-NS2-N-P-M-SH-G-F-M2-L). In order to increase the possibility
of successful recovery, exemplary shifting manipulations have been
performed in a version of RSV in which the SH gene had been deleted
(Whitehead et al., J. Virol., 73:3438-42 (1999), incorporated
herein by reference). This facilitates recovery because this virus
makes larger plaques in vitro (Bukreyev et al., J. Virol.,
71:8973-82 (1997), incorporated herein by reference). G and F were
then moved individually to position 1, or were moved together to
positions 1 and 2, respectively. Surprisingly, recombinant RSV were
readily recovered in which G or F were moved to position 1, or in
which G and F were moved to positions 1 and 2, respectively.
Similarly extensive modifications in gene order also have been
achieved with two highly attenuated vaccine candidates in which the
NS2 gene was deleted on its own, or in which the NS1 and NS2 genes
were deleted together. In these two vaccine candidates, the G and F
glycoproteins were moved together to positions 1 and 2
respectively, and the G, F and SH glycoproteins were deleted from
their original downstream position. Thus, the recovered viruses
G1F2.DELTA.NS2.DELTA.SH and G1F2/.DELTA.NS1.DELTA.NS2.DELTA.SH had
two and three genes deleted respectively in addition to the shift
of the G and F genes. To illustrate the extent of the changes
involved, the gene orders of wild type RSV
(NS1-NS2-N-P-M-SH-G-F-M2-L) and the G1F2/.DELTA.NS2.DELTA.SH virus
(G-F-NS1-N-P-M-M2-L) or the .DELTA.NS1.DELTA.NS2.DELTA.SH
(G-F-N-P-M-M2-L) can be compared. This shows that the positions of
most or all of the genes relative to the promoter were changed.
Nonetheless, these highly attenuated derivatives retained the
capacity to be grown in cell culture.
In other detailed aspects of the invention, M2 ORF2 deletion and
knock out mutants are employed as "vectors" for protective antigens
of other pathogens, particularly respiratory tract pathogens such
as parainfluenza virus (PIV). For example, recombinant RSV having a
M2 ORF2 deletion or knock out may be engineered which incorporate
sequences that encode protective antigens from PIV to produce
infectious, attenuated vaccine virus. The cloning of PIV cDNA and
other disclosure supplemental to the instant invention is provided
in United States Patent Application entitled PRODUCTION OF
PARAINFLUENZA VIRUS VACCINES FROM CLONED NUCLEOTIDE SEQUENCES,
filed May 22, 1998, Ser. No. 09/083,793 (corresponding to
International Publication No. WO 98/53078) and its priority,
provisional application filed May 23, 1997, Serial No. 60/047,575,
U.S. Provisional Patent Application Ser. No. 60/143,134 entitled
ATTENUATED HUMAN-BOVINE CHIMERIC PARAINFLUENZA VIRUS VACCINES,
filed by Baily et al. on Jul. 9, 1999 and U.S. Provisional Patent
Application Ser. No. 09/350,821 entitled RECOMBINANT PARAINFLUENZA
VIRUS VACCINES ATTENUATED BY DELETION OR ABLATION OF A
NON-ESSENTIAL GENE, filed by Durbin et al. on Jul. 9, 1999; each
incorporated herein by reference. This disclosure includes
description of the following plasmids that may be employed to
produce infectious PIV viral clones or to provide a source of PIV
genes or genome segments for use within the invention: p3/7(131)
(ATCC 97990); p3/7(131)2G (ATCC 97989); and p218(131) (ATCC 97991);
each deposited under the terms of the Budapest Treaty with the
American Type Culture Collection (ATCC) of 10801 University
Boulevard, Manassas, Va. 20110-2209, U.S.A., and granted the above
identified accession numbers.
According to this aspect of the invention, M2 ORF2 deletion and
knock out mutants RSV are provided which incorporate at least one
PIV sequence, for example a polynucleotide containing sequences
from either or both PIV1 and PIV2 or PIV1 and PIV3. Individual
genes of RSV may be replaced with counterpart genes from human PIV,
such as the F glycoprotein genes of PIV1, PIV2, or PIV3.
Alternatively, a selected, heterologous genome segment, such as a
cytoplasmic tail, transmembrane domain or ectodomain of substituted
for a counterpart genome segment in, e.g., the same gene in RSV,
within a different gene in RSV, or into a non-coding sequence of
the RSV genome or antigenome. In one embodiment, a genome segment
from an F gene of HPIV3 is substituted for a counterpart human RSV
genome segment to yield constructs encoding chimeric proteins, e.g.
fusion proteins having a cytoplasmic tail and/or transmembrane
domain of RSV fused to an ectodomain of PIV to yield a novel
attenuated virus, and/or a multivalent vaccine immunogenic against
both PIV and RSV. Alternatively, one or more PIV3 gene(s) or genome
segment(s) can be added to a partial or complete, chimeric or
non-chimeric RSV genome or antigenome.
To construct chimeric RSV, heterologous genes may be added or
substituted in whole or in part to the background genome or
antigenome. In the case of chimeras generated by substitution, a
selected gene or genome segment encoding a protein or protein
region (e.g., a cytoplasmic tail, transmembrane domain or
ectodomain, an epitopic site or region, a binding site or region,
an active site or region containing an active site, etc.) from a
human or bovine RSV is substituted for a counterpart gene or genome
segment in the background RSV genome or antigenome to yield novel
recombinants having desired phenotypic changes compared to one or
both of the respective wild-type (or mutant parent) RSV strains. As
used herein, "counterpart" genes or, genome segments refer to
counterpart polynucleotides from different RSV sources that encode
homologous or equivalent proteins or protein domains, epitopes, or
amino acid residues, or which represent homologous or equivalent
cis-acting signals which may include but are not limited to species
and allelic variants among different RSV subgroups or strains.
In other alternate embodiments, M2 ORF2 deletion and knock out RSV
designed as vectors for carrying heterologous antigenic
determinants incorporate one or more antigenic determinants of a
non-RSV pathogen, such as a human parainfluenza virus (HPIV). In
one exemplary embodiment, one or more HPIV1, HPIV2, or HPIV3
gene(s) or genome segment(s) encoding one or more HN and/or F
glycoprotein(s) or antigenic domain(s), fragment(s) or epitope(s)
thereof is/are added to or incorporated within the partial or
complete HRSV vector genome or antigenome. In more detailed
embodiments, a transcription unit comprising an open reading frame
(ORF) of an HPIV1, HPIV2, or HPIV3 HN or F gene is added to or
incorporated within the chimeric HRSV vector genome or
antigenome.
Mutations incorporated within cDNAs, vectors and viral particles of
the invention can be introduced individually or in combination into
a full-length M2 ORF2 deletion or knock out mutant and the
phenotypes of rescued virus containing the introduced mutations can
be readily determined. In exemplary embodiments, amino acid changes
displayed by attenuated, biologically-derived viruses versus a
wild-type RSV, for example changes exhibited by cpRSV or tsRSV, are
incorporated in combination within a recombinant M2 ORF2 deletion
or knock out mutant RSV to yield a desired level of attenuation for
vaccine use.
The present invention thus provides M2 ORF2 deletion and knock out
mutant RSV clones, vectors and particles which may incorporate
multiple, phenotype-specific mutations introduced in selected
combinations into the recombinant genome or antigenome to produce a
suitably attenuated, infectious virus or subviral particle. This
process, coupled with routine phenotypic evaluation, provides M2
ORF2 deletion and knock out mutants having such desired
characteristics as attenuation, temperature sensitivity, altered
immunogenicity, cold-adaptation, small plaque size, host range
restriction, etc. Mutations thus identified are compiled into a
"menu" and introduced in various combinations to calibrate a
vaccine virus to a selected level of attenuation, immunogenicity
and stability.
In yet additional aspects of the invention, M2 ORF2 deletion and
knock out mutants, with or without additional attenuating
mutations, are constructed to have a nucleotide modification to
yield a desired phenotypic, structural, or functional change.
Typically, the selected nucleotide modification will specify a
phenotypic change, for example a change in growth characteristics,
attenuation, temperature-sensitivity, cold-adaptation, plaque size,
host range restriction, or immunogenicity. Structural changes in
this context include introduction or ablation of restriction sites
into RSV encoding cDNAs for ease of manipulation and
identification.
In preferred embodiments, nucleotide changes within the genome or
antigenome of an M2 ORF2 deletion or knock out mutant include
modification of an additional viral gene by partial or complete
deletion of the gene or reduction or ablation (knock-out) of its
expression. Target genes for mutation in this context include the
attachment (G) protein, fusion (F) protein, small hydrophobic (SH),
RNA binding protein (N), phosphoprotein (P), the large polymerase
protein (L), the transcription elongation factor (M2 ORF1), the
matrix (M) protein, and two nonstructural proteins, NS1 and NS2.
Each of these proteins can be selectively deleted, substituted or
rearranged, in whole or in part, alone or in combination with other
desired modifications, to achieve novel RSV recombinants.
In one aspect of the invention, an SH, NS1, NS2, or G gene is
modified in an M2 ORF2 deletion or knock out mutant RSV. For
example, each of these genes may be deleted in whole or in part or
its expression reduced or ablated (e.g., by introduction of a stop
codon or frame shift mutation or alteration of a transcriptional or
translational start site) to alter the phenotype of the resultant
recombinant clone to improve growth, attenuation, immunogenicity or
other desired phenotypic characteristics. For example, deletion of
the SH gene in the recombinant genome or antigenome will yield a
vaccine candidate having novel phenotypic characteristics such as
enhanced growth in vitro and/or attenuation in vivo. In a related
aspect, an SH gene deletion, or deletion of another selected
non-essential gene or genome segment such as a NS1 or NS2 gene, is
constructed in an M2 ORF2 deletion or knock out mutant, alone or in
combination with one or more different mutations specifying an
attenuated phenotype, e.g., a point mutation adopted directly (or
in modified form, e.g., by introducing multiple nucleotide changes
in a codon specifying the mutation) from a biologically derived
attenuated RSV mutant. For example, the SH, NS1, NS2 or G gene may
be deleted in combination with one or more cp and/or ts mutations
adopted from cpts248/404, cpts530/1009, cpts530/030 or another
selected mutant RSV strain, to yield a recombinant RSV exhibiting
increased yield of virus, enhanced attenuation, improved
immunogenicity and genetic resistance to reversion from an
attenuated phenotype due to the combined effects of the different
mutations.
Alternative nucleotide modifications in M2 ORF2 deletion and knock
out RSV mutants of the invention can include a deletion, insertion,
addition or rearrangement of a cis-acting regulatory sequence for a
selected gene in the recombinant genome or antigenome. In one
example, a cis-acting regulatory sequence of one RSV gene is
changed to correspond to a heterologous regulatory sequence, which
may be a counterpart cis-acting regulatory sequence of the same
gene in a different RSV or a cis-acting regulatory sequence of a
different RSV gene. For example, a gene end signal may be modified
by conversion or substitution to a gene end signal of a different
gene in the same RSV strain. In other embodiments, the nucleotide
modification may comprise an insertion, deletion, substitution, or
rearrangement of a translational start site within the recombinant
genome or antigenome, e.g., to ablate an alternative translational
start site for a selected form of a protein. In one example, the
translational start site for a secreted form of the RSV G protein
is ablated to modify expression of this form of the G protein and
thereby produce desired in vivo effects.
In addition, a variety of other genetic alterations can be produced
in a RSV genome or antigenome having a deletion or knock-out of M2
ORF2, alone or together with one or more attenuating mutations
adopted from a biologically derived mutant RSV. For example, genes
or genome segments from non-RSV sources may be inserted in whole or
in part. Alternatively, the order of genes can be changed, gene
overlap removed, or an RSV genome promoter replaced with its
antigenome counterpart. Different or additional modifications in
the recombinant genome or antigenome can be made to facilitate
manipulations, such as the insertion of unique restriction sites in
various intergenic regions (e.g., a unique StuI site between the G
and F genes) or elsewhere. Nontranslated gene sequences can be
removed to increase capacity for inserting foreign sequences. In
yet additional aspects, polynucleotide molecules or vectors
encoding the recombinant RSV genome or antigenome can be modified
to encode non-RSV sequences, e.g., a cytokine, a T-helper epitope,
a restriction site marker, or a protein of a microbial pathogen
(e.g., virus, bacterium or fungus) capable of eliciting a
protective immune response in an intended host. Non-RSV genes of
interest include those encoding cytokines (e.g., IL-2 through
IL-18, especially IL-2, IL-4, IL-6 and IL-12, IL-18, etc.),
gamma-interferon, GM-CSF, chemokines and proteins rich in T helper
cell epitopes (see, e.g., U.S. Provisional Patent Application
Serial No. 60/143,425, incorporated herein by reference). This
provides the ability to modify and improve the immune responses
against RSV both quantitatively and qualitatively.
All of the foregoing modifications within a recombinant RSV genome
or antigenome, including nucleotide insertions, rearrangements,
deletions or substitutions yielding point mutations, site-specific
nucleotide changes, and changes involving entire genes or genome
segments, may be made to either a heterologous donor gene or genome
segment, or in a partial or complete recipient or background genome
or antigenome. In each case, these alterations will preferably
specify one or more phenotypic change(s) in the resulting
recombinant RSV, such as a phenotypic change that results in
attenuation, temperature-sensitivity, cold-adaptation, small plaque
size, host range restriction, alteration in gene expression, or a
change in an immunogenic epitope.
In related aspects of the invention, compositions (e.g., isolated
polynucleotides and vectors incorporating an RSV-encoding cDNA) and
methods are provided for producing an isolated infectious
recombinant RSV bearing an attenuating, M2 ORF2 deletion or knock
out mutation. Included within these aspects of the invention are
novel, isolated polynucleotide molecules and vectors incorporating
such molecules that comprise a RSV genome or antigenome which is
modified by a partial or complete deletion of M2 ORF2 or one or
more nucleotide changes that reduce or ablate expression of M2
ORF2. Also provided is the same or different expression vector
comprising one or more isolated polynucleotide molecules encoding
N, P, L and RNA polymerase elongation factor proteins. These
proteins also can be expressed directly from the genome or
antigenome cDNA. The vector(s) is/are preferably expressed or
coexpressed in a cell or cell-free lysate, thereby producing an
infectious M2 ORF2 deletion or knock out mutant RSV particle or
subviral particle.
The above methods and compositions for producing M2 ORF2 deletion
and knock out mutant RSV yield infectious viral or subviral
particles, or derivatives thereof. An infectious virus is
comparable to the authentic RSV virus particle and is infectious as
is. It can directly infect fresh cells. An infectious subviral
particle typically is a subcomponent of the virus particle which
can initiate an infection under appropriate conditions. For
example, a nucleocapsid containing the genomic or antigenomic RNA
and the N, P, L and M2(ORF1) proteins is an example of a subviral
particle which can initiate an infection if introduced into the
cytoplasm of cells. Subviral particles provided within the
invention include viral particles which lack one or more
protein(s), protein segment(s), or other viral component(s) not
essential for infectivity.
In other embodiments the invention provides a cell or cell-free
lysate containing an expression vector which comprises an isolated
polynucleotide molecule encoding an M2 ORF2 deletion or knock out
mutant RSV genome or antigenome as described above, and an
expression vector (the same or different vector) which comprises
one or more isolated polynucleotide molecules encoding the N, P, L
and RNA polymerase elongation factor proteins of RSV. One or more
of these proteins also can be expressed from the genome or
antigenome cDNA. Upon expression the genome or antigenome and N, P,
L, and RNA polymerase elongation factor proteins combine to produce
an infectious RSV viral or subviral particle.
The recombinant RSV of the invention are useful in various
compositions to generate a desired immune response against RSV in a
host susceptible to RSV infection. Attenuated M2 ORF2 deletion and
knock out mutant RSVs of the invention are capable of eliciting a
protective immune response in an infected human host, yet are
sufficiently attenuated so as to not cause unacceptable symptoms of
severe respiratory disease in the immunized host. The attenuated
virus or subviral particle may be present in a cell culture
supernatant, isolated from the culture, or partially or completely
purified. The virus may also be lyophilized, and can be combined
with a variety of other components for storage or delivery to a
host, as desired.
The invention further provides novel vaccines comprising a
physiologically acceptable carrier and/or adjuvant and an isolated
attenuated M2 ORF2 deletion or knock out mutant RSV particle or
subviral particle. In preferred embodiments, the vaccine is
comprised of an M2 ORF2 deletion or knock out mutant RSV having at
least one, and preferably two or more attenuating mutations or
other nucleotide modifications as described above to achieve a
suitable balance of attenuation and immunogenicity. The vaccine can
be formulated in a dose of 10.sup.3 to 10.sup.6 PFU or more of
attenuated virus. The vaccine may comprise attenuated M2 ORF2
deletion or knock out virus that elicits an immune response against
a single RSV strain or antigenic subgroup, e.g. A or B, or against
multiple RSV strains or subgroups. In this regard, M2 ORF2 deletion
and knock out mutant RSV can be combined in vaccine formulations
with other RSV vaccine strains or subgroups having different
immunogenic characteristics for more effective protection against
one or multiple RSV strains or subgroups.
In related aspects, the invention provides a method for stimulating
the immune system of an individual to elicit an immune response
against RSV in a mammalian subject. The method comprises
administering a formulation of an immunologically sufficient amount
of an attenuated, M2 ORF2 deletion or knock out mutant RSV in a
physiologically acceptable carrier and/or adjuvant. In one
embodiment, the immunogenic composition is a vaccine comprised of
an M2 ORF2 deletion or knock out mutant RSV having at least one,
and preferably two or more attenuating mutations or other
nucleotide modifications specifying a desired phenotype as
described above. The vaccine can be formulated in a dose of
10.sup.3 to 10.sup.6 PFU or more of attenuated virus. The vaccine
may comprise attenuated M2 ORF2 deletion or knock out mutant RSV
virus that elicits an immune response against a single RSV strain
or antigenic subgroup, e.g. A or B, or against multiple RSV strains
or subgroups. M2 ORF2 deletion and knock out mutants can be
combined with RSV having different immunogenic characteristics in a
vaccine mixture, or administered separately in a coordinated
treatment protocol, to elicit more effective protection against one
RSV strain, or against multiple RSV strains or subgroups.
Preferably the immunogenic composition is administered to the upper
respiratory tract, e.g., by spray, droplet or aerosol. Often, the
composition will be administered to an individual seronegative for
antibodies to RSV or possessing transplacentally acquired maternal
antibodies to RSV.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 depicts construction of the NdeI and KS mutations, which
interrupt M2 ORF2. Nt sequences are in positive-sense and blocked
in triplets according to amino acid coding in ORF2. Nt positions
relative to the complete 15,223-nt recombinant antigenome are in
parentheses. Other numbers refer to amino acid positions in the
194-amino acid M2-1 protein or 90-amino acid M2-2 protein. Panel A
is a diagram of the two overlapping M2 ORFs. In the sequence at the
top, the three potential translational start sites for M2-2 are
underlined and their encoded methionine residues are boxed. The
termination codon for ORF1 is also underlined. In the diagram,
restriction sites used for mutagenesis and cloning are indicated.
(SEQ ID NO. 3):
ACAAATGACCATGCCAAAAATAATGATACTACCTGACAAATATCCTTGTAGT. (SEQ ID NO.
6): TNDHAKNNDTT. Panel B depicts construction of the NdeI mutation.
The NdeI site at position 8299 in the middle of the M2-2 ORF was
opened, filled in and relegated, which added two nt (lower case) to
codon 47 of M2-2. This shifted the register to another reading
frame, which was open for 18 additional codons encoding non-M2-2
amino acids (underlined). (SEQ ID NO. 4):
AACCATATGTACTCACCGAATCAAACATTCAATGAAATCCATTGGACCTCTCAAG AATTGA.
(SEQ ID NO. 7): NHMYSPNQTFNEIHWTSQEL. (SEQ ID NO. 5):
AACCATATATGTACTCACCGAATCAAACATTCAATGAAATCCATTGGACCTCTCA AGAATTGA.
(SEQ ID NO. 8): NHICTHRIKHSMKSIGPLKN. Panel C depicts construction
of the K5 mutation. The sequence shows the junction between ORF1
and ORF2, as in Panel A. Potential ORF2 initiation codons in the wt
parent are underlined, as is the ORF1 termination codon. Nt changes
in K5 are indicated above their wt counterparts. The three
potential initiation codons for ORF2, codons 1, 3 and 7, were
changed to ACG, which had no effect on amino acid coding in ORF 1.
The next potential methionyl start site in ORF2 is at codon 30. In
addition, stop codons were introduced into all three frames
immediately downstream of the M2-1 termination codon. In
combination, these mutations had the effect of changing M2-2 amino
acid 12 from K to N and terminating at codon 13.
FIG. 2 demonstrates that the NdeI and K5 mutations each ablate the
inhibitory function of M2-2 in a reconstituted minigenome system.
In panel A, HEp-2 cells were simultaneously infected with vTF7-3 (5
plaque forming units per cell) and transfected with plasmid
encoding the negative-sense C2 minigenome cDNA (200 ng) and support
plasmids (N, 400 ng; P, 200 ng; L, 100 ng) per well of a 6-well
dish and supplemented with pTM constructs (80 ng) containing no
insert (lane 2), M2 ORF 1 (lane 4), M2 ORF 2 (lane 3), M2 ORFs 1+2
(lane 5) or the M2 ORFs 1+2 containing the NdeI (lane 6) or K5
(lane 7) mutations. Lane 1 is a negative control lacking L. Cells
were exposed to 2 .mu.g actinomycin D per ml from 24-26 h
post-infection (Feams et al., Virology 236, 188-201, 1997,
incorporated herein by reference). At 48 h post-infection, total
intracellular RNA was isolated and electrophoresed on formaldehyde
gels for Northern blot analysis (Grosfeld et al., J. Virol.
69:5677-86, 1995, incorporated herein by reference). Blots were
hybridized to a negative-sense CAT specific riboprobe to detect
both mRNA and antigenome. In panel B: HEp-2 cells were transfected
as described below with plasmid encoding positive-sense C4
mini-antigenome complemented by the N, P and L plasmids as in Panel
A. The transfection mixtures were supplemented with increasing
amounts (0.008, 0.04 and 0.2 times the relative molar ratio of
transfected pTM-N) of pTM constructs encoding M2 ORFs 1+2 (lanes 2,
3 and 4), M2 ORF 1 (lanes 4, 5 and 6), M2 ORF 2 (lanes 8, 9 and 10)
or ORFs 1+2 containing the NdeI (lanes 11, 12 and 13) or K5 (lanes
14, 15 and 16) mutation. Total intracellular RNA was analyzed by
Northern blots hybridized with a positive-sense CAT specific
riboprobe to detect genomic RNA.
FIG. 3 illustrates cytopathogenicity of the rA2-NdeI and rA2-K5
(also referred to as rA2.DELTA.M2-2), viruses compared to rA2-wt.
HEp-2 cells were infected at a moi of 1 with the indicated virus,
or mock-infected, incubated for the indicated time, and
photographed at 10.times. magnification. The 48 h micrographs are
darker due to a difference in exposure. Large syncytia are obvious
in the two mutant viruses at 48 and 72 h, and smaller ones are
evident at 24 h and in rA2-wt-infected cells at the same three time
points.
FIG. 4 illustrates kinetics of growth of rA2-wt, rA2-NdeI and
rA2-KS in cell culture. Panel A shows single step growth kinetics.
HEp-2 cells were infected with rA2-wt, rA2-NdeI or rA2-K5 at an moi
of 5 pfu per cell and the entire medium overlay was harvested at
the indicated times post-infection and flash-frozen. Viral titers
were determined by plaque assay. Panel B shows multi-cycle growth
kinetics. HEp-2 cells were infected in triplicate at an moi of 0.01
pfu per cell with the above viruses. At the indicated times
post-infection, the entire medium overlay was removed,
flash-frozen, and replaced with fresh medium. Mean virus titers
determined by plaque assay (with error bars) are shown.
FIG. 5 provides a Northern blot analysis of RNA replication and
transcription. HEp-2 cells infected with rA2-wt (a, d and g),
rA2-NdeI (b, e and h) and rA2-K5 (c, f and i) were harvested at 3 h
intervals (lanes 1-10) from the single cycle growth curve described
in FIG. 4A, and total intracellular RNA was isolated and subjected
to Northern blot analysis. Blots were hybridized with a
negative-sense N specific riboprobe (a, b and c), a negative-sense
F specific riboprobe (d, e and f) or a positive-sense F specific
riboprobe (g, h and i). Monocistronic mRNA (i.e. N or F),
polycistronic read through mRNAs (i.e. NS 1-NS2-N and G-F-M2),
antigenome and genome are indicated.
FIG. 6 provides a Western blot analysis of the accumulation of F
and G glycoproteins in HEp-2 cells infected at an moi of 5 with
rA2-wt (A, C) or rA2-K5 (B, D). Cells were harvested at the
indicated time and total cellular protein was subjected to
polyacrylamide gel electrophoresis under denaturing and reducing
conditions, transferred to nitrocellulose (Teng et al., J. Virol.
73:466-473, 1999), and reacted with rabbit anti-peptide. serum
against the cytoplasmic domain of the F (A, B) or G (C, D) protein.
Bound antibodies were detected with horseradish
peroxidase-conjugated goat anti-rabbit IgG and visualized by
enhanced chemiluminescence (Amersham).
DESCRIPTION OF THE SPECIFIC EMBODIMENTS
The present invention provides recombinant RSV (rRSV) in which
expression of M2ORF2 gene, newly characterized herein to encode a
transcription/replication regulatory factor M2-2, is reduced or
ablated to yield an assemblage of novel RSV vaccine candidates.
Expression of M2 ORF2 is reduced or ablated by modifying a
recombinant RSV genome or antigenome to incorporate a frame shift
mutation, or one or more stop codons in M2 ORF2. Other alterations
to achieve disruption of M2 ORF2 expression or M2-2 protein
expression or function to generate attenuated RSV vaccine
candidates include partial or complete deletion of the M2 ORF2
coding sequence, in whole or in part, to render the M2-2 protein
partially or entirely non-functional or terminate its expression.
Alternatively, expression of the M2-2 gene can be up-regulated or
down-regulated in a recombinant RSV, for example by placing the
M2-2 ORF in a more promoter-proximal or promoter-distal position,
respectively in the recombinant genome or antigenome. Upregulation
of M2-2 can also be achieved by constructing the genome or
antigenome to include the M2-2 ORF as a separate gene with its own
gene start end gene end signals.
RSV is generally characterized as an enveloped nonsegmented
negative strand RNA virus of the paramyxovirus family (Collins, et
al., Fields Virology 2:1313-1352, 1996, incorporated herein by
reference). Its genome, which is 15,222 nucleotides (nt) in length
for the well known strain A2, is transcribed into 10 messenger RNAs
that were previously shown to encode 10 proteins (Collins, et al.,
Fields Virology 2:1313-1352, 1996; Atreya, et al., J. Virol.
72:1452-61, 1998; Bukreyev, et al., J. Virol. 71:8973-82, 1997;
Collins, et al., Proc. Natl. Acad. Sci. USA 93:81-5, 1996; Teng and
Collins, J. Virol. 72:5707-16, 1998; Teng and Collins, J. Virol.
73:466-473, 1999; Whitehead, et al., J. Virol. 73:3438-42, 1999,
each incorporated herein by reference).
As used herein, "RSV gene" generally refers to a portion of the RSV
genome encoding an mRNA and typically begins at the upstream end
with the 10-nucleotide gene-start (GS) signal and ends at the
downstream end with the 12 to 13-nucleotide gene-end (GE) signal.
Ten such genes for use within the invention are known for RSV,
namely NS1, NS2, N, P, M, SH, G, F, M2 and L. The term "gene" is
also used herein to refer to a "translational open reading frame"
(ORF). ORF is more specifically defined as a translational open
reading frame encoding a significant RSV protein, of which 11 are
currently recognized: NS1, NS2, N, P, M, SH, G, F, M2-1
(alternatively, M2(ORF1)), M2-2 (alternatively, M2(ORF2)), and L.
Thus, the term "gene" interchangeably refers to a genomic RNA
sequence that encodes a subgenomic RNA, and to a ORF (the latter
term applies particularly in a situation such as in the case of the
RSV M2 gene, where a single mRNA contains two overlapping ORFs that
encode distinct proteins). Collins et al., J. Gen. Virol.
71:3015-3020, 1990; Bermingham and Collins, Proc. Natl. Acad. Sci.
USA 96:11259-11264, 1999; Ahmadian et al., EMBO J. 19:2681-2689,
2000; Jin et al., J. Virol. 74:74-82, 2000 (each incorporated
herein by reference). When the term "gene" is used in the context
of determining gene position relative to a promoter position, the
term ordinarily refers strictly to an mRNA-encoding sequence
bordered by transcription gene-start and gene-end signal motifs
(Collins et al., Proc. Natl. Acad. Sci. USA 83:4594-4598, 1986; Kuo
et al., J. Virol. 70:6892-6901, 1996; each incorporated herein by
reference).
By "genome segment" is meant any length of continuous nucleotides
from the RSV genome, which may be part of an ORF, a gene, or an
extragenic region, or a combination thereof.
Four of the RSV proteins presently identified are
nucleocapsid/polymerase proteins, namely the major nucleocapsid N
protein, the phosphoprotein P, and polymerase protein L, and the
transcription antitermination protein M2-1 encoded by a first open
reading frame (ORF) in the M2 gene. Three of these proteins are
surface glycoproteins, namely the attachment G protein, the fusion
F glycoprotein responsible for penetration and syncytium formation,
and the small hydrophobic SH protein of unknown function. The
matrix M protein is an internal virion protein involved in virion
formation. There are two nonstructural proteins NS1 and NS2 of
unknown function. Finally, there is a second open reading frame
(ORF) in the M2 mRNA which encodes an RNA regulatory factor
M2-2.
The G and F proteins are the major neutralization and protective
antigens (Collins, et al., Fields Virology 2:1313-1352, 1996;
Connors, et al., J. Virol. 66:1277-81, 1992). Resistance to
reinfection by RSV is largely mediated by serum and mucosal
antibodies specific against these proteins. RSV-specific cytotoxic
T cells are also induced by RSV infection and can be directed
against a number of different proteins, but this effector has not
yet been shown to be an important contributor to long term
resistance to reinfection. However, both CD8+ and CD4+ cells can be
important in regulating the immune response, and both may be
involved in viral pathogenesis (Johnson, et al., J. Virol.
72:2871-80, 1998; Srikiatkhachom and Braciale, J. Exp. Med.
186:421-32, 1997). Thus, F and G are the most important antigenic
determinants, but other proteins can also play important roles in
the immune response.
The M2 ORF2 mRNA encodes an RNA regulatory factor M2-2. The M2-2
mRNA, not found in other paramyxoviruses or rhabdoviruses, contains
two overlapping translational open reading frames (ORFs) which each
express a protein (FIG. 1A) (Collins et al., J. Gen. Virol.
71:3015-20, 1990, incorporated herein by reference). The upstream
ORF1 encodes the 194-amino acid M2-1 protein, which is a structural
component of the virion (Peeples et al., Virology 95:137-45, 1979,
incorporated herein by reference) and is an anti-termination factor
that promotes transcriptional chain elongation and also increases
the frequency of read through at gene junctions (Collins et al.,
Proc. Nat. Acad. Sci. USA 93:81-5, 1996; Feams and Collins, J.
Virol. 73:5852-5864, 1999; Collins et al. Virology 259:251-255,
1999; Hardy et al., J. Virol. 72:520-6, 1998, each incorporated
herein by reference). ORF2 of strain A2 has 3 potential start site
at codons 1, 3 and 7, all of which overlap with ORF1 (FIG. 1A).
Initiation at the first of these would give an M2-2 protein of 90
amino acids. M2 ORF2 is present in all pneumoviruses examined to
date (Collins et al., J. Gen. Virol. 71:3015-20, 1990; Ling et al.,
J. Gen. Virol. 73:1709-15, 1992; Zamora et al., J. Gen. Virol.
73:737-41, 1992, each incorporated herein by reference).
Translation of M2 mRNA in a cell-free system yielded the M2-1
protein and a second, 11 kDa protein which was of the appropriate
size to be the M2-2 protein (Collins et al., J. Gen. Virol.
71:3015-20, 1990). Coexpression of M2-2 in a model minireplicon
system was found to have a very potent down-regulatory effect on
RNA synthesis (Collins et al., Proc. Nat. Acad. Sci. USA 93:81-5,
1996; Hardy et al., J. Virol. 72:520-6, 1998). More recently, the
RSV M2-2 protein was detected as a minor species in RSV-infected
cells. Thus, several lines of evidence indicate that the M2-2 ORF
is an eleventh RSV gene. However, definitive evidence that an ORF
encodes a significant viral protein includes identification of a
biological effect mediated by expression of the ORF in an
infectious virus. This is demonstrated for M2-2 according to the
methods of the present invention by ablating or deleting all or
part of the M2-2 ORF and thereafter identifying phenotypic
changes--including a shift in the balance of RNA transcription and
replication. Although previous studies suggested that the M2-2
protein generally down-regulates transcription and RNA replication,
the instant disclosure demonstrates that M2-2 unexpectedly shifts
the balance of RNA synthesis from transcription to replication.
Expression of M2 ORF2 is preferably reduced or ablated by modifying
the recombinant RSV genome or antigenome to incorporate a frame
shift mutation or one or more stop codons in M2 ORF2. In more
detailed aspects of the invention, M2 ORF2 is subjected to
mutagenesis to generate a specific frame-shift mutation, hereafter
called the NdeI mutation (FIG. 1B). The restriction enzyme site
within ORF2 for the NdeI mutation was identified at genome position
8299, and the frame-shift mutation (2 nts added) was at codon 47 of
the predicted 90 amino acid protein (FIG. 1B). Accordingly, the
NdeI mutant (exemplified by recombinant strain rA2-NdeI) encodes
the N-terminal 46 amino acids of M2-2 fused to 18 heterologous
amino acids encoded by the frame-shift. Optional frame shift
mutations to generate M2 ORF2 knock out mutants are readily
identified.
In other more detailed aspects of the invention, a second exemplary
M2-2 knock-out mutation is described below, the K5 mutation, which
ablates expression of M2 ORF2 by altering three potential
initiation codons within M2 ORF2 (FIGS. 1A and 1C) to ACG stop
codons. A stop codon may also be added in each register following
the ORF1 termination codon, terminating M2 ORF2 at codon 13 (FIG.
1C) to minimize the possibility of reversion or non-AUG initiation.
An exemplary M2 ORF2 knock out mutant in this context is the
recombinant strain rA2-K5 (also referred to as rA2.DELTA.M2-2),
described below. Other alterations to achieve disruption of M2 ORF2
expression or M2-2 protein expression or function to generate
attenuated RSV vaccine candidates include partial or complete
deletion of the M2 ORF2 coding sequence, in whole or in part, to
render the M2-2 protein partially or entirely non-functional or
terminate its expression. Yet another method for changing the level
of expression of M2-ORF2 is to alter its translational start site
or its spacing relative to the upstream ORF1. For example, M2-ORF2
can be expressed as a separate gene at any locus in the genome or
antigenome, e.g., by insertion of the M2-ORF2 with its own gene
start and gene end signals into an intergenic or other non-coding
region of the genome or antigenome.
As noted above, the recombinant RSV of the invention bearing one or
more mutations in M2 ORF2 possess highly desirable phenotypic
characteristics for vaccine development (see also, Bermingham et
al., Proc. Natl. Acad. Sci. USA 96:11259-11264, 1999; and Jin et
al., J. Virol. 74:74-82, 2000, each incorporated herein by
reference). The modifications described herein that delete M2 ORF2,
in whole or in part, or reduce or ablate expression of M2 ORF2
specify a range of desired phenotypic changes in the resulting
virus or subviral particle. In preferred embodiments, M2 ORF2
deletion and knock out mutants exhibit attenuated viral growth
compared to growth of a corresponding wild-type or mutant parental
RSV strain. Growth, for example in cell cultures, may be reduced by
about two-fold, more often about 5-fold, and preferably about
10-fold or greater overall (e.g., as measured after a 7-8 day
period in culture) compared to growth of the corresponding
wild-type or mutant parental RSV strain. In more detailed aspects,
recombinant RSV of the invention exhibit delayed kinetics of viral
growth, wherein growth during an initial 2-5 day period is reduced
by about 100-fold and up to 1,000-fold or more compared to kinetics
of growth in the corresponding wild-type or mutant parental RSV
strain. These desirable effects are specified by reduction or
ablation of M2-2 ORF2 expression. Intermediate effects are achieved
by reduction of M2-2 protein synthesis. Furthermore, as M2-2 is a
regulatory protein, alterations in virus growth and the pattern of
gene expression can also be achieved by increasing rather than
decreasing M2-ORF2 expression. As described above, this can be
readily achieved by expressing M2-ORF2 as a separate gene and, if
necessary, moving the gene to a more promoter-proximal or
promoter-distal location.
Recombinant vaccine viruses bearing M2 ORF2 deletion and knock out
mutations also preferably exhibit a change in mRNA transcription.
One aspect of this change is delayed kinetics of viral mRNA
synthesis compared to kinetics of mRNA synthesis of a corresponding
wild-type or mutant parental RSV strain. However, after time (e.g.,
at 24 hours post-infection) the M2 ORF2 deletion and knock out
mutants exhibit an increase in cumulative mRNA synthesis. This
increase of cumulative mRNA synthesis can be achieved to levels of
about 50-100%, 100-200%, 200-300% or greater compared to mRNA
accumulation in the corresponding wild-type or mutant parental RSV
strain.
Also provided within the invention are M2 ORF2 deletion and knock
out mutants which exhibit a reduction in viral RNA replication
compared to viral RNA replication (synthesis of genome/antigenome)
of the corresponding wild-type or mutant parental RSV strain. Thus,
accumulation of genomic RNA (e.g., after a post-infection period of
24 hours) is about 25-30%, 15-25%, 10-15% or lower compared to
genomic RNA accumulation in the corresponding wild-type or mutant
parental RSV strain.
In preferred M2 ORF2 deletion and knock out mutants of the
invention, both of the foregoing changes in mRNA and genomic RNA
synthesis are observed. Thus, a cumulative molar ratio of mRNA to
genomic RNA is increased 2- to 5-fold, 5-to 10-fold, 10- to 20-fold
or greater compared to a cumulative molar ratio of mRNA to genomic
RNA observed for the corresponding wild-type or mutant parental RSV
strain.
Also provided herein are M2 ORF2 deletion and knock out mutants
exhibiting increased viral protein accumulation in infected cells
compared to viral protein accumulation in cells infected with a
corresponding wild-type or mutant parental RSV strain. Increased
viral protein levels (e.g., at 36 hours post-infection) may be
50-100%, 100-200%, 200-300% or greater. This is particularly
desirable in M2 ORF2 deletion and knock out mutants which exhibit
wherein the phenotypic change comprises increased expression of one
or more viral antigens compared to expression of the antigen(s) in
the corresponding wild-type or mutant parental RSV strain. This is
a particularly desirable phenotype considering that other
attenuating mutations for RSV typically result in reduced antigen
expression and immunogenicity.
In summary, preferred M2 ORF2 deletion and knock out mutants are
engineered to express undiminished or increased levels of selected
viral antigens while also exhibiting an attenuated phenotype. These
recombinants thus maintain immunogenic potential due to the
increased mRNA transcription and antigen expression, while
attenuation is maintained through concomitant reductions in
replication and growth. This surprising assemblage of phenotypic
traits is highly desired for vaccine development because the
vaccine candidates can be suitably attenuated without sacrificing
immunogenic potential, and may indeed exhibit increased immunogenic
activity.
The instant invention provides for development of live-attenuated
RSV vaccine candidates incorporating M2 ORF2 deletion or knock out
mutations. These recombinant viruses are constructed through a cDNA
intermediate and cDNA-based recovery system. Recombinant viruses
which are made from cDNA replicate independently and are propagated
in the same manner as if they were biologically-derived. M2 ORF2
deletion and knock out mutants can be further modified to
incorporate specific attenuating mutations, as well as a variety of
other mutations and nucleotide modifications, to yield desired
structural or phenotypic affects.
Detailed descriptions of the materials and methods for producing
recombinant RSV from cDNA, and for making and testing the full
range of mutations and nucleotide modifications disclosed herein as
supplemental aspects of the present invention, are set forth in,
e.g., U.S. Provisional Patent Application No. 60/007,083, filed
Sep. 27, 1995; U.S. Patent application Ser. No. 08/720,132, filed
Sep. 27, 1996; U.S. Provisional Patent Application No. 60/021,773,
filed Jul. 15, 1996; U.S. Provisional Patent Application No.
60/046,141, filed May 9, 1997; U.S. Provisional Patent Application
No. 60/047,634, filed May 23, 1997; U.S. Pat. No. 5,993,824, issued
Nov. 30, 1999 (corresponding to International Publication No. WO
98/02530); U.S. patent application Ser. No. 09/291,894, filed by
Collins et al. on Apr. 13, 1999; U.S. Provisional Patent
Application No. 60/129,006, filed by Murphy et al. on Apr. 13,
1999; Crowe et al., Vaccine 12: 691-699, 1994; and Crowe et al.,
Vaccine 12: 783-790, 1994; Collins, et al., Proc Nat. Acad. Sci.
USA 92:11563-11567, 1995; Bukreyev, et al., J Virol 70:6634-41,
1996, Juhasz et al., J. Virol. 71(8):5814-5819, 1997; Durbin et
al., Virology 235:323-332, 1997; Karron et al., J. Infect. Dis.
176:1428-1436, 1997); He et al. Virology 237:249-260, 1997; Baron
et al. J. Virol. 71:1265-1271, 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; Bukreyev, et al.,
Proc. Nat. Acad. Sci. USA 96:2367-2372, 1999; Bermingham and
Collins, Proc. Natl. Acad. Sci. USA 96:11259-11264, 1999 Juhasz et
al., Vaccine 17:1416-1424, 1999; Juhasz et al., J. Virol.
73:5176-5180, 1999; Teng and Collins, J. Virol. 73:466-473, 1999;
Whitehead et al., J. Virol. 73:9773-9780, 1999; Whitehead et al.,
J. Virol. 73:871-877, 1999; and Whitehead et al., J. Virol.
73:3438-3442, 1999. Exemplary methods for producing recombinant RSV
from cDNA involve intracellular coexpression, typically from
plasmids cotransfected into tissue culture cells, of an RSV
antigenomic RNA and the RSV N, P, M2-1 and L proteins. This
launches a productive infection that results in the production of
infectious cDNA-derived virus, which is termed recombinant virus.
Once generated, recombinant RSV is readily propagated in the same
manner as biologically-derived virus, and a recombinant virus and a
counterpart biologically-derived virus cannot be distinguished
unless the former had been modified to contain one or more
introduced changes as markers.
The ability to generate infectious RSV from cDNA provides a method
for introducing predetermined changes into infectious virus via the
cDNA intermediate. This method has been demonstrated to produce a
wide range of infectious, attenuated derivatives of RSV, for
example recombinant vaccine candidates containing one or more amino
acid substitutions in a viral protein, deletion of one or more
genes or ablation of gene expression, and/or one or more nucleotide
substitutions in cis-acting RNA signals yielding desired effects on
viral phenotype (see, e.g., Bukreyev et al., J. Virol.
71:8973-8982, 1997; Whitehead et al., J. Virol. 72:4467-4471, 1998;
Whitehead et al., Virology, 247:232-39, 1998; Bermingham and
Collins, Proc. Natl. Acad. Sci. USA 96:11259-11264,1999; Juhasz et
al., Vaccine 17:1416-1424, 1999; Juhasz et al., J. Virol.
73:5176-5180, 1999; Teng and Collins, J. Virol. 73:466-473, 1999;
Whitehead et al., J. Virol. 73:871-877, 1999; Whitehead et al., J.
Virol. 73:3438-3442, 1999; and Collins et al., Adv. Virus Res.
54:423-451, 1999, each incorporated herein by reference).
Exemplary of the foregoing teachings are methods and procedures
useful within the invention for mutagenizing, isolating and
characterizing RSV to obtain attenuated mutant strains (e.g.,
temperature sensitive (ts), cold passaged (cp) cold-adapted (ca),
small plaque (sp) and host-range restricted (hr) mutant strains)
and for identifying the genetic changes that specify the attenuated
phenotype. In conjunction with these methods, the foregoing
documents detail procedures for determining replication,
immunogenicity, genetic stability and protective efficacy of
biologically derived and recombinantly produced attenuated human
RSV, including human RSV A and B subgroups, in accepted model
systems, including murine and non-human primate model systems. In
addition, these documents describe general methods for developing
and testing immunogenic compositions, including monovalent and
bivalent vaccines, for prophylaxis and treatment of RSV
infection.
Methods for producing infectious recombinant RSV by construction
and expression of cDNA encoding an RSV genome or antigenome
coexpressed with essential RSV proteins are also described in the
above-incorporated documents (see, e.g., U.S. Provisional Patent
Application No. 60/007,083, filed Sep. 27, 1995; U.S. patent
application Ser. No. 08/720,132, filed Sep. 27, 1996; U.S.
Provisional Patent Application No. 60/021,773, filed Jul. 15, 1996;
U.S. Provisional Patent Application No. 60/046,141, filed May 9,
1997; U.S. Provisional Patent Application No. 60/047,634, filed May
23, 1997; U.S. patent application Ser. No. 08/892,403, filed Jul.
15, 1997 (corresponding to International Publication No. WO
98/02530)).
Also disclosed are methods for constructing and evaluating
infectious recombinant RSV that are modified to incorporate
phenotype-specific mutations identified in biologically-derived RSV
mutants, e.g., cp and ts mutations adopted in recombinant RSV from
biologically derived RSV mutants designated 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-1 cp52/2B5 (ATCC VR
2542), and RSV B-1 cp-23 (ATCC VR 2579). The recombinant RSV thus
provided may incorporate one, two, or more ts mutations from the
same, or different, biologically derived RSV mutant(s), for example
one or more of the 248/404, 248/955, 530/1009, or 530/1030
biological mutants. In the latter context, multiply attenuated
recombinants may have a combination of attenuating mutations from
two, three or more biological mutants, e.g., a combination of
attenuating mutations from the RSV mutants 530/1009/404,
248/404/1009, 248/404/1030, or 248/404/1009/1030 mutants. In
exemplary embodiments, one or more attenuating mutations specify a
temperature-sensitive substitution at amino acid Asn43, Phe521,
Gln831, Met1169, or Tyr1321 in the RSV polymerase gene or a
temperature-sensitive nucleotide substitution in the gene-start
sequence of gene M2. Preferably, these mutations involve identical
or conservative changes with the following changes identified in
biologically derived mutant RSV; Ile for Asn43, Leu for Phe521, Leu
for Gln831, Val for Met1169, and Asn for Tyr1321.
In one recent exemplary embodiment of the invention, the sequence
of RSV mutant cpts248/955 was determined, with the exception of the
first 29 nucleotides (3'-end of the genome) and the last 33
nucleotides (5'-end) of the genome. The sequence was then compared
to that of parental virus cpts248. Mutant virus cpts248/955
contained all the mutations previously identified in cspts248, as
well as the following mutations: 1. Insertion of an A residue in
the P gene-end signal at nucleotide 3236. This increases the poly-A
tract from 7 A's to 8 A's. The is the same insertion observed
previously in recombinant RSV rA2 virus preparations, which did not
effect replication levels in mice. 2. An Asn to Ile mutation of
amino acid 43 of the L polymerase due to A to U mutation at cpRSV
nucleotide (nt) 8626. It is therefore considered that the
cpts248/955 phenotype is attributed to the missense mutation at nt
8626. This is consistent with previous findings for the RSV 530,
1030, 1009, and 248 mutants.
Yet additional mutations that may be incorporated in M2 ORF2
deletion and knock out RSV mutants of the invention are mutations,
e.g., attenuating mutations, identified in heterologous RSV or more
distantly related negative stranded RNA viruses. In particular,
attenuating and other desired mutations identified in one negative
stranded RNA virus may be "transferred", e.g., copied, to a
corresponding position within the genome or antigenome of the M2
ORF2 deletion and knock out mutants. Briefly, desired mutations in
one heterologous negative stranded RNA virus are transferred to the
RSV recipient (e.g., bovine or human RSV, respectively). This
involves mapping the mutation in the heterologous virus, thus
identifying by sequence alignment the corresponding site in the
recipient RSV, and mutating the native sequence in the RSV
recipient to the mutant genotype (either by an identical or
conservative mutation), as described in International Application
No. PCT/US00/09695 filed Apr. 12, 2000 and corresponding priority
U.S. Provisional Patent Application Serial No. 60/129,006, each
incorporated herein by reference. As this disclosure teaches, it is
preferable to modify the recipient genome or antigenome to encode
an alteration at the subject site of mutation that corresponds
conservatively to the alteration identified in the heterologous
mutant virus. For example, if an amino acid substitution marks a
site of mutation in the mutant virus compared to the corresponding
wild-type sequence, then a similar substitution should be
engineered at the corresponding residue(s) in the recombinant
virus. Preferably the substitution will involve an identical or
conservative amino acid to the substitute residue present in the
mutant viral protein. However, it is also possible to alter the
native amino acid residue at the site of mutation
non-conservatively with respect to the substitute residue in the
mutant protein (e.g., by using any other amino acid to disrupt or
impair the function of the wild-type residue). Negative stranded
RNA viruses from which exemplary mutations are identified and
transferred into a recombinant RSV of the invention include other
RSVs (e.g., murine), PIV, Sendai virus (SeV), Newcastle disease
virus (NDV), simian virus 5 (SV5), measles virus (MeV), rindepest
virus, canine distemper virus (CDV), rabies virus (RaV) and
vesicular stomatitis virus (VSV). A variety of exemplary mutations
are disclosed, including but not limited to an amino acid
substitution of phenylalanine at position 521 of the RSV L protein
(corresponding to a substitution of phenylalanine at position 456
of the HPIV3 L protein). In the case of mutations marked by
deletions or insertions, these can be introduced as corresponding
deletions or insertions into the recombinant virus, however the
particular size and amino acid sequence of the deleted or inserted
protein fragment can vary.
A variety of additional types of mutations are also disclosed in
the foregoing incorporated references and can be readily engineered
into a recombinant RSV of the invention to calibrate attenuation,
immunogenicity or provide other advantageous structural and/or
phenotypic effects. For example, restriction site markers are
routinely introduced within the M2 ORF2 deletion or knock out
mutant antigenome or genome to facilitate cDNA construction and
manipulation. Also described in the incorporated references are a
wide range of nucleotide modifications other than point or
site-specific mutations that are useful within the instant
invention. For example, methods and compositions are disclosed for
producing recombinant RSV expressing an additional foreign gene,
e.g., a chloramphenicol acetyl transferase (CAT) or luciferase
gene. Such recombinants generally exhibit reduced growth associated
with the inserted gene. This attenuation appears to increase with
increasing length of the inserted gene. The finding that insertion
of a foreign gene into recombinant RSV reduces level of replication
and is stable during passage in vitro provides another effective
method for attenuating RSV for vaccine use. Similar or improved
effects can thus be achieved by insertion of other desired genes,
for example cytokines such as interferon-.gamma., interleukin-2,
interleukin-4 and GM-CSF, among others.
Additional nucleotide modifications disclosed in the foregoing
references for incorporation into M2 ORF2 deletion and knock out
RSV of the invention include partial or complete deletion or
ablation of a different RSV gene outside of M2 ORF2. Thus,
additional RSV genes or genome segments within recombinant RSV of
the invention may be deleted, including partial or complete
deletions of open reading frames and/or cis-acting regulatory
sequences of the RSV NS1, NS2, N, P, M, G, F, SH, M2(ORF1), and/or
L genes. Within this aspect of the invention nucleotide
modifications may be engineered to delete or silence a selected
gene to achieve a recombinant vaccine candidate that replicates
well in vitro but which is attenuated for replication in vivo
(Bukreyev et al., J. Virol. 71:8973-8982, 1997; 23] Teng et al., J.
Virol. 73:466-473, 1999; each incorporated herein by reference).
For example, deletion of the SH gene results in a virus,
exemplified by rA2.DELTA.SH, that replicates in vitro with an
efficiency equal to or slightly better than that of wild-type rRSV
(rA2) and which is moderately attenuated in mice and chimpanzees
(Bukreyev et al., J. Virol. 71:8973-8982, 1997; Whitehead et al.,
J. Virol. 73:3438-3442, 1999; each incorporated herein by
reference). Recombinant RSV from which the NS2 gene is deleted,
designated rA2.DELTA.NS2, exhibits reduced growth kinetics and
reduced yield of infectious virus in vitro and is markedly
attenuated in mice and chimpanzees (Teng et al., J. Virol.
73:466-473, 1999; Whitehead et al., J. Virol. 73:3438-3442, 1999;
each incorporated herein by reference). Similar in vitro properties
are disclosed for a recombinant bovine RSV from which the NS2 gene
is deleted (Buchholz et al., J. Virol. 73:251-259, 1999;
incorporated herein by reference).
In one example, a recombinant RSV was generated in which expression
of the SH gene was ablated by removal of a polynucleotide sequence
encoding the SH mRNA and protein. Deletion of the SH gene yielded
not only recoverable, infectious RSV, but one which exhibited
substantially improved growth in tissue culture based on both yield
of infectious virus and plaque size. This improved growth in tissue
culture specified by the SH deletion provides useful tools for
developing M2 ORF2 deletion and knock out mutant RSV vaccines, for
example by overcoming problems of poor RSV yields in culture.
Moreover, these deletions are highly stable against genetic
reversion, rendering RSV clones derived therefrom particularly
useful as vaccine agents.
SH-minus RSV recombinants also exhibit site-specific attenuation in
the upper respiratory tract of mice, which presents novel
advantages for vaccine development. Certain of the current RSV
strains under evaluation as live virus vaccines, for example cp
mutants, do not exhibit significantly altered growth in tissue
culture. These are host range mutations and they restrict
replication in the respiratory tract of chimpanzees and humans
approximately 100-fold in the lower respiratory tract. Another
exemplary type of mutation, ts mutations, tend to preferentially
restrict virus replication in the lower respiratory tract due to
the gradient of increasing body temperature from the upper to the
lower respiratory tract. In contrast to these cp and ts mutants,
SH-minus RSV mutants have distinct phenotypes of greater
restriction in the upper respiratory tract. This is particularly
desirable for vaccine viruses for use in very young infants,
because restriction of replication in the upper respiratory tract
is required to ensure safe vaccine administration in this
vulnerable age group whose members breathe predominantly through
the nose. Further, in any age group, reduced replication in the
upper respiratory tract will reduce morbidity from otitis media. In
addition to these advantages, the nature of SH deletion mutations,
involving e.g., nearly 400 nt and ablation of an entire mRNA,
represents a type of mutation which will be highly refractory to
reversion.
Also discussed in the context of SH gene modifications is a
comparison of SH genes among different RSVs, including human and
bovine RSVs, and other pneumoviruses to provide additional tools
and methods for generating useful RSV recombinant vaccines. For
example, the two RSV antigenic subgroups, A and B, exhibit a
relatively high degree of conservation in certain SH domains. In
two such domains, the N-terminal region and putative
membrane-spanning domains of RSV A and B display 84% identity at
the amino acid level, while the C-terminal putative ectodomains are
more divergent (approx. 50% identity). Comparison of the SH genes
of two human RSV subgroup B strains, 8/60 and 18537, identified
only a single amino acid difference (Anderson et al., supra). The
SH proteins of human versus bovine RSV are approximately 40%
identical, and share major structural features including (i) an
asymmetric distribution of conserved residues; (ii) very similar
hydrophobicity profiles; (iii) the presence of two N-linked
glycosylation sites with one site being on each side of the
hydrophobic region; and (iv) a single cysteine residue on the
carboxyterminal side of the central hydrophobic region of each SH
protein. (Anderson et al., supra). By evaluating these and other
sequence similarities and differences, selections can be made of
heterologous sequence(s) that can be substituted or inserted within
infectious M2 ORF2 deletion and knock out mutant RSV clones, for
example to yield vaccines having multi-specific immunogenic effects
or, alternatively or in addition, desirable effects such as
attenuation.
Also disclosed in the context of gene deletions are the effects of
changing gene position. For example, deletion of the SH gene
results in an effective change in downstream gene position to a
more promoter proximal position. This may be associated with an
increase in transcription of downstream genes in the recombinant
virus. Alternatively, the position of any gene can be changed to
alter expression, for example by insertion or transpostioning of
the gene to an upstream or downstream intergenic or other noncoding
region. Thus, methods are provided for altering levels of RSV gene
expression by changing gene order or position in the genome or
antigenome. Decreased levels of expression of downstream genes are
expected to specify attenuation phenotypes, whereas increased
expression can achieve the opposite effects in recombinant RSV in
permissive hosts, e.g., chimpanzees and humans.
In another example described in the above-incorporated references,
expression of the NS2. gene is ablated by introduction of stop
codons into the translational open reading frame (ORF). The rate of
release of infectious virus was reduced for this NS2 knock-out
virus compared to wild-type. In addition, comparison of the plaques
of the mutant and wild-type viruses showed that those of the NS2
knock-out were greatly reduced in size. This type of mutation can
thus be incorporated within viable recombinant RSV to yield altered
phenotypes, in this case reduced rate of virus growth and reduced
plaque size in vitro. These and other knock-out methods and mutants
will therefore provide for yet additional recombinant RSV vaccine
agents, based on the correlation between reduced plaque size in
vitro and attenuation in vivo. Expression of the NS2 gene also was
ablated by complete removal of the NS2 gene, yielding a virus with
a similar phenotype.
Other RSV genes which have been successfully deleted include the
NS1 and G genes. The former was deleted by removal of the
polynucleotide sequence encoding the respective protein, and the
latter by introducing a frame-shift or altering translational start
sites and introducing stop codons. Specifically, the NS1 gene was
deleted by removal of nucleotides 122 to 630 in the antigenomic
cDNA, thereby joining the upstream nontranslated region of NS1 to
the translational initiation codon of NS2. This virus, designated
rA2.DELTA.NS1, exhibited reduced RNA replication, plaque size,
growth kinetics and approximately 10-fold lower yield of infectious
virus in vitro. Interestingly, recovered NS1-minus virus produce
small plaques in tissue culture albeit not as small as those of the
NS2 deletion virus. The fact that the NS1-minus virus can grow,
albeit with reduced efficiency, identifies the NS1 protein as an
accessory protein, one that is dispensable to virus growth. The
plaque size of the NS1-minus virus was similar to that of NS2
knock-out virus in which expression of the NS2 protein was ablated
by introducing translational stop codons into its coding sequence.
The small plaque phenotype is commonly associated with attenuating
mutations. This type of mutation can thus be incorporated within
viable recombinant RSV to yield altered phenotypes. These and other
knock out methods and mutants will therefore provide for yet
additional recombinant RSV vaccine agents, based on the known
correlation between plaque size in vitro and attenuation in vivo.
The NS2 knock-out mutant exhibited a moderately attenuated
phenotype in the upper respiratory tract and a highly attenuated
phenotype in the lower respiratory tract in naive chimpanzees. This
mutant also elicited greatly reduced disease symptoms in chimps
while stimulating significant resistance to challenge by the
wild-type virus (Whitehead et al., J. Virol. 73:3438-3442, 1999,
incorporated herein by reference).
Yet additional methods and compositions provided within the
incorporated references and useful within the invention involve
different nucleotide modifications within M2 ORF2 deletion and
knock out mutants that alter different cis-acting regulatory
sequences within the recombinant genome or antigenome. For example,
a translational start site for a secreted form of the RSV G
glycoprotein can be deleted to disrupt expression of this form of
the G glycoprotein. The RSV G protein is synthesized in two forms:
as an anchored type II integral membrane protein and as a
N-terminally resected form which lacks essentially all of the
membrane anchor and is secreted (Hendricks et al., J. Virol.
62:2228-2233, 1988). The two forms have been shown to be derived by
translational initiation at two different start sites: the longer
form initiates at the first AUG of the G ORF, and the second
initiates at the second AUG of the ORF at codon 48 and is further
processed by proteolysis (Roberts et al., J. Virol. 68: 4538-4546
1994). The presence of this second start site is highly conserved,
being present in all strains of human, bovine and ovine RSV
sequenced to date. It has been suggested that the soluble form of
the G protein might mitigate host immunity by acting as a decoy to
trap neutralizing antibodies. Also, soluble G has been implicated
in preferential stimulation of a Th2-biased response, which in turn
appears to be associated with enhanced immunopathology upon
subsequent exposure to RSV. With regard to an RSV vaccine virus, it
is highly desirable to minimize antibody trapping or imbalanced
stimulation of the immune system, and so it would be desirable to
ablate expression of the secreted form of the G protein. This has
been achieved in recombinant virus. Thus, this mutation is
particularly useful to qualitatively and/or quantitatively alter
the host immune response elicited by the recombinant virus, rather
than to directly attenuate the virus. Also the G protein gene may
be deleted altogether. The resulting virus exhibits a host range
effect, growing inefficiently on HEp-2 cells but growing as
efficiently as wild type virus on Vero cells. Presumably,
attachment function can also be provided by another protein or can
be dispensed with altogether. Thus, the invention also provides
live-attenuated RSV vaccine virus lacking the G protein.
The incorporated references also describe modulation of the
phenotype of recombinant RSV by altering cis-acting transcription
signals of exemplary genes, e.g., NS1 and NS2. The results of these
nucleotide modifications are consistent with modification of gene
expression by altering cis-regulatory elements, for example to
decrease levels of read through mRNAs and increase expression of
proteins from downstream genes. The resulting recombinant viruses
will preferably exhibit increased growth kinetics and increased
plaque size. Exemplary modifications to cis-acting regulatory
sequences include modifications to gene end (GE) and gene start
(GS) signals associated with RSV genes. In this context, exemplary
changes include alterations of the GE signals of the NS1 and NS2
genes rendering these signals identical to the naturally-occurring
GE signal of the RSV N gene. The resulting recombinant virus
exhibits increased growth kinetics and plaque size and therefore
provide yet additional means for beneficially modifying phenotypes
of M2 ORF2 deletion and knock out mutant RSV vaccine
candidates.
Also useful within the instant invention are methods and
compositions provided in the above-incorporated references that
allow production of attenuated M2 ORF2 deletion and knock out
mutant RSV vaccine virus comprising sequences from both RSV
subgroups A and B, e.g., to yield a RSV A or B vaccine or a
bivalent RSV A/B vaccine. Thus, methods and compositions provided
in the above-incorporated references that allow production of
attenuated M2 ORF2 deletion and knock out RSV vaccine viruses
comprising sequences from both RSV subgroups A and B, e.g., to
yield a RSV A or B vaccine or a bivalent RSV A/B vaccine (see,
e.g., U.S. patent application Ser. No. 09/291,894, filed by Collins
et al. on Apr. 13, 1999, incorporated herein by reference). In one
example a RSV subgroup B-specific vaccine virus is provided in
which an attenuated subgroup A virus is used to express the F
and/or G glycoproteins of a subgroup B RSV. Because the F and G
proteins are the major protective antigens and confer most of the
RSV subgroup specificity, this chimeric virus will stimulate a
strong immune response against subgroup B. This strategy may be
implemented using two alternative approaches. One is to insert the
G glycoprotein gene of a subgroup B virus into the subgroup A
background (or vice-versa) as an additional gene. However, since
the F protein also exhibits significant subgroup-specificity, it
would be preferable to express both subgroup B glycoproteins in a
subgroup B-specific vaccine. Moreover, it is desirable to further
modify a subgroup B virus to achieve proper attenuation and
immunogenicity in accordance with the teachings herein. Thus, the
second, more desirable strategy to achieve an RSV subgroup B
vaccine is to remove the G and F genes from a subgroup A
recombinant cDNA background genome or antigenome, and replace them
with the G and F genes of a subgroup B RSV. The resulting A/B
chimeric RSV contains the internal proteins of subgroup A and the
external protective antigens of subgroup B. This virus can then be
attenuated to a desired level by systematic incorporation of
attenuating mutations as described above. For example, specific
attenuating mutations that have been incorporated into chimeric RSV
A/B viruses include: (i) three of the five cp mutations, namely the
mutation in N (V2671) and the two in L (C319Y and H1690Y), but not
the two in F since these are removed by substitution with the B1 F
gene; (ii) the 248 (Q831L), 1030 (Y1321N) and, optionally, 404-L
(D1183E) mutations which have been identified in attenuated strain
A2 viruses; (iii) the single nucleotide substitution at position 9
in the gene-start signal of the M2 gene, and (iv) deletion of the
SH gene. Other immediately available mutations in chimeric RSV A/B
include, but are not limited to, NS1, NS2, SH, or G gene deletions,
and the 530 and 1009 mutations, alone or in combination.
Desired phenotypic changes that are engineered into M2 ORF2
deletion and knock out mutant RSV of the invention include, but are
not limited to, attenuation in cell culture or in a selected host
environment, resistance to reversion from the attenuated phenotype,
enhanced immunogenic characteristics (e.g., as determined by
enhancement, or diminution, of an elicited immune response),
upregulation or downregulation of transcription and/or translation
of selected viral products, etc. In preferred aspects of the
invention, attenuated, M2 ORF2 deletion and knock out mutant RSV
are produced in which the recombinant genome or antigenome is
further modified by introducing one or more attenuating mutations
specifying an attenuating phenotype. These mutations may be
generated de novo and tested for attenuating effects according to a
rational design mutagenesis strategy as described in the
above-incorporated references. Alternatively, the attenuating
mutations can be identified in a biologically derived mutant RSV
and thereafter incorporated into the M2 ORF2 deletion and knock out
mutant RSV of the invention.
Attenuating mutations in biologically derived RSV for incorporation
within an M2 ORF2 deletion or knock out mutant RSV vaccine strain
may occur naturally or may be introduced into wild-type RSV strains
by well known mutagenesis procedures. For example, incompletely
attenuated parental RSV strains can be produced by chemical
mutagenesis during virus growth in cell cultures to which a
chemical mutagen has been added, by selection of virus that has
been subjected to passage at suboptimal temperatures in order to
introduce growth restriction mutations, or by selection of a
mutagenized virus that produces small plaques (sp) or exhibit
temperature sensitive (ts) phenotypes in cell culture, as generally
described herein and in U.S. Pat. No. 5,922,326, incorporated
herein by reference.
By "biologically derived RSV" is meant any RSV not produced by
recombinant means. Thus, biologically derived RSV include naturally
occurring RSV of all subgroups and strains, including, e.g.,
naturally occurring RSV having a wild-type genomic sequence and RSV
having genomic variations from a reference wild-type RSV sequence,
e.g., RSV having a mutation specifying an attenuated phenotype.
Likewise, biologically derived RSV include RSV mutants derived from
a parental RSV strain by, inter alia, artificial mutagenesis and
selection procedures (see, e.g., International Publication WO
93/21310, incorporated herein by reference).
The level of temperature sensitivity of replication in exemplary
attenuated RSV for use within the invention is determined by
comparing its replication at a permissive temperature with that at
several restrictive temperatures. The lowest temperature at which
the replication of the virus is reduced 100-fold or more in
comparison with its replication at the permissive temperature is
termed the shutoff temperature. In experimental animals and humans,
both the replication and virulence of RSV correlate with the
mutant's shutoff temperature. Replication of mutants with a shutoff
temperature of 39.degree. C. is moderately restricted, whereas
mutants with a shutoff of 38.degree. C. replicate less well and
symptoms of illness are mainly restricted to the upper respiratory
tract. A virus with a shutoff temperature of 35.degree. C. to
37.degree. C. will typically be fully attenuated in chimpanzees and
substantially attenuated in humans. Thus, attenuated biologically
derived mutant and M2 ORF2 deletion and knock out mutant RSV of the
invention which are ts will have a shutoff temperature in the range
of about 35.degree. C. to 39.degree. C., and preferably from
35.degree. C. to 38.degree. C. The addition of a ts mutation into a
partially attenuated strain produces a multiply attenuated virus
useful within vaccine compositions of the invention.
A number of attenuated RSV strains as candidate vaccines for
intranasal administration have been developed using multiple rounds
of chemical mutagenesis to introduce multiple mutations into a
virus which had already been attenuated during cold-passage (e.g.,
Connors et al., Virology 208: 478-484, 1995; Crowe et al., Vaccine
12: 691-699, 1994; and Crowe et al., Vaccine 12: 783-790, 1994,
incorporated herein by reference). Evaluation in rodents,
chimpanzees, adults and infants indicate that certain of these
candidate vaccine strains are relatively stable genetically, are
highly immunogenic, and may be satisfactorily attenuated.
Nucleotide sequence analysis of some of these attenuated viruses
indicates that each level of increased attenuation is associated
with specific nucleotide and amino acid substitutions. The
above-incorporated references also disclose how to routinely
distinguish between silent incidental mutations and those
responsible for phenotype differences by introducing the mutations,
separately and in various combinations, into the genome or
antigenome of infectious RSV clones. This process coupled with
evaluation of phenotype characteristics of parental and derivative
virus identifies mutations responsible for such desired
characteristics as attenuation, temperature sensitivity,
cold-adaptation, small plaque size, host range restriction,
etc.
Mutations thus identified are compiled into a "menu" and are then
introduced as desired, singly or in combination, to adjust an M2
ORF2 deletion or knock out mutant RSV vaccine virus to an
appropriate level of attenuation, immunogenicity, genetic
resistance to reversion from an attenuated phenotype, etc., as
desired. Preferably, the chimeric RSV of the invention are
attenuated by incorporation of at least one, and more preferably
two or more, attenuating mutations identified from such a menu,
which may be defined as a group of known mutations within a panel
of biologically derived mutant RSV strains. Preferred panels of
mutant RSV strains described herein are cold passaged (cp) and/or
temperature sensitive (ts) mutants, for example a panel comprised
of RSV mutants designated 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-1 cp52/2B5 (ATCC VR 2542), and RSV
B-1 cp-23 (ATCC VR 2579) (each deposited under the terms of the
Budapest Treaty with the American Type Culture Collection (ATCC) of
10801 University Boulevard, Manassas, Va. 20110-2209, U.S.A., and
granted the above identified accession numbers).
From this exemplary panel of biologically derived mutants, a large
menu of attenuating mutations are provided which can each be
combined with any other mutation(s) within the panel for
calibrating the level of attenuation in a recombinant, M2 ORF2
deletion or knock out mutant RSV for vaccine use. Additional
mutations may be derived from RSV having non-ts and non-cp
attenuating mutations as identified, e.g., in small plaque (sp),
cold-adapted (ca) or host-range restricted (hr) mutant strains.
Attenuating mutations may be selected in coding portions of a donor
or recipient RSV gene or in non-coding regions such as a
cis-regulatory sequence. For example, attenuating mutations may
include single or multiple base changes in a gene start sequence,
as exemplified by a single or multiple base substitution in the M2
gene start sequence at nucleotide 7605.
M2 ORF2 deletion and knock out mutants RSV designed and selected
for vaccine use often have at least two and sometimes three or more
attenuating mutations to achieve a satisfactory level of
attenuation for broad clinical use. In one embodiment, at least one
attenuating mutation occurs in the RSV polymerase gene and involves
a nucleotide substitution specifying an amino acid change in the
polymerase protein specifying a temperature-sensitive (ts)
phenotype. Exemplary M2 ORF2 deletion and knock out mutants in this
context incorporate one or more nucleotide substitutions in the
large polymerase gene L resulting in an amino acid change at amino
acid Asn43, Phe521, Gln831, Met1169, or Tyr1321, as exemplified by
the changes, Ile for Asn43, Leu for Phe521, Leu for Gln831, Val for
Met1169, and Asn for Tyr1321. Alternately or additionally, M2 ORF2
deletion and knock out mutant RSV of the invention may incorporate
a ts mutation in a different RSV gene, e.g., in the M2 gene.
Preferably, two or more nucleotide changes are incorporated in a
codon specifying an attenuating mutation, e.g., in a codon
specifying a ts mutation, thereby decreasing the likelihood of
reversion from an attenuated phenotype.
In accordance with the methods of the invention, M2 ORF2 deletion
and knock out mutant RSV can be readily constructed and
characterized that incorporate at least one and up to a full
complement of attenuating mutations present within a panel of
biologically derived mutant RSV strains. Thus, mutations can be
assembled in any combination from a selected panel of mutants, for
example, 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-1 cp52/2B5 (ATCC VR 2542), and RSV B-1 cp-23 (ATCC VR
2579). In this manner, attenuation of vaccine candidates can be
finely adjusted for use in one or fewer classes of patients,
including seronegative infants.
In more specific embodiments, M2 ORF2 deletion and knock out mutant
RSV for vaccine use incorporate at least one and up to a full
complement of attenuating mutations specifying a
temperature-sensitive and/or attenuating amino acid substitution at
Asn43, Phe521, Gln831, Met1169 or Tyr1321 in the RSV polymerase
gene L, or a temperature-sensitive nucleotide substitution in the
gene-start sequence of gene M2. Alternatively or additionally, the
recombinant RSV of the invention may incorporate at least one and
up to a full complement of mutations from cold-passaged attenuated
RSV, for example one or more mutations specifying an amino acid
substitution at Val267 in the RSV N gene, Glu218 or Thr523 in the
RSV F gene, Cys319 or His1690 in the RSV polymerase gene L.
In other detailed embodiments, the M2 ORF2 deletion and knock out
mutant RSV of the invention is further modified to incorporate
attenuating mutations selected from (i) a panel of mutations
specifying temperature-sensitive amino acid substitutions Gln831 to
Leu, and Tyr1321 to Asn in the RSV polymerase gene L; (ii) a
temperature-sensitive nucleotide substitution in the gene-start
sequence of gene M2; (iii) an attenuating panel of mutations
adopted from cold-passaged RSV specifying amino acid substitutions
Val267 Ile in the RSV N gene, and Cys319 to Tyr and His1690 Tyr in
the RSV polymerase gene L; or (iv) deletion or ablation of
expression of one or more of the RSV SH, NS1, NS2, G and M2-2
genes. Preferably, these and other examples of M2 ORF2 deletion and
knock out mutant RSV incorporate at least two attenuating mutations
adopted from biologically derived mutant RSV, which may be derived
from the same or different biologically derived mutant RSV strains.
Also preferably, these exemplary mutants have one or more of their
attenuating mutations stabilized by multiple nucleotide changes in
a codon specifying the mutation.
In accordance with the foregoing description, the ability to
produce infectious RSV from cDNA permits introduction of specific
engineered changes within M2 ORF2 deletion and knock out mutants.
In particular, infectious, recombinant RSV are employed for
identification of specific mutation(s) in biologically derived,
attenuated RSV strains, for example mutations which specify ts, ca,
att and other phenotypes. Desired mutations are thus identified and
introduced into recombinant, M2 ORF2 deletion and knock out mutant
RSV vaccine strains. The capability of producing virus from cDNA
allows for routine incorporation of these mutations, individually
or in various selected combinations, into a full-length cDNA clone,
whereafter the phenotypes of rescued recombinant viruses containing
the introduced mutations can be readily determined.
By identifying and incorporating specific, biologically derived
mutations associated with desired phenotypes, e.g., a cp or ts
phenotype, into infectious RSV clones, the invention provides for
other, site-specific modifications at, or within close proximity
to, the identified mutation. Whereas most attenuating mutations
produced in biologically derived RSV are single nucleotide changes,
other "site specific" mutations can also be incorporated by
recombinant techniques into biologically derived or recombinant
RSV. As used herein, site-specific mutations include insertions,
substitutions, deletions or rearrangements of from 1 to 3, up to
about 5-15 or more altered nucleotides (e.g., altered from a
wild-type RSV sequence, from a sequence of a selected mutant RSV
strain, or from a parent recombinant RSV clone subjected to
mutagenesis). Such site-specific mutations may be incorporated at,
or within the region of, a selected, biologically derived mutation.
Alternatively, the mutations can be introduced in various other
contexts within an RSV clone, for example at or near a cis-acting
regulatory sequence or nucleotide sequence encoding a protein
active site, binding site, immunogenic epitope, etc.
Site-specific RSV mutants typically retain a desired attenuating
phenotype, but may additionally exhibit altered phenotypic
characteristics unrelated to attenuation, e.g., enhanced or
broadened immunogenicity, and/or improved growth. Further examples
of desired, site-specific mutants include recombinant RSV designed
to incorporate additional, stabilizing nucleotide mutations in a
codon specifying an attenuating mutation. Where possible, two or
more nucleotide substitutions are introduced at codons that specify
attenuating amino acid changes in a parent mutant or recombinant
RSV clone, yielding a biologically derived or recombinant RSV
having genetic resistance to reversion from an attenuated
phenotype. In other embodiments, site-specific nucleotide
substitutions, additions, deletions or rearrangements are
introduced upstream (N-terminal direction) or downstream
(C-terminal direction), e.g., from 1 to 3, 5-10 and up to 15
nucleotides or more 5' or 3', relative to a targeted nucleotide
position, e.g., to construct or ablate an existing cis-acting
regulatory element.
In addition to single and multiple point mutations and
site-specific mutations, changes to M2 ORF2 deletion and knock out
mutant RSV disclosed herein include deletions, insertions,
substitutions or rearrangements of whole genes or genome segments.
These mutations may alter small numbers of bases (e.g., from 15-30
bases, up to 35-50 bases or more), large blocks of nucleotides
(e.g., 50-100, 100-300, 300-500, 500-1,000 bases), or nearly
complete or complete genes (e.g., 1,000-1,500 nucleotides,
1,500-2,500 nucleotides, 2,500-5,000, nucleotides, 5,00-6,5000
nucleotides or more) in the donor or recipient genome or
antigenome, depending upon the nature of the change (i.e., a small
number of bases may be changed to insert or ablate an immunogenic
epitope or change a small genome segment, whereas large block(s) of
bases are involved when genes or large genome segments are added,
substituted, deleted or rearranged.
In additional aspects, the invention provides for supplementation
of mutations adopted into a recombinant RSV clone from biologically
derived RSV, e.g., cp and ts mutations, with additional types of
mutations involving the same or different genes in a further
modified M2-2 deletion or ablation mutant. RSV encodes ten mRNAs
and ten or eleven proteins. Three of these are transmembrane
surface proteins, namely the attachment G rotein, fusion F protein
involved in penetration, and small hydrophobic SH protein. G and F
are the major viral neutralization and protective antigens. Four
additional proteins are associated with the viral nucleocapsid,
namely the RNA binding protein N, the phosphoprotein P, the large
polymerase protein L, and the transcription elongation factor M2
ORF 1. A second ORF in M2, the M2-2 ORF encodes an important RNA
regulatory factor. The matrix M protein is part of the inner virion
and probably mediates association between the nucleocapsid and the
envelope. Finally, there are two nonstructural proteins, NS1 and
NS2, of unknown function. Each of these proteins can be selectively
altered in terms of expression levels, or can be added deleted,
substituted or rearranged, in whole or in part, alone or in
combination with other desired modifications, to yield an M2 ORF2
deletion or knock out mutant RSV exhibiting novel vaccine
characteristics.
Thus, in addition to or in combination with attenuating mutations
adopted from biologically derived RSV mutants, the present
invention also provides a range of additional methods for
attenuating or otherwise modifying the phenotype of M2 ORF2
deletion and knock out mutant RSV based on recombinant engineering
of infectious RSV clones. A variety of alterations can be produced
in an isolated polynucleotide sequence encoding the donor gene or
genome segment or the background genome or antigenome for
incorporation into infectious clones. More specifically, to achieve
desired structural and phenotypic changes in recombinant RSV, the
invention allows for introduction of modifications which delete,
substitute, introduce, or rearrange a selected nucleotide or
plurality of nucleotides from a parent genome or antigenome, as
well as mutations which delete, substitute, introduce or rearrange
whole gene(s) or genome segment(s), within an M2 ORF2 deletion or
knock out mutant RSV clone.
Desired modifications of infectious RSV according to the invention
are typically selected to specify a desired phenotypic change,
e.g., a change in viral growth, temperature sensitivity, ability to
elicit a host immune response, attenuation, etc. These changes can
be brought about either in a donor or recipient genome or
antigenome by, e.g., mutagenesis of a parent RSV clone to ablate,
introduce or rearrange a specific gene(s) or genome region(s)
(e.g., a genome segment that encodes a protein structural domain,
such as a cytoplasmic, transmembrane or extracellular domain, an
immunogenic epitope, binding region, active site, etc. or a
cis-acting signal). Genes of interest in this regard include all of
the genes of the RSV genome: 3'-NS1-NS2-N-P-M-SH-G-F-M21/M2-2-L-5',
as well as heterologous genes from other RSV, other viruses and a
variety of other non-RSV sources as indicated herein.
Also provided are modifications in M2 ORF2 deletion and knock out
mutant RSV which simply alter or ablate expression of a selected
gene, e.g., by introducing a termination codon within a selected
RSV coding sequence, changing the position of an RSV gene relative
to an operably linked promoter, introducing or removing an upstream
start codon to alter rates of expression, modifying (e.g., by
changing position, altering an existing sequence, or substituting
an existing sequence with a heterologous sequence) GS and/or GE
transcription signals to alter phenotype (e.g., growth, temperature
restrictions on transcription, etc.), and various other deletions,
substitutions, additions and rearrangements that specify
quantitative or qualitative changes, e.g., in viral replication,
transcription of selected gene(s), or translation of selected
protein(s).
The ability to analyze and incorporate other types of attenuating
mutations into M2 ORF2 deletion and knock out mutants for vaccine
development extends to a broad assemblage of targeted changes in
RSV clones. For example, deletion of the SH gene yields a
recombinant RSV having novel phenotypic characteristics, including
enhanced growth. In the present invention, an SH, NS1, NS2 or G
gene (or any other selected, non-essential gene or genome segment)
is deleted in a recombinant RSV, which may also have one or more
additional mutations specifying an attenuated phenotype, e.g., one
or more mutation(s) adopted from a biologically derived attenuated
RSV mutant. In exemplary embodiments, an SH, NS1, NS2 or G gene is
deleted in combination with one or more cp and/or ts mutations
adopted from cpts248/404, cpts530/1009, cpts530/1030, or another
selected mutant RSV strain or with other changes determined
empirically, to yield a recombinant RSV having increased yield of
virus, enhanced attenuation, and resistance to phenotypic
reversion, due to the combined effects of the different
mutations.
Any RSV gene which is not essential for growth, for example the SH,
NS1 NS2 or G genes, can be ablated or otherwise modified in a
recombinant RSV to yield desired effects on virulence,
pathogenesis, immunogenicity and other phenotypic characters. For
example, ablation by deletion of a non-essential gene such as SH
results in enhanced viral growth in culture. Without wishing to be
bound by theory, this effect is likely due in part to a reduced
nucleotide length of the viral genome. In the case of one exemplary
SH-minus clone, the modified viral genome is 14,825 nt long, 398
nucleotides less than wild-type. By engineering similar mutations
that decrease genome size, e.g., in other coding or noncoding
regions elsewhere in the RSV genome, such as in the P, M, F and M2
genes, the invention provides several readily obtainable methods
and materials for improving RSV growth.
In addition, a variety of other genetic alterations can be produced
in a RSV genome or antigenome for incorporation into infectious M2
ORF2 deletion and knock out mutant RSV, alone or together with one
or more attenuating mutations adopted from a biologically derived
mutant RSV. Additional heterologous genes and genome segments (e.g.
from different RSV genes, different RSV strains or types, or
non-RSV sources) may be inserted in whole or in part, the order of
genes changed, gene overlap removed, an RSV genome promoter
replaced with its antigenome counterpart, portions of genes removed
or substituted, and even entire genes deleted. Different or
additional modifications in the sequence can be made to facilitate
manipulations, such as the insertion of unique restriction sites in
various intergenic regions or elsewhere. Nontranslated gene
sequences can be removed to increase capacity for inserting foreign
sequences.
Also provided within the invention are genetic modifications in an
M2 ORF2 deletion or knock out mutant RSV which alter or ablate the
expression of a selected gene or genome segment without removing
the gene or genome segment from the RSV clone. For example, this
can be achieved by introducing a frame shift mutation or
termination codon within a selected coding sequence, changing the
position of a gene or introducing an upstream start codon to alter
its rate of expression, or changing GS and/or GE transcription
signals to alter phenotype (e.g., growth, temperature restrictions
on transcription, etc.). In more detailed aspects of the invention,
M2 ORF2 deletion and knock out mutant RSV are provided in which
expression of the NS2 gene is ablated at the translational level
without deletion of the gene or of a segment thereof, by, e.g.,
introducing two tandem translational termination codons into a
translational open reading frame (ORF). This yields viable virus in
which a selected gene has been silenced at the level of translation
without deleting its gene. These forms of knock-out virus will
often exhibit reduced growth rates and small plaque sizes in tissue
culture. Thus, these methods provide yet additional, novel types of
attenuating mutations which ablate expression of a viral gene that
is not one of the major viral protective antigens. In this context,
knock-out virus phenotypes produced without deletion of a gene or
genome segment can be alternatively produced by deletion
mutagenesis, as described herein, to effectively preclude
correcting mutations that may restore synthesis of a target
protein. Several other gene knock-outs for M2 ORF2 deletion and
knock out mutants can be made using alternate designs and methods
that are well known in the art (as described, for example, in
(Kretschmer et al., Virology 216:309-316, 1996; Radicle et al.,
Virology 217:418-412, 1996; and Kato et al., EMBOSS J. 16:178-587,
1987; and Schneider et al., Virology 277:314-322, 1996, each
incorporated herein by reference).
Other mutations for incorporation into M2 ORF2 deletion and knock
out mutant RSV of the invention include mutations directed toward
cis-acting signals, which can be identified, e.g., by mutational
analysis of RSV minigenomes. For example, insertional and
deletional analysis of the leader and trailer and flanking
sequences identifies viral promoters and transcription signals and
provides a series of mutations associated with varying degrees of
reduction of RNA replication or transcription. Saturation
mutagenesis (whereby each position in turn is modified to each of
the nucleotide alternatives) of these cis-acting signals also has
identified many mutations which reduced (or in one case increased)
RNA replication or transcription. Any of these mutations can be
inserted into an M2 ORF2 deletion or knock out mutant RSV
antigenome or genome as described herein. Evaluation and
manipulation of trans-acting proteins and cis-acting RNA sequences
using the complete antigenome cDNA is assisted by the use of RSV
minigenomes (see, e.g., Grosfeld et al., J. Virol. 69: 5677-5686,
1995, incorporated herein by reference), whose helper-dependent
status is useful in the characterization of those mutants which are
too inhibitory to be recovered in replication-independent
infectious virus.
Additional mutations within M2 ORF2 deletion and knock out mutant
RSV involve replacement of the 3' end of genome with its
counterpart from antigenome, which is associated with changes in
RNA replication and transcription. In addition, the intergenic
regions (Collins et al., Proc. Natl. Acad. Sci. USA 83:4594-4598,
1986, incorporated herein by reference) can be shortened or
lengthened or changed in sequence content, and the
naturally-occurring gene overlap (Collins et al., Proc. Natl. Acad.
Sci. USA 84:5134-5138, 1987, incorporated herein by reference) can
be removed or changed to a different intergenic region by the
methods described herein. In one exemplary embodiment, the level of
expression of specific RSV proteins, such as the protective F and G
antigens, can be increased by substituting the natural sequences
with ones which have been made synthetically and designed to be
consistent with efficient translation. In this context, it has been
shown that codon usage can be a major factor in the level of
translation of mammalian viral proteins (Haas et al., Current Biol.
6:315-324, 1996, incorporated herein by reference). Examination of
the codon usage of the mRNAs encoding the F and G proteins of RSV,
which are the major protective antigens, shows that the usage is
consistent with poor expression. Thus, codon usage can be improved
by the recombinant methods of the invention to achieve improved
expression for selected genes. In another exemplary embodiment, a
sequence surrounding a translational start site (preferably
including a nucleotide in the -3 position) of a selected RSV gene
is modified, alone or in combination with introduction of an
upstream start codon, to modulate RSV gene expression by specifying
up- or down-regulation of translation.
Alternatively, or in combination with other RSV modifications
disclosed herein, M2 ORF2 deletion and knock out mutant RSV gene
expression can be modulated by altering a transcriptional GS signal
of a selected gene(s) of the virus. In one exemplary embodiment,
the GS signal of NS2 is modified to include a defined mutation
(e.g., the 404(M2) mutation described herein) to superimpose a ts
restriction on viral replication.
In alternative embodiments, levels of gene expression in the M2
ORF2 deletion and knock out mutants are modified at the level of
transcription. In one aspect, the position of a selected gene in
the RSV gene map can be changed to a more promoter-proximal or
promoter-distal position, whereby the gene will be expressed more
or less efficiently, respectively. According to this aspect,
modulation of expression for specific genes can be achieved
yielding reductions or increases of gene expression from two-fold,
more typically four-fold, up to ten-fold or more compared to
wild-type levels often attended by a commensurate decrease in
expression levels for reciprocally, positionally substituted genes.
In one example, the NS2 gene (second in order in the RSV gene map)
is substituted in position for the SH gene (sixth in order),
yielding a predicted decrease in expression of NS2. In other
exemplary embodiments, the F and G genes are transpositioned singly
or together to a more promoter-proximal or promoter-distal site
within the RSV gene map to achieve higher or lower levels of gene
expression, respectively. These and other transpositioning changes
yield novel M2 ORF2 deletion and knock out mutants of RSV having
attenuated phenotypes, for example due to decreased expression of
selected viral proteins involved in RNA replication, or having
other desirable properties such as increased antigen
expression.
Infectious M2 ORF2 deletion and knock out mutant RSV clones of the
invention can also be engineered according to the methods and
compositions disclosed herein to enhance immunogenicity and induce
a level of protection greater than that provided by infection with
a wild-type RSV or a parent RSV. For example, an immunogenic
epitope from a heterologous RSV strain or type, or from a non-RSV
source such as PIV, can be added to a recombinant clone by
appropriate nucleotide changes in the polynucleotide sequence
encoding the genome or antigenome. Alternatively, RSV can be
engineered to add or ablate (e.g., by amino acid insertion,
substitution or deletion) immunogenic proteins, protein domains, or
forms of specific proteins (such as the secreted form of G)
associated with desirable or undesirable immunological
reactions.
Within the methods of the invention, additional genes or genome
segments may be inserted into or proximate to the M2 ORF2 deletion
and knock out mutant RSV genome or antigenome. These genes may be
under common control with recipient genes, or may be under the
control of an independent set of transcription signals. Genes of
interest include the RSV genes identified above, as well as non-RSV
genes. Non-RSV genes of interest include those encoding cytokines
(e.g., IL-2 through IL-18, especially IL-2, IL-4, IL-6 and IL-12,
IL-18, etc.), gamma-interferon, GM-CSF, chemokines and proteins
rich in T helper cell epitopes. These additional proteins can be
expressed as a separate protein, or as a chimera engineered from a
second copy of one of the RSV proteins, such as SH. This provides
the ability to modify and improve the immune responses against RSV
both quantitatively and qualitatively.
In exemplary embodiments of the invention, insertion of foreign
genes or genome segments, and in some cases of noncoding nucleotide
sequences, within an M2 ORF2 deletion or knock out mutant RSV
genome results in a desired increase in genome length causing yet
additional, desired phenotypic effects. Increased genome length
results in attenuation of the resultant RSV, dependent in part upon
the length of the insert. In addition, the expression of certain
proteins, e.g. a cytokine, from a non-RSV gene inserted into M2
ORF2 deletion and knock out mutant RSV will result in attenuation
of the virus due to the action of the protein. Exemplary cytokines
that yield an infectious, attenuated viral phenotype and high level
cytokine expression from RSV transfected cells include
interleukin-2 (IL-2), IL-4, GM-CSF, and .gamma.-interferon.
Additional effects including augmentation of cellular and humoral
immune responses will also attend introduction of cytokines into
recombinant RSV of the invention.
Deletions, insertions, substitutions and other mutations involving
changes of whole viral genes or genome segments within an M2 ORF2
deletion or knock out mutants yield genetically stable vaccine
candidates, which are particularly important in the case of
immunosuppressed individuals. Many of these changes will result in
attenuation of resultant vaccine strains, whereas others will
specify different types of desired phenotypic changes. For example,
accessory (i.e., not essential for in vitro growth) genes are
excellent candidates to encode proteins that specifically interfere
with host immunity (see, e.g., Kato et al., EMBO. J. 16:578-87,
1997, incorporated herein by reference). Ablation of such genes in
vaccine viruses is expected to reduce virulence and pathogenesis
and/or improve immunogenicity.
In alternative aspects of the invention, the infectious M2 ORF2
deletion and knock out mutant RSV produced from a cDNA-expressed
genome or antigenome can be any of the RSV or RSV-like strains,
e.g., human, bovine, murine, etc., or of any pneumovirus, e.g.,
pneumonia virus of mice avian pneumovirus (previously called turkey
rhinotracheitis virus). To engender a protective immune response,
the RSV strain may be one which is endogenous to the subject being
immunized, such as human RSV being used to immunize humans. The
genome or antigenome of endogenous RSV can be modified, however, to
express RSV genes or genome segments from a combination of
different sources, e.g., a combination of genes or genome segments
from different RSV species, subgroups, or strains, or from an RSV
and another respiratory pathogen such as PIV.
In certain embodiments of the invention, M2 ORF2 deletion and knock
out mutant RSV are provided wherein genes or genome segments within
a human or bovine RSV (e.g., a human RSV background genome or
antigenome) are replaced with counterpart heterologous genes or
genome segments from a non-human, non-bovine RSV, e.g., a murine
pneumonia virus. Substitutions, deletions, and additions of RSV
genes or genome segments in this context can include part or all of
one or more of the NS1, NS2, N, P, M, SH, M2(ORF1), M2(ORF2) and L
genes, or part or all of the G and F genes which preferably does
not include the major neutralization and protective epitopes. Also,
human or bovine RSV cis-acting sequences, such as promoter or
transcription signals, can be replaced with non-human, non-bovine
counterpart sequences. Thus, infectious M2 ORF2 deletion and knock
out mutant RSV intended for administration to humans can be a human
RSV that has been modified to contain genes from a murine RSV in
addition to bovine RSV.
Replacement of a human RSV coding sequence (e.g., of NS1, NS2, SH,
or G) or non-coding sequence (e.g., a promoter, gene-end,
gene-start, intergenic or other cis-acting element) with a
counterpart bovine RSV sequence yields chimeric RSV having a
variety of possible attenuating and other phenotypic effects. In
particular, host range and other desired effects arise from
substituting a bovine RSV gene imported within a human RSV
background, wherein the bovine gene does not function efficiently
in a human cell, e.g., from incompatibility of the heterologous
sequence or protein with a biologically interactive human RSV
sequence or protein (i.e., a sequence or protein that ordinarily
cooperates with the substituted sequence or protein for viral
transcription, translation, assembly, etc.) or, more typically in a
host range restriction, with a cellular protein or some other
aspect of the cellular milieu which is different between the
permissive and less permissive host. In one such embodiment, a
chimeric bovine-human RSV incorporates a substitution of the human
RSV NP gene or genome segment with a counterpart bovine NP gene or
genome segment, which chimera can optionally be constructed to
incorporate additional genetic changes, e.g., point mutations or
gene deletions. In exemplary embodiments, bovine RSV sequences are
selected for introduction into human RSV based on known aspects of
bovine RSV structure and function, as provided in, e.g., Pastey et
al., J. Gen. Viol. 76:193-197, 1993; Pastey et al., Virus Res.
29:195-202, 1993; Zamora et al., J. Gen. Virol. 73:737-741, 1992;
Mallipeddi 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, each incorporated herein by reference, and
in accordance with the teachings disclosed herein.
In other embodiments of the invention mutations of interest for
introduction within M2 ORF2 deletion and knock out mutant RSV are
modeled after a tissue culture-adapted nonpathogenic strain of
pneumonia virus of mice (the murine counterpart of human RSV) which
lacks a cytoplasmic tail of the G protein (Randhawa et al.,
Virology 207:240-245, 1995). Accordingly, in one aspect of the
invention the cytoplasmic and/or transmembrane domains of one or
more of the human RSV glycoproteins, F, G and SH, are added,
deleted, modified, or substituted within a chimeric RSV using a
heterologous counterpart sequence (e.g., a sequence from a
cytoplasmic, or transmembrane domain of a F, G, or SH protein of a
murine pneumonia virus) to achieve a desired attenuation. As
another example, a nucleotide sequence at or near the cleavage site
of the F protein, or the putative attachment domain of the G
protein, can be modified by point mutations, site-specific changes,
or by alterations involving entire genes or genome segments to
achieve novel effects on viral growth in tissue culture and/or
infection and pathogenesis.
In related aspects of the invention, the disclosed modifications
relating to M2-2 are incorporated within chimeric human-bovine RSV,
which are recombinantly engineered to incorporate nucleotide
sequences from both human and bovine RSV strains to produce an
infectious, chimeric virus or subviral particle. Exemplary
human-bovine chimeric RSV of the invention incorporate a chimeric
RSV genome or antigenome comprising both human and bovine
polynucleotide sequences, as well as a major nucleocapsid (N)
protein, a nucleocapsid phosphoprotein (P), a large polymerase
protein (L), and a RNA polymerase elongation factor. Additional RSV
proteins may be included in various combinations to provide a range
of infectious subviral particles, up to a complete viral particle
or a viral particle containing supernumerary proteins, antigenic
determinants or other additional components.
Chimeric human-bovine RSV for use within the invention are
generally described in U.S. Patent Application entitled PRODUCTION
OF ATTENUATED, HUMAN-BOVINE CHIMERIC RESPIRATORY SYNCYTIAL VIRUS
VACCINES, filed by Bucholz et al. on Jun. 23, 2000, and in its
priority U.S. Provisional Patent Application Serial No. 60/143,132
(each incorporated herein by reference). These chimeric recombinant
RSV include a partial or complete "background" RSV genome or
antigenome derived from or patterned after a human or bovine RSV
strain or subgroup virus combined with one or more heterologous
gene(s) or genome segment(s) of a different RSV strain or subgroup
virus to form the human-bovine chimeric RSV genome or antigenome.
In certain aspects of the invention, chimeric RSV incorporate a
partial or complete bovine RSV background genome or antigenome
combined with one or more heterologous gene(s) or genome segment(s)
from a human RSV. In alternate aspects of the invention chimeric
RSV incorporate a partial or complete human RSV background genome
or antigenome combined with one or more heterologous gene(s) or
genome segment(s) from a bovine RSV.
In exemplary embodiments, the invention is directed to an
infectious M2 ORF2 deletion or knock out respiratory syncytial
viruses (RSVs) that comprise a major nucleocapsid (N) protein, a
nucleocapsid phosphoprotein (P), a large polymerase protein (L), a
RNA polymerase elongation factor, and a partial or complete RSV
background genome or antigenome of a human or bovine RSV combined
with one or more heterologous gene(s) and/or genome segment(s) of a
different RSV to form a human-bovine chimeric RSV genome or
antigenome. The heterologous gene(s) and/or genome segment(s) that
are useful within the invention include one or more RSV NS1, NS2,
N, P, M, SH, M2(ORF1), M2(ORF2), L, F or G gene(s) or genome
segment(s). Alternatively, heterologous genes and genome segments
for incorporation within human-bovine chimeric RSV may include a
leader, trailer or intergenic region of the RSV genome, or a
segment thereof. Various modifications to the M2-2 gene, including
partial or complete deletions and other modifications that reduce
or eliminate M2-2 expression can be incorporated within the
chimeric genome or antigenome.
Within more detailed embodiments, human-bovine chimeric RSV of the
invention incorporate one or more heterologous genes and/or genome
segments that encode a RSV F, G and/or SH glycoprotein or an
immunogenic domain or epitope thereof. Alternatively, the
human-bovine chimeric RSV may incorporate a chimeric glycoprotein
having both human and bovine glycoprotein domains or immunogenic
epitopes. For example, the latter type of chimera may be
constructed by incorporation into a bovine background genome or
antigenome a heterologous genome segment encoding a glycoprotein
ectodomain in proper reading frame with a genome segment encoding a
functional remaining portion of the corresponding glycoprotein in
the bovine genome or antigenome, whereby the resultant chimeric
virus expresses a functional chimeric glycoprotein.
In other alternative embodiments of the invention, human-bovine
chimeric RSV are provided wherein a human RSV is attenuated by
incorporation of a selected bovine gene, genome segment, or
plurality of genes or genome segments. In certain embodiments
selected heterologous gene sets from BRSV are coordinately
transferred into a HRSV background genome or antigenome. Exemplary
bovine RSV genes from which individual or coordinately transferred
groups of genes may be selected include the RSV N, P, NS1, NS2,
M2-1 and M genes, which may be replaced singly or in any
combination in a human RSV background genome or antigenome by one
or more heterologous gene(s) from a bovine RSV to yield an
attenuated chimeric derivative. In more detailed aspects, both N
and P genes of a human RSV are replaced coordinately by counterpart
N and P genes from a bovine RSV. This coordinate gene replacement
is facilitated by functional cooperativity between certain genes in
the RSV genome, which often arises in the case of neighboring gene
pairs in the genome. Thus, in other alternative embodiments, both
NS1 and NS2 genes of a human RSV are replaced by counterpart NS1
and NS2 genes from a bovine RSV. In yet additional embodiments, two
or more of the M2-1, M2-2 and L genes of a HRSV are replaced by
counterpart genes from a bovine RSV. For certain vaccine candidates
within the invention for which a high level of host-range
restriction is desired, each of the N, P, NS1, NS2, M2-1 and M
genes of a human RSV are replaced by counterpart N, P, NS1, NS2,
M2-1 and M genes from a bovine RSV. Within these various
constructs, any selected modification to the M2-2 gene disclosed
herein, including partial or complete deletion of the gene or other
modification of the gene (e.g., altering or ablating a cis-acting
regulatory sequence or rearranging the position of M2-2), can be
incorporated in the chimeric genome or antigenome.
Within a different aspect of the invention, human-bovine chimeric
RSV having a modification involving M2-2 as disclosed herein are
constructed wherein the chimeric genome or antigenome comprises a
partial or complete bovine RSV background genome or antigenome
combined with one or more heterologous gene(s) and/or genome
segment(s) from a human RSV. In certain embodiments, one or more
human RSV glycoprotein genes selected from F, G and SH, or one or
more genome segment(s) encoding cytoplasmic domain, transmembrane
domain, ectodomain or immunogenic epitope portion(s) of F, G,
and/or SH is/are added or substituted within a partial or complete
bovine RSV background genome or antigenome. For example, one or
both human RSV glycoprotein genes F and G may be substituted to
replace one or both counterpart F and G glycoprotein genes in a
partial bovine RSV background genome or antigenome. Within these
and related embodiments, the human-bovine chimeric genome or
antigenome can incorporate antigenic determinants from one or both
subgroup A and subgroup B human RSV. In more detailed aspects, both
human RSV glycoprotein genes F and G are substituted to replace
counterpart F and G glycoprotein genes in the bovine RSV background
genome or antigenome. An exemplary human-bovine chimeric RSV
bearing these features in the examples below is rBRSV/A2. In
combination with one or more of the modifications provided in this
chimeric virus, the invention will incorporate a selected
modification involving M2-2 as disclosed herein.
Yet additional human-bovine chimeric RSV of the invention having a
modification of M2-2 incorporate one or more human RSV glycoprotein
genes selected from F, G and SH which are added or substituted at a
position that is more promoter-proximal compared to a wild-type
gene order position of a counterpart gene or genome segment within
a partial or complete bovine RSV background genome or antigenome.
In one such embodiment, both human RSV glycoprotein genes G and F
are substituted at gene order positions 1 and 2, respectively, to
replace counterpart G and F glycoprotein genes deleted at wild type
positions 7 and 8, respectively in a partial bovine RSV background
genome or antigenome. An exemplary human-bovine chimeric RSV
bearing these features described in the above-incorporated
disclosures is rBRSV/A2-G1F2.
Coordinate gene transfers within human-bovine chimeric RSV are also
directed to introduction of human antigenic genes within a bovine
background genome or antigenome. In certain embodiments, one or
more human RSV envelope-associated genes selected from F, G, SH,
and M is/are added or substituted within a partial or complete
bovine RSV background genome or antigenome. For example, one or
more human RSV envelope-associated genes selected from F, G, SH,
and M may be added or substituted within a partial bovine RSV
background genome or antigenome in which one or more
envelope-associated genes selected from F, G, SH, and M is/are
deleted. In more detailed aspects, one or more genes from a gene
set defined as human RSV envelope-associated genes F, G, and M are
added within a partial bovine RSV background genome or antigenome
in which envelope-associated genes F, G, SH, and M are deleted. An
exemplary human-bovine chimeric RSV bearing these features
described in the incorporated references is rBRSV/A2-MGF. In
combination with one or more of the modifications provided in this
chimeric virus, the invention will incorporate a selected
modification involving M2-2 as disclosed herein.
In yet additional aspects of the invention, M2 ORF2 deletion and
knock out RSV can be readily designed as "vectors" to incorporate
antigenic determinants from different pathogens, including more
than one RSV strain or group (e.g., both human RSV A and RSV B
subgroups), human parainfluenza virus (HPIV) including HPIV3, HPIV2
and HPIV1, measles virus and other pathogens (see, e.g., U.S.
Provisional Patent Application Serial No. 60/170,195; U.S. patent
application Ser. No. 09/458,813; and U.S. patent application Ser.
No. 09/459,062, each incorporated herein by reference). Within
various embodiments, the recombinant genome or antigenome comprises
a partial or complete RSV "vector genome or antigenome" combined
with one or more heterologous genes or genome segments encoding one
or more antigenic determinants of one or more heterologous
pathogens. The heterologous pathogen may be a heterologous RSV
(i.e., a RSV of a different strain or subgroup), and the
heterologous gene or genome segment may encode a RSV NS1, NS2, N,
P, M, SH, M2(ORF1), M2(ORF2), L, F or G protein or fragment (e.g.,
a immunogenic domain or epitope) thereof. For example, the vector
genome or antigenome may be a partial or complete RSV A genome or
antigenome and the heterologous gene(s) or genome segment(s) may
encode antigenic determinant(s) of a RSV B subgroup virus.
In alternative embodiments, the RSV vector genome or antigenome is
a partial or complete bovine RSV (BRSV) genome or antigenome and
the heterologous gene(s) or genome segment(s) encoding the
antigenic determinant(s) is/are of one or more human RSVs (HRSVs).
For example, the partial or complete BRSV genome or antigenome may
incorporate one or more gene(s) or genome segment(s) encoding one
or more HRSV glycoprotein genes selected from F, G and SH, or one
or more genome segment(s) encoding cytoplasmic domain,
transmembrane domain, ectodomain or immunogenic epitope portion(s)
of F, G, and/or SH of HRSV.
In other alternate embodiments, M2 ORF2 deletion and knock out RSV
designed as "vectors" for carrying heterologous antigenic
determinants incorporate one or more antigenic determinants of a
non-RSV pathogen, such as a human parainfluenza virus (HPIV). In
one exemplary embodiment, one or more HPIV1, HPIV2, or HPIV3
gene(s) or genome segment(s) encoding one or more HN and/or F
glycoprotein(s) or antigenic domain(s), fragment(s) or epitope(s)
thereof is/are added to or incorporated within the partial or
complete HRSV vector genome or antigenome. In more detailed
embodiments, a transcription unit comprising an open reading frame
(ORF) of an HPIV1, HPIV2, or HPIV3 HN or F gene is added to or
incorporated within the chimeric HRSV vector genome or anti
genome.
In yet additional alternate embodiments, the M2 ORF2 deletion or
knock out vector genome or antigenome comprises a partial or
complete HRSV or BRSV genome or antigenome and the heterologous
pathogen is selected from measles virus, subgroup A and subgroup B
respiratory syncytial viruses, mumps virus, human papilloma
viruses, type 1 and type 2 human immunodeficiency viruses, herpes
simplex viruses, cytomegalovirus, rabies virus, Epstein Barr virus,
filoviruses, bunyaviruses, flaviviruses, alphaviruses and influenza
viruses. Based on this exemplary list of candidate pathogens, the
selected heterologous antigenic determinant(s) may be selected from
measles virus HA and F proteins, subgroup A or subgroup B
respiratory syncytial virus F, G, SH and M2 proteins, mumps virus
HN and F proteins, human papilloma virus L1 protein, type 1 or type
2 human immunodeficiency virus gp160 protein, herpes simplex virus
and cytomegalovirus gB, gC, gD, gE, gG, gH, gI, gJ, gK, gL, and gM
proteins, rabies virus G protein, Epstein Barr Virus gp350 protein;
filovirus G protein, bunyavirus G protein, Flavivirus E and NS1
proteins, and alphavirus E protein, and antigenic domains,
fragments and epitopes thereof. In one embodiment, the heterologous
pathogen is measles virus and the heterologous antigenic
determinant(s) is/are selected from the measles virus HA and F
proteins and antigenic domains, fragments and epitopes thereof. To
achieve such a chimeric construct, a transcription unit comprising
an open reading frame (ORF) of a measles virus HA gene may be added
to or incorporated within a HRSV vector genome or antigenome.
In all embodiments of the invention that involve construction of a
chimeric RSV, the addition or substitution of a heterologous or
"donor" polynucleotide to a recipient or "background" genome or
antigenome can involve only a portion of a donor gene of interest.
Commonly, non-coding nucleotides such as cis-acting regulatory
elements and intergenic sequences need not be transferred with the
donor gene coding region. Thus, a coding sequence (e.g., a partial
or complete open reading frame (ORF)) of a particular gene may be
added or substituted to the partial or complete background genome
or antigenome under control of a heterologous promoter (e.g., a
promoter existing in the background genome or antigenome) of a
counterpart gene or different gene as compared to the donor
sequence. A variety of additional genome segments provide useful
donor polynucleotides for inclusion within a chimeric genome or
antigenome to express chimeric RSV having novel and useful
properties. For example, heterologous genome segments may encode
part or all of a glycoprotein cytoplasmic tail region,
transmembrane domain or ectodomain, an epitopic site or region, a
binding site or region containing a binding site, an active site or
region containing an active site, etc., of a selected protein from
a human or bovine RSV. These and other genome segments can be added
to a complete background genome or antigenome or substituted
therein for a counterpart genome segment to yield novel chimeric
RSV recombinants. Certain recombinants will express a chimeric
protein, e.g., a protein having a cytoplasmic tail and/or
transmembrane domain of one RSV fused to an ectodomain of another
RSV.
In other detailed aspects of the invention, M2 ORF2 deletion and
knock out viruses are created or modified by shifting a relative
gene order or spatial position of one or more genes or genome
segments within a recombinant RSV genome or antigenom--to generate
a recombinant vaccine virus that is infectious, attenuated and
immunogenic in humans and other mammals (see, U.S. Provisional
Patent Application Ser. No. 60/213,708 entitled RESPIRATORY
SYNCYTIAL VIRUS VACCINES EXPRESSING PROTECTIVE ANTIGENS FROM
PROMOTOR-PROXIMAL GENES, filed by Krempl et al., Jun. 23, 2000,
incorporated herein by reference). These recombinant RSVs of the
invention typically comprise a major nucleocapsid (N) protein, a
nucleocapsid phosphoprotein (P), a large polymerase protein (L), a
RNA polymerase elongation factor, and a partial or complete
recombinant RSV genome or antigenome having one or more
positionally shifted RSV genes or genome segments within the
recombinant genome or antigenome. In certain aspects of the
invention, the recombinant RSV features one or more positionally
shifted genes or genome segments that may be shifted to a more
promoter-proximal or promoter-distal position by insertion,
deletion, or rearrangement of one or more displacement
polynucleotides within the partial or complete recombinant RSV
genome or antigenome. Displacement polynucleotides may be inserted
or rearranged into a non-coding region (NCR) of the recombinant
genome or antigenome, or may be incorporated in the recombinant RSV
genome or-antigenome as a separate gene unit (GU).
In exemplary embodiments of the invention, isolated infectious
recombinant RSV are constructed by addition, deletion, or
rearrangement of one or more displacement polynucleotides that may
be selected from one or more RSV gene(s) or genome segment(s)
selected from RSV NS1, NS2, N, P, M, SH, M2(ORF1), M2(ORF2), L, F
and G genes and genome segments and leader, trailer and intergenic
regions of the RSV genome and segments thereof. In more detailed
embodiments, polynucleotide inserts, and deleted or rearranged
elements within the recombinant RSV genome or antigenome are
selected from one or more bovine RSV (BRSV) or human RSV (HRSV)
gene(s) or genome segment(s) selected from RSV NS1, NS2, N, P, M,
SH, M2(ORF1), M2(ORF2), L, F and G gene(s) or genome segment(s) and
leader, trailer and intergenic regions of the RSV genome or
segments thereof.
In certain aspects of the invention, displacement polynucleotides
are deleted to form the recombinant RSV genome or antigenome, to
create or supplement the M2 ORF2 deletion or knock out mutation.
Deletion of a displacement polynucleotide in this manner causes a
positional shift of one or more "shifted" RSV genes or genome
segments within the recombinant genome or antigenome to a more
promoter-proximal position relative to a position of the shifted
gene(s) or genome segment(s) within a wild type RSV (e.g., HRSV A2
or BRSV kansas strain) genome or antigenome. Exemplary displacement
polynucleotides that may be deleted in this manner to form the
recombinant RSV genome or antigenome may be selected from one or
more RSV NS1, NS2, SH, M2(ORF2), or G gene(s) or genome segment(s)
thereof.
In more detailed embodiments of the invention, a displacement
polynucleotide comprising a RSV NS1 gene is deleted to form the
recombinant RSV genome or antigenome. Alternatively, a displacement
polynucleotide comprising a RSV NS2 gene may be deleted to form the
recombinant RSV genome or antigenome. Alternatively, a displacement
polynucleotide comprising a RSV SH gene may be deleted to form the
recombinant RSV genome or antigenome. Alternatively, a displacement
polynucleotide comprising RSV M2(ORF2) can be deleted to form the
recombinant RSV genome or antigenome. Alternatively, a displacement
polynucleotide comprising a RSV G gene may be deleted to form the
recombinant RSV genome or antigenome or antigenome.
In yet additional embodiments, multiple displacement
polynucleotides comprising RSV genes or genome segments may be
deleted to create or modify a M2 ORF2 deletion or knock out mutant
RSV. For example, RSV F and G genes may both be deleted to further
modify the recombinant RSV genome or antigenome or antigenome
having an M2-2 deletion or knock out mutation. Alternatively, the
RSV NS1 and NS2 genes may both be deleted to form the recombinant
RSV genome or antigenome or antigenome. Alternatively, the RSV SH
and NS2 genes may both be deleted in the recombinant RSV genome or
antigenome or antigenome. Alternatively, the RSV SH, NS1 and NS2
genes can all be deleted in the recombinant RSV genome or
antigenome or antigenome.
In different embodiments of the invention, isolated infectious
recombinant RSV having a M2 ORF2 deletion or knock out mutation are
provided wherein one or more displacement polynucleotides is/are
added, substituted, or rearranged within the recombinant RSV genome
or antigenome to cause a positional shift of one or more shifted
RSV gene(s) or genome segment(s). Among these modifications, gene
and genome segment insertions and rearrangements may introduce or
rearrange the subject genes or genome segments to a more
promoter-proximal or promoter-distal position relative to a
respective position of each subject (inserted or rearranged) gene
or genome segment within a corresponding (e.g., bovine or human)
wild type RSV genome or antigenome. Displacement polynucleotides
which may be added, substituted, or rearranged within the
recombinant RSV genome or antigenome can be selected from one or
more of the RSV NS1, NS2, SH, M2(ORF2), F, and/or G gene(s) or
genome segment(s) thereof.
In more detailed embodiments, displacement polynucleotides are
selected for insertion or rearrangement within the M2 ORF2 deletion
or knock out RSV genome or antigenome which comprises one or more
RSV genes or genome segments that encode one or more RSV
glycoproteins or immunogenic domains or epitopes of RSV
glycoproteins. In exemplary embodiments, these displacement
polynucleotides are selected from genes or genome segments encoding
RSV F, G, and/or SH glycoproteins or immunogenic domains or
epitopes thereof. For example, one or more RSV glycoprotein gene(s)
selected from F, G and SH may be added, substituted or rearranged
within the recombinant RSV genome or antigenome to a position that
is more promoter-proximal or promoter-distal compared to the wild
type gene order position of the gene(s).
In exemplary embodiments, the RSV glycoprotein gene G is rearranged
within the recombinant RSV genome or antigenome to a gene order
position that is more promoter-proximal compared to the wild type
gene order position of G. In more detailed aspects, the RSV
glycoprotein gene G is shifted to gene order position 1 within said
recombinant RSV genome or antigenome. In other exemplary
embodiments, the RSV glycoprotein gene F is rearranged within the
recombinant RSV genome or antigenome to a more promoter-proximal
position, for example by shifting the F gene to gene order position
1 within the recombinant genome or antigenome. In yet additional
exemplary embodiments, both RSV glycoprotein genes G and F are
rearranged within the recombinant RSV genome or antigenome to gene
order positions that are more promoter-proximal compared to their
respective wild type gene order positions. In more detailed
aspects, the RSV glycoprotein gene G is shifted to gene order
position 1 and the RSV glycoprotein gene F is shifted to gene order
position 2.
In yet additional constructs featuring glycoprotein gene shifts,
recombinant M2 ORF2 deletion and knock out RSV are produced having
one or more RSV glycoprotein gene(s) selected from F, G and SH, or
a genome segment thereof, added, substituted or rearranged within
the recombinant RSV genome or antigenome, wherein one or more RSV
NS1, NS2, SH, M2(ORF2), or G gene(s) or genome segment(s) thereof
is/are deleted. Thus, a gene or genome segment of RSV F, G, or SH
may be added, substituted or rearranged in a background wherein a
displacement polynucleotide comprising a RSV NS1 gene is deleted to
form the recombinant RSV genome or antigenome. Alternatively, a
gene or genome segment of RSV F, G, or SH may be added, substituted
or rearranged in a background wherein a displacement polynucleotide
comprising a RSV NS2 gene is deleted to form the recombinant RSV
genome or antigenome. Alternatively, a gene or genome segment of
RSV F, G, or SH may be added, substituted or rearranged in a
background wherein a displacement polynucleotide comprising a RSV
SH gene is deleted to form the recombinant RSV genome or
antigenome.
In one embodiment, the RSV glycoprotein gene G is rearranged within
a recombinant RSV genome or antigenome having an SH gene deletion
to a gene order position that is more promoter-proximal compared to
the wild type gene order position of G. In more detailed aspects,
the RSV glycoprotein gene G is shifted to gene order position 1
within the recombinant RSV genome or antigenome, as exemplified by
the recombinant vaccine candidate G1/.DELTA.SH. In another
embodiment, the RSV glycoprotein gene F is rearranged within a
recombinant RSV genome or antigenome having an SH gene deletion to
a more promoter-proximal proximal position. In more detailed
aspects, the F gene is shifted to gene order position 1, as
exemplified by the recombinant F1.DELTA.SH. In yet another
embodiment, both RSV glycoprotein genes G and F are rearranged
within a .DELTA.SH recombinant RSV genome or antigenome to gene
order positions that are more promoter-proximal compared to the
wild type gene order positions of G and F. In more detailed
aspects, the RSV glycoprotein gene G is shifted to gene order
position 1 and the RSV glycoprotein gene F is shifted to gene order
position 1 within the recombinant RSV genome or antigenome, as
exemplified by the recombinant G1F1/.DELTA.SH.
Yet additional examples of gene position-shifted RSV are provided
for use within the invention featuring shifts of glycoprotein
gene(s) selected from F, G and SH, which are produced within a
recombinant RSV genome or antigenome having multiple genes or
genome segments selected from RSV NS1, NS2, SH, M2(ORF2), and G
gene(s) or genome segment(s) deleted (see, U.S. Patent Application
Ser. No. 60/213,708 entitled RESPIRATORY SYNCYTIAL VIRUS VACCINES
EXPRESSING PROTECTIVE ANTIGENS FROM PROMOTOR-PROXIMAL GENES, filed
by Krempl et al., Jun. 23, 2000 incorporated herein by reference).
In one example, the RSV SH and NS2 genes are both deleted to form
the recombinant RSV genome or antigenome or antigenome, and one or
both RSV glycoprotein genes G and F are rearranged within the
recombinant RSV genome to more promoter-proximal gene order
positions. In more detailed aspects, G is shifted to gene order
position 1 and F is shifted to gene order position 2, as
exemplified by the recombinant G1F1/.DELTA.NS2.DELTA.SH. In another
example, all of the RSV SH, NS1 and NS2 genes are deleted to form
the recombinant RSV genome or antigenome or antigenome, and one or
both RSV glycoprotein genes G and F are rearranged within the
recombinant RSV genome or antigenome to more promoter-proximal
positions, as exemplified by the recombinant vaccine candidate
G1F1/.DELTA.NS2.DELTA.NS2.DELTA.SH.
In yet additional aspects of the invention, gene position-shifted
RSV having a M2 ORF2 deletion or knock out mutations are combined
with or incorporated within human-bovine chimeric RSV (see, U.S.
Patent Application entitled PRODUCTION OF ATTENUATED, HUMAN-BOVINE
CHIMERIC RESPIRATORY SYNCYTIAL VIRUS VACCINES, filed by Bucholz et
al. on Jun. 23, 2000, and in its priority U.S. Provisional Patent
Application Serial No. 60/143,132 (each incorporated herein by
reference). Within these aspects, the recombinant genome or
antigenome comprises a partial or complete human RSV (HRSV) or
bovine RSV (BRSV) background genome or antigenome combined with one
or more heterologous gene(s) or genome segment(s) from a different
RSV to for a human-bovine chimeric RSV genome or antigenome. The
heterologous gene or genome segment of the different, HRSV or BRSV
may be added or substituted at a position that is more
promoter-proximal or promoter-distal compared to a wild type gene
order position of a counterpart gene or genome segment within the
partial or complete HRSV or BRSV background genome or antigenome.
In one such example, both human RSV glycoprotein genes G and F are
substituted at gene order positions 1 and 2, respectively, to
replace counterpart G and F glycoprotein genes deleted at wild type
positions 7 and 8, respectively in a partial bovine RSV background
genome or antigenome, as exemplified by the recombinant virus
rBRSV/A2-G1F2. In other embodiments, one or more human RSV
envelope-associated genes selected from F, G, SH, and M is/are
added or substituted within a partial or complete bovine RSV
background genome or antigenome. In more detailed aspects, one or
more human RSV envelope-associated genes selected from F, G, SH,
and M is/are added or substituted within a partial bovine RSV
background genome or antigenome in which one or more
envelope-associated genes selected from F, G, SH, and M is/are
deleted. In one embodiment, human RSV envelope-associated genes F,
G, and M are added within a partial bovine RSV background genome or
antigenome in which all of the envelope-associated genes F, G, SH,
and M are deleted, as exemplified by the recombinant virus
rBRSV/A2-MGF.
In another alternate embodiment of the invention, isolated
infectious recombinant RSV having a M2 ORF2 deletion or knock out
are provided in which the RSV M2(ORF1) is shifted to a more
promoter-proximal position within the recombinant RSV genome or
antigenome. The result of this gene shift is to upregulate
transcription of the recombinant virus.
In addition to the above described modifications to M2 ORF2
deletion and knock out mutant RSV, different or additional
modifications in RSV clones can be made to facilitate
manipulations, such as the insertion of unique restriction sites in
various intergenic regions (e.g., a unique StuI site between the G
and F genes) or elsewhere. Nontranslated gene sequences can be
removed to increase capacity for inserting foreign sequences.
In another aspect of the invention, compositions (e.g., isolated
polynucleotides and vectors incorporating an M2 ORF2 deletion or
knock out-encoding cDNA) are provided for producing an isolated
infectious RSV. Using these compositions and methods, infectious
RSV are generated from a RSV genome or antigenome, a nucleocapsid
(N) protein, a nucleocapsid phosphoprotein (P), a large (L)
polymerase protein, and an RNA polymerase elongation factor. In
related aspects of the invention, compositions and methods are
provided for introducing the aforementioned structural and
phenotypic changes into a recombinant RSV to yield infectious,
attenuated vaccine viruses.
Introduction of the foregoing defined mutations into an infectious,
M2 ORF2 deletion and knock out mutant RSV clone can be achieved by
a variety of well known methods. By "infectious clone" with regard
to DNA is meant cDNA or its product, synthetic or otherwise, which
can be transcribed into genomic or antigenomic RNA capable of
serving as template to produce an infectious virus or subviral
particle. Thus, defined mutations can be introduced by conventional
techniques (e.g., site-directed mutagenesis) into a cDNA copy of
the genome or antigenome. The use of antigenome or genome cDNA
subfragments to assemble a complete antigenome or genome cDNA as
described herein has the advantage that each region can be
manipulated separately (smaller cDNAs are easier to manipulate than
large ones) and then readily assembled into a complete cDNA. Thus,
the complete antigenome or genome cDNA, or any subfragment thereof,
can be used as template for oligonucleotide-directed mutagenesis.
This can be through the intermediate of a single-stranded phagemid
form, such as using the Muta-gene.RTM. kit of Bio-Rad Laboratories
(Richmond, Calif.) or a method using a double-stranded plasmid
directly as template such as the Chameleon mutagenesis kit of
Stratagene (La Jolla, Calif.), or by the polymerase chain reaction
employing either an oligonucleotide primer or template which
contains the mutation(s) of interest. A mutated subfragment can
then be assembled into the complete antigenome or genome cDNA. A
variety of other mutagenesis techniques are known and available for
use in producing the mutations of interest in the RSV antigenome or
genome cDNA. Mutations can vary from single nucleotide changes to
replacement of large cDNA pieces containing one or more genes or
genome regions.
Thus, in one illustrative embodiment mutations are introduced by
using the Muta-gene phagemid in vitro mutagenesis kit available
from Bio-Rad. In brief, cDNA encoding a portion of an RSV genome or
antigenome is cloned into the 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 introduction of an altered nucleotide at the
desired position of the genome or antigenome. The plasmid
containing the genetically altered genome or antigenome fragment is
then amplified, its sequence is confirmed, and the mutated piece is
then reintroduced into the full-length genome or antigenome
clone.
The ability to introduce defined mutations into infectious RSV has
many applications, including the analyses of RSV molecular biology
and pathogenesis. For example, the functions of RSV proteins can be
investigated and manipulated by introducing mutations which ablate
or reduce their level of expression, or which yield mutant protein.
In one exemplary embodiment hereinbelow, recombinant RSV is
constructed in which expression of a viral gene, namely the SH
gene, is ablated by deletion of the mRNA coding sequence and
flanking transcription signals. Surprisingly, not only could this
virus be recovered, but it grew efficiently in tissue culture. In
fact, its growth was substantially increased over that of the
wild-type, based on both yield of infectious virus and on plaque
size. This improved growth in tissue culture from the SH deletion
and other RSV derivatives of the invention provides useful tools
for developing RSV vaccines, which overcome the problem of RSV's
poor yield in tissue culture that had complicated production of
vaccine virus in other systems. These deletions are highly stable
against genetic reversion, rendering the RSV clones derived
therefrom particularly useful as vaccine agents.
The invention also provides methods for producing infectious M2
ORF2 deletion and knock out mutant RSV from one or more isolated
polynucleotides, e.g., one or more cDNAs. According to the present
invention cDNA encoding a RSV genome or antigenome is constructed
for intracellular or in vitro coexpression with the necessary viral
proteins to form infectious RSV. By "RSV antigenome" is meant an
isolated positive-sense polynucleotide molecule which serves as the
template for the synthesis of progeny RSV genome. Preferably a cDNA
is constructed which is a positive-sense version of the RSV genome,
corresponding to the replicative intermediate RNA, or antigenome,
so as to minimize the possibility of hybridizing with
positive-sense transcripts of the complementing sequences that
encode proteins necessary to generate a transcribing, replicating
nucleocapsid, i.e., sequences that encode N, P, L and M2(ORF1)
protein. In an RSV minigenome system, genome and antigenome were
equally active in rescue, whether complemented by RSV or by
plasmids, indicating that either genome or antigenome can be used
and thus the choice can be made on methodologic or other
grounds.
A native RSV genome typically comprises a negative-sense
polynucleotide molecule which, through complementary viral mRNAs,
encodes eleven known species of viral proteins, i.e., the
nonstructural species NS1 and NS2, N, P, matrix (M), small
hydrophobic (SH), glycoprotein (G), fusion (F), M2(ORF1), M2(ORF2),
and L, substantially as described in (Mink et al., Virology
185:615-624, 1991; Stec et al., Virology 183:273-287, 1991; and
Connors et al., Virol. 208:478-484, 1995; Collins et al., Proc.
Nat. Acad. Sci. USA 93:81-85, 1996), each incorporated herein by
reference. It is recognized that one or more of these 11 proteins
may be expressed in structural-distinct forms which might have
functional differences, and one or more distinct protein species
may remain to be identified.
For purposes of the present invention the genome or antigenome of
the recombinant RSV of the invention need only contain those genes
or portions thereof necessary to render the viral or subviral
particles encoded thereby infectious. Further, the genes or
portions thereof may be provided by more than one polynucleotide
molecule, i.e., a gene may be provided by complementation or the
like from a separate nucleotide molecule, or can be expressed
directly from the genome or antigenome cDNA.
By recombinant RSV is meant a RSV or RSV-like viral or subviral
particle derived directly or indirectly from a recombinant
expression system or propagated from virus or subviral particles
produced therefrom. The recombinant expression system will employ a
recombinant expression vector which comprises an operably linked
transcriptional unit comprising an assembly of at least a genetic
element or elements having a regulatory role in RSV gene
expression, for example, a promoter, a structural or coding
sequence which is transcribed into RSV RNA, and appropriate
transcription initiation and termination sequences.
To produce infectious RSV from cDNA-expressed genome or antigenome,
the genome or antigenome is coexpressed with those RSV proteins
necessary to (i) produce a nucleocapsid capable of RNA replication,
and (ii) render progeny nucleocapsids competent for both RNA
replication and transcription. Transcription by the genome
nucleocapsid provides the other RSV proteins and initiates a
productive infection. Alternatively, additional RSV proteins needed
for a productive infection can be supplied by coexpression.
An RSV antigenome may be constructed for use in the present
invention by assembling cloned cDNA segments, representing in
aggregate the complete antigenome, by polymerase chain reaction
(PCR; described in, e.g., U.S. Pat. Nos. 4,683,195 and 4,683,202,
and PCR Protocols: A Guide to Methods and Applications, Innis et
al., eds., Academic Press, San Diego, 1990, incorporated herein by
reference) of reverse-transcribed copies of RSV mRNA or genome RNA.
For example, cDNAs containing the lefthand end of the antigenome,
spanning from an appropriate promoter (e.g., T7 RNA polymerase
promoter) and the leader region complement to the SH gene, are
assembled in an appropriate expression vector, such as a plasmid
(e.g., pBR322) or various available cosmid, phage, or DNA virus
vectors. The vector may be modified by mutagenesis and/or insertion
of synthetic polylinker containing unique restriction sites
designed to facilitate assembly. For example, a plasmid vector
described herein was derived from pBR322 by replacement of the
PstI-EcoRI fragment with a synthetic DNA containing convenient
restriction enzyme sites. Use of pBR322 as a vector stabilized
nucleotides 3716-3732 of the RSV sequence, which otherwise
sustained nucleotide deletions or insertions, and propagation of
the plasmid was in bacterial strain DH10B to avoid an artifactual
duplication and insertion which otherwise occurred in the vicinity
of nt 4499. For ease of preparation the G, F and M2 genes can be
assembled in a separate vector, as can be the L and trailer
sequences. The right-hand end (e.g., L and trailer sequences) of
the antigenome plasmid may contain additional sequences as desired,
such as a flanking ribozyme and tandem T7 transcriptional
terminators. The ribozyme can be hammerhead type (e.g., Grosfeld et
al., J. Virol. 69:5677-5686, 1995), which would yield a 3' end
containing a single nonviral nucleotide, or can any of the other
suitable ribozymes such as that of hepatitis delta virus (Perrotta
et al., Nature 350:434-436, 1991) which would yield a 3' end free
of non-RSV nucleotides. A middle segment (e.g., G-to-M2 piece) is
inserted into an appropriate restriction site of the leader-to-SH
plasmid, which in turn is the recipient for the
L-trailer-ribozyme-terminator piece, yielding a complete
antigenome. In an illustrative example described herein, the leader
end was constructed to abut the promoter for T7 RNA polymerase
which included three transcribed G residues for optimal activity;
transcription donates these three nonviral G's to the 5' end of the
antigenome. These three nonviral G residues can be omitted to yield
a 5' end free of nonviral nucleotides. To generate a nearly correct
3' end, the trailer end was constructed to be adjacent to a
hammerhead ribozyme, which upon cleavage would donate a single
3'-phosphorylated U residue to the 3' end of the encoded RNA.
In certain embodiments of the invention, complementing sequences
encoding proteins necessary to generate a transcribing, replicating
RSV nucleocapsid are provided by one or more helper viruses. Such
helper viruses can be wild-type or mutant. Preferably, the helper
virus can be distinguished phenotypically from the virus encoded by
the RSV cDNA. For example, it is desirable to provide monoclonal
antibodies which react immunologically with the helper virus but
not the virus encoded by the RSV cDNA. Such antibodies can be
neutralizing antibodies. In some embodiments, the antibodies can be
used to neutralize the helper virus background to facilitate
identification and recovery of the recombinant virus, or in
affinity chromatography to separate the helper virus from the
recombinant virus. Mutations can be introduced into the RSV cDNA
which render the recombinant RSV nonreactive or resistant to
neutralization with such antibodies.
A variety of nucleotide insertions and deletions can be made in the
M2 ORF2 deletion and knock out mutant RSV genome or antigenome to
generate a properly attenuated clone. The nucleotide length of the
genome of wild-type human RSV (15,222 nucleotides) is a multiple of
six, and members of the Paramyxovirus and Morbillivirus genera
typically abide by a "rule of six," i.e., genomes (or minigenomes)
replicate efficiently only when their nucleotide length is a
multiple of six (thought to be a requirement for precise spacing of
nucleotide residues relative to encapsulating NP protein).
Alteration of RSV genome length by single residue increments had no
effect on the efficiency of replication, and sequence analysis of
several different minigenome mutants following passage showed that
the length differences were maintained without compensatory
changes. Thus, RSV lacks the strict requirement of genome length
being a multiple of six, and nucleotide insertions and deletions
can be made in the RSV genome or antigenome without defeating
replication of the recombinant RSV of the present invention.
Alternative means to construct cDNA encoding an M2 ORF2 deletion
and knock out mutant RSV genome or antigenome include by reverse
transcription-PCR using improved PCR conditions (e.g., as described
in Cheng et al., Proc. Natl. Acad. Sci. USA 91:5695-5699, 1994;
Samal et al., J. Virol 70:5075-5082, 1996, each incorporated herein
by reference) to reduce the number of subunit cDNA components to as
few as one or two pieces. In other embodiments different promoters
can be used (e.g., T3, SP6) or different ribozymes (e.g., that of
hepatitis delta virus. Different DNA vectors (e.g., cosmids) can be
used for propagation to better accommodate the large size genome or
antigenome.
The N, P and L proteins, necessary for RNA replication, require an
RNA polymerase elongation factor such as the M2(ORF1) protein for
processive transcription. Thus M2(ORF1) or a substantially
equivalent transcription elongation factor for negative strand RNA
viruses is required for the production of infectious RSV and is a
necessary component of functional nucleocapsids during productive
infection. The need for the M2(ORF1) protein is consistent with its
role as a transcription elongation factor. The need for expression
of the RNA polymerase elongation factor protein for negative strand
RNA viruses is a feature of the present invention. M2(ORF1) can be
supplied by expression of the complete M2-gene, either by the
genome or antigenome or by coexpression therewith, although in this
form the second ORF2 may also be expressed and can have an
inhibitory effect on virus recovery. Therefore, for production of
infectious virus using the complete M2 gene the activities of the
two ORFs should be balanced to permit sufficient expression of
M(ORF1) to provide transcription elongation activity yet not so
much of M(ORF2) to inhibit RNA replication. Alternatively, the ORF1
protein is provided from a cDNA engineered to lack ORF2 or which
encodes a defective ORF2. Efficiency of virus production may also
be improved by co-expression of additional viral protein genes,
such as those encoding envelope constituents (i.e., SH, M, G, F
proteins).
The invention also provides novel compositions and methods for
producing purified RSV proteins. The enhanced protein synthesis of
M2-ORF 2 deletion and knock out mutant RSV renders these viruses
particularly useful as a source of purified RSV proteins, for
example to provide purified RSV antigens for preparation of subunit
vaccines. In accordance with the teachings herein, cells that are
amenable to productive infection by RSV are infected with a M2-ORF2
deletion or knock out mutant RSV of the invention and the cells are
cultured under conditions that allow for propagation of the mutant
virus. The virus is then removed from the cell culture and isolated
from cellular components, for example by well known clarification
methods. Thereafter one or more desired viral proteins, e.g., an
RSV F and/or G antigen, can be purified using conventional
chromatographic or other purification methods. By "purified RSV
protein" is meant protein that is substantially free of cellular or
viral protein and other contaminants that may render the protein
non-suitable for clinical use. In other aspects, the subject
protein, including a specific RSV protein or a combined sample of
one or more and up to a complete assemblage of RSV proteins, are
purified to about 85-90% purity, preferably 95% purity up to 98%
purity or greater.
For both the characterization and control of RSV disease, there is
a clear need for purified and partially-purified RSV antigen. In
particular, there is a need for an enriched source of the RSV G and
F glycoproteins, since these are the major protective and
neutralization antigens. RSV antigens find a number of uses. One
exemplary use is to administer the RSV antigen as an noninfectious
subunitvaccine. Another use is as an antigen to monitor antibody
responses in humans and experimental animals, such as in an
enzyme-linked immunoadsorbant assay (ELISA). The need for purified
antigens in diagnostic assays is well recognized. These exemplary
uses of purified protein, namely as a vaccine and for diagnostic
tests, is provided for means of illustration, not limitation.
A surprising aspect of the M2-2 knock out and deletion mutations is
that they confer increased expression of RSV proteins, particularly
RSV antigens. The use of a nonreplicating subunit vaccine is an
attractive strategy for certain groups of potential vaccinees, such
as immunocompromised individuals, older children who are
seropositive for RSV, health care providers, and the elderly. One
obvious advantage is the safety factor associated with the lack of
infectivity. As another potential advantage, a noninfectious
subunit vaccine would be more stable in storage and handling than
an infectious viral vaccine. Although purified subunit vaccines
have been associated with immunopathologic complications in
experimental animals (Murphy et al., Vaccine 8:497-502,1990,
incorporated herein by reference), this appears to occur only in
situations where there has not been prior exposure to RSV antigen
(Graham et al., J. Immun. 151:2032-2040, 1993, incorporated herein
by reference). Thus, a subunit vaccine would be considered
appropriate for seropositive individuals. Furthermore, there is
some controversy as to whether purified subunit vaccines actually
cause immunopathologic complications in seronegative individuals
(Corvaia et al., J. Infec. Dis. 176:560-9,1997; Plotnicky-Gilquin
et al., Virology 258:128-40, 1999, each incorporated herein by
reference). In addition, general advances in vaccine technology,
such as improved adjuvants and new modes of immunization, offer the
possibility to modulate immune responses to reduce adverse
reactions (Oien et al., Vaccine 12:731-5, 1994; Tebby et al., Viral
Immunol. 12:41-5,1999; Siegrist et al., J. Infect. Dis.
179:1326-33, 1999, each incorporated herein by reference). Although
it is generally thought that a parenteral route of immunization
would be particularly sensitive to immunosuppression due to
maternally derived antibodies and hence limit the effectiveness of
a subunit vaccine, there is evidence in rodents that this is not
the case, and also that a parenteral immunization in the very young
rodent can be highly effective (Brandt et al., J. Infect. Dis.
176:884-91, 1997, incorporated herein by reference). Thus, while a
live attenuated RSV vaccine continues to seem to be the strategy of
choice for the pediatric population, the primary target for RSV
immunopropylaxis, a noninfectious subunit vaccine also likely, will
have an important role, such as for immunization of certain
populations as noted above.
Several sources of RSV antigens currently exist. One source is the
authentic, virus-encoded proteins which are synthesized during RSV
infection of tissue culture cells such as HEp-2 or Vero cells
(Walsh et al., J. Gen. Virol. 65:761-7, 1984, and ibid, 66:409-425,
1985, incorporated herein by reference). Another source is the
expression of RSV protein by recombinant vectors, of which there
are numerous examples. One example is expression of individual RSV
proteins in mammalian cells by recombinant vaccinia virus vectors
(Elango et al., Proc. Natl. Acad. Sci. USA 83:1906-1910, 1986;
Olmsted et al., Proc. Natl. Acad. Sci. USA 83:7462-7466, 1986, each
incorporated herein by reference). Another example is infection of
insect cells with recombinant baculovirus expressing RSV antigen,
such as been described for a novel fusion protein of the F and G
proteins (Wathen et al., J. Gen. Virol. 70:2625-35, 1989,
incorporated herein by reference) as well as for complete or
truncated versions of the F protein (Wathen et al., J. Infect. Dis.
159:255-264, 1989, incorporated herein by reference). Yet another
example is expression from a recombinant vector in yeast cells
(Ding et al., Virology, 159:450-3, 1987, incorporated herein by
reference). Similarly, RSV antigens have been expressed in whole or
in part in bacteria (Martin-Gallardo et al., J. Gen. Virol.
74:453-8, 1993; Power et al., Virology 230:155-66, 1997, each
incorporated herein by reference). Another source of RSV antigen
has been synthetic peptides, such as ones representing short linear
regions of the G or F proteins (Levely et al., Cell. Immun.
125:65-78, 1990; Trudel et al., Virology 185:749-57, 1991, each
incorporated herein by reference).
RSV antigens can be purified from various sources by methods well
known to those skilled in the art. As a typical example, a
mammalian cell line can be infected with a standard wild type
strain of RSV and, at the peak of antigen accumulation, the cells
can be lysed with a mild detergent, insoluble debris can be
sedimented by centrifugation, and the resultant supernatant can be
subjected to chromatography for the purification of viral antigen.
For example, the F and G glycoproteins can be selected using
immobilized monoclonal antibody against each protein, and
subsequently eluted with a low pH buffer (Walsh et al., J. Gen.
Virol. 65:761-7, 1984, and ibid, 66:409-425, 1985, each
incorporated herein by reference). This can be supplemented or
substituted with other well known purification procedures, such as
ion exchange chromatography, reversed-phase high-performance liquid
chromatography, denaturation and protein refolding, etc. (Walsh et
al., J. Gen. Virol. 65:7617, 1984, and ibid, 66:409-425, 1985;
Wells et al., Protein Expr. Purif. 5:391-40,1994; Falsey and Walsh,
Vaccine 1 5:1130-1132, 1997, each incorporated herein by
reference). Indeed, candidate subunit RSV vaccines have been
produced by these methods using RSV-infected cells as the starting
material: namely, PFP-1, an immunoaffinity-purified vaccine
consisting of approximately 90-95% F protein (Tristram et al.,
Vaccine 12:551-556, 1994, incorporated herein by reference), and
PFP-2, and ion exchange chromatography-purified vaccine consisting
of 98% F protein (Tristram et al., J. Infect. Dis. 170:425-8, 1994,
incorporated herein by reference).
While PFP-1 and PFP-2 are derived from cells infected with wild
type RSV, the alternative approach of expression of proteins from a
recombinant source has several potential advantages. A number of
these advantages are listed here: the protein can be truncated to
include only the portions deemed relevant or the protein can be
fused to sequences which might aid in its purification, stability
or immunogenicity (Power et al., Virology 230:155-66, 1997,
incorporated herein by reference); proteins can be engineered to
facilitate recovery (Wathen et al., J. Infect. Dis. 159:255-264,
1989, incorporated herein by reference); the protein sequence
itself can be altered to improve various characteristics, such as
to increase solubility (Murby et al., Eur. J. Biochem. 230:38-44,
1995, incorporated herein by reference) or to ablate
immunopathologic reactions (Tebbey et al., J. Exp. Med.
188:1967-72, 1998, incorporated herein by reference); chimeric
proteins can be designed for the purpose of broadening the immune
response (Wathen et al., J. Gen. Virol. 70:2625-35, 1989,
incorporated herein by reference); expression of one or more viral
proteins from one or more vectors in the absence of other viral
genes ensures an absence of contamination by the other viral
proteins; recombinant vectors frees the experimenter from the
limitations of working with RSV and offers the potential of
improved expression. These are offered by means of examples and do
not encompass the full range of benefits of recombinant vectors.
The use of synthetic peptides includes many of these benefits and
also has the potential for greater purity.
Despite the many potential benefits, the use of heterologous
vectors such as baculovirus, vaccinia virus, or bacterial systems
also poses complications. For example, each method introduces
heterologous antigens which must be removed, especially in the case
of insect or bacterial cells. Expression in insect cells can
provide altered glycosylation, and expression in bacteria can yield
malfolded protein lacking disulfide bonds, phosphorylation and
glycosylation (Bialy, Biotechnology 5:883-890, 1987, incorporated
herein by reference). Also, the promise of increased levels of
expression and purity have proven to be elusive. For example, the
amounts of protein expressed in cells infected with recombinant
baculovirus or vaccinia virus have not been greater than that
expressed in cells infected with wild type RSV. Indeed, the most
promising subunit vaccines are PFP-1 and PFP-2, which are derived
from mammalian cells infected with standard RSV.
A completely unanticipated aspect of the invention is that it
provides an M2-2 knock out or deletion virus which can be used
directly in cell culture to provide improved yield of RSV protein
for isolation and purification. The M2-2 knock out and deletion
viruses of the invention express levels of viral protein, including
viral antigen (e.g., F and/or G protein(s)), which are increased
approximately 2-fold, preferably 2 to 3-fold, up to 5-fold, 10-fold
or greater compared to protein expression in wild-type or parental
mutant strains, and thus provide materials for purification that
are enriched in RSV protein(s). It is well known that even a modest
increases in protein expression can be highly advantageous in large
scale production, yielding a product of improved quantity and
quality. Thus, M2-2 knock out and deletion viruses can be used
directly to infect cells permissive to RSV infection and
propagation, such as cultured HEp-2 or Vero cells, which can then
be subjected to protein purification procedures to yield F, G or
other viral proteins. Furthermore, the greater yield observed in
the examples hereinbelow represents results under conditions which
have not been optimized for protein expression. In other aspects of
the invention, permissive cells are screened to select cells that
yield the highest level of protein, and experimental conditions are
further modified according to known methods to maximize the
viability of the over-expressing cells and thus further improve the
yield.
Furthermore, the fact that the M2-2 knock out and deletion viruses
are recombinant offers further possibilities of improvement, and
combines the benefits of recombinant expression with the
authenticity or protein products associated with expression by RSV
in permissive cells. For example, amino acids 184-198 in the G
protein have been shown to be associated with priming for enhanced
immunopathology in the mouse model, and has been confirmed in part
with T cells from human donors (Tebbey et al., J. Exp. Med.
188:1967 72, 1998, incorporated herein by reference). In a second
study, deletion of the overlapping domain of amino acids 193-200
ablated the capacity of G protein to induce immunopathology (Sparer
et al., J. Exp. Med. 187:1921-6, 1998, incorporated herein by
reference), confirming that this region of the G protein contains
one or more epitopes associated with priming for enhanced disease.
Thus, recombinant RSV has been prepared in which amino acids
187-197 are deleted (mutant 187/197) or in which this same region
was deleted and amino acids 198-200 altered by amino acid
substitution (mutant 187/200). Each virus replicates as efficiently
in vitro as wild type RSV. Thus, the 187/197 or 187/200 mutations
can be incorporated within a M2-2 knock out or deletion mutation to
prepare recombinant virus which expressing increased amounts of
viral proteins, and expressing a G protein which has been
engineered to remove a domain associated with enhanced
immunopathology.
Also within the invention, the M2-ORF 2 deletion and knock out
mutant RSV can be further modified to delete the G protein gene
altogether from recombinant RSV. The resulting G deletion virus
replicates to low titer on HEp-2 cells, but on Vero cells
replication is comparable to that of wild type virus and the G
deletion virus forms plaques. Since G is not required for virus
growth under these conditions, the G protein can be engineered
without regard for whether or not this affects its function. Thus,
it is possible to make changes to improve or alter immunogenicity,
solubility, reactogenicity, or any other feature. The resulting
recombinant virus can then be used to infect cells for the
expression of viral antigen. In addition, it has been found that
other attenuated RSV mutants, for example the cpts248/404 mutant
also exhibit increased levels of protein synthesis. Thus,
incorporation of one or more additional attenuating mutations that
specify increased protein synthesis, for example a mutation adopted
from cpts248/404, into an M2-2 knock out or deletion mutant of the
invention will provide additional advantages in terms of increased
protein expression.
To generate infectious RSV incorporating M2-ORF 2 deletion or knock
out mutations, isolated polynucleotides (e.g., cDNA) encoding the
M2 ORF2 deletion and knock out mutant RSV genome or antigenome are
expressed, separately, or in cis, including expression from the
antigenome or genome cDNA, with the N, P, L and M2(ORF1) proteins.
These polynucleotides are inserted by transfection,
electroporation, mechanical insertion, transduction or the like,
into cells which are capable of supporting a productive RSV
infection, e.g., HEp-2, FRhL-DBS2, MRC, and Vero cells.
Transfection of isolated polynucleotide sequences may be introduced
into cultured cells by, for example, calcium phosphate-mediated
transfection (Wigler et al., Cell 14:725, 1978; Corsaro and
Pearson, Somatic Cell Genetics 7:603, 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 regent, e.g., LipofectACE.RTM. (Life
Technologies) (each of the foregoing references are incorporated
herein by reference).
The N, P, L and M2(ORF1) proteins are encoded by one or more cDNAs
and expression vectors which can be the same or separate from that
which encodes the genome or antigenome, and various combinations
thereof. Furthermore, one or more proteins, and particularly the
M2-1 protein, can be supplied directly from the antigenome or
genome (Collins et al., Virology 259:251-258, 1999, incorporated
herein by reference). Additional proteins may be included as
desired, encoded by its own vector or by a vector encoding a N, P,
L, or M2(ORF1) protein and/or the complete genome or anti genome.
Expression of the genome or antigenome and proteins from
transfected plasmids can be achieved, for example, by each cDNA
being under the control of a promoter for T7 RNA polymerase, which
in turn is supplied by infection, transfection or transduction with
an expression system for the T7 RNA polymerase, e.g., a vaccinia
virus MVA strain recombinant which expresses the T7 RNA polymerase
(Wyatt et al., Virology, 210:202-205, 1995, incorporated herein by
reference). The viral proteins, and/or T7 RNA polymerase, can also
be provided from transformed mammalian cells, or by transfection of
preformed mRNA or protein.
Alternatively, synthesis of antigenome or genome can be conducted
in vitro (cell-free) in a combined transcription-translation
reaction, followed by transfection into cells. Or, antigenome or
genome RNA can be synthesized in vitro and transfected into cells
expressing RSV proteins.
To select candidate vaccine viruses according to the invention, the
criteria of viability, attenuation and immunogenicity are
determined according to well known methods. Viruses which will be
most desired in vaccines of the invention must maintain viability,
have a stable attenuation phenotype, exhibit replication in an
immunized host (albeit at lower levels), and effectively elicit
production of an immune response in a vaccinee sufficient to confer
protection against serious disease caused by subsequent infection
from wild-type virus. Clearly, the heretofore known and reported RS
virus mutants do not meet all of these criteria. Indeed, contrary
to expectations based on the results reported for known attenuated
RSV, viruses of the invention are not only viable and more
appropriately attenuated than previous mutants, but are more stable
genetically in vivo than those previously studied
mutants--retaining the ability to stimulate a protective immune
response and in some instances to expand the protection afforded by
multiple modifications, e.g., induce protection against different
viral strains or subgroups, or protection by a different
immunologic basis, e.g., secretory versus serum immunoglobulins,
cellular immunity, and the like. Prior to the invention, genetic
instability of the ts phenotype following replication in vivo has
been common for ts viruses (Murphy et al., Infect. Immun.
37:235-242, 1982).
To propagate an M2 ORF2 deletion and knock out mutants RSV virus
for vaccine use and other purposes, a number of cell lines which
allow for RSV growth may be used. RSV grows in a variety of human
and animal cells. Preferred cell lines for propagating attenuated
RS virus for vaccine use include DBS-FRhL-2, MRC-5, and Vero cells.
Highest virus yields are usually achieved with epithelial cell
lines such as Vero cells. Cells are typically inoculated with virus
at a multiplicity of infection ranging from about 0.001 to 1.0 or
more, and are cultivated under conditions permissive for
replication of the virus, e.g., at about 30-37.degree. C. and for
about 3-5 days, or as long as necessary for virus to reach an
adequate titer. Virus is removed from cell culture and separated
from cellular components, typically by well known clarification
procedures, e.g., centrifugation, and may be further purified as
desired using procedures well known to those skilled in the
art.
M2 ORF2 deletion and knock out RSV mutants which has been
satisfactorily attenuated and otherwise modified as described
herein can be tested in various well known and generally accepted
in vitro and in vivo models to confirm adequate attenuation,
resistance to phenotypic reversion, and immunogenicity for vaccine
use. In in vitro assays, the modified virus (e.g., a multiply
attenuated, biologically derived or recombinant RSV) is tested for
temperature sensitivity of virus replication, i.e. ts phenotype,
and for the small plaque phenotype. Modified viruses are further
tested in animal models of RSV infection. A variety of animal
models have been described and are summarized in (Meignier et al.,
eds., Animal Models of Respiratory Syncytial Virus Infection,
Merieux Foundation Publication, 1991, which is incorporated herein
by reference). A cotton rat model of RSV infection is described in
(U.S. Pat. No. 4,800,078 and Prince et al., Virus Res. 3:193-206,
1985), which are incorporated herein by reference, and is
considered predictive of attenuation and efficacy in humans and
non-human primates. In addition, a primate model of RSV infection
using the chimpanzee is predictive of attenuation and efficacy in
humans, as is described in detail in (Richardson et al., J. Med.
Virol. 3:91-100, 1978; Wright et al., Infect. Immun. 37:397-400,
1982; Crowe et al., Vaccine 11:1395-1404, 1993, each incorporated
herein by reference).
RSV model systems, including rodents and chimpanzees for evaluating
attenuation and infectivity of RSV vaccine candidates are widely
accepted in the art and the data obtained therefrom correlate well
with RSV infection and attenuation. The mouse and cotton rat models
are especially useful in those instances in which candidate RSV
viruses display inadequate growth in chimpanzees, for example in
the case of RSV subgroup B viruses.
In accordance with the foregoing description and based on the
Examples below, the invention also provides isolated, infectious M2
ORF2 deletion and knock out mutant RSV compositions for vaccine
use. The attenuated virus which is a component of a vaccine is in
an isolated and typically purified form. By isolated is meant to
refer to RSV which is in other than a native environment of a
wild-type virus, such as the nasopharynx of an infected individual.
More generally, isolated is meant to include the attenuated virus
as a component of a cell culture or other artificial medium where
it can be propagated and characterized in a controlled setting. For
example, attenuated RSV of the invention may be produced by an
infected cell culture, separated from the cell culture and added to
a stabilizer.
RSV vaccines of the invention contain as an active ingredient an
immunogenically effective amount of RSV produced as described
herein. Biologically derived or recombinant RSV can be used
directly in vaccine formulations, or lyophilized. Lyophilized virus
will typically be maintained at about 4.degree. C. When ready for
use the lyophilized virus is reconstituted in a stabilizing
solution, e.g., saline, SPG, Mg++ and HEPES, with or without
adjuvant, as further described below. The recombinantly modified
virus may be introduced into a host with a physiologically
acceptable carrier and/or adjuvant. Useful carriers are well known
in the art, and include, e.g., water, buffered water, 0.4% saline,
0.3% glycine, hyaluronic acid and the like. The resulting aqueous
solutions may be packaged for use as is, or lyophilized, the
lyophilized preparation being combined with a sterile solution
prior to administration, as mentioned above. The compositions may
contain pharmaceutically acceptable auxiliary substances as
required to approximate physiological conditions, such as pH
adjusting and buffering agents, tonicity adjusting agents, wetting
agents and the like, for example, sodium acetate, sodium lactate,
sodium chloride, 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.RTM.
QS-21 (Aquila Biopharmaceuticals, Inc., Farmingham, Mass.),
MPL.RTM. (3-0-deacylated monophosphoryl lipid A; RIBI ImmunoChem
Research, Inc., Hamilton, Mont.), and interleukin-12 (Genetics
Institute, Cambridge, Mass.).
Upon immunization with an M2 ORF2 deletion and knock out mutant RSV
vaccine composition as described herein, via aerosol, droplet,
oral, topical or other route, the immune system of the host
responds to the vaccine by producing antibodies specific for one or
more RSV virus proteins, e.g., F and/or G glycoproteins. As a
result of the vaccination the host becomes at least partially or
completely immune to RSV infection, or resistant to developing
moderate or severe RSV disease, particularly of the lower
respiratory tract.
M2 ORF2 deletion and knock out mutant RSV vaccines of the invention
may comprise attenuated virus that elicits an immune response
against a single RSV strain or antigenic subgroup, e.g. A or B, or
against multiple RSV strains or subgroups. In this context, the RSV
can elicit a monospecific immune response or a polyspecific immune
response against multiple RSV strains or subgroups. Alternatively,
RSV having different immunogenic characteristics can be combined in
a vaccine mixture or administered separately in a coordinated
treatment protocol to elicit more effective protection against one
RSV strain, or against multiple RSV strains or subgroups.
The host to which the vaccine is administered can be any mammal
susceptible to infection by RSV or a closely related virus and
capable of generating a protective immune response to antigens of
the vaccinizing virus. Thus, suitable hosts include humans,
non-human primates, bovine, equine, swine, ovine, caprine,
lagamorph, rodents, etc. Accordingly, the invention provides
methods for creating vaccines for a variety of human and veterinary
uses.
The vaccine compositions containing the attenuated M2 ORF2 deletion
and knock out mutant RSV of the invention are administered to a
patient susceptible to or otherwise at risk of RSV infection in an
"immunogenically effective dose" which is sufficient to induce or
enhance the individual's immune response capabilities against RSV.
In the case of human subjects, the attenuated virus of the
invention is administered according to well established human RSV
vaccine protocols, as described in, e.g., (Wright et al., Infect.
Immun. 37:397-400, 1982; Kim et al., Pediatrics 52:56-63, 1973; and
Wright et al., J. Pediatr. 88:931-936, 1976), which are each
incorporated herein by reference. Briefly, adults or children are
inoculated intranasally via droplet with an immunogenically
effective dose of RSV vaccine, typically in a volume of 0.5 ml of a
physiologically acceptable diluent or carrier. This has the
advantage of simplicity and safety compared to parenteral
immunization with a non-replicating vaccine. It also provides
direct stimulation of local respiratory tract immunity, which plays
a major role in resistance to RSV. Further, this mode of
vaccination effectively bypasses the immunosuppressive effects of
RSV-specific maternally-derived serum antibodies, which typically
are found in the very young. Also, while the parenteral
administration of RSV antigens can sometimes be associated with
immunopathologic complications, this has never been observed with a
live virus.
In all subjects, the precise amount of M2 ORF2 deletion and knock
out mutant RSV vaccine administered and the timing and repetition
of administration will be determined based on the patient's state
of health and weight, the mode of administration, the nature of the
formulation, etc. Dosages will generally range from about 10.sup.3
to about 10.sup.6 plaque forming units (PFU) or more, e.g.,
10.sup.7 to 10.sup.8 PFU of virus per patient, more commonly from
about 10.sup.4 to 10.sup.5 PFU virus per patient. In any event, the
vaccine formulations should provide a quantity of attenuated RSV of
the invention sufficient to effectively stimulate or induce an
anti-RSV immune response, e.g., as can be determined by complement
fixation, plaque neutralization, and/or enzyme-linked immunosorbent
assay, among other methods. In this regard, individuals are also
monitored for signs and symptoms of upper respiratory illness. As
with administration to chimpanzees, the attenuated virus of the
vaccine grows in the nasopharynx of vaccinees at levels
approximately 10-fold or more lower than wild-type virus, or
approximately 10-fold or more lower when compared to levels of
incompletely attenuated RSV.
In neonates and infants, multiple administration may be required to
elicit sufficient levels of immunity. Administration should begin
within the first month of life, and at intervals throughout
childhood, such as at two months, six months, one year and two
years, as necessary to maintain sufficient levels of protection
against native (wild-type) RSV infection. Similarly, adults who are
particularly susceptible to repeated or serious RSV infection, such
as, for example, health care workers, day care workers, family
members of young children, the elderly, individuals with
compromised cardiopulmonary function, may require multiple
immunizations to establish and/or maintain protective immune
responses. Levels of induced immunity can be monitored by measuring
amounts of neutralizing secretory and serum antibodies, and dosages
adjusted or vaccinations repeated as necessary to maintain desired
levels of protection. Further, different vaccine viruses may be
indicated for administration to different recipient groups. For
example, an engineered RSV strain expressing a cytokine or an
additional protein rich in T cell epitopes may be particularly
advantageous for adults rather than for infants. Alternatively, a
lower level of attenuation may be selected for older vaccinees. RSV
vaccines produced in accordance with the present invention can be
combined with viruses expressing antigens of another subgroup or
strain of RSV to achieve protection against multiple RSV subgroups
or strains. Alternatively, the vaccine virus may incorporate
protective epitopes of multiple RSV strains or subgroups engineered
into one RSV clone as described herein.
Typically when different vaccine viruses are used they will be
administered in an admixture simultaneously, but they may also be
administered separately. For example, as the F glycoproteins of the
two RSV subgroups differ by only about 10% in amino acid sequence,
this similarity is the basis for a cross-protective immune response
as observed in animals immunized with RSV or F antigen and
challenged with a heterologous strain. Thus, immunization with one
strain may protect against different strains of the same or
different subgroup. However, optimal protection probably will
require immunization against both subgroups.
The M2 ORF2 deletion and knock out mutant RSV vaccines of the
invention elicit production of an immune response that is
protective against serious lower respiratory tract disease, such as
pneumonia and bronchiolitis when the individual is subsequently
infected with wild-type RSV. While the naturally circulating virus
is still capable of causing infection, particularly in the upper
respiratory tract, there is a very greatly reduced possibility of
rhinitis as a result of the vaccination and possible boosting of
resistance by subsequent infection by wild-type virus. Following
vaccination, there are detectable levels of host engendered serum
and secretory antibodies which are capable of neutralizing
homologous (of the same subgroup) wild-type virus in vitro and in
vivo. In many instances the host antibodies will also neutralize
wild-type virus of a different, non-vaccine subgroup.
Preferred M2 ORF2 deletion and knock out mutants of the present
invention exhibit a very substantial diminution of virulence when
compared to wild-type virus that is circulating naturally in
humans. The virus is sufficiently attenuated so that symptoms of
infection will not occur in most immunized individuals. In some
instances the attenuated virus may still be capable of
dissemination to unvaccinated individuals. However, its virulence
is sufficiently abrogated such that severe lower respiratory tract
infections in the vaccinated or incidental host do not occur.
The level of attenuation of M2 ORF2 deletion and knock out mutants
may be determined by, for example, quantifying the amount of virus
present in the respiratory tract of an immunized host and comparing
the amount to that produced by wild-type RSV or other attenuated
RSV which have been evaluated as candidate vaccine strains. For
example, the attenuated virus of the invention will have a greater
degree of restriction of replication in the upper respiratory tract
of a highly susceptible host, such as a chimpanzee, compared to the
levels of replication of wild-type virus, e.g., 10- to 1000-fold or
more less. Also, the level of replication of the attenuated RSV
vaccine strain in the upper respiratory tract of the chimpanzee
should be less than that of the RSV A2 ts-1 mutant, which was
demonstrated previously to be incompletely attenuated in
seronegative human infants. In order to further reduce the
development of rhinorrhea, which is associated with the replication
of virus in the upper respiratory tract, an ideal vaccine candidate
virus should exhibit a restricted level of replication in both the
upper and lower respiratory tract. However, the attenuated viruses
of the invention must be sufficiently infectious and immunogenic in
humans to confer protection in vaccinated individuals. Methods for
determining levels of RSV in the nasopharynx of an infected host
are well known in the literature. Specimens are obtained by
aspiration or washing out of nasopharyngeal secretions and virus
quantified in tissue culture or other by laboratory procedure. See,
for example, (Belshe et al., J. Med. Virology 1:1 57-162, 1977;
Friedewald 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), each incorporated herein by
reference. The virus can conveniently be measured in the
nasopharynx of host animals, such as chimpanzees.
In some instances it may be desirable to combine the M2 ORF2
deletion and knock out mutant RSV vaccines of the invention with
vaccines which induce protective responses to other agents,
particularly other childhood viruses. For example, a chimeric RSV
vaccine of the present invention can be administered simultaneously
with PIV vaccine, such as described in Clements et al., J. Clin.
Microbiol. 29:1175-1182, 1991, incorporated herein by reference. In
another aspect of the invention the chimeric RSV can be employed as
a vector for protective antigens of other respiratory tract
pathogens, such as PIV, by incorporating the sequences encoding
those protective antigens into the RSV genome or antigenome which
is used to produce infectious RSV, as described herein.
In yet another aspect of the invention an M2 ORF2 deletion or knock
out mutant RSV is employed as a vector for transient gene therapy
of the respiratory tract. According to this embodiment the M2 ORF2
deletion and knock out mutant RSV genome or antigenome incorporates
a sequence which is capable of encoding a gene product of interest.
The gene product of interest is under control of the same or a
different promoter from that which controls RSV expression. The
infectious RSV produced by coexpressing the recombinant RSV genome
or antigenome with the N, P, L and M2(ORF1) proteins and containing
a sequence encoding the gene product of interest is administered to
a patient. This can involve a recombinant RSV which is fully
infectious (i.e., competent to infect cultured cells and produce
infectious progeny), or can be a recombinant RSV which, for
example, lacks one or more of the G, F and SH surface glycoprotein
genes and is propagated in cells which provide one or more of these
proteins in trans by stable or transient expression. In such a
case, the recombinant virus produced would be competent for
efficient infection, but would be highly inefficient in producing
infectious particles. The lack of expressed cell surface
glycoproteins also would reduce the efficiency of the host immune
system in eliminating the infected cells. These features would
increase the durability and safety of expression of the foreign
gene.
With regard to gene therapy, administration is typically by
aerosol, nebulizer, or other topical application to the respiratory
tract of the patient being treated. M2 ORF2 deletion and knock out
mutant RSV is administered in an amount sufficient to result in the
expression of therapeutic or prophylactic levels of the desired
gene product. Examples of representative gene products which are
administered in this method include those which encode, for
example, those particularly suitable for transient expression,
e.g., interleukin-2, interleukin-4, gamma-interferon, GM-CSF,
G-CSF, erythropoietin, and other cytokines, glucocerebrosidase,
phenylalanine hydroxylase, cystic fibrosis transmembrane
conductance regulator (CFTR), hypoxanthine-guanine phosphoribosyl
transferase, cytotoxins, tumor suppressor genes, antisense RNAs,
and vaccine antigens.
The following examples are provided by way of illustration, not
limitation. In brief, these examples describe a surprising
transcription/replication "switch" function of the 90-amino acid
M2-2 protein, whose function was heretofore unknown. Viable
recombinant RSV was recovered in which expression of M2-2 was
ablated, identifying it as an accessory factor dispensable for
growth in cell culture. Virus lacking a functional M2-2 protein
grew less efficiently than did the wild type parent in cell
culture, with titers that were reduced 1000-fold during the initial
2-5 days and 10-fold by days 7-8. In cells infected with M2-2
knock-out virus, the accumulation of genomic RNA, was approximately
15-18% that of wild type, while the accumulation of mRNA was
approximately 175 to 300% that of wild type. Synthesis of the F and
G glycoproteins, the major RSV neutralization and protective
antigens, was increased in proportion with the mRNA increase. In
cells infected with wild type RSV, mRNA accumulation increased
dramatically up to approximately 12-15 hours post-infection and
then leveled off, whereas accumulation continued to increase in
cells infected with the M2-2 knock-out viruses. These findings
suggest that M2-2 mediates a regulatory from transcription to RNA
replication, one which provides an initial high level of mRNA
synthesis followed by a shift in the RNA synthetic program in favor
of genomic RNA for virion assembly. For the purpose of vaccine
development within the present invention, this represents a highly
desirable phenotype in which virus growth is attenuated while gene
expression is undiminished or, more typically, concomitantly
increased. This is an unexpected and highly desirable phenotype for
vaccine development, since attenuating mutations described to date
typically are associated with a decrease in antigen expression and
a concomitant decrease in immunogenicity.
EXAMPLE I
M2-2 Mutant Plasmid Constructions
All recombinant RSV viruses and cDNA clones were based on RSV
strain A2 of antigenic subgroup A. An 805 bp MscI-BamHI fragment
(nt 7696-8501 in the complete recombinant antigenomic sequence)
containing most of M2 ORF1 and all of ORF2 was subcloned and
subjected to mutagenesis. A unique NdeI site in ORF2 was opened,
filled-in, and relegated, creating a frame-shift mutation hereafter
called the NdeI mutation (FIG. 1B). The NdeI restriction enzyme
site within ORF2 was identified at genome position 8299, and the
frame-shift mutation (2 nts added) was at codon 47 of the predicted
90 amino acid protein (FIG. 1B). Thus the NdeI mutant encoded the
N-terminal 46 amino acids of M2-2 fused to the 18 heterologous
amino acids encoded by the frame-shift.
To create a second M2-2 knock-out mutant, hereafter called the K5
mutant (rA2-K5, also referred to as rA2.DELTA.M2-2), PCR
mutagenesis (Byrappa et al., Genome Research 5:404-407, 1995,
incorporated herein by reference) was carried out on the subcloned
MscI-BamHI fragment. This mutagenesis was designed to completely
ablate expression of ORF2, by mutation of each of the three
potential initiation codons for ORF2 (FIGS. 1A and 1C) into ACG
codons. To minimize the possibility of reversion or non-AUG
initiation (Curran et al., Embo J. 7:245-51, 1988; Mol. Cell. Biol.
18:5021-31, 1998, each incorporated herein by reference), a stop
codon was also added in each register following the ORF1
termination codon, terminating M2 ORF2 at codon 13 (FIG. 1C). The
M2-1 amino acid sequence was not affected in either mutant.
The mutagenic oligonucleotides, which were 5'-phosphorylated, were
as follows:
The presence of the NdeI and K5 mutations in their respective cDNAs
was confirmed by sequencing, and each was sub-cloned into the
AflII/BamHI sites of the support plasmid pTM-M2, which encodes both
M2 ORFs and was used previously to supply both M2-1 and M2-2
proteins in a model minigenome system (Collins et al., Proc. Nat.
Acad. Sci. USA 93:81-5, 1996, incorporated herein by reference).
The same fragment was also cloned into the AflII/BamHI sites of
pUC118-FM2, which contained the F and M2 genes, and the StuI/BamHI
fragment of this plasmid was subsequently transferred to the
full-length antigenomic cDNA (D53) to create NdeI and K5
antigenomic cDNAs.
EXAMPLE II
Effects of M2-2 Knock-out Mutations on Minireplicon Transcription
and Replication
The function(s) of the M2-2 protein was not known, but it had
previously been shown to strongly inhibit minigenome RNA synthesis
(Collins et al., Proc. Nat. Acad. Sci. USA 93:81-5, 1996; Grosfeld
et al., J. Virol. 69:5677-86, 1995 Hardy et al., J. Virol.
72:520-6, 1998, each incorporated herein by reference). Therefore
this highly sensitive assay was used to verify that the NdeI and K5
mutations ablated this inhibitory effect. RSV transcription and
replication were studied using a negative-sense RSV-CAT minigenome
C2, which contains the CAT ORF under the control of RSV GS and GE
signals flanked by the 3'-leader and 5'-trailer regions of the RSV
genome (Collins et al., Proc. Nat. Acad. Sci. USA 93:81-5, 1996;
Grosfeld et al., J. Virol. 69:5677-86, 1995). Intracellular
synthesis of the C2 minigenome was driven from the transfected
plasmid by T7 RNA polymerase supplied from the recombinant vaccinia
virus vTF7-3 (Fuerst et al., Proc. Nat. Acad. Sci. USA
83:8122-8126, 1986), and RSV proteins were expressed from
cotransfected support plasmids.
When minigenome C2 was complemented by N, P and L alone, it
directed the synthesis of antigenome and CAT mRNA (FIG. 2A, lane
2), with the latter being mostly prematurely terminated as observed
previously (Collins et al., Proc. Nat. Acad. Sci. USA 93:81-5,
1996; Grosfeld et al., J. Virol. 69:5677-86, 1995). When plasmid
expressing M2-1 was added in addition, CAT mRNA was synthesized as
full-length molecules (FIG. 2A, lane 4). Coexpression of M2-2
instead of M2-1 strongly inhibited the synthesis of antigenome and
mRNA (FIG. 2A, compare lanes 1 and 3). When the M2 plasmid
contained both ORF1 and ORF2 in their native configuration, M2-1+2,
there was a significant reduction in transcription and replication
products compared to that seen with M2-1, showing that the
inhibitory activity of M2-2 predominated at this particular plasmid
concentration (compare FIG. 2A, lanes 4 and 5). In contrast, M2-1+2
containing the NdeI or the K5 mutation behaved similarly to M2-1
alone, indicating that the inhibitory activity of M2-2 had been
ablated without affecting M2-1 (compare FIG. 2A, lanes 6 and 7 with
lane 4).
Comparable results were obtained when the C2 plasmid was replaced
by the C4 plasmid, which expresses a positive-sense RNA
representing the antigenomic replicative intermediate of the C2
minigenome (Collins et al., Proc. Nat. Acad. Sci. USA 93:81-5,
1996; Grosfeld et al., J. Virol. 69:5677-86, 1995). In this case,
Northern blots were analyzed with a positive-sense riboprobe to
detect the synthesis of minigenome. Cotransfection of increasing
amounts of M2-1 plasmid (0.008, 0.04 and 0.2 times the relative
molar amount of N plasmid) had no effect on the synthesis of
minigenome (FIG. 2B, lanes 5, 6 and 7) consistent with previous
findings that it does not affect replication (Collins et al., Proc.
Nat. Acad. Sci. USA 93:81-5, 1996). However, increasing amounts of
M2-2 alone or M2-1+2 led to a progressive reduction in the amounts
of minigenome (FIG. 2B, lanes 8, 9 and 10 and lanes 2, 3 and 4),
reflecting the inhibitory activity of M2-2. In contrast, increasing
amounts of M2-1+2 containing either the NdeI and the K5 mutant did
not detectably inhibit minigenome synthesis (FIG. 2B, lanes 11, 12
and 13 for the NdeI mutant, and lanes 14, 15 and 16 for the K5
mutant), indicating that neither mutant expressed an M2-2 protein
active in this function.
EXAMPLE III
Recovery and Growth In vitro of M2 ORF 2 Mutant RSV
The NdeI and K5 mutations were individually incorporated into a
full length antigenomic cDNA and recovery of infectious rRSV was
performed as described previously (Collins et al., Proc. Natl.
Acad. Sci. USA 92:11563-11567, 1995, incorporated herein by
reference). Viruses containing either the NdeI or K5 mutations
(rA2-NdeI and rA2-K5 respectively) were recovered successfully, and
the presence of the mutations was confirmed by sequencing RT-PCR
product generated from infected cell RNA. Thus, M2-2 is an
accessory protein that is not required for RSV growth in cell
culture.
The rA2-NdeI and rA2-K5 viruses displayed a large plaque phenotype
and accelerated syncytium formation. Specifically, the plaques
which formed after 3 days were large and syncytial and resembled
those formed by the wt virus at day 6 post-infection (not shown).
When cell monolayers were infected at an moi of 1 pfu per cell,
syncytium formation was evident by 24 h and was extensive by 48 h
(FIG. 3), resembling those formed by the wt virus at day 4
post-infection. These phenotypic changes suggest that the mutant
viruses are either more fusogenic or have altered kinetics of
growth or gene expression, or a combination of these phenotypic
changes, compared to the parental strain.
To examine single-cycle growth kinetics, monolayers of HEp-2 cells
were infected with rA2-wt, rA2-NdeI or rA2-K5 at an moi of 5 pfu
per cell (FIG. 4A). Both the rA2-NdeI and rA2-K5 recombinant
viruses displayed slightly reduced growth kinetics compared to wt,
with the final virus titers being approximately 10-fold less. To
accentuate any potential differences, multi-step growth cycles were
evaluated in HEp-2 cells infected in triplicate at an moi of 0.01
pfu per cell (FIG. 4B). In cells infected with rA2-wt virus, peak
virus titers were reached at 4-5 days post infection whereupon they
leveled and began to decline. This might be due to increased
cytopathogenicity, which became evident for the wt after day 6 and
was more pronounced than for the mutants. Both of the mutant
viruses had markedly delayed and reduced growth kinetics during the
first few days of incubation (1000-fold less mutant virus released
compared to wt at days 2, 3 and 4 post-infection), but by day 8
post-infection the titers approached those of the parental strain.
However, the maximum titers of the two mutants were consistently
10-fold lower than that of wt. These results show that the large
plaque morphology exhibited by the mutant viruses was not
associated with increased virus release when compared to the
parental strain.
Northern Blot Analysis of Viral RNAs
RNA replication and transcription by the M2-2 mutant viruses were
examined. In the single step growth experiment described above,
cell monolayers from replicate plates were harvested at 3 h
intervals, and total intracellular RNA was analyzed by Northern
blot hybridization. The accumulation of antigenome and mRNAs was
monitored by hybridization with negative-sense riboprobes against
the N gene (FIG. 5 Panels a, b and c) or the F gene (Panels d, e
and f). The pattern of monocistronic, dicistronic and tricistronic
mRNAs produced by the mutant viruses was qualitatively similar to
that of the wt strain. This suggests that ablation of the
expression of M2-2 did not grossly alter the transcription
antitermination effect of M2-1. The accumulation of mRNA and
antigenome in the cells infected with the rA2-wt virus was first
detected at 6 h post-infection (FIG. 5, panels a and d, lane 3) and
increased rapidly to approximately 12-15 h post-infection, and
thereafter increased more slowly or plateaued (FIG. 5, panels a and
d, lanes 5-10). In contrast, both the rA2-NdeI and rA2-K5 viruses
displayed a marked delay in the synthesis of mRNA and antigenome,
such that these RNAs became detectable at 9-12 hours post-infection
(FIG. 5, panels b and e, lanes 4 and 5 for rA2-NdeI, panels c and
f, lanes 4-5 for rA2-KS). The mRNA levels then increased to levels
surpassing those of wt. In contrast the accumulation of antigenome
was considerably reduced compared to wt (FIG. 5, panels b and e,
lanes 7-10 for rA2-NdeI, and panels c and f, lanes 7-10 for
rA2-K5). For example, phosphorimager analysis of the blots probed
with the negative-sense F riboprobe (FIG. 5, panels d, e and f)
revealed a 1.3- to 2.0-fold increase in accumulated mRNA by 24 h
post-infection, with a simultaneous three-fold reduction in the
accumulation of antigenomic RNA.
RNA replication was further examined by hybridization of replicate
blots with a positive-sense F riboprobe, which detected the
accumulation of genomic RNA (FIG. 5, Panels g, h and i).
Phosphoimager analysis indicated that the accumulation of genome by
the mutant viruses was 15-18% that of the parental virus at 24 h.
This reduction in genomic RNA template was unexpected given the
increase the accumulation of MRNA mentioned above. Overall, the
molar ratio of mRNA to genome was approximately 7- to 18-fold
greater in the mutant viruses. This provides evidence for a
regulatory balance between transcription and RNA replication, one
which swings in favor of transcription when expression of the M2-2
protein is ablated.
Increased Expression of Major Viral Antigens
The increase in the accumulation of mRNA in cells infected with the
rA2-NdeI and rA2-K5 viruses was mirrored by an increase in the
accumulation of viral protein. FIG. 6 shows a Western blot analysis
of the F (panels A and B) and G (Panels C and D) proteins
synthesized in cells infected with rA2-wt virus or rA2-K5 virus.
The amount of F or G protein present in the harvested cells at 36 h
post-infection (panels A, B, C, and D, lane 6) was 3-fold greater
for rA2-NdeI than for rA2-wt.
Summarizing the above results, M2 ORF2 can be interrupted in rRSV
of the invention without loss of viability in cell culture.
However, there were significant alterations in the viral RNA
synthetic program, cell culture pathogenicity, and growth
characteristics. These findings demonstrate that M2 ORF2 is an
eleventh RSV gene, which is somewhat unexpected since the ORF is
located in the downstream half of the M2 mRNA, is preceded by 11
methionyl codons, and thus would not be expected to be efficiently
translated. Since ORF2 is expressed as a separate protein (Collins
et al., J. Gen. Virol. 71:3015-20, 1990), one possibility is that
it is accessed by a ribosomal stop-restart mechanism, such as
described for the second ORF of the M gene of influenza virus B
(Horvath et al., Embo J. 9:2639-47, 1990). The activity described
for M2-2 identifies an RNA regulatory protein in a negative strand
RNA virus.
The absence of M2-2 in recombinant RSV of the invention is
associated with a reduction in the accumulation of genomic and
antigenomic RNA, the products of RNA replication, and an increase
in the accumulation of mRNA, the product of transcription. This
indicates two activities for M2-2. The first is to increase RNA
replication. The second activity is to regulate transcription. In
cells infected with rA2-wt, the accumulation of mRNA increased
rapidly up to approximately 12 h and more slowly thereafter,
suggesting that transcription is down-regulated after that time.
One of the effects of ablating M2-2 expression is to delay the
appearance of mRNA. Although this may mean that M2-2 mediates
positive regulation early in infection, the simpler explanation is
that the delayed, reduced synthesis of mRNA is a consequence of
delayed, reduced synthesis of its genomic RNA template. Although
the accumulation of mRNA by the mutant viruses was delayed, it
reached wt levels by approximately 12-15 h and continued to
increase thereafter. This suggests that M2-2 mediates negative
regulation of transcription late in infection, which is alleviated
in its absence. Since the proteins of nonsegmented negative strand
RNA viruses typically increase in abundance during the course of
infection, this negative regulatory effect likely is
concentration-dependent. Thus, RSV transcription is subject to
negative autoregulation, and RNA regulation is subject to positive
regulation.
It is generally thought that there is a dynamic, reversible
"switch" between transcription and RNA replication by nonsegmented
negative strand RNA viruses. For example, the synthesis of mRNA and
antigenome ostensively involves the same promoter and genomic
nucleocapsid template and, for most viruses, the same protein
components, N, P and L. RSV is an exception in having the
additional transcription-specific factor M2-1. One widely accepted
model is that RNA synthesis switches from transcription to RNA
replication when sufficient N protein accumulates to mediate
cosynthetic encapsidation of the nascent RNA. This somehow switches
the polymerase to read through gene junctions and synthesize a
complete antigenome (Lamb et al., In Fields Virology (B. N. Fields
et al., Eds., Vol. 1, pp. 1177-1204. Lippincott-Raven,
Philadelphia, 1996). However, in earlier work we were unable to
reconstitute this switch in a model minireplicon system by
overexpression of N protein (Feams et al., Virology 236, 188-201,
1997). Unexpectedly, the present study implicates the M2-2 protein
in this switch. It remains to be determined whether the observed
effects on transcription and replication are linked rather than
independent events. In terms of a single-step growth cycle, the
present results suggest that the M2-2 protein functions around
12-15 h post infection to reduce transcription (after which the
already-made mRNAs continue to drive protein synthesis) and turn on
RNA replication, shifting the RNA synthetic program into virion
production.
It is possible that M2-2 also has other functions. However, the
other phenotypic differences observed to date for the M2-2
knock-out viruses probably can be explained by the changes in the
RNA synthetic program described above. For example, the delay and
reduction of virus production might simply be a consequence of the
delay and reduction in synthesis of progeny genome, and the initial
delay in the accumulation of mRNA. The other phenotypic difference,
accelerated plaque formation, could be a consequence of increased
synthesis of surface glycoproteins and accelerated cell-to-cell
fusion. Nonetheless, this does not preclude other activities for
M2-2.
Previous studies showed that the M2-2 protein inhibited RNA
replication and transcription by RSV model minireplicons (Collins
et al., Proc. Nat. Acad. Sci. USA 93:81-5, 1996; Hardy et al., J.
Virol. 72:520-6, 1998). M2-2-mediated inhibition of minigenome
transcription is consistent with the findings disclosed herein.
However, the previously-observed M2-2-mediated inhibition of
minireplicon RNA replication contrasts with the present findings,
where the absence rather than the presence of M2-2 is associated
with reduced RNA replication by rRSV. Thus, certain results from
the minireplicon system may be incomplete. This distinction may be
attributable to differences between the minireplicon system and an
authentic virus infection, for example: (i) the supply of proteins
would be greatly affected by regulation in an authentic infection
but not in a reconstituted minireplicon system where proteins are
supplied by transfected plasmids, (ii) the effects of M2-2 observed
to date have been in minireplicon systems in which only a subset of
viral proteins was supplied, and (iii) the relative level of M2-2
expressed in an authentic infection has not been determined but
seems to be very low, and the minireplicon studies to date might
have used levels that were too high.
The finding that M2-2 is not essential for growth defines this
species as an accessory protein. Other paramyxovirus accessory
proteins include the RSV SH, NS1 and NS2 proteins, the V and C
proteins of Sendai virus, measles virus and parainfluenza virus
type 3 (PIV3) and the D protein of PIV3 (Bukreyev et al., J. Virol.
71: 8973-82, 1997; Delenda et al., Virology 228: 55-62, 1997; He et
al., Virology 250:30-40, 1998; Kato et al., EMBO J. 16:578-587,
1997; Kurotani et al., Genes to Cells 3:111-24, 1998; Latorre et
al., J. Virol. 72:5984-93, 1998; Radecke et al., Virology
217:418-21, 1996; Schneider et al., Virology 227:314-22, 1997; Teng
et al., J. Virol. 72:5707-16, 1998; Whitehead et al., J. Virol.
73:3438-42, 1999; and U.S. patent application Ser. No. 09/350,821;
each incorporated herein by reference). Among these, the Sendai
virus C protein has been studied the most extensively. Ablation of
the expression of the V protein in recombinant Sendai virus was
associated with increases in transcription, RNA replication and
virus growth in vitro (Kato et al., Embo J. 16:578-587, 1997),
although these differences were not apparent in a separate study
(Delenda et al., Virology 228:55-62, 1997). Growth of V-minus
Sendai virus in vivo was attenuated, suggesting that the V protein
augments pathogenicity (Delenda et al., Virology 228:55-62,
1997).
The Sendai virus C protein is expressed as four proteins, namely
C', C, Y1 and Y2, which arise from translational initiation at the
first through fourth translational start sites in the C ORF,
respectively. Deletion of these proteins individually and in groups
has complex effects which are not completely defined and which are
complicated because deletion of one species can alter expression of
the another. Deletion of the C' and C proteins individually
resulted in increased synthesis of mRNA and genomic RNA, whereas
production of infectious virus was slightly reduced (Latorre et
al., J. Virol. 72:5984-93, 1998). Inexplicably, the C'-minus virus
retained virulence in vivo whereas the C-minus virus was
attenuated. This result is particularly surprising since C' and C
differ only buy the presence of 11 additional N-terminal amino
acids in C'. Deletion of both C' and C delayed the appearance of
genome and mRNA, after which these species were overproduced, and
greatly reduced the production of infectious virus (Kurotani et
al., Genes Cells 3:111-24, 1998; Latorre et al., J. Virol.
72:5984-93, 1998). Elimination of all four C-related proteins in
Sendai virus resulted in a virus that was extremely debilitated for
RNA synthesis and growth in vitro (Kurotani et al., Genes Cells
3:111-24, 1998). The complexity of these effects indicates that the
functions of the V and various C proteins cannot be explained
solely with respect to regulation of RNA synthesis and remain to be
defined.
M2 ORF2 knock out mutants of the invention are particularly useful
as candidates for development of RSV vaccines. Ablation of
expression of the M2-2 gene in the above examples yielded
attenuated virus growth in cell culture by at least 1000-fold
during the initial days of a multi-cycle growth curve in vitro,
with the final yield of infectious virus being reduced
approximately 10-fold. This level of attenuation is highly
desirable for construction of recombinant vaccine viruses of the
invention. The similarity in final yield between the wt and M2-2
knock-out viruses in cell culture indicates that this modification
in recombinant RSV is amenable to production of vaccine virus.
Surprisingly, although virus growth is attenuated in M2-2 knock out
mutants, gene expression is enhanced. Typically, RSV gene
expression is roughly proportional to virus growth, and attenuating
mutations which reduce growth reduce antigen production. Thus, one
of the long-standing problems in RSV vaccine development has been
to provide a level of attenuation which minimizes disease, yet
retains sufficient immunogenicity. The M2-2 knock out mutation
provides an important tool to resolve this problem by conferring
significant attenuation in a recombinant RSV that also exhibits a
concomitant increase, rather than decrease, in antigen expression.
Although these examples describe the effects of ablating expression
of M2-2, it is clear that intermediate effects can be achieved by
reducing rather than ablating expression. Also, expression of M2-2
can be increased to achieve other effects on virus growth and gene
expression. Alteration of the level of gene expression can be
achieved by altering the M2-2 translational start site, or by
placing the ORF under control of gene start and gene end signals
and expressing it as a separate mRNA. The level of mRNA expression
could be varied by the placement of the gene in a different gene
order than the natural gene order position; or by mutations in
cis-acting transcription elements, such as the mutation in position
9 of the gene start signal which was identified in cpts248/404 RSV
(Whitehead et al., Virology 247:232-239, 1998, incorporated herein
by reference).
EXAMPLE IV
Attenuation and Immunogenicity of NS1 or M2-2 Mutant RSV in
Chimpanzees
In the present example, recombinant RSV which cannot express the
NS1 or M2-2 protein, designated rA2.DELTA.NS1 and rA2.DELTA.M2-2,
respectively, were evaluated as live-attenuated RSV vaccines. The
rA2.notident.NS1 virus, described above, contains a large deletion
that confers genetic stability during replication in vitro and in
vivo and specifies attenuated replication in vitro that is
approximately 10-fold reduced compared to that of wild-type
recombinant RSV (rA2). The M2-2 mutant RSV, designated
rA2.DELTA.M2-2, exhibited delayed in vitro growth kinetics but
reached final titers similar to those of rA2. In the present
example, each virus was administered to the respiratory tract of
RSV-seronegative chimpanzees to assess replication, immunogenicity,
and protective efficacy in this model host.
As described in further detail below, the rA2.DELTA.NS1 and
rA2.DELTA.M2-2 viruses were 2,200- to 55,000-fold restricted in
replication in the upper and lower respiratory tracts of
chimpanzees and induced a level of RSV-neutralizing serum antibody
that was only slightly reduced compared to that of wild-type RSV.
The replication of wild-type RSV after challenge was reduced more
than 10,000-fold at each site. Importantly, rA2.DELTA.NS1 and
rA2.DELTA.M2-2 were 10-fold more restricted in replication in the
upper respiratory tract than was the cpts248/404 virus, a vaccine
candidate that retains mild reactogenicity in the upper respiratory
tract of the 1 month-old infant. Thus, either virus might be
appropriately attenuated for this age group, which is a target
population for a RSV vaccine. In addition, these results confirm
that neither NS1 nor M2-2 is essential for RSV replication in vivo,
although each is important for efficient replication.
The rA2.DELTA.M2-2 and rA2.DELTA.NS1 viruses were evaluated for
replication, immunogenicity, and protective efficacy in the upper
and lower respiratory tracts of chimpanzees. The rA2.DELTA.M2-2 and
rA2.DELTA.NS1 viruses were constructed using the antigenomic cDNA
described above (see also, Collins, et al. (Collins et al., Proc.
Natl. Acad. Sci. USA 92:11563-11567, 1995; incorporated herein by
reference), and the recombinant viruses also contained two types of
modifications described above: (i) the introduction of a set of six
translationally silent restriction site markers in the L gene,
called the "sites" mutations, and (ii) two amino acid substitutions
in the F protein, called the "HEK" mutations, which make the
recombinant virus identical at the amino acid level to the
wild-type RSV A2 parent from which the cpts248/404 series of
biological vaccine candidates was derived (see also, Juhasz et al.,
J. Virol. 71:5814-5819, 1997; Whitehead et al., Virology
247:232-39, 1998, each incorporated herein by reference). These
mutations were shown to be phenotypically silent in the chimpanzee
(Whitehead et al., J. Virol. 72:4467-4471, 1998; incorporated
herein by reference). The rA2.DELTA.NS1 virus used in this study
was reconstructed in a sites/HEK background, in preparation for
clinical evaluation, whereas the rA2.DELTA.M2-2 virus is in the
original genetic background, a difference that is irrelevant for
the present study (Bermingham et al., Proc. Natl. Acad. Sci. USA
96:11259-11264, 1999; Whitehead et al., Virology 247:232-39, 1998,
each incorporated herein by reference).
The rA2.DELTA.NS1 and rA2.DELTA.M2-2 viruses were administered
individually to juvenile RSV-seronegative chimpanzees by combined
intranasal and intratracheal inoculation, as described previously
(Crowe et al., Vaccine 12:783-790, 1994; incorporated herein by
reference). Since both viruses were attenuated in vitro, animals
were inoculated with a relatively high dose of 10.sup.5 PFU per ml
per site. To monitor virus replication in the upper and lower
respiratory tracts, respectively, nasopharyngeal swabs and tracheal
lavage samples were collected at intervals over ten days
post-infection and subsequently assayed for virus titer. The mean
peak virus titer was determined for each group (Table I). The
chimpanzees were monitored daily for rhinorrhea, a symptom of upper
respiratory tract illness, and the mean peak score was determined
for each group (Table I). Due to the limited availability of
RSV-seronegative chimpanzees, the number of animals per group was
small, necessitating comparisons with previous studies in which we
had evaluated biologically-derived RSV strain A2 (wt RSV A2),
recombinant wild-type strain A2 (rA2), rA2.DELTA.SH, rA2.DELTA.NS2,
and a recombinant version of the above-mentioned cpts248/404
vaccine candidate (rA2cp248/404) (Table I).
Replication of both rA2.DELTA.NS1 and rA2.DELTA.M2-2 was reduced
more than 2200-fold and 2800-fold, respectively, in the upper
respiratory tract compared to rA2 (Table I). Shedding of
rA2.DELTA.NS1 or rA2.DELTA.M2-2 was detected sporadically and at a
low level beginning 2 to 7 days post-inoculation and each animal
shed virus over a period of 3 to 8 days. Thus, the recovered virus
was not carried over from the initial inoculum, but represented
replication near the level of detection over a period of several
days. In the lower respiratory tract, rA2.DELTA.NS1 was reduced
more than 17,000-fold in replication compared to rA2, while
rA2.DELTA.M2-2 was undetectable at all time points (greater than
55,000-fold reduction). It is important to note that the dose of
rA2.DELTA.NS1 and rA2.DELTA.M2-2 used was 10-fold greater than that
of rA2. Furthermore, both viruses were more attenuated than
rA2cp248/404 which was given at the same dose, particularly in the
case of rA2.DELTA.M2-2 which was not recovered from the lungs of
infected chimps. In addition, both rA2.DELTA.NS1 and rA2.DELTA.M2-2
were unusual in being highly restricted in the upper as well as the
lower respiratory tract. In the upper respiratory tract, each virus
was approximately 10-fold more restricted than cpts248/404, and
175-fold more restricted than rA2.DELTA.NS2. Since upper
respiratory tract congestion was observed during clinical
evaluation of the cpts248/404 virus in 1-2 month old infants
(Wright, P. F., R. A. Karron, R. B. Belshe, J. Thompson, J. E.
Crowe Jr., T. G. Boyce, L. L. Halburnt, G. W. Reed, S. S.
Whitehead, E. L. Anderson, A. E. Wittek, R. Casey, M. Eichelberger,
B. Thumar, V. B. Randolph, S. A. Udem, R. M. Chanock, and B. R.
Murphy "Evaluation of a live, cold-passaged, temperature-sensitive,
respiratory syncytial virus (RSV) vaccine candidate in infancy,"
submitted; incorporated herein by reference) and since infants of
that age are obligate nose-breathers, mutations that confer a level
of restriction of replication in the upper respiratory tract
greater than that of cpts248/404 Will be desirable for inclusion in
a live-attenuated vaccine virus. Animals receiving rA2.DELTA.NS1 or
rA2.DELTA.M2-2 had slightly more rhinorrhea than those infected
with rA2cp248/404, though still less than animals infected with a
ten-fold smaller dose of rA2. Although it is possible that the
absence of NS1 or M2-2 resulted in a virus that retained a moderate
level of virulence but replicated poorly, this appears unlikely. It
is expected that further evaluation, including clinical studies,
will show that the amount of residual virulence associated with
rA2.DELTA.NS1 and rA2.DELTA.M2-2 is attributed to their greatly
reduced replication.
Despite the highly-restricted replication of the rA2.DELTA.NS1 and
rA2.DELTA.M2-2 viruses, immunization with either recombinant
induced a level of RSV-neutralizing serum antibody that was within
2-fold of that induced by rA2cp248/404 (Table I). Furthermore,
animals previously infected with either rA2.DELTA.NS1 or
rA2.DELTA.M2-2 were highly resistant to the replication of wt RSV
administered intranasally and intratracheally 56 days
post-immunization (Table II). The level of protection in both cases
was similar in the upper respiratory tract and somewhat less in the
lower respiratory tract to that seen with cpts248/404, both in mean
peak titer and in mean days of shedding.
TABLE I rA2.DELTA.NS1 and rA2.DELTA.M2-2 are highly attenuated and
immunogenic in both the upper and lower respiratory tracts of
chimpanzees Mean peak virus titer.sup.c (log.sub.10 Virus used to
Dose.sup.b pfu/ml .+-. SE) (Duncan grouping) Rhinorhea score.sup.d
Mean serum neutralizing antibody infect (per site, Nasopharyngeal
(range = 0-4) titer.sup.c (reciprocal log.sub.2) chimpanzees.sup.a
No. of animals log.sub.10 pfu) swab Tracheal lavage Mean peak Day 0
Day 28 wt RSV A2.sup.f 2 4.0 5.0 .+-. 0.35 (A) 5.5 .+-. 0.40 (A)
3.0 <3.3 11.2 rA2.sup.g 2 4.0 4.9 .+-. 0.15 (A) 5.4 .+-. 0.05
(A) 2.5 <3.3 10.5 rA2.DELTA.SH.sup.g 3 4.0 4.6 .+-. 0.10 (A) 3.8
.+-. 0.31 (B) 1.0 <3.3 10.2 rA2.DELTA.NS2.sup.g 4 4.0 3.8 .+-.
0.41 (B) 1.4 .+-. 0.29 (C) 1.0 3.4 10.6 rA2cp248/404.sup.g 4 5.0
2.5 .+-. 0.25 (C) 1.4 .+-. 0.37 (C) 0.8 3.4 10.6 rA2.DELTA.NS1 4
5.0 1.6 .+-. 0.12 (D) 1.2 .+-. 0.43 (C) 2.0 <3.3 9.8
rA2.DELTA.M2-2 4 5.0 1.5 .+-. 0.09 (D) <0.7 1.8 <3.3 9.1
.sup.a All recombinant-derived viruses (r) contain the sites and
HEK mutations (see text), except for rA2.DELTA.M2-2. .sup.b
Chimpanzees were inoculated by the intranasal and intratracheal
routes with the indicated amount of virus in a 1 ml inoculum per
site. .sup.c Nasopharyngeal swab samples were collected daily for
ten days and tracheal lavage samples were collected on days 2, 5,
6, 8 and 10. Mean peak titers were calculated and assigned to
statistically similar groups by Duncan's Multiple Range test
(.alpha. = 0.05). Means in each column with different letters are
significantly different. .sup.d The amount of rhinorrhea was
estimated daily and assigned a score (0 to 4) that indicated extent
and severity. Scores indicate severe [4], moderate [3], mild [2],
trace [1], or no [0] rhinorrhea. Shown are the man peak scores.
.sup.e Serum RSV-neutralizing antibody titers were determined by a
complement-enhanced 60% plaque-reduction assay using wt RSV A2 and
HEp-2 cell monolayer cultures incubated at 37.degree. C..
RSV-seronegative chimpanzee serum used as a negative control had a
neutralizing antibody titer .3 log.sub.2. Adult human serum used as
a positive control had a neutralizing antibody titer of 11.4
log.sub.2. .sup.f Historic controls from the study of Crowe, et
al., (Vaccine 13:847-855, 1995; incorporated herein by reference).
.sup.g Data from the study of Whitehead, et al. (J. Virol.
73:3438-3442, 1999; incorporated herein by reference).
TABLE II rA2.DELTA.NS1 and rA2.DELTA.M2-2 are highly protective
against challenge with wt RSV A2 in the upper and lower respiratory
tracts or chimpanzees. Replication of RSV challenge virus at the
indicated site.sup.b Nasopharynx Trachea Immunizing Inoculum
dose.sup.a No. of Mean days of Mean days of Mean peak virus
(log.sub.10 PFU/ml) animals shedding Mean peak titer.sup.c shedding
Mean peak titer.sup.c rhinorrhea score rA2.DELTA.NS1 5.0 4 2.8 .+-.
0.75 1.7 .+-. 0.46 1.0 .+-. 0.41 1.8 .+-. 0.73 1.0 rA2.DELTA.M2-2
5.0 4 3.5 .+-. 0.87 2.3 .+-. 0.71 1.0 .+-. 0.71 1.7 .+-. 0.63 1.0
rA2.DELTA.NS2.sup.d 4.0 4 ND.sup.f 1.9 .+-. 0.30 ND.sup.f 2.2 .+-.
0.77 1.0 cpts248/404.sup.e 4.7 2 3.5 .+-. 0.50 2.3 .+-. 0.25 0
<0.7 1.0 none.sup.e 2 8.5 .+-. 0.50 5.0 .+-. 0.35 6.0 .+-. 1.0
4.8 .+-. 0.30 3.0 .sup.a Each virus was initially administered at
the indicated dose in a 1.0 ml inoculum given intranasally and
intratracheally. .sup.b On day 56, chimpanzees were challenged with
wt RSV A2 administered at a dose of 10.sup.4 PFU/ml in a 1.0 ml
inoculum given intranasally and intratracheally. Nasopharyngeal
swab samples were collected daily for twelve days and tracheal
lavage samples were collected on days 2, 5, 6, 8, and 12. .sup.c
Mean peak titers (log.sub.10 PFU/ml) were calculated using the peak
virus titer achieved in each animal. .sup.d Data from the study of
Whitehead, et al. (J. Virol. 73:3438-3442, 1999; incorporated
herein by reference). .sup.e Historic controls from the study of
Crowe, et al., (Vaccine 13:847-855, 1995; incorporated herein by
reference). .sup.f ND, not determined
As noted above, deletion of NS2 and other modifications to RSV
genes yields desired phenotypic effects, including attenuation via
non-ts mutations (Whitehead et al., J. Virol. 73:3438-3442, 1999;
incorporated herein by reference). Compared to rA2.DELTA.NS2,
rA2.DELTA.NS1 and rA2.DELTA.M2-2 are substantially more attenuated,
even at a ten-fold higher dose (Table I), while providing similar
levels of protection against challenge with wt RSV (Table II).
Deletion mutants are extremely stable both in vitro and in vivo,
thus making them attractive candidates for vaccine development.
This property will be important in certain aspects of the
invention. A low level of genetic instability in a RSV vaccine
likely would not be a problem in normal individuals, particularly
considering the high prevalence of fully-virulent wild-type RSV.
However, vaccine virus might have prolonged replication in
immunocompromised individuals. Thus, it will often be desirable to
engineer recombinant vaccine viruses that contain attenuating
mutations resistant to reversion.
A principal target for a RSV vaccine is the 1 to 2 month old
infant, while a second major target is the elderly. A
live-attenuated vaccine for RSV-naive infants will need to be more
attenuated than one for use in adults (see, e.g., Gonzalez et al.,
Vaccine 18:1763-1772, 2000; incorporated herein by reference). In
this context, the rA2.DELTA.NS1 and rA2.DELTA.M2-2 viruses are
similar to cpts248/404 in their level of replication, and therefore
will most likely be useful in development of a pediatric RSV
vaccine, either as currently constructed or with the inclusion of
one or more additional attenuating mutations described herein. As
with other RSV recombinants described herein, the rA2.DELTA.NS1 and
rA2.DELTA.M2-2 viruses can be rapidly adapted as a RSV subgroup A
or subgroup B vaccine virus by replacing the F and G glycoproteins.
(Whitehead et al., J. Virol. 73:9773-80, 1999; incorporated herein
by reference).
Microorganism Deposit Information
The following materials have been deposited with the American Type
Culture Collection, 10801 University Boulevard, Manassas, Va.
20110-2209, under the conditions of the Budapest Treaty and
designated as follows:
Plasmid Accession No. Deposit Date cpts RSV 248 ATCC VR 2450 Mar.
22, 1994 cpts RSV 248/404 ATCC VR 2454 Mar. 22, 1994 cpts RSV
248/955 ATCC VR 2453 Mar. 22, 1994 cpts RSV 530 ATCC VR 2452 Mar.
22, 1994 cpts RSV 530/1009 ATCC VR 2451 Mar. 22, 1994 cpts RSV
530/1030 ATCC VR 2455 Mar. 22, 1994 RSV B-1 cp52/2B5 ATCC VR 2542
Sept. 26, 1996 RSV B-1 cp-23 ATCC VR 2579 July 15, 1997 p3/7(131)
ATCC 97990 Apr. 18, 1997 p3/7(131)2G ATCC 97989 Apr. 18, 1997
p218(131) ATCC 97991 Apr. 18, 1997
Although the foregoing invention has been described in detail by
way of example for purposes of clarity of understanding, it will be
apparent to the artisan that certain changes and modifications may
be practice within the scope of the appended claims which are
presented by way of illustration not limitation. In this context,
various publications and other references have been cited within
the foregoing disclosure for economy of description. Each of these
references are incorporated herein by reference in its entirety for
all purposes.
SEQUENCE LISTING <100> GENERAL INFORMATION: <160>
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<200> SEQUENCE CHARACTERISTICS: <210> SEQ ID NO 6
<211> LENGTH: 11 <212> TYPE: PRT <213> ORGANISM:
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<400> SEQUENCE: 6 Thr Asn Asp His Ala Lys Asn Asn Asp Thr Thr
1 5 10 <200> SEQUENCE CHARACTERISTICS: <210> SEQ ID NO
7 <211> LENGTH: 20 <212> TYPE: PRT <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
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SEQUENCE CHARACTERISTICS: <210> SEQ ID NO 8 <211>
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* * * * *