HERPES SIMPLEX VIRUS TYPE 1, APOE-

ε4 AND ALZHEIMER’S DISEASE: THE

CASE FOR ANTIVIRAL TREATMENT

Ruth Itzhaki, Matthew Wozniak, Alison Frost

Molecular Neurobiology Lab

Faculty of Life Sciences
University of Manchester
 RATIONALE FOR A VIRAL ROLE
IN AD
 Several persistent viruses can cause neurological disease (e.g.,
measles virus, HIV).

 Herpes simplex virus type 1 (HSV1) is implicated in AD because

a) it can latently infect neuronal cells life-long. Reactivated by stress,

etc.
b) it is ubiquitous
c) acute infection - herpes simplex encephalitis (HSE) – & AD affect
the same brain regions (Ball, 1982, Esiri, 2001, etc).

 HSE survivors show cognitive, memory & behavioural decline.

 MICROBES & DAMAGE
 Some microbes can remain in the body lifelong after acute
infection – they don’t just “hit and run”.

Infection can be latent, but reactivation may occur.

 “Infect” doesn’t necessarily mean “affect”, so “controls” may be

infected. Severity of damage probably depends on host genes.

Susceptibility to microbial infection, like susceptibility to

microbial damage, can depend on host genes.

 Effects of microbe may depend on tissue/cell type involved & on

microbe strain.
 HSV1 & THE NERVOUS SYSTEM

 HSV1 infects most humans in infancy. Remains latent in the PNS.

 Latency: viral DNA is present, only one abundant set of

transcripts, no viral proteins.

 Stress, immunosuppression, etc, can reactivate HSV1: whole

viruses form, i.e., acute infection occurs.

 On reactivation, 20+% of people develop herpes labialis (cold

sores); the remainder are unaffected.

 In acute infection, HSV1 subverts the cell machinery, & stimulates

cells into cycle (to G2/M). Occurs in AD too
 QUESTIONS

1. Is HSV1 present in “normal” brains?

2. Is the virus active in brain?

3. Is HSV1 in brain associated with AD?

4. Is there a causal link with AD neuropathology?

5. If a link is found, might antivirals work against AD?

 HSV1 & APOE-ε4 IN AD (& COLD SORES)
Answers to questions 1-3
 HSV1 DNA is present (latently) in many elderly normal & AD brains
(shown by solution & in situ PCR) (J Med Virol. 1991. et seq.). Subsequently, 5
other groups found HSV1 DNA in human brains.

 It has reactivated, then replicated there, perhaps recurrently (shown

by intrathecal antibodies to HSV1) (J Med Virol. 2005).

 HSV1 in brain of APOE-ε4 carriers confers a strong risk of AD,

accounting for ~ 60% of patients (Lancet, 1997; Alz Repts. 1998).

 APOE-ε4 is a risk factor for herpes labialis (Lancet, 1997; Alz Repts. 1998).
(Confirmed by Koelle et al., 2010)

Conclusion: in CNS & PNS, HSV1 damage is greater in

APOE-ε4 carriers.
 HSV1 LINKS TO AD NEUROPATHOLOGY

Previous relevant work:

HSV1 glycoprotein B peptides form fibrils ultra-structurally

identical to Aβ; HSV1 might “seed” plaques (Cribbs et al., 2000).

AβPP is associated with HSV1 during virus anterograde

transport; might affect AβPP degradation & synaptic function
(Satpute-Krishnan et al., 2003).
 HSV1 INCREASES LEVELS OF Aβ & AD-LIKE
TAU
HSV1 infection of human neural cells increases:

 intracellular Aβ 1-42 & Aβ 1-40 & also BACE & nicastrin (Neurosci.
Lett., 2007).

 tau phosphorylation at AD-relevant sites, & also PKA &

GSK3β (J. Alz. Dis., 2009).
Also, HSV1-infected mice show Aβ accumulation in brain
NB:
• BACE increase is caused by phosphorylation of eIF2α, due to activation of PKR (Ill-Raga et al.,
submitted)

• HSV1 encodes a protein kinase, US3; this activates & functionally overlaps cell kinase PKA.

• GSK3 regulates Aβ production

IN SITU PCR & THIOFLAVIN S OR IHC
Wozniak et al., J.Pathol 2009

HSV1 DNA (in situ PCR) Plaques (Thioflavin S)

20X 20X

HSV1 DNA (in situ PCR, brown) &

Aβ (IHC, purple) 20X

>90% plaques
contain viral DNA
 PERCENT AMYLOID PLAQUES CONTAINING
HSV1 DNA, AND HSV1 DNA CLUSTERS
ASSOCIATED WITH PLAQUES

*
* p<0.001
 CO-LOCALISATION OF HSV1 DNA &
PLAQUES (J Pathol. 2009).
 In AD & aged normal brains, 80-90% of amyloid plaques
contain HSV1 DNA.

 In AD brains, over 70% of the HSV1 DNA is located in plaques;

only 24% in aged normals, possibly because less produced
&/or more cleared.

 Artefactual co-localisation can be refuted.

Of course association does not prove causality, but as
HSV1 infection causes Aβ accumulation the viral DNA-
amyloid co-localisation indicates that HSV1 is a major
cause of toxic Aβ products & plaques.
 ACYCLOVIR
 ACV is a nucleoside analogue that stops viral replication.
 Its action requires HSV1 thymidine kinase (TK).
 TK phosphorylates ACV to its mono-phosphate form.
 Cell enzymes further phosphorylate to di- and tri-phosphate.
The tri-phosphate competes with dGTP as a DNA polymerase
substrate; in new DNA it prevents elongation.
 Thus ACV “finds” HSV1-infected cells & stops HSV1
replication.

NB. In practice, valacyclovir, the biodrug of ACV, is

used orally as its absorption is very much greater.
 EFFECTS OF ACYCLOVIR ON THE AD-LIKE CHANGES IN HSV1-
INFECTED CELLS

HSV1-infected HSV1-infected Uninfected Uninfected

no ACV 50uM ACV No ACV 50uM ACV

Aβ
(Aβ1-42)

P-tau
(pS214)

HSV1
 HSV1-INDUCED CHANGES IN Aβ & P-tau,
& EFFECT OF ACV
5000

4500

4000
p<0.001
3500

3000

2500

2000

1500

1000

500

0
0uM ACV 100uM ACV 0uM ACV 100uM ACV

HSV1 -infected (10pfu/cell) HSV1 -infected (10pfu/cell) Uninfected Uninfected

6000

5000
p<0.001

4000

3000

2000

1000

0
0uM ACV 100uM ACV 0uM ACV 100uM ACV

HSV1 - infected HSV1 -infected Uninfected Uninfected

(10pfu/cell) (10pfu/cell)
 ANSWERS

1. Is HSV1 present in “normal” brains?

Yes, in the elderly.

2. Is the virus active in brain?

Yes, perhaps recurrently.

3. Is HSV1 in brain associated with AD?

Yes, in APOE-ε4 carriers.

4. Is there a causal link with AD neuropathology?

Yes, with plaques and AD-like tau.

5. If a link is found, might antivirals work against AD?

Based on our cell culture work, they should reduce not
only a major cause but also Aβ and P-tau.
 FUTURE POSSIBILITIES FOR
PREVENTION OR TREATMENT OF AD

 Immunisation against HSV1 in infancy (more

feasible now, with rising age of primary infection).
 Use of antiviral agents to retard disease
progression.

N.B. Antivirals (e.g., VCV/ACV) would target only

HSV1, i.e., a cause of AD, rather than the
symptoms/features, & not host cells. In fact they
cause very few side-effects.
 PROOF OF CONCEPT TRIAL
We hope to recruit 100 HSV-seropositive patients with
mild to moderate AD, in four centres.

Treat half with VCV (2x500mg/day), half with placebo.

Follow for 12 months, investigating cognitive function

(ADAS-Cog & MMSE), activities of daily living (IDDD),
behavioural and psychological symptoms (NPI), Quality
of life (DEMQOL), & global impression of change (GIC),
assessing pre-randomisation and at 3, 6, 9 & 12 months.

Check renal function.

PAST LAB MEMBERS COLLABORATORS
Curtis Dobson Andrew Mee
Ann Cookson Gordon Wilcock
Stacey Efstathiou
Suzanne Shipley Margaret Esiri
Tom Lloyd Seth Love
Woan-Ru Lin Brian Faragher
Gordon Jamieson Roy Jennings
Dazhuang Shang Marc Combrinck
Bob Cooper
Acknowledgements include Paul Klapper
Alzheimer’s Society; Henry Chris Preston
Smith Charity. Raj Kalaria
David Mann
Nigel Hooper
 RELEVANT WORK BY OTHERS (1)
 Measles virus causes NFT in brain, & HIV causes amyloid plaques
and NFT. *

 Systemic infection causes cognitive decline in the elderly (Strandberg

et al., 2003; Holmes et al., 2003) - consistent with a viral role in AD: peripheral
infection→ brain inflammation → latent HSV1 reactivation in brain→
both inflammatory and direct damage

 Serum IgM → evidence of recent HSV reactivation (Letenneur et al.

2008).

 Genetic support for a viral role (Porcellini et al., 2010)

*HSV1 is the only relevant virus found so far in elderly

brains and so is uniquely placed to cause AD-like
damage
 RELEVANT WORK BY OTHERS (2)

 In HSV1-infected APOE-transgenic mice, viral load in brain is greater

in APOE-ε4 than APOE-ε3 animals (Burgos et al., 2003, 2006;
Bhattacharjee et al, 2008), & APOE alleles differentially affect HSV1
expression (Miller & Federoff, 2007).

 Repeated stress reactivates latent HSV1 in brains of IL-/-mice,

causing widespread lesions. HSV1 damage is greater in brains of
APOE-ε4 than of APOE-ε3 mice, & in older than in young mice (Sawtell,
IHW abstracts, 2010, & http://www.cincinnatichildrens.org/health/subscribe/ped-
insights/November2010/herpes.htm).

 HSV1 infection of primary cultures of embryonic neurons causes

functional changes, AD-like tau phosphorylation, increased Aβ, &
triggers AD-like caspase-3 activation & tau cleavage (Piacentini et al, 2010;
Santana et al., 2011; Zambrano et al., 2008; Lerchundi et al., 2010).
 SUMMARY (1)

 HSV1 DNA resides, and the virus has replicated,

in brain of many elderly people.

 HSV1 DNA in brain and APOE-ε4 confer a strong

risk of AD (cf. APOE-ε4 and herpes labialis).

 HSV1 causes Aβ accumulation & AD-like tau, &

increases levels of relevant enzymes.
 SUMMARY (2)

 HSV1 DNA co-localises with amyloid plaques

in AD brains.

 ACV reduces HSV1-induced Aβ formation

& AD-like tau phosphorylation in cell cultures.

 Vaccination of mice prevents HSV1 latency in

brain.

 APOE determines severity of damage (or

susceptibility to infection) by diverse
pathogens.
 Questions (1)
Holtzman:
Q: In animal models, do particular types of herpes virus
actually accelerate AD-type pathology and its associated
neurodegeneration? If so, what type?

A: We have been trying for 10 years – unsuccessfully - to

get funds for work on HSV1-infected mice. However, a
pilot study showed Aβ deposits in infected WT mouse
brains (Neurosci. Lett., 2007).
 Questions (2)
Holtzman:
Q: In humans, is there evidence in living humans that those
developing AD pathology and neurodegeneration (pre and post-
symptomatically) have differences (or not) in prior or current CNS
infections with different herpes or other viruses?

A: It’s not known if they are more or less prone, though many elderly
normals as well as AD patients harbour HSV1 in brain. As to overt
CNS infections, luckily these are very rare and so the sample numbers
would be very small. HSV1 causes herpes simplex encephalitis, a
very severe acute event affecting 1 to 3 people per million. Previously
most would have died but now ACV/VCV treatment prevents death -
complicating this issue.
 Questions (3)
Anonymous (!):
Is active HSV1 seen more commonly before and during the onset of
typical AD symptoms than in age-matched elderly who did not develop
AD or its precursor, mild cognitive impairment (MCI)? This could be done
by demonstrating links between HSV1 titers and AD biomarkers (e.g. low
cerebrospinal fluid Aβ42/40 ratio, high PIB signal in brain amyloid
imaging)
A: it is unknown whether active HSV1 infection is more common before
and during AD onset. HSV antibody titres (IgG) in serum are not
indicative as they differ little between reactivation and latency episodes
(probably because reactivation frequency is high). In brain, HSV1 can’t be
detected ante mortem, let alone “latent” or “reactivated” virus – except
via CSF taken weekly (hardly feasible)! However, intrathecal antibodies
(IgG) are present in many elderly controls and AD patients, so HSV1 must
have reactivated in brain, perhaps recurrently. Also, see Letenneur et al
(2008) re detection of IgM in serum of those who will develop AD –
indicating viral reactivation in PNS and possibly also in the CNS.
 Questions (4)
Schlossmacher:
Q. Is there any study in the neuropathology literature that has examined brains
of survivors of a monophasic HSV encephalitis to determine whether Aβ and
tau are dysregulated versus survivors of other encephalitides?
A. Nothing relevant has been published, but with so much damage it would
probably be hard to detect.

Q. Do patients who are on chronic antiviral treatment with Zovirax (e.g., for
prevention of recurrence of genital herpes) have a lesser risk of AD
development or AD progression?
A. We have been unable to find any data on this (or on HSE survivors) but
would expect little effect in brain as absorption of ACV is poor (the biodrug
VCV is much better absorbed).

Q. Do patients with classical herpes simplex type 1 infections carry tau-

positive tangles in their ganglion Gasseri of cranial nerve 5 at postmortem
examination?
A. Nothing published. NB, It is difficult to obtain human trigeminal ganglia.
 HSV1 DNA PRESENCE IN
PLAQUES IS NOT ARTEFACTUAL
Might pre-existing plaques attract & engulf viral DNA from other sites? No: viral DNA is large
(so movement is unlikely), & does not persist extra-cellularly.

Might microglia engulf HSV1 DNA or be infected & then carry the viral DNA to plaques (to
which they are attracted)? No: infection of microglia in brain is rare (Esiri et al. 1995).

Might pre-existing plaques reactivate HSV1 via inflammatory effects? No: that would require
all the 80-90% of plaques with viral DNA to have been located next to latently infected cells.

Might viral DNA be stuck to plaques? No: they contain no cell DNA (Ginsberg et al. 1997) though its
amount is vastly greater - & 10-20% contain no viral DNA at all.

Also, average plaque size is ~50 um diameter but sections are only 7 um thick; thus, most
sections display plaque interiors - & all reveal viral DNA throughout.
PROTECTION OF MICE FROM HSV1 LATENCY IN BRAIN
BY VACCINATION WITH HSV1 GLYCOPROTEINS (Neurobiol.
Aging., 2001)

45

41 % (N=39)
40

35

30

25

20

15

10
7% (N=41)

5
Proportion of m ice with latent infection in brain (% )
0 % (N=9)
0

HSV1 infection + PBS HSV1 infection + ISCOM vaccine ISCOM Vaccine

PROPORTION OF BRAINS HARBOURING
HERPESVIRUSES (J. Pathol. 2002)
80
N = 61
N = 50 AD
70
Normal
N = 48
60

50

N = 35
40
N = 45
N = 29

30
Virus-positive (%)

N = 35
20

N = 53

10

0
HSV1 HSV2 Virus CMV HHV6

* OR = 3.9, 95%CI 1.54-9.64

 EFFECT OF APOE ON OUTCOME OF
INFECTION
Disease Agent Risk Protective
Herpes labialis HSV1 APOE-ε4 (p<0.0001)

Genital herpes HSV2 APOE-ε4 (p=0.001)

Herpes simplex encephalitis HSV1 APOE-ε2 (p<0.0001)

HCV-induced liver disease HCV APOE-ε4d(p=0.006)

Post-herpetic neuralgia VZV APOE-ε3a(p=0.0007) APOE-ε4b(p=0.006)

APOE-ε2ε3c(p=0.02)
Shingles VZV APOE-ε4ε4a(p=0.003)
Infectious mononucleosis EBV APOE-ε4ε4b(p=0.002)

Malaria P. falciparum APOE-ε2ε2 (p=0.008)

a. whole group & females, b. females only, c. whole group only, d. whole group & males.
Conclusion: APOE is a major factor in determining outcome of infection. Allele
involved probably depends on cell type as well as pathogen type.