DNA immunization utilizing a herpes simplex virus type 2myogenic DNA vaccine protects mice from mortality and

prevents genital herpes

John R. Gebhard*, Julie Zhu, Xia Cao, John Minnick, Barbara A. Araneo

DNA Vaccines and Gene Transfer, Pharmadigm Inc, 2401 Foothill Drive, Salt Lake City, UT 84109, USA

Received 24 June 1999; received in revised form 30 August 1999; accepted 30 August 1999

Abstract

A gene transfer vector for DNA immunization was developed in which the promoter was derived from the murine musclecreatine kinase (MCK) gene; a gene expressed only in di�erentiated skeletal muscle. In vitro, we observed high-level, butunrestricted, gene expression from the cytomegalovirus (CMV) promoter unlike expression from the MCK promoter which was

weak but restricted to myo®bers. A myogenic DNA vaccine (MDV) that encoded the glycoprotein D gene from herpes simplexvirus type-2 (HSV-2) was used to DNA immunize mice. MDV immunization resulted in virus speci®c immunity that protectedHSV-2 infected mice from mortality and prevented the development of genital herpes. Therefore, we conclude that high-levelgene expression or the use of a strong transcription unit was not a prerequisite for an e�cacious DNA vaccine and the use of a

nonviral tissue speci®c promoter could su�ce. # 2000 Elsevier Science Ltd. All rights reserved.

Keywords: DNA vaccine; Tissue speci®c; Muscle; HSV-2

1. Introduction

Vaccination has resulted in global eradication ofsmallpox, near elimination of poliomyelitis, and pro-tection against a variety of human pathogens (e.g.diphtheria, measles, mumps, rubella, and chickenpox).Despite these successes, emerging diseases such asEbola virus, human immunode®ciency virus, Hanta-virus, re-emerging diseases (e.g. tuberculosis, malaria),and endemic infections (e.g. Borrelia burgdoferi, Neis-seria gonorrheae, hepatitis C virus) present new chal-lenges to the future of vaccine development. DNAimmunization is an attractive alternative to traditionalvaccination methods. Intramuscular or intradermalinjection of DNA plasmids encoding either full-lengthmicrobial proteins or de®ned epitopes of the microbe

has been demonstrated to elicit both humoral and cel-

lular based protective immunity [1±11]. To date, one

human trial has been published that has demonstrated

the production of an immune response by DNA im-

munization in a naiõÈ ve population [12].

The construction of an e�cient DNA vaccine

depends on many variables. These include, but are not

limited to, the DNA backbone, the type of promoter

(viral/nonviral, constitutive, tissue speci®c) the use of

introns [13], ancillary noncoding regions, immunosti-

mulatory sequences [14±17], the encoded antigen, the

route of delivery, and the use of delivery vehicles (e.g.

liposomes). The current dogma concerning DNA vac-

cine constructs suggests that viral promoters, as

opposed to cellular promoters, ensures high-level gene

expression ultimately augmenting and providing for a

protective immune response [18]. Most reported DNA

vaccines utilize plasmid vectors in which the promoter

is of viral origin (e.g. CMV); one investigation has stu-

Vaccine 18 (2000) 1837±1846

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

PII: S0264-410X(99 )00418 -1

www.elsevier.com/locate/vaccine

* Corresponding author. Tel.: +1 801-464-6106; fax: +1 801-464-

6116.

E-mail address: jgebhard@pharmadigm.com (J.R. Gebhard).

died the generation of an immune response throughthe use of a cellular promoter [19].

Whether a cellular promoter could be an e�cienttranscription element as part of a DNA vaccine andresult in protective immunity is unknown. Since genetranscription from the DNA vaccine is more easilymonitored and controlled if expression is tissuerestricted, we focused on the development of a tissuespeci®c promoter. Muscle is an accessible tissue forDNA immunization and is regarded as the site ofchoice for vaccination. We rationalized that the use ofa muscle speci®c promoter might be suitable in aDNA vaccine and chose the muscle creatine kinase(MCK) gene to test our hypothesis. The MCK gene iswell characterised with gene expression highlyrestricted to skeletal and to a lesser extent, cardiacmuscle [20±34]. MCK is transcriptionally inducedduring the di�erentiation of myoblasts to myo®bers[20,22,24,35,36]. Additionally, myo®bers are multinu-cleated favoring more expression events per ®ber.

HSV-2 is responsible for a�icting an estimated500,000 individuals every year [37]. Currently, 20% ofthe US population is infected with HSV-2, an increaseof 30% since the 1970s [37]. HSV is extremely patho-genic exhibiting a lytic and latent state. Physical mani-festations of the lytic stage result in genital lesions [38],whereas in the latent stage the virus is resident in theperipheral nervous system. Recurrence among individ-uals is controlled by the cell-mediated immune system(CMI), speci®cally CD4+ and CD8+ cytotoxic T-lym-phocytes (CTL) [39±41]. Numerous approaches to pro-vide a prophylactic vaccine have been employed, buthave met with little success. Thus, the need for a pro-phylactic vaccine remains paramount.

We investigated the utility of a minimal murineMCK promoter/enhancer to function as the transcrip-tional cassette of a DNA vaccine against HSV-2. Wehave termed such a vaccine construct a ``myogenicDNA vaccine''. In the present manuscript, we reportfor the ®rst time that a plasmid DNA that utilized atissue speci®c (myogenic) nonviral promoter thatencodes the HSV-2 glycoprotein D gene elicited a pro-tective antiviral immune response in a mouse modelfor HSV-2 [42,43].

2. Materials and methods

2.1. Animals

Female BALB/c mice (8 weeks) were purchasedfrom Charles River Laboratories, and used at 12±16weeks.

2.2. Virus and cell lines

In all experiments, the virulent HSV-2 strain 333was utilized. Vero cells maintained in DMEM sup-plemented with 10% fetal calf serum (Gibco-LTI) wereobtained from Robert Fujinami (University of Utah).C2C12 murine myoblasts were purchased from theAmerican Type Culture Collection (ATCC), and main-tained in the undi�erentiated state by passage at lowdensity in DMEM supplemented with 20% fetal calfserum and 0.5% chicken embryo extract. To induceformation of multinucleated myo®bers, C2C12 myo-blasts were allowed to reach 80±90% con¯uency atwhich time the medium was change to DMEM sup-plemented with 2% horse serum (Hyclone).

2.3. Virus plaque assay

HSV-2 was serially diluted ten-fold in DMEM with-out serum. The virus dilutions were applied to Verocells at 80% con¯uency. After gentle rocking at 378C,5% CO2 for 1 h, the virus innoculum was aspirated,fresh complete media was applied, and the virallyinfected cells were incubated for 48 h at 378C, 5%CO2 at which time cytopathic e�ect (CPE) was noted.Viral plaques were visualized with a stain that con-sisted of 0.1% crystal violet (Sigma) in 20% ethanol.

2.4. Plasmid DNA

The parental plasmid pMCKv.2 was derivatizedfrom pcDNA3.1 (Invitrogen) and constructed as fol-lows. The CMV promoter, the neomycin gene, and theSV40 origin and polyadenylation signal were excisedfrom pcDNA3.1. The MCK minimal enhancer/promo-ter was obtained by polymerase chain reaction (PCR)from C3H murine genomic DNA. The 1.3 kB MCKminimal enhancer/promoter was subcloned into thederivatized pcDNA3.1 vector to yield pMCKv.2. Thefull-length glycoprotein D gene (gD2) from HSV-2 (akind gift from Gary Cohen, University of Pennsylva-nia) was inserted into pMCKv.2 to yield the plasmid,pMCKv.2-gD2. Likewise, pMCK-GFP was con-structed by insertion of the GFP coding cassetteexcised from pEGFP-NI (Clontech). All plasmids werepropagated in XL-Blue (Stratagene) Escherichia colibacteria according to standard methods. EndotoxinDNA was prepared through the use of a�nity chroma-tography (Qiagen, Inc.).

2.5. Plasmid immunizations and virus challenge

Adult (3 months) female BALB/c mice were immu-nized with 100 mg of DNA (either pMCKv.2 orpMCKv.2-gD2) dissolved in 50 ml of sterile phosphatebu�ered saline (PBS). Injections with a 28 gauge nee-

J.R. Gebhard et al. / Vaccine 18 (2000) 1837±18461838

dle targeted to the left tibealis anterior muscle weregiven at 3-week intervals for a total of three injections.

Four weeks after the ®nal DNA immunization, but7 days prior to infection with HSV-2, the mice weregiven subcutaneous injections of 2 mg of Depo-Pro-vera (UpJohn Co.). Mice were anesthesized intra-peri-toneally with 2 mg/ml ketamine, and intra-vaginallyexposed to Dacron plugs soaked in ten ml of a 1� 105

pfu/ml of HSV-2 solution (32 LD50). The plugs wereremoved after 30 min. All animals were monitoreddaily for the appearance of lesions, and scored accord-ing to the following scale: 0 = no visible redness orlesions, 1 = redness or mild swelling, 2 = erosions,vesicles, or moderate swelling, 3 = several large ves-icles, 4 = large ulcers with severe maceration and/orurinary retention and/or hind limb paralysis.

2.6. Serology

At the indicated times in the text, sera was collectedinto Microtainer tubes (Becton Dickinson) by retro-orbital puncture, and clari®ed by centrifugation. Arecombinant baculovirus (Invitrogen) that secretesHSV-2 gD protein was constructed and used as thesource of antigen in the enzyme-linked immunoassay(ELISA). Ninety-six well ¯at bottom plates (Corning)were coated overnight at 48C with a dilution of gD2containing baculovirus supernatant in phosphate buf-fered saline (PBS; the exact dilution was determinedempirically). The next day, the plates were washed 4times with distilled water followed by 3 washes withPBS/0.5% Tween 20 (PBS-Tween). The plates wereblocked for 2 h at 378C, 5% CO2, with a bu�er thatconsisted of 10% FCS/PBS-Tween. Clari®ed seradiluted two-fold was applied to blocked plates andincubated overnight at 48C. After primary antibodytreatment, unreacted antibody was removed by washesof distilled water and PBS-Tween 20. Rabbit anti-mouse IgG (or in isotype pro®les IgG1 or IgG2a) con-jugated to horseradish peroxidase was applied andallowed to incubate for 2 h at room temperature. Theplates were washed, developed with a citrate bu�erthat contained the chromagen 2,2 '-Azino-bis(3-ethyl-benthiazoline)-6-sulfonic acid (ABTS). The reactionwas terminated by addition of a solution of SDS/DMF. The absorbances were determined at 405 nm ina Spectramax kinetics microplate reader (MolecularDevices, Inc.). A positive serological response and cut-o� values in the experimental group were ascertainedthrough the comparison to a positive HSV-2 gD2 spec-i®c antisera control present on each microtiter plate.

2.7. Statistical analyses

The geometric endpoint mean titer (GMT) of eachmouse was determined through the use of the statisti-

cal software program, Prizm 2.01. The statistical sig-ni®cance of the results was evaluated by the Mann±Whitney two-tailed t-test as reported in the ®gurelegends.

3. Results

3.1. The muscle speci®c DNA vaccine construct istranscriptionally restricted to di�erentiated muscle

To illustrate that modi®cation of the promoterplaced in a di�erent DNA backbone still retained tran-scriptional stringency, reporter constructs were gener-ated for use in transient transfection assays. Murinemyoblasts (C2C12) were transfected with pMCK-GFP,or a control reporter plasmid (pEGFP-NI) which uti-lized the CMV promoter. No ¯uorescence was detectedin pMCK-GFP transfected myoblasts (Fig. 1, panelA), whereas pEGFP-NI transfected myoblasts demon-strated ubiquitous and intense GFP ¯uorescence (Fig.1, panel B). After transfected myoblasts were allowedto di�erentiate to myo®bers, however, pMCK-GFP ex-pression was detected (Fig. 1, panel C). Multinucleatedmyo®ber formation was con®rmed through the use ofthe nuclear stain 4 ',6-diamidino-2-phenylindole (DAPI)(Fig. 1, panels E and F). Although GFP expressedfrom the MCK promoter was weak, ¯uorescence wasrestricted to myo®bers unlike the pEGFP-NI trans-fected muscle cells, which exhibited high-level unrest-ricted gene expression [as judged from GFP¯uorescence (Fig. 1, panels C±E)].

Similar transient tranfections were performed inmonkey kidney cells, keratinocyte cells, CHO cells,and a macrophage derived cell line. In all cases, onlythe CMV, but not the MCK, construct possessed theability to synthesize RNA based on GFP or b-galacto-sidase protein expression (data not shown).

3.2. Plasmid immunization with a muscle speci®cpromoter generates a speci®c HSV-2 glycoprotein Dantibody response

We were interested in whether the observed lowtranscription from MCK could result in the gener-ation of a speci®c antiviral immune response. Toanswer this query, we constructed a MDV thatencoded the HSV-2 gD. Mice were DNA immu-nized intramuscularly (i.m.) with either pMCKv.2(control) or with the MDV as described in Section2. Between DNA immunizations, sera collected fromthe mice were examined for the development ofHSV-2 gD speci®c antibody (total IgG) by ELISA.Fig. 2 illustrates the kinetics of seroconversion inMDV mice, where strong gD2 speci®c antibody re-sponses developed prior to HSV-2 infection. Speci®c

J.R. Gebhard et al. / Vaccine 18 (2000) 1837±1846 1839

gD2 antibody was detected after two DNA immu-

nizations in the MDV mice. After the ®nal boost,

seroconversion had reached 95% (11±12 weeks after

the ®rst DNA immunization). Infection with HSV-2

resulted in an initial decline in circulating antibody

titer in MDV mice. The change in titer was tem-

porary as we observed a consistent rise in gD2

speci®c IgG that surpassed the antibody levels

achieved pre-challenge (Fig. 2). Control immunized

mice (pMCKv.2) did not develop any detectableantibody to gD2 at any time (pre or post challenge).

In addition to IgG, gD2 speci®c serum IgA and

Fig. 1. Gene expression from the modi®ed MCK promoter is restricted to di�erentiated muscle. Undi�erentiated murine C2C12 muscle myo-

blasts were transfected with pMCK-GFP (panels A, C, and E) or pEGFP-NI (panels B, D, and F). Forty-eight hours post-transfection, ex-

pression of GFP in myoblasts was visualized by ¯uorescence (panels A and B). Di�erentiation of myoblasts to multinucleated myo®bers was

induced and GFP expression in myo®bers after 4 days was noted (panels C and D). Panels E and F are DAPI stained C2C12 cells in di�eren-

tiation medium. Note that not all cells di�erentiate, but still express GFP (panel F). In all panels, the length of photographic exposure was equiv-

alent.

J.R. Gebhard et al. / Vaccine 18 (2000) 1837±18461840

IgM were analyzed. Serum IgA was not detected pre-or post-challenge, while IgM could be detected pre-challenge and early in infection (data not shown).

3.3. DNA immunization with pMCKv.2-gD2 generated amixed but predominant Th1-like response

The IgG isotype has been utilized as a measure ofthe type of Th response (Th1 or Th2) as a result ofDNA immunization, and as an indirect correlate ofcell-mediated immunity (54). Because there is strongdata that indicates i.m. delivery of DNA results in aTh1 response and biolistics stimulates a Th2, wewanted to determine if expression directed from theMDV could e�ect either pathway.

Individual sera collected at various time points wereassayed for the presence of IgG2a and IgG1 isotypeantibodies. The results indicated that the myogenicDNA vaccine resulted in a mixed Th response sinceboth IgG2a and IgG1 isotypes were found to predomi-nate in individual mice pre-challenge (Fig. 3). Twice asmany animals demonstrated an IgG2a antibody pro®lethat suggested that parenteral DNA immunizationwith the MDV stimulated a Th1-like response. Isotypeswitching was not observed post challenge with HSV-2within animals that presented either a Th1 or Th2 likeresponse upon infection with HSV-2. It should benoted, however, that an appreciable number of animals

generated a prevalent Th2 or a balanced Th1/Th2 pro-®le.

3.4. Mice immunized with the myogenic DNA vaccineare protected from lethal challenge with HSV-2

Although mice that received the myogenic DNAvaccine generated a HSV-2 speci®c antibody response,and had acquired ``immunity'' to HSV-2, whether theimmunity was protective to virus insult was unknown.Therefore, to ascertain if protective antiviral immunityhad been achieved with the MDV, DNA immunizedmice were challenged intra-vaginally with a lethal doseof HSV-2 (32 LD50). Although morbidity was notedwithin 7 days post infection in mice immunized withthe empty DNA plasmid (pMCKv.2) (Fig. 6), no mor-tality was observed (Fig. 4). At 10 days post infection,however, marked mortality was noted in mice thatreceived the control DNA (41% survival) versus theMDV immunized mice (100% survival). Although41% of the control mice had survived at 10 days postinfection, there was considerable morbidity in thisgroup. By day 11 post infection there was a markedincrease in the control mice (26%; n = 8) that suc-cumbed in HSV-2 infection. Thus, as indicated in Fig.4, at 14 days post infection, most control DNA immu-nized mice had succumbed to HSV-2 whilst the ma-

Fig. 3. Myogenic DNA vaccination generates a heterotypic IgG pro-

®le. Animals were DNA immunized with either the control plasmid

DNA (pMCKv.2; n = 30) or either the myogenic DNA vaccine

(MDV; n=40) as described in the text. At the indicated time points,

sera was collected from all mice (unless indicated in the text) and

assayed for the presence of either HSV-gD2 speci®c IgG1 or IgG2a

antibody. The data are presented as the number of mice which

exhibited a predominant IgG1, IgG2a, an equivalent IgG1/IgG2a, or

neither an IgG1 nor IgG2a response (Nil). Only the data from the

MDV animals are presented since the control animals did not have a

detectable IgG response. This experiment is representative of one of

three that were conducted.

Fig. 2. The myogenic DNA vaccine stimulates speci®c HSV-2 IgG

antibody. Animals were DNA immunized as described in the text

with either control plasmid DNA (pMCKv.2; n = 30) or the myo-

genic DNA vaccine (MDV; n=40). Prior to infection, sera were col-

lected at two weeks post immunization. After infection with HSV-2,

sera were collected from all mice and assayed for HSV-2 gD2 speci®c

total IgG by ELISA. The data are presented as the geometric mean

titer. First, second, and third represent the antibody titer after the

indicated DNA boost, prior to infection with HSV-2. PID refers to

post infection day.

J.R. Gebhard et al. / Vaccine 18 (2000) 1837±1846 1841

jority of the myogenic DNA immunized mice survived.Although the endpoint of the assay was 14 days, wedid not observe any MDV mice succumb at later times(up to 45 days).

Viral pro®les conducted on the control mice at theindicated time points (by plaque assay) that survivedHSV-2 infection indicated that they had not been pro-ductively infected. Accordingly, in MDV immunizedmice which succumbed to HSV-2, the presence HSV-2gD speci®c antibody or a positive humoral responseswas not detected. The cell-mediated immune, or T-cell,response was not evaluated. Therefore, it is possiblethat there was a CD4+/CD8+ in the MDV mice thatdid not demonstrate a positive humoral response.

3.5. Rapid clearance of HSV-2 in DNA immunizedanimals

The results presented thus far indicate that a DNAvaccine that utilized a cellular tissue speci®c promoterelicits a virus speci®c humoral immune response thatresulted in protective antiviral immunity. To determineif the mechanism of protection (survival) was predi-cated on the ability of the host's immune system toclear infectious virus from the site of infection, HSV-2shed vaginally was assessed by plaque assay from bothcontrol and myogenic DNA immunized animals.

Early in the infectious cycle, no signi®cant di�erencein the quantity of infectious virus shed between controland DNA immunized mice was noted (Fig. 5). We didnot observe any MDV immunized mice that demon-

strated ``sterilizing'' immunity. In fact, signi®cant viraltiters were found in both groups of animals. At day 4post infection (peak HSV-2 replication), however,there was a clear and signi®cant di�erence (PE0.0001)in the quantity of virus shed from the mice thatreceived the MDV versus the control DNA (ca. 104

pfu/ml vs 106 pfu/ml). The di�erence in the titer ofinfectious virus shed intra-vaginally was further magni-®ed by day 7 post infection in which most MDVanimals had cleared HSV-2, whereas the controlimmunized animals still retained a signi®cant quantityof virus (105 pfu/ml).

As noted in the preceding section, there was exten-sive morbidity in the surviving control animals at day10 post-infection. Virus shed from these animals wasnot evaluated at day 10 and is not re¯ected in Fig. 5.Rather the day 10 values are representative of the con-trol mice that survived lethal challenge (n=4).

3.6. Myogenic DNA vaccinated mice are protected fromprimary genital disease

To determine whether the MDV could protect micefrom the physical manifestations of HSV-2 infection,i.e. genital lesions, mice were infected with a high dose(32 LD50) of HSV-2 and followed for the developmentof disease. Disease in the mouse model for HSV-2 isphysically apparent within 5±7 days post-infection.Mice that received the control DNA demonstratedsigns of genital herpes within 5 days post infection(Fig. 6). The severity of the lesions increased in thecontrol mice with each successive day and at the lattertimes of infection hind limb paralysis was observed an

Fig. 5. HSV-2 is cleared more rapidly in myogenic DNA vaccinated

animals. Mice were DNA immunized with either the control

(pMCKv.2; n = 30) or myogenic DNA vaccine (MDV; n = 40).

Three-weeks after the ®nal boost, the mice were infected intra-vagin-

ally with a lethal dose of HSV-2 (32 LD50). At the time points indi-

cated, infectious virus yield was assessed by plaque assay of vaginal

¯uid. The data are expressed as plaque forming units/ml of vaginal

wash. This experiment is representative of one of three that were

conducted.

Fig. 4. Myogenic DNA vaccinated mice are protected from a lethal

dose of HSV-2. Mice were DNA immunized with either the control

(pMCKv.2; n = 30) or myogenic DNA vaccine (MDV; n = 40).

Three-weeks after the ®nal boost, the mice were infected intra-vagin-

ally with a lethal dose of HSV-2 (32 LD50). Mortality was followed

over a 14 day time span. At day 4 post infection there was a signi®-

cant di�erence in the quantity of HSV-2 shed vaginally between the

control and the MDV groups (P E 0.0001); Mann±Whitney two-

tailed t-test). This experiment is representative of one of three that

were conducted.

J.R. Gebhard et al. / Vaccine 18 (2000) 1837±18461842

indication of peripheral nervous system involvementby HSV-2. In contrast, MDV immunized mice dem-onstrated marked resistance to the development ofdisease. Less than 5% of MDV mice developed symp-toms of disease; the severity in these mice was extre-mely mild (score of <1). Therefore, the MDV wasprotective against the development of genital herpes.

4. Discussion

The results herein demonstrate for the ®rst time thatplasmid immunization with a DNA construct, inwhich the transcription unit was both nonviral and tis-sue speci®c was su�cient to generate a protective anti-viral immune response. Importantly, the promoterutilized in this study was speci®c for muscle, a tissuereadily accessible for administering vaccines.

An attractive feature of the MCK promoter is itstranscriptional attributes since the MCK promoter isactive in di�erentiated muscle as opposed to myo-blasts. Transcriptional mapping analysis reveals a 259bp enhancer element distal to the transcription startsite of the MCK promoter which is responsible forskeletal muscle restriction [26,30,44]. MCK activationis dependent on the transcription factor, MyoD1,which is activated during di�erentiation by sequencespeci®c binding to the E box of the MCK promoter[28,45]. MyoD1, however, is inactive in smoothmuscle, while MCK regulation is more complex in car-diac muscle requiring additional elements in the 3.0 kb

promoter. The current myogenic DNA vaccine con-struct contained the MCK minimal promoter/enhancerelement and thus, would not be expected to be activein cardiac muscle.

The myogenic DNA construct appeared to be tran-scriptionally weak compared to CMV, a strong viralpromoter employed in the majority of DNA vaccines(Fig. 1). Gene expression from the immediate earlyCMV enhancer/promoter was very strong and activein both muscle and nonmuscle cells. On the otherhand, expression from MCK was weak but restrictedto di�erentiated skeletal muscle. It was possible thatMCK would be an ine�cient component of a DNAvaccine since the prevailing assumption has been thatstrong expression of the antigen is required to result inprotective immunity [36]. This assumption appeared tobe invalid in the current study. Because the inherentsensitivity of the immune system is the basis of thecell-mediated immune response (e.g. cytotoxic T cells,NK cells), other factors may be more important thanthe high-level expression of the encoded antigen. Suchfactors include the nature of the antigen [46], the routeand site of delivery, or the presence of stimulatorysequences. Indeed, it is appreciated that the very routeof DNA delivery in¯uences the development of Th1versus a Th2 like response [47].

In the present study we observed that MDV immu-nization led to a preponderance of IgG2a gD2 speci®cantibody. The generation of isotype speci®c antibodyis frequently the result of conformation dependant epi-topes produced by the live virus. DNA vaccines closelyresemble viruses in the mechanism of gene expression(e.g. conformationally correct protein production).Whether the immune response generated as a result ofMDV immunization mimicked a live HSV-2 infectionwas not analyzed; however, good protective immunityagainst HSV-2 was achieved that suggests that theMDV can elicit a vigorous immune response similar tothat attained by live viral infections. Additionally, ourresults with the MDV suggest that a strong promoterthat achieves in high-level gene expression is notalways necessary to initiate a productive and protectiveimmune response. DNA immunization with the MDVdid not prevent the replication of HSV-2 (Fig. 5), butdid provide for the generation of speci®c antiviral IgGand protected against mortality and morbidity uponHSV-2 infection. An excellent vaccine should allowviral replication but not cause disease, because viralreplication should further boost immunity and a stron-ger memory response. Such a scenario was found withMDV mice. There was an initial burst of viral replica-tion (Fig. 5), but then HSV-2 was cleared from MDVmice more rapidly than mice that did not receive theMDV presumably due to B and T-cell memory.

The MDV did not provide for sterilizing immunity.By day 4 post infection, however, the quantity of

Fig. 6. The development of genital herpes is abrogated in animals

that receive the myogenic DNA vaccine. Mice were either adminis-

tered the myogenic DNA vaccine (MDV) or the control DNA

(pMCKv.2) as described in the text. Following immunization, the

mice were infected with a lethal dose of HSV-2 and assessed visually

on a scale of 0 to 4 (4 = most severe Ð see Section 2) daily until

day 14. The data represent the mean lesion score at each representa-

tive day. This experiment is representative of one of three that were

conducted.

J.R. Gebhard et al. / Vaccine 18 (2000) 1837±1846 1843

HSV-2 shed intravaginally from MDV animals was 2logs lower than control mice when compared to day 2post infection. In fact, the control animals were neverable to attain the level of virus clearance attained bythe MDV group. Concomitant with the relatively inef-fective viral clearance in the control DNA immunizedgroup (vector alone) was the appearance of genitallesions (Fig. 6), while in the MDV group there was notan appreciable appearance of acute disease. It is tempt-ing to speculate based on the cummulative results ofFigs. 5 and 6 that there is a critical threshold of virusthat the mouse immune system can e�ectively combat;above this threshold, there is the appearance of HSV-2disease. It is documented that there is a cell-mediatedcomponent in the control of HSV-2; both arms of theimmune system need to be active in the clearance ofHSV infection [41,48,49]. In human populations, bothCD4+ and CD8+ cytotoxic T lymphocytes have beencharacterized from virus infected sites [39,41,50,51].Koelle et al. isolated from human donors, lesion-in®l-trating HSV-2 speci®c T-cells, a subset that werespeci®c for glycoprotein D [39±41,52]. We hypothesizethat the rapid clearance found between day 2 to day 4post infection is due not only to humoral, but alsocell-mediated immunity generated by the MDV. It ispossible that secondary CTL generated by the MDVare responsible for the rapid clearance of virus.Whether this hypothesis and gD2 speci®c CD4+/CD8+ T-cells are generated as a result of MDV ad-ministration is currently under investigation.

Upon antigenic stimulation, CD4+ T-helper lym-phocytes (Th) proceed down two developmental routesdependent on the cytokine milieu that result in di�er-ent e�ector functions: one leads to the development ofTh1 cells, and the other to Th2 cells [53]. Th1 cells se-crete Il-2, TNF-a, TNF-b, g-interferon, activate CD8 Tcells, macrophages, and promote the synthesis of IgGantibody class switching to the IgG2a isotype func-tions [54±56]. Th2 cells, however, synthesize IL-4, IL-5, IL-10, promote B-cell activation and the develop-ment of IgG1 antibody functions [54±56]. Addition-ally, each group of Th cells regulates the other due tothe action of cytokines secreted. Analysis of the IgGsubtypes indirectly supported that both the Th1 andTh2 pathways were stimulated as a result of MDV ad-ministration.

How might both the MHC class I and II pathwaysbe stimulated by the MDV? Recent investigations intothe mechanism of antigen presentation by DNA vac-cines suggest that bone marrow derived cells are themajor antigen presenting cells (APC) [57±59]. Dendri-tic cells (DC), the most e�cient APC, have beenshown by others to be directly transfected by DNA.Therefore, the DC would directly synthesize the anti-gen intracellularly and present the antigen via both theMHC class I and class II pathways. Although it is

possible that the MDV directly transfected the DC,there is no evidence that MyoD1 is active in cells de-rived from a myeloid lineage. Thus, we would notexpect the MDV to be operative in DC.

Muscle is known to express antigen after DNA vac-cination [60], but muscle tissue expresses little MHCclass I and no class II molecules. It has recently beendemonstrated, however, that antigen could be trans-ferred by myocytes to professional APC [61]. Otherresearch suggests that soluble protein (and perhapspeptide fragments) may be transferred to residentmacrophages or dendritic cells to culminate in a Th1or Th2 response [58]. In the present study, our prelimi-nary data with the MDV are more congruent withantigen priming due to antigen transfer. We suggestthat the glycoprotein D gene is synthesized in the myo-®bers and part or all of the protein is transferred toprofessional APC. The professional APC would be re-sponsible for presentation and activation of the CD4+

and CD8+ pathways.The demonstration that the MDV construct was e�-

cacious may indicate a use in the design of prophylac-tic and therapeutic therapies when the use of a viralpromoter is not desirous or ine�cient. Of note is therecent report that a construct containing a viral, butnot a cellular promoter, exhibited attenuation and tox-icity in response to, or in conjunction with, cytokines[62,63]. It can be envisioned that the use of a tissuespeci®c promoter such as MCK may be utilized in con-ditions where low gene expression is required (e.g.allergy). Gene expression from MCK by virtue of itsspeci®city to muscle is inherently less promiscuous andtherefore may be considered to be a safer element in aDNA vaccine construct than a viral promoter. DNAvaccines harboring viral promoters may migrate toother organs. Since viral promoters are constitutive, ifgene expression occurs in an unintended site there isa possibility for damage to the organ, possibly dueto an autoimmune reaction. Indeed, it has beendemonstrated that DNA immunization with the nucleo-protein gene from LCMV can stimulate local in¯am-mation in the muscle; the in¯ammation was due to theviral protein and not the injection [64].

In conclusion, we have demonstrated that a DNAvaccine construct that consists of the nonviral, musclespeci®c promoter, MCK, has achieved similar DNAvaccine endpoints as plasmid constructs that employviral promoters. The information gained with MCKwill contribute to the design of future nonviral DNAvaccines. As indicated in this report, it was not necess-ary to attain high-level expression of the encoded geneto achieve protective immunity indicating that ``moreis not always better'' in the construction of DNA vac-cines. Rather all facets of the DNA vaccination pro-cess must be considered (e.g. the antigen, route andsite of delivery, etc.). To our knowledge, this is the

J.R. Gebhard et al. / Vaccine 18 (2000) 1837±18461844

®rst description of the use of this class of promoter tobe used for DNA vaccination that results in protectiveantiviral immunity. Although additional studies will beneeded to understand the T-cell responses more com-pletely, the HSV-2 speci®c MDV may ®nd utility inthe clinical setting.

Acknowledgements

We thank Robert Fujinami for critical reading ofthe manuscript, Kristi Neufeld for her expertise inphotomicroscopy, and Joesph Ricigliano for the par-ental MCK plasmid.

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