I. Review of Literature VII. Discussion...I. Review of Literature VII. Discussion VIII. Conclusions...

I. Review of Literature VII. Discussion

VIII. Conclusions

X. Bibliography• Aubert, Martin e,and JohnA.Blaho. "Modu lationofApop tosisdu rin gHerpesSimp lexViru s In fection inHuman Cel ls."Microb esan d Infectio n3.10 (2 001 ):8 59-6 6.Web.• Au slander,Beth A., Fran kM .B iro ,andSu san L.Ro sen thal . "Gen ital Herpes inAdo lescen ts."Semin a rsin P edia tric Infectiou sDisea ses (20 05 ):24 -30.Web .• Bernard ,Marie-C lo tild e,Véronique Barban ,Fab rine P radezyn ski , Aymeric DeMontfo rt,RobertRyal l ,C atherin eCai l let, and P atriciaLondono -Hayes. "Immunogen ici ty,

P ro tectiveEfficacy,and Non -Rep l icativeStatu so fth eHSV-2 Vaccin eCand id ateHSV529 in M iceand Gu ineaP igs."P LoS ONE1 0.4 (201 5):n.p ag.Web .• Bern stein ,David I . , Jul ieD.Earwood ,Fern ando J.B ravo,GaryH.Cohen ,Ro selyn J. Ei senberg, Jenni ferR.Clark, JeffreyFairman ,and RhondaD.Card in. "Effectso fHerpes

Simp lexVi ru sType2 Glycop rotein Vaccin esandCLDC Ad ju van tonGen ital Herpes In fection in th eGu ineaP ig."Vaccin e 29 .1 1 (201 1):207 1-0 78.Scien ceDirect. Web .<h ttp ://www.scien ced irect.com/scien ce/article/p i i /S0 2 6 44 10X11 00 02 11>.

• Bern stein ,David I . ,Nicho lasFarley,Fern ando J. B ravo , Jul ieEarwood ,Mon icaMcneal , JeffFai rman ,and RhondaCard in. "TheAd juvan tCLDC IncreasesP ro tectiono faHerpesSimp lexType2 Glycop ro teinDVaccine in Gu in eaP igs."Vaccine 2 8.21 (2 01 0):3 74 8-75 3.ScienceDirect. Web .<h ttp ://www.scien ced irect.com/scien ce/article/p i i /S0 2 6 44 10X09 01 54 24>.

• B ickel , M . "TheRo leo fIn terleu kin -8 in In flammation and Mechan ismsofRegu lation ."Journal o fP eriodon tology (1 99 3):n .p ag.Web .• Brigh t, Helen ,David Lu ciaPerez,C lareCh risty,Pau l Co ckle,JimE.Eyles, DaisyHammond,Tan siK hodai , Su sanneLang,K ateWest, and P eterT.Loudon."TheEfficacyo f

HSV-2 Vaccin esBased onGDandGB IsEnhan ced b y theAdd itionof ICP2 7."Va ccine 30 .52 (20 12 ):75 29 -535 .Scien ceDirect. Web .<h ttp ://www.scien ced irect.com/scien ce/article/p i i /S0 2 6 44 10X12 01 49 22>.

• Brown ,ZaneA. "Effecto fSero logicStatu sandCesarean Del iveryon Tran smissionRatesofHerpesSimplexVi ru sFromMother to In fant." Jama2 89 .2 (2 00 3):2 03 .Web.• Conno l ly, Sarah A., Jul iaO. Jackson ,Theodo reS.Jardetzky,andR ich ard Longnecker. "Fu sin gStructu reandFunction :AStructu ral V iewof theHerpesvi ru sEn try

Mach in ery."Na tu reReviewsMicro biolog yNatRevM icro 9.5 (201 1):369 -81 .Web .• Co sta,X .Da,M.F.Kramer, J. Zhu ,M .A.B ro ckman ,andD.M.Knip e."Con struction,P heno typ icAnalysis, and Immunogen ici tyofa UL5/UL2 9Doub leDeletionMu tantof

HerpesSimp lexVi ru s2 ." Journ al ofVi rolog y 74 .1 7 (20 00):79 63-9 71 .Web .• Co rey, Lawren ce,And riaG.M .Langenberg,RhodaAshley,Ro seE.Seku lo vich ,Al len E. Izu, JrJohn M .Douglas, H.HunterHandsfield,Terri Warren ,Li saMarr,Stephen

Tyrin g,R ich ard Dicarlo ,Adao ra A.Ad imora,PeterLeone,Co rnel iaL. Dekker,RaeLyn Burke,Wai P in gLeong,StephenE.Strau s,and Fo rTheChironHsv Vaccin eStud yGroup . "Recomb inan tGlycopro teinVaccin e for theP reven tion ofGen ital HSV-2 Infection."Jama 2 82.4 (1 999 ).Web .

• Cunn in gham,Anthony L., Russel l J. Diefenbach ,Mon icaMiranda-Saksena,Lid i ja Bo snjak,MinKim,Cheryl Jones,and Mark W.Douglas. "TheCycleofHuman HerpesSimp lexVi ru s In fection:Vi rusTran spo rtand ImmuneContro l."Th eJo urn al o fIn fectio usDiseases J INFECTDIS (20 06 ).Web .

• Deshmane,Satish L., SergeyK remlev,Shoh reh Amini , andBassel E.Sawaya. "MonocyteChemoattractan tP ro tein-1 (MCP-1 ):AnOverview." Jou rnal of Interferon&Cyto kin eResearch 2 9 .6 (2 009 ):3 13-2 6.Web

• Diaz,Fern ando M .,and David M .Kn ipe. "Pro tection fromGen ital HerpesDisease,Sero conversion and Laten t In fection inaNon-leth al Mu rin eGen ital In fection Model b y Immun ization with anHSV-2 Rep l ication-defectiveMutan tVi rus."Virolo gy 48 8 (20 16):61 -6 7.Web .

• "DermNet NewZealand ."Herp esSimp lex. N.p ., Oct.20 15 .Web .• Dudek,Tim,and David M .K nipe. "Repl ication -defectiveVi ru sesasVaccin esand Vaccin eVecto rs."Vi rology3 4 4.1 (20 06):230 -39 .Web .• Field s, Bernard N. "Heres Simp lexVi ru ses."FieldsVi rolo gy. 6thed .P hi ladelphia,Pa. [u.a.:Lip pinco ttWi ll iams&Wi lkin s, 2 00 1.P rin t.• Freeman ,EstherE, Helen AWeiss, Ju d ith R Glynn,P amelaLC ross, JamesAWh itwo rth,and R ichard JHayes. "HerpesSimp lexViru s2 In fection IncreasesHIVAcquisition

in Men and Women :SystematicReviewand Meta-analysiso fLongi tudin al Studies."Aid s(2 00 6):7 3-83 .Web .• Hosh ino ,Y., S. K .Dalai , K .Wang,L. Pesn icak,T.Y. Lau ,D.M.Kn ip e, J. I. Cohen ,and S.E.Strau s."ComparativeEfficacyand Immunogen ici tyo fRepl ication -Defective,

Recomb in an tGlycop rotein ,andDNAVaccin es forHerpesSimplexVi rus2 In fection s in M iceand Gu ineaP igs."Journal o fV i rology7 9.7 (20 05 ):45 54 .Web .• "Gen ital Herpes."Th eAmerica n Co l leg eofObstetrician sand Gyn eco log ists. Web .<h ttp://www.acog.o rg/-/med ia/Fo r-

P atien ts/faq0 5 4 .pd f?dmc=1&ts=2 01 510 13 T1 236 21 31 97>.• "Gen ital Herpes - CDC FactSheet."Cen ters forDisea seC on trol a nd P reven tion .C enters fo rDiseaseContro landPreven tion,14Oct.2 01 5.Web .• Grauwet,K o rneel , C laud iaCan ton i, MonicaParod i, AndreaDeMaria, BertDevriendt,Dan ielaPende,Lo ren zoMo retta,Massimo Vi tale,andHerman W.Favo reel .

"Modu lation o fCD1 12 b y theAlphaherpesvi ru s GDPro teinSupp ressesDNAM-1–dependen tNKCel l -med iated Lysiso f Infected Cel ls."Pro ceed ing so fth eNation al Aca d emyo fScien cesP ro cNatl Aca d SciUSA (201 4):161 18 -612 3.Print.

• Han , Jin -Young,DerekD.Slo an ,Martin eAubert, SaraA.Mi ller, ChungH.Dang,andKei th R .Jerome."Apopto sisandAntigen Recep to rFun ction inTandBCel ls fol lowingExpo su re to HerpesSimp lexVi rus."Virolo gy 35 9.2 (20 07 ):25 3-63 .Web.

• John son ,D.C., T. W.Wisner,and C .C .Wrigh t."HerpesSimp lexVi rusGlycoprotein sGB and GDFun ction inaRedundan tFash ion ToPromo teSecondaryEn velopmen t."Jo u rn a l ofV iro lo gy 8 5.10 (20 11 ):49 10 -926 .Web .

• John ston ,Ch ristine,David M .Koel le, and AnnaWald . "HSV-2:In P ursu itofaVaccin e." Jou rna lo fC linica l Investig ation J. Cl in . Invest. 1 21.1 2 (201 1):460 0-6 09.Th eJo u rn a l ofCl inical In vestig ation .Web .<http ://www.jci .org/articles/view/5 7 1 48>.

• John ston ,Ch ristine,Sami L. Gottl ieb ,and AnnaWald ."Statu so fVaccineResearch and Developmen tofVaccin es forHerpesSimp lex Viru s."Vaccin e34 .2 6 (20 16):2948-9 5 2 .Web .

• Kuo ,Ti ffan y,Ch ristineWang,Tin aBadakh shan ,Sravya Ch i lu ku ri, and Lbach ir Benmohamed . "TheChal len gesandOpportuni ties fo r th eDevelopmen to faT-cel l Epitope-b ased HerpesSimp lexVaccin e."Va ccine 3 2.50 (2 01 4):6 73 3-74 5.Scien ceDirect. Web .<http ://www.scien ced irect.com/scien ce/article/p i i /S02 64 41 0X140 13 59 0>.

• Kwon ,Byungsu k,Byung-SYoun,andByoung SKwon."Fun ction so fNewly Iden ti fied Memberso fth eTumorNecro sisFacto rRecep to r/l igandSuperfami l ies inLymphocytes."Cu rren tOp inion in Immuno lo gy:34 0-45 .Web .

• La,Soo j in . "HerpesSimp lexVi rusType1 Glycopro tein D Inh ib itsT-cel l Pro li feration ."Molecu lesan dCel l s1 4.3 (20 02 ):39 8-4 03.PubMed. Web .<h ttp ://www.n cb i .n lm.n ih.go v/pubmed /1 25 21 30 3>.

• Lai rson ,David R ."P reven tionofHerpesSimp lexViru sEyeDisease."Arch iveso fOphthalmo logy12 1.1 (20 03 ):10 8.Web .• Lazear,Eric, J. Ch arlesWh itbeck,Yi Zuo,AndreaCarfí , GaryH.Cohen ,Roselyn J.Ei senberg,and ClaudeKrummenacher. "Induction o fCon formational Changesat theN-

terminu so fHerpesSimp lexViru sGlycopro teinDuponBinding to HVEMandNectin-1 ."Viro logy 4 48 (2 014 ):1 85-9 5.ScienceDirect. Web .h ttp ://www.scien ced irect.com/scien ce/article/p i i /S0 0 42 68 221 30 05 79 5

• Lin t, Al l i sonL.Van ,Ernesto To rres-Lopez,and David M .Kn ipe. "Immunization with aRep lication -defectiveHerpesSimp lexVi ru s2Mu tantRedu cesHerpesSimplexVi rus1 In fection andPreven tsOcu larDisease."Virolo gy 36 8.2 (20 07 ):22 7-31 .Web.

• Looker,K ath arin e. "AnEstimateo f theGlobal P revalen ceand In cid en ceofHerpesSimplexVi ru sType2 Infection ."Bul letinof theWo rldHea l thOrga nization Bul l WorldHea l th Org a n (20 08 ):80 5-1 2.Web .

• Lopez-Med in a,Eduardo, Jo sephB .Can tey,andPab lo J.Sán chez. "TheMo rtal ityo fNeonatal HerpesSimp lexVi rus Infection."The Jo urn al o fP ed ia trics 1 66.6 (20 15 ):1 5 2 9 -5 32 .Scien ceDirect. Web .<h ttp ://www.scien ced irect.com/scien ce/article/p i i/S0 0 22 347 61 50 025 16 >.

• Lö vheim,Hugo , Jonathan Gi l tho rpe,Ro lfAdol fsson,Lars-Gö ranNi lsson ,andFred rikElgh . "Reactivated HerpesSimp lex In fection Increases th eR iskOf Alzheimer' s d isease."Alzh eimer' s&Demen tia1 1.6 (20 15 ):59 3-99 .Scien ceDirect. Web .<h ttp ://www.scien ced irect.com/scien ce/article/p i i/S1 5 525 26 01 40 242 12 >.

• Owusu -Edu sei , Kwame,Elain eW.Flagg,and ThomasL.Gift. "Ho sp ital i zation Costp erCaseo fNeonatal HerpesSimp lexVi ru sIn fectionFromClaimsData." Journ al ofP ed ia tricNu rsing3 0.2 (20 15):34 6-52 .Scien ceDirect. Web .<h ttp://www.scien ced irect.com/scien ce/article/p i i /S0 88 25 96 31 400 23 83 >.

• Petro ,Ch ristopher,Pab loAGonzález, Natal iaCheshenko,Thomas Jandl , Nazan inKhajouein ejad ,Angèle Bénard ,Mayami Sengupta,BetsyCHero ld ,andWil l iamR Jacob s. "HerpesSimp lexType2 Vi rusDeleted in Glycoprotein DPro tectsagain stVagin al , Skin andNeural Disease."ELi fe (2 0 15 ).ELi fe. Web .1 6Sep t.2 01 5.<h ttp ://el i fescien ces.o rg/con ten t/4 /e0 6 05 4>.

• Reszka,Natal ia J. , Timo th yDudek,and David M.K nipe. "Con struction and P ropertiesofaHerpesSimplexViru s2Dl5 -2 9Vaccin eCandidateStrain En cod ingan HSV-1Virion Ho stShu toffP rotein ."Vaccine (2 01 0):2 75 4-76 2.ScienceDirect. Web .<h ttp ://www.scien ced irect.com/scien ce/article/p i i /S0 264 41 0X100 00 62 9>.

• Ro th ,K ri sty, V icto rH.Ferrei ra, andCharuK au shic."HSV-2Vaccin e:Curren tStateand Insigh tsinto Developmen tofaVaccin eThatTargetsGen ital Muco sal Pro tection ."Micro b ia l Path og en esis5 8 (20 13):45 -5 4.PubMed . Web .<h ttp://www.ncb i .n lm.nih.go v/pubmed /2 3 159 48 5>.

• Royer,HeatherRhea,El i zabeth C.Falk, and Su san M.Heid rich ."Gen ital HerpesBel iefs:Imp lications forSexual Heal th ." Jou rnal ofPed iatrica nd Ado lescen tGyn eco logy2 6 .2 (2 01 3):1 09 -16 .Scien ceDirect. Web .<h ttp://www.scien ced irect.com/scien ce/article/p i i /S1 08 33 188 12 00 242 2?np=y>.

• Saw,Wan Tin g,ZeneMatsuda,Roselyn J. Ei senberg,GaryH.Cohen ,and Do in a Atanasiu ."UsingaSp l it Lu ci feraseAssay (SLA) to Measu re th eKineticso fCel l–cel l FusionMed iated b yHerpesSimp lexVi rusGlycop rotein s."Metho ds. Scien ceDirect. Web.27 Sep t.20 15.<h ttp ://www.scien ced irect.com/scien ce/article/p i i /S1 0 4 62 023 15 00 227 3>.

• Szenbo rn , Leszek,Barb araK raszewska-Głomba,Teresa Jackowska,Ewa Du szczyk,EwaMajda-Stan isławska,MagdalenaMarczyń ska,Elżb ieta Ołd ak,Małgo rzataP awłowska,Wo jciech Słu żewski , JacekWyso cki , JoannaStryczyń ska-K azub ska,and ErnestK uchar."P ol i shConsen su sGu idel ineson theUseo fAcyclo vi rin th eTreatmentand P reven tion o fVZVand HSVIn fection s." Jou rna lo f Infectio nan dCh emo thera py 2 2.2 (20 16):65-7 1.Web .

• Wh itley,R ich ard J. "HerpesSimp lexViru sIn fection s."Goldman 'sCeci l Med icine 35 7.92 67 (2 00 1):2 12 5-12 8.Scien ceDirect. Web .<h ttp ://www.scien ced irect.com/scien ce/article/p i i /S0 0 4 26 822 09 00 581 9>.

• Xu ,Fu j ie, MayaR .Sternberg,Benny J. Kotti ri ,Geraldin eM.Mcqu i llan ,Fran cisK. Lee,Andre J. Nahmias,Stu artM.Berman ,andLau riE.Markowitz."Trend s in HerpesSimp lexVi ru sType1 andType2 Serop revalen ce in th eUnited States." JAMA (200 6):964 .Web.

III. Research Question

IV. Hypothesis

II. Research Rationale

Virus

Pathogenesis

Glycoproteins

Treatment & Vaccines

http://jvi.asm.org/content/

• gB & gD- viral envelope proteins• Immunodominant (Connolly et al., 2011)

• Principal target for neutraliz ing antibodies (Bright et al., 2012)

• Required for membrane fusion• Cellular receptors

• Herpes Virus Entry Mediator (HVEM)• Nectin

• Primary infection• Establishes latency (Cunningham et al., 2006)

• Structure• Double stranded DNA• Viral envelope

h ttp ://heal thd rip.com/wp-content/

IX. Future Research

• Antibiotics • Therapeutic vaccines• Prophylactic vaccines

• HSV-2 dl5-29 (Costa et al., 2000)• Glycoprotein subunit vaccines

(Roth et al., 2013)• HSV-2 ∆gD (Petro et al., 2015)

• HSV-1• Causes over 50,000 eye

infections each year (Lairsonet al., 2003)

• HSV-2• More common in

adolescents and adults• Increases risk of HIV by 3

fold (Freeman et al., 2006)

Why is protective immunity induced by HSV-2 ∆gD but not by HSV-2 dl5-29 or WT HSV-2?

HSV-2 ∆gD induces the the least cellular death and the largest anti-viral inflammatory response.

• HSV-2 ∆gD elicits antibody dependent cell mediated cytotoxicity (Petro et al., 2015)

• HSV-2 dl5-29 elicits neutralizing antibodies (Hoshino et al., 2005)• Not sufficient to provide protection

• MCP-1 • Regulates migration of monocytes and macrophages• Has an important role in immunological surveillance (Deshmane et al.,

2009) • IL-8

• Induces changes in neutrophils• The migration of cells (Bickel et al., 1993)

• TNF-𝛂 & IL- 6 • May be produced by CD4+T cells and neurons in response to HSV

infection in the nervous system (Fields et al., 2001)• HSV-2 dl5-29: “leaky vaccine” (Bernard et al., 2015)

Limitations

• Unable to repeat experiment in pbmcs• Time

• HSV-2 ∆gD and HSV-2 dl5-29 induce different pathways• HSV ∆gD: monocyte chemoattractants• HSV-2 dl5-29: pro-inflammatory cytokines

• HSV-2 dl5-29 induced the highest cytotoxic ity

• Analyze pathways in v ivo following vaccination• Quantify different innate cell populations activated by vaccination• Determine the mechanism of immune system activation following HSV-2

∆gD vaccination• Find more therapeutic targets

Genital Herpes Simplex Virus (HSV) Infections — Initial Visits to Physicians’ Offices, United States, 1966–2014

h ttp s://www.cd c.go v/std /stats15 /figu res/5 0.htm

The Role of Glycoproteins in Membrane Fusion

Vaccine Being Administered

V. Methods

VI. Results

16 24 400

50

100

150

200

Hours post-infection

TN

F-α

(p

g/m

l)

Mock

dl5-29

gD

WT***

****

**

16 24 400

500

1000

1500

Hours post-infection

IL-6

(p

g/m

l)

Mock

dl5-29

gD

WT****

********

16 24 400

500

1000

1500

2000

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MC

P-1

(p

g/m

l)

Mock

dl5-29

gD

WT

*

16 24 400

5000

10000

15000

20000

25000

Hours post-infection

IL-8

(p

g/m

l)

Mock

dl5-29

gD

WT

***

A

B

C

D

16 24 400

50

100

150

Hours post-infection

% C

ytot

oxic

ity

Mock

dl5-29

?gD

WT

****

Cellular Death

Proteins

h ttp ://www.b iolegend .com/legendplex

• Cytokine Bead Assay• LEGENDplex bead based immunoassay kit

(BioLegend, Inc.)• CANTO flow cytometer (BD Biosciences, Inc)

• LDH Assay• Cytotoxic ity Detection Kit (CloneTech)

AssaysCell Lines and Viruses Infections

• 3 virus strains grown and harvested in complementing cell lines

• 2 x 105 Human keratinocyte (HaCaT) cells/well• Multiplicity of infection (MOI) = 1• Supernatant removed and flash frozen at 4 time

points: 0 hours post infection (hpi), 16 hpi, 24 hpi, and 40 hpi

• Microscope images captured at 4 time points

* = p< 0.05** = p< 0.01*** = p< 0.001**** = p< 0.0001

• Wild type HSV-2 strain G• Vero cells (kidney cells of an African Green

Monkey)• HSV-2 ∆gD

• VD60 cells (genetically modified Vero cells)• HSV-2 dl5-29

• V529 cells (genetically modified Vero cells)

0 hpi 16 hpi 40 hpi24 hpi

Figure 3• 13 proteins were analyzed • Some proteins are not displayed because they

were either not detected or not statistically different as compared to the mock control

• Not displayed:• IL-1β, • IFN-𝛂, • IFB-ɣ, • IL-10• IL-12p70• IL-17A• IL-18• IL-23• IL-33

• Increased production of monocyte chemoattractants in response to HSV-2 ∆gD

• MCP-1• IL-8

• Increased production of pro-inflammatory cytokines in response to HSV-2 dl5-29

• TNF-α• IL-6

Figure 3. Increased late-production of monocyte chemoattractants following HSV-2 ∆gD infection

Figure 2: Increased cytotoxicity of HSV-2 dl5-29 and HSV-2 WT infected cells

Figure 1: Increased cell death of HSV-2 dl5-29 and HSV-2 WT

Statistical Analysis

• All experiments performed in triplicate • Experiments independently repeated 3-5 times• Data analyzed in Prism• Two way ANOVA• Dunnet’s post test• Vaccine strains were compared to mock group

h ttp ://wi l l iamrjacobs.org

* = p< 0.05** = p< 0.01*** = p< 0.001**** = p< 0.0001

Figure 1• HaCaT cells started out 100% confluent and were infected at an MOI of 1 Images

were taken by a Nikon Ti Microscope • Most cellular death in HSV-2 dl5-29 and HSV-2 WT

Figure 2• % cytotoxicity determined by LDH level in cellular supernatant • HSV-2 dl5-29 induced highest cytotoxicity levels

HSV-2 Virus

LEGENDplex Kit

HSV-2 ∆gD Induced Cellular Anti-Viral Response and Death of Human

Keratinocytes