Distinguishing Features of High- and Low-Dose Vaccine against Ocular HSV-1 Infection Correlates with Recognition of Specific HSV-1–Encoded Proteins

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 1x10 3 to 1x10 5 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 (1x10 4 –1x10 5 PFU) of the HSV-1 0ΔNLS–vaccinated mice, displaying reduced viral latency, ocular pathology, or inflammation in comparison with the lowest dose (1x10 3 PFU) or vehicle vaccine employed. Fifteen HSV-1–encoded proteins were uniquely recognized by antisera from high-dose (1x10 5 PFU)–vaccinated mice in comparison with low-dose (1x10 3 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. I

Full text

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. Sjoelund
Daniel J. J. Carr, Grzegorz B. Gmyrek, Adrian Filiberti, Amanda N. Berube, William P. Browne,
http://www.immunohorizons.org/content/4/10/608
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Distinguishing Features of High- and Low-Dose Vaccine
against Ocular HSV-1 Infection Correlates with Recognition of
Specific HSV-1–Encoded Proteins
Daniel J. J. Carr,*
,†
Grzegorz B. Gmyrek,* Adrian Filiberti,* Amanda N. Berube,* William P. Browne,* Brett M. Gudgel,* and
Virginie 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 0Δnuclear 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 1 310
3
to 1 310
5
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 (1 310
4
–13
10
5
PFU) of the HSV-1 0ΔNLS–vaccinated mice, displaying reduced viral latency, ocular pathology, or inflammation in comparison
with the lowest dose (1 310
3
PFU) or vehicle vaccine employed. Fifteen HSV-1–encoded proteins were uniquely recognized by
antisera from high-dose (1 310
5
PFU)–vaccinated mice in comparison with low-dose (1 310
3
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 predominantly
of collagen lamellae highly organized into interwoven fibrils in the
anterior stroma that run parallel to the cornea surface in the
posterior 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 from
environmental insult and allow passage of light to the lens and
retina. Although the eye is considered an immunologically
privileged 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 McGee
Eye Institute No. 415A, 608 Stanton L. Young Boulevard, Oklahoma City, OK 73104. E-mail address: dan-carr@ouhsc.edu
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 unrestricted
grant 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 of
Oklahoma 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 or
infection, the immune privilege dynamics of the cornea dramat-
ically change.
HSV-1 is a highly successful human pathogen that has a
seroprevalence rate above 50% worldwide but is declining in
prevalence in the United States and elsewhere (6). It continues to
be a significant ocular pathogen that can elicit immune-driven,
irreversible damage to the cornea in patients that experience
episodic reactivationof latent virus. Experimentally, in responseto
ocular infection resident and infiltrating myeloid-derived cells are
activated and/or initially recruited to the cornea, followed by NK
cells and T lymphocytes that collectively facilitate clearance of
the pathogen insult but also lead to severe inflammation and
irreversible tissue pathology, including vascularization (blood and
lymphatic vessel genesis) of the normally avascular central cornea
(7–9). In addition, the innervation of the cornea, which normally
maintains the homeostasis of the ocular surface (10), is dramatically
altered and can lead to dry eye disease (11–14). A compromised
visual axis as a result of HSV-1–mediated corneal pathology that
cannot be managed successfully often leads to corneal transplant, a
high-risk surgical procedure with frequent graft failures (15). As
there is currently no intervention to permanently alleviate patient
suffering or prevent the acquisition of HSV-1 or HSV-2, strategies
are sought to enhance patient resistance to this infection, which in
2013, had an estimated economic burden of over $90 million dollars
in the United States alone (16).
The potential to protect individuals from pathogens or
products encoded by pathogens has been realized and demon-
strated experimentally for well over 100 y (17).Early vaccine work
against HSV-1 suggested Ags associated with the envelope or the
specific subunit glycoprotein D (gD) were protective in preventing
mortality or the establishment of latency following acute infection
in mice (18, 19). Follow-up studies targeting gD or other HSV-1
subunits as prophylactic or therapeutic vaccines have demon-
strated various degrees of efficacy in the generation of sterile
immunity, reducing the establishment of latency or preventing
reactivation of latent virus (20–25). Most subunit vaccine
approaches likely generate an Ab response with modest T cell
input. As T cells and specifically CD8
+
T cells have been shown to
control HSV-1 reactivation in mice (26–28), recent studies by one
grouphave focused on prophylacticvaccinesthat elicit a protective
CD8
+
T cell response using HLA-restricted transgenic mice and
rabbits (29–31). Specifically, peptide epitopes of glycoprotein B
(gB) and the tegument proteins VP11/12 and VP13/14 identified for
polyfunctional CD8
+
T cell responses from seropositive, asymp-
tomatic HLA-A*201-01 individuals used in CpG-adjuvant vaccines
prevented HLA-A*2:01 transgenic mouse and rabbit mortality
associated with a significant drop in ocular viral replication
following acute HSV-1 challenge. A follow-up study using a
different set of HLA-A*02:-01–restricted epitopes from UL9,
UL25, and UL44 gene products in a prime/pull therapeutic
strategy found this approach significantly increased the tissue
resident effector memory CD8
+
T cell population in the ganglio n of
HSV-1 latently infected mice and prevented virus reactivation and
reduced ocular disease scores (32). Although these results hold
promise in the development of candidate prophylactic or
therapeutic vaccines against ocular HSV-1 infection, with few
exceptions, none of the studies referenced above included an
evaluation of the visual axis in terms of quantifiable pathological
changes of the cornea, including function as well as analysis of
visual performance.
Previous studies by our group have identified theapplication of
a live, attenuated HSV-1 mutant (HSV-1 0Δnuclear location signal
[NLS]) as a highly efficacious prophylactic vaccine against HSV-1
(33). HSV-1 0ΔNLS was demonstrated to be a safe vaccine using
IFNAR1-deficient mice and provided superior protection com-
pared with a subunit vaccine used inclinical trials (33, 34). Notably,
the efficacy ofthe vaccinein the control of virus replication in the
cornea was linked to early expression of complement and the
neonatal Fc receptor, which supported the correlate of protection
tobeAb(35).Finally,HSV-10ΔNLS–vaccinated mice retained
corneal function with preservation of the visual axis in the first
study to report such findings (36). In the current investigation, a
dose-response study was conducted using the HSV-1 0ΔNLS
vaccine to determine the lowest dose required to maximize the
protective efficacy. Although all doses of vaccine protected mice
from HSV-1–mediated mortality, there were significant differ-
ences in terms of level of inflammation and corneal pathology with
mice vaccinated with the high-dose inoculum showing the least
pathology in comparison with the low-dose–vaccinated mice. We
identified 15 viral-encoded proteins uniquely recognized by
antiserumfromhigh-dose–vaccinated mice that may serve as
candidates 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 infection
Female and male outbred CD-1 mice were obtained from Charles
River Laboratories (Wilmington, MA). Ai14/Rosa26-tdTomato-
Cre-reporter (Rosa) male and female mice on a C57BL/6
background were originally purchased from The Jackson Laboratory
(Bar Harbor, ME) and bred in-house. All animals were housed in a
specific pathogen-free facility at the Dean McGee Eye Institute o n
the University of Oklahoma Health Sciences Center (OUHSC)
campus. Investigators adhered to procedures approved by the
OUHSC Institutional Animal Care and Use Committee (protocol
no. 16-087-SSIC and no. 19-060-ACHIX), and animals were
handled in accordance with the National Institutes of Health’s
Guide for the Care and Use of Laboratory Animals. Mice were
anesthetized for all procedures by i.p. injection of ketamine (100
mg/kg) plus xylazine (5.0 mg/kg) and were euthanized by cardiac
perfusionof 10 ml PBS for tissue collection. Animals (6–10 wk old)
were vaccinated using a prime/boost approach via ipsilateral
footpad (s.c.) and quadriceps (i.m.) injection 3 wk later as
described (33). The immunization dose ranged from 1 310
3
to 1 3
10
5
PFU HSV-1 0ΔNLS (KOS strain) in 10 ml PBS (primer and
boost). PBS alone served as the vehicle control.
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CD-1 mice were infected 30 d following the secondary boost by
applying 1 310
3
PFU HSV-1 McKrae to each cornea following
partial epithelial debridement with a 25-gauge needle. Rosa26
mice were infected 30 d postboost by applying 1 310
4
PFU HSV-1
SC16 expressing Cre recombinase (37) to each cornea following
debridement as described above.
Serological and virological assays
Peripheral blood was obtained from the facial vein of anesthetized
mice at 30 d postboost and fractionated using Microtainer serum
separation tubes (Becton Dickinson, Franklin Lakes, NJ). Ab-
containing serum was evaluated for virus-neutralizing titers in the
presence of guinea pig complement (Rockland, Limerick, PA) on
Vero cell monolayers as described (33). To quantify virus found in
the tear film or tissue during acute infection of mice, corneas were
swabbed with cotton-tipped applicators, and tissue was excised
and homogenized, and the clarified supernatant was assayed for
infectious virus by standard plaque assay (33). HSV-1 genome copy
number was conducted by PCR on total DNA isolated from the
trigeminal ganglia (TG) of surviving mice 30 d postinfection (DPI)
using a proprietary primer-probe mixture, according to the
manufacturer’s instructions (Virusys, Taneytown, MD).
Analysis of corneal pathology
Gross corneal pathology was conducted at 7 DPI by a masked
observer examining eyes through a Kowa SL14 portable slit lamp
biomicroscope (KowaOptimed, Torrance, CA) usingthe following
scoring scheme: 0, no pathology; 1, injected eye, no opacity; 2, focal
opacity; 3, hazy opacity over entire cornea; 4, dense opacity in
central 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 was
performed 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 washes
in PBS. The tissue was then incubated in PBS containing 1% Triton
X-100 overnight. Labeling, imaging, and analysis of corneal vessels
were performed using an Olympus FV1200 confocal microscope
and MetaMorph Imaging suite software (Sunnyvale, CA) as
previously described (39).
Visualization and analysis of Cre-inducible, tomato red–
staining cells were conducted using an Olympus FV1200 confocal
microscope and MetaMorph Imaging suite software. Specifically,
the TG from vaccinated mice were harvested 30 DPI and placed in
PBS for 5 min. PBS was removed, and 4% paraformaldehyde was
added to each TG. The samples were processed in 5-mm sections
and placed onto slides by Excalibur Pathology (Norman, OK).
Slides were then imaged, and the threshold area calculated for
each section was visualized using MetaMorph Imaging suite
software.
Flow cytometry
Corneas, TG, and sub–mandibular lymph nodes (MLN) were
harvested from vaccinated mice at 3 and 7 DPI following
exsanguination. Briefly, TG or MLN pairs were macerated into
single-cell suspensions in RPMI 1640 medium containing 10%
heat-inactivated FBS, 13antibiotic/antimycotic solution, and
10 mg/ml gentamicin (Invitrogen, Carlsbad, CA) (complete media).
Corneas were digested in 0.25 W¨
umsch units of Liberase TL
enzyme(Roche Diagnostics, Indianapolis, IN) suspended in500 ml
of complete media at 37°C for1 h and exposed to triturationevery
15–20 min. Corneas, TG, and MLN were then filtered through a
40-mmnylonmeshfilter (Thermo Fisher Scientific, Waltham,
MA) prior to labeling. Cell suspensions were blocked with anti-
CD16/32 (eBioscience, San Diego, CA), labeled with the indicated
combination of Abs for 20–30 min, and washed in 13PBS
FIGURE 1. Mice vaccinated with 1 310
3
–1310
5
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 1 310
3
–1310
5
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,
Δ
p,0.05, comparing 1 310
4
–1310
5
PFU HSV-1 0ΔNLS to the 1 310
3
PFU HSV-1 0ΔNLS–vaccinated group as determined by
ANOVA and Scheffé multiple comparison test. (B) Mice vaccinated with 1 310
3
–1310
5
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 MACSQuant
10 flow cytometer with MACSQuantify software (Miltenyi Biotec,
Bergisch Gladbach, Germany). Isotype labeling and fluorescence
minus one controls were conducted to validate specificlabeling
and negate spectral overlap respectively (Supplemental Fig. 1).
Cytokine/chemokine quantification
Corneas and TG from vaccinated mice were collected 7 DPI
following exsanguination. Uninfected mouse tissue served as
baseline controls. Tissue wasweighed upon extraction and placed
in Next Advance GREEN bead lysis tubes (Averill Park, NY)
containing PBS and 13protease inhibitor mixture (Santa Cruz
Biotechnology, Dallas, TX). The samples were then homogenized
in a Next Advance Bullet Blender Storm 24 homogenizer for
10 min, sonicated in a water bath for 10 min, and subsequently
analyzed for cytokine/chemokine content using customized kits
for select analytes (MilliporeSigma, Billerica, MA) and a Bio-Plex
suspension array system to detect and quantify analytes (Bio-Rad
Laboratories, Hercules, CA). The sample contents were normal-
ized based on the wet weight of each cornea and reported in
picogram analyte per milligram cornea. Samples were diluted 1:5
prior to analysis. The limit of detection of each analyte evaluated
was as follows: eotaxin, 3.17 pg; G-CSF, 3.26 pg; GM-CSF, 20.1 pg;
IFN-g,3.22pg;IL-1a,3.78pg;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 1 310
3
–1310
5
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 6SEM. *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 ttest. **p,0.01, comparing the indicated group to the PBS-vaccinated control group,
Δ
p,0.05, comparing the
indicated groups to the low (1 310
3
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 (1 310
3
–1310
5
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 1 310
4
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 identification
using Ab from vaccinated mice
To determine the repertoire of HSV-1 proteins that are recognized
by HSV-1 0ΔNLS–vaccinated mouse serum, 1.2 310
6
Vero cells
were plated into each well of a six-well plate and infected at a
multiplicity of infection of 1.0 with HSV-1 McKrae. Twenty-four
hours postinfection (PI), the cells were collected, washed in 1.0 ml
of PBS twice, and centrifuged at 300 3g,5minforeachwash.
Following the second wash, the supernatant was removed, and the
cells were resuspended in 500 ml of 1% Triton X-100 detergent
(lysis buffer) and placed on ice for 15–20 min. Following the
incubation, cell lysates were clarified from cell debris by
centrifugation (10,000 3g, 10 min at 4°C). The supernatants
from infected and uninfected Vero cells were incubated with
4ml of serum from vaccinated mice and 100 ml of immuno-
magnetic protein G microbeads (Miltenyi Biotec) at 4°C for
30 min with gentle agitation every 5 min. The protein/Ab/
microbead complex was then added onto mMACS magnetic
bead columns (Miltenyi Biotec). The columns were washed four
times with 200 ml lysis buffer, and retained proteins were eluted
with 50 ml 100 mM glycine (pH 2.5).
Trypsin digest of immunoprecipitated prote ins was performed
according to the filter-aided sample preparation protocol (40).
Briefly,theeluatewasbuffer exchanged to 8 M urea, the proteins
were reduced with 10 mM DTT and then alkylated with 10 mM
iodoacetamide. The peptides were eluted in 10 mM ammonium
acetate (pH 8), dried, and resuspended in 10 mM ammonium
formate (pH 10). Liquid chromatography tandem mass spectrom-
etry (MS/MS) was performed by coupling a nanoAcquity Ultra-
Performance LC (UPLC; Waters, Manchester, U.K.) to a Q-TOF
SYNAPT G2S instrument (Waters). Each protein digest (;100 ng
of peptide) was delivered to a trap column (300 mm350 mm
nanoAcquity UPLC NanoEase Column 5 mmBEHC18;Waters)at
aflow rate of 2 ml/min in 99.9% solvent A (10 mM ammonium
formate pH 10, in HPLC grade water). After 3 min of loading and
washing, peptides were transferred to another trap column
(180 mm320 nanoAcquity UPLC 2G-V/MTrap 5 mmSymmetry
C18; Waters) using a gradient from 1 to 60% solvent B (100%
acetonitrile). The peptides were then eluted and separated at a
flow 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 mm3150 mm nanoAcquity UPLC 1.8 mm HSST3; Waters).
The eluent was sprayed via PicoTip Emitters (Waters) at a spray
voltage of 3.0 kV and a sampling cone voltage of 30 V and a source
offset of 60 V. The source temperature was set to 70°C. The cone
gas flow was turned off, the nano flow gas pressure was set at 0.3
bar, and the purge gas flow was set at 750 ml/h. TheSYNAPT G2S
instrument was operated in data-independent mode with ion
mobility (HDMSe). Full-scan mass spectrometry (MS) and
MS/MS (m/z50–2000) were acquired i n resolution mode (20,000
resolution FWHM at m/z 400). Tandem mass spectra were
generated in the trapping region of the ion mobility cell by using a
collisional energy ramp from 20 V (low mass, start/end) to 35 V
(high mass, start/end). A variable IMS wave velocity was used.
Wave velocity was ramped from 300 to 600 m/s (start to end), and
the ramp was applied over the full IMS cycle. A manual release
time of 500 ms was set for the mobility trapping and a trap height
of 15 V with an extract height of 0 V. The pusher/ion mobility
synchronization for the HDMSe method was performed using
MassLynx V4.1 and DriftScope v2.4. LockSpray of Glu–
fibrinopeptide-B (m/z 785.8427) was acquired every 60 s, and
lock mass correction was applied postacquisition.
Raw MS data were processed by ProteinLynx Global Server
(Waters) for peptide and protein identification. MS/MS were
searched against the Uniprot HSV-1 proteome database (release
date November 2, 2017, containing 1776 unreviewed sequences)
with the following search parameters: full tryptic specificity, up
to two missed cleavage sites, carbamidomethylation of cysteine
residues was set as a fixed modification, and N-terminal protein
acetylation and methionine oxidation were set as variable
modifications. Proteins reported were identified in two or more
out of seven samples per group.
Passive immunization
Serum obtained from terminal cardiac punctured PBS (vehicle)–
or 0ΔNLS (1 310
3
or 1 310
5
PFU)–vaccinated mice was pooled
and administered i.p. (250 ml) to naive CD-1 male or female mice
24 h prior to HSV-1 (1000 PFU/eye) challenge. Mechanosensory
function of the cornea was assessed 1–7DPIusingaCochet–
Bonnet esthesiometer as previously described (12). Mice were
monitored for cumulative survival and deaths recorded to 21 DPI.
Mice were subsequently exsanguinated, and the corneas were
assessed for opacity as previously described (41) and subsequently
processed for neovascularization (39).
Statistics
GraphPad Prism 8 was used to analyze data for statistical
significance (p,0.05) as determined using statistical tests
described in each figure. Data are presented as mean 6SEM.
RESULTS
HSV-1 0ΔNLS vaccine suppresses virus replication and
prevents HSV-1–mediated mortality in a dose-
dependent fashion
Previous studies investigating the efficacy of the HSV-1 0ΔNLS
vaccine 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
10
3
PFU HSV-1 0ΔNLS vaccine to higher-dose vaccines. (C) HSV-1 copy number in the TG of vaccinated mice (1 310
3
–1310
5
PFU 0ΔNLS) at day
30 PI (n=21–22 per group). Data were analyzed by ANOVA and Tukey post hoc ttest. *p,0.05, **p,0.01, comparing the 1 310
3
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 1 310
3
PFU HSV-1 0ΔNLS, 1 310
5
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 63, 4 62, 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 1 310
5
PFU for the primary immunization and boost
(33–36). To formalize the minimal efficacious dose of vaccine to
maximize the protective effect against subsequent challenge, mice
were immunized with doses ranging from 1 310
3
to 1 310
5
PFU
prior to challenge. Mice immunized with 1 310
4
–1310
5
PFU
HSV-1 0ΔNLS showed significantly higher neutralizing Ab titers to
HSV-1 compared with mice vaccinated with 1 310
3
PFU HSV-1
0ΔNLS (Fig. 1A). However, all doses of HSV-1 0ΔNLS used to
immunize mice were found to protect animals subsequently
challenged with HSV-1 as measured by cumulative survival (Fig. 1B).
To further compare the dose response of the HSV-1 0ΔNLS
vaccinein resistance against ocular HSV-1 challenge, the tear film,
cornea, and TG from mice immunized with 1 310
3
–1310
5
PFU
HSV-1 0ΔNLS or vehicle (PBS) were evaluated for viral content.
Mice vaccinated with any dose of HSV-1 0ΔNLS were found to
shed less virus during acute infection (i.e., 3–7 DPI) compared
with PBS-vaccinated mice (Fig. 2A). By comparison, only the 1 3
10
4
–1310
5
PFU dose of HSV-1 0ΔNLS reduced corneal virus
titer, whereas the 1 310
3
PFU dose of HSV-1 0ΔNLS did not have a
significant effectby7DPI(Fig.2B).Similartowhatwasobserved
in viral shedding, mice immunized with any dose of the HSV-1
0ΔNLS vaccine showed reduction in virus replication in the TG
compared with the PBS-vaccinated control group (Fig. 2C).
However, little to no infectious virus was recovered from the TG
of mice vaccinated with 1 310
4
–1310
5
PFU HSV-1 0ΔNLS
compared with the low (1 310
3
PFU) dose of HSV-1 0ΔNLS in
which over 50% of mice TG had detectable levels of infectious
virus (Fig. 2C).
A critical aspect of HSV-1 infection is the establishment
of latency as a result of invasion of the sensory neurons that reside
in the TG during acute infection. To determine the success of
permanently colonizing neurons in the TG, Cre-inducible
tdTomato fluorescent reporter mice vaccinated with HSV-1
0ΔNLS (1 310
3
–1310
5
PFU) were challenged with HSV-1
encoding Cre recombinase under the infected cell protein (ICP)
0 lytic gene promoter (37). Cells that survive the acute infection
are permanently “tagged”and will express tdTomato. TG from
mice immunized with 1 310
4
–1310
5
PFU HSV-1 0ΔNLS
displayed significantly fewer labeled cells as measured by
threshold area compared with mice vaccinated with 1 310
3
PFU HSV-1 0ΔNLS at 30 DPI (Fig. 3A, 3B). These results are
consistent with the genome copy number of HSV-1 recovered in
the TG of latent-infected, vaccinated mice with a significant
reduction recovered from mice immunized with 1 310
4
–1310
5
PFU HSV-1 0ΔNLS compared with the 1 310
3
PFU dose 30 DPI
(Fig. 3C). In this experiment, PBS-vaccinated mice did not survive
and, therefore, could not be assessed as a baselinepositive control.
However, a previous study found PBS-vaccinated tdTomato
fluorescent reporter mice that did survive the acute infection to
30 DPI displayed a similar phenotype as that shown with 1 310
3
PFU 0ΔNLS-vaccinatedanimals (36). In summary, all doses of the
HSV-1 0ΔNLS vaccine tested showed protection against ocular
HSV-1 challenge in terms of cumulative survival and viral spread
and replication in the TG, although the lowest dose evaluated
displayed no efficacy against HSV-1 replication in the cornea,
which correlated with a low neutralizing Ab titer and higher virus
copy number found in the TG of latent-infected mice.
Leukocyte infiltration into the cornea and TG is stymied in
HSV-1 0ΔNLS–vaccinated mice in a dose-dependent manner
HSV-1 infection of the cornea elicits a robust cellular immune
response initially with a massive onslaught of neutrophils and
activation of mast cells, followed by the infiltration of activated
monocytes/macrophages and NK cells and eventually CD4
+
and
CD8
+
Tcells(5,42–44). We evaluated T and myeloid cell
infiltration comparing vaccinated to nonvaccinated animals at
3 and 7 DPI in the cornea, the latter time point when non-
vaccinated mice begin to succumb to infection (Fig. 1B). A
representative flow plot comparing PBS-, 1 310
3
PFU 0ΔNLS
(low)–, and 1 310
5
PFU 0ΔNLS (high)–vaccinated mice for T and
B cells (Fig. 4A) and myeloid cells (Fig. 4D) is provided. At 3 DPI,
the high-dose–vaccinated mice showed significantly fewer CD4
+
and CD8
+
T cells residing in thecornea compared withthe PBS- or
low-dose–vaccinated mice (Fig. 4B). By 7 DPI, the high and low
0ΔNLS–dose-vaccinated mouse corneas retained fewer T cells
than the PBS-vaccinated counterparts (Fig. 4C). A similar finding
was 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–vaccinated mice possessing significantly fewer cells
compared with the PBS-vaccinated control animals. Significantly
fewer monocyte/macrophage populations were found in the
cornea of the high- and low-dose 0ΔNLS–vaccinated mice
compared with the PBS-vaccinated controls at 3 (Fig. 4G) and 7
(Fig. 4H) DPI as well. Although modest in number, the corneas of
infected mice contained significantly fewer CD19
+
B lymphocytes
in the high-dose–immunized mice compared with the vehicle
control–vaccinated animals at 7 DPI (Fig. 4C), but not 3 DPI (Fig.
4B). As we are unable to detect B lymphocytes in the uninfected
CD-1 mouse cornea, the infiltration of these cells following
infection is of interest relative to local Ab production. Currently,
we have not been able to detect anti–HSV-1 Ab in the tear film of
vaccinated mice prior to or following infection.
We also evaluated T and myeloid cell infiltration comparing
vaccinated to nonvaccinated animals at 3 and 7 DPI in the TG. A
ANOVA and Tukey post hoc ttest. (D) Representative flow plot corneal digest for distribution of myeloid cells in vaccinated mice designated as
1: (CD45
+
CD11b
+
Ly-6G
mid
Ly-6C
+
), 2: (CD45
+
CD11b
+
Ly-6G
high
Ly-6C
int
), and 3: (CD45
+
CD11b
+
Ly-6G
2
Ly-6C
high
). Absolute number of granulocytes
(CD45
+
CD11b
+
Ly-6G
mid
Ly-6C
+
) and (CD45
+
CD11b
+
Ly-6G
high
Ly-6C
int
)at3(E) and 7 (F) DPI. Absolute number of monocytes (CD45
+
CD11b
+
Ly-6G
2
Ly-6C
high
)at3(G) and 7 (H) DPI. *p,0.05, **p,0.01, ***p,0.001, comparing the indicated group to PBS-vaccinated group or 1 310
3
0ΔNLS
dose–vaccinated mice as determined by ANOVA and Tukey post hoc ttest.
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FIGURE 5. Diminished leukocyte infiltrate in the TG of vaccinated mice in response to HSV-1 infection.
Male and female mice were s.c. immunized with 1 310
3
PFU HSV-1 0ΔNLS, 1 310
5
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 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 51 611, 42 65, and 102 620,
respectively. *p,0.05, comparing the indicated group to PBS-vaccinated group as determined by ANOVA and Tukey post hoc ttest, 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-
6G
mid
Ly-6C
+
), 2: (CD45
+
CD11b
+
Ly-6G
high
Ly-6C
int
), and 3: (CD45
+
CD11b
+
Ly-6G
2
Ly-6C
high
). Absolute number of granulocytes (Continued)(Continued)
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representative flow plot comparing PBS-, 1 310
3
PFU 0ΔNLS
(low)–,and1310
5
PFU 0ΔNLS (high)–vaccinated mice for T and
B cells (Fig. 5A) and myeloid cells (Fig. 5D) is provided. Unlike the
cornea, 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 high
0ΔNLS–dose-vaccinated mice at 7 DPI (Fig. 5C). The granulocyte
infiltration was not impacted by the 0ΔNLS vaccination at 3 DPI
with similar numbers of CD11b
+
Ly-6G
+
Ly-6C
+/int
cells (Fig. 5E).
However, at 7 DPI, the granulocyte numbers were significantly
reduced in the TG of the high 0ΔNLS–dose-immunized mice
compared with the PBS control–vaccinated animals (Fig. 5F). By
comparison, monocyte/macrophage (CD45
+
CD11b
+
Ly-6G
2
Ly-
6C
high
) numbers were significantly lower in the TG of the high
0ΔNLS–dose-immunized mice compared with the PBS control–
vaccinated animals at 3 (Fig. 5G) and 7 (Fig. 5H) DPI. No
differences were found comparing the low-dose–vaccinated mice
to the PBS-vaccinated control group. It should also be noted there
was no change in the B lymphocyte number that resides in the TG
comparing uninfected levels to that following infection, suggesting
that unlike the cornea, B lymphocytes do not traffictotheTGof
infected mice (Fig. 5B, 5C).
Next, we surveyed the draining lymph nodes (MLN) of
vaccinated mice at 3 and 7 DPI to determine if the results found in
the infected tissue mirrored that foundin the organized lymphoid
tissue most responsible for the generation of the adaptive immune
response during acute infection. The results of surveying the MLN
at these time points found the cell numbers reflected a very similar
profile to that reported for the cornea for T lymphocytes (Fig. 6A)
and myeloid cells (Fig. 6D). Specifically, CD4
+
and CD8
+
Tcells
(Fig. 6B, 6C) as well as the granulocyte (Fig. 6E, 6F) and monocyte/
macrophage (Fig. 6G, 6H) populations were significantly reduced
in the MLN of the high 0ΔNLS–dose-vaccinated mice compared
with the PBS- and low-dose–vaccinated groups at 3 and 7 DPI. In
addition to T cells, B lymphocyte numbers in the MLN of the high
0ΔNLS–dose-vaccinated animals were considerably lower than
the low-dose–or PBS-vaccinated groups at 3 (Fig. 6B) and 7 (Fig.
6C) DPI.A similar profile was found in thecornea, TG,and MLN in
mice vaccinated with 1 310
4
PFU 0ΔNLS as that with 1 310
5
PFU
0ΔNLSat7DPI(datanotshown).The3DPItimepointwasnot
conducted with 1 310
4
PFU 0ΔNLS-vaccinated animals. With
some exceptions, the effectiveness of the vaccine in terms of
reduction in HSV-1 found in a given tissue is inversely correlated
with the leukocyte infiltrate and the lack of expansion of cells
within the draining lymph nodes. We surmise the overall
attenuated cellular response to infection in the surveyed tissue of
vaccinated mice is due to greater control of viral replication and,
therefore, fewer Ag available to drive local and regional immune
activation postchallenge.
Select cytokine and chemokines levels expressed in infected
cornea and TG are dramatically reduced in HSV-1
0ΔNLS–vaccinated mice
Chemokines including CCL2, CCL3, CCL5, CXCL1, and CXCL10
are expressed early in the cornea following HSV-1 infection
(45–47). Neutralization or loss of select chemokines or their
cognate receptor results in aberrant and, oftentimes, loss of
leukocyte infiltration during acute corneal HSV-1 infection (38,
46, 48–53). Therefore, to determine if the reduction in corneal
leukocyte infiltration in response to HSV-1 infection in the
vaccinated mice correlated with chemokine expression, the
expression 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, vaccinated
mouse corneas in accordance with 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 the
vaccinated 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,and
TNF-a, were all reduced inthe vaccinated animals, but the levels
did not achieve significant (p,0.05) differences in large part
because of sample variation (Table I).
T cell and myeloid cell infiltration into the TG was also muted
in the higher-vaccinated–dose animals following HSV-1 infection.
Such results were reflected by the expression of cytokines and
chemokines. 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 (1 310
4
–1310
5
PFU HSV-1 0ΔNLS)–vaccinated mice compared with the vehicle-
or lower-dose–immunized animals (Table II). Although expres-
sion was modest, TNF-alevels in the TG were significantly
reduced in all vaccinated mice compared with the vehicle
control–vaccinated group (Table II). Other immune mediators,
including CXCL1, G-CSF, IL-6, and M-CSF, were lower in the
higher-vaccinated mouse TG but, because of variability, did not
reach significance (Table II). Neither IL-1bnor VEGF-A was
detectable above background (Table II), whereas CXCL2 and
IL-10 showed high background levels (in uninfected animals)
and, therefore, were not included in the analysis (data not
shown). Taken together, the measurement of analytes in the
cornea and TG reveal a strong correlation with the reduction of
leukocyte infiltration, control of viral replication in these tissues,
and Ab neutralization titers observed in thehigh-dose–vaccinated
mice7DPI.
(CD45
+
CD11b
+
Ly-6G
mid
Ly-6C
+
) and (CD45
+
CD11b
+
Ly-6G
high
Ly-6C
int
)at3(E) and 7 (F) DPI. Absolute number of monocytes (CD45
+
CD11b
+
Ly-6G
2
Ly-6C
high
)at3(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 ttest.
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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.
Male and female mice were s.c. immunized with 1 310
3
PFU HSV-1 0ΔNLS, 1 310
5
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 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 386,139 613,075, 143,432 63,572, (Continued)(Continued)
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Corneal neovascularization is greatly diminished in high-
dose HSV-1 0ΔNLS–vaccinated mice
Corneal neovascularization is a hallmark of herpetic stromal
keratitis in rodents and humans alike (8). Previously, we reported a
loss of vision in mice as a result of HSV-1 infection could be linked
to severe corneal opacity and gross neovascularization (36).
Therefore, we investigated the impact of vaccine doses on the
severity of corneal blood and lymphatic vessel genesis at 30 DPI, a
time when maximum angiogenesis is evident (54). Our results
reflect the dose-response efficacy of the vaccine with the higher
vaccine doses (1 310
4
–1310
5
PFU HSV-1 0ΔNLS), preventing
corneal hem and lymph angiogenesis in comparison with the low
(1 310
3
PFU HSV-1 0ΔNLS) vaccine dose, which showed a similar
profile to vehicle-vaccinated mice (33) (Fig. 7A–D). Because the
vehicle-vaccinated mice do not survive out to 30 DPI to a
significant degree (Fig. 1B), we assessed corneal opacity in the
animalsat 7 DPI via slitlamp examination using a masked observer.
Similar to what is observed with neovascularization, mice that
received the higher vaccine doses displayed minimal corneal
opacity compared with mice that received low-dose or vehicle
vaccine (Fig. 7E). Collectively, these results concur with the data
above, demonstrating the efficacy of the HSV-1 0ΔNLS vaccine
against ocular viral challenge is greatly attenuated at immunization
doses below 1 310
4
PFU.
Serum from mice immunized with the high HSV-1 0ΔNLS
vaccine dose immunoprecipitates HSV-1 proteins not
recognized using the low HSV-1 0ΔNLS vaccine dose
Because the high vaccine dose demonstrated superior efficacy in
protecting mice against HSV-1 in comparison with mice immu-
nized with the low dose, we hypothesized viral proteins may be
uniquely recognized by Ab from the antiserum of high-dose–
vaccinated mice. To test this hypothesis, antiserum obtained from
high- and low-dose–immunized mice was evaluated for recogni-
tion of HSV-1 Ags. A total of 15 HSV-1 proteins were recognized by
antiserum from high-dose–immunized mice significantly above
that recognized by low-dose–vaccinated animals (Table III).
Furthermore, two additional HSV-1 proteins (UL18/VP23 and
UL35/VP26) were recognized by the high and low HSV-1
0ΔNLS–immunized mice that were significantly above the level
TABLE I. Cytokine/chemokine expression in cornea of HSV-1 0ΔNLS–vaccinated mice
Cytokine/Chemokine Vehicle 1 310
3
PFU 0ΔNLS 1 310
4
PFU 0ΔNLS 1 310
5
PFU 0ΔNLS
Eotaxin/CCL11 125 652 44 620
a
13 66
b
16 67
b
G-CSF 1077 6795 88 650 24 621 0 60
IFN-ɤ63 619 11 68
b
262
b
060
b
IL-1a133 685 55 618 44 617 59 625
IL-1b77 653 10 67262060
IL-6 158 678 28 618
b
363
b
060
b
IL-10 16 69262
a
060
a
060
a
IP-10/CXCL10 3539 6868 1474 6731
a
343 6154
b
260 6111
b
KC/CXCL1 1947 61261 494 6399 417 6290 46 628
LIF 106 667 40 634 3 63262
MCP1/CCL2 3069 61466 728 6514
a
235 6106
a
96 657
b
M-CSF 18 611 12 66764565
MIG/CXCL9 1188 6422 240 671
b
83 629
b
80 628
b
MIP-1a/CCL3 412 6221 96 664
a
41 613
a
39 614
a
MIP-1b/CCL4 618 6275 118 654
a
28 614
b
20 613
b
MIP-2/CXCL2 3751 63401 1599 61490 58 640 50 631
RANTES/CCL5 99 633 22 65
a
18 66
b
10 66
b
TNF-a16 610 2 62060060
VEGF-A 24 614 10 65362
a
060
a
Corneas were collected at day 7 PI and processed for cytokine/chemokine content by suspension array. Numbers reflect picograms per milligram of each indicated
analyte 6SEM, n=5–6 samples per group. Numbers in bold reflect significant differences.
a
p,0.05, comparing the indicated group to the vehicle control as determined by ANOVA and Dunnett multiple comparison test. Uninfected corneas had no
detectable analyte expressed except those noted in this study (picograms per milligram 6SD, n= 2 per analyte): eotaxin, 7 62; IL-1a,12612; IL-10, 5 65; CXCL10,
15 615; and CXCL1, 39 624.
b
p,0.01, comparing the indicated group to the vehicle control as determined by ANOVA and Dunnett multiple comparison test. Uninfected corneas had no
detectable analyte expressed except those noted in this study (picograms per milligram 6SD, n= 2 per analyte): eotaxin, 7 62; IL-1a,12612; IL-10, 5 65; CXCL10,
15 615; and CXCL1, 39 624.
and 117,423 611,108, 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 ttest. (D) Representative flow plot MLN distribution of myeloid cells in vaccinated mice
designated as 1: (CD45
+
CD11b
+
Ly-6G
mid
Ly-6C
+
), 2: (CD45
+
CD11b
+
Ly-6G
high
Ly-6C
int
), and 3: (CD45
+
CD11b
+
Ly-6G
2
Ly-6C
high
). Absolute number
of granulocytes (CD45
+
CD11b
+
Ly-6G
mid
Ly-6C
+
) and (CD45
+
CD11b
+
Ly-6G
high
Ly-6C
int
)at3(E) and 7 (F) DPI. Absolute number of monocytes
(CD45
+
CD11b
+
Ly-6G
2
Ly-6C
high
)at3(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 ttest.
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displayed by serum from the vehicle-vaccinated mice (Table III).
Additional HSV-1 proteins were precipitated by the HSV-1
0ΔNLS–vaccinated mice, but the abundance of recognition did
not reach significance compared with the vehicle-immunized
mice because of the variability from animal to animal.
Passive immunization and cornea pathology
To further characterize the relevance of differences in protein
recognition of sera from mice immunized with the high (1 310
5
PFU) versus low (1 310
3
PFU) dose of the 0ΔNLS vaccine, naive
mice were passively immunized with antiserum from the high-
and low-dose–vaccinated animals and subsequently challenged
with HSV-1. Relative to mice receiving sera from PBS-vaccinated
(naive control) animals, recipients of sera from either dose of
0ΔNLS vaccine were found to be resistant to HSV-1 challenge in
terms of cumulative survival (Fig. 8A). However, there were
marked differences between mice receiving sera from high versus
low 0ΔNLS–dose vaccine in terms of mechanosensory function
(Fig.8B),opacity(Fig.8C),andneovascularization,including
lymphangiogenesis (Fig. 8D–G). Specifically, there was minimal
corneal sensation loss in the high-dose sera recipients following
HSV-1 infection compared with the other two groups with a 44%
loss in low-dose sera recipients and 96% loss in the naive sera
recipients by 7 DPI (Fig. 8B). Likewise, HSV-1–infected mice
receiving sera from high-dose–vaccinated animals possessed
cornea opacity levels similar to uninfected mice and lower than
mice receiving sera from low-dose–or PBS-vaccinated, HSV-
1–infected animals (Fig. 8C). Equally revealing is the genesis of
blood 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 was
notablevessel growth in the corneafrom low-dose sera recipients,
albeit lower than naive sera recipients (Fig. 8D–G). Collectively,
the results clearly demonstrate differences between recipients of
sera from high- versus low-dose 0ΔNLS–vaccinated mice in terms
of corneal pathology that likely relates back to coverage of Ag
recognition by Ab from these vaccinated animals.
DISCUSSION
In the current study, we compared different doses of the HSV-1
0DNLS vaccine in mice subsequently challenged with HSV-1 to
define the minimum effective dose that affords the host protection
against ocular HSV-1 infection. Whereas all doses were found to be
highly effective in termsof cumulative survival, there was a distinct
difference in the lack of efficacy of the low dose (1 310
3
PFU)
versus higher doses (1 310
4
–1310
5
PFU) of the HSV-1 0DNLS
vaccine in nearly all other aspects of protection measured in the
infected tissue including virus replication and spread, establish-
ment of latency, inflammation including cytokine and chemokine
expression and leukocyte infiltration, and corneal pathology
including opacity and neovascularization. These findings were
inversely correlated to the neutralizing Ab titer from the vaccinated
mice: the higher the Ab titer, the lower the inflammatory profile,
and reduction in virus replication and spread. It is worth noting the
expansion of the lymphoid populationobservedinthevehicle-or
low-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 1 310
3
PFU 0ΔNLS 1 310
4
PFU 0ΔNLS 1 310
5
PFU 0ΔNLS
Eotaxin/CCL11 1435 6363 633 6157 188 682
a
94 626
b
G-CSF 562 6268 389 6231 12 612 0 60
IFN-ɤ902 627 402 6192 38 626
a
060
b
IL-1b363464060060
IL-6 744 6275 613 6290 0 60060
IP-10/CXCL10 15,194 62,777 11,528 63,919 1648 6865
a
436 6373
b
KC/CXCL1 505 6203 399 6238 27 627 6 64
LIF 155 652 71 631 464
a
060
b
MCP1/CCL2 3634 61,241 1097 6563 79 648
a
12 69
b
M-CSF 48 613 48 620 7 67060
MIG/CXCL9 2310 6423 1655 6457 444 6276
a
103 692
b
MIP-1a/CCL3 1964 6375 865 6388 75 635
a
54 630
b
MIP-1b/CCL4 3238 6656 1380 6646 41 641
b
060
b
RANTES/CCL5 430 676 263 674 28 621
b
060
c
TNF-a33 616 060
a
060
a
060
a
VEGF-A 0 60060666060
TG were collected at day 7 PI, and processed for cytokine/chemokine content by suspension array. Numbers reflect picograms per milligram of each indicated
analyte 6SEM, n=8–10 samples per group. Numbers in bold reflect significant differences.
a
p,0.05, comparing the indicated group to the vehicle control as determined by ANOVA and Dunnett multiple comparison test. Uninfected corneas had no
detectable analyte expressed except those noted in this study (picograms per milligram 6SEM, n= 3 per analyte): eotaxin, 54 654; CXCL10, 16 616; and CXCL1,
39 624.
b
p,0.01, comparing the indicated group to the vehicle control as determined by ANOVA and Dunnett multiple comparison test. Uninfected corneas had no
detectable analyte expressed except those noted in this study (picograms per milligram 6SEM, n= 3 per analyte): eotaxin, 54 654; CXCL10, 16 616; and CXCL1,
39 624.
c
p,0.001, comparing the indicated group to the vehicle control as determined by ANOVA and Dunnett multiple comparison test. Uninfected corneas had no
detectable analyte expressed except those noted in this study (picograms per milligram 6SEM, n= 3 per analyte): eotaxin, 54 654; CXCL10, 16 616; and CXCL1,
39 624.
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the higher doses, which we interpret to suggest Ag abundance is
limited in high-dose–vaccinated mice likely because of control of
the infection by Ab. This result is not to say T cells are not
involved in this process, as there is ample evidence by numerous
investigatorsusingmutantHSV-1asvaccinesthatTcellsplayarole
either directly as effector cells and/or facilitate the Ab response
against HSV-1 (24, 55, 56). However, the current study was focused
on Ab as a means to further delineate differences in vaccine doses
that might be explained by Ag recognition.
Because there was a striking difference in vaccine efficacy
measuring corneal keratitis comparing the high- to low-dose
vaccine, we considered the possibility that unique HSV-1 Ags
might be recognized by antiserum fromthe high-dose–vaccinated
mice 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 1 310
3
–1310
5
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), 1 310
3
PFU], medium [(B), 1 310
4
PFU], and high [(C), 1 310
5
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.
ΔΔ
p,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 ttest, n= 6 mice per group.
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of isolated proteins by MS from immunoprecipitation runs
identified 15 virus-encoded proteins recognized by the antiserum
from the high-dose–vaccinated group significantly beyond that of
the antiserum from the low-dose–vaccinated or naive (vehicle)–
vaccinated groups. One family of recognized proteins included six
HSV-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 the
antiserum, both recombinant gB and gH or derived peptides used
as immunogens or antagonists have been reported to suppress
HSV replication, block anterograde or retrograde spread of the
virus, and/or prevent virus-associated disease through a robust
T cell or Ab response to the Ag (23, 29, 31, 57, 58). Recombinant gC
and gD have also been evaluated as prototypical vaccines against
HSV-1 administered prophylactically or therapeutically with
reported success (19–21, 25, 59). Similar to the other HSV-1
glycoproteins, gE and gL alone or in combination with other HSV-
1 glycoproteins when used as vaccines have been reported to
protect mice from a lethal HSV-1 challenge (60–62).
The nonstructural intracellular proteins primarily recognized
bytheantiserumfromhigh-doseHSV-10ΔNLS–vaccinated mice,
which includes pUL39/ICP6, pUL29/ICP8, pUL40/RR2, pUL31/
NEC1, pUL12/NUC, and Rs1/ICP4 are all important elements in
virus replication. pUL31, a component of the nuclear egress
complex, is thought to promote the selective process in infectious
virusparticle assembly along with pUL17 and pUL25(63). ICP8 is a
ssDNA binding protein critical for efficient annealing of cDNA and,
therefore, essential for DNA replication during infection (64).
pUL12 exonuclease is important in the packaging of viral DNA into
infectious virus in which mutants in the UL12 gene show a
significant loss in the production of infectious virus (65). One of
themoreintriguingoutcomesintheproteomicanalysiswas
the recognition of pUL39 and pUL40, the former the most highly
recognized protein by the antiserum from the high-dose–
vaccinated mice. Early work reported a pUL39 null mutant
replicated poorly in vitro and in vivo and did not cause ocular
disease following cornea infection (66). Human and mouse data
also suggest this protein is a specifictargetofCD8
+
Tcellsresiding
in the TG following infection (67, 68). The other subunit of the
ribonucleotide reductase, pUL40, is recognized by T cells from
asymptomatic seropositive patients infected with HSV-2 and has
been found to be a highly protective immunogen when used to
vaccinate guinea pigs against subsequent challenge with HSV-2
(69). The other nonstructural intracellular protein, ICP4, found to
be selectivelyrecognized by antiserum from high-dose–vaccinated
mice has been found to be instrumental in driving vascular
endothelium growth factor A–induced corneal neovascularization
in response to HSV-1 infection (70). This recognition and
predicted neutralization of ICP4 are consistent with the reduced
level of corneal neovascularization in the high-dose–vaccinated
mice compared with the low-dose–vaccinated animals following
ocular HSV-1 challenge (Fig. 7).
A third group of HSV-1 proteins recognized by the antiserum
from high-dose–vaccinated mice includes the tegument and
capsid proteins pUL19/VP5, pUL48/VP16, and pUL25/CVC2.
These proteins along with the other capsid proteins recognized by
TABLE III. HSV-1 protein recognition by antiserum from vaccinated mice
HSV-1 Protein High Dose (1 310
5
PFU) Low Dose (1 310
3
PFU) Vehicle
UL39/ICP6/RR1 6,793,035 61,303,967
a
352,954 654,369 372,251 625,242
UL19/MCP/VP5 6,696,422 6890,026
a
1,843,337 6869,108 535,912 6151,606
UL29/ICP8/DBP 5,597,621 61,263,758
a
130,614 690,589 94,761 650,909
UL27/gB 4,453,171 6884,210
a
106,422 663,346 22,788 611,578
UL22/gH 2,223,914 6550,125
a
12,540 612,540 0 60
UL18/TRX2/VP23 2,335,474 6487,355
c
967,491 6392,761
d
330,052 660,015
UL40/RR2 1,773,914 6513,782
a
85,050 624,622 89,785 621,998
UL44/gC 1,329,966 6253,999
a
20,216 69945 0 60
US6/gD 1,026,939 6191,329
a
060060
UL38/TRX1/VP19c 827,451 6263,020
d
219,067 6125,881 28,706 69146
US8/gE 826,764 6169,833
a
72,489 618,495 41,676 69301
UL35/SCP/VP26 779,290 6127,433
c
239,978 6160,633
d
28,879 622,879
UL1/gL 554,211 6147,467
a
1386 61386 721 6721
UL48/VP16 584,616 6145,499
a
99,682 661,382 32,467 615,242
UL31/NEC1 584,149 6204,252
b
105,703 628,173 63,740 623,269
UL26/VP24/21 414,514 6125,223
d
130,212 672,319 41,565 69047
UL12/NUC 372,549 687,858
a
65,219 629,728 83,159 635,251
UL25/CVC2 309,826 675,562
b
104,840 649,050 51,411 625,687
RS1/ICP4 277,967 673,595
b
67,645 618,463 29,843 612,568
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 sequence
database. Numbers reflect matched peptide abundance/intensity per protein 6SEM by antiserum from HSV-1 0ΔNLS–or PBS (vehicle)–vaccinated mice (n= 7 per
group from three independent experiments).
a
p,0.01, comparing the high dose to the other groups of vaccinated mice.
b
p,0.05, comparing the high dose to the other groups of vaccinated mice.
c
p,0.01, comparing the indicated group to the vehicle group as determined by ANOVA and Scheffé multiple comparison test.
d
p,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 1 310
3
PFU HSV-1 0ΔNLS, 1 310
5
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 1 310
5
0ΔNLS-vaccinated mice to
that of recipients of sera from the 1 310
3
0ΔNLS-vaccinated animals as determined by ANOVA and Tukey ttest. (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 30 330 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 1 310
5
0ΔNLS passively immunized mice to the PBS- and 1 310
3
0ΔNLS passively immunized mice as determined by ANOVA and
Tukey ttest. Representative images for (E) PBS, (F) low 1 310
3
0ΔNLS, and (G) high 10
5
0ΔNLS passively immunized mice are shown. (E and F)
Original magnification 340.
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eitherhigh-orlow-dose–vaccinated mice, including pUL18/VP23,
UL38/VP19c, UL35/VP26, and UL26/VP24/21, are critical in
HSV-1 replication whether it be transactivation of viral immediate
early lytic genes (VP16) or incorporation and assembly of capsid
proteins into the capsid shell (71–73). Because VP5, VP19c, and
pUL25 contribute to the long-range axonal transport of the
infectious unit of the virion to the neuronal cell bodies through an
assembled capsid-associated tegument complex (74), it is quite
possible the antiserum recognizes only a few epitopes on a single
protein entity that results in the immunoprecipitation of the
majority of the complex. Consequently, one or more of these
proteins may not be a contributing member of the protective
immune repertoire of Ags recognized by the antiserum from the
high-dose 0ΔNLS–vaccinated mice. Another possibility is the
complex itself forms a structural epitope recognized by the Ab that
contributes to protection from virus spread, replication, and
establishment of latency. Thus, analysis of single viral-encoded
proteins that collectively form the capsid-associated tegument
protein complex would afford little to no protection when used as a
vaccine as the “protective”epitope if it is only formed by the
associated protein complex. Although this may be true for Ab
recognition, epitopes representing capsid or tegument proteins
have been found to generate a robust CD8
+
T cell response with
polyfunctional effector T cells that elicit a protective immune
response against HSV-1 keratitis (30, 75). Theimportance of HSV-1
protein recognition by the antisera is underscored by the passive
immunization results that show quantifiable corneal pathology is
reduced or absent in high 0ΔNLS–dose-vaccinated mice com-
pared with the low-dose–or PBS-vaccinated control following
HSV-1 challenge (Fig. 8). Even though survival was similar
between the high and low 0ΔNLS–vaccinated mice and signifi-
cantly above the PBS-vaccinated control group, the difference in
cornea pathology between the 0ΔNLS-immunized groups dem-
onstrates the need to incorporate a more encompassing approach
in evaluating the success of a vaccine to protect against an ocular
pathogen, a criteria often overlooked or only accomplished
subjectively from most laboratories in the HSV-1 field. In the
case of the current investigation, further studies are required to
clearly elucidate those specific viral-encoded tegument and
capsid proteins as well as other identified viral proteins that are
contributors to the HSV-1 0ΔNLS vaccine efficacy.
DISCLOSURES
D.J.J.C. is a member of the scientific advisory board of the
company, Rational Vaccines, Inc., that owns the patent rights
to the HSV-1 0DNLS vaccine. The other authors have no financial
conflicts of interest.
ACKNOWLEDGMENTS
We thank the Laboratory for Molecular Biology and Cytometry Research
at 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 material
resources: Brian Gebhardt, original stock of HSV-1 McKrae; Stacey
Efstathiou, rHSV-1 SC16 ICP0-Cre virus; and Rational Vaccines, Inc.,
live-attenuated HSV-1 0DNLS vaccine.
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