U.S. patent application number 13/976873 was filed with the patent office on 2014-05-22 for expression systems.
This patent application is currently assigned to OKAIROS AG. The applicant listed for this patent is Riccardo Cortese, Alfredo Nicosia, Alessandra Vitelli. Invention is credited to Riccardo Cortese, Alfredo Nicosia, Alessandra Vitelli.
Application Number | 20140141042 13/976873 |
Document ID | / |
Family ID | 45464575 |
Filed Date | 2014-05-22 |
United States Patent
Application |
20140141042 |
Kind Code |
A1 |
Vitelli; Alessandra ; et
al. |
May 22, 2014 |
Expression Systems
Abstract
The invention relates to an expression system comprising
polynucleotides encoding proteins, wherein the expression system
comprises a first polynucleotide encoding at least one protein,
peptide or variant thereof, which induces a T cell response, and a
second polynucleotide encoding at least one protein peptide or
variant thereof, which induces an anti-pathogenic B cell response.
The invention further relates to protein mixtures encoded by the
expression system and cells comprising the expression system or the
protein mixture and pharmaceutical compositions comprising the
expression system or the protein mixture. The expression system,
polynucleotides, proteins, cells, and pharmaceutical compositions
are useful in the prophylaxis or treatment of infections. The
invention further relates to nucleotide constructs which comprises,
essentially consists or consists of a polynucleotide encoding a
modified influenza hemagglutinin (HA).
Inventors: |
Vitelli; Alessandra; (Rome,
IT) ; Nicosia; Alfredo; (Naples, IT) ;
Cortese; Riccardo; (Basel, CH) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Vitelli; Alessandra
Nicosia; Alfredo
Cortese; Riccardo |
Rome
Naples
Basel |
|
IT
IT
CH |
|
|
Assignee: |
OKAIROS AG
Basel
CH
|
Family ID: |
45464575 |
Appl. No.: |
13/976873 |
Filed: |
December 30, 2011 |
PCT Filed: |
December 30, 2011 |
PCT NO: |
PCT/EP11/74307 |
371 Date: |
January 28, 2014 |
Current U.S.
Class: |
424/210.1 ;
424/204.1; 424/209.1; 424/211.1; 424/230.1; 424/232.1; 435/320.1;
435/325 |
Current CPC
Class: |
C12N 2800/22 20130101;
A61P 31/04 20180101; A61P 43/00 20180101; C12N 2760/18522 20130101;
A61P 31/12 20180101; C12N 15/85 20130101; C07K 14/005 20130101;
A61P 31/16 20180101; C12N 15/86 20130101; C12N 2710/10343 20130101;
A61K 39/145 20130101; A61K 39/155 20130101; A61K 2039/53 20130101;
A61P 31/14 20180101; C12N 2760/18534 20130101; A61K 39/12 20130101;
C12N 2840/20 20130101 |
Class at
Publication: |
424/210.1 ;
435/320.1; 424/204.1; 424/230.1; 424/211.1; 424/209.1; 424/232.1;
435/325 |
International
Class: |
C12N 15/85 20060101
C12N015/85 |
Foreign Application Data
Date |
Code |
Application Number |
Dec 30, 2010 |
EP |
PCT/EP2010/007995 |
Claims
1. An expression system comprising polynucleotides encoding
proteins, wherein the expression system comprises a first
polynucleotide encoding at least one protein, peptide or variant
thereof, which induces a T cell response, and a second
polynucleotide encoding at least one protein peptide or variant
thereof, which induces an anti-pathogenic B cell response.
2. The expression system of claim 1 wherein the first
polynucleotide and the second polynucleotide are linked such that
they are expressed as an artificial polyprotein.
3. The expression system of claim 1, wherein the proteins, peptides
or fragments encoded by the first and the second polynucleotide are
separated co- or posttranslationally.
4. The expression system of claim 1, wherein a polynucleotide which
encodes a cleavage site is positioned between the first
polynucleotide and the second polynucleotide.
5. The expression system of claim 4, wherein the cleavage site is a
self-cleaving site or an endopeptidase cleavage site.
6. (canceled)
7. The expression-system of claim 1, further comprising a third
polynucleotide encoding a protein or variant thereof.
8-13. (canceled)
14. The expression-system of claim 1, wherein the expression system
comprises at least one vector selected from the group consisting of
plasmid vectors, cosmid vectors, phage vectors, and viral vectors,
viral like particles, and bacterial spores.
15. The expression-system of claim 14, wherein the viral vector is
an adenovirus vector selected from the group consisting of PanAd1,
PanAd2, PanAd3, ChAd55, ChAd 73, ChAd83, ChAd146, ChAd147, ChAd3,
ChAd4, ChAd5, ChAd6, ChAd7, ChAd8, ChAd9, ChAd10, ChAd11, ChAd16,
ChAd17, ChAd19, ChAd20, ChAd22, ChAd24, ChAd26, ChAd30, ChAd31,
ChAd37, ChAd38, ChAd44, ChAd63 and ChAd82.
16. The expression-system of claim 14, wherein the viral vector is
selected from the group consisting of cytomegalovirus vectors,
arena virus vectors, and pox virus vectors.
17. The expression system of claim 1, wherein at least one of the
first polynucleotide or the second polynucleotide is selected from
the group consisting of a protein, peptide or variant thereof
derived from a pathogen.
18. The expression system of claim 17, wherein the pathogen is a
virus.
19. The expression system of claim 1, wherein the protein that
induces a T cell response is a non-structural and/or internal
protein of a virus, and/or the protein that induces an
anti-pathogenic B cell response is a structural and/or surface
protein of a virus.
20-23. (canceled)
24. The expression system of claim 18, wherein the virus is
selected from the group consisting of paramyxoviruses and
orthomyxoviruses.
25-26. (canceled)
27. The expression system of claim 24, wherein the paramyxovirus is
selected from the group consisting of Pneumovirus, and
Metapneumovirus, and Respirovirus.
28. (canceled)
29. The expression system of claim 19, wherein the structural
and/or surface protein of the virus is selected from the group of
paramyxovirus proteins consisting of fusion protein (F), and any of
the attachment glycoproteins G, H, and HN.
30-31. (canceled)
32. The expression system of claim 19, wherein the non-structural
and/or internal protein is selected from the group of paramyxovirus
proteins consisting of nucleoprotein N, Matrix proteins M and M2,
Phosphoprotein P, non structural proteins NS1 and NS2, and the
catalytic subunit of the polymerase (L).
33-35. (canceled)
36. The expression system of claim 24, wherein the orthomyxovirus
is selected from Influenza A virus, Influenza B virus, Influenza C
virus, Thogotovirus, Isavirus and unclassified
Orthomyxoviridae.
37. (canceled)
38. The expression system of claim 19, wherein the structural
and/or surface protein of the virus is selected from the group
orthomyxovirus proteins consisting of HA and NA.
39-40. (canceled)
41. The expression system of claim 19, wherein the non-structural
and/or internal protein(s) is selected from the group of
orthomyxovirus proteins consisting of NP, M1, M2, NS1, NS2/NEP, PA,
PB1, PB2 and PB1-F2 (PB1F2).
42-43. (canceled)
44. An isolated protein mixture encoded by the expression system of
claim 1.
45. An isolated host cell containing the expression-system
according to claim 1.
46. A composition comprising the expression-system according to
claim 1 and a pharmaceutical acceptable carrier and/or
excipient.
47-50. (canceled)
51. A method of treatment or prevention of a viral disease
comprising administration of an effective amount of the
expression-system according to claim 1.
52. The method of claim 51, wherein the disease is caused by a
virus selected from the group consisting of cytomegalovirus, arena
virus, pox virus, paramyxovirus, and orthomyxovirus.
53. (canceled)
54. A nucleic acid construct which comprises a polynucleotide
encoding a modified influenza hemagglutinin (HA), wherein the HA0
cleavage site is modified by introducing one or more basic amino
acids.
55-57. (canceled)
58. The expression system of claim 1, comprising at least one
polynucleotide encoding a modified influenza HA protein, wherein
the HAO cleavage site is modified by introducing one or more basic
amino acids.
59. An isolated protein mixture encoded by the expression system of
claim 58.
60. An isolated host cell containing the expression-system
according to claim 58.
61. A composition comprising the expression-system of claim 58 and
a pharmaceutical acceptable carrier and/or excipient.
62-63. (canceled)
64. A method of treatment or prevention of a viral disease
comprising administration of an effective amount of the
expression-system according to claim 58.
65. (canceled)
Description
FIELD OF THE INVENTION
[0001] The invention relates to an expression system comprising
polynucleotides encoding proteins, wherein the expression system
comprises a first polynucleotide encoding at least one protein,
peptide or variant thereof, which induces a T cell response, and a
second polynucleotide encoding at least one protein peptide or
variant thereof, which induces an anti-pathogenic B cell response.
The invention further relates to protein mixtures encoded by the
expression system and cells comprising the expression system or the
protein mixture and pharmaceutical compositions comprising the
expression system or the protein mixture. The expression system,
polynucleotides, proteins, cells, and pharmaceutical compositions
are useful in the prophylaxis or treatment of infections. The
invention further relates to nucleotide constructs and expression
systems encoding a modified influenza hemagglutinin (HA).
BACKGROUND OF THE INVENTION
[0002] Infectious diseases are still a major thread of mankind. One
way for preventing or treating infectious diseases is the
artificial induction of an immune response by vaccination which is
the administration of antigenic material to an individual such that
an adaptive immune response against the respective antigen is
developed. The antigenic material may be pathogens (e.g.
microorganisms or viruses) which are structurally intact but
inactivated (i.e. non-infective) or which are attenuated (i.e. with
reduced infectivity), or purified components of the pathogen that
have been found to be highly immunogenic. Another approach for
inducing an immune response against a pathogen is the provision of
expression systems comprising one or more vector encoding
immunogenic proteins or peptides of the pathogen. Such vector may
be in the form of naked plasmid DNA, or the immunogenic proteins or
peptides are delivered by using viral vectors, for example on the
basis of modified vaccinia viruses (e.g. Modified Vaccinia Ankara;
MVA) or adenoviral vectors. Such expression systems have the
advantage of comprising well-characterized components having a low
sensitivity against environmental conditions.
[0003] It is a particular aim when developing vector based
expression systems that the application of these expression systems
to a patient elicits an immune response which is protective against
the infection by the respective pathogen. However, although
inducing an immunogenic response against the pathogen, some
expression systems are not able to elicit an immune response which
is strong enough to fully protect against infections by the
pathogen. Accordingly, there is still a need for expressions
systems which are capable of inducing a protective immune response
against a pathogen, e.g. an infectious agent like a virus.
[0004] Viruses
[0005] Viruses are a group of pathogens/infectious agents which
have no own metabolism and can be considered as obligatory
endoparasites of the respective host cells using at least parts of
host's cell facilities for conducting viral protein expression and
virus replication. Viruses can be classified on the basis of the
type (DNA/RNA), the strandedness (single-stranded (ss) or
double-stranded (ds)), the sense (negative sense or positive sense)
of the nucleic acid constituting their genome and their replication
(Baltimore classification). Accordingly, viruses are generally
classified in DNA and RNA viruses. Viruses can be further
classified into single-stranded (ss) or double-stranded (ds) DNA or
RNA viruses, which genome is a single-stranded or double-stranded
nucleic acid. Some viruses have a genome which is partially
double-stranded and partially single-stranded (e.g.
hepadnaviruses). The orientation or "sense" of the genome and/or in
the manufacturing of medicament for use in the prophylaxis or
treatment of a pathogen and/or for use in methods of prophylaxis or
treatment of a pathogen, wherein the pathogen plays an important
role for the viral life cycle of viruses, in particular in the life
cycle of ssRNA viruses or ssDNA viruses. A positive sense ssRNA (+)
genome has the same orientation as a cellular mRNA and can be
directly translated into viral proteins. In the life cycle of
viruses having a negative sense single-stranded RNA genome (ssRNA
(-)), it is necessary that the genomic sequences are transcribed
into positive sense mRNA which can be translated into viral
proteins by the host cell. A single-stranded genome that contains
both positive-sense and negative-sense is called "ambisense" (e.g.
ssRNA (+/-), ssDNA(+/-)).
[0006] Although the genome of viruses may be quite large (e.g. in
the case of DNA viruses), in particular small RNA viruses have
evolutionary developed strategies for expressing their gene
products (e.g. proteins and peptides) in a very efficient manner.
One of these strategies is the expression of one or more
polyprotein encoded by the viral genome, which is co- or
posttranslationally processed into single proteins and/or peptides.
This strategy is adapted, for example, by some double-stranded (ds)
RNA viruses or single-stranded (ss) RNA viruses having a positive
sense genome. "Enveloped viruses", such as orthomyxoviruses,
paramyxoviruses, retroviruses, flaviviruses, rhabdoviruses and
alphaviruses, are surrounded by a lipid bilayer originating from
the host plasma membrane (1).
[0007] Attachment glycoproteins are found in all enveloped viruses
and mediate the initial interaction between the viral envelope and
the plasma membrane of the host cell via their binding to
carbohydrate moieties or cell adhesion domains of proteins or other
molecules on the plasma membrane of the host cell. Thereby,
attachment glycoproteins bridge the gap between the virus and the
membrane of the host cell. Attachment glycoproteins designated as
"H" possess hemagglutinin activity, glycoproteins designated as "HN
possesses hemagglutinin and neuraminidase activities. Attachment
glycoproteins are designated as "G" when they have neither
haemagglutination nor neuraminidase activity.
[0008] Paramyxoviruses
[0009] Paramyxoviruses are a family of animal viruses which
comprises a single stranded non-segmented negative-sense RNA.
Paramyxoviruses are responsible for a number of animal and human
diseases. The RNA genome of paramyxoviruses is 15-19 kilo bases
(kb) in length and encodes 6-10 genes. Each gene contains
transcription start/stop signals at the beginning and end, which
are transcribed as part of the gene. The gene sequence is conserved
across the paramyxoviruses due to a phenomenon known as
transcriptional polarity in which genes closest to the 3' end of
the genome are transcribed in greater abundance than those towards
the 5' end. After each gene is transcribed, the RNA-Dependent RNA
polymerase pauses to release the new mRNA when it encounters an
intergenic sequence. When the RNA polymerase is paused, there is a
chance that it will dissociate from the RNA genome. If it
dissociates, it must reenter the genome at the leader sequence,
rather than continuing to transcribe the length of the genome. As a
result, the further downstream genes are from the leader sequence,
the less they will be transcribed by the RNA polymerase. The genes
of paramyxoviruses are arranged in relative order of protein needed
for successful infection. The conserved gene sequence is
Nucleocapsid--Phosphoprotein--Matrix--Fusion--Attachment--Large
(polymerase).
[0010] Many Paramyxovirus genomes follow the so-called "Rule of
Six". According to this rule, the total length of the genome is
almost always a multiple of six. However, the members of the
sub-family Pneumovirinae comprising the Respiratory Syncytial Virus
(RSV) do not follow this rule.
[0011] Respiratory Syncytial Virus (RSV)
[0012] The enveloped virus designated as respiratory syncytial
virus (RSV) is the most important cause of viral lower respiratory
tract illness (LRTI) in infants and children worldwide (2). In the
United States, it is estimated that 70,000-126,000 infants are
hospitalized annually with RSV pneumonia or bronchiolitis and that
the rate of hospitalization for bronchiolitis has increased since
1980 (3). Children are infected by 2 years of age and the WHO has
estimated that RSV causes disease in approximately 64 million
children each year and 160,000 deaths. In industrialised countries,
RSV is responsible for at least 50% of hospitalisations for
respiratory disease in children, and up to 6% of all RSV infections
in children result in hospitalisation (4). RSV infection does not
provoke lasting immunity, so that human hosts experience lifelong
cycles of infection and re-infection. Although it is traditionally
regarded as a pediatric pathogen, RSV also causes severe disease in
the elderly and immuno-compromised individuals (5). The burden of
RSV disease in the elderly is comparable to that of seasonal
influenza and the economic impact of RSV-related disease in adults
is estimated to be greater than that of influenza in relation to
numbers of days lost from work (6, 7). Monoclonal antibody
prophylaxis is effective in reducing RSV hospitalisations by 50% in
infants at high risk of severe disease (8). However, there is
currently no effective RSV vaccine or anti-viral therapy.
[0013] The disastrous effect of a formalin-inactivated (FI) RSV
vaccine in infants in the 1960s has hampered vaccine development.
The vaccine failed to protect against RSV infection and induced
exacerbated respiratory disease (9) which has been attributed to
induction of high titre, poorly neutralising, low affinity
antibodies, lack of CD8+ T cell priming and induction of a
Th2-biased immune response (10, 11, and 12). There is evidence that
RSV impairs the induction of an adequate adaptive T cell immune
response (13).
[0014] There is, therefore, a clear need for an effective vaccine
not only to protect infants, but also to boost immunity in the
elderly and to reduce the circulation of RSV in siblings and
adults, who are the main source of RSV infection for infants. A RSV
vaccine capable of inducing neutralizing antibody response and
potent and broad T cell response for priming a T cell responses in
individuals who have not yet been infected with RSV (infants) or
for boosting a pre-existing T cell response in individuals who need
to `reset` the memory response to higher levels (elderly) is
especially desirable.
[0015] Orthomyxoviruses
[0016] Orthomyxoviruses are a family of RNA viruses that includes
five genera: Influenzavirus A, Influenzavirus B, Influenzavirus C,
Isavirus and Thogotovirus. A sixth genus has recently been
described. The first three genera contain viruses that cause
influenza in vertebrates, including birds, humans, and other
mammals. The three genera of influenza virus have antigenic
differences in their nucleoprotein and matrix protein.
Influenzavirus A infects humans, other mammals, and birds, and
causes all influenza pandemics. Influenzavirus B infects humans and
seals. Influenzavirus C infects humans and pigs.
[0017] Viruses of the Orthomyxovirus family contain 6 to 8 segments
of linear negative-sense single stranded RNA. The total genome
length is 12000-15000 nucleotides (nt). The largest segment
2300-2500 nt; of second largest 2300-2500 nt; of third 2200-2300
nt; of fourth 1700-1800 nt; of fifth 1500-1600 nt; of sixth
1400-1500 nt; of seventh 1000-1100 nt; of eighth 800-900 nt. Genome
sequence has terminal repeated sequences; repeated at both ends.
Terminal repeats at the 5'-end are 12-13 nucleotides long. The
nucleotide sequences of 3'-terminus are identical the same in
genera of same family; most on RNA (segments), or on all RNA
species. Terminal repeats at the 3'-end 9-11 nucleotides long.
[0018] Influenza virus is one of the most important respiratory
pathogens. In the US alone, influenza infection is responsible for
20,000-40,000 deaths and over 100,000 hospitalizations annually
(1). Infants, the elderly, and individuals with compromised
cardiac, pulmonary, or immune systems are at great risk of serious
complications following flu infection.
[0019] Immunization proves to be the most effective measure in
preventing the disease. One of the common features shared by all
current influenza vaccines consists in targeting primarily the
induction of neutralizing antibodies directed against the major
viral envelope protein, hemagglutinin (HA).
SUMMARY OF THE INVENTION
[0020] The invention provides in a first aspect an expression
system comprising a first polynucleotide encoding at least one
protein, peptide or variant thereof, which induces a T cell
response and a second polynucleotide encoding at least one protein,
peptide or variant thereof, which induces an anti-pathogenic B cell
response.
[0021] In a second aspect, the invention provides an isolated
protein mixture encoded by the expression system of the first
aspect.
[0022] In a third aspect, the invention provides an isolated host
cell containing the expression system of the first aspect and/or
the protein mixture of the second aspect.
[0023] In a fourth aspect, the present invention provides a
composition comprising the expression system of the first aspect,
or the protein mixture of the second aspect, and a pharmaceutical
acceptable carrier and/or excipient.
[0024] In a fifth aspect, the present invention provides the
expression system of the first aspect, the protein mixture of the
second aspect, the cell of the third aspect and the composition of
the fourth aspect, for the use in medicine in particular in the
treatment or prevention of infectious diseases, preferably a viral
disease.
[0025] In a sixth aspect, the present invention provides for a
method of treatment or prevention of a viral disease comprising the
administration of an effective amount of the expression system of
the first aspect, the protein mixture of the second aspect, the
cell of the third aspect and the composition of the fourth
aspect.
[0026] In a seventh aspect, the present invention provides for a
method of enhancing an immune response comprising the
administration of the expression system of the first aspect, the
protein mixture of the second aspect, the cell of the third aspect
and the composition of the fourth aspect.
[0027] In an eighth aspect, the present invention provides
nucleotide constructs encoding influenza hemagglutinin (HA), an
expression system comprising these nucleotide constructs, and
proteins or polyproteins encoded by the nucleotide constructs or
the expression system, wherein the HA0 cleavage site has a
multibasic sequence.
[0028] In a ninth aspect, the present invention provides the use of
the multibasic HA0 cleavage site for constructing expression
systems capable for expressing influenza hemagglutinin (HA) in
vitro and/or in vivo.
[0029] In a tenth aspect, the invention provides an isolated
protein mixture encoded by the expression system of the eighth
aspect.
[0030] In an eleventh aspect, the invention provides an isolated
host cell containing the nucleotide constructs, the expression
system or the proteins or polyproteins of the eighth aspect and/or
the protein mixture of the tenth aspect.
[0031] In a twelfth aspect, the present invention provides a
composition comprising the nucleotide constructs, the expression
system or the proteins or polyproteins of the eighth aspect, or the
protein mixture of the tenth aspect, and a pharmaceutical
acceptable carrier and/or excipient.
[0032] In a thirteenth aspect, the present invention provides the
nucleotide constructs, the expression system or the proteins or
polyproteins of the eighth aspect, the protein mixture of the tenth
aspect, the cell of the eleventh aspect and the composition of the
twelfth aspect, for the use in medicine in particular in the
treatment or prevention of influenza virus infections.
[0033] In a fourteenth aspect, the present invention provides for a
method of treatment or prevention of an influenza virus infections
comprising the administration of an effective amount of the
nucleotide constructs, the expression system or the proteins or
polyproteins of the eighth aspect, the protein mixture of the tenth
aspect, the cell of the eleventh aspect and the composition of the
twelfth aspect.
[0034] In a fifteenth aspect, the present invention provides for a
method of enhancing an immune response comprising the
administration of the nucleotide constructs, the expression system
or the proteins or polyproteins of the eighth aspect, the protein
mixture of the tenth aspect, the cell of the eleventh aspect and
the composition of the twelfth aspect. The above summary does not
necessarily describe all aspects of the present invention.
BRIEF DESCRIPTION OF THE FIGURES
[0035] FIG. 1: Schematic Diagram of the RSV vaccine polyprotein.
conF0=consensus sequence of the F protein, 2A=translational
cleavage site of the Foot and Mouth Disease virus, conN=consensus
sequence of the N protein, conM2-1=consensus of the M2-1
protein.
[0036] FIG. 2: The vaccine antigen F0.DELTA.TM-N-M2-1 is
efficiently processed in mammalian cells. Western Blot analysis of
lysates from HeLa cells; nt: not transfected Hela. RSV transf: HeLa
cells transfected with F0.DELTA.TM-N-M2-1. RSV inf: Hep2 cells
infected with RSV strain A
[0037] FIG. 3: The secreted F protein forms a homotrimer. Western
Blot analysis of supernatant from transfected HeLa cells; RSV:
F0.DELTA.TM-N-M2-1 transfected, F0: F0.DELTA.TM transfected, Ctrl:
empty plasmid transfected
[0038] FIG. 4: The F protein expressed from the vaccine polyprotein
is a better immunogen than the F protein alone. A. Western Blot
analysis of supernatant from HeLa cells infected with
PanAd3/F0.DELTA.TM-N-M2-1 which was probed with different dilutions
of sera from mice immunized with F0.DELTA.TM or F0.DELTA.TM-N-M2-1
B. densitometric scanning of the Western Blot on panel A. Data are
expressed as Relative Intensity of the area corresponding to the
protein band.
[0039] FIG. 5: The RSV vaccine induced potent systemic T cell
immunity in mice by a single intramuscular injection. IFNg-Elispot
assay of splenocytes of PanAd3/F0.DELTA.TM-N-M2-1 immunized Balb/C
mice using mapped immunodominant peptides from RSV F and M
proteins.
[0040] FIG. 6: Schematic diagram of the Influenza vaccine
polyprotein. NP=consensus sequence of the NP protein, M1=consensus
sequence of M1 protein, 2A=translational cleavage site of the Foot
and Mouth Disease virus, H1p=consensus sequence of the HA protein
from H1N12009.
[0041] FIG. 7 Western Blot analysis of H1p expression in
transfected HeLa cells. Total lysate of HeLa cells transfected with
PVJ-H1p (Lane 1), with PVJ-H1 (Lane 2) and not transfected CTR
(Lane 3). The arrows show the bands corresponding to the uncleaved
(70 kD) HA0 form and the cleaved (28 Kd) HA2. The polyclonal
anti-HA serum recognize epitopes in the HA2 protein fragment. It is
shown that the H1p protein is fully processed.
[0042] FIG. 8 Whole-cell FACS analysis of membrane-displayed HA
proteins. The histograms represent the median fluorescence analysis
of HeLa cells transfected with wild type HA (right upper panel) and
H1 (right lower panel). Cells were incubated with hyperimmune mouse
polyclonal serum raised against H1p and then with a secondary
anti-mouse antibody PE-conjugated. In the left upper and lower
panels, cells were incubated with mouse pre-immune serum to set the
background fluorescence level.
[0043] FIG. 9 H1p is able to induce higher antibody titers. ELISA
assay on coated recombinant HA (H1N1California 2009). Antibody
titers were measured on sera from animals immunized with H1 and
H1p. Titers were calculated by serial dilution of the sera and
represents the dilution giving an OD value three times higher than
the background.
[0044] FIG. 10 HA (H1N1Mexico2009) pseudotyped virus infection of
MDCK cells is more potently neutralized by the serum of animals
immunized with H1p. Results of an ELISA assay on coated recombinant
HA (H1N1California 2009) with the sera of animals immunized with
H1p and NPM1H1p.
[0045] FIG. 11 H1p expressed in the context of the triple antigen
is able to induce higher antibody titers. ELISA assay on coated
recombinant HA (H1N1California 2009). Antibody titers were measured
on sera from animals immunized with H1p and NPM1H1p. Titers were
calculated by serial dilution of the sera and represent the
dilution giving an OD value three times higher than the
background.
[0046] FIG. 12 Western Blot analysis of NPM1H1p antigen expression
in transfected HeLa cells shows that the protein is fully
processed. Total lysate of HeLa cells transfected with pNEB-NPM1H1p
(Lane 1), with pNEB-NPM1 (Lane 2) and mock transfected (Lane 3).
The arrow shows the band corresponding to the fusion protein NPM1
(70 kD. A monoclonal anti-NP antibody has been used to detect the
intracellular protein.
[0047] FIG. 13 H1p derived from processing of NPM1H1p is displayed
on the cell membrane and correctly folded. Whole-cell FACS analysis
of HeLa cells mock transfected (left panel) or transfected with
NPM1H1p (right panel). Cells were incubated with the mouse mAb C179
which binds to a conformational epitope in the HA stem region and
then with a secondary anti-mouse antibody PE-conjugated.
DETAILED DESCRIPTION OF THE INVENTION
[0048] Before the present invention is described in detail below,
it is to be understood that this invention is not limited to the
particular methodology, protocols and reagents described herein as
these may vary. It is also to be understood that the terminology
used herein is for the purpose of describing particular embodiments
only, and is not intended to limit the scope of the present
invention which will be limited only by the appended claims. Unless
defined otherwise, all technical and scientific terms used herein
have the same meanings as commonly understood by one of ordinary
skill in the art.
[0049] Preferably, the terms used herein are defined as described
in "A multilingual glossary of biotechnological terms: (IUPAC
Recommendations)", Leuenberger, H. G. W, Nagel, B. and Kolbl, H.
eds. (1995), Helvetica Chimica Acta, CH-4010 Basel,
Switzerland).
[0050] Several documents are cited throughout the text of this
specification. Each of the documents cited herein (including all
patents, patent applications, scientific publications,
manufacturer's specifications, instructions, GenBank Accession
Number sequence submissions etc.), whether supra or infra, is
hereby incorporated by reference in its entirety. Nothing herein is
to be construed as an admission that the invention is not entitled
to antedate such disclosure by virtue of prior invention.
DEFINITIONS
[0051] Throughout this specification and the claims which follow,
unless the context requires otherwise, the word "comprise", and
variations such as "comprises" and "comprising", will be understood
to imply the inclusion of a stated integer or step or group of
integers or steps but not the exclusion of any other integer or
step or group of integers or steps.
[0052] The abbreviations "F" or "F0" are used interchangeably
herein and refer to the Fusion protein of paramyxoviruses,
preferably of RSV.
[0053] The abbreviation "G" refers to the Glycoprotein of
paramyxoviruses, preferably of pneumovirinae, more preferably of
RSV.
[0054] The abbreviation "H" refers to the Hemagglutinin Protein of
paramyxoviruses, preferably of morbilliviruses.
[0055] The abbreviation "HN" refers to the
Hemagglutinin-Neuraminidase Protein of paramyxoviruses,
particularly of Respirovirus, Avulavirus and Rubulavirus.
[0056] The abbreviation "N" refers to the Nucleocapsid protein of
paramyxoviruses, preferably of RSV.
[0057] The abbreviation "M" refers to the glycosylated Matrix
protein of paramyxoviruses, preferably of RSV.
[0058] With respect to paramyxoviruses, the abbreviation "M2" or
"M2-1" refers to the non-glycosylated Matrix protein of
paramyxoviruses, preferably of RSV.
[0059] The abbreviation "P" refers to the Phosphoprotein of
paramyxoviruses, preferably of RSV.
[0060] With respect to paramyxoviruses, the abbreviation "NS1" and
"NS2" refer to the non-structural proteins 1 and 2 of
paramyxoviruses, preferably of RSV.
[0061] The abbreviation "L" refers to the catalytic subunit of the
polymerase of paramyxoviruses, preferably of RSV.
[0062] The abbreviation "HA" refers to the hemagglutinin of
orthomyxovirus, preferably influenzaviruses, more preferably of
influenza A virus.
[0063] The abrevation "HA0" refers to the precursor protein of
hemagglutinin subunits HA1 and HA2 of orthomyxovirus, preferably
influenzaviruses, more preferably of influenza A virus The
abrevation "H1p" refers to the modified hemagglutinin of
orthomyxovirus, preferably influenzaviruses, more preferably of
influenza A virus.
[0064] The abrevation "NA" refers to the neuraminidase of
orthomyxovirus, preferably influenzaviruses, more preferably of
influenza A virus.
[0065] The abrevation "NP" refers to the nucleoprotein of
orthomyxoviruses, preferably influenzaviruses, more preferably of
influenza A virus.
[0066] The abrevation "M1" refers to the matrixprotein 1 of
orthomyxoviruses, preferably influenzaviruses, more preferably of
influenza A virus.
[0067] With respect to orthomyxoviruses, the abbreviation "M2"
refers to the Matrix protein M2 of orthomyxoviruses, preferably
influenzaviruses, more preferably of influenza A virus.
[0068] With respect to orthomyxovirus, the abbreviation "NS1"
refers to the non-structural protein 1 of orthomyxoviruses,
preferably influenzaviruses, more preferably of influenza A
virus.
[0069] The abbreviation "NS2/NEP" refers to the non-structural
protein 2 (also referred to as NEP, nuclear export protein) of
orthomyxoviruses, preferably influenzaviruses, more preferably
influenza A virus.
[0070] The abbreviation "PA" refers to a polymerase subunit protein
of orthomyxoviruses, preferably influenzaviruses, more preferably
influenza A virus.
[0071] The abbreviation "PB1" refers to a polymerase subunit
protein of orthomyxoviruses, preferably influenzaviruses, more
preferably influenza A virus. The abbreviation "PB2" refers to a
polymerase subunit protein of orthomyxoviruses, preferably
influenzaviruses, more preferably influenza A virus.
[0072] The abbreviation "PB1-F2" or "PB1F2" refers to a protein
encoded by an alternate reading frame in the PB1 Gene segment of
orthomyxoviruses, preferably influenzaviruses, more preferably
influenza A virus.
[0073] The term "expression system" as used herein refers to a
system designed to produce one or more gene products of interest.
Typically, such system is designed "artificially", i.e. by
gene-technological means usable to produce the gene product of
interest either in vitro in cell-free systems or in vivo in
cell-based systems. It is understood that naturally occurring
expression systems such as for instance native viruses are not
encompassed by the expression system of the present invention.
[0074] The "gene product of interest" typically refers to a
macromolecule such as but not limited to RNA, peptide, polypeptide,
or protein, or segment, epitope, or fragment thereof.
[0075] In an expression system the gene product of interest is
encoded for by one or more nucleic acid molecules. Nucleic acid
molecules are understood as a polymeric macromolecules made from
nucleotide monomers. Nucleotide monomers are composed of a
nucleobase, a five-carbon sugar (such as but not limited to ribose
or 2'-deoxyribose), and one to three phosphate groups. Typically, a
polynucleotide is formed through phosphodiester bonds between the
individual nucleotide monomers. In the context of the present
invention referred to nucleic acid molecules include but are not
limited to ribonucleic acid (RNA) and deoxyribonucleic acid (DNA).
The terms "polynucleotide" and "nucleic acid" are used
interchangeably herein.
[0076] In cell-free expression systems isolated polynucleotides are
used as template for in vitro translation reactions. In cell-based
expression systems polynucleotides are comprised on one or more
vectors. As used herein, the term "vector" refers to a protein or a
polynucleotide or a mixture thereof which is capable of being
introduced or of introducing the proteins and/or nucleic acid
comprised therein into a cell. In the context of the present
invention it is preferred that the genes of interest encoded by the
introduced polynucleotide are expressed within the cell upon
introduction of the vector or vectors. Examples of suitable vectors
include but are not limited to plasmids, cosmids, phages, viruses
or artificial chromosomes.
[0077] The phrase "induction of T cell response" refers to the
generation or the re-stimulation of virus specific CD4+ or CD8+ T
cells. The expression system of the invention can induce or
re-stimulate a T cell mediated adaptive response directed to the
MHC class I or class II epitopes present in the viral proteins
expressed by the polynucleotide. Such T cell response can be
measured by art known methods, preferably by ex-vivo re-stimulation
of T cells with synthetic peptides spanning the entire viral
proteins and analysis of proliferation or Interferon-gamma
production.
[0078] The phrase "induction of B cell response" refers to the
generation or the re-stimulation of virus specific B cells
producing immunoglobulins of class IgG or IgA. The expression
system of the invention can induce or re-stimulate B cells
producing antibodies specific for pathogenic, e.g. viral, antigens
expressed by the polynucleotide. Such B cell response can be
measured by ELISA (Enzyme Linked Immuno Stained Assay) assay with
the synthetic antigen of serum or mucosal immunoglobulin.
Alternatively the induced antibody titer can be measured by virus
neutralization assays.
[0079] The phrase "induction of an anti-pathogenic B cell response"
refers to the generation or the re-stimulation of virus specific B
cells producing immunoglobulins of class IgG or IgA which
inactivates, eliminates, blocks and/or neutralizes the respective
pathogen such that the disease caused by the pathogen does not
break out and/or the symptoms are alleviated. This is also called a
"protective immune response" against the pathogen. The expression
system of the invention can induce or re-stimulate B cells
producing antibodies specific for pathogenic, e.g. viral, antigens
expressed by the polynucleotide. Such B cell response can be
measured by ELISA (Enzyme Linked Immuno Stained Assay) assay with
the synthetic antigen of serum or mucosal immunoglobulin.
Alternatively the induced antibody titer can be measured by virus
neutralization assays.
[0080] The phrase "enhancing an immune response" refers to the
strengthening or intensification of the humoral and/or cellular
immune response against an immunogen, preferably pathogens, more
preferably viruses. The enhancement of the immune response can be
measured by comparing the immune response elicited by an expression
system of the invention with the immune response of an expression
system expressing the same antigen/immunogen alone by using tests
described herein and/or tests well known in the present technical
field.
[0081] In an expression system, a gene of interest may be encoded
by a single polynucleotide or by several separate polynucleotides.
In cell-based expression systems one or more polynucleotides may be
comprised on a single or on several separate vectors. Each of these
polynucleotides may encode the whole or a part of the gene product
of interest.
[0082] Furthermore, expression systems may encompass "expression
control sequences" that regulate the expression of the gene of
interest. Typically, expression control sequences are polypeptides
or polynucleotides such as but not limited to promoters, enhancers,
silencers, insulators, or repressors.
[0083] Accordingly, a vector comprising one or more polynucleotides
encoding for one or more gene products of interest may comprise
further expression control sequences. In a vector comprising more
than one polynucleotide encoding for one or more gene products of
interest, the expression may be controlled together or separately
by one or more expression control sequences. More specifically,
each polynucleotide comprised on the vector may be control by a
separate expression control sequence or all polynucleotides
comprised on the vector may be controlled by a single expression
control sequence. Polynucleotides comprised on a single vector
controlled by a single expression control sequences preferably form
an open reading frame.
[0084] The term "expression system" further encompasses the
expression of the gene product of interest comprising the
transcription of the polynucleotides, RNA splicing, translation
into a polypeptide, and post-translational modification of a
polypeptide or protein.
[0085] The term "open reading frame" (ORF) refers to a sequence of
nucleotides, that can be translated into amino acids. Typically,
such an ORF contains a start codon, a subsequent region usually
having a length which is a multiple of 3 nucleotides, but does not
contain a stop codon (TAG, TAA, TGA, UAG, UAA, or UGA) in the given
reading frame. Typically, ORFs occur naturally or are constructed
artificially, i.e. by gene-technological means. An ORF codes for a
protein where the amino acids into which it can be translated form
a peptide-linked chain.
[0086] The terms "protein", "polypeptide" and "peptide" are used
interchangeably herein and refer to any peptide-linked chain of
amino acids, regardless of length or post-translational
modification.
[0087] The term "post-translational" used herein refers to events
that occur after the translation of a nucleotide triplet into an
amino acid and the formation of a peptide bond to the proceeding
amino acid in the sequence. Such post-translational events may
occur after the entire polypeptide was formed or already during the
translation process on those parts of the polypeptide that have
already been translated. Post-translational events typically alter
or modify the chemical or structural properties of the resultant
polypeptide. Examples of post-translational events include but are
not limited to events such as glycosylation or phosphorylation of
amino acids, or cleavage of the peptide chain, e.g. by an
endopeptidase.
[0088] The term "co-translational" used herein refers to events
that occur during the translation process of a nucleotide triplet
into an amino acid chain. Those events typically alter or modify
the chemical or structural properties of the resultant amino acid
chain. Examples of co-translational events include but are not
limited to events that may stop the translation process entirely or
interrupted the peptide bond formation resulting in two discreet
translation products.
[0089] As used herein, the terms "polyprotein" or "artificial
polyprotein" refer to an amino acid chain that comprises, or
essentially consists of or consists of two amino acid chains that
are not naturally connected to each other. The polyprotein may
comprise one or more further amino acid chains. Each amino acid
chain is preferably a complete protein, i.e. spanning an entire
ORF, or a fragment, domain or epitope thereof. The individual parts
of a polyprotein may either be permanently or temporarily connected
to each other. Parts of a polyprotein that are permanently
connected are translated from a single ORF and are not later
separated co- or post-translationally. Parts of polyproteins that
are connected temporarily may also derive from a single ORF but are
divided co-translationally due to separation during the translation
process or post-translationally due to cleavage of the peptide
chain, e.g. by an endopeptidase. Additionally or alternatively,
parts of a polyprotein may also be derived from two different ORF
and are connected post-translationally, for instance through
covalent bonds.
[0090] Proteins or polyproteins usable in the present invention
(including protein derivatives, protein variants, protein
fragments, protein segments, protein epitops and protein domains)
can be further modified by chemical modification. This means such a
chemically modified polypeptide comprises other chemical groups
than the 20 naturally occurring amino acids. Examples of such other
chemical groups include without limitation glycosylated amino acids
and phosphorylated amino acids. Chemical modifications of a
polypeptide may provide advantageous properties as compared to the
parent polypeptide, e.g. one or more of enhanced stability,
increased biological half-life, or increased water solubility.
Chemical modifications applicable to the variants usable in the
present invention include without limitation: PEGylation,
glycosylation of non-glycosylated parent polypeptides, or the
modification of the glycosylation pattern present in the parent
polypeptide. Such chemical modifications applicable to the variants
usable in the present invention may occur co- or
post-translational.
[0091] The term "segment" refers to any part of a macromolecule
(e.g. a polypeptide, protein or polyprotein) into which this
macromolecule can be divided. A macromolecule may consist of one or
more segments. Such segmentation may exist due to functional (e.g.
having immunoreactive features or membrane attachment functions) or
structural (e.g. nucleotide or amino acid sequence, or secondary or
tertiary structure) properties of the macromolecule and/or the
individual segment. In the context of the present invention it is
preferred that the term "segment" refers to a part of a protein or
polyprotein. It is particularly preferred that such segment folds
and/or functions independently of the rest of the protein or
polyprotein.
[0092] An "epitope", also known as antigenic determinant, is the
segment of a macromolecule that is recognized by the immune system,
specifically by antibodies, B cells, or T cells. Such epitope is
that part or segment of a macromolecule capable of binding to an
antibody or antigen-binding fragment thereof. In this context, the
term "binding" preferably relates to a specific binding. In the
context of the present invention it is preferred that the term
"epitope" refers to the segment of protein or polyprotein that is
recognized by the immune system. Epitopes usually consist of
chemically active surface groupings of molecules such as amino
acids or sugar side chains and usually have specific
three-dimensional structural characteristics, as well as specific
charge characteristics. Conformational and non-conformational
epitopes are distinguished in that the binding to the former but
not the latter is lost in the presence of denaturing solvents.
[0093] As used herein, the term "domain" refers to the segment of a
protein or polyprotein sequence or structure (or corresponding
nucleotide sequence) that can evolve, function, and/or exist
independently of the rest of the protein chain. Typically, a
protein consists of one or several domains with each of them being
three-dimensional structure that are stable and folded
independently of the rest of the protein chain. Such domain
typically forms an independent functional unit within the protein
(e.g. transmembrane-domains, immunoglobulin-like domains, or
DNA-binding domains).
[0094] As used herein, the term protein or segment "variant" is to
be understood as a polypeptide (or segment) which differs in
comparison to the polypeptide (or segment, epitop, or domain) from
which it is derived by one or more changes in the amino acid
sequence. The polypeptide from which a protein variant is derived
is also known as the parent polypeptide. Likewise, the segment from
which a segment variant is derived from is known as the parent
segment. Typically, a variant is constructed artificially,
preferably by gene-technological means. Typically, the parent
polypeptide is a wild-type protein or wild-type protein domain. In
the context of the present invention it is further preferred that a
parent polypeptide (or parent segment) is the consensus sequence of
two or more wild-type polypeptides (or wild-type segments).
Further, the variants usable in the present invention may also be
derived from homologs, orthologs, or paralogs of the parent
polypeptide or from artificially constructed variant, provided that
the variant exhibits at least one biological activity of the parent
polypeptide. The changes in the amino acid sequence may be amino
acid exchanges, insertions, deletions, N-terminal truncations, or
C-terminal truncations, or any combination of these changes, which
may occur at one or several sites. In preferred embodiments, a
variant usable in the present invention exhibits a total number of
up to 200 (up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35,
40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130,
140, 150, 160, 170, 180, 190, or 200) changes in the amino acid
sequence (i.e. exchanges, insertions, deletions, N-terminal
truncations, and/or C-terminal truncations). The amino acid
exchanges may be conservative and/or non-conservative. In preferred
embodiments, a variant usable in the present invention differs from
the protein or domain from which it is derived by up to 1, 2, 3, 4,
5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70,
75, 80, 85, 90, 95, or 100 amino acid exchanges, preferably
conservative amino acid changes.
[0095] Alternatively or additionally, a "variant" as used herein,
can be characterized by a certain degree of sequence identity to
the parent polypeptide or parent polynucleotide from which it is
derived. More precisely, a protein variant in the context of the
present invention exhibits at least 80% sequence identity to its
parent polypeptide. A polynucleotide variant in the context of the
present invention exhibits at least 80% sequence identity to its
parent polynucleotide. Preferably, the sequence identity of protein
variants is over a continuous stretch of 20, 30, 40, 45, 50, 60,
70, 80, 90, 100 or more amino acids. Preferably, the sequence
identity of polynucleotide variants is over a continuous stretch of
60, 90, 120, 135, 150, 180, 210, 240, 270, 300 or more
nucleotides.
[0096] The term "at least 80% sequence identity" is used throughout
the specification with regard to polypeptide and polynucleotide
sequence comparisons. This expression preferably refers to a
sequence identity of at least 80%, at least 81%, at least 82%, at
least 83%, at least 84%, at least 85%, at least 86%, at least 87%,
at least 88%, at least 89%, at least 90%, at least 91%, at least
92%, at least 93%, at least 94%, at least 95%, at least 96%, at
least 97%, at least 98%, or at least 99% to the respective
reference polypeptide or to the respective reference
polynucleotide. Preferably, the polypeptide in question and the
reference polypeptide exhibit the indicated sequence identity over
a continuous stretch of 20, 30, 40, 45, 50, 60, 70, 80, 90, 100 or
more amino acids or over the entire length of the reference
polypeptide. Preferably, the polynucleotide in question and the
reference polynucleotide exhibit the indicated sequence identity
over a continuous stretch of 60, 90, 120, 135, 150, 180, 210, 240,
270, 300 or more nucleotides or over the entire length of the
reference polypeptide.
[0097] Variants may additionally or alternatively comprise
deletions of amino acids, which may be N-terminal truncations,
C-terminal truncations or internal deletions or any combination of
these. Such variants comprising N-terminal truncations, C-terminal
truncations and/or internal deletions are referred to as "deletion
variant" or "fragments" in the context of the present application.
The terms "deletion variant" and "fragment" are used
interchangeably herein. A fragment may be naturally occurring (e.g.
splice variants) or it may be constructed artificially, preferably
by gene-technological means. Preferably, a fragment (or deletion
variant) has a deletion of up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15,
20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or
100 amino acids at its N-terminus and/or at its C-terminus and/or
internally as compared to the parent polypeptide, preferably at its
N-terminus, at its N- and C-terminus, or at its C-terminus. In case
where two sequences are compared and the reference sequence is not
specified in comparison to which the sequence identity percentage
is to be calculated, the sequence identity is to be calculated with
reference to the longer of the two sequences to be compared, if not
specifically indicated otherwise. If the reference sequence is
indicated, the sequence identity is determined on the basis of the
full length of the reference sequence indicated by SEQ ID, if not
specifically indicated otherwise. For example, a peptide sequence
consisting of 50 amino acids compared to the amino acid sequence of
protein F according to SEQ ID NO: 1 may exhibit a maximum sequence
identity percentage of 10.04% (50/498) while a sequence with a
length of 249 amino acids may exhibit a maximum sequence identity
percentage of 50.00% (249/498).
[0098] The similarity of nucleotide and amino acid sequences, i.e.
the percentage of sequence identity, can be determined via sequence
alignments. Such alignments can be carried out with several
art-known algorithms, preferably with the mathematical algorithm of
Karlin and Altschul (Karlin & Altschul (1993) Proc. Natl. Acad.
Sci. USA 90: 5873-5877), with hmmalign (HMMER package,
http://hmmer.wustl.edu/) or with the CLUSTAL algorithm (Thompson,
J. D., Higgins, D. G. & Gibson, T. J. (1994) Nucleic Acids Res.
22, 4673-80) available e.g. on http://www.ebi.ac.uk/Tools/clustalw/
or on http://www.ebi.ac.uk/Tools/clustalw2/index.html or on
http://npsa-pbil.ibcp.fr/cgi-bin/npsa_automat.pl?page=/NPSA/npsa_clustalw-
.html. Preferred parameters used are the default parameters as they
are set on http://www.ebi.ac.uk/Tools/clustalw/ or
http://www.ebi.ac.uk/Tools/clustalw2/index.html. The grade of
sequence identity (sequence matching) may be calculated using e.g.
BLAST, BLAT or BlastZ (or BlastX). A similar algorithm is
incorporated into the BLASTN and BLASTP programs of Altschul et al.
(1990) J. Mol. Biol. 215: 403-410. BLAST polynucleotide searches
are performed with the BLASTN program, score=100, word length=12,
to obtain polynucleotide sequences that are homologous to those
nucleic acids which encode F, N, or M2-1. BLAST protein searches
are performed with the BLASTP program, score=50, word length=3, to
obtain amino acid sequences homologous to the F polypeptide, N
polypeptide, or M2-1 polypeptide. To obtain gapped alignments for
comparative purposes, Gapped BLAST is utilized as described in
Altschul et al. (1997) Nucleic Acids Res. 25: 3389-3402. When
utilizing BLAST and Gapped BLAST programs, the default parameters
of the respective programs are used. Sequence matching analysis may
be supplemented by established homology mapping techniques like
Shuffle-LAGAN (Brudno M., Bioinformatics 2003b, 19 Suppl 1:154-162)
or Markov random fields. When percentages of sequence identity are
referred to in the present application, these percentages are
calculated in relation to the full length of the longer sequence,
if not specifically indicated otherwise.
[0099] The polynucleotides of the invention encodes proteins,
peptides or variants thereof which comprise amino acids which are
designated following the standard one- or three-letter code
according to WIPO standard ST.25 unless otherwise indicated. If not
indicated otherwise, the one- or three letter code is directed at
the naturally occurring L-amino acids and the amino acid sequence
is indicated in the direction from the N-terminus to the C-terminus
of the respective protein, peptide or variant thereof.
[0100] "Hybridization" can also be used as a measure of sequence
identity or homology between two nucleic acid sequences. A nucleic
acid sequence encoding a protein of the invention, or a portion of
any of these can be used as a hybridization probe according to
standard hybridization techniques. The hybridization of a
respective probe to DNA or RNA from a test source is an indication
of the presence of the target DNA or RNA, respectively, in the test
source. Hybridization conditions are known to those skilled in the
art and can be found, for example, in Current Protocols in
Molecular Biology, John Wiley & Sons, N.Y., 6.3.1-6.3.6, 1991.
"Moderate hybridization conditions" are defined as equivalent to
hybridization in 2.times. sodium chloride/sodium citrate (SSC) at
30.degree. C., followed by a wash in 1.times.SSC, 0.1% SDS at
50.degree. C. "Highly stringent conditions" are defined as
equivalent to hybridization in 6.times. sodium chloride/sodium
citrate (SSC) at 45.degree. C., followed by a wash in
0.2.times.SSC, 0.1% SDS at 65.degree. C.
[0101] Additionally or alternatively a deletion variant may occur
not due to structural deletions of the respective amino acids as
described above, but due to these amino acids being inhibited or
otherwise not able to fulfill their biological function. Typically,
such functional deletion occurs due to the insertions to or
exchanges in the amino acid sequence that changes the functional
properties of the resultant protein, such as but not limited to
alterations in the chemical properties of the resultant protein
(i.e. exchange of hydrophobic amino acids to hydrophilic amino
acids), alterations in the post-translational modifications of the
resultant protein (e.g. post-translational cleavage or
glycosylation pattern), or alterations in the secondary or tertiary
protein structure. Additionally or alternatively, a functional
deletion may also occur due to transcriptional or
post-transcriptional gene silencing (e.g. via siRNA) or the
presence or absence of inhibitory molecules such as but not limited
to protein inhibitors or inhibitory antibodies.
[0102] In the context of the present invention it is preferred that
a protein (or a segment or a domain or an epitope) being
"functionally deleted" refers to the fact that the amino acids or
nucleotides of the corresponding sequence are either deleted or
present but not fulfilling their biological function.
[0103] As used herein, the term "consensus" refers to an amino acid
or nucleotide sequence that represents the results of a multiple
sequence alignment, wherein related sequences were compared to each
other. Such consensus sequence is composed of the amino acids or
nucleotides most commonly observed at each position. In the context
of the present invention it is preferred that the sequences used in
the sequence alignment to obtain the consensus sequence are
sequences of different viral subtypes/serotypes strains isolated in
various different disease outbreaks worldwide. Each individual
sequence used in the sequence alignment is referred to as the
sequence of a particular virus "isolate". A more detailed
description of the mathematical methods to obtain such consensus is
provided in the Example section. In case that for a given position
no "consensus nucleotide" or "consensus amino acid" can be
determined, e.g. because only two isolates were compared, than it
is preferred that the amino acid of each one of the isolates is
used. The resulting protein is assessed for its respective B cell
and/or T cell inducing ability.
[0104] A "peptide linker" (or short: "linker") in the context of
the present invention refers to an amino acid sequence of between 1
and 100 amino acids. In preferred embodiments, a peptide linker
according to the present invention has a minimum length of at least
1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,
20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 amino acids. In
further preferred embodiments, a peptide linker according to the
present invention has a maximum length of 100, 95, 90, 85, 80, 75,
70, 65, 60, 55, 50, 45, 40, 35, 34, 33, 32, 31, 30, 29, 28, 27, 26,
25, 24, 23, 22, 21, 20, 19, 18, 17, 16, or 15 amino acids or less.
It is preferred that peptide linkers provide flexibility among the
two amino acid proteins, fragments, segments, epitopes and/or
domains that are linked together. Such flexibility is generally
increased if the amino acids are small. Thus, preferably the
peptide linker of the present invention has an increased content of
small amino acids, in particular of glycins, alanines, serines,
threonines, leucines and isoleucines. Preferably, more than 20%,
30%, 40%, 50%, 60% or more of the amino acids of the peptide linker
are small amino acids. In a preferred embodiment the amino acids of
the linker are selected from glycines and serines. In especially
preferred embodiments, the above-indicated preferred minimum and
maximum lengths of the peptide linker according to the present
invention may be combined, if such a combination makes
mathematically sense. In further preferred embodiments, the peptide
linker of the present invention is non-immunogenic; in particularly
preferred embodiments, the peptide linker is non-immunogenic to
humans.
[0105] The term "cleavage site" as used herein refers to an amino
acid sequence or nucleotide sequence where this sequence directs
the division, e.g. because it is recognized by a cleaving enzyme,
and/or can be divided. Typically, a polypeptide chain is cleaved by
hydrolysis of one or more peptide bonds that link the amino acids
and a polynucleotide chain is cleaved by hydrolysis of one or more
of the phosphodiester bond between the nucleotides. Cleavage of
peptide- or phosphodiester-bonds may originate from chemical or
enzymatic cleavage. Enzymatic cleavage refers to such cleavage
being attained by proteolytic enzymes including but not limited to
restriction endonuclease (e.g. type I, type II, type II, type IV or
artificial restriction enzymes) and endo- or exo-peptidases or
-proteases (e.g. serine-proteases, cysteine-proteases,
metallo-proteases, threonine proteases, aspartate proteases,
glutamic acid proteases). Typically, enzymatic cleavage occurs due
to self-cleavage or is effected by an independent proteolytic
enzyme. Enzymatic cleavage of a protein or polypeptide can happen
either co- or post-translational. Accordingly, the term
"endopeptidase cleavage site" used herein, refers to a cleavage
cite within the amino acid or nucleotide sequence where this
sequence is cleaved or is cleavable by an endopeptidase (e.g.
trypsin, pepsin, elastase, thrombin, collagenase, furin,
thermolysin, endopeptidase V8, cathepsins). Alternatively or
additionally, the polyprotein of the present invention can be
cleaved by an autoprotease, i.e. a protease which cleaves peptide
bonds in the same protein molecule which also comprises the
protease. Examples of such autoproteases are the NS2 protease from
flaviviruses or the VP4 protease of birnaviruses.
[0106] Alternatively, the term "cleavage site" refers to an amino
acid sequence or nucleotide sequence that prevents the formation of
peptide- or phosphodiester-bonds between amino acids or
nucleotides, respectively. For instance, the bond formation may be
prevented due to co-translational self-processing of the
polypeptide or polyprotein resulting in two discontinuous
translation products being derived from a single translation event
of a single open reading frame. Typically, such self-processing is
effected by a "ribosomal skip" caused by a pseudo stop-codon
sequence that induces the translation complex to move from one
codon to the next without forming a peptide bond. Examples of
sequences inducing a ribosomal skip include but are not limited to
viral 2A peptides or 2A-like peptide (herein both are collectively
referred to as "2A peptide" or interchangeably as "2A site" or "2A
cleavage site") which are used by several families of viruses,
including Picornavirus, insect viruses, Aphtoviridae, Rotaviruses
and Trypanosoma. Best known are 2A sites of rhinovirus and
foot-and-mouth disease virus of the Picornaviridae family which are
typically used for producing multiple polypeptides from a single
ORF.
[0107] Accordingly, the term "self-cleavage site" as used herein
refers to a cleavage site within the amino acid or nucleotide
sequence where this sequence is cleaved or is cleavable without
such cleavage involving any additional molecule or where the
peptide- or phosphodiester-bond formation in this sequence is
prevented in the first place (e.g. through co-translational
self-processing as described above).
[0108] It is understood that cleavage sites typically comprise
several amino acids or are encoded by several codons (e.g. in those
cases, wherein the "cleavage site" is not translated into protein
but leads to an interruption of translation). Thus, the cleavage
site may also serve the purpose of a peptide linker, i.e.
sterically separates two peptides. Thus, in some embodiments a
"cleavage site" is both a peptide linker and provides above
described cleavage function. In this embodiment the cleavage site
may encompass additional N- and/or C-terminal amino acids.
[0109] The term "host cell" as used herein refers to a cell that
harbours a vector (e.g. a plasmid or virus). Such host cell may
either be a prokaryotic (e.g. a bacterial cell) or a eukaryotic
cell (e.g. a fungal, plant or animal cell).
[0110] "Pharmaceutically acceptable" means approved by a regulatory
agency of the Federal or a state government or listed in the U.S.
Pharmacopeia or other generally recognized pharmacopeia for use in
animals, and more particularly in humans.
[0111] The term "carrier", as used herein, refers to a
pharmacologically inactive substance such as but not limited to a
diluent, excipient, or vehicle with which the therapeutically
active ingredient is administered. Such pharmaceutical carriers can
be liquid or solid. Liquid carrier include but are not limited to
sterile liquids, such as saline solutions in water and oils,
including those of petroleum, animal, vegetable or synthetic
origin, such as peanut oil, soybean oil, mineral oil, sesame oil
and the like. Saline solutions and aqueous dextrose and glycerol
solutions can also be employed as liquid carriers, particularly for
injectable solutions. A saline solution is a preferred carrier when
the pharmaceutical composition is administered intravenously or
intranasally by a nebulizer.
[0112] Suitable pharmaceutical excipients include starch, glucose,
lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel,
sodium stearate, glycerol monostearate, talc, sodium chloride,
dried skim milk, glycerol, propylene, glycol, water, ethanol and
the like.
[0113] Examples of suitable pharmaceutical carriers are described
in "Remington's Pharmaceutical Sciences" by E. W. Martin.
[0114] The term "composition" is intended to include the
formulation of the active compound with encapsulating material as a
carrier providing a capsule in which the active component with or
without other carriers, is surrounded by a carrier, which is thus,
in association with it.
[0115] The term "adjuvant" refers to agents that augment,
stimulate, activate, potentiate, or modulate the immune response to
the active ingredient of the composition at either the cellular or
humoral level, e.g. immunologic adjuvants stimulate the response of
the immune system to the actual antigen, but have no immunological
effect themselves. Examples of such adjuvants include but are not
limited to inorganic adjuvants (e.g. inorganic metal salts such as
aluminium phosphate or aluminium hydroxide), organic adjuvants
(e.g. saponins or squalene), oil-based adjuvants (e.g. Freund's
complete adjuvant and Freund's incomplete adjuvant), cytokines
(e.g. IL-1.beta., IL-2, IL-7, IL-12, IL-18, GM-CFS, and
INF-.gamma.) particulate adjuvants (e.g. immuno-stimulatory
complexes (ISCOMS), liposomes, or biodegradable microspheres),
virosomes, bacterial adjuvants (e.g. monophosphoryl lipid A, or
muramyl peptides), synthetic adjuvants (e.g. non-ionic block
copolymers, muramyl peptide analogues, or synthetic lipid A), or
synthetic polynucleotides adjuvants (e.g polyarginine or
polylysine).
[0116] The term "active ingredient" refers to the substance in a
pharmaceutical composition or formulation that is biologically
active, i.e. that provides pharmaceutical value. A pharmaceutical
composition may comprise one or more active ingredients which may
act in conjunction with or independently of each other.
[0117] The active ingredient can be formulated as neutral or salt
forms. Pharmaceutically acceptable salts include those formed with
free amino groups such as those derived from hydrochloric,
phosphoric, acetic, oxalic, tartaric acids, etc., and those formed
with free carboxyl groups such as but not limited to those derived
from sodium, potassium, ammonium, calcium, ferric hydroxides,
isopropylamine, triethylamine, 2-ethylamino ethanol, histidine,
procaine, and the like.
[0118] As used herein, a "patient" means any mammal, reptile or
bird that may benefit from a treatment with a tumour vaccine
described herein. Preferably, a "patient" is selected from the
group consisting of laboratory animals (e.g. mouse or rat),
domestic animals (including e.g. guinea pig, rabbit, horse, donkey,
cow, sheep, goat, pig, chicken, camel, cat, dog, turtle, tortoise,
snake, or lizard), or primates including chimpanzees, bonobos,
gorillas and human beings. It is particularly preferred that the
"patient" is a human being.
[0119] As used herein, "treat", "treating" or "treatment" of a
disease or disorder means accomplishing one or more of the
following: (a) reducing the severity of the disorder; (b) limiting
or preventing development of symptoms characteristic of the
disorder(s) being treated; (c) inhibiting worsening of symptoms
characteristic of the disorder(s) being treated; (d) limiting or
preventing recurrence of the disorder(s) in patients that have
previously had the disorder(s); and (e) limiting or preventing
recurrence of symptoms in patients that were previously symptomatic
for the disorder(s).
[0120] As used herein, "prevent", "preventing", "prevention", or
"prophylaxis" of a disease or disorder means preventing that such
disease or disorder occurs in patient.
[0121] As used herein, "administering" includes in vivo
administration, as well as administration directly to tissue ex
vivo, such as vein grafts.
[0122] An "effective amount" is an amount of a therapeutic agent
sufficient to achieve the intended purpose. The effective amount of
a given therapeutic agent will vary with factors such as the nature
of the agent, the route of administration, the size and species of
the animal to receive the therapeutic agent, and the purpose of the
administration. The effective amount in each individual case may be
determined empirically by a skilled artisan according to
established methods in the art.
Embodiments of the Invention
[0123] The present invention will now be further described. In the
following passages different aspects of the invention are defined
in more detail. Each aspect so defined may be combined with any
other aspect or aspects unless clearly indicated to the contrary.
In particular, any feature indicated as being preferred or
advantageous may be combined with any other feature or features
indicated as being preferred or advantageous.
[0124] In a first aspect the invention provides an expression
system comprising a first polynucleotide encoding at least one
protein, peptide or variant thereof, which induces a T cell
response and a second polynucleotide encoding at least one protein,
which induces an anti-pathogenic B cell response. One of the
advantages provided by the present invention is the fact that the B
cell response to the protein, peptide or variant thereof inducing
an antipathogenic B cell response can be enhanced, if at the same
time a protein inducing a T cell response is administered.
[0125] In the context of the present invention the term "expression
system" preferably refers to one or more polynucleotide sequences
comprising in addition to the first and second polynucleotide the
elements to direct transcription and translation of the proteins
encoded by the first and second or any further polynucleotide,
which may be included in the preferred embodiments outlined below.
Such elements included promoter and enhancer elements to direct
transcription of mRNA in a cell-free or a cell-based based system,
preferably a cell-based system. In another embodiment, wherein the
polynucleotides are provided as translatable RNAs is envisioned
that the expression system comprises those elements that are
necessary for translation and/or stabilization of RNAs encoding the
T cell and B cell inducing protein, e.g. polyA-tail, IRES, cap
structures etc.
[0126] According to a preferred embodiment of the first aspect, the
first polynucleotide encodes a protein which induces a reaction of
the immune system (i.e. immune response) in a host which is
mediated by T cells. A T cell response involves the activation of
antigen-specific T lymphocyte such as but not limited to cytotoxic
T cells (CTLs), T helper cells (T.sub.H cells), central memory T
cells (TCM cells), effector memory T cells (TEM cells), and
regulatory T cells (Treg cells). A T cell response against a
protein is induced, if peptides of the protein are processed within
the cell and presented to T cells on the surface of the cell via
the MHC I or MHC II pathway. Thus, in the context of the present
invention preferably those proteins or parts thereof are used for
inducing a T cell response that are normally not exposed to, e.g.
non structural or internal proteins or parts of structural or
internal proteins of a virus not accessible to B-cells.
[0127] The second polynucleotide encodes a protein, peptide or
variant thereof that induces an anti-pathogenic B cell response. A
B cell response is an immune response based on the activation of B
lymphocytes, which produce and secrete antigen specific antibodies.
B cells involved in such immune response include but are not
limited to plasma B cells, memory B cells and B-1 cells. Thus, in
the context of the present invention preferably those pathogenic,
e.g. viral, proteins or parts thereof are used for inducing a B
cell response that are exposed on the outside of the virus, e.g.
structural proteins or at least those parts of structural proteins
accessible to B-cells on the outside of the pathogen (virus).
[0128] In embodiments of the first aspect of the present invention,
the first and the second polynucleotide are comprised on separate
vectors or on the same vector. Accordingly, the first
polynucleotide may be comprised on one vector and the second
polynucleotide may be comprised on a second vector. Alternatively
or additionally, the first and the second polynucleotide may be
comprised on the same vector. It is preferred that the first and
the second polynucleotide are comprised on the same vector. It is
particularly preferred that the first and the second polynucleotide
comprised on the same vector are linked in such that they are
expressed as a polyprotein. Preferably, the first and the second
polynucleotide form an open reading frame.
[0129] It is preferred that the first and the second polynucleotide
are expressed as an artificial polyprotein. In the context of the
present invention the term "artificial polyprotein" is directed at
polyproteins which are not naturally occurring, e.g. which are
generated by using recombinant DNA techniques. Accordingly, the
proteins, peptides or variants thereof encoded in this artificial
polyprotein are preferably derived from pathogens which genome do
not encode a polyprotein comprising the proteins, peptides or
variants encoded by the first and second polynucleotide (and,
optionally, the third polynucleotide) of the invention. Preferably,
the first and second polynucleotides are derived from viruses,
encoding no polyprotein or a polyprotein wherein the respective
polynucleotides have a different order and/or sequence. More
preferably, the first and second polynucleotide are derived from a
virus which is selected from the group consisting of a DNA virus, a
negative sense single stranded (ssRNA(-)) RNA virus or an ambisense
RNA virus. Further preferred, the virus is selected from
negative-single stranded (ssRNA(-)) RNA virus. Even more preferred,
the virus is selected from enveloped ssRNA(-) viruses, more
preferably from the group consisting of paramyxoviruses and
orthomyxoviruses.
[0130] In preferred embodiments of the first aspect the protein,
which induces a T cell response is a non-structural and/or internal
protein of a virus, and/or the protein, which induces an
anti-pathogenic B cell response is a structural and/or surface
protein of a pathogen, preferably a virus, wherein the virus is
preferably selected from the group consisting of a DNA virus, a
negative-strand RNA virus or an ambisense RNA virus. Even more
preferred, the virus is selected from negative-single stranded
(ssRNA(-)) RNA virus. Even more preferred, the virus is selected
from enveloped ssRNA(-) viruses, more preferably from the group
consisting of paramyxoviruses and orthomyxoviruses.
[0131] It is preferred that the amino acid sequence of the
structural (surface) and/or non-structural (internal) protein
comprises consecutive segments or a consensus sequence of one or
more different virus isolates.
[0132] In the context of the present invention it is preferred that
the term "segment" refers to a part of a protein or polyprotein. It
is particularly preferred that such segment folds and/or functions
independently of the rest of the protein or polyprotein such as but
not limited to a domain, an epitope or a fragment thereof. It is
understood that a protein variant in the context of the present
invention differs in comparison to its parent polypeptide in
changes in the amino acid sequence such as amino acid exchanges,
insertions, deletions, N-terminal truncations, or C-terminal
truncations, or any combination of these changes, which may occur
at one or several sites whereby the variant exhibits at least 80%
sequence identity to its parent polypeptide.
[0133] In preferred embodiments, the structural protein, peptide or
a variant thereof is a protein or peptide exposed on the surface of
the native pathogen, e.g. a virus. It is preferred that the
structural and/or surface protein triggers a T-cell independent
immune response such as but not limited to an antibody mediated
immune response or an activation of the complement system. In a
particularly preferred embodiment, the structural and/or surface
protein induces an antibody mediated immune response. Such antibody
mediated immune response is based on the activation of B cells
which produce and secrete antigen specific antibodies. B cells
involved in such immune response include but are not limited to
plasma B cells, memory B cells and B-1 cells.
[0134] According to a preferred embodiment of the first aspect, the
second polynucleotide encodes a protein or variant thereof that
induces an anti-pathogenic B cell response. A B cell response is an
immune response based on the activation of B lymphocytes, which
produce and secrete antigen specific antibodies. B cells involved
in such immune response include but are not limited to plasma B
cells, memory B cells and B-1 cells. Thus, in the context of the
present invention preferably those proteins or parts thereof are
used for inducing a B cell response that are exposed on the outside
of a virus, e.g. structural and/or surface proteins or at least
those parts of structural and/or surface proteins accessible to
B-cells on the outside of a virus. An anti-pathogenic B cell
response is a B cell response directed against a pathogen which
inactivates, eliminates, blocks and/or neutralizes the respective
pathogen such that the disease caused by the pathogen does not
break out and/or the symptoms are alleviated. In preferred
embodiments of the invention, the anti-apthogenic B cell response
is effected by antibodies that bind to the surface of a pathogenic
organism and attract the first component of the complement cascade
with their Fc region and initiate activation of the "classical"
complement system. This results in pathogen elimination by two
mechanisms. First, the binding of the antibody and complement
molecules marks the pathogen for ingestion by phagocytes in a
process called opsonization. Secondly, some complement system
components form a membrane attack complex to assist antibodies to
destroy the pathogen directly. Alternatively, the anti-apthogenic B
cell response is effected by antibodies that bind to the pathogen's
structural proteins blocking the attachment to cellular receptors.
In this way, the antibody can neutralize the infection. As a
further alternative, the anti-apthogenic B cell response is
effected by antibodies that bind at a specialized region of the
pathogen's surface protein, the fusion peptide, which is necessary
for the entry of the pathogen into the host cell. The antibody
binding results in fixing the protein in a pre-fusion state and
blocking infection. The ability of a protein or variant to induce B
cell response which is anti-pathogenic can be determined by the
skilled person by applying tests and/or assays well known in the
art.
[0135] In a further preferred embodiment, a membrane attachment
domain of the protein exposed on the surface of the native virus or
variant thereof is functionally deleted, thus, either being
structurally deleted or structurally present but not fulfilling its
biological function. In a particularly preferred embodiment, the
amino acid sequence corresponding to the membrane attachment domain
is deleted. The deletion of the membrane attachment region serves
the purpose of ascertaining that the anti-pathogenic B cell
response inducing protein is secreted from the cell into which the
expression system of the invention has been introduced.
[0136] In a further preferred embodiment the anti-pathogenic B cell
response inducing protein comprises a secretion signal, which
targets the protein to the endoplasmatic reticulum (ER). Such
secretion signals are present preferably in the context of a
deleted membrane attachment domain. The skilled person is well
aware of various such secretion signals, which may be used as
heterologous secretion signals, e.g. added to the N-terminus of the
anti-pathogenic B cell response inducing protein. Alternatively or
additionally a naturally occurring secretion signal may be used,
which is, e.g., present in the majority of structural and/or
surface viral proteins. Thus, if naturally present in the
respective protein it is preferred that the secretion signal is
maintained in a modified version of the structural and/or surface
protein.
[0137] In embodiments of the first aspect, the non-structural
protein is a conserved internal protein suitable for inducing a T
cell mediated immune response against the pathogen, preferably the
viruses, involving the activation of antigen-specific T lymphocyte
such as but not limited to cytotoxic T cells (CTLs), T helper cells
(T.sub.H cells), central memory T cells (TCM cells), effector
memory T cells (TEM cells), and regulatory T cells (Treg cells).
Thus, preferably the T cell inducing protein of the pathogen
(virus) does not comprise a secretion signal.
[0138] In the context of the present invention, the protein,
peptide or variant thereof encoded by the first polynucleotide is
located either N- or C-terminally with respect to the protein,
peptide or variant thereof encoded by the second polynucleotide. In
a preferred embodiment, the protein, peptide or variant thereof
encoded by the second polynucleotide is located C-terminally with
respect to the protein, peptide or variant thereof encoded by the
first polynucleotide.
[0139] Accordingly, embodiments of the present invention have the
formula X-Y or Y-X, wherein "X" depicts the T cell response
inducing protein and "Y" depicts the anti-pathogenic B cell
response inducing protein and a "dash" depicts a peptide bond.
[0140] In preferred embodiments of the first aspect, a
polynucleotide encoding a cleavage site is positioned between the
first polynucleotide and the second polynucleotide. It is within
the scope of the present invention that every protein can be
combined with any other protein and that any two proteins can or
cannot be connected or linked by a cleavage site.
[0141] It is preferred that this cleavage site is either a
self-cleaving site (i.e. a cleavage site within the amino acid
sequence where this sequence is cleaved or is cleavable without
such cleavage involving any additional molecule or where the
peptide-bond formation in this sequence is prevented in the first
place) or an endopeptidase cleavage site (i.e. a cleavage cite
within the amino acid sequence where this sequence is cleaved or is
cleavable by an endopeptidase, e.g. trypsin, pepsin, elastase,
thrombin, collagenase, furin, thermolysin, endopeptidase V8,
cathepsins). More preferably, the self-cleaving site is a 2A
cleavage site selected from the group consisting of a viral 2A
peptide or 2A-like peptide of Picornavirus, insect viruses,
Aphtoviridae, Rotaviruses and Trypanosoma, preferably wherein the
2A cleavage site is the 2 A peptide of foot and mouth disease
virus. Alternatively or additionally, the polyprotein of the
present invention can be cleaved by an autoprotease, i.e. a
protease which cleaves peptide bonds in the same protein molecule
which also comprises the protease. Examples of such autoproteases
are the NS2 protease from flaviviruses or the VP4 protease of
birnaviruses
[0142] In the context of the present invention, the cleavage site
can be positioned N-terminally with respect to the protein, peptide
or variant thereof encoded by the first polynucleotide and
C-terminally with respect to the protein, peptide or variant
thereof encoded by the second polynucleotide. Alternatively the
cleavage site can be positioned C-terminally with respect to the
protein, peptide or variant thereof encoded by the first
polynucleotide and N-terminally with respect to the protein,
peptide or variant thereof encoded by the second
polynucleotide.
[0143] Accordingly, embodiments of the present invention have the
formula X-C-Y or Y-C-X, wherein X" depicts the T cell response
inducing protein and "Y" depicts the anti-pathogenic B cell
response inducing protein, "C" depicts a cleavage site, and a
"dash" depicts a peptide bond.
[0144] In a preferred embodiment of the first aspect, the
expression system further comprises a third polynucleotide encoding
a protein, peptide or a variant thereof of a pathogen.
[0145] It is preferred that the protein, peptide or variant thereof
encoded by the third polynucleotide is a protein, peptide or
variant thereof inducing a T cell response, preferably the third
polynucleotide is a protein, peptide or variant thereof which is a
non-structural or internal protein, peptide or variant thereof
inducing a T cell response.
[0146] It is preferred that the protein, peptide or variant thereof
encoded by the third polynucleotide differs from the protein,
peptide or variant thereof encoded by the first polynucleotide or
the second polynucleotide. Preferably, the proteins, peptides or
variants thereof encoded by the first, second and the third
polynucleotide differ from each other in that they comprise amino
acid sequences of different proteins.
[0147] In preferred embodiments a polynucleotide encoding a linker
is positioned between the second polynucleotide and the third
polynucleotide. It is preferred that the linker is a flexible
linker, preferably a flexible linker comprising an amino acid
sequence according to SEQ ID NO: 6
(Gly-Gly-Gly-Ser-Gly-Gly-Gly).
[0148] In preferred embodiments the third polynucleotide is
comprised on a separate or on the same vector as the first
polynucleotide and/or the second polynucleotide.
[0149] Accordingly, the first polynucleotide is comprised on one
vector and the second polynucleotide is comprised on a second
vector and the third polynucleotide is comprised on a third vector.
Alternatively or additionally, the first and the second
polynucleotide are comprised on the same vector and the third
polynucleotide is comprised on a separate vector, or the first and
the third polynucleotide are comprised on the same vector and the
second polynucleotide is comprised on a separate vector, or the
second and the third polynucleotide are comprised on the same
vector and the first polynucleotide is comprised on a separate
vector. Alternatively or additionally, the first and the second and
the third polynucleotide are comprised on the same vector. It is
preferred that the first and the second and the third
polynucleotide may be comprised on the same vector. It is
particularly preferred that the first and the second and the third
polynucleotide comprised on the same vector are linked in such that
they are expressed as a polyprotein. Preferably, the first and the
second and the third polynucleotide comprised on the same vector
form an open reading frame and, preferably, are expressed as a
polyprotein.
[0150] In preferred embodiments of this aspect, the protein encoded
by the second polynucleotide is located N-terminally with respect
to the protein encoded by the first polynucleotide and/or the
protein of the optional third polynucleotide, or the protein
encoded by the second polynucleotide is located C-terminally with
respect to the protein encoded by the first polynucleotide and/or
the protein of the optional third polynucleotide.
[0151] In even more preferred embodiments of this aspect, the first
polynucleotide is located N-terminally with respect to the protein
encoded by the second polynucleotide and/or the protein of the
optional third polynucleotide is located N-terminally with respect
to the protein encoded by the first polynucleotide or is located
between the protein encoded by the second polynucleotide and the
protein encoded by the first polynucleotide; or the protein encoded
by the first polynucleotide is located C-terminally with respect to
the protein encoded by the second polynucleotide and/or the protein
of the optional third polynucleotide is located C-terminally with
respect to the protein encoded by the first polynucleotide or is
located between the protein encoded by the second polynucleotide
and the protein encoded by the first polynucleotide.
[0152] Preferred embodiments of the present invention have the
formula X-K-Y, Y-K-X, X-K-Y-Y, Y-Y-K-X, X-Y-K-Y, Y-K-Y-X,
X-K-Y-K-Y, Y-K-Y-K-X, X-C-Y, Y-C-X, X-C-Y-Y, Y-Y-C-X, X-Y-C-Y,
Y-C-Y-X, X-C-Y-C-Y, Y-C-Y-C-X, X-K-Y-C-Y, Y-C-Y-K-X, X-C-Y-K-Y, or
Y-K-Y-C-X, wherein "X" depicts the second polynucleotide encoding
at least one protein, peptide or variant thereof, which induces an
antipathogenic B cell response and "Y" depicts the first
polynucleotide encoding at least one protein, peptide or variant
thereof, which induces an T cell response, "K" indicates that one
or more peptide linkers are present in this position, "C" indicates
that one or more cleavage sites are present in this position and a
"dash" depicts a peptide bond. Preferred arrangements are Y-K-Y-C-X
or X-C-Y-K-Y.
[0153] It is further preferred that the non-structural and/or
internal protein encoded by the third polynucleotide is a conserved
internal protein suitable for inducing a T cell mediated immune
response against the virus involving the activation of
antigen-specific T lymphocyte such as but not limited to cytotoxic
T cells (CTLs), T helper cells (T.sub.H cells), central memory T
cells (TCM cells), effector memory T cells (TEM cells), and
regulatory T cells (Treg cells).
[0154] It is within the scope of the present invention that every
protein can be combined with any other protein and that any two
proteins can or cannot be connected or linked by either a cleavage
site or a linker peptide.
[0155] In preferred embodiments, the vector or vectors comprising
the first, and the second and/or the third polynucleotide is/are
selected from the group consisting of plasmid, cosmid, phage,
virus, and artificial chromosome. More preferably, a vector
suitable for practicing the present invention is selected from the
group consisting of plasmid vectors, cosmid vectors, phage vectors,
preferably lambda phage and filamentous phage vectors, viral
vectors, adenovirus vectors (e.g., non-replicating Ad5, Ad11, Ad26,
Ad35, Ad49, ChAd3, ChAd4, ChAd5, ChAd6, ChAd7, ChAd8, ChAd9,
ChAd10, ChAd11, ChAd16, ChAd17, ChAd19, ChAd20, ChAd22, ChAd24,
ChAd26, ChAd30, ChAd31, ChAd37, ChAd38, ChAd44, ChAd55, ChAd63,
ChAd 73, ChAd82, ChAd83, ChAd146, ChAd147, PanAd1, PanAd2, and
PanAd3 vectors or replication-competent Ad4 and Ad7 vectors),
adeno-associated virus (AAV) vectors (e.g., AAV type 5 and type 2),
alphavirus vectors (e.g., Venezuelan equine encephalitis virus
(VEE), sindbis virus (SIN), semliki forest virus (SFV), and VEE-SIN
chimeras), herpes virus vectors (e.g. vectors derived from
cytomegaloviruses, like rhesus cytomegalovirus (RhCMV) (14)), arena
virus vectors (e.g. lymphocytic choriomeningitis virus (LCMV)
vectors (15)), measles virus vectors, pox virus vectors (e.g.,
vaccinia virus, modified vaccinia virus Ankara (MVA), NYVAC
(derived from the Copenhagen strain of vaccinia), and avipox
vectors: canarypox (ALVAC) and fowlpox (FPV) vectors), vesicular
stomatitis virus vectors, retrovirus, lentivirus, viral like
particles, and bacterial spores. The vectors ChAd3, ChAd4, ChAd5,
ChAd6, ChAd7, ChAd8, ChAd9, ChAd10, ChAd 11, ChAd16, ChAd17,
ChAd19, ChAd20, ChAd22, ChAd24, ChAd26, ChAd30, ChAd31, ChAd37,
ChAd38, ChAd44, ChAd63 and ChAd82 are described in detail in WO
2005/071093. The vectors PanAd1, PanAd2, PanAd3, ChAd55, ChAd73,
ChAd83, ChAd146, and ChAd147 are described in detail in WO
2010/086189. It is particularly preferred that the vector is
selected from the group consisting of MVA, ChAd63 and PanAd3.
[0156] In preferred embodiments, the expression system is for use
in medicine. In more preferred embodiments, the expression system
is for use in the prophylaxis or treatment of an infection and/or
in the manufacturing of medicament for use in the prophylaxis or
treatment of an infection and/or for use in methods of prophylaxis
or treatment of an infection, wherein the infection is preferably a
viral infection, particularly preferably for use in the prophylaxis
or treatment of a pathogen and/or in the manufacturing of
medicament for use in the prophylaxis or treatment of a pathogen
and/or for use in methods of prophylaxis or treatment of a
pathogen, wherein the pathogen preferably is a virus. Preferably,
the expression system is for use in the prophylaxis or treatment of
an infection by a virus and/or in the manufacturing of medicament
for use in the prophylaxis or treatment of a virus and/or for use
in methods of prophylaxis or treatment of a virus, wherein the
pathogen selected from the group of a DNA virus, a negative-single
stranded (ssRNA(-)) RNA virus or an ambisense RNA virus. Further
preferred the expression system is for use in the prophylaxis or
treatment and/or in the manufacturing of medicament for use in the
prophylaxis or treatment of a virus and/or for use in methods of
prophylaxis or treatment of an infection by negative sense
single-stranded (ssRNA(-)) RNA virus. Even more preferred, the
expression system is for use in the prophylaxis or treatment and/or
in the manufacturing of medicament for use in the prophylaxis or
treatment of a virus and/or for use in methods of prophylaxis or
treatment of an infection by a virus selected from enveloped
ssRNA(-) viruses, more preferably from the group consisting of
paramyxoviruses and orthomyxoviruses.
[0157] In preferred embodiments the expression system is for use in
enhancing an immune response. In more preferred embodiments, the
expression system is for use in enhancing a B cell immune response
against an immunogen, preferably a pathogen, more preferably a
virus as defined above.
[0158] According to a preferred embodiment of the first aspect, the
first polynucleotide encodes a viral protein of a paramyxovirus or
variant thereof which induces a reaction of the immune system (i.e.
immune response) in a host which is mediated by T cells, and the
second polynucleotide encodes a viral protein of a paramyxovirus or
variant thereof that induces an anti-pathogenic B cell response
against paramyxoviruses. It is preferred that the paramyxovirus
whose viral proteins are encoded for by the first and second
polynucleotide is selected from the subfamily of Pneumovirinae,
Paramyxovirinae, Fer-de-Lance-Virus, Nariva-Virus, Salem-Virus,
Tupaia-Paramyxovirus, Beilong-Virus, J-Virus, Menangle-Virus,
Mossmann-Virus, and Murayama-Virus. In even more preferred
embodiments, the Pneumovirinae is selected from the group
consisting of Pneumovirus, preferably human respiratory syncytial
virus (RSV), murine pneumonia virus, bovine RSV, ovine RSV, caprine
RSV, turkey rinotracheitis virus, and Metapneumovirus, preferably
human metapneumovirus (hMPV) and avian metapneumovirus. In even
more preferred embodiments, the Paramyxovirinae is selected from
the group consisting of Respirovirus, preferably human
parainfluenza virus 1 and 3, and Rubulavirus, preferably human
parainfluenza virus 2 and 4.
[0159] In embodiments of the first aspect of the present invention,
the first and the second polynucleotide are comprised on separate
vectors or on the same vector. Accordingly, the first
polynucleotide may be comprised on one vector and the second
polynucleotide may be comprised on a second vector. Alternatively
or additionally, the first and the second polynucleotide may be
comprised on the same vector. It is preferred that the first and
the second polynucleotide are comprised on the same vector. It is
particularly preferred that the first and the second polynucleotide
comprised on the same vector are linked in such that they are
expressed as a polyprotein. Preferably, the first and the second
polynucleotide form an open reading frame.
[0160] According to preferred embodiments of the first aspect the
first polynucleotide encodes a viral protein of a paramyxovirus or
variant thereof which induces a reaction of the immune system (i.e.
immune response) in a host which is mediated by T cells. A T cell
response involves the activation of antigen-specific T lymphocyte
such as but not limited to cytotoxic T cells (CTLs), T helper cells
(T.sub.H cells), central memory T cells (TCM cells), effector
memory T cells (TEM cells), and regulatory T cells (Treg cells). A
T cell response against a protein is induced, if peptides of the
protein are processed within the cell and presented to T cells on
the surface of the cell via the MHC I or MHC II pathway. Thus, in
the context of the present invention preferably those viral
proteins or parts thereof are used for inducing a T cell response
that are normally not exposed on the outside of the virus, e.g. non
structural or internal proteins or parts of structural or surface
proteins not accessible to B-cells on the outside of the virus.
[0161] The second polynucleotide encodes a viral protein of a
paramyxovirus or variant thereof that induces an anti-pathogenic B
cell response against the paramyxovirus. A B cell response is an
immune response based on the activation of B lymphocytes, which
produce and secrete antigen specific antibodies. B cells involved
in such immune response include but are not limited to plasma B
cells, memory B cells and B-1 cells. Thus, in the context of the
present invention preferably those viral proteins or parts thereof
are used for inducing an anti-pathogenic B cell response that are
exposed on the outside of the virus, e.g. structural proteins or at
least those parts of structural proteins accessible to B-cells on
the outside of the virus.
[0162] According to a preferred embodiment of the first aspect, the
second polynucleotide encodes a viral protein of a paramyxovirus or
variant thereof that induces an anti-pathogenic B cell response. A
B cell response is an immune response based on the activation of B
lymphocytes, which produce and secrete antigen specific antibodies.
B cells involved in such immune response include but are not
limited to plasma B cells, memory B cells and B-1 cells. Thus, in
the context of the present invention preferably those viral
proteins or parts thereof are used for inducing an anti-pathogenic
B cell response that are exposed on the outside of the virus, e.g.
structural and/or surface proteins or at least those parts of
structural and/or surface proteins accessible to B-cells on the
outside of the virus.
[0163] In preferred embodiments of the first aspect the viral
protein of a paramyxovirus, which induces a T cell response is a
non-structural and/or internal protein of a paramyxovirus, and/or
the viral protein of a paramyxovirus, which induces an
anti-pathogenic B cell response is a structural and/or surface
protein of a paramyxovirus.
[0164] It is preferred that the amino acid sequence of the
structural (surface) and/or non-structural (internal) protein
comprises consecutive segments or a consensus sequence of one or
more different paramyxovirus isolates.
[0165] In preferred embodiments, the structural protein is a
protein exposed on the surface of the native paramyxovirus or a
variant thereof. It is preferred that the structural protein
triggers a T-cell independent immune response such as but not
limited to an antibody mediated immune response or an activation of
the complement system. In a particularly preferred embodiment, the
structural and/or surface protein induces an antibody mediated
immune response. Such antibody mediated immune response is based on
the activation of B cells which produce and secrete antigen
specific antibodies. B cells involved in such immune response
include but are not limited to plasma B cells, memory B cells and
B-1 cells.
[0166] In a further preferred embodiment, the membrane attachment
domain of the protein exposed on the surface of the native
paramyxovirus or variant thereof is functionally deleted, thus,
either being structurally deleted or structurally present but not
fulfilling its biological function. In a particularly preferred
embodiment, the amino acid sequence corresponding to the membrane
attachment domain is deleted. The deletion of the membrane
attachment region serves the purpose of ascertaining that the
anti-pathogenic B cell response inducing protein is secreted from
the cell into which the expression system of the invention has been
introduced.
[0167] In a further preferred embodiment the anti-pathogenic B cell
response inducing paramyxovirus protein comprises a secretion
signal, which targets the protein to the endoplasmatic reticulum
(ER). Thus, if naturally present in the respective structural or
surface protein it is preferred that the secretion signal is
maintained in a modified version of the structural or surface
protein.
[0168] It is further preferred that the structural and/or surface
protein of the native paramyxovirus is selected from the group
consisting of fusion protein (F) and any of the attachment
glycoproteins G, H, and HN.
[0169] The attachment glycoproteins are found in all enveloped
viruses and mediate the initial interaction between the viral
envelope and the plasma membrane of the host cell via their binding
to carbohydrate moieties or cell adhesion domains of proteins or
other molecules on the plasma membrane of the host cell. Thereby,
attachment glycoproteins bridge the gap between the virus and the
membrane of the host cell. Attachment glycoproteins designated as
"H" possess hemagglutinin activity and are found in morbilliviruses
and henipaviruses, glycoproteins designated as "FIN possess
hemagglutinin and neuraminidase activities and are found in
respiroviruses, rubulaviruses and avulaviruses. Attachment
glycoproteins are designated as "G" when they have neither
haemagglutination nor neuraminidase activity. G attachment
glycoproteins can be found in all members of Pneumovirinae.
[0170] Fusion protein "F" is found in all enveloped viruses and
mediates the fusion of the viral envelope with the plasma membrane
of the host cell. F is a type I glycoprotein that recognizes
receptors present on the cell surface of the host cell to which it
binds. F consists of a fusion peptide adjacent to which the
transmembrane domains are located, followed by two heptad repeat
(HR) regions, HR1 and HR2, respectively. Upon insertion of the
fusion peptide into the plasma membrane of the host cell, the HR1
region forms a trimeric coiled coil structure into whose
hydrophobic grooves the HR2 regions folds back. Thereby, a hairpin
structure is formed that draws the viral lipid bilayer and cellular
plasma membrane even closer together and allows for the formation
of a fusion pore and consecutively the complete fusion of both
lipid bilayers enabling the virus capsid to enter into the
cytoplasm of the host cell. All of these features are common in
fusion-mediating proteins of enveloped viruses.
[0171] In a preferred embodiment of the first aspect, F comprises,
essentially consists of or consists of an amino acid sequence of F
of one RSV isolate or a consensus amino acid sequence of two or
more different RSV isolates, preferably according to SEQ ID NO: 1,
more preferably according to SEQ ID NO: 2 or a variant thereof.
[0172] In preferred embodiments of the first aspect, the
non-structural protein is a conserved internal protein of
paramyxoviruses suitable for inducing a T cell mediated immune
response against the paramyxovirus, involving the activation of
antigen-specific T lymphocyte such as but not limited to cytotoxic
T cells (CTLs), T helper cells (T.sub.H cells), central memory T
cells (TCM cells), effector memory T cells (TEM cells), and
regulatory T cells (Treg cells). Thus, preferably the T cell
inducing protein of the paramyxovirus does not comprise a secretion
signal.
[0173] Preferably, the non-structural and/or internal protein is
selected from the group consisting of nucleoprotein N, Matrix
proteins M and M2, Phosphoprotein P, non structural proteins NS1
and NS2, and the catalytic subunit of the polymerase (L).
[0174] The nucleoprotein N serves several functions which include
the encapsidation of the RNA genome into a RNAase-resistant
nucleocapsid. N also interacts with the M protein during virus
assembly and interacts with the P-L polymerase during transcription
and replication of the genome.
[0175] The matrix protein M is the most abundant protein in
paramyxovirus and is considered to be the central organizer of
viral morphology by interacting with the cytoplasmatic tail of the
integral membrane proteins and the nucleocapsid. M2 is a second
membrane-associated protein that is not glycosylated and is mainly
found in pneumovirus.
[0176] Phosphoprotein P binds to the N and L proteins and forms
part of the RNA polymerase complex in all paramyxoviruses. Large
protein L is the catalytic subunit of RNA-dependent RNA
polymerase.
[0177] The function of non-structural proteins NS1 and NS2 has not
yet been identified; however, there are indications that they are
involved in the viral replication cycle.
[0178] In preferred embodiments, N comprises an amino acid sequence
of N, of one RSV isolate or a consensus amino acid sequence of two
or more different RSV isolates, preferably according to SEQ ID NO:
3 and wherein M2 comprises an amino acid sequence of M2 of one RSV
isolate or a consensus amino acid sequence of two or more different
RSV isolates, preferably according to SEQ ID NO: 5. It is further
preferred that wherein N comprises the amino acid sequence
according to SEQ ID NO: 4 and M2 comprises the amino acid sequence
according to SEQ ID NO: 5.
[0179] In the context of the present invention, the structural
and/or surface protein encoded by the first polynucleotide is
located either N- or C-terminally with respect to the
non-structural and/or internal protein encoded by the second
polynucleotide. In a preferred embodiment, the non-structural
and/or internal protein encoded by the second polynucleotide is
located C-terminally with respect to the structural and/or surface
protein encoded by the first polynucleotide.
[0180] More specifically, N, M, M2, P, NS1, NS2, or L can be
located N- or C-terminally of F, G, H, or HN. Preferably, N, M, M2,
P, NS1, NS2, or L are located C-terminally of F, G, H, or HN. In a
more preferred embodiment N or M2 are located C-terminally of F. In
a particularly preferred embodiment N is located C-terminally of
F.
[0181] Accordingly, embodiments of the present invention have the
formula X-Y or Y-X, wherein "X" depicts F, G, H, or HN and "Y"
depicts N, M, M2, P, NS1, NS2, or L and a "dash" depicts a peptide
bond. Preferred arrangements are the following:
[0182] F-N, G-N, H-N, HN-N, F-M, G-M, H-M, HN-M, F-M2, G-M2, H-M2,
HN-M2, F-P, G-P, H-P, HN-P, F-NS1, G-NS1, H-NS1, HN-NS1, F-NS2,
G-NS2, H-NS2, HN-NS2, F-L, G-L, H-L, HN-L, N-F, N-G, N-H, N-HN,
M-F, M-G, M-H, M-HN, M2-F, M2-G, M2-H, M2-HN, P-F, P-G, P-H, P-HN,
NS1-FF, NS1-G, NS1-H, NS1-HN, NS2-F, NS2-G, NS2-H, NS2-HN, L-F,
L-G, L-H, or L-HN.
[0183] It is within the scope of the present invention that every
protein can be combined with any other protein.
[0184] In preferred embodiments of the first aspect, a
polynucleotide encoding a cleavage site is positioned between the
first polynucleotide and the second polynucleotide.
[0185] It is preferred that this cleavage site is either a
self-cleaving site (i.e. a cleavage site within the amino acid
sequence where this sequence is cleaved or is cleavable without
such cleavage involving any additional molecule or where the
peptide-bond formation in this sequence is prevented in the first
place) or an endopeptidase cleavage site (i.e. a cleavage cite
within the amino acid sequence where this sequence is cleaved or is
cleavable by an endopeptidase, e.g. trypsin, pepsin, elastase,
thrombin, collagenase, furin, thermolysin, endopeptidase V8,
cathepsins). More preferably, the self-cleaving site is a 2A
cleavage site selected from the group consisting of a viral 2A
peptide or 2A-like peptide of Picornavirus, insect viruses,
Aphtoviridae, Rotaviruses and Trypanosoma, preferably wherein the
2A cleavage site is the 2 A peptide of foot and mouth disease
virus.
[0186] In the context of the present invention, the cleavage site
can be positioned N-terminally with respect to the structural
and/or surface protein encoded by the first polynucleotide and
C-terminally with respect to the non-structural and/or internal
protein encoded by the second polynucleotide. Alternatively the
cleavage site can be positioned C-terminally with respect to the
structural and/or surface protein encoded by the first
polynucleotide and N-terminally with respect to the non-structural
and/or internal protein encoded by the second polynucleotide. More
specifically, the cleavage site can be positioned C- or
N-terminally with respect to F, G, H, or HN and C- or N-terminally
with respect to N, M, M2, P, NS1, NS2, or L. In a preferred
embodiment the cleavage site is located N-terminally with respect
to N, M, M2, P, NS1, NS2, or L and C-terminally with respect to F,
G, H, or HN. It is particularly preferred that the cleavage site is
located N-terminally with respect to N and C-terminally with
respect to F.
[0187] Accordingly, embodiments of the present invention have the
formula X-C-Y or Y-C-X, wherein "X" depicts F, G, H, or HN and "Y"
depicts N, M, M2, P, NS1, NS2, or L, "C" depicts a cleavage site,
and a "dash" depicts a peptide bond. Preferred arrangements are the
following:
[0188] F-C-N, G-C-N, H-C-N, HN-C-N, F-C-M, G-C-M, H-C-M, HN-C-M,
F-C-M2, G-C-M2, H-C-M2, HN-C-M2, F-C-P, G-C-P, H-C-P, HN-C-P,
F-C-NS1, G-C-NS1, H-C-NS1, HN-C-NS1, F-C-NS2, G-C-NS2, H-C-NS2,
HN-C-NS2, F-C-L, G-C-L, H-C-L, HN-C-L, N-C-F, N-C-G, N-C-H, N-C-HN,
M-C-F, M-C-G, M-C-H, M-C-HN, M2-C-F, M2-C-G, M2-C-H, M2-C-HN,
P-C-F, P-C-G, P-C-H, P-C-HN, NS1-C-FF, NS1-C-G, NS1-C-H, NS1-C-HN,
NS2-C-F, NS2-C-G, NS2-C-H, NS2-C-HN, L-C-F, L-C-G, L-C-H, or
L-C-HN. Particularly, preferred is F-C-N.
[0189] It is within the scope of the present invention that every
protein can be combined with any other protein and that any two
proteins can or cannot be connected or linked by a cleavage
site.
[0190] In preferred embodiment of the first aspect, the expression
system further comprises a third polynucleotide encoding a
non-structural and/or internal protein of a paramyxovirus or a
variant thereof. Preferably, the non-structural and/or internal
protein is of a paramyxovirus selected from the group consisting of
Pneumovirinae, Paramyxovirinae, Fer-de-Lance-Virus, Nariva-Virus,
Salem-Virus, Tupaia-Paramyxovirus, Beilong-Virus, J-Virus,
Menangle-Virus, Mossmann-Virus, and Murayama-Virus, more
preferably, the Pneumovirinae is selected from the group consisting
of Pneumovirus, preferably human respiratory syncytical virus
(RSV), murine pneumonia virus, bovine RSV, ovine RSV, caprine RSV,
turkey rinotracheitis and Metapneumovirus, preferably human
metapneumovirus, avian metapneumovirus, more preferably, the
Paramyxovirinae is selected from the group consisting of
Respirovirus, preferably human parainfluenza virus 1 and 3, and
Rubulavirus, preferably human parainfluenza virus 2 and 4.
[0191] In preferred embodiments the third polynucleotide is
comprised on a separate or on the same vector as the first
polynucleotide and/or the second polynucleotide.
[0192] Accordingly, the first polynucleotide is comprised on one
vector and the second polynucleotide is comprised on a second
vector and the third polynucleotide is comprised on a third vector.
Alternatively or additionally, the first and the second
polynucleotide are comprised on the same vector and the third
polynucleotide is comprised on a separate vector, or the first and
the third polynucleotide are comprised on the same vector and the
second polynucleotide is comprised on a separate vector, or the
second and the third polynucleotide are comprised on the same
vector and the first polynucleotide is comprised on a separate
vector. Alternatively or additionally, the first and the second and
the third polynucleotide are comprised on the same vector. It is
preferred that the first and the second and the third
polynucleotide may be comprised on the same vector. It is
particularly preferred that the first and the second and the third
polynucleotide comprised on the same vector are linked in such that
they are expressed as a viral polyprotein. Preferably, the first
and the second and the third polynucleotide comprised on the same
vector form an open reading frame.
[0193] It is further preferred that the non-structural and/or
internal protein encoded by the third polynucleotide is a conserved
internal protein suitable for inducing a T cell mediated immune
response against the virus involving the activation of
antigen-specific T lymphocyte such as but not limited to cytotoxic
T cells (CTLs), T helper cells (T.sub.H cells), central memory T
cells (TCM cells), effector memory T cells (TEM cells), and
regulatory T cells (Treg cells).
[0194] Preferably, the non-structural and/or internal protein is
selected from the group consisting of nucleoprotein N, Matrix
proteins M and M2, Phosphoprotein P, non structural proteins NS1
and NS2, and the catalytic subunit of the polymerase (L).
[0195] In preferred embodiments, N comprises an amino acid sequence
of N, of one RSV isolate or a consensus amino acid sequence of two
or more different RSV isolates, preferably according to SEQ ID NO:
3 and wherein M2 comprises an amino acid sequence of M2 of one RSV
isolate or a consensus amino acid sequence of two or more different
RSV isolates, preferably according to SEQ ID NO: 5. It is further
preferred that wherein N comprises the amino acid sequence
according to SEQ ID NO: 4 and M2 comprises the amino acid sequence
according to SEQ ID NO: 5.
[0196] It is preferred that the non-structural and/or internal
protein encoded by the third polynucleotide differs from the
non-structural and/or internal protein encoded by the second
polynucleotide.
[0197] The non-structural and/or internal proteins encoded by the
second and the third polynucleotide differ from each other in that
they comprise amino acid sequences of different viral proteins. For
instance, this means that the non-structural and/or internal
protein encoded by the second polynucleotide comprises the amino
acid sequence of the N protein whilst the non-structural and/or
internal protein encoded by the second polynucleotide comprises the
amino acid sequence of the M2 protein or vice versa.
[0198] The non-structural and/or internal protein encoded by the
third polynucleotide can be located either N- or C-terminally of
the non-structural and/or internal protein encoded by the second
polynucleotide. In a preferred embodiment of the first aspect, the
non-structural and/or internal protein encoded by the third
polynucleotide is located C-terminally of the non-structural and/or
internal protein encoded by the second polynucleotide. More
specifically, N or M2 can be located N- or C-terminally of N or M2,
preferably N is located C-terminally of M2.
[0199] In preferred embodiments a polynucleotide encoding a linker
is positioned between the second polynucleotide and the third
polynucleotide. It is preferred that the linker is a flexible
linker, preferably a flexible linker comprising an amino acid
sequence according to SEQ ID NO: 6.
[0200] In embodiments of the first aspect, the protein encoded by
the second polynucleotide is located N-terminally with respect to
the protein encoded by the first polynucleotide and/or the protein
of the optional third polynucleotide, or the protein encoded by the
second polynucleotide is located C-terminally with respect to the
protein encoded by the first polynucleotide and/or the protein of
the optional third polynucleotide.
[0201] In even more preferred embodiments of this aspect, the first
polynucleotide is located N-terminally with respect to the protein
encoded by the second polynucleotide and/or the protein of the
optional third polynucleotide is located N-terminally with respect
to the protein encoded by the first polynucleotide or is located
between the protein encoded by the second polynucleotide and the
protein encoded by the first polynucleotide; or the protein encoded
by the first polynucleotide is located C-terminally with respect to
the protein encoded by the second polynucleotide and/or the protein
of the optional third polynucleotide is located C-terminally with
respect to the protein encoded by the first polynucleotide or is
located between the protein encoded by the second polynucleotide
and the protein encoded by the first polynucleotide. More
specifically, F, G, H, or HN are located C- or N-terminally with
respect to N, M, M2, P, NS1, NS2, or L and N, M, M2, P, NS1, NS2,
or L are located C- or N-terminally with respect to N or M2. In a
preferred embodiment F is located N-terminally with respect to N
and M2 is located C-terminally with respect to N.
[0202] Accordingly, preferred embodiments of the present invention
have the formula X-K-Y, Y-K-X, X-K-Y-Y, Y-Y-K-X, X-Y-K-Y, Y-K-Y-X,
X-K-Y-K-Y, Y-K-Y-K-X, X-C-Y, Y-C-X, X-C-Y-Y, Y-Y-C-X, X-Y-C-Y,
Y-C-Y-X, X-C-Y-C-Y, Y-C-Y-C-X, X-K-Y-C-Y, Y-C-Y-K-X, X-C-Y-K-Y, or
Y-K-Y-C-X, wherein "X" depicts F, G, H, or HN and "Y" depicts N, M,
M2, P, NS1, NS2, or L, "K" indicates that one or more peptide
linkers are present in this position, "C" indicates that one or
more cleavage sites are present in this position and a "dash"
depicts a peptide bond. Preferred arrangements are X-C-Y-K-Y. Even
more preferred arrangements are the following:
F-K-N, G-K-N, H-K-N, HN-K-N, F-K-M, G-K-M, H-K-M, HN-K-M, F-K-M2,
G-K-M2, H-K-M2, HN-K-M2, F-K-P, G-K-P, H-K-P, HN-K-P, F-K-NS1,
G-K-NS1, H-K-NS1, HN-K-NS1, F-K-NS2, G-K-NS2, H-K-NS2, HN-K-NS2,
F-K-L, G-K-L, H-K-L, HN-K-L, N-K-F, N-K-G, N-K-H, N-K-HN, M-K-F,
M-K-G, M-K-H, M-K-HN, M2-K-F, M2-K-G, M2-K-H, M2-K-HN, P-K-F,
P-K-G, P-K-H, P-K-HN, NS1-K-FF, NS1-K-G, NS1-K-H, NS1-K-HN,
NS2-K-F, NS2-K-G, NS2-K-H, NS2-K-HN, L-K-F, L-K-G, L-K-H, L-K-HN,
F-C-N, G-C-N, H-C-N, HN-C-N, F-C-M, G-C-M, H-C-M, HN-C-M, F-C-M2,
G-C-M2, H-C-M2, HN-C-M2, F-C-P, G-C-P, H-C-P, HN-C-P, F-C-NS1,
G-C-NS1, H-C-NS1, HN-C-NS1, F-C-NS2, G-C-NS2, H-C-NS2, HN-C-NS2,
F-C-L, G-C-L, H-C-L, HN-C-L, N-C-F, N-C-G, N-C-H, N-C-HN, M-C-F,
M-C-G, M-C-H, M-C-HN, M2-C-F, M2-C-G, M2-C-H, M2-C-HN, P-C-F,
P-C-G, P-C-H, P-C-HN, NS1-C-FF, NS1-C-G, NS1-C-H, NS1-C-HN,
NS2-C-F, NS2-C-G, NS2-C-H, NS2-C-HN, L-C-F, L-C-G, L-C-H, or
L-C-HN, F-N-M, G-N-M, H-N-M, HN-N-M, F-N-M2, G-N-M2, H-N-M2,
HN-N-M2, F-N-P, G-N-P, H-N-P, HN-N-P, F-N-NS1, G-N-NS1, H-N-NS1,
HN-N-NS1, F-N-NS2, G-N-NS2, H-N-NS2, HN-N-NS2, F-N-L, G-N-L, H-N-L,
HN-N-L, F-M-N, G-M-N, H-M-N, HN-M-N, F-M-P, G-M-P, H-M-P, HN-M-P,
F-M-NS1, G-M-NS1, H-M-NS1, HN-M-NS1, F-M-NS2, G-M-NS2, H-M-NS2,
HN-M-NS2, F-M-L, G-M-L, H-M-L, HN-M-L, F-M2-N, G-M2-N, H-M2-N,
HN-M2-N, F-M2-P, G-M2-P, H-M2-P, HN-M2-P, F-M2-NS1, G-M2-NS1,
H-M2-NS1, HN-M2-NS1, F-M2-NS2, G-M2-NS2, H-M2-NS2, HN-M2-NS2,
F-M2-L, G-M2-L, H-M2-L, HN-M2-L, F-P-N, G-P-N, H-P-N, HN-P-N,
F-P-M, G-P-M, H-P-M, HN-P-M, F-P-M2, G-P-M2, H-P-M2, HN-P-M2,
F-P-NS1, G-P-NS1, H-P-NS1, HN-P-NS1, F-P-NS2, G-P-NS2, H-P-NS2,
HN-P-NS2, F-P-L, G-P-L, H-P-L, HN-P-L, F-NS1-N, G-NS1-N, H-NS1-N,
HN-NS1-N, F-NS1-M, G-NS1-M, H-NS1-M, HN-NS1-M, F-NS1-M2, G-NS1-M2,
H-NS1-M2, HN-NS1-M2, F-NS1-P, G-NS1-P, H-NS1-P, HN-NS1-P,
F-NS1-NS2, G-NS1-NS2, H-NS1-NS2, HN-NS1-NS2, F-NS1-L, G-NS1-L,
H-NS1-L, HN-NS1-L, F-NS2-N, G-NS2-N, H-NS2-N, HN-NS2-N, F-NS2-M,
G-NS2-M, H-NS2-M, HN-NS2-M, F-NS2-M2, G-NS2-M2, H-NS2-M2,
HN-NS2-M2, F-NS2-P, G-NS2-P, H-NS2-P, HN-NS2-P, F-NS2-NS1,
G-NS2-NS1, H-NS2-NS1, HN-NS2-NS1, F-NS2-L, G-NS2-L, H-NS2-L,
HN-NS2-L, F-L-N, G-L-N, H-L-N, HN-L-N, F-L-M2, G-L-M2, H-L-M2,
HN-L-M2, F-L-P, G-L-P, H-L-P, HN-L-P, F-L-NS1, G-L-NS1, H-L-NS1,
HN-L-NS1, F-L-NS2, G-L-NS2, H-L-NS2, HN-L-NS2, M-N-F, M-N-G, M-N-H,
M-N-HN, M2-N-F, M2-N-G, M2-N-H, M2-N-HN, P-N-F, P-N-G, P-N-H,
P-N-HN, NS1-N-F, NS1-N-G, NS1-N-H, NS1-N-HN, NS2-N-F, NS2-N-G,
NS2-N-H, NS2-N-HN, L-N-F, L-N-G, L-N-H, L-N-HN, N-M-F, N-M-G,
N-M-H, N-M-HN, P-M-F, P-M-G, P-M-H, P-M-HN, NS1-M-F, NS1-M-G,
NS1-M-H, NS1-M-HN, NS2-M-F, NS2-M-G, NS2-M-H, NS2-M-HN, L-M-F,
L-M-G, L-M-H, L-M-HN, N-M2-F, N-M2-G, N-M2-H, N-M2-HN, P-M2-F,
P-M2-G, P-M2-H, P-M2-HN, NS1-M2-F, NS1-M2-G, NS1-M2-H, NS1-M2-HN,
NS2-M2-F, NS2-M2-G, NS2-M2-H, NS2-M2-HN, L-M2-F, L-M2-G, L-M2-H,
L-M2-HN, N-P-F, N-P-G, N-P-H, N-P-HN, M-P-F, M-P-G, M-P-H, M-P-HN,
M2-P-F, M2-P-G, M2-P-H, M2-P-HN, NS1-P-F, NS1-P-G, NS1-P-H,
NS1-P-HN, NS2-P-F, NS2-P-G, NS2-P-H, NS2-P-HN, L-P-F, L-P-G, L-P-H,
L-P-HN, N-NS1-F, N-NS1-G, N-NS1-H, N-NS1-HN, M-NS1-F, M-NS1-G,
M-NS1-H, M-NS1-HN, M2-NS1-F, M2-NS1-G, M2-NS1-H, M2-NS1-HN,
P-NS1-F, P-NS1-G, P-NS1-H, P-NS1-HN, NS2-NS1-F, NS2-NS1-G,
NS2-NS1-H, NS2-NS1-HN, L-NS1-F, L-NS1-G, L-NS1-H, L-NS1-HN,
N-NS2-F, N-NS2-G, N-NS2-H, N-NS2-HN, M-NS2-F, M-NS2-G, M-NS2-H,
M-NS2-HN, M2-NS2-F, M2-NS2-G, M2-NS2-H, M2-NS2-HN, P-NS2-F,
P-NS2-G, P-NS2-H, P-NS2-HN, NS1-NS2-F, NS1-NS2-G, NS1-NS2-H,
NS1-NS2-HN, L-NS2-F, L-NS2-G, L-NS2-H, L-NS2-HN, N-L-F, N-L-G,
N-L-H, N-L-HN, M-L-F, M-L-G, M-L-H, M-L-HN, M2-L-F, M2-L-G, M2-L-H,
M2-L-HN, P-L-F, P-L-G, P-L-H, P-L-HN, NS1-L-F, NS1-L-G, NS1-L-H,
NS1-L-HN, NS2-L-F, NS2-L-G, NS2-L-H, NS2-L-HN, F-K-N-N, G-K-N-N,
H-K-N-N, HN-K-N-N, F-K-N-M, G-K-N-M, H-K-N-M, HN-K-N-M, F-K-N-M2,
G-K-N-M2, H-K-N-M2, HN-K-N-M2, F-K-N-P, G-K-N-P, H-K-N-P, HN-K-N-P,
F-K-N-NS1, G-K-N-NS1, H-K-N-NS1, HN-K-N-NS1, F-K-N-NS2, G-K-N-NS2,
H-K-N-NS2, HN-K-N-NS2, F-K-N-L, G-K-N-L, H-K-N-L, HN-K-N-L,
F-C-N-N, G-C-N-N, H-C-N-N, HN-C-N-N, F-C-N-M, G-C-N-M, H-C-N-M,
HN-C-N-M, F-C-N-M2, G-C-N-M2, H-C-N-M2, HN-C-N-M2, F-C-N-P,
G-C-N-P, H-C-N-P, HN-C-N-P, F-C-N-NS1, G-C-N-NS1, H-C-N-NS1,
HN-C-N-NS1, F-C-N-NS2, G-C-N-NS2, H-C-N-NS2, HN-C-N-NS2, F-C-N-L,
G-C-N-L, H-C-N-L, HN-C-N-L, F-K-M-M, G-K-M-M, H-K-M-M, HN-K-M-M,
F-K-M-M2, G-K-M-M2, H-K-M-M2, HN-K-M-M2, F-K-M-N, G-K-M-N, H-K-M-N,
HN-K-M-N, F-K-M-P, G-K-M-P, H-K-M-P, HN-K-M-P, F-K-M-NS1,
G-K-M-NS1, H-K-M-NS1, HN-K-M-NS1, F-K-M-NS2, G-K-M-NS2, H-K-M-NS2,
HN-K-M-NS2, F-K-M-L, G-K-M-L, H-K-M-L, HN-K-M-L, F-K-M2-M,
G-K-M2-M, H-K-M2-M, HN-K-M2-M, F-K-M2-N, G-K-M2-N, H-K-M2-N,
HN-K-M2-N, F-K-M2-P, G-K-M2-P, H-K-M2-P, HN-K-M2-P, F-K-M2-NS1,
G-K-M2-NS1, H-K-M2-NS1, HN-K-M2-NS1, F-K-M2-NS2, G-K-M2-NS2,
H-K-M2-NS2, HN-K-M2-NS2, F-K-M2-L, G-K-M2-L, H-K-M2-L, HN-K-M2-L,
F-K-P-N, G-K-P-N, H-K-P-N, HN-K-P-N, F-K-P-M, G-K-P-M, H-K-P-M,
HN-K-P-M, F-K-P-M2, G-K-P-M2, H-K-P-M2, HN-K-P-M2, F-K-P-P,
G-K-P-P, H-K-P-P, HN-K-P-P, F-K-P-NS1, G-K-P-NS1, H-K-P-NS1,
HN-K-P-NS1, F-K-P-NS2, G-K-P-NS2, H-K-P-NS2, HN-K-P-NS2, F-K-P-L,
G-K-P-L, H-K-P-L, HN-K-P-L, F-K-NS1-N, G-K-NS1-N, H-K-NS1-N,
HN-K-NS1-N, F-K-NS1-M, G-K-NS1-M, H-K-NS1-M, HN-K-NS1-M,
F-K-NS1-M2, G-K-NS1-M2, H-K-NS1-M2, HN-K-NS1-M2, F-K-NS1-P,
G-K-NS1-P, H-K-NS1-P, HN-K-NS1-P, F-K-NS1-NS1, G-K-NS1-NS1,
H-K-NS1-NS1, HN-K-NS1-NS1, F-K-NS1-NS2, G-K-NS1-NS2, H-K-NS1-NS2,
HN-K-NS1-NS2, F-K-NS1-L, G-K-NS1-L, H-K-NS1-L, HN-K-NS1-L,
F-K-NS2-N, G-K-NS2-N, H-K-NS2-N, HN-K-NS2-N, F-K-NS2-M, G-K-NS2-M,
H-K-NS2-M, HN-K-NS2-M, F-K-NS2-M2, G-K-NS2-M2, H-K-NS2-M2,
HN-K-NS2-M2, F-K-NS2-P, G-K-NS2-P, H-K-NS2-P, HN-K-NS2-P,
F-K-NS2-NS1, G-K-NS2-NS1, H-K-NS2-NS1, HN-K-NS2-NS1, F-K-NS2-NS2,
G-K-NS2-NS2, H-K-NS2-NS2, HN-K-NS2-NS2, F-K-NS2-L, G-K-NS2-L,
H-K-NS2-L, HN-K-NS2-L, F-K-L-N, G-K-L-N, H-K-L-N, HN-K-L-N,
F-K-L-M, G-K-L-M, H-K-L-M, HN-K-L-M, F-K-L-M2, G-K-L-M2, H-K-L-M2,
HN-K-L-M2, F-K-L-P, G-K-L-P, H-K-L-P, HN-K-L-P, F-K-L-NS1,
G-K-L-NS1, H-K-L-NS1, HN-K-L-NS1, F-K-L-NS2, G-K-L-NS2, H-K-L-NS2,
HN-K-L-NS2, F-K-L-L, G-K-L-L, H-K-L-L, HN-K-L-L, F-N-K-N, G-N-K-N,
H-N-K-N, HN-N-K-N, F-N-K-M, G-N-K-M, H-N-K-M, HN-N-K-M, F-N-K-M2,
G-N-K-M2, H-N-K-M2, HN-N-K-M2, F-N-K-P, G-N-K-P, H-N-K-P, HN-N-K-P,
F-N-K-NS1, G-N-K-NS1, H-N-K-NS1, HN-N-K-NS1, F-N-K-NS2, G-N-K-NS2,
H-N-K-NS2, HN-N-K-NS2, F-N-K-L, G-N-K-L, H-N-K-L, HN-N-K-L,
F-M-K-N, G-M-K-N, H-M-K-N, HN-M-K-N, F-M-K-M, G-M-K-M, H-M-K-M,
HN-M-K-M, F-M-K-M2, G-M-K-M2, H-M-K-M2, HN-M-K-M2, F-M-K-P,
G-M-K-P, H-M-K-P HN-M-K-P, F-M-K-NS1, G-M-K-NS1, H-M-K-NS1,
HN-M-K-NS1, F-M-K-NS2, G-M-K-NS2, H-M-K-NS2, HN-M-K-NS2, F-M-K-L,
G-M-K-L, H-M-K-L, HN-M-K-L, F-M2-K-N, G-M2-K-N, H-M2-K-N,
HN-M2-K-N, F-M2-K-M, G-M2-K-M, H-M2-K-M, HN-M2-K-M, F-M2-K-M2,
G-M2-K-M2, H-M2-K-M2, HN-M2-K-M2, F-M2-K-P, G-M2-K-P, H-M2-K-P
HN-M2-K-P, F-M2-K-NS1, G-M2-K-NS1, H-M2-K-NS1, HN-M2-K-NS1,
F-M2-K-NS2, G-M2-K-NS2, H-M2-K-NS2, HN-M2-K-NS2, F-M2-K-L,
G-M2-K-L, H-M2-K-L, HN-M2-K-L, F-P-K-N, G-P-K-N, H-P-K-N, HN-P-K-N,
F-P-K-M, G-P-K-M, H-P-K-M, HN-P-K-M, F-P-K-M2, G-P-K-M2, H-P-K-M2;
HN-P-K-M2, F-P-K-P, G-P-K-P, H-P-K-P, HN-P-K-P, F-P-K-NS1,
G-P-K-NS1, H-P-K-NS1, HN-P-K-NS1, F-P-K-NS2, G-P-K-NS2, H-P-K-NS2,
HN-P-K-NS2, F-P-K-L, G-P-K-L, H-P-K-L, HN-P-K-L, F-NS1-K-N,
G-NS1-K-N, H-NS1-K-N, HN-NS1-K-N, F-NS1-K-M, G-NS1-K-M, H-NS1-K-M;
HN-NS1-K-M, F-NS1-K-M2, G-NS1-K-M2, H-NS1-K-M2; HN-NS1-K-M2,
F-NS1-K-P, G-NS1-K-P, H-NS1-K-P, HN-NS1-K-P, F-NS1-K-NS1,
G-NS1-K-NS1, H-NS1-K-NS1, HN-NS1-K-NS1, F-NS1-K-NS2, G-NS1-K-NS2,
H-NS1-K-NS2, HN-NS1-K-NS2, F-NS1-K-L, G-NS1-K-L, H-NS1-K-L,
HN-NS1-K-L, F-NS2-K-N, G-NS2-K-N, H-NS2-K-N, HN-NS2-K-N, F-NS2-K-M,
G-NS2-K-M, H-NS2-K-M, HN-NS2-K-M, F-NS2-K-M2, G-NS2-K-M2,
H-NS2-K-M2, HN-NS2-K-M2, F-NS2-K-P, G-NS2-K-P, H-NS2-K-P,
HN-NS2-K-P, F-NS2-K-NS1, G-NS2-K-NS1, H-NS2-K-NS1, HN-NS2-K-NS1,
F-NS2-K-NS2, G-NS2-K-NS2, H-NS2-K-NS2, HN-NS2-K-NS2, F-NS2-K-L,
G-NS2-K-L, H-NS2-K-L, HN-NS2-K-L, F-C-M-M, G-C-M-M, H-C-M-M,
HN-C-M-M, F-C-M-M2, G-C-M-M2, H-C-M-M2, HN-C-M-M2, F-C-M-N,
G-C-M-N, H-C-M-N, HN-C-M-N, F-C-M-P, G-C-M-P, H-C-M-P, HN-C-M-P,
F-C-M-NS1, G-C-M-NS1, H-C-M-NS1, HN-C-M-NS1, F-C-M-NS2, G-C-M-NS2,
H-C-M-NS2, HN-C-M-NS2, F-C-M-L, G-C-M-L, H-C-M-L, HN-C-M-L,
F-C-M2-M, G-C-M2-M, H-C-M2-M, HN-C-M2-M, F-C-M2-N, G-C-M2-N,
H-C-M2-N, HN-C-M2-N, F-C-M2-P, G-C-M2-P, H-C-M2-P, HN-C-M2-P,
F-C-M2-NS1, G-C-M2-NS1, H-C-M2-NS1, HN-C-M2-NS1, F-C-M2-NS2,
G-C-M2-NS2, H-C-M2-NS2, HN-C-M2-NS2, F-C-M2-L, G-C-M2-L, H-C-M2-L,
HN-C-M2-L, F-C-P-N, G-C-P-N, H-C-P-N, HN-C-P-N, F-C-P-M, G-C-P-M,
H-C-P-M, HN-C-P-M, F-C-P-M2, G-C-P-M2, H-C-P-M2, HN-C-P-M2,
F-C-P-P, G-C-P-P, H-C-P-P, HN-C-P-P, F-C-P-NS1, G-C-P-NS1,
H-C-P-NS1, HN-C-P-NS1, F-C-P-NS2, G-C-P-NS2, H-C-P-NS2, HN-C-P-NS2,
F-C-P-L, G-C-P-L, H-C-P-L, HN-C-P-L, F-C-NS1-N, G-C-NS1-N,
H-C-NS1-N, HN-C-NS1-N, F-C-NS1-M, G-C-NS1-M, H-C-NS1-M, HN-C-NS1-M,
F-C-NS1-M2, G-C-NS1-M2, H-C-NS1-M2, HN-C-NS1-M2, F-C-NS1-P,
G-C-NS1-P, H-C-NS1-P, HN-C-NS1-P, F-C-NS1-NS1, G-C-NS1-NS1,
H-C-NS1-NS1, HN-C-NS1-NS1, F-C-NS1-NS2, G-C-NS1-NS2, H-C-NS1-NS2,
HN-C-NS1-NS2, F-C-NS1-L, G-C-NS1-L, H-C-NS1-L, HN-C-NS1-L,
F-C-NS2-N, G-C-NS2-N, H-C-NS2-N, HN-C-NS2-N, F-C-NS2-M, G-C-NS2-M,
H-C-NS2-M, HN-C-NS2-M, F-C-NS2-M2, G-C-NS2-M2, H-C-NS2-M2,
HN-C-NS2-M2, F-C-NS2-P, G-C-NS2-P, H-C-NS2-P, HN-C-NS2-P,
F-C-NS2-NS1, G-C-NS2-NS1, H-C-NS2-NS1, HN-C-NS2-NS1, F-C-NS2-NS2,
G-C-NS2-NS2, H-C-NS2-NS2, HN-C-NS2-NS2, F-C-NS2-L, G-C-NS2-L,
H-C-NS2-L, HN-C-NS2-L, F-C-L-N, G-C-L-N, H-C-L-N, HN-C-L-N,
F-C-L-M, G-C-L-M, H-C-L-M, HN-C-L-M, F-C-L-M2, G-C-L-M2, H-C-L-M2,
HN-C-L-M2, F-C-L-P, G-C-L-P, H-C-L-P, HN-C-L-P, F-C-L-NS1,
G-C-L-NS1, H-C-L-NS1, HN-C-L-NS1, F-C-L-NS2, G-C-L-NS2, H-C-L-NS2,
HN-C-L-NS2, F-C-L-L, G-C-L-L, H-C-L-L, HN-C-L-L, F-N-C-N, G-N-C-N,
H-N-C-N, HN-N-C-N, F-N-C-M, G-N-C-M, H-N-C-M, HN-N-C-M, F-N-C-M2,
G-N-C-M2, H-N-C-M2, HN-N-C-M2, F-N-C-P, G-N-C-P, H-N-C-P, HN-N-C-P,
F-N-C-NS1, G-N-C-NS1, H-N-C-NS1, HN-N-C-NS1, F-N-C-NS2, G-N-C-NS2,
H-N-C-NS2, HN-N-C-NS2, F-N-C-L, G-N-C-L, H-N-C-L, HN-N-C-L,
F-M-C-N, G-M-C-N, H-M-C-N, HN-M-C-N, F-M-C-M, G-M-C-M, H-M-C-M,
HN-M-C-M, F-M-C-M2, G-M-C-M2, H-M-C-M2, HN-M-C-M2, F-M-C-P,
G-M-C-P, H-M-C-P HN-M-C-P, F-M-C-NS1, G-M-C-NS1, H-M-C-NS1,
HN-M-C-NS1, F-M-C-NS2, G-M-C-NS2, H-M-C-NS2, HN-M-C-NS2, F-M-C-L,
G-M-C-L, H-M-C-L, HN-M-C-L, F-M2-C-N, G-M2-C-N, H-M2-C-N,
HN-M2-C-N, F-M2-C-M, G-M2-C-M, H-M2-C-M, HN-M2-C-M, F-M2-C-M2,
G-M2-C-M2, H-M2-C-M2, HN-M2-C-M2, F-M2-C-P, G-M2-C-P, H-M2-C-P
HN-M2-C-P, F-M2-C-NS1, G-M2-C-NS1, H-M2-C-NS1, HN-M2-C-NS1,
F-M2-C-NS2, G-M2-C-NS2, H-M2-C-NS2, HN-M2-C-NS2, F-M2-C-L,
G-M2-C-L, H-M2-C-L, HN-M2-C-L, F-P-C-N, G-P-C-N, H-P-C-N, HN-P-C-N,
F-P-C-M, G-P-C-M, H-P-C-M, HN-P-C-M, F-P-C-M2, G-P-C-M2, H-P-C-M2;
HN-P-C-M2, F-P-C-P, G-P-C-P, H-P-C-P, HN-P-C-P, F-P-C-NS1,
G-P-C-NS1, H-P-C-NS1, HN-P-C-NS1, F-P-C-NS2, G-P-C-NS2, H-P-C-NS2,
HN-P-C-NS2, F-P-C-L, G-P-C-L, H-P-C-L, HN-P-C-L, F-NS1-C-N,
G-NS1-C-N, H-NS1-C-N, HN-NS1-C-N, F-NS1-C-M, G-NS1-C-M, H-NS1-C-M;
HN-NS1-C-M, F-NS1-C-M2, G-NS1-C-M2, H-NS1-C-M2; HN-NS1-C-M2,
F-NS1-C-P, G-NS1-C-P, H-NS1-C-P, HN-NS1-C-P, F-NS1-C-NS1,
G-NS1-C-NS1, H-NS1-C-NS1, HN-NS1-C-NS1, F-NS1-C-NS2, G-NS1-C-NS2,
H-NS1-C-NS2, HN-NS1-C-NS2, F-NS1-C-L, G-NS1-C-L, H-NS1-C-L,
HN-NS1-C-L, F-NS2-C-N, G-NS2-C-N, H-NS2-C-N, HN-NS2-C-N, F-NS2-C-M,
G-NS2-C-M, H-NS2-C-M, HN-NS2-C-M, F-NS2-C-M2, G-NS2-C-M2,
H-NS2-C-M2, HN-NS2-C-M2, F-NS2-C-P, G-NS2-C-P, H-NS2-C-P,
HN-NS2-C-P, F-NS2-C-NS1, G-NS2-C-NS1, H-NS2-C-NS1, HN-NS2-C-NS1,
F-NS2-C-NS2, G-NS2-C-NS2, H-NS2-C-NS2, HN-NS2-C-NS2, F-NS2-C-L,
G-NS2-C-L, H-NS2-C-L, HN-NS2-C-L, F-L-K-N, G-L-K-N, H-L-K-N,
HN-L-K-N, F-L-K-M, G-L-K-M, H-L-K-M, HN-L-K-M, F-L-K-M2, G-L-K-M2,
H-L-K-M2, HN-L-K-M2, F-L-K-P, G-L-K-P, H-L-K-P, HN-L-K-P,
F-L-K-NS1, G-L-K-NS1, H-L-K-NS1, HN-L-K-NS1, F-L-K-NS2, G-L-K-NS2,
H-L-K-NS2, HN-L-K-NS2, F-L-K-L, G-L-K-L, H-L-K-L, HN-L-K-L,
F-L-C-N, G-L-C-N, H-L-C-N, HN-L-C-N, F-L-C-M, G-L-C-M, H-L-C-M,
HN-L-C-M, F-L-C-M2, G-L-C-M2, H-L-C-M2, HN-L-C-M2, F-L-C-P,
G-L-C-P, H-L-C-P, HN-L-C-P, F-L-C-NS1, G-L-C-NS1, H-L-C-NS1,
HN-L-C-NS1, F-L-C-NS2, G-L-C-NS2, H-L-C-NS2, HN-L-C-NS2, F-L-C-L,
G-L-C-L, H-L-C-L, HN-L-C-L, F-K-N-K-N, G-K-N-K-N, H-K-N-K-N,
HN-K-N-K-N, F-K-M-K-N, G-K-M-K-N, H-K-M-K-N, HN-K-M-K-N,
F-K-M2-K-N, G-K-M2-K-N, H-K-M2-K-N, HN-K-M2-K-N, F-K-P-K-N,
G-K-P-K-N, H-K-P-K-N, HN-K-P-K-N, F-K-NS1-K-N, G-K-NS1-K-N,
H-K-NS1-K-N, HN-K-NS1-K-N, F-K-NS2-K-N, G-K-NS2-K-N, H-K-NS2-K-N,
HN-K-NS2-K-N, F-K-L-K-N, G-K-L-K-N, H-K-L-K-N, HN-K-L-K-N,
F-K-N-K-M, G-K-N-K-M, H-K-N-K-M, HN-K-N-K-M, F-K-M-K-M, G-K-M-K-M,
H-K-M-K-M, HN-K-M-K-M, F-K-M2-K-M, G-K-M2-K-M, H-K-M2-K-M,
HN-K-M2-K-M, F-K-P-K-M, G-K-P-K-M, H-K-P-K-M, HN-K-P-K-M,
F-K-NS1-K-M, G-K-NS1-K-M, H-K-NS1-K-M, HN-K-NS1-K-M, F-K-NS2-K-M,
G-K-NS2-K-M, H-K-NS2-K-M, HN-K-NS2-K-M, F-K-L-K-M, G-K-L-K-M,
H-K-L-K-M, HN-K-L-K-M, F-K-N-K-M2, G-K-N-K-M2, H-K-N-K-M2,
HN-K-N-K-M2, F-K-M-K-M2, G-K-M-K-M2, H-K-M-K-M2, HN-K-M-K-M2,
F-K-M2-K-M2, G-K-M2-K-M2, H-K-M2-K-M2, HN-K-M2-K-M2, F-K-P-K-M2,
G-K-P-K-M2, H-K-P-K-M2, HN-K-P-K-M2, F-K-NS1-K-M2, G-K-NS1-K-M2,
H-K-NS1-K-M2, HN-K-NS1-K-M2, F-K-NS2-K-M2, G-K-NS2-K-M2,
H-K-NS2-K-M2, HN-K-NS2-K-M2, F-K-L-K-M2, G-K-L-K-M2, H-K-L-K-M2,
HN-K-L-K-M2, F-K-N-K-P, G-K-N-K-P, H-K-N-K-P, HN-K-N-K-P,
F-K-M-K-P, G-K-M-K-P, H-K-M-K-P, HN-K-M-K-P, F-K-M2-K-P,
G-K-M2-K-P, H-K-M2-K-P, HN-K-M2-K-P, F-K-P-K-P, G-K-P-K-P,
H-K-P-K-P, HN-K-P-K-P, F-K-NS1-K-P, G-K-NS1-K-P, H-K-NS1-K-P,
HN-K-NS1-K-P, F-K-NS2-K-P, G-K-NS2-K-P, H-K-NS2-K-P, HN-K-NS2-K-P,
F-K-L-K-P, G-K-L-K-P, H-K-L-K-P, HN-K-L-K-P, F-K-N-K-NS1,
G-K-N-K-NS1, H-K-N-K-NS1, HN-K-N-K-NS1, F-K-M-K-NS1, G-K-M-K-NS1,
H-K-M-K-NS1, HN-K-M-K-NS1, F-K-M2-K-NS1, G-K-M2-K-NS1,
H-K-M2-K-NS1, HN-K-M2-K-NS1, F-K-P-K-NS1, G-K-P-K-NS1, H-K-P-K-NS1,
HN-K-P-K-NS1, F-K-NS1-K-NS1, G-K-NS1-K-NS1, H-K-NS1-K-NS1,
HN-K-NS1-K-NS1, F-K-NS2-K-NS1, G-K-NS2-K-NS1, H-K-NS2-K-NS1,
HN-K-NS2-K-NS1, F-K-L-K-NS1, G-K-L-K-NS1, H-K-L-K-NS1,
HN-K-L-K-NS1, F-K-N-K-NS2, G-K-N-K-NS2, H-K-N-K-NS2, HN-K-N-K-NS2,
F-K-M-K-NS2, G-K-M-K-NS2, H-K-M-K-NS2, HN-K-M-K-NS2, F-K-M2-K-NS2,
G-K-M2-K-NS2, H-K-M2-K-NS2, HN-K-M2-K-NS2, F-K-P-K-NS2,
G-K-P-K-NS2, H-K-P-K-NS2, HN-K-P-K-NS2, F-K-NS1-K-NS2
G-K-NS1-K-NS2, H-K-NS1-K-NS2, HN-K-NS1-K-NS2, F-K-NS2-K-NS2
G-K-NS2-K-NS2, H-K-NS2-K-NS2, HN-K-NS2-K-NS2, F-K-L-K-NS2,
G-K-L-K-NS2, H-K-L-K-NS2, HN-K-L-K-NS2, F-K-N-K-L, G-K-N-K-L,
H-K-N-K-L, HN-K-N-K-K, F-K-M-K-L, G-K-M-K-L, H-K-M-K-L, HN-K-M-K-L,
F-K-M2-K-L, G-K-M2-K-L, H-K-M2-K-L, HN-K-M2-K-L, F-K-P-K-L,
G-K-P-K-L, H-K-P-K-L, HN-K-P-K-L, F-K-NS1-K-L, G-K-NS1-K-L,
H-K-NS1-K-L, HN-K-NS1-K-L, F-K-NS2-K-L, G-K-NS2-K-L, H-K-NS2-K-L,
HN-K-NS2-K-L, F-K-L-K-L, G-K-L-K-L, H-K-L-K-L, HN-K-L-K-L, N-K-N-F,
N-K-N-G, N-K-N-H, N-K-N-HN, M-K-N-F, M-K-N-G, M-K-N-H, M-K-N-HN,
M2-K-N-F, M2-K-N-G, M2-K-N-H, M2-K-N-HN, P-K-N-F, P-K-N-G, P-K-N-H,
P-K-N-HN, NS1-K-N-F, NS1-K-N-G, NS1-K-N-H, NS1-K-N-HN, NS2-K-N-F,
NS2-K-N-G, NS2-K-N-H, NS2-K-N-HN, L-K-N-F, L-K-N-G, L-K-N-H,
L-K-N-HN, N-K-M-F, N-K-M-G, N-K-M-H, N-K-M-HN, M-K-M-F, M-K-M-G,
M-K-M-H, M-K-M-HN, M2-K-M-F, M2-K-M-G, M2-K-M-H, M2-K-M-HN,
P-K-M-F, P-K-M-G, P-K-M-H, P-K-M-HN, NS1-K-M-F, NS1-K-M-G,
NS1-K-M-H, NS1-K-M-HN, NS2-K-M-F, NS2-K-M-G, NS2-K-M-H, NS2-K-M-HN,
L-K-M-F, L-K-M-G, L-K-M-H, L-K-M-HN, N-K-M2-F, N-K-M2-G, N-K-M2-H,
N-K-M2-HN, M-K-M2-F, M-K-M2-G, M-K-M2-H, M-K-M2-HN, M2-K-M2-F,
M2-K-M2-G, M2-K-M2-H, M2-K-M2-HN, P-K-M2-F, P-K-M2-G, P-K-M2-H,
P-K-M2-HN, NS1-K-M2-F, NS1-K-M2-G, NS1-K-M2-H, NS1-K-M2-HN,
NS2-K-M2-F, NS2-K-M2-G, NS2-K-M2-H, NS2-K-M2-HN, L-K-M2-F,
L-K-M2-G, L-K-M2-H, L-K-M2-HN, N-K-P-F, N-K-P-G, N-K-P-H, N-K-P-HN,
M-K-P-F, M-K-P-G, M-K-P-H, M-K-P-HN, M2-K-P-F, M2-K-P-G, M2-K-P-H,
M2-K-P-HN, P-K-P-F, P-K-P-G, P-K-P-H, P-K-P-HN, NS1-K-P-F,
NS1-K-P-G, NS1-K-P-H, NS1-K-P-HN, NS2-K-P-F, NS2-K-P-G, NS2-K-P-H,
NS2-K-P-HN, L-K-P-F, L-K-P-G, L-K-P-H, L-K-P-HN, N-K-NS1-F,
N-K-NS1-G, N-K-NS1-H, N-K-NS1-HN, M-K-NS1-F, M-K-NS1-G, M-K-NS1-H,
M-K-NS1-HN, M2-K-NS1-F, M2-K-NS1-G, M2-K-NS1-H, M2-K-NS1-HN,
P-K-NS1-F, P-K-NS1-G, P-K-NS1-H, P-K-NS1-HN, NS1-K-NS1-F,
NS1-K-NS1-G, NS1-K-NS1-H, NS1-K-NS1-HN, NS2-K-NS1-F, NS2-K-NS1-G,
NS2-K-NS1-H, NS2-K-NS1-HN, L-K-NS1-F, L-K-NS1-G, L-K-NS1-H,
L-K-NS1-HN, N-K-NS2-F, N-K-NS2-G, N-K-NS2-H, N-K-NS2-HN, M-K-NS2-F,
M-K-NS2-G, M-K-NS2-H, M-K-NS2-HN, M2-K-NS2-F, M2-K-NS2-G,
M2-K-NS2-H, M2-K-NS2-HN, P-K-NS2-F, P-K-NS2-G, P-K-NS2-H,
P-K-NS2-HN, NS1-K-NS2-F, NS1-K-NS2-G, NS1-K-NS2-H, NS1-K-NS2-HN,
NS2-K-NS2-F, NS2-K-NS2-G, NS2-K-NS2-H, NS2-K-NS2-HN, L-K-NS2-F,
L-K-NS2-G, L-K-NS2-H, L-K-NS2-HN, N-K-L-F, N-K-L-G, N-K-L-H,
N-K-L-HN, M-K-L-F, M-K-L-G, M-K-L2-H, M-K-L-HN, M2-K-L-F, M2-K-L-G,
M2-K-L-H, M2-K-L-HN, P-K-L-F, P-K-L-G, P-K-L-H, P-K-L-HN,
NS1-K-L-F, NS1-K-L-G, NS1-K-L-H, NS1-K-L-HN, NS2-K-L-F, NS2-K-L-G,
NS2-K-L-H, NS2-K-L-HN, L-K-L-F, L-K-L-G, L-K-L-H, L-K-L-HN,
N-N-K-F, N-N-K-G, N-N-K-H, N-N-K-HN, N-M-K-F, N-M-K-G, N-M-K-H,
N-M-K-HN, N-M2-K-F, N-M2-K-G, N-M2-K-H, N-M2-K-HN, N-P-K-F,
N-P-K-G, N-P-K-H, N-P-K-HN, N-NS1-K-F, N-NS1-K-G, N-NS1-K-H,
N-NS1-K-HN, N-NS2-K-F, N-NS2-K-G, N-NS2-K-H, N-NS2-K-HN, N-L-K-F,
N-L-K-G, N-L-K-H, N-L-K-HN, M-N-K-F, M-N-K-G, M-N-K-H, M-N-K-HN,
M-M-K-F, M-M-K-G, M-M-K-H, M-M-K-HN, M-M2-K-F, M-M2-K-G, M-M2-K-H,
M-M2-K-HN, M-P-K-F, M-P-K-G, M-P-K-H, M-P-K-HN, M-NS1-K-F,
M-NS1-K-G, M-NS1-K-H, M-NS1-K-HN, M-NS2-K-F, M-NS2-K-G, M-NS2-K-H,
M-NS2-K-HN, M-L-K-F, M-L-K-G, M-L-K-H, M-L-K-HN, M2-N-K-F,
M2-N-K-G, M2-N-K-H, M2-N-K-HN, M2-M-K-F, M2-M-K-G, M2-M-K-H,
M2-M-K-HN, M2-M2-K-F, M2-M2-K-G, M2-M2-K-H, M2-M2-K-HN, M2-P-K-F,
M2-P-K-G, M2-P-K-H, M2-P-K-HN, M2-NS1-K-F, M2-NS1-K-G, M2-NS1-K-H,
M2-NS1-K-HN, M2-NS2-K-F, M2-NS2-K-G, M2-NS2-K-H, M2-NS2-K-HN,
M2-L-K-F,
M2-L-K-G, M2-L-K-H, M2-L-K-HN, P-N-K-F, P-N-K-G, P-N-K-H, P-N-K-HN,
P-M-K-F, P-M-K-G, P-M-K-H, P-M-K-HN, P-M2-K-F, P-M2-K-G, P-M2-K-H,
P-M2-K-HN, P-P-K-F, P-P-K-G, P-P-K-H, P-P-K-HN, P-NS1-K-F,
P-NS1-K-G, P-NS1-K-H, P-NS1-K-HN, P-NS2-K-F, P-NS2-K-G, P-NS2-K-H,
P-NS2-K-HN, P-L-K-F, P-L-K-G, P-L-K-H, P-L-K-HN, NS1-N-K-F,
NS1-N-K-G, NS1-N-K-H, NS1-N-K-HN, NS1-M-K-F, NS1-M-K-G, NS1-M-K-H,
NS1-M-K-HN, NS1-M2-K-F, NS1-M2-K-G, NS1-M2-K-H, NS1-M2-K-HN,
NS1-P-K-F, NS1-P-K-G, NS1-P-K-H, NS1-P-K-HN, NS1-NS1-K-F,
NS1-NS1-K-G, NS1-NS1-K-H, NS1-NS1-K-HN, NS1-NS2-K-F, NS1-NS2-K-G,
NS1-NS2-K-H, NS1-NS2-K-HN, NS1-L-K-F, NS1-L-K-G, NS1-L-K-H,
NS1-L-K-HN, NS2-N-K-F, NS2-N-K-G, NS2-N-K-H, NS2-N-K-HN, NS2-M-K-F,
NS2-M-K-G, NS2-M-K-H, NS2-M-K-HN, NS2-M2-K-F, NS2-M2-K-G,
NS2-M2-K-H, NS2-M2-K-HN, NS2-P-K-F, NS2-P-K-G, NS2-P-K-H,
NS2-P-K-HN, NS2-NS1-K-F, NS2-NS1-K-G, NS2-NS1-K-H, NS2-NS1-K-HN,
NS2-NS2-K-F, NS2-NS2-K-G, NS2-NS2-K-H, NS2-NS2-K-HN, NS2-L-K-F,
NS2-L-K-G, NS2-L-K-H, NS2-L-K-HN, L-N-K-F, L-N-K-G, L-N-K-H,
L-N-K-HN, L-M-K-F, L-M-K-G, L-M-K-H, L-M-K-HN, L-M2-K-F, L-M2-K-G,
L-M2-K-H, L-M2-K-HN, L-L-K-F, L-P-K-G, L-P-K-H, L-P-K-HN,
L-NS1-K-F, L-NS1-K-G, L-NS1-K-H, L-NS1-K-HN, L-NS2-K-F, L-NS2-K-G,
L-NS2-K-H, L-NS2-K-HN, L-L-K-F, L-L-K-G, L-L-K-H, L-L-K-HN,
N-K-N-K-F, N-K-N-K-G, N-K-N-K-H, N-K-N-K-HN, N-K-M-K-F, N-K-M-K-G,
N-K-M-K-H, N-K-M-K-HN, N-K-M2-K-F, N-K-M2-K-G, N-K-M2-K-H,
N-K-M2-K-HN, N-K-P-K-F, N-K-P-K-G, N-K-P-K-H, N-K-P-K-HN,
N-K-NS1-K-F, N-K-NS1-K-G, N-K-NS1-K-H, N-K-NS1-K-HN, N-K-NS2-K-F,
N-K-NS2-K-G, N-K-NS2-K-H, N-K-NS2-K-HN, N-K-L-K-F, N-K-L-K-G,
N-K-L-K-H, N-K-L-K-HN, M-K-N-K-F, M-K-N-K-G, M-K-N-K-H, M-K-N-K-HN,
M-K-M-K-F, M-K-M-K-G, M-K-M-K-H, M-K-M-K-HN, M-K-M2-K-F,
M-K-M2-K-G, M-K-M2-K-H, M-K-M2-K-HN, M-K-P-K-F, M-K-P-K-G,
M-K-P-K-H, M-K-P-K-HN, M-K-NS1-K-F, M-K-NS1-K-G, M-K-NS1-K-H,
M-K-NS1-K-HN, M-K-NS2-K-F, M-K-NS2-K-G, M-K-NS2-K-H, M-K-NS2-K-HN,
M-K-L-K-F, M-K-L-K-G, M-K-L-K-H, M-K-L-K-HN, M2-K-N-K-F,
M2-K-N-K-G, M2-K-N-K-H, M2-K-N-K-HN, M2-K-M-K-F, M2-K-M-K-G,
M2-K-M-K-H, M2-K-M-K-HN, M2-K-M2-K-F, M2-K-M2-K-G, M2-K-M2-K-H,
M2-K-M2-K-HN, M2-K-P-K-F, M2-K-P-K-G, M2-K-P-K-H, M2-K-P-K-HN,
M2-K-NS1-K-F, M2-K-NS1-K-G, M2-K-NS1-K-H, M2-K-NS1-K-HN,
M2-K-NS2-K-F, M2-K-NS2-K-G, M2-K-NS2-K-H, M2-K-NS2-K-HN,
M2-K-L-K-F, M2-K-L-K-G, M2-K-L-K-H, M2-K-L-K-HN, P-K-N-K-F,
P-K-N-K-G, P-K-N-K-H, P-K-N-K-HN, P-K-M-K-F, P-K-M-K-G, P-K-M-K-H,
P-K-M-K-HN, P-K-M2-K-F, P-K-M2-K-G, P-K-M2-K-H, P-K-M2-K-HN,
P-K-P-K-F, P-K-P-K-G, P-K-P-K-H, P-K-P-K-HN, P-K-NS1-K-F,
P-K-NS1-K-G, P-K-NS1-K-H, P-K-NS1-K-HN, P-K-NS2-K-F, P-K-NS2-K-G,
P-K-NS2-K-H, P-K-NS2-K-HN, P-K-L-K-F, P-K-L-K-G, P-K-L-K-H,
P-K-L-K-HN, NS1-K-N-K-F, NS1-K-N-K-G, NS1-K-N-K-H, NS1-K-N-K-HN,
NS1-K-M-K-F, NS1-K-M-K-G, NS1-K-M-K-H, NS1-K-M-K-HN, NS1-K-M2-K-F,
NS1-K-M2-K-G, NS1-K-M2-K-H, NS1-K-M2-K-HN, NS1-K-P-K-F,
NS1-K-P-K-G, NS1-K-P-K-H, NS1-K-P-K-HN, NS1-K-NS1-K-F,
NS1-K-NS1-K-G, NS1-K-NS1-K-H, NS1-K-NS1-K-HN, NS1-K-NS2-K-F,
NS1-K-NS2-K-G, NS1-K-NS2-K-H, NS1-K-NS2-K-HN, NS1-K-L-K-F,
NS1-K-L-K-G, NS1-K-L-K-H, NS1-K-L-K-HN, NS2-K-N-K-F, NS2-K-N-K-G,
NS2-K-N-K-H, NS2-K-N-K-HN, NS2-K-M-K-F, NS2-K-M-K-G, NS2-K-M-K-H,
NS2-K-M-K-HN, NS2-K-M2-K-F, NS2-K-M2-K-G, NS2-K-M2-K-H,
NS2-K-M2-K-HN, NS2-K-P-K-F, NS2-K-P-K-G, NS2-K-P-K-H, NS2-K-P-K-HN,
NS2-K-NS1-K-F, NS2-K-NS1-K-G, NS2-K-NS1-K-H, NS2-K-NS1-K-HN,
NS2-K-NS2-K-F, NS2-K-NS2-K-G, NS2-K-NS2-K-H, NS2-K-NS2-K-HN,
NS2-K-L-K-F, NS2-K-L-K-G, NS2-K-L-K-H, NS2-K-L-K-HN, L-K-N-K-F,
L-K-N-K-G, L-K-N-K-H, L-K-N-K-HN, L-K-M-K-F, L-K-M-K-G, L-K-M-K-H,
L-K-M-K-HN, L-K-M2-K-F, L-K-M2-K-G, L-K-M2-K-H, L-K-M2-K-HN,
L-K-P-K-F, L-K-P-K-G, L-K-P-K-H, L-K-P-K-HN, L-K-NS1-K-F,
L-K-NS1-K-G, L-K-NS1-K-H, L-K-NS1-K-HN, L-K-NS2-K-F, L-K-NS2-K-G,
L-K-NS2-K-H, L-K-NS2-K-HN, L-K-L-K-F, L-K-L-K-G, L-K-L-K-H, or
L-K-L-K-HN, F-C-N-K-N, G-C-N-K-N, H-C-N-K-N, HN-C-N-K-N, F-C-M-K-N,
G-C-M-K-N, H-C-M-K-N, HN-C-M-K-N, F-C-M2-K-N, G-C-M2-K-N,
H-C-M2-K-N, HN-C-M2-K-N, F-C-P-K-N, G-C-P-K-N, H-C-P-K-N,
HN-C-P-K-N, F-C-NS1-K-N, G-C-NS1-K-N, H-C-NS1-K-N, HN-C-NS1-K-N,
F-C-NS2-K-N, G-C-NS2-K-N, H-C-NS2-K-N, HN-C-NS2-K-N, F-C-L-K-N,
G-C-L-K-N, H-C-L-K-N, HN-C-L-K-N, F-C-N-K-M, G-C-N-K-M, H-C-N-K-M,
HN-C-N-K-M, F-C-M-K-M, G-C-M-K-M, H-C-M-K-M, HN-C-M-K-M,
F-C-M2-K-M, G-C-M2-K-M, H-C-M2-K-M, HN-C-M2-K-M, F-C-P-K-M,
G-C-P-K-M, H-C-P-K-M, HN-C-P-K-M, F-C-NS1-K-M, G-C-NS1-K-M,
H-C-NS1-K-M, HN-C-NS1-K-M, F-C-NS2-K-M, G-C-NS2-K-M, H-C-NS2-K-M,
HN-C-NS2-K-M, F-C-L-K-M, G-C-L-K-M, H-C-L-K-M, HN-C-L-K-M,
F-C-N-K-M2, G-C-N-K-M2, H-C-N-K-M2, HN-C-N-K-M2, F-C-M-K-M2,
G-C-M-K-M2, H-C-M-K-M2, HN-C-M-K-M2, F-C-M2-K-M2, G-C-M2-K-M2,
H-C-M2-K-M2, HN-C-M2-K-M2, F-C-P-K-M2, G-C-P-K-M2, H-C-P-K-M2,
HN-C-P-K-M2, F-C-NS1-K-M2, G-C-NS1-K-M2, H-C-NS1-K-M2,
HN-C-NS1-K-M2, F-C-NS2-K-M2, G-C-NS2-K-M2, H-C-NS2-K-M2,
HN-C-NS2-K-M2, F-C-L-K-M2, G-C-L-K-M2, H-C-L-K-M2, HN-C-L-K-M2,
F-C-N-K-P, G-C-N-K-P, H-C-N-K-P, HN-C-N-K-P, F-C-M-K-P, G-C-M-K-P,
H-C-M-K-P, HN-C-M-K-P, F-C-M2-K-P, G-C-M2-K-P, H-C-M2-K-P,
HN-C-M2-K-P, F-C-P-K-P, G-C-P-K-P, H-C-P-K-P, HN-C-P-K-P,
F-C-NS1-K-P, G-C-NS1-K-P, H-C-NS1-K-P, HN-C-NS1-K-P, F-C-NS2-K-P,
G-C-NS2-K-P, H-C-NS2-K-P, HN-C-NS2-K-P, F-C-L-K-P, G-C-L-K-P,
H-C-L-K-P, HN-C-L-K-P, F-C-N-K-NS1, G-C-N-K-NS1, H-C-N-K-NS1,
HN-C-N-K-NS1, F-C-M-K-NS1, G-C-M-K-NS1, H-C-M-K-NS1, HN-C-M-K-NS1,
F-C-M2-K-NS1, G-C-M2-K-NS1, H-C-M2-K-NS1, HN-C-M2-K-NS1,
F-C-P-K-NS1, G-C-P-K-NS1, H-C-P-K-NS1, HN-C-P-K-NS1, F-C-NS1-K-NS1,
G-C-NS1-K-NS1, H-C-NS1-K-NS1, HN-C-NS1-K-NS1, F-C-NS2-K-NS1,
G-C-NS2-K-NS1, H-C-NS2-K-NS1, HN-C-NS2-K-NS1, F-C-L-K-NS1,
G-C-L-K-NS1, H-C-L-K-NS1, HN-C-L-K-NS1, F-C-N-K-NS2, G-C-N-K-NS2,
H-C-N-K-NS2, HN-C-N-K-NS2, F-C-M-K-NS2, G-C-M-K-NS2, H-C-M-K-NS2,
HN-C-M-K-NS2, F-C-M2-K-NS2, G-C-M2-K-NS2, H-C-M2-K-NS2,
HN-C-M2-K-NS2, F-C-P-K-NS2, G-C-P-K-NS2, H-C-P-K-NS2, HN-C-P-K-NS2,
F-C-NS1-K-NS2 G-C-NS1-K-NS2, H-C-NS1-K-NS2, HN-C-NS1-K-NS2,
F-C-NS2-K-NS2 G-C-NS2-K-NS2, H-C-NS2-K-NS2, HN-C-NS2-K-NS2,
F-C-L-K-NS2, G-C-L-K-NS2, H-C-L-K-NS2, HN-C-L-K-NS2, F-C-N-K-L,
G-C-N-K-L, H-C-N-K-L, HN-C-N-K-L, F-C-M-K-L, G-C-M-K-L, H-C-M-K-L,
HN-C-M-K-L, F-C-M2-K-L, G-C-M2-K-L, H-C-M2-K-L, HN-C-M2-K-L,
F-C-P-K-L, G-C-P-K-L, H-C-P-K-L, HN-C-P-K-L, F-C-NS1-K-L,
G-C-NS1-K-L, H-C-NS1-K-L, HN-C-NS1-K-L, F-C-NS2-K-L, G-C-NS2-K-L,
H-C-NS2-K-L, HN-C-NS2-K-L, F-C-L-K-L, G-C-L-K-L, H-C-L-K-L,
HN-C-L-K-L, N-C-N-F, N-C-N-G, N-C-N-H, N-C-N-HN, M-C-N-F, M-C-N-G,
M-C-N-H, M-C-N-HN, M2-C-N-F, M2-C-N-G, M2-C-N-H, M2-C-N-HN,
P-C-N-F, P-C-N-G, P-C-N-H, P-C-N-HN, NS1-C-N-F, NS1-C-N-G,
NS1-C-N-H, NS1-C-N-HN, NS2-C-N-F, NS2-C-N-G, NS2-C-N-H, NS2-C-N-HN,
L-C-N-F, L-C-N-G, L-C-N-H, L-C-N-HN, N-C-M-F, N-C-M-G, N-C-M-H,
N-C-M-HN, M-C-M-F, M-C-M-G, M-C-M-H, M-C-M-HN, M2-C-M-F, M2-C-M-G,
M2-C-M-H, M2-C-M-HN, P-C-M-F, P-C-M-G, P-C-M-H, P-C-M-HN,
NS1-C-M-F, NS1-C-M-G, NS1-C-M-H, NS1-C-M-HN, NS2-C-M-F, NS2-C-M-G,
NS2-C-M-H, NS2-C-M-HN, L-C-M-F, L-C-M-G, L-C-M-H, L-C-M-HN,
N-C-M2-F, N-C-M2-G, N-C-M2-H, N-C-M2-HN, M-C-M2-F, M-C-M2-G,
M-C-M2-H, M-C-M2-HN, M2-C-M2-F, M2-C-M2-G, M2-C-M2-H, M2-C-M2-HN,
P-C-M2-F, P-C-M2-G, P-C-M2-H, P-C-M2-HN, NS1-C-M2-F, NS1-C-M2-G,
NS1-C-M2-H, NS1-C-M2-HN, NS2-C-M2-F, NS2-C-M2-G, NS2-C-M2-H,
NS2-C-M2-HN, L-C-M2-F, L-C-M2-G, L-C-M2-H, L-C-M2-HN, N-C-P-F,
N-C-P-G, N-C-P-H, N-C-P-HN, M-C-P-F, M-C-P-G, M-C-P-H, M-C-P-HN,
M2-C-P-F, M2-C-P-G, M2-C-P-H, M2-C-P-HN, P-C-P-F, P-C-P-G, P-C-P-H,
P-C-P-HN, NS1-C-P-F, NS1-C-P-G, NS1-C-P-H, NS1-C-P-HN, NS2-C-P-F,
NS2-C-P-G, NS2-C-P-H, NS2-C-P-HN, L-C-P-F, L-C-P-G, L-C-P-H,
L-C-P-HN, N-C-NS1-F, N-C-NS1-G, N-C-NS1-H, N-C-NS1-HN, M-C-NS1-F,
M-C-NS1-G, M-C-NS1-H, M-C-NS1-HN, M2-C-NS1-F, M2-C-NS1-G,
M2-C-NS1-H, M2-C-NS1-HN, P-C-NS1-F, P-C-NS1-G, P-C-NS1-H,
P-C-NS1-HN, NS1-C-NS1-F, NS1-C-NS1-G, NS1-C-NS1-H, NS1-C-NS1-HN,
NS2-C-NS1-F, NS2-C-NS1-G, NS2-C-NS1-H, NS2-C-NS1-HN, L-C-NS1-F,
L-C-NS1-G, L-C-NS1-H, L-C-NS1-HN, N-C-NS2-F, N-C-NS2-G, N-C-NS2-H,
N-C-NS2-HN, M-C-NS2-F, M-C-NS2-G, M-C-NS2-H, M-C-NS2-HN,
M2-C-NS2-F, M2-C-NS2-G, M2-C-NS2-H, M2-C-NS2-HN, P-C-NS2-F,
P-C-NS2-G, P-C-NS2-H, P-C-NS2-HN, NS1-C-NS2-F, NS1-C-NS2-G,
NS1-C-NS2-H, NS1-C-NS2-HN, NS2-C-NS2-F, NS2-C-NS2-G, NS2-C-NS2-H,
NS2-C-NS2-HN, L-C-NS2-F, L-C-NS2-G, L-C-NS2-H, L-C-NS2-HN, N-C-L-F,
N-C-L-G, N-C-L-H, N-C-L-HN, M-C-L-F, M-C-L-G, M-C-L2-H, M-C-L-HN,
M2-C-L-F, M2-C-L-G, M2-C-L-H, M2-C-L-HN, P-C-L-F, P-C-L-G, P-C-L-H,
P-C-L-HN, NS1-C-L-F, NS1-C-L-G, NS1-C-L-H, NS1-C-L-HN, NS2-C-L-F,
NS2-C-L-G, NS2-C-L-H, NS2-C-L-HN, L-C-L-F, L-C-L-G, L-C-L-H,
L-C-L-HN, N-N-C-F, N-N-C-G, N-N-C-H, N-N-C-HN, N-M-C-F, N-M-C-G,
N-M-C-H, N-M-C-HN, N-M2-C-F, N-M2-C-G, N-M2-C-H, N-M2-C-HN,
N-P-C-F, N-P-C-G, N-P-C-H, N-P-C-HN, N-NS1-C-F, N-NS1-C-G,
N-NS1-C-H, N-NS1-C-HN, N-NS2-C-F, N-NS2-C-G, N-NS2-C-H, N-NS2-C-HN,
N-L-C-F, N-L-C-G, N-L-C-H, N-L-C-HN, M-N-C-F, M-N-C-G, M-N-C-H,
M-N-C-HN, M-M-C-F, M-M-C-G, M-M-C-H, M-M-C-HN, M-M2-C-F, M-M2-C-G,
M-M2-C-H, M-M2-C-HN, M-P-C-F, M-P-C-G, M-P-C-H, M-P-C-HN,
M-NS1-C-F, M-NS1-C-G, M-NS1-C-H, M-NS1-C-HN, M-NS2-C-F, M-NS2-C-G,
M-NS2-C-H, M-NS2-C-HN, M-L-C-F, M-L-C-G, M-L-C-H, M-L-C-HN,
M2-N-C-F, M2-N-C-G, M2-N-C-H, M2-N-C-HN, M2-M-C-F, M2-M-C-G,
M2-M-C-H, M2-M-C-HN, M2-M2-C-F, M2-M2-C-G, M2-M2-C-H, M2-M2-C-HN,
M2-P-C-F, M2-P-C-G, M2-P-C-H, M2-P-C-HN, M2-NS1-C-F, M2-NS1-C-G,
M2-NS1-C-H, M2-NS1-C-HN, M2-NS2-C-F, M2-NS2-C-G, M2-NS2-C-H,
M2-NS2-C-HN, M2-L-C-F, M2-L-C-G, M2-L-C-H, M2-L-C-HN, P-N-C-F,
P-N-C-G, P-N-C-H, P-N-C-HN, P-M-C-F, P-M-C-G, P-M-C-H, P-M-C-HN,
P-M2-C-F, P-M2-C-G, P-M2-C-H, P-M2-C-HN, P-P-C-F, P-P-C-G, P-P-C-H,
P-P-C-HN, P-NS1-C-F, P-NS1-C-G, P-NS1-C-H, P-NS1-C-HN, P-NS2-C-F,
P-NS2-C-G, P-NS2-C-H, P-NS2-C-HN, P-L-C-F, P-L-C-G, P-L-C-H,
P-L-C-HN, NS1-N-C-F, NS1-N-C-G, NS1-N-C-H, NS1-N-C-HN, NS1-M-C-F,
NS1-M-C-G, NS1-M-C-H, NS1-M-C-HN, NS1-M2-C-F, NS1-M2-C-G,
NS1-M2-C-H, NS1-M2-C-HN, NS1-P-C-F, NS1-P-C-G, NS1-P-C-H,
NS1-P-C-HN, NS1-NS1-C-F, NS1-NS1-C-G, NS1-NS1-C-H, NS1-NS1-C-HN,
NS1-NS2-C-F, NS1-NS2-C-G, NS1-NS2-C-H, NS1-NS2-C-HN, NS1-L-C-F,
NS1-L-C-G, NS1-L-C-H, NS1-L-C-HN, NS2-N-C-F, NS2-N-C-G, NS2-N-C-H,
NS2-N-C-HN, NS2-M-C-F, NS2-M-C-G, NS2-M-C-H, NS2-M-C-HN,
NS2-M2-C-F, NS2-M2-C-G, NS2-M2-C-H, NS2-M2-C-HN, NS2-P-C-F,
NS2-P-C-G, NS2-P-C-H, NS2-P-C-HN, NS2-NS1-C-F, NS2-NS1-C-G,
NS2-NS1-C-H, NS2-NS1-C-HN, NS2-NS2-C-F, NS2-NS2-C-G, NS2-NS2-C-H,
NS2-NS2-C-HN, NS2-L-C-F, NS2-L-C-G, NS2-L-C-H, NS2-L-C-HN, L-N-C-F,
L-N-C-G, L-N-C-H, L-N-C-HN, L-M-C-F, L-M-C-G, L-M-C-H, L-M-C-HN,
L-M2-C-F, L-M2-C-G, L-M2-C-H, L-M2-C-HN, L-L-C-F, L-P-C-G, L-P-C-H,
L-P-C-HN, L-NS1-C-F, L-NS1-C-G, L-NS1-C-H, L-NS1-C-HN, L-NS2-C-F,
L-NS2-C-G, L-NS2-C-H, L-NS2-C-HN, L-L-C-F, L-L-C-G, L-L-C-H,
L-L-C-HN, N-C-N-K-F, N-C-N-K-G, N-C-N-K-H, N-C-N-K-HN, N-C-M-K-F,
N-C-M-K-G, N-C-M-K-H, N-C-M-K-HN, N-C-M2-K-F, N-C-M2-K-G,
N-C-M2-K-H, N-C-M2-K-HN, N-C-P-K-F, N-C-P-K-G, N-C-P-K-H,
N-C-P-K-HN, N-C-NS1-K-F, N-C-NS1-K-G, N-C-NS1-K-H, N-C-NS1-K-HN,
N-C-NS2-K-F, N-C-NS2-K-G, N-C-NS2-K-H, N-C-NS2-K-HN, N-C-L-K-F,
N-C-L-K-G, N-C-L-K-H, N-C-L-K-HN, M-C-N-K-F, M-C-N-K-G, M-C-N-K-H,
M-C-N-K-HN, M-C-M-K-F, M-C-M-K-G, M-C-M-K-H, M-C-M-K-HN,
M-C-M2-K-F, M-C-M2-K-G, M-C-M2-K-H, M-C-M2-K-HN, M-C-P-K-F,
M-C-P-K-G, M-C-P-K-H, M-C-P-K-HN, M-C-NS1-K-F, M-C-NS1-K-G,
M-C-NS1-K-H, M-C-NS1-K-HN, M-C-NS2-K-F, M-C-NS2-K-G, M-C-NS2-K-H,
M-C-NS2-K-HN, M-C-L-K-F, M-C-L-K-G, M-C-L-K-H, M-C-L-K-HN,
M2-C-N-K-F, M2-C-N-K-G, M2-C-N-K-H, M2-C-N-K-HN, M2-C-M-K-F,
M2-C-M-K-G, M2-C-M-K-H, M2-C-M-K-HN, M2-C-M2-K-F, M2-C-M2-K-G,
M2-C-M2-K-H, M2-C-M2-K-HN, M2-C-P-K-F, M2-C-P-K-G, M2-C-P-K-H,
M2-C-P-K-HN, M2-C-NS1-K-F, M2-C-NS1-K-G, M2-C-NS1-K-H,
M2-C-NS1-K-HN, M2-C-NS2-K-F, M2-C-NS2-K-G, M2-C-NS2-K-H,
M2-C-NS2-K-HN, M2-C-L-K-F, M2-C-L-K-G, M2-C-L-K-H, M2-C-L-K-HN,
P-C-N-K-F, P-C-N-K-G, P-C-N-K-H, P-C-N-K-HN, P-C-M-K-F, P-C-M-K-G,
P-C-M-K-H, P-C-M-K-HN, P-C-M2-K-F, P-C-M2-K-G, P-C-M2-K-H,
P-C-M2-K-HN, P-C-P-K-F, P-C-P-K-G, P-C-P-K-H, P-C-P-K-HN,
P-C-NS1-K-F, P-C-NS1-K-G, P-C-NS1-K-H, P-C-NS1-K-HN, P-C-NS2-K-F,
P-C-NS2-K-G, P-C-NS2-K-H, P-C-NS2-K-HN, P-C-L-K-F, P-C-L-K-G,
P-C-L-K-H, P-C-L-K-HN, NS1-C-N-K-F, NS1-C-N-K-G, NS1-C-N-K-H,
NS1-C-N-K-HN, NS1-C-M-K-F, NS1-C-M-K-G, NS1-C-M-K-H, NS1-C-M-K-HN,
NS1-C-M2-K-F, NS1-C-M2-K-G, NS1-C-M2-K-H, NS1-C-M2-K-HN,
NS1-C-P-K-F, NS1-C-P-K-G, NS1-C-P-K-H, NS1-C-P-K-HN, NS1-C-NS1-K-F,
NS1-C-NS1-K-G, NS1-C-NS1-K-H, NS1-C-NS1-K-HN, NS1-C-NS2-K-F,
NS1-C-NS2-K-G, NS1-C-NS2-K-H, NS1-C-NS2-K-HN, NS1-C-L-K-F,
NS1-C-L-K-G, NS1-C-L-K-H, NS1-C-L-K-HN, NS2-C-N-K-F, NS2-C-N-K-G,
NS2-C-N-K-H, NS2-C-N-K-HN, NS2-C-M-K-F, NS2-C-M-K-G, NS2-C-M-K-H,
NS2-C-M-K-HN, NS2-C-M2-K-F, NS2-C-M2-K-G, NS2-C-M2-K-H,
NS2-C-M2-K-HN, NS2-C-P-K-F, NS2-C-P-K-G, NS2-C-P-K-H, NS2-C-P-K-HN,
NS2-C-NS1-K-F, NS2-C-NS1-K-G, NS2-C-NS1-K-H, NS2-C-NS1-K-HN,
NS2-C-NS2-K-F, NS2-C-NS2-K-G, NS2-C-NS2-K-H, NS2-C-NS2-K-HN,
NS2-C-L-K-F, NS2-C-L-K-G, NS2-C-L-K-H, NS2-C-L-K-HN, L-C-N-K-F,
L-C-N-K-G, L-C-N-K-H, L-C-N-K-HN, L-C-M-K-F, L-C-M-K-G, L-C-M-K-H,
L-C-M-K-HN, L-C-M2-K-F, L-C-M2-K-G, L-C-M2-K-H, L-C-M2-K-HN,
L-C-P-K-F, L-C-P-K-G, L-C-P-K-H, L-C-P-K-HN, L-C-NS1-K-F,
L-C-NS1-K-G, L-C-NS1-K-H, L-C-NS1-K-HN, L-C-NS2-K-F, L-C-NS2-K-G,
L-C-NS2-K-H, L-C-NS2-K-HN, L-C-L-K-F, L-C-L-K-G, L-C-L-K-H, or
L-C-L-K-HN. Most preferably the arrangement is F-C-N-K-M2.
[0204] In preferred embodiments, the expression system is for use
in the prophylaxis or treatment of viral infection, particularly
preferably for use in the prophylaxis or treatment of a
paramyxovirus infection, preferably a RSV infection and/or in the
manufacturing of medicament for use in the prophylaxis or treatment
of a paramyxovirus infection, preferably a RSV infection, and/or
for use in methods of prophylaxis or treatment of an of a
paramyxovirus infection, preferably a RSV infection.
[0205] In preferred embodiments, the expression system is for use
in enhancing an immune response, preferably a B cell immune
response against a paramyxovirus infection, preferably a RSV
infection.
[0206] According to a preferred embodiment of the first aspect, the
first polynucleotide encodes a viral protein of a orthomyxovirus or
variant thereof which induces a reaction of the immune system (i.e.
immune response) in a host which is mediated by T cells, and the
second polynucleotide encodes a viral protein of a orthomyxovirus
or variant thereof that induces an anti-pathogenic B cell response
against the orthomyxovirus. It is preferred that the orthomyxovirus
whose viral proteins are encoded for by the first and second
polynucleotide is selected from the genus of Influenzavirus A,
Influenzavirus B, Influenzavirus C, Thogotoviris and Isavirus. In
even more preferred embodiments, the orthomxyovirus is
Influenzavirus A, preferably selected from the subtypes H1N1, H1N2,
H2N2, H3N1, H3N2, H3N8, H5N1, H5N2, H5N3, H5N8, H5N9, H7N1, H7N2,
H7N3, H7N4, H7N7, H9N2, H10N7. According to a preferred embodiment
of the first aspect, the second polynucleotide encodes a viral
protein of an orthomyxovirus or variant thereof that induces an
anti-pathogenic B cell response against the orthomyxovirus.
[0207] According to preferred embodiments of the first aspect first
polynucleotide encodes a viral protein of a orthomyxovirus or
variant thereof which induces a reaction of the immune system (i.e.
immune response) in a host which is mediated by T cells. A T cell
response involves the activation of antigen-specific T lymphocyte
such as but not limited to cytotoxic T cells (CTLs), T helper cells
(T.sub.H cells), central memory T cells (TCM cells), effector
memory T cells (TEM cells), and regulatory T cells (Treg cells). A
T cell response against a protein is induced, if peptides of the
protein are processed within the cell and presented to T cells on
the surface of the cell via the MHC I or MHC II pathway. Thus, in
the context of the present invention preferably those viral
proteins or parts thereof are used for inducing a T cell response
that are normally not exposed on the outside of the virus, e.g. non
structural or internal proteins or parts of structural or surface
proteins not accessible to B-cells on the outside of the virus.
[0208] According to a preferred embodiment of the first aspect, the
second polynucleotide encodes a viral protein of an orthomyxovirus
or variant thereof that induces an anti-pathogenic B cell response.
A B cell response is an immune response based on the activation of
B lymphocytes, which produce and secrete antigen specific
antibodies. B cells involved in such immune response include but
are not limited to plasma B cells, memory B cells and B-1 cells.
Thus, in the context of the present invention preferably those
viral proteins or parts thereof are used for inducing a B cell
response that are exposed on the outside of the virus, e.g.
structural and/or surface proteins or at least those parts of
structural and/or surface proteins accessible to B-cells on the
outside of the virus.
[0209] In a preferred embodiment of the first aspect the viral
protein of an orthomyxovirus, which induces a T cell response is a
non-structural and/or internal protein of an orthomyxovirus, and/or
the viral protein of a orthomyxovirus, which induces an
anti-pathogenic B cell response is a structural and/or surface
protein of a orthomyxovirus.
[0210] It is preferred that the amino acid sequence of the
structural (surface) and/or non-structural and/or internal protein
comprises consecutive segments or a consensus sequence of one or
more different orthomyxovirus isolates.
[0211] In preferred embodiments, the structural protein is a
protein exposed on the surface of the native orthomyxovirus or a
variant thereof. It is preferred that the structural and/or surface
protein triggers a T-cell independent immune response such as but
not limited to an antibody mediated immune response or an
activation of the complement system. In a particularly preferred
embodiment, the structural and/or surface protein induces an
antibody mediated immune response. Such antibody mediated immune
response is based on the activation of B cells which produce and
secrete antigen specific antibodies. B cells involved in such
immune response include but are not limited to plasma B cells,
memory B cells and B-1 cells.
[0212] In a further preferred embodiment, the membrane attachment
domain of the protein exposed on the surface of the native
orthomyxovirus or variant thereof is functionally deleted, thus,
either being structurally deleted or structurally present but not
fulfilling its biological function. In a particularly preferred
embodiment, the amino acid sequence corresponding to the membrane
attachment domain is deleted. The deletion of the membrane
attachment region serves the purpose of ascertaining that the
anti-pathogenic B cell response inducing protein is secreted from
the cell into which the expression system of the invention has been
introduced.
[0213] It is further preferred that the viral surface proteins of
the native orthomyxovirus is selected from the group consisting of
hemagglutinin (HA) and neuraminidase (NA). It is more preferred
that the viral surface protein of the native orthomyxovirus is
hemagglutinin (HA).
[0214] In a preferred embodiment of the first aspect, HA comprises,
essentially consists of or consists of an amino acid sequence of HA
of one influenza A virus isolate or a consensus amino acid sequence
of two or more different influenza A virus isolates, preferably
according to SEQ ID NO: 8 or SEQ ID NO: 20, more preferably
according to SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 21 or a
variant of one of these sequences.
[0215] In preferred embodiments of the first aspect, the
non-structural protein is a conserved internal protein of
orthomyxoviruses suitable for inducing a T cell mediated immune
response against the paramyxovirus, involving the activation of
antigen-specific T lymphocyte such as but not limited to cytotoxic
T cells (CTLs), T helper cells (T.sub.H cells), central memory T
cells (TCM cells), effector memory T cells (TEM cells), and
regulatory T cells (Treg cells). Thus, preferably the T cell
inducing protein of the orthomyxovirus does not comprise a
secretion signal.
[0216] Preferably, the non-structural and/or internal protein is
selected from the group consisting of nucleoprotein NP, Matrix
proteins M1 and M2, non structural proteins NS1 and NS2/NEP, and
the RNA polymerases PA, PB1, PB2 and the protein PB1-F2
(PB1F2).
[0217] The nucleoprotein NP is a structural protein which
encapsidates the negative strand viral RNA. NP is one of the main
determinants of species specificity.
[0218] The protein M1 is a matrix protein of the influenza virus.
It forms a coat inside the viral envelope. The M1 protein binds to
the viral RNA. It also has multiple regulatory functions, performed
by interaction with the components of the host cell. The mechanisms
regulated include a role in the export of the viral
ribonucleoproteins from the host cell nucleus, inhibition of viral
transcription, and a role in the virus assembly and budding. The M1
protein forms a layer under the patches of host cell membrane that
are rich with the viral hemagglutinin, neuraminidase and M2
transmembrane proteins, and facilitates budding of the mature
viruses.
[0219] The non-structural NS1 protein is created by the internal
protein encoding, linear negative-sense, single stranded RNA, NS
gene segment and which also codes for the nuclear export protein or
NEP, formerly referred to as the NS2 protein, which mediates the
export of vRNPs. NS1 also binds dsRNA. As a consequence of its
binding to dsRNA, the NS1 protein blocks the activation of the
dsRNA-activated protein kinase (PKR) in vitro. This kinase
phosphorylates the alpha subunit of eukaryotic translation
initiation factor 2 (elF-2 alpha), leading to a decrease in the
rate of initiation of translation. In the absence of NS1, this
pathway is inhibited during anti-viral response to halt all protein
translation--thus stopping the synthesis of viral proteins;
however, the influenza virus' NS1 protein is an agent that
circumvents host defenses to allow viral gene transcription to
occur.
[0220] In preferred embodiments, HA comprises an amino acid
sequence of HA of one influenza A virus isolate or a consensus
amino acid sequence of two or more different influenza A virus
isolates, preferably according to SEQ ID NO: 9 or SEQ ID NO: 21, NP
comprises an amino acid sequence of NP of one influenza A virus
isolate or a consensus amino acid sequence of two or more different
influenza A virus isolates, preferably according to SEQ ID NO: 11,
and/or M1 comprises an amino acid sequence of M1 of one influenza A
virus isolate or a consensus amino acid sequence of two or more
different influenza A virus isolates, preferably according to SEQ
ID NO: 12. It is further preferred that when NP comprises the amino
acid sequence according to SEQ ID NO: 11 and M1 comprises the amino
acid sequence according to SEQ ID NO: 12.
[0221] In the context of the present invention, the structural
and/or surface protein encoded by the first polynucleotide is
located either N- or C-terminally with respect to the
non-structural and/or internal protein encoded by the second
polynucleotide. In a preferred embodiment, the non-structural
and/or internal protein encoded by the second polynucleotide is
located C-terminally with respect to the structural and/or surface
protein encoded by the first polynucleotide.
[0222] More specifically, HA or NA can be located N- or
C-terminally of NP, M1, M2, NS1, NS2/NEP, PA, PB1, PB2 or PB1-F2
(PB1F2). In a preferred embodiment M1 is located N-terminally of
HA.
[0223] Accordingly, embodiments of the present invention have the
formula X-Y or Y-X, wherein "X" depicts HA or NA, preferably HA,
and "Y" depicts NP, M1, M2, NS1, NS2/NEP, PA, PB1, PB2 or PB1-F2
(PB1F2), preferably NP or M1, and a "dash" depicts a peptide bond.
Preferred arrangements are the following:
[0224] HA-NP, HA-M1, HA-M2, HA-NS1, HA-NS2/NEP, HA-PA, HA-PB1,
HA-PB2, HA-PB1F2, NP-HA, M1-HA, M2-HA, NS1-HA, NS2/NEP-HA, PA-HA,
PB1-HA, PB2-HA, PB1F2-HA, NA-NP, NA-M1, NA-M2, NA-NS1, NA-NS2/NEP,
NA-PA, NA-PB1, NA-PB2, NA-PB1F2, NP-NA, M1-NA, M2-NA, NS1-NA,
NS2/NEP-NA, PA-NA, PB1-NA, PB2-NA or PB1F2-NA. A particularly
preferred arrangement is M1-HA.
[0225] It is within the scope of the present invention that every
protein can be combined with any other protein.
[0226] In preferred embodiments of the first aspect, a
polynucleotide encoding a cleavage site is positioned between the
first polynucleotide and the second polynucleotide.
[0227] It is preferred that this cleavage site is either a
self-cleaving site (i.e. a cleavage site within the amino acid
sequence where this sequence is cleaved or is cleavable without
such cleavage involving any additional molecule or where the
peptide-bond formation in this sequence is prevented in the first
place) or an endopeptidase cleavage site (i.e. a cleavage cite
within the amino acid sequence where this sequence is cleaved or is
cleavable by an endopeptidase, e.g. trypsin, pepsin, elastase,
thrombin, collagenase, furin, thermolysin, endopeptidase V8,
cathepsins). More preferably, the self-cleaving site is a 2A
cleavage site selected from the group consisting of a viral 2A
peptide or 2A-like peptide of Picornavirus, insect viruses,
Aphtoviridae, Rotaviruses and Trypanosoma, preferably wherein the
2A cleavage site is the 2 A peptide of foot and mouth disease
virus. Alternatively or additionally, the polyprotein of the
present invention can be cleaved by an autoprotease, i.e. a
protease which cleaves peptide bonds in the same protein molecule
which also comprises the protease. Examples of such autoproteases
are the NS2 protease from flaviviruses or the VP4 protease of
birnaviruses.
[0228] In the context of the present invention, the cleavage site
can be positioned N-terminally with respect to the structural
and/or surface protein encoded by the first polynucleotide and
C-terminally with respect to the non-structural and/or internal
protein encoded by the second polynucleotide. Alternatively the
cleavage site can be positioned C-terminally with respect to the
structural and/or surface protein encoded by the first
polynucleotide and N-terminally with respect to the non-structural
and/or internal protein encoded by the second polynucleotide. More
specifically, the cleavage site can be positioned C- or
N-terminally with respect to HA or NA and C- or N-terminally with
respect to NP, M1, M2, NS1, NS2/NEP, PA, PB1, PB2 or PB1-F2
(PB1F2). In a preferred embodiment the cleavage site is located
C-terminally with respect to NP M2, NS1, NS2/NEP, PA, PB1, PB2 or
PB1-F2 (PB1F2) and N-terminally with respect to HA or NA. It is
particularly preferred that the cleavage site is located
N-terminally with respect to HA and C-terminally with respect to
M1.
[0229] Accordingly, embodiments of the present invention have the
formula X-C-Y or Y-C-X, wherein "X" depicts HA or NA, preferably,
HA and "Y" depicts NP, M1, M2, NS1, NS2/NEP, PA, PB1, PB2 or PB1-F2
(PB1F2), preferably NP or M1, "C" depicts a cleavage site, and a
"dash" depicts a peptide bond.
[0230] Preferred arrangements are the following:
[0231] HA-C-NP, HA-C-M1, HA-C-M2, HA-C-NS1, HA-C-NS2/NEP, HA-C-PA,
HA-C-PB1, HA-C-PB2, HA-C-PB1F2, NP-C-HA, M1-C-HA, M2-C-HA,
NS1-C-HA, NS2/NEP-C-HA, PA-C-HA, PB1-C-HA, PB2-C-HA, PB1F2-C-HA,
NA-C-NP, NA-C-M1, NA-C-M2, NA-C-NS1, NA-C-NS2/NEP, NA-C-PA,
NA-C-PB1, NA-C-PB2, NA-C-PB1F2, NP-C-NA, M1-C-NA, M2-C-NA,
NS1-C-NA, NS2/NEP-C-NA, PA-C-NA, PB1-C-NA, PB2-C-NA or PB1F2-C-NA.
A particularly preferred arrangement is M1-C-HA.
[0232] It is within the scope of the present invention that every
protein can be combined with any other protein and that any two
proteins can or cannot be connected or linked by a cleavage
site.
[0233] In preferred embodiment of the first aspect, the expression
system further comprises a third polynucleotide encoding a
non-structural and/or internal protein of an orthomyxovirus or a
variant thereof. Preferably, the non-structural and/or internal
protein is of a orthomyxovirus selected from the genus of
Influenzavirus A, Influenzavirus B, Influenzavirus C, Thogotoviris
and Isavirus. In even more preferred embodiments, the
orthomxyovirus is Influenzavirus A, preferably selected from the
influenza A virus subtypes H1N1, H1N2, H2N2, H3N1, H3N2, H3N8,
H5N1, H5N2, H5N3, H5N8, H5N9, H7N1, H7N2, H7N3, H7N4, H7N7, H9N2,
H10N7, more preferably the influenza A virus subtype H1N1.
[0234] In preferred embodiments the third polynucleotide is
comprised on a separate or on the same vector as the first
polynucleotide and/or the second polynucleotide.
[0235] Accordingly, the first polynucleotide is comprised on one
vector and the second polynucleotide is comprised on a second
vector and the third polynucleotide is comprised on a third vector.
Alternatively or additionally, the first and the second
polynucleotide are comprised on the same vector and the third
polynucleotide is comprised on a separate vector, or the first and
the third polynucleotide are comprised on the same vector and the
second polynucleotide is comprised on a separate vector, or the
second and the third polynucleotide are comprised on the same
vector and the first polynucleotide is comprised on a separate
vector. Alternatively or additionally, the first and the second and
the third polynucleotide are comprised on the same vector. It is
preferred that the first and the second and the third
polynucleotide may be comprised on the same vector. It is
particularly preferred that the first and the second and the third
polynucleotide comprised on the same vector are linked in such that
they are expressed as a viral polyprotein. Preferably, the first
and the second and the third polynucleotide comprised on the same
vector form an open reading frame.
[0236] It is further preferred that the non-structural and/or
internal protein encoded by the third polynucleotide is a conserved
internal protein suitable for inducing a T cell mediated immune
response against the virus involving the activation of
antigen-specific T lymphocyte such as but not limited to cytotoxic
T cells (CTLs), T helper cells (T.sub.H cells), central memory T
cells (TCM cells), effector memory T cells (TEM cells), and
regulatory T cells (Treg cells).
[0237] Preferably, the non-structural and/or internal protein is
selected from the group consisting of NP, M1, M2, NS1, NS2/NEP, PA,
PB1, PB2 or PB1-F2 (PB1F2), more preferably NP or M1.
[0238] It is preferred that the non-structural and/or internal
protein encoded by the third polynucleotide differs from the
non-structural and/or internal protein encoded by the second
polynucleotide.
[0239] The non-structural and/or internal proteins encoded by the
second and the third polynucleotide differ from each other in that
they comprise amino acid sequences of different viral proteins. For
instance, this means that the non-structural and/or internal
protein encoded by the second polynucleotide comprises the amino
acid sequence of the M1 protein whilst the non-structural and/or
internal protein encoded by the third polynucleotide comprises the
amino acid sequence of the NP protein or vice versa.
[0240] The non-structural and/or internal protein encoded by the
third polynucleotide can be located either N- or C-terminally of
the non-structural and/or internal protein encoded by the second
polynucleotide. In a preferred embodiment of the first aspect, the
non-structural and/or internal protein encoded by the third
polynucleotide is located C-terminally of the non-structural and/or
internal protein encoded by the second polynucleotide.
[0241] In preferred embodiments a polynucleotide encoding a linker
is positioned between the second polynucleotide and the third
polynucleotide. It is preferred that the linker is a flexible
linker, preferably a flexible linker comprising an amino acid
sequence according to SEQ ID NO: 6.
[0242] In embodiments of the first aspect, the protein encoded by
the second polynucleotide is located N-terminally with respect to
the protein encoded by the first polynucleotide and/or the protein
of the optional third polynucleotide, or the protein encoded by the
second polynucleotide is located C-terminally with respect to the
protein encoded by the first polynucleotide and/or the protein of
the optional third polynucleotide.
[0243] In even more preferred embodiments of this aspect, the first
polynucleotide is located N-terminally with respect to the protein
encoded by the second polynucleotide and/or the protein of the
optional third polynucleotide is located N-terminally with respect
to the protein encoded by the first polynucleotide or is located
between the protein encoded by the second polynucleotide and the
protein encoded by the first polynucleotide; or the protein encoded
by the first polynucleotide is located C-terminally with respect to
the protein encoded by the second polynucleotide and/or the protein
of the optional third polynucleotide is located C-terminally with
respect to the protein encoded by the first polynucleotide or is
located between the protein encoded by the second polynucleotide
and the protein encoded by the first polynucleotide. More
specifically, HA, or NA are located C- or N-terminally with respect
to NP, M1, M2, NS1, NS2/NEP, PA, PB1, PB2 or PB1-F2 (PB1F2).
[0244] In a preferred embodiment HA is located C-terminally with
respect to M1 and NP is located N-terminally with respect to
M1.
[0245] Accordingly, preferred embodiments of the present invention
have the formula X-K-Y, Y-K-X, X-K-Y-Y, Y-Y-K-X, X-Y-K-Y, Y-K-Y-X,
X-K-Y-K-Y, Y-K-Y-K-X, X-C-Y, Y-C-X, X-C-Y-Y, Y-Y-C-X, X-Y-C-Y,
Y-C-Y-X, X-C-Y-C-Y, Y-C-Y-C-X, X-K-Y-C-Y, Y-C-Y-K-X, X-C-Y-K-Y, or
Y-K-Y-C-X, wherein "X" depicts HA, or NA, preferably HA, and "Y"
depicts NP, M1, M2, NS1, NS2/NEP, PA, PB1, PB2 or PB1-F2 (PB1F2),
preferably NP or M1, "K" indicates that one or more peptide linkers
are present in this position, "C" indicates that one or more
cleavage sites are present in this position and a "dash" depicts a
peptide bond. Preferred arrangements are Y-K-Y-C-X. Even more
preferred arrangements are the following: HA-K-NP, NP-K-HA,
HA-K-NP-NP, NP-NP-K-HA, HA-NP-K-NP, NP-K-NP-HA, HA-K-NP-K-NP,
NP-K-NP-K-HA, HA-C-NP, NP-C-HA, HA-C-NP-NP, NP-NP-C-HA, HA-NP-C-NP,
NP-C-NP-HA, HA-C-NP-C-NP, NP-C-NP-C-HA, HA-K-NP-C-NP, NP-C-NP-K-HA,
HA-C-NP-K-NP, NP-K-NP-C-HA, HA-K-NP-M1, HA-K-M1-NP, NP-M1-K-HA,
M1-NP-K-HA, HA-NP-K-M1, HA-M1-K-NP, NP-K-M1-HA, M1-K-NP-HA,
HA-K-NP-K-M1, HA-K-M1-K-NP, NP-K-M1-K-HA, M1-K-NP-K-HA, HA-C-NP-M1,
HA-C-M1-NP, NP-M1-C-HA, M1-NP-C-HA, HA-NP-C-M1, HA-M1-C-NP,
NP-C-M1-HA, M1-C-NP-HA, HA-C-NP-C-M1, HA-C-M1-C-NP, NP-C-M1-C-HA,
M1-C-NP-C-HA, HA-K-NP-C-M1, HA-K-M1-C-NP, NP-C-M1-K-HA,
M1-C-NP-K-HA, HA-C-NP-K-M1, HA-C-M1-K-NP, NP-K-M1-C-HA,
M1-K-NP-C-HA, HA-K-NP-M2, HA-K-M2-NP, NP-M2-K-HA, M2-NP-K-HA,
HA-NP-K-M2, HA-M2-K-NP, NP-K-M2-HA, M2-K-NP-HA, HA-K-NP-K-M2,
HA-K-M2-K-NP, NP-K-M2-K-HA, M2-K-NP-K-HA, HA-C-NP-M2, HA-C-M2-NP,
NP-M2-C-HA, M2-NP-C-HA, HA-NP-C-M2, HA-M2-C-NP, NP-C-M2-HA,
M2-C-NP-HA, HA-C-NP-C-M2, HA-C-M2-C-NP, NP-C-M2-C-HA, M2-C-NP-C-HA,
HA-K-NP-C-M2, HA-K-M2-C-NP, NP-C-M2-K-HA, M2-C-NP-K-HA,
HA-C-NP-K-M2, HA-C-M2-K-NP, NP-K-M2-C-HA, M2-K-NP-C-HA,
HA-K-NP-NS1, HA-K-NS1-NP, NP-NS1-K-HA, NS1-NP-K-HA, HA-NP-K-NS1,
HA-NS1-K-NP, NP-K-NS1-HA, NS1-K-NP-HA, HA-K-NP-K-NS1,
HA-K-NS1-K-NP, NP-K-NS1-K-HA, NS1-K-NP-K-HA, HA-C-NP-NS1,
HA-C-NS1-NP, NP-NS1-C-HA, NS1-NP-C-HA, HA-NP-C-NS1, HA-NS1-C-NP,
NP-C-NS1-HA, NS1-C-NP-HA, HA-C-NP-C-NS1, HA-C-NS1-C-NP,
NP-C-NS1-C-HA, NS1-C-NP-C-HA, HA-K-NP-C-NS1, HA-K-NS1-C-NP,
NP-C-NS1-K-HA, NS1-C-NP-K-HA, HA-C-NP-K-NS1, HA-C-NS1-K-NP,
NP-K-NS1-C-HA, NS1-K-NP-C-HA, HA-K-NP-NS2/NEP, HA-K-NS2/NEP-NP,
NP-NS2/NEP-K-HA, NS2/NEP-NP-K-HA, HA-NP-K-NS2/NEP, HA-NS2/NEP-K-NP,
NP-K-NS2/NEP-HA, NS2/NEP-K-NP-HA, HA-K-NP-K-NS2/NEP,
HA-K-NS2/NEP-K-NP, NP-K-NS2/NEP-K-HA, NS2/NEP-K-NP-K-HA,
HA-C-NP-NS2/NEP, HA-C-NS2/NEP-NP, NP-NS2/NEP-C-HA, NS2/NEP-NP-C-HA,
HA-NP-C-NS2/NEP, HA-NS2/NEP-C-NP, NP-C-NS2/NEP-HA, NS2/NEP-C-NP-HA,
HA-C-NP-C-NS2/NEP, HA-C-NS2/NEP-C-NP, NP-C-NS2/NEP-C-HA,
NS2/NEP-C-NP-C-HA, HA-K-NP-C-NS2/NEP, HA-K-NS2/NEP-C-NP,
NP-C-NS2/NEP-K-HA, NS2/NEP-C-NP-K-HA, HA-C-NP-K-NS2/NEP,
HA-C-NS2/NEP-K-NP, NP-K-NS2/NEP-C-HA, NS2/NEP-K-NP-C-HA,
HA-K-NP-PA, HA-K-PA-NP, NP-PA-K-HA, PA-NP-K-HA, HA-NP-K-PA,
HA-PA-K-NP, NP-K-PA-HA, PA-K-NP-HA, HA-K-NP-K-PA, HA-K-PA-K-NP,
NP-K-PA-K-HA, PA-K-NP-K-HA, HA-C-NP-PA, HA-C-PA-NP, NP-PA-C-HA,
PA-NP-C-HA, HA-NP-C-PA, HA-PA-C-NP, NP-C-PA-HA, PA-C-NP-HA,
HA-C-NP-C-PA, HA-C-PA-C-NP, NP-C-PA-C-HA, PA-C-NP-C-HA,
HA-K-NP-C-PA, HA-K-PA-C-NP, NP-C-PA-K-HA, PA-C-NP-K-HA,
HA-C-NP-K-PA, HA-C-PA-K-NP, NP-K-PA-C-HA, PA-K-NP-C-HA,
HA-K-NP-PB1, HA-K-PB1-NP, NP-PB1-K-HA, PB1-NP-K-HA, HA-NP-K-PB1,
HA-PB1-K-NP, NP-K-PB1-HA, PB1-K-NP-HA, HA-K-NP-K-PB1,
HA-K-PB1-K-NP, NP-K-PB1-K-HA, PB1-K-NP-K-HA, HA-C-NP-PB1,
HA-C-PB1-NP, NP-PB1-C-HA, PB1-NP-C-HA, HA-NP-C-PB1, HA-PB1-C-NP,
NP-C-PB1-HA, PB1-C-NP-HA, HA-C-NP-C-PB1, HA-C-PB1-C-NP,
NP-C-PB1-C-HA, PB1-C-NP-C-HA, HA-K-NP-C-PB1, HA-K-PB1-C-NP,
NP-C-PB1-K-HA, PB1-C-NP-K-HA, HA-C-NP-K-PB1, HA-C-PB1-K-NP,
NP-K-PB1-C-HA, PB1-K-NP-C-HA, HA-K-NP-PB2, HA-K-PB2-NP,
NP-PB2-K-HA, PB2-NP-K-HA, HA-NP-K-PB2, HA-PB2-K-NP, NP-K-PB2-HA,
PB2-K-NP-HA, HA-K-NP-K-PB2, HA-K-PB2-K-NP, NP-K-PB2-K-HA,
PB2-K-NP-K-HA, HA-C-NP-PB2, HA-C-PB2-NP, NP-PB2-C-HA, PB2-NP-C-HA,
HA-NP-C-PB2, HA-PB2-C-NP, NP-C-PB2-HA, PB2-C-NP-HA, HA-C-NP-C-PB2,
HA-C-PB2-C-NP, NP-C-PB2-C-HA, PB2-C-NP-C-HA, HA-K-NP-C-PB2,
HA-K-PB2-C-NP, NP-C-PB2-K-HA, PB2-C-NP-K-HA, HA-C-NP-K-PB2,
HA-C-PB2-K-NP, NP-K-PB2-C-HA, PB2-K-NP-C-HA, HA-K-NP-PB1F2,
HA-K-PB1F2-NP, NP-PB1F2-K-HA, PB1F2-NP-K-HA, HA-NP-K-PB1F2,
HA-PB1F2-K-NP, NP-K-PB1F2-HA, PB1F2-K-NP-HA, HA-K-NP-K-PB1F2,
HA-K-PB1F2-K-NP, NP-K-PB1F2-K-HA, PB1F2-K-NP-K-HA, HA-C-NP-PB1F2,
HA-C-PB1F2-NP, NP-PB1F2-C-HA, PB1F2-NP-C-HA, HA-NP-C-PB1F2,
HA-PB1F2-C-NP, NP-C-PB1F2-HA, PB1F2-C-NP-HA, HA-C-NP-C-PB1F2,
HA-C-PB1F2-C-NP, NP-C-PB1F2-C-HA, PB1F2-C-NP-C-HA, HA-K-NP-C-PB1F2,
HA-K-PB1F2-C-NP, NP-C-PB1F2-K-HA, PB1F2-C-NP-K-HA, HA-C-NP-K-PB1F2,
HA-C-PB1F2-K-NP, NP-K-PB1F2-C-HA, PB1F2-K-NP-C-HA, HA-K-M1,
M1-K-HA, HA-K-M1-M1, M1-M1-K-HA, HA-M1-K-M1, M1-K-M1-HA,
HA-K-M1-K-M1, M1-K-M1-K-HA, HA-C-M1, M1-C-HA, HA-C-M1-M1,
M1-M1-C-HA, HA-M1-C-M1, M1-C-M1-HA, HA-C-M1-C-M1, M1-C-M1-C-HA,
HA-K-M1-C-M1, M1-C-M1-K-HA, HA-C-M1-K-M1, M1-K-M1-C-HA, HA-K-M1-M2,
HA-K-M2-M1, M1-M2-K-HA, M2-M1-K-HA, HA-M1-K-M2, HA-M2-K-M1,
M1-K-M2-HA, M2-K-M1-HA, HA-K-M1-K-M2, HA-K-M2-K-M 1, M1-K-M2-K-HA,
M2-K-M 1-K-HA, HA-C-M1-M2, HA-C-M2-M1, M1-M2-C-HA, M2-M 1-C-HA,
HA-M1-C-M2, HA-M2-C-M1, M1-C-M2-HA, M2-C-M1-HA, HA-C-M1-C-M2,
HA-C-M2-C-M1, M1-C-M2-C-HA, M2-C-M1-C-HA, HA-K-M1-C-M2,
HA-K-M2-C-M1, M1-C-M2-K-HA, M2-C-M1-K-HA, HA-C-M1-K-M2,
HA-C-M2-K-M1, M1-K-M2-C-HA, M2-K-M1-C-HA, HA-K-M1-NS1, HA-K-NS1-M1,
M1-NS1-K-HA, NS1-M1-K-HA, HA-M1-K-NS1, HA-NS1-K-M1, M1-K-NS1-HA,
NS1-K-M1-HA, HA-K-M1-K-NS1, HA-K-NS1-K-M1, M1-K-NS1-K-HA,
NS1-K-M1-K-HA, HA-C-M1-NS1, HA-C-NS1-M1, M1-NS1-C-HA, NS1-M1-C-HA,
HA-M1-C-NS1, HA-NS1-C-M1, M1-C-NS1-HA, NS1-C-M1-HA, HA-C-M1-C-NS1,
HA-C-NS1-C-M1, M1-C-NS1-C-HA, NS1-C-M1-C-HA, HA-K-M1-C-NS1,
HA-K-NS1-C-M1, M1-C-NS1-K-HA, NS1-C-M1-K-HA, HA-C-M1-K-NS1,
HA-C-NS1-K-M1, M1-K-NS1-C-HA, NS1-K-M1-C-HA, HA-K-M1-NS2/NEP,
HA-K-NS2/NEP-M1, M1-NS2NEP-K-HA, NS2/NEP-M1-K-HA, HA-M1-K-NS2/NEP,
HA-NS2/NEP-K-M1, M1-K-NS2/NEP-HA, NS2/NEP-K-M1-HA,
HA-K-M1-K-NS2/NEP, HA-K-NS2/NEP-K-M1, M1-K-NS2/NEP-K-HA,
NS2/NEP-K-M1-K-HA, HA-C-M1-NS2/NEP, HA-C-NS2/NEP-M1,
M1-NS2/NEP-C-HA, NS2/NEP-M1-C-HA, HA-M1-C-NS2/NEP, HA-NS2/NEP-C-M1,
M1-C-NS2/NEP-HA, NS2/NEP-C-M1-HA, HA-C-M1-C-NS2/NEP,
HA-C-NS2/NEP-C-M1, M1-C-NS2/NEP-C-HA, NS2/NEP-C-M1-C-HA,
HA-K-M1-C-NS2/NEP, HA-K-NS2/NEP-C-M1, M1-C-NS2NEP-K-HA,
NS2/NEP-C-M1-K-HA, HA-C-M1-K-NS2/NEP, HA-C-NS2/NEP-K-M1,
M1-K-NS2/NEP-C-HA, NS2/NEP-K-M1-C-HA, HA-K-M1-PA, HA-K-PA-M1,
M1-PA-K-HA, PA-M1-K-HA, HA-M1-K-PA, HA-PA-K-M1, M1-K-PA-HA,
PA-K-M1-HA, HA-K-M1-K-PA, HA-K-PA-K-M1, M1-K-PA-K-HA, PA-K-M1-K-HA,
HA-C-M1-PA, HA-C-PA-M1, M1-PA-C-HA, PA-M1-C-HA, HA-M1-C-PA,
HA-PA-C-M1, M1-C-PA-HA, PA-C-M1-HA, HA-C-M1-C-PA, HA-C-PA-C-M1,
M1-C-PA-C-HA, PA-C-M1-C-HA, HA-K-M1-C-PA, HA-K-PA-C-M1,
M1-C-PA-K-HA, PA-C-M1-K-HA, HA-C-M1-K-PA, HA-C-PA-K-M1,
M1-K-PA-C-HA, PA-K-M1-C-HA, HA-K-M1-PB1, HA-K-PB1-M1, M1-PB1-K-HA,
PB1-M1-K-HA, HA-M1-K-PB1, HA-PB1-K-M1, M1-K-PB1-HA, PB1-K-M1-HA,
HA-K-M1-K-PB1, HA-K-PB1-K-M1, M1-K-PB1-K-HA, PB1-K-M1-K-HA,
HA-C-M1-PB1, HA-C-PB1-M1, M1-PB1-C-HA, PB1-M1-C-HA, HA-M1-C-PB1,
HA-PB1-C-M1, M1-C-PB1-HA, PB1-C-M1-HA, HA-C-M1-C-PB1,
HA-C-PB1-C-M1, M1-C-PB1-C-HA, PB1-C-M1-C-HA, HA-K-M1-C-PB1,
HA-K-PB1-C-M1, M1-C-PB1-K-HA, PB1-C-M1-K-HA, HA-C-M1-K-PB1,
HA-C-PB1-K-M1, M1-K-PB1-C-HA, PB1-K-M1-C-HA, HA-K-M1-PB2,
HA-K-PB2-M1, M1-PB2-K-HA, PB2-M1-K-HA, HA-M1-K-PB2, HA-PB2-K-M1,
M1-K-PB2-HA, PB2-K-M1-HA, HA-K-M1-K-PB2, HA-K-PB2-K-M1,
M1-K-PB2-K-HA, PB2-K-M1-K-HA, HA-C-M1-PB2, HA-C-PB2-M1,
M1-PB2-C-HA, PB2-M1-C-HA, HA-M1-C-PB2, HA-PB2-C-M1, M1-C-PB2-HA,
PB2-C-M1-HA, HA-C-M1-C-PB2, HA-C-PB2-C-M1, M1-C-PB2-C-HA,
PB2-C-M1-C-HA, HA-K-M1-C-PB2, HA-K-PB2-C-M1, M1-C-PB2-K-HA,
PB2-C-M1-K-HA, HA-C-M1-K-PB2, HA-C-PB2-K-M1, M1-K-PB2-C-HA,
PB2-K-M1-C-HA, HA-K-M1-PB1F2, HA-K-PB1F2-M1, M1-PB1F2-K-HA,
PB1F2-M1-K-HA, HA-M1-K-PB1F2, HA-PB1F2-K-M1, M1-K-PB1F2-HA,
PB1F2-K-M1-HA, HA-K-M1-K-PB1F2, HA-K-PB1F2-K-M1, M1-K-PB1F2-K-HA,
PB1F2-K-M1-K-HA, HA-C-M1-PB1F2, HA-C-PB1F2-M1, M1-PB1F2-C-HA,
PB1F2-M1-C-HA, HA-M1-C-PB1F2, HA-PB1F2-C-M1, M1-C-PB1F2-HA,
PB1F2-C-M1-HA, HA-C-M1-C-PB1F2, HA-C-PB1F2-C-M1, M1-C-PB1F2-C-HA,
PB1F2-C-M1-C-HA, HA-K-M1-C-PB1F2, HA-K-PB1F2-C-M1, M1-C-PB1F2-K-HA,
PB1F2-C-M1-K-HA, HA-C-M1-K-PB1F2, HA-C-PB1F2-K-M1, M1-K-PB1F2-C-HA,
PB1F2-K-M1-C-HA, HA-K-M2, M2-K-HA, HA-K-M2-M2, M2-M2-K-HA,
HA-M2-K-M2, M2-K-M2-HA, HA-K-M2-K-M2, M2-K-M2-K-HA, HA-C-M2,
M2-C-HA, HA-C-M2-M2, M2-M2-C-HA, HA-M2-C-M2, M2-C-M2-HA,
HA-C-M2-C-M2, M2-C-M2-C-HA, HA-K-M2-C-M2, M2-C-M2-K-HA,
HA-C-M2-K-M2, M2-K-M2-C-HA, HA-K-M2-NS1, HA-K-NS1-M2, M2-NS1-K-HA,
NS1-M2-K-HA, HA-M2-K-NS1, HA-NS1-K-M2, M2-K-NS1-HA, NS1-K-M2-HA,
HA-K-M2-K-NS1, HA-K-NS1-K-M2, M2-K-NS1-K-HA, NS1-K-M2-K-HA,
HA-C-M2-NS1, HA-C-NS1-M2, M2-NS1-C-HA, NS1-M2-C-HA, HA-M2-C-NS1,
HA-NS1-C-M2, M2-C-NS1-HA, NS1-C-M2-HA, HA-C-M2-C-NS1,
HA-C-NS1-C-M2, M2-C-NS1-C-HA, NS1-C-M2-C-HA, HA-K-M2-C-NS1,
HA-K-NS1-C-M2, M2-C-NS1-K-HA, NS1-C-M2-K-HA, HA-C-M2-K-NS1,
HA-C-NS1-K-M2, M2-K-NS1-C-HA, NS1-K-M2-C-HA, HA-K-M2-NS2/NEP,
HA-K-NS2/NEP-M2, M2-NS2/NEP-K-HA, NS2/NEP-M2-K-HA, HA-M2-K-NS2/NEP,
HA-NS2/NEP-K-M2, M2-K-NS2/NEP-HA, NS2/NEP-K-M2-HA,
HA-K-M2-K-NS2/NEP, HA-K-NS2/NEP-K-M2, M2-K-NS2/NEP-K-HA,
NS2/NEP-K-M2-K-HA, HA-C-M2-NS2/NEP, HA-C-NS2/NEP-M2,
M2-NS2/NEP-C-HA, NS2/NEP-M2-C-HA, HA-M2-C-NS2/NEP, HA-NS2/NEP-C-M2,
M2-C-NS2/NEP-HA, NS2/NEP-C-M2-HA, HA-C-M2-C-NS2/NEP,
HA-C-NS2/NEP-C-M2, M2-C-NS2/NEP-C-HA, NS2/NEP-C-M2-C-HA,
HA-K-M2-C-NS2/NEP, HA-K-NS2/NEP-C-M2, M2-C-NS2/NEP-K-HA,
NS2/NEP-C-M2-K-HA, HA-C-M2-K-NS2/NEP, HA-C-NS2/NEP-K-M2,
M2-K-NS2/NEP-C-HA, NS2/NEP-K-M2-C-HA, HA-K-M2-PA, HA-K-PA-M2,
M2-PA-K-HA, PA-M2-K-HA, HA-M2-K-PA, HA-PA-K-M2, M2-K-PA-HA,
PA-K-M2-HA, HA-K-M2-K-PA, HA-K-PA-K-M2, M2-K-PA-K-HA, PA-K-M2-K-HA,
HA-C-M2-PA, HA-C-PA-M2, M2-PA-C-HA, PA-M2-C-HA, HA-M2-C-PA,
HA-PA-C-M2, M2-C-PA-HA, PA-C-M2-HA, HA-C-M2-C-PA, HA-C-PA-C-M2,
M2-C-PA-C-HA, PA-C-M2-C-HA, HA-K-M2-C-PA, HA-K-PA-C-M2,
M2-C-PA-K-HA, PA-C-M2-K-HA, HA-C-M2-K-PA, HA-C-PA-K-M2,
M2-K-PA-C-HA, PA-K-M2-C-HA, HA-K-M2-PB1, HA-K-PB1-M2, M2-PB1-K-HA,
PB1-M2-K-HA, HA-M2-K-PB1, HA-PB1-K-M2, M2-K-PB1-HA, PB1-K-M2-HA,
HA-K-M2-K-PB1, HA-K-PB1-K-M2, M2-K-PB1-K-HA, PB1-K-M2-K-HA,
HA-C-M2-PB1, HA-C-PB1-M2, M2-PB1-C-HA, PB1-M2-C-HA, HA-M2-C-PB1,
HA-PB1-C-M2, M2-C-PB1-HA, PB1-C-M2-HA, HA-C-M2-C-PB1,
HA-C-PB1-C-M2, M2-C-PB1-C-HA, PB1-C-M2-C-HA, HA-K-M2-C-PB1,
HA-K-PB1-C-M2, M2-C-PB1-K-HA, PB1-C-M2-K-HA, HA-C-M2-K-PB1,
HA-C-PB1-K-M2, M2-K-PB1-C-HA, PB1-K-M2-C-HA, HA-K-M2-PB2,
HA-K-PB2-M2, M2-PB2-K-HA, PB2-M2-K-HA, HA-M2-K-PB2, HA-PB2-K-M2,
M2-K-PB2-HA, PB2-K-M2-HA, HA-K-M2-K-PB2, HA-K-PB2-K-M2,
M2-K-PB2-K-HA, PB2-K-M2-K-HA, HA-C-M2-PB2, HA-C-PB2-M2,
M2-PB2-C-HA, PB2-M2-C-HA, HA-M2-C-PB2, HA-PB2-C-M2, M2-C-PB2-HA,
PB2-C-M2-HA, HA-C-M2-C-PB2, HA-C-PB2-C-M2, M2-C-PB2-C-HA,
PB2-C-M2-C-HA, HA-K-M2-C-PB2, HA-K-PB2-C-M2, M2-C-PB2-K-HA,
PB2-C-M2-K-HA, HA-C-M2-K-PB2, HA-C-PB2-K-M2, M2-K-PB2-C-HA,
PB2-K-M2-C-HA, HA-K-M2-PB1F2, HA-K-PB1F2-M2, M2-PB1F2-K-HA,
PB1F2-M2-K-HA, HA-M2-K-PB1F2, HA-PB1F2-K-M2, M2-K-PB1F2-HA,
PB1F2-K-M2-HA, HA-K-M2-K-PB1F2, HA-K-PB1F2-K-M2, M2-K-PB1F2-K-HA,
PB1F2-K-M2-K-HA, HA-C-M2-PB1F2, HA-C-PB1F2-M2, M2-PB1F2-C-HA,
PB1F2-M2-C-HA, HA-M2-C-PB1F2, HA-PB1F2-C-M2, M2-C-PB1F2-HA,
PB1F2-C-M2-HA, HA-C-M2-C-PB1F2, HA-C-PB1F2-C-M2, M2-C-PB1F2-C-HA,
PB1F2-C-M2-C-HA, HA-K-M2-C-PB1F2, HA-K-PB1F2-C-M2, M2-C-PB1F2-K-HA,
PB1F2-C-M2-K-HA, HA-C-M2-K-PB1F2, HA-C-PB1F2-K-M2, M2-K-PB1F2-C-HA,
PB1F2-K-M2-C-HA, HA-K-NS1, NS1-K-HA, HA-K-NS1-NS1, NS1-NS1-K-HA,
HA-NS1-K-NS1, NS1-K-NS1-HA, HA-K-NS1-K-NS1, NS1-K-NS1-K-HA,
HA-C-NS1, NS1-C-HA, HA-C-NS1-NS1, NS1-NS1-C-HA, HA-NS1-C-NS1,
NS1-C-NS1-HA, HA-C-NS1-C-NS1, NS1-C-NS1-C-HA, HA-K-NS1-C-NS1,
NS1-C-NS1-K-HA, HA-C-NS1-K-NS1, NS1-K-NS1-C-HA, HA-K-NS1-NS2/NEP,
HA-K-NS2/NEP-NS1, NS1-NS2/NEP-K-HA, NS2/NEP-NS1-K-HA,
HA-NS1-K-NS2/NEP, HA-NS2/NEP-K-NS1, NS1-K-NS2/NEP-HA,
NS2/NEP-K-NS1-HA, HA-K-NS1-K-NS2/NEP, HA-K-NS2/NEP-K-NS1,
NS1-K-NS2/NEP-K-HA, NS2/NEP-K-NS1-K-HA, HA-C-NS1-NS2/NEP,
HA-C-NS2/NEP-NS1, NS1-NS2/NEP-C-HA, NS2/NEP-NS1-C-HA,
HA-NS1-C-NS2/NEP, HA-NS2/NEP-C-NS1, NS1-C-NS2/NEP-HA,
NS2/NEP-C-NS1-HA, HA-C-NS1-C-NS2/NEP, HA-C-NS2/NEP-C-NS1,
NS1-C-NS2/NEP-C-HA, NS2/NEP-C-NS1-C-HA, HA-K-NS1-C-NS2/NEP,
HA-K-NS2/NEP-C-NS1, NS1-C-NS2/NEP-K-HA, NS2/NEP-C-NS1-K-HA,
HA-C-NS1-K-NS2/NEP, HA-C-NS2/NEP-K-NS1, NS1-K-NS2/NEP-C-HA,
NS2/NEP-K-NS1-C-HA, HA-K-NS1-PA, HA-K-PA-NS1, NS1-PA-K-HA,
PA-NS1-K-HA, HA-NS1-K-PA, HA-PA-K-NS1, NS1-K-PA-HA, PA-K-NS1-HA,
HA-K-NS1-K-PA, HA-K-PA-K-NS1, NS1-K-PA-K-HA, PA-K-NS1-K-HA,
HA-C-NS1-PA, HA-C-PA-NS1, NS1-PA-C-HA, PA-NS1-C-HA, HA-NS1-C-PA,
HA-PA-C-NS1, NS1-C-PA-HA, PA-C-NS1-HA, HA-C-NS1-C-PA,
HA-C-PA-C-NS1, NS1-C-PA-C-HA, PA-C-NS1-C-HA, HA-K-NS1-C-PA,
HA-K-PA-C-NS1, NS1-C-PA-K-HA, PA-C-NS1-K-HA, HA-C-NS1-K-PA,
HA-C-PA-K-NS1, NS1-K-PA-C-HA, PA-K-NS1-C-HA, HA-K-NS1-PB1,
HA-K-PB1-NS1, NS1-PB1-K-HA, PB1-NS1-K-HA, HA-NS1-K-PB1,
HA-PB1-K-NS1, NS1-K-PB1-HA, PB1-K-NS1-HA, HA-K-NS1-K-PB1,
HA-K-PB1-K-NS1, NS1-K-PB1-K-HA, PB1-K-NS1-K-HA, HA-C-NS1-PB1,
HA-C-PB1-NS1, NS1-PB1-C-HA, PB1-NS1-C-HA, HA-NS1-C-PB1,
HA-PB1-C-NS1, NS1-C-PB1-HA, PB1-C-NS1-HA, HA-C-NS1-C-PB1,
HA-C-PB1-C-NS1, NS1-C-PB1-C-HA, PB1-C-NS1-C-HA, HA-K-NS1-C-PB1,
HA-K-PB1-C-NS1, NS1-C-PB1-K-HA, PB1-C-NS1-K-HA, HA-C-NS1-K-PB1,
HA-C-PB1-K-NS1, NS1-K-PB1-C-HA, PB1-K-NS1-C-HA, HA-K-NS1-PB2,
HA-K-PB2-NS1, NS1-PB2-K-HA, PB2-NS1-K-HA, HA-NS1-K-PB2,
HA-PB2-K-NS1, NS1-K-PB2-HA, PB2-K-NS1-HA, HA-K-NS1-K-PB2,
HA-K-PB2-K-NS1, NS1-K-PB2-K-HA, PB2-K-NS1-K-HA, HA-C-NS1-PB2,
HA-C-PB2-NS1, NS1-PB2-C-HA, PB2-NS1-C-HA, HA-NS1-C-PB2,
HA-PB2-C-NS1, NS1-C-PB2-HA, PB2-C-NS1-HA, HA-C-NS1-C-PB2,
HA-C-PB2-C-NS1, NS1-C-PB2-C-HA, PB2-C-NS1-C-HA, HA-K-NS1-C-PB2,
HA-K-PB2-C-NS1, NS1-C-PB2-K-HA, PB2-C-NS1-K-HA, HA-C-NS1-K-PB2,
HA-C-PB2-K-NS1, NS1-K-PB2-C-HA, PB2-K-NS1-C-HA, HA-K-NS1-PB1F2,
HA-K-PB1F2-NS1, NS1-PB1F2-K-HA, PB1F2-NS1-K-HA, HA-NS1-K-PB1F2,
HA-PB1F2-K-NS1, NS1-K-PB1F2-HA, PB1F2-K-NS1-HA, HA-K-NS1-K-PB1F2,
HA-K-PB1F2-K-NS1, NS1-K-PB1F2-K-HA, PB1F2-K-NS1-K-HA,
HA-C-NS1-PB1F2, HA-C-PB1F2-NS1, NS1-PB1F2-C-HA, PB1F2-NS1-C-HA,
HA-NS1-C-PB1F2, HA-PB1F2-C-NS1, NS1-C-PB1F2-HA, PB1F2-C-NS1-HA,
HA-C-NS1-C-PB1F2, HA-C-PB1F2-C-NS1, NS1-C-PB1F2-C-HA,
PB1F2-C-NS1-C-HA, HA-K-NS1-C-PB1F2, HA-K-PB1F2-C-NS1,
NS1-C-PB1F2-K-HA, PB1F2-C-NS1-K-HA, HA-C-NS1-K-PB1F2,
HA-C-PB1F2-K-NS1, NS1-K-PB1F2-C-HA, PB1F2-K-NS1-C-HA, HA-K-NS2/NEP,
NS2/NEP-K-HA, HA-K-NS2/NEP-NS2/NEP, NS2/NEP-NS2/NEP-K-HA,
HA-NS2/NEP-K-NS2/NEP, NS2/NEP-K-NS2/NEP-HA, HA-K-NS2/NEP-K-NS2/NEP,
NS2/NEP-K-NS2/NEP-K-HA, HA-C-NS2/NEP, NS2/NEP-C-HA,
HA-C-NS2/NEP-NS2/NEP, NS2/NEP-NS2/NEP-C-HA, HA-NS2/NEP-C-NS2/NEP,
NS2/NEP-C-NS2/NEP-HA, HA-C-NS2/NEP-C-NS2/NEP,
NS2/NEP-C-NS2/NEP-C-HA, HA-K-NS2/NEP-C-NS2/NEP,
NS2/NEP-C-NS2/NEP-K-HA, HA-C-NS2/NEP-K-NS2/NEP,
NS2/NEP-K-NS2/NEP-C-HA, HA-K-NS2/NEP-PA, HA-K-PA-NS2/NEP,
NS2/NEP-PA-K-HA, PA-NS2/NEP-K-HA, HA-NS2/NEP-K-PA, HA-PA-K-NS2/NEP,
NS2/NEP-K-PA-HA, PA-K-NS2/NEP-HA, HA-K-NS2/NEP-K-PA,
HA-K-PA-K-NS2/NEP, NS2/NEP-K-PA-K-HA, PA-K-NS2/NEP-K-HA,
HA-C-NS2/NEP-PA, HA-C-PA-NS2/NEP, NS2/NEP-PA-C-HA, PA-NS2/NEP-C-HA,
HA-NS2/NEP-C-PA, HA-PA-C-NS2/NEP, NS2/NEP-C-PA-HA, PA-C-NS2/NEP-HA,
HA-C-NS2/NEP-C-PA, HA-C-PA-C-NS2/NEP, NS2/NEP-C-PA-C-HA,
PA-C-NS2/NEP-C-HA, HA-K-NS2/NEP-C-PA, HA-K-PA-C-NS2/NEP,
NS2/NEP-C-PA-K-HA, PA-C-NS2/NEP-K-HA, HA-C-NS2/NEP-K-PA,
HA-C-PA-K-NS2/NEP, NS2/NEP-K-PA-C-HA, PA-K-NS2/NEP-C-HA,
HA-K-NS2/NEP-PB1, HA-K-PB1-NS2/NEP, NS2/NEP-PB1-K-HA,
PB1-NS2/NEP-K-HA, HA-NS2/NEP-K-PB1, HA-PB1-K-NS2/NEP,
NS2/NEP-K-PB1-HA, PB1-K-NS2/NEP-HA, HA-K-NS2/NEP-K-PB1,
HA-K-PB1-K-NS2/NEP, NS2/NEP-K-PB1-K-HA, PB1-K-NS2/NEP-K-HA,
HA-C-NS2/NEP-PB1, HA-C-PB1-NS2/NEP, NS2/NEP-PB1-C-HA,
PB1-NS2/NEP-C-HA, HA-NS2/NEP-C-PB1, HA-PB1-C-NS2/NEP,
NS2/NEP-C-PB1-HA, PB1-C-NS2/NEP-HA, HA-C-NS2/NEP-C-PB1,
HA-C-PB1-C-NS2/NEP, NS2/NEP-C-PB1-C-HA, PB1-C-NS2/NEP-C-HA,
HA-K-NS2/NEP-C-PB1, HA-K-PB1-C-NS2/NEP, NS2/NEP-C-PB1-K-HA,
PB1-C-NS2/NEP-K-HA, HA-C-NS2/NEP-K-PB1, HA-C-PB1-K-NS2/NEP,
NS2/NEP-K-PB1-C-HA, PB1-K-NS2/NEP-C-HA, HA-K-NS2/NEP-PB2,
HA-K-PB2-NS2/NEP, NS2/NEP-PB2-K-HA, PB2-NS2/NEP-K-HA,
HA-NS2/NEP-K-PB2, HA-PB2-K-NS2/NEP, NS2/NEP-K-PB2-HA,
PB2-K-NS2/NEP-HA, HA-K-NS2/NEP-K-PB2, HA-K-PB2-K-NS2/NEP,
NS2/NEP-K-PB2-K-HA, PB2-K-NS2/NEP-K-HA, HA-C-NS2/NEP-PB2,
HA-C-PB2-NS2/NEP, NS2/NEP-PB2-C-HA, PB2-NS2/NEP-C-HA,
HA-NS2/NEP-C-PB2, HA-PB2-C-NS2/NEP, NS2/NEP-C-PB2-HA,
PB2-C-NS2/NEP-HA, HA-C-NS2/NEP-C-PB2, HA-C-PB2-C-NS2/NEP,
NS2/NEP-C-PB2-C-HA, PB2-C-NS2/NEP-C-HA, HA-K-NS2/NEP-C-PB2,
HA-K-PB2-C-NS2/NEP, NS2/NEP-C-PB2-K-HA, PB2-C-NS2/NEP-K-HA,
HA-C-NS2/NEP-K-PB2, HA-C-PB2-K-NS2/NEP, NS2/NEP-K-PB2-C-HA,
PB2-K-NS2/NEP-C-HA, HA-K-NS2/NEP-PB1F2, HA-K-PB1F2-NS2/NEP,
NS2/NEP-PB1F2-K-HA, PB1F2-NS2/NEP-K-HA, HA-NS2/NEP-K-PB1F2,
HA-PB1F2-K-NS2/NEP, NS2/NEP-K-PB1F2-HA, PB1F2-K-NS2/NEP-HA,
HA-K-NS2/NEP-K-PB1F2, HA-K-PB1F2-K-NS2/NEP, NS2/NEP-K-PB1F2-K-HA,
PB1F2-K-NS2/NEP-K-HA, HA-C-NS2/NEP-PB1F2, HA-C-PB1F2-NS2/NEP,
NS2/NEP-PB1F2-C-HA, PB1F2-NS2/NEP-C-HA, HA-NS2/NEP-C-PB1F2,
HA-PB1F2-C-NS2/NEP, NS2/NEP-C-PB1F2-HA,
PB1F2-C-NS2/NEP-HA, HA-C-NS2/NEP-C-PB1F2, HA-C-PB1F2-C-NS2/NEP,
NS2/NEP-C-PB1F2-C-HA, PB1F2-C-NS2/NEP-C-HA, HA-K-NS2/NEP-C-PB1F2,
HA-K-PB1F2-C-NS2/NEP, NS2/NEP-C-PB1F2-K-HA, PB1F2-C-NS2/NEP-K-HA,
HA-C-NS2/NEP-K-PB1F2, HA-C-PB1F2-K-NS2/NEP, NS2/NEP-K-PB1F2-C-HA,
PB1F2-K-NS2/NEP-C-HA, HA-K-PA, PA-K-HA, HA-K-PA-PA, PA-PA-K-HA,
HA-PA-K-PA, PA-K-PA-HA, HA-K-PA-K-PA, PA-K-PA-K-HA, HA-C-PA,
PA-C-HA, HA-C-PA-PA, PA-PA-C-HA, HA-PA-C-PA, PA-C-PA-HA,
HA-C-PA-C-PA, PA-C-PA-C-HA, HA-K-PA-C-PA, PA-C-PA-K-HA,
HA-C-PA-K-PA, PA-K-PA-C-HA, HA-K-PA-PB1, HA-K-PB1-PA, PA-PB1-K-HA,
PB1-PA-K-HA, HA-PA-K-PB1, HA-PB1-K-PA, PA-K-PB1-HA, PB1-K-PA-HA,
HA-K-PA-K-PB1, HA-K-PB1-K-PA, PA-K-PB1-K-HA, PB1-K-PA-K-HA,
HA-C-PA-PB1, HA-C-PB1-PA, PA-PB1-C-HA, PB1-PA-C-HA, HA-PA-C-PB1,
HA-PB1-C-PA, PA-C-PB1-HA, PB1-C-PA-HA, HA-C-PA-C-PB1,
HA-C-PB1-C-PA, PA-C-PB1-C-HA, PB1-C-PA-C-HA, HA-K-PA-C-PB1,
HA-K-PB1-C-PA, PA-C-PB1-K-HA, PB1-C-PA-K-HA, HA-C-PA-K-PB1,
HA-C-PB1-K-PA, PA-K-PB1-C-HA, PB1-K-PA-C-HA, HA-K-PA-PB2,
HA-K-PB2-PA, PA-PB2-K-HA, PB2-PA-K-HA, HA-PA-K-PB2, HA-PB2-K-PA,
PA-K-PB2-HA, PB2-K-PA-HA, HA-K-PA-K-PB2, HA-K-PB2-K-PA,
PA-K-PB2-K-HA, PB2-K-PA-K-HA, HA-C-PA-PB2, HA-C-PB2-PA,
PA-PB2-C-HA, PB2-PA-C-HA, HA-PA-C-PB2, HA-PB2-C-PA, PA-C-PB2-HA,
PB2-C-PA-HA, HA-C-PA-C-PB2, HA-C-PB2-C-PA, PA-C-PB2-C-HA,
PB2-C-PA-C-HA, HA-K-PA-C-PB2, HA-K-PB2-C-PA, PA-C-PB2-K-HA,
PB2-C-PA-K-HA, HA-C-PA-K-PB2, HA-C-PB2-K-PA, PA-K-PB2-C-HA,
PB2-K-PA-C-HA, HA-K-PA-PB1F2, HA-K-PB1F2-PA, PA-PB1F2-K-HA,
PB1F2-PA-K-HA, HA-PA-K-PB1F2, HA-PB1F2-K-PA, PA-K-PB1F2-HA,
PB1F2-K-PA-HA, HA-K-PA-K-PB1F2, HA-K-PB1F2-K-PA, PA-K-PB1F2-K-HA,
PB1F2-K-PA-K-HA, HA-C-PA-PB1F2, HA-C-PB1F2-PA, PA-PB1F2-C-HA,
PB1F2-PA-C-HA, HA-PA-C-PB1F2, HA-PB1F2-C-PA, PA-C-PB1F2-HA,
PB1F2-C-PA-HA, HA-C-PA-C-PB1F2, HA-C-PB1F2-C-PA, PA-C-PB1F2-C-HA,
PB1F2-C-PA-C-HA, HA-K-PA-C-PB1F2, HA-K-PB1F2-C-PA, PA-C-PB1F2-K-HA,
PB1F2-C-PA-K-HA, HA-C-PA-K-PB1F2, HA-C-PB1F2-K-PA, PA-K-PB1F2-C-HA,
PB1F2-K-PA-C-HA, HA-K-PB1, PB1-K-HA, HA-K-PB1-PB1, PB1-PB1-K-HA,
HA-PB1-K-PB1, PB1-K-PB1-HA, HA-K-PB1-K-PB1, PB1-K-PB1-K-HA,
HA-C-PB1, PB1-C-HA, HA-C-PB1-PB1, PB1-PB1-C-HA, HA-PB1-C-PB1,
PB1-C-PB1-HA, HA-C-PB1-C-PB1, PB1-C-PB1-C-HA, HA-K-PB1-C-PB1,
PB1-C-PB1-K-HA, HA-C-PB1-K-PB1, PB1-K-PB1-C-HA, HA-K-PB1-PB2,
HA-K-PB2-PB1, PB1-PB2-K-HA, PB2-PB1-K-HA, HA-PB1-K-PB2,
HA-PB2-K-PB1, PB1-K-PB2-HA, PB2-K-PB1-HA, HA-K-PB1-K-PB2,
HA-K-PB2-K-PB1, PB1-K-PB2-K-HA, PB2-K-PB1-K-HA, HA-C-PB1-PB2,
HA-C-PB2-PB1, PB1-PB2-C-HA, PB2-PB1-C-HA, HA-PB1-C-PB2,
HA-PB2-C-PB1, PB1-C-PB2-HA, PB2-C-PB1-HA, HA-C-PB1-C-PB2,
HA-C-PB2-C-PB1, PB1-C-PB2-C-HA, PB2-C-PB1-C-HA, HA-K-PB1-C-PB2,
HA-K-PB2-C-PB1, PB1-C-PB2-K-HA, PB2-C-PB1-K-HA, HA-C-PB1-K-PB2,
HA-C-PB2-K-PB1, PB1-K-PB2-C-HA, PB2-K-PB1-C-HA, HA-K-PB1-PB1F2,
HA-K-PB1F2-PB1, PB1-PB1F2-K-HA, PB1F2-PB1-K-HA, HA-PB1-K-PB1F2,
HA-PB1F2-K-PB1, PB1-K-PB1F2-HA, PB1F2-K-PB1-HA, HA-K-PB1-K-PB1F2,
HA-K-PB1F2-K-PB1, PB1-K-PB1F2-K-HA, PB1F2-K-PB1-K-HA,
HA-C-PB1-PB1F2, HA-C-PB1F2-PB1, PB1-PB1F2-C-HA, PB1F2-PB1-C-HA,
HA-PB1-C-PB1F2, HA-PB1F2-C-PB1, PB1-C-PB1F2-HA, PB1F2-C-PB1-HA,
HA-C-PB1-C-PB1F2, HA-C-PB1F2-C-PB1, PB1-C-PB1F2-C-HA,
PB1F2-C-PB1-C-HA, HA-K-PB1-C-PB1F2, HA-K-PB1F2-C-PB1,
PB1-C-PB1F2-K-HA, PB1F2-C-PB1-K-HA, HA-C-PB1-K-PB1F2,
HA-C-PB1F2-K-PB1, PB1-K-PB1F2-C-HA, PB1F2-K-PB1-C-HA, HA-K-PB2,
PB2-K-HA, HA-K-PB2-PB2, PB2-PB2-K-HA, HA-PB2-K-PB2, PB2-K-PB2-HA,
HA-K-PB2-K-PB2, PB2-K-PB2-K-HA, HA-C-PB2, PB2-C-HA, HA-C-PB2-PB2,
PB2-PB2-C-HA, HA-PB2-C-PB2, PB2-C-PB2-HA, HA-C-PB2-C-PB2,
PB2-C-PB2-C-HA, HA-K-PB2-C-PB2, PB2-C-PB2-K-HA, HA-C-PB2-K-PB2,
PB2-K-PB2-C-HA, HA-K-PB2-PB1F2, HA-K-PB1F2-PB2, PB2-PB1F2-K-HA,
PB1F2-PB2-K-HA, HA-PB2-K-PB1F2, HA-PB1F2-K-PB2, PB2-K-PB1F2-HA,
PB1F2-K-PB2-HA, HA-K-PB2-K-PB1F2, HA-K-PB1F2-K-PB2,
PB2-K-PB1F2-K-HA, PB1F2-K-PB2-K-HA, HA-C-PB2-PB1F2, HA-C-PB1F2-PB2,
PB2-PB1F2-C-HA, PB1F2-PB2-C-HA, HA-PB2-C-PB1F2, HA-PB1F2-C-PB2,
PB2-C-PB1F2-HA, PB1F2-C-PB2-HA, HA-C-PB2-C-PB1F2, HA-C-PB1F2-C-PB2,
PB2-C-PB1F2-C-HA, PB1F2-C-PB2-C-HA, HA-K-PB2-C-PB1F2,
HA-K-PB1F2-C-PB2, PB2-C-PB1F2-K-HA, PB1F2-C-PB2-K-HA,
HA-C-PB2-K-PB1F2, HA-C-PB1F2-K-PB2, PB2-K-PB1F2-C-HA,
PB1F2-K-PB2-C-HA, HA-K-PB1F2, PB1F2-K-HA, HA-K-PB1F2-PB1F2,
PB1F2-PB1F2-K-HA, HA-PB1F2-K-PB1F2, PB1F2-K-PB1F2-HA,
HA-K-PB1F2-K-PB1F2, PB1F2-K-PB1F2-K-HA, HA-C-PB1F2, PB1F2-C-HA,
HA-C-PB1F2-PB1F2, PB1F2-PB1F2-C-HA, HA-PB1F2-C-PB1F2,
PB1F2-C-PB1F2-HA, HA-C-PB1F2-C-PB1F2, PB1F2-C-PB1F2-C-HA,
HA-K-PB1F2-C-PB1F2, PB1F2-C-PB1F2-K-HA, HA-C-PB1F2-K-PB1F2,
PB1F2-K-PB1F2-C-HA, NA-K-NP, NP-K-NA, NA-K-NP-NP, NP-NP-K-NA,
NA-NP-K-NP, NP-K-NP-NA, NA-K-NP-K-NP, NP-K-NP-K-NA, NA-C-NP,
NP-C-NA, NA-C-NP-NP, NP-NP-C-NA, NA-NP-C-NP, NP-C-NP-NA,
NA-C-NP-C-NP, NP-C-NP-C-NA, NA-K-NP-C-NP, NP-C-NP-K-NA,
NA-C-NP-K-NP, NP-K-NP-C-NA, NA-K-NP-M1, NA-K-M1-NP, NP-M1-K-NA,
M1-NP-K-NA, NA-NP-K-M1, NA-M1-K-NP, NP-K-M1-NA, M1-K-NP-NA,
NA-K-NP-K-M1, NA-K-M1-K-NP, NP-K-M1-K-NA, M1-K-NP-K-NA, NA-C-NP-M1,
NA-C-M1-NP, NP-M1-C-NA, M1-NP-C-NA, NA-NP-C-M1, NA-M1-C-NP,
NP-C-M1-NA, M1-C-NP-NA, NA-C-NP-C-M1, NA-C-M1-C-NP, NP-C-M1-C-NA,
M1-C-NP-C-NA, NA-K-NP-C-M1, NA-K-M1-C-NP, NP-C-M1-K-NA,
M1-C-NP-K-NA, NA-C-NP-K-M1, NA-C-M1-K-NP, NP-K-M1-C-NA,
M1-K-NP-C-NA, NA-K-NP-M2, NA-K-M2-NP, NP-M2-K-NA, M2-NP-K-NA,
NA-NP-K-M2, NA-M2-K-NP, NP-K-M2-NA, M2-K-NP-NA, NA-K-NP-K-M2,
NA-K-M2-K-NP, NP-K-M2-K-NA, M2-K-NP-K-NA, NA-C-NP-M2, NA-C-M2-NP,
NP-M2-C-NA, M2-NP-C-NA, NA-NP-C-M2, NA-M2-C-NP, NP-C-M2-NA,
M2-C-NP-NA, NA-C-NP-C-M2, NA-C-M2-C-NP, NP-C-M2-C-NA, M2-C-NP-C-NA,
NA-K-NP-C-M2, NA-K-M2-C-NP, NP-C-M2-K-NA, M2-C-NP-K-NA,
NA-C-NP-K-M2, NA-C-M2-K-NP, NP-K-M2-C-NA, M2-K-NP-C-NA,
NA-K-NP-NS1, NA-K-NS1-NP, NP-NS1-K-NA, NS1-NP-K-NA, NA-NP-K-NS1,
NA-NS1-K-NP, NP-K-NS1-NA, NS1-K-NP-NA, NA-K-NP-K-NS1,
NA-K-NS1-K-NP, NP-K-NS1-K-NA, NS1-K-NP-K-NA, NA-C-NP-NS1,
NA-C-NS1-NP, NP-NS1-C-NA, NS1-NP-C-NA, NA-NP-C-NS1, NA-NS1-C-NP,
NP-C-NS1-NA, NS1-C-NP-NA, NA-C-NP-C-NS1, NA-C-NS1-C-NP,
NP-C-NS1-C-NA, NS1-C-NP-C-NA, NA-K-NP-C-NS1, NA-K-NS1-C-NP,
NP-C-NS1-K-NA, NS1-C-NP-K-NA, NA-C-NP-K-NS1, NA-C-NS1-K-NP,
NP-K-NS1-C-NA, NS1-K-NP-C-NA, NA-K-NP-NS2/NEP, NA-K-NS2/NEP-NP,
NP-NS2/NEP-K-NA, NS2/NEP-NP-K-NA, NA-NP-K-NS2/NEP, NA-NS2/NEP-K-NP,
NP-K-NS2/NEP-NA, NS2/NEP-K-NP-NA, NA-K-NP-K-NS2/NEP,
NA-K-NS2/NEP-K-NP, NP-K-NS2/NEP-K-NA, NS2/NEP-K-NP-K-NA,
NA-C-NP-NS2/NEP, NA-C-NS2/NEP-NP, NP-NS2/NEP-C-NA, NS2/NEP-NP-C-NA,
NA-NP-C-NS2/NEP, NA-NS2/NEP-C-NP, NP-C-NS2/NEP-NA, NS2/NEP-C-NP-NA,
NA-C-NP-C-NS2/NEP, NA-C-NS2/NEP-C-NP, NP-C-NS2/NEP-C-NA,
NS2/NEP-C-NP-C-NA, NA-K-NP-C-NS2/NEP, NA-K-NS2/NEP-C-NP,
NP-C-NS2/NEP-K-NA, NS2/NEP-C-NP-K-NA, NA-C-NP-K-NS2/NEP,
NA-C-NS2/NEP-K-NP, NP-K-NS2/NEP-C-NA, NS2/NEP-K-NP-C-NA,
NA-K-NP-PA, NA-K-PA-NP, NP-PA-K-NA, PA-NP-K-NA, NA-NP-K-PA,
NA-PA-K-NP, NP-K-PA-NA, PA-K-NP-NA, NA-K-NP-K-PA, NA-K-PA-K-NP,
NP-K-PA-K-NA, PA-K-NP-K-NA, NA-C-NP-PA, NA-C-PA-NP, NP-PA-C-NA,
PA-NP-C-NA, NA-NP-C-PA, NA-PA-C-NP, NP-C-PA-NA, PA-C-NP-NA,
NA-C-NP-C-PA, NA-C-PA-C-NP, NP-C-PA-C-NA, PA-C-NP-C-NA,
NA-K-NP-C-PA, NA-K-PA-C-NP, NP-C-PA-K-NA, PA-C-NP-K-NA,
NA-C-NP-K-PA, NA-C-PA-K-NP, NP-K-PA-C-NA, PA-K-NP-C-NA,
NA-K-NP-PB1, NA-K-PB1-NP, NP-PB1-K-NA, PB1-NP-K-NA, NA-NP-K-PB1,
NA-PB1-K-NP, NP-K-PB1-NA, PB1-K-NP-NA, NA-K-NP-K-PB1,
NA-K-PB1-K-NP, NP-K-PB1-K-NA, PB1-K-NP-K-NA, NA-C-NP-PB1,
NA-C-PB1-NP, NP-PB1-C-NA, PB1-NP-C-NA, NA-NP-C-PB1, NA-PB1-C-NP,
NP-C-PB1-NA, PB1-C-NP-NA, NA-C-NP-C-PB1, NA-C-PB1-C-NP,
NP-C-PB1-C-NA, PB1-C-NP-C-NA, NA-K-NP-C-PB1, NA-K-PB1-C-NP,
NP-C-PB1-K-NA, PB1-C-NP-K-NA, NA-C-NP-K-PB1, NA-C-PB1-K-NP,
NP-K-PB1-C-NA, PB1-K-NP-C-NA, NA-K-NP-PB2, NA-K-PB2-NP,
NP-PB2-K-NA, PB2-NP-K-NA, NA-NP-K-PB2, NA-PB2-K-NP, NP-K-PB2-NA,
PB2-K-NP-NA, NA-K-NP-K-PB2, NA-K-PB2-K-NP, NP-K-PB2-K-NA,
PB2-K-NP-K-NA, NA-C-NP-PB2, NA-C-PB2-NP, NP-PB2-C-NA, PB2-NP-C-NA,
NA-NP-C-PB2, NA-PB2-C-NP, NP-C-PB2-NA, PB2-C-NP-NA, NA-C-NP-C-PB2,
NA-C-PB2-C-NP, NP-C-PB2-C-NA, PB2-C-NP-C-NA, NA-K-NP-C-PB2,
NA-K-PB2-C-NP, NP-C-PB2-K-NA, PB2-C-NP-K-NA, NA-C-NP-K-PB2,
NA-C-PB2-K-NP, NP-K-PB2-C-NA, PB2-K-NP-C-NA, NA-K-NP-PB1F2,
NA-K-PB1F2-NP, NP-PB1F2-K-NA, PB1F2-NP-K-NA, NA-NP-K-PB1F2,
NA-PB1F2-K-NP, NP-K-PB1F2-NA, PB1F2-K-NP-NA, NA-K-NP-K-PB1F2,
NA-K-PB1F2-K-NP, NP-K-PB1F2-K-NA, PB1F2-K-NP-K-NA, NA-C-NP-PB1F2,
NA-C-PB1F2-NP, NP-PB1F2-C-NA, PB1F2-NP-C-NA, NA-NP-C-PB1F2,
NA-PB1F2-C-NP, NP-C-PB1F2-NA, PB1F2-C-NP-NA, NA-C-NP-C-PB1F2,
NA-C-PB1F2-C-NP, NP-C-PB1F2-C-NA, PB1F2-C-NP-C-NA, NA-K-NP-C-PB1F2,
NA-K-PB1F2-C-NP, NP-C-PB1F2-K-NA, PB1F2-C-NP-K-NA, NA-C-NP-K-PB1F2,
NA-C-PB1F2-K-NP, NP-K-PB1F2-C-NA, PB1F2-K-NP-C-NA, NA-K-M1,
M1-K-NA, NA-K-M1-M1, M1-M1-K-NA, NA-M1-K-M1, M1-K-M1-NA,
NA-K-M1-K-M1, M1-K-M1-K-NA, NA-C-M1, M1-C-NA, NA-C-M1-M1,
M1-M1-C-NA, NA-M1-C-M1, M1-C-M1-NA, NA-C-M1-C-M1, M1-C-M1-C-NA,
NA-K-M1-C-M1, M1-C-M1-K-NA, NA-C-M1-K-M1, M1-K-M1-C-NA, NA-K-M1-M2,
NA-K-M2-M 1, M1-M2-K-NA, M2-M 1-K-NA, NA-M1-K-M2, NA-M2-K-M 1,
M1-K-M2-NA, M2-K-M1-NA, NA-K-M1-K-M2, NA-K-M2-K-M 1, M1-K-M2-K-NA,
M2-K-M1-K-NA, NA-C-M1-M2, NA-C-M2-M1, M1-M2-C-NA, M2-M1-C-NA,
NA-M1-C-M2, NA-M2-C-M1, M1-C-M2-NA, M2-C-M1-NA, NA-C-M1-C-M2,
NA-C-M2-C-M1, M1-C-M2-C-NA, M2-C-M1-C-NA, NA-K-M1-C-M2,
NA-K-M2-C-M1, M1-C-M2-K-NA, M2-C-M1-K-NA, NA-C-M1-K-M2,
NA-C-M2-K-M1, M1-K-M2-C-NA, M2-K-M1-C-NA, NA-K-M1-NS1, NA-K-NS1-M1,
M1-NS1-K-NA, NS1-M1-K-NA, NA-M1-K-NS1, NA-NS1-K-M1, M1-K-NS1-NA,
NS1-K-M1-NA, NA-K-M1-K-NS1, NA-K-NS1-K-M1, M1-K-NS1-K-NA,
NS1-K-M1-K-NA, NA-C-M1-NS1, NA-C-NS1-M1, M1-NS1-C-NA, NS1-M1-C-NA,
NA-M1-C-NS1, NA-NS1-C-M1, M1-C-NS1-NA, NS1-C-M1-NA, NA-C-M1-C-NS1,
NA-C-NS1-C-M1, M1-C-NS1-C-NA, NS1-C-M1-C-NA, NA-K-M1-C-NS1,
NA-K-NS1-C-M1, M1-C-NS1-K-NA, NS1-C-M1-K-NA, NA-C-M1-K-NS1,
NA-C-NS1-K-M1, M1-K-NS1-C-NA, NS1-K-M1-C-NA, NA-K-M1-NS2/NEP,
NA-K-NS2/NEP-M1, M1-NS2NEP-K-NA, NS2/NEP-M1-K-NA, NA-M1-K-NS2/NEP,
NA-NS2/NEP-K-M1, M1-K-NS2/NEP-NA, NS2/NEP-K-M1-NA,
NA-K-M1-K-NS2/NEP, NA-K-NS2/NEP-K-M1, M1-K-NS2/NEP-K-NA,
NS2/NEP-K-M1-K-NA, NA-C-M1-NS2/NEP, NA-C-NS2/NEP-M1,
M1-NS2NEP-C-NA, NS2/NEP-M1-C-NA, NA-M1-C-NS2/NEP, NA-NS2/NEP-C-M1,
M1-C-NS2/NEP-NA, NS2/NEP-C-M1-NA, NA-C-M1-C-NS2/NEP,
NA-C-NS2/NEP-C-M1, M1-C-NS2NEP-C-NA, NS2/NEP-C-M1-C-NA,
NA-K-M1-C-NS2/NEP, NA-K-NS2/NEP-C-M1, M1-C-NS2NEP-K-NA,
NS2/NEP-C-M1-K-NA, NA-C-M1-K-NS2/NEP, NA-C-NS2/NEP-K-M1,
M1-K-NS2/NEP-C-NA, NS2/NEP-K-M1-C-NA, NA-K-M1-PA, NA-K-PA-M1,
M1-PA-K-NA, PA-M1-K-NA, NA-M1-K-PA, NA-PA-K-M1, M1-K-PA-NA,
PA-K-M1-NA, NA-K-M1-K-PA, NA-K-PA-K-M1, M1-K-PA-K-NA, PA-K-M1-K-NA,
NA-C-M1-PA, NA-C-PA-M1, M1-PA-C-NA, PA-M1-C-NA, NA-M1-C-PA,
NA-PA-C-M1, M1-C-PA-NA, PA-C-M1-NA, NA-C-M1-C-PA, NA-C-PA-C-M1,
M1-C-PA-C-NA, PA-C-M1-C-NA, NA-K-M1-C-PA, NA-K-PA-C-M1,
M1-C-PA-K-NA, PA-C-M1-K-NA, NA-C-M1-K-PA, NA-C-PA-K-M1,
M1-K-PA-C-NA, PA-K-M1-C-NA, NA-K-M1-PB1, NA-K-PB1-M1, M1-PB1-K-NA,
PB1-M1-K-NA, NA-M1-K-PB1, NA-PB1-K-M1, M1-K-PB1-NA, PB1-K-M1-NA,
NA-K-M1-K-PB1, NA-K-PB1-K-M1, M1-K-PB1-K-NA, PB1-K-M1-K-NA,
NA-C-M1-PB1, NA-C-PB1-M1, M1-PB1-C-NA, PB1-M1-C-NA, NA-M1-C-PB1,
NA-PB1-C-M1, M1-C-PB1-NA, PB1-C-M1-NA, NA-C-M1-C-PB1,
NA-C-PB1-C-M1, M1-C-PB1-C-NA, PB1-C-M1-C-NA, NA-K-M1-C-PB1,
NA-K-PB1-C-M1, M1-C-PB1-K-NA, PB1-C-M1-K-NA, NA-C-M1-K-PB1,
NA-C-PB1-K-M1, M1-K-PB1-C-NA, PB1-K-M1-C-NA, NA-K-M1-PB2,
NA-K-PB2-M1, M1-PB2-K-NA, PB2-M1-K-NA, NA-M1-K-PB2, NA-PB2-K-M1,
M1-K-PB2-NA, PB2-K-M1-NA, NA-K-M1-K-PB2, NA-K-PB2-K-M1,
M1-K-PB2-K-NA, PB2-K-M1-K-NA, NA-C-M1-PB2, NA-C-PB2-M1,
M1-PB2-C-NA, PB2-M1-C-NA, NA-M1-C-PB2, NA-PB2-C-M1, M1-C-PB2-NA,
PB2-C-M1-NA, NA-C-M1-C-PB2, NA-C-PB2-C-M1, M1-C-PB2-C-NA,
PB2-C-M1-C-NA, NA-K-M1-C-PB2, NA-K-PB2-C-M1, M1-C-PB2-K-NA,
PB2-C-M1-K-NA, NA-C-M1-K-PB2, NA-C-PB2-K-M1, M1-K-PB2-C-NA,
PB2-K-M1-C-NA, NA-K-M1-PB1F2, NA-K-PB1F2-M1, M1-PB1F2-K-NA,
PB1F2-M1-K-NA, NA-M1-K-PB1F2, NA-PB1F2-K-M1, M1-K-PB1F2-NA,
PB1F2-K-M1-NA, NA-K-M1-K-PB1F2, NA-K-PB1F2-K-M1, M1-K-PB1F2-K-NA,
PB1F2-K-M1-K-NA, NA-C-M1-PB1F2, NA-C-PB1F2-M1, M1-PB1F2-C-NA,
PB1F2-M1-C-NA, NA-M1-C-PB1F2, NA-PB1F2-C-M1, M1-C-PB1F2-NA,
PB1F2-C-M1-NA, NA-C-M1-C-PB1F2, NA-C-PB1F2-C-M1, M1-C-PB1F2-C-NA,
PB1F2-C-M1-C-NA, NA-K-M1-C-PB1F2, NA-K-PB1F2-C-M1, M1-C-PB1F2-K-NA,
PB1F2-C-M1-K-NA, NA-C-M1-K-PB1F2, NA-C-PB1F2-K-M1, M1-K-PB1F2-C-NA,
PB1F2-K-M1-C-NA, NA-K-M2, M2-K-NA, NA-K-M2-M2, M2-M2-K-NA,
NA-M2-K-M2, M2-K-M2-NA, NA-K-M2-K-M2, M2-K-M2-K-NA, NA-C-M2,
M2-C-NA, NA-C-M2-M2, M2-M2-C-NA, NA-M2-C-M2, M2-C-M2-NA,
NA-C-M2-C-M2, M2-C-M2-C-NA, NA-K-M2-C-M2, M2-C-M2-K-NA,
NA-C-M2-K-M2, M2-K-M2-C-NA, NA-K-M2-NS1, NA-K-NS1-M2, M2-NS1-K-NA,
NS1-M2-K-NA, NA-M2-K-NS1, NA-NS1-K-M2, M2-K-NS1-NA, NS1-K-M2-NA,
NA-K-M2-K-NS1, NA-K-NS1-K-M2, M2-K-NS1-K-NA, NS1-K-M2-K-NA,
NA-C-M2-NS1, NA-C-NS1-M2, M2-NS1-C-NA, NS1-M2-C-NA, NA-M2-C-NS1,
NA-NS1-C-M2, M2-C-NS1-NA, NS1-C-M2-NA, NA-C-M2-C-NS1,
NA-C-NS1-C-M2, M2-C-NS1-C-NA, NS1-C-M2-C-NA, NA-K-M2-C-NS1,
NA-K-NS1-C-M2, M2-C-NS1-K-NA, NS1-C-M2-K-NA, NA-C-M2-K-NS1,
NA-C-NS1-K-M2, M2-K-NS1-C-NA, NS1-K-M2-C-NA, NA-K-M2-NS2/NEP,
NA-K-NS2/NEP-M2, M2-NS2/NEP-K-NA, NS2/NEP-M2-K-NA, NA-M2-K-NS2/NEP,
NA-NS2/NEP-K-M2, M2-K-NS2/NEP-NA, NS2/NEP-K-M2-NA,
NA-K-M2-K-NS2/NEP, NA-K-NS2/NEP-K-M2, M2-K-NS2/NEP-K-NA,
NS2/NEP-K-M2-K-NA, NA-C-M2-NS2/NEP, NA-C-NS2/NEP-M2,
M2-NS2/NEP-C-NA, NS2/NEP-M2-C-NA, NA-M2-C-NS2/NEP, NA-NS2/NEP-C-M2,
M2-C-NS2/NEP-NA, NS2/NEP-C-M2-NA, NA-C-M2-C-NS2/NEP,
NA-C-NS2/NEP-C-M2, M2-C-NS2/NEP-C-NA, NS2/NEP-C-M2-C-NA,
NA-K-M2-C-NS2/NEP, NA-K-NS2/NEP-C-M2, M2-C-NS2/NEP-K-NA,
NS2/NEP-C-M2-K-NA, NA-C-M2-K-NS2/NEP, NA-C-NS2/NEP-K-M2,
M2-K-NS2/NEP-C-NA, NS2/NEP-K-M2-C-NA, NA-K-M2-PA, NA-K-PA-M2,
M2-PA-K-NA, PA-M2-K-NA, NA-M2-K-PA, NA-PA-K-M2, M2-K-PA-NA,
PA-K-M2-NA, NA-K-M2-K-PA, NA-K-PA-K-M2, M2-K-PA-K-NA, PA-K-M2-K-NA,
NA-C-M2-PA, NA-C-PA-M2, M2-PA-C-NA, PA-M2-C-NA, NA-M2-C-PA,
NA-PA-C-M2, M2-C-PA-NA, PA-C-M2-NA, NA-C-M2-C-PA, NA-C-PA-C-M2,
M2-C-PA-C-NA, PA-C-M2-C-NA, NA-K-M2-C-PA, NA-K-PA-C-M2,
M2-C-PA-K-NA, PA-C-M2-K-NA, NA-C-M2-K-PA, NA-C-PA-K-M2,
M2-K-PA-C-NA, PA-K-M2-C-NA, NA-K-M2-PB1, NA-K-PB1-M2, M2-PB1-K-NA,
PB1-M2-K-NA, NA-M2-K-PB1, NA-PB1-K-M2, M2-K-PB1-NA, PB1-K-M2-NA,
NA-K-M2-K-PB1, NA-K-PB1-K-M2, M2-K-PB1-K-NA, PB1-K-M2-K-NA,
NA-C-M2-PB1, NA-C-PB1-M2, M2-PB1-C-NA, PB1-M2-C-NA, NA-M2-C-PB1,
NA-PB1-C-M2, M2-C-PB1-NA, PB1-C-M2-NA, NA-C-M2-C-PB1,
NA-C-PB1-C-M2, M2-C-PB1-C-NA, PB1-C-M2-C-NA, NA-K-M2-C-PB1,
NA-K-PB1-C-M2, M2-C-PB1-K-NA, PB1-C-M2-K-NA, NA-C-M2-K-PB1,
NA-C-PB1-K-M2, M2-K-PB1-C-NA, PB1-K-M2-C-NA, NA-K-M2-PB2,
NA-K-PB2-M2, M2-PB2-K-NA, PB2-M2-K-NA, NA-M2-K-PB2, NA-PB2-K-M2,
M2-K-PB2-NA, PB2-K-M2-NA, NA-K-M2-K-PB2, NA-K-PB2-K-M2,
M2-K-PB2-K-NA, PB2-K-M2-K-NA, NA-C-M2-PB2, NA-C-PB2-M2,
M2-PB2-C-NA, PB2-M2-C-NA, NA-M2-C-PB2, NA-PB2-C-M2, M2-C-PB2-NA,
PB2-C-M2-NA, NA-C-M2-C-PB2, NA-C-PB2-C-M2, M2-C-PB2-C-NA,
PB2-C-M2-C-NA, NA-K-M2-C-PB2, NA-K-PB2-C-M2, M2-C-PB2-K-NA,
PB2-C-M2-K-NA, NA-C-M2-K-PB2, NA-C-PB2-K-M2, M2-K-PB2-C-NA,
PB2-K-M2-C-NA, NA-K-M2-PB1F2, NA-K-PB1F2-M2, M2-PB1F2-K-NA,
PB1F2-M2-K-NA, NA-M2-K-PB1F2, NA-PB1F2-K-M2, M2-K-PB1F2-NA,
PB1F2-K-M2-NA, NA-K-M2-K-PB1F2, NA-K-PB1F2-K-M2, M2-K-PB1F2-K-NA,
PB1F2-K-M2-K-NA, NA-C-M2-PB1F2, NA-C-PB1F2-M2, M2-PB1F2-C-NA,
PB1F2-M2-C-NA, NA-M2-C-PB1F2, NA-PB1F2-C-M2, M2-C-PB1F2-NA,
PB1F2-C-M2-NA, NA-C-M2-C-PB1F2, NA-C-PB1F2-C-M2, M2-C-PB1F2-C-NA,
PB1F2-C-M2-C-NA, NA-K-M2-C-PB1F2, NA-K-PB1F2-C-M2, M2-C-PB1F2-K-NA,
PB1F2-C-M2-K-NA, NA-C-M2-K-PB1F2, NA-C-PB1F2-K-M2, M2-K-PB1F2-C-NA,
PB1F2-K-M2-C-NA, NA-K-NS1, NS1-K-NA, NA-K-NS1-NS1, NS1-NS1-K-NA,
NA-NS1-K-NS1, NS1-K-NS1-NA, NA-K-NS1-K-NS1, NS1-K-NS1-K-NA,
NA-C-NS1, NS1-C-NA, NA-C-NS1-NS1, NS1-NS1-C-NA, NA-NS1-C-NS1,
NS1-C-NS1-NA, NA-C-NS1-C-NS1, NS1-C-NS1-C-NA, NA-K-NS1-C-NS1,
NS1-C-NS1-K-NA, NA-C-NS1-K-NS1, NS1-K-NS1-C-NA, NA-K-NS1-NS2/NEP,
NA-K-NS2/NEP-NS1, NS1-NS2/NEP-K-NA, NS2/NEP-NS1-K-NA,
NA-NS1-K-NS2/NEP, NA-NS2/NEP-K-NS1, NS1-K-NS2/NEP-NA,
NS2/NEP-K-NS1-NA, NA-K-NS1-K-NS2/NEP, NA-K-NS2/NEP-K-NS1,
NS1-K-NS2/NEP-K-NA, NS2/NEP-K-NS1-K-NA, NA-C-NS1-NS2/NEP,
NA-C-NS2/NEP-NS1, NS1-NS2/NEP-C-NA, NS2/NEP-NS1-C-NA,
NA-NS1-C-NS2/NEP, NA-NS2/NEP-C-NS1, NS1-C-NS2/NEP-NA,
NS2/NEP-C-NS1-NA, NA-C-NS1-C-NS2/NEP, NA-C-NS2/NEP-C-NS1,
NS1-C-NS2/NEP-C-NA, NS2/NEP-C-NS1-C-NA, NA-K-NS1-C-NS2/NEP,
NA-K-NS2/NEP-C-NS1, NS1-C-NS2/NEP-K-NA, NS2/NEP-C-NS1-K-NA,
NA-C-NS1-K-NS2/NEP, NA-C-NS2/NEP-K-NS1, NS1-K-NS2/NEP-C-NA,
NS2/NEP-K-NS1-C-NA, NA-K-NS1-PA, NA-K-PA-NS1, NS1-PA-K-NA,
PA-NS1-K-NA, NA-NS1-K-PA, NA-PA-K-NS1, NS1-K-PA-NA, PA-K-NS1-NA,
NA-K-NS1-K-PA, NA-K-PA-K-NS1, NS1-K-PA-K-NA, PA-K-NS1-K-NA,
NA-C-NS1-PA, NA-C-PA-NS1, NS1-PA-C-NA, PA-NS1-C-NA, NA-NS1-C-PA,
NA-PA-C-NS1, NS1-C-PA-NA, PA-C-NS1-NA, NA-C-NS1-C-PA,
NA-C-PA-C-NS1, NS1-C-PA-C-NA, PA-C-NS1-C-NA, NA-K-NS1-C-PA,
NA-K-PA-C-NS1, NS1-C-PA-K-NA, PA-C-NS1-K-NA, NA-C-NS1-K-PA,
NA-C-PA-K-NS1, NS1-K-PA-C-NA, PA-K-NS1-C-NA, NA-K-NS1-PB1,
NA-K-PB1-NS1, NS1-PB1-K-NA, PB1-NS1-K-NA, NA-NS1-K-PB1,
NA-PB1-K-NS1, NS1-K-PB1-NA, PB1-K-NS1-NA, NA-K-NS1-K-PB1,
NA-K-PB1-K-NS1, NS1-K-PB1-K-NA, PB1-K-NS1-K-NA, NA-C-NS1-PB1,
NA-C-PB1-NS1, NS1-PB1-C-NA, PB1-NS1-C-NA, NA-NS1-C-PB1,
NA-PB1-C-NS1, NS1-C-PB1-NA, PB1-C-NS1-NA, NA-C-NS1-C-PB1,
NA-C-PB1-C-NS1, NS1-C-PB1-C-NA, PB1-C-NS1-C-NA, NA-K-NS1-C-PB1,
NA-K-PB1-C-NS1, NS1-C-PB1-K-NA, PB1-C-NS1-K-NA, NA-C-NS1-K-PB1,
NA-C-PB1-K-NS1, NS1-K-PB1-C-NA, PB1-K-NS1-C-NA, NA-K-NS1-PB2,
NA-K-PB2-NS1, NS1-PB2-K-NA, PB2-NS1-K-NA, NA-NS1-K-PB2,
NA-PB2-K-NS1, NS1-K-PB2-NA, PB2-K-NS1-NA, NA-K-NS1-K-PB2,
NA-K-PB2-K-NS1, NS1-K-PB2-K-NA, PB2-K-NS1-K-NA, NA-C-NS1-PB2,
NA-C-PB2-NS1, NS1-PB2-C-NA, PB2-NS1-C-NA, NA-NS1-C-PB2,
NA-PB2-C-NS1, NS1-C-PB2-NA, PB2-C-NS1-NA, NA-C-NS1-C-PB2,
NA-C-PB2-C-NS1, NS1-C-PB2-C-NA, PB2-C-NS1-C-NA, NA-K-NS1-C-PB2,
NA-K-PB2-C-NS1, NS1-C-PB2-K-NA, PB2-C-NS1-K-NA, NA-C-NS1-K-PB2,
NA-C-PB2-K-NS1, NS1-K-PB2-C-NA, PB2-K-NS1-C-NA, NA-K-NS1-PB1F2,
NA-K-PB1F2-NS1, NS1-PB1F2-K-NA, PB1F2-NS1-K-NA, NA-NS1-K-PB1F2,
NA-PB1F2-K-NS1, NS1-K-PB1F2-NA, PB1F2-K-NS1-NA, NA-K-NS1-K-PB1F2,
NA-K-PB1F2-K-NS1, NS1-K-PB1F2-K-NA, PB1F2-K-NS1-K-NA,
NA-C-NS1-PB1F2, NA-C-PB1F2-NS1, NS1-PB1F2-C-NA, PB1F2-NS1-C-NA,
NA-NS1-C-PB1F2, NA-PB1F2-C-NS1, NS1-C-PB1F2-NA, PB1F2-C-NS1-NA,
NA-C-NS1-C-PB1F2, NA-C-PB1F2-C-NS1, NS1-C-PB1F2-C-NA,
PB1F2-C-NS1-C-NA, NA-K-NS1-C-PB1F2, NA-K-PB1F2-C-NS1,
NS1-C-PB1F2-K-NA, PB1F2-C-NS1-K-NA, NA-C-NS1-K-PB1F2,
NA-C-PB1F2-K-NS1, NS1-K-PB1F2-C-NA, PB1F2-K-NS1-C-NA, NA-K-NS2/NEP,
NS2/NEP-K-NA, NA-K-NS2/NEP-NS2/NEP, NS2/NEP-NS2/NEP-K-NA,
NA-NS2/NEP-K-NS2/NEP, NS2/NEP-K-NS2/NEP-NA, NA-K-NS2/NEP-K-NS2/NEP,
NS2/NEP-K-NS2/NEP-K-NA, NA-C-NS2/NEP, NS2/NEP-C-NA,
NA-C-NS2/NEP-NS2/NEP, NS2/NEP-NS2/NEP-C-NA, NA-NS2/NEP-C-NS2/NEP,
NS2/NEP-C-NS2/NEP-NA, NA-C-NS2/NEP-C-NS2/NEP,
NS2/NEP-C-NS2/NEP-C-NA, NA-K-NS2/NEP-C-NS2/NEP,
NS2/NEP-C-NS2/NEP-K-NA, NA-C-NS2/NEP-K-NS2/NEP,
NS2/NEP-K-NS2/NEP-C-NA, NA-K-NS2/NEP-PA, NA-K-PA-NS2/NEP,
NS2/NEP-PA-K-NA, PA-NS2/NEP-K-NA, NA-NS2/NEP-K-PA, NA-PA-K-NS2/NEP,
NS2/NEP-K-PA-NA, PA-K-NS2/NEP-NA, NA-K-NS2/NEP-K-PA,
NA-K-PA-K-NS2/NEP, NS2/NEP-K-PA-K-NA, PA-K-NS2/NEP-K-NA,
NA-C-NS2/NEP-PA, NA-C-PA-NS2/NEP, NS2/NEP-PA-C-NA, PA-NS2/NEP-C-NA,
NA-NS2/NEP-C-PA, NA-PA-C-NS2/NEP, NS2/NEP-C-PA-NA, PA-C-NS2/NEP-NA,
NA-C-NS2/NEP-C-PA, NA-C-PA-C-NS2/NEP, NS2/NEP-C-PA-C-NA,
PA-C-NS2/NEP-C-NA, NA-K-NS2/NEP-C-PA, NA-K-PA-C-NS2/NEP,
NS2/NEP-C-PA-K-NA, PA-C-NS2/NEP-K-NA, NA-C-NS2/NEP-K-PA,
NA-C-PA-K-NS2/NEP, NS2/NEP-K-PA-C-NA, PA-K-NS2/NEP-C-NA,
NA-K-NS2/NEP-PB1, NA-K-PB1-NS2/NEP, NS2/NEP-PB1-K-NA,
PB1-NS2/NEP-K-NA, NA-NS2/NEP-K-PB1, NA-PB1-K-NS2/NEP,
NS2/NEP-K-PB1-NA, PB1-K-NS2/NEP-NA, NA-K-NS2/NEP-K-PB1,
NA-K-PB1-K-NS2/NEP, NS2/NEP-K-PB1-K-NA, PB1-K-NS2/NEP-K-NA,
NA-C-NS2/NEP-PB1, NA-C-PB1-NS2/NEP, NS2/NEP-PB1-C-NA,
PB1-NS2/NEP-C-NA, NA-NS2/NEP-C-PB1, NA-PB1-C-NS2/NEP,
NS2/NEP-C-PB1-NA, PB1-C-NS2/NEP-NA, NA-C-NS2/NEP-C-PB1,
NA-C-PB1-C-NS2/NEP, NS2/NEP-C-PB1-C-NA, PB1-C-NS2/NEP-C-NA,
NA-K-NS2/NEP-C-PB1, NA-K-PB1-C-NS2/NEP, NS2/NEP-C-PB1-K-NA,
PB1-C-NS2/NEP-K-NA, NA-C-NS2/NEP-K-PB1, NA-C-PB1-K-NS2/NEP,
NS2/NEP-K-PB1-C-NA, PB1-K-NS2/NEP-C-NA, NA-K-NS2/NEP-PB2,
NA-K-PB2-NS2/NEP, NS2/NEP-PB2-K-NA, PB2-NS2/NEP-K-NA,
NA-NS2/NEP-K-PB2, NA-PB2-K-NS2/NEP, NS2/NEP-K-PB2-NA,
PB2-K-NS2/NEP-NA, NA-K-NS2/NEP-K-PB2, NA-K-PB2-K-NS2/NEP,
NS2/NEP-K-PB2-K-NA, PB2-K-NS2/NEP-K-NA, NA-C-NS2/NEP-PB2,
NA-C-PB2-NS2/NEP, NS2/NEP-PB2-C-NA, PB2-NS2/NEP-C-NA,
NA-NS2/NEP-C-PB2, NA-PB2-C-NS2/NEP, NS2/NEP-C-PB2-NA,
PB2-C-NS2/NEP-NA, NA-C-NS2/NEP-C-PB2, NA-C-PB2-C-NS2/NEP,
NS2/NEP-C-PB2-C-NA, PB2-C-NS2/NEP-C-NA, NA-K-NS2/NEP-C-PB2,
NA-K-PB2-C-NS2/NEP, NS2/NEP-C-PB2-K-NA, PB2-C-NS2/NEP-K-NA,
NA-C-NS2/NEP-K-PB2, NA-C-PB2-K-NS2/NEP, NS2/NEP-K-PB2-C-NA,
PB2-K-NS2/NEP-C-NA, NA-K-NS2/NEP-PB1F2, NA-K-PB1F2-NS2/NEP,
NS2/NEP-PB1F2-K-NA, PB1F2-NS2/NEP-K-NA, NA-NS2/NEP-K-PB1F2,
NA-PB1F2-K-NS2/NEP, NS2/NEP-K-PB1F2-NA, PB1F2-K-NS2/NEP-NA,
NA-K-NS2/NEP-K-PB1F2, NA-K-PB1F2-K-NS2/NEP, NS2/NEP-K-PB1F2-K-NA,
PB1F2-K-NS2/NEP-K-NA, NA-C-NS2/NEP-PB1F2, NA-C-PB1F2-NS2/NEP,
NS2/NEP-PB1F2-C-NA, PB1F2-NS2/NEP-C-NA, NA-NS2/NEP-C-PB1F2,
NA-PB1F2-C-NS2/NEP, NS2/NEP-C-PB1F2-NA, PB1F2-C-NS2/NEP-NA,
NA-C-NS2/NEP-C-PB1F2, NA-C-PB1F2-C-NS2/NEP, NS2/NEP-C-PB1F2-C-NA,
PB1F2-C-NS2/NEP-C-NA, NA-K-NS2/NEP-C-PB1F2, NA-K-PB1F2-C-NS2/NEP,
NS2/NEP-C-PB1F2-K-NA, PB1F2-C-NS2/NEP-K-NA, NA-C-NS2/NEP-K-PB1F2,
NA-C-PB1F2-K-NS2/NEP, NS2/NEP-K-PB1F2-C-NA, PB1F2-K-NS2/NEP-C-NA,
NA-K-PA, PA-K-NA, NA-K-PA-PA, PA-PA-K-NA, NA-PA-K-PA, PA-K-PA-NA,
NA-K-PA-K-PA, PA-K-PA-K-NA, NA-C-PA, PA-C-NA, NA-C-PA-PA,
PA-PA-C-NA, NA-PA-C-PA, PA-C-PA-NA, NA-C-PA-C-PA, PA-C-PA-C-NA,
NA-K-PA-C-PA, PA-C-PA-K-NA, NA-C-PA-K-PA, PA-K-PA-C-NA,
NA-K-PA-PB1, NA-K-PB1-PA, PA-PB1-K-NA, PB1-PA-K-NA, NA-PA-K-PB1,
NA-PB1-K-PA, PA-K-PB1-NA, PB1-K-PA-NA, NA-K-PA-K-PB1,
NA-K-PB1-K-PA, PA-K-PB1-K-NA, PB1-K-PA-K-NA, NA-C-PA-PB1,
NA-C-PB1-PA, PA-PB1-C-NA, PB1-PA-C-NA, NA-PA-C-PB1, NA-PB1-C-PA,
PA-C-PB1-NA, PB1-C-PA-NA, NA-C-PA-C-PB1, NA-C-PB1-C-PA,
PA-C-PB1-C-NA, PB1-C-PA-C-NA, NA-K-PA-C-PB1, NA-K-PB1-C-PA,
PA-C-PB1-K-NA, PB1-C-PA-K-NA, NA-C-PA-K-PB1, NA-C-PB1-K-PA,
PA-K-PB1-C-NA, PB1-K-PA-C-NA, NA-K-PA-PB2, NA-K-PB2-PA,
PA-PB2-K-NA, PB2-PA-K-NA, NA-PA-K-PB2, NA-PB2-K-PA, PA-K-PB2-NA,
PB2-K-PA-NA, NA-K-PA-K-PB2, NA-K-PB2-K-PA, PA-K-PB2-K-NA,
PB2-K-PA-K-NA, NA-C-PA-PB2, NA-C-PB2-PA, PA-PB2-C-NA, PB2-PA-C-NA,
NA-PA-C-PB2, NA-PB2-C-PA, PA-C-PB2-NA, PB2-C-PA-NA, NA-C-PA-C-PB2,
NA-C-PB2-C-PA, PA-C-PB2-C-NA, PB2-C-PA-C-NA, NA-K-PA-C-PB2,
NA-K-PB2-C-PA, PA-C-PB2-K-NA, PB2-C-PA-K-NA, NA-C-PA-K-PB2,
NA-C-PB2-K-PA, PA-K-PB2-C-NA, PB2-K-PA-C-NA, NA-K-PA-PB1F2,
NA-K-PB1F2-PA, PA-PB1F2-K-NA, PB1F2-PA-K-NA, NA-PA-K-PB1F2,
NA-PB1F2-K-PA, PA-K-PB1F2-NA, PB1F2-K-PA-NA, NA-K-PA-K-PB1F2,
NA-K-PB1F2-K-PA, PA-K-PB1F2-K-NA, PB1F2-K-PA-K-NA, NA-C-PA-PB1F2,
NA-C-PB1F2-PA, PA-PB1F2-C-NA, PB1F2-PA-C-NA, NA-PA-C-PB1F2,
NA-PB1F2-C-PA, PA-C-PB1F2-NA, PB1F2-C-PA-NA, NA-C-PA-C-PB1F2,
NA-C-PB1F2-C-PA, PA-C-PB1F2-C-NA, PB1F2-C-PA-C-NA, NA-K-PA-C-PB1F2,
NA-K-PB1F2-C-PA, PA-C-PB1F2-K-NA, PB1F2-C-PA-K-NA, NA-C-PA-K-PB1F2,
NA-C-PB1F2-K-PA, PA-K-PB1F2-C-NA, PB1F2-K-PA-C-NA, NA-K-PB1,
PB1-K-NA, NA-K-PB1-PB1, PB1-PB1-K-NA, NA-PB1-K-PB1, PB1-K-PB1-NA,
NA-K-PB1-K-PB1, PB1-K-PB1-K-NA, NA-C-PB1, PB1-C-NA, NA-C-PB1-PB1,
PB1-PB1-C-NA, NA-PB1-C-PB1, PB1-C-PB1-NA, NA-C-PB1-C-PB1,
PB1-C-PB1-C-NA, NA-K-PB1-C-PB1, PB1-C-PB1-K-NA, NA-C-PB1-K-PB1,
PB1-K-PB1-C-NA, NA-K-PB1-PB2, NA-K-PB2-PB1, PB1-PB2-K-NA,
PB2-PB1-K-NA, NA-PB1-K-PB2, NA-PB2-K-PB1, PB1-K-PB2-NA,
PB2-K-PB1-NA, NA-K-PB1-K-PB2, NA-K-PB2-K-PB1, PB1-K-PB2-K-NA,
PB2-K-PB1-K-NA, NA-C-PB1-PB2, NA-C-PB2-PB1, PB1-PB2-C-NA,
PB2-PB1-C-NA, NA-PB1-C-PB2, NA-PB2-C-PB1, PB1-C-PB2-NA,
PB2-C-PB1-NA, NA-C-PB1-C-PB2, NA-C-PB2-C-PB1, PB1-C-PB2-C-NA,
PB2-C-PB1-C-NA, NA-K-PB1-C-PB2, NA-K-PB2-C-PB1, PB1-C-PB2-K-NA,
PB2-C-PB1-K-NA, NA-C-PB1-K-PB2, NA-C-PB2-K-PB1, PB1-K-PB2-C-NA,
PB2-K-PB1-C-NA, NA-K-PB1-PB1F2, NA-K-PB1F2-PB1, PB1-PB1F2-K-NA,
PB1F2-PB1-K-NA, NA-PB1-K-PB1F2, NA-PB1F2-K-PB1, PB1-K-PB1F2-NA,
PB1F2-K-PB1-NA, NA-K-PB1-K-PB1F2, NA-K-PB1F2-K-PB1,
PB1-K-PB1F2-K-NA, PB1F2-K-PB1-K-NA, NA-C-PB1-PB1F2, NA-C-PB1F2-PB1,
PB1-PB1F2-C-NA, PB1F2-PB1-C-NA, NA-PB1-C-PB1F2, NA-PB1F2-C-PB1,
PB1-C-PB1F2-NA, PB1F2-C-PB1-NA, NA-C-PB1-C-PB1F2, NA-C-PB1F2-C-PB1,
PB1-C-PB1F2-C-NA, PB1F2-C-PB1-C-NA, NA-K-PB1-C-PB1F2,
NA-K-PB1F2-C-PB1, PB1-C-PB1F2-K-NA, PB1F2-C-PB1-K-NA,
NA-C-PB1-K-PB1F2, NA-C-PB1F2-K-PB1, PB1-K-PB1F2-C-NA,
PB1F2-K-PB1-C-NA, NA-K-PB2, PB2-K-NA, NA-K-PB2-PB2, PB2-PB2-K-NA,
NA-PB2-K-PB2, PB2-K-PB2-NA, NA-K-PB2-K-PB2, PB2-K-PB2-K-NA,
NA-C-PB2, PB2-C-NA, NA-C-PB2-PB2, PB2-PB2-C-NA, NA-PB2-C-PB2,
PB2-C-PB2-NA, NA-C-PB2-C-PB2, PB2-C-PB2-C-NA, NA-K-PB2-C-PB2,
PB2-C-PB2-K-NA, NA-C-PB2-K-PB2, PB2-K-PB2-C-NA, NA-K-PB2-PB1F2,
NA-K-PB1F2-PB2, PB2-PB1F2-K-NA, PB1F2-PB2-K-NA, NA-PB2-K-PB1F2,
NA-PB1F2-K-PB2, PB2-K-PB1F2-NA, PB1F2-K-PB2-NA, NA-K-PB2-K-PB1F2,
NA-K-PB1F2-K-PB2, PB2-K-PB1F2-K-NA, PB1F2-K-PB2-K-NA,
NA-C-PB2-PB1F2, NA-C-PB1F2-PB2, PB2-PB1F2-C-NA, PB1F2-PB2-C-NA,
NA-PB2-C-PB1F2, NA-PB1F2-C-PB2, PB2-C-PB1F2-NA, PB1F2-C-PB2-NA,
NA-C-PB2-C-PB1F2, NA-C-PB1F2-C-PB2, PB2-C-PB1F2-C-NA,
PB1F2-C-PB2-C-NA, NA-K-PB2-C-PB1F2, NA-K-PB1F2-C-PB2,
PB2-C-PB1F2-K-NA, PB1F2-C-PB2-K-NA, NA-C-PB2-K-PB1F2,
NA-C-PB1F2-K-PB2, PB2-K-PB1F2-C-NA, PB1F2-K-PB2-C-NA, NA-K-PB1F2,
PB1F2-K-NA, NA-K-PB1F2-PB1F2, PB1F2-PB1F2-K-NA, NA-PB1F2-K-PB1F2,
PB1F2-K-PB1F2-NA, NA-K-PB1F2-K-PB1F2, PB1F2-K-PB1F2-K-NA,
NA-C-PB1F2, PB1F2-C-NA, NA-C-PB1F2-PB1F2, PB1F2-PB1F2-C-NA,
NA-PB1F2-C-PB1F2, PB1F2-C-PB1F2-NA, NA-C-PB1F2-C-PB1F2,
PB1F2-C-PB1F2-C-NA, NA-K-PB1F2-C-PB1F2, PB1F2-C-PB1F2-K-NA,
NA-C-PB1F2-K-PB1F2 or PB1F2-K-PB1F2-C-NA. Most preferably, the
arrangement is NP-K-M1-C-HA.
[0246] It is within the scope of the present invention that every
protein can be combined with any other protein and that any two
proteins can or cannot be connected or linked by either a cleavage
site or a linker peptide.
[0247] In preferred embodiments, the expression system is for use
in the prophylaxis or treatment of viral infection, particularly
preferably for use in the prophylaxis or treatment of an
orthomyxovirus infection, preferably an influenza virus infection,
more preferably an influenza A virus infection, and/or in the
manufacturing of medicament for use in the prophylaxis or treatment
of an orthomyxovirus infection, preferably an influenza virus
infection, more preferably an influenza A virus infection, and/or
for use in methods of prophylaxis or treatment of an orthomyxovirus
infection, preferably an influenza virus infection, more preferably
an influenza A virus infection, preferably an influenza virus
infection, more preferably an influenza A virus infection.
[0248] In preferred embodiments, the expression system is for use
in enhancing an immune response, preferably a B cell immune
response an orthomyxovirus infection, preferably an influenza virus
infection, more preferably an influenza A virus infection.
[0249] In a preferred embodiment of this aspect, HA is defined
according to the eighth aspect.
[0250] It is particularly preferred that the viral polyprotein
encoded by the first, the second and the third polynucleotide has
an amino acid according to SEQ ID NO: 13 or a variant thereof
and/or is encoded by a polynucleotide having the nucleic acid
sequence of SEQ ID NO: 14 or a variant thereof. Preferably, the
encoded triple antigen protein NP-M1-H1p is processed into a
cytoplasmic NP-M1 fusion protein and a membrane spanning H1p
protein by the 2A sequence.
[0251] In preferred embodiments, the expression system is for use
in the prophylaxis or treatment of an orthomyxovirus infection,
preferably an influenza A virus infection.
[0252] In more preferred embodiments, the expression system is for
use in enhancing an immune response, preferably a B cell immune
response against a an orthomyxovirus protein, preferably an
influenza A virus protein.
[0253] In preferred embodiments, the vector or vectors comprising
the first, and the second and/or the third polynucleotide is/are
selected from the group consisting of plasmid, cosmid, phage,
virus, and artificial chromosome. More preferably, a vector
suitable for practicing the present invention is selected from the
group consisting of plasmid vectors, cosmid vectors, phage vectors,
preferably lambda phage and filamentous phage vectors, viral
vectors, adenovirus vectors (e.g., non-replicating Ad5, Ad11, Ad26,
Ad35, Ad49, ChAd3, ChAd4, ChAd5, ChAd6, ChAd7, ChAd8, ChAd9,
ChAd10, ChAd11, ChAd16, ChAd17, ChAd19, ChAd20, ChAd22, ChAd24,
ChAd26, ChAd30, ChAd31, ChAd37, ChAd38, ChAd44, ChAd55, ChAd63,
ChAd 73, ChAd82, ChAd83, ChAd146, ChAd147, PanAd1, PanAd2, and
PanAd3 vectors or replication-competent Ad4 and Ad7 vectors),
adeno-associated virus (AAV) vectors (e.g., AAV type 5 and type 2),
alphavirus vectors (e.g., Venezuelan equine encephalitis virus
(VEE), sindbis virus (SIN), semliki forest virus (SFV), and VEE-SIN
chimeras), herpes virus vectors (e.g. vectors derived from
cytomegaloviruses, like rhesus cytomegalovirus (RhCMV) (14)), arena
virus vectors (e.g. lymphocytic choriomeningitis virus (LCMV)
vectors (15)), measles virus vectors, pox virus vectors (e.g.,
vaccinia virus, modified vaccinia virus Ankara (MVA), NYVAC
(derived from the Copenhagen strain of vaccinia), and avipox
vectors: canarypox (ALVAC) and fowlpox (FPV) vectors), vesicular
stomatitis virus vectors, retrovirus, lentivirus, viral like
particles, and bacterial spores. The vectors ChAd3, ChAd4, ChAd5,
ChAd6, ChAd7, ChAd8, ChAd9, ChAd10, ChAd11, ChAd16, ChAd17, ChAd19,
ChAd20, ChAd22, ChAd24, ChAd26, ChAd30, ChAd31, ChAd37, ChAd38,
ChAd44, ChAd63 and ChAd82 are described in detail in WO
2005/071093. The vectors PanAd1, PanAd2, PanAd3, ChAd55, ChAd73,
ChAd83, ChAd146, and ChAd147 are described in detail in WO
2010/086189. It is particularly preferred that the vector is
selected from the group consisting of MVA, ChAd63 and PanAd3.
[0254] In preferred embodiments, the expression system is for use
in medicine. In more preferred embodiments, the expression system
is for use in the prophylaxis or treatment of viral infection,
particularly preferably for use in the prophylaxis or treatment of
RSV infection.
[0255] In a second aspect, the present invention provides an
isolated protein mixture encoded by the expression system of the
first aspect. Preferably, the isolated protein mixture contains,
essentially contains or comprises one or more of the viral proteins
encoded by the expression system of the first aspect.
[0256] In preferred embodiments, the isolated protein mixture is
for use in medicine. In particularly preferred embodiments, the
isolated protein mixture is for use in the prophylaxis or treatment
of viral infection, particularly preferably for use in the
prophylaxis or treatment of RSV infection or in the prophylaxis or
treatment of influenza A infection.
[0257] In a third aspect, the present invention provides an
isolated host cell containing the expression system of the first
aspect and/or the protein mixture of the second aspect. It is
understood that such host cell includes but is not limited to
prokaryotic (e.g. a bacterial cell) or eukaryotic cells (e.g. a
fungal, plant or animal cell).
[0258] In preferred embodiments, the host cell is for use in
medicine. In particularly preferred embodiments, the host cell is
for use in the prophylaxis or treatment of viral infection,
particularly preferably for use in the prophylaxis or treatment of
RSV infection or in the prophylaxis or treatment of influenza A
infection.
[0259] In a fourth aspect, the present invention provides a
composition comprising the expression system of the first aspect or
the protein mixture of the second aspect and a pharmaceutical
acceptable carrier and/or excipient. Preferably, such composition
is a pharmaceutical composition.
[0260] The composition of the fourth aspect contains a
therapeutically effective amount of the compound, preferably in
purified form, together with a suitable amount of carrier so as to
provide the form for proper administration to the patient. The
formulation should suit the mode of administration.
[0261] The compositions can take the form of solutions,
suspensions, emulsion, tablets, pills, capsules, powders,
sustained-release formulations and the like. The composition can be
formulated as a suppository, with traditional binders and carriers
such as triglycerides.
[0262] For preparing pharmaceutical compositions of the present
invention, pharmaceutically acceptable carriers can be either solid
or liquid.
[0263] Solid form compositions include powders, tablets, pills,
capsules, lozenges, cachets, suppositories, and dispersible
granules. A solid excipient can be one or more substances, which
may also act as diluents, flavoring agents, binders, preservatives,
tablet disintegrating agents, or an encapsulating material. In
powders, the excipient is preferably a finely divided solid, which
is in a mixture with the finely divided inhibitor of the present
invention. In tablets, the active ingredient is mixed with the
carrier having the necessary binding properties in suitable
proportions and compacted in the shape and size desired. Suitable
excipients are magnesium carbonate, magnesium stearate, talc,
sugar, lactose, pectin, dextrin, starch, gelatin, tragacanth,
methylcellulose, sodium carboxymethylcellulose, a low melting wax,
cocoa butter, and the like. For preparing suppositories, a low
melting wax, such as a mixture of fatty acid glycerides or cocoa
butter, is first melted and the active component is dispersed
homogeneously therein, as by stirring. The molten homogeneous
mixture is then poured into convenient sized molds, allowed to
cool, and thereby to solidify. Tablets, powders, capsules, pills,
cachets, and lozenges can be used as solid dosage forms suitable
for oral administration.
[0264] Liquid form composition include solutions, suspensions, and
emulsions, for example, water, saline solutions, aqueous dextrose,
glycerol solutions or water/propylene glycol solutions. A saline
solution is a preferred carrier when the pharmaceutical composition
is administered intravenously or intranasally by a nebulizer. For
parenteral injection, liquid preparations can be formulated in
solution in, e.g. aqueous polyethylene glycol solution.
[0265] In a particularly preferred embodiment of this aspect, the
pharmaceutical composition is in the form of a solution,
suspension, or emulsion and is administered intranasally by a
nebulizer.
[0266] Preferably, the pharmaceutical composition is in unit dosage
form. In such form the composition may be subdivided into unit
doses containing appropriate quantities of the active component.
The unit dosage form can be a packaged composition, the package
containing discrete quantities of the composition, such as packeted
tablets, capsules, and powders in vials or ampoules. Also, the unit
dosage form can be a capsule, an injection vial, a tablet, a
cachet, or a lozenge itself, or it can be the appropriate number of
any of these in packaged form.
[0267] The composition, if desired, can also contain minor amounts
of wetting or emulsifying agents, or pH buffering agents.
[0268] Furthermore, such composition may also comprise other
pharmacologically active substance such as but not limited to
adjuvants and/or additional active ingredients.
[0269] Adjuvants in the context of the present invention include
but are not limited to Examples of such adjuvants include but are
not limited to inorganic adjuvants, organic adjuvants, oil-based
adjuvants, cytokines, particulate adjuvants, virosomes, bacterial
adjuvants, synthetic adjuvants, or synthetic polynucleotides
adjuvants.
[0270] Additional active ingredients include but are not limited to
other vaccine compounds or compositions. Preferably, the additional
active ingredient is another viral vaccine, more preferably a
vaccine against a DNA virus, a negative sense single stranded
(ssRNA(-)) RNA virus or an ambisense RNA virus. Further preferred,
the virus is selected from negative-single stranded (ssRNA(-)) RNA
virus. Even more preferred, the virus is selected from enveloped
ssRNA(-) viruses, more preferably from the group consisting of
paramyxoviruses and orthomyxoviruses. Preferably, the additional
active ingredient is a vaccine against paramyxoviruses, preferably
selected from the group consisting of Pneumovirinae,
Paramyxovirinae, Fer-de-Lance-Virus, Nariva-Virus, Salem-Virus,
Tupaia-Paramyxovirus, Beilong-Virus, J-Virus, Menangle-Virus,
Mossmann-Virus, and Murayama-Virus. It is particularly preferred
that the Pneumovirinae is selected from the group consisting of
Pneumovirus, (e.g. human respiratory syncytical virus (RSV), murine
pneumonia virus, bovine RSV, ovine RSV, caprine RSV) and
Metapneumovirus, (e.g. human metapneumovirus, avian
metapneumovirus). It is particularly preferred that the
Paramyxovirinae is selected from the group consisting of
Respirovirus (e.g. human parainfluenza virus 1 and 3), and
Rubulavirus, (e.g. human parainfluenza virus 2 and 4).
Alternatively or additionally, the additional active ingredient is
preferably another viral vaccine against an orthomyxovirus, more
preferably selected from the genus of Influenzavirus A,
Influenzavirus B, Influenzavirus C, Thogotoviris and Isavirus. In
even more preferred embodiments, the orthomxyovirus is
Influenzavirus A, preferably selected from the influenza A virus
subtypes H1N1, H1N2, H2N2, H3N1, H3N2, H3N8, H5N1, H5N2, H5N3,
H5N8, H5N9, H7N1, H7N2, H7N3, H7N4, H7N7, H9N2, H10N7, more
preferably the influenza A virus subtype H1N1.
[0271] In a fifth aspect the present invention provides for an
expression system of the first aspect, the isolated protein mixture
of the second aspect, the isolated host cell of the third aspect or
the composition of the fourth aspect, for the use in the treatment
or prevention of a viral disease.
[0272] In preferred embodiments of this aspect, the viral disease
is caused by a DNA virus, a negative sense single stranded
(ssRNA(-)) RNA virus or an ambisense RNA virus. Further preferred,
the virus is selected from negative-single stranded (ssRNA(-)) RNA
virus. Even more preferred, the virus is selected from enveloped
ssRNA(-) viruses, more preferably from the group consisting of
paramyxoviruses and orthomyxoviruses. The paramyxovirus is
preferably selected from the group consisting of Pneumovirinae,
Paramyxovirinae, Fer-de-Lance-Virus, Nariva-Virus, Salem-Virus,
Tupaia-Paramyxovirus, Beilong-Virus, J-Virus, Menangle-Virus,
Mossmann-Virus, and Murayama-Virus. Even more preferably, the
Pneumovirinae is selected from the group consisting of Pneumovirus,
(e.g. human respiratory syncytical virus (RSV), murine pneumonia
virus, bovine RSV, ovine RSV, caprine RSV) and Metapneumovirus,
(e.g. human metapneumovirus, avaian metapneumovirus). Even more
preferably, the Paramyxovirinae is selected from the group
consisting of Respirovirus (e.g. human parainfluenza virus 1 and
3), and Rubulavirus, (e.g. human parainfluenza virus 2 and 4).
Alternatively or additionally, the viral disease is preferably
caused by an orthomyxovirus, more preferably selected from the
genus of Influenzavirus A, Influenzavirus B, Influenzavirus C,
Thogotoviris and Isavirus. In even more preferred embodiments, the
orthomxyovirus is Influenzavirus A, preferably selected from the
influenza A virus subtypes H1N1, H1N2, H2N2, H3N1, H3N2, H3N8,
H5N1, H5N2, H5N3, H5N8, H5N9, H7N1, H7N2, H7N3, H7N4, H7N7, H9N2,
H10N7, more preferably the influenza A virus subtype H1N1.
[0273] In a sixth aspect, the present invention provides for a
method of treatment or prevention of a viral disease comprising the
administration of effective amounts of the expression system of the
first aspect, the isolated protein mixture of the second aspect,
the isolated host cell of the third aspect or the composition of
the fourth aspect for the use in the treatment or prevention of a
viral disease.
[0274] In preferred embodiments of this aspect, the viral disease
is caused by a DNA virus, a negative sense single stranded
(ssRNA(-)) RNA virus or an ambisense RNA virus. Further preferred,
the virus is selected from negative-single stranded (ssRNA(-)) RNA
virus. Even more preferred, the virus is selected from enveloped
ssRNA(-) viruses, more preferably from the group consisting of
paramyxoviruses and orthomyxoviruses. The paramyxovirus is
preferably selected from the group consisting of Pneumovirinae,
Paramyxovirinae, Fer-de-Lance-Virus, Nariva-Virus, Salem-Virus,
Tupaia-Paramyxovirus, Beilong-Virus, J-Virus, Menangle-Virus,
Mossmann-Virus, and Murayama-Virus. Even more preferably, the
Pneumovirinae is selected from the group consisting of Pneumovirus,
(e.g. human respiratory syncytical virus (RSV), murine pneumonia
virus, bovine RSV, ovine RSV, caprine RSV) and Metapneumovirus,
(e.g. human metapneumovirus, avaian metapneumovirus). Even more
preferably, the Paramyxovirinae is selected from the group
consisting of Respirovirus (e.g. human parainfluenza virus 1 and
3), and Rubulavirus, (e.g. human parainfluenza virus 2 and 4).
Alternatively or additionally, the viral disease is preferably
caused by an orthomyxovirus, more preferably selected from the
genus of Influenzavirus A, Influenzavirus B, Influenzavirus C,
Thogotoviris and Isavirus. In even more preferred embodiments, the
orthomxyovirus is Influenzavirus A, preferably selected from the
influenza A virus subtypes H1N1, H1N2, H2N2, H3N1, H3N2, H3N8,
H5N1, H5N2, H5N3, H5N8, H5N9, H7N1, H7N2, H7N3, H7N4, H7N7, H9N2,
H10N7, more preferably the influenza A virus subtype H1N1.
[0275] In a seventh aspect, the present invention provides for a
method of enhancing an immune response against an immunogen
comprising the administration of the expression system of the first
aspect, the protein mixture of the second aspect, the cell of the
third aspect and the composition of the fourth aspect.
[0276] In preferred embodiments of this aspect, the immunogen is a
pathogen, more preferred the immunogen is a virus. Preferably, the
virus is selected from the group consisting of a DNA virus, a
negative sense single stranded (ssRNA(-)) RNA virus or an ambisense
RNA virus. Further preferred, the virus is selected from
negative-single stranded (ssRNA(-)) RNA virus. Even more preferred,
the virus is selected from enveloped ssRNA(-) viruses, more
preferably from the group consisting of paramyxoviruses and
orthomyxoviruses. The paramyxovirus is preferably selected from the
group consisting of Pneumovirinae, Paramyxovirinae,
Fer-de-Lance-Virus, Nariva-Virus, Salem-Virus,
Tupaia-Paramyxovirus, Beilong-Virus, J-Virus, Menangle-Virus,
Mossmann-Virus, and Murayama-Virus. Even more preferably, the
Pneumovirinae is selected from the group consisting of Pneumovirus,
(e.g. human respiratory syncytical virus (RSV), murine pneumonia
virus, bovine RSV, ovine RSV, caprine RSV) and Metapneumovirus,
(e.g. human metapneumovirus, avaian metapneumovirus). Even more
preferably, the Paramyxovirinae is selected from the group
consisting of Respirovirus (e.g. human parainfluenza virus 1 and
3), and Rubulavirus, (e.g. human parainfluenza virus 2 and 4).
Alternatively or additionally, the viral disease is preferably
caused by an orthomyxovirus, more preferably selected from the
genus of Influenzavirus A, Influenzavirus B, Influenzavirus C,
Thogotoviris and Isavirus. In even more preferred embodiments, the
orthomxyovirus is Influenzavirus A, preferably selected from the
influenza A virus subtypes H1N1, H1N2, H2N2, H3N1, H3N2, H3N8,
H5N1, H5N2, H5N3, H5N8, H5N9, H7N1, H7N2, H7N3, H7N4, H7N7, H9N2,
H10N7, more preferably the influenza A virus subtype H1N1.
[0277] In an eighth aspect, the present invention provides
nucleotide constructs encoding influenza hemagglutinin (HA), an
expression system comprising these nucleotide constructs, and
proteins or polyproteins encoded by the nucleotide constructs,
wherein the HA0 cleavage site has a multibasic sequence.
[0278] The nucleic acid construct of this aspect comprises,
essentially consists or consists of a polynucleotide encoding a
modified influenza hemagglutinin (HA), wherein the HA0 cleavage
site is modified by introducing one or more basic amino acids.
Preferably, in the modified HA, the HA0 cleavage site of the
consensus HA gene was substituted with the multibasic cleavage site
of H5N1 that is cleaved by ubiquitous proteases to obtain a fully
processed HA (H1p).
[0279] Influenza hemagglutinin (HA) is a protein belonging to the
group of viral hemagglutinins found on the surface of the influenza
viruses. It is an antigenic glycoprotein which is responsible for
binding the virus to the cell that is being infected. HA proteins
like influenza hemagglutinin bind to cells with sialic acid on the
membranes, such as cells in the upper respiratory tract or
erythrocytes. There are at least 16 different HA antigens. These
serotypes or subtypes are named H1 through H16.
[0280] HA has two functions:
[0281] 1. Recognition of target cells by binding to sialic
acid-containing receptors.
[0282] 2. Mediating the entry of the viral genome into the target
cells by causing the fusion of host endosomal membrane with the
viral membrane (envelop) In detail, HA binds to the monosaccharide
sialic acid which is present on the surface of its target cells.
The cell membrane then engulfs the virus and the portion of the
membrane that encloses the virus forms an endosome. Then the
endosome is acidified and being transformed it into a lysosome. As
soon as the pH within the endosome drops to about 6.0, the original
folded structure of the HA molecule becomes unstable, causing it to
partially unfold, releasing a hydrophobic portion of its peptide
chain. This fusion peptide acts inserts into the endosomal
membrane. Then, the rest of the HA molecule refolds into a new
structure and causes the fusion of the viral membrane with the
endosomal membrane such that the contents of the virus, including
its RNA genome, are released into the cytoplasm of the cell.
[0283] To acquire its membrane fusion potential, HA0 must be
cleaved into HA1 and HA2 by host cell proteases. Cleavage occurs at
a linker sequence connecting the HA1 and HA2 subunits, which is
located on a partially surface exposed loop. The H0 cleavage site
of the influenza HA is located at about aa 340 in the H1N1 subtype
(aa 339 to 344 of SEQ ID NO: 8), the H5N1 subtype (aa 337 to 346 of
SEQ ID NO: 10) and H3N2 (aa 340 to 350 of SEQ ID NO: 20). The
position of the H0 cleavage site in other subtypes of Influenza
virus HA can be determined by the skilled person by conducting
sequence alignments and analysing the homology of the sequences by
methods well-known in this technical field. Influenza A virus HA of
subtype H1N1 and H3N2 require cleavage by host cell proteases to
transit into a fusion-competent state. Proteolytic activation of
influenza viruses can occur in the Golgi apparatus or at the plasma
membrane of infected cells, as well as in the extracellular space
and in target cell vesicles, so the nature of the cleavage site and
the respective activating proteases have important implications for
the biological properties of influenza virus as well as for
therapeutic intervention. HA of subtype H5N1 were shown to harbour
several arginine and lysine residues at the cleavage site, with an
R-X-R/K-R consensus sequence being indispensable for efficient
cleavage. In addition, evidence was obtained that cleavage of HA
might occur in the trans-Golgi network (TGN). It has been
demonstrated that these viruses are activated by furin.
[0284] The amino acid sequence of SEQ ID NO: 8 is a consensus
sequence derived from the alignment of 829 sequences of the H1N1
subtype annotated in the NCBI Influenza Virus Resource Database,
circulating worldwide from April to September 2009. The amino acid
sequence of SEQ ID NO: 9 is identical to SEQ ID NO: 8 with the
exception that the natural H0 protease cleavage site has been
substituted with a multibasic site derived from H5N1. The amino
acid sequence of SEQ ID NO: 10 is a consensus derived from the
alignment of 259 sequences of the H5N1 subtype annotated in the
NCBI Influenza Virus Resource Database, infecting humans worldwide
from 1990 to 2009. The amino acid sequence SEQ ID NO: 20 is the
sequence of HA of influenza A virus subtype H3N2, strain
A/Wellington/01/2004(H3N2). The amino acid sequence of SEQ ID NO:
21 is based on SEQ ID NO: 20 wherein the natural H0 protease
cleavage site has been substituted with a multibasic site derived
from H5N1
[0285] The amino acid sequence of SEQ ID NO: 11 is a NP consensus
sequence which was designed on the basis of the alignment of the
different influenza subtype consensus sequences. Further, the NP
sequence of SEQ ID NO: 11 lacks the Nuclear Localization Signal
residing in aa 6-8 (TRK to AAA) to increase cytoplasmic
expression.
[0286] The amino acid sequence of SEQ ID NO: 12 is a M1 consensus
sequence which was derived by alignment of different consensus
sequences which were aligned and the most common amino acid at each
position was chosen.
[0287] In a preferred embodiment of this aspect, the nucleic acid
construct and/or the expression system comprising this nucleic acid
construct, comprises elements to direct transcription and
translation of the HA and the optional further proteins encoded by
the nucleic acid construct and/or the expression system, which may
be included in the preferred embodiments outlined below. Such
elements included promoter and enhancer elements to direct
transcription of mRNA in a cell-free or a cell-based based system,
preferably a cell-based system. In another embodiment, wherein the
polynucleotides are provided as translatable RNAs is envisioned
that the expression system comprises those elements that are
necessary for translation and/or stabilization of RNAs encoding the
HA and/or the T cell inducing protein(s), e.g. polyA-tail, IRES,
cap structures etc.
[0288] In a preferred embodiment of this aspect, the nucleic acid
construct encodes a HA protein, peptide or variant thereof
comprising a modified H0 cleavage site, wherein the HA is selected
from the group of HA subtypes consisting of H1, H2, H3, H4, H6, H7,
H8, H9, H10, H11, H12, H13, H14, H15, H16 or a variant thereof or a
consensus sequence thereof, or a variant thereof or a consensus
sequence of one or more of the HA subtypes selected from the group
of HA subtypes consisting of H1, H2, H3, H4, H5, H6, H7, H8, H9,
H10, H11, H12, H13, H14, H15, H16. Preferably, the HA subtypes are
selected from the group consisting of H1, H2, H3, H7, H9, H10, or a
variant thereof or a consensus sequence thereof, or a variant
thereof or a consensus sequence of one or more of the HA subtypes
selected from the group of HA subtypes consisting of H1, H2, H3,
H5, H7, H9, H10. More preferred, the HA protein, peptide or variant
thereof which comprises a modified H0 cleavage site, is a HA from
subtype H1 or a variant thereof. Most preferred, the polynucleotide
encoding the HA of the nucleotide construct has the sequence of SEQ
ID NO: 9.
[0289] In a preferred embodiment of this aspect, the nucleic acid
construct encodes a HA protein, peptide or variant thereof, wherein
H0 cleavage site is modified by substituting at least one non-basic
amino acid by a basic amino acid and/or by introducing at least one
basic amino acid into the sequence of the H0 cleavage site.
Preferably, the basic amino acid is selected from the group
consisting of arginine (Arg; R), lysine (Lys; K) and histidine
(His, H). More preferably, the basic amino acid is selected from
the group consisting of arginine (Arg; R) and lysine (Lys; K).
[0290] Preferably, the cleavage site comprises a sequence of 6 to
12 amino acids, more preferably 10-12 amino-acids.
[0291] Preferably, at least 20%, at least 30%, at least 40%, at
least 50%, at least 60%, at least 70%, at least 80%, at least 90%,
or 100% to the amino acids of the polypeptide forming the H0
cleavage site are basic amino acids.
[0292] Preferably, the HA0 cleavage site of has a sequence selected
from the group consisting of PQRERRRKKR (SEQ ID NO: 15), PQRESRRKKR
(SEQ ID NO: 16), PQGERRRKKR (SEQ ID NO: 17), PLRERRRKR (SEQ ID NO:
18) and PQRETR (SEQ ID NO: 19). Most preferred, the HA0 cleavage
site of has the sequence PQRERRRKKR (SEQ ID NO: 15).
[0293] In Table 1 the sequences of the H0 cleavage site of H1N1
strain and the sequences of the H0 cleavage site of H5N1 strain are
compared in their respective context of the HA amino acid chain. In
the first line, the consensus sequence of the H0 polybasic cleavage
site of the highly pathogenic H5N1 subtype is indicated. In the
second line, the amino acid sequence of the H0 cleavage site of the
HA of the H1N1 wild-type strain is indicated. In the third line the
engineered sequence of the polybasic cleavage site in the H1p
protein, replacing the natural sequence.
TABLE-US-00001 TABLE 1 H5N1 cleavage LATGLRNS PQRERRRKKR site
GLFGAIA . . . H1N1 cleavage LATGLRNV PSIQSR site GLFGAIA . . . H1p
cleavage LATGLRNI PQRERRRKKR site GLFGAIA . . .
[0294] In embodiments of the eighth aspect of the present
invention, the nucleic acid construct is part of an expression
system encoding the modified HA and a second polynucleotide. In
this expression system the polynucleotide encoding the modified HA
and the second polynucleotide are comprised on separate vectors or
on the same vector. Accordingly, the polynucleotide encoding the
modified HA may be comprised on one vector and the second
polynucleotide may be comprised on a second vector. Alternatively
or additionally, the polynucleotide encoding the modified HA and
the second polynucleotide may be comprised on the same vector. It
is preferred that the polynucleotide encoding the modified HA and
the second polynucleotide are comprised on the same vector. It is
particularly preferred that the polynucleotide encoding the
modified HA and the second polynucleotide comprised on the same
vector are linked in such that they are expressed as a polyprotein.
Preferably, the polynucleotide encoding the modified HA and the
second polynucleotide form an open reading frame.
[0295] It is preferred that the polynucleotide encoding the
modified HA and the second polynucleotide are expressed as an
artificial polyprotein. In the context of the present invention the
term "artificial polyprotein" is directed at polyproteins which are
not naturally occurring, e.g. which are generated by using
recombinant DNA techniques. Accordingly, the proteins, peptides or
variants thereof encoded in this artificial polyprotein are
preferably derived from pathogens which genome do not encode a
polyprotein comprising the proteins, peptides or variants encoded
by the polynucleotide encoding the modified HA and second
polynucleotide of the invention. Preferably, the polynucleotide
encoding the modified HA and the second polynucleotide are both
derived from influenza A viruses.
[0296] In preferred embodiments of the eighth aspect, the second
polynucleotide encodes a protein or variant thereof, which induces
a T cell response, and which is, preferably, a non-structural
and/or internal protein of influenza A virus. Preferably, the
non-structural and/or internal protein encoded by the second
polynucleotide is selected from the group consisting of NP, M1, M2,
NS1, NS2/NEP, PA, PB1, PB2 or PB1-F2 (PB1F2).
[0297] It is preferred that the amino acid sequence of the modified
HA and/or the non-structural (internal) protein encoded by the
second polynucleotide comprises consecutive segments or a consensus
sequence of one or more different virus isolates.
[0298] In the context of the present invention it is preferred that
the term "segment" refers to a part of a protein or polyprotein. It
is particularly preferred that such segment folds and/or functions
independently of the rest of the protein or polyprotein such as but
not limited to a domain, an epitope or a fragment thereof. It is
understood that a protein variant in the context of the present
invention differs in comparison to its parent polypeptide in
changes in the amino acid sequence such as amino acid exchanges,
insertions, deletions, N-terminal truncations, or C-terminal
truncations, or any combination of these changes, which may occur
at one or several sites whereby the variant exhibits at least 80%
sequence identity to its parent polypeptide.
[0299] In a further preferred embodiment, a membrane attachment
domain of the modified HA or a variant thereof is functionally
deleted, thus, either being structurally deleted or structurally
present but not fulfilling its biological function. In a
particularly preferred embodiment, the amino acid sequence
corresponding to the membrane attachment domain is deleted. The
deletion of the membrane attachment region serves the purpose of
ascertaining that the anti-pathogenic B cell response inducing
protein is secreted from the cell into which the expression system
of the invention has been introduced.
[0300] In a further preferred embodiment of this aspect the
modified HA comprises a secretion signal, which targets the protein
to the endoplasmatic reticulum (ER). Such secretion signals are
present preferably in the context of a deleted membrane attachment
domain. The skilled person is well aware of various such secretion
signals, which may be used as heterologous secretion signals, e.g.
added to the N-terminus of the modified HA. Alternatively or
additionally a naturally occurring secretion signal may be used,
which is, e.g., present in the majority of structural and/or
surface viral proteins. Thus, if naturally present in the
respective protein, it is preferred that the secretion signal is
maintained in a modified version of the structural and/or surface
protein.
[0301] In embodiments of the eighth aspect, the non-structural
protein is a conserved internal protein suitable for inducing a T
cell mediated immune response against the pathogen involving the
activation of antigen-specific T lymphocyte such as but not limited
to cytotoxic T cells (CTLs), T helper cells (T.sub.H cells),
central memory T cells (TCM cells), effector memory T cells (TEM
cells), and regulatory T cells (Treg cells). Thus, preferably the T
cell inducing protein of the pathogen does not comprise a secretion
signal.
[0302] In the context of the present invention, the modified HA or
variant thereof is located either N- or C-terminally with respect
to the protein, peptide or variant thereof encoded by the second
polynucleotide. In a preferred embodiment, the protein, peptide or
variant thereof encoded by the second polynucleotide is located
N-terminally with respect to the modified HA or variant
thereof.
[0303] In preferred embodiments of the eighth aspect, a
polynucleotide encoding a cleavage site is positioned between the
modified HA or variant thereof and the second polynucleotide. It is
within the scope of the present invention that that any two
proteins can or cannot be connected or linked by a cleavage
site.
[0304] It is preferred that this cleavage site is either a
self-cleaving site (i.e. a cleavage site within the amino acid
sequence where this sequence is cleaved or is cleavable without
such cleavage involving any additional molecule or where the
peptide-bond formation in this sequence is prevented in the first
place) or an endopeptidase cleavage site (i.e. a cleavage cite
within the amino acid sequence where this sequence is cleaved or is
cleavable by an endopeptidase, e.g. trypsin, pepsin, elastase,
thrombin, collagenase, furin, thermolysin, endopeptidase V8,
cathepsins). More preferably, the self-cleaving site is a 2A
cleavage site selected from the group consisting of a viral 2A
peptide or 2A-like peptide of Picornavirus, insect viruses,
Aphtoviridae, Rotaviruses and Trypanosoma, preferably wherein the
2A cleavage site is the 2 A peptide of foot and mouth disease
virus. Alternatively or additionally, the polyprotein of the
present invention can be cleaved by an autoprotease, i.e. a
protease which cleaves peptide bonds in the same protein molecule
which also comprises the protease. Examples of such autoproteases
are the NS2 protease from flaviviruses or the VP4 protease of
birnaviruses
[0305] In the context of the present invention, the cleavage site
can be positioned N-terminally with respect to the modified HA or
variant thereof and C-terminally with respect to the protein,
peptide or variant thereof encoded by the second polynucleotide.
Alternatively the cleavage site can be positioned C-terminally with
respect to the modified HA or variant thereof and N-terminally with
respect to the protein, peptide or variant thereof encoded by the
second polynucleotide.
[0306] In preferred embodiment of the eighth aspect, the expression
system further comprises a third polynucleotide encoding a protein,
peptide or a variant thereof of a pathogen.
[0307] It is preferred that the protein, peptide or variant thereof
encoded by the third polynucleotide differs from the modified HA or
variant thereof or from the protein, peptide or variant thereof
encoded by the second polynucleotide. Preferably, the proteins,
peptides or variants thereof encoded by the first, second and the
third polynucleotide differ from each other in that they comprise
amino acid sequences of different proteins.
[0308] In preferred embodiments of the eighth aspect, the third
polynucleotide encodes a protein or variant thereof, which induces
a T cell response, and which is, preferably, a non-structural
and/or internal protein of influenza A virus. Preferably, the
non-structural and/or internal protein encoded by the third
polynucleotide is selected from the group consisting of NP, M1, M2,
NS1, NS2/NEP, PA, PB1, PB2 or PB1-F2 (PB1F2).
[0309] In preferred embodiments a polynucleotide encoding a linker
is positioned between the second polynucleotide and the third
polynucleotide. It is preferred that the linker is a flexible
linker, preferably a flexible linker comprising an amino acid
sequence according to SEQ ID NO: 6
(Gly-Gly-Gly-Ser-Gly-Gly-Gly).
[0310] In preferred embodiments the third polynucleotide is
comprised on a separate or on the same vector as the polynucleotide
encoding the modified HA or variant thereof and/or the second
polynucleotide.
[0311] Accordingly, the polynucleotide encoding the modified HA or
variant thereof is comprised on one vector and the second
polynucleotide is comprised on a second vector and the third
polynucleotide is comprised on a third vector. Alternatively or
additionally, the polynucleotide encoding the modified HA or
variant thereof and the second polynucleotide are comprised on the
same vector and the third polynucleotide is comprised on a separate
vector, or the polynucleotide encoding the modified HA or variant
thereof and the third polynucleotide are comprised on the same
vector and the second polynucleotide is comprised on a separate
vector, or the second and the third polynucleotide are comprised on
the same vector and the polynucleotide encoding the modified HA or
variant thereof is comprised on a separate vector. Alternatively or
additionally, the polynucleotide encoding the modified HA or
variant thereof and the second and the third polynucleotide are
comprised on the same vector. It is preferred that the
polynucleotide encoding the modified HA or variant thereof and the
second and the third polynucleotide may be comprised on the same
vector. It is particularly preferred that the polynucleotide
encoding the modified HA or variant thereof and the second and the
third polynucleotide comprised on the same vector are linked in
such that they are expressed as a polyprotein. Preferably, the
polynucleotide encoding the modified HA or variant thereof and the
second and the third polynucleotide comprised on the same vector
form an open reading frame.
[0312] In preferred embodiments of this aspect, the vector or
vectors comprising the polynucleotide encoding the modified HA or
variant thereof, and the second and/or the third polynucleotide
is/are selected from the group consisting of plasmid, cosmid,
phage, virus, and artificial chromosome. More preferably, a vector
suitable for practicing the present invention is selected from the
group consisting of plasmid vectors, cosmid vectors, phage vectors,
preferably lambda phage and filamentous phage vectors, viral
vectors, adenovirus vectors (e.g., non-replicating Ad5, Ad11, Ad26,
Ad35, Ad49, ChAd3, ChAd4, ChAd5, ChAd6, ChAd7, ChAd8, ChAd9,
ChAd10, ChAd11, ChAd16, ChAd17, ChAd19, ChAd20, ChAd22, ChAd24,
ChAd26, ChAd30, ChAd31, ChAd37, ChAd38, ChAd44, ChAd55, ChAd63,
ChAd 73, ChAd82, ChAd83, ChAd146, ChAd147, PanAd1, PanAd2, and
PanAd3 vectors or replication-competent Ad4 and Ad7 vectors),
adeno-associated virus (AAV) vectors (e.g., AAV type 5 and type 2),
alphavirus vectors (e.g., Venezuelan equine encephalitis virus
(VEE), sindbis virus (SIN), semliki forest virus (SFV), and VEE-SIN
chimeras), herpes virus vectors (e.g. vectors derived from
cytomegaloviruses, like rhesus cytomegalovirus (RhCMV) (14)), arena
virus vectors (e.g. lymphocytic choriomeningitis virus (LCMV)
vectors (15)), measles virus vectors, pox virus vectors (e.g.,
vaccinia virus, modified vaccinia virus Ankara (MVA), NYVAC
(derived from the Copenhagen strain of vaccinia), and avipox
vectors: canarypox (ALVAC) and fowlpox (FPV) vectors), vesicular
stomatitis virus vectors, retrovirus, lentivirus, viral like
particles, and bacterial spores. The vectors ChAd3, ChAd4, ChAd5,
ChAd6, ChAd7, ChAd8, ChAd9, ChAd10, ChAd11, ChAd16, ChAd17, ChAd19,
ChAd20, ChAd22, ChAd24, ChAd26, ChAd30, ChAd31, ChAd37, ChAd38,
ChAd44, ChAd63 and ChAd82 are described in detail in WO
2005/071093. The vectors PanAd1, PanAd2, PanAd3, ChAd55, ChAd73,
ChAd83, ChAd146, and ChAd147 are described in detail in WO
2010/086189. It is particularly preferred that the vector is
selected from the group consisting of MVA, ChAd63 and PanAd3.
[0313] In preferred embodiments of this aspect, the nucleotide
construct or the expression system or the vector or vectors
comprising the polynucleotide of the nucleotide construct or the
expression system may encompass "expression control sequences" that
regulate the expression of the gene of interest. Typically,
expression control sequences are polypeptides or polynucleotides
such as but not limited to promoters, enhancers, silencers,
insulators, or repressors.
[0314] In a particularly preferred embodiment of this aspect, the
expression system is defined according to the embodiments of the
first aspect of the present invention directed at expressing
systems comprising polynucleotides encoding proteins, peptides or
variants thereof from orthomyxovirus, preferably proteins, peptides
or variants from influenza A viruses.
[0315] In preferred embodiments, the nucleic acid construct and/or
the expression system of the eighth aspect is for use in medicine.
In more preferred embodiments, the nucleic acid constructs, the
expression systems or the proteins of this aspect are for use in
the prophylaxis or treatment of an influenza A virus infection
and/or in the manufacturing of medicament for use in the
prophylaxis or treatment of an influenza A virus infection and/or
for use in methods of prophylaxis or treatment of an influenza A
virus infection.
[0316] In preferred embodiments the expression system is for use in
enhancing an immune response. In more preferred embodiments, the
expression system is for use in enhancing an anti-pathogenic B cell
immune response against an influenza A virus infection, more
preferably an influenza A virus as defined in the first aspect of
the invention.
[0317] In a ninth aspect, the present invention provides the use of
the multibasic HA0 cleavage site as defined in the eighth aspect
for constructing a nucleic acid construct or an expression systems
capable of expressing the modified influenza hemagglutinin (HA) of
the eighth aspect in vitro and/or in vivo. Furthermore, this aspect
provides the isolated protein mixture, the protein and/or
polyprotein encoded by the nucleic acid construct or expression
system constructed according to this aspect.
[0318] In a tenth aspect, the invention provides an isolated
protein mixture encoded by the expression system of the eighth
aspect. Preferably, the isolated protein mixture contains,
essentially contains or comprises one or more of the proteins or
polyproteins encoded by the nucleic acid construct or the
expression system of the eighth aspect. In preferred embodiments,
the isolated protein mixture is for use in medicine. In
particularly preferred embodiments, the isolated protein mixture is
for use in the prophylaxis or treatment of a viral infection,
particularly preferably for use in the prophylaxis or treatment of
an influenza A virus infection and/or in the manufacturing of
medicament for use in the prophylaxis or treatment of an influenza
A virus infection and/or for use in methods of prophylaxis or
treatment of an influenza A virus infection.
[0319] In an eleventh aspect, the invention provides an isolated
host cell containing the nucleotide constructs, the expression
system or the proteins or polyproteins of the eighth aspect and/or
the protein mixture of the tenth aspect. It is understood that such
host cell includes but is not limited to prokaryotic (e.g. a
bacterial cell) or eukaryotic cells (e.g. a fungal, plant or animal
cell). In preferred embodiments of this aspect, the host cell is
for use in medicine. In particularly preferred embodiments, the
host cell is for use in the prophylaxis or treatment of an
influenza A virus infection and/or in the manufacturing of
medicament for use in the prophylaxis or treatment of an influenza
A virus infection and/or for use in methods of prophylaxis or
treatment of an influenza A virus infection.
[0320] In a twelfth aspect, the present invention provides a
composition comprising the nucleotide constructs, the expression
system or the proteins or polyproteins of the eighth aspect, or the
protein mixture of the tenth aspect, and a pharmaceutical
acceptable carrier and/or excipient. Preferably, such composition
is a pharmaceutical composition.
[0321] The composition of the twelfth aspect contains a
therapeutically effective amount of the compound, preferably in
purified form, together with a suitable amount of carrier so as to
provide the form for proper administration to the patient. The
formulation should suit the mode of administration.
[0322] The compositions can take the form of solutions,
suspensions, emulsion, tablets, pills, capsules, powders,
sustained-release formulations and the like. The composition can be
formulated as a suppository, with traditional binders and carriers
such as triglycerides.
[0323] For preparing pharmaceutical compositions of the present
invention, pharmaceutically acceptable carriers can be either solid
or liquid.
[0324] Solid form compositions include powders, tablets, pills,
capsules, lozenges, cachets, suppositories, and dispersible
granules. A solid excipient can be one or more substances, which
may also act as diluents, flavoring agents, binders, preservatives,
tablet disintegrating agents, or an encapsulating material. In
powders, the excipient is preferably a finely divided solid, which
is in a mixture with the finely divided inhibitor of the present
invention. In tablets, the active ingredient is mixed with the
carrier having the necessary binding properties in suitable
proportions and compacted in the shape and size desired. Suitable
excipients are magnesium carbonate, magnesium stearate, talc,
sugar, lactose, pectin, dextrin, starch, gelatin, tragacanth,
methylcellulose, sodium carboxymethylcellulose, a low melting wax,
cocoa butter, and the like. For preparing suppositories, a low
melting wax, such as a mixture of fatty acid glycerides or cocoa
butter, is first melted and the active component is dispersed
homogeneously therein, as by stirring. The molten homogeneous
mixture is then poured into convenient sized molds, allowed to
cool, and thereby to solidify. Tablets, powders, capsules, pills,
cachets, and lozenges can be used as solid dosage forms suitable
for oral administration.
[0325] Liquid form composition include solutions, suspensions, and
emulsions, for example, water, saline solutions, aqueous dextrose,
glycerol solutions or water/propylene glycol solutions. A saline
solution is a preferred carrier when the pharmaceutical composition
is administered intravenously or intranasally by a nebulizer. For
parenteral injection, liquid preparations can be formulated in
solution in, e.g. aqueous polyethylene glycol solution.
[0326] In a particularly preferred embodiment of this aspect, the
pharmaceutical composition is in the form of a solution,
suspension, or emulsion and is administered intranasally by a
nebulizer.
[0327] Preferably, the pharmaceutical composition is in unit dosage
form. In such form the composition may be subdivided into unit
doses containing appropriate quantities of the active component.
The unit dosage form can be a packaged composition, the package
containing discrete quantities of the composition, such as packeted
tablets, capsules, and powders in vials or ampoules. Also, the unit
dosage form can be a capsule, an injection vial, a tablet, a
cachet, or a lozenge itself, or it can be the appropriate number of
any of these in packaged form.
[0328] The composition, if desired, can also contain minor amounts
of wetting or emulsifying agents, or pH buffering agents.
[0329] Furthermore, such composition may also comprise other
pharmacologically active substance such as but not limited to
adjuvants and/or additional active ingredients.
[0330] Adjuvants in the context of the present invention include
but are not limited to Examples of such adjuvants include but are
not limited to inorganic adjuvants, organic adjuvants, oil-based
adjuvants, cytokines, particulate adjuvants, virosomes, bacterial
adjuvants, synthetic adjuvants, or synthetic polynucleotides
adjuvants.
[0331] Additional active ingredients include but are not limited to
other vaccine compounds or compositions. Preferably, the additional
active ingredient is another viral vaccine, more preferably a
vaccine against a DNA virus, a negative sense single stranded
(ssRNA(-)) RNA virus or an ambisense RNA virus. Further preferred,
the virus is selected from negative-single stranded (ssRNA(-)) RNA
virus. Even more preferred, the virus is selected from enveloped
ssRNA(-) viruses, more preferably from the group consisting of
paramyxoviruses and orthomyxoviruses. Preferably, the additional
active ingredient is a vaccine against paramyxoviruses, preferably
selected from the group consisting of Pneumovirinae,
Paramyxovirinae, Fer-de-Lance-Virus, Nariva-Virus, Salem-Virus,
Tupaia-Paramyxovirus, Beilong-Virus, J-Virus, Menangle-Virus,
Mossmann-Virus, and Murayama-Virus. It is particularly preferred
that the Pneumovirinae is selected from the group consisting of
Pneumovirus, (e.g. human respiratory syncytical virus (RSV), murine
pneumonia virus, bovine RSV, ovine RSV, caprine RSV) and
Metapneumovirus, (e.g. human metapneumovirus, avaian
metapneumovirus). It is particularly preferred that the
Paramyxovirinae is selected from the group consisting of
Respirovirus (e.g. human parainfluenza virus 1 and 3), and
Rubulavirus, (e.g. human parainfluenza virus 2 and 4).
Alternatively or additionally, the additional active ingredient is
preferably another viral vaccine against an orthomyxovirus, more
preferably selected from the genus of Influenzavirus A,
Influenzavirus B, Influenzavirus C, Thogotoviris and Isavirus. In
even more preferred embodiments, the orthomxyovirus is
Influenzavirus A, preferably selected from the influenza A virus
subtypes H1N1, H1N2, H2N2, H3N1, H3N2, H3N8, H5N1, H5N2, H5N3,
H5N8, H5N9, H7N1, H7N2, H7N3, H7N4, H7N7, H9N2, H10N7, more
preferably the influenza A virus subtype H1N1.
[0332] In a thirteenth aspect, the present invention provides the
nucleotide constructs, the expression system or the proteins or
polyproteins of the eighth aspect, the protein mixture of the tenth
aspect, the cell of the eleventh aspect and the composition of the
twelfth aspect, for the use in medicine in particular in the
treatment or prevention of influenza A virus infections. The
influenza A virus is preferably selected from the influenza A virus
subtypes H1N1, H1N2, H2N2, H3N1, H3N2, H3N8, H5N1, H5N2, H5N3,
H5N8, H5N9, H7N1, H7N2, H7N3, H7N4, H7N7, H9N2, H10N7, more
preferably the influenza A virus subtype H1N1.
[0333] In a fourteenth aspect, the present invention provides for a
method of treatment or prevention of an influenza A virus
infections comprising the administration of an effective amount of
the nucleotide constructs, the expression system or the proteins or
polyproteins of the eighth aspect, the protein mixture of the tenth
aspect, the cell of the eleventh aspect and the composition of the
twelfth aspect. The influenza A virus is preferably selected from
the influenza A virus subtypes H1N1, H1N2, H2N2, H3N1, H3N2, H3N8,
H5N1, H5N2, H5N3, H5N8, H5N9, H7N1, H7N2, H7N3, H7N4, H7N7, H9N2,
H10N7, more preferably the influenza A virus subtype H1N1.
[0334] In a fifteenth aspect, the present invention provides for a
method of enhancing an immune response comprising the
administration of the nucleotide constructs or the expression
system or the proteins or polyproteins of the eighth aspect, the
protein mixture of the tenth aspect, the cell of the eleventh
aspect and the composition of the twelfth aspect. In a preferred
embodiment of this aspect, the method enhances an immune response
against influenza A virus. The influenza A virus is preferably
selected from the influenza A virus subtypes H1N1, H1N2, H2N2,
H3N1, H3N2, H3N8, H5N1, H5N2, H5N3, H5N8, H5N9, H7N1, H7N2, H7N3,
H7N4, H7N7, H9N2, H10N7, more preferably the influenza A virus
subtype H1N1.
[0335] The following examples are merely illustrative of the
present invention and should not be construed to limit the scope of
the invention as indicated by the appended claims in any way.
EXAMPLES
Example 1
Design and Synthesis of DNA Encoding Optimised RSV Antigen
[0336] Consensus Vaccine
[0337] Several computational alternatives to isolate-based vaccine
design exist. One approach is reconstruction of the most recent
common ancestor (MRCA) sequence (9). In this type of analysis, the
ancestral state is an estimate of the actual sequence that existed
in the past (i.e., it comes directly from the reconstructed
history). Another type of computational analysis is a center of the
tree (COT) approach. The COT approach identifies a point on the
unrooted phylogeny, where the average evolutionary distance from
that point to each tip on the phylogeny is minimized. Advocates of
this approach state that because the COT is a point on the
phylogeny, the estimated COT sequence will have the same advantages
as the estimated ancestral sequence. See, for example, U.S.
Application 2005/0137387 A1. However, this COT approach is
sufficiently complex that reducing it to practice for a large and
heterologous data set such as the Influenza sequence database is
not practical with technology. Overall, the MRCA and COT approaches
are impractical for application to the complex Influenza sequence
database.
[0338] A third type of computational analysis is the consensus
sequence approach. Because the consensus sequence is composed of
the amino acid most commonly observed at each position, it likely
represents the most-fit state of the virus. Thus, effective evasion
of the immune response by selection of a sequence divergent from
consensus may result in a less fit virus from a replicative
standpoint. The consensus sequence approach favors heavily sampled
sublineages and deemphasizes outliers. As such, the approaches
utilized herein are far more straightforward than the other types
of computational analyses. Furthermore, these approaches can use
the entire data set for RSV. One advantage of the consensus
sequence is that it minimizes the genetic differences between
vaccine strains and contemporary isolates, effectively reducing the
extent of diversity by half, and thus it may have enhanced
potential for eliciting cross-reactive responses.
[0339] Vaccine Design
[0340] To design the vaccine antigen of the present invention,
protein sequences of the F0-, N-, and M2-1-proteins of RSV were
retrieved from the National Center for Biotechnology Information
(NCBI) RSV Resource database (http://www.ncbi.nlm.nih.gov). Protein
sequences were chosen from different RSV subtype A strains.
[0341] A F0 consensus sequence was derived by alignment of all
non-identical sequences of the F-protein using MUSCLE version 3.6
and applying the majority rule. The vaccine's F0 consensus sequence
was designed on the basis of the alignment of the different RSV
sequences. The sequence similarity of the vaccine consensus F0
sequence was measured performing BLAST analysis, which stands for
Basic Local Alignment Search Tool and is publicly available through
the NCBI. The highest average similarity of the consensus sequence,
calculated compared to all RSV sequences in the database, was 100%
with respect to the human respiratory syncytial virus A2
strain.
[0342] Further, the vaccine's F0 sequence lacks the transmembrane
region residing in amino acids 525 to 574 to allow for the
secretion of F0.DELTA.TM.
[0343] Finally, the vaccine F0.DELTA.TM sequence was
codon-optimized for expression in eukaryotic cells.
[0344] The vaccine's N consensus sequence was derived by alignment
of all non-identical sequences of the N-protein using MUSCLE
version 3.6 and applying the majority rule. BLAST analysis of the N
consensus sequence found the best alignment with the human
respiratory syncytial virus A2 strain. The vaccine's N sequence was
then codon-optimized for expression in eukaryotic cells.
[0345] A M2-1 consensus sequence was derived by alignment of all
non-identical sequences of the M2-1-protein using MUSCLE version
3.6 and applying the majority rule. BLAST analysis of the M2-1
consensus sequence found the best alignment with the human
respiratory syncytial virus A2 strain. Finally, the vaccine M2-1
sequence was codon-optimized for expression in eukaryotic
cells.
[0346] The vaccines F0.DELTA.TM sequence and N sequence were spaced
by the cleavage sequence 2A of the Foot and Mouth Disease virus.
The vaccines N sequence and M2-1 sequence were separated by a
flexible linker (GGGSGGG; SEQ ID NO: 6).
[0347] Finally, the codon-optimized viral genes were cloned as the
single open reading frame F0.DELTA.TM-N-M2-1. A schematic diagram
of the antigen composition is given in FIG. 1.
[0348] Generation of DNA Plasmids Encoding F0.DELTA.TM and
F0.DELTA.TM-N-M2-1
[0349] Consensus F0.DELTA.TM, N and M2-1 sequences were optimized
for mammalian expression, including the addition of a Kozak
sequence and codon optimization. The DNA sequence encoding the
multi-antigen vaccine was chemically synthesized and then
sub-cloned by suitable restriction enzymes EcoRV and NotI into the
pVJTetOCMV shuttle vector under the control of the CMV
promoter.
[0350] Generation of PanAd3 Viral-Vectored RSV Vaccine
[0351] A viral-vectored RSV vaccine PanAd3/F0.DELTA.TM-N-M2-1 was
generated which contains a 809 aa polyprotein coding for the
consensus F0.DELTA.TM, N and M2-1 proteins fused by a flexible
linker.
[0352] Bonobo Adenovirus type 3 (PanAd3) is a novel adenovirus
strain with improved seroprevalence and has been described
previously.
[0353] Cloning of F0.DELTA.TM-N-M2-1 from the plasmid vector
pVJHTetOCMV/F0.DELTA.TM-N-M2-1 into the PanAd3 pre-Adeno vector was
performed by cutting out the antigen sequences flanked by
homologous regions and enzymatic in vitro recombination.
[0354] Analysis of Antigen Expression in Mammalian Cells
[0355] To control that the unique combination of viral antigens was
efficiently expressed and correctly processed into mammalian cells,
Hela cells were transfected with 10 .mu.g of DNA plasmid encoding
the F0.DELTA.TM-N-M2-1 antigen. Cells were cultured for 36 hours
before the supernatant was collected and cell lysates were
prepared. Proteins were separated by SDS-PAGE and blotted onto
nylon filters. A mouse monoclonal antibody (mAb8) raised against
the M viral protein (gift from Dr. Geraldine Taylor) was used to
reveal the expressed proteins.
[0356] As shown in FIGS. 2 and 3, the fused viral protein N-M2-1 is
very efficiently released from the polyprotein by the 2A cleavage
site and recognized as a major band by mAb8. Very few high
molecular weight precursor is present at steady-state in the cells.
Lysates of Hep2-cells infected with RSV strain A were used as
control.
[0357] Non-Reducing SDS-PAGE and Western blot analysis of the cell
culture medium showed that the F-protein deleted of the
trans-membrane region is secreted into the supernatant (see FIG. 3,
lane RSV). The molecular weight of the F-protein in the supernatant
is consistent with homotrimeric F-protein, which is its native
configuration.
Example 2
Vaccine Immunogenicity in Mice
[0358] Anti-F Antibodies by DNA Immunization
[0359] DNA plasmids encoding F0.DELTA.TM-N-M2-1 or F0.DELTA.TM
alone were used to immunize mice by DNA plasmid injection and
electroporation (GET) with a regimen of priming and boosting at
three weeks post prime. Sera of immunized mice were collected two
weeks after boosting and pooled.
[0360] Supernatants from Hela cells infected with
PanAd3/F0.DELTA.TM-N-M2-1 at MOI 250 were separated on non-reducing
SDS-PAGE, blotted onto nylon filters and probed with different
dilutions of sera from mice immunized with F0.DELTA.TM or
F0.DELTA.TM-N-M2-1.
[0361] As shown in FIGS. 4A and B, the antibody titers raised by
the F-protein expressed in the context of the vaccine antigen are
at least 30 times higher than those elicited by the F-protein
alone. Thus, the F0.DELTA.TM-N-M2-1 antigen has superior
immunogenic properties in inducing B-cell responses in mice.
[0362] T Cell Response
[0363] The immunological potency of the chimpanzee adenoviral
vector PanAd3 bearing the RSV vaccine antigen F0.DELTA.TM-N-M2-1
was evaluated in mice.
[0364] Groups of Balb/C mice were immunized by intramuscular
injection in the quadriceps with increasing dose of
PanAd3/F0.DELTA.TM-N-M2-1. 4 weeks after vaccination mice were
sacrificed and splenocytes were subjected to IFN.gamma.-Elispot
assay using mapped immunodominant peptides from RSV F- and
M-proteins (peptide GWYTSVITIELSNIKE (F aa 51-66) peptide KYKNAVTEL
(F aa 85-93) and peptide SYIGSINNI (M aa 282-290)).
[0365] As shown in FIG. 5, a potent T cell response was observed
against known Balb/C immunodominant epitopes against RSV F and M
proteins.
Example 3
Induction of Neutralizing Antibodies by H1p
[0366] The novel chimeric H1p protein engineered to contain the
multibasic HA0 cleavage site from H5N1 is efficiently expressed and
fully cleaved in transfected HeLa cells. The equivalent protein
with the wild type cleavage site, H1, is not cleaved in HeLa cells,
as shown in FIG. 7.
[0367] In order to control that the chimeric H1p protein is
correctly displayed on the cell membrane, a whole-cell FACS binding
assay has been performed using a polyclonal anti-HA serum to reveal
the transfected protein on the cell surface. As shown in FIG. 8,
H1p is exposed on the cell membrane as efficiently as the
corresponding wild type HA protein.
[0368] To measure the immunological potency of H1p, Balb/C mice
were immunized with plasmid DNA vectors encoding the modified H1p
and the unmodified H1 (PW-H1p and PVJ-H1, respectively). The sera
from immunized animals have been analyzed by ELISA on purified
recombinant HA protein (H1N1California2009). The anti-HA titers
elicited by the engineered H1p protein were surprisingly higher
than those elicited by the HA bearing the wild type protease
cleavage site (FIG. 9).
[0369] To confirm and expand these results, the sera from H1 and
H1p immunized animals were tested for their capacity to neutralize
the infection of retroviral vectors pseudotyped with the Flu HA
protein in a cell culture based assay. Pseudovirions are infectious
for a single cycle of infection in which they express the reporter
gene luciferase. FIG. 10 shows the serum neutralization capacity in
a HA (H1N1Mexico2009) pseudotyped virus particles infection assay
on MDCK cells. The result confirms that the antibodies elicited by
H1p have greater neutralizing activity than those induced by H1
protein.
Example 4
Enhanced Antibody Titer by a Polyprotein Comprising NP, M1 and
H1p
[0370] Head to head comparison of the immunological potency of the
H1p and NPM1H1p revealed that the HA protein expressed in the
context of the triple antigen induces higher antibody titer than HA
alone. FIG. 11 shows the results of an ELISA assay where a
recombinant HA (H1N1California 2009) was coated on the bottom of 96
well plate. Serial dilutions of sera of animals immunized with H1p
and NPM1H1p were put on the plate and the bound IgG were revealed
with an anti-mouse IgG secondary antibody. As already observed for
the RSV antigen, the co-expression of internal antigens (NP and M1)
with the surface exposed antigen (HA) improves the humoral response
directed to the latter protein.
Example 5
Processing of the Novel FLU Antigen Composed of NP, M1 and H1p
[0371] To control the expression and processing of the vaccine
antigen proteins, HeLa cells have been transfected with an
expression plasmid containing NPM1H1p under the control of the CMV
promoter. Cells were harvested 48 hours after transfection. Half
cells were lysed for Western Blot analysis (FIG. 12) and half were
incubated with a commercially available antibody C179 (Okuno Y, JVI
1994), which binds the stem region of the HA protein (FIG. 13) and
analysed by FACS. Western blot analysis of the total cell lysate
shows a unique 70 kDa band which correspond to the NPM1 fusion
protein (FIG. 12). This indicates that the antigen is fully and
correctly processed out of the 2A cleavage site. FIG. 13 shows that
the released H1p protein is then displayed on the cell membrane and
correctly folded, as detected by the use of a
conformation-dependent antibody C179 which binds to the HA stem
region. Accordingly, the novel FLU antigen composed of NP, M1 and
H1p is correctly processed and the released HA protein is displayed
on the cell surface and recognized by a conformational
antibody.
REFERENCES
[0372] 1) Ono & Freed, (2005), Adv. Virus Res., 273:5419-5442
[0373] 2) Collins P et al., (1996). Parainfluenza viruses,
1205-1241. In Fields et al. (ed.), Fields Virology. Lippincot-Raven
Publishers, Philadelphia (PA) USA [0374] 3) Shay D K et al.,
(1999), Bronchiolitis-Associated Hospitalizations Among US
Children, 1980-1996. JAMA 282:1440-1446 [0375] 4) Simoes EA &
Carbonell-Estrany X (2003), Pediatr Infect Dis J 22:S13-8;
discussion S 18-20. [0376] 5) Collins P L & Graham BS (2008), J
Virol 82:2040-55 [0377] 6) Falsey A R et al., (2005), N Engl J Med
352:1749-59 [0378] 7) Fleming D M & Elliot A J (2007), Eur
Respir J 30:1029-31 [0379] 8) Cardenas S et al., (2005), Expert Rev
Anti Infect Ther 3:719-26 [0380] 9) Kim H W et al., (1969), Amer J
Epidemiol 89:422-434 [0381] 10) Delgado M F et al., (2009), Nat Med
15:34-41 [0382] 11) Graham B S et al., (1993), J Immunol
151:2032-2040 [0383] 12) Polack F P et al., (2002), J Exp Med
196:859-865 [0384] 13) Castilow E M & Varga S M, (2008), Future
Virol 3:445-454 [0385] 14) Hansen S et al., (2011), Nature
473:523-527 [0386] 15) Flatz, L et al. (2010), Nature Med
16:339-345
SEQUENCE LISTING
Free Text Information
[0386] [0387] SEQ ID NO: 1 F protein minimal sequence [0388] SEQ ID
NO: 2 F0.DELTA.TM [0389] SEQ ID NO: 3 N protein minimal sequence
[0390] SEQ ID NO: 4 N protein of RSV [0391] SEQ ID NO: 5 M2-1
protein of RSV [0392] SEQ ID NO: 6 peptide linker [0393] SEQ ID NO:
7 F0.DELTA.TM-N-M2-1 [0394] SEQ ID NO: 8 HA subtype H1 consensus
sequence [0395] SEQ ID NO: 9 HA subtype H1 consensus sequence
modified H0 cleavage site [0396] SEQ ID NO: 10 HA subtype H5
consensus sequence [0397] SEQ ID NO: 11 NP consensus sequence
[0398] SEQ ID NO: 12 M1 consensus sequence [0399] SEQ ID NO: 13
NP-M1-HA (amino acid sequence) [0400] SEQ ID NO: 14 NP-M1-HA
(nucleic acid sequence) [0401] SEQ ID NO: 15 H0 cleavage site
sequence [0402] SEQ ID NO: 16 H0 cleavage site sequence [0403] SEQ
ID NO: 17 H0 cleavage site sequence [0404] SEQ ID NO: 18 H0
cleavage site sequence [0405] SEQ ID NO: 19 H0 cleavage site
sequence [0406] SEQ ID NO: 20 HA subtype H3 strain
A/Wellington/01/2004(H3N2) [0407] SEQ ID NO: 21 HA subtype H3
strain A/Wellington/01/2004(H3N2) modified H0 cleavage site
Sequence CWU 1
1
211360PRTArtificial SequenceSynthetic Polypeptide 1Val Leu His Leu
Glu Gly Glu Val Asn Lys Ile Lys Ser Ala Leu Leu 1 5 10 15 Ser Thr
Asn Lys Ala Val Val Ser Leu Ser Asn Gly Val Ser Val Leu 20 25 30
Thr Ser Lys Val Leu Asp Leu Lys Asn Tyr Ile Asp Lys Gln Leu Leu 35
40 45 Pro Ile Val Asn Lys Gln Ser Cys Ser Ile Ser Asn Ile Glu Thr
Val 50 55 60 Ile Glu Phe Gln Gln Lys Asn Asn Arg Leu Leu Glu Ile
Thr Arg Glu 65 70 75 80 Phe Ser Val Asn Ala Gly Val Thr Thr Pro Val
Ser Thr Tyr Met Leu 85 90 95 Thr Asn Ser Glu Leu Leu Ser Leu Ile
Asn Asp Met Pro Ile Thr Asn 100 105 110 Asp Gln Lys Lys Leu Met Ser
Asn Asn Val Gln Ile Val Arg Gln Gln 115 120 125 Ser Tyr Ser Ile Met
Ser Ile Ile Lys Glu Glu Val Leu Ala Tyr Val 130 135 140 Val Gln Leu
Pro Leu Tyr Gly Val Ile Asp Thr Pro Cys Trp Lys Leu 145 150 155 160
His Thr Ser Pro Leu Cys Thr Thr Asn Thr Lys Glu Gly Ser Asn Ile 165
170 175 Cys Leu Thr Arg Thr Asp Arg Gly Trp Tyr Cys Asp Asn Ala Gly
Ser 180 185 190 Val Ser Phe Phe Pro Gln Ala Glu Thr Cys Lys Val Gln
Ser Asn Arg 195 200 205 Val Phe Cys Asp Thr Met Asn Ser Leu Thr Leu
Pro Ser Glu Val Asn 210 215 220 Leu Cys Asn Val Asp Ile Phe Asn Pro
Lys Tyr Asp Cys Lys Ile Met 225 230 235 240 Thr Ser Lys Thr Asp Val
Ser Ser Ser Val Ile Thr Ser Leu Gly Ala 245 250 255 Ile Val Ser Cys
Tyr Gly Lys Thr Lys Cys Thr Ala Ser Asn Lys Asn 260 265 270 Arg Gly
Ile Ile Lys Thr Phe Ser Asn Gly Cys Asp Tyr Val Ser Asn 275 280 285
Lys Gly Val Asp Thr Val Ser Val Gly Asn Thr Leu Tyr Tyr Val Asn 290
295 300 Lys Gln Glu Gly Lys Ser Leu Tyr Val Lys Gly Glu Pro Ile Ile
Asn 305 310 315 320 Phe Tyr Asp Pro Leu Val Phe Pro Ser Asp Glu Phe
Asp Ala Ser Ile 325 330 335 Ser Gln Val Asn Glu Lys Ile Asn Gln Ser
Leu Ala Phe Ile Arg Lys 340 345 350 Ser Asp Glu Leu Leu His Asn Val
355 360 2524PRTArtificial SequenceSynthetic Polypeptide 2Met Glu
Leu Leu Ile Leu Lys Ala Asn Ala Ile Thr Thr Ile Leu Thr 1 5 10 15
Ala Val Thr Phe Cys Phe Ala Ser Gly Gln Asn Ile Thr Glu Glu Phe 20
25 30 Tyr Gln Ser Thr Cys Ser Ala Val Ser Lys Gly Tyr Leu Ser Ala
Leu 35 40 45 Arg Thr Gly Trp Tyr Thr Ser Val Ile Thr Ile Glu Leu
Ser Asn Ile 50 55 60 Lys Glu Asn Lys Cys Asn Gly Thr Asp Ala Lys
Val Lys Leu Ile Lys 65 70 75 80 Gln Glu Leu Asp Lys Tyr Lys Asn Ala
Val Thr Glu Leu Gln Leu Leu 85 90 95 Met Gln Ser Thr Pro Ala Thr
Asn Asn Arg Ala Arg Arg Glu Leu Pro 100 105 110 Arg Phe Met Asn Tyr
Thr Leu Asn Asn Ala Lys Lys Thr Asn Val Thr 115 120 125 Leu Ser Lys
Lys Arg Lys Arg Arg Phe Leu Gly Phe Leu Leu Gly Val 130 135 140 Gly
Ser Ala Ile Ala Ser Gly Val Ala Val Ser Lys Val Leu His Leu 145 150
155 160 Glu Gly Glu Val Asn Lys Ile Lys Ser Ala Leu Leu Ser Thr Asn
Lys 165 170 175 Ala Val Val Ser Leu Ser Asn Gly Val Ser Val Leu Thr
Ser Lys Val 180 185 190 Leu Asp Leu Lys Asn Tyr Ile Asp Lys Gln Leu
Leu Pro Ile Val Asn 195 200 205 Lys Gln Ser Cys Ser Ile Ser Asn Ile
Glu Thr Val Ile Glu Phe Gln 210 215 220 Gln Lys Asn Asn Arg Leu Leu
Glu Ile Thr Arg Glu Phe Ser Val Asn 225 230 235 240 Ala Gly Val Thr
Thr Pro Val Ser Thr Tyr Met Leu Thr Asn Ser Glu 245 250 255 Leu Leu
Ser Leu Ile Asn Asp Met Pro Ile Thr Asn Asp Gln Lys Lys 260 265 270
Leu Met Ser Asn Asn Val Gln Ile Val Arg Gln Gln Ser Tyr Ser Ile 275
280 285 Met Ser Ile Ile Lys Glu Glu Val Leu Ala Tyr Val Val Gln Leu
Pro 290 295 300 Leu Tyr Gly Val Ile Asp Thr Pro Cys Trp Lys Leu His
Thr Ser Pro 305 310 315 320 Leu Cys Thr Thr Asn Thr Lys Glu Gly Ser
Asn Ile Cys Leu Thr Arg 325 330 335 Thr Asp Arg Gly Trp Tyr Cys Asp
Asn Ala Gly Ser Val Ser Phe Phe 340 345 350 Pro Gln Ala Glu Thr Cys
Lys Val Gln Ser Asn Arg Val Phe Cys Asp 355 360 365 Thr Met Asn Ser
Leu Thr Leu Pro Ser Glu Val Asn Leu Cys Asn Val 370 375 380 Asp Ile
Phe Asn Pro Lys Tyr Asp Cys Lys Ile Met Thr Ser Lys Thr 385 390 395
400 Asp Val Ser Ser Ser Val Ile Thr Ser Leu Gly Ala Ile Val Ser Cys
405 410 415 Tyr Gly Lys Thr Lys Cys Thr Ala Ser Asn Lys Asn Arg Gly
Ile Ile 420 425 430 Lys Thr Phe Ser Asn Gly Cys Asp Tyr Val Ser Asn
Lys Gly Val Asp 435 440 445 Thr Val Ser Val Gly Asn Thr Leu Tyr Tyr
Val Asn Lys Gln Glu Gly 450 455 460 Lys Ser Leu Tyr Val Lys Gly Glu
Pro Ile Ile Asn Phe Tyr Asp Pro 465 470 475 480 Leu Val Phe Pro Ser
Asp Glu Phe Asp Ala Ser Ile Ser Gln Val Asn 485 490 495 Glu Lys Ile
Asn Gln Ser Leu Ala Phe Ile Arg Lys Ser Asp Glu Leu 500 505 510 Leu
His Asn Val Asn Ala Gly Lys Ser Thr Thr Asn 515 520
3170PRTArtificial SequenceSynthetic Polypeptide 3Ile Lys Ile Leu
Arg Asp Ala Gly Tyr His Val Lys Ala Asn Gly Val 1 5 10 15 Asp Val
Thr Thr His Arg Gln Asp Ile Asn Gly Lys Glu Met Lys Phe 20 25 30
Glu Val Leu Thr Leu Ala Ser Leu Thr Thr Glu Ile Gln Ile Asn Ile 35
40 45 Glu Ile Glu Ser Arg Lys Ser Tyr Lys Lys Met Leu Lys Glu Met
Gly 50 55 60 Glu Val Ala Pro Glu Tyr Arg His Asp Ser Pro Asp Cys
Gly Met Ile 65 70 75 80 Ile Leu Cys Ile Ala Ala Leu Val Ile Thr Lys
Leu Ala Ala Gly Asp 85 90 95 Arg Ser Gly Leu Thr Ala Val Ile Arg
Arg Ala Asn Asn Val Leu Lys 100 105 110 Asn Glu Met Lys Arg Tyr Lys
Gly Leu Leu Pro Lys Asp Ile Ala Asn 115 120 125 Ser Phe Tyr Glu Val
Phe Glu Lys Tyr Pro His Phe Ile Asp Val Phe 130 135 140 Val His Phe
Gly Ile Ala Gln Ser Ser Thr Arg Gly Gly Ser Arg Val 145 150 155 160
Glu Gly Ile Phe Ala Gly Leu Phe Met Asn 165 170 4391PRTArtificial
SequenceSynthetic Polypeptide 4Met Ala Leu Ser Lys Val Lys Leu Asn
Asp Thr Leu Asn Lys Asp Gln 1 5 10 15 Leu Leu Ser Ser Ser Lys Tyr
Thr Ile Gln Arg Ser Thr Gly Asp Ser 20 25 30 Ile Asp Thr Pro Asn
Tyr Asp Val Gln Lys His Ile Asn Lys Leu Cys 35 40 45 Gly Met Leu
Leu Ile Thr Glu Asp Ala Asn His Lys Phe Thr Gly Leu 50 55 60 Ile
Gly Met Leu Tyr Ala Met Ser Arg Leu Gly Arg Glu Asp Thr Ile 65 70
75 80 Lys Ile Leu Arg Asp Ala Gly Tyr His Val Lys Ala Asn Gly Val
Asp 85 90 95 Val Thr Thr His Arg Gln Asp Ile Asn Gly Lys Glu Met
Lys Phe Glu 100 105 110 Val Leu Thr Leu Ala Ser Leu Thr Thr Glu Ile
Gln Ile Asn Ile Glu 115 120 125 Ile Glu Ser Arg Lys Ser Tyr Lys Lys
Met Leu Lys Glu Met Gly Glu 130 135 140 Val Ala Pro Glu Tyr Arg His
Asp Ser Pro Asp Cys Gly Met Ile Ile 145 150 155 160 Leu Cys Ile Ala
Ala Leu Val Ile Thr Lys Leu Ala Ala Gly Asp Arg 165 170 175 Ser Gly
Leu Thr Ala Val Ile Arg Arg Ala Asn Asn Val Leu Lys Asn 180 185 190
Glu Met Lys Arg Tyr Lys Gly Leu Leu Pro Lys Asp Ile Ala Asn Ser 195
200 205 Phe Tyr Glu Val Phe Glu Lys Tyr Pro His Phe Ile Asp Val Phe
Val 210 215 220 His Phe Gly Ile Ala Gln Ser Ser Thr Arg Gly Gly Ser
Arg Val Glu 225 230 235 240 Gly Ile Phe Ala Gly Leu Phe Met Asn Ala
Tyr Gly Ala Gly Gln Val 245 250 255 Met Leu Arg Trp Gly Val Leu Ala
Lys Ser Val Lys Asn Ile Met Leu 260 265 270 Gly His Ala Ser Val Gln
Ala Glu Met Glu Gln Val Val Glu Val Tyr 275 280 285 Glu Tyr Ala Gln
Lys Leu Gly Gly Glu Ala Gly Phe Tyr His Ile Leu 290 295 300 Asn Asn
Pro Lys Ala Ser Leu Leu Ser Leu Thr Gln Phe Pro His Phe 305 310 315
320 Ser Ser Val Val Leu Gly Asn Ala Ala Gly Leu Gly Ile Met Gly Glu
325 330 335 Tyr Arg Gly Thr Pro Arg Asn Gln Asp Leu Tyr Asp Ala Ala
Lys Ala 340 345 350 Tyr Ala Glu Gln Leu Lys Glu Asn Gly Val Ile Asn
Tyr Ser Val Leu 355 360 365 Asp Leu Thr Ala Glu Glu Leu Glu Ala Ile
Lys His Gln Leu Asn Pro 370 375 380 Lys Asp Asn Asp Val Glu Leu 385
390 5194PRTArtificial SequenceSynthetic Polypeptide 5Met Ser Arg
Arg Asn Pro Cys Lys Phe Glu Ile Arg Gly His Cys Leu 1 5 10 15 Asn
Gly Lys Arg Cys His Phe Ser His Asn Tyr Phe Glu Trp Pro Pro 20 25
30 His Ala Leu Leu Val Arg Gln Asn Phe Met Leu Asn Arg Ile Leu Lys
35 40 45 Ser Met Asp Lys Ser Ile Asp Thr Leu Ser Glu Ile Ser Gly
Ala Ala 50 55 60 Glu Leu Asp Arg Thr Glu Glu Tyr Ala Leu Gly Val
Val Gly Val Leu 65 70 75 80 Glu Ser Tyr Ile Gly Ser Ile Asn Asn Ile
Thr Lys Gln Ser Ala Cys 85 90 95 Val Ala Met Ser Lys Leu Leu Thr
Glu Leu Asn Ser Asp Asp Ile Lys 100 105 110 Lys Leu Arg Asp Asn Glu
Glu Leu Asn Ser Pro Lys Ile Arg Val Tyr 115 120 125 Asn Thr Val Ile
Ser Tyr Ile Glu Ser Asn Arg Lys Asn Asn Lys Gln 130 135 140 Thr Ile
His Leu Leu Lys Arg Leu Pro Ala Asp Val Leu Lys Lys Thr 145 150 155
160 Ile Lys Asn Thr Leu Asp Ile His Lys Ser Ile Thr Ile Asn Asn Pro
165 170 175 Lys Glu Ser Thr Val Ser Asp Thr Asn Asp His Ala Lys Asn
Asn Asp 180 185 190 Thr Thr 67PRTArtificial SequenceSynthetic
Polypeptide 6Gly Gly Gly Ser Gly Gly Gly 1 5 71146PRTArtificial
SequenceSynthetic Polypeptide 7Met Glu Leu Leu Ile Leu Lys Ala Asn
Ala Ile Thr Thr Ile Leu Thr 1 5 10 15 Ala Val Thr Phe Cys Phe Ala
Ser Gly Gln Asn Ile Thr Glu Glu Phe 20 25 30 Tyr Gln Ser Thr Cys
Ser Ala Val Ser Lys Gly Tyr Leu Ser Ala Leu 35 40 45 Arg Thr Gly
Trp Tyr Thr Ser Val Ile Thr Ile Glu Leu Ser Asn Ile 50 55 60 Lys
Glu Asn Lys Cys Asn Gly Thr Asp Ala Lys Val Lys Leu Ile Lys 65 70
75 80 Gln Glu Leu Asp Lys Tyr Lys Asn Ala Val Thr Glu Leu Gln Leu
Leu 85 90 95 Met Gln Ser Thr Pro Ala Thr Asn Asn Arg Ala Arg Arg
Glu Leu Pro 100 105 110 Arg Phe Met Asn Tyr Thr Leu Asn Asn Ala Lys
Lys Thr Asn Val Thr 115 120 125 Leu Ser Lys Lys Arg Lys Arg Arg Phe
Leu Gly Phe Leu Leu Gly Val 130 135 140 Gly Ser Ala Ile Ala Ser Gly
Val Ala Val Ser Lys Val Leu His Leu 145 150 155 160 Glu Gly Glu Val
Asn Lys Ile Lys Ser Ala Leu Leu Ser Thr Asn Lys 165 170 175 Ala Val
Val Ser Leu Ser Asn Gly Val Ser Val Leu Thr Ser Lys Val 180 185 190
Leu Asp Leu Lys Asn Tyr Ile Asp Lys Gln Leu Leu Pro Ile Val Asn 195
200 205 Lys Gln Ser Cys Ser Ile Ser Asn Ile Glu Thr Val Ile Glu Phe
Gln 210 215 220 Gln Lys Asn Asn Arg Leu Leu Glu Ile Thr Arg Glu Phe
Ser Val Asn 225 230 235 240 Ala Gly Val Thr Thr Pro Val Ser Thr Tyr
Met Leu Thr Asn Ser Glu 245 250 255 Leu Leu Ser Leu Ile Asn Asp Met
Pro Ile Thr Asn Asp Gln Lys Lys 260 265 270 Leu Met Ser Asn Asn Val
Gln Ile Val Arg Gln Gln Ser Tyr Ser Ile 275 280 285 Met Ser Ile Ile
Lys Glu Glu Val Leu Ala Tyr Val Val Gln Leu Pro 290 295 300 Leu Tyr
Gly Val Ile Asp Thr Pro Cys Trp Lys Leu His Thr Ser Pro 305 310 315
320 Leu Cys Thr Thr Asn Thr Lys Glu Gly Ser Asn Ile Cys Leu Thr Arg
325 330 335 Thr Asp Arg Gly Trp Tyr Cys Asp Asn Ala Gly Ser Val Ser
Phe Phe 340 345 350 Pro Gln Ala Glu Thr Cys Lys Val Gln Ser Asn Arg
Val Phe Cys Asp 355 360 365 Thr Met Asn Ser Leu Thr Leu Pro Ser Glu
Val Asn Leu Cys Asn Val 370 375 380 Asp Ile Phe Asn Pro Lys Tyr Asp
Cys Lys Ile Met Thr Ser Lys Thr 385 390 395 400 Asp Val Ser Ser Ser
Val Ile Thr Ser Leu Gly Ala Ile Val Ser Cys 405 410 415 Tyr Gly Lys
Thr Lys Cys Thr Ala Ser Asn Lys Asn Arg Gly Ile Ile 420 425 430 Lys
Thr Phe Ser Asn Gly Cys Asp Tyr Val Ser Asn Lys Gly Val Asp 435 440
445 Thr Val Ser Val Gly Asn Thr Leu Tyr Tyr Val Asn Lys Gln Glu Gly
450 455 460 Lys Ser Leu Tyr Val Lys Gly Glu Pro Ile Ile Asn Phe Tyr
Asp Pro 465 470 475 480 Leu Val Phe Pro Ser Asp Glu Phe Asp Ala Ser
Ile Ser Gln Val Asn 485 490 495 Glu Lys Ile Asn Gln Ser Leu Ala Phe
Ile Arg Lys Ser Asp Glu Leu 500 505 510 Leu His Asn Val Asn Ala Gly
Lys Ser Thr Thr Asn Arg Lys Arg Arg 515 520 525 Ala Pro Val Lys Gln
Thr Leu Asn Phe Asp Leu Leu Lys Leu Ala Gly 530 535 540 Asp Val Glu
Ser Asn Pro Gly Pro Met Ala Leu Ser Lys Val Lys Leu 545 550 555 560
Asn Asp Thr Leu Asn Lys Asp Gln Leu Leu Ser Ser Ser Lys Tyr Thr 565
570 575 Ile Gln Arg Ser Thr Gly Asp Ser Ile Asp Thr Pro Asn Tyr Asp
Val 580 585 590 Gln Lys His Ile Asn Lys Leu Cys Gly Met Leu Leu Ile
Thr Glu Asp 595 600 605 Ala Asn His Lys Phe Thr Gly Leu Ile Gly Met
Leu
Tyr Ala Met Ser 610 615 620 Arg Leu Gly Arg Glu Asp Thr Ile Lys Ile
Leu Arg Asp Ala Gly Tyr 625 630 635 640 His Val Lys Ala Asn Gly Val
Asp Val Thr Thr His Arg Gln Asp Ile 645 650 655 Asn Gly Lys Glu Met
Lys Phe Glu Val Leu Thr Leu Ala Ser Leu Thr 660 665 670 Thr Glu Ile
Gln Ile Asn Ile Glu Ile Glu Ser Arg Lys Ser Tyr Lys 675 680 685 Lys
Met Leu Lys Glu Met Gly Glu Val Ala Pro Glu Tyr Arg His Asp 690 695
700 Ser Pro Asp Cys Gly Met Ile Ile Leu Cys Ile Ala Ala Leu Val Ile
705 710 715 720 Thr Lys Leu Ala Ala Gly Asp Arg Ser Gly Leu Thr Ala
Val Ile Arg 725 730 735 Arg Ala Asn Asn Val Leu Lys Asn Glu Met Lys
Arg Tyr Lys Gly Leu 740 745 750 Leu Pro Lys Asp Ile Ala Asn Ser Phe
Tyr Glu Val Phe Glu Lys Tyr 755 760 765 Pro His Phe Ile Asp Val Phe
Val His Phe Gly Ile Ala Gln Ser Ser 770 775 780 Thr Arg Gly Gly Ser
Arg Val Glu Gly Ile Phe Ala Gly Leu Phe Met 785 790 795 800 Asn Ala
Tyr Gly Ala Gly Gln Val Met Leu Arg Trp Gly Val Leu Ala 805 810 815
Lys Ser Val Lys Asn Ile Met Leu Gly His Ala Ser Val Gln Ala Glu 820
825 830 Met Glu Gln Val Val Glu Val Tyr Glu Tyr Ala Gln Lys Leu Gly
Gly 835 840 845 Glu Ala Gly Phe Tyr His Ile Leu Asn Asn Pro Lys Ala
Ser Leu Leu 850 855 860 Ser Leu Thr Gln Phe Pro His Phe Ser Ser Val
Val Leu Gly Asn Ala 865 870 875 880 Ala Gly Leu Gly Ile Met Gly Glu
Tyr Arg Gly Thr Pro Arg Asn Gln 885 890 895 Asp Leu Tyr Asp Ala Ala
Lys Ala Tyr Ala Glu Gln Leu Lys Glu Asn 900 905 910 Gly Val Ile Asn
Tyr Ser Val Leu Asp Leu Thr Ala Glu Glu Leu Glu 915 920 925 Ala Ile
Lys His Gln Leu Asn Pro Lys Asp Asn Asp Val Glu Leu Gly 930 935 940
Gly Gly Gly Ser Gly Gly Gly Gly Met Ser Arg Arg Asn Pro Cys Lys 945
950 955 960 Phe Glu Ile Arg Gly His Cys Leu Asn Gly Lys Arg Cys His
Phe Ser 965 970 975 His Asn Tyr Phe Glu Trp Pro Pro His Ala Leu Leu
Val Arg Gln Asn 980 985 990 Phe Met Leu Asn Arg Ile Leu Lys Ser Met
Asp Lys Ser Ile Asp Thr 995 1000 1005 Leu Ser Glu Ile Ser Gly Ala
Ala Glu Leu Asp Arg Thr Glu Glu 1010 1015 1020 Tyr Ala Leu Gly Val
Val Gly Val Leu Glu Ser Tyr Ile Gly Ser 1025 1030 1035 Ile Asn Asn
Ile Thr Lys Gln Ser Ala Cys Val Ala Met Ser Lys 1040 1045 1050 Leu
Leu Thr Glu Leu Asn Ser Asp Asp Ile Lys Lys Leu Arg Asp 1055 1060
1065 Asn Glu Glu Leu Asn Ser Pro Lys Ile Arg Val Tyr Asn Thr Val
1070 1075 1080 Ile Ser Tyr Ile Glu Ser Asn Arg Lys Asn Asn Lys Gln
Thr Ile 1085 1090 1095 His Leu Leu Lys Arg Leu Pro Ala Asp Val Leu
Lys Lys Thr Ile 1100 1105 1110 Lys Asn Thr Leu Asp Ile His Lys Ser
Ile Thr Ile Asn Asn Pro 1115 1120 1125 Lys Glu Ser Thr Val Ser Asp
Thr Asn Asp His Ala Lys Asn Asn 1130 1135 1140 Asp Thr Thr 1145
8566PRTArtificial SequenceArtificial Sequence 8Met Lys Ala Ile Leu
Val Val Leu Leu Tyr Thr Phe Ala Thr Ala Asn 1 5 10 15 Ala Asp Thr
Leu Cys Ile Gly Tyr His Ala Asn Asn Ser Thr Asp Thr 20 25 30 Val
Asp Thr Val Leu Glu Lys Asn Val Thr Val Thr His Ser Val Asn 35 40
45 Leu Leu Glu Asp Lys His Asn Gly Lys Leu Cys Lys Leu Arg Gly Val
50 55 60 Ala Pro Leu His Leu Gly Lys Cys Asn Ile Ala Gly Trp Ile
Leu Gly 65 70 75 80 Asn Pro Glu Cys Glu Ser Leu Ser Thr Ala Ser Ser
Trp Ser Tyr Ile 85 90 95 Val Glu Thr Ser Ser Ser Asp Asn Gly Thr
Cys Tyr Pro Gly Asp Phe 100 105 110 Ile Asp Tyr Glu Glu Leu Arg Glu
Gln Leu Ser Ser Val Ser Ser Phe 115 120 125 Glu Arg Phe Glu Ile Phe
Pro Lys Thr Ser Ser Trp Pro Asn His Asp 130 135 140 Ser Asn Lys Gly
Val Thr Ala Ala Cys Pro His Ala Gly Ala Lys Ser 145 150 155 160 Phe
Tyr Lys Asn Leu Ile Trp Leu Val Lys Lys Gly Asn Ser Tyr Pro 165 170
175 Lys Leu Ser Lys Ser Tyr Ile Asn Asp Lys Gly Lys Glu Val Leu Val
180 185 190 Leu Trp Gly Ile His His Pro Ser Thr Ser Ala Asp Gln Gln
Ser Leu 195 200 205 Tyr Gln Asn Ala Asp Ala Tyr Val Phe Val Gly Ser
Ser Arg Tyr Ser 210 215 220 Lys Lys Phe Lys Pro Glu Ile Ala Ile Arg
Pro Lys Val Arg Asp Gln 225 230 235 240 Glu Gly Arg Met Asn Tyr Tyr
Trp Thr Leu Val Glu Pro Gly Asp Lys 245 250 255 Ile Thr Phe Glu Ala
Thr Gly Asn Leu Val Val Pro Arg Tyr Ala Phe 260 265 270 Ala Met Glu
Arg Asn Ala Gly Ser Gly Ile Ile Ile Ser Asp Thr Pro 275 280 285 Val
His Asp Cys Asn Thr Thr Cys Gln Thr Pro Lys Gly Ala Ile Asn 290 295
300 Thr Ser Leu Pro Phe Gln Asn Ile His Pro Ile Thr Ile Gly Lys Cys
305 310 315 320 Pro Lys Tyr Val Lys Ser Thr Lys Leu Arg Leu Ala Thr
Gly Leu Arg 325 330 335 Asn Val Pro Ser Ile Gln Ser Arg Gly Leu Phe
Gly Ala Ile Ala Gly 340 345 350 Phe Ile Glu Gly Gly Trp Thr Gly Met
Val Asp Gly Trp Tyr Gly Tyr 355 360 365 His His Gln Asn Glu Gln Gly
Ser Gly Tyr Ala Ala Asp Leu Lys Ser 370 375 380 Thr Gln Asn Ala Ile
Asp Glu Ile Thr Asn Lys Val Asn Ser Val Ile 385 390 395 400 Glu Lys
Met Asn Thr Gln Phe Thr Ala Val Gly Lys Glu Phe Asn His 405 410 415
Leu Glu Lys Arg Ile Glu Asn Leu Asn Lys Lys Val Asp Asp Gly Phe 420
425 430 Leu Asp Ile Trp Thr Tyr Asn Ala Glu Leu Leu Val Leu Leu Glu
Asn 435 440 445 Glu Arg Thr Leu Asp Tyr His Asp Ser Asn Val Lys Asn
Leu Tyr Glu 450 455 460 Lys Val Arg Ser Gln Leu Lys Asn Asn Ala Lys
Glu Ile Gly Asn Gly 465 470 475 480 Cys Phe Glu Phe Tyr His Lys Cys
Asp Asn Thr Cys Met Glu Ser Val 485 490 495 Lys Asn Gly Thr Tyr Asp
Tyr Pro Lys Tyr Ser Glu Glu Ala Lys Leu 500 505 510 Asn Arg Glu Glu
Ile Asp Gly Val Lys Leu Glu Ser Thr Arg Ile Tyr 515 520 525 Gln Ile
Leu Ala Ile Tyr Ser Thr Val Ala Ser Ser Leu Val Leu Val 530 535 540
Val Ser Leu Gly Ala Ile Ser Phe Trp Met Cys Ser Asn Gly Ser Leu 545
550 555 560 Gln Cys Arg Ile Cys Ile 565 9570PRTArtificial
SequenceSynthetic Polypeptide 9Met Lys Ala Ile Leu Val Val Leu Leu
Tyr Thr Phe Ala Thr Ala Asn 1 5 10 15 Ala Asp Thr Leu Cys Ile Gly
Tyr His Ala Asn Asn Ser Thr Asp Thr 20 25 30 Val Asp Thr Val Leu
Glu Lys Asn Val Thr Val Thr His Ser Val Asn 35 40 45 Leu Leu Glu
Asp Lys His Asn Gly Lys Leu Cys Lys Leu Arg Gly Val 50 55 60 Ala
Pro Leu His Leu Gly Lys Cys Asn Ile Ala Gly Trp Ile Leu Gly 65 70
75 80 Asn Pro Glu Cys Glu Ser Leu Ser Thr Ala Ser Ser Trp Ser Tyr
Ile 85 90 95 Val Glu Thr Ser Ser Ser Asp Asn Gly Thr Cys Tyr Pro
Gly Asp Phe 100 105 110 Ile Asp Tyr Glu Glu Leu Arg Glu Gln Leu Ser
Ser Val Ser Ser Phe 115 120 125 Glu Arg Phe Glu Ile Phe Pro Lys Thr
Ser Ser Trp Pro Asn His Asp 130 135 140 Ser Asn Lys Gly Val Thr Ala
Ala Cys Pro His Ala Gly Ala Lys Ser 145 150 155 160 Phe Tyr Lys Asn
Leu Ile Trp Leu Val Lys Lys Gly Asn Ser Tyr Pro 165 170 175 Lys Leu
Ser Lys Ser Tyr Ile Asn Asp Lys Gly Lys Glu Val Leu Val 180 185 190
Leu Trp Gly Ile His His Pro Ser Thr Ser Ala Asp Gln Gln Ser Leu 195
200 205 Tyr Gln Asn Ala Asp Ala Tyr Val Phe Val Gly Ser Ser Arg Tyr
Ser 210 215 220 Lys Lys Phe Lys Pro Glu Ile Ala Ile Arg Pro Lys Val
Arg Asp Gln 225 230 235 240 Glu Gly Arg Met Asn Tyr Tyr Trp Thr Leu
Val Glu Pro Gly Asp Lys 245 250 255 Ile Thr Phe Glu Ala Thr Gly Asn
Leu Val Val Pro Arg Tyr Ala Phe 260 265 270 Ala Met Glu Arg Asn Ala
Gly Ser Gly Ile Ile Ile Ser Asp Thr Pro 275 280 285 Val His Asp Cys
Asn Thr Thr Cys Gln Thr Pro Lys Gly Ala Ile Asn 290 295 300 Thr Ser
Leu Pro Phe Gln Asn Ile His Pro Ile Thr Ile Gly Lys Cys 305 310 315
320 Pro Lys Tyr Val Lys Ser Thr Lys Leu Arg Leu Ala Thr Gly Leu Arg
325 330 335 Asn Ile Pro Gln Arg Glu Arg Arg Arg Lys Lys Arg Gly Leu
Phe Gly 340 345 350 Ala Ile Ala Gly Phe Ile Glu Gly Gly Trp Thr Gly
Met Val Asp Gly 355 360 365 Trp Tyr Gly Tyr His His Gln Asn Glu Gln
Gly Ser Gly Tyr Ala Ala 370 375 380 Asp Leu Lys Ser Thr Gln Asn Ala
Ile Asp Glu Ile Thr Asn Lys Val 385 390 395 400 Asn Ser Val Ile Glu
Lys Met Asn Thr Gln Phe Thr Ala Val Gly Lys 405 410 415 Glu Phe Asn
His Leu Glu Lys Arg Ile Glu Asn Leu Asn Lys Lys Val 420 425 430 Asp
Asp Gly Phe Leu Asp Ile Trp Thr Tyr Asn Ala Glu Leu Leu Val 435 440
445 Leu Leu Glu Asn Glu Arg Thr Leu Asp Tyr His Asp Ser Asn Val Lys
450 455 460 Asn Leu Tyr Glu Lys Val Arg Ser Gln Leu Lys Asn Asn Ala
Lys Glu 465 470 475 480 Ile Gly Asn Gly Cys Phe Glu Phe Tyr His Lys
Cys Asp Asn Thr Cys 485 490 495 Met Glu Ser Val Lys Asn Gly Thr Tyr
Asp Tyr Pro Lys Tyr Ser Glu 500 505 510 Glu Ala Lys Leu Asn Arg Glu
Glu Ile Asp Gly Val Lys Leu Glu Ser 515 520 525 Thr Arg Ile Tyr Gln
Ile Leu Ala Ile Tyr Ser Thr Val Ala Ser Ser 530 535 540 Leu Val Leu
Val Val Ser Leu Gly Ala Ile Ser Phe Trp Met Cys Ser 545 550 555 560
Asn Gly Ser Leu Gln Cys Arg Ile Cys Ile 565 570 10568PRTArtificial
SequenceSynthetic Polypeptide 10Met Glu Lys Ile Val Leu Leu Leu Ala
Ile Val Ser Leu Val Lys Ser 1 5 10 15 Asp Gln Ile Cys Ile Gly Tyr
His Ala Asn Asn Ser Thr Glu Gln Val 20 25 30 Asp Thr Ile Met Glu
Lys Asn Val Thr Val Thr His Ala Gln Asp Ile 35 40 45 Leu Glu Lys
Thr His Asn Gly Lys Leu Cys Asp Leu Asp Gly Val Lys 50 55 60 Pro
Leu Ile Leu Arg Asp Cys Ser Val Ala Gly Trp Leu Leu Gly Asn 65 70
75 80 Pro Met Cys Asp Glu Phe Ile Asn Val Pro Glu Trp Ser Tyr Ile
Val 85 90 95 Glu Lys Ala Asn Pro Ala Asn Asp Leu Cys Tyr Pro Gly
Asn Phe Asn 100 105 110 Asp Tyr Glu Glu Leu Lys His Leu Leu Ser Arg
Ile Asn His Phe Glu 115 120 125 Lys Ile Gln Ile Ile Pro Lys Ser Ser
Trp Ser Asp His Glu Ala Ser 130 135 140 Ser Gly Val Ser Ser Ala Cys
Pro Tyr Gln Gly Arg Ser Ser Phe Phe 145 150 155 160 Arg Asn Val Val
Trp Leu Ile Lys Lys Asn Ser Thr Tyr Pro Thr Ile 165 170 175 Lys Arg
Ser Tyr Asn Asn Thr Asn Gln Glu Asp Leu Leu Val Leu Trp 180 185 190
Gly Ile His His Pro Asn Asp Ala Ala Glu Gln Thr Arg Leu Tyr Gln 195
200 205 Asn Pro Thr Thr Tyr Ile Ser Val Gly Thr Ser Thr Leu Asn Gln
Arg 210 215 220 Leu Val Pro Lys Ile Ala Thr Arg Ser Lys Val Asn Gly
Gln Ser Gly 225 230 235 240 Arg Met Glu Phe Phe Trp Thr Ile Leu Lys
Pro Asn Asp Ala Ile Asn 245 250 255 Phe Glu Ser Asn Gly Asn Phe Ile
Ala Pro Glu Tyr Ala Tyr Lys Ile 260 265 270 Val Lys Lys Gly Asp Ser
Thr Ile Met Lys Ser Glu Leu Glu Tyr Gly 275 280 285 Asn Cys Asn Thr
Lys Cys Gln Thr Pro Met Gly Ala Ile Asn Ser Ser 290 295 300 Met Pro
Phe His Asn Ile His Pro Leu Thr Ile Gly Glu Cys Pro Lys 305 310 315
320 Tyr Val Lys Ser Asn Arg Leu Val Leu Ala Thr Gly Leu Arg Asn Ser
325 330 335 Pro Gln Arg Glu Arg Arg Arg Lys Lys Arg Gly Leu Phe Gly
Ala Ile 340 345 350 Ala Gly Phe Ile Glu Gly Gly Trp Gln Gly Met Val
Asp Gly Trp Tyr 355 360 365 Gly Tyr His His Ser Asn Glu Gln Gly Ser
Gly Tyr Ala Ala Asp Lys 370 375 380 Glu Ser Thr Gln Lys Ala Ile Asp
Gly Val Thr Asn Lys Val Asn Ser 385 390 395 400 Ile Ile Asp Lys Met
Asn Thr Gln Phe Glu Ala Val Gly Arg Glu Phe 405 410 415 Asn Asn Leu
Glu Arg Arg Ile Glu Asn Leu Asn Lys Lys Met Glu Asp 420 425 430 Gly
Phe Leu Asp Val Trp Thr Tyr Asn Ala Glu Leu Leu Val Leu Met 435 440
445 Glu Asn Glu Arg Thr Leu Asp Phe His Asp Ser Asn Val Lys Asn Leu
450 455 460 Tyr Asp Lys Val Arg Leu Gln Leu Arg Asp Asn Ala Lys Glu
Leu Gly 465 470 475 480 Asn Gly Cys Phe Glu Phe Tyr His Lys Cys Asp
Asn Glu Cys Met Glu 485 490 495 Ser Val Arg Asn Gly Thr Tyr Asp Tyr
Pro Gln Tyr Ser Glu Glu Ala 500 505 510 Arg Leu Lys Arg Glu Glu Ile
Ser Gly Val Lys Leu Glu Ser Ile Gly 515 520 525 Thr Tyr Gln Ile Leu
Ser Ile Tyr Ser Thr Val Ala Ser Ser Leu Ala 530 535 540 Leu Ala Ile
Met Val Ala Gly Leu Ser Leu Trp Met Cys Ser Asn Gly 545 550 555 560
Ser Leu Gln Cys Arg Ile Cys Ile 565 11498PRTArtificial
SequenceSynthetic Polypeptide 11Met Ala Ser Gln Gly Ala Ala Ala Ser
Tyr Glu Gln Met Glu Thr Asp 1 5 10 15 Gly Asp Arg Gln Asn Ala Thr
Glu Ile Arg Ala Ser Val Gly Lys Met 20 25 30 Ile Asp Gly Ile
Gly
Arg Phe Tyr Ile Gln Met Cys Thr Glu Leu Lys 35 40 45 Leu Ser Asp
Tyr Glu Gly Arg Leu Ile Gln Asn Ser Leu Thr Ile Glu 50 55 60 Lys
Met Val Leu Ser Ala Phe Asp Glu Arg Arg Asn Arg Tyr Leu Glu 65 70
75 80 Glu His Pro Ser Ala Gly Lys Asp Pro Lys Lys Thr Gly Gly Pro
Ile 85 90 95 Tyr Arg Arg Val Asp Gly Lys Trp Met Arg Glu Leu Val
Leu Tyr Asp 100 105 110 Lys Glu Glu Ile Arg Arg Ile Trp Arg Gln Ala
Asn Asn Gly Glu Asp 115 120 125 Ala Thr Ala Gly Leu Thr His Met Met
Ile Trp His Ser Asn Leu Asn 130 135 140 Asp Thr Thr Tyr Gln Arg Thr
Arg Ala Leu Val Arg Thr Gly Met Asp 145 150 155 160 Pro Arg Met Cys
Ser Leu Met Gln Gly Ser Thr Leu Pro Arg Arg Ser 165 170 175 Gly Ala
Ala Gly Ala Ala Val Lys Gly Ile Gly Thr Met Val Met Glu 180 185 190
Leu Ile Arg Met Ile Lys Arg Gly Ile Asn Asp Arg Asn Phe Trp Arg 195
200 205 Gly Glu Asn Gly Arg Lys Thr Arg Ser Ala Tyr Glu Arg Met Cys
Asn 210 215 220 Ile Leu Lys Gly Lys Phe Gln Thr Ala Ala Gln Arg Ala
Met Met Asp 225 230 235 240 Gln Val Arg Glu Ser Arg Asn Pro Gly Asn
Ala Glu Ile Glu Asp Leu 245 250 255 Ile Phe Leu Ala Arg Ser Ala Leu
Ile Leu Arg Gly Ser Val Ala His 260 265 270 Lys Ser Cys Leu Pro Ala
Cys Val Tyr Gly Pro Ala Val Ala Ser Gly 275 280 285 Tyr Asp Phe Glu
Lys Glu Gly Tyr Ser Leu Val Gly Ile Asp Pro Phe 290 295 300 Lys Leu
Leu Gln Asn Ser Gln Val Tyr Ser Leu Ile Arg Pro Asn Glu 305 310 315
320 Asn Pro Ala His Lys Ser Gln Leu Val Trp Met Ala Cys His Ser Ala
325 330 335 Ala Phe Glu Asp Leu Arg Leu Ser Ser Phe Ile Arg Gly Thr
Lys Val 340 345 350 Ser Pro Arg Gly Lys Leu Ser Thr Arg Gly Val Gln
Ile Ala Ser Asn 355 360 365 Glu Asn Met Asp Asn Met Gly Ser Ser Thr
Leu Glu Leu Arg Ser Arg 370 375 380 Tyr Trp Ala Ile Arg Thr Arg Ser
Gly Gly Asn Thr Asn Gln Gln Arg 385 390 395 400 Ala Ser Ala Gly Gln
Ile Ser Val Gln Pro Thr Phe Ser Val Gln Arg 405 410 415 Asn Leu Pro
Phe Glu Lys Ser Thr Ile Met Ala Ala Phe Thr Gly Asn 420 425 430 Thr
Glu Gly Arg Thr Ser Asp Met Arg Ala Glu Ile Ile Arg Met Met 435 440
445 Glu Ser Ala Lys Pro Glu Glu Val Ser Phe Arg Gly Arg Gly Val Phe
450 455 460 Glu Leu Ser Asp Glu Lys Ala Thr Asn Pro Ile Val Pro Ser
Phe Asp 465 470 475 480 Met Ser Asn Glu Gly Ser Tyr Phe Phe Gly Asp
Asn Ala Glu Glu Tyr 485 490 495 Asp Asn 12252PRTArtificial
SequenceSynthetic Polypeptide 12Met Ser Leu Leu Thr Glu Val Glu Thr
Tyr Val Leu Ser Ile Val Pro 1 5 10 15 Ser Gly Pro Leu Lys Ala Glu
Ile Ala Gln Arg Leu Glu Asp Val Phe 20 25 30 Ala Gly Lys Asn Thr
Asp Leu Glu Ala Leu Met Glu Trp Leu Lys Thr 35 40 45 Arg Pro Ile
Leu Ser Pro Leu Thr Lys Gly Ile Leu Gly Phe Val Phe 50 55 60 Thr
Leu Thr Val Pro Ser Glu Arg Gly Leu Gln Arg Arg Arg Phe Val 65 70
75 80 Gln Asn Ala Leu Asn Gly Asn Gly Asp Pro Asn Asn Met Asp Lys
Ala 85 90 95 Val Lys Leu Tyr Arg Lys Leu Lys Arg Glu Ile Thr Phe
His Gly Ala 100 105 110 Lys Glu Ile Ala Leu Ser Tyr Ser Ala Gly Ala
Leu Ala Ser Cys Met 115 120 125 Gly Leu Ile Tyr Asn Arg Met Gly Ala
Val Thr Thr Glu Val Ala Phe 130 135 140 Gly Leu Val Cys Ala Thr Cys
Glu Gln Ile Ala Asp Ser Gln His Arg 145 150 155 160 Ser His Arg Gln
Met Val Thr Thr Thr Asn Pro Leu Ile Arg His Glu 165 170 175 Asn Arg
Met Val Leu Ala Ser Thr Thr Ala Lys Ala Met Glu Gln Met 180 185 190
Ala Gly Ser Ser Glu Gln Ala Ala Glu Ala Met Glu Ile Ala Ser Gln 195
200 205 Ala Arg Gln Met Val Gln Ala Met Arg Thr Ile Gly Thr His Pro
Ser 210 215 220 Ser Ser Thr Gly Leu Arg Asp Asp Leu Leu Glu Asn Leu
Gln Thr Tyr 225 230 235 240 Gln Lys Arg Met Gly Val Gln Met Gln Arg
Phe Lys 245 250 131357PRTArtificial SequenceSynthetic Polypeptide
13Met Ala Ser Gln Gly Ala Ala Ala Ser Tyr Glu Gln Met Glu Thr Asp 1
5 10 15 Gly Asp Arg Gln Asn Ala Thr Glu Ile Arg Ala Ser Val Gly Lys
Met 20 25 30 Ile Asp Gly Ile Gly Arg Phe Tyr Ile Gln Met Cys Thr
Glu Leu Lys 35 40 45 Leu Ser Asp Tyr Glu Gly Arg Leu Ile Gln Asn
Ser Leu Thr Ile Glu 50 55 60 Lys Met Val Leu Ser Ala Phe Asp Glu
Arg Arg Asn Arg Tyr Leu Glu 65 70 75 80 Glu His Pro Ser Ala Gly Lys
Asp Pro Lys Lys Thr Gly Gly Pro Ile 85 90 95 Tyr Arg Arg Val Asp
Gly Lys Trp Met Arg Glu Leu Val Leu Tyr Asp 100 105 110 Lys Glu Glu
Ile Arg Arg Ile Trp Arg Gln Ala Asn Asn Gly Glu Asp 115 120 125 Ala
Thr Ala Gly Leu Thr His Met Met Ile Trp His Ser Asn Leu Asn 130 135
140 Asp Thr Thr Tyr Gln Arg Thr Arg Ala Leu Val Arg Thr Gly Met Asp
145 150 155 160 Pro Arg Met Cys Ser Leu Met Gln Gly Ser Thr Leu Pro
Arg Arg Ser 165 170 175 Gly Ala Ala Gly Ala Ala Val Lys Gly Ile Gly
Thr Met Val Met Glu 180 185 190 Leu Ile Arg Met Ile Lys Arg Gly Ile
Asn Asp Arg Asn Phe Trp Arg 195 200 205 Gly Glu Asn Gly Arg Lys Thr
Arg Ser Ala Tyr Glu Arg Met Cys Asn 210 215 220 Ile Leu Lys Gly Lys
Phe Gln Thr Ala Ala Gln Arg Ala Met Met Asp 225 230 235 240 Gln Val
Arg Glu Ser Arg Asn Pro Gly Asn Ala Glu Ile Glu Asp Leu 245 250 255
Ile Phe Leu Ala Arg Ser Ala Leu Ile Leu Arg Gly Ser Val Ala His 260
265 270 Lys Ser Cys Leu Pro Ala Cys Val Tyr Gly Pro Ala Val Ala Ser
Gly 275 280 285 Tyr Asp Phe Glu Lys Glu Gly Tyr Ser Leu Val Gly Ile
Asp Pro Phe 290 295 300 Lys Leu Leu Gln Asn Ser Gln Val Tyr Ser Leu
Ile Arg Pro Asn Glu 305 310 315 320 Asn Pro Ala His Lys Ser Gln Leu
Val Trp Met Ala Cys His Ser Ala 325 330 335 Ala Phe Glu Asp Leu Arg
Leu Ser Ser Phe Ile Arg Gly Thr Lys Val 340 345 350 Ser Pro Arg Gly
Lys Leu Ser Thr Arg Gly Val Gln Ile Ala Ser Asn 355 360 365 Glu Asn
Met Asp Asn Met Gly Ser Ser Thr Leu Glu Leu Arg Ser Arg 370 375 380
Tyr Trp Ala Ile Arg Thr Arg Ser Gly Gly Asn Thr Asn Gln Gln Arg 385
390 395 400 Ala Ser Ala Gly Gln Ile Ser Val Gln Pro Thr Phe Ser Val
Gln Arg 405 410 415 Asn Leu Pro Phe Glu Lys Ser Thr Ile Met Ala Ala
Phe Thr Gly Asn 420 425 430 Thr Glu Gly Arg Thr Ser Asp Met Arg Ala
Glu Ile Ile Arg Met Met 435 440 445 Glu Ser Ala Lys Pro Glu Glu Val
Ser Phe Arg Gly Arg Gly Val Phe 450 455 460 Glu Leu Ser Asp Glu Lys
Ala Thr Asn Pro Ile Val Pro Ser Phe Asp 465 470 475 480 Met Ser Asn
Glu Gly Ser Tyr Phe Phe Gly Asp Asn Ala Glu Glu Tyr 485 490 495 Asp
Asn Gly Gly Gly Gly Ser Gly Gly Gly Gly Met Ser Leu Leu Thr 500 505
510 Glu Val Glu Thr Tyr Val Leu Ser Ile Val Pro Ser Gly Pro Leu Lys
515 520 525 Ala Glu Ile Ala Gln Arg Leu Glu Asp Val Phe Ala Gly Lys
Asn Thr 530 535 540 Asp Leu Glu Ala Leu Met Glu Trp Leu Lys Thr Arg
Pro Ile Leu Ser 545 550 555 560 Pro Leu Thr Lys Gly Ile Leu Gly Phe
Val Phe Thr Leu Thr Val Pro 565 570 575 Ser Glu Arg Gly Leu Gln Arg
Arg Arg Phe Val Gln Asn Ala Leu Asn 580 585 590 Gly Asn Gly Asp Pro
Asn Asn Met Asp Lys Ala Val Lys Leu Tyr Arg 595 600 605 Lys Leu Lys
Arg Glu Ile Thr Phe His Gly Ala Lys Glu Ile Ala Leu 610 615 620 Ser
Tyr Ser Ala Gly Ala Leu Ala Ser Cys Met Gly Leu Ile Tyr Asn 625 630
635 640 Arg Met Gly Ala Val Thr Thr Glu Val Ala Phe Gly Leu Val Cys
Ala 645 650 655 Thr Cys Glu Gln Ile Ala Asp Ser Gln His Arg Ser His
Arg Gln Met 660 665 670 Val Thr Thr Thr Asn Pro Leu Ile Arg His Glu
Asn Arg Met Val Leu 675 680 685 Ala Ser Thr Thr Ala Lys Ala Met Glu
Gln Met Ala Gly Ser Ser Glu 690 695 700 Gln Ala Ala Glu Ala Met Glu
Ile Ala Ser Gln Ala Arg Gln Met Val 705 710 715 720 Gln Ala Met Arg
Thr Ile Gly Thr His Pro Ser Ser Ser Thr Gly Leu 725 730 735 Arg Asp
Asp Leu Leu Glu Asn Leu Gln Thr Tyr Gln Lys Arg Met Gly 740 745 750
Val Gln Met Gln Arg Phe Lys Arg Lys Arg Arg Ala Pro Val Lys Gln 755
760 765 Thr Leu Asn Phe Asp Leu Leu Lys Leu Ala Gly Asp Val Glu Ser
Asn 770 775 780 Pro Gly Pro Met Lys Ala Ile Leu Val Val Leu Leu Tyr
Thr Phe Ala 785 790 795 800 Thr Ala Asn Ala Asp Thr Leu Cys Ile Gly
Tyr His Ala Asn Asn Ser 805 810 815 Thr Asp Thr Val Asp Thr Val Leu
Glu Lys Asn Val Thr Val Thr His 820 825 830 Ser Val Asn Leu Leu Glu
Asp Lys His Asn Gly Lys Leu Cys Lys Leu 835 840 845 Arg Gly Val Ala
Pro Leu His Leu Gly Lys Cys Asn Ile Ala Gly Trp 850 855 860 Ile Leu
Gly Asn Pro Glu Cys Glu Ser Leu Ser Thr Ala Ser Ser Trp 865 870 875
880 Ser Tyr Ile Val Glu Thr Ser Ser Ser Asp Asn Gly Thr Cys Tyr Pro
885 890 895 Gly Asp Phe Ile Asp Tyr Glu Glu Leu Arg Glu Gln Leu Ser
Ser Val 900 905 910 Ser Ser Phe Glu Arg Phe Glu Ile Phe Pro Lys Thr
Ser Ser Trp Pro 915 920 925 Asn His Asp Ser Asn Lys Gly Val Thr Ala
Ala Cys Pro His Ala Gly 930 935 940 Ala Lys Ser Phe Tyr Lys Asn Leu
Ile Trp Leu Val Lys Lys Gly Asn 945 950 955 960 Ser Tyr Pro Lys Leu
Ser Lys Ser Tyr Ile Asn Asp Lys Gly Lys Glu 965 970 975 Val Leu Val
Leu Trp Gly Ile His His Pro Ser Thr Ser Ala Asp Gln 980 985 990 Gln
Ser Leu Tyr Gln Asn Ala Asp Ala Tyr Val Phe Val Gly Ser Ser 995
1000 1005 Arg Tyr Ser Lys Lys Phe Lys Pro Glu Ile Ala Ile Arg Pro
Lys 1010 1015 1020 Val Arg Asp Gln Glu Gly Arg Met Asn Tyr Tyr Trp
Thr Leu Val 1025 1030 1035 Glu Pro Gly Asp Lys Ile Thr Phe Glu Ala
Thr Gly Asn Leu Val 1040 1045 1050 Val Pro Arg Tyr Ala Phe Ala Met
Glu Arg Asn Ala Gly Ser Gly 1055 1060 1065 Ile Ile Ile Ser Asp Thr
Pro Val His Asp Cys Asn Thr Thr Cys 1070 1075 1080 Gln Thr Pro Lys
Gly Ala Ile Asn Thr Ser Leu Pro Phe Gln Asn 1085 1090 1095 Ile His
Pro Ile Thr Ile Gly Lys Cys Pro Lys Tyr Val Lys Ser 1100 1105 1110
Thr Lys Leu Arg Leu Ala Thr Gly Leu Arg Asn Ile Pro Gln Arg 1115
1120 1125 Glu Arg Arg Arg Lys Lys Arg Gly Leu Phe Gly Ala Ile Ala
Gly 1130 1135 1140 Phe Ile Glu Gly Gly Trp Thr Gly Met Val Asp Gly
Trp Tyr Gly 1145 1150 1155 Tyr His His Gln Asn Glu Gln Gly Ser Gly
Tyr Ala Ala Asp Leu 1160 1165 1170 Lys Ser Thr Gln Asn Ala Ile Asp
Glu Ile Thr Asn Lys Val Asn 1175 1180 1185 Ser Val Ile Glu Lys Met
Asn Thr Gln Phe Thr Ala Val Gly Lys 1190 1195 1200 Glu Phe Asn His
Leu Glu Lys Arg Ile Glu Asn Leu Asn Lys Lys 1205 1210 1215 Val Asp
Asp Gly Phe Leu Asp Ile Trp Thr Tyr Asn Ala Glu Leu 1220 1225 1230
Leu Val Leu Leu Glu Asn Glu Arg Thr Leu Asp Tyr His Asp Ser 1235
1240 1245 Asn Val Lys Asn Leu Tyr Glu Lys Val Arg Ser Gln Leu Lys
Asn 1250 1255 1260 Asn Ala Lys Glu Ile Gly Asn Gly Cys Phe Glu Phe
Tyr His Lys 1265 1270 1275 Cys Asp Asn Thr Cys Met Glu Ser Val Lys
Asn Gly Thr Tyr Asp 1280 1285 1290 Tyr Pro Lys Tyr Ser Glu Glu Ala
Lys Leu Asn Arg Glu Glu Ile 1295 1300 1305 Asp Gly Val Lys Leu Glu
Ser Thr Arg Ile Tyr Gln Ile Leu Ala 1310 1315 1320 Ile Tyr Ser Thr
Val Ala Ser Ser Leu Val Leu Val Val Ser Leu 1325 1330 1335 Gly Ala
Ile Ser Phe Trp Met Cys Ser Asn Gly Ser Leu Gln Cys 1340 1345 1350
Arg Ile Cys Ile 1355 144079DNAArtificial SequenceSynthetic
Oligonucleotide 14atggcctctc agggcgcagc cgccagctac gagcagatgg
aaaccgacgg cgacagacag 60aacgccaccg agatcagagc cagcgtgggc aagatgatcg
acggcatcgg ccggttctac 120atccagatgt gcaccgagct gaagctgtcc
gactacgagg gccggctgat ccagaacagc 180ctgaccatcg agaagatggt
gctgtccgcc ttcgacgagc ggcggaatag atacctggaa 240gaacacccca
gcgccggcaa ggaccctaag aaaaccggcg gacccatcta cagacgggtg
300gacggcaagt ggatgcggga gctggtgctg tacgacaaag aggaaatcag
aagaatctgg 360cggcaggcca acaatggcga ggatgccaca gccggcctga
cccacatgat gatctggcac 420agcaacctga acgacaccac ctaccagcgg
acaagagccc tcgtgcggac cggcatggac 480ccccggatgt gcagcctgat
gcagggcagc acactgccta gaagatctgg cgccgctggc 540gctgccgtga
agggcatcgg caccatggtc atggaactga tccggatgat caagcggggc
600atcaacgacc ggaacttttg gagaggcgag aacggcagaa agaccagatc
cgcctacgag 660cggatgtgca acatcctgaa gggcaagttc cagacagccg
cccagagggc aatgatggac 720caggtccggg agagcagaaa ccccggcaac
gccgagatcg aggacctgat cttcctggcc 780agaagcgccc tgatcctgag
aggcagcgtg gcccacaaga gctgcctgcc cgcctgtgtg 840tatggaccag
ccgtggctag cggctacgac ttcgagaaag agggctacag cctcgtcggc
900atcgacccct tcaagctgct gcagaacagc caggtgtaca gcctgatccg
gcccaacgag 960aaccctgccc acaagtccca gctcgtgtgg atggcctgcc
acagcgccgc cttcgaggat 1020ctgcggctgt ccagcttcat ccggggcacc
aaggtgtccc ccagaggcaa gctgtctacc 1080agaggcgtgc agatcgccag
caacgagaac atggacaaca tgggcagcag caccctggaa 1140ctgcggagcc
ggtactgggc catcagaacc agaagcggcg gcaacaccaa ccagcagaga
1200gccagcgccg gacagatcag cgtgcagccc accttctccg tgcagcggaa
tctgcccttc 1260gagaagtcca
ccatcatggc cgccttcacc ggcaataccg agggccggac cagcgacatg
1320cgggccgaga tcatcagaat gatggaaagc gccaagcccg aggaagtgtc
cttccggggc 1380agaggcgtgt tcgagctgtc cgatgagaag gccaccaacc
ccatcgtgcc cagcttcgac 1440atgagcaacg agggcagcta cttcttcggc
gacaacgccg aggaatacga caatggcggc 1500ggaggatctg ggggcggagg
catgagtctg ctgactgaag tcgagacata cgtgctgtcc 1560atcgtgccta
gcggccctct gaaggccgag atcgcccagc ggctggaaga tgtgttcgcc
1620ggcaagaaca ccgacctgga agccctgatg gaatggctga aaacccggcc
catcctgagc 1680cctctgacca agggcatcct gggcttcgtg ttcaccctga
ccgtgccctc tgagcggggc 1740ctgcagcgga gaagattcgt gcagaacgcc
ctgaacggca acggcgaccc caacaacatg 1800gataaggccg tgaagctgta
ccggaagctg aagcgggaga tcaccttcca cggcgccaaa 1860gagatcgccc
tgtcctactc tgccggcgct ctggccagct gcatgggcct catctacaac
1920cggatgggcg ccgtgaccac agaggtggcc ttcggcctcg tgtgtgccac
ttgcgagcag 1980atcgccgaca gccagcaccg gtcccacaga cagatggtca
ccaccaccaa ccctctgatc 2040cggcacgaga accggatggt gctcgcctct
accaccgcca aggccatgga acagatggcc 2100ggcagcagcg aacaggccgc
cgaagccatg gaaatcgcca gccaggccag gcagatggtc 2160caggccatgc
ggaccatcgg cacccaccct agcagctcca ccggcctgcg ggacgatctg
2220ctggaaaatc tgcagaccta tcagaaacgg atgggggtgc agatgcagcg
gttcaagagg 2280aaaagacgtg cgccagtaaa gcagacatta aactttgatt
tgctgaaact tgcaggtgat 2340gtagagtcaa atccaggtcc aatgaaggcc
atcctggtgg tgctgctgta caccttcgcc 2400accgccaacg ccgacaccct
gtgcattggg tatcatgcca acaactccac cgacaccgtg 2460gataccgtgc
tggaaaagaa tgtgacagtc acccacagcg tgaacctgct ggaagataag
2520cacaatggga agctgtgcaa gctgcggggc gtggcccctc tgcacctggg
caagtgcaat 2580atcgccggct ggatcctggg aaatcctgag tgcgagagcc
tgagcaccgc cagcagctgg 2640tcttacatcg tcgagacaag cagcagcgac
aacggcacct gttaccccgg cgatttcatt 2700gattatgagg aactgcgcga
gcagctgtcc tccgtgtcca gcttcgagag attcgagatc 2760ttccctaaga
ccagctcctg gcccaaccac gactccaaca agggcgtgac cgccgcctgt
2820cctcacgctg gcgccaagag cttctacaag aacctgatct ggctcgtcaa
gaagggcaac 2880tcctacccca agctgagcaa gagctacatc aacgacaagg
gcaaagaggt cctcgtcctc 2940tggggaattc atcaccctag caccagcgcc
gaccagcaga gcctgtatca gaacgccgac 3000gcctacgtgt tcgtgggcag
cagccggtac agcaagaagt tcaagcctga gatcgccatc 3060agacccaaag
tgcgggacca ggaaggccgg atgaactact actggaccct ggtggaaccc
3120ggcgacaaga tcaccttcga ggccaccggc aatctggtgg tgcccagata
cgccttcgcc 3180atggaacgga acgccggcag cggcatcatc atcagcgaca
cccccgtgca cgactgcaat 3240accacctgtc agacacctaa gggggccatc
aacacctccc tgcctttcca gaatatccac 3300cctatcacca tcgggaagtg
tcctaaatat gtcaagagca ccaagctgcg gctggccaca 3360ggactgagaa
acatccccca gagagagcgg agacggaaga agcggggcct gttcggcgct
3420atcgctggat tcattgaggg cggatggacc ggcatggtcg atgggtggta
tgggtatcat 3480caccagaatg agcagggctc cggatatgcc gccgatctga
agtccactca gaacgccatc 3540gacgagatca caaacaaagt caactccgtg
atcgagaaaa tgaatactca gttcaccgcc 3600gtcgggaaag agttcaatca
cctggagaag cgcatcgaga atctcaacaa aaaggtcgac 3660gacggcttcc
tcgacatctg gacatacaat gctgaactgc tcgtgctgct ggagaatgag
3720agaaccctgg attaccacga ttccaacgtc aagaatctct atgagaaagt
gcggagccag 3780ctgaagaaca atgccaaaga aatcgggaat ggctgttttg
agttttatca caagtgtgat 3840aacacctgta tggaatccgt gaagaatggc
acatacgatt accctaagta ctccgaagag 3900gccaagctga acagagaaga
gatcgacggg gtcaagctgg aaagcacccg gatctatcag 3960atcctggcca
tctattctac cgtggcctcc tccctggtgc tggtggtgtc cctgggcgct
4020atcagcttct ggatgtgctc caatggctcc ctgcagtgca gaatctgtat
ttgataatt 40791510PRTArtificial SequenceSynthetic Polypeptide 15Pro
Gln Arg Glu Arg Arg Arg Lys Lys Arg 1 5 10 1610PRTArtificial
SequenceSynthetic Polypeptide 16Pro Gln Arg Glu Ser Arg Arg Lys Lys
Arg 1 5 10 1710PRTArtificial SequenceSynthetic Polypeptide 17Pro
Gln Gly Glu Arg Arg Arg Lys Lys Arg 1 5 10 189PRTArtificial
SequenceSynthetic Polypeptide 18Pro Leu Arg Glu Arg Arg Arg Lys Arg
1 5 196PRTArtificial SequenceSynthetic Polypeptide 19Pro Gln Arg
Glu Thr Arg 1 5 20566PRTArtificial SequenceSynthetic Polypeptide
20Met Lys Thr Ile Ile Ala Leu Ser Tyr Ile Leu Cys Leu Val Phe Ala 1
5 10 15 Gln Lys Leu Pro Gly Asn Asp Asn Ser Thr Ala Thr Leu Cys Leu
Gly 20 25 30 His His Ala Val Pro Asn Gly Thr Ile Val Lys Thr Ile
Thr Asn Asp 35 40 45 Gln Ile Glu Val Thr Asn Ala Thr Glu Leu Val
Gln Ser Ser Ser Thr 50 55 60 Gly Gly Ile Cys Asp Ser Pro His Gln
Ile Leu Asp Gly Glu Asn Cys 65 70 75 80 Thr Leu Ile Asp Ala Leu Leu
Gly Asp Pro Gln Cys Asp Gly Phe Gln 85 90 95 Asn Lys Lys Trp Asp
Leu Phe Val Glu Arg Ser Lys Ala Tyr Ser Asn 100 105 110 Cys Tyr Pro
Tyr Asp Val Pro Asp Tyr Ala Ser Leu Arg Ser Leu Val 115 120 125 Ala
Ser Ser Gly Thr Leu Glu Phe Asn Asn Glu Ser Phe Asn Trp Thr 130 135
140 Gly Val Thr Gln Asn Gly Thr Ser Ser Ala Cys Lys Arg Arg Ser Asn
145 150 155 160 Lys Ser Phe Phe Ser Arg Leu Asn Trp Leu Thr His Leu
Lys Phe Lys 165 170 175 Tyr Pro Ala Leu Asn Val Thr Met Pro Asn Asn
Glu Lys Phe Asp Lys 180 185 190 Leu Tyr Ile Trp Gly Val His His Pro
Gly Thr Asp Asn Asp Gln Ile 195 200 205 Ser Leu Tyr Ala Gln Ala Ser
Gly Arg Ile Thr Val Ser Thr Lys Arg 210 215 220 Ser Gln Gln Thr Val
Ile Pro Asn Ile Gly Ser Arg Pro Arg Val Arg 225 230 235 240 Asp Val
Pro Ser Arg Ile Ser Ile Tyr Trp Thr Ile Val Lys Pro Gly 245 250 255
Asp Ile Leu Leu Ile Asn Ser Thr Gly Asn Leu Ile Ala Pro Arg Gly 260
265 270 Tyr Phe Lys Ile Arg Ser Gly Lys Ser Ser Ile Met Arg Ser Asp
Ala 275 280 285 Pro Ile Gly Lys Cys Asn Ser Glu Cys Ile Thr Pro Asn
Gly Ser Ile 290 295 300 Pro Asn Asp Lys Pro Phe Gln Asn Val Asn Arg
Ile Thr Tyr Gly Ala 305 310 315 320 Cys Pro Arg Tyr Val Lys Gln Asn
Thr Leu Lys Leu Ala Thr Gly Met 325 330 335 Arg Asn Val Pro Glu Lys
Gln Thr Arg Gly Ile Phe Gly Ala Ile Ala 340 345 350 Gly Phe Ile Glu
Asn Gly Trp Glu Gly Met Val Asp Gly Trp Tyr Gly 355 360 365 Phe Arg
His Gln Asn Ser Glu Gly Thr Gly Gln Ala Ala Asp Leu Lys 370 375 380
Ser Thr Gln Ala Ala Ile Asn Gln Ile Asn Gly Lys Leu Asn Arg Leu 385
390 395 400 Ile Gly Lys Thr Asn Glu Lys Phe His Gln Ile Glu Lys Glu
Phe Ser 405 410 415 Glu Val Glu Gly Arg Ile Gln Asp Leu Glu Lys Tyr
Val Glu Asp Thr 420 425 430 Lys Ile Asp Leu Trp Ser Tyr Asn Ala Glu
Leu Leu Val Ala Leu Glu 435 440 445 Asn Gln His Thr Ile Asp Leu Thr
Asp Ser Glu Met Asn Lys Leu Phe 450 455 460 Glu Arg Thr Lys Lys Gln
Leu Arg Glu Asn Ala Glu Asp Met Gly Asn 465 470 475 480 Gly Cys Phe
Lys Ile Tyr His Lys Cys Asp Asn Ala Cys Ile Gly Ser 485 490 495 Ile
Arg Asn Gly Thr Tyr Asp His Asp Val Tyr Arg Asp Glu Ala Leu 500 505
510 Asn Asn Arg Phe Gln Ile Lys Gly Val Glu Leu Lys Ser Gly Tyr Lys
515 520 525 Asp Trp Ile Leu Trp Ile Ser Phe Ala Ile Ser Cys Phe Leu
Leu Cys 530 535 540 Val Ala Leu Leu Gly Phe Ile Met Trp Ala Cys Gln
Lys Gly Asn Ile 545 550 555 560 Arg Cys Asn Ile Cys Ile 565
21566PRTArtificial SequenceSynthetic Polypeptide 21Met Lys Thr Ile
Ile Ala Leu Ser Tyr Ile Leu Cys Leu Val Phe Ala 1 5 10 15 Gln Lys
Leu Pro Gly Asn Asp Asn Ser Thr Ala Thr Leu Cys Leu Gly 20 25 30
His His Ala Val Pro Asn Gly Thr Ile Val Lys Thr Ile Thr Asn Asp 35
40 45 Gln Ile Glu Val Thr Asn Ala Thr Glu Leu Val Gln Ser Ser Ser
Thr 50 55 60 Gly Gly Ile Cys Asp Ser Pro His Gln Ile Leu Asp Gly
Glu Asn Cys 65 70 75 80 Thr Leu Ile Asp Ala Leu Leu Gly Asp Pro Gln
Cys Asp Gly Phe Gln 85 90 95 Asn Lys Lys Trp Asp Leu Phe Val Glu
Arg Ser Lys Ala Tyr Ser Asn 100 105 110 Cys Tyr Pro Tyr Asp Val Pro
Asp Tyr Ala Ser Leu Arg Ser Leu Val 115 120 125 Ala Ser Ser Gly Thr
Leu Glu Phe Asn Asn Glu Ser Phe Asn Trp Thr 130 135 140 Gly Val Thr
Gln Asn Gly Thr Ser Ser Ala Cys Lys Arg Arg Ser Asn 145 150 155 160
Lys Ser Phe Phe Ser Arg Leu Asn Trp Leu Thr His Leu Lys Phe Lys 165
170 175 Tyr Pro Ala Leu Asn Val Thr Met Pro Asn Asn Glu Lys Phe Asp
Lys 180 185 190 Leu Tyr Ile Trp Gly Val His His Pro Gly Thr Asp Asn
Asp Gln Ile 195 200 205 Ser Leu Tyr Ala Gln Ala Ser Gly Arg Ile Thr
Val Ser Thr Lys Arg 210 215 220 Ser Gln Gln Thr Val Ile Pro Asn Ile
Gly Ser Arg Pro Arg Val Arg 225 230 235 240 Asp Val Pro Ser Arg Ile
Ser Ile Tyr Trp Thr Ile Val Lys Pro Gly 245 250 255 Asp Ile Leu Leu
Ile Asn Ser Thr Gly Asn Leu Ile Ala Pro Arg Gly 260 265 270 Tyr Phe
Lys Ile Arg Ser Gly Lys Ser Ser Ile Met Arg Ser Asp Ala 275 280 285
Pro Ile Gly Lys Cys Asn Ser Glu Cys Ile Thr Pro Asn Gly Ser Ile 290
295 300 Pro Asn Asp Lys Pro Phe Gln Asn Val Asn Arg Ile Thr Tyr Gly
Ala 305 310 315 320 Cys Pro Arg Tyr Val Lys Gln Asn Thr Leu Lys Leu
Ala Thr Gly Met 325 330 335 Arg Asn Val Ile Pro Gln Arg Glu Arg Arg
Arg Lys Lys Arg Ile Ala 340 345 350 Gly Phe Ile Glu Asn Gly Trp Glu
Gly Met Val Asp Gly Trp Tyr Gly 355 360 365 Phe Arg His Gln Asn Ser
Glu Gly Thr Gly Gln Ala Ala Asp Leu Lys 370 375 380 Ser Thr Gln Ala
Ala Ile Asn Gln Ile Asn Gly Lys Leu Asn Arg Leu 385 390 395 400 Ile
Gly Lys Thr Asn Glu Lys Phe His Gln Ile Glu Lys Glu Phe Ser 405 410
415 Glu Val Glu Gly Arg Ile Gln Asp Leu Glu Lys Tyr Val Glu Asp Thr
420 425 430 Lys Ile Asp Leu Trp Ser Tyr Asn Ala Glu Leu Leu Val Ala
Leu Glu 435 440 445 Asn Gln His Thr Ile Asp Leu Thr Asp Ser Glu Met
Asn Lys Leu Phe 450 455 460 Glu Arg Thr Lys Lys Gln Leu Arg Glu Asn
Ala Glu Asp Met Gly Asn 465 470 475 480 Gly Cys Phe Lys Ile Tyr His
Lys Cys Asp Asn Ala Cys Ile Gly Ser 485 490 495 Ile Arg Asn Gly Thr
Tyr Asp His Asp Val Tyr Arg Asp Glu Ala Leu 500 505 510 Asn Asn Arg
Phe Gln Ile Lys Gly Val Glu Leu Lys Ser Gly Tyr Lys 515 520 525 Asp
Trp Ile Leu Trp Ile Ser Phe Ala Ile Ser Cys Phe Leu Leu Cys 530 535
540 Val Ala Leu Leu Gly Phe Ile Met Trp Ala Cys Gln Lys Gly Asn Ile
545 550 555 560 Arg Cys Asn Ile Cys Ile 565
* * * * *
References