Delayed Spread and Reduction in Virus Titer after Anterior Chamber Inoculation of a Recombinant of...

Delayed Spread and Reduction in Virus Titer afterAnterior Chamber Inoculation of a Recombinant ofHSV-1 Expressing IL-16

Nancy M. Archin,1,2 Lennard van den Boom,3 Ludmila Perelygina,4 Julia M. Hilliard,4 andSally S. Atherton1,2,3

PURPOSE. The timing of T-cell infiltration of the hypothalamus iscrucial in the prevention of bilateral retinitis in mice inoculatedwith HSV-1 through the anterior chamber (AC). In H129-in-fected mice, T-cells are recruited to the suprachiasmatic nucleiof the hypothalamus too late to protect infected mice fromdevelopment of bilateral retinitis. The purpose of these studieswas to determine whether alteration of T-cell recruitment tothe hypothalamus would affect the timing and pattern of virusspread after AC inoculation.

METHODS. A recombinant of the H129 strain of HSV-1 express-ing IL-16, a cytokine with lymphocytic and monocytic che-moattractant properties, was constructed, and mice were in-oculated in the AC with H129wt, H129wt and H129/IL-16, orH129wt and H129/pGal10 (a recombinant virus containingvector only).

RESULTS. AC inoculation of BALB/c mice with H129wt andH129/IL-16 resulted in a delay of virus spread to the hypothal-amus and the contralateral retina, and this delay correlatedwith decreased virus titers in infected tissues, compared withmice infected with H129wt or mice infected with H129wt andH129/pGal10. Although the number of infiltrating T-cells in thebrains of mice infected with H129wt, H129wt and H129/IL-16,or H129wt and H129/pGal10 was similar, more Mac-1–positivecells were detected early (postinoculation day 2) in the in-jected eyes of mice infected with H129wt and H129/IL-16 thanin mice infected with H129wt and/or H129wt and H129/pGal10.

CONCLUSIONS. These results suggest that early recruitment ofMac-1–positive cells to the injected eye may play a role indelaying virus spread in mice infected with H129wt and theIL-16–expressing recombinant virus. IL-16 delivery vectorscould be exploited to prevent or delay HSV-1 infection of thehypothalamus, allowing development of the antiviral immuneresponse and subsequent inhibition of virus spread into the

optic nerve and retina. (Invest Ophthalmol Vis Sci. 2003;44:3066–3076) DOI:10.1167/iovs.02-1071

Acute retinal necrosis (ARN) syndrome was first describedseveral decades ago.1 Although rare, the disease has since

been recognized to be a cause of blindness in otherwisehealthy individuals throughout the world.2–8 ARN is character-ized by necrotizing retinitis, vitritis, retinal arteritis, and, insome cases, optic neuropathy.9–11 Untreated, the disease israpidly progressive and often leads to vision loss in the affectedeye. Herpes simplex virus (HSV) types 1 and 2, varicella-zostervirus, and, to a lesser extent, cytomegalovirus have all beenimplicated as causes of ARN in humans.2–8

In a mouse model of ARN, anterior chamber (AC) inocula-tion of BALB/c mice with the KOS strain of HSV-1 results inretinal necrosis in the uninoculated eye only.12–14 Althoughthe AC of the injected eye is infected, the retina is spared. Bycontrast, AC inoculation of BALB/c mice with H129, a neuro-invasive and neurovirulent strain of HSV-1, results in bilateralretinal necrosis.15 After AC inoculation, both strains of virusspread retrogradely through the central nervous system (CNS)to infect the suprachiasmatic nuclei (SCN) of the hypothala-mus13,15 from which virus can then spread to the retinathrough the optic nerve. However, although both H129 andKOS are able to spread from the ipsilateral SCN to the retina ofthe contralateral eye, only H129 is also able to spread from thecontralateral SCN to infect the retina of the injected, ipsilateraleye.13,15

Recent investigations have shown that the timing of virusentry and T-lymphocyte infiltration of the SCN differ for thetwo strains of virus and that these differences appear to deter-mine whether bilateral or unilateral retinitis develops in theinfected mice.16,17 H129 is detected in both the ipsilateral andcontralateral SCN at postinoculation (PI) day 4, whereas KOS isnot detected in the ipsilateral SCN until PI day 5 nor in thecontralateral SCN until PI day 7.13,15 In H129-infected mice,infiltration of the SCN by T-cells and cytokine production arenot observed until PI day 5, by which time virus has alreadyspread to the retina of both eyes.16 By contrast, in KOS-infected mice, T-cells are detected in the ipsilateral SCN con-comitant with virus and in the contralateral SCN 2 days beforevirus.17 Several studies support the idea that T-cells in thecontralateral SCN of KOS-infected mice limit the spread ofvirus into the optic nerve of the injected eye thereby sparingthe retina of that eye from virus infection. Bilateral retinitisdevelops in thymectomized, CD4� and CD8� T-cell–depletedmice after AC inoculation of KOS, whereas it does not developin mice with depletion of only CD4� or CD8� T cells.18

Furthermore, in thymectomized, CD4� and CD8� T-cell–de-pleted mice, virus is detected in the ipsilateral SCN a day earlierthan in normal, nondepleted mice and in the contralateral SCN2 days earlier.19 Adoptive transfer of KOS-specific immuneeffector T cells protects mice from retinitis after AC inoculationby preventing virus spread into the SCN.20

From the 1Department of Microbiology, University of TexasHealth Science Center at San Antonio, San Antonio, Texas; the 2De-partment of Cellular Biology and Anatomy, Medical College of Georgia,Augusta, Georgia; the 3Department of Cellular and Structural Biology,University of Texas Health Science Center at San Antonio, San Antonio,Texas; and the 4Viral Immunology Center, Department of Biology,Georgia State University, Atlanta, Georgia.

Supported by the National Eye Institute Grants EY06753 (NMA)and National Institutes of Health Grant EY06012 (SSA).

Submitted for publication October 18, 2002; revised January 25,2003; accepted February 7, 2003.

Disclosure: N.M. Archin, None; L. van den Boom, None; L.Perelygina, None; J.M. Hilliard, None; S.S. Atherton, None

The publication costs of this article were defrayed in part by pagecharge payment. This article must therefore be marked “advertise-ment” in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Corresponding author: Sally S. Atherton, Department of CellularBiology and Anatomy, Medical College of Georgia, R and E Building,Room CB2915, Augusta, GA 30912; [email protected].

Investigative Ophthalmology & Visual Science, July 2003, Vol. 44, No. 73066 Copyright © Association for Research in Vision and Ophthalmology

Because H129-infected mice have a delay in T-cell recruit-ment to the SCN and develop bilateral retinitis after AC inoc-ulation, it was hypothesized that if T-cell recruitment to thehypothalamus was altered, the timing and/or pattern of virusspread would be affected. To test this hypothesis, a recombi-nant of H129 expressing IL-16 was constructed. IL-16, formerlylymphocyte chemoattractant factor or LCF,21,22 is a proinflam-matory cytokine that is chemotactic for all CD4� lymphoidcells, including CD4� T-lymphocytes, monocytes, eosinophils,and dendritic cells.23–26 IL-16 induces expression of IL-2 recep-tor and major histocompatibility class (MHC) class II in humanCD4� T cells and increases intracellular Ca2� and inositol-1,4,5-triphosphate in these cells.24,27 Sources of IL-16 include,but are not limited to, lymphocytes, macrophages, eosinophils,dendritic cells, and mast cells.28 IL-16 is synthesized as a pre-cursor molecule (pro-IL-16) composed of approximately 631amino acid residues.29 In CD8� T-cells, pro-IL-16 is cleaved bycaspase-3 to produce functional IL-16.30

Although the role of IL-16 in HSV-1 infection is not known,recombinant IL-16 has been shown to repress HIV-1 replicationin peripheral blood mononuclear cells (PBMCs), macrophages,and dendritic cells.31–33 In addition, human CD4� cells trans-fected with IL-16 cDNA are resistant to HIV infection.34 Resis-tance is believed to occur at the level of mRNA expression andmost likely involves inhibition of HIV-1 promoter activity.34,35

The sequence and general structure of IL-16 are conservedacross species. Both the human and mouse IL-16 genes com-prise seven exons and six introns.36 Furthermore, human andmouse IL-16 display 82.1% homology at the C-terminal region,thus exhibiting conservation in both structure and func-tion.37,38

The purpose of these studies was to determine whetherexpression of IL-16 by HSV-1 strain H129 would affect theroute of spread, timing, and/or extent of infection after unioc-ular AC inoculation of BALB/c mice. Studies were also con-ducted to determine the pattern and timing of inflammatorycell infiltration in animals infected with H129wt, with H129wt,and an IL-16 expressing recombinant virus (H129/IL-16) orwith H129wt and a recombinant virus containing the vectoronly (H129/pGal10).

MATERIALS AND METHODS

Viruses

H129, a neurovirulent strain of HSV-1 (a gift from Richard D. Dix, PhD,University of Miami School of Medicine, Miami, FL) was originallyisolated from a patient with encephalitis.39 Stocks of all viruses used inthis study were propagated on Vero cells (ATCC, Manassas, VA) grownin complete Dulbecco’s modified Eagle’s medium (DMEM) containing5% fetal bovine serum (FBS) and antibiotics. The titer of virus stockswas determined by standard plaque assay on Vero cells and titers wereexpressed as plaque-forming units (PFU) per milliliter. Stocks werestored at �70°C in 1-mL aliquots, and a fresh aliquot of stock virus wasthawed and used for each experiment. The titers of virus stocks usedin these studies were 1 � 108 PFU/mL (H129/IL-16), 2.25 � 108

PFU/mL (H129wt), and 3.87 � 108 PFU/mL (H129/pGal10).

Plasmids

pRc/CMV-IL16, a plasmid that contains an open reading frame of 390base pairs and that codes for the C-terminal (functional) region of thehuman IL-16 gene, was a gift from Paul Zhou, PhD (Southwest Foun-dation for Biomedical Research, San Antonio, TX). The IL-16 cDNA wasexcised from pRc/CMV-IL16 by restriction enzyme digestion. HindIIIand SalI restriction sites were added to the cDNA by using PCRmethods and the primers that are listed at the end of the paragraph.The cDNA was then cloned into the vector pGal10 (a gift from JerryWeir, PhD, U.S. Food and Drug Administration, Bethesda, MD) at the

HindIII and SalI site to generate the vector pGal10/IL-16. In this vector,the IL-16 gene is located downstream from the HSV immediate early110K promoter. The pGal10 vector was designed specifically for thegeneration of recombinant HSVs.40 It contains the lacZ gene under theHSV gC promoter and provides a marker for selecting recombinants.The vector also contains flanking thymidine kinase (tk) sequences todirect homologous recombination. However, because recombinationdisrupts the HSV genomic tk locus, all recombinants generated withthis vector are TK deficient (tk�). As a consequence, recombinantscannot replicate in cells that have no cellular tk. Primers (Genosys, TheWoodlands, TX) used for generating pGal10/IL-16 were as follows:IL16-forward, 5�-GTCGACCATGCCTGACCTCAACTCCT-3�, which hasa SalI site; and IL16-reverse, 5�-AAGCTTCTAGGAGTCTCCAGCAGCTG-3�, which has a HindIII site.

Generation of H129/IL-16

Recombinant viruses were generated from H129wt. H129 virus waspropagated in Vero cells. H129 virions were isolated by centrifugationthrough a sucrose gradient, and DNA was extracted from purifiedvirions. pGal10/IL-16 DNA was amplified by transforming Escherichiacoli HB109 cells (Life Technologies, Rockville, MD). Plasmid DNA waspurified using a plasmid extraction kit (Wizard; Promega, Madison, WI)and digested with EcoRI (Promega). Vero cells were then cotransfectedwith H129 and digested plasmid DNA by calcium phosphate precipi-tation (Invitrogen, Carlsbad, CA) or by using a lipophilic transfectionagent (Lipofectin; Life Technologies). To screen for recombinants,transfectants were incubated with complete DMEM containing 5% fetalbovine serum, antibiotics, 1% methylcellulose (Sigma-Aldrich, St. Louis,MO), 18 �g/mL acyclovir (Sigma-Aldrich), and 250 �g/mL X-gal (LifeTechnologies) at 37°C in 5% CO2 for several days. Blue plaques indic-ative of recombinant viruses were selected and plaque purified bythree or four passages through cell culture before growing to hightiter. Recombinants were then assayed for the presence of the IL-16insert by Southern blot hybridization and for production of IL-16 byELISA. In the H129/IL-16 recombinant, IL-16 was expressed as animmediate early gene product.

Generation of H129/pGal10

pGal10 plasmid DNA alone, without IL-16 cDNA, was amplified inHB109 cells by the process described earlier. Vero cells were thentransfected with H129 and EcoRI-digested pGal10 DNA, and recombi-nants were screened and purified using the procedures described inthe prior section.

Southern Blot Hybridization

DNA was isolated from recombinant and wild-type viruses, as de-scribed herein. Viral DNA and pGal10/IL-16 plasmid DNA were di-gested with SalI and HindIII. DNA from pRc-CMV/IL-16 was digestedwith HindIII. Digested DNAs were then electrophoretically separatedon a 1% agarose gel containing ethidium bromide and visualized undera UV lamp. The DNA was then transferred to a nylon membrane(Nytran Supercharge; Schleicher & Schuell, Keene, NH), using a rapiddownward transfer system (Schleicher & Schuell) under neutral con-ditions. The blot was hybridized with IL-16 DNA probe that had beenlabeled with digoxigenin (DIG) using a kit (DIG-High Prime; RocheApplied Science, Indianapolis, IN). The membrane was then reactedwith anti-Dig alkaline phosphatase antibody (Roche Applied Science)and visualized colorimetrically with BCP/NBP (Roche Applied Sci-ence).

Production of IL-16 in Cultured Cells

Vero cells were infected with H129/IL-16, H129wt, or H129/pGal10 ata multiplicity of infection (MOI) of 0.01 PFU and incubated at 37°C in5% CO2. At 4, 8, 12, 18, 24, and 48 hours after infection. Infected cellsupernatant was collected and stored at �80°C. The supernatant wasthen assayed for IL-16 using a anti-human IL-16 ELISA kit (Cytoscreen;

IOVS, July 2003, Vol. 44, No. 7 Pathogenesis of IL-16 Expressing an HSV-1 Recombinant 3067

Biosource, Camarillo, CA). The results were read by plate reader (AT400; SLT, Hillsborough, NC) and plate reader software (DeltaSoft,Princeton, NJ). Uninfected cell supernatant was used as the negativecontrol. The results were plotted graphically on computer (Delta-Graph; DeltaPoint, Inc., Monterey, CA).

In Vitro Single-Cycle Growth Kinetics of Viruses

Cell-culture dishes (35 mm) were seeded with 2 � 106 Vero cells/dishand incubated overnight at 37°C under 5% CO2. Cells were theninoculated with H129wt, H129/IL-16, or H129/pGal10 at an MOI of 5PFU/cell and incubated at 37°C under 5% CO2 for 1 hour to allow virusto adsorb. Plates were then washed with PBS to remove any unad-sorbed virus, overlaid with 2 mL DMEM containing 5% FBS and antibi-otics, and incubated as described earlier. Samples were collected at 0,2, 4,6, 12, and 24 hours postinfection and stored at �80°C. Sampleswere then subjected to several cycles of freeze-thaw and centrifuged.The titer of virus in the supernatants was determined by plaque assayon duplicate cultures of Vero cells.

Animals

Adult female BALB/c mice 8 to 12 weeks old (Taconic, Germantown,NY) were used in all experiments. The mice were housed in accor-dance with National Institutes of Health guidelines, and all studyprocedures conformed to the ARVO Statement for the Use of Animalsin Ophthalmic and Vision Research. All mice were maintained on a12-hour light–dark cycle and were given unrestricted access to foodand water. For all intraocular injections and perfusions, mice wereanesthetized intramuscularly with a mixture of 0.08 mg xylazine and0.9 mg ketamine per 25 g body mass.

Inoculations

Mice were anesthetized and inoculated through the AC route as fol-lows: The right eye was proptosed, aqueous humor was removed byparacentesis, and 2 �L containing 5 � 103 PFU of H129wt virus or 2 �Lcontaining a mixture of 5 � 103 PFU of H129wt virus and 1 � 105 PFUof either H129/IL-16 or H129/pGal10 was injected into the AC with a30-gauge needle attached to a 100-�L microsyringe (Hamilton, Reno,NV). The inoculum was prepared by diluting virus stock in DMEMcontaining antibiotics. Control mice were inoculated in the AC with 2�L of either phosphate-buffered saline (PBS) or DMEM from mock-infected cells.

Perfusions

Mice were deeply anesthetized and perfused transcardially with PBSfor approximately 3 minutes. After perfusion, the brain, both eyes, andboth trigeminal ganglia were quickly removed, snap frozen on dry ice,and stored at �80°C.

Plaque Assays

The brain, eyes, and trigeminal ganglia from mice infected withH129wt only, H129wt and H129/IL-16, or H129wt and H129/pGal10were homogenized separately in 500 �L of DMEM containing antibi-otics. Each homogenate was serially diluted and plated on Vero cellsthat were 80% confluent. Adsorption of virus was performed for 1 hourat 37°C in a CO2 incubator. After adsorption, the cells were overlaidwith a 1:1 solution of 2� DMEM containing 10% serum and antibioticsand 1% low-melting-point agarose (Life Technologies). After 5 days at37°C, the cells were fixed with 0.5% glutaraldehyde (Sigma-Aldrich)and incubated overnight with X-gal (Life Technologies). Blue and clearplaques were counted, and the titer of virus (total PFU per sample) wascalculated and plotted on computer (DeltaGraph; DeltaPoint, Inc.).

Immunohistochemistry

Virus, T Cells, and IL-16 Single Labeling. Serial frozen 8- to10-�m sections were collected on positively charged slides (FisherScientific, Pittsburgh, PA). Sections for immunohistochemistry were

fixed with acetone and blocked with normal goat serum (VectorLaboratories, Burlingame, CA). Sections to be stained for T cells orIL-16 were also blocked against endogenous biotin, with an avidin-biotin blocking kit (ABC; Vector Laboratories) used according to themanufacturer’s instructions. Sections were then incubated with (1) arabbit anti-HSV-1 polyclonal antibody (Accurate Chemicals, Westbury,NY); (2) a hamster anti-mouse �/� TCR monoclonal antibody (CloneH57-597; BD PharMingen, San Diego, CA); (3) a biotinylated rat anti-mouse CD4 monoclonal antibody (clone RM4-5; BD PharMingen); (4)a biotinylated rat, anti-mouse CD8 monoclonal antibody (Clone 53-6.7;BD PharMingen); (5) biotinylated rabbit, anti-human IL-16 polyclonalantibody (Biosource); or (6) FITC-labeled anti-CD11b (clone M1/70,Mac-1 Ab; BD PharMingen). After incubation with the CD11b primaryantibody, labeled sections were washed, mounted with anti-fade me-dium containing 4�,6�-diamino-2-phenylindole (DAPI; VectaShield; Vec-tor Laboratories) and examined under a fluorescent microscope forCD11-positive, fluorescent cells. After incubation with all other pri-mary antibodies, sections were washed with PBS several times andincubated with the appropriate biotinylated secondary antibody, ifnecessary. Sections were then reacted with 0.3% hydrogen peroxide toeliminate endogenous peroxidase, washed several times, and reactedwith avidin-biotin solution (Vector Laboratories). Diaminobenzidine(DAB; Sigma-Aldrich) was used as the chromogen for sections stainedfor HSV-1. DAB reaction was intensified by adding 0.04% nickel chlo-ride (Sigma-Aldrich) to the DAB solution. Sections stained for T cellsand IL-16 were reacted with purple dye and peroxidase substrates,respectively (Vector VIP Purple and Vector SG, respectively; VectorLaboratories). All sections (except those stained for IL-16) were thencounterstained with methyl green (Sigma-Aldrich), dehydrated in agraded ethanol series, cleared with xylene, coverslipped, and exam-ined microscopically for the purple-black–stained cells indicative ofvirus infection or purple-stained T lymphocytes. IL-16 sections werecounterstained with nuclear red dye (Nuclear Fast Red; Vector Labo-ratories), processed as described earlier, and examined microscopi-cally for gray-stained IL-16–expressing cells.

Virus, T Cells, and IL-16-Double Labeling. Frozen sectionswere processed as described in the prior section and incubated witheither biotinylated or nonbiotinylated rabbit anti-human IL-16 (Bio-source). Sections were washed with PBS and incubated with avidinD-Texas red or 7-amino-4-methylcoumarin-3-acetic acid (AMCA)–la-beled-anti-rabbit IgG (Vector Laboratories) and washed again. Sectionswere then incubated overnight with one of the following secondprimary antibodies: (1) FITC-labeled anti-CD11b, (2) FITC-labeled anti-CD8a, (3) FITC-labeled anti-�/� TCR (all from BD PharMingen), or (4)FITC-labeled anti-HSV-1 (Dako, Carpinteria, CA) for 1 hour at 37°C.Sections were washed, mounted with antifade medium containingDAPI (VectaShield; Vector Laboratories), and examined with a fluores-cence microscope for red IL-16–expressing cells; green CD11b-, CD8-,�/� TCR–, or HSV-1–positive cells, and orange or blue-green cellscoexpressing IL-16 and any of the listed antigens.

�-Galactosidase Staining

Frozen sections were prepared as described earlier. Sections adjacentto those used for immunohistochemistry were fixed with glutaralde-hyde and stained with the �-galactosidase substrate X-gal, using previ-ously reported methods.13 Sections were then counterstained withSafranin-O (Sigma-Aldrich), dehydrated, cleared with xylene, cover-slipped, and examined microscopically for blue-stained areas indicat-ing sites of recombinant virus infection.

RESULTS

Construction of Recombinant Viruses

Recombinant HSV-1 (H129/IL-16) was constructed by first in-serting the cDNA coding for functional IL-16 into pGal10, avector that has previously been used to generate HSV-1 recom-binants expressing the cytokine IFN-�.40 The resultant plas-

3068 Archin et al. IOVS, July 2003, Vol. 44, No. 7

mid, pGal10-IL-16, was then used to generate H129/IL-16 (Fig.1A), as described in the Methods section. To control for theeffect of the pGal10 vector in these studies, a recombinantH129 virus containing the pGal10 vector alone, H129/pGal10(Fig. 1B), was also constructed.

To confirm the presence of IL-16 cDNA in H129/IL-16, viralDNAs were isolated and digested with HindIII and SalI andevaluated by Southern blot hybridization. As expected, IL-16DNA sequences were detected only in the vectors in whichIL-16 cDNA had been inserted (i.e., pRc-CMV-IL-16 and pGal10-

FIGURE 1. Construction of H129/IL-16 and H129/pGal10. (A) Diagrams of HSV-1 (H129), pGal10/IL-16, and H129/IL-16. a, b, and c denote theinverted repeat sequences in the HSV genome. Plasmid pGal10/IL-16 was constructed by inserting the cDNA coding for functional IL-16 into theSalI and HindIII site of plasmid pGal10. IL-16 was placed under the control of the 110k HSV promoter (k). pGal10 contains the lacZ gene underthe control of the HSV gC promoter (g) and is flanked by tk sequences to direct homologous recombination by disrupting the HSV tk locus.H129/IL-16 was produced after cotransfection of Vero cells with H129 and EcoRI-digested pGal10/IL-16 DNA. IL-16�, tk�, �-gal� virus wasscreened. (B) Diagrams of HSV-1 (H129), pGal10, and H129/pGal10. H129/pGal10 was produced by cotransfection of Vero cells with H129 andEcoRI-linearized pGal10 DNA. �-gal, tk� virus was screened. E, EcoRI; H, HindIII; S, SalI; X, XbaI.


IL-16) and in the recombinant virus, H129/IL-16 (Fig. 2). IL-16cDNA was not detected in wild-type virus or in recombinantvirus containing vector alone (H129/pGal10).

To determine whether cells infected with H129/IL-16 ex-pressed IL-16, ELISA was performed on infected Vero cellsupernatants, by using an anti-human IL-16 ELISA kit (Bio-source). IL-16 was detected as early as 8 hours after infection.The highest level of IL-16 was 15 ng per 5 � 106 PFU/mL at 48hours after infection (Fig. 3). IL-16 was not detected in super-natants of Vero cells infected with wild-type virus or H129/pGal10.

In Vitro Growth Kinetics of VirusesTo assay the in vitro replication of H129wt, H129/IL-16, andH129/pGal10, single-cycle growth experiments were per-

formed. As shown in Figure 4, the kinetics of viral replicationwere similar for all viruses.

Complementation of Recombinant Viruses withWild-Type Virus

Construction of HSV-1 recombinants using the pGal10 vectorinvolved homologous recombination at the HSV tk locus. Allrecombinants generated using this vector were tk� and toreplicate in cells without tk, such as neurons, H129/IL-16 andH129/pGal10 were complemented with wild-type virus, aspreviously described.13,41

To determine the ratio of wild-type to recombinant viruswith the highest complementation, mixtures of H129/IL-16and H129wt or H129/pGal10 and H129wt were prepared atratios of 1:1 to 20:1 (recombinant/wild type). Two groups of

FIGURE 2. Southern blot hybridization confirming the presence ofIL-16 in H129/IL-16. Viral DNAs were isolated and digested with Hin-dIII and SalI, plasmid pGal10 was also digested with HindIII and SalI,and plasmid pRc/CMV/Il-16 was digested with HindIII. DNAs wereelectrophoretically separated, and Southern blot hybridization wasperformed. (A) Photomicrograph showing separation of digested viralDNA. (B) Blot of transferred DNA after probing with DIG-labeled IL-16probe. The predicted IL-16 fragment size is indicated by the arrow andwas detected only in H129/IL-16 and plasmid-digested DNAs. Ladder:DNA Marker VIII ladder (Roche Applied Science, Indianapolis, IN).

FIGURE 3. In vitro production ofIL-16 by H129/IL-16. Supernatantfrom Vero cells infected with H129/IL-16 was evaluated for production ofIL-16 by ELISA. Supernatants fromH129wt- and H129/pGal10-infectedVero cells were also studied. Thehighest level of IL-16 by H129/IL-16was detected at 48 hours per 5 � 106

PFU/mL of virus. IL-16 was not de-tected in the supernatant of eitherH129wt- or H129/pGal10–infectedcells. Bkgrd, background.

FIGURE 4. Single-cycle growth kinetics of H129wt, H129/IL-16, andH129/pGal10. Vero cells were infected at an MOI of 5 PFU/cell.Samples were collected at different time points, and the titer of virusin each sample was determined by standard plaque assays on Verocells. The kinetics of viral growth was similar for all three viruses. Thelimit of the detection was 0.7 log10 PFU/2 � 106 cells.


BALB/c mice were then inoculated with a mixture of wild-typeand recombinant virus in one AC. Control mice were inocu-lated with 5 � 103 PFU of H129wt, 1 � 105 PFU of H129/IL-16,or 1 � 105 PFU of H129/pgal10. On postinfection (PI) days 6and 8, the contralateral eye was removed, and a standardplaque assay was performed on Vero cells, using homogenatesof the contralateral eyes. Plaques were stained with X-gal todifferentiate blue (recombinant) from clear (wild type)plaques. Although the AC of the inoculated eye of mice in-jected with H129/IL-16 or H129/pGal10 was infected, viruswas not recovered from the contralateral eye of these mice. Incontrast, high titers of virus (�1 � 106 PFU/mL at PI day 8)were recovered from the contralateral eye of mice infectedwith H129wt alone. Virus was also recovered from the con-tralateral eye of mice infected with a mixture of H129wt andH129/IL-16 or H129wt and H129/pGal10. The ratio of recom-binant to wild-type virus that resulted in the highest titer ofboth viruses was 20:1. At this ratio, an average of 1.43 � 106

PFU/mL of H129/IL-16 and 6.40 � 106 PFU/mL of H129wt wasrecovered from the uninoculated eye at PI day 8 The ratio of20:1 (recombinant/wild type) was therefore used for all studieswith recombinant viruses.

Expression of Viral Antigen and Recovery ofVirus in the Brain

After AC inoculation with H129wt, virus spreads through thebrain to infect the retina of both the inoculated and uninocu-

lated eye.15 Therefore, the titer of virus in the brain of miceinfected with recombinant viruses was compared with the titerof virus recovered from mice infected with H129wt to deter-mine whether infection with H129/IL-16 affected the spread ofvirus into the brain. As shown in Figure 5A, the titer of virusrecovered from the ipsilateral half of the brain of mice infectedwith H129wt and H129/IL-16 at PI day 4 was significantlylower (P � 0.05) than that recovered from mice infected witheither H129wt or with H129wt and H129/pGal10. Althoughless virus was also recovered from the contralateral half of thebrain of mice infected with H129wt and H129/IL-16 than frommice infected with H129wt, the difference between the titer ofvirus recovered from the contralateral brain of H129/IL-16–and H129/pGal10–infected mice was not significant (notshown).

Compared with mice infected with H129wt or withH129wt and H129/pGal10, virus infection of the SCN wasdelayed in mice infected with H129wt and H129/IL-16. At PIday 4, virus was observed in the ipsilateral SCN of three of nineH129/IL-16–infected mice, compared with four of six miceinfected with H129/pGal10 and six of six mice infected withH129wt (Fig. 5B). In the contralateral SCN, at PI day 4, viruswas detected in one of nine mice infected with H129/IL-16compared with three of six pGal10-infected mice and five ofsix of H129wt-infected mice (not shown). At PI day 5, less viralantigen was detected in both the ipsilateral and contralateralSCN of mice infected with H129/IL-16 compared with miceinfected with H129wt or H129/pGal10 (Fig. 6).

FIGURE 5. (A) Titers (average PFU� SEM) of virus recovered from theipsilateral brain of mice inoculatedwith H129wt, H129wt and H129/IL-16 (H129/IL-16), or H129wt andH129/pGal10 (H129/pGal10). Micewere inoculated in the AC of theright eye with 5 � 103 PFU ofH129wt alone or a mixture of 5 �103 PFU of H129wt and 1 � 105 PFUof either H129/IL-16 or H129/pGal10. Mice were killed on the daysindicated, and the brain from eachmouse was removed and snap fro-zen. Brains were separated into right(ipsilateral) and left (contralateral)halves. A standard plaque assay wasperformed on tissue homogenates.Plaques were stained with X-gal.Clear and blue plaques were countedseparately and the data combinedand analyzed for statistical signifi-cance. Virus titers were determinedin duplicate. Results are combinedfrom two separate experiments: n �

3 and n � 5 mice per day, per group. *Significantly different from H129wt and H129/pGal10 (P � 0.05; Student-Newman-Keuls test). The limitof the detection was 0.7 log10 PFU/tissue. (B) Delayed infection of the ipsilateral SCN of H129/IL-16–infected mice. Mice were inoculated in theAC with H129wt, H129wt and H129/IL-16 (H129/IL-16), or H129wt and H129/pGal10 (H129/pGal10). The brain from each mouse was removedand processed for immunohistochemistry to detect viral antigen.

FIGURE 6. Photomicrograph of viralinfection of the SCN of mice infectedwith H129wt and H129/IL-16 (A),H129wt and H129/pGal10 (B), orH129wt (C) at PI day 5. At this time,less viral antigen was observed in theSCN of H129/IL-16–infected mice thanin H129wt- or H129/pGal10–infectedmice. (✽ ) Midline of the brain; OC; opticchiasma. Original magnification, �99.


Expression of Viral Antigen and Recovery ofVirus in the Eye

To determine whether delayed virus infection of the SCN andthe decrease of virus titer in the brain affects the spread ofvirus into the eyes, virus recovery studies were performed onthe eyes of mice inoculated with H129wt, H129wt, and H129/IL-16, or H129wt and H129/pGal10. Although the titer of virusrecovered from the injected eye was similar for all three virusesat all time points (not shown), at PI day 6, the titer of virusrecovered from the contralateral eye of H129/IL-16–infectedmice was significantly lower (P � 0.05, Student-Newman-Keulstest) than from mice infected with H129wt (Fig. 7A). Thisdifference appeared to correlate with the presence of IL-16;the titer of virus recovered from the contralateral eye of miceinfected with H129/IL-16 was also significantly lower (P �0.05) than the titer recovered from mice infected with H129/pGal10 (Fig. 7A).

Irrespective of the infecting virus, the ipsilateral retina ofmice became infected at PI day 5 (not shown). However, virusinfection of the contralateral retina was delayed in H129/IL-16–infected mice. Virus was observed in the contralateralretina of mice infected with either H129wt or H129/pGal10 atPI day 5 (Figs. 7B, 8B, 8C), whereas in mice infected withH129/IL-16, virus was not detected in the contralateral retinauntil PI day 6 (Figs. 7B, 8A). Although the contralateral retina ofH129/pGal10–infected mice was infected at PI day 5, less viralantigen was detected than in the retina of mice infected withwild-type virus (compare Figs. 8B and 8C).

Expression of IL-16 in the Eyes and Brain

In the injected eyes of H129/IL-16–infected mice, IL-16 wasobserved mainly in the anterior chamber along the iris andciliary body at PI day 2 (Fig. 9A) A few IL-16–positive cellswere also observed in the iris and ciliary body of H129wt- andH129/pGal10-infected mice at PI day 2. However, the expres-sion of IL-16 in the injected eyes of H129wt- and H129/pGal10-infected mice was less than that observed in H129/IL-16–infected mice (compare Fig. 9A with Figs. 9B, 9C).

To determine whether IL-16 was expressed in the brain ofH129/IL-16–infected mice, brain sections adjacent to sectionsthat contained viral antigen were stained for IL-16. The staining

pattern for IL-16 correlated with the pattern of viral antigenexpression (Figs. 9D, 9E), suggesting that some or all of theIL-16 was produced by virus-infected neurons. To confirm thisobservation, H129/IL-16–infected brain sections were stainedfor IL-16 and HSV-1, for IL-16 and �/� T cells, for IL-16 andCD8� T cells, or for IL-16– and Mac-1–positive cells. Althougha few CD8� T cells, �/� T cells, and Mac-1–positive cellscolabeled with IL-16 antigen (not shown), the majority of cellsexpressing IL-16 in the brain appeared to be infected neurons(Figs. 9D–F). However, not all infected neuronal cells ex-pressed IL-16.

Brain sections from H129/pGal10- and H129wt-infectedmice were also immunolabeled for IL-16. IL-16 was also de-tected in the brain of these mice, and the staining pattern forIL-16 correlated with the pattern of viral antigen expression aswell. However, IL-16 expression in H129/pGal10- and H129wt-infected mice was less extensive than that observed in thebrain of H129/IL-16–infected mice (not shown). Double immu-nofluorescent labeling of brain tissues from H129/pGal10- andH129wt-infected mice showed that infected neurons as well asT cells and Mac-1–positive cells were sources of IL-16 in thebrain of these mice (not shown). IL-16 was not detected in thebrain of mock-infected mice.

Inflammatory Cell Infiltration

Because IL-16 is a chemoattractant for several types of immunecells, including CD4� T cells, monocytes, dendritic cells andeosinophils, sections from the brain and inoculated eyes ofH129wt-, H129wt- and H129/IL-16–, or H129wt- and H129/pGal10–infected mice were immunolabeled for CD4� T cells,�/� T cells, CD8� T cells, and Mac-1–positive cells to deter-mine whether IL-16 affects recruitment of these cells to in-fected tissues. Results from H129wt- and H129/IL-16–infectedmice were compared with results from mice infected withH129wt or with H129wt and H129/pGal10. At PI days 2 and 3,only a few T cells (three to five mice) were detected in the AC,and the vitreous of the injected eyes of mice inoculated withH129wt or with either of the two recombinant viruses (notshown). However, more Mac-1–positive cells were detected inthe injected eyes of mice infected with H129/IL-16 at PI day 2compared with the injected eyes of H129/pGal10- or H129wt-

FIGURE 7. (A) Titers (average PFU� SEM) of virus recovered from thecontralateral retina of mice inocu-lated with H129wt, H129wt andH129/IL-16 (H129/IL-16), or H129wtand H129/pGal10 (H129/pGal10).Mice were inoculated in the AC ofthe right eye with 5 � 103 PFU ofH129wt alone, or a mixture of 5 �103 PFU of H129wt plus 1 � 105 PFUof either H129/IL-16 or H129/pGal10. Mice were killed on the daysindicated, and the eyes of eachmouse were removed and snap fro-zen. A standard plaque assay wasperformed on tissue homogenates.Plaques were stained with X-gal.Clear and blue plaques were countedseparately and the data combinedand analyzed for statistical signifi-cance. Virus titers were determinedin duplicate. Results are combinedfrom two separate experiments: n �3 and n � 5 mice per day, per group. *Significantly different from H129wt and H129/pGal10 (P � 0.05 by Student-Newman-Keuls test). The limitof the detection is 0.7 log10 PFU/ tissue. (B) Delayed infection of the contralateral retina of H129/IL-16–infected mice. Mice were inoculated in theAC with H129wt, H129wt and H129/IL-16 (H129/IL-16), or H129wt and H129/pGal10 (H129/pGal10). The eyes of each mouse was removed andprocessed for immunohistochemistry to detect viral antigen.


infected mice (Figs. 10A–C). Mac-1–positive infiltrates werelocated primarily in the AC and the vitreous and the ganglioncell layer.

Early after inoculation (PI days 2–4), T cells were detectedin the brain mainly in the root entry zone of the oculomotornerve and the ventricles, irrespective of the virus type. At thistime, the number of infiltrating T lymphocytes in the brain ofmice infected with either wild-type or recombinant viruses wassimilar (not shown). At PI day 6, many infiltrating T-cells wereobserved in the brain of H129/IL-16, especially the mesenceph-alon, when the titer of recombinant virus in the brain was alsothe highest and many �-gal–positive cells were observed (notshown). Because there was an abundance of viral antigen andT-cells at PI day 6,16 it was not possible to observe a differencebetween mice infected with wild-type virus and mice infectedwith recombinant virus. However, a difference was observedin the number of Mac-1–positive cells in the SCN at PI day 6.There were more Mac-1–positive cells in the SCN of H129/IL-16–infected mice than in the SCN of mice infected with H129/pGal10 or with H129wt (Figs. 10D–F). Because the Mac-1antigen is expressed on microglia, it was not surprising thatMac-1–positive cells were also observed in the brain of mock-infected mice. However, the number of Mac-1–positive cells inthe brain of mock-infected mice was lower than was observedin the brain of virus-infected mice (not shown).

DISCUSSION

In this study, a recombinant H129 virus expressing IL-16, acytokine with lymphocytic and monocytic chemoattractantproperties, was constructed to determine whether productionof IL-16 would affect the timing or route of virus spread afterAC inoculation and whether mice infected with IL-16–express-ing recombinant virus would be protected from bilateral reti-nitis. Several key differences were observed in mice infectedwith H129/IL-16 compared with mice infected with H129wtalone or with mice infected with H129/pGal10. Infection ofthe SCN was delayed in mice infected with H129/IL-16 com-pared with mice infected with H129/pGal10 or H129wt. Be-tween PI days 3 and 5, less viral antigen was detected in thebrains of H129/IL-16–infected mice than in mice inoculatedwith H129wt or H129/pGal10. The lower amount of viralantigen correlated with recovery of significantly lower titers ofvirus from the brains of H129/IL-16–infected mice. AlthoughH129/IL-16–infected mice developed bilateral retinitis, reduc-tion in the amount of virus in the brain and delayed infectionof the ipsilateral SCN correlated with a delay in infection of thecontralateral retina and with recovery of significantly lower

FIGURE 8. Photomicrograph of viral infection of the contralateral ret-ina of mice infected with H129wt and H129/IL-16 (A), H129wt andH129/pGal10 (B), or H129wt (C) at PI day 5. Viral antigen was notdetected in the retina of mice infected with H129/IL-16 until PI day 6,whereas viral antigen (arrows) was detected in the contralateral retinaof H129wt and H129/pGal10–infected mice at PI day 5. Originalmagnification, �99.

FIGURE 9. Photomicrograph of IL-16expression in the iris of the injectedeyes of mice infected with H129wtand H129/IL-16 (A), H129wt andH129/pGal10 (B), or H129wt (C) atPI day 2. IL-16 expression in the in-jected eyes of H129/IL-16–infectedmice was more extensive (arrows)than that observed in H129/pGal10-or H129wt-infected mice. Viral anti-gen (D) and IL-16 (E) in the oculo-motor nucleus of H129/IL-16–in-fected mice at PI day 6. Colabeling ofviral antigen and IL-16 in the samearea (F) showed that many infectedneurons also expressed IL-16 (blue-green stained cells). Arrows: virus in-fected IL-16–expressing cells and/orcells expressing both antigens. Orig-inal magnification, �198.


titers of virus in the uninoculated eye. Finally, more Mac-1–positive cells were observed in the injected eyes of H129/IL-16–infected mice early after infection compared withH129wt– or H129/pGal10–infected mice.

The results in mice infected with H129/pGal10 were inter-mediate between those of mice infected with H129wt andmice infected with H129/IL-16 virus. For example, althoughvirus was observed in the SCN of H129wt- and H129/pGal10-infected mice on the same day, less viral antigen was observedin the H129/pGal10-infected mice. Similarly, although viruswas observed in the contralateral eye of H129/pGal10- andH129wt-infected mice at the same time, less viral antigen andlower titers of virus were recovered from H129/pGal10-in-fected mice. These results suggest that in vivo complementa-tion and/or the vector used in the construction of the recom-binant viruses influenced the spread and extent of viralinfection. However, the observations that the titers of comple-mented virus were essentially the same for both H129/pGal10and H129/IL-16 and that H129/IL-16 and H129/pGal10 repli-cated to similar titers in vitro suggest that the effect of thevector was similar in the recombinant viruses. Therefore, thepresence of the vector alone does not account for the differ-ences in the timing of viral infection in H129/IL-16–infectedmice compared with H129wt- or H129/pGal10-infected miceand supports the idea that production of IL-16 in H129/IL-16–infected mice affects the timing of virus infection. Neverthe-less, the results presented in this study demonstrated a de-crease in virulence of H129/pGal10 compared with H129wt.Factors that may influence the virulence and therefore thespread of H129/pGal10 include a reduced efficiency of repli-cation in certain cell types such as neurons, the ability toinduce immunomodulatory molecules such as IFN-� andTNF-�, and/or the capacity for the induction of inflammatorycells, all of which may limit the spread of H129/pGal10 in vivo.

The mechanism by which IL-16 delays spread of virus in thebrain and to the contralateral retina in H129/IL-16–infectedmice is not known. However, delay of virus spread into thebrain and contralateral retina most likely begins in the injectedeye. At PI day 2, more Mac-1–positive cells were detected inthe injected eyes of H129/IL-16–inoculated mice than in miceinoculated with H129wt or H129/pGal10. Although at PI day 2,H129/IL-16 virus and IL-16 were detected only in AC struc-tures, such as the iris and ciliary body, many Mac-1–positivecells were also detected in the vitreous and along the ganglioncell layer of the retina. The mechanism by which production ofIL-16 in the AC might induce infiltration of Mac-1 positive cells

in the posterior segment of the eye is not clear. The Mac-1antigen is expressed on macrophages, microglia, dendriticcells, and natural killer (NK) cells at various levels and is rapidlyupregulated on neutrophils after activation.42–44 Mac-1, alsoknown as complement receptor 3 (CR3), is a member of the�2-intergrin family, which plays an important role in lympho-cyte and leukocyte migration into tissues. Mac-1 mediates ad-hesion to c3bi and intracellular adhesion molecule (ICAM)-1.43,45,46 The Mac-1–expressing cells infiltrating the injectedeye probably include cells of monocytic lineage as well asdendritic cells. The ciliary body and iris contain resident mac-rophages and dendritic cells,47,48 which may be induced tomigrate by IL-16 present in these structures. Similarly, retinalmicroglia located in the ganglion cell layer, the inner plexiformlayer, and the subretinal space49,50 could migrate as a result ofIL-16 in the injected eye. The precise role of Mac-1–positivecells in H129/IL-16 infection remains to be deciphered, but itmay involve nonspecific antiviral mechanisms, such as secre-tion of type 1 interferons or other nonspecific immunomodu-latory factors.

In these studies, although virus was not detected in thecontralateral SCN of most H129/IL-16–infected mice until PIday 5, the ipsilateral retina of all mice contained virus. InH129wt-infected mice, virus normally spreads from the con-tralateral SCN, which is infected at PI day 4, to infect the retinaof the ipsilateral eye at PI day 5.15 Thus, the results in H129/IL-16–infected mice suggest that in addition to spreadingthrough the optic nerve, virus may also spread directly fromthe anterior to the posterior segment of the injected eye. Directanterior-to-posterior spread of virus in H129/IL-16–infectedmice may explain the observation that the ipsilateral retina isinfected concomitant with the infection of the contralateralSCN. Because early direct spread of virus from the anterior tothe posterior segment of the eye is controlled by NK cells,51

early production of IL-16 may downregulate IFN-� and othermolecules required for NK cell activation.52

Finally, IL-16 was detected in the brain of mice infectedwith wild-type or recombinant viruses. However, in mice in-fected with H129/IL-16, expression of IL-16 was more exten-sive than that observed in mice infected with either H129wt orH129/pGal10. This observation suggests that some of the IL-16detected in H129/IL-16–infected mice was of recombinantorigin. However, because of the limitations of the methodsused to detect IL-16, additional studies are needed to distin-guish endogenous IL-16 from IL-16 made by recombinant virus.

FIGURE 10. Mac-1–positive cells inthe retina and vitreous of the in-jected eyes of mice inoculated withH129wt and H129/IL-16 (A), H129wtand H129/pGal10 (B), or H129wt (C)at PI day 2. More Mac-1–positive cellswere detected in the injected eyes ofH129/IL-16–infected mice than inH129wt- or H129/pGal10–infectedmice at PI day 2. Mac-1–positive cellswere located in the vitreous (✽✽ )and the ganglion cell layer (arrows)of the retina. A few Mac-1–positivecells were also observed in the innerplexiform layer (double arrows). Afew Mac-1–positive cells were ob-served in the vitreous of H129wt-in-fected mice (arrowheads). Mac-1–positive cells were also detected inthe AC (not shown). Mac-1–positivecells in the SCN of H129/IL-16 (D),H129/pGal10 (E), and H129wt (F) at PI day 6. More Mac-1–positive cells (arrows) were detected in the SCN of H129/IL-16–infected mice thanin H129wt- or H129/pGal10–infected mice at PI day 6. (✽ ) Midline of the brain. Original magnification, �198.


Although the initial goal of these studies was to delay and/orprevent the spread of virus into the eyes by promoting earlyT-cell infiltration of the hypothalamus, the number of T cellsobserved in infected tissues of H129/IL-16–injected mice early(days 2–4) during infection was not significantly different fromthat observed in mice infected with H129wt or H129/pGal10.The lack of difference in T-cell recruitment most likely reflectsdelayed infection of the hypothalamus in H129/IL-16–infectedmice and a lower amount of IL-16 available to participate in thechemotaxis of T-cells. Indeed, many infiltrating T-cells wereobserved in the hypothalamus of H129-IL-16–infected mice atPI day 6, when the highest titer of recombinant virus and thegreatest number of recombinant virus-infected cells were alsoobserved. More Mac-1–positive cells were also observed in theSCN of H129/IL-16–infected mice at PI day 6 than in miceinfected with either wild-type virus or H129/pGal10. However,these cells did not appear to play a role in delaying virus spreadto the contralateral retina, because virus had already spread tothe contralateral retina at PI day 6.

In conclusion, AC inoculation of BALB/c mice with H129/IL-16 resulted in a decrease of virus in infected tissues and adelay in virus infection of the SCN and of the contralateralretina. Although the mechanism by which virus infection wasdelayed is not clear, the results presented herein suggest thatearly recruitment of Mac-1–positive cells to the injected eyeaffects virus spread. Additional studies are needed to differen-tiate recombinant virus–produced IL-16 from endogenouslyproduced IL-16 and to define the mechanism by which IL-16retards virus spread after uniocular AC inoculation of H129/IL-16.

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Delayed Spread and Reduction in Virus Titer after Anterior Chamber Inoculation of a Recombinant of...

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