Immunity induced by DNA immunization with herpes simplex virus type 2 glycoproteins B and C

Vaccine 18 (2000) 875±883

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Immunity induced by DNA immunization with herpes simplex virus type 2 glycoproteins B and C Joseph C. Mester 1, Tara A. Twomey, Eric T. Tepe, David I. Bernstein* Division of Infectious Diseases, Children's Hospital Medical Center, 3333 Burnet Avenue, Cincinnati, OH 45229-3039, USA Received 7 April 1999; received in revised form 14 May 1999; accepted 21 May 1999

Abstract The complete sequence of herpes simplex virus type 2 (HSV-2) glycoproteins B and C (gB & gC) were cloned into plasmid expression vectors and evaluated in murine and guinea pig genital HSV-2 models. Balb/c mice were immunized with either pgB2 or pgC-2 plasmids intramuscularly (IM) or intradermally (ID). The vaccines induced HSV-2-speci®c neutralizing and ELISA IgG antibody, but little or no enhancement of viral clearance from the vagina was detected following intravaginal challenge. Immunization of guinea pigs with pgB-2 or pgC-2 induced ELISA IgG antibody; however, antibody titers were approximately one log10 unit lower than that seen in HSV-2 convalescent sera. IM immunization of guinea pigs with either plasmid also did not decrease vaginal viral shedding following vaginal challenge, but the severity of the acute disease and the subsequent number of recurrent lesion days were reduced in animals immunized with pgB-2. Lastly, IM immunization of latently infected guinea pigs with a combined gB-2 and gC-2 plasmid vaccine signi®cantly reduced the number of subsequent HSV-2 recurrences. DNA vectors expressing gB-2 or gC-2 were both immunogenic, although the gB-2 plasmid induced higher titers of antibody and signi®cantly reduced primary and recurrent herpetic disease in the guinea pig model. These results also suggest that immunotherapy with plasmid expression vectors may be e€ective against recurrent genital HSV-2 disease. # 1999 Elsevier Science Ltd. All rights reserved. Keywords: Genital herpes; HSV vaccine; DNA immunization

1. Introduction Primary infection by herpes simplex virus type 2 (HSV-2) through mucocutaneous `portals of entry' results in the virus establishing a long-term latent infection of the host's nervous system. Symptomatic and asymptomatic viral reactivation results in spread of infection with approximately 1 million new cases of genital HSV infections per year in the US [1]. Preclinical HSV vaccine approaches have included live and killed whole HSV, recombinant adenoviruses

* Corresponding author. Tel.: +1-513-636-4578; fax: +1-513-6367682. E-mail address: [email protected] (D.I. Bernstein) 1 Present Address: GeneMedicine Inc., 8301 New Trails Dr., The Woodlands, TX 77381

or vaccinia viruses expressing HSV proteins and whole protein or peptide subunit vaccines in adjuvants, reviewed in Ref. [2]. Most recently, live attenuated, replication impaired viruses and plasmid-based vaccines have been shown to be both immunogenic and protective in animal models [3±10]. The ideal antigen(s) or vaccine strategy for HSV has not been identi®ed. Individual recombinant viral proteins administered in adjuvant or expressed via adenovirus or vaccinia virus recombinants have been shown to confer substantial levels of protection in various animal model evaluations [11±15]. Recombinant proteins in adjuvant have also shown promise as therapeutic vaccines in animal models of recurrent genital herpes [16,17]. In man, however, prophylactic and therapeutically administered protein subunit vaccines have proven immunogenic but minimally protective [18±21]. At present it is unclear whether a vaccine

0264-410X/99/$ - see front matter # 1999 Elsevier Science Ltd. All rights reserved. PII: S 0 2 6 4 - 4 1 0 X ( 9 9 ) 0 0 3 2 5 - 4

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composed of a single, several or many viral proteins would confer the ultimate level of immunity against HSV. Additionally, whether the optimum components of a therapeutic vaccine would be identical to that of a prophylactic vaccine is unknown. Herpes simplex virus genomic DNA codes for approximately 80 proteins. Most or all are expressed during lytic infection, many are found in intact virions and at least 11 have been identi®ed as envelope glycoproteins. Many are targets for the human immune system, as revealed by immunoprecipitation and Western blot analysis of convalescent sera. These include the envelope glycoproteins B, C, D, E and G, virion components VP5 (capsid) and VP16 (tegument) and the ICP35 capsid assembly protein [22]. Human HSVimmune T-cell reactivity has been documented against gB, gC, gD, VP16, VP22 and UL21 tegument proteins and the viral dUTPase (UL50) [23,24]. DNA-based vaccines have shown ecacy in many animal models of infectious disease [25]. In general, these vaccines are easily and cheaply produced and are able to generate the full spectrum of adaptive immune responses. For a complex virus such as HSV, genetic immunization is well suited for the rational determination of the optimal viral protein components of an e€ective vaccine. To date, HSV-1 gB, gC, gD and gE have been shown to e€ective genetic immunogens in animal models of type-1 herpetic disease [7,25,26]. Of these, gB and gD appear to be superior to gC and gE. For HSV-2, gD and the combination of a truncated gB and full-length gD have also been shown to be e€ective using animal models of genital herpes [3,4]. When delivered intramuscularly in saline or bupivacaine, plasmids encoding gD-2 led to the development of HSV-2-speci®c immunity that signi®cantly lowered but did not completely block virus replication in the vagina of infected guinea pigs [3,10]. Additionally, the symptoms of both primary and recurrent HSV-2 disease were signi®cantly reduced in guinea pigs immunized with the gD-2 plasmids. To further assess the immunogenicity and protective potential of plasmid-expressed HSV-2 proteins in vaginal challenge models, the complete gB-2 and gC-2 genes were cloned into expression plasmids. The ability of a combination of the gB-2 and gC-2 expression plasmids to reduce recurrences in latently infected guinea pigs was also examined. 2. Methods 2.1. Viruses and cell lines HSV-2 strains 333 and 186 were propagated in Vero cells maintained on MEM with 5% FCS (Life Technologies, Gaithersburg MD). HSV-2 strain MS

was propagated in low passage rabbit kidney cells prepared from New Zealand White rabbits (Hazelton Research Products, Denver PA) maintained on Eagle's Basal medium with 10% FCS. Titration on Vero cell monolayers revealed titers of 108±109 pfu/ml. 2.2. Plasmid construction All reagents used were from Life Technologies unless otherwise noted. Vectors were digested and dephosphorylated with bacterial alkaline phophatase before use in cloning reactions. DNA ligations were done overnight at room temperature with T4 DNA ligase at 1:1 or 1:3 molar ratios of insert to vector. Transformations of dH5a CaCl2 competent cells were performed with 10±100 ng of ligated material. A single transformation colony was used to inoculate 2 ml of 2X YT media (Difco Laboratories, Detroit, MI) supplemented with 50 mg/ml ampicillin (Sigma Chemical Co., St. Louis, MO) and grown overnight at 378C with shaking. Plasmid DNA was isolated from the overnight cultures using a version of the rapid boil method [27] and inserts veri®ed by restriction enzyme analysis. Large cultures (1±2 l) were grown in 2X YT media with 50 mg/ml ampicillin and processed via standard techniques [28]. 2.2.1. Subcloning of glycoprotein B Five microgram of viral genomic DNA prepared from HSV-2 strain 333 was digested with KpnI as recommended by the supplier for 2±3 h at 378C. Following electrophoresis into low melt 0.5% agarose, the 8 kb KpnI `J' fragment was excised from the gel and subcloned into the KpnI site of pUC19. The KpnI `J' clone KJ11 was digested with BamHI and the 5 kb BamHI subfragment cloned into the BamHI site of pUC19. This clone, designated KJ11BamHI, was digested with MaeI (Boehringer Mannheim, Indianapolis IN) to isolate the glycoprotein B (gB) gene. The 2.8 kb MaeI fragment was subcloned into a modi®ed pSP72 vector designated pSP72(-NdeI)NdeI. This vector was constructed by deletion of the single NdeI site of pSP72 by digestion with NdeI, blunt-ending with Klenow and recircularization with T4 DNA ligase. A NdeI site linker (Genosys, The Woodlands TX) was ligated into SmaI-digested pSP72. A 2.8ckb KpnI-XbaI fragment containing the gB gene was then cloned into expression vector pcDNA3 (Invitrogen, San Diego, CA). The gB gene was cloned with 39 bp upstream of the translation start site. 2.2.2. Subcloning of glycoprotein C The HSV-2 333 strain 10 kb BamHI `A' fragment from pTYL302 (kindly provided by Joseph Glorioso) was digested with SalI and the 2.9 kb fragment containing the gC gene was cloned into the SalI site of

J.C. Mester et al. / Vaccine 18 (2000) 875±883

pUC19. The gC gene was excised from this clone as a 2 kb BssHII fragment and subcloned into the BssHII site of pUC19BssHII (BssHII linkers (Genosys) ligated into the SmaI site of pUC19). A 2 kb HindIII±XbaI fragment containing the gC gene was then cloned into expression vector pcDNA1 (Invitrogen). The gC gene was subcloned with 40 bp upstream of the translation start site. Genetic immunogens pgB-2 (gB in pcDNA3) and pgC-2 (gC in pcDNA1) were puri®ed by centrifugation over CsCl gradients. The integrity and purity of plasmid DNA for immunization was veri®ed by agarose gel electrophoresis. Plasmid DNA was bound to 2.6 mm gold beads and loaded into Tefzel tubing (McMaster-Carr, Chicago, IL) as recommended by the Accell gene gun manufacturer (PowderJect, Middleton, WI). The amount of plasmid coated to gold beads for gene gun inoculation was assessed by washing the bound plasmid o€ of the beads and reading the OD of the eluent at 260 nm. 2.3. Veri®cation of plasmid expression Expression of full-length gB-2 and gC-2 from the plasmid constructs was veri®ed by polyacrylamide gel electrophoresis following in vitro transcription and translation (using the TNT System from Promega, Madison, WI). In vivo expression of immunologically relevant gB-2 and gC-2 was veri®ed by Western blot reactivity and immunoprecipitation of the relevant HSV-2 glycoprotein by murine plasmid-immune sera. 2.4. Immunization of mice and guinea pigs Groups of 10±12 female Balb/c mice (Harlan Sprague Dawley, Indianapolis, IN) were inoculated. Plasmid DNA's at 0.4 mg/ml in sterile saline were injected bilaterally in the hind leg muscles so that each mouse received a total of 80 mg DNA. For gene gun inoculation, the ventral ¯ank of the mouse was shaved and two nonoverlapping injections of 0.8 mg DNA were delivered at a helium discharge pressure of 400 psi. Positive control mice received UV-inactivated HSV-2 in sterile saline bilaterally injected in hind leg muscles so that each mouse received a total of 107 pfu before inactivation. A second `booster' immunization was done in all groups 4 weeks after the initial immunization. For prophylaxis, female Hartley guinea pigs (Charles River, Wilmington, MA) were inoculated bilaterally in hind leg muscles with pgB-2 or pgC-2 plasmid DNA formulated in sterile saline at 0.5 mg/ml so that each animal received a total of 100 mg DNA. Two immunizations were given on days 0 and 112 for the initial experiment and three immunizations on days 0,

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14 and 63 for the second experiment. Control animals received mock injections of sterile saline. 2.5. ELISA and neutralization assays Sera were obtained prior to challenge and HSV-2speci®c antibody detected by ELISA (mouse and guinea pig) and plaque neutralization (mouse). ELISA assays for HSV-speci®c antibody were performed as previously described utilizing lectin-puri®ed HSV-2 glycoprotein as the solid phase antigen [10]. Neutralization assays were performed in 96-well tissue culture plates by incubating 100 pfu HSV-2 strain 333 with serial 3-fold dilutions of sera in the presence of complement (1:15 ®nal of Low-Tox M Rabbit complement, Accurate Chemical, Westbury, NY) for 3 h at 378C. Vero cells (2  104 per well) were then added and viral cytopathic e€ect assessed 24±48 h later. 2.6. HSV-2 vaginal challenge of mice and guinea pigs Animals were inoculated as previously described [3,29]. Brie¯y, mice were pre-treated with methylhydroxy-progesterone acetate (Sigma Chemical Co.) and the vaginal vault swabbed before installation of 4  104 pfu HSV-2 strain 186 in 20 ml [29]. Vaginal swabs were obtained on days 1, 2, 4, 6 and 8 postinfection and immediately titered on Vero cells. Guinea pigs were infected twice (within a 2-h period) with 5  105 pfu HSV-2 strain MS in 100 ml after swabbing the vaginal vault [3,10]. Vaginal swab samples were collected on days 1, 2, 4, 6, 8 and 10 postinoculation and titered on Vero cells. Guinea pigs were evaluated daily and the severity of primary genital skin disease quanti®ed on a scale of 0 (none) to 4 (severe vesiculoulcerative disease of the perineum) [3,10]. After recovery from primary disease, guinea pigs were observed daily from days 15±60 postinfection for evidence of spontaneous recurrent herpetic lesions [3,10]. Statistical signi®cance between vaccinated and unvaccinated groups was determined by two-tailed t-tests with Bonferroni correction for multiple comparisons. 2.7. Immunotherapy of HSV-2-infected guinea pigs The e€ect of pgB-2 and pgC-2 plasmid immunization of guinea pigs latently infected with HSV-2 was assessed as previously described [16]. Groups of 15 female guinea pigs were initially inoculated intravaginally with 5  105 pfu HSV-2 strain MS and the primary disease scored daily for 14 days. The guinea pigs were then randomized to equalize primary disease scores and then vaccinated on days 14 and 35 postinoculation. Fifty microgram of each plasmid immunogen (100 mg total) in sterile saline was injected bilaterally into the hind leg muscles. Negative control animals

Titers in responding mice. p < 0.05 compared to PBS IM group. c Immune response and protection of mice following two immunizations of plasmid expressing HSV-2 glycoproteins B or C (gB or gC). Groups of 10±12 female Balb/c mice were inoculated twice intradermally (ID) via the gene gun with 1.6 mg of DNA, or twice intramuscularly (IM) via a needle with 80 mg of DNA, as described in Methods. ELISA and neutralization assays were performed on sera drawn at week 7. Animals were challenged intravaginally with HSV-2 strain 186 at week 8 and vaginal swabs assayed for infectious virus on days 2, 4 and 6 postchallenge.

a

b

4.0 20.3 3.3 20.2b 3.4 20.3 3.9 20.3 (3.92 0.3)a 2.4 20.2b 3.9 20.1 4.720.4 4.820.3 3.520.4b 5.220.3 (5.420.3)a 3.620.3b 4.920.3 2.020.1 2.520.1 1.020.2 < 1.5 (0.42 0.4)a 3.320.1 < 1.5 10/10 9/10 9/10 4/10 12/12 0/12

50% neutralization titer

gB-2 gene gun gB-2 IM gC-2 gene gun gC-2 IM UV-HSV-2 IM PBS IM

2.420.03 2.320.1 2.020.2 1.120.4 (2.720.03)a 3.320.1 < 1.5

Day 2 ELISA titer

Day 4

Vaginal viral titers (log10) following infectious challenge with HSV-2 (186) HSV-2-speci®c antibody titers post-vaccination/prechallenge (log10) No. of responders per group Vaccine group

Table 1 Antibody titers in mice after immunization and vaginal viral titers after HSV-2 challengec

3.420.4 2.720.3 2.620.4 3.820.3 (3.420.4)a 1.420.3b 3.320.3

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Day 6

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received mock injections of 100 ml of sterile saline. Recurrent lesions were examined daily from day 14 through day 60 postinoculation. Statistical signi®cance between the vaccinated and unvaccinated group was determined by a two-tailed t-test. 3. Results Both murine and guinea pig models of genital herpes were used to assess the immunogenicity and protective potential of plasmid immunogens encoding full length HSV-2 gB and gC. In the murine model, female Balb/c mice were immunized twice intradermally (ID) via the gene gun with 1.6 mg of DNA, or twice intramuscularly (IM) via a needle with 80 mg of DNA. Intramuscular saline (vehicle) injections were performed for the negative controls, as preliminary results indicated that equivalent doses of `null' plasmid IM in saline did not elicit detectable HSV-speci®c immunity (data not shown). As shown in Table 1, positive control mice immunized with two doses of UV-inactivated HSV-2 IM generated an average of 3.3 log10 units of ELISA and HSV-2-neutralizing antibody. Mice immunized with the gB-2 plasmid (pgB-2), by either the IM or ID route, generated an average of from 2.0 to 2.5 log10 units of ELISA and HSV-2-neutralizing antibody. Gene gun immunization of mice with the gC-2 plasmid pgC-2 resulted in 90% seroconversion, with average log10 antibody titers of 2.0 (ELISA) and 1.0 (neutralization). Intramuscular immunization of mice with pgC-2 resulted in 40% seroconversion. The IMimmunized gC-2 responder mice had average HSV-2speci®c ELISA titers of 2.7 log10 units, with little or no detectable neutralizing activity. On day 56, all mice were challenged intravaginally with HSV-2 strain 186 and viral shedding monitored on days 2, 4 and 6 postchallenge. Immunization with UV-inactivated HSV-2 signi®cantly ( p < 0.05) reduced vaginal virus titers compared to the control group on all days examined. Among the plasmid immunized mice, signi®cant ( p < 0.05) reductions in vaginal virus titers were evident on day 2 for pgC-2 gene gun-immunized mice (given 3.2 mg total DNA) and on day 4 for pgB-2 IM-immunized mice (given 160 mg total DNA). Among the individual plasmid immune mice, there was no correlation between higher antibody titers (ELISA or neutralization) and enhanced clearance of virus from the vagina (data not shown). In guinea pigs, an initial experiment evaluated the immunogenicity and ecacy of 100 mg of pgB-2 administered IM in saline on days 0 and 112. Sera obtained on days 49 and 142 demonstrated average log10 HSV2-speci®c ELISA titers of 1.5 and 2.8, respectively (Table 2). Following intravaginal challenge on day 154 (approximately ®ve months from the initial immuniz-

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Table 2 Antibody response in guinea pigs immunized with pgB-2a Group

Day postinoculation

No. of responders per group

HSV-2-speci®c IgG ELISA titers (log10)

gB-2 plasmid immunized

49 142 35 46 35 and 46

13/15 15/15 5/5

1.52 0.2 2.82 0.07 3.32 0.02 3.42 0.06 < 1.4

HSV-2-infected Uninfected/unimmunized

0/15

a ELISA titers of pgB-2 plasmid-immunized and HSV-2-immune guinea pigs. Female Hartley guinea pigs were inoculated twice IM with 100 mg of pgB-2. Negative control animals received mock injections of sterile saline. Convalescent sera from unimmunized animals challenged intravaginally with HSV-2 strain MS was used as a positive control.

ation) with HSV-2 strain MS, the pgB-2-immunized group experienced a signi®cantly milder course of primary disease than the PBS-immunized negative controls (Fig. 1(A)). Mean lesion scores were reduced on days 4±7 postinfection ( p < 0.001). Viral shedding from the vagina through day 8 postinfection, however, was not decreased (Fig. 1(B)). The cumulative incidence of recurrent disease, monitored through day 60 postinfection, was reduced in the pgB-2-immunized group by approximately 50% (Fig. 1(C), p < 0.005 versus control). To compare pgB-2 and pgC-2 prophylactic immunization in guinea pigs, groups of 12 animals were immunized IM with 100 mg of either plasmid in saline on days 0, 14 and 63. Animals were challenged intravaginally with HSV-2 on day 91 and the course of primary and recurrent disease followed daily. ELISA antibody titers following the second and third immunization were comparable for pgB-2 and pgC-2 immunized animals, although the pgB-2 titers were slightly higher at both time points (Table 3). The course of primary disease was not altered by pgC-2 immunization, but was signi®cantly lower in pgB-2-immunized animals (Fig. 2(A), p < 0.05 for days 4±7). Vaginal viral shedding after challenge was similar in the plasmid-immunized and unimmunized control groups (data not shown). Recurrent disease was decreased in both plasmid-immunized groups, from a mean of 9.2 recurrences per con-

trol animal to a mean of 7.3 recurrences for pgC-2immunized animals ( p = NS) and 4.6 recurrences for pgB-2-immunized animals ( p < 0.01) (Fig. 2(B)). To evaluate the ability of plasmids to act as therapeutic immunogens, guinea pigs were ®rst infected intravaginally with HSV-2. Primary disease was monitored for 14 days. Animals received of a combination of 50 mg of each pgB-2 and pgC-2 (for a total of 100 mg DNA given IM in saline) on days 14 and 35 following HSV-2 inoculation. As shown in Fig. 3, the combined gB-2+gC-2 plasmid immunotherapy signi®cantly decreased the number of days recurrent lesions observed ( p < 0.001).

4. Discussion Viral envelope glycoproteins are prime prospects for protein subunit and DNA-based vaccines. They are expressed on free virions and in infected cells and are targeted by both humoral and cellular mechanisms of immunity. HSV gB is a major target of virus-neutralizing antibody in convalescent patient sera and has been shown to elicit CD4+ T-cells following natural infection [22]. HSV gC is also a target of antibody and Tcells following natural infection [22]. Additionally, HSV gC is an important mediator of viral pathogenesis. By binding complement component C3, gC aids

Table 3 Antibody response in guinea pigs immunized with pgB-2 or pgC-2a Group

No. of responders per group

Day postinoculation

HSV-2-speci®c IgG ELISA titers (log10)

gB-2 plasmid immunized

12/12

gC-2 plasmid immunized

12/12

56 84 56 84 35 46 35 and 46

2.32 0.1 2.82 0.06 1.72 0.4 2.52 0.05 3.42 0.07 3.92 0.2 < 1.4

HSV-2-infected Uninfected/unimmunized

6/6 0/12

a ELISA titers of pgB-2 and pgC-2 plasmid-immunized guinea pigs. Female Hartley guinea pigs were inoculated three times IM with 100 mg of pgB-2 or pgC-2 plasmids. Negative control animals received mock injections of sterile saline. Convalescent sera from unimmunized animals challenged intravaginally with HSV-2 strain MS was used as a positive control.

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Fig. 2. Primary and recurrent herpetic disease in pgB-2 and pgC-2immunized and control guinea pigs. (A) Primary disease, (B) recurrent disease. Female Hartley guinea pigs were inoculated three times IM with 100 mg of plasmid DNA. Negative control animals received mock injections of sterile saline. Animals were challenged intravaginally with HSV-2 and genital herpetic disease scored as described in Methods.

Fig. 1. Primary and recurrent herpetic disease in pgB-2-immunized and control guinea pigs. (A) Primary disease, (B) viral shedding from the vagina, (C) recurrent disease. Female Hartley guinea pigs were inoculated twice IM with 100 mg of pgB-2. Negative control animals received mock injections of sterile saline, as described in Methods. Primary disease was observed daily and scored on a scale of scale of 0 (none) to 4 (severe vesiculoulcerative disease of the perineum). Vaginal swab samples were collected on days 1, 2, 4, 6, 8 and 10 postinfection and titered on Vero cells. After recovery from primary disease, guinea pigs were observed daily from days 15±60 postinfection for evidence of spontaneous recurrent herpetic lesions.

the virus in evading the immune system [30]. In the alphaherpesvirus pseudorabies virus (PrV) system, gC is a major protective target of virus-speci®c cytotoxic T-cells (CTLs) in the natural host [31]. Genetic immunization with PrV gC is protective against PrV-induced Aujeszky's disease [32].

Fig. 3. Immunotherapy of recurrent herpetic disease with pgB-2 and pgC-2. Groups of 15 female guinea pigs recovering from primary HSV-2 infection were given immunotherapeutic inoculations of 50 mg each of pgB-2 and pgC-2 (100 mg total) on days 14 and 35 postinfection. Negative control animals received mock injections of 100 ml of sterile saline. Recurrent lesions were examined daily from days 15± 60.

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In non-genital murine models, genetic immunogens encoding HSV gB and gC have been shown to be protective. A full length gB-1 plasmid elicited neutralizing antibody and CD4+ T-cells able to protect against zosteriform spread of HSV-1 [7], while genetic immunization with a truncated/secretory form of gB-2 induced neutralizing antibody and protection from a lethal intraperitoneal (IP) challenge [4]. Immunization with a gC-1 plasmid yielded virus-speci®c antibody and protection from lethal IP challenge when high plasmid doses were given to mice [26]. In this report, genetic immunogens encoding full length HSV-2 gB and gC were constructed and analyzed in guinea pig and murine models of genital herpes. The result of two prophylactic immunizations given at a 4-week interval was examined in mice. Preliminary experiments indicated that at least two injections of 50 mg of plasmid in saline were required for complete seroconversion following IM inoculation in mice (data not shown). This investigation utilized two 80-mg doses for IM inoculation. For gene gun inoculation of mice, the 1.6 mg dose administered was the maximum amount of DNA able to be loaded onto gold beads and administered as two nonoverlapping ID inoculations. For the guinea pig experiments, some variation in immunization schedule was explored, with either two or three prophylactic immunizations performed. In both cases a 100 mg immunizing dose was utilized. Prior work with a gD-2 expression plasmid demonstrated little di€erence in outcome following immunization of guinea pigs with 50, 100 or 250 mgs of plasmid given IM in saline [3]. Two or three immunizations with pgB-2 or pgC-2 elicited HSV-speci®c serum IgG in either mice or guinea pigs. The titers elicited by the genetic immunogens approached but were approximately 1 log10 unit lower than that induced by immunization with whole virus. In general, the IgG ELISA titers elicited by immunization with pgB-2 were higher but comparable to that elicited by immunization with pgC-2. In mice, HSV-2speci®c neutralizing antibody was elicited by immunization with gB-2, with titers approximately 1 log10 unit lower than that following immunization with whole HSV-2. Both IM and ID routes were used to vaccinate mice with pgB-2 and pgC-2. Vaccination via either route led to the development of HSV-2-speci®c antibody and similar neutralizing titers following immunization with pgB-2. However, only 4 of 10 mice immunized with pgC-2 by the IM route seroconverted, whereas 9 out of 10 seroconverted after gene gun administration of pgC-2. Variability in the number of immune responders following IM immunization of plasmid in saline has been documented by other investigators [26,33]. Advantages of the gene gun to deliver genetic immunogens intradermally versus IM inoculation of plasmid in

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saline may therefore be the more consistent immune response and the requirement for relatively lower doses of plasmid [33]. Alternative plasmid formulations, such as bupivacaine [34], cationic lipids [35] or synthetic polymers [36], may enhance the frequency and extent of the immune response to genetic immunogens delivered IM. Vaccination of mice by either the IM or ID route failed to confer signi®cant protection from vaginal replication of HSV-2. This may be due to the high virulence in mice of the HSV-2 186 strain used for challenge. Higher or more frequent doses of the genetic immunogens by either route may have elicited higher serum IgG titers and enhanced protection from infectious challenge. Other investigators have also reported the absence of `sterilizing immunity' to HSV following vaccination [3,10]. Related reports have also shown that genetic immunization with gB-1 was able to protect against a low but not a high vaginal challenge dose of HSV-1 strain McKrae [37]. While no reduction was seen in the amount of viral shedding from the vagina in pgB-2 or pgC-2 immunized guinea pigs following viral challenge, signi®cant protection against the development of primary genital lesions was observed in animals vaccinated with pgB-2. Additionally, prophylactic vaccination with pgB-2 led to a decrease in the number of recurrent lesions following resolution of primary disease. These results suggest that genetic immunization with gB-2 may be e€ective against HSV-2. Reports from several investigators have suggested that gD may be the best single prophylactic immunogen for HSV [11,14,26]. Still, a combination of gD with other structural proteins like gB or gC and nonstructural viral proteins may confer optimum levels of protection [14]. When the pgB-2 and pgC-2 plasmids were combined and administered immunotherapeutically during the course of recurrent disease, a signi®cant reduction in the number of lesions was seen. Previous evaluations of gB and or gD subunit vaccines have shown that immunization can reduce recurrences in the guinea pig model of genital herpes [16,17], but this is the ®rst indication that plasmid vaccines may also be e€ective. When gD encoding plasmid vaccines have been evaluated in this model, however, they have not been e€ective except when combined with unique adjuvants [38] (L. Stanberry, personal communication). DNA-based vaccines may be ideal for rapid preclinical analysis of combinations of immunogenic/protective viral sequences. Their immunogenicity may be further enhanced by molecular augmentation with cytokine expression plasmids [39], targeting of professional APCs in vivo [40] or other molecular methods for rerouting the expressed antigen [41,42]. They may also ®nd quick application for immunotherapy of cancer and infectious diseases.

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References [1] Wald A, Corey L. The clinical features and diagnostic evaluation of genital herpes. In: Stanberry LR, editor. Genital and neonatal herpes. NY: John Wiley and Sons, 1996. p. 109±37. [2] Bernstein DI, Stanberry LR. Herpes simplex vaccines. Vaccine 1999;17:1681±9. [3] Bourne N, Stanberry LR, Bernstein DI, Lew D. DNA immunization against experimental genital herpes simplex virus infection. J Inf Dis 1996;173:800±7. [4] McClements WL, Armstrong ME, Keys RD, Liu MA. Immunization with DNA vacccines encoding glycoprotein D or glycoprotein B, alone or in combination, induces protective immunity in animal models of herpes simplex virus-2 disease. PNAS USA 1996;93:11414±20. [5] Boursnell MEG, Entwisle C, Blakeley D, et al. A genetically inactivated herpes simplex virus type 2 (HSV-2) vaccine provides e€ective protection against primary and recurrent HSV-2 disease. J Inf Dis 1997;175:16±25. [6] Hariharan MJ, Driver DA, Townsend K, et al. DNA immunization against herpes simplex virus: enhanced ecacy using a sindbis virus-based vector. J Virol 1998;72:950±8. [7] Manickan E, Rouse RJ, Yu Z, et al. Genetic immunization against herpes simplex virus: protection is mediated by CD4+ T lymphocytes. J Immunol 1995;155:259±65. [8] Spector FC, Kern ER, Palmer J, Kaiwar R, Cha TA, Brown P, Spaete RR. Evaluation of a live attenuated recombinant virus RAV 9395 as a herpes simplex virus type 2 vaccine in guinea pigs. J Inf Dis 1998;177:1143±54. [9] Walker J, Laycock KA, Pepose JS, Leib DA. Postexposure vaccination with a virion host shuto€ defective mutant reduces UV-B radiation-induced ocular herpes simplex virus shedding in mice. Vaccine 1998;16:6±8. [10] Bernstein DI, Tepe ER, Mester JC, Arnold RL, Stanberry LR, Higgins T. E€ects of DNA immunization formulated with bupivacaine in murine and guinea pig models of genital herpes simplex virus infection. Vaccine 1999;9:1964±9. [11] Blacklaws BA, Krishna S, Minson AC, Nash AA. Immunogenicity of herpes simplex virus type 1 glycoproteins expressed in vaccinia virus recombinants. Virology 1990;177:727±36. [12] Gallichan WS, Johnson DC, Graham FL, Rosenthal KL. Mucosal immunity and protection after intranasal immunization with recombinant adenovirus expressing herpes simplex virus glycoprotein B. J Inf Dis 1993;168:622±9. [13] Mester JC, Rouse BT. The mouse model and understanding immunity to HSV. Rev Inf Dis 1991;13:S935±S945. [14] Ghiasi H, Nesburn AB, Wechsler SL. Vaccination with a cocktail of seven recombinantly expressed HSV-1 glycoproteins protects against ocular HSV-1 challenge more eciently than vaccination with any individual glycoprotein. Vaccine 1996;14:107±12. [15] Manickan E, Francotte M, Kuklin N, et al. Vaccination with recombinant vaccinia viruses expressing ICP27 induces protective immunity against herpes simplex virus through CD4+ Th1+ T-cells. J Virol 1995;69:4711±6. [16] Bernstein DI, Harrison CJ, Jenski LJ, et al. Cell-mediated immunologic responses and recurrent genital herpes in the guinea pig. J Immunol 1991;146:3571±7. [17] Stanberry LR. The concept of immune-based therapies in chronic viral infections. J AIDS 1994;7:S1±S5. [18] Mertz GJ, Ashley R, Burke RL, et al. Double-blind placebo controlled trial of a herpes simplex virus type 2 glycoprotein vaccine in persons at high risk for genital herpes infection. J Inf Dis 1990;161:653±60. [19] Langenberg AGM, Burke RL, Adair SF, et al. A recombinant

[20] [21]

[22] [23]

[24] [25] [26] [27] [28] [29] [30] [31] [32] [33] [34] [35]

[36]

[37] [38]

[39]

[40]

glycoprotein vaccine for herpes simplex type 2: safety and ecacy. Ann Int Med 1995;122:889±98. Bishopp F. Chiron's stock dives on news of herpes vaccine failure. Bioworld 1996;7:1±4. Straus SE, Wald A, Kost RG, et al. Immunotherapy of recurrent genital herpes with recombinant herpes simplex virus type 2 glycoproteins D and B: results of a placebo-controlled vaccine trial. J Inf Dis 1997;176:1129±34. Mester JC, Milligan GN, Bernstein DI. The immunobiology of herpes simplex virus. In: Stanberry LR, editor. Genital and neonatal herpes. NY: John Wiley and Sons, 1996. p. 49±91. Koelle DM, Frank JM, Johnson ML, Kwok WW. Recognition of herpes simplex virus type 2 tegument proteins by CD4 T-cells in®ltrating human genital herpes lesions. J Virol 1998;72:7476± 83. Koelle DM, Corey L, Burke RL, et al. Antigenic speci®cities of human CD4+ T-cell clones recovered from recurrent genital herpes simplex virus type 2 lesions. J Virol 1994;68:2803±10. Manickan E, Karem KL, Rouse BT. DNA vaccines: a modern gimmick or a boon to vaccinology? Crit Rev Immunol 1997;17:139±54. Nass PH, Elkins KL, Weir JP. Antibody response and protective capacity of plasmid vaccines expressing three di€erent herpes simplex virus glycoproteins. J Inf Dis 1998;178:611±7. Holmes DS, Quigley M. A rapid boiling method for the preparation of bacterial plasmids. Anal Biochem 1981;114:193±7. Godson GN, Vapnek D. A simple method of preparing large amounts of FX174 RF I supercoiled DNA. Biochim Biophys Acta 1973;299:516±20. Milligan GN, Bernstein DI. Interferon gamma (IFNg) enhances resolution of herpes simplex virus type 2 (HSV-2) infection of the murine genital tract. Virology 1997;229:259±68. Lubinski JM, Wang L, Soulika AM, et al. Herpes simplex virus type 1 glycoprotein gC mediates immune evasion in vivo. J Virol 1998;72:8257±63. Mettenleiter TC. Immunobiology of pseudorabies (Aujeszky's disease). Vet Immunol Immunopathol 1996;54:221±9. Gerdts V, Jons A, Makoschey B, Visser N, Mettenleiter TC. Protection of pigs against Aujeszky's disease by DNA vaccination. J Gen Virol 1997;78:2139±46. Barry MA, Johnston SA. Biological features of genetic immunization. Vaccine 1997;15:788±91. Wells DJ. Improved gene transfer by direct plasmid injection associated with regeneration in mouse skeletal muscle. FEBS Lett 1993;332:179±82. Ishii N, Fukushima J, Kaneko T, Okada E, et al. Cationic liposomes are a strong adjuvant for a DNA vaccine of human immunode®ciency virus type 1. AIDS Res Human Retroviruses 1997;13:1421±8. Mumper RJ, Duguid JG, Anwer K, Barron MK, Nitta H, Rolland AP. Polyvinyl derivatives as novel interactive polymers for controlled gene delivery to muscle. Pharm Res 1996;13:701± 9. Kuklin N, Daheshia M, Karem K, Manickan E, Rouse BT. Induction of mucosal immunity against herpes simplex virus by plasmid DNA immunization. J Virol 1997;71:3138±45. Harrison CJ, Miller RL, Bernstein DI. E€ect of S28463 as an adjuvant for an immunotherapeutic HSV glycoprotein D (gD) DNA vaccine: Reduction of recurrent genital HSV disease in guinea pigs. New Orleans, LA: ICAAC, 1996. Kim JJ, Trivedi NN, Nottingham LK, et al. Modulation of amplitude and direction of in vivo immune responses by coadministration of cytokine gene expression cassettes with DNA immunogens. Eur J Immunol 1998;28:1089±103. Boyle JS, Brady JL, Lew AM. Enhanced responses to a DNA vaccine encoding a fusion antigen that is directed to sites of immune induction. Nature 1998;392:408±11.

J.C. Mester et al. / Vaccine 18 (2000) 875±883 [41] Rodriguez F, Zhang J, Whitton JL. DNA immunization: ubiquitination of a viral protein enhances cytotoxic T-lymphocyte induction and antiviral protection but abrogates antibody induction. J Virol 1997;71:8497±503.

883

[42] Wu TC, Guarnieri FG, Staveley-O'Carroll KF, et al. Engineering an intracellular pathway for major histocompatibility complex class II presentation of antigens. PNAS USA 1995;92:11671±5.