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Applications of the RG
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Study of molecular biology of
RNA viruses
Pathogenesis
Vaccine development
Vectors
Other applications….
VPM 700
May’06
Molecular Biology of RNA viruses

Provide insights into the many aspects that
control the virus life cycle
- the location and boundaries of the
cis-acting elements
- the secondary structure of promotor regions
- the functional domains of the trans-acting
proteins
- the gene stop-start signals
- AND MANY MORE……
Pathogenesis

Genetic determinant of virulence by H5N1
virus in mice (Hatta et al., Science, 2001)
A/HongKong/483/97 (HK483) – Highly Pathogenic
A/HongKong/486/97 (HK486) – Mildly Pathogenic
Prepared the single-gene reassortant viruses
In vivo characterization
PB2 Protein is responsible for the
difference in virulence
Amino acid residue at position 627
influence the outcome of infection
H7N2 influenza in the U.S.
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H7N2 LPAI virus circulation in NY and NJ LBM’s since 1994
LBM H7N2 virus served as source of infection for commercial
poultry operation
- 1997-1998, Pennsylvania
- 2001-2002, Pennsylvania
- 2002, Virginia, West Virginia, North Carolina
- 2003, Connecticut
- 2004, Delaware, Maryland
LBM H7N2 virus may serve as source of infection for human
- 2003, 1st human case of H7N2 infection in New York
Determinant of pathogenicity
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Not totally defined
An optimal constellation of viral genes is required for
pathogenicity expression
Hemagglutinin (HA) structure is a primary determinant
of pathogenicity in chickens and turkeys
“HA0 cleavage into HA1 and HA2”
- Necessary for fusion peptide to be exposed
- Essential for virus to become infectious
All AI viruses : trypsin-like enzyme (respiratory, intestinal)
HPAI viruses : ubiquitous protease (systemic)
Key features for HA cleavability
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The amino acid
sequence at the
cleavage site
- multiple basic a.a.
The carbohydrate in
the vicinity of the
cleavage site
- no glycosylation at
Asn11
Receptor binding
site
Asn11
Cleavage site
Molecular features of H7N2 virus
1994-1995, 1999
Cleavage site sequence
(HA1/HA2)*
P-E-N-P-K-T-R/G
1995-1999, 2002
P-E-N-P-K-P-R/G
23
1995, 1998
P-E-K-P-K-T-R/G
2
1998-2005
P-E-K-P-K-P-R/G
426
2002, 2004-2005
P-E-K-P-K-K-R/G
82
Year observed
No. of isolates
* Basic amino acids (Lysine(K) and Arginine(R)) are in bold character.
Is H7N2 virus progress toward HPAI virus ?
What additional changes are required ?
6
Pathogenic Potential of H7N2 Virus
Virus
Parent
Mutant
Mutant
Mutant
Mutant
Mutant
Mutant
Mutant
Mutant
Mutant
Mutant
Mutant
Mutant
sequence
In vitro
In vivo
replication plaquing
in ECE
efficiency
PEKP---KKR/G
1
PEKR---KKR/G
2
PEKK---KKR/G---- inefficient
3
PEKP--RKKR/G
4
PEKP--KKKR/G---- inefficient
5
PEKR--RKKR/G
6
PEKR--KKKR/G---- inefficient
7
PEKP-KKKKR/G
8
PEIP-KKKKR/G
9
PEKPKKRKKR/G
10
PEIPKKRKKR/G
11
PENPKKRKKR/G
12
PETPKKRKKR/G
Positive control (CK/Chile/02 (H7N3))
mortality
virulence
-
0/8
-
-
0/8
-
-
0/8
-
+
0/8
++
-
0/8
-
++
2/8
+++
-
0/8
-
+
0/8
+
+
0/8
+
+++
3/8
++++
+++
4/8
++++
+++
5/8
++++
+++
5/8
++++
+++
1/8
+++
Transcription Plasmids (8)
Rescue of viruses
carrying mutated
HA genes
P
Avian – PB2
T
P
Avian – PB1
T
P
Avian – PA
T
P
Mutated H7
T
P
Avian – NP
T
P
Avian – N1
T
P
Avian – M
T
P
Avian – NS
T
Expression Plasmids (4)
+
P
PB2
A
P
PB1
A
P
PA
A
P
NP
A
Co-transfection
293T
Amplification
10-day
eggs
Reassortant H7N1 strains
with different H7 gene
Reverse genetics using helper virus
4 Expression Plasmids
P
PB2
A
P
PB1
A
P
PA
A
P
NP
A
+
Transcription Plasmids
P
H7 HA
T
Co-transfection
Selection of HP reassortant by
plaque assay in CEF
293T
24hr incubation
HP
reassortant
293T
Helper virus infection
(wild type H7 virus)
48hr incubation
no HP
reassortant
4 Expression Plasmids
P
PB2
A
P
PB1
A
P
PA
A
P
NP
A
+
Transcription Plasmids
P
H7 HA
T
Co-transfection
M 3 PEKP--RKKR/G
M 5 PEKR--RKKR/G
M 7 PEKP-KKKKR/G
M 8 PEIP-KKKKR/G
M 9 PEKPKKRKKR/G
M 10 PEIPKKRKKR/G
M 11 PENPKKRKKR/G
M 12 PETPKKRKKR/G
CK/Chile/02-H7
Egret/HK/02-H5
CK/Indonesia/04-H5
√
√
√
√
Selection of HP reassortant by
plaque assay in CEF
293T
24hr incubation
293T
Helper virus infection
(wild type H7 virus)
Chile/02, Egret/02,
Indonesia/04,
H7-M3, M5, M9, M12
reassortants
48hr incubation
All other H7 mutants
Pathogenicity of reassortants rescued
by helper virus rescue system
ECE
Virus
CEF
Chickens
Cleavage site sequence
EID50/ml
MDT
0.3ug trypisn
no trypsin
Mortality
MDT
Virus titer
h-M-3
PEKP--RKKR
7.9
<2
4X10^6 (1mm)
2X10^6 (>2mm)
8/8
2.9+2.0
6.4+0.1(2)
h-M-5
PEKR--RKKR
9.5
<2
5X10^7 (1mm)
3X10^6 (>2mm)
8/8
1.9+0.8
-
h-M-9
PEKPKKRKKR
9.2
<2
3X10^7 (1-2mm) 8X10^6 (>2mm)
8/8
2.9+1.2
6.3+1.3 (2)
h-M-12
PETPKKRKKR
8.5
<2
2X10^7 (1-2mm) 1X10^7 (>2mm)
8/8
3.6+0.7
5.6+0.5(4)
PEKPKTCSPLSRCRETR
8.2
<2
8/8
3.6+1.3
5.8+0.8(4)
h-M-chile
2X10^8 (2mm)
2X10^7 (>2mm)
MDT: Mean death time (average days taken to kill the embryo or chickens
b Number of birds that died / number of birds inoculated.
c The virus titer is express as the log
10 EID50 per milliliter ± standard deviation.
d No birds survived
a
Vaccine Development
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Non-structural (NS1) protein, a novel
target for the development of influenza
vaccines
Functions of the NS1 protein
- Inhibit the early production of cellular
antiviral mRNA
- display host unique PL (PDZ domain
ligand) motifs at the C-terminus for PDZ
domains involved in cell signaling
pathways (Science, 2006)
Genomes
RNA segments (bp)
Protein (aa)
1. Polymerase (basic) 2 (2341)
PB2 (759)
2. Polymerase (basic) 1 (2341)
PB1 (757), *PB1-F2
3. Polymerase (acidic) (2233)
PA (716)
4. Hemagglutinin (1775)
HA (565)
5. Nucleoprotein (1565)
NP (498)
6. Neuraminidase (1413)
NA (454)
7. Matrix (1027)
M1 (252)
Virion
constituents
M2 (97)
8. Nonstructural (890)
NS2(NEP)(121)
NS1 (230)
Infected cells
A/turkey/Oregon/71 (H7N3)
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Low virulence AI isolate from a turkey flock with mild
respiratory illness
Genetically distinct stocks exist
TK/OR/71-SEPRL (Suarez et al., 1999)
- Low passage stock of the field isolates
- Encode a full length NS1 protein (230aa)
TK/OR/71-del (Norton et al., 1987)
- Unknown passage history
- 10 nt deletion in the NS gene resulting in a truncated NS1 protein
TK/OR/71-D-dels (Lee et al., 2005)
- Derived from TK/OR/71-del stock by passaging in old ECE
- Different size of deletion in the middle of the NS gene
stop
*
SEPRL
NS genes
*
del
880 bp
705 bp
644 bp
487 bp
890bp
880bp
380-389 (10nt)
*
D-del 1
705bp
267-451 (185nt)
*
D-del 2
644bp
218-463 (246nt)
*
D-del 3
204-606 (403nt)
487bp
translation
RNA binding
SEPRL
Effector domain
1
230
73
230 aa
117
▼
del
NS1
124 aa
80
▼
D-del 1
64
D-del 2
▼
90 aa
160
▼
148 aa
59
D-del 3
▼
144 aa
Transcription Plasmids (8)
Rescue of viruses
carrying different
NS genes
P
A/WSN/33 – PB2
T
P
A/WSN/33 – PB1
T
P
A/WSN/33 - PA
T
P
Avian – H5
T
P
A/WSN/33 - NP
T
P
Avian – N1
T
P
A/WSN/33 - M
T
P
NS
T
SEPRL (890bp)
del (880bp)
D-del 1 (705bp)
D-del 2 (689bp)
D-del 3 (644bp)
D-del 4 (487bp)
Expression Plasmids (4)
+
P
PB2
A
P
PB1
A
P
PA
A
P
NP
A
Co-transfection
293T
Amplification
10-day
eggs
Reassortant H5N1 strains
with different NS gene
DIVA strategy
(Differentiating Infected from Vaccinated Animals)
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Use of heterologous strain containing the same hemagglutinin
subtype as the field virus but a different neuraminidase in
an oil-emulsion vaccine (Beard, 1986; Stone, 1987)
H7N3 subtype vaccine was used to control H7N1 LPAI
virus in Italy (2000-2002)
H7N3 subtype vaccine is being used to control H7N2 LPAI
virus in Connecticut
Limitations
1) availability of heterologous vaccine
(The H7N3 outbreak in Northern Italy in 2002.)
2) degree of homology between the hemagglutinin gene in
vaccine and challenge virus
Reverse genetics for rapid generation
of influenza vaccine banks
WSN-PB2
WSN-PB1
WSN-PA
P
P
P
T
WSN-NP
P
T
T
WSN-M
P
WSN-PB1
WSN-PA
WSN-NP
T
WSN-NS
P
T
WSN-PB2
T
6 transcription plasmids from WSN/33 (H1N1)
4 expression plasmids
+
H5: A/CK/PA/13609/93 (H5N2)
H7: A/TK/VA/55/02 (H7N2)
H5 or H7
N1 or N8
P
P
T
T
N1: A/DK/Anyang/01 (H5N1)
N8: A/DK/NY/191255-59/02 (H5N8)
Transfection
293T
102.0-4.0 EID50 / ml
2 days
ECE
Amplification
2-3 days
rH5N1, rH7N1
rH5N8, rH7N8
Reassortant vaccine strains
108.0 EID50 / ml
Use of Reverse Genetics for Development of
DIVA Vaccines (Lee et al., Vaccine 2004)
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Use of plasmid-based reverse genetic system to produce viruses
with specific HA and NA subtypes
Post-Vaccination
Groups
*HI titer (s.d.)
Post-Challenge
**NI test
Number of positive birds
***Virus titer (s.d.)
N1
N2
N8
3dpi
5dpi
3dpi
5dpi
H5N2
6.5 (0.9)
0/10
10/10
0/10
4/10
0/10
0.4 (0.6)
0
rH5N1
6.7 (0.8)
10/10
0/10
0/10
4/10
0/10
0.6 (1.1)
0
10/10
9/10
4.5 (0.5)
3.4 (0.6)
Control
H7N2
6.8 (0.9)
0/10
10/10
0/10
0/10
0/10
0
0
rH7N8
6.8 (1.1)
0/10
0/10
10/10
0/10
0/10
0
0
10/10
10/10
4.9 (0.4)
3.9 (0.9)
Control
*HI titer is expressed as log2 reciprocal of the endpoint in twofold sera dilution
**NI test were performed with NI, N2 and N8 antigen and the number of positive samples
were indicated
***Virus titer is expressed as log10 50% egg infective doses per milliliter
Differentiation by indirect IFA assay
H5N2 infected
H5N2 vaccinated
rH5N1 vaccinated
H7N2 infected
H7N2 vaccinated
rH7N8 vaccinated
An alternative DIVA approach on the basis
of antibodies to NS1
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The NS1 protein is nonstructural
Killed vaccines for influenza are primarily made with
whole virion
The NS1 DIVA approach works well with horses
receiving purified vaccines (Ozaki et al., 2001).
Vaccinated chickens produce low levels of antibody to
the NS1 protein, but virus-infected chickens will
produce higher levels of NS1 antibody (Tumpey et al.,
2005).
Application – DIVA vaccine
RNA binding
SEPRL
1
Effector domain
73
230
230 aa
D-del 1
90 aa
D-del 2
163 aa

DIVA strategy
- truncated NS1 recombinant protein
produced in E. coli can be used as coating
antigen in ELISA format
- seroconversion will be observed in infected,
but not in vaccinated birds
Application - Vector
Insert foreign sequences
*
705bp
D-del 1
D-del-GFP

267-451
CRS
NS1
Expression of foreign gene
- GFP
- HIS tag
- Foreign antigenic epitope
GFP
338 aa
Application – Live Vaccine
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
For humans,
- cold-adapted live vaccine
- FluMistTM was approved for use in the U.S.
Potential problems of the use of live influenza
vaccines in poultry
- bird-to-bird transmission
- vaccine-induced respiratory disease
- potential for recombination with newly
introduced AIV
Application – Live Vaccines
Genetically engineered live attenuating virus
- changes in the PB2 gene
- exchanging promoter region of the NA gene
- generating viruses that have truncated NS1
 TK/OR/71-del virus
- attenuation in pathogenicity
- decreased ability to replicate in chickens
- inefficient bird-to-bird transmission
=> In ovo vaccination with attenuated live influenza
virus that induces strong immune response, but has
no infectious virus present in the hatched chicks.

Other Application of RG
- Antigenic drift study
A
194
154
188 158
123
126
160
181
B
218
Antigenic drift occurs away from
the vaccine strain with avian
influenza virus
(Lee et al., J Virology, 2004)
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The genetic changes of the HA1
gene were concentrated in or
near the predicted antigenic
sites in the protein.
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We also found a mutation that
created a glycosylation site near
the antigenic site
=> Using a mapping approach,
amino acid changes that are
important for immunity will be
identified

Receptor
binding site
136137
175
113
71
270
C
40
275
VPM 700
May’06