HERPES SIMPLEX VIRUS TYPE 1, APOEε4 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
(Confirmed by Koelle et al., 2010) (Lancet, 1997; Alz Repts. 1998).

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) 20X

Plaques (Thioflavin S) 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 DNAamyloid 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 HSV1INFECTED CELLS
HSV1-infected no ACV HSV1-infected 50uM ACV Uninfected No ACV Uninfected 50uM ACV

Aβ (Aβ1-42)

P-tau (pS214)

HSV1

HSV1-INDUCED CHANGES IN Aβ & P-tau, & EFFECT OF ACV
5000 4500 4000 3500 3000 2500 2000 1500 1000 500 0 0uM ACV 100uM ACV 0uM ACV Uninfected 100uM ACV Uninfected p<0.001

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

6000 5000 p<0.001 4000 3000 2000 1000 0 0uM ACV HSV1 - infected (10pfu/cell) 100uM ACV HSV1 -infected (10pfu/cell) 0uM ACV Uninfected 100uM ACV Uninfected

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

Curtis Dobson Ann Cookson Suzanne Shipley Tom Lloyd Woan-Ru Lin Gordon Jamieson Dazhuang Shang
Acknowledgements include Alzheimer’s Society; Henry Smith Charity.

COLLABORATORS Andrew Mee Gordon Wilcock Stacey Efstathiou Margaret Esiri Seth Love Brian Faragher Roy Jennings Marc Combrinck Bob Cooper Paul Klapper Chris Preston 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
2008). (Letenneur et al.

 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/pedinsights/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 postsymptomatically) 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 taupositive 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

0 % (N=9) ISCOM Vaccine

HSV1 infection + PBS

HSV1 infection + ISCOM vaccine

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

50 N = 35 N = 45 N = 29

40

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 Herpes labialis Genital herpes Herpes simplex encephalitis HCV-induced liver disease Post-herpetic neuralgia Shingles Infectious mononucleosis Malaria Agent HSV1 HSV2 HSV1 HCV VZV VZV EBV
P. falciparum

Risk APOE-ε4 (p<0.0001) APOE-ε4 (p=0.001) APOE-ε2 (p<0.0001)

Protective

APOE-ε4d(p=0.006) APOE-ε3a(p=0.0007) APOE-ε4ε4a(p=0.003) APOE-ε4ε4b(p=0.002) APOE-ε2ε2 (p=0.008) APOE-ε4b(p=0.006) APOE-ε2ε3c(p=0.02)

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.

Sign up to vote on this title
UsefulNot useful