Reduced Pathology and Improved Behavioral Performance in Alzheimer's Disease Mice Vaccinated With HSV Amplicons Expressing Amyloid-beta and Interleukin-4

Reduced Pathology and Improved BehavioralPerformance in Alzheimer’s Disease MiceVaccinated With HSV Amplicons ExpressingAmyloid-

β

and Interleukin-4

Maria E Frazer

1

,

Jennifer E Hughes

1

,

Michael A Mastrangelo

1

,

Jennifer L Tibbens

1

,

Howard J Federoff

1–3,

*

and

William J

Bowers

1–3

1

Center or Aging and Developmental Biology, University o Rochester School o Medicine and Dentistry, Rochester, New York, USA;

2

Department o Neurology, University o Rochester School o Medicine and Dentistry, Rochester, New York, USA;

3

Department o Microbiology and Immunology, University o Rochester School o Medicine and Dentistry, Rochester, New York, USA

Immunotherapeutics designed to dissolve existing amyloidplaques or to interrupt amyloid-

β

(A

β

) accumulation maybe easible or treatment and/or prevention o Alzheimer’sdisease (AD). “Shaping” the immune responses elicitedagainst A

β

is requisite toward generating an ecacious andsae outcome; this can be achieved by minimizing the pos-sibility o deleterious infammatory reactions in the brain asobserved in clinical testing o A

β

peptide/adjuvant-basedmodalities. Herpes simplex virus (HSV)-based ampliconscan coexpress multiple antigens and/or immunomodula-tory genes due to their large genetic size capacity, thereby acilitating antigen-specic immune response shaping. We have constructed an amplicon (HSV

IE

A

β

CMV

IL-4) thatco-delivers A

β

1–42

with interleukin-4 (IL-4), a cytokine thatpromotes the generation o Th2-like T-cell responses,which are avored in the setting o AD immunotherapy.Triple-transgenic AD (3xTg-AD) mice, which progressivelydevelop both amyloid and neurobrillary tangle pathol-ogy, were vaccinated thrice with HSV

IE

A

β

CMV

IL-4, or a seto control amplicon vectors. Increased Th2-related, A

β

-specic antibodies, improved learning and unctioningo memory, and prevention o AD-related amyloid andtau pathological progression were observed signicantlymore in the HSV

IE

A

β

CMV

IL-4 vaccinated mice as comparedto the other experimental groups. Our study underscoresthe potential o A

β

immunotherapy or AD and highlightsthe potency o amplicons in acilitating the immuneresponse modulation to a disease-relevant antigen.

Received 20 September 2007; accepted 14 February 2008; published online 11 March 2008. doi:10.1038/mt.2008.39

IntroductIon

Alzheimer’s disease (AD) is a progressive neurodegenerativedisorder commonly associated with dementia and a decline inperormance o normal age-related activities. As the diseaseprogresses, cognitive decline in episodic and semantic memory, aswell as decision making, judgment, language, and spatial orienta-tion become prooundly evident (reviewed in re. 1). Hallmark ea-tures o AD pathology include, but are not limited to, extracellularplaques o the peptide amyloid-

β

(A

β

) and hyperphosphorylationo tau protein, presenting as intraneuronal neurobrillary tangles,that proceeds in a spatial and temporal pattern with diminishingsynaptic density and eventually leads to marked neuronal loss.

2,3

Te societal burden imparted by this debilitating disease callsor the derivation and testing o new natural history modiyingtherapeutic approaches. Currently available therapeutic agentsemployed in treating AD are designed to manage the cognitive andemotional symptoms o the disease (reviewed in re. 4). Tese inter- ventions, however, provide only transient symptomatic benet, asthey are not designed to specically target pathologic mechanismsunderlying the genesis o AD. Te amyloid cascade hypothesis sug-gests modalities which will abrogate the proteolytic generation o A

β

peptides, prevent A

β

brillogenesis, and/or amyloid depositionthat would in turn impact the onset and/or severity o AD.

5

A promising, yet inherently complex, means o inhibitingA

β

brillogenesis and/or deposition relates to the use o the hostimmune system to mount specic immune responses againstthe sel-peptide, A

β

. Te ndings rom a variety o immune-based strategies speak to the promise o this approach,

6–10

butalso reveal the potential or morbid complications o AD immu-notherapy.

11–13

Given these mixed clinical results, A

β

-directedimmunotherapy warrants urther study particularly i an opti-mal immune response/antibody can be developed. Helper -cellunction, normally required or an eective antibody response, isbelieved to exclusively require T2-biased responses in the set-ting o AD immunotherapy, as a strong T1 response carries therisk o inducing a local inammatory response to the amyloidprecursor protein (APP) antigen should the A

β

-primed  cellspenetrate the blood/brain barrier. Although at present untested in

*Current address

: Oce o the Executive Vice President or Health Sciences, Georgetown University Medical Center, Washington, District o Columbia, USA

Correspondence:

William J. Bowers, Department o Neurology, Center or Aging and Developmental Biology, University o Rochester School o Medicine and Dentistry, 601 Elmwood Avenue, Box 645, Rochester, New York 14642, USA. E-mail :william_bowers@urmc.rochester.edu

original article

Molecular Therapy

vol. 16 no. 5, 845–853 may 2008

845

846

www.moleculartherapy.org

vol. 16 no. 5 may 2008

Immunomodulating HSV Amplicons as AD Vaccines

human clinical trials, gene-based vaccination technology due toits inherent versatility may allow or greater precision in antigenpresentation and immunomodulation that could lead to saer andmore ecacious vaccines or AD.

14,15

Given its ease o manipulation, absence o immunosuppres-sive viral genes, ability to eciently transduce antigen present-ing cells, and large transgene capacity, the herpes simplex virus(HSV) amplicon represents a well-positioned platorm on whichto build an A

β

-directed AD vaccine.

16–19

Our laboratory has previ-ously employed amplicons to elicit distinctive immune responsesagainst A

β

using a used molecular adjuvant approach.

15

Herein,we describe the derivation o an A

β

-expressing amplicon vac-cine (HSV

IE

A

β

CMV

IL-4) that co-delivers interleukin-4 (IL-4), acytokine expressed by T2  cells and demonstrated to promoteT2-biased immune responses.

20

HSV

IE

A

β

CMV

IL-4-mediated vaccination o triple-transgenic AD (3xg-AD) mice,

21,22

whichdevelop both amyloid deposition and tau-related dysunctionthat mirrors the spatial and temporal progression o pathology observed in human AD, resulted in suppression o the AD-relatedpathological hallmark appearance and as well as a concomitantimprovement in learning and unctioning o memory.

results

Tree HSV amplicon vectors were constructed(

Figure 1a

): pHS-V

IE

1

CMV

2 served as a no-insert control and harbored only theHSV origin o replication and packaging signal along with empty HSV immediate-early 4/5 gene promoter and cytomegalovirus(CMV) immediate-early promoter-driven transcription cassettes;pHSV

IE

A

β

CMV

2 contained the DNA sequence or human A

β

1–42

peptide under the transcriptional control o the HSV immediate-early 4/5 gene promoter; and pHSV

IE

A

β

CMV

IL-4 harbored A

β

1–42

downstream o the HSV IE4/5 gene promoter and murine IL-4under the transcriptional control o the CMV promoter. estingo pHSV

IE

A

β

CMV

IL-4

in vitro

by transection o baby hamster kid-ney cells and enzyme-linked immunosorbent assay analysis o cellculture supernatants demonstrated that this amplicon expressedhigh levels o secreted murine IL-4 (

Figure 1b

). Te integrity o the A

β

1–42

expression cassette was conrmed previously.

15

Te amplicon plasmids were subsequently packaged into helper virus–ree virus stocks and titered using previously describedmethodologies.

23,24

wo-month-old male 3xg-AD mice each received threeinjections o 1 × 10

6

transduction units o a designated amplicon

IL-4 ELISA

P B S

0100200300500600700800400

m I L - 4 c o n c e n t r a t i o n ( p g / m l )

b

2Vaccination #1(SQ)42 weeksBlood drawBlood draw6

Blood draw9ICC w/stereologyAge(months)Barnes maze(Baseline)2 weeksBarnes maze11Barnes maze

Experimental timeline

c

MCSA

�

42pApAIE4/5CMVori“a”

MCSpApAIE4/5CMVori“a”MCS

HSV amplicon plasmids

a

IL-4pApAIE4/5CMVori“a”A

�

422 weekspHSV

IE

1

CMV

2pHSV

IE

A

�

CMV

2pHSV

IE

A

�

CMV

IL-4Transfected amplicon plasmid

p H S V

I E

A

�

C M V

I L - 4 p H S V

I E

A

�

C M V

2 p H S V

I E

1

C M V

2

3Vaccination #2(SQ)Vaccination #3(SQ)

Fi 1 shmai pai    ampi v,

in vitro

fmai  ampi-mia mi ii-4(Il-4) xpi, a  i.

(

a

) Three kanamycin-resistant herpes simplex virus (HSV) amplicon plasmids were constructed: one thatserved as an empty vector control (pHSV

IE

1

CMV

2) with HSV origin o replication (ori) and HSV packaging signal (“a”), a second (pHSV

IE

A

β

CMV

2) thatexpressed the A

β

1–42

peptide derived rom human amyloid precursor protein under the transcriptional control o the HSV immediate-early (IE) 4/5gene promoter and SV40 polyadenylation signal (pA), and a third (pHSV

IE

A

β

CMV

IL-4) that expressed A

β

1–42

via the immediate-early 4/5 promoter/SV40pA transcription unit and murine IL-4 under the separate transcriptional control o the cytomegalovirus (CMV) immediate-early promoter and bovinegrowth hormone polyadenylation signal. All amplicons were packaged using a previously described helper virus–ree method.

23

(

b

) Each ampliconplasmid was transiently transected into baby hamster kidney cells and culture supernatants were analyzed by enzyme-linked immunosorbent assay(ELISA) to assess murine IL-4 expression (pg/ml) rom the pHSV

IE

A

β

CMV

IL-4 amplicon (

N

= 4/experimental group). A phosphate-buered saline (PBS)group served as a no-vector control condition. (

c

) A Barnes maze behavioral assessment was perormed to determine baseline learning and memory unctioning at 2 months o age. Each packaged vector (1 × 10

6

transduction units) was delivered subcutaneously (SQ) to a randomized cohort o male3xTg-AD mice

22

(

N

= 6/experimental group). Amplicons were administered to each animal thrice, and humoral assessments were perormed 2 weeksater each vaccination. An intermediate Barnes maze assessment was perormed at 6 months o age. Antibody isotype analysis was perormed onsera obtained at the 9-month post-initial vaccination time point. Vaccinated mice were killed at 11 months o age at which time endpoint behavioral,histological, and stereological analyses were perormed. 3xTg-AD mice, triple-transgenic Alzheimer’s disease mice; ICC, immunocytochemistry.

Molecular Terapy

vol. 16 no. 5 may 2008

847

Immunomodulating HSV Amplicons as AD Vaccines

vaccine. Te timing o vaccinations, blood draws, behavioralassessments by Barnes maze, and immunohistochemical analysesare depicted schematically in

Figure 1c

.For these experiments,we chose to allow primed mice to “rest” or 120 days beore thenal boost. Since HSV-1 amplicon vectors induce high and tran-sient levels o transgene (antigen) expression,

25

we predicted thatthe overwhelming majority o A

β

-specic  and B cells present atthe time o the nal boost would consist o memory immune cells(reviewed by re. 26).

HsV

Ie

A

β

cMV

Il-4 vaia 3xt-A mi xhibiha v  A

β

1–42

pif aibi haxhibi th2 bia

Afer the rst and second set o immunizations, mice receivingHSV

IE

A

β

CMV

2, HSV

IE

A

β

CMV

IL-4, or HSV

IE

1

CMV

2 exhibited nosignicant dierences in anti-A

β

antibody levels(

Figure 2a

).Following the nal immunization, however, HSV

IE

A

β

CMV

IL-4 vaccinated mice harbored signicantly higher levels o circulatingA

β

-specic antibodies than 3xg-AD mice receiving eitherHSV

IE

A

β

CMV

2 or the empty vector control HSV

IE

1

CMV

2 amplicon,indicating that co-delivery o IL-4 during amplicon vaccinationaugmented the overall A

β

-directed humoral response (

Figure 2a

).Te delivery o HSV

IE

A

β

CMV

2 amplicon was surprisingly unableto break tolerance in 3xg-AD mice, as levels o

α

-A

β

antibodiesdid not increase over those ound in mice vaccinated with theHSV

IE

1

CMV

2 control vector. It is also o note that HSV

IE

1

CMV

2- vaccinated mice did exhibit detectable anti-A

β

antibodies. Tisis not surprising since the age-related development o anti-A

β

antibodies in nonvaccinated AD mice has been describedpreviously.

27

Serum samples obtained at the 9-month timepoint were urther examined to isotype the anti-A

β

antibod-ies elaborated as a result o amplicon vaccination. Sera romHSV

IE

A

β

CMV

IL-4-immunized 3xg-AD mice possessed a higherrepresentation o anti-A

β

specic antibodies o the T2-derivedIgG1 isotype and suppressed levels o T1-related IgG2b, IgG2c,and IgG3 isotypes, indicating that the coexpression o IL-4 led toa T2-like immune response bias.

HsV

Ie

A

β

cMV

Il-4 vaia mi xhibi impvpma  h Ba maz

Long-term spatial memory, which is adversely aected in AD andis partially dependent upon hippocampal circuitry unction, wasassessed in 3xg-AD mice using the Barnes maze spatial memory paradigm.

28

Te mice need to learn and remember the relation-ship among distal spatial cues to navigate to the escape box. Micewere tested in the Barnes maze at 2 months o age to establishbaseline data and at 6 and 11 months o age to gauge the eectsthat amplicon-mediated vaccination may have on the various out-put measures o the Barnes maze paradigm. Te ollowing mea-sures were assessed: distance traveled by each mouse to reach thegoal box, errors made during the search or the correct escapehole, and the time required by the mouse to enter the escape box(latency). I the A

β

-directed amplicon vaccines imparted theintended humoral response, we would expect to observe shorterdistances traveled, ewer errors made, and shorter latency times.However, should amplicon-based A

β

vaccination elicit deleteri-ous immune responses, such adverse events may aect cogni-tive unctioning, where HSV

IE

A

β

CMV

2 and/or HSV

IE

A

β

CMV

IL-4injected mice perorm less optimally as compared to HSV

IE

1

CMV

2control amplicon–vaccinated mice.Measurements o distance, errors, and latency amongst theamplicon-immunized groups were highly similar at the 2-monthbaseline time point and at the 6-month midpoint o the study,when AD-related pathologies are minimally detectable in 3xg-AD mice(

Figure 3

;res. 21,22). At the 11-month time point, just prior to being killed, HSV

IE

A

β

CMV

IL-4 vaccinated miceexhibited improved perormance in each o the Barnes mazeparameters assessed. Briey, HSV

IE

A

β

CMV

IL-4 mice traveledless distance than HSV

IE

1

CMV

2 vaccinated mice (

Figure 3a

;

P

<0.05), made ewer errors(

Figure 3b

)and completed the mazewith lower latency times(

Figure 3c

)than HSV

IE

1

CMV

2 (

P

< 0.05)and HSV

IE

A

β

CMV

2 (

P

< 0.05) vaccinated mice. In aggregate, thesedata indicate that HSV

IE

A

β

CMV

IL-4 vaccination improved spatiallearning and unctioning o memory in 3xg-AD mice presum-ably because o preventing A

β

accumulation in the brain and

A

�

42-specific Isotype ELISA at 9 months of age

b

00.10.20.30.40.50.60.70.80.9

I g G 1

I g G 2 a

I g G 2 b

I g G 3

I g A

I g M

k

l

I g G 2 c

*****************************

C o r r e c t e d a b s o r b a n c e a t 4 5 0 n m

A

�

42-specific ELISA

a

**

E n d p o i n t t i t e r

HSV

IE

1

CMV

2HSV

IE

A

�

CMV

2HSV

IE

A

�

CMV

IL-4*Isotypes05001,0001,5002,0002,500239Age (months)

Fi 2 eiiai  iiiv hma p i hpimpx vi (HsV) ampi v–vaia 3xt-Ad mi.

(

a

) Helper virus–ree HSV

IE

A

β

CMV

2 (black bars), HSV

IE

A

β

CMV

IL-4 (grey bars),and HSV

IE

1

CMV

2 (open bars) were delivered subcutaneously thrice to3xTg-AD mice beginning at 2 months o age (1 × 10

6

transduction units/vaccination). Serum was obtained rom each vaccinated mouse accord-ing to the schema illustrated in

Figure 1

and serum samples were an-alyzed or levels o antibodies binding specically to the A

β

1–42

peptidein quadruplicate by enzyme-linked immunosorbent assay (ELISA). Levelso A

β

-specic antibodies arising rom each vaccination were correctedusing serum isolated rom control mice, and are expressed as endpointtiters. (

b

) Isotypes o

α

-A

β

1–42

antibodies were determined by ELISA usingsera obtained at the 9-month time point rom vaccinated 3xTg-ADmice. Levels o A

β

-specic antibody isotypes arising rom each vaccina-tion were corrected using serum isolated rom control mice, and areexpressed as “corrected absorbance at 450 nm”. Error bars represent SD.*

P

< 0.05, **

P

< 0.01, and ***

P

< 0.001 as determined by two-wayanalysis o variance with Bonerroni

post-hoc

analysis. CMV, cytomega-lovirus; IE, immediate-early; Ig, immunoglobulin; IL-4, interleukin-4;3x Tg-AD mice, triple-transgenic Alzheimer’s disease mice.