distinguishing features of high- and low-dose vaccine ... · ocular pathology in hsv-1–challenged...

Encoded Proteins−of Specific HSV-1 against Ocular HSV-1 Infection Correlates with Recognition

Distinguishing Features of High- and Low-Dose Vaccine

Brett M. Gudgel and Virginie H. SjoelundDaniel J. J. Carr, Grzegorz B. Gmyrek, Adrian Filiberti, Amanda N. Berube, William P. Browne,

http://www.immunohorizons.org/content/4/10/608https://doi.org/10.4049/immunohorizons.2000060doi:

2020, 4 (10) 608-626ImmunoHorizons

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Distinguishing Features of High- and Low-Dose Vaccineagainst Ocular HSV-1 Infection Correlates with Recognition ofSpecific HSV-1–Encoded Proteins

Daniel J. J. Carr,*,† Grzegorz B. Gmyrek,* Adrian Filiberti,* Amanda N. Berube,* William P. Browne,* Brett M. Gudgel,* andVirginie H. Sjoelund‡

*Department of Ophthalmology, University of Oklahoma Health Sciences Center, Oklahoma City, OK 73104; †Department of Microbiology and

Immunology, University of Oklahoma Health Sciences Center, Oklahoma City, OK 73104; and ‡Laboratory for Molecular Biology and Cytometry

Research, University of Oklahoma Health Sciences Center, Oklahoma City, OK 73104

ABSTRACT

The protective efficacy of a live-attenuated HSV type 1 (HSV-1) vaccine, HSV-1 0Δ nuclear location signal (NLS), was evaluated in mice

prophylactically in response to ocular HSV-1 challenge. Mice vaccinated with the HSV-1 0ΔNLS were found to be more resistant to

subsequent ocular virus challenge in terms of viral shedding, spread, the inflammatory response, and ocular pathology in a dose-

dependent fashion. Specifically, a strong neutralizing Ab profile associated with low virus titers recovered from the cornea and

trigeminal ganglia was observed in vaccinated mice in a dose-dependent fashion with doses ranging from 1 3 103 to 1 3 105 PFU

HSV-1 0ΔNLS. This correlation also existed in terms of viral latency in the trigeminal ganglia, corneal neovascularization, and

leukocyte infiltration and expression of inflammatory cytokines and chemokines in infected tissue with the higher doses (13 104–13

105 PFU) of the HSV-1 0ΔNLS–vaccinated mice, displaying reduced viral latency, ocular pathology, or inflammation in comparison

with the lowest dose (1 3 103 PFU) or vehicle vaccine employed. Fifteen HSV-1–encoded proteins were uniquely recognized by

antisera from high-dose (1 3 105 PFU)–vaccinated mice in comparison with low-dose (1 3 103 PFU)– or vehicle-vaccinated animals.

Passive immunization using high-dose–vaccinated, but not low-dose–vaccinated, mouse sera showed significant efficacy against

ocular pathology in HSV-1–challenged animals. In summary, we have identified the minimal protective dose of HSV-1 0ΔNLS vaccine

in mice to prevent HSV-mediated disease and identified candidate proteins that may be useful in the development of a noninfectious

prophylactic vaccine against the insidious HSV-1 pathogen. ImmunoHorizons, 2020, 4: 608–626.

INTRODUCTION

The normal corneal stroma of the eye is composed predominantlyof collagen lamellae highly organized into interwovenfibrils in theanterior stroma that run parallel to the cornea surface in theposterior stroma (1). This architectural arrangement along with

the organization of the epithelial and endothelial layers pro-vides a durable cover to protect the remainder of the eye fromenvironmental insult and allow passage of light to the lens andretina. Although the eye is considered an immunologicallyprivileged organ (2), resident leukocytes, including macro-phages, dendritic cells, and mast cells, populate primarily the

Received for publication June 25, 2020. Accepted for publication September 23, 2020.

Address correspondence and reprint requests to: Dr. Daniel J.J. Carr, Department of Ophthalmology, University of Oklahoma Health Sciences Center, Dean McGeeEye Institute No. 415A, 608 Stanton L. Young Boulevard, Oklahoma City, OK 73104. E-mail address: [email protected]

ORCIDs: 0000-0003-1954-2478 (D.J.J.C.); 0000-0002-5134-0190 (A.F.); 0000-0002-7929-5950 (V.H.S.).

This work was supported by National Institutes of Health Grants R01 AI053108, P20 GM103477, and P30 EY021725. Additional support was provided by an unrestrictedgrant from Research to Prevent Blindness.

Abbreviations used in this article: DPI, day postinfection; gB, glycoprotein B; gC, glycoprotein C; gD, glycoprotein D; gH, glycoprotein H; gL, glycoprotein L; ICP,infected cell protein; MLN, mandibular lymph node; MS, mass spectrometry; MS/MS, tandem mass spectrometry; NLS, nuclear location signal; OUHSC, University ofOklahoma Health Sciences Center; PI, postinfection; Rosa, Ai14/Rosa26-tdTomato-Cre-reporter; TG, trigeminal ganglia; UPLC, UltraPerformance LC.

The online version of this article contains supplemental material.

This article is distributed under the terms of the CC BY 4.0 Unported license.

Copyright © 2020 The Authors

608 https://doi.org/10.4049/immunohorizons.2000060

RESEARCH ARTICLE

Infectious Disease

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peripheral cornea or limbal arcade proximal to the vasculature(3–5). In response to environmental stimuli including trauma orinfection, the immune privilege dynamics of the cornea dramat-ically change.

HSV-1 is a highly successful human pathogen that has aseroprevalence rate above 50% worldwide but is declining inprevalence in the United States and elsewhere (6). It continues tobe a significant ocular pathogen that can elicit immune-driven,irreversible damage to the cornea in patients that experienceepisodic reactivationof latent virus.Experimentally, in response toocular infection resident and infiltratingmyeloid-derived cells areactivated and/or initially recruited to the cornea, followed by NKcells and T lymphocytes that collectively facilitate clearance ofthe pathogen insult but also lead to severe inflammation andirreversible tissue pathology, including vascularization (blood andlymphatic vessel genesis) of the normally avascular central cornea(7–9). In addition, the innervation of the cornea, which normallymaintains thehomeostasis of the ocular surface (10), is dramaticallyaltered and can lead to dry eye disease (11–14). A compromisedvisual axis as a result of HSV-1–mediated corneal pathology thatcannot bemanaged successfully often leads to corneal transplant, ahigh-risk surgical procedure with frequent graft failures (15). Asthere is currently no intervention to permanently alleviate patientsuffering or prevent the acquisition of HSV-1 or HSV-2, strategiesare sought to enhance patient resistance to this infection, which in2013, hadanestimatedeconomicburdenof over$90milliondollarsin the United States alone (16).

The potential to protect individuals from pathogens orproducts encoded by pathogens has been realized and demon-strated experimentally forwell over 100 y (17). Early vaccineworkagainst HSV-1 suggested Ags associated with the envelope or thespecific subunit glycoproteinD (gD)wereprotective inpreventingmortality or the establishment of latency following acute infectionin mice (18, 19). Follow-up studies targeting gD or other HSV-1subunits as prophylactic or therapeutic vaccines have demon-strated various degrees of efficacy in the generation of sterileimmunity, reducing the establishment of latency or preventingreactivation of latent virus (20–25). Most subunit vaccineapproaches likely generate an Ab response with modest T cellinput. As T cells and specifically CD8+ T cells have been shown tocontrol HSV-1 reactivation in mice (26–28), recent studies by onegrouphave focusedonprophylacticvaccines that elicit aprotectiveCD8+ T cell response using HLA-restricted transgenic mice andrabbits (29–31). Specifically, peptide epitopes of glycoprotein B(gB) and the tegument proteins VP11/12 andVP13/14 identified forpolyfunctional CD8+ T cell responses from seropositive, asymp-tomaticHLA-A*201-01 individuals used in CpG-adjuvant vaccinesprevented HLA-A*2:01 transgenic mouse and rabbit mortalityassociated with a significant drop in ocular viral replicationfollowing acute HSV-1 challenge. A follow-up study using adifferent set of HLA-A*02:-01–restricted epitopes from UL9,UL25, and UL44 gene products in a prime/pull therapeuticstrategy found this approach significantly increased the tissueresident effectormemoryCD8+Tcell population in the ganglionofHSV-1 latently infectedmice and prevented virus reactivation and

reduced ocular disease scores (32). Although these results holdpromise in the development of candidate prophylactic ortherapeutic vaccines against ocular HSV-1 infection, with fewexceptions, none of the studies referenced above included anevaluation of the visual axis in terms of quantifiable pathologicalchanges of the cornea, including function as well as analysis ofvisual performance.

Previous studiesby our grouphave identified the application ofa live, attenuatedHSV-1mutant (HSV-1 0Δ nuclear location signal[NLS]) as a highly efficacious prophylactic vaccine against HSV-1(33). HSV-1 0ΔNLS was demonstrated to be a safe vaccine usingIFNAR1-deficient mice and provided superior protection com-paredwith a subunit vaccineused inclinical trials (33, 34).Notably,the efficacy of the vaccine in the control of virus replication in thecornea was linked to early expression of complement and theneonatal Fc receptor, which supported the correlate of protectionto be Ab (35). Finally, HSV-1 0ΔNLS–vaccinated mice retainedcorneal function with preservation of the visual axis in the firststudy to report such findings (36). In the current investigation, adose-response study was conducted using the HSV-1 0ΔNLSvaccine to determine the lowest dose required to maximize theprotective efficacy. Although all doses of vaccine protected micefrom HSV-1–mediated mortality, there were significant differ-ences in terms of level of inflammation andcorneal pathologywithmice vaccinated with the high-dose inoculum showing the leastpathology in comparison with the low-dose–vaccinated mice. Weidentified 15 viral-encoded proteins uniquely recognized byantiserum from high-dose–vaccinated mice that may serve ascandidates for further testing as surrogate vaccines for the live-attenuated HSV-1 0ΔNLS used as a vaccine in the current study.

MATERIALS AND METHODS

Mice, vaccination procedure, and ocular infectionFemale and male outbred CD-1 mice were obtained from CharlesRiver Laboratories (Wilmington, MA). Ai14/Rosa26-tdTomato-Cre-reporter (Rosa) male and female mice on a C57BL/6backgroundwereoriginally purchased fromTheJacksonLaboratory(BarHarbor,ME) andbred in-house. All animalswerehoused in aspecific pathogen-free facility at theDeanMcGeeEye Institute onthe University of Oklahoma Health Sciences Center (OUHSC)campus. Investigators adhered to procedures approved by theOUHSC Institutional Animal Care and Use Committee (protocolno. 16-087-SSIC and no. 19-060-ACHIX), and animals werehandled in accordance with the National Institutes of Health’sGuide for the Care and Use of Laboratory Animals. Mice wereanesthetized for all procedures by i.p. injection of ketamine (100mg/kg) plus xylazine (5.0mg/kg) andwere euthanized by cardiacperfusionof 10ml PBS for tissue collection. Animals (6–10wkold)were vaccinated using a prime/boost approach via ipsilateralfootpad (s.c.) and quadriceps (i.m.) injection 3 wk later asdescribed (33). The immunization dose ranged from 13 103 to 13105 PFU HSV-1 0ΔNLS (KOS strain) in 10 ml PBS (primer andboost). PBS alone served as the vehicle control.


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CD-1micewere infected 30 d following the secondary boost byapplying 1 3 103 PFU HSV-1 McKrae to each cornea followingpartial epithelial debridement with a 25-gauge needle. Rosa26micewere infected 30 d postboost by applying 13 104 PFUHSV-1SC16 expressing Cre recombinase (37) to each cornea followingdebridement as described above.

Serological and virological assaysPeripheral bloodwas obtained from the facial vein of anesthetizedmice at 30 d postboost and fractionated using Microtainer serumseparation tubes (Becton Dickinson, Franklin Lakes, NJ). Ab-containing serumwas evaluated for virus-neutralizing titers in thepresence of guinea pig complement (Rockland, Limerick, PA) onVero cell monolayers as described (33). To quantify virus found inthe tear film or tissue during acute infection of mice, corneaswereswabbed with cotton-tipped applicators, and tissue was excisedand homogenized, and the clarified supernatant was assayed forinfectious virus by standard plaque assay (33).HSV-1 genome copynumber was conducted by PCR on total DNA isolated from thetrigeminal ganglia (TG) of survivingmice 30 d postinfection (DPI)using a proprietary primer-probe mixture, according to themanufacturer’s instructions (Virusys, Taneytown, MD).

Analysis of corneal pathologyGross corneal pathology was conducted at 7 DPI by a maskedobserver examining eyes through a Kowa SL14 portable slit lampbiomicroscope (KowaOptimed,Torrance,CA) using the followingscoring scheme: 0, no pathology; 1, injected eye, no opacity; 2, focalopacity; 3, hazy opacity over entire cornea; 4, dense opacity incentral cornea with remainder haze; 5, same as 4 but with ulcer;and 6, corneal perforation as previously described (38).

Visualization of blood and lymphatic vessel genesis wasperformed in which corneas from enucleated eyes of euthanized

mice were fixed in a 4% solution of paraformaldehyde (Sigma-Aldrich, St. Louis, MO) for 30 min, followed by two 5-min washesinPBS. The tissuewas then incubated in PBS containing 1%TritonX-100 overnight. Labeling, imaging, and analysis of corneal vesselswere performed using an Olympus FV1200 confocal microscopeand MetaMorph Imaging suite software (Sunnyvale, CA) aspreviously described (39).

Visualization and analysis of Cre-inducible, tomato red–staining cells were conducted using an Olympus FV1200 confocalmicroscope and MetaMorph Imaging suite software. Specifically,theTG fromvaccinatedmicewere harvested 30DPI andplaced inPBS for 5 min. PBS was removed, and 4% paraformaldehyde wasadded to each TG. The samples were processed in 5-mm sectionsand placed onto slides by Excalibur Pathology (Norman, OK).Slides were then imaged, and the threshold area calculated foreach section was visualized using MetaMorph Imaging suitesoftware.

Flow cytometryCorneas, TG, and sub–mandibular lymph nodes (MLN) wereharvested from vaccinated mice at 3 and 7 DPI followingexsanguination. Briefly, TG or MLN pairs were macerated intosingle-cell suspensions in RPMI 1640 medium containing 10%heat-inactivated FBS, 13 antibiotic/antimycotic solution, and10mg/ml gentamicin (Invitrogen,Carlsbad,CA) (completemedia).Corneas were digested in 0.25 Wumsch units of Liberase TLenzyme (RocheDiagnostics, Indianapolis, IN) suspended in500mlof complete media at 37°C for 1 h and exposed to trituration every15–20 min. Corneas, TG, and MLN were then filtered through a40-mm nylon mesh filter (Thermo Fisher Scientific, Waltham,MA) prior to labeling. Cell suspensions were blocked with anti-CD16/32 (eBioscience, San Diego, CA), labeled with the indicatedcombination of Abs for 20–30 min, and washed in 13 PBS

FIGURE 1. Mice vaccinated with 13 103–13 105 PFU HSV-1 0ΔNLS are protected against HSV-1–mediated mortality but show differences in Ab

neutralization titers.

(A) Male and female mice (n = 11–17 per group) were s.c. immunized with 1 3 103–1 3 105 PFU HSV-1 0DNLS vaccine, followed by an i.m. boost

3 wk later. Blood was collected 30 d postboost and assessed for neutralization titers to HSV-1. **p , 0.01, compared with the vehicle (PBS)

vaccinated group, Δp , 0.05, comparing 1 3 104–1 3 105 PFU HSV-1 0ΔNLS to the 1 3 103 PFU HSV-1 0ΔNLS–vaccinated group as determined by

ANOVA and Scheffé multiple comparison test. (B) Mice vaccinated with 1 3 103–1 3 105 PFU HSV-1 0ΔNLS (n = 17–19 per group) were challenged

with 1000 PFU HSV-1 McKrae/eye 30 d following the final immunization. Cumulative survival was recorded over 30 d postinfection (DPI). **p ,

0.001, comparing the HSV-1 0ΔNLS–vaccinated mice to vehicle (PBS) control–vaccinated animals as determined by ANOVA and Wilcoxon test.


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containing 1% BSA. All samples were analyzed on a MACSQuant10flowcytometerwithMACSQuantify software (Miltenyi Biotec,Bergisch Gladbach, Germany). Isotype labeling and fluorescenceminus one controls were conducted to validate specific labelingand negate spectral overlap respectively (Supplemental Fig. 1).

Cytokine/chemokine quantificationCorneas and TG from vaccinated mice were collected 7 DPIfollowing exsanguination. Uninfected mouse tissue served asbaseline controls. Tissue wasweighed upon extraction and placedin Next Advance GREEN bead lysis tubes (Averill Park, NY)containing PBS and 13 protease inhibitor mixture (Santa CruzBiotechnology, Dallas, TX). The samples were then homogenizedin a Next Advance Bullet Blender Storm 24 homogenizer for10 min, sonicated in a water bath for 10 min, and subsequently

analyzed for cytokine/chemokine content using customized kitsfor select analytes (MilliporeSigma, Billerica, MA) and a Bio-Plexsuspension array system to detect and quantify analytes (Bio-RadLaboratories, Hercules, CA). The sample contents were normal-ized based on the wet weight of each cornea and reported inpicogram analyte per milligram cornea. Samples were diluted 1:5prior to analysis. The limit of detection of each analyte evaluatedwas as follows: eotaxin, 3.17 pg; G-CSF, 3.26 pg; GM-CSF, 20.1 pg;IFN-g, 3.22 pg; IL-1a, 3.78 pg; IL-1b, 3.30 pg; IL-2, 3.27 pg; IL-3,3.19 pg; IL-4, 3.23 pg; IL-5, 3.04 pg; IL-6, 3.25 pg; IL-7, 3.07 pg; IL-10, 3.26 pg; IL-12p40, 2.93 pg; IL-12p70, 2.77 pg; IL-13, 8.53 pg; IL-15, 2.63 pg; IL-17, 3.28 pg; CXCL10, 3.23 pg; CXCL1, 3.09 pg; LIF,3.24 pg; CCL2, 2.99 pg; M-CSF, 3.58 pg; CXCL9, 3.05 pg; CCL3,17.25 pg; CCL4, 19.11 pg; CXCL2, 20.29 pg; CCL5, 3.2 pg; TNF-a,3.22 pg; and VEGF-A, 3.24 pg.

FIGURE 2. HSV-1 0ΔNLS vaccine suppresses viral replication and spread in the cornea and TG in a dose-dependent manner.

Male and female mice were s.c. immunized with 1 3 103–1 3 105 PFU HSV-1 0DNLS or vehicle (PBS) vaccine, followed by an i.m. boost 3 wk later.

Vaccinated mice (n = 17–19 per group) were challenged with 1000 PFU HSV-1 McKrae/eye 30 d following the final immunization. (A) Mouse

corneas (five to seven mice per group per time point) were swabbed from vaccinated mice at the indicated day (1–7) PI and assayed for viral content

by plaque assay. Data are presented as mean 6 SEM. *p , 0.05, **p , 0.01, comparing the indicated group to PBS-vaccinated control. (B) Mouse

corneas were harvested at day 7 PI, processed, and assayed for viral content by plaque assay. The experiment was repeated twice with five to six

mice per group. *p , 0.05, **p , 0.01, comparing the indicated group to the PBS-vaccinated control group. (C) Mouse TG were harvested at day 7

PI, processed, and assayed for viral content by plaque assay. The experiment was repeated twice with 9–11 mice per group. Data were analyzed by

ANOVA and Tukey post hoc t test. **p , 0.01, comparing the indicated group to the PBS-vaccinated control group, Δp , 0.05, comparing the

indicated groups to the low (1 3 103 PFU 0ΔNLS)–dose-vaccinated group.




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FIGURE 3. HSV-1 0ΔNLS vaccine reduces viral load in the TG of immunized mice during latency.

Latent virus was analyzed in TG from vaccinated mice following ocular infection. (A) Representative sections of confocal images from vaccinated

mice (1 3 103–1 3 105 PFU HSV-1 0ΔNLS) expressing the Cre-inducible tdTomato reporter construct on the Rosa26 locus on a C57BL/6

background 30 d after ocular challenge with 1 3 104 PFU/eye Cre-expressing HSV1 SC16. Neurons successfully infected with the virus are

permanently labeled and express the tdTomato reporter. Scale bar, 100 mm. (B) Summary threshold area of tdTomato expression (Continued)(Continued)




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Immunoprecipitation and HSV-1 protein identificationusing Ab from vaccinated miceTodetermine the repertoire ofHSV-1 proteins that are recognizedby HSV-1 0ΔNLS–vaccinated mouse serum, 1.2 3 106 Vero cellswere plated into each well of a six-well plate and infected at amultiplicity of infection of 1.0 with HSV-1 McKrae. Twenty-fourhours postinfection (PI), the cellswere collected, washed in 1.0mlof PBS twice, and centrifuged at 300 3 g, 5 min for each wash.Following the secondwash, the supernatantwas removed, and thecells were resuspended in 500 ml of 1% Triton X-100 detergent(lysis buffer) and placed on ice for 15–20 min. Following theincubation, cell lysates were clarified from cell debris bycentrifugation (10,000 3 g, 10 min at 4°C). The supernatantsfrom infected and uninfected Vero cells were incubated with4 ml of serum from vaccinated mice and 100 ml of immuno-magnetic protein G microbeads (Miltenyi Biotec) at 4°C for30 min with gentle agitation every 5 min. The protein/Ab/microbead complex was then added onto mMACS magneticbead columns (Miltenyi Biotec). The columns werewashed fourtimes with 200 ml lysis buffer, and retained proteins were elutedwith 50 ml 100 mM glycine (pH 2.5).

Trypsin digest of immunoprecipitatedproteinswas performedaccording to the filter-aided sample preparation protocol (40).Briefly, the eluate was buffer exchanged to 8 M urea, the proteinswere reduced with 10 mM DTT and then alkylated with 10 mMiodoacetamide. The peptides were eluted in 10 mM ammoniumacetate (pH 8), dried, and resuspended in 10 mM ammoniumformate (pH 10). Liquid chromatography tandemmass spectrom-etry (MS/MS) was performed by coupling a nanoAcquity Ultra-Performance LC (UPLC; Waters, Manchester, U.K.) to a Q-TOFSYNAPT G2S instrument (Waters). Each protein digest (;100 ngof peptide) was delivered to a trap column (300 mm 3 50 mmnanoAcquity UPLCNanoEase Column 5mmBEHC18;Waters) ata flow rate of 2 ml/min in 99.9% solvent A (10 mM ammoniumformate pH 10, in HPLC grade water). After 3 min of loading andwashing, peptides were transferred to another trap column(180 mm3 20 nanoAcquity UPLC 2G-V/MTrap 5 mm SymmetryC18; Waters) using a gradient from 1 to 60% solvent B (100%acetonitrile). The peptides were then eluted and separated at aflow rate of 200 nl/min using a gradient from 1 to 40% solvent B(0.1% FA in acetonitrile) for 60 min on an analytical column(7.5 mm 3 150 mm nanoAcquity UPLC 1.8 mm HSST3; Waters).The eluent was sprayed via PicoTip Emitters (Waters) at a sprayvoltage of 3.0 kV and a sampling cone voltage of 30V and a sourceoffset of 60 V. The source temperature was set to 70°C. The conegas flow was turned off, the nano flow gas pressure was set at 0.3bar, and the purge gasflowwas set at 750ml/h. The SYNAPTG2Sinstrument was operated in data-independent mode with ionmobility (HDMSe). Full-scan mass spectrometry (MS) and

MS/MS(m/z50–2000)wereacquired in resolutionmode (20,000resolution FWHM at m/z 400). Tandem mass spectra weregenerated in the trapping region of the ionmobility cell by using acollisional energy ramp from 20 V (low mass, start/end) to 35 V(high mass, start/end). A variable IMS wave velocity was used.Wave velocitywas ramped from300 to 600m/s (start to end), andthe ramp was applied over the full IMS cycle. A manual releasetime of 500ms was set for the mobility trapping and a trap heightof 15 V with an extract height of 0 V. The pusher/ion mobilitysynchronization for the HDMSe method was performed usingMassLynx V4.1 and DriftScope v2.4. LockSpray of Glu–fibrinopeptide-B (m/z 785.8427) was acquired every 60 s, andlock mass correction was applied postacquisition.

Raw MS data were processed by ProteinLynx Global Server(Waters) for peptide and protein identification. MS/MS weresearched against the Uniprot HSV-1 proteome database (releasedate November 2, 2017, containing 1776 unreviewed sequences)with the following search parameters: full tryptic specificity, upto two missed cleavage sites, carbamidomethylation of cysteineresidues was set as a fixed modification, and N-terminal proteinacetylation and methionine oxidation were set as variablemodifications. Proteins reportedwere identified in two ormoreout of seven samples per group.

Passive immunizationSerum obtained from terminal cardiac punctured PBS (vehicle)–or 0ΔNLS (1 3 103 or 1 3 105 PFU)–vaccinated mice was pooledand administered i.p. (250 ml) to naive CD-1 male or female mice24 h prior to HSV-1 (1000 PFU/eye) challenge. Mechanosensoryfunction of the cornea was assessed 1–7 DPI using a Cochet–Bonnet esthesiometer as previously described (12). Mice weremonitored for cumulative survival and deaths recorded to 21 DPI.Mice were subsequently exsanguinated, and the corneas wereassessed for opacity as previously described (41) and subsequentlyprocessed for neovascularization (39).

StatisticsGraphPad Prism 8 was used to analyze data for statisticalsignificance (p , 0.05) as determined using statistical testsdescribed in each figure. Data are presented as mean 6 SEM.

RESULTS

HSV-1 0ΔNLS vaccine suppresses virus replication andprevents HSV-1–mediated mortality in a dose-dependent fashionPrevious studies investigating the efficacy of the HSV-1 0ΔNLSvaccine against ocular HSV-1 challenge were conducted using a

by cells in TG sections (n = 7 per group) from immunized mice (n = 3 per group). Data are displayed as threshold area, ***p , 0.001, comparing 1 3

103 PFU HSV-1 0ΔNLS vaccine to higher-dose vaccines. (C) HSV-1 copy number in the TG of vaccinated mice (1 3 103–1 3 105 PFU 0ΔNLS) at day

30 PI (n = 21–22 per group). Data were analyzed by ANOVA and Tukey post hoc t test. *p, 0.05, **p, 0.01, comparing the 13 103 PFU dose to the

higher doses of HSV-1 0ΔNLS.




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FIGURE 4. Diminished leukocyte infiltrate in the cornea of vaccinated mice in response to HSV-1 infection.

Male and female mice were s.c. immunized with 1 3 103 PFU HSV-1 0ΔNLS, 1 3 105 PFU HSV-1 0DNLS, or vehicle (PBS) vaccine, followed by an i.m.

boost 3 wk later. Mice (n = 5 per group) were challenged with 1000 PFU HSV-1 McKrae/eye 30 d following the final immunization. Mice were

euthanized at 3 or 7 DPI, and the corneas were removed and enzymatically processed to single-cell suspensions. (A) Representative flow plot

corneal digest for distribution of CD4+ and CD8+ T cells in vaccinated mice. (B) Absolute number of CD4+ and CD8+ T cells and CD19+ B

lymphocytes residing in the cornea of vaccinated mice 3 DPI. (C) Absolute number of CD4+ and CD8+ T cells and CD19+ B lymphocytes residing in

the cornea of vaccinated mice 7 DPI. Uninfected absolute number of CD4+ and CD8+ T cells and CD19+ B lymphocytes were 9 6 3, 4 6 2, and 4 6

3, respectively. *p , 0.05, **p , 0.01, ***p , 0.001, comparing the indicated group to PBS-vaccinated group as determined by (Continued)(Continued)




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dose of 1 3 105 PFU for the primary immunization and boost(33–36). To formalize the minimal efficacious dose of vaccine tomaximize the protective effect against subsequent challenge,micewere immunized with doses ranging from 13 103 to 13 105 PFUprior to challenge. Mice immunized with 1 3 104–1 3 105 PFUHSV-10ΔNLSshowedsignificantlyhigherneutralizingAb titers toHSV-1 compared with mice vaccinated with 1 3 103 PFU HSV-10ΔNLS (Fig. 1A). However, all doses of HSV-1 0ΔNLS used toimmunize mice were found to protect animals subsequentlychallengedwithHSV-1 asmeasured by cumulative survival (Fig. 1B).

To further compare the dose response of the HSV-1 0ΔNLSvaccine in resistance against ocularHSV-1 challenge, the tear film,cornea, and TG from mice immunized with 13 103–13 105 PFUHSV-1 0ΔNLS or vehicle (PBS) were evaluated for viral content.Mice vaccinated with any dose of HSV-1 0ΔNLS were found toshed less virus during acute infection (i.e., 3–7 DPI) comparedwith PBS-vaccinated mice (Fig. 2A). By comparison, only the 13104–1 3 105 PFU dose of HSV-1 0ΔNLS reduced corneal virustiter,whereas the 13 103 PFUdose ofHSV-1 0ΔNLSdid not have asignificant effect by 7 DPI (Fig. 2B). Similar to what was observedin viral shedding, mice immunized with any dose of the HSV-10ΔNLS vaccine showed reduction in virus replication in the TGcompared with the PBS-vaccinated control group (Fig. 2C).However, little to no infectious virus was recovered from the TGof mice vaccinated with 1 3 104–1 3 105 PFU HSV-1 0ΔNLScompared with the low (1 3 103 PFU) dose of HSV-1 0ΔNLS inwhich over 50% of mice TG had detectable levels of infectiousvirus (Fig. 2C).

A critical aspect of HSV-1 infection is the establishmentof latency as a result of invasion of the sensory neurons that residein the TG during acute infection. To determine the success ofpermanently colonizing neurons in the TG, Cre-inducibletdTomato fluorescent reporter mice vaccinated with HSV-10ΔNLS (1 3 103–1 3 105 PFU) were challenged with HSV-1encoding Cre recombinase under the infected cell protein (ICP)0 lytic gene promoter (37). Cells that survive the acute infectionare permanently “tagged” and will express tdTomato. TG frommice immunized with 1 3 104–1 3 105 PFU HSV-1 0ΔNLSdisplayed significantly fewer labeled cells as measured bythreshold area compared with mice vaccinated with 1 3 103

PFU HSV-1 0ΔNLS at 30 DPI (Fig. 3A, 3B). These results areconsistent with the genome copy number of HSV-1 recovered inthe TG of latent-infected, vaccinated mice with a significantreduction recovered from mice immunized with 13 104–13 105

PFU HSV-1 0ΔNLS compared with the 13 103 PFU dose 30 DPI(Fig. 3C). In this experiment, PBS-vaccinatedmice did not surviveand, therefore, couldnot be assessed as a baselinepositive control.

However, a previous study found PBS-vaccinated tdTomatofluorescent reporter mice that did survive the acute infection to30 DPI displayed a similar phenotype as that shown with 13 103

PFU0ΔNLS-vaccinated animals (36). In summary, all doses of theHSV-1 0ΔNLS vaccine tested showed protection against ocularHSV-1 challenge in terms of cumulative survival and viral spreadand replication in the TG, although the lowest dose evaluateddisplayed no efficacy against HSV-1 replication in the cornea,which correlatedwith a lowneutralizingAb titer andhigher viruscopy number found in the TG of latent-infected mice.

Leukocyte infiltration into the cornea and TG is stymied inHSV-1 0ΔNLS–vaccinated mice in a dose-dependent mannerHSV-1 infection of the cornea elicits a robust cellular immuneresponse initially with a massive onslaught of neutrophils andactivation of mast cells, followed by the infiltration of activatedmonocytes/macrophages and NK cells and eventually CD4+ andCD8+ T cells (5, 42–44). We evaluated T and myeloid cellinfiltration comparing vaccinated to nonvaccinated animals at3 and 7 DPI in the cornea, the latter time point when non-vaccinated mice begin to succumb to infection (Fig. 1B). Arepresentative flow plot comparing PBS-, 1 3 103 PFU 0ΔNLS(low)–, and 13 105 PFU0ΔNLS (high)–vaccinatedmice for T andB cells (Fig. 4A) and myeloid cells (Fig. 4D) is provided. At 3 DPI,the high-dose–vaccinated mice showed significantly fewer CD4+

andCD8+Tcells residing in the corneacomparedwith thePBS-orlow-dose–vaccinated mice (Fig. 4B). By 7 DPI, the high and low0ΔNLS–dose-vaccinated mouse corneas retained fewer T cellsthan the PBS-vaccinated counterparts (Fig. 4C). A similar findingwas observed in analysis of granulocytic populations at 3 (Fig. 4E)and 7 (Fig. 4F) DPI, with either the high-dose or high- and low-dose 0ΔNLS–vaccinatedmice possessing significantly fewer cellscompared with the PBS-vaccinated control animals. Significantlyfewer monocyte/macrophage populations were found in thecornea of the high- and low-dose 0ΔNLS–vaccinated micecompared with the PBS-vaccinated controls at 3 (Fig. 4G) and 7(Fig. 4H)DPI as well. Althoughmodest in number, the corneas ofinfectedmice contained significantly fewer CD19+ B lymphocytesin the high-dose–immunized mice compared with the vehiclecontrol–vaccinated animals at 7 DPI (Fig. 4C), but not 3 DPI (Fig.4B). As we are unable to detect B lymphocytes in the uninfectedCD-1 mouse cornea, the infiltration of these cells followinginfection is of interest relative to local Ab production. Currently,we have not been able to detect anti–HSV-1 Ab in the tear film ofvaccinated mice prior to or following infection.

We also evaluated T and myeloid cell infiltration comparingvaccinated to nonvaccinated animals at 3 and 7 DPI in the TG. A

ANOVA and Tukey post hoc t test. (D) Representative flow plot corneal digest for distribution of myeloid cells in vaccinated mice designated as

1: (CD45+CD11b+Ly-6GmidLy-6C+), 2: (CD45+CD11b+Ly-6GhighLy-6Cint), and 3: (CD45+CD11b+Ly-6G2Ly-6Chigh). Absolute number of granulocytes

(CD45+CD11b+Ly-6GmidLy-6C+) and (CD45+CD11b+Ly-6GhighLy-6Cint) at 3 (E) and 7 (F) DPI. Absolute number of monocytes (CD45+CD11b+Ly-6G2

Ly-6Chigh) at 3 (G) and 7 (H) DPI. *p , 0.05, **p , 0.01, ***p , 0.001, comparing the indicated group to PBS-vaccinated group or 1 3 103 0ΔNLS

dose–vaccinated mice as determined by ANOVA and Tukey post hoc t test.




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FIGURE 5. Diminished leukocyte infiltrate in the TG of vaccinated mice in response to HSV-1 infection.



euthanized at 3 or 7 DPI, and the TG were removed and enzymatically processed to single-cell suspensions. (A) Representative flow plot TG digest

for distribution of CD4+ and CD8+ T cells in vaccinated mice. (B) Absolute number of CD4+ and CD8+ T cells and CD19+ B lymphocytes residing

in the TG of vaccinated mice 3 DPI. (C) Absolute number of CD4+ and CD8+ T cells and CD19+ B lymphocytes residing in the cornea of

vaccinated mice 7 DPI. Uninfected absolute number of CD4+ and CD8+ T cells and CD19+ B lymphocytes were 51 6 11, 42 6 5, and 102 6 20,

respectively. *p , 0.05, comparing the indicated group to PBS-vaccinated group as determined by ANOVA and Tukey post hoc t test, n = 5 mice

per group. (D) Representative flow plot corneal digest for distribution of myeloid cells in vaccinated mice designated as 1: (CD45+CD11b+Ly-

6GmidLy-6C+), 2: (CD45+CD11b+Ly-6GhighLy-6Cint), and 3: (CD45+CD11b+Ly-6G2Ly-6Chigh). Absolute number of granulocytes (Continued)(Continued)




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representative flow plot comparing PBS-, 1 3 103 PFU 0ΔNLS(low)–, and 13 105 PFU 0ΔNLS (high)–vaccinatedmice for T andB cells (Fig. 5A) andmyeloid cells (Fig. 5D) is provided. Unlike thecornea, only the high-dose–vaccinated mice possessed signifi-cantly fewer CD4+ T cells compared with the PBS control–vaccinated group at 3 DPI (Fig. 5B), whereas both CD4+ and CD8+

T cell numbers were significantly reduced in the TG of high0ΔNLS–dose-vaccinated mice at 7 DPI (Fig. 5C). The granulocyteinfiltration was not impacted by the 0ΔNLS vaccination at 3 DPIwith similar numbers of CD11b+Ly-6G+Ly-6C+/int cells (Fig. 5E).However, at 7 DPI, the granulocyte numbers were significantlyreduced in the TG of the high 0ΔNLS–dose-immunized micecompared with the PBS control–vaccinated animals (Fig. 5F). Bycomparison, monocyte/macrophage (CD45+CD11b+Ly-6G2Ly-6Chigh) numbers were significantly lower in the TG of the high0ΔNLS–dose-immunized mice compared with the PBS control–vaccinated animals at 3 (Fig. 5G) and 7 (Fig. 5H) DPI. Nodifferences were found comparing the low-dose–vaccinated miceto the PBS-vaccinated control group. It should also be noted therewas no change in the B lymphocyte number that resides in the TGcomparing uninfected levels to that following infection, suggestingthat unlike the cornea, B lymphocytes do not traffic to the TG ofinfected mice (Fig. 5B, 5C).

Next, we surveyed the draining lymph nodes (MLN) ofvaccinatedmice at 3 and 7 DPI to determine if the results found inthe infected tissuemirrored that found in the organized lymphoidtissuemost responsible for the generation of the adaptive immuneresponse during acute infection. The results of surveying theMLNat these timepoints found the cell numbers reflected avery similarprofile to that reported for the cornea for T lymphocytes (Fig. 6A)and myeloid cells (Fig. 6D). Specifically, CD4+ and CD8+ T cells(Fig. 6B, 6C) aswell as the granulocyte (Fig. 6E, 6F) andmonocyte/macrophage (Fig. 6G, 6H) populations were significantly reducedin the MLN of the high 0ΔNLS–dose-vaccinated mice comparedwith the PBS- and low-dose–vaccinated groups at 3 and 7 DPI. Inaddition to T cells, B lymphocyte numbers in theMLN of the high0ΔNLS–dose-vaccinated animals were considerably lower thanthe low-dose– or PBS-vaccinated groups at 3 (Fig. 6B) and 7 (Fig.6C)DPI.Asimilarprofilewas found in thecornea,TG, andMLNinmice vaccinatedwith 13 104 PFU0ΔNLS as thatwith 13 105 PFU0ΔNLS at 7 DPI (data not shown). The 3 DPI time point was notconducted with 1 3 104 PFU 0ΔNLS-vaccinated animals. Withsome exceptions, the effectiveness of the vaccine in terms ofreduction in HSV-1 found in a given tissue is inversely correlatedwith the leukocyte infiltrate and the lack of expansion of cellswithin the draining lymph nodes. We surmise the overallattenuated cellular response to infection in the surveyed tissue ofvaccinated mice is due to greater control of viral replication and,

therefore, fewer Ag available to drive local and regional immuneactivation postchallenge.

Select cytokine and chemokines levels expressed in infectedcornea and TG are dramatically reduced in HSV-10ΔNLS–vaccinated miceChemokines including CCL2, CCL3, CCL5, CXCL1, and CXCL10are expressed early in the cornea following HSV-1 infection(45–47). Neutralization or loss of select chemokines or theircognate receptor results in aberrant and, oftentimes, loss ofleukocyte infiltration during acute corneal HSV-1 infection (38,46, 48–53). Therefore, to determine if the reduction in cornealleukocyte infiltration in response to HSV-1 infection in thevaccinated mice correlated with chemokine expression, theexpression of 10 chemokines was evaluated by suspension array.The results show a significant drop in CCL2, CCL3, CCL4, CCL5,CCL11, CXCL9, and CXCL10 in HSV-1–infected, vaccinatedmouse corneas in accordancewith the vaccine dose administered(Table I). Proinflammatory factors, including IL-6 and IFN-g,were also found to be reduced, as was IL-10 and VEGF-A in thevaccinated mouse cornea in a dose-dependent fashion (Table I).Other chemokines and inflammatory molecules investigated,including CXCL1, CXCL2, IL-1a, IL-1b, G-CSF, LIF,M-CSF, andTNF-a, were all reduced in the vaccinated animals, but the levelsdid not achieve significant (p , 0.05) differences in large partbecause of sample variation (Table I).

T cell and myeloid cell infiltration into the TGwas also mutedin the higher-vaccinated–dose animals following HSV-1 infection.Such results were reflected by the expression of cytokines andchemokines. Specifically, CCL2, CCL3, CCL4, CCL5, CCL11,CXCL9, CXCL10, IFN-g, and LIF expression were also signifi-cantly reduced in the TG from the higher-dose (1 3 104–1 3 105

PFUHSV-1 0ΔNLS)–vaccinatedmice compared with the vehicle-or lower-dose–immunized animals (Table II). Although expres-sion was modest, TNF-a levels in the TG were significantlyreduced in all vaccinated mice compared with the vehiclecontrol–vaccinated group (Table II). Other immune mediators,including CXCL1, G-CSF, IL-6, and M-CSF, were lower in thehigher-vaccinated mouse TG but, because of variability, did notreach significance (Table II). Neither IL-1b nor VEGF-A wasdetectable above background (Table II), whereas CXCL2 andIL-10 showed high background levels (in uninfected animals)and, therefore, were not included in the analysis (data notshown). Taken together, the measurement of analytes in thecornea and TG reveal a strong correlation with the reduction ofleukocyte infiltration, control of viral replication in these tissues,andAbneutralization titers observed in thehigh-dose–vaccinatedmice 7 DPI.

(CD45+CD11b+Ly-6GmidLy-6C+) and (CD45+CD11b+Ly-6GhighLy-6Cint) at 3 (E) and 7 (F) DPI. Absolute number of monocytes (CD45+CD11b+Ly-6G2

Ly-6Chigh) at 3 (G) and 7 (H) DPI. *p , 0.05, **p , 0.01, comparing the indicated group to PBS-vaccinated group as determined by ANOVA and

Tukey post hoc t test.




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FIGURE 6. Absence of lymphocyte expansion in draining lymph nodes of HSV-1–infected mice vaccinated with high-dose HSV-1 0ΔNLS.



euthanized at 3 or 7 DPI, and the MLN were removed and processed to single-cell suspensions. (A) Representative flow plot MLN distribution of

CD4+ and CD8+ T cells in vaccinated mice. (B) Absolute number of CD4+ and CD8+ T cells and CD19+ B lymphocytes residing in the MLN of

vaccinated mice 3 DPI. (C) Absolute number of CD4+ and CD8+ T cells and CD19+ B lymphocytes residing in the MLN of vaccinated mice 7 DPI.

Uninfected absolute number of CD4+ and CD8+ T cells and CD19+ B lymphocytes were 386,139 6 13,075, 143,432 6 3,572, (Continued)(Continued)




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Corneal neovascularization is greatly diminished in high-dose HSV-1 0ΔNLS–vaccinated miceCorneal neovascularization is a hallmark of herpetic stromalkeratitis in rodents andhumans alike (8). Previously,we reported aloss of vision inmice as a result of HSV-1 infection could be linkedto severe corneal opacity and gross neovascularization (36).Therefore, we investigated the impact of vaccine doses on theseverity of corneal blood and lymphatic vessel genesis at 30 DPI, atime when maximum angiogenesis is evident (54). Our resultsreflect the dose-response efficacy of the vaccine with the highervaccine doses (1 3 104–1 3 105 PFU HSV-1 0ΔNLS), preventingcorneal hem and lymph angiogenesis in comparison with the low(13 103 PFUHSV-1 0ΔNLS) vaccine dose,which showed a similarprofile to vehicle-vaccinated mice (33) (Fig. 7A–D). Because thevehicle-vaccinated mice do not survive out to 30 DPI to asignificant degree (Fig. 1B), we assessed corneal opacity in theanimalsat 7DPIvia slit lampexaminationusingamaskedobserver.Similar to what is observed with neovascularization, mice thatreceived the higher vaccine doses displayed minimal cornealopacity compared with mice that received low-dose or vehicle

vaccine (Fig. 7E). Collectively, these results concur with the dataabove, demonstrating the efficacy of the HSV-1 0ΔNLS vaccineagainst ocular viral challenge is greatly attenuatedat immunizationdoses below 13 104 PFU.

Serum from mice immunized with the high HSV-1 0ΔNLSvaccine dose immunoprecipitates HSV-1 proteins notrecognized using the low HSV-1 0ΔNLS vaccine doseBecause the high vaccine dose demonstrated superior efficacy inprotecting mice against HSV-1 in comparison with mice immu-nized with the low dose, we hypothesized viral proteins may beuniquely recognized by Ab from the antiserum of high-dose–vaccinated mice. To test this hypothesis, antiserum obtained fromhigh- and low-dose–immunized mice was evaluated for recogni-tion ofHSV-1Ags. A total of 15HSV-1 proteinswere recognizedbyantiserum from high-dose–immunized mice significantly abovethat recognized by low-dose–vaccinated animals (Table III).Furthermore, two additional HSV-1 proteins (UL18/VP23 andUL35/VP26) were recognized by the high and low HSV-10ΔNLS–immunized mice that were significantly above the level

TABLE I. Cytokine/chemokine expression in cornea of HSV-1 0ΔNLS–vaccinated mice

Cytokine/Chemokine Vehicle 1 3 103 PFU 0ΔNLS 1 3 104 PFU 0ΔNLS 1 3 105 PFU 0ΔNLS

Eotaxin/CCL11 125 6 52 44 6 20a 13 6 6b 16 6 7b

G-CSF 1077 6 795 88 6 50 24 6 21 0 6 0IFN-ɤ 63 6 19 11 6 8b 2 6 2b 0 6 0b

IL-1a 133 6 85 55 6 18 44 6 17 59 6 25IL-1b 77 6 53 10 6 7 2 6 2 0 6 0IL-6 158 6 78 28 6 18b 3 6 3b 0 6 0b

IL-10 16 6 9 2 6 2a 0 6 0a 0 6 0a

IP-10/CXCL10 3539 6 868 1474 6 731a 343 6 154b 260 6 111b

KC/CXCL1 1947 6 1261 494 6 399 417 6 290 46 6 28LIF 106 6 67 40 6 34 3 6 3 2 6 2MCP1/CCL2 3069 6 1466 728 6 514a 235 6 106a 96 6 57b

M-CSF 18 6 11 12 6 6 7 6 4 5 6 5MIG/CXCL9 1188 6 422 240 6 71b 83 6 29b 80 6 28b

MIP-1a/CCL3 412 6 221 96 6 64a 41 6 13a 39 6 14a

MIP-1b/CCL4 618 6 275 118 6 54a 28 6 14b 20 6 13b

MIP-2/CXCL2 3751 6 3401 1599 6 1490 58 6 40 50 6 31RANTES/CCL5 99 6 33 22 6 5a 18 6 6b 10 6 6b

TNF-a 16 6 10 2 6 2 0 6 0 0 6 0VEGF-A 24 6 14 10 6 5 3 6 2a 0 6 0a

Corneas were collected at day 7 PI and processed for cytokine/chemokine content by suspension array. Numbers reflect picograms per milligram of each indicatedanalyte 6 SEM, n = 5–6 samples per group. Numbers in bold reflect significant differences.ap , 0.05, comparing the indicated group to the vehicle control as determined by ANOVA and Dunnett multiple comparison test. Uninfected corneas had nodetectable analyte expressed except those noted in this study (picograms per milligram 6 SD, n = 2 per analyte): eotaxin, 7 6 2; IL-1a, 12 6 12; IL-10, 5 6 5; CXCL10,15 6 15; and CXCL1, 39 6 24.bp , 0.01, comparing the indicated group to the vehicle control as determined by ANOVA and Dunnett multiple comparison test. Uninfected corneas had nodetectable analyte expressed except those noted in this study (picograms per milligram 6 SD, n = 2 per analyte): eotaxin, 7 6 2; IL-1a, 12 6 12; IL-10, 5 6 5; CXCL10,15 6 15; and CXCL1, 39 6 24.

and 117,423 6 11,108, respectively. *p , 0.05, **p , 0.01, ***p , 0.001, comparing the high 0ΔNLS–vaccinated dose group to PBS-vaccinated

group as determined by ANOVA and Tukey post hoc t test. (D) Representative flow plot MLN distribution of myeloid cells in vaccinated mice

designated as 1: (CD45+CD11b+Ly-6GmidLy-6C+), 2: (CD45+CD11b+Ly-6GhighLy-6Cint), and 3: (CD45+CD11b+Ly-6G2Ly-6Chigh). Absolute number

of granulocytes (CD45+CD11b+Ly-6GmidLy-6C+) and (CD45+CD11b+Ly-6GhighLy-6Cint) at 3 (E) and 7 (F) DPI. Absolute number of monocytes

(CD45+CD11b+Ly-6G2Ly-6Chigh) at 3 (G) and 7 (H) DPI. *p , 0.05, **p , 0.01, comparing the high 0ΔNLS–vaccinated dose to PBS-vaccinated

group as determined by ANOVA and Tukey post hoc t test.




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displayed by serum from the vehicle-vaccinated mice (Table III).Additional HSV-1 proteins were precipitated by the HSV-10ΔNLS–vaccinated mice, but the abundance of recognition didnot reach significance compared with the vehicle-immunizedmice because of the variability from animal to animal.

Passive immunization and cornea pathologyTo further characterize the relevance of differences in proteinrecognition of sera from mice immunized with the high (1 3 105

PFU) versus low (13 103 PFU) dose of the 0ΔNLS vaccine, naivemice were passively immunized with antiserum from the high-and low-dose–vaccinated animals and subsequently challengedwith HSV-1. Relative to mice receiving sera from PBS-vaccinated(naive control) animals, recipients of sera from either dose of0ΔNLS vaccine were found to be resistant to HSV-1 challenge interms of cumulative survival (Fig. 8A). However, there weremarked differences betweenmice receiving sera from high versuslow 0ΔNLS–dose vaccine in terms of mechanosensory function(Fig. 8B), opacity (Fig. 8C), and neovascularization, includinglymphangiogenesis (Fig. 8D–G). Specifically, there was minimalcorneal sensation loss in the high-dose sera recipients followingHSV-1 infection compared with the other two groups with a 44%loss in low-dose sera recipients and 96% loss in the naive serarecipients by 7 DPI (Fig. 8B). Likewise, HSV-1–infected micereceiving sera from high-dose–vaccinated animals possessedcornea opacity levels similar to uninfected mice and lower thanmice receiving sera from low-dose– or PBS-vaccinated, HSV-1–infected animals (Fig. 8C). Equally revealing is the genesis ofblood and lymphatic vessels in the cornea in response to HSV-1.

High-dose sera recipients displayed little to no cornea neo-vascularization as a result of HSV-1 challenge, whereas there wasnotable vessel growth in the cornea from low-dose sera recipients,albeit lower than naive sera recipients (Fig. 8D–G). Collectively,the results clearly demonstrate differences between recipients ofsera fromhigh- versus low-dose 0ΔNLS–vaccinatedmice in termsof corneal pathology that likely relates back to coverage of Agrecognition by Ab from these vaccinated animals.

DISCUSSION

In the current study, we compared different doses of the HSV-10DNLS vaccine in mice subsequently challenged with HSV-1 todefine theminimumeffective dose that affords the host protectionagainst ocularHSV-1 infection.Whereas all doseswere found tobehighlyeffective in termsof cumulative survival, therewasadistinctdifference in the lack of efficacy of the low dose (1 3 103 PFU)versus higher doses (1 3 104–1 3 105 PFU) of the HSV-1 0DNLSvaccine in nearly all other aspects of protection measured in theinfected tissue including virus replication and spread, establish-ment of latency, inflammation including cytokine and chemokineexpression and leukocyte infiltration, and corneal pathologyincluding opacity and neovascularization. These findings wereinversely correlated to theneutralizingAb titer fromthevaccinatedmice: the higher the Ab titer, the lower the inflammatory profile,and reduction in virus replication and spread. It isworth noting theexpansion of the lymphoid population observed in the vehicle- orlow-dose–vaccinated mice was not evident in mice that received

TABLE II. Cytokine/chemokine expression in trigeminal ganglion of HSV-1 0ΔNLS–vaccinated mice

Cytokine/Chemokine Vehicle 1 3 103 PFU 0ΔNLS 1 3 104 PFU 0ΔNLS 1 3 105 PFU 0ΔNLS

Eotaxin/CCL11 1435 6 363 633 6 157 188 6 82a 94 6 26b

G-CSF 562 6 268 389 6 231 12 6 12 0 6 0IFN-ɤ 902 6 27 402 6 192 38 6 26a 0 6 0b

IL-1b 3 6 3 4 6 4 0 6 0 0 6 0IL-6 744 6 275 613 6 290 0 6 0 0 6 0IP-10/CXCL10 15,194 6 2,777 11,528 6 3,919 1648 6 865a 436 6 373b

KC/CXCL1 505 6 203 399 6 238 27 6 27 6 6 4LIF 155 6 52 71 6 31 4 6 4a 0 6 0b

MCP1/CCL2 3634 6 1,241 1097 6 563 79 6 48a 12 6 9b

M-CSF 48 6 13 48 6 20 7 6 7 0 6 0MIG/CXCL9 2310 6 423 1655 6 457 444 6 276a 103 6 92b

MIP-1a/CCL3 1964 6 375 865 6 388 75 6 35a 54 6 30b

MIP-1b/CCL4 3238 6 656 1380 6 646 41 6 41b 0 6 0b

RANTES/CCL5 430 6 76 263 6 74 28 6 21b 0 6 0c

TNF-a 33 6 16 0 6 0a 0 6 0a 0 6 0a

VEGF-A 0 6 0 0 6 0 6 6 6 0 6 0

TG were collected at day 7 PI, and processed for cytokine/chemokine content by suspension array. Numbers reflect picograms per milligram of each indicatedanalyte 6 SEM, n = 8–10 samples per group. Numbers in bold reflect significant differences.ap , 0.05, comparing the indicated group to the vehicle control as determined by ANOVA and Dunnett multiple comparison test. Uninfected corneas had nodetectable analyte expressed except those noted in this study (picograms per milligram 6 SEM, n = 3 per analyte): eotaxin, 54 6 54; CXCL10, 16 6 16; and CXCL1,39 6 24.bp , 0.01, comparing the indicated group to the vehicle control as determined by ANOVA and Dunnett multiple comparison test. Uninfected corneas had nodetectable analyte expressed except those noted in this study (picograms per milligram 6 SEM, n = 3 per analyte): eotaxin, 54 6 54; CXCL10, 16 6 16; and CXCL1,39 6 24.cp , 0.001, comparing the indicated group to the vehicle control as determined by ANOVA and Dunnett multiple comparison test. Uninfected corneas had nodetectable analyte expressed except those noted in this study (picograms per milligram 6 SEM, n = 3 per analyte): eotaxin, 54 6 54; CXCL10, 16 6 16; and CXCL1,39 6 24.




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the higher doses, which we interpret to suggest Ag abundance islimited in high-dose–vaccinated mice likely because of control ofthe infection by Ab. This result is not to say T cells are notinvolved in this process, as there is ample evidence by numerousinvestigators usingmutantHSV-1 as vaccines thatT cells play a roleeither directly as effector cells and/or facilitate the Ab responseagainstHSV-1 (24, 55, 56). However, the current studywas focused

on Ab as a means to further delineate differences in vaccine dosesthat might be explained by Ag recognition.

Because there was a striking difference in vaccine efficacymeasuring corneal keratitis comparing the high- to low-dosevaccine, we considered the possibility that unique HSV-1 Agsmight be recognizedbyantiserum fromthehigh-dose–vaccinatedmice not recognized by low-dosed–vaccinated animals. Analysis

FIGURE 7. Corneal neovascularization and opacity are significantly reduced in vaccinated mice in a dose-dependent fashion after HSV-1

challenge.

Male and female CD-1 mice were s.c. immunized with 1 3 103–1 3 105 PFU HSV-1 0DNLS or vehicle (PBS) vaccine, followed by an i.m. boost 3 wk

later. Mice (n = 6 per group) were challenged with 1000 PFU HSV-1 McKrae/eye 30 d following the final immunization. Thirty DPI, the mice were

initially evaluated for opacity using a masked observer and then subsequently euthanized. The corneas were removed and processed for whole

mount staining for blood and lymphatic vessels. (A–C) Representative z-stacked corneal images depicting blood (red) and lymphatic (green) vessels

comparing low [(A), 1 3 103 PFU], medium [(B), 1 3 104 PFU], and high [(C), 1 3 105 PFU] HSV-1 0ΔNLS–vaccinated mouse corneas. Scale bar,

100 mm. (D) Metamorph quantification of corneal area covered by lymphatic or blood vessels comparing each group of vaccinated animals.

*p , 0.05, comparing the high vaccine dose to the low vaccine dose for lymphatic vessels, **p , 0.01, comparing the high and medium vaccine

dose to the low vaccine dose for blood vessels. (E) Opacity score of corneas from each groups of vaccinated mice. ΔΔp , 0.01, comparing the

indicated groups to the low vaccine dose group, **p , 0.01, comparing the indicated groups to the vehicle (PBS)–treated group as determined by

ANOVA and Tukey post hoc t test, n = 6 mice per group.




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of isolated proteins by MS from immunoprecipitation runsidentified 15 virus-encoded proteins recognized by the antiserumfrom the high-dose–vaccinated group significantly beyond that ofthe antiserum from the low-dose–vaccinated or naive (vehicle)–vaccinated groups.One family of recognized proteins included sixHSV-1 encoded glycoproteins: gB, glycoprotein H (gH), glyco-protein C (gC), gD, glycoprotein E (gE), and glycoprotein L (gL).Of the two glycoproteins with the highest reactivity score to theantiserum, both recombinant gB and gH or derived peptides usedas immunogens or antagonists have been reported to suppressHSV replication, block anterograde or retrograde spread of thevirus, and/or prevent virus-associated disease through a robustT cell orAb response to theAg (23, 29, 31, 57, 58). Recombinant gCand gD have also been evaluated as prototypical vaccines againstHSV-1 administered prophylactically or therapeutically withreported success (19–21, 25, 59). Similar to the other HSV-1glycoproteins, gEandgLaloneor incombinationwith otherHSV-1 glycoproteins when used as vaccines have been reported toprotect mice from a lethal HSV-1 challenge (60–62).

The nonstructural intracellular proteins primarily recognizedby the antiserum from high-dose HSV-1 0ΔNLS–vaccinated mice,which includes pUL39/ICP6, pUL29/ICP8, pUL40/RR2, pUL31/NEC1, pUL12/NUC, and Rs1/ICP4 are all important elements invirus replication. pUL31, a component of the nuclear egresscomplex, is thought to promote the selective process in infectiousvirusparticleassemblyalongwithpUL17 andpUL25 (63). ICP8 isassDNAbindingproteincritical forefficient annealingof cDNAand,therefore, essential for DNA replication during infection (64).

pUL12 exonuclease is important in the packaging of viralDNA intoinfectious virus in which mutants in the UL12 gene show asignificant loss in the production of infectious virus (65). One ofthe more intriguing outcomes in the proteomic analysis wasthe recognition of pUL39 and pUL40, the former the most highlyrecognized protein by the antiserum from the high-dose–vaccinated mice. Early work reported a pUL39 null mutantreplicated poorly in vitro and in vivo and did not cause oculardisease following cornea infection (66). Human and mouse dataalso suggest this protein is a specific target of CD8+ T cells residingin the TG following infection (67, 68). The other subunit of theribonucleotide reductase, pUL40, is recognized by T cells fromasymptomatic seropositive patients infected with HSV-2 and hasbeen found to be a highly protective immunogen when used tovaccinate guinea pigs against subsequent challenge with HSV-2(69). The other nonstructural intracellular protein, ICP4, found tobe selectively recognizedbyantiserumfromhigh-dose–vaccinatedmice has been found to be instrumental in driving vascularendothelium growth factor A–induced corneal neovascularizationin response to HSV-1 infection (70). This recognition andpredicted neutralization of ICP4 are consistent with the reducedlevel of corneal neovascularization in the high-dose–vaccinatedmice compared with the low-dose–vaccinated animals followingocular HSV-1 challenge (Fig. 7).

A third group of HSV-1 proteins recognized by the antiserumfrom high-dose–vaccinated mice includes the tegument andcapsid proteins pUL19/VP5, pUL48/VP16, and pUL25/CVC2.These proteins alongwith the other capsid proteins recognized by

TABLE III. HSV-1 protein recognition by antiserum from vaccinated mice

HSV-1 Protein High Dose (1 3 105 PFU) Low Dose (1 3 103 PFU) Vehicle

UL39/ICP6/RR1 6,793,035 6 1,303,967a 352,954 6 54,369 372,251 6 25,242UL19/MCP/VP5 6,696,422 6 890,026a 1,843,337 6 869,108 535,912 6151,606UL29/ICP8/DBP 5,597,621 6 1,263,758a 130,614 6 90,589 94,761 6 50,909UL27/gB 4,453,171 6 884,210a 106,422 6 63,346 22,788 6 11,578UL22/gH 2,223,914 6 550,125a 12,540 6 12,540 0 6 0UL18/TRX2/VP23 2,335,474 6 487,355c 967,491 6 392,761d 330,052 6 60,015UL40/RR2 1,773,914 6 513,782a 85,050 6 24,622 89,785 6 21,998UL44/gC 1,329,966 6 253,999a 20,216 6 9945 0 6 0US6/gD 1,026,939 6 191,329a 0 6 0 0 6 0UL38/TRX1/VP19c 827,451 6 263,020d 219,067 6 125,881 28,706 6 9146US8/gE 826,764 6 169,833a 72,489 6 18,495 41,676 6 9301UL35/SCP/VP26 779,290 6 127,433c 239,978 6 160,633d 28,879 6 22,879UL1/gL 554,211 6 147,467a 1386 6 1386 721 6 721UL48/VP16 584,616 6 145,499a 99,682 6 61,382 32,467 615,242UL31/NEC1 584,149 6 204,252b 105,703 6 28,173 63,740 6 23,269UL26/VP24/21 414,514 6 125,223d 130,212 6 72,319 41,565 6 9047UL12/NUC 372,549 6 87,858a 65,219 6 29,728 83,159 6 35,251UL25/CVC2 309,826 6 75,562b 104,840 6 49,050 51,411 6 25,687RS1/ICP4 277,967 6 73,595b 67,645 6 18,463 29,843 6 12,568

Serum from naive WT or HSV-1 0ΔNLS–immunized mice (high and low dose) was used to immunoprecipitate viral-encoded proteins from HSV-1–infected Vero cells.Precipitated proteins were analyzed by MS. Proteins derived from HSV-1 were identified by cross-referencing derivative peptide ions with a reference sequencedatabase. Numbers reflect matched peptide abundance/intensity per protein 6 SEM by antiserum from HSV-1 0ΔNLS– or PBS (vehicle)–vaccinated mice (n = 7 pergroup from three independent experiments).ap , 0.01, comparing the high dose to the other groups of vaccinated mice.bp , 0.05, comparing the high dose to the other groups of vaccinated mice.cp , 0.01, comparing the indicated group to the vehicle group as determined by ANOVA and Scheffé multiple comparison test.dp , 0.05, comparing the indicated group to the vehicle group as determined by ANOVA and Scheffé multiple comparison test.WT, wild-type.




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FIGURE 8. Passively immunized mice with sera from high 0ΔNLS dose–vaccinated mice protect against HSV-1–mediated corneal pathology.

Sera (250 ml) from male and female mice (n = 10 per group) that were immunized and boosted with 1 3 103 PFU HSV-1 0ΔNLS, 1 3 105 PFU HSV-1

0DNLS, or vehicle (PBS) was administered i.p. to naive recipients 24 h prior to infection with HSV-1 (1000 PFU/cornea). (A) Mice were monitored for

cumulative survival out to 21 DPI. **p, 0.01, comparing the recipients of sera from 0ΔNLS-vaccinated groups to the PBS-vaccinated sera recipients

as determined by the Mantel–Cox test. (B) Over the course of the first 7 DPI, cornea sensation was evaluated using a Cochet–Bonnet esthesiometer

comparing the recipients of sera from 0ΔNLS-vaccinated groups to each other and to the PBS sera recipients. Only five mice from the PBS sera

recipient groups could be evaluated at 7 DPI because of mortality. *p , 0.05, **p , 0.01, comparing the recipients of sera from 0ΔNLS-vaccinated

mice to the recipients of sera from PBS-vaccinated mice, ^p , 0.05, comparing the recipient of sera from the 1 3 105 0ΔNLS-vaccinated mice to

that of recipients of sera from the 1 3 103 0ΔNLS-vaccinated animals as determined by ANOVA and Tukey t test. (C) The corneas of passively

immunized mice infected with HSV-1 were surgically removed from exsanguinated animals that survived out to 21 DPI and assessed for opacity

measuring the OD at 500 nm wavelength in a 30 3 30 matrix over the cornea surface. Uninfected mouse corneas served as the baseline control

(dotted line). (D) The corneas from C were then stained for lymphatic (LYVE-1) and blood (CD31) vessels. Metamorph quantification of corneal area

containing LYVE-1+ and CD31+ vessels. **p , 0.01, *p , 0.05, comparing the PBS serum–immunized group to all other groups, ##p , 0.01,

comparing the 1 3 105 0ΔNLS passively immunized mice to the PBS- and 1 3 103 0ΔNLS passively immunized mice as determined by ANOVA and

Tukey t test. Representative images for (E) PBS, (F) low 1 3 103 0ΔNLS, and (G) high 105 0ΔNLS passively immunized mice are shown. (E and F)

Original magnification 340.




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either high- or low-dose–vaccinatedmice, including pUL18/VP23,UL38/VP19c, UL35/VP26, and UL26/VP24/21, are critical inHSV-1 replicationwhether it be transactivation of viral immediateearly lytic genes (VP16) or incorporation and assembly of capsidproteins into the capsid shell (71–73). Because VP5, VP19c, andpUL25 contribute to the long-range axonal transport of theinfectious unit of the virion to the neuronal cell bodies through anassembled capsid-associated tegument complex (74), it is quitepossible the antiserum recognizes only a few epitopes on a singleprotein entity that results in the immunoprecipitation of themajority of the complex. Consequently, one or more of theseproteins may not be a contributing member of the protectiveimmune repertoire of Ags recognized by the antiserum from thehigh-dose 0ΔNLS–vaccinated mice. Another possibility is thecomplex itself forms a structural epitope recognizedby theAb thatcontributes to protection from virus spread, replication, andestablishment of latency. Thus, analysis of single viral-encodedproteins that collectively form the capsid-associated tegumentproteincomplexwouldafford little tonoprotectionwhenusedas avaccine as the “protective” epitope if it is only formed by theassociated protein complex. Although this may be true for Abrecognition, epitopes representing capsid or tegument proteinshave been found to generate a robust CD8+ T cell response withpolyfunctional effector T cells that elicit a protective immuneresponseagainstHSV-1keratitis (30, 75).The importanceofHSV-1protein recognition by the antisera is underscored by the passiveimmunization results that show quantifiable corneal pathology isreduced or absent in high 0ΔNLS–dose-vaccinated mice com-pared with the low-dose– or PBS-vaccinated control followingHSV-1 challenge (Fig. 8). Even though survival was similarbetween the high and low 0ΔNLS–vaccinated mice and signifi-cantly above the PBS-vaccinated control group, the difference incornea pathology between the 0ΔNLS-immunized groups dem-onstrates the need to incorporate a more encompassing approachin evaluating the success of a vaccine to protect against an ocularpathogen, a criteria often overlooked or only accomplishedsubjectively from most laboratories in the HSV-1 field. In thecase of the current investigation, further studies are required toclearly elucidate those specific viral-encoded tegument andcapsid proteins as well as other identified viral proteins that arecontributors to the HSV-1 0ΔNLS vaccine efficacy.

DISCLOSURES

D.J.J.C. is a member of the scientific advisory board of thecompany, Rational Vaccines, Inc., that owns the patent rightsto the HSV-1 0DNLS vaccine. The other authors have no financialconflicts of interest.

ACKNOWLEDGMENTS

We thank the Laboratory for Molecular Biology and Cytometry Researchat OUHSC for use of the Core Facility, which provided proteomic services.We thank Renee Sallack for technical help in processing tissue. We

acknowledge the following individuals/entities for providing materialresources: Brian Gebhardt, original stock of HSV-1 McKrae; StaceyEfstathiou, rHSV-1 SC16 ICP0-Cre virus; and Rational Vaccines, Inc.,live-attenuated HSV-1 0DNLS vaccine.

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