Murine response to DNA encoding herpes simplex virus type-1glycoprotein D targeted to the liver

James V. Rogersa, Barbara E. Hullb, Pamela S. Finkc, Henry C. Chioud, NancyJ. Bigleyc,*

aBiomedical Sciences Ph.D. Program, Wright State University, Dayton, OH 45435, USAbDepartment of Biological Sciences, Wright State University, Dayton, OH 45435, USA

cDepartment of Microbiology and Immunology, Wright State University, Dayton, OH 45435, USAdThe Immune Response Corporation, Carlsbad, CA 92008, USA

Received 27 December 1998; accepted 17 September 1999

Abstract

Plasmid DNA encoding herpes simplex virus type-1 glycoprotein D (gD-1) was complexed with asialoorosomucoid conjugated

to poly-L-lysine. Following its intravenous injection into BALB/c mice, this complex was targeted to the liver. Liver cellsexpressing gD-1 were detected immunohistochemically through day 6 post-immunization, while gD-1 DNA was detectablethrough 14 days post-immunization. Decline of gD-1 expression and detectable gD-1 DNA in the liver correlated with in¯ux of

T cells, predominantly CD4+. The ASOR-poly-L-lysine DNA carrier system promotes hepatic expression of gD-1 and may beuseful in vaccination against herpes simplex virus type-1. # 2000 Elsevier Science Ltd. All rights reserved.

Keywords: Asialoorosomucoid; Liver; Vaccine

1. Introduction

Herpes simplex virus type-1 (HSV-1) causes recur-rent lesions in the oral mucosa of individuals world-wide. These lesions follow reactivation of the latentvirus from the trigeminal ganglion and serve as amajor site for HSV-1 transmission. HSV-1 infectionsstimulate cytotoxic T lymphocytes (CTLs), primarilyCD4+ T cells, leading to viral clearance [1±6]. A vac-cine against HSV-1 could limit the frequency andseverity of herpetic lesions.

Immunization with DNA encoding antigenic pro-teins can induce cellular and/or humoral responses byproviding prolonged antigen expression in vivo [7,8].Intracellular synthesis of DNA encoded antigens pos-sessing a conformation similar or identical to the wild-

type protein stimulate the immune system [8±10].Plasmid constructs of HSV DNA encoding either gly-coproteins B (gB) or D (gD) generate both antibodyand T cell responses. These two glycoproteins areexpressed during the early stages of HSV infection[11±14]. Immunization routes have included directinjection into footpad and skeletal muscle, intranasaladministration and injection of transfected dendriticcells [15±20]. Injection of animals with plasmids encod-ing gB and gD stimulated cell-mediated and humoralresponses, leading to protection against viral challengeof the vagina and skin [15±20].

A soluble, DNA carrier system consisting of asia-loorosomucoid (ASOR)-poly-L-lysine complexed witha plasmid DNA resists nuclease degradation and tar-gets foreign DNA to cells such as hepatocytes thatexpress receptors for ASOR [21±23]. Other cells withasialoglycoprotein receptors (ASGPr) include macro-phages and enterocytes [24±26]. Five minutes post-intravenous injection, ASOR/ASGPr complexes inter-nalize via receptor-mediated endocytosis, with 92±95%

Vaccine 18 (2000) 1522±1530

0264-410X/00/$ - see front matter # 2000 Elsevier Science Ltd. All rights reserved.

PII: S0264-410X(99 )00438 -7

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* Corresponding author. Tel.: +1-937-775-2824; fax: +1-937-775-

2012.

E-mail address: nancy.bigley@wright.edu (N.J. Bigley).

of the ASOR/ASGPr complex entering the endoso-mal±lysosomal pathway [27±29]. About 80±85% ofhepatocytes endocytose ASOR-poly-L-lysine DNAcomplexes [30], leading to the production of membranebound, intracellular and secreted proteins [22,31,32].These proteins are maximally expressed by 24±48 hand are detectable up to 6 days post-injection. Uptakeof ASOR-poly-L-lysine DNA complexes and sub-sequent transgene expression is inhibited in vivo and invitro by excess ASOR [30,33]. Partial hepatectomyprior to injection of ASOR-PL-DNA prolongs ex-pression for 11 weeks, since targeted DNA constructsremain in cytoplasmic vesicles which are not trans-ported to the nucleus [29,34].

Immunization with the ASOR-PL-DNA carrier sys-tem encoding gD-1 generated CTL responses inBALB/c (H-2d) and C3H (H-2k) mice [35]. Followingintravenous injection of mice with gD-ASOR, maximalgD-1 expression was detected in isolated liver cells at 3days post-immunization; these expression levelsdropped signi®cantly by day 7, persisting at low levelsthrough 14 days [35]. The purpose of this study was tomonitor expression of gD-1 by cells in the liver, heartand spleen and to characterize the in¯ux of T cells intothe liver following intravenous injection of nuclease-insensitive plasmid DNA encoding the gD-1 gene.

2. Materials and methods

2.1. Animals

Eight week-old female BALB/c (H-2d) mice werepurchased from Harlan Sprague-Dawley, Inc.(Indianapolis, IN) and housed in groups of 2±3 percage. Mice were housed at least one week prior to im-munization. Animal use was approved by theLaboratory Animal Care and Use Committee atWright State University.

2.2. Immunization of mice

As previously described [22,30], the pBK-CMV vec-tor expressing gD-1 [35] was complexed with asialoor-osomucoid (ASOR) conjugated to poly-L-lysine.Although the pBK-CMV vector contains the SV40eukaryotic origin of replication, this plasmid will onlyreplicate in cells infected with SV40 or expressingSV40 TAG (Stratagene, La Jolla, CA). Wilson et al.[34] characterized DNA targeted to the liver as non-replicating and remaining extrachromosomal with>95% of rescued plasmids maintaining restriction pat-terns indistinguishable from the input plasmid.Consequently, the pBK-CMV vector encoding gD-1 isnot likely to be integrated chromosomally. Mice wereimmunized intravenously via the tail vein with 1.0 ml

of phosphate-bu�ered saline (pH 7.3) containing 10 mgof DNA encoding gD-1 complexed with ASOR-poly-L-lysine (gD-ASOR). Control mice were injected withDNA encoding gD-1, but not complexed with ASOR(gD-control). Animals immunized with pBK-ASOR (aplasmid lacking the gD-1 gene, but complexed withASOR) or uninjected were used as negative controls.At 2, 4, 6, 8, 14, 21 and 28 days post-immunization,three mice were euthanized and samples of liver, spleenand heart were removed, snap frozen in liquid nitrogenand stored at ÿ708C for future DNA analysis.Additional samples of liver, spleen and heart werein®ltrated with Tissue-Tek O.C.T. embedding media(Miles Inc., Elkhart, IN) and frozen in isopentanecooled with liquid nitrogen or were ®xed in 2% buf-fered formalin (pH 7.4) and embedded in para�n.

2.3. Immunohistochemistry

Seven micron thick para�n sections of liver andheart tissues were incubated with polyclonal rabbitantibodies against HSV-1 gD, CD3 (Dako,Carpinteria, CA), or P-selectin (PharMingen, SanDiego, CA); gD-1 expression was also localized inspleen sections. The primary antibodies were detectedusing biotinylated goat anti-rabbit secondary anti-bodies from the ImmunoPure ABC AlkalinePhosphatase Staining kit, generating a red substrate(Pierce, Rockford, IL). Liver cryosections 7 mm thickwere cut at ÿ208C, air dried at room temperature and®xed for 10 min in acetone at 48C. These sections wereincubated with rat monoclonal antibodies against CD4or CD8 (Caltag Laboratories, Burlingame, CA), fol-lowed by biotinylated rabbit anti-rat secondary anti-bodies (Pierce, Rockford, IL) and localized asdescribed above. Control sections were incubated withnon-immune serum instead of primary antibody. Allslides were counterstained with 1% fast green.

2.4. DNA analysis

Frozen tissue samples were thawed and chromoso-mal DNA was isolated using TRI REAGENT1

(Molecular Research Center, Inc., Cincinnati, OH)according to the manufacturer's protocol. To detectthe presence of HSV-1 gD DNA in the tissues, thepolymerase chain reaction (PCR) was used to amplifya 309 base pair region of the HSV-1 gD gene with thefollowing primers: sense, 5 '-TTGGCGGATGCCTCTCTCAAG-3 '; antisense, 5 '-ACAGTTGCCTCCCATCCGAAAC-3 '. As an in-ternal control, a 195 base pair PCR product for glycer-aldehyde-3-phosphate-dehydrogenase (GAPDH) wasampli®ed with the following primers: sense, 5 '-CCATGGAGAAGGCTGGGG-3 '; antisense, 5 '-CAAAGTTGTCATGGATGACC-3 '. The PCR mix-

J.V. Rogers et al. / Vaccine 18 (2000) 1522±1530 1523

tures (Life Technologies, Gaithersburg, MD) contained1.0 mM MgCl2, 100 pmol of each primer and 20 ng ofchromosomal DNA. Reactions were carried out usinga Perkin Elmer GeneAmp System 2400 thermocycler.gD-1 was ampli®ed during 35 cycles with denaturation(958C), annealing (588C) and extension (728C) stepslasting 1 min, 30 s and 1 min, respectively. TheGAPDH annealing was carried out at 578C, with allsteps lasting 30 s through 25 cycles of ampli®cation.PCR products were analyzed by agarose gel (1.2%)electrophoresis followed by ethidium bromide staining.Fragments were transferred to nitrocellulose mem-branes and hybridized with an oligonucleotide probespeci®c for an internal region of the PCR product.The probe for gD-1 was 5 '-TTGCCGCGAAAGCGATTGGG-3 ' and 5 '-CTAAGCATGTGGTGGTGCA-3 ' for GAPDH.Probes were end-labeled with digoxigenin-11-dUTP/dATP using the 3 ' tailing reaction according to theGenius System User's Guide (Boehringer Mannheim,Indianapolis, IN). DNA was hybridized overnight at608C for the gD-1 probe and 428C for the GAPDHprobe. The Genius nonradioactive detection kit(Boehringer Mannheim, Indianapolis, IN) was usedaccording to the manufacturer's protocol to detectbound probes.

2.5. Quantitative data

The number of immunopositive cells for gD-1, CD3,CD4, CD8, or P-selectin was quanti®ed in 10 continu-ous ®elds from each of three tissue sections per animalper time point using an ocular grid that enclosed anarea of approximately 0.0625 mm2. P-selectin ex-pression was determined as a proportion of the 25quadrants within the ocular grid containing at leastone immunopositive cell or platelet aggregate.Quantitative data were expressed as mean2S.E.M.The Alternate Welch t test was used to comparemeans, using P < 0.05 as the level for signi®cance.

3. Results

3.1. Immunohistochemistry

Both parenchymal and non-parenchymal cellsexpressed gD-1 (Fig. 1). Parenchymal cells thatexpressed gD-1 resembled hepatocytes, while gD-1+

non-parenchymal cells spanning the sinusoidal capil-laries appeared to be Kup�er cells. Punctate immuno-positive staining of parenchymal cells surrounded thenucleus (Fig. 1A), while non-parenchymal cells showedpunctate cytoplasmic and cell surface staining (Fig.1B). At 2, 4 and 6 days post-immunization, the aver-age number of gD-1+ cells per ®eld (0.0625 mm2) ran-

Fig. 1. Light micrographs demonstrating immunohistochemical local-

ization of gD-1 in liver, spleen and heart at 6 days post-immuniz-

ation with gD-ASOR. Bar � 25mm: (A) Cytoplasmic localization of

gD-1 (arrow) within liver parenchymal cells (between the arrow-

heads). (B) Non-parenchymal liver cell (arrow) within a sinusoidal

capillary (asterisk) expressing gD-1 throughout the cytoplasm. (C)

Arrow indicates a cell expressing gD-1 within the white pulp of the

spleen. (D) Cardiac cell (between the arrowheads) contains cyto-

plasmic gD-1+ material (arrow).

J.V. Rogers et al. / Vaccine 18 (2000) 1522±15301524

ged from 1.50 to 2.0 in the liver parenchyma and from0.30 to 0.50 in the non-parenchyma (Fig. 2). The aver-age number of gD-1+ parenchymal cells dropped sig-ni®cantly �P � 0:01� from 6 to 8 days (00.60) post-immunization and remained low through 28 days post-immunization. Since gD-1 expression was highest inthe liver parenchyma through 6 days post-immuniz-ation, we compared this time period with tissues fromcontrol mice. The expression of gD-1 was detectable at6 days post-injection in liver parenchymal and non-parenchymal cells (0.15 and 0.03, respectively) in miceinjected with gD-control, although these numbers weresigni®cantly lower �P � 0:01� than observed in gD-ASOR injected animals (Fig. 2B). The expression ofgD-1 could not be detected in unimmunized mice ormice immunized with pBK-ASOR.

Immunopositive staining for gD-1 was observed inspleen and heart samples (Fig. 1C and D). Splenic cells

Fig. 2. (A) Average numbers of liver parenchymal and non-parench-

ymal cells expressing gD-1 following intravenous injection with gD-

ASOR. The mean values at 2, 4 and 6 days post-immunization are

signi®cantly higher (asterisks; P < 0.05) than mean values at 8

through 28 days post-immunization. (B) Average numbers of liver

parenchymal and non-parenchymal cells expressing gD-1 at 6 days

post-immunization with gD-ASOR, gD-control, pBK-ASOR, or

unimmunized. Asterisks: mean values signi®cantly higher than the

corresponding controls.

Fig. 3. Immunolocalization of P-selectin, CD4 and CD8 in liver

samples 6 days post-immunization with gD-ASOR. Bar � 25mm: (A)

Arrows indicate P-selectin+ areas within the liver following gD-

ASOR immunization. (B) Localization of CD4+ cells (B; arrows)

and CD8+ cells (C; arrows) in®ltrating the liver following immuniz-

ation with gD-ASOR.

J.V. Rogers et al. / Vaccine 18 (2000) 1522±1530 1525

contained gD-1 positive material in both the cytoplasmand cell periphery, while cardiac cells expressed gD-1in the cytoplasm. The average number of gD-1+ cellsranged from 0.04 to 0.17 in the white pulp and from 0to 0.02 in the red pulp, while the average number ofgD-1+ cells in the heart ranged from 0.03 to 0.33. gD-1 expression in the spleen or heart could not be di�er-entiated from the background staining in tissue sec-tions from gD-control immunized animals.

The in¯ux of T cells into the liver following immu-nization was characterized immunohistochemically bycomparing the cellular expression of P-selectin, CD3,CD4 and CD8 in treated and control mice. In liversections, the proportion of ocular grids per observed®eld containing P-selectin+ cells in the vasculature,including Kup�er cells and endothelial cells as well asplatelet aggregates (Fig. 3A), ranged from 12 to 60%(Fig. 4). This proportion increased signi®cantly

�P � 0:02� between 2 and 4 days post-immunization.P-selectin+ expression remained above 26% through-out 21 days of observation and declined to about 12%by 28 days. A signi®cantly higher �P � 0:02� pro-portion of P-selectin+ cells were found in the livercompared with controls, but no signi®cant di�erenceswere observed in the heart samples (Fig. 4B).

Throughout the observed time course, the averagenumber of CD3+ cells per 0.0625 mm2 in liver sectionsof mice injected with gD-ASOR ranged from 0.54 to5.18, with a maximum at 14 d post-immunization (Fig.5A). At 2 and 4 days post-immunization, the averagenumber of CD3+ cells detected was 0.54 and 1.09, re-spectively. This number increased signi®cantly �P �0:01� from 4 to 6 days post-immunization. In animalsimmunized with gD-ASOR, signi®cantly more �P �0:01� CD3+ cells in®ltrated the liver compared withcontrol animals (Fig. 5B). The average number ofCD3+ cells per 0.0625 mm2 of heart in mice injected

Fig. 4. (A) Proportion of P-selectin expression per 0.0625 mm2 in

liver and heart following immunization with gD-ASOR. Mean values

at 4 through 21 days post-immunization are signi®cantly higher

(asterisks; P < 0.05) than mean values at 2 and 28 days post-immu-

nization. No signi®cant expression of P-selectin can be detected in

the heart following gD-ASOR injection. (B) Average number of liver

and heart endothelial cells expressing P-selectin (2S.E.M.) 6 days

post-immunization with gD-ASOR, gD-control, pBK-ASOR, or

unimmunized. Asterisk indicates the mean value is signi®cantly

higher (P < 0.05) than the respective control.

Fig. 5. (A) Average number of CD3+ cells in liver and heart follow-

ing immunization with gD-ASOR. Asterisks indicate mean values at

6 through 28 days post-immunization are signi®cantly higher

(P < 0.05) than mean values at 2 and 4 days post-immunization. No

signi®cant in¯ux of CD3+ cells occurs in the heart following immu-

nization. (B) Average number of CD3+ cells in liver and heart at 6

days post-immunization with gD-ASOR, gD-control, pBK-ASOR,

or unimmunized. Asterisk indicates the mean value is signi®cantly

higher (P< 0.05) than the respective control.

J.V. Rogers et al. / Vaccine 18 (2000) 1522±15301526

with gD-ASOR ranged from 0.07 to 0.26; experimentaland control animals did not di�er signi®cantly (Fig.5B). During the peak periods of T cell in¯ux (6, 8 and14 days post-immunization), we observed three-foldhigher numbers of CD4+ cells than CD8+ cells (Figs.3 and 6).

3.2. DNA analysis

Southern analysis con®rmed the predicted sizes ofthe PCR products from liver, spleen and heart samplesof BALB/c mice as 309 bp for gD-1 and 195 bp forGAPDH. In liver samples of mice injected with gD-ASOR, DNA encoding gD-1 could be identi®ed up to14 days, but could not be detected at 21 and 28 dayspost-immunization (Fig. 7A). The GAPDH controlswere uniformly positive throughout the examined timecourse. Low, but detectable levels of DNA encodinggD-1 in liver samples occurred only at 2 days post-im-munization following gD-control immunization; gD-1DNA was not identi®ed in control liver samples ofunimmunized mice or animals injected with pBK-ASOR (Fig. 7B). The gD-1 gene was detectable inspleen and heart samples of mice injected with gD-ASOR through 8 days and 2 days post-immunization,respectively (Fig. 7C and D).

4. Discussion

In vitro and in vivo, ASGPr+ hepatocytes expresstransgenes following delivery of DNA complexed toasialoglycoprotein-poly-L-lysine [21,22,30±32,34,35].

Our results show that intravenous injection of the gD-ASOR complex leads to signi®cant expression of gD-1by parenchymal and non-parenchymal cells. Uptake ofthe gD-ASOR complex by hepatocytes presumablyre¯ects receptor-mediated endocytosis, as demon-strated with other ASOR-poly-L-lysine DNA com-plexes [21,22,30±35]. This complex could also be takenup by Kup�er cells via phagocytosis of larger aggre-gates of the ASOR-poly-L-lysine DNA complex [22].

Southern blots of PCR products ampli®ed fromliver DNA isolates demonstrated that the observedloss of gD-1 DNA leads to a decrease in the numberof hepatocytes expressing gD-1. We observed that Tcell in¯ux into the liver correlates with the decrease ingD-1 expression. In adenovirus-transduced livers ofimmunocompetent mice, transgene expression anddetectable viral DNA declines when lymphocyte in®l-tration and the number apoptotic cells increases [36±39]. These adenovirus-transduced cells are suggested tobe destroyed by the immune system and replaced byproliferating hepatocytes which do not express theforeign gene [36±39].

During T cell in¯ux, we observed signi®cantly higherexpression of P-selectin in gD-ASOR immunized ani-mals, consistent with the role P-selectin plays in leuko-cyte homing to the liver [40±44]. The T cells weobserved in®ltrating the liver are predominantlyCD4+, a subtype of T cells which respond to gD-1 inBALB/c mice [5,6,12,14,45]. We observed a 3:1 ratioof CD4+:CD8+ T cells during the ®rst two weekspost-immunization. Cruz et al. [35] also observed aCTL response in BALB/c mice immunized with gD-ASOR that was generated against cells expressingMHC class II, but not against cells expressing onlyMHC class I molecules. The signi®cant increase in Tcells in®ltrating the liver occurring as gD-1 expressiondeclines in gD-ASOR treated, but not control mice,suggests the observed T cell response may contributeto the decrease in gD-1 expression and detectable gD-1DNA.

In addition to the liver, we observed low levels ofuptake of gD-ASOR in the spleen and the heart. Inthe spleen, macrophages could phagocytize the gD-ASOR complex, resulting in low numbers of gD-1+

cells. gD-1 DNA could not be detected in heartsamples beyond 2 days post-immunization with gD-ASOR, presumably re¯ecting low levels of non-speci®cuptake of the DNA complex. Although in vivo ex-pression of DNA encoding a foreign gene by cardiaccells can occur following direct injection [46], onlyabout 0.4% of the total radiolabeled ASOR-poly-L-lysine DNA complex injected intravenously could bedetected in heart samples [30]. This low uptake of theDNA complex uptake presumably accounts for thelow number of cardiac cells expressing gD-1.

This targeted delivery system may be useful in vacci-

Fig. 6. Average number of CD4+ and CD8+ cells in liver at 6, 8

and 14 days post-immunization with gD-ASOR. Note the consis-

tantly higher proportion of CD4+ cells than CD8+ cells.

J.V. Rogers et al. / Vaccine 18 (2000) 1522±1530 1527

nation against pathogenic micro-organisms since trans-

gene expression within the liver may induce immuno-

logical memory. Our study shows that intravenous

immunization with plasmid DNA encoding gD-1 com-

plexed with asialoorosomucoid-poly-L-lysine results in

transient expression of gD-1 in the liver. The decrease

in gD-1 expression and detectable gD-1 DNA corre-

lates with signi®cant T cell in¯ux into the liver in gD-

ASOR treated, but not control mice. We are currently

investigating the potential of immunization with gD-

ASOR as a vaccine against HSV-1.

Acknowledgements

This research was supported by the NationalInstitutes of Dental Research Grant 1R03 DE12521-01A1 (N.J.B.) and a Research Challenge Grant fromWright State University (B.E.H.).

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Fig. 7. Southern blots of gD-1 and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) PCR products ampli®ed from DNA isolated from the

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