Unexpected Dynamics of Helicase Primase Resistance in Herpes Simplex Virus1

1
Unexpected Dynamics of Helicase Primase
Resistance in Herpes Simplex Virus-1
Hugh J Field Reader in Comparative Virology,
Department of Veterinary Medicine
2
Herpes simplex virus -1 (HSV-1) Chemotherapy has
been widely used since 1980
acyclovir - valaciclovir penciclovir -
famciclovir
Nucleoside analogues activated by HSV-1
thymidine kinase (TK) inhibit HSV-1 DNA
polymerase
3
HSV-1 Genome Map
Helicase (UL5)
Accessory Protein of Helicase Primase (UL8)
Primase (UL52)
Thymidine kinase (UL23)
DNA-polymerase (UL30)
Adapted from The Homepage of the late Dr. Edward
K. Wagner. (http//darwin.bio.uci.edu/faculty/w
agner/hsv7f.html accessed Aug 2007).
4
Novel Helicase - Primase Inhibitors of Herpes
Simplex Virus
Helicase-primase inhibitors appear to be
extremely active in tissue culture various
animal models Several e.g. BAY 57-1293 are being
developed for use in man
BAY 57-1293 Mol.wt. 402 Da
H-P inhibitors claimed advantage of low
resistance selection
5
Antiviral Compounds Inhibitors of DNA Synthesis
HSV ds DNA
xxx reported site for BAY57-1293 resistance
mutations
6
Viruses for Study
HSV-1 strains
1) SC16 or PDK "crude" (Working stocks)
3 times plaque-purified in absence of inhibitor
SC16 "cl-2" or PDK "cl-1" (clones)
2) Ten clinical isolates (A-J)
7
Plaque Reduction Assay in Vero cells
Sensitivity of Various HSV-1 Viruses to BAY57-1293
140
SC16 cl-2
PDK cl-1
120
100
80
Plaques () control
ED50
60
40
20
0
-3
-2
-1
0
1
2
3
BAY57-1293 (log10)?g/ml
8
Sensitivity of HSV-1 Viruses to BAY57-1293 by
plaque-reduction assay in Vero cells
9
BAY 57-1293 is an example of an HPI
extremely active compound with low toxicity

• I will show
• Highly efficacious in murine infection model
• (including immunosuppressed)
• 2. Resistance is an issue that requires
  investigation

10
Neck - Ear Zosteriform infection Model
Mice Balb/c
Day 0
Ear swelling
Virus HSV 105
p.f.u.
Day 3
0
2
4
6
8
10
Time p.i. (days)
Ear swelling
(inflammation)
Day 7
Ear virus (Log10)
Measure
Ear virus
0
2
4
6
8
10
Time p.i.
(days)
Assess ganglia for Latency
at 1- 2 months
Awan et al, 1998, Antivir. Res.
11
Murine Zosteriform Model
12
Progress of Lesions Following Neck Inoculation
Virus SC16 (1 x 105 p.f.u.)
(No therapy)
Day 0
Day 3
13
Progress of Lesions Following Neck Inoculation
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Comparison of BAY 57-1293 with Famciclovir
Mice 3-4 week old, female, Balb/C Virus
HSV-1 (SC16) scarification at the neck,
5x104 p.f.u. in 100ml Therapy Placebo, gavage,
OD days 1-4 p.i. BAY 57-1293 _at_ 18mg/kg,
oral gavage, OD day 1-4 p.i. BAY 57-1293 _at_
18mg/kg i.p. OD day 1-4 p.i. Famvir _at_ 1
mg/ml (gt150 mg/kg), drinking water day
1-7
15
Infection Parameters for Assessing Antiviral
Efficacy
measure daily (n5) survival weight
changes ear thickness virology on days
1,3,5,8 p.i. (n3) skin at inoculation
site skin of ear pinna brain stem
16
Effect of BAY 57-1293 on Weight Gain in Balb/c
Mice
Weight gain ()
Treatment days 1-4
17
Effect of BAY 57-1293 on Survival in Balb/c Mice
Effect of HelicasePrimase Inhibitor on HSV-1 in
Balb/c Mice
ii
Survival
100
treated FAM or Bay 57-1293 n10
infected control n5
75
Surviving mice ()
Surviving mice (
50
25
3
0
6
0
1
2
4
5
7
8
9
Time p.i. (days)
18
Effect of BAY 57-1293 on Ear Swelling

100
75
Uninfected
Infected placebo
50
Ear thickness increase ()
Oral Bay 57 -1293
25
i.p. Bay 57 -1293
Fam drinking water
0
BAY 57 1293 days 1-4
6
-25
0
1
2
3
4
5
7
8
9
Time p.i. (days)
Doses

Bay 15-1293 18 mg/Kg po or ip once per day 1-4
Fam approx. 120 mg/Kg drinking water days 1-7
19
Effect of BAY 57-1293 on Infectious Virus in Skin
at the Inoculation Site
20
Infectious virus Log10 p.f.u./ear
limit of detection
21
Effect of BAY 57-1293 on HSV-1 in Balb/C Mice on
Infectious Virus in the CNS
5
Infectious virus Log10 b.f.u./brain
4
Placebo treated
3
Bay 57-1293 po
Virus in brain stem (log10 pfu/brain)
Bay 57-1293 pi.
2
Fam drinking water
1
Limit of sensitivity
0
1
3
5
7
Time p.i. (days)
22
Efficacy of BAY 57-1293 in the Immunocompromised
Host
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Mice Athymic nude on a Balb/c
background 12 mice available (two mice per
group) Virus HSV-1 SC16 cl-2
Inoculation Neck skin 104 p.f.u. Therapy
Once per day on day 3 only or 3 4 only
i.p. 15mg/Kg
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Effect of BAY 57-1293 on HSV-1 Infection in
Athymic (Nude) Balb/c Mice
i. Weight change
doses
10
8
6
4
INFECTED CONTROL
2
Bay 57-1293 one dose
0
Bay 57-1293 two doses
Weight change ()
Weight change ()
UNINFECTED CONTROL
-2
-4
-6
-8
-10
0
1
2
3
4
5
6
7
Days post-infection
25
Effect of BAY 57-1293 on HSV-1 Infection in
Athymic Mice
26

• Conclusions
• BAY57-1293 is efficacious in the murine
  zosteriform HSV-1 infection model
• The compound appeared to be effective a
  reducing both clinical signs of infection
• and infectious virus in the tissues using this
  lower dose (18mg/kg) compared with published
  and historical data using common nucleosides
• A once/day oral dose was equally or more
  effective than oral famciclovir
• (given at higher dose)
• Therapy was started after virus replication
  had commenced and a marked beneficial effect
  was seen within two days of starting treatment
• There was no evidence of "rebound" on
  cessation of therapy
• The compound was also effective when given
  i.p.

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The Issue of Drug Resistance
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Nucleoside analogue drug resistance In cell
culture DNA pol resistance Fewer than 1 in
106 TK resistance More than 1 in 104 (but
mutants are attenuated) Frequent TK resistance
mutations explained by GGGG
CCCC strings Most TK mutants are
frame-shift truncated polypeptides.
In clinical practice resistance very very rare
(except immunosuppressed)
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Unexpected Dynamics of HPI Resistance inHerpes
Simplex Virus-1
BAY 57-1293-resistant viruses readily isolated
from virus working stocks SC16 PDK (Biswas et
al, 2007, Antivir Chem Chemother) Resistant
mutants appear to be present at 10-4 Viruses
detected with continuous presence of
inhibitor Plaque-pure isolates rate drops to
10-6 (similar to published reports)
30
Screening HSV-1 clinical isolates for
HPI-sensitivity
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HSV-1 Clinical Isolates Addenbrookes Hospital
10 random HSV-1 isolates Swab MRC5 cells
-700
Vet School -700
detection virus typing
Test for sensitivity to BAY 57-1293 in VERO cells
32
Plaque Reduction Assay in Vero cells
Sensitivity of Various HSV-1 Viruses to BAY57-1293
33
Sensitivity of HSV-1 Viruses to BAY57-1293 by
plaque-reduction assay in Vero cells
34
Sensitivity of HSV-1 Viruses to BAY57-1293 by
plaque-reduction assay in Vero cells
35
Are HPI-resistant mutants present in Clinical
Isolates?
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HSV-1 Isolate
YES!
An Important Control
Unexpectedly high frequency of BAY
57-1293 resistant virus in clinical isolates
Laboratory isolates
(Biswas et al, 2007, J Antimicrob Chemother)
38
BAY 57-1293-resistant mutants selected from Clin
F and G tested by PRA in Vero cells
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Drug resistance mutations Amino Acid
Substitutions in UL5
italics high frequency occurring
43
Patients F and G Appear to be Unrelated
Both isolates are 2006 HSV-1 from genital ulcers
Specimen F female, 25 years, vulval swab,
11.04.06 Addenbrooke's Hospital Specimen G
male, 33 years, penile swab, 13.04.06 West
Suffolk Hospital
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Are virus isolates from F and G related?
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Nucleotide Polymorphisms in UL5 gene (including
silent mutations) in relation to the published
sequence of Strain 17
Clin G-rm-1 1
A200?G 2 3 T614 ?C 4 5
6 G1347 ?T T2069 ?G

• SC16 cl-2
• A41 ?G
• A200?G
• A483 ?G
• T614 ?C
• C984 ?G
• C1158 ?A
• G1347 ?T
• T2069 ?G
• C2484 ?T

PDK cl-1 G77 ?A G168 ?A A200?G G1279
?A G2163 ?A
Clin F-rm-1 A41 ?G A200?G A477
?G A483 ?G T614 ?C C984 ?G G1086 ?A
C1158 ?A G1347 ?T T2069 ?G
(excluding the drug resistance mutation)
46
Amino acid polymorphism between isolates F and G
BAY-G-r1


polymorphism
I
II
III
IV
V
VI
882aa
F351G N L M K 356
N342NKRCVEHE350
K356(AAG)?N(AAT)
Strain 17 TFEHQKLRCSVRQSENVLTYLICNRTLREYARLSYSW
AIFINNKRCVEHEFGNLMKVLEY 360
PDK cl-1 TFEHQKLRCSVRQSENVLTYLICNRTLREYARLSYSWA
IFINNKRCVEHEFGNLMKVLEY 360
SC16 cl-2 TFEHQKLRCSVRQSENVLTYLICNRTLREYARLSYSW
AIFINNKRCVEHEFGNLMKVLEY 360
BAY-F-r1 TFEHQKLRCSVRQSENVLTYLICNRTLREYARLSYSWA
IFINNKRCVEHEFGNLMNVLEY 360
BAY-G-r1 TFEHQKLRCSVRQSENVLTYLICNRTLREYARLSYSWA
IFINNKRCVEHEFGNLMNVLEY 360


Resistance mutation
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Genetic Clustering based on UL5
a) Nucleotide seq.-based
Analysis using MEGA software
b) Amino acid seq.-based
Analysis using MEGA software
48

• Conclusions
• Helicase-primase inhibitor BAY 57-1293 extremely
  active
• in animal models compares favourably with
  nucleosides
• Brief therapy effective in normal and
  immunocompromised mice
• Some HSV-1 laboratory isolates contain BAY
  57-1293-resistant variants at unexpectedly high
  frequency
• 4. Also true for recent HSV-1 clinical
  isolates from untreated patients

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Subhajit Biswas - Chipping Campden - August 2006
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Acknowledgements
Graduate Student Subhajit Biswas Technica
l help Liz Lay Collaborations Ken Powell
(Arrow Therapeutics) Lynn Jennens (Arrow
Therapeutics) Laurence Tiley
(Cambridge) Jonathan Lyall (Cambridge) Jesus
Aguirre-Hernandez (Cambridge) Islam Tohamy
Mohammad (Cambridge) Mihai Swift (NST part II
student) Chris Smith (Pathology
Department) Funding and scholarships
Cambridge Nehru Scholarship ORS Award
(Universities UK) Materials grant in aid Arrow
Therapeutics, London